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  • C12N2310/00—Structure or type of the nucleic acid
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  • C12N2320/35—Special therapeutic applications based on a specific dosage / administration regimen

Definitions

• This invention relates to novel microRNA-29 (miR-29) compounds, compositions comprising the same, and their use in therapy.
• miR-29 compounds and compositions that are particularly effective and safe in the treatment of diseases that involve localised collagen dysregulation, such as tendon damage including tendinopathy, and tissue fibrosis including Peyronie’s disease and Dupuytren’s disease.
• advantageous dosages and treatment regimens for miR-29 compounds are also provided.
• MicroRNAs are small, non-coding RNAs that work alongside the RNA-Induced Silencing Complex (RISC) to reduce or prevent the translation of messenger RNAs (mRNA) containing complementary target sequences.
• RISC RNA-Induced Silencing Complex
• miR-29 Members of the miR-29 family have been implicated as post-transcriptional regulators of the extracellular matrix, e.g., collagen.
• Dysregulated collagen synthesis in particular the overproduction of one or more types of collagen, contributes to the pathology seen in certain diseases, such as tendon damage including tendinopathy, and tissue fibrosis including Peyronie’s disease and Dupuytren’s disease.
• miR-29 regulates key disease pathways in collagen production in tendon damage.
• miR-29 e.g., miR-29a, also referred herein as miR29a
• collagen 3 col3
• tendon disease there is a reduction of miR-29 levels and an overproduction of col3, which leads to mechanically inferior tendon tissue that is prone to further damage.
• collagen 3 This encompasses conditions that involve localised collagen dysregulation and require reducing or inhibiting expression of one or more types of collagen, such as collagen 3, including but not limited to tendon damage including tendinopathy (e.g., lateral epicondylitis, also known as tennis elbow), and tissue fibrosis such as Peyronie’s disease and Dupuytren’s disease.
• tendon damage including tendinopathy (e.g., lateral epicondylitis, also known as tennis elbow), and tissue fibrosis such as Peyronie’s disease and Dupuytren’s disease.
• tendon damage e.g., lateral epicondylitis, also known as tennis elbow
• tissue fibrosis such as Peyronie’s disease and Dupuytren’s disease.
• the compounds described herein mimic the activity of natural miR-29 while certain chemical modifications provide improved pharmacological properties, such as increased activity, stability and cellular uptake, yet without causing immunogenicity issues.
• a miR-29 compound in the treatment of diseases such as tendon damage including tendinopathy, and tissue fibrosis such as Peyronie’s disease and Dupuytren’s disease.
• the inventors have shown that administering by local (intra-tendon) injection of a miR-29 compound to a damaged tendon improves tendon healing and tendon fibre quality, e.g., in a mouse model of tendinopathy.
• therapeutic efficacy can be achieved with only a single administration (i.e., a single dose).
• Dosages suitable for therapy e.g., human therapy
• Data are also available to support therapeutic efficacy of miR-29 compounds in tissue fibrosis such as Dupuytren’s and Peyronie’s disease.
• the miR-29 compounds can be administered locally at the site of the pathology (intralesionally) in a simple aqueous carrier.
• Data provided herein show that there is negligible systemic exposure or distribution into other tissues following local intralesional administration, thereby providing a particularly safe therapy.
• a single dose can be therapeutically effective, only one visit to a healthcare professional is required, thereby enabling a simple, cost-effective and fast yet effective treatment.
• the present invention provides miR-29 compounds, compositions comprising the same, and their use in therapy.
• the invention therefore provides a miR-29 compound or a salt thereof comprising a guide (antisense) strand and a passenger (sense) strand, which mimics the biological activity of native miR-29a, for example the downregulation of collagen 3 expression.
• the compounds of the invention are therefore capable of reducing or inhibiting collagen 3 expression.
• the miR-29 compounds provided herein can also mimic the biological activity of the other miR-29 family members.
• the miR-29 compound can therefore also be referred to as a miR-29 mimic, or a miR-29 mimetic compound.
• the guide and passenger strands of the miR-29 compounds are oligonucleotides, i.e., they contain nucleosides linked by intemucleoside linkages.
• the guide and passenger strands form a duplex, typically containing a number of mismatches and an overhang in at least one strand.
• the miR-29 compounds described herein are typically chemically modified, which means that at least one sugar, intemucleoside linkage or nucleobase has a chemical structure that differs from that in naturally-occurring miR-29.
• the chemical modification(s) can be selected to improve the pharmacological properties of the miR-29 compound, e.g. , by enhancing its activity and/or stability.
• exemplary modified sugars are 2’-fluoro ribose, 2’-O-methyl ribose or 2’-deoxy ribose.
• An exemplary modified intemucleoside linkage is a phosphorothioate intemucleoside linkage.
• An exemplary modified nucleobase is a 5-methyl cytosine.
• the miR-29 compounds may also be conjugated (i.e., covalently linked) to a lipophilic moiety such as a cholesterol moiety, typically at the 3’ end of the passenger strand. Such conjugation to a lipophilic moiety may improve the properties of the miR-29 compound for in vivo use, e.g., by improving its cellular uptake.
• the invention therefore provides a miR-29 compound or a salt thereof comprising:
• a guide strand comprising a nucleobase sequence which differs from 5’-UAGCACCAUCUGAAAUCGGUUA-3’ (SEQ ID NO: 1) at no more than five (or no more than four, three, two, or one) positions outside of the sequence 5’-AGCACCA-3’ (SEQ ID NO: 8, which is the seed sequence), and optionally at no more than three (or no more than two, or one) positions within the seed sequence; wherein at least one nucleoside comprises a 2’ -fluoro ribose and optionally wherein at least one nucleoside comprises a 2’-O-methyl ribose; and
• a passenger strand comprising a nucleobase sequence that differs from 5’-ACUGAUUUCUUUUGGUGUUCAG-3’ (SEQ ID NO: 2) at no more than five (or no more than four, three, two, or one; for example at no more than three) positions; and wherein at least one nucleoside comprises a 2’-O-methyl ribose and at least one nucleoside comprises an unmodified ribose.
• a nucleobase sequence “differs” from another nucleobase sequence if there are different, additional, or missing nucleobases in corresponding positions.
• a nucleobase sequence that differs from another nucleobase sequence at no more than a certain number of positions also encompasses a nucleobase sequence that differs at zero positions, i.e., the nucleobase sequences may be identical.
• nucleobase sequence of the guide strand may differ from the nucleobase sequence of native miR-29a (SEQ ID NO: 1) outside of the seed sequence of native miR-29a (AGCACCA, SEQ ID NO: 8) at no more than five positions, e.g., at no more than four positions, no more than three positions, no more than two positions, or at no more than one position, preferably at no more than three positions.
• nucleobase sequence of the guide strand outside of the seed sequence is identical to the corresponding positions in SEQ ID NO: 1.
• the seed sequence of the guide strand of the miR-29 compound may differ from the seed sequence of native miR-29a (AGCACCA, SEQ ID NO: 8) at no more than three positions, e.g., at no more than two positions, or at no more than one position.
• the seed sequence is identical to SEQ ID NO: 8.
• the nucleobase sequence of the passenger strand may differ from 5’-ACUGAUUUCUUUUGGUGUUCAG-3’ (SEQ ID NO: 2) at no more than five positions, for example at no more than four positions. In preferred embodiments, the nucleobase sequence of the passenger strand differs from SEQ ID NO: 2 at no more than three positions. In other embodiments, the nucleobase sequence of the passenger strand differs from SEQ ID NO: 2 at no more than two positions. In a specific embodiment, the nucleobase sequence of the passenger strand differs from SEQ ID NO: 2 at no more than one position.
• nucleobase sequence of the passenger strand has the sequence of SEQ ID NO: 2 or a sequence that has one different, additional, or missing nucleobase relative to SEQ ID NO: 2.
• SEQ ID NO: 3 has one missing nucleobase relative to SEQ ID NO: 2.
• SEQ ID NO: 2 and SEQ ID NO: 3 differ only in one position, also provided herein are embodiments wherein the passenger strand differs from SEQ ID NO: 3 in no more than five etc. positions, as above.
• the passenger strand comprises a nucleobase sequence that differs from 5 ’ - ACUGAUUUCUUUUGGUGUUC AG-3 ’ (SEQ ID NO: 2) at no more than five (e.g, no more than four, three, two, or one; such as no more than three) positions outside of the sequence 5’-UUU- 3’ and/or 5’-UUCA-3’ underlined in SEQ ID NO: 2 above, which correspond to positions 10-12 and 18-21 of SEQ ID NO: 2.
• the nucleobase sequence of the passenger strand differs from SEQ ID NO: 2 only outside of the sequences 5’-UUU-3’ and 5’-UUCA-3’.
• the nucleobase sequence of the passenger strand may differ from SEQ ID NO: 2 at no more than two positions outside of the sequences 5’-UUU-3’ and 5’-UUCA-3’.
• the nucleobase sequence of the passenger strand may differ from SEQ ID NO: 2 at no more than one or two positions outside of the sequences 5’-UUU-3’ and 5’-UUCA-3’, for example at no more than one position.
• such a passenger strand is combined with a fluorinated guide strand as described herein (e.g., comprising one or more 2 ’-fluoro ribonucleosides, such as wherein at least 50% of the nucleosides are 2’-fluoro ribonucleosides, or wherein all nucleosides except one or two 2’-O-methyl ribose nucleosides, preferably located at the 3’ end, are 2’-fluoro ribonucleosides).
• a fluorinated guide strand as described herein (e.g., comprising one or more 2 ’-fluoro ribonucleosides, such as wherein at least 50% of the nucleosides are 2’-fluoro ribonucleosides, or wherein all nucleosides except one or two 2’-O-methyl ribose nucleosides, preferably located at the 3’ end, are 2’-fluoro rib
• the guide strand has a nucleobase sequence comprising at least 7 consecutive nucleobases of the nucleobase sequence of SEQ ID NO: 1.
• the guide strand may have a nucleobase sequence comprising at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or at least 21 consecutive nucleobases of the nucleobase sequence of SEQ ID NO: 1.
• the guide strand has a nucleobase sequence comprising at least 18 consecutive nucleobases of the nucleobase sequence of SEQ ID NO: 1.
• the passenger strand may have a nucleobase sequence comprising at least 7 consecutive nucleobases of the nucleobase sequence of SEQ ID NO: 2.
• the passenger strand may have a nucleobase sequence comprising at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or at least 21 consecutive nucleobases of the nucleobase sequence of SEQ ID NO: 2.
• the passenger strand has a nucleobase sequence comprising at least 18 consecutive nucleobases of the nucleobase sequence of SEQ ID NO: 2.
• the guide strand comprises a nucleobase sequence that is at least 85% identical across the entire length of SEQ ID NO: 1.
• the guide strand may comprise a nucleobase sequence that is at least 90%, 95%, 96%, 97%, 98%, at least 99%, or 100% identical across the entire length of SEQ ID NO: 1.
• the guide strand has a nucleobase sequence as defined in the preceding sentence and it comprises the sequence 5’-AGCACCA-3’ (SEQ ID NO: 8) at positions that correspond to positions 2-8 of SEQ ID NO: 1.
• the nucleobase sequence of the guide strand may differ from SEQ ID NO: 1 only outside of positions 2-8 of SEQ ID NO: 1. The difference may be defined by percentage identity or by the number of identical/different nucleobases, as described above.
• the passenger strand may comprise a nucleobase sequence that is at least 85% identical across the entire length of SEQ ID NO: 2 or 3.
• the passenger strand may comprise a nucleobase sequence that is at least 90%, 95%, 96%, 97%, 98%, at least 99%, or 100% identical across the entire length of SEQ ID NO: 2 or 3.
• the passenger strand has a nucleobase sequence as defined in the preceding sentence and it comprises the sequence 5’-UUU-3’ and/or 5’-UUCA-3’ at positions that correspond to positions 10-12 and 18-21 of SEQ ID NO: 2.
• the nucleobase sequence of the passenger strand may differ from SEQ ID NO: 2 only outside of positions 10-12 and/or 18-21 of SEQ ID NO: 2.
• the nucleobase sequence of the passenger strand may differ from SEQ ID NO: 2 only outside of positions 10-12 and 18-21 of SEQ ID NO: 2. The difference may be defined by percentage identity or by the number of identical/different nucleobases, as described above.
• such a passenger strand is combined with a fluorinated guide strand as described herein (e.g., comprising one or more 2’ -fluoro ribonucleosides, such as wherein at least 50% of the nucleosides are 2’-fluoro ribonucleosides, or wherein all nucleosides except one or two 2’-O-methyl ribose nucleosides, preferably located at the 3’ end, are a 2’-fluoro ribonucleosides).
• a fluorinated guide strand as described herein (e.g., comprising one or more 2’ -fluoro ribonucleosides, such as wherein at least 50% of the nucleosides are 2’-fluoro ribonucleosides, or wherein all nucleosides except one or two 2’-O-methyl ribose nucleosides, preferably located at the 3’ end, are a 2’-flu
• the guide strand and/or the passenger strand may consist of the nucleobase sequence as indicated above.
• the guide strand comprises or consists of the nucleobase sequence 5’-UAGCACCAUCUGAAAUCGGUUA-3’ (SEQ ID NO: 1)
• the passenger strand comprises or consists of the nucleobase sequence 5’-ACUGAUUUCUUUUGGUGUUCAG-3’ (SEQ ID NO: 2) or
• the guide strand comprises or consists of the nucleobase sequence 5’-UAGCACCAUCUGAAAUCGGUUA-3’ (SEQ ID NO: 1), and/or the passenger strand comprises or consists of the nucleobase sequence 5 ’ -ACUGAUUUCUUUUGGUGUUCA-3 ’ (SEQ ID NO: 3).
• the guide strand comprises nucleobase sequence 5’-UAGCACCAUCUGAAAUCGGUUA-3’ (SEQ ID NO: 1)
• the passenger strand comprises the nucleobase sequence 5 ’-ACUGAUUUCUUUUGGUGUUCA-3’ (SEQ ID NO: 3).
• the guide strand consists of the nucleobase sequence 5’-UAGCACCAUCUGAAAUCGGUUA-3’ (SEQ ID NO: 1)
• the passenger strand consists of the nucleobase sequence 5 ’-ACUGAUUUCUUUUGGUGUUCA-3’ (SEQ ID NO: 3).
• the term “consists of’ does not exclude the presence of other (non-nucleoside) moieties attached to the guide strand or to the passenger strand, e.g., the passenger strand may be linked to a lipophilic moiety such as a cholesterol moiety, for example via a linker, typically at the 3 ’ end of the passenger strand.
• the nucleobase at the 3’ end of the passenger strand is an A or a G In certain embodiments, the nucleobase at the 3’ end of the passenger strand is an A.
• At least or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or 21 nucleosides of the guide strand may comprises a 2’-fluoro ribose.
• at least or about 11, 12, 13, 14, 15, 16, 17, 18 or 19 nucleosides of the guide strand are 2’-fluoro ribose nucleosides.
• no more than 20 or no more than 21 nucleosides of the guide strand are 2’ -fluoro ribose nucleosides.
• At least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94% or at least 95% of the nucleosides of the guide strand comprise a 2’- fluoro ribose.
• at least one nucleoside of the guide strand contains a sugar (which may be a modified sugar) other than a 2’-fluoro ribose.
• At least 50% (or at least 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94% or at least 95%) of the nucleosides of the guide strand comprise a 2’-fluoro ribose, and at least one nucleoside of the guide strand comprises a 2’-O-methyl ribose.
• at least 50% (or at least 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94% or at least 95%) of the nucleosides of the guide strand comprise a 2 ’-fluoro ribose, and at least two nucleosides of the guide strand comprise a 2’-O-methyl ribose.
• one or two nucleosides of the guide strand may comprise a 2’-O-methyl ribose.
• the nucleosides of the guide strand that comprise a 2’-O-methyl ribose may be located at the 3 ’ end of the guide strand.
• at least 50%, 60%, 70%, 80% or 90% of the nucleosides of the guide strand are 2’-fluoro ribose nucleosides, and/or no more than 91%, or no more than 95%, of the nucleosides of the guide strand are 2’-fluoro ribose nucleosides.
• at least 50% and no more than 91% of the nucleosides of the guide strand are 2’-fluoro ribose nucleosides.
• the nucleosides of the passenger strand that comprise a 2’-O-methyl ribose and an unmodified ribose alternate.
• at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or at least 20 contiguous nucleosides in the passenger strand may be alternately a 2’-O-methyl ribose nucleoside and a nucleoside with an unmodified ribose moiety.
• nucleosides in the passenger strand there are at least 18 contiguous nucleosides in the passenger strand that are alternately a 2’-O-methyl ribose nucleoside and a nucleoside with an unmodified ribose moiety.
• One or more nucleosides of the passenger strand may comprise a deoxy ribose. Such nucleosides may be located at the 3 ’ end of the passenger strand.
• At least 5, 6, 7, 8 or 9 nucleosides of the passenger strand are 2’-O-methyl ribose nucleosides, and/or no more than 10 nucleosides of the passenger strand are 2’-O-methyl ribose nucleosides.
• At least 10%, 20%, 30%, 40% or 45% of the nucleosides of the passenger strand are 2’-O-methyl ribose nucleotides, and/or no more than 50% of the oligonucleotide of the passenger strand are 2’-O-methyl ribose nucleotides.
• the invention thus provides a miR-29 compound or a salt thereof that includes a guide strand as described in any of the embodiments herein, and a passenger strand as described in any of the embodiments herein.
• the invention provides a miR-29 compound or a salt thereof, comprising:
• a guide strand comprising the nucleobase sequence 5’-UAGCACCAUCUGAAAUCGGUUA-3’ (SEQ ID NO: 1), or a nucleobase sequence which differs from SEQ ID NO: 1 at no more than five positions outside of the sequence 5’-AGCACCA-3’ (SEQ ID NO: 8), and optionally at no more than three positions within that sequence; wherein at least 50% of the nucleosides comprise a 2’-fluoro ribose and wherein one or two nucleosides, preferably at the 3’ end, each comprise a 2’-O-methyl ribose; and
• a passenger strand comprising the nucleobase sequence 5’-ACUGAUUUCUUUUGGUGUUCAG-3’ (SEQ ID NO: 2) or
• SEQ ID NO: 3 5’-ACUGAUUUCUUUUGGUGUUCA-3’ (SEQ ID NO: 3), or comprising a nucleobase sequence that differs from SEQ ID NO: 2 at no more than three positions; and wherein at least 10 contiguous nucleosides are alternately a 2’-O-methyl ribose nucleoside and a nucleoside with an unmodified ribose moiety.
• the invention provides a miR-29 compound or a salt thereof, comprising:
• a guide strand comprising the nucleobase sequence 5’-UAGCACCAUCUGAAAUCGGUUA-3’ (SEQ ID NO: 1), or a nucleobase sequence which differs from SEQ ID NO: 1 at no more than three positions outside of the sequence 5’-AGCACCA-3’ (SEQ-ID NO: 8); wherein at least 50% of the nucleosides comprise a 2’ fluoro ribose and wherein one or two nucleosides at the 3’ end each comprise a 2’-O-methyl ribose; and
• SEQ ID NO: 3 5’-ACUGAUUUCUUUUGGUGUUCA-3’ (SEQ ID NO: 3), or comprising a nucleobase sequence that differs from SEQ ID NO: 2 at no more than three positions; and wherein at least 10 contiguous nucleosides are alternately a 2’-O-methyl ribose nucleoside and a nucleoside with an unmodified ribose moiety.
• Exemplary compounds according to this embodiment are Tenol5, Teno 18, Tenol9, Teno20, Teno29, Teno32, Teno33 and Teno34, which are described in the Examples below.
• At least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or at least 20 contiguous nucleosides in the passenger strand may be alternately a 2’-O-methyl ribose nucleoside and a nucleoside with an unmodified ribose moiety.
• at least 12 contiguous nucleosides in the passenger strand are alternately a 2’-O-methyl ribose nucleoside and a nucleoside with an unmodified ribose moiety.
• at least 14 contiguous nucleosides in the passenger strand are alternately a 2’- O-methyl ribose nucleoside and a nucleoside with an unmodified ribose moiety.
• At least 16 contiguous nucleosides in the passenger strand are alternately a 2’-O-methyl ribose nucleoside and a nucleoside with an unmodified ribose moiety.
• at least 18 contiguous nucleosides in the passenger strand are alternately a 2’-O-methyl ribose nucleoside and a nucleoside with an unmodified ribose moiety.
• the at least one or two nucleosides in the passenger strand that each comprise a 2’-O-methyl ribose are located at the 3’ end of the passenger strand.
• one or two nucleosides at the 3 ’ end of the guide strand may each comprise a 2’-O-methyl ribose and (i) each remaining nucleoside of the guide strand that comprises a 2’-modified ribose is a 2’-fluoro ribose nucleoside, or (ii) each remaining nucleoside of the guide strand is a 2’-fluoro ribose nucleoside or an unmodified ribose moiety.
• each nucleoside of the guide strand comprises a 2’-modified ribose
• each nucleoside of the guide strand comprises a 2’-O-methyl ribose or a 2’-fluoro ribose, or
• one or two nucleosides at the 3’ end of the guide strand each comprise a 2’-O-methyl ribose and each remaining nucleoside of the guide strand is a 2’ -fluoro ribose nucleoside.
• none of the intemucleoside linkages of the guide strand and/or the passenger are chemically modified.
• all of the intemucleoside linkages of the guide strand and/or the passenger strand are phosphodiester intemucleoside linkages.
• none of the nucleobases of the guide strand and/or the passenger are chemically modified.
• none of the nucleobases of the guide strand and/or the passenger strand contain a 5-methyl cytosine.
• nucleobase sequence of the guide strand is as defined in SEQ ID NO: 1
• nucleobase sequence of the passenger strand is as defined in SEQ ID NO: 2 or SEQ ID NO: 3.
• nucleobase sequence of the guide strand is as defined in SEQ ID NO: 1
• nucleobase sequence of the passenger strand is as defined in SEQ ID NO: 3.
• the guide strand is
• An exemplary compound according to this embodiment is Teno 18, which is described in the Examples below.
• the guide strand is
• An exemplary compound according to this embodiment is Teno20, which is described in the Examples below.
• the guide strand is
• An exemplary compound according to this embodiment is Teno33, which is described in the
• the miR-29 compound or salt thereof may further comprise a cholesterol moiety covalently attached to the 3’ end of the passenger strand.
• the cholesterol moiety may be a tri ethyleneglycol cholesterol moiety.
• An exemplary cholesterol moiety is 3’phosphopropy-2-ol,2,2'-[Ethane-l,2-diylbis(oxy)]di(ethan-l-ol),10-O-[l-propyl-3-N- carbamoyl cholesteryl].
• phosphate group at the 5’ position of the sugar of the nucleoside at the 5’ end of the guide strand (‘5’Phos’).
• the guide strand is 5’-PhosfUfAfGfCfAfCfCfAfUfCfUfGfAfAfUfCfGfGfUmUmA-3’; and the passenger strand is 5’-mArCmUrGmArUmUrUmCrUmUrUmUrGmGrUmGrUmUrCdA-3’; wherein all internucleoside linkages of the guide strand and of the passenger strand are phosphodiester intemucleoside linkages.
• An exemplary compound according to this embodiment is Teno 18, which is described in the Examples below.
• the guide strand is
• An exemplary compound according to this embodiment is cholesterol- conjugated T enol 8 (CWT-001), which is described in the Examples below.
• the invention also provides a pharmaceutical composition comprising a miR-29 compound or salt thereof as described herein.
• the pharmaceutical composition typically comprises a pharmaceutically acceptable carrier or diluent.
• the pharmaceutically acceptable carrier or diluent is a sterile solution.
• the pharmaceutically acceptable carrier or diluent is a parenterally acceptable aqueous solution.
• the pharmaceutically acceptable carrier or diluent may be phosphate buffered saline (PBS).
• the pharmaceutically acceptable carrier or diluent may comprise additional substances such as EDTA, for example at a final concentration of 0.001% - 0.005%, e.g., 0.003% (100 ⁇ M).
• the pharmaceutical composition does not include non-liquid carriers, e.g., the miR-29 compound or salt thereof is not comprised in, or administered in, to or with solid carriers such as implantable scaffolds.
• the invention further provides a container comprising a miR-29 compound or salt thereof as described herein.
• the miR-29 compound or salt thereof is typically provided in a therapeutically effective amount.
• a therapeutically effective amount is an amount of the miR-29 compound or salt thereof that when administered to a subject reduces or inhibits collagen 3 and typically results in an improvement in disease symptoms, e.g., reduced pain or disability, improved functionality, and/or slower disease progression.
• the container typically comprises a label affixed to the container.
• the container may be a glass vial (e.g, a single use glass vial) containing the pharmaceutical composition described herein.
• the container may be a syringe or an injector.
• the invention also provides a miR-29 compound or salt thereof, or a pharmaceutical composition as described herein, for use in therapy.
• a miR-29 compound or salt thereof or the use of a pharmaceutical composition as described herein, for the manufacture of a medicament.
• Also provided is a method of treating a disease or condition comprising administering a therapeutically effective amount of a miR-29 compound or salt thereof, or a pharmaceutical composition as described herein, to a subject in need thereof.
• a miR-29 compound or a salt thereof for use in therapy for example comprising intralesional administration (e.g., a single intralesional administration) by inj ection of the miR-29 compound or salt thereof, wherein the miR-29 compound or salt thereof comprises the guide strand
• the treatment is for collagen dysregulation, in particular collagen overproduction, for example collagen 3 overproduction.
• overproduction refers to increased collagen production relative to corresponding healthy tissue. The overproduction is typically locally.
• tendon damage such as tendinopathy involves local collagen 3 overproduction. Fibrotic conditions such as Peyronie’s disease or Dupuytren’s disease also involve local collagen 3 overproduction.
• the miR-29 compound or salt thereof, or the pharmaceutical composition described herein is for use in treating tendon damage such as tendinopathy, or tissue fibrosis such as Peyronie’s disease or Dupuytren’s disease.
• the miR-29 compound or salt thereof, or the pharmaceutical composition described herein is for use in treating tendon damage.
• the tendon damage may be tendinopathy.
• the tendon damage may be chronic tendinopathy.
• the tendon damage may be lateral epicondylitis.
• the subject is typically a human.
• another preferred subject is a horse, particularly a horse that has been bred for equine sport.
• the invention provides dosages and treatment regimens for miR-29 compounds, salts thereof, and pharmaceutical compositions comprising the same.
• the dosages and treatment regimens provided herein are suitable for a range of miR-29 compounds, i.e., the applicability of the disclosure of dosages and treatment regimens herein includes but is not limited to the novel miR-29 compounds provided herein. Nevertheless, the dosages and treatment regimens preferably involve the novel miR-29 compounds, because the inventors have found that the dosages and treatment regimens are particularly suitable for these compounds.
• the miR-29 compound for use with the dosage and treatment regimens disclosed herein can be chemically modified, as described above.
• the miR-29 compound or salt thereof comprises modified sugars but does not comprise modified intemucleoside linkages or modified nucleobases (the miR-29 compound or salt thereof does not comprise modified intemucleoside linkages and it does not comprise modified nucleobases).
• all of the intemucleoside linkages of the guide strand and/or the passenger strand can be phosphodiester intemucleoside linkages.
• the miR- 29 compounds may also be conjugated (i.e., covalently linked) to a lipophilic moiety such as a cholesterol moiety, typically at the 3’ end of the passenger strand.
• a lipophilic moiety such as a cholesterol moiety
• the miR-29 compound or salt thereof may be any one of the specific compounds or salts described herein, or it can be a different miR-29 compound or salt thereof.
• the invention therefore provides a miR-29 compound or a salt thereof for use in therapy comprising a single intralesional administration by injection of a dose of the miR-29 compound or the salt thereof.
• the corresponding use for the manufacture of a medicament, and the corresponding method of treatment, are also provided, as described above and below.
• treatment comprises no more than two, or no more than three, intralesional administrations by injection of the miR-29 compound or the salt thereof. In other embodiment, the treatment comprises a single intralesional administration by injection of the miR-29 compound or the salt thereof.
• the treatment may be for tendon damage (e.g., tendinopathy) and the administration is into the damaged area of the tendon (intra-tendon, or intratendinous).
• tendon damage e.g., tendinopathy
• administration is into the damaged area of the tendon (intra-tendon, or intratendinous).
• the treatment may be for Peyronie’s disease and the administration is into the affected area of the tunica albuginea of the subject.
• the treatment may be for Dupuytren’s disease and the administration is into the affected area of the palmar fascia of the subject.
• a single dose of the miR-29 compound may be between 10 ⁇ g and 5000 ⁇ g of the miR-29 compound, or the equivalent dose of the salt thereof.
• the dose refers to the weight of the oligonucleotide portion of the guide and passenger strand (i.e., excluding any conjugate groups such as a cholesterol moiety), as a free acid.
• a single dose may therefore be between 10 ⁇ g and 4500 ⁇ g, 10 ⁇ g and 4000 ⁇ g, 10 ⁇ g and 3500 ⁇ g, 10 ⁇ g and 3000 ⁇ g, or between 10 ⁇ g and 2500 ⁇ g of the miR-29 compound, or the equivalent dose of the salt thereof.
• a single dose may be between 47 ⁇ g and 4740 ⁇ g of the miR-29 compound, or the equivalent dose of the salt thereof.
• a single dose may be between 47 ⁇ g and 4270 ⁇ g of the miR-29 compound, or the equivalent dose of the salt thereof.
• a single dose may be between 47 ⁇ g and 1900 ⁇ g of the miR-29 compound, or the equivalent dose of the salt thereof.
• a single dose may be between 47 ⁇ g and 1420 ⁇ g of the miR-29 compound, or the equivalent dose of the salt thereof.
• a single dose may be between 47 ⁇ g and 475 ⁇ g of the miR-29 compound, or the equivalent dose of the salt thereof.
• a single dose may be between 95 ⁇ g and 4740 ⁇ g of the miR-29 compound, or the equivalent dose of the salt thereof.
• a single dose may be between 95 ⁇ g and 4270 ⁇ g of the miR-29 compound, or the equivalent dose of the salt thereof.
• a single dose may be between 95 ⁇ g and 1900 ⁇ g of the miR-29 compound, or the equivalent dose of the salt thereof.
• a single dose may be between 95 ⁇ g and 1420 ⁇ g of the miR-29 compound, or the equivalent dose of the salt thereof.
• a single dose may be between 95 ⁇ g and 475 ⁇ g of the miR-29 compound, or the equivalent dose of the salt thereof.
• a single dose may be between 190 ⁇ g and 4740 ⁇ g of the miR-29 compound, or the equivalent dose of the salt thereof.
• a single dose may be between 190 ⁇ g and 4270 ⁇ g of the miR-29 compound, or the equivalent dose of the salt thereof.
• a single dose may be between 190 ⁇ g and 1900 ⁇ g of the miR-29 compound, or the equivalent dose of the salt thereof.
• a single dose may be between 190 ⁇ g and 1420 ⁇ g of the miR-29 compound, or the equivalent dose of the salt thereof.
• a single dose may be between 190 ⁇ g and 475 ⁇ g of the miR-29 compound, or the equivalent dose of the salt thereof.
• a single dose may be between 475 ⁇ g and 4740 ⁇ g of the miR-29 compound, or the equivalent dose of the salt thereof.
• a single dose may be between 475 ⁇ g and 4270 ⁇ g of the miR-29 compound, or the equivalent dose of the salt thereof.
• a single dose may be between 475 ⁇ g and 1900 ⁇ g of the miR-29 compound, or the equivalent dose of the salt thereof.
• a single dose may be between 475 ⁇ g and 1420 ⁇ g of the miR-29 compound, or the equivalent dose of the salt thereof.
• a single dose may be between 1420 ⁇ g and 4740 ⁇ g of the miR-29 compound, or the equivalent dose of the salt thereof.
• a single dose may be between 1420 ⁇ g and 4270 ⁇ g of the miR-29 compound, or the equivalent dose of the salt thereof.
• a single dose may be between 1420 ⁇ g and 1900 ⁇ g of the miR-29 compound, or the equivalent dose of the salt thereof.
• a single dose is between 190 ⁇ g and 1420 ⁇ g of the miR-29 compound, or the equivalent dose of the salt thereof.
• a single dose may be about 190 ⁇ g, 475 ⁇ g, 1420 ⁇ g or 4270 ⁇ g of the miR-29 compound, or the equivalent dose of the salt thereof.
• a single dose may be about 190 ⁇ g, 475 ⁇ g or 1420 ⁇ g of the miR-29 compound, or the equivalent dose of the salt thereof.
• This means that a single dose may be about 190 ⁇ g of the miR-29 compound, or the equivalent dose of the salt thereof.
• a single dose may be about 475 ⁇ g of the miR-29 compound, or the equivalent dose of the salt thereof.
• a single dose may be about or 1420 ⁇ g of the miR-29 compound, or the equivalent dose of the salt thereof.
• a single dose is between about 190 ⁇ g and about 475 ⁇ g of the miR-29 compound, or the equivalent dose of the salt thereof.
• a single dose may be about 190 ⁇ g of the miR-29 compound, or the equivalent dose of the salt thereof.
• a single dose may also be about 475 ⁇ g of the miR-29 compound, or the equivalent dose of the salt thereof.
• a single dose is about 190 ⁇ g of the miR-29 compound, or the equivalent dose of the salt thereof.
• the invention provides a miR-29 compound or a salt thereof for use in the treatment of tendon damage in a subject, wherein the treatment comprises a single administration by injection of a dose of at least 47 ⁇ g of the miR-29 compound, or the equivalent dose of the salt thereof, into the damaged tendon of the subject.
• the invention provides a miR-29 compound or a salt thereof for use in the treatment of Peyronie’s disease in a subject, wherein the treatment comprises a single administration by injection of a dose of at least 47 ⁇ g of the miR-29 compound, or the equivalent dose of the salt thereof, into the affected area of the tunica albuginea of the subject.
• the invention provides a miR-29 compound or a salt thereof for use in the treatment of Dupuytren’s disease in a subject, wherein the treatment comprises a single administration by injection of a dose of at least 47 ⁇ g of the miR-29 compound, or the equivalent dose of the salt thereof, into the affected area of the palmar fascia of the subject.
• the single dose of the miR-29 compound is at least 95 ⁇ g, at least 190 ⁇ g, at least 475 ⁇ g, at least 1420 ⁇ g, at least 1900 ⁇ g, at least 4270 ⁇ g, or the equivalent dose of the salt thereof.
• the single dose of the miR-29 compound may be at least 95 ⁇ g (or at least 190 ⁇ g, at least 475 ⁇ g, at least 1420 ⁇ g, at least 1900 ⁇ g, at least 4270 ⁇ g) and no more than 4740 ⁇ g of the miR-29 compound, or the equivalent dose of the salt thereof.
• a single dose may be within a certain range as described above.
• a single dose of the miR-29 compound may be at least or about, 10 ⁇ g, 15 ⁇ g, 20 ⁇ g, 25 ⁇ g, 30 ⁇ g, 35 ⁇ g, 40 ⁇ g, 45 ⁇ g, 47 ⁇ g, 50 ⁇ g, 55 ⁇ g, 60 ⁇ g, 75 ⁇ g, 80 ⁇ g, 95 ⁇ g, 100 ⁇ g, 110 ⁇ g, 120 ⁇ g, 130 ⁇ g, 140 ⁇ g, 150 ⁇ g, 160 ⁇ g, 170 ⁇ g, 180 ⁇ g, 190 ⁇ g, 200 ⁇ g, 220 ⁇ g, 240 ⁇ g, 260 ⁇ g, 280 ⁇ g, 300 ⁇ g, 320 ⁇ g, 340 ⁇ g, 350 ⁇ g, 360 ⁇ g, 380 ⁇ g, 400 ⁇ g, 420 ⁇ g, 440 ⁇ g, 460 ⁇ g, 470 ⁇ g, 475 ⁇ g, 480 ⁇ g, 500 ⁇ g,
• a single dose of the miR-29 compound is no more than 190 ⁇ g, 200 ⁇ g, 220 ⁇ g, 240 ⁇ g, 260 ⁇ g, 280 ⁇ g, 300 ⁇ g, 320 ⁇ g, 340 ⁇ g, 350 ⁇ g, 360 ⁇ g, 380 ⁇ g, 400 ⁇ g, 420 ⁇ g, 440 ⁇ g, 460 ⁇ g, 470 ⁇ g, 475 ⁇ g, 480 ⁇ g, 500 ⁇ g, 550 ⁇ g, 600 ⁇ g, 650 ⁇ g, 700 ⁇ g, 750 ⁇ g, 800 ⁇ g, 850 ⁇ g, 900 ⁇ g, 950 ⁇ g, 1000 ⁇ g, 1100 ⁇ g, 1200 ⁇ g, 1300 ⁇ g, 1400 ⁇ g, 1420 ⁇ g, 1500 ⁇ g, 1600 ⁇ g, 1700 ⁇ g, 1800 ⁇ g, 1900 ⁇ g, 1900
• a single dose of the miR-29 compound is no more than 190 ⁇ g, or the equivalent dose of the salt thereof. In a specific embodiments a single dose of the miR-29 compound is no more than 475 ⁇ g, or the equivalent dose of the salt thereof. In a specific embodiment, a single dose of the miR-29 compound is no more than 1420 ⁇ g, or the equivalent dose of the salt thereof. In a specific embodiment, a single dose of the miR-29 compound is no more than 1900 ⁇ g, or the equivalent dose of the salt thereof. In a specific embodiment, a single dose of the miR-29 compound is no more than 4270 ⁇ g, or the equivalent dose of the salt thereof.
• the tendon damage may be tendinopathy.
• the tendon damage may be chronic tendinopathy.
• the tendon damage may be lateral epicondylitis, medial epicondylitis, rotator cuff tendinopathy, common extensor origin tendinopathy, common flexor origin tendinopathy, gluteal tendinopathy (greater trochanteric pain syndrome, GTPS), patellar tendinopathy, jumper’s knee, plantar fasciitis, Achilles tendinopathy, peroneal tendinopathy, supraspinatus syndrome, or a combination thereof.
• the tendon damage is upper limb tendinopathy (including lateral epicondylitis, golfer’s elbow, rotator cuff tendinopathy and De Quervain’s disease).
• the tendon damage is lateral epicondylitis.
• the subject is typically a human.
• another preferred subject is a horse, particularly a horse that has been bred for equine sport.
• the miR-29 compound or salt thereof is administered in a volume of 0.5 ml-5 ml, e.g., 0.5 ml-1.5 ml, such as 0.8 ml-1.2 ml, for example 0.9 ml-1.1 ml.
• the volume may be about 0.5 ml, 0.75 ml, 1 ml, 1.25 ml, 1.5 ml, 1.75 ml, 2 ml, 2.5 ml, 3 ml, 3.5 ml, 4 ml, 4.5 ml or about 5 ml.
• the volume is about 1 ml.
• the miR-29 compound or salt thereof is administered in a volume of about 1 ml of a sterile aqueous solution.
• the miR-29 compound or salt thereof may be administered at a concentration that corresponds to any of the dosages indicated above.
• the dosage is 190 ⁇ g of the miR-29 compound
• the compound may be administered at a concentration of 190 ⁇ g/ml.
• the miR-29 compound or salt thereof is any one of the specific miR-29 compounds of salts thereof as provided herein.
• the invention therefore provides a miR-29 compound or a salt thereof for use in therapy comprising a single intralesional administration by injection of a dose of the miR-29 compound or the salt thereof, wherein the guide strand is 5’-fUfAfGfCfAfCfCfAfUfCfUfGfAfAfUfCfGfUmUmA-3’, the passenger strand is 5’-mArCmUrGmArUmUrUmCrUmUrUmUrGmGrUmGrUmUrCdA-3’ and comprises a cholesterol moiety covalently attached to the 3’ end of the passenger strand (5’-mArCmUrGmArUmUrUmCrUmUrGmGrUmGrUmUrCdAchol-3’), and the dose of the miR-29 compound (including the weight of the cholesterol moiety) is between 50
• the dose of the miR-29 compound is between about 200 ⁇ g and about 500 ⁇ g, or the equivalent dose of the salt thereof.
• the dose of the miR-29 compound may be about 200 ⁇ g, or the equivalent dose of the salt thereof.
• the dose of the miR-29 compound (including the weight of the cholesterol moiety) may also be about 500 ⁇ g, or the equivalent dose of the salt thereof.
• the dose of the miR-29 compound (including the weight of the cholesterol moiety) is about 200 ⁇ g, or the equivalent dose of the salt thereof.
• the guide strand of the miR-29 compound or salt thereof is 5’-fUfAfGfCfAfCfCfAfUfCfUfGfAfAfUfCfGfUmUmA-3’, the passenger strand of the miR-29 compound or salt thereof is
• the dose of the miR-29 compound including the weight of the cholesterol moiety is between 50 ⁇ g and 5000 ⁇ g, for example 200 ⁇ g, 500 ⁇ g, 1500 ⁇ g or 4500 ⁇ g, or the equivalent dose of the salt thereof.
• the dose of the miR-29 compound including the weight of the cholesterol moiety may be about 200 ⁇ g, or the equivalent dose of the salt thereof.
• the dose of the miR-29 compound including the weight of the cholesterol moiety may be about 500 ⁇ g, or the equivalent dose of the salt thereof.
• the dose of the miR-29 compound including the weight of the cholesterol moiety may be about 1500 ⁇ g, or the equivalent dose of the salt thereof.
• the dose of the miR-29 compound including the weight of the cholesterol moiety may be about 4500 ⁇ g, or the equivalent dose of the salt thereof.
• the dose of the miR-29 compound (including the weight of the cholesterol moiety) is between about 200 ⁇ g and about 500 ⁇ g, or the equivalent dose of the salt thereof.
• the dose of the miR-29 compound (including the weight of the cholesterol moiety) may be about 200 ⁇ g, or the equivalent dose of the salt thereof.
• the dose of the miR-29 compound may also be about 500 ⁇ g, or the equivalent dose of the salt thereof. In a specific embodiment, the dose of the miR-29 compound (including the weight of the cholesterol moiety) is about 200 ⁇ g, or the equivalent dose of the salt thereof.
• the therapy may be for tissue fibrosis, such as Peyronie’s disease, or Dupuytren’s disease.
• the therapy may be for tendon damage.
• the tendon damage may be tendinopathy.
• the tendon damage may be chronic tendinopathy.
• the tendon damage may be lateral epicondylitis.
• the miR- 29 compound may be administered as a salt, for example as a sodium salt.
• the carrier may be a sterile aqueous solution that is parenterally acceptable, for example PBS.
• the invention provides a process for making a miR-29 compound in accordance with the invention.
• the invention also provides a process for making a pharmaceutical composition, comprising providing a miR-29 compound in accordance with the invention and combining the compound with a pharmaceutically acceptable carrier or diluent.
• a “microRNA-29” compound as used herein refers to an oligonucleotide that has the biological function of a natural endogenous miR-29, which includes miR-29a, miR-29b (which includes miR- 29b- 1 and miR-29b-2) and miR-29c. That function includes the targeting of mRNA by complementarily binding (hybridising) to the 3' untranslated region (3'UTR) of any target mRNA that contains one or more binding sites for miR-29 (e.g., one or more binding sites for miR-29a), thereby causing its degradation and/or translational repression.
• Target mRNAs that harbour binding sites for a miR-29 typically encode certain extracellular matrix proteins, such as collagen, for example collagen 3 (col3al).
• the miR-29 compounds described herein mimic the biological function of a natural miR-29, but may be modified relative to a natural miR-29 (e.g., natural miR- 29a) so as to, e.g., enhance its activity, stability and/or cellular uptake, but without causing immunogenicity. Modifications may include nucleobase sequence modifications, and/or chemical modifications at one or more sugar moieties, intemucleoside linkages, and/or nucleobases. Modifications also include conjugation, e.g., to a lipophilic moiety.
• the compound has the biological function of human mature miR-29a, i. e. , it is a mimic of human mature miR-29a.
• the miR-29 compounds described herein are capable of reducing or inhibiting the expression of a protein that is encoded by an mRNA that harbours one or more binding sites for miR-29, e.g., one or more binding sites for miR-29a.
• the miR-29 compounds described herein are capable of reducing or inhibiting collagen expression, for example collagen 3 (col3al) expression.
• the miR-29 compounds may reduce collagen expression to a greater extent than native miR-29a.
• the miR-29 compounds may reduce collagen expression by at least 10%, at least 20%, or at least 30% or more compared to native miR-29a.
• Suitable assays to assess reduction in collagen (col3al) expression are known to the person skilled in the art, such as the luciferase assay or the qPCR assay described in Example 1 below. Protein levels may also be assessed by methods well known in the art (e.g., Western Blot).
• the miR-29 compounds described herein are capable of cellular uptake, for example they can be taken up in vivo by target cells (such as tenocytes, or fibroblasts) without the use of transfection agents or other transfection vehicles such as liposomes. Conjugation of the miR-29 compound with a lipophilic moiety such as a cholesterol moiety enhances cellular uptake. Capability of cellular uptake can be assessed by methods known to the skilled person in the art, for example measuring miR-29 activity in the in vitro collagen 3 luciferase reporter assay.
• the miR-29 compounds described herein are stable. A stable miR-29 compound substantially retains activity in the in vitro collagen 3 luciferase reporter assay after storage at 37°C for three months.
• miR-29 compounds described herein are stable also in the sense that they have a longer in vivo half-life as a result of 2’ sugar modifications, compared to unmodified (natural) miR-29.
• Double-stranded RNA can stimulate innate immune responses in mammals.
• miR-29a can bind to receptors of the Toll-like receptor (TLR) family in immune cells and trigger an inflammatory response (ref. 5). Immune stimulation could significantly limit the in vivo application of miRNA therapy.
• TLR Toll-like receptor
• the miR-29 compounds provided herein are non-immunogenic when administered to a subject, e.g., a human subject. Suitable assays are known to the person skilled in the art to assess non -immunogenicity, such as the TNF-alpha assay using human macrophages as described in Example 1 below.
• Preferred compounds do not induce the expression of TNF-alpha, or they induce the expression of no more than lO ⁇ g/ml TNF- alpha, as measured in the supernatants of cultured primary human macrophages 24 hours after treatment with the compound.
• the compounds described herein do not induce an undesirable immune response, the compounds do not need to be protected from recognition by the immune system by encapsulation into liposomes or similar means.
• the compounds described herein are therefore effective in vivo even when administered in a simple aqueous carrier, e.g., PBS. This significantly simplifies preparation and administration of the therapy and avoids the introduction of further foreign components into the subject which could raise safety concerns.
• the compounds described herein therefore have the advantage of effectively mimicking the function of natural miR-29 (e.g., miR-29a) but having a higher activity and stability yet being non-immunogenic.
• the Examples also show that the compounds can be anti-inflammatory.
• the exemplary miR-29 compounds described in the Examples are therefore chemically synthesised mimics of miR-29 which have improved stability, activity and cellular uptake, while being non- immunogenic.
• the nucleobase sequence of the compounds is closest to that of miR-29a, however as noted above functional mimicry can extend to other miR-29 family members.
• the miR-29 compounds are stable in an aqueous carrier and remain active even after storage at 37°C for three months.
• certain miR-29 compounds have been chemically modified via the introduction of 2’ -fluoro and 2’-O-methyl groups in the guide strand, and 2’-O- methyl groups in the passenger strand.
• the passenger strand may additionally contain a 3’ cholesterol group to increase cellular uptake.
• the miR-29 compounds described herein have a well-defined mode of action.
• the compounds directly target the key pathways in collagen production associated with a range of pathogenic conditions, including tendon damage such as tendinopathy and other diseases that require a reduction or inhibition of collagen (in particular collagen 3) production, e.g., tissue fibrosis.
• tendon damage such as tendinopathy and other diseases that require a reduction or inhibition of collagen (in particular collagen 3) production, e.g., tissue fibrosis.
• treatment with the miR-29 compounds described herein does not require invasive procedures and can be delivered at the point of initial diagnosis, thereby initiating recovery at the very earliest time.
• miR-29 compounds described herein are able to significantly reduce the expression of collagen 3, for example in tenocytes and fibroblasts.
• the Examples also show that a single intra-tendon injection of miR-29 compound significantly reduced collagen 3 expression and improved tendon structure in the mouse tendon injury models.
• double-stranded miR-29 compounds (comprising a guide and a passenger strand) are more active than single-stranded miR-29 compounds (comprising a guide strand only; e.g., Teno36 in Example 1).
• the miR-29 compounds described herein are therefore typically double-stranded.
• the two strands are substantially complementary to each other such that they form a duplex.
• the guide strand may be between 20 and 24 linked nucleosides in length.
• the guide strand may be 20, 21, 22, 23 or 24 linked nucleosides in length.
• the guide strand is between 20 and 23 linked nucleosides in length.
• the guide strand is between 20 and 22 linked nucleosides in length.
• the guide strand is between 21 and 23 linked nucleosides in length.
• the guide strand is 21 or 22 linked nucleosides in length.
• the guide strand is 22 or 23 linked nucleosides in length.
• the guide strand is 22 linked nucleosides in length.
• the passenger strand may be between 19 and 23, or between 20 and 24 linked nucleosides in length.
• the passenger strand may be 19, 20, 21, 22, 23 or 24 linked nucleosides in length.
• the passenger strand is between 19 and 22 linked nucleosides in length.
• the passenger strand is between 19 and 21 linked nucleosides in length.
• the passenger strand is 20 or 21 linked nucleosides in length.
• the passenger strand is 21 or 22 linked nucleosides in length.
• the passenger strand is 21 linked nucleosides in length.
• the guide and passenger strands of the miR-29 compound form a duplex due to portions of sequence complementarity between the respective oligonucleotides of the guide and passenger strand.
• the guide and passenger strand may form a duplex such that the guide strand has a 3 ’ overhang, e.g. , of two nucleosides, relative to the passenger strand, and the passenger strand has a 3’ overhang, e.g., of one nucleoside, relative to the guide strand.
• the guide and passenger strands may form a duplex such that the guide strand has a 3’ overhang, e.g., of two nucleosides, relative to the passenger strand.
• mismatches between corresponding nucleobases of the guide strand and the passenger strand when aligned, i.e. , when in a duplex.
• a mismatch exists where the corresponding nucleobases are not complementary.
• Complementary nucleobases are U and A, and C and G.
• there may be 3 to 8 mismatches for example 3, 4, 5, 6, 7 or 8 mismatches.
• there are 6 mismatches may be at positions 3, 10 to 12, 18 and 20 (numbering relative to the passenger strand).
• the inventors have surprisingly found that miR-29 compounds that contain mismatches between the guide and the passenger strand have a higher activity (e.g., as determined in the collagen 3 luciferase assay) compared to miR-29 compounds that have fewer or no mismatches.
• the miR-29 compounds described herein may comprise one or more conjugate groups covalently attached to the oligonucleotide(s) of the compound.
• An exemplary conjugate group is a cholesterol moiety, although other conjugate moieties that improve the pharmacological properties (e.g., the cellular uptake) of the oligonucleotide are known in the art and can be used instead of a cholesterol moiety.
• the conjugate groups are therefore typically lipophilic moieties.
• the miR-29 compound comprises a cholesterol moiety covalently attached to one or more oligonucleotide strands of the miR-29 compound.
• the cholesterol moiety is covalently attached to the passenger strand.
• the cholesterol moiety is covalently attached to the passenger strand, and the guide strand is unconjugated.
• the cholesterol moiety is covalently attached to the 3’ end of the passenger strand.
• the cholesterol moiety may be attached via a linker, e.g., a triethyleneglycol.
• the cholesterol moiety is a triethyleneglycol cholesterol moiety.
• An exemplary cholesterol moiety is 3’phosphopropy-2-ol,2,2'-[Ethane-l,2- diylbis(oxy)]di(ethan-l-ol), 10-O-[l-propyl-3-N-carbamoylcholesteryl],
• the miR-29 compounds described herein can be provided as a salt.
• the term “compound” therefore includes a salt of the compound.
• the salt is a pharmaceutically acceptable salt, i. e. , the salt retains the biological activity of the compound and does not impart toxicological effects thereto.
• pharmaceutically acceptable salts include sodium salts and potassium salt.
• the miR-29 compound is a sodium salt.
• a particular miR-29 compound described in the Examples is CWT-001.
• CWT-001 is a doublestranded chemically modified mimic of at least miR-29a, comprising a guide (antisense) and a passenger (sense) strand at a 1 : 1 ratio.
• the nucleobase sequences and chemical modifications of the guide and passenger strands correspond to those of ‘Teno 18’ (T18).
• CWT-001 inhibits the expression of at least the same target genes as naturally-occurring miR-29a, including collagen 3.
• the sugar residues have been chemically modified via the introduction of 2’-fluoro and 2’-O-methyl groups, which increase stability and activity of the compound.
• the guide strand of CWT-001 has the same nucleobase sequence as the naturally -occurring guide strand of human mature miR-29a (“hsa-miR-29a-3p”), which is:
• Each nucleoside is a 2 ’-substituted nucleoside, which means that the nucleoside comprises a sugar moiety that comprises at least one 2’ -substituent group other than H or OH.
• the guide strand of CWT-001 has the following formula:
• f denotes a nucleoside containing a fluorine at the 2’ position of the ribose ring
• m denotes a nucleoside containing an O-methyl group at the 2’ position of the ribose ring.
• the intemucleoside linkages are phosphodiester intemucleoside linkages throughout.
• RNA 5 ’Phospho (2 ’ deoxy-2 ’ Fluoro)U-(2 ’ deoxy-2 ’Fluoro) A-(2 ’ deoxy-2 ’ Fluoro)G-(2 ’ deoxy -
• CWT-001 passenger strand The passenger strand of CWT-001 has the same nucleobase sequence as the naturally-occurring passenger strand of human mature miR-29a (“hsa-miR-29a-5p”) except that the G at the 3’ end is not present in CWT-001.
• the nucleobase sequence of the CWT-001 passenger strand therefore is:
• the passenger strand contains nucleosides comprising a modified sugar moiety (2’-O-methyl), nucleosides comprising an unmodified sugar moiety, and a nucleoside comprising a deoxy ribose sugar moiety.
• the passenger strand of CWT-001 has the following formula:
• m denotes a nucleoside containing an O-methyl group at the 2’ position of the ribose ring
• r denotes a ribonucleoside, i.e., containing an OH group at the 2’ position of the ribose ring
• d denotes a deoxy ribose nucleoside
• the intemucleoside linkages are phosphodiester intemucleoside linkages throughout.
• the passenger strand of CWT-001 comprises a cholesterol moiety, e.g., a triethyleneglycol cholesterol moiety, covalently attached to the 3 ’ end of the passenger strand.
• the cholesterol moiety improves the pharmacological properties of the miR-29a compound, e.g., the cholesterol moiety increases the cellular uptake of the oligonucleotide.
• the chemical name of the CWT-001 passenger strand, as a sodium salt, is:
• RNA ((2’OMethyl)A-C-((2’OMethyl)U-G-(2’OMethyl)A-U-(2’OMethyl)U-U-(2’OMethyl)C-U- (2 ’ OMethyl)U-U-(2 ’ OMethyl)U-G-(2 ’ OMethyl)G-U-(2 ’ OMethyl)G-U-(2 ’ OMethyl)U-C-
• the structural formula of the CWT-001 passenger strand as a free acid is shown below:
• the structural formula of the CWT-001 passenger strand in ionised form i.e., the anion of a salt is shown below:
• CWT-001 Further information on CWT-001 is provided in the following table A.
• Methods of treatment and therapeutic uses of the ndcroRNA-29 compounds and compositions are particularly suitable in the treatment of diseases that involve localised collagen dysregulation, characterised by a local overproduction of one or more types of collagen fibre.
• the miR-29 compounds described herein reduce or inhibit the expression of collagen 3, for example as assessed in a luciferase assay.
• the miR-29 compounds described herein therefore may be useful in the treatment of diseases that require the reduction or inhibition of expression of one or more types of collagen fibres, for example collagen 3 (col3al).
• tendon damage in particular tendinopathy
• Peyronie in particular tendinopathy
• Dupuytren Dupuytren
• Tendons are the connective tissue attaching muscle to bone. Tendon tissue comprises around 30% collagen and 2% elastin (wet weight) embedded in an extracellular matrix containing various types of cells, most notably tenocytes.
• the predominant collagen is type 1 collagen, which has a large diameter (40-60 nm) and links together to form tight fibre bundles.
• Type 3 collagen is also present and is smaller in diameter (10-20 nm), forming looser reticular bundles.
• a healthy tendon contains primarily well-ordered bundles of collagen 1 (coll) fibres (-95%) interspersed with smaller amounts of biomechanically inferior collagen 3 (col3) fibres (-5%).
• miR-29 In healthy tenocytes, miR-29 (e.g., miR-29a) directly suppresses collagen 3 overproduction, neovascularisation, hyper-proliferation of tenocytes, and adhesion formation, by targeting Col3al, VEGFA, AKT3 and TGF-B2, respectively.
• the sequence of miR-29a and its binding sites in these target mRNAs are conserved in mammalian species.
• biomechanical properties of tendon are related to cross-sectional area (i.e., thickness), collagen content, and the ratio between different types of collagen.
• a healthy tendon contains primarily well-ordered bundles of collagen 1 (coll) fibres (-95%) interspersed with smaller amounts of biomechanically inferior collagen 3 (col3) fibres (-5%).
• Col3 biomechanically inferior collagen 3
• Tendon damage may be caused by or associated with numerous factors including but not limited to external trauma, mechanical stress (including over-use), degeneration, inflammation, and combinations of these.
• Tendinopathy is the medical name for diseases associated with overuse and injury of the tendon.
• Overuse tendinopathy is a complex multi-faceted pathology of the tendon, clinically diagnosed after gradual onset of activity-related pain, decreased function and sometimes with localized swelling of the tendon.
• Over-use, repetitive strain or trauma leads to the breakdown of collagen fibres in the tendon.
• tenocytes initiate a repair response, where they initially produce high levels of collagen 3 which acts as a ‘patch’ to quickly restore tendon function.
• collagen 3 will be gradually replaced by collagen 1.
• collagen 3 is biomechanically inferior to collagen 1
• the tendon is prone to further damage, causing more collagen 3 production.
• a vicious cycle of further damage and inferior healing results in the formation of lesions characterised by increased collagen 3 (-30%), cellularity, vascularisation, inflammation and adhesion formation.
• FIG. 4 shows that treatment with an exemplary miR-29 compound according to the invention (CWT-001) significantly improved tendon fibre alignment and collagen production via the suppression of collagen 3 expression in tendon disease.
• SDFT equine collagenase-induced tendinopathy
• a single intralesional injection of a miR-29 compound reduced collagen 3 expression and lesion size, and improved tendon structure (see ref. 3). Together, these data provide a rationale for safe and effective human therapy using miR-29 compounds, including the miR-29 compounds provided herein. In light of the experiments described herein (see the Examples), doses in the ⁇ g and low mg range are expected to be well tolerated in animal species and can be administered in volumes that can easily and safely be injected (e.g, around 1 ml).
• the miR-29 compounds and compositions of the invention may therefore be used in the treatment of tendinopathy, such as overuse tendinopathy.
• the subject may be a human subject.
• the subject may have sub-acute tendinopathy.
• Treatment with the compounds and compositions of this invention may reduce progression to chronic tendinopathy.
• the subject may have chronic tendinopathy.
• the subject may have active tendinopathy.
• the subject may have a partially -tom tendon. Treatment may reduce progression to a fully-torn tendon.
• the methods of the invention may be applied to any damaged tendon.
• the main tendons affected by tendinopathy in humans are the Achilles tendon, the supraspinatus tendon, the common flexor tendons, the common extensor tendons, the patellar tendon and the gluteal tendons.
• the main tendon affected by tendinopathy in equine subjects is the superficial flexor tendon.
• one or more of these tendons are targets for treatment.
• the tendon damage may therefore be lateral epicondylitis, medial epicondylitis, rotator cuff tendinopathy, common extensor origin tendinopathy, common flexor origin tendinopathy, gluteal tendinopathy (greater trochanteric pain syndrome, GTPS), patellar tendinopathy, jumper’s knee, plantar fasciitis, Achilles tendinopathy, peroneal tendinopathy, supraspinatus syndrome, or a combination thereof.
• the tendon damage is upper limb tendinopathy (including lateral epicondylitis, golfer’s elbow, rotator cuff tendinopathy and De Quervain’s disease).
• the tendon damage is lateral epicondylitis.
• Treatment with a miR-29 compound promotes the regeneration or repair of the afflicted tissue, e.g. , in the tendon, tunica albuginea or plantar fascia.
• Treatment with a miR-29 compound reduces the expression of collagen, e.g., collagen 3, at the site of treatment.
• the methods of the invention may be applied at any stage of tendon damage, or at any stage of the healing process of a damaged tendon.
• the miR-29 compounds and compositions of the invention are administered to the subject at the point of initial diagnosis.
• the subject for example the human subject, may have failed to respond to, or had an inadequate response to, or was intolerant to a prior treatment. This means that the subject’s symptoms have persisted despite the treatment.
• a prior tendinopathy treatment may include one or more of: physical therapy, splinting, treatment with a nonsteroidal anti-inflammatory drug (NSAID), and local corticosteroid injection into the affected tendon.
• the subject may have failed conservative treatment, i.e., the symptoms persisted despite treatment, for a period of 3 months or more.
• Efficacy of miR-29 treatment in tendon damage can be assessed on the basis of one or more of (see also Example 7): - reduction of pain, for example measured using Visual Analogue Scale (VAS), e.g., at 14 days, 28 days and/or 90 days following administration of a miR-29 compound or salt thereof; - improvement in disability and symptoms, for example measured using the Disabilities of the Arm, Shoulder, and Hand (Quick DASH) Score, and/or the American Shoulder and Elbow Surgeons Elbow (ASES-E), e.g., at 14 days, 28 days and/or 90 days following administration of a miR-29 compound or salt thereof; improvement in pain and disability, for example measured by the Patient Rated Tennis Elbow Evaluation (PRTEE), e.g., at 14 days, 28 days and/or 90 days following administration of a miR-29 compound or salt thereof; and improvement in the treated tendon(s) as measured by ultrasound assessment, e
• VAS Visual Analogue Scale
• PRTEE Patient Rated
• Dupuytren’s disease affects the fibrous layer of tissue that lies underneath the skin in the palm and fingers, also known as the ‘fascia’ or ‘palmar fascia’.
• the fascia thickens, then tightens over time. This causes the fingers to be pulled inward, towards the palm, resulting in what is known as a Dupuytren's contracture.
• Subjects with DD may be unable to perform certain daily activities.
• nonsurgical e.g., steroid or collagenase injection
• surgical treatment options are available to help slow the progression of the disease and improve motion in the affected fingers, these can have significant side effects or complications.
• DD is characterised by an abnormal fibroblast proliferation process and an overproduction of collagens, including collagen 3 (ref. 6).
• the miR-29 compounds described herein are thought to be efficacious in the treatment of Dupuytren’s disease on the basis of their ability to inhibit particularly effectively and safely local collagen (e.g., collagen 3) expression in the fascia, therefore countering the formation of fibrotic tissue that characterises this disease.
• Efficacy of miR-29 treatment in Dupuytren’s disease can be assessed on the basis of improvement in disability and symptoms, for example measured using the Disabilities of the Arm, Shoulder, and Hand (Quick DASH) Score, symptoms and/or reduction of pain (VAS scoring), and clinical assessment of reduction in Dupuytren’s nodule and cord formation (for example by improvement in the Tubiana grading system). See, e.g., ref. 7.
• Peyronie’s disease is a progressive fibrotic disorder characterised by the formation of scars or plaques within the tunica albuginea (TA) of the penis.
• the condition is highly debilitating on both a physical as well as an emotional level due to penile pain and the development of penile deformities.
• Fibrotic tissue is associated with a shift in the normal collagen 3/1 ratio toward an increase in the content of collagen 3.
• the collagen 3/1 ratio was higher in the PD group compared with control groups (P ⁇ .05) (ref. 8).
• the miR-29 compounds described herein are thought to be efficacious in the treatment of Peyronie’s disease on the basis of their ability to inhibit particularly effectively and safely local collagen 3 expression in the TA, therefore countering the formation of fibrotic tissue that characterises this disease.
• Efficacy of miR-29 treatment in Peyronie’s disease can be assessed on the basis of improvement in disability, symptoms and/or reduction of pain, improvement or lack of progression of penile curvature and improvement in duplex ultrasonography.
• the subject is typically a human.
• the methods of the invention may extend to any other mammals, including other primates (especially great apes such as gorilla, chimpanzee and orangutan, but also Old World and New World monkeys) as well as rodents (including mice and rats), and other common laboratory, domestic and agricultural animals (including but not limited to rabbits, dogs, cats, horses, cows, sheep, goats, etc.).
• Another preferred subject is a horse, particularly a horse that has been bred for equine sport.
• the miR-29 compounds or salts thereof described herein can be formulated in pharmaceutical compositions. These compositions may comprise, in addition to the miR-29 compound or salt thereof, a pharmaceutically acceptable excipient, carrier, buffer, stabiliser or other materials well known to those skilled in the art. Such materials are non-toxic and do not interfere with the efficacy of the miR-29 compound.
• the pharmaceutical composition comprises a miR-29 compound or a salt thereof and a pharmaceutically acceptable carrier or diluent, such as a sterile liquid carrier, e.g, an injectable solution.
• a pharmaceutically acceptable carrier or diluent may comprise saline.
• the pharmaceutically acceptable carrier or diluent may be a sterile aqueous solution, for example phosphate buffered saline (PBS).
• PBS phosphate buffered saline
• the pharmaceutically acceptable carrier or diluent may be a sterile aqueous solution, for example a sodium chloride solution such as isotonic saline (e.g, 0.9% w/v NaCl).
• the pharmaceutical composition comprises a miR-29 compound or a salt thereof, and further comprises PBS and a sodium chloride solution such as isotonic saline (e.g, 0.9% w/v NaCl).
• a sodium chloride solution such as isotonic saline (e.g, 0.9% w/v NaCl).
• local administration for example by local injection, is particularly suitable.
• the compounds and compositions of the invention are therefore typically administered locally at the site of affliction (intra-lesionally).
• the injection may be delivered into the affected tendon. Injection into the affected tendon is also known as intratendinous administration.
• the miR-29 compound can be comprised in a pharmaceutical composition further comprising a parenterally acceptable aqueous solution.
• the solution is sterile.
• the solution is pyrogen-free and has suitable pH, isotonicity and stability.
• isotonic vehicles such as phosphate buffered saline, sodium chloride solution, Ringer's solution, Lactated Ringer's solution.
• Preservatives, stabilisers, buffers, antioxidants and/or other additives may be included, as required.
• EDTA may added, e.g., at a final concentration of 0.001% - 0.005%, e.g., 0.003% (100 pM).
• a particular formulation of the compounds of the invention is an injectable solution, for example a solution comprising a miR-29 compound or salt of the invention and PBS, optionally further comprising EDTA.
• Injection into the tendon is typically guided by ultrasound, for example using the “peppering technique” (ref. 11).
• the “peppering technique” is an injection method whereby multiple small injections are performed by withdrawing, redirecting and reinserting the needle without emerging from the skin. In the injured/ chronic tendon situation this may be advantageous in order to stimulate a healing response.
• a single dose of the miR-29 compound is administered into the tendon in the form of multiple needle inj ections at different sites within the damaged tendon.
• a dose of a miR-29 compound as indicated herein refers to the weight of the oligonucleotide portion of the guide and passenger strand (z.e. , excluding any conjugate groups such as a cholesterol moiety), as a free acid.
• the reference compound is CWT-001, as a free acid, and excluding the triethyleneglycol cholesterol moiety.
• Equivalent doses of miR-29 compounds that may have a different molecular weight, of salts, and/or of conjugated forms can be calculated by the skilled person. Equivalent doses may also be indicated in molar amounts (e.g., nmoles) or molar concentrations (e.g. , nM). The skilled person knows how to convert between these different units.
• the indicated dosages refer to the free acid of the cholesterol -conjugated form of the compounds. Therefore, where the Examples and Figures refer to 200 ⁇ g or 200 ⁇ g/ml, this corresponds to about 190 ⁇ g or 190 ⁇ g/ml of the compound as a free acid excluding the cholesterol moiety; where the Examples refer to 500 ⁇ g or 500 ⁇ g/ml, this corresponds to about 475 ⁇ g or 475 ⁇ g/ml of the compound as a free acid excluding the cholesterol moiety; where the Examples refer to 1500 ⁇ g or 1500 ⁇ g/ml, this corresponds to about 1420 ⁇ g or 1420 ⁇ g/ml of the compound as a free acid excluding the cholesterol moiety; where the Examples refer to 2000 ⁇ g or 2000 ⁇ g/ml, this corresponds to about 1900 ⁇ g or 1900 ⁇ g/ml of the compound as a free acid excluding the cholesterol moiety, etc. Where the cholesterol moiety is excluded, this exclude
• a therapeutically effective amount is an amount of the miR-29 compound or salt thereof that when administered to a subject results in an improvement in disease symptoms, e.g., reduced pain or disability, improved functionality, and/or prevented or slower disease progression.
• a miR-29 compound can be administered directly into the damaged tissue, i.e., intralesionally. Administration is typically by injection, for example using a needle.
• a therapeutic effect may be achieved with only a single administration of the doses described herein.
• a “single” administration means that only one dose of the compound is required to achieve a therapeutic effect. The subject therefore typically does not need to return for a second dose in order to experience an improvement in disease symptoms at the site of administration of the first dose.
• composition “comprising” encompasses “including” as well as “consisting”, e.g., a composition “comprising” X may consist exclusively of X or may include something additional, e.g., X + Y.
• references to a percentage sequence identity between two oligonucleotide sequences means that, when aligned, that percentage of nucleobases is the same in comparing the two sequences, across the full length of those sequences.
• Figure 1 (A) Structure of the CWT-001 guide (antisense) strand, as a sodium salt. (B) Structure of the CWT-001 passenger (sense) strand, as a sodium salt.
• FIG. 3 Selected miR29 compounds were tested for potential immunogenicity by assessing their capacity to induce the production of pro-inflammatory cytokine TNF-alpha in primary human macrophages. Untreated cells (“untreated”) were used as negative control; cells treated with 2.5 ⁇ g/ml imiquimod (“IMQ 2.5”) were used as a positive control. (B) Selected miR29 compounds were tested for their ability to knock-down the expression of col3al in human tenocytes. Scrambled miR29a (“scr”), and untreated cells (“untr”) were used as a negative control.
• scr Scrambled miR29a
• untreated cells untreated cells
• (C) Luciferase activity of HEK293 cells co-transfected with luciferase plasmid containing human collagen 3 miR29- binding sites and selected miR-29 compounds as indicated. Scrambled miR29a (“scr”) was used as a negative control. Significance was assessed using one-way ANOVA test. ***P ⁇ 0.001 compared to Teno20, n 3.
• Figure 4 Histological and molecular analysis of mouse tendons following induction of tendon injury followed immediately by treatment with CWT-001.
• B Representative H&E stained sections of injured tendons after 7 days of treatment with CWT-001 (right) or PBS control (left).
• Figure 5 Levels of pro -inflammatory cytokine tumour necrosis factor alpha (TNF-a) in the supernatants of cultured primary human macrophages 24 hours after treatment with CWT-001, scrambled negative control or poly IC positive control, measured by ELISA.
• TNF-a pro -inflammatory cytokine tumour necrosis factor alpha
• Figure 7 Expression levels of interferon-inducible genes MX1 (A), OAS2 (B), IFITM1 (C) and IRF9 (D) in mouse tendon at 0, 1, 3 and 7 days after intra-tendon injection of CWT-001 or PBS negative control, measured by qPCR and expressed as absolute copy number of interferon response genes per 106 copies of 18S.
• FIG. 10 Levels of miR29a in mouse kidney (A), spleen (B), liver (C), serum (D), lung (E), muscle (F) and heart (G) harvested 0, 1, 4 and 7 days after intravenous injection of CWT-001, measured by qPCR.
• Figure 12 Levels of miR29a in mouse serum (A), lung (B), skin proximal to tendon injection (C), kidney (D), spleen (E), muscle (F), liver (G), heart (H) and bone marrow (I) harvested 7 days after intravenous injection of 4, 20, 40 and 50 mg/ml of CWT-001, or PBS control, measured by qPCR.
• Figure 13 Study design for a phase 1, randomised, double-blind, placebo-controlled study evaluating the safety, tolerability and pharmacokinetics of single ascending doses of CWT-001 injections in human subjects with lateral epicondylitis.
• Figure 14 Ultrasound images (A and B) illustrating probe placement during administration of CWT-001 to the common extensor tendon. The radial head (r), radiohumeral joint (j), lateral epicondyle, and common extensor tendon are visualised. The probe is placed along the lateral aspect of the elbow parallel to the longitudinal axis of the common extensor tendon.
• FIG 16 Levels of collagen 1 (A) and collagen 3 (B) in human Dupuytren’s fibroblasts following transfection with 5 pM of a miR29a mimic, a miR29b mimic, a miR29c mimic, CWT-001, a miR29a antagomir or a scrambled miR29a control, measured by qPCR.
• VAS 100 mm visual analogue scale
• the patient rated tennis elbow evaluation (PRTEE) was used to measure perceived pain and disability in study subjects, and the change in PRTEE score was plotted from pre-dose on Day 1, to Day 14, Day 28 and Day 90.
• Ultrasound tissue characterisation (UTC) was used to visualise tendon structure and to quantify grey-scale tendon matrix changes into 4 different echotypes related to tendon integrity.
• Types I and II represent an organised matrix; Types III and IV represent a disorganised matrix (A).
• Representative ultrasound images of the lateral elbow are provided for a subject that had received the placebo at pre-inj ection (B), Day 28 (C) and Day 90 (D), and for a subject that had received 200 ⁇ g CWT-001 at pre-inj ection (E), Day 20 (F) and Day 90 (G).
• the percentages of UTC Type I, II, III and IV tendon tissue and the total percentage of reparative tendon (UTC Types I and II) vs degenerative tendon (UTC types III and IV) were quantified for the subjects that had received a 200 ⁇ g (H, I), 500 ⁇ g (J, K) or 1500 ⁇ g (L, M) dose of CWT-001 or matched placebo.
• a library of miR-29 mimic compounds containing various chemical modifications (2 ’-fluoro ribose, 2’-O-Methyl ribose, deoxyribose, and/or phosphorothioate) in different combinations was created. All compounds with the exception of one (Teno36) are double-stranded compounds containing a guide strand and a passenger strand.
• the guide strand of each compound has a nucleobase sequence that is identical to the guide strand of natural human mature miR-29a (hsa-miR-29a-3p; SEQ ID NO: 1).
• the passenger strand has a nucleobase sequence that is either identical to the passenger strand of natural human mature miR-29a (hsa-miR-29a-5p; SEQ ID NO: 2), or it may lack the 3’ G nucleotide (SEQ ID NO: 3), and/or it may have one or more nucleotide substitutions relative to the sequence of the natural miR-29a passenger strand (SEQ ID NOs: 4-6), see Table lb.
• a negative control passenger strand was also tested, representing the reverse sequence of SEQ ID NO: 2 (SEQ ID NO: 7).
• the nucleobase sequences together with any sugar and intemucleoside linkage (backbone) modifications are shown in Table la.
• the first row indicates the guide strand
• the second row indicates the passenger strand. All sequences are shown in the 5’ to 3’ direction.
• the SEQ ID NOs indicate the nucleobase sequence (i.e., excluding the chemical modifications).
• Teno46 does not contain any chemical modifications and therefore corresponds to native mature miR-29a.
• Teno39 was previously described (ref. 2).
• the structure of an exemplary compound, Teno 18, as a sodium salt and wherein the passenger strand is conjugated at the 3’ end to a cholesterol moiety, is shown in Figure 1A (guide strand) and IB (passenger strand).
• A adenosine deoxyribonucleoside
• G guanine deoxyribonucleoside
• U uracil deoxyribonucleoside
• SEQ ID NO: 2 represents the native human miR-29a passenger strand sequence.
• SEQ ID NO: 3 differs from SEQ ID NO: 2 at one position, as shown in underlining.
• SEQ ID NO: 4 differs from SEQ ID NO: 2 at five positions.
• SEQ ID NO: 5 differs from SEQ ID NO: 2 at six positions.
• SEQ ID NO: 6 differs from SEQ ID NO: 2 at seven positions.
• SEQ ID NO: 7 is the reverse sequence of SEQ ID NO: 2 and represents a negative control.
• Table 1c below shows the combination of 2’ sugar modifications, nucleobase sequence (as SEQ ID NOs), and the intemucleoside linkages in each compound.
• Tenol-Teno49 contain one of five types of guide strand (indicated as G1-G5 below), and one of 11 types of passenger strand (indicated as Pl-Pl 1 below).
• Teno36 is an exception as it does not contain a passenger strand. All sequences are shown in the 5’ to 3’ direction.
• Table 1c shows that it contains the “Gl” guide strand, which has the nucleobase sequence of SEQ ID NO: 1, wherein three nucleosides at the 3’ end are 2’-O-methyl ribonucleosides and the remaining nucleosides are unmodified ribonucleosides, and all intemucleoside linkages are phosphodiester.
• the passenger strand is “Pl”, having the nucleobase sequence of SEQ ID NO: 2 and wherein all nucleosides are alternately 2’-O-methyl and unmodified ribonucleosides, and all intemucleoside linkages are phosphodiester.
• Teno2 contains the same guide strand as Tenol (“Gl”), but a different passenger strand (“P2”), etc.
• m 2’-0-methyl ribonucleotide (-O-Me);
• f 2’-deoxy-fluoro ribonucleotide (-F);
• d 2’-deoxyribonucleotide (-H);
• MiR-29 compounds - grouped by passenger strand The compounds were tested for activity relative to native human mature miR29a using an in vitro collagen 3 luciferase reporter assay.
• the basis of this assay is a luciferase reporter gene containing the miR29a binding sites from collagen 3.
• luciferase activity is inversely proportional to miR29a activity. This means that high miR29a activity more strongly suppresses collagen 3 expression, which results in a lower luciferase signal.
• the luciferase assays were performed in HEK293 cells, after transfection of the miR-29 compounds with transfection agent.
• a selection of compounds (Teno2, Teno5, Teno6, Teno7, Tenol l, Teno 12, Tenol3, Tenol5, Teno 18, Tenol9, Teno20, Teno25, Teno26, Teno27, Teno32, Teno33 and Teno34) was tested for potential immunogenicity by testing their capacity to induce the production of pro-inflammatory cytokine TNF-alpha in primary human macrophages.
• Imiquimod a TLR7/8 agonist
• IMQ Imiquimod
• TNF-alpha a TLR7/8 agonist
• Results are shown in Figure 3 A.
• Compounds that induced the expression of more than lO ⁇ g/ml TNF-alpha were not taken forward.
• Teno 12 was taken forward as it represented a miR-29 compound with the lowest immunogenicity amongst those tested that contained a guide strand with phosphorothioate internucleoside linkages (Tenol l, Teno 12 and Tenol3).
• T enol 8 and Teno33 were taken forward for in vivo activity testing in human tenocytes and a mouse tendon injury model.
• a cholesterol moiety was conjugated to the 3’ end of the passenger strand of each compound (e.g., see Fig. IB for Teno 18).
• Both T enol 8 and Teno33 effectively knocked down col3al expression in human tenocytes (Figure 3D) and in a mouse tendon injury model (Figure 3E).
• Treatment with Teno 18 knocked down col3al expression most effectively at the day 5 time point in human tenocytes (Figure 3D), and at the 1 day and 3 day time points in the mouse tendon injury model (Figure 3E).
• Teno 18 was therefore selected as the clinical candidate for further in vivo testing, described in the Examples that follow.
• the drug substance (cholesterol-conjugated Teno 18, designated CWT-001) is synthesised using routine methods (solid phase synthesis) and provided as a lyophilised powder.
• the drug product can be provided as a sterile solution containing 5mg/mL CWT-001, typically at pH 7.3, in clear glass vials, which is diluted with saline for injection prior to administration.
• CWT- 001 can be provided as a sterile solution at the final concentration for administration (e.g., about 200 ⁇ g/ml, 500 ⁇ g/ml, 1500 ⁇ g/ml, 2000 ⁇ g/ml, 4500 ⁇ g/ml or 5000 ⁇ g/ml, such as about 200 ⁇ g/ml, 500 ⁇ g/ml, 1500 ⁇ g/ml, or 4500 ⁇ g/ml).
• EDTA can be added, e.g., at a final concentration of 0.001% - 0.005%, e.g., about 0.003% (100 pM).
• the drug product vials can be stored and transported at -20°C and are intended for single use. Frozen vials appear cloudy white. Upon thawing to room temperature, the active pharmaceutical ingredient (API) will solubilise and become a clear colourless solution.
• the structure of the CWT-001 drug substance (as a sodium salt) is shown in Figure 1.
• Figure 1A shows the guide strand
• Figure IB shows the passenger strand, conjugated to a cholesterol moiety at the 3 ’ end.
• a validated mouse tendon injury model (ref. 12) was selected to test if treatment with CWT-001 significantly reduced collagen 3 expression and improved tendon healing.
• tendon injury was induced using a 0.75mm diameter biopsy needle followed immediately by injection of 0.35 ⁇ g/ml CWT-001 into the site of injury. Mice were allowed to recover for 1, 3 or 7 days and tendons removed for histological and molecular analysis.
• MicroRNAs by their nature interact with large numbers of target mRNA species in most cases via base-pairing of a 6-8 nucleotide seed sequence.
• the sequence of CWT-001’s guide strand is identical to that of natural miR29a meaning that CWT-001 will target the same mRNAs as the natural molecule via a RISC-dependent mechanism.
• a bioinformatics analysis of both guide and passenger strands was conducted as recommended by the Oligonucleotide Safety Working Group guidance (see ref. 13). This analysis generated a list of 52 genes detailed below, which could theoretically hybridise with CWT-001’s guide and passenger strands. Mutations in 13 of these genes were associated with known human diseases, as also detailed below.
• Q-PCR analysis was performed on the expression levels of miRNAs in primary human tenocytes after treatment with CWT-001.
• Potential cross-hybridization targets were identified as hsa-miR29a, hsa-miR29c, hsa-mir-1302-4, hsa-mir-3657, hsa-mir-3657 and hsa-mir-4647.
• the top hits in the sense (+) orientation is the target microRNA miR29a and miR29c (miR29b was not identified in light of the bioinformatics parameters used for this analysis).
• Potential cross-hybridization targets are in the complementary (-) orientation.
• microRNA sequences are poorly characterized microRNA hsa-mir-3657 and hsa-mir-4647. Both these microRNAs may represent in silico artefacts with no function. Moreover, both these sequences share a limited region of full interrupted identity (lObp) and are unlikely to have enough affinity to be strongly modulated by steric inhibition by miR29a mimics at physiological concentrations in vivo.
• the top complementary microRNA sequences are hsa-mir-548at, hsa-mir-1283-2 and hsa-mir-515 family. All these sequences share a limited region of full interrupted identity (1 Ibp or less) and are unlikely to have enough affinity to be strongly modulated by steric inhibition by CWT-001 at physiological concentrations in vivo.
• RNA sequences qPCR analysis was performed on the expression levels of potential off-target genes in primary human tenocytes after treatment with CWT-001.
• the data shows that treatment with CWT-001 does not produce off-target effects in primary human tenocytes.
• Double-stranded RNA may elicit an immune response (ref. 14).
• ds-RNA Double-stranded RNA
• CWT-001 might elicit an immune response
• TNFoc pro-inflammatory cytokine tumour necrosis factor-alpha
• RT-QPCR reverse transcription quantitative PCR
• RNA QC Pool samples for each matrix were prepared as endogenous controls to assess endogenous baseline levels. During sample analysis, RNA QC Pool samples are used for data normalisation of the QC samples and to determine the expected Ct values and determined copies numbers. RNA QC Pool samples are set to be within the following values:
• CWT-001 In the event that CWT-001 entered the systemic circulation, the duration of CWT-001 ’s detectability against background levels of endogenous miR29a in human serum was determined.
• CWT-001 was diluted to concentrations equivalent to those predicted immediately following intravenous (IV) delivery in an adult human (1 : 5,000 in 1 ml of serum, based on average adult blood volume of 5 litres).
• IV intravenous
• Samples of 0, 2, 20 and 100 ⁇ g/ml CWT-001 were incubated with fresh human serum at 37°C for 0, 6, 12 and 24 hrs.
• mice were injected with 50pl of 50mg/ml CWT-001, and serum, heart, liver, kidney, spleen and muscle were collected after 0, 1, 4 and 7 days.
• CWT-001 levels were measured using a miR29a qPCR assay, values were normalised to GAPDH and shown as relative fold-change compared to untreated controls.
• CWT-001 has negligible distribution in tissues following intravenous administration, even when administered at 25 times of the level of a typical clinical dose (z.e., 25 times of 50pl of 2mg/ml, which is the mouse equivalent of a human clinical dose of 1ml of 2mg/ml CWT-001).
• Example 4d Systemic exposure of CWT-001 in circulation after intra-tendon injection
• CWT-001 Less than 1% of the CWT-001 is ultimately taken up by cells via the serum. Overall, this means that only very low absolute levels of administered CWT-001 will enter non-target cells via systemic exposure. The observed systemic increase in CWT-001 is therefore unlikely to exert any pharmacodynamic action in cells apart from the target cells within the tissue into which CWT-001 is administered (e.g., the tendon).
• Rats were treated with a 30pL injection of saline control, 10 mg/ml or 50 mg/ml (i.e., 0, 0.3 or 1.5 mg total dose) of CWT-001.
• Blood samples were collected from the jugular vein from 3 animals per sex per group at 0.5, 1, 2, 4, 8 and 24 hours post dose and plasma prepared. Plasma samples were analysed for the concentration of CWT-001 using a validated analytical procedure (RT-QPCR).
• Toxicokinetic parameters were estimated using a non-compartmental approach consistent with the intra-tendon route of administration in this study. A summary of the toxicokinetic parameters is provided in Table 2 below. Table 2 - Toxicokinetic Parameters of CWT-001 in Male and Female Rat Plasma
• Plasma CWT-001 concentration vs time profiles displayed a rapid absorption phase (T max WHS 0.5 hours in all profiles).
• Plasma CWT-001 concentrations decreased in a multi -phasic manner up to 4 hours post-dose, after which, in females, concentrations slightly increased up to 8 hours (last quantifiable concentration, 0.3 mg) or remained at a relatively steady state until the end of the sampling period (24 hours) (1.5 mg).
• concentrations at both doses remained at a steady state after 4 hours until the end of the sampling period (24 hours).
• systemic exposure to CWT-001 increased in a generally less than dose-proportional manner in females, and greater than dose-proportional manner in males.
• Systemic exposure to CWT-001 was greater in males than in females at both 0.3 mg and 1.5 mg dose levels.
• Toxicokinetic assessment was incorporated into the design of the single dose toxicity study of CWT-001 by intra-tendinous injection in dogs with a 14 day recovery period (Example 5d). Dogs were treated with a 250pL injection of saline control, 10 mg/ml or 50 mg/ml of CWT-001 (i.e., 0, 2.5 or 12.5 mg total dose). Blood samples were collected from the jugular vein from 3 animals per sex per group at 0.5, 1, 2, 4, 6 and 48 hours post dose and plasma prepared. Plasma samples were analysed for the concentration of CWT-001 using a validated analytical procedure (RT-QPCR). Toxicokinetic parameters were estimated using a non-compartmental approach consistent with the intra-tendon route of administration in this study. A summary of the toxicokinetic parameters is provided in the following Table.
• Plasma CWT-001 concentration versus time profiles displayed a rapid absorption phase (median Tmax of 0.5 or 1 hours). This was followed by a bi-phasic decline in concentrations firstly in a rapid manner up to 1 or 2 hours post dose, and secondly at a slower rate up to the last quantifiable concentration (except for females dosed at 12.5 mg, which showed a slight increase in concentrations between 4 and 6 hours post dose).
• systemic exposure to CWT-001 increased in a generally dose-proportional manner in females, and less than dose-proportionally in males. Systemic exposure to CWT-001 was greater in males than females at a dose level of 2.5 mg, but comparable between sexes at 12.5 mg.
• the objectives of this study were to determine the potential toxicity of CWT-001 when given to rats by intra-tendinous injection, with the aim of establishing and confirming a maximum feasible dose (MFD).
• MFD maximum feasible dose
• Each animal received a single intra-tendinous administration (30 ⁇ L) of CWT-001 into the right Achilles tendon using a 0.5 mL insulin syringe.
• Animals were anaesthetised using isoflurane, and the tendon was exposed using a small surgical incision to the hindlimb to allow placement of CWT- 001. Animals were sutured closed after the dose was administered.
• the highest dose level (1.5 mg) represented the maximum feasible dose that could be administered by this route, limited by the solubility of the test item and the volume that could reasonably be injected into the tendon.
• the following parameters and end points were evaluated in this study: clinical observations, body weights and gross necropsy findings. There were no clinical signs, changes in body weight or gross necropsy findings that were considered to be related to CWT-001.
• the objectives of this study were to determine the potential toxicity of CWT-001 when given as a single intra-tendinous injection to dogs and to provide data to support dose level selection for a follow-on 14 day toxicity study.
• the study design is set out in Table 5 below.
• Each animal received a single intra-tendinous administration (250 ⁇ L) of CWT-001 into the right Achilles tendon using a syringe.
• Animals were anaesthetised using isoflurane, and the tendon was exposed using a small surgical incision to the hindlimb to allow placement of CWT-001. Animals were sutured closed after the dose was administered.
• the dose level used (12.5 mg) represented the maximum feasible dose that could be administered by this route, limited by the solubility of the test item and the volume that could reasonably be injected into the tendon.
• the following parameters and end points were evaluated in this study: clinical observations, body weights, food consumption, clinical pathology parameters (haematology, coagulation and clinical chemistry), gross necropsy findings and organ weights.
• Example 5c A single dose toxicity study of CWT-001 by intra-tendinous injection in rats with a 14 day recovery peri.od.
• the highest dose level (1.5 mg) represented the maximum feasible dose that could be administered by this route, limited by the solubility of the test item and the volume that could reasonably be injected into the tendon of a rat (see Example 5a). All formulation analysis results were found to be within or equal to the acceptance criteria of ⁇ 10% (individual values within or equal to ⁇ 15%) of their theoretical concentrations.
• CWT -001 was considered to be the No Observed Adverse Effect Level (NOAEL) in rats.
• NOAEL No Observed Adverse Effect Level
• the objectives of this study were to determine the potential toxicity of CWT-001 when given as a single intra-tendinous injection to dogs, and to evaluate the potential reversibility of any findings. In addition, the toxi co kinetic characteristics of CWT-001 were determined.
• the study design is set out in Table 7 below.
• the highest dose level (12.5 mg) represented the maximum feasible dose that could be administered by this route, limited by the solubility of the test item and the volume that could reasonably be injected into the tendon of a dog (see Example 5b). All formulation analysis results were found to be within or equal to the acceptance criteria of ⁇ 10% (individual values within or equal to ⁇ 15%) of their theoretical concentrations.
• Example 6a Central nervous system (modified Irwin observations) in rats
• the first 6 male main study animals from each group were observed at 2, 24 and 48 hours post dose. The following parameters were included in the observations:
• Pupil size measured using a guidance chart to estimate the size in millimetres (pre-dose pupil size data was recorded on the day of dosing).
• Body temperature measured by using a probe inserted approximately 2 cm past the anal sphincter. Pre-dose body temperature was also recorded. To reduce measurement variability a temperature habituation trial was conducted by inserting the temperature probe prior to the recording of the predose measurements, however, no body temperature data was collected during this trial.
• mice On the test day, animals were removed from their home cage and placed in a whole body plethysmograph chamber for approximately 45 minutes, with the last 15 minutes of this period classified as the control period. The animals were then removed from the chambers for dosing of control or test item. Measurements were then taken from 2-4, 24 and 51 hours post dose, animals were placed in the chamber to acclimate for at least 30 minutes before any measurements. The effects of chamber temperature and humidity on tidal volume were compensated for within the plethysmograph chamber. Ventilatory parameters were determined from the respiratory signal. Sampling frequency was fixed at 500 Hz.
• Cardiovascular assessments were measured pre-treatment (prior to Day 1) for a period at least 26 hours, on Day 2 of the dosing period for at least 20 hours (acquiring data between 24 hours to 44 hours post dose) and on Day 14 of the recovery period.
• the ECG and blood pressure of each animal was recorded by Jacketed External Telemetry (JETTM) System in conjunction with PA-C10-TOX-LA transmitter (Data Sciences International (DSITM), St Pauls, MN, USA).
• JETTM Jacketed External Telemetry
• PA-C10-TOX-LA transmitter Data Sciences International (DSITM), St Pauls, MN, USA).
• DSITM Data Sciences International
• St Pauls St Pauls, MN, USA
• ECG and BP recordings were carried out for at least 26 hours.
• pretreatment telemetry data acquisitions of at least 26 hours duration were recorded from all animals. Sections of these pretreatment signals were reviewed, as deemed necessary, for any ECG morphology abnormalities, quality of signal and normality of heart rate, arterial blood pressure and ECG values (including QTca calculations [QT corrected for heart rate]). The following parameters were reported:
• SBP Systolic
• DBP diastolic
• MAP mean arterial blood pressure
• HR heart rate
• ECG parameters RR, PR, QRS, QT corrected for individual animals, QTca intervals QA interval [derived from the blood pressure and ECG waveforms]
• the results from the examples described above indicate that the miR-29 compounds provided herein are safe and effective in reducing or inhibiting collagen 3 expression in vivo, including in mouse models of tendinopathy. Additionally, the inventors have previously shown that an equine collagenase-induced tendinopathy (SDFT) model reflects human tendinopathy, with reduced expression of miR-29a after tendon injury and a concomitant increase in col3:coll ratio. A single intralesional injection of a miR-29 compound (100 nM, which translates to around 2 ⁇ g/ml in a total volume of 1.5 ml, i.e., a total dose of 3 ⁇ g) reduced collagen 3 expression and lesion size, and improved tendon structure (see ref. 3). Together, these data provide a rationale for safe and effective human therapy using miR-29 compounds, including the miR-29 compounds provided herein.
• SDFT equine collagenase-induced tendinopathy
• the current 2nd line (following physiotherapy) standard of care treatment of lateral epicondylitis remains local injection of corticosteroid to the area of tendon pain, even though the long-term benefit remains questionable.
• a local injection of CWT-001 to the area of tendon damage of the common extensor origin of the elbow is therefore in keeping with current standard of care but shows improved safety and efficacy.
• An ultrasound guided injection to the area of tendon damage is used, as ultrasound examination is often used as the imaging modality of choice not only to assess the status of the tendons but also to guide injection therapy (ref. 15).
• the primary objective of the study is to determine the safety and tolerability of single ascending doses of CWT-001 in human subjects with lateral epicondylitis.
• the secondary objective of the study is to determine the single dose pharmacokinetics (PK) of CWT-001 administration in human subjects with lateral epicondylitis, and to assess the efficacy of CWT-001 administration in human subjects with lateral epicondylitis.
• PK pharmacokinetics
• the primary endpoint of the study is the comparison of safety data between CWT-001 versus placebo as measured by incidence of adverse events (AEs), clinical laboratory abnormalities, changes in vital signs (blood pressure, temperature, respiratory rate, and pulse rate), 12-lead electrocardiogram (ECG) parameters, physical examinations and skin score assessment at 14 days post injection.
• AEs adverse events
• vital signs blood pressure, temperature, respiratory rate, and pulse rate
• ECG electrocardiogram
• the secondary endpoints of the study are:
• VAS Visual Analogue Scale
• miR-29 doses in the ⁇ g and low mg range are expected to be effective and well tolerated in animal species, and can be administered in volumes that can easily and safely be injected (e.g., around 1 ml). These dose levels also confirm the mechanism of action targeting collagens, e.g., in improving tendon healing in the animal models used.
• Single dose extended toxicity studies via intra-tendinous administration were conducted with CWT-001 in the rat (up to 1.5 mg, see Example 5a) and dog (up to 12.5 mg, see Example 5b) and the no-observed- adverse-effect level for each study was the maximum feasible dose.
• Preclinical assessments of single intra-tendon administration of CWT-001 did not result in adverse findings at levels that were 41 and 25 times the respective doses in rat and dog compared to a human clinical dose of 2 mg, for example.
• Subjects were divided into cohorts (8 subjects per cohort) and randomised to receive a single 1ml dose of CWT-001 (a 5 mg/mL solution of CWT-001 in PBS and EDTA was diluted to the various final concentrations using normal saline, i.e. 0.9% w/v NaCl) or matched placebo (normal saline, i.e. 0.9% w/v NaCl) (3: 1) administered via intra-tendon injection.
• the starting dose of CWT-001 is 200 ⁇ g/mL.
• the dose was escalated to 500 ⁇ g/mL and then 1500 ⁇ g/mL.
• An additional dose of (up to) 4500 ⁇ g/mL was planned to be administered if no improvement in VAS pain assessment was seen, however this was not necessary.
• Doses were administered in an escalating manner, following satisfactory review of all safety, tolerability, plasma PK, and efficacy data (where available) from lower doses. Doses were determined based on the ongoing evaluation of the safety, tolerability, plasma PK, and efficacy data (where available). All doses were given as a 1ml injection under ultrasound guidance into the affected area.
• Ultrasonography was applied using a 5-7.5 MHz linear array transducer on aLOGIQ E9 Ultrasound System.
• the probe was placed along the lateral aspect of the elbow parallel to the longitudinal axis of the common extensor tendon.
• the radial head, radiohumeral joint, lateral epicondyle, and common extensor tendon was visualised.
• the skin was sterilised and the USG probe enclosed in a sterile cover. After visualisation of the tip of the needle at the exact site (hypointense area), ImL of CWT-001 or placebo was injected (see Figure 14).
• CWT-001 was administered to the tendon using the “peppering technique” (ref. 11).
• the “peppering technique” is an injection method whereby after the needle is inserted into the tender area, multiple small injections are performed by withdrawing, redirecting and reinserting the needle without emerging from the skin. In the injured/ chronic tendon situation this may be preferable to stimulate a healing response.
• the study population included 24 subjects. Each subject met all the inclusion criteria and none of the exclusion criteria for this study.
• Subject is male or female, of any ethnic origin.
• Subject is aged between 18 to 70 years, inclusive, with no evidence of skeletal immaturity.
• Subject has a body mass index (BMI) of 18 to 35 kg/m 2 , inclusive.
• Subject is >50 kg.
• Subject has a clinical diagnosis of lateral epicondylitis.
• the subject is otherwise healthy as determined by a responsible physician, based on medical history, physical examinations, concomitant medication, vital signs, 12-lead ECGs and clinical laboratory evaluations. Laboratory values may be re-tested once at the discretion of the Investigator.
• Subject s symptoms can be reproduced with resisted supination or wrist dorsiflexion (as confirmed by tenderness at lateral epicondyle and positive pick up back of chair sign).
• Subject s symptoms have persisted for at least 6 weeks to 6 months, despite conservative treatment that includes one or a combination of: a. Physical therapy b. Splinting c. NSAIDs
• Subject is independent, ambulatory, and can comply with all post-injection evaluations and visits.
• Female subjects of childbearing potential must agree to use a highly effective method of contraception from 1 month pre-dose in combination with male partner’s use of a condom during the trial and for 3 months post-dose. Subjects must have a negative pregnancy test at screening and Day 1.
• Subject has undergone previous corticosteroid injection therapy to the affected elbow in less than 6 months prior to enrolment.
• Subject uses or has recent use of medications known to affect the skeleton e.g., glucocorticoid usage >5 mg/day, fluoroquinolone antibiotics).
• Subject has undergone surgical intervention to the affected elbow for the treatment of lateral epicondylitis.
• Subject has a positive medical history of any of the following: a. Medial epicondylitis b. Radial tunnel syndrome c. Carpal tunnel syndrome d. Septic or gouty arthritis in the affected elbow within the previous 2 years prior to enrolment e. Cervical radiculopathy within the previous 6 months prior to enrolment f. Trauma to the affected elbow within the past 6 weeks g. Neuromuscular or primary/secondary muscular deficiency, which limits the ability to perform functional measurement (e.g., grip strength test) h. Coincidental rheumatologic, inflammatory diseases, including, but not limited to axial spondyloarthritis, psoriatic arthritis and rheumatoid arthritis i. Osteoarthritis of the affected elbow j. Fibromyalgia judged sufficient to compromise the evaluation of response
• Subject currently has an acute infection at the injection site.
• lymphoproliferative disease or any known malignancy or history of malignancy of any organ system except for basal cell carcinoma, squamous cell carcinoma or actinic keratoses of the skin that have been treated with no evidence of recurrence in the past 5 years, carcinoma in situ of the cervix or non-invasive malignant colon polyps that have been removed), treated or untreated within the past 5 years prior to baseline regardless of whether there is evidence of local recurrence or metastases.
• Subject is physically or mentally compromised (e.g., currently being treated for a psychiatric disorder, senile dementia, Alzheimer’s disease, etc) to the extent that the Investigator judges the subject to be unable or unlikely to remain compliant. 10. Subject has received an investigational therapy or approved therapy for investigational use within 30 days of injection procedure or during the follow-up phase of the study.
• Subjects should abstain from strenuous exercise for 48 hours prior to each visit, and while in the CRU. Subjects are also not allowed to operate any motor vehicle for at least 24 hours after injection of the investigational medicinal product (IMP).
• IMP investigational medicinal product
• Subjects should abstain from tobacco and ni cotine-containing products (including e- cigarettes) for 90 days prior to dosing, throughout the study until completion of the study.
• Subjects should refrain from consuming poppy seeds 48 hours prior to screening and each visit to avoid a positive result on the drugs of abuse screen.
• Subjects were fasted overnight for 8 hours pre-dose. In addition, subjects are advised to fast at least 4 hours prior to any visit where clinical laboratory evaluations were carried out.
• the ultrasound assessment at screening was conducted at any time during the 4-week screening period and includes a scan of the contra-lateral elbow. At least 1 week prior to dosing, subjects were required to have discontinued any use of opiate or NS AID medications they have been using for pain management. From Day -7 to Day -1, subjects were allowed to take paracetamol (less than or equal to 4 g/day) or use an ice pack to manage pain.
• Subjects attended the CRU on Day -2/-1 and pre-dose on Day 1 to confirm eligibility and undergo safety and efficacy baseline assessments. Following confirmation of eligibility, subjects were randomised and dosed with CWT-001 or placebo. After the injection, the subject was rested for 30 minutes. Subjects underwent post-dose safety and PK assessments and were discharged from the CRU on the same day. Subjects returned to the CRU on Day 2 for PK sampling assessments, and for safety and PK assessments on Day 7. Subjects returned to the CRU on Day 14, Day 28 and Day 90 for safety and efficacy assessments (further details provided below). Subjects underwent ultrasound assessment on Day 28 and Day 90. The duration of the study for each subject is approximately 16 weeks.
• tendinopathy pain intensity VAS
• ASES-E score SES-E score
• PRTEE score ultrasound assessment of tendon at days 28 and 90 post-injection. Stopping criteria include:
• a ‘serious’ adverse reaction in 1 subject i.e., a serious adverse event (SAE, defined below) that is considered at least possibly related to IMP administration.
• SAE serious adverse event
• Severe non-serious adverse reaction, i. e. , a severe non-serious AE considered as at least possibly related to IMP administration, in 2 subjects in the same cohort, independent of within, or not within, the same system-organ class (SOC).
• SOC system-organ class
• An AE is any untoward medical occurrence in a study subject which either emerges, or worsens from screening, during the clinical study, regardless of its potential relationship to the medicinal product.
• An AE therefore, can be any unfavourable or unintended sign, including a clinically- significant abnormal laboratory finding, symptom, or disease.
• An SAE is an untoward medical occurrence that, at any dose:
• hospitalisation signifies that the subject has been detained (usually involving at least an overnight stay) at the hospital or emergency ward for observation and/or treatment that would not have been appropriate in the physician’s office or out setting.
• Complications that occur during hospitalisation are AEs. If complication prolongs hospitalisation or fulfils any other serious criteria, the event is serious. When in doubt whether ‘hospitalisation’ occurred or was necessary, the AE should be considered serious.
• disperability means a substantial disruption of a person’s ability to conduct normal life functions. This definition is not intended to include experiences of relatively minor medical significance such as uncomplicated headache, nausea, vomiting, diarrhoea, influenza and accidental trauma (e.g., sprained ankle) which may interfere or prevent everyday life functions but do not constitute a substantial disruption.
• severe is often used to describe the intensity (severity) of a specific event (as in mild, moderate or severe myocardial infarction); the event itself, however, may be of relatively minor medical significance (such as severe headache). This is not the same as “serious”, which is based on subject/event outcome or action criteria described above and are usually associated with events that pose a threat to a subject’s life or functioning. A severe AE does not necessarily need to be considered serious. For example, persistent nausea of several hours duration may be considered severe nausea but not an SAE. On the other hand, a stroke resulting in only a minor degree of disability may be considered mild but would be defined as an SAE based on the above criteria. Seriousness (not severity) serves as a guide for defining regulatory reporting obligations
• a baseline PK blood sample is collected within 30 minutes pre-dose. Blood samples for determination of plasma concentrations of miR29a were taken at the timepoints stated in Table 9 below. Plasma concentration-time data are analysed by noncompartmental methods. Nominal times were used for interim decisions, however actual sampling times were used for final calculations. Approximate sampling volumes are provided in Table 8. Table 8 - Sampling summary
• Pharmacokinetic plasma samples are collected and miR29a is quantified using a validated quantitative real time PCR method developed at Thermo Fisher.
• VAS visual analogue scale
• Randomized controlled trials (RCTs) in orthopaedics have evaluated the use of normal saline (NS) injections as a placebo/control group, expecting no physiological effect of this substance.
• NS normal saline
• a recent study (ref. 22) of intra-articular saline injections into knee joints in patients suffering from osteoarthritis suggested plausible and potential beneficial effects of saline such as pain relief and osmolality-related changes.
• the ASES score is developed by the Society of the American Shoulder and Elbow Surgeons for the assessment of shoulder and elbow function (ref. 26).
• the ASES-E score is self-administered and has 17 questions in the areas of symptoms and functions. The severity of pain was graded on a 10 cm visual analogue scale that ranges from 0 (no pain at all) to 10 (pain as bad as it can be). The recall period is how does the subject feel at this point in time. This validated tool has been chosen as it combines subject reported outcomes with a physician assessment in change in elbow range of movement and strength (ref. 27). Efficacy assessment - quick disability of the arm, should, and hand (DASH) score
• the Quick DASH is an abbreviated form of DASH, a subject reported outcome tool, developed by the American Academy of Orthopaedic Surgeons along with the Institute for Work & Health (Toronto, Ontario, Canada).
• the Quick DASH Index is self-administered and uses 11 items to measure physical function and symptoms in subjects with any or multiple musculoskeletal disorders of the upper limb.
• This validated outcome measure (ref. 27) provides core information of the change in subject functionality post CWT-001 injection.
• the mean change in QuickDASH score for subjects that had received any dose of CWT-001 also exceeded the established MCID for DASH in patients with upper-limb musculoskeletal disorders, which is -20 points (ref. 28).
• the mean improvement in QuickDASH score at Day 90 relative to the pre-dose score for subjects that had received any dose of CWT-001 was -53 ⁇ 5 points.
• the PRTEE formerly called the Patient- Rated Forearm Evaluation Questionnaire, is a 15 -item selfreported questionnaire to measure perceived pain and disability in people with tennis elbow. It has three subscales: pain, usual activities and specific activities. The pain subscale has five items about the intensity of pain during various activities. The specific activities subscale has six items tapping into the difficulty experienced while performing specific activities, like lifting a coffee cup. The four items in the ‘usual activities’ subscale capture the difficulty experienced in performing usual daily roles including those performed during work and recreation.
• the PRTEE is an excellent example of a disease-specific self-report measure. This is very helpful in capturing aspects of pain and function that are more specific to tennis elbow than a generic joint-specific or region-specific measure. This validated outcome measure (ref. 27) provides core information of the change in subject functionality post CWT-001 injection.
• the mean change in PRTEE score for all subjects that received any dose of CWT-001 was determined and compared with placebo (Figure 19D).
• the mean improvement in PRTEE score at Day 90 was 71 ⁇ 6 % of the baseline pre-dose score for subjects that had received any dose of CWT-001. This exceeds the MCID for clinical significance defined as “much better” or “completely recovered” for PRTEE, which is an improvement of 37% of baseline score (ref. 29)
• the lateral tendon complex at the level of the elbow is easily accessible to ultrasonographic examination because it is located immediately under the subcutaneous tissue and the fascia. Normally, the complex is visualized as a hyperechoic linear structure aligned between the corresponding attachments. Characteristic ultrasound finding of lateral elbow tendinopathy include focal hypoechoic areas within the tendon, which are associated with loss of the normal internal collagen fibre alignment pattern. Importantly studies have shown that sonographic features are consistent with disease process and demonstrate resolution with improvement of symptoms using VAS and DASH scores post therapeutic treatment (ref. 30). Based on preclinical models, ultrasound assessment was conducted at Days 28 and 90 post-injection. Importantly, this provides data on the mechanism of action of CWT-001 (improvement in collagen structure), return to normal tendon structure post treatment which is beneficial for subject recovery and durability of the treatment.
• UTC ultrasound tissue characterisation
• Types I (green) and II (blue) represent an organised matrix
• Types III (red) and IV (black) represent a disorganised matrix ( Figure 20A).
• the advantage of this tool compared to conventional ultrasound, is that it captures a three-dimensional ultrasound image of the tendon and uses standardised parameters (transducer tilt angle, depth and gain settings). Bilateral scans (both lateral elbows) were performed by a single examiner with experience performing UTC scans.
• Representative ultrasound images of the lateral elbow are provided for a subject that had received the placebo at pre-inj ection (Figure 20B), Day 28 (Figure 20C) and Day 90 (Figure 20D), and for a subject that had received 200 ⁇ g CWT-001 at pre-inj ection (Figure 20E), Day 20 (Figure 20F) and Day 90 ( Figure 20G).
• UTC Type I, II, III and IV tendon tissue The percentages of UTC Type I, II, III and IV tendon tissue and the total percentage of reparative tendon (UTC Types I and II) vs degenerative tendon (UTC types III and IV) were quantified for the subjects.
• the percentage of reparative tendon tissue, in particular intact UTC type I tendon increased from Day 0, to Day 28 to Day 90 for subjects that received CWT-001, and was much higher for subjects that received CWT-001 than for subjects that received the placebo.
• the mean percentages of reparative tendon for all subjects that received any dose of CWT-001 increased significantly from Day 0 to Day 28 (P ⁇ 0.05) and to Day 90 (P ⁇ 0.01), while no significant change was observed for the subjects that received the placebo.
• the subjects that had received CWT-001 also had significantly higher levels of reparative tendon than the subjects that had received the placebo at Day 28 (P ⁇ 0.05) and Day 90 (P ⁇ 0.01), and had significantly lower levels of degenerative tendon at Day 28 (P ⁇ 0.05) and Day 90 (P ⁇ 0.01).
• the UTC data demonstrate a quantifiable improvement in tendon structure for human subjects with lateral epicondylitis following treatment with a single 200 ⁇ g, 500 ⁇ g or 1500 ⁇ g dose of CWT-001.
• Example 8 - Dupuytren’s Disease ( Figures 15 and 16) qPCR analysis of miR29a expression in healthy and Dupuytren’s fascia tissue was performed (4 samples for the healthy group, 3 samples for the Dupuytren’s group). The results showed that the levels of miR29a were significantly reduced in samples from Dupuytren’s tissue relative to healthy controls undergoing carpal tunnel release surgery ( Figure 15). These experiments showed that the fibrotic growth observed in Dupuytren’s diseases is associated with a reduction in miR29a expression. This observation indicated that miR-29 replacement therapy may prevent or reduce the collagen overproduction seen in Dupuytren’s diseases.
• CWT-001 is administered to subjects with Peyronie’s Disease as a single intra-lesional (intra- penile) injection into the tunica albuginea of the penis.
• Subjects are males with early stage (acute or active phase) Peyronie’s Disease, with a penis curvature of ⁇ 30° and/or palpable plaque ⁇ 2cm in extent (based on Kelami classification, ref. 32). Subjects are followed up for 1 year post-injection.
• Efficacy assessment includes curvature of the penis, penile duplex Doppler ultrasound, pain VAS, IEFF questionnaire, and PDQ questionnaire.

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