WO2018141025A1 - Réactifs pour le traitement de maladies ou affections oculaires associées à la néovascularisation, et leur utilisation - Google Patents

Réactifs pour le traitement de maladies ou affections oculaires associées à la néovascularisation, et leur utilisation Download PDF

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WO2018141025A1
WO2018141025A1 PCT/AU2018/050075 AU2018050075W WO2018141025A1 WO 2018141025 A1 WO2018141025 A1 WO 2018141025A1 AU 2018050075 W AU2018050075 W AU 2018050075W WO 2018141025 A1 WO2018141025 A1 WO 2018141025A1
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seq
set forth
shmir
sequence
sequence set
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David Suhy
Shih-Chu KAO
Peter ROELVINK
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Benitec Biopharma Limited
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C12N15/1136—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against growth factors, growth regulators, cytokines, lymphokines or hormones
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/14—Type of nucleic acid interfering N.A.
    • C12N2310/141—MicroRNAs, miRNAs
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/50—Physical structure
    • C12N2310/51—Physical structure in polymeric form, e.g. multimers, concatemers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2330/00—Production
    • C12N2330/50—Biochemical production, i.e. in a transformed host cell
    • C12N2330/51—Specially adapted vectors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/16011—Human Immunodeficiency Virus, HIV
    • C12N2740/16041—Use of virus, viral particle or viral elements as a vector
    • C12N2740/16043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector

Definitions

  • RNA interference RNA interference
  • AMD age-related macular degeneration
  • Age related macular degeneration is the leading cause of irreversible vision loss in the United States and many other industrialised countries. "Dry” AMD is the most common type of macular degeneration and affects 90% of the people who have the condition.
  • the dry form is characterized by the formation of drusen within the macula, a specialized structural region of the retina which capture the light that enters the eye.
  • drusen is formed under the retinal pigment epithelial (RPE) cells and its presence is thought to lead to atrophy of photoreceptors due to a breakdown or thinning of the RPE layer of that supports the photoreceptor cells. It is also thought that persistence of drusen within the retina leads to a persistent inflammatory reaction and results in a cascade of secondary responses that eventually can lead to wet AMD.
  • RPE retinal pigment epithelial
  • the "wet" form of AMD is characterized by an abnormal outgrowth of blood vessels from the vasculature situated behind the retina in a process that is often referred to as choroidal neovascularization (CNV). While not as prevalent as the dry form, it has a more rapid onset and is more severe phenotype, often leading to reduction of a substantial portion of the visual field.
  • CNV choroidal neovascularization
  • the current standard of care for wet AMD is Ranibizumab (RAN), a monoclonal antibody fragment with strong affinity to the vascular endothelial growth factor-A (VEGF- A), a molecular moiety secreted from cells and known to cause the formation or growth of nascent blood vessels.
  • RAN Ranibizumab
  • VEGF- A vascular endothelial growth factor-A
  • RAN binds to and inhibits the biologic activity of VEGF-A, thereby preventing the interaction of VEGF-A with its receptors (VEGFR1 and VEGFR2) on the surface of endothelial cells. This results in a reduction in endothelial cell proliferation, less vascular leakage, and a reduction in new blood vessel formation characteristic of CNV.
  • RAN acts as a molecular sponge to mop-up secreted VEGF-A. Inefficiencies in this process may be one reason why vision is only stabilized, not improved in most patients. In other words, it treats the symptoms but not the cause.
  • Aflibercept is a recombinant fusion protein consisting of VEGF- binding portions from the extracellular domains of human VEGF receptors 1 and 2, that are fused to the Fc portion of the human IgGl immunoglobulin. As well as binding all isomers of the VEGF-A family, it also binds VEGF-B and placental growth factor.
  • Aflibercept is deleivered as an intravitreal injection, and because (like RAN) it has a relatively short half-life, it must be administered regularly (one injection per month for three consecutive months, followed by one injection every 2 months).
  • AAV2-sFLT01 is a gene therapy vector that expresses a modified soluble Fltl receptor coupled to a human IgGl Fc.
  • AAV2-sFLT01 functions to neutralize the pro-angiogenic activities of VEGF for treatment of wet AMD via an intravitreal injection. (Wasworth et al. Molecular Therapy vol. 19 no. 2 Feb. 2011; 326- 334).
  • the use of an AAV vector is expected to ensure long-term expression, lasting for many months or even years, from a single injection.
  • the molecule PF-04523655 is a 19 nucleotide siRNA that inhibits the expression of the hypoxia-inducible gene RTP801 (Nguyen et al. Ophthalmology. 2012 Sep;119(9): 1867- 73).
  • RTP801 hypoxia-inducible gene
  • siRNAs are transmembrane proteins that play a key role in the innate immune system. Often positioned on either the cell surface or on intracellular vesicles such as the endosome, some family members of this family recognize double stranded RNA, not normally present in the endogenous cell, as foreign substance and triggers a cascade of molecule responses. This leads to interferon activation, which has a transitory therapeutic effect in mouse models. However interferon has a much lower efficacy in humans which explains the poor efficacy of this treatment in human clinical testing.
  • Retinostat is an equine infectious anaemia virus (EIAV) based lentivirus vector expressing angiostatin and endostatin, both of which are naturally occurring angiogenesis inhibitors in the ocular compartment. Endostatin blocks VEGF signalling, reduces vascular permeability, decreases cell matrix adhesion and promotes endothelial cell apoptosis.
  • EIAV equine infectious anaemia virus
  • Angiostatin prevents endothelial cell proliferation and migration.
  • the genes are delivered via a subretinal injection and inhibit the formation of new blood vessels.
  • Sub-retinal delivery however requires an intensive surgical procedure, which, unlike intravitreal delivery, does not lend itself to outpatient treatments or treatment at a local doctor.
  • the present disclosure is based, in part, on the recognition that existing therapeutic agents for treatment of AMD are limited in their efficacy, have undesirable side-effects and/or require frequent adminstration, typically involving monthly injections into the eye.
  • the inventors recognised a need for further therapeutic agents for treatment of AMD e.g., wet AMD, preferably with longer lasting effects and/or which can reverse the symptoms with greater efficacy and minimal side-effects.
  • ddRNAi DNA-directed RNA interference
  • shmiRs short hairpin micro-RNAs
  • VEGFb vascular endothelial growth factor B
  • PGF placental growth factor
  • shmiRs of the disclosure comprise effector sequences capable of inhibiting or reducing expression of VEGFb or PGF gene transcripts, and can be expressed the ddRNAi construct alone or together, including in combination with shmiRs targeting conserved regions of RNA transcripts produced by vascular endothelial growth factor A (VEGFa).
  • VEGFa vascular endothelial growth factor A
  • exemplary ddRNAi construct of the disclosure expresses three shmiRs targeting conserved regions of RNA transcripts produced by VEGFb, VEGFa and PGF, respectively.
  • exemplary shmiRs of the disclosure comprise effector sequences which are capable of inhibiting or reducing expression of gene transcripts from the respective AMD-associated gene i.e., VEGFb, VEGFa or PGF, in vitro when expressed together from a ddRNAi construct.
  • an exemplary ddRNAi construct expressing three shmiRs targeting conserved regions of RNA transcripts produced by VEGFb, VEGFa and PGF respectively, is capable of inhibiting or reducing expression of gene transcripts from VEGFb and VEGFa independently when introduced to ARPE-19 cells (a human retinal pigment epithelial cell line).
  • the same exemplary ddRNAi construct expressing three shmiRs targeting conserved regions of RNA transcripts produced by VEGFb, VEGFa and PGF respectively is capable of inhibiting or reducing expression of gene transcripts from PGF when introduced to JEG- cells (a human placental choriocarcinoma cell lined that expresses high levels of PGF).
  • JEG- cells a human placental choriocarcinoma cell lined that expresses high levels of PGF.
  • the present disclosure provides a nucleic acid comprising a DNA sequence which encodes a short hairpin micro-RNA (shmiR), said shmiR comprising:
  • pri-miRNA primary micro RNA
  • the effector sequence is substantially complementary to a region of equivalent length in an RNA transcript of VEGFb set forth set forth in any one of SEQ ID NOs: 1-10 or a region of equivalent length in an RNA transcript of PGF set forth in any one of SEQ ID NOs: 11-22.
  • the effector sequence will be less than 30 nucleotides in length.
  • a suitable effector sequence may be in the range of 17-29 nucleotides in length.
  • the effector sequence will be 20 nucleotides in length. More preferably, the effector sequence will be 21 nucleotides in length and the effector complement sequence will be 20 nucleotides in length.
  • the effector sequence may comprise 6 base pair mismatches relative to the region of equivalent length in the sequence set forth in any one of SEQ ID NOs: 1-22 to which the effector sequence is substantially complementary.
  • the effector sequence comprises 5 base pair mismatches relative to the region of equivalent length in the sequence set forth in any one of SEQ ID NOs: 1-22 to which the effector sequence is substantially complementary.
  • the effector sequence comprises 4 base pair mismatches relative to the region of equivalent length in the sequence set forth in any one of SEQ ID NOs: 1-22 to which the effector sequence is substantially complementary.
  • the effector sequence comprises 3 base pair mismatches relative to the region of equivalent length in the sequence set forth in any one of SEQ ID NOs: 1-22 to which the effector sequence is substantially complementary.
  • the effector sequence comprises 2 base pair mismatches relative to the region of equivalent length in the sequence set forth in any one of SEQ ID NOs: 1-22 to which the effector sequence is substantially complementary.
  • the effector sequence comprises 1 base pair mismatch relative to the region of equivalent length in the sequence set forth in any one of SEQ ID NOs: 1-22 to which the effector sequence is substantially complementary.
  • the effector sequence is 100% complementary to a region of equivalent length within a sequence set forth in any one of SEQ ID NOs: 1-22. Where mismatches are present, it is preferred that they are not located within the region corresponding to the seed region of the shmiR i.e., nucleotides 2-8 of the effector sequence.
  • the nucleic acid described herein may comprise a DNA sequence encoding a shmiR selected from the group consisting of:
  • VEGFb_shmiR- 1 comprising: (i) an effector sequence which is substantially complementary to the sequence set forth in SEQ ID NO:23 with the exception of 1, 2, 3, 4, 5 or 6 base mismatches, provided that the effector sequence is capable of forming a duplex with a sequence set forth in SEQ ID NO:23; and (ii) an effector complement sequence comprising a sequence which is substantially complementary to the effector sequence;
  • VEGFb_shmiR-2 comprising: (i) an effector sequence which is substantially complementary to the sequence set forth in SEQ ID NO:25 with the exception of 1, 2, 3, 4, 5 or 6 base mismatches, provided that the effector sequence is capable of forming a duplex with a sequence set forth in SEQ ID NO:25; and (ii) an effector complement sequence comprising a sequence which is substantially complementary to the effector sequence;
  • VEGFb_shmiR-3 comprising: (i) an effector sequence which is substantially complementary to the sequence set forth in SEQ ID NO:27 with the exception of 1, 2, 3, 4, 5 or 6 base mismatches, provided that the effector sequence is capable of forming a duplex with a sequence set forth in SEQ ID NO:27; and (ii) an effector complement sequence comprising a sequence which is substantially complementary to the effector sequence;
  • VEGFb_shmiR-4 comprising: (i) an effector sequence which is substantially complementary to the sequence set forth in SEQ ID NO:29 with the exception of 1, 2, 3, 4, 5 or 6 base mismatches, provided that the effector sequence is capable of forming a duplex with a sequence set forth in SEQ ID NO:29; and (ii) an effector complement sequence comprising a sequence which is substantially complementary to the effector sequence;
  • VEGFb_shmiR-5 comprising: (i) an effector sequence which is substantially complementary to the sequence set forth in SEQ ID NO:31 with the exception of 1, 2, 3, 4, 5 or 6 base mismatches, provided that the effector sequence is capable of forming a duplex with a sequence set forth in SEQ ID NO:31; and (ii) an effector complement sequence comprising a sequence which is substantially complementary to the effector sequence;
  • VEGFb_shmiR-6 comprising: (i) an effector sequence which is substantially complementary to the sequence set forth in SEQ ID NO:33 with the exception of 1, 2, 3, 4, 5 or 6 base mismatches, provided that the effector sequence is capable of forming a duplex with a sequence set forth in SEQ ID NO:33; and (ii) an effector complement sequence comprising a sequence which is substantially complementary to the effector sequence;
  • VEGFb_shmiR-7 comprising: (i) an effector sequence which is substantially complementary to the sequence set forth in SEQ ID NO:35 with the exception of 1, 2, 3, 4, 5 or 6 base mismatches, provided that the effector sequence is capable of forming a duplex with a sequence set forth in SEQ ID NO:35; and (ii) an effector complement sequence comprising a sequence which is substantially complementary to the effector sequence;
  • VEGFb_shmiR-8 comprising: (i) an effector sequence which is substantially complementary to the sequence set forth in SEQ ID NO:37 with the exception of 1, 2, 3, 4, 5 or 6 base mismatches, provided that the effector sequence is capable of forming a duplex with a sequence set forth in SEQ ID NO:37; and (ii) an effector complement sequence comprising a sequence which is substantially complementary to the effector sequence;
  • VEGFb_shmiR-9 comprising: (i) an effector sequence which is substantially complementary to the sequence set forth in SEQ ID NO:39 with the exception of 1, 2, 3, 4, 5 or 6 base mismatches, provided that the effector sequence is capable of forming a duplex with a sequence set forth in SEQ ID NO:39; and (ii) an effector complement sequence comprising a sequence which is substantially complementary to the effector sequence;
  • VEGFb_shmiR-10 comprising: (i) an effector sequence which is substantially complementary to the sequence set forth in SEQ ID NO:41 with the exception of 1, 2, 3, 4, 5 or 6 base mismatches, provided that the effector sequence is capable of forming a duplex with a sequence set forth in SEQ ID NO:41; and (ii) an effector complement sequence comprising a sequence which is substantially complementary to the effector sequence;
  • PGF_shmiR-l comprising: (i) an effector sequence which is substantially complementary to the sequence set forth in SEQ ID NO:43 with the exception of 1, 2, 3, 4, 5 or 6 base mismatches, provided that the effector sequence is capable of forming a duplex with a sequence set forth in SEQ ID NO:43; and (ii) an effector complement sequence comprising a sequence which is substantially complementary to the effector sequence;
  • PGF_shmiR-2 comprising: (i) an effector sequence which is substantially complementary to the sequence set forth in SEQ ID NO:45 with the exception of 1, 2, 3, 4, 5 or 6 base mismatches, provided that the effector sequence is capable of forming a duplex with a sequence set forth in SEQ ID NO:45; and (ii) an effector complement sequence comprising a sequence which is substantially complementary to the effector sequence;
  • PGF_shmiR-3 comprising: (i) an effector sequence which is substantially complementary to the sequence set forth in SEQ ID NO:47 with the exception of 1, 2, 3, 4, 5 or 6 base mismatches, provided that the effector sequence is capable of forming a duplex with a sequence set forth in SEQ ID NO:47; and (ii) an effector complement sequence comprising a sequence which is substantially complementary to the effector sequence;
  • PGF_shmiR-4 comprising: (i) an effector sequence which is substantially complementary to the sequence set forth in SEQ ID NO:49 with the exception of 1, 2, 3, 4, 5 or 6 base mismatches, provided that the effector sequence is capable of forming a duplex with a sequence set forth in SEQ ID NO:49; and (ii) an effector complement sequence comprising a sequence which is substantially complementary to the effector sequence;
  • PGF_shmiR-5 comprising: (i) an effector sequence which is substantially complementary to the sequence set forth in SEQ ID NO:51 with the exception of 1, 2, 3, 4, 5 or 6 base mismatches, provided that the effector sequence is capable of forming a duplex with a sequence set forth in SEQ ID NO:51; and (ii) an effector complement sequence comprising a sequence which is substantially complementary to the effector sequence;
  • PGF_shmiR-6 comprising: (i) an effector sequence which is substantially complementary to the sequence set forth in SEQ ID NO:53 with the exception of 1, 2, 3, 4, 5 or 6 base mismatches, provided that the effector sequence is capable of forming a duplex with a sequence set forth in SEQ ID NO:53; and (ii) an effector complement sequence comprising a sequence which is substantially complementary to the effector sequence;
  • PGF_shmiR-7 comprising: (i) an effector sequence which is substantially complementary to the sequence set forth in SEQ ID NO:55 with the exception of 1, 2, 3, 4, 5 or 6 base mismatches, provided that the effector sequence is capable of forming a duplex with a sequence set forth in SEQ ID NO:55; and (ii) an effector complement sequence comprising a sequence which is substantially complementary to the effector sequence;
  • PGF_shmiR-8 comprising: (i) an effector sequence which is substantially complementary to the sequence set forth in SEQ ID NO:57 with the exception of 1, 2, 3, 4, 5 or 6 base mismatches, provided that the effector sequence is capable of forming a duplex with a sequence set forth in SEQ ID NO:57; and (ii) an effector complement sequence comprising a sequence which is substantially complementary to the effector sequence;
  • PGF_shmiR-9 comprising: (i) an effector sequence which is substantially complementary to the sequence set forth in SEQ ID NO:59 with the exception of 1, 2, 3, 4, 5 or 6 base mismatches, provided that the effector sequence is capable of forming a duplex with a sequence set forth in SEQ ID NO:59; and (ii) an effector complement sequence comprising a sequence which is substantially complementary to the effector sequence;
  • PGF_shmiR-10 comprising: (i) an effector sequence which is substantially complementary to the sequence set forth in SEQ ID NO:61 with the exception of 1, 2, 3, 4, 5 or 6 base mismatches, provided that the effector sequence is capable of forming a duplex with a sequence set forth in SEQ ID NO:61; and (ii) an effector complement sequence comprising a sequence which is substantially complementary to the effector sequence;
  • PGF_shmiR-l l comprising: (i) an effector sequence which is substantially complementary to the sequence set forth in SEQ ID NO:63 with the exception of 1, 2, 3, 4, 5 or 6 base mismatches, provided that the effector sequence is capable of forming a duplex with a sequence set forth in SEQ ID NO:63; and (ii) an effector complement sequence comprising a sequence which is substantially complementary to the effector sequence; and
  • PGF_shmiR-12 comprising: (i) an effector sequence which is substantially complementary to the sequence set forth in SEQ ID NO:65 with the exception of 1, 2, 3, 4, 5 or 6 base mismatches, provided that the effector sequence is capable of forming a duplex with a sequence set forth in SEQ ID NO:65; and (ii) an effector complement sequence comprising a sequence which is substantially complementary to the effector sequence.
  • nucleic acid described herein may comprise a DNA sequence encoding a shmiR selected from the group consisting of:
  • VEGFb_shmiR- 1 comprising an effector sequence set forth in SEQ ID NO:24 and an effector complement sequence which is substantially complementary to the sequence set forth in SEQ ID NO:24 and capable of forming a duplex therewith;
  • VEGFb_shmiR-2 comprising an effector sequence set forth in SEQ ID NO:26 and an effector complement sequence which is substantially complementary to the sequence set forth in SEQ ID NO:26 and capable of forming a duplex therewith;
  • VEGFb_shmiR-3 comprising an effector sequence set forth in SEQ ID NO:28 and an effector complement sequence which is substantially complementary to the sequence set forth in SEQ ID NO:28 and capable of forming a duplex therewith;
  • VEGFb_shmiR-4 comprising an effector sequence set forth in SEQ ID NO:30 and an effector complement sequence which is substantially complementary to the sequence set forth in SEQ ID NO:30 and capable of forming a duplex therewith;
  • VEGFb_shmiR-5 comprising an effector sequence set forth in SEQ ID NO:32 and an effector complement sequence which is substantially complementary to the sequence set forth in SEQ ID NO:32 and capable of forming a duplex therewith
  • VEGFb_shmiR-6 comprising an effector sequence set forth in SEQ ID NO:34 and an effector complement sequence which is substantially complementary to the sequence set forth in SEQ ID NO:34 and capable of forming a duplex therewith;
  • VEGFb_shmiR-7 comprising an effector sequence set forth in SEQ ID NO:36 and an effector complement sequence which is substantially complementary to the sequence set forth in SEQ ID NO:36 and capable of forming a duplex therewith;
  • VEGFb_shmiR-8 comprising an effector sequence set forth in SEQ ID NO:38 and an effector complement sequence which is substantially complementary to the sequence set forth in SEQ ID NO:38 and capable of forming a duplex therewith;
  • VEGFb_shmiR-9 comprising an effector sequence set forth in SEQ ID NO:40 and an effector complement sequence which is substantially complementary to the sequence set forth in SEQ ID NO:40 and capable of forming a duplex therewith
  • VEGFb_shmiR-10 comprising an effector sequence set forth in SEQ ID NO:42 and an effector complement sequence which is substantially complementary to the sequence set forth in SEQ ID NO:42 and capable of forming a duplex therewith;
  • PGF_shmiR-l comprising an effector sequence set forth in SEQ ID NO:44 and an effector complement sequence which is substantially complementary to the sequence set forth in SEQ ID NO:44 and capable of forming a duplex therewith;
  • PGF_shmiR-2 comprising an effector sequence set forth in SEQ ID NO:46 and an effector complement sequence which is substantially complementary to the sequence set forth in SEQ ID NO:46 and capable of forming a duplex therewith;
  • PGF_shmiR-3 comprising an effector sequence set forth in SEQ ID NO:48 and an effector complement sequence which is substantially complementary to the sequence set forth in SEQ ID NO:48 and capable of forming a duplex therewith;
  • PGF_shmiR-4 comprising an effector sequence set forth in SEQ ID NO: 50 and an effector complement sequence which is substantially complementary to the sequence set forth in SEQ ID NO:50 and capable of forming a duplex therewith;
  • PGF_shmiR-5 comprising an effector sequence set forth in SEQ ID NO: 52 and an effector complement sequence which is substantially complementary to the sequence set forth in SEQ ID NO:52 and capable of forming a duplex therewith;
  • PGF_shmiR-6 comprising an effector sequence set forth in SEQ ID NO: 54 and an effector complement sequence which is substantially complementary to the sequence set forth in SEQ ID NO:54 and capable of forming a duplex therewith;
  • PGF_shmiR-7 comprising an effector sequence set forth in SEQ ID NO: 56 and an effector complement sequence which is substantially complementary to the sequence set forth in SEQ ID NO:56 and capable of forming a duplex therewith;
  • PGF_shmiR-8 comprising an effector sequence set forth in SEQ ID NO:58 and an effector complement sequence which is substantially complementary to the sequence set forth in SEQ ID NO:58 and capable of forming a duplex therewith;
  • PGF_shmiR-9 comprising an effector sequence set forth in SEQ ID NO: 60 and an effector complement sequence which is substantially complementary to the sequence set forth in SEQ ID NO:60 and capable of forming a duplex therewith
  • PGF_shmiR-10 comprising an effector sequence set forth in SEQ ID NO:62 and an effector complement sequence which is substantially complementary to the sequence set forth in SEQ ID NO:62 and capable of forming a duplex therewith;
  • PGF_shmiR-l l comprising an effector sequence set forth in SEQ ID NO:64 and an effector complement sequence which is substantially complementary to the sequence set forth in SEQ ID NO:64 and capable of forming a duplex therewith;
  • PGF_shmiR-12 comprising an effector sequence set forth in SEQ ID NO:66 and an effector complement sequence which is substantially complementary to the sequence set forth in SEQ ID NO:66 and capable of forming a duplex therewith.
  • the shmiR encoded by the nucleic acid described herein may comprise an effector complement sequence comprising 1, 2, 3, 4, 5 or 6 mismatches relative to the corresponding effector sequence, provided that the cognate effector and effector
  • complement sequences are capable of forming a duplex region.
  • nucleic acid described herein may comprise a DNA sequence encoding a shmiR selected from the group consisting of:
  • VEGFb_shmiR- 1 comprising an effector sequence set forth in SEQ ID NO:24 and an effector complement sequence set forth in SEQ ID NO:23;
  • VEGFb_shmiR-2 comprising an effector sequence set forth in SEQ ID NO:26 and an effector complement sequence set forth in SEQ ID NO:25;
  • VEGFb_shmiR-3 comprising an effector sequence set forth in SEQ ID NO:28 and an effector complement sequence set forth in SEQ ID NO:27;
  • VEGFb_shmiR-4 comprising an effector sequence set forth in SEQ ID NO:30 and an effector complement sequence set forth in SEQ ID NO:29;
  • VEGFb_shmiR-5 comprising an effector sequence set forth in SEQ ID NO:32 and an effector complement sequence set forth in SEQ ID NO:31;
  • VEGFb_shmiR-6 comprising an effector sequence set forth in SEQ ID NO:34 and an effector complement sequence set forth in SEQ ID NO:33;
  • VEGFb_shmiR-7 comprising an effector sequence set forth in SEQ ID NO:36 and an effector complement sequence set forth in SEQ ID NO:35;
  • VEGFb_shmiR-8 comprising an effector sequence set forth in SEQ ID NO:38 and an effector complement sequence set forth in SEQ ID NO:37
  • VEGFb_shmiR-9 comprising an effector sequence set forth in SEQ ID NO:40 and an effector complement sequence set forth in SEQ ID NO:39;
  • VEGFb_shmiR-10 comprising an effector sequence set forth in SEQ ID NO:42 and an effector complement sequence set forth in SEQ ID NO:41;
  • PGF_shmiR-l comprising an effector sequence set forth in SEQ ID NO:44 and an effector complement sequence set forth in SEQ ID NO:43;
  • PGF_shmiR-2 comprising an effector sequence set forth in SEQ ID NO:46 and an effector complement sequence set forth in SEQ ID NO:45;
  • PGF_shmiR-3 comprising an effector sequence set forth in SEQ ID NO:48 and an effector complement sequence set forth in SEQ ID NO:47;
  • PGF_shmiR-4 comprising an effector sequence set forth in SEQ ID NO: 50 and an effector complement sequence set forth in SEQ ID NO:49;
  • PGF_shmiR-5 comprising an effector sequence set forth in SEQ ID NO: 52 and an effector complement sequence set forth in SEQ ID NO:51;
  • PGF_shmiR-6 comprising an effector sequence set forth in SEQ ID NO: 54 and an effector complement sequence set forth in SEQ ID NO:53;
  • PGF_shmiR-7 comprising an effector sequence set forth in SEQ ID NO: 56 and an effector complement sequence set forth in SEQ ID NO:55;
  • PGF_shmiR-8 comprising an effector sequence set forth in SEQ ID NO:58 and an effector complement sequence set forth in SEQ ID NO:57;
  • PGF_shmiR-9 comprising an effector sequence set forth in SEQ ID NO:60 and an effector complement sequence set forth in SEQ ID NO:59;
  • PGF_shmiR-10 comprising an effector sequence set forth in SEQ ID NO:62 and an effector complement sequence set forth in SEQ ID NO:61;
  • PGF_shmiR-l 1 comprising an effector sequence set forth in SEQ ID NO:64 and an effector complement sequence set forth in SEQ ID NO:63;
  • PGF_shmiR-12 comprising an effector sequence set forth in SEQ ID NO:66 and an effector complement sequence set forth in SEQ ID NO:65.
  • the shmiR encoded by the nucleic acid of the disclosure may comprise, in a 5' to 3' direction:
  • the effector complement sequence the stemloop sequence
  • the shmiR encoded by the nucleic acid of the disclosure may comprise, in a 5' to 3' direction:
  • Suitable loop sequences may be selected from those known in the art. However, an exemplary stemloop sequence is set forth in SEQ ID NO: 67.
  • Suitable primary micro RNA (pri-miRNA or pri-R) backbones for use in a nucleic acid of the disclosure may be selected from those known in the art.
  • the pri- miRNA backbone may be selected from a pri-miR-30a backbone, a pri-miR-155 backbone, a pri-miR-21 backbone and a pri-miR-136 backbone.
  • the pri-miRNA backbone is a pri-miR-30a backbone.
  • the 5' flanking sequence of the pri-miRNA backbone is set forth in SEQ ID NO: 68 and the 3' flanking sequence of the pri-miRNA backbone is set forth in SEQ ID NO: 69.
  • the nucleic acid described herein comprises a DNA sequence selected from the sequence set forth in any one of SEQ ID NOs: 92-113.
  • a shmiR encoded by the nucleic acid of the disclosure may comprise a sequence set forth in any one of SEQ ID NOs: 70-91.
  • a nucleic acid described herein which encodes a shmiR targeting VEGFb may comprise a DNA sequence selected from the sequence set forth in any one of SEQ ID NOs: 92-101 and encode a shmiR comprising or consisting of a sequence set forth in any one of SEQ ID NOs: 70-79.
  • a nucleic acid described herein which encodes a shmiR targeting PGF may comprise a DNA sequence selected from the sequence set forth in any one of SEQ ID NOs:
  • shRNA short hairpin RNA
  • the effector sequence is substantially complementary to a region of equivalent length in an RNA transcript of VEGFb set forth set forth in any one of SEQ ID NOs: 1-10 or a region of equivalent length in an RNA transcript of PGF set forth set forth in any one of SEQ ID NOs: 11-22.
  • the effector sequence will be less than 30 nucleotides in length.
  • a suitable effector sequence may be in the range of 17-29 nucleotides in length.
  • effector and effector complement sequence combinations are described herein in the context of shmiRs of the disclosure targeting VEGFb and PGF and shall be taken to apply mutatis mutandis to each example in which a shRNA targeting the corresponding region of a transcript of VEGFb of PGF is described, including nucleic acids encoding such shRNAs.
  • the shRNA will comprise a stem loop sequence positioned between the effector sequence and the effector complement sequence.
  • the shRNA encoded by the nucleic acid of the disclosure may comprise, in a 5' to 3' direction:
  • the shRNA encoded by the nucleic acid of the disclosure may comprise, in a 5' to 3' direction:
  • Suitable loop sequences are described herein in the context of shmiRs and shall be taken to apply mutatis mutandis to each example in which a shRNA is described.
  • an exemplary stemloop sequence is set forth in SEQ ID NO: 67.
  • nucleic acids in accordance with the present disclosure may be combined or used in conjunction with one or more agents for treating AMD. Accordingly, the present disclosure provides a plurality of nucleic acids comprising:
  • a nucleic acid comprising a DNA sequence encoding a shmiR targeting VEGFa (VEGFa_shmiR) comprising:
  • pri-miRNA primary micro RNA
  • effector sequence of the shmiR at (b)(ii) is substantially complementary to a region of equivalent length in the RNA transcript set forth in SEQ ID NO: 114;
  • shmiRs encoded by the nucleic acids at (a) and (b) comprise different effector sequences.
  • the effector sequence of VEGFa_shmiR at (b)(ii) which is substantially complementary to a region of equivalent length in the RNA sequence set forth in SEQ ID NO: 114 will be less than 30 nucleotides in length.
  • a suitable effector sequence of the shRNA may be in the range of 17-29 nucleotides in length.
  • the effector sequence will be 20 nucleotides in length. More preferably, the effector sequence will be 21 nucleotides in length and the effector complement sequence will be 20 nucleotides in length.
  • the effector sequence of the VEGFa_shmiR at (b)(ii) may comprise 6 base pair mismatches relative to the sequence set forth in SEQ ID NO: 114 to which the effector sequence is substantially complementary.
  • the effector sequence of VEGFa_shmiR at (b)(ii) may comprise 5 base pair mismatches relative to the sequence set forth in SEQ ID NO: 114 to which the effector sequence is substantially complementary.
  • the effector sequence of VEGFa_shmiR at (b)(ii) may comprise 4 base pair mismatches relative to the sequence set forth in SEQ ID NO: 114 to which the effector sequence is substantially complementary.
  • the effector sequence of VEGFa_shmiR at (b)(ii) may comprise 3 base pair mismatches relative to the sequence set forth in SEQ ID NO: 114 to which the effector sequence is substantially complementary.
  • the effector sequence of VEGFa_shmiR at (b)(ii) may comprise 2 base pair mismatches relative to the sequence set forth in SEQ ID NO: 114 to which the effector sequence is substantially complementary.
  • the effector sequence of VEGFa_shmiR at (b)(ii) may comprise 1 base pair mismatch relative to the sequence set forth in SEQ ID NO: 114 to which the effector sequence is substantially complementary.
  • the effector sequence of VEGFa_shmiR is 100% complementary to a region of equivalent length within the sequence set forth in SEQ ID NO: 114. Where mismatches are present, it is preferred that they are not located within the region
  • VEGFa_shmiR corresponding to the seed region of VEGFa_shmiR i.e., nucleotides 2-8 of the effector sequence.
  • VEGFa_shmiR comprises: (i) an effector sequence which is substantially complementary to the sequence set forth in SEQ ID NO: 114 with the exception of 1, 2, 3, 4, 5 or 6 base mismatches, provided that the effector sequence is capable of forming a duplex with a sequence set forth in SEQ ID NO: 114; and (ii) an effector complement sequence comprising a sequence which is substantially complementary to the effector sequence.
  • VEGFa_shmiR comprises an effector sequence set forth in SEQ ID NO: 115 and an effector complement sequence which is substantially complementary to the sequence set forth in SEQ ID NO: 115 and capable of forming a duplex therewith.
  • VEGFa_shmiR may comprise an effector sequence set forth in SEQ ID NO: 115 and an effector complement sequence set forth in SEQ ID NO: 114.
  • sequence of VEGFa_shmiR may comprise, in a 5' to 3' direction:
  • sequence of VEGFa_shmiR may comprise, in a 5' to 3' direction::
  • VEGFa_shmiR Any suitable loop sequence known in the art may be included in VEGFa_shmiR.
  • an exemplary stemloop sequence is set forth in SEQ ID NO: 67.
  • the pri-miRNA backbone included in VEGFa_shmiR may be selected from those known in the art, such as those already described herein.
  • the pri-miRNA backbone is a pri-miR-30a backbone.
  • the 5' flanking sequence of the pri-miRNA backbone of VEGFa_shmiR is set forth in SEQ ID NO: 68 and the 3 ' flanking sequence of the pri-miRNA backbone of VEGFa_shmiR is set forth in SEQ ID NO: 69.
  • VEGFa_shmiR comprises or consists of the sequence set forth in SEQ ID NO: 116.
  • VEGFa_shmiR comprises a DNA sequence comprising or consisting of the sequence set forth in SEQ ID NO: 117.
  • a plurality of nucleic acids in accordance with the present disclosure may comprise up to 10 nucleic acids, each encoding a shmiR or shRNA as described herein, such as two nucleic acids or three nucleic acids or four nucleic acids or five nucleic acids or six nucleic acids or seven nucleic acids or eight nucleic acids or nine nucleic acids or ten nucleic acids.
  • the plurality of nucleic acids comprises two nucleic acids of the disclosure, each encoding a shmiR or shRNA as described herein.
  • the plurality of nucleic acids comprises three nucleic acids of the disclosure, each encoding a shmiR or shRNA as described herein.
  • the plurality of nucleic acids comprises four nucleic acids of the disclosure, each encoding a shmiR or shRNA as described herein. In one example, the plurality of nucleic acids comprises five nucleic acids of the disclosure, each encoding a shmiR or shRNA as described herein. In one example, the plurality of nucleic acids comprises six nucleic acids of the disclosure, each encoding a shmiR or shRNA as described herein. In one example, the plurality of nucleic acids comprises seven nucleic acids of the disclosure, each encoding a shmiR or shRNA as described herein.
  • the plurality of nucleic acids comprises eight nucleic acids of the disclosure, each encoding a shmiR or shRNA as described herein. In one example, the plurality of nucleic acids comprises nine nucleic acids of the disclosure, each encoding a shmiR or shRNA as described herein. In one example, the plurality of nucleic acids comprises ten nucleic acids of the disclosure, each encoding a shmiR or shRNA as described herein.
  • two or more of the nucleic acids may form separate parts of the same polynucleotide.
  • two or more of the nucleic acids in the plurality form parts of different
  • the or each nucleic acid in accordance with the present disclosure may comprise, or be in operable linkage with, one or more transcriptional terminator sequences.
  • the or each nucleic acid may comprise a transcriptional terminator sequence at the 3 ' terminus of the sequence encoding the shmiR or shRNA.
  • Such sequences will depend on the choice of promoter and will be known to a person of skill in the art.
  • a transcriptional terminator sequence may include ' ⁇ ' or ' XXXXX'.
  • each nucleic acid in accordance with the present disclosure may comprise, or be in operable linkage with, a transcription initiator sequence.
  • a transcription initiator sequence Suitable transcription initiator sequences will be known to a skilled person.
  • the or each nucleic acid in accordance with the present disclosure may comprise one or more restriction sites e.g., to facilitate cloning of the nucleic acid(s) into cloning or expression vectors.
  • the nucleic acids described herein may include a restriction site upstream and/or downstream of the DNA sequence encoding a shmiR or shRNA of the disclosure. Suitable restriction enzyme recognition sequences will be known to a person of skill in the art.
  • the plurality of nucleic acids comprises at least one nucleic acid encoding a shmiR targeting VEGFb and at least one nucleic acid encoding a shmiR targeting PGF.
  • Exemplary nucleic acids encoding shmiRs targeting VEGFb and PGF respectively are described herein and shall be taken to apply mutatis mutandis to this example.
  • the shmiR targeting VEGFb may be selected from VEGFb_shmiR-l, VEGFb_shmiR-4 and VEGFb_shmiR-10 and the shmiR targeting PGF may be selected from PGF_shmiR-3, PGF_shmiR-7 and PGF_shmiR-10.
  • the plurality of nucleic acids comprises a nucleic acid encoding VEGFb_shmiR-l and a nucleic acid encoding PGF_shmiR-3.
  • the plurality of nucleic acids comprises a nucleic acid encoding
  • VEGFb_shmiR-l and a nucleic acid encoding PGF_shmiR-7.
  • the plurality of nucleic acids comprises at least one nucleic acid encoding a shmiR targeting VEGFb and at least one nucleic acid encoding a shmiR targeting VEGFa.
  • Exemplary nucleic acids encoding shmiRs targeting VEGFb and VEGFa respectively are described herein and shall be taken to apply mutatis mutandis to this example.
  • the shmiR targeting VEGFb may be selected from VEGFb_shmiR- 1, VEGFb_shmiR-4 and VEGFb_shmiR-10 and the shmiR targeting VEGFa is
  • the plurality of nucleic acids comprises a nucleic acid encoding VEGFb_shmiR-l and a nucleic acid encoding VEGFa_shmiR.
  • the plurality of nucleic acids comprises at least one nucleic acid encoding a shmiR targeting PGF and at least one nucleic acid encoding a shmiR targeting VEGFa.
  • Exemplary nucleic acids encoding shmiRs targeting PGF and VEGFa respectively are described herein and shall be taken to apply mutatis mutandis to this example.
  • the shmiR targeting PGF may be selected from PGF_shmiR-3, PGF_shmiR-7 and PGF_shmiR-10 and the shmiR targeting VEGFa is VEGFa_shmiR.
  • the plurality of nucleic acids comprises a nucleic acid encoding PGF_shmiR-3 and a nucleic acid encoding VEGFa_shmiR.
  • the plurality of nucleic acids comprises a nucleic acid encoding PGF_shmiR-7 and a nucleic acid encoding VEGFa_shmiR.
  • the plurality of nucleic acids comprises at least one nucleic acid encoding a shmiR targeting VEGFb, at least one nucleic acid encoding a shmiR targeting VEGFa, and at least one nucleic acid encoding a shmiR targeting PGF.
  • Exemplary nucleic acids encoding shmiRs targeting VEGFb, PGF and VEGFa respectively are described herein and shall be taken to apply mutatis mutandis to this example.
  • the shmiR targeting VEGFb may be selected from VEGFb_shmiR-l, VEGFb_shmiR-4 and VEGFb_shmiR-10
  • the shmiR targeting VEGFa is VEGFa_shmiR
  • the shmiR targeting PGF may be selected from PGF_shmiR-3, PGF_shmiR-7 and PGF_shmiR-10.
  • the plurality of nucleic acids comprises a nucleic acid encoding VEGFb_shmiR-l, a nucleic acid encoding VEGFa_shmiR, and a nucleic acid encoding PGF_shmiR-3.
  • the plurality of nucleic acids comprises a nucleic acid encoding VEGFb_shmiR-l, a nucleic acid encoding VEGFa_shmiR, and a nucleic acid encoding PGF_shmiR-7.
  • a nucleic acid in accordance with the present disclosure may also be provided in the form of, or be comprised in, a DNA-directed RNA interference (ddRNAi) construct which is capable of expressing one or more shmiRs or shRNAs which is/are encoded by the nucleic acid(s) of the present disclosure.
  • ddRNAi DNA-directed RNA interference
  • one or more ddRNAi constructs comprising a nucleic acid of the disclosure is also provided.
  • ddRNAi constructs each comprising a nucleic acid encoding a shmiR or shRNA as described herein is provided, wherein:
  • At least one of the plurality of ddRNAi constructs comprises a first nucleic acid of the plurality of nucleic acids as described herein;
  • At least one of the plurality of ddRNAi constructs comprises a second nucleic acid of the plurality of nucleic acids described herein;
  • first and second nucleic acids encode shmiRs or shRNAs comprising effector sequences that are different to one another.
  • the effector sequences of the respective shmiRs or shRNAs target different gene transcripts.
  • the plurality of ddRNAi constructs described herein may comprise up to 10 ddRNAi constructs, each comprising one or more nucleic acids encoding a shmiR or shRNA as described herein, such as two ddRNAi constructs or three ddRNAi constructs or four ddRNAi constructs or five ddRNAi constructs or six ddRNAi constructs or seven ddRNAi constructs or eight ddRNAi constructs or nine ddRNAi constructs or ten ddRNAi constructs of the disclosure.
  • a ddRNAi construct of the disclosure comprises a plurality of nucleic acids as described herein, and thus encodes a plurality of shmiRs or shRNA targeting AMD-associated genes, wherein each of the shmiRs or shRNA are different to one another.
  • the effector sequences of the respective shmiRs or shRNAs target different gene transcripts.
  • the ddRNAi construct comprises at least two nucleic acids of the disclosure, wherein each of the nucleic acids encode different shmiRs.
  • One exemplary ddRNAi construct comprising a plurality of nucleic acids of the disclosure comprises, preferably in a 5' to 3' direction:
  • the ddRNAi construct comprises, preferably in a 5' to 3' direction:
  • the ddRNAi construct of the disclosure comprises, preferably in a 5' to 3' direction:
  • Another exemplary ddRNAi construct comprising a plurality of nucleic acids of the disclosure comprises, preferably in a 5' to 3' direction:
  • the ddRNAi construct comprises, preferably in a 5' to 3' direction:
  • the ddRNAi construct of the disclosure comprises, preferably in a 5' to 3' direction:
  • a ddRNAi construct as described herein comprises a single promoter which is operably-linked to the or each nucleic acid encoding a shmiR or shRNA of the disclosure.
  • each nucleic acid encoding a shmiR or shRNA of the disclosure is operably-linked to a separate promoter.
  • the promoter(s) is(are) positioned upstream of the respective nucleic acid(s) encoding the shmiR(s) or shRNA(s).
  • the promoters may be the same or different.
  • Exemplary promoters are RNA pol
  • U6 and HI promoters such as for example, the U6 and HI promoters.
  • Exemplary U6 promoters are
  • the ddRNAi construct of the disclosure comprises, preferably in a 5' to 3' direction:
  • the ddRNAi construct may comprise, preferably in a 5' to 3' direction:
  • the ddRNAi construct may comprise, preferably in a 5' to 3' direction:
  • the ddRNAi construct of the disclosure comprises, preferably in a
  • the ddRNAi construct may comprise, preferably in a 5' to 3' direction:
  • the ddRNAi construct of the disclosure may comprise, preferably in a 5' to 3' direction:
  • one or more of the promoters may be operably-linked to a proximal sequence element 7 (PSE7) i.e., rather than the proximal sequence element corresponding to the respective promoter.
  • PSE7 proximal sequence element 7
  • the ddRNAi construct of the disclosure comprises, preferably in a 5' to 3' direction:
  • the ddRNAi construct may comprise, preferably in a 5' to 3' direction:
  • the ddRNAi construct may comprise, preferably in a 5' to 3' direction:
  • the ddRNAi construct of the disclosure comprises, preferably in a
  • the ddRNAi construct may comprise, preferably in a 5' to 3' direction:
  • the ddRNAi construct of the disclosure may comprise, preferably in a 5' to 3' direction:
  • the present disclosure also provides an expression vector, comprising a ddRNAi construct of the disclosure.
  • the present disclosure also provides plurality of expression vectors each of which comprises a ddRNAi construct of the disclosure.
  • one or more of the plurality of expression vectors comprises a plurality of ddRNAi constructs as disclosed herein.
  • each of the plurality of expression vectors comprises a plurality of ddRNAi constructs as disclosed herein.
  • each of the plurality of expression vectors comprises a single ddRNAi construct as described herein.
  • the plurality of expression vectors may collectively express a plurality of shmiRs or shRNAs in accordance with the present disclosure.
  • the or each expression vector is a plasmid or a minicircle.
  • the plasmid or minicircle or expression vector or ddRNAi construct is complexed with a cationic DNA binding polymer.
  • the or each expression vector is a viral vector.
  • the viral vector is selected from the group consisting of an adeno-associated viral (AAV) vector, a retroviral vector, an adenoviral vector (AdV) and a lentiviral (LV) vector.
  • AAV adeno-associated viral
  • AdV adenoviral vector
  • LV lentiviral
  • the present disclosure also provides a composition comprising a ddRNAi construct and/or a plurality of ddRNAi constructs and/or expression vector and/or a plurality of expression vectors as described herein.
  • the composition may also comprise one or more pharmaceutically acceptable carriers and/or diluents.
  • the present disclosure also provides a method of treating or preventing an ocular disease or disorder characterised by, or associated with, undesired neovascularization, said method comprising administering to the subject a therapeutically effective amount of a nucleic acid, a plurality of nucleic acids, a ddRNAi construct, a plurality of ddRNAi constructs, an expression vector, a plurality of expression vectors and/or composition described herein to the subject.
  • the ocular disease or disorder to be treated or prevented is selected from the group consisting of AMD e.g., wet AMD or dry AMD, diabetic retinopathy,
  • Diabetic Macular Edema DME
  • corneal neovascularization choroidal neovascularization
  • cyclitis Hippel-Lindau Disease
  • retinopathy of prematurity pterygium
  • histoplasmosis iris neovascularization
  • macular edema macular edema
  • glaucoma-associated neovascularization Purtscher's retinopathy and Retinal Vein Occlusion (RVO).
  • the disclosure provides a method of treating age-related macular degeneration (AMD) in a subject, said method comprising administering to the subject a therapeutically effective amount of a nucleic acid, a plurality of nucleic acids, a ddRNAi construct, a plurality of ddRNAi constructs, an expression vector, a plurality of expression vectors and/or composition described herein to the subject.
  • AMD age-related macular degeneration
  • the AMD is wet AMD.
  • the AMD is dry AMD.
  • treatment may comprise arresting or slowing progression of dry AMD to wet AMD.
  • the disclosure provides a method of treating diabetic retinopathy in a subject, said method comprising administering to the subject a therapeutically effective amount of a nucleic acid, plurality of nucleic acids, a ddRNAi construct, a plurality of ddRNAi constructs, an expression vector, a plurality of expression vectors or a composition as described herein.
  • the disclosure provides a method of treating Diabetic Macular Edema (DME) in a subject, said method comprising administering to the subject a therapeutically effective amount of a nucleic acid, plurality of nucleic acids, a ddRNAi construct, a plurality of ddRNAi constructs, an expression vector, a plurality of expression vectors or a composition as described herein.
  • DME Diabetic Macular Edema
  • the disclosure provides a method of reducing or inhibiting corneal neovascularization in a subject, said method comprising administering to the subject a therapeutically effective amount of a nucleic acid, plurality of nucleic acids, a ddRNAi construct, a plurality of ddRNAi constructs, an expression vector, a plurality of expression vectors or a composition as described herein.
  • the present disclosure also provides a method of reducing or inhibiting choroidal neovascularisation (CNV) in a subject, said method comprising administering to the subject a therapeutically effective amount of a nucleic acid, a plurality of nucleic acids, a ddRNAi construct, a plurality of ddRNAi constructs, an expression vector, a plurality of expression vectors and/or composition described herein to the subject.
  • CNV choroidal neovascularisation
  • the subject who is in need of reduction or inhibition of CNV is suffering from AMD, developing AMD or predisposed to AMD.
  • the AMD may be wet AMD.
  • the disclosure provides a method of treating cyclitis in a subject, said method comprising administering to the subject a therapeutically effective amount of a nucleic acid, plurality of nucleic acids, a ddRNAi construct, a plurality of ddRNAi constructs, an expression vector, a plurality of expression vectors or a composition as described herein.
  • the disclosure provides a method of treating Hippel-Lindau Disease in a subject, said method comprising administering to the subject a therapeutically effective amount of a nucleic acid, plurality of nucleic acids, a ddRNAi construct, a plurality of ddRNAi constructs, an expression vector, a plurality of expression vectors or a composition as described herein.
  • the disclosure provides a method of treating retinopathy of prematurity in a subject, said method comprising administering to the subject a
  • nucleic acid a nucleic acid, plurality of nucleic acids, a ddRNAi construct, a plurality of ddRNAi constructs, an expression vector, a plurality of expression vectors or a composition as described herein.
  • the disclosure provides a method of treating pterygium in a subject, said method comprising administering to the subject a therapeutically effective amount of a nucleic acid, plurality of nucleic acids, a ddRNAi construct, a plurality of ddRNAi constructs, an expression vector, a plurality of expression vectors or a composition as described herein.
  • the disclosure provides a method of treating histoplasmosis in a subject, said method comprising administering to the subject a therapeutically effective amount of a nucleic acid, plurality of nucleic acids, a ddRNAi construct, a plurality of ddRNAi constructs, an expression vector, a plurality of expression vectors or a composition as described herein.
  • the disclosure provides a method of reducing or inhibiting iris neovascularization in a subject, said method comprising administering to the subject a therapeutically effective amount of a nucleic acid, plurality of nucleic acids, a ddRNAi construct, a plurality of ddRNAi constructs, an expression vector, a plurality of expression vectors or a composition as described herein.
  • the disclosure provides a method of treating macular edema in a subject, said method comprising administering to the subject a therapeutically effective amount of a nucleic acid, plurality of nucleic acids, a ddRNAi construct, a plurality of ddRNAi constructs, an expression vector, a plurality of expression vectors or a composition as described herein.
  • the disclosure provides a method of treating glaucoma-associated neovascularization in a subject, said method comprising administering to the subject a therapeutically effective amount of a nucleic acid, plurality of nucleic acids, a ddRNAi construct, a plurality of ddRNAi constructs, an expression vector, a plurality of expression vectors or a composition as described herein.
  • the disclosure provides a method of treating Purtscher's retinopathy in a subject, said method comprising administering to the subject a therapeutically effective amount of a nucleic acid, plurality of nucleic acids, a ddRNAi construct, a plurality of ddRNAi constructs, an expression vector, a plurality of expression vectors or a composition as described herein.
  • the disclosure provides a method of treating or preventing retinal vein occlusion (RVO) in a subject, said method comprising administering to the subject a therapeutically effective amount of a nucleic acid, plurality of nucleic acids, a ddRNAi construct, a plurality of ddRNAi constructs, an expression vector, a plurality of expression vectors or a composition as described herein.
  • RVO retinal vein occlusion
  • the administration to the subject is by intravitreal injection or subretinal injection.
  • administration of the nucleic acid, the plurality of nucleic acids, the ddRNAi construct, the plurality of ddRNAi constructs, the expression vector, the plurality of expression vectors and/or the composition described herein to the subject is performed in conjunction with one or more other treatments known to be suitable for treatment of the ocular disease or condition associated with or
  • the administration may occur in combination with administration of one or more other agents known for treatment of AMD e.g., ranibizumab, aflibercept, bevacizumab, pegaptanib sodium and/or verteporfin.
  • agents known for treatment of AMD e.g., ranibizumab, aflibercept, bevacizumab, pegaptanib sodium and/or verteporfin.
  • a nucleic acid, a plurality of nucleic acids, a ddRNAi construct, a plurality of ddRNAi constructs, an expression vector, a plurality of expression vectors and/or a composition of the present disclosure is provided in the form of a kit.
  • a nucleic acid, a plurality of nucleic acids, a ddRNAi construct, a plurality of ddRNAi constructs, an expression vector, a plurality of expression vectors and/or a composition of the present disclosure may be packaged together with one or more other therapeutic agents known for treating the ocular disease or condition associated with, or characterised by, neovascularisation as described herein. Such other therapeutic agents will be known to a person of skill in the art.
  • a nucleic acid, a plurality of nucleic acids, a ddRNAi construct, a plurality of ddRNAi constructs, an expression vector, a plurality of expression vectors and/or a composition of the present disclosure may be packaged with instruction for use in a method of the disclosure.
  • the present disclosure also provides use of a nucleic acid, a plurality of nucleic acids, a ddRNAi construct, a plurality of ddRNAi constructs, an expression vector, a plurality of expression vectors and/or a composition described herein in the preparation of a medicament for treating or preventing an ocular disease or disorder characterised by, or associated with, undesired neovascularization.
  • the ocular disease or disorder to be treated or prevented is selected from the group consisting of AMD e.g., wet AMD or dry AMD, diabetic retinopathy, Diabetic Macular Edema (DME), corneal neovascularization, choroidal neovascularization, cyclitis, Hippel-Lindau Disease, retinopathy of prematurity, pterygium, histoplasmosis, iris neovascularization, macular edema, glaucoma-associated AMD e.g., wet AMD or dry AMD, diabetic retinopathy, Diabetic Macular Edema (DME), corneal neovascularization, choroidal neovascularization, cyclitis, Hippel-Lindau Disease, retinopathy of prematurity, pterygium, histoplasmosis, iris neovascularization, macular edema, glaucoma-associated
  • AMD e.g.
  • the disclosure provides for use of a nucleic acid, a plurality of nucleic acids, a ddRNAi construct, a plurality of ddRNAi constructs, an expression vector, a plurality of expression vectors and/or a composition described herein in the preparation of a medicament for treating or preventing AMD.
  • the AMD is wet AMD.
  • the AMD is dry AMD.
  • treatment or prevention may comprise arresting or slowing progression of dry AMD to wet AMD.
  • the disclosure provides for use of a nucleic acid, a plurality of nucleic acids, a ddRNAi construct, a plurality of ddRNAi constructs, an expression vector, a plurality of expression vectors and/or a composition described herein in the preparation of a medicament for treating or preventing diabetic retinopathy in a subject in need thereof.
  • the disclosure provides for use of a nucleic acid, a plurality of nucleic acids, a ddRNAi construct, a plurality of ddRNAi constructs, an expression vector, a plurality of expression vectors and/or a composition described herein in the preparation of a medicament for treating or preventing Diabetic Macular Edema (DME) in a subject in need thereof.
  • a nucleic acid a plurality of nucleic acids
  • a ddRNAi construct a plurality of ddRNAi constructs
  • an expression vector a plurality of expression vectors and/or a composition described herein in the preparation of a medicament for treating or preventing Diabetic Macular Edema (DME) in a subject in need thereof.
  • DME Diabetic Macular Edema
  • the disclosure also provides use of a nucleic acid, a plurality of nucleic acids, a ddRNAi construct, a plurality of ddRNAi constructs, an expression vector, a plurality of expression vectors and/or a composition described herein in the preparation of a medicament for reducing or inhibiting choroidal neovascularisation (CNV) in a subject in need thereof.
  • CNV choroidal neovascularisation
  • a subject who is in need of reduction or inhibition of CNV may be suffering from AMD, developing AMD or predisposed to AMD.
  • the AMD may be wet AMD.
  • the disclosure provides for use of a nucleic acid, a plurality of nucleic acids, a ddRNAi construct, a plurality of ddRNAi constructs, an expression vector, a plurality of expression vectors and/or a composition described herein in the preparation of a medicament for treating or preventing cyclitis in a subject in need thereof.
  • the disclosure provides for use of a nucleic acid, a plurality of nucleic acids, a ddRNAi construct, a plurality of ddRNAi constructs, an expression vector, a plurality of expression vectors and/or a composition described herein in the preparation of a medicament for treating or preventing Hippel-Lindau Disease in a subject in need thereof.
  • the disclosure provides for use of a nucleic acid, a plurality of nucleic acids, a ddRNAi construct, a plurality of ddRNAi constructs, an expression vector, a plurality of expression vectors and/or a composition described herein in the preparation of a medicament for treating or preventing retinopathy of prematurity in a subject in need thereof.
  • the disclosure provides for use of a nucleic acid, a plurality of nucleic acids, a ddRNAi construct, a plurality of ddRNAi constructs, an expression vector, a plurality of expression vectors and/or a composition described herein in the preparation of a medicament for treating or preventing pterygium in a subject in need thereof.
  • the disclosure provides for use of a nucleic acid, a plurality of nucleic acids, a ddRNAi construct, a plurality of ddRNAi constructs, an expression vector, a plurality of expression vectors and/or a composition described herein in the preparation of a medicament for treating or preventing histoplasmosis in a subject in need thereof.
  • the disclosure provides for use of a nucleic acid, a plurality of nucleic acids, a ddRNAi construct, a plurality of ddRNAi constructs, an expression vector, a plurality of expression vectors and/or a composition described herein in the preparation of a medicament for treating or preventing iris neovascularization in a subject in need thereof.
  • the disclosure provides for use of a nucleic acid, a plurality of nucleic acids, a ddRNAi construct, a plurality of ddRNAi constructs, an expression vector, a plurality of expression vectors and/or a composition described herein in the preparation of a medicament for treating or preventing macular edema in a subject in need thereof.
  • the disclosure provides for use of a nucleic acid, a plurality of nucleic acids, a ddRNAi construct, a plurality of ddRNAi constructs, an expression vector, a plurality of expression vectors and/or a composition described herein in the preparation of a medicament for treating or preventing glaucoma-associated neovascularization in a subject in need thereof.
  • the disclosure provides for use of a nucleic acid, a plurality of nucleic acids, a ddRNAi construct, a plurality of ddRNAi constructs, an expression vector, a plurality of expression vectors and/or a composition described herein in the preparation of a medicament for treating or preventing Purtscher's retinopathy in a subject in need thereof.
  • the disclosure provides for use of a nucleic acid, a plurality of nucleic acids, a ddRNAi construct, a plurality of ddRNAi constructs, an expression vector, a plurality of expression vectors and/or a composition described herein in the preparation of a medicament for treating or preventing retinal vein occlusion (RVO) in a subject in need thereof.
  • a nucleic acid a plurality of nucleic acids
  • a ddRNAi construct a plurality of ddRNAi constructs
  • an expression vector a plurality of expression vectors and/or a composition described herein in the preparation of a medicament for treating or preventing retinal vein occlusion (RVO) in a subject in need thereof.
  • RVO retinal vein occlusion
  • the present disclosure also provides a nucleic acid, a plurality of nucleic acids, a ddRNAi construct, a plurality of ddRNAi constructs, an expression vector, a plurality of expression vectors and/or composition described herein for use in therapy of an ocular disease or condition associated with, or characterised by, neovascularisation.
  • the nucleic acid, the plurality of nucleic acids, the ddRNAi construct, a plurality of ddRNAi constructs, the expression vector, the plurality of expression vectors and/or the composition may be for use in treating or preventing an ocular disease or disorder selected from the group consisting of AMD e.g., wet AMD or dry AMD, diabetic retinopathy, Diabetic Macular Edema (DME), corneal neovascularization, choroidal neovascularization, cyclitis, Hippel-Lindau Disease, retinopathy of prematurity, pterygium, histoplasmosis, iris neovascularization, macular edema, glaucoma-associated neovascularization, Purtscher's retinopathy and Retinal Vein Occlusion (RVO).
  • AMD e.g., wet AMD or dry AMD
  • DME Diabetic Macular Edema
  • corneal neovascularization
  • the nucleic acid, the plurality of nucleic acids, the ddRNAi construct, a plurality of ddRNAi constructs, the expression vector, the plurality of expression vectors and/or the composition may be for use in treating or preventing AMD in a subject e.g., wet AMD.
  • the nucleic acid, the plurality of nucleic acids, the ddRNAi construct, a plurality of ddRNAi constructs, the expression vector, the plurality of expression vectors and/or the composition may be for use in reducing or inhibiting CNV in a subject, such as in a subject suffering from AMD, who is developing AMD or who is predisposed to AMD. For example, wet AMD.
  • Figure 1 shows (A) a map of the of pSilencer 2.1-U6 hygro vector, (B) an insert of VEGFb shmiR-1 in such construct, and (C) a predicted RNA folding structure of VEGFb shmiR-1.
  • Figure 2 illustrates: (A) strand preference activities of VEGFb shmiRs in a lucif erase reporter assay; and (B) strand preference activities of PGF shmiRs in a luciferase reporter assay. shmiRs showing higher anti-sense strand activities over sense strand activities were identified as preferred drug candidates.
  • Figure 3 illustrates: (A) hyperfunctional activities of selective VEGFb shmiRs; and (B) hyperfunctional activities of selective PGF shmiRs.
  • VEGFb and PGF shmiRs inhibited luciferase protein expression in a dose dependent manner.
  • Figure 4 shows down-regulation of endogenous VEGFb and PGF target expression by selected single shmiR constructs: (A) VEGFb shmiR knockdown activities in ARPE-19 cells; and (B) PGF shmiR knockdown activities in BeWo cells. Precent inhibition was calculated as relative activities to pSilencer negative control.
  • Figures 5 provides an schematic of four ddRNAi constructs expressing three different shmiRs under control of three separate U6 promoters for simultaneously down regulation of VEGFa, VEGFb and PGF expression.
  • FIG. 6 illustrates that when expressed by a triple construct, VEGFa shmiR-8 was capable of inhibiting VEGFa target expression in a dose dependent manner in ARPE-19 cells: (A) VEGFa mRNA inhibition levels determined using RT-qPCR analysis; (B) VEGFa protein inhibition levels analyzed using ELISA assay; and (C) VEGFa shmiR-8 expression levels analyzed using RT-qPCR assay. Precent inhibition was calculated as relative activities to TT034 negative control.
  • Figure 7 illustrates that when expressed by a triple construct, VEGFb shmiR- 1 efficiently inhibited VEGFb target expression in ARPE-19 cells; (A) VEGFb mRNA inhibition levels determined using RT-qPCR analysis and (b) VEGFb shmiR- 1 expression levels analyzed using RT-qPCR assay. Precent inhibition was calculated as relative activities to TT034 negative control.
  • Figure 8 illustrates that when expressed by a triple construct, PGF shmiR-3 and shmiR-7 could both independently down-regulate expression of endogenous PGF in JEG-3 cells: (A) PGF mRNA inhibition levels determined using RT-qPCR analysis; and (B, C) PGF shmiR-3 and PGF shmiR-7 expression levels analyzed using RT-qPCR assay. Precent inhibition was calculated as relative activities to TT034 negative control.
  • FIG. 9 illustrates that there is high level expression of all three shmiRs (VEGFa- shmiR-8, VEGFb-shmiR-1 and PGF-shmiR-7) in retina layer of the eye following delivery of the AAV-based ddRNAi construct, CapVarl-BB201, to retina tissues. This figure also shows that expression of the shmiRs was detectable in RPE/Choroid layer. Key to the Sequence Listing
  • SEQ ID NO: 1 Target region 1 w thin RNA transcript of VEGFb (VEGFb- 1).
  • SEQ ID NO: 2 Target region 2 w thin RNA transcript of VEGFb (VEGFb-2).
  • SEQ ID NO: 3 Target region 3 w thin RNA transcript of VEGFb (VEGFb-3).
  • SEQ ID NO: 4 Target region 4 w thin RNA transcript of VEGFb (VEGFb-4).
  • SEQ ID NO: 6 Target region 6 w thin RNA transcript of VEGFb (VEGFb-6).
  • SEQ ID NO: 7 Target region 7 w thin RNA transcript of VEGFb (VEGFb-7).
  • SEQ ID NO: 8 Target region 8 w thin RNA transcript of VEGFb (VEGFb-8).
  • SEQ ID NO: 9 Target region 9 w thin RNA transcript of VEGFb (VEGFb-9).
  • SEQ ID NO: 10 Target region 10 within RNA transcript of VEGFb (VEGFb-10).
  • SEQ ID NO: 11 Target region 1 w thin RNA transcript of PGF (PGF-1).
  • SEQ ID NO: 12 Target region 2 w thin RNA transcript of PGF (PGF-2).
  • SEQ ID NO: 15 Target region 5 w thin RNA transcript of PGF (PGF-5).
  • SEQ ID NO: 16 Target region 6 w thin RNA transcript of PGF (PGF-6).
  • SEQ ID NO: 18 Target region 8 w thin RNA transcript of PGF (PGF-8).
  • SEQ ID NO: 20 Target region 10 within RNA transcript of PGF (PGF- 10).
  • SEQ ID NO: 21 Target region 11 within RNA transcript of PGF (PGF-11).
  • SEQ ID NO: 22 Target region 12 within RNA transcript of PGF (PGF- 12).
  • VEGFb_shmiR-l VEGFb_shmiR-l.
  • SEQ ID NO: 24 RNA effector sequence for shmiR designated VEGFb_shmiR- 1.
  • SEQ ID NO: 25 RNA effector complement sequence for shmiR designated
  • VEGFb_shmiR-2 VEGFb_shmiR-2.
  • SEQ ID NO: 26 RNA effector sequence for shmiR designated VEGFb_shmiR-2.
  • SEQ ID NO: 27 RNA effector complement sequence for shmiR designated
  • SEQ ID NO: 28 RNA effector sequence for shmiR designated VEGFb_shmiR-3.
  • SEQ ID NO: 29 RNA effector complement sequence for shmiR designated VEGFb_shmiR-4.
  • SEQ ID NO: 30 RNA effector sequence for shmiR designated VEGFb_shmiR-4.
  • SEQ ID NO: 31 RNA effector complement sequence for shmiR designated
  • SEQ ID NO: 32 RNA effector sequence for shmiR designated VEGFb_shmiR-5.
  • SEQ ID NO: 33 RNA effector complement sequence for shmiR designated
  • VEGFb_shmiR-6 VEGFb_shmiR-6.
  • SEQ ID NO: 34 RNA effector sequence for shmiR designated VEGFb_shmiR-6.
  • SEQ ID NO: 35 RNA effector complement sequence for shmiR designated
  • VEGFb_shmiR-7 VEGFb_shmiR-7.
  • SEQ ID NO: 36 RNA effector sequence for shmiR designated VEGFb_shmiR-7.
  • SEQ ID NO: 37 RNA effector complement sequence for shmiR designated
  • VEGFb_shmiR-8 VEGFb_shmiR-8.
  • SEQ ID NO: 38 RNA effector sequence for shmiR designated VEGFb_shmiR-8.
  • SEQ ID NO: 39 RNA effector complement sequence for shmiR designated
  • VEGFb_shmiR-9 VEGFb_shmiR-9.
  • SEQ ID NO: 40 RNA effector sequence for shmiR designated VEGFb_shmiR-9.
  • SEQ ID NO: 41 RNA effector complement sequence for shmiR designated
  • VEGFb_shmiR-10 VEGFb_shmiR-10.
  • SEQ ID NO: 42 RNA effector sequence for shmiR designated VEGFb_shmiR- 10.
  • SEQ ID NO: 43 RNA effector complement sequence for shmiR designated PGF_shmiR- 1.
  • SEQ ID NO: 44 RNA effector sequence for shmiR designated PGF_shmiR-l.
  • SEQ ID NO: 45 RNA effector complement sequence for shmiR designated PGF_shmiR-
  • SEQ ID NO: 46 RNA effector sequence for shmiR designated PGF_shmiR-2.
  • SEQ ID NO: 47 RNA effector complement sequence for shmiR designated PGF_shmiR- 3.
  • SEQ ID NO: 48 RNA effector sequence for shmiR designated PGF_shmiR-3.
  • SEQ ID NO: 49 RNA effector complement sequence for shmiR designated PGF_shmiR- 4.
  • SEQ ID NO: 50 RNA effector sequence for shmiR designated PGF_shmiR-4.
  • SEQ ID NO: 51 RNA effector complement sequence for shmiR designated PGF_shmiR-
  • SEQ ID NO: 52 RNA effector sequence for shmiR designated PGF_shmiR-5.
  • SEQ ID NO: 53 RNA effector complement sequence for shmiR designated PGF_shmiR-
  • SEQ ID NO: 54 RNA effector sequence for shmiR designated PGF_shmiR-6.
  • SEQ ID NO: 55 RNA effector complement sequence for shmiR designated PGF_shmiR-
  • SEQ ID NO: 56 RNA effector sequence for shmiR designated PGF_shmiR-7.
  • SEQ ID NO: 57 RNA effector complement sequence for shmiR designated PGF_shmiR-
  • SEQ ID NO: 58 RNA effector sequence for shmiR designated PGF_shmiR-8.
  • SEQ ID NO: 59 RNA effector complement sequence for shmiR designated PGF_shmiR-
  • SEQ ID NO: 60 RNA effector sequence for shmiR designated PGF_shmiR-9.
  • SEQ ID NO: 61 RNA effector complement sequence for shmiR designated PGF_shmiR-
  • SEQ ID NO: 62 RNA effector sequence for shmiR designated PGF_shmiR-10.
  • SEQ ID NO: 63 RNA effector complement sequence for shmiR designated PGF_shmiR-
  • SEQ ID NO: 64 RNA effector sequence for shmiR designated PGF_shmiR-l 1.
  • SEQ ID NO: 65 RNA effector complement sequence for shmiR designated PGF_shmiR-
  • SEQ ID NO: 66 RNA effector sequence for shmiR designated PGF_shmiR-12.
  • SEQ ID NO: 67 stemloop RNA sequence for shmiRs
  • SEQ ID NO: 68 5' flanking sequence of pri-miR-30a backbone.
  • SEQ ID NO: 69 3' flanking sequence of pri-miR-30a backbone.
  • SEQ ID NO: 70 RNA sequence for shmiR designated VEGFb_shmiR-l.
  • SEQ ID NO: 71 RNA sequence for shmiR designated VEGFb_ shmiR-2.
  • SEQ ID NO: 72 RNA sequence for shmiR designated VEGFb_shmiR-3.
  • SEQ ID NO: 73 RNA sequence for shmiR designated VEGFb_shmiR-4.
  • SEQ ID NO: 74 RNA sequence for shmiR designated VEGFb_shmiR-5.
  • SEQ ID NO: 75 RNA sequence for shmiR designated VEGFb_shmiR-6.
  • SEQ ID NO: 76 RNA sequence for shmiR designated VEGFb_shmiR-7.
  • SEQ ID NO: 77 RNA sequence for shmiR designated VEGFb_shmiR-8.
  • SEQ ID NO: 78 RNA sequence for shmiR designated VEGFb_shmiR-9.
  • SEQ ID NO: 79 RNA sequence for shmiR designated VEGFb_shmiR-10.
  • SEQ ID NO: 80 RNA sequence for shmiR designated PGF_shmiR-l.
  • SEQ ID NO: 81 RNA sequence for shmiR designated PGF_shmiR-2.
  • SEQ ID NO: 82 RNA sequence for shmiR designated PGF_shmiR-3.
  • SEQ ID NO: 83 RNA sequence for shmiR designated PGF_shmiR-4.
  • SEQ ID NO: 84 RNA sequence for shmiR designated PGF_shmiR-5.
  • SEQ ID NO: 85 RNA sequence for shmiR designated PGF_shmiR-6.
  • SEQ ID NO: 86 RNA sequence for shmiR designated PGF_shmiR-7.
  • SEQ ID NO: 87 RNA sequence for shmiR designated PGF_shmiR-8.
  • SEQ ID NO: 88 RNA sequence for shmiR designated PGF_shmiR-9.
  • SEQ ID NO: 89 RNA sequence for shmiR designated PGF_shmiR-10.
  • SEQ ID NO: 90 RNA sequence for shmiR designated PGF_shmiR-l 1.
  • SEQ ID NO: 91 RNA sequence for shmiR designated PGF_shmiR-12.
  • SEQ ID NO: 92 DNA sequence coding for shmiR designated VEGFb_shmiR- 1.
  • SEQ ID NO: 93 DNA sequence coding for shmiR designated VEGFb_shmiR- 2.
  • SEQ ID NO: 94 DNA sequence coding for shmiR designated VEGFb_shmiR- 3.
  • SEQ ID NO: 95 DNA sequence coding for shmiR designated VEGFb_shmiR- 4.
  • SEQ ID NO: 96 DNA sequence coding for shmiR designated VEGFb_shmiR- 5.
  • SEQ ID NO: 97 DNA sequence coding for shmiR designated VEGFb_shmiR- 6.
  • SEQ ID NO: 98 DNA sequence coding for shmiR designated VEGFb_shmiR- 7.
  • SEQ ID NO: 99 DNA sequence coding for shmiR designated VEGFb_shmiR- 8.
  • SEQ ID NO: 100 DNA sequence coding for shmiR designated VEGFb_shmiR- 9.
  • SEQ ID NO: 101 DNA sequence coding for shmiR designated VEGFb_shmiR- 10.
  • SEQ ID NO: 102 DNA sequence coding for shmiR designated PGF_shmiR-l.
  • SEQ ID NO: 103 DNA sequence coding for shmiR designated PGF_shmiR-2.
  • SEQ ID NO: 104 DNA sequence coding for shmiR designated PGF_shmiR-3.
  • SEQ ID NO: 105 DNA sequence coding for shmiR designated PGF_shmiR-4.
  • SEQ ID NO: 106 DNA sequence coding for shmiR designated PGF_shmiR-5.
  • SEQ ID NO: 107 DNA sequence coding for shmiR designated PGF_shmiR-6.
  • SEQ ID NO: 108 DNA sequence coding for shmiR designated PGF_shmiR-7.
  • SEQ ID NO: 109 DNA sequence coding for shmiR designated PGF_shmiR-8.
  • SEQ ID NO: 110 DNA sequence coding for shmiR designated PGF_shmiR-9.
  • SEQ ID NO: 111 DNA sequence coding for shmiR designated PGF_shmiR-10.
  • SEQ ID NO: 112 DNA sequence coding for shmiR designated PGF_shmiR-l l.
  • SEQ ID NO: 113 DNA sequence coding for shmiR designated PGF_shmiR-12.
  • SEQ ID NO: 114 RNA effector complement sequence for shmiR designated
  • SEQ ID NO: 115 RNA effector sequence for shmiR designated VEGFa_shmiR.
  • SEQ ID NO: 116 RNA sequence for shmiR designated VEGFa_shmiR.
  • SEQ ID NO: 117 DNA sequence coding for shmiR designated VEGFa_shmiR.
  • SEQ ID NO: 118 DNA sequence for primer designated VEGFb-shmiRl_fwd primer seq.
  • SEQ ID NO: 119 DNA sequence for primer designated VEGFa-shmiR8_fwd primer seq.
  • SEQ ID NO: 120 DNA sequence for primer designated PGF-shmiR7_fwd primer seq.
  • SEQ ID NO: 121 DNA sequence for primer designated PGF-shmiR3_fwd primer seq.
  • SEQ ID NO: 122 DNA sequence for primer designated VEGFb-shmiRl_RNA standard oligo seq.
  • SEQ ID NO: 123 DNA sequence for primer designated VEGFa-shmIR8_RNA standard oligo seq.
  • SEQ ID NO: 124 DNA sequence for primer designated PGF-shmiR7_RNA standard olig seq.
  • SEQ ID NO: 125 DNA sequence for primer designated PGF-shmiR3_RNA standard olig seq.
  • the recombinant DNA, recombinant protein, cell culture, and immunological techniques utilized in the present disclosure are standard procedures, well known to those skilled in the art. Such techniques are described and explained throughout the literature in sources such as, J. Perbal, A Practical Guide to Molecular Cloning, John Wiley and Sons (1984), J. Sambrook et al. Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press (1989), T.A. Brown (editor), Essential Molecular Biology: A Practical Approach, Volumes 1 and 2, IRL Press (1991), D.M. Glover and B.D. Hames (editors), DNA Cloning: A Practical Approach, Volumes 1-4, IRL Press (1995 and 1996), and F.M.
  • RNA is meant a molecule comprising at least one ribonucleotide residue.
  • ribonucleotide is meant a nucleotide with a hydroxyl group at the 2' position of a ⁇ -D-ribo- furanose moiety.
  • the terms include double-stranded RNA, single-stranded RNA, isolated RNA such as partially purified RNA, essentially pure RNA, synthetic RNA, recombinantly- produced RNA, as well as altered RNA that differs from naturally occurring RNA by the addition, deletion, substitution and/or alteration of one or more nucleotides.
  • Such alterations can include addition of non-nucleotide material, such as to the end(s) of the RNA or internally, for example at one or more nucleotides of the RNA.
  • Nucleotides in the RNA molecules of the instant disclosure can also comprise non-standard nucleotides, such as non- naturally occurring nucleotides or chemically synthesized nucleotides or deoxynucleotides. These altered RNAs can be referred to as analogs or analogs of naturally-occurring RNA.
  • RNA interference refers generally to RNA-dependent silencing of gene expression initiated by double stranded RNA (dsRNA) molecules in a cell's cytoplasm.
  • dsRNA double stranded RNA
  • the dsRNA molecule reduces or inhibits transcription products of a target nucleic acid sequence, thereby silencing the gene or reducing expression of that gene.
  • double stranded RNA or “dsRNA” refers to a RNA molecule having a duplex structure and comprising an effector sequence and an effector complement sequence which are of similar length to one another.
  • the effector sequence and the effector complement sequence can be in a single RNA strand or in separate RNA strands.
  • the "effector sequence” (often referred to as a “guide strand”) is substantially complementary to a region of a target sequence, which in the present case, is a transcript of VEGFb, PGF or VEGFa.
  • the "effector sequence” can also be referred to as the "antisense sequence”.
  • effector complement sequence will be of sufficient complementary to the effector sequence such that it can anneal to the effector sequence to form a duplex.
  • the effector complement sequence will be substantially homologous to a region of target sequence.
  • effector complement sequence can also be referred to as the "complement of the effector sequence” or the sense sequence.
  • duplex refers to regions in two complementary or substantially complementary nucleic acids (e.g., RNAs), or in two complementary or substantially complementary regions of a single- stranded nucleic acid (e.g., RNA), that form base pairs with one another, either by Watson-Crick base pairing or any other manner that allows for a stabilized duplex between the nucleotide sequences that are complementary or substantially complementary. It will be understood by the skilled person that within a duplex region, 100% complementarity is not required; substantial complementarity is allowable. Substantial complementarity includes may include 69% or greater
  • a single mismatch in a duplex region consisting of 19 base pairs results in 94.7% complementarity, rendering the duplex region substantially complementary.
  • two mismatches in a duplex region consisting of 19 base pairs results in 89,5% complementarity, rendering the duplex region substantially complementary.
  • three mismatches in a duplex region consisting of 19 base pairs results in 84.2% complementarity, rendering the duplex region substantially complementary, and so on.
  • the dsRNA may be provided as a hairpin or stem loop structure, with a duplex region comprised of an effector sequence and effector complement sequence linked by at least 2 nucleotide sequence which is termed a stem loop.
  • a dsRNA is provided as a hairpin or stem loop structure it can be referred to as a "hairpin RNA” or “short hairpin RNAi agent” or “shRNA”.
  • Pre-miRNA shRNAs can be naturally produced from pri-miRNA by the action of the enzymes Drosha and Pasha which recognize and release regions of the primary miRNA transcript which form a stem-loop structure.
  • the pri-miRNA transcript can be engineered to replace the natural stem-loop structure with an artificial/recombinant stem- loop structure. That is, an artificial/recombinant stem-loop structure may be inserted or cloned into a pri-miRNA backbone sequence which lacks its natural stem-loop structure.
  • dsRNA molecules produced using this approach are known as "shmiRNAs", “shmiRs” or “microRNA framework shRNAs”.
  • the term "complementary" with regard to a sequence refers to a complement of the sequence by Watson-Crick base pairing, whereby guanine (G) pairs with cytosine (C), and adenine (A) pairs with either uracil (U) or thymine (T).
  • a sequence may be complementary to the entire length of another sequence, or it may be complementary to a specified portion or length of another sequence.
  • U may be present in RNA
  • T may be present in DNA. Therefore, an A within either of a RNA or DNA sequence may pair with a U in a RNA sequence or T in a DNA sequence.
  • the term "substantially complementary” is used to indicate a sufficient degree of complementarity or precise pairing such that stable and specific binding occurs between nucleic acid sequences e.g., between the effector sequence and the effector complement sequence or between the effector sequence and the target sequence. It is understood that the sequence of a nucleic acid need not be 100% complementary to that of its target or complement.
  • the term encompasses a sequence complementary to another sequence with the exception of an overhang. In some cases, the sequence is complementary to the other sequence with the exception of 1-2 mismatches. In some cases, the sequences are complementary except for 1 mismatch. In some cases, the sequences are complementary except for 2 mismatches. In other cases, the sequences are complementary except for 3 mismatches. In yet other cases, the sequences are complementary except for 4 mismatches.
  • a nucleic acid that encodes a shRNA or shmiR of the disclosure will comprise a DNA sequence which serves as a template for transcription of the respective shRNA or shmiR.
  • DNA-directed RNAi construct refers to a nucleic acid comprising DNA sequence which, when transcribed produces a shRNA or shmiR molecule which elicits RNAi.
  • the ddRNAi construct may comprise a nucleic acid which is transcribed as a single RNA that is capable of self-annealing into a hairpin structure with a duplex region linked by a stem loop of at least 2 nucleotides i.e., shRNA or shmiR, or as a single RNA with multiple shRNAs or shmiRs, or as multiple RNA transcripts each capable of folding as a single shRNA or shmiR respectively.
  • the ddRNAi construct may be within an expression vector i.e., "ddRNAi expression construct", e.g., operably-linked to a promoter.
  • operably-linked or “operable linkage” (or similar) means that a. coding nucleic acid sequence is linked to, or in association with, a regulatory sequence, e.g., a promoter, in a manner which facilitates expression of the coding sequence.
  • a regulatory sequence e.g., a promoter
  • Regulatory sequences include promoters, enhancers, and other expression control elements that are art-recognized and are selected to direct expression of the coding sequence.
  • a “vector” will be understood to mean a vehicle for introducing a nucleic acid into a cell.
  • Vectors include, but are not limited to, p!asmids, phagemids, viruses, bacteria, and vehicles derived from viral or bacterial sources.
  • a "plasmid” is a circular, double-stranded DNA molecule.
  • a useful type of vector for use in accordance with the present disclosure is a viral vector, wherein heterologous DN A sequences are inserted into a viral genome that can be modified to delete one or more viral genes or parts thereof.
  • Certain vectors are capable of autonomous replication in a host cell (e.g., vectors having an origin of replication that functions in the host cell). Other vectors can be stably integrated into the genome of a host cell, and are thereby replicated along with the host genome.
  • the term "expression vector” will be understood to mean a vector capable of expressing a RNA molecule of the disclosure.
  • treating refers to clinical intervention designed to alter the natural course of the individual or cell being treated during the course of clinical pathology. Desirable effects of treatment include decreasing the rate of disease progression, ameliorating or palliating the disease state, and remission or improved prognosis.
  • a “therapeutically effective amount” is at least the minimum concentration or amount required to effect a measurable improvement of a particular disease or disorder (e.g., an ocular disease or disorder characterised by, or associated with, neovascularisation, such as AMD).
  • a therapeutically effective amount herein may vary according to factors such as the disease state, age, sex, and weight of the patient, and the ability of the shmiR, nucleic acid encoding same, ddRNAi or expression construct to elicit a desired response in the individual.
  • a therapeutically effective amount is also one in which any toxic or detrimental effects of the shRNA or shmiR, nucleic acid encoding same, ddRNAi or expression construct are outweighed by the therapeutically beneficial effects.
  • the "subject” or “patient” can be a human or non-human animal suffering from or predisposed to an ocular disease or disorder characterised by, or associated with, neovascularisation, such as AMD.
  • the "non-human animal” may be a primate, livestock (e.g. sheep, horses, cattle, pigs, donkeys), companion animal (e.g. pets such as dogs and cats), laboratory test animal (e.g. mice, rabbits, rats, guinea pigs), performance animal (e.g. racehorses, camels, greyhounds) or captive wild animal.
  • livestock e.g. sheep, horses, cattle, pigs, donkeys
  • companion animal e.g. pets such as dogs and cats
  • laboratory test animal e.g. mice, rabbits, rats, guinea pigs
  • performance animal e.g. racehorses, camels, greyhounds
  • the subject or patient is a mammal.
  • the subject or patient is a prim
  • reduced expression refers to the absence or an observable decrease in the level of protein and/or mRNA product from the target gene(s) e.g., VEGFb, PGF and/or VEGFa.
  • the decrease does not have to be absolute, but may be a partial decrease sufficient for there to a detectable or observable change as a result of the RNAi effected by the shmiR encoded by the nucleic acid of the disclosure.
  • the decrease can be measured by determining a decrease in the level of mRNA and/or protein product from a target nucleic acid relative to a cell lacking the shmiR or shRNA, nucleic acid encoding same, ddRNAi construct or expression construct, and may be as little as 1 %, 5% or 10%, or may be absolute i.e., 100% inhibition.
  • the effects of the decrease may be determined by examination of the outward properties i.e., quantitative and/or qualitative phenotype of the cell or organism, and may also include an assessment of vision or impairment thereof, rate of growth of abnormal blood vessels under the macula and/or fluid leakage from those blood vessels, following administration of a ddRNAi construct of the disclosure or a composition comprising same.
  • the present disclosure provides a nucleic acid comprising a DNA sequence which encodes a short hairpin micro-RNA (shmiR), said shmiR comprising: an effector sequence of at least 17 nucleotides in length;
  • the effector sequence is substantially complementary to a region of equivalent length in an RNA transcript of VEGFb set forth set forth in any one of SEQ ID NOs: 1-10 or a region of equivalent length in an RNA transcript of PGF set forth set forth in any one of SEQ ID NOs: 11-22.
  • the effector sequence will be less than 30 nucleotides in length.
  • a suitable effector sequence may be in the range of 17-29 nucleotides in length.
  • the effector sequence will be 21 nucleotides in length. More preferably, the effector sequence will be 21 nucleotides in length and the effector complement sequence will be 20 nucleotides in length.
  • the shmiR comprises an effector sequence which is substantially complementary to a region of equivalent length within an RNA transcript of VEGFb comprising or consisting of the sequence set forth in SEQ ID NO: 1
  • a shmiR in accordance with this example is hereinafter designated "VEGFb_shmiR-l".
  • the effector sequence may be substantially complementary to a region of equivalent length within an RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 1 and contain 6 mismatch bases relative thereto.
  • the effector sequence may be substantially complementary to a region of equivalent length within an RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 1 and contain 5 mismatch bases relative thereto.
  • the effector sequence may be substantially
  • the effector sequence may be substantially complementary to a region of equivalent length within an RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 1 and contain 4 mismatch bases relative thereto.
  • the effector sequence may be substantially complementary to a region of equivalent length within an RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 1 and contain 3 mismatch bases relative thereto.
  • the effector sequence may be substantially complementary to a region of equivalent length within an RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 1 and contain 2 mismatch bases relative thereto.
  • the effector sequence may be substantially complementary to a region of equivalent length within an RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 1 and contain 1 mismatch base relative thereto.
  • the effector sequence may be 100% complementary to a region of equivalent length within an RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 1.
  • the shmiR comprises an effector sequence which is substantially complementary to a region of equivalent length within an RNA transcript of VEGFb comprising or consisting of the sequence set forth in SEQ ID NO: 2.
  • a shmiR in accordance with this example is hereinafter designated "VEGFb_shmiR-2".
  • the effector sequence may be substantially complementary to a region of equivalent length within an RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 2 and contain 6 mismatch bases relative thereto.
  • the effector sequence may be substantially complementary to a region of equivalent length within an RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 2 and contain 5 mismatch bases relative thereto.
  • the effector sequence may be substantially
  • the effector sequence may be substantially identical to a region of equivalent length within an RNA transcript of VEGFb comprising or consisting of the sequence set forth in SEQ ID NO: 2 and contain 4 mismatch bases relative thereto.
  • the effector sequence may be substantially identical to a region of equivalent length within an RNA transcript of VEGFb comprising or consisting of the sequence set forth in SEQ ID NO: 2 and contain 4 mismatch bases relative thereto.
  • the effector sequence may be substantially
  • the effector sequence may be substantially complementary to a region of equivalent length within an RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 2 and contain 3 mismatch bases relative thereto.
  • the effector sequence may be substantially complementary to a region of equivalent length within an RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 2 and contain 2 mismatch bases relative thereto.
  • the effector sequence may be substantially complementary to a region of equivalent length within an RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 2 and contain 1 mismatch base relative thereto.
  • the effector sequence may be 100% complementary to a region of equivalent length within an RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 2.
  • the shmiR comprises an effector sequence which is substantially complementary to a region of equivalent length within an RNA transcript of VEGFb comprising or consisting of the sequence set forth in SEQ ID NO: 3.
  • a shmiR in accordance with this example is hereinafter designated "VEGFb_shmiR-3".
  • the effector sequence may be substantially complementary to a region of equivalent length within an RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 3 and contain 6 mismatch bases relative thereto.
  • the effector sequence may be substantially complementary to a region of equivalent length within an RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 3 and contain 5 mismatch bases relative thereto.
  • the effector sequence may be substantially
  • the effector sequence may be substantially complementary to a region of equivalent length within an RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 3 and contain 4 mismatch bases relative thereto.
  • the effector sequence may be substantially complementary to a region of equivalent length within an RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 3 and contain 3 mismatch bases relative thereto.
  • the effector sequence may be substantially complementary to a region of equivalent length within an RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO:
  • the effector sequence may be substantially complementary to a region of equivalent length within an RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 3 and contain 1 mismatch base relative thereto.
  • the effector sequence may be 100% complementary to a region of equivalent length within an RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 3.
  • the shmiR comprises an effector sequence which is substantially complementary to a region of equivalent length within an RNA transcript of VEGFb comprising or consisting of the sequence set forth in SEQ ID NO: 4.
  • a shmiR in accordance with this example is hereinafter designated "VEGFb_shmiR-4".
  • the effector sequence may be substantially complementary to a region of equivalent length within an RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO:
  • the effector sequence may be substantially complementary to a region of equivalent length within an RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 4 and contain 5 mismatch bases relative thereto.
  • the effector sequence may be substantially
  • the effector sequence may be substantially complementary to a region of equivalent length within an RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 4 and contain 4 mismatch bases relative thereto.
  • the effector sequence may be substantially complementary to a region of equivalent length within an RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 4 and contain 3 mismatch bases relative thereto.
  • the effector sequence may be substantially complementary to a region of equivalent length within an RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 4 and contain 2 mismatch bases relative thereto.
  • the effector sequence may be substantially complementary to a region of equivalent length within an RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 4 and contain 1 mismatch base relative thereto.
  • the effector sequence may be 100% complementary to a region of equivalent length within an RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 4.
  • the shmiR comprises an effector sequence which is substantially complementary to a region of equivalent length within an RNA transcript of VEGFb comprising or consisting of the sequence set forth in SEQ ID NO: 5.
  • a shmiR in accordance with this example is hereinafter designated "VEGFb_shmiR-5".
  • the effector sequence may be substantially complementary to a region of equivalent length within an RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 5 and contain 6 mismatch bases relative thereto.
  • the effector sequence may be substantially complementary to a region of equivalent length within an RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 5 and contain 5 mismatch bases relative thereto.
  • the effector sequence may be substantially
  • the effector sequence may be substantially complementary to a region of equivalent length within an RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 5 and contain 4 mismatch bases relative thereto.
  • the effector sequence may be substantially complementary to a region of equivalent length within an RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 5 and contain 3 mismatch bases relative thereto.
  • the effector sequence may be substantially complementary to a region of equivalent length within an RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 5 and contain 2 mismatch bases relative thereto.
  • the effector sequence may be substantially complementary to a region of equivalent length within an RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 5 and contain 1 mismatch base relative thereto.
  • the effector sequence may be 100% complementary to a region of equivalent length within an RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 5.
  • the shmiR comprises an effector sequence which is substantially complementary to a region of equivalent length within an RNA transcript of VEGFb comprising or consisting of the sequence set forth in SEQ ID NO: 6.
  • a shmiR in accordance with this example is hereinafter designated "VEGFb_shmiR-6".
  • the effector sequence may be substantially complementary to a region of equivalent length within an RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 6 and contain 6 mismatch bases relative thereto.
  • the effector sequence may be substantially complementary to a region of equivalent length within an RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 6 and contain 5 mismatch bases relative thereto.
  • the effector sequence may be substantially
  • the effector sequence may be substantially complementary to a region of equivalent length within an RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 6 and contain 4 mismatch bases relative thereto.
  • the effector sequence may be substantially complementary to a region of equivalent length within an RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 6 and contain 3 mismatch bases relative thereto.
  • the effector sequence may be substantially complementary to a region of equivalent length within an RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO:
  • the effector sequence may be substantially complementary to a region of equivalent length within an RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 6 and contain 1 mismatch base relative thereto.
  • the effector sequence may be 100% complementary to a region of equivalent length within an RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 6.
  • the shmiR comprises an effector sequence which is substantially complementary to a region of equivalent length within an RNA transcript of VEGFb comprising or consisting of the sequence set forth in SEQ ID NO: 7.
  • a shmiR in accordance with this example is hereinafter designated "VEGFb_shmiR-7".
  • the effector sequence may be substantially complementary to a region of equivalent length within an RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO:
  • the effector sequence may be substantially complementary to a region of equivalent length within an RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 7 and contain 5 mismatch bases relative thereto.
  • the effector sequence may be substantially
  • the effector sequence may be substantially complementary to a region of equivalent length within an RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 7 and contain 4 mismatch bases relative thereto.
  • the effector sequence may be substantially complementary to a region of equivalent length within an RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 7 and contain 3 mismatch bases relative thereto.
  • the effector sequence may be substantially complementary to a region of equivalent length within an RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 7 and contain 2 mismatch bases relative thereto.
  • the effector sequence may be substantially complementary to a region of equivalent length within an RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 7 and contain 1 mismatch base relative thereto.
  • the effector sequence may be 100% complementary to a region of equivalent length within an RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 7.
  • the shmiR comprises an effector sequence which is substantially complementary to a region of equivalent length within an RNA transcript of VEGFb comprising or consisting of the sequence set forth in SEQ ID NO: 8.
  • a shmiR in accordance with this example is hereinafter designated "VEGFb_shmiR-8".
  • the effector sequence may be substantially complementary to a region of equivalent length within an RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 8 and contain 6 mismatch bases relative thereto.
  • the effector sequence may be substantially complementary to a region of equivalent length within an RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 8 and contain 5 mismatch bases relative thereto.
  • the effector sequence may be substantially
  • the effector sequence may be substantially complementary to a region of equivalent length within an RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 8 and contain 4 mismatch bases relative thereto.
  • the effector sequence may be substantially complementary to a region of equivalent length within an RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 8 and contain 3 mismatch bases relative thereto.
  • the effector sequence may be substantially complementary to a region of equivalent length within an RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 8 and contain 2 mismatch bases relative thereto.
  • the effector sequence may be substantially complementary to a region of equivalent length within an RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 8 and contain 1 mismatch base relative thereto.
  • the effector sequence may be 100% complementary to a region of equivalent length within an RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 8.
  • the shmiR comprises an effector sequence which is substantially complementary to a region of equivalent length within an RNA transcript of VEGFb comprising or consisting of the sequence set forth in SEQ ID NO: 9.
  • a shmiR in accordance with this example is hereinafter designated "VEGFb_shmiR-9".
  • the effector sequence may be substantially complementary to a region of equivalent length within an RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO:
  • the effector sequence may be substantially complementary to a region of equivalent length within an RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 9 and contain 5 mismatch bases relative thereto.
  • the effector sequence may be substantially
  • the effector sequence may be substantially complementary to a region of equivalent length within an RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 9 and contain 4 mismatch bases relative thereto.
  • the effector sequence may be substantially complementary to a region of equivalent length within an RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 9 and contain 3 mismatch bases relative thereto.
  • the effector sequence may be substantially complementary to a region of equivalent length within an RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 9 and contain 2 mismatch bases relative thereto.
  • the effector sequence may be substantially complementary to a region of equivalent length within an RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 9 and contain 1 mismatch base relative thereto.
  • the effector sequence may be 100% complementary to a region of equivalent length within an RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 9.
  • the shmiR comprises an effector sequence which is substantially complementary to a region of equivalent length within an RNA transcript of VEGFb comprising or consisting of the sequence set forth in SEQ ID NO: 10.
  • a shmiR in accordance with this example is hereinafter designated "VEGFb_shmiR-10".
  • the effector sequence may be substantially complementary to a region of equivalent length within an RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO:
  • the effector sequence may be substantially complementary to a region of equivalent length within an RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 10 and contain 5 mismatch bases relative thereto.
  • the effector sequence may be substantially complementary to a region of equivalent length within an RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 10 and contain 4 mismatch bases relative thereto.
  • the effector sequence may be substantially complementary to a region of equivalent length within an RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 10 and contain 3 mismatch bases relative thereto.
  • the effector sequence may be substantially complementary to a region of equivalent length within an RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 10 and contain 2 mismatch bases relative thereto.
  • the effector sequence may be substantially complementary to a region of equivalent length within an RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO:
  • the effector sequence may be 100% complementary to a region of equivalent length within an RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 10.
  • the shmiR comprises an effector sequence which is substantially complementary to a region of equivalent length within an RNA transcript of PGF comprising or consisting of the sequence set forth in SEQ ID NO: 11.
  • a shmiR in accordance with this example is hereinafter designated "PGF_shmiR-l".
  • the effector sequence may be substantially complementary to a region of equivalent length within an RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO:
  • the effector sequence may be substantially complementary to a region of equivalent length within an RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 11 and contain 5 mismatch bases relative thereto.
  • the effector sequence may be substantially complementary to a region of equivalent length within an RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 11 and contain 4 mismatch bases relative thereto.
  • the effector sequence may be substantially complementary to a region of equivalent length within an RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 11 and contain 3 mismatch bases relative thereto.
  • the effector sequence may be substantially complementary to a region of equivalent length within an RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 11 and contain 2 mismatch bases relative thereto.
  • the effector sequence may be substantially complementary to a region of equivalent length within an RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 11 and contain 1 mismatch base relative thereto.
  • the effector sequence may be 100% complementary to a region of equivalent length within an RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 11.
  • the shmiR comprises an effector sequence which is substantially complementary to a region of equivalent length within an RNA transcript of PGF comprising or consisting of the sequence set forth in SEQ ID NO: 12.
  • a shmiR in accordance with this example is hereinafter designated "PGF_shmiR-2".
  • the effector sequence may be substantially complementary to a region of equivalent length within an RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 12 and contain 6 mismatch bases relative thereto.
  • the effector sequence may be substantially complementary to a region of equivalent length within an RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 12 and contain 5 mismatch bases relative thereto.
  • the effector sequence may be substantially complementary to a region of equivalent length within an RNA transcript of VEGFb comprising or consisting of the sequence set forth in SEQ ID NO: 12 and contain 4 mismatch bases relative thereto.
  • the effector sequence may be substantially complementary to a region of equivalent length within an RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 12 and contain 3 mismatch bases relative thereto.
  • the effector sequence may be substantially complementary to a region of equivalent length within an RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 12 and contain 2 mismatch bases relative thereto.
  • the effector sequence may be substantially complementary to a region of equivalent length within an RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 12 and contain 1 mismatch base relative thereto.
  • the effector sequence may be 100% complementary to a region of equivalent length within an RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 12.
  • the shmiR comprises an effector sequence which is substantially complementary to a region of equivalent length within an RNA transcript of PGF comprising or consisting of the sequence set forth in SEQ ID NO: 13.
  • a shmiR in accordance with this example is hereinafter designated "PGF_shmiR-3".
  • the effector sequence may be substantially complementary to a region of equivalent length within an RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 13 and contain 6 mismatch bases relative thereto.
  • the effector sequence may be substantially complementary to a region of equivalent length within an RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 13 and contain 5 mismatch bases relative thereto.
  • the effector sequence may be substantially complementary to a region of equivalent length within an RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 13 and contain 4 mismatch bases relative thereto.
  • the effector sequence may be substantially complementary to a region of equivalent length within an RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 13 and contain 3 mismatch bases relative thereto.
  • the effector sequence may be substantially complementary to a region of equivalent length within an RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 13 and contain 2 mismatch bases relative thereto.
  • the effector sequence may be substantially complementary to a region of equivalent length within an RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO:
  • the effector sequence may be 100% complementary to a region of equivalent length within an RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: l 3.
  • the shmiR comprises an effector sequence which is substantially complementary to a region of equivalent length within an RNA transcript of PGF comprising or consisting of the sequence set forth in SEQ ID NO: 14.
  • a shmiR in accordance with this example is hereinafter designated "PGF_shmiR-4".
  • the effector sequence may be substantially complementary to a region of equivalent length within an RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO:
  • the effector sequence may be substantially complementary to a region of equivalent length within an RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 14 and contain 5 mismatch bases relative thereto.
  • the effector sequence may be substantially complementary to a region of equivalent length within an RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 14 and contain 4 mismatch bases relative thereto.
  • the effector sequence may be substantially complementary to a region of equivalent length within an RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 14 and contain 3 mismatch bases relative thereto.
  • the effector sequence may be substantially complementary to a region of equivalent length within an RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 14 and contain 2 mismatch bases relative thereto.
  • the effector sequence may be substantially complementary to a region of equivalent length within an RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 14 and contain 1 mismatch base relative thereto.
  • the effector sequence may be 100% complementary to a region of equivalent length within an RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 14.
  • the shmiR comprises an effector sequence which is substantially complementary to a region of equivalent length within an RNA transcript of PGF comprising or consisting of the sequence set forth in SEQ ID NO: 15.
  • a shmiR in accordance with this example is hereinafter designated "PGF_shmiR-5".
  • the effector sequence may be substantially complementary to a region of equivalent length within an RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 15 and contain 6 mismatch bases relative thereto.
  • the effector sequence may be substantially complementary to a region of equivalent length within an RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 15 and contain 5 mismatch bases relative thereto.
  • the effector sequence may be substantially complementary to a region of equivalent length within an RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 15 and contain 4 mismatch bases relative thereto.
  • the effector sequence may be substantially complementary to a region of equivalent length within an RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 15 and contain 3 mismatch bases relative thereto.
  • the effector sequence may be substantially complementary to a region of equivalent length within an RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 15 and contain 2 mismatch bases relative thereto.
  • the effector sequence may be substantially complementary to a region of equivalent length within an RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 15 and contain 1 mismatch base relative thereto.
  • the effector sequence may be 100% complementary to a region of equivalent length within an RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 15.
  • the shmiR comprises an effector sequence which is substantially complementary to a region of equivalent length within an RNA transcript of PGF comprising or consisting of the sequence set forth in SEQ ID NO: 16.
  • a shmiR in accordance with this example is hereinafter designated "PGF_shmiR-6".
  • the effector sequence may be substantially complementary to a region of equivalent length within an RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 16 and contain 6 mismatch bases relative thereto.
  • the effector sequence may be substantially complementary to a region of equivalent length within an RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 16 and contain 5 mismatch bases relative thereto.
  • the effector sequence may be substantially complementary to a region of equivalent length within an RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 16 and contain 4 mismatch bases relative thereto.
  • the effector sequence may be substantially complementary to a region of equivalent length within an RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 16 and contain 3 mismatch bases relative thereto.
  • the effector sequence may be substantially complementary to a region of equivalent length within an RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 16 and contain 2 mismatch bases relative thereto.
  • the effector sequence may be substantially complementary to a region of equivalent length within an RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO:
  • the effector sequence may be 100% complementary to a region of equivalent length within an RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 16.
  • the shmiR comprises an effector sequence which is substantially complementary to a region of equivalent length within an RNA transcript of PGF comprising or consisting of the sequence set forth in SEQ ID NO: 17.
  • a shmiR in accordance with this example is hereinafter designated "PGF_shmiR-7".
  • the effector sequence may be substantially complementary to a region of equivalent length within an RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 17 and contain 6 mismatch bases relative thereto.
  • the effector sequence may be substantially complementary to a region of equivalent length within an RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 17 and contain 5 mismatch bases relative thereto.
  • the effector sequence may be substantially complementary to a region of equivalent length within an RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 17 and contain 4 mismatch bases relative thereto.
  • the effector sequence may be substantially complementary to a region of equivalent length within an RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 17 and contain 3 mismatch bases relative thereto.
  • the effector sequence may be substantially complementary to a region of equivalent length within an RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 17 and contain 2 mismatch bases relative thereto.
  • the effector sequence may be substantially complementary to a region of equivalent length within an RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 17 and contain 1 mismatch base relative thereto.
  • the effector sequence may be 100% complementary to a region of equivalent length within an RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 17.
  • the shmiR comprises an effector sequence which is substantially complementary to a region of equivalent length within an RNA transcript of PGF comprising or consisting of the sequence set forth in SEQ ID NO: 18.
  • a shmiR in accordance with this example is hereinafter designated "PGF_shmiR-8".
  • the effector sequence may be substantially complementary to a region of equivalent length within an RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO:
  • the effector sequence may be substantially complementary to a region of equivalent length within an RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 18 and contain 5 mismatch bases relative thereto.
  • the effector sequence may be substantially complementary to a region of equivalent length within an RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 18 and contain 4 mismatch bases relative thereto.
  • the effector sequence may be substantially complementary to a region of equivalent length within an RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 18 and contain 3 mismatch bases relative thereto.
  • the effector sequence may be substantially complementary to a region of equivalent length within an RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 18 and contain 2 mismatch bases relative thereto.
  • the effector sequence may be substantially complementary to a region of equivalent length within an RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO:
  • the effector sequence may be 100% complementary to a region of equivalent length within an RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 18.
  • the shmiR comprises an effector sequence which is substantially complementary to a region of equivalent length within an RNA transcript of PGF comprising or consisting of the sequence set forth in SEQ ID NO: 19.
  • a shmiR in accordance with this example is hereinafter designated "PGF_shmiR-9".
  • the effector sequence may be substantially complementary to a region of equivalent length within an RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 19 and contain 6 mismatch bases relative thereto.
  • the effector sequence may be substantially complementary to a region of equivalent length within an RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 19 and contain 5 mismatch bases relative thereto.
  • the effector sequence may be substantially complementary to a region of equivalent length within an RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 19 and contain 4 mismatch bases relative thereto.
  • the effector sequence may be substantially complementary to a region of equivalent length within an RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 19 and contain 3 mismatch bases relative thereto.
  • the effector sequence may be substantially complementary to a region of equivalent length within an RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 19 and contain 2 mismatch bases relative thereto.
  • the effector sequence may be substantially complementary to a region of equivalent length within an RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO:
  • the effector sequence may be 100% complementary to a region of equivalent length within an RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 19.
  • the shmiR comprises an effector sequence which is substantially complementary to a region of equivalent length within an RNA transcript of PGF comprising or consisting of the sequence set forth in SEQ ID NO: 20.
  • a shmiR in accordance with this example is hereinafter designated "PGF_shmiR-10".
  • the effector sequence may be substantially complementary to a region of equivalent length within an RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 20 and contain 6 mismatch bases relative thereto.
  • the effector sequence may be substantially complementary to a region of equivalent length within an RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 20 and contain 5 mismatch bases relative thereto.
  • the effector sequence may be substantially complementary to a region of equivalent length within an RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 20 and contain 4 mismatch bases relative thereto.
  • the effector sequence may be substantially complementary to a region of equivalent length within an RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 20 and contain 3 mismatch bases relative thereto.
  • the effector sequence may be substantially complementary to a region of equivalent length within an RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 20 and contain 2 mismatch bases relative thereto.
  • the effector sequence may be substantially complementary to a region of equivalent length within an RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 20 and contain 1 mismatch base relative thereto.
  • the effector sequence may be 100% complementary to a region of equivalent length within an RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 20.
  • the shmiR comprises an effector sequence which is substantially complementary to a region of equivalent length within an RNA transcript of PGF comprising or consisting of the sequence set forth in SEQ ID NO: 21.
  • a shmiR in accordance with this example is hereinafter designated "PGF_shmiR-l l".
  • the effector sequence may be substantially complementary to a region of equivalent length within an RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 21 and contain 6 mismatch bases relative thereto.
  • the effector sequence may be substantially complementary to a region of equivalent length within an RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 21 and contain 5 mismatch bases relative thereto.
  • the effector sequence may be substantially complementary to a region of equivalent length within an RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 21 and contain 4 mismatch bases relative thereto.
  • the effector sequence may be substantially complementary to a region of equivalent length within an RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 21 and contain 3 mismatch bases relative thereto.
  • the effector sequence may be substantially complementary to a region of equivalent length within an RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 21 and contain 2 mismatch bases relative thereto.
  • the effector sequence may be substantially complementary to a region of equivalent length within an RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO:
  • the effector sequence may be 100% complementary to a region of equivalent length within an RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 21.
  • the shmiR comprises an effector sequence which is substantially complementary to a region of equivalent length within an RNA transcript of PGF comprising or consisting of the sequence set forth in SEQ ID NO: 22.
  • a shmiR in accordance with this example is hereinafter designated "PGF_shmiR-12".
  • the effector sequence may be substantially complementary to a region of equivalent length within an RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO:
  • the effector sequence may be substantially complementary to a region of equivalent length within an RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 22 and contain 5 mismatch bases relative thereto.
  • the effector sequence may be substantially complementary to a region of equivalent length within an RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 22 and contain 4 mismatch bases relative thereto.
  • the effector sequence may be substantially complementary to a region of equivalent length within an RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 22 and contain 3 mismatch bases relative thereto.
  • the effector sequence may be substantially complementary to a region of equivalent length within an RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 22 and contain 2 mismatch bases relative thereto.
  • the effector sequence may be substantially complementary to a region of equivalent length within an RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 22 and contain 1 mismatch base relative thereto.
  • the effector sequence may be 100% complementary to a region of equivalent length within an RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 22.
  • the effector sequence of a shmiR of the disclosure is substantially complementary to a RNA transcript of VEGFb or PGF as described herein and contains 1, 2, 3, 4, 5 or 6 mismatch base(s) relative thereto, it is preferred that the mismatch(es) are not located within the region corresponding to the seed region of the shmiR i.e., nucleotides 2-8 of the effector sequence.
  • the nucleic acid as described herein comprises a DNA sequence encoding VEGFb_shmiR-l comprising: (i) an effector sequence which is substantially complementary to the sequence set forth in SEQ ID NO:23 with the exception of 1, 2, 3, 4, 5 or 6 base mismatches, provided that the effector sequence is capable of forming a duplex with a sequence set forth in SEQ ID NO:23; and (ii) an effector complement sequence comprising a sequence which is substantially complementary to the effector sequence.
  • VEGFb_shmiR- 1 encoded by the nucleic acid may comprise an effector sequence set forth in SEQ ID NO:24 and an effector complement sequence which is substantially complementary to the sequence set forth in SEQ ID NO:24 and capable of forming a duplex therewith.
  • the effector complement sequence which is substantially complementary to the sequence set forth in SEQ ID NO:24 may be the sequence set forth in SEQ ID NO:23.
  • a shmiR in accordance with this example is hereinafter designated "VEFGb shmiR- '.
  • the nucleic acid as described herein comprises a DNA sequence encoding VEGFb_shmiR-2 comprising: (i) an effector sequence which is substantially complementary to the sequence set forth in SEQ ID NO:25 with the exception of 1, 2, 3, 4, 5 or 6 base mismatches, provided that the effector sequence is capable of forming a duplex with a sequence set forth in SEQ ID NO:25; and (ii) an effector complement sequence comprising a sequence which is substantially complementary to the effector sequence.
  • VEGFb_shmiR-2 encoded by the nucleic acid may comprise an effector sequence set forth in SEQ ID NO:26 and an effector complement sequence which is substantially complementary to the sequence set forth in SEQ ID NO:26 and capable of forming a duplex therewith.
  • the effector complement sequence which is substantially complementary to the sequence set forth in SEQ ID NO:26 may be the sequence set forth in SEQ ID NO:25.
  • a shmiR in accordance with this example is hereinafter designated "VEGFb_shmiR-2".
  • the nucleic acid as described herein comprises a DNA sequence encoding VEGFb_shmiR-3 comprising: (i) an effector sequence which is substantially complementary to the sequence set forth in SEQ ID NO:27 with the exception of 1, 2, 3, 4, 5 or 6 base mismatches, provided that the effector sequence is capable of forming a duplex with a sequence set forth in SEQ ID NO:27; and (ii) an effector complement sequence comprising a sequence which is substantially complementary to the effector sequence.
  • VEGFb_shmiR-3 encoded by the nucleic acid may comprise an effector sequence set forth in SEQ ID NO:28 and an effector complement sequence which is substantially complementary to the sequence set forth in SEQ ID NO:28 and capable of forming a duplex therewith.
  • the effector complement sequence which is substantially complementary to the sequence set forth in SEQ ID NO:28 may be the sequence set forth in SEQ ID NO:27.
  • a shmiR in accordance with this example is hereinafter designated "VEGFb_shmiR-3".
  • the nucleic acid as described herein comprises a DNA sequence encoding VEGFb_shmiR-4 comprising: (i) an effector sequence which is substantially complementary to the sequence set forth in SEQ ID NO:29 with the exception of 1, 2, 3, 4, 5 or 6 base mismatches, provided that the effector sequence is capable of forming a duplex with a sequence set forth in SEQ ID NO:29; and (ii) an effector complement sequence comprising a sequence which is substantially complementary to the effector sequence.
  • VEGFb_shmiR-4 encoded by the nucleic acid may comprise an effector sequence set forth in SEQ ID NO:30 and an effector complement sequence which is substantially complementary to the sequence set forth in SEQ ID NO:30 and capable of forming a duplex therewith.
  • the effector complement sequence which is substantially complementary to the sequence set forth in SEQ ID NO:30 may be the sequence set forth in SEQ ID NO:29.
  • a shmiR in accordance with this example is hereinafter designated "VEGFb_shmiR-4".
  • the nucleic acid as described herein comprises a DNA sequence encoding VEGFb_shmiR-5 comprising: (i) an effector sequence which is substantially complementary to the sequence set forth in SEQ ID NO:31 with the exception of 1, 2, 3, 4, 5 or 6 base mismatches, provided that the effector sequence is capable of forming a duplex with a sequence set forth in SEQ ID NO:31 ; and (ii) an effector complement sequence comprising a sequence which is substantially complementary to the effector sequence.
  • VEGFb_shmiR-5 encoded by the nucleic acid may comprise an effector sequence set forth in SEQ ID NO:32 and an effector complement sequence which is substantially complementary to the sequence set forth in SEQ ID NO:32 and capable of forming a duplex therewith.
  • the effector complement sequence which is substantially complementary to the sequence set forth in SEQ ID NO:32 may be the sequence set forth in SEQ ID NO:31.
  • a shmiR in accordance with this example is hereinafter designated "VEGFb_shmiR-5".
  • the nucleic acid as described herein comprises a DNA sequence encoding VEGFb_shmiR-6 comprising: (i) an effector sequence which is substantially complementary to the sequence set forth in SEQ ID NO:33 with the exception of 1, 2, 3, 4, 5 or 6 base mismatches, provided that the effector sequence is capable of forming a duplex with a sequence set forth in SEQ ID NO:33; and (ii) an effector complement sequence comprising a sequence which is substantially complementary to the effector sequence.
  • VEGFb_shmiR-6 encoded by the nucleic acid may comprise an effector sequence set forth in SEQ ID NO:34 and an effector complement sequence which is substantially complementary to the sequence set forth in SEQ ID NO:34 and capable of forming a duplex therewith.
  • the effector complement sequence which is substantially complementary to the sequence set forth in SEQ ID NO:34 may be the sequence set forth in SEQ ID NO:33.
  • a shmiR in accordance with this example is hereinafter designated "VEGFb_shmiR-6".
  • the nucleic acid as described herein comprises a DNA sequence encoding VEGFb_shmiR-7 comprising: (i) an effector sequence which is substantially complementary to the sequence set forth in SEQ ID NO:35 with the exception of 1, 2, 3, 4, 5 or 6 base mismatches, provided that the effector sequence is capable of forming a duplex with a sequence set forth in SEQ ID NO:35; and (ii) an effector complement sequence comprising a sequence which is substantially complementary to the effector sequence.
  • VEGFb_shmiR-7 encoded by the nucleic acid may comprise an effector sequence set forth in SEQ ID NO:36 and an effector complement sequence which is substantially complementary to the sequence set forth in SEQ ID NO:36 and capable of forming a duplex therewith.
  • the effector complement sequence which is substantially complementary to the sequence set forth in SEQ ID NO:36 may be the sequence set forth in SEQ ID NO:35.
  • a shmiR in accordance with this example is hereinafter designated "VEGFb_shmiR-7".
  • the nucleic acid as described herein comprises a DNA sequence encoding VEGFb_shmiR-8 comprising: (i) an effector sequence which is substantially complementary to the sequence set forth in SEQ ID NO:37 with the exception of 1, 2, 3, 4, 5 or 6 base mismatches, provided that the effector sequence is capable of forming a duplex with a sequence set forth in SEQ ID NO:37; and (ii) an effector complement sequence comprising a sequence which is substantially complementary to the effector sequence.
  • VEGFb_shmiR-8 encoded by the nucleic acid may comprise an effector sequence set forth in SEQ ID NO:38 and an effector complement sequence which is substantially complementary to the sequence set forth in SEQ ID NO:38 and capable of forming a duplex therewith.
  • the effector complement sequence which is substantially complementary to the sequence set forth in SEQ ID NO:38 may be the sequence set forth in SEQ ID NO:37.
  • a shmiR in accordance with this example is hereinafter designated "VEGFb_shmiR-8".
  • the nucleic acid as described herein comprises a DNA sequence encoding VEGFb_shmiR-9 comprising: (i) an effector sequence which is substantially complementary to the sequence set forth in SEQ ID NO:39 with the exception of 1, 2, 3, 4, 5 or 6 base mismatches, provided that the effector sequence is capable of forming a duplex with a sequence set forth in SEQ ID NO:39; and (ii) an effector complement sequence comprising a sequence which is substantially complementary to the effector sequence.
  • VEGFb_shmiR-9 encoded by the nucleic acid may comprise an effector sequence set forth in SEQ ID NO:40 and an effector complement sequence which is substantially complementary to the sequence set forth in SEQ ID NO:40 and capable of forming a duplex therewith.
  • the effector complement sequence which is substantially complementary to the sequence set forth in SEQ ID NO:40 may be the sequence set forth in SEQ ID NO:39.
  • a shmiR in accordance with this example is hereinafter designated "VEGFb_shmiR-9".
  • the nucleic acid as described herein comprises a DNA sequence encoding VEGFb_shmiR-10 comprising: (i) an effector sequence which is substantially complementary to the sequence set forth in SEQ ID NO:41 with the exception of 1, 2, 3, 4, 5 or 6 base mismatches, provided that the effector sequence is capable of forming a duplex with a sequence set forth in SEQ ID NO:41; and (ii) an effector complement sequence comprising a sequence which is substantially complementary to the effector sequence.
  • VEGFb_shmiR-10 encoded by the nucleic acid may comprise an effector sequence set forth in SEQ ID NO:42 and an effector complement sequence which is substantially complementary to the sequence set forth in SEQ ID NO:42 and capable of forming a duplex therewith.
  • the effector complement sequence which is substantially complementary to the sequence set forth in SEQ ID NO:42 may be the sequence set forth in SEQ ID NO:41.
  • a shmiR in accordance with this example is hereinafter designated "VEGFb_shmiR-10".
  • the nucleic acid as described herein comprises a DNA sequence encoding PGF_shmiR-l comprising: (i) an effector sequence which is substantially complementary to the sequence set forth in SEQ ID NO:43 with the exception of 1, 2, 3, 4, 5 or 6 base mismatches, provided that the effector sequence is capable of forming a duplex with a sequence set forth in SEQ ID NO:43; and (ii) an effector complement sequence comprising a sequence which is substantially complementary to the effector sequence.
  • PGF_shmiR-l encoded by the nucleic acid may comprise an effector sequence set forth in SEQ ID NO:44 and an effector complement sequence which is substantially complementary to the sequence set forth in SEQ ID NO:44 and capable of forming a duplex therewith.
  • the effector complement sequence which is substantially complementary to the sequence set forth in SEQ ID NO:44 may be the sequence set forth in SEQ ID NO:41.
  • a shmiR in accordance with this example is hereinafter designated "PGF_shmiR-l".
  • the nucleic acid as described herein comprises a DNA sequence encoding PGF_shmiR-2 comprising: (i) an effector sequence which is substantially complementary to the sequence set forth in SEQ ID NO:45 with the exception of 1, 2, 3, 4, 5 or 6 base mismatches, provided that the effector sequence is capable of forming a duplex with a sequence set forth in SEQ ID NO:45; and (ii) an effector complement sequence comprising a sequence which is substantially complementary to the effector sequence.
  • PGF_shmiR-2 encoded by the nucleic acid may comprise an effector sequence set forth in SEQ ID NO:46 and an effector complement sequence which is substantially complementary to the sequence set forth in SEQ ID NO:46 and capable of forming a duplex therewith.
  • the effector complement sequence which is substantially complementary to the sequence set forth in SEQ ID NO:46 may be the sequence set forth in SEQ ID NO:45.
  • a shmiR in accordance with this example is hereinafter designated "PGF_shmiR-2".
  • the nucleic acid as described herein comprises a DNA sequence encoding PGF_shmiR-3 comprising: (i) an effector sequence which is substantially complementary to the sequence set forth in SEQ ID NO:47 with the exception of 1, 2, 3, 4, 5 or 6 base mismatches, provided that the effector sequence is capable of forming a duplex with a sequence set forth in SEQ ID NO:47; and (ii) an effector complement sequence comprising a sequence which is substantially complementary to the effector sequence.
  • PGF_shmiR-3 encoded by the nucleic acid may comprise an effector sequence set forth in SEQ ID NO:48 and an effector complement sequence which is substantially complementary to the sequence set forth in SEQ ID NO:48 and capable of forming a duplex therewith.
  • the effector complement sequence which is substantially complementary to the sequence set forth in SEQ ID NO:48 may be the sequence set forth in SEQ ID NO:47.
  • a shmiR in accordance with this example is hereinafter designated "PGF_shmiR-3".
  • the nucleic acid as described herein comprises a DNA sequence encoding PGF_shmiR-4 comprising: (i) an effector sequence which is substantially complementary to the sequence set forth in SEQ ID NO:49 with the exception of 1, 2, 3, 4, 5 or 6 base mismatches, provided that the effector sequence is capable of forming a duplex with a sequence set forth in SEQ ID NO:49; and (ii) an effector complement sequence comprising a sequence which is substantially complementary to the effector sequence.
  • PGF_shmiR-4 encoded by the nucleic acid may comprise an effector sequence set forth in SEQ ID NO:50 and an effector complement sequence which is substantially complementary to the sequence set forth in SEQ ID NO:50 and capable of forming a duplex therewith.
  • the effector complement sequence which is substantially complementary to the sequence set forth in SEQ ID NO:50 may be the sequence set forth in SEQ ID NO:49.
  • a shmiR in accordance with this example is hereinafter designated "PGF_shmiR-4".
  • the nucleic acid as described herein comprises a DNA sequence encoding PGF_shmiR-5 comprising: (i) an effector sequence which is substantially complementary to the sequence set forth in SEQ ID NO:51 with the exception of 1, 2, 3, 4, 5 or 6 base mismatches, provided that the effector sequence is capable of forming a duplex with a sequence set forth in SEQ ID NO:51; and (ii) an effector complement sequence comprising a sequence which is substantially complementary to the effector sequence.
  • PGF_shmiR-5 encoded by the nucleic acid may comprise an effector sequence set forth in SEQ ID NO:52 and an effector complement sequence which is substantially complementary to the sequence set forth in SEQ ID NO:52 and capable of forming a duplex therewith.
  • the effector complement sequence which is substantially complementary to the sequence set forth in SEQ ID NO:52 may be the sequence set forth in SEQ ID NO:51.
  • a shmiR in accordance with this example is hereinafter designated "PGF_shmiR-5".
  • the nucleic acid as described herein comprises a DNA sequence encoding PGF_shmiR-6 comprising: (i) an effector sequence which is substantially complementary to the sequence set forth in SEQ ID NO:53 with the exception of 1, 2, 3, 4, 5 or 6 base mismatches, provided that the effector sequence is capable of forming a duplex with a sequence set forth in SEQ ID NO:53; and (ii) an effector complement sequence comprising a sequence which is substantially complementary to the effector sequence.
  • PGF_shmiR-6 encoded by the nucleic acid may comprise an effector sequence set forth in SEQ ID NO:54 and an effector complement sequence which is substantially complementary to the sequence set forth in SEQ ID NO:54 and capable of forming a duplex therewith.
  • the effector complement sequence which is substantially complementary to the sequence set forth in SEQ ID NO:54 may be the sequence set forth in SEQ ID NO:53.
  • a shmiR in accordance with this example is hereinafter designated "PGF_shmiR-6".
  • the nucleic acid as described herein comprises a DNA sequence encoding PGF_shmiR-7 comprising: (i) an effector sequence which is substantially complementary to the sequence set forth in SEQ ID NO:55 with the exception of 1, 2, 3, 4, 5 or 6 base mismatches, provided that the effector sequence is capable of forming a duplex with a sequence set forth in SEQ ID NO:55; and (ii) an effector complement sequence comprising a sequence which is substantially complementary to the effector sequence.
  • PGF_shmiR-7 encoded by the nucleic acid may comprise an effector sequence set forth in SEQ ID NO:56 and an effector complement sequence which is substantially complementary to the sequence set forth in SEQ ID NO:56 and capable of forming a duplex therewith.
  • the effector complement sequence which is substantially complementary to the sequence set forth in SEQ ID NO:56 may be the sequence set forth in SEQ ID NO:55.
  • a shmiR in accordance with this example is hereinafter designated "PGF_shmiR-7".
  • the nucleic acid as described herein comprises a DNA sequence encoding PGF_shmiR-8 comprising: (i) an effector sequence which is substantially complementary to the sequence set forth in SEQ ID NO:57 with the exception of 1, 2, 3, 4, 5 or 6 base mismatches, provided that the effector sequence is capable of forming a duplex with a sequence set forth in SEQ ID NO:57; and (ii) an effector complement sequence comprising a sequence which is substantially complementary to the effector sequence.
  • PGF_shmiR-8 encoded by the nucleic acid may comprise an effector sequence set forth in SEQ ID NO:58 and an effector complement sequence which is substantially complementary to the sequence set forth in SEQ ID NO:58 and capable of forming a duplex therewith.
  • the effector complement sequence which is substantially complementary to the sequence set forth in SEQ ID NO:58 may be the sequence set forth in SEQ ID NO:57.
  • a shmiR in accordance with this example is hereinafter designated "PGF_shmiR-8".
  • the nucleic acid as described herein comprises a DNA sequence encoding PGF_shmiR-9 comprising: (i) an effector sequence which is substantially complementary to the sequence set forth in SEQ ID NO:59 with the exception of 1, 2, 3, 4, 5 or 6 base mismatches, provided that the effector sequence is capable of forming a duplex with a sequence set forth in SEQ ID NO:59; and (ii) an effector complement sequence comprising a sequence which is substantially complementary to the effector sequence.
  • PGF_shmiR-9 encoded by the nucleic acid may comprise an effector sequence set forth in SEQ ID NO:60 and an effector complement sequence which is substantially complementary to the sequence set forth in SEQ ID NO:60 and capable of forming a duplex therewith.
  • the effector complement sequence which is substantially complementary to the sequence set forth in SEQ ID NO:60 may be the sequence set forth in SEQ ID NO:59.
  • a shmiR in accordance with this example is hereinafter designated "PGF_shmiR-9".
  • the nucleic acid as described herein comprises a DNA sequence encoding PGF_shmiR-10 comprising: (i) an effector sequence which is substantially complementary to the sequence set forth in SEQ ID NO:61 with the exception of 1, 2, 3, 4, 5 or 6 base mismatches, provided that the effector sequence is capable of forming a duplex with a sequence set forth in SEQ ID NO:61 ; and (ii) an effector complement sequence comprising a sequence which is substantially complementary to the effector sequence.
  • PGF_shmiR-10 encoded by the nucleic acid may comprise an effector sequence set forth in SEQ ID NO:62 and an effector complement sequence which is substantially complementary to the sequence set forth in SEQ ID NO:62 and capable of forming a duplex therewith.
  • the effector complement sequence which is substantially complementary to the sequence set forth in SEQ ID NO:62 may be the sequence set forth in SEQ ID NO:61.
  • a shmiR in accordance with this example is hereinafter designated "PGF_shmiR-10".
  • the nucleic acid as described herein comprises a DNA sequence encoding PGF_shmiR-l l comprising: (i) an effector sequence which is substantially complementary to the sequence set forth in SEQ ID NO:63 with the exception of 1, 2, 3, 4, 5 or 6 base mismatches, provided that the effector sequence is capable of forming a duplex with a sequence set forth in SEQ ID NO:63; and (ii) an effector complement sequence comprising a sequence which is substantially complementary to the effector sequence.
  • PGF_shmiR-l l encoded by the nucleic acid may comprise an effector sequence set forth in SEQ ID NO:64 and an effector complement sequence which is substantially complementary to the sequence set forth in SEQ ID NO:64 and capable of forming a duplex therewith.
  • the effector complement sequence which is substantially complementary to the sequence set forth in SEQ ID NO:64 may be the sequence set forth in SEQ ID NO:63.
  • a shmiR in accordance with this example is hereinafter designated "PGF_shmiR-H".
  • the nucleic acid as described herein comprises a DNA sequence encoding PGF_shmiR-12 comprising: (i) an effector sequence which is substantially complementary to the sequence set forth in SEQ ID NO:65 with the exception of 1, 2, 3, 4, 5 or 6 base mismatches, provided that the effector sequence is capable of forming a duplex with a sequence set forth in SEQ ID NO:65; and (ii) an effector complement sequence comprising a sequence which is substantially complementary to the effector sequence.
  • PGF_shmiR-12 encoded by the nucleic acid may comprise an effector sequence set forth in SEQ ID NO:66 and an effector complement sequence which is substantially complementary to the sequence set forth in SEQ ID NO:66 and capable of forming a duplex therewith.
  • the effector complement sequence which is substantially complementary to the sequence set forth in SEQ ID NO:66 may be the sequence set forth in SEQ ID NO:65.
  • a shmiR in accordance with this example is hereinafter designated "PGF_shmiR-12".
  • the shmiR encoded by the nucleic acid of the disclosure may comprise, in a 5' to 3' direction:
  • the shmiR encoded by the nucleic acid of the disclosure may comprise, in a 5' to 3' direction:
  • Suitable loop sequences may be selected from those known in the art. However, an exemplary stemloop sequence is set forth in SEQ ID NO: 67.
  • Suitable primary micro RNA (pri-miRNA or pri-R) backbones for use in a nucleic acid of the disclosure may be selected from those known in the art.
  • the pri- miRNA backbone may be selected from a pri-miR-30a backbone, a pri-miR-155 backbone, a pri-miR-21 backbone and a pri-miR-136 backbone.
  • the pri-miRNA backbone is a pri-miR-30a backbone.
  • the nucleic acid encoding the shmiRs of the disclosure may comprise DNA sequence encoding the sequence set forth in SEQ ID NO: 68 and DNA sequence encoding the sequence set forth in SEQ ID NO: 69.
  • the nucleic acid described herein may comprise a DNA sequence selected from the sequence set forth in any one of SEQ ID NOs: 92-113.
  • the nucleic acid may comprise a DNA sequence selected from the sequence set forth in any one of SEQ ID NOs: 92-101.
  • the nucleic acid described herein comprises or consists of a DNA sequence set forth in SEQ ID NO: 92 and encodes a shmiR (VEGFb_shmiR-l) comprising or consisting of the sequence set forth in SEQ ID NO: 70.
  • the nucleic acid described herein comprises or consists of a DNA sequence set forth in SEQ ID NO: 93 and encodes a shmiR (VEGFb_shmiR-2) comprising or consisting of the sequence set forth in SEQ ID NO: 71.
  • the nucleic acid described herein comprises or consists of a DNA sequence set forth in SEQ ID NO: 94 and encodes a shmiR (VEGFb_shmiR-3) comprising or consisting of the sequence set forth in SEQ ID NO: 72.
  • the nucleic acid described herein comprises or consists of a DNA sequence set forth in SEQ ID NO: 95 and encodes a shmiR (VEGFb_shmiR-4) comprising or consisting of the sequence set forth in SEQ ID NO: 73.
  • the nucleic acid described herein comprises or consists of a DNA sequence set forth in SEQ ID NO: 96 and encodes a shmiR (VEGFb_shmiR-5) comprising or consisting of the sequence set forth in SEQ ID NO: 74.
  • the nucleic acid described herein comprises or consists of a DNA sequence set forth in SEQ ID NO: 97 and encodes a shmiR (VEGFb_shmiR-6) comprising or consisting of the sequence set forth in SEQ ID NO: 75.
  • the nucleic acid described herein comprises or consists of a DNA sequence set forth in SEQ ID NO: 98 and encodes a shmiR (VEGFb_shmiR-7) comprising or consisting of the sequence set forth in SEQ ID NO: 76.
  • the nucleic acid described herein comprises or consists of a DNA sequence set forth in SEQ ID NO: 99 and encodes a shmiR (VEGFb_shmiR-8) comprising or consisting of the sequence set forth in SEQ ID NO: 77.
  • the nucleic acid described herein comprises or consists of a DNA sequence set forth in SEQ ID NO: 100 and encodes a shmiR (VEGFb_shmiR-9) comprising or consisting of the sequence set forth in SEQ ID NO: 78.
  • the nucleic acid described herein comprises or consists of a DNA sequence set forth in SEQ ID NO: 101 and encodes a shmiR (VEGFb_shmiR-10) comprising or consisting of the sequence set forth in SEQ ID NO: 79.
  • Exemplary nucleic acids targeting VEGFb encode a shmiR selected from
  • VEGFb_shmiR-l VEGFb_shmiR-4, and VEGFb_shmiR-10 as described herein.
  • the nucleic acid described herein encodes a shmiR targeting PGF
  • the nucleic acid may comprise a DNA sequence selected from the sequence set forth in any one of SEQ ID NOs: 102-113.
  • the nucleic acid described herein comprises or consists of a DNA sequence set forth in SEQ ID NO: 102 and encodes a shmiR (PGF_shmiR-l) comprising or consisting of the sequence set forth in SEQ ID NO: 80.
  • the nucleic acid described herein comprises or consists of a DNA sequence set forth in SEQ ID NO: 103 and encodes a shmiR (PGF_shmiR-2) comprising or consisting of the sequence set forth in SEQ ID NO: 81.
  • the nucleic acid described herein comprises or consists of a DNA sequence set forth in SEQ ID NO: 104 and encodes a shmiR (PGF_shmiR-3) comprising or consisting of the sequence set forth in SEQ ID NO: 82.
  • the nucleic acid described herein comprises or consists of a DNA sequence set forth in SEQ ID NO: 105 and encodes a shmiR (PGF_shmiR-4) comprising or consisting of the sequence set forth in SEQ ID NO: 83.
  • the nucleic acid described herein comprises or consists of a DNA sequence set forth in SEQ ID NO: 106 and encodes a shmiR (PGF_shmiR-5) comprising or consisting of the sequence set forth in SEQ ID NO: 84.
  • the nucleic acid described herein comprises or consists of a DNA sequence set forth in SEQ ID NO: 107 and encodes a shmiR (PGF_shmiR-6) comprising or consisting of the sequence set forth in SEQ ID NO: 85.
  • the nucleic acid described herein comprises or consists of a DNA sequence set forth in SEQ ID NO: 108 and encodes a shmiR (PGF_shmiR-7) comprising or consisting of the sequence set forth in SEQ ID NO: 86.
  • the nucleic acid described herein comprises or consists of a DNA sequence set forth in SEQ ID NO: 109 and encodes a shmiR (PGF_shmiR-8) comprising or consisting of the sequence set forth in SEQ ID NO: 87.
  • the nucleic acid described herein comprises or consists of a DNA sequence set forth in SEQ ID NO: 110 and encodes a shmiR (PGF_shmiR-9) comprising or consisting of the sequence set forth in SEQ ID NO: 88.
  • the nucleic acid described herein comprises or consists of a DNA sequence set forth in SEQ ID NO: 111 and encodes a shmiR (PGF_shmiR-10) comprising or consisting of the sequence set forth in SEQ ID NO: 89.
  • the nucleic acid described herein comprises or consists of a DNA sequence set forth in SEQ ID NO: 112 and encodes a shmiR (PGF_shmiR-l 1) comprising or consisting of the sequence set forth in SEQ ID NO: 90.
  • the nucleic acid described herein comprises or consists of a DNA sequence set forth in SEQ ID NO: 113 and encodes a shmiR (PGF_shmiR-12) comprising or consisting of the sequence set forth in SEQ ID NO: 91.
  • Exemplary nucleic acids targeting PGF encode a shmiR selected from PGF_shmiR-
  • PGF_shmiR-7 and PGF_shmiR-10 as described herein.
  • effector/effector complement sequence combinations encoded by the nucleic acids described herein may also be expressed and thus provided in the form of a short hairpin RNA (shRNA).
  • shRNA short hairpin RNA
  • the present disclosure also provides a nucleic acid comprising a DNA sequence encoding a shRNA comprising:
  • an effector sequence of at least 17 nucleotides in length a stemloop sequence
  • the effector sequence is substantially complementary to an RNA transcript of VEGFb set forth set forth in any one of SEQ ID NOs: 1-10 or an RNA transcript of PGF set forth set forth in any one of SEQ ID NOs: 11-22.
  • the effector sequence will be less than 30 nucleotides in length.
  • a suitable effector sequence may be in the range of 17-29 nucleotides in length.
  • effector and effector complement sequence combinations are described herein in the context of shmiRs of the disclosure targeting VEGFb and PGF and shall be taken to apply mutatis mutandis to each example in which a shRNA targeting the corresponding region of a transcript of VEGFb of PGF is described, including nucleic acids encoding such shRNAs.
  • the shRNA will comprise a stem loop sequence positioned between the effector sequence and the effector complement sequence.
  • the shRNA encoded by the nucleic acid of the disclosure may comprise, in a 5' to 3' direction:
  • the shRNA encoded by the nucleic acid of the disclosure may comprise, in a 5' to 3' direction:
  • Suitable loop sequences are described herein in the context of shmiRs and shall be taken to apply mutatis mutandis to each example in which a shRNA is described.
  • an exemplary stemloop sequence is set forth in SEQ ID NO: 67.
  • nucleic acid in accordance with the present disclosure may be combined or used in conjunction with other therapeutic agents for treating AMD or inhibiting CNV.
  • the present disclosure provides a nucleic acid comprising a DNA sequence encoding a shmiR as described herein (e.g., one or shmiRs designated VEGFb_shmiRl to VEGFb_shmiR-10 or PGF_shmiRl to PGF_shmiR- 12 described herein) in combination with one or more other agents for treating AMD or inhibiting CNV.
  • a plurality of nucleic acids are provided comprising: (a) at least one nucleic acid as described herein; and
  • nucleic acid comprising a DNA sequence encoding a shmiR as described herein;
  • VEGFa_shmiR a nucleic acid comprising a DNA sequence encoding a shmiR targeting VEGFa (designated herein as "VEGFa_shmiR”) comprising:
  • pri-miRNA primary micro RNA
  • effector sequence is substantially complementary to a region of corresponding length in the RNA transcript set forth in SEQ ID NO: 114;
  • shmiRs encoded by the nucleic acids at (a) and (b) comprise different effector sequences.
  • the effector sequence of VEGFa_shmiR will be less than 30 nucleotides in length.
  • a suitable effector sequence of VEGFa_shmiR may be in the range of 17-29 nucleotides in length.
  • the effector sequence of VEGFa_shmiR will be 21 nucleotides in length. More preferably, the effector sequence of VEGFa_shmiR will be 21 nucleotides in length and the corresponding effector complement sequence will be 20 nucleotides in length.
  • VEGFa_shmiR will comprise an effector sequence which is substantially complementary to a region of corresponding length within an RNA transcript of VEGFa comprising or consisting of the sequence set forth in SEQ ID NO: 114.
  • the effector sequence of VEGFa_shmiR may be substantially complementary to a region of corresponding length within an RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 114 and contain 6 mismatch bases relative thereto.
  • the effector sequence of VEGFa_shmiR may be substantially complementary to a region of corresponding length within an RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 114 and contain 5 mismatch bases relative thereto.
  • the effector sequence of VEGFa_shmiR may be substantially complementary to a region of corresponding length within an RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 114 and contain 4 mismatch bases relative thereto.
  • the effector sequence of VEGFa_shmiR may be substantially complementary to a region of corresponding length within an RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 114 and contain 3 mismatch bases relative thereto.
  • the effector sequence of VEGFa_shmiR may be substantially complementary to a region of corresponding length within an RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 114 and contain 2 mismatch bases relative thereto.
  • the effector sequence of VEGFa_shmiR may be substantially complementary to a region of corresponding length within an RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 114 and contain 1 mismatch base relative thereto.
  • the effector sequence of VEGFa_shmiR may be 100% complementary to a region of corresponding length within an RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 114.
  • the effector sequence of VEGFa_shmiR is substantially complementary to SEQ ID NO: 114 and contains 1, 2, 3, 4, 5 or 6 mismatch base(s) relative thereto, it is preferred that the mismatch(es) are not located within the region corresponding to the seed region of the shmiR i.e., nucleotides 2-8 of the effector sequence.
  • VEGFa_shmiR as described herein comprises: (i) an effector sequence which is substantially complementary to the sequence set forth in SEQ ID NO: 114 with the exception of 1, 2, 3, 4, 5 or 6 base mismatches, provided that the effector sequence is capable of forming a duplex with a sequence set forth in SEQ ID NO: 114; and (ii) an effector complement sequence comprising a sequence which is substantially complementary to the effector sequence.
  • the shmiR encoded by the nucleic acid may comprise an effector sequence set forth in SEQ ID NO:l 15 and an effector complement sequence which is substantially complementary to the sequence set forth in SEQ ID NO: 115 and capable of forming a duplex therewith.
  • the effector complement sequence which is substantially complementary to the sequence set forth in SEQ ID NO: 115 may be the sequence set forth in SEQ ID NO: 115.
  • a suitable loop sequence for VEGFa_shmiR may be selected from those known in the art.
  • an exemplary stemloop sequence is set forth in SEQ ID NO: 67.
  • suitable primary micro RNA (pri-miRNA or pri-R) backbones for use in VEGFa_shmiR may be selected from those known in the art, such as those already described herein.
  • the pri-miRNA backbone may be a pri-miR-30a backbone, in which the 5' flanking sequence is set forth in SEQ ID NO: 68 and the 3 ' flanking sequence is set forth in SEQ ID NO: 69.
  • a nucleic acid encoding the VEGFa_shmiR may comprise DNA sequence encoding the sequence set forth in SEQ ID NO: 68 and DNA sequence encoding the sequence set forth in SEQ ID NO: 69.
  • the nucleic acid encoding VEGFa_shmiR comprises or consists of a DNA sequence set forth in SEQ ID NO: 117 and encodes a shmiR comprising or consisting of the sequence set forth in SEQ ID NO: 116.
  • a plurality of nucleic acids as described herein may comprise two or more nucleic acids encoding shmiRs as described herein, such as two, or three, or four, or five, or six, or seven, or eight, or nine, or ten nucleic acids encoding shmiRs as described herein.
  • the plurality of nucleic acids described herein comprises at least one nucleic acid encoding a shmiR targeting VEGFb as described herein (i.e., VEGFb_shmiR-l to VEGFb_shmiR- 10) and at least one nucleic acid encoding a shmiR targeting PGF (i.e., PGF_shmiR- l to PGF_shmiR- 12).
  • a plurality of nucleic acids of the disclosure may comprise:
  • a nucleic acid comprising a DNA sequence encoding one of VEGFb_shmiR-l, VEGFb_shmiR-4 or VEGFb_shmiR- 10 as described herein;
  • nucleic acid comprising a DNA sequence encoding one of PGF_shmiR-3,
  • the plurality of nucleic acids described herein comprises at least one nucleic acid encoding a shmiR targeting VEGFb as described herein (i.e.,
  • a plurality of nucleic acids of the disclosure may comprise:
  • a nucleic acid comprising a DNA sequence encoding one of VEGFb_shmiR-l, VEGFb_shmiR-4 or VEGFb_shmiR- 10 as described herein;
  • nucleic acid comprising a DNA sequence encoding VEGFa_shmiR as described herein.
  • the plurality of nucleic acids described herein comprises at least one nucleic acid encoding a shmiR targeting PGF (i.e., PGF_shmiR-l to PGF_shmiR-12) and at least one nucleic acid encoding a shmiR targeting VEGFa (i.e., VEGFa_shmiR).
  • a plurality of nucleic acids of the disclosure may comprise:
  • nucleic acid comprising a DNA sequence encoding one of PGF_shmiR-3,
  • nucleic acid comprising a DNA sequence encoding VEGFa_shmiR as described herein.
  • the plurality of nucleic acids described herein comprises at least one nucleic acid encoding a shmiR targeting VEGFb as described herein (i.e.,
  • VEGFb_shmiR-l to VEGFb_shmiR-10 at least one nucleic acid encoding a shmiR targeting PGF (i.e., PGF_shmiR-l to PGF_shmiR-12) and at least one nucleic acid encoding a shmiR targeting VEGFa (i.e., VEGFa_shmiR).
  • a plurality of nucleic acids of the disclosure may comprise:
  • nucleic acid comprising a DNA sequence encoding one of VEGFb_shmiR-l
  • VEGFb_shmiR-4 or VEGFb_shmiR-10 as described herein;
  • nucleic acid comprising a DNA sequence encoding one of PGF_shmiR-3,
  • nucleic acid comprising a DNA sequence encoding VEGFa_shmiR as described herein.
  • the plurality of nucleic acids of the disclosure comprises:
  • a nucleic acid comprising a DNA sequence encoding VEGFa_shmiR.
  • the plurality of nucleic acids of the disclosure comprises:
  • a nucleic acid comprising a DNA sequence encoding VEGFb_shmiR- 1 ;
  • the plurality of nucleic acids of the disclosure comprises:
  • nucleic acid comprising a DNA sequence encoding PGF-shmiR-7.
  • exemplary nucleic acids of the disclosure encoding shmiRs designated VEGFb_shmiR-l, VEGFb_shmiR-4, VEGFb_shmiR-10, PGF_shmiR-3, PGF_shmiR-7, PGF_shmiR-10 and VEGFa_shmiR are described herein and shall be taken to apply mutatis mutandis to each example in which a plurality of nucleic acids of the disclosure is described
  • the plurality of nucleic acids of the disclosure comprises:
  • a nucleic acid comprising a DNA sequence encoding VEGFb_shmiR- 1 comprising an effector sequence consisting of the sequence set forth in SEQ ID NO: 24 and an effector complement sequence consisting of the sequence set forth in SEQ ID NO: 23;
  • nucleic acid comprising a DNA sequence encoding VEGFa_shmiR comprising an effector sequence consisting of the sequence set forth in SEQ ID NO: 115 and an effector complement sequence consisting of the sequence set forth in SEQ ID NO: 114.
  • the plurality of nucleic acids of the disclosure comprises:
  • a nucleic acid comprising a DNA sequence encoding VEGFb_shmiR- 1 comprising an effector sequence consisting of the sequence set forth in SEQ ID NO: 24 and an effector complement sequence consisting of the sequence set forth in SEQ ID NO: 23;
  • nucleic acid comprising a DNA sequence encoding VEGFa_shmiR comprising an effector sequence consisting of the sequence set forth in SEQ ID NO: 115 and an effector complement sequence consisting of the sequence set forth in SEQ ID NO: 114;
  • nucleic acid comprising a DNA sequence encoding a PGF_shmiR-3 comprising an effector sequence consisting of the sequence set forth in SEQ ID NO: 48 and an effector complement sequence consisting of the sequence set forth in SEQ ID NO:47.
  • the plurality of nucleic acids of the disclosure comprises:
  • a nucleic acid comprising a DNA sequence encoding VEGFb_shmiR- 1 comprising an effector sequence consisting of the sequence set forth in SEQ ID NO: 24 and an effector complement sequence consisting of the sequence set forth in SEQ ID NO: 23;
  • nucleic acid comprising a DNA sequence encoding VEGFa_shmiR comprising an effector sequence consisting of the sequence set forth in SEQ ID NO: 115 and an effector complement sequence consisting of the sequence set forth in SEQ ID NO: 114; and (iii) a nucleic acid comprising a DNA sequence encoding a PGF_shmiR-7 comprising an effector sequence consisting of the sequence set forth in SEQ ID NO: 56 and an effector complement sequence consisting of the sequence set forth in SEQ ID NO: 55.
  • the plurality of nucleic acids of the disclosure comprises:
  • nucleic acid comprising a DNA sequence comprising or consisting of the sequence set forth in SEQ ID NO: 92 (encoding a shmiR comprising or consisting of the sequence set forth in SEQ ID NO: 70);
  • nucleic acid comprising a DNA sequence comprising or consisting of the sequence set forth in SEQ ID NO: 117 (encoding a shmiR comprising or consisting of the sequence set forth in SEQ ID NO: 116).
  • the plurality of nucleic acids of the disclosure comprises:
  • nucleic acid comprising a DNA sequence comprising or consisting of the sequence set forth in SEQ ID NO: 92 (encoding a shmiR comprising or consisting of the sequence set forth in SEQ ID NO: 70);
  • nucleic acid comprising a DNA sequence comprising or consisting of the sequence set forth in SEQ ID NO: 117 (encoding a shmiR comprising or consisting of the sequence set forth in SEQ ID NO: 116);
  • nucleic acid comprising a DNA sequence comprising or consisting of the sequence set forth in SEQ ID NO: 104 (encoding a shmiR comprising or consisting of the sequence set forth in SEQ ID NO: 82).
  • the plurality of nucleic acids of the disclosure comprises:
  • nucleic acid comprising a DNA sequence comprising or consisting of the sequence set forth in SEQ ID NO: 92 (encoding a shmiR comprising or consisting of the sequence set forth in SEQ ID NO: 70);
  • nucleic acid comprising a DNA sequence comprising or consisting of the sequence set forth in SEQ ID NO: 117 (encoding a shmiR comprising or consisting of the sequence set forth in SEQ ID NO: 116);
  • nucleic acid comprising a DNA sequence comprising or consisting of the sequence set forth in SEQ ID NO: 108 (encoding a shmiR comprising or consisting of the sequence set forth in SEQ ID NO: 86).
  • two or more of the nucleic acids may form separate parts of the same polynucleotide.
  • two or more of the nucleic acids in the plurality form parts of different
  • the plurality of nucleic acids described herein are provided as multiple components e.g., multiple compositions.
  • each of the nucleic acids of the plurality may be provided separately.
  • at least one of the nucleic acids may be provided separately and two or more of the plurality provided together.
  • the or each nucleic acid in accordance with the present disclosure may comprise, or be in operable linkage with, additional elements e.g., to facilitate transcription of the RNA.
  • the or each nucleic acid may comprise a promoter operably-linked to the sequence encoding a shmiR described herein.
  • Other regulatory elements e.g., transcriptional terminators and initiators, are known in the art and/or described herein.
  • the or each nucleic acid in accordance with the present disclosure may comprise one or more restriction sites e.g., to facilitate cloning of the nucleic acid(s) into cloning or expression vectors.
  • the nucleic acids described herein may include a restriction site upstream and/or downstream of the sequence encoding a shmiR or shRNA of the disclosure. Suitable restriction enzyme recognition sequences will be known to a person of skill in the art.
  • the nucleic acid(s) of the disclosure may include a BamHl restriction site (GGATCC) at the 5' terminus i.e., upstream of the sequence encoding the shmiR, and a Hindlll restriction site (AAGCTT) at the 3 ' terminus i.e., downstream of the sequence encoding the shmiR.
  • GGATCC BamHl restriction site
  • AAGCTT Hindlll restriction site
  • the or each nucleic acid of the disclosure is provided in the form of, or is comprised in, a DNA-directed RNAi (ddRNAi) construct.
  • ddRNAi DNA-directed RNAi
  • the present disclosure provides a ddRNAi construct comprising a nucleic acid as described herein.
  • the present disclosure provides a ddRNAi construct comprising a plurality of nucleic acids described herein. Exemplary nucleic acids encoding shmiRs targeting VEGFb and PGF, provided alone or in combination with a nucleic acid encoding a shmiR targeting VEGFa, are described herein and shall be taken to apply mutatis mutandis to this example of the disclosure.
  • the ddRNAi construct comprises a nucleic acid of the disclosure operably-linked to a promoter.
  • each of the nucleic acids may be operably-linked to a promoter.
  • the nucleic acids in the ddRNAi construct may be operably- linked to the same promoter.
  • the nucleic acids in the ddRNAi construct may be operably-linked to different promoters.
  • a ddRNAi of the disclosure comprises a nucleic acid comprising or consisting of a DNA sequence set forth in SEQ ID NO: 92 and encodes a shmiR
  • the ddRNAi construct may comprise one or more further nucleic acids of the disclosure, such as a nucleic acid comprising a DNA sequence set forth in any one of SEQ ID NOs: 102-113 or 117.
  • the further nucleic acids comprise a DNA sequence selected from the sequences set forth in SEQ ID NOs: 104, 108, 111 and 117.
  • a ddRNAi of the disclosure comprises a nucleic acid comprising or consisting of a DNA sequence set forth in SEQ ID NO: 93 and encodes a shmiR
  • the ddRNAi construct may comprise one or more further nucleic acids of the disclosure, such as a nucleic acid comprising a DNA sequence set forth in any one of SEQ ID NOs: 102-113 or 117.
  • the further nucleic acids comprise a DNA sequence selected from the sequences set forth in SEQ ID NOs: 104, 108, 111 and 117.
  • a ddRNAi of the disclosure comprises a nucleic acid comprising or consisting of a DNA sequence set forth in SEQ ID NO: 94 and encodes a shmiR
  • the ddRNAi construct may comprise one or more further nucleic acids of the disclosure, such as a nucleic acid comprising a DNA sequence set forth in any one of SEQ ID NOs: 102-113 or 117.
  • the further nucleic acids comprise a DNA sequence selected from the sequences set forth in SEQ ID NOs: 104, 108, 111 and 117.
  • a ddRNAi of the disclosure comprises a nucleic acid comprising or consisting of a DNA sequence set forth in SEQ ID NO: 95 and encodes a shmiR
  • the ddRNAi construct may comprise one or more further nucleic acids of the disclosure, such as a nucleic acid comprising a DNA sequence set forth in any one of SEQ ID NOs: 102-113 or 117.
  • the further nucleic acids comprise a DNA sequence selected from the sequences set forth in SEQ ID NOs: 104, 108, 111 and 117.
  • a ddRNAi of the disclosure comprises a nucleic acid comprising or consisting of a DNA sequence set forth in SEQ ID NO: 96 and encodes a shmiR
  • the ddRNAi construct may comprise one or more further nucleic acids of the disclosure, such as a nucleic acid comprising a DNA sequence set forth in any one of SEQ ID NOs: 102-113 or 117.
  • the further nucleic acids comprise a DNA sequence selected from the sequences set forth in SEQ ID NOs: 104, 108, 111 and 117.
  • a ddRNAi of the disclosure comprises a nucleic acid comprising or consisting of a DNA sequence set forth in SEQ ID NO: 97 and encodes a shmiR
  • VEGFb_shmiR-6 comprising or consisting of the sequence set forth in SEQ ID NO: 75.
  • the ddRNAi construct may comprise one or more further nucleic acids of the disclosure, such as a nucleic acid comprising a DNA sequence set forth in any one of SEQ ID NOs:
  • the further nucleic acids comprise a DNA sequence selected from the sequences set forth in SEQ ID NOs: 104, 108, 111 and 117.
  • a ddRNAi of the disclosure comprises a nucleic acid comprising or consisting of a DNA sequence set forth in SEQ ID NO: 98 and encodes a shmiR
  • the ddRNAi construct may comprise one or more further nucleic acids of the disclosure, such as a nucleic acid comprising a DNA sequence set forth in any one of SEQ ID NOs: 102-113 or 117.
  • the further nucleic acids comprise a DNA sequence selected from the sequences set forth in SEQ ID NOs: 104, 108, 111 and 117.
  • a ddRNAi of the disclosure comprises a nucleic acid comprising or consisting of a DNA sequence set forth in SEQ ID NO: 99 and encodes a shmiR
  • the ddRNAi construct may comprise one or more further nucleic acids of the disclosure, such as a nucleic acid comprising a DNA sequence set forth in any one of SEQ ID NOs: 102-113 or 117.
  • the further nucleic acids comprise a DNA sequence selected from the sequences set forth in SEQ ID NOs: 104, 108, 111 and 117.
  • a ddRNAi of the disclosure comprises a nucleic acid comprising or consisting of a DNA sequence set forth in SEQ ID NO: 100 and encodes a shmiR
  • the ddRNAi construct may comprise one or more further nucleic acids of the disclosure, such as a nucleic acid comprising a DNA sequence set forth in any one of SEQ ID NOs: 102-113 or 117.
  • the further nucleic acids comprise a DNA sequence selected from the sequences set forth in SEQ ID NOs: 104, 108, 111 and 117.
  • a ddRNAi of the disclosure comprises a nucleic acid comprising or consisting of a DNA sequence set forth in SEQ ID NO: 101 and encodes a shmiR
  • the ddRNAi construct may comprise one or more further nucleic acids of the disclosure, such as a nucleic acid comprising a DNA sequence set forth in any one of SEQ ID NOs: 102-113 or 117.
  • the further nucleic acids comprise a DNA sequence selected from the sequences set forth in SEQ ID NOs: 104, 108, 111 and 117.
  • a ddRNAi of the disclosure comprises a nucleic acid comprising or consisting of a DNA sequence set forth in SEQ ID NO: 102 and encodes a shmiR
  • the ddRNAi construct may comprise one or more further nucleic acids of the disclosure, such as a nucleic acid comprising a DNA sequence set forth in any one of SEQ ID NOs: 92-101 or 117.
  • the further nucleic acids comprise a DNA sequence selected from the sequences set forth in SEQ ID NOs: 92, 95, 101 and 117.
  • a ddRNAi of the disclosure comprises a nucleic acid comprising or consisting of a DNA sequence set forth in SEQ ID NO: 103 and encodes a shmiR
  • the ddRNAi construct may comprise one or more further nucleic acids of the disclosure, such as a nucleic acid comprising a DNA sequence set forth in any one of SEQ ID NOs: 92-101 or 117.
  • the further nucleic acids comprise a DNA sequence selected from the sequences set forth in SEQ ID NOs: 92, 95, 101 and 117.
  • a ddRNAi of the disclosure comprises a nucleic acid comprising or consisting of a DNA sequence set forth in SEQ ID NO: 104 and encodes a shmiR
  • the ddRNAi construct may comprise one or more further nucleic acids of the disclosure, such as a nucleic acid comprising a DNA sequence set forth in any one of SEQ ID NOs: 92-101 or 117.
  • the further nucleic acids comprise a DNA sequence selected from the sequences set forth in SEQ ID NOs: 92, 95, 101 and 117.
  • a ddRNAi of the disclosure comprises a nucleic acid comprising or consisting of a DNA sequence set forth in SEQ ID NO: 105 and encodes a shmiR
  • the ddRNAi construct may comprise one or more further nucleic acids of the disclosure, such as a nucleic acid comprising a DNA sequence set forth in any one of SEQ ID NOs: 92-101 or 117.
  • the further nucleic acids comprise a DNA sequence selected from the sequences set forth in SEQ ID NOs: 92, 95, 101 and 117.
  • a ddRNAi of the disclosure comprises a nucleic acid comprising or consisting of a DNA sequence set forth in SEQ ID NO: 106 and encodes a shmiR
  • the ddRNAi construct may comprise one or more further nucleic acids of the disclosure, such as a nucleic acid comprising a DNA sequence set forth in any one of SEQ ID NOs: 92-101 or 117.
  • the further nucleic acids comprise a DNA sequence selected from the sequences set forth in SEQ ID NOs: 92, 95, 101 and 117.
  • a ddRNAi of the disclosure comprises a nucleic acid comprising or consisting of a DNA sequence set forth in SEQ ID NO: 107 and encodes a shmiR
  • the ddRNAi construct may comprise one or more further nucleic acids of the disclosure, such as a nucleic acid comprising a DNA sequence set forth in any one of SEQ ID NOs: 92-101 or 117.
  • the further nucleic acids comprise a DNA sequence selected from the sequences set forth in SEQ ID NOs: 92, 95, 101 and 117.
  • a ddRNAi of the disclosure comprises a nucleic acid comprising or consisting of a DNA sequence set forth in SEQ ID NO: 108 and encodes a shmiR
  • the ddRNAi construct may comprise one or more further nucleic acids of the disclosure, such as a nucleic acid comprising a DNA sequence set forth in any one of SEQ ID NOs: 92-101 or 117.
  • the further nucleic acids comprise a DNA sequence selected from the sequences set forth in SEQ ID NOs: 92, 95, 101 and 117.
  • a ddRNAi of the disclosure comprises a nucleic acid comprising or consisting of a DNA sequence set forth in SEQ ID NO: 109 and encodes a shmiR
  • the ddRNAi construct may comprise one or more further nucleic acids of the disclosure, such as a nucleic acid comprising a DNA sequence set forth in any one of SEQ ID NOs: 92-101 or 117.
  • the further nucleic acids comprise a DNA sequence selected from the sequences set forth in SEQ ID NOs: 92, 95, 101 and 117.
  • a ddRNAi of the disclosure comprises a nucleic acid comprising or consisting of a DNA sequence set forth in SEQ ID NO: 110 and encodes a shmiR
  • the ddRNAi construct may comprise one or more further nucleic acids of the disclosure, such as a nucleic acid comprising a DNA sequence set forth in any one of SEQ ID NOs: 92-101 or 117.
  • the further nucleic acids comprise a DNA sequence selected from the sequences set forth in SEQ ID NOs: 92, 95, 101 and 117.
  • a ddRNAi of the disclosure comprises a nucleic acid comprising or consisting of a DNA sequence set forth in SEQ ID NO: 111 and encodes a shmiR
  • the ddRNAi construct may comprise one or more further nucleic acids of the disclosure, such as a nucleic acid comprising a DNA sequence set forth in any one of SEQ ID NOs: 92-101 or 117.
  • the further nucleic acids comprise a DNA sequence selected from the sequences set forth in SEQ ID NOs: 92, 95, 101 and 117.
  • a ddRNAi of the disclosure comprises a nucleic acid comprising or consisting of a DNA sequence set forth in SEQ ID NO: 112 and encodes a shmiR
  • the ddRNAi construct may comprise one or more further nucleic acids of the disclosure, such as a nucleic acid comprising a DNA sequence set forth in any one of SEQ ID NOs: 92-101 or 117.
  • the further nucleic acids comprise a DNA sequence selected from the sequences set forth in SEQ ID NOs: 92, 95, 101 and 117.
  • a ddRNAi of the disclosure comprises a nucleic acid comprising or consisting of a DNA sequence set forth in SEQ ID NO: 113 and encodes a shmiR
  • the ddRNAi construct may comprise one or more further nucleic acids of the disclosure, such as a nucleic acid comprising a DNA sequence set forth in any one of SEQ ID NOs: 92-101 or 117.
  • the further nucleic acids comprise a DNA sequence selected from the sequences set forth in SEQ ID NOs: 92, 95, 101 and 117.
  • An exemplary ddRNAi construct comprising a two nucleic acids of the disclosure comprises:
  • the present disclosure also provides a ddRNAi construct comprising at least three nucleic acids described herein, each of which encode shmiRs targeting different genes.
  • the disclosure provides a ddRNAi construct comprising:
  • (c) a nucleic acid comprising or consisting of the sequence set forth in SEQ ID NO: 104 (encoding PGF_shmiR-3 comprising or consisting of the sequence set forth in SEQ ID NO:82).
  • (a) to (c) are configured in a 5' to 3' direction relative to one another within the ddRNAi construct.
  • the disclosure provides a ddRNAi construct comprising:
  • nucleic acid comprising or consisting of the sequence set forth in SEQ ID NO: 92 (encoding VEGFb_shmiR- 1 comprising or consisting of the sequence set forth in SEQ ID NO:70);
  • SEQ ID NO: 116 SEQ ID NO: 116
  • a nucleic acid comprising or consisting of the sequence set forth in SEQ ID NO: 108 (encoding PGF_shmiR-7 comprising or consisting of the sequence set forth in SEQ ID NO:86).
  • (a) to (c) are configured in a 5' to 3' direction relative to one another within the ddRNAi construct.
  • the or each nucleic acid comprised therein may be operably-linked to a promoter.
  • the ddRNAi construct as described herein may comprise a single promoter which is operably-linked to the or each nucleic acid comprised therein e.g., to drive expression of one or more shmiRs from the ddRNAi construct.
  • each shmiR-encoding nucleic acid comprised in the ddRNAi construct is operably-linked to a separate promoter.
  • the promoters can be the same or different.
  • the construct may comprise multiple copies of the same promoter with each copy operably-linked to a different nucleic acid of the disclosure.
  • each promoter operably-linked to a shmiR-encoding nucleic acid of the disclosure is different.
  • each nucleic acids encoding one of respective shmiRs is operably-linked to a different promoter.
  • a ddRNAi construct encodes three or more shmiRs
  • two (or more) of the nucleic acids encoding the shmiRs are linked to the same promoter and one (or more) of the nucleic acids encoding the other shmiR(s) is/are linked to a different promoter(s).
  • the or each promoter is a constitutive promoter.
  • constitutive when made in reference to a promoter means that the promoter is capable of directing transcription of an operably-linked nucleic acid sequence in the absence of a specific stimulus (e.g., heat shock, chemicals, light, etc.). Typically, constitutive promoters are capable of directing expression of a coding sequence in substantially any cell and any tissue.
  • the promoters used to transcribe shmiRs from the nucleic acid(s) of the disclosure include promoters for ubiquitin, CMV, ⁇ -actin, histone H4, EF-la or pgk genes controlled by RNA polymerase II, or promoter elements controlled by RNA polymerase I.
  • a Pol II promoter such as CMV, SV40, Ul, ⁇ -actin or a hybrid Pol II promoter is employed.
  • a promoter controlled by RNA polymerase III is used, such as a U6 promoter (U6-1, U6-8, U6-9), HI promoter, 7SL promoter, a human Y promoter (hYl, hY3, hY4 (see Maraia, et al, Nucleic Acids Res 22(15):3045-52(1994)) and hY5 (see Maraia, et al, Nucleic Acids Res 24(18):3552-59(1994)), a human MRP-7-2 promoter, an Adenovirus VA1 promoter, a human tRNA promoter, or a 5s ribosomal RNA promoter.
  • U6 promoter U6-1, U6-8, U6-9
  • HI promoter 7SL promoter
  • a human Y promoter hYl, hY3, hY4 (see Maraia, et al, Nucleic Acids Res 22(15):3045-52(
  • Suitable promoters for use in a ddRNAi construct of the disclosure are described in US Patent No. 8,008,468 and US Patent No. 8, 129,510.
  • the promoter is a RNA pol III promoter.
  • the promoter is a U6 promoter (e.g., a U6-1, U6-8 or U6-9 promoter).
  • the promoter is a HI promoter.
  • each of the nucleic acids in the ddRNAi construct is operably-linked to a U6 promoter e.g., each operably-linked to a separate U6 promoter.
  • the ddRNAi construct comprises a U6- 1 promoter.
  • the ddRNAi construct comprises a U6-8 promoter.
  • the ddRNAi construct comprises a U6-9 promoter.
  • promoters of variable strength are employed.
  • use of two or more strong promoters may tax the cell, by, e.g., depleting the pool of available nucleotides or other cellular components needed for transcription.
  • use of several strong promoters may cause a toxic level of expression of RNAi agents e.g., shmiRs, in the cell.
  • RNAi agents e.g., shmiRs
  • one or more of the promoters in the multiple -promoter ddRNAi construct is weaker than other promoters in the construct, or all promoters in the construct may express the shmiRs at less than a maximum rate.
  • Promoters may also be modified using various molecular techniques, or otherwise, e.g., through modification of various regulatory elements, to attain weaker levels or stronger levels of transcription.
  • One means of achieving reduced transcription is to modify sequence elements within promoters known to control promoter activity.
  • the Proximal Sequence Element (PSE) is known to affect the activity of human U6 promoters (see Domitrovich, et al., Nucleic Acids Res 31: 2344-2352 (2003).
  • tissue specific refers to a promoter that is capable of directing selective transcription of a nucleic acid of interest to a specific type of tissue (e.g., ocular tissues) in the relative absence of expression of the same nucleotide sequence of interest in a different type of tissue (e.g., liver).
  • tissue-specific as applied to a promoter refers to a promoter which is capable of directing selective
  • a ddRNAi construct of the disclosure may additionally comprise one or more enhancers to increase expression of the shmiRs encoded by the nucleic acids described herein. Enhancers appropriate for use in examples of the present disclosure will be known to those skilled in the art.
  • a ddRNAi construct of the disclosure may comprise a
  • the transcriptional terminator linked to a nucleic acid encoding a shmiR of the disclosure can be the same or different.
  • the terminator may be a contiguous stretch of 4 or more or 5 or more or 6 or more T residues.
  • the terminators can be different and are matched to the promoter from the gene from which the terminator is derived.
  • Such terminators include the SV40 poly A, the AdV VAl gene, the 5S ribosomal RNA gene, and the terminators for human t-RNAs.
  • promoters and terminators may be mixed and matched, as is commonly done with RNA pol II promoters and terminators.
  • the promoter and terminator combinations used for each nucleic acid in a ddRNAi construct comprising a plurality of nucleic acids is different to decrease the likelihood of DNA recombination events between components.
  • One exemplary ddRNAi construct of the disclosure comprises a nucleic acid comprising or consisting of the sequence set forth in SEQ ID NO:92 (encoding
  • VEGFb_shmiR-l comprising or consisting of the sequence set forth in SEQ ID NO:70) operably-linked to a U6 promoter e.g., a U6-9 promoter.
  • a U6 promoter e.g., a U6-9 promoter.
  • Another exemplary ddRNAi construct of the disclosure comprises a nucleic acid comprising or consisting of the sequence set forth in SEQ ID NO: 104 (encoding
  • PGF_shmiR-3 comprising or consisting of the sequence set forth in SEQ ID NO: 82) operably-linked to a U6 promoter e.g., a U6-8 promoter.
  • Another exemplary ddRNAi construct of the disclosure comprises a nucleic acid comprising or consisting of the sequence set forth in SEQ ID NO: 108 (encoding
  • PGF_shmiR-7 comprising or consisting of the sequence set forth in SEQ ID NO: 86) operably-linked to a U6 promoter e.g., a U6-8 promoter.
  • the ddRNAi construct of the disclosure comprises a plurality of nucleic acids described herein
  • the ddRNAi construct may comprise:
  • a nucleic acid comprising or consisting of the sequence set forth in SEQ ID NO: 92 (encoding VEGFb_shmiR- 1 comprising or consisting of the sequence set forth in SEQ ID NO:70) operably-linked to a U6 promoter e.g., a U6-9 promoter; and
  • SEQ ID NO: 116 operably-linked to a U6 promoter e.g., a U6-1 promoter.
  • the ddRNAi construct of the disclosure comprises a plurality of nucleic acids described herein
  • the ddRNAi construct may comprise:
  • SEQ ID NO:70 operably-linked to a U6 promoter e.g., a U6-9 promoter;
  • nucleic acid comprising or consisting of the sequence set forth in SEQ ID NO: 104 (encoding PGF_shmiR-3 comprising or consisting of the sequence set forth in SEQ ID NO:82) operably-linked to a U6 promoter e.g., a U6-8 promoter.
  • the ddRNAi construct of the disclosure comprises a plurality of nucleic acids described herein
  • the ddRNAi construct may comprise:
  • a nucleic acid comprising or consisting of the sequence set forth in SEQ ID NO: 92 (encoding VEGFb_shmiR- 1 comprising or consisting of the sequence set forth in SEQ ID NO:70) operably-linked to a U6 promoter e.g., a U6-9 promoter; and
  • the ddRNAi construct of the disclosure comprises at least three nucleic acids described herein, the ddRNAi construct may comprise:
  • a nucleic acid comprising or consisting of the sequence set forth in SEQ ID NO: 92 (encoding VEGFb_shmiR- 1 comprising or consisting of the sequence set forth in SEQ ID NO:70) operably-linked to a U6 promoter e.g., a U6-9 promoter;
  • nucleic acid comprising or consisting of the sequence set forth in SEQ ID NO: 117 (encoding VEGFa_shmiR comprising or consisting of the sequence set forth in SEQ ID NO: 116) operably-linked to a U6 promoter e.g., a U6-1 promoter;
  • nucleic acid comprising or consisting of the sequence set forth in SEQ ID NO: 104 (encoding PGF_shmiR-3 comprising or consisting of the sequence set forth in SEQ ID NO:82) operably-linked to a U6 promoter e.g., a U6-8 promoter.
  • the ddRNAi construct of the disclosure comprises at least three nucleic acids described herein
  • the ddRNAi construct may comprise:
  • SEQ ID NO:70 operably-linked to a U6 promoter e.g., a U6-9 promoter;
  • nucleic acid comprising or consisting of the sequence set forth in SEQ ID NO: 117 (encoding VEGFa_shmiR comprising or consisting of the sequence set forth in SEQ ID NO: 116) operably-linked to a U6 promoter e.g., a U6-1 promoter;
  • An exemplary ddRNAi construct of the disclosure comprises, in a 5' to 3' direction:
  • Another exemplary ddRNAi construct of the disclosure comprises, in a 5' to 3' direction: (a) U6-9 promoter upstream of a nucleic acid comprising or consisting of the sequence set forth in SEQ ID NO:92:
  • the present disclosure also provides a plurality of ddRNAi constructs comprising two or more ddRNAi constructs, each comprising nucleic acid encoding a shmiR of the disclosure operably-linked to a suitable promoter as described herein.
  • the plurality of ddRNAi constructs comprises:
  • a ddRNAi construct which comprises a nucleic acid comprising or consisting of the sequence set forth in SEQ ID NO:92 (encoding VEGFb_shmiR- 1 comprising or consisting of the sequence set forth in SEQ ID NO:70) operably-linked to a U6 promoter e.g., a U6-9 promoter; and
  • a ddRNAi construct which comprises a nucleic acid comprising or consisting of the sequence set forth in SEQ ID NO: 117 (encoding VEGFa_shmiR comprising or consisting of the sequence set forth in SEQ ID NO: 116) operably-linked to a U6 promoter e.g., a U6-1 promoter.
  • the plurality of ddRNAi constructs comprises:
  • a ddRNAi construct which comprises a nucleic acid comprising or consisting of the sequence set forth in SEQ ID NO:92 (encoding VEGFb_shmiR- 1 comprising or consisting of the sequence set forth in SEQ ID NO:70) operably-linked to a U6 promoter e.g., a U6-9 promoter; and
  • a ddRNAi construct which comprises a nucleic acid comprising or consisting of the sequence set forth in SEQ ID NO: 104 (encoding PGF_shmiR-3 comprising or consisting of the sequence set forth in SEQ ID NO:82) operably-linked to a U6 promoter e.g., a U6-8 promoter.
  • the plurality of ddRNAi constructs comprises:
  • a ddRNAi construct which comprises a nucleic acid comprising or consisting of the sequence set forth in SEQ ID NO:92 (encoding VEGFb_shmiR- 1 comprising or consisting of the sequence set forth in SEQ ID NO:70) operably-linked to a U6 promoter e.g., a U6-9 promoter; and
  • a ddRNAi construct which comprises a nucleic acid comprising or consisting of the sequence set forth in SEQ ID NO: 108 (encoding PGF_shmiR-7 comprising or consisting of the sequence set forth in SEQ ID NO:86) operably-linked to a U6 promoter e.g., a U6-8 promoter.
  • the plurality of ddRNAi constructs comprises three ddRNAi constructs
  • the plurality of ddRNAi constructs comprises:
  • a ddRNAi construct which comprises a nucleic acid comprising or consisting of the sequence set forth in SEQ ID NO:92 (encoding VEGFb_shmiR- 1 comprising or consisting of the sequence set forth in SEQ ID NO:70) operably-linked to a U6 promoter e.g., a U6-9 promoter;
  • a ddRNAi construct which comprises a nucleic acid comprising or consisting of the sequence set forth in SEQ ID NO: 117 (encoding VEGFa_shmiR comprising or consisting of the sequence set forth in SEQ ID NO: 116) operably-linked to a U6 promoter e.g., a U6-1 promoter; and
  • a ddRNAi construct which comprises a nucleic acid comprising or consisting of the sequence set forth in SEQ ID NO: 104 (encoding PGF_shmiR-3 comprising or consisting of the sequence set forth in SEQ ID NO:82) operably-linked to a U6 promoter e.g., a U6-8 promoter.
  • the plurality of ddRNAi constructs comprises three ddRNAi constructs
  • the plurality of ddRNAi constructs comprises:
  • a ddRNAi construct which comprises a nucleic acid comprising or consisting of the sequence set forth in SEQ ID NO:92 (encoding VEGFb_shmiR- 1 comprising or consisting of the sequence set forth in SEQ ID NO:70) operably-linked to a U6 promoter e.g., a U6-9 promoter;
  • a ddRNAi construct which comprises a nucleic acid comprising or consisting of the sequence set forth in SEQ ID NO: 117 (encoding VEGFa_shmiR comprising or consisting of the sequence set forth in SEQ ID NO: 116) operably-linked to a U6 promoter e.g., a U6-1 promoter; and
  • a ddRNAi construct which comprises a nucleic acid comprising or consisting of the sequence set forth in SEQ ID NO: 108 (encoding PGF_shmiR-7 comprising or consisting of the sequence set forth in SEQ ID NO:86) operably-linked to a U6 promoter e.g., a U6-8 promoter.
  • the or each ddRNAi construct can comprise one or more multiple cloning sites and/or unique restriction sites that are located strategically, such that the promoter, nucleic acid encoding the shmiR and/or other regulator elements are easily removed or replaced.
  • the or each ddRNAi construct can be assembled from smaller oligonucleotide components using strategically located restriction sites and/or complementary sticky ends.
  • the base vector for one approach according to the present disclosure comprises plasmids with a multilinker in which all sites are unique (though this is not an absolute requirement). Sequentially, each promoter is inserted between its designated unique sites resulting in a base cassette with one or more promoters, all of which can have variable orientation.
  • Sequentially, again, annealed primer pairs are inserted into the unique sites downstream of each of the individual promoters, resulting in a single-, double- or multiple-expression cassette construct.
  • the insert can be moved into, e.g. an AdV backbone or an AAV backbone using two unique restriction enzyme sites (the same or different ones) that flank the single-, double- or multiple-expression cassette insert.
  • the construct can be accomplished using any suitable genetic engineering techniques known in the art, including without limitation, the standard techniques of PCR, oligonucleotide synthesis, restriction endonuclease digestion, ligation, transformation, plasmid purification, and DNA sequencing.
  • the construct comprises, for example, sequences necessary to package the ddRNAi construct into viral particles and/or sequences that allow integration of the ddRNAi construct into the target cell genome.
  • the or each viral construct additionally contains genes that allow for replication and propagation of virus, however such genes will be supplied in trans.
  • the or each viral construct cam contain genes or genetic sequences from the genome of any known organism incorporated in native form or modified.
  • a viral construct may comprise sequences useful for replication of the construct in bacteria.
  • the or each construct also may contain additional genetic elements.
  • additional genetic elements may include a reporter gene, such as one or more genes for a fluorescent marker protein such as GFP or RFP; an easily assayed enzyme such as beta-galactosidase, luciferase, beta-glucuronidase, chloramphenical acetyl transferase or secreted embryonic alkaline phosphatase; or proteins for which immunoassays are readily available such as hormones or cytokines.
  • genes that may find use in embodiments of the present disclosure include those coding for proteins which confer a selective growth advantage on cells such as adenosine deaminase, aminoglycodic phosphotransferase, dihydrofolate reductase, hygromycin-B-phosphotransferase, drug resistance, or those genes coding for proteins that provide a biosynthetic capability missing from an auxotroph.
  • a reporter gene is included along with the or each construct, an internal ribosomal entry site (IRES) sequence can be included.
  • the additional genetic elements are operably linked with and controlled by an independent promoter/enhancer.
  • a suitable origin of replication for propagation of the construct in bacteria may be employed. The sequence of the origin of replication generally is separated from the ddRNAi construct and other genetic sequences. Such origins of replication are known in the art and include the pUC, ColEl, 2-micron or SV40 origins of replication.
  • a ddRNAi construct of the disclosure is included within an expression vector.
  • each ddRNAi construct may be included within its own expression vector.
  • the or each expression vector is a plasmid, e.g., as is known in the art.
  • a suitable plasmid expression vector is a Psh vector e.g., with a U6 promoter and proximal sequence element 7 (PSE7).
  • the expression vector is mini-circle DNA.
  • Mini-circle DNA is described in U.S. Patent Publication No. 2004/0214329.
  • Mini-circle DNA are useful for persistently high levels of nucleic acid transcription.
  • the circular vectors are characterized by being devoid of expression-silencing bacterial sequences.
  • mini-circle vectors differ from bacterial plasmid vectors in that they lack an origin of replication, and lack drug selection markers commonly found in bacterial plasmids, e.g. ⁇ -lactamase, tet, and the like. Consequently, minicircle DNA becomes smaller in size, allowing more efficient delivery.
  • the expression vector is a viral vector.
  • a viral vector based on any appropriate virus may be used to deliver a nucleic acid or ddRNAi construct of the disclosure.
  • hybrid viral systems may be of use. The choice of viral delivery system will depend on various parameters, such as the tissue targeted for delivery, transduction efficiency of the system, pathogenicity, immunological and toxicity concerns, and the like.
  • Commonly used classes of viral systems used in gene therapy can be categorized into two groups according to whether their genomes integrate into host cellular chromatin (oncoretroviruses and lentiviruses) or persist in the cell nucleus predominantly as extrachromosomal episomes (adeno-associated virus, adenoviruses and herpesviruses).
  • a viral vector of the disclosure integrates into a host cell's chromatin.
  • a viral vector of the disclosure persists in a host cell's nucleus as an extrachomosomal episome.
  • a viral vector is an adenoviral (AdV) vector.
  • Adenoviruses are medium-sized double-stranded, non-enveloped DNA viruses with linear genomes that is between 26-48 Kbp. Adenoviruses gain entry to a target cell by receptor-mediated binding and internalization, penetrating the nucleus in both non-dividing and dividing cells.
  • Adenoviruses are heavily reliant on the host cell for survival and replication and are able to replicate in the nucleus of vertebrate cells using the host's replication machinery.
  • a viral vector is from the Parvoviridae family.
  • the Parvoviridae is a family of small single-stranded, non-enveloped DNA viruses with genomes approximately 5000 nucleotides long. Included among the family members is adeno-associated virus (AAV).
  • AAV adeno-associated virus
  • a viral vector of the disclosure is an AAV.
  • AAV is a dependent parvovirus that generally requires co-infection with another virus (typically an adenovirus or herpesvirus) to initiate and sustain a productive infectious cycle. In the absence of such a helper virus, AAV is still competent to infect or transduce a target cell by receptor-mediated binding and internalization, penetrating the nucleus in both non-dividing and dividing cells. Because progeny virus is not produced from AAV infection in the absence of helper virus, the extent of transduction is restricted only to the initial cells that are infected with the virus. It is this feature which makes AAV a desirable vector for the
  • AAV appears to lack human pathogenicity and toxicity (Kay, et ah, Nature. 424: 251 (2003)). Since the genome normally encodes only two genes it is not surprising that, as a delivery vehicle, AAV is limited by a packaging capacity of 4.5 single stranded kilobases (kb). However, although this size restriction may limit the genes that can be delivered for replacement gene therapies, it does not adversely affect the packaging and expression of shorter sequences such as shmiRs and shRNAs.
  • Retroviruses comprise single- stranded RNA animal viruses that are characterized by two unique features. First, the genome of a retrovirus is diploid, consisting of two copies of the RNA. Second, this RNA is transcribed by the virion-associated enzyme reverse transcriptase into double- stranded DNA. This double- stranded DNA or provirus can then integrate into the host genome and be passed from parent cell to progeny cells as a stably-integrated component of the host genome.
  • a viral vector is a lentivirus.
  • Lentivirus vectors are often pseudotyped with vesicular steatites virus glycoprotein (VSV-G), and have been derived from the human immunodeficiency virus (HIV); visan-maedi, which causes encephalitis (visna) or pneumonia in sheep; equine infectious anemia virus (EIAV), which causes autoimmune hemolytic anemia and encephalopathy in horses; feline immunodeficiency virus (FIV), which causes immune deficiency in cats; bovine immunodeficiency virus (BIV) which causes lymphadenopathy and lymphocytosis in cattle; and simian immunodeficiency virus (SIV), which causes immune deficiency and encephalopathy in non-human primates.
  • VSV-G vesicular steatites virus glycoprotein
  • Vectors that are based on HIV generally retain ⁇ 5% of the parental genome, and ⁇ 25% of the genome is incorporated into packaging constructs, which minimizes the possibility of the generation of reverting replication-competent HIV.
  • Biosafety has been further increased by the development of self-inactivating vectors that contain deletions of the regulatory elements in the downstream long-terminal-repeat sequence, eliminating transcription of the packaging signal that is required for vector mobilization.
  • One of the main advantages to the use of lentiviral vectors is that gene transfer is persistent in most tissues or cell types, even following cell division of the transduced cell.
  • a lentiviral-based construct used to express shmiRs and/or shRNAs from the nucleic acids and ddRNAi constructs of the disclosure comprises sequences from the 5' and 3' long terminal repeats (LTRs) of a lentivirus.
  • the viral construct comprises an inactivated or self-inactivating 3' LTR from a lentivirus.
  • the 3' LTR may be made self- inactivating by any method known in the art.
  • the U3 element of the 3' LTR contains a deletion of its enhancer sequence, e.g., the TATA box, Spl and NF-kappa B sites.
  • the provirus that is integrated into the host genome will comprise an inactivated 5' LTR.
  • the LTR sequences may be LTR sequences from any lentivirus from any species.
  • the lentiviral-based construct also may incorporate sequences for MMLV or MSCV, RSV or mammalian genes.
  • the U3 sequence from the lentiviral 5' LTR may be replaced with a promoter sequence in the viral construct. This may increase the titer of virus recovered from the packaging cell line. An enhancer sequence may also be included.
  • viral or non-viral systems known to those skilled in the art may be used to deliver the ddRNAi or nucleic acid of the present invention to cells of interest, including but not limited to gene-deleted adenovirus-transposon vectors (see Yant, et al., Nature Biotech. 20:999-1004 (2002)); systems derived from Sindbis virus or Semliki forest virus (see Perri, et al, J. Virol. 74(20):9802-07 (2002)); systems derived from Newcastle disease virus or Sendai virus.
  • Exemplary cell culture -based methods which are useful for determining the ability of nucleic acids, ddRNAi constructs, expression constructs or compositions of the disclosure to reduce or inhibit expression of AMD-associated genes are described in Examples 2-8 herein.
  • rodent, pig and non-human primate models established by laser or direct mechanical/surgical injury to the RPE/Bruch's membrane complex, alteration of the RPE and surrounding environment by external interventions (such as exogenous compounds injected in the subretinal space), VEGF-eluting scleral pellets, or internally (such as genetic knock-out models).
  • rodent models are reviewed in Pennesi et al., (2012) Mol. Aspects Med., 33(4): 487- 509 and Grossniklaus et al., (2010) Prog. Retin.
  • one or more of the nucleic acids or ddRNAi constructs or expression vectors of the disclosure is/are provided in a composition with a carrier.
  • the carrier is a lipid-based carrier, cationic lipid, or liposome nucleic acid complex, a liposome, a micelle, a virosome, a lipid nanoparticle or a mixture thereof.
  • the carrier is a polymer-based carrier such as a cationic polymer- nucleic acid complex.
  • the carrier is a cyclodextrin -based carrier such as a cyclodextrin polymer- nucleic acid complex.
  • the carrier is a protein-based carrier such as a cationic peptide- nucleic acid complex.
  • the carrier is a lipid nanoparticle.
  • Exemplary nanoparticles are described, for example, in US7514099.
  • one or more of the nucleic acids or ddRNAi constructs or expression vectors of the disclosure is/are formulated with a lipid nanoparticle composition comprising a cationic lipid/Cholesterol/PEG-C-DMA/DSPC (e.g., in a 40/48/2/10 ratio), a cationic lipid/Cholesterol/PEG-DMG/DSPC (e.g., in a 40/48/2/10 ratio), or a cationic lipid/Cholesterol/PEG-DMG (e.g., in a 60/38/2 ratio).
  • the cationic lipid is Octyl CL in DMA, DL in DMA, L-278, DLinKC2DMA, or MC3.
  • one or more of the nucleic acids or ddRNAi constructs or expression vectors of the disclosure is/are formulated with any of the cationic lipid formulations described in WO 2010/021865; WO 2010/080724; WO 2010/042877; WO 2010/105209 or WO 2011/022460.
  • one or more of the nucleic acids or ddRNAi constructs or expression vectors of the disclosure is/are conjugated to or complexed with another compound, e.g., to facilitate delivery of the RNA or ddRNAi or expression construct.
  • another compound e.g., to facilitate delivery of the RNA or ddRNAi or expression construct.
  • 2004/0162260 e.g., CDM-LBA, CDM-Pip-LBA, CDM-PEG, CDM-NAG, etc.
  • polyethylene glycol is covalently attached to a nucleic acid or ddRNAi construct or expression construct of the disclosure.
  • the attached PEG can be any molecular weight, e.g.,. from about 100 to about 50,000 daltons (Da).
  • one or more of the nucleic acids or ddRNAi constructs or expression vectors of the disclosure is/are formulated with a carrier comprising surface- modified liposomes containing poly(ethylene glycol) lipids (PEG-modified, or long- circulating liposomes or stealth liposomes), such as is disclosed in for example, WO 96/10391; WO 96/10390; or WO 96/10392.
  • a carrier comprising surface- modified liposomes containing poly(ethylene glycol) lipids (PEG-modified, or long- circulating liposomes or stealth liposomes), such as is disclosed in for example, WO 96/10391; WO 96/10390; or WO 96/10392.
  • the nucleic acids or ddRNAi constructs or expression constructs of the disclosure can also be formulated or complexed with polyethyleneimine or a derivative thereof, such as polyethyleneimine -polyethyleneglycol-N-acetylgalactosamine (PEI-PEG- GAL) or polyethyleneimine -polyethyleneglycol-tri-N-acetylgalactosamine (PEI-PEG- triGAL) derivatives.
  • PEI-PEG- GAL polyethyleneimine -polyethyleneglycol-N-acetylgalactosamine
  • PEI-PEG- triGAL polyethyleneimine -polyethyleneglycol-tri-N-acetylgalactosamine
  • one or more of the nucleic acids or ddRNAi constructs or expression vectors of the disclosure is/are complexed with membrane disruptive agents such as those described in U.S. Patent Application Publication No. 2001/0007666.
  • compositions and methods of treatment are provided.
  • One or more nucleic acids, ddRNAi constructs or expression vectors of the disclosure may be formulated in a pharmaceutical composition for preventing or treating a disease or disorder of the eye characterised by undesired neovascularization.
  • disease or disorder of the eye characterized by undesired neovascularization refers to any disease or disorder in which neovascularization causes or contributes to damage to the eye or a particular structure of the eye (e.g., retina, macula, rods, cones, retinal pigment epithelium, Bruch's membrane, etc.) or causes or contributes to impairment of vision from the eye.
  • Diseases and disorders of the eye which are contemplated by this term include, but are not limited to, wet AMD, diabetic retinopathy, Diabetic Macular Edema (DME), corneal neovascularization, choroidal neovascularization, cyclitis, Hippel-Lindau Disease, retinopathy of prematurity, pterygium, histoplasmosis, iris neovascularization, macular edema, glaucoma-associated neovascularization, Purtscher's retinopathy, Retinal Vein Occlusion (RVO), and the like.
  • dry AMD is not primarily characterized by neovascularization
  • the fact that patients who develop the wet form of AMD are believed to have had the dry form of AMD first supports the conclusion that the treatments described herein will be beneficial in the treatment of dry AMD e.g., to arrest or slow its progress to wet AMD, and that dry AMD may be included in this disease category.
  • ocular diseases in which cellular degeneration has been implicated and for which the composition of the disclosure may be useful include retinal detachment, chorioretinal degenerations, retinal degenerations, photoreceptor degenerations, RPE degenerations, mucopolysaccharidoses, rod-cone dystrophies, cone-rod dystrophies and cone degenerations, particularly when such diseases or conditions are associated with a disease or disorder of the eye characterized by undesired neovascularization as described herein.
  • the disclosure provides a method of treating AMD in a subject, said method comprising administering to the subject a therapeutically effective amount of a nucleic acid, plurality of nucleic acids, a ddRNAi construct, a plurality of ddRNAi constructs, an expression vector, a plurality of expression vectors or a composition as described herein.
  • the AMD is wet AMD.
  • the AMD is dry AMD.
  • treatment may comprise arresting or slowing progression of dry AMD to wet AMD.
  • the disclosure provides a method of treating diabetic retinopathy in a subject, said method comprising administering to the subject a therapeutically effective amount of a nucleic acid, plurality of nucleic acids, a ddRNAi construct, a plurality of ddRNAi constructs, an expression vector, a plurality of expression vectors or a composition as described herein.
  • the disclosure provides a method of treating Diabetic Macular Edema
  • DME in a subject, said method comprising administering to the subject a therapeutically effective amount of a nucleic acid, plurality of nucleic acids, a ddRNAi construct, a plurality of ddRNAi constructs, an expression vector, a plurality of expression vectors or a composition as described herein.
  • the disclosure provides a method of reducing or inhibiting corneal neovascularization in a subject, said method comprising administering to the subject a therapeutically effective amount of a nucleic acid, plurality of nucleic acids, a ddRNAi construct, a plurality of ddRNAi constructs, an expression vector, a plurality of expression vectors or a composition as described herein.
  • the disclosure provides a method of reducing or inhibiting choroidal neovascularisation (CNV) in a subject, said method comprising administering to the subject a therapeutically effective amount of a nucleic acid, plurality of nucleic acids, a ddRNAi construct, a plurality of ddRNAi constructs, an expression vector, a plurality of expression vectors or a composition as described herein.
  • CNV choroidal neovascularisation
  • the disclosure provides a method of treating cyclitis in a subject, said method comprising administering to the subject a therapeutically effective amount of a nucleic acid, plurality of nucleic acids, a ddRNAi construct, a plurality of ddRNAi constructs, an expression vector, a plurality of expression vectors or a composition as described herein.
  • the disclosure provides a method of treating Hippel-Lindau Disease in a subject, said method comprising administering to the subject a therapeutically effective amount of a nucleic acid, plurality of nucleic acids, a ddRNAi construct, a plurality of ddRNAi constructs, an expression vector, a plurality of expression vectors or a composition as described herein.
  • the disclosure provides a method of treating retinopathy of prematurity in a subject, said method comprising administering to the subject a
  • nucleic acid a nucleic acid, plurality of nucleic acids, a ddRNAi construct, a plurality of ddRNAi constructs, an expression vector, a plurality of expression vectors or a composition as described herein.
  • the disclosure provides a method of treating pterygium in a subject, said method comprising administering to the subject a therapeutically effective amount of a nucleic acid, plurality of nucleic acids, a ddRNAi construct, a plurality of ddRNAi constructs, an expression vector, a plurality of expression vectors or a composition as described herein.
  • the disclosure provides a method of treating histoplasmosis in a subject, said method comprising administering to the subject a therapeutically effective amount of a nucleic acid, plurality of nucleic acids, a ddRNAi construct, a plurality of ddRNAi constructs, an expression vector, a plurality of expression vectors or a composition as described herein.
  • the disclosure provides a method of reducing or inhibiting iris neovascularization in a subject, said method comprising administering to the subject a therapeutically effective amount of a nucleic acid, plurality of nucleic acids, a ddRNAi construct, a plurality of ddRNAi constructs, an expression vector, a plurality of expression vectors or a composition as described herein.
  • the disclosure provides a method of treating macular edema in a subject, said method comprising administering to the subject a therapeutically effective amount of a nucleic acid, plurality of nucleic acids, a ddRNAi construct, a plurality of ddRNAi constructs, an expression vector, a plurality of expression vectors or a composition as described herein.
  • the disclosure provides a method of treating glaucoma-associated neovascularization in a subject, said method comprising administering to the subject a therapeutically effective amount of a nucleic acid, plurality of nucleic acids, a ddRNAi construct, a plurality of ddRNAi constructs, an expression vector, a plurality of expression vectors or a composition as described herein.
  • the disclosure provides a method of treating Purtscher's retinopathy in a subject, said method comprising administering to the subject a therapeutically effective amount of a nucleic acid, plurality of nucleic acids, a ddRNAi construct, a plurality of ddRNAi constructs, an expression vector, a plurality of expression vectors or a composition as described herein.
  • the disclosure provides a method of treating or preventing retinal vein occlusion (RVO) in a subject, said method comprising administering to the subject a therapeutically effective amount of a nucleic acid, plurality of nucleic acids, a ddRNAi construct, a plurality of ddRNAi constructs, an expression vector, a plurality of expression vectors or a composition as described herein.
  • RVO retinal vein occlusion
  • the therapeutic compositions of the disclosure may be used alone or in combination with one or more agents or compositions known to be suitable for treatment of the disease or condition.
  • the therapeutic compositions of the disclosure may be used alone or in combination with one or more agents or compositions known to be suitable for treatment of AMD, such as, for example, ranibizumab, aflibercept, bevacizumab, pegaptanib sodium and/or verteporfin.
  • compositions will desirably include materials that increase the biological stability of the nucleic acids, ddRNAi constructs or expression vectors of the disclosure and/or materials that increase the ability of the compositions to penetrate the eye, in particular, cells in and around the macula.
  • the therapeutic compositions of the disclosure may be administered in pharmaceutically acceptable carriers (e.g., physiological saline), which are selected on the basis of the mode and route of administration, and standard pharmaceutical practice.
  • pharmaceutically acceptable carriers e.g., physiological saline
  • One having ordinary skill in the art can readily formulate a pharmaceutical composition that comprises one or more nucleic acids, ddRNAi constructs or expression vectors of the disclosure. In some cases, an isotonic formulation is used.
  • additives for isotonicity can include sodium chloride, dextrose, mannitol, sorbitol and lactose.
  • isotonic solutions such as phosphate buffered saline are preferred.
  • Stabilizers include gelatin and albumin.
  • a vasoconstriction agent is added to the formulation.
  • the compositions according to the present disclosure are provided sterile and pyrogen free. Suitable pharmaceutical carriers, as well as pharmaceutical necessities for use in
  • Routes of administration include, but are not limited to, intravitreal injection, periocular injection and/or subretinal injection.
  • An exemplary and preferred route of administration is intravitreal injection.
  • Targeted transfection of the eye in vivo for delivery of nucleic acids, ddRNAi constructs or expression vectors of the disclosure may be accomplished through intravitreal injection or subretinal injection with a composition comprising one or more nucleic acids, ddRNAi constructs or expression vectors as described herein complexed with a suitable carrier.
  • Such compositions are useful for pharmaceutical applications and may readily be formulated in a suitable sterile, non-pyrogenic vehicle, e.g., buffered saline for injection, for intravitreal, periocular and/or subretinal injection.
  • the volume, concentration, and formulation of the pharmaceutical composition as well as the dosage regimen may be tailored specifically to maximize cellular delivery while minimizing toxicity such as an inflammatory response.
  • an injection volume of 0.05ml is commonly used for intravitreal injection of existing point of care drugs for AMD, but this may be varied between about 0.01ml to about 0.2ml to achieve the optimal volume, concentration, and formulation combination. Kits
  • the present disclosure also provides the nucleic acids, ddRNAi constructs, expression vectors and/or compositions of the disclosure in a kit form.
  • the kit may comprise a container.
  • the kit typically contains one or more nucleic acids, ddRNAi constructs, expression vectors or compositions of the disclosure with instructions for administration.
  • the kit contains more than one nucleic acids, ddRNAi constructs, expression vectors or composition of the disclosure.
  • the kit contains more than one nucleic acids, ddRNAi constructs, expression vectors or compositions of the disclosure packed together with one or more other compounds for treatment of AMD, such as, for example, ranibizumab, aflibercept, bevacizumab, pegaptanib sodium and/or verteporfin.
  • Example 1 Design and generation of ddRNAi constructs targeting VEGFb and PGF
  • mRNA sequences corresponding to VEGFb and PGF from human and macaca species were aligned, and conserved regions identified from the sequence alignments. Potential target regions for design of ddRNAi constructs were identified from those regions which were conserved across the alignments. Ten target regions were identified for VEGFb (Table 1) and twelve target regions were identified for PGF (Table 2).
  • DNA-directed RNA interference (ddRNAi) constructs coding for short-hairpin microRNAs (shmiRs) (also referred to hereinafter as “shmiR constructs”) were then designed based on the target regions identified for VEGFb and PGF.
  • shmiR constructs DNA sequences coding for the respective effector and effector complement sequence combinations corresponding to the target regions for VEGFb and PGF were generated (set forth in Tables 3 and 4 respectively) and incorporated into a pre-miRNA backbone.
  • Each shmiR construct comprised a DNA sequence coding for: a 5' flanking region of the pri-miRNA backbone (SEQ ID NO: 68); an effector complement sequence (see column 2 of Tables 3 and 4); a stem/loop junction sequence (SEQ ID NO 67); an effector sequence (see column 4 of Tables 3 and 4); and a 3 ' flanking region of the pri-miRNA backbone (SEQ ID NO: 69).
  • the shmiR constructs coding for shmiRs targeting VEGFb and PGF are set forth in Tables 5 and 6 respectively, and the shmiR encoded by those shmiR constructs are set forth in Tables 7 and 8, respectively.
  • Each shmiR construct was cloned between the BamHI / Hindlll sites of pSilencer 2.1-U6 hygro vector (ThermoFisher), which contained a human U6-1 promoter to drive expression of the shmiR.
  • a map of the vector and an insert for a representative shmiR construct (designated "VEGFb shmiR- 1") are shown in Figures 1A and IB respectively.
  • the secondary structure of the expressed VEGFb shmiR-1 predicted using m-Fold program is shown in Figures 1C.
  • VEGFb_shmiR-l AGGAAAGUGGUGUCAUGGAU SEQ ID NO: 23 AUCCAUGACACCACUUUCCUC : SEQ ID NO: 24
  • VEGFb_shmiR-2 AUGGGCACCGUGGCCAAACA SEQ ID NO: 25 UGUUUGGCCACGGUGCCCAU ( - SEQ ID NO: 26
  • VEGFb_shmiR-3 GCACCAAGUCCGGAUGCAGA SEQ ID NO: 27 UCUGCAUCCGGACUUGGUGC1 j SEQ ID NO: 28
  • VEGFb_shmiR-4 GGGAGAUGUCCCUGGAAGAA SEQ ID NO: 29 UUCUUCCAGGGACAUCUCCCC : SEQ ID NO: 30
  • VEGFb_shmiR-5 GGCUUAGAGCUCAACCCAGA SEQ ID NO: 31
  • VEGFb_shmiR-6 AACAAAGAGGAGCCUGGUAA SEQ ID NO: 33 UUACCAGGCUCCUCUUUGUUi 2 SEQ ID NO: 34
  • VEGFb_shmiR-7 AAGACCUCAGCCCAGGCAGA SEQ ID NO: 35 UCUGCCUGGGCUGAGGUCUU 3 SEQ ID NO: 36
  • VEGFb_shmiR-8 AUCAUCAAACAGGACAGAGU SEQ ID NO: 37 ACUCUGUCCUGUUUGAUGAU Q SEQ ID NO: 38
  • VEGFb_shmiR-9 AGGACAGAGUUGGAAGAGGA SEQ ID NO: 39 UCCUCUUCCAACUCUGUCCUC j SEQ ID NO: 40
  • VEGFb_shmiR-10 GGAUUUGGGCUUUGGUACAA SEQ ID NO: 41
  • VEGFb_shmiR-l GGTATATTGCTGTTGACAGTGAGCGAAGGAAAGTGGTGTCATGGATAC TGTGAAGCAGATGG SEQ ID NO: 92
  • VEGFb_shmiR-2 GGTATATTGCTGTTGACAGTGAGCGAATGGGCACCGTGGCCAAACAAC TGTGAAGCAGATGG SEQ ID NO: 93
  • VEGFb_shmiR-3 GGTATATTGCTGTTGACAGTGAGCGTGCACCAAGTCCGGATGCAGAAC r rGTGAAGCAGATGG SEQ ID NO: 94
  • VEGFb_shmiR-4 GGTATATTGCTGTTGACAGTGAGCGAGGGAGATGTCCCTGGAAGAAAC TGTGAAGCAGATGG SEQ ID NO: 95
  • VEGFb_shmiR-5 GGTATATTGCTGTTGACAGTGAGCGAGGCTTAGAGCTCAACCCAGAAC r rGTGAAGCAGATGG SEQ ID NO: 96
  • VEGFb_shmiR-6 GGTATATTGCTGTTGACAGTGAGCGAAACAAAGAGGAGCCTGGTAAAC TGTGAAGCAGATG SEQ ID NO: 97
  • VEGFb_shmiR-7 GGTATATTGCTGTTGACAGTGAGCGAAAGACCTCAGCCCAGGCAGAAC TGTGAAGCAGATGG SEQ ID NO: 98
  • VEGFb_shmiR-8 GGTATATTGCTGTTGACAGTGAGCGAATCATCAAACAGGACAGAGTAC TGTGAAGCAGATGG SEQ ID NO: 99
  • VEGFb_shmiR-9 GGTATATTGCTGTTGACAGTGAGCGAAGGACAGAGTTGGAAGAGGAAC TGTGAAGCAGATG SEQ ID NO: 100
  • VEGFb_shmiR-10 GGTATATTGCTGTTGACAGTGAGCGAGGATTTGGGCTTTGGTACAAACl GTGAAGCAGATGG SEQ ID NO: 101
  • PGF_shmiR-2 GGTATATTGCTGTTGACAGTGAGCGACCAGAAGATGCTCGAACC :ACACTGTGAAGCAGATGG SEQ ID NO: 103
  • PGF_shmiR-4 GGTATATTGCTGTTGACAGTGAGCGAACCATGCAGCTCCTAAAC rATACTGTGAAGCAGATGG SEQ ID NO: 105
  • PGF_shmiR-7 GGTATATTGCTGTTGACAGTGAGCGACAGGAATTCAGTGCCTTC AAACTGTGAAGCAGATGG SEQ ID NO: 108
  • PGF_shmiR-8 GGTATATTGCTGTTGACAGTGAGCGAAAAGAGAAGCCAGCC ACAACTGTGAAGCAGATG SEQ ID NO: 109
  • PGF_shmiR-9 GGTATATTGCTGTTGACAGTGAGCGATGCTACCTGTTCTTGGGC( TACTGTGAAGCAGATGGG SEQ ID NO: 110
  • PGF_shmiR-10 GGTATATTGCTGTTGACAGTGAGCGAAGAACATTCAGCTCTGG ⁇ LGAACTGTGAAGCAGATGG SEQ ID NO: 111
  • PGF_shmiR-2 GGUAUAUUGCUGUUGACAGUGAGCGACCAGAAGAUGCUCGAA CCACACUGUGAAGCAGAU SEQ ID NO: 81
  • PGF_shmiR-3 GGUAUAUUGCUGUUGACAGUGAGCGAGGUCAUGAGGCUGUUC CCUUACUGUGAAGCAGAU SEQ ID NO: 82
  • PGF_shmiR-4 GGUAUAUUGCUGUUGACAGUGAGCGAACCAUGCAGCUCCUAA AGAUACUGUGAAGCAGAU SEQ ID NO: 83
  • PGF_shmiR-7 GGUAUAUUGCUGUUGACAGUGAGCGACAGGAAUUCAGUGCCU UCAAACUGUGAAGCAGAU SEQ ID NO: 86

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  • Physics & Mathematics (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

Abstract

L'invention concerne des réactifs d'interférence ARN (ARNi) destinés au traitement ou à la prévention de maladies ou affections oculaires associées à la néovascularisation, telles que la dégénérescence maculaire liée à l'âge (AMD). Elle concerne des compositions comprenant ces réactifs, et leur utilisation pour traiter des individus souffrant de, ou prédisposés à des maladies ou affections oculaires associées à la néovascularisation, telles que la DMLA.
PCT/AU2018/050075 2017-02-03 2018-02-05 Réactifs pour le traitement de maladies ou affections oculaires associées à la néovascularisation, et leur utilisation WO2018141025A1 (fr)

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Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2014107763A1 (fr) * 2013-01-08 2014-07-17 Benitec Biopharma Limited Traitement de la dégénérescence maculaire liée à l'âge

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2014107763A1 (fr) * 2013-01-08 2014-07-17 Benitec Biopharma Limited Traitement de la dégénérescence maculaire liée à l'âge

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
BRUCE BARNETT: "Biotech Showcase", BENITEC BIOPHARMA, 9 January 2017 (2017-01-09), pages 1 - 46, XP055533840, Retrieved from the Internet <URL:https://benitec.com/wp-content/uploads/2017/09/201701_Benitec_Biopharma_Biotech_Showcase.pdf.> [retrieved on 20180321] *
FISCHER, C ET AL.: "FLT1 and its ligands VEGFB and PlGF: drug targets for anti-angiogenic therapy?", NATURE REVIEWS CANCER, vol. 8, no. 12, 2008, pages 942 - 956, XP055533844 *

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