WO2020028562A1 - Snail sirna-loaded mesoporous silica nanoparticles - Google Patents

Snail sirna-loaded mesoporous silica nanoparticles Download PDF

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Publication number
WO2020028562A1
WO2020028562A1 PCT/US2019/044496 US2019044496W WO2020028562A1 WO 2020028562 A1 WO2020028562 A1 WO 2020028562A1 US 2019044496 W US2019044496 W US 2019044496W WO 2020028562 A1 WO2020028562 A1 WO 2020028562A1
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snail
sirna
subject
cancer
composition
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PCT/US2019/044496
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French (fr)
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Juli UNTERNAEHRER
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Loma Linda University
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/713—Double-stranded nucleic acids or oligonucleotides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6921—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere
    • A61K47/6923—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being an inorganic particle, e.g. ceramic particles, silica particles, ferrite or synsorb
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6921—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere
    • A61K47/6927—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores
    • A61K47/6929—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y5/00—Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/14—Type of nucleic acid interfering N.A.
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2320/00—Applications; Uses
    • C12N2320/10—Applications; Uses in screening processes
    • C12N2320/11—Applications; Uses in screening processes for the determination of target sites, i.e. of active nucleic acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2320/00—Applications; Uses
    • C12N2320/30—Special therapeutic applications
    • C12N2320/32—Special delivery means, e.g. tissue-specific

Definitions

  • Ovarian cancer is the most lethal gynecological cancer and fifth leading cause of cancer deaths among women (Siegel et al., 2017, C4 Cancer J Clin., 67(l):7-30).
  • Current treatment options involve surgery and chemotherapy but are ineffective due to recurrent malignant tumor growth (Coleman et al., 2013, Nat Rev Clin Oncol. 10(4):211-24).
  • Relapse is attributed in part to a small population of cancer stem cells (CSC) found within the tumor (Bati!e and C!evers., 2017, Nat Med. 23(10):1124-34).
  • CSC cancer stem cells
  • Snail epithelial mesenchymal transition regulator
  • siRNA loaded mesoporous silica nanoparticles as a suitable delivery vehicle to downregulate expression of the TWIST gene, associated with ovarian cancer.
  • Conjugating hyaluronic acid to the anti-TWIST siRNA loaded mesoporous silica nanoparticles (MSNs) was found to enhance delivery of the siRNAs to a specific receptor (CD44) in the ovarian cancer cells (Shahin et al., 2018, Nanomedicine: Nanotechnology, Biology, and Medicine , 14(4) : 1381-1394).
  • compositions comprising a SNAIL inhibitor bound to a delivery vehicle are provided.
  • a siRNA is provided having a nucleic acid sequence of any one of SEQ ID NOs: 1-20, 27 and 28.
  • DNA sequences encoding an siRNA sequence comprising the nucleic acid sequence of any one of SEQ ID NOs: 1-20, 27 and 28.
  • a method of treating ovarian cancer in a subject includes administering to the subject a therapeutically effective amount of a SNAIL inhibitor bound to a delivery vehicle.
  • a method of reducing or inhibiting ovarian cancer metastasis in a subject includes administering a therapeutically effective amount of a SNAIL inhibitor bound to a delivery vehicle to the subject.
  • FIG. 1A and FIG. IB show reduced relative expression of Snail in 293T ceils after transfection of various small inhibitory RNAs (siRNAs) Twelve different siRNAs were tested, including anti-Snail siRNAs referred to herein as siSnail-1 through siSnail-10.
  • the siRNAs were transfected into the 293T cells at three different concentrations (1 nM, 3 nM and 10 nM). 24 hours after transfection, the level of Snail RNA was determined by qRT-PCR using a 5' (FIG. 1A) or 3’ (FIG. IB) primer pair. Non-transfection (Non Tr) is shown. HPRT was also targeted and is shown as a positive control.
  • siNC non -specific control
  • ail samples are normalized to siNC.
  • FIG. 2 shows relative expression of HPRT in 293T ceils after transfection of siRNAs (siSnail-1 through siSnail-10) provided at three different concentrations (1 nM, 3 nM and 10 nM). 24 hours after transfection, the level of HPRT RNA was determined by qRT-PCR. Non transfection (Non Tr) is shown. siHPRT is shown as a positive control. siNC (non-specific control) is shown as a negative control.
  • FIG. 3 is a graph showing decreased Snail expression in OvcarS cells at 24 to 72 hours after transfection with siRNA-loaded hyaluronic acid conjugated mesoporous silica nanoparticles (HA-MSNs). After 24 hours, a 97% decrease in Snail expression was observed. At 48 hours, a 92% decrease was noted and at 72 hours there was an 87% decrease in Snail expression. A positive control siRNA is included for comparison (siScramble). DETAILED DESCRIPTION OF THE INVENTION
  • compositions comprising SNAIL inhibitors bound to a delivery vehicle and methods of treating ovarian cancer in a subject, or preventing metastasis of ovarian cancer cells in a subject using the same.
  • target cells e.g., cancer cells
  • siRNAs in the absence of a delivery vehicle do not enter cells in vitro (see, e.g., Roberts et al., (2017), Nanomedicine: Nanotechnology , Biology , and Medicine , 13:965-976).
  • the disclosure provides novel compositions for targeting SNAIL and methods for treating ovarian cancer or reducing metastasis of ovarian cancer cells through the downregulation of SNAIL.
  • polypeptide peptide
  • protein protein
  • amino acid polymers in which one or more amino acid residue is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers and non-naturally occurring amino acid polymers.
  • nucleic acid As used herein, the terms“nucleic acid,” “oligonucleotide,”“nucleic acid sequence,” “nucleic acid fragment” and “polynucleotide” are used interchangeably and are intended to include, but are not limited to, a polymeric form of nucleotides covalently linked together that may have various lengths, either deoxyribonucleotides or ribonucleotides, or analogs, derivatives or modifications thereof. Different polynucleotides may have different three- dimensional structures, and may perform various functions, known or unknown.
  • Non-limiting examples of polynucleotides include a gene, a gene fragment, an exon, an intron, intergenic DNA, messenger RNA (rnRNA), transfer RNA, ribosomal RNA, a ribozyme, cDNA, a recombinant polynucleotide, a branched polynucleotide, a plasmid, a vector, isolated DNA of a sequence, isolated RNA of a sequence, a nucleic acid probe, and a primer.
  • Polynucleotides useful for the invention may comprise natural nucleic acid sequences and variants thereof, artificial nucleic acid sequences, or a combination of such sequences.
  • a polynucleotide is typically composed of a specific sequence of four nucleotide bases: adenine (A); cytosine (C); guanine (G); and thymine (T) (uracil (U) for thymine (T) when the polynucleotide is RNA).
  • Polynucleotides may optionally include one or more non-standard nucleotide(s), nucleotide analog(s) and/or modified nucleotides.
  • Percentage of sequence identity is determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide or polypeptide sequence in the comparison window may comprise additions or deletions (i.e., gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity.
  • nucleic acids or polypeptide sequences refers to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same (i.e., about 60% identity, preferably 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher identity over a specified region, when compared and aligned for maximum correspondence over a comparison window or designated region) as measured using a BLAST or BLAST 2.0 sequence comparison algorithms with default parameters described below, or by manual alignment and visual inspection (see, e.g., NCBI web site http://www.ncbi.nlm.nih.gov/BLAST/or the like).
  • sequences are then said to be "substantially identical.”
  • This definition also refers to, or may be applied to, the compliment of a test sequence.
  • the definition also includes sequences that have deletions and/or additions, as well as those that have substitutions.
  • Algorithms such as BLAST, can account for gaps.
  • identity exists over a region that is at least about 15 amino acids or nucleotides in length, or more preferably over a region that is at least 20 amino acids or nucleotides in length.
  • the disclosure includes nucleic acid sequences that are substantially identical ⁇ e.g., at least 90% identical) to any of the nucleic acid sequences set forth as SEQ ID NOs: 1-20, 27 and 28.
  • the named protein includes any of the protein's naturally occurring forms, or variants that maintain the protein transcription factor activity (e.g., within at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% activity compared to the native protein).
  • variants have at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino add sequence identity across the whole sequence or a portion of the sequence (e.g. a 50, 100, 150 or 200 continuous amino acid portion) compared to a naturally occurring form.
  • the protein is the protein as identified by its NCBI sequence reference.
  • the protein is the protein as identified by its NCBI sequence reference or functional fragment thereof.
  • SNA!l or“SNA!L” gene refers to any of the recombinant or naturally- occurring forms of the gene encoding zinc finger protein SNA!l or SNA!L, homologs or variants thereof that maintain SNAIL protein activity (e.g. within at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% activity as compared to native SNAIL).
  • the SNA!l gene is conserved in chimpanzee, rhesus monkey, dog, cow, mouse and rat.
  • At least 160 organisms are known to have orthologs to the human SNAI1 gene (i.e , NCBI Gene ID: 6615).
  • the SNA!l gene is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleic acid identified by the NCBI Gene ID: 6615 (NM__005985.3).
  • the SNAI1 gene is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleic acid identified by the NCBI Gene ID: 10415.
  • the SNAI1 gene is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the mouse nucleic acid identified by the NCBI Gene ID: 20613 (NMJ311427).
  • SNAI2 or " SNAIL2” refers to any of the recombinant or naturally- occurring forms of the gene encoding zinc finger protein 5NAI2, homologs or variants thereof that maintain SNAI2 protein activity (e.g. within at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% activity as compared to native SMAI2).
  • the SNAI2 gene is conserved in humans, chimpanzee, rhesus monkey, dog, cow, chicken, frog, zebrafish and rat.
  • At least 220 organisms are known to have orthoiogs to the human SNAI2 gene (i.e., NCB! Gene ID: 6591).
  • the SNAI2 gene is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleic acid identified by the NCBI Gene ID: 6591 (NM_003068.4).
  • the SNAI2 gene is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the mouse nucleic acid identified by the NCBI Gene ID: 20583 (NM_011415).
  • ZEB1 refers to any of the recombinant or naturally-occurring forms of the gene encoding zinc finger protein ZEB1, homologs or variants thereof that maintain Zebl protein activity (e.g. within at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% activity as compared to native ZEB1).
  • the ZEB1 gene is conserved in chimpanzee, rhesus monkey, dog, cow, chicken, mouse, frog, zebrafish and rat. At least 230 organisms are known to have orthologs to the human ZEB1 gene (i.e., NCBI Gene ID: 6935).
  • the ZEB1 gene is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleic acid identified by the NCBI Gene ID: 6935 (e.g., NM 301128128; NM_0Q1174093; MJ301174G94; and NM 301323649).
  • the ZEB1 gene is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the mouse nucleic acid identified by the NCBI Gene ID: 21417 (e.g., NM 001360981; NM J301360982; and NM_ . 011546).
  • ZEB2 refers to any of the recombinant or naturally-occurring forms of the gene encoding zinc finger protein ZEB2, homologs or variants thereof that maintain Zeb2 protein activity (e.g. within at least 50%, 60%, 7G? , 80%, 90%, 91%, 92%, 93%, 94%, 95?4, 96%, 97%, 98%, 99% or 100% activity as compared to native ZEB2).
  • the ZEB2 gene is conserved in chimpanzee, rhesus monkey, dog, chicken, mouse, frog, and zebrafish. At least 230 organisms are known to have orthoiogs to the human ZEB2 gene (i.e., NCBI Gene ID: 9839).
  • the ZE82 gene is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleic acid identified by the NCBI Gene ID: 9839 (e.g., NM_001171653 and NM_014795).
  • the Z£B2 gene is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the mouse nucleic acid identified by the NCB! Gene ID: 24136 (e.g., NM_ . 001289521; NM_ 001355288; MM .. 001355291; MM ... 015753 and MM ... 001355289).
  • Snail or Snail protein refer to any recombinant or naturally-occurring form of a protein encoded by the SNA!l gene, homologs or variants thereof that maintain Snail protein activity (e.g , within at least 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity as compared to native Snail).
  • Snail or Snail is a Zinc Finger protein encoded by the SNAil gene.
  • Snail belongs to a family of transcription factors that promote the repression of adhesion molecule E-cadherin to regulate epithelial cells becoming mesenchymal cells.
  • variants of a Snail have at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100? amino acid sequence identity across the whole sequence or a portion of the sequence (e.g. a 50, 100, 150 or 200 continuous amino acid portion) compared to a naturally occurring Snail polypeptide (e.g., NCBI reference number: fsiP__005976) or NCBI Gene 10:6615 or Gene 10:10415 .
  • a naturally occurring Snail polypeptide e.g., NCBI reference number: fsiP__005976
  • NCBI Gene 10:6615 or Gene 10:10415 NCBI reference number: fsiP__005976
  • variants of the Snail protein have at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity across the whole sequence or a portion of the sequence (e.g. a 50, 100, 150 or 200 continuous amino acid portion) compared to a naturally occurring mouse Snail polypeptide (e.g., NCBI reference number: NP_035557) or NCBI Gene 10:20613.
  • a naturally occurring mouse Snail polypeptide e.g., NCBI reference number: NP_035557
  • NCBI Gene 10:20613 e.g., NCBI Gene 10:20613.
  • Snai2 or “Snail2” protein refer to any recombinant or naturally-occurring form of a protein encoded by the SNAI2 gene, homologs or variants thereof that maintain Snail protein activity (e.g., within at least 50%, 60? , 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity as compared to native Snail2).
  • Snai2 is a Zinc Finger protein encoded by the SNAI2 gene.
  • Snai2 belongs to a family of transcription factors that promote the repression of adhesion molecule E-cadherin to regulate epithelial cells becoming mesenchymal cells.
  • variants of a Snai2 have at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 1QQ% amino acid sequence identity across the whole sequence or a portion of the sequence (e.g. a 50, 100, 150 or 200 continuous amino add portion) compared to a naturally occurring Snai2 polypeptide (e.g., MCBI reference number: NP_003059) or MCBI Gene !D:6591.
  • a naturally occurring Snai2 polypeptide e.g., MCBI reference number: NP_003059
  • variants of the Snai2 protein have at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity across the whole sequence or a portion of the sequence (e.g. a 50, 100, 150 or 200 continuous amino add portion) compared to a naturally occurring mouse 5nai2 polypeptide (e.g., NCBI reference number: NP__035545) or NCBI Gene ID: 20583.
  • a naturally occurring mouse 5nai2 polypeptide e.g., NCBI reference number: NP__035545
  • NCBI Gene ID NCBI Gene ID: 20583.
  • Zebl protein refers to any recombinant or naturally- occurring form of a protein encoded by the ZEB1 gene, bomo!ogs or variants thereof that maintain Zebl protein activity (e.g., within at least 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity as compared to native Zebl).
  • Zebl is a Zinc Finger protein encoded by the ZEB1 gene.
  • variants of a Zebl have at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino add sequence identity across the whole sequence or a portion of the sequence (e.g. a 50, 100, 150 or 200 continuous amino acid portion) compared to a naturally occurring Zebl polypeptide (e.g., NCBI reference number: NP_001121600; NP_001167564 and NP_001167565) or NCBI Gene ID:6935.
  • a naturally occurring Zebl polypeptide e.g., NCBI reference number: NP_001121600; NP_001167564 and NP_001167565
  • variants of the Zebl protein have at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity across the whole sequence or a portion of the sequence (e.g. a 50, 100, 150 or 200 continuous amino acid portion) compared to a naturally occurring mouse Zebl polypeptide (e.g., NCBI reference number: NP_001347910; NP 301347911 and NP_0355676) or NCBI Gene ID: 21417.
  • a naturally occurring mouse Zebl polypeptide e.g., NCBI reference number: NP_001347910; NP 301347911 and NP_0355676
  • NCBI Gene ID NCBI Gene ID: 21417.
  • Zeb2 protein refers to any recombinant or naturally- occurring form of a protein encoded by the ZEB2 gene, homoiogs or variants thereof that maintain Zeb2 protein activity (e.g., within at least 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity as compared to native Zeb2).
  • Zeb2 is a Zinc Finger protein encoded by the ZEB2 gene.
  • variants of a Zeb2 have at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity across the whole sequence or a portion of the sequence (e.g.
  • variants of the Zeb2 protein have at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity across the whole sequence or a portion of the sequence (e.g. a 50, 100, 150 or 200 continuous a ino acid portion) compared to a naturally occurring mouse Zeb2 polypeptide (e.g., NCBI reference number: NP 001276450; MR 001342217; MR .. 001342218; and NP .. 001342219 ⁇ or NCBI Gene ID: 24136.
  • siRNA refers to a nucleic acid that forms a double stranded RNA, which double stranded RNA has the ability to reduce or inhibit expression of a target gene when the siRNA is present (e.g. expressed) in the same cell as the target gene.
  • the siRNA is typically about 5 to about 100 nucleotides in length, more typically about 10 to about 50 nucleotides in length, more typically about 15 to about 40 nucleotides in length, most typically about 20-30 base nucleotides, or about 20-25 or about 24-29 nucleotides in length, e.g., 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length.
  • siRNA molecules and methods of generating them are described in, e.g., Bass, 2001, Nature, 411, 428-429; Elbashir et a!., 2001, Nature , 411, 494-498; Patent Application Publication Nos: WO 00/44895; WO 01/36646; WO 99/32619; WO 00/01846; WO 01/29058; WO 99/07409; and WO 00/44914.
  • a DNA molecule that transcribes siRNA also provides RNA interference (RNAi).
  • DNA molecules for transcribing siRNA are disclosed in for example, U.S. Pat. No. 6,573,099, and U.S. Patent Application Publication Nos.
  • siRNA can be delivered to a target cell using a vector (e.g., a plasmid, bacterial or viral vector) or by oligonucleotide (see, e.g., Subramanya et al., (2010) Expert Opin Biol Ther., 10(2)201-13).
  • a vector e.g., a plasmid, bacterial or viral vector
  • oligonucleotide see, e.g., Subramanya et al., (2010) Expert Opin Biol Ther., 10(2)201-13).
  • delivery vehicle refers to a support structure that transfers a component of genetic material or a protein to or into a cell.
  • Genetic material includes, but is not limited to DNA, RNA (e.g., siRNAs), proteins, polypeptides, or fragments thereof.
  • a "cell” as used herein, refers to prokaryotic and eukaryotic cells and include cells derived from mammals. In some embodiments, a cell is an ovarian cancer cell.
  • the term "compound” refers to any molecule, either naturally occurring or synthetic, e.g., peptide, protein, oligopeptide (e.g., from about 5 to about 25 amino acids in length, preferably from about 10 to 20 or 12 to 18 a ino acids in length, preferably 12, 15, or 18 amino acids in length), small organic molecule, polysaccharide, peptide, circular peptide, peptidomimetic, lipid, fatty acid, siRNA, polynucleotide, oligonucleotide, etc.
  • peptide protein
  • oligopeptide e.g., from about 5 to about 25 amino acids in length, preferably from about 10 to 20 or 12 to 18 a ino acids in length, preferably 12, 15, or 18 amino acids in length
  • small organic molecule polysaccharide, peptide, circular peptide, peptidomimetic, lipid, fatty acid, siRNA, polynucleotide, oligonucleotide, etc
  • the terms “inhibitor,” “repressor”, or “downregu!ator”, interchangeably refer to a compound that results in a detectabiy lower expression or activity level as compared to a control.
  • the inhibited expression or activity can be 10%, 20%, 30%, 40%, 50?4, 60%, 70%, 80%, 90% or less than that in a control.
  • the inhibition is 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, or more in comparison to a control.
  • the inhibitor is a siRNA that inhibits cellular function (e.g., replication) by binding, partially or totally blocking stimulation, decreases, prevents, or delays activation, or inactivates, desensitize, or down-regulates signal transduction, gene expression, or enzymatic activity necessary for protein activity.
  • cellular function e.g., replication
  • a "Snail inhibitor” refers to a compound, such as a siRIMA, that results in a detectabiy lower expression of SNAIL genes (e.g., SNAIL1 and SNAIL2) or Snail proteins (e.g., Snai!l and Snail2) or lower activity level of Snail proteins as compared to those levels without such compound.
  • a Snail inhibitor is a compound, such as a siRNA, that results in a detectabiy lower expression of ZEB genes (e.g., ZEB1 and ZEB2) or Zeb proteins (e.g., Zebl and Zeb2) or lower activity level of Zeb proteins as compared to those levels without such compound in some embodiments, a Snail inhibitor is an anti-SNA!L siRNA.
  • a Snail inhibitor is a composition (e.g., a Snail inhibitor bound to a delivery vehicle or an anti-SNAIL siRNA bound to a esoporous silica nanoparticle bound to hyaluronic acid or folic acid) described herein.
  • a Snail inhibitor is a pharmaceutical composition described herein.
  • a "pharmaceutical composition” is a formulation containing the composition (e.g., a Snail inhibitor bound to a nanoparticle) described herein in a form suitable for administration to a subject in some embodiments, the pharmaceutical composition is in bulk or in unit dosage form.
  • the unit dosage form is any of a variety of forms, including, for example, a capsule, an IV bag, a tablet, or a vial.
  • the quantity of active ingredient (e.g., SNAIL inhibitor bound to a nanoparticle) in a unit dose of composition is an effective amount and is varied according to the particular treatment involved.
  • active ingredient e.g., SNAIL inhibitor bound to a nanoparticle
  • composition is mixed under sterile conditions with a pharmaceutically acceptable carrier, and any preservatives, buffers, or propellants that are required.
  • the phrase "pharmaceutically acceptable” refers to compounds, anions, cations, materials, compositions, carriers, and/or dosage forms which are, within the scope of medical judgment, suitable for use in contact with tissues of humans and animals without excessive toxicity, irritation, allergic response, or other complications, commensurate with a reasonable benefit/risk ratio.
  • “Pharmaceutically acceptable excipient” means an excipient that is useful in preparing a pharmaceutical composition that is generally safe, non-toxic and neither biologically nor otherwise undesirable, and includes excipient that is acceptable for veterinary use as well as human pharmaceutical use.
  • Pharmaceutically acceptable excipients in therapeutic compositions may contain liquids such as water, saline, glycerol and ethanol. Additionally, auxiliary substances, such as wetting or emulsifying agents, pH buffering substances, and the like, may be present in such vehicles.
  • monotherapy refers to the administration of a single therapeutic compound to a subject in need thereof.
  • monotherapy involves administration of a therapeutically effective amount of a composition described herein (e.g., a SNAIL inhibitor bound to a nanoparticle).
  • monotherapy can include administration of a composition of the present invention to a subject to treat cancer.
  • monotherapy may be contrasted with combination therapy, in which a combination of multiple compositions (e.g., a SNAIL inhibitor bound to a delivery vehicle and an anti-cancer agent) are administered, preferably with each component of the combination present in a therapeutically effective amount.
  • an anti-cancer agent is used in accordance with its plain ordinary meaning and refers to a composition (e.g. compound, drug, antagonist, inhibitor, modulator) having antineoplastic properties or the ability to inhibit the growth or proliferation of ceils in some embodiments, an anti-cancer agent is a chemotherapeutic.
  • an anti-cancer agent is an agent identified herein having utility in methods of treating cancer.
  • an anti-cancer agent is an agent approved by the FDA or similar regulatory agency for treating cancer.
  • an anti-cancer agent is an agent having utility in methods of treating ovarian cancer.
  • Other anti-cancer agents, and combinations of anti-cancer agents are well known in the art (see, e.g., www.cancer.org/docroot/cdg/cdg__0.asp).
  • the terms “subject” or “patient” are used interchangeably to refer to a subject having cancer or a subject having a precancerous condition.
  • a subject has cancer.
  • a "subject” includes a mammal.
  • the mammal can include a human or appropriate non-human mammal, such as a primate, mouse, rat, dog, cat, cow, or horse.
  • the methods disclosed herein are applicable to both human therapy and veterinary applications.
  • the subject has ovarian cancer.
  • Subjects with ovarian cancer include subjects with one or more signs or symptoms of ovarian cancer.
  • a subject may have an increased risk of developing ovarian cancer relative to the population at large (e.g., a female subject 30 years old or older, obese, no reproductive history, fertility drug use for longer than one year, androgen use, estrogen therapy, hormone therapy, a family history or personal history of ovarian cancer).
  • a subject with an increased risk of developing ovarian cancer relative to the population at large is a female subject having a germ-line or spontaneous mutation in BRCA1 or BRCA2, or both.
  • refractory cancer refers to cancer that does not respond to treatment.
  • the cancer may be resistant at the beginning of treatment or may become resistant during treatment in some embodiments, the subject has cancer recurrence following remission on most recent therapy. In some embodiments, the subject received and failed to respond to a known effective therapy for the cancer treatment.
  • cancer refers to ail types of cancer, neoplasm or malignant tumors found in mammals, including metastatic cancers.
  • cancer refers to ovarian cancer or other cancers that involve with epithelial to mesenchymal transition (EMT)
  • EMT epithelial to mesenchymal transition
  • the terms“metastasis” and “metastatic” can be used interchangeably and refer to the spread of a proliferative disease, e.g., cancer, from one organ or another non- adjacent organ or body part. Cancer may occur at an originating site, e.g., ovaries, which is referred to as a primary tumor, e.g., primary ovarian cancer.
  • metastatic cancer refers to a disease in which a subject has or had a primary tumor and has one or more secondary tumors.
  • non-metastatic cancer refers to subjects having a primary tumor but not one or more secondary tumors.
  • an effective amount or "a therapeutically effective amount” as provided herein refers to an amount effective to achieve its intended purpose.
  • the actual amount effective for a particular application will depend, inter alia, on the condition being treated.
  • the pharmaceutical compositions described herein will contain an active compound (e.g., anti-SNAIL siRNA bound to a nanoparticle) and optionally, an anti -cancer agent to achieve the desired result, e.g., reducing, eliminating, or slowing the progression of disease symptoms (e.g., ovarian cancer).
  • an active compound e.g., anti-SNAIL siRNA bound to a nanoparticle
  • an anti -cancer agent e.g., reducing, eliminating, or slowing the progression of disease symptoms (e.g., ovarian cancer).
  • the effect can be detected by any assay method known in the art.
  • compositions selected for administration will depend upon the subject's body weight, size, and health; the nature and extent of the condition; and the composition or combination of compositions selected for administration. Therapeutically effective amounts for a given situation can be determined by routine experimentation that is within the skill of a treating clinician.
  • treatment refers to any indicia of success in the treatment or amelioration of an injury, disease, or condition, including any objective or subjective parameter such as abatement; remission; diminishing of symptoms or making the injury, disease, or condition more tolerable to the subject; slowing in the rate of degeneration or decline (e.g., cognitive impairment); making the final point of degeneration less debilitating; and/or improving a subject's physical or mental well-being.
  • the administration of compositions or pharmaceutical compositions of the disclosure may or can lead to the elimination of a sign or symptom, however, elimination is not required. ill.
  • SNAIL inhibitors are inhibitors of expression of a SNAIL gene (e.g., SNAIL1 and SNAIL2) or a Snail protein (e.g., Snaill and Snaii2) in mammals.
  • a SNAIL inhibitor includes the expression of one or more genes or proteins from ZEB1 or ZEB2.
  • a SNAIL inhibitor is a siRNA.
  • a SNAIL inhibitor comprises one or more siRNAs targeted against expression of SNAIL1 or SNAIL2 or a Snail transcript (e.g., Snaill and Snail 2 mRNA).
  • siRNA molecules of the disclosure include isolated siRNA molecules that bind to a single stranded RNA molecule, which is a messenger RNA (mRNA) that encodes Snaill or Snai!2 (also referred to herein, as Snail and Snai2, respectively).
  • siRNA molecules of the disclosure also include isolated siRNA molecules that bind to a single stranded RNA molecule that encodes Zebl or Zeb2.
  • Snail l, Snail2, Zebl and Zeb2 are zinc finger proteins that belongs to a family of proteins associated with epithelial to mesenchymal transition (EMT).
  • Snail protein is encoded by the SNAIL gene (e.g., NCBI Gene ID: 6615).
  • Snail protein and gene sequences are publicly available. For example, an amino acid sequence for a Snail protein in humans can be found at NP 005976 and a nucleotide sequence for the corresponding mRNA sequence can be found at: NM 305985.
  • Snail2 protein and gene sequences are publicly available.
  • an amino acid sequence for a Snai2 protein in humans can be found at NP__003059 and a nucleotide sequence for the corresponding mRNA sequence can be found at: NM 303G68
  • Zebl protein and gene sequences are publicly available.
  • an a ino acid sequence for a Zebl protein in humans can be found at NP_001121600 and a nucleotide sequence for the corresponding mRNA sequence can be found at: NM 001128128.
  • Zeb2 protein and gene sequences are publicly available.
  • an amino acid sequence for a Zeb2 protein in humans can be found at MR 001165124 and a nucleotide sequence for the corresponding mRNA sequence can be found at: NM 001171653.
  • Snaill and S n a ⁇ 12 nucleic acids and proteins can vary from those publicly available, such as polymorphisms resulting in one or more substitutions, deletions, insertions, or combinations thereof, while still retaining Snaill or Snail2 biological activity.
  • the amino acid sequence of Snaill or Snail2 may be about 80%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to a Snaill or Snail2 sequence publicly available.
  • a nucleic acid sequence of a SNAIL inhibitor may be at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% complementary to a publicly available SNAil, SNAI2, ZEB1 or ZEB2 sequence.
  • the SNAIL inhibitor bound to the delivery vehicle takes effect when the expression level of SNAIL1 or SNAIL2 gene or Snaill or Snail2 protein or the activity level of Snaill or Snail2 protein is less than 90% of an initia l level, less than 80% of an initial level, less than 70% of an initial level, less than 60% of an initial level, less than 50% of an initial level, less than 40% of an initial level, less than 30? of an initial level, less than 20% of an initial level or less than 10% of an initial level.
  • a SNAIL inhibitor is an anti-SNAIL siRNA.
  • anti-SNAIL siRNA includes all forms of anti-SNAIL siRNA, including variants, modifications and derivatives thereof.
  • the anti-SNAIL siRNA molecule is an oligonucleotide with a length of about 15 to about 40 base pairs (e.g., about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26 27, 28, 29, 30, 31, 32 33, 34, 35, 36, 37, 38, 39, or about 40 base pairs).
  • the anti-SNAIL siRNA molecule is an oligonucleotide with a length of about 15 to about 30 base pairs.
  • the molecule is an oligonucleotide with a length of about 20 to about 25 base pairs.
  • the anti-SNAIL siRNA molecule may have blunt ends at both ends, or sticky ends at both ends, or a blunt end at one end and a sticky end at the other.
  • an anti-SNAIL siRNA targets SNAIL or variants or homologs thereof.
  • an anti-SNAIL siRNA targets SNAI2 or variants or homologs thereof.
  • an anti-SNAIL siRNA targets both SNA!l and SNAI2, or their variants and homologs in some embodiments, an anti-SNAIL siRNA targets ZEB1 and/or ZEB2, or their variants and homo!ogs.
  • Exemplary anti-SNAIL siRNA sequences include, but are not limited to, any one or more of SEQ ID NOs:l- 20, 27 and 28.
  • the SNAIL siRNA bound to the delivery vehicle is siSNAIL- 7 (i.e., SEQ ID NOs:13 and 14).
  • anti-SNAIL siRNAs of the invention include nucleic acid sequences having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to any one or more of SEQ ID NOs:l-20, 27 and 28.
  • An siRNA molecule of the disclosure may comprise naturally occurring nucleotides or a nucleic acid modification.
  • nucleic acid modifications include chemical substitutions at the ribose and/or phosphate and/or base positions.
  • Modified nucleotides are described in U.S. Pat. No. 5,660,985, which describes oligonucleotides containing nucleotide derivatives chemically modified at the 2' position of ribose, 5 position of pyrimidines, and 8 position of purines.
  • U.S. Pat. No. 5,756,703 describes oligonucleotides containing various 2'- modified pyrimidines.
  • 5,580,737 describes highly specific nucleic acid ligands containing one or more nucleotides modified with 2'-amino (2'-NH2), 2'-fluoro (2'-F), and/or 2'-0-methyi ⁇ 2'-GMe) substituents.
  • Modification of siRNAs contemplated in this disclosure include, but are not limited to, those which provide other chemical groups that incorporate additional charge, polarizability, hydrophobicity, hydrogen bonding, electrostatic interaction, and fluxionality to the siRNA bases or to the siRNA sequence as a whole.
  • nucleic acid modifications include, but are not limited to, 2'-position sugar modifications, 5-position pyrimidine modifications, 8-position purine modifications, modifications at exocyclic amines, substitution of 4-thiouridine, substitution of 5-bromo or 5-iodo-uracil; backbone modifications, phosphorothioate or alkyl phosphate modifications, methylations, unusual base-pairing combinations such as the isobases isocytidine and isoguanidine and the like. Nucleic acid modifications also include 2'-Q-methy!
  • Nucleic acid modifications can also include 3' and 5' modifications such as capping.
  • an antisense sequence (guide strand) and a sense (passenger strand) can contain different nucleic acid modifications.
  • a passenger strand may contain nucleic acid modifications that promote loading of the guide strand onto mRNA cleavage machinery, modifications that prevent the passenger sequence loading into a RISC complex, modifications that prevent nuclease-mediated degradation, modifications that reduce immunogenicity mediated by toll-like receptors and RIG-1, or a combination thereof.
  • the passenger strand may contain inverted abasic riboses, 2'-0-methyl base (e.g., methyiuracil), or combination thereof in some embodiments, the guide strand may contain nucleic add modifications that increase their loading efficiency to a RISC complex. In some embodiments, a guide strand may contain a 2-thio-deoxyuracil.
  • the disclosure provides a composition comprising a SNAIL inhibitor bound to a delivery vehicle.
  • the delivery vehicle is a nanoparticle.
  • the delivery vehicle is a lipid vehicle.
  • Delivery vehicles include, but are not limited to, vectors such as viruses (e.g., retroviruses, adenoviruses, and adeno-associated viruses), virus capsids, liposomes or liposomal vesicles, lipoplexes, polyplexes, dendrimers, macrophages, artificial chromosomes, nanoparticles, polymers and also hybrid particles, examples of which include virosomes.
  • Delivery vehicles may have multiple surfaces and compartments for attachment and storage of components (e.g., anti-cancer agents). These include, but are not limited to, outer surfaces and pores.
  • the delivery vehicle is a nanoparticle, a lipid particle or a viral vector.
  • Any nanoparticle known for siRNA delivery can be used.
  • Numerous nanoparticles (NPs) prepared from polymers, liposomes, protein based NPs and inorganic NPs have been developed and a variety of nanoparticles are currently being evaluated in clinical studies.
  • One advantage of NPs is that they offer targeted tissue and/or site delivery. Their nanoscale size allows NPs to escape through blood vessels at the tissue site through leaky vascular structure (enhanced permeability and retention effect) in addition to this passive mechanism, a variety of targeting moieties can be attached to NPs (e.g., bound) to confer active targeting capability.
  • nanopartides for delivering compositions described herein include, but are not limited to, solid nanopartides (e.g., metals such as silver, gold, iron, titanium), non-metal nanopartides, lipid-based solids (e.g., liposome), polymers (e.g., polyethylenimene, dendrimer), suspensions of nanopartides, or combinations thereof (e.g., polyethylenimene-liposome, dendrisome).
  • solid nanopartides e.g., metals such as silver, gold, iron, titanium
  • non-metal nanopartides e.g., lipid-based solids (e.g., liposome)
  • polymers e.g., polyethylenimene, dendrimer
  • suspensions of nanopartides e.g., polyethylenimene-liposome, dendrisome.
  • MSNs Mesoporous Silica Nanopartides
  • a delivery vehicle comprises a mesoporous silica nanopartide (MSN).
  • MSNs are inorganic NPs suitable for delivery of anticancer drugs and siRNAs.
  • MSNs are typically synthesized by the sol -gel method (see, Finlay et ai., 2015, Nanomedicine: 11:1657-66), which enables preparation of homogeneous nanoparticles with diameters as small as 40 nm or as large as desired in the nanopartide scale.
  • MSNs of the disclosure are prepared such that their average diameter is between about 40 nm and about 900 nm in diameter, about 50 nm and about 800 nm in diameter, about 75 n and about 600 nm in diameter, about 100 n and about 500 nm in diameter, o about 150 nm and about 400 nm in diameter. In some embodiments, MSNs of the disclosure are about 100 nm in diameter, about 200 nm in diameter, about 300 nm in diameter, about 400 nm in diameter, about 500 nm, about 600 nm in diameter, about 700 nm in diameter, about 800 nm in dimeter, or about 900 nm in diameter.
  • a single MSN can include thousands of pores that provide significant storage space for drugs (e.g., anti-cancer drugs) and other compounds.
  • MSNs of the disclosure are prepared such that they contain more than 1,000 pores, more than 2,000 pores, more than 3,000 pores, more than 4,000 pores, more than 5,000 pores, more than 10,000 pores, more than 20,000 pores, more than 30,000 pores, more than 40,000 pores, more than 50,000 pores, or more than 100,000 pores.
  • MSNs are biocompatible and their safety has been demonstrated in a number of animal experiments (see, e.g., Roberts et al., (2017), Nanomedicine: Nanotechnology , Biology , and Medicine, 13:965-976; Lu et al., (2010), Small, 6:1794-1805 and Wang et al., (2015), Nanomedicine: Nanotechnology, Biology and Medicine. ll(2):313-327).
  • MSNs can be coated (e.g., conjugated) with one or more targeting moieties that bind to a receptor on a target cell (e.g., cancer cells), thereby enhancing nanoparticle uptake by the target cell, and optionally, a nanovalve that controls release of an anti-cancer drug in the MSN resulting in apoptosis. Additional guidance for making and using MSNs can be found in at least U.S. Patent Publications 2012/0207795 and 2017/0233733, the contents of each are incorporated herein in their entireties in some embodiments, MSNs can be prepared according to the method set forth in Roberts et al., (2017).
  • the nanoparticle is porous.
  • the porous structure of a nanoparticle may allow both the binding of nucleotides on the surface of the nanoparticle as well as encapsulation of small molecules (e.g., drugs) within the nanoparticle.
  • one or more anti-cancer agents are encapsulated within the pores of a nanoparticle.
  • a nanoparticle comprises a nanovalve, as described below.
  • release of an anti-cancer agent from a nanoparticle e.g., a MSN
  • Nanovalves can provide an open/ciose function so that an anti-cancer agent stored in the pores of the nanoparticle is only released when the nanoparticle encounters a specific condition (e.g., change in pH, exposure to magnetic field or light) that transitions the nanovalve from closed to open, thereby releasing the anti-cancer agent from the pores of the MSN.
  • a specific condition e.g., change in pH, exposure to magnetic field or light
  • the surface of a nanoparticle can be chemically modified.
  • a silica surface e.g., MSN
  • a silica surface may be converted into positive charge to bind DNA or siRNA.
  • Methods for introducing cationic charge to inorganic materials typically involve surface grafting with amine groups and coating with cationic polymers (e.g. polyethylenimine, polyamidoamine, polylysine) through either covalent or non-covending (e.g. electrostatic) association (see, e.g., Radu et al., (2004) 1. Am. Chem.
  • a nanopartide e.g. MSN
  • PEI polyethylenimine
  • Any method known in the art to bind, conjugate, coat, or attach PEI to nanoparticles is suitable for use with the invention.
  • chemically modified MSNs can be prepared according to the method set forth in Finlay et al., (2015) Nanomedicine , 11:1657-66.
  • the surface of a nanopartide can be bound (e.g., coated) with a positively charged compound or chemical moiety (e.g. polyethyleneimine (PEI)), which non- covalently binds to negatively charged nucleic acids (e.g., anti-SNAIL siRNA) via electrostatic interaction.
  • a positively charged compound or chemical moiety e.g. polyethyleneimine (PEI)
  • PEI polyethyleneimine
  • the positively charged compound also referred to herein as a positively charged non-covalent linker
  • the positively charged non-covalent linker compacts DNA and siRNA into complexes that are effectively taken up in cells (see., Roberts et al., 2017).
  • the positively charged non-covalent linker is attached to the nanopartide surface through covalent (e.g., using bioconjugate techniques as disclosed herein or known in the art) and electrostatic interactions.
  • PEI is attached to a nanopartide surface through covalent and electrostatic interactions.
  • PEI polymers of different size can be used to prepare compositions of the invention in some embodiments, PEI polymers range in molecular weight (MW) from 0.6 KD to 25 KD. in some embodiments, a nanopartide can be modified using a low MW multi-branched PEI (e.g., less than or equal to a 1200 Da).
  • the surface of a delivery vehicle comprises one or more targeting moieties.
  • a targeting moiety e.g., on a nanopartide refers to a moiety that targets and binds to a receptor found on the surface of a target ceil, for example, a cancer cell.
  • a delivery vehicle comprises a targeting moiety that binds to a cell receptor found in higher abundance in cancer cells as compared to non- cancerous cells.
  • the targeting moiety targets and binds to a receptor such as, but not limited to, CD44, CD34, CD117, CD99MIC2, CD133, MMP-2, folate receptors, and follicle stimulating hormone receptor and other hormone receptors
  • the delivery vehicle comprises a targeting moiety known to bind to a ceil receptor associated with cancer such as, but not limited to, HER2, progesterone-receptors (PR+) and estrogen receptors (ER+)).
  • the delivery vehicle comprises a targeting moiety known to bind to a tumor-associated carbohydrate antigen (TACAs) (see, Cazet et al., 2010, Breast Cancer Research, 12:204).
  • TACAs tumor-associated carbohydrate antigen
  • the delivery vehicle comprises a targeting moiety known to bind to a ceil receptor associated with ovarian cancer such as, but not limited to, CD151, mesothelin, Cancer Antigen 125 (CAI25), Carcinoembryonic antigen (CEA), M2-PK, and Sialyl-Tn.
  • a targeting moiety known to bind to a ceil receptor associated with ovarian cancer such as, but not limited to, CD151, mesothelin, Cancer Antigen 125 (CAI25), Carcinoembryonic antigen (CEA), M2-PK, and Sialyl-Tn.
  • the delivery vehicle comprises at least one targeting moiety that targets and binds to a receptor found on an ovarian cancer ceil (e.g., CD44 or a folate receptor) in some embodiments, the delivery vehicle (e.g. MSN) comprises two or more surface modifications, such as attachment of PEI and a targeting moiety (e.g., CD44).
  • the targeting moiety is hyaluronic acid (HA).
  • the targeting moiety is folic acid (FA).
  • compositions provided herein include a SNAIL inhibitor bound to a delivery vehicle having the following components: HA- PEI-MSN-anti-SNA!L siRNA-anti-cancer agent or FA-PE!-MSN-anti-SNAIL siRNA-anti-cancer agent.
  • a delivery vehicle e.g., MSN
  • MSN delivery vehicle
  • Nanovalves are chemical moieties (e.g., rotaxanes and pseudorotaxanes) that include a stalk and a moving part.
  • the moving part acts as a gatekeeper of the MSN pore openings. Pore openings can be triggered by recognition events such as, but not limited to, pH, redox, charge, metal ions biomolecules (such as enzymes) and external control (such as magnetic field and light).
  • An exemplary nanova!ve is triggered by low pH (around pH 6).
  • One exemplary nanova!ve comprises a stalk in which cyclodextrin is attached.
  • a delivery vehicle can comprise a nanovalve that is activated by an external stimulus.
  • a thermosensitive nanovalve For example, a thermosensitive nanovalve.
  • Nanoparticles containing nanovalves that are activated by an external stimulus are an attractive method for drug delivery as they provide a nan-invasive treatment having refined control over a target area, exposure time, and hence dosage.
  • magnetic core nanoparticies e.g., Magnet-MSN also referred to herein as MSN-B
  • a magnetic nanoparticle when exposed to an oscillating magnetic field, can produce heat (temperature in excess of 42°C) that stimulates a thermally responsive nanovalve (such as, thermosensitive pseudorotaxane). Additional guidance regarding making and using magnetic nanoparticies can be found in the U.S. Patent Publication 2010/0255103, the content of which is incorporated herein in its entirety. Additional guidance regarding nanovalves triggered by light can be found in U.5. Patent Publication 2010/0284924, the content of which is incorporated herein in its entirety.
  • bioconjugate refers to the association between atoms or molecules.
  • the association can be direct or indirect.
  • a bioconjugate between a first moiety e.g. — NH2, — COOH, — N- hydroxysuccinimide, or— maleimide
  • a second moiety e.g., sulfhydryl, sulfur-containing amino acid
  • covalent bond or linker e.g. a first linker or second linker
  • non-covalent bond e.g. electrostatic interactions
  • bioconjugates are formed using conjugate chemistry including, but not limited to, nucleophilic substitutions (e.g., reactions of amines and alcohols with acyl halides, active esters), electrophilic substitutions (e.g., enamine reactions) and additions to carbon-carbon and carbon-heteroatom multiple bonds (e.g., Michael reaction, Diels-Alder addition).
  • nucleophilic substitutions e.g., reactions of amines and alcohols with acyl halides, active esters
  • electrophilic substitutions e.g., enamine reactions
  • additions to carbon-carbon and carbon-heteroatom multiple bonds e.g., Michael reaction, Diels-Alder addition.
  • a first moiety e.g., a SNAIL inhibitor
  • a second moiety on a delivery vehicle e.g., a nanoparticle
  • a non-covalent chemical linker or covalent chemical linker formed by a reaction between the first moiety [e.g., a SNAIL inhibitor) and the second moiety on the delivery vehicle.
  • the first moiety includes one or more reactive moieties, e.g., a covalent reactive moiety, as described herein (e.g., alkyne, azide, amine, ester, N-hydroxy-succinimide, maieimide or thiol reactive moiety).
  • the first moiety includes a linker (e.g., first linker) with one or more reactive moieties, e.g., a covalent reactive moiety, as described herein (e.g., alkyne, azide, amine, ester, N-hydroxy-succinimide, maieimide or thiol reactive moiety).
  • the delivery vehicle includes one or more reactive moieties, e.g., a covalent reactive moiety, as described herein (e.g., alkyne, azide, amine, ester, N-hydroxy-succinimide, maieimide or thiol reactive moiety).
  • the delivery vehicle includes a linker with one or more reactive moieties, e.g., a covalent reactive moiety, as described herein (e.g., alkyne, azide, amine, ester, N-hydroxy-succinimide, maieimide or thiol reactive moiety).
  • a linker with one or more reactive moieties e.g., a covalent reactive moiety, as described herein (e.g., alkyne, azide, amine, ester, N-hydroxy-succinimide, maieimide or thiol reactive moiety).
  • reactive functional groups e.g., reactive groups such as bioconjugate or bioconjugate reactive groups
  • conjugate chemistries herein include, for example:
  • haloalkyi groups wherein the halide can be later displaced with a nucleophilic group such as, for example, an amine, a carboxylate anion, thiol anion, carbanion, or an alkoxide ion, thereby resulting in the covalent attachment of a new group at the site of the halogen atom;
  • a nucleophilic group such as, for example, an amine, a carboxylate anion, thiol anion, carbanion, or an alkoxide ion
  • dienophi!e groups which are capable of participating in Diels-Alder reactions such as, for example, maleimido or maieimide groups;
  • aldehyde or ketone groups such that subsequent derivatization is possible via formation of carbonyl derivatives such as, for example, imines, hydrazones, semicarbazones or oximes, or via such mechanisms as Grignard addition or aikyilithium addition;
  • su!fonyi halide groups for subsequent reaction with amines, for example, to form sulfonamides;
  • thiol groups which can be converted to disulfides, reacted with acyl halides, or bonded to metals such as gold, or react with maleimides;
  • amine or sulfhydryi groups (e.g., present in cysteine), which can be, for example, acylated, alkylated or oxidized;
  • alkenes which can undergo, for example, cycloadditions, acylation, Michael addition, etc;
  • the present invention also provides siRNA comprising a nucleic acid sequence of any one or more of SEQ ID NOs: 1-20, 27 and 28.
  • the siRNA can include at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any one of SEQ ID NGs:l-2G, 27 and 28.
  • a siRNA of the disclosure can be included in a vector for transfection and expression of the siRNA.
  • the present invention also provides a DNA sequence encoding a siRNA nucleic acid sequence of any one of SEQ ID NQs: 1-20, 27 and 28. Such DNA sequences can be included in a vector for transfection and expression of the siRNA.
  • the present invention also provides a DNA sequence encoding an SNAIL inhibitor in some embodiments, the SNAIL inhibitor corresponds to a nucleic acid sequence encoding a protein, polynucleotide, or fragment thereof that reduces or inhibits expression of a SNAIL gene or Snail protein in vivo or in vitro in some embodiments, reduction in Snail protein or gene expression is at least 10%, 20%, 30? , 40%, 50%, 60%, 70%, 80%, 90& or more, as compared to native Snail expression under the same or comparable conditions.
  • the invention further provides methods of using the compositions described herein.
  • methods of treating a subject diagnosed with cancer e.g., ovarian cancer
  • the method comprises administering to a subject in need thereof a therapeutically effective amount of a SNAIL inhibitor bound to a delivery vehicle.
  • the methods described herein relate to treating ovarian cancer.
  • the methods described herein relate to treating epithelial ovarian cancer in some embodiments, the method comprises treating a subject with an effective amount of a composition disclosed herein.
  • the method comprises treating a subject diagnosed with ovarian cancer with an effective amount of an anti-SNAIL siRNA bound to a delivery vehicle.
  • the anti-SNA!L siRNA is selected from one or more of SEQ ID NOs:l-20, 27 and 28.
  • the method comprises administering to a subject in need thereof a therapeutically effective amount of a SNAIL inhibitor bound to a delivery vehicle.
  • the methods described herein relate to inhibiting ovarian cancer metastasis.
  • the methods described herein relate to inhibiting epithelial ovarian cancer metastasis.
  • the method comprises treating a subject with an effective amount of a composition disclosed herein.
  • the method comprises administering to a subject diagnosed with ovarian cancer an effective amount of an anti-SNAIL siRNA bound to a delivery vehicle.
  • the anti-SNAIL siRNA is selected from one or more of SEQ ID NOs:l-20, 27 and 28.
  • the therapeutic methods disclosed herein comprise administering a SNAIL inhibitor bound to a delivery vehicle i n
  • the SNAIL inhibitor is a protein or polynucleotide that reduces or inhibits expression of a SNAIL1 , SNAIL2, ZEB1 or ZEB2 gene or a Snaill, Snail2, Zebl or Zebl protein (e.g., in vitro or in vivo).
  • a SNAIL inhibitor comprises a compound that inhibits or reduces the production of a Snaill, Snaii2, Zebl, or Zeb2 protein or mRNA transcript (e.g., in vitro or in vivo).
  • a SNAIL inhibitor bound to a delivery vehicle inhibits or reduces the expression of a SNA!Ll or SNAIL2 gene or Snaill or Snail2 protein (e.g., in an in vitro or in vivo assay) by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or more as compared to the level of expression of the corresponding Snaill or Snail2 protein or corresponding SNAIL1 or SNAIL2 gene in the absence of the SNAIL inhibitor bound to the delivery vehicle.
  • the SNAIL inhibitor comprises a siRNA.
  • the SNAIL inhibitor comprises a siRNA selected from SEQ ID NGs:l-20, 27 and 28.
  • the SNAIL inhibitor comprises siSNAIL-7 (i.e., SEQ ID NGs:13 and 14).
  • determining whether a compound is a SNAIL inhibitor can be accomplished by measuring the expression of a Snail protein or mRNA transcript in the presence of the compound and detecting a decrease in expression of the Snail protein or mRNA transcript.
  • determining whether a compound is a SNAIL inhibitor can be performed by measuring the relative reduction in protein expression of a Snail protein by incubating the 'test' compound and 'control' samples under test conditions.
  • the expression of proteins can be routinely detected using Western blot, and mRNA transcripts can be detected using quantitative reverse transcriptase PCR or Northern blot.
  • a compound that reduces expression of a Snail protein or mRNA transcript by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50?4, or more, is a SNAIL inhibitor.
  • a SNAIL inhibitor bound to a delivery vehicle e.g., HA-PEi- MSN-SEQ ID NOs: 13 and 14 or FA-PEi-MSN-SEQ ID NGs:l and 2 is administered in a therapeutically effective amount in some embodiments, a daily dose range of about 0.01 mg/kg to about 500 mg/kg, or about Q.l mg/kg to about 200 mg/kg, or about 1 mg/kg to about 100 mg/kg, or about 10 mg/kg to about 50 mg/kg, can be used.
  • the dosages may be varied depending upon the requirements of the patient, the severity of the condition being treated (e.g., early stage or advanced cancer), and the agent being employed.
  • the dose will also be determined by the existence, nature, and extent of any adverse side -effects that accompany the administration of a particular agent in a particular patient. Determination of the proper dosage for a particular situation is within the skill of the practitioner. In some embodiments, treatment is initiated with smaller dosages which are less than the optimum dose. Thereafter, the dosage is increased by small increments until the optimum effect under the circumstances is reached. For convenience, the total daily dosage may be divided and administered in portions during the day, if desired in some embodiments, the SNAIL inhibitor bound to the delivery vehicle fe.g., HA-MSN-SEQ.
  • ID NOs:13 and 14 is administered to a subject for a period of at least 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, or longer.
  • the SNAIL inhibitor bound to the delivery vehicle is administered for an indefinite period of time. In some embodiments, the SNAIL inhibitor bound to the delivery vehicle is administered for the rest of the subject's life or until administration of the Snail inhibitor no longer provides a therapeutic benefit.
  • a SNAIL inhibitor bound to a delivery vehicle can be administered according to any suitable method.
  • Methods of administration include, for example, intravenous, intraperitoneal, intramuscular, subcutaneous, or oral administration.
  • treatment with a SNAIL inhibitor bound to a delivery vehicle as disclosed herein is combined with one or more other therapies (e.g., combination therapy).
  • treatment with a SNAIL inhibitor bound to a delivery vehicle is combined with one or more anti-cancer agents.
  • the subject to be treated is a human in some embodiments, the subject is an adult human at least 30 years of age. In some embodiments, the subject is a human who has been diagnosed with cancer or who is suspected of having cancer. In some embodiments, the subject is an adult female human (e.g., diagnosed with or suspected of having ovarian cancer). In some embodiments, the subject is an adult female human who has been diagnosed with or is suspected of having epithelial ovarian cancer.
  • the methods disclosed herein comprise treating a subject having or suspected of having cancer in some embodiments, the subject has refractory cancer.
  • treatment efficacy can be measured using one or more tests for measuring remission, diminishment, or elimination of cancer from the subject, e.g., as described below. Any suitable method for determining treatment efficacy may be used and such methods are considered within the scope of a treating physician in some embodiments, treatment of a subject having cancer or suspected of having cancer is measured by detecting the level of Snail expression in the subject e.g , as described below.
  • treatment of a subject having cancer or suspected of having cancer is measured by imaging analysis, e.g., as described below in another embodiment, treatment of a subject with cancer or suspected of having cancer can be determined by measuring tumor size and/or volume. In another embodiment, treatment of a subject with cancer or suspected of having cancer can be determined by measuring survival rate of the subject as compared to other patients having the same form of cancer (e.g., ovarian cancer).
  • imaging analysis e.g., as described below
  • treatment of a subject with cancer or suspected of having cancer can be determined by measuring tumor size and/or volume.
  • treatment of a subject with cancer or suspected of having cancer can be determined by measuring survival rate of the subject as compared to other patients having the same form of cancer (e.g., ovarian cancer).
  • treatment of a subject having cancer is measured by assessing a target area of a subject (e.g., liver, lungs, ovaries) using magnetic resonance imaging (MRi), computed tomography (CT), or positron emission tomography (PET) imaging in some embodiments, treatment is measured by comparing the target area of the subject having cancer (e.g., MRI of the liver of the subject) prior to administration of a composition disclosed herein to the target area of the subject after administration of the composition (e.g., measured between 1 and 6 months after the composition is first administered).
  • MRi magnetic resonance imaging
  • CT computed tomography
  • PET positron emission tomography
  • treatment of a subject can be measured by comparing the level of Snail expression in a sample from the subject after administration of the composition (e.g., HA-PEI-MSN-SEQ ID NGs:13 and 14) to the subject (e.g., between 1 and 6 months after first administration) to the level of the Snail expression in a sample from a similarly situated subject (e.g., same gender, age, and ethnicity) who has not been administered with the composition.
  • the composition e.g., HA-PEI-MSN-SEQ ID NGs:13 and 14
  • the subject e.g., between 1 and 6 months after first administration
  • a sample from a similarly situated subject e.g., same gender, age, and ethnicity
  • treating the subject can result in a reduction in tumor size.
  • a reduction in tumor size is also referred to as "tumor regression".
  • tumor size would be reduced by about 5% or greater relative to tumor size prior to treatment with a composition disclosed herein; more preferably, tumor size is reduced by about 10% or greater; more preferably, reduced by about 20% or greater; more preferably, reduced by about 30% or greater; more preferably, reduced by about 40% or greater; even more preferably, reduced by about 50% or greater; and most preferably, reduced by greater than about 75% or greater.
  • Tumor size may be measured by any reproducible means of measurement. For example, tumor size may be measured as a diameter of the tumor.
  • treating cancer can result in a reduction in tumor volume.
  • tumor volume would be reduced by about 5% or greater relative to tumor size prior to treatment with a composition disclosed herein; more preferably, tumor volume is reduced by about 10% or greater; more preferably, reduced by about 20% or greater; more preferably, reduced by about 30% or greater; more preferably, reduced by about 40% or greater; even more preferably, reduced by about 50% or greater; and most preferably, reduced by greater than about 75% or greater.
  • Tumor volume may be measured by any reproducible means of measurement.
  • treating a subject with cancer using a composition disclosed herein can result in an increase in average survival time of a population of treated subjects in comparison to a population of untreated subjects.
  • the average survival time is increased by more than 30 days; more preferably, by more than 60 days; more preferably, by more than 90 days; and most preferably, by more than 120 days.
  • An increase in average survival time of a population may be measured by any reproducible means. For example, by calculating for a population the average length of survival following initiation of treatment or alternatively by calculating for a population the average length of survival following completion of a first round of treatment.
  • treating cancer with a composition disclosed herein can result in a decrease in mortality rate of a population of treated subjects in comparison to an untreated population in some embodiments, the mortality rate is decreased by more than 2%; more preferably, by more than 5%; more preferably, by more than 10%; and most preferably, by more than 25%.
  • a decrease in mortality rate may be measured by any reproducible means. For example, by calculating for a population the average number of disease-related deaths per unit time following initiation of treatment or alternatively by calculating for a population the average number of disease-related deaths per unit time following completion of a first round of treatment.
  • the disclosure provides methods for inhibiting or reducing cancer metastasis in a subject in some embodiments, administration of a SNAIL inhibitor bound to a delivery vehicle in an effective amount to a subject (e.g., FA-PEI-MSN-SEQ ID NOs:13 and 14) inhibits or reduces cancer metastasis in the subject inhibition or reduction in cancer metastasis is determined using one or more techniques known in the art (e.g., an imaging technique, protein or mRNA transcript measurement, as described herein) over a period of time (e.g., between 1 and 6 months or between 1 month and 1 year), after administration of the composition to the subject.
  • a SNAIL inhibitor bound to a delivery vehicle in an effective amount to a subject e.g., FA-PEI-MSN-SEQ ID NOs:13 and 14
  • an imaging technique e.g., protein or mRNA transcript measurement, as described herein
  • a period of time e.g., between 1 and 6 months or between 1 month and 1 year
  • reducing or inhibiting metastasis can be determined by the observation of an absence of secondary cancer sites (i.e., metastatic cancer cells) in the subject by an attending physician or veterinarian following treatment. For example, by measuring a decrease in the number of metastatic lesions in other tissues or organs distant from the primary tumor site.
  • the number of metastatic lesions would be reduced by about 5% or greater relative to number prior to treatment; more preferably, the number of metastatic lesions is reduced by about 10% or greater; more preferably, reduced by about 20% or greater; more preferably, reduced by about 30% or greater; more preferably, reduced by about 40% or greater; even more preferably, reduced by about 50? or greater; and most preferably, reduced by greater than about 75%.
  • the number of metastatic lesions may be measured by any reproducible means of measurement.
  • the number of metastatic lesions may be measured by counting metastatic lesions visible to the naked eye or at a specified magnification (e.g., IOc, lOOx, or 50Qx magnification).
  • magnification e.g., IOc, lOOx, or 50Qx magnification.
  • reduction or inhibition of metastasis in a subject can be determined by an absence of secondary cancers in the subject after 6 months or more, following treatment of the subject in some embodiments, reduction or inhibition in metastasis can be monitored in the subject at each time point the subject receives an additional treatment or a pre-determined point thereafter.
  • the efficacy of cancer treatment is measured by imaging an area of the subject contemplated for treatment.
  • imaging an area of the subject contemplated for treatment For example, in some embodiments, magnetic resonance imaging (MRI), computed tomography (CT), or positron emission tomography (PET) is performed to evaluate the presence of a tumor in the subject (e.g., liver, lung, ovaries, prostate, etc.,) and observation of the tumor's decrease in size and/or volume as a result of treatment.
  • MRI magnetic resonance imaging
  • CT computed tomography
  • PET positron emission tomography
  • treatment of cancer in a subject is determined by evaluating the anatomical or organ structure of the subject using MRI, e.g., structural MR! (sMR!), after the subject is treated with a SNAIL inhibitor bound to a delivery vehicle in some embodiments, the results of the imaging are compared to a baseline, e.g., from a control subject or from the subject prior to treatment.
  • MRI structural MR!
  • the method comprises measuring in a sample the level of expression of one or more Snail proteins or rrsRNA transcripts from the SNAIL gene.
  • the Snail protein corresponds to a full-length protein having at least 90% identity to NCBI : NM 005985.3 or NCBI : NP 005976.
  • treatment of cancer in a subject is determined by measuring the level of Snail protein or Snail rrsRNA transcript in a sample from a subject being treated with a SNAIL inhibitor bound to a delivery vehicle (e.g., HA-PEI-MSN- SEQ ID NOs:13 and 14).
  • a delivery vehicle e.g., HA-PEI-MSN- SEQ ID NOs:13 and 14
  • the level of expression of Snail mRNA or protein in the sample is decreased by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or more as compared to a reference value (e.g., the level of Snail expression in a sample from the subject prior to treatment).
  • a reference value e.g., the level of Snail expression in a sample from the subject prior to treatment.
  • polynucleotide (e.g., mRNA) expression is measured using routine techniques such as reverse transcription polymerase chain reaction (RT-PCR), Real- Time reverse transcription polymerase chain reaction (Real-Time RT-PCR), semi-quantitative RT-PCR, quantitative polymerase chain reaction (qPCR), quantitative RT-PCR (qRT-PCR), multiplexed branched DIMA (bDNA) assay, microarray hybridization, or sequence analysis (e.g., RNA sequencing ("RNA-Seq”)).
  • RT-PCR reverse transcription polymerase chain reaction
  • Real-Time RT-PCR Real-Time reverse transcription polymerase chain reaction
  • qPCR quantitative polymerase chain reaction
  • qRT-PCR quantitative RT-PCR
  • bDNA multiplexed branched DIMA
  • microarray hybridization or sequence analysis (e.g., RNA sequencing ("RNA-Seq”)).
  • Quantitative PCR and RT-PCR assays for measuring gene expression are also commercially available (e.g., TaqMan® Gene Expression Assays, ThermoFisber Scientific). Other techniques for quantitating transcription are well known in the art (e.g., sequencing and mass spectrometry).
  • a detectable moiety can be used in the assays described.
  • a wide variety of detectable moieties can be used, with the choice of label depending on, for example, sensitivity required, ease of conjugation, stability requirements, and available instrumentation.
  • Suitable detectable moieties include, but are not limited to, radionuclides, fluorescent dyes ⁇ e.g., fluorescein, fluorescein isothiocyanate (FITC), Oregon GreenTM, rhodamine, Texas red, tetrarhodimine isothiocynate (TRITC), Cy3, Cy5, etc. ⁇ , fluorescent markers ⁇ e.g., green fluorescent protein (GFP), phycoerythrin, etc. ⁇ , autoquenched fluorescent compounds that are activated by tumor-associated proteases, enzymes ⁇ e.g., iuciferase, horseradish peroxidase, alkaline phosphatase, etc.), biotin, digoxigenin, metals, and the like.
  • a signal from a detectable moiety can be analyzed, for example, using a spectrophotometer to detect color from a chromogenic substrate; a radiation counter to detect radiation; or a fluorometer to detect fluorescence in the presence of light of a certain wavelength.
  • a quantitative analysis can be made using a spectrophotometer in some embodiments, the amount of signal can be quantified using an automated high-content imaging system ⁇ e.g., ! ageXpress, Molecular Devices Inc., Sunnyvale, CA).
  • a measurement from a subject is compared to a reference value.
  • a unit of measurement e.g., MR! or mRNA transcript
  • a reference value for a unit of measurement as described herein is determined for the subject prior to treatment with a SNAIL inhibitor bound to a delivery vehicle.
  • the subject is assessed prior to treatment to determine a "baseline value" for the subject, against which the subject is compared at one or more time points after treatment.
  • a reference value for an imaging technique is determined by imaging a target area of a normal subject or population of subjects (e.g., subjects known not to have cancer) to determine characteristic features of a normal target area (e.g., size, volume, shape, etc.,).
  • a reference value for Snail expression is determined by assessing the level of Snail expression in samples (e.g., by mass spectrometry) from a normal subject or population of subjects (e.g., subjects known not to have cancer).
  • a reference value is determined for a population of subjects (e.g., 10, 20, 50, 100, 200, 500 subjects or more) known not to have cancer.
  • a reference value for a unit of measurement is determined for a subject or population of subjects diagnosed as having the same form of cancer as the test subject (e.g., ovarian cancer).
  • a reference value for an imaging analysis is determined by imaging a target area of a subject or population of subjects diagnosed as having ovarian cancer to determine characteristics for the disease
  • a reference value for Snail expression is determined by assessing the level of Snail expression in samples from a subject or population of subjects diagnosed as having ovarian cancer.
  • a reference value is determined for a population of subjects (e.g., 10, 20, 50, 100, 200, 500 subjects or more) ail having ovarian cancer.
  • the population of subjects is matched to a test subject according to one or more patient characteristics (e.g., age, sex, ethnicity, or other criteria) in some embodiments, the reference value is established using the same type of sample from the population of subjects (e.g., a sample comprising epithelial ceils from an ovary).
  • patient characteristics e.g., age, sex, ethnicity, or other criteria
  • the reference value is established using the same type of sample from the population of subjects (e.g., a sample comprising epithelial ceils from an ovary).
  • compositions and kits comprising a SNAIL inhibitor bound to a delivery vehicle are provided.
  • the pharmaceutical compositions and kits are for use in treating a subject having cancer (e.g., ovarian cancer).
  • the pharmaceutical compositions and kits are for use reducing or inhibiting metastasis of cancer.
  • compositions comprising a SNAIL inhibitor bound to a delivery vehicle are provided in some embodiments, the SNAIL inhibitor is a protein or fragment thereof that reduces expression of a SNAIL gene or Snail protein as compared to a control not treated with the Snail inhibitor in some embodiments, the SNAIL inhibitor is an anti-SNAIL siRNA bound to a delivery vehicle i n some embodiments, the anti- SNAIL siRNA bound to the delivery vehicle is any one of SEQ ID NOs:l-20, 27 and 28. In some embodiments, the siRNA bound to the delivery vehicle is SEQ ID NOs:13 and 14.
  • the siRNA includes a nucleic acid sequence having at least 9Q%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to any one or more of SEQ ID NOs:l-20, 27 and 28.
  • the delivery vehicle is a nanoparticie or a lipid particle.
  • the nanoparticie is a MSN or MSN coated with one or more surface modifications such as, but not limited to, PEI and/or a targeting moiety (e.g., HA or FA).
  • the delivery vehicle is a MSN having a nanovalve that is triggered under specific conditions to release one or more anti-cancer agents from pores of the MSN.
  • compositions can further comprise one or more pharmaceutically acceptable carriers, adjuvants, and/or vehicles appropriate for the particular route of administration for which the composition is to be employed in some embodiments, the carrier, adjuvant, and/or vehicle is suitable for intravenous, intramuscular, oral, intraperitoneal or subcutaneous administration.
  • pharmaceutically acceptable carriers are well-known in the art.
  • Examples of pharmaceutically acceptable carriers include, but are not limited to, aqueous solutions, e.g., water or physiologically compatible buffers such as Hanks’s solution, Ringer's solution, or physiological saline buffer.
  • a pharmaceutical composition for use in in vivo administration is sterile. Sterilization can be accomplished according to methods known in the art, e.g., heat sterilization, steam sterilization, sterile filtration, or irradiation.
  • compositions may vary depending on the particular use envisioned. Determination of an appropriate dosage or route of administration is well within the skill of one in the art. Suitable dosages are also described in Section III above.
  • a composition or a pharmaceutical composition provided herein may, if desired, be presented in a kit (e.g., a pack or dispenser device) which may contain one or more unit dosage forms containing the composition or the pharmaceutical composition, for example (1) a SNAIL inhibitor bound to a delivery vehicle, (2) a composition including an anti-cancer drug and a SNAIL inhibitor bound to a delivery vehicle, or (3) a pharmaceutical composition including a pharmaceutically acceptable excipient and a composition described herein.
  • the pack may, for example, comprise metal or plastic foil, such as a blister pack.
  • the pack or dispenser device may be accompanied by instructions for administration.
  • Compositions described herein may be formulated in a compatible pharmaceutical carrier, placed in an appropriate container, and labeled for treatment of an indicated condition.
  • Instructions for use may also be provided instructions (e.g., written, tape, VCR, CD-ROM, etc.) for carrying out an assay to determine efficacy or activity of the pharmaceutical formulation may also be included in the kit.
  • the assay may for example be in the form of qRT-PCR, Western or Northern blot analysis, sequencing and mass spectrometry as known in the art. V. Examples
  • This example describes an exemplary experimental protocol for testing the effect of SNAIL inhibitors bound to a delivery vehicle in vitro .
  • siRNAs were designed to include a region of about 25 bp complementarity between the siRNA and Snail. Additionally, siRNAs were preferentially selected that were complementary to the 5’ end of the gene, in exons, and in regions that did not include complementary over a substantial portion (e.g., about 20 bp) to other Snai members (e.g., Snai2). in some instances, siRNAs were designed to include homology to mouse.
  • siRNAs Dicer-substrate small inhibitory RNAs (siRNAs) (siSNAIL-1 through siSNAIL-10, CrossReact siSnail.l and 1.2, siHPRT and siNC) were screened for their effectiveness at SNAIL knockdown in human embryonic kidney ceils (293T cells; Takara Bio, USA, Catalog No: 632180). See Table 1 and FIG. 1A and IB. The gene location provided in Table 1 refers to the start position of the corresponding siSNAIL RNA in the human SNAI1 gene. As is evident from FIG. 1A and FIG. IB, siSNAIL-7 gave a dose response for SNAil knockdown across all three concentrations.
  • siRNAs can be designed, for example using the criteria set forth in this example, and within about 100 bp, about 5Qbp, about 40 bp, about 30 bp, or about 20 bp upstream or downstream of the gene location of siSNA!L-7 that will also provide knockdown of SNAil.
  • Table 1 Exemplary anti-SNAIL siRNAs
  • r corresponds to RNA bases (as opposed to DNA)
  • the anti-SNAIL siRNAs contained one or more nucleic acid modifications, such as a 2'- O-methyl base and/or an inverted abasic ribose as indicated.
  • Anti-SMA!L siRNA duplexes were formed by placing equal molar volumes of each anti-SNAIL siRNA together and heating at 100°C for 10 minutes followed by cooling to room temperature over several hours.
  • 293T cells were obtained from Takara Bio, USA, Catalog No:632180. The 293T cells were plated at a concentration of 100 thousand cells in a 6 well plate. Transient transfection of each Anti-SNAIL siRNA was achieved using UPQFECTAMINE ® RNAiMax (Ther oFisher Scientific, Waltham, MA ⁇ at a concentration of 1 nm, 3 nm, or 10 nM per siRNA or siScramb!e. Transfection of the Anti-SNAIL siRNA was carried out by incubation at 37°C, 5% CO ? for 24 hours.
  • Snail 1 (forward): AAG CAT TTC AAC GCC TCC AAA
  • Probe 56-FAM/ CCC CAA TCG / ZEN / GAA GCC TAA CTA CAG C / BIABkFQ Snail 2 (forward): GGC TGC TAG AAG GCC AT
  • siSNA!L-7 was chosen for further evaluation due to its effective dose response for Snail knockdown (see, FIGS. 1A and IB).
  • Nanopartides leads to knockdown of SNAIL in ovarian cancer cells in vitro siRNAs
  • siRNAs were prepared, essentially as set forth in Example 1.
  • MSNs were prepared according to a sol-gel method known in the art (see., U.S. Patent Application 2017/0233733). The addition of cationic PEI coating to the MSNs was prepared as previously described (see, Finlay et a!., (2015), Nanomedicine: 11:1657-66).
  • MSNs having a targeting moiety for ovarian cancer cells (e.g., a targeting moiety for folate receptors or CD44) was prepared as previously described (see, Shahin et a!., (2016), Nanomedicine: Nanotechnology , Biology and Medicine , 14:1381-1394 and Lu et ai., (2012) Nanomedicine: Nanotechnology , Biology and Medicine, 8(2):212-2G, both of which are incorporated herein by reference in their entireties).
  • siSNAIL-7 (i.e., SEQ ID NOs:13 and 14) was loaded onto HA-PEI-MSNs prepared as described in U.S. Patent Application 2017/0233733, incorporated herein by reference.
  • the siSNAIL-7-HA-PEI-MSNs were incubated overnight at 4°C on a roller. The following day, an aliquot of the siSNAIL-7-HA-PEi-MSNs was added to each well of a 6-well plate containing GVCAR8 ovarian cancer cells (obtained from Cariotta Glackin's laboratory, City of Hope, CA). After 24, 48, 72, or 168 hours, Snail expression was detected by qRT-PCR, as set forth in Example 1, in comparison to a control siRNA (siScramble) (see FIG. 3).
  • a time dependent SNAIL knockdown was observed with greater than 95? SNAIL protein reduction at 24 hours following transfection (FIG. 3). The SNAIL knockdown continued at a rate of greater than 85% for at least 72 hours post-transfection.
  • Example 3 Treatment of a subject having ovarian cancer using HA-MSN-Anti-SNAIL siRNAs in vivo
  • siRNAs are prepared essentially as set forth in Example 1.
  • a delivery vehicle comprising MSNs is prepared, essentially as set forth in Example 2.
  • the MSNs can include a targeting moiety (e.g , folic acid or hyaluronic acid) for ovarian cancer cells (e.g., a targeting moiety for CD44 or folate receptors).
  • a targeting moiety e.g , folic acid or hyaluronic acid
  • ovarian cancer cells e.g., a targeting moiety for CD44 or folate receptors.
  • the siR A and delivery vehicle are bound together, essentially as set forth in Example 2.
  • composition e.g., HA-PEI-MSN-SEQ ID NOS:13 and 14
  • a subject having ovarian cancer e.g., via intravenous or intraperitoneal route, once or twice a week.
  • an appropriate dosage may include 15 n ol siRNA per 25 g mouse (approximately 100 ug/kg) or 600 nmol siRNA per kg of adult human (approximately 100 ug/kg).
  • cells from the subject can be harvested (e.g., a biopsy) and assessed for expression of SNAIL, for example using qRT-PCR, essentially as set forth in Example 1. Additionally, the subject can be monitored for characteristics indicative of a reduction in tumor load, such as decrease in tumor size, volume, or increased survival rate.
  • the subject can be administered with one or more subsequent treatments (e.g., HA-PEI-MSN- SEQ ID NGS:13 and 14), for example, until such time that the subject is deemed in remission.

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Abstract

The present invention relates to compositions comprising SNAIL inhibitors bound to a delivery vehicle and optionally one or more anti-cancer agents, and methods of using the compositions for the treatment of ovarian cancer.

Description

SNAIL SIR A-LOADED MESOPOROUS SILICA NANOPARTICLES
BACKGROUND OF THE INVENTION
[0001] Ovarian cancer is the most lethal gynecological cancer and fifth leading cause of cancer deaths among women (Siegel et al., 2017, C4 Cancer J Clin., 67(l):7-30). Current treatment options involve surgery and chemotherapy but are ineffective due to recurrent malignant tumor growth (Coleman et al., 2013, Nat Rev Clin Oncol. 10(4):211-24). Relapse is attributed in part to a small population of cancer stem cells (CSC) found within the tumor (Bati!e and C!evers., 2017, Nat Med. 23(10):1124-34). Expression of an epithelial mesenchymal transition regulator called Snail (Snail) within cancer cells has been linked to the acquisition of stem cell-like phenotypes (Shibue and Weinberg, 2017, Nat Rev Clin Oncol., 14(10):611-29 and Hojo et al., 2018, Sci Rep., 8(1):8704, both of which are incorporated by reference in their entireties).
[0002] Researchers have recently identified siRNA loaded mesoporous silica nanoparticles (MSNs) as a suitable delivery vehicle to downregulate expression of the TWIST gene, associated with ovarian cancer (Roberts et al., 2017, Nanomedicine, 13(3):965-976). Conjugating hyaluronic acid to the anti-TWIST siRNA loaded mesoporous silica nanoparticles (MSNs) was found to enhance delivery of the siRNAs to a specific receptor (CD44) in the ovarian cancer cells (Shahin et al., 2018, Nanomedicine: Nanotechnology, Biology, and Medicine , 14(4) : 1381-1394).
[0003] No current ovarian cancer therapies specifically target cancer stem cells. Thus, additional treatments for recurrent ovarian tu ors containing cancer stem cells are needed.
BRIEF SUMMARY OF THE INVENTION
[0004] In one aspect, compositions comprising a SNAIL inhibitor bound to a delivery vehicle are provided. [0005] In another aspect, a siRNA is provided having a nucleic acid sequence of any one of SEQ ID NOs: 1-20, 27 and 28.
[0006] In yet another aspect, DNA sequences encoding an siRNA sequence comprising the nucleic acid sequence of any one of SEQ ID NOs: 1-20, 27 and 28.
[0007] In another aspect, a method of treating ovarian cancer in a subject is provided. The method includes administering to the subject a therapeutically effective amount of a SNAIL inhibitor bound to a delivery vehicle.
[0008] In yet another aspect, a method of reducing or inhibiting ovarian cancer metastasis in a subject is provided. The method includes administering a therapeutically effective amount of a SNAIL inhibitor bound to a delivery vehicle to the subject.
BRIEF DESCRIPTION OF THE DRAWINGS
[0009] FIG. 1A and FIG. IB show reduced relative expression of Snail in 293T ceils after transfection of various small inhibitory RNAs (siRNAs) Twelve different siRNAs were tested, including anti-Snail siRNAs referred to herein as siSnail-1 through siSnail-10. The siRNAs were transfected into the 293T cells at three different concentrations (1 nM, 3 nM and 10 nM). 24 hours after transfection, the level of Snail RNA was determined by qRT-PCR using a 5' (FIG. 1A) or 3’ (FIG. IB) primer pair. Non-transfection (Non Tr) is shown. HPRT was also targeted and is shown as a positive control. siNC (non -specific control) is shown as a negative control; ail samples are normalized to siNC.
[0010] FIG. 2 shows relative expression of HPRT in 293T ceils after transfection of siRNAs (siSnail-1 through siSnail-10) provided at three different concentrations (1 nM, 3 nM and 10 nM). 24 hours after transfection, the level of HPRT RNA was determined by qRT-PCR. Non transfection (Non Tr) is shown. siHPRT is shown as a positive control. siNC (non-specific control) is shown as a negative control.
[0011] FIG. 3 is a graph showing decreased Snail expression in OvcarS cells at 24 to 72 hours after transfection with siRNA-loaded hyaluronic acid conjugated mesoporous silica nanoparticles (HA-MSNs). After 24 hours, a 97% decrease in Snail expression was observed. At 48 hours, a 92% decrease was noted and at 72 hours there was an 87% decrease in Snail expression. A positive control siRNA is included for comparison (siScramble). DETAILED DESCRIPTION OF THE INVENTION
I. INTRODUCTION
[0012] Disclosed herein are compositions comprising SNAIL inhibitors bound to a delivery vehicle and methods of treating ovarian cancer in a subject, or preventing metastasis of ovarian cancer cells in a subject using the same. It is known in the art that delivery of siRNAs to target cells (e.g., cancer cells) is hampered by the rapid digestion of siRNAs by nucleases and that siRNAs in the absence of a delivery vehicle do not enter cells in vitro (see, e.g., Roberts et al., (2017), Nanomedicine: Nanotechnology , Biology , and Medicine , 13:965-976). In order to improve delivery of siRNAs as therapeutic compounds, various chemical modifications (see, Behlke et al., (2008) Oligonucleotides , 18(4):305-19 and Czauderna et al., (2003), Nucleic Acids Research, 31(11):2705016) and delivery vehicles have been proposed. Recently, mesoporous silica nanoparticles loaded with TWIST siRNAs have been delivered in vitro in amounts capable of downregulating expression of TWIST in ovarian cancer ceils, suggesting a potential method for the treatment of ovarian cancer in vivo (Roberts et al., 2017). Yet other methods for the treatment of ovarian cancer, particularly ovarian tumors having cancer stem cell phenotype like cells within an ovarian tumor, are needed.
[0013] Thus, in one aspect, the disclosure provides novel compositions for targeting SNAIL and methods for treating ovarian cancer or reducing metastasis of ovarian cancer cells through the downregulation of SNAIL.
II. DEFINITIONS
[0014] While various embodiments and aspects of the present invention are shown and described herein, it will be obvious to those skilled in the art that such embodiments and aspects are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention it should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention.
[0015] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described. Ail documents, or portions of documents, cited in the application including, without limitation, patents, patent applications, articles, books, manuals, and treatises are hereby expressly incorporated by reference in their entirety for any purpose. [0016] Unless defined otherwise, all technical and scientific terms used herein have the meaning commonly understood by a person skilled in the art to which this invention belongs. The following references provide one of skill with a general definition of many of the terms used in this invention: Singleton et al., Dictionary of Microbiology and Molecular Biology (2nd ed. 1994); The Cambridge Dictionary of Science and Technology (Walker ed., 1988); The Glossary of Genetics, 5th Ed., R. Rieger et al. (eds.), Springer Verlag (1991); and Hale & Marham, The Harper Collins Dictionary of Biology (1991). As used herein, the following terms have the meanings ascribed to them unless specified otherwise.
[0017] The use of a singular indefinite or definite article (e.g., "a," "an," "the," etc.) in this disclosure and in the following claims follows the traditional approach in patents of meaning "at least one" unless in a particular instance it is clear from context that the term is intended in that particular instance to mean specifically one and only one. Likewise, the term "comprising" is open ended, not excluding additional items, features, components, etc. References identified herein are expressly incorporated herein by reference in their entireties unless otherwise indicated.
[0018] The terms "comprise," "include,” and "have," and the derivatives thereof, are used herein interchangeably as comprehensive, open-ended terms. For example, use of "comprising," "including," or "having" means that whatever element is comprised, had, or included, is not the only element encompassed by the subject of the clause that contains the verb.
[0019] The terms "polypeptide," "peptide" and "protein" are used interchangeably herein to refer to a polymer of amino acid residues, wherein the polymer may In embodiments be conjugated to a moiety that does not consist of amino acids. The terms apply to amino acid polymers in which one or more amino acid residue is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers and non-naturally occurring amino acid polymers.
[0020] As used herein, the terms“nucleic acid," "oligonucleotide,"“nucleic acid sequence," "nucleic acid fragment" and "polynucleotide" are used interchangeably and are intended to include, but are not limited to, a polymeric form of nucleotides covalently linked together that may have various lengths, either deoxyribonucleotides or ribonucleotides, or analogs, derivatives or modifications thereof. Different polynucleotides may have different three- dimensional structures, and may perform various functions, known or unknown. Non-limiting examples of polynucleotides include a gene, a gene fragment, an exon, an intron, intergenic DNA, messenger RNA (rnRNA), transfer RNA, ribosomal RNA, a ribozyme, cDNA, a recombinant polynucleotide, a branched polynucleotide, a plasmid, a vector, isolated DNA of a sequence, isolated RNA of a sequence, a nucleic acid probe, and a primer. Polynucleotides useful for the invention may comprise natural nucleic acid sequences and variants thereof, artificial nucleic acid sequences, or a combination of such sequences. A polynucleotide is typically composed of a specific sequence of four nucleotide bases: adenine (A); cytosine (C); guanine (G); and thymine (T) (uracil (U) for thymine (T) when the polynucleotide is RNA). Polynucleotides may optionally include one or more non-standard nucleotide(s), nucleotide analog(s) and/or modified nucleotides.
[0021] "Percentage of sequence identity" is determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide or polypeptide sequence in the comparison window may comprise additions or deletions (i.e., gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity.
[0022] As used herein, the terms "identical" or percent "identity," in the context of two or more nucleic acids or polypeptide sequences, refers to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same (i.e., about 60% identity, preferably 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher identity over a specified region, when compared and aligned for maximum correspondence over a comparison window or designated region) as measured using a BLAST or BLAST 2.0 sequence comparison algorithms with default parameters described below, or by manual alignment and visual inspection (see, e.g., NCBI web site http://www.ncbi.nlm.nih.gov/BLAST/or the like). Such sequences are then said to be "substantially identical." This definition also refers to, or may be applied to, the compliment of a test sequence. The definition also includes sequences that have deletions and/or additions, as well as those that have substitutions. Algorithms, such as BLAST, can account for gaps. Preferably, identity exists over a region that is at least about 15 amino acids or nucleotides in length, or more preferably over a region that is at least 20 amino acids or nucleotides in length. In some instances, the disclosure includes nucleic acid sequences that are substantially identical {e.g., at least 90% identical) to any of the nucleic acid sequences set forth as SEQ ID NOs: 1-20, 27 and 28.
[0023] For specific proteins described herein (e.g., Snail), the named protein includes any of the protein's naturally occurring forms, or variants that maintain the protein transcription factor activity (e.g., within at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% activity compared to the native protein). In some embodiments, variants have at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino add sequence identity across the whole sequence or a portion of the sequence (e.g. a 50, 100, 150 or 200 continuous amino acid portion) compared to a naturally occurring form. In other embodiments, the protein is the protein as identified by its NCBI sequence reference. In other embodiments, the protein is the protein as identified by its NCBI sequence reference or functional fragment thereof.
[0024] As used herein, "SNA!l" or“SNA!L” gene refers to any of the recombinant or naturally- occurring forms of the gene encoding zinc finger protein SNA!l or SNA!L, homologs or variants thereof that maintain SNAIL protein activity (e.g. within at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% activity as compared to native SNAIL). The SNA!l gene is conserved in chimpanzee, rhesus monkey, dog, cow, mouse and rat. At least 160 organisms are known to have orthologs to the human SNAI1 gene (i.e , NCBI Gene ID: 6615). In some embodiments, the SNA!l gene is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleic acid identified by the NCBI Gene ID: 6615 (NM__005985.3). in some embodiments, the SNAI1 gene is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleic acid identified by the NCBI Gene ID: 10415. in some embodiments, the SNAI1 gene is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the mouse nucleic acid identified by the NCBI Gene ID: 20613 (NMJ311427).
[0025] As used herein, "SNAI2" or " SNAIL2 " refers to any of the recombinant or naturally- occurring forms of the gene encoding zinc finger protein 5NAI2, homologs or variants thereof that maintain SNAI2 protein activity (e.g. within at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% activity as compared to native SMAI2). The SNAI2 gene is conserved in humans, chimpanzee, rhesus monkey, dog, cow, chicken, frog, zebrafish and rat. At least 220 organisms are known to have orthoiogs to the human SNAI2 gene (i.e., NCB! Gene ID: 6591). In some embodiments, the SNAI2 gene is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleic acid identified by the NCBI Gene ID: 6591 (NM_003068.4). In some embodiments, the SNAI2 gene is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the mouse nucleic acid identified by the NCBI Gene ID: 20583 (NM_011415).
[0026] As used herein,“ZEB1" refers to any of the recombinant or naturally-occurring forms of the gene encoding zinc finger protein ZEB1, homologs or variants thereof that maintain Zebl protein activity (e.g. within at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% activity as compared to native ZEB1). The ZEB1 gene is conserved in chimpanzee, rhesus monkey, dog, cow, chicken, mouse, frog, zebrafish and rat. At least 230 organisms are known to have orthologs to the human ZEB1 gene (i.e., NCBI Gene ID: 6935). in some embodiments, the ZEB1 gene is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleic acid identified by the NCBI Gene ID: 6935 (e.g., NM 301128128; NM_0Q1174093; MJ301174G94; and NM 301323649). In some embodiments, the ZEB1 gene is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the mouse nucleic acid identified by the NCBI Gene ID: 21417 (e.g., NM 001360981; NM J301360982; and NM_ .011546).
[0027] As used herein,“ZEB2" refers to any of the recombinant or naturally-occurring forms of the gene encoding zinc finger protein ZEB2, homologs or variants thereof that maintain Zeb2 protein activity (e.g. within at least 50%, 60%, 7G? , 80%, 90%, 91%, 92%, 93%, 94%, 95?4, 96%, 97%, 98%, 99% or 100% activity as compared to native ZEB2). The ZEB2 gene is conserved in chimpanzee, rhesus monkey, dog, chicken, mouse, frog, and zebrafish. At least 230 organisms are known to have orthoiogs to the human ZEB2 gene (i.e., NCBI Gene ID: 9839). In some embodiments, the ZE82 gene is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleic acid identified by the NCBI Gene ID: 9839 (e.g., NM_001171653 and NM_014795). In some embodiments, the Z£B2 gene is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the mouse nucleic acid identified by the NCB! Gene ID: 24136 (e.g., NM_ .001289521; NM_ 001355288; MM..001355291; MM...015753 and MM...001355289).
[0028] As used herein, the terms "Snail" or "Snail" protein refer to any recombinant or naturally-occurring form of a protein encoded by the SNA!l gene, homologs or variants thereof that maintain Snail protein activity (e.g , within at least 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity as compared to native Snail). Snail or Snail is a Zinc Finger protein encoded by the SNAil gene. Snail belongs to a family of transcription factors that promote the repression of adhesion molecule E-cadherin to regulate epithelial cells becoming mesenchymal cells. Transcription factors, such as Snail, are difficult to target with small molecule drugs due to their nuclear localization (Bobbin and Rossi, 2016, Anna. Rev. Pharmacol. Toxicol., 56:103-22). In some embodiments, variants of a Snail have at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100? amino acid sequence identity across the whole sequence or a portion of the sequence (e.g. a 50, 100, 150 or 200 continuous amino acid portion) compared to a naturally occurring Snail polypeptide (e.g., NCBI reference number: fsiP__005976) or NCBI Gene 10:6615 or Gene 10:10415 . In some embodiments, variants of the Snail protein have at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity across the whole sequence or a portion of the sequence (e.g. a 50, 100, 150 or 200 continuous amino acid portion) compared to a naturally occurring mouse Snail polypeptide (e.g., NCBI reference number: NP_035557) or NCBI Gene 10:20613.
[0029] As used herein, the terms "Snai2" or "Snail2" protein refer to any recombinant or naturally-occurring form of a protein encoded by the SNAI2 gene, homologs or variants thereof that maintain Snail protein activity (e.g., within at least 50%, 60? , 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity as compared to native Snail2). Snai2 is a Zinc Finger protein encoded by the SNAI2 gene. Snai2 belongs to a family of transcription factors that promote the repression of adhesion molecule E-cadherin to regulate epithelial cells becoming mesenchymal cells. In some embodiments, variants of a Snai2 have at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 1QQ% amino acid sequence identity across the whole sequence or a portion of the sequence (e.g. a 50, 100, 150 or 200 continuous amino add portion) compared to a naturally occurring Snai2 polypeptide (e.g., MCBI reference number: NP_003059) or MCBI Gene !D:6591. In some embodiments, variants of the Snai2 protein have at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity across the whole sequence or a portion of the sequence (e.g. a 50, 100, 150 or 200 continuous amino add portion) compared to a naturally occurring mouse 5nai2 polypeptide (e.g., NCBI reference number: NP__035545) or NCBI Gene ID: 20583.
[0030] As used herein, the terms "Zebl" protein refers to any recombinant or naturally- occurring form of a protein encoded by the ZEB1 gene, bomo!ogs or variants thereof that maintain Zebl protein activity (e.g., within at least 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity as compared to native Zebl). Zebl is a Zinc Finger protein encoded by the ZEB1 gene. In some embodiments, variants of a Zebl have at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino add sequence identity across the whole sequence or a portion of the sequence (e.g. a 50, 100, 150 or 200 continuous amino acid portion) compared to a naturally occurring Zebl polypeptide (e.g., NCBI reference number: NP_001121600; NP_001167564 and NP_001167565) or NCBI Gene ID:6935. in some embodiments, variants of the Zebl protein have at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity across the whole sequence or a portion of the sequence (e.g. a 50, 100, 150 or 200 continuous amino acid portion) compared to a naturally occurring mouse Zebl polypeptide (e.g., NCBI reference number: NP_001347910; NP 301347911 and NP_0355676) or NCBI Gene ID: 21417.
[0031] As used herein, the terms "Zeb2" protein refers to any recombinant or naturally- occurring form of a protein encoded by the ZEB2 gene, homoiogs or variants thereof that maintain Zeb2 protein activity (e.g., within at least 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity as compared to native Zeb2). Zeb2 is a Zinc Finger protein encoded by the ZEB2 gene. In some embodiments, variants of a Zeb2 have at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity across the whole sequence or a portion of the sequence (e.g. a 50, 100, 150 or 200 continuous amino acid portion) compared to a naturally occurring Zeb2 polypeptide (e.g., NCBI reference number: NP_001165124 and NP_055610) or NCBI Gene !D:9839 in some embodiments, variants of the Zeb2 protein have at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity across the whole sequence or a portion of the sequence (e.g. a 50, 100, 150 or 200 continuous a ino acid portion) compared to a naturally occurring mouse Zeb2 polypeptide (e.g., NCBI reference number: NP 001276450; MR 001342217; MR..001342218; and NP..001342219} or NCBI Gene ID: 24136.
[0032] An“siRNA" refers to a nucleic acid that forms a double stranded RNA, which double stranded RNA has the ability to reduce or inhibit expression of a target gene when the siRNA is present (e.g. expressed) in the same cell as the target gene. The siRNA is typically about 5 to about 100 nucleotides in length, more typically about 10 to about 50 nucleotides in length, more typically about 15 to about 40 nucleotides in length, most typically about 20-30 base nucleotides, or about 20-25 or about 24-29 nucleotides in length, e.g., 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length. siRNA molecules and methods of generating them are described in, e.g., Bass, 2001, Nature, 411, 428-429; Elbashir et a!., 2001, Nature , 411, 494-498; Patent Application Publication Nos: WO 00/44895; WO 01/36646; WO 99/32619; WO 00/01846; WO 01/29058; WO 99/07409; and WO 00/44914. A DNA molecule that transcribes siRNA also provides RNA interference (RNAi). DNA molecules for transcribing siRNA are disclosed in for example, U.S. Pat. No. 6,573,099, and U.S. Patent Application Publication Nos. 2002/0160393 and 2003/0027783, and Tuschl and Borkhardt, Molecular Interventions , 2:158 (2002). siRNA can be delivered to a target cell using a vector (e.g., a plasmid, bacterial or viral vector) or by oligonucleotide (see, e.g., Subramanya et al., (2010) Expert Opin Biol Ther., 10(2)201-13).
[0033] As used herein, the term "delivery vehicle", refers to a support structure that transfers a component of genetic material or a protein to or into a cell. Genetic material includes, but is not limited to DNA, RNA (e.g., siRNAs), proteins, polypeptides, or fragments thereof.
[0034] A "cell" as used herein, refers to prokaryotic and eukaryotic cells and include cells derived from mammals. In some embodiments, a cell is an ovarian cancer cell.
[0035] As used herein, the term "compound" refers to any molecule, either naturally occurring or synthetic, e.g., peptide, protein, oligopeptide (e.g., from about 5 to about 25 amino acids in length, preferably from about 10 to 20 or 12 to 18 a ino acids in length, preferably 12, 15, or 18 amino acids in length), small organic molecule, polysaccharide, peptide, circular peptide, peptidomimetic, lipid, fatty acid, siRNA, polynucleotide, oligonucleotide, etc. [0036] As used herein, the terms "inhibitor," "repressor", or “downregu!ator", interchangeably refer to a compound that results in a detectabiy lower expression or activity level as compared to a control. The inhibited expression or activity can be 10%, 20%, 30%, 40%, 50?4, 60%, 70%, 80%, 90% or less than that in a control. In some embodiments, the inhibition is 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, or more in comparison to a control. In some embodiments, the inhibitor is a siRNA that inhibits cellular function (e.g., replication) by binding, partially or totally blocking stimulation, decreases, prevents, or delays activation, or inactivates, desensitize, or down-regulates signal transduction, gene expression, or enzymatic activity necessary for protein activity.
[0037] A "Snail inhibitor" refers to a compound, such as a siRIMA, that results in a detectabiy lower expression of SNAIL genes (e.g., SNAIL1 and SNAIL2) or Snail proteins (e.g., Snai!l and Snail2) or lower activity level of Snail proteins as compared to those levels without such compound. In some embodiments, a Snail inhibitor is a compound, such as a siRNA, that results in a detectabiy lower expression of ZEB genes (e.g., ZEB1 and ZEB2) or Zeb proteins (e.g., Zebl and Zeb2) or lower activity level of Zeb proteins as compared to those levels without such compound in some embodiments, a Snail inhibitor is an anti-SNA!L siRNA. In some embodiments, a Snail inhibitor is a composition (e.g., a Snail inhibitor bound to a delivery vehicle or an anti-SNAIL siRNA bound to a esoporous silica nanoparticle bound to hyaluronic acid or folic acid) described herein. In some embodiments, a Snail inhibitor is a pharmaceutical composition described herein.
[0038] A "pharmaceutical composition" is a formulation containing the composition (e.g., a Snail inhibitor bound to a nanoparticle) described herein in a form suitable for administration to a subject in some embodiments, the pharmaceutical composition is in bulk or in unit dosage form. The unit dosage form is any of a variety of forms, including, for example, a capsule, an IV bag, a tablet, or a vial. The quantity of active ingredient (e.g., SNAIL inhibitor bound to a nanoparticle) in a unit dose of composition is an effective amount and is varied according to the particular treatment involved. One skilled in the art will appreciate that it is sometimes necessary to make routine variations to the dosage depending on the age and condition of the patient. One skilled in the art can determine the appropriate dosage and route of administration to a subject. A variety of routes are contemplated, including oral, rectal, parenteral, subcutaneous, intravenous, intramuscular, intraperitoneal, and the like. In embodiments, the composition is mixed under sterile conditions with a pharmaceutically acceptable carrier, and any preservatives, buffers, or propellants that are required.
[0039] As used herein, the phrase "pharmaceutically acceptable" refers to compounds, anions, cations, materials, compositions, carriers, and/or dosage forms which are, within the scope of medical judgment, suitable for use in contact with tissues of humans and animals without excessive toxicity, irritation, allergic response, or other complications, commensurate with a reasonable benefit/risk ratio.
[0040] "Pharmaceutically acceptable excipient" means an excipient that is useful in preparing a pharmaceutical composition that is generally safe, non-toxic and neither biologically nor otherwise undesirable, and includes excipient that is acceptable for veterinary use as well as human pharmaceutical use. A discussion of pharmaceutically acceptable excipients is available in REMINGTON'S PHARMACEUTICAL SCIENCES (Mack Pub. Co., N.J. 1991). Pharmaceutically acceptable excipients in therapeutic compositions may contain liquids such as water, saline, glycerol and ethanol. Additionally, auxiliary substances, such as wetting or emulsifying agents, pH buffering substances, and the like, may be present in such vehicles.
[0041] As used herein, "monotherapy" refers to the administration of a single therapeutic compound to a subject in need thereof. Preferably, monotherapy involves administration of a therapeutically effective amount of a composition described herein (e.g., a SNAIL inhibitor bound to a nanoparticle). For example, monotherapy can include administration of a composition of the present invention to a subject to treat cancer. Monotherapy may be contrasted with combination therapy, in which a combination of multiple compositions (e.g., a SNAIL inhibitor bound to a delivery vehicle and an anti-cancer agent) are administered, preferably with each component of the combination present in a therapeutically effective amount.
[0042] As used herein, "anti-cancer agent" is used in accordance with its plain ordinary meaning and refers to a composition (e.g. compound, drug, antagonist, inhibitor, modulator) having antineoplastic properties or the ability to inhibit the growth or proliferation of ceils in some embodiments, an anti-cancer agent is a chemotherapeutic. In some embodiments, an anti-cancer agent is an agent identified herein having utility in methods of treating cancer. In some embodiments, an anti-cancer agent is an agent approved by the FDA or similar regulatory agency for treating cancer. In some embodiments, an anti-cancer agent is an agent having utility in methods of treating ovarian cancer. Other anti-cancer agents, and combinations of anti-cancer agents are well known in the art (see, e.g., www.cancer.org/docroot/cdg/cdg__0.asp).
[0043] As used herein, the terms "subject" or "patient" are used interchangeably to refer to a subject having cancer or a subject having a precancerous condition. In some embodiments, a subject has cancer. A "subject" includes a mammal. The mammal can include a human or appropriate non-human mammal, such as a primate, mouse, rat, dog, cat, cow, or horse. Thus, the methods disclosed herein are applicable to both human therapy and veterinary applications. In some embodiments, the subject has ovarian cancer. Subjects with ovarian cancer include subjects with one or more signs or symptoms of ovarian cancer. Signs and symptoms of ovarian cancer include bloating, pelvic or abdominal pain, trouble eating or feeling full quickly, urinary symptoms such as urgency or frequency, fatigue, upset stomach, back pain, constipation, menstrual changes, and/or abdominal swelling with weight loss. In some embodiments, a subject may have an increased risk of developing ovarian cancer relative to the population at large (e.g., a female subject 30 years old or older, obese, no reproductive history, fertility drug use for longer than one year, androgen use, estrogen therapy, hormone therapy, a family history or personal history of ovarian cancer). In some embodiments, a subject with an increased risk of developing ovarian cancer relative to the population at large is a female subject having a germ-line or spontaneous mutation in BRCA1 or BRCA2, or both.
[0044] As used herein, "refractory cancer" refers to cancer that does not respond to treatment. The cancer may be resistant at the beginning of treatment or may become resistant during treatment in some embodiments, the subject has cancer recurrence following remission on most recent therapy. In some embodiments, the subject received and failed to respond to a known effective therapy for the cancer treatment.
[0045] As used herein, the term "cancer" refers to ail types of cancer, neoplasm or malignant tumors found in mammals, including metastatic cancers. In some embodiments, cancer refers to ovarian cancer or other cancers that involve with epithelial to mesenchymal transition (EMT) [0046] As used herein, the terms“metastasis" and "metastatic" can be used interchangeably and refer to the spread of a proliferative disease, e.g., cancer, from one organ or another non- adjacent organ or body part. Cancer may occur at an originating site, e.g., ovaries, which is referred to as a primary tumor, e.g., primary ovarian cancer. Some cancer cells in the primary tumor may acquire the ability to penetrate and infiltrate surrounding normal tissue in the local area and/or the ability to penetrate the walls of the lymphatic system or vascular system circulating through the system to other sites in the body (i.e., metastasize). A second clinically detectable tumor formed from cancer cells of a primary tumor is referred to as a metastatic or secondary tumor. When cancer cells metastasize, the metastatic tumor cells are presumed to be similar to those of the original tumor. Thus, if lung cancer metastasizes to the breast, the secondary tumor at the site of the breast consists of abnormal lung cells and not abnormal breast cells. The secondary tumor in the breast is referred to as metastatic lung cancer. Thus, metastatic cancer refers to a disease in which a subject has or had a primary tumor and has one or more secondary tumors. The phrase non-metastatic cancer refers to subjects having a primary tumor but not one or more secondary tumors.
[0047] "An effective amount" or "a therapeutically effective amount" as provided herein refers to an amount effective to achieve its intended purpose. The actual amount effective for a particular application will depend, inter alia, on the condition being treated. When administered in methods to treat a disease, the pharmaceutical compositions described herein will contain an active compound (e.g., anti-SNAIL siRNA bound to a nanoparticle) and optionally, an anti -cancer agent to achieve the desired result, e.g., reducing, eliminating, or slowing the progression of disease symptoms (e.g., ovarian cancer). The effect can be detected by any assay method known in the art. The precise effective amount for a subject will depend upon the subject's body weight, size, and health; the nature and extent of the condition; and the composition or combination of compositions selected for administration. Therapeutically effective amounts for a given situation can be determined by routine experimentation that is within the skill of a treating clinician.
[0048] As used herein, the terms "treatment," "treating," and "treat" refer to any indicia of success in the treatment or amelioration of an injury, disease, or condition, including any objective or subjective parameter such as abatement; remission; diminishing of symptoms or making the injury, disease, or condition more tolerable to the subject; slowing in the rate of degeneration or decline (e.g., cognitive impairment); making the final point of degeneration less debilitating; and/or improving a subject's physical or mental well-being. The administration of compositions or pharmaceutical compositions of the disclosure may or can lead to the elimination of a sign or symptom, however, elimination is not required. ill. COMPOSITIONS
SNAIL inhibitors
[0049] In one aspect, provided herein are SNAIL inhibitors. In some embodiments, SNAIL inhibitors are inhibitors of expression of a SNAIL gene (e.g., SNAIL1 and SNAIL2) or a Snail protein (e.g., Snaill and Snaii2) in mammals. In some embodiments, a SNAIL inhibitor includes the expression of one or more genes or proteins from ZEB1 or ZEB2.
[0050] In some embodiments, a SNAIL inhibitor is a siRNA. in some embodiments, a SNAIL inhibitor comprises one or more siRNAs targeted against expression of SNAIL1 or SNAIL2 or a Snail transcript (e.g., Snaill and Snail 2 mRNA). siRNA molecules of the disclosure include isolated siRNA molecules that bind to a single stranded RNA molecule, which is a messenger RNA (mRNA) that encodes Snaill or Snai!2 (also referred to herein, as Snail and Snai2, respectively). siRNA molecules of the disclosure also include isolated siRNA molecules that bind to a single stranded RNA molecule that encodes Zebl or Zeb2. Snail l, Snail2, Zebl and Zeb2 are zinc finger proteins that belongs to a family of proteins associated with epithelial to mesenchymal transition (EMT). Snail protein is encoded by the SNAIL gene (e.g., NCBI Gene ID: 6615). Snail protein and gene sequences are publicly available. For example, an amino acid sequence for a Snail protein in humans can be found at NP 005976 and a nucleotide sequence for the corresponding mRNA sequence can be found at: NM 305985. Snail2 protein and gene sequences are publicly available. For example, an amino acid sequence for a Snai2 protein in humans can be found at NP__003059 and a nucleotide sequence for the corresponding mRNA sequence can be found at: NM 303G68 Zebl protein and gene sequences are publicly available. For example, an a ino acid sequence for a Zebl protein in humans can be found at NP_001121600 and a nucleotide sequence for the corresponding mRNA sequence can be found at: NM 001128128. Zeb2 protein and gene sequences are publicly available. For example, an amino acid sequence for a Zeb2 protein in humans can be found at MR 001165124 and a nucleotide sequence for the corresponding mRNA sequence can be found at: NM 001171653.
[0051] One skilled in the art will appreciate that Snaill and S n a ί 12 nucleic acids and proteins can vary from those publicly available, such as polymorphisms resulting in one or more substitutions, deletions, insertions, or combinations thereof, while still retaining Snaill or Snail2 biological activity. Accordingly, in various embodiments, the amino acid sequence of Snaill or Snail2 may be about 80%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to a Snaill or Snail2 sequence publicly available.
[0052] In some embodiments, a nucleic acid sequence of a SNAIL inhibitor may be at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% complementary to a publicly available SNAil, SNAI2, ZEB1 or ZEB2 sequence.
[0053] In some embodiments, the SNAIL inhibitor bound to the delivery vehicle takes effect when the expression level of SNAIL1 or SNAIL2 gene or Snaill or Snail2 protein or the activity level of Snaill or Snail2 protein is less than 90% of an initia l level, less than 80% of an initial level, less than 70% of an initial level, less than 60% of an initial level, less than 50% of an initial level, less than 40% of an initial level, less than 30? of an initial level, less than 20% of an initial level or less than 10% of an initial level. Methods for determining the expression level of a Snail gene or a Snail protein or the activity level of a Snail protein is well known in the art (see., e.g., Finlay et a!., 2015, Nanomedicine: 11:1657-66; Roberts et a!., 2017, Nanomedicine, 13{3):965-976); and Shahin et al., 2018, Nanomedicine: Nanotechnology, Biology , and Medicine, 14(4}:1381-1394).
[0054] in some embodiments, a SNAIL inhibitor is an anti-SNAIL siRNA. As used herein, the term "anti-SNAIL siRNA" includes all forms of anti-SNAIL siRNA, including variants, modifications and derivatives thereof. In one embodiment, the anti-SNAIL siRNA molecule is an oligonucleotide with a length of about 15 to about 40 base pairs (e.g., about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26 27, 28, 29, 30, 31, 32 33, 34, 35, 36, 37, 38, 39, or about 40 base pairs). In another embodiment, the anti-SNAIL siRNA molecule is an oligonucleotide with a length of about 15 to about 30 base pairs. In yet another embodiment, the molecule is an oligonucleotide with a length of about 20 to about 25 base pairs. In some embodiments, the anti-SNAIL siRNA molecule may have blunt ends at both ends, or sticky ends at both ends, or a blunt end at one end and a sticky end at the other. In one embodiment, an anti-SNAIL siRNA targets SNAIL or variants or homologs thereof. In one embodiment, an anti-SNAIL siRNA targets SNAI2 or variants or homologs thereof. In yet another embodiment, an anti-SNAIL siRNA targets both SNA!l and SNAI2, or their variants and homologs in some embodiments, an anti-SNAIL siRNA targets ZEB1 and/or ZEB2, or their variants and homo!ogs. Exemplary anti-SNAIL siRNA sequences include, but are not limited to, any one or more of SEQ ID NOs:l- 20, 27 and 28. in some embodiments, the SNAIL siRNA bound to the delivery vehicle is siSNAIL- 7 (i.e., SEQ ID NOs:13 and 14).
[0055] One skilled in the art will appreciate that anti-SNAIL siRNAs of the invention include nucleic acid sequences having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to any one or more of SEQ ID NOs:l-20, 27 and 28.
[0056] An siRNA molecule of the disclosure may comprise naturally occurring nucleotides or a nucleic acid modification. Examples of nucleic acid modifications include chemical substitutions at the ribose and/or phosphate and/or base positions. Modified nucleotides are described in U.S. Pat. No. 5,660,985, which describes oligonucleotides containing nucleotide derivatives chemically modified at the 2' position of ribose, 5 position of pyrimidines, and 8 position of purines. U.S. Pat. No. 5,756,703 describes oligonucleotides containing various 2'- modified pyrimidines. U.S. Pat. No. 5,580,737 describes highly specific nucleic acid ligands containing one or more nucleotides modified with 2'-amino (2'-NH2), 2'-fluoro (2'-F), and/or 2'-0-methyi {2'-GMe) substituents. Modification of siRNAs contemplated in this disclosure include, but are not limited to, those which provide other chemical groups that incorporate additional charge, polarizability, hydrophobicity, hydrogen bonding, electrostatic interaction, and fluxionality to the siRNA bases or to the siRNA sequence as a whole. Such nucleic acid modifications include, but are not limited to, 2'-position sugar modifications, 5-position pyrimidine modifications, 8-position purine modifications, modifications at exocyclic amines, substitution of 4-thiouridine, substitution of 5-bromo or 5-iodo-uracil; backbone modifications, phosphorothioate or alkyl phosphate modifications, methylations, unusual base-pairing combinations such as the isobases isocytidine and isoguanidine and the like. Nucleic acid modifications also include 2'-Q-methy! modifications, 2'-0-metbyi modified ribose sugars with terminal phospborothioates and a cholesterol group at the 3' end, 2'-0- metboxyethyi (2'-MOE) modifications, 2'-f!uoro modifications, and 2', 4' methylene modifications (referred to as "locked nucleic acids" or LNAs). Nucleic acid modifications can also include 3' and 5' modifications such as capping.
[0057] In some embodiments, an antisense sequence (guide strand) and a sense (passenger strand) can contain different nucleic acid modifications. In some embodiments, a passenger strand may contain nucleic acid modifications that promote loading of the guide strand onto mRNA cleavage machinery, modifications that prevent the passenger sequence loading into a RISC complex, modifications that prevent nuclease-mediated degradation, modifications that reduce immunogenicity mediated by toll-like receptors and RIG-1, or a combination thereof. In some embodiments, the passenger strand may contain inverted abasic riboses, 2'-0-methyl base (e.g., methyiuracil), or combination thereof in some embodiments, the guide strand may contain nucleic add modifications that increase their loading efficiency to a RISC complex. In some embodiments, a guide strand may contain a 2-thio-deoxyuracil.
Delivery Vehicle
[0058] The disclosure provides a composition comprising a SNAIL inhibitor bound to a delivery vehicle. In some embodiments, the delivery vehicle is a nanoparticle. in one embodiment, the delivery vehicle is a lipid vehicle. Delivery vehicles include, but are not limited to, vectors such as viruses (e.g., retroviruses, adenoviruses, and adeno-associated viruses), virus capsids, liposomes or liposomal vesicles, lipoplexes, polyplexes, dendrimers, macrophages, artificial chromosomes, nanoparticles, polymers and also hybrid particles, examples of which include virosomes. Delivery vehicles may have multiple surfaces and compartments for attachment and storage of components (e.g., anti-cancer agents). These include, but are not limited to, outer surfaces and pores.
[0059] In some embodiments, the delivery vehicle is a nanoparticle, a lipid particle or a viral vector. Any nanoparticle known for siRNA delivery can be used. Numerous nanoparticles (NPs) prepared from polymers, liposomes, protein based NPs and inorganic NPs have been developed and a variety of nanoparticles are currently being evaluated in clinical studies. One advantage of NPs is that they offer targeted tissue and/or site delivery. Their nanoscale size allows NPs to escape through blood vessels at the tissue site through leaky vascular structure (enhanced permeability and retention effect) in addition to this passive mechanism, a variety of targeting moieties can be attached to NPs (e.g., bound) to confer active targeting capability. The ability of nanopartides to target delivery of anti-cancer drugs to tumors also results in decreased chemotherapy-related off-target toxicity in patients. Exemplary nanopartides for delivering compositions described herein include, but are not limited to, solid nanopartides (e.g., metals such as silver, gold, iron, titanium), non-metal nanopartides, lipid-based solids (e.g., liposome), polymers (e.g., polyethylenimene, dendrimer), suspensions of nanopartides, or combinations thereof (e.g., polyethylenimene-liposome, dendrisome). Additional information regarding suitable nanopartides can be found in Coelho et al., (2013) N Engi J Med., 369:819-29; Tabernero et al., (2013) Cancer Discovery, 3(4):363-470; Zuris et al., (2015) Nat Biotechnol., 33(l):73-80; and Published Patent Application WO/2015089419 A2, each of which is incorporated herein by reference in its entirety.
Mesoporous Silica Nanopartides (MSNs)
[0060] In one embodiment, a delivery vehicle comprises a mesoporous silica nanopartide (MSN). MSNs are inorganic NPs suitable for delivery of anticancer drugs and siRNAs. MSNs are typically synthesized by the sol -gel method (see, Finlay et ai., 2015, Nanomedicine: 11:1657-66), which enables preparation of homogeneous nanoparticles with diameters as small as 40 nm or as large as desired in the nanopartide scale.
[0061] In some embodiments, MSNs of the disclosure are prepared such that their average diameter is between about 40 nm and about 900 nm in diameter, about 50 nm and about 800 nm in diameter, about 75 n and about 600 nm in diameter, about 100 n and about 500 nm in diameter, o about 150 nm and about 400 nm in diameter. In some embodiments, MSNs of the disclosure are about 100 nm in diameter, about 200 nm in diameter, about 300 nm in diameter, about 400 nm in diameter, about 500 nm, about 600 nm in diameter, about 700 nm in diameter, about 800 nm in dimeter, or about 900 nm in diameter.
[0062] A single MSN can include thousands of pores that provide significant storage space for drugs (e.g., anti-cancer drugs) and other compounds. In some embodiments, MSNs of the disclosure are prepared such that they contain more than 1,000 pores, more than 2,000 pores, more than 3,000 pores, more than 4,000 pores, more than 5,000 pores, more than 10,000 pores, more than 20,000 pores, more than 30,000 pores, more than 40,000 pores, more than 50,000 pores, or more than 100,000 pores. MSNs are biocompatible and their safety has been demonstrated in a number of animal experiments (see, e.g., Roberts et al., (2017), Nanomedicine: Nanotechnology , Biology , and Medicine, 13:965-976; Lu et al., (2010), Small, 6:1794-1805 and Wang et al., (2015), Nanomedicine: Nanotechnology, Biology and Medicine. ll(2):313-327).
[0063] MSNs can be coated (e.g., conjugated) with one or more targeting moieties that bind to a receptor on a target cell (e.g., cancer cells), thereby enhancing nanoparticle uptake by the target cell, and optionally, a nanovalve that controls release of an anti-cancer drug in the MSN resulting in apoptosis. Additional guidance for making and using MSNs can be found in at least U.S. Patent Publications 2012/0207795 and 2017/0233733, the contents of each are incorporated herein in their entireties in some embodiments, MSNs can be prepared according to the method set forth in Roberts et al., (2017).
[0064] In some embodiments, the nanoparticle is porous. The porous structure of a nanoparticle may allow both the binding of nucleotides on the surface of the nanoparticle as well as encapsulation of small molecules (e.g., drugs) within the nanoparticle. in some embodiments, one or more anti-cancer agents are encapsulated within the pores of a nanoparticle. In some embodiments, a nanoparticle comprises a nanovalve, as described below. In some embodiments, release of an anti-cancer agent from a nanoparticle (e.g., a MSN) can be controlled by nanovaives within the nanoparticle. Nanovalves can provide an open/ciose function so that an anti-cancer agent stored in the pores of the nanoparticle is only released when the nanoparticle encounters a specific condition (e.g., change in pH, exposure to magnetic field or light) that transitions the nanovalve from closed to open, thereby releasing the anti-cancer agent from the pores of the MSN.
Chemically modified MSNs
[0065] In some embodiments, the surface of a nanoparticle (e.g. a MSN) can be chemically modified. In some embodiments, to maximize delivery of negatively charged nucleic acids to cells, a silica surface (e.g., MSN) may be converted into positive charge to bind DNA or siRNA. Methods for introducing cationic charge to inorganic materials, which include silica, iron oxide, and gold, typically involve surface grafting with amine groups and coating with cationic polymers (e.g. polyethylenimine, polyamidoamine, polylysine) through either covalent or non-covaient (e.g. electrostatic) association (see, e.g., Radu et al., (2004) 1. Am. Chem. Soc., 126:13216-17; Bharali et al., (2005) Proc. Natl. Acad. Sci. U.S.A., 102:11539-44; Bonoiu et aL, (2009) Proc. Natl. Acad. Sci. U.S.A., 106:5546-50; Elbakry et al., (2009) Nano Lett., 9:2059-64; Fuller et al., (2008) Biomaterials, 29:1526-32; Kneuer et aL, (2000) Bioconjugate Chem., 11:926-932; McBain et al., (2007) J. Mater. Chem., 17:2561-65; and Zbu et al., (2004) Biotechnol. Appl. Biochem., 39:179-87). In some embodiments, a nanopartide (e.g. MSN) is bound to polyethylenimine (PEI) to form PEI-MSN. Any method known in the art to bind, conjugate, coat, or attach PEI to nanoparticles is suitable for use with the invention. In one embodiment, chemically modified MSNs can be prepared according to the method set forth in Finlay et al., (2015) Nanomedicine , 11:1657-66.
[0066] In some embodiments, the surface of a nanopartide can be bound (e.g., coated) with a positively charged compound or chemical moiety (e.g. polyethyleneimine (PEI)), which non- covalently binds to negatively charged nucleic acids (e.g., anti-SNAIL siRNA) via electrostatic interaction. In some embodiments, the positively charged compound (also referred to herein as a positively charged non-covalent linker) is a synthetic cationic polymer. In embodiments, the positively charged non-covalent linker compacts DNA and siRNA into complexes that are effectively taken up in cells (see., Roberts et al., 2017). in some embodiments, the positively charged non-covalent linker is attached to the nanopartide surface through covalent (e.g., using bioconjugate techniques as disclosed herein or known in the art) and electrostatic interactions. In some embodiments, PEI is attached to a nanopartide surface through covalent and electrostatic interactions. PEI polymers of different size can be used to prepare compositions of the invention in some embodiments, PEI polymers range in molecular weight (MW) from 0.6 KD to 25 KD. in some embodiments, a nanopartide can be modified using a low MW multi-branched PEI (e.g., less than or equal to a 1200 Da).
MSN Targeting Moieties
[0067] In some embodiments, the surface of a delivery vehicle comprises one or more targeting moieties. As used herein, a targeting moiety (e.g., on a nanopartide) refers to a moiety that targets and binds to a receptor found on the surface of a target ceil, for example, a cancer cell. In some embodiments, a delivery vehicle comprises a targeting moiety that binds to a cell receptor found in higher abundance in cancer cells as compared to non- cancerous cells. In some embodiments, the targeting moiety targets and binds to a receptor such as, but not limited to, CD44, CD34, CD117, CD99MIC2, CD133, MMP-2, folate receptors, and follicle stimulating hormone receptor and other hormone receptors in some embodiments, the delivery vehicle comprises a targeting moiety known to bind to a ceil receptor associated with cancer such as, but not limited to, HER2, progesterone-receptors (PR+) and estrogen receptors (ER+)). In some embodiments, the delivery vehicle comprises a targeting moiety known to bind to a tumor-associated carbohydrate antigen (TACAs) (see, Cazet et al., 2010, Breast Cancer Research, 12:204). in some embodiments, the delivery vehicle comprises a targeting moiety known to bind to a ceil receptor associated with ovarian cancer such as, but not limited to, CD151, mesothelin, Cancer Antigen 125 (CAI25), Carcinoembryonic antigen (CEA), M2-PK, and Sialyl-Tn.
[0068] In some embodiments, the delivery vehicle comprises at least one targeting moiety that targets and binds to a receptor found on an ovarian cancer ceil (e.g., CD44 or a folate receptor) in some embodiments, the delivery vehicle (e.g. MSN) comprises two or more surface modifications, such as attachment of PEI and a targeting moiety (e.g., CD44). In some embodiments, the targeting moiety is hyaluronic acid (HA). In some embodiments, the targeting moiety is folic acid (FA). In some embodiments, compositions provided herein include a SNAIL inhibitor bound to a delivery vehicle having the following components: HA- PEI-MSN-anti-SNA!L siRNA-anti-cancer agent or FA-PE!-MSN-anti-SNAIL siRNA-anti-cancer agent.
Nanovalves
[0069] In some embodiments, a delivery vehicle (e.g., MSN) can comprise a nanovalve. Nanovalves are chemical moieties (e.g., rotaxanes and pseudorotaxanes) that include a stalk and a moving part. The moving part acts as a gatekeeper of the MSN pore openings. Pore openings can be triggered by recognition events such as, but not limited to, pH, redox, charge, metal ions biomolecules (such as enzymes) and external control (such as magnetic field and light). An exemplary nanova!ve is triggered by low pH (around pH 6). One exemplary nanova!ve comprises a stalk in which cyclodextrin is attached. Cyclodextrin binding causes the nanova!ve to be closed. Upon exposure to low pH (such as in some tumor tissues), cydodextrin is released, thus releasing the contents of the MSN pores (e.g., an anti-cancer agent). The pH threshold can be adjusted by chemically modifying the nanovalve. Additional guidance regarding nanovalves triggered by low pH can be found in U.S. Patent Publication 2010/0310465, the content of which is incorporated herein in its entirety. [0070] In some embodiments, a delivery vehicle can comprise a nanovalve that is activated by an external stimulus. For example, a thermosensitive nanovalve. Nanoparticles (e.g., MSNs) containing nanovalves that are activated by an external stimulus are an attractive method for drug delivery as they provide a nan-invasive treatment having refined control over a target area, exposure time, and hence dosage. In some embodiments, magnetic core nanoparticies (e.g., Magnet-MSN also referred to herein as MSN-B) can be utilized. For example, when exposed to an oscillating magnetic field, a magnetic nanoparticle can produce heat (temperature in excess of 42°C) that stimulates a thermally responsive nanovalve (such as, thermosensitive pseudorotaxane). Additional guidance regarding making and using magnetic nanoparticies can be found in the U.S. Patent Publication 2010/0255103, the content of which is incorporated herein in its entirety. Additional guidance regarding nanovalves triggered by light can be found in U.5. Patent Publication 2010/0284924, the content of which is incorporated herein in its entirety.
Bioconjugafes
[0071] As used herein, the term "bioconjugate" or "bioconjugate linker" refers to the association between atoms or molecules. The association can be direct or indirect. For example, a bioconjugate between a first moiety (e.g. — NH2, — COOH, — N- hydroxysuccinimide, or— maleimide) and a second moiety (e.g., sulfhydryl, sulfur-containing amino acid) can be direct, e.g., by covalent bond or linker (e.g. a first linker or second linker), or indirect, e.g., by non-covalent bond (e.g. electrostatic interactions (e.g. ionic bond, hydrogen bond, halogen bond), van der Waals interactions (e.g. dipole-dipole, dipole-induced dipole, London dispersion), ring stacking (pi effects), hydrophobic interactions and the like) in some embodiments, bioconjugates are formed using conjugate chemistry including, but not limited to, nucleophilic substitutions (e.g., reactions of amines and alcohols with acyl halides, active esters), electrophilic substitutions (e.g., enamine reactions) and additions to carbon-carbon and carbon-heteroatom multiple bonds (e.g., Michael reaction, Diels-Alder addition). These and other useful reactions are discussed in, for example, March, ADVANCED ORGANIC CHEMISTRY, 3rd Ed., John Wiley & Sons, New York, 1985; Hermanson, BIOCONJUGATE TECHNIQUES, Academic Press, San Diego, 1996; and Feeney et ai., MODIFICATION OF PROTEINS; Advances in Chemistry Series, Vol. 198, American Chemical Society, Washington, D.C., 1982. [0072] In embodiments, a first moiety (e.g., a SNAIL inhibitor) is non-covalently attached to a second moiety on a delivery vehicle [e.g., a nanoparticle) through a non-covalent chemical linker or covalent chemical linker formed by a reaction between the first moiety [e.g., a SNAIL inhibitor) and the second moiety on the delivery vehicle. In some embodiments, the first moiety includes one or more reactive moieties, e.g., a covalent reactive moiety, as described herein (e.g., alkyne, azide, amine, ester, N-hydroxy-succinimide, maieimide or thiol reactive moiety). In some embodiments, the first moiety includes a linker (e.g., first linker) with one or more reactive moieties, e.g., a covalent reactive moiety, as described herein (e.g., alkyne, azide, amine, ester, N-hydroxy-succinimide, maieimide or thiol reactive moiety). In some embodiments, the delivery vehicle includes one or more reactive moieties, e.g., a covalent reactive moiety, as described herein (e.g., alkyne, azide, amine, ester, N-hydroxy-succinimide, maieimide or thiol reactive moiety). In some embodiments, the delivery vehicle includes a linker with one or more reactive moieties, e.g., a covalent reactive moiety, as described herein (e.g., alkyne, azide, amine, ester, N-hydroxy-succinimide, maieimide or thiol reactive moiety). Useful reactive functional groups (e.g., reactive groups such as bioconjugate or bioconjugate reactive groups) used for conjugate chemistries herein include, for example:
(a) carboxyl groups and various derivatives thereof including, but not limited to, N- hydroxysuccinimide esters, N-hydroxybenztriazole esters, acid halides, acyl imidazoles, thioesters, p-nitrophenyl esters, alkyl, alkenyl, alkynyl and aromatic esters;
(b) hydroxyl groups which can be converted to esters, ethers, aldehydes, etc.
(c) haloalkyi groups wherein the halide can be later displaced with a nucleophilic group such as, for example, an amine, a carboxylate anion, thiol anion, carbanion, or an alkoxide ion, thereby resulting in the covalent attachment of a new group at the site of the halogen atom;
(d) dienophi!e groups which are capable of participating in Diels-Alder reactions such as, for example, maleimido or maieimide groups;
(e) aldehyde or ketone groups such that subsequent derivatization is possible via formation of carbonyl derivatives such as, for example, imines, hydrazones, semicarbazones or oximes, or via such mechanisms as Grignard addition or aikyilithium addition;
(f) su!fonyi halide groups for subsequent reaction with amines, for example, to form sulfonamides; (g) thiol groups, which can be converted to disulfides, reacted with acyl halides, or bonded to metals such as gold, or react with maleimides;
(h) amine or sulfhydryi groups (e.g., present in cysteine), which can be, for example, acylated, alkylated or oxidized;
(i) alkenes, which can undergo, for example, cycloadditions, acylation, Michael addition, etc;
(j) epoxides, which can react with, for example, amines and hydroxyl compounds;
(k) phosphoramidif.es and other standard functional groups useful in nucleic acid synthesis;
(L) metal silicon oxide bonding;
(m) metal bonding to reactive phosphorus groups (e.g. phosphines) to form, for example, phosphate diester bonds; and
(n) azides coupled to alkynes using copper catalyzed cycloaddition click chemistry.
[0073] The present invention also provides siRNA comprising a nucleic acid sequence of any one or more of SEQ ID NOs: 1-20, 27 and 28. In some instances the siRNA can include at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any one of SEQ ID NGs:l-2G, 27 and 28. in some embodiments, a siRNA of the disclosure can be included in a vector for transfection and expression of the siRNA.
[0074] The present invention also provides a DNA sequence encoding a siRNA nucleic acid sequence of any one of SEQ ID NQs: 1-20, 27 and 28. Such DNA sequences can be included in a vector for transfection and expression of the siRNA.
[0075] The present invention also provides a DNA sequence encoding an SNAIL inhibitor in some embodiments, the SNAIL inhibitor corresponds to a nucleic acid sequence encoding a protein, polynucleotide, or fragment thereof that reduces or inhibits expression of a SNAIL gene or Snail protein in vivo or in vitro in some embodiments, reduction in Snail protein or gene expression is at least 10%, 20%, 30? , 40%, 50%, 60%, 70%, 80%, 90& or more, as compared to native Snail expression under the same or comparable conditions.
III. THERAPEUTIC METHODS
[0076] The invention further provides methods of using the compositions described herein. [0077] In one aspect, methods of treating a subject diagnosed with cancer (e.g., ovarian cancer) are provided in some embodiments, the method comprises administering to a subject in need thereof a therapeutically effective amount of a SNAIL inhibitor bound to a delivery vehicle. In some embodiments, the methods described herein relate to treating ovarian cancer. In some embodiments, the methods described herein relate to treating epithelial ovarian cancer in some embodiments, the method comprises treating a subject with an effective amount of a composition disclosed herein. In some embodiments, the method comprises treating a subject diagnosed with ovarian cancer with an effective amount of an anti-SNAIL siRNA bound to a delivery vehicle. In some embodiments, the anti-SNA!L siRNA is selected from one or more of SEQ ID NOs:l-20, 27 and 28.
[0078] In another aspect, methods of inhibiting cancer metastasis in a subject are provided in some embodiments, the method comprises administering to a subject in need thereof a therapeutically effective amount of a SNAIL inhibitor bound to a delivery vehicle. In some embodiments, the methods described herein relate to inhibiting ovarian cancer metastasis. In some embodiments, the methods described herein relate to inhibiting epithelial ovarian cancer metastasis. In some embodiments, the method comprises treating a subject with an effective amount of a composition disclosed herein. In some embodiments, the method comprises administering to a subject diagnosed with ovarian cancer an effective amount of an anti-SNAIL siRNA bound to a delivery vehicle. In some embodiments, the anti-SNAIL siRNA is selected from one or more of SEQ ID NOs:l-20, 27 and 28.
SNAIL inhibitors
[0079] in some embodiments, the therapeutic methods disclosed herein comprise administering a SNAIL inhibitor bound to a delivery vehicle i n some embodiments, the SNAIL inhibitor is a protein or polynucleotide that reduces or inhibits expression of a SNAIL1 , SNAIL2, ZEB1 or ZEB2 gene or a Snaill, Snail2, Zebl or Zebl protein (e.g., in vitro or in vivo). in some embodiments, a SNAIL inhibitor comprises a compound that inhibits or reduces the production of a Snaill, Snaii2, Zebl, or Zeb2 protein or mRNA transcript (e.g., in vitro or in vivo).
[0080] in some embodiments, a SNAIL inhibitor bound to a delivery vehicle inhibits or reduces the expression of a SNA!Ll or SNAIL2 gene or Snaill or Snail2 protein (e.g., in an in vitro or in vivo assay) by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or more as compared to the level of expression of the corresponding Snaill or Snail2 protein or corresponding SNAIL1 or SNAIL2 gene in the absence of the SNAIL inhibitor bound to the delivery vehicle.
[0081] in some embodiments, the SNAIL inhibitor comprises a siRNA. in some embodiments, the SNAIL inhibitor comprises a siRNA selected from SEQ ID NGs:l-20, 27 and 28. In some embodiments, the SNAIL inhibitor comprises siSNAIL-7 (i.e., SEQ ID NGs:13 and 14).
[0082] in some embodiments, determining whether a compound is a SNAIL inhibitor can be accomplished by measuring the expression of a Snail protein or mRNA transcript in the presence of the compound and detecting a decrease in expression of the Snail protein or mRNA transcript. For example, in some e bodiments, determining whether a compound is a SNAIL inhibitor can be performed by measuring the relative reduction in protein expression of a Snail protein by incubating the 'test' compound and 'control' samples under test conditions. For example, the expression of proteins can be routinely detected using Western blot, and mRNA transcripts can be detected using quantitative reverse transcriptase PCR or Northern blot. In some embodiments, a compound that reduces expression of a Snail protein or mRNA transcript by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50?4, or more, is a SNAIL inhibitor.
[0083] in some embodiments, a SNAIL inhibitor bound to a delivery vehicle (e.g., HA-PEi- MSN-SEQ ID NOs: 13 and 14 or FA-PEi-MSN-SEQ ID NGs:l and 2) is administered in a therapeutically effective amount in some embodiments, a daily dose range of about 0.01 mg/kg to about 500 mg/kg, or about Q.l mg/kg to about 200 mg/kg, or about 1 mg/kg to about 100 mg/kg, or about 10 mg/kg to about 50 mg/kg, can be used. The dosages, however, may be varied depending upon the requirements of the patient, the severity of the condition being treated (e.g., early stage or advanced cancer), and the agent being employed. The dose will also be determined by the existence, nature, and extent of any adverse side -effects that accompany the administration of a particular agent in a particular patient. Determination of the proper dosage for a particular situation is within the skill of the practitioner. In some embodiments, treatment is initiated with smaller dosages which are less than the optimum dose. Thereafter, the dosage is increased by small increments until the optimum effect under the circumstances is reached. For convenience, the total daily dosage may be divided and administered in portions during the day, if desired in some embodiments, the SNAIL inhibitor bound to the delivery vehicle fe.g., HA-MSN-SEQ. ID NOs:13 and 14) is administered to a subject for a period of at least 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, or longer. In some embodiments, the SNAIL inhibitor bound to the delivery vehicle is administered for an indefinite period of time. In some embodiments, the SNAIL inhibitor bound to the delivery vehicle is administered for the rest of the subject's life or until administration of the Snail inhibitor no longer provides a therapeutic benefit.
[0084] In the practice of therapeutic methods described herein, a SNAIL inhibitor bound to a delivery vehicle can be administered according to any suitable method. Methods of administration include, for example, intravenous, intraperitoneal, intramuscular, subcutaneous, or oral administration.
[0085] in some embodiments, treatment with a SNAIL inhibitor bound to a delivery vehicle as disclosed herein is combined with one or more other therapies (e.g., combination therapy). For example, in some embodiments, treatment with a SNAIL inhibitor bound to a delivery vehicle is combined with one or more anti-cancer agents.
Subject Populations
[0086] in some embodiments, the subject to be treated is a human in some embodiments, the subject is an adult human at least 30 years of age. In some embodiments, the subject is a human who has been diagnosed with cancer or who is suspected of having cancer. In some embodiments, the subject is an adult female human (e.g., diagnosed with or suspected of having ovarian cancer). In some embodiments, the subject is an adult female human who has been diagnosed with or is suspected of having epithelial ovarian cancer.
Treating Cancers
[0087] in some embodiments, the methods disclosed herein comprise treating a subject having or suspected of having cancer in some embodiments, the subject has refractory cancer. In some embodiments, treatment efficacy can be measured using one or more tests for measuring remission, diminishment, or elimination of cancer from the subject, e.g., as described below. Any suitable method for determining treatment efficacy may be used and such methods are considered within the scope of a treating physician in some embodiments, treatment of a subject having cancer or suspected of having cancer is measured by detecting the level of Snail expression in the subject e.g , as described below. In some embodiments, treatment of a subject having cancer or suspected of having cancer is measured by imaging analysis, e.g., as described below in another embodiment, treatment of a subject with cancer or suspected of having cancer can be determined by measuring tumor size and/or volume. In another embodiment, treatment of a subject with cancer or suspected of having cancer can be determined by measuring survival rate of the subject as compared to other patients having the same form of cancer (e.g., ovarian cancer).
[0088] In some instances, treatment of a subject having cancer is measured by assessing a target area of a subject (e.g., liver, lungs, ovaries) using magnetic resonance imaging (MRi), computed tomography (CT), or positron emission tomography (PET) imaging in some embodiments, treatment is measured by comparing the target area of the subject having cancer (e.g., MRI of the liver of the subject) prior to administration of a composition disclosed herein to the target area of the subject after administration of the composition (e.g., measured between 1 and 6 months after the composition is first administered).
[0089] In some instances, treatment of a subject can be measured by comparing the level of Snail expression in a sample from the subject after administration of the composition (e.g., HA-PEI-MSN-SEQ ID NGs:13 and 14) to the subject (e.g., between 1 and 6 months after first administration) to the level of the Snail expression in a sample from a similarly situated subject (e.g., same gender, age, and ethnicity) who has not been administered with the composition.
Tumor Size
[0090] in some embodiments, treating the subject can result in a reduction in tumor size. A reduction in tumor size is also referred to as "tumor regression". Preferably, after treatment, tumor size would be reduced by about 5% or greater relative to tumor size prior to treatment with a composition disclosed herein; more preferably, tumor size is reduced by about 10% or greater; more preferably, reduced by about 20% or greater; more preferably, reduced by about 30% or greater; more preferably, reduced by about 40% or greater; even more preferably, reduced by about 50% or greater; and most preferably, reduced by greater than about 75% or greater. Tumor size may be measured by any reproducible means of measurement. For example, tumor size may be measured as a diameter of the tumor.
Tumor Volume
[0091] In some embodiments, treating cancer can result in a reduction in tumor volume. Preferably, after treatment, tumor volume would be reduced by about 5% or greater relative to tumor size prior to treatment with a composition disclosed herein; more preferably, tumor volume is reduced by about 10% or greater; more preferably, reduced by about 20% or greater; more preferably, reduced by about 30% or greater; more preferably, reduced by about 40% or greater; even more preferably, reduced by about 50% or greater; and most preferably, reduced by greater than about 75% or greater. Tumor volume may be measured by any reproducible means of measurement.
Survival and Mortality Rates
[0092] In some embodiments, treating a subject with cancer using a composition disclosed herein can result in an increase in average survival time of a population of treated subjects in comparison to a population of untreated subjects. Preferably, the average survival time is increased by more than 30 days; more preferably, by more than 60 days; more preferably, by more than 90 days; and most preferably, by more than 120 days. An increase in average survival time of a population may be measured by any reproducible means. For example, by calculating for a population the average length of survival following initiation of treatment or alternatively by calculating for a population the average length of survival following completion of a first round of treatment.
[0093] In some embodiments, treating cancer with a composition disclosed herein can result in a decrease in mortality rate of a population of treated subjects in comparison to an untreated population in some embodiments, the mortality rate is decreased by more than 2%; more preferably, by more than 5%; more preferably, by more than 10%; and most preferably, by more than 25%. A decrease in mortality rate may be measured by any reproducible means. For example, by calculating for a population the average number of disease-related deaths per unit time following initiation of treatment or alternatively by calculating for a population the average number of disease-related deaths per unit time following completion of a first round of treatment.
Reducing or inhibiting Metastasis
[0094] in some embodiments, the disclosure provides methods for inhibiting or reducing cancer metastasis in a subject in some embodiments, administration of a SNAIL inhibitor bound to a delivery vehicle in an effective amount to a subject (e.g., FA-PEI-MSN-SEQ ID NOs:13 and 14) inhibits or reduces cancer metastasis in the subject inhibition or reduction in cancer metastasis is determined using one or more techniques known in the art (e.g., an imaging technique, protein or mRNA transcript measurement, as described herein) over a period of time (e.g., between 1 and 6 months or between 1 month and 1 year), after administration of the composition to the subject. In some embodiments, reducing or inhibiting metastasis can be determined by the observation of an absence of secondary cancer sites (i.e., metastatic cancer cells) in the subject by an attending physician or veterinarian following treatment. For example, by measuring a decrease in the number of metastatic lesions in other tissues or organs distant from the primary tumor site. Preferably, after treatment, the number of metastatic lesions would be reduced by about 5% or greater relative to number prior to treatment; more preferably, the number of metastatic lesions is reduced by about 10% or greater; more preferably, reduced by about 20% or greater; more preferably, reduced by about 30% or greater; more preferably, reduced by about 40% or greater; even more preferably, reduced by about 50? or greater; and most preferably, reduced by greater than about 75%. The number of metastatic lesions may be measured by any reproducible means of measurement. The number of metastatic lesions may be measured by counting metastatic lesions visible to the naked eye or at a specified magnification (e.g., IOc, lOOx, or 50Qx magnification). Thus, in some instances, reduction or inhibition of metastasis in a subject can be determined by an absence of secondary cancers in the subject after 6 months or more, following treatment of the subject in some embodiments, reduction or inhibition in metastasis can be monitored in the subject at each time point the subject receives an additional treatment or a pre-determined point thereafter.
Imaging analysis
[0095] In some embodiments, the efficacy of cancer treatment is measured by imaging an area of the subject contemplated for treatment. For example, in some embodiments, magnetic resonance imaging (MRI), computed tomography (CT), or positron emission tomography (PET) is performed to evaluate the presence of a tumor in the subject (e.g., liver, lung, ovaries, prostate, etc.,) and observation of the tumor's decrease in size and/or volume as a result of treatment. The use of imaging technologies for the clinical evaluation of cancer is described in the art. See, e.g., Kim et al., 2015, Imaging , 42(2):247-60; Fischerova and Burgetova, 2014, Best Prac Res Clin Obsiet Gynaecol , 28(5)697-720; and Xia et al., 2015, Cancer Imaging, 15:19.
[0096] in some embodiments, treatment of cancer in a subject is determined by evaluating the anatomical or organ structure of the subject using MRI, e.g., structural MR! (sMR!), after the subject is treated with a SNAIL inhibitor bound to a delivery vehicle in some embodiments, the results of the imaging are compared to a baseline, e.g., from a control subject or from the subject prior to treatment.
Protein or Transcript Analysis
[0097] In some embodiments, the method comprises measuring in a sample the level of expression of one or more Snail proteins or rrsRNA transcripts from the SNAIL gene. In some embodiments, the Snail protein corresponds to a full-length protein having at least 90% identity to NCBI : NM 005985.3 or NCBI : NP 005976.
[0098] In some embodiments, treatment of cancer (e.g., ovarian cancer) in a subject is determined by measuring the level of Snail protein or Snail rrsRNA transcript in a sample from a subject being treated with a SNAIL inhibitor bound to a delivery vehicle (e.g., HA-PEI-MSN- SEQ ID NOs:13 and 14). In some embodiments, the level of expression of Snail mRNA or protein in the sample is decreased by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or more as compared to a reference value (e.g., the level of Snail expression in a sample from the subject prior to treatment).
[0099] in some embodiments, polynucleotide (e.g., mRNA) expression is measured using routine techniques such as reverse transcription polymerase chain reaction (RT-PCR), Real- Time reverse transcription polymerase chain reaction (Real-Time RT-PCR), semi-quantitative RT-PCR, quantitative polymerase chain reaction (qPCR), quantitative RT-PCR (qRT-PCR), multiplexed branched DIMA (bDNA) assay, microarray hybridization, or sequence analysis (e.g., RNA sequencing ("RNA-Seq")). Methods of quantifying polynucleotide expression are described, e.g., in Fassbinder-Orth, Integrative and Comparative Biology, 2014, 54:396 -406; Thellin et aL, Biotechnology Advances, 2009, 27:323-333; and Zheng et aL, Clinical Chemistry, 2006, 52:7 (doi: 10/1373/clinchem.2005.065078). In some embodiments, quantitative RT- PCR is used to measure the level of a polynucleotide (e.g., mRIMA) in a biological sample. See, e.g., Nolan et a!., Nat. Protoc, 2006, 1:1559-1582; Wong et aL, BioTechniques, 2005, 39:75- 75. Quantitative PCR and RT-PCR assays for measuring gene expression are also commercially available (e.g., TaqMan® Gene Expression Assays, ThermoFisber Scientific). Other techniques for quantitating transcription are well known in the art (e.g., sequencing and mass spectrometry).
[0100] A detectable moiety can be used in the assays described. A wide variety of detectable moieties can be used, with the choice of label depending on, for example, sensitivity required, ease of conjugation, stability requirements, and available instrumentation. Suitable detectable moieties include, but are not limited to, radionuclides, fluorescent dyes {e.g., fluorescein, fluorescein isothiocyanate (FITC), Oregon Green™, rhodamine, Texas red, tetrarhodimine isothiocynate (TRITC), Cy3, Cy5, etc.}, fluorescent markers {e.g., green fluorescent protein (GFP), phycoerythrin, etc.}, autoquenched fluorescent compounds that are activated by tumor-associated proteases, enzymes {e.g., iuciferase, horseradish peroxidase, alkaline phosphatase, etc.), biotin, digoxigenin, metals, and the like.
[0101] A signal from a detectable moiety can be analyzed, for example, using a spectrophotometer to detect color from a chromogenic substrate; a radiation counter to detect radiation; or a fluorometer to detect fluorescence in the presence of light of a certain wavelength. For detection of enzyme-linked antibodies, a quantitative analysis can be made using a spectrophotometer in some embodiments, the amount of signal can be quantified using an automated high-content imaging system {e.g., ! ageXpress, Molecular Devices Inc., Sunnyvale, CA).
Reference Values
[0102] In some embodiments, for assessing treatment of a subject with cancer as disclosed herein, a measurement from a subject (e.g., imaging or protein/mRNA transcript analysis) is compared to a reference value. A variety of methods can be used to determine the reference value for a unit of measurement (e.g., MR! or mRNA transcript) as described herein.
[0103] In one embodiment, a reference value for a unit of measurement as described herein is determined for the subject prior to treatment with a SNAIL inhibitor bound to a delivery vehicle. In some embodiments, the subject is assessed prior to treatment to determine a "baseline value" for the subject, against which the subject is compared at one or more time points after treatment.
[0104] in another embodiment, a reference value for an imaging technique is determined by imaging a target area of a normal subject or population of subjects (e.g., subjects known not to have cancer) to determine characteristic features of a normal target area (e.g., size, volume, shape, etc.,). In another embodiment, a reference value for Snail expression is determined by assessing the level of Snail expression in samples (e.g., by mass spectrometry) from a normal subject or population of subjects (e.g., subjects known not to have cancer). As a non-limiting example, in one embodiment, a reference value is determined for a population of subjects (e.g., 10, 20, 50, 100, 200, 500 subjects or more) known not to have cancer.
[0105] In another embodiment, a reference value for a unit of measurement is determined for a subject or population of subjects diagnosed as having the same form of cancer as the test subject (e.g., ovarian cancer). For example, a reference value for an imaging analysis is determined by imaging a target area of a subject or population of subjects diagnosed as having ovarian cancer to determine characteristics for the disease in some embodiments, a reference value for Snail expression is determined by assessing the level of Snail expression in samples from a subject or population of subjects diagnosed as having ovarian cancer. As a non-limiting example, in one embodiment, a reference value is determined for a population of subjects (e.g., 10, 20, 50, 100, 200, 500 subjects or more) ail having ovarian cancer.
[0106] in some embodiments, the population of subjects is matched to a test subject according to one or more patient characteristics (e.g., age, sex, ethnicity, or other criteria) in some embodiments, the reference value is established using the same type of sample from the population of subjects (e.g., a sample comprising epithelial ceils from an ovary).
[0107] Determination of particular threshold values for identifying a test subject as having cancer (e.g., ovarian cancer) are within the skill of those in the art guided by this disclosure. It will be understood that standard statistical methods may be employed by the practitioner in making such determinations. See, e.g., Principles of Biostatistics by Marcello Pagano et al. (Brook Cole; 2000); and Fundamentals of Biostatistics by Bernard Rosner (Duxbury Press, 5th Ed, 1999).
IV. PHARMACEUTICAL COMPOSITIONS AND KITS
[0108] In another aspect, pharmaceutical compositions and kits comprising a SNAIL inhibitor bound to a delivery vehicle are provided. In some embodiments, the pharmaceutical compositions and kits are for use in treating a subject having cancer (e.g., ovarian cancer). In some embodiments, the pharmaceutical compositions and kits are for use reducing or inhibiting metastasis of cancer.
Pharmaceutical Compositions
[0109] In some embodiments, pharmaceutical compositions comprising a SNAIL inhibitor bound to a delivery vehicle are provided in some embodiments, the SNAIL inhibitor is a protein or fragment thereof that reduces expression of a SNAIL gene or Snail protein as compared to a control not treated with the Snail inhibitor in some embodiments, the SNAIL inhibitor is an anti-SNAIL siRNA bound to a delivery vehicle i n some embodiments, the anti- SNAIL siRNA bound to the delivery vehicle is any one of SEQ ID NOs:l-20, 27 and 28. In some embodiments, the siRNA bound to the delivery vehicle is SEQ ID NOs:13 and 14. In some embodiments, the siRNA includes a nucleic acid sequence having at least 9Q%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to any one or more of SEQ ID NOs:l-20, 27 and 28. in some embodiments, the delivery vehicle is a nanoparticie or a lipid particle. In some embodiments, the nanoparticie is a MSN or MSN coated with one or more surface modifications such as, but not limited to, PEI and/or a targeting moiety (e.g., HA or FA). In some embodiments, the delivery vehicle is a MSN having a nanovalve that is triggered under specific conditions to release one or more anti-cancer agents from pores of the MSN.
[0110] Guidance for preparing formulations can be found in any number of handbooks for pharmaceutical preparation and formulation that are known to those of skill in the art. See, e.g., Remington: The Science and Practice of Pharmacy, 21st Edition, Philadelphia, PA. Lippincott Williams & Wilkins, 2005. [0111] The pharmaceutical compositions can further comprise one or more pharmaceutically acceptable carriers, adjuvants, and/or vehicles appropriate for the particular route of administration for which the composition is to be employed in some embodiments, the carrier, adjuvant, and/or vehicle is suitable for intravenous, intramuscular, oral, intraperitoneal or subcutaneous administration. Pharmaceutically acceptable carriers are well-known in the art. See, e.g., Handbook of Pharmaceutical Excipients (5th ed , Ed. Rowe ef ai., Pharmaceutical Press, Washington, D.C.). Examples of pharmaceutically acceptable carriers include, but are not limited to, aqueous solutions, e.g., water or physiologically compatible buffers such as Hanks’s solution, Ringer's solution, or physiological saline buffer.
[0112] Typically, a pharmaceutical composition for use in in vivo administration is sterile. Sterilization can be accomplished according to methods known in the art, e.g., heat sterilization, steam sterilization, sterile filtration, or irradiation.
[0113] The dosage of pharmaceutical compositions may vary depending on the particular use envisioned. Determination of an appropriate dosage or route of administration is well within the skill of one in the art. Suitable dosages are also described in Section III above.
Kits
[0114] A composition or a pharmaceutical composition provided herein may, if desired, be presented in a kit (e.g., a pack or dispenser device) which may contain one or more unit dosage forms containing the composition or the pharmaceutical composition, for example (1) a SNAIL inhibitor bound to a delivery vehicle, (2) a composition including an anti-cancer drug and a SNAIL inhibitor bound to a delivery vehicle, or (3) a pharmaceutical composition including a pharmaceutically acceptable excipient and a composition described herein. The pack may, for example, comprise metal or plastic foil, such as a blister pack. The pack or dispenser device may be accompanied by instructions for administration. Compositions described herein may be formulated in a compatible pharmaceutical carrier, placed in an appropriate container, and labeled for treatment of an indicated condition. Instructions for use may also be provided instructions (e.g., written, tape, VCR, CD-ROM, etc.) for carrying out an assay to determine efficacy or activity of the pharmaceutical formulation may also be included in the kit. The assay may for example be in the form of qRT-PCR, Western or Northern blot analysis, sequencing and mass spectrometry as known in the art. V. Examples
[0115] The following examples are offered to illustrate, but not to limit, the claimed invention.
Example 1: Transfection of anti-SNAIL siRNA leads to knockdown of
SNAIL in 293T cells in vitro
[0116] This example describes an exemplary experimental protocol for testing the effect of SNAIL inhibitors bound to a delivery vehicle in vitro . siRNA Design
[0117] Here, siRNAs were designed to include a region of about 25 bp complementarity between the siRNA and Snail. Additionally, siRNAs were preferentially selected that were complementary to the 5’ end of the gene, in exons, and in regions that did not include complementary over a substantial portion (e.g., about 20 bp) to other Snai members (e.g., Snai2). in some instances, siRNAs were designed to include homology to mouse.
[0118] Twelve Dicer-substrate small inhibitory RNAs (siRNAs) (siSNAIL-1 through siSNAIL-10, CrossReact siSnail.l and 1.2, siHPRT and siNC) were screened for their effectiveness at SNAIL knockdown in human embryonic kidney ceils (293T cells; Takara Bio, USA, Catalog No: 632180). See Table 1 and FIG. 1A and IB. The gene location provided in Table 1 refers to the start position of the corresponding siSNAIL RNA in the human SNAI1 gene. As is evident from FIG. 1A and FIG. IB, siSNAIL-7 gave a dose response for SNAil knockdown across all three concentrations. We anticipate that additional siRNAs can be designed, for example using the criteria set forth in this example, and within about 100 bp, about 5Qbp, about 40 bp, about 30 bp, or about 20 bp upstream or downstream of the gene location of siSNA!L-7 that will also provide knockdown of SNAil. Table 1: Exemplary anti-SNAIL siRNAs
Figure imgf000040_0001
Figure imgf000041_0001
"r" corresponds to RNA bases (as opposed to DNA)
"m" corresponds to 2'-0-methyl bases
[0119] The anti-SNAIL siRNAs contained one or more nucleic acid modifications, such as a 2'- O-methyl base and/or an inverted abasic ribose as indicated. Anti-SMA!L siRNA duplexes were formed by placing equal molar volumes of each anti-SNAIL siRNA together and heating at 100°C for 10 minutes followed by cooling to room temperature over several hours.
Cell Culture and Transfection
[0120] 293T cells were obtained from Takara Bio, USA, Catalog No:632180. The 293T cells were plated at a concentration of 100 thousand cells in a 6 well plate. Transient transfection of each Anti-SNAIL siRNA was achieved using UPQFECTAMINE® RNAiMax (Ther oFisher Scientific, Waltham, MA} at a concentration of 1 nm, 3 nm, or 10 nM per siRNA or siScramb!e. Transfection of the Anti-SNAIL siRNA was carried out by incubation at 37°C, 5% CO? for 24 hours.
Quantitative RT-PCR
[0121] After 24 hours, levels of Snail RNA were determined with qRT-PCR using 5' (Snail-1) and 3’ (Snail-2) primer pairs (FIG. 1A and FIG. IB). As a positive control, HPRT was targeted (see, FIG. 2).
[0122] Total cellular RNA was isolated. cDNA was synthesized from total RNA and Quantitative multiplex RT-PCR was performed. Thermocycling was conducted in a thermocyder for 40 cycles as follows: (95°C 15 sec, 60°C 30 sec, 72°C 30 sec), followed by melt curve analysis. Data was analyzed using data analysis software. Primers used for qRT- PCR were:
Snail 1 (forward): AAG CAT TTC AAC GCC TCC AAA
Snail 1 (reverse): AGG ATC TCT GGT TGT GGT ATG AC
Probe: 56-FAM/ CCC CAA TCG / ZEN / GAA GCC TAA CTA CAG C / BIABkFQ Snail 2 (forward): GGC TGC TAG AAG GCC AT
Snail 2 (reverse): GCA CTG GTA CTT CTT GAC ATC T
Probe: 5 HEX/ TTC GCT GAC / ZEN / CGC TCC AAC CT / SIABkFQ
Results
[0123] A time dependent SNAIL knockdown was observed having greater than 40% SNAIL protein reduction at 24 hours following transfection (FIGS 1A and IB).
[0124] siSNA!L-7 was chosen for further evaluation due to its effective dose response for Snail knockdown (see, FIGS. 1A and IB).
Example 2: Delivery of L-7 bound to hyaluronic acid Mesoporous Silica
Figure imgf000042_0001
Nanopartides leads to knockdown of SNAIL in ovarian cancer cells in vitro siRNAs
[0125] siRNAs were prepared, essentially as set forth in Example 1.
MSN Production
[0126] MSNs were prepared according to a sol-gel method known in the art (see., U.S. Patent Application 2017/0233733). The addition of cationic PEI coating to the MSNs was prepared as previously described (see, Finlay et a!., (2015), Nanomedicine: 11:1657-66). MSNs having a targeting moiety (e.g., folic acid or hyaluronic acid) for ovarian cancer cells (e.g., a targeting moiety for folate receptors or CD44) was prepared as previously described (see, Shahin et a!., (2018), Nanomedicine: Nanotechnology , Biology and Medicine , 14:1381-1394 and Lu et ai., (2012) Nanomedicine: Nanotechnology , Biology and Medicine, 8(2):212-2G, both of which are incorporated herein by reference in their entireties).
[0127] siSNAIL-7 (i.e., SEQ ID NOs:13 and 14) was loaded onto HA-PEI-MSNs prepared as described in U.S. Patent Application 2017/0233733, incorporated herein by reference. The siSNAIL-7-HA-PEI-MSNs were incubated overnight at 4°C on a roller. The following day, an aliquot of the siSNAIL-7-HA-PEi-MSNs was added to each well of a 6-well plate containing GVCAR8 ovarian cancer cells (obtained from Cariotta Glackin's laboratory, City of Hope, CA). After 24, 48, 72, or 168 hours, Snail expression was detected by qRT-PCR, as set forth in Example 1, in comparison to a control siRNA (siScramble) (see FIG. 3).
[0128] A time dependent SNAIL knockdown was observed with greater than 95? SNAIL protein reduction at 24 hours following transfection (FIG. 3). The SNAIL knockdown continued at a rate of greater than 85% for at least 72 hours post-transfection.
Example 3: Treatment of a subject having ovarian cancer using HA-MSN-Anti-SNAIL siRNAs in vivo
[0129] Here, we present a prophetic example demonstrating treatment of a subject having ovarian cancer using compositions and methods of the disclosure. siRNAs
[0130] siRNAs are prepared essentially as set forth in Example 1.
Figure imgf000043_0001
[0131] A delivery vehicle comprising MSNs is prepared, essentially as set forth in Example 2. The MSNs can include a targeting moiety (e.g , folic acid or hyaluronic acid) for ovarian cancer cells (e.g., a targeting moiety for CD44 or folate receptors). The siR A and delivery vehicle are bound together, essentially as set forth in Example 2.
Treatment
[0132] The composition (e.g., HA-PEI-MSN-SEQ ID NOS:13 and 14) is administered to a subject having ovarian cancer (e.g., via intravenous or intraperitoneal route, once or twice a week). While not being bound by the following, an appropriate dosage may include 15 n ol siRNA per 25 g mouse (approximately 100 ug/kg) or 600 nmol siRNA per kg of adult human (approximately 100 ug/kg).
Evaluation
[0133] After administration, cells from the subject can be harvested (e.g., a biopsy) and assessed for expression of SNAIL, for example using qRT-PCR, essentially as set forth in Example 1. Additionally, the subject can be monitored for characteristics indicative of a reduction in tumor load, such as decrease in tumor size, volume, or increased survival rate. The subject can be administered with one or more subsequent treatments (e.g., HA-PEI-MSN- SEQ ID NGS:13 and 14), for example, until such time that the subject is deemed in remission.
[0134] All publications and patents cited in this specification are herein incorporated by reference as if each individual publication or patent were specifically and individually indicated to be incorporated by reference and are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited
[0135] The inventions have been described broadly and generica!iy herein. Each of the narrower species and subgeneric groupings falling within the generic disclosure also form part of the invention. In addition, where features or aspects of the invention are described in terms of Markush groups, those skilled in the art will recognize that the invention is also thereby described in terms of any individual member or subgroup of members of the Markush group.
[0136] It should be understood that although the present invention has been specifically disclosed by certain aspects, embodiments, and optional features, modification, improvement and variation of such aspects, e bodiments, and optional features can be resorted to by those skilled in the art, and that such modifications, improvements and variations are considered to be within the scope of this disclosure.

Claims

Claims What is claimed is:
1. A composition comprising a siRMA bound to a delivery vehicle.
2. The composition of claim 1, wherein the siRMA is an anti-SNAIL siRMA.
3. The composition of claim 2, wherein the anti-SNAIL siRNA comprises any one of SEQ ID Mos: 1-20, 27 and 28.
4. The composition of claim 3, wherein the anti-SNAIL siRNA comprises a nucleic acid modification.
5. The composition of claim 4, wherein the nucleic acid modification is a 2'-0-methyl base or inverted abasic deoxyribose.
6. The composition of claim 4, wherein the nucleic acid modification is a 2-thio-deoxyuracil.
7. The composition of claim 1, wherein the delivery vehicle is a nanoparticle.
8. The composition of claim 7, wherein the nanoparticle is a mesoporous silica nanoparticle (MSN).
9. The composition of claim 8, wherein the MSN is bound to polyethyleneimine (PEI).
10. The composition of claim 8, wherein the MSN further comprises a targeting moiety.
11. The composition of claim 10, wherein the targeting moiety comprises hyaluronic acid or folic acid.
12. The composition of claim 1, further comprising a pharmaceutically acceptable excipient to form a pharmaceutical composition.
13. An siRNA comprising a nucleic acid sequence of any one of SEQ ID NOs: 1-20, 27 and 28.
14. A DNA sequence encoding an siRNA sequence comprising the nucleic acid sequence of any one of SEQ ID NOs: 1-20, 27 and 28.
15. A method of treating ovarian cancer in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a siRNA bound to a delivery vehicle.
16. The method of claim 15, wherein the ovarian cancer is epithelial ovarian cancer (EQC).
17. The method of claim 15, wherein the siRNA bound to the delivery vehicle is prepared according to any one or claims 1-11.
18. The method of claim 15, wherein the therapeutically effective amount of the siRNA bound to the delivery vehicle is administered intravenously or subcutaneously.
19. A method of reducing or inhibiting ovarian cancer metastasis in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a siRNA bound to a delivery vehicle.
20. The method of claim 19, wherein the ovarian cancer is epithelial ovarian cancer (EOC).
21. The method of claim 19, wherein the siRNA bound to the delivery vehicle is prepared according to any one or claims 1-11.
22. The method of claim 19, wherein the therapeutically effective amount of the siRNA bound to the delivery vehicle is administered intravenously or subcutaneously.
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