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WO2025052098A1 - Compositions de petits arn activateurs (sarna) ciblant tmem173 et procédés d'utilisation - Google Patents

Compositions de petits arn activateurs (sarna) ciblant tmem173 et procédés d'utilisation Download PDF

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Publication number
WO2025052098A1
WO2025052098A1 PCT/GB2024/052261 GB2024052261W WO2025052098A1 WO 2025052098 A1 WO2025052098 A1 WO 2025052098A1 GB 2024052261 W GB2024052261 W GB 2024052261W WO 2025052098 A1 WO2025052098 A1 WO 2025052098A1
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sarna
cancer
inhibitor
sequence
tmem173
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Nagy Adly Habib
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Mina Therapeutics Ltd
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Mina Therapeutics Ltd
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    • 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
    • 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
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2818—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against CD28 or CD152
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • 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 nucleic acids [NA]
    • 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
    • C12N2320/31—Combination therapy

Definitions

  • the disclosure relates to oligonucleotide, specifically saRNA, compositions for modulating gene expression and to the methods of using the compositions in diagnostic and therapeutic applications.
  • RNAs increased gene expression by targeting ncRNAs that overlap gene promoters (Janowski et al., Nature Chemical Biology, vol.3: 166-173 (2007), the contents of which are incorporated herein by reference in their entirety).
  • Any short RNA which leads to up-regulation of the expression of a target gene by any mechanism is termed a short activating RNA or small activating RNA (saRNA).
  • the present disclosure provides synthetic isolated small activating RNAs (saRNAs) which up-regulate the expression of a target gene, wherein the target gene is TMEM173 (STING).
  • saRNAs synthetic isolated small activating RNAs
  • STING TMEM173
  • Pharmaceutical compositions comprising the saRNAs are also provided.
  • FIG. 1 A and IB show murine mRNA levels after transfections with muSTING and controls.
  • FIG. 2A-2D show tumor volume changes after treatment with MTL-STING, MTL- muSTING, MTL-STING + anti-PD-1, or MTL-muSTING + anti-PD-1.
  • compositions, methods and kits for modulating target gene expression and/or function for therapeutic purposes comprise at least one saRNA that upregulates the expression of the target gene.
  • One aspect of the present disclosure provides a method to design and synthesize saRNA.
  • small activating RNA means a single-stranded or double-stranded RNA that upregulates or has a positive effect on the expression of a specific gene.
  • the saRNA may be single-stranded of 14 to 30 nucleotides, such as 19, 20, 21, 22, or 23 nucleotides.
  • the saRNA may also be double-stranded, each strand comprising 14 to 30 nucleotides, such as 19, 20, 21, 22, or 23 nucleotides.
  • the gene is called the target gene of the saRNA.
  • the target gene is a double-stranded DNA comprising a coding strand and a template strand.
  • an saRNA that upregulates the expression of the TMEM173 gene is called an “TMEM173- saRNA” and the TMEM173 gene is the target gene of the TMEM173-saRNA.
  • a target gene may be any gene of interest.
  • a target gene has a promoter region on the template strand.
  • the RNA transcript transcribed from the target gene is referred to thereafter as the target transcript.
  • the target transcript may be an mRNA of the target gene.
  • the target transcript may exist in the mitochondria.
  • the saRNAs of the present disclosure may have a downstream effect on a biological process or activity.
  • a saRNA targeting a first transcript may have an effect (either upregulating or downregulating) on a second, non-target transcript.
  • the saRNA comprises an antisense strand that is at least 80% complementary to a region on the template strand or coding strand of the target gene. This region on the template strand or coding strand, where the strand of the saRNA hybridizes or binds to, is referred to as the “targeted sequence” or “target site”. In some embodiments, the target region is on the coding strand. In some embodiments, the target region is on the template strand.
  • the term “complementary to” in the context means being able to hybridize under stringent conditions. It is to be understood that thymidine of the DNA is replaced by uridine in RNA and that this difference does not alter the understanding of the term “complementarity”.
  • the antisense strand of the saRNA may be at least 80%, 90%, 95%, 98%, 99% or 100% identical with the reverse complement of the targeted sequence. Thus, the reverse complement of the antisense strand of the saRNA has a high degree of sequence identity with the targeted sequence.
  • the targeted sequence may have the same length, i.e., the same number of nucleotides, as the saRNA and/or the reverse complement of the saRNA.
  • the targeted sequence comprises at least 14 and less than 30 nucleotides.
  • the targeted sequence has 19, 20, 21, 22, or 23 nucleotides.
  • the location of the targeted sequence is situated within a promoter area of the template strand.
  • the targeted sequence is located within a TSS (transcription start site) core of the template stand.
  • TSS core or “TSS core sequence” as used herein, refers to a region between 2000 nucleotides upstream and 2000 nucleotides downstream of the TSS (transcription start site). Therefore, the TSS core comprises 4001 nucleotides and the TSS is located at position 2001 from the 5’ end of the TSS core sequence.
  • transcription start site means a nucleotide on the template strand of a gene corresponding to or marking the location of the start of transcription. The TSS may be located within the promoter region on the template strand of the gene.
  • the targeted sequence is located between 1000 nucleotides upstream and 1000 nucleotides downstream of the TSS.
  • the targeted sequence is located between 500 nucleotides upstream and 500 nucleotides downstream of the TSS.
  • the targeted sequence is located between 250 nucleotides upstream and 250 nucleotides downstream of the TSS.
  • the targeted sequence is located between 100 nucleotides upstream and 100 nucleotides downstream of the TSS.
  • the targeted sequence is located upstream of the TSS in the TSS core.
  • the targeted sequence may be less than 2000, less than 1000, less than 500, less than 250, or less than 100 nucleotides upstream of the TSS.
  • the targeted sequence is located downstream of the TSS in the TSS core.
  • the targeted sequence may be less than 2000, less than 1000, less than 500, less than 250, or less than 100 nucleotides downstream of the TSS.
  • the targeted sequence is located +/- 50 nucleotides surrounding the TSS of the TSS core. In some embodiments, the targeted sequence substantially overlaps the TSS of the TSS core. In some embodiments, the targeted sequence begins or ends at the TSS of the TSS core. In some embodiments, the targeted sequence overlaps the TSS of the TSS core by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 or 19 nucleotides in either the upstream or downstream direction.
  • the location of the targeted sequence on the template strand is defined by the location of the 5’ end of the targeted sequence.
  • the 5’ end of the targeted sequence may be at any position of the TSS core and the targeted sequence may start at any position selected from position 1 to position 4001 of the TSS core.
  • the targeted sequence when the 5’ end of the targeted sequence is located between position 1 to position 2000 of the TSS core, the targeted sequence is considered upstream of the TSS and when the 5’ end of the targeted sequence is from position 2002 to 4001, the targeted sequence is considered downstream of the TSS.
  • the targeted sequence is considered to be a TSS centric sequence and is neither upstream nor downstream of the TSS.
  • the targeted sequence when the 5’ end of the targeted sequence is at position 1600 of the TSS core, i.e., it is the 1600 th nucleotide of the TSS core, the targeted sequence starts at position 1600 of the TSS core and is considered to be upstream of the TSS.
  • the saRNA of the present disclosure is a single-stranded saRNA.
  • the single-stranded saRNA may be at least 14, or at least 18, e.g., 19, 20, 21, 22 or 23 nucleotides in length since oligonucleotide duplex exceeding this length may have an increased risk of inducing the interferon response.
  • the length of the single-stranded saRNA is less than 30 nucleotides.
  • the length of the single-stranded saRNA is 19 to 25 nucleotides.
  • the single-stranded saRNA may be exactly 19 nucleotides in length.
  • the single-stranded saRNA may be exactly 20 nucleotides in length. In another embodiment, the single-stranded saRNA may be exactly 21 nucleotides in length. In another embodiment, the single-stranded saRNA may be exactly 22 nucleotides in length. In another embodiment, the single-stranded saRNA may be exactly 23 nucleotides in length. In some embodiments, the single-stranded saRNA of the present disclosure comprises a sequence of at least 14 nucleotides and less than 30 nucleotides, which has at least 80%, 90%, 95%, 98%, 99% or 100% complementarity to the targeted sequence. In one embodiment, the sequence which has at least 80%, 90%, 95%, 98%, 99% or 100% complementarity to the targeted sequence is at least 15, 16, 17, 18 or 19 nucleotides in length, or 18 to 22, or 19 to 21, or exactly 19.
  • the saRNA of the present disclosure has two strands that form a duplex, one strand being an antisense or guide strand.
  • the saRNA duplex is also called a double-stranded saRNA.
  • a double-stranded saRNA or saRNA duplex, as used herein, is a saRNA that includes more than one, and preferably, two, strands in which interstrand hybridization can form a region of duplex structure.
  • the two strands of a double-stranded saRNA are referred to as an antisense strand or a guide strand, and a sense strand or a passenger strand.
  • Each strand of the duplex may be at least 14, or at least 18, e.g., 19, 20, 21 or 22 nucleotides in length.
  • the duplex may be hybridized over a length of at least 12, or at least 15, or at least 17, or at least 19 nucleotides.
  • Each strand may be exactly 19, 20, 21, 22, or 23 nucleotides in length.
  • the length of each strand of the saRNA is less than 30 nucleotides since oligonucleotide duplex exceeding this length may have an increased risk of inducing the interferon response. In one embodiment, the length of each strand of the saRNA is 19 to 25 nucleotides.
  • the antisense strand of the saRNA of the present disclosure comprises a sequence of at least 14 nucleotides and less than 30 nucleotides, such as exactly 19, 20, 21, 22, or 23 nucleotides in length, which has at least 80%, 90%, 95%, 98%, 99% or 100% complementarity to the targeted sequence.
  • the sequence which has at least 80%, 90%, 95%, 98%, 99% or 100% complementarity to the targeted sequence is at least 15, 16, 17, 18 or 19 nucleotides in length, or 18 to 22, or 19 to 21, or exactly 19.
  • the antisense strand may have no more than 5, or no more than 4 or 3, or no more than 2, or no more than 1 , or no mismatches with the targeted sequence on the template strand. Therefore, the antisense strand has a high degree of complementarity to the targeted sequence on the template strand.
  • the sense strand of the saRNA duplex has a high degree of sequence identity with the targeted sequence on the template strand.
  • a “strand” in the context of the present disclosure means a contiguous sequence of nucleotides, including non-naturally occurring or modified nucleotides. Two or more strands may be, or each form a part of, separate molecules, or they may be connected covalently, e.g., by a linker such as a polyethyleneglycol linker. At least one strand of a saRNA may comprise a region that is complementary to a region on the guide strand of the target gene (targeted sequence) and has sequence identity with a region on the coding strand of the target gene. Such a strand is called an antisense or guide strand of the saRNA duplex. A second strand of a saRNA that comprises a region complementary to the antisense strand of the saRNA is called a sense or passenger strand.
  • a saRNA duplex may also be formed from a single molecule that is at least partly self-complementary forming a hairpin structure, including a duplex region.
  • the term “strand” refers to one of the regions of the saRNA that is complementary to another internal region of the saRNA.
  • the guide strand of the saRNA will have no more than 5, or no more than 4 or 3, or no more than 2, or no more than 1, or no mismatches with the sequence within the region on the template strand of the target gene (targeted sequence).
  • the passenger strand of a saRNA may comprise at least one nucleotide that is not complementary to the corresponding nucleotide on the guide strand, called a mismatch with the guide strand.
  • the mismatch with the guide strand may encourage preferential loading of the guide strand (Wu et al., PLoS ONE, vol.6 (12):e28580 (2011), the contents of which are incorporated herein by reference in their entirety).
  • the at least one mismatch with the guide strand may be at 3 ’ end of the passenger strand.
  • the 3’ end of the passenger strand may comprise 1-5 mismatches with the guide strand.
  • the 3’ end of the passenger strand may comprise 2-3 mismatches with the guide strand.
  • the 3’ end of the passenger strand may comprise 6-10 mismatches with the guide strand.
  • a saRNA duplex may have siRNA-like complementarity to the targeted sequence on the template strand; that is, 100% complementarity between nucleotides 2-6 from the 5' end of the guide strand in the saRNA duplex and a region on the targeted sequence.
  • Other nucleotides of the saRNA may, in addition, have at least 80%, 90%, 95%, 98%, 99% or 100% complementarity to a region of the targeted sequence.
  • nucleotides 7 (counted from the 5' end) until the 3' end of the saRNA may have least 80%, 90%, 95%, 98%, 99% or 100% complementarity to a region of the targeted sequence.
  • small interfering RNA or “siRNA” in the context mean a double-stranded RNA typically 20-25 nucleotides long involved in the RNA interference (RNAi) pathway and interfering with or inhibiting the expression of a specific gene.
  • the gene is the target gene of the siRNA.
  • a siRNA is usually about 21 nucleotides long, with 3' overhangs (e.g., 2 nucleotides) at each end of the two strands.
  • the saRNA may comprise a number of unpaired nucleotides at the 3' end of each strand forming 3' overhangs or tails.
  • the number of unpaired nucleotides forming the 3' overhang of each strand may be in the range of 1 to 5 nucleotides, or 1 to 3 nucleotides, or 2 nucleotides.
  • the saRNA of the present disclosure may be singlestranded and consists of (i) a sequence having at least 80% complementarity to a targeted sequence on the template strand of the target gene; and (ii) a 3' tail (overhang) of 1 -5 nucleotides, which may comprise uracil residues, such as UU, UUU, or mUmU (m strands for 2’-0Me modification).
  • the saRNA of the present disclosure may be double-stranded and consists of a first strand comprising (i) a first sequence having at least 80% complementarity to a targeted sequence on the template strand of the target gene; and (ii) a 3' overhang of 1 -5 nucleotides; and a second strand comprising (i) a second sequence that forms a duplex with the first sequence and (ii) a 3’ overhang of 1-5 nucleotides.
  • a 3’ tail (overhang) shall not be regarded as mismatches with regard to determine complementarity between the saRNA antisense strand and the targeted sequence.
  • the saRNA of the present disclosure may have complementarity to the targeted sequence over its whole length, except for the 3' tail (overhang), if present.
  • the saRNA of the present disclosure may contain a flanking sequence.
  • the flanking sequence may be inserted in the 3’ end or 5’ end of the saRNA of the present disclosure.
  • the flanking sequence is the sequence of a miRNA, rendering the saRNA to have miRNA configuration and may be processed with Drosha and Dicer.
  • the saRNA of the present disclosure has two strands and is cloned into a microRNA precursor, e.g., miR-30 backbone flanking sequence.
  • the saRNA of the present disclosure may comprise a restriction enzyme substrate or recognition sequence.
  • the restriction enzyme recognition sequence may be at the 3’ end or 5’ end of the saRNA of the present disclosure.
  • restriction enzymes include Notl and Asci.
  • the antisense strand of the saRNA has a high degree of sequence identity with the reverse complement of the targeted sequence.
  • the antisense strand of the saRNA of the present disclosure may also be defined as having “identity” to a region on the coding strand of the target gene. Therefore, the genomic sequence of the target gene may be used to design saRNAs.
  • the target gene of the saRNAs of the present disclosure is TMEM173 (SUNG). Sequences of the target gene, protein and mRNA encoded by the target genes, and TSS cores of the target gene is provided in Table 1. Table 1 Sequences of the target gene and protein and mRNA encoded by the target gene
  • Table 2 describes the saRNAs’ targeted sequences, the genomic location of the targeted sequences, and the relative location of saRNAs with no 3’ overhang.
  • the targeted sequence is defined as a region on the template strand of the target gene.
  • the relative location is the distance from the 5’ end of the targeted sequence to the TSS.
  • a negative number represents a location upstream of the TSS and a positive number represents a location downstream of the TSS.
  • the saRNAs may be single-stranded and comprise 14-30 nucleotides.
  • the sequence of a single-stranded saRNA may have at least 60%, 70%, 80% or 90% identity with a sequence selected from the sequences of the antisense strands in Table 3.
  • the singlestranded saRNA comprises a sequence selected from the sequences of the antisense strands in Table 3.
  • the single-stranded saRNA may have a 3’ tail (overhang).
  • the sequence of a single-stranded saRNA with a 3’ tail (overhang) may have at least 60%, 70%, 80% or 90% identity with a sequence selected from the sequences of the antisense strands in Table 4.
  • the single-stranded saRNA comprises a sequence selected from the sequences of the antisense strands in Table 4.
  • the saRNAs may be double-stranded.
  • the two strands form a duplex and each strand comprises 14-30 nucleotides.
  • the first strand of a double-stranded saRNA may have at least 60%, 70%, 80% or 90% identity with a sequence selected from the sequences of the antisense strands in Table 3.
  • the first strand of the double-stranded saRNA comprises a sequence selected from the sequences of the antisense strands in Table 3.
  • the second strand of a double-stranded saRNA may have at least 60%, 70%, 80% or 90% identity with a sequence selected from the sequences of the sense strands in Table 3.
  • the second strand of the double-stranded saRNA comprises a sequence selected from the sequences of the sense strands in Table 3.
  • the double-stranded saRNA may have a 3’ overhang on each strand.
  • the first strand of a double-stranded saRNA may have at least 60%, 70%, 80% or 90% identity with a sequence selected from the sequences of the antisense strands in Table 4.
  • the first strand of the double-stranded saRNA comprises a sequence selected from the sequences of the antisense strands in Table 4.
  • the second strand of a double-stranded saRNA may have at least 60%, 70%, 80% or 90% identity with a sequence selected from the sequences of the sense strands in Table 4.
  • the second strand of the double-stranded saRNA comprises a sequence selected from the sequences of the sense strands in Table 4.
  • the saRNAs may be modified or unmodified.
  • - u means 2’0-methyl-uracil (2’-OMe).
  • the 3 ’ overhang, uu, in the sequences may be replaced with any other 3 ’ overhang, such as UU (unmodified uracils) or UUU.
  • 5 ’ overhangs such as dT, ddT, or inv Ab can also be added to the 5’ position.
  • N A, C, G or U
  • the saRNA of the present disclosure may be produced by any suitable method, for example synthetically or by expression in cells using standard molecular biology techniques which are well-known to a person of ordinary skill in the art.
  • the saRNA of the present disclosure may be chemically synthesized or recombinantly produced using methods known in the art.
  • compositions comprising a small activating RNA (saRNA) that upregulates a target gene, and at least one pharmaceutically acceptable carrier.
  • saRNA small activating RNA
  • any conventional excipient medium may be contemplated within the scope of the present disclosure, except insofar as any conventional excipient medium may be incompatible with a substance or its derivatives, such as by producing any undesirable biological effect or otherwise interacting in a deleterious manner with any other component(s) of the pharmaceutical composition.
  • compositions are administered to humans, human patients or subjects.
  • active ingredient generally refers to saRNA to be delivered as described herein.
  • Subjects to which administration of the pharmaceutical compositions is contemplated include, but are not limited to, humans and/or other primates; mammals, including commercially relevant mammals such as cattle, pigs, horses, sheep, cats, dogs, mice, and/or rats; and/or birds, including commercially relevant birds such as poultry, chickens, ducks, geese, and/or turkeys.
  • Formulations of the pharmaceutical compositions described herein may be prepared by any method known or hereafter developed in the art of pharmacology. In general, such preparatory methods include the step of bringing the active ingredient into association with an excipient and/or one or more other accessory ingredients, and then, if necessary and/or desirable, dividing, shaping and/or packaging the product into a desired single- or multi-dose unit.
  • a pharmaceutical composition in accordance with the disclosure may be prepared, packaged, and/or sold in bulk, as a single unit dose, and/or as a plurality of single unit doses.
  • a “unit dose” is discrete amount of the pharmaceutical composition comprising a predetermined amount of the active ingredient.
  • the amount of the active ingredient is generally equal to the dosage of the active ingredient which would be administered to a subject and/or a convenient fraction of such a dosage such as, for example, one-half or one-third of such a dosage.
  • compositions in accordance with the disclosure will vary, depending upon the identity, size, and/or condition of the subject treated and further depending upon the route by which the composition is to be administered.
  • the composition may comprise between 0.1% and 100%, e.g., between .5 and 50%, between 1-30%, between 5-80%, at least 80% (w/w) active ingredient.
  • the formulations described herein may contain at least one saRNA.
  • the formulations may contain 1, 2, 3, 4 or 5 saRNAs with different sequences.
  • the formulation contains at least three saRNAs with different sequences.
  • the formulation contains at least five saRNAs with different sequences.
  • the saRNA of the present disclosure can be formulated using one or more excipients to: (1) increase stability; (2) increase cell transfection; (3) permit the sustained or delayed release (e.g., from a depot formulation of the saRNA); (4) alter the biodistribution (e.g., target the saRNA to specific tissues or cell types); (5) increase the translation of encoded protein in vivo; and/or (6) alter the release profile of encoded protein in vivo.
  • the pharmaceutical compositions may be formulated in liposomes such as, but not limited to, DiLa2 liposomes (Marina Biotech, Bothell, WA), SMARTICLESO/NOV340 (Manna Biotech, Bothell, WA), neutral DOPC (1,2-dioleoyl-sn- glycero-3 -phosphocholine) based liposomes (e.g., siRNA delivery for ovarian cancer (Landen et al. Cancer Biology & Therapy 2006 5(12)1708-1713); the contents of which is herein incorporated by reference in its entirety) and hyaluronan-coated liposomes (Quiet Therapeutics, Israel).
  • DiLa2 liposomes Marina Biotech, Bothell, WA
  • SMARTICLESO/NOV340 Manna Biotech, Bothell, WA
  • neutral DOPC 1,2-dioleoyl-sn- glycero-3 -phosphocholine
  • siRNA delivery for ovarian cancer Lianden
  • the saRNA of the present disclosure is encapsulated into liposomes, wherein the liposomes comprise l-palmitoyl-2-oleoyl-sn-glycero-3 -phosphocholine (POPC), l,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), cholesteryl-hemisuccinate (CHEMS), and 4-(2-aminoethyl)-morpholino-cholesterol hemisuccinate (MOCHOL).
  • POPC l-palmitoyl-2-oleoyl-sn-glycero-3 -phosphocholine
  • DOPE l,2-dioleoyl-sn-glycero-3-phosphoethanolamine
  • CHEMS cholesteryl-hemisuccinate
  • MOCHOL 4-(2-aminoethyl)-morpholino-cholesterol hemisuccinate
  • the liposome is SMARTICLES®/NOV340 which
  • the nanoparticles are anionic at physiological pH, and their specific lipid ratio imparts a “pH-tunable” character and a charge to the liposomes, which changes depending upon the surrounding pH of the microenvironment to facilitate movement across physiologic membranes.
  • SMARTICLES® nanoparticles are sized to avoid extensive immediate hepatic sequestration, with an average diameter of approximately about 50 - about 150 nm, or about 100 - about 120 nm, facilitating more prolonged systemic distribution and improved serum stability after i.v. injection leading to broader tissue distribution with high levels in liver, spleen and bone marrow reported.
  • the present disclosure provides a pharmaceutical composition comprising the saRNAs and a liposome, wherein the liposome comprises POPC, DOPE, CHEMS and MOCHOL in the molar ratio of 6:24:23:47.
  • the saRNAs may be encapsulated into the liposome by the step of: dissolving the saRNA in a first buffer to form a saRNA solution, filtering the saRNA solution through a 0.2 pm filter, mixing the filtered saRNA solution with a lipid solution in an injection module to form a liposome formulation, and adding a second buffer to the liposome formulation.
  • the first buffer may comprise sodium acetate and sucrose.
  • the pH for the saRNA solution may be about 4.0.
  • the concentration of the saRNA in the saRNA solution may be about 2.38 mg/mL.
  • the lipid solution comprises l-palmitoyl-2-oleoyl-sn-glycero-3- phosphocholine (POPC), l,2-dioleoyl-sn-glycero-3 -phosphoethanolamine (DOPE), cholesteryl- hemisuccinate (CHEMS), and 4-(2-aminoethyl)-morpholino-cholesterol hemisuccinate (MOCHOL).
  • POPC l-palmitoyl-2-oleoyl-sn-glycero-3- phosphocholine
  • DOPE l,2-dioleoyl-sn-glycero-3 -phosphoethanolamine
  • CHEMS cholesteryl- hemisuccinate
  • MOCHOL 4-(2-aminoethyl)-morpholino-cholesterol hemisuccinate
  • the molar ratio of POPC:DOPE:CHEMS:MOCHOL may be about 6:24:23:47.
  • the second buffer may
  • the present disclosure encompasses the delivery of saRNA for any of therapeutic, prophylactic, pharmaceutical, diagnostic or imaging by any appropriate route taking into consideration likely advances in the sciences of drug delivery. Delivery may be naked or formulated.
  • the saRNA of the present disclosure may be delivered to a cell naked.
  • naked refers to delivering saRNA free from agents which promote transfection.
  • the saRNA delivered to the cell may contain no modifications.
  • the naked saRNA may be delivered to the cell using routes of administration known in the art and described herein.
  • the saRNA of the present disclosure may be formulated, using the methods described herein.
  • the formulations may contain saRNA which may be modified and/or unmodified.
  • the formulations may further include, but are not limited to, cell penetration agents, a pharmaceutically acceptable carrier, a delivery agent, a bioerodible or biocompatible polymer, a solvent, and a sustained-release delivery depot.
  • the formulated saRNA may be delivered to the cell using routes of administration known in the art and described herein.
  • compositions may also be formulated for direct delivery to an organ or tissue in any of several ways in the art including, but not limited to, direct soaking or bathing, via a catheter, by gels, powder, ointments, creams, gels, lotions, and/or drops, by using substrates such as fabric or biodegradable materials coated or impregnated with the compositions, and the like.
  • the saRNA of the present disclosure may also be cloned into a retroviral replicating vector (RRV) and transduced to cells.
  • RRV retroviral replicating vector
  • the saRNA of the present disclosure may be administered by any route which results in a therapeutically effective outcome. These include, but are not limited to enteral, gastroenteral, epidural, oral, transdermal, epidural (peridural), intracerebral (into the cerebrum), intracerebroventricular (into the cerebral ventricles), epicutaneous (application onto the skin), intradermal, (into the skin itself), subcutaneous (under the skin), nasal administration (through the nose), intravenous (into a vein), intraarterial (into an artery), intramuscular (into a muscle), intracardiac (into the heart), intraosseous infusion (into the bone marrow), intrathecal (into the spinal canal), intraperitoneal, (infusion or injection into the peritoneum), intravesical infusion, intravitreal, (through the eye), intracavernous injection, ( into the base of the penis), intravaginal administration, intrauterine, extra-amniotic administration, transdermatitis,
  • compositions may be administered in a way which allows them cross the blood-brain barrier, vascular barrier, or other epithelial barrier.
  • Routes of administration disclosed in International Publication WO 2013/090648 filed December 14, 2012, the contents of which are incorporated herein by reference in their entirety, may be used to administer the saRNA of the present disclosure.
  • the saRNAs of the present disclosure are delivered intratumorally.
  • a pharmaceutical composition described herein can be formulated into a dosage form described herein, such as a topical, intranasal, intratracheal, or injectable (e.g., intravenous, intraocular, intravitreal, intramuscular, intracardiac, intraperitoneal, subcutaneous).
  • injectable e.g., intravenous, intraocular, intravitreal, intramuscular, intracardiac, intraperitoneal, subcutaneous.
  • Liquid dosage forms, injectable preparations, pulmonary forms, and solid dosage forms described in International Publication WO 2013/090648 filed December 14, 2012, the contents of which are incorporated herein by reference in their entirety may be used as dosage forms for the saRNA of the present disclosure.
  • One aspect of the present disclosure provides methods of using saRNA of the present disclosure and pharmaceutical compositions comprising the saRNA and at least one pharmaceutically acceptable carrier.
  • the saRNA of the present disclosure modulates the expression of its target gene.
  • a method of regulating the expression of a target gene in vitro and/or in vivo comprising administering the saRNA of the present disclosure.
  • the expression of the target gene is increased by at least 5, 10, 20, 30, 40%, or at least 45, 50, 55, 60, 65, 70, 75%, or at least 80% in the presence of the saRNA of the present disclosure compared to the expression of the target gene in the absence of the saRNA of the present disclosure.
  • the expression of the target gene is increased by a factor of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, or by a factor of at least 15, 20, 25, 30, 35, 40, 45, 50, or by a factor of at least 60, 70, 80, 90, 100, in the presence of the saRNA of the present disclosure compared to the expression of the target gene in the absence of the saRNA of the present disclosure.
  • One aspect of the present application provides a method of modulating the expression of the SUNG (Stimulator Of Interferon Response CGAMP Interactor; STING1; TMEM173) gene comprising administering TMEM173-saRNA of the present disclosure.
  • the expression of the TMEM173 gene is increased by at least 20, 30, 40%, or at least 45, 50, 55, 60, 65, 70, 75%, or at least 80% in the presence of the TMEM173-saRNA of the present disclosure compared to the expression of the TMEM173 gene in the absence of the TMEM173- saRNA of the present disclosure.
  • the expression of the TMEM173 gene is increased by a factor of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, or by a factor of at least 15, 20, 25, 30, 35, 40, 45, 50, or by a factor of at least 60, 70, 80, 90, 100, in the presence of the TMEM173- saRNA of the present disclosure compared to the expression of the STING gene in the absence of the TMEM173 -saRNA of the present disclosure.
  • the modulation of the expression of the TMEM173 gene may be reflected or determined by the change of the TMEM173 mRNA levels.
  • the TMEM173 gene encodes an endoplasmic reticulum adaptor protein critical for innate immune signalling.
  • cGAMP cyclic GMP-AMP
  • cGAMP cyclic GMP-AMP
  • STING is downregulated in various type of tumours by promoter hypermethylation. Restoration of SUNG expression by DNA methylation inhibitors improve control of tumour growth (Kitajima et al., Cancer Discovery, vol.9(l):34 (2019)).
  • TMEM173-saRNAs of the present disclosure may be used to prevent or treat diseases or disorders associated with STING.
  • TMEM173 -saRNA of the present disclosure is used to prevent or treat diseases such as cancer, TMEM173-associated vasculopathy, infantile-onset and familial chilblain lupus.
  • saRNAs of the present invention may be used to treat any disease associated with the TMEM173 gene.
  • methods for treating a subject comprising administering a therapeutically-effective amount of the saRNAs of the present disclosure, to the subject having cancer, suspected of having cancer, or having a predisposition to a cancer.
  • cancer embraces any disease or malady characterized by uncontrolled cell proliferation, e.g., hyperproliferation.
  • Cancers may be characterized by tumors, e.g., solid tumors or any neoplasm. In some embodiments, the cancer is a solid tumor.
  • saRNAs of the present invention are effective for inhibiting tumor growth, whether measured as a net value of size (weight, surface area or volume) or as a rate over time, in multiple types of tumors.
  • the size of a tumor is reduced by about 60 % or more after treatment with saRNAs of the present invention. In some embodiments, the size of a tumor is reduced by at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 100%, by a measure of weight, and/or area and/or volume.
  • cancers include, but are not limited to, acoustic neuroma, acute leukemia, acute lymphocytic leukemia, acute myelocytic leukemia (monocytic, myeloblastic, adenocarcinoma, angiosarcoma, astrocytoma, myelomonocytic and promyelocytic), acute T-cell leukemia, basal cell carcinoma, bile duct carcinoma, bladder cancer, brain cancer, breast cancer, bronchogenic carcinoma, cervical cancer, chondrosarcoma, chordoma, choriocarcinoma, chronic leukemia, chronic lymphocytic leukemia, chronic myelocytic (granulocytic) leukemia, chronic myelogenous leukemia, colon cancer, colorectal cancer, craniopharyngioma, cystadenocarcinoma, diffuse large B-cell lymphoma, Burkitt’s lymphoma, dyspro
  • cancers include primary cancer, metastatic cancer, oropharyngeal cancer, hypopharyngeal cancer, liver cancer, gall bladder cancer, bile duct cancer, small intestine cancer, urinary tract cancer, kidney cancer, urothelium cancer, female genital tract cancer, uterine cancer, gestational trophoblastic disease, male genital tract cancer, seminal vesicle cancer, testicular cancer, germ cell tumors, endocrine gland tumors, thyroid cancer, adrenal cancer, pituitary gland cancer, hemangioma, sarcoma arising from bone and soft tissues, Kaposi’s sarcoma, nerve cancer, ocular cancer, meningial cancer, glioblastomas, neuromas, neuroblastomas, Schwannomas, solid tumors arising from hematopoietic malignancies such as leukemias, metastatic melanoma, recurrent or persistent ovarian epithelial cancer, fallopian tube cancer, primary peritoneal cancer,
  • the cancers treatable by methods of the present disclosure generally occur in mammals.
  • Mammals include, for example, humans, non-human primates, dogs, cats, rats, mice, rabbits, ferrets, guinea pigs, horses, pigs, sheep, goats, and cattle.
  • the subject in need thereof receives a dose of less than about 2.5 - 5.0mg of TMEM173-saRNAs.
  • TMEM173-saRNAs may be administered in a solution of about 1 to 2ml, wherein the solution comprises NOV340 Smarticles and buffer.
  • the saRNA of the present invention may be provided in combination with additional active agents or therapies known to have an effect in the particular method being considered.
  • the combination therapy comprising saRNA and additional active agents or therapies may be given to any patient in need thereof to treat any disorder described herein, including metabolics regulation, surgical care, hyperproliferative disorders, and/or stem cell regulation.
  • the saRNA of the present invention in combination with additional active agents or therapies may be used to treat a cancer.
  • the additional active agents may be administered simultaneously or sequentially with the saRNA.
  • the additional active agents may be administered in a mixture with the saRNA or be administered separately from the saRNA.
  • administered simultaneously is not specifically restricted and means that the components of the combination therapy, i.e., saRNA of the present invention and the additional active agents, are substantially administered at the same time, e.g. as a mixture or in immediate subsequent sequence.
  • the term “administered sequentially” as used herein is not specifically restricted and means that the components of the combination therapy, i.e., saRNA of the present invention and the additional active agents, are not administered at the same time but one after the other, or in groups, with a specific time interval between administrations.
  • the time interval may be the same or different between the respective administrations of the components of the combination therapy and may be selected, for example, from the range of 2 minutes to 96 hours, 1 to 7 days or one, two or three weeks.
  • the time interval between the administrations may be in the range of a few minutes to hours, such as in the range of 2 minutes to 72 hours, 30 minutes to 24 hours, or 1 to 12 hours.
  • saRNA of the present invention is administered before the additional active agents.
  • additional active agents are administered before the saRNA of the present invention.
  • the molar ratio of the saRNA of the present invention and the additional active agents is not particularly restricted.
  • the molar ratio between the two components may be in the range of 1 : 500 to 500: 1 , or of 1 : 100 to 100: 1, or of 1:50 to 50: 1, or of 1:20 to 20:1, or of 1 :5 to 5: 1, or 1: 1. Similar molar ratios apply when more than two components are combined in a composition.
  • Each component may comprise, independently, a predetermined molar weight percentage from about 1% to 10%, or about 10% to about 20%, or about 20% to about 30%, or about 30% to 40%, or about 40% to 50%, or about 50% to 60%, or about 60% to 70%, or about 70% to 80%, or about 80% to 90%, or about 90% to 99% of the composition.
  • the saRNA and/or compositions of the present application may be combined with at least one additional therapy, such as surgical treatment, radiation therapy, chemotherapy, immunotherapy, gene therapy (e.g., CRISPR therapies, mRNA-based therapies, other oligonucleotide therapies) and/or with any other antineoplastic treatment method.
  • additional therapy such as surgical treatment, radiation therapy, chemotherapy, immunotherapy, gene therapy (e.g., CRISPR therapies, mRNA-based therapies, other oligonucleotide therapies) and/or with any other antineoplastic treatment method.
  • the treatments may be in any order at appropriate dosing and frequency for each treatment.
  • the saRNA and/or compositions of the present application may be combined with at least one radiation therapy.
  • the saRNA and/or compositions of the present application may be combined with at least one chemotherapy.
  • the saRNA and/or compositions of the present application may be combined with radiation therapy and chemotherapy.
  • the saRNA and/or compositions of the present application may be combined with at least one immunotherapy.
  • immunotherapy refers to any therapy that can provoke and/or enhance an immune response to destroy tumor cells in a subject.
  • the saRNA and/or compositions of the present application may be combined with cancer vaccines.
  • the term “vaccine” refers to a composition for generating immunity for the prophylaxis and/or treatment of diseases.
  • the saRNA and/or compositions of the present application may be combined with immunotherapeutics such as immune checkpoint inhibitors.
  • the checkpoint inhibitor may be an antagonist agent against CTLA-4 such as an antibody, a functional fragment of the antibody, a polypeptide, or a functional fragment of the polypeptide, or a peptide, which can bind to CTLA-4 with high affinity and prevent the interaction of B7-1/2 (CD80/86) with CTLA-4.
  • the CTLA-4 antagonist is an antagonistic antibody, or a functional fragment thereof.
  • Suitable anti-CTLA-4 antagonistic antibody include, without limitation, anti-CTLA-4 antibodies, human anti-CTLA-4 antibodies, mammalian anti-CTLA-4 antibodies, humanized anti-CTLA-4 antibodies, monoclonal anti- CTLA-4 antibodies, polyclonal anti-CTLA-4 antibodies, chimeric anti-CTLA-4 antibodies, MDX-010 (ipilimumab), tremelimumab (fully humanized), anti-CD28 antibodies, anti-CTLA-4 adnectins, anti-CTLA-4 domain antibodies, single chain anti-CTLA-4 antibody fragments, heavy chain anti-CTLA-4 fragments, light chain anti-CTLA-4 fragments, and the antibodies disclosed in U.S.
  • Additional anti-CTLA-4 antagonist agents include, but are not limited to, any inhibitors that are capable of disrupting the ability of CTLA-4 to bind to the ligands CD80/86.
  • the checkpoint inhibitor may be agents used for blocking the PD-1 pathway include antagonistic peptides/antibodies and soluble PD-L1 ligands (See Table 5).
  • the saRNAs of the present invention may be used in combination with one or more immune checkpoint blockade (ICB) agent.
  • the combination may have synergistic effect on preventing and/or treating any cancer, such as but not limited to HCC.
  • the ICB is a small inhibiting RNA (siRNA).
  • siRNA may be single stranded or double stranded.
  • the ICB is an antibody.
  • the ICB is a small molecule.
  • the ICB is any agent in checkpoint inhibitor in Table 5.
  • the ICB is Pembroluzimab, Tremelimumab, Durvalumab or
  • the saRNAs and/or compositions of the present application may be combined with a gene therapy, such as CRISPR (Clustered Regularly Interspaced Short Palidromic Repeats) therapy.
  • CRISPR Clustered Regularly Interspaced Short Palidromic Repeats
  • CRISPR therapy refers to any treatment that involves CRISPR-Cas system for gene editing.
  • kits for conveniently and/or effectively carrying out methods of the present disclosure.
  • kits will comprise sufficient amounts and/or numbers of components to allow a user to perform multiple treatments of a subject(s) and/or to perform multiple experiments.
  • kits for regulate the expression of genes in vitro or in vivo comprising saRNA of the present disclosure or a combination of saRNA of the present disclosure, saRNA modulating other genes, siRNAs, miRNAs or other oligonucleotide molecules.
  • the kit may further comprise packaging and instructions and/or a delivery agent to form a formulation composition.
  • the delivery agent may comprise a saline, a buffered solution, a lipidoid, a dendrimer or any delivery agent disclosed herein.
  • the buffer solution may include sodium chloride, calcium chloride, phosphate and/or EDTA.
  • the buffer solution may include, but is not limited to, saline, saline with 2mM calcium, 5% sucrose, 5% sucrose with 2mM calcium, 5% Mannitol, 5% Mannitol with 2mM calcium, Ringer’s lactate, sodium chloride, sodium chloride with 2mM calcium and mannose (See U.S. Pub. No. 20120258046; herein incorporated by reference in its entirety).
  • the buffer solutions may be precipitated or it may be lyophilized. The amount of each component may be varied to enable consistent, reproducible higher concentration saline or simple buffer formulations. The components may also be varied in order to increase the stability of saRNA in the buffer solution over a period of time and/or under a variety of conditions.
  • the present disclosure provides for devices which may incorporate saRNA of the present disclosure. These devices contain in a stable formulation available to be immediately delivered to a subject in need thereof, such as a human patient.
  • Non-limiting examples of the devices include a pump, a catheter, a needle, a transdermal patch, a pressurized olfactory delivery device, iontophoresis devices, multi-layered microfluidic devices.
  • the devices may be employed to deliver saRNA of the present disclosure according to single, multi- or split-dosing regiments.
  • the devices may be employed to deliver saRNA of the present disclosure across biological tissue, intradermal, subcutaneously, or intramuscularly. More examples of devices suitable for delivering oligonucleotides are disclosed in International Publication WO 2013/090648 filed December 14, 2012, the contents of which are incorporated herein by reference in their entirety.
  • Administered in combination means that two or more agents are administered to a subject at the same time or within an interval such that there may be an overlap of an effect of each agent on the patient. In some embodiments, they are administered within about 60, 30, 15, 10, 5, or 1 minute of one another. In some embodiments, the administrations of the agents are spaced sufficiently close together such that a combinatorial (e.g., a synergistic) effect is achieved.
  • Animal As used herein, the term “animal” refers to any member of the animal kingdom. In some embodiments, “animal” refers to humans at any stage of development.
  • animal refers to non-human animals at any stage of development.
  • the non-human animal is a mammal (e.g., a rodent, a mouse, a rat, a rabbit, a monkey, a dog, a cat, a sheep, cattle, a primate, or a pig).
  • animals include, but are not limited to, mammals, birds, reptiles, amphibians, fish, and worms.
  • the animal is a transgenic animal, genetically-engineered animal, or a clone.
  • association means that the moieties are physically associated or connected with one another, either directly or via one or more additional moieties that serves as a linking agent, to form a structure that is sufficiently stable so that the moieties remain physically associated under the conditions in which the structure is used, e.g., physiological conditions.
  • An “association” need not be strictly through direct covalent chemical bonding. It may also suggest ionic or hydrogen bonding or a hybridization based connectivity sufficiently stable such that the “associated” entities remain physically associated.
  • biologically active refers to a characteristic of any substance that has activity in a biological system and/or organism. For instance, a substance that, when administered to an organism, has a biological effect on that organism, is considered to be biologically active.
  • the saRNA of the present disclosure may be considered biologically active if even a portion of the saRNA is biologically active or mimics an activity considered biologically relevant.
  • cancer in an individual refers to the presence of cells possessing characteristics typical of cancer-causing cells, such as uncontrolled proliferation, immortality, metastatic potential, rapid growth and proliferation rate, and certain characteristic morphological features. Often, cancer cells will be in the form of a tumor, but such cells may exist alone within an individual, or may circulate in the blood stream as independent cells, such as leukemic cells.
  • Cell growth is principally associated with growth in cell numbers, which occurs by means of cell reproduction (i.e. proliferation) when the rate of the latter is greater than the rate of cell death (e.g. by apoptosis or necrosis), to produce an increase in the size of a population of cells, although a small component of that growth may in certain circumstances be due also to an increase in cell size or cytoplasmic volume of individual cells.
  • An agent that inhibits cell growth can thus do so by either inhibiting proliferation or stimulating cell death, or both, such that the equilibrium between these two opposing processes is altered.
  • Cell type refers to a cell from a given source (e.g., a tissue, organ) or a cell in a given state of differentiation, or a cell associated with a given pathology or genetic makeup.
  • Chromosome As used herein, the term “chromosome” refers to an organized structure of DNA and protein found in cells.
  • nucleic acids refers to hybridization or base pairing between nucleotides or nucleic acids, such as, for example, between the two strands of a double-stranded DNA molecule or between an oligonucleotide probe and a target are complementary.
  • Condition refers to the status of any cell, organ, organ system or organism. Conditions may reflect a disease state or simply the physiologic presentation or situation of an entity. Conditions may be characterized as phenotypic conditions such as the macroscopic presentation of a disease or genotypic conditions such as the underlying gene or protein expression profiles associated with the condition. Conditions may be benign or malignant.
  • Controlled Release refers to a pharmaceutical composition or compound release profile that conforms to a particular pattern of release to effect a therapeutic outcome.
  • Cytostatic refers to inhibiting, reducing, suppressing the growth, division, or multiplication of a cell (e.g., a mammalian cell (e.g., a human cell)), bacterium, virus, fungus, protozoan, parasite, prion, or a combination thereof.
  • Cytotoxic refers to killing or causing injurious, toxic, or deadly effect on a cell (e.g., a mammalian cell (e.g., a human cell)), bacterium, virus, fungus, protozoan, parasite, prion, or a combination thereof.
  • Delivery refers to the act or manner of delivering a compound, substance, entity, moiety, cargo or payload.
  • Delivery Agent refers to any substance which facilitates, at least in part, the in vivo delivery of a saRNA of the present disclosure to targeted cells.
  • Encapsulate As used herein, the term “encapsulate” means to enclose, surround or encase.
  • Engineered As used herein, embodiments of the disclosure are “engineered” when they are designed to have a feature or property, whether structural or chemical, that varies from a starting point, wild type or native molecule.
  • Equivalent subject may be e.g. a subject of similar age, sex and health such as liver health or cancer stage, or the same subject prior to treatment according to the disclosure.
  • the equivalent subject is "untreated” in that he does not receive treatment with a saRNA according to the disclosure. However, he may receive a conventional anti-cancer treatment, provided that the subject who is treated with the saRNA of the disclosure receives the same or equivalent conventional anti-cancer treatment.
  • Exosome is a vesicle secreted by mammalian cells.
  • Expression refers to one or more of the following events: (1) production of an RNA template from a DNA sequence (e.g., by transcription); (2) processing of an RNA transcript (e.g., by splicing, editing, 5’ cap formation, and/or 3’ end processing); (3) translation of an RNA into a polypeptide or protein; and (4) post-translational modification of a polypeptide or protein.
  • Feature As used herein, a “feature” refers to a characteristic, a property, or a distinctive element.
  • Formulation ' includes at least one saRNA of the present disclosure and a delivery agent.
  • Fragment refers to a portion.
  • fragments of proteins may comprise polypeptides obtained by digesting full-length protein isolated from cultured cells.
  • Fragments of oligonucleotides may comprise nucleotides, or regions of nucleotides.
  • a “functional” biological molecule is a biological molecule in a form in which it exhibits a property and/or activity by which it is characterized.
  • Gene refers to a nucleic acid sequence that comprises control and most often coding sequences necessary for producing a polypeptide or precursor. Genes, however, may not be translated and instead code for regulatory or structural RNA molecules.
  • a gene may be derived in whole or in part from any source known to the art, including a plant, a fungus, an animal, a bacterial genome or episome, eukaryotic, nuclear or plasmid DNA, cDNA, viral DNA, or chemically synthesized DNA.
  • a gene may contain one or more modifications in either the coding or the untranslated regions that could affect the biological activity or the chemical structure of the expression product, the rate of expression, or the manner of expression control. Such modifications include, but are not limited to, mutations, insertions, deletions, and substitutions of one or more nucleotides.
  • the gene may constitute an uninterrupted coding sequence or it may include one or more introns, bound by the appropriate splice junctions.
  • Gene expression refers to the process by which a nucleic acid sequence undergoes successful transcription and in most instances translation to produce a protein or peptide.
  • measurements may be of the nucleic acid product of transcription, e.g., RNA or mRNA or of the amino acid product of translation, e.g., polypeptides or peptides. Methods of measuring the amount or levels of RNA, mRNA, polypeptides and peptides are well known in the art.
  • Genome The term “genome” is intended to include the entire DNA complement of an organism, including the nuclear DNA component, chromosomal or extrachromosomal DNA, as well as the cytoplasmic domain (e.g., mitochondrial DNA).
  • homology refers to the overall relatedness between polymeric molecules, e.g. between nucleic acid molecules (e.g. DNA molecules and/or RNA molecules) and/or between polypeptide molecules.
  • polymeric molecules are considered to be “homologous” to one another if their sequences are at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical or similar.
  • the term “homologous” necessarily refers to a comparison between at least two sequences (polynucleotide or polypeptide sequences).
  • two polynucleotide sequences are considered to be homologous if the polypeptides they encode are at least about 50%, 60%, 70%, 80%, 90%, 95%, or even 99% for at least one stretch of at least about 20 amino acids.
  • homologous polynucleotide sequences are characterized by the ability to encode a stretch of at least 4-5 uniquely specified amino acids. For polynucleotide sequences less than 60 nucleotides in length, homology is determined by the ability to encode a stretch of at least 4-5 uniquely specified amino acids.
  • two protein sequences are considered to be homologous if the proteins are at least about 50%, 60%, 70%, 80%, or 90% identical for at least one stretch of at least about 20 amino acids.
  • hyperproliferative cell may refer to any cell that is proliferating at a rate that is abnormally high in comparison to the proliferating rate of an equivalent healthy cell (which may be referred to as a "control").
  • An "equivalent healthy” cell is the normal, healthy counterpart of a cell. Thus, it is a cell of the same type, e.g., from the same organ, which performs the same functions(s) as the comparator cell. For example, proliferation of a hyperproliferative hepatocyte should be assessed by reference to a healthy hepatocyte, whereas proliferation of a hyperproliferative prostate cell should be assessed by reference to a healthy prostate cell.
  • an "abnormally high" rate of proliferation it is meant that the rate of proliferation of the hyperproliferative cells is increased by at least 20, 30, 40%, or at least 45, 50, 55, 60, 65, 70, 75%, or at least 80%, as compared to the proliferative rate of equivalent, healthy (non- hyperproliferative) cells.
  • the "abnormally high” rate of proliferation may also refer to a rate that is increased by a factor of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, or by a factor of at least 15, 20, 25, 30, 35, 40, 45, 50, or by a factor of at least 60, 70, 80, 90, 100, compared to the proliferative rate of equivalent, healthy cells.
  • Hyperproliferative disorder may be any disorder which involves hyperproliferative cells as defined above.
  • hyperproliferative disorders include neoplastic disorders such as cancer, psoriatic arthritis, rheumatoid arthritis, gastric hyperproliferative disorders such as inflammatory bowel disease, skin disorders including psoriasis, Reiter's syndrome, pityriasis rubra pilaris, and hyperproliferative variants of the disorders of keratinization.
  • hyperproliferative cell The presence of hyperproliferative cells within an animal may be identifiable using scans such as X- rays, MRI or CT scans.
  • the hyperproliferative cell may also be identified, or the proliferation of cells may be assayed, through the culturing of a sample in vitro using cell proliferation assays, such as MTT, XTT, MTS or WST-1 assays.
  • Cell proliferation in vitro can also be determined using flow cytometry.
  • Identity refers to the overall relatedness between polymeric molecules, e.g., between oligonucleotide molecules (e.g. DNA molecules and/or RNA molecules) and/or between polypeptide molecules. Calculation of the percent identity of two polynucleotide sequences, for example, can be performed by aligning the two sequences for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second nucleic acid sequences for optimal alignment and non-identical sequences can be disregarded for comparison purposes).
  • the length of a sequence aligned for comparison purposes is at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or 100% of the length of the reference sequence.
  • the nucleotides at corresponding nucleotide positions are then compared. When a position in the first sequence is occupied by the same nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position.
  • the percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which needs to be introduced for optimal alignment of the two sequences.
  • the comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm.
  • the percent identity between two nucleotide sequences can be determined using methods such as those described in Computational Molecular Biology, Lesk, A. M., ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D. W., ed., Academic Press, New York, 1993; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; Computer Analysis of Sequence Data, Part I, Griffin, A. M., and Griffin, H. G, eds., Humana Press, New Jersey, 1994; and Sequence Analysis Primer, Gribskov, M.
  • Methods commonly employed to determine percent identity between sequences include, but are not limited to those disclosed in Carillo, H., and Lipman, D., SIAM J Applied Math., 48:1073 (1988); incorporated herein by reference. Techniques for determining identity are codified in publicly available computer programs. Exemplary computer software to determine homology between two sequences include, but are not limited to, GCG program package, Devereux, J., et al. , Nucleic Acids Research, 12(1), 387 (1984)), BLASTP, BLASTN, and FASTA Altschul, S. F. etal., J. Molec. Biol., 215, 403 (1990)).
  • Inhibit expression of a gene means to cause a reduction in the amount of an expression product of the gene.
  • the expression product can be an RNA transcribed from the gene (e.g., an mRNA) or a polypeptide translated from an mRNA transcribed from the gene.
  • a reduction in the level of an mRNA results in a reduction in the level of a polypeptide translated therefrom.
  • the level of expression may be determined using standard techniques for measuring mRNA or protein.
  • in vitro refers to events that occur in an artificial environment, e.g., in a test tube or reaction vessel, in cell culture, in a Petri dish, etc., rather than within an organism (e.g., animal, plant, or microbe).
  • Isolated refers to a substance or entity that has been separated from at least some of the components with which it was associated (whether in nature or in an experimental setting). Isolated substances may have varying levels of purity in reference to the substances from which they have been associated. Isolated substances and/or entities may be separated from at least about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or more of the other components with which they were initially associated.
  • isolated agents are more than about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or more than about 99% pure.
  • a substance is “pure” if it is substantially free of other components.
  • substantially isolated is meant that the compound is substantially separated from the environment in which it was formed or detected. Partial separation can include, for example, a composition enriched in the compound of the present disclosure.
  • Substantial separation can include compositions containing at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 97%, or at least about 99% by weight of the compound of the present disclosure, or salt thereof. Methods for isolating compounds and their salts are routine in the art.
  • Metastasis means the process by which cancer invades and spreads from the place at which it first arose as a primary tumor to distant locations in the body. Metastasis also refers to cancers resulting from the spread of the primary tumor. For example, someone with breast cancer may show metastases in their lymph system, liver, bones or lungs.
  • Modified refers to a changed state or structure of a molecule of the disclosure. Molecules may be modified in many ways including chemically, structurally, and functionally. In one embodiment, the saRNAs of the present disclosure are modified by the introduction of non-natural nucleosides and/or nucleotides.
  • Naturally occurring As used herein, “naturally occurring” means existing in nature without artificial aid.
  • nucleic acid refers to a molecule comprised of one or more nucleotides, i.e., ribonucleotides, deoxyribonucleotides, or both.
  • the term includes monomers and polymers of ribonucleotides and deoxyribonucleotides, with the ribonucleotides and/or deoxyribonucleotides being bound together, in the case of the polymers, via 5' to 3' linkages.
  • the ribonucleotide and deoxyribonucleotide polymers may be single or double-stranded.
  • linkages may include any of the linkages known in the art including, for example, nucleic acids comprising 5' to 3' linkages.
  • the nucleotides may be naturally occurring or may be synthetically produced analogs that are capable of forming base-pair relationships with naturally occurring base pairs.
  • Examples of non-naturally occurring bases that are capable of forming base-pairing relationships include, but are not limited to, aza and deaza pyrimidine analogs, aza and deaza purine analogs, and other heterocyclic base analogs, wherein one or more of the carbon and nitrogen atoms of the pyrimidine rings have been substituted by heteroatoms, e.g., oxygen, sulfur, selenium, phosphorus, and the like.
  • Patient refers to a subject who may seek or be in need of treatment, requires treatment, is receiving treatment, will receive treatment, or a subject who is under care by a trained professional for a particular disease or condition.
  • Peptide As used herein, “peptide” is less than or equal to 50 amino acids long, e.g., about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids long.
  • compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.
  • compositions refers any ingredient other than the compounds described herein (for example, a vehicle capable of suspending or dissolving the active compound) and having the properties of being substantially nontoxic and non-inflammatory in a patient.
  • Excipients may include, for example: antiadherents, antioxidants, binders, coatings, compression aids, disintegrants, dyes (colors), emollients, emulsifiers, fillers (diluents), film formers or coatings, flavors, fragrances, glidants (flow enhancers), lubricants, preservatives, printing inks, sorbents, suspensing or dispersing agents, sweeteners, and waters of hydration.
  • antiadherents antioxidants, binders, coatings, compression aids, disintegrants, dyes (colors), emollients, emulsifiers, fillers (diluents), film formers or coatings, flavors, fragrances, glidants (flow enhancers), lubricants, preservatives, printing inks, sorbents, suspensing or dispersing agents, sweeteners, and waters of hydration.
  • excipients include, but are not limited to: butylated hydroxytoluene (BHT), calcium carbonate, calcium phosphate (dibasic), calcium stearate, croscarmellose, crosslinked polyvinyl pyrrolidone, citric acid, crospovidone, cysteine, ethylcellulose, gelatin, hydroxypropyl cellulose, hydroxypropyl methylcellulose, lactose, magnesium stearate, maltitol, mannitol, methionine, methylcellulose, methyl paraben, microcrystalline cellulose, polyethylene glycol, polyvinyl pyrrolidone, povidone, pregelatinized starch, propyl paraben, retinyl palmitate, shellac, silicon dioxide, sodium carboxymethyl cellulose, sodium citrate, sodium starch glycolate, sorbitol, starch (corn), stearic acid, sucrose, talc, titanium dioxide, vitamin A, vitamin E, vitamin C,
  • compositions described herein also includes pharmaceutically acceptable salts of the compounds described herein.
  • pharmaceutically acceptable salts refers to derivatives of the disclosed compounds wherein the parent compound is modified by converting an existing acid or base moiety to its salt form (e.g., by reacting the free base group with a suitable organic acid).
  • examples of pharmaceutically acceptable salts include, but are not limited to, mineral or organic acid salts of basic residues such as amines; alkali or organic salts of acidic residues such as carboxylic acids; and the like.
  • Representative acid addition salts include acetate, adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, fumarate, glucoheptonate, glycerophosphate, hemisulfate, heptonate, hexanoate, hydrobromide, hydrochloride, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pe
  • alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, and the like, as well as nontoxic ammonium, quaternary ammonium, and amine cations, including, but not limited to ammonium, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, triethylamine, ethylamine, and the like.
  • the pharmaceutically acceptable salts of the present disclosure include the conventional non-toxic salts of the parent compound formed, for example, from non-toxic inorganic or organic acids.
  • the pharmaceutically acceptable salts of the present disclosure can be synthesized from the parent compound which contains a basic or acidic moiety by conventional chemical methods.
  • such salts can be prepared by reacting the free acid or base forms of these compounds with a stoichiometric amount of the appropriate base or acid in water or in an organic solvent, or in a mixture of the two; generally, nonaqueous media like ether, ethyl acetate, ethanol, isopropanol, or acetonitrile are preferred.
  • nonaqueous media like ether, ethyl acetate, ethanol, isopropanol, or acetonitrile are preferred.
  • Lists of suitable salts are found in Remington ’s Pharmaceutical Sciences, 17 th ed., Mack Publishing Company, Easton, Pa., 1985, p. 1418, Pharmaceutical Salts: Properties, Selection, and Use, P.H. Stahl and C.G. Wermuth (eds.), Wiley-VCH, 2008, and Berge et al., Journal of Pharmaceutical Science, 66, 1- 19 (1977), each of which is incorporated herein by reference in its entirety.
  • solvate means a compound of the disclosure wherein molecules of a suitable solvent are incorporated in the crystal lattice.
  • a suitable solvent is physiologically tolerable at the dosage administered.
  • solvates may be prepared by crystallization, recrystallization, or precipitation from a solution that includes organic solvents, water, or a mixture thereof.
  • solvents examples include ethanol, water (for example, mono-, di-, and tri-hydrates), N- methylpyrrolidinone (NMP), dimethyl sulfoxide (DMSO), A, A ’-dimethylformamide (DMF), A, A ’-dimethylacetamide (DMAC), l,3-dimethyl-2-imidazolidinone (DMEU), 1,3-dimethyl- 3,4,5,6-tetrahydro-2-(lH)-pyrimidinone (DMPU), acetonitrile (ACN), propylene glycol, ethyl acetate, benzyl alcohol, 2-pyrrolidone, benzyl benzoate, and the like.
  • the solvent When water is the solvent, the solvate is referred to as a “hydrate.”
  • Pharmacologic effect is a measurable biologic phenomenon in an organism or system which occurs after the organism or system has been contacted with or exposed to an exogenous agent. Pharmacologic effects may result in therapeutically effective outcomes such as the treatment, improvement of one or more symptoms, diagnosis, prevention, and delay of onset of disease, disorder, condition or infection. Measurement of such biologic phenomena may be quantitative, qualitative or relative to another biologic phenomenon. Quantitative measurements may be statistically significant. Qualitative measurements may be by degree or kind and may be at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more different. They may be observable as present or absent, better or worse, greater or less.
  • Exogenous agents when referring to pharmacologic effects are those agents which are, in whole or in part, foreign to the organism or system. For example, modifications to a wild type biomolecule, whether structural or chemical, would produce an exogenous agent. Likewise, incorporation or combination of a wild type molecule into or with a compound, molecule or substance not found naturally in the organism or system would also produce an exogenous agent.
  • the saRNA of the present disclosure comprises exogenous agents.
  • Examples of pharmacologic effects include, but are not limited to, alteration in cell count such as an increase or decrease in neutrophils, reticulocytes, granulocytes, erythrocytes (red blood cells), megakaryocytes, platelets, monocytes, connective tissue macrophages, epidermal langerhans cells, osteoclasts, dendritic cells, microglial cells, neutrophils, eosinophils, basophils, mast cells, helper T cells, suppressor T cells, cytotoxic T cells, natural killer T cells, B cells, natural killer cells, or reticulocytes.
  • alteration in cell count such as an increase or decrease in neutrophils, reticulocytes, granulocytes, erythrocytes (red blood cells), megakaryocytes, platelets, monocytes, connective tissue macrophages, epidermal langerhans cells, osteoclasts, dendritic cells, microglial cells, neutrophils, eosinophils, bas
  • Pharmacologic effects also include alterations in blood chemistry, pH, hemoglobin, hematocrit, changes in levels of enzymes such as, but not limited to, liver enzymes AST and ALT, changes in lipid profiles, electrolytes, metabolic markers, hormones or other marker or profile known to those of skill in the art.
  • Physicochemical means of or relating to a physical and/or chemical property.
  • the term “preventing” refers to partially or completely delaying onset of an infection, disease, disorder and/or condition; partially or completely delaying onset of one or more symptoms, features, or clinical manifestations of a particular infection, disease, disorder, and/or condition; partially or completely delaying onset of one or more symptoms, features, or manifestations of a particular infection, disease, disorder, and/or condition; partially or completely delaying progression from an infection, a particular disease, disorder and/or condition; and/or decreasing the risk of developing pathology associated with the infection, the disease, disorder, and/or condition.
  • Prognosing means a statement or claim that a particular biologic event will, or is very likely to, occur in the future.
  • progression As used herein, the term “progression” or “cancer progression” means the advancement or worsening of or toward a disease or condition.
  • Proliferate As used herein, the term “proliferate” means to grow, expand or increase or cause to grow, expand or increase rapidly. “Proliferative” means having the ability to proliferate. “Anti-proliferative” means having properties counter to or inapposite to proliferative properties.
  • Protein means a polymer of amino acid residues linked together by peptide bonds.
  • a protein may be naturally occurring, recombinant, or synthetic, or any combination of these.
  • a protein may also comprise a fragment of a naturally occurring protein or peptide.
  • a protein may be a single molecule or may be a multi-molecular complex. The term protein may also apply to amino acid polymers in which one or more amino acid residues are an artificial chemical analogue of a corresponding naturally occurring amino acid.
  • Protein expression refers to the process by which a nucleic acid sequence undergoes translation such that detectable levels of the amino acid sequence or protein are expressed.
  • Purified As used herein, “purify,” “purified,” “purification” means to make substantially pure or clear from unwanted components, material defilement, admixture or imperfection.
  • regression As used herein, the term “regression” or “degree of regression” refers to the reversal, either phenotypically or genotypically, of a cancer progression. Slowing or stopping cancer progression may be considered regression.
  • sample refers to a subset of its tissues, cells or component parts (e.g. body fluids, including but not limited to blood, mucus, lymphatic fluid, synovial fluid, cerebrospinal fluid, saliva, amniotic fluid, amniotic cord blood, urine, vaginal fluid and semen).
  • body fluids including but not limited to blood, mucus, lymphatic fluid, synovial fluid, cerebrospinal fluid, saliva, amniotic fluid, amniotic cord blood, urine, vaginal fluid and semen).
  • a sample further may include a homogenate, lysate or extract prepared from a whole organism or a subset of its tissues, cells or component parts, or a fraction or portion thereof, including but not limited to, for example, plasma, serum, spinal fluid, lymph fluid, the external sections of the skin, respiratory, intestinal, and genitourinary tracts, tears, saliva, milk, blood cells, tumors, organs.
  • a sample further refers to a medium, such as a nutrient broth or gel, which may contain cellular components, such as proteins or nucleic acid molecule.
  • Single unit dose is a dose of any therapeutic administered in one dose/at one time/single route/single point of contact, i.e., single administration event.
  • Similarity refers to the overall relatedness between polymeric molecules, e.g. between polynucleotide molecules (e.g. DNA molecules and/or RNA molecules) and/or between polypeptide molecules. Calculation of percent similarity of polymeric molecules to one another can be performed in the same manner as a calculation of percent identity, except that calculation of percent similarity takes into account conservative substitutions as is understood in the art.
  • split dose is the division of single unit dose or total daily dose into two or more doses.
  • Stable refers to a compound that is sufficiently robust to survive isolation to a useful degree of purity from a reaction mixture, and in one embodiment, capable of formulation into an efficacious therapeutic agent.
  • Stabilized As used herein, the term “stabilize”, “stabilized,” “stabilized region” means to make or become stable.
  • Suffering from' An individual who is “suffering from” a disease, disorder, and/or condition has been diagnosed with or displays one or more symptoms of a disease, disorder, and/or condition.
  • Susceptible to An individual who is “susceptible to” a disease, disorder, and/or condition has not been diagnosed with and/or may not exhibit symptoms of the disease, disorder, and/or condition but harbors a propensity to develop a disease or its symptoms.
  • an individual who is susceptible to a disease, disorder, and/or condition may be characterized by one or more of the following: (1) a genetic mutation associated with development of the disease, disorder, and/or condition; (2) a genetic polymorphism associated with development of the disease, disorder, and/or condition; (3) increased and/or decreased expression and/or activity of a protein and/or nucleic acid associated with the disease, disorder, and/or condition; (4) habits and/or lifestyles associated with development of the disease, disorder, and/or condition; (5) a family history of the disease, disorder, and/or condition; and (6) exposure to and/or infection with a microbe associated with development of the disease, disorder, and/or condition.
  • an individual who is susceptible to a disease, disorder, and/or condition will develop the disease, disorder, and/or condition. In some embodiments, an individual who is susceptible to a disease, disorder, and/or condition will not develop the disease, disorder, and/or condition.
  • Synthetic means produced, prepared, and/or manufactured by the hand of man. Synthesis of polynucleotides or polypeptides or other molecules of the present disclosure may be chemical or enzymatic.
  • Targeted cells refers to any one or more cells of interest.
  • the cells may be found in vitro, in vivo, in situ or in the tissue or organ of an organism.
  • the organism may be an animal, in one embodiment, a mammal, or a human and in one embodiment, a patient.
  • therapeutic agent refers to any agent that, when administered to a subject, has a therapeutic, diagnostic, and/or prophylactic effect and/or elicits a desired biological and/or pharmacological effect.
  • Therapeutically effective amount means an amount of an agent to be delivered (e.g., nucleic acid, drug, therapeutic agent, diagnostic agent, prophylactic agent, etc) that is sufficient, when administered to a subject suffering from or susceptible to an infection, disease, disorder, and/or condition, to treat, improve symptoms of, diagnose, prevent, and/or delay the onset of the infection, disease, disorder, and/or condition.
  • agent to be delivered e.g., nucleic acid, drug, therapeutic agent, diagnostic agent, prophylactic agent, etc
  • the term “therapeutically effective outcome” means an outcome that is sufficient in a subject suffering from or susceptible to an infection, disease, disorder, and/or condition, to treat, improve symptoms of, diagnose, prevent, and/or delay the onset of the infection, disease, disorder, and/or condition.
  • Total daily dose As used herein, a “total daily dose” is an amount given or prescribed in 24 hour period. It may be administered as a single unit dose.
  • treating refers to partially or completely alleviating, ameliorating, improving, relieving, delaying onset of, inhibiting progression of, reducing severity of, and/or reducing incidence of one or more symptoms or features of a particular infection, disease, disorder, and/or condition.
  • “treating” cancer may refer to inhibiting survival, growth, and/or spread of a tumor.
  • Treatment may be administered to a subject who does not exhibit signs of a disease, disorder, and/or condition and/or to a subject who exhibits only early signs of a disease, disorder, and/or condition for the purpose of decreasing the risk of developing pathology associated with the disease, disorder, and/or condition.
  • a method of treating when applied to, for example, cancer refers to a procedure or course of action that is designed to reduce, eliminate or prevent the number of cancer cells in an individual, or to alleviate the symptoms of a cancer.
  • a method of treating does not necessarily mean that the cancer cells or other disorder will, in fact, be completely eliminated, that the number of cells or disorder will, in fact, be reduced, or that the symptoms of a cancer or other disorder will, in fact, be alleviated.
  • a method of treating cancer will be performed even with a low likelihood of success, but which, given the medical history and estimated survival expectancy of an individual, is nevertheless deemed an overall beneficial course of action.
  • Tumor growth As used herein, the term “tumor growth” or “tumor metastases growth”, unless otherwise indicated, is used as commonly used in oncology, where the term is principally associated with an increased mass or volume of the tumor or tumor metastases, primarily as a result of tumor cell growth.
  • Unmodified refers to any substance, compound or molecule prior to being changed in any way. Unmodified may, but does not always, refer to the wild type or native form of a biomolecule. Molecules may undergo a series of modifications whereby each modified molecule may serve as the “unmodified” starting molecule for a subsequent modification.
  • articles such as “a,” “an,” and “the” may mean one or more than one unless indicated to the contrary or otherwise evident from the context. Claims or descriptions that include “or” between one or more members of a group are considered satisfied if one, more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process unless indicated to the contrary or otherwise evident from the context.
  • the disclosure includes embodiments in which exactly one member of the group is present in, employed in, or otherwise relevant to a given product or process.
  • the disclosure includes embodiments in which more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process.
  • any particular embodiment of the present disclosure that falls within the prior art may be explicitly excluded from any one or more of the claims. Since such embodiments are deemed to be known to one of ordinary skill in the art, they may be excluded even if the exclusion is not set forth explicitly herein. Any particular embodiment of the compositions of the disclosure (e.g., any nucleic acid or protein encoded thereby; any method of production; any method of use; etc.) can be excluded from any one or more claims, for any reason, whether or not related to the existence of prior art.
  • Sense and antisense strands of saRNA were synthesized. They were first annealed in a Tris-EDTA based buffer following a denaturing step at 90°C, followed by a gradual anneal step to room temperature. Cells were seeded at 0.25 to 1x10 5 per well in a 24-well plate and transfected using Lipofectamine 2000 (Life Technologies). Transfection was performed immediately after seeding with the indicated oligonucleotide concentration using luL of Lipofectamine 2000. Cells were then transfected 24 hours later and harvested for analysis 48 hours and 72 hours after seeding.
  • QIAGEN Quantitech Reverse transcription kit
  • saRNAs of the present disclosure such as TMEM173-Pr-70-invAb-Se-ml or TMEM173-Pr-70-ml-emod51, were encapsulated in a liposome with methods disclosed in Example 17 of WO2016170349, the contents of which are incorporated herein by reference in their entirety.
  • TMEM173-Pr-70-invAb-Se-ml or TMEM173-Pr-70-ml-emod51 were encapsulated with the following steps: dissolving the saRNA in a first buffer to form a saRNA solution, filtering the saRNA solution through a 0.2 pm filter, mixing the filtered saRNA solution with a lipid solution in an injection module to form a liposome formulation, and adding a second buffer to the liposome formulation,
  • the first buffer may be a sodium acetate/sucrose buffer and the pH value for the saRNA solution may be about 4.0.
  • the concentration of the saRNA in the saRNA solution is about 2.38 mg/mL.
  • the lipid solution may comprise l-palmitoyl-2-oleoyl-sn-glycero-3 -phosphocholine (POPC), l,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), cholesteryl-hemisuccinate (CHEMS), and 4-(2-aminoethyl)-morpholino-cholesterol hemisuccinate (MOCHOL).
  • POPC l-palmitoyl-2-oleoyl-sn-glycero-3 -phosphocholine
  • DOPE l,2-dioleoyl-sn-glycero-3-phosphoethanolamine
  • CHEMS cholesteryl-hemisuccinate
  • MOCHOL 4-(2-aminoethyl)-morpholino-cholesterol hemisuccinate
  • the second buffer may have a pH of about 9 and may be a sodium chloride/sodium phosphate buffer.
  • Example 1 In Vivo Efficacy Studies of TMEM173-saRNA Compositions using humanized mouse models
  • compositions comprising TMEM173-saRNAs and NOV340 Smarticles are tested in humanized mouse models that are most representative of the human immune system (for example BRGSF-HIS mice). Stem cells from 2 - 5 human donors are engrafted. Once the hemopoietic system is established, animals are subcutaneously implanted with patient-derived xenograft. Tumours are allowed to reach a volume of 100 mm 3 and intra-tumour dosing of the animals commence.
  • Treatment groups include vehicle control, TMEM173-saRNA composition monotherapy group, TMEM173-saRNA composition in combination with other cancer treatment modalities such as but not limited to checkpoint inhibitors, PARP inhibitors, chemotherapy and radiotherapy.
  • Intra-tumour dosing of TMEM173-saRNA composition is carried out at 2.4 mg/kg or 60 pg per tumour twice a week until experimental endpoint.
  • the schedules are based on data determined by previous studies.
  • TMEM173-saRNAs within the human genome is introduced to the murine genomic locus of the Tmeml73 gene to generate a mouse with 100 % homology to the targeting region of the human TMEM173.
  • the mouse model is assessed for upregulation of the murine Tmeml73 gene after treatment with TMEM-saRNA composition.
  • the engineered mice are used for study of the response of syngeneic tumour models to TMEM173-saRNA composition as a monotherapy and TMEM- saRNA composition in combination with other cancer treatment modalities such as but not limited to checkpoint inhibitors, PARP inhibitors, chemotherapy and radiotherapy.
  • tumour is tested as either a single or dual flank subcutaneous implant.
  • a murine surrogate molecule (muSTING) of TMEM173-Pr-70-invAb-Se-ml was designed. muSTING upregulates murine TMEM173 mRNA by targeting a similar location within the TMEM173 gene. Similar to TMEM173-Pr-70-invAb-Se-ml, the surrogate molecule targets the first intron of the murine STING gene.
  • TMEM173-Pr-70-invAb-Se-ml and muSTING were formulated in NOV340 Smarticles (referred to as MTL-STING and MTL-muSTING, respectively) according to the method disclosed herein.
  • the pharmacology of MTL-STING and MTL-muSTING was assessed by intrathecal route (LT.) dosing of subcutaneous MC38 tumour model (murine colorectal cancer) in female C57B1/6 mice. Some mice further received PD-1 antibody (10 mg/kg via intraperitoneal injection (I P.)).
  • the treatment agents are listed in Table 6 below.
  • Tumours were measured three times weekly by digital calipers and volume of tumours calculated using elliptical formula (TT/6 X width x width x length). Animals were weighed at the same time as the tumour measurement. All animals were observed for Jackpot hour post dosing and any adverse effects noted. Tumour growth inhibition (%TGI) from the start of treatment was assessed by comparison of the geometric mean change in tumor volume for the control and treated groups. Statistical significance was evaluated using a one-tailed t test. The results are shown in Table 8 below.

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Abstract

La divulgation concerne des saRNA utiles pour réguler à la hausse l'expression d'un gène cible et des compositions thérapeutiques comprenant ces saRNA, le gène cible étant TMEM173. La divulgation concerne également des procédés d'utilisation de ces saRNA et des compositions thérapeutiques comprenant lesdits saRNA.
PCT/GB2024/052261 2023-09-08 2024-08-30 Compositions de petits arn activateurs (sarna) ciblant tmem173 et procédés d'utilisation Pending WO2025052098A1 (fr)

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