EP4587563A1 - Compositions and methods for treating cancer - Google Patents

Compositions and methods for treating cancer

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Publication number
EP4587563A1
EP4587563A1 EP23864205.2A EP23864205A EP4587563A1 EP 4587563 A1 EP4587563 A1 EP 4587563A1 EP 23864205 A EP23864205 A EP 23864205A EP 4587563 A1 EP4587563 A1 EP 4587563A1
Authority
EP
European Patent Office
Prior art keywords
rabla
gdp
cancer
cell
seq
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
EP23864205.2A
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German (de)
French (fr)
Inventor
Kuo-Tang Tseng
Mohsen Amir Alipour
Zemin Yao
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Reliable Holdings Co Ltd
Motigenix Singapore Pte Ltd
Original Assignee
Reliable Holdings Co Ltd
Motigenix Singapore Pte Ltd
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Application filed by Reliable Holdings Co Ltd, Motigenix Singapore Pte Ltd filed Critical Reliable Holdings Co Ltd
Publication of EP4587563A1 publication Critical patent/EP4587563A1/en
Pending legal-status Critical Current

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/46—Hydrolases (3)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/51—Nanocapsules; Nanoparticles
    • A61K9/5107—Excipients; Inactive ingredients
    • A61K9/5123—Organic compounds, e.g. fats, sugars
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/04—Antineoplastic agents specific for metastasis
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/82—Translation products from oncogenes
    • 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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y306/00—Hydrolases acting on acid anhydrides (3.6)
    • C12Y306/05—Hydrolases acting on acid anhydrides (3.6) acting on GTP; involved in cellular and subcellular movement (3.6.5)
    • C12Y306/05002—Small monomeric GTPase (3.6.5.2)

Definitions

  • proteins, lipids, and/or glycogens may be associated with a variety of important diseases, disorders, and conditions.
  • Methods for targeting such proteins, lipids, and/or glycogens are desirable.
  • a microautophagyenhancing agent comprising a GDP-bound form of Rabi a, such as Rabla S25N , Rabla N1241 , Rabla D41N , Rabla D47N , or another dominant negative (DN) GDP -bound form of Rabi a, or one or more expressible nucleic acids encoding such a Rabla GDP , may be used to decrease cell viability and/or cause cell death in a wide variety of different cancer cell types.
  • a microautophagyenhancing agent comprising a GDP-bound form of Rabi a, such as Rabla S25N , Rabla N1241 , Rabla D41N , Rabla D47N , or another dominant negative (DN) GDP -bound form of Rabi a, or one or more expressible nucleic acids encoding such a Rabla GDP , may be used to decrease cell viability and/or cause cell death in a wide variety of different cancer cell types.
  • DN dominant negative
  • a method for reducing cell viability of a cancer cell, or for preventing or treating cancer, in a subject in need thereof comprising: treating a cancer cell of the subject with a GDP -bound form of Rabi a (Rabla GDP ), one or more expressible nucleic acids encoding Rabla GDP , or a combination thereof; thereby increasing a cellular level of Rabla GDP in the cancer cell, resulting in decreased cell viability or death of the cancer cell.
  • SEQ ID NO: 18 Human Rabla D47N ; or a polypeptide having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with any of these sequences and preferentially binding GDP.
  • SEQ ID NO: 18 Human Rabla D47N ; or a polypeptide having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with any of these sequences and preferentially binding GDP.
  • the Rabla GDP may comprise or consist of the amino acid sequence:
  • the Rabla GDP may be in the form of a fusion protein, wherein the Rabla GDP is fused or otherwise directly or indirectly linked, optionally via a linker, with a signaling or targeting peptide, a fluorescent peptide or other marker or tracer, or another peptide or non-peptide moiety for targeted delivery, facilitating cell uptake, increasing stability or in vivo half-life, or improving another therapeutic, diagnostic, or in vivo property of the Rabla GDP .
  • Luciferase Tag (SEQ ID NO: 20, MG-008 ORF mRNA Sequence with 5’ Luciferase Tag); or a nucleic acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity therewith and encoding a Rabla GDP preferentially binding GDP; or a nucleic acid sequence equivalent to any of the above sequences due to codon redundancy.
  • a use of a GDP -bound form of Rabi a (Rabla GDP ), one or more expressible nucleic acids encoding Rabla GDP , or a combination thereof, for reducing cell viability of a cancer cell, or for preventing or treating cancer, in a subject in need thereof.
  • RablaGDP GDP -bound form of Rabi a
  • one or more expressible nucleic acids encoding RablaGDP or a combination thereof, for reducing cell viability of certain cancer cells in vitro or in vivo.
  • a use of a GDP -bound form of Rabi a (Rabla GDP ), one or more expressible nucleic acids encoding Rabla GDP , or a combination thereof, in the manufacture of a medicament for reducing cell viability of certain cancer cells, or for preventing or treating cancer, in a subject in need thereof.
  • Rabla GDP a GDP -bound form of Rabi a
  • one or more expressible nucleic acids encoding Rabla GDP or a combination thereof, in the manufacture of a medicament for reducing cell viability of certain cancer cells in vitro or in vivo.
  • the Rabla GDP may be or may comprise Rabla S25N , Rabla N1241 , Rabla D41N , Rabla D47N , or another dominant negative (DN) GDP-bound form of Rabi a.
  • SEQ ID NO: 18 Human Rabla D47N ; or a polypeptide having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with any of these sequences and preferentially binding GDP.
  • the Rabla GDP may comprise or consist of the amino acid sequence: MSSMNPEYDYLFKLLLIGDSGVGKNCLLLRFADDTYTESYISTIGVDFKIRTIELDGK TIKLQIWDTAGQERFRTITSSYYRGAHGIIVVYDVTDQESFNNVKQWLQEIDRYASE NVNKLLVGNKCDLTTKKVVDYTTAKEFADSLGIPFLETSAKNATNVEQSFMTMAAE IKKRMGPGATAGGAEKSNVKIQSTPVKQSGGGCC (SEQ ID NO: 6; Human Rabla S25N ); or a polypeptide having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity therewith and preferentially binding GDP.
  • the Rabla GDP may be in the form of a fusion protein, wherein the Rabla GDP is fused or otherwise directly or indirectly linked, optionally via a linker, with a signaling or targeting peptide, a fluorescent peptide or other marker or tracer, or another peptide or non-peptide moiety for targeted delivery, facilitating cell uptake, increasing stability or in vivo half-life, or improving another therapeutic, diagnostic, or in vivo property of the Rabla GDP .
  • the fusion protein may comprise the amino acid sequence:
  • the Rabla GDP may be in the form of a fusion protein, and may comprise the amino acid sequence: MEDAKNIKKGPAPFYPLEDGTAGEQLHKAMKRYALVPGTIAFTDAHIEVNITYAEYF EMSVRLAEAMKRYGLNTNHRIVVCSENSLQFFMPVLGALFIGVAVAPANDIYNERE LLNSMNISQPTVVFVSKKGLQKILNVQKKLPIIQKIIIMDSKTDYQGFQSMYTFVTSHL PPGFNEYDF VPESFDRDKTIALIMNS SGSTGLPKGVALPHRT ACVRF SHARDPIFGNQI IPDTAILSVVPFHHGFGMFTTLGYLICGFRVVLMYRFEEELFLRSLQDYKIQSALLVPT LFSFFAKSTLIDKYDLSNLHEIASGGAPLSKEVGEAVAKRFHLPGIRQGYGLTETTSAI LITPEGDDKPGAVGKVVPFFEAKVVDLD
  • the one or more expressible nucleic acids may encode one or more Rabla GDP s as defined herein.
  • macroautophagy may work with mitotic catastrophe to get rid of cancer cells that escape apoptosis (Simon HU, Friis R. ATG5: a distinct role in the nucleus. Autophagy. 2014 Jan; 10(1): 176-7). As a tumor suppression mechanism, macroautophagy also maintains genome stability, induces senescence and possibly autophagic cell death (Gozuacik D, Kimchi A. Autophagy as a cell death and tumor suppressor mechanism. Oncogene. 2004 Apr 12;23 (16) :2891 - 906).
  • ROS Reactive oxygen species
  • ROS reactive oxygen species
  • ROS Reactive oxygen species
  • OH hydroxyl radicals
  • alkoxyl radicals such as hydroxyl radicals (OH), alkoxyl radicals, superoxide anion (02 -), singlet oxygen (102) and hydrogen peroxide (H2O2)
  • H2O2 hydrogen peroxide
  • Tumors often arise from sites of chronic irritation, infection, or inflammation; virtually all cancer cells are believed to stem from cells that are functionally defective accompanied with metabolic reprogramming, as well as elevated baseline ROS production.
  • alterations in cellar macroautophagic process(es) have been observed in several cancers.
  • Macroautophagy regulatory machinery also declines with age and environmental/nutritional stress, leading to more dysfunctional or cancerous cells (Martinez -Lopez N, Athonvarangkul D, Singh R. Autophagy and aging. Adv Exp Med Biol. 2015;847:73-87). Cancer cells often have much lowered activity of basal macroautophagy than normal cells.
  • Stimulation of microautophagy (which in certain embodiments may involve piecemeal engulfment of target membrane(s)) by promoting lysosome movement toward the target membranes) may be particularly desirable for treatment of a variety of diseases and/or disorders and/or conditions.
  • Specific lysosome positioning inside the cells may be associated with different types of lysosome activity.
  • lysosome positioning may correlate with the activity of mTOR and may regulate autophagic flux.
  • mTORCl may be inactive and lysosomes may be accumulated in the perinuclear region of the cells, which may perform macroautophagy by stimulation of fusion of encased target membrane with lysosomes.
  • lysosomes may move in a bidirectional manner along the microtubules, and such a lysosomal motility is governed by different sets of motor proteins that are recruited through different mechanisms.
  • microautophagy may involve direct engulfment of cytoplasmic cargo at a boundary membrane by autophagic tubes, which may mediate both invagination and vesicle scission into the lumen of lysosomes (see Li, W.-W., Li, J. & Bao, J.-K. Microautophagy: lesser- known self-eating. Cell. Mol. Life Sci. 69, 1125-1136 (2011)).
  • Direct lysosomal degradation of target substrates may occur for DNA, for example (referred to as a piecemeal autophagy - see Fujiwara, Y. et al. Direct uptake and degradation of DNA by lysosomes. - PubMed - NCBI.
  • Lysosomal fusion and SNARE function are impaired by cholesterol accumulation in lysosomal storage disorders.
  • the EMBO Journal 29, 3607-3620 (2010); and Pankiv, S. et al. FYCO1 is a Rab7 effector that binds to LC3 and PI3P to mediate microtubule plus end-directed vesicle transport. J. Cell Biol. 188, 253-269 (2010)). Movement of lysosomes toward the cell periphery and their positioning within the cells may be associated with signaling (see R. & Bonifacino, J. S. Lysosome Positioning Influences mT0RC2 and AKT Signaling. Molecular Cell 75, 26-38. e3 (2019)).
  • mTORCl, mTORC2 and AKT activation may be important for lysosomal peripheral distribution (see Poiis, C. & Codogno, P. Lysosome positioning coordinates mTORCl activity and autophagy. Nature Cell Biology 13, 342-344 (2011); and Cabukusta, B. & Neefjes, J. Mechanisms of lysosomal positioning and movement. Traffic 19, 761-769 (2016)).
  • microautophagy and/or piecemeal degradation of target substrates and/or membranes may involve movement of lysosomes toward the peripheral and target membrane in cytosol (e.g. direct interaction with target membrane) (see Pu, J., Guardia, C. M., Keren-Kaplan, T. & Bonifacino, J. S. Mechanisms and functions of lysosome positioning. J. Cell. Sci. 129, 4329- 4339 (2016); and Katherine R Parzych, D. J. K. An Overview of Autophagy: Morphology, Mechanism, and Regulation. Antioxid. Redox Signal. 20, 460-473 (2014)).
  • movement of lysosomes toward the cell periphery (from perinuclear region of the cell) and interaction with target membranes/ substrates (e.g. glycogen, lipid, protein) in cell periphery may be associated with the activation of mTORCl/mTORC2 (see Rabanal-Ruiz, Y. & Korol chuk, V. I. mTORCl and Nutrient Homeostasis: The Central Role of the Lysosome. Int J Mol Sci 19, 818 (2016); Jia, R. & Bonifacino, J. S. Lysosome Positioning Influences mTORC2 and AKT Signaling. Molecular Cell 75, 26-38. e3 (2019)).
  • target membranes/ substrates e.g. glycogen, lipid, protein
  • Rabi a DN (dominant negative form of Rabi a) may simulate lysosome peripheral distribution (from perinuclear region) through activating mTORCl/mTORC2 proteins inside the cells without the need of external (or extracellular) signal for their activation (to support lysosome peripheral distribution).
  • methods as described herein may be in vitro methods, in vivo methods, or both.
  • a method for reducing cell viability of a cancer cell, or for preventing or treating cancer, in a subject in need thereof comprising: treating a cancer cell of the subject with a GDP -bound form of Rabi a (Rabla GDP ), one or more expressible nucleic acids encoding Rabla GDP , or a combination thereof; thereby increasing a cellular level of Rabla GDP in the cancer cell, resulting in decreased cell viability or death of the cancer cell.
  • a method for reducing cell viability of a cancer cell in vitro or in vivo comprising: treating the cancer cell with a GDP -bound form of Rabi a (Rabla GDP ), one or more expressible nucleic acids encoding Rabla GDP , or a combination thereof; thereby increasing a cellular level of Rabla GDP in the cancer cell, resulting in decreased cell viability or death of the cancer cell.
  • the Rabla GDP may comprise the amino acid sequence: MSSMNPEYDYLFKLLLIGDSGVGKNCLLLRFADDTYTESYISTIGVDFKIRTIELDGK
  • SEQ ID NO: 18 Human Rabla D47N ; or a polypeptide having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with any of these sequences and preferentially binding GDP.
  • the Rabla GDP may consist of the amino acid sequence:
  • the Rabla GDP may be in the form of a fusion protein, wherein the Rabla GDP is fused or otherwise directly or indirectly linked, optionally via a linker, with a signaling or targeting peptide, a fluorescent peptide or other marker or tracer, or another peptide or non-peptide moiety for targeted delivery, facilitating cell uptake, increasing stability or in vivo half-life, or improving another therapeutic, diagnostic, or in vivo property of the Rabla GDP .
  • the fusion protein can comprise any protein or tag, for example, GFP, YFP, mCherry, luciferase specific antibody, aptamer for identification or delivery to a specific organ or the like.
  • the fusion protein may comprise the amino acid sequence:
  • the Rabla GDP may be in the form of a fusion protein, and may comprise the amino acid sequence:
  • the one or more expressible nucleic acids may encode any one or more of the Rabla GDP s as defined herein.
  • the one or more expressible nucleic acids may be DNA-based, or RNA-based.
  • the one or more expressible nucleic acids may transiently express the Rabla GDP in the cell, or wherein the one or more expressible nucleic acids may integrate in the cell genome and express the Rabla GDP in the cell.
  • the one or more expressible nucleic acids may comprise one or more expression vectors, plasmids, or mRNAs encoding and capable of expressing the Rabla GDP inside the cell.
  • the one or more expressible nucleic acids may comprise a nucleic acid sequence of:
  • the one or more expressible nucleic acids may comprise a nucleic acid sequence of:
  • Luciferase Tag (SEQ ID NO: 20, MG-008 ORF mRNA Sequence with 5’ Luciferase Tag); or a nucleic acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity therewith and encoding a Rabla GDP preferentially binding GDP; or a nucleic acid sequence equivalent to any of the above sequences due to codon redundancy.
  • a use of a GDP -bound form of Rabi a (Rabla GDP ), one or more expressible nucleic acids encoding Rabla GDP , or a combination thereof, for reducing cell viability of a cancer cell, or for preventing or treating cancer, in a subject in need thereof.
  • RablaGDP GDP -bound form of Rabi a
  • one or more expressible nucleic acids encoding RablaGDP or a combination thereof, for reducing cell viability of a cancer cell in vitro or in vivo.
  • a use of a GDP -bound form of Rabi a (Rabla GDP ), one or more expressible nucleic acids encoding Rabla GDP , or a combination thereof, in the manufacture of a medicament for reducing cell viability of a cancer cell, or for preventing or treating cancer, in a subject in need thereof.
  • the Rabla GDP may consist of the amino acid sequence:
  • the Rabla GDP may be in the form of a fusion protein, wherein the Rabla GDP is fused or otherwise directly or indirectly linked, optionally via a linker, with a signaling or targeting peptide, a fluorescent peptide or other marker or tracer, or another peptide or non-peptide moiety for targeted delivery, facilitating cell uptake, increasing stability or in vivo half-life, or improving another therapeutic, diagnostic, or in vivo property of the Rabla GDP .
  • the Rabla GDP may be in the form of a fusion protein, and may comprise the amino acid sequence: MEDAKNIKKGPAPFYPLEDGTAGEQLHKAMKRYALVPGTIAFTDAHIEVNITYAEYF EMSVRLAEAMKRYGLNTNHRIVVCSENSLQFFMPVLGALFIGVAVAPANDIYNERE LLNSMNISQPTVVFVSKKGLQKILNVQKKLPIIQKIIIMDSKTDYQGFQSMYTFVTSHL PPGFNEYDF VPESFDRDKTIALIMNS SGSTGLPKGVALPHRT ACVRF SHARDPIFGNQI IPDTAILSVVPFHHGFGMFTTLGYLICGFRVVLMYRFEEELFLRSLQDYKIQSALLVPT LFSFFAKSTLIDKYDLSNLHEIASGGAPLSKEVGEAVAKRFHLPGIRQGYGLTETTSAI LITPEGDDKPGAVGKVVPFFEAKVVDLD
  • the one or more expressible nucleic acids may comprise a nucleic acid sequence of:
  • Luciferase Tag (SEQ ID NO: 20, MG-008 ORF mRNA Sequence with 5’ Luciferase Tag); or a nucleic acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity therewith and encoding a Rabla GDP preferentially binding GDP; or a nucleic acid sequence equivalent to any of the above sequences due to codon redundancy.
  • polypeptide comprising the amino acid sequence:
  • the cancer or cancer cell may be any of a wide variety of cancer types.
  • results from testing indicate that anti-cancer effects may be observed in a wide variety of different cancer cell lines, supporting broad anti-cancer applicability.
  • Anticancer effects were also observed in vivo, as described in Example 2 below, further supporting the applicability for the treatment of cancer in subjects.
  • the cancer may be breast adenocarcinoma, cholangiocarcinoma, colon adenocarcinoma, colorectal carcinoma, prostate carcinoma, embryonal rhabdomyosarcoma, gastric carcinoma, glioblastoma, hepatocellular carcinoma, invasive ductal carcinoma, lung cancer adenocarcinoma, pancreatic ductal adenocarcinoma, papillomavirus-related endocervical adenocarcinoma, squamous cell carcinoma of the oral cavity, or squamous cell carcinoma of the oral tongue.
  • the cancer cell lines may be selected from the group consisting of A549 (lung adenocarcinoma), HCT116 (colorectal carcinoma), HT29 (colon adenocarcinoma), HuCCTl (cholangiocarcinoma), PC-3 (prostate carcinoma), RD (rhabdomyosarcoma), SC-M1 (gastric carcinoma), U-87MG (glioblastoma), SW480 (colon adenocarcinoma), PANC-1 (pancreatic adenocarcinoma), OECM- 1 (oral carcinoma), OC2 (oral carcinoma), MIA PaCa-2 (pancreatic adenocarcinoma), MDA-MB- 468 (breast adenocarcinoma), MDA-MB-231 (breast adenocarcinoma), MCF-7 (ductal carcinoma), Mahlavu (hepatocellular carcinoma), HSC-3 (S)
  • references to increasing lysosome-mediated microautophagy may refer to an increase in the rate, extent, capacity, or efficacy of the lysosome-mediated microautophagy process in a cell as compared to baseline levels of the cell, or as compared with a corresponding treated or untreated control cell, or as compared to levels in a reference diseased cell or reference cell having accumulation of a protein, lipid, or glycogen substrate.
  • An increase in lysosomal association-dissociation events may refer to an increase in the rate, extent, or efficacy of lysosomal association-dissociation events in a cell as compared to baseline levels of a corresponding treated or untreated control cell, for example an identical cell treated under identical conditions but without a microautophagy-modulating agent or with a compound or composition that is known not to affect the process.
  • a microautophagy-enhancing agent may be any suitable agent which increases or facilitates the rate, activity, extent, or efficacy of lysosome-mediated microautophagy in a cell, or that increases lysosomal motility or bidirectional motility.
  • a suitable microautophagyenhancing agent may be or comprise a GDP -bound form of Rabi a (Rabla GDP ), one or more expressible nucleic acids encoding Rabla GDP , or a combination thereof.
  • lysosome-mediated microautophagy of a target protein, lipid, or glycogen substrate in a cell may be increased by treatment with a microautophagy-enhancing agent comprising a GDP -bound form of Rabla, such as Rabla S25N , Rab 1 a N1241 , Rab 1 a D41N , Rab 1 a D47N , or another dominant negative (DN) GDP -bound form of Rab 1 a.
  • a microautophagy-enhancing agent comprising a GDP -bound form of Rabla, such as Rabla S25N , Rab 1 a N1241 , Rab 1 a D41N , Rab 1 a D47N , or another dominant negative (DN) GDP -bound form of Rab 1 a.
  • the specific amino acid or nucleic acid sequence of a particular gene may vary from species to species.
  • the human Rabla amino acid sequence may have homologs in other species having sequence variation from the human sequence.
  • the general effect for example, the phenotypic effect
  • the general effect of a homolog sequence may be substantially similar to the effect of the wild-type sequence in a given cell or subject.
  • Suitable functional equivalents of Rabla S25N , Rabla D41N , Rabla D47N , and Rabla N1241 may include, for example, suitable Rab la variants or mutants having at least 80% (or >85%, or >90%, or >95%, or >99%) sequence identity to Rabla WT or Rabla S25N or Rabla N1241 or Rabla D41N or Rabla D47N and which preferentially bind GDP over GTP while also retaining the relevant cellular/biochemical functions of Rabla S25N or Rabla N1241 or Rabla D41N or Rabla D47N as described in detail herein.
  • a microautophagy-enhancing agent may be, or comprise, one or more expressible nucleic acids encoding Rabla GDP , such as any suitable nucleic acid/expression vector (i.e. vector, cassette, mRNA, modified mRNA, plasmid, for example) which encodes for/expresses a GDP -bound form of Rabla (Rabla GDP ) such as, for example, Rabla S25N or Rabla N1241 or Rabla D41N or Rabla D47N , or a functional equivalent thereof.
  • any suitable nucleic acid/expression vector i.e. vector, cassette, mRNA, modified mRNA, plasmid, for example
  • Rabla GDP a GDP -bound form of Rabla
  • sequences are mainly described herein with reference to human and/or mouse homologs. It will be understood that functional equivalents and/or variants may be found in a variety of different species, such as among different mammals. References herein to particular sequence modifications and/or mutants providing for Rabla GDP (DN) or Rabla GTP (DA) forms often provide position and modification/mutation information (e.g.
  • Rabl Q67L is a modification/mutation of a mouse sequence, and there is no Q at the 67 th position of human Rabla; rather, there is a Q in the human sequence at the 63 rd position, and so the modification/mutation with reference to the human sequence is Rabla Q63L .
  • expression of a particular protein within a cell may refer to the production of a polypeptide from a nucleic acid sequence encoding the polypeptide.
  • Gene expression may include both transcription and translation processes, and so gene expression may refer to production of a nucleic acid sequence such as an mRNA (i.e. transcription), production of a protein (i.e. translation), or both.
  • overexpression of a particular gene in a cell may refer to increasing the expression of a particular gene within a cell as compared to wildtype, baseline, or untreated levels. Overexpression, or introduction of a mutant gene, into cells may be accomplished using any of several methods known in the art.
  • a vector (either viral, plasmid, or other) comprising one or more copies of the particular gene each driven by a suitable promoter sequence (for example, a constitutive or inducible promoter), or an mRNA or chemically modified version thereof may be introduced into cells via transfection, electroporation, or viral infection, or another suitable method know in the art.
  • suitable expression vector techniques for overexpressing or introducing a particular gene into a cell are known in the art (see, for example, Molecular Cloning: A Laboratory Manual (4th Ed.), 2012, Cold Spring Harbor Laboratory Press).
  • nucleic acids for expressing a particular gene may encode or include features as described in "Genes VII", Lewin, B. Oxford University Press (2000) or “Molecular Cloning: A Laboratory Manual”, Sambrook et al., Cold Spring Harbor Laboratory, 3rd edition (2001).
  • a nucleotide sequence encoding a polypeptide or protein may be incorporated into a suitable vector, such as a commercially available vector.
  • Vectors may also be individually constructed or modified using standard molecular biology techniques, as outlined, for example, in Sambrook et al. (Cold Spring Harbor Laboratory, 3rd edition (2001)).
  • Biomolecules, compounds, and/or compositions as described herein may include one or more pharmaceutically acceptable excipients, diluents, and/or carriers.
  • a pharmaceutically acceptable carrier, diluent, or excipient may include any suitable carrier, diluent, or excipient known to the person of skill in the art.
  • Examples of pharmaceutically acceptable excipients may include, but are not limited to, cellulose derivatives, sucrose, and starch.
  • pharmaceutically acceptable excipients may include suitable fillers, binders, lubricants, buffers, glidants, and disentegrants known in the art (see, for example, Remington: The Science and Practice of Pharmacy (2006)).
  • Examples of pharmaceutically acceptable carriers, diluents, and excipients may be found in, for example, Remington's Pharmaceutical Sciences (2000 — 20th edition) and in the United States Pharmacopeia: TheNational Formulary (USP 24 NF19) published in 1999.
  • a conservative amino acid substitution may include one in which an amino acid is substituted for another amino acid having similar properties such that the folding, activity, or other functionality of the protein is not significantly affected.
  • aromatic amino acids which may be substitutable, may include phenylalanine, tryptophan, and tyrosine.
  • interchangeable hydrophobic amino acids which may be substitutable, may include leucine, isoleucine, methionine, and valine.
  • interchangeable polar amino acids which may be substitutable, may include glutamine and asparagine.
  • interchangeable basic amino acids which may be substitutable, may include arginine, lysine, and histidine.
  • dominant-negative (DN) Rabi a e.g. GDP -bound Rabi a, Rabla GDP
  • DN dominant-negative
  • Rabla GDP any suitable Rabla DN or Rabla GDP may be used.
  • any suitable dominant negative form of Rabla i.e. a constant/locked Rabla in its GDP form
  • Rabla constant GDP -bound form is generally not available in normal physiological conditions, in which the native protein Rabla is constantly shifting between its GTP and GDP forms. It is contemplated that in certain embodiments, genetic mutation and/or amino acid substitution/modification may be used to lock this GTPase in its GDP -bound form (or constant GTP form), and prevent it from going to its GTP state (or GDP state).
  • any suitable modification(s) that can substantially keep the integrity of the GDP form/state of GTPases may promote microautophagy and degradation of target substrate(s) through lysosomal piecemeal engulfment.
  • Rabla GDP may be administered to a parti culalr cell type or subject in need thereof in generally any suitable manner which may be selected to suit the particular cell type, subject, and/or indication.
  • a nucleic acid sequences encoding and capable of expressing the Rabla GDP may be administered to the subject or introduced to the cell type via any suitable transfection or nucleic acid delivery approach as will be known to the person of skill in the art having regard to the teachings herein.
  • delivery may be based on DNA or RNA transfection (for example, using common transfection regent(s) such as lipofectamine (Invitrogen), FuGENE (Rosche), using DNA adenovirus (gene therapy), or using Modified RNA (i.e.
  • protein may be administered or delivered to cells in need thereof, optionally assisted with any suitable technique or delivery vehicle for facilitating protein delivery to a cell or cells.
  • LNPs are an advanced non-viral gene delivery system. LNPs allow someone skilled in the art to safely and effectively deliver nucleic acids to cells in vitro or in vivo, which has applications in gene editing, rapid vaccine development, immuno-oncology and treatment of rare genetic and undruggable diseases, without being limiting.
  • LNPs are being used more frequently in the art due to their advantageous properties, including their controlled and sustained-release properties, low toxicity, biocompatibility with tissues and cells, low immune response, increased deliverable gene size, cell free manufacturing, high nucleic acid encapsulation efficiency, potent transfection, and improved penetration into tissues to deliver therapeutics.
  • Rabla DN (GDP form) Induces Cancer Cell Death and Stimulates Microautophagy
  • Various types of cancer cells were treated with Rabla DN (in this example, Rabla S25N , and the Rabla DN was introduced into cells as either a transfected mRNA nucleic acid, or as a transfected DNA expression plasmid, that is capable of expressing Rabla S25N or a fusion protein comprising Rabla S25N and an N-terminal Luciferase tag in the cancer cells.
  • the Rabla DN is also referred to as MG-008.
  • Amino acid sequence of the fusion protein comprising Rabla S25N and an N-terminal Luciferase tag, which is expressed in cells following transfection with either the DNA expression plasmid or the mRNA described above, is shown in Figure 36 as SEQ ID NO: 21.
  • mRNA expressing Rabla DN also referred to as MG-008, comprising the ORF shown in Figure 36 as SEQ ID NO: 20; in this example, mRNA expressing a fusion protein comprising Rab 1 aS25N and an N-terminal Luciferase tag was used
  • mRNA expressing a fusion protein comprising RablaS25N and an N-terminal Luciferase tag was used.
  • the protocol was performed according to the following transfection protocol:
  • Figures 1-35 Data for each cell type is shown in Figures 1-35, each of which provide representative phase contrast microscopy images of cells treated with control, transfection vehicle, MG-008, or MG-008-Luc treated cells, along with a bar graph showing measured cell viability % (by crystal violet stain) of each.
  • Rabla S25N Various types of cancer cells were treated with Rabla DN (in this example, Rabla S25N , and the Rabla DN was introduced into cells as either a transfected mRNA nucleic acid, or as a transfected DNA expression plasmid, that is capable of expressing Rabla S25N or a fusion protein comprising Rabla S25N and an N-terminal Luciferase tag in the cancer cells.
  • Results show a selectivity for decreasing cell viability of cancer cells versus normal cells, and an anti-cancer effect was observed across many different cancer cell types, supporting broad anticancer applicability.
  • Example 2 In vivo reduction in tumor cell growth and metastasis following treatment with lipid nanoparticle-encapsulated Rabla DN
  • Fl 1 is Rabla DN encapsulated in lipid nanoparticles (LNPs).
  • mice bodyweight was monitored to determine if there were any detrimental health effects from the Fl l treatment.
  • the Balb/c mice were injected with 4T1 tumor cells as described above.
  • day 0 the mouse was given a single dose of Fl 1 intravenously (IV) or PBS as the control.
  • Body weight was measured until the end of the experiment (total of 7 weeks post-IV treatment).
  • the body weight of the PBS control mouse did not change much before it died at day 35 ([blue line, lower line).
  • the Fl 1 treated mouse showed bodyweight gain throughout the entire experiment (orange line, upper line

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Abstract

Compounds, compositions, uses, and methods for reducing cell viability of a cancer cell, or for preventing or treating cancer, are provided herein. In certain examples, methods for reducing cell viability of a cancer cell and/or for preventing or treating cancer in a subject in need thereof are provided which may include a step of treatment with a GDP-bound form of Rab1a (Rab1aGDP), one or more expressible nucleic acids encoding Rab1aGDP, or a combination thereof.

Description

COMPOSITIONS AND METHODS FOR TREATING CANCER
FIELD OF INVENTION
The present invention relates generally to treatment of cancer. More specifically, the present invention relates to compositions and methods for enhancing microautophagy to treat various cancers and related diseases or disorders.
BACKGROUND
There are three known cellular processes, in eukaryotes, through which the cytosolic content and/or subcellular organelles can be delivered to or acquired by lysosomes for degradation: namely macroautophagy (commonly known as autophagy), microautophagy, and chaperone-mediated autophagy. Microautophagy, unlike macroautophagy or chaperone-mediated autophagy, is a type of autophagic process that is mediated by direct lysosomal (in mammals) or vacuolar (in plants and fungi) piecemeal engulfment of a cytoplasmic target or cargo (e.g. protein, lipid, glycogen or pathogen).
Microautophagy may contribute to cytosolic protein degradation via late endosomes (MVBs). In addition, microautophagy may also support direct glycogen delivery to lysosomes and its degradation. In this regard, it is contemplated that malfunctioning or insufficient microautophagy may be associated with development of a variety of metabolic and/or neurological diseases, for example.
Clearly, accumulation of particular proteins, lipids, and/or glycogens may be associated with a variety of important diseases, disorders, and conditions. Methods for targeting such proteins, lipids, and/or glycogens are desirable.
Cancer is a major ongoing health concern, leading to many deaths annually. Cancer includes a large group of diseases having a wide range of underlying causes. Each cancer may differ in biology and pathophysiology, but virtually all cancers are believed to be associated with membrane trafficking dysregulation (Parachoniak CA, Park M. Dynamics of receptor trafficking in tumorigenicity. Trends Cell Biol. 2012 May;22(5):231-40). Unfortunately, cancer and related diseases or disorders remain a significant problem, and anti-cancer treatments are highly desirable.
Alternative, additional, and/or improved anti-cancer agents, compositions, and/or methods for treating cancer and related diseases or disorders are desirable.
SUMMARY OF INVENTION
As described in detail herein, it has now been identified that treatment with a microautophagyenhancing agent comprising a GDP-bound form of Rabi a, such as RablaS25N, RablaN1241, RablaD41N, RablaD47N, or another dominant negative (DN) GDP -bound form of Rabi a, or one or more expressible nucleic acids encoding such a RablaGDP, may be used to decrease cell viability and/or cause cell death in a wide variety of different cancer cell types.
In an embodiment, there is provided herein a method for reducing cell viability of a cancer cell, or for preventing or treating cancer, in a subject in need thereof, said method comprising: treating a cancer cell of the subject with a GDP -bound form of Rabi a (RablaGDP), one or more expressible nucleic acids encoding RablaGDP, or a combination thereof; thereby increasing a cellular level of RablaGDP in the cancer cell, resulting in decreased cell viability or death of the cancer cell.
In another embodiment, there is provided herein a method for reducing cell viability of a cancer cell in vitro or in vivo, said method comprising: treating the cancer cell with a GDP -bound form of Rabi a (RablaGDP), one or more expressible nucleic acids encoding RablaGDP, or a combination thereof; thereby increasing a cellular level of RablaGDP in the cancer cell, resulting in decreased cell viability or death of the cancer cell. In another embodiment of any of the above method or methods, the RablaGDP may be or may comprise RablaS25N, RablaN1241, RablaD41N, RablaD47N, or another dominant negative (DN) GDP- bound form of Rabi a.
In still another embodiment of any of the above method or methods, the RablaGDP may comprise the amino acid sequence:
MSSMNPEYDYLFKLLLIGDSGVGKNCLLLRFADDTYTESYISTIGVDFKIRTIELDGK TIKLQIWDTAGQERFRTITSSYYRGAHGIIVVYDVTDQESFNNVKQWLQEIDRYASE NVNKLLVGNKCDLTTKKVVDYTTAKEFADSLGIPFLETSAKNATNVEQSFMTMAAE IKKRMGPGATAGGAEKSNVKIQSTPVKQSGGGCC (SEQ ID NO: 6; Human RablaS25N);
MGDYKDDDDKGGSGGSSMNPEYDYLFKLLLIGDSGVGKSCLLLRFADDTYTESYIS TIGVDFKIRTIELDGKTIKLQIWDTAGQERFRTITSSYYRGAHGIIVVYDVTDQESFNN VKQWLQEIDRYASENVNKLLVGIKCDLTTKKVVDYTTAKEFADSLGIPFLETSAKNA TNVEQSFMTMAAEIKKRMGPGATAGGAEKSNVKIQSTPVKQSGGGCC (SEQ ID NO: 9; Mouse RablN1241); the amino acid sequence of human RablaD41N; or
MSSMNPEYDYLFKLLLIGDSGVGKSCLLLRFADDTYTESYISTIGVNFKIRTIELDGKT
IKLQIWDTAGQERFRTITSSYYRGAHGIIVVYDVTDQESFNNVKQWLQEIDRYASEN VNKLLVGNKCDLTTKKVVDYTTAKEFADSLGIPFLETSAKNATNVEQSFMTMAAEI KKRMGPGATAGGAEKSNVKIQSTPVKQSGGGCC
(SEQ ID NO: 18; Human RablaD47N); or a polypeptide having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with any of these sequences and preferentially binding GDP.
In yet another embodiment of any of the above method or methods, the RablaGDP may consist of the amino acid sequence: MSSMNPEYDYLFKLLLIGDSGVGKNCLLLRFADDTYTESYISTIGVDFKIRTIELDGK
TIKLQIWDTAGQERFRTITSSYYRGAHGIIVVYDVTDQESFNNVKQWLQEIDRYASE
NVNKLLVGNKCDLTTKKVVDYTTAKEFADSLGIPFLETSAKNATNVEQSFMTMAAE
IKKRMGPGATAGGAEKSNVKIQSTPVKQSGGGCC
(SEQ ID NO: 6; Human RablaS25N);
MGDYKDDDDKGGSGGSSMNPEYDYLFKLLLIGDSGVGKSCLLLRFADDTYTESYIS
TIGVDFKIRTIELDGKTIKLQIWDTAGQERFRTITSSYYRGAHGIIVVYDVTDQESFNN
VKQWLQEIDRYASENVNKLLVGIKCDLTTKKVVDYTTAKEFADSLGIPFLETSAKNA
TNVEQSFMTMAAEIKKRMGPGATAGGAEKSNVKIQSTPVKQSGGGCC
(SEQ ID NO: 9; Mouse RablN1241); the amino acid sequence of human RablaD41N; or
MSSMNPEYDYLFKLLLIGDSGVGKSCLLLRFADDTYTESYISTIGVNFKIRTIELDGKT
IKLQIWDTAGQERFRTITSSYYRGAHGIIVVYDVTDQESFNNVKQWLQEIDRYASEN
VNKLLVGNKCDLTTKKVVDYTTAKEFADSLGIPFLETSAKNATNVEQSFMTMAAEI KKRMGPGATAGGAEKSNVKIQSTPVKQSGGGCC
(SEQ ID NO: 18; Human RablaD47N); or a polypeptide having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with any of these sequences and preferentially binding GDP.
In another embodiment of any of the above method or methods, the RablaGDP may comprise or consist of the amino acid sequence:
MSSMNPEYDYLFKLLLIGDSGVGKNCLLLRFADDTYTESYISTIGVDFKIRTIELDGK
TIKLQIWDTAGQERFRTITSSYYRGAHGIIVVYDVTDQESFNNVKQWLQEIDRYASE
NVNKLLVGNKCDLTTKKVVDYTTAKEFADSLGIPFLETSAKNATNVEQSFMTMAAE IKKRMGPGATAGGAEKSNVKIQSTPVKQSGGGCC
(SEQ ID NO: 6; Human RablaS25N); or a polypeptide having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity therewith and preferentially binding GDP.
In still another embodiment of any of the above method or methods, the RablaGDP may be in the form of a fusion protein, wherein the RablaGDP is fused or otherwise directly or indirectly linked, optionally via a linker, with a signaling or targeting peptide, a fluorescent peptide or other marker or tracer, or another peptide or non-peptide moiety for targeted delivery, facilitating cell uptake, increasing stability or in vivo half-life, or improving another therapeutic, diagnostic, or in vivo property of the RablaGDP.
In yet another embodiment of any of the above method or methods, the fusion protein may comprise the amino acid sequence:
MSSMNPEYDYLFKLLLIGDSGVGKNCLLLRFADDTYTESYISTIGVDFKIRTIELDGK TIKLQIWDTAGQERFRTITSSYYRGAHGIIVVYDVTDQESFNNVKQWLQEIDRYASE NVNKLLVGNKCDLTTKKVVDYTTAKEFADSLGIPFLETSAKNATNVEQSFMTMAAE IKKRMGPGATAGGAEKSNVKIQSTPVKQSGGGCC (SEQ ID NO: 6; Human RablaS25N); or a polypeptide having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity therewith and preferentially binding GDP.
In another embodiment of any of the above method or methods, the RablaGDP may be in the form of a fusion protein, and may comprise the amino acid sequence:
MEDAKNIKKGPAPFYPLEDGTAGEQLHKAMKRYALVPGTIAFTDAHIEVNITYAEYF EMSVRLAEAMKRYGLNTNHRIVVCSENSLQFFMPVLGALFIGVAVAPANDIYNERE LLNSMNISQPTVVFVSKKGLQKILNVQKKLPIIQKIIIMDSKTDYQGFQSMYTFVTSHL PPGFNEYDF VPESFDRDKTIALIMNS SGSTGLPKGVALPHRT ACVRF SHARDPIFGNQI IPDTAILSVVPFHHGFGMFTTLGYLICGFRVVLMYRFEEELFLRSLQDYKIQSALLVPT LFSFFAKSTLIDKYDLSNLHEIASGGAPLSKEVGEAVAKRFHLPGIRQGYGLTETTSAI LITPEGDDKPGAVGKVVPFFEAKVVDLDTGKTLGVNQRGELCVRGPMIMSGYVNNP EATNALIDKDGWLHSGDIAYWDEDEHFFIVDRLKSLIKYKGYQVAPAELESILLQHP NIFDAGVAGLPDDDAGELPAAVVVLEHGKTMTEKEIVDYVASQVTTAKKLRGGVV FVDEVPKGLTGKLDARKIREILIKAKKGGKSKLMSSMNPEYDYLFKLLLIGDSGVGK NCLLLRFADDTYTESYISTIGVDFKIRTIELDGKTIKLQIWDTAGQERFRTITSSYYRG AHGIIVVYDVTDQESFNNVKQWLQEIDRYASENVNKLLVGNKCDLTTKKVVDYTT AKEFADSLGIPFLETSAKNATNVEQSFMTMAAEIKKRMGPGATAGGAEKSNVKIQST PVKQSGGGCC
(SEQ ID NO: 21); or a polypeptide having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity therewith and preferentially binding GDP.
In still another embodiment of any of the above method or methods, the one or more expressible nucleic acids may encode any one or more of the RablaGDPs as defined herein.
In yet another embodiment of any of the above method or methods, the one or more expressible nucleic acids may be DNA-based, or RNA-based.
In another embodiment of any of the above method or methods, the one or more expressible nucleic acids may transiently express the RablaGDP in the cell, or wherein the one or more expressible nucleic acids may integrate in the cell genome and express the RablaGDP in the cell.
In still another embodiment of any of the above method or methods, the one or more expressible nucleic acids may comprise one or more expression vectors, plasmids, or mRNAs encoding and capable of expressing the RablaGDP inside the cell.
In yet another embodiment of any of the above method or methods, the one or more expressible nucleic acids may comprise a nucleic acid sequence of:
ATGTCCAGCATGAATCCCGAATATGATTATTTATTCAAGTTACTTCTGATTGGCG ACTCAGGGGTTGGAAAGAATTGCCTTCTTCTTAGGTTTGCAGATGATACATATAC AGAAAGCTACATCAGCACAATTGGTGTGGATTTCAAAATAAGAACTATAGAGTT
AGACGGGAAAACAATCAAGCTTCAAATATGGGACACAGCAGGCCAGGAAAGAT
TTCGAACAATCACCTCCAGTTATTACAGAGGAGCCCATGGCATCATAGTTGTGTA
TGATGTGACAGATCAGGAGTCCTTCAATAATGTTAAACAGTGGCTGCAGGAAAT
AGATCGTTATGCCAGTGAAAATGTCAACAAATTGTTGGTAGGGAACAAATGTGA
TCTGACCACAAAGAAAGTAGTAGACTACACAACAGCGAAGGAATTTGCTGATTC
CCTTGGAATTCCGTTTTTGGAAACCAGTGCTAAGAATGCAACGAATGTAGAACA
GTCTTTCATGACGATGGCAGCTGAGATTAAAAAGCGAATGGGTCCCGGAGCAAC
AGCTGGTGGTGCTGAGAAGTCCAATGTTAAAATTCAGAGCACTCCAGTCAAGCA GTCAGGTGGAGGTTGCTGCTAA
(Human RablaS25N ORF Codon Sequence, SEQ ID NO: 5);
ATGGGGGACTACAAGGACGACGATGACAAGGGGGGTAGCGGTGGATCCAGCAT
GAATCCCGAATATGATTATTTATTCAAGTTACTTCTGATTGGCGATTCTGGGGTT
GGAAAGTCCTGCCTTCTCCTTAGGTTTGCAGATGATACGTATACGGAAAGCTACA
TCAGCACAATTGGTGTGGATTTCAAGATACGAACTATAGAGTTAGATGGGAAAA
CAATCAAGCTACAGATATGGGACACAGCAGGCCAGGAAAGATTTCGAACAATCA
CTTCCAGTTATTACAGAGGAGCCCATGGCATCATAGTTGTGTATGATGTGACAGA
TCAGGAGTCCTTCAATAACGTTAAACAGTGGCTGCAGGAGATAGATCGCTACGC
CAGTGAAAATGTCAACAAGTTGTTGGTAGGGATCAAATGTGACCTGACCACAAA
GAAAGTAGTAGACTACACAACAGCAAAGGAATTTGCAGATTCCCTTGGAATTCC
ATTTTTGGAAACCAGTGCTAAGAACGCAACGAATGTAGAACAGTCTTTCATGAC
GATGGCAGCTGAGATTAAAAAGCGAATGGGTCCTGGAGCTACAGCTGGTGGTGC
CGAGAAGTCCAATGTTAAAATCCAGAGCACTCCAGTCAAGCAGTCAGGTGGAGG CTGCTGCTAA
(Mouse RablaN1241 ORF Codon Seqeunce, SEQ ID NO: 8); or
ATGTCCAGCATGAATCCCGAATATGATTATTTATTCAAGTTACTTCTGATTGGCGACT
CAGGGGTTGGAAAGTCTTGCCTTCTTCTTAGGTTTGCAGATGATACATATACAGAAA GCTACATCAGCACAATTGGTGTGAACTTCAAAATAAGAACTATAGAGTTAGACGGG AAAACAATCAAGCTTCAAATATGGGACACAGCAGGCCAGGAAAGATTTCGAACAAT CACCTCCAGTTATTACAGAGGAGCCCATGGCATCATAGTTGTGTATGATGTGACAGA TCAGGAGTCCTTCAATAATGTTAAACAGTGGCTGCAGGAAATAGATCGTTATGCCAG TGAAAATGTCAACAAATTGTTGGTAGGGAACAAATGTGATCTGACCACAAAGAAAG TAGTAGACTACACAACAGCGAAGGAATTTGCTGATTCCCTTGGAATTCCGTTTTTGG AAACCAGTGCTAAGAATGCAACGAATGTAGAACAGTCTTTCATGACGATGGCAGCT GAGATTAAAAAGCGAATGGGTCCCGGAGCAACAGCTGGTGGTGCTGAGAAGTCCAA TGTTAAAATTCAGAGCACTCCAGTCAAGCAGTCAGGTGGAGGTTGCTGCTAA
(Human RablaD47N ORF Codon Sequence, SEQ ID NO: 17); or a nucleic acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity therewith and encoding a RablaGDP preferentially binding GDP; or a nucleic acid sequence equivalent to any of the above sequences due to codon redundancy.
In another embodiment of any of the above method or methods, the one or more expressible nucleic acids may comprise a nucleic acid sequence of:
ATGGAAGACGCCAAAAACATAAAGAAAGGCCCGGCGCCATTCTATCCGCTAGAG
GATGGAACCGCTGGAGAGCAACTGCATAAGGCTATGAAGAGATACGCCCTGGTT
CCTGGAACAATTGCTTTTACAGATGCACATATCGAGGTGAACATCACGTACGCG
GAATACTTCGAAATGTCCGTTCGGTTGGCAGAAGCTATGAAACGATATGGGCTG
AATACAAATCACAGAATCGTCGTATGCAGTGAAAACTCTCTTCAATTCTTTATGC CGGTGTTGGGCGCGTTATTTATCGGAGTTGCAGTTGCGCCCGCGAACGACATTTA TAATGAACGTGAATTGCTCAACAGTATGAACATTTCGCAGCCTACCGTAGTGTTT GTTTCCAAAAAGGGGTTGCAAAAAATTTTGAACGTGCAAAAAAAATTACCAATA ATCCAGAAAATTATTATCATGGATTCTAAAACGGATTACCAGGGATTTCAGTCGA TGTACACGTTCGTCACATCTCATCTACCTCCCGGTTTTAATGAATACGATTTTGTA CCAGAGTCCTTTGATCGTGACAAAACAATTGCACTGATAATGAACTCCTCTGGAT CTACTGGGTTACCTAAGGGTGTGGCCCTTCCGCATAGAACTGCCTGCGTCAGATT CTCGCATGCCAGAGATCCTATTTTTGGCAATCAAATCATTCCGGATACTGCGATT TTAAGTGTTGTTCCATTCCATCACGGTTTTGGAATGTTTACTACACTCGGATATTT
GATATGTGGATTTCGAGTCGTCTTAATGTATAGATTTGAAGAAGAGCTGTTTTTA
CGATCCCTTCAGGATTACAAAATTCAAAGTGCGTTGCTAGTACCAACCCTATTTT
CATTCTTCGCCAAAAGCACTCTGATTGACAAATACGATTTATCTAATTTACACGA
AATTGCTTCTGGGGGCGCACCTCTTTCGAAAGAAGTCGGGGAAGCGGTTGCAAA
ACGCTTCCATCTTCCAGGGATACGACAAGGATATGGGCTCACTGAGACTACATC
AGCTATTCTGATTACACCCGAGGGGGATGATAAACCGGGCGCGGTCGGTAAAGT
TGTTCCATTTTTTGAAGCGAAGGTTGTGGATCTGGATACCGGGAAAACGCTGGGC
GTTAATCAGAGAGGCGAATTATGTGTCAGAGGACCTATGATTATGTCCGGTTATG
TAAACAATCCGGAAGCGACCAACGCCTTGATTGACAAGGATGGATGGCTACATT
CTGGAGACATAGCTTACTGGGACGAAGACGAACACTTCTTCATAGTTGACCGCTT
GAAGTCTTTAATTAAATACAAAGGATACCAGGTGGCCCCCGCTGAATTGGAGTC
GATATTGTTACAACACCCCAACATCTTCGACGCGGGCGTGGCAGGTCTTCCCGAC
GATGACGCCGGTGAACTTCCCGCCGCCGTTGTTGTTTTGGAGCACGGAAAGACG
ATGACGGAAAAAGAGATCGTGGATTACGTCGCCAGTCAAGTAACAACCGCCAAA
AAGTTGCGCGGAGGAGTTGTGTTTGTGGACGAAGTACCGAAAGGTCTTACCGGA
AAACTCGACGCAAGAAAAATCAGAGAGATCCTCATAAAGGCCAAGAAGGGCGG
AAAGTCCAAATTGATGTCCAGCATGAATCCCGAATATGATTATTTATTCAAGTTA
CTTCTGATTGGCGACTCAGGGGTTGGAAAGAATTGCCTTCTTCTTAGGTTTGCAG
ATGATACATATACAGAAAGCTACATCAGCACAATTGGTGTGGATTTCAAAATAA
GAACTATAGAGTTAGACGGGAAAACAATCAAGCTTCAAATATGGGACACAGCAG
GCCAGGAAAGATTTCGAACAATCACCTCCAGTTATTACAGAGGAGCCCATGGCA
TCATAGTTGTGTATGATGTGACAGATCAGGAGTCCTTCAATAATGTTAAACAGTG
GCTGCAGGAAATAGATCGTTATGCCAGTGAAAATGTCAACAAATTGTTGGTAGG
GAACAAATGTGATCTGACCACAAAGAAAGTAGTAGACTACACAACAGCGAAGG
AATTTGCTGATTCCCTTGGAATTCCGTTTTTGGAAACCAGTGCTAAGAATGCAAC
GAATGTAGAACAGTCTTTCATGACGATGGCAGCTGAGATTAAAAAGCGAATGGG
TCCCGGAGCAACAGCTGGTGGTGCTGAGAAGTCCAATGTTAAAATTCAGAGCAC
TCCAGTCAAGCAGTCAGGTGGAGGTTGCTGCTAA
£SEQ ID NO: 19, MG-008 ORF DNA Sequence with 5’ Luciferase Tag); or AUGGAAGACGCCAAAAACAUAAAGAAAGGCCCGGCGCCAUUCUAUCCGCUAG
AGGAUGGAACCGCUGGAGAGCAACUGCAUAAGGCUAUGAAGAGAUACGCCCU
GGUUCCUGGAACAAUUGCUUUUACAGAUGCACAUAUCGAGGUGAACAUCACG
UACGCGGAAUACUUCGAAAUGUCCGUUCGGUUGGCAGAAGCUAUGAAACGAU
AUGGGCUGAAUACAAAUCACAGAAUCGUCGUAUGCAGUGAAAACUCUCUUCA
AUUCUUUAUGCCGGUGUUGGGCGCGUUAUUUAUCGGAGUUGCAGUUGCGCCC
GCGAACGACAUUUAUAAUGAACGUGAAUUGCUCAACAGUAUGAACAUUUCGC
AGCCUACCGUAGUGUUUGUUUCCAAAAAGGGGUUGCAAAAAAUUUUGAACGU
GCAAAAAAAAUUACCAAUAAUCCAGAAAAUUAUUAUCAUGGAUUCUAAAACG
GAUUACCAGGGAUUUCAGUCGAUGUACACGUUCGUCACAUCUCAUCUACCUCC
CGGUUUUAAUGAAUACGAUUUUGUACCAGAGUCCUUUGAUCGUGACAAAACA
AUUGCACUGAUAAUGAACUCCUCUGGAUCUACUGGGUUACCUAAGGGUGUGG
CCCUUCCGCAUAGAACUGCCUGCGUCAGAUUCUCGCAUGCCAGAGAUCCUAUU
UUUGGCAAUCAAAUCAUUCCGGAUACUGCGAUUUUAAGUGUUGUUCCAUUCC
AUCACGGUUUUGGAAUGUUUACUACACUCGGAUAUUUGAUAUGUGGAUUUCG
AGUCGUCUUAAUGUAUAGAUUUGAAGAAGAGCUGUUUUUACGAUCCCUUCAG
GAUUACAAAAUUCAAAGUGCGUUGCUAGUACCAACCCUAUUUUCAUUCUUCG
CCAAAAGCACUCUGAUUGACAAAUACGAUUUAUCUAAUUUACACGAAAUUGC
UUCUGGGGGCGCACCUCUUUCGAAAGAAGUCGGGGAAGCGGUUGCAAAACGC
UUCCAUCUUCCAGGGAUACGACAAGGAUAUGGGCUCACUGAGACUACAUCAG
CUAUUCUGAUUACACCCGAGGGGGAUGAUAAACCGGGCGCGGUCGGUAAAGU
UGUUCCAUUUUUUGAAGCGAAGGUUGUGGAUCUGGAUACCGGGAAAACGCUG
GGCGUUAAUCAGAGAGGCGAAUUAUGUGUCAGAGGACCUAUGAUUAUGUCCG
GUUAUGUAAACAAUCCGGAAGCGACCAACGCCUUGAUUGACAAGGAUGGAUG
GCUACAUUCUGGAGACAUAGCUUACUGGGACGAAGACGAACACUUCUUCAUA
GUUGACCGCUUGAAGUCUUUAAUUAAAUACAAAGGAUACCAGGUGGCCCCCG
CUGAAUUGGAGUCGAUAUUGUUACAACACCCCAACAUCUUCGACGCGGGCGU
GGCAGGUCUUCCCGACGAUGACGCCGGUGAACUUCCCGCCGCCGUUGUUGUUU
UGGAGCACGGAAAGACGAUGACGGAAAAAGAGAUCGUGGAUUACGUCGCCAG
UCAAGUAACAACCGCCAAAAAGUUGCGCGGAGGAGUUGUGUUUGUGGACGAA GUACCGAAAGGUCUUACCGGAAAACUCGACGCAAGAAAAAUCAGAGAGAUCC UCAUAAAGGCCAAGAAGGGCGGAAAGUCCAAAUUGAUGUCCAGCAUGAAUCC CGAAUAUGAUUAUUUAUUCAAGUUACUUCUGAUUGGCGACUCAGGGGUUGGA AAGAAUUGCCUUCUUCUUAGGUUUGCAGAUGAUACAUAUACAGAAAGCUACA UCAGCACAAUUGGUGUGGAUUUCAAAAUAAGAACUAUAGAGUUAGACGGGAA AACAAUCAAGCUUCAAAUAUGGGACACAGCAGGCCAGGAAAGAUUUCGAACA AUCACCUCCAGUUAUUACAGAGGAGCCCAUGGCAUCAUAGUUGUGUAUGAUG UGACAGAUCAGGAGUCCUUCAAUAAUGUUAAACAGUGGCUGCAGGAAAUAGA UCGUUAUGCCAGUGAAAAUGUCAACAAAUUGUUGGUAGGGAACAAAUGUGAU CUGACCACAAAGAAAGUAGUAGACUACACAACAGCGAAGGAAUUUGCUGAUU CCCUUGGAAUUCCGUUUUUGGAAACCAGUGCUAAGAAUGCAACGAAUGUAGA ACAGUCUUUCAUGACGAUGGCAGCUGAGAUUAAAAAGCGAAUGGGUCCCGGA GCAACAGCUGGUGGUGCUGAGAAGUCCAAUGUUAAAAUUCAGAGCACUCCAG UCAAGCAGUCAGGUGGAGGUUGCUGCUAA
(SEQ ID NO: 20, MG-008 ORF mRNA Sequence with 5’ Luciferase Tag); or a nucleic acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity therewith and encoding a RablaGDP preferentially binding GDP; or a nucleic acid sequence equivalent to any of the above sequences due to codon redundancy.
In another embodiment, there is provided herein a use of a GDP -bound form of Rabi a (RablaGDP), one or more expressible nucleic acids encoding RablaGDP, or a combination thereof, for reducing cell viability of a cancer cell, or for preventing or treating cancer, in a subject in need thereof.
In another embodiment, there is provided herein a use of a GDP -bound form of Rabi a (RablaGDP), one or more expressible nucleic acids encoding RablaGDP, or a combination thereof, for reducing cell viability of certain cancer cells in vitro or in vivo.
In another embodiment, there is provided herein a use of a GDP -bound form of Rabi a (RablaGDP), one or more expressible nucleic acids encoding RablaGDP, or a combination thereof, in the manufacture of a medicament for reducing cell viability of certain cancer cells, or for preventing or treating cancer, in a subject in need thereof.
In another embodiment, there is provided herein a use of a GDP -bound form of Rabi a (RablaGDP), one or more expressible nucleic acids encoding RablaGDP, or a combination thereof, in the manufacture of a medicament for reducing cell viability of certain cancer cells in vitro or in vivo.
In another embodiment of any of the above use or uses, the RablaGDP may be or may comprise RablaS25N, RablaN1241, RablaD41N, RablaD47N, or another dominant negative (DN) GDP-bound form of Rabi a.
In still another embodiment of any of the above use or uses, the Rab 1 aGDP may comprise the amino acid sequence:
MSSMNPEYDYLFKLLLIGDSGVGKNCLLLRFADDTYTESYISTIGVDFKIRTIELDGK TIKLQIWDTAGQERFRTITSSYYRGAHGIIVVYDVTDQESFNNVKQWLQEIDRYASE NVNKLLVGNKCDLTTKKVVDYTTAKEFADSLGIPFLETSAKNATNVEQSFMTMAAE IKKRMGPGATAGGAEKSNVKIQSTPVKQSGGGCC
(SEQ ID NO: 6; Human RablaS25N);
MGDYKDDDDKGGSGGSSMNPEYDYLFKLLLIGDSGVGKSCLLLRFADDTYTESYIS
TIGVDFKIRTIELDGKTIKLQIWDTAGQERFRTITSSYYRGAHGIIVVYDVTDQESFNN VKQWLQEIDRYASENVNKLLVGIKCDLTTKKVVDYTTAKEFADSLGIPFLETSAKNA TNVEQSFMTMAAEIKKRMGPGATAGGAEKSNVKIQSTPVKQSGGGCC
(SEQ ID NO: 9; Mouse RablN1241); the amino acid sequence of human RablaD41N; or
MSSMNPEYDYLFKLLLIGDSGVGKSCLLLRFADDTYTESYISTIGVNFKIRTIELDGKT IKLQIWDTAGQERFRTITSSYYRGAHGIIVVYDVTDQESFNNVKQWLQEIDRYASEN VNKLLVGNKCDLTTKKVVDYTTAKEFADSLGIPFLETSAKNATNVEQSFMTMAAEI KKRMGPGATAGGAEKSNVKIQSTPVKQSGGGCC
(SEQ ID NO: 18; Human RablaD47N); or a polypeptide having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with any of these sequences and preferentially binding GDP.
In yet another embodiment of any of the above use or uses, the RablaGDP may consist of the amino acid sequence:
MSSMNPEYDYLFKLLLIGDSGVGKNCLLLRFADDTYTESYISTIGVDFKIRTIELDGK TZKLQIWDTAGQERFRTITSSYYRGAHGIIVVYDVTDQESFNNVKQWLQEIDRYASE NVNKLLVGNKCDLTTKKVVDYTTAKEFADSLGIPFLETSAKNATNVEQSFMTMAAE IKKRMGPGATAGGAEKSNVKIQSTPVKQSGGGCC
(SEQ ID NO: 6; Human RablaS25N);
MGDYKDDDDKGGSGGSSMNPEYDYLFKLLLIGDSGVGKSCLLLRFADDTYTESYIS TIGVDFKIRTIELDGKTIKLQIWDTAGQERFRTITSSYYRGAHGIIVVYDVTDQESFNN VKQWLQEIDRYASENVNKLLVGIKCDLTTKKVVDYTTAKEFADSLGIPFLETSAKNA TNVEQSFMTMAAEIKKRMGPGATAGGAEKSNVKIQSTPVKQSGGGCC (SEQ ID NO: 9; Mouse RablN1241); the amino acid sequence of human RablaD41N; or
MSSMNPEYDYLFKLLLIGDSGVGKSCLLLRFADDTYTESYISTIGVNFKIRTIELDGKT
IKLQIWDTAGQERFRTITSSYYRGAHGIIVVYDVTDQESFNNVKQWLQEIDRYASEN VNKLLVGNKCDLTTKKVVDYTTAKEFADSLGIPFLETSAKNATNVEQSFMTMAAEI KKRMGPGATAGGAEKSNVKIQSTPVKQSGGGCC
(SEQ ID NO: 18; Human RablaD47N); or a polypeptide having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with any of these sequences and preferentially binding GDP.
In another embodiment of any of the above use or uses, the RablaGDP may comprise or consist of the amino acid sequence: MSSMNPEYDYLFKLLLIGDSGVGKNCLLLRFADDTYTESYISTIGVDFKIRTIELDGK TIKLQIWDTAGQERFRTITSSYYRGAHGIIVVYDVTDQESFNNVKQWLQEIDRYASE NVNKLLVGNKCDLTTKKVVDYTTAKEFADSLGIPFLETSAKNATNVEQSFMTMAAE IKKRMGPGATAGGAEKSNVKIQSTPVKQSGGGCC (SEQ ID NO: 6; Human RablaS25N); or a polypeptide having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity therewith and preferentially binding GDP.
In still another embodiment of any of the above use or uses, the RablaGDP may be in the form of a fusion protein, wherein the RablaGDP is fused or otherwise directly or indirectly linked, optionally via a linker, with a signaling or targeting peptide, a fluorescent peptide or other marker or tracer, or another peptide or non-peptide moiety for targeted delivery, facilitating cell uptake, increasing stability or in vivo half-life, or improving another therapeutic, diagnostic, or in vivo property of the RablaGDP.
In yet another embodiment of any of the above use or uses, the fusion protein may comprise the amino acid sequence:
MSSMNPEYDYLFKLLLIGDSGVGKNCLLLRFADDTYTESYISTIGVDFKIRTIELDGK TIKLQIWDTAGQERFRTITSSYYRGAHGIIVVYDVTDQESFNNVKQWLQEIDRYASE NVNKLLVGNKCDLTTKKVVDYTTAKEFADSLGIPFLETSAKNATNVEQSFMTMAAE IKKRMGPGATAGGAEKSNVKIQSTPVKQSGGGCC (SEQ ID NO: 6; Human RablaS25N); or a polypeptide having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity therewith and preferentially binding GDP.
In another embodiment of any of the above use or uses, the RablaGDP may be in the form of a fusion protein, and may comprise the amino acid sequence: MEDAKNIKKGPAPFYPLEDGTAGEQLHKAMKRYALVPGTIAFTDAHIEVNITYAEYF EMSVRLAEAMKRYGLNTNHRIVVCSENSLQFFMPVLGALFIGVAVAPANDIYNERE LLNSMNISQPTVVFVSKKGLQKILNVQKKLPIIQKIIIMDSKTDYQGFQSMYTFVTSHL PPGFNEYDF VPESFDRDKTIALIMNS SGSTGLPKGVALPHRT ACVRF SHARDPIFGNQI IPDTAILSVVPFHHGFGMFTTLGYLICGFRVVLMYRFEEELFLRSLQDYKIQSALLVPT LFSFFAKSTLIDKYDLSNLHEIASGGAPLSKEVGEAVAKRFHLPGIRQGYGLTETTSAI LITPEGDDKPGAVGKVVPFFEAKVVDLDTGKTLGVNQRGELCVRGPMIMSGYVNNP EATNALIDKDGWLHSGDIAYWDEDEHFFIVDRLKSLIKYKGYQVAPAELESILLQHP NIFDAGVAGLPDDDAGELPAAVVVLEHGKTMTEKEIVDYVASQVTTAKKLRGGVV FVDEVPKGLTGKLDARKIREILIKAKKGGKSKLMSSMNPEYDYLFKLLLIGDSGVGK NCLLLRFADDTYTESYISTIGVDFKIRTIELDGKTIKLQIWDTAGQERFRTITSSYYRG AHGIIVVYDVTDQESFNNVKQWLQEIDRYASENVNKLLVGNKCDLTTKKVVDYTT AKEFADSLGIPFLETSAKNATNVEQSFMTMAAEIKKRMGPGATAGGAEKSNVKIQST PVKQSGGGCC
(SEQ ID NO: 21); or a polypeptide having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity therewith and preferentially binding GDP.
In still another embodiment of any of the above use or uses, the one or more expressible nucleic acids may encode one or more RablaGDPs as defined herein.
In yet another embodiment of any of the above use or uses, the one or more expressible nucleic acids may be DNA-based, or RNA-based.
In another embodiment of any of the above use or uses, the one or more expressible nucleic acids may transiently express the RablaGDP in the cell, or wherein the one or more expressible nucleic acids may integrate in the cell genome and express the RablaGDP in the cell.
In another embodiment of any of the above use or uses, the one or more expressible nucleic acids may comprise one or more expression vectors, plasmids, or mRNAs encoding and capable of expressing the RablaGDP inside the cell.
In another embodiment of any of the above use or uses, the one or more expressible nucleic acids may comprise a nucleic acid sequence of:
ATGTCCAGCATGAATCCCGAATATGATTATTTATTCAAGTTACTTCTGATTGGCG
ACTCAGGGGTTGGAAAGAATTGCCTTCTTCTTAGGTTTGCAGATGATACATATAC
AGAAAGCTACATCAGCACAATTGGTGTGGATTTCAAAATAAGAACTATAGAGTT
AGACGGGAAAACAATCAAGCTTCAAATATGGGACACAGCAGGCCAGGAAAGAT
TTCGAACAATCACCTCCAGTTATTACAGAGGAGCCCATGGCATCATAGTTGTGTA
TGATGTGACAGATCAGGAGTCCTTCAATAATGTTAAACAGTGGCTGCAGGAAAT
AGATCGTTATGCCAGTGAAAATGTCAACAAATTGTTGGTAGGGAACAAATGTGA
TCTGACCACAAAGAAAGTAGTAGACTACACAACAGCGAAGGAATTTGCTGATTC
CCTTGGAATTCCGTTTTTGGAAACCAGTGCTAAGAATGCAACGAATGTAGAACA
GTCTTTCATGACGATGGCAGCTGAGATTAAAAAGCGAATGGGTCCCGGAGCAAC
AGCTGGTGGTGCTGAGAAGTCCAATGTTAAAATTCAGAGCACTCCAGTCAAGCA GTCAGGTGGAGGTTGCTGCTAA
(Human RablaS25N ORF Codon Sequence, SEQ ID NO: 5);
ATGGGGGACTACAAGGACGACGATGACAAGGGGGGTAGCGGTGGATCCAGCAT
GAATCCCGAATATGATTATTTATTCAAGTTACTTCTGATTGGCGATTCTGGGGTT
GGAAAGTCCTGCCTTCTCCTTAGGTTTGCAGATGATACGTATACGGAAAGCTACA
TCAGCACAATTGGTGTGGATTTCAAGATACGAACTATAGAGTTAGATGGGAAAA
CAATCAAGCTACAGATATGGGACACAGCAGGCCAGGAAAGATTTCGAACAATCA
CTTCCAGTTATTACAGAGGAGCCCATGGCATCATAGTTGTGTATGATGTGACAGA
TCAGGAGTCCTTCAATAACGTTAAACAGTGGCTGCAGGAGATAGATCGCTACGC
CAGTGAAAATGTCAACAAGTTGTTGGTAGGGATCAAATGTGACCTGACCACAAA
GAAAGTAGTAGACTACACAACAGCAAAGGAATTTGCAGATTCCCTTGGAATTCC
ATTTTTGGAAACCAGTGCTAAGAACGCAACGAATGTAGAACAGTCTTTCATGAC
GATGGCAGCTGAGATTAAAAAGCGAATGGGTCCTGGAGCTACAGCTGGTGGTGC
CGAGAAGTCCAATGTTAAAATCCAGAGCACTCCAGTCAAGCAGTCAGGTGGAGG CTGCTGCTAA
(Mouse RablaN1241 ORF Codon Seqeunce, SEQ ID NO: 8); or
ATGTCCAGCATGAATCCCGAATATGATTATTTATTCAAGTTACTTCTGATTGGCGACT
CAGGGGTTGGAAAGTCTTGCCTTCTTCTTAGGTTTGCAGATGATACATATACAGAAA GCTACATCAGCACAATTGGTGTGAACTTCAAAATAAGAACTATAGAGTTAGACGGG AAAACAATCAAGCTTCAAATATGGGACACAGCAGGCCAGGAAAGATTTCGAACAAT CACCTCCAGTTATTACAGAGGAGCCCATGGCATCATAGTTGTGTATGATGTGACAGA TCAGGAGTCCTTCAATAATGTTAAACAGTGGCTGCAGGAAATAGATCGTTATGCCAG TGAAAATGTCAACAAATTGTTGGTAGGGAACAAATGTGATCTGACCACAAAGAAAG TAGTAGACTACACAACAGCGAAGGAATTTGCTGATTCCCTTGGAATTCCGTTTTTGG AAACCAGTGCTAAGAATGCAACGAATGTAGAACAGTCTTTCATGACGATGGCAGCT GAGATTAAAAAGCGAATGGGTCCCGGAGCAACAGCTGGTGGTGCTGAGAAGTCCAA TGTTAAAATTCAGAGCACTCCAGTCAAGCAGTCAGGTGGAGGTTGCTGCTAA
(Human RablaD47N ORF Codon Sequence, SEQ ID NO: 17); or a nucleic acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity therewith and encoding a RablaGDP preferentially binding GDP; or a nucleic acid sequence equivalent to any of the above sequences due to codon redundancy.
In still another embodiment of any of the above use or uses, the one or more expressible nucleic acids may comprise a nucleic acid sequence of:
ATGGAAGACGCCAAAAACATAAAGAAAGGCCCGGCGCCATTCTATCCGCTAGAG
GATGGAACCGCTGGAGAGCAACTGCATAAGGCTATGAAGAGATACGCCCTGGTT
CCTGGAACAATTGCTTTTACAGATGCACATATCGAGGTGAACATCACGTACGCG
GAATACTTCGAAATGTCCGTTCGGTTGGCAGAAGCTATGAAACGATATGGGCTG
AATACAAATCACAGAATCGTCGTATGCAGTGAAAACTCTCTTCAATTCTTTATGC
CGGTGTTGGGCGCGTTATTTATCGGAGTTGCAGTTGCGCCCGCGAACGACATTTA TAATGAACGTGAATTGCTCAACAGTATGAACATTTCGCAGCCTACCGTAGTGTTT
GTTTCCAAAAAGGGGTTGCAAAAAATTTTGAACGTGCAAAAAAAATTACCAATA
ATCCAGAAAATTATTATCATGGATTCTAAAACGGATTACCAGGGATTTCAGTCGA
TGTACACGTTCGTCACATCTCATCTACCTCCCGGTTTTAATGAATACGATTTTGTA
CCAGAGTCCTTTGATCGTGACAAAACAATTGCACTGATAATGAACTCCTCTGGAT
CTACTGGGTTACCTAAGGGTGTGGCCCTTCCGCATAGAACTGCCTGCGTCAGATT
CTCGCATGCCAGAGATCCTATTTTTGGCAATCAAATCATTCCGGATACTGCGATT
TTAAGTGTTGTTCCATTCCATCACGGTTTTGGAATGTTTACTACACTCGGATATTT
GATATGTGGATTTCGAGTCGTCTTAATGTATAGATTTGAAGAAGAGCTGTTTTTA
CGATCCCTTCAGGATTACAAAATTCAAAGTGCGTTGCTAGTACCAACCCTATTTT
CATTCTTCGCCAAAAGCACTCTGATTGACAAATACGATTTATCTAATTTACACGA
AATTGCTTCTGGGGGCGCACCTCTTTCGAAAGAAGTCGGGGAAGCGGTTGCAAA
ACGCTTCCATCTTCCAGGGATACGACAAGGATATGGGCTCACTGAGACTACATC
AGCTATTCTGATTACACCCGAGGGGGATGATAAACCGGGCGCGGTCGGTAAAGT
TGTTCCATTTTTTGAAGCGAAGGTTGTGGATCTGGATACCGGGAAAACGCTGGGC
GTTAATCAGAGAGGCGAATTATGTGTCAGAGGACCTATGATTATGTCCGGTTATG
TAAACAATCCGGAAGCGACCAACGCCTTGATTGACAAGGATGGATGGCTACATT
CTGGAGACATAGCTTACTGGGACGAAGACGAACACTTCTTCATAGTTGACCGCTT
GAAGTCTTTAATTAAATACAAAGGATACCAGGTGGCCCCCGCTGAATTGGAGTC
GATATTGTTACAACACCCCAACATCTTCGACGCGGGCGTGGCAGGTCTTCCCGAC
GATGACGCCGGTGAACTTCCCGCCGCCGTTGTTGTTTTGGAGCACGGAAAGACG
ATGACGGAAAAAGAGATCGTGGATTACGTCGCCAGTCAAGTAACAACCGCCAAA
AAGTTGCGCGGAGGAGTTGTGTTTGTGGACGAAGTACCGAAAGGTCTTACCGGA
AAACTCGACGCAAGAAAAATCAGAGAGATCCTCATAAAGGCCAAGAAGGGCGG
AAAGTCCAAATTGATGTCCAGCATGAATCCCGAATATGATTATTTATTCAAGTTA
CTTCTGATTGGCGACTCAGGGGTTGGAAAGAATTGCCTTCTTCTTAGGTTTGCAG
ATGATACATATACAGAAAGCTACATCAGCACAATTGGTGTGGATTTCAAAATAA
GAACTATAGAGTTAGACGGGAAAACAATCAAGCTTCAAATATGGGACACAGCAG
GCCAGGAAAGATTTCGAACAATCACCTCCAGTTATTACAGAGGAGCCCATGGCA
TCATAGTTGTGTATGATGTGACAGATCAGGAGTCCTTCAATAATGTTAAACAGTG GCTGCAGGAAATAGATCGTTATGCCAGTGAAAATGTCAACAAATTGTTGGTAGG
GAACAAATGTGATCTGACCACAAAGAAAGTAGTAGACTACACAACAGCGAAGG
AATTTGCTGATTCCCTTGGAATTCCGTTTTTGGAAACCAGTGCTAAGAATGCAAC
GAATGTAGAACAGTCTTTCATGACGATGGCAGCTGAGATTAAAAAGCGAATGGG
TCCCGGAGCAACAGCTGGTGGTGCTGAGAAGTCCAATGTTAAAATTCAGAGCAC
TCCAGTCAAGCAGTCAGGTGGAGGTTGCTGCTAA
(SEQ ID NO: 19, MG-008 ORF DNA Sequence with 5’ Luciferase Tag); or
AUGGAAGACGCCAAAAACAUAAAGAAAGGCCCGGCGCCAUUCUAUCCGCUAG
AGGAUGGAACCGCUGGAGAGCAACUGCAUAAGGCUAUGAAGAGAUACGCCCU
GGUUCCUGGAACAAUUGCUUUUACAGAUGCACAUAUCGAGGUGAACAUCACG
UACGCGGAAUACUUCGAAAUGUCCGUUCGGUUGGCAGAAGCUAUGAAACGAU
AUGGGCUGAAUACAAAUCACAGAAUCGUCGUAUGCAGUGAAAACUCUCUUCA
AUUCUUUAUGCCGGUGUUGGGCGCGUUAUUUAUCGGAGUUGCAGUUGCGCCC
GCGAACGACAUUUAUAAUGAACGUGAAUUGCUCAACAGUAUGAACAUUUCGC
AGCCUACCGUAGUGUUUGUUUCCAAAAAGGGGUUGCAAAAAAUUUUGAACGU
GCAAAAAAAAUUACCAAUAAUCCAGAAAAUUAUUAUCAUGGAUUCUAAAACG
GAUUACCAGGGAUUUCAGUCGAUGUACACGUUCGUCACAUCUCAUCUACCUCC
CGGUUUUAAUGAAUACGAUUUUGUACCAGAGUCCUUUGAUCGUGACAAAACA
AUUGCACUGAUAAUGAACUCCUCUGGAUCUACUGGGUUACCUAAGGGUGUGG
CCCUUCCGCAUAGAACUGCCUGCGUCAGAUUCUCGCAUGCCAGAGAUCCUAUU
UUUGGCAAUCAAAUCAUUCCGGAUACUGCGAUUUUAAGUGUUGUUCCAUUCC
AUCACGGUUUUGGAAUGUUUACUACACUCGGAUAUUUGAUAUGUGGAUUUCG
AGUCGUCUUAAUGUAUAGAUUUGAAGAAGAGCUGUUUUUACGAUCCCUUCAG
GAUUACAAAAUUCAAAGUGCGUUGCUAGUACCAACCCUAUUUUCAUUCUUCG
CCAAAAGCACUCUGAUUGACAAAUACGAUUUAUCUAAUUUACACGAAAUUGC
UUCUGGGGGCGCACCUCUUUCGAAAGAAGUCGGGGAAGCGGUUGCAAAACGC
UUCCAUCUUCCAGGGAUACGACAAGGAUAUGGGCUCACUGAGACUACAUCAG
CUAUUCUGAUUACACCCGAGGGGGAUGAUAAACCGGGCGCGGUCGGUAAAGU
UGUUCCAUUUUUUGAAGCGAAGGUUGUGGAUCUGGAUACCGGGAAAACGCUG GGCGUUAAUCAGAGAGGCGAAUUAUGUGUCAGAGGACCUAUGAUUAUGUCCG GUUAUGUAAACAAUCCGGAAGCGACCAACGCCUUGAUUGACAAGGAUGGAUG GCUACAUUCUGGAGACAUAGCUUACUGGGACGAAGACGAACACUUCUUCAUA GUUGACCGCUUGAAGUCUUUAAUUAAAUACAAAGGAUACCAGGUGGCCCCCG CUGAAUUGGAGUCGAUAUUGUUACAACACCCCAACAUCUUCGACGCGGGCGU GGCAGGUCUUCCCGACGAUGACGCCGGUGAACUUCCCGCCGCCGUUGUUGUUU UGGAGCACGGAAAGACGAUGACGGAAAAAGAGAUCGUGGAUUACGUCGCCAG UCAAGUAACAACCGCCAAAAAGUUGCGCGGAGGAGUUGUGUUUGUGGACGAA GUACCGAAAGGUCUUACCGGAAAACUCGACGCAAGAAAAAUCAGAGAGAUCC UCAUAAAGGCCAAGAAGGGCGGAAAGUCCAAAUUGAUGUCCAGCAUGAAUCC CGAAUAUGAUUAUUUAUUCAAGUUACUUCUGAUUGGCGACUCAGGGGUUGGA AAGAAUUGCCUUCUUCUUAGGUUUGCAGAUGAUACAUAUACAGAAAGCUACA UCAGCACAAUUGGUGUGGAUUUCAAAAUAAGAACUAUAGAGUUAGACGGGAA AACAAUCAAGCUUCAAAUAUGGGACACAGCAGGCCAGGAAAGAUUUCGAACA AUCACCUCCAGUUAUUACAGAGGAGCCCAUGGCAUCAUAGUUGUGUAUGAUG UGACAGAUCAGGAGUCCUUCAAUAAUGUUAAACAGUGGCUGCAGGAAAUAGA UCGUUAUGCCAGUGAAAAUGUCAACAAAUUGUUGGUAGGGAACAAAUGUGAU CUGACCACAAAGAAAGUAGUAGACUACACAACAGCGAAGGAAUUUGCUGAUU CCCUUGGAAUUCCGUUUUUGGAAACCAGUGCUAAGAAUGCAACGAAUGUAGA ACAGUCUUUCAUGACGAUGGCAGCUGAGAUUAAAAAGCGAAUGGGUCCCGGA GCAACAGCUGGUGGUGCUGAGAAGUCCAAUGUUAAAAUUCAGAGCACUCCAG UCAAGCAGUCAGGUGGAGGUUGCUGCUAA
(SEQ ID NO: 20, MG-008 ORF mRNA Sequence with 5’ Luciferase Tag); or a nucleic acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity therewith and encoding a RablaGDP preferentially binding GDP; or a nucleic acid sequence equivalent to any of the above sequences due to codon redundancy.
In another embodiment, there is provided herein a polypeptide comprising the amino acid sequence:
MSSMNPEYDYLFKLLLIGDSGVGKNCLLLRFADDTYTESYISTIGVDFKIRTIELD
GKTIKLQIWDTAGQERFRTITSSYYRGAHGIIVVYDVTDQESFNNVKQWLQEIDR YASENVNKLLVGNKCDLTTKKVVDYTTAKEFADSLGIPFLETSAKNATNVEQSF MTMAAEIKKRMGPGATAGGAEKSNVKIQSTPVKQSGGGCC (SEQ ID NO: 6; Human RablaS25N);
MGDYKDDDDKGGSGGSSMNPEYDYLFKLLLIGDSGVGKSCLLLRFADDTYTES
YISTIGVDFKIRTIELDGKTIKLQIWDTAGQERFRTITSSYYRGAHGIIVVYDVTDQ ESFNNVKQWLQEIDRYASENVNKLLVGIKCDLTTKKVVDYTTAKEFADSLGIPFL ETSAKNATNVEQSFMTMAAEIKKRMGPGATAGGAEKSNVKIQSTPVKQSGGGC C
(SEQ ID NO: 9; Mouse RablN1241); the amino acid sequence of human RablaD41N; or
MSSMNPEYDYLFKLLLIGDSGVGKSCLLLRFADDTYTESYISTIGVNFKIRTIELD
GKTIKLQIWDTAGQERFRTITSSYYRGAHGIIVVYDVTDQESFNNVKQWLQEIDR YASENVNKLLVGNKCDLTTKKVVDYTTAKEFADSLGIPFLETSAKNATNVEQSF MTMAAEIKKRMGPGATAGGAEKSNVKIQSTPVKQSGGGCC
(SEQ ID NO: 18; Human RablaD47N); or a polypeptide having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with any of these sequences and preferentially binding GDP; for use in reducing cell viability of certain cancer cells, or for preventing or treating cancer, in a subject in need thereof; or for use in reducing cell viability of certain cancer cells in vitro or in vivo.
In another embodiment, there is provided herein a pharmaceutical composition comprising a GDP -bound form of Rabi a (RablaGDP), one or more expressible nucleic acids encoding RablaGDP, or a combination thereof; and another anti-cancer agent.
In yet another embodiment, there is provided herein a kit comprising any one or more of: a GDP -bound form of Rabi a (RablaGDP); one or more expressible nucleic acids encoding RablaGDP; an anti-cancer agent; instructions for performing any of the method or methods as described herein; or any combinations thereof.
In still another embodiment of any of the above methods, uses, or polypeptides for use, the cancer may be brain cancer, breast cancer, cervical cancer, colon cancer, ductal cancer, gastric cancer, liver cancer, lung cancer, oral cancer, pancreatic cancer, or prostate cancer.
In yet another embodiment of any of the above methods, uses, or polypeptides for use, the cancer may be breast adenocarcinoma, cholangiocarcinoma, colon adenocarcinoma, colorectal carcinoma, prostate carcinoma, embryonal rhabdomyosarcoma, gastric carcinoma, glioblastoma, hepatocellular carcinoma, invasive ductal carcinoma, lung cancer adenocarcinoma, pancreatic ductal adenocarcinoma, papillomavirus-related endocervical adenocarcinoma, squamous cell carcinoma of the oral cavity, or squamous cell carcinoma of the oral tongue.
In another embodiment of any of the above methods, uses, or polypeptides for use, the cancer cell lines may be selected from the group consisting of A549 (lung adenocarcinoma), HCT116 (colorectal carcinoma), HT29 (colon adenocarcinoma), HuCCTl (cholangiocarcinoma), PC-3 (prostate carcinoma), RD (rhabdomyosarcoma), SC-M1 (gastric carcinoma), U-87MG (glioblastoma), SW480 (colon adenocarcinoma), PANC-1 (pancreatic adenocarcinoma), OECM- 1 (oral carcinoma), OC2 (oral carcinoma), MIA PaCa-2 (pancreatic adenocarcinoma), MDA-MB- 468 (breast adenocarcinoma), MDA-MB-231 (breast adenocarcinoma), MCF-7 (ductal carcinoma), Mahlavu (hepatocellular carcinoma), HSC-3 cells (Squamous cell carcinoma of the oral tongue), HeLa (cervical carcinoma), HCT116 (colorectal carcinoma), U-87MG (glioblastoma), among others.
BRIEF DESCRIPTION OF DRAWINGS
These and other features will become further understood having regard to the following drawings in which various cancer cells were treated with mRNA-encoded RablaGDP:
FIGURE 1 shows that transfection of A549 cells (lung adenocarcinoma) with mRNA encoding MG-008 (100 ng per well) resulted in cell death as determined, 72 h post-transfection, by phase contrast microscopy (panels A (Control), B (Lipofectamine MessengerMAX), and C (MG-008)) and by crystal violet stain (D), as described in Example 1;
FIGURE 2 shows that transfection of HCT116 cells (colorectal carcinoma) with mRNA encoding MG-008 (100 ng per well) resulted in cell death as determined, 72 h post-transfection, by phase contrast microscopy (panels A (Control), B (Lipofectamine MessengerMAX), and C (MG-008)) and by crystal violet stain (D), as described in Example 1;
FIGURE 3 shows that transfection of HT29 cells (colon adenocarcinoma) with mRNA encoding MG-008 (100 ng per well) resulted in cell death as determined, 72 h post-transfection, by phase contrast microscopy (panels A (Control), B (Lipofectamine MessengerMAX), and C (MG-008)) and by crystal violet stain (D), as described in Example 1;
FIGURE 4 shows that transfection of HuCCTl cells (cholangiocarcinoma) with mRNA encoding MG-008 (100 ng per well) resulted in cell death as determined, 72 h post-transfection, by phase contrast microscopy (panels A (Control), B (Lipofectamine MessengerMAX), and C (MG-008)) and by crystal violet stain (D), as described in Example 1;
FIGURE 5 shows that transfection of PC-3 cells (prostate carcinoma) with mRNA encoding MG- 008 (100 ng per well) resulted in cell death as determined, 72 h post-transfection, by phase contrast microscopy (panels A (Control), B (Lipofectamine MessengerMAX), and C (MG-008)) and by crystal violet stain (D), as described in Example 1; FIGURE 6 shows that transfection of RD cells (rhabdomyosarcoma) with mRNA encoding MG- 008 (100 ng per well) resulted in cell death as determined, 72 h post-transfection, by phase contrast microscopy (panels A (Control), B (Lipofectamine MessengerMAX), and C (MG-008)) and by crystal violet stain (D), as described in Example 1;
FIGURE 7 shows that transfection of SC-M1 cells (gastric carcinoma) with mRNA encoding MG- 008 (100 ng per well) resulted in cell death as determined, 72 h post-transfection, by phase contrast microscopy (panels A (Control), B (Lipofectamine MessengerMAX), and C (MG-008)) and by crystal violet stain (D), as described in Example 1;
FIGURE 8 shows that transfection of Vero cells (a non-cancerous cell line) with mRNA encoding MG-008 (100 ng per well) did not result in cell death as determined, 72 h post-transfection, by phase contrast microscopy (panels A (Control), B (Lipofectamine MessengerMAX), and C (MG- 008)) and by crystal violet stain (D), as described in Example 1;
FIGURE 9 shows that transfection of U-87MG cells (glioblastoma) with mRNA encoding MG- 008 (100 ng per well) resulted in cell death as determined, 72 h post-transfection, by phase contrast microscopy (panels A (Control), B (Lipofectamine MessengerMAX), and C (MG-008)) and by crystal violet stain (D), as described in Example 1;
FIGURE 10 shows that transfection of SW480 cells (colon adenocarcinoma) with mRNA encoding MG-008 (100 ng per well) resulted in cell death as determined, 72 h post-transfection, by phase contrast microscopy (panels A (Control), B (Lipofectamine MessengerMAX), and C (MG-008)) and by crystal violet stain (D), as described in Example 1;
FIGURE 11 shows that transfection of PANC-1 cells (pancreatic adenocarcinoma) with mRNA encoding MG-008 (100 ng per well) resulted in cell death as determined, 72 h post-transfection, by phase contrast microscopy (panels A (Control), B (Lipofectamine MessengerMAX), and C (MG-008)) and by crystal violet stain (D), as described in Example 1;
FIGURE 12 shows that transfection of OECM-1 cells (oral carcinoma) with mRNA encoding MG- 008 (100 ng per well) resulted in cell death as determined, 72 h post-transfection, by phase contrast microscopy (panels A (Control), B (Lipofectamine MessengerMAX), and C (MG-008)) and by crystal violet stain (D), as described in Example 1;
FIGURE 13 shows that transfection of OC2 cells (oral carcinoma) with mRNA encoding MG-008 (100 ng per well) resulted in cell death as determined, 72 h post-transfection, by phase contrast microscopy (panels A (Control), B (Lipofectamine MessengerMAX), and C (MG-008)) and by crystal violet stain (D), as described in Example 1;
FIGURE 14 shows that transfection of MIA PaCa-2 cells (pancreatic adenocarcinoma) with mRNA encoding MG-008 (100 ng per well) resulted in cell death as determined, 72 h posttransfection, by phase contrast microscopy (panels A (Control), B (Lipofectamine MessengerMAX), and C (MG-008)) and by crystal violet stain (D), as described in Example 1;
FIGURE 15 shows that transfection of MDA-MB-468 cells (breast adenocarcinoma) with mRNA encoding MG-008 (100 ng per well) resulted in cell death as determined, 72 h post-transfection, by phase contrast microscopy (panels A (Control), B (Lipofectamine MessengerMAX), and C (MG-008)) and by crystal violet stain (D), as described in Example 1;
FIGURE 16 shows that transfection of MDA-MB-231 cells (breast adenocarcinoma) with mRNA encoding MG-008 (100 ng per well) resulted in cell death as determined, 72 h post-transfection, by phase contrast microscopy (panels A (Control), B (Lipofectamine MessengerMAX), and C (MG-008)) and by crystal violet stain (D), as described in Example 1;
FIGURE 17 shows that transfection of MCF-7 cells (ductal carcinoma) with mRNA encoding MG- 008 (100 ng per well) resulted in cell death as determined, 72 h post-transfection, by phase contrast microscopy (panels A (Control), B (Lipofectamine MessengerMAX), and C (MG-008)) and by crystal violet stain (D), as described in Example 1;
FIGURE 18 shows that transfection of Mahlavu cells (hepatocellular carcinoma) with mRNA encoding MG-008 (100 ng per well) resulted in cell death as determined, 72 h post-transfection, by phase contrast microscopy (panels A (Control), B (Lipofectamine MessengerMAX), and C (MG-008)) and by crystal violet stain (D), as described in Example 1;
FIGURE 19 shows that transfection of HSC-3 cells (Squamous cell carcinoma of the oral tongue) with mRNA encoding MG-008 (100 ng per well) resulted in cell death as determined, 72 h posttransfection, by phase contrast microscopy (panels A (Control), B (Lipofectamine MessengerMAX), and C (MG-008)) and by crystal violet stain (D), as described in Example 1;
FIGURE 20 shows that transfection of HeLa cells (cervical carcinoma) with mRNA encoding MG- 008 (100 ng per well) resulted in cell death as determined, 72 h post-transfection, by phase contrast microscopy (panels A (Control), B (Lipofectamine MessengerMAX), and C (MG-008)) and by crystal violet stain (D), as described in Example 1;
FIGURE 21 shows that transfection of HCT116 cells (colorectal carcinoma) with mRNA encoding MG-008 (100 ng per well) resulted in cell death as determined, 72 h post-transfection, by phase contrast microscopy (panels A (Control), B (Lipofectamine MessengerMAX), and C (MG-008)) and by crystal violet stain (D), as described in Example 1;
FIGURE 22 shows that transfection of Vero cells (a non-cancerous cell line) with plasmid DNA encoding MG-008 or MG-008-Luc (100 ng per well) did not result in cell death as determined, 48h post-transfection, by phase contrast microscopy (panels A (Control), B (Lipofectamine 2000), C (+ MG-008), and D (+ MG-008-Luc) and by crystal violet stain (E), as described in Example 1;
FIGURE 23 shows that transfection of U-87MG cells (glioblastoma) with plasmid DNA encoding MG-008 or MG-008-Luc (100 ng per well) resulted in cell death as determined, 48 h posttransfection, by phase contrast microscopy (panels A (Control), B (Lipofectamine 2000), C (+ MG-008), and D (+ MG-008-Luc)) and by crystal violet stain (E), as described in Example 1;
FIGURE 24 shows that transfection of SW480 cells (colon adenocarcinoma) with plasmid DNA encoding MG-008 or MG-008-Luc (100 ng per well) resulted in cell death as determined, 48 h post-transfection, by phase contrast microscopy (panels A (Control), B (Lipofectamine 2000), C (+ MG-008), and D (+ MG-008-Luc)) and by crystal violet stain (E), as described in Example 1;
FIGURE 25 shows that transfection of SC-M1 cells (gastric carcinoma) with plasmid DNA encoding MG-008 or MG-008-Luc (100 ng per well) resulted in cell death as determined, 72 h post-transfection, by phase contrast microscopy (panels A (Control), B (Lipofectamine 2000), C (+ MG-008), and D (+ MG-008-Luc)) and by crystal violet stain (E), as described in Example 1; FIGURE 26 shows that transfection of PC-3 cells (prostate carcinoma) with plasmid DNA encoding MG-008 or MG-008-Luc (100 ng per well) resulted in cell death as determined, 72 h post-transfection, by phase contrast microscopy (panels A (Control), B (Lipofectamine 2000), C (+ MG-008), and D (+ MG-008-Luc)) and by crystal violet stain (E), as described in Example 1.
FIGURE 27 shows that transfection of PANC-1 cells (pancreatic adenocarcinoma) with plasmid DNA encoding MG-008 or MG-008-Luc (100 ng per well) resulted in cell death as determined, 48 h post-transfection, by phase contrast microscopy (panels A (Control), B (Lipofectamine 2000), C (+ MG-008), and D (+ MG-008-Luc)) and by crystal violet stain (E), as described in Example 1;
FIGURE 28 shows that transfection of MIA PaCa-2 cells (pancreatic adenocarcinoma) with plasmid DNA encoding MG-008 or MG-008-Luc (100 ng per well) resulted in cell death as determined, 48 h post-transfection, by phase contrast microscopy (panels A (Control), B (Lipofectamine 2000), C (+ MG-008), and D (+ MG-008-Luc)) and by crystal violet stain (E), as described in Example 1;
FIGURE 29 shows that transfection of MDA-MB-468 cells (breast adenocarcinoma) with plasmid DNA encoding MG-008 or MG-008-Luc (100 ng per well) resulted in cell death as determined, 48 h post-transfection, by phase contrast microscopy (panels A (Control), B (Lipofectamine 2000), C (+ MG-008), and D (+ MG-008-Luc)) and by crystal violet stain (E), as described in Example 1;
FIGURE 30 shows that transfection of MDA-MB-231 cells (breast adenocarcinoma) with plasmid DNA encoding MG-008 or MG-008-Luc (100 ng per well) resulted in cell death as determined, 48 h post-transfection, by phase contrast microscopy (panels A (Control), B (Lipofectamine 2000), C (+ MG-008), and D (+ MG-008-Luc)) and by crystal violet stain (E), as described in Example 1;
FIGURE 31 shows that transfection of MCF-7 cells (ductal carcinoma) with plasmid DNA encoding MG-008 or MG-008-Luc (100 ng per well) resulted in cell death as determined, 48 h post-transfection, by phase contrast microscopy (panels A (Control), B (Lipofectamine 2000), C (+ MG-008), and D (+ MG-008-Luc)) and by crystal violet stain (E), as described in Example 1; FIGURE 32 shows that transfection of Mahlavu cells (hepatocellular carcinoma) with plasmid DNA encoding MG-008 or MG-008-Luc (100 ng per well) resulted in cell death as determined, 48 h post-transfection, by phase contrast microscopy (panels A (Control), B (Lipofectamine 2000), C (+ MG-008), and D (+ MG-008-Luc)) and by crystal violet stain (E), as described in Example 1;
FIGURE 33 shows that transfection of HeLa cells (cervical carcinoma) with plasmid DNA encoding MG-008 or MG-008-Luc (100 ng per well) resulted in cell death as determined, 48 h post-transfection, by phase contrast microscopy (panels A (Control), B (Lipofectamine 2000), C (+ MG-008), and D (+ MG-008-Luc)) and by crystal violet stain (E), as described in Example 1;
FIGURE 34 shows that transfection of HCT116 cells (colorectal carcinoma) with plasmid DNA encoding MG-008 or MG-008-Luc (100 ng per well) resulted in cell death as determined, 72 h post-transfection, by phase contrast microscopy (panels A (Control), B (Lipofectamine 2000), C (+ MG-008), and D (+ MG-008-Luc)) and by crystal violet stain (E), as described in Example 1;
FIGURE 35 shows that transfection of A549 cells (lung adenocarcinoma) with plasmid DNA encoding MG-008 or MG-008-Luc (100 ng per well) resulted in cell death as determined, 72 h post-transfection, by phase contrast microscopy (panels A (Control), B (Lipofectamine 2000), C (+ MG-008), and D (+ MG-008-Luc)) and by crystal violet stain (E), as described in Example 1; and
FIGURE 36 shows certain amino acid sequences and nucleic acid sequences as described herein.
FIGURE 37 shows a female Balb/c mouse (7 weeks old) that was injected, into the left 4th mammary inguinal gland, with 1 x 1064T1-Fluc-Neo cells (a mouse mammary carcinoma cell line expressing luciferase). One week later, the mouse was given a single intravenous (IV) dose of Fl 1 at 0.05 mpk (in 100 pl PBS). The untreated control mouse was injected with PBS alone. Subsequently, the |in vivo imaging system (IVIS)j[Ai] images of bioluminescence derived from the 4T1 tumor cells were documented weekly for the next consecutive 7 weeks.
FIGURE 38 shows the quantification of total luciferin flux obtained from the experiment in FIG. 37. Metastasis was observed in the untreated control mouse at day 30 (FIG. 38A, left panel), and the mice died at day 37. In mouse injected with Fl 1, no metastasis was observed throughout the entire 7 weeks of the experiment (FIG. 38B, right panel).
FIGURE 39 shows a female Balb/c mouse that was injected into the left 4th mammary inguinal gland with 4T1 tumor cells as described in FIG. 37. One week later, the mouse was given a single dose of Fl l intravenously (IV), and the control mouse was injected with PBS alone. Morphological changes at the 4T1 injection site were monitored. In control mouse, tumor lump was palpable at day 14 post-IV treatment, and it grew further until the mouse died at day 35 (images in the upper panel). In the Fl 1 treatment mouse, the tumor lump was undetectable until day 35 post-IV treatment (images in the lower panel).
FIGURE 40 shows the bodyweight changes in Balb/c mice after being injected with 4T1 tumor cells (as described in above for FIG. 37) and a single dose of either Fl l or PBS (control) given one week later (day 0) intravenously (IV). Body weight was measured until the end of the experiment (total of 7 weeks post-IV treatment). The bodyweight of the PBS control mouse did not change much before it died at day 35 (blue line). The Fl l treated mouse showed bodyweight gain throughout the entire experiment (orange line).
DETAILED DESCRIPTION
Described herein are compounds, compositions, uses, and methods for reducing cell viability of a cancer cell in vitro or in vivo, and/or for preventing or treating cancer in a cell or subject in need thereof. It will be appreciated that embodiments and examples are provided for illustrative purposes intended for those skilled in the art and are not meant to be limiting in any way.
Cancer is a major ongoing health concern, leading to many deaths annually. Cancer includes a large group of diseases having a wide range of underlying causes. Each cancer may differ in biology and pathophysiology, but virtually all cancers are believed to be associated with membrane trafficking dysregulation (Parachoniak CA, Park M. Dynamics of receptor trafficking in tumorigenicity. Trends Cell Biol. 2012 May;22(5):231-40). In cancer initiation and progression, mitochondria and lysosomes have an important role because of their relevance in energy homeostasis and cell death (Anderson RG, Ghiraldeli LP, Pardee TS. Mitochondria in cancer metabolism, an organelle whose time has come? Biochim Biophys Acta Rev Cancer. 2018 Aug;1870(l):96-102). Indeed, functional dysregulation of lysosomes has been found to play important roles in human cancer tumorigenesis and its progression (Davidson SM, Vander Heiden MG. Critical Functions of the Lysosome in Cancer Biology. Annu Rev Pharmacol Toxicol. 2017 Jan 6;57:481-507).
Cancer development and progression induces fundamental changes in lysosomal function, which in turn imposes profound effects on tumor invasion, metastasis, and in its sensitivity to anticancer treatments. Rapidly dividing cells, such as cancer cells, are highly dependent on lysosomal function, and dramatic changes in lysosomal volume, composition, and subcellular localization occur during transformation and cancer progression (Zhitomirsky B, Assaraf YG. Lysosomes as mediators of drug resistance in cancer. Drug Resist Updat. 2016 Jan;24:23-33). For example, Cathepsins, which are lysosomal proteases, have important roles in cancer progression. Activity of cathepsins outside of the cell is linked to tumor growth and invasion, whereas such activity inside the cell is linked to tumor growth inhibition (Rudzinska M, Parodi A, Soond SM, Vinarov AZ, Korolev DO, Morozov AO, Daglioglu C, Tutar Y, Zamyatnin AA Jr. The Role of Cysteine Cathepsins in Cancer Progression and Drug Resistance. Int J Mol Sci. 2019 Jul 23;20(14):3602).
Autophagy is a major intracellular degradation system that derives its degradative abilities from the lysosome. It is a homeostatic and evolutionarily conserved mechanism of self-digestion by which the cells degrade and recycle long-lived proteins and excess or damaged organelles. Autophagy is a highly regulated process by which misfolded proteins and organelles reach lysosomes for their degradation (Kim KH, Lee MS. Autophagy— a key player in cellular and body metabolism. Nat Rev En-docrinol. 2014 Jun;10(6):322-37). There are 3 different types of autophagy, namely macroautophagy (commonly known as autophagy), chaperon mediated autophagy, and microautophagy. Lysosomal degradation of substrates in chaperon mediate autophagy and microautophagy occurs directly within the lysosomes (Parzych KR, Klionsky DJ. An overview of autophagy: morphology, mechanism, and regulation. Antioxid Redox Signal. 2014 Jan 20;20(3):460-73). The first evidence of a suppressive role of autophagy in cancer was the observation that heterozygous loss of the Beclinl encoding gene (Becnl) associated with chromosomal instability in breast, ovarian and prostate cancer. During macroautophagy, the cancer cell's cytoplasm, proteins, and organelles are surrounded by double-walled membranes known as autophagosome vesicles. Subsequently, the autophagosome vesicles are fused with lysosomes to form autophagolysosomes, the contents of which are then degraded by lysosomal enzymes (Gong C, Bauvy C, Tonelli G, Yue W, Delomenie C, Nicolas V, Zhu Y, Domergue V, Marin-Esteban V, Tharinger H, Delbos L, Gary-Gouy H, Morel AP, Ghavami S, Song E, Codogno P, Mehrpour M. Beclin 1 and autophagy are required for the turn origeni city of breast cancer stem-like/progenitor cells. Oncogene. 2013 May 2;32(18):2261-72, 2272e.l-l 1). In addition, macroautophagy has been shown to activate and mediate senescence in cultured human lung fibroblast cells and primary biliary cirrhosis by inducing cell cycle arrest in transformed cells to prevent tumorigenesis (Mathiassen SG, De Zio D, Cecconi F. Autophagy and the Cell Cycle: A Complex Landscape. Front Oncol. 2017 Mar 31;7:51).
Under certain conditions, macroautophagy may work with mitotic catastrophe to get rid of cancer cells that escape apoptosis (Simon HU, Friis R. ATG5: a distinct role in the nucleus. Autophagy. 2014 Jan; 10(1): 176-7). As a tumor suppression mechanism, macroautophagy also maintains genome stability, induces senescence and possibly autophagic cell death (Gozuacik D, Kimchi A. Autophagy as a cell death and tumor suppressor mechanism. Oncogene. 2004 Apr 12;23 (16) :2891 - 906). Besides autophagic cells death, tumor suppressor mechanism of macroautophagy may include some or all of the following aspects: inhibiting abnormal cells proliferation, inhibiting chronic necrosis of cancer cells, and reducing the DNA damage (Pathania AS, Guru SK, Kumar S, Kumar A, Ahmad M, Bhushan S, Sharma PR, Mahajan P, Shah BA, Sharma S, Nargotra A, Vishwakarma R, Korkaya H, Malik F. Interplay between cell cycle and autophagy induced by boswellic acid analog. Sci Rep. 2016 Sep 29;6:33146).
Cell death may include or involve different factors such as change in morphology of the cells or their functions. There are three major types of cell death: apoptosis (programmed cell death type 1), autophagy (programmed cell death type 2), and necroptosis (programmed cell death type 3) (Fuchs Y, Steller H. Programmed cell death in animal development and disease. Cell. 2011 Nov 11;147(4):742-58). In general, autophagy functions in cell-protective events. However, it may also be utilized as a cell-suicide mechanism, which is known as “autophagic cell death”. The process of autophagic cell death thus differs from apoptotic or necroptotic programmed cell death (Kroemer G, Levine B. Autophagic cell death: the story of a misnomer. Nat Rev Mol Cell Biol. 2008 Dec;9(12): 1004-10). In addition, autophagic cell death is the major mechanism that is associated with tumor suppression effect of macroautophagy. In this regard, myeloma cells may avoid cell death by restricting the autophagic activity through the cleavage of autophagic inducer, BCL2 -interacting protein BCLAF1, by caspase- 10 (Rosebeck S, Alonge MM, Kandarpa M, Mayampurath A, Volchenboum SL, Jasielec J, Dytfeld D, Maxwell SP, Kraftson SJ, McCauley D, Shacham S, Kauffman M, Jakubowiak AJ. Synergistic Myeloma Cell Death via Novel Intracellular Activation of Caspase- 10-Dependent Apoptosis by Carfilzomib and Selinexor. Mol Cancer Ther. 2016 Jan;15(l):60-71). Autophagic cell death is frequently induced in cells that lack apoptotic machinery, such as p53-deficient cancer cells (Scherz-Shouval R, Weidberg H, Gonen C, Wilder S, Elazar Z, Oren M. p53-dependent regulation of autophagy protein LC3 supports cancer cell survival under prolonged starvation. Proc Natl Acad Sci U S A. 2010 Oct 26;107(43): 18511-6).
Reactive oxygen species (ROS) have been shown that may play an important role in autophagic cell death. Excessive ROS production promotes cytochrome c to be released from mitochondria into the cytoplasm and triggers programmed cell death (Filomeni G, De Zio D, Cecconi F. Oxidative stress and autophagy: the clash between damage and metabolic needs. Cell Death Differ. 2015 Mar;22(3):377-88). An elevated basal level of ROS has been observed in cancer cells, and the high endogenous ROS levels in cancer cells make them vulnerable and susceptible to the ROS- induced cell death (Liou GY, Storz P. Reactive oxygen species in cancer. Free Radic Res. 2010 May;44(5):479-96). Although the reasons for elevated ROS production in cancer cells are not fully understood, it is generally believed that it most likely associated with augmented bioenergetics in the cells. Because proliferating cancer cells require tremendous amount of energy (i.e. ATP), the bioenergetic process is massively upregulated to sustain cell proliferation, cell differentiation, and cell migrations (i.e. metastasis). Unfortunately, ROS is an inevitable byproduct of the bioenergetic process, which is most produced in the mitochondria during the ATP synthesis process known as oxidative phosphorylation. Reactive oxygen species (ROS) generally include chemically reactive molecules containing oxygen, which may be produced as a result of cellular metabolism. Reactive oxygen species (ROS) includes a group of ions and molecules, such as hydroxyl radicals ( OH), alkoxyl radicals, superoxide anion (02 -), singlet oxygen (102) and hydrogen peroxide (H2O2) (Auten RL, Davis JM. Oxygen toxicity and reactive oxygen species: the devil is in the details. Pediatr Res. 2009 Aug;66(2): 121-7). Many anticancer therapies have been developed based on the ability of ROS to kill cancer cells. This includes, for example, Methoxyestradiol, Buthionine sulfoximine, Imexon, Cisplatin, Doxorubicin, Motexafin gadolinium, tert-Butylhydroquinone, Seleno-compounds, among others. (Perillo B, Di Donato M, Pezone A, Di Zazzo E, Giovannelli P, Galasso G, Castoria G, Migliaccio A. ROS in cancer therapy: the bright side of the moon. Exp Mol Med. 2020 Feb;52(2): 192-203). The elevated level of ROS in cancer cells results from an increased ROS production or a decreased ROS elimination, or both. In either case, endolysosomal activities are intricately involved in the maintenance of cellular ROS homeostasis, especially in cancer settings.
Tumors often arise from sites of chronic irritation, infection, or inflammation; virtually all cancer cells are believed to stem from cells that are functionally defective accompanied with metabolic reprogramming, as well as elevated baseline ROS production. At cell biology levels, alterations in cellar macroautophagic process(es) have been observed in several cancers. Macroautophagy regulatory machinery also declines with age and environmental/nutritional stress, leading to more dysfunctional or cancerous cells (Martinez -Lopez N, Athonvarangkul D, Singh R. Autophagy and aging. Adv Exp Med Biol. 2015;847:73-87). Cancer cells often have much lowered activity of basal macroautophagy than normal cells. Many of the oncogenes and tumor-suppressor genes are closely associated with macroautophagy. For instance, the well-known PTEN tumor-suppressor gene blocks PI3K/Akt thus activating macroautophagy. Mutations to PTEN, found very commonly in cancers, are associated with lowered levels of macroautophagy and increased risk of cancer (Tan MH, Mester JL, Ngeow J, Rybicki LA, Orloff MS, Eng C. Lifetime cancer risks in individuals with germline PTEN mutations. Clin Cancer Res. 2012 Jan 15;18(2):400-7). Interestingly, many anticancer agents have been reported to induce macroautophagy as well. Some anticancer agents that can induce macroautophagy are: tamoxifen, rapamycin, arsenic trioxide, temozolomide, histone deacetylase inhibitors, ionizing radiation, vitamin D analogues, and etoposide (Liu EY, Xu N, O'Prey J, Lao LY, Joshi S, Long JS, O'Prey M, Croft DR, Beaumatin F, Baudot AD, Mrschtik M, Rosenfeldt M, Zhang Y, Gillespie DA, Ryan KM. Loss of autophagy causes a synthetic lethal deficiency in DNA repair. Proc Natl Acad Sci U S A. 2015 Jan 20;l 12(3):773-8).
In this regard, the present inventors recognized that in addition to apoptosis (programmed cell death) and necroptosis, autophagic lysosome-mediated cell death, particularly the process of microautophagy, may be used for developing an effective strategy for anticancer therapy.
Stimulation of microautophagy (which in certain embodiments may involve piecemeal engulfment of target membrane(s)) by promoting lysosome movement toward the target membranes) may be particularly desirable for treatment of a variety of diseases and/or disorders and/or conditions. Specific lysosome positioning inside the cells may be associated with different types of lysosome activity. In addition, lysosome positioning may correlate with the activity of mTOR and may regulate autophagic flux. During macroautophagy, mTORCl may be inactive and lysosomes may be accumulated in the perinuclear region of the cells, which may perform macroautophagy by stimulation of fusion of encased target membrane with lysosomes. In contrast, in the process of microautophagy, piecemeal engulfment of target membrane(s) by lysosomes may be happening through lysosome movement and direct interaction with target membrane(s) without need of formation of autophagosome(s). Lysosomes may move in a bidirectional manner along the microtubules, and such a lysosomal motility is governed by different sets of motor proteins that are recruited through different mechanisms.
In certain embodiments, microautophagy may involve direct engulfment of cytoplasmic cargo at a boundary membrane by autophagic tubes, which may mediate both invagination and vesicle scission into the lumen of lysosomes (see Li, W.-W., Li, J. & Bao, J.-K. Microautophagy: lesser- known self-eating. Cell. Mol. Life Sci. 69, 1125-1136 (2011)). Direct lysosomal degradation of target substrates may occur for DNA, for example (referred to as a piecemeal autophagy - see Fujiwara, Y. et al. Direct uptake and degradation of DNA by lysosomes. - PubMed - NCBI. Autophagy 9, 1167-1171 (2014)). In addition, lysosomes may be able to move toward the different organelles and/or membranes substrates (e.g. plasma membrane, mitochondria) and may interact with them directly by recruitment of motor proteins and SNAREs proteins (see Andrews, N. W. Lysosomes and the plasma membrane. J. Cell Biol. 158, 389-394 (2002); Hofmann, I. & Munro, S. An N-terminally acetylated Arf-like GTPase is localised to lysosomes and affects their motility. J. Cell. Sci. 119, 1494-1503 (2006); Fraldi, A. et al. Lysosomal fusion and SNARE function are impaired by cholesterol accumulation in lysosomal storage disorders. The EMBO Journal 29, 3607-3620 (2010); and Pankiv, S. et al. FYCO1 is a Rab7 effector that binds to LC3 and PI3P to mediate microtubule plus end-directed vesicle transport. J. Cell Biol. 188, 253-269 (2010)). Movement of lysosomes toward the cell periphery and their positioning within the cells may be associated with signaling (see R. & Bonifacino, J. S. Lysosome Positioning Influences mT0RC2 and AKT Signaling. Molecular Cell 75, 26-38. e3 (2019)). In this regard, it is contemplated that mTORCl, mTORC2 and AKT activation may be important for lysosomal peripheral distribution (see Poiis, C. & Codogno, P. Lysosome positioning coordinates mTORCl activity and autophagy. Nature Cell Biology 13, 342-344 (2011); and Cabukusta, B. & Neefjes, J. Mechanisms of lysosomal positioning and movement. Traffic 19, 761-769 (2018)).
In certain embodiments, microautophagy and/or piecemeal degradation of target substrates and/or membranes may involve movement of lysosomes toward the peripheral and target membrane in cytosol (e.g. direct interaction with target membrane) (see Pu, J., Guardia, C. M., Keren-Kaplan, T. & Bonifacino, J. S. Mechanisms and functions of lysosome positioning. J. Cell. Sci. 129, 4329- 4339 (2016); and Katherine R Parzych, D. J. K. An Overview of Autophagy: Morphology, Mechanism, and Regulation. Antioxid. Redox Signal. 20, 460-473 (2014)). In certain embodiments, movement of lysosomes toward the cell periphery (from perinuclear region of the cell) and interaction with target membranes/ substrates (e.g. glycogen, lipid, protein) in cell periphery may be associated with the activation of mTORCl/mTORC2 (see Rabanal-Ruiz, Y. & Korol chuk, V. I. mTORCl and Nutrient Homeostasis: The Central Role of the Lysosome. Int J Mol Sci 19, 818 (2018); Jia, R. & Bonifacino, J. S. Lysosome Positioning Influences mTORC2 and AKT Signaling. Molecular Cell 75, 26-38. e3 (2019)).
Without wishing to be bound by theory, it is contemplated that Rabi a DN (dominant negative form of Rabi a) may simulate lysosome peripheral distribution (from perinuclear region) through activating mTORCl/mTORC2 proteins inside the cells without the need of external (or extracellular) signal for their activation (to support lysosome peripheral distribution).
The use of Rab 1 aGDP or Rab 1 aGTP in reducing cancer cell viability and/or causing cancer cell death: As described in detail herein, it has now been identified that treatment with a microautophagyenhancing agent comprising a GDP -bound form of Rabi a, such as RablaS25N, RablaN124I(a mouse Rabi sequence), RablaD41N, RablaD47N, or another dominant negative (DN) GDP -bound form of Rabi a, or one or more expressible nucleic acids encoding such a RablaGDP, may be used to decrease cell viability and/or cause cell death in a wide variety of different cancer cell types.
As will be understood, in certain embodiments, methods as described herein may be in vitro methods, in vivo methods, or both.
In an embodiment, there is provided herein a method for reducing cell viability of a cancer cell, or for preventing or treating cancer, in a subject in need thereof, said method comprising: treating a cancer cell of the subject with a GDP -bound form of Rabi a (RablaGDP), one or more expressible nucleic acids encoding RablaGDP, or a combination thereof; thereby increasing a cellular level of RablaGDP in the cancer cell, resulting in decreased cell viability or death of the cancer cell.
In another embodiment, there is provided herein a method for reducing cell viability of a cancer cell in vitro or in vivo, said method comprising: treating the cancer cell with a GDP -bound form of Rabi a (RablaGDP), one or more expressible nucleic acids encoding RablaGDP, or a combination thereof; thereby increasing a cellular level of RablaGDP in the cancer cell, resulting in decreased cell viability or death of the cancer cell.
In another embodiment of any of the above method or methods, the RablaGDP may be or may comprise RablaS25N, RablaN1241, RablaD41N, RablaD47N, or another dominant negative (DN) GDP- bound form of Rabi a.
In still another embodiment of any of the above method or methods, the RablaGDP may comprise the amino acid sequence: MSSMNPEYDYLFKLLLIGDSGVGKNCLLLRFADDTYTESYISTIGVDFKIRTIELDGK
TIKLQIWDTAGQERFRTITSSYYRGAHGIIVVYDVTDQESFNNVKQWLQEIDRYASE
NVNKLLVGNKCDLTTKKVVDYTTAKEFADSLGIPFLETSAKNATNVEQSFMTMAAE IKKRMGPGATAGGAEKSNVKIQSTPVKQSGGGCC
(SEQ ID NO: 6; Human RablaS25N);
MGDYKDDDDKGGSGGSSMNPEYDYLFKLLLIGDSGVGKSCLLLRFADDTYTESYIS
TIGVDFKIRTIELDGKTIKLQIWDTAGQERFRTITSSYYRGAHGIIVVYDVTDQESFNN
VKQWLQEIDRYASENVNKLLVGIKCDLTTKKVVDYTTAKEFADSLGIPFLETSAKNA
TNVEQSFMTMAAEIKKRMGPGATAGGAEKSNVKIQSTPVKQSGGGCC
(SEQ ID NO: 9; Mouse RablN1241); the amino acid sequence of human RablaD41N; or
MSSMNPEYDYLFKLLLIGDSGVGKSCLLLRFADDTYTESYISTIGVNFKIRTIELDGKT
IKLQIWDTAGQERFRTITSSYYRGAHGIIVVYDVTDQESFNNVKQWLQEIDRYASEN VNKLLVGNKCDLTTKKVVDYTTAKEFADSLGIPFLETSAKNATNVEQSFMTMAAEI KKRMGPGATAGGAEKSNVKIQSTPVKQSGGGCC
(SEQ ID NO: 18; Human RablaD47N); or a polypeptide having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with any of these sequences and preferentially binding GDP.
In yet another embodiment of any of the above method or methods, the RablaGDP may consist of the amino acid sequence:
MSSMNPEYDYLFKLLLIGDSGVGKNCLLLRFADDTYTESYISTIGVDFKIRTIELDGK
TIKLQIWDTAGQERFRTITSSYYRGAHGIIVVYDVTDQESFNNVKQWLQEIDRYASE
NVNKLLVGNKCDLTTKKVVDYTTAKEFADSLGIPFLETSAKNATNVEQSFMTMAAE IKKRMGPGATAGGAEKSNVKIQSTPVKQSGGGCC
(SEQ ID NO: 6; Human RablaS25N); MGDYKDDDDKGGSGGSSMNPEYDYLFKLLLIGDSGVGKSCLLLRFADDTYTESYIS
TIGVDFKIRTIELDGKTIKLQIWDTAGQERFRTITSSYYRGAHGIIVVYDVTDQESFNN
VKQWLQEIDRYASENVNKLLVGIKCDLTTKKVVDYTTAKEFADSLGIPFLETSAKNA
TNVEQSFMTMAAEIKKRMGPGATAGGAEKSNVKIQSTPVKQSGGGCC
(SEQ ID NO: 9; Mouse RablN1241); the amino acid sequence of human RablaD41N; or
MSSMNPEYDYLFKLLLIGDSGVGKSCLLLRFADDTYTESYISTIGVNFKIRTIELDGKT
IKLQIWDTAGQERFRTITSSYYRGAHGIIVVYDVTDQESFNNVKQWLQEIDRYASEN VNKLLVGNKCDLTTKKVVDYTTAKEFADSLGIPFLETSAKNATNVEQSFMTMAAEI KKRMGPGATAGGAEKSNVKIQSTPVKQSGGGCC
(SEQ ID NO: 18; Human RablaD47N); or a polypeptide having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with any of these sequences and preferentially binding GDP.
In another embodiment of any of the above method or methods, the RablaGDP may comprise or consist of the amino acid sequence:
MSSMNPEYDYLFKLLLIGDSGVGKNCLLLRFADDTYTESYISTIGVDFKIRTIELDGK
TIKLQIWDTAGQERFRTITSSYYRGAHGIIVVYDVTDQESFNNVKQWLQEIDRYASE
NVNKLLVGNKCDLTTKKVVDYTTAKEFADSLGIPFLETSAKNATNVEQSFMTMAAE IKKRMGPGATAGGAEKSNVKIQSTPVKQSGGGCC
(SEQ ID NO: 6; Human RablaS25N); or a polypeptide having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity therewith and preferentially binding GDP.
In still another embodiment of any of the above method or methods, the RablaGDP may be in the form of a fusion protein, wherein the RablaGDP is fused or otherwise directly or indirectly linked, optionally via a linker, with a signaling or targeting peptide, a fluorescent peptide or other marker or tracer, or another peptide or non-peptide moiety for targeted delivery, facilitating cell uptake, increasing stability or in vivo half-life, or improving another therapeutic, diagnostic, or in vivo property of the RablaGDP. In an embodiment that is not meant to be limiting in any manner, the fusion protein can comprise any protein or tag, for example, GFP, YFP, mCherry, luciferase specific antibody, aptamer for identification or delivery to a specific organ or the like.
In yet another embodiment of any of the above method or methods, the fusion protein may comprise the amino acid sequence:
MSSMNPEYDYLFKLLLIGDSGVGKNCLLLRFADDTYTESYISTIGVDFKIRTIELDGK
TIKLQIWDTAGQERFRTITSSYYRGAHGIIVVYDVTDQESFNNVKQWLQEIDRYASE NVNKLLVGNKCDLTTKKVVDYTTAKEFADSLGIPFLETSAKNATNVEQSFMTMAAE IKKRMGPGATAGGAEKSNVKIQSTPVKQSGGGCC
(SEQ ID NO: 6; Human RablaS25N); or a polypeptide having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity therewith and preferentially binding GDP.
In another embodiment of any of the above method or methods, the RablaGDP may be in the form of a fusion protein, and may comprise the amino acid sequence:
MEDAKNIKKGPAPFYPLEDGTAGEQLHKAMKRYALVPGTIAFTDAHIEVNITYAEYF EMSVRLAEAMKRYGLNTNHRIVVCSENSLQFFMPVLGALFIGVAVAPANDIYNERE LLNSMNISQPTVVFVSKKGLQKILNVQKKLPIIQKIIIMDSKTDYQGFQSMYTFVTSHL PPGFNEYDF VPESFDRDKTIALIMNS SGSTGLPKGVALPHRT ACVRF SHARDPIFGNQI IPDTAILSVVPFHHGFGMFTTLGYLICGFRVVLMYRFEEELFLRSLQDYKIQSALLVPT LFSFFAKSTLIDKYDLSNLHEIASGGAPLSKEVGEAVAKRFHLPGIRQGYGLTETTSAI LITPEGDDKPGAVGKVVPFFEAKVVDLDTGKTLGVNQRGELCVRGPMIMSGYVNNP EATNALIDKDGWLHSGDIAYWDEDEHFFIVDRLKSLIKYKGYQVAPAELESILLQHP
NIFDAGVAGLPDDDAGELPAAVVVLEHGKTMTEKEIVDYVASQVTTAKKLRGGVV FVDEVPKGLTGKLDARKIREILIKAKKGGKSKLMSSMNPEYDYLFKLLLIGDSGVGK NCLLLRFADDTYTESYISTIGVDFKIRTIELDGKTIKLQIWDTAGQERFRTITSSYYRG
AHGIIVVYDVTDQESFNNVKQWLQEIDRYASENVNKLLVGNKCDLTTKKVVDYTT AKEFADSLGIPFLETSAKNATNVEQSFMTMAAEIKKRMGPGATAGGAEKSNVKIQST PVKQSGGGCC
(SEQ ID NO: 21); or a polypeptide having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity therewith and preferentially binding GDP.
In still another embodiment of any of the above method or methods, the one or more expressible nucleic acids may encode any one or more of the RablaGDPs as defined herein.
In yet another embodiment of any of the above method or methods, the one or more expressible nucleic acids may be DNA-based, or RNA-based.
In another embodiment of any of the above method or methods, the one or more expressible nucleic acids may transiently express the RablaGDP in the cell, or wherein the one or more expressible nucleic acids may integrate in the cell genome and express the RablaGDP in the cell.
In still another embodiment of any of the above method or methods, the one or more expressible nucleic acids may comprise one or more expression vectors, plasmids, or mRNAs encoding and capable of expressing the RablaGDP inside the cell.
In yet another embodiment of any of the above method or methods, the one or more expressible nucleic acids may comprise a nucleic acid sequence of:
ATGTCCAGCATGAATCCCGAATATGATTATTTATTCAAGTTACTTCTGATTGGCG
ACTCAGGGGTTGGAAAGAATTGCCTTCTTCTTAGGTTTGCAGATGATACATATAC
AGAAAGCTACATCAGCACAATTGGTGTGGATTTCAAAATAAGAACTATAGAGTT
AGACGGGAAAACAATCAAGCTTCAAATATGGGACACAGCAGGCCAGGAAAGAT TTCGAACAATCACCTCCAGTTATTACAGAGGAGCCCATGGCATCATAGTTGTGTA TGATGTGACAGATCAGGAGTCCTTCAATAATGTTAAACAGTGGCTGCAGGAAAT AGATCGTTATGCCAGTGAAAATGTCAACAAATTGTTGGTAGGGAACAAATGTGA
TCTGACCACAAAGAAAGTAGTAGACTACACAACAGCGAAGGAATTTGCTGATTC
CCTTGGAATTCCGTTTTTGGAAACCAGTGCTAAGAATGCAACGAATGTAGAACA
GTCTTTCATGACGATGGCAGCTGAGATTAAAAAGCGAATGGGTCCCGGAGCAAC
AGCTGGTGGTGCTGAGAAGTCCAATGTTAAAATTCAGAGCACTCCAGTCAAGCA GTCAGGTGGAGGTTGCTGCTAA
(Human RablaS25N ORF Codon Sequence, SEQ ID NO: 5);
ATGGGGGACTACAAGGACGACGATGACAAGGGGGGTAGCGGTGGATCCAGCAT
GAATCCCGAATATGATTATTTATTCAAGTTACTTCTGATTGGCGATTCTGGGGTT
GGAAAGTCCTGCCTTCTCCTTAGGTTTGCAGATGATACGTATACGGAAAGCTACA
TCAGCACAATTGGTGTGGATTTCAAGATACGAACTATAGAGTTAGATGGGAAAA
CAATCAAGCTACAGATATGGGACACAGCAGGCCAGGAAAGATTTCGAACAATCA
CTTCCAGTTATTACAGAGGAGCCCATGGCATCATAGTTGTGTATGATGTGACAGA
TCAGGAGTCCTTCAATAACGTTAAACAGTGGCTGCAGGAGATAGATCGCTACGC
CAGTGAAAATGTCAACAAGTTGTTGGTAGGGATCAAATGTGACCTGACCACAAA
GAAAGTAGTAGACTACACAACAGCAAAGGAATTTGCAGATTCCCTTGGAATTCC
ATTTTTGGAAACCAGTGCTAAGAACGCAACGAATGTAGAACAGTCTTTCATGAC
GATGGCAGCTGAGATTAAAAAGCGAATGGGTCCTGGAGCTACAGCTGGTGGTGC
CGAGAAGTCCAATGTTAAAATCCAGAGCACTCCAGTCAAGCAGTCAGGTGGAGG CTGCTGCTAA
(Mouse RablaN1241 ORF Codon Seqeunce, SEQ ID NO: 8); or
ATGTCCAGCATGAATCCCGAATATGATTATTTATTCAAGTTACTTCTGATTGGCGACT
CAGGGGTTGGAAAGTCTTGCCTTCTTCTTAGGTTTGCAGATGATACATATACAGAAA
GCTACATCAGCACAATTGGTGTGAACTTCAAAATAAGAACTATAGAGTTAGACGGG
AAAACAATCAAGCTTCAAATATGGGACACAGCAGGCCAGGAAAGATTTCGAACAAT
CACCTCCAGTTATTACAGAGGAGCCCATGGCATCATAGTTGTGTATGATGTGACAGA TCAGGAGTCCTTCAATAATGTTAAACAGTGGCTGCAGGAAATAGATCGTTATGCCAG TGAAAATGTCAACAAATTGTTGGTAGGGAACAAATGTGATCTGACCACAAAGAAAG TAGTAGACTACACAACAGCGAAGGAATTTGCTGATTCCCTTGGAATTCCGTTTTTGG AAACCAGTGCTAAGAATGCAACGAATGTAGAACAGTCTTTCATGACGATGGCAGCT GAGATTAAAAAGCGAATGGGTCCCGGAGCAACAGCTGGTGGTGCTGAGAAGTCCAA TGTTAAAATTCAGAGCACTCCAGTCAAGCAGTCAGGTGGAGGTTGCTGCTAA
(Human RablaD47N ORF Codon Sequence, SEQ ID NO: 17); or a nucleic acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity therewith and encoding a RablaGDP preferentially binding GDP; or a nucleic acid sequence equivalent to any of the above sequences due to codon redundancy.
In another embodiment of any of the above method or methods, the one or more expressible nucleic acids may comprise a nucleic acid sequence of:
ATGGAAGACGCCAAAAACATAAAGAAAGGCCCGGCGCCATTCTATCCGCTAGAG
GATGGAACCGCTGGAGAGCAACTGCATAAGGCTATGAAGAGATACGCCCTGGTT
CCTGGAACAATTGCTTTTACAGATGCACATATCGAGGTGAACATCACGTACGCG
GAATACTTCGAAATGTCCGTTCGGTTGGCAGAAGCTATGAAACGATATGGGCTG
AATACAAATCACAGAATCGTCGTATGCAGTGAAAACTCTCTTCAATTCTTTATGC CGGTGTTGGGCGCGTTATTTATCGGAGTTGCAGTTGCGCCCGCGAACGACATTTA TAATGAACGTGAATTGCTCAACAGTATGAACATTTCGCAGCCTACCGTAGTGTTT GTTTCCAAAAAGGGGTTGCAAAAAATTTTGAACGTGCAAAAAAAATTACCAATA ATCCAGAAAATTATTATCATGGATTCTAAAACGGATTACCAGGGATTTCAGTCGA TGTACACGTTCGTCACATCTCATCTACCTCCCGGTTTTAATGAATACGATTTTGTA CCAGAGTCCTTTGATCGTGACAAAACAATTGCACTGATAATGAACTCCTCTGGAT CTACTGGGTTACCTAAGGGTGTGGCCCTTCCGCATAGAACTGCCTGCGTCAGATT CTCGCATGCCAGAGATCCTATTTTTGGCAATCAAATCATTCCGGATACTGCGATT TTAAGTGTTGTTCCATTCCATCACGGTTTTGGAATGTTTACTACACTCGGATATTT GATATGTGGATTTCGAGTCGTCTTAATGTATAGATTTGAAGAAGAGCTGTTTTTA CGATCCCTTCAGGATTACAAAATTCAAAGTGCGTTGCTAGTACCAACCCTATTTT CATTCTTCGCCAAAAGCACTCTGATTGACAAATACGATTTATCTAATTTACACGA AATTGCTTCTGGGGGCGCACCTCTTTCGAAAGAAGTCGGGGAAGCGGTTGCAAA
ACGCTTCCATCTTCCAGGGATACGACAAGGATATGGGCTCACTGAGACTACATC
AGCTATTCTGATTACACCCGAGGGGGATGATAAACCGGGCGCGGTCGGTAAAGT
TGTTCCATTTTTTGAAGCGAAGGTTGTGGATCTGGATACCGGGAAAACGCTGGGC
GTTAATCAGAGAGGCGAATTATGTGTCAGAGGACCTATGATTATGTCCGGTTATG
TAAACAATCCGGAAGCGACCAACGCCTTGATTGACAAGGATGGATGGCTACATT
CTGGAGACATAGCTTACTGGGACGAAGACGAACACTTCTTCATAGTTGACCGCTT
GAAGTCTTTAATTAAATACAAAGGATACCAGGTGGCCCCCGCTGAATTGGAGTC
GATATTGTTACAACACCCCAACATCTTCGACGCGGGCGTGGCAGGTCTTCCCGAC
GATGACGCCGGTGAACTTCCCGCCGCCGTTGTTGTTTTGGAGCACGGAAAGACG
ATGACGGAAAAAGAGATCGTGGATTACGTCGCCAGTCAAGTAACAACCGCCAAA
AAGTTGCGCGGAGGAGTTGTGTTTGTGGACGAAGTACCGAAAGGTCTTACCGGA
AAACTCGACGCAAGAAAAATCAGAGAGATCCTCATAAAGGCCAAGAAGGGCGG
AAAGTCCAAATTGATGTCCAGCATGAATCCCGAATATGATTATTTATTCAAGTTA
CTTCTGATTGGCGACTCAGGGGTTGGAAAGAATTGCCTTCTTCTTAGGTTTGCAG
ATGATACATATACAGAAAGCTACATCAGCACAATTGGTGTGGATTTCAAAATAA
GAACTATAGAGTTAGACGGGAAAACAATCAAGCTTCAAATATGGGACACAGCAG
GCCAGGAAAGATTTCGAACAATCACCTCCAGTTATTACAGAGGAGCCCATGGCA
TCATAGTTGTGTATGATGTGACAGATCAGGAGTCCTTCAATAATGTTAAACAGTG
GCTGCAGGAAATAGATCGTTATGCCAGTGAAAATGTCAACAAATTGTTGGTAGG
GAACAAATGTGATCTGACCACAAAGAAAGTAGTAGACTACACAACAGCGAAGG
AATTTGCTGATTCCCTTGGAATTCCGTTTTTGGAAACCAGTGCTAAGAATGCAAC
GAATGTAGAACAGTCTTTCATGACGATGGCAGCTGAGATTAAAAAGCGAATGGG
TCCCGGAGCAACAGCTGGTGGTGCTGAGAAGTCCAATGTTAAAATTCAGAGCAC
TCCAGTCAAGCAGTCAGGTGGAGGTTGCTGCTAA
(SEQ ID NO: 19, MG-008 ORF DNA Sequence with 5’ Luciferase Tag); or
AUGGAAGACGCCAAAAACAUAAAGAAAGGCCCGGCGCCAUUCUAUCCGCUAG
AGGAUGGAACCGCUGGAGAGCAACUGCAUAAGGCUAUGAAGAGAUACGCCCU
GGUUCCUGGAACAAUUGCUUUUACAGAUGCACAUAUCGAGGUGAACAUCACG UACGCGGAAUACUUCGAAAUGUCCGUUCGGUUGGCAGAAGCUAUGAAACGAU
AUGGGCUGAAUACAAAUCACAGAAUCGUCGUAUGCAGUGAAAACUCUCUUCA
AUUCUUUAUGCCGGUGUUGGGCGCGUUAUUUAUCGGAGUUGCAGUUGCGCCC
GCGAACGACAUUUAUAAUGAACGUGAAUUGCUCAACAGUAUGAACAUUUCGC
AGCCUACCGUAGUGUUUGUUUCCAAAAAGGGGUUGCAAAAAAUUUUGAACGU
GCAAAAAAAAUUACCAAUAAUCCAGAAAAUUAUUAUCAUGGAUUCUAAAACG
GAUUACCAGGGAUUUCAGUCGAUGUACACGUUCGUCACAUCUCAUCUACCUCC
CGGUUUUAAUGAAUACGAUUUUGUACCAGAGUCCUUUGAUCGUGACAAAACA
AUUGCACUGAUAAUGAACUCCUCUGGAUCUACUGGGUUACCUAAGGGUGUGG
CCCUUCCGCAUAGAACUGCCUGCGUCAGAUUCUCGCAUGCCAGAGAUCCUAUU
UUUGGCAAUCAAAUCAUUCCGGAUACUGCGAUUUUAAGUGUUGUUCCAUUCC
AUCACGGUUUUGGAAUGUUUACUACACUCGGAUAUUUGAUAUGUGGAUUUCG
AGUCGUCUUAAUGUAUAGAUUUGAAGAAGAGCUGUUUUUACGAUCCCUUCAG
GAUUACAAAAUUCAAAGUGCGUUGCUAGUACCAACCCUAUUUUCAUUCUUCG
CCAAAAGCACUCUGAUUGACAAAUACGAUUUAUCUAAUUUACACGAAAUUGC
UUCUGGGGGCGCACCUCUUUCGAAAGAAGUCGGGGAAGCGGUUGCAAAACGC
UUCCAUCUUCCAGGGAUACGACAAGGAUAUGGGCUCACUGAGACUACAUCAG
CUAUUCUGAUUACACCCGAGGGGGAUGAUAAACCGGGCGCGGUCGGUAAAGU
UGUUCCAUUUUUUGAAGCGAAGGUUGUGGAUCUGGAUACCGGGAAAACGCUG
GGCGUUAAUCAGAGAGGCGAAUUAUGUGUCAGAGGACCUAUGAUUAUGUCCG
GUUAUGUAAACAAUCCGGAAGCGACCAACGCCUUGAUUGACAAGGAUGGAUG
GCUACAUUCUGGAGACAUAGCUUACUGGGACGAAGACGAACACUUCUUCAUA
GUUGACCGCUUGAAGUCUUUAAUUAAAUACAAAGGAUACCAGGUGGCCCCCG
CUGAAUUGGAGUCGAUAUUGUUACAACACCCCAACAUCUUCGACGCGGGCGU
GGCAGGUCUUCCCGACGAUGACGCCGGUGAACUUCCCGCCGCCGUUGUUGUUU
UGGAGCACGGAAAGACGAUGACGGAAAAAGAGAUCGUGGAUUACGUCGCCAG
UCAAGUAACAACCGCCAAAAAGUUGCGCGGAGGAGUUGUGUUUGUGGACGAA
GUACCGAAAGGUCUUACCGGAAAACUCGACGCAAGAAAAAUCAGAGAGAUCC
UCAUAAAGGCCAAGAAGGGCGGAAAGUCCAAAUUGAUGUCCAGCAUGAAUCC
CGAAUAUGAUUAUUUAUUCAAGUUACUUCUGAUUGGCGACUCAGGGGUUGGA AAGAAUUGCCUUCUUCUUAGGUUUGCAGAUGAUACAUAUACAGAAAGCUACA UCAGCACAAUUGGUGUGGAUUUCAAAAUAAGAACUAUAGAGUUAGACGGGAA AACAAUCAAGCUUCAAAUAUGGGACACAGCAGGCCAGGAAAGAUUUCGAACA AUCACCUCCAGUUAUUACAGAGGAGCCCAUGGCAUCAUAGUUGUGUAUGAUG UGACAGAUCAGGAGUCCUUCAAUAAUGUUAAACAGUGGCUGCAGGAAAUAGA UCGUUAUGCCAGUGAAAAUGUCAACAAAUUGUUGGUAGGGAACAAAUGUGAU CUGACCACAAAGAAAGUAGUAGACUACACAACAGCGAAGGAAUUUGCUGAUU CCCUUGGAAUUCCGUUUUUGGAAACCAGUGCUAAGAAUGCAACGAAUGUAGA ACAGUCUUUCAUGACGAUGGCAGCUGAGAUUAAAAAGCGAAUGGGUCCCGGA GCAACAGCUGGUGGUGCUGAGAAGUCCAAUGUUAAAAUUCAGAGCACUCCAG UCAAGCAGUCAGGUGGAGGUUGCUGCUAA
(SEQ ID NO: 20, MG-008 ORF mRNA Sequence with 5’ Luciferase Tag); or a nucleic acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity therewith and encoding a RablaGDP preferentially binding GDP; or a nucleic acid sequence equivalent to any of the above sequences due to codon redundancy.
In another embodiment, there is provided herein a use of a GDP -bound form of Rabi a (RablaGDP), one or more expressible nucleic acids encoding RablaGDP, or a combination thereof, for reducing cell viability of a cancer cell, or for preventing or treating cancer, in a subject in need thereof.
In another embodiment, there is provided herein a use of a GDP -bound form of Rabi a (RablaGDP), one or more expressible nucleic acids encoding RablaGDP, or a combination thereof, for reducing cell viability of a cancer cell in vitro or in vivo.
In another embodiment, there is provided herein a use of a GDP -bound form of Rabi a (RablaGDP), one or more expressible nucleic acids encoding RablaGDP, or a combination thereof, in the manufacture of a medicament for reducing cell viability of a cancer cell, or for preventing or treating cancer, in a subject in need thereof. In another embodiment, there is provided herein a use of a GDP -bound form of Rabi a (RablaGDP), one or more expressible nucleic acids encoding RablaGDP, or a combination thereof, in the manufacture of a medicament for reducing cell viability of a cancer cell in vitro or in vivo.
In another embodiment of any of the above use or uses, the RablaGDP may be or may comprise RablaS25N, RablaN1241, RablaD41N, RablaD47N, or another dominant negative (DN) GDP-bound form of Rabi a.
In still another embodiment of any of the above use or uses, the Rab 1 aGDP may comprise the amino acid sequence:
MSSMNPEYDYLFKLLLIGDSGVGKNCLLLRFADDTYTESYISTIGVDFKIRTIELDGK TIKLQIWDTAGQERFRTITSSYYRGAHGIIVVYDVTDQESFNNVKQWLQEIDRYASE NVNKLLVGNKCDLTTKKVVDYTTAKEFADSLGIPFLETSAKNATNVEQSFMTMAAE IKKRMGPGATAGGAEKSNVKIQSTPVKQSGGGCC
(SEQ ID NO: 6; Human RablaS25N);
MGDYKDDDDKGGSGGSSMNPEYDYLFKLLLIGDSGVGKSCLLLRFADDTYTESYIS TIGVDFKIRTIELDGKTIKLQIWDTAGQERFRTITSSYYRGAHGIIVVYDVTDQESFNN VKQWLQEIDRYASENVNKLLVGIKCDLTTKKVVDYTTAKEFADSLGIPFLETSAKNA TNVEQSFMTMAAEIKKRMGPGATAGGAEKSNVKIQSTPVKQSGGGCC (SEQ ID NO: 9; Mouse RablN1241); the amino acid sequence of human RablaD41N; or
MSSMNPEYDYLFKLLLIGDSGVGKSCLLLRFADDTYTESYISTIGVNFKIRTIELDGKT
IKLQIWDTAGQERFRTITSSYYRGAHGIIVVYDVTDQESFNNVKQWLQEIDRYASEN VNKLLVGNKCDLTTKKVVDYTTAKEFADSLGIPFLETSAKNATNVEQSFMTMAAEI KKRMGPGATAGGAEKSNVKIQSTPVKQSGGGCC
(SEQ ID NO: 18; Human RablaD47N); or a polypeptide having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with any of these sequences and preferentially binding GDP.
In yet another embodiment of any of the above use or uses, the RablaGDP may consist of the amino acid sequence:
MSSMNPEYDYLFKLLLIGDSGVGKNCLLLRFADDTYTESYISTIGVDFKIRTIELDGK
TZKLQIWDTAGQERFRTITSSYYRGAHGIIVVYDVTDQESFNNVKQWLQEIDRYASE
NVNKLLVGNKCDLTTKKVVDYTTAKEFADSLGIPFLETSAKNATNVEQSFMTMAAE IKKRMGPGATAGGAEKSNVKIQSTPVKQSGGGCC
(SEQ ID NO: 6; Human RablaS25N);
MGDYKDDDDKGGSGGSSMNPEYDYLFKLLLIGDSGVGKSCLLLRFADDTYTESYIS
TIGVDFKIRTIELDGKTIKLQIWDTAGQERFRTITSSYYRGAHGIIVVYDVTDQESFNN
VKQWLQEIDRYASENVNKLLVGIKCDLTTKKVVDYTTAKEFADSLGIPFLETSAKNA TNVEQSFMTMAAEIKKRMGPGATAGGAEKSNVKIQSTPVKQSGGGCC
(SEQ ID NO: 9; Mouse RablN1241); the amino acid sequence of human RablaD41N; or
MSSMNPEYDYLFKLLLIGDSGVGKSCLLLRFADDTYTESYISTIGVNFKIRTIELDGKT
IKLQIWDTAGQERFRTITSSYYRGAHGIIVVYDVTDQESFNNVKQWLQEIDRYASEN VNKLLVGNKCDLTTKKVVDYTTAKEFADSLGIPFLETSAKNATNVEQSFMTMAAEI KKRMGPGATAGGAEKSNVKIQSTPVKQSGGGCC
(SEQ ID NO: 18; Human RablaD47N); or a polypeptide having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with any of these sequences and preferentially binding GDP.
In another embodiment of any of the above use or uses, the RablaGDP may comprise or consist of the amino acid sequence: MSSMNPEYDYLFKLLLIGDSGVGKNCLLLRFADDTYTESYISTIGVDFKIRTIELDGK TIKLQIWDTAGQERFRTITSSYYRGAHGIIVVYDVTDQESFNNVKQWLQEIDRYASE NVNKLLVGNKCDLTTKKVVDYTTAKEFADSLGIPFLETSAKNATNVEQSFMTMAAE IKKRMGPGATAGGAEKSNVKIQSTPVKQSGGGCC (SEQ ID NO: 6; Human RablaS25N); or a polypeptide having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity therewith and preferentially binding GDP.
In still another embodiment of any of the above use or uses, the RablaGDP may be in the form of a fusion protein, wherein the RablaGDP is fused or otherwise directly or indirectly linked, optionally via a linker, with a signaling or targeting peptide, a fluorescent peptide or other marker or tracer, or another peptide or non-peptide moiety for targeted delivery, facilitating cell uptake, increasing stability or in vivo half-life, or improving another therapeutic, diagnostic, or in vivo property of the RablaGDP.
In yet another embodiment of any of the above use or uses, the fusion protein may comprise the amino acid sequence:
MSSMNPEYDYLFKLLLIGDSGVGKNCLLLRFADDTYTESYISTIGVDFKIRTIELDGK TIKLQIWDTAGQERFRTITSSYYRGAHGIIVVYDVTDQESFNNVKQWLQEIDRYASE NVNKLLVGNKCDLTTKKVVDYTTAKEFADSLGIPFLETSAKNATNVEQSFMTMAAE IKKRMGPGATAGGAEKSNVKIQSTPVKQSGGGCC (SEQ ID NO: 6; Human RablaS25N); or a polypeptide having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity therewith and preferentially binding GDP.
In another embodiment of any of the above use or uses, the RablaGDP may be in the form of a fusion protein, and may comprise the amino acid sequence: MEDAKNIKKGPAPFYPLEDGTAGEQLHKAMKRYALVPGTIAFTDAHIEVNITYAEYF EMSVRLAEAMKRYGLNTNHRIVVCSENSLQFFMPVLGALFIGVAVAPANDIYNERE LLNSMNISQPTVVFVSKKGLQKILNVQKKLPIIQKIIIMDSKTDYQGFQSMYTFVTSHL PPGFNEYDF VPESFDRDKTIALIMNS SGSTGLPKGVALPHRT ACVRF SHARDPIFGNQI IPDTAILSVVPFHHGFGMFTTLGYLICGFRVVLMYRFEEELFLRSLQDYKIQSALLVPT LFSFFAKSTLIDKYDLSNLHEIASGGAPLSKEVGEAVAKRFHLPGIRQGYGLTETTSAI LITPEGDDKPGAVGKVVPFFEAKVVDLDTGKTLGVNQRGELCVRGPMIMSGYVNNP EATNALIDKDGWLHSGDIAYWDEDEHFFIVDRLKSLIKYKGYQVAPAELESILLQHP NIFDAGVAGLPDDDAGELPAAVVVLEHGKTMTEKEIVDYVASQVTTAKKLRGGVV FVDEVPKGLTGKLDARKIREILIKAKKGGKSKLMSSMNPEYDYLFKLLLIGDSGVGK NCLLLRFADDTYTESYISTIGVDFKIRTIELDGKTIKLQIWDTAGQERFRTITSSYYRG AHGIIVVYDVTDQESFNNVKQWLQEIDRYASENVNKLLVGNKCDLTTKKVVDYTT AKEFADSLGIPFLETSAKNATNVEQSFMTMAAEIKKRMGPGATAGGAEKSNVKIQST PVKQSGGGCC
(SEQ ID NO: 21); or a polypeptide having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity therewith and preferentially binding GDP.
In still another embodiment of any of the above use or uses, the one or more expressible nucleic acids may encode one or more RablaGDPs as defined herein.
In yet another embodiment of any of the above use or uses, the one or more expressible nucleic acids may be DNA-based, or RNA-based.
In another embodiment of any of the above use or uses, the one or more expressible nucleic acids may transiently express the RablaGDP in the cell, or wherein the one or more expressible nucleic acids may integrate in the cell genome and express the RablaGDP in the cell.
In another embodiment of any of the above use or uses, the one or more expressible nucleic acids may comprise one or more expression vectors, plasmids, or mRNAs encoding and capable of expressing the RablaGDP inside the cell.
In another embodiment of any of the above use or uses, the one or more expressible nucleic acids may comprise a nucleic acid sequence of:
ATGTCCAGCATGAATCCCGAATATGATTATTTATTCAAGTTACTTCTGATTGGCG
ACTCAGGGGTTGGAAAGAATTGCCTTCTTCTTAGGTTTGCAGATGATACATATAC
AGAAAGCTACATCAGCACAATTGGTGTGGATTTCAAAATAAGAACTATAGAGTT
AGACGGGAAAACAATCAAGCTTCAAATATGGGACACAGCAGGCCAGGAAAGAT
TTCGAACAATCACCTCCAGTTATTACAGAGGAGCCCATGGCATCATAGTTGTGTA
TGATGTGACAGATCAGGAGTCCTTCAATAATGTTAAACAGTGGCTGCAGGAAAT
AGATCGTTATGCCAGTGAAAATGTCAACAAATTGTTGGTAGGGAACAAATGTGA
TCTGACCACAAAGAAAGTAGTAGACTACACAACAGCGAAGGAATTTGCTGATTC
CCTTGGAATTCCGTTTTTGGAAACCAGTGCTAAGAATGCAACGAATGTAGAACA
GTCTTTCATGACGATGGCAGCTGAGATTAAAAAGCGAATGGGTCCCGGAGCAAC
AGCTGGTGGTGCTGAGAAGTCCAATGTTAAAATTCAGAGCACTCCAGTCAAGCA GTCAGGTGGAGGTTGCTGCTAA
(Human RablaS25N ORF Codon Sequence, SEQ ID NO: 5);
ATGGGGGACTACAAGGACGACGATGACAAGGGGGGTAGCGGTGGATCCAGCAT
GAATCCCGAATATGATTATTTATTCAAGTTACTTCTGATTGGCGATTCTGGGGTT
GGAAAGTCCTGCCTTCTCCTTAGGTTTGCAGATGATACGTATACGGAAAGCTACA
TCAGCACAATTGGTGTGGATTTCAAGATACGAACTATAGAGTTAGATGGGAAAA
CAATCAAGCTACAGATATGGGACACAGCAGGCCAGGAAAGATTTCGAACAATCA
CTTCCAGTTATTACAGAGGAGCCCATGGCATCATAGTTGTGTATGATGTGACAGA
TCAGGAGTCCTTCAATAACGTTAAACAGTGGCTGCAGGAGATAGATCGCTACGC
CAGTGAAAATGTCAACAAGTTGTTGGTAGGGATCAAATGTGACCTGACCACAAA
GAAAGTAGTAGACTACACAACAGCAAAGGAATTTGCAGATTCCCTTGGAATTCC
ATTTTTGGAAACCAGTGCTAAGAACGCAACGAATGTAGAACAGTCTTTCATGAC
GATGGCAGCTGAGATTAAAAAGCGAATGGGTCCTGGAGCTACAGCTGGTGGTGC
CGAGAAGTCCAATGTTAAAATCCAGAGCACTCCAGTCAAGCAGTCAGGTGGAGG CTGCTGCTAA
(Mouse RablaN1241 ORF Codon Seqeunce, SEQ ID NO: 8); or
ATGTCCAGCATGAATCCCGAATATGATTATTTATTCAAGTTACTTCTGATTGGCGACT
CAGGGGTTGGAAAGTCTTGCCTTCTTCTTAGGTTTGCAGATGATACATATACAGAAA GCTACATCAGCACAATTGGTGTGAACTTCAAAATAAGAACTATAGAGTTAGACGGG AAAACAATCAAGCTTCAAATATGGGACACAGCAGGCCAGGAAAGATTTCGAACAAT CACCTCCAGTTATTACAGAGGAGCCCATGGCATCATAGTTGTGTATGATGTGACAGA TCAGGAGTCCTTCAATAATGTTAAACAGTGGCTGCAGGAAATAGATCGTTATGCCAG TGAAAATGTCAACAAATTGTTGGTAGGGAACAAATGTGATCTGACCACAAAGAAAG TAGTAGACTACACAACAGCGAAGGAATTTGCTGATTCCCTTGGAATTCCGTTTTTGG AAACCAGTGCTAAGAATGCAACGAATGTAGAACAGTCTTTCATGACGATGGCAGCT GAGATTAAAAAGCGAATGGGTCCCGGAGCAACAGCTGGTGGTGCTGAGAAGTCCAA TGTTAAAATTCAGAGCACTCCAGTCAAGCAGTCAGGTGGAGGTTGCTGCTAA
(Human RablaD47N ORF Codon Sequence, SEQ ID NO: 17); or a nucleic acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity therewith and encoding a RablaGDP preferentially binding GDP; or a nucleic acid sequence equivalent to any of the above sequences due to codon redundancy.
In still another embodiment of any of the above use or uses, the one or more expressible nucleic acids may comprise a nucleic acid sequence of:
ATGGAAGACGCCAAAAACATAAAGAAAGGCCCGGCGCCATTCTATCCGCTAGAG
GATGGAACCGCTGGAGAGCAACTGCATAAGGCTATGAAGAGATACGCCCTGGTT
CCTGGAACAATTGCTTTTACAGATGCACATATCGAGGTGAACATCACGTACGCG
GAATACTTCGAAATGTCCGTTCGGTTGGCAGAAGCTATGAAACGATATGGGCTG
AATACAAATCACAGAATCGTCGTATGCAGTGAAAACTCTCTTCAATTCTTTATGC
CGGTGTTGGGCGCGTTATTTATCGGAGTTGCAGTTGCGCCCGCGAACGACATTTA TAATGAACGTGAATTGCTCAACAGTATGAACATTTCGCAGCCTACCGTAGTGTTT
GTTTCCAAAAAGGGGTTGCAAAAAATTTTGAACGTGCAAAAAAAATTACCAATA
ATCCAGAAAATTATTATCATGGATTCTAAAACGGATTACCAGGGATTTCAGTCGA
TGTACACGTTCGTCACATCTCATCTACCTCCCGGTTTTAATGAATACGATTTTGTA
CCAGAGTCCTTTGATCGTGACAAAACAATTGCACTGATAATGAACTCCTCTGGAT
CTACTGGGTTACCTAAGGGTGTGGCCCTTCCGCATAGAACTGCCTGCGTCAGATT
CTCGCATGCCAGAGATCCTATTTTTGGCAATCAAATCATTCCGGATACTGCGATT
TTAAGTGTTGTTCCATTCCATCACGGTTTTGGAATGTTTACTACACTCGGATATTT
GATATGTGGATTTCGAGTCGTCTTAATGTATAGATTTGAAGAAGAGCTGTTTTTA
CGATCCCTTCAGGATTACAAAATTCAAAGTGCGTTGCTAGTACCAACCCTATTTT
CATTCTTCGCCAAAAGCACTCTGATTGACAAATACGATTTATCTAATTTACACGA
AATTGCTTCTGGGGGCGCACCTCTTTCGAAAGAAGTCGGGGAAGCGGTTGCAAA
ACGCTTCCATCTTCCAGGGATACGACAAGGATATGGGCTCACTGAGACTACATC
AGCTATTCTGATTACACCCGAGGGGGATGATAAACCGGGCGCGGTCGGTAAAGT
TGTTCCATTTTTTGAAGCGAAGGTTGTGGATCTGGATACCGGGAAAACGCTGGGC
GTTAATCAGAGAGGCGAATTATGTGTCAGAGGACCTATGATTATGTCCGGTTATG
TAAACAATCCGGAAGCGACCAACGCCTTGATTGACAAGGATGGATGGCTACATT
CTGGAGACATAGCTTACTGGGACGAAGACGAACACTTCTTCATAGTTGACCGCTT
GAAGTCTTTAATTAAATACAAAGGATACCAGGTGGCCCCCGCTGAATTGGAGTC
GATATTGTTACAACACCCCAACATCTTCGACGCGGGCGTGGCAGGTCTTCCCGAC
GATGACGCCGGTGAACTTCCCGCCGCCGTTGTTGTTTTGGAGCACGGAAAGACG
ATGACGGAAAAAGAGATCGTGGATTACGTCGCCAGTCAAGTAACAACCGCCAAA
AAGTTGCGCGGAGGAGTTGTGTTTGTGGACGAAGTACCGAAAGGTCTTACCGGA
AAACTCGACGCAAGAAAAATCAGAGAGATCCTCATAAAGGCCAAGAAGGGCGG
AAAGTCCAAATTGATGTCCAGCATGAATCCCGAATATGATTATTTATTCAAGTTA
CTTCTGATTGGCGACTCAGGGGTTGGAAAGAATTGCCTTCTTCTTAGGTTTGCAG
ATGATACATATACAGAAAGCTACATCAGCACAATTGGTGTGGATTTCAAAATAA
GAACTATAGAGTTAGACGGGAAAACAATCAAGCTTCAAATATGGGACACAGCAG
GCCAGGAAAGATTTCGAACAATCACCTCCAGTTATTACAGAGGAGCCCATGGCA
TCATAGTTGTGTATGATGTGACAGATCAGGAGTCCTTCAATAATGTTAAACAGTG GCTGCAGGAAATAGATCGTTATGCCAGTGAAAATGTCAACAAATTGTTGGTAGG
GAACAAATGTGATCTGACCACAAAGAAAGTAGTAGACTACACAACAGCGAAGG
AATTTGCTGATTCCCTTGGAATTCCGTTTTTGGAAACCAGTGCTAAGAATGCAAC
GAATGTAGAACAGTCTTTCATGACGATGGCAGCTGAGATTAAAAAGCGAATGGG
TCCCGGAGCAACAGCTGGTGGTGCTGAGAAGTCCAATGTTAAAATTCAGAGCAC
TCCAGTCAAGCAGTCAGGTGGAGGTTGCTGCTAA
(SEQ ID NO: 19, MG-008 ORF DNA Sequence with 5’ Luciferase Tag); or
AUGGAAGACGCCAAAAACAUAAAGAAAGGCCCGGCGCCAUUCUAUCCGCUAG
AGGAUGGAACCGCUGGAGAGCAACUGCAUAAGGCUAUGAAGAGAUACGCCCU
GGUUCCUGGAACAAUUGCUUUUACAGAUGCACAUAUCGAGGUGAACAUCACG
UACGCGGAAUACUUCGAAAUGUCCGUUCGGUUGGCAGAAGCUAUGAAACGAU
AUGGGCUGAAUACAAAUCACAGAAUCGUCGUAUGCAGUGAAAACUCUCUUCA
AUUCUUUAUGCCGGUGUUGGGCGCGUUAUUUAUCGGAGUUGCAGUUGCGCCC
GCGAACGACAUUUAUAAUGAACGUGAAUUGCUCAACAGUAUGAACAUUUCGC
AGCCUACCGUAGUGUUUGUUUCCAAAAAGGGGUUGCAAAAAAUUUUGAACGU
GCAAAAAAAAUUACCAAUAAUCCAGAAAAUUAUUAUCAUGGAUUCUAAAACG
GAUUACCAGGGAUUUCAGUCGAUGUACACGUUCGUCACAUCUCAUCUACCUCC
CGGUUUUAAUGAAUACGAUUUUGUACCAGAGUCCUUUGAUCGUGACAAAACA
AUUGCACUGAUAAUGAACUCCUCUGGAUCUACUGGGUUACCUAAGGGUGUGG
CCCUUCCGCAUAGAACUGCCUGCGUCAGAUUCUCGCAUGCCAGAGAUCCUAUU
UUUGGCAAUCAAAUCAUUCCGGAUACUGCGAUUUUAAGUGUUGUUCCAUUCC
AUCACGGUUUUGGAAUGUUUACUACACUCGGAUAUUUGAUAUGUGGAUUUCG
AGUCGUCUUAAUGUAUAGAUUUGAAGAAGAGCUGUUUUUACGAUCCCUUCAG
GAUUACAAAAUUCAAAGUGCGUUGCUAGUACCAACCCUAUUUUCAUUCUUCG
CCAAAAGCACUCUGAUUGACAAAUACGAUUUAUCUAAUUUACACGAAAUUGC
UUCUGGGGGCGCACCUCUUUCGAAAGAAGUCGGGGAAGCGGUUGCAAAACGC
UUCCAUCUUCCAGGGAUACGACAAGGAUAUGGGCUCACUGAGACUACAUCAG
CUAUUCUGAUUACACCCGAGGGGGAUGAUAAACCGGGCGCGGUCGGUAAAGU
UGUUCCAUUUUUUGAAGCGAAGGUUGUGGAUCUGGAUACCGGGAAAACGCUG GGCGUUAAUCAGAGAGGCGAAUUAUGUGUCAGAGGACCUAUGAUUAUGUCCG GUUAUGUAAACAAUCCGGAAGCGACCAACGCCUUGAUUGACAAGGAUGGAUG GCUACAUUCUGGAGACAUAGCUUACUGGGACGAAGACGAACACUUCUUCAUA GUUGACCGCUUGAAGUCUUUAAUUAAAUACAAAGGAUACCAGGUGGCCCCCG CUGAAUUGGAGUCGAUAUUGUUACAACACCCCAACAUCUUCGACGCGGGCGU GGCAGGUCUUCCCGACGAUGACGCCGGUGAACUUCCCGCCGCCGUUGUUGUUU UGGAGCACGGAAAGACGAUGACGGAAAAAGAGAUCGUGGAUUACGUCGCCAG UCAAGUAACAACCGCCAAAAAGUUGCGCGGAGGAGUUGUGUUUGUGGACGAA GUACCGAAAGGUCUUACCGGAAAACUCGACGCAAGAAAAAUCAGAGAGAUCC UCAUAAAGGCCAAGAAGGGCGGAAAGUCCAAAUUGAUGUCCAGCAUGAAUCC CGAAUAUGAUUAUUUAUUCAAGUUACUUCUGAUUGGCGACUCAGGGGUUGGA AAGAAUUGCCUUCUUCUUAGGUUUGCAGAUGAUACAUAUACAGAAAGCUACA UCAGCACAAUUGGUGUGGAUUUCAAAAUAAGAACUAUAGAGUUAGACGGGAA AACAAUCAAGCUUCAAAUAUGGGACACAGCAGGCCAGGAAAGAUUUCGAACA AUCACCUCCAGUUAUUACAGAGGAGCCCAUGGCAUCAUAGUUGUGUAUGAUG UGACAGAUCAGGAGUCCUUCAAUAAUGUUAAACAGUGGCUGCAGGAAAUAGA UCGUUAUGCCAGUGAAAAUGUCAACAAAUUGUUGGUAGGGAACAAAUGUGAU CUGACCACAAAGAAAGUAGUAGACUACACAACAGCGAAGGAAUUUGCUGAUU CCCUUGGAAUUCCGUUUUUGGAAACCAGUGCUAAGAAUGCAACGAAUGUAGA ACAGUCUUUCAUGACGAUGGCAGCUGAGAUUAAAAAGCGAAUGGGUCCCGGA GCAACAGCUGGUGGUGCUGAGAAGUCCAAUGUUAAAAUUCAGAGCACUCCAG UCAAGCAGUCAGGUGGAGGUUGCUGCUAA
(SEQ ID NO: 20, MG-008 ORF mRNA Sequence with 5’ Luciferase Tag); or a nucleic acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity therewith and encoding a RablaGDP preferentially binding GDP; or a nucleic acid sequence equivalent to any of the above sequences due to codon redundancy.
In another embodiment, there is provided herein a polypeptide comprising the amino acid sequence:
MSSMNPEYDYLFKLLLIGDSGVGKNCLLLRFADDTYTESYISTIGVDFKIRTIELD
GKTIKLQIWDTAGQERFRTITSSYYRGAHGIIVVYDVTDQESFNNVKQWLQEIDR YASENVNKLLVGNKCDLTTKKVVDYTTAKEFADSLGIPFLETSAKNATNVEQSF MTMAAEIKKRMGPGATAGGAEKSNVKIQSTPVKQSGGGCC (SEQ ID NO: 6; Human RablaS25N);
MGDYKDDDDKGGSGGSSMNPEYDYLFKLLLIGDSGVGKSCLLLRFADDTYTES
YISTIGVDFKIRTIELDGKTIKLQIWDTAGQERFRTITSSYYRGAHGIIVVYDVTDQ ESFNNVKQWLQEIDRYASENVNKLLVGIKCDLTTKKVVDYTTAKEFADSLGIPFL ETSAKNATNVEQSFMTMAAEIKKRMGPGATAGGAEKSNVKIQSTPVKQSGGGC C
(SEQ ID NO: 9; Mouse RablN1241); the amino acid sequence of human RablaD41N; or
MSSMNPEYDYLFKLLLIGDSGVGKSCLLLRFADDTYTESYISTIGVNFKIRTIELD
GKTIKLQIWDTAGQERFRTITSSYYRGAHGIIVVYDVTDQESFNNVKQWLQEIDR YASENVNKLLVGNKCDLTTKKVVDYTTAKEFADSLGIPFLETSAKNATNVEQSF MTMAAEIKKRMGPGATAGGAEKSNVKIQSTPVKQSGGGCC
(SEQ ID NO: 18; Human RablaD47N); or a polypeptide having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with any of these sequences and preferentially binding GDP; for use in reducing cell viability of a cancer cell, or for preventing or treating cancer, in a subject in need thereof; or for use in reducing cell viability of a cancer cell in vitro or in vivo.
In another embodiment, there is provided herein a pharmaceutical composition comprising a GDP -bound form of Rabi a (RablaGDP), one or more expressible nucleic acids encoding RablaGDP, or a combination thereof; and another anti-cancer agent.
In yet another embodiment, there is provided herein a kit comprising any one or more of: a GDP -bound form of Rabi a (RablaGDP); one or more expressible nucleic acids encoding RablaGDP; an anti-cancer agent; instructions for performing any of the method or methods as described herein; or any combinations thereof.
As will be understood, in certain embodiments of any of the methods, uses, or polypeptides for use as described herein, the cancer or cancer cell may be any of a wide variety of cancer types. As described in Example 1 below, results from testing indicate that anti-cancer effects may be observed in a wide variety of different cancer cell lines, supporting broad anti-cancer applicability. Anticancer effects were also observed in vivo, as described in Example 2 below, further supporting the applicability for the treatment of cancer in subjects.
In still another embodiment of any of the above methods, uses, or polypeptides for use, the cancer may be brain cancer, breast cancer, cervical cancer, colon cancer, ductal cancer, gastric cancer, liver cancer, lung cancer, oral cancer, pancreatic cancer, or prostate cancer.
In yet another embodiment of any of the above methods, uses, or polypeptides for use, the cancer may be breast adenocarcinoma, cholangiocarcinoma, colon adenocarcinoma, colorectal carcinoma, prostate carcinoma, embryonal rhabdomyosarcoma, gastric carcinoma, glioblastoma, hepatocellular carcinoma, invasive ductal carcinoma, lung cancer adenocarcinoma, pancreatic ductal adenocarcinoma, papillomavirus-related endocervical adenocarcinoma, squamous cell carcinoma of the oral cavity, or squamous cell carcinoma of the oral tongue.
In another embodiment of any of the above methods, uses, or polypeptides for use, the cancer cell lines may be selected from the group consisting of A549 (lung adenocarcinoma), HCT116 (colorectal carcinoma), HT29 (colon adenocarcinoma), HuCCTl (cholangiocarcinoma), PC-3 (prostate carcinoma), RD (rhabdomyosarcoma), SC-M1 (gastric carcinoma), U-87MG (glioblastoma), SW480 (colon adenocarcinoma), PANC-1 (pancreatic adenocarcinoma), OECM- 1 (oral carcinoma), OC2 (oral carcinoma), MIA PaCa-2 (pancreatic adenocarcinoma), MDA-MB- 468 (breast adenocarcinoma), MDA-MB-231 (breast adenocarcinoma), MCF-7 (ductal carcinoma), Mahlavu (hepatocellular carcinoma), HSC-3 (Squamous cell carcinoma of the oral tongue), HeLa (cervical carcinoma), and HCT116 (colorectal carcinoma).
As will be understood, lysosome-mediated microautophagy may refer to a cellular process in which a cellular lipid or protein or glycogen substrate, or a portion thereof, is engulfed and degraded by a lysosome. In this particular regard, lysosome-mediated microautophagy is an integral component of cell’s bioenergetic process (i.e. ATP generation) as well as biosynthesis process (i.e. synthesizing new biomass through recycling existing “old” materials).
It will also be understood that references to increasing lysosome-mediated microautophagy may refer to an increase in the rate, extent, capacity, or efficacy of the lysosome-mediated microautophagy process in a cell as compared to baseline levels of the cell, or as compared with a corresponding treated or untreated control cell, or as compared to levels in a reference diseased cell or reference cell having accumulation of a protein, lipid, or glycogen substrate.
It will further be understood that increasing lysosome-mediated microautophagy may include restoring or increasing lysosomal motility or lysosomal bidirectional motility in a cell, and/or may include enhancing, increasing, activating, or otherwise restoring or rescuing lysosomal motility activity in a cell. Such restoration or rescue may result in an increase in autophagy (such as, for example, micro- and/or macro- autophagy) and/or lysosomal degradation capacity. As detailed herein, there are several diseases, conditions, and cellular states in which cellular lysosomal motility may be impaired, reduced, blocked, or suppressed. As lysosome-mediated microautophagy may involve lysosomal motility and kiss-and-run events, microautophagyenhancing agents may restore or increase lysosomal motility in a cell. Lysosomal motility may play an important role in several cellular functions, including lysosome-mediated microautophagy, lysosome-mediated macroautophagy, lysosomal regeneration, and lysosomal maturation processes. References to restoring lysosomal motility and/or lysosomal bidirectional motility may refer to modulating cellular lysosomal motility/bidirectional motility levels back to those of corresponding normal or healthy control cells having baseline levels of lysosomal motility/bidirectional motility. Such modulation may, in certain embodiments, also modulate degradation capacity (i.e. phagy) levels back to those of normal or healthy cells.
It will be understood that lysosomal association-dissociation events between lysosomes and the lipid or protein or glycogen substrate may refer to events where a lysosome associates with a lipid or protein or glycogen substrate (i.e. a lipid droplet or a protein aggregate, for example), acquires at least a portion of the lipid or protein or glycogen substrate, and then dissociates from the lipid or protein or glycogen substrate. Lysosomal association-dissociation (i.e. the "on" and "off) events between lysosomes and the lipid or protein or glycogen substrate may be considered as "kiss-and- run"-type events. As part of the association (or "kiss") event, at least a small piece of substrate (i.e. lipid) may be "grabbed" or engulfed by the lysosome from the substrate (i.e. cytosolic lipid droplets, or CLD, for example). In the case of lipid droplet substrates, this may be achieved through the formation of a fusion pore between lysosome and the CLD. As part of the dissociation (or "run") event, dissociation of the lysosome from the substrate (i.e. CLD) may occur. An increase in lysosomal association-dissociation events may refer to an increase in the rate, extent, or efficacy of lysosomal association-dissociation events in a cell as compared to baseline levels of a corresponding treated or untreated control cell, for example an identical cell treated under identical conditions but without a microautophagy-modulating agent or with a compound or composition that is known not to affect the process.
It will be understood that in certain embodiments, microautophagy-enhancing agents may be used to correct a microautophagy deficiency in a cell, or a cellular condition in which microautophagy is decreased.
The person of skill in the art having regard to the teachings herein will understand that a microautophagy-enhancing agent may be any suitable agent which increases or facilitates the rate, activity, extent, or efficacy of lysosome-mediated microautophagy in a cell, or that increases lysosomal motility or bidirectional motility. In an embodiment, a suitable microautophagyenhancing agent may be or comprise a GDP -bound form of Rabi a (RablaGDP), one or more expressible nucleic acids encoding RablaGDP, or a combination thereof.
Ras-related protein Rab-IA (i.e. Rabla) is a protein that in humans is encoded by the RAB1A gene. It may control vesicle transport from the endoplasmic reticulum (ER) to the Golgi compartment and on to the cell surface, and may play an important role in IL-8 and growth hormone secretion. In addition, it may play a role in autophagosome assembly in macroautophagy and cellular defense reactions against pathogenic bacteria when it is in its GTP -bound form. It also may regulate motility of endocytic compartment(s).
As described in detail herein, it has now been identified that lysosome-mediated microautophagy of a target protein, lipid, or glycogen substrate in a cell may be increased by treatment with a microautophagy-enhancing agent comprising a GDP -bound form of Rabla, such as RablaS25N, Rab 1 aN1241, Rab 1 aD41N, Rab 1 aD47N, or another dominant negative (DN) GDP -bound form of Rab 1 a.
It will be understood that the specific amino acid or nucleic acid sequence of a particular gene may vary from species to species. By way of example, the human Rabla amino acid sequence may have homologs in other species having sequence variation from the human sequence. In some embodiments, although homologous sequences may vary between species, the general effect (for example, the phenotypic effect) of a homolog sequence may be substantially similar to the effect of the wild-type sequence in a given cell or subject.
In particular embodiments, a microautophagy-enhancing agent may be, or comprise, a GDP -bound form of Rabla (RablaGDP) such as, for example, RablaS25N, RablaN1241, RablaD41N, RablaD47N, or a functional equivalent thereof, or another dominant negative (DN) GDP -bound form of Rabla. Sequences for human and/or mouse RablaWT and certain GDP -bound forms thereof are shown in Figure 36. Suitable GDP-bound forms of Rabla may include any suitable Rabla variant which is "dominant negative", or which preferentially binds GDP over GTP. Such RablaGDP variants may be identified using techniques known in the art (see, for example, Chan, C.-C. et al. Systematic Discovery of Rab GTPases with Synaptic Functions in Drosophila. Current Biology 21, 1704-1715 (2011); Tabancay, A. P. et al. Identification of dominant negative mutants of Rheb GTPase and their use to implicate the involvement of human Rheb in the activation of p70S6K. J. Biol. Chem. 278, 39921-39930 (2003); and Dumas, J. J., Zhu, Z., Connolly, J. L. & Lambright, D. G. Structural basis of activation and GTP hydrolysis in Rab proteins. Structure 7, 413 — s2 (1999), each of which is herein incorporated by reference in its entirety).
Figure 36 shows certain sequences of nucleic acids and amino acids/proteins as described herein. In Figure 36, SEQ ID NOs: l-3 provide human RablaWT DNA gene sequence, ORF codon sequence, and amino acid sequence, respectively; SEQ ID NOs:4-6 provide human RablaS25N DNA gene sequence, ORF codon sequence, and amino acid sequence, respectively; SEQ ID NOs:7-9 provide mouse RablN1241 DNA gene sequence, ORF codon sequence, and amino acid sequence, respectively; SEQ ID NOs: 10-12 provide human RablaQ70L DNA gene sequence, ORF codon sequence, and amino acid sequence, respectively; SEQ ID NOs: 13-15 provide human RablaQ63L DNA gene sequence, ORF codon sequence, and amino acid sequence, respectively; SEQ ID NOs: 16-18 provide human RablaD47N DNA gene sequence, ORF codon sequence, and amino acid sequence, respectively; and SEQ ID NOs: 19-21 provide MG-008 ORF DNA sequence with 5’ luciferase tag, MG-008 ORF mRNA sequence with 5’ Luciferase tag, and MG-008 Protein sequence with N-terminal luciferase tag (see Example 1 for further discussion of SEQ ID NOs: 19-21), respectively. Sequence of human RablaD41N may be found at https://www.addgene.org/49581/ (which is herein incorporated by reference in its entirety), and is commercially available. In certain embodiments, there is provided herein a nucleic acid or amino acid comprising any of these sequences. In certain embodiments, there is provided herein a nucleic acid or amino acid comprising a nucleic acid sequence or amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with any of these sequences (i.e. with any of SEQ ID NOs: 1-21 or the sequence of RablaD41N) or an active fragment thereof.
Suitable functional equivalents of RablaS25N, RablaD41N, RablaD47N, and RablaN1241 may include, for example, suitable Rab la variants or mutants having at least 80% (or >85%, or >90%, or >95%, or >99%) sequence identity to RablaWT or RablaS25N or RablaN1241 or RablaD41N or RablaD47N and which preferentially bind GDP over GTP while also retaining the relevant cellular/biochemical functions of RablaS25N or RablaN1241 or RablaD41N or RablaD47N as described in detail herein. In further embodiments, it will be understood that a microautophagy-enhancing agent may be, or comprise, one or more expressible nucleic acids encoding RablaGDP, such as any suitable nucleic acid/expression vector (i.e. vector, cassette, mRNA, modified mRNA, plasmid, for example) which encodes for/expresses a GDP -bound form of Rabla (RablaGDP) such as, for example, RablaS25N or RablaN1241 or RablaD41N or RablaD47N, or a functional equivalent thereof.
As will be understood, sequences are mainly described herein with reference to human and/or mouse homologs. It will be understood that functional equivalents and/or variants may be found in a variety of different species, such as among different mammals. References herein to particular sequence modifications and/or mutants providing for RablaGDP (DN) or RablaGTP (DA) forms often provide position and modification/mutation information (e.g. S25N, D41N, D47N, N124I; Q70L, Q67L, Q63L) with reference to human and/or mouse homolog/sequence for convenience; however, it will be understood that equivalent DN and/or DA forms may be implemented in homologous sequences from other species and/or other sequences related to the human and/or mouse sequences provided herein, although the positioning and/or nature of the modification/mutation may vary somewhat depending on the particular sequence of interest. By way of example, RablN1241 is medication/mutation of a mouse sequence. By way of another example, RablQ67L is a modification/mutation of a mouse sequence, and there is no Q at the 67th position of human Rabla; rather, there is a Q in the human sequence at the 63rd position, and so the modification/mutation with reference to the human sequence is RablaQ63L.
A person of skill in the art will understand that a microautophagy-reducing agent may be or comprise any one or more of a GTP -bound form of Rabla (RablaGTP), one or more expressible nucleic acids encoding RablaGTP, Rabla wild-type (RablaWT), or one or more expressible nucleic acids encoding RablaWT. In certain embodiments, a RablaGTP may be or comprise RablaQ70L, RablaQ67L(in mouse sequence), RablaQ63L (in human Rabla sequence), or a functional equivalent thereof, or another dominant active (DA) GTP-bound form of Rabla. GTP-bound forms of Rabla may include any Rabla variant which is "dominant active", or which preferentially binds GTP over GDP. Such RablaGTP variants may be identified using techniques known in the art (see, for example, Chan, C.-C. et al. Systematic Discovery of Rab GTPases with Synaptic Functions in Drosophila. Current Biology 21, 1704-1715 (2011); Tabancay, A. P. et al. Identification of dominant negative mutants of Rheb GTPase and their use to implicate the involvement of human Rheb in the activation of p70S6K. J. Biol. Chem. 278, 39921-39930 (2003); and Dumas, J. J., Zhu, L., Connolly, J. L. & Lambright, D. G. Structural basis of activation and GTP hydrolysis in Rab proteins. Structure 7, 413 — s2 (1999), each of which is herein incorporated by reference in its entirety), such as those used to identify RablaQ70L and/or RablaQ67L and/or RablaQ63L.
It will be understood that in certain embodiments, treatment with a microautophagy-enhancing agent, such as RablaGDP, may comprise introducing the RablaGDP protein to a cell, expressing RablaGDP within the cell, or both, for example.
It will be understood that expression of a particular protein within a cell may refer to the production of a polypeptide from a nucleic acid sequence encoding the polypeptide. Gene expression may include both transcription and translation processes, and so gene expression may refer to production of a nucleic acid sequence such as an mRNA (i.e. transcription), production of a protein (i.e. translation), or both. It will further be understood that overexpression of a particular gene in a cell may refer to increasing the expression of a particular gene within a cell as compared to wildtype, baseline, or untreated levels. Overexpression, or introduction of a mutant gene, into cells may be accomplished using any of several methods known in the art. By way of example, a vector (either viral, plasmid, or other) comprising one or more copies of the particular gene each driven by a suitable promoter sequence (for example, a constitutive or inducible promoter), or an mRNA or chemically modified version thereof may be introduced into cells via transfection, electroporation, or viral infection, or another suitable method know in the art. Suitable expression vector techniques for overexpressing or introducing a particular gene into a cell are known in the art (see, for example, Molecular Cloning: A Laboratory Manual (4th Ed.), 2012, Cold Spring Harbor Laboratory Press). The skilled person having regard to the teachings herein will be aware of a wide variety of expressible nucleic acids encoding a particular protein, such as RablaGDP, that may be prepared for introduction to a cell (either transiently or long-term via integration into the genome, for example) to provide expression of the protein of interest.
It will be understood that compounds and/or compositions comprising or consisting of one or more of the nucleic acids and/or proteins as described herein may be used. Compositions may additionally comprise one or more pharmaceutically acceptable diluents, carriers, excipients, or buffers. Compositions may be used for administering one or more nucleic acids and/or proteins to a cell in vitro or in vivo. Introduction of a gene, in the context of inserting a nucleic acid sequence into a cell, may refer to "transfection", "transformation", or "transduction", and may include the incorporation or introduction of a nucleic acid sequence into a eukaryotic cell where the nucleic acid sequence may optionally be incorporated into the genome of the cell, or transiently expressed (for example, transfected mRNA). A protein or enzyme may be introduced into a cell by delivering the protein or enzyme itself into the cell, or by expressing an mRNA encoding the protein or enzyme within the cell, leading to its translation.
As will be known to one of skill in the art, expressible nucleic acids for expressing a particular gene may encode or include features as described in "Genes VII", Lewin, B. Oxford University Press (2000) or "Molecular Cloning: A Laboratory Manual", Sambrook et al., Cold Spring Harbor Laboratory, 3rd edition (2001). A nucleotide sequence encoding a polypeptide or protein may be incorporated into a suitable vector, such as a commercially available vector. Vectors may also be individually constructed or modified using standard molecular biology techniques, as outlined, for example, in Sambrook et al. (Cold Spring Harbor Laboratory, 3rd edition (2001)). The person of skill in the art will recognize that a vector may include nucleotide sequences encoding desired elements that may be operably linked to a nucleotide sequence encoding a polypeptide or protein. Such nucleotide sequences encoding desired elements may include transcriptional promoters, transcriptional enhancers, transcriptional terminators, translational initiators, translational terminators, ribosome binding sites, 5'- untranslated region, 3'- untranslated regions, cap structure, poly A tail, and/or an origin of replication. Selection of a suitable vector may depend upon several factors, including, without limitation, the size of the nucleic acid to be incorporated into the vector, the type of transcriptional and translational control elements desired, the level of expression desired, copy number desired, whether chromosomal integration is desired, the type of selection process that is desired, or the host cell or the host range that is intended to be transformed.
The person of skill in the art will understand that biomolecules and/or compounds described herein may be provided in pharmaceutical compositions together with a pharmaceutically acceptable diluent, carrier, or excipient, and/or together with one or more separate active agents or drugs as part of a pharmaceutical combination or pharmaceutical composition. In certain embodiments, the biomolecules, compounds, and/or pharmaceutical compositions may be administered in a treatment regimen simultaneously, sequentially, or in combination with other drugs or pharmaceutical compositions, either separately or as a combined formulation or combination.
Biomolecules, compounds, and/or compositions as described herein may include one or more pharmaceutically acceptable excipients, diluents, and/or carriers. A pharmaceutically acceptable carrier, diluent, or excipient may include any suitable carrier, diluent, or excipient known to the person of skill in the art. Examples of pharmaceutically acceptable excipients may include, but are not limited to, cellulose derivatives, sucrose, and starch. The person of skill in the art will recognize that pharmaceutically acceptable excipients may include suitable fillers, binders, lubricants, buffers, glidants, and disentegrants known in the art (see, for example, Remington: The Science and Practice of Pharmacy (2006)). Examples of pharmaceutically acceptable carriers, diluents, and excipients may be found in, for example, Remington's Pharmaceutical Sciences (2000 — 20th edition) and in the United States Pharmacopeia: TheNational Formulary (USP 24 NF19) published in 1999.
It will also be understood that one or more conservative amino acid substitutions may be possible. As will be recognized, a conservative amino acid substitution may include one in which an amino acid is substituted for another amino acid having similar properties such that the folding, activity, or other functionality of the protein is not significantly affected. Examples of aromatic amino acids, which may be substitutable, may include phenylalanine, tryptophan, and tyrosine. Examples of interchangeable hydrophobic amino acids, which may be substitutable, may include leucine, isoleucine, methionine, and valine. Examples of interchangeable polar amino acids, which may be substitutable, may include glutamine and asparagine. Examples of interchangeable basic amino acids, which may be substitutable, may include arginine, lysine, and histidine. Examples of interchangeable acidic amino acids, which may be substitutable, may include aspartic acid and glutamic acid. Finally, examples of interchangeable small amino acids, which may be substitutable, may include alanine, serine, threonine, cysteine, and glycine.
As described in detail herein, dominant-negative (DN) Rabi a (e.g. GDP -bound Rabi a, RablaGDP) may be used for increasing microautophagy, which may provide for degradation of a cellular target of interest such as a disease-related protein, glycogen, or lipid. As will be understood, any suitable Rabla DN or RablaGDP may be used. A variety of Rabi a DN proteins will be known to the person of skill in the art having regard to the teachings herein. In certain embodiments, generally any suitable dominant negative form of Rabla (i.e. a constant/locked Rabla in its GDP form) may be used to promote lysosomal movement toward the cytosol and peripheral of the cells (from perinuclear region of the cells), and stimulate direct lysosome interaction with target substrates, for example.
In certain embodiments, it is contemplated that expression of Rabla DN (GDP form) may stimulate activation of mT0RCl/mT0RC2 and AKT based on their effect on lysosomal positioning and promoting lysosome movement toward the peripheral and toward the target substrates (see also Jia, R. & Bonifacino, J. S. Lysosome Positioning Influences mT0RC2 and AKT Signaling. Molecular Cell 75, 26-38. e3 (2019)).
It is contemplated herein that different genetic modifications of Rabla which lock this protein in its constant GDP -bound state may be used to promote direct lysosome interaction and piecemeal engulfment of different target substrate(s) (such as one or more protein, lipid, and/or glycogen target(s)). Rabla constant GDP -bound form (DN) is generally not available in normal physiological conditions, in which the native protein Rabla is constantly shifting between its GTP and GDP forms. It is contemplated that in certain embodiments, genetic mutation and/or amino acid substitution/modification may be used to lock this GTPase in its GDP -bound form (or constant GTP form), and prevent it from going to its GTP state (or GDP state). It is contemplated that in certain embodiments, any suitable modification(s) that can substantially keep the integrity of the GDP form/state of GTPases (which may exert an impact on signaling as well as an effect on lysosome movement to peripheral/cytosol) may promote microautophagy and degradation of target substrate(s) through lysosomal piecemeal engulfment.
In certain embodiments, RablaGDP may be administered to a parti culalr cell type or subject in need thereof in generally any suitable manner which may be selected to suit the particular cell type, subject, and/or indication. In certain embodiments, a nucleic acid sequences encoding and capable of expressing the RablaGDP may be administered to the subject or introduced to the cell type via any suitable transfection or nucleic acid delivery approach as will be known to the person of skill in the art having regard to the teachings herein. In certain embodiments, delivery may be based on DNA or RNA transfection (for example, using common transfection regent(s) such as lipofectamine (Invitrogen), FuGENE (Rosche), using DNA adenovirus (gene therapy), or using Modified RNA (i.e. stabilized RNA) delivery to body (using, for example, virus or microvesicle, exosome, or ectosome). In certain embodiments, protein may be administered or delivered to cells in need thereof, optionally assisted with any suitable technique or delivery vehicle for facilitating protein delivery to a cell or cells.
In certain embodiments, |RablaGDP or RablaDN j[A2]may be administered to a particular cell type, such as cancer cells, using lipid nanoparticles (LNPs). It is envisioned that LNPs would encapsulate RablaGDP or RablaDN, nucleic acids capable of expressing RablaGDP or RablaDN, or a combination thereof.
LNPs are an advanced non-viral gene delivery system. LNPs allow someone skilled in the art to safely and effectively deliver nucleic acids to cells in vitro or in vivo, which has applications in gene editing, rapid vaccine development, immuno-oncology and treatment of rare genetic and undruggable diseases, without being limiting.
LNPs are being used more frequently in the art due to their advantageous properties, including their controlled and sustained-release properties, low toxicity, biocompatibility with tissues and cells, low immune response, increased deliverable gene size, cell free manufacturing, high nucleic acid encapsulation efficiency, potent transfection, and improved penetration into tissues to deliver therapeutics.
LNP formulations may be a combination of a number of lipids/molecules including, but not limited to, ionizable cationic lipids, neutral lipids, helper lipids, phospholipids, poly (lactic-co-glycolic acid) (PLGA), l,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), cholesterol, and polyethylene glycol (PEG)-lipid conjugates. The LNPs used may be custom made or purchased from commercially available sources, such as, but not limited to, TriLink BioTechnologies or Precision Nano System.
It is also envisioned that LNPs could be replaced by (liposomes or polymer nanoparti cles][A3] .
EXAMPLE 1: RablaDN (GDP form) Induces Cancer Cell Death and Stimulates Microautophagy A series of experiments were performed to investigate the anti-cancer effects of Rab 1 aDN on a wide variety of different cancer cell types. Various types of cancer cells were treated with RablaDN (in this example, RablaS25N, and the RablaDN was introduced into cells as either a transfected mRNA nucleic acid, or as a transfected DNA expression plasmid, that is capable of expressing RablaS25N or a fusion protein comprising RablaS25N and an N-terminal Luciferase tag in the cancer cells. In this Example, the RablaDN is also referred to as MG-008. Sequence of the ORF used in the DNA expression plasmid expressing a fusion protein comprising RablaS25N and an N-terminal Luciferase tag used in these studies is shown in Figure 36 as SEQ ID NO: 19. Sequence of the ORF of the mRNA expressing a fusion protein comprising Rab 1 aS25N and an N-terminal Luciferase tag used in these studies is shown in Figure 36 as SEQ ID NO: 20.
Amino acid sequence of the fusion protein comprising RablaS25N and an N-terminal Luciferase tag, which is expressed in cells following transfection with either the DNA expression plasmid or the mRNA described above, is shown in Figure 36 as SEQ ID NO: 21.
Treatment with mRNA:
Transfection of cancer cells with mRNA expressing RablaDN (also referred to as MG-008, comprising the ORF shown in Figure 36 as SEQ ID NO: 20; in this example, mRNA expressing a fusion protein comprising Rab 1 aS25N and an N-terminal Luciferase tag was used) in this example, mRNA expressing a fusion protein comprising RablaS25N and an N-terminal Luciferase tag was used. The protocol was performed according to the following transfection protocol:
Reagent.
Lipofectamine™ MessengerMAX™ Transfection Reagent (Thermo Fisher Scientific)
Protocol'.
1. Seed cells (5000 cell/well)
2. Incubation at 37 °C, 16 hours
3. Transfection of mRNA using Lipofectamine™ MessengerMAX™ Transfection Reagent (96 well plate)
(a) Dilute MessengerMAX™ reagent in Opti-MEM™ Medium (each well: 5 pL Opti-MEM™ Medium with 0.3 pL MessengerMAX™ reagent)
(b) Incubate diluted MessengerMAX™ Reagent in Opti-MEM™ Medium for 10 minutes at room temperature
(c) Dilute mRNA in Opti-MEM™ Medium (each well: 5 pL Opti-MEM™ Medium with 0.1 pg mRNA)
(d) Add diluted mRNA to diluted MessengerMAX™ Reagent, Incubate for 5 minutes at room temperature.
(e) Remove 50 pL cell culture medium (96 well)
(f) Add mRNA-lipid complex to cells (10 pL for each well)
(g) Add fresh 50 pL culture medium to each well
4. Incubation at 37 °C, 24 hours
5. Remove the transfection reagent containing medium
6. Wash by PBS twice
7. Add fresh 100 pL culture medium to each well
8. Incubation at 37 °C, 48 hours
9. Analyze the cell viability by crystal violet staining
Several different cancer cell types were tested, as shown in the accompanying Figures. For each cell-type, cell viability following treatment with mRNA expressing MG-008 was compared with cell viability of comparator control cells, and with cell viability of comparator cells treated with Lipofectamine MessengerMAX delivery vehicle (but no active MG-008, as another control comparator). For comparison, Vero cells (chlorocebus sabaeus (green monkey)) were also tested, showing selectivity of MG-008 for decreasing cell viability of cancer cells versus non-cancer cells.
The following cancer cell lines were tested in this study: A549 cells (lung cancer adenocarcinoma), HCT116 cells (colorectal carcinoma), HT29 cells (colon adenocarcinoma), HuCCTl (cholangiocarcinoma), PC-3 cells (prostate carcinoma), RD cells (embryonal rhabdomyosarcoma), SC-M1 cells (gastric carcinoma), U-87MG cells (glioblastoma), SW480 cells (colon adenocarcinoma), PANC-1 cells (pancreatic ductal adenocarcinoma), OECM-1 cells (squamous cell carcinoma of the oral cavity), OC2 cells (squamous cell carcinoma of the oral cavity), MIA PaCa-2 cells (pancreatic ductal adenocarcinoma), MDA-MB-468 cells (breast adenocarcinoma), MDA-MB-231 cells (breast adenocarcinoma), MCF-7 cells (invasive ductal carcinoma), Mahlavu cells (hepatocellular carcinoma), HSC-3 (squamous cell carcinoma of the oral tongue), HeLa cells (papillomavirus-related endocervical adenocarcinoma), and HCT116 cells (colorectal carcinoma). Data is shown in the Figures.
For all the cancer cells tested, treatment with mRNA expressing RablaDN, which was RablaS25N in this example, resulted in a marked decrease in cell viability of the cancer cells, as compared with the controls. Vero cells (chlorocebus sabaeus (green monkey)) were also tested (Figure 8), showing selectivity of MG-008/RablaDN for decreasing cell viability of cancer cells versus noncancer cells. Data for each cell type is shown in the Figures, each of which provide representative phase contrast microscopy images of cells treated with control, transfection vehicle, and MG-008 treated cells, along with a bar graph showing measured cell viability % (by crystal violet stain) of each.
Treatment with DNA Plasmid:
Transfection of a variety of different cancer cells with two different DNA plasmids expressing RablaDN was tested. A DNA plasmid expressing a fusion protein comprising RablaS25N and an N-terminal Luciferase tag (also referred to as MG-008-Luc), comprising the ORF shown in Figure 36 as SEQ ID NO: 19, was used. A DNA plasmid expressing the RablaS25N, but without the N- terminal Luciferase tag (also referred to as MG-008) was also tested. Transfection was performed according to the following transfection protocol: Reagent.
Lipofectamine™ 2000 Transfection Reagent (Thermo Fisher Scientific)
Protocol'.
1. Seed cells (5000 cell/well)
2. Incubation at 37 °C, 16 hours
3. Transfect plasmid DNA by Lipofectamine™ 2000 Transfection Reagent (96 well plate)
(a) Dilute Lipofectamine™ 2000 reagent in Opti-MEM™ Medium (each well:
5 pL Opti-MEM™ Medium with 0.3 pL Lipofectamine™ 2000 reagent)
(b) Dilute plasmid in Opti-MEM™ Medium (each well: 5 pL Opti-MEM™ Medium with 0.1 pg plasmid DNA)
(c) Add diluted plasmid DNA to diluted Lipofectamine™ 2000 Reagent, Incubate for 5 minutes at room temperature.
(d) Remove 50 pL cell culture medium (96 well)
(e) Add plasmid DNA-lipid complex to cells (10 pL for each well)
(f) Add fresh 50 pL culture medium to each well
4. Incubation at 37 °C, 24 hours
5. Remove the transfection reagent containing medium
6. Wash by PBS twice
7. Add fresh 100 pL culture medium to each well
8. Incubation at 37 °C, 48 hours 9. Analyze the cell viability by crystal violet staining
Several different cancer cell types were tested, as shown in Figures 24-40. For each cell-type, cell viability following treatment with DNA plasmid expressing MG-008 or MG-008-Luc was compared with cell viability of comparator control cells, and with cell viability of comparator cells treated with Lipofectamine 2000 delivery vehicle (but no active MG-008, as another control comparator). For comparison, Vero cells (chlorocebus sabaeus (green monkey)) were also tested, showing selectivity of MG-008 for decreasing cell viability of cancer cells versus non-cancer cells.
The following cancer cell lines were tested in this study: U-87MG cells (glioblastoma), SW480 cells (colon adenocarcinoma), SC-M1 (gastric carcinoma), PC-3 cells (prostate carcinoma), PANC-1 cells (pancreatic ductal adenocarcinoma), OC2 cells (squamous cell carcinoma of the oral cavity), MIA PaCa-2 cells (pancreatic ductal adenocarcinoma), MDA-MB-468 cells (breast adenocarcinoma), MDA-MB-231 cells (breast adenocarcinoma), MCF-7 cells (invasive ductal carcinoma), Mahlavu cells (hepatocellular carcinoma), HuCCTl cells (cholangiocarcinoma), HT29 cells (colon adenocarcinoma), HeLa cells (papillomavirus-related endocervical adenocarcinoma), HCT116 cells (colorectal carcinoma), and A549 cells (lung adenocarcinoma). Data is shown in Figures 1-35.
For all the cancer cells tested, treatment with DNA plasmid expressing RablaDN, which was RablaS25N in this example (either with or without an N-terminal Luciferase tag), resulted in a marked decrease in cell viability of the cancer cells, as compared with the controls. Vero cells (chlorocebus sabaeus (green monkey)) were also tested, showing selectivity of MG-008/RablaDN for decreasing cell viability of cancer cells versus non-cancer cells. Data for each cell type is shown in Figures 1-35, each of which provide representative phase contrast microscopy images of cells treated with control, transfection vehicle, MG-008, or MG-008-Luc treated cells, along with a bar graph showing measured cell viability % (by crystal violet stain) of each.
The data obtained in these studies supports anti-cancer effect of RablaDN on a wide variety of different cancer cell types. Various types of cancer cells were treated with RablaDN (in this example, RablaS25N, and the RablaDN was introduced into cells as either a transfected mRNA nucleic acid, or as a transfected DNA expression plasmid, that is capable of expressing RablaS25N or a fusion protein comprising RablaS25N and an N-terminal Luciferase tag in the cancer cells. Results show a selectivity for decreasing cell viability of cancer cells versus normal cells, and an anti-cancer effect was observed across many different cancer cell types, supporting broad anticancer applicability.
One or more illustrative embodiments have been described by way of example. It will be understood to persons skilled in the art that a number of variations and modifications can be made without departing from the scope of the invention as defined in the claims.
Example 2: In vivo reduction in tumor cell growth and metastasis following treatment with lipid nanoparticle-encapsulated RablaDN
A series of experiments were performed to investigate the in vivo anti-cancer effects of Fl 1. Fl 1, as mentioned in the following paragraphs and in Figures 37-40, is RablaDN encapsulated in lipid nanoparticles (LNPs).
In this example, the efficacy of Fl l treatment in Balb/c mice that had been injected with 4T1 cancer cells was established. Each female Balb/c mouse (7 weeks old) was injected with 1 x 106 4T1-Fluc-Neo cells (a mouse mammary carcinoma cell line expressing luciferase) into the left 4th mammary inguinal gland. One week later, the mouse was given a single intravenous (IV) dose of Fl l at 0.05 mpk (in 100 pl PBS). The untreated control mouse was injected with PBS alone. Subsequently, the in vivo imaging system (IVIS) images of bioluminescence derived from the 4T1 tumor cells were documented weekly for the next consecutive 7 weeks (FIG. 37). The total luciferin flux obtained from this experiment was then quantified (FIG. 38). Metastasis was observed in the untreated control mouse at day 30 (FIG. 38A), and the mice died at day 37. In the mouse injected with Fl 1, no metastasis was observed throughout the entire 7 weeks of experiment (FIG. 38B). These data suggest that a single IV treatment with Fl l could effectively suppress 4T1 metastasis in this mouse model.
A further experiment was conducted to determine the morphological changes that occurred at the FT1 injection site in Balb/c mice following Fl l or PBS (control) administration. Again, each female Balb/c mouse was injected with 4T1 cancer cells into the left 4th mammary inguinal gland, as described above. One week later, the mouse was given a single dose of F 11 intravenously (IV), and the control mouse was injected with PBS alone. Morphological changes at the 4T1 injection site were monitored. In the control mouse, a tumor lump was palpable at day 14 post-IV treatment, and it grew further until the mouse died at day 35 (images in the upper panel of FIG. 39). In the Fl 1 treatment mouse, the tumor lump was undetectable until day 35 post-IV treatment (images in the lower panel of FIG. 39). These data suggest that a single Fl l treatment could effectively attenuate 4T1 tumor cell growth in the mice.
To supplement the treatment efficacy data, mice bodyweight was monitored to determine if there were any detrimental health effects from the Fl l treatment. For this experiment, the Balb/c mice were injected with 4T1 tumor cells as described above. One week later (day 0), the mouse was given a single dose of Fl 1 intravenously (IV) or PBS as the control. Body weight was measured until the end of the experiment (total of 7 weeks post-IV treatment). The body weight of the PBS control mouse did not change much before it died at day 35 ([blue line, lower line). The Fl 1 treated mouse showed bodyweight gain throughout the entire experiment (orange line, upper line|[A4]). These data suggest that Fl l treatment exerted no detrimental effect on the overall health conditions in this mouse model.
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W02017/008141 - Lysosomal Degradation of Lipids and Proteins and Method of Use Thereof
All references cited herein and throughout this specification are herein incorporated by reference in their entireties.

Claims

WHAT IS CLAIMED IS:
1. A method for reducing cell viability of a cancer cell, or for preventing or treating cancer, in a subject in need thereof, said method comprising: treating a cancer cell of the subject with a GDP -bound form of Rabi a (RablaGDP), one or more expressible nucleic acids encoding RablaGDP, or a combination thereof; thereby increasing a cellular level of RablaGDP in the cancer cell, resulting in decreased cell viability or death of the cancer cell.
2. A method for reducing cell viability of a cancer cell in vitro or in vivo, said method comprising: treating the cancer cell with a GDP -bound form of Rabi a (RablaGDP), one or more expressible nucleic acids encoding RablaGDP, or a combination thereof; thereby increasing a cellular level of RablaGDP in the cancer cell, resulting in decreased cell viability or death of the cancer cell.
3. The method of claim 1 or 2, wherein the RablaGDP is or comprises RablaS25N, RablaN1241, RablaD41N, RablaD47N, or another dominant negative (DN) GDP -bound form of Rabi a.
4. The method of any one of claims 1-3, wherein the RablaGDP comprises the amino acid sequence:
MSSMNPEYDYLFKLLLIGDSGVGKNCLLLRFADDTYTESYISTIGVDFKIRTIELDGK TIKLQIWDTAGQERFRTITSSYYRGAHGIIVVYDVTDQESFNNVKQWLQEIDRYASE NVNKLLVGNKCDLTTKKVVDYTTAKEFADSLGIPFLETSAKNATNVEQSFMTMAAE IKKRMGPGATAGGAEKSNVKIQSTPVKQSGGGCC
(SEQ ID NO: 6; Human RablaS25N);
MGDYKDDDDKGGSGGSSMNPEYDYLFKLLLIGDSGVGKSCLLLRFADDTYTESYIS TIGVDFKIRTIELDGKTIKLQIWDTAGQERFRTITSSYYRGAHGIIVVYDVTDQESFNN VKQWLQEIDRYASENVNKLLVGIKCDLTTKKVVDYTTAKEFADSLGIPFLETSAKNA TNVEQSFMTMAAEIKKRMGPGATAGGAEKSNVKIQSTPVKQSGGGCC (SEQ ID NO: 9; Mouse RablN1241); the amino acid sequence of human RablaD41N; or
MSSMNPEYDYLFKLLLIGDSGVGKSCLLLRFADDTYTESYISTIGVNFKIRTIELDGKT
IKLQIWDTAGQERFRTITSSYYRGAHGIIVVYDVTDQESFNNVKQWLQEIDRYASEN VNKLLVGNKCDLTTKKVVDYTTAKEFADSLGIPFLETSAKNATNVEQSFMTMAAEI KKRMGPGATAGGAEKSNVKIQSTPVKQSGGGCC
(SEQ ID NO: 18; Human RablaD47N); or a polypeptide having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with any of these sequences and preferentially binding GDP. The method of any one of claims 1-4, wherein the RablaGDP consists of the amino acid sequence:
MSSMNPEYDYLFKLLLIGDSGVGKNCLLLRFADDTYTESYISTIGVDFKIRTIELDGK
TIKLQIWDTAGQERFRTITSSYYRGAHGIIVVYDVTDQESFNNVKQWLQEIDRYASE
NVNKLLVGNKCDLTTKKVVDYTTAKEFADSLGIPFLETSAKNATNVEQSFMTMAAE IKKRMGPGATAGGAEKSNVKIQSTPVKQSGGGCC
(SEQ ID NO: 6; Human RablaS25N);
MGDYKDDDDKGGSGGSSMNPEYDYLFKLLLIGDSGVGKSCLLLRFADDTYTESYIS
TIGVDFKIRTIELDGKTIKLQIWDTAGQERFRTITSSYYRGAHGIIVVYDVTDQESFNN
VKQWLQEIDRYASENVNKLLVGIKCDLTTKKVVDYTTAKEFADSLGIPFLETSAKNA
TNVEQSFMTMAAEIKKRMGPGATAGGAEKSNVKIQSTPVKQSGGGCC
(SEQ ID NO: 9; Mouse RablN1241); the amino acid sequence of human RablaD41N; or
MSSMNPEYDYLFKLLLIGDSGVGKSCLLLRFADDTYTESYISTIGVNFKIRTIELDGKT
IKLQIWDTAGQERFRTITSSYYRGAHGIIVVYDVTDQESFNNVKQWLQEIDRYASEN VNKLLVGNKCDLTTKKVVDYTTAKEFADSLGIPFLETSAKNATNVEQSFMTMAAEI KKRMGPGATAGGAEKSNVKIQSTPVKQSGGGCC
(SEQ ID NO: 18; Human RablaD47N); or a polypeptide having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with any of these sequences and preferentially binding GDP. The method of any one of claims 1 -4, wherein the Rab 1 aGDP comprises or consists of the amino acid sequence:
MSSMNPEYDYLFKLLLIGDSGVGKNCLLLRFADDTYTESYISTIGVDFKIRTIELDGK TIKLQIWDTAGQERFRTITSSYYRGAHGIIVVYDVTDQESFNNVKQWLQEIDRYASE NVNKLLVGNKCDLTTKKVVDYTTAKEFADSLGIPFLETSAKNATNVEQSFMTMAAE IKKRMGPGATAGGAEKSNVKIQSTPVKQSGGGCC
(SEQ ID NO: 6; Human RablaS25N); or a polypeptide having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity therewith and preferentially binding GDP. The method of any one of claims 1-4, wherein the RablaGDP is in the form of a fusion protein, wherein the RablaGDP is fused or otherwise directly or indirectly linked, optionally via a linker, with a signalling or targeting peptide, a fluorescent peptide or other marker or tracer, or another peptide or non-peptide moiety for targeted delivery, facilitating cell uptake, increasing stability or in vivo half-life, or improving another therapeutic, diagnostic, or in vivo property of the RablaGDP. The method of claim 7, wherein the fusion protein comprises the amino acid sequence:
MSSMNPEYDYLFKLLLIGDSGVGKNCLLLRFADDTYTESYISTIGVDFKIRTIELDGK TIKLQIWDTAGQERFRTITSSYYRGAHGIIVVYDVTDQESFNNVKQWLQEIDRYASE NVNKLLVGNKCDLTTKKVVDYTTAKEFADSLGIPFLETSAKNATNVEQSFMTMAAE IKKRMGPGATAGGAEKSNVKIQSTPVKQSGGGCC
(SEQ ID NO: 6; Human RablaS25N); or a polypeptide having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity therewith and preferentially binding GDP. The method of claim 7 or 8, wherein the RablaGDP is in the form of a fusion protein, and comprises the amino acid sequence:
MEDAKNIKKGPAPFYPLEDGTAGEQLHKAMKRYALVPGTIAFTDAHIEVNITYAEYF EMSVRLAEAMKRYGLNTNHRIVVCSENSLQFFMPVLGALFIGVAVAPANDIYNERE LLNSMNISQPTVVFVSKKGLQKILNVQKKLPIIQKIIIMDSKTDYQGFQSMYTFVTSHL PPGFNEYDF VPESFDRDKTIALIMNS SGSTGLPKGVALPHRT ACVRF SHARDPIFGNQI IPDTAILSVVPFHHGFGMFTTLGYLICGFRVVLMYRFEEELFLRSLQDYKIQSALLVPT LFSFFAKSTLIDKYDLSNLHEIASGGAPLSKEVGEAVAKRFHLPGIRQGYGLTETTSAI LITPEGDDKPGAVGKVVPFFEAKVVDLDTGKTLGVNQRGELCVRGPMIMSGYVNNP EATNALIDKDGWLHSGDIAYWDEDEHFFIVDRLKSLIKYKGYQVAPAELESILLQHP NIFDAGVAGLPDDDAGELPAAVVVLEHGKTMTEKEIVDYVASQVTTAKKLRGGVV FVDEVPKGLTGKLDARKIREILIKAKKGGKSKLMSSMNPEYDYLFKLLLIGDSGVGK NCLLLRFADDTYTESYISTIGVDFKIRTIELDGKTIKLQIWDTAGQERFRTITSSYYRG AHGIIVVYDVTDQESFNNVKQWLQEIDRYASENVNKLLVGNKCDLTTKKVVDYTT
AKEFADSLGIPFLETSAKNATNVEQSFMTMAAEIKKRMGPGATAGGAEKSNVKIQST PVKQSGGGCC
(SEQ ID NO: 21); or a polypeptide having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity therewith and preferentially binding GDP. The method of claim 1 or 2, wherein the one or more expressible nucleic acids encode one or more RablaGDP as defined in any one of claims 3-9. The method of claim 10, wherein the one or more expressible nucleic acids are DNA-based, or RNA-based. The method of claim 10 or 11, wherein the one or more expressible nucleic acids transiently express the Rab 1 aGDP in the cell, or wherein the one or more expressible nucleic acids integrate in the cell genome and express the RablaGDP in the cell. The method of any one of claims 1, 2, or 10, wherein the one or more expressible nucleic acids comprise one or more expression vectors, plasmids, or mRNAs encoding and capable of expressing the RablaGDP inside the cell. The method of claim 13, wherein the one or more expressible nucleic acids comprise a nucleic acid sequence of
ATGTCCAGCATGAATCCCGAATATGATTATTTATTCAAGTTACTTCTGATTGGCG ACTCAGGGGTTGGAAAGAATTGCCTTCTTCTTAGGTTTGCAGATGATACATATAC AGAAAGCTACATCAGCACAATTGGTGTGGATTTCAAAATAAGAACTATAGAGTT AGACGGGAAAACAATCAAGCTTCAAATATGGGACACAGCAGGCCAGGAAAGAT TTCGAACAATCACCTCCAGTTATTACAGAGGAGCCCATGGCATCATAGTTGTGTA TGATGTGACAGATCAGGAGTCCTTCAATAATGTTAAACAGTGGCTGCAGGAAAT AGATCGTTATGCCAGTGAAAATGTCAACAAATTGTTGGTAGGGAACAAATGTGA TCTGACCACAAAGAAAGTAGTAGACTACACAACAGCGAAGGAATTTGCTGATTC CCTTGGAATTCCGTTTTTGGAAACCAGTGCTAAGAATGCAACGAATGTAGAACA GTCTTTCATGACGATGGCAGCTGAGATTAAAAAGCGAATGGGTCCCGGAGCAAC AGCTGGTGGTGCTGAGAAGTCCAATGTTAAAATTCAGAGCACTCCAGTCAAGCA GTCAGGTGGAGGTTGCTGCTAA
(Human RablaS25N ORF Codon Sequence, SEQ ID NO: 5);
ATGGGGGACTACAAGGACGACGATGACAAGGGGGGTAGCGGTGGATCCAGCAT GAATCCCGAATATGATTATTTATTCAAGTTACTTCTGATTGGCGATTCTGGGGTT GGAAAGTCCTGCCTTCTCCTTAGGTTTGCAGATGATACGTATACGGAAAGCTACA
TCAGCACAATTGGTGTGGATTTCAAGATACGAACTATAGAGTTAGATGGGAAAA
CAATCAAGCTACAGATATGGGACACAGCAGGCCAGGAAAGATTTCGAACAATCA
CTTCCAGTTATTACAGAGGAGCCCATGGCATCATAGTTGTGTATGATGTGACAGA
TCAGGAGTCCTTCAATAACGTTAAACAGTGGCTGCAGGAGATAGATCGCTACGC
CAGTGAAAATGTCAACAAGTTGTTGGTAGGGATCAAATGTGACCTGACCACAAA
GAAAGTAGTAGACTACACAACAGCAAAGGAATTTGCAGATTCCCTTGGAATTCC
ATTTTTGGAAACCAGTGCTAAGAACGCAACGAATGTAGAACAGTCTTTCATGAC
GATGGCAGCTGAGATTAAAAAGCGAATGGGTCCTGGAGCTACAGCTGGTGGTGC
CGAGAAGTCCAATGTTAAAATCCAGAGCACTCCAGTCAAGCAGTCAGGTGGAGG CTGCTGCTAA
(Mouse RablaN1241 ORF Codon Seqeunce, SEQ ID NO: 8); or
ATGTCCAGCATGAATCCCGAATATGATTATTTATTCAAGTTACTTCTGATTGGCGACT
CAGGGGTTGGAAAGTCTTGCCTTCTTCTTAGGTTTGCAGATGATACATATACAGAAA GCTACATCAGCACAATTGGTGTGAACTTCAAAATAAGAACTATAGAGTTAGACGGG AAAACAATCAAGCTTCAAATATGGGACACAGCAGGCCAGGAAAGATTTCGAACAAT CACCTCCAGTTATTACAGAGGAGCCCATGGCATCATAGTTGTGTATGATGTGACAGA TCAGGAGTCCTTCAATAATGTTAAACAGTGGCTGCAGGAAATAGATCGTTATGCCAG TGAAAATGTCAACAAATTGTTGGTAGGGAACAAATGTGATCTGACCACAAAGAAAG TAGTAGACTACACAACAGCGAAGGAATTTGCTGATTCCCTTGGAATTCCGTTTTTGG AAACCAGTGCTAAGAATGCAACGAATGTAGAACAGTCTTTCATGACGATGGCAGCT GAGATTAAAAAGCGAATGGGTCCCGGAGCAACAGCTGGTGGTGCTGAGAAGTCCAA TGTTAAAATTCAGAGCACTCCAGTCAAGCAGTCAGGTGGAGGTTGCTGCTAA
(Human RablaD47N ORF Codon Sequence, SEQ ID NO: 17); or a nucleic acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity therewith and encoding a RablaGDP preferentially binding GDP; or a nucleic acid sequence equivalent to any of the above sequences due to codon redundancy. The method of claim 13, wherein the one or more expressible nucleic acids comprise a nucleic acid sequence of:
ATGGAAGACGCCAAAAACATAAAGAAAGGCCCGGCGCCATTCTATCCGCTAGAG GATGGAACCGCTGGAGAGCAACTGCATAAGGCTATGAAGAGATACGCCCTGGTT CCTGGAACAATTGCTTTTACAGATGCACATATCGAGGTGAACATCACGTACGCG GAATACTTCGAAATGTCCGTTCGGTTGGCAGAAGCTATGAAACGATATGGGCTG AATACAAATCACAGAATCGTCGTATGCAGTGAAAACTCTCTTCAATTCTTTATGC CGGTGTTGGGCGCGTTATTTATCGGAGTTGCAGTTGCGCCCGCGAACGACATTTA TAATGAACGTGAATTGCTCAACAGTATGAACATTTCGCAGCCTACCGTAGTGTTT GTTTCCAAAAAGGGGTTGCAAAAAATTTTGAACGTGCAAAAAAAATTACCAATA ATCCAGAAAATTATTATCATGGATTCTAAAACGGATTACCAGGGATTTCAGTCGA TGTACACGTTCGTCACATCTCATCTACCTCCCGGTTTTAATGAATACGATTTTGTA CCAGAGTCCTTTGATCGTGACAAAACAATTGCACTGATAATGAACTCCTCTGGAT CTACTGGGTTACCTAAGGGTGTGGCCCTTCCGCATAGAACTGCCTGCGTCAGATT CTCGCATGCCAGAGATCCTATTTTTGGCAATCAAATCATTCCGGATACTGCGATT TTAAGTGTTGTTCCATTCCATCACGGTTTTGGAATGTTTACTACACTCGGATATTT GATATGTGGATTTCGAGTCGTCTTAATGTATAGATTTGAAGAAGAGCTGTTTTTA CGATCCCTTCAGGATTACAAAATTCAAAGTGCGTTGCTAGTACCAACCCTATTTT CATTCTTCGCCAAAAGCACTCTGATTGACAAATACGATTTATCTAATTTACACGA AATTGCTTCTGGGGGCGCACCTCTTTCGAAAGAAGTCGGGGAAGCGGTTGCAAA ACGCTTCCATCTTCCAGGGATACGACAAGGATATGGGCTCACTGAGACTACATC AGCTATTCTGATTACACCCGAGGGGGATGATAAACCGGGCGCGGTCGGTAAAGT TGTTCCATTTTTTGAAGCGAAGGTTGTGGATCTGGATACCGGGAAAACGCTGGGC
GTTAATCAGAGAGGCGAATTATGTGTCAGAGGACCTATGATTATGTCCGGTTATG
TAAACAATCCGGAAGCGACCAACGCCTTGATTGACAAGGATGGATGGCTACATT CTGGAGACATAGCTTACTGGGACGAAGACGAACACTTCTTCATAGTTGACCGCTT GAAGTCTTTAATTAAATACAAAGGATACCAGGTGGCCCCCGCTGAATTGGAGTC GATATTGTTACAACACCCCAACATCTTCGACGCGGGCGTGGCAGGTCTTCCCGAC GATGACGCCGGTGAACTTCCCGCCGCCGTTGTTGTTTTGGAGCACGGAAAGACG ATGACGGAAAAAGAGATCGTGGATTACGTCGCCAGTCAAGTAACAACCGCCAAA AAGTTGCGCGGAGGAGTTGTGTTTGTGGACGAAGTACCGAAAGGTCTTACCGGA
AAACTCGACGCAAGAAAAATCAGAGAGATCCTCATAAAGGCCAAGAAGGGCGG
AAAGTCCAAATTGATGTCCAGCATGAATCCCGAATATGATTATTTATTCAAGTTA
CTTCTGATTGGCGACTCAGGGGTTGGAAAGAATTGCCTTCTTCTTAGGTTTGCAG
ATGATACATATACAGAAAGCTACATCAGCACAATTGGTGTGGATTTCAAAATAA
GAACTATAGAGTTAGACGGGAAAACAATCAAGCTTCAAATATGGGACACAGCAG
GCCAGGAAAGATTTCGAACAATCACCTCCAGTTATTACAGAGGAGCCCATGGCA
TCATAGTTGTGTATGATGTGACAGATCAGGAGTCCTTCAATAATGTTAAACAGTG
GCTGCAGGAAATAGATCGTTATGCCAGTGAAAATGTCAACAAATTGTTGGTAGG
GAACAAATGTGATCTGACCACAAAGAAAGTAGTAGACTACACAACAGCGAAGG
AATTTGCTGATTCCCTTGGAATTCCGTTTTTGGAAACCAGTGCTAAGAATGCAAC
GAATGTAGAACAGTCTTTCATGACGATGGCAGCTGAGATTAAAAAGCGAATGGG
TCCCGGAGCAACAGCTGGTGGTGCTGAGAAGTCCAATGTTAAAATTCAGAGCAC
TCCAGTCAAGCAGTCAGGTGGAGGTTGCTGCTAA
(SEQ ID NO: 19, MG-008 ORF DNA Sequence with 5’ Luciferase Tag); or
AUGGAAGACGCCAAAAACAUAAAGAAAGGCCCGGCGCCAUUCUAUCCGCUAG
AGGAUGGAACCGCUGGAGAGCAACUGCAUAAGGCUAUGAAGAGAUACGCCCU
GGUUCCUGGAACAAUUGCUUUUACAGAUGCACAUAUCGAGGUGAACAUCACG
UACGCGGAAUACUUCGAAAUGUCCGUUCGGUUGGCAGAAGCUAUGAAACGAU
AUGGGCUGAAUACAAAUCACAGAAUCGUCGUAUGCAGUGAAAACUCUCUUCA
AUUCUUUAUGCCGGUGUUGGGCGCGUUAUUUAUCGGAGUUGCAGUUGCGCCC
GCGAACGACAUUUAUAAUGAACGUGAAUUGCUCAACAGUAUGAACAUUUCGC
AGCCUACCGUAGUGUUUGUUUCCAAAAAGGGGUUGCAAAAAAUUUUGAACGU
GCAAAAAAAAUUACCAAUAAUCCAGAAAAUUAUUAUCAUGGAUUCUAAAACG
GAUUACCAGGGAUUUCAGUCGAUGUACACGUUCGUCACAUCUCAUCUACCUCC
CGGUUUUAAUGAAUACGAUUUUGUACCAGAGUCCUUUGAUCGUGACAAAACA
AUUGCACUGAUAAUGAACUCCUCUGGAUCUACUGGGUUACCUAAGGGUGUGG
CCCUUCCGCAUAGAACUGCCUGCGUCAGAUUCUCGCAUGCCAGAGAUCCUAUU
UUUGGCAAUCAAAUCAUUCCGGAUACUGCGAUUUUAAGUGUUGUUCCAUUCC AUCACGGUUUUGGAAUGUUUACUACACUCGGAUAUUUGAUAUGUGGAUUUCG
AGUCGUCUUAAUGUAUAGAUUUGAAGAAGAGCUGUUUUUACGAUCCCUUCAG
GAUUACAAAAUUCAAAGUGCGUUGCUAGUACCAACCCUAUUUUCAUUCUUCG
CCAAAAGCACUCUGAUUGACAAAUACGAUUUAUCUAAUUUACACGAAAUUGC
UUCUGGGGGCGCACCUCUUUCGAAAGAAGUCGGGGAAGCGGUUGCAAAACGC
UUCCAUCUUCCAGGGAUACGACAAGGAUAUGGGCUCACUGAGACUACAUCAG
CUAUUCUGAUUACACCCGAGGGGGAUGAUAAACCGGGCGCGGUCGGUAAAGU
UGUUCCAUUUUUUGAAGCGAAGGUUGUGGAUCUGGAUACCGGGAAAACGCUG
GGCGUUAAUCAGAGAGGCGAAUUAUGUGUCAGAGGACCUAUGAUUAUGUCCG
GUUAUGUAAACAAUCCGGAAGCGACCAACGCCUUGAUUGACAAGGAUGGAUG
GCUACAUUCUGGAGACAUAGCUUACUGGGACGAAGACGAACACUUCUUCAUA
GUUGACCGCUUGAAGUCUUUAAUUAAAUACAAAGGAUACCAGGUGGCCCCCG
CUGAAUUGGAGUCGAUAUUGUUACAACACCCCAACAUCUUCGACGCGGGCGU
GGCAGGUCUUCCCGACGAUGACGCCGGUGAACUUCCCGCCGCCGUUGUUGUUU
UGGAGCACGGAAAGACGAUGACGGAAAAAGAGAUCGUGGAUUACGUCGCCAG
UCAAGUAACAACCGCCAAAAAGUUGCGCGGAGGAGUUGUGUUUGUGGACGAA
GUACCGAAAGGUCUUACCGGAAAACUCGACGCAAGAAAAAUCAGAGAGAUCC
UCAUAAAGGCCAAGAAGGGCGGAAAGUCCAAAUUGAUGUCCAGCAUGAAUCC
CGAAUAUGAUUAUUUAUUCAAGUUACUUCUGAUUGGCGACUCAGGGGUUGGA
AAGAAUUGCCUUCUUCUUAGGUUUGCAGAUGAUACAUAUACAGAAAGCUACA
UCAGCACAAUUGGUGUGGAUUUCAAAAUAAGAACUAUAGAGUUAGACGGGAA
AACAAUCAAGCUUCAAAUAUGGGACACAGCAGGCCAGGAAAGAUUUCGAACA
AUCACCUCCAGUUAUUACAGAGGAGCCCAUGGCAUCAUAGUUGUGUAUGAUG
UGACAGAUCAGGAGUCCUUCAAUAAUGUUAAACAGUGGCUGCAGGAAAUAGA
UCGUUAUGCCAGUGAAAAUGUCAACAAAUUGUUGGUAGGGAACAAAUGUGAU
CUGACCACAAAGAAAGUAGUAGACUACACAACAGCGAAGGAAUUUGCUGAUU
CCCUUGGAAUUCCGUUUUUGGAAACCAGUGCUAAGAAUGCAACGAAUGUAGA
ACAGUCUUUCAUGACGAUGGCAGCUGAGAUUAAAAAGCGAAUGGGUCCCGGA
GCAACAGCUGGUGGUGCUGAGAAGUCCAAUGUUAAAAUUCAGAGCACUCCAG
UCAAGCAGUCAGGUGGAGGUUGCUGCUAA (SEQ ID NO: 20, MG-008 ORF mRNA Sequence with 5’ Luciferase Tag); or a nucleic acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity therewith and encoding a RablaGDP preferentially binding GDP; or a nucleic acid sequence equivalent to any of the above sequences due to codon redundancy. Use of a GDP -bound form of Rabi a (RablaGDP), one or more expressible nucleic acids encoding RablaGDP, or a combination thereof, for reducing cell viability of a cancer cell, or for preventing or treating cancer, in a subject in need thereof. Use of a GDP -bound form of Rabi a (RablaGDP), one or more expressible nucleic acids encoding RablaGDP, or a combination thereof, for reducing cell viability of a cancer cell in vitro or in vivo. Use of a GDP -bound form of Rabi a (RablaGDP), one or more expressible nucleic acids encoding Rab 1 aGDP, or a combination thereof, in the manufacture of a medicament for reducing cell viability of a cancer cell, or for preventing or treating cancer, in a subject in need thereof. Use of a GDP -bound form of Rab la (RablaGDP), one or more expressible nucleic acids encoding RablaGDP, or a combination thereof, in the manufacture of a medicament for reducing cell viability of a cancer cell in vitro or in vivo. The use of any one of claims 16-19, wherein the RablaGDP is or comprises RablaS25N, RablaN1241, RablaD41N, RablaD47N, or another dominant negative (DN) GDP -bound form of Rab la. The use of any one of claims 16-20, wherein the RablaGDP comprises the amino acid sequence:
MSSMNPEYDYLFKLLLIGDSGVGKNCLLLRFADDTYTESYISTIGVDFKIRTIELDGK TIKLQIWDTAGQERFRTITSSYYRGAHGIIVVYDVTDQESFNNVKQWLQEIDRYASE NVNKLLVGNKCDLTTKKVVDYTTAKEFADSLGIPFLETSAKNATNVEQSFMTMAAE IKKRMGPGATAGGAEKSNVKIQSTPVKQSGGGCC (SEQ ID NO: 6; Human RablaS25N);
MGDYKDDDDKGGSGGSSMNPEYDYLFKLLLIGDSGVGKSCLLLRFADDTYTESYIS
TIGVDFKIRTIELDGKTIKLQIWDTAGQERFRTITSSYYRGAHGIIVVYDVTDQESFNN
VKQWLQEIDRYASENVNKLLVGIKCDLTTKKVVDYTTAKEFADSLGIPFLETSAKNA TNVEQSFMTMAAEIKKRMGPGATAGGAEKSNVKIQSTPVKQSGGGCC
(SEQ ID NO: 9; Mouse RablN1241); the amino acid sequence of human RablaD41N; or
MSSMNPEYDYLFKLLLIGDSGVGKSCLLLRFADDTYTESYISTIGVNFKIRTIELDGKT
IKLQIWDTAGQERFRTITSSYYRGAHGIIVVYDVTDQESFNNVKQWLQEIDRYASEN VNKLLVGNKCDLTTKKVVDYTTAKEFADSLGIPFLETSAKNATNVEQSFMTMAAEI KKRMGPGATAGGAEKSNVKIQSTPVKQSGGGCC
(SEQ ID NO: 18; Human RablaD47N); or a polypeptide having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with any of these sequences and preferentially binding GDP. The use of any one of claims 16-21, wherein the RablaGDP consists of the amino acid sequence:
MSSMNPEYDYLFKLLLIGDSGVGKNCLLLRFADDTYTESYISTIGVDFKIRTIELDGK
TIKLQIWDTAGQERFRTITSSYYRGAHGIIVVYDVTDQESFNNVKQWLQEIDRYASE
NVNKLLVGNKCDLTTKKVVDYTTAKEFADSLGIPFLETSAKNATNVEQSFMTMAAE
IKKRMGPGATAGGAEKSNVKIQSTPVKQSGGGCC
(SEQ ID NO: 6; Human RablaS25N);
MGDYKDDDDKGGSGGSSMNPEYDYLFKLLLIGDSGVGKSCLLLRFADDTYTESYIS
TIGVDFKIRTIELDGKTIKLQIWDTAGQERFRTITSSYYRGAHGIIVVYDVTDQESFNN
VKQWLQEIDRYASENVNKLLVGIKCDLTTKKVVDYTTAKEFADSLGIPFLETSAKNA TNVEQSFMTMAAEIKKRMGPGATAGGAEKSNVKIQSTPVKQSGGGCC
(SEQ ID NO: 9; Mouse RablN1241); the amino acid sequence of human RablaD41N; or
MSSMNPEYDYLFKLLLIGDSGVGKSCLLLRFADDTYTESYISTIGVNFKIRTIELDGKT IKLQIWDTAGQERFRTITSSYYRGAHGIIVVYDVTDQESFNNVKQWLQEIDRYASEN VNKLLVGNKCDLTTKKVVDYTTAKEFADSLGIPFLETSAKNATNVEQSFMTMAAEI KKRMGPGATAGGAEKSNVKIQSTPVKQSGGGCC
(SEQ ID NO: 18; Human RablaD47N); or a polypeptide having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with any of these sequences and preferentially binding GDP. The use of any one of claims 16-21, wherein the RablaGDP comprises or consists of the amino acid sequence:
MSSMNPEYDYLFKLLLIGDSGVGKNCLLLRFADDTYTESYISTIGVDFKIRTIELDGK TIKLQIWDTAGQERFRTITSSYYRGAHGIIVVYDVTDQESFNNVKQWLQEIDRYASE NVNKLLVGNKCDLTTKKVVDYTTAKEFADSLGIPFLETSAKNATNVEQSFMTMAAE IKKRMGPGATAGGAEKSNVKIQSTPVKQSGGGCC
(SEQ ID NO: 6; Human RablaS25N); or a polypeptide having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity therewith and preferentially binding GDP. The use of any one of claims 16-21, wherein the RablaGDP is in the form of a fusion protein, wherein the RablaGDP is fused or otherwise directly or indirectly linked, optionally via a linker, with a signalling or targeting peptide, a fluorescent peptide or other marker or tracer, or another peptide or non-peptide moiety for targeted delivery, facilitating cell uptake, increasing stability or in vivo half-life, or improving another therapeutic, diagnostic, or in vivo property of the RablaGDP. The use of claim 24, wherein the fusion protein comprises the amino acid sequence: MSSMNPEYDYLFKLLLIGDSGVGKNCLLLRFADDTYTESYISTIGVDFKIRTIELDGK TIKLQIWDTAGQERFRTITSSYYRGAHGIIVVYDVTDQESFNNVKQWLQEIDRYASE NVNKLLVGNKCDLTTKKVVDYTTAKEFADSLGIPFLETSAKNATNVEQSFMTMAAE IKKRMGPGATAGGAEKSNVKIQSTPVKQSGGGCC (SEQ ID NO: 6; Human RablaS25N); or a polypeptide having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity therewith and preferentially binding GDP. The use of claim 24 or 25, wherein the RablaGDP is in the form of a fusion protein, and comprises the amino acid sequence:
MEDAKNIKKGPAPFYPLEDGTAGEQLHKAMKRYALVPGTIAFTDAHIEVNITYAEYF EMSVRLAEAMKRYGLNTNHRIVVCSENSLQFFMPVLGALFIGVAVAPANDIYNERE LLNSMNISQPTVVFVSKKGLQKILNVQKKLPIIQKIIIMDSKTDYQGFQSMYTFVTSHL PPGFNEYDF VPESFDRDKTIALIMNS SGSTGLPKGVALPHRT ACVRF SHARDPIFGNQI IPDTAILSVVPFHHGFGMFTTLGYLICGFRVVLMYRFEEELFLRSLQDYKIQSALLVPT LFSFFAKSTLIDKYDLSNLHEIASGGAPLSKEVGEAVAKRFHLPGIRQGYGLTETTSAI LITPEGDDKPGAVGKVVPFFEAKVVDLDTGKTLGVNQRGELCVRGPMIMSGYVNNP EATNALIDKDGWLHSGDIAYWDEDEHFFIVDRLKSLIKYKGYQVAPAELESILLQHP NIFDAGVAGLPDDDAGELPAAVVVLEHGKTMTEKEIVDYVASQVTTAKKLRGGVV FVDEVPKGLTGKLDARKIREILIKAKKGGKSKLMSSMNPEYDYLFKLLLIGDSGVGK NCLLLRFADDTYTESYISTIGVDFKIRTIELDGKTIKLQIWDTAGQERFRTITSSYYRG AHGIIVVYDVTDQESFNNVKQWLQEIDRYASENVNKLLVGNKCDLTTKKVVDYTT
AKEFADSLGIPFLETSAKNATNVEQSFMTMAAEIKKRMGPGATAGGAEKSNVKIQST PVKQSGGGCC
(SEQ ID NO: 21); or a polypeptide having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity therewith and preferentially binding GDP. The use of any one of claims 16-19, wherein the one or more expressible nucleic acids encode one or more RablaGDP as defined in any one of claims 20-26. The use of claim 27, wherein the one or more expressible nucleic acids are DNA-based, or RNA-based. The use of claim 27 or 28, wherein the one or more expressible nucleic acids transiently express the RablaGDP in the cell, or wherein the one or more expressible nucleic acids integrate in the cell genome and express the RablaGDP in the cell. The use of any one of claims 16-19 or 27, wherein the one or more expressible nucleic acids comprise one or more expression vectors, plasmids, or mRNAs encoding and capable of expressing the RablaGDP inside the cell. The use of claim 30, wherein the one or more expressible nucleic acids comprise a nucleic acid sequence of:
ATGTCCAGCATGAATCCCGAATATGATTATTTATTCAAGTTACTTCTGATTGGCG ACTCAGGGGTTGGAAAGAATTGCCTTCTTCTTAGGTTTGCAGATGATACATATAC AGAAAGCTACATCAGCACAATTGGTGTGGATTTCAAAATAAGAACTATAGAGTT AGACGGGAAAACAATCAAGCTTCAAATATGGGACACAGCAGGCCAGGAAAGAT TTCGAACAATCACCTCCAGTTATTACAGAGGAGCCCATGGCATCATAGTTGTGTA TGATGTGACAGATCAGGAGTCCTTCAATAATGTTAAACAGTGGCTGCAGGAAAT AGATCGTTATGCCAGTGAAAATGTCAACAAATTGTTGGTAGGGAACAAATGTGA TCTGACCACAAAGAAAGTAGTAGACTACACAACAGCGAAGGAATTTGCTGATTC CCTTGGAATTCCGTTTTTGGAAACCAGTGCTAAGAATGCAACGAATGTAGAACA GTCTTTCATGACGATGGCAGCTGAGATTAAAAAGCGAATGGGTCCCGGAGCAAC AGCTGGTGGTGCTGAGAAGTCCAATGTTAAAATTCAGAGCACTCCAGTCAAGCA GTCAGGTGGAGGTTGCTGCTAA
(Human RablaS25N ORF Codon Sequence, SEQ ID NO: 5);
ATGGGGGACTACAAGGACGACGATGACAAGGGGGGTAGCGGTGGATCCAGCAT GAATCCCGAATATGATTATTTATTCAAGTTACTTCTGATTGGCGATTCTGGGGTT
GGAAAGTCCTGCCTTCTCCTTAGGTTTGCAGATGATACGTATACGGAAAGCTACA
TCAGCACAATTGGTGTGGATTTCAAGATACGAACTATAGAGTTAGATGGGAAAA
CAATCAAGCTACAGATATGGGACACAGCAGGCCAGGAAAGATTTCGAACAATCA
CTTCCAGTTATTACAGAGGAGCCCATGGCATCATAGTTGTGTATGATGTGACAGA
TCAGGAGTCCTTCAATAACGTTAAACAGTGGCTGCAGGAGATAGATCGCTACGC
CAGTGAAAATGTCAACAAGTTGTTGGTAGGGATCAAATGTGACCTGACCACAAA
GAAAGTAGTAGACTACACAACAGCAAAGGAATTTGCAGATTCCCTTGGAATTCC
ATTTTTGGAAACCAGTGCTAAGAACGCAACGAATGTAGAACAGTCTTTCATGAC
GATGGCAGCTGAGATTAAAAAGCGAATGGGTCCTGGAGCTACAGCTGGTGGTGC
CGAGAAGTCCAATGTTAAAATCCAGAGCACTCCAGTCAAGCAGTCAGGTGGAGG CTGCTGCTAA
(Mouse RablaN1241 ORF Codon Seqeunce, SEQ ID NO: 8); or
ATGTCCAGCATGAATCCCGAATATGATTATTTATTCAAGTTACTTCTGATTGGCGACT
CAGGGGTTGGAAAGTCTTGCCTTCTTCTTAGGTTTGCAGATGATACATATACAGAAA GCTACATCAGCACAATTGGTGTGAACTTCAAAATAAGAACTATAGAGTTAGACGGG AAAACAATCAAGCTTCAAATATGGGACACAGCAGGCCAGGAAAGATTTCGAACAAT CACCTCCAGTTATTACAGAGGAGCCCATGGCATCATAGTTGTGTATGATGTGACAGA TCAGGAGTCCTTCAATAATGTTAAACAGTGGCTGCAGGAAATAGATCGTTATGCCAG TGAAAATGTCAACAAATTGTTGGTAGGGAACAAATGTGATCTGACCACAAAGAAAG TAGTAGACTACACAACAGCGAAGGAATTTGCTGATTCCCTTGGAATTCCGTTTTTGG AAACCAGTGCTAAGAATGCAACGAATGTAGAACAGTCTTTCATGACGATGGCAGCT GAGATTAAAAAGCGAATGGGTCCCGGAGCAACAGCTGGTGGTGCTGAGAAGTCCAA TGTTAAAATTCAGAGCACTCCAGTCAAGCAGTCAGGTGGAGGTTGCTGCTAA
(Human RablaD47N ORF Codon Sequence, SEQ ID NO: 17); or a nucleic acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity therewith and encoding a RablaGDP preferentially binding GDP; or a nucleic acid sequence equivalent to any of the above sequences due to codon redundancy. The use of claim 30, wherein the one or more expressible nucleic acids comprise a nucleic acid sequence of:
ATGGAAGACGCCAAAAACATAAAGAAAGGCCCGGCGCCATTCTATCCGCTAGAG GATGGAACCGCTGGAGAGCAACTGCATAAGGCTATGAAGAGATACGCCCTGGTT CCTGGAACAATTGCTTTTACAGATGCACATATCGAGGTGAACATCACGTACGCG GAATACTTCGAAATGTCCGTTCGGTTGGCAGAAGCTATGAAACGATATGGGCTG AATACAAATCACAGAATCGTCGTATGCAGTGAAAACTCTCTTCAATTCTTTATGC CGGTGTTGGGCGCGTTATTTATCGGAGTTGCAGTTGCGCCCGCGAACGACATTTA TAATGAACGTGAATTGCTCAACAGTATGAACATTTCGCAGCCTACCGTAGTGTTT GTTTCCAAAAAGGGGTTGCAAAAAATTTTGAACGTGCAAAAAAAATTACCAATA ATCCAGAAAATTATTATCATGGATTCTAAAACGGATTACCAGGGATTTCAGTCGA TGTACACGTTCGTCACATCTCATCTACCTCCCGGTTTTAATGAATACGATTTTGTA CCAGAGTCCTTTGATCGTGACAAAACAATTGCACTGATAATGAACTCCTCTGGAT CTACTGGGTTACCTAAGGGTGTGGCCCTTCCGCATAGAACTGCCTGCGTCAGATT CTCGCATGCCAGAGATCCTATTTTTGGCAATCAAATCATTCCGGATACTGCGATT TTAAGTGTTGTTCCATTCCATCACGGTTTTGGAATGTTTACTACACTCGGATATTT GATATGTGGATTTCGAGTCGTCTTAATGTATAGATTTGAAGAAGAGCTGTTTTTA CGATCCCTTCAGGATTACAAAATTCAAAGTGCGTTGCTAGTACCAACCCTATTTT CATTCTTCGCCAAAAGCACTCTGATTGACAAATACGATTTATCTAATTTACACGA AATTGCTTCTGGGGGCGCACCTCTTTCGAAAGAAGTCGGGGAAGCGGTTGCAAA ACGCTTCCATCTTCCAGGGATACGACAAGGATATGGGCTCACTGAGACTACATC AGCTATTCTGATTACACCCGAGGGGGATGATAAACCGGGCGCGGTCGGTAAAGT TGTTCCATTTTTTGAAGCGAAGGTTGTGGATCTGGATACCGGGAAAACGCTGGGC GTTAATCAGAGAGGCGAATTATGTGTCAGAGGACCTATGATTATGTCCGGTTATG TAAACAATCCGGAAGCGACCAACGCCTTGATTGACAAGGATGGATGGCTACATT CTGGAGACATAGCTTACTGGGACGAAGACGAACACTTCTTCATAGTTGACCGCTT GAAGTCTTTAATTAAATACAAAGGATACCAGGTGGCCCCCGCTGAATTGGAGTC GATATTGTTACAACACCCCAACATCTTCGACGCGGGCGTGGCAGGTCTTCCCGAC GATGACGCCGGTGAACTTCCCGCCGCCGTTGTTGTTTTGGAGCACGGAAAGACG
ATGACGGAAAAAGAGATCGTGGATTACGTCGCCAGTCAAGTAACAACCGCCAAA
AAGTTGCGCGGAGGAGTTGTGTTTGTGGACGAAGTACCGAAAGGTCTTACCGGA
AAACTCGACGCAAGAAAAATCAGAGAGATCCTCATAAAGGCCAAGAAGGGCGG
AAAGTCCAAATTGATGTCCAGCATGAATCCCGAATATGATTATTTATTCAAGTTA
CTTCTGATTGGCGACTCAGGGGTTGGAAAGAATTGCCTTCTTCTTAGGTTTGCAG
ATGATACATATACAGAAAGCTACATCAGCACAATTGGTGTGGATTTCAAAATAA
GAACTATAGAGTTAGACGGGAAAACAATCAAGCTTCAAATATGGGACACAGCAG
GCCAGGAAAGATTTCGAACAATCACCTCCAGTTATTACAGAGGAGCCCATGGCA
TCATAGTTGTGTATGATGTGACAGATCAGGAGTCCTTCAATAATGTTAAACAGTG
GCTGCAGGAAATAGATCGTTATGCCAGTGAAAATGTCAACAAATTGTTGGTAGG
GAACAAATGTGATCTGACCACAAAGAAAGTAGTAGACTACACAACAGCGAAGG
AATTTGCTGATTCCCTTGGAATTCCGTTTTTGGAAACCAGTGCTAAGAATGCAAC
GAATGTAGAACAGTCTTTCATGACGATGGCAGCTGAGATTAAAAAGCGAATGGG
TCCCGGAGCAACAGCTGGTGGTGCTGAGAAGTCCAATGTTAAAATTCAGAGCAC
TCCAGTCAAGCAGTCAGGTGGAGGTTGCTGCTAA
£SEQ ID NO: 19, MG-008 ORF DNA Sequence with 5’ Luciferase Tag); or
AUGGAAGACGCCAAAAACAUAAAGAAAGGCCCGGCGCCAUUCUAUCCGCUAG
AGGAUGGAACCGCUGGAGAGCAACUGCAUAAGGCUAUGAAGAGAUACGCCCU
GGUUCCUGGAACAAUUGCUUUUACAGAUGCACAUAUCGAGGUGAACAUCACG
UACGCGGAAUACUUCGAAAUGUCCGUUCGGUUGGCAGAAGCUAUGAAACGAU
AUGGGCUGAAUACAAAUCACAGAAUCGUCGUAUGCAGUGAAAACUCUCUUCA
AUUCUUUAUGCCGGUGUUGGGCGCGUUAUUUAUCGGAGUUGCAGUUGCGCCC
GCGAACGACAUUUAUAAUGAACGUGAAUUGCUCAACAGUAUGAACAUUUCGC
AGCCUACCGUAGUGUUUGUUUCCAAAAAGGGGUUGCAAAAAAUUUUGAACGU
GCAAAAAAAAUUACCAAUAAUCCAGAAAAUUAUUAUCAUGGAUUCUAAAACG
GAUUACCAGGGAUUUCAGUCGAUGUACACGUUCGUCACAUCUCAUCUACCUCC
CGGUUUUAAUGAAUACGAUUUUGUACCAGAGUCCUUUGAUCGUGACAAAACA
AUUGCACUGAUAAUGAACUCCUCUGGAUCUACUGGGUUACCUAAGGGUGUGG CCCUUCCGCAUAGAACUGCCUGCGUCAGAUUCUCGCAUGCCAGAGAUCCUAUU
UUUGGCAAUCAAAUCAUUCCGGAUACUGCGAUUUUAAGUGUUGUUCCAUUCC
AUCACGGUUUUGGAAUGUUUACUACACUCGGAUAUUUGAUAUGUGGAUUUCG
AGUCGUCUUAAUGUAUAGAUUUGAAGAAGAGCUGUUUUUACGAUCCCUUCAG
GAUUACAAAAUUCAAAGUGCGUUGCUAGUACCAACCCUAUUUUCAUUCUUCG
CCAAAAGCACUCUGAUUGACAAAUACGAUUUAUCUAAUUUACACGAAAUUGC
UUCUGGGGGCGCACCUCUUUCGAAAGAAGUCGGGGAAGCGGUUGCAAAACGC
UUCCAUCUUCCAGGGAUACGACAAGGAUAUGGGCUCACUGAGACUACAUCAG
CUAUUCUGAUUACACCCGAGGGGGAUGAUAAACCGGGCGCGGUCGGUAAAGU
UGUUCCAUUUUUUGAAGCGAAGGUUGUGGAUCUGGAUACCGGGAAAACGCUG
GGCGUUAAUCAGAGAGGCGAAUUAUGUGUCAGAGGACCUAUGAUUAUGUCCG
GUUAUGUAAACAAUCCGGAAGCGACCAACGCCUUGAUUGACAAGGAUGGAUG
GCUACAUUCUGGAGACAUAGCUUACUGGGACGAAGACGAACACUUCUUCAUA
GUUGACCGCUUGAAGUCUUUAAUUAAAUACAAAGGAUACCAGGUGGCCCCCG
CUGAAUUGGAGUCGAUAUUGUUACAACACCCCAACAUCUUCGACGCGGGCGU
GGCAGGUCUUCCCGACGAUGACGCCGGUGAACUUCCCGCCGCCGUUGUUGUUU
UGGAGCACGGAAAGACGAUGACGGAAAAAGAGAUCGUGGAUUACGUCGCCAG
UCAAGUAACAACCGCCAAAAAGUUGCGCGGAGGAGUUGUGUUUGUGGACGAA
GUACCGAAAGGUCUUACCGGAAAACUCGACGCAAGAAAAAUCAGAGAGAUCC
UCAUAAAGGCCAAGAAGGGCGGAAAGUCCAAAUUGAUGUCCAGCAUGAAUCC
CGAAUAUGAUUAUUUAUUCAAGUUACUUCUGAUUGGCGACUCAGGGGUUGGA
AAGAAUUGCCUUCUUCUUAGGUUUGCAGAUGAUACAUAUACAGAAAGCUACA
UCAGCACAAUUGGUGUGGAUUUCAAAAUAAGAACUAUAGAGUUAGACGGGAA
AACAAUCAAGCUUCAAAUAUGGGACACAGCAGGCCAGGAAAGAUUUCGAACA
AUCACCUCCAGUUAUUACAGAGGAGCCCAUGGCAUCAUAGUUGUGUAUGAUG
UGACAGAUCAGGAGUCCUUCAAUAAUGUUAAACAGUGGCUGCAGGAAAUAGA
UCGUUAUGCCAGUGAAAAUGUCAACAAAUUGUUGGUAGGGAACAAAUGUGAU
CUGACCACAAAGAAAGUAGUAGACUACACAACAGCGAAGGAAUUUGCUGAUU
CCCUUGGAAUUCCGUUUUUGGAAACCAGUGCUAAGAAUGCAACGAAUGUAGA
ACAGUCUUUCAUGACGAUGGCAGCUGAGAUUAAAAAGCGAAUGGGUCCCGGA GCAACAGCUGGUGGUGCUGAGAAGUCCAAUGUUAAAAUUCAGAGCACUCCAG UCAAGCAGUCAGGUGGAGGUUGCUGCUAA
(SEQ ID NO: 20, MG-008 ORF mRNA Sequence with 5’ Luciferase Tag); or a nucleic acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity therewith and encoding a RablaGDP preferentially binding GDP; or a nucleic acid sequence equivalent to any of the above sequences due to codon redundancy. A polypeptide comprising the amino acid sequence:
MSSMNPEYDYLFKLLLIGDSGVGKNCLLLRFADDTYTESYISTIGVDFKIRTIELD
GKTZKLQIWDTAGQERFRTITSSYYRGAHGIIVVYDVTDQESFNNVKQWLQEIDR YASENVNKLLVGNKCDLTTKKVVDYTTAKEFADSLGIPFLETSAKNATNVEQSF MTMAAEIKKRMGPGATAGGAEKSNVKIQSTPVKQSGGGCC (SEQ ID NO: 6; Human RablaS25N);
MGDYKDDDDKGGSGGSSMNPEYDYLFKLLLIGDSGVGKSCLLLRFADDTYTES
YISTIGVDFKIRTIELDGKTIKLQIWDTAGQERFRTITSSYYRGAHGIIVVYDVTDQ ESFNNVKQWLQEIDRYASENVNKLLVGIKCDLTTKKVVDYTTAKEFADSLGIPFL ETSAKNATNVEQSFMTMAAEIKKRMGPGATAGGAEKSNVKIQSTPVKQSGGGC C
(SEQ ID NO: 9; Mouse RablN1241); the amino acid sequence of human RablaD41N; or
MSSMNPEYDYLFKLLLIGDSGVGKSCLLLRFADDTYTESYISTIGVNFKIRTIELD
GKTIKLQIWDTAGQERFRTITSSYYRGAHGIIVVYDVTDQESFNNVKQWLQEIDR YASENVNKLLVGNKCDLTTKKVVDYTTAKEFADSLGIPFLETSAKNATNVEQSF MTMAAEIKKRMGPGATAGGAEKSNVKIQSTPVKQSGGGCC
(SEQ ID NO: 18; Human RablaD47N); or a polypeptide having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with any of these sequences and preferentially binding GDP; for use in reducing cell viability of a cancer cell, or for preventing or treating cancer, in a subject in need thereof; or for use in reducing cell viability of a cancer cell in vitro or in vivo. A pharmaceutical composition comprising a GDP -bound form of Rabi a (RablaGDP), one or more expressible nucleic acids encoding RablaGDP, or a combination thereof; and another anti-cancer agent. A kit comprising any one or more of: a GDP -bound form of Rabi a (RablaGDP); one or more expressible nucleic acids encoding RablaGDP; an anti-cancer agent; instructions for performing a method as defined in any one of claims 1-15; or any combinations thereof. The method of any one of claims 1-15, the use of any one of claims 16-32, or the polypeptide for use of claim 33, wherein the cancer is lung cancer, colon cancer, prostate cancer, gastric cancer, brain cancer, pancreatic cancer, oral cancer, breast cancer, ductal cancer, liver cancer, or cervical cancer. The method of any one of claims 1-15, the use of any one of claims 16-32, or the polypeptide for use of claim 33, wherein the cancer is lung cancer adenocarcinoma, colorectal carcinoma, colon adenocarcinoma, cholangiocarcinoma, prostate carcinoma, embryonal rhabdomyosarcoma, gastric carcinoma, glioblastoma, colon adenocarcinoma, pancreatic ductal adenocarcinoma, squamous cell carcinoma of the oral cavity, squamous cell carcinoma of the oral cavity, pancreatic ductal adenocarcinoma, breast adenocarcinoma, breast adenocarcinoma, invasive ductal carcinoma, hepatocellular carcinoma, cholangiocarcinoma, squamous cell carcinoma of the oral tongue, papillomavirus-related endocervical adenocarcinoma, colorectal carcinoma, or lung adenocarcinoma. The method of any one of claims 1-15, the use of any one of claims 16-32, or the polypeptide for use of claim 33, wherein the cancer cell is selected from the group consisting of A549 cells, HCT116 cells, HT29 cells, HuCCTl, PC-3 cells, RD cells, SC-M1 cells, U-87MG cells, SW480 cells, PANC-1 cells, OECM-1 cells, OC2 cells, MIA PaCa-2 cells, MDA-MB-468 cells, MDA-MB-231 cells, MCF-7 cells, Mahlavu cells, HuCCTl cells, HSC-3, HeLa cells, HCT116 cells, or A549 cells. A method for reducing cell viability of a cancer cell, or for preventing or treating cancer, in a subject in need thereof, said method comprising: treating a cancer cell of the subject with lipid nanoparticles encapsulating RablaDN, one or more expressible nucleic acids encoding RablaDN, or a combination thereof; thereby increasing a cellular level of RablaDN in the cancer cell, resulting in decreased cell viability or death of the cancer cell. Use of RablaDN encapsulated lipid nanoparticles for reducing cell viability of a cancer cell in vitro or in vivo. Use of RablaDN encapsulated lipid nanoparticles for the treatment of cancer. Use of RablaDN encapsulated lipid nanoparticles for reducing metastases in a patient.
EP23864205.2A 2022-09-12 2023-09-12 Compositions and methods for treating cancer Pending EP4587563A1 (en)

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