WO2017008065A2 - Constructions de vecteurs et procédés pour obtenir ou améliorer la localisation de protéines à la surface de cellules - Google Patents

Constructions de vecteurs et procédés pour obtenir ou améliorer la localisation de protéines à la surface de cellules Download PDF

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WO2017008065A2
WO2017008065A2 PCT/US2016/041659 US2016041659W WO2017008065A2 WO 2017008065 A2 WO2017008065 A2 WO 2017008065A2 US 2016041659 W US2016041659 W US 2016041659W WO 2017008065 A2 WO2017008065 A2 WO 2017008065A2
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vector
utr
polypeptide
cell
cells
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WO2017008065A3 (fr
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Christine MAYR
Weirui MA
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Memorial Sloan Kettering Cancer Center
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Memorial Sloan Kettering Cancer Center
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70596—Molecules with a "CD"-designation not provided for elsewhere
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/02—Fusion polypeptide containing a localisation/targetting motif containing a signal sequence
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/03—Fusion polypeptide containing a localisation/targetting motif containing a transmembrane segment
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/035—Fusion polypeptide containing a localisation/targetting motif containing a signal for targeting to the external surface of a cell, e.g. to the outer membrane of Gram negative bacteria, GPI- anchored eukaryote proteins
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/60—Fusion polypeptide containing spectroscopic/fluorescent detection, e.g. green fluorescent protein [GFP]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/70—Fusion polypeptide containing domain for protein-protein interaction
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/85—Fusion polypeptide containing an RNA binding domain

Definitions

  • the present application includes a sequence listing in electronic form as a txt file in ascii format titled "7704-0027-Z_ST25[5][4].txt" and having a size of 51.8 kb. The contents of this txt file are incorporated by reference herein.
  • This disclosure generally relates to membrane localization of proteins regulated by certain 3'UTR isoforms. More specifically, this disclosure relates to achieving or enhancing protein localization on a cell membrane using components of 3'UTRs.
  • the disclosure is directed to a recombinant vector comprising a promoter, the vector being adapted for introduction of a first polynucleotide encoding a first polypeptide of interest to be expressed in a target cell and localized on the surface of the target cell and a second polynucleotide segment comprising a fragment of a longer 3 'UTR or nucleic acid encoding a fragment of a longer 3 'UTR, the fragment derived from nucleic acid encoding a second polypeptide that is substantially localized to the surface of a cell in which it is expressed in nature, said fragment comprising an HuR binding site.
  • the polynucleotide segment is flanked by restriction sites.
  • a CAC motif is interposed between a first (5') restriction site and the fragment.
  • the fragment is up to about 1000 nucleotides long and in some embodiments up to about 500 nucleotides long.
  • the segment has a sequence selected from the group consisting of SEQ ID NO:3 ; SEQ ID NO: 56; SEQ ID NO: 57and SEQ ID NO: 58 or a sequence at least 90% homologous to any of the foregoing.
  • the vector further comprises an insert containing an exogenous polynucleotide encoding the polypeptide of interest, wherein upon delivery of the vector to a target cell, the polypeptide is expressed and substantially localized on the target cell surface.
  • the first polynucleotide encoding the first polypeptide of interest is or is encoded by an open reading frame (ORF) in turn encoding a polypeptide normally expressed on a cell surface; in other embodiments, the polynucleotide encodes a polypeptide that is not normally substantially localized on the target cell surface.
  • ORF open reading frame
  • the insert further comprises a polynucleotide encoding a signal peptide and a polynucleotide encoding a transmembrane domain derived from a polypeptide expressed on a cell surface.
  • the insert also comprises a polynucleotide encoding a C- terminal of a polypeptide that is normally expressed on the target cell or a fragment thereof encoding a SET binding site.
  • the transmembrane domain also includes a SET binding site.
  • one or both of the signal peptide and transmembrane domain are derived from CD47.
  • the transmembrane domain includes the entire
  • the polypeptide of interest is GFP.
  • the first polypeptide of interest is selected from the group consisting of taste receptors and glutamate receptors.
  • the first polypeptide of interest is selected from the group consisting of INSR, IGFR, TGFBR1, EPHB2, BMPR1A, EGFR, FZD5, CD44, and PDGFR.
  • the insert comprises the polynucleotide encoding the signal peptide of CD47 followed by the polynucleotide encoding the polypeptide of interest followed by the transmembrane domain of CD47 including the C-terminal thereof and said insert is 5' to the 3'UTR.
  • the promoter is selected from the group consisting of ubiquitous promoters or tissue-specific promoters.
  • the vector is selected from the group consisting of plasmids and viral vectors.
  • the vector is a viral vector selected from the group consisting of selected from the group consisting of an adenoviral vector, an adeno associated vector (AAV), a baculo viral vector and a retroviral vector.
  • the promoter is part of an expression cassette comprised by the vector.
  • the fragment is preceded by a CA-rich motif or a GA-rich motif or an AU-rich sequence interposed between the fragment and a first (5') restriction site.
  • the disclosure relates to a recombinant polynucleotide construct comprising (i) a first segment having a sequence encoding a polypeptide that is not normally substantially localized on the surface of a target cell, (ii) a second heterologous segment having a sequence that comprises a fragment of a 3'UTR comprising an HuR binding site or nucleic acid encoding said fragment, wherein said construct has the property of effecting localization of at least a substantial fraction of said polypeptide to the cell surface upon expression of the polypeptide in a cell harboring the construct.
  • the polypeptide comprises a transmembrane domain sequence and a peptide signal sequence of a membrane-associated polypeptide.
  • the transmembrane domain includes the C- terminal of said membrane-associated polypeptide or at least a fragment of said C-terminal encoding a SET binding site.
  • the 3'UTR fragment has a sequence comprising a nucleic acid having SEQ ID NO: 3 or SEQ ID NO: 56 or SEQ ID NO: 57 or SEQ ID NO: 58 or a sequence having at least 90% homology to any of the foregoing.
  • the present disclosure relates to a polynucleotide construct comprising the sequence SEQ ID NO 3, SEQ ID NO 56, SEQ ID NO 57 and SEQ ID NO: 58 or a sequence having at least 90% homology to any of the foregoing flanked between two restriction sites.
  • the construct comprises at least one of a CA-rich motif, a GA-rich motif and an AU-rich motif following the first restriction site.
  • the present disclosure is directed to a method for increasing a fraction of a polypeptide of interest expressed within a cell that is localized on the surface of the cell, comprising transfecting the cell with one of the foregoing vectors under conditions that permit expression of the polypeptide, wherein HuR, SET and RAC1 are present within the cell, have access to the polypeptide at least post-translationally, and effect localization of the polypeptide to the cell surface.
  • the present disclosure relates to methods for increasing a fraction of a polypeptide expressed within a cell that is transported to the surface of the cell, comprising introducing in the cell a construct described above in this section under conditions that permit expression of the polypeptide, wherein HuR, SET and RAC1 are present within the cell, have access to the polypeptide at least post-translationally, and effect localization of the polypeptide to the cell surface.
  • Figure 1 The long 3' UTR of CD47 localizes GFP-TM protein to the plasma membrane, whereas the short 3' UTR localizes it to the endoplasmic reticulum, a,
  • IC intracellular
  • b 3'-seq analysis of naive B cells shows two 3'UTR isoforms of CD47 mRNA (short 3'UTR (SU) and long 3'UTR (LU)). Shown is the last exon of the gene model CD47 with 5 transmembrane domains. Isoform abundance shown in transcripts per million (TPM).
  • TPM transcripts per million
  • RNA gel is shown as loading control, d, Staining of U20S cells as in a after transfection of a control shRNA (sh Co) or an shRNA against the long CD47 3'UTR isoform.
  • CD47 protein contains an N-terminal signal peptide, ECD, five TMDs and a cytoplasmic C terminus.
  • ECD was replaced with GFP and either fused with the long (GFP-TM- LU) or the short CD47 3'UTR (GFP-TM-SU).
  • Constructs are drawn to scale, f, Fluorescence confocal microscopy of fixed U20S cells after transfection of GFP-TM-LU or GFP-TM-SU. Bottom, with additional staining of the endoplasmic reticulum with anti-calnexin.
  • g FACS analysis of GFP expression in transfected U20S cells with (black lines, detection of total expression) and without permeabilization (dark grey lines (top left and bottom right), detection of surface (surf.) expression). Values for mean fluorescence intensity (MFI) are shown in parentheses. Unstained cells are shown in light grey.
  • FIG. 1 3'UTR-dependent protein localization (UDPL) depends on HuR, SET and RAC1, and mediates surface localization of membrane proteins, a, Model of UDPL.
  • HuR binds to the long 3' UTR and recruits SET.
  • SET binds to the newly translated cytoplasmic domains of CD47. This step probably requires energy.
  • SET interacts with RAC1 and active RAC1 translocates SET and CD47 to the plasma membrane, b, FACS analysis of endogenous CD47 protein expression in HEK293 cells.
  • FIG. 1 Left panel is after transfection of control shRNA (shCo) or shRNAs against HuR (shown are all GFP + cells). Middle and right panels depict cells stably expressing the indicated shRNAs. Surface CD47 (top) and total CD47 protein (bottom) were measured, c, 3'-seq analysis for CD44 in HEK293 cells and for TNFRSF13C in B-LCL cells, as shown in Fig. lb.
  • CD47 protein has different functions depending on whether it was generated by the 5i7 or LU isoform.
  • GFP was inserted in frame between the signal peptide and the rest of the CD47 open reading frame.
  • GFP-CD47 was fused with either the long or short CD47 3'UTR, called CD47-LU and CD47-SU, respectively.
  • b FACS analysis of surface (surf.; light grey) and total (black) GFP-CD47 expression in transfected JinB8 cells. Shown as in Fig. Ig.
  • FIG. 5 Expression of the long CD47 3' UTR isoform correlates with cell surface expression of CD47 protein
  • a Fluorescence confocal microscopy of cells shown as in Fig. la. Representative images out of hundreds of cells are shown. Scale bars, 10 ⁇ .
  • b FACS analysis of endogenous CD47 expression in cells shown in Fig. la and a.
  • Unstained cells are shown in light grey, c, Left, quantification of mean fluorescence intensity (MFI) values from b. Right, fraction of surface and intracellular CD47 levels in cells lines from b. Intracellular CD47 was calculated by subtracting CD47 surface values from total CD47 values, d, Northern blot of HEK293 cells stably expressing the indicated shRNAs and hybridized for CD47. The shRNAs against CD47-LU target only the long 3'UTR isoforms of CD47. The blot and corresponding RNA gel are shown as in Fig. lc. e, Quantification of CD47 total mRNA and 3'UTR isoform levels in U20S cells by qRT-PCR.
  • MFI mean fluorescence intensity
  • the total amount of CD47 mRNA after transfection of sh Co was set to 1.
  • FIG. 6 UDPL depends on HuR, SET and RAC1 and mediates surface localization of membrane proteins
  • a Western blot of HEK293 cells transiently transfected (left, middle) or stably expressing (right) sh Co or shRNAs against HuR. The blot shows reduced HuR protein expression after HuR knockdown, but no change in protein expression of CD47, TSPAN13, CD44 or SET. Actin was used as loading control
  • b Quantification of CD47 total mRNA and 3'UTR isoform levels in HEK293 cells by qRT-PCR. GAPDH- normalized values after transfection of sh2 HuR or sh Co are shown.
  • n 3 biological replicates.
  • the total amount of CD47 mRNA after transfection of sh Co was set to 1.
  • 3'UTR isoforms that encode proteins using UDPL contain uridine- rich elements. Shown are the 3'UTR sequences of CD47, CD44, HuR-BS and HuR-BSA. Dark grey, ApA signals. Light grey, uridine-rich elements with the potential to be HuR- binding sites.
  • FIG. 8 3'UTR isoforms that encode proteins using UDPL contain uridine- rich elements. Shown are the 3'UTR sequences of ITGAl, TNFRSF13C and TSPAN13. Dark grey, ApA signals. Light grey, uridine-rich elements with the potential to be HuR-binding sites.
  • FIG. 9 Local recruitment of SET to the site of translation is required for UDPL.
  • a Western blot of cells used in Fig. 3b shows the amount of overexpression achieved by transfection of MS2-mC-SET or MS2-mC-HuR (for constructs, see b).
  • anti-SET detects endogenous expression of SET as well as overexpressed SET.
  • anti-HuR detects endogenous HuR and overexpressed HuR. Actin was used as loading control.
  • Anti-HuR and anti-SET were used on the same blot. Actin as loading control was performed once. The marker is shown in kDa. Asterisk indicates unspecific band.
  • the top construct depicts GFP-TM-SU (Fig. le) and the bottom construct shows a fusion of MS2 coat protein (MS2), mC (red) and HuR or SET, respectively.
  • MS2 coat protein MS2 coat protein
  • mC red
  • HuR or SET HuR or SET
  • Overexpression of HuR or SET compared with expression of MS2- mC alone does not change surface or total GFP expression, when co-transfected with GFP-TM-SU (without the addition of MS2-binding sites to the SU isoform) as shown by FACS analysis.
  • MS2-binding sites (MS2-BS, RNA stem loops) were added to GFP-TM-SU (and the proximal polyadenylation signal was mutated) to obtain GFP-TM-SU-MS2-BS.
  • tethering of HuR or SET to the short 3'UTR of GFPTM localizes GFP to the cell surface without changing total GFP expression. Histograms are shown as in b.
  • CD47 contains at least two SET-binding sites in its cytoplasmic domains, a, FACS analysis of surface GFP expression after transfection of GFP-TM-LU (black line) and GFP-TM-LU constructs containing a single point mutation in the cytoplasmic C terminus (light grey line, adjacent to black line), K290A (left), K297A (middle), K304A (right) in HEK293 cells. Partial destruction of a single SET-binding site results in up to 37% reduction in GFP surface expression. Values for MFI are shown in parentheses. Unstained cells are shown in dark grey.
  • CD47 protein has different functions depending on whether it was generated by the 5i7 or LU isoform.
  • a Left, western blot of HEK293 cells after transfection of the indicated constructs shows GFP-CD47 expression using an anti-GFP antibody. Actin was used as loading control.
  • Right as in left panel after transfection of CD47-SU and CD47- LU into HEK293 (left) or JinB8 cells (right).
  • GFP-CD47 expression was quantified after normalization with respect to actin using Image J. Shown is the fold change in GFP-CD47 expression of CD47-SU after setting CD47-LU to L b, The experiment is similar to Fig. 3b and Fig.
  • Top row shows values of constructs without the ECD and bottom row shows values of constructs containing the full coding regions of the indicated proteins.
  • the fold increase in surface GFP expression was calculated from MFI (LU)/MFI (SU).
  • the contribution of the ECD domain for surface expression of BAFFR is 1.2-fold (3.8/3.1).
  • CFSE carboxyfluorescein succinimidyl ester
  • f The fraction of surviving cells (TO-PR03 negative) as measured by FACS analysis at day 3 (d3) after increasing doses of ⁇ -irradiation is shown for Jurkat, JinB8 (CD47 ⁇ / ⁇ ) and the GFP + fraction after nucleofection of JinB8 cells with either CD47-SU or CD47-LU.
  • ELAVL1 encodes HuR.
  • the data set from Lianoglou et al. was analyzed to obtain the TPM values (Lianoglou et al. Genes D ⁇ ?v.2013 Nov l;27(21):2380-96).
  • 'HuR targets' consist of the union of HuR targets identified previously (Lebedeva et al. Mol. Cell 2011 Aug 5;43(3):340-52; Uren et al. J Biol Chem. 2011 Oct 28;286(43):37063-6).
  • Membrane proteins consist of all the proteins that contain the tag "membrane” using gene ontology analysis. The fraction of membrane proteins found is consistent with the fraction of membrane proteins found in yeast (Stagljar et al. Trends Biochem Sci. 2002 Nov;27(l l):559-63). Fisher's exact test shows no enrichment or depletion of membrane proteins among the HuR targets.
  • FIG. 13 All tested UDPL candidates have potential SET binding sites in their cytoplasmic domains. Shown are the amino acid sequences of the TMDs and cytoplasmic domains of the membrane proteins studied. The TMDs are shown in grey.
  • FIG. 14 UDPL sequence candidates to be tested. Shown are the nucleotide sequences of UTR fragments of indicated proteins that have or will be tested for their ability to facilitate the expression of proteins on the surface of the cell.
  • 2x HuR-BS refers to a repeat of a 3'UTR fragment encompassing the HuR binding site and derived from CD47.
  • Figure 15. 3'UTR sequences that comprise an HuR- binding site of TNF alpha increase surface localization of proteins.
  • Figure 15 A shows the top three motifs that are enriched in the 3 'UTRs of human membrane proteins. The motifs were determined using the search engine HOMER (homer.salk.edu).
  • Figure 15B, 15C, and 15D show fold change in 3'UTR mediated increase in CD47 surface localization using UTR-1 (SEQ ID NO: 56, UTR- 2 (SEQ ID NO: 57), and UTR-3 (SEQ ID NO: 58) sequences. Each 3'UTR was tested in HeLa (figure 15B), U20S (figure 15C), and HEK293 (figure 15D) cells. Shown is a mean ⁇ s.d. of biological replicates.
  • Vector generally is a DNA or RNA molecule, such as a plasmid, virus, phagemid, cosmid or other nucleic acid construct that (a) contains or is adapted to contain a heterologous nucleic acid segment and (b) serves as a vehicle for delivering a polynucleotide of interest to the interior of a target organism such as a target bacterium or target cell.
  • Polypeptide is used interchangeably with “protein” and means a polymer having a primary linear structure of about 100 or more amino acid residues linked one to the other in a chain by peptide bonds and forming part of (or the whole of) a molecule.
  • fusion protein refers to a polypeptide comprising a polypeptide or fragment thereof coupled via a peptide bond to one or more heterologous amino acid sequences.
  • Transmembrane domain or "TM” means a segment of a protein that spans the membrane bilayer of a cell and thus crosses the cell membrane.
  • a protein may have more than one transmembrane domains, which together may constitute a transmembrane region.
  • CD47 has 5 transmembrane domains (Rebres et al. J Biol Chem. 2001 ;276:34607-34616).
  • transmembrane domain addition means the incorporation in a vector or polynucleotide construct of nucleic acid encoding one or more transmembrane domains of a membrane-associated polypeptide.
  • the transmembrane domains may for example be separated by surface or cytoplasmic side segments of the original protein, such as for example a segment encompassing the binding site for SET.
  • operably linked refers to a functional relationship between two or more polynucleotide (e.g., DNA) segments.
  • the term refers to the functional relationship of a transcriptional regulatory sequence to a sequence to be transcribed.
  • a promoter or enhancer sequence is operably linked to a coding sequence if it stimulates or modulates the transcription of the coding sequence in an appropriate host cell or other expression system.
  • promoter transcriptional regulatory sequences that are operably linked to a transcribable sequence are contiguous to the transcribable sequence, i.e., they are cis-acting.
  • some transcriptional regulatory sequences, such as enhancers need not be physically contiguous or even located in close proximity to the coding sequences whose transcription they enhance.
  • Promoter refers to a sequence that regulates transcription of an operably- linked gene, or nucleotide sequence encoding a protein, etc. Promoters provide the sequence sufficient to direct transcription, as well as, the recognition sites for RNA polymerase and other transcription factors required for efficient transcription and can direct cell specific expression.
  • “Expression cassette” refers to a polynucleotide segment comprising a promoter and nucleic acid encoding a polypeptide to be expressed.
  • the promoter is operably linked to the nucleic acid to be expressed.
  • the cassette optionally also encompasses other elements that regulate transcription or translation such as enhancers, termination codons, polyadenylation site, IRES, selectable markers and other inducible elements.
  • 3'UTR means the untranslated region of mRNA (or the transcribed corresponding region of DNA) that follows the translation termination codon. This region is known to contain regulatory segments that can influence polyadenylation, translation efficiency, as well as localization and stability of the mRNA.
  • the 3'UTR is also known to contain binding sites for regulatory proteins and for microRNAs (miRNAs). Many 3'UTRs also contain AU-rich elements (AREs). Proteins bind AREs to affect the stability or decay rate of transcripts in a localized manner or to affect translation initiation.
  • the 3'UTR contains the sequence AAUAAA that directs addition of several hundred adenine residues called the poly(A) tail to the end of the mRNA transcript.
  • Poly(A) binding protein binds to this tail, contributing to regulation of mRNA translation, stability, and export.
  • the 3'UTR can also contain sequences that attract proteins to associate the mRNA with the cytoskeleton, transport it to or from the cell nucleus, or perform other types of RNA localization.
  • sequences within the 3'UTR the physical characteristics of the region, including its length and secondary structure, contribute to translation regulation.
  • GFP green fluorescent protein, a protein originally isolated from the jellyfish Aequorea Victoria which fluoresces with a bright green color when exposed to light in the blue to UV range, and similarly fluorescing proteins from other marine species such as the sea pansy.
  • CD47 Cluster of Differentiation 47 also known as integrin associated protein (IAP) is a transmembrane protein that in humans is encoded by the CD47 gene. CD47 is ubiquitously expressed in human cells but it is not normally particularly localized on the cell surface. CD47 belongs to the immunoglobulin superfamily, partners with membrane integrins and also binds the ligands thrombospondin- 1 (TSP-1) and signal-regulatory protein alpha (SIRPa) which is expressed in phagocytic cells. CD47 is involved in a range of cellular processes, including apoptosis, proliferation, adhesion, and migration.
  • TSP-1 thrombospondin- 1
  • SIRPa signal-regulatory protein alpha
  • CD47 functions as a marker of self ("don't eat me signal") for phagocytic cells, and downregulates phagocytic activity.
  • CD47 has also been implicated in processes such as, for example, apoptosis, survival, proliferation, adhesion, migration, and regulation of angiogenesis, blood pressure, tissue perfusion, and/or platelet homeostasis.
  • CD47 inhibits dendritic cell (DC) maturation and activation.
  • CD47 has been implicated in cancer (immune escape).
  • CD47 is overexpressed in various hematological and solid malignancies.
  • CD47 is a documented cancer stem cell/tumor initiating cell marker. It is thought that CD47 overexpression may help tumor cells to escape immune surveillance and killing by innate immune cells.
  • CD47 High levels of CD47 are also associated with poor clinical outcome in cancers such as, for example, leukemias, lymphomas, breast cancer, colon cancer, ovarian cancer, bladder cancer, prostate cancer, and/or glioma. Because of its normally relatively poor expression on the cell surface, CD47 was arbitrarily chosen as a test protein by the present inventors to test the ability of the present methods to augment the proportion of a protein that is localized on the cell surface.
  • Human R or “human antigen R” denotes an RNA binding protein that modulates the stability and translational efficiency of mRNA.
  • SET or "protein SET” is a protein that in humans is encoded by the SET gene.
  • SET contains a long acidic C-terminus that interacts with the RRM 3 (RNA recognition motif 3) of HuR.
  • Nuclear SET binds to histone tails and prevents acetylation, but phosphorylated SET localizes to the cytoplasm and the surface of the endoplasmic reticulum.
  • SET was shown to interact with Racl, which results in translocation of SET to the plasma membrane upon Racl activation. Whereas Racl -GDP is mostly localized to the cytoplasm, Racl-GTP (active Racl) localizes to the leading edge of the plasma membrane (ten Klooster et al. EMBO J 26, 336-345 (2007)).
  • the inventors have demonstrated that in addition to HuR, SET and RAC1 are also necessary for 3' UTR-dependent protein localization (Example 3).
  • RAC1 or "ras-related C3 botulinum toxin substrate 1," a protein encoded by the RAC1 gene (a protooncogene), is a GTPase belonging to the RAS superfamily of small GTP- binding proteins controlling cell growth, cell motility, cytoskeletal organization and protein kinase activation.
  • RaCl regulates many cellular processes and is implicated in cancer metastasis. Certain activating RaCl mutations have been associated with certain cancers. Orthologs exist in rodents, primates and many other species. There are differently spliced versions of this protein that have different functions.
  • substantially when used in the context of protein localization on the cell surface means that a significant fraction of a protein being expressed is directed to the surface.
  • significant fraction 1 ⁇ 2, 1 ⁇ 2, 1 ⁇ 4, etc.
  • substantiality of localization when using long 3 'UTR or a fragment thereof containing an HuR binding site can be assessed by reference to the fraction of a polypeptide of interest that is directed to the cell surface without the use of such 3 'UTR or fragment.
  • Examples of a substantial increase in the fraction of a polypeptide directed to the cell surface include without limitation 1.2 or more the amount localized on the cell surface in the absence of the long 3 'UTR or fragment.
  • the present invention is based in part on the discovery that different isoforms of 3'UTR act as scaffolds to regulate (enhance or curb) membrane protein cell surface localization.
  • compositions and methods for enhancing the cell surface localization of proteins whether these are already membrane-associated proteins (in which case their localization is enhanced) or proteins which are not expressed on a cell's surface to any substantial extent but which are desired to be so expressed, such as G-protein coupled receptor proteins (Dunham, J.H., et al, "Enhancement of the surface expression of G protein-coupled receptors" Trends in Biotechnology 27(9): 541 (2009)) and antigens to be presented on the surface of immune system cells.
  • G-protein coupled receptor proteins unham, J.H., et al, "Enhancement of the surface expression of G protein-coupled receptors" Trends in Biotechnology 27(9): 541 (2009)
  • Membrane-associated polypeptide or “membrane-associated protein” or “membrane protein” or “membrane polypeptide” means a polypeptide that is present on the membrane of a cell and includes without limitation membrane, transmembrane and cell surface polypeptides, receptor polypeptides, envelope polypeptides and the like.
  • Membrane proteins are proteins which in their native state are associated with lipid membranes (e.g., nuclear membrane, cellular membrane, mitochondrial membranes, liposomal membranes, endoplasmic reticulum membranes, chloroplast membranes, etc.).
  • membrane proteins includes transmembrane proteins, and proteins which are partially or fully embedded in membranes in their native state or simply residing on the surface of such membranes.
  • membrane proteins includes both extrinsic and intrinsic proteins. Extrinsic membrane proteins are generally located entirely outside of the membrane, but are bound to the membrane by weak molecular attractions (such as, for example, ionic, hydrogen, and/or Van der Waals bonds). Intrinsic membrane proteins are, generally, embedded in the membrane. Intrinsic membrane proteins include, but are not limited to proteins which extend from one side of the membrane to the other, i.e., transmembrane proteins.
  • incorporation of the 74 nt fragment of the long 3'UTR of CD47 (SEQ ID NO: 3) in a construct or vector according to the present disclosure comprising a polypeptide of interest will result in substantial localization of the polypeptide to the cell surface without needing to incorporate the entire long 3'UTR region of CD47.
  • incorporation of the 53 nt artificial fragment (SEQ ID NO: 56) in a construct or vector according to the present disclosure comprising a polypeptide of interest will result in substantial localization of the polypeptide to the cell surface.
  • incorporation of the 117 nt artificial fragment (SEQ ID NO: 57) in a construct or vector according to the present disclosure comprising a polypeptide of interest will result in substantial localization of the polypeptide to the cell surface.
  • incorporation of the 384 nt artificial fragment (SEQ ID NO: 58) in a construct or vector according to the present disclosure comprising a polypeptide of interest will result in substantial localization of the polypeptide to the cell surface.
  • the present disclosure relates to vectors that facilitate increased expression of a protein (more accurately, expression of a higher proportion of a protein) on the surface of a cell.
  • proteins can include any protein, whether it can penetrate the lipid bilayer, or reside on a surface layer of the cell membrane.
  • Nonlimiting examples include: receptors of enzymes, peptide hormones, and local hormones, sugar acceptors/carriers, and cell membrane antigens.
  • membrane protein examples include, but are not limited to, receptors for growth factors (e.g., vascular endothelial growth factor (VEGF), transforming growth factor (TGF), fibroblast growth factor (FF), platelet derived growth factor (PDGF), insulin- like growth factor), insulin receptor, MHC proteins (e.g., VEGF, vascular endothelial growth factor (VEGF), transforming growth factor (TGF), fibroblast growth factor (FF), platelet derived growth factor (PDGF), insulin- like growth factor), insulin receptor, MHC proteins (e.g.
  • growth factors e.g., vascular endothelial growth factor (VEGF), transforming growth factor (TGF), fibroblast growth factor (FF), platelet derived growth factor (PDGF), insulin- like growth factor
  • VEGF vascular endothelial growth factor
  • TGF transforming growth factor
  • FF fibroblast growth factor
  • PDGF platelet derived growth factor
  • insulin receptor e.g.
  • cytokine receptors e.g., interleukin (IL)-l, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15 receptors
  • IL-l interleukin
  • IL-6 interleukin-6
  • IL-7 IL-8
  • IL-9 IL-10
  • IL-11 interleukin-12
  • IL-13 IL-13
  • IL-14 IL-15 receptors
  • tyrosine-kinase-associated receptors such as Src, Yes, Fgr, Lck, Fit, Lyn, Hck, and Blk
  • G-protein coupled receptors such as receptors for the hormone relaxin (LGR7 and LGR8) and chemokine receptor (e.g., CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CCR
  • the present disclosure relates to vectors that facilitate increased localization of G- protein coupled receptors (GPCRs) on the surface of the cell.
  • GPCRs constitute the largest family of transmembrane proteins and play an important part in signal transduction by converting extracellular stimuli including light, smells, neurotransmitters and hormones, into intracellular signals.
  • GPCRs exhibit low and hence not substantial natural expression on the cell surface.
  • an expression system that permits higher localization of proteins of interest on the cell surface would facilitate the testing of putative drugs and hence the drug discovery process.
  • the present disclosure relates to vectors that facilitate increased localization of receptor and other surface proteins such as INSR, IGFR, TGFBR1, EPHB2, BMPR1A, EGFR, FZD5, CD44, and PDGFR.
  • receptor and other surface proteins such as INSR, IGFR, TGFBR1, EPHB2, BMPR1A, EGFR, FZD5, CD44, and PDGFR.
  • the vectors, constructs and methods of the present disclosure facilitate the study of membrane proteins.
  • the vectors, constructs and methods of the present disclosure can be used to determine which membrane proteins completely depend on their 3'UTRs and which membrane proteins only partially, or not at all depend on their 3'UTRs for surface expression.
  • vectors, constructs, and methods of the present disclosure can be used in drug discovery for identifying agents that can modulate the activity of membrane proteins.
  • the vectors, constructs, and methods of the present disclosure can be used in immunotherapy.
  • vectors, constructs, and methods of the present disclosure can be used for expressing a chimeric antigen receptor (CAR) on the surface of a T cell.
  • CARs are artificial molecules that, when present at the surface of immune effector cells, enable them to recognize a desired protein, or antigen, and trigger the killing of cells harboring this antigen at their surface (target cells).
  • Primary T cells can be isolated and engineered to express CARs— receptors that combine an extracellular, single-chain antibody domain, which recognizes a specific tumor-associated antigen, with intracellular signaling domains from the T cell receptor (TCR) and costimulatory receptors.
  • TCR T cell receptor
  • CAR T cells Upon antigen ligand engagement, CAR T cells execute multiple key therapeutic functions, including production of antitumor cytokines and killing of target tumor cells.
  • use of specific 3'UTR sequences of the present disclosure can promote and/or enhance sustained surface expression and localization of CARs on T cells.
  • T cells require two signals to become fully activated.
  • a first signal which is antigen-specific, is provided through the T cell receptor which interacts with peptide-MHC molecules on the membrane of antigen presenting cells (APC).
  • a second signal is antigen nonspecific and is provided by the interaction between co- stimulatory molecules expressed on the membrane of APC and the T cell.
  • Co-stimulatory molecules are a heterogeneous group of cell surface molecules that act to amplify or counteract the initial activating signals provided to T cells from the T cell receptor following its interaction with an antigen/major histocompatibility complex.
  • vectors, constructs, and methods of the present disclosure can be used to increase the cell surface expression of co- stimulatory molecules, which could lead to an overall increase of the immune response.
  • the vectors, constructs and methods of the present disclosure facilitate the localization on the cell surface of proteins fused to a fluorescent protein as a detectable marker.
  • Fusion proteins are useful because they can be constructed to contain two or more desired functional elements that can be from two or more different proteins. Fusions that include larger polypeptides, such as an IgG Fc region, and even entire proteins, such as the green fluorescent protein (“GFP”) chromophore-containing proteins, have particular utility. Fusion proteins can be produced recombinantly by constructing a nucleic acid sequence which encodes the polypeptide or a fragment thereof in frame with a nucleic acid sequence encoding a different protein or peptide and then expressing the fusion protein.
  • GFP green fluorescent protein
  • the short 3'UTR appropriately linked (functionally tethered) to either nucleic acid encoding HuR or nucleic acid encoding SET serves as a surrogate for the long 3'UTR which by containing an HuR binding site recruits HuR in turn recruiting SET and drives the expressed polypeptide of interest to the cell surface.
  • a polynucleotide sufficient to facilitate localization of membrane proteins to the cell surface is 74- nucleotide sequence SEQ ID NO 3.
  • sequence sufficient to facilitate localization of membrane proteins to the cell surface is at least 90% or at least 92% or at least 95% or at least 98% homologous (i.e., having the foregoing percent sequence identity) to the 74- nucleotide sequence SEQ ID NO 3.
  • Percent sequence identity between nucleic acid sequences can be determined using FASTA with its default parameters (a word size of 6 and the NOP AM factor for the scoring matrix) or using Gap with its default parameters as provided in GCG Version 6.1.
  • sequences can be compared using the computer program, BLAST (Altschul et al., J. Mol. Biol. 215:403-410 (1990); Gish and States, Nature Genet.
  • one or more uridine nucleotides within the 74-nucleotide can be replaced by uridine analogs, wherein uridine analog refers to a compound which bares structural similarity with uridine, or a metabolic precursor thereof.
  • Uridine analogs according to the invention include, but are not limited to: uracil, uridine monophosphate, uridine biphosphate, uridine triphosphate; thymine, thymidine, thymidine monophosphate, thymidine biphosphate, thymidine triphosphate; -D-uridine-5'- bis(SATE)phosphodiester, a prodrug of -D-uridine-5'-monophosphate; acylated uridine nucleosides, triacetyluridine (TAU), 5-(phenylthio)acyclouridine (PTAU), or any other uridine analog which can be used for substitution of uridine.
  • TAU triacetyluridine
  • PTAU 5-(phenylthio)acyclouridine
  • a polynucleotide sufficient to facilitate localization of membrane proteins to the cell surface is a 53-nucleotide artificial sequence SEQ ID NO: 56.
  • a sequence sufficient to facilitate localization of membrane proteins to the cell surface is at least 90% or at least 92% or at least 95% or at least 98% homologous (i.e., having the foregoing percent sequence identity) to the 53- nucleotide sequence SEQ ID NO 56.
  • a polynucleotide sufficient to facilitate localization of membrane proteins to the cell surface is a 117 -nucleotide artificial sequence SEQ ID NO: 57.
  • the sequence sufficient to facilitate localization of membrane proteins to the cell surface is at least 90% or at least 92% or at least 95% or at least 98% homologous (i.e., having the foregoing percent sequence identity) to the 117- nucleotide sequence SEQ ID NO 57.
  • a polynucleotide sufficient to facilitate localization of membrane proteins to the cell surface is a 384-nucleotide artificial sequence SEQ ID NO: 58.
  • the sequence sufficient to facilitate localization of membrane proteins to the cell surface is at least 90% or at least 92% or at least 95% or at least 98% homologous (i.e., having the foregoing percent sequence identity) to the 384- nucleotide sequence SEQ ID NO 58.
  • the polynucleotide sufficient to facilitate localization of membrane proteins to the cell comprises a sequence comprising an HuR binding site from TNF-cc: TTGTGATTATTTATTATTTATTTATTATTTATTTATTTA (SEQ ID NO: 59).
  • the polynucleotide sufficient to facilitate localization of membrane proteins to the cell comprises a sequence comprising an HuR binding site from TNF-cc (SEQ ID NO: 59) and one or more sequence elements, including but not limited to restriction site sequences, sequences found in the long 3'UTR of CD47, and sequence motifs enriched in the 3'UTRs of membrane proteins.
  • compositions and methods of the present disclosure facilitate expression of non-membrane proteins on the surface of the cell.
  • non-membrane proteins can include proteins that are routinely used as markers in molecular biology. The most frequently used markers are fluorescent proteins and epitope tags. Fluorescent markers such as green fluorescent protein (GFP), yellow fluorescent protein (YFP), mCherry, and cyan fluorescent protein (CFP) enable the study of cellular processes by fluorescent microscopy.
  • GFP green fluorescent protein
  • YFP yellow fluorescent protein
  • CFP cyan fluorescent protein
  • compositions and methods of the present disclosure facilitate the expression of immunogenic portions of antigens from foreign organisms or other pathogenic agents (e.g., cancerous cells) on the cell surface.
  • Antigens generally include, but are not limited to, proteins, polypeptides, peptides, polysaccharides such as glycans, polysaccharide conjugates, peptide and non-peptide mimics of polysaccharides and other molecules, small molecules, lipids, glycolipids, and carbohydrates.
  • Antigens which can be localized on the cell surface using the methods and compositions of the present disclosure will generally comprise a peptide or polypeptide component or moiety.
  • the expression of antigens on the cell surface can directly or indirectly stimulate one or more components of the immune response.
  • compositions and methods of the present disclosure facilitate expression of secretory proteins on the surface of a cell.
  • protein hormones include cytokines, lymphokines, neurotrophic hormones and adenohypophyseal polypeptide hormones such as growth hormone, prolactin, placental lactogen, luteinizing hormone, follicle-stimulating hormone, thyrotropin, chorionic gonadotropin, corticotropin, a or ⁇ -melanocyte-stimulating hormone, ⁇ -lipotropin, ⁇ -lipotropin and the endorphins; hypothalamicmic release-inhibiting hormones such as corticotropin-release factor, growth hormone release-inhibiting hormone, growth hormone-release factor; and other polypeptide hormones.
  • the present disclosure is directed to targeting cell surface (or membrane or transmembrane) proteins to reduce or eliminate their localization on the cell surface.
  • the present disclosure provides inhibitors of 3'UTR localization function (such as shRNA, RNAi, or CRISPR system against DNA encoding long 3'UTR) that will block access by HuR to the HuR binding site within 3'UTR (see, e.g. Figure 1, Figure 5 and Example 1).
  • the present disclosure provides compositions and methods for enhancing the cell surface localization of proteins.
  • Observed and expected cell surface localization enhancements obtained by use of the compositions and methods of the present disclosure commonly include about 1.2 fold, about 1.3 fold, about 1.4 fold, about 1.5 fold, about 1.6 fold, about 1.7 fold, about 1.8 fold, about 1.9 fold, or about 2.0 fold or more increase in the proportion of a protein expressed on a cell surface compared to negative control (expression on the surface of the same type of cell in the absence of the present compositions and methods).
  • Vectors according to the invention include plasmids and viruses. Many such vectors and expression systems are well known and documented in the art. Numerous vectors are known in the art including, but not limited to, linear polynucleotides, transposons, polynucleotides associated with ionic or amphiphilic compounds, plasmids, bacteriophages and viruses. Examples of viral vectors include, but are not limited to, vaccinia and attenuated vaccinia vectors such as MVA, adenoviral vectors, adeno-associated virus vectors, retroviral vectors such as herpes or lentivirus derived vectors, and the like.
  • the vector is a plasmid, including plasmids designed to integrate into the host chromosome, and plasmids that are autonomously replicating in the host cell.
  • the plasmid may be maintained in the host cell at various levels, and therefore, the vector of the invention may be a low copy-number plasmid or high copy-number plasmid.
  • the vector may be a viral vector (commercially available from Addgene). Commercially available plasmids can be purchased from numerous vendors, including Addgene and New England Biolabs.
  • suitable vectors can be chosen or constructed for expression and localization of the polypeptides of interest of the present disclosure, containing the appropriate regulatory sequences, including promoter sequences, terminator sequences, polyadenylation sequences, enhancer sequences, marker genes and other sequences as appropriate.
  • appropriate regulatory sequences including promoter sequences, terminator sequences, polyadenylation sequences, enhancer sequences, marker genes and other sequences as appropriate.
  • Many known techniques and protocols for manipulation of nucleic acid for example, in the preparation of nucleic acid constructs, mutagenesis, sequencing, introduction of DNA into cells and gene expression, and analysis of proteins, are described in detail in Short Protocols in Molecular Biology: a compendium of methods from Current protocols in molecular biology, Volume 2, F. M. Ausubel et al, eds., John Wiley & Sons, Inc., fifth edition, 2002.
  • the choice of the vector will depend on several factors, including the compatibility of the vector with the cell into which the vector is to be introduced (e.g., a mammalian cell for expression, or another host cell such as a bacterial cell, useful for propagating or amplifying the vector), and the ability of the vector to integrate into the mammalian or host cell genome.
  • the vector can be a viral vector, a phage, a phagemid, a cosmid, a fosmid, a bacteriophage, an artificial chromosome, a cloning vector, a shuttle vector, a plasmid (linear or closed circular), or the like.
  • Vectors can include chromosomal, non-chromosomal and synthetic DNA sequences.
  • vectors of the present disclosure are commercially available and can be obtained from sources such as Addgene, Sigma Aldrich, Promega, Invitrogen, etc.
  • Vectors of the present disclosure can comprise any of a number of promoters known to the art, wherein the promoter is constitutive, regulatable or inducible, cell type specific, tissue-specific, or species specific. Further specific examples include, e.g., tetracycline-responsive promoters (Gossen M, Bujard H, Proc Natl Acad Sci USA. 1992,15;89(12):5547-51). In addition to the sequence sufficient to direct transcription, a promoter sequence of the invention can also include sequences of other regulatory elements that are involved in modulating transcription (e.g.: enhancers, kozak sequences and introns).
  • promoter/regulatory sequences useful for driving constitutive expression of a gene include, but are not limited to, for example, the cytomegalovirus immediate early promoter enhancer sequence, the SV40 early promoter, the immunoglobulin promoter, as well as the Rous sarcoma virus promoter, and the like.
  • inducible and tissue specific expression of an RNA, transmembrane proteins, or other proteins can be accomplished by placing the nucleic acid encoding such a molecule under the control of an inducible or tissue specific promoter/regulatory sequence.
  • tissue specific or inducible promoter/regulatory sequences which are useful for this purpose include, but are not limited to, the MMTV LTR inducible promoter, the SV40 late enhancer/promoter, synapsin 1 promoter, ET hepatocyte promoter, GS glutamine synthase promoter and many others.
  • tissue-specific promoters can be found at http://www.invivogen.com/prom-a-list.
  • promoters which are well known in the art can be induced in response to inducing agents such as metals, glucocorticoids, tetracycline, hormones, and the like, are also contemplated for use with the invention.
  • the present disclosure includes the use of any promoter/regulatory sequence known in the art that is capable of driving expression of the desired protein operably linked thereto. Expression of vectors can be transient or stable.
  • Truncated promoters may also be generated from promoter fragments or by mixing and matching fragments of known regulatory elements as is practiced in the art (Andersen et al. Mol Biotechnol. 2011 Jun;48(2): 128-37).
  • Vectors of the present disclosure can be delivered to cells of any type, including but not limited to neural cells (including cells of the peripheral and central nervous systems, in particular, brain cells), lung cells, retinal cells, epithelial cells (e.g., gut and respiratory epithelial cells), muscle cells, pancreatic cells (including islet cells), hepatic cells, myocardial cells, bone cells (e.g., bone marrow stem cells), hematopoietic stem cells, spleen cells, keratinocytes, fibroblasts, endothelial cells, prostate cells, germ cells, immune system cells, for example T-cells, and the like.
  • the cell may be any progenitor cell.
  • the cell can be a stem cell.
  • the cells can be from any species of origin.
  • Transfection refers to the taking up of a vector by a host cell whether or not any coding sequences are in fact expressed. Numerous methods of transfection are known to the ordinarily skilled artisan, for example, lipofectamine, calcium phosphate co-precipitation, electroporation, DEAE-dextran treatment, microinjection, viral infection, and other methods known in the art. Transduction refers to entry of a virus into the cell and expression (e.g., transcription and/or translation) of sequences delivered by the viral vector genome. In the case of a recombinant vector, "transduction" generally refers to entry of the recombinant viral vector into the cell and expression of a nucleic acid of interest delivered by the vector genome.
  • CD47 as an Example of a Cell Surface Protein Localized by Long 3'UTR
  • CD47 is best known as a ubiquitous cell surface molecule that acts as a marker of self and protects cells from phagocytosis by macrophages.
  • the inventors show that CD47 protein is expressed on the cell surface, as well as intracellularly.
  • the CD47 gene produces alternative 3'UTRs (SEQ ID NO 1), wherein the two isoforms differ in the length of the 3'UTR. Further, the association of two 3'UTR isoforms of different length with the CD47 molecule phenomenon appears to be universal for CD47, as the presence of two 3' UTR isoforms was detected in every cell line tested
  • the present disclosure demonstrates the ability of the long 3' UTR of CD47 and fragments thereof (Example 5) to control cell surface localization of CD47 and to facilitate cell surface localization of proteins other than CD47.
  • Example 5 transcription of a construct containing nucleic acid encoding green fluorescent protein (GFP) containing the long 3'UTR, along with the signal peptide and the C-terminal of CD47 containing one or more transmembrane domains of CD47 including the SET binding site (named GFP-TM-LU), followed by translation results in GFP localized on the cell surface.
  • GFP green fluorescent protein
  • GFP encoded by an mRNA containing the peptide signal and transmembrane domain of CD47 including its C-terminal but only the short 3'UTR of CD47 (named GFP-TM-SU) localized mostly in the endoplasmic reticulum (ER).
  • GFP-TM-SU short 3'UTR of CD47
  • the long 3'UTR isoform of CD47 encodes information necessary for cell surface expression independently of RNA localization.
  • HuR 3'UTR-dependent protein localization is dependent on an RNA-binding protein (RBP) Embryonic Lethal Abnormal Vision (ELAV) Ll/Human antigen R (HuR) (Ma et al. / Biol Chem. 1996 Apr 5; 271(14):8144-51). HuR belongs to the ELAV/Hu family of proteins.
  • RBP RNA-binding protein
  • ELAV Embryonic Lethal Abnormal Vision
  • HuR Ll/Human antigen R
  • the ELAV/Hu proteins possess three RNA-recognition motifs through which they bind with high affinity and specificity to target mRNAs containing AU- and U-rich sequence elements (ARE) found within 3 '-untranslated regions (3'-UTRs) and modify their expression by altering their stability, translation, or both. Since the long 3 'UTR of CD47 contains many uridine-rich elements (illustrated in the printed version of SEQ ID NO: 1) which could potentially be bound by HuR, the inventors tested whether HuR was needed in 3 'UTR-dependent protein localization.
  • ARE AU- and U-rich sequence elements
  • the long 3'UTR of CD47 requires the activity of HuR; an effective segment of the 3'UTR should include the HuR binding site, which can be found within or immediately adjacent to and overlapping with one or more uridine-rich regions of the long 3'UTR.
  • HuR In addition to its role as an RBP, HuR has been shown to interact with various proteins through protein-protein interactions, including SET, ANP32A, and ANP32B (Brennan et al. /. Cell Biol. 151, 1-14 (2000).
  • SET contains a long acidic C-terminus that interacts with the RRM 3 (RNA recognition motif 3) of HuR. While nuclear SET binds to histone tails and prevents acetylation, phosphorylated SET localizes to the cytoplasm and the surface of the endoplasmic reticulum (ten Klooster et al. EMBO J 26, 336-345 (2007); Fan et al. EMBO J 17, 3448- 3460 (1998)).
  • SET was shown to interact with Racl, which results in translocation of SET to the plasma membrane upon Racl activation.
  • Racl-GDP is mostly localized to the cytoplasm
  • Racl-GTP active Racl
  • the inventors have demonstrated that in addition to HuR, SET and RAC1 are also needed for 3'UTR-dependent protein localization (Example 3). Indeed, if the C- terminus of the membrane-associated protein is not included in the polynucleotide construct, the SET binding site will be necessarily omitted and localization will be substantially reduced.
  • the entire C-terminus may not be necessary as long as the SET-binding site is included.
  • Blazemarks for SET binding sites are provided in Fig. 13 and Table 1 and its description herein (Example 7) or various membrane-associated proteins: CD47 (SEQ ID NO 8), CD44 (SEQ ID NO 9), ITGA1 (SEQ ID NO 10), BAFFR (SEQ ID NO 11) and TSPAN13 (SEQ ID NO 12).
  • 3'UTR-dependent protein localization is a widespread mechanism for surface expression of membrane proteins. Examination of numerous transmembrane proteins that are derived from mRNAs with 3'UTR isoforms that can be bound by HuR (Kishore, S. et al. Nat Methods 8, 559-564 (2011); Lebedeva, S. et al. Mol Cell 43, 340-352 (2011); Mukherjee, N. et al. Mol Cell 43, 327-339 (2011)) showed that their cell surface localization is also dependent on HuR (Example 4).
  • the inventors generated GFP-fused long UTR (LU) and short UTR (SU) constructs for transmembrane proteins, including transmembrane proteins CD44, ITGA1, and TNFRSF13C, as well as their respective transmembrane domains (TMDs), C-termini, and 3'UTR.
  • LU long UTR
  • SU short UTR
  • TMDs transmembrane domains
  • 3'UTR increased surface localization of GFP-TM (Example 4).
  • the present disclosure provides a smaller 3'UTR segment of the longer 3'UTR sufficient for surface localization of transmembrane proteins.
  • a 3'UTR (74 nucleotide long) with only a few HuR-binding sites (HuR-BS) was adequate to mediate the cell surface localization.
  • deletion of the 31- nucleotide uridine-rich sequence (HuR-BSA) abolished the ability of 74- nucleotide long sequence to mediate cell surface protein localization.
  • a uridine-rich sequence embodying an HuR binding site is necessary and sufficient for surface localization by UDPL.
  • the uridine-rich sequence can be much shorter than the long 3UTR itself as illustrated not only by the 74-nt segment but also by even shorter polynucleotides described below.
  • AU rich elements can be separated into three classes (Chen et al, 1995): Class I AREs contain several dispersed copies of an AUUUA motif within U-rich regions. C-My c and MyoD contain class 1 AREs. Class II AREs possess two or more overlapping UUAUUUA(U/A)(U/A) nonamers. Molecules containing this type of AREs include GM- CSF and TNF-oc.
  • Class III ARES are less well defined (Peng et al. Mol. Cell. Biol. 16: 1490- 1499 (1996). These AU rich regions do not contain an AUUUA motif. c-Jun and Myogenin are two well-studied examples of this class. HuR binds to AREs of all the three classes. Thus, according to the methods of present disclosure, engineering the HuR specific binding sites into the 3 ' UTR of nucleic acid molecules is anticipated to lead to increased surface localization of proteins.
  • the minimal sequence containing an HuR binding site that is sufficient for surface localization of proteins comprises one stretch of two uridines. In some embodiments, the minimal sequence sufficient for surface localization of proteins comprises a stretch of three uridines. In some embodiments, the minimal sequence sufficient for surface localization of proteins comprises a stretch of five uridines.
  • the minimal sequence containing an HuR binding site that is sufficient for surface localization of proteins comprises two or more stretches of 2-7 uridines within a stretch of 100 nt.
  • HuR binds to the HuR-BS and to the LU but not to the SU isoform of CD47 (Example 6).
  • SET also associates with the long 3' UTR of CD47, which is dependent on HuR.
  • SET or HuR overexpression in the cell was insufficient to localize GFP-TM-SU to the cell surface.
  • tethering of SET or HuR to the short 3'UTR isoform of CD47 was sufficient to redirect GFP-TM localization from the endoplasmic reticulum to the plasma membrane.
  • HuR-binding sites are highly abundant as HuR binds to thousands of mRNAs (Kishore et al., Nature Methods 8, 559-564 (2011); Lebedeva et al., Mol. Cell 43, 340-352 (2011); Mukherjee, N et al., Mol. Cell 43, 327-339 (2011)), with a third of them being membrane proteins (Fig. 12b).
  • Fig. 3d, e and Fig. 10 Although the precise SET-binding motif has not been elucidated, the inventors (Fig. 3d, e and Fig. 10) and others have shown that SET binds to positively charged amino acids in histone tails or cytoplasmic domains of membrane proteins (Schneider et al. /. Biol. Chem.
  • the SET binding site comprises of one or more positively charged amino acids, such as lysine or arginine and has to be located in the cytoplasmic domain of a membrane protein.
  • the cytoplasmic domains of membrane proteins are enriched in positively charged amino acids for topological reasons (Nillson et al. Proteins 60, 606-616 (2005)). Therefore, potential SET-binding sites in cytoplasmic domains of membrane proteins are very widespread.
  • cytoplasmic domain C- terminal
  • Figure 13 and Table lillustrate fragments of cytoplasmic domains of various proteins harboring SET- binding sites and indicate lysine and arginine residues potentially contained within such SET binding sites. These could be used in vectors, constructs and methods of the present disclosure to increase surface localization of a polypeptide of interest.
  • CD47 expressed in the ER.
  • the majority of known functions of CD47 involve its expression on the cell surface, where, in addition to protecting cells from phagocytosis, it also plays an important role in cell adhesion and migration (Jaiswal et al. Cell 138, 271-285 (2009); Ridley et al., Cell 70, 401-410 (1992); Oldenborg et al. ISRN hematology 2013, 614619 (2013)).
  • CD47 also functions in the regulation of apoptosis (Lamy et al., / Biol Chem 278, 23915-23921 (2003)), as CD47 deficient Jurkat cells (called JinB8 cells, Reinhold et al., International immunology 11, 707-718 (1999)) and cells or tissues from CD47 knockout mice (Lindberg et al. Science 274, 795-798 (1996)) fail to undergo apoptosis after ⁇ - irradiation (Isanberg et al., The American journal of pathology 173, 1100-1112 (2008); Soto- Pantoja et al. Journal of genetic syndrome & gene therapy 2 (2011).
  • CD47 promotes a pro-apoptotic response to ⁇ -irradiation has remained unknown.
  • expression of CD47-SU in JinB8 cells restored apoptosis, but expression of CD47-LU did not affect the loss- of-apoptosis phenotype (Fig. 4e and Fig. 1 If, g).
  • transcriptional upregulation alone would be non-optimal as it would confer increased susceptibility to apoptosis.
  • ApA-generated 3' UTR isoforms allow independent regulation of differentially localized and functionally distinct CD47 protein.
  • a polypeptide of interest can be expressed and directed to the cell surface where it might exert one type of functionality without undesirable effects that may accompany wholesale upregulation of transcription of all forms (and increase of all effects) of the polypeptide of interest.
  • CD47 protein localized to the same cellular compartment can exert different functions depending on whether it was produced by the SU or LU mRNA isoform.
  • UTR sequences comprising HuR binding site from the 3 'UTR of TNF-oc enhance surface localization of proteins
  • the present disclosure provides smaller 3 'UTR segments that comprise an HuR binding site from TNF-oc.
  • TNF-oc As shown in Example 12, several artificial UTRs (UTR-1, UTR-2, and UTR-3) were able to increase surface localization of CD47 ( Figure 15B, 15C, and 15D).
  • Each of the three artificial UTRs comprises a sequence comprising an HuR binding site from TNF-oc:
  • TTGTGATTATTTATTATTTATTTATTTATTTA (SEQ ID NO: 59).
  • the inventors identified three motifs that are enriched in the 3 'UTRs of human membrane proteins ( Figure 15A). These motifs (such as CACACA and GAGAGA) were then used as additional sequences in the design and generation of artificial 3 'UTR-2. Given that these motifs are enriched in the 3 'UTRs of membrane proteins, it is likely that the presence of one or more of these motifs in the 3 'UTR serves to facilitate and/or increase surface localization of proteins.
  • artificial 3'UTR sequences of the present disclosure can comprise restriction site sequences, such as GC-rich sequences shown in Example 12. While GC-rich sequences are restriction sites, they make sure that the AU-rich sequences in between are accessible.
  • the sequences of artificial 3 'UTRs of the present disclosure can also comprise sequences found in the long 3'UTR of CD47. Addition of 3'UTR sequences of CD47 to the artificial 3'UTR of the present disclosure was carried out in UTR-2 and UTR-3 (see example 12, Table 2).
  • the artificial 3'UTR sequences of the present disclosure can comprise one or more of additional elements including, but not limiting to restriction site sequences, sequences found in the long 3'UTR of CD47, sequence motifs enriched in the 3 'UTRs of membrane proteins, and additional sequences that are likely to further enhance surface localization of proteins in at least one cell type.
  • AU-rich HuR binding sites found within the 3'UTR of TNF-oc are efficient in enhancing protein surface localization.
  • sequences found in the 3 'UTRs of genes that contain AU-rich elements can also be used to promote and/or enhance protein surface localization.
  • Genes that contain AU-rich elements within their 3 'UTR include, but are not limited to ELAVL1, HNRNPD, IL1B, IL3, IFNB1, TNF, CSF2RA, VEGFA, FOS, JUNB, JUN, MYC, TP53, CDKN1A, CCNA2, CCNB1 and CCND1.
  • kits for conveniently and/or effectively carrying out methods of the present disclosure.
  • kits will comprise sufficient amounts of components to allow a user to perform multiple experiments.
  • Such experiments include enhancing cell surface localization of a protein or fragment thereof, as well as promoting cell surface localization of a protein or a fragment thereof that otherwise does not localize to cell surface naturally.
  • kits comprising the polynucleotides, vectors and constructs comprising one or more polynucleotides of the disclosure.
  • the kits of the present disclosure comprise fragments of the 3 'UTR sequences, wherein the fragments comprise an HuR binding site.
  • the kits can further comprise a vector into which said fragments of the 3 'UTR sequences can be inserted.
  • the kit further comprises reagents such as a transfection reagent that can be used to introduce the vector after a polynucleotide of interest has been incorporated in it into cells. Instructions for use may also be optionally included.
  • the kit comprises a vector and instructions for its use.
  • MCF7 breast cancer
  • HeLa cervical cancer
  • HEK293 epidermal growth factor
  • Caov-3 ovarian carcinoma
  • NTERA2 epidermal carcinoma
  • THP-1 cells monocytic leukaemia
  • B-LCL Epstein Barr virus (EBV)- immortalized human B cells described earlier (Lianoglou et al. Genes Dev. 27, 2380-2396 (2013)).
  • U20S cells were a gift from T. Brummelkamp, Toledo (B-cell lymphoma) cells were a gift from M. Mueschen, U251 (glioblastoma) cells were a gift from I.
  • pSUPERretropuro was modified by cloning IRES::GFP (derived from pMSCVpig) (Mayr et al. Cell 138, 673-684 (2009)) downstream of puromycin to obtain pSUPERretropuro containing enhanced (e) GFP (shRNA-GFP).
  • IRES::GFP derived from pMSCVpig
  • shRNA-GFP enhanced GFP
  • the following DNA oligonucleotides served as shRNA precursors and were cloned into pSUPER-GFP or pSUPER.
  • the proximal polyadenylation site was mutated from AAUAAA to ACUCAA using the QuikChange Multi Site Directed Mutagenesis Kit (Agilent).
  • the resulting plasmids were used to test qPCR primers for accuracy in measuring short to long 3'UTR isoform ratios (see later).
  • the short 3'UTR of CD47 used in the MS2-BS construct was cloned from the plasmid containing the long 3'UTR with the mutated proximal polyadenylation site.
  • eGFP was PCR- amplified from pMSCV-pig and inserted upstream of each CD47 3'UTR (BamHI, NotI).
  • the signal peptide of CD47 was generated by annealing two DNA oligonucleotides that were inserted into the BamHI site.
  • the sequence of the TMDs and C-terminal tail of CD47 (the longest isoform, isoform 4; ref. Reinhold et al. /. Cell Sci. 108, 3419-3425 (1995)) were cloned fromToledo cDNA and inserted downstream of eGFP (BsrgI, NotI).
  • Isoform 4 was chosen as it is the most abundant isoform in Jurkat cells (data not shown).
  • the two nucleotides of eGFP that occur after the BsrgI site were included in the forward primer.
  • the ECD, TMDs and C terminus of CD47 were PCR amplified from Toledo cDNA using the TM reverse primer and the CDS forward primer and inserted downstream of eGFP (BsrgI, NotI).
  • the sequence of HuR-BS and HuR-BSA are shown in Fig. 7 and replaced the long 3'UTR in GFP-TM-LU.
  • the GFP constructs containing the TMDs and C termini fused to either the long or short 3'UTRs of CD44 (SEQ ID NO 2), 1TGA1 (SEQ ID NO 5) and TNFRSF13C (SEQ ID NO 6) were generated as follows.
  • TMDs, C termini and short 3'UTRs of CD44, ITGA1 and TNFRSF13C were cloned from Toledo, SHSY-5Y and B-LCL cDNA respectively and inserted downstream of eGFP (BsrgI, Xbal).
  • Short 3'UTRs consisted of the first 122 nucleotides of CD44, the first 45 nucleotides of ITGA1 and the first 337 nucleotides of TNFRSF13C.
  • Long 3' UTRs: 3,068 nucleotides after the stop codon of CD44 and 3,996 nucleotides after the stop codon of ITGA1 were used.
  • TNFRSF13C 1,221 nucleotides after the stop codon together with the last 600 nucleotides of the 3'UTR (nucleotides 2,712-3,311 after the stop codon containing the majority of HuR-binding sites) were used as long 3'UTR and were cloned from genomic DNA.
  • the complete open reading frame of TNFRSF13C was amplified from human B-cell cDNA and cloned using Gibson Assembly Cloning (NEB) into pcDNA3.1 vector used above downstream of eGFP.
  • CD44-SU and -LU the open reading frame of CD44 (without the start codon) was amplified from cDNA of the human breast cancer cell line MDA-MB231 and cloned using Gibson Assembly Cloning (NEB) into pcDNA3.1 vector used above downstream of eGFP.
  • NEB Gibson Assembly Cloning
  • To generate the GFP-TM-LUAC construct the sequence of just the TMDs of CD47 was cloned from Toledo cDNA using the TM forward primer and TMAC reverse primer and inserted downstream of eGFP (Bsrgl, Notl). The 24 MS2-binding sites were cloned from a plasmid obtained from J.Gerst (Slobodin et al.
  • CD47UTRF 5 ' - ATGCGCGGCCGCAGTGAAGTGATGGACTCCGATT- 3' ;
  • SEQ ID NO 24 CD47shortUTRR: 5' -
  • CD47CDSF 5 ' - ATGCTGTAC AAGC AGCTACTATTTAATA AAAC AA-3 ' ;
  • SEQ ID NO 33 TMDCR: 5 ' - ATGCGCGGCCGCTTATTTCATATAAACTAGTCCAAGTAA-3 ' ;
  • SEQ ID NO 34 MS2-BSF: 5 ' - ATGCTCTAGAGGGCCCTATATATCGATCCTAAG-3 ' ;
  • SEQ ID NO 35 MS2-BSR: 5 ' - ATGCGGGCCCTTTATTATGCTTGGTACCGAGCTCG-3 ' .
  • SEQ ID NO 36 HuRF: 5 ' - ATGCCGTACGAGTCTAATGGTTATGAAG ACCAC A-3 ' ;
  • SEQ ID NO 37 HuRR: 5 ' - ATGCTCTAGATTATTTGTGGGACTTGTTGGT-3 ' ;
  • SEQ ID NO 38 SETF: 5 ' - ATGCTGTACAAGTCGGCGCCGGCGGCCAAA-3 ' ;
  • SEQ ID NO 39 SETR: 5' - ATGCTCTAGATTAGTCATCTTCTCCTTCATCCTC-3 ' ;
  • SEQ ID NO 40 SETR: 5' - ATGCTCTAGATTAGTCATCTTCTCCTTCATCCTC-3 ' ;
  • StopF 5 ' -GTACAAGTAATAATAAT-3 ' ;
  • SEQ ID NO 41 StopR: 5'- CTAGATTATTATTACTT-3' ;
  • SEQ ID NO 42 mCherryF: 5'- ATGCGGATCCGTGAGCAAGGGCGAGGAG-3 ' ;
  • SEQ ID NO 43 mCherryR: 5'- TTACTTGTACAGCTCGTCCATGC-3 ' .
  • the N17RAC1 construct was provided by A. Hall (Ridley et al. Cell 70, 401-410 (1992)). Transfections
  • mice were incubated with mouse anti-CD47-PerCy5.5 (BD Biosciences, 561261), mouse anti-CD44-PE (BD Biosciences, 561858), chicken anti-GFP (Abeam, abl3970), mouse anti-ITGAl-PE (BD Biosciences, 555749), mouse anti- BAFFR-PE (BD Biosciences, 554680) or rabbit anti- TSPAN13 (Genetex, GTX52155) in FACS buffer A (0.5% FBS in PBS) for 30 min at 4°C, and then washed twice in FACS Buffer A.
  • mouse anti-CD47-PerCy5.5 BD Biosciences, 561261
  • mouse anti-CD44-PE BD Biosciences, 561858
  • chicken anti-GFP Abeam, abl3970
  • mouse anti-ITGAl-PE BD Biosciences, 555749
  • mouse anti- BAFFR-PE BD Biosciences, 554680
  • rabbit anti- TSPAN13 Genetex, GTX52
  • FACS intracellular FACS
  • cells were fixed for 15 min at room temperature in fixation buffer (4% PFA, 0.02% sodium azide, and 0.1% Tween 20 in PBS), washed in FACS buffer B (0.02% sodium azide, 0.1% Tween 20 in PBS), permeabilized for 10 min at 4°C in permeabilization buffer (0.02% sodium azide, 0.1% Tween 20 and 10% dimethyl sulfoxide in PBS), washed, re-fixed for 5 min at room temperature in fixation buffer, and washed again.
  • Cells were incubated with the same primary and secondary antibodies as for surface FACS in FACS buffer B for 30 min at 4°C, and then washed twice in FACS Buffer B.
  • FACS live/dead analysis by FACS
  • TO-PR03 in FACS buffer A (0.5% FBS in PBS) for 10 min at 4°C, and then washed twice in FACS Buffer A. Cells were analysed is the same manner as for surface FACS.
  • CD47 For surface staining of CD47, cells were fixed for 15 min at room temperature in fixation buffer A (4% PFA and 0.02% sodium azide in PBS), washed with PBS, blocked for 15 min at 4°C in 5% Normal Goat Serum (Invitrogen, PCN5000) in PBS and then incubated with mouse anti-human CD47 (Santa Cruz, sc-59079) primary antibody for 1 h at 4°C in PBS.
  • fixation buffer A 4% PFA and 0.02% sodium azide in PBS
  • Alexa Fluor 594 and 680 were pseudo-coloured red and endogenous GFP and mCherry were imaged as they appear, without any antibody.
  • Custom Stellaris FISH Probes (Biosearch Technologies) were designed for the open reading frame of eGFP using the Stellaris Probe designer website, and with the assistance of Biosearch Technologies staff. The probes were conjugated to the Quasar 670 fluorochrome. Staining was carried out according to the manufacturer's protocols. Briefly, 24 h after transfection of the GFP-TM constructs cells were trypsinized and plated on Millicell EZ glass slides (Millipore) and allowed to grow overnight. Cells were washed in PBS, fixed in 4% PFA at room temperature for 10 min and permeabilized in 70% ethanol at 4°C for 2 h.
  • 3'-seq. 3'-seq reads of naive B cells, B-LCL and HEK293 cells were analysed and visualized as described previously 3 .
  • SEQ ID NO 44 CD47 probe F: 5 ' -TTGATGGAGCTCTAAACAAGTCC-3 ' ;
  • SEQ ID NO 45 CD47 probe R: 5 ' - GAATAACCAATATGGCAATGACG-3 ' ;
  • SEQ ID NO 46 GFP probe F: 5'-TAAACGGCCACAAGTTCAGC-3' ;
  • SEQ ID NO 47 GFP probe R: 5'- CTTGTACAGCTCGTCCATGC-3 ' .
  • CD47TotalF 5 ' -AGTGATGGACTCCGATTTGG-3' ; CD47TotalR: 5'- GGGTCTCATAGGTGACAACCA-3 ' ; CD47LongF: 5 ' - AAGAG AACTCC AGTGTTGCT- 3'; CD47LongR: 5 ' -ACGGTAACACAGCTGTAAAACA-3 ' ; GAPDHF: 5'- ACAACTTTGGTATCGTGGAAGG-3' ; GAPDHR: 5'- TATTTGGCAGGTTTTTCTAGACG-3 ' .
  • GFP-TM-LU, GFPTM- SU, GFP-TM-HuR-BS and GFP-TM-HuR-BSA were transfected into HEK293 cells and immunoprecipitation of protein-RNA complexes was carried out as previously described 37 .
  • RNA-immunoprecipitations were performed with crosslinking to prevent re-association of HuR with mRNA after lysis 38. Briefly, cells were harvested and washed twice in cold PBS. Formaldehyde was added to a final concentration of 1% (v/v) and the cells were incubated at room temperature for 10 min. The reaction was quenched by addition of glycine to a final concentration of 0.25M and incubated at room temperature for 5 min.
  • the cell pellet was washed twice in cold PBS and resuspended in RIPA buffer (25mM Tris-HCl (pH 7.4), 150mM NaCl, 1% NP-40, 1% Na- deoxycholate, 0.1% SDS, lmM EDTA, protease inhibitor cocktail (Roche, 04693124001)).
  • RIPA buffer 25mM Tris-HCl (pH 7.4), 150mM NaCl, 1% NP-40, 1% Na- deoxycholate, 0.1% SDS, lmM EDTA, protease inhibitor cocktail (Roche, 04693124001)
  • the mRNPs were solubilized by three rounds of sonication for 15 s each in a Misonix Ultrasonic Processor S-4000 at an output of 8-9 W. Insoluble material was removed by centrifugation.
  • Lysates were pre-cleared by addition of magnetic protein A beads (Millipore, LSKMAGAIO) and incubation for 30 min at 4°C with constant mixing.
  • the pre-cleared lysate was divided into three parts. One portion was retained for the input control and anti- HuR (Millipore, 07-1735) or IgG (Santa Cruz, sc-2025) were added to the two other portions and incubated at 4°C for 2 h.
  • Magnetic protein A beads (Millipore, LSKMAGAIO) were added and incubated at 4°C for 1 h.
  • qRT-PCR was carried out using primers for GAPDH (see earlier) and GFP, so as not to amplify endogenous CD47.
  • the primers used were as follows: SEQ ID NO 48: GFPF: 5 ' -TAA ACGGCC ACAAGTC AGC-3 ' ; SEQ ID NO 49: GFPR: 5'- AAGTCGTGCTGCTTCATGTG-3 ' .
  • HEK293 cells transiently transfected with sh2 HuR or sh Co were used to assess the presence of SET on endogenous CD47 mRNA and the requirement of HuR for this association. Cells were transfected and treated with high dose puromycin for 3 days. FACS analysis was used to ensure greater than 90% of surviving cells expressed the shRNA. RNA- immunoprecipitation was carried out as described earlier, but using anti-SET (Abeam, abl81990) or IgG (Santa Cruz, sc-2025).
  • CD47F 5 ' - AAGAGAACTCCAGTGTTGCT-3 '
  • CD47R 5'- ACGGTA AC AC AGCTGT A AA AC A- 3 ' .
  • HEK293 cells stably expressing sh3 HuR, sh4 HuR, shl SET, sh2 SET, sh2 Racl, sh3 Racl or sh Co, as well as HEK293 cells transiently transfected with sh2 HuR, CD47-SU, CD47-LU, CD47-LUlN17Racl, CD47-LU2Km, CD47-LUAC or CD47-LUACL, were lysed in Laemmli buffer (Sigma, S3401), boiled for 7 min and then cooled on ice. Lysates were run on NuPAGE Novex 4-12% Bis-Tris Gel (Invitrogen, NP0322BOX) and transferred to PVDF membrane (Bio-rad, 162-0177).
  • the antibodies were diluted in Odyssey Blocking Buffer containing 0.1% Tween 20 and the blots were incubated overnight at 4°C. After four washes in PBST (PBS plus 0.1% Tween 20) the blots were incubated for 1 h at room temperature in Odyssey Blocking Buffer containing 0.1% Tween 20 and 0.01% SDS and the following secondary antibodies: donkey anti-mouse IRDye 700 (Rockland Immunochemicals, 610-730-002), donkey antirabbit IRDye 680 (Li-Cor Biosciences, 926-68073), donkey anti-rabbit IRDye 800 (Li-Cor Biosciences, 926-32213), donkey anti-mouse IRDye 800 (Li-Cor Biosciences, 926-32212) and rabbit anti-chicken IRDye 800 (Rockland Immunochemicals, 603-432-002). The blots were washed four times in PBST and then thoroughly soaked in PBS
  • CD47-SU, CD47-LU, CD47-LUAC and CD47- LUACL were transfected into HEK293 cells and the cells were lysed in ice cold RIPA buffer ((25mMTris-HCl (pH 7.4), 150mM NaCl, 1% NP-40, 1% Na-deoxycholate, 0.1% SDS, 1 mM EDTA, protease inhibitor cocktail (Roche, 04693124001)) for 5 min on ice. After the cells were spun down at 20,000g for 20 min the supernatant was pre-cleared as described earlier. The lysate was divided in two equal parts (a small portion was removed to be used as the input control).
  • RIPA buffer (25mMTris-HCl (pH 7.4), 150mM NaCl, 1% NP-40, 1% Na-deoxycholate, 0.1% SDS, 1 mM EDTA, protease inhibitor cocktail (Roche, 04693124001)
  • Anti-GFP Invitrogen, A-6455
  • IgG Santa Cruz, sc-2025
  • Chicken anti-GFP (Abeam, abl3970) antibody was used to confirm immunoprecipitation of GFP constructs and rabbit anti-SET (Abeam, ab 181990) antibody was used to assess co-immunoprecipitation of SET protein.
  • CD47-SU, CD47-LU and pcDNA 3.1 vector alone were transfected into HEK293 cells.
  • the levels of RAC1-GTP were assessed using an Active Racl Detection Kit (Cell Signaling Technology, 8815) following the manufacturer's protocol. Briefly, cells were lysed in ice cold lysis buffer and centrifuged at 16,000g for 15 min at 4°C. The supernatant was added to GST-PAK1-PBD and glutathione resin and incubated with rocking for 1 h at 4°C (a small portion was removed to be used as the input control). The resin was washed three times with lysis buffer and then SDS sample buffer was added to elute the bound proteins. Western blotting was carried out as described earlier. Mouse anti-RACl (Cell signaling Technology, 8631S) antibody was used to detect RAC1- GTP as well as to assess total RAC1 in the samples.
  • Human macrophages were obtained by differentiation of THP-1 cells with 25 ngml "1 phorbol 12-myristate 13-acetate (PMA; Sigma) for 3 days. On day 3, Jurkat, JinB8, or transfected JinB8 cells were treated with 10 ⁇ g ml "1 Mitomycin C for 2.5 h at 37°C and washed three times in media. Mitocycin C treatment halts cell division and allows for a more accurate assessment of the percentage of cells that are phagocytosed. For JinB8 cells transfected with CD47-LU or CD47-SU the percentage of GFP + cells was determined by FACS before co-culture. The cells were either cultured alone or co-cultured with fully differentiated macrophages.
  • PMA phorbol 12-myristate 13-acetate
  • the long 3'UTR of CD47 localizes CD47 protein to the cell surface
  • shRNA-mediated knock-down (KD) of the longer 3'UTR isoform resulted in a decrease of CD47 surface expression without a change in intracellular expression when compared to the effects of a control shRNA (sh Co) (Fig. Id and Fig. 5d-g). These results demonstrate that the long 3'UTR isoform primarily facilitates cell surface localization of CD47 protein.
  • KD shRNA-mediated knock-down
  • GFP green fluorescent protein
  • S signal peptide
  • TMD transmembrane domain
  • GFP-TM This newly generated GFP was referred to as GFP- TM (Fig. le).
  • GFP-TM-LU mRNA containing the long 3'UTR of CD47
  • GFP-TM-SU GFP-TM encoded by an mRNA with the short 3'UTR of CD47
  • HuR, SET, and RACl mediate 3' UTR-dependent protein localization
  • UDPL 3'UTR-dependent protein localization
  • shRNAs was used to knockdown (KD) HuR in HEK293 cells (Fig. 6a).
  • HuR KD did not affect CD47 mRNA or 3'UTR isoform levels (Fig. 5d, 6b), nor did it affect total CD47 protein levels (Fig. 2b, bottom panel, Fig. 6a, 6c).
  • KD of HuR reduced CD47 cell surface expression (Fig. 2b, top panel, Fig. 2c). Accordingly, these observations show that in the case of CD47, HuR neither affects mRNA stability nor translation, but instead mediates CD47 protein localization post-translationally.
  • HuR interacts with a number of proteins through protein-protein interaction, including SET.
  • SET further interacts with Racl, which results in translocation of SET to the plasma membrane upon Racl activation.
  • Racl Racl
  • both proteins were knocked down (separately) in HEK293 cells.
  • KD of SET or Racl by shRNAs reduced surface expression of CD47 without affecting overall CD47 levels (Fig. 2b, Fig. 6c, 6d).
  • the long 3'UTR of CD44 contains a strong HuR-BS and enabled higher GFP surface expression than GFP generated from the short 3'UTR isoforms of ITGA1 or TNFRSF13C which do not contain HuR-BS (Fig. 2d).
  • the longer 3'UTR increases surface localization of GFP (Fig. If, h, 2d).
  • the 3'UTR of CD47 contains over 30 putative HuR-binding sites (HuR-BS) (Fig. 7).
  • HuR-BS putative HuR-binding sites
  • Fig. 7 a 3'UTR with only a few HuR-BS was tested for the ability to mediate surface localization (Fig. 7) and SEQ ID NO 3.
  • a single HuR-BS containing uridine-rich sequence was sufficient for surface localization of GFP-TM.
  • a single HuR-BS containing uridine-rich sequence was less potent than the full-length 3'UTR of CD47, it still achieved 75% of the potency of the full-length 3'UTR.
  • CD47 is a known HuR target (Kishore, S. et al. Nat Methods 8, 559-564 (2011); Lebedeva, S. et al. Mol Cell 43, 340-352 (2011); Mukherjee, N. et al. Mol Cell 43, 327-339 (2011)).
  • RNA immunoprecipitation RNA-IP
  • HuR was able to bind the single HuR-BS, which was abolished after mutation of the site (Fig. 3 a).
  • MS2 binding sites were added to the short 3'UTR of GFP-TM (Fig. 3b).
  • MS2-BS are derived from the bacteriophage MS2 and form RNA stem loops (Bertrand et al. Mol Cell 2, 437- 445 (1998)).
  • the capsid protein of MS2 here, called simply MS2 specifically recognizes these MS2 stem loops. Constructs were generated containing MS2 fused to mCherry and then either HuR, SET or with no further coding sequence (Fig. 3b).
  • the cytoplasmic domains of CD47 contain two separate SET-BS.
  • SET interacts with the CD47-LU protein to mediate 3' UTR-dependent protein localization.
  • CD47-LU Localizes to the Cell Surface via UDPL mediated by active RAC1
  • CD47-LU efficiently localized to the cell surface via UDPL mediated by active RAC1, since expression of a dominant-negative Racl (N17Racl, which has a higher affinity to Racl-GDP) (Ridley et al. Cell 70, 401-410 (1992)) decreased surface localization of GFP (Fig. 4b, c, and Fig. l ib).
  • CD47-SU primarily localizes to the endoplasmic reticulum, but also localizes partially to the cell surface, but independently of active RAC1 (Fig. 4b, c).
  • Membrane proteins rely on 3 " UTR-dependent protein localization for surface expression to varying degrees
  • Respective ECDs were added to CD44 and BAFFR (TNFRSF13C encodes the BAFF receptor, BAFFR), which increased surface expression of their SU isoforms compared with their GFP-TM isoforms, but to a lesser extent than was observed for CD47 (Fig. 1 lc, d).
  • Di- or multimerization of cell surface receptor subunits, which often occurs through their ECDs, is a common strategy for overcoming endoplasmic reticulum retention, because it results in masking of endoplasmic reticulum retention signals (Zerangue et al., Neuron, 22, 537-548 (1999)).
  • CD47-SU, CD44-SU and BAFFR-SU might use such a mechanism (although the multimerization partners are unknown) for their partial surface expression.
  • BAFFR the ECD only increased surface expression by 1.2-fold, indicating that BAFFR strongly depends on UDPL for surface expression (Fig. l id). This is supported by the absence of BAFFR on B cells in Rac-deficient mice (Walmsley et al. Science 302, 459-462 (2003)).
  • the data presented in this example suggests that membrane proteins rely on UDPL for surface expression to varying degrees.
  • Example 10 CD47 protein has different functions regarding cell survival depending on whether it was generated by the short or long 3 'UTR isoform
  • CD47ER CD47ER due to mis-localization or if CD47ER actually has an independent intracellular function. All cell types analyzed expressed higher amounts of the short 3'UTR isoform of CD47 (Fig. lc), which is associated with ER expression.
  • CD47 also functions in the regulation of apoptosis (Ridley et al., Cell 70, 401-410 (1992)).
  • Expression of GFP-CD47-SU (Fig. 4a) in JinB8 cells primarily leads to expression of CD47ER (Fig. 4b, left panel) and resulted in increased cell death after ⁇ -irradiation, such that it rescued the loss-of-apoptosis phenotype of CD47 deficient cells (Fig. 4e,).
  • GFP-CD47- LU (Fig. 4a) lead to comparable CD47 protein expression (Fig. 4b, Fig. 11a), but produced exclusively surface CD47 (Fig. 4b, right panel).
  • GFP-CD47-LU did not rescue the loss-of- apoptosis phenotype of JinB8 cells (Fig. 4e, Fig. 1 If ).
  • CD47-SU As the surface localization of CD47-SU is RACl -independent, it also did not co- localize with RACl at the plasma membrane (Fig. 4f). In contrast, CD47-LU showed strong co-localization with RACl at lamellipodia (Fig. 4f). Both activated RACl and CD47 are necessary for efficient cell migration and activated RACl localizes to the leading edge of migrating cells (ten Klooster et al. EMBO J. 26, 336-345 (2007); Lindberg et al. Science 21 A, 795-798 (1996); Frazier et al. UCSD Nature Molecule Pages (University of California, San Diego, 2010)).
  • CD47-LU resulted in changes in cell morphology with the generation of lamellipodia at the leading edge of cells.
  • CD47-LU but not CD47-SU, resulted in increased active RACl (Fig. 4g), which suggests that CD47-LU may cooperate with RACl during cell migration.
  • Artificial UTR-1 comprises the HuR-binding site from TNF-a. Previous studies have shown that HuR binds AU-rich elements, where the AU-rich element can have a core sequence of AUUUA or UUAUUUA(U/A)(U/A). Furthermore, it is known that binding of HuR to such an element regulates mRNA stability (in either a positive or negative manner, dependent on the context).
  • the artificial UTR-1 sequence also comprises GC-rich sequence at each end of. The GC-rich sequences are restriction sites, but they make sure that the AU-rich sequence in between is accessible.
  • UTR- 1 also contains a CAC sequence derived from TNF alpha which the inventors also considered important for surface localization.
  • the inventors carried out motif analysis using the search engine HOMER (available from the Salk Institute for Biological Studies). The inventors searched for the 8-mers that are bound by RNA-binding proteins. The motif analysis demonstrated that CACACA and GAGAGA sequences may also be important for surface localization ( Figure 15 A). In consideration of the results of the motif analysis, the inventors designed artificial UTR-2, which contains restriction sites, but also endogenous elements from the CD47 3'UTR that resemble CACACA and GAGAGA, flanking the HuR binding element. Finally, the inventors also generated artificial UTR-3, which comprises the HuR binding element of TNF- ⁇ as well as a piece of the CD47 3'UTR. Table 2 provides UTR-1, UTR-2, and UTR-3 sequences, where each sequence element and/or fragment within UTR-1, UTR-2, and UTR-3 is marked using normal letters, underlined letters, capital letters, and bold capital letters (see below).
  • sequences in bold capital letters indicate sequence comprising HuR- binding site from TNF-a; sequences in normal lower-case letters correspond to restriction sites; underlined lower-case sequences represent sequences found in the long 3 'UTR of CD47 (UTR-2 comprises nucleotides 2798-2813 and 3558-3598 of long 3'UTR of CD47, while UTR-3 comprises nucleotides 996-1317 of long 3'UTR of CD47); and normal capital letters (non-bold capital letters) correspond to artificial CACA and CCAACCTC sequences.
  • each artificial 3'UTR sequence was capable of increasing protein localization to the cell surface. Furthermore, while the effect of each UTR-1, UTR-2, and UTR-3 is comparable to that one of the long 3'UTR of CD47, it is possible that potencies for other membrane proteins or for different cell types can be further optimized for each of UTR-1, UTR-2, and UTR-3.
  • the use of polynucleotide sequences of the present disclosure is not limited to human cells, and can be used for surface localization of proteins in other organisms, as well as for localizing onto the surface non-human proteins.
  • the HuR-BS of TNF-a is completely conserved between mouse and human.
  • the artificial UTR sequences of the present disclosure which comprise the HuR-BS of TNF-a can be used in mouse cells or for surface localization of mouse proteins.
  • Example 5 As described in Example 5, the 74-nt region was identified containing HuR binding site (HuR-BS), which was successful in effecting surface localization of proteins.
  • HuR-BS HuR binding site
  • GFP green fluorescent protein
  • a signal peptide, the transmembrane domain and C-terminus of a transmembrane protein was incorporated into the vector engineered to express GFP-TM- containing 2xHuR-BS.
  • the resulting vector was tested for its ability to localize to the plasma membrane in numerous cell types, including HEK293 cells.
  • the inventors did not observe an increase in surface localization of protein when two tandem HuR-BS (2xHuR-BS) were used instead of one.
  • inventors tested the ability of more than two consecutive HuR-BS sequences (4xHuR-BS or 6xHuR-BS) no increase in surface localization was observed.
  • the inventors will also test 2x HuR-BS with spacer (SEQ ID NO 51) for its ability to increase cell surface localization.
  • 3'UTR sequences including sequences found within other transmembrane proteins will be evaluated.
  • 3'UTR sequences found within various other transmembrane proteins including CXCR4 (SEQ ID NO 52), FAS (SEQ ID NO 53), TNF (SEQ ID NO 54), and PDGFA (SEQ ID NO 55) will be evaluated in the similar manner (see Figure 14).
  • Shorter fragments of 3'UTR sequences of transmembrane proteins listed in Figure 14 will also be evaluated for the ability to localize proteins to the plasma membrane. It is anticipated that fragments of these UTR sequences containing an HuR binding domain and optionally additional elements based on the foregoing motif analysis will be effective in achieving increased localization of proteins of interest on a cell surface.

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Abstract

La présente invention concerne des fragments de 3'UTR qui comprennent un site de liaison de HuR et qui favorisent la localisation de protéines en surface, même des protéines qui ne sont normalement pas sensiblement localisées sur une surface cellulaire. L'invention concerne également des vecteurs comprenant de tels fragments et des procédés permettant d'augmenter la localisation d'un polypeptide sur la surface d'une cellule.
PCT/US2016/041659 2015-07-08 2016-07-08 Constructions de vecteurs et procédés pour obtenir ou améliorer la localisation de protéines à la surface de cellules Ceased WO2017008065A2 (fr)

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