US20230332159A1 - Materials and methods for the delivery of therapeutic nucleic acids to tissues - Google Patents

Materials and methods for the delivery of therapeutic nucleic acids to tissues Download PDF

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US20230332159A1
US20230332159A1 US17/985,683 US202217985683A US2023332159A1 US 20230332159 A1 US20230332159 A1 US 20230332159A1 US 202217985683 A US202217985683 A US 202217985683A US 2023332159 A1 US2023332159 A1 US 2023332159A1
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tissue
aptamer
construct
islets
sarna
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Paolo Serafini
Dimitri Van Simaeys
Adriana De La Fuente
Alessia Zoso
Silvio Bicciato
Jimmy Caroli
Cristian Taccioli
Andrea Grilli
Midhat Abdulreda
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Modena And Reggio Emilia Unimore, University of
University of Miami
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University of Miami
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    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/115—Aptamers, i.e. nucleic acids binding a target molecule specifically and with high affinity without hybridising therewith ; Nucleic acids binding to non-nucleic acids, e.g. aptamers
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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    • C12Q1/6813—Hybridisation assays
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    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
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    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
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    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
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    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56966—Animal cells
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
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Definitions

  • the present disclosure provides materials and methods for the delivery of therapeutic nucleic cells (and imaging agents) to tissues.
  • Diabetes is a group of metabolic disorders in which there are high blood sugar levels over a prolonged period caused by the insufficiency of the hormone insulin produced by the pancreatic beta cells.
  • type 1 diabetes is caused by the progressive autoimmune destruction of beta cells whereas in type 2 diabetes insulin is not produced in quantities sufficient for the body needs.
  • beta cell loss was debated, in the last decade it became clear that a loss of beta cells is involved also in the pathophysiology of type 2 diabetes (Rojas et al., Journal of Diabetes Research, vol. 2018, Article ID 9601801, 19 pages, 2018; Donath et al., Diabetes. 2005 Dec;54 Suppl 2:S108-13).
  • beta cell beta cell mass
  • methods to determine diabetes progression rely mostly on the indirect measurement (i.e the determination of glucose or c-peptide concentration in the blood) and cannot discriminate whether many cells produce little insulin or few cells produce large quantities of this hormone. Thus, these methods cannot measure the progressive beta cell loss in patients with diabetes.
  • the lack of adequate marker specific for beta cell make also impossible to deliver therapeutics specifically to beta cells to halt or reverse beta cell loss.
  • RNA aptamers have emerged as effective delivery vehicles for siRNAs in the treatment of many human diseases because they actively enhance the intracellular accumulation of therapeutic cargo by receptor-mediated internalization or by clathrin-mediated endocytosis (35-39,43-74).
  • the use of aptamers to deliver the therapeutic RNA of interest to the ⁇ cells offers advantage over the use of viral vectors such for example the transient modulation of the gene of interest, the lack of immunogenicity, and a great safety profile.
  • adenoviral vectors have been mostly used for efficient delivery of genes and siRNA to primary pancreatic islets in vitro (79-84).
  • the disclosure provides a method of delivering one or more agents to a tissue comprising contacting the tissue with a construct comprising an aptamer that is specific for the tissue conjugated to the agent.
  • the tissue is from an organ selected from the group consisting of pancreas, heart, lung, kidney, stomach, skin and brain.
  • the tissue is pancreatic islets.
  • the agent is a therapeutic nucleic acid.
  • the therapeutic nucleic acid is a therapeutic RNA.
  • the therapeutic RNA is selected from the group consisting of siRNA and saRNA.
  • the agent is an imaging reagent.
  • imaging reagents include, but are not limited to, fluorochromes, Positron emission tomography tracer such as Fluorine-18, oxygen-15, gallium 68, magnetic resonance imaging contrast agents such as gadolinium, iron oxide, iron platinum and manganese.
  • the contacting step can occur in vitro or in vivo.
  • the disclosure provides a construct comprising an aptamer conjugated to a small activating RNA (saRNA).
  • saRNA small activating RNA
  • the aptamer is specific for human pancreatic islets.
  • the aptamer is selected from the group consisting of M12-3773 and 1-717.
  • the aptamer is specific for clusterin (CLU, gene id 1191). In some embodiments, the aptamer is specific for “Transmembrane emp24 domain-containing protein 6” (TMED6, gene id 146456).
  • the tissue is adrenal tissue or bone marrow and the aptamer is is 173-2273, 107-901 or m6-3239.
  • the tissue is breast tissue, lung tissue or lymph node tissue and the aptamer is 107-901 and m6-3239.
  • the tissue is brain cerebellum and the aptamer is 173-2273, 107-901, m1-2623 or m6-3239.
  • the tissue is brain cerebral cortex tissue, pituitary tissue, colon tissue, endothelium tissue, esophagus tissue, heart tissue or kidney tissue and the aptamer is 107-901, m1-2623 or m6-3239.
  • the tissue is fallopian tube tissue and the aptamer is m6-3239.
  • the tissue is liver tissue and the aptamer is 166-279, 107-901, m1-2623 or m6-3239.
  • the tissue is ovarian tissue and the aptamer is 107-901.
  • the tissue is placenta tissue and the aptamer is 166-270, 173-2273, 107-901, m1-2623 or m6-3239.
  • the tissue is prostate tissue and the aptamer is 173-2273 or 107-901.
  • the tissue is spinal cord tissue and the aptamer is 166-279 or 173-2273.
  • the tissue is testis tissue and the aptamer is 166-279, 173-2273, 107-901, m1-2623, m6-3239 and m12-3773.
  • the tissue is thymus tissue and the aptamer is 173-2273, 107-901 or mf-2623.
  • the tissue is thyroid tissue and the aptamer is m1-2623.
  • the tissue is ureter tissue and the aptamer is 107-901.
  • tissue is cervical tissue and the aptamer is 166-279.
  • the tissue is islets of Langerhans or pancreatic tissue and the aptamer is 166-279, 173-2273, 107-901, 1-717, m1-2623, m6-3239 or m12-3773.
  • the disclosure provides a method of delivering one or more agents to pancreatic islets comprising contacting the islets with a construct comprising an aptamer that is specific for islets conjugated to the agent.
  • the agent is a therapeutic nucleic acid.
  • the therapeutic nucleic acid is a therapeutic RNA.
  • the therapeutic RNA is selected from the group consisting of siRNA and saRNA.
  • the agent is an imaging reagent.
  • imaging reagents include, but are not limited to, fluorochromes, Positron emission tomography tracer such as Fluorine-18, oxygen-15, gallium 68, magnetic resonance imaging contrast agents such as gadolinium, iron oxide, iron platinum and manganese.
  • the contacting step can occur in vitro or in vivo .
  • the aptamer is selected from the group consisting of M12-3773 and 1-717. In some embodiments, the aptamer is specific for clusterin (CLU, gene id 1191). In some embodiments, the aptamer is specific for “Transmembrane emp24 domain-containing protein 6” (TMED6, gene id 146456).
  • CLU clusterin
  • TMED6 Transmembrane emp24 domain-containing protein 6
  • the disclosure provides a method of measuring beta cell mass comprising contacting the beta cell with a construct comprising an aptamer conjugated to an imaging reagent in an amount effective to measure the mass of the beta cell.
  • the aptamer is selected from the group consisting of M12-3773 and 1-717.
  • the imaging reagent is a fluorochrome.
  • the imaging reagent is a PET tracer.
  • the imaging reagent is a MRI contrast reagent.
  • the imaging reagent can be conjugated to the aptamer via chelators.
  • the disclosure provides a method of modulating proliferation of beta cell comprising contacting the beta cell with a construct comprising an aptamer conjugated to a therapeutic nucleic acid in an amount effective to modulate proliferation of the beta cell.
  • the aptamer is selected from the group consisting of M12-3773 and 1-717.
  • the therapeutic nucleic acid is a therapeutic RNA.
  • the therapeutic RNA is selected from the group consisting of siRNA and saRNA.
  • the contacting step can occur in vitro or in vivo .
  • the disclosure provides a method for inhibiting beta cell apoptosis comprising contacting the beta cell with a construct comprising an aptamer conjugated to a therapeutic nucleic acid in an amount effective to inhibit apoptosis of the beta cell.
  • the aptamer is selected from the group consisting of M12-3773 and 1-717.
  • the therapeutic nucleic acid is a therapeutic RNA.
  • the therapeutic RNA is selected from the group consisting of siRNA and saRNA.
  • the therapeutic RNA upregulate the protein XIAP (X-linked inhibitor of apoptosis gene id 331).
  • the contacting step can occur in vitro or in vivo .
  • the disclosure provides a method for inhibiting tissue graft apoptosis in a subject in need thereof comprising contacting the tissue graft with a construct comprising an aptamer conjugated to a therapeutic nucleic acid in an amount effective to inhibit apoptosis of the tissue graft.
  • the therapeutic nucleic acid is a therapeutic RNA.
  • the therapeutic RNA is selected from the group consisting of siRNA and saRNA.
  • the therapeutic RNA upregulates the protein XIAP (X-linked inhibitor of apoptosis gene id 331).
  • the contacting step can occur in vitro or in vivo.
  • the tissue graft is from an organ selected from the group consisting of pancreas, heart, lung, kidney, stomach and skin.
  • the aptamer is a muscle specific aptamer and the tissue is heart tissue.
  • the tissue is contacted with the therapeutic RNA that upregulates the protein XIAP in the absence of an aptamer.
  • the disclosure provides a method for inhibiting tissue graft apoptosis in a subject in need thereof comprising contacting the tissue graft with a therapeutic RNA that upregulates the protein XIAP (X-linked inhibitor of apoptosis gene id 331) in an amount effective to inhibit apoptosis of the tissue graft.
  • the contacting step can occur in vitro or in vivo .
  • the tissue graft is from an organ selected from the group consisting of pancreas, heart, lung, kidney, stomach and skin.
  • the disclosure provides a method for protecting a beta cell from T-cell mediated cytotoxicity of the beta cell comprising contacting the beta cell with a construct comprising an aptamer conjugated to a therapeutic nucleic acid in an amount effective to inhibit T cell mediated cytotoxicity of the beta cell.
  • the aptamer is selected from the group consisting of M12-3773 and 1-717.
  • the therapeutic nucleic acid is able to increase immune checkpoint.
  • the therapeutic nucleic acid is a therapeutic RNA.
  • the therapeutic RNA is selected from the group consisting of siRNA and saRNA.
  • the therapeutic RNA upregulate the protein CD274 (Programmed death-ligand 1, PDL1, gene id 29126). The contacting step can occur in vitro or in vivo .
  • the disclosure provides a method of treating diabetes in a subject in need thereof comprising administering to the subject a construct comprising an aptamer conjugated to a small activating RNA (saRNA) in an amount effective to treat diabetes in the subject.
  • saRNA small activating RNA
  • the aptamer is selected from the group consisting of M12-3773 and 1-717.
  • one or more aptamers specific for the beta cells can be used in combination to increase delivery of the therapeutic agent or imaging reagents.
  • the aptamers are selected from the group consisting of M12-3773 and 1-717.
  • an aptamer comprising a nucleotide sequence set forth in SEQ ID NO: 264 or 259 is also contemplated.
  • the aptamer is conjugated to an saRNA.
  • the saRNA upregulates the protein XIAP (X-linked inhibitor of apoptosis gene id 331).
  • saRNA upregulates the protein CD274 (Programmed death-ligand 1, PDL1, gene id 29126,
  • FIG. 1 is a flow chart showing HT-cluster SELEX as an unsupervised strategy for the isolation of aptamers specific for human islets using human islets and acinar tissue.
  • FIGS. 2 A and 2 B provide a flow chart showing HT-Toggle-cluster SELEX to isolate islets specific aptamers crossreacting between mouse and human. 8 cycles of HT-cluster SELEX were performed as described in FIG. 1 using as negative and positive selectors mouse acinar tissues and mouse islets respectively ( FIG. 2 A ). The resulting polyclonal aptamer from cycle 8 was used for two additional cycle of selection using either acinar tissue and islets from mice or acinar tissue and islets isolated from cadaveric donors ( FIG. 2 B ).
  • FIG. 3 shows images resulting from HT-Toggle-cluster SELEX to isolate islets specific aptamers crossreacting between mouse and human.
  • FIG. 4 shows images resulting from HT-Toggle-cluster SELEX identifying monoclonal aptamers that rcognize human islets but not human acinar tissue.
  • FIGS. 5 A and 5 B show that aptamers 1-717 ( FIG. 5 A ) and M12-3772 ( FIG. 5 B ) show an extraordinary specificity for human islets.
  • FIG. 5 C is a table showing the results of various tissue staining with various selected aptamers.
  • FIG. 6 shows that aptamers 1-717 and M12-3772 recognize mouse islets and other mouse tissues.
  • FIGS. 7 A- 7 D shows that aptamers 1-717 ( FIGS. 7 B and 7 C ) and M12-3773 ( FIGS. 7 A and 7 D ) recognize preferentially human beta cells.
  • FIGS. 8 A- 8 C show that clusterin is a possible target for aptamer m12-3773.
  • FIG. 8 A provides the strategy for an aptamer based mass spectrometry and mascot-based analysis.
  • Clusterin (UniProtKB - P10909) ( FIG. 8 B ) had a high Mascot score (236).
  • FIG. 8 C shows that silencing of Clusterin reduced the capacity of aptamer m12-7337, but not of aptamer 1-717, to bind to beta cells.
  • FIGS. 9 A- 9 C shows that TMED6 is the putative target for aptamer 1-717.
  • FIG. 9 A shows the experimental strategy to detect the target for aptamer 1-717.
  • FIG. 9 B provides results of the binding array assay described in Example 1.
  • FIG. 9 C is a graph showing that competitive assays confirm the specificity of aptamer 1-717 for TMED6
  • FIGS. 10 A and 10 B shows that a mixture of aptamer 1-717 and m12-3773 recognize human islets in vivo better than the individual clones.
  • a cumulative-synergistic signal was observed in the EFP region when the mixture of both aptamers was used possibly because different islet epitopes were targeted by each aptamer ( FIG. 10 A ).
  • 4 hour later fluorescence signal in epididymal fat pad region was measure by “In vivo imaging system (IVIS)”.
  • IVIS In vivo imaging system
  • FIGS. 11 A- 11 C show that Aptamer 1-717 and M12-3773 allow the measurement of human beta cell mass in vivo.
  • FIG. 11 A is a schematic of the experiment performed in Example 2.
  • FIGS. 11 B and 11 C show that fluorescence signal in the EFP region was proportional to the number of engrafted islets indicating that these aptamers can be used to measure ⁇ cell mass in vivo
  • FIG. 11 D Syngeneic (Balb/c) or allogeneic (C57B1 ⁇ 6) ilsets were transplanted subcutaneously (in the right and left flank respectively) of immunocompetent Balb/c mice.
  • Rejection was longitudinally monitored by injecting AF750-conjugated aptamer intravenously and by performing IVIS 5 hours later. Data show that rejection of the allogeneic C57B16 islet graft can be measured over time as seen by the loss of signal on the left flank. Instead signal (right) of the syngeneic islet graft is maintained over time indicating graft survival.
  • FIG. 12 is a schematic diagram aptamer chimera for the delivery of therapeutic RNA via islets specific aptamers.
  • FIGS. 13 A and 13 B show that islets specific aptamer chimera allows for the delivery of therapeutic RNA via islets specific aptamers.
  • FIG. 13 A is a schematic of the experimental procedure.
  • FIG. 13 B shows that the aptamer chimera significantly downregulate the expression of the target gene.
  • FIGS. 14 A- 14 C shows that p57kip2-siRNA-islet specific aptamer chimera induce human beta cell proliferation in vivo.
  • FIG. 14 A shows the experimental procedure: Streptozotocin-treated, immune deficient NSG mice were transplanted with a suboptimal quantity (250 IEQ) of human islets in the anterior chamber of the eye.
  • FIG. 14 B provides immunofluorescence pictures of the graft from mice treated with control chimera or p57kip2-siRNA/aptamer chimera. Glucagon and insulin staining is depicted in dark gray as pseudocolor whereas BrdU staining as measure of cell proliferation is depicted in white as pseudocolor.
  • FIG. 14 A shows the experimental procedure: Streptozotocin-treated, immune deficient NSG mice were transplanted with a suboptimal quantity (250 IEQ) of human islets in the anterior chamber of the eye.
  • FIG. 14 B provides immunofluorescence pictures of the graft from mice treated with
  • FIG. 15 A provides a schematic of the method described in Example 4.
  • FIG. 15 B shows the identification of small activating RNA (saRNA) specific for the human “X-Linked Inhibitor Of Apoptosis” (Xiap, Gene ID: 331).
  • saRNA small activating RNA
  • FIGS. 16 A- 16 C Xiap-saRNA aptamer chimera protect human beta cells from cytokine induced apoptosis.
  • FIG. 16 A shows the experimental procedure described in Example 5.
  • FIG. 16 B shows the flow cytometry analysis of single cell suspension of islets treated with scrambled saRNA chimera (CTRL chimera) or XIAP-saRNA/aptamer chimera (Xiap Chimera) and later challenged with cytokines (CTK) or left untreated (No CTK).
  • CTRL chimera scrambled saRNA chimera
  • Xiap Chimera XIAP-saRNA/aptamer chimera
  • FIG. 16 C is a spaghetti plot from 5 independent experiments each with islets from a different cadaveric donor using chimera generated with either aptamer m12-3773 or aptamer 1-717.
  • FIGS. 17 A and B show that the Xiap-saRNA/islet specific aptamer chimera protect beta cells from primary nonfunction.
  • FIG. 17 A is a schematic for the experiment described in Example 5.
  • FIGS. 18 A- 18 B are a schematic of the protocol described in Example 6.
  • FIG. 18 B is a graph showing the identification of small activating RNA (saRNA) specific for the human “PDL1” (CD274, Twelve saRNAs (provided in Table 5) were found to upregulate Xiap expression more than 10 times (range 10.4-74.8) over scrambled saRNA. Gene ID: 29126).
  • saRNA small activating RNA
  • FIGS. 19 A - 19 B PDL1-saRNA/islet specific aptamer chimera upregulate PDL1 on human beta cells.
  • FIG. 19 A is a schematic showing that PDL1-saRNA/aptamer chimera were added to non-dissociated human islets from cadaveric donor. 48h later, islets were dissociated, labelled with anti-insulin, anti-glucagon and anti-PDL1 antibodies and analyzed by flow cytometry. Results of the flow cytometery is shown in FIG. 19 B .
  • FIGS. 20 A- 20 C PDL1-saRNA/aptamer chimera upregulate PDL1 in vivo.
  • FIG. 20 A provides a schematic of a protocol where immune deficient NSG mice were transplanted in the anterior chamber of the eye with human islets from a cadaveric donor. 3 weeks later, mice were treated with PDL1-saRNA(636)/1-717-aptamer chimera. Scramble-saRNA/aptamer chimera was used as control (CTRL chimera).
  • FIG. 20 B are images showing PDL1 expression in tissues.
  • FIG. 20 C provides graphs summarizing of PDL1 expression on the engrafted islets at baseline or 5 days after treatment with PDL1-saRNA/aptamer chimera or scrambled-saRNA/aptamer chimera.
  • RNA/aptamer chimeras were generated to modulate gene expression in human ⁇ cells in vivo to induce their transient proliferation and improve their resistance to auto/alloimmunity.
  • islet-specific aptamers to deliver: A) siRNA against p57kip2 to induce ⁇ cell proliferation, B) saRNA promoting Xiap expression to protect islets from apoptosis, and C) saRNA promoting PDL1 expression to protect ⁇ cells from T cell cytotoxicity. Because of the absence of reliable humanized mouse model of autoimmune T1D, our approach is based on the use of NSG or humanized NSG mice transplanted with human islets before aptamer treatment.
  • human islets The use of human islets is dictated by species specific difference in p57kip2 biology (14) and by the specificity of PDL1 and Xiap saRNAs for the human genes. Ex vivo and innovative in vivo techniques are employed to quantify the response to in vivo treatment through imaging of ⁇ cell proliferation, apoptosis, and interaction with the immune system. We envision the use of these aptamers as mono or multimodal approach where difference genes can be modulated simultaneously.
  • RNA aptamers have emerged as effective delivery vehicles for siRNAs and other drugs to specific cell subsets or tissues for the treatment of many human diseases (60,62-75). Indeed, through the interactions between the aptamer and its cellular membrane target, aptamers actively enhance the intracellular accumulation of therapeutic agents (37-39,43-61). Some aptamer drugs are FDA-approved and more than 30 are being tested in clinical trials (16-24).
  • aptamers that do not find a specific target are rapidly eliminated via the kidney; those that find their target in tissues or cells remain detectable for up two weeks.
  • Their bioavailability, plasma half-life, and pharmacokinetic properties can be easily engineered by increasing their size by the addition of Polyethylene glycol (PEG) during synthesis, or by conjugation with nanoparticles (60,62-74).
  • Aptamers can be conjugated to siRNA, miRNA or saRNA to deliver the desirable therapeutic effect in specific targets.
  • the ability to directly engineer aptamers with high specificity and defined functions is a distinct advantage over antibodies and other small molecules.
  • Unsupervised toggled-SELEX was performed starting with a polyclonal aptamer library against mouse islets and using islet depleted human acinar cells and handpicked human islets from 4 different cadaveric donors as negative and positive selectors, respectively. This allowed for the depletion of non-specific (acinar tissue binding) RNA aptamers and enrich the library for those aptamers specific for mouse and human islets.
  • HT-cluster SELEX was used as an unsupervised strategy for the isolation of aptamers specific for human islets using human islets and acinar tissue.
  • a random aptamer library was generated by PCR and Durascribe T7 RNA transcription from a cDNA random library (TCT CGG ATC CTC AGC GAG TCG TC TG (N40) CCG CAT CGT CCT CCC TA (SEQ ID NO: 413), comprising a 40-nt variable region flanked by two constant region.
  • RNA aptamer library (Table 1), enriched for islets specific aptamers, was used for new selection cycle. A total of 8 selection cycles was performed using islets and acinar tissue from 4 unrelated cadaveric donors.
  • HT-Toggle-cluster SELEX was used to isolate islet-specific aptamers crossreacting between mouse and human. 8 cycles of HT-cluster SELEX were performed as described in FIG. 1 using as negative and positive selectors mouse acinar tissues and mouse islets respectively ( FIG. 2 A ). The resulting polyclonal aptamer from cycle 8 was used for two additional cycle of selection using either acinar tissue and islets from mice or acinar tissue and islets isolated from cadaveric donors ( FIG. 2 B ). The resulting polyclonal aptamer library underwent HT-sequencing and bio-informatic analysis ( FIG.
  • monoclonal aptamers corresponding to the sequences identified by the bio-informatic analysis were generated by PCR and T7 RNA polymerase using overlapping oligonucleotides as template.
  • the resulting monoclonal aptamers were labelled with Cyanin-3 and used as fluorescent probes on sections of human pancreas from cadaveric donor. Clone number is reported with the prefix “m” indicating the identification of aptamer from the HT-Toggle cluster SELEX.
  • FIG. 4 In order to evaluate further the specificity of the chosen monoclonal aptamers, the best performers of FIG. 4 (166-279, 173-2273, 107-901, 1-717, m1-2623, m5-3239, and m12-3773) were used to stained FDA approved tissues microarrays. Each of these arrays contains sections from 30 human tissues (with each tissue replicated from 3 different donors) and are usually used for antibodies screening but have not yet been used for aptamer evaluation. Briefly, these tissues microarrays were stained with the chosen cy3 labelled RNA aptamers of irrelevant aptamers as control. Each tissue was then analyzed by immunofluorescence microscopy.
  • aptamers 1-717 FIG. 5 A
  • aptamer m12-3773 FIG. 5 B
  • FIG. 5 C While most of the aptamers show binding not only as expected in the pancreatic islet but also in other tissues ( FIG. 5 C ), aptamer 1-717 ( FIG. 5 A ) and aptamer m12-3773 ( FIG. 5 B ) show an extraordinary specificity for the pancreatic islets and negligible binding to the other tissues evaluated (adrenal, bone marrow, breast, brain, colon, endothelial, esophagus, fallopian tube, heart, kidney, liver, lung, lymph node, Ovary, placenta, prostate, skin, spinal cord, spleen, muscle, stomach, testis, thymus, thyroid, ureter, uterus, and testis).
  • confocal microscopy FIG. 7 A and FIG. 7 B
  • flow cytometry FIG. 7 C and FIG. 7 D
  • confocal microscopy sections of human pancreas were stained with cy-3 labelled aptamer M12-3773 ( FIG. 7 A ) or cy3-labeled aptamer 1-717 ( FIG. 7 B ). Sections were counterstained with antibodies against insulin and glucagon, and DAPI and evaluated by confocal microscopy. Data show binding of both aptamers to both alpha and beta cells but with an higher signal on beta cells.
  • Clusterin is a possible target for aptamer m12-3773.
  • 3′biotin-aptamer m12-3773 was synthetized with a oligo synthesizer and used to label single cell suspension from human islets. Cells were washed and their cytoplasm lysed with tween20/BSA solution. Aptamers bound to their ligand recovered with magnetic beads and magnetic separation. Capture ligands were released by the aptamer-beads complex at 95° C. in SDS and run in SDS page. Bands were cut and subjected to mass spectrometry and mascot-based analysis ( FIG. 8 A ). Clusterin (UniProtKB - P10909) ( FIG.
  • FIGS. 9 shows that TMED6 is the putative target for aptamer 1-717.
  • FIG. 9 A shows the experimental strategy to detect the target for aptamer 1-717.
  • protein arrays HuProtTM v2.0, Arrayit
  • These protein arrays contains more than 19,000 human recombinant proteins allowing, by informatics analysis, the identification of the cognate protein of antibodies, peptides, or protein. This technology has been adapted for the identification of aptamer’s ligands. Briefly, pre-blocked arrays were hybridized with 1 ⁇ g of cy3 labeled 1-717 or m12-3773 in blocking buffer for 30′ at RT.
  • Arrays were washed, read in triplicate on a Genepix microarray reader and analyzed by an ad hoc generated software.
  • This software 1) acquires the data from the gpr file, 2) adds a description column with (GeneID, Control, blank, ND), 2) use a optimized “plotArray” function modified in several points (the script was implemented for a specific protein array, plus some problem in UTF file format), 3) performs a quality control that includes a microarray image rebuilding the generation of MA plots, 4) normalized the data and substracts the background; and 5) analyze the differential expression between arrays via graphs of p-value distribution, volcano plot, and analysis of significant modulation by t-test.
  • TMED6 (NM144676.1, protein id Q8WW62) as the most likely ligand of aptamer 1-717. See FIG. 9 B .
  • Competitive assays ( FIG. 9 C ) confirm the specificity of this target.
  • aptamer 1-717 and m12-3773 can recognize human islets in vivo.
  • aptamer 1-717 and aptamer m12-3773 in which each monoclonal aptamer is biotinylated and complexed with streptavidin to form a tetrameric nanoparticle (hereafter called tetraptamer).
  • This formulation has a superior pharmacokinetic and better affinity than the corresponding monomeric aptamer.
  • Biotin/streptavidin Alexafluor (AF750)-labeled aptamers (amptamer 1-717 or aptamer m12-3773, or an equimolar mixture of the two aptamers) were injected intravenously in immunodeficient NSG mice (engrafted with human islets in the epididymal fat pad (EFP)) to evaluate whether m12-3773 and 1-717 can recognize human islets in vivo.
  • EFP epididymal fat pad
  • aptamers m12-3773 and 1-717 can be used to measure ⁇ cells mass in vivo.
  • immune deficient NSG mice were transplanted with different quantities (range 62.5-500 IEQ) of human islets in the epididymal fatpad. 21 days later, mice were injected iv with Alexafluor 750 tetraptamer generated by the complexation of an equimolar mixture of aptamer 1-717 and m12-3773 to streptavidin. 4 hours later signal was quantified by IVIS.
  • FIG. 11 A Aptamers m12-3773 and 1-717 recognized both the mouse endogenous islets and the human islets transplanted in the EFP ( FIG. 11 B ).
  • fluorescence signal in the EFP region was proportional to the number of engrafted islets indicating that these aptamers can be used to measure ⁇ cell mass in vivo ( FIGS. 11 B and 11 C ).
  • the signal from the islets persisted for 10 days after injection (not shown).
  • rejection of the allogeneic C57B16 islet graft can be measured over time as seen by the loss of signal on the left flank. Instead signal (right panel of FIG. 11 D ) of the syngeneic graft is maintained over time indicating graft survival.
  • the selected aptamers m12-3773 and 1-717 bind mouse and human ⁇ cells with good specificity in vitro and in vivo and thus may be useful in targeting therapeutics to human ⁇ cells in vivo.
  • Example 3 Aptamera Chimera Can Deliver Therapeutic RNA to Islets
  • islet-specific RNA aptamers 1-717 and m12-3773 can be easily conjugated to therapeutic RNA by prolonging their 3′ end with a trinucleotide linker region (i.e. GGG) and the passanger strand (passanger tail) of the desired therapeutic RNA.
  • GGG trinucleotide linker region
  • the therapeutic RNA guide strand is then simply annealed to the modified aptamer by admixing equimolar quantities of the two RNAs at 70° C. and allowing the mixture to slowly cool down at room temperature.
  • FIG. 13 A is a schematic of the experimental procedure. Islets specific aptamer chimera were generated as detailed in FIG. 12 by conjugating aptamer 1-717 or aptamer m12-3773 with siRNA specific for mouse insulin1 ⁇ 2 (INS1 ⁇ 2) or the inhibitor of cell proliferation human p57kip2 (uniprot P49918, alias CDN1c).
  • the INS 112-siRNA/aptamer chimera was added to non-dissociated mouse islets whereas the p57kip2-siRNA/aptamer chimera was added to human islets from a cadaveric donor. Scramble siRNA/aptamer chimera were used as negative controls. 72 hours later, expression of INS 1 ⁇ 2 and p57kip2 was quantified by qRT-PCR on transfected mouse islets and transfected human islets respectively. As shown in FIG. 13 B , the aptamer chimera significantly downregulate the expression of the target gene.
  • FIGS. 14 p57kip2-siRNA-islet specific aptamer chimera induce human beta cell proliferation in vivo.
  • FIG. 14 A shows the experimental procedure: Streptozotocin-treated, immune deficient NSG mice were transplanted with a suboptimal quantity (250 IEQ) of human islets in the anterior chamber of the eye. Mice were maintained euglycemic by s.c. implantation of insulin pellet.
  • mice 21 days later, when islets were vascularized, insulin pellet was removed to allow the development of hyperglycemia, mice were fed with BrdU for 7 days to evaluate cell proliferation, and treated with i) scramble-siRNA/aptamer chimera, or ii) p57kip2-siRNA/aptamer chimera.
  • mice were humanely euthanized, and beta and alpha cell proliferation was evaluated by immune fluorescence microscopy after labeling the graft sections with antibodies against insulin, glucagon, and BrDU (white).
  • FIG. 14 B provides immunofluorescence pictures of the graft from mice treated with control chimera or p57kip2-siRNA/aptamer chimera.
  • FIG. 14 C shows quantification of proliferating beta and alpha cells. Taken together these data indicate that p57kip2-siRNA/aptamer chimera can induce in vivo human beta cell proliferation in a hyperglycemic setting that mimic T1 and T2 diabetes.
  • P57kip2 silencing in ⁇ cells has important therapeutic implications. Indeed, mutations of p57Kip2 are associated with focal hyperinsulinism of infancy (FHI), a clinical syndrome characterized by a dramatic non-neoplastic clonal expansion of ⁇ cells (14), overproduction of insulin, and severe uncontrollable hypoglycemia (89,90). FHI’s focal lesions are characterized by excessive ⁇ cell proliferation that correlates with p57kip2 loss (91,92). Although the pro-proliferative activity of p57kip2 silencing is not desirable in FHI and in cancers, a temporally defined silencing might be useful to promote adult ⁇ cell proliferation in T1D.
  • FHI focal hyperinsulinism of infancy
  • adenoviral-shRNA mediated silencing of p57kip2 in human islets obtained from deceased adult organ donors increased ⁇ cell replication by more than 3-fold once the islets were transplanted into hyperglycemic, immune-deficient mice (14).
  • the newly replicated cells retained properties of mature ⁇ cells, such as expression of insulin, PDX1, and NKX6.114.
  • no ⁇ cell proliferation was observed in normoglycemic mice indicating that hyperglycemia may provide additional pro-proliferative signals (93).
  • p57Kip2 is frequently downregulated in human cancers (94) and has been proposed as a tumor suppressor gene since its ectopic expression is sufficient to halt neoplastic cell proliferation (94).
  • a temporally controlled modulation of p57kip2 through aptamer delivery may be important in diabetes to increase ⁇ cell proliferation in a temporally controlled manner. This might be sufficient to increase ⁇ cell mass during timed administrations while avoiding the safety concerns with non-controllable, neoplastic-like proliferation of ⁇ cells that may results with stable silencing.
  • Example 4 Upregulation of XIAP Via saRNA-Aptamer Chimera Inhibits Apoptosis in ⁇ Cells.
  • Apoptotic cell death is a hallmark in the loss of insulin-producing ⁇ cells in all forms of diabetes (99-101).
  • Leukocytes infiltration and activation as well as high glycemia within the islets leads to high local concentrations of apoptotic trigger including inflammatory cytokines, chemokines, and reactive oxygen species99.
  • Most of these apoptotic pathways converge onto caspase (CASP) 3 and 7 activation leading to genetic reprogramming, phosphatidylserine flip, and apoptotic bodies formation (102).
  • ⁇ cell apoptosis can further feed the autoimmune process by stimulating self-antigen presentation and autoreactive T cell activation (103).
  • primary non-function i.e. the partial but significant and sometimes total loss of the grafted islet mass, which occurs early after transplantation (104-106).
  • ⁇ cell apoptosis initiates during the isolation procedure and upon transplantation is exacerbated by hypoxia and hyperglycemia as well as pro-coagulatory and proinflammatory cascades (107).
  • Primary-non-function accounts for more than 50% of the functional islet mass loss occurring during the first 48 hours after transplantation (106).
  • blocking even temporally apoptotic ⁇ cell death is highly desirable not only to preserve ⁇ cell mass in type 1 diabetes (T1D) and in islet transplantation but also to reduce auto-reactive T cell activation and further immune damage.
  • This protein is most potent member of the apoptosis-inhibitor family and prevents the activation of CASP 3, 7 and 9(108); ii) Xiap overexpression using viral vector improved ⁇ cell viability, prevented their cytokine- or hypoxia-induced apoptosis(109-111), iii) Xiap transduced human islets prolonged normoglycemia when are transplanted in diabetic NOD-SCID mice (11).
  • a controlled Xiap activation via saRNA delivered with islets specific aptamers can be useful alternative to reduce primary nonfunction, prevent ⁇ cell loss and the self-feeding autoimmune process in T1D.
  • RNAs Small activating RNAs
  • saRNAs are oligonucleotides that exert their action in specific promoter regions and upregulate mRNA and protein expression for up to 4 weeks (depending on cell replication, mRNA and protein turn-over) (112-122).
  • saRNA-mediated gene upregulation through mechanisms still not fully understood but is thought to involve epigenetic changes or down-modulation of inhibitory RNA (123-125).
  • saRNAs provide safe, specific, and temporary gene activation without the insertion of DNA elements since their specificity is comparable to that of gRNA in CRISP/CAS9 system but no irreversible DNA modification are induced 126. While therapeutic saRNAs are being investigated for cancer treatment, to our knowledge no studies have been performed in T1D (127-130).
  • saRNAs capable of specifically upregulating the anti-apoptotic gene XIAP were identified. Briefly, we have first examined the human XIAP promoter using the previously described algorithms (112,131). This analysis that includes genome blast analysis to avoid non-specific sequences, returned more than 156 putative saRNA target regions. We synthetized the 96 putative saRNA with highest scores and tested them for their capacity to upregulate Xiap by transfecting the human epithelial cell line A549. This cell line was used because it is easily transfectable, has low basal expression of PDL1 and Xiap.
  • qRT-PCR was performed 96 hours after transfection and results were normalized on the same cell line transfected with scrambled saRNA ( FIGS. 15 ). Twelve saRNAs (provided in Table 5) were found to upregulate Xiap expression more than 10 times (range 10.4-74.8) over scrambled saRNA.
  • Xiap-saRNA aptamer chimeras were generated as described in FIG. 12 by conjugating the identified Xiap saRNA (-449, table 2) to either aptamer m12-3773 or aptamer 1-717.
  • FIG. 12 In vitro proof of principle experiments were performed using human islets isolated from cadaveric donors to determine if Xiap-saRNA delivered by aptamer can protect ⁇ cell from apoptosis.
  • Xiap-saRNA chimeras were generated as described in FIG. 12 by conjugating the identified Xiap saRNA (-449, table 2) to either aptamer m12-3773 or aptamer 1-717.
  • FIG. 16 A shows the experimental procedure: Freshly-isolated, non-dissociated, human islets (200IEQ) from cadaveric donor were transfected with Xiap-saRNA by adding the Xiap/saRNA aptamer chimera (5ug) to the culture. Scramble saRNA/aptamer chimera were used as negative control. 48 hours later, half of the wells were challenged with inflammatory cytokines (IFNg, TNFa, and IL1b) to induce beta cell apoptosis. Beta cell death was evaluated by flow cytometry 24 hours after cytokine challenge by measuring beta/alpha cell ratio after staining for insulin and glucagon.
  • IFNg inflammatory cytokines
  • FIG. 16 B shows the flow cytometry analysis of single cell suspension of islets treated with scrambled saRNA chimera (CTRL chimera) or XIAP-saRNA/aptamer chimera (Xiap Chimera) and later challenged with cytokines (CTK) or left untreated (No CTK).
  • CTRL chimera scrambled saRNA chimera
  • Xiap Chimera XIAP-saRNA/aptamer chimera
  • FIG. 16 C is a spaghetti plot from 5 independent experiments each with islets from a different cadaveric donor using chimera generated with either aptamer m12-3773 or aptamer 1-717. Paired T test value is reported. Data show that Xiap-saRNA aptamer chimera protect beta cells from cytokine-induced apoptosis.
  • FIG. 17 B Provided in FIG. 17 B is the schematic the Xiap-saRNA/aptamer chimera for graft preservation.
  • human Islets were cultured in media where chimera was added at 48h, 24h and on the day of transplantation 600 IEQ were transplanted per mouse in the left kidney capsule of streptozotocin diabetic NOG mice.
  • Data showed that pretreatment of human islets with aptamer chimera greatly improve the efficacy of islet transplantation with approximately 80% of mice becoming normoglycemic by day 2.
  • Example 6 Protect Islets From Allo- and Auto-immunity in Humanized Mice Via PDL1-saRNA/Aptamer Chimera
  • PDL1-PD1 antagonists in cancer (135). Engagement of PD1 by PDL1 down-regulates effector T cell proliferation and activation, induces T cell cycle arrest and apoptosis, and promotes IL10-producing Treg (136-139). Interestingly, one of the emerging side effect anti-PD1 treatment is T1D140. This suggests that PDL1/PD1 may play an important role in controlling T cell tolerance against ⁇ cells. Indeed, in NOD mice PDL1 blockade accelerate T1D in female mice and induce it in male (13).
  • saRNAs specific for PDL1 FIGS. 18 . Briefly, putative candidate sequence of small activating RNA for PDL1 were identified by scanning the PDL1 promoter using publically available algorithms. This analysis return more than 200 putative target saRNA target regions. The 95 putative saRNA with higher score were synthetized and tested for their capacity to up-regulate PDL1 by transfecting the human epithelial cell line A549. qRT-PCR was performed 96 hours after transfection and results normalized on the same cell line transfected with scrambled saRNA. 19 saRNAs were found able to upregulate Xiap expression more than 3 times (range 3.01-63.27) over scrambled saRNA (Table 6).
  • PDL1 expression was evaluated by gating on insulin positive beta cells or glucagon positive alpha cells. While treatment with control chimera does not modify PDL1 expression, treatment with PDL1-saRNA/aptamer significantly upregulate the expression of this important immune modulatory protein on beta cells ( FIG. 19 B ). Interestingly no changes were observed in alpha cells confirming indirectly the preferential binding of this aptamer to beta cells.
  • FIG. 20 A immune deficient NSG mice were transplanted in the anterior chamber of the eye with human islets from a cadaveric donor. 3 weeks later, mice were treated with PDL1-saRNA(636)/1-717-aptamer chimera generated as described in FIG. 12 and FIGS. 19 . Scramble-saRNA/aptamer chimera was used as control (CTRL chimera). Five days after treatment, PDL1 expression (white) on the islets (dark gray) was quantified by in vivo labelling with anti-PDL1 antibody and in vivo microscopy ( FIG. 20 B ). Summary of PDL1 expression on the engrafted islets at baseline or 5 days after treatment with PDL1-saRNA/aptamer chimera or scrambled-saRNA/aptamer chimera FIG. 20 C ).
  • NSG mice will be engrafted with human islets in the ACE.
  • mice Three weeks after transplant, mice will be treated with Xiap saRNA-aptamer chimera(s) or control chimera.
  • human islet grafts will be challenged by intraocular injection of IL1 ⁇ , TNF- ⁇ , and IFN ⁇ to induce apoptosis in ⁇ cells via activation of caspase 3 and 7.
  • Caspase 3 and 7 activity will be evaluated in vivo by our intraocular imaging system using CASP3/7 Green Detection Reagent.
  • This cell-permeant reagent consists of a four-amino acid peptide (DEVD) conjugated to a nucleic acid-binding dye.
  • DEVD four-amino acid peptide conjugated to a nucleic acid-binding dye.
  • caspase 3 and 7 cleave the probe, allow the dye to bind to the DNA, and emit a bright, fluorogenic signal that can be detected at the cellular level in the ACE28.
  • in vivo staining with anti-Annexin V antibodies will be used to directly measured islet cell apoptosis in vivo ( FIGS. 7 ).
  • mice will be transplanted with 500 IEQ human islets in the ACE or EFP. 3 weeks later mice will be treated with Xiap chimera(s) or scrambled controls. Treatment will be repeated as determine in Aim2b.
  • mice will receive CFSE labelled human T cells mismatched for HLA to the islet. Without any treatment, the adoptive transfer of allogeneic T cells results in graft loss and return to hyperglycemia within 3 weeks.
  • the chimera identified in the EndoC-BH3 cells will be further validated using primary human islets from 6 cadaveric donors; this will provide 88% of power to detect 1.25SD difference from control in one tailed paired t-test.
  • 3 different readout methods qPCR, western blot, and enzymatic assay
  • at least 3 independent repetitions will be performed for each experiment using human islets from 3 different cadaveric donors.
  • NSG mice transplanted with 500 IEQ human islets in the EFP will be treated i.v. or s.c. with different doses (6, 20, and 60 pmoles/g) of islets-specific aptamers conjugated with p57kip2siRNA or scrambled siRNA (control-chimera) as negative control.
  • mice will be used adenovirus encoding the same p57kip2 shRNA-transfected islets as positive control (14).
  • grafts will be harvested, and p57kip2 expression quantified by i) qRT-PCR on laser captured islets, and ii) by quantitative computer assisted immunofluorescence analysis95. Both techniques are optimized at the Diabetes Research Institute (96,97) and in the laboratories of the PIs95. To evaluate possible dose-dependent toxicity, sera and organs of interest (spleen, liver, lymph nodes, lung, kidney, and brain) will be collected and sent to the mouse pathology laboratory of University of Miami for histopathological evaluation.
  • mice will be transplanted with 500 IEQ human islets in the ACE.
  • mice will be treated i.v. or by intraocular injection (i.o) with different doses (6, 20, 60pm/g) of our aptamer-chimera loaded with p57kip2 siRNA or AF647-scrambled siRNA (control-chimera) as negative control.
  • i.o intraocular injection
  • In vivo transfection efficiency of the AF647 siRNA will be evaluated with our intraocular imaging system 2, 3, 4, 8, and 24 hours after injection (28).
  • graft will be removed and p57kip2 expression quantified by qRT-PCR on islets explanted from the ACE and by i) qRT-PCR on laser capture islets and ii) by quantitative computer assisted immunofluorescence microscopy analysis (95).
  • mice Once the optimal dose and route of administration are identified and the kinetics of p57kip2 silencing evaluated, we will determine the number of administrations of p57kip2siRNA-aptamer chimera needed to induce substantial changes (i.e., ⁇ 100% increase) in ⁇ cell mass. Since p57kip2 silencing was shown to induce ⁇ cell proliferation only in hyperglycemic mice14, sub-marginal human islet mass (250 IEQ) will be transplanted in the EFP or ACE of NSG mice. 21 days after transplant, mice will be rendered hyperglycemic by streptozotocin (STZ) treatment. STZ selectively eliminates mouse islets as human ⁇ cells are considerably more resistant (98).
  • mice will receive 1, 2, 3, or 4 administration of islet-specific or control aptamer chimeras.
  • the frequency of the aptamer administration will be determined based on the time course established in Example 5.
  • BrdU will be administered in drinking water for ex vivo determination of proliferation.
  • ⁇ cell mass in the EPF group will be evaluated longitudinally (baseline, during treatment, 5 and 10 days after the last treatment) by IVIS ( FIGS. 2 ).
  • islets mass will be evaluated by in vivo imaging and quantitative analysis of islet volume (28).
  • Ten days after the last treatment, grafts will be harvested and analyzed by immunostaining to determine (i) ⁇ cell proliferation via BrdU and Ki76 staining and (ii) ⁇ to ⁇ cell ratio.
  • Example 10 Determine if Aptamer Mediated Silencing Can Restore Normoglycemia in Diabetic Mice Transplanted With Sub-Marginal Islet Mass
  • the purpose of this Example is to test if aptamer mediated p57kip2 silencing can restore normoglycemia in diabetic mice transplanted with suboptimal number of human islets.
  • STZ-diabetic NSG mice maintained on insulin therapy will be transplanted with different quantities of human islets (50, 150, 350 IEQ) in the ACE.
  • insulin pellets will be removed, and mice will be treated with p57kip2siRNA-aptamer chimera or scrambled control, locally or systemically.
  • two additional groups of mice will be treated locally with adenoviral vector encoding for p57kip2shRNA or RFP as control. Pilot experiments using RFP encoding adenovirus will be performed in the ACE to determine the minimal dose necessary for transducing at least 90% of the islets.
  • Transduction efficiency will be quantified using our in vivo imaging system (28).
  • blood glucose will be used as readout for treatment efficacy in addition to intravital imaging and volume analysis of the ACE islet grafts.
  • the varied sub-marginal islet mass in the different groups may also reveal the degree of the hyperglycemic drive on human islet proliferation.
  • STZ-diabetic mice will be transplanted in the EFP with the same sub-marginal human islet masses (50, 150, 350 IEQ) and maintained on insulin during the engraftment period. 3 weeks later insulin pellet will be removed and mice will be treated with p57kip2siRNA-aptamer chimera or the scrambled control. We will monitor glycemia and ⁇ cell mass by IVIS longitudinally as readouts.
  • glucose tolerance tests will be performed in mice with restored normoglycemia to further evaluate the islet function under stress conditions.
  • mice per experimental group 3 in each repetition
  • 12 mice per group will be used to accounting for the higher expected variation of the read-out.
  • the same phenomenon will be measured with at least 2 independent methods.

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Abstract

The present disclosure provides materials and methods for the delivery of therapeutic nucleic cells (and imaging agents) to tissues.

Description

    CROSS-REFERENCE TO RELATED APPLICATIONS
  • The present application is a divisional of U.S. Application No. 17/053,193 filed Nov. 5, 2020 (now U.S. Pat. No. 11,584,935), which is a national phase application of International Application No. PCT/US19/31346 filed May 8, 2019, and which claims the benefit of priority to U.S. Provisional Application No. 62/668,463 filed May 8, 2018, the disclosures of which are incorporated herein by reference in their entirety.
    • FIELD OF THE INVENTION
  • The present disclosure provides materials and methods for the delivery of therapeutic nucleic cells (and imaging agents) to tissues.
    • STATEMENT OF GOVERNMENT SUPPORT
  • This invention was made with government support under grant number DK116241 awarded by the National Institutes of Health. The government has certain rights in the invention.
    • INCORPORATION BY REFERENCE OF MATERIALS SUBMITTED ELECTRONICALLY
  • This application contains, as a separate part of the disclosure, a Sequence Listing in computer readable form (Filename: 53048B_Seqlisting.xml; Size: 3,380,995 bytes; Created: May 22, 2023, which is incorporated by reference in its entirety.
    • BACKGROUND
  • Diabetes is a group of metabolic disorders in which there are high blood sugar levels over a prolonged period caused by the insufficiency of the hormone insulin produced by the pancreatic beta cells. In particular, type 1 diabetes is caused by the progressive autoimmune destruction of beta cells whereas in type 2 diabetes insulin is not produced in quantities sufficient for the body needs. Although for many years the contribution of beta cell loss to type 2 diabetes was debated, in the last decade it became clear that a loss of beta cells is involved also in the pathophysiology of type 2 diabetes (Rojas et al., Journal of Diabetes Research, vol. 2018, Article ID 9601801, 19 pages, 2018; Donath et al., Diabetes. 2005 Dec;54 Suppl 2:S108-13). Despite this knowledge to date there are no available methods to directly measure the number of beta cell (beta cell mass) in vivo or to deliver therapeutics specifically to these important cells. Indeed, methods to determine diabetes progression rely mostly on the indirect measurement (i.e the determination of glucose or c-peptide concentration in the blood) and cannot discriminate whether many cells produce little insulin or few cells produce large quantities of this hormone. Thus, these methods cannot measure the progressive beta cell loss in patients with diabetes. The lack of adequate marker specific for beta cell make also impossible to deliver therapeutics specifically to beta cells to halt or reverse beta cell loss.
  • RNA aptamers have emerged as effective delivery vehicles for siRNAs in the treatment of many human diseases because they actively enhance the intracellular accumulation of therapeutic cargo by receptor-mediated internalization or by clathrin-mediated endocytosis (35-39,43-74). The use of aptamers to deliver the therapeutic RNA of interest to the β cells offers advantage over the use of viral vectors such for example the transient modulation of the gene of interest, the lack of immunogenicity, and a great safety profile. To date, adenoviral vectors have been mostly used for efficient delivery of genes and siRNA to primary pancreatic islets in vitro (79-84). In vivo, however, beside the technical difficulties in using of viral vectors, their inherent immunogenicity and possible recombination with wild type virus raises serious safety concerns. Indeed, viral vectors can induce strong immune responses with secondary complications that may include multi-organs failure and even death (85). The advent of lentiviral vectors alleviated some of the immunogenicity concerns, but lentiviruses are not as efficient as adenoviruses in transducing intact human islets (86,87); although current in vitro protocols are being optimized88. Nevertheless, lentiviral integration in the genome still raises safety concerns, risks of insertional mutagenesis and recombination with wild type viruses.
    • SUMMARY
  • In one aspect, the disclosure provides a method of delivering one or more agents to a tissue comprising contacting the tissue with a construct comprising an aptamer that is specific for the tissue conjugated to the agent. In some embodiments, the tissue is from an organ selected from the group consisting of pancreas, heart, lung, kidney, stomach, skin and brain. In some embodiments, the tissue is pancreatic islets. In some embodiments, the agent is a therapeutic nucleic acid. In some embodiments, the therapeutic nucleic acid is a therapeutic RNA. In some embodiments, the therapeutic RNA is selected from the group consisting of siRNA and saRNA. In some embodiments, the agent is an imaging reagent. Exemplary imaging reagents include, but are not limited to, fluorochromes, Positron emission tomography tracer such as Fluorine-18, oxygen-15, gallium 68, magnetic resonance imaging contrast agents such as gadolinium, iron oxide, iron platinum and manganese. The contacting step can occur in vitro or in vivo.
  • In another aspect, the disclosure provides a construct comprising an aptamer conjugated to a small activating RNA (saRNA). In some embodiments, the aptamer is specific for human pancreatic islets. In some embodiments, the aptamer is selected from the group consisting of M12-3773 and 1-717.
  • In some embodiments, the aptamer is specific for clusterin (CLU, gene id 1191). In some embodiments, the aptamer is specific for “Transmembrane emp24 domain-containing protein 6” (TMED6, gene id 146456).
  • In some embodiments, the tissue is adrenal tissue or bone marrow and the aptamer is is 173-2273, 107-901 or m6-3239. In some embodiments, the tissue is breast tissue, lung tissue or lymph node tissue and the aptamer is 107-901 and m6-3239. In some embodiments, the tissue is brain cerebellum and the aptamer is 173-2273, 107-901, m1-2623 or m6-3239. In some embodiments, the tissue is brain cerebral cortex tissue, pituitary tissue, colon tissue, endothelium tissue, esophagus tissue, heart tissue or kidney tissue and the aptamer is 107-901, m1-2623 or m6-3239. In some embodiments, the tissue is fallopian tube tissue and the aptamer is m6-3239. In some embodiments, the tissue is liver tissue and the aptamer is 166-279, 107-901, m1-2623 or m6-3239. In some embodiments, the tissue is ovarian tissue and the aptamer is 107-901. In some embodiments, the tissue is placenta tissue and the aptamer is 166-270, 173-2273, 107-901, m1-2623 or m6-3239. In some embodiments, the tissue is prostate tissue and the aptamer is 173-2273 or 107-901. In some embodiments, the tissue is spinal cord tissue and the aptamer is 166-279 or 173-2273. In some embodiments, the tissue is testis tissue and the aptamer is 166-279, 173-2273, 107-901, m1-2623, m6-3239 and m12-3773. In some embodiments, the tissue is thymus tissue and the aptamer is 173-2273, 107-901 or mf-2623. In some embodiments, the tissue is thyroid tissue and the aptamer is m1-2623. In some embodiments, the tissue is ureter tissue and the aptamer is 107-901. In some embodiments, tissue is cervical tissue and the aptamer is 166-279. In some embodiments, the tissue is islets of Langerhans or pancreatic tissue and the aptamer is 166-279, 173-2273, 107-901, 1-717, m1-2623, m6-3239 or m12-3773.
  • In another aspect, the disclosure provides a method of delivering one or more agents to pancreatic islets comprising contacting the islets with a construct comprising an aptamer that is specific for islets conjugated to the agent. In some embodiments, the agent is a therapeutic nucleic acid. In some embodiments, the therapeutic nucleic acid is a therapeutic RNA. In some embodiments, the therapeutic RNA is selected from the group consisting of siRNA and saRNA. In some embodiments, the agent is an imaging reagent. Exemplary imaging reagents include, but are not limited to, fluorochromes, Positron emission tomography tracer such as Fluorine-18, oxygen-15, gallium 68, magnetic resonance imaging contrast agents such as gadolinium, iron oxide, iron platinum and manganese. The contacting step can occur in vitro or in vivo.
  • In some embodiments, the aptamer is selected from the group consisting of M12-3773 and 1-717. In some embodiments, the aptamer is specific for clusterin (CLU, gene id 1191). In some embodiments, the aptamer is specific for “Transmembrane emp24 domain-containing protein 6” (TMED6, gene id 146456).
  • In another aspect, the disclosure provides a method of measuring beta cell mass comprising contacting the beta cell with a construct comprising an aptamer conjugated to an imaging reagent in an amount effective to measure the mass of the beta cell. In some embodiments, the aptamer is selected from the group consisting of M12-3773 and 1-717. In some embodiments, the imaging reagent is a fluorochrome. In some embodiments, the imaging reagent is a PET tracer. In some embodiments, the imaging reagent is a MRI contrast reagent. In some embodiments, the imaging reagent can be conjugated to the aptamer via chelators.
  • In another aspect, the disclosure provides a method of modulating proliferation of beta cell comprising contacting the beta cell with a construct comprising an aptamer conjugated to a therapeutic nucleic acid in an amount effective to modulate proliferation of the beta cell. In some embodiments, the aptamer is selected from the group consisting of M12-3773 and 1-717. In some embodiments, the therapeutic nucleic acid is a therapeutic RNA. In some embodiments, the therapeutic RNA is selected from the group consisting of siRNA and saRNA. The contacting step can occur in vitro or in vivo.
  • In another aspect, the disclosure provides a method for inhibiting beta cell apoptosis comprising contacting the beta cell with a construct comprising an aptamer conjugated to a therapeutic nucleic acid in an amount effective to inhibit apoptosis of the beta cell. In some embodiments, the aptamer is selected from the group consisting of M12-3773 and 1-717. In some embodiments, the therapeutic nucleic acid is a therapeutic RNA. In some embodiments, the therapeutic RNA is selected from the group consisting of siRNA and saRNA. In some embodiments the therapeutic RNA upregulate the protein XIAP (X-linked inhibitor of apoptosis gene id 331). The contacting step can occur in vitro or in vivo.
  • In another aspect, the disclosure provides a method for inhibiting tissue graft apoptosis in a subject in need thereof comprising contacting the tissue graft with a construct comprising an aptamer conjugated to a therapeutic nucleic acid in an amount effective to inhibit apoptosis of the tissue graft. In some embodiments, the therapeutic nucleic acid is a therapeutic RNA. In some embodiments, the therapeutic RNA is selected from the group consisting of siRNA and saRNA. In some embodiments, the therapeutic RNA upregulates the protein XIAP (X-linked inhibitor of apoptosis gene id 331). The contacting step can occur in vitro or in vivo. In some embodiments, the tissue graft is from an organ selected from the group consisting of pancreas, heart, lung, kidney, stomach and skin. In some embodiments, the aptamer is a muscle specific aptamer and the tissue is heart tissue.
  • In some embodiments, the tissue is contacted with the therapeutic RNA that upregulates the protein XIAP in the absence of an aptamer. For example, in another aspect, the disclosure provides a method for inhibiting tissue graft apoptosis in a subject in need thereof comprising contacting the tissue graft with a therapeutic RNA that upregulates the protein XIAP (X-linked inhibitor of apoptosis gene id 331) in an amount effective to inhibit apoptosis of the tissue graft. The contacting step can occur in vitro or in vivo. In some embodiments, the tissue graft is from an organ selected from the group consisting of pancreas, heart, lung, kidney, stomach and skin.
  • In another aspect, the disclosure provides a method for protecting a beta cell from T-cell mediated cytotoxicity of the beta cell comprising contacting the beta cell with a construct comprising an aptamer conjugated to a therapeutic nucleic acid in an amount effective to inhibit T cell mediated cytotoxicity of the beta cell. In some embodiments, the aptamer is selected from the group consisting of M12-3773 and 1-717. In some embodiments, the therapeutic nucleic acid is able to increase immune checkpoint. In some embodiments, the therapeutic nucleic acid is a therapeutic RNA. In some embodiments, the therapeutic RNA is selected from the group consisting of siRNA and saRNA. In some embodiments the therapeutic RNA upregulate the protein CD274 (Programmed death-ligand 1, PDL1, gene id 29126). The contacting step can occur in vitro or in vivo.
  • In another aspect, the disclosure provides a method of treating diabetes in a subject in need thereof comprising administering to the subject a construct comprising an aptamer conjugated to a small activating RNA (saRNA) in an amount effective to treat diabetes in the subject. In some embodiments, the aptamer is selected from the group consisting of M12-3773 and 1-717.
  • In another aspect, one or more aptamers specific for the beta cells can be used in combination to increase delivery of the therapeutic agent or imaging reagents. In some embodiments, the aptamers are selected from the group consisting of M12-3773 and 1-717.
  • An aptamer comprising a nucleotide sequence set forth in SEQ ID NO: 264 or 259 is also contemplated. In some embodiments, the aptamer is conjugated to an saRNA. In some embodiments, the saRNA upregulates the protein XIAP (X-linked inhibitor of apoptosis gene id 331). In some embodiments, saRNA upregulates the protein CD274 (Programmed death-ligand 1, PDL1, gene id 29126,
    • BRIEF DESCRIPTION OF THE FIGURES
  • FIG. 1 is a flow chart showing HT-cluster SELEX as an unsupervised strategy for the isolation of aptamers specific for human islets using human islets and acinar tissue.
  • FIGS. 2A and 2B provide a flow chart showing HT-Toggle-cluster SELEX to isolate islets specific aptamers crossreacting between mouse and human. 8 cycles of HT-cluster SELEX were performed as described in FIG. 1 using as negative and positive selectors mouse acinar tissues and mouse islets respectively (FIG. 2A). The resulting polyclonal aptamer from cycle 8 was used for two additional cycle of selection using either acinar tissue and islets from mice or acinar tissue and islets isolated from cadaveric donors (FIG. 2B).
  • FIG. 3 shows images resulting from HT-Toggle-cluster SELEX to isolate islets specific aptamers crossreacting between mouse and human.
  • FIG. 4 shows images resulting from HT-Toggle-cluster SELEX identifying monoclonal aptamers that rcognize human islets but not human acinar tissue.
  • FIGS. 5A and 5B show that aptamers 1-717 (FIG. 5A) and M12-3772 (FIG. 5B) show an extraordinary specificity for human islets. FIG. 5C is a table showing the results of various tissue staining with various selected aptamers.
  • FIG. 6 shows that aptamers 1-717 and M12-3772 recognize mouse islets and other mouse tissues.
  • FIGS. 7A-7D shows that aptamers 1-717 (FIGS. 7B and 7C) and M12-3773 (FIGS. 7A and 7D) recognize preferentially human beta cells.
  • FIGS. 8A-8C show that clusterin is a possible target for aptamer m12-3773. FIG. 8A provides the strategy for an aptamer based mass spectrometry and mascot-based analysis. Clusterin (UniProtKB - P10909) (FIG. 8B) had a high Mascot score (236). FIG. 8C shows that silencing of Clusterin reduced the capacity of aptamer m12-7337, but not of aptamer 1-717, to bind to beta cells.
  • FIGS. 9A-9C shows that TMED6 is the putative target for aptamer 1-717. FIG. 9A shows the experimental strategy to detect the target for aptamer 1-717. FIG. 9B provides results of the binding array assay described in Example 1. FIG. 9C is a graph showing that competitive assays confirm the specificity of aptamer 1-717 for TMED6
  • FIGS. 10A and 10B shows that a mixture of aptamer 1-717 and m12-3773 recognize human islets in vivo better than the individual clones. A cumulative-synergistic signal was observed in the EFP region when the mixture of both aptamers was used possibly because different islet epitopes were targeted by each aptamer (FIG. 10A). 4 hour later fluorescence signal in epididymal fat pad region was measure by “In vivo imaging system (IVIS)”. The data in FIG. 10B shows that both aptamer 1-717 and aptamer m12-3773 can recognize the islets in vivo.
  • FIGS. 11A-11C show that Aptamer 1-717 and M12-3773 allow the measurement of human beta cell mass in vivo. FIG. 11A is a schematic of the experiment performed in Example 2. FIGS. 11B and 11C show that fluorescence signal in the EFP region was proportional to the number of engrafted islets indicating that these aptamers can be used to measure β cell mass in vivo FIG. 11D: Syngeneic (Balb/c) or allogeneic (C57B⅙) ilsets were transplanted subcutaneously (in the right and left flank respectively) of immunocompetent Balb/c mice. Rejection was longitudinally monitored by injecting AF750-conjugated aptamer intravenously and by performing IVIS 5 hours later. Data show that rejection of the allogeneic C57B16 islet graft can be measured over time as seen by the loss of signal on the left flank. Instead signal (right) of the syngeneic islet graft is maintained over time indicating graft survival.
  • FIG. 12 is a schematic diagram aptamer chimera for the delivery of therapeutic RNA via islets specific aptamers.
  • FIGS. 13A and 13B show that islets specific aptamer chimera allows for the delivery of therapeutic RNA via islets specific aptamers. FIG. 13A is a schematic of the experimental procedure. FIG. 13B shows that the aptamer chimera significantly downregulate the expression of the target gene.
  • FIGS. 14A-14C shows that p57kip2-siRNA-islet specific aptamer chimera induce human beta cell proliferation in vivo. FIG. 14A shows the experimental procedure: Streptozotocin-treated, immune deficient NSG mice were transplanted with a suboptimal quantity (250 IEQ) of human islets in the anterior chamber of the eye. FIG. 14B provides immunofluorescence pictures of the graft from mice treated with control chimera or p57kip2-siRNA/aptamer chimera. Glucagon and insulin staining is depicted in dark gray as pseudocolor whereas BrdU staining as measure of cell proliferation is depicted in white as pseudocolor. FIG. 14C shows quantification of proliferating beta and alpha cells. Taken together these data indicate that p57kip2-siRNA/aptamer chimera can induce in vivo human beta cell proliferation in a hyperglycemic setting that mimic T1 and T2 diabetes.
  • FIG. 15A provides a schematic of the method described in Example 4. FIG. 15B shows the identification of small activating RNA (saRNA) specific for the human “X-Linked Inhibitor Of Apoptosis” (Xiap, Gene ID: 331).
  • FIGS. 16A-16C. Xiap-saRNA aptamer chimera protect human beta cells from cytokine induced apoptosis. FIG. 16A shows the experimental procedure described in Example 5. FIG. 16B shows the flow cytometry analysis of single cell suspension of islets treated with scrambled saRNA chimera (CTRL chimera) or XIAP-saRNA/aptamer chimera (Xiap Chimera) and later challenged with cytokines (CTK) or left untreated (No CTK). FIG. 16C is a spaghetti plot from 5 independent experiments each with islets from a different cadaveric donor using chimera generated with either aptamer m12-3773 or aptamer 1-717.
  • FIGS. 17A and B show that the Xiap-saRNA/islet specific aptamer chimera protect beta cells from primary nonfunction. FIG. 17A is a schematic for the experiment described in Example 5. FIG. 17B: human Islets were cultured in media where chimera was added at 48h, 24h and on the day of transplantation 600 IEQ were transplanted per mouse in the left kidney capsule of streptozotocin diabetic NOG mice. Data showed that pretreatment of human islets with aptamer chimera greatly improve the efficacy of islet transplantation with approximately 80% of mice becoming normoglycemic by day 2. In contrast only 50% of mice engrafted with islets (P=0.02; nchimera treated= 10; nuntreated = 8) reverse diabetes and with a delayed kinetic.
  • FIGS. 18A-18B. FIG. 18A provides a schematic of the protocol described in Example 6. FIG. 18B is a graph showing the identification of small activating RNA (saRNA) specific for the human “PDL1” (CD274, Twelve saRNAs (provided in Table 5) were found to upregulate Xiap expression more than 10 times (range 10.4-74.8) over scrambled saRNA. Gene ID: 29126).
  • FIGS. 19A -19B PDL1-saRNA/islet specific aptamer chimera upregulate PDL1 on human beta cells. FIG. 19A is a schematic showing that PDL1-saRNA/aptamer chimera were added to non-dissociated human islets from cadaveric donor. 48h later, islets were dissociated, labelled with anti-insulin, anti-glucagon and anti-PDL1 antibodies and analyzed by flow cytometry. Results of the flow cytometery is shown in FIG. 19B.
  • FIGS. 20A-20C. PDL1-saRNA/aptamer chimera upregulate PDL1 in vivo. FIG. 20A provides a schematic of a protocol where immune deficient NSG mice were transplanted in the anterior chamber of the eye with human islets from a cadaveric donor. 3 weeks later, mice were treated with PDL1-saRNA(636)/1-717-aptamer chimera. Scramble-saRNA/aptamer chimera was used as control (CTRL chimera). FIG. 20B are images showing PDL1 expression in tissues. FIG. 20C provides graphs summarizing of PDL1 expression on the engrafted islets at baseline or 5 days after treatment with PDL1-saRNA/aptamer chimera or scrambled-saRNA/aptamer chimera.
    •  DETAILED DESCRIPTION
  • As described in the Examples, therapeutic RNA/aptamer chimeras were generated to modulate gene expression in human β cells in vivo to induce their transient proliferation and improve their resistance to auto/alloimmunity. In particular, we have optimized and validate the use of islet-specific aptamers to deliver: A) siRNA against p57kip2 to induce β cell proliferation, B) saRNA promoting Xiap expression to protect islets from apoptosis, and C) saRNA promoting PDL1 expression to protect β cells from T cell cytotoxicity. Because of the absence of reliable humanized mouse model of autoimmune T1D, our approach is based on the use of NSG or humanized NSG mice transplanted with human islets before aptamer treatment. The use of human islets is dictated by species specific difference in p57kip2 biology (14) and by the specificity of PDL1 and Xiap saRNAs for the human genes. Ex vivo and innovative in vivo techniques are employed to quantify the response to in vivo treatment through imaging of β cell proliferation, apoptosis, and interaction with the immune system. We envision the use of these aptamers as mono or multimodal approach where difference genes can be modulated simultaneously.
  • The in vivo use of RNA aptamers is particularly appealing because this class of molecules has low immunogenicity, high capacity to penetrate deep into the tissues, and ability to recognize the cognate target with high affinity and specificity. The fluorinated backbone of the aptamers make them resistant to RNAse degradation and incapable to trigger TLR signaling (41,42). RNA aptamers have emerged as effective delivery vehicles for siRNAs and other drugs to specific cell subsets or tissues for the treatment of many human diseases (60,62-75). Indeed, through the interactions between the aptamer and its cellular membrane target, aptamers actively enhance the intracellular accumulation of therapeutic agents (37-39,43-61). Some aptamer drugs are FDA-approved and more than 30 are being tested in clinical trials (16-24). When administered in vivo, aptamers that do not find a specific target are rapidly eliminated via the kidney; those that find their target in tissues or cells remain detectable for up two weeks. Their bioavailability, plasma half-life, and pharmacokinetic properties can be easily engineered by increasing their size by the addition of Polyethylene glycol (PEG) during synthesis, or by conjugation with nanoparticles (60,62-74). Aptamers can be conjugated to siRNA, miRNA or saRNA to deliver the desirable therapeutic effect in specific targets. The ability to directly engineer aptamers with high specificity and defined functions is a distinct advantage over antibodies and other small molecules.
    • EXAMPLES Example 1 - Isolation of Monoclonal RNA Aptamer Specific for Human Islets
  • Unsupervised toggled-SELEX was performed starting with a polyclonal aptamer library against mouse islets and using islet depleted human acinar cells and handpicked human islets from 4 different cadaveric donors as negative and positive selectors, respectively. This allowed for the depletion of non-specific (acinar tissue binding) RNA aptamers and enrich the library for those aptamers specific for mouse and human islets.
  • As shown in FIG. 1 , HT-cluster SELEX was used as an unsupervised strategy for the isolation of aptamers specific for human islets using human islets and acinar tissue. A random aptamer library was generated by PCR and Durascribe T7 RNA transcription from a cDNA random library (TCT CGG ATC CTC AGC GAG TCG TC TG (N40) CCG CAT CGT CCT CCC TA (SEQ ID NO: 413), comprising a 40-nt variable region flanked by two constant region. 5 ug (~8.3×1013 aptamers) of this random library were depleted for aptamers binding the acinar tissue using islets depleted pancreata (negative selector) from cadaveric donors. Unbound aptamers were then incubated with hand-picked islets (100-300 IEQ as positive selector) from cadaveric donors. Islets were washed with PBS and islets-bound aptamers were recovered by RNA extraction and re-amplified by RT-PCR and T7-RNA polymerase using 2′-Fluorine-dCTP (2′-F-dCTP) and 2′-Fluorine-dUTP (2′-F-dUTP), ATP, and GTP for improved RNAse resistance. The resulting RNA aptamer library (Table 1), enriched for islets specific aptamers, was used for new selection cycle. A total of 8 selection cycles was performed using islets and acinar tissue from 4 unrelated cadaveric donors. Library from each cycles were HT sequenced and subject to bio-informatic analysis to perform frequency and cluster analysis and identify those monoclonal aptamer and family of aptamers enriched during the selection process. The most frequent monoclonal aptamers among the most frequent families present on the library from cycle 8 were chosen for empirical testing.
  • TABLE 2
    Putative human islet specific aptamers isolated via cluster SELEX (from FIG. 1 )
    Putative human islet specific aptamers isolated via cluster SELEX (from FIG. 1 )
    aptamer name SEQ ID NO. sequence
    279 1 GGAGGAGCUACGAUGCGGCCGAUUUCGUCAUCCUCCAUA CCAUCGCCUUACCGUUCCGCGUCAGACGACUCGCUGAGGAUCCG ACA
    2529 2 GGAGGAGCUACGAUGCGGCCGUCUCGCUCUCAUCCCAUGCA CGAAACCUCUCUCACUGCACAGACGACUCGCUGAGGAUCCGACA
    2031 3 GGAGGAGCUACGAUGCGGUGAAUUCUUCCGGCACUUUGUCA UCUUCACCCCCAUGCUGCACAGACGACUCGCUGAGGAUCCGACA
    1134 4 GGAGGAGCUACGAUGCGGCCGAUUUCGUCAUCCUCCAUACCA UCGCCUCACCGUUCCGCGUCAGACGACUCGCUGAGGAUCCGACA
    664 5 GGAGGAGCUACGAUGCGGCCGAUUUCGUCAUCCUCCACACCAUC G CCUUACCGUUCCGCGUCAGACGACUCGCUGAGGAUCCGACA
    877 6 GGAGGAGCUACGAUGCGGCCGAUUUCGUCAUCCUCCGUACCAUC GC CUUACCGUUCCGCGUCAGACGACUCGCUGAGGAUCCGACA
    2437 7 GGAGGAGCUACGAUGCGGCCCAUCACUCCCGCGUAUUGCGAACG CAU CGUUAUUUAGCCGUCAGACGACUCGCUGAGGAUCCGACA
    1131 8 GGAGGAGCUACGAUGCGGUCGAUUUCGUCAUCCUCCAUACCAUC GCC UUACCGUUCCGCGUCAGACGACUCGCUGAGGAUCCGACA
    436 9 GGAGGAGCUACGAUGCGGCCGAUUUCGUCAUCCUCCAUACCAUC GUC UUACCGUUCCGCGUCAGACGACUCGCUGAGGAUCCGACA
    19 10 GGAGGAGCUACGAUGCGGCCGAUUUCGUCAUCCUCCAUACCAUC GC CUUACCGCUCCGCGUCAGACGACUCGCUGAGGAUCCGACA
    665 11 GGAGGAGCUACGAUGCGGCCGAUUUCGUCAUCCUCCAUGCCAUC GCC UUACCGUUCCGCGUCAGACGACUCGCUGAGGAUCCGACA
    280 12 GGAGGAGCUACGAUGCGGCCGAUUUCGUCAUCCUCCAUACCAUC GCCCUACCGUUCCGCGUCAGACGACUCGCUGAGGAUCCGACA
    79 13 GGAGGAGCUACGAUGCGGCCGAUUUCGUCAUCCUCCAUACCAUC GCCUUGCCGUUCCGCGUCAGACGACUCGCUGAGGAUCCGACA
    278 14 GGAGGAGCUACGAUGCGGCCGAUUUCGUCAUCCUCCAUACCAUC GCCUUACCAUUCCGCGUCAGACGACUCGCUGAGGAUCCGACA
    658 15 GGAGGAGCUACGAUGCGGCCGAUUUCGUCAUCCCCCAUACCAUC GCCUUACCGUUCCGCGUCAGACGACUCGCUGAGGAUCCGACA
    37 16 GGAGGAGCUACGAUGCGGCCGAUAUCGUCAUCCUCCAUACCAUC GCCUUACCGUUCCGCGUCAGACGACUCGCUGAGGAUCCGACA
    485 17 GGAGGAGCUACGAUGCGGCCGAUUUCGUCAUCCUCCAUACCAUC GCCUACCGUUCCGCGUCAGACGACUCGCUGAGGAUCCGACA
    2617 18 GGAGGAGCUACGAUGCGGUGUACACUGAUUGCCUUUGUGUUAUG AGCGACAGAUCUGCCAGACAGACGACUCGCUGAGGAUCCGACA
    2273 19 GGAGGAGCUACGAUGCGGACCUUGUUUUCCUCUGUACCCCACUU CCCCAUUUCUCCCUGCUCAGACGACUCGCUGAGGAUCCGACA
    146 20 GGAGGAGCUACGAUGCGGCCGAUCUCGUCAUCCUCCAUACCAUC GCCUUACCGUUCCGCGUCAGACGACUCGCUGAGGAUCCGACA
    657 21 GGAGGAGCUACGAUGCGGCCGAUUUCGUCAUCCUCCAUACCAUC GCCUUACCGUCCCGCGUCAGACGACUCGCUGAGGAUCCGACA
    141 22 GGAGGAGCUACGAUGCGGCCGAUUUCGUCAUCCUCCAUACCGUC GCCUUACCGUUCCGCGUCAGACGACUCGCUGAGGAUCCGACA
    2048 23 GGAGGAGCUACGAUGCGGCCGAUUUCGUCGUCCUCCAUACCAUC GCCUUACCGUUCCGCGUCAGACGACUCGCUGAGGAUCCGACA
    901 24 GGAGGAGCUACGAUGCGGGGAAGCAACACUUAGUCGCGAUUGAU ACGUGCGCAGUCAUCAGACAGACGACUCGCUGAGGAUCCGACA
    268 25 GGAGGAGCUACGAUGCGGCCGAUUUCGCCAUCCUCCAUACCAUC GCCUUACCGUUCCGCGUCAGACGACUCGCUGAGGAUCCGACA
    683 26 GGAGGAGCUACGAUGCGGCCGAUUUCGUCAUCCUCCAUACCAUC GCCUUCCGUUCCGCGUCAGACGACUCGCUGAGGAUCCGACA
    655 27 GGAGGAGCUACGAUGCGGCCGAUUUCGUCAUCCUCCAUAUCAUC GCCUUACCGUUCCGCGUCAGACGACUCGCUGAGGAUCCGACA
    1427 28 GGAGGAGCUACGAUGCGGCCGAUUUCGUCACCCUCCAUACCAUC GCCUUACCGUUCCGCGUCAGACGACUCGCUGAGGAUCCGACA
    457 29 GGAGGAGCUACGAUGCGGCCGACUUCGUCAUCCUCCAUACCAUC GCCUUACCGUUCCGCGUCAGACGACUCGCUGAGGAUCCGACA
    1141 30 GGAGGAGCUACGAUGCGGCCGAUUUCGUCAUCCUCCAUACCAUC GCCUUACCUUCCGCGUCAGACGACUCGCUGAGGAUCCGACA
    149 31 GGAGGAGCUACGAUGCGGCCGAUUUCGUCAUCCUCCAUACCAUC GCCUUACCGUUCCGCAGUCAGACGACUCGCUGAGGAUCCGACA
    1759 32 GGAGGAGCUACGAUGCGGCCGAUUUCGUCAUCCUCCUUACCAUC GCCUUACCGUUCCGCGUCAGACGACUCGCUGAGGAUCCGACA
    264 33 GGAGGAGCUACGAUGCGGCCGAUUUCGUCAUCCUUCAUACCAUC GCCUUACCGUUCCGCGUCAGACGACUCGCUGAGGAUCCGACA
    259 34 GGAGGAGCUACGAUGCGGCCGAUUUCGUCAUCCUCCAUACUAUC GCCUUACCGUUCCGCGUCAGACGACUCGCUGAGGAUCCGACA
    1130 35 GGAGGAGCUACGAUGCGGCCGAUUUCAUCAUCCUCCAUACCAUC GCCUUACCGUUCCGCGUCAGACGACUCGCUGAGGAUCCGACA
    453 36 GGAGGAGCUACGAUGCGGCCGAUUUCGUCAUCCUCCAUACCAUC GCCUUACCUUUCCGCGUCAGACGACUCGCUGAGGAUCCGACA
    1133 37 GGAGGAGCUACGAUGCGGCCGAUUUCGUCAUCCUCUAUACCAUC GCCUUACCGUUCCGCGUCAGACGACUCGCUGAGGAUCCGACA
    883 38 GGAGGAGCUACGAUGCGGCCGAUUUCGUCAUCCUCCAAACCAUC GCCUUACCGUUCCGCGUCAGACGACUCGCUGAGGAUCCGACA
    155 39 GGAGGAGCUACGAUGCGGCCGAUUUCGUCAUCCUCCAUACCAUC GCCUUAUCGUUCCGCGUCAGACGACUCGCUGAGGAUCCGACA
    75 40 GGAGGAGCUACGAUGCGGCCGAUUCCGUCAUCCUCCAUACCAUC GCCUUACCGUUCCGCGUCAGACGACUCGCUGAGGAUCCGACA
    2049 41 GGAGGAGCUACGAUGCGGCUGAUUUCGUCAUCCUCCAUACCAUC GCCUUACCGUUCCGCGUCAGACGACUCGCUGAGGAUCCGACA
    1103 42 GGAGGAGCUACGAUGCGGCCGAUUUUCGUCAUCCUCCAUACCAU CGCCUUACCGUUCCGCGUCAGACGACUCGCUGAGGAUCCGACA
    885 43 GGAGGAGCUACGAUGCGGCCGAUUUCGUCAUCCUCAAUACCAUC GCCUUACCGUUCCGCGUCAGACGACUCGCUGAGGAUCCGACA
    281 44 GGAGGAGCUACGAUGCGGCCGAUUUCGUCAUCCUCCAUACCAUC GCCUUCCCGUUCCGCGUCAGACGACUCGCUGAGGAUCCGACA
    2381 45 GGAGGAGCUACGAUGCGGAUUACCAACUUGAACGCCGAGAGUGU GGUCACGUGUUCUGCAGACAGACGACUCGCUGAGGAUCCGACA
    879 46 GGAGGAGCUACGAUGCGGCCGAUUUCGUCAUCCUCCAUACCAUC GCCUUACCGUUUCGCGUCAGACGACUCGCUGAGGAUCCGACA
    292 47 GGAGGAGCUACGAUGCGGCCGAUUUCGUAUCCUCCAUACCAUCG CCUUACCGUUCCGCGUCAGACGACUCGCUGAGGAUCCGACA
    1511 48 GGAGGAGCUACGAUGCGGUUAUGCGUUUAAGUCAUUGACGCGUU ACACUGGAGGGGGCCAGACAGACGACUCGCUGAGGAUCCGACA
    148 49 GGAGGAGCUACGAUGCGGCCGAUUUCGUCAUCCUCCAUACCAUC GCCUUACCGUUCCGCAUCAGACGACUCGCUGAGGAUCCGACA
    878 50 GGAGGAGCUACGAUGCGGCCGAUUUCGUCAUCCUCCAUAACAUC GCCUUACCGUUCCGCGUCAGACGACUCGCUGAGGAUCCGACA
    156 51 GGAGGAGCUACGAUGCGGCCGAUUUCGUCAUCCUCCUACCAUCG CCUUACCGUUCCGCGUCAGACGACUCGCUGAGGAUCCGACA
    266 52 GGAGGAGCUACGAUGCGGCCGAUUUCGUUAUCCUCCAUACCAUC GCCUUACCGUUCCGCGUCAGACGACUCGCUGAGGAUCCGACA
    459 53 GGAGGAGCUACGAUGCGGCCGAUUUCGUCAUCCUCCAUACCAUC GCCAUACCGUUCCGCGUCAGACGACUCGCUGAGGAUCCGACA
    668 54 GGAGGAGCUACGAUGCGGCCGAUUUCGUCAUCCUCCAUACCAUC GCCUAACCGUUCCGCGUCAGACGACUCGCUGAGGAUCCGACA
    1760 55 GGAGGAGCUACGAUGCGGCCGAUUUCGUCAUCCUCCAUACCAUC GCUUUACCGUUCCGCGUCAGACGACUCGCUGAGGAUCCGACA
    661 56 GGAGGAGCUACGAUGCGGCCGAUUUCGUCAUCCUCCAUACCAUU GCCUUACCGUUCCGCGUCAGACGACUCGCUGAGGAUCCGACA
    1129 57 GGAGGAGCUACGAUGCGGCCGAUUUCGUCAUACUCCAUACCAUC GCCUUACCGUUCCGCGUCAGACGACUCGCUGAGGAUCCGACA
    438 58 GGAGGAGCUACGAUGCGGCCGAUUUCGUCAUCCUCCAUAGCAUC GCCUUACCGUUCCGCGUCAGACGACUCGCUGAGGAUCCGACA
    277 59 GGAGGAGCUACGAUGCGGCCGAUUUCGUCAUCCUCCAUACCAUC GCCUUACCCUUCCGCGUCAGACGACUCGCUGAGGAUCCGACA
    18 60 GGAGGAGCUACGAUGCGGCCGAUUUCGUAAUCCUCCAUACCAUC GCCUUACCGUUCCGCGUCAGACGACUCGCUGAGGAUCCGACA
    152 61 GGAGGAGCUACGAUGCGGCCGAUUUCGUCAUCCUCCAUACCAUC ACCUUACCGUUCCGCGUCAGACGACUCGCUGAGGAUCCGACA
    460 62 GGAGGAGCUACGAUGCGGCCGAUUUCGUCAUCCUCCAUACCAUC GCCUUUCCGUUCCGCGUCAGACGACUCGCUGAGGAUCCGACA
    1370 63 GGAGGAGCUACGAUGCGGCCCAUCCCUCCCGCGUAUUGCGAACG CAUCGUUAUUUAGCCGUCAGACGACUCGCUGAGGAUCCGACA
    717 64 GGAGGAGCUACGAUGCGGUAAUUCUCAGGAGGUGCGGAACGGGA UAUGGAUUGUUCGCCAGACAGACGACUCGCUGAGGAUCCGACA
    456 65 GGAGGAGCUACGAUGCGGCCGAUUUCGUCAUCCUCCAUCCCAUC GCCUUACCGUUCCGCGUCAGACGACUCGCUGAGGAUCCGACA
    876 66 GGAGGAGCUACGAUGCGGCCGAUUUCGUCAUCAUCCAUACCAUC GCCUUACCGUUCCGCGUCAGACGACUCGCUGAGGAUCCGACA
    391 67 GGAGGAGCUACGAUGCGGUGAAUUCUUCCGGCACUUUGUCAUCU UCACUCCCAUGCUGCACAGACGACUCGCUGAGGAUCCGACA
    659 68 GGAGGAGCUACGAUGCGGCCGAUUUCGUCAUCCUACAUACCAUC GCCUUACCGUUCCGCGUCAGACGACUCGCUGAGGAUCCGACA
    437 69 GGAGGAGCUACGAUGCGGCCGAUUUCGUCAUCCUCCAUACCAUC CCCUUACCGUUCCGCGUCAGACGACUCGCUGAGGAUCCGACA
    143 70 GGAGGAGCUACGAUGCGGCCGAUUUCGUCAUCCACCAUACCAUC GCCUUACCGUUCCGCGUCAGACGACUCGCUGAGGAUCCGACA
    802 71 GGAGGAGCUACGAUGCGGCCGUCUCGCUCUCAUCCCGUGCACGA AACCUCUCUCACUGCACAGACGACUCGCUGAGGAUCCGACA
    2192 72 GGAGGAGCUACGAUGCGGCAACAAACUAAUCAGACACGAGACAGA GAGAUAGAUCUGCCAGACAGACGACUCGCUGAGGAUCCGACA
    1736 73 GGAGGAGCUACGAUGCGGUGAAUUCUUCCGGCACUUUGUCAUCU UCACCCCCCUGCUGCACAGACGACUCGCUGAGGAUCCGACA
    462 74 GGAGGAGCUACGAUGCGGCCGAUUUCGUCAUCCUCCAUACCAUC GCCUUAGCGUUCCGCGUCAGACGACUCGCUGAGGAUCCGACA
    882 75 GGAGGAGCUACGAUGCGGCCGAUUUCGUCAUCCUCCAUACCAUA GCCUUACCGUUCCGCGUCAGACGACUCGCUGAGGAUCCGACA
    140 76 GGAGGAGCUACGAUGCGGCCGAUUUCGUCAUCCUCCAUACAAUC GCCUUACCGUUCCGCGUCAGACGACUCGCUGAGGAUCCGACA
    363 77 GGAGGAGCUACGAUGCGGCCCAUCACUCCCGCGUACUGCGAACG CAUCGUUAUUUAGCCGUCAGACGACUCGCUGAGGAUCCGACA
    275 78 GGAGGAGCUACGAUGCGGCCGAUUUCGUCAUCCUCCAUACCAUC GCCUUACCGAUCCGCGUCAGACGACUCGCUGAGGAUCCGACA
    667 79 GGAGGAGCUACGAUGCGGCCGAAUUCGUCAUCCUCCAUACCAUC GCCUUACCGUUCCGCGUCAGACGACUCGCUGAGGAUCCGACA
    36 80 GGAGGAGCUACGAUGCGGCCGAUUUCGUCAUCCUCCAUACCAUC GACUUACCGUUCCGCGUCAGACGACUCGCUGAGGAUCCGACA
    441 81 GGAGGAGCUACGAUGCGGCCGAUUUCGUCAUCCUGCAUACCAUC GCCUUACCGUUCCGCGUCAGACGACUCGCUGAGGAUCCGACA
    1383 82 GGAGGAGCUACGAUGCGGUCCUUGUUUUCCUCUGUACCCCACUU CCCCAUUUCUCCCUGCUCAGACGACUCGCUGAGGAUCCGACA
    1429 83 GGAGGAGCUACGAUGCGGCCGAUUUCUUCAUCCUCCAUACCAUC GCCUUACCGUUCCGCGUCAGACGACUCGCUGAGGAUCCGACA
    262 84 GGAGGAGCUACGAUGCGGCCGAUUUCGUCAUCCUCCAUACCAUC UCCUUACCGUUCCGCGUCAGACGACUCGCUGAGGAUCCGACA
    451 85 GGAGGAGCUACGAUGCGGCCGAUUUCGUCAUCCUCCAUACCAUG GCCUUACCGUUCCGCGUCAGACGACUCGCUGAGGAUCCGACA
    446 86 GGAGGAGCUACGAUGCGGCCGAUUUCGGCAUCCUCCAUACCAUC GCCUUACCGUUCCGCGUCAGACGACUCGCUGAGGAUCCGACA
    265 87 GGAGGAGCUACGAUGCGGCCGAUUUCGUGAUCCUCCAUACCAUC GCCUUACCGUUCCGCGUCAGACGACUCGCUGAGGAUCCGACA
    880 88 GGAGGAGCUACGAUGCGGCCGAUUUCGUCAUCCUCCAUACCAUC GCCUUACCGUACCGCGUCAGACGACUCGCUGAGGAUCCGACA
    323 89 GGAGGAGCUACGAUGCGGACGGAGGAUAGUUGCUAAUCGAGCCC UGCCGACGCUUCAGACAGACGACUCGCUGAGGAUCCGACA
    458 90 GGAGGAGCUACGAUGCGGCCGAUUUCGUCAUCCUCCGUACCAUC GCCUCACCGUUCCGCGUCAGACGACUCGCUGAGGAUCCGACA
    662 91 GGAGGAGCUACGAUGCGGCCGAUUUCGUCAUCCUCCAUACCAUC GCCUUACAGUUCCGCGUCAGACGACUCGCUGAGGAUCCGACA
    682 92 GGAGGAGCUACGAUGCGGCCGAUUUCGUCAUCCUCCAUACCAUC CCUUACCGUUCCGCGUCAGACGACUCGCUGAGGAUCCGACA
    154 93 GGAGGAGCUACGAUGCGGCCGAUUUCGUCAUCCUCCAUACCAUC GCCUGACCGUUCCGCGUCAGACGACUCGCUGAGGAUCCGACA
    282 94 GGAGGAGCUACGAUGCGGCCGAUUUCGUCAUCCUCCAUACCAUC GCCUUAACGUUCCGCGUCAGACGACUCGCUGAGGAUCCGACA
    449 95 GGAGGAGCUACGAUGCGGCCGAUUUCGUCAUCCUCCAUACCAUC GCCUUACCGGUCCGCGUCAGACGACUCGCUGAGGAUCCGACA
    16 96 GGAGGAGCUACGAUGCGGCCGAUUCGUCAUCCUCCAUACCAUCG CCUUACCGUUCCGCGUCAGACGACUCGCUGAGGAUCCGACA
    900 97 GGAGGAGCUACGAUGCGGCCGAUUUCGUCAUCCUCCAUACCAUC GCCUUACCGUCCGCGUCAGACGACUCGCUGAGGAUCCGACA
    2032 98 GGAGGAGCUACGAUGCGGUGAAUUCUUCCGGCACUUUGUCAUCU UCACCCCCAUGCUGCGCAGACGACUCGCUGAGGAUCCGACA
    267 99 GGAGGAGCUACGAUGCGGCCGAUUUCGUCAUCCUCCAUACCAAC GCCUUACCGUUCCGCGUCAGACGACUCGCUGAGGAUCCGACA
    72 100 GGAGGAGCUACGAUGCGGCCGAUUUCGUCAUCCUCCAUACCCUC GCCUUACCGUUCCGCGUCAGACGACUCGCUGAGGAUCCGACA
    1075 101 GGAGGAGCUACGAUGCGGUGAAUUCUUCCGGCACUUUGUCAUCU UCACCUCCAUGCUGCACAGACGACUCGCUGAGGAUCCGACA
    261 102 GGAGGAGCUACGAUGCGGCCGAUUUCGUCAUCCUCCAUACCAUC GGCUUACCGUUCCGCGUCAGACGACUCGCUGAGGAUCCGACA
    801 103 GGAGGAGCUACGAUGCGGCCGUCUCGCUCUCAUCCCAUGCACGA AACCUCUCUCGCUGCACAGACGACUCGCUGAGGAUCCGACA
    291 104 GGAGGAGCUACGAUGCGGCCGAUUUGUCAUCCUCCAUACCAUCG CCUUACCGUUCCGCGUCAGACGACUCGCUGAGGAUCCGACA
    599 105 GGAGGAGCUACGAUGCGGUGAAUUCUUCCGGCACUUUGUCAUCU UCACCCCCACGCUGCACAGACGACUCGCUGAGGAUCCGACA
    272 106 GGAGGAGCUACGAUGCGGCCGAUUACGUCAUCCUCCAUACCAUC GCCUUACCGUUCCGCGUCAGACGACUCGCUGAGGAUCCGACA
    447 107 GGAGGAGCUACGAUGCGGCCGAUUUCGCCAUCCUCCAUACCAUC GCCUCACCGUUCCGCGUCAGACGACUCGCUGAGGAUCCGACA
    74 108 GGAGGAGCUACGAUGCGGCCGAUUUCGACAUCCUCCAUACCAUC GCCUUACCGUUCCGCGUCAGACGACUCGCUGAGGAUCCGACA
    674 109 GGAGGAGCUACGAUGCGGCCGAUUUCGUCAUCCUCCAUACAUCG CCUUACCGUUCCGCGUCAGACGACUCGCUGAGGAUCCGACA
    4 110 GGAGGAGCUACGAUGCGGCCGAUUUCGUCAUCCUCCAUACGAUC GCCUUACCGUUCCGCGUCAGACGACUCGCUGAGGAUCCGACA
    455 111 GGAGGAGCUACGAUGCGGCCGAUUUCGUCAUCCUCCGUACCAUC GCCUUACCAUUCCGCGUCAGACGACUCGCUGAGGAUCCGACA
    890 112 GGAGGAGCUACGAUGCGGCCGAUUUCGUCAUCCCCAUACCAUCG CCUUACCGUUCCGCGUCAGACGACUCGCUGAGGAUCCGACA
    260 113 GGAGGAGCUACGAUGCGGCCGAUUUCGUCAUCCUCCAUACCUUC GCCUUACCGUUCCGCGUCAGACGACUCGCUGAGGAUCCGACA
    39 114 GGAGGAGCUACGAUGCGGCCGAUUUCGUCAUGCUCCAUACCAUC GCCUUACCGUUCCGCGUCAGACGACUCGCUGAGGAUCCGACA
    57 115 GGAGGAGCUACGAUGCGGCCCAUCACUCCCGCGCAUUGCGAACG CAUCGUUAUUUAGCCGUCAGACGACUCGCUGAGGAUCCGACA
    889 116 GGAGGAGCUACGAUGCGGCCGAUUUCGUCAUCUCCAUACCAUCG CCUUACCGUUCCGCGUCAGACGACUCGCUGAGGAUCCGACA
    828 117 GGAGGAGCUACGAUGCGGUGAAUUCUUCCGGCACUUUGUCAUCU UCACCCCCAUGCCGCACAGACGACUCGCUGAGGAUCCGACA
    2016 118 GGAGGAGCUACGAUGCGGCCCAUCACUCCCGCGUAUUGCGAACG CCUCGUUAUUUAGCCGUCAGACGACUCGCUGAGGAUCCGACA
    666 119 GGAGGAGCUACGAUGCGGUCGAUUUCGUCAUCCUCCGUACCAUC GCCUUACCGUUCCGCGUCAGACGACUCGCUGAGGAUCCGACA
    1738 120 GGAGGAGCUACGAUGCGGUGAAUUCUUCCGGCACUUUGUCAUCU UCAUCCCCAUGCUGCACAGACGACUCGCUGAGGAUCCGACA
    656 121 GGAGGAGCUACGAUGCGGCCGAUUUCGUCAUCCUCCAUACCAUC GCCUUACCGUUACGCGUCAGACGACUCGCUGAGGAUCCGACA
    654 122 GGAGGAGCUACGAUGCGGCCGAUUUCGUCAACCUCCAUACCAUC GCCUUACCGUUCCGCGUCAGACGACUCGCUGAGGAUCCGACA
    440 123 GGAGGAGCUACGAUGCGGCCGAUUUCGUCAUCCGCCAUACCAUC GCCUUACCGUUCCGCGUCAGACGACUCGCUGAGGAUCCGACA
    370 124 GGAGGAGCUACGAUGCGGCCGUCUCGCUCUCAUCCCAUGCACGA AACCUCUCUCCCUGCACAGACGACUCGCUGAGGAUCCGACA
    881 125 GGAGGAGCUACGAUGCGGCCGAUUUCGUCAUCCUCCACACCAUC GCCUUACCGCUCCGCGUCAGACGACUCGCUGAGGAUCCGACA
    150 126 GGAGGAGCUACGAUGCGGCCGAUUUCGUCAUCCUCCAUACCAUC GCCUUACCGUUCCGCUUCAGACGACUCGCUGAGGAUCCGACA
    73 127 GGAGGAGCUACGAUGCGGCCGAUUUCGUCAUCGUCCAUACCAUC GCCUUACCGUUCCGCGUCAGACGACUCGCUGAGGAUCCGACA
    670 128 GGAGGAGCUACGAUGCGGCCGAUUUCGUCAUCCUCCAUACCAUC GCCGUACCGUUCCGCGUCAGACGACUCGCUGAGGAUCCGACA
    263 129 GGAGGAGCUACGAUGCGGCCGAUUUCGUCAUCCUCCAUACCAUC GCCUUACCGUUGCGCGUCAGACGACUCGCUGAGGAUCCGACA
    270 130 GGAGGAGCUACGAUGCGGCCGAUGUCGUCAUCCUCCAUACCAUC GCCUUACCGUUCCGCGUCAGACGACUCGCUGAGGAUCCGACA
    1137 131 GGAGGAGCUACGAUGCGGCCGAUAUCGUCAUCCUCCAUACCAUC GCCUUCCGUUCCGCGUCAGACGACUCGCUGAGGAUCCGACA
    238 132 GGAGGAGCUACGAUGCGGCCGAUUUCGUCAUCCUCCAUACCAUC GCCUUACCGUUCGCGUCAGACGACUCGCUGAGGAUCCGACA
    603 133 GGAGGAGCUACGAUGCGGUGAAUUCUUCCGGCACUUUGUCAUCU UCACCCCAUGCUGCACAGACGACUCGCUGAGGAUCCGACA
    827 134 GGAGGAGCUACGAUGCGGUGAAUUCUUCCGGCACUUUGUCAUCU UCGCCCCCAUGCUGCACAGACGACUCGCUGAGGAUCCGACA
    1192 135 GGAGGAGCUACGAUGCGGCAGGUGCGGGAUCUAAUGCGUAGACA GCCAUAUACUGACACAGACAGACGACUCGCUGAGGAUCCGACA
    117 136 GGAGGAGCUACGAUGCGGCCGUCUCGCUCUCAUCCCAUGCACGA ACCCUCUCUCACUGCACAGACGACUCGCUGAGGAUCCGACA
    448 137 GGAGGAGCUACGAUGCGGCCGAUUGCGUCAUCCUCCAUACCAUC GCCUUACCGUUCCGCGUCAGACGACUCGCUGAGGAUCCGACA
    1739 138 GGAGGAGCUACGAUGCGGUGAAUUCUUCCGGCACUUUGUCAUCU UCACCCCCAUGCUGCUCAGACGACUCGCUGAGGAUCCGACA
    576 139 GGAGGAGCUACGAUGCGGCCGUCUCGCUCUCAUCCCAUGCACGA AACCUCUCUCACUGCGCAGACGACUCGCUGAGGAUCCGACA
    185 140 GGAGGAGCUACGAUGCGGACGGAAGGAUAGUUGCUAAUCGAGCC CUGCCGACGCUUCAGACAGACGACUCGCUGAGGAUCCGACA
    2131 141 GGAGGAGCUACGAUGCGGCAAAAACUGAUAAACACAGGUCCGGCA UUUGAGCGUACACCCAGACAGACGACUCGCUGAGGAUCCGACA
    823 142 GGAGGAGCUACGAUGCGGUGAAUUCUUCCGGCACUUUGUCAUCU UCACCCCCGUGCUGCACAGACGACUCGCUGAGGAUCCGACA
    40 143 GGAGGAGCUACGAUGCGGCCGAUUUCGUCAUCCUCCAUACCUCG CCUUACCGUUCCGCGUCAGACGACUCGCUGAGGAUCCGACA
    38 144 GGAGGAGCUACGAUGCGGCCGAUUUCGUCAUCCUCCAUACCAUC GCCUUACCGUUCCGCCUCAGACGACUCGCUGAGGAUCCGACA
    560 145 GGAGGAGCUACGAUGCGGCCCAUCACUCCCGCGUGUUGCGAACG CAUCGUUAUUUAGCCGUCAGACGACUCGCUGAGGAUCCGACA
    1183 146 GGAGGAGCUACGAUGCGGCUUCCCUAUUCCAAAGGAGGUGCGGU ACGUUUUGUUACGCCAGACAGACGACUCGCUGAGGAUCCGACA
    435 147 GGAGGAGCUACGAUGCGGCCGAUUUCGUCAUCCUCCAUACUAUC GCCCUACCGUUCCGCGUCAGACGACUCGCUGAGGAUCCGACA
    273 148 GGAGGAGCUACGAUGCGGCCGAUUUCGUCAUCCUCCACACCAUC GCCUUACCGUUCCGCAUCAGACGACUCGCUGAGGAUCCGACA
    439 149 GGAGGAGCUACGAUGCGGCCGAUUUCGUCAUCCUCCAUACCAUC GCCUUACCGUGCCGCGUCAGACGACUCGCUGAGGAUCCGACA
    1082 150 GGAGGAGCUACGAUGCGGUGAAUUCUUCCGGCACCUUGUCAUCU UCACCCCCAUGCUGCACAGACGACUCGCUGAGGAUCCGACA
    321 151 GGAGGAGCUACGAUGCGGUGUACCCUGAUUGCCUUUGUGUUAUG AGCGACAGAUCUGCCAGACAGACGACUCGCUGAGGAUCCGACA
    562 152 GGAGGAGCUACGAUGCGGCCCACCACUCCCGCGUAUUGCGAACG CAUCGUUAUUUAGCCGUCAGACGACUCGCUGAGGAUCCGACA
    1735 153 GGAGGAGCUACGAUGCGGUGAAUUCUUCCGGCACUUUGUCGUCU UCACCCCCAUGCUGCACAGACGACUCGCUGAGGAUCCGACA
    106 154 GGAGGAGCUACGAUGCGGUACACUCAGUCACGUAGCACCGCAGU GACCCUUUGUACCGCAGACAGACGACUCGCUGAGGAUCCGACA
    1487 155 GGAGGAGCUACGAUGCGGCCAGCCACACUUUGACCGAAUUGGCA AGCGCGGGCAAAUCGAACAGACGACUCGCUGAGGAUCCGACA
    581 156 GGAGGAGCUACGAUGCGGCCGUCUCGCUCUCAUCCCAUGCACGA CACCUCUCUCACUGCACAGACGACUCGCUGAGGAUCCGACA
    1063 157 GGAGGAGCUACGAUGCGGUCGUCUCGCUCUCAUCCCAUGCACGA AACCUCUCUCACUGCACAGACGACUCGCUGAGGAUCCGACA
    480 158 GGAGGAGCUACGAUGCGGCCGAUUUCGUCAUCCUCCAUACCAUG CCUUACCGUUCCGCGUCAGACGACUCGCUGAGGAUCCGACA
    1061 159 GGAGGAGCUACGAUGCGGCCGUCUCGCUCUCCUCCCAUGCACGA AACCUCUCUCACUGCACAGACGACUCGCUGAGGAUCCGACA
    1479 160 GGAGGAGCUACGAUGCGGGCUGUGCCGGCCCUGCUCUGGUCGC CAUUGUCAGUCUGUGCAGACAGACGACUCGCUGAGGAUCCGACA
    1392 161 GGAGGAGCUACGAUGCGGUGAAUUCUCCCGGCACUUUGUCAUCU UCACCCCCAUGCUGCACAGACGACUCGCUGAGGAUCCGACA
    225 162 GGAGGAGCUACGAUGCGGACCUUGUUUUUCCUCUGUACCCCACU UCCCCAUUUCUCCCUGCUCAGACGACUCGCUGAGGAUCCGACA
    1856 163 GGAGGAGCUACGAUGCGGAUUAUUGUUUGACGUAUUCCAAGUGA GAUUACGCACGCACCAGACAGACGACUCGCUGAGGAUCCGACA
    269 164 GGAGGAGCUACGAUGCGGCCGAUAUCGUCAUCCUCCAUACCAUC GCCUUACCGUCCCGCGUCAGACGACUCGCUGAGGAUCCGACA
    829 165 GGAGGAGCUACGAUGCGGUGAAUUCUUCCGGCACUUUGUCAUCU UCCCCCCAUGCUGCACAGACGACUCGCUGAGGAUCCGACA
    800 166 GGAGGAGCUACGAUGCGGCCGUCUCGCUCUUAUCCCAUGCACGA AACCUCUCUCACUGCACAGACGACUCGCUGAGGAUCCGACA
    389 167 GGAGGAGCUACGAUGCGGUGAAUUCUUCCGGCACUUUGUCAUCU UCACCCUCAUGCUGCACAGACGACUCGCUGAGGAUCCGACA
    28 168 GGAGGAGCUACGAUGCGGCCCAUCACUCCCGCGUAUUGCGAACG CAUCGUUGUUUAGCCGUCAGACGACUCGCUGAGGAUCCGACA
    1737 169 GGAGGAGCUACGAUGCGGUGAAUUCUUCCGGCACUUUGUCAUCC UCACCCCCAUGCUGCACAGACGACUCGCUGAGGAUCCGACA
    1052 170 GGAGGAGCUACGAUGCGGCCCAUCACUCCCACGUAUUGCGAACG CAUCGUUAUUUAGCCGUCAGACGACUCGCUGAGGAUCCGACA
    405 171 GGAGGAGCUACGAUGCGGCCGAUUUCGUCAUCCUCCAUACCAUC GCCUUACCGUUUCCGCGUCAGACGACUCGCUGAGGAUCCGACA
    317 172 GGAGGAGCUACGAUGCGGGGAAGCAACACUUAGUCGGGAUUGAU ACGUGCCCAGUCAGCAGUCAGACGACUCGCUGAGGAUCCGACA
    1716 173 GGAGGAGCUACGAUGCGGCCGAUCACUCCCGCGUAUUGCGAACG CAUCGUUAUUUAGCCGUCAGACGACUCGCUGAGGAUCCGACA
    623 174 GGAGGAGCUACGAUGCGGCCGAAUUUCGUCAUCCUCCAUACCAU CGCCUUACCGUUCCGCGUCAGACGACUCGCUGAGGAUCCGACA
    305 175 GGAGGAGCUACGAUGCGGCCGAUUUCGUCAUCCUCCAUACCAUC GCUUACCGUUCCGCGUCAGACGACUCGCUGAGGAUCCGACA
    686 176 GGAGGAGCUACGAUGCGGCCGAUUUCGUCAUCCUCCAUACCAUC GCCCACCGUUCCGCGUCAGACGACUCGCUGAGGAUCCGACA
    151 177 GGAGGAGCUACGAUGCGGCCGAUUUCGUCAUCCUCCAUACCAUC GCCUUACCGUUCCGUCAGACGACUCGCUGAGGAUCCGACA
    178 178 GGAGGAGCUACGAUGCGGGGAAGCACCACUUAGUCGCGAUUGAU ACGUGCGCAGUCAUCAGACAGACGACUCGCUGAGGAUCCGACA
    1085 179 GGAGGAGCUACGAUGCGGUGAAUUCUUCCGGCACUUUGUCAUCU UCACGCCCAUGCUGCACAGACGACUCGCUGAGGAUCCGACA
    1428 180 GGAGGAGCUACGAUGCGGCCGAUUUCGUCAUCCUCCAAGCCAUC GCCUUACCGUUCCGCGUCAGACGACUCGCUGAGGAUCCGACA
    1401 181 GGAGGAGCUACGAUGCGGUGAAUUCUUCCGACACUUUGUCAUCU UCACCCCCAUGCUGCACAGACGACUCGCUGAGGAUCCGACA
    1 182 GGAGGAGCUACGAUGCGGGGAAGCCACACUUAGUCGCGAUUGAU ACGUGCGCAGUCAUCAGACAGACGACUCGCUGAGGAUCCGACA
    799 183 GGAGGAGCUACGAUGCGGCCGUCUCGUUCUCAUCCCAUGCACGA AACCUCUCUCACUGCACAGACGACUCGCUGAGGAUCCGACA
    98 184 GGAGGAGCUACGAUGCGGACGGAGGAUAGUUGCUAAUCGAGCCC UGCCGACGCUUCAGUCAGACGACUCGCUGAGGAUCCGACA
    550 185 GGAGGAGCUACGAUGCGGACGGUUUCACCUCUAGGAGCACUGAA AGCCAACCUUCGCGCACAGACGACUCGCUGAGGAUCCGACA
    2279 186 GGAGGAGCUACGAUGCGGUGAAUUCCUCCGGCACUUUGUCAUCU UCACCCCCAUGCUGCACAGACGACUCGCUGAGGAUCCGACA
    2047 187 GGAGGAGCUACGAUGCGGCCGAUUUCGUCAUCCUCCACAUCAUC GCCUUACCGUUCCGCGUCAGACGACUCGCUGAGGAUCCGACA
    490 188 GGAGGAGCUACGAUGCGGCCGAUUUCGUCAUCCCCCAUACCAUC GCCUUACCUUCCGCGUCAGACGACUCGCUGAGGAUCCGACA
    606 189 GGAGGAGCUACGAUGCGGUGAAUUCUUCCGGCACUUGUCAUCUU CACCCCCAUGCUGCACAGACGACUCGCUGAGGAUCCGACA
    2019 190 GGAGGAGCUACGAUGCGGCCGUCUCGCUCUCAUCCCAUGCGCGA AACCUCUCUCACUGCACAGACGACUCGCUGAGGAUCCGACA
    1393 191 GGAGGAGCUACGAUGCGGUGAAUUCUUCCGGCACUUUGUCAUCU UCACCCCUAUGCUGCACAGACGACUCGCUGAGGAUCCGACA
    678 192 GGAGGAGCUACGAUGCGGCCGAUUUUCGUCAUCCUCCAUACCAU CGCCCUACCGUUCCGCGUCAGACGACUCGCUGAGGAUCCGACA
    1051 193 GGAGGAGCUACGAUGCGGCCCAUCACUCCCGCGUAUUGCGAACG CAUCGUUAUUUAGCUGUCAGACGACUCGCUGAGGAUCCGACA
    109 194 GGAGGAGCUACGAUGCGGCCCAUCGCUCCCGCGUAUUGCGAACG CAUCGUUAUUUAGCCGUCAGACGACUCGCUGAGGAUCCGACA
    145 195 GGAGGAGCUACGAUGCGGCCGAUUUCGGCAUCCUCCACACCAUC GCCUUACCGUUCCGCGUCAGACGACUCGCUGAGGAUCCGACA
    469 196 GGAGGAGCUACGAUGCGGCCGAUUUCGUCAUCCUCCAUCAUCGC CUUACCGUUCCGCGUCAGACGACUCGCUGAGGAUCCGACA
    1076 197 GGAGGAGCUACGAUGCGGUGAACUCUUCCGGCACUUUGUCAUCU UCACCCCCAUGCUGCACAGACGACUCGCUGAGGAUCCGACA
    1373 198 GGAGGAGCUACGAUGCGGCCCAUCACUCCCGCGUAUUGCGAACG CAUCGUUAUUCAGCCGUCAGACGACUCGCUGAGGAUCCGACA
    2272 199 GGAGGAGCUACGAUGCGGCCGUCUCGCUCUCAUCCCAUGCACAA AACCUCUCUCACUGCACAGACGACUCGCUGAGGAUCCGACA
    1100 200 GGAGGAGCUACGAUGCGGCCGAUUUCGUCAUCCCCCAUACCAUC GCCUUACCGUUCCGCAGUCAGACGACUCGCUGAGGAUCCGACA
    452 201 GGAGGAGCUACGAUGCGGCCGAUUUCGUCAUCCUCCACACCAUC GCCUUACUGUUCCGCGUCAGACGACUCGCUGAGGAUCCGACA
    1720 202 GGAGGAGCUACGAUGCGGCCGUCUCGCUCUCAUCCCAUGCACGA AAUCUCUCUCACUGCACAGACGACUCGCUGAGGAUCCGACA
    1374 203 GGAGGAGCUACGAUGCGGCCCAUCACUCCCGCGUAUUGCGAACG CAUCGCUAUUUAGCCGUCAGACGACUCGCUGAGGAUCCGACA
    283 204 GGAGGAGCUACGAUGCGGCCGAUUUCGUCAUCCUCAUACCAUCG CCUUACCGUUCCGCGUCAGACGACUCGCUGAGGAUCCGACA
    1724 205 GGAGGAGCUACGAUGCGGACCUUGUUUCCCUCUGUACCCCACUU CCCCAUUUCUCCCUGCUCAGACGACUCGCUGAGGAUCCGACA
    1083 206 GGAGGAGCUACGAUGCGGUGAAUUCUUCCGGCACUUUGUCAUCU CCACCCCCAUGCUGCACAGACGACUCGCUGAGGAUCCGACA
    2282 207 GGAGGAGCUACGAUGCGGUCGAUUUCGUCAUCCUCCAUACCAUC GCCUUACUGUUCCGCGUCAGACGACUCGCUGAGGAUCCGACA
    663 208 GGAGGAGCUACGAUGCGGCCGAUUUCGUCAUCCUCCAUACCAUC GUCUUACCUUUCCGCGUCAGACGACUCGCUGAGGAUCCGACA
    172 209 GGAGGAGCUACGAUGCGGCCGAUUUCGUCAUCCUCCAUACCAUC GCCUUACGUUCCGCGUCAGACGACUCGCUGAGGAUCCGACA
    153 210 GGAGGAGCUACGAUGCGGCCGAUUUCGUCAUCCUCCAUACCAUC GCCUUACCACUCCGCGUCAGACGACUCGCUGAGGAUCCGACA
    324 211 GGAGGAGCUACGAUGCGGACGGAGGAUAGUUGCUAAUCGAGCCC UGCUGACGCUUCAGACAGACGACUCGCUGAGGAUCCGACA
    2132 212 GGAGGAGCUACGAUGCGGUGUACACUGAUUGCCUUUGUGUUAUG GGCGACAGAUCUGCCAGACAGACGACUCGCUGAGGAUCCGACA
    1390 213 GGAGGAGCUACGAUGCGGUGAAUCCUUCCGGCACUUUGUCAUCU UCACCCCCAUGCUGCACAGACGACUCGCUGAGGAUCCGACA
    1400 214 GGAGGAGCUACGAUGCGGUGAAUUCUUCCGGCACUUUGCCAUCU UCACCCCCAUGCUGCACAGACGACUCGCUGAGGAUCCGACA
    1380 215 GGAGGAGCUACGAUGCGGACCUCGUUUUCCUCUGUACCCCACUU CCCCAUUUCUCCCUGCUCAGACGACUCGCUGAGGAUCCGACA
    1721 216 GGAGGAGCUACGAUGCGGCCGUCUCGCUCUCAUCCCAUGCACGA AACCUCUCUAACUGCACAGACGACUCGCUGAGGAUCCGACA
    375 217 GGAGGAGCUACGAUGCGGCCGUCUCGCUCUCAUCCCAUGCACGA AACCUCCCUCACUGCACAGACGACUCGCUGAGGAUCCGACA
    1064 218 GGAGGAGCUACGAUGCGGCCGUCUCGCUCUCAUCCCAUGCACGA AACCUCUCUCACCGCACAGACGACUCGCUGAGGAUCCGACA
    787 219 GGAGGAGCUACGAUGCGGCCCAUCACUCCCGCGUAUCGCGAACG CAUCGUUAUUUAGCCGUCAGACGACUCGCUGAGGAUCCGACA
    848 220 GGAGGAGCUACGAUGCGGCCGAUUUUUCGUCAUCCUCCAUACCA UCGCCUUACCGUUCCGCGUCAGACGACUCGCUGAGGAUCCGACA
    575 221 GGAGGAGCUACGAUGCGGCCGUCUCGCUCUCAUCCCAUGCACGA AACCCCUCUCACUGCACAGACGACUCGCUGAGGAUCCGACA
    240 222 GGAGGAGCUACGAUGCGGCAGAUUUCGUCAUCAUCCAUACCAUC GCCUUACCGUUCCGCGUCAGACGACUCGCUGAGGAUCCGACA
    210 223 GGAGGAGCUACGAUGCGGCCCAUCACUCCCGCGCACUGCGAACG CAUCGUUAUUUAGCCGUCAGACGACUCGCUGAGGAUCCGACA
    351 224 GGAGGAGCUACGAUGCGGCCCAUCCCUCCCGCGUAUUGCGAACG CCUCGUUAUUUAGCCGUCAGACGACUCGCUGAGGAUCCGACA
    554 225 GGAGGAGCUACGAUGCGGAAUCUCCCGAACGCAUUAGUCAGUCC CAUACCCGUGUGCCGCGUCAGACGACUCGCUGAGGAUCCGACA
    789 226 GGAGGAGCUACGAUGCGGCCCAUCACUCCCGUGUAUUGCGAACG CAUCGUUAUUUAGCCGUCAGACGACUCGCUGAGGAUCCGACA
    288 227 GGAGGAGCUACGAUGCGGCCGAUUUCGUCAUCCUCCAACCAUCG CCUUACCGUUCCGCGUCAGACGACUCGCUGAGGAUCCGACA
    785 228 GGAGGAGCUACGAUGCGGCCCAUCACUCCCGCGUAUUGCGAACG CAUCGUUAUCUAGCCGUCAGACGACUCGCUGAGGAUCCGACA
    1430 229 GGAGGAGCUACGAUGCGGACGAUUUCGUCAUCCUCCAUACCAUC GCCUUACCGUUCCGAGUCAGACGACUCGCUGAGGAUCCGACA
    1053 230 GGAGGAGCUACGAUGCGGCCCAUCACUCCCGCGUAUUGCGAACG UAUCGUUAUUUAGCCGUCAGACGACUCGCUGAGGAUCCGACA
    2158 231 GGAGGAGCUACGAUGCGGAUUACCAACUUGAACGCCGAGAGUGU GGUCAUGUGUUCUGCAGACAGACGACUCGCUGAGGAUCCGACA
    892 232 GGAGGAGCUACGAUGCGGCCGAUUUUCGUCAUCCUCCAUGCCAU CGCCUUACCGUUCCGCGUCAGACGACUCGCUGAGGAUCCGACA
    596 233 GGAGGAGCUACGAUGCGGUGGAUUCUUCCGGCACUUUGUCAUCU UCACCCCCAUGCUGCACAGACGACUCGCUGAGGAUCCGACA
    454 234 GGAGGAGCUACGAUGCGGCCGAUUUCGUCAUCCUCCACACCAUC GCCUUACCCUUCCGCGUCAGACGACUCGCUGAGGAUCCGACA
    1763 235 GGAGGAGCUACGAUGCGGCCGAUUUCGUCAUCCUCCAUACCAUC GUCUUACCGUUCUGCGUCAGACGACUCGCUGAGGAUCCGACA
    605 236 GGAGGAGCUACGAUGCGGUGAAUUCUUCCGGCACUUUGUCAUCU UCACCCCCCAUGCUGCACAGACGACUCGCUGAGGAUCCGACA
    1073 237 GGAGGAGCUACGAUGCGGACCUUGUUUUCCUCUGUACCCCACUU CCCAUUUCUCCCUGCUCAGACGACUCGCUGAGGAUCCGACA
    791 238 GGAGGAGCUACGAUGCGGCCCAUCACUCCCGCGUAUUGCGAGCG CAUCGUUAUUUAGCCGUCAGACGACUCGCUGAGGAUCCGACA
    77 239 GGAGGAGCUACGAUGCGGCCGAUUUCGUCAUCCUCCAUACCAUC GCCUUACCGUUCCGAGACAGACGACUCGCUGAGGAUCCGACA
    568 240 GGAGGAGCUACGAUGCGGCCCAUCACUCCCGCGAAUUGCGAACG CAUCGUUAUUUAGCCGUCAGACGACUCGCUGAGGAUCCGACA
    803 241 GGAGGAGCUACGAUGCGGCCGUCUCGCUCCCAUCCCAUGCACGA AACCUCUCUCACUGCACAGACGACUCGCUGAGGAUCCGACA
    571 242 GGAGGAGCUACGAUGCGGCCCAUCACUCCCGCGUAUUGCGAACG CAUCGUUAUUAGCCGUCAGACGACUCGCUGAGGAUCCGACA
    585 243 GGAGGAGCUACGAUGCGGACCUUGUUUUCCUCCGUACCCCACUU CCCCAUUUCUCCCUGCUCAGACGACUCGCUGAGGAUCCGACA
    851 244 GGAGGAGCUACGAUGCGGCUGAUUUCGUCAUCCCCCAUACCAUC GCCUUACCGUUCCGCGUCAGACGACUCGCUGAGGAUCCGACA
    601 245 GGAGGAGCUACGAUGCGGUGAAUUCUUCCGGCACUUUGUCAUCU UCACCCCCAUGCGGCACAGACGACUCGCUGAGGAUCCGACA
    706 246 GGAGGAGCUACGAUGCGGACGGAGGAUAGUUGCUAAUCGAGCCC UGCGGACGCUUCAGACAGACGACUCGCUGAGGAUCCGACA
    1391 247 GGAGGAGCUACGAUGCGGUGAAUUCUUCCGGCACUUUGUCAUCU UCACCCCCAGGCUGCACAGACGACUCGCUGAGGAUCCGACA
    471 248 GGAGGAGCUACGAUGCGGCCGAUUUCGUAUCCUCCGUACCAUCG CCUUACCGUUCCGCGUCAGACGACUCGCUGAGGAUCCGACA
    116 249 GGAGGAGCUACGAUGCGGCCGUCUCGAUCUCAUCCCAUGCACGA AACCUCUCUCACUGCACAGACGACUCGCUGAGGAUCCGACA
    47 250 GGAGGAGCUACGAUGCGGCCGAUUUCGUCAUCCUCCAUACCAUC GCCUCCGUUCCGCGUCAGACGACUCGCUGAGGAUCCGACA
  • As shown in FIGS. 2 , HT-Toggle-cluster SELEX was used to isolate islet-specific aptamers crossreacting between mouse and human. 8 cycles of HT-cluster SELEX were performed as described in FIG. 1 using as negative and positive selectors mouse acinar tissues and mouse islets respectively (FIG. 2A). The resulting polyclonal aptamer from cycle 8 was used for two additional cycle of selection using either acinar tissue and islets from mice or acinar tissue and islets isolated from cadaveric donors (FIG. 2B). The resulting polyclonal aptamer library underwent HT-sequencing and bio-informatic analysis (FIG. 2C) to determine the frequency of each monoclonal aptamer present in the library selected using mouse or human tissues. Monoclonal aptamers (Table 3) enriched in the human library (putative aptamers against human islets, rectangular selection) were chosen for empirical testing. Table 3 provides also putative aptamers against human islets.
  • TABLE 3
    aptamer sequences specific for human islets
    name SEQ ID NO Sequence
    166-279 251 GGAGGACGAUGCGGCCGAUUUCGUCAUCCUCCAUACC AUCGCCUUACCGUUCCGCGUCAGACGACUCGCUGAGG AUCCGAGA
    109-2031 252 GGAGGACGAUGCGGUGAAUUCUUCCGGCACUUUGUCA UCUUCACCCCCAUGCUGCACAGACGACUCGCUGAGGAU CCGAGA
    208-2529 253 GGAGGACGAUGCGGCCGUCUCGCUCUCAUCCCAUGCA CGAAACCUCUCUCACUGCACAGACGACUCGCUGAGGAU CCGAGA
    64-2437 254 GGAGGACGAUGCGGCCCAUCACUCCCGCGUAUUGCGA ACGCAUCGUUAUUUAGCCGUCAGACGACUCGCUGAGG AUCCGAGA
    173-2273 255 GGAGGACGAUGCGGACCUUGUUUUCCUCUGUACCCCA CUUCCCCAUUUCUCCCUGCUCAGACGACUCGCUGAGGA UCCGAGA
    12-2617 256 GGAGGACGAUGCGGUGUACACUGAUUGCCUUUGUGU UAUGAGCGACAGAUCUGCCAGACGACUCGCUGAGGAU CCGAGA
    107-901 257 GGAGGACGAUGCGGGGAAGCAACACUUAGUCGCGAUU GAUACGUGCGCAGUCAUCAGACGACUCGCUGAGGAUC CGAGA
    155-1103 258 GGAGGACGAUGCGGCCGAUUUUCGUCAUCCUCCAUAC CAUCGCCUUACCGUUCCCAGACGACUCGCUGAGGAUCC GAGA
    1-717 259 GGAGGACGAUGCGGUAAUUCUCAGGAGGUGCGGAAC GGGAUAUGGAUUGUUCGCCAGACGACUCGCUGAGGAU CCGAGA
    m1-2623 260 GGAGGACGAUGCGGUACACUCAGUCACGUAGCACCGC AGUGACCCUUUGUACCGCAGACGACUCGCUGAGGAUC CGAGA
    m5-3229 261 GGAGGACGAUGCGGCCUAGUACAAAAGCCUGAUCUCU
    GUGAGCAGACACUAGAACAGACGACUCGCUGAGGAUC CGAGA
    m7-2539 262 GGAGGACGAUGCGGAUUACCAACUUGAACGCCGAGAG UGUGGUCACGUGUUCUGCAGACGACUCGCUGAGGAUC CGAGA
    m9-3076 263 GGAGGACGAUGCGGGGAAGCAACACUUAGUCGCGAUU GAUACGUGCGCAGUCAUCAGACGACUCGCUGAGGAUC CGAGA
    m12-3773 264 GGAGGACGAUGCGGCAACAAACUAAUCAGACACGAGAC AGAGAGAUAGAUCUGCCAGACGACUCGCUGAGGAUCC GAGA
    m24-3219 265 GGAGGACGAUGCGGCAGGUGCGGGAUCUAAUGCGUA GACAGCCAUAUACUGACACAGACGACUCGCUGAGGAUC CGAGA
  • TABLE 4
    Putative human islet specific aptamers isolated via toggle-cluster SELEX (from FIGS. 2 )
    Putative human islet specific aptamers isolated via toggle-cluster SELEX (from FIGS. 2 )
    aptamer name SEQ ID NO sequence
    m2-1 266 GGAGGAGCUACGAUGCGGCAGGUGCGGGGUCUAAUGCGUAGACAG CCAUAUACUGACACAGACAGACGACUCGCUGAGGAUCCGACA
    m2-2 267 GGAGGAGCUACGAUGCGGCAGGGGCGGGGUCUAAUGCGUAGACAG CCAUAUACUGACACAGACAGACGACUCGCUGAGGAUCCGACA
    m322-3 268 GGAGGAGCUACGAUGCGGGGAAGCAACACUUAGUCGCGAUUGAUGC GUGCGCAGUCAUCAGACAGACGACUCGCUGAGGAUCCGACA
    m323-4 269 GGAGGAGCUACGAUGCGGGGAAGCAACACUUAGUCGCGAUUGAUAU GUGCGCAGUCAUCAGACAGACGACUCGCUGAGGAUCCGACA
    m630-5 270 GGAGGAGCUACGAUGCGGGGAAGCAACACUUAGUCGCGAUUGAUAC GUGCGUAGUCAUCAGACAGACGACUCGCUGAGGAUCCGACA
    m631-6 271 GGAGGAGCUACGAUGCGGGGAAGCAACACUUAGUCGCGAUUGGUAC GUGCGCAGUCAUCAGACAGACGACUCGCUGAGGAUCCGACA
    m635-7 272 GGAGGAGCUACGAUGCGGGGAAGCAACACUUAGUCGCGAUUGAUAC GUGCGCGGUCAUCAGACAGACGACUCGCUGAGGAUCCGACA
    m636-8 273 GGAGGAGCUACGAUGCGGGGAAGCAACGCUUAGUCGCGAUUGAUAC GUGCGCAGUCAUCAGACAGACGACUCGCUGAGGAUCCGACA
    m685-9 274 GGAGGAGCUACGAUGCGGUAAUUCUCAGGAGGUGCGGAACGGGAU AUGGUUUGUUCGCCAGUCAGACGACUCGCUGAGGAUCCGACA
    m703-10 275 GGAGGAGCUACGAUGCGGUAAUUCUCAGGAGGUGCGGAACGGGAU AUGGAUUGUCCGCCAGUCAGACGACUCGCUGAGGAUCCGACA
    m705-11 276 GGAGGAGCUACGAUGCGGUAAUUCUCAGGAGGUGCGGAACGGGAU AUGGAAUGUUCGCCAGUCAGACGACUCGCUGAGGAUCCGACA
    m706-12 277 GGAGGAGCUACGAUGCGGUAAUUCUCAGGAGGUGCGGAGCGGGAU AUGGAUUGUUCGCCAGUCAGACGACUCGCUGAGGAUCCGACA
    m1024-13 278 GGAGGAGCUACGAUGCGGACCAUCGCUCCCGCGUAUUGCGAACGCA UCGUUAUUUAGCCGUCAGACGACUCGCUGAGGAUCCGACA
    m1028-14 279 GGAGGAGCUACGAUGCGGCCCAUCACUCCCGCGUAUUGCGUACGCA UCGUUAUUUAGCCGUCAGACGACUCGCUGAGGAUCCGACA
    m1146-15 280 GGAGGAGCUACGAUGCGGCCGGAGGCAGUCACUAAUCUUCACUUCC CUUAGACAUGCGCAGACAGACGACUCGCUGAGGAUCCGACA
    m1157-16 281 GGAGGAGCUACGAUGCGGGGAAGCAACACUUAGUCGCGAUUGAUAC GUGUGCAGUCAUCAGACAGACGACUCGCUGAGGAUCCGACA
    m1161-17 282 GGAGGAGCUACGAUGCGGGGAAGCAACAUUUAGUCGCGAUUGAUAC GUGCGCAGUCAUCAGACAGACGACUCGCUGAGGAUCCGACA
    m1164-18 283 GGAGGAGCUACGAUGCGGGGAGGCAACACUUAGUCGCGAUUGAUAC GUGCGCAGUCAUCAGACAGACGACUCGCUGAGGAUCCGACA
    m1164-19 284 GGAGGAGCUACGAUGCGGGGGAGCAACACUUAGUCGCGAUUGAUAC GUGCGCAGUCAUCAGACAGACGACUCGCUGAGGAUCCGACA
    m1166-20 285 GGAGGAGCUACGAUGCGGGGAAGCAAUACUUAGUCGCGAUUGAUAC GUGCGCAGUCAUCAGACAGACGACUCGCUGAGGAUCCGACA
    m1170-21 286 GGAGGAGCUACGAUGCGGGGAAGCAACACUUAGUCGGGAUUGAUAC GUGCCCAGUCAUCAGACAGACGACUCGCUGAGGAUCCGACA
    m1200-22 287 GGAGGAGCUACGAUGCGGCCGUCUCGCUCUCAUCCCAUGCACGACA CCUCUCUCACUGCACAGACGACUCGCUGAGGAUCCGACA
    m1233-23 288 GGAGGAGCUACGAUGCGGUAAUUCUCAGGAGGUACGGAACGGGAUA UGGAUUGUUCGCCAGUCAGACGACUCGCUGAGGAUCCGACA
    m1234-24 289 GGAGGAGCUACGAUGCGGUAAUUCUCAGGAGGUGCGGAACGGGAU AUGGAUGGUUCGCCAGUCAGACGACUCGCUGAGGAUCCGACA
    m1246-25 290 GGAGGAGCUACGAUGCGGUAAUUCUCAGGAGGUGCGGAACGGGAU AUGGGUUGUUCGCCAUACAGACGACUCGCUGAGGAUCCGACA
    m1259-26 291 GGAGGAGCUACGAUGCGGUAAUUCCCAGGAGGUGCGGAACGGGAU AUGGAUUGUUCGCCAGUCAGACGACUCGCUGAGGAUCCGACA
    m1260-27 292 GGAGGAGCUACGAUGCGGUAAUUCACAGGAGGUGCGGAACGGGAU AUGGAUUGUUCGCCAGUCAGACGACUCGCUGAGGAUCCGACA
    m1303-28 293 GGAGGAGCUACGAUGCGGCCGAUUGCGUCAUCCUCCAUACCAUCGC CUUACCGUUCCGCGUCAGACGACUCGCUGAGGAUCCGACA
    m1617-29 294 GGAGGAGCUACGAUGCGGCCCAUCACUCACGCGUAGUGCGAACGCA UCGUUAUUUAGCCGUCAGACGACUCGCUGAGGAUCCGACA
    m1684-30 295 GGAGGAGCUACGAUGCGGUGUACACUGAUUGCCUUUGGGUUAAGA GCGACAGAUCCGGCAGACAGACGACUCGCUGAGGAUCCGACA
    m1721-31 296 GGAGGAGCUACGAUGCGGGGAAGCGACACUUAGUCGCGAUUGAUAC GUGCGCAGUCAUCAGACAGACGACUCGCUGAGGAUCCGACA
    m1723-32 297 GGAGGAGCUACGAUGCGGGGAAGCAACACUUAGUCGGGAUUGAUAC GUGCCCAGUCAGCAGACAGACGACUCGCUGAGGAUCCGACA
    m1793-33 298 GGAGGAGCUACGAUGCGGUAAUUCUCAGGAGGCGCGGAACGGGAU AUGGAUUGUUCGCCAGUCAGACGACUCGCUGAGGAUCCGACA
    m1794-34 299 GGAGGAGCUACGAUGCGGUAAUUCUCAGGAGGAGCGGAACGGGAU AUGGAUUGUUCGCCAGUCAGACGACUCGCUGAGGAUCCGACA
    m1800-35 300 GGAGGAGCUACGAUGCGGUAAUUCUCAGGAGGUGCGGAACGGGAU ACGGAUUGUUCGCCAGUCAGACGACUCGCUGAGGAUCCGACA
    m1800-36 301 GGAGGAGCUACGAUGCGGUAAUUCUCAGGAGGUGCGGAACGGGAU AAGGAUUGUUCGCCAGUCAGACGACUCGCUGAGGAUCCGACA
    m1808-37 302 GGAGGAGCUACGAUGCGGUAAUUCUCAGGAGGUGCGGAACGGGAU AUGGAUUGCUCGCCAGUCAGACGACUCGCUGAGGAUCCGACA
    m1809-38 303 GGAGGAGCUACGAUGCGGUAAUUCUCAGGAGGUGCGGAACGGGAU AUGGAUUAUUCGCCAGUCAGACGACUCGCUGAGGAUCCGACA
    m1810-39 304 GGAGGAGCUACGAUGCGGUAAUUCUCAGGAGGUGCGGAACGGGAU AUGGAUUGUGCGCCAGUCAGACGACUCGCUGAGGAUCCGACA
    m1811-40 305 GGAGGAGCUACGAUGCGGUAAUUCUCAGGAGGUGCGGAACGGGAU AUGGAUUGUACGCCAGUCAGACGACUCGCUGAGGAUCCGACA
    m1812-41 306 GGAGGAGCUACGAUGCGGUAAUUCUCAGGAGGUGCGGAACGGGAU AUGGAUUGUUCGCAAGUCAGACGACUCGCUGAGGAUCCGACA
    m1820-42 307 GGAGGAGCUACGAUGCGGUAAUUCUCAGGAGGUGCGGAAUGGGAU AUGGAUUGUUCGCCAGUCAGACGACUCGCUGAGGAUCCGACA
    m1823-43 308 GGAGGAGCUACGAUGCGGUAAUUCGCAGGAGGUGCGGAACGGGAU AUGGAUUGUUCGCCAGACAGACGACUCGCUGAGGAUCCGACA
    m2124-44 309 GGAGGAGCUACGAUGCGGCCCAUCACUCCCGCGUAUUGCGACCGCA UCGUUAUUUAGCCGUCAGACGACUCGCUGAGGAUCCGACA
    m2149-45 310 GGAGGAGCUACGAUGCGGAUUACCAACUUGAACGCCGAAAGUGGGG UCACGUUUUCCGCAGACAGACGACUCGCUGAGGAUCCGACA
    m2219-46 311 GGAGGAGCUACGAUGCGGGGAAGCAACACUUAGUCGCGAUUGAUAC GUGCGCAGUCAUCGGACAGACGACUCGCUGAGGAUCCGACA
    m2272-47 312 GGAGGAGCUACGAUGCGGUAAUUCUCAGGUGGUGCGGAACGGGAU AUGGAUUGUUCGCCAGUCAGACGACUCGCUGAGGAUCCGACA
    m2284-48 313 GGAGGAGCUACGAUGCGGUGAUUCUCAGGAGGUGCGGAACGGGAU AUGGAUUGUUCGCCAGUCAGACGACUCGCUGAGGAUCCGACA
    m2288-49 314 GGAGGAGCUACGAUGCGGUAAUUCUCAGGAGGUGCGGAACGGGAU AUGGGUUGUUCGCCAGUCAGACGACUCGCUGAGGAUCCGACA
    m2502-50 315 GGAGGAGCUACGAUGCGGCCCAUCACUCCCGCGUAUUGCGAACGCA UCGUUAUUUAGCCAGACAGACGACUCGCUGAGGAUCCGACA
    m2514-51 316 GGAGGAGCUACGAUGCGGCCCAUCACUCACGCGAAUUGCGAACGCA UCGUUAUUUAGCCGUCAGACGACUCGCUGAGGAUCCGACA
    m2548-52 317 GGAGGAGCUACGAUGCGGCGCAUCACUCCCGCGUAUUGCGAACGCA UCGUUAUUUAGCCGUCAGACGACUCGCUGAGGAUCCGACA
    m2569-53 318 GGAGGAGCUACGAUGCGGAUUACCAACUUGAACGCCGAGAGUGUGG UCACGUGUUCUGCAGACAGACGACUCGCUGAGGAUCCGACA
    m2578-54 319 GGAGGAGCUACGAUGCGGCACAUACUGACAAUGGUUACCAGAGCAG GUCCGGCACAUCCAGACAGACGACUCGCUGAGGAUCCGACA
    m2581-55 320 GGAGGAGCUACGAUGCGGUUACGCGUUUAAGUCAUUGACGCGUUAC ACUGGAGGGGGCCAGACAGACGACUCGCUGAGGAUCCGACA
    m2623-56 321 GGAGGAGCUACGAUGCGGUACACUCAGUCACGUAGCACCGCAGUGA CCCUUUGUACCGCAGACAGACGACUCGCUGAGGAUCCGACA
    m2675-57 322 GGAGGAGCUACGAUGCGGGGAAGCAACACUUAGUCGUGAUUGAUAC GUGCGCAGUCAUCAGACAGACGACUCGCUGAGGAUCCGACA
    m2708-58 323 GGAGGAGCUACGAUGCGGUAAUUCUCGGGAGGUGCGGAACGGGAU AUGGAUUGUUCGCCAGUCAGACGACUCGCUGAGGAUCCGACA
    m2715-59 324 GGAGGAGCUACGAUGCGGUAAUUCUCAGGAGGUGCAGAACGGGAUA UGGAUUGUUCGCCAGUCAGACGACUCGCUGAGGAUCCGACA
    m2726-60 325 GGAGGAGCUACGAUGCGGUAAUUCUCAGGAGGUGCGGAACGGGAU AUGGAUUGUUGGCCAGUCAGACGACUCGCUGAGGAUCCGACA
    m2728-61 326 GGAGGAGCUACGAUGCGGUAAUUCUCAGGAGGUGCGGAACGGGAU AUGGAUUGUUAGCCAGUCAGACGACUCGCUGAGGAUCCGACA
    m2856-62 327 GGAGGAGCUACGAUGCGGCGGAUCACUCCCGCGUAUUGCGAACGC AUCGUUAUUUAGCCGUCAGACGACUCGCUGAGGAUCCGACA
    m2908-63 328 GGAGGAGCUACGAUGCGGCCCAUCACUCCCGCGUAUUGCGAACGCA UCGUUAUUGAGCCGUCAGACGACUCGCUGAGGAUCCGACA
    m2913-64 329 GGAGGAGCUACGAUGCGGCCCAUCACUCGCGCGUAUUGCGAACGCA UAGUUAUUUAGCCGUCAGACGACUCGCUGAGGAUCCGACA
    m2929-65 330 GGAGGAGCUACGAUGCGGCAACAAACUAAUCAGACACGAGGCAGAA AGAUAGGUCCGGCAGACAGACGACUCGCUGAGGAUCCGACA
    m2951-66 331 GGAGGAGCUACGAUGCGGUGUAGCGAGAAUCGCGUUGUUGGGUGG UCUGUUGUCAGACGACUCGCUGAGGAUCCGACA
    m3075-67 332 GGAGGAGCUACGAUGCGGGGAAGCAACACUUAGUCGCGAUUGAUAC GUGCGCAGUUAUCAGACAGACGACUCGCUGAGGAUCCGACA
    m3076-68 333 GGAGGAGCUACGAUGCGGGGAAGCAACACUUAGUCGCGAUUGAUAC GUGCGCAGUCAUCAGACAGACGACUCGCUGAGGAUCCGACA
    m3076-69 334 GGAGGAGCUACGAUGCGGUGAAGCAACACUUAGUCGCGAUUGAUAC GUGCGCAGUCAUCAGACAGACGACUCGCUGAGGAUCCGACA
    m3076-70 335 GGAGGAGCUACGAUGCGGGGAAGCAACACUUGGUCGCGAUUGAUAC GUGCGCAGUCAUCAGACAGACGACUCGCUGAGGAUCCGACA
    m3076-71 336 GGAGGAGCUACGAUGCGGGGAAGCAACACUUAGUCGCGAUUGAUAC GUGCGCAGUCAUCAGGCAGACGACUCGCUGAGGAUCCGACA
    m3076-72 337 GGAGGAGCUACGAUGCGGGAAGCAACACUUAGUCGCGAUUGAUACG UGCGCAGUCAUCAGACAGACGACUCGCUGAGGAUCCGACA
    m3076-73 338 GGAGGAGCUACGAUGCGGGGAAGCAGCACUUAGUCGCGAUUGAUAC GUGCGCAGUCAUCAGACAGACGACUCGCUGAGGAUCCGACA
    m3078-74 339 GGAGGAGCUACGAUGCGGGGAAGUAACACUUAGUCGCGAUUGAUAC GUGCGCAGUCAUCAGACAGACGACUCGCUGAGGAUCCGACA
    m3092-75 340 GGAGGAGCUACGAUGCGGUAACUCUCAGGAGGUGCGGAACGGGAU AUGGAUUGUUCGCCAGUCAGACGACUCGCUGAGGAUCCGACA
    m3100-76 341 GGAGGAGCUACGAUGCGGUAAUUCUCAGGAGGUGUGGAACGGGAU AUGGAUUGUUCGCCAGUCAGACGACUCGCUGAGGAUCCGACA
    m3101-77 342 GGAGGAGCUACGAUGCGGUAAUUCUCAGGAGGUGCGGAACGGGUU AUGGAUUGUUCGCCAGUCAGACGACUCGCUGAGGAUCCGACA
    m3104-78 343 GGAGGAGCUACGAUGCGGUAAUUCUCAGGAGGUGCGGAACGGGAU AUGGAUUGUUCGCGAGUCAGACGACUCGCUGAGGAUCCGACA
    m3117-79 344 GGAGGAGCUACGAUGCGGCCGAUUUCGUCAUGCUCCAUACCAUCGC CUUACCGUUCCGCGUCAGACGACUCGCUGAGGAUCCGACA
    m3211-80 345 GGAGGAGCUACGAUGCGGCCCAUCACUCGCGCGUAUUGCGAACGCA UCGUUAUUUAGCCGUCAGACGACUCGCUGAGGAUCCGACA
    m3219-81 346 GGAGGAGCUACGAUGCGGCAGGUGCGGGAUCUAAUGCGUAGACAG CCAUAUACUGACACAGACAGACGACUCGCUGAGGAUCCGACA
    m3219-82 347 GGAGGAGCUACGAUGCGGCAGGGGCGGGAUCUAAUGCGUAGACAG CCAUAUACUGACACAGACAGACGACUCGCUGAGGAUCCGACA
    m3229-83 348 GGAGGAGCUACGAUGCGGCCUAGUACAAAAGCCUGAUCUCUGUGAG CAGACACUAGAACAGACAGACGACUCGCUGAGGAUCCGACA
    m3248-84 349 GGAGGAGCUACGAUGCGGUGUACACUGAUUGCCUUUGUGUUAUGA GCGACAGAUCUGCCAGACAGACGACUCGCUGAGGAUCCGACA
    m3250-85 350 GGAGGAGCUACGAUGCGGCAUACACACUUGACUUUAGGGAACGAAC CUCUAGCCGUGGCCAGACAGACGACUCGCUGAGGAUCCGACA
    m3265-86 351 GGAGGAGCUACGAUGCGGACGGAGGAUAGUUGCUAAUCGAGCCCU GCUGACGCUUCAGACAGACGACUCGCUGAGGAUCCGACA
    m3428-87 352 GGAGGAGCUACGAUGCGGCCGUCUCGCUCUCAUCCCAUGCACGAAA CCUCUCUCAGUGCACAGACGACUCGCUGAGGAUCCGACA
    m3435-88 353 GGAGGAGCUACGAUGCGGUAAUUCUCAGGGGGUGCGGAACGGGAU AUGGAUUGUUCGCCAGUCAGACGACUCGCUGAGGAUCCGACA
    m3435-89 354 GGAGGAGCUACGAUGCGGUAAUUCUCAGGAGGUGCGGAACGGGAC AUGGAUUGUUCGCCAGUCAGACGACUCGCUGAGGAUCCGACA
    m3435-90 355 GGAGGAGCUACGAUGCGGUAAUUCUCAGGAGGUGCGGAACGAGAUA UGGAUUGUUCGCCAGUCAGACGACUCGCUGAGGAUCCGACA
    m3435-91 356 GGAGGAGCUACGAUGCGGUAAUUCUCAGGAGGUGCGGAACGGGAU AUGAAUUGUUCGCCAGUCAGACGACUCGCUGAGGAUCCGACA
    m3435-92 357 GGAGGAGCUACGAUGCGGUAAUUCUCAGGAAGUGCGGAACGGGAUA UGGAUUGUUCGCCAGUCAGACGACUCGCUGAGGAUCCGACA
    m3500-93 358 GGAGGAGCUACGAUGCGGCCCAUCACUCCCGCGUAUGGCGAACGCA UCGUUAUUUAGCCGUCAGACGACUCGCUGAGGAUCCGACA
    m3523-94 359 GGAGGAGCUACGAUGCGGUCAUGGAUUCAUUACAGGAGGUGCGGU GCUAUAUGCACGCCAGACAGACGACUCGCUGAGGAUCCGACA
    m3546-95 360 GGAGGAGCUACGAUGCGGCCAGCCACACUUUGACCGAAUUGGCAAG CGCGGGCAAAUCGAACAGACGACUCGCUGAGGAUCCGACA
    m3548-96 361 GGAGGAGCUACGAUGCGGCCUAGUACAAAAGCCUGAUCUUUGGGAA CCGACCCUAGGACAGACAGACGACUCGCUGAGGAUCCGACA
    m3550-97 362 GGAGGAGCUACGAUGCGGCUUACAGCUCACCAUUUAUGGGAGGCCC GGUGUUGUGUUCCAGACAGACGACUCGCUGAGGAUCCGACA
    m3565-98 363 GGAGGAGCUACGAUGCGGAUUAUUGUUUGACGUAUUCCAAGUGAGA UUACGCACGCACCAGACAGACGACUCGCUGAGGAUCCGACA
    m3568-99 364 GGAGGAGCUACGAUGCGGAACAGCUUAAUCGCCAGUCGAUACGCGC CAUACAUCAUCACAGACAGACGACUCGCUGAGGAUCCGACA
    m3745-100 365 GGAGGAGCUACGAUGCGGUAAUUCUCAGGAGAUGCGGAACGGGAUA UGGAUUGUUCGCCAGUCAGACGACUCGCUGAGGAUCCGACA
    m3745-101 366 GGAGGAGCUACGAUGCGGUAAUUCUCAGGAGGUGCGAAACGGGAUA UGGAUUGUUCGCCAGUCAGACGACUCGCUGAGGAUCCGACA
    m3748-102 367 GGAGGAGCUACGAUGCGGACGAUUUCGUCAUCCUCCAUACCAUCGC CUUACCGUUCAGCGUCAGACGACUCGCUGAGGAUCCGACA
    m3773-103 368 GGAGGAGCUACGAUGCGGCAACAAACUAAUCAGACACGAGACAGAG AGAUAGAUCUGCCAGACAGACGACUCGCUGAGGAUCCGACA
    m3788-104 369 GGAGGAGCUACGAUGCGGUUAUGCGUUUAAGUCAUUGACGCGUUAC ACUGGAGGGGGCCAGACGACUCAGACGACUCGCUGAGGAUCCGACA
    m3823-105 370 GGAGGAGCUACGAUGCGGACGGAGGAUAGUUGCUAAUCGAGCCCU GCGGACGCUUCAGACAGACGACUCGCUGAGGAUCCGACA
    m3831-106 371 GGAGGAGCUACGAUGCGGCUUACAGCUCACCAUUUUUGGGAGGCC CGGUGUUGUGUUCCAGACAGACGACUCGCUGAGGAUCCGACA
    m3845-107 372 GGAGGAGCUACGAUGCGGACGGAAGGAUAGUUGCUAAUCGAGCCCU GCCGACGCUUCAGACAGACGACUCGCUGAGGAUCCGACA
    m3997-108 373 GGAGGAGCUACGAUGCGGUAAUUCUCAGGAGGUGCGGAACGGGAU AUAGAUUGUUCGCCAGUCAGACGACUCGCUGAGGAUCCGACA
    m3997-109 374 GGAGGAGCUACGAUGCGGUAAUUCUCAGAAGGUGCGGAACGGGAUA UGGAUUGUUCGCCAGUCAGACGACUCGCUGAGGAUCCGACA
    m3997-110 375 GGAGGAGCUACGAUGCGGUAAUUCUCAGGAGGUGCGGAACGGGAU AUGGAUUGUUCGCCCGUCAGACGACUCGCUGAGGAUCCGACA
    m3997-111 376 GGAGGAGCUACGAUGCGGUAAUUCUCAAGAGGUGCGGAACGGGAUA UGGAUUGUUCGCCAGUCAGACGACUCGCUGAGGAUCCGACA
    m4097-112 377 GGAGGAGCUACGAUGCGGCAAAAACUGAUAAACACAGGUCCGGCAU UUGAGCGUACACCCAGACAGACGACUCGCUGAGGAUCCGACA
    m4110-113 378 GGAGGAGCUACGAUGCGGUCGGAGGAUAGUUGCUAAUCGAGCCCU GCCGACGCUUCAGACAGACGACUCGCUGAGGAUCCGACA
    m4275-114 379 GGAGGAGCUACGAUGCGGUUAUGCGUUUAAGUCAUUGACGCGUUAC ACUGGAGGGGGCCAGACAGACGACUCGCUGAGGAUCCGACA
    m4347-115 380 GGAGGAGCUACGAUGCGGCAAAAAACUGAUAAACACAGGUCCGGCA UUUGAGCGUACACCCAGACAGACGACUCGCUGAGGAUCCGACA
    m4365-116 381 GGAGGAGCUACGAUGCGGACGGAGGAUAGUUGCUAAUCGAGCCCU GCCGACGCUUCAGACAGACGACUCGCUGAGGAUCCGACA
  • Next, the specificity for human islets of the identified monoclonal aptamers were tested with the two high throughput cluster SELEX strategies described in FIG. 1 and FIGS. 2 . Briefly, monoclonal aptamers corresponding to the sequences identified by the bio-informatic analysis were generated by PCR and T7 RNA polymerase using overlapping oligonucleotides as template. The resulting monoclonal aptamers were labelled with Cyanin-3 and used as fluorescent probes on sections of human pancreas from cadaveric donor. Clone number is reported with the prefix “m” indicating the identification of aptamer from the HT-Toggle cluster SELEX. Data show that while some aptamers (m166-279, m5-3229, 107-901, m7-2537, m9-3076, 1-717, 173-2273, m12-3772) show a good specificity for the islets, others label also the acinar tissue (12-2617, 109-2031, m1-2623, m24-3219), and others did not show any staining (208-2529, 155-1113, 64-2437).
  • In order to evaluate further the specificity of the chosen monoclonal aptamers, the best performers of FIG. 4 (166-279, 173-2273, 107-901, 1-717, m1-2623, m5-3239, and m12-3773) were used to stained FDA approved tissues microarrays. Each of these arrays contains sections from 30 human tissues (with each tissue replicated from 3 different donors) and are usually used for antibodies screening but have not yet been used for aptamer evaluation. Briefly, these tissues microarrays were stained with the chosen cy3 labelled RNA aptamers of irrelevant aptamers as control. Each tissue was then analyzed by immunofluorescence microscopy. While most of the aptamers show binding not only as expected in the pancreatic islet but also in other tissues (FIG. 5C), aptamer 1-717 (FIG. 5A) and aptamer m12-3773 (FIG. 5B) show an extraordinary specificity for the pancreatic islets and negligible binding to the other tissues evaluated (adrenal, bone marrow, breast, brain, colon, endothelial, esophagus, fallopian tube, heart, kidney, liver, lung, lymph node, Ovary, placenta, prostate, skin, spinal cord, spleen, muscle, stomach, testis, thymus, thyroid, ureter, uterus, and testis).
  • To evaluate if aptamer 1-717 and m12-3772 can recognize not only human islets but also the mouse counterpart, staining with these two Cy-3 labelled monoclonal aptamers were performed on tissues microarrays each containing 11 tissues from healthy mice. These experiments show that both aptamer 1-717 and aptamer m12-3773 can also recognize mouse islets. See FIG. 6 . However, in contrast to what observed on human tissues, both aptamers can recognize at different level not only the pancreatic tissue but also the spleen, the stomach, and the jejunum. These data suggest that differences in the distribution of the cognate targets may exist between the two species.
  • To evaluate better the specificity of the aptamers within the islets, two different techniques were employed: confocal microscopy (FIG. 7A and FIG. 7B) and flow cytometry (FIG. 7C and FIG. 7D). For confocal microscopy, sections of human pancreas were stained with cy-3 labelled aptamer M12-3773 (FIG. 7A) or cy3-labeled aptamer 1-717 (FIG. 7B). Sections were counterstained with antibodies against insulin and glucagon, and DAPI and evaluated by confocal microscopy. Data show binding of both aptamers to both alpha and beta cells but with an higher signal on beta cells.
  • For flow cytometry, single cell suspension of human islets were stained with Cy3 labelled aptamer M12-3773 (FIG. 7C) or cy3-labeled aptamer 1-717 (FIG. 7D), counterstained with vital dye and antibodies specific for insulin and glucagon, and analyzed. Aptamer signal (open histograms) was quantified on the alpha (top histograms) or beta cells (right histograms) after gating respectively on glucagon positive or insulin positive cells (contour plot). An irrelevant aptamer (filled histograms) was used as negative control.
  • Clusterin is a possible target for aptamer m12-3773. 3′biotin-aptamer m12-3773 was synthetized with a oligo synthesizer and used to label single cell suspension from human islets. Cells were washed and their cytoplasm lysed with tween20/BSA solution. Aptamers bound to their ligand recovered with magnetic beads and magnetic separation. Capture ligands were released by the aptamer-beads complex at 95° C. in SDS and run in SDS page. Bands were cut and subjected to mass spectrometry and mascot-based analysis (FIG. 8A). Clusterin (UniProtKB - P10909) (FIG. 9B) was one of the protein with the higher score (236), had an elevated sequence covered from peptides identified by mass spectrometry, had a molecular weight compatible to the one of the band cut from the SDS page, and more importantly, was the only one of the tested one whose silencing reduced the capacity of aptamer m12-7337, but not of aptamer 1-717, to bind to beta cells (FIG. 8C).
  • FIGS. 9 shows that TMED6 is the putative target for aptamer 1-717. FIG. 9A shows the experimental strategy to detect the target for aptamer 1-717. To identify the target of aptamer 1-717, protein arrays (HuProt™ v2.0, Arrayit) were used. These protein arrays contains more than 19,000 human recombinant proteins allowing, by informatics analysis, the identification of the cognate protein of antibodies, peptides, or protein. This technology has been adapted for the identification of aptamer’s ligands. Briefly, pre-blocked arrays were hybridized with 1 µg of cy3 labeled 1-717 or m12-3773 in blocking buffer for 30′ at RT. Arrays were washed, read in triplicate on a Genepix microarray reader and analyzed by an ad hoc generated software. This software 1) acquires the data from the gpr file, 2) adds a description column with (GeneID, Control, blank, ND), 2) use a optimized “plotArray” function modified in several points (the script was implemented for a specific protein array, plus some problem in UTF file format), 3) performs a quality control that includes a microarray image rebuilding the generation of MA plots, 4) normalized the data and substracts the background; and 5) analyze the differential expression between arrays via graphs of p-value distribution, volcano plot, and analysis of significant modulation by t-test. This analysis proposed TMED6 (NM144676.1, protein id Q8WW62) as the most likely ligand of aptamer 1-717. See FIG. 9B. Competitive assays (FIG. 9C) confirm the specificity of this target. As shown in FIG. 9C, competitive assays confirm the specificity of aptamer 1-717 for TMED6. Briefly, serial sections of human pancreas were stained with Cy-3 labelled aptamer 1-717 in the presence of different concentration of recombinant TMED6 protein (i.e., molar ratio aptamer/recombinant protein range= 1/1-⅒). Images were acquired by a fluorescence microscope and aptamer binding quantified by cellprofiler. Data show that addition of recombinant TMED6 inhibit aptamer 1-717 binding in a dose dependent manner strongly suggesting that TMED6 is the target of this aptamer.
    • Example 2 - Identified Aptamers Were Islet Specific in Vivo
  • To evaluate whether aptamer 1-717 and m12-3773 can recognize human islets in vivo, we employed immunodeficient NSG mice engrafted with human islets in the epydidimal fatpad. Additionally, we use a new formulation of aptamer 1-717 and aptamer m12-3773 in which each monoclonal aptamer is biotinylated and complexed with streptavidin to form a tetrameric nanoparticle (hereafter called tetraptamer). This formulation has a superior pharmacokinetic and better affinity than the corresponding monomeric aptamer.
  • Biotin/streptavidin Alexafluor (AF750)-labeled aptamers (amptamer 1-717 or aptamer m12-3773, or an equimolar mixture of the two aptamers) were injected intravenously in immunodeficient NSG mice (engrafted with human islets in the epididymal fat pad (EFP)) to evaluate whether m12-3773 and 1-717 can recognize human islets in vivo. A cumulative-synergistic signal was observed in the EFP region when the mixture of both aptamers was used possibly because different islet epitopes were targeted by each aptamer (FIG. 10A). 4 hour later fluorescence signal in epididymal fat pad region was measure by “In vivo imaging system (IVIS)”. The data in FIG. 10B shows that both aptamer 1-717 and aptamer m12-3773 can recognize the islets in vivo. Additionally this experiment reveals that the use of an equimolar mixture of the two aptamers significantly increase the signal to background ratio.
  • To determine if aptamers m12-3773 and 1-717 can be used to measure β cells mass in vivo, immune deficient NSG mice were transplanted with different quantities (range 62.5-500 IEQ) of human islets in the epididymal fatpad. 21 days later, mice were injected iv with Alexafluor 750 tetraptamer generated by the complexation of an equimolar mixture of aptamer 1-717 and m12-3773 to streptavidin. 4 hours later signal was quantified by IVIS. FIG. 11A. Aptamers m12-3773 and 1-717 recognized both the mouse endogenous islets and the human islets transplanted in the EFP (FIG. 11B). Importantly, fluorescence signal in the EFP region was proportional to the number of engrafted islets indicating that these aptamers can be used to measure β cell mass in vivo (FIGS. 11B and 11C). The signal from the islets persisted for 10 days after injection (not shown). As shown in FIG. 11D, rejection of the allogeneic C57B16 islet graft can be measured over time as seen by the loss of signal on the left flank. Instead signal (right panel of FIG. 11D) of the syngeneic graft is maintained over time indicating graft survival.
  • In summary, the selected aptamers m12-3773 and 1-717 bind mouse and human β cells with good specificity in vitro and in vivo and thus may be useful in targeting therapeutics to human β cells in vivo.
    • Example 3 - Aptamera Chimera Can Deliver Therapeutic RNA to Islets
  • As shown in FIG. 12 , islet-specific RNA aptamers 1-717 and m12-3773 can be easily conjugated to therapeutic RNA by prolonging their 3′ end with a trinucleotide linker region (i.e. GGG) and the passanger strand (passanger tail) of the desired therapeutic RNA. The therapeutic RNA guide strand is then simply annealed to the modified aptamer by admixing equimolar quantities of the two RNAs at 70° C. and allowing the mixture to slowly cool down at room temperature.
  • To evaluate if aptamers can be a non-viral alternative for transfecting β cells, we conducted proof of principle experiments aimed to knockdown via aptamer delivery insulin (INS) 1 and 2 in non-dissociated mouse islets. FIG. 13A is a schematic of the experimental procedure. Islets specific aptamer chimera were generated as detailed in FIG. 12 by conjugating aptamer 1-717 or aptamer m12-3773 with siRNA specific for mouse insulin½ (INS½) or the inhibitor of cell proliferation human p57kip2 (uniprot P49918, alias CDN1c). The INS 112-siRNA/aptamer chimera was added to non-dissociated mouse islets whereas the p57kip2-siRNA/aptamer chimera was added to human islets from a cadaveric donor. Scramble siRNA/aptamer chimera were used as negative controls. 72 hours later, expression of INS ½ and p57kip2 was quantified by qRT-PCR on transfected mouse islets and transfected human islets respectively. As shown in FIG. 13B, the aptamer chimera significantly downregulate the expression of the target gene.
  • As shown in FIGS. 14 , p57kip2-siRNA-islet specific aptamer chimera induce human beta cell proliferation in vivo. FIG. 14A shows the experimental procedure: Streptozotocin-treated, immune deficient NSG mice were transplanted with a suboptimal quantity (250 IEQ) of human islets in the anterior chamber of the eye. Mice were maintained euglycemic by s.c. implantation of insulin pellet. 21 days later, when islets were vascularized, insulin pellet was removed to allow the development of hyperglycemia, mice were fed with BrdU for 7 days to evaluate cell proliferation, and treated with i) scramble-siRNA/aptamer chimera, or ii) p57kip2-siRNA/aptamer chimera. Nine days after treatment, mice were humanely euthanized, and beta and alpha cell proliferation was evaluated by immune fluorescence microscopy after labeling the graft sections with antibodies against insulin, glucagon, and BrDU (white). FIG. 14B provides immunofluorescence pictures of the graft from mice treated with control chimera or p57kip2-siRNA/aptamer chimera. Glucagon and insulin staining is depicted in dark gray as pseudocolor whereas BrdU staining as measure of cell proliferation is depicted in white as pseudocolor. FIG. 14C shows quantification of proliferating beta and alpha cells. Taken together these data indicate that p57kip2-siRNA/aptamer chimera can induce in vivo human beta cell proliferation in a hyperglycemic setting that mimic T1 and T2 diabetes.
  • P57kip2 silencing in β cells has important therapeutic implications. Indeed, mutations of p57Kip2 are associated with focal hyperinsulinism of infancy (FHI), a clinical syndrome characterized by a dramatic non-neoplastic clonal expansion of β cells (14), overproduction of insulin, and severe uncontrollable hypoglycemia (89,90). FHI’s focal lesions are characterized by excessive β cell proliferation that correlates with p57kip2 loss (91,92). Although the pro-proliferative activity of p57kip2 silencing is not desirable in FHI and in cancers, a temporally defined silencing might be useful to promote adult β cell proliferation in T1D. Indeed, adenoviral-shRNA mediated silencing of p57kip2 in human islets obtained from deceased adult organ donors increased β cell replication by more than 3-fold once the islets were transplanted into hyperglycemic, immune-deficient mice (14). The newly replicated cells retained properties of mature β cells, such as expression of insulin, PDX1, and NKX6.114. Interestingly, no β cell proliferation was observed in normoglycemic mice indicating that hyperglycemia may provide additional pro-proliferative signals (93). These findings opened the possibility for a new therapeutic intervention to restore an adequate β cell mass in patients with T1D and/or to reduce the number of islets needed during transplantation. However, to date the translatability of these finding was hindered by safety concerns associated with use of viral vectors and neoplasm formation as a result of stable p57Kip2silencing. Indeed, p57Kip2 is frequently downregulated in human cancers (94) and has been proposed as a tumor suppressor gene since its ectopic expression is sufficient to halt neoplastic cell proliferation (94). However, a temporally controlled modulation of p57kip2 through aptamer delivery may be important in diabetes to increase β cell proliferation in a temporally controlled manner. This might be sufficient to increase β cell mass during timed administrations while avoiding the safety concerns with non-controllable, neoplastic-like proliferation of β cells that may results with stable silencing.
    • Example 4 - Upregulation of XIAP Via saRNA-Aptamer Chimera Inhibits Apoptosis in Β Cells.
  • Apoptotic cell death is a hallmark in the loss of insulin-producing β cells in all forms of diabetes (99-101). Leukocytes infiltration and activation as well as high glycemia within the islets leads to high local concentrations of apoptotic trigger including inflammatory cytokines, chemokines, and reactive oxygen species99. Most of these apoptotic pathways converge onto caspase (CASP) 3 and 7 activation leading to genetic reprogramming, phosphatidylserine flip, and apoptotic bodies formation (102).
  • β cell apoptosis can further feed the autoimmune process by stimulating self-antigen presentation and autoreactive T cell activation (103). Similarly, in islets transplantation setting, primary non-function, i.e. the partial but significant and sometimes total loss of the grafted islet mass, which occurs early after transplantation (104-106). β cell apoptosis initiates during the isolation procedure and upon transplantation is exacerbated by hypoxia and hyperglycemia as well as pro-coagulatory and proinflammatory cascades (107). Primary-non-function accounts for more than 50% of the functional islet mass loss occurring during the first 48 hours after transplantation (106).
  • Thus, blocking even temporally apoptotic β cell death is highly desirable not only to preserve β cell mass in type 1 diabetes (T1D) and in islet transplantation but also to reduce auto-reactive T cell activation and further immune damage.
  • This protein is most potent member of the apoptosis-inhibitor family and prevents the activation of CASP 3, 7 and 9(108); ii) Xiap overexpression using viral vector improved β cell viability, prevented their cytokine- or hypoxia-induced apoptosis(109-111), iii) Xiap transduced human islets prolonged normoglycemia when are transplanted in diabetic NOD-SCID mice (11). However, since Xiap is upregulated in many cancers, its stable overexpression raise important safety concerns. Therefore, a controlled Xiap activation via saRNA delivered with islets specific aptamers can be useful alternative to reduce primary nonfunction, prevent β cell loss and the self-feeding autoimmune process in T1D.
  • Small activating RNAs (saRNAs) are oligonucleotides that exert their action in specific promoter regions and upregulate mRNA and protein expression for up to 4 weeks (depending on cell replication, mRNA and protein turn-over) (112-122). saRNA-mediated gene upregulation through mechanisms still not fully understood but is thought to involve epigenetic changes or down-modulation of inhibitory RNA (123-125). saRNAs provide safe, specific, and temporary gene activation without the insertion of DNA elements since their specificity is comparable to that of gRNA in CRISP/CAS9 system but no irreversible DNA modification are induced 126. While therapeutic saRNAs are being investigated for cancer treatment, to our knowledge no studies have been performed in T1D (127-130).
  • Therefore, we have identified saRNAs capable of specifically upregulating the anti-apoptotic gene XIAP. Briefly, we have first examined the human XIAP promoter using the previously described algorithms (112,131). This analysis that includes genome blast analysis to avoid non-specific sequences, returned more than 156 putative saRNA target regions. We synthetized the 96 putative saRNA with highest scores and tested them for their capacity to upregulate Xiap by transfecting the human epithelial cell line A549. This cell line was used because it is easily transfectable, has low basal expression of PDL1 and Xiap. qRT-PCR was performed 96 hours after transfection and results were normalized on the same cell line transfected with scrambled saRNA (FIGS. 15 ). Twelve saRNAs (provided in Table 5) were found to upregulate Xiap expression more than 10 times (range 10.4-74.8) over scrambled saRNA.
  • TABLE 5
    saRNA sequences to upregulate human Xiap
    Position Fold change Xiap saRNA sequence SEQ ID NO
    -234 74.8083 UAGCUGAAGUUCAUCUCUCuu 382
    -1134 46.7026 UUUCAGCCUUAAGGAUGGUuu 383
    -449 37.1938 UUUAUUCUCCCCUUGGGUGuu 384
    -344 18.7146 UACUCCCUCUGCCUAUGUGuu 385
    -121 15.4365 UUUACUGUUUUGGCUGGGCuu 386
    -682 13.9281 AAAAUGCUGGUCAUACCCUuu 387
    -354 13.1961 UUGUUCAAACUACUCCCUCuu 388
    -374 12.5789 UUUUCCUGCCUUCCGCUAAuu 389
    -593 11.9908 UUACAGGGUAAUGUGGUGAuu 390
    -758 11.0947 GAUUGGGAGGUGAAGGGAAuu 391
    -680 10.6792 AAUGCUGGUCAUACCCUGGuu 392
    -531 10.5239 UACAAGAUAUGAUCCUCCCuu 393
  • In vitro proof of principle experiments were performed using human islets isolated from cadaveric donors to determine if Xiap-saRNA delivered by aptamer can protect β cell from apoptosis. Xiap-saRNA aptamer chimeras were generated as described in FIG. 12 by conjugating the identified Xiap saRNA (-449, table 2) to either aptamer m12-3773 or aptamer 1-717. FIG. 16A shows the experimental procedure: Freshly-isolated, non-dissociated, human islets (200IEQ) from cadaveric donor were transfected with Xiap-saRNA by adding the Xiap/saRNA aptamer chimera (5ug) to the culture. Scramble saRNA/aptamer chimera were used as negative control. 48 hours later, half of the wells were challenged with inflammatory cytokines (IFNg, TNFa, and IL1b) to induce beta cell apoptosis. Beta cell death was evaluated by flow cytometry 24 hours after cytokine challenge by measuring beta/alpha cell ratio after staining for insulin and glucagon. FIG. 16B shows the flow cytometry analysis of single cell suspension of islets treated with scrambled saRNA chimera (CTRL chimera) or XIAP-saRNA/aptamer chimera (Xiap Chimera) and later challenged with cytokines (CTK) or left untreated (No CTK). FIG. 16C is a spaghetti plot from 5 independent experiments each with islets from a different cadaveric donor using chimera generated with either aptamer m12-3773 or aptamer 1-717. Paired T test value is reported. Data show that Xiap-saRNA aptamer chimera protect beta cells from cytokine-induced apoptosis.
  • Interestingly, untreated islets in the absence of cytokines showed higher proportion in α cells (β/α cell ratio=0.8) in the presence of CTRL-chimera (FIG. 16B) and in absence of any chimera (data not shown), suggesting that β cell viability may be affected more than α cells during islet isolation. Addition of cytokines further reduced β cell proportion (β/α cell ratio~0.5). Notably, incubation with Xiap-saRNA/chimera not only prevented the CTKs-induced decrease in β cells (β/α cell ratio~1.6) but also prevented β cell loss associated with islets isolation. These data indicate that saRNA-chimeras can be used to modulate Xiap expression in human islets.
    • Example 5 - Use of Xiap-saRNA/Aptamer Chimera to Prevent Primary Nonfunction
  • Human islets from cadaveric donors were transfected with Xiap-saRNA aptamer chimera or control-chimera as detailed in FIG. 17A Twenty-four hours after transfection, islets were transplanted in the anterior chamber of the eye of immune deficient NSG mice. Islets cell apoptosis was evaluate longitudinally by in vivo annexin V (ANXA5) staining and in vivo microscopy. Data show that treatment with Xiap-saRNA/aptamer chimera before transplantation drastically reduce apoptosis (ANXA5), and thus cellular loss of the graft.
  • Provided in FIG. 17B is the schematic the Xiap-saRNA/aptamer chimera for graft preservation. As shown in FIG. 17C, human Islets were cultured in media where chimera was added at 48h, 24h and on the day of transplantation 600 IEQ were transplanted per mouse in the left kidney capsule of streptozotocin diabetic NOG mice. Data showed that pretreatment of human islets with aptamer chimera greatly improve the efficacy of islet transplantation with approximately 80% of mice becoming normoglycemic by day 2. In contrast only 50% od mice engrafted with islets (P=0.02; nchimera treated= 10; nuntreated = 8) reverse diabetes and with a delayed kinetic.
    • Example 6 - Protect Islets From Allo- and Auto-immunity in Humanized Mice Via PDL1-saRNA/Aptamer Chimera
  • The clinical importance of PDL1 expression in the maintenance of tissue specific tolerance is highlighted by the success of PDL1-PD1 antagonists in cancer (135). Engagement of PD1 by PDL1 down-regulates effector T cell proliferation and activation, induces T cell cycle arrest and apoptosis, and promotes IL10-producing Treg (136-139). Interestingly, one of the emerging side effect anti-PD1 treatment is T1D140. This suggests that PDL1/PD1 may play an important role in controlling T cell tolerance against β cells. Indeed, in NOD mice PDL1 blockade accelerate T1D in female mice and induce it in male (13). Conversely, PDL1 ectopic expression in syngeneic transplanted islets protects NOD mice against T1D recurrence (12,13). NOD transgenic mice expressing PDL1 under control of the insulin promoter shows delayed incidence in diabetes, reduction T1D incidence, and a systemic, islet specific, T cell anergy (141). In humans, PDL1 polymorphisms is associated with T1D (OR=1.44) (142).
  • Given the importance that PDL1 expression might play in controlling T cell reactivity to β cells, we identified saRNAs specific for PDL1 (FIGS. 18 ). Briefly, putative candidate sequence of small activating RNA for PDL1 were identified by scanning the PDL1 promoter using publically available algorithms. This analysis return more than 200 putative target saRNA target regions. The 95 putative saRNA with higher score were synthetized and tested for their capacity to up-regulate PDL1 by transfecting the human epithelial cell line A549. qRT-PCR was performed 96 hours after transfection and results normalized on the same cell line transfected with scrambled saRNA. 19 saRNAs were found able to upregulate Xiap expression more than 3 times (range 3.01-63.27) over scrambled saRNA (Table 6).
  • TABLE 6
    saRNA sequences to upregulate human PDL1
    Position fold change PDL1-saRNA SEQ ID NO
    -261 63.2769 UUUAUCAGAAAGGCGUCCCuu 394
    -583 14.1907 UUAAGGCUGCGGAAGCCUAuu 395
    -739 13.0165 UUGACCUCAAGUGAUCCGCuu 396
    -461 11.5844 GACUUCCUCAAAGUUCCUCuu 397
    -584 7.7063 UAAGGCUGCGGAAGCCUAUuu 398
    -349 5.8792 UAAAAAGUCAGCAGCAGACuu 399
    -353 5.2152 AAGUCAGCAGCAGACCCAUuu 400
    -608 5.0249 GUGAGGGUUAAGAAAGCCCuu 401
    -881 4.833 CUGCAGUUCAAAAUACUGCuu 402
    -637 4.1477 UUUGGGUUAGUGAAUGGGCuu 403
    -683 3.9179 UUUACUUAAGUAUUAUCCCuu 404
    -594 3.7109 GAAGCCUAUUCUAGGUGAGuu 405
    -352 3.6316 AAAGUCAGCAGCAGACCCAuu 406
    -351 3.3859 AAAAGUCAGCAGCAGACCCuu 407
    -609 3.3669 UGAGGGUUAAGAAAGCCCUuu 408
    -713 3.3464 CUAGGUGCUCUCUUUUCUCuu 409
    -636 3.28 CUUUGGGUUAGUGAAUGGGuu 410
    -460 3.0587 UGACUUCCUCAAAGUUCCUuu 411
    -464 3.0192 UUCCUCAAAGUUCCUCGACuu 412
  • Next whether the islet-specific-aptamers described herein can effectively deliver PDL1-saRNAs to human islets and upregulate PDL1 expression was tested. Aptamer-PDL1-saRNA chimeras were generated by conjugating aptamer 1-717 to PDL1-saRNA-636 (Table 6) as described in FIG. 12 . As shown in FIG. 19A, these PDL1-saRNA/aptamer chimera were added to non-dissociated human islets from cadaveric donor. 48h later, islets were dissociated, labelled with anti-insulin, anti-glucagon and anti-PDL1 antibodies and analyzed by flow cytometry (FIG. 19B). PDL1 expression was evaluated by gating on insulin positive beta cells or glucagon positive alpha cells. While treatment with control chimera does not modify PDL1 expression, treatment with PDL1-saRNA/aptamer significantly upregulate the expression of this important immune modulatory protein on beta cells (FIG. 19B). Interestingly no changes were observed in alpha cells confirming indirectly the preferential binding of this aptamer to beta cells. These proof of principle data indicate that aptamers can be effectively used to deliver functional PDL1-saRNA into human β cells in vitro.
  • Next, the ability of PDL1-saRNA/aptamer chimera to upregulate PDL1 in vivo was assessed. As shown in FIG. 20A, immune deficient NSG mice were transplanted in the anterior chamber of the eye with human islets from a cadaveric donor. 3 weeks later, mice were treated with PDL1-saRNA(636)/1-717-aptamer chimera generated as described in FIG. 12 and FIGS. 19 . Scramble-saRNA/aptamer chimera was used as control (CTRL chimera). Five days after treatment, PDL1 expression (white) on the islets (dark gray) was quantified by in vivo labelling with anti-PDL1 antibody and in vivo microscopy (FIG. 20B). Summary of PDL1 expression on the engrafted islets at baseline or 5 days after treatment with PDL1-saRNA/aptamer chimera or scrambled-saRNA/aptamer chimera FIG. 20C).
  • These results indicated that: i) it is possible to detect PDL1 in human islet cells in vivo, ii) our aptamer chimeras transfect human islets in vivo, and iii) it is possible to upregulate PDL1 in human islets in vivo via aptamer chimera.
    • Example 7 - Assess Β Cell Protection From Apoptosis by Aptamer Mediated Xiap Upregulation
  • In the first set of experiments, NSG mice will be engrafted with human islets in the ACE. Three weeks after transplant, mice will be treated with Xiap saRNA-aptamer chimera(s) or control chimera. At different time points, human islet grafts will be challenged by intraocular injection of IL1β, TNF-α, and IFNγ to induce apoptosis in β cells via activation of caspase 3 and 7. Caspase 3 and 7 activity will be evaluated in vivo by our intraocular imaging system using CASP3/7 Green Detection Reagent. This cell-permeant reagent consists of a four-amino acid peptide (DEVD) conjugated to a nucleic acid-binding dye. Upon activation, caspase 3 and 7 cleave the probe, allow the dye to bind to the DNA, and emit a bright, fluorogenic signal that can be detected at the cellular level in the ACE28. Additionally, in vivo staining with anti-Annexin V antibodies will be used to directly measured islet cell apoptosis in vivo (FIGS. 7 ).
  • The second set of experiment aims to evaluate the effect of Xiap modulation on anti-islet allo-immunity. Briefly, STZ-diabetic NSG mice will be transplanted with 500 IEQ human islets in the ACE or EFP. 3 weeks later mice will be treated with Xiap chimera(s) or scrambled controls. Treatment will be repeated as determine in Aim2b. One week after the first treatment, mice will receive CFSE labelled human T cells mismatched for HLA to the islet. Without any treatment, the adoptive transfer of allogeneic T cells results in graft loss and return to hyperglycemia within 3 weeks. Thus, we will assess the protective effect of Xiap chimera treatment on the human islet allograft survival using as readouts: i) glycemia, ii) human c-peptide plasma levels and, in the ACE group, iii) the longitudinal evaluation of T cell infiltration and volumetric analysis of engrafted islets as we showed in (77,78).
  • To ensure data reproducibility of Xiap chimera effect among individuals, the chimera identified in the EndoC-BH3 cells will be further validated using primary human islets from 6 cadaveric donors; this will provide 88% of power to detect 1.25SD difference from control in one tailed paired t-test. To avoid artifacts, 3 different readout methods (qPCR, western blot, and enzymatic assay) will be used and at least 3 independent repetitions will be performed for each experiment using human islets from 3 different cadaveric donors. In transplantation studies, a total of 9 mice per group (3 in each repetition) will be used to ensure 90% of power (ANOVA, α=0.05) and detect 1.6SD difference to control.
    • Example 8 - Optimize the Dose for in Vivo Silencing of P57kip2
  • In a first set of experiments, NSG mice transplanted with 500 IEQ human islets in the EFP will be treated i.v. or s.c. with different doses (6, 20, and 60 pmoles/g) of islets-specific aptamers conjugated with p57kip2siRNA or scrambled siRNA (control-chimera) as negative control. We will use adenovirus encoding the same p57kip2 shRNA-transfected islets as positive control (14). At predefined time-points (e.g., day 1, 2, 3, 4, and 5 after administration), grafts will be harvested, and p57kip2 expression quantified by i) qRT-PCR on laser captured islets, and ii) by quantitative computer assisted immunofluorescence analysis95. Both techniques are optimized at the Diabetes Research Institute (96,97) and in the laboratories of the PIs95. To evaluate possible dose-dependent toxicity, sera and organs of interest (spleen, liver, lymph nodes, lung, kidney, and brain) will be collected and sent to the mouse pathology laboratory of University of Miami for histopathological evaluation.
  • In the second set of experiments, NSG mice will be transplanted with 500 IEQ human islets in the ACE. Three weeks later, mice will be treated i.v. or by intraocular injection (i.o) with different doses (6, 20, 60pm/g) of our aptamer-chimera loaded with p57kip2 siRNA or AF647-scrambled siRNA (control-chimera) as negative control. In vivo transfection efficiency of the AF647 siRNA will be evaluated with our intraocular imaging system 2, 3, 4, 8, and 24 hours after injection (28). At selected time-points (e.g., 2, 3, 4, and 7 days after treatment), graft will be removed and p57kip2 expression quantified by qRT-PCR on islets explanted from the ACE and by i) qRT-PCR on laser capture islets and ii) by quantitative computer assisted immunofluorescence microscopy analysis (95).
    • Example 9 - Optimize Treatment Length and Frequency for Aptamer-Chimera Administration
  • Once the optimal dose and route of administration are identified and the kinetics of p57kip2 silencing evaluated, we will determine the number of administrations of p57kip2siRNA-aptamer chimera needed to induce substantial changes (i.e., ≥100% increase) in β cell mass. Since p57kip2 silencing was shown to induce β cell proliferation only in hyperglycemic mice14, sub-marginal human islet mass (250 IEQ) will be transplanted in the EFP or ACE of NSG mice. 21 days after transplant, mice will be rendered hyperglycemic by streptozotocin (STZ) treatment. STZ selectively eliminates mouse islets as human β cells are considerably more resistant (98). Once the mice become hyperglycemic (usually 5-6 days after treatment), mice will receive 1, 2, 3, or 4 administration of islet-specific or control aptamer chimeras. The frequency of the aptamer administration will be determined based on the time course established in Example 5. BrdU will be administered in drinking water for ex vivo determination of proliferation. β cell mass in the EPF group will be evaluated longitudinally (baseline, during treatment, 5 and 10 days after the last treatment) by IVIS (FIGS. 2 ). In the ACE group, islets mass will be evaluated by in vivo imaging and quantitative analysis of islet volume (28). Ten days after the last treatment, grafts will be harvested and analyzed by immunostaining to determine (i) β cell proliferation via BrdU and Ki76 staining and (ii) α to β cell ratio.
    • Example 10 - Determine if Aptamer Mediated Silencing Can Restore Normoglycemia in Diabetic Mice Transplanted With Sub-Marginal Islet Mass
  • The purpose of this Example is to test if aptamer mediated p57kip2 silencing can restore normoglycemia in diabetic mice transplanted with suboptimal number of human islets.
  • In the first set of experiments, STZ-diabetic NSG mice maintained on insulin therapy (s.c pellet implant for sustained insulin release) will be transplanted with different quantities of human islets (50, 150, 350 IEQ) in the ACE. Three weeks later, insulin pellets will be removed, and mice will be treated with p57kip2siRNA-aptamer chimera or scrambled control, locally or systemically. To compare this treatment with today gold standard for islets transfection, two additional groups of mice will be treated locally with adenoviral vector encoding for p57kip2shRNA or RFP as control. Pilot experiments using RFP encoding adenovirus will be performed in the ACE to determine the minimal dose necessary for transducing at least 90% of the islets. Transduction efficiency will be quantified using our in vivo imaging system (28). In the experimental groups (which received 50, 150, and 350 IEQ), blood glucose will be used as readout for treatment efficacy in addition to intravital imaging and volume analysis of the ACE islet grafts. The varied sub-marginal islet mass in the different groups may also reveal the degree of the hyperglycemic drive on human islet proliferation.
  • In the second set of experiments, STZ-diabetic mice will be transplanted in the EFP with the same sub-marginal human islet masses (50, 150, 350 IEQ) and maintained on insulin during the engraftment period. 3 weeks later insulin pellet will be removed and mice will be treated with p57kip2siRNA-aptamer chimera or the scrambled control. We will monitor glycemia and β cell mass by IVIS longitudinally as readouts.
  • In both sets of experiments, glucose tolerance tests (GTTs) will be performed in mice with restored normoglycemia to further evaluate the islet function under stress conditions.
  • To ensure reproducibility in the results, at least 3 independent repetitions will be performed for each experiment using human islets from 3 different cadaveric donors. The use a total of 9 mice per experimental group (3 in each repetition) gives 90% of power (One way ANOVA, α=0.05) to detect an effect size of 1.6SD to control. 12 mice per group will be used to accounting for the higher expected variation of the read-out. To minimize readout-specific artifacts, the same phenomenon will be measured with at least 2 independent methods.
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Claims (23)

What is claimed is:
1. A method of measuring beta cell mass comprising contacting the beta cell with a construct comprising an aptamer conjugated to an imaging reagent in an amount effective to measure the mass of the beta cell.
2. The method of claim 1, wherein the aptamer is M12-3773 or 1-717.
3. The method of claim 1, wherein the imaging agent is a fluorochrome, a PET tracer or a MRI contrast reagent.
4. The method of claim 1, wherein the aptamer is conjugated to the imaging reagent via chelators.
5-16. (canceled)
17. An aptamer comprising the nucleotide sequence set forth in SEQ ID NO: 264 or SEQ ID NO: 259.
18. (canceled)
19. A construct comprising the aptamer of claim 17 conjugated to a saRNA.
20. The construct of claim 19, wherein the saRNA upregulates the protein XIAP (X-linked inhibitor of apoptosis gene id 331) or the protein CD274 (Programmed death-ligand 1, PDL1, gene id 29126)..
21. (canceled)
22. A construct comprising an aptamer conjugated to a small activating RNA (saRNA).
23. The construct of claim 22, wherein the aptamer is specific for a tissue.
24. The construct of claim 23, wherein the tissue is from an organ.
25. The construct of claim 24, wherein the organ is pancreas, heart, lung, kidney, stomach, skin or brain.
26. The construct of claim 23, wherein the tissue is pancreatic islets.
27. The construct of claim 22, wherein the construct further comprises a detectable label.
28. The construct of claim 27, wherein the detectable label is a fluorochrome, Positron emission tomography tracer such as Fluorine-18, oxygen-15, gallium 68, magnetic resonance imaging contrast agents such as gadolinium, iron oxide, iron platinum or manganese.
29. The construct of claim 22, wherein the aptamer is M12-3773 or 1-717.
30. The construct of claim 22, wherein the aptamer is specific for clusterin (CLU, gene id 1191).
31. The construct of claim 22, wherein the aptamer is specific for “Transmembrane emp24 domain-containing protein 6” (TMED6, gene id 146456).
32. The construct of claim 22, wherein the tissue is adrenal tissue,bone marrow, breast tissue, lung tissue or lymph node tissue, brain cerebellum, is brain cerebral cortex tissue, pituitary tissue, colon tissue, endothelium tissue, esophaqus tissue, heart tissue, kidney tissue, fallopian tube tissue, liver tissue, ovarian tissue, placenta tissue, prostate tissue, spinal cord tissue, testis tissue, thymus tissue, thyroid tissue, ureter tissue, cervical tissue, islets of Langerhans or pancreatic tissue.
33. The construct of claim 32, wherein the aptamer is
(a) 173-2273, 107-901 or m6-3239;
(b) 107-901 or m6-3239;
(c) 173-2273, 107-901, m1-2623 or m6-3239;
(d) 107-901, m1-2623 or m6-3239;
(e) m6-3239;
(f) 166-279, 107-901, m1-2623 or m6-3239;
(g) 107-901;
(h) 166-270, 173-2273, 107-901, m1-2623 or m6-3239;
(i) 173-2273 or 107-901;
(j) 166-279 or 173-2273;
(k) 166-279, 173-2273, 107-901, m1-2623, m6-3239 and m12-3773;
(l) 173-2273, 107-901 or mf-2623;
(m) m1-2623;
(n) 166-279; or
(o) 166-279, 173-2273, 107-901, 1-717, m1-2623, m6-3239 or m12-3773 .
34-64. (canceled)
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