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WO2024165924A2 - Procédé de purification de cellules in situ hautement efficace basé sur un ou plusieurs régulateurs d'arnm synthétiques et un peptide synthétique - Google Patents

Procédé de purification de cellules in situ hautement efficace basé sur un ou plusieurs régulateurs d'arnm synthétiques et un peptide synthétique Download PDF

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WO2024165924A2
WO2024165924A2 PCT/IB2024/000077 IB2024000077W WO2024165924A2 WO 2024165924 A2 WO2024165924 A2 WO 2024165924A2 IB 2024000077 W IB2024000077 W IB 2024000077W WO 2024165924 A2 WO2024165924 A2 WO 2024165924A2
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cell
cells
formula
mrna
alp
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WO2024165924A3 (fr
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Yi Kuang
Cheuk Yin LI
Zhenghua LIANG
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Hong Kong University of Science and Technology
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Hong Kong University of Science and Technology
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)

Definitions

  • Cell purification describes the process of separating unwanted cells from a biological sample to eliminate external influences on downstream results. It is an important method to help scientists study specific cell types. Biological samples often contain multiple cell types at different stages in their development processes and these unwanted or off-target cells can have negative impacts in scientific analyses.
  • stem cell technologies especially the induced pluripotent stem cells (iPSCs) technologies
  • differentiated cells generated from stem cells offer previously unattainable sources of cells for various purposes, including but not limited to disease modelling, cell therapy, tissue engineering, personalized drug screening.
  • iPSCs induced pluripotent stem cells
  • stem cells differentiation is rarely 100% successful: undifferentiated stem cells can exist; wrong type of cells can be generated.
  • the subject invention pertains to a cell purification method.
  • the method uses novel synthetic mRNA switches and at least one synthetic peptide.
  • the synthetic mRNA switch can encode a cell surface alkaline phosphatase sequence operably linked to a sequence targeting cytosolic cell type marker molecule.
  • the synthetic peptide is a phosphorylated D-amino acid formed peptide with N terminus modified with aromatic rings.
  • the synthetic mRNA switch can be introduced to a cell.
  • the switch can distinguish target cells from non-target cells based on the difference in the level of the cytosolic cell type marker molecules.
  • the alkaline phosphatase can be expressed only on the non-target cell surface.
  • a synthetic peptide can be added into culture media containing the cell.
  • the alkaline phosphatase triggers the dephosphorylation of the synthetic peptide. In certain embodiments, when the alkaline phosphatase is highly expressed, the dephosphorylation rate is high.
  • self-assembly of the dephosphorylated synthetic peptide can form a nanonet/hydrogel around the surface of the cells to induce viability loss of these cells.
  • the dephosphorylation rate is low. “Leaky expression” means basal level ALP expression when the translation of the mRNA is suppressed.
  • the dephosphorylated synthetic peptide can diffuse into media and the cells will not be damaged.
  • the subject methods can be used to remove stem cells.
  • the removal of stem cells prevents stem-cell caused tumor generation or colony formation and also reduces the difficulty level for selecting a marker molecule.
  • the subject methods yield a high purity and high recovery of target cells.
  • the method can be used for sensing different cytosolic marker molecules; for sensing one or more molecules; for eliminating cells with or without cytosolic marker molecules; and for purifying one or two types of cells at the same time.
  • FIGs. 1A-1B The scheme of the subject cell purification method.
  • FIG. IB The chemical structures of the peptides synthesized.
  • FIGs. 2A-2B Alkaline phosphatase activities of HEK293 at 24 hours posttransfection of different amounts of ALP mRNA and 201B7 without transfection.
  • FIG. 3 Observation of ALP triggered nanonet/hydrogel formation by the peptides.
  • the HEK293 cells were transfected with or without ALP encoding mRNA.
  • the cells were incubated with peptides at 500 pM at 37°C for 2 hours, the dishes were placed in a 4°C room for 5 min. The dishes were tilted and agitated to collect the detached nanonets/hydrogels in media. Using a wide mouth transfer pipette, the media were collected into 1.5 mL Eppendorf tubes and centrifuged at 7500 rpm for 1 minutes to reveal the nanonets/hydrogels at the bottom of the tubes.
  • FIGs. 4A-4E FIG. 4A Illustration of miR-21-ALP switch and miR-21-Bim switch.
  • FIG. 4B Relative viability of HEK293 cells 24 hours post-treatment with miR-21-ALP + D3 or miR-21-Bim, co-transfected with different amount of microRNA-21 mimic.
  • FIG. 4C Relative viability of HEK293, MDA-MB-231 and HepG2 cells 24 hours post-treatment with miR-21-ALP + D3 or miR-21-Bim (FIGs. 4D-4E) Co-culture of HEK293-CFP, MDA-MB- 231 and HepG2-RFP treated by miR-21-ALP + D3 or miR-21-Bim.
  • FIG. 4D Relative cell number of each cell line.
  • FIGs. 6A-6D FIG. 6A Illustration of neuron cell differentiation.
  • FIG. 6B Relative viability of 201B7, Neuron and 201B7dl4 cells 24 hours post-treatment with miR-31-ALP + D3, ALP mRNA + D3 or D3 only (FIGs. 6C-6D) Co-culture of 201B7, Neuron and 201B7dl4 cells.
  • FIG. 6C Representative flow cytometry analysis of the co-culture 24 hours post-treatment, cells are stained with anti-Nestin or anti-Oct3/4 antibody
  • SEQ ID NO: 2 4x a nti-miR-21-5'-UTR
  • SEQ ID NO: 5 2xanti-miR-132-5'-UTR
  • SEQ ID NO: 8 4x a nti-miR-21-3’-UTR
  • compositions containing amounts of ingredients where the term “about” is used, these compositions contain the stated amount of the ingredient with a variation (error range) of 0-10% around the value (X ⁇ 10%). In other contexts, the term “about” is providing a variation (error range) of 0- 10% around a given value (X ⁇ 10%).
  • this variation represents a range that is up to 10% above or below a given value, for example, X ⁇ 1%, X ⁇ 2%, X ⁇ 3%, X ⁇ 4%, X ⁇ 5%, X ⁇ 6%, X ⁇ 7%, X ⁇ 8%, X ⁇ 9%, or X ⁇ 10%.
  • ranges are stated in shorthand to avoid having to set out at length and describe each and every value within the range. Any appropriate value within the range can be selected, where appropriate, as the upper value, lower value, or the terminus of the range.
  • a range of 0.1-1.0 represents the terminal values of 0.1 and 1.0, as well as the intermediate values of 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, and all intermediate ranges encompassed within 0.1-1.0, such as 0.2-0.5, 0.2-0.8, 0.7-1.0, etc.
  • a range of 5-10 indicates all the values between 5.0 and 10.0 as well as between 5.00 and 10.00 including the terminal values.
  • ranges are used herein, combinations and subcombinations of ranges (e.g., subranges within the disclosed range) and specific embodiments therein are explicitly included.
  • peptide and “protein” are used interchangeably herein to refer to a polymer of amino acids.
  • the terms apply to amino acid polymers in which one or more amino acid residues are artificial chemical mimetics of corresponding naturally occurring amino acids, as well as to naturally occurring amino acid polymers and non- naturally occurring amino acid polymers.
  • the terms encompass amino acid chains of any length, including full-length proteins, wherein the amino acid residues are linked by covalent peptide bonds.
  • amino acid refers to standard nomenclature, amino acid residue as denominated by either a three letter or a single letter code as indicated as follows: Alanine (Ala, A), Arginine (Arg, R), Asparagine (Asn, N), Aspartic Acid (Asp, D), Cysteine (Cys, C), Glutamine (Gin, Q), Glutamic Acid (Glu, E), Glycine (Gly, G), Histidine (His, H), Isoleucine (He, I), Leucine (Leu, L), Lysine (Lys, K), Methionine (Met, M), Phenylalanine (Phe, F), Proline (Pro, P), Serine (Ser, S), Threonine (Thr, T), Tryptophan (Trp, W), Tyrosine (Tyr, Y), and Valine (Vai, V).
  • nucleic acid or “polynucleotide” refers to deoxyribonucleic acids (DNA) or ribonucleic acids (RNA) and polymers thereof in either single- or doublestranded form. Unless specifically limited, the term encompasses nucleic acids containing known analogs of natural nucleotides that have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides.
  • nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions), alleles, orthologs, single nucleotide polymorphisms (SNPs), and complementary sequences as well as the sequence explicitly indicated.
  • degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and/or deoxyinosine residues (Batzer et al., Nucleic Acid Res. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608 (1985); and Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)).
  • nucleic acid is used interchangeably with gene, cDNA, and mRNA encoded by a gene.
  • nucleotide probe used in the method of this invention has at least 70% sequence identity, preferably 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity, to a target sequence or complementary sequence thereof), when compared and aligned for maximum correspondence over a comparison window, or designated region as measured using one of the following sequence comparison algorithms or by manual alignment and visual inspection. Such sequences are then said to be “substantially identical”. With regard to polynucleotide sequences, this definition also refers to the complement of a test sequence.
  • a nucleotide sequence is referred to as “operably linked” when it is placed into a functional relationship with another nucleotide segment (for example, an mRNA sequence encoding a cell surface alkaline phosphatase linked to a nucleotide sequence targeting a cytosolic cell type marker molecule).
  • Two or more operably linked nucleotide sequences can be separated by 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, about 25, about 30, about 45, about 50, about 75, about 100, about 150, or more nucleotides. Linking can be accomplished by ligation at convenient restriction sites or at adapters or linkers inserted in lieu thereof.
  • reduces is meant a negative alteration of at least 1%, 5%, 10%, 25%, 50%, 75%, or 100%.
  • compositions or methods provided herein can be combined with one or more of any of the other compositions and methods provided herein.
  • the subject invention pertains to a cell purification method.
  • the method uses novel synthetic mRNA switches of from about 1600 to about 3000 nucleotides in length and at least one synthetic peptide.
  • the synthetic mRNA switch can encode a cell surface placental alkaline phosphatase sequence (NCBI Reference Sequence: NP 001623.3) operably linked to a sequence targeting cytosolic cell type marker molecule or an mRNA sequence encoding a cell surface alkaline phosphatase operably linked to a nucleotide sequence targeting a viral coat protein and an mRNA sequence encoding a viral coat protein operably linked to a nucleotide sequence targeting a cytosolic cell type marker molecule.
  • the mRNA switch is an RNA oligonucleotide.
  • the cytosolic cell type marker molecule is a microRNA (miR), preferably a high expressing miRNA selected from those well-known in the art, as can be found, for example, at miRbase (https://mirbase.org).
  • the miR is miR-31, miR-21, or miR-132.
  • the RNA binding protein is bacteriophage MS2 coat protein (MCP), L7Ae (Uniplot: Q8U160), or PP7 coat protein (NP 042305.1).
  • the sequence encoding the cell surface alkaline phosphatase sequence is separated from the sequence targeting cytosolic cell type marker molecule or the sequence targeting RNA binding protein by at 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, about 25, about 30, about 45, about 50, about 75, about 100, about 150, or more nucleotides.
  • the synthetic peptide is a phosphorylated D-amino acid formed peptide with N terminus modified with aromatic rings.
  • the phosphorylated D-amino acid formed peptide with an N terminus modified with an aromatic ring is D-l (Formula (I)), D-2 (Formula (II)), D-3 (Formula (III)), D-4 (Formula (IV)), D-5 (Formula (V)), D-6 (Formula (VI)), D-7 (Formula (VII)), or D-8 (Formula (VIII)):
  • the phosphorylated D-amino acid formed peptide with an N terminus modified with an aromatic ring is D-3 (Formula (III)).
  • the synthetic mRNA switch can be introduced to a cell, preferably at a concentration of at least 200pM.
  • the switch can distinguish target cells from non-target cells based on the difference in the level of the cytosolic cell type marker molecules.
  • the alkaline phosphatase can be expressed only on the non-target cell surface.
  • a synthetic peptide can be added into culture media containing the cell, preferably at a concentration of at least 200pM.
  • the alkaline phosphatase triggers the dephosphorylation of the synthetic peptide. In certain embodiments, when the alkaline phosphatase is highly expressed, the dephosphorylation rate is high.
  • the target cell type is a neuron.
  • the non-target cell type is any cell type without the miRNA of the target. In certain embodiments, the non-target cell type is a stem cell.
  • self-assembly of the dephosphorylated synthetic peptide can form a nanonet/hydrogel around the surface of the non-target cells to induce viability loss of these non-target cells.
  • the dephosphorylation rate is low.
  • the dephosphorylated synthetic peptide can diffuse into media and the cells will not be damaged.
  • Stem cells naturally express alkaline phosphatase on the surface; therefore, in certain embodiments, the subject methods can be used to remove stem cells. In certain embodiments, the removal of stem cells prevents stem-cell caused tumor generation or colony formation and also reduces the difficulty level for selecting a marker molecule. In certain embodiments, there is no need to consider the marker molecule level in stem cells.
  • the subject methods yield a high purity and high recovery of target cells.
  • the method can be used for sensing different cytosolic marker molecules; for sensing one or more molecules; for eliminating cells with or without cytosolic marker molecules; and for purifying one or two types of cells at the same time.
  • This invention describes a method for in situ cell purification, which comprises the following steps:
  • the non-target cell is a stem cell. In certain embodiments, at least about 75%, about 80%, about 85%, about 90%, about 95%, about 97.5%, about 99%, about 99.9%, about 99.99% of the non-target cells are eliminated.
  • Table 1 shows the nucleotide sequences of UTRs used in all the samples. All the UTR sequences were incorporated into the DNA templates encoding for alkaline phosphatase (ALP), bcl-2 interacting mediator of cell death (Bim), puromycin resistant gene (PuroR), enhanced green fluorescent protein (EGFP) or bacteriophage MS2 coat protein (MCP) using PCR (Q5® High-Fidelity 2X Master Mix). The purified PCR products were directly used for in vitro synthesis of mRNAs using MEGAscriptTM T7 Transcription Kit (Invitrogen (Carlsbad, California, USA)).
  • ALP alkaline phosphatase
  • Bim bcl-2 interacting mediator of cell death
  • PuroR puromycin resistant gene
  • EGFP enhanced green fluorescent protein
  • MCP bacteriophage MS2 coat protein
  • the mRNAs were synthesized with adenosine triphosphate, cytidine triphosphate, N 1 methylpseudouridine-5’ -triphosphate, guanosine triphosphate and Anti Reverse Cap Analog (TriLink BioTechnologies (San Diego, California, USA)) with a molar ratio at 5:5:5: 1 :4. All peptides (FIGs. 1A-1B) were synthesized by standard solidphase peptide synthesis. All the cells are cultured and passaged using standard media and standard Trypsin protocol. LipofectamineTM MessengerMaxTM (Therm ofisher(Waltham, Massachusetts, USA)) was used for all the transfections of mRNAs, following the manufacturer’s protocol.
  • HEK293 and 201B7 cells were seeded in 24-well plate 24 hours before transfection at 0.5 x 10 5 cells/well.
  • HEK293 were transfected with 90, 170, 260, 340, 430, 510, 600, 680, 770 or 860 nM of ALP mRNA and co-transfected with 90 nM PuroR mRNA for positively transfected cells selection.
  • cell lysates were collected using CelLytic M (Sigma- Aldrich) following manufacturer’s protocol.
  • the ALP activities were determined by alkaline phosphatase assay kit (Abeam (Cambridge, United Kingdom)) following manufacturer’s protocol.
  • HEK293 were transfected with 600 nM of ALP mRNA and co-transfected with 90 nM PuroR mRNA for positively transfected cells selection.
  • peptide treatment described in method, of D-l, D-2, D-3, D-4, D-5, D-6, D-7, D-8, or D-9 was performed.
  • MTT based cell viability assay was performed following manufacturer’s protocol.
  • FIGs. 1A-1B The ALP activities increased with the amount of mRNA transfected (FIG. 2A).
  • FIG. 2A The ALP activities plateaued above 510 nM mRNA transfected with ALP activity of around 70 mU/ng which is comparable to high ALP activity cell 201B7.
  • HEK293, MDA-MB-231 and HepG2 cells were seeded in 24-well plate 24 hours before transfection at 0.5 x 10 5 cells/well.
  • HEK293-CFP, MDA-MB-231 and HepG2-RFP were seeded in 6-well plate 24 hours before transfection at 1 x 10 5 , 2 x 10 5 and 2 x 10 5 cells/well respectively. All cells were transfected with 600nM of miR-21-ALP or miR-21-Bim, and co-transfected with 90 nM PuroR mRNA for positively transfected cells selection.
  • peptide treatment with D-3 peptide was performed for cells transfected by ALP mRNA. After that, MTT based cell viability assay was performed following manufacturer’s protocol. For coculture experiment, the cells were resuspended in culture medium with trypsin and analysis by flow cytometry with BD FACSAria III (BD biosciences (Franklin Lakes, New lersey, USA)).
  • the Bim protein is a cytotoxic protein and Bim expressing synthetic mRNA switches are previously described methods for cell purification. Both of the switches were designed to sense microRNA-21 to reduce protein expression level.
  • the performance of both switches were increased amount of artificial microRNA-21 mimic.
  • the viability of cells treated with miR-21-ALP switch + D-3 reached above 80% at 1 nM mimic, while that of the cells treated with miR-21-Bim switch remained around 50% even at 4 nM mimic (FIG. 4B). This showed that the miR-21-ALP switch +D-3 is more sensitive to low microRNA concentration as shown in the increase of viability at low mimic concentration.
  • HEK293 was effectively removed in both group while the viability of MD A-MB-231 and HepG2 was only recovered in miR-21-ALP switch + D3 group.
  • miR-21-Bim group the viability for MD A-MB-231 remained below 20% and the viability for HepG2 could only recover up to 70%. This further provide evidence for miR-21-ALP switch + D3 being more sensitive on microRNA.
  • the cells treated with miR-21-Bim switch remained at low cell viability even at high artificial mimic concentration, and in high microRNA-21 activity cell lines. This might suggest leaky expression of cytotoxic protein.
  • the miR-21- ALP + D3 group we observed viability recovery up to >80% for artificial mimic, -100% for high microRNA-21 activity cell lines and even in co-culture condition. Therefore, our method could effectively reduce the effect of leaky expression from microRNA switches.
  • HEK293 were seeded in 24-well plate 24 hours before transfection at 0.5 x 10 5 cells/well.
  • the cells were transfected with 600 nM of miR-21-ALP, miR-IDT- ALP, miR-21/IDT-ALP or both miR-21-ALP and miR-IDT-ALP (noted as miR-21- ALP/miR-IDT-ALP).
  • the cells were co-transfected with 2 nM microRNA mimics and 90 nM PuroR mRNA or 90 nM PuroR mRNA only for the group “w/o mimic”.
  • peptide treatment with D-3 peptide was performed. After that, MTT based cell viability assay was performed following manufacturer’s protocol.
  • FIG. 5A ALP expressing mRNA switches that sense miR-21, miR-IDT, or both
  • FIG. 5B the cell viabilities response to each switch and the co-transfected microRNA mimic as expected.
  • a drop in viability for target cell especially in miR-IDT-ALP switch group can also be observed.
  • multi-input logic circuits more precise and criteria of cell purification can be achieved.
  • 201B7, Neuron and 201B7dl4 were seeded in 24-well plate 24 hours before transfection at 0.5 x 10 5 cells/well.
  • differentiated neuron culture and 201B7dl4 were seeded in 6-well plate 24 hours before transfection at 1 x 10 5 and 1 x 10 5 cells/well respectively.
  • the cells were transfected with 600 nM of miR-31- ALP or ALP mRNA and co-transfected with 90 nM PuroR mRNA for positively transfected cells selection.
  • peptide treatment with D-3 peptide was performed. After that, MTT based cell viability assay was performed following manufacturer’s protocol.
  • the cells were either analysis by antibody staining or alkaline phosphatase staining.
  • antibody staining the cells were resuspended in culture medium with trypsin and stained with anti-Nestin antibody (Abeam) and anti-OCT3/4 antibody Alexa Fluor 488 (Thermofisher) following standard antibody staining protocol. The result was analyzed by flow cytometry with Attune NxT (Invitrogen).
  • alkaline phosphatase staining the cells were kept culturing until day 7 after peptide treatment. The alkaline phosphatase of the cells was stained by Alkaline Phosphatase stain kit (Thermofisher) following manufacturer’s protocol.
  • iPSC 201B7 into neuron cells (FIG. 6A) and construct miR-31-ALP switch, where microRNA-31 is the marker microRNA for neuron cells.
  • miR-31 is the marker microRNA for neuron cells.
  • 201B7dl4 we differentiated the 201B7 into randomly differentiated tissue nonspecific cells, 201B7dl4. As shown in FIG. 6B, the viability of neuron remained >90% for both miR-31 ALP switch + D3 and D3 only.
  • the undifferentiated 201B7 had high level of ALP activity as stated in the first section, was effectively removed in all condition with D3 treatment, while the randomly differentiated 201B7dl4 was only eliminated in the present of either miR-31 -ALP switch or ALP mRNA. Then, we differentiated the neuron cells, added with 201B7dl4 and purified it with our method. As shown in FIG. 6C, the treated cells successfully restored neuron population (Nestin+) from -50% to 90% while the undifferentiated population is reduced from -13% to 2%. Furthermore, we continued incubating the co-culture for a week and the undifferentiated cells were stained with ALP stain. As shown in FIG. 6D, due to the difference of growth rate, majority of control group was undifferentiated cells. For the treated group, surprisingly, no undifferentiated cells are observed. This showed that our method can successfully purify cells during differentiation.
  • HEK293 were seeded in 24-well plate 24 hours before transfection at 0.5xl0 5 cells/well.
  • the cells were transfected with 600 nM of MS2-ALP or MS2-Bim.
  • the cells were co-transfected with 160 nM MCP mRNA and 90 nM PuroR mRNA or PuroR mRNA only.
  • peptide treatment with D-3 peptide was performed for cells transfected by ALP mRNA. After that, MTT based cell viability assay was performed following manufacturer’s protocol.

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Abstract

La présente invention concerne un procédé de purification de cellules in situ. Plus spécifiquement, le procédé comprend un régulateur d'ARNm synthétique et un peptide synthétique. Le régulateur d'ARNm synthétique contient une séquence codant pour une phosphatase alcaline de surface cellulaire liée de manière fonctionnelle à une séquence ciblant une molécule marqueur de type cellule cytosolique ou une protéine d'enveloppe virale et/ou une séquence ciblant une molécule marqueur de type cellule cytosolique et une protéine d'enveloppe virale. Le peptide synthétique est un peptide formé par un acide aminé D phosphorylé avec une terminaison N modifiée par des cycles aromatiques.
PCT/IB2024/000077 2023-02-06 2024-02-06 Procédé de purification de cellules in situ hautement efficace basé sur un ou plusieurs régulateurs d'arnm synthétiques et un peptide synthétique Ceased WO2024165924A2 (fr)

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