Efficient in-situ cell purification method based on synthetic mRNA switch and synthetic peptide

Abstract

The invention relates to an in-situ cell purification method. More specifically, the method includes synthesizing an mRNA switch and synthesizing a peptide. The synthetic mRNA switch includes a sequence encoding a cell surface alkaline phosphatase that can be linked to a sequence of a targeted cytosolute cell type marker molecule or a viral coat protein and / or a sequence of a targeted cytosolute cell type marker molecule and a viral coat protein. The synthetic peptide is a peptide formed by phosphorylating a D-amino acid and having an N-terminal modified by an aromatic ring.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of U.S. Provisional Application Serial No. 63 / 483,519, filed February 6, 2023, the entire contents of which are incorporated herein by reference.

[0003] Sequence Listing

[0004] The sequence listing of this application is named "HKUS187XPCT.xml", which was created on February 5, 2024, and is 8,662 bytes in length. The entire content of this sequence listing is incorporated herein by reference in its entirety. Background Art

[0005] Cell purification describes the process of isolating unwanted cells from a biological sample to eliminate extraneous influences on downstream results. Cell purification is an important method that helps scientists study specific cell types. Biological samples often contain multiple cell types at different stages of their development, and these unwanted, or non-target, cells can negatively impact scientific analysis.

[0006] With the development of stem cell technology, particularly induced pluripotent stem cell (iPSC) technology, differentiated cells derived from stem cells have provided previously unavailable cell sources for a variety of purposes, including but not limited to disease modeling, cell therapy, tissue engineering, and personalized drug screening. However, one obstacle to its application is the purity of the target cells. Stem cell differentiation is rarely 100% successful: undifferentiated stem cells may exist, and the wrong cell type may be produced.

[0007] There are many methods for purifying cell samples, but the two most popular methods are magnetic bead-based cell sorting and fluorescence-activated cell sorting. Currently, competing products and services for purifying cells focus mainly on the use of antibodies for cell identification. Although this method can effectively purify cells based on cell type-specific surface antigens, such methods are limited by the availability of cell type-specific surface antigens and the need to convert attached cells into suspension, which damages the target cells and results in low recovery rates. Existing in situ methods include chemical or peptide-based in situ cell purification and synthetic mRNA switch-based in situ cell purification. Chemical or peptide-based methods are designed to target only specific cells and lack universal applicability. The recovery rate of target cells for purification based on synthetic mRNA switches is low, and additional consideration is required to remove stem cells. Therefore, new methods are needed to more effectively purify in situ cells. Summary of the Invention

[0008] The present invention relates to a cell purification method. The method utilizes a novel synthetic mRNA switch and at least one synthetic peptide. In some embodiments, the synthetic mRNA switch can encode a cell surface alkaline phosphatase sequence that can be linked to a sequence targeting a cytosolic cell type marker molecule. In some embodiments, the synthetic peptide is formed from phosphorylated D-amino acids and has an N-terminus modified with an aromatic ring.

[0009] In some embodiments, a synthetic mRNA switch can be introduced into cells. In some embodiments, the switch can distinguish target cells from non-target cells based on differences in the levels of cytosolic cell type marker molecules. In some embodiments, alkaline phosphatase can be expressed only on the surface of non-target cells. In some embodiments, after the switch is introduced, the synthetic peptide can be added to the culture medium containing the cells. In some embodiments, alkaline phosphatase triggers dephosphorylation of the synthetic peptide. In some embodiments, when alkaline phosphatase is highly expressed, the dephosphorylation rate is high.

[0010] In some embodiments, the self-assembly of dephosphorylated synthetic peptides can form a nanomesh / hydrogel around the surface of cells, resulting in reduced survival of these cells. In some embodiments, the dephosphorylation rate is low when leaky expression of alkaline phosphatase (ALP) occurs. "Leaky expression" refers to the basal level of ALP expression when the translation of mRNA is inhibited. In some embodiments, the dephosphorylated synthetic peptides can diffuse into the culture medium and the cells will not be damaged.

[0011] Stem cells naturally express alkaline phosphatase on their surface; therefore, in some embodiments, the subject methods can be used to remove stem cells. In some embodiments, removing stem cells prevents tumor formation or colony formation caused by stem cells and also reduces the difficulty level of selecting marker molecules. In some embodiments, the level of marker molecules in stem cells does not need to be considered.

[0012] In some embodiments, the subject methods provide high purity and high recovery of target cells. In some embodiments, the methods can be used to sense different cytosolic marker molecules; to sense one or more molecules; to remove cells with or without cytosolic marker molecules; and to purify one or two types of cells simultaneously. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] This patent or patent application file contains at least one drawing drawn in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0014] Figure 1A to Figure 1B . Figure 1A Schematic diagram of the subject cell purification method. Figure 1B Chemical structures of synthetic peptides.

[0015] Figures 2A to 2B . Figure 2A Alkaline phosphatase activity of HEK293 and untransfected 201B7 24 h after transfection with different amounts of ALP mRNA. Figure 2B Survival rate of HEK293 cells 24 hours after treatment with peptides transfected with or without ALP mRNA (n=3; data are expressed as mean ± SD).

[0016] Figure 3 Observe the formation of nanomesh / hydrogels triggered by ALP with the peptide. HEK293 cells were transfected with or without mRNA encoding ALP. On the second day, the cells were incubated with 500 μM of the peptide at 37°C for 2 hours, and the culture dish was placed in a 4°C room for 5 minutes. The culture dish was tilted and agitated to collect the nanomesh / hydrogel that had been shed from the culture medium. Using a wide-mouth pipette, the culture medium was collected into a 1.5 mL Eppendorf tube and centrifuged at 7500 rpm for 1 minute to reveal the nanomesh / hydrogel at the bottom of the tube.

[0017] Figures 4A to 4E . Figure 4A Schematic representation of the miR-21-ALP switch and miR-21-Bim switch. Figure 4B After 24 hours of treatment with miR-21-ALP+D3 or miR-21-Bim,

[0018] Relative survival rate of HEK293 cells co-transfected with the same amount of microRNA-21 mimics. Figure 4C Relative viability of HEK293, MDA-MB-231, and HepG2 cells 24 hours after treatment with miR-21-ALP+D3 or miR-21-Bim. Figures 4D to 4E ) Co-cultures of HEK293-CFP, MDA-MB-231, and HepG2-RFP treated with miR-21-ALP+D3 or miR-21-Bim. Figure 4D Relative cell numbers of each cell line. Figure 4E Representative flow cytometry dot plots (n=3; data are expressed as mean ± SD).

[0019] Figure 5Graphs of the miR-21-ALP switch, miR-IDT-ALP switch, and miR-21 / IDT-ALP switch. Relative viability of HEK293 cells 24 hours after treatment with D3. Twenty-four hours before treatment with D3, cells were transfected with the miR-21-ALP switch, miR-IDT-ALP switch, miR-21 / IDT-ALP switch, and miR-21-ALP + miR-IDT-ALP switch, and co-transfected with microRNA-21 mimics, microRNA-IDT mimics, microRNA-21 + microRNA-IDT mimics, or without mimics (n = 3; data are presented as mean ± SD).

[0020] 6A to 6D . Figure 6A Schematic representation of neuronal cell differentiation. Figure 6B Relative survival rates of 201B7, neurons, and 201B7d14 cells 24 hours after treatment with miR-31-ALP+D3, ALP mRNA+D3, or D3 alone. ( Figures 6C to 6D )

[0021] Co-culture of 201B7, neuronal, and 201B7d14 cells. Figure 6C Representative flow cytometric analysis of co-cultures 24 hours after treatment, cells were stained with anti-nestin or anti-Oct3 / 4 antibodies. Figure 6D Co-cultures 7 days after treatment were stained for ALP (n=3; data are expressed as mean ± SD).

[0022] Figure 7 A switch encoding ALP, a protein that senses MCP proteins, treated with D-3 peptide effectively eliminated cells expressing MCP proteins. Cells without MCP protein expression experienced minimal loss. Cell purification performance was significantly superior to that of a switch encoding Bim, a protein that senses MCP proteins. (n = 3; data are presented as mean ± SD).

[0023] A brief description of the sequence

[0024] SEQ ID NO:1:5′-UTR

[0025] SEQ ID NO:2:4×anti-miR-21-5′-UTR

[0026] SEQ ID NO:3:4×anti-IDT-5′-UTR

[0027] SEQ ID NO:4:2×anti-31-5′-UTR

[0028] SEQ ID NO:5:2x anti-miR-132-5′-UTR

[0029] SEQ ID NO:6:2xMS2-5′UTR

[0030] SEQ ID NO:7:3′-UTR

[0031] SEQ ID NO:8:4×anti-miR-21-3′-UTR DETAILED DESCRIPTION

[0032] Selected definition

[0033] As used herein, the singular forms "a," "an," and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise. Furthermore, to the extent that the terms "include," "comprises," "includes," "has," "with," or variations thereof are used in the detailed description and / or claims, such terms are intended to be inclusive in a manner similar to the term "comprising." The transitional terms / phrases (and any grammatical variations thereof) "comprising," "including," "consisting essentially of," and "consisting of" may be used interchangeably.

[0034] The term "about" means within an acceptable error range for a particular value as determined by one of ordinary skill in the art, the error resulting from which depends in part on how the value is measured, i.e., the limitations of the measurement system. In the case of compositions containing a certain amount of ingredients for which the term "about" is used, these compositions contain the ingredients in that amount with a variation (error range) of 0% to 10% (X ± 10%) around the value. In other cases, the term "about" provides a variation (error range) of 0% to 10% (X ± 10%) around a given value. Clearly, this variation represents a range of 10% above or below a given value, such as X ± 1%, X ± 2%, X ± 3%, X ± 4%, X ± 5%, X ± 6%, X ± 7%, X ± 8%, X ± 9%, or X ± 10%.

[0035] In the present disclosure, scope is stated in abbreviated form to avoid having to state in detail and describe each value and all values in the scope. Where appropriate, any suitable value in the scope can be selected as the upper limit, lower limit or end value of the scope. For example, scope 0.1 to 1.0 represents and includes end value 0.1 and 1.0 and intermediate value 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9 and all intermediate ranges included in 0.1 to 1.0, such as 0.2 to 0.5, 0.2 to 0.8, 0.7 to 1.0, etc. It is conceivable that, in a range, there are the values of at least two significant figures, such as, scope 5 to 10 represents between 5.0 and 10.0 and all values between 5.00 and 10.00, including end value. When using scope herein, the combination and sub-combination (such as, the sub-range in the disclosed scope) and specific embodiments thereof are clearly included in scope.

[0036] In this application, the terms "peptide" and "protein" are used interchangeably herein to refer to polymers of amino acids. The term applies to amino acid polymers in which one or more amino acid residues is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as naturally occurring amino acid polymers and non-naturally occurring amino acid polymers. As used herein, the term encompasses amino acid chains of any length, including full-length proteins, in which the amino acid residues are linked by covalent peptide bonds.

[0037] As used herein, the term "amino acid" refers to the standard nomenclature, with amino acid residues represented by either a three-letter or one-letter code as follows: alanine (Ala, A), arginine (Arg, R), asparagine (Asn, N), aspartic acid (Asp, D), cysteine (Cys, C), glutamine (Gln, Q), glutamic acid (Glu, E), glycine (Gly, G), histidine (His, H), isoleucine (Ile, 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 (Val, V).

[0038] As used herein, the term "nucleic acid" or "polynucleotide" refers to a polymer of deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) and single-stranded or double-stranded forms thereof. Unless otherwise specified, the term encompasses those nucleic acids comprising known analogs of natural nucleotides, which have binding properties similar to the nucleic acids mentioned and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise specified, a particular nucleic acid sequence also implies and encompasses conservatively modified variants (e.g., degenerate codon substitutions), alleles, homologous sequences, single nucleotide polymorphisms (SNPs), and complementary sequences, as well as sequences explicitly noted. Specifically, degenerate codon substitution can be achieved by generating the following sequences: wherein the third position of one or more selected (or all) codons is substituted with mixed base and / or deoxyinosine residues (see Batzer et al., Nucleic Acids 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)). The term nucleic acid can be used interchangeably with gene, cDNA, and mRNA encoded by a gene.

[0039] As used herein, in the context of describing two or more polynucleotide or amino acid sequences, the term "identical" or percentage "identity" refers to two or more identical or specified percentages of amino acid residues or identical nucleotide sequences or subsequences (e.g., the nucleotide probes used in the methods of the present invention have at least 70% sequence identity, preferably 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the target sequence or its complementary sequence when compared and aligned for maximum correspondence over a comparison window, or using one of the following sequence comparison algorithms, or by manual alignment and visual inspection. Such sequences are referred to as "substantially identical." With respect to polynucleotide sequences, this definition also refers to the complementary sequence of a test sequence.

[0040] When a nucleotide sequence establishes a functional relationship with another nucleotide fragment (e.g., an mRNA sequence encoding a cell surface alkaline phosphatase connected to a nucleotide sequence targeting a cytosolic cell type marker molecule), the nucleotide sequence is referred to as "capable of connecting." Two or more nucleotide sequences capable of connecting can be spaced apart 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. Connection can be achieved by carrying out a ligation reaction at a suitable restriction site or at a connector or linker inserted in place of the restriction site.

[0041] "Decrease" refers to a negative change of at least 1%, 5%, 10%, 25%, 50%, 75%, or 100%.

[0042] By "increase" is meant a positive change of at least 1%, 5%, 10%, 25%, 50%, 75%, or 100%.

[0043] Any composition or method provided herein can be combined with one or more of any other compositions and methods provided herein.

[0044] Other features and advantages of the invention will be apparent from the following description of preferred embodiments of the invention, and from the claims.

[0045] All references cited herein are incorporated by reference in their entirety.

[0046] Cell purification methods

[0047] The present invention relates to a method for cell purification. The method uses a new synthetic mRNA switch of about 1600 to about 3000 nucleotides in length and at least one synthetic peptide. In some embodiments, the synthetic mRNA switch can encode a cell surface placental alkaline phosphatase sequence (NCBI reference sequence: NP_001623.3) that can be connected to a sequence targeting a cytosolic cell type marker molecule, or an mRNA sequence encoding a cell surface alkaline phosphatase that can be connected to a nucleotide sequence targeting a viral coat protein, and an mRNA sequence encoding a viral coat protein that can be connected to a nucleotide sequence targeting a cytosolic cell type marker molecule. In some embodiments, the mRNA switch is an RNA oligonucleotide. In some embodiments, the cytosolic cell type marker molecule is a microRNA (miR), preferably a highly expressed miRNA, which is selected from highly expressed miRNAs known in the art, such as those found in (for example) miRbase (https: / / mirbase.org). In some embodiments, the miR is miR-31, miR-21, or miR-132. In some embodiments, the RNA binding protein is bacteriophage MS2 coat protein (MCP), L7Ae (Uniplot: Q8U160) or PP7 coat protein (NP_042305.1). In some embodiments, the sequence encoding the cell surface alkaline phosphatase sequence is separated from the sequence targeting the cytosolic cell type marker molecule or the sequence targeting the RNA binding protein 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.

[0048] In some embodiments, the synthetic peptide is a peptide formed from phosphorylated D-amino acids and having an N-terminus modified by an aromatic ring. In some embodiments, the peptide formed from phosphorylated D-amino acids and having an N-terminus modified by an aromatic ring is D-1 (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)):

[0049] Formula (I)

[0050]

[0051] Formula (II)

[0052]

[0053] Formula (III)

[0054]

[0055] Formula (IV)

[0056]

[0057] Formula (V)

[0058]

[0059] Formula (VI)

[0060]

[0061] Formula (VII)

[0062]

[0063] Formula (VIII)

[0064]

[0065] In a preferred embodiment, the peptide formed from phosphorylated D-amino acids and having an N-terminus modified with an aromatic ring is D-3 (Formula (III)).

[0066] In some embodiments, the synthetic mRNA switch can be introduced into the cell at a concentration of preferably at least 200 pM. In some embodiments, the switch can distinguish target cells from non-target cells based on the difference in the levels of cytosolic cell type marker molecules. In some embodiments, alkaline phosphatase can be expressed only on the surface of non-target cells. In some embodiments, after the switch is introduced, the synthetic peptide can be added to the culture medium containing the cells at a concentration of preferably at least 200 μM. In some embodiments, alkaline phosphatase triggers dephosphorylation of the synthetic peptide. In some embodiments, when alkaline phosphatase is highly expressed, the dephosphorylation rate is high. In some embodiments, the target cell type is a neuron. The non-target cell type is any cell type that does not contain the target miRNA. In some embodiments, the non-target cell type is a stem cell.

[0067] In some embodiments, the self-assembly of the dephosphorylated synthetic peptides can form a nanomesh / hydrogel around the surface of non-target cells, thereby reducing the survival rate of these non-target cells. In some embodiments, when leaky expression of alkaline phosphatase occurs, the dephosphorylation rate is low. In some embodiments, the dephosphorylated synthetic peptides can diffuse into the culture medium without damaging the cells.

[0068] Stem cells naturally express alkaline phosphatase on their surface; therefore, in some embodiments, the subject methods can be used to remove stem cells. In some embodiments, removing stem cells prevents tumor formation or colony formation caused by stem cells and also reduces the difficulty level of selecting marker molecules. In some embodiments, the level of marker molecules in stem cells does not need to be considered.

[0069] In some embodiments, the subject methods produce target cells of high purity and high recovery. In some embodiments, the methods can be used to sense different cytosolic marker molecules; to sense one or more molecules; to remove cells with or without cytosolic marker molecules; and to purify one or two types of cells simultaneously.

[0070] The present invention describes a method for in situ cell purification, comprising the following steps:

[0071] introducing into cells an mRNA having a sequence encoding a cell surface alkaline phosphatase that can be linked to a sequence targeting a cytosolic cell type marker molecule; or introducing into cells an mRNA having a sequence encoding a cell surface alkaline phosphatase that can be linked to a sequence targeting a viral coat protein and an mRNA having a sequence encoding a viral coat protein that can be linked to a sequence targeting a cytosolic cell type marker molecule; and

[0072] The cells are incubated in a medium containing a peptide formed from phosphorylated D-amino acids and having an N-terminus modified with an aromatic ring.

[0073] In some embodiments, the non-target cells are stem cells. In some 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 removed.

[0074] Materials and methods

[0075] Table 1 shows the nucleotide sequences of the UTRs used in all samples. High-Fidelity 2X Master Mix) was used to introduce the entire UTR sequence into DNA templates encoding alkaline phosphatase (ALP), bcl-2 cell death interaction mediator (Bim), puromycin resistance gene (PuroR), enhanced green fluorescent protein (EGFP), or bacteriophage MS2 coat protein (MCP). The purified PCR products were directly used for PCR using MEGAscript TMmRNA was synthesized in vitro using the T7 transcription kit (Invitrogen, Carlsbad, CA, USA). ATP, cytidine, and N-terminal ATP were used in a molar ratio of 5:5:5:1:4. 1 mRNA was synthesized using methylpseudouridine-5'-triphosphate, guanosine triphosphate, and anti-reverse cap analogs (TriLink BioTechnologies (San Diego, CA, USA)). All peptides were synthesized by standard solid-phase peptide synthesis ( Figure 1A to Figure 1B All cells were cultured and passaged using standard culture medium and a standard trypsin protocol. TM MessengerMax TM (Thermofisher (Waltham, MA, USA)) was used for transfection of all mRNAs. For all transfections, 90 nM of PuroR mRNA was co-transfected for positive transfection cell selection. Positive transfection cells were selected by adding puromycin to a final concentration of 4 μg / mL 4 hours after transfection. Positive transfection cell selection was performed in all experiments. All peptide treatments were performed by first dissolving the peptide in water and NaOH to 10 mM. The peptide solution was diluted in culture medium to a final concentration of 500 μM. The cell culture medium was replaced with culture medium containing D-3 and kept for 2 hours. After incubation for 2 hours, the culture medium was replaced with fresh culture medium and incubated for an additional 22 hours before further analysis.

[0076] Table 1. UTR sequences and samples containing them.

[0077]

[0078]

[0079] All patents, patent applications, provisional applications, and publications referenced or cited herein, including all figures and tables thereof, are incorporated herein by reference in their entirety to the extent they are not inconsistent with the explicit teachings of this specification.

[0080] The following are examples illustrating the methods of practicing the present application. These examples should not be construed as limiting. Unless otherwise indicated, all percentages are by weight, and all solvent mixtures are by volume.

[0081] Example 1 - Cell purification method can effectively remove cells

[0082] HEK293 and 201B7 cells were cultured at 0.5 × 10 5Cells / well were seeded into 24-well plates and transfected 24 hours later. HEK293 were transfected with 90nM, 170nM, 260nM, 340nM, 430nM, 510nM, 600nM, 680nM, 770nM or 860nM ALP mRNA and co-transfected with 90nM PuroR mRNA for positive transfection cell selection. 24 hours after transfection, cell lysates were collected using CelLytic M (Sigma-Aldrich) according to the manufacturer's protocol. ALP activity was determined by alkaline phosphatase assay kit (Abcam (Cambridge, UK)) according to the manufacturer's protocol. For peptide comparison, HEK293 were transfected with 600nM ALP mRNA and co-transfected with 90nM PuroR mRNA for positive transfection cell selection. As described in the methods of the present invention, peptide treatment was performed on D-1, D-2, D-3, D-4, D-5, D-6, D-7, D-8 or D-9 24 hours after transfection. An MTT-based cell viability assay was performed according to the manufacturer's protocol.

[0083] We optimized the mRNA concentration and peptide type for cell purification. We synthesized a series of phosphate-conjugated peptides ( Figure 1A to Figure 1B To determine the concentration for the following tests, we transfected HEK293 cells with increasing amounts of ALP mRNA. ALP activity increased with increasing amounts of transfected mRNA ( Figure 2A We observed that ALP activity stabilized when the transfected mRNA concentration was higher than 510 nM, with an ALP activity of approximately 70 mU / ng, which was comparable to that of the high ALP activity cell line 201B7.

[0084] Next, we treated cells transfected with 600 nM ALP mRNA with a series of peptides. Among the peptides tested, D-3 was the most effective in cell removal, with a cell viability of <10% ( Figure 2B ). In addition, when cells were not transfected with ALP mRNA, cell viability remained at 100%. This shows that peptides without ALP do not cause any toxic effects, which is ideal for cell purification to increase yield.

[0085] Example 2—Cell purification method is highly sensitive to microRNAs

[0086] In the microRNA sensing experiments, HEK293, MDA-MB-231, and HepG2 cells were cultured at 0.5 × 10 5 Cells / well were seeded into 24-well plates and transfected 24 hours later. For co-culture experiments, HEK293-CFP, MDA-MB-231, and HepG2-RFP were each inoculated at 1×105 cells / well, 2×10 5 cells / well and 2×10 5 Cells / well were seeded into 6-well plates and transfected 24 hours later. All cells were transfected with 600 nM miR-21-APL or miR-21-Bim and co-transfected with 90 nM PuroR mRNA for positive transfection cell selection. 24 hours after transfection, cells transfected with ALP mRNA were treated with D-3 peptide. Afterwards, MTT-based cell viability assays were performed according to the manufacturer's protocol. For co-culture experiments, cells were resuspended in trypsin-containing culture medium and analyzed by flow cytometry with a BD FACSAria III (BD biosciences (Franklin Lakes, NJ, USA)).

[0087] We synthesized two mRNA switches expressing ALP and Bim, respectively ( Figure 4A ). The Bim protein is a cytotoxic protein, and the synthetic mRNA switch expressing Bim is what was used in the aforementioned cell purification method. These two switches were designed to sense microRNA-21 to reduce protein expression levels. We first compared the performance of the two switches with increasing amounts of artificial microRNA-21 mimics. We observed that cells treated with the miR-21-APL switch + D-3 achieved a survival rate of over 80% at 1 nM mimic, while cells treated with the miR-21-Bim switch maintained a survival rate of approximately 50% even at 4 nM mimics ( Figure 4B This suggests that the miR-21-ALP switch + D-3 is more sensitive to low microRNA concentrations, as shown by the increased survival rate at low mimic concentrations.

[0088] Next, we compared the responses of the two groups to endogenous microRNA activity. We treated the two groups with three different cell lines: HEK293 with low microRNA-21 activity, MDA-MB-231 with high microRNA-21 activity, and HepG2. Figure 4C As shown, HEK293 cells were effectively eliminated in both groups, while the survival rates of MDA-MB-231 and HepG2 cells were restored only in the miR-21-ALP switch + D3 group. In the miR-21-Bim group, the survival rate of MDA-MB-231 cells remained below 20%, and the survival rate of HepG2 cells could only be restored to 70%. This further demonstrates that the miR-21-ALP switch + D3 is more sensitive to microRNAs.

[0089] Finally, we compared the performance of selective cell removal of the two groups under co-culture conditions to simulate real-world applications. To visualize the cell population, we used fluorescent cell lines: HEK293-CFP and HepG2-RFP. Figure 4D and Figure 3 In Figure E, relative cell numbers are expressed as percentages of cell counts in the treated groups relative to the untreated control group. For the miR-21-ALP+D3 group, effective depletion of HEK293-CFP was observed, while relative cell counts for the other two highly active microRNA-21 cell lines were >90%. For the miR-21-Bim group, not only was a significant loss of the two highly active microRNA-21 cell lines observed, but a significant amount of HEK293-CFP was also observed in the dot plot.

[0090] Example 3—Multi-signal amplification minimizes the impact of leaky expression

[0091] As shown in the previous section, cells treated with the miR-21-Bim switch maintained low cell viability even at high concentrations of the mimic and in a cell line with high microRNA-21 activity. This could indicate leaky expression of the cytotoxic protein. For the mimic, we observed >80% viability recovery in the miR-21-ALP+D3 group and ~100% viability recovery in the cell line with high microRNA-21 activity, even in co-culture conditions. Therefore, our approach effectively mitigates the effects of leaky expression of the microRNA switch.

[0092] Example 4—Cell purification methods can be incorporated into mRNA logic gates

[0093] In the experiment, HEK293 cells were cultured at 0.5 × 10 5 Cells were seeded into 24-well plates at 400 cells / well and transfected 24 hours later. Cells were transfected with 600 nM miR-21-APL, miR-IDT-ALP, miR-21 / IDT-ALP, or both miR-21-ALP and miR-IDT-ALP (denoted as miR-21-ALP / miR-IDT-ALP). Cells were co-transfected with 2 nM microRNA mimics and 90 nM PuroR mRNA, or with 90 nM PuroR mRNA alone for the "w / o mimics" group. 24 hours after transfection, peptide treatment was performed with D-3 peptide. Subsequently, an MTT-based cell viability assay was performed according to the manufacturer's protocol.

[0094] We constructed ALP-expressing mRNA switches that sense miR-21, miR-IDT, or both ( Figure 5 A), as a model decision-making intelligent mRNA for targeted delivery. Figure 5 As shown in Figure B, cell viability responded as expected to each switch and co-transfected microRNA mimic. However, decreased target cell viability was observed, particularly in the miR-IDT-ALP switch group. By introducing a multi-input logic circuit, more precise and standardized cell purification was achieved.

[0095] Example 5 - Cell purification method confirms the differentiation of neuronal cells

[0096] In the experiment, 201B7, neuron and 201B7d14 were added at 0.5×10 5 Cells / well were seeded into 24-well plates and transfected 24 hours later. For co-culture experiments, differentiated neuronal cultures and 201B7d14 were cultured at 1×10 5 cells / well and 1×10 5 Cells / well were seeded into 6-well plates and transfected 24 hours later. The cells were transfected with 600nM miR-31-APL or ALP mRNA and co-transfected with 90nM PuroR mRNA for positive transfection cell selection. 24 hours after transfection, peptide treatment was performed with D-3 peptide. Afterwards, MTT-based cell viability analysis was performed according to the manufacturer's protocol. For co-culture experiments, cells were analyzed by antibody staining or alkaline phosphatase staining. For antibody staining, cells were resuspended in culture medium containing trypsin and stained with anti-nestin antibody (Abcam) and anti-OCT3 / 4 antibody Alexa Fluor 488 (Thermofisher) according to standard antibody staining protocols. The results were analyzed by flow cytometry using Attune NxT (Invitrogen). For alkaline phosphatase staining, cells were kept in culture until the 7th day after peptide treatment. Cells were stained for alkaline phosphatase using an alkaline phosphatase staining kit (Thermofisher) according to the manufacturer's protocol.

[0097] During differentiation, cell cultures typically produce a heterogeneous mixture of target cells, undifferentiated cells, and misdifferentiated cells. Therefore, we demonstrated the purification effect of our method for differentiation. First, we differentiated iPSC 201B7 into neuronal cells ( Figure 6A ), and constructed a miR-31-ALP switch, wherein microRNA-31 is a neuronal cell marker microRNA. In order to represent incorrectly differentiated cells, we differentiated 201B7 into randomly differentiated tissue-nonspecific cells 201B7d14. Figure 6BAs shown, neuronal survival remained >90% for both the miR-31ALP switch + D3 and D3 alone. Undifferentiated 201B7, which had high levels of ALP activity as shown in the first column, was effectively eliminated in all cases treated with D3, while randomly differentiated 201B7d14 was eliminated only in the presence of either the miR-31ALP switch or ALP mRNA.

[0098] We then differentiated the neuronal cells, added 201B7d14, and purified the cells using our method. Figure 6C As shown, the treated cells successfully restored the neuronal population (Nestin+) from 50% to 90%, while the undifferentiated population decreased from 13% to 2%. In addition, we continued to incubate the co-culture for one week and stained the undifferentiated cells with ALP stain. Figure 6D As shown, due to the difference in growth rate, the majority of the control group was undifferentiated cells. Surprisingly, no undifferentiated cells were observed in the treated group. This shows that our method can successfully purify cells during the differentiation process.

[0099] Example 6 - Cell Purification Methods: Protein Sensing to Purify Cells

[0100] In this experiment, HEK293 cells were cultured at 0.5 × 10 5 Cells were seeded into 24-well plates at 400 cells / well and transfected 24 hours later. Cells were transfected with 600 nM of MS2-ALP or MS2-Bim. Cells were co-transfected with 160 nM of MCP mRNA and 90 nM of PuroR mRNA, or PuroR mRNA alone. Twenty-four hours after transfection, cells transfected with ALP mRNA were treated with D-3 peptide. Subsequently, an MTT-based cell viability assay was performed according to the manufacturer's protocol.

[0101] We constructed an ALP mRNA switch and a Bim mRNA switch that sense MCP protein. Figure 7 The switch was transfected into HEK293 cells with or without co-transfection of mRNA encoding MCP as indicated. Figure 7 As shown, the ALP switch treated with D-3 restored nearly 100% of MCP-expressing cells while eliminating most of the MCP-non-expressing cells. The purification performance of our method was significantly better than that of the Bim switch excision method.

[0102] It should be understood that the examples and embodiments described herein are for illustrative purposes only, and that various modifications or changes will occur to those skilled in the art, and that these modifications or changes are intended to be included within the spirit and scope of this application and within the scope of the appended claims. In addition, any element or limitation of any invention or embodiment disclosed herein may be combined with any and / or all other elements or limitations disclosed herein (alone or in any combination) or any other invention or embodiment, and all such combinations are within the scope of the present invention without limitation.

Claims

1. A method for purifying cells, comprising: i) applying to the cells an mRNA sequence encoding a cell surface alkaline phosphatase that can be linked to a nucleotide sequence targeting a cytosolic cell type marker molecule, or an mRNA sequence encoding a cell surface alkaline phosphatase that can be linked to a nucleotide sequence targeting a viral coat protein, and an mRNA sequence encoding a viral coat protein that can be linked to a nucleotide sequence targeting a cytosolic cell type marker molecule; as well as ii) incubating the cells in culture medium. 2 . The method according to claim 1 , wherein the culture medium contains a peptide formed from phosphorylated D-amino acids and having an N-terminus modified with an aromatic ring.

3. The method according to claim 2, wherein the peptide formed by phosphorylated D-amino acids and having an N-terminus modified by an aromatic ring is D-1 (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)): Formula (I) Formula (II) Formula (III) Formula (IV) Formula (V) Formula (VI) Formula (VII) Formula (VIII) 4. The method according to claim 3, wherein the peptide formed by phosphorylated D-amino acids and having an N-terminus modified by an aromatic ring is D-3 (Formula (III)): Formula (III) The method of claim 1 , wherein the cells are neurons. The method of claim 1 , wherein the cells do not express surface alkaline phosphatase. The method according to claim 1 , wherein the cytosolic cell type marker is microRNA-21, microRNA-31 or microRNA-132.

8. The method according to claim 1, wherein the viral coat protein is bacteriophage MS2 coat protein (MCP).

9. The method of claim 1, wherein the cell surface alkaline phosphatase sequence is separated from the sequence targeting the cytosolic cell type marker molecule or the sequence encoding the viral coat protein by 0 to about 150 nucleotides.

10. The method of claim 1, wherein the sequence targeting the cytosolic cell type marker molecule is separated from the sequence encoding the viral coat protein by 0 to about 150 nucleotides.

11. The method of claim 1, wherein the mRNA sequence encoding cell surface alkaline phosphatase that can be linked to a nucleotide sequence targeting a cytosolic cell type marker molecule is about 1600 nucleotides to about 3000 nucleotides in length.

12. The method of claim 1, wherein the mRNA sequence encoding cell surface alkaline phosphatase that can be linked to a nucleotide sequence targeting a viral coat protein is about 1600 nucleotides to about 3000 nucleotides in length.

13. The method of claim 1, wherein the mRNA sequence encoding the viral coat protein that can be linked to the nucleotide sequence targeting the cytosolic cell type marker molecule is about 400 nucleotides to about 2000 nucleotides in length.

Images