Patent Application
20230127548
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Methods for intracellular transport of nucleic acids are much sought after in the context of both in vitro delivery reagents and in vivo therapeutics. Recently, it was found that micellar assemblies of hundreds of amphiphiles consisting of single-stranded DNA which has been covalently linked to a hydrophobic polymer, referred to as DNA-polymer amphiphile nanoparticles or DPANPs, can readily access the cytosol of cells where they modulate mRNA expression of target genomes without transfection or other helper reagents, making them potential therapeutic nucleic acid carriers. However, despite their effective uptake properties and efficacy in the cytosol, it was unknown how these polyanionic structures can enter cells. Indeed, generally, bottlenecks in understanding and achieving delivery and uptake remain a forefront issue in translatability of macromolecular and nanomaterials-based therapeutics generally, including with respect to nucleic acid therapies. However, elucidating these mechanisms is typically done through a collection of poor, low throughput choices that include stepwise knockouts of genes postulated to be potential candidates (e.g. anion scavengers), the use of pharmacological inhibitors that often have broad side-effects, or membrane disruption methods that raise questions as to biological relevance. Thus, unbiased screening technologies for the identification of molecular regulators of uptake would dramatically improve the ability to develop and understand synthetic molecule and material uptake and transport.
Detailed herein are methods of identifying a cell uptake modulator of a molecule (e.g., micellar assemblies, DPANPs) in a cell. In some embodiments, a method of identifying a cell uptake modulator of a molecule can include (a) contacting a plurality of cells of a cell-containing biological sample with a plurality of gene-editing agents, wherein a gene-editing agent from the plurality of gene-editing agents recognizes and alters a target gene of at least one cell of the plurality of cells; (b) contacting the plurality of cells with a plurality of molecules, wherein at least one molecule of the plurality of molecules is transported into at least one cell of the plurality of cells; and (c) detecting a presence of the at least one molecule in the plurality of cells, thereby identifying the cell uptake modulator of the molecule.
Various non-limiting aspects of these methods are described herein, and can be used in any combination without limitation. Additional aspects of various components of methods for identifying a cell uptake modulator or cell uptake mechanism are known in the art.
It must be noted that, as used in the specification and the appended claims, the singular forms “a,” “an” and “the” include plural referents unless the context clearly dictates otherwise.
As used herein, “biological sample” can refer to a sample generally including cells and/or other biological material. A biological sample can be obtained from a mammalian organism. For example, a biological sample can be obtained from a human. A biological sample can be obtained from a non-human mammal (e.g., a dog, a cat, a monkey, a mouse, or a rat). A biological sample can be obtained from non-mammalian organisms (e.g., a plants, an insect, an arachnid, a nematode), a fungi, an amphibian, or a fish (e.g., zebrafish). A biological sample can be obtained from a prokaryote such as a bacterium, e.g., Escherichia coli, Staphylococci or Mycoplasma pneumoniae; an archaea; a virus such as Hepatitis C virus or human immunodeficiency virus; or a viroid. A biological sample can be obtained from a eukaryote, such as a patient derived organoid (PDO) or patient derived xenograft (PDX). Biological samples can be derived from a homogeneous culture or population of organisms or alternatively from a collection of several different organisms, for example, in a community or ecosystem.
The biological sample can include any number of macromolecules, for example, cellular macromolecules and organelles (e.g., mitochondria and nuclei). The biological sample can be a nucleic acid sample and/or protein sample. The biological sample can be a carbohydrate sample or a lipid sample. The biological sample can be obtained as a tissue sample, such as a tissue section, biopsy, a core biopsy, needle aspirate, or fine needle aspirate. The sample can be a fluid sample, such as a blood sample, urine sample, or saliva sample. The sample can be a skin sample, a colon sample, a cheek swab, a histology sample, a histopathology sample, a plasma or serum sample, a tumor sample, living cells, cultured cells, a clinical sample such as, for example, whole blood or blood-derived products, blood cells, or cultured tissues or cells, including cell suspensions.
In some embodiments, the biological sample can be a tissue sample. In some embodiments, the tissue sample can include live cells from a cell culture. In some embodiments, the tissue sample can be a fresh, frozen tissue sample. In some embodiments, the fresh, frozen tissue sample is cryoground into powder. In some embodiments, the biological sample can be live cells on standard tissue culture dishes. In some embodiments, the biological sample can be flash, frozen tissues that have been cryoground into powder and placed on tissue culture dishes, pre-chilled on dry ice.
As used herein, a “cell” can refer to either a prokaryotic or eukaryotic cell, optionally obtained from a subject or a commercially available source.
As used herein, “detecting” can refer to a method used to discover, determine, or confirm the existence or presence of a compound and/or substance (e.g., DNA, RNA, a protein, a fluorophore). In some embodiments, a detecting method can be used to detect a detectable label. In some embodiments, detecting can include chemiluminescence or fluorescence techniques. In some embodiments, detecting can include immunological-based methods (e.g., quantitative enzyme-linked immunosorbent assays (ELISA), Western blotting, or dot blotting) wherein antibodies are used to react specifically with entire proteins or specific epitopes of a protein. In some embodiments, detecting can include immunoprecipitation of the protein.
As used herein, “modulating” can refer to modifying, regulating, or altering the effects of receptor or transporter systems in a cell. In some embodiments, modulating cell uptake can include transporting a molecule or compound (e.g., a labeled molecule, neurotransmitter, metabolite, drugs, proteins, or nanoparticles) into a cell. In some embodiments, modulating cell uptake can include efflux of a molecule or compound from a cell.
As used herein, “nucleic acid” is used to include any compound and/or substance that comprise a polymer of nucleotides. In some embodiments, a polymer of nucleotides are referred to as polynucleotides. Exemplary nucleic acids or polynucleotides can include, but are not limited to, ribonucleic acids (RNAs), deoxyribonucleic acids (DNAs), threose nucleic acids (TNAs), glycol nucleic acids (GNAs), peptide nucleic acids (PNAs), locked nucleic acids (LNAs, including LNA having a β-D-ribo configuration, α-LNA having an α-L-ribo configuration (a diastereomer of LNA), 2′-amino-LNA having a 2′-amino functionalization, and 2-amino-α-LNA having a 2′-amino functionalization) or hybrids thereof. Naturally-occurring nucleic acids generally have a deoxyribose sugar (e.g., found in deoxyribonucleic acid (DNA)) or a ribose sugar (e.g., found in ribonucleic acid (RNA)).
A nucleic acid can contain nucleotides having any of a variety of analogs of these sugar moieties that are known in the art. A deoxyribonucleic acid (DNA) can have one or more bases selected from the group consisting of adenine (A), thymine (T), cytosine (C), or guanine (G), and a ribonucleic acid (RNA) can have one or more bases selected from the group consisting of uracil (U), adenine (A), cytosine (C), or guanine (G).
Provided herein are methods of identifying a cell uptake modulator of a molecule that include (a) contacting a plurality of cells of a cell-containing biological sample with a plurality of gene-editing agents, wherein a gene-editing agent from the plurality of gene-editing agents recognizes and alters a target gene of at least one cell of the plurality of cells; (b) contacting the plurality of cells with a plurality of molecules, wherein at least one molecule of the plurality of molecules is transported into at least one cell of the plurality of cells; and (c) detecting a presence of the at least one molecule in the plurality of cells, thereby identifying the cell uptake modulator of the molecule.
As used herein, the term “cell uptake modulator” can refer to a ligand that modulates or regulates the mechanism of transporting a molecule across the cell membrane either into or out of a cell. In some embodiments, a cell uptake modulator can transport a molecule into the cell. In some embodiments, a cell uptake modulator can transport a molecule out of the cell. In some embodiments, a cell uptake mechanism can be an active process (e.g., pump transport, vesicle transport, exocytosis, endocytosis, pinocytosis, or phagocytosis). In some embodiments, a cell uptake mechanism can be a passive process (e.g., simple diffusion, osmosis, or facilitated diffusion).
In some embodiments, the method can include delivering a plurality of gene-editing agents into a cell. In some embodiments, the plurality of gene-editing agents can include one or more of the same gene-editing agent. In some embodiments, the plurality of gene-editing agents can include one or more of different gene-editing agents. In some embodiments, a gene-editing agent from the plurality of gene-editing agents comprises a single guide RNA (sgRNA) corresponding to a gene from a gene library, wherein the plurality of gene-editing agents comprises different gene-editing agents comprising different sgRNAs from the gene library.
In some embodiments, the delivering comprises lipofection. In some embodiments, the delivering comprises a virus-based delivery. In some embodiments, the virus-based delivery comprises adeno-associated virus or lentivirus. In some embodiments, the gene-editing agent is a lentivirus. In some embodiments, the gene-editing agent comprises CRISPR/Cas9 components. In some embodiments, the gene-editing agent comprises a single guide RNA (sgRNA), wherein the sgRNA is targeted to the target gene of a cell of the biological sample.
As used herein, the term “gene-editing agent” can refer to an agent that allows for changing the DNA or RNA (e.g., mRNA) in the genome. In some embodiments, gene-editing can include insertion, deletion, modification, or replacement of the DNA or RNA. In some embodiments, a gene-editing agent can include a nuclease-based gene editing platform. In some embodiments, a gene-editing agent can include zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), engineered meganucleases, or a clustered regularly interspaced short palindromic repeats (CRISPR) system. In some embodiments, a gene-editing agent can include RNA interference (e.g., short hairpin RNA (shRNA), small interfering RNA (siRNA), antisense oligonucleotide (ASO), or microRNA mimics). In some embodiments, the gene-editing agent can include CRISPR components. For example, in some embodiments, CRISPR components can include, but are not limited to, a guide RNA and a CRISPR-associated endonuclease (Cas protein). In some embodiments, the gene-editing agent can include a guide RNA (e.g., gRNA or sgRNA) and a CRISPR-associated endonuclease (Cas protein). In some embodiments, the gene-editing agent comprises shRNAs, siRNAs, ASOs, or microRNa mimics.
As used herein, the term “CRISPR” refers to a technique of sequence specific genetic manipulation relying on the clustered regularly interspaced short palindromic repeats pathway, which unlike RNA interference regulates gene expression at a transcriptional level. The term “gRNA” or “guide RNA” refers to the guide RNA sequences used to target specific genes for correction employing the CRISPR technique. Techniques of designing gRNAs and donor therapeutic polynucleotides for target specificity are well known in the art. For example, Doench, J., et al. Nature biotechnology 2014;32(12):1262-7 and Graham, D., et al. Genome Biol. 2015; 16: 260. The term “Single guide RNA” or “sgRNA” is a specific type of gRNA that combines tracrRNA (transactivating RNA), which binds to Cas9 to activate the complex to create the necessary strand breaks, and crRNA (CRISPR RNA), comprising complimentary nucleotides to the tracrRNA, into a single RNA construct. Exemplary methods of employing the CRISPR technique are described in WO 2017/091630, which is incorporated by reference in its entirety. Non-limiting aspects of CRISPR/Cas9 screening with a gene library containing sgRNAs are described in Wang et al., Science (2014); 343(6166):80-4, WO 2018/005691, and WO 2016/196805, the entire contents of each of which are incorporated herein by reference.
In some embodiments, a single guide RNA can recognize a target gene (e.g., a target RNA), for example, by hybridizing to the target gene. In some embodiments, the single guide RNA comprises a sequence that is complementary to the target gene. In some embodiments, the sgRNA can include one or more modified nucleotides. In some embodiments, the sgRNA has a length that is about 10 nt (e.g., about 20 nt, about 30 nt, about 40 nt, about 50 nt, about 60 nt, about 70 nt, about 80 nt, about 90 nt, about 100 nt, about 120 nt, about 140 nt, about 160 nt, about 180 nt, about 200 nt, about 300 nt, about 400 nt, about 500 nt, about 600 nt, about 700 nt, about 800 nt, about 900 nt, about 1000 nt, or about 2000 nt).
In some embodiments, a single guide RNA can recognize a variety of target genes. In some embodiments, a single guide RNA can recognize a variety of RNA targets. For example, a target RNA can be messenger RNA (mRNA), ribosomal RNA (rRNA), signal recognition particle RNA (SRP RNA), transfer RNA (tRNA), small nuclear RNA (snRNA), small nucleolar RNA (snoRNA), antisense RNA (aRNA), long noncoding RNA (IncRNA), microRNA (miRNA), piwi-interacting RNA (piRNA), small interfering RNA (siRNA), short hairpin RNA (shRNA), retrotransposon RNA, viral genome RNA, or viral noncoding RNA. In some embodiments, a target gene can be a gene involved in pathogenesis of conditions such as cancers, neurodegeneration, cutaneous conditions, endocrine conditions, intestinal diseases, infectious conditions, neurological conditions, liver diseases, heart disorders, or autoimmune diseases. In some embodiments, a target gene can be a therapeutic target for conditions such as cancers, neurodegeneration, cutaneous conditions, endocrine conditions, intestinal diseases, infectious conditions, neurological conditions, liver diseases, heart disorders, or autoimmune diseases.
In some embodiments, a method described herein comprises contacting a plurality of cells with a plurality of molecules, wherein the molecule is a DNA polymer micelle. As used herein, a “DNA polymer micelle” refers to a molecule that can be arranged in a spherical form in an aqueous solution, wherein the DNA polymer micelle includes an aggregate of amphiphiles that have a nonpolar hydrophobic region and a polar hydrophilic region. In some embodiments, a DNA polymer micelle comprises a plurality of amphiphilic compounds, wherein an amphiphilic compound of the plurality of amphiphilic compounds comprises a hydrophobic polymer, a DNA sequence, and a detectable label.
In some embodiments, the DNA sequence comprises a single-stranded DNA sequence. In some embodiments, the DNA sequence comprises about 20 nucleotides. In some embodiments, the DNA sequence comprises about 30 nucleotides. In some embodiments, the DNA sequence comprises about 40 nucleotides. In some embodiments, the DNA sequence comprises about 10 to about 50 (e.g., about 15 to about 50, about 20 to about 50, about 25 to about 50, about 30 to about 50, about 35 to about 50, about 40 to about 50, about 45 to about 50, about 10 to about 45, about 15 to about 45, about 20 to about 45, about 25 to about 45, about 30 to about 45, about 35 to about 45, about 40 to about 45, about 10 to about 40, about 15 to about 40, about 20 to about 40, about 25 to about 40, about 30 to about 40, about 35 to about 40, about 10 to about 35, about 15 to about 35, about 20 to about 35, about 25 to about 35, about 30 to about 35, about 10 to about 30, about 15 to about 30, about 20 to about 30, about 25 to about 30, about 10 to about 25, about 15 to about 25, about 20 to about 25, about 10 to about 20, about 15 to about 20, or about 10 to about 15) nucleotides.
In some embodiments, the DNA sequence comprises a spacer sequence. In some embodiments, the spacer sequence comprises a thymine (T) nucleotide and/or an adenine (A) nucleotide. In some embodiments, the DNA sequence is complementary to a sequence that is not expressed in mammalian cells (e.g., GFP).
In some embodiments, the DNA sequence is covalently linked to the hydrophobic polymer. In some embodiments, the hydrophobic polymer can be a Ring-opening metathesis polymerization (ROMP) polymer.
In some embodiments, the amphiphilic compound includes a detectable label. As used herein, a “detectable label” refer to a directly or indirectly detectable moiety that is associated with (e.g., conjugated to) a molecule to be detected. The detectable label can be directly detectable by itself (e.g., radioisotope labels or fluorescent labels) or, in the case of an enzymatic label, can be indirectly detectable, e.g., by catalyzing chemical alterations of a chemical substrate compound or composition, which chemical substrate compound or composition is directly detectable. Detectable labels can be suitable for small scale detection and/or suitable for high-throughput screening. For example, suitable detectable labels can include, but are not limited to, radioisotopes, fluorophores, chemiluminescent compounds, bioluminescent compounds, and dyes.
In some embodiments, a detectable label can be attached to a composition to be detected. For example, detectable labels can be incorporated during nucleic acid polymerization or amplification (e.g., Cy5®-labelled nucleotides, such as Cy5®-dCTP). Any suitable detectable label can be used. In some embodiments, the detectable label is a fluorophore. For example, the fluorophore can be from a group that includes: 7-AAD (7-Aminoactinomycin D), Acridine Orange (+DNA), Acridine Orange (+RNA), Alexa Fluor® 350, Alexa Fluor® 430, Alexa Fluor® 488, Alexa Fluor® 532, Alexa Fluor® 546, Alexa Fluor® 555, Alexa Fluor® 568, Alexa Fluor® 594, Alexa Fluor® 633, Alexa Fluor® 647, Alexa Fluor® 660, Alexa Fluor® 680, Alexa Fluor® 700, Alexa Fluor® 750, Allophycocyanin (APC), AMCA / AMCA-X, 7-Aminoactinomycin D (7-AAD), 7-Amino-4-methylcoumarin, 6-Aminoquinoline, Aniline Blue, ANS, APC-Cy7, ATTO-TAG™ CBQCA, ATTO-TAG™ FQ, Auramine O-Feulgen, BCECF (high pH), BFP (Blue Fluorescent Protein), BFP / GFP FRET, BOBO™-1 / BO-PRO™-1, BOBO™-3 / BO-PRO™-3, BODIPY® FL, BODIPY® TMR, BODIPY® TR-X, BODIPY® 530/550, BODIPY® 558/568, BODIPY® 564/570, BODIPY® 581/591, BODIPY® 630/650-X, BODIPY® 650-665-X, BTC, Calcein, Calcein Blue, Calcium Crimson™, Calcium Green-1™, Calcium Orange™, Calcofluor® White, 5-Carboxyfluoroscein (5-FAM), 5-Carboxynaphthofluoroscein, 6-Carboxyrhodamine 6G, 5-Carboxytetramethylrhodamine (5-TAMRA), Carboxy-X-rhodamine (5-ROX), Cascade Blue®, Cascade Yellow™, CCF2 (GeneBLAzer™), CFP (Cyan Fluorescent Protein), CFP / YFP FRET, Chromomycin A3, Cl-NERF (low pH), CPM, 6-CR 6G, CTC Formazan, Cy2®, Cy3®, Cy3.5®, Cy5®, Cy5.5®, Cy7®, Cychrome (PE-Cy5), Dansylamine, Dansyl cadaverine, Dansylchloride, DAPI, Dapoxyl, DCFH, DHR, DiA (4-Di-16-ASP), DiD (DilC18(5)), DIDS, Dil (DilC18(3)), DiO (DiOC18(3)), DiR (DilC18(7)), Di-4 ANEPPS, Di-8 ANEPPS, DM-NERF (4.5-6.5 pH), DsRed (Red Fluorescent Protein), EBFP, ECFP, EGFP, ELF® -97 alcohol, Eosin, Erythrosin, Ethidium bromide, Ethidium homodimer-1 (EthD-1), Europium (III) Chloride, 5-FAM (5-Carboxyfluorescein), Fast Blue, Fluorescein-dT phosphoramidite, FITC, Fluo-3, Fluo-4, FluorX®, Fluoro-Gold™ (high pH), Fluoro-Gold™ (low pH), Fluoro-Jade, FM® 1-43, Fura-2 (high calcium), Fura-2 / BCECF, Fura Red™ (high calcium), Fura Red™ / Fluo-3, GeneBLAzer™ (CCF2), GFP Red Shifted (rsGFP), GFP Wild Type, GFP / BFP FRET, GFP / DsRed FRET, Hoechst 33342 & 33258, 7-Hydroxy-4-methylcoumarin (pH 9), 1,5 IAEDANS, Indo-1 (high calcium), Indo-1 (low calcium), Indodicarbocyanine, Indotricarbocyanine, JC-1, 6-JOE, JOJO™-1 / JO-PRO™-1, LDS 751 (+DNA), LDS 751 (+RNA), LOLO™-1 / LO-PRO™-1, Lucifer Yellow, LysoSensor™ Blue (pH 5), LysoSensor™ Green (pH 5), LysoSensor™ Yellow/Blue (pH 4.2), LysoTracker® Green, LysoTracker® Red, LysoTracker® Yellow, Mag-Fura-2, Mag-Indo-1, Magnesium Green™, Marina Blue®, 4-Methylumbelliferone, Mithramycin, MitoTracker® Green, MitoTracker® Orange, MitoTracker® Red, NBD (amine), Nile Red, Oregon Green® 488, Oregon Green® 500, Oregon Green® 514, Pacific Blue, PBF1, PE (R-phycoerythrin), PE-Cy5, PE-Cy7, PE-Texas Red, PerCP (Peridinin chlorphyll protein), PerCP-Cy5.5 (TruRed), PharRed (APC-Cy7), C-phycocyanin, R-phycocyanin, R-phycoerythrin (PE), PI (Propidium Iodide), PKH26, PKH67, POPO™-1 / PO-PRO™-1, POPO™-3 / PO-PRO™-3, Propidium Iodide (PI), PyMPO, Pyrene, Pyronin Y, Quantam Red (PE-Cy5), Quinacrine Mustard, R670 (PE-Cy5), Red 613 (PE-Texas Red) , Red Fluorescent Protein (DsRed), Resorufin, RH 414, Rhod-2, Rhodamine B, Rhodamine Green™, Rhodamine Red™, Rhodamine Phalloidin, Rhodamine 110, Rhodamine 123, 5-ROX (carboxy-X-rhodamine), S65A, S65C, S65L, S65T, SBFI, SITS, SNAFL®-1 (high pH), SNAFL®-2, SNARF®-1 (high pH), SNARF®-1 (low pH), Sodium Green™, SpectrumAqua®, SpectrumGreen® #1, Spectrum Green® #2, SpectrumOrange®, SpectrumRed®, SYTO® 11, SYTO® 13, SYTO® 17, SYTO® 45, SYTOX® Blue, SYTOX® Green, SYTOX® Orange, 5-TAMRA (5-Carboxytetramethylrhodamine), Tetramethylrhodamine (TRITC), Texas Red® / Texas Red®-X, Texas Red®-X (NHS Ester), Thiadicarbocyanine, Thiazole Orange, TOTO®-1 / TO-PRO®-1, TOTO®-3 / TO-PRO®-3, TO-PRO®-5, Tri-color (PE-Cy5), TRITC (Tetramethylrhodamine), TruRed (PerCP-Cy5.5), WW 781, X-Rhodamine (XRITC) , Y66F, Y66H, Y66W, YFP (Yellow Fluorescent Protein), YOYO®-1 / YO-PRO®-1, YOYO®-3 / YO-PRO®-3, 6-FAM (Fluorescein), 6-FAM (NHS Ester), 6-FAM (Azide), HEX, TAMRA (NHS Ester), Yakima Yellow, MAX, TET, TEX615, ATTO 488, ATTO 532, ATTO 550, ATTO 565, ATTO Rho101, ATTO 590, ATTO 633, ATTO 647N, TYE 563, TYE 665, TYE 705, 5′ IRDye® 700, 5′ IRDye® 800, 5′ IRDye® 800 CW (NHS Ester), WellRED D4 Dye, WellRED D3 Dye, WellRED D2 Dye, Lightcycler® 640 (NHS Ester), and Dy 750 (NHS Ester). In some embodiments, the fluorophore is Cy5.
In some embodiments, a detectable label is or includes a luminescent or chemiluminescent moiety. Common luminescent/chemiluminescent moieties include, but are not limited to, peroxidases such as horseradish peroxidase (HRP), soybean peroxidase (SP), alkaline phosphatase, and luciferase. These protein moieties can catalyze chemiluminescent reactions given the appropriate chemical substrates (e.g., an oxidizing reagent plus a chemiluminescent compound). Non-limiting examples of chemiluminescent compound families include 2,3-dihydro-l,4-phthalazinedione luminol, 5-amino-6,7,8-trimethoxy- and the dimethylamino[ca]benz analog. These compounds can luminesce in the presence of alkaline hydrogen peroxide or calcium hypochlorite and base. Other examples of chemiluminescent compound families include, e.g., 2,4,5-triphenylimidazoles, para-dimethylamino and - methoxy substituents, oxalates such as oxalyl active esters, p-nitrophenyl, N-alkyl acridinum esters, luciferins, lucigenins, or acridinium esters.
In some embodiments, the detectable label is located at the 3′ end of the DNA sequence. In some embodiments, the detectable label is conjugated to the DNA sequence via a phosphodiester linkage.
In some embodiments, a DNA polymer micelle comprises a plurality of amphiphilic compounds. In some embodiments, the DNA polymer micelle comprises about 20 amphiphilic compounds. In some embodiments, the DNA polymer micelle comprises about 10 to about 100 (e.g., about 15 to about 100, about 20 to about 100, about 25 to about 100, about 30 to about 100, about 35 to about 100, about 40 to about 100, about 45 to about 100, about 50 to about 100, about 55 to about 100, about 60 to about 100, about 65 to about 100, about 70 to about 100, about 75 to about 100, about 80 to about 100, about 85 to about 100, about 90 to about 100, about 95 to about 100, about 10 to about 95, about 15 to about 95, about 20 to about 95, about 25 to about 95, about 30 to about 95, about 35 to about 95, about 40 to about 95, about 45 to about 95, about 50 to about 95, about 55 to about 95, about 60 to about 95, about 65 to about 95, about 70 to about 95, about 75 to about 95, about 80 to about 95, about 85 to about 95, about 90 to about 95, about 10 to about 90, about 15 to about 90, about 20 to about 90, about 25 to about 90, about 30 to about 90, about 35 to about 90, about 40 to about 90, about 45 to about 90, about 50 to about 90, about 55 to about 90, about 60 to about 90, about 65 to about 90, about 70 to about 90, about 75 to about 90, about 80 to about 90, about 85 to about 90, about 10 to about 85, about 15 to about 85, about 20 to about 85, about 25 to about 85, about 30 to about 85, about 35 to about 85, about 40 to about 85, about 45 to about 85, about 50 to about 85, about 55 to about 85, about 60 to about 85, about 65 to about 85, about 70 to about 85, about 75 to about 85, about 80 to about 85, about 10 to about 80, about 15 to about 80, about 20 to about 80, about 25 to about 80, about 30 to about 80, about 35 to about 80, about 40 to about 80, about 45 to about 80, about 50 to about 80, about 55 to about 80, about 60 to about 80, about 65 to about 80, about 70 to about 80, about 75 to about 80, about 10 to about 75, about 15 to about 75, about 20 to about 75, about 25 to about 75, about 30 to about 75, about 35 to about 75, about 40 to about 75, about 45 to about 75, about 50 to about 75, about 55 to about 75, about 60 to about 75, about 65 to about 75, about 70 to about 75, about 10 to about 70, about 15 to about 70, about 20 to about 70, about 25 to about 70, about 30 to about 70, about 35 to about 70, about 40 to about 70, about 45 to about 70, about 50 to about 70, about 55 to about 70, about 60 to about 70, about 65 to about 70, about 10 to about 65, about 15 to about 65, about 20 to about 65, about 25 to about 65, about 30 to about 65, about 35 to about 65, about 40 to about 65, about 45 to about 65, about 50 to about 65, about 55 to about 65, about 60 to about 65, about 10 to about 60, about 15 to about 60, about 20 to about 60, about 25 to about 60, about 30 to about 60, about 35 to about 60, about 40 to about 60, about 45 to about 60, about 50 to about 60, about 55 to about 60, about 10 to about 55, about 15 to about 55, about 20 to about 55, about 25 to about 55, about 30 to about 55, about 35 to about 55, about 40 to about 55, about 45 to about 55, about 50 to about 55, about 10 to about 50, about 15 to about 50, about 20 to about 50, about 25 to about 50, about 30 to about 50, about 35 to about 50, about 40 to about 50, about 45 to about 50, about 10 to about 45, about 15 to about 45, about 20 to about 45, about 25 to about 45, about 30 to about 45, about 35 to about 45, about 40 to about 45, about 10 to about 40, about 15 to about 40, about 20 to about 40, about 25 to about 40, about 30 to about 40, about 35 to about 40, about 10 to about 35, about 15 to about 35, about 20 to about 35, about 25 to about 35, about 30 to about 35, about 10 to about 30, about 15 to about 30, about 20 to about 30, about 25 to about 30, about 10 to about 25, about 15 to about 25, about 20 to about 25, about 10 to about 20, about 15 to about 20, or about 10 to about 15) amphiphilic compounds.
In some embodiments, the DNA polymer micelle can further comprise a therapeutic cargo. As used herein, the term “therapeutic cargo” refers to a reagent or substance that is related to the treatment of disease or disorder. For example, a therapeutic cargo can include, but is not limited to, nanoparticles, therapeutic agents, small RNA and oligonucleotides (e.g., miRNA and siRNA), small molecule drugs, DNA and plasmids, proteins (e.g., enzymes and inhibitors), or gene-editing complexes). In some embodiments, the therapeutic cargo comprises a nucleic acid.
In some embodiments, a method provided herein includes detecting a presence of at least one molecule in the plurality of cells, and thereby identifying a cell uptake modulator of a molecule. In some embodiments, the detecting further comprises: selecting a molecule uptake-deficient cell from the plurality of cells; isolating an sgRNA fragment from the molecule uptake-deficient cell; and profiling the isolated sgRNA fragment to identify the target gene of the isolated sgRNA fragment, wherein the target gene is the identified cell uptake modulator.
In some embodiments, the method can include detecting an absence of at least one molecule in the plurality of cells. In some embodiments, the method can include detecting a presence and/or an absence of a plurality of molecules. In some embodiments, the method can include discriminating between cells based on a predetermined parameter. In some embodiments, a molecule of the plurality of molecules can include a detectable label. In some embodiments, a molecule can include a plurality of detectable labels. In some embodiments, the plurality of detectable labels can include the same detectable label. In some embodiments, the plurality of detectable labels can include different labels.
In some embodiments, the selecting of a molecule uptake-deficient cell can comprise cell sorting through which a specific cell type is separated from other cells in a biological sample, wherein the cell sorting is based on the physical or biological properties (e.g., size, morphological parameters, viability, or extracellular and intracellular protein expression). In some embodiments, the selecting of a molecule uptake-deficient cell can comprise immunomagnetic cell sorting (MACS). In some embodiments, the selecting of a molecule uptake-deficient cell can comprise using microfluidic devices. In some embodiments, the selecting of a molecule uptake-deficient cell can comprise antibody-based methods of cell separation. In some embodiments, the selecting of a molecule uptake-deficient cell can comprise fluorescence activated cell sorting (FACS).
In some embodiments, the isolating of the sgRNA fragment can include selective enrichment by using an RNA oligonucleotide with a sequence complementary to the sgRNA fragment. In some embodiments, the RNA oligonucleotide can comprises a biotinylated RNA oligonucleotide with a sequence complementary to the sgRNA fragment, wherein the biotinylated RNA oligonucleotide can be selected suing biotinylation-streptavidin affinity using any of a variety of methods known to the field (e.g., streptavidin beads). In some embodiments, the RNA oligonucleotides comprise a sequence complementary to a constant region flanking the sgRNA fragment. In some embodiments, the RNA oligonucleotide comprises SEQ ID NO: 1 and SEQ ID NO: 2. In some embodiments, the RNA oligonucleotide comprises SEQ ID NO: 2 and SEQ ID NO: 3.
In some embodiments, the profiling of the isolated sgRNA fragment includes sequencing of the isolated sgRNA fragment. A wide variety of different sequencing methods can be used to profile the sgRNA fragment. In some embodiments, sequencing of polynucleotides can be performed by various commercial systems. More generally, sequencing can be performed using nucleic acid amplification, polymerase chain reaction (PCR) (e.g., digital PCR and droplet digital PCR (ddPCR), quantitative PCR, real time PCR, multiplex PCR, PCR-based singleplex methods, emulsion PCR), and/or isothermal amplification.
Other examples of methods for sequencing genetic material include, but are not limited to, DNA hybridization methods (e.g., Southern blotting), restriction enzyme digestion methods, Sanger sequencing methods, next-generation sequencing methods (e.g., single-molecule real-time sequencing, nanopore sequencing, and Polony sequencing), ligation methods, and microarray methods. Additional examples of sequencing methods that can be used include targeted sequencing, single molecule real-time sequencing, exon sequencing, electron microscopy-based sequencing, panel sequencing, transistor-mediated sequencing, direct sequencing, random shotgun sequencing, Sanger dideoxy termination sequencing, whole-genome sequencing, sequencing by hybridization, pyrosequencing, capillary electrophoresis, gel electrophoresis, duplex sequencing, cycle sequencing, single-base extension sequencing, solid-phase sequencing, high-throughput sequencing, massively parallel signature sequencing, co-amplification at lower denaturation temperature-PCR (COLD-PCR), sequencing by reversible dye terminator, paired-end sequencing, near-term sequencing, exonuclease sequencing, sequencing by ligation, short-read sequencing, single-molecule sequencing, sequencing-by-synthesis, real-time sequencing, reverse-terminator sequencing, nanopore sequencing, MS-PET sequencing, and any combinations thereof. In some embodiments, the profiling of the isolated sgRNA fragment includes high-throughput sequencing.
Described herein are methods for identifying a cell uptake modulator to provide insights into mechanisms of transport of therapeutically relevant materials and molecules, thereby creating opportunities for further therapeutic development.
In some embodiments, a method can identify a cell uptake modulator in a cell, wherein the cell uptake modulator is an effector of DNA polymer micelle uptake into the cell. In some embodiments, the cell uptake modulator can include a direct interactor. In some embodiments, the cell uptake modulator can include an indirect interactor. In some embodiments, the cell uptake modulator can include SLC18B1, SLC26A11, miR-4476, KLRC3, USP17L5, HENMT1, ARMC4, NPTX1, MOS, MSR1, SCARB1, or NTC. In some embodiments, the identified cell uptake modulator is SLC18B1.
Also described herein are methods of delivering a therapeutic cargo into a cell that include (a) contacting a plurality of cells of a cell-containing biological sample with a gene-editing agent, wherein the gene-editing agent recognizes and alters a target gene of at least one cell of the plurality of cells; (b) contacting the plurality of cells with a plurality of molecules, wherein at least one molecule of the plurality of molecules is transported into at least one cell of the plurality of cells, and wherein a molecule of the plurality of molecules comprises the therapeutic cargo; and (c) detecting a presence of the at least one molecule in the plurality of cells, thereby delivering the therapeutic cargo into the cell.
Also provided herein are methods of targeting specific tissue for cellular uptake of a DNA polymer micelle that include (a) contacting a plurality of cells of a cell-containing biological sample with a gene-editing agent, wherein the gene-editing agent recognizes and alters a target gene of at least one cell of the plurality of cells; (b) contacting the plurality of cells with a plurality of molecules, wherein at least one molecule of the plurality of molecules is transported into at least one cell of the plurality of cells; (c) detecting a presence of the at least one molecule in the plurality of cells; (d) selecting a molecule uptake-deficient cell from the plurality of cells; (e) isolating an sgRNA fragment from the molecule uptake-deficient cell; (f) profiling the isolated sgRNA fragment to identify the target gene of the isolated sgRNA fragment, thereby identifying a cell uptake modulator of the molecule; and (g) identifying a specific tissue where the target gene is expressed and thereby targeting the specific tissue to uptake the DNA polymer micelle.
Also provided herein are methods of high-throughput screening for a target gene encoding a cell uptake modulator in a cell that include (a) contacting a plurality of cells of a cell-containing biological sample with a plurality of gene-editing agents, wherein a gene-editing agent from the plurality of gene-editing agents recognizes and alters a target gene of at least one cell of the plurality of cells; (b) contacting the plurality of cells with a plurality of molecules, wherein at least one molecule of the plurality of molecules is transported into at least one cell of the plurality of cells; (c) selecting a population of cells having a modulated phenotype; (d) identifying and characterizing an sgRNA in the population of cells, thereby identifying the target gene encoding the cell uptake modulator.
Reagents were purchased from commercial sources and used without further purification. The hydrophobic polymer portion of the DNA-polymer amphiphile was prepared via ROMP using a norbornene-phenyl monomer ((N-benzyl)-5-norbornene-exo-2,3-dicarboximide), carboxylic acid chain transfer agent (4,4′-(but-2-ene-1,4-diylbis(oxy))dibenzoic acid), and the ruthenium initiator [(IMesH2)(C5H5N)2(Cl)2Ru=CHPh]. Monomer (500 - 600 mg) and initiator (⅟20 molar ratio) were added to separate, dry Schlenk flasks with stir bars charged with N2 and dissolved in dry CDCl3 (3 mL and 0.5 mL, respectively). Once dissolved, the initiator solution was added to the monomer solution via cannulate with stirring. After 20 minutes, a 50 µL aliquot was removed and set aside for characterization by SEC-MALS. To the remaining reaction, 2 equivalents of chain transfer agent (with respect to initiator) in dry DMF was added and allowed to react for 45 minutes. The polymer was purified by precipitation three times in cold MeOH, then the pellet was redissolved in DCM and centrifuged to remove excess chain transfer agent. The supernatant was concentrated to dryness and run on a column (mobile phase 4% MeOH in DCM) and fractions containing the polymer product were combined and concentrated to dryness. Polymer dispersity and molecular weight were measured using a Phenomenex Phenogel 5 µ 10, 10 k-1000 k, 300 × 7.80 mm (0.05 M LiBr in DMF)) using a Shimadzu LC-AT-VP pump equipped with a multiangle light scattering detector (DAWN-HELIOS: Wyatt Technology), a refractive index detector (Wyatt Optilab T-rEX), and a UV-vis detector (Shimadzu SPD-10 AVP) normalized to a polystyrene standard. Polymer dispersity was measured to be 1.005 ; Mn = 5212 g/mol. Successful cross-metathesis with the chain transfer agent was monitored by 1 H NMR spectroscopy on a Varian Mercury Plus spectrometer (400 MHz) by observing the shift of the alkylidene proton.
The hydrophilic DNA component of the amphiphile was synthesized using standard phosphoramidite coupling on an Applied Biosciences 394 automated synthesizer with a 1000 Å CPG column (Glen Research) on a 1 µmol scale. Phosphoramidite monomers (bz-dA-CE #10-1000-10, Ac-dC-CE #10-1015-10, dmf-dG-CE #10-1029-10, dT-CE #10-1030-10, Fluorescein-dT-CE #10-1056-90 and Cyanine 5 Phosphoramidite #10-5915-95) were purchased from Glen Research and dissolved in dry solvent according to manufacturer instructions. Synthesis conditions include activator 4,5-Dicyanoimidazole (Glen Reasearch), 0.02 M iodine in THF/pyridine/water (Glen Research) as the oxidizer, 3% Trichloroacetic acid (Glen Research) as the deblocking solution, and capping mixtures THF/Pyridine/Ac2O (Glen Research) and 16% 1-Methylimidazole in THF (Glen Research). A 5′- amino modifier (Glen Research) was coupled onto the end of sequences for subsequent conjugation with the polymer. Aliquots of each sequence (2-5 mg) were cleaved from the solid support and deprotected (concentrated ammonia for 24-36 hr) for analysis. The MMT protecting group of the amino modifier was left intact to act as a drag-tag for HPLC purification. HPLC purified sequences were desalted using C18 resin (Ziptips, Millipore) and confirmed by MALDI-TOF using 2’,4',6′-Trihydroxyacetophenone/ammonium citrate and 3-hydroxypicolinic acid as a matrix.
DNA was then conjugated to polymer on solid support. Polymer (30 mg) was dissolved in 100 µL dry DMF with 5 µL DIPEA. Coupling agent (HATU, 0.9 equivalents with respect to polymer) was added to the solution and allowed to activate for 10 minutes. Concurrently, the MMT protecting group on the 5′-amino terminus of the DNA was deprotected using trichloroacetic acid followed by rinsing and drying the DNA on solid support with DCM and Ar, respectively. The DNA on CPG beads were then added to the activated polymer solution in a microcentrifuge tube and allowed to react at room temperature on a shaker. After two hours, the reaction was centrifuged to pool the CPGs with DNA-polymer conjugates and the supernatant was removed. CPGs were washed three times with 1 mL DMF by centrifugation, then a freshly activated solution of polymer (prepared in identical fashion as above) was added to the washed beads for a second reaction, which was left on a shaker overnight. The conjugated CPG was then washed in the same fashion as described previously, and on the last wash the beads were returned to the manufacturer CPG column and washed by flowing 15 mL DMF, followed by 15 mL DCM through the column to remove any unreacted polymer. After drying under Ar, the CPG beads were transferred to 2 mL plastic vials and cleaved/deprotected with 0.5 mL concentrated ammonia for 24-36 hours. Micelles from the DNA-polymer conjugate are spontaneously formed in the aqueous deprotection solution, and after removing the beads by filtration and washing with H2O, DMSO, and formamide, the filtrate containing micelles and any unreacted DNA was dialyzed into nanopure H2O using 10 k MWCO snakeskin dialysis tubing (Thermo Scientific). Crude micelle solution was then concentrated to 1 mL under reduced pressure, and injected on SEC-FPLC (HiPrep 26/60 Sephacryl S-200 High Resolution packed column with mobile phase of 50 mM Tris pH 8.5 on an Akta purifier (Pharmacia Biotech) running a P-900 pump and a UV-900 UV-Vis multi-wavelength detector) to purify. Pure micelles were then dialyzed into nanopure H2O and concentrated under reduced pressure. Characterization of micelles was performed by denaturing PAGE (15% separating, 7% stacking, 1 ×TBE, 8 M Urea) to confirm purity, and TEM to confirm morphology. TEM samples were prepared by glow-discharging grids (formvar/carbon-coated, 400 mesh copper, Ted Pella) at 20 mA for 90 seconds using a s4 Emitech K350 glow discharge unit followed by treatment with 250 mM CaCl2. After blotting away excess salt solution, 3.5 µL micelle sample was added to the grid and allowed to dry for 10 minutes and then washed by passing 3 drops of glass distilled H2O over the grid followed by 3 drops of 1% w/w uranyl acetate stain, the excess of which was immediately blotted away using filter paper. The sample-loaded grid was then loaded onto the microscope for imaging.
HEK293T cells were cultured at 37° C. and 5% CO2 in complete medium (Dulbecco’s Modified Eagle Medium, Life Technologies) supplemented with 10% fetal bovine serum (OmegaScientific), and 1X penicillin/streptomycin (Corning). Cells were maintained in 10 cm petri dishes and passaged at ~75 - 90% confluency every 3-5 days.
To amplify the A sub-pool, 400 ng of pooled plasmid DNA was electroporated into Endura (Lucigen) E. coli (performed as 4 electroporations of 2 ul (100 ng) pooled plasmid DNA into 25 uL cells), 2 mL of recovery buffer (Lucigen) was added, and cells were recovered at 37° C. for 1 hour. Cells were then pooled, plated onto 245 mm bioassay plates, and incubated at 33° C. for ~16 hours. Cells were then scraped and maxiprepped (Qiagen) with a maximum of 450 mg bacteria per column. This same procedure was repeated for the B sub-pool. Colony counting estimated total recovery of 1.44×108 and 9.6×107 colonies for the A and B libraries respectively.
To prepare pooled library lentivirus, 6×15 cm plates of HEK293XT cells were grown to 40% confluency and transitioned to Opti-MEM reduced serum media for one hour. For each plate, transfection mix was prepared as one mixture of 2.5 mL Opti-MEM, 124 µL Lipofectamine PLUS reagent (ThermoFisher), 14.4 µg Gecko v2 plasmid DNA, 6.2 µg pMD2.G, and 9.3 µg psPAX2, and a second mixture of 2.5 mL Opti-MEM with 62.2 µL Lipofectamine 2000. The two mixtures were incubated for 5 minutes at room temperature, mixed, incubated for 10 minutes at room temperature, and then the 5 mL mixture was added dropwise to the 15 cm plate. Six plates each were transfected for both the A and B library sub-pools. After 6 hours, media was replaced with standard growth media (DMEM with 10% FBS).
Lentivirus was harvested 56 hours post-transfection by filtering media through 0.45 µm PES filter. Filtered media was then ultracentrifuged at 20,000 rpm for 2 hours at 4° C., and 100× concentrated lentivirus was resuspended in DMEM with 10% FBS, incubated overnight at 4° C., and aliquoted and stored at -80° C.
To test lentiviral titer, 1.5 × 106 HEK293XT cells were plated per well of a 24 well plate in DMEM with 10% FBS supplemented with 8 ug/mL protamine sulfate. After 30 minutes, varying amounts of pooled library lentivirus was added and spinfection was performed by centrifuging plates at 37° C. for 2 hours at 2,000 rpm, after which media was changed. The following day, each well was split into paired wells with and without puromycin. After 48 hours of selection, cell survival was determined by hemocytometer count, and viral amounts equivalent to multiplicity of 0.3 were identified and used for following experiments.
For full screen experiments, 24 wells of 3×106 HEK293T were plated in 12 well plates in DMEM supplemented with 10% FBS and 8 µg/mL protamine sulfate. After 30 minute incubation, 3 µL lentivirus (equivalent to 0.3 MOI) was diluted in 100 µL of Opti-MEM and added dropwise to each well. Plates were then centrifuged at 2,000 rpm at 37° C. for 2 hours, after which media was changed to standard DMEM supplemented with 10% FBS. 30 hours post-transduction, cells were passaged into media containing 1 µg/mL Puromycin. Cells were passaged for 10 days maintaining at least 65×106 cells (1000× coverage) per passage. For library preparation experiments, cell pellets were frozen for gDNA extraction and library preparation at the indicated timepoints.
For uptake screening, at 10 days post transduction, cells were treated with 0.1 nM (replicate 1) or 0.2 nM (replicate 2) Cy5-labeled micelles and incubated for 2 hours. To identify uptake-deficient cells, Fluorescent Activated Cell Sorting (FACS) was performed with gates were set to isolate the ~1% (~6.9× 105 cells out of 56 million sorted; replicate 1) and ~3% (1.7× 106 cells out of 96.2 million sorted; replicate 2) of cells with the lowest Cy5 signal (e.g., see
To generate antisense probes for targeted enrichment, two templates with T7 promoter sequences were generated by performing PCR amplification (Q5 Polymerase, NEB) off of GeCKO v2.0 plasmid DNA using H1T7 and H1R primers for probe 1 and primers T2RT7 and T2F for probe 2. Probe 1 hybridizes to the negative strand of upstream sequence, whereas probe 2 hybridizes to the forward strand of the downstream sequence (
gDNA was isolated using the DNeasy Blood and Tissue Kit (Qiagen), including RNAse A treatment. gDNA was quantified by TapeStation 2200 (Agilent), and then fragmented by sonication (either Q800R2 Sonicator (Qsonica) or Bioruptor Plus (Diagenode)) to 800-1000 bp fragments. After gDNA was denatured, 10% of biotinylated probes (by mass; e.g. 10 µg probes for 100 µg gDNA) were pre-coupled to Streptavidin beads (ThermoFisher) using the manufacturer recommended protocol, and were then hybridized to vector-containing gDNA fragments in 1 × LiCl/Urea Buffer (the final 1 × Buffer contains 25 mM Tris pH 7.4, 5 mM EDTA, 400 mM LiCl, 0.1% NP40, 0.1% SDS, 0.1% sodium deoxycholate, 1M urea) for 3 hours at 60° C. After hybridization, beads were rinsed with 1× LiCl/Urea Buffer at 45° C. for 2-3 minutes. sgRNA-containing gDNA fragments were eluted by digesting RNA probes with Ambion® RNase Cocktail™ (ThermoFisher) and by degrading RNA with NaOH (Sigma). gDNA fragments were purified using the DNA Clean & Concentrator-5 kit (Zymo Research).
Purified gDNA fragments were then PCR amplified (NEBNext Ultra II Q5, NEB) with FLI1F and FLIR primers in PCR1 (denaturation at 98° C. for 30 seconds, followed by 6-9 cycles of denaturation at 98° C. for 12 seconds, annealing at 69° C. for 60 seconds, and extension at 72° C. for 30 seconds, followed by a final elongation at 72° C. for 60 seconds). PCR1 products were purified using AMPure XP beads at 1.6× ratio by volume and manufacturer protocol. After purification, PCR2 was performed with NEBNext Ultra II Q5 (NEB) using standard Illumina indexed primers (e.g. D501 and D701) as follows: denaturation at 98° C. for 30 seconds, followed by 7-8 cycles of denaturation at 98° C. for 10 seconds and annealing and elongation at 72° C. for 40 seconds, followed by a final elongation at 72° C. for 60 seconds). PCR2 products were purified using AMPure XP beads at 1.3× ratio by volume and manufacturer protocol. Libraries were quantified by Tapestation 2200 (Agilent) and sequenced on the HiSeq 2500 or 4000 platforms (Illumina). As these libraries contain identical sequences upstream of the variable sgRNA region, pools submitted for sequencing contained at most 20% FLI-seq libraries, with the remainder of the pool containing high-diversity samples (e.g. standard RNA-seq).
Sequencing reads were first processed by requiring the presence of flanking upstream (GTGGAAAGGACGAAACACCG (SEQ ID NO: 1)) and downstream (GTTTT (SEQ ID NO: 2)) sequences flanking a 20nt region. These 20nt regions were tallied, and then queried against sgRNA sequences present in the Gecko v2 “A” or “B” libraries. Per-sgRNA Reads Per Million (RPM) were calculated by adding a pseudocount of 1 read and normalizing against the sum of all sequenced reads that contained an exact sequence match to a sgRNA sequence in the queried library. Pair-wise correlations in log2(RPM) were calculated using only sgRNAs with RPM>1 in at least one of the two datasets.
For identification of differentially enriched sgRNAs upon micelle treatment, a pseudocount of one read added to both values and then fold-change was calculated between RPM values in Cy5-low sorted population relative to unsorted. Next, for each sgRNA a z-score was calculated by comparing the fold-change against the distribution of fold-changes observed for 1000 non-targeting sgRNAs. A gene-level z-score was then calculated using the Stouffer’s Z-score method. As an alternative approach, sgRNA counts (as described above) were used as input to the MAGeCK (v0.5.9.3) analysis tool.
To compare against previously published CRISPR/Cas9 screen reproducibility, sgRNA read counts were obtained for the TKO library, including 65 total pair-wise replicate comparisons (3 comparisons in DLD1 cells, 3 timepoints in GBM cells, 2 libraries with 5 timepoints in HCT116 (experiment 1), 2 libraries with 4 timepoints and 3 comparisons per timepoint in HCT116 (experiment 2), 2 libraries with 4 timepoints and 3 comparisons per timepoint in HeLa (excluding T18 for library 1, which was discarded due to abnormal correlation with T15), and 4 timepoints in RPE1 cells. Additionally, sequencing data for screening performed with the Brunello library in A375 cells was obtained. Processing was performed identically as described above by identifying reads containing flanking upstream (CGAAACACCG (SEQ ID NO: 3)) and downstream (GTTT (SEQ ID NO: 2)) sequences and zero mismatch alignment to an sgRNA in the Brunello library. Final read density was calculated by adding a pseudocount of 1 read and normalizing to reads per million based on the total number of zero-mismatch reads in the sample. Pair-wise correlations in log2(RPM) were calculated using only sgRNAs with RPM>1 in at least one of the two datasets.
LentiCRISPR v2 plasmids were prepared and amplified. Sequences for the gRNAs were chosen based on: 1) highest enrichment in the pooled screen, or 2) an optimized sequence from the Brunello library. Five non-targeting control guides were also prepared as controls. Insert oligos were purchased from Integrated DNA Technologies (IDT). Post-amplification plasmid sequence was confirmed by Sanger sequencing (Genewiz) of the insert DNA using the LKO0.15’ primer.
HEK293Ts were seeded in 24-well format at 100 k cells/well one day prior to transfection. Immediately before transfection, medium was removed and replaced with antibiotic-free medium. Packaging plasmid (psPAX2), envelope plasmid (pMD2.G), and transfer plasmid (LentiCRISPRv2) were mixed with Lipofectamine® 2000 (ThermoFisher) in OptiMEM (ThermoFisher) according to the manufacturer protocol and incubated for five minutes prior to adding to wells. A total of 600 ng DNA was transfected per well, with a plasmid ratio of 4:3:1 (wt/wt/wt) packaging:envelope:transfer in a total volume of 50 µL OptiMEM. Virus was harvested at 48 hr and 72 hr post-transfection and filtered through 0.45 µm PVDF syringe filter units (Millipore).
HEK293Ts were seeded in 24-well format at 100 k cells/well one day prior to transduction. Immediately before transduction, medium was removed and replaced with antibiotic-free medium. To each well, 200 µL freshly filtered virus was added. The same transduction procedure was repeated with freshly harvested and filtered virus the following day. At ~ 95% confluency, transduced cells were seeded into 6-well format in 1.5 µg/mL puromycin (Life Technologies) containing medium. Cells were allowed to select in puromycin for 9 days, replacing medium or passaging every 3-4 days.
From the validation knockout populations generated in the previous section, cells were harvested and seeded in 10 cm petri dishes at a density of 800 - 1000 cells/plate. After 3-5 days, five colonies from each knockout were selected and gently removed from the bottom of the dish using a 200 µL pipet and transferred to individual wells of a 96-well plate. At ~ 80% confluency, the cells were passaged. At the next passage, cells were transferred to 48-well plates. mRNA was extracted from the cells and treated with DNaseI (NEB) followed by reverse transcription with Superscript III (Invitrogen) using dT(20) primers according the manufacturer’s instructions. The abundance of target cDNA relative to the housekeeping control GAPDH was measured using qPCR. Cells that demonstrated successful and complete knockout of the target gene were maintained in culture and used in material uptake studies.
Transduced HEK293Ts were seeded in 96-well format at 30 k cells/well one day prior to treatment with micelles. Medium was replaced with 50 µL micelle-containing OptiMEM at a concentration of 0.1 nM (or 0.0 nM for control wells) with respect to micelle (assuming ~ 200 unimers per particle). Treated cells were incubated for 2 hr at 37° C., then the OptiMEM was removed and cells were washed with DPBS and trypsonized. Cells were resuspended in100 µL ice cold DPBS and immediately analyzed by FACS. FACS was performed on a BD FACSCanto and laser settings remained consistent between all wells/runs.
To develop a method for readout of pooled CRISPR/Cas9 screens, a genome-wide CRISPR/Cas9 knockout screen was performed in 293T cells using the published GECKO v2 ‘A’ and ‘B’ libraries, which contains 3 guides targeting each of 19,050 genes for a total of 63,950 and 56,869 unique guides respectively (including 1,000 non-targeting controls (NTCs)) (
To address this challenge, the Fast Library of Inserts (FLI-seq) approach was developed to remove the majority of unwanted non-sgRNA genomic DNA (
Although this approach significantly decreases the necessary number of PCR reactions and handling complexity, it was critical that the approach properly enriches for sgRNAs that are enriched in the cellular pool and does not introduce high levels of technical irreproducibility. First, to query whether a true signal was recovered, library preparation and high-throughput sequencing of gDNA was performed from D0 and D10 post-infection of HEK293T cells. It was observed that guides targeting essential genes showed significant depletion by D10, confirming successful integration and excision by Cas9 and successful library generation (
Next, to assay reproducibility, two replicate structures were defined: infection replicates (in which two replicate infections were performed and cells were maintained separately through the entire experiment) and technical replicates (in which after 10 days in culture, isolated genomic DNA was split in half and independently enriched and amplified into library) (
To confirm that the FLI-seq approach decreases variability compared to previous approaches, its reproducibility was compared to that observed in previously published CRISPR lethality screens. Considering 65 replicate pairs from TKO library and 3 from Brunello library, a median Pearson correlation of 0.65 (
The ability of DPANPs to carry functional nucleic acids into the cytosol of cells and modulate mRNA expression of target genomes without transfection or other helper reagents makes them potential therapeutic nucleic acid carriers, but little is known about how they are able to access the cell interior (
Two replicate infections of the Gecko ‘A’ library in 293T cells were performed, and after 10-12 days of selection cells were treated with Cy5-labeled micelles followed by fluorescence activated cell sorting (FACS). As cells with the lowest Cy5 signal would contain sgRNA targeted to genes that mediate uptake of the nanomaterials, the lowest 1-3% were selected (Replicate 1; 694 k out of 56 M total input cells, Replicate 2; 1.71 M out of 96.2 M total input cells) as the ‘uptake deficient’ Cy5-low population (
Next, knockout cell populations were prepared for the top candidates to verify their respective influence on uptake. Two separate gRNAs were chosen per gene to prepare knockouts for validation: the most highly enriched guide from the uptake screen (GeCKO V2 ‘A’ library) and, when available, an independent guide from the more recently optimized Brunello human sgRNA library, which was not included in the initial screen. In cases where the Brunello guide was not available, the second most enriched guide from the screen was included. In addition to candidate hits from the screen, knockouts were prepared for two scavenger receptor proteins, MSR1 (rank 7976) and SCARB1 (rank 1904). These receptor proteins have been implicated previously in uptake of similarly structured nanoparticles by other methods, but did not appear to have significant influence in the screen. After transduction and selection for infection, cells were incubated with micelles and fluorescence of each knockout was determined by flow cytometry. During this analysis, it became apparent that the highest ranked gene candidate, SLC18B1, displayed a significant (ca. 40-60%) reduction in fluorescence after material incubation. The other genes examined, as well as the genes implicated in the literature, showed no significant effect (
To further confirm the effect of SLC18B1, an additional three guides were tested (including guides not included in the original screen) which also showed a significant reduction in uptake compared to wildtype and non-targeting guide controls (
As one of the key advantages of DPANPs is to carry nucleic acids or other cargos into cells, the ability to differentially target cells based on SLC18B1 expression would present a potential avenue for future development of targeted therapies. To explore the potential for such approaches, it was queried whether altered SLC18B1 expression was associated with disease progression in the Cancer Genome Atlas (TGCA) resource. Notably, it was observed that higher expression of SLC18B1 served as a significant positive prognostic marker for head and neck cancer (
It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.
This application claims priority to U.S. Provisional Application Serial No. 63/249,892, filed on Sep. 29, 2021. The disclosure of the prior application is considered part of the disclosure of this application, and is incorporated herein by reference in its entirety.
This invention was made with Government support under Grant Nos. HG004659 and NS103172 awarded by the National Institutes of Health, and under DMR1710105 awarded by the National Science Foundation. The Government has certain rights in the invention.
| Number | Date | Country | |
|---|---|---|---|
| 63249892 | Sep 2021 | US |
