Patent Application
20240183842
Provided herein are compositions comprising positively supercharged proteins (+scProteins), cell membrane-derived vesicles (extracellular vesicles (EV), virus-like particles (VLP)s, and lentiviral vectors (LVV)), and cargo, and methods of use thereof to deliver the cargo to the intracellular space of target cells. Also provided are reporter constructs that can be used to detect genome editing events, and methods of use thereof.
Delivery of biomolecules and other cargo to intracellular spaces has been a challenge. Cell membrane-based biovesicles (BVs) are important candidate drug delivery vehicles and comprise extracellular vesicles, virus-like particles, and lentiviral vectors. Also described are compositions and methods for detecting CRISPR-based nucleic acid modifications.
Provided herein are compositions comprising biovesicles, wherein the biovesicles comprise a lipid bilayer surrounding an aqueous lumen; a positively supercharged protein; and one or more cargo molecules complexed with the positively supercharged protein. In some embodiments, the biovesicles are extracellular vesicles (EVs), lentiviral vectors (LVVs), or virus-like particles (VLPs).
In some embodiments, the EVs are obtained from a biofluid or tissue obtained from a living animal, preferably a mammal, more preferably a human.
In some embodiments, the EVs are obtained from media in a cell culture (i.e., a cell culture of donor cells that “donate” the EVs). In some embodiments, the positively supercharged protein comprises supercharged eGFP or a variant thereof, preferably +scmCerulean3 or +scGFP. In some embodiments, the +scmCerulean3 is at least 95% identical to SEQ ID NO:4, and has a predicted surface charge of at least +11. In some embodiments, the +scGFP is at least 95% identical to SEQ ID NO:4, and has a predicted surface charge of at least +11.
In some embodiments, the cargo comprises a nucleic acid, a protein, a Ribonucleoprotein (RNP), a combination of DNA and protein, or a small molecules. In some embodiments, the nucleic acid is DNA or RNA. In some embodiments, the cargo is a protein. In some embodiments, the protein is a genome editing or epigenome modulating protein. In some embodiments, the genome editing or epigenome modulating protein is or comprises a CRISPR based nuclease, zinc finger (ZF) or a TALE, optionally fused to an epigenome modulator.
In some embodiments, the positively supercharged protein and the protein cargo are in a fusion protein, optionally wherein the positively supercharged protein is fused to the N or C terminus of the cargo, optionally with a polypeptide linker therebetween.
In some embodiments, the protein is or comprises a CRISPR based nuclease, and the cargo further comprises at least one guide RNA complexed with the CRISPR based nuclease.
Also provided herein are methods for delivering a cargo to a cell or tissues, the method comprising contacting the cell or tissue with a composition as described herein. The cell or tissue can be in vivo (in a living animal), or can be ex vivo or in vitro.
Additionally, provided herein are reporter constructs that comprise a transmembrane domain protein; at least one reporter gene fused to an intracellular portion of the transmembrane domain protein; an affinity tag fused to an extracellular portion of the transmembrane domain protein; optionally at least one reporter gene fused to an extracellular portion of the transmembrane domain protein; and a nucleotide sequence complementary to a target gRNA sequence, preferably comprising a variation, e.g., a pathological variation such as a frame shift or a premature stop codon, wherein the sequence prevents expression of the reporter construct.
In some embodiments, the transmembrane domain protein comprises at least two transmembrane domains and at least one extracellular loop, and wherein the affinity tag is disposed in the at least one extracellular loop. In some embodiments, the transmembrane domain is CD63 tetraspanin.
In some embodiments, the reporter genes comprise one or both of a fluorescent tag and a bioluminescent tag; optionally the fluorescent tag is intracellular and the bioluminescent tag is extracellular.
For example, in some embodiments two gRNAs can be used to cut out a sequence from the extracellular loop. That sequence can have a stop codon or a poly A sequence in-between the 2 gRNAs targets and interrupts construct translation. In that case you don't need a frame shift or stop codon, all guide RNA sequences would work and only when the two gRNAs cut simultaneously would the construct be expressed.
Also provided are methods for detecting CRISPR editing of a target sequence, e.g., a target sequence comprising a variation such as a premature stop codon, in a genome of a cell, the method comprising: expressing in the cell with a reporter construct as described herein; isolating extracellular vesicles EVs from the cell; and detecting expression of the reporter construct by detecting a signal from the reporter gene. In some embodiments, the method further comprises isolating the EVs or cells expressing the construct by contacting the cells or EVs with an affinity-tag binding reagent, and isolating cells or EVs that comprise the affinity tag.
The term “about” is used herein to mean ±10%. In some embodiments, about means ±5%. When about is present before a series of numbers or a range, it is understood that “about” can modify each of the numbers in the series or range. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Methods and materials are described herein for use in the present invention; other, suitable methods and materials known in the art can also be used. The materials, methods, and examples are illustrative only and not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control.
Other features and advantages of the invention will be apparent from the following detailed description and figures, and from the claims.
Data are presented with mean and SEM (error bars) and analyzed with unpaired t-test. **** and * represent p-values of <0.0001 and <0.5, respectively. R2 represents the statistical measure of how close the data are to the fitted regression line.
Data is presented as mean and SEM (error bars) and analyzed with unpaired t-test or one-way ANOVA. ** , *** and **** represent a p-value of ≤0.01, <0.0001 to 0.001 and <0.0001, respectively.
Fluorescence in time measurements was normalized to t=0. Data is presented as mean and SEM (error bars) and analyzed with unpaired t-test, Kruskal-Wallis test or one-way anova test. **** , *** , ** and * represent a p-value of <0.0001, <0.001, <0.01, and <0.05, respectively. R2 represents the statistical measure of how close the data are to the fitted regression line.
Data presented with mean and SEM (error bars) and analyzed with unpaired t-test, or one-way anova test. When needed, data was transformed to log10 to qualify for test assumptions. **** , *** , ** and * represent a p-value of <0.0001, <0.001, <0.01, and <0.05, respectively.
Data presented with mean and SEM (error bars) and analyzed with unpaired t-test, or one-way anova test. **** , *** , ** and * represent a p-value of <0.0001, <0.001, <0.01, and <0.05, respectively.
Cell-derived biovesicles (BVs) comprise a large group of bioentities present in the extracellular space ranging from extracellular vesicles (EVs), including exosomes and microvesicles1, viral-like particles to enveloped viruses2, including lentiviral vectors (LVVs). The common elements among all these BV types are their natural capacity to incorporate biomolecules (endogenous cargo or endocargo) from a parent cell, their subcellular scale (nano to micro), their release into the extracellular space, and protection of their luminal contents by a lipid membrane3.4.
Non-infectious EVs have gained more and more attention as a potential therapeutic-delivery vehicle option5-7. EVs have the potential to counter major drawbacks of virus-based vectors, such as immunogenicity, potentially allele-disruptive genome integration, small capacity for additional non-viral encoded biocargo, and potential cytotoxicity. Biomarker studies have demonstrated that the host tolerates high numbers of EVs produced by virtually every cell in the body in biofluids and extracellular spaces, including both from diseased and normal tissues8. Limiting factors in EV therapeutic development are our understanding of defined mechanisms of biomolecule sorting from the donor cells into EVs and their fate in recipient cells3,9. The bioactivity of EV cargo acting upon recipient cells has been the subject of debate in recent years, due in part to experimental limitations at the single EV level10 and quantitation of functional cargo11. The overall observed effect of the EV endocargo in recipient cells is seemingly low and might need additional boost signals to improve therapeutic relevance or multiple EV exposures.
Here, we explore a strategy that is not limited by the donor cell's ability to package a desired payload into EVs. This avenue is called exogenous EV loading, which others have pursued for loading synthetic RNAs and proteins with lipofection agents or electroporation12,13. Our strategy exploits synthetically reprogrammed proteins with positively charged amino acids, such as arginine and lysine, exposed on the outside of the protein structure, while their functional amino acids remain unchanged from their parental structure14. The biophysical properties of these positively supercharged proteins (+scProteins) enable association with and migration through the cell membranes of living cells15. Here we demonstrate that +scProteins utilize EV properties to load negatively charged cargo, including DNA and RNA species, as well as fused proteins and aid in the functional delivery of the these latter.
In some of the present methods and composition, +scProteins and EV, VLP, and/or LVV membranes are used to achieve combinatorial functional delivery of cargo to recipient cells. Larger EVs were more prone to +scProtein loading compared to smaller ones, indicating that more luminal volume and interaction surface promote incorporation of +scProteins. The surface of EVs consists of negatively charged lipids, such as ceramides24 and phosphatidylserine34 restricting association with negative supercharged scProteins (−scProteins) and non-supercharged proteins (scaffold). This ensures that larger membrane-encompassed vesicles exert a higher negative charge (from −12.3.0 mV to −16.0 mV), compared to smaller EVs (−9.0 m V to −12.3 mV) and non-membranous exomeres (−2.7 mV to −9.7 mV)35. As an important factor in cargo uptake by EVs, we noticed that our +scProtein did not associate with −11 mV synthetic liposomes in contrast to SEC-purified EVs. This observation is in line with earlier reports that the charge of lipids is less important for docking of Arg-rich peptides in contrast with sugar moieties on biomembranes36. It is known that the EV surface contains heavily glycosylated proteins that influences their uptake by cells37. EV adopt sugars from their originating both plasma membrane and endocytic cell membrane compartments38. In this regard, the extracellular domain of tetraspanins are equipped with N-linked glycosylation sites39 important for endocytic membrane trafficking40,41. We've demonstrated that deglycosylated EVs were not able to integrate +scProteins and a high number of tetraspanins on the EV surface is propitious for +scProtein association. In terms of the % EVs loaded in a SEC purified EV sample, we expect that our supercharging method could be improved by +scProtein loading of EV subpopulations rich in tetraspanins such as CD63+CD81+EVs, with large particle size, with a high glycosylation status, and with a negative surface charge.
Supercharging of HEK293T EVs did not influence uptake but did increase +scProtein half-life following uptake by HEK293T cells. Improving scEV uptake through using scEVs from different donor cell sources did not increase +scProtein levels when exposed to the same cell type, indicating the scProtein half-life is dependent on processes dictated by the recipient cell. Through lysotracker red experiments scEVs were found to be taken up into low pH cell compartments, indicating that scEVs are exposed to endolysosomal conditions. Without wishing to be bound by theory, it was hypothesized that the stability of scEV assembly inside cells aids in the scEV-mediated delivery of pDNA and other cargo to a recipient cell. Supercharging of proteins protects them against proteolysis and other physical stresses, such as denaturation by temperature or denaturation and aggregation by chemicals like 2,2,2-trifluoroethanol15,30. The resilience to many hazardous factors and tolerance of scProteins42 compared to amphipathic cell penetrating peptides43 makes them ideal in delivery of associated cargo into a living cell or organism. As demonstrated herein, assembly formation through supercharging of EVs protects the EVs against deglycosylation and protects their cargo against DNAse activity and degradation in recipient cells. Delivery of scEV assemblies containing a higher level of +scProteins, such as large scEVs (220 nm to 100 nm) compared to smaller scEVs (<100 nm) was accompanied with an increased half-life of cargo. Longer stability implies longer interaction with the endosomal compartments, therefore boosting the +scProteins ability to escape from endosomal compartments44,45. We confirmed nuclear translocation of the +scProtein, as well as delayed cytosolic +scProtein degradation, using the 26S proteasome inhibitor Bort in combination with Chloroquine (CHL), Bafilomycin A1 (Baf), and concanamycin A (conA). More importantly, pDNA delivery and expression after cell uptake was increased with pDNA-scEVs compared to +scProtein and pDNA alone.
Formation of pDNA-scEVs is built upon the potential of +scProteins to adhere to/form complexes with genetic material mainly through their lysine residues46, while at the same time, protecting the associated DNA from degradation15. We utilized this +scProtein property to piggyback DNA-scProtein complexes for entrance into EVs. Nanoluc expression in HEK293T cells confirmed transgene delivery by pDNA-scEVs through the detection of bioluminescence. We adapted this readout to corroborate whether supercharging of BVs might be applicable for delivery of multiple types of biomolecules through scLVVs. pDNA-scLVVs generated a nanoluc signal whereby two components CRE and a FLEx-OFF reporter were delivered to the same cell generating bioluminescent readout. The LVV component provided viral RNA encoding a non-active floxed reporter, while the pDNA component encoded a CRE enzyme able to activate the floxed reporter. Apart from nanoluc activity, secondary downstream analysis with qPCR confirmed FLEx reporter editing by Cre. This functional delivery of multiple biomolecule types by means of a single carrier was not only shown in cell culture but substantiated by activation of the Ai9 reporter in mouse brain cells.
Nanoscopic vesicles derived from cells in culture provide a valuable route for supercharging to enhance cargo loading, cellular uptake and functional delivery of cargo. Delivery of multiple types of biomolecules47 is a highly valuable tool for next-generation research and modern medicine9. The supercharging methods described herein can overcome some of the hurdles seen with packaging EVs through natural and transgenic routes with donor cells, including payload inconsistency, reduced options for multicargo transport, limited control over the loading process, and the need for oversaturating recipient cells with loaded EVs to achieve functional responses48.
Described herein are customizable BV, and BV-loading technique that can be:
Thus, provided herein are compositions comprising positively supercharged proteins (+scProteins), membrane-bound vesicles (EV, VLP, and/or LVV membranes), and cargo, and methods of use thereof to deliver the cargo to the intracellular space of target cells.
The present methods and compositions can include the use of any glycosylated enclosed biovesicles (i.e., naturally-derived biovesicles, such as extracellular vesicles (EVs), lentiviral vectors (LVVs), or virus-like particles (VLPs), e.g., GAG-VLPs, VSVG-VLPs GAG/VSV G-VLPs). VSVG VLPs are described, e.g., in Campbell et al., Mol Ther. 2019 Jan 2;27(1): 151-163. GAG GLPs are described, e.g., in Ashley et al., Cell. 2018 Jan 11;172(1-2):262-274.e11. In general, the exosomes are about 30 nm-150 nm; microvesicles are about 100nm-1 um; and lentiviral vectors are about 80-120 nm in diameter. The biovesicles include a bilayer, e.g., similar to a cell membrane, that surrounds an inner aqueous lumen that can contain soluble components. The bilayer is surrounded by a glycocalyx comprising membrane-anchored negatively charged glycoproteins. For example, extracellular vesicles can be comprised of a phospholipid bilayer (and associated glycocalyx) obtained from a donor cell, e.g., an animal cell, e.g., a mammalian cell, or a bacterial cell, comprising various negatively charged glycoproteins. See, e.g., Thery et al., Nat Rev Immunol. 2009 Aug;9(8):581-93; Wang et al. Nature Communications 9, 1-7 (2018); Lainscek et al. ACS Synthetic Biology 7, 2715-2725 (2018)). Methods known in the art for obtaining biovesicles from donor cells can be used. For example, the biovesicles can be obtained from culture media in which donor cells, e.g., primary or immortalized cultured cells, are maintained in vitro. Wang et al., STAR Protocols, Volume 2, Issue 1, 19 March 2021, 100295. Biovesicles can also be obtained from biofluids, e.g., blood, urine, cerebrospinal fluid (CSF), tears, saliva, breast milk, ascites, etc. or from tissues (e.g., from brain, lung, tumor, or adipose tissue-derived mesenchymal stem/stromal cells; see, e.g., Hurwitz et al., J Vis Exp. 2019 Feb 7; (144): 10.3791/59143; Kaur et al., Int. J. Mol. Sci. 2021, 22, 11830; Lee et al., Int J Mol Sci. 2020 Jul; 21(13): 4774). Biovesicles can be concentrated or isolated from a biological sample using size exclusion chromatography, density gradient centrifugation, differential centrifugation, nanomembrane ultrafiltration, immunoabsorbent capture, affinity purification, microfluidic separation, anion exchange exodisk, asymmetrical flow field-flow fractionation, tangential flow and/or gel permeation chromatography (for example, as described in U.S. Pat. Nos. 6,899,863 and 6,812,023), iodixanol and sucrose density gradients, organelle electrophoresis (for example, as described in U.S. Pat. No. 7,198,923), magnetic activated cell sorting (MACS), with a nanomembrane ultrafiltration concentrator, polymer-based precipitation, or immunological separation; see, e.g., Yakimchik, Exosomes: isolation and characterization methods and specific markers, 2016-11-30, dx.doi.org/10.13070/mm.en.5.1450, and references cited therein. An exemplary polymer based exosome precipitation system is the ExoQuick from System Biosciences. Various combinations of isolation or concentration methods can be used. See, e.g., Witwer et al., J Extracell Vesicles. 2021 Dec; 10(14):e12182 for EV nomenclature, sample collection and pre-processing, EV separation and concentration, characterization, functional studies, and reporting requirements/exceptions.
+scProteins
The present methods and compositions include positively supercharged proteins, which have, or have been engineered to have, a surface charge of at least +11; in some embodiments, the protein is at least +15, +20, +25, +27, +30, +32, +35, +36, or +38. Surface charge can be predicted using bioinformatics, including RaptorX (Xu et al., Nature Machine Intelligence 3:601-609 (2021), available at raptorx.uchicago.edu) and I-TASSER(Yang et al., Nature Methods, 12: 7-8 (2015), and Yang et al., Nucleic Acids Research, 43: W174-W181 (2015), available at zhanggroup.org//I-TASSER/) that can be used to generate a Predicted Solvent Accessible (PSA) Amino Acids based on a or PSA score. Exemplary positively supercharged proteins can include +scmCerulean3 and +scGFP (sequences provided below). The surface charge of a protein can be increased by the addition of amino acids with a positive charge, e.g., amino acids that are presented on the surface of the protein. For example, the solvent exposed surface of a beta-barrel protein, such as mCerulean3, can be engineered to contain increased levels of positively charged amino acids—Lysine, Arginine or Histidine—on the outside of the barrel. Proteins with beta-barrel structures include human retinol-binding protein, porins, lipocalins, and translocases. Other enzymes or proteins with a functional site can also be supercharged if the positively charged amino acid residues are incorporated at distal sites to the catalytic cleft on the solvent exposed protein surface. Example of such functional proteins include Cas9, Cre, Talen and zinc finger nucleases.
In some embodiments, the +scProteins comprise +scmCerulean3 (sequence provided below). Also provided herein are the +scProteins themselves, e.g., isolated recombinant +scmCerulean3, as well as nucleic acids encoding the +scmCerulean3, vectors comprising the nucleic acids, and host cells comprising and optionally expressing the +scmCerulean3. A vector is a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked and can include a plasmid, cosmid or viral vector. The vector can be capable of translation (RNA), autonomous replication (RNA or DNA) or it can integrate into a host DNA. Viral vectors include, e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses.
A vector can include a +scmCerulean3 nucleic acid in a form suitable for expression of the nucleic acid in a host cell. Preferably, the recombinant expression vector includes one or more regulatory sequences operatively linked to the nucleic acid sequence to be expressed. The regulatory sequences can include promoters, enhancers and other expression control elements (e.g., polyadenylation signals). Regulatory sequences can include those that direct constitutive expression of a nucleotide sequence, as well as tissue-specific regulatory and/or inducible sequences. The design of the expression vector can depend on such factors as the choice of the host cell to be transformed, the level of expression of protein desired, and the like. The expression vectors of the invention can be introduced into host cells to thereby produce proteins or polypeptides, including fusion proteins or polypeptides, encoded by +scmCerulean3-encoding nucleic acids as described herein (e.g., +scmCerulean3 proteins, fusion proteins, and the like). The expression vectors described here for +scmCerulean3 can also be used for other purposes herein.
AA sequence eGFP=GFP
AA sequence positive supercharged eGFP=+scGFP
AA sequence negative supercharged eGFP=−scGFP
AA sequence of supercharged mCerulean3=+scmCerulean3
AA sequence of wild type mCerulean3
In some embodiments, the +ScProtein is at least 80%, e.g., at least 85%, 90%, 95%, 97%, or 98% identical, or is 100% identical, to a reference sequence provided herein (e.g., SEQ ID NO: 2 or 4). To determine the percent identity of two amino acid sequences, or of two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second amino acid or nucleic acid sequence for optimal alignment and non-homologous sequences can be disregarded for comparison purposes). In a preferred embodiment, the length of a reference sequence aligned for comparison purposes is at least 80% of the length of the reference sequence, and in some embodiments is at least 90% or 100%. The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position (as used herein amino acid or nucleic acid “identity” is equivalent to amino acid or nucleic acid “homology”). The percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences.
The comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm. For example, the percent identity between two amino acid sequences can determined using the Needleman and Wunsch ((1970) J. Mol. Biol. 48:444-453) algorithm which has been incorporated into the GAP program in the GCG software package (available on the world wide web at gcg.com), using the default parameters, e.g., a Blossum 62 scoring matrix with a gap penalty of 12, a gap extend penalty of 4, and a frameshift gap penalty of 5.
In some embodiments, additional positive charge can be added by the addition of one or more positively charged peptides such as a nuclear localization signal (NLS). Exemplary NLS include SV40 NLS (PKKKRKV; SEQ ID NO:6) or nucleoplasmin NLS (KRPAATKKAGQAKKKK; SEQ ID NO:7). Other NLSs are known in the art; see, e.g., Cokol et al., EMBO Rep. 2000 Nov 15; 1(5): 411-415; Freitas and Cunha, Curr Genomics. 2009 Dec; 10(8): 550-557. The +scProteins can be produced recombinantly using methods known in the art. For example, +scProteins can be expressed in E. coli, insect cells (e.g., using baculovirus expression vectors), yeast cells or mammalian cells. Suitable host cells, methods of expression, and methods of purification are known in the art, see, e.g., Goeddel, (1990) Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, CA; Wingfield, Curr Protoc Protein Sci. 2015; 80: 6.1.1-6.1.35; Young et al., Biotechnol J. 2012 May;7(5):620-34; Rosane and Ceccarelli, Front. Microbiol., 17 April 2014, doi.org/10.3389/fmicb.2014.00172. In some embodiments, the +scPRotein includes an affinity tag attached at the N- or C-terminus, and the tag is used in purification; see, e.g., Mishra, Curr Protein Pept Sci. 2020;21(8):821-830. Affinity tags can include FLAG, hemagglutinin, myc, streptavidin, polyhistidine (e.g., hexahistidine), GST, and so on. Alternatively, the recombinant expression vector can be transcribed and translated in vitro, e.g., using T7 promoter regulatory sequences and T7 polymerase.
“Cargo” can include biomolecules, e.g., nucleic acids (e.g., DNA, RNA), a combination of DNA and RNA, Ribonucleoproteins (RNPs), a combination of DNA and proteins, proteins, or natural or synthetic small or large molecules. The cargo can be used or intended for use, e.g., as a therapeutic or diagnostic; in some embodiments, the cargo is used for the applications of genome editing, epigenome modulation, and/or transcriptome modulation. In some embodiments, plasmids of up to 11, 12, 13, 14, or 15 kB are delivered with this method. Several different kinds of cargo can be included, e.g., RNA or DNA, proteins, and small or large molecules.
One of skill in the art will appreciate that these are examples and are non-limiting. In some embodiments, the cargo is for genome editing, epigenome modulation, and/or transcriptome or translational modulation, and includes delivering proteins, or nucleic acids encoding proteins, that can effect genome editing, epigenome modulation, and/or transcriptome or translational modulation, e.g., Clustered Regularly Interspaced Palindromic Repeat (CRISPR) based nucleases or nickases, base editors, and other proteins that comprise CRISPR based nucleases to direct an effector protein to target DNA, e.g., that comprise a CRISPR based nuclease (as used herein, “CRISPR based nucleases” includes proteins that have no nuclease activity, or have only nickase activity). Exemplary CRISPR based nucleases include Cas9 (e.g., SpCas9 or SaCas9), xCas9, Cas12a (Cpf1), Cas13, and others. RNA in this context can be, e.g., a single guide RNA (sgRNA), CRISPR RNA (crRNA) and tracrRNA (e.g., for use with CRISPR based nucleases, and/or mRNA coding for cargo. Cargo developed for applications of genome editing also includes, e.g., nucleases and base editors. CRISPR based nucleases are described, for example, in United States Patent Publications U.S. Pat. No. 8,697,359B1; US20180208976A1; International Publications WO2014093661A2; WO2017184786A8; and Anzalone et al., Nature Biotechnology 38:824-844 (2020). Other nucleases include, e.g., FokI and Acul zinc finger nucleases (ZFNs) and Transcription activator-like effector nucleases (TALENs). ZFNs are described, for example, in United States Patent Publications 20030232410; 20050208489; 20050026157; 20050064474; 20060188987; 20060063231; and International Publication WO 07/014275. TALENs are described, for example, in United States Patent Publication U.S. Pat. No. 9,393,257B2; and International Publication WO2014134412A1. Base editors or prime editors can also be delivered, include any CRISPR based nuclease orthologs (wild type, nickase, or catalytically inactive (CI)), e.g., fused at the N-terminus to a deaminase or a functional derivative thereof with or without a fusion at the C-terminus to one or multiple uracil glycosylase inhibitors (UGIs) using polypeptide linkers of variable length. Base editors are described, for example, in United States Patent Publications US20150166982; US20180312825; U.S. Pat. No. 10,113,163; and International Publications WO2015089406; WO2018218188; WO2017070632; WO2018027078; and WO2018165629. Prime editors are described, e.g., in WO2020191248, Anzalone et al., Nat. Biotechnol., 38: 824-844 (2020); Anzalone et al., Nature, 576: 149-157 (2019); Song et al., Nature Communications 12:5617 (2021), and Chen et al., Cell 184(22): 5635-5652.e29, 28 October 2021. sgRNAs can optionally be complexed with genome editing reagents during production within producer cells. Cargo could refer to AAV (e.g., AAV protein capsid and ITR-flanked DNA cargo). Cargo designed for the purposes of epigenome modulation can include CRISPR based nucleases, zinc fingers (ZFs) and TALEs fused to an epigenome modulator or combination of epigenome modulators or a functional derivative thereof connected together by one or more variable length polypeptide linkers. Cargo designed for the purposes of transcriptome editing can include CRISPR based nucleases or any functional derivatives thereof or CRISPR based nucleases or any functional derivatives thereof fused to deaminases by one or more variable length polypeptide linkers.
The cargo can also include any therapeutically or diagnostically useful nucleic acid, DNA, RNA, protein, RNP, or combination of DNA, protein and/or RNP. See, e.g., WO2014005219; U.S. Pat. No. 10,137,206; US20180339166; U.S. Pat. No. 5,892,020A; EP2134841B1; WO2007020965A1. Exemplary RNAs can include miRNAs, mRNAs, small guide RNA (sgRNA), long non-coding RNAs (IncRNAs), and In some embodiments the cargo is an inhibitory nucleic acid, e.g., antisense oligonucleotides, ribozymes, external guide sequence (EGS) oligonucleotides, siRNA compounds, single- or double-stranded RNA interference (RNAi) compounds such as siRNA compounds, modified bases/locked nucleic acids (LNAs), peptide nucleic acids (PNAs), and other oligomeric compounds or oligonucleotide mimetics which hybridize to at least a portion of the target nucleic acid and modulate its function. In some embodiments, the inhibitory nucleic acids include antisense RNA, antisense DNA, chimeric antisense oligonucleotides, antisense oligonucleotides comprising modified linkages, interference RNA (RNAi), short interfering RNA (siRNA); a micro, interfering RNA (miRNA); a small, temporal RNA (stRNA); or a short, hairpin RNA (shRNA); small RNA-induced gene activation (RNAa); small activating RNAs (saRNAs), or combinations thereof.
Preferably, the cargo is complexed with the +scProtein, e.g., by a charge interaction (e.g., the cargo is negatively charged), or by being conjugated to the +scProtein, e.g., by a chemical bond or other affinity interaction, or (in the case of protein cargo) by being expressed as a fusion protein with the +scProtein. In some embodiments, the cargo comprises a protein for genome editing or epigenome modulation, and the +scProtein can be fused to the N or C terminus of the cargo, optionally with a polypeptide linker therebetween. The cargo complex then flips/is loaded across the EV membrane.
Methods of Loading the Cargo into Biovesicles
Further provided herein are methods for loading cargo into nanosized cell membrane derived biovesicles as described herein. The methods including contacting a sample comprising biovesicles with +scProtein/cargo complexes, under conditions that allow the +scProtein/cargo complexes to be taken across the lipid bilayer into the biovesicles. The methods can include mixing the +scProteins and the cargo to form +scProtein/cargo complexes, e.g., in cases where the cargo and +scProteins are not expressed as a fusion protein. As one example, a fusion protein comprising a CRISPR based nuclease (or a protein comprising a CRISPR based nuclease such as a base editor) linked to a +scProtein as described herein is expressed and purified, e.g., in a bacterial or mammalian expression system, and then mixed with a desired guide RNA, and formation of ribonucleoprotein (RNP) complexes comprising the guide RNA and CRISPR based nuclease is allowed to occur. The RNP complexes are then combined with biovesicles as described herein, and complexes are translocated into the biovesicles into the biovesicle lumen. The methods can include contacting the biovesicles with +scProtein/cargo complexes for at least 15 minutes, e.g., at least 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 18 hours, or 24 hours, e.g., up to 24, 36, or 48 hours or more. In some embodiments, where the methods include contacting the biovesicles with +scProtein/cargo complexes for 15 minutes to 2 hours, the contacting is performed at 25-27° C.; where the contacting step is performed for longer, e.g., for 18-24 hours, the contacting is performed at about 4° C.
The methods can further include a step of purification to remove any complexes that are not taken into the biovesicles. For example, where the biovesicles containing the +scProteins and/or cargo include an affinity tag exposed on the membrane surface, immunoaffinity purification using an antibody directed to the tag, e.g., affixed to beads or a solid surface, can be used.
Generally speaking, the loading methods can be performed under physiological or near-physiological conditions, e.g., at a pH of 6.5-8, or 6.5-7.5; in the absence of detergents (e.g., Tween, triton ×100, digitonin, and/or saponin); in isotonic solutions (e.g., 280-320 mOsm/L, preferably 280-315, 280-312, 300-312, or 300-310 mOsm/L, for biovesicles derived from mammals), or in solutions that are not hypotonic (e.g., at least 280, 285, 290, 295, or 300 mOsm/L). In some embodiments, the methods do not include sonication or other mechanical disruption of the biovesicles.
In some embodiments, the biovesicles are obtained from a subject who is in need of a treatment that would beneficially be delivered using a method described herein, and the methods can include obtaining biovesicles from the subject, loading the biovesicles with +scProtein/cargo complexes, and then re-administering the loaded biovesicles back to the subject, wherein the cargo is a therapeutic agent that is useful in treating the subject. Thus provided are methods of delivering a cargo, e.g., as described herein, e.g., a cargo comprising a therapeutic or diagnostic agent, to a subject in need thereof.
Gene editing is a very powerful technique, but it is somewhat hampered by biological and technical requirements. For example, all components (gene editing enzyme, sgRNA, genomic target sequence) need to be spatially and temporally present/expressed in one cell. Therefore, gene editing is often an inherently low occurrence event. Rare cells that underwent the desired mutation are hard to select out of a pool of wild type cells. Gene editing is often impaired by off-target effects, and genomic repair mechanisms post-cutting by Cas9 and Cas9-like gene editing enzymes can induce undesired frame-shifts or deletions.
To counter these restrictions, single cell sorting is commonly used to select out the desired genetically engineered clones. In this technique, each sorted single cell has to be grown to a high number of cells so as to lyse and subsequently isolate gDNA for sequencing. The latter is necessary to retrieve cells with only the desired genomic correction and low-to-no off-target effects in other regions of the genome. This has proven to be a lengthy method that is concomitant with the loss of many appropriately gene-edited cells.
To facilitate this process, we designed a construct that doesn't require flow cytometry sorting, reports if gene editing events have occurred in a pool of unsorted cells, and preselects for cells that have a significantly higher chance to contain the desired correction in the genome. To accomplish these premises, we designed a transmembrane construct, e.g., comprising a transmembrane domain such as CD63 tetraspanin, that contains a fluorescent tag, bioluminescent tag, and an affinity tag. Expression of these tags is interrupted by a nucleotide sequence complementary to the target gRNA sequence. Our data confirms that when the novel CD63 construct is expressed, the transgenic protein is present in the cell membrane and in extracellular vesicles. On the extracellular side of the membrane the bioluminescent tag and the affinity tag are present while on the intracellular side a fluorescent and/or bioluminescent tag resides. The fluorescent tag enables detection with microscopy or in fixed tissues. The bioluminescent tag, such as nanoluc luciferase or -to a lesser extent firefly luciferase-enables very high discrimination of EVs in the cells or media or biofluids derived from such cells that express the construct. The affinity tags, such as Flag-tag or HA-tag, enable selection of construct-expressing cells with affinity columns or construct-expressing EV selection with affinity Dynabeads (sigma) or affinity resin (sigma). The construct has been shown to allow EV selection from specific cell populations in vivo in both dissociated tissues and biofluids, and to discriminate them from EVs produced by other cells that don't contain the construct. For example, glioma cells or hNPCs (human neuronal progenitor cells) implanted in the mouse brain and expressing the construct release reporter-containing EVs. Rare tumor-derived EVs are detectable in blood, liver, lung, etc. by use of the affinity-tag and the luminescent protein. Similarly, when injected in a mouse, reporter-expressing EVs are trackable with their affinity/bioluminescent tag in blood, liver, lung, heart etc. in vivo and postmortem in harvested organs. Release or recovery of construct expressing EVs or construct expressing cells from beads is possible with high affinity peptides such as Flag-tag peptide or HA-peptide.
In some embodiments, an interchangeable cassette in the construct expressing the CD63 construct can be used to introduce sequences complementary to gRNA targeting sequences. This cassette contains a stop codon and impedes expression of the affinity, bioluminescent, and fluorescent tag. As shown herein, only if the Cas9 and a specific sgRNA to the gRNA targeting sequence is functional in the recipient cell, the stop codon is altered and only if the construct is correctly reassembled post-gene-editing, will the cells become bioluminescent. Extra or fewer nucleotides can optionally be introduced in the sgRNA sequence to select for cells that implemented frameshifts post-gene editing. Moreover, by using both firefly and nanoluc luciferase constructs it is possible to perform multiplexing assays as both luciferases can be discriminated from each other in a single assay. Similarly, antibodies against the incorporated affinity tags can immobilize (for example, printed antibodies on a microfluidic chip) or enable detection (detection antibody with fluorescent dye) of the EVs, without the need for compromising the EV membrane layer.
When both reporter and sgRNA are oriented to gDNA targets, when cells are transfected with a plasmid that encodes both the reporter and the sgRNA (the gene editing enzyme can be introduced as protein, mRNA and/or plasmid) the following events will occur, preferably in the following order:
In some embodiments, this whole selection process can be performed in 48 h-72 h. The sooner the immune affinity selection occurs, the lower the chances that there will be off-target gene editing events. The technology is also adaptable to preclinical animal models. In this case, delivery methods for sgRNA/gene editing enzymes can be tested in animals. For example, transgenic tumor cells can be implanted in animals or transgenic animals can be made with the reporter construct. Hereby, researchers can anticipate the time and number of correct CRISPR/Cas9 events in a living animal.
Thus, described herein is a transmembranal protein construct that can be used to detect and isolate EV-producing cells that harbor specific gene-editing events in their genome, comprising a transmembrane domain protein, such as CD63 tetraspanin, that contains a reporter gene, e.g., a fluorescent tag and/or a bioluminescent tag; an affinity tag; and a nucleotide sequence complementary to the target gRNA sequence.
A number of transmembrane proteins can be used in the present constructs, including any of the tetraspanins, e.g., CD63, CD9, CD81. As shown in
In addition, the sequence encoding the reporter construct includes a sequence that is identical or complementary to a gRNA targeting sequence in the genome of a cell, wherein the gene editing target harbors a variation, e.g., a pathological variation, e.g., a stop codon, e.g., a premature stop codon, that results in a pathology, e.g., a loss of expression or a loss or gain of function. Multiple guide sequences targeting the EV construct sequence can also be used. For example, in some embodiments two gRNAs can be used to cut out a sequence from the extracellular loop. That sequence can have a stop codon or a poly A sequence in-between the 2 gRNAs targets and interrupts construct translation. In that case you don't need a frame shift or stop codon, all guide RNA sequences would work and only when the two gRNAs cut simultaneously would the construct be expressed.
The construct includes at least one reporter gene that is expressed on the inside and/or at least one reporter gene that is expressed on the outside of the cell, e.g., a fluorescent and/or bioluminescent reporter gene, and thus on the inside of EVs that are produced by the cell. Fluorescent reporter genes can include green fluorescent protein or a derivative thereof, cyan fluorescent protein (CFP), red fluorescent protein (RFP), mCherry, Tag-RFP, etc.). Useful fluorescent proteins also include mutants and spectral variants of these proteins that retain the ability to fluoresce. See e.g., Shaner et al., Nat. Biotech. 22:1567 (2004), Tag-RFP (Shaner, N. C. et al., 2008 Nature Methods, 5(6), 545-551), Other fluorescent proteins that can be used in the methods described include, but are not limited to, AcGFP, AcGFP1, AmCyan, copGFP, CyPet, dKeima-Tandem, DsRed, dsRed-Express, DsRed-Monomer, DsRed2, AmCyan1, AQ143, AsRed2, Azami Green, Azurite, BFP, Cerulean, CFP, CGFP, Citrine, dTomato, dTomato-Tandem, EBFP, EBFP2, ECFP, EGFP, Emerald, EosFP, EYFP, GFP, mBanana, mCerulean, mCFP, mCherry, mCitrine, mECFP, mEmerald, mGrape1, mGrape2, mHoneydew, Midori-Ishi Cyan, mKeima, mKO, mOrange, mOrange2, mPlum, mRaspberry, mRFP1, mRuby, mStrawberry, mTagBFP, mTangerine, mTeal, mTomato, mTurquoise, mWasabi, PhiYFP, ReAsH, Sapphire, Superfolder GFP, T- HcRed-Tandem, HcRedl, JRed, Katuska, Kusabira Orange, Kusabira Orange2, mApple, Sapphire, TagCFP, TagGFP, TagRFP, TagRFP-T, TagYFP, tdTomato, Topaz, TurboGFP, Venus, YFP, YPet, ZsGreen, and ZsYellowl, all of which are known in the art, e.g., described in the literature or otherwise commercially available. Bioluminescent proteins include aequorin, and firefly or nanoluc luciferase, and variants thereof (see, e.g., Rowe et al., Anal Chem. 2009 Nov 1; 81(21): 8662-8668; Wang et al., ACS Chem. Neurosci. 2018, 9, 4, 639-650).
Also provided methods that can include expressing the reporter construct in cells, along with a CRISPR based nuclease and guide RNA, e.g., by contacting the cells with a vector or vectors comprising sequences encoding the reporter construct, nuclease, and gRNA, or a vector or vectors comprising sequences encoding the reporter construct and nuclease/gRNA RNP, or a vector or vectors comprising sequences encoding the reporter construct and gRNA and nuclease proteins. The cells are maintained until editing can occur, and then production of EVs comprising the reporter construct is monitored, e.g., by assaying culture media (for cells or tissues in vitro) or biofluids (for cells or tissues in vivo). Affinity purification using the affinity tags can be used to isolate cells and/or EVs that comprise the reporter construct.
The invention is further described in the following examples, which do not limit the scope of the invention described in the claims.
Cell membrane-based biovesicles (BVs) are important candidate drug delivery vehicles and comprise extracellular vesicles, virus-like particles, and lentiviral vectors.
Here, we introduce a non-enzymatic assembly of purified BVs, supercharged proteins, and plasmid DNA called pDNA-scBVs. This multicomponent vehicle results from the interaction of negative sugar moieties on BVs and supercharged proteins that contain positively charged amino acids on their surface to enhance their affinity for pDNA. pDNA-scBVs were demonstrated to mediate floxed reporter activation in culture by delivering a Cre transgene. We introduced pDNA-scBVs containing both a CRE-encoding plasmid and a BV-packaged floxed reporter into the brains of Ai9 mice. Successful delivery of both payloads by pDNA-scBVs was confirmed with reporter signal in the striatal brain region.
Overall, we developed a more efficient method to load isolated BVs with cargo that functionally modified recipient cells. Augmenting the natural properties of BVs opens avenues for adoptive extracellular interventions using therapeutic loaded cargo.
CELL CULTURE. Human embryonic kidney 293 (HEK293T), GL261 cells and HeLa cells were obtained from the American Type Culture Collection and were cultured at 37° C. in a 5% CO2 humidified incubator. Culture media was comprised of Dulbecco's modified essential medium (DMEM) with L-glutamine (Corning) supplemented with penicillin (100 units/ml), streptomycin (100 mg/ml) (P/S) (Corning) and 10% fetal bovine serum (FBS) (Gemini Bioproducts). Stock cells were passaged 2-3 times/week with 1:4 split ratio and used within 8 passages. Cells were monthly tested for mycoplasma contamination (Mycoplasma PCR Detection Kit, abm G238) and found negative. Cells grown for EV isolation were cultured in media supplemented with 5% EV-depleted FBS (FBS was depleted of EVs by 16 hrs centrifugation at 160,000×g).
EV AND BIOVESICLE ISOLATION FROM CELLS WITH SIZE EXCLUSION CHROMATOGRAPHY (qEV) COLUMN. EVs isolated from thirty ml of conditioned medium were collected from cells cultured at 70% confluency in two 100 mm plates after 72 h (seeding density 2.2×106 cells/plate). The conditioned media was centrifuged at 300×g for 10 min to remove intact cells, dead cells and cell debris. The medium was then concentrated using a centrifugal concentrator with a 100,000 molecular-weight cutoff (Amicon®Ultra-15 Centrifugal filters), yielding about 0.5 ml concentrate (two spins of 15 ml at 6000×g for 10 min). This concentrate was resolved by passing through IZON qEV original size exclusion columns (SEC) followed by 15 ml of double filtered (0.2 μm) PBS. Five-hundred-microliter fractions were collected. High particle/low protein fractions (from 7 to 11) were pooled and concentrated using Amicon®Ultra-0.5 Centrifugal filters to a final volume of 200 μL at 10,000g for 30 min. The typical yield of an EV isolation was approximately 7.1×107±3.2×107 particles/ml. This method was adapted to isolate EVs, LVVs (transgene plasmid, psPAX2 (Addgene #12260) and pMD2.G (#12259), VSV G-VLPs (pMD2.G (Addgene #12259), and GAG-VLPs (psPAX2 (Addgene #12260)) before being exposed to scProteins. LVVs purified from media of 2.5 million HEK293Tcells transfected with psPAX2 (Addgene #12260) and pMD2.G (Addgene #12259) were isolated with SEC.
LOADING OF BIOVESICLES WITH scPROTEIN. 5×1012 concentrated EVs (based on Nanosight measurement) were loaded in 50 μl with 283 nM recombinant scProtein and incubated for 15-45 min with gentle agitation on a HulaMixer™ at room temperature. Then 50 μl Ni-NTA agarose resin (Qiagen) or NEBExpress Ni-NTA Magnetic Beads (NEB) in PBS were added, incubated for 15-30 min to 24 h on a HulaMixer™ and compared based on fluorescence to a control of scProtein and agarose resin (Qiagen) without EV suspension. After centrifugation for 30 sec at 14,000×g, the supernatant was collected leaving the resin with the bound scProtein that was not associated with the EVs. Fluorescence in suspension was visually inspected using a UV lamp with black background and quantified with a microplate reader (Synergy H1 Hybrid Multi-Mode Reader, BioTek) at an excitation wavelength of 485 nm for GFP or 433 nm for mCerulean3. Similarly, +scProtein solution was exposed to liposomes (100-200 nm vesicles based on Nanosight) at a concentration of 6.29×108 particles/ml that were kindly provided by Dr. Van Solinge. The liposomes were diluted to match our EV sample with 7.5×107±1.2×107 particles/ml (based on Nanosight). The liposomes used were negatively charged DPPC-PEG(2000)-DSPE-cholesterol liposomes, which have been characterized in depth by Deshantri et al. 201949. This method was also adapted for loading LVVs, GAG-VLPs, VSV G-VLPs GAG/VSV G-VLPs. The carrier LVV was concentrated with spin filters (see above) and 30 μl of this suspension was loaded with both plasmid (1 μg) and scProtein (2.2 mol). The total solution of 90 μl was incubated with gentle agitation on a HulaMixer™ at 4° C. overnight to generate scLVV. Then 90 μl samples were added to cells at a density of 50,000 cells in each well of a 12-well plate. Genomic viral RNA (vRNA)-carrying EVs, GAG-VLPs and VSV G-VLPs were generated similarly to scLVV, but were generated either from cells expressing psPAX2 encoding pol and GAG (GAG-VLPs), or pMD2.G encoding for VSV G (VSV G-VLPs).
scEV CHARACTERIZATION WITH EXOVIEW. A sample of EVs purified from HEK293T cells (see above) was concentrated with spin filter columns (Milipore 30 kDa) to a final volume of 30 μl. These EVs were either loaded with 20 μl 283 nM scProtein (see procedure above) or remained unloaded at 4° C. overnight. According to the guidelines provided by NanoView Biosciences (USA), the samples were incubated on the ExoView Tetraspanin Chip for 16 h at room temperature. After washing the chips three times in 1 ml PBS for 3 min, they were incubated with ExoView Tetraspanin labeling antibodies (1:500 in PBST) with 2% BSA for 2 h. The chips were rinsed with PBS and then imaged with the ExoView R100 reader. Procedure and initial analysis were performed by the ExoView representative.
scPROTEIN PRODUCTION. One Shot® RBL21 Star™ (DE3; Invitrogen) bacteria were transformed with plasmids encoding the scProtein of interest and plated on LB agar plates with appropriate antibiotic (kanamycin 50 μg/mL, Sigma). After overnight incubation at 37° C., a medium sized, isolated colony was picked and inoculated into a 5 mL overnight seed culture of LB media. The seed culture was diluted 1:20 and grown in 2×YT media (Sigma) supplemented with 0.2 um filtered 40 mM MgSO4 (Sigma), 2.5 mM KCI (Sigma), and 20 mM glucose (Sigma) until obtaining OD600 ˜0.1-0.3 and then, for protein expression, the bacteria were induced by adding 0.5 mM isopropyl β-d-1-thiogalactopyranoside (IPTG; Sigma) and incubated overnight at 37° C. Cultures were harvested by centrifugation at 3000×g for 10 min and pellets stored at −80° C. or processed immediately.
BIO-BEADS PULL-DOWN OF scEVs. Bio-Beads SM-2 resin (Biorad) were suspended in PBS to 0.2 g/ml. Subsequently, 50 μl of the Bio-Bead solution was added to a 500 μl scEV suspension generated from a mixture of 7.5×107+/−1.2×107 EV particles/ml and 10 pmol of +scProtein or a scProtein solution without EVs. The samples were incubated overnight at 4° C. degrees with gentle agitation on a HulaMixer™. As a control for Bio-Bead pull-down, a 10% Triton X-100 solution was incubated under the same conditions. The following day, the Bio-Beads were spun down (6000×g for 1 min) and resuspended in 50 μl PBS. 50 μl of the supernatant and the Bio-Bead solution were measured with a Synergy H1 Hybrid Multi-Mode Reader (Biotek).
REMOVAL OF N-GLYCOSYLATED MOIETIES. 7.5×106 EVs in 100 μl solution prior or after scProtein loading were incubated with 10 μl Heparinase I/II/III seperately from Flavobacterium heparinum (Sigma) or with PNGase F (New England Biolabs). For optimal enzyme activity, a buffer solution was added provided by the supplier and the samples were incubated for 24 h at 37° C. or 25° C., respectively, in a HulaMixer Sample Mixer (Invitrogen). Ni-NTA affinity resin was added and pelleted (16,000 g for 5 min) to exclude unloaded scProtein. scProtein fluorescence was measured after incubation for 15-30 min to 24 h in Ni-NTA supernatant and Ni-NTA resin pellet.
CD63 IMMUNOAFFINITY BEADS FOR scEV CHARACTERIZATION. 20 μl of antiCD63 beads (invitrogen) were washed twice with 0.1% BSA inPBS and provided with 100 μl suspensions of HEK293T scEVs, HEK293T EVs, scProteins, HEK293T Bodipy TR labelled scEVs, or HEK293T EVs. The samples were incubated overnight at 4° C. with gentle agitation on a HulaMixer™. Beads were washed twice with 0.1% BSA/PBS and when needed incubated with antiCD63-APC (MEM-259, Invitrogen) in a 100 μl volume with 0.1% BSA/PBS for 1 h at 4° C. with gentle agitation. Samples were washed twice by centrifugation (300 g for 15 min) and dissolved in 250 μl 0.1% BSA/PBS for flow cytometry measurement.
LABELING OF scEVs AND UPTAKE. 1.8-2.0×107 EVs from HEK293T, HeLa, or GL261 cells in 30 μl PBS each were labelled with 10 μM Bodipy™TR-ceramide (ThermoFisher) for 1 h on a HulaMixer at 37° C. (
For inhibitor treatment before scEV uptake, HEK293T cells (50,000 cells per well) were seeded in 24-well culture plates (Falcon) for 24 h in DMEM with serum. The cells were then pre-incubated with the inhibitors in Optimem without serum. The following inhibitors were used: 200 nM bafilomycin A1 (Sigma-Aldrich), 200 nM concanamycin A (Sigma-Aldrich), 100 μChloroquine (Sigma-Aldrich) or 5 μBortezomib (Millipore). The labelled EVs (107-108 particles) were added each well for 24 h in the presence of the indicated inhibitors. Following incubation, cells were trypsinized and analyzed based on scProtein and Bodipy TR fluorescence on a Beckman SORP 5 Laser BD Fortessa Flow Cytometer in the MGH core facility.
For uptake of different EV sizes, 3×108 per 0.5 ml BODIPY-TR labelled scEVs were serial filtered through 450 nm (Costar), 220 nm (Costar), 100 nm (Costar), 300 kDa (˜30 nm, Millipore), 100 kDa (˜10 nm, Millipore), and 30 kDa (˜3 nm, Millipore) cut-off filters. Images were acquired every 10 min for 200 min using a Synergy H1 Hybrid Multi-Mode Reader (BioTek). The average fluorescent intensity of scProtein and Bodipy TR was normalized to the starting value.
NUCLEUS ISOLATION POST-scEV EXPOSURE OF HEK293T CELLS. 107-108 scEVs and EVs derived from HEK293T, HeLa, and GL261 cells were incubated with 50,000 HEK293T cells per 24-well. After 4 days of incubation, cells were trypsinized and washed with PBS. Cell fractionation kit (Abcam, 109718) was used to extract cytosolic, mitochondrial, and nuclear proteins. scProtein fluorescence was measured with a Synergy H1 Hybrid Multi-Mode Reader (BioTek).
ASSEMBLY OF scEVs WITH PLASMID DNA (pDNA). 107-108 HEK293T EVs, GL261 EVs, HeLa EVs, VSV G-VLPs, GAG-VLPs, VSV G/GAG-VLPs and LVVs were incubated with 50-100 pmol scProtein and 1 μg pDNA overnight at 4° C. Binding of pDNA to complexes was verified on 1% agarose gels. To test the stability of assembly, 2 units of TURBO Dnase I (Invitrogen) was added at 37° C. for 15 min and compared to pDNA/scProtein or pDNA with and without DNase treatment. pDNA used for this experiment has been summarized in Table 1.
BIOLUMINESCENT AND FLUORESCENT ASSAYS. Recipient cells were trypsinized and seeded in 24-well plates (50,000 cells/well) in 500 μl complete DMEM media. After 24 h, 100 μl of the pDNA-scEV suspension was added to the cultured cells. Each day nanoluciferase37was monitored by removing 50 to 100 μl of media. Nanoluciferase expression was analyzed with the addition of furimazine (Nano-Glo®Luciferase, Promega) diluted in 1×PBS in a range from 1:250 to 1:500. Samples were incubated with the reagent for at least 3 min prior to reading on Synergy H1 Hybrid Multi-Mode Reader (BioTek). For luminescent readings, samples were loaded into white 96-well culture plates (Lumitrac 200). For fluorescent readings, the samples were loaded into black 96-well culture plates (10,000 cells/well). Each sample was loaded in triplicate with a volume of 100 μl in each well. Biovesicles were loaded with pDNA as listed above. Coronal tissue samples from mouse brain corresponding to 150 μm thick sections were homogeneized in 500 μl Nano-Glo® Luciferase Assay Buffer. Bioluminescence were analysed by adding 100 μl sample and 100 μl 1:250 furimazine (Nano-Glo® Luciferase, Promega) in 1×PBS. The excitation laser was shut off, and the emitted light was measured at two different gains: 135 and 200.
ANIMALS. All animal experiments were conducted under the oversight of the MGB Institution Animal Care and Use Committee. Ai9 mice31 were maintained with unlimited access to water and food under a 12-hour light/dark cycle. Male and female Ai9 mice ranging from 8-10 weeks in age were randomly assigned to experimental groups (N=5 treated, N=4 control).
STEREOTAXIC INJECTIONS INTO STRIATUM. Mice were all stereotactically injected into the right striatum (coordinates: anteroposterior: +0.6 mm, lateral: ±1.8 mm, ventral: −3.3 mm) with nanobiologicals in a final volume of 4 μl containing scProtein, LVV encoding FLEx-reporter and/or mammalian CRE plasmid. Control animals were injected with 4 μl containing scProtein, LVV encoding FLEx-reporter and bacterial CRE plasmid, all at an infusion rate of 0.25 mL/min using a 10 mL Hamilton syringe. Five min after the infusion was completed, the needle was retracted 0.3 mm and allowed to remain in place for an additional 3 min prior to its complete removal from the mouse brain35.
MOUSE TISSUE PREPARATION FOR IMMUNOHISTOCHEMISTRY, BIOLUMINESCENCE AND RT-PCR. Mice were sacrificed with a 100-200 μl bolus of ketamine (17.5 mg/ml) and Xylazine (2.5 mg/ml) intraperitoneally followed by an intracardiac perfusion with 50 ml PBS. Brains were collected and frozen at −80° C. Coronal sections of the striatum at 16 μm thickness were obtained using a cryostat (LEICA CM3050S, Leica Microsystems). Sections were alternately collected for immunohistochemistry, bioluminescence and RT-PCR.
STATISTICAL ANALYSIS AND REPRODUCIBILITY. Data were analyzed using GraphPad Prism 9, version 9.1.0 (GraphPad Software Inc., La Jolla, CA). All statistical tests were two-sided and a p-value of less than 0.05 was considered statistically significant. Data were presented as the mean ±S.E.M. The statistical tests used are indicated in the figure legends. Multiple comparisons of significance between groups were performed using the Tukey procedure for ANOVA or Dunn's multiple comparison test for Kruskal-Wallis, as indicated in the corresponding figure legends. The statistical analyses for uptake of EVs was modeled with non-linear regression using a one-phase association or one phase-decay equation. Graphical illustrations in figures were done in Adobe Illustrator 26.0.2.
TRANSMISSION ELECTRON MICROSCOPY (TEM). A 20 μl HEK293T EV sample was directly applied on the grid. After 1 min absorbance onto the grid, the excess liquid is blotted off the film surface using a filter paper (Whatman). The grid is floated on a small drop (˜5 μl) of staining solution (0.75% uranyl formate, 1% uranyl acetate or 1-2% PTA). After 20 seconds, the excess stain is blotted off and the sample is air dried briefly before it's examined in the TEM. Images were captured at the HMS electron microscopy core facility using Tecnai G2 Spirit Bio TWIN transmission electron microscope.
BULK BIOVESICLE NUMBER ESTIMATION WITH NANOPARTICLE TRACKING ANALYSIS. Number of BVs diluted in PBS was assayed using Nanoparticle Tracking Analysis Version 2.2 Build 0375 instrument (Nano Sight). Particles were measured for 60 s and the number of particles (30-800 nm) was determined using NTA Software 2.2. Samples were diluted 1:1000 in PBS prior to analysis. The following photographic conditions were used: frames processed (1498 of 1498 or 1499 of 1499); frames per second (24.97 or 24.98 f/s); calibration (190 nm/pixel); and detection threshold (6 or 7 multi).
PLASMID CONSTRUCTION. For production of bacteria expressing recombinant recombinant proteins construct were cloned into the pET28a vector (Addgene #85492). The plasmid was restricted with Ncol and Xhol and used as backbone for Gibson assembly (New England Biolabs) to insert the gBlock (IDT) expression cassettes of scProteins (+scGFP, −scGFP, GFP, +scCER, +NLS−scCER, see amino acid sequences in supplementary data). Each assembly reaction contained approximately 100 ng insert and 50 ng expression vector and was incubated at 50° C. for 30 min-4 hrs following the manufacturer's protocol. After the assembly reaction, the reaction mix was transformed into NEB 5-alpha competent E. coli strain (New England Biolabs) or One Shot®TOP10 Competent Cells (ThermoFisher). After overnight growth at 37° C. on Kanamycin 50 μg/mL (Sigma) containing agar plates [10 ml Bacto agar (Sigma) with LB medium in a 60 mm dish], single colonies were selected and grown with Kanamycin 50 μg/mL (Sigma) containing LB broth (Sigma). Single colony suspensions containing respective plasmid with insert were extracted using a QIAprep Spin Miniprep Kit (Qiagen). To confirm correct insertion, a restriction digest was performed, and fragments electrophoresed in a 1.5% agarose gel and stained with arose gel and stained with GelRed (Biotium). Images were acquired under UV light using Azure Biosystems c300 Image. When correct profiles were detected, complete plasmid sequencing using next-generation sequencing technology (MGH CCIB DNA Core) was performed to validate plasmid integrity. Vectors generated following this procedure and used for this manuscript are summarized in Table 1.
ScPROTEIN PURIFICATION. The protein purification protocol is an adaptation of Thompson et al., 2008. Frozen pellets were thawed and resuspended in 50 mL PBS with 2 M NaCl, 20 mM imidazole, pH 7.5, with one tablet of EDTA-free Complete Protease Inhibitor (Roche). The resuspended pellets were divided into two fractions and lysed by sonication in a Sonic Dismembrator 550 (Fisher Scientific) for 5 min. Cell debris was removed by centrifugation at 4000×g for 10 min. The supernatant was transferred to a new 50 mL conical tube and 1 mL of settled Ni-NTA agarose resin (Qiagen) was added to the bacterial lysate and incubated at 4° C. for 30-45 min with a HulaMixer™. Then the supernatant and the Ni-NTA resin were transferred to a column. The packed resin was rinsed first with 20 mL PBS and 2 M NaCl, then with 15 mL of PBS, 2 M NaCl, and 20 mM imidazole, and finally eluted with 2 mL PBS, 2 M NaCl, and 500 mM imidazole. To remove the imidazole from the protein solution it was dialyzed against 1 L PBS at 4° C. for 1 hr, and subsequently dialyzed overnight with 2 L fresh PBS buffer. Proteins were quantified via fluorescence and BSA protein (Pierce™, ThermoFisher) assay. Purity and protein size was confirmed by SDS-PAGE and nitrocellulose blot transfer. Protein bands were visualized with Pierce™ Reversible Protein Stain Kit for Nitrocellulose (Thermo Scientific). Aliquots were stored at −80° C.
FLOW CYTOMETRY. Flow cytometric analysis was performed on the Beckman SORP 5 Laser BD Fortessa Flow Cytometer in the MGH core facility. Forward and side scatter signals were used to distinguish live cells from polystyrene beads. Proper gating was performed to identify positive fluorescent signals compared to non-stained or single stained controls.
LENTIVIRAL VECTOR (LVV) PRODUCTION AND CELL TRANSDUCTION. LVVs were produced in HEK293T cells with a three-plasmid system, following Addgene recommendations. Briefly, cells were seeded and 24 hrs later, transfected with psPAX2 (#12260) and pMD2.G (#12259) packaging plasmids and the transgene of interest flanked by LTRs. Six hrs after transfection, cells were rinsed with PBS and media was replaced. Lentiviral isolation was performed 72 hrs later by ultracentrifugation at 70,000×g and the pellet was re-suspended in 1% BSA in PBS36. The viral particle content was evaluated by assessing HIV-1 p24 antigen levels by ELISA (Retro Tek, Gentaur, Paris, France). Concentrated viral stocks were stored at −80° C. until use. LVVs were used for generating stable cell lines after selection by either antibiotic resistance or flow sorting. HEK293T and HELA cells were transduced 24 hrs after plating with LVVs (400 ng of P24 HIV antigen per 200,000 cells). Twenty-four hours later, the medium was replaced with DMEM media with antibiotics and FBS, and cells were cultured and expanded under standard conditions. Stable cell lines were obtained and mCherry or GFP fluorescence was monitored at every passage.
IMMUNOHISTOCHEMISTRY. Sections were fixed with 4% paraformaldehyde for 10 min and incubated with blocking solution [0.1% Triton X-100 containing 10% normal goat serum (SigmaAldrich) in PBS] and then incubated overnight at 4° C. in blocking solution with primary antibody: rabbit anti-RFP antibody (1:250Invitrogen, polyclonal). Sections were rinsed with PBS 5 times and incubated for 2 hr at room temperature with the secondary antibody: goat anti-rabbit TRITC 594 (1:1000) diluted in blocking solution. The sections were washed and mounted in VECTASHIELD® Antifade Mounting Medium (Vector Labs Cat# H-1000) on gelatin-coated slides. Immunoreactivity of mouse sections was visualized and analyzed in Keyence BZ-X810 All-in-one Fluorescence imaging microscope.
DNA AND RNA EXTRACTIONS. The DNeasy Blood & Tissue Kit (Qiagen) was used for genomic DNA extraction from cells. QIAprep Spin Miniprep Kit (Qiagen) and EndoFree MaxiPlasmid Kits (Qiagen) were used for plasmid extraction from bacteria. RNA was extracted using the miRNeasy Mini Kit (Qiagen), according to manufacturer's protocol. The RNA concentration and integrity (RIN score) were determined using the Nanodrop (ThermoFischer Scientific) and Agilent 2100 Bioanalyzer Pico-chips (Agilent Technologies), following manufacturer's protocol.
CONFOCAL MICROSCOPY. 10,000 cells were seeded on a 35 mm optical Petri dish (Thermofisher) and incubated with approx. 10{circumflex over ( )}6 scEVs for 5 days at 37° C. Prior to imaging, LysoTracker Red DND-99 (Invitrogen) was added to the media to a final working concentration of 50 nM for 1 h. The cells were washed twice with fresh media prior to imaging on a Zeiss LSM710 Laser Scanning Confocal of the MGH Cancer Center Translational Imaging Core. Of note, Lysotracker Red DND-99 was incubated under similar conditions with isolated EVs and did not stain our scEVs. To detect nuclear translocation 10,000 cells were seeded on an optical Petri dish (theromofisher) and incubated with 10{circumflex over ( )}6 scEVs for 5days at 37° C. Cells were fixed with paraformaldehyde for 15 min at RT, washed twice with PBS and incubated with a AF594-Phalloidin (Invitrogen) according to manufacturer instructions. After twice washing with PBS, Dapi was provided through VECTASHIELD® Antifade Mounting Medium (Vector Labs Cat# H-1000). ImageJ version 2.1.0/1.53c was used for analysis.
Green fluorescent protein (GFP) has a beta barrel scaffold with a center chromophore16. Through site-specific mutagenesis of the center structure, the fluorescent spectrum of this fluorescent protein can be switched from green emission (EX485-EM538) to cyan emission (EX433-EM475)17 (
The unique affinity of isolated EVs for +scProteins was further demonstrated by the dependency of +scGFP capture on the number of EVs in solution. When a higher number of SEC-purified HEK293T EVs were provided to the same amount of +scGFP (15 pmol), an increase in +scGFP fluorescence was observed (
Our results support the ability of positively charged residues of +scProtein to associate with BVs, such as EVs. Here, we investigated whether EV properties also aid in +scProtein association. Bio-Beads SM2 are nonpolar polystyrene adsorbents which are not expected to bind to hydrophilic +scGFP (
We explored whether the lipids in EV membranes are the main determinant underlying association with +scProtein. Therefore, we compared +scProtein association with liposomes consisting of dipalmitoylphosphatidylcholine-polyethylene glycol 2000—distearoylphosphatidylethanolamine-cholesterol with a known surface charge of −11±1 mV to that of EVs (
Uptake of EVs by recipient cells can be monitored with membrane dye labelled EVs23. To explore the uptake of scEVs by recipient cells, we fluorescently labelled scEVs with a Bodipy TR ceramide membrane dye24 (
When exposing HEK293T Bodipy TR-scEVs to HEK293T cells cultured in EV-free media, an increase in Bodipy TR cell-associated fluorescence was observed over a 200 min (1 measurement/10 min) monitoring experiment (
To examine the potential mechanisms of HEK293T scEV uptake, we pretreated HEK293T cells with inhibitors targeting the endolysosomal and autophagic pathways before adding Bodipy TR-scEVs. v-ATPase inhibitors concanamycin A (ConA, 200 nM) and Bafilomycin A1 (Baf, 200 nM) were used to investigate whether scEVs act through a potential pH-dependent uptake mechanism. Inhibitor pretreated HEK293T cells (1 h) were investigated 24 h post-exposure to Bodipy TR-scEVs with flow cytometry. No significant decrease in Bodipy TR was observed compared to sham-pretreated cells (
In contrast to the use of homologous scEVs, Bodipy TR-scEVs from other cell sources were tested for their internalization potential on common recipient HEK293T cells. HeLa EVs and GL261 EVs had a similar Nanosight Analysis profile before being labelled with Bodipy-TR, supercharged with +scProtein, and exposed to HEK293T cells (
To verify whether in addition to the origin of EVs, viral factors could influence the uptake of supercharged BVs, virus-like particles (VLPs) were generated from HEK293T cells by overexpression of VSV G, GAG, or both VSV G and GAG (
Altogether, our data indicates that scEV uptake and scProtein kinetics in recipient cells can be influenced by particle size and EV source, but not by endogenous EV loading of viral components.
Our previous observations with inhibitors of endolysosomal function suggested this route of EV internalization by cells. Here, we marked the low pH compartments in a scEV-recipient HEKT293T cell with LysoTracker Red29 and investigated its position compared to +scProtein (
It has been reported that plasmid DNA (pDNA) can be piggybacked into cells with scProteins15. We tested whether EVs associated with both +scProtein and pDNA would generate a pDNA encoded signal in the pDNA-scEVs recipient cells (
Our previous findings indicate that pDNA-scL VVs deliver both LVV- and pDNA-encoded transgenes using mCherry and a Nanoluc reporter, respectively. Here, we tested whether both transgenes (pDNA-transgene and LVV-transgene) can successfully be delivered to the same recipient cell and collaboratively generate a bioluminescent signal (
We revisited our FLEx model to test whether pDNA-scL VV can co-deliver two payloads (pDNA transgene and LVV transgene) to brain cells (
Since its discovery, gene editing has provided the ability to meticulously change genes with a profound effect on both therapeutics and molecular research. Even with new tools constantly being developed to increase efficiency and precision of the technique, the repair mechanisms post-gene editing are still error prone making it critical to detect and/or select a desired gene corrected cell clone. Since the contents of extracellular vesicles (EVs) reflect the cells that produced them, if a gene editing event occurs, the EV cargo should contain the gene corrected products, such as a protein or RNA species. The catch lays in the fact that EVs are by their nature very heterogeneous and only a small fraction of the population may harbor the gene edited products.
We designed a CD63 construct with a genomic DNA target sequence for detection of a desired gene editing event. See
As shown in
Two exemplary CD63 constructs were produced that enable easy (e.g., a two-step procedure) isolation and detection of EVs derived from the media/biofluids of a low number of cells (approx 10e3-10e4 cells). The CD63 construct has been shown to work in vivo (with GL261 glioma tumor cells in mice) and in vitro. When glioma cells with the CD63-reporter were injected in the brain of mice, the EVs released from the implanted tumor were trackable in mouse biofluids. Injecting EVs containing these construct were trackable in a mouse using the bioluminscence tag even in difficult to reach organs such as the brain of mice. gRNA targeting sequences against a stop codon in the genome can easily be implemented in the CD63 construct to detect CRISPR/Cas9 events altering a stop codon. If an mRNA coding sequence is targeted in the genome of the reporter cell, the affected mRNA post-gene editing can be retrieved in the reporter EVs.
Thus, a CD63 transgenic reporter protein contained in the membrane of cells and EVs can be used to detect and select out correctly gene edited EV-donor cells early on, reducing effort in avoiding cells with off targets effects.
NoMi sequence to isolate EVs and cells expressing modified CD63 construct:
Example sequence of CRISPR reporter lentiviral vector:
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 the benefit of U.S. Provisional Application Ser. Nos. 63/178,444, filed on Apr. 22, 2021, and 63/180,489, filed on Apr. 27, 2021. The entire contents of the foregoing are incorporated herein by reference.
This invention was made with Government support under Grant Nos. CA232103 and CA069246 awarded by the National Institutes of Health. The Government has certain rights in the invention.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2022/071873 | 4/22/2022 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 63180489 | Apr 2021 | US | |
| 63178444 | Apr 2021 | US |
