WO2023018938A1 - Procédés de génération de transcrits d'arn précis - Google Patents

Procédés de génération de transcrits d'arn précis Download PDF

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WO2023018938A1
WO2023018938A1 PCT/US2022/040166 US2022040166W WO2023018938A1 WO 2023018938 A1 WO2023018938 A1 WO 2023018938A1 US 2022040166 W US2022040166 W US 2022040166W WO 2023018938 A1 WO2023018938 A1 WO 2023018938A1
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rna
protein
expression
encoding
cells
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Christof Fellmann
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The J. David Gladstone Institutes, A Testamentary Trust Established Under The Will Of J. David Gladstone
The Regents Of The University Of California
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    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
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    • C12N9/1241—Nucleotidyltransferases (2.7.7)
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    • C12N2310/00—Structure or type of the nucleic acid
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/35—Nature of the modification
    • C12N2310/351—Conjugate
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    • C12N2330/00—Production
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    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/16011—Human Immunodeficiency Virus, HIV
    • C12N2740/16041—Use of virus, viral particle or viral elements as a vector
    • C12N2740/16043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector

Definitions

  • RNA polymerase II is a multiprotein complex that transcribes DNA into precursors of messenger RNA (mRNA) often referred to as primary RNAs or pre-mRNAs.
  • mRNA messenger RNA
  • pre-mRNAs undergo processing in the nucleus and transportation into the cytoplasm before they are translated into protein.
  • processing and/or transportation is not desirable.
  • a 5’ cap or a poly(A) tail may interfere with its ability to complex with a Cas protein and perform targeted genomic modifications.
  • Pol-II promoters are specific for particular tissues or stages of development. Pol-II promoters can also be inducible. Hence, Pol-II promoters can be useful for providing expression at the time and place where it may be needed. But in some cases, the processing that the Pol-II generated transcripts undergo is not needed or is not desired.
  • RNA transcripts by using engineered mascRNA (MALAT1 -associated small cytoplasmic RNA) elements to specifically excise a portion of a given primary RNA transcript.
  • the primary RNA transcript can be a transcript expressed from an RNA polymerase II (Pol-II) promoter.
  • RNA polymerase II (Pol- II) promoter operably linked to a nucleic acid segment encoding at least one RNA, wherein the nucleic acid segment is flanked by mascRNA-encoding elements.
  • the mascRNA-encoding elements can separately include RNA sequences with at least 90% sequence identity to any of SEQ ID NOs: 1-6.
  • the RNA polymerase II (Pol-II) promoter can be an inducible promoter, a cell-specific promoter, a tissue-specific promoter, or a developmentally regulated promoter.
  • the nucleic acid segment encoding at least one RNA can encode one or more guide RNAs, single-guide RNAs (sgRNAs), interfering RNAs (RNAi), antisense RNAs, small hairpin RNAs (shRNAs), retrons, modified retrons, RNA templates, reverse transcriptase (RT) templates, templates for homology dependent repair (HDR), donor DNAs, donor RNAs, primers, or inhibitory nucleic acids.
  • the expression system can further include at least one expression cassette, each expression cassette having a promoter operably linked to a protein-encoding segment.
  • such proteinencoding segments can encode a reverse transcriptase, a Cas nuclease, a Cas protein, a transposase, a fluorescent protein, a marker segment encoding a signal-producing protein, or a combination thereof.
  • the protein encoding expression cassette can further include one or more 3’ triple-helix motifs that stabilize 3 ’-ends of non-polyadenylated transcripts.
  • the expression system include one or more viral expression vectors that can be or have been packaged by packaging cells.
  • the RNA polymerase II (Pol-II) promoter, the nucleic acid segment encoding at least one RNA, the mascRNA-encoding elements, a protein encoding segment, or a combination thereof is encoded on an antisense strand of the viral expression vector.
  • Such expression systems can further include an expression cassette or expression vector providing a promoter operably linked to a B2 nucleic acid segment encoding a Nodamura virus protein B2 (NovB2).
  • Host cells that include such expression systems are also described herein.
  • Such host cells can endogenously express RNase P, RNase Z, or a combination thereof; such host cells can also be modified to express RNase P, RNase Z, or a combination thereof.
  • the host cells can also express at least one heterologous reverse transcriptase, Cas nuclease, Cas protein, transposase, fluorescent protein, signal-producing protein, Nodamura virus protein B2 (NovB2), or a combination thereof.
  • the host cells can have expression systems that include viral expression vectors that can be or has been packaged by a packaging cell.
  • such viral expression vectors can have the RNA polymerase II (Pol-II) promoter, the nucleic acid segment encoding at least one RNA, the mascRNA- encoding elements, a protein encoding segment, or a combination thereof encoded on an antisense strand of the viral expression vector.
  • RNA polymerase II Polymerase II
  • Expression of at least one heterologous signal-producing protein, protein, RNA, mascRNA-encoding element, or a combination thereof from the expression system in the host cell can be detected in a variety of ways.
  • any of the heterologous signal -producing protein, protein, RNA, mascRNA-encoding element, or a combination thereof from the expression system in the host cell can be detected by measuring or detecting the RNA, protein, encoding DNA, or a signal produced by the heterologous signal-producing protein.
  • the methods can include transducing one or more host cells with an expression system that includes at least one expression cassette, each expression cassette having an RNA polymerase II (Pol-II) promoter operably linked to a nucleic acid segment encoding at least one RNA, wherein the nucleic acid segment is flanked by mascRNA-encoding elements.
  • the host cells used in the methods can in some cases express at least one heterologous reverse transcriptase, Cas nuclease, Cas protein, transposase, fluorescent protein, signal-producing protein, Nodamura virus protein B2 (NovB2), or a combination thereof.
  • the nucleic acid segment encoding at least one RNA encodes one or more guide RNAs, single-guide RNAs (sgRNAs), interfering RNAs (RNAi), antisense RNAs, small hairpin RNAs (shRNAs), retrons, modified retrons, RNA templates, reverse transcriptase (RT) templates, templates for homology dependent repair (HDR), donor DNAs, donor RNAs, primers, or inhibitory nucleic acids.
  • sgRNAs single-guide RNAs
  • RNAi interfering RNAs
  • shRNAs small hairpin RNAs
  • retrons modified retrons
  • RNA templates reverse transcriptase (RT) templates
  • HDR homology dependent repair
  • donor DNAs donor RNAs
  • primers primers
  • inhibitory nucleic acids bind to a target site in a genomic DNA; such methods can include evaluating the genome of one or more of the host cells for genomic modifications.
  • the expression systems used in the methods can include viral vector expression systems that can be or has been packaged in packaging cells, for example, where the RNA polymerase II (Pol-II) promoter, the nucleic acid segment encoding at least one RNA, the mascRNA-encoding elements, a protein encoding segment, or a combination thereof is encoded on an antisense strand of the viral expression vector.
  • the methods can also include use of at least one protein encoding expression cassette each having a promoter operably linked to at least one proteinencoding segment.
  • Such protein-encoding segments an for example encode at least one reverse transcriptase, Cas nuclease, Cas protein, transposase, fluorescent protein, signal-producing protein, Nodamura virus protein B2 (NovB2), or a combination thereof.
  • the specifically excised portion can be a guide RNA such as a single-guide RNA (sgRNA) for a CRISPR system.
  • sgRNA single-guide RNA
  • Most CRISPR/Cas9 guide RNA expression systems use RNA polymerase-III (Pol-III) promoters such as U6, which are efficient. But such Pol-III promoters act in a constitutive fashion.
  • Pol-III promoters act in a constitutive fashion.
  • Use of Pol-II promoters as described herein allows the sgRNA to be expressed in a more regulated fashion.
  • a sgRNA can be expressed by the same Pol-II promoter as for a target gene so that the sgRNA becomes available when the target gene is expressed.
  • RNA flanking regions allow an RNA to be excised so that the 5 ’cap and/or a 3’ poly (A) tail are not present and do not adversely affect the structure or function of the sgRNA.
  • Guide RNAs can therefore be expressed in the right cells, at the right development stage, and under the same transcriptional controls as the target gene.
  • the expression systems and methods described herein generates guide RNAs that are less prone to degradation, providing more efficient genomic modifications.
  • the methods and compositions described herein are useful for Pol-II promoter-dependent tissue-specific, cell type-specific, and pathway-specific sgRNA expression and CRISPR activity.
  • sgRNA expression and CRISPR activity can also be inducible and reversible.
  • Applications include, but are not limited to, CRISPR cutting (single-strand or double-strand cuts) and CRISPR-based genome modifications, such as CRISPR interference (CRISPRi), CRISPR activation (CRISPRa), base editing (BE), PRIME editing (PE), CRISPR epigenetic modifications, CRISPR-based molecular recording, and any other applications of CRISPR systems involving use of a guide RNA.
  • CRISPR cutting single-strand or double-strand cuts
  • CRISPR-based genome modifications such as CRISPR interference (CRISPRi), CRISPR activation (CRISPRa), base editing (BE), PRIME editing (PE), CRISPR epigenetic modifications, CRISPR-based molecular recording, and any other applications of CRISPR systems involving use of a guide RNA.
  • CRISPRi CRISPR interference
  • CRISPRa CRISPR activation
  • BE base editing
  • PE PRIME
  • the specifically excised portion can be an RNA transcript that serves as a template for another RNA (RNA template).
  • RNA template RNA template
  • the cell can naturally express a reverse transcriptase or be modified to express a reverse transcriptase.
  • a reverse transcriptase can generate a DNA from the RNA template.
  • such an reverse transcribed DNA can serve as the basis for homology-directed repair (HDR), similar to approaches used in PRIME editing.
  • HDR homology-directed repair
  • the specifically excised portion can in some cases be a retron.
  • Retrons are bacteria-derived elements that undergo reverse transcription to produces fragments of single-stranded DNA (ssDNA).
  • ssDNA single-stranded DNA
  • Such ssDNA can also serve as a donor DNA for genomic editing.
  • FIG. 1A-1G illustrate that mascRNA-based guide RNA biogenesis enables Pol-II promoter-controlled genome editing of a fluorescent protein (e.g. a green fluorescent protein or a blue fluorescent protein (BFP) edited to produce a blue fluorescent protein or a green fluorescent protein (GFP)).
  • a fluorescent protein e.g. a green fluorescent protein or a blue fluorescent protein (BFP) edited to produce a blue fluorescent protein or a green fluorescent protein (GFP)
  • FIG. 1A shows the structure and sequence (SEQ ID NO: 1) of a natural mascRNA that can be cleaved at the 5’-end by RNase P and at the 3 ’-end by RNase Z.
  • SEQ ID NO: 1 shows the structure and sequence (SEQ ID NO: 1) of a natural mascRNA that can be cleaved at the 5’-end by RNase P and at the 3 ’-end by RNase Z.
  • IB is a schematic illustrating guide RNA biogenesis of RNA polymerase II (Pol-II) expressed precursor guide (pre-guide) RNA transcripts that were processed by RNase P and/or RNase Z (RNase P/Z) cleavage into mature CRISPR-Cas9 single-guide RNAs (sgRNAs).
  • the mascRNA can be processed by either RNase P or RNase Z on both sides.
  • MascRNAs are tRNA- like structures that are recognized and processed by the endogenous RNase P/Z machinery.
  • TH triple-helix motif that stabilizes the 3 ’-end of a non-polyadenylated transcript.
  • FP fluorescent protein (e.g. BFP, GFP, or mCherry).
  • FIG. 1C illustrates the structure of an RNase P/Z guide RNA (PZG) system, composed of a segment having one or more sgRNA sequences flanked by mascRNA elements (e.g., mascRNA engineered derivatives maR-X and maR-M).
  • mascRNA elements e.g., mascRNA engineered derivatives maR-X and maR-M.
  • the mascRNA sequence shown in FIG. 1 A can also be used.
  • the mascRNAs elements shown have altered stem sequences to decrease homology and minimize recombination during combined use in lentiviral vectors.
  • the PZG system can also include a spacer (the N nucleotides, with a length and a sequence that can vary depending upon the guide RNA selected). Note that after pre-guide RNA processing, no flanking mascRNA or other sequences remain that could encumber sgRNA performance. However, at least part of the spacer can be part of the mature RNA (e.g., part of the guide RNA) (SEQ ID NO: 10, where n is a, g, c, t or u). FIG. ID shows results of a comparison of genome editing efficiency of Pol-III versus Pol-II promoter expressed sgRNAs.
  • HEK293T cells encoding a transgenic GFP were transiently transfected with a plasmid expressing a Pol-III (U6 promoter) driven sgRNA and Cas9 (pCF123) or a set of two plasmids expressing a Pol-II (LTR promoter) driven sgRNA (PZG system, pCF206) and Cas9 (pCF210). Genome editing was analyzed at day 10 post-transfection using T7E1 assay and quantified by densitometry.
  • FIG. IE graphically illustrates the percent editing of Pol-III versus Pol-II promoter expressed sgRNAs as detected from densitometry of the gel shown in FIG.
  • FIG. IF is a schematic illustrating the BFP-to-GFP conversion and showing the sequences edited.
  • Cells were co-transfected with plasmids expressing Cas9 and the indicated sgRNAs, either from a Pol-III (U6) promoter or Pol-II PZG system, as well as ssDNA homology directed repair (HDR) templates.
  • HDR ssDNA homology directed repair
  • FIG. 2A-2D illustrate use of anti-sense lentiviral vectors and Nodamura virus protein B2 (NovB2) for efficient delivery and biogenesis of Pol-II guide RNAs (sgRNAs).
  • FIG. 2A shows two constructs for comparing sense-strand encoded guide RNA expression from either a Pol-III promoter or a Pol-II promoter.
  • the U6-sgRNA (pCF221) construct included the sgRNA operably linked to a Pol-III promoter (U6), while the Sense-pzgRNA (pCF212) construct included a coding region for the sgRNA that was flanked by mascRNAs, where expression was driven by a Pol-II promoter.
  • FIG. 2B graphically illustrates that percentage of HEK-RT1-Cas9 cells that expressed red fluorescent protein (RFP) at day 5 post-transduction with the constructs shown in FIG. 2A.
  • FIG. 2C shows that co-transfection of Nodamura virus protein B2 (NovB2) and the mascRNA-guide RNA construct on the antisense strand of a viral vector solves the RNase/degradation problem illustrated in FIG. 2B when using a lentiviral vector and a Pol-II promoter.
  • the pCF561 construct contained a 5’ LTR on the sense strand and an active promoter driving mCherry on the antisense strand.
  • the sense strand is packaged allowing expression of the mascRNA-guide RNA, which can then be processed to provide the sgRNA.
  • FIG. 2D is a schematic illustrating lentiviral delivery strategies for RNase P/ RNase Z mature RNA systems (e.g., a PZG system).
  • Encoding recombinant DNA on the anti-sense strand prevents cleavage of the viral genome by RNase P / RNase Z in packaging cells.
  • sense and anti-sense transcripts can form double-stranded RNA (dsRNA) species that are degraded by cellular machinery. This problem was solved by co-transfection of Nodamura virus protein B2 (NovB2) to prevent such degradation, thereby enabling high-titer virion production.
  • NovB2 Nodamura virus protein B2
  • FIG. 3 graphically illustrates guide RNA (e.g., single guide RNA) expression from an optimized mascRNA-based Pol-II promoter system provides genome editing efficiencies comparable to those of standard U6 RNA polymerase III (Pol-III) promoter methods.
  • guide RNA e.g., single guide RNA
  • FIG. 4A-4I illustrate a single or multiplexed all-in-one doxycycline controlled RNA expression (e.g., a PZG) system that provides inducible mature RNA (e.g., single guide RNA) production, and when the mature RNA is a guide RNA, inducible editing at endogenous loci can be achieved.
  • FIG. 4A is a schematic of the structures of doxycycline-inducible (dox-inducible) sgRNA expression vectors, where mascRNA elements flank the segment encoding the guide RNAs (sgRNA).
  • All vectors featured an all-in-one Tet-On system encoded on the lentiviral antisense strand, and either a puromycin (pCF621), hygromycin B (pCF622), or blasticidin S (pCF623) resistance marker.
  • the symbol TH refers to a triple-helix motif
  • the UCOE refers to a ubiquitous chromatin opening element
  • the EFS refers to a EF la-short promoter
  • the SFFV refers to a spleen focus-forming virus long terminal repeat promoter
  • BSD blasticidin S deaminase.
  • FIG. 4B shows a doxycycline response curve where mCherry induction was used to quantify doxycycline-induced expression.
  • FIG. 4C graphically illustrates the median fluorescence intensity (MFI) of mCherry-positive cells from the data shown in FIG. 4B.
  • MFI median fluorescence intensity
  • FIG. 4D illustrates robust doxycycline-controlled genome editing at the PCSK9 endogenous locus as detected by T7 endonuclease 1 (T7E1) assays of genomic DNA from U251-Cas9 (pCF226) cells stably transduced with a vector (pCF622).
  • T7E1 T7 endonuclease 1
  • FIG. 4E illustrates robust doxycycline-controlled genome editing at the PTEN endogenous locus as detected by T7 endonuclease 1 (T7E1) assays of genomic DNA from U251- Cas9 (pCF226) cells stably transduced with a vector (pCF622). As shown, editing gave rise to smaller genomic fragments when doxycycline induced expressed of the guide RNA encoded in the pCF226 vector. One representative replicate is shown. Arrows highlight the cleaved bands in the respective lane.
  • T7E1 T7 endonuclease 1
  • FIG. 4F illustrates robust doxy cy cline-controlled genome editing at the GABPB 1 endogenous locus as detected by T7 endonuclease 1 (T7E1) assays of genomic DNA from U251-Cas9 (pCF226) cells stably transduced with a vector (pCF622 or pCF623).
  • T7E1 T7 endonuclease 1
  • the guide RNAs sgRNAs
  • editing gave rise to smaller T7E1 assay fragments when doxycycline induced expression of the guide RNA encoded in the pCF226 vector.
  • One representative replicate is shown. Arrows highlight the cleaved bands in the respective lane.
  • 4G is a schematic illustrating cell killing by lethal guide RNAs (sgCIDEs) that target repetitive genomic sequence elements when the lethal guide RNA (sgCIDEs) are expressed in a doxycycline- inducible manner from the expression mascRNA flanked systems described herein.
  • the assay was performed in Cas9 expressing glioblastoma (GBM) target cells with wild-type cells as controls for normalization.
  • the expression / fluorescence of mCherry was used to detect live cells, and its relative absence (compared to control cells) indicated cell death.
  • FIG. 4H graphically illustrates the percentage of nontargeting control (PZG-sgNT) cells and lethal guide RNA expressing (PZG-sgCIDE) cells that express mCherry from the pCF622 vector.
  • PZG-sgNT nontargeting control
  • PZG-sgCIDE lethal guide RNA expressing
  • FIG. 5A-5C illustrate that the optimized Pol-II expression systems enable sgRNA multiplexing and controlled removal of target sites (e.g. removal of an exon, or removal of any desired genomic region).
  • FIG. 5A is a schematic illustrating the structure of optimized Pol-II controlled expression of two sgRNAs.
  • the expression system can provide doxycycline-inducible (dox-inducible) sgRNA expression to produce two sgRNAs that can target and remove PCSK9 exon 4 variants as illustrated below the expression construct.
  • FIG. 5B illustrates precise dox-induced excision of a genomic locus (exon 4 of the PCSK9 gene).
  • a genotyping gel is shown demonstrating simultaneous cutting by two sgRNAs and removal of the intervening genomic sequence in U251-Cas9 (pCF226) cells stably transduced with a pCF622 vector that enables dox-induced expression of the pair of guide RNAs.
  • Transduced cells were selected on hygromycin before treatment with or without doxycycline (1 pg/ml) for four days. One representative replicate is shown. In the marker lanes, the heavier bands represent the 500 bp and 1 kb reference markers.
  • C refers to a water control.
  • FIG. 5C graphically illustrates the percent of cells with exon 4 of the PCSK9 gene removed, illustrating quantification of dual-cut excision efficiency. The data represent the mean and standard deviation of triplicates from samples partially shown in FIG. 5B.
  • RNAs from Pol-II promoters by using engineered mascRNA (MALAT1 -associated small cytoplasmic RNA) elements to specifically excise the desired mature RNA portion from a given primary RNA transcript.
  • engineered mascRNA MALAT1 -associated small cytoplasmic RNA
  • use of Pol-II promoters provides improved, regulated control of RNA expression.
  • Pol-II transcripts are processed to include extra nucleotides (5’ caps, poly(A) tails) that can interfere with the function of certain RNAs such as CRISPR guide RNAs, retrons, RNAi transcripts, RNA templates for reverse transcription, and the like.
  • the expression systems and methods described herein allow Pol-II regulated, inducible expression of such precisely tailored RNAs that are immediately useful (without the necessity to remove 5’caps, poly(A) tails, etc.).
  • RNAs are generated as longer transcripts where the RNA of interest is flanked on its 5’ and 3’ sides by mascRNA elements.
  • MascRNAs are removed by RNase P/Z in a manner similar to transfer RNAs (tRNAs), leading to their excision from longer precursor transcripts.
  • tRNAs transfer RNAs
  • the result is an RNA without any undesirable features such as a 5’cap, a poly(A) tail, or a mascRNA (see, e.g., FIG. IB).
  • Nuclear ribonuclease P (RNase P) is a ubiquitous endoribonuclease, found in archaea, bacteria and eukaryotes as well as chloroplasts and mitochondria. Its best characterized enzyme activity is the generation of mature 5 '-ends of tRNAs by cleaving the 5 '-leader elements of precursor-tRNAs. However, RNase P readily cleaves mascRNAs from primary RNAs that include RNA segments (e.g., guide RNAs) of interest.
  • RNA segments e.g., guide RNAs
  • RNase Z is an endonuclease that generally removes 3' extensions from tRNA precursors, which is an essential step in tRNA biogenesis.
  • Human cells contain a long form (RNase Z(L)) encoded by ELAC2, and a short form (RNase Z(S); ELAC1).
  • RNase Z(S) was found in the cytosol, whereas RNase Z(L) localized to the nucleus and mitochondria.
  • MALAT1 metalastasis associated lung adenocarcinoma transcript 1; see NCBI accession nos. NR_002819.4, NR_144567, and NR_144568.1.
  • the MALATI gene is also referred to as HCN; NEAT2; PRO2853; LINC00047; and NCRNA00047.
  • a sequence for a mascRNA is shown below (SEQ ID NO: 1; NCBI Accession no. HG975416.1) and its structure is also shown in FIG. 1.
  • a mascRNA is shown below as being a nucleotide sequence segment (positions 8356 - 8413) of the Homo sapiens nuclear enriched abundant transcript 2 (NEAT2) mRNA (NCBI accession no. EFl 77381.1; SEQ ID NO:2).
  • a mascRNA is shown below as being a nucleotide sequence segment (positions 7519-7576) of the Homo sapiens metastasis associated in lung adenocarcinoma transcript 1 long isoform, a transcribed non-coding RNA (NCBI accession no. BK001418.1; SEQ ID NO:3)
  • nucleotide sequence segment (positions 7753-7810) of the Homo sapiens alpha gene sequence (NCBI accession no. AF203815.1; SEQ ID NO:4).
  • the maR-X derivative of mascRNA that can be used in the expression systems described herein has the following sequence (SEQ ID NO:5).
  • the maR-M derivative of mascRNA that can be used in the expression systems described herein has the following sequence (SEQ ID NO: 6).
  • the mascRNAs can have sequence variations that do not adversely affect their structures.
  • the mascRNAs have one or more nucleotide differences from the sequences from the SEQ ID NO: 1-6 sequences.
  • the mascRNAs used in the compositions and methods described herein can, for example, have two, three, four, five, six, seven, eight, nine, ten, or more nucleotide differences from the sequence shown as for SEQ ID NO: 1-6.
  • the mascRNAs used in the methods and compositions described herein can be a DNA or RNA with at least 70%, or at least 75%, or at least 80%, or at least 85%, or at least 90%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or at least 99.5% sequence identity to any of SEQ ID NO: 1-6.
  • MascRNAs can be processed by RNase P, RNase Z, or a combination thereof.
  • RNase P and RNase Z are ubiquitously expressed endogenously.
  • Either one of the RNase P and RNase Z enzymes can cleave the mature RNA (e.g., guide RNA) from the primary RNA that is expressed from a Pol-II promoter.
  • RNase P and RNase Z each (separately) form ribonucleoprotein complexes/enzymes and are needed in any cell undergoing tRNA expression and protein production.
  • the mechanism for mascRNA excision is similar to that for transfer RNAs (tRNAs).
  • tRNAs transfer RNAs
  • mature mascRNAs are not aminoacylated and do not contain an anticodon loop.
  • mascRNA do not affect cellular tRNA homeostasis when exogenously expressed, and the aminoacyl group(s) and anticodon loop would not interfere with mascRNA processing.
  • Pol-II driven guide RNA expression enables the design of polycistronic constructs containing a fluorescence marker for easier experimental calibration and spatiotemporal quantification of expressed precisely structured mature RNAs.
  • a triple-helix motif can be included for stabilization of the messenger RNA (mRNA) after guide excision.
  • mRNA messenger RNA
  • guide RNA is used generally to embrace both crispr RNA (crRNA), a trans-activating CRISPR RNA (tracrRNA), and a single guide RNA (sgRNA) that includes both the crRNA and the tracrRNA.
  • the crRNA is typically a 17-20 nucleotide sequence complementary to the target DNA, while a tracrRNA base pairs with the crRNA to facilitate Cas endonuclease activity.
  • a single guide RNA (sgRNA) can be engineered to include the tracrRNA that as well as the crRNA.
  • the expression systems, constructs, host cells and methods described herein include or utilize single guide RNAs (sgRNAs).
  • RNAs e.g. guide RNAs, sgRNAs, RNAi, shRNAs, and retrons
  • RNAi e.g. guide RNAs, sgRNAs, RNAi, shRNAs, and retrons
  • RNAi e.g. guide RNAs, sgRNAs, RNAi, shRNAs, and retrons
  • RNAi e.g. guide RNAs, sgRNAs, RNAi, shRNAs, and retrons
  • RNA-II promoter- driven system that involves a Pol-II promoter operably linked to an RNA-coding region flanked by mascRNA elements.
  • mascRNA elements are readily removed by endogenous enzymes (RNaseP/Z)
  • RNaseP/Z endogenous enzymes
  • this expression system produced precise sequences of RNAs (e.g. guide RNAs) to be produced in a controlled, regulated fashion.
  • RNaseP/Z enzymes are ubiquitous because they are involved in tRNA processing.
  • the expression systems and methods described herein result in scarless biogenesis of mature RNAs, including guide RNAs.
  • FIG. IB illustrates mascRNA-based guide RNA biogenesis from Pol-II promoter precursor constructs, using transfected plasmid vectors in human cells.
  • Pol-II based sgRNA expression led to similar editing efficiency compared to a construct based on a state-of- the-art Pol-III promoter (U6) (FIG. 1D-1G).
  • U6 state-of- the-art Pol-III promoter
  • Such constructs and methods provide a robust platform enabling Pol-II mediated expression of mature RNAs such as guide RNAs, retrons, RNAi, shRNAs, and RNA templates for a reverse transcriptase.
  • RNase P and RNase Z are core fitness genes essential to cell survival (Hart et al. Cell 163: 1515-26 (2015)). However, in some cases RNase P and/or RNase Z can degrade desirable RNAs.
  • the Pol-II guide RNA expression system including the mascRNA elements could be encoded on the antisense strand that is not needed for viral packaging. But long double-stranded RNA species produced by transcription in the sense and antisense directions could still be degraded by cellular responses. This problem was overcome by also expressing a viral auxiliary protein - Nodamura virus protein B2 (NovB2) that has evolved to suppress antiviral responses in infected cells. A sequence for the Nodamura viral B2 protein is shown below (NCBI accession no. NP_077731.1; SEQ ID N0:7).
  • a nucleotide sequence for this B2 Nodamura viral protein is available from the NCBI database as accession no. NC_002690.1 and AF174533.1.
  • a nucleotide sequence coding a B2 Nodamura viral protein (available at NCBI NC_002690.1).
  • Nucleotides 2744-3157 of the NCBI NC_002690.1 encode the B2 Nodamura viral protein and are shown below as SEQ ID NO: 8.
  • a nucleotide sequence coding a B2 Nodamura viral protein (available at NCBI AF174533.1). Nucleotides 2744-3157 encoding a B2 Nodamura viral protein are shown below as SEQ ID NO:9.
  • B2 Nodamura viral protein and nucleotide sequences can have sequence variations that do not adversely affect their structures or functions.
  • the B2 Nodamura viral protein and nucleotide sequences can have one or more nucleotide or amino acid differences from the sequences from the SEQ ID NO:7-9 sequences.
  • the B2 Nodamura viral protein and nucleotide sequences used in the compositions and methods described herein can, for example, have two, three, four, five, six, seven, eight, nine, ten, or more nucleotide or amino acid differences from the sequences shown as for SEQ ID NO:7-9.
  • the B2 Nodamura viral protein and nucleotide sequences used in the methods and compositions described herein can have at least 70%, or at least 75%, or at least 80%, or at least 85%, or at least 90%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or at least 99.5% sequence identity to any of SEQ ID NO:7-9.
  • RNA polymerase II is a 12-subunit DNA-dependent RNA polymerase that transcribes nuclear genes encoding messenger RNAs and several small nuclear RNAs.
  • Pol-II promoters contain combinations of DNA sequences, which include core or basal promoter elements, promoter proximal elements, and distal enhancer elements. Transcription initiation by pol II is precisely regulated by transcription factors that interact with each other and with the Pol-II promoter elements.
  • Core Pol-II promoters in higher eukaryotes span from approximately 40 base pairs upstream to 40 base pairs downstream of the transcription start site.
  • Core promoter elements such as the TATA box, Initiator, BRE, MTE, DPE, and/or DCE can be found in focused core promoters.
  • Pol-II promoters can have one or more of these promoter elements, or two or more of these promoter elements, or three or more of these promoter elements, or four or more of these promoter elements. Many Pol-II promoters do not have all core promoter elements.
  • the AT -rich sequence is the AT -rich sequence called the TATA box, which is located from approximately 24- 31 base pairs upstream of the transition start site.
  • the consensus TATA box sequence is TATAWAAR (nontemplate strand, using IUPAC symbols). This sequence is conserved from archaebacteria to humans.
  • the IUPAC symbols for degenerate nucleotide bases are shown in Table 1 below.
  • Thymine or Uracil
  • the Initiator element includes the transcription start site.
  • the Initiator has consensus sequence YYANWYY (nontemplate strand using IUPAC symbols) in humans
  • TATAWAAR The upstream T nucleotide in the TATA box consensus sequence TATAWAAR is most commonly at -31 or -30 relative to the A+l (or G+l) in the Initiator sequence.
  • TATA-binding protein (TBP) of the transcription factor IID (TFIID) multisubunit complex binds the TATA box sequence and bends the DNA to commence recruitment of the transcription machinery.
  • TATA- containing promoters make up only 10-15% of the mammalian promoters.
  • the DPE downstream core promoter element
  • the DPE consensus sequence is RGWYV (using IUPAC symbols) and is conserved from Drosophila to humans. It is a downstream TFIID recognition sequence that is important for basal transcription activity.
  • the motif ten element is found just upstream of the DPE from +18 to +27 relative to the A+l in the Inr. and has a consensus sequence of CSARCSSAAC (nontemplate strand; using IUPAC symbols)
  • the BRE (TFIIB recognition element) was originally identified as a TFIIB- binding sequence that is immediately upstream of a subset of TATA boxes.
  • TFIIB can bind upstream or downstream of the TATA box at the BRE U (upstream BRE, which is the same as the original BRE) or BRE d (downstream BRE) sequences.
  • the BRE U consensus sequence is SSRCGCC (using IUPAC symbols).
  • BRE d is located immediately downstream of the TATA box and has a consensus of RTDKKKK (using IUPAC symbols).
  • the BRE U and BRE d can act in either a positive or negative manner.
  • the DCE (downstream core element) occurs frequently with the TATA box and appears to be distinct from the DPE.
  • the DCE consists of three sub-elements: the Si sub-element with sequence CTTC at +6 to +11; the Sn sub-element with sequence CTGT from +16 to +21; and the Sm sub-element with sequence AGC from +30 to +34.
  • auxiliary proteins Two groups of auxiliary proteins facilitate and control Pol II promoter transcription.
  • protein-protein interactions that regulate pol II activity involve components of the preinitiation complex (TBP, TAFus, TFIIB, pol II, TFIIF, TFIIE, and TFIIH) and transcriptional activators (bound either to promoter proximal or distal enhancer elements).
  • TATA box, Inr, MTE, DPE, and DCE are recognition sites for the binding of transcription factor TFIID.
  • TFIID transcription factor
  • BRE U and BRE d interact with TFIIB.
  • core promoter elements are not universal; rather, each is present in only a subset of core promoters. Moreover, some core promoters appear to lack all of the known core promoter elements.
  • TATA box and BRE are the most ancient of the core promoter motifs.
  • the TATA box and BRE along with their cognate protein factors, TBP (TATA boxbinding protein) and TFIIB (transcription factor IIB), are conserved from Archaea to humans.
  • TBP TATA boxbinding protein
  • TFIIB transcription factor IIB
  • the expression systems described herein can include a nucleic acid encoding a guide RNA that specifically recognizes a target nucleic acid.
  • Guide RNAs gRNAs
  • sgRNAs single guide RNAs
  • the guide RNA and the enzyme form complexes before they bind to the targeted RNA or DNA site. Once bound to the target site, the RNA-targeting or DNA-targeting nucleases can cleave or otherwise alter the targeted RNA or DNA.
  • Guide RNAs typically include short RNAs composed of a scaffold sequence necessary for Cas protein-binding and a user- defined ⁇ 20 nucleotide recognition sequence that binds to the target nucleic acid to be recognized and/or modified.
  • Guide RNAs can have sequences complementary to the target sequence.
  • the genomic target of the Cas protein or Cas nuclease can be changed simply by changing the target recognition sequence in the guide RNA.
  • the target sequence (and hence the guide RNA sequence) may need to be near a protospacer adjacent motif (PAM) sequence.
  • PAM protospacer adjacent motif
  • guide RNA sequences are generally selected that are complementary to a target sequence that is near or that includes a PAM sequence.
  • PAM sequences recognized by various Cas nucleases are shown in Table 2 below.
  • SpCas9 VQR variant 3’ NGAN or NGNG xCas9 3' NG, GA A, or GAT
  • NM Neisseria meningitidis
  • ST Streptococcus thermophilus
  • TD Treponema denticola
  • the target nucleic acid can be selected by identifying all PAM sequences within the genetic region to be targeted. If there are no PAM sequences for a particular Cas nuclease within the selected target, an alternative Cas enzyme can be used. Once possible PAM sequences and possible target sites have been identified, a guide RNA can be prepared, or a nucleic acid segment encoding the guide RNA sequence can be prepared and incorporated into one of the expression systems described herein so that the segments encoding the mascRNAs flank the guide RNA coding region.
  • Cas protein employed has nuclease activity (e.g., endonuclease activity), but in other cases the Cas protein employed has reduced nuclease activity or undetectable levels of nuclease activity.
  • the term Cas protein includes deactivated or partially deactivated Cas proteins (Cas nickases, dead Cas proteins). Such dead Cas proteins can be used in CRISPR interference (CRISPRi) as well as CRISPR activation (CRISPRa). In CRISPRi, dead Cas proteins bind to its DNA target but does not cleave it. The binding of the dead Cas protein alone will prevent the cell’s transcription machinery from accessing the promoter, hence inhibiting the gene expression.
  • a dead Cas protein s ability to bind target DNA can be exploited for activation, i.e. CRISPRa.
  • a transcriptional activator can be fused to a dead Cas protein to activate gene expression without changing the target DNA sequence.
  • the expression systems described herein can include a nucleic acid encoding an inhibitory nucleic acid that specifically recognizes a target nucleic acid (e.g., a target RNA).
  • a target nucleic acid e.g., a target RNA
  • Such expression systems can include a Pol-II promoter operably linked to a nucleic acid segment flanked by mascRNAs, where the nucleic acid segment encodes an inhibitory nucleic acid.
  • the inhibitory nucleic acid binds to a target nucleic acid and inhibits the transcription, translation, or splicing of the target nucleic acid.
  • An inhibitory nucleic acid can have at least one segment that will hybridize to a target nucleic acid under intracellular or stringent conditions.
  • the inhibitory nucleic acid can reduce expression of target nucleic acid.
  • An inhibitory nucleic acid may hybridize to a genomic DNA, a messenger RNA, or a combination thereof.
  • An inhibitory nucleic acid may be single stranded or double stranded, circular or linear.
  • An inhibitory nucleic acid is a polymer of ribose nucleotides or deoxyribose nucleotides having more than 13 nucleotides in length.
  • An inhibitory nucleic acid can reduce the expression and/or activity of a target nucleic acid.
  • Such an inhibitory nucleic acid may be completely complementary to a segment of an endogenous target nucleic acid (e.g., an RNA). Alternatively, some variability is permitted in the inhibitory nucleic acid sequences relative to target nucleic acid sequences.
  • An inhibitory nucleic acid can hybridize to a target nucleic acid under intracellular conditions or under stringent hybridization conditions and is sufficiently complementary to inhibit expression of the endogenous target nucleic acid. Intracellular conditions refer to conditions such as temperature, pH and salt concentrations typically found inside a cell, e.g. an animal or mammalian cell.
  • stringent hybridization conditions are selected to be about 5°C lower than the thermal melting point (T m ) for the specific sequence at a defined ionic strength and pH.
  • stringent conditions encompass temperatures in the range of about 1°C to about 20 °C lower than the thermal melting point of the selected sequence, depending upon the desired degree of stringency as otherwise qualified herein.
  • Inhibitory nucleic acid can have, for example, 2, 3, 4, or 5 or more stretches of contiguous nucleotides that are precisely complementary to a target nucleic acid coding sequence, each separated by a stretch of contiguous nucleotides that are not complementary to adjacent coding sequences, can inhibit the function of one or more target nucleic acids.
  • each stretch of contiguous nucleotides is at least 4, 5, 6, 7, or 8 or more nucleotides in length.
  • Non-complementary intervening sequences may be 1, 2, 3, or 4 nucleotides in length.
  • Inhibitory nucleic acids of the invention include, for example, a short hairpin RNA, a small interfering RNA, a ribozyme or an antisense nucleic acid molecule.
  • the inhibitory nucleic acid molecule may be single or double stranded and may function in an enzyme-dependent manner or by steric blocking.
  • Inhibitory nucleic acid molecules that function in an enzyme-dependent manner include forms dependent on RNase H activity to degrade target mRNA. These include singlestranded DNA, RNA, and phosphorothioate molecules, as well as the double-stranded RNAi/siRNA system that involves target mRNA recognition through sense-antisense strand pairing followed by degradation of the target mRNA by the RNA-induced silencing complex.
  • Steric blocking inhibitory nucleic acids which are RNase-H independent, interfere with gene expression or other mRNA-dependent cellular processes by binding to a target mRNA and getting in the way of other processes.
  • Small interfering RNAs may be used to specifically reduce translation of target nucleic acids such that translation of the encoded target polypeptide is reduced.
  • SiRNAs mediate post-transcriptional gene silencing in a sequence-specific manner. See, for example, website at invitrogen.com/site/us/en/home/Products-and-Services/Applications/ mai.html. Once incorporated into an RNA-induced silencing complex, siRNA mediate cleavage of the homologous endogenous mRNA transcript by guiding the complex to the homologous mRNA transcript, which is then cleaved by the complex.
  • the siRNA may be homologous and/or complementary to any region of the target transcript.
  • the region of homology may be 30 nucleotides or less in length, preferable less than 25 nucleotides, and more preferably about 21 to 23 nucleotides in length.
  • SiRNA is typically double stranded and may have two-nucleotide 3’ overhangs, for example, 3’ overhanging UU dinucleotides.
  • Methods for designing siRNAs are known to those skilled in the art. See, for example, Elbashir et al. Nature 411: 494-498 (2001); Harborth et al. Antisense Nucleic Acid Drug Dev. 13: 83-106 (2003).
  • the construction of the siRNA expression plasmid can involve the selection of a target region of an mRNA. Elbashir et al. have provided guidelines for making siRNAs (Elbashir, S.M., et al., Analysis of gene function in somatic mammalian cells using small interfering RNAs. Methods, 2002. 26(2): p. 199-213).
  • a target region may be selected that can be 50 to 100 nucleotides downstream of the start codon. The 5' and 3' untranslated regions and regions close to the start codon are often avoided because these may be blocked by regulatory proteins bound to their binding sites.
  • siRNA can begin with AA, have 3' UU overhangs for both the sense and antisense siRNA strands, and have an approximate 50 % G/C content.
  • An example of a sequence for a synthetic siRNA is 5'-AA(N19)UU, where N is any nucleotide in the mRNA sequence and should be approximately 50% G-C content.
  • the selected sequence(s) can be compared to others in the human genome database to minimize homology to other known coding sequences (e.g., by Blast search, for example, through the NCBI website).
  • the insert encoding the siRNA may be expressed as an RNA transcript that folds into an siRNA hairpin.
  • the RNA transcript may include a sense siRNA sequence that is linked to its reverse complementary antisense siRNA sequence by a spacer sequence that forms the loop of the hairpin as well as, optionally, a string of U’s at the 3’ end.
  • the loop of the hairpin may be of any appropriate lengths, for example, 3 to 30 nucleotides in length, preferably, 3 to 23 nucleotides in length, and may be of various nucleotide sequences including, AUG, CCC, UUCG, CCACC, CTCGAG, AAGCUU, CCACACC and UUCAAGAGA.
  • SiRNAs can be produced in vivo by cleavage of double-stranded RNA introduced directly or via a transgene or virus. .
  • a segment encoding an inhibitory nucleic acid such as a short hairpin RNA siRNA or an antisense oligonucleotide can be inserted between mascRNA elements within the expression systems described herein.
  • retrons in the expression systems and methods described herein can be unmodified retrons or engineered retrons.
  • a retron is a distinct DNA sequence found in the genome of many bacteria species that codes for reverse transcriptase and a unique single-stranded DNA/RNA hybrid called multicopy single-stranded DNA (msDNA).
  • Retron msr RNA is a non-coding RNA produced by retron elements and is the immediate precursor to the synthesis of msDNA. The retron msr RNA folds into a characteristic secondary structure that contains a conserved guanosine residue at the end of a stem loop.
  • RNA/RNA chimera which is composed of small single-stranded DNA linked to small single-stranded RNA.
  • the RNA strand is joined to the 5' end of the DNA chain via a 2'- 5 ' phosphodiester linkage that occurs from the 2' position of the conserved internal guanosine residue.
  • a retron operon carries a promoter sequence P that controls the synthesis of an RNA transcript carrying three loci: msr, msd, and ret.
  • the ret gene product, a reverse transcriptase processes the msd/msr portion of the RNA transcript into msDNA.
  • Retron elements are about 2 kb long. They contain a single operon controlling the synthesis of an RNA transcript carrying three loci, msr, msd, and ret, that are involved in msDNA synthesis.
  • the DNA portion of msDNA is encoded by the msd gene
  • the RNA portion is encoded by the msr gene
  • the product of the ret gene is a reverse transcriptase similar to the RTs produced by retroviruses and other types of retroelements.
  • the retron RT contains seven regions of conserved amino acids, including a highly conserved tyr-ala-asp-asp (YADD) sequence associated with the catalytic core.
  • the ret gene product is responsible for processing the msd/msr portion of the RNA transcript into msDNA.
  • Engineered retrons are modified to include additional nucleotides, to include fewer nucleotides, and/or to enhance production of msDNA in a cell.
  • Expression cassettes/expression vectors and expression systems that can express the precisely structured RNAs described herein can be administered to subjects.
  • Cells that have been modified to include the expression cassettes/expression vectors and expression systems can also be administered to subjects.
  • Such expression cassettes, expression vectors, expression systems, and cells generated as described herein can be employed for treatment or prevention of diseases and conditions in a human patient or other subjects. Patients or subjects can be in need of such treatment. In some cases, the patients or subjects may not yet exhibit any symptoms of a condition or disease or another medical condition.
  • the expression cassettes, expression vectors, expression systems, and cells are administered in a manner that permits them to be incorporated into, graft or migrate to a specific tissue site. Such expression systems, expression cassettes, expression vectors, and cells can reconstitute or regenerate functionally deficient areas of tissues. Devices are available that can be adapted for administering cells to selected tissue sites. For therapy, expression systems, expression cassettes, expression vectors, and/or cells can be administered locally or systemically. Administration can be by injection, catheter, implantable device, or the like. The expression systems, expression cassettes, expression vectors, and cells can be administered in any physiologically acceptable excipient or carrier that does not adversely affect the subject. For example, the expression systems, expression cassettes, expression vectors, and cells can be administered intravenously.
  • Methods of administering expression systems, expression cassettes, expression vectors, and/or cells to subjects, particularly human subjects include injection or implantation of the expression systems, expression cassettes, expression vectors, and cells into target sites.
  • the administering expression systems, expression cassettes, expression vectors, and/or cells can be inserted into a delivery device which facilitates introduction, uptake, incorporation, or implantation of the expression systems, expression cassettes, expression vectors, and cells.
  • a delivery devices include tubes, e.g., catheters, for introducing cells, expression vectors, and fluids into the body of a recipient subject.
  • the tubes can additionally include a needle, e.g., a syringe, through which the cells of the invention can be introduced into the subject at a desired location. Multiple injections may be made using this procedure.
  • the term "solution” includes a carrier or diluent in which the expression system, expression cassettes, expression vectors, and cells of the invention remain viable and/or functional.
  • Carriers and diluents that can be used include saline, aqueous buffer solutions, solvents and/or dispersion media. The use of such carriers and diluents are available.
  • the solution is preferably sterile and fluid to the extent that easy syringability exists.
  • the expression systems, expression cassettes, expression vectors, and cells can also be embedded in a support matrix.
  • suitable ingredients include matrix proteins that support or promote the incorporation of adhesion of the expression systems, expression cassettes, expression vectors, and modified cells.
  • the composition may include physiologically acceptable matrix scaffolds. Such physiologically acceptable matrix scaffolds can be resorbable and/or biodegradable.
  • expression systems, expression cassettes, expression vectors can be incorporated into cells to generate modified cells.
  • an expression vector encoding a nuclease e.g., a Cas nuclease
  • a nuclease e.g., a Cas nuclease
  • a population of modified cells generated by the methods described herein can include low percentages of non-modified cells (e.g., other cardiac cells and/or endothelial cells).
  • a population of modified cells for use in compositions and for administration to subjects can have less than about 90% non-modified cells, less than about 85% non-modified cells, less than about 80% non-modified cells, less than about 75% non-modified cells, less than about 70% non-modified cells, less than about 65% non-modified cells, less than about 60% non-modified cells, less than about 55% non-modified cells, less than about 50% non-modified cells, less than about 45% non-modified cells, less than about 40% non-modified cells, less than about 35% non-modified cells, less than about 30% non-modified cells, less than about 25% non-modified cells, less than about 20% non-modified cells, less than about 15% non-modified cells, less than about 12% non-modified cells, less than about 10% non-modified cells, less than about
  • modified cells are capable of migrating to an appropriate site for regeneration and differentiation within a subject.
  • the modified cells can first be tested in a suitable animal model. At one level, modified cells are assessed for their ability to survive and maintain their phenotype in vivo. Modified cells can also be assessed to ascertain whether they migrate to diseased or injured sites in vivo, or to determine an appropriate number, or dosage, of cells to be administered.
  • Cell compositions can be administered to immunodeficient animals (such as nude mice, or animals rendered immunodeficient chemically or by irradiation). Tissues can be harvested from the experimental animal after a period of regrowth and the harvested tissues can be assessed to determine whether the administered cells or progeny thereof are still present, are alive, and/or have migrated to desired or undesired locations.
  • Injected cell types can be traced by a variety of methods.
  • cells containing or expressing a detectable label such as green fluorescent protein, or betagalactosidase
  • the cells can be pre-labeled, for example, with BrdU or [ 3 H]-thymidine, or by introduction of an expression cassette that can express green fluorescent protein, or beta-galactosidase.
  • the modified cells can be detected by their expression of a cell marker that is not expressed by the animal employed for testing (for example, a human-specific antigen when injecting cells into an experimental animal).
  • the presence and phenotype of the administered population of modified cells can be assessed by fluorescence microscopy (e.g., for green fluorescent protein, or beta-galactosidase), by immunohistochemistry (e.g., using an antibody against a human antigen), by ELISA (using an antibody against a human antigen), or by RT-PCR analysis using primers and hybridization conditions that cause amplification to be specific for an expressed RNA.
  • fluorescence microscopy e.g., for green fluorescent protein, or beta-galactosidase
  • immunohistochemistry e.g., using an antibody against a human antigen
  • ELISA using an antibody against a human antigen
  • RT-PCR analysis using primers and hybridization conditions that cause amplification to be specific for an expressed RNA.
  • Modified cells can be included in the compositions in varying amounts depending upon the extent of disease or the condition of the subject.
  • the compositions can be prepared in liquid form for local or systemic administration containing about 10 3 to about 10 12 modified cells, or about 10 4 to about IO 10 modified cells, or about 10 5 to about 10 8 modified cells.
  • One or more expression vectors that can express one or more RNAs, nucleases, or a combination thereof can also be administered to subjects with or without the cells.
  • the expression systems, guide RNAs, nucleases, and/or other expression cassettes/vectors may be administered in a composition as a single dose, in multiple doses, in a continuous or intermittent manner, depending, for example, upon the recipient's physiological condition, whether the purpose of the administration is in response to a stressful event or for more sustained therapeutic purposes, and other factors known to skilled practitioners.
  • the administration of the compositions of the invention may be as a single dose, or essentially continuous over a preselected period of time, or it may be in a series of spaced doses. Both local and systemic administration is contemplated.
  • the amounts of expression vectors, RNPs, guide RNAs, nucleases, and/or cells for use in treatment will vary not only with the particular carrier selected but also with the route of administration, the nature of the condition being treated and the age and condition of the patient. Ultimately, the attendant health care provider may determine proper dosage.
  • nucleic acid or “a protein” or “a cell” includes a plurality of such nucleic acids, proteins, or cells (for example, a solution or dried preparation of nucleic acids or expression cassettes, a solution of proteins, or a population of cells), and so forth.
  • the term “or” is used to refer to a nonexclusive or, such that “A or B” includes “A but not B,” “B but not A,” and “A and B,” unless otherwise indicated.
  • RNase P/Z is defined as “RNase P and/or RNase Z”. Either of the RNase P or RNase Z enzymes can cleave mascRNA from an intervening nucleic acid segment.
  • PZG is defined as an “RNase P and/or RNase Z guide RNA” construct.
  • PZG system is defined as an “RNase P and/or RNase Z guide RNA system.”
  • Recombinant as used herein to describe a nucleic acid molecule means a polynucleotide of genomic, cDNA, bacterial, semi synthetic, or synthetic origin which, by virtue of its origin or manipulation, is not associated with all or a portion of the polynucleotide with which it is associated in nature.
  • recombinant as used with respect to a protein or polypeptide means a polypeptide produced by expression of a recombinant polynucleotide.
  • the gene of interest is cloned and then expressed in transformed organisms, as described further below.
  • the host organism expresses the foreign gene to produce the protein under expression conditions.
  • a "cell” refers to any type of cell.
  • the cell can be in an organism or it can be maintained outside of an organism.
  • the cell can be within a living organism and be in its normal (native) state.
  • the term “cell” includes an individual cell or a group or population of cells.
  • the cell(s) can be a prokaryotic, eukaryotic, or archaeon cell(s), such as a bacterial, archaeal, fungal, protist, plant, or animal cell(s).
  • the cell(s) can be from or be within tissues, organs, and biopsies.
  • the cell(s) can be a recombinant cell(s), a cell(s) from a cell line cultured in vitro.
  • the cell(s) can include cellular fragments, cell components, or organelles comprising nucleic acids. In some cases, the cell(s) are human cells.
  • cell(s) also encompasses artificial cells, such as nanoparticles, liposomes, polymersomes, or microcapsules encapsulating nucleic acids.
  • artificial cells such as nanoparticles, liposomes, polymersomes, or microcapsules encapsulating nucleic acids.
  • the methods described herein can be performed, for example, on a sample comprising a single cell or a population of cells.
  • the term also includes genetically modified cells.
  • transformation refers to the insertion of an exogenous polynucleotide (e.g., an engineered retron) into a host cell, irrespective of the method used for the insertion. For example, direct uptake, transfection, transduction or f- mating are included.
  • exogenous polynucleotide may be maintained as a nonintegrated vector, for example, a plasmid, or alternatively, may be integrated into the host genome.
  • Recombinant host cells refer to cells which can be, or have been, used as recipients for recombinant vector or other transferred DNA, and include the original progeny of the original cell which has been transfected.
  • a "coding sequence” or a sequence which "encodes" a selected RNA or a selected polypeptide is a nucleic acid molecule which is transcribed (in the case of DNA) and translated (in the case of mRNA) into a polypeptide in vivo when placed under the control of appropriate regulatory sequences (or “control elements”).
  • the boundaries of the coding sequence can be determined by a start codon at the 5' (amino) terminus and a translation stop codon at the 3' (carboxy) terminus.
  • a coding sequence can include, but is not limited to, cDNA from viral, prokaryotic or eukaryotic mRNA, genomic DNA sequences from viral or prokaryotic DNA, and even synthetic DNA sequences.
  • a transcription termination sequence may be located 3' to the coding sequence.
  • Typical "control elements,” include, but are not limited to, transcription promoters, transcription enhancer elements, transcription termination signals, polyadenylation sequences (located 3' to the translation stop codon), sequences for optimization of initiation of translation (located 5’ to the coding sequence), and translation termination sequences.
  • “Operably linked” refers to an arrangement of elements wherein the components so described are configured so as to perform their usual function.
  • a given promoter operably linked to a coding sequence is capable of effecting the expression of the coding sequence when the proper enzymes are present.
  • the promoter need not be contiguous with the coding sequence, so long as it functions to direct the expression thereof.
  • intervening untranslated yet transcribed sequences can be present between the promoter sequence and the coding sequence and the promoter sequence can still be considered “operably linked" to the coding sequence.
  • Encoded by refers to a nucleic acid sequence which codes for a polypeptide or RNA sequence.
  • the polypeptide sequence or a portion thereof contains an amino acid sequence of at least 3 to 5 amino acids, more preferably at least 8 to 10 amino acids, and even more preferably at least 15 to 20 amino acids from a polypeptide encoded by the nucleic acid sequence.
  • the RNA sequence or a portion thereof contains a nucleotide sequence of at least 3 to 5 nucleotides, more preferably at least 8 to 10 nucleotides, and even more preferably at least 15 to 20 nucleotides.
  • isolated refers to material that is free to varying degrees from components which normally accompany it as found in its native state.
  • Isolate denotes a degree of separation from original source or surroundings.
  • Purify denotes a degree of separation that is higher than isolation.
  • a “purified” or “biologically pure” protein is sufficiently free of other materials such that any impurities do not materially affect the biological properties of the protein or cause other adverse consequences. That is, a nucleic acid or peptide of this invention is purified if it is substantially free of cellular material, viral material, or culture medium when produced by recombinant DNA techniques, or chemical precursors or other chemicals when chemically synthesized.
  • Purity and homogeneity are typically determined using analytical chemistry techniques, for example, polyacrylamide gel electrophoresis or high-performance liquid chromatography.
  • the term "purified" can denote that a nucleic acid or protein gives rise to essentially one band in an electrophoretic gel.
  • modifications for example, phosphorylation or glycosylation, different modifications may give rise to different isolated proteins, which can be separately purified.
  • “Expression” refers to detectable production of a gene product by a cell.
  • the gene product may be a transcription product (i.e., RNA), which may be referred to as “gene expression”, or the gene product may be a translation product of the transcription product (i.e., a protein), depending on the context.
  • Polynucleotide refers to a polynucleotide of interest or fragment thereof which is essentially free, e.g., contains less than about 50%, preferably less than about 70%, and more preferably less than about at least 90%, of the protein and/or nucleic acids with which the polynucleotide is naturally associated.
  • Techniques for purifying polynucleotides of interest include, for example, disruption of the cell containing the polynucleotide with a chaotropic agent and separation of the polynucleotide(s) and proteins by ion-exchange chromatography, affinity chromatography and sedimentation according to density.
  • substantially purified generally refers to isolation of a substance (compound, polynucleotide, protein, polypeptide, peptide composition) such that the substance comprises the majority percent of the sample in which it resides.
  • a substantially purified component comprises 50%, preferably 80%-85%, more preferably 90-95% of the sample.
  • Techniques for purifying polynucleotides and polypeptides of interest are well-known in the art and include, for example, ionexchange chromatography, affinity chromatography and sedimentation according to density.
  • transfection is used to refer to the uptake of foreign DNA by a cell.
  • a cell has been "transfected” when exogenous DNA has been introduced inside the cell membrane.
  • transfection techniques are generally known in the art. See, e.g., Graham et al. (1973) Virology, 52:456, Sambrook et al. (2001) Molecular Cloning, a laboratory manual, 3rd edition, Cold Spring Harbor Laboratories, New York, Davis et al. (1995) Basic Methods in Molecular Biology, 2nd edition, McGraw-Hill, and Chu et al. (1981) Gene 13: 197.
  • Such techniques can be used to introduce one or more exogenous DNA moieties into suitable host cells.
  • the term refers to both stable and transient uptake of the genetic material and includes uptake of peptide-linked or antibody-linked DNAs.
  • transduction refers to the introduction of foreign nucleic acid to a cell through a replication-incompetent viral vector.
  • a “vector” is capable of transferring nucleic acid sequences to target cells (e.g., viral vectors, non-viral vectors, particulate carriers, and liposomes).
  • target cells e.g., viral vectors, non-viral vectors, particulate carriers, and liposomes.
  • vector construct means any nucleic acid construct capable of directing the expression of a nucleic acid of interest and which can transfer nucleic acid sequences to target cells.
  • the term includes cloning and expression vehicles, as well as viral vectors.
  • “Mammalian cell” refers to any cell derived from a mammalian subject suitable for transfection with an engineered vector system comprising an expression system described herein.
  • the cell may be xenogeneic, autologous, or allogeneic.
  • the cell can be a primary cell obtained directly from a mammalian subject.
  • the cell may also be a cell derived from the culture and expansion of a cell obtained from a mammalian subject. Immortalized cells are also included within this definition.
  • the cell has been genetically engineered to express a recombinant protein and/or nucleic acid.
  • subject includes animals, including both vertebrates and invertebrates, including, without limitation, invertebrates such as arthropods, mollusks, annelids, and cnidarians; and vertebrates such as amphibians, including frogs, salamanders, and caecillians; reptiles, including lizards, snakes, turtles, crocodiles, and alligators; fish; mammals, including human and non-human mammals such as nonhuman primates, including chimpanzees and other apes and monkey species; laboratory animals such as mice, rats, rabbits, hamsters, guinea pigs, and chinchillas; domestic animals such as dogs and cats; farm animals such as sheep, goats, pigs, horses and cows; and birds such as domestic, wild and game birds, including chickens, turkeys and other gallinaceous birds, ducks, geese, and the like.
  • the disclosed methods find use in
  • Gene transfer refers to methods or systems for reliably inserting DNA or RNA of interest into a host cell. Such methods can result in transient expression of non-integrated transferred DNA, extrachromosomal replication and expression of transferred replicons (e.g., episomes), or integration of transferred genetic material into the genomic DNA of host cells.
  • Gene delivery expression vectors include, but are not limited to, vectors derived from bacterial plasmid vectors, viral vectors, non- viral vectors, alphaviruses, pox viruses and vaccinia viruses.
  • the term "derived from” is used herein to identify the original source of a molecule but is not meant to limit the method by which the molecule is made which can be, for example, by chemical synthesis or recombinant means.
  • a polynucleotide "derived from" a designated sequence refers to a polynucleotide sequence which comprises a contiguous sequence of approximately at least about 6 nucleotides, preferably at least about 8 nucleotides, more preferably at least about 10-12 nucleotides, and even more preferably at least about 15-20 nucleotides corresponding, i.e., identical or complementary to, a region of the designated nucleotide sequence.
  • the derived polynucleotide will not necessarily be derived physically from the nucleotide sequence of interest, but may be generated in any manner, including, but not limited to, chemical synthesis, replication, reverse transcription or transcription, which is based on the information provided by the sequence of bases in the region(s) from which the polynucleotide is derived. As such, it may represent either a sense or an antisense orientation of the original polynucleotide.
  • complementary refers to polynucleotides that are able to form base pairs with one another. Base pairs are typically formed by hydrogen bonds between nucleotide units in an anti-parallel orientation between polynucleotide strands. Complementary polynucleotide strands can base pair in a Watson-Crick manner (e.g., A to T, A to U, C to G), or in any other manner that allows for the formation of duplexes. As persons skilled in the art are aware, when using RNA as opposed to DNA, uracil (U) rather than thymine (T) is the base that is considered to be complementary to adenosine.
  • uracil when uracil is denoted in the context of the present invention, the ability to substitute a thymine is implied, unless otherwise stated.
  • “Complementarity” may exist between two RNA strands, two DNA strands, or between an RNA strand and a DNA strand. It is generally understood that two or more polynucleotides may be “complementary” and able to form a duplex despite having less than perfect or less than 100% complementarity. Two sequences are "perfectly complementary” or " 100% complementary” if at least a contiguous portion of each polynucleotide sequence, comprising a region of complementarity, perfectly base pairs with the other polynucleotide without any mismatches or interruptions within such region.
  • Two or more sequences are considered “perfectly complementary” or “100% complementary” even if either or both polynucleotides contain additional non-complementary sequences as long as the contiguous region of complementarity within each polynucleotide is able to perfectly hybridize with the other.
  • "Less than perfect” complementarity refers to situations where less than all of the contiguous nucleotides within such region of complementarity are able to base pair with each other. Determining the percentage of complementarity between two polynucleotide sequences is a matter of ordinary skill in the art.
  • Cas9 encompasses type II clustered regularly interspaced short palindromic repeats (CRISPR) system Cas9 endonucleases from any species, and also includes biologically active fragments, variants, analogs, and derivatives thereof that retain Cas9 endonuclease activity (i.e., catalyze site-directed cleavage of DNA to generate double-strand breaks) or minimal Cas target DNA or RNA binding activity.
  • CRISPR clustered regularly interspaced short palindromic repeats
  • a gRNA may comprise a sequence "complementary" to a target sequence (e.g., major or minor allele), capable of sufficient base-pairing to form a duplex (i.e., the gRNA hybridizes with the target sequence). Additionally, the gRNA may comprise a sequence complementary to a PAM sequence, wherein the gRNA also hybridizes with the PAM sequence in a target DNA.
  • a target sequence e.g., major or minor allele
  • the gRNA may comprise a sequence complementary to a PAM sequence, wherein the gRNA also hybridizes with the PAM sequence in a target DNA.
  • a “target site” or “target sequence” is the nucleic acid sequence recognized (i.e., sufficiently complementary for hybridization) by a guide RNA (gRNA) or a homology arm of a donor polynucleotide.
  • the target site may be allele-specific (e.g., a major or minor allele).
  • a target site can be a genomic site that is intended to be modified such as by insertion of one or more nucleotides, replacement of one or more nucleotides, deletion of one or more nucleotides, or a combination thereof.
  • a CRISPR adaptation system refers collectively to transcripts and other elements involved in the expression of or directing the activity of CRISPR- associated (“Cas") genes, including sequences encoding a Cas gene, and a CRISPR array nucleic acid sequence including a leader sequence and at least one repeat sequence.
  • CRISPR-associated genes including sequences encoding a Cas gene, and a CRISPR array nucleic acid sequence including a leader sequence and at least one repeat sequence.
  • one or more elements of a CRISPR adaption system are derived from a type I, type II, or type III CRISPR system.
  • Casl and Cas2 are found in all three types of CRISPR-Cas systems, and they are involved in spacer acquisition. In the I-E system of E. coh. Casl and Cas2 form a complex where a Cas2 dimer bridges two Casl dimers.
  • Cas2 performs a non-enzymatic scaffolding role, binding double-stranded fragments of invading DNA, while Casl binds the singlestranded flanks of the DNA and catalyzes their integration into CRISPR arrays.
  • one or more elements of a CRISPR system is derived from a particular organism comprising an endogenous CRISPR system, such as Streptococcus pyogenes.
  • a CRISPR system is characterized by elements that promote the formation of a CRISPR complex at the site of a target sequence (also referred to as a protospacer in the context of an endogenous CRISPR system).
  • a vector comprises a regulatory element operably linked to an enzyme-coding sequence encoding a CRISPR enzyme, such as a Cas protein.
  • Cas proteins include Casl, CaslB, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csnl and Csxl2), CaslO, Csyl, Csy2, Csy3, Csel, Cse2, Cscl, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmrl, Cmr3, Cmr4, Cmr5, Cmr6, Csbl, Csb2, Csb3, Csxl7, Csxl4, CsxlO, Csxl6, CsaX, Csx3, Csxl, Csxl5, Csfl, Csf2, Csf3, Csf4, homo
  • the disclosure provides protospacers that are adjacent to short (3 - 5 bp) DNA sequences termed protospacer adjacent motifs (PAM).
  • PAMs are important for type I and type II systems during acquisition.
  • type I and type II systems protospacers are excised at positions adjacent to a PAM sequence, with the other end of the spacer is cut using a ruler mechanism, thus maintaining the regularity of the spacer size in the CRISPR array.
  • the conservation of the PAM sequence differs between CRISPR-Cas systems and may be evolutionarily linked to Casl and the leader sequence.
  • the protospacer is a defined synthetic DNA.
  • the defined synthetic DNA is at least 3, 5,10, 20, 30, 40, or 50 nucleotides, or between 3-50, or between 10-100, or between 20-90, or between 30-80, or between 40-70, or between 50-60, nucleotides in length.
  • the oligo nucleotide sequence or the defined synthetic DNA includes a modified "AAG" protospacer adjacent motif (PAM).
  • a regulatory element is operably linked to one or more elements of a CRISPR system so as to drive expression of the one or more elements of the CRISPR system.
  • CRISPRs Clustered Regularly Interspaced Short Palindromic Repeats
  • SPIDRs Sacer Interspersed Direct Repeats
  • the CRISPR locus comprises a distinct class of interspersed short sequence repeats (SSRs) that were recognized in E. coli (Ishino et al, J. BactenoL, 169:5429-5433 (1987); and Nakata et al., J.
  • the CRISPR loci typically differ from other SSRs by the structure of the repeats, which have been termed short regularly spaced repeats (SRSRs) (Janssen et al, OMICS J. Integ. Biol., 6:23-33 (2002); and Mojica et al, Mol. Microbiol., 36:244-246 (2000)).
  • SRSRs short regularly spaced repeats
  • the repeats are short elements that occur in clusters that are regularly spaced by unique intervening sequences with a substantially constant length (Mojica et al., (2000), supra).
  • the repeat sequences are highly conserved between strains, the number of interspersed repeats and the sequences of the spacer regions typically differ from strain to strain (van Embden et al., J.
  • CRISPR loci have been identified in more than 40 prokaryotes (See e.g., Jansen et al, Mol. Microbiol., 43: 1565-1575 (2002); and Mojica et al, (2005)) including, but not limited to Aeropyrum, Pyrobaculum, Sulfolobus, Archaeoglobus, Halocarcula, Methanobacteriumn, Methanococcus, Methanosarcina, Methanopyrus, Pyrococcus, Picrophilus, Thernioplasnia, Corynebacterium, Mycobacterium, Streptomyces, Aquifrx, Porphvromonas, Chlorobium, Thermus, Bacillus, Listeria, Staphylococcus, Clostridium, Thermoanaerobacter, Mycoplasma, Fusobacterium, Azarcus, Chromobacterium, Neisseria, Nitro
  • an enzyme coding sequence encoding a CRISPR enzyme is codon optimized for expression in particular cells, such as eukaryotic cells.
  • the eukaryotic cells may be those of or derived from a particular organism, such as a mammal, including but not limited to human, mouse, rat, rabbit, dog, or non-human primate.
  • codon optimization refers to a process of modifying a nucleic acid sequence for enhanced expression in the host cells of interest by replacing at least one codon (e.g. about one or more than about 1, 2, 3, 4, 5, 10, 15, 20, 25, 50, or more codons) of the native sequence with codons that are more frequently or most frequently used in the genes of that host cell while maintaining the native amino acid sequence.
  • Codon bias differs in codon usage between organisms
  • mRNA messenger RNA
  • tRNA transfer RNA
  • the predominance of selected tRNAs in a cell is generally a reflection of the codons used most frequently in peptide synthesis. Accordingly, genes can be tailored for optimal gene expression in a given organism based on codon optimization. Codon usage tables are readily available, for example, at the "Codon Usage Database", and these tables can be adapted in a number of ways.
  • codon optimizing a particular sequence for expression in a particular host cell are also available, such as Gene Forge (Aptagen; Jacobus, Pa.), are also available.
  • one or more codons e.g. 1, 2, 3, 4, 5, 10, 15, 20, 25, 50, or more, or all codons
  • one or more codons in a sequence encoding a CRISPR enzyme correspond to the most frequently used codon for a particular amino acid.
  • Example 1 Pol-II promoter-controlled genome editing by mascRNA-based guide RNAs
  • This example illustrates that mascRNA-based guide RNA synthesis enables Pol-II promoter-controlled genome editing.
  • FIG. 1A A structure for a mascRNA with the following sequence (SEQ ID NO: 1) is shown in FIG. 1A.
  • FIG. IB A schematic of a mascRNA-based guide RNA construct is shown in FIG. IB, which also illustrates processing by RNase P and/or RNase Z (RNase P/Z) cleavage to generate a mature CRISPR-Cas9 single-guide RNA (sgRNA).
  • FIG. 1C illustrates the structure of RNase P/Z guide RNA (PZG) system, composed of engineered mascRNA derivatives, maR-X and maR-M, flanking the sgRNA sequence.
  • FIG. 1A shows the structure and sequence (SEQ ID NO: 1) of an mascRNA that was cleaved at the 5 ’-end by RNase P and at the 3 ’-end by RNase Z.
  • IB is a schematic illustrating guide RNA biogenesis of RNA polymerase II (Pol-II) expressed precursor guide (pre-guide) RNA transcripts that are processed by RNase P/Z cleavage into mature CRISPR-Cas9 single-guide RNAs (sgRNAs). Scissors symbols indicate approximate RNase P/Z cleavage sites.
  • a marker such as a fluorescent protein (FP) can be encoded in the construct, which can have a triple-helix motif (TH) to stabilize the 3 ’-end of a non-polyadenylated transcript.
  • FP fluorescent protein
  • TH triple-helix motif
  • mascRNAs elements have altered stem sequences to decrease homology and minimize recombination during combined use in lentiviral vectors. Note that after pre-guide RNA processing, no flanking sequences remain that could encumber sgRNA performance.
  • HEK293T cells encoding a transgenic GFP were transiently transfected with a plasmid expressing a Pol-III (U6 promoter) driven sgRNA and Cas9 (pCF123).
  • HEK293T cells encoding a transgenic GFP were transiently transfected with a set of two plasmids expressing a Pol-II (LTR promoter) driven sgRNA (PZG system, pCF206) and Cas9 (pCF210).
  • Genome editing was analyzed at day 10 posttransfection by genome DNA extraction, PCR amplification, T7E1 treatment, and quantification by densitometry of the gel (FIG. ID).
  • the efficiency of genome editing efficiency of Pol- III versus Pol-II promoter expressed sgRNAs is approximately the same.
  • genomic site encoding blue fluorescent protein (BFP) was targeted for modification using a Pol-II expressed sgRNA to produce a genomic site that produced a green fluorescent protein (GFP).
  • BFP blue fluorescent protein
  • Cells were cotransfected with plasmids expressing Cas9 and the indicated sgRNAs, either from a Pol-III (U6) promoter or Pol-II PZG system, as well as ssDNA homology directed repair (HDR) templates (see FIG. IF).
  • HDR ssDNA homology directed repair
  • 1G graphically illustrates the percent homology directed repair (HDR) as detected by flow cytometry (percent GFP expressing cells).
  • T target indicates an oligonucleotide that can hybridize to the non-target strand
  • NT nontarget indicates a sequence homologous to the non-target strand.
  • the numbers indicate the length of the HDR template for the section upstream of the Cas9 cleavage site, and the length of the downstream (PAM proximal) region, respectively.
  • Pol-II expressed sgRNAs generated GFP-expressing cells via homology-directed repair (HDR) at least as efficiently as the Pol-III expressed sgRNAs.
  • HDR homology-directed repair
  • Example 2 Lentiviral expression vectors for Pol-II driven single-guide RNA (sgRNA) systems
  • FIG. 2A is a schematic diagram of these Pol-III and Pol-II constructs.
  • HEK293T packaging cells were transfected with these constructs and the supernatant from the transfected cells was collected for evaluation by exposing HEK- RT1-Cas9 target cells to the supernatants.
  • the HEK-RT1-Cas9 target cells are transduced by the virions.
  • the percentage of HEK-RT1-Cas9 that expressed a red fluorescent protein was used as a measure of whether the constructs could support viable lentiviral and sgRNA production.
  • FIG. 2B little or no RFP-producing HEK-RT1-Cas9 cells were detected when the guide RNA flanked by mascRNAs was in the sense orientation and expressed from a Pol-II promoter (LTR).
  • LTR Pol-II promoter
  • RFP-producing HEK-RT1-Cas9 cells were only observed with the guide RNA expressed from the Pol-III promoter (U6) (FIG. 2B).
  • Constructs that included the sgRNA in the sense orientation (and flanked by mascRNAs with expression driven by a Pol-II promoter) were either degraded by RNase or underwent convergent transcription to produce a double-stranded RNA (dsRNA) that was degraded within the cells.
  • RNase P was at least partially responsible for the degradation of constructs that included the sgRNA with flanking mascRNAs in the sense orientation, where sgRNA expression was driven by a Pol-II promoter.
  • RNase P and RNase Z are core fitness genes essential to cell survival (Hart et al. Cell 163: 1515-26 (2015)).
  • the Pol-II guide RNA expression system including the mascRNA elements could be encoded on the antisense strand that is not needed for viral packaging.
  • Nodamura viral B2 A sequence for the Nodamura viral B2 protein is shown below (NCBI accession no. NP_077731.1; SEQ ID NO:7).
  • a nucleotide sequence for this B2 Nodamura viral protein is available from the NCBI database as described herein and provided as SEQ ID NOs:8-9.
  • lentiviral constructs were again used with a Pol-II promoter (5 ’-LTR), but the mCherry sequence was in the antisense orientation (pCF561 construct) along with a separate NovB2 lentiviral helper vector (pCF802/807) encoding NovB2.
  • HEK293T packaging cells were transfected with the transfer vectors and helper plasmids indicated in FIG. 2D, including the sense and antisense mascRNA- guide RNA. The supernatant was harvested from the transfected cells to evaluate whether virions were produced that could modify HEK-RT1-Cas9 target cells to produce a fluorescent signal.
  • FIG. 2D is a schematic illustrating lentiviral delivery strategies for RNase P/Z mature RNA systems.
  • Encoding recombinant DNA on the anti-sense strand prevents cleavage of the viral genome by RNase P/Z in packaging cells.
  • sense and anti-sense transcripts can still form double-stranded RNA (dsRNA) species that are degraded by cellular machinery.
  • dsRNA double-stranded RNA
  • this expression system prevented cellular cleavage of the provirus and rescued viral production by packaging cells.
  • Example 3 Guide RNAs produced from the optimized mascRNA-based Pol-II promoter system has editing efficiencies comparable to Pol-III
  • the optimized mascRNA-based Pol-II guide RNA expression systems with the NovB2-expressing helper vectors described in Example 2 were evaluated to determine with the guide RNAs produced could provide genome editing as efficiently as guide RNAs expressed from Pol-III expression vectors.
  • HEK293T packaging cells were transfected with the lentiviral vectors with mascRNA-based Pol-II guide RNA on the antisense strand as well as the NovB2- expressing helper vectors. Supernatants were collected from the cells, and the supernatants were assessed in a HEK-RT1 GFP reporter cells to ascertain whether the encoded guide RNAs could efficiently knockout green fluorescent protein (GFP). Lentivirus were successfully produced by the packaging cells that enabled the stable integration of Pol-II sgRNA systems into HEK-RT1 GFP reporter cells. The percentage GFP+ cells was assessed at day 3 and day 5 post-transduction. As illustrated in FIG. 3, genome editing efficiencies of the optimized Pol-II guide RNA systems were comparable to those of standard U6 RNA polymerase III (Pol-III) promoter methods.
  • Pol-III U6 RNA polymerase III
  • Pol-II promoters as well as from constitutive Pol-II promoters.
  • Pol-III expression is constitutive and is not easily regulated.
  • Pol-II expression allows the production of guide RNAs in tissuespecific, cell type-specific, and pathway-specific ways that are inducible and reversible.
  • the flexibility and control provided by use of the optimized Pol-II expression systems described herein is superior to the currently available Pol-III expression systems.
  • Example 4 All-in-one doxycycline controlled expression system allows inducible editing at endogenous loci.
  • This example illustrates controlled, inducible editing of several different genomic loci using the single or multiplexed expression systems described herein.
  • doxycycline-inducible (dox-inducible) sgRNA expression vectors used are shown in FIG. 4A. All variants featured an all-in-one Tet-On system encoded on the lentiviral antisense strand, and either a puromycin (pCF621), hygromycin B (pCF622) or blasticidin S (pCF623) resistance marker.
  • pCF621 puromycin
  • pCF622 hygromycin B
  • blasticidin S pCF623 resistance marker
  • U251 cell lines were established that stably expressed the pCF621, pCF622 or pCF623 recipient (reci) vectors, and selected on puromycin (pCF621), hygromycin B (pCF622) or blasticidin S (pCF623).
  • FIG. 4B graphically illustrates the percentage of mCherry-positive cells
  • FIG. 4C graphically illustrates the median fluorescence intensity (MFI) of mCherry- positive cells at the levels of doxycycline shown in FIG. 4B.
  • MFI median fluorescence intensity
  • T7 endonuclease 1 (T7E1) assays were performed genomic DNA from in U251-Cas9 (pCF226) cells to evaluate whether the guide RNAs encoded by the pCF622 vector could perform genomic editing.
  • FIG. 4G illustrates a competitive proliferation assay for targeting repetitive genomic elements and killing cells that have such repetitive genomic elements (e.g., certain types of cancer cells).
  • the competitive proliferation assay involved expressing lethal guide RNAs (sgCIDEs, named sgCIDE-1/2/3) that targeted repetitive genomic elements and, for comparison, non-targeting control (sgNT-1/2/3) guide RNAs.
  • sgCIDEs lethal guide RNAs
  • sgCIDE-1/2/3 that targeted repetitive genomic elements
  • sgNT-1/2/3 guide RNAs expressing non-targeting control (sgNT-1/2/3) guide RNAs.
  • GBM glioblastoma
  • the LN229 glioblastoma cells were stably transduced with lentiviral vectors pCF622 or pCF623 to provide doxycycline (dox)-controlled guide RNA expression. Changes in ratios of sgRNA-transduced cells (mCherry+) upon dox treatment over time were monitored by flow cytometry.
  • FIGs. 4H-4I expression of the sgCIDE-1/2/3 guide RNAs enabled glioblastoma cell depletion by genome shredding.
  • the pCF623-sgCIDEs led to slightly more rapid cell depletion compared to pCF622-sgCIDEs (FIG. 4H) indicating more efficient functional guide RNA induction.
  • the optimized Pol-II expression system was used to provide inducible guide RNA expression to provide precisely tailored sgRNAs that target PCSK9 exon 4.
  • the PCSK9 gene is implicated in familial hypercholesterolemia (FH). Gain and loss-of- function variations in PCSK9 result in high and low levels of LDL-C, respectively. Genetically determined decreases in LDL-C levels, including 28 percent decreases in African Americans and 15 percent decreases in white subjects, have been associated with substantial reductions in coronary heart disease risk as compared with LDL-C levels in noncamers of the PCSK9-genetic variations. These findings have triggered a race to develop PCSK9 inhibitors to reduce LDL-C for prevention of adverse atherosclerotic cardiovascular events.
  • FIG. 5A illustrates the structure of the sgPCSK9 (pCF622) multiplexed construct, showing that the sgRNA are flanked by mascRNA-encoding elements.
  • the sgPCSK9 (pCF622) multiplexed construct provides doxycycline- inducible (dox-inducible) expression of two guide RNAs (sgPCSK9-5 and sgPCSK9- 7) that can excise part of exon 4 of the PCSK9 gene in cells that express Cas9.
  • the pCF622 vector is an all-in-one Tet-On system encoded on the lentiviral antisense strand with a hygromycin B resistance marker.
  • the pCF622 expression system includes mascRNA-encoding elements that flank the sgRNA coding regions, a triplehelix motif (TH), ubiquitous chromatin opening element (UCOE), an EF la-short promoter (EFS), and spleen focus-forming virus (SFFV) long terminal repeat promoter.
  • TH triplehelix motif
  • UOE ubiquitous chromatin opening element
  • EFS EF la-short promoter
  • SFFV spleen focus-forming virus
  • U251-Cas9 cells were stably transduced with the pCF622 vectors to provide dox-induced expression of the sgPCSK9-5 and sgPCSK9-7 guide RNAs.
  • Transduced cells were selected on hygromycin before treatment with (+) or without (-) doxycycline (1 pg/ml) for four days.
  • FIG. 5B shows a representative gel where one band in a lane identifies an unedited PCSK9 genomic segment, or where two bands indicate the unedited and a shortened (excised) PCSK9 genomic segment (successful editing). As shown in FIG. 5B, shorter dual-cut bands are only present when the cells inducibly expressed both of the sgPCSK9-5 and sgPCSK9-7 guide RNAs.
  • FIG. 5C graphically illustrates the percent of cells with dual -cut genomes, indicating the percent of cells without part of exon 4 of the PCSK9 gene.
  • Jinek, M. et al. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science 337, 816-21 (2012).
  • An expression system comprising at least one expression cassette, each expression cassette comprising an RNA polymerase II (Pol-II) promoter operably linked to a nucleic acid segment encoding at least one RNA, wherein the nucleic acid segment is flanked by mascRNA-encoding elements.
  • RNA polymerase II RNA polymerase II
  • mascRNA-encoding elements comprise RNA sequences with at least 70% sequence identity, or 90% sequence identity to any of SEQ ID NO: 1-6.
  • RNA polymerase II (Pol-II) promoter is an inducible promoter.
  • RNA polymerase II (Pol-II) promoter is a cell-specific or tissue-specific promoter.
  • RNA polymerase II (Pol-II) promoter is a developmentally regulated promoter.
  • nucleic acid segment encoding at least one RNA encodes at least two guide RNAs, at least three guide RNAs, at least four guide RNAs, or at least five guide RNAs.
  • nucleic acid segment encoding at least one RNA comprises at least one retron nucleic acid.
  • nucleic acid segment encoding at least one RNA encodes at least two retrons, at least three retrons, at least four retrons, or at least five retrons.
  • nucleic acid segment encoding at least one RNA encodes at least one RNAi, antisense RNA, or shRNA.
  • RNAi antisense RNAs or shRNAs encodes at least two RNAi, antisense RNAs or shRNAs, at least three RNAi, antisense RNAs or shRNAs, at least four RNAi, antisense RNAs or shRNA, or at least five RNAi antisense RNAs or shRNA.
  • nucleic acid segment encoding at least one RNA encodes at least one template for a reverse transcriptase (RT), or template for homology dependent repair (HDR; e.g., a donor DNA).
  • RT reverse transcriptase
  • HDR homology dependent repair
  • the nucleic acid segment encoding at least one RNA encodes at least two, or at least three, or at least four, or at least five templates for a reverse transcriptase (RT), or at least two, or at least three, or at least four, or at least five templates for homology dependent repair (HDR; e.g., at least 2-5 donor DNAs).
  • a host cell comprising the expression system of any one of statements 1-20.
  • the host cell of any one of statements 21-23 modified to express at least one heterologous Cas nuclease, Cas protein, reverse transcriptase, transposase, fluorescent protein, marker segment encoding a signal-producing protein.
  • a method comprising transducing one or more host cells with an expression system comprising at least one expression cassette, each expression cassette comprising an RNA polymerase II (Pol-II) promoter operably linked to a nucleic acid segment encoding at least one RNA, wherein the nucleic acid segment is flanked by mascRNA-encoding elements.
  • RNA polymerase II RNA polymerase II
  • mascRNA-encoding elements comprise RNA sequences with at least 70% sequence identity, or 90% sequence identity to any of SEQ ID NO: 1-6.
  • any one of statements 26-30 wherein the one or more host cells express at least one heterologous Cas nuclease, Cas protein, reverse transcriptase, transposase, fluorescent protein, marker segment encoding a signal-producing protein.
  • the nucleic acid segment encoding at least one RNA encodes at least one guide RNA (e.g., single-guide RNA).
  • nucleic acid segment encoding at least one RNA encodes at least two guide RNAs, at least three guide RNAs, at least four guide RNAs, or at least five guide RNAs.
  • nucleic acid segment encoding at least one RNA encodes at least two retrons, at least three retrons, at least four retrons, or at least five retrons.
  • RNAi antisense RNAs or shRNAs encodes at least two RNAi, antisense RNAs or shRNAs, at least three RNAi, antisense RNAs or shRNAs, at least four RNAi, antisense RNAs or shRNA, or at least five RNAi antisense RNAs or shRNA.
  • nucleic acid segment encoding at least one RNA encodes at least one template for a reverse transcriptase (RT), or template for homology dependent repair (HDR).
  • RT reverse transcriptase
  • HDR homology dependent repair
  • nucleic acid segment encoding at least one RNA encodes at least two, or at least three, or at least four, or at least five templates for a reverse transcriptase (RT), or at least two, or at least three, or at least four, or at least five templates for homology dependent repair (HDR).
  • RT reverse transcriptase
  • HDR homology dependent repair

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Abstract

L'invention concerne des procédés et des compositions pour générer des transcrits d'ARN précis à l'aide d'éléments mascRNA (petit ARN cytoplasmique associé à MALAT1) pour découper et retirer spécifiquement une partie souhaitée d'un transcrit d'ARN primaire donné. Le transcrit d'ARN primaire peut être un transcrit exprimé à partir d'un promoteur d'ARN polymérase II (Pol-II) pour permettre une régulation et un contrôle transcriptionnels.
PCT/US2022/040166 2021-08-12 2022-08-12 Procédés de génération de transcrits d'arn précis WO2023018938A1 (fr)

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20120232130A1 (en) * 2009-04-16 2012-09-13 Cepko Constance L Methods for inhibiting starvation of a cell
US20170022499A1 (en) * 2014-04-03 2017-01-26 Massachusetts Institute Of Techology Methods and compositions for the production of guide rna
US20190106693A1 (en) * 2015-05-13 2019-04-11 President And Fellows Of Harvard College Methods of Making and Using Guide RNA for Use With Cas9 Systems

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20120232130A1 (en) * 2009-04-16 2012-09-13 Cepko Constance L Methods for inhibiting starvation of a cell
US20170022499A1 (en) * 2014-04-03 2017-01-26 Massachusetts Institute Of Techology Methods and compositions for the production of guide rna
US20190106693A1 (en) * 2015-05-13 2019-04-11 President And Fellows Of Harvard College Methods of Making and Using Guide RNA for Use With Cas9 Systems

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
MAETZIG ET AL.: "Mechanisms controlling titer and expression of bidirectional lentiviral and gammaretroviral vectors", GENE THER, vol. 17, no. 3, March 2010 (2010-03-01), pages 400 - 411, XP055659955, DOI: 10.1038/gt.2009.129 *

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