WO2022109275A2 - Vectors, systems and methods for eukaryotic gene editing - Google Patents

Vectors, systems and methods for eukaryotic gene editing Download PDF

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WO2022109275A2
WO2022109275A2 PCT/US2021/060099 US2021060099W WO2022109275A2 WO 2022109275 A2 WO2022109275 A2 WO 2022109275A2 US 2021060099 W US2021060099 W US 2021060099W WO 2022109275 A2 WO2022109275 A2 WO 2022109275A2
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protein
abe
sequence
cells
coding sequence
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WO2022109275A3 (en
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Baisong Lu
Anthony Atala
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Wake Forest University Health Sciences
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Wake Forest University Health Sciences
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Priority to AU2021381397A priority patent/AU2021381397A1/en
Priority to US18/037,708 priority patent/US20230405116A1/en
Priority to CA3196996A priority patent/CA3196996A1/en
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K40/00—Cellular immunotherapy
    • A61K40/10—Cellular immunotherapy characterised by the cell type used
    • A61K40/11—T-cells, e.g. tumour infiltrating lymphocytes [TIL] or regulatory T [Treg] cells; Lymphokine-activated killer [LAK] cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/0008—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition
    • A61K48/0025—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition wherein the non-active part clearly interacts with the delivered nucleic acid
    • A61K48/0041—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition wherein the non-active part clearly interacts with the delivered nucleic acid the non-active part being polymeric
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    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
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    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/115—Aptamers, i.e. nucleic acids binding a target molecule specifically and with high affinity without hybridising therewith ; Nucleic acids binding to non-nucleic acids, e.g. aptamers
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    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
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    • C12N9/14—Hydrolases (3)
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    • C12Y305/04—Hydrolases acting on carbon-nitrogen bonds, other than peptide bonds (3.5) in cyclic amidines (3.5.4)
    • C12Y305/04004—Adenosine deaminase (3.5.4.4)
    • C—CHEMISTRY; METALLURGY
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    • C07K2319/00—Fusion polypeptide
    • C07K2319/70—Fusion polypeptide containing domain for protein-protein interaction
    • C07K2319/735—Fusion polypeptide containing domain for protein-protein interaction containing a domain for self-assembly, e.g. a viral coat protein (includes phage display)
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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
    • C12N2310/10—Type of nucleic acid
    • C12N2310/20—Type of nucleic acid involving clustered regularly interspaced short palindromic repeats [CRISPR]
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/35—Nature of the modification
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    • C12N2310/3519—Fusion with another nucleic acid
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    • 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

  • BACKGROUND [0004] Fusion of adenine deaminases to nuclease-deficient type II CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated 9) creates adenine base editors (ABEs) that can edit genomic DNA without double-stranded DNA cleavage. Base editing generates precise point mutations in genomic DNA without generating double strand breaks. Further, adenine base editing does not require a DNA donor template and does not rely on cellular homologous directed repair. Thus, it has great potential as a gene therapy for genetic diseases caused by transition mutations, which account for 61% of disease-causing point mutations.
  • ABEs adenine base editors
  • ABEs Adenine base editors
  • a mammalian expression plasmid comprising a eukaryotic promoter operably linked to a non-viral nucleic acid sequence
  • the non-viral nucleic acid sequence comprises: (i) a nucleic acid sequence encoding an adenosine base pair editor (ABE), wherein the ABE is a fusion protein comprising an adenosine deaminase and a catalytically impaired CRISPR-associated endonuclease; and (ii) a guide RNA (gRNA) coding sequence, wherein the gRNA coding sequence comprises at least one aptamer coding sequence.
  • ABE adenosine base pair editor
  • gRNA guide RNA
  • the catalytically impaired CRISPR-associated endonuclease coding sequence encodes a Cas9 D10A protein.
  • the adenine base editor is ABE7.10 or ABE8.
  • the at least one aptamer coding sequence encodes an aptamer sequence bound specifically by an ABP selected from the group consisting of MS2 coat protein, PP7 coat protein, lambda N RNA-binding domain, or Com protein.
  • the aptamer is an MS2 aptamer sequence or a com aptamer sequence.
  • the sgRNA coding sequence comprises at least one aptamer inserted into the tetraloop or the ST2 loop of the sgRNA coding sequence. In some embodiments, the sgRNA coding comprises at least one com aptamer inserted into the ST2 loop of the gRNA coding sequence.
  • a lentiviral packaging system comprising: (a) a packaging plasmid comprising a eukaryotic promoter operably linked to a Gag nucleotide sequence, wherein the Gag nucleotide sequence comprises a nucleocapsid (NC) coding sequence and a matrix protein (MA) coding sequence, wherein one or both of the NC coding sequence or the MA coding sequence comprises at least one non-viral aptamer-binding protein (ABP) nucleotide sequence, and wherein the packaging plasmid does not encode a functional integrase protein; (b) at least one mammalian expression plasmid provided herein; and (c) an envelope plasmid comprising an envelope glycoprotein coding sequence.
  • a packaging plasmid comprising a eukaryotic promoter operably linked to a Gag nucleotide sequence, wherein the Gag nucleotide sequence comprises a nucleocapsid (NC) coding sequence and
  • the packaging plasmid further comprises a Rev nucleotide sequence and a Tat nucleotide sequence.
  • the system further comprises a second packaging plasmid comprising a Rev nucleotide sequence.
  • the at least one non-viral ABP nucleotide sequence encodes MS2 coat protein, PP7 coat protein, lambda N peptide, or Com protein.
  • a lentivirus-like particle comprising: (a) a fusion protein comprising a nucleocapsid (NC) protein or a matrix (MA) protein wherein the NC protein or MA protein comprises at least one non-viral aptamer binding protein (ABP); and (b) a ribonucleotide protein (RNP) complex comprising: (i) an adenine base editor (ABE), wherein the ABE is a fusion polypeptide comprising an adenine base editor and a catalytically impaired CRISPR-associated endonuclease; and (ii) a gRNA, wherein the lentivirus-like particle does not comprise a functional integrase protein.
  • NC nucleocapsid
  • MA matrix
  • RNP ribonucleotide protein
  • the catalytically impaired CRISPR-associated endonuclease is a catalytically impaired Cas9 protein, a catalytically impaired Cpf1 protein, or a derivative of either.
  • the adenine base editor is ABE 7.10 or ABE 8.
  • a method of producing a lentivirus-like particle comprising: (a) transfecting a plurality of eukaryotic cells with the packaging plasmid, the at least one mammalian expression plasmid, and the envelope plasmid of any of the systems described herein; and (b) culturing the transfected eukaryotic cells for sufficient time for lentivirus-like particles to be produced.
  • the lentivirus-like particle produced comprises a RNP comprising: (i) an adenine base editor (ABE), wherein the ABE is a fusion polypeptide comprising an adenosine deaminase and a catalytically impaired CRISPR-associated endonuclease; and (ii) a guide RNA.
  • ABE adenine base editor
  • the plurality of eukaryotic cells are mammalian cells.
  • a method of modifying a genomic target sequence in a cell comprising transducing a plurality of eukaryotic cells with a plurality of viral particles described herein, wherein the RNP binds to the genomic target sequence in genomic DNA of the cell and the ABE deaminates an adenine at the genomic target sequence, thereby modifying the genomic target sequence.
  • the plurality of eukaryotic cells are mammalian cells.
  • the plurality of eukaryotic cells are cells present in subject.
  • the subject is a human subject.
  • the subject is injected with the plurality of viral particles.
  • cells comprising any of the plasmids, lentiviral packaging systems or lentivirus-like particles described herein. Cells modified by any of the methods provided herein are also provided. [0013] Further provided is a method for treating a disease in a subject comprising: (a) obtaining cells from the subject; and (b) modifying the cells of the subject using any of the genomic editing methods described herein; and administering the modified cells to the subject.
  • the disease is cancer.
  • the disease is sickle cell anemia.
  • the cells are T cells. DESCRIPTION OF THE FIGURES [0014] The present application includes the following figures.
  • FIG. 1A is a diagram showing the predicted ABE off-target hotspot in human USP38 mRNA according to aspects of this disclosure. The predicted hotspot (red) and the primers used for PCR amplification are indicated.
  • FIG.1B shows the results of RT-PCR and targeted NGS which detected high levels of A to G changes in a 440 nt region of USP38 mRNA region after ABE DNA transfection according to aspects of this disclosure.
  • FIG.1C shows the sequences of the most frequent NGS reads (SEQ ID NOs: 108- 117) from cells transfected with plasmid DNA expressing ABE targeting ABE-site 1 according to aspects of this disclosure.
  • the predicted RNA off-target hotspot is underlined (highest peak). The A to G changes are shown.
  • FIG.1D shows the results of next generation sequence (NGS) analysis of on-target base editing at ABE site 1 according to aspects of this disclosure.
  • NGS next generation sequence
  • FIG. 2A is an exemplary modification to an sgRNA scaffold for ABE RNP packaging according to aspects of this disclosure (SEQ ID NO: 121).
  • the Tetraloop (GAAA) and the ST2 loop are indicated by dashed boxes.
  • the core aptamer sequences are underlined and the additional linkers are not underlined.
  • Vertical lines indicate complementary base ST2 loop can be replaced with an MS2 aptamer sequence (SEQ ID NO: 122).
  • the tetraloop or the ST2 loop can be replaced with a com aptamer sequence (SEQ ID NO: 123).
  • FIG.2B shows the results of qPCR to detect ABE-g1 RNP activity on ABE site 1 according to aspects of this disclosure.
  • a total of 200 ng p24 of various LV capsids were used to transduce 2.5x10 4 HEK293T cells.
  • the gDNA was used for qPCR with primers matching edited sequences. *** indicates p ⁇ 0.0001, Tukey's multiple comparison test following one-way analysis of variance (ANOVA). Error bars indicate s.e.m, of three replicates.
  • FIG.2C shows the results of qPCR to detect ABE-g5 RNP activity on ABE site 5 according to aspects of this disclosure.
  • FIG.2D shows NGS analysis of capsid-RNP-mediated base editing at ABE site 5 according to aspects of this disclosure.
  • Capsid-RNPs (108 ng p24) were used to transduce 2.5x10 4 HEK293T cells.
  • SEQ ID NO: 124 is a reference sequence Alleles with base editing frequencies of >0.2% are listed (SEQ ID NOs: 125-133) and frequencies with A>G changes at different positions are shown at the bottom.
  • FIG. 3 shows NGS analysis of capsid-RNP mediated base editing at ABE site 1 according to aspects of this disclosure.
  • Capsid-RNPs in the amount of 200 ng p24 were used to transduce 2.5x10 4 HEK293T cells.
  • SEQ ID NO: 134 is a Reference sequence. The alleles with base editing frequencies of >0.1% were listed (SEQ ID NOs: 134-139) and the frequencies with A>G changes at different positions are shown at the bottom.
  • FIG. 4A shows that aptamer/(aptamer binding protein (ABP) interaction is necessary for functional ABE packaging in lentiviral capsids according to aspects of this disclosure.
  • ABSP aptamer/(aptamer binding protein
  • FIG. 4B shows estimates of ABE protein amounts in LV capsids according to aspects of this disclosure.
  • FIG.4C shows the results of qPCR detection of base editing activities of ABE-g5 RNP capsids and ABE-g5 ST2-com RNP capsids according to aspects of this disclosure.2.5x10 4 HEK293T cells were treated with 200 ng p24 of capsids-RNPs. 48 hours later gDNA was extracted and analyzed by qPCR to detect base editing at site 5. DNA from cells treated with ABE-g1 ST2-com RNP capsids (from FIG.3B) was used as the control to show site specificity.
  • FIG. 4D shows the results of qPCR using known concentrations of plasmid DNA to examine the effects of com addition on PCR detection according to aspects of this disclosure.
  • FIG.4E shows RT-qPCR comparison of sgRNA levels in ABE-g5 RNP and ABE- g5 ST2-com RNP capsids treated with and without TritonTM X-100 according to aspects of this disclosure. *** indicates p ⁇ 0.0001 in Bonferroni post hoc tests following ANOVA.
  • FIG. 5A is a Western blot of ABE levels after transducing HEK293T cells according to aspects of this disclosure. Gel images of ABE and ⁇ -actin are shown. The arrow indicates position of the full-length ABE bands. The ⁇ -actin image demonstrates that all samples have lysate input. Normalization was not attempted since the RNP amount was independent of cell proliferation.
  • FIG. 5A is a Western blot of ABE levels after transducing HEK293T cells according to aspects of this disclosure. Gel images of ABE and ⁇ -actin are shown. The arrow indicates position of the full-length ABE bands. The ⁇ -actin image demonstrates that all samples have
  • FIG. 6 is an NGS analysis of RNA off-targets in capsid-RNP treated cells at the hotspot in USP38 mRNA according to aspects of this disclosure. Substitution rates in capsid- RNP (targeting ABE site 1) treated cells (peaks above the X-axis) and in negative control cells treated with nickase (peaks below the X-axis) showed no difference. A to G change rates at both peaks were of background level. The position of the predicted hotspot is indicated.
  • nucleic acid or “nucleotide” refers to deoxyribonucleic acids (DNA) or ribonucleic acids (RNA) and polymers thereof in either single- or double-stranded form. It is understood that when an RNA is described, its corresponding DNA is also described, wherein uridine is represented as thymidine. Similarly, when a DNA is described, its corresponding RNA is also described wherein thymidine is represented by uridine.
  • nucleic acids containing known analogues of natural nucleotides that have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides.
  • a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences as well as the sequence explicitly indicated.
  • degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and/or deoxyinosine residues (Batzer et al., Nucleic Acid Res.19:5081 (1991); Ohtsuka et al., J. Biol. Chem.260:2605-2608 (1985); and Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)).
  • the polynucleotides of the invention also encompass all forms of sequences including, but not limited to, single-stranded forms, double-stranded forms, hairpins, stem-and-loop structures, and the like.
  • the term “gene” can refer to the segment of DNA involved in producing or encoding a polypeptide chain. It may include regions preceding and following the coding region (leader and trailer) as well as intervening sequences (introns) between individual coding segments (exons). Alternatively, the term “gene” can refer to the segment of DNA involved in producing or encoding a non-translated RNA, such as an rRNA, tRNA, guide RNA, or micro RNA.
  • Treating refers to any indicia of success in the treatment or amelioration or prevention of the disease, condition, or disorder, including any objective or subjective parameter such as abatement; remission; diminishing of symptoms or making the disease condition more tolerable to the patient; slowing in the rate of degeneration or decline; or making the final point of degeneration less debilitating.
  • the treatment or amelioration of symptoms can be based on objective or subjective parameters; including the results of an examination by a physician.
  • the term “treating” includes the administration of the compounds, lentivirus-like particles or agents of the present disclosure to prevent or delay, to alleviate, or to arrest or inhibit development of the symptoms or conditions associated with a disease, condition or disorder as described herein.
  • therapeutic effect refers to the reduction, elimination, or prevention of the disease, symptoms of the disease, or side effects of the disease in the subject.
  • “Treating” or “treatment” using the methods of the present disclosure includes preventing the onset of symptoms in a subject that can be at increased risk of a disease or disorder associated with a disease, condition or disorder as described herein, but does not yet experience or exhibit symptoms, inhibiting the symptoms of a disease or disorder (slowing or arresting its development), providing relief from the symptoms or side effects of a disease (including palliative treatment), and relieving the symptoms of a disease (causing regression).
  • Treatment can be prophylactic (to prevent or delay the onset of the disease, or to prevent the manifestation of clinical or subclinical symptoms thereof) or therapeutic suppression or alleviation of symptoms after the manifestation of the disease or condition.
  • treatment includes preventative (e.g., prophylactic), curative, or palliative treatment.
  • a “promoter” is defined as one or more a nucleic acid control sequences that direct transcription of a nucleic acid.
  • a promoter includes necessary nucleic acid sequences near the start site of transcription, such as, in the case of a polymerase II type promoter, a TATA element.
  • a promoter also optionally includes distal enhancer or repressor elements, which can be located as much as several thousand base pairs from the start site of transcription.
  • “Polypeptide,” “peptide,” and “protein” are used interchangeably herein to refer to a polymer of amino acid residues. All three terms apply to amino acid polymers in which one or more amino acid residue is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers and non-naturally occurring amino acid polymers. As used herein, the terms encompass full-length proteins, truncated proteins, and fragments thereof, and amino acid chains, wherein the amino acid residues are linked by covalent peptide bonds.
  • fusion polypeptide or “fusion protein” is a polypeptide comprising two or more proteins or fragments thereof.
  • a linker comprising about 3 to 10 amino acids can be positioned between any two proteins or fragments thereof to help facilitate proper folding of the proteins upon expression.
  • identity or “substantial identity”, as used in the context of a polynucleotide or polypeptide sequence described herein, refers to a sequence that has at least 60% sequence identity to a reference sequence. Alternatively, percent identity can be any integer from 60% to 100%.
  • Exemplary embodiments include at least: 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, as compared to a reference sequence using the programs described herein; preferably BLAST using standard parameters, as described below. It is understood that sequences having at 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to any nucleotide or polypeptide sequence set forth herein, for example, any one of SEQ ID NOs: 1- 48, can be used in the compositions and methods provided herein.
  • nucleic acid sequence can comprise, consist of, or consist essentially of any nucleic acid sequence described herein.
  • polypeptide can comprise, consist of, or consist essentially of, any polypeptide sequence described herein.
  • sequence comparison typically one sequence acts as a reference sequence to which test sequences are compared.
  • test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Default program parameters can be used, or alternative parameters can be designated.
  • sequence comparison algorithm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters.
  • a “comparison window”, as used herein, includes reference to a segment of any one of the number of contiguous positions selected from the group consisting of from 20 to 600, about 20 to 50, about 20 to 100, about 50 to about 200 or about 100 to about 150, in which a sequence may be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned.
  • Methods of alignment of sequences for comparison are well-known in the art.
  • Optimal alignment of sequences for comparison may be conducted by the local homology algorithm of Smith and Waterman Add. APL. Math.2:482 (1981), by the homology alignment algorithm of Needleman and Wunsch J. Mol.
  • the algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence.
  • T is referred to as the neighborhood word score threshold (Altschul et al, supra).
  • M forward score for a pair of matching residues; always >0
  • N penalty score for mismatching residues; always ⁇ 0).
  • Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached.
  • the BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment.
  • the BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin & Altschul, Proc. Nat'l. Acad.
  • a nucleic acid is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid to the reference nucleic acid is less than about 0.01, more preferably less than about 10 -5 , and most preferably less than about 10 -20 .
  • subject is meant an individual.
  • the subject is a mammal, such as a primate, and, more specifically, a human.
  • Non-human primates are subjects as well.
  • subject includes domesticated animals, such as cats, dogs, etc., livestock (for example, cattle, horses, pigs, sheep, goats, etc.) and laboratory animals (for example, ferret, chinchilla, mouse, rabbit, rat, gerbil, guinea pig, etc.).
  • livestock for example, cattle, horses, pigs, sheep, goats, etc.
  • laboratory animals for example, ferret, chinchilla, mouse, rabbit, rat, gerbil, guinea pig, etc.
  • patient or subject may be used interchangeably and can refer to a subject afflicted with a disease or disorder.
  • An “expression cassette” is a nucleic acid construct, generated recombinantly or synthetically, with a series of specified nucleic acid elements that permit transcription of a particular polynucleotide sequence in a host cell.
  • An expression cassette may be part of a plasmid, viral genome, or nucleic acid fragment.
  • an expression cassette includes a polynucleotide to be transcribed, operably linked to a promoter, followed by a transcription termination signal sequence.
  • An expression cassette may or may not include specific regulatory sequences, such as 5’ or 3’ untranslated regions from human globin genes.
  • a “reporter gene” encodes proteins that are readily detectable due to their biochemical characteristics, such as enzymatic activity or chemifluorescent features. These reporter proteins can be used as selectable markers.
  • One specific example of such a reporter is green fluorescent protein. Fluorescence generated from this protein can be detected with various commercially-available fluorescent detection systems. Other reporters can be detected by staining.
  • the reporter can also be an enzyme that generates a detectable signal when contacted with an appropriate substrate.
  • the reporter can be an enzyme that catalyzes the formation of a detectable product. Suitable enzymes include, but are not limited to, proteases, nucleases, lipases, phosphatases and hydrolases.
  • the reporter can encode an enzyme whose substrates are substantially impermeable to eukaryotic plasma membranes, thus making it possible to tightly control signal formation.
  • suitable reporter genes that encode enzymes include, but are not limited to, CAT (chloramphenicol acetyl transferase; Alton and Vapnek (1979) Nature 282: 864-869); luciferase (lux); ⁇ -galactosidase; LacZ; ⁇ .- glucuronidase; and alkaline phosphatase (Toh, et al. (1980) Eur. J. Biochem. 182: 231-238; and Hall et al. (1983) J. Mol. Appl.
  • the CRISPR-associated endonuclease is a catalytically impaired nuclease.
  • catalytically impaired refers to decreased CRISPR-associated endonuclease enzymatic activity for cleaving one or both strands of DNA.
  • catalytically impaired CRISPR-associated endonucleases include but are not limited to catalytically impaired Cas9, catalytically impaired Cpf1 and catalytically impaired C2c2.
  • the catalytically impaired CRISPR-associated endonuclease is a the catalytically impaired Cas9, for example Cas9 D10A, which cleaves or nicks only one strand of DNA.
  • the CRISPR-associated endonuclease may be a catalytically impaired CRISPR-associated endonuclease, wherein the endonuclease cannot cleave both strands of a double-stranded DNA molecule, i.e., cannot make a double-stranded break.
  • Modifications include, but are not limited to, altering one or more amino acids to inactivate the nuclease activity or the nuclease domain.
  • D10A and/or H840A mutations can be made in Cas9 from Streptococcus pyogenes to reduce or inactivate Cas9 nuclease activity.
  • a catalytically impaired Cas9 may include polypeptide sequences modified to reduce nuclease activity or removal of a polypeptide sequence or sequences to reduce nuclease activity.
  • the catalytically impaired Cas9 retains the ability to bind to DNA even though the nuclease activity has been inactivated.
  • a catalytically impaired Cas9 includes the polypeptide sequence or sequences required for DNA binding but includes modified nuclease sequences or lacks nuclease sequences responsible for nuclease activity.
  • the Cas9 protein is a full- length Cas9 sequence from S. pyogenes lacking the polypeptide sequence of the RuvC nuclease domain and/or the HNH nuclease domain and retaining the DNA binding function.
  • the Cas9 protein sequences have at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98% or 99% identity to Cas9 polypeptide sequences lacking the RuvC nuclease domain and/or the HNH nuclease domain and retains DNA binding function.
  • CRISPR-associate endonucleases that can be catalytically impaired include, but are not limited to, nucleases present in any bacterial species that encodes a Type II or a Type V CRISPR/Cas system.
  • the “CRISPR/Cas” system refers to a widespread class of bacterial systems for defense against foreign nucleic acid. CRISPR/Cas systems are found in a wide range of eubacterial and archaeal organisms. CRISPR/Cas systems include type I, II, and III sub-types. The CRISPR/Cas system classification as described in by Makarova, et al.
  • Type II CRISPR/Cas system (Nat Rev Microbiol.2015 Nov; 13(11):722-36) defines five types and 16 subtypes based on shared characteristics and evolutionary similarity. These are grouped into two large classes based on the structure of the effector complex that cleaves genomic DNA.
  • the Type II CRISPR/Cas system was the first used for genome engineering, with Type V following in 2015. Wild-type type II CRISPR/Cas systems utilize an RNA-mediated nuclease Cas protein or homolog (referred to herein as a “CRISPR-associated endonuclease”) in complex with guide RNA to recognize and cleave foreign nucleic acid. Cas9 proteins also use an activating RNA (also referred to as a transactivating or tracr RNA).
  • RNAs having the activity of either a guide RNA or both a guide RNA and an activating RNA, depending on the type of CRISPR- associated endonuclease used therewith, are also known in the art. In some cases, such dual activity guide RNAs are referred to as a single guide RNA (sgRNA). Synthetic guide RNAs that do not contain an activating RNA sequence may also be referred to as sgRNAs. In this disclosure, the terms sgRNA and gRNA are used interchangeably to refer to an RNA molecule that complexes with a CRISPR-associated endonuclease and localizes the ribonucleoprotein complex to a target DNA sequence.
  • the CRISPR-associated endonuclease can be a Cas9 polypeptide (Type II) or a Cpf1 polypeptide (Type V).
  • Type II Cas9 polypeptide
  • Type V Cpf1 polypeptide
  • Abudayyeh et al. Science 2016 August 5; 353(6299):aaf5573; Fonfara et al. Nature 532: 517-521 (2016), and Zetsche et al., Cell 163(3): p.759-771, 22 October 2015.
  • the term “Cas9 polypeptide” means a Cas9 protein, or a fragment or derivative thereof, identified in any bacterial species that encodes a Type II CRISPR/Cas system.
  • CRISPR-associated endonucleases such as Cas9 and Cas9 homologs
  • Cas9 and Cas9 homologs are found in a wide variety of eubacteria, including, but not limited to bacteria of the following taxonomic groups: Actinobacteria, Aquificae, Bacteroidetes- Chlorobi, Chlamydiae-Verrucomicrobia, Chlroflexi, Cyanobacteria, Firmicutes, Proteobacteria, Spirochaetes, and Thermotogae.
  • An exemplary Cas9 protein is the Streptococcus pyogenes Cas9 protein (SpCas9).
  • Another exemplary Cas9 protein is the Staphylococcus aureus Cas9 protein (SaCas9). Additional Cas9 proteins and homologs thereof are described in, e.g., Chylinksi, et al., RNA Biol.2013 May 1; 10(5): 726–737 ; Nat. Rev. Microbiol. 2011 June; 9(6): 467-477; Hou, et al., Proc Natl Acad Sci USA.
  • CRISPR-associated endonucleases include Cpf1 (See, e.g., Zetsche et al., Cell, Volume 163, Issue 3, p.759–771, 22 October 2015) and homologs thereof.
  • Full-length Cas9 is an endonuclease comprising a recognition domain and two nuclease domains (HNH and RuvC, respectively) that creates double-stranded breaks in DNA sequences.
  • HNH is linearly continuous
  • RuvC is separated into three regions, one left of the recognition domain, and the other two right of the recognition domain flanking the HNH domain.
  • Cas9 is targeted to a genomic site in a cell by interacting with a guide RNA that hybridizes to a 20-nucleotide DNA sequence that immediately precedes an NGG motif recognized by Cas9. This results in a double-strand break in the genomic DNA of the cell.
  • a Cas9 nuclease that requires an NGG protospacer adjacent motif (PAM) immediately 3’ of the region targeted by the guide RNA can be utilized.
  • Cas9 proteins with orthogonal PAM motif requirements can be utilized to target sequences that do not have an adjacent NGG PAM sequence.
  • Exemplary Cas9 proteins with orthogonal PAM sequence specificities include, but are not limited to those described in Esvelt et al., Nature Methods 10: 1116–1121 (2013).
  • Various Cas9 nucleases can be utilized in the methods described herein.
  • a Cas9 nuclease that requires an NGG protospacer adjacent motif (PAM) immediately 3’ of the region targeted by the guide RNA, such as SpCas9 can be utilized.
  • Such Cas9 nucleases can be targeted to any region of a genome that contains an NGG sequence.
  • a Cas9 nuclease that requires an NNGRRT (SEQ ID NO:79) or NNGRR(N) (SEQ ID NO: 80) PAM immediately 3’ of the region targeted by the guide RNA, such as SaCas9 can be utilized.
  • Cas9 proteins with orthogonal PAM motif requirements can be utilized to target sequences that do not have an adjacent NGG PAM sequence.
  • Exemplary Cas9 proteins with orthogonal PAM sequence specificities include, but are not limited to those described in Esvelt, K.M., et al., Nature Methods 10(11): 1116–1121 (2013) and those described in Zetsche et al., Cell, Volume 163, Issue 3, p.759–771, 22 October 2015. [0037]
  • the catalytically impaired CRISPR-associated endonuclease is a Cas9 nickase, for example, Cas9 D10A .
  • the Cas9 10A in the ABE is encoded by SEQ ID NO: 29.
  • the Cas910A comprises SEQ ID NO: 30.
  • a Cas9 nickase is bound to target nucleic acid as part of a complex with a guide RNA, a single strand break or nick is introduced into the target nucleic acid.
  • a pair of Cas9 nickases, each bound to a structurally different guide RNA, can be targeted to two proximal sites of a target genomic region.
  • Exemplary Cas9 nickases include Cas9 nucleases having a D10A or H840A mutation.
  • the CRISPR-associated endonuclease is a catalytically impaired Cpf1 polypeptide.
  • Cpf1 protein is a Class II, Type V CRISPR/Cas system protein.
  • Cpf1 is a smaller and simpler endonuclease than Cas9 (such as the spCas9).
  • the Cpf1 protein has a RuvC-like endonuclease domain that is similar to the RuvC domain of Cas9 but does not have a HNH endonuclease domain.
  • the N-terminal domain of Cpf1 also does not have the alpha-helical recognition lobe like the Cas9 protein.
  • Cpf1 introduces a sticky-end-like DNA double- stranded break with a 4 or 5 nucleotide overhang.
  • the Cpf1 protein does not need a tracrRNA; rather, the Cpf1 protein functions with only a crRNA.
  • the sgRNA does not comprise a tracr sequence.
  • the sgRNA used with the Cpf1 protein may comprise only a crRNA sequence (constant region).
  • a Cpf1 protein that requires an TTTN or TTN PAM (depending on the species, where “N” is an nucleobase) immediately 5’ of the region targeted by the guide RNA can be utilized.
  • TTTN or TTN PAM depending on the species, where “N” is an nucleobase
  • the CRISPR-associated endonuclease is FnCpf1p and the PAM is 5′ TTN, where N is A/C/G or T.
  • the CRISPR-associated endonuclease is PaCpf1p and the PAM is 5′ TTTV, where V is A/C or G
  • the CRISPR-associated endonuclease is FnCpf1p and the PAM is 5′ TTN, where N is A/C/G or T, and the PAM is located upstream of the 5′ end of the protospacer.
  • the CRISPR-associated endonuclease is FnCpf1p and the PAM is 5′ CTA and is located upstream of the 5′ end of the protospacer or the target locus.
  • the CRISPR-associated endonuclease is AsCpf1p and the PAM is 5′ TTTN.
  • activity in the context of sgRNA activity, or RNP activity, i.e., RNP activity of a complex comprising: (1) a gRNA and (2) a fusion protein comprising ABE and a catalytically impaired CRISPR-associated endonuclease, refers to the ability of a sgRNA to bind to a target genetic element.
  • activity also refers to the ability of an ABE RNP (i.e., an sgRNA complexd with an ABE) to edit base pairs, i.e., perform an A to G change in one strand of DNA.
  • the phrase “editing” in the context of editing of a genome of a cell refers to inducing a structural change in the sequence of the genome at a target genomic region, for example, editing performed by an ABE.
  • the editing can take the form of an A to G change in one strand of DNA (or a T to C change on the opposite strand of DNA) at a target genomic region.
  • the nucleotide sequence can encode a polypeptide or a fragment thereof. See, for example, Gaudelli et al., “Programmable base editing of A-T to G-C in genomic DNA without DNA cleavage,” Nature 551: 464-471 (2017).
  • an adenine base editor or “ABE” refers to a fusion protein comprising an adenosine deaminase and a catalytically impaired CRISPR-associated endonuclease.
  • the adenosine deaminase is a tadA enzyme that deaminates adenine on a single-strand of DNA to form inosine. See, Gaudelli et al, (2017).
  • the ABE is a fusion protein comprising a catalytically impaired CRISPR-associated endonuclease and one or more copies, for example, two, three, four copies, etc.
  • the term “ribonucleoprotein complex,” “RNPs”, and the like refers to a complex between: (1) an ABE and a crRNA (e.g., guide RNA or single guide RNA), (2) an ABE and a trans-activating crRNA (tracrRNA), (3) an ABE, a catalytically impaired CRISPR-associated endonuclease (e.g., Cas9), and a guide RNA, or (4) a combination thereof (e.g., a complex containing the ABE and the catalytically impaired CRISPR-associated endonuclease, a tracrRNA, and a crRNA guide).
  • a crRNA e.g., guide RNA or single guide RNA
  • tracrRNA trans-activating crRNA
  • Cas9 a catalytically impaired CRISPR-associated endonuclease
  • a guide RNA e.g., Cas9
  • a combination thereof e.g., a complex containing
  • a “cell” can be any eukaryotic cell, for example, human T cell or a cell capable of differentiating into a T cell, for example, a T cell that expresses a TCR receptor molecule. These include hematopoietic stem cells and cells derived from hematopoietic stem cells. Populations of cells, for example, populations of cells comprising viral particles or genetically modified cells made by any of the genomic editing methods provided herein, are also provided. [0044] As used herein, the phrase “hematopoietic stem cell” refers to a type of stem cell that can give rise to a blood cell.
  • Hematopoietic stem cells can give rise to cells of the myeloid or lymphoid lineages, or a combination thereof. Hematopoietic stem cells are predominantly found in the bone marrow, although they can be isolated from peripheral blood, or a fraction thereof. Various cell surface markers can be used to identify, sort, or purify hematopoietic stem cells. In some cases, hematopoietic stem cells are identified as c-kit + and lin-. In some cases, human hematopoietic stem cells are identified as CD34 + , CD59 + , Thy1/CD90 + , CD38 lo/- , C- kit/CD117 + , lin-.
  • human hematopoietic stem cells are identified as CD34-, CD59 + , Thy1/CD90 + , CD38 lo/- , C-kit/CD117 + , lin-.
  • human hematopoietic stem cells are identified as CD133 + , CD59 + , Thy1/CD90 + , CD38 lo/- , C-kit/CD117 + , lin-.
  • mouse hematopoietic stem cells are identified as CD34 lo/- , SCA-1 + , Thy1 +/lo , CD38 + , C- kit + , lin-.
  • the hematopoietic stem cells are CD150 + CD48-CD244-.
  • hematopoietic cell refers to a cell derived from a hematopoietic stem cell.
  • the hematopoietic cell may be obtained or provided by isolation from an organism, system, organ, or tissue (e.g., blood, or a fraction thereof).
  • an hematopoietic stem cell can be isolated and the hematopoietic cell obtained or provided by differentiating the stem cell.
  • Hematopoietic cells include cells with limited potential to differentiate into further cell types.
  • hematopoietic cells include, but are not limited to, multipotent progenitor cells, lineage-restricted progenitor cells, common myeloid progenitor cells, granulocyte-macrophage progenitor cells, or megakaryocyte-erythroid progenitor cells.
  • Hematopoietic cells include cells of the lymphoid and myeloid lineages, such as lymphocytes, erythrocytes, granulocytes, monocytes, and thrombocytes.
  • the hematopoietic cell is an immune cell, such as a T cell, B cell, macrophage, a natural killer (NK) cell or dendritic cell.
  • NK natural killer
  • the cell is an innate immune cell.
  • T cell refers to a lymphoid cell that expresses a T cell receptor molecule.
  • T cells include human alpha beta ( ⁇ ) T cells and human gamma delta ( ⁇ ) T cells.
  • T cells include, but are not limited to, na ⁇ ve T cells, stimulated T cells, primary T cells (e.g., uncultured), cultured T cells, immortalized T cells, helper T cells, cytotoxic T cells, memory T cells, regulatory T cells, natural killer T cells, combinations thereof, or sub- populations thereof.
  • T cells can be CD4 + , CD8 + , or CD4 + and CD8 + .
  • T cells can also be CD4-, CD8-, or CD4- and CD8 -.
  • T cells can be helper cells, for example helper cells of type TH1, TH2, T H 3, T H 9, T H 17, or T FH .
  • T cells can be cytotoxic T cells.
  • Regulatory T cells can be FOXP3 + or FOXP3-.
  • T cells can be alpha/beta T cells or gamma/delta T cells. In some cases, the T cell is a CD4 + CD25 hi CD127 lo regulatory T cell.
  • the T cell is a regulatory T cell selected from the group consisting of type 1 regulatory (Tr1), TH3, CD8+CD28-, Treg17, and Qa-1 restricted T cells, or a combination or sub-population thereof.
  • the T cell is a FOXP3 + T cell.
  • the T cell is a CD4 + CD25 lo CD127 hi effector T cell.
  • the T cell is a CD4 + CD25 lo CD127 hi CD45RA hi CD45RO- na ⁇ ve T cell.
  • a T cell can be a recombinant T cell that has been genetically manipulated.
  • the phrase “primary” in the context of a primary cell is a cell that has not been transformed or immortalized. Such primary cells can be cultured, sub-cultured, or passaged a limited number of times (e.g., cultured 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 times). In some cases, the primary cells are adapted to in vitro culture conditions. In some cases, the primary cells are isolated from an organism, system, organ, or tissue, optionally sorted, and utilized directly without culturing or sub-culturing. In some cases, the primary cells are stimulated, activated, or differentiated.
  • primary T cells can be activated by contact with (e.g., culturing in the presence of) CD3, CD28 agonists, IL-2, IFN- ⁇ , or a combination thereof.
  • CD3, CD28 agonists, IL-2, IFN- ⁇ , or a combination thereof e.g., CD3, CD28 agonists, IL-2, IFN- ⁇ , or a combination thereof.
  • compositions, systems, methods of manufacture, and methods for efficient delivery of adenine base editors (ABEs) to eukaryotic cells using viral particles are provided herein.
  • ABEs adenine base editors
  • components, systems, methods of manufacture, and methods for efficient delivery to cells of RNPs comprising (1) an adenosine base pair editor (ABE), wherein the ABE is a fusion protein comprising an adenosine deaminase and a catalytically impaired CRISPR-associated endonuclease; and (2) an sgRNA, via lentivirus-like particles, are provided.
  • ABE adenosine base pair editor
  • sgRNA via lentivirus-like particles
  • Mammalian Expression Plasmids Provided herein are mammalian expression plasmids that are used to deliver CRISPR component coding sequences, i.e., an sgRNA and an ABE, into mammalian cells being used to generate the lentivirus-like particles of this disclosure.
  • CRISPR component coding sequences i.e., an sgRNA and an ABE
  • a mammalian expression plasmid comprising a eukaryotic promoter operably linked to a non-viral nucleic acid sequence, wherein the non-viral nucleic acid sequence comprises; (i) a nucleic acid sequence encoding an adenosine base pair editor (ABE), wherein the ABE is a fusion protein comprising an adenosine deaminase and a catalytically impaired CRISPR- associated endonuclease; and (ii) a guide RNA (gRNA) coding sequence, wherein the gRNA coding sequence comprises at least one aptamer coding sequence.
  • ABE adenosine base pair editor
  • gRNA guide RNA
  • one or more copies of an ABE can be fused or linked to a catalytically impaired CRISPR-associate endonuclease.
  • the site-directed nuclease is linked to the adenine base editor via a peptide linker.
  • the linker can be between about 2 and about 25 amino acids in length.
  • the adenine base editor can be an ABE7 (for example, ABE7.10 (Gaudelli et al.
  • the mammalian expression plasmids provided herein comprise CRISPR component coding sequences, e.g., the coding sequence for a catalytically impaired CRISPR- associated endonuclease and a gRNA.
  • the gRNA coding sequence comprises at least one aptamer coding sequence.
  • the at least one aptamer coding sequence may be positioned at the 5’ end or the 3’ end of the gRNA. In some instances, the at least one aptamer coding sequence may be inserted at an internal position within the gRNA such as, for example, at one or more of the loops formed in the folded gRNA. For example, where the gRNA is for the Cas9 protein, the at least one aptamer coding sequence may be positioned at the tetra loop, the stem loop 2 (ST2), or the 3’ end of the gRNA.
  • a spacer of 1-30 nucleotides may be positioned between the gRNA the at least one aptamer coding sequence, or flanking the at least one aptamer coding sequence.
  • the mammalian expression vector comprises at least one aptamer coding sequence that encodes an aptamer sequence that is bound specifically by an aptamer- binding protein (ABP).
  • an aptamer sequence is an RNA sequence that forms a tertiary loop structure that is specifically bound by an ABP.
  • ABPs are RNA-binding proteins or RNA-binding protein domains.
  • Suitable aptamer coding sequences include polynucleotide sequences that encode known bacteriophage aptamer sequences.
  • Exemplary aptamer coding sequences include those encoding the aptamer sequences provided above in Table 1.
  • the aptamers are bound by a dimer of ABP.
  • These aptamer sequences are RNA sequences known to be bound specifically by bacteriophage proteins.
  • the at least one aptamer coding sequence encodes an aptamer sequence bound specifically by an ABP selected from the group consisting of MS2 coat protein, PP7 coat protein, lambda N RNA-binding domain, or Com protein. Table 1.
  • the mammalian expression vector comprises a sgRNA that comprises one aptamer coding sequence downstream thereof.
  • the gRNA may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 aptamer coding sequences.
  • the gRNA may comprise two aptamer coding sequences in tandem.
  • a sgRNA is a single guide RNA sequence that interacts with a CRISPR-associated endonuclease (a CRISPR site-directed nuclease) and specifically binds to or hybridizes to a target nucleic acid within the genome of a cell (genomic target sequence), such that the sgRNA and the CRISPR-associated endonuclease co-localize to the target nucleic acid in the genome of the cell.
  • Each sgRNA includes a DNA targeting sequence or protospacer sequence of about 10 to 50 nucleotides in length that specifically binds to or hybridizes to a target DNA sequence in the genome.
  • the DNA targeting sequence may be about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleotides in length.
  • the DNA targeting sequence may be about 15-30 nucleotides, about 15-25 nucleotides, about 10-25 nucleotides, or about 18-23 nucleotides.
  • the DNA targeting sequence is about 20 nucleotides.
  • the sgRNA comprises a crRNA sequence and a transactivating crRNA (tracrRNA) sequence.
  • the sgRNA does not comprise a tracrRNA sequence.
  • the DNA targeting sequence is designed to complement (e.g., perfectly complement) or substantially complement (e.g., having 1-4 mismatches) to the target DNA sequence.
  • the DNA targeting sequence can incorporate wobble or degenerate bases to bind multiple genetic elements.
  • the 19 nucleotides at the 3’ or 5’ end of the binding region are perfectly complementary to the target genetic element or elements.
  • the binding region can be altered to increase stability. For example, non-natural nucleotides, can be incorporated to increase RNA resistance to degradation.
  • the binding region can be altered or designed to avoid or reduce secondary structure formation in the binding region.
  • the binding region can be designed to optimize G-C content.
  • G-C content is preferably between about 40% and about 60% (e.g., 40%, 45%, 50%, 55%, 60%).
  • the binding region can be selected to begin with a sequence that facilitates efficient transcription of the sgRNA.
  • the binding region can begin at the 5’ end with a G nucleotide.
  • the binding region can contain modified nucleotides such as, without limitation, methylated or phosphorylated nucleotides.
  • the term “complementary” or “complementarity” refers to base pairing between nucleotides or nucleic acids, for example, and not to be limiting, base pairing between a sgRNA and a target sequence.
  • Complementary nucleotides are, generally, A and T (or A and U), and G and C.
  • the guide RNAs described herein can comprise sequences, for example, DNA targeting sequence that are perfectly complementary or substantially complementary (e.g., having 1-4 mismatches) to a genomic sequence.
  • the sgRNA includes a sgRNA constant region that interacts with or binds to the CRISPR-associated endonuclease.
  • the constant region of an sgRNA can be from about 75 to 250 nucleotides in length.
  • the constant region is a modified constant region comprising one, two, three, four, five, six, seven, eight, nine, ten or more nucleotide substitutions in the stem, the stem loop, a hairpin, a region in between hairpins, and/or the nexus of a constant region.
  • a modified constant region that has at least 80%, 85%, 90%, or 95% activity, as compared to the activity of the natural or wild-type sgRNA constant region from which the modified constant region is derived, may be used in the constructs described herein.
  • the mammalian expression plasmids comprise a eukaryotic promoter operably linked to the non-viral nucleic acid sequence.
  • a RNA polymerase II promoter is operably linked to the catalytically impaired CRISPR-associated endonuclease coding sequence and a RNA polymerase III promoter is operably linked to the gRNA coding sequence.
  • the RNA polymerase II promoter sequence is selected from a mammalian species.
  • the RNA polymerase III promoter sequences is selected from a mammalian species. For example, these promoter sequences can be selected from a human, cow, sheep, buffalo, pig, or mouse, to name a few.
  • the RNA polymerase II promoter sequence is a CMV, FE1 ⁇ , or SV40 sequence.
  • the RNA polymerase III promoter sequence is a U6 or an H1 sequence.
  • the RNA polymerase II sequence is a modified RNA polymerase II sequence.
  • the RNA polymerase II sequences having at least 80%, 85%, 90%, 95%, or 99% identity to a wild-type RNA polymerase II promoter sequence from any mammalian species can be used in the constructs provided herein.
  • the RNA polymerase III sequence is a modified RNA polymerase III sequence.
  • the RNA polymerase III sequences having at least 80%, 85%, 90%, 95%, or 99% identity to a wild-type RNA polymerase III promoter sequence from any mammalian species can be used in the constructs provided herein.
  • Those of skill in the art readily understand how to determine the identity of two polypeptides or nucleic acids.
  • the identity can be calculated after aligning the two sequences so that the identity is at its highest level.
  • Another way of calculating identity can be performed by published algorithms. For example, optimal alignment of sequences for comparison can be conducted using the algorithm of Needleman and Wunsch, J. Mol. Biol.48(3): 443-453 (1970).
  • the eukaryotic promoter is an inducible or regulatable promoter.
  • Coding sequences transcribed from a RNA pol II promoter include a poly(A) signal and a transcription terminator sequence downstream of the coding sequence.
  • RNA pol III promoter a sequence-based element
  • the role of the terminator, a sequence-based element, is to define the end of a transcriptional unit (such as a gene) and initiate the process of releasing the newly synthesized RNA from the transcription machinery. Terminators are found downstream of the gene to be transcribed, and typically occur directly after any 3’ regulatory elements, such as the polyadenylation or poly(A) signal.
  • the mammalian expression plasmid may also include at least one polynucleotide sequence encoding a RNA-stabilizing sequence positioned downstream of the CRISPR component coding sequence or the aptamer coding sequence if positioned downstream of the CRISPR component coding sequence.
  • the polynucleotide sequence encoding the RNA-stabilizing sequence is transcribed downstream of the CRISPR/Cas system component coding sequence and stabilizes the longevity of the transcribed RNA sequence.
  • the polynucleotide sequence encoding the RNA-stabilizing sequence is positioned downstream of the catalytically impaired CRISPR-associated endonuclease coding sequence.
  • RNA-stabilizing sequence is positioned downstream of the gRNA coding sequence.
  • An exemplary RNA- stabilizing sequence is the sequence of the 3’ UTR of human beta globin gene as set forth in SEQ ID NO:17 (DNA) and SEQ ID NO:18 (RNA).
  • SEQ ID NO: 34 Another example of an RNA-stabilizing sequence is SEQ ID NO: 34 which comprises two copies of SEQ ID NO: 17.
  • Other RNA- stabilizing sequences are described in Hayashi, T. et al., Developmental Dynamics 239(7):2034-2040 (2010) and Newbury, S. et al., Cell 48(2):297-310 (1987).
  • a spacer of 1-30 nucleotides may be positioned between the CRISPR component coding sequence and the at least one polynucleotide sequence encoding RNA-stabilizing sequence.
  • the mammalian expression plasmid may comprise one or more expression cassettes.
  • the mammalian expression plasmid comprises a first expression cassette that encodes the ABE and a second expression cassette that encodes the gRNA comprising at least one aptamer.
  • the mammalian expression plasmid may also comprise a reporter gene.
  • Another aspect of this disclosure are lentiviral packaging systems. Such systems include the mammalian expression plasmids described in this disclosure.
  • the system includes a lentiviral packaging plasmid comprising a eukaryotic promoter operably linked to a viral sequence, for example, a Gag nucleotide sequence, wherein the Gag nucleotide sequence comprises a nucleocapsid (NC) coding sequence and a matrix protein (MA) coding sequence, wherein one or both of the NC coding sequence or the MA coding sequence comprise at least one non-viral aptamer-binding protein (ABP) nucleotide sequence, and wherein the packaging plasmid does not encode a functional integrase protein.
  • a lentiviral packaging plasmid comprising a eukaryotic promoter operably linked to a viral sequence, for example, a Gag nucleotide sequence, wherein the Gag nucleotide sequence comprises a nucleocapsid (NC) coding sequence and a matrix protein (MA) coding sequence, wherein one or both of the NC coding sequence or the MA
  • a lentiviral packaging system comprising: (a) a packaging plasmid comprising a eukaryotic promoter operably linked to a Gag nucleotide sequence, wherein the Gag nucleotide sequence comprises a nucleocapsid (NC) coding sequence and a matrix protein (MA) coding sequence, wherein one or both of the NC coding sequence or the MA coding sequence comprises at least one non-viral aptamer-binding protein (ABP) nucleotide sequence, and wherein the packaging plasmid does not encode a functional integrase protein; (b) at least one mammalian expression plasmid comprising (i) a nucleic acid sequence encoding an adenosine base pair editor (ABE), wherein the ABE is a fusion protein comprising an adenosine deaminase and a catalytically impaired CRISPR-associated endonuclease and (
  • the system may include a second generation packaging plasmid or third generation packaging plasmids or modified versions thereof.
  • the packaging plasmid includes the Gag nucleotide sequence as described above and further comprises a Rev nucleotide sequence and a Tat nucleotide sequence.
  • the system includes a first packaging plasmid including a Gag nucleotide sequence as described above and a second packaging plasmid comprising a Rev nucleotide sequence.
  • the viral protein coding sequences are operably linked to a eukaryotic promoter for example, each individually or one promoter for multiple protein coding sequences.
  • the system may include a second generation packaging plasmid or third generation packaging plasmids or modified versions thereof.
  • the ABP coding sequence is at the 5’ end or 3’ end of the viral protein coding sequence, i.e., at the 5’ end or the 3’ end of the NC or MA coding sequence.
  • the ABP coding sequence may be inserted into the viral protein coding sequence such that the encoded ABP is fused to the viral protein.
  • the ABP coding sequence may be inserted in frame at an internal position within the viral protein coding sequence.
  • the ABP coding sequence When positioned in frame at an internal position near the 5’ or 3’ end of the viral protein coding sequence, the ABP coding sequence is positioned so as not to disrupt processing sequences such as those described in Tritch, R.J. et al., J. Virol.65(2):922-30 (1991) and Scarlata, S. and Carter, C., Biochimica et Biophysica Acta – Biomembranes 1614(1):62-72 (2003), which are incorporated herein by reference in their entirety.
  • the Gag nucleotide sequence encodes, inter alia, the NC coding sequence and the MA coding sequence, and the Gag precursor protein is processed by proteolytic cleavage into separate mature viral proteins.
  • nucleotides in the viral protein coding sequence may be replaced with the ABP protein coding sequence.
  • a linker sequence encoding 3-6 amino acids may be positioned between the viral protein coding sequence and the ABP coding sequence, or flanking the ABP coding sequence, to help facilitate proper folding of the protein domains upon expression.
  • the modified viral protein is NC and the ABP coding sequence is inserted at the 5’ end or the 3’ end of the NC coding sequence.
  • the modified viral protein is NC and the ABP coding sequence is inserted before or after one of the zinc finger (ZF) domains.
  • the ABP coding sequence may be inserted after the last codon of the second ZF (ZF2) domain.
  • the ABP coding sequence may be inserted before the first codon of the ZF2 domain.
  • the ABP coding sequence may be inserted before the first codon of the first ZF (ZF1) domain.
  • the ABP coding sequence may be inserted after the last codon of the first ZF (ZF1) domain.
  • the ABP coding sequence is inserted into the NC coding sequence in a manner that does not disrupt the highly positive stretch of amino acids in the NC protein.
  • the modified viral protein is MA and the ABP coding sequence is inserted at the 5’ end or the 3’ end of the MA coding sequence.
  • the ABP coding sequence is inserted in frame at an internal position within the MA coding sequence.
  • nucleotides in the MA coding sequence may be replaced with the ABP protein coding sequence.
  • nucleotides encoding amino acids 44-132 of the MA protein may be replaced with the ABP coding sequence.
  • the ABP coding sequence is inserted prior to the codon encoding amino acid 44 of the MA protein.
  • the ABP coding sequence is inserted after the codon encoding amino acid 132 of the MA protein.
  • the system includes a packaging plasmid comprising a eukaryotic promoter operably linked to a NEF coding sequence or a VPR coding sequence, wherein the NEF coding sequence or the VPR coding sequence comprises at least one non- viral ABP nucleotide sequence.
  • the system may include a second generation packaging plasmid or third generation packaging plasmids or modified versions thereof.
  • the packaging plasmid includes a Gag nucleotide sequence, a Rev nucleotide sequence, and a Tat nucleotide sequence.
  • the system includes a first packaging plasmid including a Gag nucleotide sequence and a second packaging plasmid comprising a Rev nucleotide sequence.
  • the modified viral protein is VPR and the ABP coding sequence is inserted at the 5’ end or the 3’ end of the VPR coding sequence. In one example, the ABP coding sequence is inserted at the 5’ end of the VPR coding sequence.
  • the modified viral protein is NEF and the ABP coding sequence is inserted at the 5’ end or the 3’ end of the NEF coding sequence. In one example, the ABP coding sequence is inserted at the 3’ end of the NEF coding sequence.
  • the coding sequence of the viral protein may be one of SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, or SEQ ID NO:25.
  • the amino acid sequence of the the viral protein may be one of SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, or SEQ ID NO:26.
  • the lentiviral packaging plasmid comprises a sequence encoding at least one of SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, or SEQ ID NO:26 operably linked to a eukaryotic promoter.
  • the polypeptide may comprise three mutations that enhances packaging in the viral capsid such as, for example, the following substitution mutations: G3C, V153L, and E177G.
  • the plasmids may encode one or more viral proteins that comprise two or more aptamer-binding proteins fused thereto.
  • the Gag nucleotide sequence of the lentiviral packaging plasmid may comprise a NC coding sequence and a MA coding sequence and where one or both of the NC coding sequence or the MA coding sequence comprises a first non-viral ABP nucleotide sequence and a second non-viral ABP nucleotide sequence.
  • the first non-viral ABP nucleotide sequence and the second non-viral ABP nucleotide sequence may both encode the same ABP.
  • the first non-viral ABP nucleotide sequence and the second non-viral ABP nucleotide sequence encode different ABPs.
  • the Gag nucleotide sequence of the lentiviral packaging plasmid may comprise a NC coding sequence comprising at least one first non-viral ABP nucleotide sequence and a MA coding sequence comprising at least one second non-viral ABP nucleotide sequence .
  • the at least one first non-viral ABP nucleotide sequence and the at least one second non-viral ABP nucleotide sequence may both encode the same ABP.
  • the at least one first non-viral ABP nucleotide sequence and the at least one second non-viral ABP nucleotide sequence encode different ABPs.
  • the packaging plasmid may encode a VPR coding sequence or a NEF coding sequence and where the VPR coding sequence or the NEF coding sequence comprises a first non-viral ABP nucleotide sequence and a second non-viral ABP nucleotide sequence.
  • the first non-viral ABP nucleotide sequence and the second non-viral ABP nucleotide sequence may both encode the same ABP.
  • the first non-viral ABP nucleotide sequence and the second non-viral ABP nucleotide sequence encode different ABPs.
  • a non-viral aptamer-binding protein (ABP) nucleotide sequence encodes a polypeptide sequence that binds to an RNA aptamer sequence.
  • suitable ABPs include bacteriophage RNA- binding proteins that bind specifically to RNA sequences that form stem-loop structures referred to as RNA aptamer sequences.
  • non-viral aptamer binding protein examples include MS2 coat protein, PP7 coat protein, lambda N peptide, and Com (Control of mom) protein.
  • the lambda N peptide may be amino acids 1-22 of the lambda N protein, which are the RNA- binding domain of the protein.
  • the ABPs bind to their aptamers as dimers. Information about these ABP and the aptamer sequences to which they bind is provided in Table 1.
  • the at least one non-viral ABP nucleotide sequence encodes a polypeptide having the sequence set forth in any of SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, or SEQ ID NO:16.
  • the at least one non-viral ABP nucleotide sequence comprises any of SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, or SEQ ID NO:15.
  • a feature of the lentiviral packaging plasmids provided herein is that they may not encode a functional integrase protein. When the packaging plasmids do not encode a functional integrase protein and they are used in the systems and methods described herein, there is substantially reduced risk the nucleic acid molecules carried by the lentivirus-like particles produced using these packaging plasmids will integrate into the genome of the transduced eukaryotic cell.
  • the lentiviral packaging plasmid comprises an integrase coding sequence with an integrase-inactivating mutation therein.
  • the integrase- inactivating mutation may be an aspartic acid to valine mutation at amino acid position 64 (D64V) of the integrase protein encoded by the integrase coding sequence.
  • the lentiviral packaging plasmid comprises a deletion of all or a portion of an integrase coding sequence.
  • the lentiviral packaging plasmids comprise a eukaryotic promoter operably linked to the Gag nucleotide sequence.
  • the mammalian expression plasmids comprise a eukaryotic promoter operably linked to the VPR coding sequence or the NEF coding sequence.
  • the eukaryotic promoter is a RNA polymerase II promoter.
  • the RNA polymerase II promoter sequence is selected from a mammalian species.
  • the promoter sequence can be selected from a human, cow, sheep, buffalo, pig, or mouse, to name a few.
  • the RNA polymerase II promoter sequence is a CMV, FE1 ⁇ , or SV40 sequence.
  • the RNA polymerase II sequence is a modified RNA polymerase II sequence.
  • RNA polymerase II sequences having at least 80%, 85%, 90%, 95%, or 99% identity to a wild-type RNA polymerase II promoter sequence from any mammalian species can be used in the constructs provided herein.
  • identity can be calculated after aligning the two sequences so that the identity is at its highest level.
  • Another way of calculating identity can be performed by published algorithms. For example, optimal alignment of sequences for comparison can be conducted using the algorithm of Needleman and Wunsch, J. Mol. Biol.48: 443 (1970).
  • the eukaryotic promoter is an inducible promoter.
  • Coding sequences transcribed from a RNA pol II promoter include a poly(A) signal and a transcription terminator sequence downstream of the coding sequence.
  • Commonly used mammalian terminators e.g., SV40, hGH, BGH, and rbGlob
  • sequence motif AAUAAA which promotes both polyadenylation and termination.
  • the role of the terminator, a sequence-based element, is to define the end of a transcriptional unit (such as a gene) and initiate the process of releasing the newly synthesized RNA from the transcription machinery. Terminators are found downstream of the gene to be transcribed, and typically occur directly after any 3’ regulatory elements, such as the polyadenylation or poly(A) signal.
  • the lentiviral packaging plasmids may comprise one or more expression cassettes.
  • the system also can include an envelope plasmid having an envelope coding sequence that encodes a viral envelope glycoprotein.
  • the Env nucleotide sequence may encode VSV-G.
  • the envelope coding sequence is operably linked to a eukaryotic promoter. Appropriate eukaryotic promoters are described above. In some instances, the eukaryotic promoter is a RNA pol II promoter.
  • the system can comprise any of the packaging plasmids, envelope plasmids and mammalian expression plasmids, i.e., a mammalian expresson plasmid comprising (i) a nucleic acid sequence encoding an ABE; and (ii) a gRNA comprising at least one aptamer, described herein.
  • kits include the components of the systems described in this disclosure.
  • kits include one or more of the plasmids described herein.
  • Lentivirus-like Particles for example, lentivirus-like particles made by any of the methods described herein.
  • a lentivirus-like particle is multiprotein structure that mimics the organization and conformation of authentic native viruses but lacks the viral genome.
  • a plurality of lentivirus-like particles are also provided.
  • the lentivirus-like particles contain a modified lentiviral protein that is a fusion protein in which at least one aptamer-binding protein is fused to one or more viral proteins.
  • the modified viral protein may be structural or non-structural.
  • Exemplary structural proteins are lentiviral nucleocapsid (NC) protein and matrix (MA) protein.
  • Exemplary non-structural proteins are viral protein R (VPR) and negative regulatory factor (NEF).
  • the particles contain a fusion protein comprising a NC protein and a MA protein where one or both thereof are fused with at least one non-viral aptamer binding protein (ABP).
  • the NC protein of the particles may have two functional zinc finger protein domains. In particular, retention of the second NC zinc finger domain may preserve the efficiency of viral assembly and budding.
  • the particles contain a fusion protein comprising a VPR protein or a NEF protein where the VPR protein or the NEF protein are fused with at least one non-viral ABP.
  • the particles also contain an RNP comprising: (i) an adenosine base pair editor (ABE), wherein the ABE is a fusion protein comprising an adenosine deaminase and a catalytically impaired CRISPR-associated endonuclease; and (ii) a gRNA.
  • ABE adenosine base pair editor
  • Any of the mammalian expression plasmids described herein comprising a non-viral nucleic acid sequence, wherein at least one aptamer is attached or inserted into the gRNA sequence, can be used to generated lentivirus-like particles containing RNPs.
  • the lentivirus-like particles do not contain a functional integrase protein.
  • the particles may comprise a viral fusion protein comprising one or more ABPs.
  • the particles contain a NC protein, a MA protein, or both, where one or both of the NC protein or MA protein are fused with one or more non-viral ABP.
  • lentivirus-like particles comprise a NC protein fused with at least one non-viral ABP.
  • lentivirus-like particles comprise a MA protein fused with at least one non-viral ABP.
  • the lentivirus-like particles may comprise a NC protein and a MA protein, where one or both of the NC protein or the MA protein may be fused with two non- viral ABP proteins, a first non-viral ABP and a second non-viral ABP fused to a C’ terminal end of the first non-viral ABP (i.e. in tandem).
  • the particles may contain one or both of a NC protein or a MA protein fused with a first non-viral ABP and a second non-viral ABP.
  • the lentivirus-like particle contains a VPR protein or a NEF protein, where the VPR protein or the NEF protein is fused to one or more non-viral ABP.
  • the lentivirus-like particle contains a VPR protein or a NEF protein fused to two non-viral ABP, a first non-viral ABP and a second non-viral ABP fused to a C’ terminal end of the first non-viral ABP (i.e. in tandem).
  • the lentivirus-like particle contains a VPR protein or a NEF protein fused to a first non-viral ABP and a second non-viral ABP.
  • the first non-viral ABP and the second non-viral ABP may both be the same ABP.
  • the first non-viral ABP and the second non-viral ABP may be different ABPs.
  • the lentivirus-like particles may comprise a NC protein with at least one first non-viral ABP fused to MA protein with at least one second non-viral ABP fused to its C’ terminal end.
  • the at least one first non-viral ABP and the at least one second non-viral ABP both be the same ABP.
  • the at least one first non-viral ABP protein and the at least one second non-viral ABP may be different ABPs.
  • the first non-viral ABP and the second non-viral ABP may both be the same ABP.
  • the first non-viral ABP and the second non-viral ABP may be different ABPs.
  • a non-viral ABP is a polypeptide sequence that binds to an RNA aptamer sequence.
  • suitable ABPs include bacteriophage RNA-binding proteins that bind specifically to known RNA aptamer sequences, which are RNA sequences that form stem-loop structures.
  • Exemplary non-viral aptamer binding protein include MS2 coat protein, PP7 coat protein, lambda N peptide, and Com (Control of mom) protein.
  • the lambda N peptide may be amino acids 1-22 of the lambda N protein, which are the RNA-binding domain of the protein.
  • the lentivirus-like particles may comprise various lentiviral proteins. However, in some instances, the lentivirus-like particles do not comprise all of the types of proteins or nucleic acids found in native lentiviruses. In some instances, the particles may contain NC, MA, CA, SP1, SP2, P6, POL, ENV, TAT, REV, VIF, VPU, VPR, and/or NEF proteins, or a derivative, combination, or portion of any thereof. In some instances, the particles may contain NC, MA, CA, SP1, SP2, P6, and POL.
  • the lentivirus-like particles may comprise only those proteins that form the viral shell (capsid). In some instances, one or more lentiviral proteins may be excluded in full or in part from the lentivirus-like particles.
  • the lentivirus-like particles may not contain a POL protein or may comprise a non-functional version of a POL protein such as, for example, a POL protein with an inactivating point mutation or an inactivating truncation.
  • the lentivirus- like particles may not contain an integrase protein or may comprise a non-functional version of an integrase protein such as, for example, an integrase protein with an inactivating point mutation or an inactivating truncation.
  • the lentivirus-like particle may contain a non-functional integrase protein comprising an aspartic acid to valine mutation at amino acid position 64 (D64V).
  • the lentivirus-like particles may not contain a reverse transcriptase protein or may comprise a non-functional version of a reverse transcriptase protein such as, for example, a reverse transcriptase protein with an inactivating point mutation or an inactivating truncation.
  • gRNA generally comprises a DNA targeting sequence and a constant region that interacts with the CRISPR-associated endonuclease.
  • the gRNA may comprise a transactivating crRNA (tracrRNA) sequence.
  • the gRNA may comprise a tracrRNA where it is to be used in conjunction with a Cas9 protein or derivative.
  • the gRNA does not comprise a tracrRNA sequence.
  • the gRNA may not comprise a tracrRNA sequence where it is to be used in conjunction with a Cpf1 protein or derivative.
  • the gRNA comprises at least one aptamer sequence.
  • the at least one aptamer sequence may be positioned at the 5’ end or the 3’ end of the gRNA. In some instances, the at least one aptamer sequence may be inserted at an internal position within the gRNA such as, for example, at one or more of the loops formed in the folded gRNA. For example, where the gRNA is for a Cas9 protein, the at least one aptamer sequence may be positioned at the tetra loop, the stem loop 2 (ST2), or the 3’ end of the gRNA. In some instances, a spacer of 1-30 ribonucleotides may be positioned between the gRNA and the at least one aptamer sequence, or flanking the at least one aptamer sequence.
  • At least one aptamer sequence does not interfere with lentivirus-like particle transduction of eukaryotic cells.
  • at least one non-viral ABP fused to one or more of the NC protein, the MA protein, the VPR protein, or the NEF protein may not interfere with lentivirus- like particle transduction of eukaryotic cells.
  • eukaryotic cells comprising a target genomic sequence of interest to be modified are transduced with lentivirus-like particles that contain a viral fusion protein comprising a viral protein fused to at least one aptamer-binding protein (ABP) and an RNP comprising (1) a gRNA and (2) an adenosine base pair editor (ABE), wherein the ABE is a fusion protein comprising an adenosine deaminase and a catalytically impaired CRISPR-associated endonuclease.
  • ABP aptamer-binding protein
  • ABE adenosine base pair editor
  • An advantage of the provided methods is reduced guide independent RNA off- target gene editing events associated with ABEs.
  • guide-independent RNA off-target activity can be reduced by at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 90%, 95%, 99% or greater, as compared to RNA off-target activity when RNPs are delivered using non-lentiviral delivery.
  • guide independent DNA off-target gene editing events are also reduced.
  • guide-dependent DNA off-target activity can be reduced by at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 90%, 95%, 99% or greater when RNPs are delivered using non- lentiviral delivery.
  • the transduced eukaryotic cells are mammalian cells. In some instances, the eukaryotic cells may be in vitro cultured cells.
  • the eukaryotic cells may be ex vivo cells obtained from a subject. In other instances, the eukaryotic cells are present in a subject.
  • subject is meant an individual.
  • the subject is a mammal, such as a primate, and, more specifically, a human.
  • Non-human primates are subjects as well.
  • subject includes domesticated animals, such as cats, dogs, etc., livestock (for example, cattle, horses, pigs, sheep, goats, etc.) and laboratory animals (for example, ferret, chinchilla, mouse, rabbit, rat, gerbil, guinea pig, etc.).
  • livestock for example, cattle, horses, pigs, sheep, goats, etc.
  • laboratory animals for example, ferret, chinchilla, mouse, rabbit, rat, gerbil, guinea pig, etc.
  • veterinary uses and medical uses and formulations are contemplated herein.
  • lentivirus-like particles may be administered to the subject, for example, injected into a subject, according to known, routine methods.
  • Exemplary modes of administration include oral, rectal, transmucosal, topical, intranasal, inhalation (e.g., via an aerosol), buccal (e.g., sublingual), vaginal, intrathecal, intraocular, transdermal, intradermal, intrapleural, intracerebral, and intraarticular), topical, and the like, as well as direct tissue or organ injection. Administration can also be to a tumor. The most suitable route in any given case will depend on the nature and severity of the condition being treated and on the nature of the particular lentivirus-like particle that is being used.
  • the lentivirus-like particles are injected intravenously (IV), intraperitoneally (IP), intramuscularly, or into a specific organ or tissue.
  • IV intravenously
  • IP intraperitoneally
  • more than one administration e.g., two, three, four or more administrations
  • An effective amount of any of the recombinant lentivirus-like particles described herein will vary and can be determined by one of skill in the art through experimentation and/or clinical trials.
  • an effective dose can be from about 10 6 to about 10 15 lentivirus- like particles, for example, from about 10 6 to about 10 14 , from about 10 6 to about 10 13 , from about 10 6 to about 10 12 lentivirus-like particles, from about 10 6 to about 10 12 , from about 10 6 to about 10 11 , or from about 10 6 to about 10 11 lentivirus-like particles.
  • Other effective dosages can be readily established by one of ordinary skill in the art through routine trials establishing dose response curves. See, for example, Mangeot et al. “Genome editing in primary cells and in vivo using viral-derived Nanoblades loaded with Cas9-sgRNA ribonucleoproteins,” Nat Commun 10, 45 (2019).
  • the provided methods are for modifying a target locus of interest, the method comprising transducing a plurality of eukaryotic cells with a plurality of viral particles, wherein the plurality of viral particles comprise (i) a fusion protein comprising a viral protein, for example, NC, MA, VRP, or NEF, wherein the viral protein comprises at least one non-viral aptamer binding protein (ABP); and (ii) a ribonucleotide protein (RNP) complex comprising (1) a gRNA and (2) an ABE, wherein the RNP is capable of binding (e.g., preferentially binding) via the gRNA, to the genomic target sequence in genomic DNA of the cell and the ABE alters the genomic DNA of the cell.
  • a fusion protein comprising a viral protein, for example, NC, MA, VRP, or NEF
  • the viral protein comprises at least one non-viral aptamer binding protein (ABP)
  • RNP ribonucleotide protein
  • the RNPs are packaged into the viral particles via the interaction of an aptamer sequence attached to or inserted into a gRNA sequence that forms a complex with the catalytically impaired CRISPR- associated endonuclease.
  • the methods described can be used with any catalytically impaired CRISPR- associated endonuclease that requires a constant region of an sgRNA for function. These include, but are not limited to RNA-guided site-directed nucleases. Examples include nucleases present in any bacterial species that encodes a Type II or V CRISPR/Cas system. Suitable CRISPR-associated endonucleases are described throughout this disclosure.
  • the site-directed nuclease can be a catalytically impaired Cas9 polypeptide, a catalytically impaired Cpf1 polypeptide, a catalytically impaired Cas9 nickase, or derivatives of any thereof.
  • the sgRNA is targeted to specific regions at or near a gene.
  • the sgRNA can be targeted to a region where single base changes are necessary, for example, to correct a single base mutation in the human beta-globin gene that causes sickle cell anemia.
  • the sgRNA allows the RNPs described herein to a specific site in the genomic sequence of a cell.
  • the adenine base editor catalyzes adenosine (A) to inosine formation in one strand, while the catalytically impaired endonuclease, for example, Cas9 D10A nicks the opposite strand, i.e., the non-edited strand.
  • endonuclease for example, Cas9 D10A
  • inosine is read as guanosine by polymerase enzymes
  • DNA repair and replication mechanims replace the original A-T base pair with a G-C base pair at the target site. See, Gaudelli et al. (2017).
  • the modifications to the system components as described in this disclosure do not impair how the system components function following transduction into eukaryotic cells. Rather, the components may function similarly or better than unmodified components upon transduction into eukaryotic cells.
  • the viral fusion proteins in the lentivirus-like particles may not interfere with the lentivirus-like particle transduction of eukaryotic cells.
  • the RNPs packaged in the lentivirus-like particles comprise at least one aptamer sequence
  • the at least one aptamer sequence may not interfere with the lentivirus-like particle transduction of eukaryotic cells.
  • the lentivirus-like proteins containing viral fusion protein may result in greater gene editing upon transduction into eukaryotic cells relative to lentivirus-like particles that do not comprise a viral fusion protein.
  • the viral fusion protein may be a NC-ABP fusion protein, such as a NC-MS2 fusion protein or NC-PP7 fusion protein.
  • the NC fusion protein is fused to one or two ABPs, such as one or two MS2 proteins, one or two PP7 proteins, or one MS2 protein and one PP7 protein.
  • the eukaryotic cells can be in vitro, ex vivo or in vivo.
  • the cell is a primary cell (isolated from a subject).
  • a primary cell is a cell that has not been transformed or immortalized. Such primary cells can be cultured, sub-cultured, or passaged a limited number of times (e.g., cultured 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 times). In some cases, the primary cells are adapted to in vitro culture conditions. In some cases, the primary cells are isolated from an organism, system, organ, or tissue, optionally sorted, and utilized directly without culturing or sub-culturing. In some cases, the primary cells are stimulated, activated, or differentiated. In some embodiments, the cells are cultured under conditions effective for expanding the population of modified cells. In some embodiments, cells modified by any of the methods provided herein are purified.
  • cells are removed from a subject, modified using any of the methods described herein and re- administered to the patient.
  • the cells are cultured for a sufficient amount of time to allow for gene editing to occur, such that a pool of cells expressing a detectable phenotype can be selected from the plurality of transduced cells.
  • the phenotype can be, for example, cell growth, survival, or proliferation.
  • the phenotype is cell growth, survival, or proliferation in the presence of an agent, such as a cytotoxic agent, an oncogene, a tumor suppressor, a transcription factor, a kinase (e.g., a receptor tyrosine kinase), a gene (e.g., an exogenous gene) under the control of a promoter (e.g., a heterologous promoter), a checkpoint gene or cell cycle regulator, a growth factor, a hormone, a DNA damaging agent, a drug, or a chemotherapeutic.
  • the phenotype can also be protein expression, RNA expression, protein activity, or cell motility, migration, or invasiveness.
  • the selecting the cells on the basis of the phenotype comprises fluorescence activated cell sorting, affinity purification of cells, or selection based on cell motility.
  • the selecting the cells comprises analysis of the genomic DNA of the cells such as by amplification, sequencing, SNP analysis, etc.
  • Sequencing methods include, but are not limited to, shotgun sequencing, bridge PCR, Sanger sequencing (including microfluidic Sanger sequencing), pyrosequencing, massively parallel signature sequencing, nanopore DNA sequencing, single molecule real-time sequencing (SMRT) (Pacific Biosciences, Menlo Park, CA), ion semiconductor sequencing, ligation sequencing, sequencing by synthesis (Illumina, San Diego, Ca), Polony sequencing, 454 sequencing, solid phase sequencing, DNA nanoball sequencing, heliscope single molecule sequencing, mass spectroscopy sequencing, pyrosequencing, Supported Oligo Ligation Detection (SOLiD) sequencing, DNA microarray sequencing, RNAP sequencing, tunneling currents DNA sequencing, and any other DNA sequencing method identified in the future.
  • SMRT single molecule real-time sequencing
  • ion semiconductor sequencing ligation sequencing
  • ligation sequencing sequencing by synthesis
  • Polony sequencing 454 sequencing
  • solid phase sequencing DNA nanoball sequencing
  • heliscope single molecule sequencing sequencing by synthesis
  • high throughput sequencing refers to all methods related to sequencing nucleic acids where more than one nucleic acid sequence is sequenced at a given time.
  • Methods of Treatment Any of the methods and compositions described herein can be used to treat a disease (e.g., cancer, a blood disorder (for example, sickle cell anemia or beta thalassemia), an infectious disease, an autoimmune disease, transplantation rejection, graft vs. host disease or other inflammatory disorder) in a subject.
  • a disease e.g., cancer, a blood disorder (for example, sickle cell anemia or beta thalassemia), an infectious disease, an autoimmune disease, transplantation rejection, graft vs. host disease or other inflammatory disorder
  • the cancer to be treated is selected from a cancer of B-cell origin, breast cancer, gastric cancer, neuroblastoma, osteosarcoma, lung cancer, colon cancer, chronic myeloid cancer, leukemia (e.g., acute myeloid leukemia, chronic lymphocytic leukemia (CLL) or acute lymphocytic leukemia (ALL)), prostate cancer, colon cancer, renal cell carcinoma, liver cancer, kidney cancer, ovarian cancer, stomach cancer, testicular cancer, rhabdomyosarcoma, and Hodgkin's lymphoma.
  • leukemia e.g., acute myeloid leukemia, chronic lymphocytic leukemia (CLL) or acute lymphocytic leukemia (ALL)
  • prostate cancer colon cancer
  • renal cell carcinoma liver cancer
  • kidney cancer ovarian cancer
  • stomach cancer testicular cancer
  • rhabdomyosarcoma rhabdomyosarcoma
  • Hodgkin's lymphoma Hod
  • the cancer of B-cell origin is selected from the group consisting of B-lineage acute lymphoblastic leukemia, B-cell chronic lymphocytic leukemia, and B-cell non-Hodgkin's lymphoma [0106]
  • the cells of the subject are modified in vivo.
  • the method of treating a disease in a subject comprises: a) obtaining cells from the subject; b) modifying the cells using any of the methods provided herein; and c) administering the modified cells to the subject. See, for example, Milone and O’Doherty “Clinical sue of lentiviral vectors,” Leukemia 32, 1529-1541 (2016).
  • the disease is selected from the group consisting of cancer, a blood disorder (for example, sickle cell anemia or beta thalassemia), an infectious disease, an autoimmune disease, transplantation rejection, graft vs. host disease or other inflammatory disorder in a subject.
  • the cells obtained from the subject are modified to express a tumor specific antigen.
  • tumor-specific antigen means an antigen that is unique to cancer cells or is expressed more abundantly in cancer cells than in in non-cancerous cells.
  • the cells obtained from the subject are T cells.
  • the modified cells are expanded prior to administration to the subject.
  • the lentivirus-like particles or cells described herein can be formulated as a pharmaceutical composition.
  • a pharmaceutical composition comprising any of the lentivirus-like particles described herein.
  • a pharmaceutical composition comprising any of the modified cells described herein
  • the pharmaceutical composition can further comprise a carrier.
  • carrier means a compound, composition, substance, or structure that, when in combination with lentivirus-like particles or cells, aids or facilitates preparation, storage, administration, delivery, effectiveness, selectivity, or any other feature of the lentivirus-like particles or cells for its intended use or purpose.
  • a carrier can be selected to minimize any degradation of the active ingredient and to minimize any adverse side effects in the subject.
  • Such pharmaceutically acceptable carriers include sterile biocompatible pharmaceutical carriers, including, but not limited to, saline, buffered saline, artificial cerebral spinal fluid, dextrose, and water.
  • pharmaceutically acceptable is meant a material that is not biologically or otherwise undesirable, which can be administered to an individual along with the selected agent without causing unacceptable biological effects or interacting in a deleterious manner with the other components of the pharmaceutical composition in which it is contained.
  • the plasmid for expressing ABE7.10 in E. coli has been described earlier (Kim et al., Nat Biotechnol 2019, 37 (4), 430- 435). Other plasmids were generated, as shown in Table 2. Gene synthesis was done by GenScript Inc. All constructs generated were confirmed by Sanger sequencing. Sequence information for primers and oligonucleotides are listed in Table 3. ABE target sequences and the oligos used for making the sgRNA expression constructs are listed in Table 4. It is understood that the sequences for the components of the plasmids listed in Table 2 can be separated by nucleic acid linkers, for example, linkers of about 2 to 100 bases.
  • any of the constructs described herein can include one or more introns, for example, between the promoter sequence and a nucleic acid encoding a polypeptide sequence (e.g., an ABE), to facilitate expression of one or more polypeptides sequences in the construct.
  • SNU-ABE plasmid which encodes codon optimized ABE 7.10 linked to an N- terminal His tag, was first transformed into BL21-star (DE3) competent cells, which were then plated on a Luria-Bertani (LB)-agar plate containing 50 ⁇ g ml ⁇ 1 kanamycin. After incubation overnight at 37°C, a single colony was selected and grown overnight at 37 °C (pre-culture) in LB broth containing 50 ⁇ g ml ⁇ 1 kanamycin and 10 ⁇ M ZnCl2 to maintain ABE catalytic activity.
  • LB Luria-Bertani
  • the culture was put on ice for about 1 h.
  • 1 mM isopropyl ⁇ -D-1- thiogalactopyranoside GoldBio, St. Louis, MO
  • the later steps in the purification procedure were all carried out at 0-4°C.
  • the cells Prior to cell lysis, the cells were harvested by centrifugation at 5,000g for 10 min, after which they were resuspended in 8 ml lysis buffer per 400 ml inoculants [50 mM sodium phosphate (Sigma- Aldrich, St. Louis, MO), 500 mM 1% Triton X-100 (Sigma-Aldrich), 20% glycerol, 1 mM phenylmethylsulfonyl fluoride (Sigma-Aldrich), 1 mg ml ⁇ 1 lysozyme from chicken egg white (Sigma-Aldrich), 10 ⁇ M ZnCl2 (Sigma-Aldrich), pH 8.0].
  • inoculants 50 mM sodium phosphate (Sigma- Aldrich, St. Louis, MO), 500 mM 1% Triton X-100 (Sigma-Aldrich), 20% glycerol, 1 mM phenylmethylsulfonyl fluoride
  • lysis cells were frozen in liquid nitrogen and thawed at 37°C for a total of three times.
  • cells were sonicated (3 min total, 5s on, 10s off), after which they were centrifuged at 13,000 rpm to clear the lysate.
  • the supernatant was mixed with 10 ml Ni-NTA agarose beads (QIAGEN) and the resin-lysate mixture was gently rotated for 1 h and then loaded onto a column.
  • the column was washed three times each with 50 ml nickel wash buffer [50 mM sodium phosphate (Sigma-Aldrich), 150 mM NaCl (Sigma-Aldrich), 35 mM imidazole (Sigma-Aldrich), 1 mM DTT (GoldBio), 10 ⁇ M ZnCl2 (Sigma-Aldrich), pH 8.0] and then the proteins were eluted with 20 ml nickel elution buffer (50 mM sodium phosphate, 150 mM NaCl, 250 mM imidazole, 20% glycerol, 1 mM DTT, 10 ⁇ M ZnCl2, pH 8.0).
  • 50 ml nickel wash buffer [50 mM sodium phosphate (Sigma-Aldrich), 150 mM NaCl (Sigma-Aldrich), 35 mM imidazole (Sigma-Aldrich), 1 mM DTT (GoldBio), 10 ⁇ M ZnCl2 (
  • the eluted proteins were further purified with 5 ml heparin Sepharose beads (GE Healthcare) in another column.
  • the column was washed with 50 ml heparin wash buffer (50 mM sodium phosphate, 150 mM NaCl, 1 mM DTT, 10 ⁇ M ZnCl2, pH 8.0) three times and proteins were eluted with 20 ml heparin elution buffer (50 mM sodium phosphate, 750 mM NaCl, 20% glycerol, 1 mM DTT, 10 ⁇ M ZnCl2, pH 8.0).
  • 50 ml heparin wash buffer 50 mM sodium phosphate, 150 mM NaCl, 1 mM DTT, 10 ⁇ M ZnCl2, pH 8.0
  • 20 ml heparin elution buffer 50 mM sodium phosphate, 750 mM NaCl, 20% glycerol, 1 mM DTT, 10
  • the eluted proteins were concentrated and the buffer changed to ABE storage buffer (200 mM NaCl, 20 mM HEPES, 1 mM DTT, 40% glycerol, PH 7.5) by centrifugation through an Amicon Ultra-4 column with a 100,000 kDa cutoff (Millipore) at 6,000xg.
  • ABE storage buffer 200 mM NaCl, 20 mM HEPES, 1 mM DTT, 40% glycerol, PH 7.5
  • ABE- and Endo V-mediated in vitro digestion of amplified target site [0118] The region spanning the ABE site 1 (Hek2) was amplified using polymerase chain reaction (PCR, chr5:+87944480-87944802) with primers HEK2-F and HEK2-R.2 ⁇ g of the resulting amplicon was then incubated with 4 ⁇ g ABE 7.10 protein and 3 ⁇ g sgRNA (targeting ABE site 1) in 200 ⁇ l ABE reaction buffer [50 mM Tris-HCl (Sigma-Aldrich), 25 mM KCl (Sigma-Aldrich), 2.5 mM MgSO4 (Sigma-Aldrich), 0.1 mM Ethylenediaminetetraacetic acid (EDTA: Sigma-Aldrich), 2 mM DTT (GoldBio), 10 mM ZnCl2 (Sigma-Aldrich), 20% glycerol] at 37 °C for 1-2
  • ABE protein and sgRNA were removed by incubation with 80 ⁇ g Proteinase K and 400 ⁇ g RNase A (both from Qiagen), respectively, for 10 min.
  • the amplicons were purified using a PCR purification kit (MGmed).1 ⁇ g of the purified amplicons were incubated with 10 units of Endo V enzyme (NEB) for 1 h. Next, the mixture was incubated with 80 ⁇ g Proteinase K, and again purified with a PCR purification kit (MGmed). Finally, the DNA fragments were imaged following electrophoresis on a 2% agarose gel.
  • RNP reconstitution and electroporation were performed following the IDT Inc. instructions.
  • a total of 2x10 5 HEK293T cells were used for each electroporation with the Amaxa Nucleofector system (Lonza, Basel, Switzerland).
  • the cells were re-suspended in 100 ⁇ l of nucleofection buffer from the Cell Line NucleofectorTM Kit V (Catalog # VCA-1003, Lonza), and placed in the electroporation cuvette.
  • 1 ⁇ l of Alt-R® Cas9 Electroporation Enhancer and 5 ⁇ l of reconstituted ABE RNPs were added to the cells in the cuvette. Finally, the cells were given an electrical shock with protocol Q-001.
  • Lentiviral capsid-RNP production Lentiviral capsids packaged with ABE RNPs were produced by a three plasmid transfection procedure.
  • ABP-modified packaging plasmid pspAX2-D64V-NC-ABP can be MCP (MS2 coat protein, binding to RNA aptamer MS2) (Peabody et al., Nucleic Acids Res 1992, 20 (7): 1649-55) or Com (binding to RNA aptamer com)) (Hattmanet al., P Natl Acad Sci USA 1991, 88 (22):10027-10031), 6 ⁇ g envelope plasmid (pMD2.G), and 16 ⁇ g plasmid DNA co-expressing ABE, and the corresponding aptamer-modified sgRNA were mixed in 1 ml Opti-MEM.
  • Opti-MEM® Reduced-Serum Medium 76 ul of 1 mg/ml polyethylenimine (PEI, Polysciences Inc., Bellevue, WA) was mixed in 1 ml Opti-MEM® Reduced-Serum Medium. The DNA mixture and the PEI mixture were then mixed and incubated at room temperature for 15 mins. The DNA/PEI mixture was then added to the cells in Opti-MEM® medium.24h after transfection, the medium was changed into 15 ml Opti-MEM® medium and the ABE RNP laden virus-like particles (VLP) were collected 48h and 72h after transfection. The supernatant was spun for 10 min at 500 g to remove cell debris. The cleared supernatant can be used directly or be further concentrated as described below.
  • PEI polyethylenimine
  • Transfection can also be done in 10-cm dishes or 6-well plates with Fugene HD (Promega, Madison, WI). DNA amounts were proportionally scaled based on vessel surface area.
  • Concentrating ABE RNP-laden VLPs [0120] The supernatant containing ABE RNP-laden VLPs was concentrated with the KrosFlo® Research 2i (KR2i) Tangential Flow Filtration System (Spectrum Lab, Cat. No. SYR2-U20) using the concentration-diafiltration-concentration mode. Briefly, 150-300 ml supernatant was first concentrated to about 50 ml, diafiltrated with 500 ml to 1000 ml PBS, and finally concentrated to about 8 ml.
  • KR2i KrosFlo® Research 2i
  • SYR2-U20 Tangential Flow Filtration System
  • the hollow fiber filter modules were made from modified polyethersulfone, with a molecular weight cut-off of 500 kDa.
  • the flow rate and the pressure limit were 80 ml/min and 8 psi for the filter module D02-E500-05-N, and 10 ml/min and 5 psi for the filter module C02-E500-05-N.
  • VLP quantification Concentration of VLPs was determined by p24 (lentiviral capsid protein CA) based ELISA (Cell Biolabs, QuickTiterTM Lentivirus Titer Kit Catalog Number VPK-107, San Diego, CA). When un-concentrated samples were assayed, the VLPs were precipitated according to the manufacturer’s instructions so that the soluble p24 protein was not detected.
  • VLPs were centrifuged with a Sorvall T-890 rotor (2 h at 120,000 g) through step gradients containing a 1 ml layer of 10% sucrose in STE [100 mM NaCl, 50 mM Tris/HCl (pH 7.5), 1 mM EDTA] with or without 0.5% Triton X-100, and a cushion of 2 ml 20% sucrose in STE solution.
  • the pelleted VLP particles were directly lysed in 100 ⁇ l of 1x Laemmli sample buffer for Western blotting or for purifying RNA for RT-qPCR analysis. [0123]
  • the proteins in each sample were separated on SDS-PAGE gels and analyzed by Western blotting.
  • the antibodies used include mouse monoclonal anti-SpCas9 antibody (ThermoFisher, CRISPR-Cas9 Monoclonal Antibody 7A9-3A3, Catalog # MA1-201, 1:1000), and p24 mouse monoclonal antibody for capsid protein (Cell Biolabs, Cat No.310810, 1:1000).
  • HRP-conjugated anti-Mouse IgG (H+L) ThermoFisher Scientific, Waltham, MA, Cat No. 31430, 1:5000
  • HRP-conjugated anti-Rabbit IgG (H+L) ThermoFisher, Cat No.31460, 1:5000
  • SpCas9 RNP standards were GenCrispr NLS-Cas9-NLS Nuclease from GenScript (Piscataway, NJ, Cat # Z03389S). Chemiluminescent reagents (Pierce, Dallas, TX) were used to visualize the protein signals in the LAS-3000 system (Fujifilm, Tokyo, Japan). Densitometry (NIH ImageJ software) was used to quantify protein amounts. RNA isolation and RT-qPCR analysis [0124] A miRNeasy Mini Kit (QIAGEN, Hilden, Germany, Cat No.217004) was used to isolate RNA from concentrated capsids or cells.
  • the QuantiTect Reverse Transcription Kit (QIAGEN) was used to reverse-transcribe the RNA to cDNA.
  • QIAGEN QuantiTect Reverse Transcription Kit
  • 0.6 ⁇ l random primers provided in the kit and 0.4 ⁇ l sgRNA-specific primer (Sp- sgRNA-R1, gcaccgactcggtgccactt (SEQ ID NO: 82), 20 ⁇ M) were used for reverse transcription.
  • guide specific forward primer ABE-g5-F (Table 2) were used together with Sp-sgRNA-R1 in SybrGreen based RT-qPCR to detect sgRNA.
  • VLP transduction [0125] VLPs (in the amount of about 10-300 ng p24 protein were added to 2.5x10 4 cells grown in 24-well plates, with 8 ⁇ g/ml polybrene. Unconcentrated supernatant of VLPs was diluted with fresh medium at a 1:1 ratio to transduce cells. The cells were incubated with the VLP-containing medium for 12-24 hours, after which the medium was replaced with normal medium.
  • the densitometry data were used to determine protein half-life using the two-phase decay method of GraphPad Prism 5.0 (Graphpad, San Diego, CA).
  • Next-generation sequencing and data analysis [0127] The regions and primers used to amplify target DNA for next generation sequencing are listed in Table 4.
  • the proofreading HotStart® ReadyMix from KAPA Biosystems (Wilmington, MA) was used for PCR.
  • the amplicons were sequenced by GeneWiz’s Amplicon-EZ service. Usually 50,000 reads/amplicon were obtained.
  • RNA hotspots for detecting ABE RNA off-target activities [0129] The major goal of this study was to find an ABE delivery method with short activity duration and minimal RNA off-target activities, for which a sensitive RNA off-target detection method is useful.
  • high-depth RNA sequencing is used to detect ABE RNA off- targets (Grunewald et al., Nature 2019, 569 (7756): 433-437) which is time-consuming and expensive.
  • RNA motif CUACGAA SEQ ID NO: 75
  • was the most efficient ABE RNA off-target was the most efficient ABE RNA off-target (Grunewald et al., Nat Biotechnol 2019, 37 (9): 1041- 1048).
  • HEK293T cells were transfected with plasmid DNA expressing Cas9 nickase (negative control), or plasmid DNA expressing ABE and sgRNA targeting ABE site 1 (Gaudelli et a l., Nature 2017, 551 (7681): 464-471).444 bp of the USP38 cDNA spanning the predicted hotspot (primers F1 and R1 in FIG.2A) were amplified for targeted next-generation sequencing (NGS).
  • NGS next-generation sequencing
  • ABE RNPs delivered by electroporation showed undetectable RNA off-target activities 24 hours after delivery [0133] Once an ABE RNA off-target hotspot was confirmed, whether or not delivering ABE RNPs by electroporation showed reduced RNA off-target activity compared with DNA transfection was studied.
  • Recombinant ABE RNPs were prepared, as previouly described (Kim et al., Nat Biotechnol 2019, 37 (4), 430-435) and their activities confirmed in an in vitro assay. 20, 10, 5, 2.5, 1.25, and 0.625 ⁇ g of ABE RNPs (targeting ABE site 1) were delivered into 2x10 5 HEK293T cells by electroporation.
  • RNA off-target activities were examined at the USP38 hotspot.
  • the aptamer com was chosen since it was the most efficient aptamer in mediating SaCas9 RNP packaging into LV capsids. One copy of the aptamer was tested, since more than one copy greatly decreases RNA stability.
  • ABE-RNP was packaged into LV capsids by co-transfecting three plasmids into HEK293T cells: the envelope plasmid pMD2.G expressing the VSV-G protein, the target plasmid co-expressing ABE and various target-specific aptamer-modified sgRNAs, and the packaging plasmids modified by the corresponding ABPs (pspAX2-D64V-NC-MS2 for MS2 modified sgRNA and pspAX2-D64V-NC-com for com modified sgRNAs), as described recently.
  • the supernatants containing capsid/ABE RNPs were used to transduce HEK293T cells. Then base editing activities with qPCR, were compared. [0138] Single guide RNA sgRNA g1 and g5 were used to target ABE sites 1 and 5, respectively. These were the two sites previously shown to be successfully edited after transfecting the corresponding ABE expressing plasmid DNA (Gaudelli et al.). qPCR was used to detect the base editing activities of capsid/ABE RNPs, packaged with sgRNA containing 2xMS2, Tetra-com, and ST2-com, respectively.
  • ABE-g5 RNP treated cells as controls for ABE-g1 RNP-treated cells and vice versa
  • All three types of ABE RNPs were functional (FIG.2B, FIG.2C).
  • 2xMS2 modification showed the least base editing activity.
  • the activities of single copy-com modified sgRNAs showed similar activities at the Tetraloop and ST2 loop locations.
  • ST2-com modified RNPs performed significantly better than Tetra-com modified RNPs (P ⁇ 0.0001). ST2-com modification of sgRNA was used for further experiments.
  • the aptamer/ABP strategy was able to package and deliver functional ABE RNPs to human cells.
  • the base editing activity of the ABE RNP VLPs was examined by NGS. When targeting ABE site 1 in 2.5x104 HEK293T cells, 200 ng p24 of capsid-ABE RNPs generated A to G editing in 31.85% alleles (FIG. 3). When targeting ABE site 5 in 2.5x10 4 HEK293T cells, 108 ng p24 of capsid-ABE RNPs (non-concentrated supernatant) generated A to G editing in 87.5% of all alleles (FIG.2D).
  • ABE protein content in capsids with ABE-g5 RNP (unmodified g5 sgRNA) and ABE-g5ST2-com RNP (ST2-com modified g5 sgRNA) was compared.
  • ABE protein associated with vesicles or the particle membrane we transiently treated the particles with 0.5% TritonTM X-100 buffer. This procedure reduced capsid protein p24 by over 100% (FIG.4A).
  • ABE protein was then examined by Western blotting with an SpCas9 antibody. ABE was only detected in capsids with ABE-g5ST2-com RNPs, but not in capsids with ABE- g5 RNPs (FIG. 4A).
  • TritonTM X-100 treatment decreased ABE amounts by 30 ⁇ 50%.
  • the ABE amount in Triton-treated capsids was about 100 pg ABE/ng p24 (FIG.4B, only considering the full-length ABE with an asterisk). Assuming 1.25x10 7 capsids per ng p24, the ABE molecule numbers per capsid were estimated at 30 molecules per capsid.
  • VLPs enable transient expression of ABE RNPs in human cells [0145] To determine the expression duration of ABE RNPs in human cells, transduced ABE-g5ST2-com RNP-laden VLPs and ABE-g5 RNP-laden VLPs (each 100 ng p24/well) were transduced into HEK293T cells and ABE protein levels were measured every 12 hours.
  • ABE levels were half of those at 12 hours post-transduction (FIG.5A, 5B).
  • FIG.5A ABE was not detected in ABE-g5 RNP VLPs.
  • ABE-g5 RNP VLPs were subjected to an ultracentrifugation in a buffer without TritonTM X-100 and VLPs used to transduce cells were not centrifuged. It is likely that, the low background ABE in cells transduced with ABE-g5 RNP VLPs were the ABE in the capsid preparation.
  • ABE RNPs delivered by VLPs showed undetectable guide-independent RNA off-target activities
  • ABE site 1 was targeted by ABE RNP-laden VLPs and plasmid DNA transfection. The conditions for the two delivery methods were determined, giving similar on- target base editing efficiencies. On-target and off-target activities were examined 24 hours after treatment, since that was the time point with the highest ABE level after VLP treatment.
  • NGS was performed on ABE site 1 genomic DNA and USP38 cDNA (amplified with F3 and R1 in FIG.1A).
  • ABE site 1 DNA had a slightly higher on-target A to G base editing rate in capsid-RNP transduced cells (14.5%) than in plasmid DNA-transfected cells (9.2%, Table 6). Table 6.
  • On-target base editing and RNA off-targets at the hotspot [0148] RNA off-targets around the USP38 hotspot were analyzed.
  • RNA off-targets in this experiment could have been caused by two non-exclusive mechanisms: 1) less DNA was transfected (250 ng versus 500 ng), and 2) RNA off-target activity was detected 24 hours rather than 48 hours after transfection. Nevertheless, delivering ABE RNPs by LV capsids did not result in detectable RNA off-targets, even though the on-target DNA base editing level was 56% higher than in cells treated with DNA transfection. [0149] RNP off-target activities were examined 24 hours after VLP delivery because the ABE RNP expression duration data showed that ABE RNPs were highest 24 hours after transduction (FIG.5A).
  • RNA off-targets were also examined at 48 hours after VLP delivery and no RNA off-target activities were observed at the hotspot (FIG. 6). Since ABE protein levels decreased quickly after this time point, it is unlikely that further RNA off-target activities could be detected later. Thus, RNA off-target activity for ABE RNPs delivered by LV capsids was below the detection limit of the assay. [0150] This work attempted to find an ABE delivery method with short activity duration, high base editing efficiency, and minimal RNA off-target activity.
  • ABE RNP-laden VLPs were developed and packaged ( ⁇ 30 ABE RNP molecules into each capsid particle).
  • ABE RNP electroporation resulted in ⁇ 5% base editing efficiency at 5 pg/cell (10 ⁇ g RNPs for 2x105 cells), whereas ABE RNP VLP transduction resulted in >30% base editing efficiency at 0.8 pg/cell ( ⁇ 20 ng RNPs for 2.5x104 cells).
  • ABE RNP-laden VLPs resulted in much more efficient base editing, although much less ABE protein was used.
  • This novel, ABE RNP-laden VLP is the first ABE RNP delivery vehicle demonstrating high base editing activity and low RNA off-target activity..
  • RNA off-target activity In addition to the high capsid assembly efficiency and base editing efficiency (>80% editing efficiency with unconcentrated VLPs), no RNA off-target activities were observed 24 hours after VLP delivery. RNA off-target generation before detection cannot be ruled out. However, typically, the earliest time to observe gene editing activity after delivering VLPs is about 16 hours post-transduction. Since escaping from the endosome system is a similar process to VLPs entering recipient cells, a comparable time should be needed for ABE RNPs to become functional after delivery. RNA off-targets, if any, could have been generated 16 to 24 hours after RNP delivery. This short time window could greatly reduce the chances of generating enough erroneous proteins to be harmful to the cells.
  • VLP is an efficient ABE RNP delivery vehicle with minimal RNA off-target activity, without the need to use the ABE mutants with reduced RNA off-target activities.
  • ABEs do not show detectable guide-independent DNA off-target activities. This development greatly reduces the safety risks caused by ABE’s guide-independent RNA off-target activities, and enables efficient and safe delivery of ABE RNPs.
  • VLP- mediated ABE RNP delivery method delivers as little as 1/10 RNPs to each cell compared with current typical RNP electroporation protocols. This low amount of transiently expressed ABE RNPs delivered by VLPs should also achieve reduced guide- dependent DNA off-target activities.
  • ABE RNPs show guide-dependent DNA base editing but undetectable guide-independent RNA off-target activities. ABE RNPs can be efficiently and functionally packaged into lentiviral capsids. VLP-delivered ABE RNPs show high on-target DNA base editing activities and undetectable RNA off-target activities.
  • Exemplary Embodiments [0156] Embodiment 1.
  • a mammalian expression plasmid comprising a eukaryotic promoter operably linked to a non-viral nucleic acid sequence
  • the non-viral nucleic acid sequence comprises: (i) a nucleic acid sequence encoding an adenosine base pair editor (ABE), wherein the ABE is a fusion protein comprising an adenosine deaminase and a catalytically impaired CRISPR-associated endonuclease; and (ii) a guide RNA (gRNA) coding sequence, wherein the gRNA coding sequence comprises at least one aptamer coding sequence.
  • ABE adenosine base pair editor
  • gRNA guide RNA
  • Embodiment 3. The mammalian expression plasmid of embodiment 1 or 2, wherein the adenine base editor is ABE 7.10 or ABE8.
  • Embodiment 4. The mammalian expression plasmid of any one of embodiments 1- 3, wherein the at least one aptamer coding sequence encodes an aptamer sequence bound specifically by an ABP selected from the group consisting of MS2 coat protein, PP7 coat protein, lambda N RNA-binding domain, or Com protein.
  • Embodiment 6. The mammalian expression plasmid of any one of embodiments 1- 5, wherein the sgRNA coding sequence comprises at least one aptamer inserted into the tetraloop or the ST2 loop of the sgRNA coding sequence.
  • Embodiment 7. The mammalian expression plasmid of embodiment 6, wherein the sgRNA coding comprises at least one com aptamer inserted into the ST2 loop of the gRNA coding sequence.
  • a lentiviral packaging system comprising: a) a packaging plasmid comprising a eukaryotic promoter operably linked to a Gag nucleotide sequence, wherein the Gag nucleotide sequence comprises a nucleocapsid (NC) coding sequence and a matrix protein (MA) coding sequence, wherein one or both of the NC coding sequence or the MA coding sequence comprises at least one non-viral aptamer-binding protein (ABP) nucleotide sequence, and wherein the packaging plasmid does not encode a functional integrase protein; b) at least one mammalian expression plasmid of any one of claims 1-7; and c) an envelope plasmid comprising an envelope glycoprotein coding sequence.
  • a packaging plasmid comprising a eukaryotic promoter operably linked to a Gag nucleotide sequence, wherein the Gag nucleotide sequence comprises a nucleocapsid (NC)
  • Embodiment 9 The lentiviral packaging system of embodiment 8, wherein the packaging plasmid further comprises a Rev nucleotide sequence and a Tat nucleotide sequence.
  • Embodiment 10 The lentiviral packaging system of embodiments 8 or 9, further comprising a second packaging plasmid comprising a Rev nucleotide sequence.
  • Embodiment 11 The lentiviral packaging system of any one of embodiments 8-10, wherein the at least one non-viral ABP nucleotide sequence encodes MS2 coat protein, PP7 coat protein, lambda N peptide, or Com protein.
  • a lentivirus-like particle comprising: a) a fusion protein comprising a nucleocapsid (NC) protein or a matrix (MA) protein wherein the NC protein or MA protein comprises at least one non-viral aptamer binding protein (ABP); and b) a ribonucleotide protein (RNP) complex comprising: (i) an adenine base editor (ABE), wherein the ABE is a fusion polypeptide comprising an adenine base editor and a catalytically impaired CRISPR-associated endonuclease; and (ii) a gRNA, wherein the lentivirus-like particle does not comprise a functional integrase protein.
  • ABE adenine base editor
  • gRNA gRNA
  • Embodiment 14 The lentivirus-like particle of embodiments 12 or 13, wherein the adenine base editor is ABE 7.10 or ABE 8.
  • a method of producing a lentivirus-like particle comprising: a) transfecting a plurality of eukaryotic cells with the packaging plasmid, the at least one mammalian expression plasmid, and the envelope plasmid of the system of any one of claims 8-11; and b) culturing the transfected eukaryotic cells for sufficient time for lentivirus-like to be produced.
  • the lentivirus-like particle comprises a ribonucleotide protein (RNP) complex comprising: (i) an adenine base editor (ABE), wherein the ABE is a fusion polypeptide comprising an adenosine deaminase and a catalytically impaired CRISPR-associated endonuclease; and (ii) a guide RNA.
  • RNP ribonucleotide protein
  • ABE adenine base editor
  • the plurality of eukaryotic cells are mammalian cells.
  • Embodiment 18 A lentivirus-like particle made by the method of any one of embodiments 15-17.
  • a method of modifying a genomic target sequence in a cell comprising transducing a plurality of eukaryotic cells with a plurality of viral particles, wherein the plurality of viral particles comprise a lentivirus-like particle according embodiment 12, wherein the RNP binds to the genomic target sequence in genomic DNA of the cell and the ABE deaminates an adenine at the genomic target sequence, thereby modifying the genomic target sequence.
  • Embodiment 20 The method of embodiment 19, wherein the plurality of eukaryotic cells are mammalian cells.
  • Embodiment 21 The method of any one of embodiments 19 or 20, wherein the plurality of eukaryotic cells are cells present in subject.
  • Embodiment 22 The method of embodiment 21, wherein the subject is a human subject.
  • Embodiment 23 The method of embodiment 22, wherein the subject is injected with the plurality of viral particles.
  • Embodiment 24 A cell containing the plasmid of any one of embodiments 1-7.
  • Embodiment 25 A cell containing the lentiviral packaging system of any one of embodiments 8-11.
  • Embodiment 26 A cell containing the lentivirus-like particle of any one of embodiments 12-14.
  • Embodiment 27 A cell modified using the method of any one of embodiments 19- 23.
  • Embodiment 28 The method of any one of embodiments 19- 23.
  • a method for treating a disease in a subject comprising: a) obtaining cells from the subject; b) modifying the cells of the subject using the method of any one of embodiments 19-23; and c) administering the modified cells to the subject.
  • Embodiment 29 The method of embodiment 28, wherein the disease is cancer.
  • Embodiment 30 The method of embodiment 29, wherein the disease is sickle cell anemia.
  • Embodiment 31 The method of any one of embodiments 28-30, wherein the cells are T cells. Sequences

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US12351814B2 (en) 2019-06-13 2025-07-08 The General Hospital Corporation Engineered human-endogenous virus-like particles and methods of use thereof for delivery to cells
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