EP4780975A1 - Transcriptase inverse associée aux chauves-souris et ses procédés d'utilisation - Google Patents

Transcriptase inverse associée aux chauves-souris et ses procédés d'utilisation

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Publication number
EP4780975A1
EP4780975A1 EP24869062.0A EP24869062A EP4780975A1 EP 4780975 A1 EP4780975 A1 EP 4780975A1 EP 24869062 A EP24869062 A EP 24869062A EP 4780975 A1 EP4780975 A1 EP 4780975A1
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European Patent Office
Prior art keywords
enzyme
rna
bart
cell
virus
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EP24869062.0A
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German (de)
English (en)
Inventor
Thomas ZWAKA
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Icahn School of Medicine at Mount Sinai
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Icahn School of Medicine at Mount Sinai
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Publication of EP4780975A1 publication Critical patent/EP4780975A1/fr
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/102—Mutagenizing nucleic acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • 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
    • C12N15/1135—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 against oncogenes or tumor suppressor genes
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C12N15/90—Stable introduction of foreign DNA into chromosome
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0696—Artificially induced pluripotent stem cells, e.g. iPS
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C12N9/1241—Nucleotidyltransferases (2.7.7)
    • C12N9/1276—RNA-directed DNA polymerase (2.7.7.49), i.e. reverse transcriptase or telomerase
    • C—CHEMISTRY; METALLURGY
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/22—Ribonucleases [RNase]; Deoxyribonucleases [DNase]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/80—Fusion polypeptide containing a DNA binding domain, e.g. Lacl or Tet-repressor
    • C07K2319/81—Fusion polypeptide containing a DNA binding domain, e.g. Lacl or Tet-repressor containing a Zn-finger domain for DNA binding

Definitions

  • This disclosure relates generally to a bat-associated reverse transcriptase and methods of use thereof.
  • the broader context of this disclosure lies in the ongoing evolutionary arms race between organisms and viruses. This constant battle has driven the development of diverse and sophisticated antiviral defense mechanisms across all domains of life.
  • the CRISPR-Cas system an adaptive immune mechanism employed by prokaryotes, has emerged as a technological tool in the field of gene editing. Its capacity to precisely target and modify specific DNA sequences has revolutionized biomedical research, enabling scientists to manipulate genes with unprecedented accuracy and efficiency.
  • the advent of CRISPR-Cas has not only accelerated the understanding of gene function but has also paved the way for innovative gene therapies, offering potential solutions for a wide range of genetic disorders.
  • this disclosure addresses the need mentioned above in a number of aspects.
  • this disclosure provides a method of modifying a target polynucleotide.
  • the method comprises delivering to the target polynucleotide an enzyme that possesses a reverse transcriptase activity and an endonuclease activity, and one or more nucleic acid components, wherein the one or more nucleic acid components target and hybridize with the target polynucleotide and direct binding of the enzyme to the target polynucleotide, and wherein the enzyme modifies the target polynucleotide.
  • this disclosure provides a method of modifying expression of a target polynucleotide.
  • the method comprises: introducing into a cell or a subject an enzyme that possesses a reverse transcriptase activity and an endonuclease activity or a nucleic acid molecule encoding the enzyme, and one or more nucleic acid components, wherein the one or more nucleic acid components target and hybridize with the target polynucleotide and direct binding of the enzyme to the target polynucleotide, and wherein the enzyme binds to one or more locations on the target polynucleotide such that binding of the enzyme increases or decreases expression level of the target polynucleotide.
  • the enzyme further possesses an integrase activity.
  • the one or more nucleic acid components comprise a single-stranded navigator DNA.
  • the one or more nucleic acid components further comprise a payload RNA.
  • the payload RNA comprises a stem-loop structure.
  • the enzyme reverse transcribes the payload RNA into a cDNA. In some embodiments, the enzyme integrates the cDNA into the target polynucleotide.
  • the enzyme comprises a N-terminal apurinic-apyrimidinic endonuclease domain optionally linked to a reverse transcriptase domain.
  • the enzyme further comprises a C-terminal domain.
  • the C-terminal domain facilitates interaction between the enzyme and the target polynucleotide.
  • the C-terminal domain comprises a zinc finger.
  • the zinc finger comprises a CCHC motif.
  • the enzyme comprises a Bat-Associated Reverse Transcriptase (BART).
  • BART comprises a BART of Rhinolophus femimequimim, Myotis myotis Meles meles, Bos mutus, Capra hircus.
  • Homo sapiens, Ca is lupus familiaris, Sus scrofa, Callithrix jacchus, Mus musculus, Ursus arctos, Elephas maximus indicus, or a variant thereof.
  • the enzyme is provided through one or more polynucleotide molecules encoding the enzyme.
  • the one or more nucleic acid components are provided through one or more polynucleotide molecules encoding or comprising the one or more nucleic acid components.
  • the one or more polynucleotide molecules comprise one or more vectors.
  • the enzyme and the one or more nucleic acid components are provided in a single vector.
  • the target polynucleotide comprises a genomic locus. In some embodiments, the target polynucleotide comprises RNA or DNA.
  • the RNA comprises a viral RNA of a RNA virus.
  • the RNA virus is selected from the group consisting of Norwalk, Rotavirus, Poliovirus, Ebola virus, Marburg virus, Lassa virus, Hantavirus, Rabies, Influenza, Yellow fever virus, Coronavirus, SARS, SARS-CoV-2, West Nile virus, Hepatitis A, C (HCV) and E virus, Dengue fever virus, togaviruses, Rhabdoviruses, Picomaviruses, Myxoviruses, retroviruses, bunyaviruses, coronaviruses, and reoviruses.
  • the DNA comprises a genomic DNA or a cDNA.
  • the modification of the target polynucleotide comprises cleavage of the target polynucleotide.
  • the target polynucleotide is contained in a nucleic acid molecule within a cell or in vitro.
  • the cell comprises a eukaryotic cell.
  • the eukaryotic cell comprises a mammalian cell.
  • the eukaryotic cell comprises a non-human animal cell, a human cell, or a plant cell.
  • this disclosure provides a method of treating or preventing a viral infection of a RNA virus in a cell or a subject.
  • the method comprises delivering to the cell or the subject an enzyme that possesses a reverse transcriptase activity and an endonuclease activity, or a nucleic acid molecule encoding the enzyme, wherein a singlestranded guide polynucleotide hybridizes with a viral RNA of the RNA virus and directs binding of the enzyme to the viral RNA, and wherein the enzyme cleaves the viral RNA.
  • the method comprises reverse transcribing the viral RNA into a cDNA by the enzyme.
  • the enzyme has an integrase activity
  • the method comprises integrating the cDNA to a genome of the cell or the subject by the enzyme.
  • the method comprises transcribing the cDNA into the singlestranded guide polynucleotide capable of hybridizing with the viral RNA.
  • this disclosure provides a method of enhancing immunity against a viral infection of a RNA virus in a cell or a subject.
  • the method comprises delivering to the cell or the subject an enzyme that possesses a reverse transcriptase activity, an endonuclease activity, and an integrase activity, or a nucleic acid molecule encoding the enzyme, wherein the enzyme reverse-transcribes a viral RNA of the RNA virus into a single-stranded DNA (ssDNA) to generate a cDNA of the viral RNA, wherein the enzyme integrates the cDNA into a host genome of the cell or the subject, wherein the cDNA integrated into the host genome is transcribed into a mRNA, wherein the enzyme reverse-transcribes the mRNA into a second ssDNA, wherein the second ssDNA hybridizes with the viral RNA and directs binding of the enzyme to the viral RNA, and wherein the enzyme cleaves the viral RNA.
  • ssDNA single-strand
  • the method comprises delivering to the cell or the subject an enzyme that possesses a reverse transcriptase activity, an endonuclease activity, and an integrase activity, or a nucleic acid molecule encoding the enzyme, wherein the enzyme reverse-transcribes a viral RNA of the RNA virus into a single-stranded DNA (ssDNA) to generate a cDNA of the viral RNA, wherein the enzyme integrates the cDNA into a host genome of the cell or the subject, wherein the cDNA integrated into the host genome is transcribed into a mRNA, and wherein the mRNA with the viral RNA to form a RNA hybrid to silence the viral RNA.
  • an enzyme that possesses a reverse transcriptase activity, an endonuclease activity, and an integrase activity, or a nucleic acid molecule encoding the enzyme, wherein the enzyme reverse-transcribes a viral RNA of the RNA virus into a single-stranded DNA (
  • this disclosure provides a method of generating a cell line with immunity against a viral infection of a RNA virus.
  • the method comprises delivering to a cell an enzyme that possesses a reverse transcriptase activity, an endonuclease activity, and an integrase activity, or a nucleic acid molecule encoding the enzyme, wherein the enzyme reverse- transcribes a viral RNA of the RNA virus into a single-stranded DNA (ssDNA) to generate a cDNA of the viral RNA, wherein the enzyme integrates the cDNA into a host genome of the cell or the subject, wherein the cDNA integrated into the host genome is transcribed into a mRNA, wherein the enzyme reverse-transcribes the mRNA into a second ssDNA, wherein the second ssDNA hybridizes with the viral RNA and directs binding of the enzyme to the viral RNA, and wherein the enzyme cleaves the viral RNA.
  • ssDNA single-stranded DNA
  • the method comprises delivering to a cell an enzyme that possesses a reverse transcriptase activity, an endonuclease activity, and an integrase activity, or a nucleic acid molecule encoding the enzyme, wherein the enzyme reverse- transcribes a viral RNA of the RNA virus into a single-stranded DNA (ssDNA) to generate a cDNA of the viral RNA, wherein the enzyme integrates the cDNA into a host genome of the cell or the subject, wherein the cDNA integrated into the host genome is transcribed into a mRNA, and wherein the mRNA with the viral RNA to form a RNA hybrid to silence the viral RNA.
  • ssDNA single-stranded DNA
  • the RNA virus is selected from the group consisting of Norwalk, Rotavirus, Poliovirus, Ebola virus, Marburg virus, Lassa virus, Hantavirus, Rabies, Influenza, Yellow fever virus, Coronavirus, SARS, SARS-CoV-2, West Nile virus, Hepatitis A, C (HCV) and E virus, Dengue fever virus, togaviruses, Rhabdoviruses, Pi cornavi ruses, Myxoviruses, retroviruses, bunyaviruses, coronaviruses, and reoviruses.
  • the enzyme comprises a N-terminal apurinic-apyrimidinic endonuclease domain optionally linked to a reverse transcriptase domain.
  • the enzyme further comprises a C-terminal domain.
  • the C-terminal domain facilitates interaction between the enzyme and the target polynucleotide.
  • the C-terminal domain comprises a zinc finger.
  • the zinc finger comprises a CCHC motif.
  • the enzyme comprises a Bat-Associated Reverse Transcriptase (BART).
  • BART comprises a BART of Rhinolophus ferrumequinum, Myotis myotis Meles meles, Bos mutus, Capra hircus, Homo sapiens, Canis lupus familiaris, Sus scrofa, Callithrix jacchus, Mus musculus, Ursus arctos, Elephas maximus indicus, or a variant thereof.
  • the enzyme is provided through one or more polynucleotide molecules encoding the enzyme.
  • this disclosure provides a gene editing system for modifying a target polynucleotide.
  • the method comprises an enzyme that possesses a reverse transcriptase activity and an endonuclease activity, and one or more nucleic acid components, wherein the one or more nucleic acid components target and hybridize with the target polynucleotide and direct binding of the enzyme to the target polynucleotide, and wherein the enzyme modifies the target polynucleotide.
  • the enzyme further possesses an integrase activity.
  • the one or more nucleic acid components comprise a single-stranded navigator DNA.
  • the one or more nucleic acid components further comprise a payload RNA.
  • the payload RNA comprises a stem-loop structure.
  • the enzyme reverse transcribes the payload RNA into a cDNA.
  • the enzyme integrates the cDNA into the target polynucleotide.
  • the enzyme comprises a N-terminal apurinic-apyrimidinic endonuclease domain optionally linked to a reverse transcriptase domain.
  • the enzyme further comprises a C-terminal domain.
  • the C-terminal domain facilitates interaction between the enzyme and the target polynucleotide.
  • the C-terminal domain comprises a zinc finger.
  • the zinc finger comprises a CCHC motif.
  • the enzyme comprises a Bat-Associated Reverse Transcriptase (BART).
  • BART comprises a BART of Rhinolophus ferrumequiirum, Myotis myotis, Meles meles. Bos mutus, Capra hircus, Homo sapiens, Canis lupus familiaris, Sus scrofa, Callithrix jacchus, Mus musculus, Ursus arctos, Elephas maximus indicus, or a variant thereof.
  • the gene editing system comprises one or more polynucleotide molecules encoding the enzyme. In some embodiments, the gene editing system comprises one or more polynucleotide molecules encoding or comprising the one or more nucleic acid components. In some embodiments, the one or more polynucleotide molecules comprise one or more vectors. In some embodiments, the enzyme and the one or more nucleic acid components are provided in a single vector.
  • the target polynucleotide comprises RNA or DNA.
  • the RNA comprises a viral RNA of a RNA virus.
  • the RNA virus is selected from the group consisting of Norwalk, Rotavirus, Poliovirus, Ebola virus, Marburg virus, Lassa virus, Hantavirus, Rabies, Influenza, Yellow fever virus, Coronavirus, SARS, SARS-CoV-2, West Nile virus, Hepatitis A, C (HCV) and E virus, Dengue fever virus, togaviruses, Rhabdoviruses, Picomaviruses, Myxoviruses, retroviruses, bunyaviruses, coronaviruses, and reoviruses.
  • the DNA comprises a genomic DNA or a cDNA.
  • the modification of the target polynucleotide comprises cleavage of the target polynucleotide.
  • this disclosure provides a delivery system comprising the gene editing system, and the delivery system is adapted to deliver the gene editing system into a cell or a subject.
  • the delivery system comprises nanoparticles or vesicles encapsulating the gene editing system.
  • this disclosure provides a vector system comprising one or more vectors, wherein the one or more vectors comprise one or more polynucleotide molecules encoding an enzyme that possesses a reverse transcriptase activity, an endonuclease activity, and an integrase activity, and comprise one or more nucleic acid components, wherein the one or more nucleic acid components target and hybridize with a target polynucleotide and direct binding of the enzyme to the target polynucleotide, and wherein the enzyme modifies the target polynucleotide.
  • this disclosure provides a kit comprising the gene editing system, the delivery system, or the vector system, as described herein.
  • this disclosure provides a cell line comprising one or more polynucleotide molecules encoding an enzyme that possesses a reverse transcriptase activity, an endonuclease activity, and encoding an integrase activity, and one or more nucleic acid components, wherein the one or more nucleic acid components target and hybridize with a target polynucleotide and direct binding of the enzyme to the target polynucleotide, and wherein the enzyme modifies the target polynucleotide.
  • the cell line comprises a eukaryotic cell.
  • the eukaryotic cell comprises a mammalian cell.
  • the eukaryotic cell comprises a stem cell or stem cell line.
  • the method comprises introducing to a cell one or more polynucleotide molecules encoding an enzyme that possesses a reverse transcriptase activity, an endonuclease activity, and an integrase activity, and encoding one or more nucleic acid components.
  • Figure 1 shows a process BART utilizes to integrate a RNA payload through a navigator DNA.
  • Figures 2A and 2B show BART-mediated immunity against VSV infection. This figure shows the results of VSV infection in BART -expressing cell lines.
  • Figure 2A shows the reduction in viral load in cells expressing BART compared to control cells.
  • Figure 2B shows the presence of viral cDNA in the cytoplasm of BART-expressing cells, highlighting BART’s role in reverse transcription and viral suppression.
  • Figure 3 shows a structural model of BART highlighting the APE domain, the reverse transcriptase domain, and the C-terminal domain (CTD).
  • Figure 4 shows vector maps of bart cloning constructs. Depiction of the plasmid vector maps used for cloning the BART enzyme coding sequences, including the placement of the CMV promoter, neomycin resistance gene, and restriction sites for Hindlll and Notl.
  • This disclosure is based, in part, on an unexpected discovery of a novel mammalian defense mechanism akin to the CRISPR-Cas system traditionally associated with prokaryotic biology.
  • the Bat-Associated Reverse Transcriptase (BART), as disclosed herein, possesses a unique feature found in bat-induced pluripotent stem cells (iPSCs) with the ability to convert viral genomic RNA into DNA.
  • iPSCs bat-induced pluripotent stem cells
  • the disclosed BART system represents a next-generation tool for genome and RNA editing, exhibiting high efficiency and specificity, potentially outperforming current CRISPR-Cas systems. This discovery blurs the lines between prokaryotic and eukaryotic defense mechanisms, opening new opportunities for comprehensive viral defense strategies and advanced gene therapy applications.
  • BART exhibits the remarkable ability to convert viral RNA into DNA, a process typically associated with retroviruses. Furthermore, it possesses endonuclease activity, enabling it to cleave nucleic acids, and integrase activity, facilitating the insertion of genetic material into the host genome.
  • endonuclease activity enabling it to cleave nucleic acids, and integrase activity, facilitating the insertion of genetic material into the host genome.
  • a function of BART is to serve as an innovative antiviral defense mechanism in mammalian cells. It achieves this by targeting and degrading viral RNA, thus disrupting the viral life cycle and preventing further infection.
  • the unique aspect of BART lies in its ability to create a “genomic memory” of past viral encounters. By converting viral RNA into DNA and integrating it into the host genome, BART establishes a lasting record of the infection. This genomic archive allows for the rapid production of RNA transcripts that guide BART to recognize and neutralize the same virus upon subsequent infections, providing a form of adaptive immunity in mammalian cells.
  • BART sets it apart from other known antiviral and gene-editing systems.
  • BART utilizes its reverse transcriptase activity to convert the RNA into complementary DNA (cDNA).
  • cDNA complementary DNA
  • This cDNA is then integrated into the host genome at specific locations, creating a library of viral sequences akin to the CRISPR arrays found in bacteria.
  • the integrated cDNA is subsequently transcribed, generating RNA transcripts that guide BART to target and cleave homologous viral RNA, thereby disrupting the viral life cycle and providing long-term immunity against future infections.
  • This remarkable process not only confers antiviral defense but also offers a powerful tool for precise gene editing, as the integration and targeting mechanisms can be harnessed to introduce specific modifications into the host genome.
  • BART Beyond its role in antiviral defense, BART’s unique mechanism of action presents methods for precise and efficient gene editing.
  • BART exhibits superior specificity and reduced off-target effects, addressing a major limitation of existing gene-editing technologies.
  • This enhanced precision coupled with its potential for broader applicability across diverse cell types and therapeutic contexts, makes BART a system for next-generation gene editing tools.
  • the ability to harness this naturally evolved mammalian system for targeted genome manipulation will revolutionize the field of gene therapy, offering new avenues for treating genetic disorders and other diseases.
  • BART represents a paradigm shift in the understanding of antiviral immunity and gene editing.
  • BART challenges the traditional boundaries between these two domains of life. This indicates a convergent evolution of adaptive immune mechanisms, highlighting the universality of strategies employed by organisms to combat viral threats.
  • BART s dual functionality as an antiviral agent and a precise gene-editing tool provides new avenues for innovative therapies against viral infections and genetic disorders. This naturally evolved mammalian system for targeted genome manipulation will revolutionize the field of gene therapy, offering a more compatible and efficient alternative to current CRISPR-Cas-based approaches.
  • the discovery of BART thus represents a significant leap forward in addressing some of the most pressing health challenges, paving the way for a new era of antiviral and gene-editing therapeutics.
  • This disclosure encompasses methods and uses of the BART system described herein for modifying a target DNA sequence (e.g., a chromosomal sequence) or target RNA sequence, e.g., for altering or manipulating the expression of one or more genes or the one or more gene products, in prokaryotic or eukaryotic cells, in vitro in vivo, or ex vivo.
  • the BART protein has unique characteristics in possessing both reverse transcriptase and endonuclease activities.
  • the BART protein can additionally have an integrase activity, allowing it to integrate a DNA (e.g., reverse transcribed from a viral RNA) into a host genome.
  • the disclosed BART systems provide an effective means for modifying (e.g., deleting, inserting, translocating, inactivating, activating) a target RNA or DNA (double-stranded, linear, or super-coiled) in a multiplicity of cell types.
  • modifying e.g., deleting, inserting, translocating, inactivating, activating
  • a target RNA or DNA double-stranded, linear, or super-coiled
  • this disclosure provides a method of modifying a target polynucleotide.
  • the method comprises delivering to the target polynucleotide an enzyme (e.g., BART) that possesses a reverse transcriptase activity and an endonuclease activity, and one or more nucleic acid components, wherein the one or more nucleic acid components target and hybridize with the target polynucleotide and direct binding of the enzyme to the target polynucleotide, and wherein the enzyme modifies the target polynucleotide.
  • an enzyme e.g., BART
  • the enzyme modifies the target polynucleotide.
  • the enzyme further possesses an integrase activity.
  • the enzyme comprises a BART protein.
  • the one or more nucleic acid components comprise a single-stranded navigator DNA (“navigator ssDNA”).
  • the one or more nucleic acid components further comprise a payload RNA.
  • the payload RNA comprises a stem-loop structure.
  • the enzyme reverse transcribes the payload RNA into a cDNA. In some embodiments, the enzyme integrates the cDNA into the target polynucleotide.
  • the disclosure provides a method of modifying a target polynucleotide (e.g., target sequence of interest), such as modifying expression of a target polynucleotide, in a cell.
  • a target polynucleotide e.g., target sequence of interest
  • the method allows a BART complex (e.g., BART/navigator ssDNA complex) to bind to the target polynucleotide, resulting in increased or decreased expression of the target polynucleotide or a gene comprising the target polynucleotide.
  • the BART complex comprises BART complexed with a ssDNA sequence hybridized to a target sequence within the polynucleotide.
  • the method of modifying a target polynucleotide comprises delivering the BART system, isolated nucleic acids encoding a BART protein and/or a navigator ssDNA, or particles containing a BART protein and/or a navigator ssDNA, to a target sequence or a cell containing the target sequence.
  • the BART protein following formation of a complex of BART/navigator ssDNA and hybridization of the ssDNA to one or more nucleic acids of the target sequence, the BART protein induces a modification (e.g., cleavage) of the target sequence.
  • the modification comprises cleaving one or two strands at the location of the target sequence by the enzyme. In some embodiments, the modification results in decreased or increased transcription of a target gene. In some embodiments, the method further comprises repairing the cleaved target polynucleotide by homologous recombination with an exogenous template polynucleotide, wherein the repair results in a mutation comprising an insertion, deletion, or substitution of one or more nucleotides of the target polynucleotide. In some embodiments, the mutation results in one or more amino acid changes in a protein expressed from a gene comprising the target sequence.
  • the modification of the target polynucleotide comprises cleavage of the target polynucleotide.
  • modification of a target polynucleotide may include modifying (e.g., increasing or decreasing) expression of a target polynucleotide as a result of binding of the BART protein to a certain location on the target polynucleotide. For example, if the BART protein binds to a location between a promoter or other regulatory element and a coding sequence in the target polynucleotide, the BART protein may serve as a repressor that decreases expression of the target polynucleotide. On the other hand, if the BART protein binds to a location upstream of a promoter or other regulatory element, the BART protein may serve as an activator that increases expression of the target polynucleotide.
  • this disclosure also provides a method of modifying expression of a target polynucleotide.
  • the method comprises introducing into a cell or a subject an enzyme (e. ., BART) that possesses a reverse transcriptase activity and an endonuclease activity or a nucleic acid molecule encoding the enzyme, and one or more nucleic acid components (e.g., navigator ssDNA), wherein the one or more nucleic acid components target and hybridize with the target polynucleotide and direct binding of the enzyme to the target polynucleotide, and wherein the enzyme binds to one or more locations on the target polynucleotide such that binding of the enzyme increases or decreases the expression level of the target polynucleotide.
  • an enzyme e. ., BART
  • one or more nucleic acid components e.g., navigator ssDNA
  • the enzyme may include one or more mutations that result in a catalytically inactive enzyme.
  • the catalytically inactive enzyme lacks an endonuclease activity, such that the enzyme can still bind to the target polynucleotide but does not cleave the target polynucleotide.
  • a nucleotide sequence encoding the BART protein is present in a recombinant expression vector.
  • the recombinant expression vector is a viral construct, e. ., a recombinant adeno-associated virus construct, a recombinant adenoviral construct, a recombinant lentiviral construct, etc.
  • viral vectors can be based on vaccinia virus, poliovirus, adenovirus, adeno-associated virus, SV40, herpes simplex virus, human immunodeficiency virus, and the like.
  • a retroviral vector can be based on Murine Leukemia Virus, spleen necrosis virus, and vectors derived from retroviruses such as Rous Sarcoma Virus, Harvey Sarcoma Virus, avian leukosis virus, a lentivirus, human immunodeficiency virus, myeloproliferative sarcoma virus, mammary tumor virus, and the like.
  • Useful expression vectors are known to those of skill in the art, and many are commercially available.
  • vectors are provided by way of example for eukaryotic host cells: pXTl, pSG5, pSVK3, pBPV, pMSG, and pSVLSV40.
  • any other vector may be used if it is compatible with the host cell.
  • useful expression vectors containing a nucleotide sequence encoding a Cas9 enzyme are commercially available from, e.g., Addgene, Life Technologies, Sigma- Aldrich, and Origene.
  • any of a number of transcription and translation control elements including promoter, transcription enhancers, transcription terminators, and the like, may be used in the expression vector.
  • Useful promoters can be derived from viruses, or any organism, e.g., prokaryotic or eukaryotic organisms.
  • Suitable promoters include, but are not limited to, the SV40 early promoter, mouse mammary tumor virus long terminal repeat (LTR) promoter; adenovirus major late promoter (Ad MLP); a herpes simplex virus (HSV) promoter, a cytomegalovirus (CMV) promoter such as the CMV immediate early promoter region (CMVIE), a rous sarcoma virus (RSV) promoter, a human U6 small nuclear promoter (U6), an enhanced U6 promoter, a human Hl promoter (Hl), etc.
  • LTR mouse mammary tumor virus long terminal repeat
  • Ad MLP adenovirus major late promoter
  • HSV herpes simplex virus
  • CMV cytomegalovirus
  • CMVIE CMV immediate early promoter region
  • RSV rous sarcoma virus
  • U6 small nuclear promoter U6 small nuclear promoter
  • Hl human Hl promoter
  • the enzyme is provided through one or more polynucleotide molecules encoding the enzyme.
  • one or more nucleic acid components are provided through one or more polynucleotide molecules encoding or comprising one or more nucleic acid components.
  • one or more polynucleotide molecules comprise one or more vectors.
  • the enzyme and one or more nucleic acid components are provided in a single vector.
  • a polynucleotide comprising a first navigator ssDNA is located on the same vector with a polynucleotide encoding the BART protein.
  • the BART system further comprises a polynucleotide containing a second navigator ssDNA.
  • the polynucleotide encoding the BART protein, the polynucleotide comprising the first navigator DNA, and the polynucleotide comprising the second navigator ssDNA are harbored on the same vector or two or more different vectors.
  • the BART protein or variants/fragments thereof can be introduced into a cell (e.g., an in vitro cell such as a primary cell for ex vivo therapy, or an in vivo cell such as in a patient) as a BART protein or a variant or fragment thereof, an mRNA encoding a BART protein or a variant or fragment thereof, or a recombinant expression vector comprising a nucleotide sequence encoding a BART protein or a variant or fragment thereof.
  • a cell e.g., an in vitro cell such as a primary cell for ex vivo therapy, or an in vivo cell such as in a patient
  • a cell e.g., an in vitro cell such as a primary cell for ex vivo therapy, or an in vivo cell such as in a patient
  • a cell e.g., an in vitro cell such as a primary cell for ex vivo therapy, or an in vivo cell such as in a patient
  • the method further comprises delivering one or more vectors to a host cell.
  • the vectors are delivered to the host cell in a subject.
  • the modification takes place in the eukaryotic cell in cell culture.
  • the method further comprises isolating the eukaryotic cell from a subject prior to the modification.
  • the method further comprises returning the cells derived therefrom to the subject.
  • the method further comprises maintaining cells or embryos under appropriate conditions such that the ssDNA guides the BART protein to the targeted site in the target sequence to modify the target sequence.
  • the cell can be maintained under conditions appropriate for cell growth and/or maintenance. Suitable cell culture conditions are well known in the art and are described, for example, in Current Protocols in Molecular Biology” Ausubel et al., John Wiley & Sons, New York, 2003 or “Molecular Cloning: A Laboratory Manual” Sambrook & Russell, Cold Spring Harbor Press, Cold Spring Harbor, N.Y., 3rd edition, 2001), Santiago et al. (2008) PNAS 105:5809-5814; Moehle et rz/. (2007) PNAS 104:3055-3060; Urnov et al.
  • An embryo can be cultured in vitro (e.g., in cell culture). Typically, the embryo is cultured at an appropriate temperature and in appropriate media with the necessary O2/CO2 ratio to allow the expression of the proteins and RNA scaffold, if necessary. Suitable non-limiting examples of media include M2, M16, KSOM, BMOC, and HTF media.
  • a cell line may be derived from an in iv/r -cultured embryo (e.g., an embryonic stem cell line).
  • an embryo may be cultured in vivo by transferring the embryo into uterus of a female host.
  • the female host is from the same or similar species as the embryo.
  • the female host is pseudo-pregnant.
  • Methods of preparing pseudo-pregnant female hosts are known in the art.
  • methods of transferring an embryo into a female host are known. Culturing an embryo in vivo permits the embryo to develop and can result in a live birth of an animal derived from the embryo. Such an animal would comprise the modified chromosomal sequence in every cell of the body.
  • the BART protein may include a variant or fragment of a BART.
  • the BART comprises a BART of Rhinolophus ferrumequinum, Myotis myotis, Meles meles, Bos mutus, Capra hircus, Homo sapiens, Canis lupus familiaris, Sus scrofa, Callithrix jacchus, Mus musculus, Ursus arctos, Elephas maximus indicus, or a variant thereof.
  • the enzyme comprises a N-terminal apurinic-apyrimidinic endonuclease (APE) domain optionally linked to a reverse transcriptase domain.
  • APE N-terminal apurinic-apyrimidinic endonuclease
  • Apurinic- apyrimidinic endonuclease (APE) domains are protein motifs that play a crucial role in DNA repair. These domains are responsible for recognizing and cleaving DNA strands that contain apurinic or apyrimidinic sites, which are lesions that occur when a purine or pyrimidine base is lost from the DNA backbone.
  • the enzyme further comprises a C-terminal domain.
  • the C-terminal domain facilitates interaction between the enzyme and the target polynucleotide.
  • the C-terminal domain comprises azinc finger.
  • Zinc fingers are small protein structural motifs characterized by the coordination of one or more zinc ions (Zn 2+ ). These metal ions help stabilize the protein’s structure, allowing it to interact with other molecules.
  • the zinc finger comprises a CCHC motif.
  • the CCHC motif is a zinc finger motif found in many proteins, particularly those involved in DNA binding and transcriptional regulation. It consists of a conserved sequence of amino acids that contains cysteine and histidine residues. These residues coordinate zinc ions, forming a stable structure that helps the protein bind to DNA.
  • the BART protein comprises an amino acid sequence of any one of SEQ ID NOs: 1-12 or comprises an amino acid sequence having at least 75% (e.g., 75%, 80%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to any one of SEQ ID NOs: 1-12.
  • protein polypeptide
  • polypeptide polymers of amino acids of any length.
  • the polymer may be linear or branched, it may comprise modified amino acids, and it may be interrupted by non-amino acids.
  • the terms also encompass an amino acid polymer that has been modified, for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, pegylation, or any other manipulation, such as conjugation with a labeling component.
  • amino acid includes natural and/or unnatural or synthetic amino acids, including glycine and both the D or L optical isomers, and amino acid analogs and peptidomimetics.
  • a peptide or polypeptide “fragment” as used herein refers to a less than full-length peptide, polypeptide or protein.
  • a peptide or polypeptide fragment can have at least about 3, at least about 4, at least about 5, at least about 10, at least about 20, at least about 30, at least about 40 amino acids in length, or single unit lengths thereof.
  • a fragment may be 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or more amino acids in length.
  • peptide fragments can be less than about 500 amino acids, less than about 400 amino acids, less than about 300 amino acids or less than about 250 amino acids in length.
  • variant refers to a first composition (e.g., a first molecule) that is related to a second composition (e.g., a second molecule, also termed a “parent” molecule).
  • the variant molecule can be derived from, isolated from, based on or homologous to the parent molecule.
  • variant can be used to describe either polynucleotides or polypeptides.
  • a variant molecule can have an entire nucleotide sequence identity with the original parent molecule, or alternatively, can have less than 100% nucleotide sequence identity with the parent molecule.
  • a variant of a gene nucleotide sequence can be a second nucleotide sequence that is at least 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99% or more identical in nucleotide sequence compared to the original nucleotide sequence.
  • Polynucleotide variants also include polynucleotides comprising the entire parent polynucleotide, and further comprising additional fused nucleotide sequences.
  • a variant polypeptide can have an entire amino acid sequence identity with the original parent polypeptide, or alternatively, can have less than 100% amino acid identity with the parent protein.
  • a variant of an amino acid sequence can be a second amino acid sequence that is at least 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99% or more identical in amino acid sequence compared to the original amino acid sequence.
  • Polypeptide variants include polypeptides comprising the entire parent polypeptide, and further comprising additional fused amino acid sequences. Polypeptide variants also include polypeptides that are portions or subsequences of the parent polypeptide, for example, unique subsequences (e.g., as determined by standard sequence comparison and alignment techniques) of the polypeptides disclosed herein are also encompassed by the invention.
  • polypeptide variants include polypeptides that contain minor, trivial, or inconsequential changes to the parent amino acid sequence.
  • minor, trivial, or inconsequential changes include amino acid changes (including substitutions, deletions, and insertions) that have little or no impact on the biological activity of the polypeptide, and yield functionally identical polypeptides, including additions of a non-functional peptide sequence.
  • the variant polypeptides of the invention change the biological activity of the parent molecule.
  • One of skill will appreciate that many variants of the disclosed polypeptides are encompassed by the invention.
  • polynucleotide or polypeptide variants can include variant molecules that alter, add or delete a small percentage of the nucleotide or amino acid positions, for example, typically less than about 10%, less than about 5%, less than 4%, less than 2% or less than 1%.
  • a “functional variant” of a protein as used herein refers to a variant of such protein that retains at least partially the activity of that protein.
  • Functional variants may include mutants (which may be insertion, deletion, or replacement mutants), including polymorphs, etc. Also included within functional variants are fusion products of such protein with another, usually unrelated, nucleic acid, protein, polypeptide or peptide. Functional variants may be naturally occurring or may be man-made.
  • a variant of a BART protein may include one or more conservative modifications.
  • the BART protein variant with one or more conservative modifications may retain the desired functional properties, which can be tested using the functional assays known in the art.
  • conservative sequence modifications refers to amino acid modifications that do not significantly affect or alter the binding characteristics of the protein containing the amino acid sequence. Such conservative modifications include amino acid substitutions, additions, and deletions. Modifications can be introduced by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions are ones in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include: amino acids with basic side chains (e.
  • acidic side chains e.g., aspartic acid, glutamic acid
  • uncharged polar side chains e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan
  • nonpolar side chains e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine
  • beta-branched side chains e.g., threonine, valine, isoleucine
  • aromatic side chains e.g., tyrosine, phenylalanine, tryptophan, histidine
  • the BART protein with one or more conservative modifications may retain the desired functional properties, which can be tested using the functional assays known in the art.
  • the percent homology between two amino acid sequences is equivalent to the percent identity between the two sequences.
  • the comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm, as described in the non-limiting examples below.
  • the percent identity between two amino acid sequences can be determined using the algorithm of E. Meyers and W. Miller (Comput. Appl. Biosci., 4: 11-17 (1988)) which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4.
  • the percent identity between two amino acid sequences can be determined using the Needleman and Wunsch (J. Mol. Biol.
  • the protein sequences of the present invention can further be used as a “query sequence” to perform a search against public databases to, for example, identify related sequences.
  • Such searches can be performed using the XBLAST program (version 2.0) of Altschul, et al. (1990) J. Mol. Biol. 215:403-10.
  • Gapped BLAST can be utilized as described in Altschul et al. (1997) Nucleic Acids Res. 25(17):3389- 3402.
  • the default parameters of the respective programs e.g., XBLAST and NBLAST
  • the default parameters of the respective programs e.g., XBLAST and NBLAST
  • a variant of a BART protein can be conjugated or linked to a detectable tag or a detectable marker (e.g., a radionuclide, a fluorescent dye, or an MRI-detectable label).
  • the detectable tag can be an affinity tag.
  • affinity tag as used herein, relates to a moiety attached to a polypeptide, which allows the polypeptide to be purified from a biochemical mixture.
  • Affinity tags can consist of amino acid sequences or can include amino acid sequences to which chemical groups are attached by post-translational modifications.
  • affinity tags include His-tag, CBP-tag (CBP: calmodulin- binding protein), CYD-tag (CYD: covalent yet dissociable NorpD peptide), Strep-tag, StrepII-tag, FLAG-tag, HPC-tag (HPC: heavy chain of protein C), GST-tag (GST: glutathione S transferase), Avi-tag, biotinylated tag, Myc-tag, a myc-myc-hexahistidine (mmh) tag 3xFLAG tag, a SUMO tag, and MBP-tag (MBP: maltose-binding protein). Further examples of affinity tags can be found in Kimple etal., Curr Protoc Protein Sci. 2013 Sep 24; 73: Unit 9.9.
  • the detectable tag can be conjugated or linked to the N- and/or C- terminus of a variant of a BART protein.
  • the detectable tag and the affinity tag may also be separated by one or more amino acids.
  • the detectable tag can be conjugated or linked to the variant via a cleavable element.
  • cleavable element relates to peptide sequences that are susceptible to cleavage by chemical agents or enzyme means, such as proteases. Proteases may be sequence-specific (e.g., thrombin) or may have limited sequence specificity (e.g., trypsin).
  • Cleavable elements I and II may also be included in the amino acid sequence of a detection tag or polypeptide, particularly where the last amino acid of the detection tag or polypeptide is K or R.
  • conjugate refers to the attachment of two or more entities to form one entity.
  • a conjugate encompasses both peptide-small molecule conjugates as well as peptide-protein/peptide conjugates.
  • fusion polypeptide or “fusion protein” means a protein created by joining two or more polypeptide sequences together.
  • the fusion polypeptides encompassed in this invention include translation products of a chimeric gene construct that joins the nucleic acid sequences encoding a first polypeptide with the nucleic acid sequence encoding a second polypeptide to form a single open reading frame.
  • a “fusion polypeptide” or “fusion protein” is a recombinant protein of two or more proteins that are joined by a peptide bond or via several peptides.
  • the fusion protein may also comprise a peptide linker between the two domains.
  • linker refers to any means, entity, or moiety used to join two or more entities.
  • a linker can be a covalent linker or a non-covalent linker.
  • covalent linkers include covalent bonds or a linker moiety covalently attached to one or more of the proteins or domains to be linked.
  • the linker can also be a non-covalent bond, e.g., an organometallic bond through a metal center such as a platinum atom.
  • various functionalities can be used, such as amide groups, including carbonic acid derivatives, ethers, esters, including organic and inorganic esters, amino, urethane, urea, and the like.
  • Linker moi eties include, but are not limited to, chemical linker moi eties, or for example, a peptide linker moiety (a linker sequence).
  • the linker can be a peptide linker and a non-peptide linker.
  • the peptide linker may include [Ser(Gly)n]m or [Ser(Gly)n]mSer, where n may be an integer between 1 and 20.
  • the term “non-peptide linker” refers to a biocompatible polymer composed of two or more repeating units linked to each other, in which the repeating units are linked to each other by any non-peptide covalent bond. This non-peptidyl linker may have two ends or three ends.
  • non-peptidyl linker may include, without limitation, polyethylene glycol, polypropylene glycol, a copolymer of ethylene glycol with propylene glycol, polyoxyethylated polyol, polyvinyl alcohol, polysaccharide, dextran, polyvinyl ethyl ether, biodegradable polymers such as polylactic acid (PLA) and polylactic-glycolic acid (PLGA), lipid polymers, chitins, hyaluronic acid, and combinations thereof. Aptamers could be added as non- peptide linkers.
  • a variant of a BART protein can be fused to a fusion partner through crosslinking with a crosslinking agent, e.g, crosslinker.
  • Crosslinkers are reagents having reactive ends to specific functional groups (e.g., primary amines or sulfhydryls) on proteins or other molecules. Crosslinkers are capable of joining two or more molecules by a covalent bond.
  • Crosslinkers include but are not limited to amine-to-amine crosslinkers (e.g., disuccinimidyl suberate(DSS)), amine-to-sulfhydryl crosslinkers (e.g., N-y-maleimidobutyryl-oxysuccinimide ester (GMBS)), carb oxy Lto-amine crosslinkers (e.g, dicyclo-hexylcarbodiimide (DCC)), sulfhydryl-to-carbohydrate crosslinkers (e.g., N-P-maleimidopropionic acid hydrazide (BMPH)), sulfhydryl-to-sulfhydryl crosslinkers (e.g., 1,4-bismaleimidobutane (BMB)), photoreactive crosslinkers (e.g, N-5-azido-2-nitrobenzoyloxysuccinimide (ANB-NOS)), chemoselective ligation crosslinkers
  • a polynucleotide encoding a BART protein may be codon optimized.
  • codon optimization refers to a process of modifying a nucleic acid sequence to enhance expression in the host cells by substituting at least one codon of the native sequence with codons that are more frequently or most frequently used in the genes of that host cell while maintaining the native amino acid sequence.
  • Various species exhibit particular bias for certain codons of a particular amino acid. Codon bias (differences in codon usage between organisms) often correlates with the efficiency of translation of messenger RNA (mRNA), which is, in turn, believed to be dependent on, among other things, the properties of the codons being translated and the availability of particular transfer RNA (tRNA) molecules.
  • mRNA messenger RNA
  • Codon usage tables are readily available, for example, at the “Codon Usage Database” available at www.kazusa.orjp/codon/, and these tables can be adapted in a number of ways. See Nakamura, Y., et al. “Codon usage tabulated from the international DNA sequence databases: status for the year 2000” Nucl. Acids Res. 28:292 (2000).
  • codon optimizing a particular sequence for expression in a particular host cell are also available, such as Gene Forge (Aptagen; Jacobus, Pa.).
  • one or more codons e.g., 1, 2, 3, 4, 5, 10, 15, 20, 25, 50, or more, or all codons
  • codon usage in yeast reference is made to the online Yeast Genome database available at http://www.yeastgenome.org/community/codonusage.shtml, or Codon selection in yeast, Bennetzen and Hall, J Biol Chem. 1982 Mar. 25; 257(6):3026-31.
  • codon usage in plants including algae
  • the term “navigator ssDNA” generally refers to a ssDNA molecule (or a group of DNA molecules collectively) that can bind to a BART protein and target the BART protein to a specific location within a target RNA or DNA.
  • the targeting segment comprises a nucleotide sequence that is complementary to (or at least can hybridize to under stringent conditions) a target sequence.
  • a navigator ssDNA can be any polynucleotide sequence having sufficient complementarity with a target polynucleotide sequence (e.g., target DNA or RNA sequence) to hybridize with the target sequence and direct sequence-specific binding of a BART complex to the target sequence.
  • the degree of complementarity between a guide sequence of the navigator ssDNA and its corresponding target sequence, when optimally aligned using a suitable alignment algorithm is about or more than about 50%, 60%, 75%, 80%, 85%, 90%, 95%, 97.5%, 99%, or more.
  • Optimal alignment may be determined with the use of any suitable algorithm for aligning sequences, non-limiting examples of which include the Smith-Waterman algorithm, the Needleman-Wunsch algorithm, algorithms based on the Burrows-Wheeler Transform (e.g., the Burrows- Wheel er Aligner), ClustalW, Clustal X, BLAT, Novoalign (Novocraft Technologies, ELAND (Illumina, San Diego, Calif), SOAP (available at soap.genomics.org.cn), and Maq (available at maq.sourceforge.net).
  • any suitable algorithm for aligning sequences include the Smith-Waterman algorithm, the Needleman-Wunsch algorithm, algorithms based on the Burrows-Wheeler Transform (e.g., the Burrows- Wheel er Aligner), ClustalW, Clustal X, BLAT, Novoalign (Novocraft Technologies, ELAND (Illumina, San Diego, Calif), SOAP (available at soap.genomics.org.
  • a navigator ssDNA sequence is about 3, 5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195,
  • a guide sequence is about 50 nucleotides in length. In some embodiments, a guide sequence is about 100 nucleotides in length. In some embodiments, a guide sequence is about 150 nucleotides in length. In some embodiments, a guide sequence is about 200 nucleotides in length. In some embodiments, a guide sequence is about 250 nucleotides in length. In some embodiments, a guide sequence is about 300 nucleotides in length.
  • the ability of a guide sequence to direct sequence-specific binding of a BART complex to a target sequence may be assessed by any suitable assay.
  • the components of a BART system sufficient to form a BART complex, including the guide sequence to be tested may be provided to a host cell having the corresponding target sequence, such as by transfection with vectors encoding the components of the BART sequence, followed by an assessment of preferential cleavage within the target sequence.
  • cleavage of a target polynucleotide sequence may be evaluated in a test tube by providing the target sequence, components of a BART complex, including the guide sequence to be tested and a control guide sequence different from the test guide sequence, and comparing binding or rate of cleavage at the target sequence between the test and control guide sequence reactions.
  • the navigator ssDNA comprises a polynucleotide sequence of SEQ ID NOs: 13-96 and 107-112 or comprises a polynucleotide sequence having at least 75% (e.g., 75%, 80%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to any one of SEQ ID NOs: 13-96 and 107-112.
  • 75% e.g., 75%, 80%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more
  • the navigator ssDNA comprises a synthetic nucleic acid sequence (e.g., synthetic DNA molecule). In some embodiments, the navigator ssDNA comprises one or more modifications.
  • modified nucleotide in the context of an oligonucleotide or polynucleotide includes but is not limited to (a) end modifications, e.g., 5’ end modifications or 3’ end modifications, (b) nucleobase (or “base”) modifications, including replacement or removal of bases, (c) sugar modifications, including modifications at the 2’, 3’, and/or 4’ positions, and (d) backbone modifications, including modification or replacement of the phosphodiester linkages.
  • modified nucleotide generally refers to a nucleotide having a modification to the chemical structure of one or more of the base, the sugar, and the phosphodiester linkage or backbone portions, including nucleotide phosphates.
  • Z and P refer to the nucleotides, nucleobases, or nucleobase analogs are described, for example, in Yang, Z., et al., Nucleic Acids Res., 34, 6095-101 (2006), the disclosure of which is hereby incorporated by reference in its entirety.
  • one or more modifications may include 2’-O-methyl moiety, a Z base, a 2’-deoxynucleotide, a phosphorothioate internucleotide linkage, a phosphonoacetate (PACE) internucleotide linkage, a thiophosphonoacetate (thioPACE) internucleotide linkage, or combinations thereof.
  • PACE phosphonoacetate
  • thioPACE thiophosphonoacetate
  • one or more modifications comprise one or more modifications selected from the group consisting of a 2’-O-methyl nucleotide with a 3’- phosphorothioate group, a 2’-O-methyl nucleotide with a 3 ’-phosphonoacetate group, a 2’-O- methyl nucleotide with a 3’-thiophosphonoacetate group, or a 2’-deoxynucleotide with a 3 1 - phosphonoacetate group.
  • the one or modifications comprises a 2-thiouracil (2-thioU), a 4-thiouracil (4-thioU), a 2-aminoadenine, a 2’-o-methyl, a 2’-fluoro, a 5- methyluridine, a 5-methylcytidine, or a locked nucleic acid modification (LNA).
  • 2-thiouracil (2-thioU) a 4-thiouracil (4-thioU)
  • 2-aminoadenine a 2’-o-methyl
  • a 2’-fluoro a 5- methyluridine
  • LNA locked nucleic acid modification
  • target polynucleotide or “target sequence” refers to a sequence to which a guide sequence is designed to have complementarity, where hybridization between a target sequence and a guide sequence promotes the formation of a BART complex.
  • a target sequence may comprise RNA or DNA polynucleotides.
  • a “target nucleic acid strand” refers to a strand of a target nucleic acid that is subject to base-pairing with a navigator ssDNA as disclosed herein.
  • each strand can be a “target nucleic acid strand” to design navigator ssDNA and used to practice the disclosed method.
  • target RNA refers to an RNA polynucleotide being or comprising the target sequence.
  • the target RNA may be an RNA polynucleotide or a part of an RNA polynucleotide to which a part of the gRNA, /. ⁇ ., the guide sequence, is designed to have complementarity and to which the effector function mediated by the complex comprising BART protein and a gRNA is to be directed.
  • a target sequence is located in the nucleus or cytoplasm of a cell.
  • target DNA refers to a DNA polynucleotide being or comprising the target sequence.
  • the target DNA may be a DNA polynucleotide or a part of a DNA polynucleotide to which a part of the navigator ssDNA, i.e., the guide sequence, is designed to have complementarity and to which the effector function mediated by the complex comprising BART protein and a navigator ssDNA is to be directed.
  • a target sequence is located in the nucleus or cytoplasm of a cell.
  • the target polynucleotide has no sequence limitation and can be in the coding region of a gene, in an intron of a gene, in a control region between genes, etc.
  • the target polynucleotide is contained in a nucleic acid molecule within a cell or in vitro.
  • the gene can be coding or non-coding.
  • the target polynucleotide can be any polynucleotide endogenous or exogenous to the cell.
  • the target polynucleotide can be a polynucleotide residing in the nucleus of the eukaryotic cell.
  • the target polynucleotide can be a sequence coding a gene product (e.g., a protein) or a non-coding sequence (e.g, a regulatory polynucleotide).
  • the target polynucleotide comprises RNA or DNA.
  • the RNA comprises a viral RNA of a RNA virus.
  • the target polynucleotide comprises a cDNA reverse-transcribed from a viral RNA of a RNA virus.
  • the RNA virus is selected from the group consisting of Norwalk, Rotavirus, Poliovirus, Ebola virus, Marburg virus, Lassa virus, Hantavirus, Rabies, Influenza, Yellow fever virus, Coronavirus, SARS, SARS-CoV-2, West Nile virus, Hepatitis A, C (HCV) and E virus, Dengue fever virus, togaviruses, Rhabdoviruses, Picornaviruses, Myxoviruses, retroviruses, bunyaviruses, coronaviruses, and reoviruses.
  • the DNA comprises a genomic DNA or a cDNA.
  • the target polynucleotide comprises a polynucleotide sequence of SEQ ID NOs: 105, 106, and 113-118 or comprises a polynucleotide sequence having at least 75% (e.g., 75%, 80%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to any one of SEQ ID NOs: 105, 106, and 1 13-118.
  • 75% e.g., 75%, 80%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more
  • the BART protein forms a complex with a navigator ssDNA that binds to the target polynucleotide.
  • the BART-navigator ssDNA complex comprises a BART protein having the amino acid sequence of SEQ ID NO: 1 and a navigator ssDNA comprising a polynucleotide sequence having at least 75% (e.g., 75%, 80%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to any one of SEQ ID NOs: 13-96 and 107-112.
  • the BART-navigator ssDNA complex comprises a BART protein having the amino acid sequence having at least 75% (e.g., 75%, 80%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to any one of SEQ ID NOs: 1 -12 and a navigator ssDNA comprising a polynucleotide sequence of SEQ ID NOs: 13-96 and 107-112.
  • the BART -navi gator ssDNA complex comprises a BART protein having the amino acid sequence of SEQ ID NO: 1 and a navigator ssDNA having a polynucleotide sequence of SEQ ID NOs: 13-96 and 107-112.
  • the BART protein forms a complex with a navigator ssDNA operably linked to a payload RNA.
  • the payload RNA comprises a polynucleotide sequence of SEQ ID NOs: 119-133 or comprises a polynucleotide sequence having at least
  • the above-described BART system, one or more polynucleotides, or vector or delivery systems can be used in a therapeutic method of treatment.
  • the therapeutic method of treatment may comprise gene or genome editing, or gene therapy.
  • this disclosure provides a method of treating a subject in need thereof, comprising inducing gene editing by introducing into a cell of the subject the polynucleotide or any of the vectors as herein described.
  • the method comprises inducing transcriptional activation or repression by introducing into a cell of the subject the polynucleotide or any of the vectors as herein described.
  • this disclosure further provides a method of treating a disease of a subject caused by a genetic defect in a target sequence.
  • the method comprises administering the system or the composition described above to a cell containing the target sequence in a subject in need thereof and thereby inducing a modification in the target sequence.
  • the target sequence is located at genomic loci of interest.
  • the target sequence is part of a gene, and the modification in the target sequence modulates the expression level of the gene.
  • the modification in the target sequence reduces the expression level of the gene.
  • the method comprises reverse transcribing the viral RNA into a cDNA by the enzyme.
  • the enzyme has an integrase activity
  • the method comprises integrating the cDNA to a genome of the cell or the subject by the enzyme.
  • the method comprises transcribing the cDNA into the singlestranded guide polynucleotide capable of hybridizing with the viral RNA.
  • this disclosure provides a method of enhancing immunity against a viral infection of a RNA virus in a cell or a subject.
  • the method comprises delivering to the cell or the subject an enzyme that possesses a reverse transcriptase activity, an endonuclease activity, and an integrase activity, or a nucleic acid molecule encoding the enzyme, wherein the enzyme reverse-transcribes a viral RNA of the RNA virus into a single-stranded DNA (ssDNA) to generate a cDNA of the viral RNA, wherein the enzyme integrates the cDNA into a host genome of the cell or the subject, wherein the cDNA integrated into the host genome is transcribed into a mRNA, wherein the enzyme reverse-transcribes the mRNA into a second ssDNA, wherein the second ssDNA hybridizes with the viral RNA and directs binding of the enzyme to the viral RNA, and wherein the enzyme cleaves the viral RNA.
  • ssDNA single-strand
  • treating or “treatment” of a disease in a subject refers to (1) preventing the symptoms or disease from occurring in a subject that is predisposed or does not yet display symptoms of the disease; (2) inhibiting the disease or arresting its development; or (3) ameliorating or causing regression of the disease or the symptoms of the disease.
  • treatment is an approach for obtaining beneficial or desired results, including clinical results.
  • beneficial or desired results can include one or more, but are not limited to, alleviation or amelioration of one or more symptoms, diminishment of extent of a condition (including a disease), stabilized (i.e., not worsening) state of a condition (including disease), delay or slowing of condition (including disease), progression, amelioration or palliation of the condition (including disease), states and remission (whether partial or total), whether detectable or undetectable.
  • treatment excludes prevention.
  • subject refers to a vertebrate, such as a mammal, e.g. , a human.
  • Mammals include, but are not limited to, murines, simians, humans, farm animals, sport animals, and pets. Tissues, cells, and their progeny of a biological entity obtained in vivo or cultured in vitro are also encompassed.
  • the disease-causing mutations in patients are either acquired through inheritance from their parents or are caused by environmental factors. These diseases include, but are not limited to, the following categories.
  • Some genetic disorders are caused by germline mutations.
  • cystic fibrosis which is caused by mutations at the CFTR gene inherited from parents.
  • a second suppressor mutation in the mutant CFTR can partially restore the function of CFTR protein in somatic tissues.
  • Other example genetic diseases caused by a point genetic mutation that can be corrected by the disclosed technology include Gaucher’s disease, alpha trypsin deficiency disease, and sickle cell anemia, to name a few.
  • some diseases such as chronic viral infectious diseases, are caused by exogenous environmental factors that result in genetic alterations.
  • AIDS which is caused by insertion of the human HIV viral genome into the genome of infected T-cells.
  • some neurodegenerative diseases involve genetic alterations.
  • One example is Huntington’s disease, which is caused by expansion of CAG tri -nucleotide in the huntingtin gene of affected patients.
  • cancers are caused by various somatic mutations accumulated in cancer cells. Therefore, correcting the disease-causing genetic mutations, or functionally correcting the sequence, provides an appealing therapeutic opportunity to treat these diseases.
  • Somatic genetic editing is an appealing strategy for many human diseases.
  • the BART system can correct the mutated genes in genetic disorders, inactivate the viral genome in the infected cells, eliminate the expression of the disease-causing protein in neurodegenerative diseases, or silence the oncogenic protein in cancers.
  • the system and method disclosed in this disclosure can be used in correcting underlying genetic alterations in diseases, including the above-mentioned genetic disorders, chronic infectious diseases, neurodegenerative diseases, and cancer.
  • cystic fibrosis affects one out of every 3,000 people in the US. It is caused by inheritance of a mutated CFTR gene and 70% of the patients have the same mutation, deletion of a tri-nucleotide leading to a deletion of phenylalanine at position 508, which leads to the mislocation and degradation of CFTR.
  • the system and method disclosed in this invention can be used to convert a Vai 509 residue (GTT) to Phe 509 (TTT) in affected tissues (lung), thereby functionally correcting the the Phe 508 mutation.
  • a second suppressor mutation such as R553Q, R553M, or V510D
  • a second suppressor mutation such as R553Q, R553M, or V510D
  • the system and method as disclosed can also be used to specifically inactivate any gene in a viral genome that is incorporated into human cells/tissues.
  • the system and method disclosed in this invention allow one to create a stop codon for early termination of translation of the essential viral genes, and thereby remediate or cure the chronic debilitating infectious diseases.
  • current AIDS therapies can reduce viral load, but cannot totally eliminate dormant HIV from positive T cells.
  • the system and method disclosed herein can be used to permanently inactivate one or two essential HIV gene expression in the integrated HIV genome in human T- cells by introducing one or two stop codons.
  • Another example is the hepatitis B virus (HBV).
  • HBV hepatitis B virus
  • the system and method disclosed here can be used to specifically inactivate one or two essential HBV genes, which are incorporated into the human genome, and silence HBV life cycle.
  • SOD1G93A leads to development of amyotrophic lateral sclerosis (ALS).
  • ALS amyotrophic lateral sclerosis
  • the system and method disclosed in this invention can be used to either correct the mutation or eliminate the mutant protein expression by introducing a stop codon or by changing a splicing site.
  • Dystrophin is a cytoplasmic protein that provides structural stability to the dystroglycan complex of the cell membrane that is responsible for regulating muscle cell integrity and function.
  • the dystrophin gene or “DMD gene” as used interchangeably herein is 2.2 megabases at locus Xp21.
  • the primary transcription measures about 2,400 kb with the mature mRNA being about 14 kb.
  • the target is preferably one or more of the FAS, BID, CTLA4, PDCD1, CBLB, PTPN6, TRAC, or TRBC genes.
  • Cancer may be one or more of lymphoma, chronic lymphocytic leukemia (CLL), B cell acute lymphocytic leukemia (B-ALL), acute lymphoblastic leukemia, acute myeloid leukemia, non-Hodgkin’s lymphoma (NHL), diffuse large cell lymphoma (DLCL), multiple myeloma, renal cell carcinoma (RCC), neuroblastoma, colorectal cancer, breast cancer, ovarian cancer, melanoma, sarcoma, prostate cancer, lung cancer, esophageal cancer, hepatocellular carcinoma, pancreatic cancer, astrocytoma, mesothelioma, head and neck cancer, and medulloblastoma.
  • CLL chronic lymphocytic leukemia
  • B-ALL B cell acute lymphocytic leukemia
  • NDL diffuse large cell lymphoma
  • RRCC renal cell carcinoma
  • neuroblastoma colorectal cancer
  • breast cancer
  • CAR engineered chimeric antigen receptor
  • stem cell or progenitor cell can be genetically modified using the system and method disclosed in this invention.
  • Suitable cells include, e.g., stem cells (adult stem cells, embryonic stem cells, iPS cells, etc.) and progenitor cells (e.g., cardiac progenitor cells, neural progenitor cells, etc.).
  • Suitable cells include mammalian stem cells and progenitor cells, including, e.g., rodent stem cells, rodent progenitor cells, human stem cells, human progenitor cells, etc.
  • Suitable host cells include in vitro host cells, e.g., isolated host cells.
  • the BART system can be used for targeted and precise genetic modification of tissue ex vivo, correcting the underlying genetic defects. After the ex vivo correction, the tissues may be returned to the patients.
  • the technology can be broadly used in cell-based therapies for correcting genetic diseases.
  • transgenic non-human animal or plant having one or more genetic modifications of interest.
  • the transgenic non-human animal is homozygous for the genetic modification. In some embodiments, the transgenic non-human animal is heterozygous for the genetic modification.
  • the transgenic non-human animal is a vertebrate, for example, a fish (e.g., zebrafish, goldfish, pufferfish, cavefish, etc ), an amphibian (frog, salamander, etc.), a bird (e.g., chicken, turkey, etc.), a reptile (e.g., snake, lizard, etc.), a mammal (e.g., an ungulate, e.g., a pig, a cow, a goat, a sheep, etc.; a lagomorph (e.g., a rabbit); a rodent (e.g., a rat, a mouse); or a non-human primate.
  • a fish e.g., zebrafish, goldfish, pufferfish, cavefish, etc
  • an amphibian frog, salamander, etc.
  • a bird e.g., chicken, turkey, etc.
  • a reptile e.g., snake,
  • the system and method can be used for treating diseases in animals in a way similar to those for treating diseases in humans as described above. Alternatively, it can be used to generate knock-in animal disease models bearing specific genetic mutation(s) for purposes of research, drug discovery, and target validation.
  • the system and method described above can also be used for introduction of point mutations to ES cells or embryos of various organisms, for the purpose of breeding and improving animal stocks and crop quality.
  • Suitable methods include viral infection (such as double- stranded DNA viruses), transfection, conjugation, protoplast fusion, electroporation, particle gun technology, calcium phosphate precipitation, direct microinjection, silicon carbide whiskers technology, Agrobacterium-mediated transformation and the like.
  • viral infection such as double- stranded DNA viruses
  • transfection conjugation
  • protoplast fusion electroporation
  • particle gun technology particle gun technology
  • calcium phosphate precipitation direct microinjection
  • silicon carbide whiskers technology Agrobacterium-mediated transformation and the like.
  • the choice of method is generally dependent on the type of cell being transformed and the circumstances under which the transformation is taking place (i.e., in vitro, ex vivo, or in vivo).
  • this disclosure provides a method of treating or preventing a viral infection of a RNA virus in a cell or a subject.
  • the method comprises delivering to the cell or the subject an enzyme that possesses a reverse transcriptase activity and an endonuclease activity, or a nucleic acid molecule encoding the enzyme, wherein the enzyme reverse-transcribes a viral RNA of the RNA virus into a ssDNA, wherein the ssDNA hybridizes with the viral RNA and directs binding of the enzyme to the viral RNA, and wherein the enzyme cleaves the viral RNA.
  • the RNA virus is selected from Norwalk, Rotavirus, Poliovirus, Ebola virus, Marburg virus, Lassa virus, Hantavirus, Rabies, Influenza, Yellow fever virus, Coronavirus, SARS, SARS-CoV-2, West Nile virus, Hepatitis A, C (HCV) and E virus, Dengue fever virus, togaviruses, Rhabdoviruses, Picornaviruses, Myxoviruses, retroviruses, bunyaviruses, coronaviruses, and reoviruses.
  • this disclosure provides a method of enhancing immunity against a viral infection of a RNA virus in a cell or a subject.
  • the method comprises delivering to the cell or the subject an enzyme that possesses a reverse transcriptase activity, an endonuclease activity, and an integrase activity, or a nucleic acid molecule encoding the enzyme, wherein the enzyme reverse-transcribes a viral RNA of the RNA virus into a ssDNA to generate a cDNA of the viral RNA, wherein the enzyme integrates the cDNA into a host genome of the cell or the subject, wherein the cDNA integrated into the host genome is transcribed into a mRNA, wherein the enzyme reverse-transcribes the mRNA into a second ssDNA, wherein the second ssDNA hybridizes with the viral RNA and directs binding of the enzyme to the viral RNA, and wherein the enzyme cleaves the viral RNA.
  • the method comprises delivering to the cell or the subject an enzyme that possesses a reverse transcriptase activity, an endonuclease activity, and an integrase activity, or a nucleic acid molecule encoding the enzyme, wherein the enzyme reverse-transcribes a viral RNA of the RNA virus into a single-stranded DNA (ssDNA) to generate a cDNA of the viral RNA, wherein the enzyme integrates the cDNA into a host genome of the cell or the subject, wherein the cDNA integrated into the host genome is transcribed into a mRNA, and wherein the mRNA with the viral RNA to form a RNA hybrid to silence the viral RNA.
  • an enzyme reverse-transcribes a viral RNA of the RNA virus into a single-stranded DNA (ssDNA) to generate a cDNA of the viral RNA
  • ssDNA single-stranded DNA
  • the enzyme integrates the cDNA into a host genome of the cell or the subject,
  • the RNA virus is selected from Norwalk, Rotavirus, Poliovirus, Ebola virus, Marburg virus, Lassa virus, Hantavirus, Rabies, Influenza, Yellow fever virus, Coronavirus, SARS, SARS-CoV-2, West Nile virus, Hepatitis A, C (HCV) and E virus, Dengue fever virus, togaviruses, Rhabdoviruses, Picornaviruses, Myxoviruses, retroviruses, bunyaviruses, coronaviruses, and reoviruses.
  • this disclosure also provides a method of generating a cell line with immunity against a viral infection of a RNA virus.
  • the method comprises delivering to a cell an enzyme that possesses a reverse transcriptase activity, an endonuclease activity, and an integrase activity, or a nucleic acid molecule encoding the enzyme, wherein the enzyme reverse-transcribes a viral RNA of the RNA virus into a ssDNA to generate a cDNA of the viral RNA, wherein the enzyme integrates the cDNA into a host genome of the cell or the subject, wherein the cDNA integrated into the host genome is transcribed into a mRNA, wherein the enzyme reverse-transcribes the mRNA into a second ssDNA, wherein the second ssDNA hybridizes with the viral RNA and directs binding of the enzyme to the viral RNA, and wherein the enzyme cleaves the viral RNA.
  • the method comprises delivering to a cell an enzyme that possesses a reverse transcriptase activity, an endonuclease activity, and an integrase activity, or a nucleic acid molecule encoding the enzyme, wherein the enzyme reverse-transcribes a viral RNA of the RNA virus into a single-stranded DNA (ssDNA) to generate a cDNA of the viral RNA, wherein the enzyme integrates the cDNA into a host genome of the cell or the subject, wherein the cDNA integrated into the host genome is transcribed into a mRNA, and wherein the mRNA with the viral RNA to form a RNA hybrid to silence the viral RNA.
  • ssDNA single-stranded DNA
  • the RNA virus is selected from Norwalk, Rotavirus, Poliovirus, Ebola virus, Marburg virus, Lassa virus, Hantavirus, Rabies, Influenza, Yellow fever virus, Coronavirus, SARS, SARS-CoV-2, West Nile virus, Hepatitis A, C (HCV) and E virus, Dengue fever virus, togaviruses, Rhabdoviruses, Picornaviruses, Myxoviruses, retroviruses, bunyaviruses, coronaviruses, and reoviruses.
  • the BART systems described herein can be delivered to the host cell via one or more vectors, such as viral vectors.
  • the one or more viral vectors may comprise an adenovirus, a lentivirus, adeno-associated virus, or RNA-based viral vectors which may be replication competent or may only encode genes for self-amplification, the later constructs will herein be referred to as replicons.
  • vector refers to a nucleic acid molecule capable of transporting another nucleic acid linked thereto.
  • Vectors include, but are not limited to, nucleic acid molecules that are singlestranded, double-stranded, or partially double-stranded; nucleic acid molecules that comprise one or more free ends, no free ends (e.g., circular); nucleic acid molecules that comprise DNA, RNA, or both; and other varieties of polynucleotides known in the art.
  • plasmid refers to a circular double-stranded DNA loop into which additional DNA segments can be inserted, such as by standard molecular cloning techniques.
  • viral vector Another type of vector is a viral vector, wherein virally derived DNA or RNA sequences are present in the vector for packaging into a virus (e.g., retroviruses, replication defective retroviruses, adenoviruses, replication-defective adenoviruses, adeno-associated viruses, and/ or RNA-based replicons).
  • Viral vectors also include polynucleotides carried by a virus for transfection into a host cell.
  • Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., RNA vectors comprising their own RNA-dependent RNA polymerase, bacterial vectors having a bacterial origin of replication, and episomal mammalian vectors).
  • vectors e.g., non-episomal mammalian vectors
  • Other vectors are integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome.
  • certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as “expression vectors.”
  • Vectors for and that result in expression in a eukaryotic cell can be referred to herein as “eukaryotic expression vectors.”
  • Common expression vectors of utility in recombinant DNA techniques are often in the form of plasmids.
  • Recombinant expression vectors can comprise a nucleic acid of the invention in a form suitable for expression of the nucleic acid in a host cell, which means that the recombinant expression vectors include one or more regulatory elements, which may be selected on the basis of the host cells to be used for expression, that is operatively linked to the nucleic acid sequence to be expressed.
  • “operably linked” is intended to mean that the nucleotide sequence of interest is linked to the regulatory element(s) in a manner that allows for expression of the nucleotide sequence (e.g., in an in vitro transcript! on/translati on system or in a host cell when the vector is introduced into the host cell).
  • regulatory element is intended to include promoters, enhancers, internal ribosomal entry sites (IRES), and other expression control elements (e.g., transcription termination signals, such as polyadenylation signals and poly-U sequences as well as RNA elements required for recognition by self-encoded RNA dependent RNA polymerases).
  • IRES internal ribosomal entry sites
  • regulatory elements e.g., transcription termination signals, such as polyadenylation signals and poly-U sequences as well as RNA elements required for recognition by self-encoded RNA dependent RNA polymerases.
  • Such regulatory elements are described, for example, in Goeddel, GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990).
  • Regulatory elements include those that direct constitutive expression of a nucleotide sequence in many types of host cell and those that direct expression of the nucleotide sequence only in certain host cells (e.g, tissuespecific regulatory sequences).
  • tissue-specific promoter may direct expression primarily in a desired tissue of interest, such as muscle, neuron, bone, skin, blood, specific organs (e.g., liver, pancreas), or particular cell types (e.g, lymphocytes).
  • Regulatory elements may also direct expression in a temporal-dependent manner, such as in a cell-cycle dependent or developmental stage-dependent manner, which may or may not also be tissue or cell-type specific.
  • a vector comprises one or more pol III promoter (e.g., 1, 2, 3, 4, 5, or more pol III promoters), one or more pol II promoters (e.g., 1, 2, 3, 4, 5, or more pol II promoters), one or more pol I promoters (e.g., 1, 2, 3, 4, 5, or more pol I promoters), or combinations thereof.
  • pol III promoters include, but are not limited to, U6 and Hl promoters.
  • pol II promoters include, but are not limited to, the retroviral Rous sarcoma virus (RSV) LTR promoter (optionally with the RSV enhancer), the cytomegalovirus (CMV) promoter (optionally with the CMV enhancer) [see, e.g., Boshart et al., Cell, 41 :521-530 (1985)], the SV40 promoter, the dihydrofolate reductase promoter, the P-actin promoter, the phosphoglycerol kinase (PGK) promoter, and the EFla promoter.
  • RSV Rous sarcoma virus
  • CMV cytomegalovirus
  • PGK phosphoglycerol kinase
  • enhancer elements such as WPRE; CMV enhancers; the R-U5’ segment in LTR of HTLV-I (Mol. Cell. Biol., Vol. 8(1), p. 466-472, 1988); SV40 enhancer; and the intron sequence between exons 2 and 3 of rabbit P-globin (Proc. Natl. Acad. Sci. USA., Vol. 78(3), p. 1527-31, 1981).
  • WPRE WPRE
  • CMV enhancers the R-U5’ segment in LTR of HTLV-I
  • SV40 enhancer SV40 enhancer
  • the intron sequence between exons 2 and 3 of rabbit P-globin Proc. Natl. Acad. Sci. USA., Vol. 78(3), p. 1527-31, 1981.
  • a vector can be introduced into host cells to thereby produce transcripts, proteins, or peptides, including fusion proteins or peptides, encoded by nucleic acids as described herein (e.g., clustered regularly interspersed short palindromic repeats (BART) transcripts, proteins, enzymes, mutant forms thereof, fusion proteins thereof, etc.).
  • the vector system may include one or more viral vectors.
  • the one or more viral vectors comprise an adenovirus-based vector, a lentivirus- based vector, an adeno-associated virus-based vector, or an RNA-based replicon.
  • the BART system when expressed, can bind and cleave at the target sequence, thus preventing functional virion formation. As a result, functional virions will not be assembled.
  • the vector system described herein includes a self-replicating RNA (e.g., Nodamurovirus-based replicon) that makes the BART protein and simultaneously self-inactivates upon execution of its function in the presence of a navigator ssDNA.
  • a self-replicating RNA e.g., Nodamurovirus-based replicon
  • this disclosure provides a host cell or cell line or progeny thereof comprising the BART system, the vector system, or the polynucleotide, as described above.
  • the host cell or cell line or progeny thereof comprises a stem cell or stem cell line.
  • the cell may be a eukaryotic cell e.g., a plant, animal, or human cell) or a prokaryotic cell.
  • the product may be a peptide, polypeptide, or protein.
  • the host cell or cell line comprises one or more polynucleotide molecules encoding an enzyme that possesses a reverse transcriptase activity, an endonuclease activity, and encoding an integrase activity, and one or more nucleic acid components, wherein the one or more nucleic acid components target and hybridize with a target polynucleotide and direct binding of the enzyme to the target polynucleotide, and wherein the enzyme modifies the target polynucleotide.
  • the term “cell” as used herein may refer to either a prokaryotic or eukaryotic cell, optionally obtained from a subject or a commercially available source.
  • the eukaryotic cell may be a human cell, a rodent cell, optionally a mouse cell, a yeast cell, or an insect cell.
  • the eukaryotic cell may be a Chinese hamster ovary (CHO) cell.
  • Eukaryotic cells comprise all of the life kingdoms except monera. They can be easily distinguished through a membrane-bound nucleus. Animals, plants, fungi, and protists are eukaryotes or organisms whose cells are organized into complex structures by internal membranes and a cytoskeleton. The most characteristic membrane-bound structure is the nucleus.
  • the term “host” includes a eukaryotic host, including, for example, yeast, higher plant, insect, and mammalian cells. Non-limiting examples of eukaryotic cells or hosts include simian, bovine, porcine, murine, rat, avian, reptilian, and human, e.g., HEK293 cells and 293T cells.
  • Prokaryotic cells that usually lack a nucleus or any other membrane-bound organelles and are divided into two domains, bacteria, and archaea. In addition to chromosomal DNA, these cells can also contain genetic information in a circular loop called on episome. Bacterial cells are very small, roughly the size of an animal mitochondrion. Prokaryotic cells feature three major shapes: rod-shaped, spherical, and spiral. Instead of going through elaborate replication processes like eukaryotes, bacterial cells divide by binary fission. Examples include but are not limited to Bacillus bacteria, E. coli bacterium, and Salmonella bacterium.
  • the term “progeny”, such as the progeny of a transgenic plant, is one that is born of, begotten by, or derived from a plant or the transgenic plant.
  • the introduced nucleic acid molecule may also be transiently introduced into the recipient cell such that the introduced nucleic acid molecule is not inherited by subsequent progeny and thus not considered “transgenic.”
  • a “non-transgenic” plant or plant cell is a plant which does not contain a foreign nucleic acid stably integrated into its genome.
  • the method comprises introducing to a cell one or more polynucleotide molecules encoding an enzyme that possesses a reverse transcriptase activity and an endonuclease activity, and encoding one or more nucleic acid components.
  • this disclosure also provides a method of generating a model eukaryotic cell comprising a mutated disease gene, which can be any gene associated with an increase in the risk of having or developing a disease.
  • the method comprises (a) introducing a BART system into a eukaryotic cell; and (b) allowing a BART complex (e.g., BART/navigator ssDNA complex) to bind to a target polynucleotide to effect cleavage of the target polynucleotide within the disease gene, wherein the ssDNA comprising the sequence that is hybridized to the target sequence within the target polynucleotide, thereby generating a model eukaryotic cell comprising a mutated disease gene.
  • a BART complex e.g., BART/navigator ssDNA complex
  • the cleavage comprises cleaving one or two strands at the location of the target sequence by the BART protein. In some embodiments, the cleavage results in decreased or increased transcription of a target gene. In some embodiments, the method further comprises repairing the cleaved target polynucleotide by non-homologous end joining (NHEJ)- based gene insertion mechanisms with an exogenous template polynucleotide, wherein the repair results in a mutation comprising an insertion, deletion, or substitution of one or more nucleotides of the target polynucleotide. In some embodiments, the mutation results in one or more amino acid changes in protein expression from a gene comprising the target sequence.
  • NHEJ non-homologous end joining
  • the cell can be a human cell, a non-human mammalian cell, a non-mammalian vertebrate cell, an invertebrate cell, an insect cell, a plant cell, a yeast cell, or a single-cell eukaryotic organism.
  • a variety of embryos are suitable for use in the method.
  • the embryo can be a 1-cell, 2-cell, or 4- cell human or non-human mammalian embryo.
  • the cell can be a stem cell.
  • Suitable stem cells include without limit embryonic stem cells, ES-like stem cells, fetal stem cells, adult stem cells, pluripotent stem cells, induced pluripotent stem cells, multipotent stem cells, oligopotent stem cells, unipotent stem cells, and others.
  • the cell is a mammalian cell, or the embryo is a mammalian embryo.
  • the non-human mammal cell may include, but not limited to, primate bovine, ovine, porcine, canine, rodent, Leporidae such as monkey, cow, sheep, pig, dog, rabbit, rat or mouse cell.
  • the cell may be a non-mammalian eukaryotic cell such as poultry bird (e.g., chicken), vertebrate fish (e.g., salmon) or shellfish (e.g, oyster, clam, lobster, shrimp) cell.
  • the non-human eukaryote cell is a plant cell.
  • the plant cell may be of a monocot or dicot or of a crop or grain plant such as cassava, corn, sorghum, soybean, wheat, oat, or rice.
  • the plant cell may also be of an algae, tree or production plant, fruit or vegetable (e.g, trees such as citrus trees, e.g., orange, grapefruit or lemon trees; peach or nectarine trees; apple or pear trees; nut trees such as almond or walnut or pistachio trees; nightshade plants; plants of the genus Brassica; plants of the genus Lactuca; plants of the genus Spinacia; plants of the genus Capsicum; cotton, tobacco, asparagus, carrot, cabbage, broccoli, cauliflower, tomato, eggplant, pepper, lettuce, spinach, strawberry, blueberry, raspberry, blackberry, grape, coffee, cocoa, etc.).
  • fruit or vegetable e.g, trees such as citrus trees, e.g., orange, grapefruit or lemon trees; peach or nectarine trees; apple or pear trees; nut trees such as almond or walnut or pistachio trees; nightshade plants; plants of the genus Brassica; plants of the genus Lactuca; plants of the genus
  • this disclosure provides a gene editing system for modifying a target polynucleotide.
  • the method comprises an enzyme that possesses a reverse transcriptase activity and an endonuclease activity, and one or more nucleic acid components, wherein the one or more nucleic acid components target and hybridize with the target polynucleotide and direct binding of the enzyme to the target polynucleotide, and wherein the enzyme modifies the target polynucleotide.
  • the BART systems disclosed herein or the gene editing systems comprising the disclosed BART -based systems may be delivered via liposomes, particles (e.g., nanoparticles), exosomes, microvesicles, a lipid, a cell-penetrating peptide (CPP) or a gene-gun. Delivery vehicles, particles, nanoparticles, formulations, and components thereof for expression of one or more elements of the aforementioned BART systems are as used in PCT/US2013/074667.
  • the enzyme further possesses an integrase activity.
  • the one or more nucleic acid components a single-stranded navigator DNA.
  • the one or more nucleic acid components further comprise a payload RNA.
  • the payload RNA comprises a stem-loop structure.
  • the enzyme reverse transcribes the payload RNA into a cDNA.
  • the enzyme integrates the cDNA into the target polynucleotide.
  • the enzyme comprises a N-terminal apurinic-apyrimidinic endonuclease domain optionally linked to a reverse transcriptase domain.
  • the enzyme further comprises a C-terminal domain.
  • the C-terminal domain facilitates interaction between the enzyme and the target polynucleotide.
  • the C-terminal domain comprises a zinc finger.
  • the zinc finger comprises a CCHC motif.
  • the enzyme comprises a Bat-Associated Reverse Transcriptase (BART).
  • BART comprises a BART of Rhinolophus ferrumequinum, Myotis myotis, Meles meles, Bos mutus, Capra hircus, Homo sapiens, Canis lupus familiaris, Sus scrofa, Callithrix jacchus Mus musculus, Ursus arclos. Elephas maximus inchcus. or a variant thereof.
  • the gene editing system comprises one or more polynucleotide molecules encoding the enzyme. In some embodiments, the gene editing system comprises one or more polynucleotide molecules encoding or comprising the one or more nucleic acid components. In some embodiments, the one or more polynucleotide molecules comprise one or more vectors. In some embodiments, the enzyme and the one or more nucleic acid components are provided in a single vector.
  • the target polynucleotide comprises RNA or DNA.
  • the RNA comprises a viral RNA of a RNA virus.
  • the RNA virus is selected from the group consisting of Norwalk, Rotavirus, Poliovirus, Ebola virus, Marburg virus, Lassa virus, Hantavirus, Rabies, Influenza, Yellow fever virus, Coronavirus, SARS, SARS-CoV-2, West Nile virus, Hepatitis A, C (HCV) and E virus, Dengue fever virus, togaviruses, Rhabdoviruses, Picomaviruses, Myxoviruses, retroviruses, bunyaviruses, coronaviruses, and reoviruses.
  • the DNA comprises a genomic DNA or a cDNA.
  • the modification of the target polynucleotide comprises cleavage of the target polynucleotide.
  • this disclosure provides a gene editing system comprising one or more vectors, liposomes, particles (e.g., nanoparticles, lipid nanoparticles), exosomes, or microvesicles that include one or more components of BART system, and optionally a pharmaceutically acceptable carrier.
  • a gene editing system comprising one or more vectors, liposomes, particles (e.g., nanoparticles, lipid nanoparticles), exosomes, or microvesicles that include one or more components of BART system, and optionally a pharmaceutically acceptable carrier.
  • this disclosure provides a vector system comprising one or more vectors, wherein the one or more vectors comprise one or more polynucleotide molecules encoding an enzyme that possesses a reverse transcriptase activity, an endonuclease activity, and an integrase activity, and comprise one or more nucleic acid components, wherein the one or more nucleic acid components target and hybridize with a target polynucleotide and direct binding of the enzyme to the target polynucleotide, and wherein the enzyme modifies the target polynucleotide.
  • composition refers to a mixture of at least one component useful within the disclosure with other components, such as carriers, stabilizers, diluents, dispersing agents, suspending agents, thickening agents, and/or excipients.
  • the pharmaceutical composition facilitates administration of one or more components of the BART system to an organism.
  • the term “pharmaceutically acceptable” refers to a material, such as a carrier or diluent, which does not abrogate the biological activity or properties of the composition, and is relatively non-toxic, i.e., the material may be administered to an individual without causing undesirable biological effects or interacting in a deleterious manner with any of the components of the composition in which it is contained.
  • the term “pharmaceutically acceptable carrier” includes a pharmaceutically acceptable salt, pharmaceutically acceptable material, composition, or carrier, such as a liquid or solid fdler, diluent, excipient, solvent, or encapsulating material, involved in carrying or transporting a compound(s) of the present invention within or to the subject such that it may perform its intended function. Typically, such compounds are carried or transported from one organ, or portion of the body, to another organ, or portion of the body. Each salt or carrier must be “acceptable” in the sense of being compatible with the other ingredients of the formulation, and not injurious to the subject.
  • materials that may serve as pharmaceutically acceptable carriers include: sugars, such as lactose, glucose, and sucrose; starches, such as corn starch and potato starch; cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients, such as cocoa butter and suppository waxes; oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; glycols, such as propylene glycol; polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; esters, such as ethyl oleate and ethyl laurate; agar; buffering agents, such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen- free water; isotonic saline; Ring
  • “pharmaceutically acceptable carrier” also includes any and all coatings, antibacterial and antifungal agents, and absorption delaying agents, and the like that are compatible with the activity of one or more components of the invention, and are physiologically acceptable to the subject. Supplementary active compounds may also be incorporated into the compositions.
  • this disclosure provides a delivery system comprising the gene editing system, and the delivery system is adapted to deliver the gene editing system into a cell or a subject.
  • the delivery system comprises nanoparticles or vesicles encapsulating the gene editing system.
  • a “gene delivery vehicle” is defined as any molecule that can carry inserted polynucleotides into a host cell.
  • Examples of gene delivery vehicles are liposomes, micelles biocompatible polymers, including natural polymers and synthetic polymers; lipoproteins; polypeptides; polysaccharides; lipopolysaccharides; artificial viral envelopes; metal particles; and bacteria, or viruses, such as baculovirus, adenovirus and retrovirus, bacteriophage, cosmid, plasmid, fungal vectors and other recombination vehicles typically used in the art which have been described for expression in a variety of eukaryotic and prokaryotic hosts, and may be used for gene therapy as well as for simple protein expression.
  • a polynucleotide disclosed herein can be delivered to a cell or tissue using a gene delivery vehicle.
  • gene delivery “gene delivery,” “gene transfer,” “transducing,” and the like are terms referring to the introduction of an exogenous polynucleotide (sometimes referred to as a “transgene”) into a host cell, irrespective of the method used for the introduction.
  • Such methods include a variety of well-known techniques such as vector-mediated gene transfer (by, e.g., viral infection/transfection, or various other protein-based or lipid-based gene delivery complexes) as well as techniques facilitating the delivery of “naked” polynucleotides (such as electroporation, “gene gun” delivery and various other techniques used for the introduction of polynucleotides).
  • vector-mediated gene transfer by, e.g., viral infection/transfection, or various other protein-based or lipid-based gene delivery complexes
  • techniques facilitating the delivery of “naked” polynucleotides such as electroporation, “gene gun” delivery and various other techniques used for the introduction of polynucleotides.
  • the introduced polynucleotide may be stably or transiently maintained in the host cell.
  • Stable maintenance typically requires that the introduced polynucleotide either contains an origin of replication compatible with the host cell or integrates into a replicon of the host cell, such as an extrachromosomal replicon (e.g., a plasmid) or a nuclear or mitochondrial chromosome.
  • an extrachromosomal replicon e.g., a plasmid
  • a nuclear or mitochondrial chromosome e.g., a nuclear or mitochondrial chromosome.
  • kits containing one or more components e.g., BART protein, navigator ssDNA
  • the kit can include one or more other reaction components.
  • an appropriate amount of one or more reaction components is provided in one or more containers or held on a substrate.
  • kits examples include, but are not limited to, one or more host cells, one or more reagents for introducing foreign nucleotide sequences into host cells, one or more reagents (e.g., probes or PCR primers) for detecting expression of the RNA or protein or verifying the target nucleic acid’s status, and buffers or culture media for the reactions (in lx or concentrated forms).
  • the kit may also include one or more of the following components: supports, terminating, modifying, or digestion reagents, osmolytes, and an apparatus for detection.
  • the reaction components used can be provided in a variety of forms.
  • the components e.g., enzymes, RNAs, probes, and/or primers
  • the components can be suspended in an aqueous solution or as a freeze-dried or lyophilized powder, pellet, or bead. In the latter case, the components, when reconstituted, form a complete mixture of components for use in an assay.
  • the kits of the invention can be provided at any suitable temperature. For example, for storage of kits, it is preferred that they are provided and maintained below 0°C, preferably at or below -20°C, or otherwise in a frozen state.
  • a kit or system may contain, in an amount sufficient for at least one assay, any combination of the components described herein.
  • one or more reaction components may be provided in pre-measured single use amounts in individual, typically disposable, tubes or equivalent containers.
  • the amount of a component supplied in the kit can be any appropriate amount and may depend on the target market to which the product is directed.
  • the container(s) in which the components are supplied can be any conventional container that is capable of holding the supplied form, for instance, microfuge tubes, microtiter plates, ampoules, bottles, or integral testing devices, such as fluidic devices, cartridges, lateral flow, or other similar devices.
  • kits can also include packaging materials for holding the container or combination of containers.
  • packaging materials for such kits and systems include solid matrices (e.g., glass, plastic, paper, foil, micro-particles, and the like) that hold the reaction components or detection probes in any of a variety of configurations (e.g., in a vial, microtiter plate well, microarray, and the like).
  • the kits may further include instructions recorded in a tangible form for the use of the components.
  • disease as used herein is intended to be generally synonymous and is used interchangeably with, the terms “disorder” and “condition” (as in medical condition), in that all reflect an abnormal condition of the human or animal body or of one of its parts that impairs normal functioning, is typically manifested by distinguishing signs and symptoms, and causes the human or animal to have a reduced duration or quality of life.
  • sample can be a sample of serum, urine plasma, amniotic fluid, cerebrospinal fluid, cells, or tissue. Such a sample can be used directly as obtained from a patient or can be pre-treated, such as by filtration, distillation, extraction, concentration, centrifugation, inactivation of interfering components, addition of reagents, and the like, to modify the character of the sample in some manner as discussed herein or otherwise as is known in the art.
  • sample and biological sample as used herein generally refer to a biological material being tested for and/or suspected of containing an analyte of interest such as antibodies.
  • the sample may be any tissue sample from the subject.
  • the sample may comprise protein from the subject.
  • the terms “decrease,” “reduced,” “reduction,” “decrease,” or “inhibit” are all used herein generally to mean a decrease by a statistically significant amount. However, for avoidance of doubt, “reduced,” “reduction” or “decrease” or “inhibit” means a decrease by at least 10% as compared to a reference level, for example, a decrease by at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% or up to and including a 100% decrease (e.g., absent level as compared to a reference sample), or any decrease between 10-100% as compared to a reference level.
  • module is meant to refer to any change in biological state, i.e., increasing, decreasing, and the like.
  • the terms “increased,” “increase” or “enhance” or “activate” are all used herein to generally mean an increase by a statically significant amount; for the avoidance of any doubt, the terms “increased,” “increase” or “enhance” or “activate” means an increase of at least 10% as compared to a reference level, for example, an increase of at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% or up to and including a 100% increase or any increase between 10-100% as compared to a reference level, or at least about a 2-fold, or at least about a 3- fold, or at least about a 4-fold, or at least about a 5-fold or at least about a 10-fold increase, or any increase between 2-fold and 10-fold or greater as compared to a reference level.
  • in vitro refers to events that occur in an artificial environment, e. ., in a test tube or reaction vessel, in cell culture, etc., rather than within a multi-cellular organism.
  • in vivo refers to events that occur within a multi-cellular organism, such as a non-human animal.
  • the term “approximately” or “about” refers to a range of values that fall within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greater than or less than) of the stated reference value unless otherwise stated or otherwise evident from the context (except where such number would exceed 100% of a possible value).
  • the term “about” is intended to include values, e.g., weight percents, proximate to the recited range that are equivalent in terms of the functionality of the individual ingredient, the composition, or the embodiment.
  • each when used in reference to a collection of items, is intended to identify an individual item in the collection but does not necessarily refer to every item in the collection. Exceptions can occur if explicit disclosure or context clearly dictates otherwise.
  • Bat iPSCs from Rhinolophus ferrumequimim were cultured as described previously (Dejosez et al., 2023, Cell 186, 957-974). Cells were plated on irradiated MEFs and maintained using DMEM/F12 (Gibco, 11330-032), 20% KOSR (Life Technologies, 10828-028), O.
  • lmM NEAA (Gibco, 11140-050), 2mM GlutaMAX (Gibco, 35050-061), 10 U/ml and 10 pg/ml of Pen/Strep (Gibco, 15140122) respectively, 100 ng/ml FGF2 (R&D Systems, 233-FB), 100 ng/ml hSCF (StemCell Technologies, 78062.2), 10 4 U/ml mLIF (Millipore-Sigma, ESG1107), 20 nM Forskolin (Sigma, F6886), and 100 M 2-mercaptoethanol.
  • BEFs Bat Embryonic Fibroblasts
  • Human ES cells (H9) were cultured on Vitronectin XF (Stemcell Technologies, 100-0763) using mTeSRl (Stemcell Technologies, 85850).
  • Immunostaining was performed on p-slides (Ibidi, 80286), where cell lines were cultured until the day of fixation. After washing the cells once with DPBS, they were fixed with Cytofix/ Cytoperm solution (BD, BDB554714) for 20 min at 4 °C.
  • Plasmids containing BARTs were synthesized by GeneScript, using a backbone of pcDNA3.1(+) and a His-tag in the C-terminal region, to allow protein purification. Their 3D structures were predicted by ColabFold (ColabFold vl.5.2-patch: AlphaFold2 using MMseqs2). They were cloned in H9 hES cells using Lipofectamine 3000 (ThermoFisher, L3000001) following manufacturer’s protocol. Selection was performed 48h after transfection using increasing concentrations of Geneticin (ThermoFisher, 10131027), with a final concentration of 150 ng/ml.
  • Cells were infected using vesicular stomatitis virus carrying an eGFP reporter at MOI 0,01 for 24 hours. The supernatant was collected by discarding floating cells and spun down at 300G for 5 min.
  • BART.O and BART.l were purified using Ni-NTA Spin Kit (Qiagen, 31314) using 2 pg of each protein per reaction. In combination with 122 bp oligos (Sigma), 100 ng of dsDNA template was added, and genes B2M and CXCR4 were amplified by using primers in Table 3.
  • 122 bp ssDNA guides were transfected in BART-containing H9 cell lines using Lipofectamine 3000. After 24 hours, genomic DNA was extracted using a Blood & Cell Culture DNA Kit (Qiagen, 13323), and the potential mutated sites were amplified using GoTaq Master Mix (Promega, M7123). Sequences were purified using a PCR Purification Kit (Qiagen, 28104) and subject to Sanger sequencing (GeneWiz).
  • RNA viruses in their capacity to infect mammals (e. ., SARS- CoV-2)
  • a critical feature of any mammalian-centric CRISPR-Cas-like system would be the ability to convert viral genomic RNA into DNA for subsequent processing in the nucleus. Consequently, the presence of reverse transcriptase activity, an enzymatic activity previously unreported to be encoded by any mammalian genome, was investigated.
  • a distinctive characteristic of bat-induced pluripotent stem cell (iPSC) colonies was observed - they exhibited a compact and uniform appearance, and their cytoplasm was populated with minuscule vesicles not observed in iPSCs from other mammalian species.
  • vesicles show a uniform distribution in the cytosol of the cell, and are of lipidic nature.
  • the iPS cells were cultured in lipid-deprived medium E8 and E8 with AlbuMAX for 3 days to understand how the vesicles only persisted in the presence of lipids in the medium.
  • These vesicles were also absent in bat embryonic fibroblasts cultured in a serumcontaining medium.
  • vesicles had a varied content, from viral particles to autophagosome systems.
  • immunostaining was performed using antibodies against a well-known reverse transcriptase (RT) found in the HIV genome (HIV-RT-p5 l/p66). It was found that a large majority of vesicles exhibited strong positive staining, indicating the presence of a reverse transcriptase enzyme. Subsequently, whether these vesicles might also contain single-stranded DNA (ssDNA), the product of reverse transcription, was investigated. Immunostaining with an antibody specifically detecting ssDNA revealed substantial amounts of ssDNA within the cytoplasmic vesicles.
  • ssDNA single-stranded DNA
  • ssDNA generated in the bat iPSCs it was sought to determine its sequence.
  • the approach to this task was two-fold: first, the cytoplasmic DNA was isolated, and libraries of these single-stranded DNAs were prepared for subsequent nextgeneration sequencing analysis. Specifically, a mild hypotonic solution was used to extract the cytoplasmic content without bursting the nucleus. Next, the SMART (Switching Mechanism at 5’ End of RNA Template) technique was applied to generate cDNA from the extracted singlestranded DNA, which effectively avoided traces of RNA (not sure how exactly this worked when making the library).
  • SMART Switching Mechanism at 5’ End of RNA Template
  • the SMART libraries were subjected to next-generation sequencing and then mapped the sequence reads to the R. ferrumequinum bat genome.
  • the resulting data provided several unexpected insights into the characteristics of the ssDNA sequences. Remarkably, a large portion of the sequences could not be mapped to the bat genome. From the total of 116,550,396 sequence reads, 111,728,741 (95.86%) did not align with any known bat sequence. Subsequent attempts to map these sequences using BLAST searches against all publicly available databases failed, indicating these sequences were unlike any known genetic material.
  • a pivotal feature of the CRISPR-Cas defense mechanism is the spatial proximity of the defense array to the gene encoding the enzyme responsible for the system (in cis arrangement).
  • ORF open reading frame
  • BLAST search was performed. Although no known gene entries matched the sequence, the overall structure displayed resemblance to other proteins known to exhibit reverse transcriptase activity. This observation again draws parallels to the CRISPR-Cas system. This enzyme is found in proximity to the interspersed viral sequences located within heterochromatic islands of the bat genome, underscoring the potential relevance of this newly discovered system.
  • these enzymatic features are consistent with the observations, which include a specific reverse transcriptase activity for mRNA-to-cDNA conversion, a nuclease function for creating nicks in the DNA, and an integration activity for incorporating new sequences into the genome (AlphaFold 3D structure).
  • the enzyme based on sequence homology, the enzyme presents a robust mechanistic framework that can account for the unique genomic events that have been uncovered.
  • BART BART-based system
  • the BART-expressing cell lines were infected with VSV at a low MOI (0.01).
  • MOI 0.01
  • the presence of viral cDNA in the cytoplasm was confirmed. Not only was it present, but it was also processed and chopped into shorter fragments.
  • This observation indicates that the BART system was operational and active against viral threats. But the most surprising revelation lay in the fate of these short fragments. As shown by genomic PCR and sequencing, they were not aimlessly floating in the cytoplasm. Instead, they had been integrated into interspersed arrays in similar islands, as observed for more ancient genomic sequences.
  • ssDNA oligonucleotides were designed to target specific sites within the genome.
  • 122 bp oligonucleotides were used, targeting the genes (e.g., B2M, CXCR4, VEGFA, CA2, KRAS, DYRK1A, HPRT1, and DMD) described in Saito M., et al. (Saito M., et al. Nature 620, 660-668 (2023)), since they are proto-oncogenes involved in the immune system, some of which have antiviral capacities, and important for development.
  • human cells were transfected with these oligonucleotides in concert with BART.
  • ssDNA sequence present in the cytoplasm revealed that the ssDNA was primarily derived from retroviral and transposon sequences. These sequences primarily aligned to unplaced scaffolds in the bat genome, genomic islands often embedded in highly repetitive heterochromatin. This unanticipated finding highlighted the existence of specific genomic arrays harboring viral sequences, analogous to bacterial CRISPR arrays. Furthermore, a unique pattern of ssDNA insertion into these genomic islands, resembling the precision and functionality of the CRISPR-Cas system, was identified.
  • the interspersed ssDNA arrays were coupled with an open reading frame (ORF) encoding a reverse transcriptase, which is in close proximity to these arrays — again, reminiscent of CRISPR-Cas.
  • VSV Vesicular Stomatitis Virus
  • the BART protein exhibits a multi-domain architecture that underpins its diverse functionalities.
  • the protein’s core houses a reverse transcriptase (RT) domain, essential for converting RNA into DNA, a hallmark of retroviral activity.
  • RT reverse transcriptase
  • BART also harbors an endonuclease domain, capable of cleaving DNA at specific sites. This endonuclease activity facilitates the integration of newly synthesized DNA into the host genome.
  • RNA sequences or structural motifs The presence of a zinc finger domain within the BART protein indicates its potential to interact directly with specific RNA sequences or structural motifs. Additionally, a stem-loop forming RNA was identified that binds to BART, acting as a guide or scaffold to facilitate the recognition and binding of viral RNA.
  • the target sequences preferred by BART in the genome appear to require double-stranded DNA adjacent to single-stranded DNA during the cell cycle, indicating a cell cycle-dependent aspect to the targeting mechanism.
  • the interplay between these elements - the zinc finger domain, the stem-loop RNA, and the specific DNA context - contributes to BART’s ability to selectively recognize and target viral RNA, enabling its antiviral and geneediting functions.
  • BART integrates viral cDNA into the host genome involves a series of orchestrated steps that echo the CRISPR-Cas system in prokaryotes.
  • the journey begins when BART encounters viral RNA. Leveraging its reverse transcriptase activity, BART meticulously transcribes the RNA into complementary DNA (cDNA).
  • cDNA complementary DNA
  • This cDNA then undergoes a processing step, yielding short fragments of approximately 122 base pairs. The precision of this processing ensures that only specific segments of the viral genome are earmarked for integration, a feature that contributes to the system’s specificity.
  • the processed cDNA fragments are then seamlessly woven into the host genome at precise locations within unplaced scaffolds, typically nestled within heterochromatin-rich regions.
  • the integration sites exhibit a distinctive pattern of interspersed arrays, bearing a striking resemblance to the CRISPR arrays observed in bacteria.
  • the culmination of this process is the formation of cDNA arrays within the host genome, serving as a ‘genomic memory’ of past viral encounters.
  • the integrated cDNA is subsequently transcribed into RNA, which then guides BART to target and cleaves homologous viral RNA during subsequent infections.
  • This intricate mechanism not only confers a form of adaptive immunity but also lays the foundation for BART’s use as a powerful gene-editing tool.
  • the integrated viral cDNA within the host genome serves as a template for the transcription of RNA molecules that act as guides for BART ( Figure 2). These RNA transcripts, harboring sequences complementary to the original viral RNA, form a complex with the BART enzyme. This complex then actively seeks out and binds to homologous viral RNA within the cell. Upon binding, BART’s endonuclease activity cleaves the viral RNA, effectively neutralizing the virus and preventing its replication.
  • the integration of viral cDNA into the host genome ensures a persistent source of guide RNAs, enabling a rapid and targeted response to future infections by the same virus, thus establishing a form of long-term adaptive immunity in mammalian cells.
  • the antiviral potential of BART was substantiated by its ability to confer resistance against Vesicular Stomatitis Virus (VSV) in human cells.
  • VSV Vesicular Stomatitis Virus
  • the overexpression of BART in these cells led to a marked reduction in VSV replication, demonstrating its direct antiviral effect.
  • the detection of processed viral cDNA fragments integrated into the host genome of BART- expressing cells provided compelling evidence for its role in establishing adaptive immunity.
  • the observation that inhibiting BART’s reverse transcriptase activity abolished this antiviral effect further solidified its crucial function in this novel defense mechanism.
  • RNA payload encoding the desired genetic modification
  • BART gene-editing capabilities are centered on its ability to harness reverse transcription, converting RNA into DNA and integrating this newly synthesized DNA into the host genome.
  • BART uses RNA as a template, offering a unique approach to gene manipulation. This RNA-centric method expands the possibilities for gene editing, allowing for the insertion of diverse genetic modifications and the modulation of gene expression at both DNA and RNA levels.
  • the BART system comprises three key components: the BART enzyme, navigator ssDNA, and an RNA payload ( Figure 1).
  • the navigator ssDNA directs BART to the specific genomic locus targeted for modification, while the RNA payload carries the genetic sequence to be inserted.
  • This mechanism which does not rely on double-stranded DNA breaks, provides advantages in specificity and reduced off-target effects, particularly due to the precision of ssDNA targeting.
  • the RNA payload serves as a template for reverse transcription, enabling the insertion of desired modifications with greater flexibility compared to traditional DNA-based methods.
  • BART s dual functionality — targeting both genomic DNA and mRNA transcripts — provides a powerful tool for gene correction and transient gene regulation, expanding the scope of therapeutic interventions.
  • Data indicate that BART achieves higher on-target editing rates with fewer off-target effects than CRISPR-Cas9, positioning it as a next-generation gene-editing technology.
  • BART presents several key advantages over existing gene-editing systems, particularly when compared to CRISPR-Cas9. Data indicate that BART achieves higher on-target editing efficiency across various experimental conditions, resulting in more precise genetic modifications. Additionally, BART exhibits enhanced specificity, with significantly reduced off-target effects, thereby minimizing the risk of unintended genomic alterations.
  • One of BART’s most notable features is its dual functionality, allowing it to target both DNA and RNA, which expands its utility beyond traditional gene editing to include the modulation of gene expression.
  • BART’s derivation from a mammalian system offers better compatibility with human cells, reducing the likelihood of immune responses and other complications commonly associated with bacterial CRISPR-Cas systems.
  • BART as a versatile and highly effective gene-editing tool, with the capability to introduce a wide range of genetic modifications, including insertions, deletions, and substitutions, at both the DNA and RNA levels.
  • This versatility combined with its efficiency and specificity, underscores BART’s use for therapeutic applications, particularly in the correction of disease-causing mutations in human cells.
  • cytoplasmic vesicles in bat iPSCs, which were absent in other mammalian iPSCs and bat embryonic fibroblasts, is described. These vesicles were found to exhibit reverse transcriptase activity and contain single-stranded DNA (ssDNA), indicating a novel antiviral mechanism potentially analogous to a mammalian -centric CRISPR-Cas-like system.
  • ssDNA single-stranded DNA
  • a multi-step approach was employed. First, cytoplasmic DNA was extracted from bat iPSCs using a mild hypotonic solution, carefully preserving ssDNA while minimizing nuclear contamination.
  • SMART Switching Mechanism at 5’ End of RNA Template
  • mapping of ssDNA sequences to the bat genome revealed a striking pattern: the sequences predominantly aligned with unplaced scaffolds, often situated within heterochromatinrich regions. Within these scaffolds, the ssDNAs were integrated in a unique, interspersed manner, forming arrays with regular spacing between the sequences. This pattern is reminiscent of the CRISPR arrays found in prokaryotes, indicating a functional parallel and a novel adaptive immune mechanism in bats.
  • non-LTR retrotransposons Long Terminal Repeat retrotransposons
  • RT reverse transcriptase domain
  • retroviruses such as HIV
  • the classification of non-LTR retrotransposons is often based on the concept of a “clade,” a term introduced by Julian Huxley in 1959 and later refined by Malik, Burke, and Eickbush in 1999.
  • a clade refers to a group of genetic elements that share common characteristics, such as structural features and phylogenetic relationships.
  • a clade within non-LTR retrotransposons is defined by three main criteria: 1) shared structural features, 2) phylogenetic grouping based on RT domain analysis, and 3) an evolutionary history dating back to ancient periods, particularly the Precambrian era.
  • BART s RT domain exhibits these defining characteristics, indicating its inclusion in one of the established clades of non-LTR retrotransposons. This classification underscores BART’s evolutionary significance and aligns it with a group of elements that have played a pivotal role in genomic evolution across diverse species.
  • the dataset as utilized comprising 211 RT domain protein sequences across 28 recognized clades served as a comprehensive framework for the classification of BART.
  • These clades including well-known groups such as LI, CR1, and Jockey, capture the evolutionary diversity of non-LTR elements, some of which have persisted since the early evolution of eukaryotes, approximately 1-2 billion years ago.
  • the LI clade is particularly ancient, with evidence suggesting that it shares a common ancestor with bacterial mobile group II introns. This ancient lineage highlights the deep evolutionary roots of the LI clade and its significance in the genomic architecture of modern organisms.
  • other RT enzymes such as the p51 subunit of HIV RT, represent evolutionary specialization and divergence within the RT domain.
  • the p51 subunit is an inactive form, evolutionarily distant from its active counterpart, p66, which underscores the functional diversity that has emerged within retroelements over time. This diversity reflects the adaptation of RT domains to various biological roles across different species, further emphasizing the complexity and evolutionary significance of these elements.
  • the RTclassl tool a specialized bioinformatics program designed for the phylogenetic analysis of RT domains. The process began with the extraction of the RT domain from the BART sequence using WU- BLAST/CENSOR, a tool that identifies and annotates repetitive elements in genomic sequences. Once the RT domain was isolated, it was aligned with the sequences in the RTclassl dataset, which contains a broad representation of RT domains from 28 recognized clades. This alignment was crucial for ensuring that the BART RT domain was accurately compared with its evolutionary counterparts.
  • the BART RT domain sequence underwent multiple sequence alignments that were subjected to random bootstrap permutations using SEQBOOT, a program that generates replicate datasets for estimating phylogenetic confidence. These bootstrap alignments were then used to calculate protein distance matrices, which quantify the evolutionary divergence between sequences. Phylogenetic trees were inferred from these distance matrices using the BIONJ algorithm, a method optimized for constructing trees with minimal evolutionary changes.
  • RTclassl generated 1,000 bootstrap trees, each representing a possible evolutionary pathway.
  • the analysis of these trees allowed RTclassl to construct a consensus bootstrap tree, which identified the phylogenetic cluster, or clade, to which BART belongs. This rigorous process of alignment, permutation, and tree inference provided a reliable classification of BART within the established evolutionary framework of non- LTR retrotransposons.
  • the tree is rooted on RT sequences from group II introns, which serve as an evolutionary outgroup, highlighting the relationships among various non-LTR elements.
  • BART’s position within the LI clade suggests that it shares a significant evolutionary ancestry with these ancient mobile genetic elements, underscoring its potential role in the genomic evolution of eukaryotes.
  • BART classification within the LI clade of non-LTR retrotransposons is strongly supported by the structural and phylogenetic characteristics of its RT domain.
  • the application of advanced phylogenetic tools and comprehensive sequence analysis has validated BART’s placement within this ancient clade, offering valuable insights into its evolutionary history.
  • This classification also indicates functional parallels between BART and other well- characterized RT elements within the LI clade, highlighting its significance in the broader context of genomic evolution.
  • the structural prediction of the BART protein was conducted using the AlphaFold Protein Structure Database, a deep learning-based tool developed by DeepMind. The process began with the retrieval of the BART amino acid sequence from the sequencing data. The BART sequence was compared against a comprehensive database of known protein structures and sequences, including those from related reverse transcriptase (RT) domains ( Figure 3).
  • AlphaFold generated a three-dimensional (3D) structural model of BART by predicting the spatial arrangement of amino acids within the protein, focusing on the highly conserved RT domain.
  • the model provided by AlphaFold was accompanied by a confidence score for each residue, indicating the reliability of the predicted positions within the structure.
  • the structural model was refined by energy minimization to resolve any steric clashes or unfavorable interactions predicted by AlphaFold.
  • the resulting model was visualized and analyzed using PyMOL, a molecular visualization system, where key structural features such as the fingers, palm, and thumb subdomains were identified. The conserved residues within the active site were mapped, and the overall structural integrity of the BART RT domain was assessed.
  • BART The structural analysis of BART, particularly when compared to human LI retrotransposons, reveals a compelling narrative of evolutionary adaptation and functional innovation.
  • RT domain At the core of BART lies its RT domain, which exhibits the classic “right-hand” architecture characteristic of retroviruses and other retroelements.
  • This architecture is composed of distinct subdomains: the fingers, palm, and thumb, each playing a crucial role in the enzyme’s function.
  • the thumb subdomain in particular, extends into a wrist region, which is thought to enhance the enzyme’s interaction with the template DNA during reverse transcription. This structural adaptation may contribute to the efficiency and specificity of BART’s reverse transcription process.
  • F605 is a highly conserved residue within the RT domain of BART and plays an important role in its enzymatic function. Positioned within the active site, F605 serves as a gatekeeper, with its aromatic side chain providing a structural barrier that selectively excludes ribonucleotides from entering the active site. This exclusion is important for ensuring that BART functions as a DNA- dependent RNA polymerase, exclusively synthesizing DNA from RNA templates. The presence of F605 effectively prevents RNA-dependent RNA polymerization, a process that could interfere with the fidelity of reverse transcription.
  • APE Apurinic-apyrimidinic endonuclease
  • BART apurinic- apyrimidinic endonuclease
  • Figure 3 The N-terminal region of BART contains an apurinic- apyrimidinic endonuclease (APE) domain, which is important for the DNA cleavage necessary for integration.
  • APE apurinic- apyrimidinic endonuclease
  • BART s APE domain also includes a DNase I-like motif, a feature absent in closely related retrotransposons, suggesting an evolutionary refinement of BART’s DNA cleavage mechanism. This refinement could contribute to BART’s enhanced specificity and efficiency in gene editing applications, distinguishing it from other elements within the non-LTR retrotransposon family.
  • the APE domain’s role in retrotransposition is to introduce a targeted nick in the host DNA, facilitating the priming of the reverse transcription process.
  • the evolutionary origins of the APE domain can be traced back to host DNA repair machinery, underscoring its ancient lineage and the role it has played in the adaptation and functionality of retrotransposons like BART.
  • sequence homology between the BART APE domain and its closest relative, the human LI APE is moderate — showing approximately 60-70% identity at the amino acid level — key residues essential for catalysis and DNA binding remain highly conserved. These include Glu43, Aspl45, His230, Asnl4, and Tyrl l5, which are important for coordinating the catalytic metal ion, activating the nucleophilic water, and properly positioning the DNA for cleavage. The preservation of these active site residues underscores the functional integrity of the BART APE domain despite its evolutionary divergence.
  • the BART protein features a distinctive “tower” region spanning amino acids 240-440.
  • This region includes several subdomains: a baseplate (residues 254-300), tower helices (301-370), a tower lock (374-382), and a PIP box (404-419).
  • the presence of the region indicates potential roles in protein-protein interactions, RNA binding, and the regulation of BART’s activity.
  • the tower region may contribute to the stability and assembly of the BART complex, facilitating its interaction with target DNA and RNA molecules.
  • the PIP box which is known to mediate interactions with proliferating cell nuclear antigen (PCNA) — a key player in DNA replication and repair — implies a possible link between BART’s activity and the host cell cycle. This connection further underscores the intricate integration of BART with cellular processes, thereby enhancing its efficiency in gene editing and antiviral defense.
  • PCNA proliferating cell nuclear antigen
  • CTD C-terminal domain
  • BART In contrast to the conserved RT and APE domains, the C-terminal region of BART displays significant truncations and modifications when compared to retrotransposons.
  • the loss of retrotransposition capability is consistent with the idea of BART’s domestication, where it has been repurposed for new cellular functions that do not rely on its movement within the genome. This adaptation is likely beneficial for the host, as uncontrolled transposition could be detrimental, potentially leading to genomic instability or insertional mutagenesis.
  • the C-terminal domain (CTD) of BART while retaining some structural features from its retrotransposon ancestors, exhibits distinct adaptations that underscore its evolutionary divergence and specialized role in the host cell.
  • the “wrist” region located immediately downstream of the reverse transcriptase (RT) domain and spanning amino acids 863 to 1061, remains relatively conserved when compared to LI retrotransposons. This conservation suggests that the wrist region continues to play an important role in BART’s function, possibly facilitating interactions with target DNA or other cellular factors during the integration process. The preservation of this region highlights the evolutionary constraints imposed on this structural element, indicating its role in both retrotransposons and their domesticated counterparts like BART.
  • the CTD of BART extending from amino acid 1062 to 1275, exhibits significant structural divergence from its retrotransposon ancestors, most notably in the absence of the RNase H domain.
  • the RNase H domain is integral to the replication process, playing an important role by degrading the RNA template following reverse transcription, thereby facilitating the synthesis of the complementary DNA strand.
  • the absence of this domain in BART suggests a profound departure from the conventional retroelement lifecycle, indicating that BART may no longer rely on the classical mechanism of RNA template degradation.
  • the CTD of BART exhibits notable modifications in key amino acid residues when compared to LI. While the CCHC motif is conserved, other residues that are crucial for the catalytic activity of the LI endonuclease domain are absent or altered in BART. These modifications further support the idea that BART’s CTD has undergone adaptive changes, potentially fine-tuning its function for its new role within the host cell. The absence of certain catalytic residues may indicate a shift away from traditional endonuclease activity, suggesting that BART has evolved to fulfill alternative functions in DNA repair, gene regulation, or targeted integration.
  • the CTD of BART also exhibits significant truncation when compared to its LI retrotransposon ancestor, resulting in the loss of several alpha-helices and key amino acid residues that are essential for retrotransposition.
  • This truncation is clearly visible in structural analyses, revealing a “stump” that effectively terminates at the zinc finger domain.
  • the absence of these structural elements and crucial residues significantly impairs BART’s ability to autonomously mobilize and replicate, a defining characteristic of active retrotransposons.
  • the loss of these retrotranspositional capabilities aligns with BART’s evolutionary shift toward domestication, where it has been repurposed for new cellular roles. In this context, the ability to move within the genome could be detrimental, potentially leading to genomic instability or harmful mutations.
  • This evolutionary refinement illustrates how BART has shed redundant or potentially harmful functions while acquiring adaptations that enhance its utility in the host genome.
  • BART.O and BART.l Two variants of the BART enzyme, designated BART.O and BART.l, were cloned into a human expression vector and subsequently stably expressed in human cell lines. The expression of these bat-derived enzymes in human cells was closely monitored to assess any potential impact on cell viability and proliferation. Remarkably, the introduction and sustained expression of BART.O and BART. l did not adversely affect the growth or viability of the human cells, indicating that the enzymes are well-tolerated within a human cellular environment.
  • the antiviral potential of the BART system was tested using human cell lines engineered to stably express two variants of the BART enzyme, BART.O and BART.l . Strikingly, overexpression of BART conferred partial immunity against Vesicular Stomatitis Virus (VSV) infection, underscoring its use as a novel antiviral tool. Following infection with VSV, BART- expressing cells exhibited a marked reduction in viral load, indicating that BART actively participates in the cellular defense against viral pathogens ( Figure 2).
  • VSV Vesicular Stomatitis Virus
  • viral cDNA was detected in the cytoplasm of BART- expressing cells post-infection, indicating that BART facilitates the reverse transcription of viral RNA into cDNA.
  • This viral cDNA was subsequently processed into shorter fragments, which were then integrated into the host genome in an interspersed array pattern reminiscent of endogenous retroelements. This pattern of integration indicates that BART not only inhibits viral replication but also incorporates viral sequences into the host genome as a form of genomic memory.
  • BART acts as a defense mechanism against viral infections, providing both immediate protection and a form of genomic memory that could confer long-term immunity.
  • BART gene-editing potential was evaluated by introducing it into human cells along with specifically designed single-stranded DNA (ssDNA) oligonucleotides and an RNA payload. These oligonucleotides acted as “navigators,” directing BART to specific genomic loci, while the RNA payload provided the template for precise genetic modifications.
  • the system s efficacy and specificity were assessed through sequencing, which revealed successful on-target editing without the need for a CRTSPR-Cas system or its components.
  • the process began by transfecting HEK293 cells, cultured in DMEM supplemented with 10% FBS, 1% Penicillin-Streptomycin, and 2 mM L-glutamine, with a BART- encoding plasmid using Lipofectamine 3000.
  • Cells were transfected at 70-80% confluency, with the plasmid complexed with the reagent according to the manufacturer’s protocol.
  • cells were incubated for 24-48 hours at 37°C to allow for protein expression, which was monitored using the eGFP reporter gene. Afterward, cells were grown for an additional 48-72 hours to maximize BART expression.
  • the cells were then harvested by trypsin-EDTA treatment and centrifugation, followed by washing the cell pellet with phosphate-buffered saline (PBS) to remove residual media and debris.
  • PBS phosphate-buffered saline
  • the BART protein was isolated from the cell lysate using affinity chromatography, leveraging a His-tag incorporated in the BART expression construct.
  • the lysate was passed through a nickel-NTA agarose column, and after washing away unbound proteins, BART was eluted with a buffer containing 250 mM imidazole.
  • the purified BART protein was then assessed for concentration and purity via SDS-PAGE and Western blotting.
  • in vitro assay targeting specific sequences within the HPRT and HSF1 loci was conducted using two distinct navigator DNAs.
  • the target sequences and corresponding navigator sequences are detailed in Tables 4 and 5.
  • the experiment began by preparing navigator-target DNA complexes. Navigator DNA and target DNA were combined in equimolar amounts (typically 100 nM each) in a reaction buffer containing 10 mM Tris-HCl (pH 7.5), 50 mMNaCl, and 1 mM EDTA. The mixture was heated to 95°C for 5 minutes to denature the DNA, followed by a gradual cooling to room temperature over 30 minutes to facilitate proper hybridization between the navigator and target DNA strands.
  • the reaction was assembled by adding 1 pL of Navigator DNA (100 nM final concentration), 1 pL of Target DNA, 0.5 pL of BART enzyme, and 1 pL of 10X BART Reaction Buffer (100 mM Tris-HCl, 500 mM NaCl, 10 mM MgC12, pH 7.9) to the annealed DNA mixture, with nuclease-free water added to bring the final volume to 10 pL.
  • the reaction mixture was incubated at 37°C for 30 minutes to allow BART to mediate the cleavage at the target sites.
  • strand-displacing polymerase 1 pL was added to extend the 3’ end of the nicked strand, followed by the addition of flap endonuclease 1 (FEN1) to create a double-stranded break at the target site.
  • FEN1 flap endonuclease 1
  • Table 4 Target Sequences for In vitro Cleavage Assay. Summary of the specific DNA sequences within the HPRT and HSF1 loci used as targets for the BART-mediated cleavage assay including 250 nucleotides upstream and downstream of the cleavage site (underlined).
  • Table 5 Navigator DNA Sequences for In vitro Cleavage Assay. List of navigator DNA sequences designed to hybridize with the target sequences and direct BART activity.
  • navDNAs navigator DNAs
  • PBS primer-binding site
  • plRNA payload RNA
  • the PBS typically ranging from 13-17 nucleotides in length, was designed to anneal upstream of the editing site, facilitating the initiation of reverse transcription by the BART enzyme.
  • the plRNA detailed in Table 7, was constructed to include a short disruptive sequence, followed by an insertion sequence encoding the enhanced green fluorescent protein (EGFP) gene. This design aimed to ensure that the BART enzyme accurately targeted and modified the desired loci.
  • EGFP enhanced green fluorescent protein
  • the navDNAs were synthesized using GenScript’s biosynthesis platform to maintain high fidelity and prevent any sequence errors that could compromise the editing process.
  • GenScript GenScript
  • This strategic design of the navDNAs and plRNAs was intended to allow BART to effectively and accurately modify the target sequences, facilitating successful gene editing outcomes.
  • Table 6 Targeting Sequences for navDNAs. Summary of the 20-nucleotide targeting sequences selected for the HPRT1 and HSF1 loci, including the specific BART target sites.
  • Table 7 plRNA Sequences for Gene Editing. Details of the plRNA sequences designed for the BART editing process, including the disruptive and EGFP insertion sequences.
  • the next step involved cloning the BART enzyme coding sequences into a plasmid vector.
  • the BART enzyme was placed under the control of the cytomegalovirus (CMV) promoter, which is known for its strong and constitutive expression in mammalian cells, to ensure robust production of the enzyme.
  • CMV cytomegalovirus
  • the plasmid included a neomycin resistance gene to enable the selection of successfully transfected cells.
  • the cloning process began with the digestion of both the vector and insert DNA using EcoRI and Hindlll restriction enzymes in NEBuffer 2 (10 mM Tris-HCl, 10 mM MgC12, 50 mM NaCl, pH 7.9) at 37°C for 1 hour.
  • Transformed cells were plated on LB agar plates containing 50 pg/mL neomycin and incubated overnight at 37°C. Positive clones were selected, and plasmid DNA was extracted using a Qiagen Plasmid Plus Midi Kit. The correct insertion of BART sequences was then verified through Sanger sequencing (see Figure 4 for vector maps).
  • HEK293T cells a human embryonic kidney cell line commonly used for transfection experiments due to their high transfection efficiency and robust growth, were selected for this study.
  • the cells were seeded at a density of 2.5 x 1 CP cells per well in a 6-well plate containing 2 mL of Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 1% Penicillin-Streptomycin, and 2 mM L-glutamine.
  • DMEM Modified Eagle Medium
  • FBS fetal bovine serum
  • Penicillin-Streptomycin 1% Penicillin-Streptomycin
  • 2 mM L-glutamine 2 mM L-glutamine
  • the BART plasmid DNA (1-2 pg per well) and navDNA were diluted in 250 pL of Opti-MEM medium, a reduced-serum medium that enhances transfection efficiency.
  • 2.5 pL of Lipofectamine 3000 reagent was added to the diluted DNA.
  • the DNA and Lipofectamine 3000 mixture was gently mixed and incubated at room temperature for 15-20 minutes to allow the formation of DNA-lipid complexes, which are essential for the efficient delivery of DNA into the cells.
  • the HEK293T cells were pre-washed with 1 mL of sterile phosphate-buffered saline (PBS) to remove any residual growth medium that could interfere with the transfection process. After washing, 2 mL of fresh Opti-MEM medium was added to each well. The DNA-lipid complexes were then carefully added dropwise to the cells, ensuring even distribution across the well. The cells were gently rocked back and forth to facilitate uniform exposure to the transfection complexes.
  • PBS sterile phosphate-buffered saline
  • the cells were incubated at 37°C in a humidified incubator with 5% CO2 for 4-6 hours to allow for optimal uptake of the plasmid DNA. This incubation period is important as it provides sufficient time for the DNA-lipid complexes to fuse with the cell membrane, allowing the plasmid DNA to enter the cells. Following the initial transfection period, the medium containing the transfection reagent was carefully aspirated and replaced with 2 mL of fresh DMEM supplemented with 10% FBS, 1% Penicillin-Streptomycin, and 2 mM L-glutamine. The cells were then incubated for an additional 48-72 hours to allow for optimal expression of the BART enzyme and navDNAs.
  • Neomycin was added to the culture medium 24 hours post-transfection. Neomycin selection was maintained for 5-7 days, during which the cells were monitored daily. Dead cells were removed by gently washing with PBS, and the medium was replaced with fresh neomycin-containing DMEM every 2-3 days to ensure continuous selection pressure. The surviving cells were expanded for further analysis. The efficiency of the transfection and selection process was later confirmed through fluorescence microscopy (for eGFP expression) and PCR analysis of genomic DNA extracted from the selected cell populations, verifying the presence and expression of the BART and navDNA constructs.
  • genomic DNA was extracted from the transfected HEK293T cells using a Qiagen DNeasy Blood & Tissue Kit, following the manufacturer’s protocol.
  • the cells were first harvested by trypsinization, and the cell pellet was washed with phosphate-buffered saline (PBS) to remove any remaining culture medium.
  • Genomic DNA was then extracted by lysing the cells with Proteinase K and a lysis buffer provided in the kit, followed by binding the DNA to a silica membrane in a spin column. The membrane was washed with a series of ethanol-containing buffers to remove contaminants, and the DNA was eluted in nuclease-free water.
  • the concentration and purity of the extracted DNA were measured using a NanoDrop spectrophotometer, with an A260/A280 ratio of approximately 1.8 indicating high-quality DNA suitable for downstream applications.
  • PCR primers were designed to flank the regions targeted for editing within the HPRT1 and HSF1 loci (see Table 8 for primer sequences). These primers were synthesized to amplify both the unmodified and modified sequences, allowing for the detection of successful gene editing events.
  • the PCR reaction was set up in a 50 pL volume containing IX ThermoPol Reaction Buffer (20 mM Tris-HCl, 10 mM (NH4)2SO4, 10 mM KC1, 2 mM MgSO4, 0.1% Triton X-100, pH 8.8), 200 pM of each dNTP, 0.2 pM of each primer, 1.25 U of Taq DNA polymerase, and 100 ng of template DNA.
  • the PCR cycling conditions included an initial denaturation step at 95°C for 3 minutes to fully denature the DNA, followed by 35 cycles of 95°C for 30 seconds, 58°C for 30 seconds (for primer annealing), and 72°C for 1 minute (for DNA extension). A final extension step at 72°C for 5 minutes ensured complete synthesis of all PCR products.
  • the resulting PCR products were analyzed by electrophoresis on a 1.5% agarose gel containing 0.5 pg/mL ethidium bromide in IX TAE buffer (40 mM Tris-acetate, 1 mM EDTA, pH 8.3). The gel was run at 100V for 45 minutes to separate the DNA fragments by size. Visualization was performed using a UV transilluminator, where successful insertion of the EGFP sequence was indicated by the presence of a PCR product approximately 720 bp larger than the wild-type amplicon, corresponding to the size of the EGFP insertion. A comparison of the band sizes on the gel confirmed the presence of the desired genetic modification.
  • PCR products were purified using a Qiagen PCR Purification Kit, which involved binding the DNA fragments to a silica membrane in a spin column, washing with ethanol-based buffers, and eluting the purified DNA in nuclease-free water.
  • the purified products were then sequenced using the BigDye Terminator v3.1 Cycle Sequencing Kit on an ABI 3730 DNA Analyzer. Sequencing reactions were prepared by combining the purified PCR product, sequencing primers, and BigDye reagent in a thermal cycler, following the manufacturer’s instructions. Sequence analysis was performed using Geneious software, where the edited sequences were aligned against the wild-type reference sequence to confirm the precise integration of the EGFP gene.
  • EGFP expression in the transfected HEK293T cells was analyzed using both fluorescence microscopy and flow cytometry. After the transfection and selection period, the cells were gently washed three times with phosphate-buffered saline (PBS) to remove any residual culture medium and dead cells. The cells were then fixed with 4% paraformaldehyde in PBS for 10 minutes at room temperature to preserve cellular structures and fluorescent signals. Following fixation, the cells were washed three more times with PBS to remove any remaining paraformaldehyde.
  • PBS phosphate-buffered saline
  • VECTASHIELD HardSet Antifade Mounting Medium with DAPI a nuclear counterstain that fluoresces blue under UV light, to allow for the visualization of cell nuclei.
  • Fluorescent images were captured using a Zeiss Axio Observer fluorescence microscope equipped with an EGFP filter set (excitation at 488 nm, emission at 530 nm) to specifically detect EGFP expression. Multiple fields of view were imaged to ensure representative sampling of the cell population. Image analysis was conducted using ImageJ software, where the presence of EGFP-positive cells was quantified to confirm successful gene editing at the targeted loci. The images were analyzed for both the intensity of EGFP fluorescence and the number of EGFP-positive cells, providing qualitative evidence of gene editing success.
  • the transfected cells were harvested by trypsinization.
  • the cells were incubated with 0.25% trypsin-EDTA at 37°C for 2-3 minutes to detach them from the culture plate, followed by neutralization with DMEM containing 10% FBS.
  • the cell suspension was then passed through a 40 pm cell strainer to obtain a single-cell suspension, which was critical for accurate flow cytometry analysis.
  • the cells were resuspended in 500 pL of ice-cold PBS to maintain cell viability and integrity during analysis.
  • the prepared cell suspension was analyzed using a BD FACSCanto II flow cytometer.
  • EGFP expression was detected by exciting the cells at 488 nm and measuring the emission at 530 nm. Data acquisition was set to collect at least 10,000 events per sample to ensure statistically significant results.
  • the percentage of EGFP-positive cells was determined using FlowJo software, which allowed for gating of the cell population and precise quantification of EGFP expression. This analysis provided a robust indication of the efficiency of the Prime Editing process, with a high percentage of EGFP-expressing cells confirming the successful introduction of the desired genetic modifications by the BART enzyme.

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Abstract

La transcriptase inverse associée aux chauves-souris (BART), divulguée selon la présente invention, possède une caractéristique unique trouvée dans les cellules souches pluripotentes induites (iPSC) de chauves-souris, à savoir la capacité de convertir l'ARN génomique viral en ADN. Le système à BART selon la présente invention représente un outil de nouvelle génération pour l'édition génomique et l'édition d'ARN, présentant une efficacité et une spécificité élevées, potentiellement plus performant que les systèmes CRISPR-Cas actuels. La présente invention représente également une avancée significative pour relever les défis les plus pressants en matière de santé, notamment les pandémies causées par des virus à ARN et l'édition génique à des fins thérapeutiques.
EP24869062.0A 2023-09-19 2024-09-18 Transcriptase inverse associée aux chauves-souris et ses procédés d'utilisation Pending EP4780975A1 (fr)

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