WO2022090153A1 - Transcriptional synchronization of two or more functional transcription products - Google Patents

Transcriptional synchronization of two or more functional transcription products Download PDF

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WO2022090153A1
WO2022090153A1 PCT/EP2021/079536 EP2021079536W WO2022090153A1 WO 2022090153 A1 WO2022090153 A1 WO 2022090153A1 EP 2021079536 W EP2021079536 W EP 2021079536W WO 2022090153 A1 WO2022090153 A1 WO 2022090153A1
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nucleotide sequence
sequence coding
self
cell
transcription product
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PCT/EP2021/079536
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French (fr)
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Mathias Schmidt
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Universität Hamburg
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Priority claimed from LU102162A external-priority patent/LU102162B1/en
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    • C—CHEMISTRY; METALLURGY
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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
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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
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8216—Methods for controlling, regulating or enhancing expression of transgenes in plant cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8216—Methods for controlling, regulating or enhancing expression of transgenes in plant cells
    • C12N15/8218—Antisense, co-suppression, viral induced gene silencing [VIGS], post-transcriptional induced gene silencing [PTGS]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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

Definitions

  • the invention relates to a cell being genetically engineered to have two or more functional transcription products transcriptionally synchronized with each other, and a method for the transcriptional synchronization of two or more functional transcription products. Further, the invention relates to a vector or vector system, and to a transgenic plant or non-human animal.
  • Genetic modification of cells is well known and is widely used to create cells with desired properties. Genetic engineering methods are, for example, frequently used to introduce foreign genes into cells, e.g. plant cells, in order to make the cells resistant to particular pathogens.
  • Such genetically engineered cells may, for example, be able to produce one or more proteins or an RNA product, for example an antisense RNA or small interfering RNA (siRNA) counteracting infestation by a pathogen.
  • RNA product for example an antisense RNA or small interfering RNA (siRNA) counteracting infestation by a pathogen.
  • Genetically manipulating cells has not only been performed with prokaryotic cells, plant cells or non-human animal cells, for example, but has also been done or at least suggested in relation to mammalian cells, including human cells, to treat or prevent diseases like genetic diseases or cancer.
  • the invention relates to a genetically engineered cell, the cell being genetically engineered to comprise a transcription unit with a promotor and a termination signal, the transcription unit comprising, between the promotor and the termination signal, a) a first nucleotide sequence coding for a first functional transcription product, b) a second nucleotide sequence coding for a second functional transcription product, c) a third nucleotide sequence coding for a first self-cleaving ribozyme, and d) a fourth nucleotide sequence coding for second self-cleaving ribozyme, the second nucleotide sequence coding for the second functional transcription product being flanked by the third and fourth nucleotide sequence coding for the first and second self-cleaving ribozyme.
  • the genetically engineered cell of the invention is genetically engineered in a way that the cell has a first functional transcription product, for example an endogenous gene product, transcriptionally synchronized with a second functional transcription product, e.g. a functional non-coding RNA molecule.
  • Both the first and the second functional transcription product are encoded in the same transcription unit, i.e. the DNA coding for the first and the second functional transcription product are transcribed under the same transcription regulatory element(s). Consequently, if the first functional transcription product, for example an endogenous gene is transcribed the DNA coding for the second functional transcription product is also transcribed in the same amount and rate as the endogenous gene.
  • transcriptionally synchronize a first functional transcription product e.g. an endogenous gene
  • more than one further functional transcription products e.g. two, three or more further functional transcription products which may be identical or different from each other.
  • the invention can provide a plethora of different cells with different properties.
  • the properties of the cell can be adapted to a variety of requirements and uses, including, but not limited to the protection against pathogens and cancerous transformation.
  • the core idea of the invention is to place a first functional transcription product and a second transcription product under exactly the same transcriptional regime, i.e. the same transcription regulatory element(s) of a given transcription unit, in order to have both transcription products transcribed in a synchronized manner.
  • a genomic gene e.g. an endogenous gene
  • a nucleic acid preferably a DNA, encoding a functional RNA molecule
  • both the first and second functional transcription product are contained in a common pre-mRNA molecule.
  • the second functional transcription product is flanked by suitable self-cleaving ribozymes such that the second functional transcription product is automatically released from a pre-mRNA transcribed from the transcription unit containing the first functional transcription product and the second functional transcription product.
  • the invention can, for example, be used for creating disease and pathogen resistance mechanisms, analysis of gene function, or for other regulatory applications in eukaryotic and prokaryotic cells.
  • an mRNA as first or second functional transcription product i.e.
  • the synchronization mechanism is configured in a way that elements necessary for the translation of the mRNA, e.g. a poly-A tail or 5' cap, are either appended to the mRNA by the endogenous natural cell machinery or are otherwise provided for, e.g. by including a synthetic poly-A tail sequence or a Lariat capping ribozyme.
  • Transcriptional synchronization can, for example, be used to synchronize the transcription of a genomic gene, e.g. endogenous gene, with a synthetically introduced “output”.
  • This output can be a functional non-coding RNA or protein.
  • Synchronization means, that all effects, which are influencing the transcription rate of a first functional transcription product, e.g. endogenous gene, are similarly affecting the synthetic output too. Such effects include, for example, promoter activity, regulation by enhancers and silencers, intronic and epigenetic effects, as well as other effects.
  • the transcription rate of the synthetic output essentially equals the transcription rate of the first functional transcription product, e.g. endogenous gene.
  • the mechanism enables the use of a precise IF-THEN logic on the transcriptional level.
  • This can, for example, be used as a precise sensor for the activity of a synchronized genomic gene.
  • it can be used to detect transcriptional dysregulation in the case of diseases via the synchronization itself, and also to enable a therapeutic answer via the synchronized output.
  • This output could be the production of a therapeutic molecule by the cell itself.
  • transcriptional synchronization can be used to create disease-resistant cells for virtually every genetic disease, viral infection and bacterial infection that impacts transcription in any way (including cancer and Alzheimer’s disease).
  • transcriptional synchronization is further ideal due to its safety aspects:
  • the output is only produced if the condition for its production is met, which is, for example, the transcription of a genomic gene, e.g. endogenous gene, being transcriptionally synchronized with the output. Also, it cannot be produced in a larger amount than the product of the gene it is synchronized with.
  • the overall strength of the produced output is dependent on the transcription rate of the gene. Thus, the stronger or longer the gene is expressed, the stronger the output is. This can be used for further processing the output as described further below for cancer.
  • the first regulation takes place via the initial synchronization, while the second layer of regulation can be introduced by using a signaling molecule as output, which is set off against another signaling output from another synchronized gene or a gene product. Depending on which signal is stronger, a final output is produced or not. Therefore, transcriptional synchronization can be used to create multiple layers of processing, or, also in combination with other mechanisms (e.g. molecular logic gates), to further enhance safety. Transcriptional synchronization is thus ideal for creating precise and safe cellular resistance mechanisms against genetic diseases and viral/bacterial infections, for example.
  • Transcriptional synchronization according to the invention can also be used for the transfection of multicellular organisms, e.g. a non-human animal or a human, without resulting mosaic mutants, i.e. organisms with transfected and non-transfected cells, as will be described in more detail further below.
  • the term “genetically engineered” in relation to a cell or organism means that the genome of the cell or organism has been changed using biotechnological means and methods.
  • the term “genetically engineering” is not restricted to a manipulation of, for example, nuclear DNA, but also encompasses manipulation of extranuclear DNA or the introduction of non-chromosomal genetic elements, e.g. plasmids.
  • the term also includes epigenetic alterations of the molecular and genetic setup of a cell or organism, or parts of these.
  • the term “cell” as used herein relates to a living biological, e.g. prokaryotic or eukaryotic cell.
  • Non-limiting examples of eukaryotic cells include plant cells, fungal cells, mammalian cell, non-human animal cells or human cells, preferably with the proviso that the cell is not a human germ cell.
  • the eukaryotic cell may, for example, be a blood cell, epithelial cell or a stem cell (e.g. embryonic stem cells, induced pluripotent stem cell, hematopoietic stem cell).
  • the cell may be a mammalian cell of the orders rodenta (mice, rats, hamsters), lagomorpha (rabbits), carnivora (cats, dogs), and artiodactyla (cows, pigs, sheep, goats, horses).
  • the cell may be from any organism, for example plant, non-human animal, human, non-human primate, mouse, rat, rabbit, cat or dog, preferably with the proviso that it is not a human germ cell.
  • the cell may be isolated or may be part of an organism (e.g., subject), but preferably with the proviso that it is not a human germ cell.
  • Procaryotic cells include, for example, bacterial cells and archaean cells.
  • transcriptionally synchronized as used herein relates to the concomitant transcription of at least two separate nucleic acid sections in the same transcription unit under the same transcription regulatory element(s), e.g. the same promoter, such that, if a first nucleic acid encoding, for example, a gene, is transcribed, the second nucleic acid section, e.g. coding for a coding or non-coding RNA molecule, is preferably also transcribed in exactly the same quantity and rate as the first nucleic acid.
  • “Transcriptional synchronization” thus in particular means the formation of a single pre mRNA transcribed from a single transcription unit containing the at least two separate nucleic acid sections, wherein the at least two separate nucleic acid sections are later released from the common pre-mRNA, such that the transcription products of the first and second nucleic acid sections are separated.
  • the transcription product of the first nucleic acid section may be an mRNA coding for a gene of a cell
  • the transcription product of the second nucleic acid section may be a coding or non-coding RNA molecule, e.g. an mRNA coding for a different protein, a reporter protein, an antisense RNA, siRNA etc.
  • transcription unit relates to a sequence of nucleotides in DNA that codes for a single RNA molecule, along with the sequences necessary for its transcription, for example a promoter, an RNA-coding sequence, and a termination signal (TS, also terminator or termination sequence).
  • transcription regulatory element relates to any element of a transcription unit regulating the quantity and rate of transcription of the transcription unit. Examples of such elements are promoters and enhancers, i.e. segments of DNA containing sequences capable of providing promoter and enhancer functions.
  • the expression according to which two separate nucleic acid sections or nucleotide sequences coding for a transcription product are “under the same transcription regulatory element(s)” means that any transcription regulatory element having an impact on the transcription of the first nucleic acid section has essentially the same impact, e.g. in terms of transcription quantity and rate, on the second nucleic acid section.
  • the term also relates to any epigenetic element or modification that takes part in regulating the transcription of the transcription unit.
  • promoter relates to a sequence of DNA to which proteins bind that initiate transcription of a DNA sequence into an RNA. Usually, a promoter is arranged towards the 5' end of the sense strand of the DNA.
  • the term “enhancer” relates to regulatory DNA sequences that enhance the transcription of an associated gene when bound by specific proteins (transcription factors).
  • terminal sequence refers to a nucleotide sequence that marks the end of a gene or operon in genomic DNA during transcription.
  • mRNA messenger RNA
  • messenger RNA relates to a single-stranded RNA molecule comprising an RNA molecule that is complementary to one of the DNA strands, i.e. the sense strand, of a gene, and, if present, regulatory elements like a 5' cap or a poly-A tail.
  • An mRNA encodes a polypeptide and is translated by the cell’s translation machinery comprising ribosomes and other components into the polypeptide.
  • pre-mRNA relates to the primary transcript from a transcription unit.
  • the term encompasses, but is not restricted to, the primary transcript from a transcription unit of eukaryotic genes containing introns and exons, which is further processed (e.g. spliced) to an mRNA containing only exon sequences, which is translated into a polypeptide (protein).
  • the term also encompasses a primary transcript of a transcription unit comprising only one or more functional non-coding RNA sequences or a transcription unit comprising further coding or non-coding sequences besides sequences encoding an mRNA.
  • the term thus encompasses any primary RNA transcript from a transcription unit comprising noncoding and/or coding RNA molecules.
  • ORF open reading frame
  • the term “open reading frame”, ORF relates to a DNA section coding for a transcription product that is directly or indirectly translatable into a protein, indirectly meaning translatable after further cellular processing, e.g. splicing of a pre mRNA.
  • the term thus not only encompasses coding DNA sequences with a number of codons divisible by three and flanked by a start and stop codon but also eukaryotic genes composed of exons and introns, i.e. nucleotide sequences that are actually translated (exons) and nucleotide sequences that are actually not translated (introns), but are removed from a pre mRNA to form an mRNA directly translatable into a protein.
  • endogenous or “homologous” is used herein with respect to a biologically functional element such as a gene, RNA, enzyme or protein to refer to it as an original or native element, i.e. a gene, RNA, enzyme or protein naturally occurring in the target cell, in contrast to a foreign (heterologous) element.
  • genomic gene refers to a gene, i.e. a nucleic acid encoding a polypeptide, of the genome of a cell.
  • genomic refers to the genetic material of a cell or organism and encompasses genetic material of chromosomal genetic material and extra-chromosomal genetic material, e.g. genetic material of mitochondria, chloroplasts or plasmids. The term includes endogenous genes and genes stably introduced into the genome of a cell.
  • genomic gene product refers to a transcription product of a genomic gene, i.e. an mRNA or pre-mRNA, which is translated into a polypeptide (protein).
  • genomic gene product also encompasses a product resulting from translation, i.e. a protein.
  • endogenous gene relates to a gene that is native to a given cell, i.e. is naturally occurring in the cell. The term does not exclude that the gene has been introduced into the cell, i.e. that a gene being identical to or comprising an endogenous gene, has been introduced into the cell.
  • endogenous gene product relates to a transcription product of an endogenous gene, i.e. an mRNA or pre-mRNA.
  • the term also encompasses a product resulting from translation, i.e. a protein.
  • heterologous refers to the foreign origin of an element, for example a gene, RNA, enzyme or other protein. “Foreign” means that the element does not naturally occur in the target cell, and for example originates from a cell or an organism with different genetic makeup, such as an organism of a different species.
  • expression is meant the conversion of a genetic information into a functional product, for example the formation of a protein or a nucleic acid, e.g. functional RNA, on the basis of the genetic information.
  • the term encompasses the biosynthesis of a protein, e.g. an enzyme, based on genetic information including previous processes such as transcription, i.e. the formation of mRNA based on a DNA template, or the synthesis of a functional RNA molecule, for example a self-cleaving ribozyme.
  • RNA molecules transcribed from a transcription unit, i.e. an RNA molecule being directly (itself) or indirectly (i.e. after modification, e.g. splicing) functional in or outside the cell.
  • the term encompasses coding RNA molecules, i.e. RNA molecules that are translated into a polypeptide, e.g. mRNA or pre-mRNA containing introns and exons, and non-coding RNA molecules, i.e. RNA that is not translated into a protein, like, for example, antisense RNA, rRNA, piRNA, miRNA, tRNA, gRNA, exRNA, etc.
  • RNA gene may be used for a DNA sequence from which a functional non-coding RNA is transcribed.
  • output or “output molecule” in relation to the functional RNA molecule relate to the intended end product to be produced in the cell of the invention. If the functional RNA is an mRNA coding for a protein the intended “output” normally is a protein. In other cases, however, the output molecule may be an RNA molecule itself (other than mRNA) having a specific intended function, for example an antisense RNA, siRNA or the like.
  • ribozyme ribonucleic acid enzymes
  • SCRz self-cleaving ribozyme
  • Classes of self-cleaving ribozymes include hepatitis delta virus (HDV)-like, hammerhead, hairpin, Varkud Satellite (VS), glmS, twister, twister sister, pistol, and hatchet ribozymes (see, for example, Lee, Ki-Young & Lee, Bong-Jin. (2017). Structural and Biochemical Properties of Novel Self-Cleaving Ribozymes. Molecules (Basel, Switzerland). 22. 10.3390/molecules22040678; Weinberg Z, Kim PB, Chen TH, et al. New classes of self-cleaving ribozymes revealed by comparative genomics analysis. Nat Chem Biol. 2015; 11(8):606-610.
  • the term “5' self-cleaving ribozyme” relates to self-cleaving ribozymes that are catalytically active towards their 3 '-end. They are cleaving near the 5 '-end of the sequence that is situated downstream of the ribozyme and are typically (but not exclusively) situated upstream (towards the 5 '-end) of the sequence, which is supposed to be cut off.
  • the term “3' self-cleaving ribozyme” relates to self-cleaving ribozymes that are catalytically active towards their 5 '-end.
  • Examples of 3' self-cleaving ribozymes are Hepatitis delta virus (HDV) ribozyme or the HDV- like CPEB3 ribozyme.
  • Examples of 5' self-cleaving ribozymes are hammerhead ribozymes (HHRz), Type-P5 twister ribozymes (Roth A, Weinberg Z, Chen AG, Kim PB, Ames TD, Breaker RR.
  • HHRz hammerhead ribozymes
  • Type-P5 twister ribozymes Roth A, Weinberg Z, Chen AG, Kim PB, Ames TD, Breaker RR.
  • a widespread self-cleaving ribozyme class is revealed by bioinformatics. Nat Chem Biol. 2014;10(l):56-60.
  • Lariat-capping ribozyme (LC ribozyme, LCrz, formerly GIRI branching enzyme) relates to a ribozyme catalyzing cleavage by a 2', 5' branching reaction, leaving the 3' product with a 3-nt lariat cap that functionally substitutes for a conventional mRNA cap (m 7 GpppN cap) in the downstream pre-mRNA (see, e.g., Meyer M, Nielsen H, Olieric V, et al. Speciation of a group I intron into a lariat capping ribozyme. Proc Natl Acad Sci U S A.
  • Lariat capping ribozyme is a ribozyme from the myxomycete Didymium iridis. formerly known as GIRI ribozyme (see RCSB PDB 6GYV released 2018-08-22).
  • RNA targeting sequences relate to first ribonucleic acids specifically binding to a second RNA, e.g. to an mRNA, pre-mRNA, miRNA or tRNA, or sequences coding for a protein binding to said second RNA sequence, e.g. an RNA or pre- mRNA binding protein, i.e. a protein specifically binding to an mRNA.
  • mRNA binding sequences are anti-sense RNA, i.e. an RNA hybridizing with an mRNA in order to prevent translation of the mRNA into a protein, microRNA (miRNA), i.e. short (21-25 nt) noncoding single-stranded RNAs, or shRNA (small hairpin RNA).
  • miRNA microRNA
  • shRNA small hairpin RNA
  • the expression, according to which the second nucleotide sequence coding for a second functional transcription product is “flanked by the third and fourth nucleotide sequences coding for the first and second self-cleaving ribozyme” means that a nucleotide sequence coding for a self-cleaving ribozyme is attached at the 3' end and at the 5' end of the second nucleotide sequence.
  • the first nucleotide sequence codes preferably for an mRNA encoding a first protein, preferably a genomic protein of the cell, further preferred an endogenous protein of the cell, or a first functional non-coding RNA molecule, preferably an endogenous functional non-coding RNA molecule of the cell.
  • the first nucleotide sequence coding for the first functional transcription product is preferably a copy of a naturally occurring or previously introduced functional transcription product being part of a naturally occurring or previously introduced transcription unit of the cell.
  • the naturally occurring or previously introduced transcription unit of the cell is preferably replaced with a transcription unit with the configuration of transcription elements as described herein, such that the naturally occurring or previously introduced functional transcription product is still transcribed or transcribably inserted in the cell, but now transcriptionally synchronized with at least one second functional transcription product.
  • the first and second nucleotide sequence code for different functional transcription products e.g. that, in case the first nucleotide sequence codes for a coding RNA molecule, e.g. an mRNA coding for a first protein, the second nucleotide sequence codes either for a different coding RNA, e.g. an mRNA coding for a second protein differing from the first protein, or a non-coding RNA molecule, e.g. siRNA, tRNA or antisense RNA. It is further preferred that not both the first and second functional nucleotide sequence code for a gRNA, i.e. a CRISPR guide RNA.
  • the second functional nucleotide sequence does not code for a gRNA, further preferred neither the first nor the second functional nucleotide sequence code for a gRNA.
  • the first nucleotide sequence codes for an endogenous gene product, preferably an endogenous protein
  • the second nucleotide sequence codes for a functional coding or non- coding RNA molecule, the functional coding RNA molecule being different from an mRNA coding for the first protein.
  • the transcription unit may comprise more than one second nucleotide sequences coding for a second functional transcription product, e.g. two, three, four or more second nucleotide sequences, such that all these second functional transcription products are transcriptionally synchronized with the first nucleotide sequence coding for the first functional transcription product. It is possible that two or more of the second nucleotide sequences code for the same functional transcription product. It is preferred, however, that the second nucleotide sequences code for different functional transcription products, e.g. several different mRNA coding for different proteins, and/or several different non-coding functional RNAs.
  • each of the further second nucleotide sequences will also be flanked by suitable self-cleaving ribozymes.
  • the skilled person will, depending on the nature and purpose of the respective further second functional transcription product, choose a suitable combination of the flanking ribozymes for each of the further second functional transcription products.
  • the second nucleotide sequence codes for a functional non-coding RNA or an mRNA encoding a protein.
  • the second nucleotide sequence may thus code for a coding RNA, mRNA, such that a protein is produced by the normal cellular protein synthesizing machinery as an “output”, or code for a non-coding functional RNA, for example an antisense RNA or siRNA.
  • the second nucleotide sequence may, for example, code for an RNA binding sequence, e.g. an mRNA binding sequence like an anti-sense RNA, microRNA or shRNA.
  • the third nucleotide sequence flanking the second nucleotide sequence codes for a 5' self-cleaving ribozyme and the fourth nucleotide sequence codes for a 3' self-cleaving ribozyme.
  • the order of the second to fourth nucleotide sequence is thus (from promoter to terminations sequence, or 5' to 3'): 5' self-cleaving ribozyme - second nucleotide sequence coding for a first functional transcription product - 3' self-cleaving ribozyme.
  • a preferred 5' self-cleaving ribozyme is a hammerhead ribozyme (HHRz).
  • the respective DNA is preferably modified in a way that a Lariatcapping ribozyme is situated at the 5 '-end of the mRNA and a synthetic poly-A-tail is situated at the 3 '-end of the mRNA.
  • the arrangement composed of the second nucleotide sequence coding for the second functional transcription product and the flanking third and fourth nucleotide sequence each coding for a self-cleaving ribozyme can either be placed upstream, i.e. 5', of the first nucleotide sequence coding for the first functional transcription product, e.g. an ORF, or downstream, i.e. 3', of the first nucleotide sequence coding for the first functional transcription product, e.g. ORF.
  • the skilled person will readily be able to adapt the flanking self-cleaving ribozyme readily to the intended purpose, depending, for example, on the nature and function (e.g. non-coding or coding) of the functional transcription product to be produced.
  • the basic configuration is more complex and comprises essentially two different embodiments.
  • a first embodiment herein also called “3' arrangement” or “3' design”
  • the second nucleotide sequence coding for the second functional transcription product and flanked by the third and fourth nucleotide sequence coding for a first and second selfcleaving ribozyme is arranged after the first nucleotide sequence coding for the first functional transcription product, the first nucleotide sequence coding for the first functional transcription product preferably (e.g.
  • the first functional transcription product is a mRNA
  • the fifth nucleotide sequence encoding a Poly A tail preferably being followed by a sixth nucleotide sequence coding for a third self-cleaving ribozyme, preferably a 3' self-cleaving ribozyme, the terms “after”, “before”, “preceded”, “followed by” or “following” relating to the direction from the promoter to the termination signal.
  • the second nucleotide sequence coding for the second functional transcription product and flanked by the third and fourth nucleotide sequence coding for a first and second self-cleaving ribozyme is arranged before the first nucleotide sequence coding for the first functional transcription product, the first nucleotide sequence coding for the first functional transcription product preferably being preceded by a seventh nucleotide sequence coding for a Lariat capping ribozyme, the terms “after”, “before”, “preceded”, “followed by” or “following” relating to the direction from the promoter to the termination signal.
  • the second nucleotide sequence coding for the second functional transcription product and flanked by the third and fourth nucleotide sequence coding for a first and second self-cleaving ribozyme is arranged after the first nucleotide sequence coding for the first functional transcription product, the third nucleotide sequence coding for a first self-cleaving ribozyme is preceded by a spacer sequence.
  • the second nucleotide sequence coding for the second functional transcription product and flanked by the third and fourth nucleotide sequence coding for a first and second self-cleaving ribozyme is arranged before the first nucleotide sequence coding for the first functional transcription product, the fourth nucleotide sequence coding for a second self-cleaving ribozyme is followed by a spacer sequence.
  • the spacer sequence is particularly useful in the 3' design, and facilitates folding of the selfcleaving ribozymes into their catalytically active state, and is preferably configured to have least possible interaction with neighboring sequences.
  • the spacer can have any suitable length and may also vary depending on the intended use and may, for example, be a stretch of several adenine nucleotides (oligo- or poly-A).
  • the spacer may have, for example, a length of 5 to 100 nucleotides, preferably 8 to 80 nt, e.g. adenine nucleotides.
  • An example for a short spacer variant is a spacer having a length of 8 nt.
  • a long variant of the spacer may have 72 nt.
  • a spacer of intermediate length may, for example have a length of 45-55, e.g. 48, nucleotides, for example adenine nucleotides. Other lengths are, however, possible.
  • 3' or 5' regulatory elements 3' or 5' untranslated regions
  • a functional transcription product e.g. mRNA molecule
  • the skilled person will configure the genomic elements accordingly and, taking his or her common general knowledge, provide for any nucleic acid elements necessary for a correct processing of a functional transcription product in the cell.
  • a synthetic Poly A tail is arranged downstream of the first nucleotide sequence coding for the first functional transcription product, if the first functional transcription product is to be an mRNA, the Poly A tail being encoded by a fifth nucleotide sequence, and the Poly A tail being immediately followed by a sixth nucleotide sequence coding for a third self-cleaving ribozyme, preferably a 3' self-cleaving ribozyme.
  • “Synthetic” in relation to the poly A tail means that adenine nucleotides are not added by the cell’s transcriptional machinery to the mRNA, but have to be encoded in the DNA. In the 5' design such a Poly A tail is not necessary, because the arrangement of elements allows for the addition of the poly A tail by the normal cellular transcriptional machinery. In the 5' design, however, a seventh nucleotide sequence encoding a Lariat capping ribozyme is preferably additionally present immediately in front of the first nucleotide sequence coding for the first functional transcription product in order to produce an mRNA having a Lariat cap as a substitute for the normal 5' m 7 G cap.
  • the order of the first functional transcription product, the fifth nucleotide sequence coding for a Poly A tail, the sixth nucleotide sequence coding for a third self-cleaving ribozyme, the spacer, and the second nucleotide sequence coding for the second functional transcription product flanked by the first and second self-cleaving ribozyme, between the promoter and the termination sequence is, in the direction from the promoter to the termination signal, as follows: First functional transcription product - fifth nucleotide sequence encoding a Poly A tail - sixth nucleotide sequence coding for the third self-cleaving ribozyme, preferably a 3' self- cleaving ribozyme - spacer sequence - third nucleotide sequence coding for the first selfcleaving ribozyme, preferably a 5' self-cleaving ribozyme - second nucleo
  • the order of the first functional transcription product, the third nucleotide sequence coding for the first self-cleaving ribozyme, the fourth nucleotide sequence coding for the second self-cleaving ribozyme, the second nucleotide sequence coding for the second functional transcription product, and, if present, the seventh nucleotide sequence coding for a Lariat capping ribozyme and the spacer sequence, between the promoter and the termination sequence, is, in the direction from the promoter to the termination signal, as follows: Third nucleotide sequence coding for the first self-cleaving ribozyme, preferably a 5' self-cleaving ribozyme - second nucleotide sequence coding for the second functional transcription product - fourth nucleotide
  • Lariat capping ribozyme is only necessary if a similar natural transcription product of the cell would have had or needed a 5' cap.
  • the Lariat capping ribozyme introduces a 5' cap functioning as a substitute to a natural 5' m 7 G cap.
  • the cell can be any cell, for example a eucaryotic or procaryotic cell.
  • the cell is a plant cell, an insect cell, a mammalian cell, a non-human animal cell, or a human cell.
  • the cell is preferably not a human embryonic stem cell obtained by a method involving the destruction of a human embryo, and preferably the cell is not a human germ cell or human zygote.
  • the cell is preferably genetically engineered according to the invention such that the cell has a resistance against bacteriophages.
  • the cell may be a pluripotent stem cell, preferably induced pluripotent stem cell (iPSC), i.e. a pluripotent stem cell generated directly from a somatic cell, e.g. a human (induced) pluripotent stem cell, including a human embryonic stem cell.
  • iPSC induced pluripotent stem cell
  • a genetically engineered bacterial cell is preferably engineered in a manner that the first nucleotide sequence coding for the first transcription product is transcribed or more transcribed at some point of or during the entire infection of the bacterium by a bacteriophage.
  • the application of the invention in bacteria is not restricted to counteracting resistances against bacteriophages, but can, for example, also be used for the stoichiometrically exactly defined production of two or more functional RNAs.
  • the invention also relates to a transgenic plant or non-human animal comprising a cell of the first aspect of the invention.
  • the present invention provides for a nucleic acid based genomic system or mechanism for transcriptionally synchronizing a first functional transcription product, for example an mRNA that is finally translated into a protein, product with at least one second functional transcription product (“output”), first nucleotide sequence coding for the first functional transcription product can be any suitable sequence for which transcriptional synchronization with a second functional transcription product holds any desired value.
  • the first nucleotide sequence is preferably included in a nucleic acid that is introduced in the genome of a cell or organism, along with the preferred design for transcriptional synchronization as described here.
  • the sequences coding for the functional transcription products are preferably (proto-)oncogenes such as ERBB2, HRAS, MYC, SRC, TERT, BCL2, EGFR, etc., or tumor suppressor genes such as TP53, RBI, APC, BRCA1, BRCA2, HLA-A, etc.
  • a respective endogenous or synthetic sequence already present in the target genome will preferably be deleted and replaced with a suitable design for transcriptional synchronization according to the invention, including the respective sequences coding for the respective functional transcription products.
  • Transcriptional synchronization can, for example, also be used to transfect multicellular organisms under avoidance of mosaic mutants, i.e. a mixture of transfected and non-transfected cells.
  • induced pluripotent stem cells iPSCs
  • a modified cell cycle related gene e.g. cyclin A/cdk2
  • a cell-to-cell mobile inhibitor of the unmodified variant of this gene e.g. a shRNA, siRNA, transcription factor
  • the cell cycle related gene is, for example, encoded by the first nucleotide sequence, and the gene inhibitor is encoded by a second nucleotide sequence.
  • the synchronized cell cycle gene is modified to be resistant to said inhibitor.
  • modified iPSCs would be implanted into a patient’s respective stem cell niche. There, native and modified stem cells would receive proliferation signals alike, resulting in an activation of cell cycle genes.
  • cyclin A/cdk2 is transcribed
  • the iPSCs would also transcribe the inhibitor in a synchronized manner according to the invention. While the cyclin A/cdk2 variant of the iPSCs is designed to be resistant to the inhibitor, the native variant is not.
  • the iPSCs engineered according to the invention are unaffected by the inhibitor and are free to proliferate.
  • the modified iPSCs gain an advantage over the native stem cell population and can proliferate at a higher rate, outcompeting their native counterpart. Over time, this would result in a stem cell niche that is exclusively comprised of the modified iPSCs, which in turn would replenish their associated tissue with new and modified differentiated cells. This use of transcriptional synchronization would result in a regulated stem cell niche replacement that also avoids creating a mosaic mutant stem cell niche.
  • This system can be further used to create a iPSC niche that is “updatable”: After a first generation iPSC niche has been established, a second generation of iPSCs could have the same cell cycle gene synchronized with an inhibitor being designed to target the former resistant cell cycle gene of the first generation iPSCs, thus leading to the same effect as described above for the native stem cell niche compared with the first generation of modified iPSCs.
  • This can be used for making adjustments if, for example, gene therapy results in unforeseen side effects, which would be impossible to correct using other current delivery methods for gene therapy. It can also be used to implement new or improved gene modifications. A useful application of this system would be to refresh stem cell populations if their genes have amassed detrimental mutations over time.
  • the invention relates to a vector or vector system for the transfection, preferably stable transfection, of a cell, the vector(-system) comprising: a) a first nucleotide sequence coding for a first functional transcription product, b) a second nucleotide sequence coding for a second functional transcription product, c) a third nucleotide sequence coding for a first self-cleaving ribozyme, and d) a fourth nucleotide sequence coding for a second self-cleaving ribozyme, the second nucleotide sequence coding for the second functional transcription product being flanked by the third and fourth nucleotide sequences coding for the first and second selfcleaving ribozyme, wherein the vector or vector system is configured to introduce the nucleotide sequences of a) to d) into a transcription unit of the cell between a promoter and a termination signal of the transcription unit.
  • the vector or vector system (vector system meaning a plurality of vectors acting together) is configured in such a way to produce a cell of the first aspect of the invention when introduced into the cell.
  • the skilled person is aware of different vector constructs or vector systems suitable for this purpose, and will choose a suitable vector or vector system, and adapt it, if necessary.
  • the vector or vector system of the invention thus is designed to introduce, into a cell, a transcription unit as described above. Consequently, any disclosure of a feature of the transcription unit described above in relation to the genetically engineered cell also applies to the vector or vector system of the invention.
  • the vector or vector system thus preferably comprises all nucleotide sequences according to a) to d) above, and, if necessary, further functional nucleic acid elements to insert the nucleotide sequences of a) to d) above and to replace a genomic sequence encoding a functional transcription product with the mechanism of the invention necessary for transcriptional synchronization.
  • the vector or vector system also comprises an element or multiple elements that are necessary for the deletion of the sequence encoding the genomic functional transcription product (e.g. sgRNA and Cas9).
  • the vector or vector system preferably includes an element or elements enabling expression of the aforementioned elements inside a target cell (e.g. a plasmid backbone comprising necessary regulatory elements such as promoters etc.).
  • the vector or vector system of the invention may also include an element or multiple elements that are necessary to guide and transport the vector/vector system to its site of intended action (e.g. viral delivery systems, such as a Lentiviral vector).
  • the vector or vector system of the invention may only comprise the nucleotide sequences of features b) to d) above, i.e. lack feature a.
  • the vector or vector system is configured to introduce the nucleotide sequences of features b) to d) above into a transcription unit of the cell between a promoter and a termination signal of the transcription unit, the transcription unit already comprising feature a), i.e. a first nucleotide sequence coding for a first functional transcription product, for example an endogenous gene coding for an endogenous protein or a nucleotide sequence coding for a functional RNA other than an mRNA, e.g. tRNA, siRNA, antisense RNA etc.
  • This vector or vector system is particularly suitable for genetic modification of a transcription unit of a cell for establishing a transcription synchronization mechanism of the invention, where the first nucleic acid coding for the first functional transcription product, e.g. an endogenous gene, shall not be replaced with an identical nucleic acid coding for the same functional transcription product.
  • the vector or vector system of the invention codes for an mRNA encoding a first protein, preferably an endogenous protein of the cell, or a first functional non-coding RNA molecule, preferably an endogenous functional non-coding RNA molecule of the cell.
  • the third nucleotide sequence codes for a functional non-coding RNA or an mRNA encoding a protein.
  • the third nucleotide sequence codes for a 5' self-cleaving ribozyme and the fourth nucleotide sequence codes for a 3' self-cleaving ribozyme.
  • At least the second functional nucleotide sequence does not code for a gRNA. It is further preferred that neither the first nor the second functional nucleotide sequence code for a gRNA.
  • the vector or vector system being particular useful for the transfection of induced pluripotent stem cells (iPSCs), the first nucleotide sequence encodes a modified cell cycle related gene, and the second nucleotide sequence encodes an inhibitor of the unmodified (native) variant of said gene, the cell cycle related gene being modified to be resistant to the inhibitor, and the inhibitor being cell-to-cell mobile, i.e. being able to move from cell to cell.
  • iPSCs induced pluripotent stem cells
  • a) the second nucleotide sequence coding for the second functional transcription product and flanked by the third and fourth nucleotide sequence coding for a first and second self-cleaving ribozyme is arranged after the first nucleotide sequence coding for the first functional transcription product, the first nucleotide sequence coding for the first functional transcription product preferably being followed by a fifth nucleotide sequence encoding a Poly A tail, the fifth nucleotide sequence encoding a Poly A tail preferably being followed by a sixth nucleotide sequence coding for a third self-cleaving ribozyme, preferably a 3' self-cleaving ribozyme, or b) the second nucleotide sequence coding for the second functional transcription product and flanked by the third and fourth nucleotide sequence coding for a first and second self-cleaving ribozyme, is arranged before the first nucleotide sequence coding for the first functional transcription product, the first nu
  • nucleic acid sequences are present: a) first nucleotide sequence coding for the first functional transcription product - fifth nucleotide sequence encoding a Poly A tail - sixth nucleotide sequence coding for the third self-cleaving ribozyme, preferably a 3' self-cleaving ribozyme - spacer sequence - third nucleotide sequence coding for the first self-cleaving ribozyme, preferably a 5' self-cleaving ribozyme - second nucleotide sequence coding for the second functional transcription product - fourth nucleotide sequence coding for the second self-cleaving ribozyme, preferably a 3' selfcleaving ribozyme, or b) third nucleotide sequence coding for the first self-cleaving ribozyme, preferably a 5' selfcleaving ribozyme - second nu
  • the invention relates to a method for the transcriptional synchronization of two or more functional transcription products of a cell, the method comprising:
  • the transcription unit comprising a promotor and a termination signal i. a first nucleotide sequence coding for a first functional transcription product, ii. a second nucleotide sequence coding for a second functional transcription product, iii. a third nucleotide sequence coding for a first self-cleaving ribozyme, and iv. a fourth nucleotide sequence coding for a second self-cleaving ribozyme, the second nucleotide sequence coding for the second functional transcription product being flanked by the third and fourth nucleotide sequences coding for the first and second selfcleaving ribozyme.
  • the method of the invention is preferably used to essentially replace an existing transcription unit of a cell, i.e. a transcription unit naturally present in the cell or previously introduced into the genome of the cell, or at least the relevant elements between the promoter and the termination signal of the transcription unit with a transcription unit having the elements according to features i. to iv. above.
  • the first nucleotide sequence coding for the first functional transcription product e.g. an ORF of a protein
  • the first functional transcription product is identical to and replaces the nucleotide sequence naturally present or previously introduced into that transcription unit.
  • the present invention also encompasses a variant of the method where the first nucleotide sequence coding for a first functional transcription product is not separately introduced into the cell (step i. above), but already present in the genome of the cell.
  • the first nucleotide sequence coding for a first functional transcription product is already present (either naturally or previously introduced) between the promotor and the termination signal of the transcription unit, and only steps ii. to iv. are performed.
  • the first nucleotide sequence codes for an mRNA encoding a first protein, preferably a genomic or endogenous protein of the cell, or a first functional non-coding RNA molecule, preferably an endogenous functional noncoding RNA molecule of the cell.
  • the first and second nucleotide sequence code for different functional transcription products e.g. that, in case the first nucleotide sequence codes for a coding RNA molecule, e.g. an mRNA coding for a first protein, the second nucleotide sequence codes either for a different coding RNA, e.g. an mRNA coding for a second protein differing from the first protein, or a non-coding RNA molecule, e.g. siRNA, tRNA or antisense RNA. It is further preferred that not both the first and second functional nucleotide sequence code for a gRNA, i.e. a CRISPR guide RNA.
  • the second functional nucleotide sequence does not code for a gRNA, further preferred neither the first nor the second functional nucleotide sequence code for a gRNA.
  • the first nucleotide sequence codes for an endogenous gene product, preferably an endogenous protein
  • the second nucleotide sequence codes for a functional coding or non-coding RNA molecule, the functional coding RNA molecule being different from an mRNA coding for the first protein.
  • the transcription unit may comprise more than one second nucleotide sequences coding for a second functional transcription product, e.g. two, three, four or more second nucleotide sequences, such that all these second functional transcription products are transcriptionally synchronized with the first nucleotide sequence coding for the first functional transcription product. It is possible that two or more of the second nucleotide sequences code for the same functional transcription product. It is preferred, however, that the second nucleotide sequences code for different functional transcription products, e.g. several different mRNA coding for different proteins, and/or several different non-coding functional RNAs.
  • each of the further second nucleotide sequences will also be flanked by suitable self-cleaving ribozymes.
  • the skilled person will, depending on the nature and purpose of the respective further second functional transcription product, choose a suitable combination of the flanking ribozymes for each of the further second functional transcription products.
  • the second nucleotide sequence codes for a functional non-coding RNA or an mRNA encoding a protein.
  • the functional non-coding RNA does not code for a gRNA.
  • the third nucleotide sequence codes for a 5' selfcleaving ribozyme and the fourth nucleotide sequence codes for a 3' self-cleaving ribozyme.
  • the third nucleotide sequence may, for example, code for a Hammerhead ribozyme
  • the fourth nucleotide sequence may, for example, code for a Hepatitis delta virus (HDV) ribozyme.
  • HDV Hepatitis delta virus
  • the cell is a eukaryotic cell, wherein, in the direction from the promoter to the termination signal, the second nucleotide sequence coding for the second functional transcription product and flanked by the third and fourth nucleotide sequence coding for a first and second self-cleaving ribozyme, is introduced after the first nucleotide sequence coding for a first functional transcription product, wherein a fifth nucleotide sequence encoding a Poly A tail is preferably introduced after the first nucleotide sequence coding for a first functional transcription product, if the first functional transcription product is an mRNA, and wherein a sixth nucleotide sequence coding for a third self-cleaving ribozyme, preferably a 3' self-cleaving ribozyme, is preferably introduced after the fifth nucleotide sequence encoding a Poly A tail. If a functional transcription product does not need a Poly A tail, the Poly A tail is not included.
  • the second nucleotide sequence coding for the second functional transcription product and flanked by the third and fourth nucleotide sequence coding for a first and second self-cleaving ribozyme is introduced after the first nucleotide sequence coding for a first functional transcription product, a spacer sequence is preferably introduced before the third nucleotide sequence coding for the first self-cleaving ribozyme.
  • the cell is a eukaryotic cell, wherein, in the direction from the promoter to the termination signal, the second nucleotide sequence coding for the second functional transcription product and flanked by the third and fourth nucleotide sequence coding for a first and second self-cleaving ribozyme, is introduced before the first nucleotide sequence coding for a first functional transcription product, wherein a seventh nucleotide sequence coding for a Lariat capping ribozyme, is preferably introduced directly before the first nucleotide sequence coding for a first functional transcription product.
  • a Lariat capping ribozyme is only included, if the first functional transcription product or any further functional transcription product needs a 5' capping-like mechanism in order to function as intended.
  • a spacer sequence is preferably introduced after the fourth nucleotide sequence coding for the second self-cleaving ribozyme.
  • the order of the first nucleotide sequence coding for a first functional transcription product, the third nucleotide sequence coding for the first self-cleaving ribozyme, the fourth nucleotide sequence coding for the second self-cleaving ribozyme, the second nucleotide sequence coding for the second functional transcription product, the seventh nucleotide sequence coding for the Lariat capping ribozyme (if necessary), and the spacer sequence are introduced in such a way between the promoter and the terminations signal that the following order results:
  • Third nucleotide sequence coding for the first self-cleaving ribozyme preferably a 5' selfcleaving ribozyme - second nucleotide sequence coding for the second functional transcription product - fourth nucleotide sequence coding for the second self-cleaving ribozyme, preferably a 3' self-cleaving ribozyme - spacer sequence - seventh nucleotide sequence coding for the Lariat capping ribozyme (if necessary) - first nucleotide sequence coding for a first functional transcription product.
  • the cell is a plant cell, an insect cell, a mammalian cell, a non-human animal cell, or a human cell.
  • the cell is a human cell
  • the method of the invention is preferably not used to modify the germ line genetic identity of a human being.
  • the cell may be a pluripotent stem cell, e.g. a human pluripotent stem cell, including a human embryonic stem cell.
  • the cell is an induced pluripotent stem cell (iPSC), i.e. a pluripotent stem cell generated directly from a somatic cell.
  • iPSC induced pluripotent stem cell
  • the cell is a prokaryotic cell, e.g. a bacterial or archaeal cell.
  • the method is preferably used for the introduction of a resistance against bacteriophages.
  • the method of the invention is used for treating or preventing a disease, preferably a genetic disease or cancer, in a subject, e.g. a human subject.
  • a disease preferably a genetic disease or cancer
  • the invention thus also relates to a method of treatment or prevention of a disease, for example a genetic disease, in a subject, comprising the steps of
  • the transcription unit comprising a promotor and a termination signal i. a first nucleotide sequence coding for a first functional transcription product, preferably an open reading frame, ORF, coding for an endogenous gene product, ii. a second nucleotide sequence coding for a second functional transcription product, iii. a third nucleotide sequence coding for a first self-cleaving ribozyme, and vi.
  • a fourth nucleotide sequence coding for a second self-cleaving ribozyme the second nucleotide sequence coding for a second functional transcription product being flanked by the third and fourth nucleotide sequences coding for the first and second selfcleaving ribozyme, such that the first functional transcription product of the cell is transcriptionally synchronized with the second functional transcription product.
  • the cell is not a human embryonic stem cell obtained by a method involving the destruction of a human embryo. Further, it is preferred that the cell is not a human gamete or human zygote. Further, the method of the invention is preferably not used to modify the germ line genetic identity of a human being.
  • the cell may be a pluripotent stem cell, e.g. a human pluripotent stem cell, including a human embryonic stem cell.
  • the cell is an induced pluripotent stem cell (iPSC), i.e. a pluripotent stem cell generated directly from a somatic cell.
  • iPSC induced pluripotent stem cell
  • the method being particular useful for genetic engineering of induced pluripotent stem cells (iPSCs), for example as part of gene therapy
  • the first nucleotide sequence encodes a modified cell cycle related gene
  • the second nucleotide sequence encodes an inhibitor of the unmodified (native) variant of said gene
  • the cell cycle related gene being modified to be resistant to the inhibitor
  • the inhibitor being cell-to-cell mobile, i.e. being able to move from cell to cell.
  • FIG. 1 Schematic illustration of two alternative embodiments of the invention related to eukaryotic cells, called “3' design” and “5' design”, respectively.
  • ORF open reading frame
  • SCRz self-cleaving ribozyme
  • TS terminating sequence
  • a) The eukaryotic 3' design has a variable output that is flanked by a 5' and a 3' self-cleaving ribozyme. After transcription, the output is cut from the pre-mRNA by the ribozymes and can fulfill its intended function. The pre-mRNA is also cut by another 3' self-cleaving ribozyme, which releases the transcribed ORF together with a synthetic poly-A tail.
  • the eukaryotic 5' design is similar to the 3' design, however, the box comprising the output flanked by self-cleaving ribozymes is located directly after the promoter.
  • the design also includes a lariat capping ribozyme (LCrz), which is separated from the 3' self-cleaving ribozyme by a spacer. After transcription, the output is cut from the pre-mRNA and can function as intended.
  • the mRNA, containing the ORF is capped at its 5' end by the LCrz. It is polyadenylated like every other mRNA.
  • Figure 2 Molecular circuit of a resistance mechanism against cancer development based on transcriptional synchronization according to the invention.
  • both STAR and AntiSTAR are flanked by a 5' and 3' self-cleaving ribozyme (SCRz).
  • the 5' SCRz here is a hammerhead ribozyme (HHRz), while the 3' SCRz could be a hepatitis delta virus (HDV) ribozyme.
  • FIG. 3 Synchronization cassette inserted in plasmid pEGFP-Nl and used in experiments with HeLa cells.
  • Underlined nucleotides Synthetic poly-A tail, double underlined nucleotides: Hepatitis delta virus ribozyme; framed nucleotides (48 adenines): Spacer; Underlined with dashed lines: Hammerhead ribozyme (adjusted to target shThr2); bold: shThr2.
  • the 6nt sequences flanking the spacer sequence are restriction sites of restriction enzymes.
  • Figure 1 schematically shows the genomic structure of preferred embodiments of two alternative embodiments (also called “3' or 5' designs” in the following) of a eukaryotic cell of the invention.
  • One design uses a synthetic poly adenine tail, enabling a box composed of a nucleotide sequence coding for a variable output (i.e. variable second functional transcription product, variable functional RNA molecule) flanked by nucleotide sequences encoding selfcleaving ribozymes (“Rz-vO-Rz box”; may also be called “device” in the following) to be placed between an open reading frame (ORF) and a corresponding transcription terminating sequence (TS).
  • ORF open reading frame
  • TS transcription terminating sequence
  • an ORF is only an example for a first nucleotide sequence coding for a first functional transcription product. This could also be replaced with other transcription product encoding sequence, e.g. a sequence coding for a tRNA.
  • This is called the 3' design, because the device is closer to the 3' end of the DNA sequence.
  • no poly A tail is used.
  • the alternative eukaryotic version is called 5' design, where the Rz-vO-Rz box is situated closer to the 5' end of the DNA sequence.
  • a LCrz is used to enable the device to be placed between a promoter and a corresponding ORF.
  • no LCrz is used.
  • first transcription product (a copy of the genomic version, here an ORF)
  • the self-cleaving ribozyme may itself be meant (e.g. in the context of an mRNA) or the nucleic acid sequence coding for self-cleaving ribozyme is meant (e.g. in the context of the coding DNA).
  • the term “output” used here may, depending on the context, not only relate to the immediate transcription product, i.e. an mRNA or a functional non-coding RNA, but also to the product resulting from a translation of the mRNA.
  • the Rz-vO-Rz box On the DNA level, the Rz-vO-Rz box is inserted into a naturally occurring gene between its ORF and transcription terminating sequence.
  • the naturally occurring first nucleotide sequence coding for the first transcription product here an ORF
  • the ORF the naturally occurring first nucleotide sequence coding for the first transcription product
  • the ORF the ORF
  • the Rz-vO-Rz box comprises a synthetic poly-A tail, followed by a 3' self-cleaving ribozyme, a spacer, a 5' self-cleaving ribozyme, a variable output and another 3' self-cleaving ribozyme.
  • a pre-mRNA is synthesized, everything except the promoter and a part of the terminating sequence is transcribed. After transcription, the ribozymes on the pre-mRNA undergo self-cleavage and the ORF gets a 5' cap. The part containing the ORF is now a functional mRNA and can be exported and translated into a protein due to the 5' cap and synthetic poly-A tail.
  • the synthetic poly-A tail enables the nuclear export and cytoplasmic translation of the mRNA. If no synthetic poly-A tail would be added, the ribozymes would be likely to prevent natural polyadenylation of the mRNA, which would inhibit nuclear export and cytoplasmic translation.
  • the part that contains the 3' and 5' self-cleaving ribozyme as well as the spacer will be degraded by nucleases.
  • the spacer is advantageous to give the ribozymes enough space to assume their active secondary structure without interfering with each other.
  • the DNA sequence of the spacer was chosen to be poly A to minimize secondary structure formation and interaction with neighboring sequences. Adenine was chosen for the spacer, so that an interaction with the poly-A tail is as unlikely as possible after transcription.
  • Thymine on the mRNA would interact readily with the poly-A tail and act as an antisense inhibitor. Cytosine and guanine might hamper the transcription by being more prone to stay doublestranded. Thus, adenine is preferably used for the spacer on the DNA sequence.
  • the output can function independently after being cut out by the ribozymes. The last 3' self-cleaving ribozyme and the attached part of the transcribed terminating sequence are degraded by nucleases.
  • the stability of the synchronized part of the 3' design can be additionally influenced by varying the length of the synthetic poly-A tail: The shorter the tail, the earlier the synchronized part is degraded by the cell.
  • FIG. lb An embodiment of the eukaryotic 5' design is depicted in figure lb. Differences compared to the 3' design are:
  • the Rz-vO-Rz box is located behind the promoter and contains a LCrz (if necessary), which creates a 5' cap on the mRNA of the synchronized gene (e.g. needed for an ORF). Polyadenylation is done naturally by the cell’s proteins.
  • LCrz is used to imitate 5' capping of the mRNA.
  • a preferred embodiment of the eukaryotic 3' design comprises on the DNA level: A poly-A sequence (if necessary) followed by a 3' self-cleaving ribozyme, a spacer, a 5' selfcleaving ribozyme, a variable output and a 3' self-cleaving ribozyme.
  • the 5' design preferably comprises on the DNA level of a 5' self-cleaving ribozyme, a variable output, a 3' self-cleaving ribozyme, a spacer and a LCrz (if necessary).
  • a preferred prokaryotic design comprises at least one Rz-vO-Rz box composed of 5' self-cleaving ribozyme, variable output and 3' self-cleaving ribozyme.
  • the box can be placed upstream or downstream of an ORF or any other suitable first sequence coding for a first transcription product.
  • RNA sequences are given.
  • Lariat capping ribozyme (LCrz) (SEQ ID NO: 01):
  • Hepatitis delta virus ribozyme (SEQ ID NO: 02):
  • N’s in bold in the sequences depend on the bases that are downstream of the HHRz.
  • Type-P5 twister ribozyme SEQ ID NO: 08:
  • Twister sister ribozyme (SEQ ID NO: 09): ggACCCGCAAGGCCGACGGCAUCCGCCGCCGCUGGUGCAAGUCCAGCCGCCCCGG GGCGGGCGCUCAUGGGUAAAC
  • AAAAAAAAAAAAAAAAAAA ( 72 nt)
  • AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA AAA ( 300 nt) Note: The stability of the mRNA can be modulated by choosing different lengths of the synthetic poly-A tail of the DNA. Other lengths than the given ones are also possible.
  • tumor suppressor genes In cancer cells, the transcription of tumor suppressor genes is downregulated by genetic/epigenetic means (Wang et al., 2018; Liu et al, 2016) or is even suspended through gene deletions (Dong, 2001; Cai and Sablina, 2016; Cooper, 2000). All this, while the expression of proto-oncogenes is increased, thus, creating oncogenes (Cooper, 2000).
  • a resistance-mechanism for cancer cells based on the invention relies on sensing these genetic alterations by comparing the transcriptional activity of (proto-)oncogenes and tumor suppressor genes (see Figure 2).
  • This is possible by adding a DNA sequence for STAR (Short Transcription Activating RNA) after a common (proto-)oncogene DNA sequence (e.g. after an ORF), thus, synchronizing the (proto-)oncogene with STAR (see Fig. 2A).
  • STAR is able to activate the transcription of a target gene by binding to an upstream transcription terminator t500, which is located in front of the gene right after its promoter (Chappell et al., 2015).
  • the terminator t500 prevents the transcription of the gene behind it via a stem-loop (Chappell et al., 2015). Yet, if a STAR molecule binds to the t500 terminator, transcription is enabled (Chappell et al., 2015).
  • a 5' hammerhead ribozyme (Gao and Zhao, 2014) is added to the 5' end of the STAR DNA sequence.
  • the 3' end of STAR has a 3' self-cleaving ribozyme added, e.g. the hepatitis delta virus ribozyme (HDVRz) (Gao and Zhao, 2014).
  • Flanking the STAR sequence with self-cleaving ribozymes results in a STAR transcript that is independently mobile from the oncogene and has no overhang from the transcription terminator of the gene, which is situated at the outermost 3' end of the gene.
  • Anti STAR DNA sequences are added in a similar way to (multiple) tumor suppressor genes. They, too, will be insulated from the normal transcript and terminator of the gene by flanking self-cleaving ribozymes at the 5' and 3' end of the AntiSTAR sequence.
  • the endogenous ORFs of the (proto-)oncogenes and tumor suppressor genes will have poly-A sequence added directly downstream to enable nuclear export and cytoplasmic translation.
  • STAR and AntiSTAR inhibit each other via antisense base pairing.
  • the mechanism is further depending on an overall equal or higher transcriptional activity of tumor suppressor genes as long as the cell is healthy.
  • tumor suppressor genes for every (proto-)oncogene that is modified in the above- mentioned way, there should be one or more tumor suppressor genes chosen, which (always) have a (combined) much higher transcription activity than the (proto-)oncogene.
  • the default state of a cell harboring this mechanism will be that the STAR-activity is quenched by the much more abundant Anti-STAR molecules.
  • This sensing mechanism qualifies as a first control and safety mechanism.
  • the transcription of these genes can be induced by STAR.
  • a second safety mechanism could be constituted by the therapeutic output itself, which can be induced by STAR: After transcriptional activation via STAR, a proton channel could be produced by the cell (see Fig. 2C). This proton channel would locate to the cell membrane and enable passive proton transport across the membrane. Thus, in an acidic tumor microenvironment, the channel will import protons into the cell. This will acidify cancer cells, while also reducing the extracellular proton concentration. This should result in cancer cell termination due to the now acidic cellular milieu, futile ATP consumption for proton export (Whitton et al., 2018), glucose deprivation (Xun et al., 2017) and a reduced capability for tumor invasion (Estrella et al., 2013).
  • the proton channel could be e.g. the otopetrin proton channels Otopl or Otop3 (Saotome et al, 2019) or a modified viral M2 proton channel (Cady et al., 2009). If this mechanism is used prior to the onset of cancer, it might prevent any later onset by denying the cancerous cell the ability to effectively unload excess protons, while also keeping a microenvironment that is more favorable for an immune response.
  • V-ATPase is used by cancer cells to unload excess protons and prevent intracellular acidification (Whitton et al., 2018).
  • inhibiting either transcription, translation or protein function of V-ATPase should acidify and kill cancer cells, while other proton unloading enzymes (e.g. monocarboxylate transporters, Na + /H + exchanger (Aoi and Marunaka, 2014) and carbonic anhydrases (Spugnini et. al, 2015) might have to be targeted too.
  • AntiSTAR genes should be located as close as possible to STAR (for direct quenching of STAR) and to the t500 terminator (close-target quenching). Thus, quenching STAR activity is less a matter of chance. Instead, it would be much more related to differences in quantity between STAR and AntiSTAR, while stochastic problems caused by diffusion area are reduced. Only a much higher STAR production or much lower AntiSTAR production would be sufficient for STAR-target activation. Also, the regulation via the RNA-based STAR/AntiSTAR-system should be quickly reversible due to fast RNA degradation (Chappell et al., 2017). The system described above is depicted in figure 2. It is to be noted, that the figure shows a simplified version of the mechanism for transcriptional synchronization, which does not contain poly-A tails.
  • Transcriptional synchronization according to the invention can also be used to create synthetic pathogen resistance in plants, e.g. crop plants.
  • any gene, which transcription rate is influenced by the pathogen can be transcriptionally synchronized with a resistance output against said pathogen.
  • the FAD7 gene could be transcriptionally synchronized with an RNAi against fungal infections: If a plant cell is wounded, FAD7 expression is upregulated (Nishiuchi et al., 1999). Fungi need to penetrate the plant cell for infection, thus upregulating the expression of FAD7 (Kirsch et al., 1997). If FAD7 is now transcriptionally synchronized with e.g.
  • RNAi against the respective fungi the expression of the RNAi is triggered upon infection.
  • Using transcriptional synchronization against plant pathogens has at least two distinctive advantages compared to using either constitutive or additional endogenous promoters coupled with a resistance mechanism: First, a constitutive promoter for a resistance is always active - even if the pathogen is not present. This means, that the plant cell is wasting its resources to produce a resistance that is most of the time not needed. This again will influence the growth and harvest yield of crop plants in a negative way, depending on how many resistances are used in the plant. Second, if the resistance is controlled by using an endogenous promoter of the plant (e.g.
  • the promoter is not solely controlling the transcription rate of a gene, but the genetic context is also important, including enhancer and silencer sequences, intronic effects, etc.
  • the promoter would be necessary to test every resistance, which is only regulated by an endogenous promoter, if its expression is regulated as intended. Depending on the number of needed resistances, this can become time- and cost-intensive.
  • Transcriptional synchronization according to the invention solves both problems, by using the existing transcriptional regulation pathways of the cell to express the synthetic resistance only if needed.
  • the endogenous gene can be synchronized with a transcription activating output, which in turn activates the transcription of multiple resistance genes.
  • the synchronization would hardly influence the transcription time for the synchronized endogenous gene, while still being able to activate a plethora of resistance genes.
  • multiple endogenous genes can be synchronized with one (or more) synthetic resistance genes so that one pathogen triggers one (or more) resistance gene(s) by upregulating the expression of all the synchronized endogenous genes.
  • Using multiple inputs (synchronized endogenous genes) and multiple outputs (synthetic resistances) would make it near impossible for any pathogen to adapt to the synthetic resistances by evolutionary means. All this, while having a relatively low impact on the plant metabolism.
  • the number of genetic modifications can be reduced significantly compared to introducing the same number of resistances by using other methods, making transcriptional synchronization very cost-efficient.
  • HeLa cells were transfected with a plasmid consisting of the commercially available plasmid pEGFP-Nl (for sequence see SEQ ID NO: 18; see also GenBank entry U55762.1) as a backbone and containing a synchronization cassette shown in Fig. 3 (see SEQ ID NO: 19).
  • plasmid consisting of the commercially available plasmid pEGFP-Nl (for sequence see SEQ ID NO: 18; see also GenBank entry U55762.1) as a backbone and containing a synchronization cassette shown in Fig. 3 (see SEQ ID NO: 19).
  • transfection, RNA extraction and qPCR protocols see, for example, Kirchner et al. 2017.
  • the backbone pEGFP-Nl included the first synchronized output (EGFP), i.e. the first functional transcription product, which is a fluorescent protein.
  • the shRNA (shThr2) of the synchronization cassette was supposed to target threonine tRNA of HeLa cells.
  • Table 1 Fluorescence values of experiment 1 for treated and untreated HeLa cells using three biological replicates split in four physical replicates each. Unit of measurement was RFU (Relative Fluorescence Units).
  • Table 2 Fluorescence values of experiment 2 for treated and untreated HeLa cells using three biological replicates split in four physical replicates each. Unit of measurement was RFU (Relative Fluorescence Units).
  • threonine tRNA levels were measured using RT-qPCR. After reverse transcription of the total RNA of 3 biological replicates, two technical replicates were used for each biological replicate. The tRNA Thr (CGU) levels were analyzed using the QuantiFast SYBR Green RT-PCR kit (from Qiagen).
  • the tRNA Thr (CGU) and 5S rRNA were amplified by using the following primers: tRNA Thr (CGU) forward (GGCCAAGTGGTAAGGC) and tRNA Thr (CGU) reverse (AGGCACGGACGGG), 5S rRNA forward (CCATACCACCCTGAACGC) and 5S rRNA reverse (GTATTCCCAGGCGGTCTC).
  • the tRNA Ct values were normalized to 5S rRNA Ct values.
  • the fold change was calculated using the double delta Ct (ddCt) analysis method.
  • CGU tRNAThr
  • Table 3 Ct values for experiment 1 obtained by RT-qPCR.

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Abstract

The invention relates to the transcriptional synchronization of two or more functional transcription products. In the invention a first functional transcription product and a second transcription product are placed under exactly the same transcriptional regime, i.e. the same transcription regulatory element(s) of a given transcription unit, in order to have both transcription products transcribed in a synchronized manner.

Description

TRANSCRIPTIONAL SYNCHRONIZATION OF TWO OR MORE FUNCTIONAL TRANSCRIPTION PRODUCTS
The invention relates to a cell being genetically engineered to have two or more functional transcription products transcriptionally synchronized with each other, and a method for the transcriptional synchronization of two or more functional transcription products. Further, the invention relates to a vector or vector system, and to a transgenic plant or non-human animal.
Genetic modification of cells is well known and is widely used to create cells with desired properties. Genetic engineering methods are, for example, frequently used to introduce foreign genes into cells, e.g. plant cells, in order to make the cells resistant to particular pathogens.
Such genetically engineered cells may, for example, be able to produce one or more proteins or an RNA product, for example an antisense RNA or small interfering RNA (siRNA) counteracting infestation by a pathogen. Genetically manipulating cells has not only been performed with prokaryotic cells, plant cells or non-human animal cells, for example, but has also been done or at least suggested in relation to mammalian cells, including human cells, to treat or prevent diseases like genetic diseases or cancer.
Although numerous approaches are known in the prior art for altering cells on the genomic level in order to equip the cells with properties that they naturally do not have or at least have only insufficiently, there is still a need for a generally and reliably applicable approach for equipping cells with desired properties, for example for protecting cells, and organisms comprising these cells, against external dangers such as pathogens or an internal transformation, e.g. to a tumor cell.
It is an object of the invention to provide such an approach.
In a first aspect the invention relates to a genetically engineered cell, the cell being genetically engineered to comprise a transcription unit with a promotor and a termination signal, the transcription unit comprising, between the promotor and the termination signal, a) a first nucleotide sequence coding for a first functional transcription product, b) a second nucleotide sequence coding for a second functional transcription product, c) a third nucleotide sequence coding for a first self-cleaving ribozyme, and d) a fourth nucleotide sequence coding for second self-cleaving ribozyme, the second nucleotide sequence coding for the second functional transcription product being flanked by the third and fourth nucleotide sequence coding for the first and second self-cleaving ribozyme.
The genetically engineered cell of the invention is genetically engineered in a way that the cell has a first functional transcription product, for example an endogenous gene product, transcriptionally synchronized with a second functional transcription product, e.g. a functional non-coding RNA molecule. Both the first and the second functional transcription product are encoded in the same transcription unit, i.e. the DNA coding for the first and the second functional transcription product are transcribed under the same transcription regulatory element(s). Consequently, if the first functional transcription product, for example an endogenous gene is transcribed the DNA coding for the second functional transcription product is also transcribed in the same amount and rate as the endogenous gene. It is also possible to transcriptionally synchronize a first functional transcription product, e.g. an endogenous gene, with more than one further functional transcription products, e.g. two, three or more further functional transcription products which may be identical or different from each other.
The invention can provide a plethora of different cells with different properties. The properties of the cell can be adapted to a variety of requirements and uses, including, but not limited to the protection against pathogens and cancerous transformation.
The core idea of the invention is to place a first functional transcription product and a second transcription product under exactly the same transcriptional regime, i.e. the same transcription regulatory element(s) of a given transcription unit, in order to have both transcription products transcribed in a synchronized manner. For example, a genomic gene, e.g. an endogenous gene, and a nucleic acid, preferably a DNA, encoding a functional RNA molecule, can be placed under exactly the same transcriptional regime, i.e. the same transcription regulatory element(s) of a given transcription unit, in order to have both the gene product and the functional RNA molecule transcribed in a synchronized manner. As a consequence, both the first and second functional transcription product are contained in a common pre-mRNA molecule. The second functional transcription product is flanked by suitable self-cleaving ribozymes such that the second functional transcription product is automatically released from a pre-mRNA transcribed from the transcription unit containing the first functional transcription product and the second functional transcription product. The invention can, for example, be used for creating disease and pathogen resistance mechanisms, analysis of gene function, or for other regulatory applications in eukaryotic and prokaryotic cells. In case of an mRNA as first or second functional transcription product, i.e. if a protein is finally to be produced in the cell from the first or second functional transcription product, the synchronization mechanism is configured in a way that elements necessary for the translation of the mRNA, e.g. a poly-A tail or 5' cap, are either appended to the mRNA by the endogenous natural cell machinery or are otherwise provided for, e.g. by including a synthetic poly-A tail sequence or a Lariat capping ribozyme.
Transcriptional synchronization can, for example, be used to synchronize the transcription of a genomic gene, e.g. endogenous gene, with a synthetically introduced “output”. This output can be a functional non-coding RNA or protein. Synchronization means, that all effects, which are influencing the transcription rate of a first functional transcription product, e.g. endogenous gene, are similarly affecting the synthetic output too. Such effects include, for example, promoter activity, regulation by enhancers and silencers, intronic and epigenetic effects, as well as other effects. Ideally, the transcription rate of the synthetic output essentially equals the transcription rate of the first functional transcription product, e.g. endogenous gene. In any case the transcription rate of the output cannot be greater than the transcription rate of the first functional transcription product. It can only be smaller, depending on the cleavage efficiency of the ribozymes used. This is, for example, important for therapeutic applications, because this mechanism cannot produce an unintentionally stronger output (= more output transcript), which could be otherwise detrimental.
Using the synchronization mechanism of the invention, a wide range of applications becomes possible. For example, the mechanism enables the use of a precise IF-THEN logic on the transcriptional level. This can, for example, be used as a precise sensor for the activity of a synchronized genomic gene. Thus, it can be used to detect transcriptional dysregulation in the case of diseases via the synchronization itself, and also to enable a therapeutic answer via the synchronized output. This output could be the production of a therapeutic molecule by the cell itself. Thus, transcriptional synchronization can be used to create disease-resistant cells for virtually every genetic disease, viral infection and bacterial infection that impacts transcription in any way (including cancer and Alzheimer’s disease).
For medical applications, transcriptional synchronization is further ideal due to its safety aspects: The output is only produced if the condition for its production is met, which is, for example, the transcription of a genomic gene, e.g. endogenous gene, being transcriptionally synchronized with the output. Also, it cannot be produced in a larger amount than the product of the gene it is synchronized with. Further, the overall strength of the produced output is dependent on the transcription rate of the gene. Thus, the stronger or longer the gene is expressed, the stronger the output is. This can be used for further processing the output as described further below for cancer. This enables the use of a second layer of regulation: The first regulation takes place via the initial synchronization, while the second layer of regulation can be introduced by using a signaling molecule as output, which is set off against another signaling output from another synchronized gene or a gene product. Depending on which signal is stronger, a final output is produced or not. Therefore, transcriptional synchronization can be used to create multiple layers of processing, or, also in combination with other mechanisms (e.g. molecular logic gates), to further enhance safety. Transcriptional synchronization is thus ideal for creating precise and safe cellular resistance mechanisms against genetic diseases and viral/bacterial infections, for example.
Transcriptional synchronization according to the invention can also be used for the transfection of multicellular organisms, e.g. a non-human animal or a human, without resulting mosaic mutants, i.e. organisms with transfected and non-transfected cells, as will be described in more detail further below.
The term “genetically engineered” in relation to a cell or organism means that the genome of the cell or organism has been changed using biotechnological means and methods. The term “genetically engineering” is not restricted to a manipulation of, for example, nuclear DNA, but also encompasses manipulation of extranuclear DNA or the introduction of non-chromosomal genetic elements, e.g. plasmids. The term also includes epigenetic alterations of the molecular and genetic setup of a cell or organism, or parts of these. The term “cell” as used herein relates to a living biological, e.g. prokaryotic or eukaryotic cell. Non-limiting examples of eukaryotic cells include plant cells, fungal cells, mammalian cell, non-human animal cells or human cells, preferably with the proviso that the cell is not a human germ cell. The eukaryotic cell may, for example, be a blood cell, epithelial cell or a stem cell (e.g. embryonic stem cells, induced pluripotent stem cell, hematopoietic stem cell). The cell may be a mammalian cell of the orders rodenta (mice, rats, hamsters), lagomorpha (rabbits), carnivora (cats, dogs), and artiodactyla (cows, pigs, sheep, goats, horses). The cell may be from any organism, for example plant, non-human animal, human, non-human primate, mouse, rat, rabbit, cat or dog, preferably with the proviso that it is not a human germ cell. The cell may be isolated or may be part of an organism (e.g., subject), but preferably with the proviso that it is not a human germ cell. Procaryotic cells include, for example, bacterial cells and archaean cells.
The term “transcriptionally synchronized” as used herein relates to the concomitant transcription of at least two separate nucleic acid sections in the same transcription unit under the same transcription regulatory element(s), e.g. the same promoter, such that, if a first nucleic acid encoding, for example, a gene, is transcribed, the second nucleic acid section, e.g. coding for a coding or non-coding RNA molecule, is preferably also transcribed in exactly the same quantity and rate as the first nucleic acid. “Transcriptional synchronization” thus in particular means the formation of a single pre mRNA transcribed from a single transcription unit containing the at least two separate nucleic acid sections, wherein the at least two separate nucleic acid sections are later released from the common pre-mRNA, such that the transcription products of the first and second nucleic acid sections are separated. The transcription product of the first nucleic acid section may be an mRNA coding for a gene of a cell, and the transcription product of the second nucleic acid section may be a coding or non-coding RNA molecule, e.g. an mRNA coding for a different protein, a reporter protein, an antisense RNA, siRNA etc.
The term “transcription unit” relates to a sequence of nucleotides in DNA that codes for a single RNA molecule, along with the sequences necessary for its transcription, for example a promoter, an RNA-coding sequence, and a termination signal (TS, also terminator or termination sequence). The term “transcription regulatory element” relates to any element of a transcription unit regulating the quantity and rate of transcription of the transcription unit. Examples of such elements are promoters and enhancers, i.e. segments of DNA containing sequences capable of providing promoter and enhancer functions. The expression according to which two separate nucleic acid sections or nucleotide sequences coding for a transcription product are “under the same transcription regulatory element(s)” means that any transcription regulatory element having an impact on the transcription of the first nucleic acid section has essentially the same impact, e.g. in terms of transcription quantity and rate, on the second nucleic acid section. The term also relates to any epigenetic element or modification that takes part in regulating the transcription of the transcription unit.
The term “promoter” relates to a sequence of DNA to which proteins bind that initiate transcription of a DNA sequence into an RNA. Usually, a promoter is arranged towards the 5' end of the sense strand of the DNA.
The term “enhancer” relates to regulatory DNA sequences that enhance the transcription of an associated gene when bound by specific proteins (transcription factors).
The terms “termination sequence”, “termination signal” or “terminator” relate to a nucleotide sequence that marks the end of a gene or operon in genomic DNA during transcription.
The term “mRNA” (messenger RNA) relates to a single-stranded RNA molecule comprising an RNA molecule that is complementary to one of the DNA strands, i.e. the sense strand, of a gene, and, if present, regulatory elements like a 5' cap or a poly-A tail. An mRNA encodes a polypeptide and is translated by the cell’s translation machinery comprising ribosomes and other components into the polypeptide.
The term pre-mRNA (precursor mRNA) as used herein relates to the primary transcript from a transcription unit. The term encompasses, but is not restricted to, the primary transcript from a transcription unit of eukaryotic genes containing introns and exons, which is further processed (e.g. spliced) to an mRNA containing only exon sequences, which is translated into a polypeptide (protein). The term also encompasses a primary transcript of a transcription unit comprising only one or more functional non-coding RNA sequences or a transcription unit comprising further coding or non-coding sequences besides sequences encoding an mRNA. The term thus encompasses any primary RNA transcript from a transcription unit comprising noncoding and/or coding RNA molecules.
The term “open reading frame”, ORF, relates to a DNA section coding for a transcription product that is directly or indirectly translatable into a protein, indirectly meaning translatable after further cellular processing, e.g. splicing of a pre mRNA. The term thus not only encompasses coding DNA sequences with a number of codons divisible by three and flanked by a start and stop codon but also eukaryotic genes composed of exons and introns, i.e. nucleotide sequences that are actually translated (exons) and nucleotide sequences that are actually not translated (introns), but are removed from a pre mRNA to form an mRNA directly translatable into a protein.
The terms “endogenous” or “homologous” is used herein with respect to a biologically functional element such as a gene, RNA, enzyme or protein to refer to it as an original or native element, i.e. a gene, RNA, enzyme or protein naturally occurring in the target cell, in contrast to a foreign (heterologous) element.
The term “genomic gene” refers to a gene, i.e. a nucleic acid encoding a polypeptide, of the genome of a cell. The term “genome” refers to the genetic material of a cell or organism and encompasses genetic material of chromosomal genetic material and extra-chromosomal genetic material, e.g. genetic material of mitochondria, chloroplasts or plasmids. The term includes endogenous genes and genes stably introduced into the genome of a cell.
The term “genomic gene product” refers to a transcription product of a genomic gene, i.e. an mRNA or pre-mRNA, which is translated into a polypeptide (protein). The term also encompasses a product resulting from translation, i.e. a protein.
The term “endogenous gene” relates to a gene that is native to a given cell, i.e. is naturally occurring in the cell. The term does not exclude that the gene has been introduced into the cell, i.e. that a gene being identical to or comprising an endogenous gene, has been introduced into the cell.
The term “endogenous gene product” relates to a transcription product of an endogenous gene, i.e. an mRNA or pre-mRNA. The term also encompasses a product resulting from translation, i.e. a protein.
The term “heterologous” refers to the foreign origin of an element, for example a gene, RNA, enzyme or other protein. “Foreign” means that the element does not naturally occur in the target cell, and for example originates from a cell or an organism with different genetic makeup, such as an organism of a different species.
By “expression” is meant the conversion of a genetic information into a functional product, for example the formation of a protein or a nucleic acid, e.g. functional RNA, on the basis of the genetic information. The term encompasses the biosynthesis of a protein, e.g. an enzyme, based on genetic information including previous processes such as transcription, i.e. the formation of mRNA based on a DNA template, or the synthesis of a functional RNA molecule, for example a self-cleaving ribozyme.
The term “functional transcription product” relates to a functional RNA molecule transcribed from a transcription unit, i.e. an RNA molecule being directly (itself) or indirectly (i.e. after modification, e.g. splicing) functional in or outside the cell. The term encompasses coding RNA molecules, i.e. RNA molecules that are translated into a polypeptide, e.g. mRNA or pre-mRNA containing introns and exons, and non-coding RNA molecules, i.e. RNA that is not translated into a protein, like, for example, antisense RNA, rRNA, piRNA, miRNA, tRNA, gRNA, exRNA, etc. The terms “functional RNA” (fRNA) or “functional RNA molecule” may synonymously be used here for the term “functional transcription product” The term “RNA gene” may be used for a DNA sequence from which a functional non-coding RNA is transcribed. The terms “output” or “output molecule” in relation to the functional RNA molecule relate to the intended end product to be produced in the cell of the invention. If the functional RNA is an mRNA coding for a protein the intended “output” normally is a protein. In other cases, however, the output molecule may be an RNA molecule itself (other than mRNA) having a specific intended function, for example an antisense RNA, siRNA or the like.
The term “ribozyme” (ribonucleic acid enzymes) relates to RNA molecules that have catalytic activities similar to protein enzymes, and are able to catalyze specific biochemical reactions, e.g. RNA splicing reactions in gene expression. The term “self-cleaving ribozyme” (SCRz) relates to ribozymes capable of self-scission, of carrying out site-specific intramolecular cleavage (self-cleavage) reactions. Classes of self-cleaving ribozymes include hepatitis delta virus (HDV)-like, hammerhead, hairpin, Varkud Satellite (VS), glmS, twister, twister sister, pistol, and hatchet ribozymes (see, for example, Lee, Ki-Young & Lee, Bong-Jin. (2017). Structural and Biochemical Properties of Novel Self-Cleaving Ribozymes. Molecules (Basel, Switzerland). 22. 10.3390/molecules22040678; Weinberg Z, Kim PB, Chen TH, et al. New classes of self-cleaving ribozymes revealed by comparative genomics analysis. Nat Chem Biol. 2015; 11(8):606-610. doi : 10.1038/nchembio.1846). The term “5' self-cleaving ribozyme” relates to self-cleaving ribozymes that are catalytically active towards their 3 '-end. They are cleaving near the 5 '-end of the sequence that is situated downstream of the ribozyme and are typically (but not exclusively) situated upstream (towards the 5 '-end) of the sequence, which is supposed to be cut off. The term “3' self-cleaving ribozyme” relates to self-cleaving ribozymes that are catalytically active towards their 5 '-end. They are cleaving near the 3 '-end of the sequence that is situated upstream of the ribozyme and are typically (but not exclusively) situated downstream (towards the 3 '-end) of the sequence, which is supposed to be cut off. In both cases, the sequence that has been cut off is no longer connected to the ribozyme or at least the full sequence of the ribozyme, (see, for example, Gao Y, Zhao Y (2014). Self-processing of ribozyme-flanked RNAs into guide RNAs in vitro and in vivo for CRISPR-mediated genome editing. Journal of Integrative Plant Biology, 56(4), 343-349. doi: 10.1111/jipb.12152).
Examples of 3' self-cleaving ribozymes are Hepatitis delta virus (HDV) ribozyme or the HDV- like CPEB3 ribozyme. Examples of 5' self-cleaving ribozymes are hammerhead ribozymes (HHRz), Type-P5 twister ribozymes (Roth A, Weinberg Z, Chen AG, Kim PB, Ames TD, Breaker RR. A widespread self-cleaving ribozyme class is revealed by bioinformatics. Nat Chem Biol. 2014;10(l):56-60. doi: 10.1038/nchembio.1386), twister-sister ribozymes (Weinberg Z, Kim PB, Chen TH, et al. New classes of self-cleaving ribozymes revealed by comparative genomics analysis. Nat Chem Biol. 2015;l l(8):606-610. doi : 10.1038/nchembio.1846) or hatchet ribozymes (Li S, Liinse CE, Harris KA, Breaker RR. Biochemical analysis of hatchet self-cleaving ribozymes. RNA. 2015;21(11): 1845-1851. doi: 10.1261/ma.052522.115).
The term “Lariat-capping ribozyme” (LC ribozyme, LCrz, formerly GIRI branching enzyme) relates to a ribozyme catalyzing cleavage by a 2', 5' branching reaction, leaving the 3' product with a 3-nt lariat cap that functionally substitutes for a conventional mRNA cap (m7GpppN cap) in the downstream pre-mRNA (see, e.g., Meyer M, Nielsen H, Olieric V, et al. Speciation of a group I intron into a lariat capping ribozyme. Proc Natl Acad Sci U S A. 2014, 111 (21): 7659- 7664, doi: 10.1073/pnas.1322248111; Krogh N, Pietschmann M, Schmid M, Jensen TH, Nielsen H., Lariat capping as a tool to manipulate the 5' end of individual yeast mRNA species in vivo, RNA, 2017, 23(5):683-695. doi: 10.1261/rna.059337.116; Tang Y, Nielsen H, Masquida B, Gardner PP, Johansen SD, Molecular characterization of a new member of the lariat capping twin-ribozyme introns, Mob DNA. 2014, 5:25, doi: 10.1186/1759-8753-5-25). An example for a Lariat capping ribozyme is a ribozyme from the myxomycete Didymium iridis. formerly known as GIRI ribozyme (see RCSB PDB 6GYV released 2018-08-22).
The terms “RNA targeting sequences” or “RNA binding sequences”, relate to first ribonucleic acids specifically binding to a second RNA, e.g. to an mRNA, pre-mRNA, miRNA or tRNA, or sequences coding for a protein binding to said second RNA sequence, e.g. an RNA or pre- mRNA binding protein, i.e. a protein specifically binding to an mRNA. Examples of mRNA binding sequences are anti-sense RNA, i.e. an RNA hybridizing with an mRNA in order to prevent translation of the mRNA into a protein, microRNA (miRNA), i.e. short (21-25 nt) noncoding single-stranded RNAs, or shRNA (small hairpin RNA). An example of a tRNA binding sequence is a tRNA binding shRNA.
The expression, according to which the second nucleotide sequence coding for a second functional transcription product is “flanked by the third and fourth nucleotide sequences coding for the first and second self-cleaving ribozyme” means that a nucleotide sequence coding for a self-cleaving ribozyme is attached at the 3' end and at the 5' end of the second nucleotide sequence. Terms like “after”, “before”, “preceded”, “followed by” or “following” in relation to the order of nucleotide sequences are to be understood as meaning that the sequences are preferably directly connected to each other at their respective 3' or 5' end, or vice versa, without any intervening nucleotide or nucleotide sequence. For example, an expression according to which a second sequence “follows” a first sequence or is “arranged after” the first sequence thus means that the second sequence is, in the direction explicitly given and/or in or 5 '-3' direction, preferably directly attached to the end of the first sequence.
The first nucleotide sequence codes preferably for an mRNA encoding a first protein, preferably a genomic protein of the cell, further preferred an endogenous protein of the cell, or a first functional non-coding RNA molecule, preferably an endogenous functional non-coding RNA molecule of the cell. The first nucleotide sequence coding for the first functional transcription product is preferably a copy of a naturally occurring or previously introduced functional transcription product being part of a naturally occurring or previously introduced transcription unit of the cell. The naturally occurring or previously introduced transcription unit of the cell is preferably replaced with a transcription unit with the configuration of transcription elements as described herein, such that the naturally occurring or previously introduced functional transcription product is still transcribed or transcribably inserted in the cell, but now transcriptionally synchronized with at least one second functional transcription product.
It is preferred that the first and second nucleotide sequence code for different functional transcription products, e.g. that, in case the first nucleotide sequence codes for a coding RNA molecule, e.g. an mRNA coding for a first protein, the second nucleotide sequence codes either for a different coding RNA, e.g. an mRNA coding for a second protein differing from the first protein, or a non-coding RNA molecule, e.g. siRNA, tRNA or antisense RNA. It is further preferred that not both the first and second functional nucleotide sequence code for a gRNA, i.e. a CRISPR guide RNA. Particularly preferred, at least the second functional nucleotide sequence does not code for a gRNA, further preferred neither the first nor the second functional nucleotide sequence code for a gRNA. In a particular preferred embodiment of the cell of the invention, the first nucleotide sequence codes for an endogenous gene product, preferably an endogenous protein, and the second nucleotide sequence codes for a functional coding or non- coding RNA molecule, the functional coding RNA molecule being different from an mRNA coding for the first protein.
The transcription unit may comprise more than one second nucleotide sequences coding for a second functional transcription product, e.g. two, three, four or more second nucleotide sequences, such that all these second functional transcription products are transcriptionally synchronized with the first nucleotide sequence coding for the first functional transcription product. It is possible that two or more of the second nucleotide sequences code for the same functional transcription product. It is preferred, however, that the second nucleotide sequences code for different functional transcription products, e.g. several different mRNA coding for different proteins, and/or several different non-coding functional RNAs. In case of the presence of more than one second nucleotide sequence coding for a second functional transcription product in the transcription unit, it is preferred that not all second nucleotide sequence code for gRNA. It is to be understood that each of the further second nucleotide sequences will also be flanked by suitable self-cleaving ribozymes. The skilled person will, depending on the nature and purpose of the respective further second functional transcription product, choose a suitable combination of the flanking ribozymes for each of the further second functional transcription products.
In a preferred embodiment of the invention, the second nucleotide sequence codes for a functional non-coding RNA or an mRNA encoding a protein. The second nucleotide sequence may thus code for a coding RNA, mRNA, such that a protein is produced by the normal cellular protein synthesizing machinery as an “output”, or code for a non-coding functional RNA, for example an antisense RNA or siRNA. The second nucleotide sequence may, for example, code for an RNA binding sequence, e.g. an mRNA binding sequence like an anti-sense RNA, microRNA or shRNA.
Preferably, the third nucleotide sequence flanking the second nucleotide sequence codes for a 5' self-cleaving ribozyme and the fourth nucleotide sequence codes for a 3' self-cleaving ribozyme. Preferably, the order of the second to fourth nucleotide sequence is thus (from promoter to terminations sequence, or 5' to 3'): 5' self-cleaving ribozyme - second nucleotide sequence coding for a first functional transcription product - 3' self-cleaving ribozyme. A preferred 5' self-cleaving ribozyme is a hammerhead ribozyme (HHRz).
If the second nucleotide sequence is chosen to code for an mRNA, which is supposed to be translated into a protein, the respective DNA is preferably modified in a way that a Lariatcapping ribozyme is situated at the 5 '-end of the mRNA and a synthetic poly-A-tail is situated at the 3 '-end of the mRNA.
For the application in procaryotes the arrangement composed of the second nucleotide sequence coding for the second functional transcription product and the flanking third and fourth nucleotide sequence each coding for a self-cleaving ribozyme can either be placed upstream, i.e. 5', of the first nucleotide sequence coding for the first functional transcription product, e.g. an ORF, or downstream, i.e. 3', of the first nucleotide sequence coding for the first functional transcription product, e.g. ORF. The skilled person will readily be able to adapt the flanking self-cleaving ribozyme readily to the intended purpose, depending, for example, on the nature and function (e.g. non-coding or coding) of the functional transcription product to be produced.
For eukaryotic cells, the basic configuration is more complex and comprises essentially two different embodiments. In a first embodiment, herein also called “3' arrangement” or “3' design”, the second nucleotide sequence coding for the second functional transcription product and flanked by the third and fourth nucleotide sequence coding for a first and second selfcleaving ribozyme, is arranged after the first nucleotide sequence coding for the first functional transcription product, the first nucleotide sequence coding for the first functional transcription product preferably (e.g. in case the first functional transcription product is a mRNA) being followed by a fifth nucleotide sequence encoding a Poly A tail, the fifth nucleotide sequence encoding a Poly A tail preferably being followed by a sixth nucleotide sequence coding for a third self-cleaving ribozyme, preferably a 3' self-cleaving ribozyme, the terms “after”, “before”, “preceded”, “followed by” or “following” relating to the direction from the promoter to the termination signal. In a second embodiment, herein also called “5' arrangement” or “5' design”, the second nucleotide sequence coding for the second functional transcription product and flanked by the third and fourth nucleotide sequence coding for a first and second self-cleaving ribozyme, is arranged before the first nucleotide sequence coding for the first functional transcription product, the first nucleotide sequence coding for the first functional transcription product preferably being preceded by a seventh nucleotide sequence coding for a Lariat capping ribozyme, the terms “after”, “before”, “preceded”, “followed by” or “following” relating to the direction from the promoter to the termination signal.
In the “3' design” of a eukaryotic cell of the invention, it is preferred that where, in the direction from the promoter to the termination signal, the second nucleotide sequence coding for the second functional transcription product and flanked by the third and fourth nucleotide sequence coding for a first and second self-cleaving ribozyme, is arranged after the first nucleotide sequence coding for the first functional transcription product, the third nucleotide sequence coding for a first self-cleaving ribozyme is preceded by a spacer sequence. In the “5' design” of a eukaryotic cell of the invention, it is preferred that where, in the direction from the promoter to the termination signal, the second nucleotide sequence coding for the second functional transcription product and flanked by the third and fourth nucleotide sequence coding for a first and second self-cleaving ribozyme, is arranged before the first nucleotide sequence coding for the first functional transcription product, the fourth nucleotide sequence coding for a second self-cleaving ribozyme is followed by a spacer sequence.
The spacer sequence is particularly useful in the 3' design, and facilitates folding of the selfcleaving ribozymes into their catalytically active state, and is preferably configured to have least possible interaction with neighboring sequences. The spacer can have any suitable length and may also vary depending on the intended use and may, for example, be a stretch of several adenine nucleotides (oligo- or poly-A). The spacer may have, for example, a length of 5 to 100 nucleotides, preferably 8 to 80 nt, e.g. adenine nucleotides. An example for a short spacer variant is a spacer having a length of 8 nt. A long variant of the spacer may have 72 nt. A spacer of intermediate length may, for example have a length of 45-55, e.g. 48, nucleotides, for example adenine nucleotides. Other lengths are, however, possible.
The skilled person is aware of the fact that 3' or 5' regulatory elements (3' or 5' untranslated regions) like poly-A tails or 5' caps may be necessary for a correct processing of a functional transcription product, e.g. mRNA molecule, in a cell, in particular in case of a eukaryotic cell. The skilled person will configure the genomic elements accordingly and, taking his or her common general knowledge, provide for any nucleic acid elements necessary for a correct processing of a functional transcription product in the cell.
In a preferred embodiment of a eukaryotic cell of the invention according to the embodiment denoted above as “3' design”, a synthetic Poly A tail is arranged downstream of the first nucleotide sequence coding for the first functional transcription product, if the first functional transcription product is to be an mRNA, the Poly A tail being encoded by a fifth nucleotide sequence, and the Poly A tail being immediately followed by a sixth nucleotide sequence coding for a third self-cleaving ribozyme, preferably a 3' self-cleaving ribozyme. “Synthetic” in relation to the poly A tail means that adenine nucleotides are not added by the cell’s transcriptional machinery to the mRNA, but have to be encoded in the DNA. In the 5' design such a Poly A tail is not necessary, because the arrangement of elements allows for the addition of the poly A tail by the normal cellular transcriptional machinery. In the 5' design, however, a seventh nucleotide sequence encoding a Lariat capping ribozyme is preferably additionally present immediately in front of the first nucleotide sequence coding for the first functional transcription product in order to produce an mRNA having a Lariat cap as a substitute for the normal 5' m7G cap.
In a preferred embodiment of the 3' design of a eukaryotic cell according to the invention, the order of the first functional transcription product, the fifth nucleotide sequence coding for a Poly A tail, the sixth nucleotide sequence coding for a third self-cleaving ribozyme, the spacer, and the second nucleotide sequence coding for the second functional transcription product flanked by the first and second self-cleaving ribozyme, between the promoter and the termination sequence, is, in the direction from the promoter to the termination signal, as follows: First functional transcription product - fifth nucleotide sequence encoding a Poly A tail - sixth nucleotide sequence coding for the third self-cleaving ribozyme, preferably a 3' self- cleaving ribozyme - spacer sequence - third nucleotide sequence coding for the first selfcleaving ribozyme, preferably a 5' self-cleaving ribozyme - second nucleotide sequence coding for the second functional transcription product - fourth nucleotide sequence coding for the second self-cleaving ribozyme, preferably a 3' self-cleaving ribozyme. As stated above, a Poly A tail is only necessary if a similar naturally occurring transcription product of the cell would have had or needed one too. In a preferred embodiment of the 5' design of a eukaryotic cell according to the invention, the order of the first functional transcription product, the third nucleotide sequence coding for the first self-cleaving ribozyme, the fourth nucleotide sequence coding for the second self-cleaving ribozyme, the second nucleotide sequence coding for the second functional transcription product, and, if present, the seventh nucleotide sequence coding for a Lariat capping ribozyme and the spacer sequence, between the promoter and the termination sequence, is, in the direction from the promoter to the termination signal, as follows: Third nucleotide sequence coding for the first self-cleaving ribozyme, preferably a 5' self-cleaving ribozyme - second nucleotide sequence coding for the second functional transcription product - fourth nucleotide sequence coding for the second self-cleaving ribozyme, preferably a 3' self-cleaving ribozyme - spacer sequence - seventh nucleotide sequence coding for the Lariat capping self-cleaving ribozyme - first functional transcription product. Again, a Lariat capping ribozyme is only necessary if a similar natural transcription product of the cell would have had or needed a 5' cap. The Lariat capping ribozyme introduces a 5' cap functioning as a substitute to a natural 5' m7G cap.
The cell can be any cell, for example a eucaryotic or procaryotic cell. Preferably, the cell is a plant cell, an insect cell, a mammalian cell, a non-human animal cell, or a human cell. If the cell is a human cell, the cell is preferably not a human embryonic stem cell obtained by a method involving the destruction of a human embryo, and preferably the cell is not a human germ cell or human zygote. In case of a bacterial cell, for example, the cell is preferably genetically engineered according to the invention such that the cell has a resistance against bacteriophages. The cell may be a pluripotent stem cell, preferably induced pluripotent stem cell (iPSC), i.e. a pluripotent stem cell generated directly from a somatic cell, e.g. a human (induced) pluripotent stem cell, including a human embryonic stem cell.
A genetically engineered bacterial cell is preferably engineered in a manner that the first nucleotide sequence coding for the first transcription product is transcribed or more transcribed at some point of or during the entire infection of the bacterium by a bacteriophage. The application of the invention in bacteria is not restricted to counteracting resistances against bacteriophages, but can, for example, also be used for the stoichiometrically exactly defined production of two or more functional RNAs.
In a further aspect, the invention also relates to a transgenic plant or non-human animal comprising a cell of the first aspect of the invention.
The present invention provides for a nucleic acid based genomic system or mechanism for transcriptionally synchronizing a first functional transcription product, for example an mRNA that is finally translated into a protein, product with at least one second functional transcription product (“output”), first nucleotide sequence coding for the first functional transcription product can be any suitable sequence for which transcriptional synchronization with a second functional transcription product holds any desired value. The first nucleotide sequence is preferably included in a nucleic acid that is introduced in the genome of a cell or organism, along with the preferred design for transcriptional synchronization as described here. In the case of a use of the invention to treat or prevent cancer, for example, the sequences coding for the functional transcription products are preferably (proto-)oncogenes such as ERBB2, HRAS, MYC, SRC, TERT, BCL2, EGFR, etc., or tumor suppressor genes such as TP53, RBI, APC, BRCA1, BRCA2, HLA-A, etc. A respective endogenous or synthetic sequence already present in the target genome will preferably be deleted and replaced with a suitable design for transcriptional synchronization according to the invention, including the respective sequences coding for the respective functional transcription products.
Transcriptional synchronization according to the invention can, for example, also be used to transfect multicellular organisms under avoidance of mosaic mutants, i.e. a mixture of transfected and non-transfected cells. For this purpose, induced pluripotent stem cells (iPSCs) are genetically modified ex vivo to exhibit transcriptional synchronization of, for example, a modified cell cycle related gene (e.g. cyclin A/cdk2) with a cell-to-cell mobile inhibitor of the unmodified variant of this gene (e.g. a shRNA, siRNA, transcription factor) that is able to impose a cytostatic but not cytotoxic effect. In an embodiment of the invention, the cell cycle related gene is, for example, encoded by the first nucleotide sequence, and the gene inhibitor is encoded by a second nucleotide sequence. The synchronized cell cycle gene is modified to be resistant to said inhibitor. Such modified iPSCs would be implanted into a patient’s respective stem cell niche. There, native and modified stem cells would receive proliferation signals alike, resulting in an activation of cell cycle genes. As soon as e.g. cyclin A/cdk2 is transcribed, the iPSCs would also transcribe the inhibitor in a synchronized manner according to the invention. While the cyclin A/cdk2 variant of the iPSCs is designed to be resistant to the inhibitor, the native variant is not. Consequently, cell-to-cell transport of the inhibitor results in the inhibition of the native stem cells’ cell cycle progression, causing cell cycle arrest and no further proliferation of the native stem cell population. Yet, the iPSCs engineered according to the invention are unaffected by the inhibitor and are free to proliferate. Thus, the modified iPSCs gain an advantage over the native stem cell population and can proliferate at a higher rate, outcompeting their native counterpart. Over time, this would result in a stem cell niche that is exclusively comprised of the modified iPSCs, which in turn would replenish their associated tissue with new and modified differentiated cells. This use of transcriptional synchronization would result in a regulated stem cell niche replacement that also avoids creating a mosaic mutant stem cell niche. This system can be further used to create a iPSC niche that is “updatable”: After a first generation iPSC niche has been established, a second generation of iPSCs could have the same cell cycle gene synchronized with an inhibitor being designed to target the former resistant cell cycle gene of the first generation iPSCs, thus leading to the same effect as described above for the native stem cell niche compared with the first generation of modified iPSCs. This can be used for making adjustments if, for example, gene therapy results in unforeseen side effects, which would be impossible to correct using other current delivery methods for gene therapy. It can also be used to implement new or improved gene modifications. A useful application of this system would be to refresh stem cell populations if their genes have amassed detrimental mutations over time.
In a further aspect the invention relates to a vector or vector system for the transfection, preferably stable transfection, of a cell, the vector(-system) comprising: a) a first nucleotide sequence coding for a first functional transcription product, b) a second nucleotide sequence coding for a second functional transcription product, c) a third nucleotide sequence coding for a first self-cleaving ribozyme, and d) a fourth nucleotide sequence coding for a second self-cleaving ribozyme, the second nucleotide sequence coding for the second functional transcription product being flanked by the third and fourth nucleotide sequences coding for the first and second selfcleaving ribozyme, wherein the vector or vector system is configured to introduce the nucleotide sequences of a) to d) into a transcription unit of the cell between a promoter and a termination signal of the transcription unit.
The vector or vector system (vector system meaning a plurality of vectors acting together) is configured in such a way to produce a cell of the first aspect of the invention when introduced into the cell. The skilled person is aware of different vector constructs or vector systems suitable for this purpose, and will choose a suitable vector or vector system, and adapt it, if necessary. The vector or vector system of the invention thus is designed to introduce, into a cell, a transcription unit as described above. Consequently, any disclosure of a feature of the transcription unit described above in relation to the genetically engineered cell also applies to the vector or vector system of the invention.
The vector or vector system thus preferably comprises all nucleotide sequences according to a) to d) above, and, if necessary, further functional nucleic acid elements to insert the nucleotide sequences of a) to d) above and to replace a genomic sequence encoding a functional transcription product with the mechanism of the invention necessary for transcriptional synchronization. Preferably the vector or vector system also comprises an element or multiple elements that are necessary for the deletion of the sequence encoding the genomic functional transcription product (e.g. sgRNA and Cas9). The vector or vector system preferably includes an element or elements enabling expression of the aforementioned elements inside a target cell (e.g. a plasmid backbone comprising necessary regulatory elements such as promoters etc.). The vector or vector system of the invention may also include an element or multiple elements that are necessary to guide and transport the vector/vector system to its site of intended action (e.g. viral delivery systems, such as a Lentiviral vector).
In an alternative embodiment, the vector or vector system of the invention may only comprise the nucleotide sequences of features b) to d) above, i.e. lack feature a. In this embodiment, the vector or vector system is configured to introduce the nucleotide sequences of features b) to d) above into a transcription unit of the cell between a promoter and a termination signal of the transcription unit, the transcription unit already comprising feature a), i.e. a first nucleotide sequence coding for a first functional transcription product, for example an endogenous gene coding for an endogenous protein or a nucleotide sequence coding for a functional RNA other than an mRNA, e.g. tRNA, siRNA, antisense RNA etc. This vector or vector system is particularly suitable for genetic modification of a transcription unit of a cell for establishing a transcription synchronization mechanism of the invention, where the first nucleic acid coding for the first functional transcription product, e.g. an endogenous gene, shall not be replaced with an identical nucleic acid coding for the same functional transcription product.
In a preferred embodiment, the vector or vector system of the invention according, the first nucleotide sequence codes for an mRNA encoding a first protein, preferably an endogenous protein of the cell, or a first functional non-coding RNA molecule, preferably an endogenous functional non-coding RNA molecule of the cell.
Preferably, the third nucleotide sequence codes for a functional non-coding RNA or an mRNA encoding a protein.
Further preferred, the third nucleotide sequence codes for a 5' self-cleaving ribozyme and the fourth nucleotide sequence codes for a 3' self-cleaving ribozyme.
In the vector or vector system of the invention it is preferred, that at least the second functional nucleotide sequence does not code for a gRNA. It is further preferred that neither the first nor the second functional nucleotide sequence code for a gRNA.
In an embodiment of the vector or vector system of the invention, the vector or vector system being particular useful for the transfection of induced pluripotent stem cells (iPSCs), the first nucleotide sequence encodes a modified cell cycle related gene, and the second nucleotide sequence encodes an inhibitor of the unmodified (native) variant of said gene, the cell cycle related gene being modified to be resistant to the inhibitor, and the inhibitor being cell-to-cell mobile, i.e. being able to move from cell to cell. In a further preferred embodiment of the vector or vector system according to the invention, a) the second nucleotide sequence coding for the second functional transcription product and flanked by the third and fourth nucleotide sequence coding for a first and second self-cleaving ribozyme, is arranged after the first nucleotide sequence coding for the first functional transcription product, the first nucleotide sequence coding for the first functional transcription product preferably being followed by a fifth nucleotide sequence encoding a Poly A tail, the fifth nucleotide sequence encoding a Poly A tail preferably being followed by a sixth nucleotide sequence coding for a third self-cleaving ribozyme, preferably a 3' self-cleaving ribozyme, or b) the second nucleotide sequence coding for the second functional transcription product and flanked by the third and fourth nucleotide sequence coding for a first and second self-cleaving ribozyme, is arranged before the first nucleotide sequence coding for the first functional transcription product, the first nucleotide sequence coding for the first functional transcription product preferably being preceded by a seventh nucleotide sequence coding for a Lariat capping ribozyme.
In a further preferred embodiment of the vector or vector system according to the invention, a) where the second nucleotide sequence coding for the second functional transcription product and flanked by the third and fourth nucleotide sequence coding for a first and second selfcleaving ribozyme, is arranged after the first nucleotide sequence coding for the first functional transcription product, the third nucleotide sequence coding for a first self-cleaving ribozyme is preceded by a spacer sequence, or b) where the second nucleotide sequence coding for the second transcription product and flanked by the third and fourth nucleotide sequence coding for a first and second self-cleaving ribozyme, is arranged before the first nucleotide sequence coding for the first functional transcription product, the fourth nucleotide sequence coding for a second self-cleaving ribozyme is followed by a spacer sequence.
In a preferred embodiment of the vector or vector system according to the invention, the following order of nucleic acid sequences is present: a) first nucleotide sequence coding for the first functional transcription product - fifth nucleotide sequence encoding a Poly A tail - sixth nucleotide sequence coding for the third self-cleaving ribozyme, preferably a 3' self-cleaving ribozyme - spacer sequence - third nucleotide sequence coding for the first self-cleaving ribozyme, preferably a 5' self-cleaving ribozyme - second nucleotide sequence coding for the second functional transcription product - fourth nucleotide sequence coding for the second self-cleaving ribozyme, preferably a 3' selfcleaving ribozyme, or b) third nucleotide sequence coding for the first self-cleaving ribozyme, preferably a 5' selfcleaving ribozyme - second nucleotide sequence coding for the second functional transcription product - fourth nucleotide sequence coding for the second self-cleaving ribozyme, preferably a 3' self-cleaving ribozyme - spacer sequence - seventh nucleotide sequence coding for the Lariat capping self-cleaving ribozyme - first nucleotide sequence coding for the first functional transcription product.
In a still further aspect, the invention relates to a method for the transcriptional synchronization of two or more functional transcription products of a cell, the method comprising:
Introducing into a transcription unit of the cell, the transcription unit comprising a promotor and a termination signal i. a first nucleotide sequence coding for a first functional transcription product, ii. a second nucleotide sequence coding for a second functional transcription product, iii. a third nucleotide sequence coding for a first self-cleaving ribozyme, and iv. a fourth nucleotide sequence coding for a second self-cleaving ribozyme, the second nucleotide sequence coding for the second functional transcription product being flanked by the third and fourth nucleotide sequences coding for the first and second selfcleaving ribozyme.
The method of the invention is preferably used to essentially replace an existing transcription unit of a cell, i.e. a transcription unit naturally present in the cell or previously introduced into the genome of the cell, or at least the relevant elements between the promoter and the termination signal of the transcription unit with a transcription unit having the elements according to features i. to iv. above. It is particularly preferred that the first nucleotide sequence coding for the first functional transcription product (e.g. an ORF of a protein) is identical to and replaces the nucleotide sequence naturally present or previously introduced into that transcription unit. It is thus intended to genetically engineer a cell in such a manner, that an already existing transcription unit be replaced with a transcription unit containing a nucleotide sequence coding for essentially the same main functional transcription product (e.g. the same protein), however, transcriptionally synchronized with another functional transcription product that was not part of the original transcription unit. It should be noted, however, that the present invention also encompasses a variant of the method where the first nucleotide sequence coding for a first functional transcription product is not separately introduced into the cell (step i. above), but already present in the genome of the cell. In this embodiment of the method of the invention the first nucleotide sequence coding for a first functional transcription product is already present (either naturally or previously introduced) between the promotor and the termination signal of the transcription unit, and only steps ii. to iv. are performed.
In a preferred embodiment of the method of the invention, the first nucleotide sequence codes for an mRNA encoding a first protein, preferably a genomic or endogenous protein of the cell, or a first functional non-coding RNA molecule, preferably an endogenous functional noncoding RNA molecule of the cell.
In the method of the invention, it is preferred that the first and second nucleotide sequence code for different functional transcription products, e.g. that, in case the first nucleotide sequence codes for a coding RNA molecule, e.g. an mRNA coding for a first protein, the second nucleotide sequence codes either for a different coding RNA, e.g. an mRNA coding for a second protein differing from the first protein, or a non-coding RNA molecule, e.g. siRNA, tRNA or antisense RNA. It is further preferred that not both the first and second functional nucleotide sequence code for a gRNA, i.e. a CRISPR guide RNA. Particularly preferred, at least the second functional nucleotide sequence does not code for a gRNA, further preferred neither the first nor the second functional nucleotide sequence code for a gRNA. In a particular preferred embodiment of the cell of the invention, the first nucleotide sequence codes for an endogenous gene product, preferably an endogenous protein, and the second nucleotide sequence codes for a functional coding or non-coding RNA molecule, the functional coding RNA molecule being different from an mRNA coding for the first protein.
The transcription unit may comprise more than one second nucleotide sequences coding for a second functional transcription product, e.g. two, three, four or more second nucleotide sequences, such that all these second functional transcription products are transcriptionally synchronized with the first nucleotide sequence coding for the first functional transcription product. It is possible that two or more of the second nucleotide sequences code for the same functional transcription product. It is preferred, however, that the second nucleotide sequences code for different functional transcription products, e.g. several different mRNA coding for different proteins, and/or several different non-coding functional RNAs. In case of the presence of more than one second nucleotide sequence coding for a second functional transcription product in the transcription unit, it is preferred that not all second nucleotide sequence code for gRNA. It is to be understood that each of the further second nucleotide sequences will also be flanked by suitable self-cleaving ribozymes. The skilled person will, depending on the nature and purpose of the respective further second functional transcription product, choose a suitable combination of the flanking ribozymes for each of the further second functional transcription products.
In a preferred embodiment of the method of the invention, the second nucleotide sequence codes for a functional non-coding RNA or an mRNA encoding a protein. Further preferred, the functional non-coding RNA does not code for a gRNA.
Preferably, in the method of the invention, the third nucleotide sequence codes for a 5' selfcleaving ribozyme and the fourth nucleotide sequence codes for a 3' self-cleaving ribozyme. The third nucleotide sequence may, for example, code for a Hammerhead ribozyme, and the fourth nucleotide sequence may, for example, code for a Hepatitis delta virus (HDV) ribozyme.
In a preferred embodiment of the method of the invention the cell is a eukaryotic cell, wherein, in the direction from the promoter to the termination signal, the second nucleotide sequence coding for the second functional transcription product and flanked by the third and fourth nucleotide sequence coding for a first and second self-cleaving ribozyme, is introduced after the first nucleotide sequence coding for a first functional transcription product, wherein a fifth nucleotide sequence encoding a Poly A tail is preferably introduced after the first nucleotide sequence coding for a first functional transcription product, if the first functional transcription product is an mRNA, and wherein a sixth nucleotide sequence coding for a third self-cleaving ribozyme, preferably a 3' self-cleaving ribozyme, is preferably introduced after the fifth nucleotide sequence encoding a Poly A tail. If a functional transcription product does not need a Poly A tail, the Poly A tail is not included.
In this embodiment of the method of the invention, it is preferred that where, in the direction from the promoter to the termination signal, the second nucleotide sequence coding for the second functional transcription product and flanked by the third and fourth nucleotide sequence coding for a first and second self-cleaving ribozyme, is introduced after the first nucleotide sequence coding for a first functional transcription product, a spacer sequence is preferably introduced before the third nucleotide sequence coding for the first self-cleaving ribozyme.
Further, in this embodiment of the method of the invention, it is preferred that, in the direction from the promoter to the termination signal, the order of the first nucleotide sequence coding for a first functional transcription product, the third nucleotide sequence coding for the first self-cleaving ribozyme, the fourth nucleotide sequence coding for the second self-cleaving ribozyme, the second nucleotide sequence coding for the second functional transcription product, the fifth nucleotide sequence encoding a Poly A tail, the sixth nucleotide sequence coding for the third self-cleaving ribozyme and the spacer sequence, if necessary, are introduced in such a way between the promoter and the terminations signal that the following order results:
First nucleotide sequence coding for a first functional transcription product - fifth nucleotide sequence encoding a Poly A tail (if necessary, as explained further above) - sixth nucleotide sequence coding for the third self-cleaving ribozyme, preferably a 3' self-cleaving ribozyme - spacer sequence - third nucleotide sequence coding for the first self-cleaving ribozyme, preferably a 5' self-cleaving ribozyme - second nucleotide sequence coding for the second functional transcription product - fourth nucleotide sequence coding for the second selfcleaving ribozyme, preferably a 3' self-cleaving ribozyme.
In an alternative embodiment of the method of the invention the cell is a eukaryotic cell, wherein, in the direction from the promoter to the termination signal, the second nucleotide sequence coding for the second functional transcription product and flanked by the third and fourth nucleotide sequence coding for a first and second self-cleaving ribozyme, is introduced before the first nucleotide sequence coding for a first functional transcription product, wherein a seventh nucleotide sequence coding for a Lariat capping ribozyme, is preferably introduced directly before the first nucleotide sequence coding for a first functional transcription product. A Lariat capping ribozyme is only included, if the first functional transcription product or any further functional transcription product needs a 5' capping-like mechanism in order to function as intended.
In this alternative embodiment of the method of the invention, it is preferred that where, in the direction from the promoter to the termination signal, the second nucleotide sequence coding for the second functional transcription product and flanked by the third and fourth nucleotide sequence coding for a first and second self-cleaving ribozyme, is introduced before the first nucleotide sequence coding for a first functional transcription product, a spacer sequence is preferably introduced after the fourth nucleotide sequence coding for the second self-cleaving ribozyme.
Further, in this alternative embodiment of the method of the invention, it is preferred that, in the direction from the promoter to the termination signal, the order of the first nucleotide sequence coding for a first functional transcription product, the third nucleotide sequence coding for the first self-cleaving ribozyme, the fourth nucleotide sequence coding for the second self-cleaving ribozyme, the second nucleotide sequence coding for the second functional transcription product, the seventh nucleotide sequence coding for the Lariat capping ribozyme (if necessary), and the spacer sequence are introduced in such a way between the promoter and the terminations signal that the following order results:
Third nucleotide sequence coding for the first self-cleaving ribozyme, preferably a 5' selfcleaving ribozyme - second nucleotide sequence coding for the second functional transcription product - fourth nucleotide sequence coding for the second self-cleaving ribozyme, preferably a 3' self-cleaving ribozyme - spacer sequence - seventh nucleotide sequence coding for the Lariat capping ribozyme (if necessary) - first nucleotide sequence coding for a first functional transcription product.
Preferably, in the method of the invention, the cell is a plant cell, an insect cell, a mammalian cell, a non-human animal cell, or a human cell. Where the cell is a human cell, it is preferred that the cell is not a human embryonic stem cell obtained by a method involving the destruction of a human embryo, and preferably with the proviso that the cell is not a human gamete or human zygote. Further, the method of the invention is preferably not used to modify the germ line genetic identity of a human being. The cell may be a pluripotent stem cell, e.g. a human pluripotent stem cell, including a human embryonic stem cell. In a preferred embodiment of the method of the invention, the cell is an induced pluripotent stem cell (iPSC), i.e. a pluripotent stem cell generated directly from a somatic cell.
In a further embodiment of the method of the invention, the cell is a prokaryotic cell, e.g. a bacterial or archaeal cell. In case of a bacterial cell, for example, the method is preferably used for the introduction of a resistance against bacteriophages.
In a particular preferred embodiment, the method of the invention is used for treating or preventing a disease, preferably a genetic disease or cancer, in a subject, e.g. a human subject. In one aspect, the invention thus also relates to a method of treatment or prevention of a disease, for example a genetic disease, in a subject, comprising the steps of
Introducing into a transcription unit of a cell of the subject, the transcription unit comprising a promotor and a termination signal i. a first nucleotide sequence coding for a first functional transcription product, preferably an open reading frame, ORF, coding for an endogenous gene product, ii. a second nucleotide sequence coding for a second functional transcription product, iii. a third nucleotide sequence coding for a first self-cleaving ribozyme, and vi. a fourth nucleotide sequence coding for a second self-cleaving ribozyme, the second nucleotide sequence coding for a second functional transcription product being flanked by the third and fourth nucleotide sequences coding for the first and second selfcleaving ribozyme, such that the first functional transcription product of the cell is transcriptionally synchronized with the second functional transcription product.
In this method, it is preferred that, where the cell is a human cell, the cell is not a human embryonic stem cell obtained by a method involving the destruction of a human embryo. Further, it is preferred that the cell is not a human gamete or human zygote. Further, the method of the invention is preferably not used to modify the germ line genetic identity of a human being. The cell may be a pluripotent stem cell, e.g. a human pluripotent stem cell, including a human embryonic stem cell. In a preferred embodiment of the method of the invention, the cell is an induced pluripotent stem cell (iPSC), i.e. a pluripotent stem cell generated directly from a somatic cell.
In an embodiment of the method of the invention, the method being particular useful for genetic engineering of induced pluripotent stem cells (iPSCs), for example as part of gene therapy, the first nucleotide sequence encodes a modified cell cycle related gene, and the second nucleotide sequence encodes an inhibitor of the unmodified (native) variant of said gene, the cell cycle related gene being modified to be resistant to the inhibitor, and the inhibitor being cell-to-cell mobile, i.e. being able to move from cell to cell.
In the following, the invention will be described in further detail by way of example only with reference to the accompanying figures.
Figure 1. Schematic illustration of two alternative embodiments of the invention related to eukaryotic cells, called “3' design” and “5' design”, respectively. Abbreviations: open reading frame (ORF), self-cleaving ribozyme (SCRz), terminating sequence (TS). a) The eukaryotic 3' design has a variable output that is flanked by a 5' and a 3' self-cleaving ribozyme. After transcription, the output is cut from the pre-mRNA by the ribozymes and can fulfill its intended function. The pre-mRNA is also cut by another 3' self-cleaving ribozyme, which releases the transcribed ORF together with a synthetic poly-A tail. This part can be further modified by the cellular machinery and become a normal mRNA. b) The eukaryotic 5' design is similar to the 3' design, however, the box comprising the output flanked by self-cleaving ribozymes is located directly after the promoter. The design also includes a lariat capping ribozyme (LCrz), which is separated from the 3' self-cleaving ribozyme by a spacer. After transcription, the output is cut from the pre-mRNA and can function as intended. The mRNA, containing the ORF, is capped at its 5' end by the LCrz. It is polyadenylated like every other mRNA.
Figure 2. Molecular circuit of a resistance mechanism against cancer development based on transcriptional synchronization according to the invention. A) Shown is the function of the mechanism in a healthy cell: Multiple tumor suppressor genes (here two), which are coupled with AntiSTAR sequences, quench the transcription-activating capability of STAR. No therapeutic output (proton channel) is produced. B) In a cancerous cell, proto-oncogenes become oncogenes and experience an increased transcription. Contrary, tumor suppressor gene transcription is reduced. Thus, in a cancerous cell, the inhibition of STAR by AntiSTAR would be reduced. C) Without AntiSTAR, STAR is able to induce the transcription of the proton channel (therapeutic output). Note: The therapeutic output is here displayed as a proton channel. However, it could also be a V-ATPase inhibitor or something else. Also, both STAR and AntiSTAR are flanked by a 5' and 3' self-cleaving ribozyme (SCRz). The 5' SCRz here is a hammerhead ribozyme (HHRz), while the 3' SCRz could be a hepatitis delta virus (HDV) ribozyme.
Figure 3. Synchronization cassette inserted in plasmid pEGFP-Nl and used in experiments with HeLa cells. Underlined nucleotides: Synthetic poly-A tail, double underlined nucleotides: Hepatitis delta virus ribozyme; framed nucleotides (48 adenines): Spacer; Underlined with dashed lines: Hammerhead ribozyme (adjusted to target shThr2); bold: shThr2. The 6nt sequences flanking the spacer sequence are restriction sites of restriction enzymes.
Examples
Figure 1 schematically shows the genomic structure of preferred embodiments of two alternative embodiments (also called “3' or 5' designs” in the following) of a eukaryotic cell of the invention. One design uses a synthetic poly adenine tail, enabling a box composed of a nucleotide sequence coding for a variable output (i.e. variable second functional transcription product, variable functional RNA molecule) flanked by nucleotide sequences encoding selfcleaving ribozymes (“Rz-vO-Rz box”; may also be called “device” in the following) to be placed between an open reading frame (ORF) and a corresponding transcription terminating sequence (TS). It is to be noted here, that an ORF is only an example for a first nucleotide sequence coding for a first functional transcription product. This could also be replaced with other transcription product encoding sequence, e.g. a sequence coding for a tRNA. This is called the 3' design, because the device is closer to the 3' end of the DNA sequence. In case the ORF is replaced with a transcription product encoding sequence that does not need 3' polyadenylation, no poly A tail is used. The alternative eukaryotic version is called 5' design, where the Rz-vO-Rz box is situated closer to the 5' end of the DNA sequence. For this, a LCrz is used to enable the device to be placed between a promoter and a corresponding ORF. In case the ORF is replaced by transcription product encoding sequence that does not need 5' capping, no LCrz is used.
The core components of the two alternative DNA sequences for the eukaryotic version shown in Figure 1 are:
For the 3' design:
1) 5' and 3' self-cleaving ribozymes flanking a variable functional RNA molecule output (“Rz- vO-Rz box”)
2) another 3' self-cleaving ribozyme
3) a spacer sequence
4) Synthetic poly adenine tail, if needed
5) first transcription product (a copy of the genomic version, here an ORF)
For the 5' design:
1) 5' and 3' self-cleaving ribozymes flanking a variable functional RNA molecule output (“Rz- vO-Rz box”)
2) a spacer sequence
3) Lariat capping ribozyme (LCrz), if needed
4) first transcription product (a copy of the genomic version, here an ORF)
It should be noted here that, for easier understanding, reference is made directly to the encoded elements also in relation to the coding DNA. For example, depending on the context, if reference is made to a self-cleaving ribozyme, the self-cleaving ribozyme may itself be meant (e.g. in the context of an mRNA) or the nucleic acid sequence coding for self-cleaving ribozyme is meant (e.g. in the context of the coding DNA). It should further be noted that the term “output” used here may, depending on the context, not only relate to the immediate transcription product, i.e. an mRNA or a functional non-coding RNA, but also to the product resulting from a translation of the mRNA. An embodiment of the eukaryotic 3' design and schematic function is shown in figure la: On the DNA level, the Rz-vO-Rz box is inserted into a naturally occurring gene between its ORF and transcription terminating sequence. For reasons of clarity, it should be noted here that the naturally occurring first nucleotide sequence coding for the first transcription product (here an ORF) can also be deleted, and reintroduced together with a copy of the first nucleotide sequence coding for the first transcription product (here the ORF), ensuring that no alteration of the transcription product encoding sequence is created by simply introducing the Rz-vO-Rz box. The Rz-vO-Rz box comprises a synthetic poly-A tail, followed by a 3' self-cleaving ribozyme, a spacer, a 5' self-cleaving ribozyme, a variable output and another 3' self-cleaving ribozyme. When a pre-mRNA is synthesized, everything except the promoter and a part of the terminating sequence is transcribed. After transcription, the ribozymes on the pre-mRNA undergo self-cleavage and the ORF gets a 5' cap. The part containing the ORF is now a functional mRNA and can be exported and translated into a protein due to the 5' cap and synthetic poly-A tail. The synthetic poly-A tail enables the nuclear export and cytoplasmic translation of the mRNA. If no synthetic poly-A tail would be added, the ribozymes would be likely to prevent natural polyadenylation of the mRNA, which would inhibit nuclear export and cytoplasmic translation. The part that contains the 3' and 5' self-cleaving ribozyme as well as the spacer will be degraded by nucleases. The spacer is advantageous to give the ribozymes enough space to assume their active secondary structure without interfering with each other. The DNA sequence of the spacer was chosen to be poly A to minimize secondary structure formation and interaction with neighboring sequences. Adenine was chosen for the spacer, so that an interaction with the poly-A tail is as unlikely as possible after transcription. Thymine (on the mRNA) would interact readily with the poly-A tail and act as an antisense inhibitor. Cytosine and guanine might hamper the transcription by being more prone to stay doublestranded. Thus, adenine is preferably used for the spacer on the DNA sequence. The output can function independently after being cut out by the ribozymes. The last 3' self-cleaving ribozyme and the attached part of the transcribed terminating sequence are degraded by nucleases. The stability of the synchronized part of the 3' design can be additionally influenced by varying the length of the synthetic poly-A tail: The shorter the tail, the earlier the synchronized part is degraded by the cell. An embodiment of the eukaryotic 5' design is depicted in figure lb. Differences compared to the 3' design are: The Rz-vO-Rz box is located behind the promoter and contains a LCrz (if necessary), which creates a 5' cap on the mRNA of the synchronized gene (e.g. needed for an ORF). Polyadenylation is done naturally by the cell’s proteins. LCrz is used to imitate 5' capping of the mRNA.
Depending on the intended output, more modifications might be necessary: If a protein is chosen as output, an additional LCrz and poly-A tail might be needed for synthetic 5' capping and 3' polyadenylation. Based on common general knowledge the skilled person will readily be able to make the necessary adaptations to the above designs. It should also be noted that the above designs are not limited to only one output. Rather, two or more outputs can be transcriptionally synchronized with the endogenous gene of interest.
In summary, a preferred embodiment of the eukaryotic 3' design comprises on the DNA level: A poly-A sequence (if necessary) followed by a 3' self-cleaving ribozyme, a spacer, a 5' selfcleaving ribozyme, a variable output and a 3' self-cleaving ribozyme. The 5' design preferably comprises on the DNA level of a 5' self-cleaving ribozyme, a variable output, a 3' self-cleaving ribozyme, a spacer and a LCrz (if necessary).
In prokaryotes, transcriptional synchronization is less complicated, due to the lack of 5' capping and 3' polyadenylation. A preferred prokaryotic design comprises at least one Rz-vO-Rz box composed of 5' self-cleaving ribozyme, variable output and 3' self-cleaving ribozyme. The box can be placed upstream or downstream of an ORF or any other suitable first sequence coding for a first transcription product.
Sequences
In the following, exemplary sequences, e.g. for suitable ribozymes etc., are presented.
Ribozymes
In the following, examples of ribozyme sequences are listed. The RNA sequences are given. Lariat capping ribozyme (LCrz) (SEQ ID NO: 01):
GGUUGGGUUGGGAAGUAUCAUGGCUAAUCACCAUGAUGCAAUCGGGUUGAACAC UUAAUUGGGUUAAAACGGUGGGGGACGAUCCCGUAACAUCCGUCCUAACGGCGA CAGACUGCACGGCCCUGCCUCUUAGGUGUGUUCAAUGAACAGUCGUUCCGAAAG GAAGCAUCCGGUAUCCCAAGACAAUC
See, for example, Meyer M, Nielsen H, Olieric V, et al. Speciation of a group I intron into a lariat capping ribozyme. Proc Natl Acad Sci U S A. 2014; 11 l(21):7659-7664. doi: 10.1073/pnas.1322248111).
Hepatitis delta virus ribozyme (HDVRz) (SEQ ID NO: 02):
CCGGCCGUACCAGGGUCGGAGGAGCGACCGCGGCCGACCCGUUGUACGAAGCCG UACCGCUUACCCUG
Alternative version (SEQ ID NO: 03):
AGGGUCGGCAUGGCAUCUCCACCUCCUCGCGGUCCGACCUGGGCUACUUCGGUA
GGCUAAGGGAGAAGCUUGGCACUGGCCGUCGUUU
See, for example, Gao Y, Zhao Y (2014). Self-processing of ribozyme-flanked RNAs into guide RNAs in vitro and in vivo for CRISPR-mediated genome editing. Journal of Integrative Plant Biology, 56(4), 343-349. doi: 10.1111/jipb.12152.
CPEB3 ribozyme (SEQ ID NO: 04)
GGGGGCCACAGCAGAAGCGUUCACGUCGCAGCCCCUGUCAGAUUCUGGUGAAUC UGCGAAUUCUGCU
See, for example, Chadalavada DM, Gratton EA, Bevilacqua PC. The human HDV-like CPEB3 ribozyme is intrinsically fast-reacting. Biochemistry. 2010;49(25):5321-5330, doi: 10.1021/bi 100434c. Also from Rfam Seed sequence alignment for RF00622 (http://rfam.xfam.org/family/RF00622/alignment/html); AL158040.14/122218-122141 (Homo sapiens); corresponding sequence in bold (SEQ ID NO: 05):
AGGGGAUAACAGGGGGCCACAGCAGAAGCGUUCACGUCGCAGCCCCUGUCAG
AUUCUGGUGAAUCUGCGAAUUCUGCU
Hammerhead ribozyme (HHRz) (SEQ ID NO: 06):
NNNNNNGACUACUCAGGCACUCCUGCUUUGCUCAUUCGAGCAG
Alternative sequence (SEQ ID NO: 07):
GGGAGNNNNNNNNCUGAUGAGUCCGUGAGGACGAAACGGUACCCGGUACCGUC
See, for example, Gao Y, Zhao Y (2014). Self-processing of ribozyme-flanked RNAs into guide RNAs in vitro and in vivo for CRISPR-mediated genome editing. Journal of Integrative
Plant Biology, 56(4), 343-349. doi: 10.1111/jipb.12152.
It is to be noted that the N’s (in bold) in the sequences depend on the bases that are downstream of the HHRz.
Other HHRz variants can, for example, be found at: http://rfam.xf am. org/family/RF02277#tabview=tab0
Hammerhead 1
Hammerhead 3
Hammerhead HH10
Hammerhead HH9
Hammerhead II
Type-P5 twister ribozyme (SEQ ID NO: 08):
CUUGUAAUGCGGCCGUGUAAAUAUUUACACGUCGGUCUCAAGCCCGAUAAACGC AGAGAGCAAG See, for example, Roth A, Weinberg Z, Chen AG, Kim PB, Ames TD, Breaker RR. A widespread self-cleaving ribozyme class is revealed by bioinformatics. Nat Chem Biol. 2014;10(l):56-60. doi:10.1038/nchembio,1386.
Other variants can, for example, be found at: https://rfam.org/family/RF02684/alignment/html
Twister sister ribozyme (SEQ ID NO: 09): ggACCCGCAAGGCCGACGGCAUCCGCCGCCGCUGGUGCAAGUCCAGCCGCCCCGG GGCGGGCGCUCAUGGGUAAAC
See, for example, Weinberg Z, Kim PB, Chen TH, et al. New classes of self-cleaving ribozymes revealed by comparative genomics analysis. Nat Chem Biol. 2015; 11(8):606-610. doi : 10.1038/nchembio.1846.
Alternative versions (RF02681 from Rfam):
>ADJS01013948.1/250-330 (SEQ ID NO: 10)
GCAACCCGCAAGGCCGACGCACAACGCGCCGCCGGUGCAAGCCCGGCCACCCUGC AAGGGGUGGGCGCUCAUGGGUACACA
>BABG01005008.1/780-696 (SEQ ID NO: 11)
GAAACCCGCUAGGCCGACAGCCUCACCGCUGCCGCUGGUGCAAGCCCAGCCGCCC CAGACCGGGGCGGGCGCUCAUGGGUAACAG
>ADJS01013948.1/577-657 (SEQ ID NO: 12)
ACGACCCGCAAGGCCGACGCAUAACGCGCCGCCGGUGCAAGCCCGGCCACCCCAC AUGGGGCGGGCGCUCAUGGGUACACA
>FP929046.1/2708602-2708521 (SEQ ID NO: 13)
AUGACCCGCAAGGCCGACGGCAUCCCGCCGCCGCUGGUGCAAGCCCAGCCGCCCC GCCAGGGCGGGCGCUCAUGGGUCCACA Hatchet ribozyme (SEQ ID NO: 14):
AAUCGUUCUUACUGAUAUCAGUGACAAACAUGUGGGGCUUAUCUAAUCUUCGGA
UUAGUAUUAGUGCAGACGUUAAAACCAUGU
See, for example, Li S, Liinse CE, Harris KA, Breaker RR. Biochemical analysis of hatchet self-cleaving ribozymes. RNA. 2015;21 (11): 1845-1851. doi:10.1261/ma.052522.115.
Alternative sequences can be found at: https://rfam.org/family/RF02678/alignment/html
Spacer sequence:
Short variant:
AAAAAAAA (= 8 nt)
Long variant (SEQ ID NO: 15):
AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA
AAAAAAAAAAAAAAAAAAA (= 72 nt)
Note: Other lengths are also possible.
Synthetic poly-A tail variant:
Short variant (SEQ ID NO: 16):
AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA (= 48 nt)
Long variant (SEQ ID NO: 17):
AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA
AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA
AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA
AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA
AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAA AAA (= 300 nt) Note: The stability of the mRNA can be modulated by choosing different lengths of the synthetic poly-A tail of the DNA. Other lengths than the given ones are also possible.
In the following, the invention is exemplified below for illustration purposes by way of the description of an application relating to resistance against human cancer and an application relating to the resistance against plant pathogens.
Resistance against human cancer
In cancer cells, the transcription of tumor suppressor genes is downregulated by genetic/epigenetic means (Wang et al., 2018; Liu et al, 2016) or is even suspended through gene deletions (Dong, 2001; Cai and Sablina, 2016; Cooper, 2000). All this, while the expression of proto-oncogenes is increased, thus, creating oncogenes (Cooper, 2000).
A resistance-mechanism for cancer cells based on the invention relies on sensing these genetic alterations by comparing the transcriptional activity of (proto-)oncogenes and tumor suppressor genes (see Figure 2). This is possible by adding a DNA sequence for STAR (Short Transcription Activating RNA) after a common (proto-)oncogene DNA sequence (e.g. after an ORF), thus, synchronizing the (proto-)oncogene with STAR (see Fig. 2A). STAR is able to activate the transcription of a target gene by binding to an upstream transcription terminator t500, which is located in front of the gene right after its promoter (Chappell et al., 2015). By default, the terminator t500 prevents the transcription of the gene behind it via a stem-loop (Chappell et al., 2015). Yet, if a STAR molecule binds to the t500 terminator, transcription is enabled (Chappell et al., 2015). To enable maximum efficiency of STAR, a 5' hammerhead ribozyme (Gao and Zhao, 2014) is added to the 5' end of the STAR DNA sequence. The 3' end of STAR has a 3' self-cleaving ribozyme added, e.g. the hepatitis delta virus ribozyme (HDVRz) (Gao and Zhao, 2014). Flanking the STAR sequence with self-cleaving ribozymes results in a STAR transcript that is independently mobile from the oncogene and has no overhang from the transcription terminator of the gene, which is situated at the outermost 3' end of the gene. Furthermore, Anti STAR DNA sequences are added in a similar way to (multiple) tumor suppressor genes. They, too, will be insulated from the normal transcript and terminator of the gene by flanking self-cleaving ribozymes at the 5' and 3' end of the AntiSTAR sequence. The endogenous ORFs of the (proto-)oncogenes and tumor suppressor genes will have poly-A sequence added directly downstream to enable nuclear export and cytoplasmic translation.
STAR and AntiSTAR inhibit each other via antisense base pairing. The mechanism is further depending on an overall equal or higher transcriptional activity of tumor suppressor genes as long as the cell is healthy. Thus, for every (proto-)oncogene that is modified in the above- mentioned way, there should be one or more tumor suppressor genes chosen, which (always) have a (combined) much higher transcription activity than the (proto-)oncogene. This way, the default state of a cell harboring this mechanism will be that the STAR-activity is quenched by the much more abundant Anti-STAR molecules.
This sensing mechanism qualifies as a first control and safety mechanism. However, it is necessary to allow STAR to induce the transcription of a therapeutic output as soon as the cell becomes cancerous (see Fig. 2B). This is possible by choosing tumor suppressor genes that are often deleted or silenced in cancer cells, e.g. the gene for MHC 1 (Garrido et al., 2016). As soon as the transcription of these genes is significantly downregulated and AntiSTAR is produced in a lower quantity than STAR, the transcription of the therapeutic output can be induced by STAR.
A second safety mechanism could be constituted by the therapeutic output itself, which can be induced by STAR: After transcriptional activation via STAR, a proton channel could be produced by the cell (see Fig. 2C). This proton channel would locate to the cell membrane and enable passive proton transport across the membrane. Thus, in an acidic tumor microenvironment, the channel will import protons into the cell. This will acidify cancer cells, while also reducing the extracellular proton concentration. This should result in cancer cell termination due to the now acidic cellular milieu, futile ATP consumption for proton export (Whitton et al., 2018), glucose deprivation (Xun et al., 2017) and a reduced capability for tumor invasion (Estrella et al., 2013). Also, it should cause an improved immune answer due to the less acidic tumor microenvironment (Huber et al., 2017). The proton channel could be e.g. the otopetrin proton channels Otopl or Otop3 (Saotome et al, 2019) or a modified viral M2 proton channel (Cady et al., 2009). If this mechanism is used prior to the onset of cancer, it might prevent any later onset by denying the cancerous cell the ability to effectively unload excess protons, while also keeping a microenvironment that is more favorable for an immune response.
Choosing a proton unloading enzyme inhibitor instead of the proton channel as therapeutic output would be likely to be even more efficient: The cancerous cell would be unable to unload their excess protons right after the pathological gene alterations have taken place. V-ATPase is used by cancer cells to unload excess protons and prevent intracellular acidification (Whitton et al., 2018). Thus, inhibiting either transcription, translation or protein function of V-ATPase should acidify and kill cancer cells, while other proton unloading enzymes (e.g. monocarboxylate transporters, Na+/H+ exchanger (Aoi and Marunaka, 2014) and carbonic anhydrases (Spugnini et. al, 2015) might have to be targeted too.
Also, AntiSTAR genes should be located as close as possible to STAR (for direct quenching of STAR) and to the t500 terminator (close-target quenching). Thus, quenching STAR activity is less a matter of chance. Instead, it would be much more related to differences in quantity between STAR and AntiSTAR, while stochastic problems caused by diffusion area are reduced. Only a much higher STAR production or much lower AntiSTAR production would be sufficient for STAR-target activation. Also, the regulation via the RNA-based STAR/AntiSTAR-system should be quickly reversible due to fast RNA degradation (Chappell et al., 2017). The system described above is depicted in figure 2. It is to be noted, that the figure shows a simplified version of the mechanism for transcriptional synchronization, which does not contain poly-A tails.
Resistance against plant pathogens
Transcriptional synchronization according to the invention can also be used to create synthetic pathogen resistance in plants, e.g. crop plants. For this, any gene, which transcription rate is influenced by the pathogen, can be transcriptionally synchronized with a resistance output against said pathogen. For example, the FAD7 gene could be transcriptionally synchronized with an RNAi against fungal infections: If a plant cell is wounded, FAD7 expression is upregulated (Nishiuchi et al., 1999). Fungi need to penetrate the plant cell for infection, thus upregulating the expression of FAD7 (Kirsch et al., 1997). If FAD7 is now transcriptionally synchronized with e.g. an RNAi against the respective fungi, the expression of the RNAi is triggered upon infection. Using transcriptional synchronization against plant pathogens has at least two distinctive advantages compared to using either constitutive or additional endogenous promoters coupled with a resistance mechanism: First, a constitutive promoter for a resistance is always active - even if the pathogen is not present. This means, that the plant cell is wasting its resources to produce a resistance that is most of the time not needed. This again will influence the growth and harvest yield of crop plants in a negative way, depending on how many resistances are used in the plant. Second, if the resistance is controlled by using an endogenous promoter of the plant (e.g. the FAD7 promoter), the aforementioned problem is not reliably solved: The promoter is not solely controlling the transcription rate of a gene, but the genetic context is also important, including enhancer and silencer sequences, intronic effects, etc. Thus, it would be necessary to test every resistance, which is only regulated by an endogenous promoter, if its expression is regulated as intended. Depending on the number of needed resistances, this can become time- and cost-intensive. Transcriptional synchronization according to the invention solves both problems, by using the existing transcriptional regulation pathways of the cell to express the synthetic resistance only if needed.
These advantages enable another possibility: Instead of directly synchronizing only one resistance output with an endogenous gene, the endogenous gene can be synchronized with a transcription activating output, which in turn activates the transcription of multiple resistance genes. Thus, the synchronization would hardly influence the transcription time for the synchronized endogenous gene, while still being able to activate a plethora of resistance genes. Further, multiple endogenous genes can be synchronized with one (or more) synthetic resistance genes so that one pathogen triggers one (or more) resistance gene(s) by upregulating the expression of all the synchronized endogenous genes. Using multiple inputs (synchronized endogenous genes) and multiple outputs (synthetic resistances) would make it near impossible for any pathogen to adapt to the synthetic resistances by evolutionary means. All this, while having a relatively low impact on the plant metabolism. Also, the number of genetic modifications can be reduced significantly compared to introducing the same number of resistances by using other methods, making transcriptional synchronization very cost-efficient. Experimental results
HeLa cells were transfected with a plasmid consisting of the commercially available plasmid pEGFP-Nl (for sequence see SEQ ID NO: 18; see also GenBank entry U55762.1) as a backbone and containing a synchronization cassette shown in Fig. 3 (see SEQ ID NO: 19). For suitable cell culture, transfection, RNA extraction and qPCR protocols see, for example, Kirchner et al. 2017.
The backbone pEGFP-Nl included the first synchronized output (EGFP), i.e. the first functional transcription product, which is a fluorescent protein. The synchronization cassette containing the second synchronized output (shThr2), i.e. the second functional transcription product flanked by ribozymes (Hammerhead ribozyme and Hepatitis delta virus ribozyme in this case) and was placed directly after the sequence for EGFP. The shRNA (shThr2) of the synchronization cassette was supposed to target threonine tRNA of HeLa cells.
A functional synchronization would result in a synchronized transcription of both outputs (EGFP and shThr2), i.e. both outputs would be transcribed at the same time, resulting in a detectable fluorescence and reduced threonine tRNA levels. To test this, HeLa cells transfected with the aforementioned plasmid and HeLa cells that where not transfected were compared. First, fluorescence was measured using a Tecan spark plate reader (see tables 1 and 2 below). This was done in two independent experiments. The fluorescence in the first experiment was on average 62.35 times greater in treated (=transfected) cells compared to untreated (not transfected) cells. In the second experiment it was on average 71.3 times greater in treated cells compared to untreated.
Table 1 : Fluorescence values of experiment 1 for treated and untreated HeLa cells using three biological replicates split in four physical replicates each. Unit of measurement was RFU (Relative Fluorescence Units).
Figure imgf000043_0001
Figure imgf000044_0001
Table 2: Fluorescence values of experiment 2 for treated and untreated HeLa cells using three biological replicates split in four physical replicates each. Unit of measurement was RFU (Relative Fluorescence Units).
Figure imgf000044_0002
Figure imgf000045_0001
Further, in both experiments the threonine tRNA levels (and 5S rRNA levels for normalization) were measured using RT-qPCR. After reverse transcription of the total RNA of 3 biological replicates, two technical replicates were used for each biological replicate. The tRNAThr (CGU) levels were analyzed using the QuantiFast SYBR Green RT-PCR kit (from Qiagen). The tRNAThr (CGU) and 5S rRNA were amplified by using the following primers: tRNAThr(CGU) forward (GGCCAAGTGGTAAGGC) and tRNAThr(CGU) reverse (AGGCACGGACGGG), 5S rRNA forward (CCATACCACCCTGAACGC) and 5S rRNA reverse (GTATTCCCAGGCGGTCTC). The tRNA Ct values were normalized to 5S rRNA Ct values. The fold change was calculated using the double delta Ct (ddCt) analysis method. The first experiment resulted in a 67.4% reduction of tRNAThr (CGU) levels (ddCt = 0.326), while the reduction in the second experiment was 10% (ddCt = 0.9). The Ct values used for the calculation of the ddCt values are shown in tables 3 and 4.
Table 3: Ct values for experiment 1 obtained by RT-qPCR.
Figure imgf000045_0002
Table 4: Ct values for experiment 2 gained by RT-qPCR.
Figure imgf000046_0001
Both experiments show that the treated (transfected) cells exhibited much higher fluorescence values than the untreated cells. This shows that the first synchronized output (EGFP) is functional. Also, in both experiments the treated cells had reduced levels of threonine tRNA, which shows that the second synchronized output (shThr2) is also functional. Thus, both synchronized outputs are functional.
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Liu B, Song J, Luan J, Sun X, Bai J, Wang H, Li A, Zhang L, Feng X, Du Z (2016). Promoter methylation status of tumor suppressor genes and inhibition of expression of DNA methyltransferase 1 in non-small cell lung cancer. Exp Biol Med (Maywood). 2016 Aug;241(14):1531-9. doi: 10.1177/1535370216645211. Epub 2016 Apr 26. PMID: 27190263; PMCID: PMC4994907.
Kirchner S, Cai Z, Rauscher R, Kastelic N, Anding M, et al. (2017) Alteration of protein function by a silent polymorphism linked to tRNA abundance. PLOS Biology 15(5): e2000779. https://doi.org/10.1371/joumal.pbio.2000779.
Meyer M, Nielsen H, Olieric V, et al. (2014). Speciation of a group I intron into a lariat capping ribozyme. Proc Natl Acad Sci U S A.;l 11(21):7659-7664. doi: 10.1073/pnas,1322248111.
Nishiuchi T, Kodama H, Yanagisawa S, Iba K (1999). Wound-induced expression of the FAD7 gene is mediated by different regulatory domains of its promoter in leaves/stems and roots. Plant Physiol.; 121(4): 1239-1246. doi: 10.1104/pp.121.4.1239.
Saotome K, Teng B, Tsui CCA, Lee WH, Tu YH, Kaplan JP, Sansom MSP, Liman ER, Ward AB (2019): Structures of the otopetrin proton channels Otopl and Otop3. Nature Structural & Molecular Biology, VOL 26, 518-525. https://doi.org/10.1038/s41594-019-0235-9.
Spugnini EP, Sonveaux P, Stock C, Perez-Sayans M, De Milito A, Avnet S, . . ., Fais S (2015). Proton channels and exchangers in cancer. Biochimica et Biophysica Acta (BBA) - Biomembranes, 1848(10), 2715-2726. doi: 10.1016/j.bbamem.2014.10.015.
Wang LH, Wu CF, Rajasekaran N, Shin YK (2018): Loss of Tumor Suppressor Gene Function in Human Cancer: An Overview. Cell Physiol Biochem 2018;51 :2647-2693. https://doi.org/10.1159/000495956. Whitton B, Okamoto H, Packham G, Crabb SJ (2018). Vacuolar ATPase as a potential therapeutic target and mediator of treatment resistance in cancer. Cancer Med. 2018 Aug;7(8):3800-3811. doi: 10.1002/cam4.1594. Epub 2018 Jun 21. PMID: 29926527; PMCID: PMC6089187.
Xun H, Ming C, Hao Wu (2017): Central role of lactate and proton in cancer cell resistance to glucose deprivation and its clinical translation. Signal Transduction and Targeted Therapy (2017) 2, el6047; doi: 10.1038/sigtrans.2016.47; published online 10 March 2017.

Claims

- 48 - CLAIMS
1. A genetically engineered cell, the cell being genetically engineered to comprise a transcription unit with a promotor and a termination signal, the transcription unit comprising, between the promotor and the termination signal, a) a first nucleotide sequence coding for a first functional transcription product, b) a second nucleotide sequence coding for a second functional transcription product, c) a third nucleotide sequence coding for a first self-cleaving ribozyme, and d) a fourth nucleotide sequence coding for a second self-cleaving ribozyme, the second nucleotide sequence coding for the second functional transcription product being flanked by the third and fourth nucleotide sequence coding for the first and second self-cleaving ribozyme.
2. The cell according to claim 1, wherein the first nucleotide sequence codes for an mRNA encoding a first protein, preferably an endogenous protein of the cell, or a first functional noncoding RNA molecule, preferably an endogenous functional non-coding RNA molecule of the cell.
3. The cell according to claim 1 or 2, wherein the second nucleotide sequence codes for a functional non-coding RNA or an mRNA encoding a protein.
4. The cell according to one of claims 1 to 3, wherein the third nucleotide sequence codes for a 5' self-cleaving ribozyme and the fourth nucleotide sequence codes for a 3' self-cleaving ribozyme.
5. The cell according to one of the preceding claims, wherein the cell is a eukaryotic cell and wherein, in the direction from the promoter to the termination signal a) the second nucleotide sequence coding for the second functional transcription product and flanked by the third and fourth nucleotide sequence coding for a first and second self-cleaving ribozyme, is arranged after the first nucleotide sequence coding for the first functional transcription product, the first nucleotide sequence coding for the first functional transcription product preferably being followed by a fifth nucleotide sequence encoding a Poly A tail, the - 49 - fifth nucleotide sequence encoding a Poly A tail preferably being followed by a sixth nucleotide sequence coding for a third self-cleaving ribozyme, preferably a 3' self-cleaving ribozyme, or b) the second nucleotide sequence coding for the second functional transcription product and flanked by the third and fourth nucleotide sequence coding for a first and second self-cleaving ribozyme, is arranged before the first nucleotide sequence coding for the first functional transcription product, the first nucleotide sequence coding for the first functional transcription product preferably being preceded by a seventh nucleotide sequence coding for a Lariat capping ribozyme.
6. The cell according to claim 5, wherein, a) where, in the direction from the promoter to the termination signal, the second nucleotide sequence coding for the second functional transcription product and flanked by the third and fourth nucleotide sequence coding for a first and second self-cleaving ribozyme, is arranged after the first nucleotide sequence coding for the first functional transcription product, the third nucleotide sequence coding for a first self-cleaving ribozyme is preceded by a spacer sequence, or b) where, in the direction from the promoter to the termination signal, the second nucleotide sequence coding for the second transcription product and flanked by the third and fourth nucleotide sequence coding for a first and second self-cleaving ribozyme, is arranged before the first nucleotide sequence coding for the first functional transcription product, the fourth nucleotide sequence coding for a second self-cleaving ribozyme is followed by a spacer sequence.
7. The cell according to claim 6, wherein, in the direction from the promoter to the termination signal, the first nucleotide sequence coding for the first functional transcription product, the third nucleotide sequence coding for the first self-cleaving ribozyme, the fourth nucleotide sequence coding for the second self-cleaving ribozyme, the second nucleotide sequence coding for the second functional transcription product, and, if present, the fifth nucleotide sequence encoding a Poly A tail, the sixth nucleotide sequence coding for the third self-cleaving ribozyme, the seventh nucleotide sequence coding for the Lariat capping ribozyme and the spacer sequence are arranged between the promoter and the terminations signal in the following order: - 50 - a) first nucleotide sequence coding for the first functional transcription product - fifth nucleotide sequence encoding a Poly A tail - sixth nucleotide sequence coding for the third self-cleaving ribozyme, preferably a 3' self-cleaving ribozyme - spacer sequence - third nucleotide sequence coding for the first self-cleaving ribozyme, preferably a 5' self-cleaving ribozyme - second nucleotide sequence coding for the second functional transcription product - fourth nucleotide sequence coding for the second self-cleaving ribozyme, preferably a 3' selfcleaving ribozyme, or b) third nucleotide sequence coding for the first self-cleaving ribozyme, preferably a 5' selfcleaving ribozyme - second nucleotide sequence coding for the second functional transcription product - fourth nucleotide sequence coding for the second self-cleaving ribozyme, preferably a 3' self-cleaving ribozyme - spacer sequence - seventh nucleotide sequence coding for the Lariat capping self-cleaving ribozyme - first nucleotide sequence coding for the first functional transcription product.
8. The cell according to one of the preceding claims, wherein the cell is a plant cell, an insect cell, a mammalian cell, a non-human animal cell, or a human cell.
9. The cell according to one of claims 1 to 4, wherein the cell is a prokaryotic cell.
10. A transgenic plant or non-human animal comprising a cell of one of claims 1 to 8.
11. Vector or vector system for the transfection, preferably stable transfection, of a cell, the vector comprising: a) a first nucleotide sequence coding for a first functional transcription product, b) a second nucleotide sequence coding for a second functional transcription product, c) a third nucleotide sequence coding for a first self-cleaving ribozyme, and d) a fourth nucleotide sequence coding for a second self-cleaving ribozyme, the second nucleotide sequence coding for the second functional transcription product being flanked by the third and fourth nucleotide sequence coding for the first and second self-cleaving ribozyme, - 51 - wherein the vector or vector system is configured to introduce the nucleotide sequences of a) to d) into a transcription unit of the cell between a promoter and a termination signal of the transcription unit.
12. A method for the transcriptional synchronization of two or more functional transcription products of a cell, the method comprising:
Introducing into a transcription unit of the cell, the transcription unit comprising a promotor and a termination signal i. a first nucleotide sequence coding for a first functional transcription product, ii. a second nucleotide sequence coding for a second functional transcription product, iii. a third nucleotide sequence coding for a first self-cleaving ribozyme, and iv. a fourth nucleotide sequence coding for a second self-cleaving ribozyme, the second nucleotide sequence coding for the second functional transcription product being flanked by the third and fourth nucleotide sequences coding for the first and second selfcleaving ribozyme.
13. The method according to claim 12, wherein the first nucleotide sequence codes for an mRNA encoding a first protein, preferably an endogenous protein of the cell, or a first functional non-coding RNA molecule, preferably an endogenous functional non-coding RNA molecule of the cell.
14. The method according to one of claims 12 or 13, wherein the second nucleotide sequence codes for a functional non-coding RNA or an mRNA encoding a protein
15. The method according to one of claims 12 or 13, wherein the third nucleotide sequence codes for a 5' self-cleaving ribozyme and the fourth nucleotide sequence codes for a 3' selfcleaving ribozyme.
16. The method according to one of claims 12 to 15, wherein the cell is a eukaryotic cell and wherein, in the direction from the promoter to the termination signal a) the second nucleotide sequence coding for the second functional transcription product and flanked by the third and fourth nucleotide sequence coding for a first and second self-cleaving ribozyme, is introduced after the first nucleotide sequence coding for a first functional transcription product, wherein a fifth nucleotide sequence encoding a Poly A tail is preferably introduced after the first nucleotide sequence coding for a first functional transcription product, and wherein a sixth nucleotide sequence coding for a third self-cleaving ribozyme, preferably a 3' self-cleaving ribozyme, is preferably introduced after the fifth nucleotide sequence encoding a Poly A tail, or b) the second nucleotide sequence coding for the second functional transcription product and flanked by the third and fourth nucleotide sequence coding for a first and second self-cleaving ribozyme, is introduced before the first nucleotide sequence coding for a first functional transcription product, wherein a seventh nucleotide sequence coding for a Lariat capping ribozyme is preferably introduced directly before the first nucleotide sequence coding for a first functional transcription product.
17. The method according to claim 16, wherein, a) where, in the direction from the promoter to the termination signal, the second nucleotide sequence coding for the second functional transcription product and flanked by the third and fourth nucleotide sequence coding for a first and second self-cleaving ribozyme, is introduced after the first nucleotide sequence coding for a first functional transcription product, a spacer sequence is introduced before the third nucleotide sequence coding for the first self-cleaving ribozyme, or b) where, in the direction from the promoter to the termination signal, the second nucleotide sequence coding for the second functional transcription product and flanked by the third and fourth nucleotide sequence coding for a first and second self-cleaving ribozyme, is introduced before the first nucleotide sequence coding for a first functional transcription product, a spacer sequence is introduced after the fourth nucleotide sequence coding for the second self-cleaving ribozyme.
18. The method according to claim 17, wherein, in the direction from the promoter to the termination signal, the first nucleotide sequence coding for a first functional transcription product, the third nucleotide sequence coding for the first self-cleaving ribozyme, the fourth nucleotide sequence coding for the second self-cleaving ribozyme, the second nucleotide sequence coding for the second functional transcription product, and, optionally, the fifth nucleotide sequence encoding a Poly A tail, the sixth nucleotide sequence coding for the third self-cleaving ribozyme, the seventh nucleotide sequence coding for the Lariat capping ribozyme, and the spacer sequence are introduced in such a way between the promoter and the terminations signal that the following order results: a) first nucleotide sequence coding for the first functional transcription product - fifth nucleotide sequence encoding a Poly A tail - sixth nucleotide sequence coding for the third self-cleaving ribozyme, preferably a 3' self-cleaving ribozyme - spacer sequence - third nucleotide sequence coding for the first self-cleaving ribozyme, preferably a 5' self-cleaving ribozyme - second nucleotide sequence coding for second functional transcription product - fourth nucleotide sequence coding for the second self-cleaving ribozyme, preferably a 3' selfcleaving ribozyme, or b) third nucleotide sequence coding for the first self-cleaving ribozyme, preferably a 5' selfcleaving ribozyme - second nucleotide sequence coding for the second functional transcription product - fourth nucleotide sequence coding for the second self-cleaving ribozyme, preferably a 3' self-cleaving ribozyme - spacer sequence - seventh nucleotide sequence coding for the Lariat capping ribozyme -first nucleotide sequence coding for a first functional transcription product.
19. The method according to one of claims 12 to 18, wherein the cell is a plant cell, an insect cell, a mammalian cell, a non-human animal cell, or a human cell.
20. The method according to one of claims 12 to 15, wherein the cell is a prokaryotic cell.
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