LU102162B1

LU102162B1 - Transcriptional synchronization of two or more functional transcription products

Info

Publication number: LU102162B1
Application number: LU102162A
Authority: LU
Inventors: Mathias Schmidt
Original assignee: Univ Hamburg
Priority date: 2020-10-26
Filing date: 2020-10-26
Publication date: 2022-04-27

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

PAT 1793 LU -1- LU102162

TRANSCRIPTIONAL SYNCHRONIZATION OF TWO OR MORE FUNCTIONAL TRANSCRIPTION PRODUCTS

DESCRIPTION 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, y

| PAT 1793 LU —2- LU102162 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 product, 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

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PAT 1793 LU -3- LU102162 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 ofa synchronized genomic gene.

Thus, it can be used to detect transcriptional dysregulation in the i#

PAT 1793 LU —4- LU102162 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. 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, y

PAT 1793 LU

= LU102162 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

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PAT 1793 LU

—6- LU102162 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

PAT 1793 LU -7- LU102162 term thus encompasses any primary RNA transcript from a transcription unit comprising non- | coding 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.

PAT 1793 LU -8- LU102162 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.

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PAT 1793 LU —9- LU102162 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 S'-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(1):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;1 1(8):606-610. doi:10.1038/nchembio.1846) or hatchet ribozymes (Li S, Lünse CE, Harris KA, Breaker RR. Biochemical analysis of hatchet self-cleaving ribozymes. RNA. 2015;21(11):1 845-1851. doi:10.1261/rna.052522.115).

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PAT 1793 LU —10— LU102162 The term “Lariat-capping ribozyme” (LC ribozyme, LCrz, formerly GIR1 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’GpppN cap) in the downstream pre-mRNA (see, e.g., Meyer M, Nielsen H, Oliéric V, et al. Speciation of a group I intron into a lariat capping ribozyme. Proc Natl Acad Sci US 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 GIR1 ribozyme (see RCSB PDB 6GYV released 2018-08-22). | 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

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PAT 1793 LU —11- LU102162 — 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. 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

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PAT 1793 LU -12- LU102162 — 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.

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 (HHRZz). If the second nucleotide sequence is chosen to code for a mRNA, which is supposed to be translated into a protein, the respective DNA is preferably modified in a way that a Lariat- capping ribozyme is situated at the S'-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. i

PAT 1793 LU -13- LU102162 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 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 (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.

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PAT 1793 LU —14- LU102162 The spacer sequence is particularly useful in the 3’ design, and facilitates folding of the self- cleaving 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.

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' m’G cap. 1s

PAT 1793 LU -15- LU102162 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 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.

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’ m’G cap. | ih

PAT 1793 LU -16- LU102162 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. 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, RB1, APC, BRCAI, 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. .

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PAT 1793 LU —17- LU102162 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 self- cleaving 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 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).

I

PAT 1793 LU -18- LU102162 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 in 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 a further preferred embodiment of the vector or vector system according 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 qu

PAT 1793 LU

— 19— LU102162 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 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 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 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 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" self- cleaving ribozyme, or b) 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

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PAT 1793 LU -20- LU102162 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 self- cleaving 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

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PAT 1793 LU -21- LU102162 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 non- coding 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.

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 y”

PAT 1793 LU —22— LU102162 | 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.

Preferably, in the method of the invention, the third nucleotide sequence codes for a 5’ self- cleaving 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.

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PAT 1793 LU

—23- LU102162 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 self- cleaving 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 dé

PAT 1793 LU -24- LU102162 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’ 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 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. In a further embodiment of the method of the invention, the cell is a prokaryotic cell, e.g. a bacterial or archaean 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.

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PAT 1793 LU 257 LU102162 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 self- cleaving 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.

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 br

PAT 1793 LU

-26- LU102162 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.

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 self- cleaving 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 br

| PAT 1793 LU -27- LU102162 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 . or

| PAT 1793 LU -28— LU102162 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 1a: 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 double- stranded.

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 1

| PAT 1793 LU -29- LU102162 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 1b.

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' self- cleaving 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.

| PAT 1793 LU —30— LU102162 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, Oliéric 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). 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

PAT 1793 LU -31- LU102162 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/bi100434c. 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.xfam.org/family/RF02277#tabview=tab0 Hammerhead 1 Hammerhead 3 Hammerhead HH10 Hammerhead HH9 Hammerhead II Type-P5 twister ribozyme (SEQ ID NO: 08):

PAT 1793 LU -32- LU102162

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(1):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)

PAT 1793 LU -33- LU102162

AUGACCCGCAAGGCCGACGGCAUCCCGCCGCCGCUGGUGCAAGCCCAGCCGCCCC GCCAGGGCGGGCGCUCAUGGGUCCACA | Hatchet ribozyme (SEQ ID NO: 14): | AAUCGUUCUUACUGAUAUCAGUGACAAACAUGUGGGGCUUAUCUAAUCUUCGGA

UUAGUAUUAGUGCAGACGUUAAAACCAUGU See, for example, Li S, Lünse CE, Harris KA, Breaker RR. Biochemical analysis of hatchet self-cleaving ribozymes. RNA. 2015;21(11):1845-1851. doi:10.1261/rna.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

PAT 1793 LU _34_ LU102162 | AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA

AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA (= 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 Bu

PAT 1793 LU —35— LU102162 overhang from the transcription terminator of the gene, which is situated at the outermost 3’ end of the gene.

Furthermore, AntiSTAR 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 q

| PAT 1793 LU -36- LU102162 less acidic tumor microenvironment (Huber et al., 201 7). The proton channel could be e.g. the otopetrin proton channels Otop1 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

SZ

PAT 1793 LU

-37- LU102162 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

"

PAT 1793 LU -38- LU102162 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. References: Aoi W, Marunaka Y (2014). Importance of pH homeostasis in metabolic health and diseases: crucial role of membrane proton transport. Biomed Res Int.; 2014:598986. doi:

10.1155/2014/598986. Epub 2014 Sep 11. PMID: 25302301; PMCID: PMC4180894.

Cady SD, Luo W, Hu F, Hong M (2009). Structure and function of the influenza A M2 proton channel. Biochemistry. 2009 Aug 11;48(31):7356-64. doi: 10.1021/bi9008837. PMID: 19601584; PMCID: PMC2879269.

Cai Y, Sablina AA (2016). Cancer-associated chromosomal deletions: Size makes a difference. Cell Cycle. 2016 Nov;15(21):2850-2851. doi: 10.1080/15548627.2016.1204869. Epub 2016 Jul

26. PMID: 27458787; PMCID: PMCS105910.

Chappell J, Takahashi MK, Lucks JB (2015): Creating small transcription activating RNAs. Nature Chemical Biology volumel 1, pages214—220. doi: 10.1038/nCHeMBIO.1737 Chappell J, Westbrook A, Verosloff, M et al. (2017): Computational design of small transcription activating RNAs for versatile and dynamic gene regulation. Nat Commun 8, 1051. doi:10.1038/s41467-017-01082-6 Cooper GM (2000). The Cell: A Molecular Approach. 2nd edition. Sunderland (MA): Sinauer Associates. Tumor Suppressor Genes. Available from: https://www.ncbi.nlm.nih.gov/books/NBK9894/ Dong JT (2001): Chromosomal deletions and tumor suppressor genes in prostate cancer. Cancer Metastasis Rev. 2001;20(3-4):173-93.

| PAT 1793 LU —39— LU102162 Estrella V, Chen T, Lloyd M, Wojtkowiak J, Cornnell HH, Ibrahim-Hashim A, Bailey K, Balagurunathan Y, Rothberg JM, Sloane BF, Johnson J, Gatenby RA, Gillies RJ (2013). Acidity generated by the tumor microenvironment drives local invasion. Cancer Res. 2013 Mar 1;73(5):1524-35. doi: 10.1158/0008-5472.CAN-12-2796. Epub 2013 Jan 3. PMID: 23288510; PMCID: PMC3594450. 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 Garrido F, Aptsiauri N, Doorduijn EM, Lora AMG, van Hall T (2016): The urgent need to recover MHC class I in cancers for effective immunotherapy. Curr Opin Immunol. 2016 Apr; 39: 44-51. doi: 10.1016/j.coi.2015.12.007 Huber V, Camisaschi C, Berzi A, Ferro S, Lugini L, Triulzi T, Tuccitto A, Tagliabue E, Castelli C, Rivoltini L (2017): Cancer acidity: An ultimate frontier of tumor immune escape and a novel target of immunomodulation. Seminars in Cancer Biology, Volume 43, April 2017, Pages 74-

89. https://doi.org/10.1016/j.semcancer.2017.03.001. Kirsch C, Takamiya-Wik M, Reinold S, Hahlbrock K, Somssich IE (1997). Rapid, transient, and highly localized induction of plastidial omega-3 fatty acid desaturase mRNA at fungal infection sites in Petroselinum crispum. Proc Natl Acad Sci U S A.; 94(5):2079-2084. doi:10.1073/pnas.94.5.2079 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. Meyer M, Nielsen H, Oliéric V, et al. (2014). Speciation of a group I intron into a lariat capping ribozyme. Proc Natl Acad Sci US A.;111(21):7659-7664. doi:10.1073/pnas.1322248111

| PAT 1793 LU — 40 — LU102162 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 Otop1 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, e16047; doi:10.1038/sigtrans.2016.47; published online 10 March 2017.

| PAT 1793 LU —41- LU102162

SEQUENCE LISTING <110> Universitaet Hamburg <120> Transcriptional synchronization of two or more functional transcription products <130> PAT 1793 LU <160> 17 <170> BiSSAP 1.3.6 <210> 1 <211> 188 <212> RNA <213> Artificial Sequence <220> <223> Lariat capping ribozyme (LCrz) <400> 1 gguuggguug ggaaguauca uggcuaauca ccaugaugca aucggguuga acacuuaauu 60 ggguuaaaac ggugggggac gaucccguaa cauccguccu aacggcgaca gacugcacgg 120 CCCUGCCUCU uagguguguu caaugaacag ucguuccgaa aggaagcauc cgguauccca 180 agacaauc 188 <210> 2 <211> 68 <212> RNA <213> Artificial Sequence <220>

| PAT 1793 LU —42- LU102162 <223> Hepatitis delta virus ribozyme (HDVRz) varl <400> 2 ccggccguac cagggucgga ggagcgaccg cggccgacce guuguacgaa gecguaccgc 60 uuacccug 68 <210> 3 <211> 88 <212> RNA <213> Artificial Sequence <220> <223> Hepatitis delta virus ribozyme (HDVRz) var2 <400> 3 agggucggca uggcaucuce accuccucgc gguccgaccu gggcuacuuc gguaggcuaa 60 gggagaagcu uggcacuggc cgucguuu 88 <210> 4 <211> 67 <212> RNA <213> Artificial Sequence <220> <223> CPEB3 ribozyme <400> 4 gggggccaca gcagaagcgu ucacgucgca gccccuguca gauucuggug aaucugcgaa 60 uucugcu 67 <210> 5

PAT 1793 LU —43- LU102162 <211> 78 <212> RNA <213> Artificial Sequence <220> <223> CPEB3 ribozyme (RF00622) <400> 5 aggggauaac agggggccac agcagaageg uucacgucgc agceccugue agauucuggu 60 gaaucugcga auucugcu 78 <210> 6 <211> 43 <212> RNA <213> Artificial Sequence <220> <223> Hammerhead ribozyme (HHRZ) <220> <221> misc feature <222> 1..6 <223> /note="n = a, g, c, or u" <400> 6 nnnnnngacu acucaggcac uccugcuuug cucauucgag cag 43 <210> 7 <211> 53 <212> RNA <213> Artificial Sequence <220>

PAT 1793 LU —44- LU102162 <223> Hammerhead ribozyme (HHRz) <220> <221> misc_feature <222> 6..13 <223> /note="n = a, g, c, or u" <400> 7 gggagnnnnn nnncugauga guccgugagg acgaaacggu acccgguacc guc 53 <210> 8 <211> 64 <212> RNA <213> Artificial Sequence <220> <223> Type-P5 twister ribozyme <400> 8 cuuguaaugc ggccguguaa auauuuacac gucggucuca agcccgauaa acgcagagag 60 caag 64 <210> 9 <211> 76 <212> RNA <213> Artificial Sequence <220> <223> Twister sister ribozyme <400> 9 ggacccgcaa ggccgacgge auccgccgee gecuggugcaa guccagecgc cccggggegg 60 gcgcucaugg guaaac 76

PAT 1793 LU —45- LU102162 <210> 10 <211> 81 <212> RNA <213> Artificial Sequence <220> <223> Twister sister ribozyme <400> 10 gcaacccgca aggccgacgce acaacgegec gccggugcaa gcccggccac ccugcaaggg 60 gugggcgcuc auggguacac a 81 <210> 11 <211> 85 <212> RNA <213> Artificial Sequence <220> <223> Twister sister ribozyme <400> 11 gaaacccgcu aggccgacag ccucaccgcu gccgcuggug caageccage cgccccagac 60 cggggcgggc gcucaugggu aacag 85 <210> 12 <211> 81 <212> RNA <213> Artificial Sequence <220>

PAT 1793 LU —46 — LU102162 <223> Twister sister ribozyme <400> 12 acgaccegca aggocgacge auaacgcgcc gccggugcaa goccggecac cccacauggg 60 gcgggcgcuc auggguacac a 81 <210> 13 <211> 82 <212> RNA <213> Artificial Sequence <220> <223> Twister sister ribozyme <400> 13 augacccgca aggccgacgg caucccgecg ccgcuggugc aagcccagcc GECCCgECAG 60 ggcgggcgcu caugggucca ca 82 <210> 14 <211> 84 <212> RNA <213> Artificial Sequence <220> <223> Hatchet ribozyme <400> 14 aaucquucuu acugauauca gugacaaaca uguggggcuu aucuaaucuu cggauuagua 60 uuagugcaga cquuaaaacc augu 84 <210> 15

PAT 1793 LU —-47 - LU102162

<211> 72

<212> RNA

<213> Artificial Sequence

<220>

<223> Spacer sequence (long)

<400> 15 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 60 aaaaaaaaaa aa 72 <210> 16

<211> 48

<212> RNA

<213> Artificial Sequence

<220>

<223> Synthetic Poly-A tail (short)

<400> 16 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaa 48 <210> 17

<211> 300 <212> RNA <213> Artificial Sequence <220> <223> Synthetic Poly-A tail (long) <400> 17 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 60

PAT 1793 LU —48 — LU102162 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 120 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 180 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 240 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 300 +

Claims

essen PAT 1793 LU _ 49 — LU102162 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 |, 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.

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 8 ——-————{{

aaa PAT 1793 LU — 50 — LU102162 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:

W ee ——_——

eee PAT 1793 LU _51— LU102162 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' self- cleaving ribozyme, or b) 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 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, y

EE ee PAT 1793 LU —_52— LU102162 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 self- cleaving 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' self- cleaving 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

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PAT 1793 LU _ 53 — LU102162 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

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EEE EEE PAT 1793 LU _ 54 — LU102162 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' self- cleaving ribozyme, or b) 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 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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