Invented by Raymond Kaempfer, Farhat Osman, Nayef Jarrous, Yitzhak Ben-Asouli, Yissum Research Development Co of Hebrew University of Jerusalem

The field of gene regulation has seen significant advancements in recent years, with the emergence of mRNA-splicing as a promising tool for controlling gene expression. mRNA-splicing refers to the process of removing certain sections of the mRNA molecule before it is translated into a protein. This process can alter the final protein product, allowing for precise control over gene expression. As a result, the market for regulation and use of mRNA-splicing in gene regulation is rapidly expanding. One of the key drivers of this market is the growing demand for targeted gene therapies. Traditional gene therapies involve introducing a functional copy of a gene into a patient’s cells to replace a defective or missing gene. However, this approach is limited by the size and complexity of the gene, as well as the difficulty of delivering the gene to the correct cells. mRNA-splicing offers a more precise approach, allowing for the selective targeting of specific genes and the modification of their expression. Another factor driving the market for mRNA-splicing is the increasing understanding of the role of alternative splicing in disease. Alternative splicing refers to the process of generating different mRNA isoforms from a single gene, which can result in different protein products. Aberrant splicing has been linked to a range of diseases, including cancer, neurological disorders, and genetic disorders. By targeting specific splicing events, mRNA-splicing therapies have the potential to treat these diseases at the genetic level. The market for mRNA-splicing is also benefiting from advances in technology. The development of CRISPR-Cas9 gene editing has enabled precise manipulation of the genome, including the ability to target specific splicing events. Additionally, advances in RNA sequencing and bioinformatics have allowed for the identification of splicing events that are specific to certain diseases, providing new targets for therapy development. Despite these promising developments, there are still challenges to be overcome in the regulation and use of mRNA-splicing. One of the main challenges is the delivery of the therapy to the correct cells. mRNA is a fragile molecule that is easily degraded, and delivering it to the correct cells can be difficult. Additionally, there are concerns about the safety of mRNA-splicing therapies, as altering gene expression could have unintended consequences. In conclusion, the market for regulation and use of mRNA-splicing in gene regulation is rapidly expanding, driven by the growing demand for targeted gene therapies, the increasing understanding of the role of alternative splicing in disease, and advances in technology. While there are still challenges to be overcome, the potential benefits of mRNA-splicing therapies make this an exciting area of research and development.

The Yissum Research Development Co of Hebrew University of Jerusalem invention works as follows

The trans-acting gene is a RNA-activated protein kinase that can phosphorylate the?-subunit of the eukaryotic initiation factor 2. The trans-acting gene is an RNA activated protein kinase capable of phosphorylating eukaryotic initiator factor 2’s?-subunit. Preferably, the trans-acting protein kinase is used. The 3? nucleotide can be used to derive the cis-acting sequence. The cis-acting nucleotide sequence can be derived from the 3? The untranslated region of the human tumor necrosis factor?

Background for Regulation and use of mRNA-splicing in gene regulation

In this application, references to various publications will be made by Arab numbers within square brackets. The full citations of these references can be found immediately before the claims at the end the specification. These publications are hereby fully disclosed in this application to describe the current state of the art, as it was known by those skilled in the field as of the date the invention claimed and described herein.

When producing recombinant protein in eukaryotic cell for biopharmaceutical or biotechnological uses, the level expression is an important parameter that controls the economics of the project. If the protein of interest is found in low concentrations in the culture medium, or the cellular extract, recovery will require more purification. This can lead to a lower yield. High constitutive expression may in some cases lead to the counterselection during cell proliferation of expressing cells, which can prevent high-level secreted proteins from being expressed in cell culture. There is therefore a need for efficient, regulated expression vectors.

The often suboptimal expression levels observed in clinical trials demonstrate the importance of improving the design of expression vectors. Transgenic animals are an ideal model to test gene therapy constructs in this context.

Transgenic animals and plants may provide an alternative for large-scale cell culture in the future to produce massive quantities of proteins, or other gene products like RNA. This production will require careful control of gene expression. The development of gene transfer technologies that enable the generation of transgenic plants or animals has led to the production of transgenic livestock as a strategy for alternative protein production systems. The secretion of protein in the milk from large mammals, for example, could be a cost-effective way to produce large quantities of valuable proteins. This technology is in its early stages of development, needs to be optimized and there are general requirements for ways to increase productivity.

Regulated Expression could be achieved in several ways. In order to design expression vectors that are suitable for biotechnological applications, efforts have been focused on transcriptional controls [1- 4] to control both transcription levels and externally regulated transcription. Translation has also been controlled to a certain extent. The use of constitutive expression vectors at high levels is generally used to produce biopharmaceuticals on a large scale [5, 6]. A regulated expression system can be used to express heterologous proteins which are toxic or have a negative impact on cell growth. It will be possible to increase the density of production cells before allowing the expression of desired proteins. This was achieved previously using vectors that induced transcription [7- 11]. The range of expression control is limited in most systems.

This invention focuses on another level of gene control that has been overlooked up until now. mRNA Splicing is the process of converting precursor transcripts to mature mRNA, containing only exons, through the excision of introns in the nucleus of the cell. This mRNA splicing process is an excellent candidate for controlling gene expression, as there are many evidences that it works in vivo. Introns are commonly included in expression vectors used in pharmaceuticals [5,6]. In the case of complementary DNA (cDNA), the intron’s contribution to the final product seems to be cDNA specific, but the mechanism by which the intron acts is still largely unknown. To date, there has been little effort directed towards regulating gene expression for biotechnological uses or gene therapy. “Regulation of mRNAsplicing could be used to regulate expression of genes transferred into cell lines, germlines or somatic tissue.

Expression is highly regulated by splicing precursor transcripts of several cytokine gene [12, 13?29]. So, soon after the onset induction of interleukin-2 and interleukin-1 (human)? (IL-1?) The flow of nuclear precursor transcriptions into mature mRNA is blocked by genes. This occurs despite the fact the transcription continues unabated after an inducer. Expression of IL-2? In the presence of translation inhibitors, mRNA superinduction is two orders of magnitude higher without any significant increase in transcription or mRNA stabilty. The splicing process of precursor transcripts, however, is greatly enhanced [13,27].

(TNF-?) (TNF-?) The gene (TNF-?) is also controlled at the splicing level [13]. 2-Aminopurine (22-AP) inhibits expression of TNF? 2-Aminopurine (2-AP) inhibits expression of TNF-? Adenine isomer 2-AP inhibits specific Kinases that phosphorylate eukaryotic Translation initiation Factor 2 (eIF2?) [17], including RNA-activated proteins kinase PKR [30]. 2-AP does NOT inhibit the transcription of human TNF? gene expression at transcription, nor does it affect mRNA stability. Splicing of TNF-? When 2-AP is present, the splicing of short-lived TNF-? The stability of TNF-? precursor transcripts remains unaffected. 2-AP prevents splicing TNF-? The splicing of TNF-? Neither human IL-1 or TNF-? Neither the human IL-1? This regulation is not found in TNF-? “A 2-AP-sensitive component expressed in functional form prior to induction regulates the splicing TNF-? “A 2-AP-sensitive component, expressed in functional form before induction, regulates splicing of TNF-?

PKR is an RNA activated Ser/Thr Protein Kinase. It is a major negative regulatory of translation [14-15]. PKR expression is constitutively expressed in many cells, but it is induced by viruses (dsRNA), interferons and double-stranded RNA. Activation of PKR requires its trans-autophosphorylation which is facilitated by RNA, especially by dsRNA [15]. PKR phosphorylates eIF2?, blocking the GDP/GTP transfer [17] and preventing eIF2 recycling between rounds of initiation translation [18]. Activation of PKR inhibits protein synthesis. Dominant-negative mutants of PKR have been described that inhibit trans-autophosphorylation of the wild type enzyme, obligatory for its activation [19?21].

Activation requires dimerization of PKR on RNA[22, 23]” and thus depends critically on the binding to RNA[23]. PKR has two tandem double-stranded RNA-binding motifs that are found in proteins like Drosophila Staufen, ribosomal S5 and E. coliRNase III [26]. In vitro, dsRNAs with the perfect A conformation and certain other RNAs such as hepatitis Delta Agent RNA, reovirus 3?UTR, and human? -tropomyosin?-UTR, activate PKR. However adenovirus RNA containing the VA conformation and Alu RNA bind PKR to inhibit its activation. Both activation and inhibition of PKR require highly ordered RNA structure, not a specific sequence. Some highly structured RNAs are activators or inhibitors of PKR, even if they have base-paired domains that are not perfectly matched. Examples include human delta hepatitis RNA [26] and VA RNA. The RNA-binding site in PKR requires 11-13 bp of dsRNA to bind [22,37,38] and can tolerate non Watson-Crick structures. Moreover, short helices that are not contiguous can work together to activate PKR by binding it.

PKR in the cell nucleoplasm was found in an underphosphorylated form [40]. Upon interferon induction, aggregates of PKR colocalize with interchromatin granules [41] which contain significant amounts spliceosomal component and are involved in spliceosome sorting, recycling and assembly [42]. These clusters recruit modified splicing factor into the perichromatin fibers where gene transcription takes place, facilitating cotransciptional processing of RNA [42]. However, there was no functional link between PKRs and splicing reported before this invention.

The TNF-?3-untranslated regions (3?-UTRs) have multiple roles in the regulation of expression of TNF? mRNA. It inhibits the murine TNF? It downregulates the murine TNF-? This AU motif is involved in translational inhibition and translation activation by IL4 [45] as well as lipopolysaccharide (14), which leads to the formation of protein complexes bind specifically to UUAUUUAUU. Prior to this invention, however, there was no report of the TNF-3?UTR or a 3?UTR in general playing a role in the regulation mRNA splicing.

This invention relates to the introduction of a novel cis element, preferably in an expression construct, within a gene of concern, so as to make splicing mRNA produced by this gene dependent upon the activation an RNA activated eIF2? The novel cis acting sequence element is used to regulate the mRNA splicing of this gene by RNA activated eIF2 kinase. This can be achieved through the manipulation of an expression vector and the application of known methods in the art for modulating the expression or activity of RNA activated eIF2 kinase. Kinase, in the recipient cell or organism. The invention offers a novel way to achieve a regulated production system of the proteins of interest, and optimize expression and yield.

The present invention is a cis acting nucleic acids sequences that make the removal of an intron/s during the production of the mRNA encoded by the gene containing at least one of these cis nucleic acids sequences, dependent on activation of a RNA activated protein kinase which is capable of phosphorylating eIF2 ?).

In specific embodiments, the RNA activated protein kinase can be referred to as the RNA activated protein kinase.

In a preferred implementation, the cis acting nucleic acids sequence of the invention are derived from 3? “In a preferred embodiment, the cis-acting nucleic acid sequence of the invention is derived from 3? gene (TNF-?3?-UTR).

In particularly preferred embodiments, the cis acting nucleic acid of the invention comprises a nucleotide series substantially denoted by the SEQ ID No:1 sequence. The invention relates also to biologically-functional fragments (derivatives), mutants, and homologues of the sequence, and to nucleotide segments which can hybridize to complementary nucleotide sytles of SEQ NO:1 as well as these biologically-functional fragments, mutations, and homologues.

In a preferred embodiment, the cis acting nucleic acids sequence of the invention includes SEQ ID No:2 as well as biologically functional derivatives, mutants, and homologues.

In Table 1, you will find the SEQ ID No:1 and SEQ NO:2 listed.

The invention states that “The cis acting nucleic acids according to the invention can render the removal of intron/s during the production mRNA from a gene harboring at least one of these cis acting nucleic acids, dependent on activation of a RNA-activated kinase which is capable of phosphorylating eukaryotic initiator factor 2’s?-subunit (eIF2?)”. The gene could be any gene, such as a gene with therapeutic, industrial or agricultural value, or a gene that encodes eIF2? or other proteins of commercial or therapeutic value.

The invention also relates to DNA constructs that include a gene with at least one intron, and a cis acting nucleotide which can render the removal of the intron/s during the production of the mRNA encoded by the gene. This gene contains at least a single cis nucleotide, and this occurs when the gene produces mRNA.

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