STABLE mRNA COMPOSITION AND A METHOD FOR PREPARATION THEREOF

Abstract

The present invention relates to stabilization of mRNA by substituting 5' cap with novel genetic elements. Especially, the present invention provides a method for increasing the stability of mRNA by introducing poly A at 5' end of mRNA. The disclosed capless mRNA of the present invention offers a faster, more cost-effective and scalable solution, addressing the key challenges faced by existing mRNA manufacturing technologies.

Description

[0001] Stable mRNA composition and a method for preparation thereof

[0002] FIELD OF THE INVENTION

[0003] The present invention generally relates to the field of nucleic acid. In particular, the present invention relates to stabilization of mRNA by substituting 5’ cap with novel genetic elements. Furthermore, the present invention provides a method for increasing the stability and also enhancement of expression.

[0004] BACKGROUND OF THE INVENTION

[0005] Therapeutic ribonucleic acid (RNA) molecules represent a promising class of drugs. RNA-based therapeutics include mRNA molecules encoding antigens for use as vaccines (Fotin-Mleczek et al. (2012) J. Gene Med. 14(6): 428-439). Especially, mRNA has proved to be a great platform for vaccine development. In addition, it is envisioned to use RNA molecules for replacement therapies, e.g. providing missing proteins such as growth factors or enzymes to patients (Kariko et al. (2012) Mol. Ther. 20(5):948-953; Kormann et al. (2012) Nat. Biotechnol. 29(2): 154-157).

[0006] However, there exist two major challenges for mRNA as therapeutic agent first is low levels of translation efficiency and second is immunogenicity of the administered mRNA molecules.

[0007] Several efforts have been made to address these challenges to produce a stable mRNA molecule / s. One of the major advancements has been the chemical modification of mRNA to reduce its immunogenic properties. Specifically, researchers found that incorporating modifications like pseudouridine and 5- methylcytosine into mRNA molecules decreased their immuno stimulatory effects, allowing for a more tolerable immune response. These modifications also showed potential in enhancing translation efficiency and mRNA stability.

[0008] Further, researchers have made developments by modifying the 5' and 3' untranslated regions (UTRs) of mRNA molecules, as well as optimizing the capping process at the 5' end to obtain stable mRNA molecule. The 5' cap, a 7- methylguanosine structure, is critical for mRNA stability, nuclear export, and translation initiation. Enhancements to the 5' cap, including the introduction of 1, 2, and 3 modifications, have shown improvements in translation efficiency. However, despite these advancements, the process of 5' capping remains complex and often leads to heterogeneous products. This heterogeneity can reduce the overall efficiency of translation and negatively impact the stability of the mRNA. Additionally, the use of different types of 5' and 3' UTRs, while beneficial in some contexts, adds further complexity. 5' UTRs (untranslated regions) present several limitations in mRNA translation. Highly stable secondary structures like hairpins can hinder ribosome recruitment and scanning. Additionally, canonical and noncanonical start codons (AUG and CUG) in the 5' UTR negatively impact translation. The role of UTRs may also vary by cell type, sometimes leading to undesirable interactions, such as miRNA recognition, making their optimization challenging.

[0009] In vitro transcription (IVT) is a commonly used technique for synthesizing mRNA in the lab, which relies on bacteriophage DNA-dependent RNA polymerases, such as SP6, T3, and T7, to produce mRNA transcripts from DNA templates. However, IVT reactions are susceptible to several issues that include the generation of truncated or abortive transcripts, polyadenylation variants leading to 3' heterogeneity, and mutated transcripts which can compromise the quality and functionality of the resulting mRNA. In vitro 5’ capping is essential for IVT-RNA to be used as therapeutic as mammalian translations are cap dependent. And it is also necessary to keep the mRNA stable. Hence, it is essential to have correct capping at 5’end to produce a stable mRNA. Inefficient capping process leads to higher production costs as it would require recapping or reprocessing to produce a good quality mRNA molecule / s. Further, it would also require more quality control tests to identify heterogeneity and variability in mRNA that would require significant additional cost during manufacturing mRNA based therapeutic. Further, inefficient capping will have an impact on mRNA vaccine efficacy and development timeline and a significant challenge in obtaining regulatory approval.

[0010] Hence, there remains a critical unmet need for developing stable mRNA. In particular, there is a need for methods that can stabilize mRNA and enhance its translation efficiency without relying on the conventional 5' cap structure.

[0011] SUMMARY OF THE INVENTION

[0012] The present invention addresses the inherent limitations of traditional capping methods by introducing a novel approach to stabilize capless mRNA and improve its translation efficiency. The invention specifically employs addition of a poly A tail to the 5' end of capless mRNA, which serves as a stabilizing element by binding to poly A-binding proteins (PABPs). The poly A tail protects the mRNA from degradation by exonucleases, particularly those that degrade RNA. Poly A-binding proteins (PABPs) bind to the poly A tail, forming a protective complex that shields the mRNA from exonucleolytic attack and also facilitate translation by interacting with other proteins involved in the translation initiation process.

[0013] The interaction between the poly A tail and PABPs is essential for maintaining the integrity of mRNA within the cytoplasm. PABPs not only prevent degradation by exonucleases. In particular, PABPs play a role in forming a closed-loop structure in the mRNA, which enhances translation by promoting ribosome recycling.

[0014] The invention discloses a novel method for stabilizing capless mRNA by adding a poly A tail at the 5' end. This strategy mimics the protective function of the 3’ polyA by recruiting PABPs to the 3' end of the mRNA, forming a protective barrier against exonucleases that would otherwise degrade the mRNA from the 3' end. The addition of a 5' poly A tail effectively substitutes for the 5' cap by stabilizing the mRNA and allowing it to persist in the cytoplasm for extended periods. This stabilization is particularly important for therapeutic applications, where high levels of protein expression are required over long durations. The present invention also specially employs incorporation of poly A tails at both the 5' and 3' ends of the mRNA as protection form ribonucleases is required at both the ends of mRNA and 5’ and 3’ poly A will also facilitate the formation of a closed-loop structure. This loop is formed by interactions between PABPs bound to both ends of the mRNA. The closed-loop configuration enhances mRNA stability by preventing degradation and also improves translation efficiency by promoting ribosome recycling.

[0015] The capless mRNA of the current invention further comprises t-RNA which increases the stability of capless mRNA.

[0016] BRIEF DESCRIPTION OF DRAWINGS

[0017] Fig. 1: illustrates the DNA template (Fig. 1A) and IVT-mRNA (Fig. IB) utilized to investigate the effects of 5’ Poly N (15) sequences in capless RNA (clRNA) on the stability and expression of green fluorescent protein (GFP) in CHO cells.

[0018] Fig. 1A: illustrates the DNA template for the IVT reaction. Each DNA template contains a T7 RNA Polymerase promoter, a 5’ anchor sequence (3 bases), a 5’ Poly N (15 bases), internal ribosome entry site (IRES), GFP, and a 3’ Poly A (15 bases). The constructs differ in their 5’ Poly N sequences: 1A (I) has Poly A, 1A (II) has Poly T, and 1A (III) has Poly C.

[0019] Fig. IB: Shows the IVT-RNA. All IVT-RNA include a 5’ anchor sequence (3 bases), a 5’ Poly N (15 bases), IRES, GFP, and a 3’ Poly A (15 bases). Variations in the 5’ Poly N are as follows: IB (I) has Poly A, IB (II) has Poly T, and IB (III) has Poly C.

[0020] Fig. 2: Illustrates the percentage of GFP-expressing cells (Fig. 2A) and mean GFP fluorescence (Fig. 2B) for the analysis of different 5’ Poly N (15) sequences in capless RNA (clRNA) regarding stability and expression of GFP in CHO cells. The experiment was conducted according to Example 6, with data collected 18-24 hours post-transfection.

[0021] Fig. 2A: Depicts the impact of Poly N on the percentage of GFP-expressing CHO cells in capless RNA.

[0022] Fig. 2B: Displays the effect of Poly N on mean GFP fluorescence in capless RNA. Fig. 3: Shows the DNA template (Fig. 3A) and IVT-mRNA (Fig. 3B) employed to investigate the impact of the length of the 5’ anchor sequence in relation to the 5’ Poly A in capless RNA (clRNA) on the stability and expression of GFP in CHO cells.

[0023] Fig. 3A: Illustrates the DNA template for the IVT reaction. All DNA templates contain a T7 RNA Polymerase promoter, a 5’ anchor sequence, a 5’ Poly A (15 bases), IRES, GFP, and a 3’ Poly A (15 bases). The constructs differ in the length of the 5’ anchor, with variations as follows: 3A (I) has a 3 bp anchor and a 15 bp Poly A, 3 A (II) has a 3 bp anchor, 3 A (III) has an 8 bp anchor and a 15 bp Poly A, 3 A (IV) has an 8 bp anchor, 3 A (V) has a 16 bp anchor and a 15 bp Poly A, and 3 A (VI) has a 16 bp anchor.

[0024] Fig. 3B: Shows the IVT-RNA. All IVT -RNA constructs include a 5’ anchor sequence, a 5’ Poly A (15 bases), IRES, GFP, and a 3’ Poly A (15 bases). Variations are consistent with the DNA template: 3B (I) has a 3 bp anchor and a 15 bp Poly A, 3B (II) has a 3 bp anchor, 3B (III) has an 8 bp anchor and a 15 bp Poly A, 3B (IV) has an 8 bp anchor, 3B (V) has a 16 bp anchor and a 15 bp Poly A, and 3B (VI) has a 16 bp anchor.

[0025] Fig. 4: Illustrates the percentage of GFP-expressing cells (Fig. 4A) and mean GFP fluorescence (Fig. 4B) utilized to study the effect of the length of the 5’ anchor sequence in relation to the 5’ Poly A in capless RNA (clRNA) on the stability and expression of GFP in CHO cells. The experiment was conducted according to Example 7, with data collected 18-24 hours post-transfection.

[0026] Fig. 4A: Depicts the effect of the length of the 5’ anchor sequence relative to the 5’ Poly A on the percentage of GFP-expressing CHO cells in capless RNA.

[0027] Fig. 4B: Illustrates the impact of the length of the 5’ anchor sequence relative to the 5’ Poly A on mean GFP fluorescence in capless RNA.

[0028] Fig. 5: Shows the DNA template (Fig. 5A) and IVT-mRNA (Fig. 5B) used to study the effect of the length of the 5’ and 3’ Poly A in capless RNA (clRNA) on the stability and expression of GFP in CHO cells.

[0029] Fig. 5A: Illustrates the DNA template for the IVT reaction. All templates feature a T7 RNA Polymerase promoter, a 16 bp 5’ anchor sequence, a 5’ Poly A, IRES, GFP, and a 3’ Poly A. Variations include different lengths of 5’ and 3’ Poly A as follows: 5 A (I) has 20 bp (5’ Poly A) and 20 bp (3’ Poly A), 5 A (II) has 20 bp (5’ Poly A) and 40 bp (3’ Poly A), 5 A (III) has 20 bp (5’ Poly A) and 60 bp (3’ Poly A), 5 A (IV) has 40 bp (5’ Poly A) and 20 bp (3’ Poly A), 5 A (V) has 40 bp (5’ Poly A) and 40 bp (3’ Poly A), 5 A (VI) has 40 bp (5’ Poly A) and 60 bp (3’ Poly A), 5 A (VII) has 60 bp (5’ Poly A) and 20 bp (3’ Poly A), 5 A (VIII) has 60 bp (5’ Poly A) and 40 bp (3’ Poly A), and 5 A (IX) has 60 bp (5’ Poly A) and 60 bp (3’ Poly A).

[0030] Fig. 5B: Shows the IVT-RNA. All IVT-RNA constructs contain a 16 bp 5’ anchor sequence, a 5’ Poly A, IRES, GFP, and a 3’ Poly A. Variations in lengths of 5’ and 3’ Poly A are consistent with the DNA template: 5B (I) has 20 bp (5’ Poly A) and 20 bp (3’ Poly A), 5B (II) has 20 bp (5’ Poly A) and 40 bp (3’ Poly A), 5B (III) has 20 bp (5’ Poly A) and 60 bp (3’ Poly A), 5B (IV) has 40 bp (5’ Poly A) and 20 bp (3’ Poly A), 5B (V) has 40 bp (5’ Poly A) and 40 bp (3’ Poly A), 5B (VI) has 40 bp (5’ Poly A) and 60 bp (3’ Poly A), 5B (VII) has 60 bp (5’ Poly A) and 20 bp (3’ Poly A), 5B (VIII) has 60 bp (5’ Poly A) and 40 bp (3’ Poly A), and 5B (IX) has 60 bp (5’ Poly A) and 60 bp (3’ Poly A).

[0031] Fig. 6: Depicts the percentage of GFP-expressing cells (Fig. 6A) and mean GFP fluorescence (Fig. 6B) to study the effects of the lengths of 5’ and 3’ Poly A in capless RNA (clRNA) on the stability and expression of GFP in CHO cells. The experiment was conducted according to Example 8, with data collected 18-24 hours post-transfection.

[0032] Fig. 6A: Illustrates the impact of the lengths of 5’ and 3’ Poly A on the percentage of GFP-expressing cells in capless RNA.

[0033] Fig. 6B: Shows the effect of the lengths of 5’ and 3’ Poly A on mean GFP fluorescence in capless RNA.

[0034] Fig. 7: Illustrates the DNA template (Fig. 7A) and IVT-mRNA (Fig. 7B) used to investigate the expression of single and multiple proteins via capless RNA.

[0035] Fig. 7A: Depicts the DNA template for the IVT reaction. Monocistronic DNA templates, used for the expression of a single protein, contain the T7 RNA Polymerase Promoter, a 16 bp 5’ Anchor Sequence, a 40 bp 5’ Poly A tail, IRES, a Reporter Protein, and a 40 bp 3’ Poly A tail. In contrast, the bicistronic DNA template, utilized for the expression of two proteins, contains the T7 RNA Polymerase Promoter, a 16 bp Anchor Sequence, a 40 bp 5’ Poly A tail, IRES, a Reporter Protein, another IRES, a second Reporter Protein, and a 40 bp 3’ Poly A tail. Specifically, 7 A (I) represents a monocistronic DNA template featuring an Attenuated IRES and GFP, 7A (II) contains a monocistronic DNA template with a Wildtype IRES and GFP, 7A (III) contains a monocistronic DNA template with a Wildtype IRES and mCherry, and 7A (IV) depicts a multicistronic DNA template with Wildtype IRES-GFP-Wildtype IRES-mCherry.

[0036] Fig. 7B: Illustrates the IVT-RNA. The monocistronic IVT-RNA, used for the expression of a single protein, includes a 16 bp 5’ Anchor Sequence, a 40 bp 5’ Poly A tail, IRES, a Reporter Protein, and a 40 bp 3’ Poly A tail. The multicistronic IVT-RNA, intended for the expression of two proteins, includes a 16 bp Anchor Sequence, a 40 bp 5’ Poly A tail, IRES, a Reporter Protein, another IRES, a second Reporter Protein, and a 40 bp 3’ Poly A tail. Specifically, 7B (I) corresponds to a monocistronic IVT-RNA with an Attenuated IRES and GFP, 7B

[0037] (II) corresponds to a monocistronic IVT-RNA with a Wildtype IRES and GFP, 7B

[0038] (III) corresponds to a monocistronic IVT-RNA with a Wildtype IRES and mCherry, and 7B (IV) corresponds to a multicistronic IVT-RNA with Wildtype IRES-GFP-Wildtype IRES-mCherry.

[0039] Fig. 7C: Illustrates the DNA template for the IVT synthesis of 5’ capped mRNA, used as a control for the capless RNA study. This DNA template includes the T7 RNA Polymerase Promoter, a 5’ UTR, GFP, a 3’ UTR, and Poly A.

[0040] Fig. 7D: Illustrates the IVT-RNA following 5’ capping. The IVT-RNA comprises a 5’ Cap, a 5’ UTR, GFP, a 3’ UTR, and Poly A.

[0041] Fig. 8: Illustrates the percentage of expressing cells (Fig. 8 A) and the mean fluorescence intensity (Fig. 8B) of monocistronic and multicistronic capless RNA (clRNA) in CHO cells. The experiment was conducted according to Example 9, with data collected 18-24 hours post-transfection.

[0042] Fig. 8A: Displays the percentage of GFP or mCherry expressing cells using monocistronic and multicistronic clRNA in CHO cells. Fig. 8B: Shows the mean GFP fluorescence of monocistronic and multicistronic clRNA in CHO cells.

[0043] Fig. 9: Illustrates the structure of tRNA and its application in capless RNA to enhance the stability and expression of GFP in CHO cells.

[0044] Fig. 9A: Depicts the secondary structures of tRNA and its post-transcriptional modifications.

[0045] Fig. 9B: Illustrates capless RNA (clRNA) without tRNA.

[0046] Fig. 9C: Illustrates clRNA containing tRNA.

[0047] Fig. 10: Depicts the DNA template (Fig. 10A) and IVT-mRNA (Fig. 10B) used to assess the effect of tRNA on the stability and expression of clRNA.

[0048] Fig. 10A: Illustrates the DNA template for the IVT reaction. In 10A (I), the template lacks tRNA sequences and includes the T7 RNA Polymerase Promoter, a 16 bp 5’ Anchor Sequence, a 40 bp 5’ Poly A tail, IRES, GFP, and a 40 bp 3’ Poly A tail. In contrast, 10A (II) incorporates two tRNA sequences and contains the T7 RNA Polymerase Promoter, a 16 bp 5’ Anchor Sequence, a 40 bp 5’ Poly A tail, tRNA, IRES, GFP, tRNA, and a 3’ Poly A tail.

[0049] Fig. 10B: Illustrates IVT-RNA. In 10B (I), the IVT-RNA lacks tRNA and includes a 16 bp 5’ Anchor Sequence, a 40 bp 5’ Poly A tail, IRES, GFP, and a 40 bp 3’ Poly A tail. In contrast, 10B (II) features IVT-RNA with two tRNA sequences, incorporating a 16 bp 5’ Anchor Sequence, a 40 bp 5’ Poly A tail, tRNA, IRES, GFP, tRNA, and a 3’ Poly A tail.

[0050] Fig. 10C: Illustrates the DNA template for the IVT synthesis of 5’ capped mRNA, serving as a control for the clRNA study. The template includes the T7 RNA Polymerase Promoter, a 5’ UTR, GFP, a 3’ UTR, and Poly A.

[0051] Fig. 10D: Illustrates the IVT-RNA following capping. The IVT-RNA comprises a 5’ Cap, a 5’ UTR, GFP, a 3’ UTR, and Poly A.

[0052] Fig. 11: Illustrates the percentage of expressing cells (Fig. 11 A) and the mean fluorescence intensity (Fig. 11B) of 5’ Cap mRNA, clRNA and clRNA with tRNA in CHO cells. The experiment was conducted according to Example 10, with data collected 18-24 hours post-transfection. Fig. 11A: Displays the percentage of GFP expressing cells using 5’ Cap mRNA, clRNA and clRNA with tRNA in CHO cells.

[0053] Fig. 11B: Shows the mean GFP fluorescence of 5’ Cap mRNA, clRNA and clRNA with tRNA in CHO cells.

[0054] Fig. 12: Comparison of stability between 5’ cap mRNA, clRNA without 5’ polyA, clRNA with 40bp 5’ polyA and 40bp 3’ polyA and clRNA with 40bp 5’ polyA, 40bp 3’ polyA and 2 tRNA genes: The figure illustrates percentage GFP expressing cells at different time points following transfection in to CHO cells. GFP expressing cells were captured under Evos M7000 fluorescence microscope (Thermofisher Scientifics) and percentage GFP expression population was determined using (Evos Software Analyser). This experiment was also conducted in accordance with Example 11, with data collected 2, 12, 36, 60, 84, 108, 132 hours post-transfection.

[0055] BRIEF DESCRIPTION OF THE INVENTION

[0056] The foregoing has outlined rather broadly the features and technical advantages of the present invention so that those skilled in the art may better understand the detailed description of the invention that follows. Additional features and advantages of the invention will be described hereinafter that form the subject of the claims of the invention. Those skilled in the art should appreciate that they may readily use the conception, and the specific embodiment disclosed as a basis for modifying or designing other structures for carrying out the same purposes of the present invention. Those skilled in the art should also realize that such equivalent constructions do not depart from the spirit and scope of the invention in its broadest form.

[0057] Definitions:

[0058] Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and "comprising", will be understood to imply the inclusion of a stated member, integer or step or group of members, integers or steps but not the exclusion of any other member, integer or step or group of members, integers or steps.

[0059] The terms "a" and "an" and "the" and similar reference used in the context of describing the invention (especially in the context of the claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context.

[0060] In the context of the present invention, the term "RNA" relates to a molecule which comprises at least one ribonucleotide residue.

[0061] The term "mRNA" means "messenger-RNA" and relates to a "transcript" which is generated by using a DNA template and encodes a peptide or protein. Typically, an mRNA comprises a 5'-UTR, a protein coding region and a 3'-UTR. mRNA only possesses limited half-life in cells and in vitro. In the context of the present invention, mRNA may be generated by in vitro transcription from a DNA template. The in vitro transcription methodology is known to the skilled person. For example, there is a variety of in vitro transcription kits commercially available. In. the context of the present invention, the mRNA, preferably the mRNA, lacks 5’ cap.

[0062] The term "5 '-cap" refers to a cap structure found on the 5 '-end of an mRNA molecule and generally consists of a guanosine nucleotide connected to the mRNA via an unusual 5' to 5' triphosphate linkage. The "conventional 5 '-cap" refers to a naturally occurring RNA 5 '-cap, preferably to the 7- methylguanosine cap (m7G).

[0063] The term, "poly A tail" used herein refers to a chain of adenine nucleotides. The term can refer to poly A tail that is to be added to an RNA transcript at 5’ end and 3’ end. The length of poly A tail is typically 5-300 nucleotides.

[0064] The term "in vitro" refers to events that occur in an artificial environment, e.g., in a test tube or reaction vessel, in cell culture, in a Petri dish, etc., rather than within an organism (e.g., animal, plant, or microbe).

[0065] The term “clRNA” used herein refers to a capless mRNA wherein both 5’ and 3’ end of mRNA comprises poly A tail. The term “anchor sequence” used herein refers to a nucleotide sequence added to the 5’ end of 5’ polyA in clRNA. The anchor sequence increases the efficiency of 5’ polyA in cap less RNA. Essentially, it helps in efficient binding of polyA binding protein to 5’ polyA tail.

[0066] The term, “Internal Ribosome Entry Sites (IRES)” used herein refers to a specific sequences found within mRNA that allow ribosomes to initiate translation without the usual need for a 5' cap structure. These sequences are particularly useful in certain conditions where cap-dependent translation is compromised, such as during stress or viral infection. IRES elements enable the ribosome to bind directly to the mRNA and start protein synthesis, ensuring that essential proteins are still produced. IRES elements are crucial for the design of multicistronic RNA constructs, such as those used in in-vitro transcription (IVT) systems. By incorporating IRES between different open reading frames (ORFs), multiple proteins can be expressed from a single RNA molecule. This is particularly useful for expressing complex protein assemblies or for co-expressing multiple therapeutic proteins, such as in gene therapy or vaccine development.

[0067] The term “coding sequence” is the portion of an mRNA molecule that encodes peptides or protein. It starts with a start codon (usually AUG) and ends with a stop codon (UAA, UAG, or UGA). This sequence determines the amino acid sequence of the protein being synthesized.

[0068] The term “monocistronic mRNA” used herein refers to mRNA molecules that code for a single protein. This is typical in eukaryotes, where each mRNA carries the genetic information for just one gene product. It allows for more precise regulation of gene expression, as each mRNA is translated into only one type of protein. This type of mRNA contains one open reading frames (ORFs), which can be translated into one type of protein with the use of IRES.

[0069] The term “Multicistronic mRNA” used herein refers to mRNA molecules that encode multiple proteins. This type of mRNA contains several open reading frames (ORFs), each of which can be translated into a different protein with the use of IRES. The term “tRNA” used in this invention refers to transfer RNA, which is a type of RNA molecule that plays a crucial role in protein synthesis. It acts as an adaptor, carrying specific amino acids to the ribosome, where proteins are assembled. . Transfer RNA (tRNA) is a small, stable RNA molecule with a halflife of more than 72hr, they plays a key role in translating mRNA sequences into amino acids during protein synthesis. tRNAs are more stable than other RNA types due to their unique secondary structure and extensive post-transcriptional modifications. tRNA folds into a cloverleaf-like structure with four distinct regions: the acceptor stem, D-loop, anticodon loop, and T\| / C loop. This structure is stabilized by extensive intra-molecular hydrogen bonding, which forms hairpin loops. The folded tRNA further adopts an L-shaped 3D conformation, making it more resistant to degradation. tRNAs undergo extensive chemical modifications at specific bases after transcription. These include methylation, pseudouridylation, and thiolation, which enhance tRNA stability by preventing misfolding, increasing resistance to RNases, and reducing susceptibility to chemical damage. These features, along with the compact and highly conserved structure, make tRNA one of the most stable RNA molecules in the cell.

[0070] In this invention, tRNA serves a unique and critical function by enhancing the stability of RNA molecules apart from it’s conventional role in facilitating translation. Due to its inherent secondary structures and post-transcriptional modifications, tRNA provides increased resilience to degradation, thereby supporting the stability and longevity of the RNA within the system. This characteristic makes tRNA an essential component in the novel application of this invention, where RNA stability is enhanced for improved performance in target applications.

[0071] Before undertaking the detailed description of the invention below it may be advantageous to set forth definitions of certain words and phrases used throughout this patent document: the terms “include” and “comprise ” as well as derivatives thereof mean inclusion without limitation; the term “or ” is inclusive meaning and / or; the phrases “associated with” and “associated therewith ” as well as derivatives thereof may mean to include be included within interconnect with contain be contained within connect to or with couple to or with be communicable with cooperate with interleave juxtapose be proximate to be bound to or with have a property of or the like; and the term .

[0072] In one embodiment, the present invention provides a stable capless mRNA structure wherein the mRNA comprises a poly A tail at 5’ end.

[0073] In the above mentioned embodiment, the poly A tail at 5 ’end in the mRNA binds with poly A-binding proteins (PABPs), which stabilize the mRNA and protect it from degradation by exonucleases.

[0074] In a further embodiment, the 5' poly A tail effectively substitutes for the 5' cap by stabilizing the mRNA and allowing it to persist in the cytoplasm for extended periods.

[0075] In another embodiment, the invention discloses a stable capless mRNA comprises poly A tails at both the 5' and 3' ends of the mRNA. This is further facilitates the formation of a closed-loop structure.

[0076] In a further embodiment, the stable mRNA further comprises anchor sequence, wherein the length of the sequence is from 3 base pair (bp) to 1000 base pair (bp).

[0077] In yet another embodiment, the capless mRNA further comprises IRES.

[0078] In another embodiment, the capless mRNA comprises a coding sequence.

[0079] In any of the above mentioned embodiments, the length of poly A tail at 5’ end and 3’ end is between 1-1000 bp.

[0080] In an embodiment the invention discloses a capless mRNA comprising; a) 5’ anchor sequence, b) 5’ Poly A, c) one or more Internal Ribosome Entry Sites (IRES) d) one or more coding sequence, and e) 3 ’ Poly A sequence. In the above mentioned embodiment, the 5’ anchor sequence length is 3-1000 base pairs.

[0081] In the above mentioned embodiment of the aforesaid embodiment, the length of poly A tail at 5’ end and 3’ end is between 1-1000 bp.

[0082] In another embodiment, the invention discloses a stable capless mRNA comprising; a. 5’ anchor sequence varying in length from 3-1000 bp b. 5 ’ poly A seq with varying length form 1 - 1000 bp c. one or more IRES d. one or more coding sequence e. 3’ poly A sequence with varying length form 1-1000 bp.

[0083] In any of the above mentioned embodiments, the stable mRNA is a mono- cistronic or multi cistronic.

[0084] In any of the above mentioned embodiments, the capless mRNA further comprises an immunomodulatory protein along with antigen of interest.

[0085] In any of the above mentioned embodiments, the capless mRNA further comprises a protein based adjuvant along with antigen of interest.

[0086] In another embodiment, the invention discloses a capless mRNA comprising; a. 5’ anchor sequence varying in length from 3-1000 bp b. 5’ poly A seq with varying length form 1-1000 bp c. one or more t-RNA d. one or more IRES e. one or more coding sequence f. 3’ poly A sequence with varying length form 1-1000 bp.

[0087] In another embodiment, t-RNA present in the capless RNA increases the stability and expression of the protein

[0088] In another embodiment, the invention discloses a capless mRNA comprising; a. 5’ anchor sequence varying in length from 3-1000 bp b. 5 ’ poly A seq with varying length form 1 - 1000 bp c. one or more t-RNA d. one or more IRES e. one or more coding sequence f. one or more t-RNA g. 3’ poly A sequence with varying length form 1-1000 bp.

[0089] In any of the above mentioned embodiments, 5’ poly A tail length is same or higher than the poly A tail present at 3 ’ end.

[0090] In any of the above mentioned embodiments of the invention, the anchor sequence length is 3 bp to 1000 bp, more specifically 8 bp to 1000 bp; preferably 16bp to 1000 bp.

[0091] In any of the above mentioned embodiments, the length of poly A tail present at both the ends 5’ and 3’ end of capless mRNA is at least 20 bp.

[0092] In another embodiment, the invention discloses a process of obtaining a stable capless mRNA wherein the method comprises steps of: a) designing a DNA template b) in-vitro transcription of the template c) incorporation of poly-A tail d) purification of capless mRNA.

[0093] In the above mentioned embodiment, the DNA template is a plasmid or polymerase chain reaction (PCR) product.

[0094] In the above mentioned embodiment of the invention, the DNA plasmid template comprises Promoter, 5’ anchor sequence, 5’ polyA sequence, IRES-gene of interest (GOI), polyA sequence, and unique type Ils restriction site.

[0095] In any of the above mentioned embodiments of the invention, the DNA plasmid template comprising Promoter, 5’ anchor, 5’ polyA, tRNA, IRES-Coding Sequence, tRNA, polyA, and unique type Ils Restriction site.

[0096] In of the above mentioned embodiments of the invention, the poly-A tail is either part of DNA template or added enzymatically to IVT-RNA at the 5’ and 3’ end of clRNA. In any of the above mentioned embodiments of the invention, IRES is a wild type or mutant IRES. Use of the IRES with varying strengths to regulate the dosage of protein produced. clRNA with varying genetic elements (poly A, tRNA or any such elements) which can result in enhanced expression and increase stability of clRNA.

[0097] In any of the above mentioned embodiments of the invention, the use of t- RNA increases the stability of capless mRNA and the number of tRNAs used for one clRNA transcript can vary from 1 - 20. The tRNA can be placed at the end of in between the sequences without affecting protein translation.

[0098] In any of the above mentioned embodiments, t-RNA can be replaced with any genetic elements which are know to have high secondary structures and post transcriptional modification. -The genetic elements used herein in this invention to increase stability of clRNA are equally applicable in linear 5’ cap mRNA, linear 5’ cap self amplifiable mRNA and circular RNA.

[0099] In any of the above mentioned embodiments, 5’ poly A sequence can be replaced with any genetic element which is bound by host cell protein and prevents degradation by ribonuclease should also be covered.

[0100] In any of the above mentioned embodiments of the invention, the disclosed stable capless mRNA is used as therapeutic agents such as vaccines, antibody production, CAR-T cell therapy, CRISPR-cas9 based genome editing. Further, the mRNA can be used in kits for research and development use.

[0101] In any of the above mentioned embodiments of the invention, the disclosed clRNA can be used to express proteins in any eukaryotic cells or organism (eg. yeast, mammalian, insect, plant or animal).

[0102] The invention discloses in the present invention has following advantages. First advantage is it is a simple design. Elimination of 5’ cap there by reducing the number of steps, cutting down the production time and simplifying the IVT process. Second, the capless mRNA obtained by addition of poly-A tail at 5’ end is more stable and further protects from degradation from exonucleases. Third, the addition of tRNA to capless mRNA increased the stability of capless mRNA. Fourth, the capless mRNA expresses multiple proteins from a single transcript, providing a versatile tool for multiple protein expression. Incorporation of modified nucleotides (eg. Pseudouridine) into the capless mRNA structure further improves translational efficiency and stability, even without the 5' cap.

[0103] The method disclosed herein to synthesize a stable capless mRNA offers a faster, more cost-effective, and scalable solution, addressing the key challenges faced by existing mRNA manufacturing technologies. The potential applications are vast, particularly in therapeutic protein production, gene therapy, and other biotechnology fields.

[0104] Certain specific aspects and embodiments of the invention are more fully described by reference to the following examples. However, these examples should not be construed as limiting the scope of the invention in any manner. Examples

[0105] Example 1: Construction of Plasmids for clRNA Study

[0106] The plasmids pJET1.2-Wildtype (W)-IRES-GFP, pJET 1.2- Attenuated (A)-IRES-GFP, pJET1.2-W-IRES-mCherry, pJET1.2-W-IRES-GFP-W-IRES- mCherry, pJET1.2-tRNA-W-IRES-GFP-tRNA, and pJET1.2-5’UTR-GFP- 3’UTR-PolyA were constructed through polymerase chain reaction (PCR) amplification of respective gene sequences. The amplified fragments, W-IRES- GFP, A-IRES-GFP, W-IRES-mCherry, W-IRES-GFP-W-IRES-mCherry, tRNA- W-IRES-GFP-tRNA, and 5’UTR-GFP-3’UTR-PolyA, were subsequently cloned into the pJET1.2 blunt-end cloning plasmid.

[0107] Example 2: Preparation of Templates for In Vitro Transcription (IVT)

[0108] Templates for the IVT reactions were generated using the Forward (F) and Reverse (R) primers specified in Table 1. PCR amplification was performed with 5 pmol of each primer and 30 ng of plasmid template in a total reaction volume of 100 pF, utilizing Pfu DNA polymerase at an annealing temperature of 55°C. Following amplification, the PCR products were purified with a PCR cleanup kit (MN) to eliminate residual primers and contaminants. The purified products were quantified for use in subsequent IVT reactions. Table 1: List of Oligos for preparation Of DNA template for IVT reaction

[0109] Example 3: In Vitro Transcription (IVT) Reaction

[0110] IVT reactions were conducted in a 10 pL volume using the Takara IVTpro T7 mRNA Synthesis Kit. The reactions were incubated at 37°C for 2 hours to facilitate complete transcription. After IVT, DNA templates were eliminated by treatment with DNase. The resultant RNA was purified using lithium chloride precipitation. RNA purity and concentration were assessed using a Nanodrop spectrophotometer, and RNA integrity was evaluated through analysis on a reducing agarose gel.

[0111] Example 4: Capping of IVT mRNA

[0112] In this example, in vitro transcribed (IVT) mRNA was capped using the Vaccinia Virus Capping Enzyme to enhance its stability and translation efficiency. Briefly, the IVT RNA, purified as described in Example 3, was mixed with the capping enzyme, guanosine 5 '-triphosphate (GTP), and buffer. The reaction mixture was incubated at 37°C for 30 minutes to facilitate the transfer of the guanylyl group from GTP to the 5' end of the RNA, resulting in a 7- methylguanylate cap structure.

[0113] Following the incubation, the capping reaction was terminated, and the capped RNA was purified using phenol-chloroform extraction. RNA purity and concentration were assessed using a Nanodrop spectrophotometer, and RNA integrity was evaluated through analysis on a reducing agarose gel. Example 5: Transient Transfection of mRNA Using Lipofectamine MessengerMAX

[0114] All the transfections were carried out in a 24 well flat-bottomed plate. Briefly, CHO cells were seeded at 1.2xl05cells / well with 400ul of DMEM / 10% FBS one day before transfection to attain 70% confluence on the day of transfection. Just before transfection cells were washed with IxPBS and 400ul of optiMEM was added. mRNA: Lipofectamine MessengerMAX complexes were prepared as per the manufacturer’s instructions using 500 ng of mRNA and added to cells at 70% confluence. Eighteen to twenty-four hours post transfection cells were analysed using FACS analyzer (Gallios).

[0115] Example 6: Impact of Poly (N) on Stability and Expression of GFP in Capless RNA (clRNA)

[0116] In this experiment, clRNA was evaluated for stability in the presence of Poly A, Poly T, and Poly C by transfecting IVT-RNA into CHO cells. Posttransfection, cells were analyzed for the percentage of GFP-expressing cells and mean GFP fluorescence using flow cytometry, 18-24 hours after transfection. The DNA templates (Fig. 1A) utilized were amplified as outlined in Example 2, and the IVT-RNA (Fig. IB) was synthesized according to Example 3. A total of 500 nanograms of IVT-RNA was transfected into CHO cells at 70% confluence using Lipofectamine MessengerMAX (Thermo Fisher Scientific). The results indicated that clRNA containing 5’ Poly A exhibited a significantly higher percentage of GFP-expressing cells (Fig. 2 A) and mean GFP fluorescence (Fig. 2B) compared to those with Poly T and Poly C. This suggests that Poly A may serve as an effective substitute for a 5’ cap, potentially due to its protective role against degradation by ribonucleases.

[0117] Example 7: Influence of 5' Anchor Length on Stability and Expression of GFP in clRNA

[0118] In Example 5, a 3 bp 5’ anchor sequence was utilized for the Poly N study. To further investigate the role of the anchor length in binding affinity for Poly A Binding Protein, clRNA constructs were synthesized with and without Poly A and anchor lengths of 3, 8, and 16 bp. The DNA templates for IVT (Fig. 3A) and IVT- RNA (Fig. 3B) were prepared as detailed in Examples 2 and 3. A total of 500 nanograms of IVT-RNA was transfected into CHO cells, and the analysis was performed as described in Example 5.

[0119] All Poly A-containing clRNA variants outperformed those without Poly A. Notably, the percentage of GFP-expressing cells (Fig. 4A) and mean fluorescence (Fig. 4B) increased with longer 5’ anchor sequences, suggesting that the efficiency of Poly A binding proteins is enhanced by the presence of an anchor sequence, with the 16 bp anchor demonstrating superior performance over the 8 bp and 3 bp sequences.

[0120] Example 8: Effect of 5' and 3' Poly A Length on Expression and Stability of GFP in clRNA

[0121] Building on Examples 4 and 5, where 15 bp lengths of 5’ and 3’ Poly A were utilized, this example aimed to evaluate optimal Poly A lengths given prior findings indicating Poly A Binding Protein binding to longer Poly A stretches. The IVT templates (Fig. 5 A) and IVT-RNA (Fig. 5B) were synthesized as outlined in Examples 2 and 3, maintaining a constant anchor length of 16 bp. A total of 500 nanograms of IVT-RNA was transfected into CHO cells at 70% confluence, and analysis was performed as per Example 5.

[0122] Results from this study is revealed a positive correlation between increased Poly A length and the percentage of GFP-expressing cells (Fig. 6A) as well as mean fluorescence (Fig. 6B). A doubling in the percentage of GFP- expressing cells was observed with >40 bp of 5’ Poly A and >20 bp of 3’ Poly A compared to 20 bp 5’ Poly A. Additionally, a fourfold increase in mean fluorescence was noted with >40 bp of 5’ Poly A alongside 20 bp of 3’ Poly A. These findings underscore the critical role of >40 bp 5’ Poly A in enhancing stability compared to varying lengths of 3’ Poly A. Example 9: Analysis of clRNA for Expression of Single and Multiple Proteins from a Single IVT RNA Transcript

[0123] In this study evaluated the efficacy of clRNA in expressing single and multiple proteins from a single transcript, concurrently comparing the strength of the IRES elements from both attenuated and wildtype sources, along with differing reporter proteins GFP (green) and mCherry (red). The DNA templates for IVT (Fig. 7A) and IVT-RNA (Fig. 7B) were synthesized as specified in Examples 2 and 3 using pJET1.2-A-IRES-GFP for monocistronic IVT-RNA with Attenuated IRES-GFP, and pJET1.2-W-IRES-GFP and pJET1.2-W-IRES- mCherry for multicistronic IVT-RNA. A total of 500 nanograms of IVT-RNA was transfected into CHO cells, and analysis was performed as described in Example 5.

[0124] Results from this study shows that the percentage of expressing cells (Fig. 8 A) and mean fluorescence (Fig. 8B) were comparable between monocistronic and multicistronic constructs for both GFP and mCherry. Notably, the fold increase in expressing cells and mean fluorescence in clRNA was consistent with results from Example 6, with Attenuated IRES-GFP demonstrating expression levels nearly equivalent to those of 5’ cap mRNA. These differences indicate that protein expression can be effectively regulated within clRNA, making it a valuable tool for varied expression levels in multicistronic expression vectors.

[0125] Example 10: Effect of tRNA on Stability and Expression of clRNA

[0126] This study investigated the impact of tRNA on the stability and expression levels of clRNA, leveraging the inherent stability of tRNA due to its secondary structures and post-transcriptional modifications (Fig. 9A). The construct pJET1.2-tRNA (Glu)-W-IRES-GFP-tRNA (Gin) was designed for this purpose. The DNA templates for IVT (Fig. 10A) and IVT-RNA (Fig. 10B) were synthesized as described in Examples 2 and 3, utilizing pJET1.2-tRNA-IRES- GFP-tRNA as a template with primers listed in Table 1. ClRNA without tRNA (Fig. 10A (I)) and 5’ cap mRNA were included as controls. A total of 500 nanograms of IVT-RNA was transfected into CHO cells, and analysis was performed as in Example 5.

[0127] Results indicated that clRNA incorporating tRNA exhibited significantly higher percentages of GFP-expressing cells and mean fluorescence. This increase in expression is likely attributable to the enhanced stability of clRNA conferred by the tRNA elements. These findings support the notion that integrating elements with robust secondary structures and host-derived post-transcriptional modifications can substantially enhance the stability of clRNA.

[0128] Example 11:

[0129] In this study, we compared the expression and stability of 5' cap mRNA (from Example 9) with various clRNA constructs — clRNA without 5' Poly A but with a 16 bp anchor (Example 7), clRNA with tRNA (Example 10), and clRNA without tRNA. CHO cells were transfected with 500 ng of each IVT RNA, and GFP expression was monitored at different time points: 2, 12, 36, 60, 84, 108, 132 hr. GFP images were taken at each time point, and the percentage of GFP- expressing cells was determined using Evos Analysis software.

[0130] Results showed that 5' cap mRNA showed high percentage expressing cells at 36hr and sharply decreased within the next 24hr., Whereas clRNA with and without tRNA showed a gradual decrease from day 2hr to 132hr. clRNA with tRNA showed significantly improved in stability compared to clRNA, indicating tRNA's role in enhancing clRNA stability.

Claims

CLAIMSWe claim:

1. A stable capless mRNA comprising5’ anchor sequence,5’ Poly A,Internal Ribosome Entry Sites (IRES) coding Sequence, and 3’ Poly A.

2. A stable capless mRNA comprising a. 5’ anchor sequence varying in length from 3-1000 bp b. 5 ’ poly A sequence with varying length form 1-1000 bp c. one or more IRES d. one or more coding sequence e. 3’ poly A seq with varying length form 1-1000 bp.

3. The capless mRNA as claimed in claim 1, wherein the coding sequence is codon optimized or native sequence.

4. The capless mRNA as claimed in claim 1, is a mono-cistronic or multi cistronic.

5. The capless mRNA as claimed in claim 1 or 2, further comprises self-amplifiable elements for replication of RNA in cells.

6. The capless mRNA as claimed in claim 1 or 2, further comprises an immune modulatory protein along with antigen of interest.

7. The capless mRNA as claimed in claim 1 or 2, further comprises t-RNA.

8. The capless mRNA as claimed in claim 7, wherein the number of t-RNA used for one clRNA transcript varies from 1 - 20.

9. A method of preparation of a stable capless mRNA comprising the steps of ; a) Designing a DNA template b) In-vitro transcription of the template c) Incorporation of poly- A tail at 5’ and 3’ end d) purification of mRNA.

10. The method as claimed in claim 9, the DNA template is a plasmid or PCR amplified product.

11. The capless mRNA as claimed in claim 1, used as therapeutic agent such as vaccines, antibody production, protein supplementation, CAR-T cell therapy, CRISPR-cas9 based genome editing.

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