mRNA based CFTR precursor and preparation method thereof

Abstract

The present disclosure discloses a messenger ribonucleic acid (mRNA) molecule encoding for a cystic fibrosis transmembrane conductance regulator (CFTR) protein. The mRNA molecule is encoded by SEQ ID No. 25 or a sequence having 95% identity to SEQ ID No. 25.

Description

[0001] MRNA BASED CFTR PRECURSOR AND PREPARATION METHOD THEREOF

[0002] FIELD OF INVENTION

[0003] [1] The present disclosure relates to a CFTR replacement therapy. More particularly, the present disclosure relates to an mRNA-based CFTR precursor for treating cystic fibrosis.

[0004] BACKGROUND OF INVENTION

[0005] [2] CFTR (cystic fibrosis transmembrane conductance regulator) is an ATP-gated anion channel mainly conducting Cl- ions across the apical plasma membranes of many different epithelial cell. There are about 2000 mutations in CFTR protein resulting in low expression of CFTR protein, expression of truncated or non-functional CFTR protein. These mutations result in compromised anion conductance at the apical plasma membrane of secretory epithelia with variable disease severity. Common phenotypic symptoms of the mutated CFTR protein include accumulation of thick mucus in the lungs, digestive tract and other parts of the body leading to cystic fibrosis (CF) disease.

[0006] [3] Current treatment for CF includes small molecule therapies, antibiotics, anti-inflammatory drugs, decongestants, bronchodilators, high protein and fat diet, and / or vitamin supplements to control the symptoms. However, they fail to treat the root cause of the disease.

[0007] [4] Small molecule therapies include treatment with lumacaftor / ivacaftor for those who have two copies of F508del mutation in their CFTR gene. Trikafta is another therapy which is a combination drug of elexacaftor / tezacaftor / ivacaftor acting as a CFTR modulator. However, 90% of CFTR mutations cannot be corrected by any available small-molecule therapies. Further, a small-molecule therapy consisting of Lumacaftor-lvacaftor is not well tolerated by some patients leading to ocular and / or hepatic side effects, or dyspnoea in others.

[0008] [5] In adverse situations, lung transplants are performed, however, reliable control of the disease is not guaranteed.

[0009] [6] Thus, there arises a need to develop new therapies to ameliorate the CF disease symptoms.

[0010] SUMMARY OF INVENTION

[0011] [7] Particular embodiments of the present disclosure are described herein below with reference to the accompanying drawings, however, it is to be understood that the disclosed embodiments are mere examples of the disclosure, which may be embodied in various forms. Well-known functions or constructions are not described in detail to avoid obscuring the present disclosure in unnecessary detail. Therefore, specific structural and functional details disclosed herein are not to be interpreted as limiting, but merely as a basis for the claims and as a representative basis for teaching one skilled in the art to variously employ the present disclosure in virtually any appropriately detailed structure.

[0012] [8] In an embodiment, the present disclosure relates to a messenger ribonucleic acid (mRNA) molecule encoding for a cystic fibrosis transmembrane conductance regulator (CFTR) protein. The mRNA molecule is encoded by SEQ ID No. 25 or a sequence having 95% identity to SEQ ID No. 25.

[0013] [9] In an embodiment, the present disclosure relates to a messenger ribonucleic acid (mRNA) molecule as claimed in any of claims 1-2.

[0014]

[0010] In another embodiment, the present disclosure relates to a precursor including messenger ribonucleic acid (mRNA) molecules. The mRNA molecules are as described above.

[0015]

[0011] In another embodiment, the present disclosure relates to a precursor as claimed in any of claims 3-5.

[0016]

[0012] In another embodiment, the present disclosure relates to a recombinant construct including at least one first promoter region, and an open reading frame encoded by SEQ ID No. 4 or a sequence having 95% identity to SEQ ID No. 4. The open reading frame is disposed downstream of all the first promoter regions. The open reading frame encodes for an mRNA molecule as described above.

[0017]

[0013] In another embodiment, the present disclosure relates to a recombinant construct as claimed in any of claims 6-13.

[0018]

[0014] In another embodiment, the present disclosure relates to a method to prepare a precursor. The method commences by ligating at least one recombinant nucleic acid molecule having an open reading frame encoded by SEQ ID No. 4 or a sequence having 95% identity to SEQ ID No. 4 to a vector to obtain a recombinant circular construct (10). The recombinant circular construct is digested using at least one restriction enzyme to obtain a recombinant linear construct (10a). The recombinant linear construct (10a) is transcribed to obtain a plurality of mRNA molecules. Each mRNA molecule is as described above.

[0019]

[0015] In another embodiment, the present disclosure relates to a method to prepare a precursor as claimed in any of claims 14-21. BRIEF DESCRIPTION OF DRAWINGS

[0020]

[0016] The summary above, as well as the following detailed description of illustrative embodiments, is better understood when read in conjunction with the apportioned drawings. For the purpose of illustrating the present disclosure, exemplary constructions of the disclosure are shown in the drawings. However, the disclosure is not limited to specific methods and instrumentality disclosed herein. Moreover, those in the art will understand that the drawings are not to scale.

[0021]

[0017] Fig. 1 depicts a method 100 to prepare a CFTR precursor, according to an embodiment of the present disclosure.

[0022]

[0018] Fig. la depicts a recombinant circular construct 10, according to an embodiment of the present disclosure.

[0023]

[0019] Fig. lai depicts the recombinant circular construct 10, according to another embodiment of the present disclosure.

[0024]

[0020] Fig. lb depicts a recombinant nucleic acid molecule 1, according to an embodiment of the present disclosure.

[0025]

[0021] Fig. lc depicts a recombinant linear construct 10a, according to an embodiment of the present disclosure.

[0026]

[0022] Figs. 2 - 6 depict experimental data of the CFTR precursor prepared by the method 100, according to an embodiment of the present disclosure.

[0027] DETAILED DESCRIPTION OF THE DRAWINGS

[0028]

[0023] Prior to describing the invention in detail, definitions of certain words or phrases used throughout this patent document will be defined: the terms "include" and "comprise", as well as derivatives thereof, mean inclusion without limitation; the term "or" is inclusive, meaning and / or; the phrases "coupled 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. Definitions of certain words and phrases are provided throughout this patent document, and those of ordinary skill in the art will understand that such definitions apply in many, if not most, instances to prior as well as future uses of such defined words and phrases.

[0024] Reference throughout this specification to "one embodiment," "an embodiment," or similar language means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment. Thus, appearances of the phrases "in one embodiment," "in an embodiment," and similar language throughout this specification may, but do not necessarily, all refer to the same embodiment, but mean "one or more but not all embodiments" unless expressly specified otherwise. The terms "including," "comprising," "having," and variations thereof mean "including but not limited to" unless expressly specified otherwise. An enumerated listing of items does not imply that any or all of the items are mutually exclusive and / or mutually inclusive, unless expressly specified otherwise. The terms "a," "an," and "the" also refer to "one or more" unless expressly specified otherwise.

[0029]

[0025] Although the operations of exemplary embodiments of the disclosed method may be described in a particular, sequential order for convenient presentation, it should be understood that the disclosed embodiments can encompass an order of operations other than the particular, sequential order disclosed. For example, operations described sequentially may in some cases be rearranged or performed concurrently. Further, descriptions and disclosures provided in association with one particular embodiment are not limited to that embodiment, and may be applied to any embodiment disclosed herein. Moreover, for the sake of simplicity, the attached figures may not show the various ways in which the disclosed system, method, and apparatus can be used in combination with other systems, methods, and apparatuses.

[0030]

[0026] Furthermore, the described features, advantages, and characteristics of the embodiments may be combined in any suitable manner. One skilled in the relevant art will recognize that the embodiments may be practiced without one or more of the specific features or advantages of a particular embodiment. In other instances, additional features and advantages may be recognized in certain embodiments that may not be present in all embodiments. These features and advantages of the embodiments will become more fully apparent from the following description and apportioned claims, or may be learned by the practice of embodiments as set forth hereinafter.

[0031]

[0027] The present disclosure relates to a CFTR precursor (or precursor) to treat and / or manage cystic fibrosis (CF) disease. The precursor helps to complement a genetic defect in cystic fibrosis transmembrane conductance regulator (CFTR) independent of the mutations in the corresponding gene. In other words, irrespective of the mutations in the CFTR gene that causes the CF disease, the precursor of the present disclosure helps to express functional recombinant CFTR proteins.

[0032]

[0028] The precursor is a composition containing at least a plurality of messenger ribonucleic acid (mRNA) molecules that encode one or more polypeptides and then to proteins of the CFTR. The mRNAs are transcripts of a portion of a recombinant nucleic acid molecule encoding the one or more polypeptides and then the proteins of the CFTR. The CFTR protein(s) restore activity of the CFTR anion channels thereby managing symptoms of CF in an individual receiving the precursor. The precursor provides functional CFTR protein to restore anion conductance across apical plasma membrane of the epithelial cells. The precursor provides long-term CFTR protein expression and requires less frequent dosing. In an exemplary embodiment, the precursor includes the mRNA molecules for the CFTR protein.

[0033]

[0029] Alternatively, the composition may include at least a plurality of complementary DNAs (cDNAs) of the plurality of mRNAs.

[0034]

[0030] Additionally or optionally, a delivery system (or a carrier system) may be used to deliver the mRNA molecules in the precursor. For example, the delivery system may be a plurality of lipid nanoparticles encapsulating the mRNA molecules.

[0035]

[0031] The precursor may be administered to an individual via any technique selected from intramuscular, subcutaneous, intradermal, intravenous, or a combination thereof. The precursor is safe for administration as the mRNA does not integrate with the host cell thus, preserving the genetic integrity of the host cell. Further, since polypeptide / protein is not required for preparation, the precursor of the present disclosure is safe and cost-effective to prepare.

[0036]

[0032] Now referring to the figures, Fig. 1 depicts an exemplary method 100 to prepare a precursor. The precursor includes a composition containing at least a plurality of messenger RNAs (mRNAs) that encode one or more polypeptides and / or proteins of the cystic fibrosis transmembrane conductance regulator (CFTR).

[0037]

[0033] The method 100 commences at step 101, where in an embodiment, at least one recombinant nucleic acid molecule 1 is ligated to a vector to obtain a recombinant circular construct 10 (or recombinant construct), as shown in Fig. la. The recombinant circular construct 10 is a substantially circular shaped polynucleotide molecule. The recombinant construct facilitates production of mRNA molecules to form a precursor used to treat or manage cystic fibrosis (CF) disease.

[0038]

[0034] A portion of the recombinant nucleic acid molecule 1 may be transcribe to at least one mRNA molecule that is translated to one or more polypeptides and / or proteins of CFTR. The recombinant nucleic acid molecule 1 may be a single stranded DNA (ssDNA) or a double stranded DNA (dsDNA). In an exemplary embodiment, the recombinant nucleic acid molecule 1 is encoded by SEQ ID No. 1. In another exemplary embodiment, the recombinant nucleic acid molecule 1 is encoded by SEQ ID No. 2. In an exemplary embodiment, the recombinant nucleic acid molecule 1 has a dsDNA structure. In an embodiment, the recombinant nucleic acid molecule 1 is synthetically synthesized (from Genscript).

[0039]

[0035] Fig. lb depicts an exemplary embodiment of a single strand of the dsDNA structure of the recombinant nucleic acid molecule 1. As depicted in Fig. lb, the recombinant nucleic acid molecule defines a 5' end and a 3' end. The recombinant nucleic acid molecule 1 includes an open reading frame (ORF) la that encodes the mRNA molecule of the CFTR protein. The ORF la is encoded by one of SEQ ID No. 3 or SEQ ID No. 4, or a sequence having 95% identity to that of SEQ ID No. 3 or SEQ ID No. 4.

[0040]

[0036] The recombinant nucleic acid molecule 1 may optionally include non-coding regions (for example, untranslated regions (UTRs) and introns). The untranslated regions (UTR) of the recombinant nucleic acid molecule 1 are transcribed to the mRNA molecule but are not translated into the protein. In an embodiment, the recombinant nucleic acid molecule 1 includes a 3'-UTR lb encoded by SEQ ID No. 5 and a 5'-UTR lc encoded by SEQ ID No. 6 based on Betaglobin. In another embodiment, the recombinant nucleic acid molecule 1 includes a 3'-UTR lb encoded by SEQ ID No. 7 and a 5'-UTR lc encoded by SEQ ID No. 8 based on Apolipoprotein E (APOE). In yet another embodiment, the recombinant nucleic acid molecule 1 includes a 3'-UTR lb encoded by SEQ ID No. 9 and a 5'-UTR lc encoded by SEQ ID No. 10 based on Albumin. The 5'-UTR lc and the 3'UTR lb are located upstream of the start codon (i.e., ATG) and downstream of the stop codon of the ORF la, respectively. The 3'-UTR lb and the 5'-UTR lc control post- transcriptional regulation and help to increase the translation of the recombinant nucleic acid molecule 1.

[0041]

[0037] Alternatively, the recombinant nucleic acid molecule 1 does not include any non-coding regions.

[0042]

[0038] The polypeptides and then the proteins obtained from the recombinant nucleic acid molecule 1 (i.e., the ORF la) are the CFTR protein. In an embodiment, the sequence of the polypeptide and protein of the CFTR is available on National Center for Biotechnology Information (NCBI) database with ID:NP_000483.3.

[0039] Additionally, or optionally, a Kozac sequence Id is disposed between the 5'-UTR lc and the start codon of the ORF la, i.e., upstream of the ORF la. The Kozac sequence acts as a ribosome binding site, guiding the ribosome to the correct start codon on the mRNA molecule. This ensures that the protein is synthesized from the correct open reading frame of the mRNA molecule. In an exemplary embodiment, the Kozac sequence is encoded by 'GCCGCCACC'.

[0043]

[0040] Fig. lb depicts exemplary restriction sites le of the recombinant nucleic acid molecule 1, disposed at respective 5' and 3' ends of the recombinant nucleic molecule 1. In an exemplary embodiment, the restriction site at the 5' end of the recombinant nucleic acid molecule 1 is recognized by the Hindlll restriction enzyme and the restriction site at the 3' end of the recombinant nucleic acid molecule 1 is recognized by the Kpnl restriction enzyme.

[0044]

[0041] Additionally, or optionally, the recombinant nucleic acid molecule 1 includes a Poly A tail region If (as shown in Fig. lb). The poly A tail region If may be disposed downstream of the ORF la, or the 3'-UTR lb. In the depicted embodiment, as shown in Fig. lb, the poly A tail region If is disposed downstream of the 3'-UTR lb. The poly A tail region If may have a length ranging from 80 nucleotides to 200 nucleotides. In an exemplary embodiment, the Poly A tail region If includes 120 nucleotides of adenosine. The poly A tail helps in maintaining stability of the mRNA molecule by preventing degradation of the mRNA molecule and exporting the mature mRNA molecule from the nucleus to the cytoplasm for its translation.

[0045]

[0042] The vector may include at least one origin of replication region 12, at least one first promoter region 14, at least one terminator region 14a, one or more selectable markers 16, a plurality of restriction sites (i.e., pre-defined nucleotide sequences that are recognized by restriction enzymes), etc. The origin of replication region 12 helps the vector to replicate inside a host cell. Alternatively, the vector may include only the first promoter region 14. In an embodiment, the vector includes one first promoter region 14.

[0046]

[0043] The first promoter region 14 may have a binding affinity to at least one RNA polymerase enzyme including at least one of T7 RNA polymerase, T3 RNA polymerase, SP6 RNA polymerase, etc. The RNA polymerase enzymes help to produce mRNA molecules from the recombinant nucleic acid molecule 1 via transcription. The first promoter region 14 may be one of T7 promoter encoded by SEQ ID No. 11 or SEQ ID No. 12, T3 promoter encoded by SEQ ID No. 13, SP6 promoter encoded by SEQ ID No. 14, etc. The recombinant nucleic acid molecule 1 is disposed downstream of at least one of the first promoter regions 14 to enable transcription of the recombinant nucleic acid molecule 1 by the first promoter region 14. In an exemplary embodiment, the first promoter region 14 includes T7 promoter encoded by SEQ ID No. 12.

[0047]

[0044] The terminator region 14a helps the RNA polymerase enzyme to fall off the recombinant nucleic acid molecule 1, thereby stopping the transcription of the recombinant nucleic acid molecule 1. In an exemplary embodiment, the terminator region 14a includes T7 terminator encoded by SEQ ID No. 15. In the depicted embodiment, as shown in Fig. la, the terminator region 14a is disposed downstream of the recombinant nucleic acid molecule 1.

[0048]

[0045] Additionally, or alternatively, the first promoter region 14 and the terminator region 14a are provided with the recombinant nucleic acid molecule 1 (as shown in Fig. lb). The first promoter region 14 is disposed upstream of the 5'-UTR lc. The ORF la is disposed downstream of the all first promoter region(s) 14. The terminator region 14a is disposed downstream of the 3'-UTR lb.

[0049]

[0046] The selectable markers 16 may include resistance gene(s) of, for example, ampicillin encoded by SEQ ID No. 16, kanamycin encoded by SEQ ID No. 17, gentamycin, neomycin, etc. In an exemplary embodiment, the vector includes resistance gene of ampicillin as the selectable marker 16. The resistance gene(s) of the selectable marker 16 may be disposed downstream of at least one second promoter region that facilitates the expression of the resistance gene(s). For example, AmpR promoter encoded by SEQ ID No. 18 and KanR promoter encoded by SEQ ID No. 19 for expression of ampicillin resistance gene and kanamycin resistance gene, respectively. The selectable marker 16 helps the host cell having the vector to resist the effects of the corresponding antibiotic.

[0050]

[0047] The recombinant nucleic acid molecule 1 may be ligated to the vector, such as, without limitation, pUC57 encoded by SEQ ID No. 20, pGEM®-T encoded by SEQ ID No. 21, pT7 encoded by SEQ ID No. 22, pBR322 encoded by SEQ ID No. 23, etc. In an exemplary embodiment, the vector is pUC57.

[0051]

[0048] At an optional step 101a, if the vector has a circular shaped structure, the vector is digested to obtain a linearized structure of the vector. The vector may be digested using one or two restriction enzymes based on the restriction sites the vector has and a position within the vector where the recombinant nucleic acid molecule 1 is to be ligated. The restriction digestion enzyme is at least one of Notl, BspQl, Hindi II, Kpnl, etc. The restriction enzyme is configured to introduce double-stranded breaks and cut the vector at one or more of the restriction sites, thereby obtaining the linearized structure of the vector. In an embodiment, the structure of the vector is linearized using 5-10 Units of Notl restriction enzyme (procured from New England Biolabs) per lpg of vector. In another embodiment, the structure of the vector is linearized using 5-10 Units of Hindi! I and Kpnl restriction enzymes (procured from New England Biolabs), each per lpg of vector. Depending upon the restriction enzyme used to linearize the structure of the vector, the linearized structure of the vector defines sticky overhang at each of the two (free) ends of the vector.

[0052]

[0049] Additionally, or optionally, the recombinant nucleic acid molecule 1 may be digested using the same restriction enzyme(s) with which the vector is digested in step 101a. Digesting the recombinant nucleic acid molecule create sticky overhang at each of the two (free) ends of the recombinant nucleic acid molecule 1. This ensures that two free ends of the recombinant nucleic acid molecule 1 has affinity to bind with respective free ends of the vector. If two different restriction enzymes are used for digesting the vector and the recombinant nucleic acid molecule 1, then the orientation of the recombinant nucleic acid molecule 1 is ensured with respect to the vector. Using two different restriction enzymes prevents the vector to self-circularize (and selfligate).

[0053]

[0050] In an embodiment, as shown in Fig. la, the recombinant construct includes the recombinant nucleic acid molecule 1 ligated to the vector having the first promoter region 14, the terminator region 14a, the origin of replication region 12, and the selectable marker 16. In another exemplary embodiment, as shown in Fig. lai, the recombinant construct includes the open reading frame la ligated to the vector having the first promoter region 14.

[0054]

[0051] At step 101b, the recombinant nucleic acid molecule 1 is ligated to the linearized structure of the vector to obtain a recombinant circular construct 10. Specifically, two free ends of the recombinant nucleic acid molecule 1 is ligated to the respective free ends of the linearized structure of the vector. In an exemplary embodiment, each of the recombinant nucleic acid molecules 1 are ligated to a respective (linearized) vector to obtain a plurality of recombinant circular constructs 10. The recombinant nucleic acid molecule 1 is ligated to the vector such that the first promoter region 14 of the vector is disposed upstream of the recombinant nucleic acid molecule 1. The recombinant nucleic acid molecule 1 may be ligated with the vector using a ligase enzyme, such as, without limitation, T4 DNA Ligase, T3 DNA ligase, Quick ligase (New England Biolabs), Gibson assembly, etc. In an exemplary embodiment, the recombinant nucleic acid molecule 1 is ligated to the vector using 40 Units of T3 DNA ligase (procured from New England Biolabs) per lpg of the vector.

[0052] At an optional step 103, the recombinant circular constructs 10 obtained in step 101b are amplified to increase their number. Increasing the number of the recombinant circular construct 10 is one of the factors to increase the yield of the mRNA molecules, for example, the mRNA molecule for the CFTR.

[0055]

[0053] In an exemplary embodiment, the recombinant circular constructs 10 are amplified using natural replication mechanism of a competent host. The competent host may include Escherichia coli (E. coli) DH5a, E. coli DH1, E. coli C600, E. coli DH10B, E. coli ToplO or strains thereof. In an exemplary embodiment, the host cell is E. coli DH5a.

[0056]

[0054] The recombinant circular constructs 10 are introduced inside the competent host cell via a technique selected form electroporation technique, heat shock technique, calcium chloride technique, etc. In an exemplary embodiment, the recombinant circular construct 10 is introduced inside the competent host cell by heat shock technique to obtain a transformed host cell. The transformed host cell replicates the recombinant circular construct(s) 10 to amplify them, i.e., to increase the number of recombinant circular constructs 10 per transformed host cell.

[0057]

[0055] The transformed host cells are cultured at a pre-defined temperature and predefined rotations per minute (RPM) for a predefined time period, to increase their number. In an exemplary embodiment, the transformed host cells are cultured at 37 °C by shaking the transformed host cells at 120 - 150 RPM inside a shaking incubator for 14-16 hours.

[0058]

[0056] The transformed host cells may be cultured in a pre-defined nutrient medium with at least one antibiotic. In an exemplary embodiment, the transformed host cells are cultured in Luria- Bertani (LB) broth (procured from Himedia) including at least one antibiotic based on the selectable marker 16. In an exemplary embodiment, the LB broth includes lOg / L of Tryptone, 5g / L of yeast extract, and lOg / L of sodium chloride. In an exemplary embodiment, the nutrient medium is supplemented with 50 mg / mL of kanamycin (procured from Duschefa). Other functionally equivalent mediums are also within the scope of the teachings of the present disclosure. Increasing the number of transformed host cells also increases the total number of recombinant circular constructs 10.

[0059]

[0057] Although the method 100 is described with natural replication of the recombinant circular constructs 10 with the help of transformed host cells, the recombinant circular constructs 10 (or portions thereof) may be synthetically amplified using technique such as polymerase chain reaction (PCR), etc. The same is within the scope of the teachings of the present disclosure.

[0058] At step 105, the plurality of recombinant circular constructs 10 are extracted from the transformed host cell(s). Each of the transformed host cells are subjected to cell lysis to obtain a cell lysate. In an exemplary embodiment, the transformed host cells are lysed by chemical lysis technique such as alkaline lysis. Other functionally equivalent technique to obtain the cell lysate is within the scope of the teachings of the present disclosure.

[0060]

[0059] The cell lysate is subjected to a purification technique to separate the recombinant circular constructs 10 from the cell lysate. The purification technique may be selected from one of column-based techniques, bead-based techniques, etc. In an exemplary embodiment, the recombinant circular constructs 10 are obtained by purifying the cell lysate using silica columnbased purification.

[0061]

[0060] At step 107, each of the recombinant circular constructs 10 is digested to obtain a recombinant linear construct 10a, as shown in Fig. lc. Similar to the recombinant circular construct 10, the recombinant linear construct 10a includes the recombinant nucleic acid molecule 1, the origin of replication region 12, the first promoter region 14, the selectable markers 16, the second promoter region, the restriction sites, etc. The recombinant linear construct 10a is a substantially linear shaped polynucleotide molecule. The recombinant circular construct 10 and the recombinant linear construct 10a are commonly termed as recombinant constructs in the context of present disclosure.

[0062]

[0061] The recombinant circular constructs 10 may be digested using at least one restriction enzyme based on the vector used to create the recombinant circular constructs 10. The restriction enzyme may be one of Hindlll, Kpnl, Notl, BamHl, EcoRI, and BspQI, etc. The restriction enzyme is configured to introduce one double strand break and cut the recombinant circular constructs 10, thereby obtaining the linear restriction constructs 10. In an embodiment, the recombinant circular construct 10 is cut downstream of the recombinant nucleic acid molecule 1 to obtain the recombinant linear construct 10a. In an embodiment, the vector is linearized using 5-10 Units of Notl (procured from New England Biolabs) restriction enzyme per lpg of the vector digested. The recombinant nucleotide molecule 1 is disposed at an end of the recombinant linear construct 10a. And, the first promoter region 14 of the vector is disposed upstream of the recombinant nucleic acid molecule 1 in the recombinant linear construct 10a.

[0063]

[0062] At step 109, the recombinant linear construct 10a is transcribed to obtain a transcript. The transcript includes a messenger RNA (mRNA) molecule of a portion of the recombinant nucleic acid molecule 1.

[0063] In an exemplary embodiment, the recombinant linear constructs 10a are transcribed using in vitro transcription technique to obtain a plurality of transcripts, i.e., the mRNA molecule. The recombinant linear construct 10a may be added to a buffer containing at least a pre-defined amount of one RNA polymerase and a plurality of nucleotide triphosphates (NTPs). The at least one RNA polymerase may be one of T7 RNA polymerase, T3 RNA polymerase and SP6 RNA polymerase, etc. The pre-defined amount of RNA polymerase ranges from 5000 U / ml to 20000 U / ml. In an embodiment, 5000 U / ml of T7 RNA polymerase (procured from New England Biolabs) is added to the buffer. The NTPs include equal ratios of at least adenosine, guanosine, cytidine, and uridine, etc. The amount of NTPs ranges from 1 mM to 5 mM.

[0064]

[0064] The buffer may have a pre-defined pH ranging from 7.5 to 8.0. In an exemplary embodiment, the buffer is Tris buffer having a pH of 7.9. The buffer along with the recombinant linear constructs 10a may be kept at a pre-defined temperature ranging from 37 °C to 40 °C for a pre-defined time period ranging from 10 min to 360 min. In an exemplary embodiment, the buffer along with the recombinant linear constructs 10a is kept at 37 °C for 180 mins. The RNA polymerase binds with the first promoter region 14 of the recombinant linear construct 10a and transcribes the recombinant nucleic acid molecule 1 disposed downstream of the first promoter region 14. The RNA polymerase polymerizes the NTPs to produce the mRNA molecules encoding for the CFTR protein. The sequence of the mRNA molecule corresponds to the sequence of a portion of the recombinant nucleic acid molecule 1. A portion of the mRNA molecule is encoded at least by SEQ ID No. 24, SEQ ID No. 25, or a sequence having 95% identity to SEQ ID No. 24 or SEQ ID NO. 25. In an embodiment, the sequence of the mRNA molecule is encoded by SEQ ID No. 25. In yet another embodiment, the mRNA molecule includes the sequence of the '5-UTR lc, the Kozac sequence Id, the ORF la, the 3'-UTR lb, the Poly A tail region If of the recombinant nucleic acid molecule 1. The mRNA molecule is a single stranded RNA molecule extending between a 5' end and a 3' end. The parameters described above are all one of the factors that helps to increase the yield of the mRNA molecules.

[0065]

[0065] Additionally, or optionally, the mRNA molecule produced at step 109 includes one or more modified nucleotides, a 5' cap, and / or a 3' poly A tail. The modified nucleotide may be at least one of 2-thiouridine (s2U), pseudouridine (UJ), N1-methylpseudouridine (m1^), N6- methyladenosine (m6A), 5-methylcytosine (m5C), Nl-Methylpseudouridine-5'-Triphosphate, and Pseudouridine-5'-Triphosphates. The modified nucleotides help to improve stability and translation efficiency of the mRNA molecule.

[0066] The 5' cap may be added to the 5' end of the mRNA molecule either during transcription of the mRNA molecule (i.e., Co-transcriptional capping) or after transcription of the mRNA molecule (i.e., Post-transcriptional Enzymatic capping). In an exemplary embodiment, the 5' cap has a Capl structure. The 5' cap helps to protect the mRNA molecule from degradation, recruitment of mRNA processing complexes, mRNA export and translation initiation, and avoiding recognition of cellular mRNAs by innate immune system as "non-self".

[0066]

[0067] The poly A tail may be added to the 3' end of the mRNA molecule either during transcription of the mRNA molecule (i.e., Co-transcriptional poly A tail addition) or after transcription of the mRNA molecule (i.e., Post-transcriptional Enzymatic poly A tail addition by poly A polymerase). The poly A tail may have a length ranging from 80 nucleotide to 200 nucleotide. In an exemplary embodiment, the Poly A tail includes 120 nucleotides of adenosine. The poly A tail helps in maintaining stability of the mRNA molecule by preventing degradation of the mRNA molecule and exporting the mature mRNA molecule from the nucleus to the cytoplasm for its translation.

[0067]

[0068] At an optional step 111, the mRNA molecules obtained at the step 109 are purified. The mRNA molecules may be purified by using a purification technique selected from one of beadbased techniques, cellulose based chromatography, precipitation, etc. In an exemplary embodiment, the mRNA molecules are purified using precipitation-based technique.

[0068]

[0069] The mRNA molecules obtained from the method 100 forms the precursor for the CFTR protein that may be administered to an individual to manage symptoms of and / or treat CF. The composition of the precursor containing the mRNA molecule(s) may be administered to an individual via any technique selected from intramuscular, subcutaneous, intravenous, intranasal, intrapulmonary, or a combination thereof. Based on the toxicity and safety criteria, a route of administration may be decided for an individual.

[0069]

[0070] In an exemplary embodiment, the mRNA molecule obtained from the method 100 is encapsulated in a plurality of lipid nanoparticles (LNPs) and purified thereafter. The lipid nanoparticles are then aerosolized and delivered within an individual. Alternatively, the lipid nanoparticles are injected within an individual. The lipid nanoparticle delivers the mRNA molecule within the cells of the individual where the mRNA molecules are translated to a plurality of polypeptides and then to proteins of the CFTR. The CFTR proteins help to restore anion conductance across apical plasma membrane of the epithelial cells thereby managing symptoms and / or treating CF in an individual.

[0071] An exemplary lipid nanoparticle is described in Indian Patent application number 202321061936, which is incorporated herein by reference and forms a part of this specification as if set forth herein in their entirety.

[0070]

[0072] Other functionally equivalent delivery systems apart from the above-described lipid nanoparticles to deliver the mRNA molecule within the cell of an individual are within the scope of the teachings of the present disclosure.

[0071]

[0073] The precursor, containing the mRNA molecule(s) or the mRNA molecules encapsulated inside a lipid nanoparticle, optionally includes at least one of a buffer, one or more stabilizers, one or more preservatives, one or more tonicity agents, etc. The pH of the buffer ranges from 7.0 to 8.0. In an exemplary embodiment, the buffer is phosphate buffer saline having a pH of 7.4. The buffer helps in maintaining the pH of the composition.

[0072]

[0074] The stabilizer helps in stabilizing the structure of the lipid nanoparticles encapsulating the mRNA molecules. The stabilizer includes one or more cryoprotectants, one or more antioxidants, one or more amino acids, etc. The cryoprotectant helps in protecting the structure of the lipid nanoparticles encapsulating the mRNA molecules during storage (freezing / drying cycles). The cryoprotectant may be one of sucrose, trehalose, mannitol, or a combination thereof. The antioxidant prevents lipid oxidation of the lipid nanoparticles encapsulating the mRNA molecules. The antioxidant may be one of ascorbic acid, alpha-tocopherol, or a combination thereof. The amino acid helps in stabilizing the structure of the mRNA molecules in the buffer and / or inside the lipid nanoparticle. The amino acid may be at least one of arginine, glycine, histidine, etc. In an exemplary embodiment, composition includes 20-50 mg / ml of mannitol, 0.1-1.0 mg / ml of alpha-tocopherol, and 1-5 mg / ml of arginine.

[0073]

[0075] The preservative prevents microbial growth in the composition. The preservatives may be one of phenol, benzyl alcohol, or a combination thereof. In an exemplary embodiment, the composition includes 0.5-2.0 % (v / v) of benzyl alcohol.

[0074]

[0076] The tonicity agent helps in maintaining osmotic balance of the composition to match with that of physiological fluids (i.e., blood). The tonicity agent may be one of sodium chloride (NaCI), glycerol, mannitol, etc., or a combination thereof. In an exemplary embodiment, the composition includes 5-10 mg / ml of NaCI.

[0075]

[0077] Additionally, or optionally, the precursor further may include one or more targeting moieties. The targeting moiety helps in site-specific delivery of the lipid nanoparticle encapsulating the mRNA molecules. The target moiety may be one or more peptides or one or more antibodies specific for ciliated epithelial cells, thus improving diffusion or facilitate dispersion of the lipid nanoparticles. In an exemplary embodiment, an outer surface of the lipid nanoparticle is conjugated with antibodies specific to ciliated epithelial cells.

[0076]

[0078] The precursor disclosed above will now be described with the help of the following examples.

[0077]

[0079] Example 1: Method to prepare the CFTR precursor of the present disclosure

[0078]

[0080] lpg of a recombinant nucleic acid molecule 1 encoded by SEQ ID No. 2 (having ORF la encoded by SEQ ID No. 4) and lpg of the pUC57 vector encoded by SEQ ID No. 20 (procured from Genscript) were respectively digested using 5-10 Units of Hind III and Kpnl restriction enzymes (procured from New England Biolabs). After digesting, the recombinant nucleic acid molecule 1 was ligated to the pUC57 vector by using 40 Units of T4 DNA ligase enzyme (procured from New England Biolabs) to obtain the recombinant circular construct 10. The recombinant circular construct 10 was then introduced into competent E. coli DH5a cells (procured from Invitrogen) by incubating the two together at 42°C for 30 seconds (heat shock technique). The transformed E. coli DH5a cells were suspended in Luria-Bertani (LB) broth (procured from Himedia) having 50 mg / ml Kanamycin (procured from Duschefa) and incubated for 16 hours in a shaker incubator ( I BS-R-19-1, from Esco). The shaker incubator was kept at a temperature of 37 °C and at 200 RPM. After 16 hours, the transformed E. coli DH5a cells were centrifuged at 6000 xg and lysed using a lysis solution having sodium hydroxide (NaOH) and sodium dodecyl sulfate (SDS). The NaOH denatured the DNA, and the SDS disrupted the cell membrane.

[0079]

[0081] The cell lysate was passed through a silica column, in the presence of a chatropic salt. The plasmid (i.e., the recombinant circular constructs 10) was bound to the silica matrix of the column. The column was then washed with buffers and alcohol-based solutions to remove contaminants such as proteins, RNA and other cellular debris. Thereafter, the plasmid was eluted from the column using a Tris buffer, which rehydrated the DNA and released it from the silica matrix. The plasmid was then washed with isopropanol and resuspended in Tris-EDTA buffer to obtain the purified recombinant circular constructs 10. The purified recombinant circular constructs 10 was then linearized using 5-10 U / pg of the recombinant circular constructs 10 of Notl restriction enzyme (procured from New England Biolabs) to obtain the recombinant linear construct 10a. The recombinant linear construct 10a was then suspended in a T7 polymerase reaction buffer (procured from New England Biolabs) containing 5000 U / ml of T7 RNA polymerase enzyme (procured from New England Biolabs) and 0.5 mM of NTPs (i.e., adenosine, guanosine, cytidine, and uridine) (procured from New England Biolabs). The buffer was maintained at a pH of 7.9. The T7 polymerase reaction buffer was incubated at 37 °C for 180 minutes to obtain the mRNA molecules. A lithium-chloride solution was added to the T7 polymerase reaction buffer and incubated at -20 °C for 30 minutes, to precipitate the mRNA molecules. A pellet of the mRNA molecules was then obtained by centrifuging the T7 polymerase buffer at high speed in a microcentrifuge. After centrifugation, the supernatant was carefully removed and the pellet of the mRNA molecule was washed with ice-cold 70% ethanol to remove residual salts and contaminants and get purified mRNA molecules.

[0080]

[0082] The purified mRNA molecules were suspended in Tris-EDTA buffer / nuclease free water and frozen at -20 °C for safe storage. The mRNA molecules were encoded by SEQ ID No. 25.

[0081]

[0083] Example 2: Administration of the precursor of the present disclosure to HEK293T cells

[0082]

[0084] A first group of 0.5 x 106to 0.6 x 106human embryonic kidney epithelial cells (HEK293T) cells (procured from American Type Culture Collection (ATCC)) were grown in Dulbecco's Modified Eagle Medium (DMEM) (procured from Gibco), supplemented with 10% v / vfetal bovine serum (FBS) (procured from Gibco) for 24 hours, at 37°C under 5% CO2. The HEK293T cells were transfected with different concentrations (1.0 pg, 2.0 pg, 3.0 pg and 6.0 pg) of the mRNA molecules obtained from Example 1 above using Lipofectamine MessengerMAX Transfection Reagent (procured from Thermo Scientific) as per the manufacturer's protocol. The transfected HEK293T cells as described above are referred to as Group A of cells.

[0083]

[0085] A second group of 0.5 x 106to 0.6 x 106human embryonic kidney epithelial cells (HEK293T) cells (procured from American Type Culture Collection (ATCC)) were grown in Dulbecco's Modified Eagle Medium (DMEM) (procured from Gibco), supplemented with 10% v / vfetal bovine serum (FBS) (procured from Gibco) for 24 hours, at 37°C under 5% CO2. Different concentrations (1.0 pg, 2.0 pg, 3.0 pg and 6.0 pg) of the mRNA molecules obtained from Example 1 above were encapsulated in lipid nanoparticles (LNPs) to obtain formulation of mRNA-LNPs (as described in Indian Patent Application number 202321061936). The HEK293T cells were transfected with the formulations of the mRNA-LNPs by incubating them together at 37 °C. A LNP formulation without any mRNA molecule was used as the negative control. The transfected HEK293T cells as described above are referred to as Group B of cells.

[0084]

[0086] Example 3: Estimation of the level of expression of CFTR protein in HEK293T cells via Western blot analysis

[0085]

[0087] After 24 hours of transfection, the transfected Group A of cells and Group B of cells were harvested and lysed using Pierce IP Lysis Buffer (procured from Thermo Scientific) supplemented with complete protease inhibitor cocktail (procured from Roche). Lysates from the respective group of cells containing equal protein concentrations were resolved using Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto a polyvinylidene fluoride (PVDF) membrane (procured from Bio-Rad). The PVDF membrane was then blocked with 5% bovine serum albumin (BSA) (procured from MP Biomedicals) prepared in IX tris-buffered saline with tween 20 (TBS-T) buffer.

[0086]

[0088] The membranes were incubated for 2 hours at room temperature with primary antibodies. The primary antibodies used were CFTR clone (procured from Sigma) at 1:1000 dilution in 5% BSA and anti-beta-actin (procured from Invitrogen) at 1:4000 dilution in 5% BSA. After incubating the membranes, the membranes were washed three times with TBS-T and then again incubated for 2 hours at room temperature with horseradish peroxidase (HRP)-conjugated secondary antibodies. The secondary antibodies used were Goat Anti-Mouse IgG (H + L)-HRP Conjugate (procured from Biorad) at 1:4000 dilution in 1% BSA. The CFTR protein and the P-Acti n protein (which served as a loading control) was visualized using enhanced chemiluminescence (ECL) substrate (procured from Pierce) and iBright imaging system (procured from Thermo Scientific).

[0087]

[0089] Fig. 2a depicts the western blot analysis of the Group A of cells and Fig. 2b depicts the western blot analysis of the Group B of the cells. The CFTR protein and P-Actin protein had average molecular weight of 178 kD and 42 kD, respectively. The first lane in the figures depict the control (i.e ., untranslated cells) and the subsequent lanes depict the different concentrations (1.0 pg, 2.0 pg, 3.0 pg and 6.0 pg) of the mRNA molecules used in Example 2 above. In fig. 2b, the second lane represents the LNP formulation without any mRNA molecule (i.e., negative control).

[0088]

[0090] It was confirmed from the western blot analysis that the mRNA molecule was introduced in the HEK293T cells and translated into CFTR protein. The expression of the mRNA molecule (relative to the respective band intensity in the Figs. 2a and 2b) increased with increase in the concentration of the mRNA molecule introduced inside the HEK293T cells. Further, it was evident that the expression of the CFTR protein saturated after concentration of 3.0 pg of mRNA.

[0089]

[0091] Fig. 2b depicts expression of the mRNA molecule (relative to the respective band intensity) increased with increase in the concentration of the mRNA molecule introduced inside the HEK293T cells.

[0090]

[0092] Example 4: Time dependent expression analysis of the mRNA molecule in HEK293T Cells

[0091]

[0093] The Group B of cells transfected with the formulation of 3 pg of the mRNA molecule encapsulated in LNP (mRNA-LNP) and LNP without any mRNA molecule (i.e. negative control) were harvested after 12 hours, 24 hours, 36 hours and 48 hours post-transfection. The lysates of the harvested HEK293T cells were obtained and analyzed via western blot analysis as described in Example 3 above. Fig. 3 depicts the western blot analysis. The analysis confirmed that the expression of the mRNA molecule is detected up to 48 hours post transfection.

[0092]

[0094] Example 5: Concentration-dependent and time-dependent cytotoxicity (cell viability) analysis in HEK293T cells

[0093]

[0095] For concentration-dependent cytotoxicity analysis, the Group B of cells transfected with the formulations of (1 pg, 2 pg, 3 pg and 6 pg of the mRNA molecule encapsulated in LNP (mRNA- LNP)) and LNP without any mRNA molecule (i.e., negative control) were harvested 24 hours of incubation, as described in Example 3. The harvested cells were incubated with 3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT reagent) (procured from Roche Cell Proliferation Kit I,) for 4 hours at 37°C. The MTT reagent was added to respective wells containing the Group B of cells to make a final concentration of 0.5 mg / mL of the MTT reagent. Postincubation, a supernatant from each of the wells were aspirated out and the formazan crystals therein were dissolved in 200pl dimethyl sulfoxide (DMSO) (procured from Sigma-Aldrich). The absorbance of the formazan crystals dissolved in DMSO in the respective well were measured using a plate reader (procured from Thermo, Model: Variyoskan LUX) at 570 nm.

[0094]

[0096] As shown in Fig. 4, increasing concentrations of CFTR mRNA-LNPs resulted in a concentration-dependent decline in cell viability. At 1 pg and 2 pg, there were only moderate reductions in cell viability compared to the control (**p < 0.01), indicating low cytotoxicity at these concentrations. However, at 3 pg and 6 pg, a significant decrease in cell viability was observed, especially at 6 pg, which showed the lowest cell viability among all groups (***p < 0.001). The data points in the plot are presented as mean ± standard deviation, with statistical significance indicated (**p < 0.01, ***p < 0.001).

[0095]

[0097] For time-dependent cytotoxicity analysis, the Group B of cells transfected with the formulation of 3 pg of the mRNA molecule encapsulated in LNP (mRNA-LNP) were harvested after 0, 12, 24, 36, and 48 hours post-transfection, as described in Example 3. The cells harvested at 0 hour served as the control. The harvested cells were incubated with 3-(4,5-dimethylthiazol-2-yl)- 2,5-diphenyltetrazolium bromide (MTT reagent) (procured from Roche Cell Proliferation Kit I) for 4 hours at 37°C. The MTT reagent was added to respective wells containing the Group B of cells to make a final concentration of 0.5 mg / mL of the MTT reagent. Post-incubation, a supernatant from each of the wells were aspirated out and the formazan crystals therein were dissolved in 200pl dimethyl sulfoxide (DMSO) (procured from Sigma-Aldrich). The absorbance of the formazan crystals dissolved in DMSO in the respective well were measured using a plate reader (procured from Thermo Scientific, Model: Variyoskan LUX) at 570 nm.

[0096]

[0098] As shown in Fig. 5, cell viability gradually decreased over time following transfection with CFTR mRNA-LNPs. At 0 hour, cell viability was close to 100%. There was a slight but statically significant decrease observed in cell viability between 12 hours and 24 hours (**p < 0.01). More substantial reductions occurred at 48 hours and 72 hours, with the cell viability dropping to 80% and 70%, respectively. The changes were highly significant compared to the 0 hour control (***p < 0.001). The data points in the plot are presented as mean ± standard deviation, with statistical significance indicated (**p < 0.01, ***p < 0.001).

[0097]

[0099] Example 6: Time dependent analysis of surface-localized CFTR expression in HEK293 Cells

[0098]

[0100] The Group B of cells transfected with the formulation of 3 pg of the mRNA molecule encapsulated in LNP (mRNA-LNP) and LNP without any mRNA molecule (i.e., negative control) were harvested after 0, 12, 24, 36, and 48 hours post-transfection, as described in Example 3. The respective cell lysates were analyzed for surface-localized (CFTR) by sandwich ELISA using the KBH4110 kit (procured form KRISGEN Bio Systems) and corresponding absorbance was measured at 450 nm.

[0099]

[0101] As shown in Fig. 6, a gradual increase in surface-localized CFTR protein on HEK293T cells, with signal intensities rising progressively from 12 hours, reaching maximum levels at 36 to 48 hours post-transfection. This suggests that CFTR protein, after synthesis, traffics to and accumulates at the plasma membrane over time. The data points in the plot are presented as mean ± standard deviation, with statistical significance indicated (**p < 0.01).

[0100]

[0102] The experimental data obtained in time dependent expression analysis of the mRNA molecule in HEK293T (Example 4) and time dependent analysis of surface-localized CFTR expression in HEK293 Cells (Example 5) together provides a comprehensive temporal profile of CFTR expression dynamics following mRNA-LNP transfection. The data reveals distinct phases of CFTR protein expression following mRNA-LNP transfection in HEK293T cells. The intracellular pool of mature CFTR, peaks early (12-24 hours) reflecting active mRNA translation. In contrast, surface-localized CFTR increases progressively, peaking at 36-48 hours, indicating protein trafficking and membrane localization lagging behind synthesis.

[0101]

[0103] The scope of the invention is only limited by the appended patent claims. More generally, those skilled in the art will readily appreciate that all parameters, dimensions, materials, and configurations described herein are meant to be exemplary and that the actual parameters, dimensions, materials, and / or configurations will depend upon the specific application or applications for which the teachings of the present invention is / are used.

Claims

WE CLAIM:

1. A messenger ribonucleic acid (mRNA) molecule encoding for a cystic fibrosis transmembrane conductance regulator (CFTR) protein, wherein the mRNA molecule is encoded by SEQ ID No. 25 or a sequence having 95% identity to SEQ ID No. 25.

2. The mRNA molecule as claimed in claim 1, wherein the mRNA molecule includes at least one of a 5'-untranslated region, 3'-untranslated region, Kozac sequence, 5' cap, and a 3' poly A tail.

3. A precursor comprising messenger ribonucleic acid (mRNA) molecules, the mRNA molecules as claimed in any of the claims 1-2.

4. The precursor as claimed in claim 3, wherein the mRNA molecules is encapsulated within lipid nanoparticles (LNPs).

5. The precursor as claimed in claim 3, wherein the precursor includes at least one buffer, one or more stabilizers, one or more preservatives, one or more tonicity agents, and one or more targeting moieties.

6. A recombinant construct comprising: a. at least one first promoter region (14); and b. an open reading frame (la) encoded by SEQ ID No. 4 or a sequence having 95% identity to SEQ ID No. 4, the open reading frame (la) is disposed downstream of all the first promoter regions (14), the open reading frame (la) encodes an mRNA molecule as claimed in claim 1.

7. The recombinant construct as claimed in claim 6, wherein the first promoter region (14) is at least one of T7 promoter encoded by one of SEQ ID No. 11 or SEQ ID No. 12, T3 promoter encoded by SEQ ID No. 13, or SP6 promoter encoded by SEQ ID No. 14.

8. The recombinant construct as claimed in claim 6, wherein the recombinant construct includes a vector having at least one origin of replication region (12), one or more selectable markers (16), and a plurality of restriction sites, optionally the vector being one of pUC57 encoded by SEQ ID No. 20, pGEM®-T encoded by SEQ ID No. 21, pT7 encoded by SEQ ID No. 22, or pBR322 encoded by SEQ ID No. 23.

9. The recombinant construct as claimed in claim 8, wherein the one or more selectable markers (16) include resistance gene(s) of ampicillin encoded by SEQ ID No. 16, or kanamycin encodedby SEQ ID No. 17, optionally the resistance gene(s) are disposed downstream of a second promoter region including AmpR promoter encoded by SEQ ID No. 18 or KanR promoter encoded by SEQ ID No. 19.

10. The recombinant construct as claimed in claim 6, wherein the recombinant construct includes a recombinant nucleic acid molecule (1) having a 5'-untranslated region (lc) located upstream of the open reading frame (la) and a 3'-untranslated region (lb) located downstream of the open reading frame (la), optionally the first promoter region (14) is disposed upstream of the 5'-untranslated region (lc) and a terminator region (14a) is disposed downstream of the 3'-untranslated region (lb).

11. The recombinant construct as claimed in claim 8, wherein a Kozac sequence (Id) is disposed upstream of the open reading frame (la).

12. The recombinant construct as claimed in any of the preceding claims 6-11, wherein the recombinant construct is a recombinant circular construct (10) having a substantially circular shaped polynucleotide molecule.

13. The recombinant construct as claimed in any of the preceding claims 6-11, wherein the recombinant construct is a recombinant linear construct (10a) having a substantially linear shaped polynucleotide molecule.

14. A method (100) to prepare a precursor, comprising: a. ligating at least one recombinant nucleic acid molecule (1) having an open reading frame (la) encoded by SEQ ID No. 4 or a sequence having 95% identity to SEQ ID No. 4 to a vector to obtain a recombinant circular construct (10); b. digesting the recombinant circular construct (10) using at least one restriction enzyme to obtain a recombinant linear construct (10a); and c. transcribing the recombinant linear construct (10a) to obtain a plurality of mRNA molecules, each mRNA molecule as claimed in any of the claims 1-2.

15. The method (100) as claimed in claim 14, wherein after obtaining the recombinant circular construct (10) at step a., the method (100) includes synthetically amplifying the recombinant circular construct (10) to increase their number or naturally replicating the recombinant circular construct (10) by: a. introducing the recombinant circular constructs (10) inside a competent host cell(s)to obtain a transformed host cell(s), b. culturing the transformed host cells in a pre-defined nutrient medium supplemented with at least one antibiotic at a pre-defined temperature for a pre-defined time period, to increase their number, and c. extracting the recombinant circular constructs (10) from the transformed host cell(s).

16. The method (100) as claimed in claim 15, wherein the step of culturing the transformed host cells includes culturing the transformed host cells in Luria-Bertani (LB) broth supplemented with the antibiotic at 37 °C by for 14-16 hours.

17. The method (100) as claimed in claim 14, wherein before the step of ligating the recombinant nucleic acid molecule (1) to the vector includes digesting the recombinant nucleic acid molecule (1) and the vector using at least one of Notl, BspQl, Hindi! I, and Kpnl restriction enzymes, thereby creating sticky overhangs at each of the two free ends of the recombinant nucleic acid molecule (1) and the vector.

18. The method (100) as claimed in claim 14, wherein the step of ligating the recombinant nucleic acid molecule (1) to the vector includes ligating the recombinant nucleic acid molecule (1) to the vector using T4 DNA Ligase, or T3 DNA ligase.

19. The method (100) as claimed in claim 14, wherein the step of transcribing the recombinant linear construct (10a) includes adding a poly A tail to a 3'end of the mRNA molecule and a 5' cap to a 5'end of the mRNA molecule during transcription of the mRNA molecule or after transcription of the mRNA molecule.

20. The method (100) as claimed in claim 14, wherein after obtaining the plurality of mRNA molecules, the method (100) includes purifying the mRNA molecules using a purification technique selected from one of bead-based techniques, cellulose based chromatography, and precipitation.

21. The method (100) as claimed in claim 14, wherein after obtaining the plurality of mRNA molecules, the method (100) includes encapsulating the mRNA molecules within a plurality of lipid nanoparticles (LNPs).

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