Recombinant DNA molecules and constructs encoding Β- glucocerebrosidase (GCASE) enzyme, and methods thereof

Abstract

The present invention relates to recombinant DNA molecule encoding β-glucocerebrosidase (GCase) enzyme. The present invention also relates to recombinant DNA construct, recombinant vector, recombinant host cell, mRNA molecule, delivery system, pharmaceutical compositions and method of treating or managing symptoms of Gaucher disease in a subject.

Description

RECOMBINANT DNA MOLECULES AND CONSTRUCTS ENCODING B- GLUCOCEREBROSIDASE (GCASE) ENZYME, AND METHODS THEREOF FIELD OF THE INVENTION

[0001] The present invention broadly relates to the field of biotechnology and enzyme replacement. More specifically, the invention pertains to mRNA-based therapeutic compositions and methods for treating Gaucher disease through delivery of glucocerebrosidase enzyme precursors.BACKGROUND OF THE INVENTION

[0002] Metabolic pathways in living organisms involve intricate biochemical networks essential for maintaining cellular homeostasis and sustaining vital life processes. These pathways rely on the coordinated action of specific enzymes, substrates, and cofactors to ensure proper cellular function. Disruption of even a single enzymatic step can have cascading effects throughout the metabolic network, leading to severe pathological consequences.

[0003] Inborn Errors of Metabolism (IEMS) represent a diverse group of inherited disorders characterized by deficiencies in specific enzymatic activities within metabolic pathways. These conditions, typically inherited in an autosomal recessive manner, result from mutations that impair enzyme function, leading to substrate accumulation, product deficiency, or toxic metabolite formation. Among the most clinically significant IEMs are the lysosomal storage disorders (LSDs), which affect the function of lysosomal enzymes responsible for the degradation of complex macromolecules

[0004] Gaucher disease (GD) stands as the most prevalent lysosomal storage disorder, with an estimated incidence of 1 in 40,000-60,000 in the general population. GD results from deficiency of the lysosomal enzyme P-glucocerebrosidase (GCase, also known as glucosylceramidase or acid P-glucosidase, EC 3.2.1.45), which is encoded by the GBA1 gene.

[0005] GCase catalyzes the hydrolytic cleavage of glucosylceramide (glucocerebroside) into ceramide and glucose, a critical step in the catabolism of membrane glycosphingolipids. Enzyme deficiency leads to the progressive accumulation of glucosylceramide and related glycolipids, particularly glucosylsphingosine (lyso-Gbl), within lysosomes of macrophages and other cell types.

[0006] The clinical spectrum of Gaucher disease is traditionally classified into three mainphenotypes based on the presence and severity of neurological involvement. Phenotypically, GD type 1 (GDI) is the most common form and typically causes no neurological damage, whereas GD type 2 (GD2) and GD type 3 (GD3) are characterized by neurological impairment. GDI is manifested by splenomegaly, blood disorders, orthopedic complications 3 and / or lack of neurological symptoms. GD2 is manifested by hepatosplenomegaly and central nervous system involvement within the first year after birth. GD3 is manifested by neurological impairments during childhood. These forms of the disease share the same defect in the enzyme glucocerebrosidase; however, the distinct subtypes of GD help in establishing the correct diagnosis and subsequent treatment plan.

[0007] The therapeutic landscape for Gaucher disease has evolved significantly since the introduction of enzyme replacement therapy in the 1990s, yet substantial limitations persist across all available treatment modalities. ERT represents the current standard of care for Type 1 Gaucher disease and involves intravenous administration of recombinant human glucocerebrosidase. Three ERT formulations are currently approved: imiglucerase (Cerezyme®), velaglucerase alfa (VPRIV®), and taliglucerase alfa (Elelyso®). While ERT has demonstrated efficacy in reducing organomegaly, improving hematological parameters, and preventing skeletal complications, several critical limitations constrain its therapeutic utility. Apart from being very expensive, the ERT does not correct the underlying genetic defect and only provides relief to non -neurological symptoms associated with GDI . Moreover, ERT cannot change the enzyme deficiency in the brain due to its inability to cross the blood-brain barrier and consequently, it is ineffective in treating neurological problems associated with GD2 and GD3.

[0008] SRT employs small-molecule inhibitors to reduce the biosynthesis of glucosylceramide, thereby decreasing substrate accumulation. Two SRT agents are approved: eliglustat (Cerdelga®) and miglustat (Zavesca®). However, SRT applications are limited to mild to moderate cases of GDI in adults. As the efficacy of SRT is limited due to its relatively low inhibitory effect on glucosylceramide synthase at doses that are not associated with unacceptable side effects that include diarrhea, weight loss, tremors, and paresthesia.

[0009] Thus, there arises a need to develop new alternatives that can overcome the challenges associated with existing treatment to improve the management of GD.SUMMARY OF THE INVENTION

[0010] In an aspect of the present disclosure, there is provided a recombinant DNA moleculeencoding P-glucocerebrosidase (GCase) enzyme, comprising a nucleic acid sequence selected from a group consisting of: (a) a sequence having at least 80% identity (b) to a sequence selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4; (b) a sequence as set forth in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4; and (c) sequence complementary to (a) or (b).

[0011] In another aspect of the present disclosure, there is provided a recombinant DNA construct comprising the recombinant DNA molecule as disclosed herein, operably linked to a heterologous promoter.

[0012] In an aspect of the present disclosure, there is provided a recombinant vector comprising the recombinant DNA construct as disclosed herein.

[0013] In an aspect of the present disclosure, there is provided a recombinant host cell comprising the recombinant DNA construct or the recombinant vector as disclosed herein.

[0014] In another aspect of the present disclosure, there is provided an mRNA molecule transcribed from the recombinant DNA molecule as disclosed herein, the recombinant DNA construct as disclosed herein, or the recombinant vector as disclosed herein.

[0015] In another aspect of the present disclosure, there is provided a delivery system comprising the mRNA molecule as disclosed herein.

[0016] In another aspect of the present disclosure, there is provided a process of preparing the mRNA molecule as disclosed herein, comprising amplifying the recombinant DNA molecule as disclosed herein to obtain a plurality of DNA molecules, transcribing the plurality of DNA molecules obtain a plurality of mRNA molecules, and optionally modifying the mRNA molecules.

[0017] In yet another aspect of the present disclosure, there is provided a pharmaceutical composition comprising the mRNA molecule as disclosed herein.

[0018] In an aspect of the present disclosure, there is provided a method of treating or managing symptoms of Gaucher disease in a subject, comprising administering the mRNA molecule as disclosed herein, the delivery system as disclosed herein or the pharmaceutical composition as disclosed herein.

[0019] These and other features, aspects, and advantages of the present subject matter will be better understood with reference to the following description. This summary is provided tointroduce a selection of concepts in a simplified form. This summary is not intended to identify key features or essential features of the claimed subject matter, nor is it intended to be used to limit the scope of the claimed subject matter.BRIEF DESCRIPTION OF THE DRAWINGS

[0020] The following drawings form a part of the present specification and are included to further illustrate aspects of the present disclosure. The disclosure may be better understood by reference to the drawings in combination with the detailed description of the specific embodiments presented herein.

[0021] Figure 1 depicts western blot analysis image of GBA GCase protein expression in HEK293T cells transfected with GBA GCase mRNA using Lipofectamine™ MessengerMAX™ (molecular weights are indicated in kilodaltons (kDa), in accordance with the embodiments herein.

[0022] Figure 2 provides a western blot analysis image of GBA GCase expression in HEK293T cells transfected with GBA GCase mRNA-LNPs, in accordance with the embodiments herein.

[0023] Figure 3 depicts time-dependent expression profile of GBA GCase protein in HEK293T cells transfected with GBA GCase mRNA-LNPs, in accordance with the embodiments herein.

[0024] Figure 4 illustrates concentration-dependent cytotoxicity profile of GBA GCase mRNA-LNPs in HEK293T cells, in accordance with the embodiments herein. Data are represented as mean ± SEM, with statistical significance indicated (**p < 0.01).

[0025] Figure 5 provides time-dependent cytotoxicity profile of GBA GCase mRNA-LNPs in HEK293T cells, in accordance with the embodiments herein. (**p < 0.01, ***p < 0.001).

[0026] Figure 6 depicts a graph showing fold change in CFTR expression at different time points post-treatment, in accordance with the embodiments herein. . Data represent mean ± SEM (n=3). Statistical significance compared with control: **p < 0.01, ***p < 0.001.

[0027] Figure 7 shows enzymatic activity of GBA GCase in HEK293T cells transfected with GBA GCase mRNA-LNP, in accordance with the embodiments herein. Data represent mean ± SEM (n=3). Statistical significance compared with untreated control: **p < 0.01, ***p < 0.001.

[0028] Figure 8 depicts the process of preparing the mRNA molecules of the present disclosure, in accordance with the embodiments herein.DETAILED DESCRIPTION OF THE INVENTION

[0029] At the very outset of the detailed description, it may be understood that the ensuing description only illustrates a particular form of this invention. However, such a particular form is only exemplary embodiment, and without intending to imply any limitation on the scope of this invention. Accordingly, the description is to be understood as an exemplary embodiment and teaching of invention and not intended to be taken restrictively.

[0030] Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skilled in the art to which this invention belongs. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular.Definitions:

[0031] For convenience, before further description of the present disclosure, certain terms employed in the specification, and examples are delineated here. These definitions should be read in the light of the remainder of the disclosure and understood as by a person of skill in the art. The terms used herein have the meanings recognized and known to those of skill in the art, however, for convenience and completeness, particular terms and their meanings are set forth below.

[0032] The articles “a”, “an” and “the” are used to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article.

[0033] The terms “comprise” and “comprising” are used in the inclusive, open sense, meaning that additional elements may be included. It is not intended to be construed as “consists of only”.

[0034] Throughout this specification, 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 element or step or group of elements or steps but not the exclusion of any other element or step or group of elements or steps.

[0035] 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 inassociation 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.

[0036] Furthermore, the described features, advantages, and characteristics of 5 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 10 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.

[0037] As used herein, “sequence identity” means the extent to which two nucleotide or amino acid sequences are invariant. “Sequence alignment” means the process of lining up two or more sequences to achieve maximal levels of identity (and, in the case of amino acid sequences, conservation) for the purpose of assessing the degree of similarity. Numerous methods for aligning sequences and assessing similarity / identity are known in the art such as, for example, the Cluster Method, wherein similarity is based on the MEGALIGN algorithm, as well as BLASTN, BLASTP, and FASTA. When using any of these programs, the settings may be selected that result in the highest sequence similarity.

[0038] As used herein, the term “promoter” is defined as a DNA sequence recognized by the synthetic machinery of the cell, or introduced synthetic machinery, required to initiate the specific transcription of a gene. In some embodiments, the promoter is a developmentally regulated promoter. As used herein, the term “operably linked,” is defined as the promoter is in the correct location and orientation in relation to the nucleic acid to control RNA polymerase initiation and expression of the gene. In some examples, one or more polypeptides are said to be “operatively linked.” In general, the term “operably linked” is meant to indicate that an element, is functionally linked to a coding sequence which may be located downstream to such element in a polynucleotide or a nucleotide sequence.

[0039] Promoters may be selected that are appropriate for the vector used to express the CARs and other polypeptides provided herein. Promoters, and other regulatory elements, are selected such that they are functional in the desired cells or tissue. In addition, this list of promotersshould not be construed to be exhaustive or limiting; other promoters that are used in conjunction with the promoters and methods disclosed herein.

[0040] As used herein, the terms “pharmaceutically” or “pharmacologically acceptable” refers to molecular entities and compositions that do not produce adverse, allergic, or other untoward reactions when administered to an animal or a human.

[0041] As used herein, “pharmaceutically acceptable carrier” includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the vectors or cells presented herein, its use in therapeutic compositions is contemplated. Supplementary active ingredients also can be incorporated into the compositions. In some embodiments, the subject is a mammal.

[0042] As used herein, the term “subject” refers to an animal, and preferably a mammal. According to particular embodiments, the subject is a mammal including a non-primate (e.g., a camel, donkey, zebra, cow, pig, horse, goat, sheep, cat, dog, rat, rabbit, guinea pig or mouse) or a primate (e.g., a monkey, chimpanzee, or human). In particular embodiments, the subject is a human.

[0043] As used herein, the term “therapeutically effective amount” refers to an amount of an active ingredient or component that elicits the desired biological or medicinal response in a subject. A therapeutically effective amount can be determined empirically and in a routine manner, in relation to the stated purpose.

[0044] As used herein, the terms “treat,” “treating,” and “treatment” are all intended to refer to an amelioration or reversal of at least one measurable physical parameter related to a cancer or autoimmunity, which is not necessarily discernible in the subject, but can be discernible in the subject. The terms “treat,” “treating,” and “treatment,” can also refer to causing regression, preventing the progression, or at least slowing down the progression of the disease, disorder, or condition. In a particular embodiment, “treat,” “treating,” and “treatment” refer to an alleviation, prevention of the development or onset, or reduction in the duration of one or more symptoms associated with the disease, disorder, or condition. In a particular embodiment, “treat,” “treating,” and “treatment” refer to prevention of the recurrence of the disease, disorder, or condition. In a particular embodiment, “treat,” “treating,” and “treatment” refer to an increase in the survival of a subject having the disease, disorder, or condition. In a particular embodiment,“treat,” “treating,” and “treatment” refer to elimination of the disease, disorder, or condition in the subject.

[0045] Ratios, concentrations, amounts, and other numerical data may be presented herein in a range format. It is to be understood that such range format is used merely for convenience and brevity and should be interpreted flexibly to include not only the numerical values explicitly recited as the limits of the range, but also to include all the individual numerical values or subranges encompassed within that range as if each numerical value and sub-range is explicitly recited.

[0046] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the disclosure, the preferred methods, and materials are now described. All publications mentioned herein are incorporated herein by reference.

[0047] The preferred embodiments of the present invention are further described and illustrated in the Figures and Examples of the present application. All aspects disclosed in the Figures or the examples, respectively, relate to the present invention unless expressly excluded. The single features of the present invention as disclosed in the experimental part can be combined unless there are technical reasons which speak against such combination.

[0048] Gaucher disease (GD) is a rare inherited lysosomal storage disorder caused by deficiency of the glucocerebrosidase (GCase) enzyme, resulting from mutations in the GBA1 gene. This enzyme deficiency leads to the pathological accumulation of glucosylceramide (GlcCer) and other glycolipids in various organs and tissues, causing visceral, hematologic, skeletal, and in some cases, severe neurological manifestations. Current treatment options for Gaucher disease are significantly limited and present substantial therapeutic challenges.

[0049] Existing enzyme replacement therapy (ERT) involves administration of recombinant enzymes but suffers from critical limitations: (1) it does not correct the underlying genetic defect and only provides symptomatic relief; (2) it is ineffective for neurological symptoms associated with GD types 2 and 3 because the recombinant enzymes cannot cross the blood-brain barrier; (3) it requires frequent intravenous infusions, creating patient compliance issues; and (4) it is extremely expensive to manufacture and administer. Substrate reduction therapy (SRT) using small-molecule inhibitors is limited to mild-to-moderate cases of GD type 1 in adults and has relatively low efficacy due to insufficient inhibitory effects at tolerable doses, while causingunacceptable side effects including diarrhea, weight loss, tremors, and paresthesia.

[0050] Furthermore, both current therapeutic approaches fail to address the fundamental issue of providing sustained, endogenous enzyme production within the patient's own cells. There exists an urgent unmet medical need for a safe, effective, and cost-efficient therapeutic approach that can overcome the blood-brain barrier limitations, provide sustained enzyme production, and potentially address all types of Gaucher disease including the severe neurological forms.

[0051] The present invention addresses these critical limitations by providing a novel mRNA-based enzyme precursor (mRNA molecules) encoding functional glucocerebrosidase enzyme, for treating Gaucher disease. The invention comprises recombinant DNA molecules encoding functional glucocerebrosidase enzyme (SEQ ID NOs: 1-4), which are transcribed into modified mRNA molecules that serve as enzyme precursors. These mRNA molecules may be formulated in lipid nanoparticle (LNP) delivery systems to facilitate efficient cellular uptake and intracellular delivery.

[0052] The invention further incorporates several technical innovations to optimize therapeutic efficacy: modified nucleotides (pseudouridine, N1-methylpseudouridine, etc.) to enhance mRNA stability and reduce immunogenicity; codon optimization for human cell expression; incorporation of stabilizing UTR sequences from highly expressed genes (albumin, hemoglobin, apolipoprotein); and advanced LNP formulation (delivery system) for targeted delivery. The dose-dependent expression profile (demonstrated at 1-7 pg concentrations) allows for precise therapeutic dosing, while the transient nature of mRNA expression provides an additional safety margin.

[0053] This comprehensive mRNA molecule or enzyme precursor as disclosed herein represents a paradigm shift in Gaucher disease treatment, potentially addressing the limitations of current therapies while offering improved patient outcomes, enhanced safety, and broader therapeutic applicability across all types of Gaucher disease.

[0054] The present invention encompasses recombinant DNA molecules encoding P-glucocerebrosidase (GCase) enzyme. In an embodiment of the present disclosure, there is provided a recombinant DNA molecule encoding P-glucocerebrosidase (GCase) enzyme, comprising a nucleic acid sequence selected from a group consisting of: (a) a sequence having at least 80% identity to a sequence selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4; (b) a sequence as set forth in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4; and sequence complementary to (a) or (b). In alternative embodiments, thesequence has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to any of SEQ ID NOs: 1-4.

[0055] In a preferred embodiment of the present disclosure, the recombinant DNA molecule comprising a nucleic acid sequence as set forth in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4. In further embodiments, the recombinant DNA molecule comprises sequences complementary to the aforementioned sequences. The recombinant DNA molecule of the present disclosure may be single-stranded DNA (ssDNA) or double-stranded DNA (dsDNA). In specific embodiments, the molecule is codon-optimized for expression in human cells, mammalian cells, or other eukaryotic cells. The codon optimization may be done for human codon usage optimization, GC content optimization (40-60%), avoidance of rare codons, elimination of cryptic splice sites and or removal of premature polyadenylation signals.

[0056] In certain embodiments, the recombinant DNA molecule further comprises one or more untranslated regions (UTRs) to enhance mRNA stability and translation efficiency. The UTRs may be positioned upstream (5' UTR) and / or downstream (3' UTR) of the coding sequence. In an embodiment of the present disclosure there is provided a recombinant DNA molecule as described herein, wherein the DNA molecule further comprises one or more non-coding regions and / or untranslated regions (UTRs). In another embodiment, the recombinant DNA molecule does not comprise any non-coding regions. In an alternate embodiment, the recombinant DNA molecule comprises a plurality of non-coding regions. In certain embodiments, the non-coding region is an intron.

[0057] The UTRs help in improving stability of the mRNA, controls gene expression and translation of the mRNA, and localization of the mRNA. In an embodiment, the UTRs are positioned upstream (5' UTR) and / or downstream (3' UTR) of the nucleic acid sequence.

[0058] Accordingly, in an embodiment, the 5' UTR comprises a sequence selected from SEQ ID NO: 5 (Hemoglobin Subunit Beta (HBB)), SEQ ID NO: 7 (Albumin), SEQ ID NO: 9 (Apolipoprotein), or SEQ ID NO: 11 (Hemoglobin subunit alpha 1). In one or more embodiments, the 3' UTR comprises a sequence selected from SEQ ID NO: 6 (Hemoglobin Subunit Beta (HBB)), SEQ ID NO: 8 (Albumin), or SEQ ID NO: 10 (Apolipoprotein), or SEQ ID NO: 12 (Hemoglobin subunit alpha 1). In a preferred embodiment, the 5' UTR comprises a sequence as set forth in SEQ ID NO: 7 (Albumin), and / or the 3' UTR comprises a sequence as set forth in SEQ ID NO: 8 (Albumin). In a further preferred embodiment, the 5' UTR comprises a sequence as set forth in SEQ ID NO: 7 (Albumin), and the 3' UTR comprises a sequence asset forth in SEQ ID NO: 8 (Albumin).

[0059] In an embodiment of the present disclosure there is provided a recombinant DNA construct comprising the recombinant DNA molecule as disclosed herein, operably linked to a heterologous promoter. The heterologous promoter may be selected from but is not limited to T7 promoter, T3 promoter, SP6 promoter, CMV promoter, or SV40 promoter, EFla promoter. In a preferred embodiment, the promoter is a T7 promoter (SEQ ID NO: 13).

[0060] In an embodiment of the present disclosure there is provided a recombinant DNA construct as disclosed herein, wherein the recombinant DNA construct comprises a sequence selected from SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, or SEQ ID NO: 19.

[0061] In an embodiment of the present disclosure there is provided a recombinant vector comprising the recombinant DNA construct as disclosed herein. The vector may be any suitable vector capable of accepting the recombinant DNA molecule, such as a plasmid vector. In another embodiment, the recombinant vector further comprises at least one origin of replication and one or more selectable markers. The origin of replication may be selected from but not limited to ColEl, pUC, or pBR322 origins. The selectable markers may be selected from Ampicillin resistance gene (AmpR), Kanamycin resistance gene (KanR), Chloramphenicol resistance gene, Tetracycline resistance gene, Neomycin resistance gene, or Hygromycin resistance gene. Preferred embodiments include Ampicillin resistance gene (AmpR) with promoter (SEQ ID NO: 14), or Kanamycin resistance gene (KanR) with promoter (SEQ ID NO: 15). A selectable marker is a gene included in the recombinant DNA construct that allows identification and selection of successfully transformed host cells.

[0062] In some embodiments, the recombinant vector further comprises multiple cloning sites and / or transcription terminators.

[0063] In one or more embodiments, the recombinant vector is selected from pUC57 vector, pUC19 vector, pBR322 vector, pET vectors, pBAD vectors. Certain embodiments may also use custom vectors comprising the above elements including but not limited to at least one origin of replication and / or one or more selectable markers. In a preferred embodiment, the recombinant vector is pUC57 vector

[0064] In an embodiment of the present disclosure there is provided a recombinant host cell comprising the recombinant DNA construct as disclosed herein, or the recombinant vector as disclosed herein. A “host cell” is used herein to receive and replicate / amplify recombinant DNA.

[0065] In another embodiment, the host cell is selected from Escherichia coli DH5a, E. coli DH1, E. coli C600, E. coli TOPIO, E. coli Stbl3 , E. coli HST08, E. coli BL21, E. coli XL1-Blue, or any other competent bacterial strain. In an exemplary embodiment, the host cell is E. coli DH5a. In another exemplary embodiment, the host cell is E. coli Stbl3.

[0066] In certain embodiments, the recombinant DNA molecule is transformed into the host cell thus forming a recombinant host cell. Any known transformation method can be used including but not limited to electroporation, heat shock transformation, chemical transformation (CaCh, RbCl methods), or microinjection.

[0067] In an embodiment of the present disclosure there is provided an mRNA molecule transcribed from the recombinant DNA molecule as disclosed herein, the recombinant DNA construct as disclosed herein, or the recombinant vector as disclosed herein. The term “enzyme precursor” may be used interchangeably with “mRNA molecule” in the present disclosure. In another embodiment of the present disclosure there is provided an enzyme precursor transcribed from the recombinant DNA molecule as disclosed herein, the recombinant DNA construct as disclosed herein, or the recombinant vector as disclosed herein

[0068] In one or more embodiments, there is provided an mRNA molecule as disclosed herein wherein the mRNA molecule further comprises one or more modified nucleotides or nucleosides. Typically, the mRNA molecule may comprise modification on a naturally occurring nucleotide (adenine (A), guanine (G), cytosine (C), and uridine (U), including thymine (T)).

[0069] In a non-limiting embodiment the modified nucleotide is selected from pyridin-4-one ribonucleoside, 5-aza-uridine, 2-thio-5-aza-uridine, 2-thiouridine, 4-thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxyuridine, 3 -methyluridine, 5-carboxymethyl-uridine, 1-carboxymethyl-pseudouridine, 5-propynyl-uridine, 1-propynyl-pseudouridine, 5-taurinomethyluridine, 1-taurinomethyl-pseudouridine, 5-taurinomethyl -2 -thio-uridine, 1-taurinomethyl-4-thio-uridine, 5-methyl-uridine, 1-methyl-pseudouridine, 4-thio-l -methylpseudouridine, 2-thio-l-methyl-pseudouridine, 1 -methyl- 1-deaza-pseudouri dine, 2-thio-l-m ethyl- 1-deaza-pseudouri dine, dihydrouridine, dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-dihydropseudouridine, 2-methoxyuridine, 2-methoxy-4-thio-uridine, 4-methoxy-pseudouridine, 4-m ethoxy -2 -thio-pseudouri dine, pseudouridine, N1 -methylpseudouridine, or 2'-O-methyluridine.

[0070] In another non-limiting embodiment the modified nucleotide is selected from 5-aza-cytidine, pseudoisocytidine, 3-methyl-cytidine, N4-acetylcytidine, 5-formylcytidine, N4-methylcytidine, 5-hydroxymethylcytidine, 1-methyl-pseudoisocytidine, pyrrolo-cytidine, pyrrolo-pseudoisocytidine, 2-thio-cytidine, 2-thio-5-methyl-cytidine, 4-thio-pseudoisocytidine, 4-thio- 1 -methyl-pseudoisocytidine, 4-thio- 1 -methyl- 1 -deaza-pseudoisocytidine, 1 -methyl- 1 -deaza-pseudoisocytidine, zebularine, 5-aza-zebularine, 5-methyl-zebularine, 5-aza-2-thio-zebularine, 2-thio-zebularine, 2-methoxy-cytidine, 2-methoxy-5-methyl-cytidine, 4-methoxy-pseudoisocytidine, 4-methoxy-l-methyl-pseudoisocytidine, 2'-O-methylcytidine, or 5-methylcytosine.

[0071] In yet another non-limiting embodiment the modified nucleotide is selected from 2-aminopurine, 2,6-diaminopurine, 7-deaza-adenine, 7-deaza-8-aza-adenine, 7-deaza-2-aminopurine, 7-deaza-8-aza-2-aminopurine, 7-deaza-2,6-diaminopurine, 7-deaza-8-aza-2,6-diaminopurine, 1 -methyladenosine, N6-methyladenosine, N6-isopentenyladenosine, N6-(cis-hydroxyisopentenyl)adenosine, 2-methylthio-N6-(cis-hydroxyisopentenyl)adenosine, N6-glycinylcarbamoyladenosine, N6-threonylcarbamoyladeno sine, 2-methylthio-N6-threonyl carbamoyladenosine, N6,N6-dimethyladenosine, 7-methyladenine, 2-methylthio-adenine, 2-methoxy-adenine, or 2'-O-methyladenosine.

[0072] In some other non-limiting embodiment, the modified nucleotide is selected from inosine, 1-methyl-inosine, wyosine, wybutosine, 7-deaza-guanosine, 7-deaza-8-aza-guanosine, 6-thio-guanosine, 6-thio-7-deaza-guanosine, 6-thio-7-deaza-8-aza-guanosine, 7-methyl-guanosine, 6-thio-7-methyl-guanosine, 7-methylinosine, 6-methoxy -guanosine, 1-methylguanosine, N2-methylguanosine, N2,N2-dimethylguanosine, 8-oxo-guanosine, 7-methyl-8-oxo-guanosine, l-methyl-6-thio-guanosine, N2-methyl-6-thio-guanosine, N2,N2-dimethyl-6-thio-guanosine, or 2'-O-methylguanosine.

[0073] In a preferred embodiment, the modified nucleotide is selected from 2-thiouridine (s2U), pseudouridine ( ), N1 -methylpseudouridine (ml'P), N6-methyladenosine (m6A), 5-methylcytosine (m5C), 2'-O-methyluridine, 2'-O-methylcytidine, 2'-O-methyladenosine, or 2'-O-methylguanosine.

[0074] In yet another embodiment, the mRNA molecule further comprises 5’ cap structure and / or polyadenylated tail. In a preferred embodiment of the present disclosure, the mRNA molecule comprises 5’ cap structure and polyadenylated tail. The 5' cap structures may be selected from Cap 0 structure (7 -methylguanosine), Cap 1 structure (7-methylguanosine with 2'-O-methylation), Cap 2 structure, CleanCap® analogs, or Anti-reverse cap analogs (ARCA).In another embodiment, the polyadenylation may be selected from poly(A) tail length of 50-200 nucleotides, preferably 80-150 nucleotides, optimally 100-120 nucleotides.

[0075] In an embodiment, the 5’ cap may be added to 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 in efficient translation, stabilization, and transportation of mRNAs in eukaryotic cells. Similarly, the poly A tail may be added to 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 helps in maintaining stability of mRNA, prevention of degradation of mRNA and efficient translation of mRNA.

[0076] In a non-limiting embodiment of the present disclosure, the mRNA molecule as disclosed herein is formulated in a delivery system for enhanced cellular uptake and stability. Preferably a lipid nanoparticle (LNP) delivery system may be used. Accordingly, the embodiments herein provide delivery system comprising the mRNA molecule as disclosed herein. In another non-limiting embodiment, the delivery system is selected from lipid nanoparticles, liposomes, polymeric nanoparticles, or viral vectors. In a preferred embodiment, the delivery system is a lipid nanoparticle (LNP) delivery system. Typically a lipid nanoparticle comprises ionizable lipids such as DLin-MC3-DMA, ALC-0315, SM-102, DLin-KC2-DMA, C12-200, or DHA-1; phospholipids, such as DSPC (l,2-distearoyl-sn-glycero-3-phosphocholine), DPPC (l,2-dipalmitoyl-sn-glycero-3 -phosphocholine), DOPE (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine) or DOPC (l,2-dioleoyl-sn-glycero-3-phosphocholine); Sterols, such as cholesterol, cholesterol derivatives or plant sterols; PEG-lipids such as DMG-PEG2000, DSPE-PEG2000, DMG-PEG5000, or other PEG-lipid conjugates.

[0077] A preferable LNP may have size in between 1-250 nm, preferably 50-130 nm; poly dispersity index of <0.4, preferably <0.3; zeta potential of -60 to +60 mV, and encapsulation efficiency: >80%, preferably >90%.

[0078] Alternate delivery systems include but are not limited to Liposomes, Polymeric nanoparticles (PLGA, chitosan, PEI), lipoplexes, viral vectors (lentiviral, adenoviral, AAV), cell-penetrating peptides, electroporation-based delivery, or microinjection.

[0079] An exemplary lipid nanoparticle is described in Indian Patent application number202321061936, which is incorporated herein by reference and will form a part of this specification as if set forth herein in their entirety. In an exemplary embodiment, the lipid nanoparticle comprises one or more cationic lipid (ionizable lipid) (such as DHA-1 (N-[(2-Hydroxyethyl)oxyethyl]azanediyl)bis(hexane-6,l-diyl) bis(2 -hexyldecanoate); one or more neutral lipid (phospholipid) such as DSPC (l,2-Distearoyl-sn-glycero-3-phosphocholine); one or more helper lipid (sterols) such as cholesterol; and one or more of polyether (PEG-lipid) such as DMG-PEG2000 ((Azane;[3-(2-methoxyethoxy)-2-tetradecanoyloxypropyl] tetradecanoate).

[0080] In a preferred embodiment, the LNP comprising the mRNA molecule or the delivery system may be referred to as mRNA-LNP Formulation. In an embodiment of the present disclosure, there is provided a process of preparing the delivery system as described herein. In an exemplary embodiment, one or more mRNA molecules as described herein is encapsulated in the LNP to prepare the mRNA-LNP Formulation or the delivery system as described herein.

[0081] In an embodiment of the present disclosure, there is provided a process of preparing the mRNA molecule as disclosed herein, comprising amplifying the recombinant DNA molecule as disclosed herein to obtain a plurality of DNA molecules, transcribing the plurality of DNA molecules obtain a plurality of mRNA molecules, and optionally modifying the mRNA molecule.

[0082] In another embodiment, amplifying the recombinant DNA molecule is using a method selected from Polymerase Chain Reaction (PCR), Loop-Mediated Isothermal Amplification (LAMP), Recombinase Polymerase Amplification (RPA), or by culturing the recombinant host cell as disclosed herein in a suitable culture media. In yet another embodiment, the transcribing may be performed using appropriate RNA polymerases: T7, T3, SP6, Buffer pH: 7.0-8.5, preferably 7.5-8.0; Temperature: 30-55°C, preferably 37-42°C; Incubation time: 10 minutes to 8 hours, preferably 1-3 hours; Nucleotide concentrations: 1-10 mM eachNTP.

[0083] Embodiments herein provide a process of preparing the mRNA molecule as disclosed herein, wherein the process further comprises a step of purifying the mRNA molecules. In certain embodiments, the purification may be performed using method selected from oligo(dT) bead-based purification, cellulose-based chromatography, silica column purification, precipitation methods (LiCl, ethanol), HPLC purification, or magnetic bead separation.

[0084] A preferred process is illustrated in Figure 8 as method 100 for preparation of the mRNA molecule of the present disclosure. The method 100 commences at step 101, where at least one recombinant nucleic acid molecule is ligated to a vector. At an optional step 103, therecombinant circular constructs obtained in step 101 are amplified to increase their number as described herein, preferably transforming a host cell. At step 105, the recombinant circular constructs are extracted from the transformed hosts. At step 107, the recombinant circular constructs are digested to obtain recombinant linear constructs. The linearization of the recombinant circular constructs to obtain the recombinant linear constructs may be performed using at least one restriction enzyme or a functionally similar molecular tool that can cleave the recombinant circular construct at a specific site (sequence). The recombinant nucleotide molecule is disposed at an end of the recombinant linear construct. The promoter of the vector is disposed upstream of the recombinant nucleotide molecule in the recombinant linear construct. At step 109, the recombinant linear constructs are transcribed to obtain a plurality of messenger RNA (mRNA) molecules of the present disclosure. At step 111, the mRNA obtained in step 109 is purified. The mRNA molecule may be purified by using a purification technique selected from one of bead -based techniques, cellulose based chromatography, precipitation, etc. In an exemplary embodiment, the mRNA molecule is purified using oligo dT bead-based technique. At an optional step 113, the structural integrity of the purified mRNA molecule obtained at either step 111 or step 109 is verified by at least one of a gel electrophoresis technique, a spectrophotometry technique and a sequencing technique. The verification of the purified mRNA provides quality and stability assurance of the purified mRNA for its use in managing GD.

[0085] Embodiments of the present disclosure provide a pharmaceutical composition comprising the mRNA molecule as disclosed herein, and a pharmaceutically acceptable carrier. In specific embodiments, the pharmaceutically acceptable carrier is an inert component or excipients added in an appropriate amount to the pharmaceutical composition.

[0086] In one or more embodiments, the pharmaceutical composition is selected from phosphate buffered saline (PBS), tris-buffered saline, normal saline, Dextrose solutions, Lactated Ringer's solution, mannitol, sucrose, trehalose or Sorbitol; excipients such as sorbitol stabilizers (sucrose, trehalose, mannitol), antioxidants (ascorbic acid, tocopherol), preservatives (benzyl alcohol, phenol), pH adjusters (sodium hydroxide, hydrochloric acid), or tonicity adjusters (sodium chloride, mannitol).

[0087] The pharmaceutical composition may be formulated as injectable solutions, lyophilized powders for reconstitution, frozen liquid formulations, nebulizable formulations or topical formulations.

[0088] In an embodiment of the present disclosure, there is provided a method of treating or managing symptoms of Gaucher disease in a subject, comprising administering a therapeutically effective amount of mRNA molecule as disclosed herein, the delivery system as disclosed herein or the pharmaceutical composition as disclosed herein, to the subject. In another embodiment of the present disclosure, administering is via a route selected from intramuscular, subcutaneous, intradermal, intravenous, intranasal, or combinations thereof. In a preferred embodiment, the subject is a mammal, preferably human. The subjects may be classified as under Gaucher disease Type 1 patients, Gaucher disease Type 2 patients, Gaucher disease Type 3 patients, pediatric patients, adult patients, treatment-naive patients or patients previously treated with ERT or SRT. Once inside the subject, the mRNAs are translated to a plurality of polypeptides and / or proteins. The polypeptides and / or proteins catalyzes the hydrolysis of GlcCer into ceramide and glucose, thereby managing symptoms and / or treating GD in a subject.

[0089] Table 1 depicts the details of exemplary sequences according to the present disclosure as also provided in the sequence listing.Table 1:SEQ ID Sequence Type Sequence descriptionNO.:1 Nucleotide GCase Sequence 12 Nucleotide GCase Sequence 23 Nucleotide GCase Sequence 34 Nucleotide GCase Sequence 45 Nucleotide Betaglobin or Hemoglobin Subunit Beta (HBB) towards the 5’ end6 Nucleotide Betaglobin or Hemoglobin Subunit Beta (HBB) towards the 3’ end7 Nucleotide Albumin (ALB) towards the 5’ end8 Nucleotide Albumin (ALB) towards the 3 ’ end9 Nucleotide Apolipoprotein (APOE) towards the 5’ end10 Nucleotide Apolipoprotein (APOE) towards the 3 ’ end11 Nucleotide Hemoglobin subunit alpha 1 (HBA1) towards the 5’ end 12 Nucleotide Hemoglobin subunit alpha 1 (HBA1) towards the 3’ end 13 Nucleotide T7 promoter (TAATACGACTCACTATAAG)14 Nucleotide AmpR promoter15 Nucleotide KanR promoter16 Nucleotide Construct 117 Nucleotide Construct 218 Nucleotide Construct 319 Nucleotide Construct 4

[0090] Although the subject matter has been described with reference to specific embodiments, this description is not meant to be construed in a limiting sense. Various modifications of the disclosed embodiments, as well as alternate embodiments of the subject matter, will become apparent to persons skilled in the art upon reference to the description of the subject matter. It is therefore contemplated that such modifications can be made without departing from the spirit or scope of the present subject matter as defined.EXAMPLES

[0091] The disclosure will now be illustrated with following examples, which is intended to illustrate the working of disclosure and not intended to take restrictively to imply any limitations on the scope of the present disclosure. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this disclosure belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice of the disclosed methods and compositions, the exemplary methods, devices and materials are described herein. It is to be understood that this disclosure is not limited to particular methods, and experimental conditions described, as such methods and conditions may vary.Example 1: Method of preparing the mRNA molecules encoding the p-glucocerebrosidase (GCase) enzyme:

[0092] Four different recombinant DNA molecules encoding P-glucocerebrosidase or GBA GCase protein were synthesized with codon optimization for human expression. Each sequence (SEQ ID NOs: 1-4) was designed to encode the full-length mature GCase enzyme. The DNA molecules were flanked with appropriate restriction sites for cloning into the pUC57 vector. The pUC57 vector was linearized using appropriate restriction enzymes, and the glucocerebrosidase coding sequences were ligated using T4 DNA ligase according to manufacturer's protocols. The ligation reactions were incubated at 16°C overnight to obtain the recombinant vectors comprising a recombinant DNA construct (SEQ ID NOs: 16 to 19).

[0093] The vector was then electroporated into competent E. coli DH5a cells. The transformed cells were suspended in Luria-Bertani broth having 1% ampicillin and cultured for 16 hours in a shaker incubator. The shaker incubator was kept at a temperature of 37 °C and at 140 RPM. After 16 hours, the cells were centrifuged and lysed. The cell lysate was subjected to column purification to get the purified vector. The purified vector was then linearized using one of thefollowing restriction enzymes: Hindlll, Kpnl, Notl, BamHl, EcoRI, BspQI based on the sequence design. The linearized vector was then suspended in a buffer containing T7 RNA polymerase enzyme and NTPs (i.e., adenosine, guanosine, cytidine, and uridine). The buffer was incubated at 37 °C for 120 minutes. The buffer was then subjected to column purification to get purified mRNA molecules or enzyme precursors of the present disclosure. The purified mRNAs were suspended in Tris-EDTA buffer / nuclease free water and frozen at -20 °C for safe storage or until further use. The resulting mRNA molecules or enzyme precursors were used in different studies. The examples provided hereinbelow are with mRNA molecule obtained from Construct 1 represented by SEQ ID NO: 16 (comprising the recombinant DNA molecule as set forth in SEQ ID NO:3).Example 2: Lipid Nanoparticle Formulation (delivery system)

[0094] The lipid nanoparticle (LNPs) used for preparing the delivery system of the present disclosure was prepared by method as described in Indian Patent application number 202321061936. Briefly, 45 mol% (12.7 mM) of DHA-1 (cationic lipid), 10 mol% (2.62 mM) DSPC (neutral lipid), 41.5 mol% (11.6 mM) cholesterol (helper lipid), and 2.5 mol% (0.623 mM) PEG2k-DMG (poly ether) were dissolved in 100% ethanol to prepare a first mixture. A second mixture was prepared by dissolving mRNA molecules of the present disclosure in 20 mM acetate buffer (pH 4.5).

[0095] One part of the first mixture was mixed with three parts of the second mixture by T-mixing using a 0.2-inch ID Tee (T-tube) and syringe pump at differential flow rates to obtain the LNPs. The LNPs were then collected and diluted in Tris buffer saline (TBS) at 1:1 ratio. The diluted LNPs were then exchanged with 100 times volume of TBS overnight at 4°C, under gentle stirring using 10 kDa slide-a-lyzer G2 dialysis cassette. After dialysis, the LNPs were filtered using 0.2 p sterile filter and stored at 2-8°C. The N / P ratio of the LNPs was 5. The following LNP (designated as LNP003 in Indian Application number 202321061936) was used in the examples of the present disclosure.Table 2: LNPLNP Cationic lipid Neutral lipid Helper lipid PolyetherLNP003 45 mol% 10 mol% 41.5 mol% 2.5 mol%DHA-1 DSPC cholesterol DMG-PEG2kExample 3: Concentration-Dependent Expression Analysis for mRNA Molecules Post Transfection with Standard Transfection Reagent in HEK293T Cells

[0096] HEK293T cells (Source: ATCC, Catalog No.: CRL-11268) were cultured in Dulbecco’s Modified Eagle Medium (DMEM) (Make: Gibco, Catalog No.: 10569) supplemented with 10% fetal bovine serum (FBS) (Make: Gibco, CatalogNo.: 26140079). Cells were transfected with various concentrations (1.0, 3.0, 5.0, and 7.0 pg) of in-vitro synthesized mRNA molecules of Example 1 using Lipofectamine™ MessengerMAX™ Transfection Reagent (Make: Thermo Scientific, Catalog No.: LMRNA008) as per the manufacturer’s protocol. At 24 hours post-transfection, cells were harvested and lysed using Pierce™ IP Lysis Buffer (Make: Thermo Scientific™, Catalog No.: 87787) supplemented with complete™ Protease Inhibitor Cocktail (Make: Roche, Catalog No.: 11697498001). Protein lysates with equal protein concentration were resolved using SDS-PAGE and transferred onto a polyvinylidene fluoride (PVDF) membrane (Make: Bio-Rad, Catalog No.: 1620177). Membranes were blocked with 5% Bovine Serum Albumin (Make: MP Biomedicals, Catalog No.: 8810025) prepared in IX Tris-buffered saline with Tween 20 (TBS-T) buffer. The membranes were incubated with primary antibodies: GBA GCase GBA mAb (Make: Invitrogen, Catalog No.: 88162, diluted 1:1000 in 5% BSA) and anti-beta-actin (Make: Invitrogen, Catalog No.: AM4302, diluted 1 :4000 in 5% BSA) for 2 hours at room temperature. Following three washes, membranes were incubated with horseradish peroxidase (HRP)-conjugated secondary antibody: Goat Anti-Mouse IgG (H + L)-HRP Conjugate (Make: BioRad, Catalog No. : 1706516). Protein bands were visualized using enhanced chemiluminescence (ECL) substrate (Make: Pierce, Catalog No.: 32132) and detected using the iB right Imaging System (Make: Thermo Scientific).

[0097] Results: As depicted in Figure 1, the western blot analysis of protein expression revealed that HEK293T cells transfected with in vitro transcribed GBA GCase mRNA molecules exhibited a clear, dose-dependent increase in GBA GCase protein expression. Minimal expression was observed at 1 pg mRNA, which progressively increased with higher doses (3, 5, and 7 pg). At 5 pg and 7 pg, GBA GCase expression was robust and reached saturation levels. No GBA GCase expression was detected in the non-transfected control (OC). P-Actin levels remained constant across all samples, confirming equal protein loading.

[0098] Conclusion: These findings demonstrate that transfection of GBA GCase mRNA into HEK293T cells results in efficient, dose-dependent expression of the target protein. The results validate the successful delivery and translation of synthetic mRNA, with optimal proteinexpression achieved at higher mRNA concentrations (>5 pg).Example 3: Concentration-Dependent Expression Analysis for mRNA Molecules Post Transfection with GCase mRNA-LNP Formulation in HEK293T Cells

[0099] HEK293T cells were cultured in DMEM (Make: Gibco, Catalog No.: 10569) supplemented with 10% FBS (Make: Gibco, Catalog No.: 26140079). Cells were transfected with various concentrations (1.0, 3.0, 5.0, and 7.0 pg) of in vitro synthesized GBA GCase mRNA encapsulated in lipid nanoparticles (LNP) formulation by direct incubation with the mRNA-LNPs. A blank LNP (BL) formulation was used as a negative control. At 24 hours posttransfection, cells were harvested and lysed using Pierce™ IP Lysis Buffer (Make: Thermo Scientific™, Catalog No.: 87787) supplemented with cOmplete™ Protease Inhibitor Cocktail (Make: Roche, Catalog No.: 11697498001). Equal protein concentrations from each sample were resolved via SDS-PAGE and transferred onto PVDF membranes (Make: Bio-Rad, Catalog No.: 1620177). Membranes were blocked with 5% BSA (Make: MP Biomedicals, Catalog No.: 8810025) prepared in IX TBS-T buffer. Primary antibodies used were GBA GCase GBA mAb (Make: Invitrogen, Catalog No.: 88162, diluted 1:1000 in 5% BSA) and anti -beta-actin (Make: Invitrogen, Catalog No.: AM4302, at 1:4000 dilution in 5% BSA), incubated for 2 hours at room temperature. After washing, membranes were incubated with HRP-conjugated secondary antibody: Goat Anti-Mouse IgG (H + L)-HRP Conjugate (Make: Bio-Rad, Catalog No.: 1706516). Detection was performed using ECL substrate (Make: Pierce, Catalog No.: 32132) and visualized on the iBright Imaging System (Make: Thermo Scientific).

[0100] Results: Western blot analysis as shown in Figure 2 revealed successful expression of GBA GCase protein in HEK293T cells following transfection with GBA GCase mRNA delivered via lipid nanoparticles (LNPs). No GBA GCase signal was observed in the untreated control (OC) & blank LNP (BL) lanes, confirming the specificity of mRNA-mediated expression. In contrast, cells transfected with increasing doses of GBA GCase mRNA-LNPs (1, 3, 5, and 7 pg) showed a dose-dependent increase in GBA GCase protein (~59 kDa). Expression was minimal at 1 pg, moderate at 3 pg, and maximal at 5-7 pg. P-Actin (~42 kDa) levels remained constant across all lanes, verifying equal protein loading.

[0101] Conclusion: These findings demonstrate that GBA GCase mRNA formulated in LNPs enables efficient delivery and translation in HEK293T cells. The data confirm dose-dependent expression of GBA GCase, with robust protein production at 5-7 pg. Importantly, no background expression was observed in control or blank LNP groups, indicating the specificityand effectiveness of LNP -mediated mRNA transfection.Example 4: Time-dependent expression of GBA GCase protein in HEK293T cells following transfection with GBA GCase mRNA-LNPs

[0102] HEK293T cells were cultured in DMEM (Make: Gibco, Catalog No.: 10569) supplemented with 10% FBS (Make: Gibco, Catalog No.: 26140079). Cells were transfected with 3 pg of in vitro synthesized GBA GCase mRNA encapsulated in LNP formulation by incubation with the mRNA-LNPs. A blank LNP served as a negative control. Transfected cells were harvested at multiple time-points: 12, 24, 48, and 72 hours, post-transfection. Cell lysates were prepared using Pierce™ IP Lysis Buffer (Make: Thermo Scientific™, Catalog No. : 87787) supplemented with cOmplete™ Protease Inhibitor Cocktail (Make: Roche, Catalog No.: 11697498001). Equal amounts of protein were resolved on SDS-PAGE and transferred onto PVDF membranes (Make: Bio-Rad, Catalog No. : 1620177). Membranes were blocked with 5% BSA (Make: MP Biomedicals, Catalog No.: 8810025) in TBS-T and incubated with primary antibodies: GBA GCase mAb (Make: Invitrogen, Catalog No.: 88162, diluted 1:1000 in 5% BSA) and anti -beta-actin (Make: Invitrogen, Catalog No.: AM4302, 1:4000 dilution in 5% BSA) for 2 hours at room temperature. After three washes, membranes were incubated with HRP-conjugated Goat Anti-Mouse IgG (H + L) secondary antibody (Make: Bio-Rad, Catalog No.: 1706516). Signal detection was performed using ECL substrate (Make: Pierce, Catalog No.: 32132) and visualized using the iBright Imaging System (Make: Thermo Scientific).

[0103] Results: Western blot analysis as depicted in Figure 3 revealed time-dependent expression of GBA GCase protein (~59 kDa) in HEK293T cells following transfection with GBA GCase mRNA-LNPs. GBA GCase expression was detectable at 12 h, sustained at 24 h, and remained evident at 48 h post-transfection. By 72 h, GBA GCase expression was no longer detectable, indicating a decline in mRNA-driven protein translation over time. P-Actin (~42 kDa) levels were constant across all samples, confirming equal protein loading.

[0104] Conclusion: These results demonstrate that GBA GCase mRNA-LNPs mediate efficient protein translation in HEK293T cells in a time-dependent manner. GBA GCase protein expression was initiated within 12 h post-transfection and maintained for at least 48 h, indicating robust and sustained protein production from the delivered mRNA.Example 5: Concentration-Dependent and Time-Dependent Cytotoxicity Analysis Using MTT Assay in HEK293T Cells Transfected with GBA GCase mRNA-LNP

[0105] Cell Culture: HEK293T cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM) (Make: Gibco, Catalog No.: 10569) supplemented with 10% fetal bovine serum (FBS) (Make: Gibco, Catalog No.: 26140079).

[0106] Transfection: Cells were transfected with various concentrations (1.0, 3.0, 5.0, and 7.0 pg) of in vitro synthesized GBA GCase mRNA encapsulated in lipid nanoparticle (LNP) formulation. For time-dependent analysis, cells were transfected with 3.0 pg of GBA GCase mRNA-LNP. Blank LNP formulation was used as a negative control.

[0107] MTT Assay (Concentration-Dependent Analysis): After 24 hours of incubation, the Roche Cell Proliferation Kit I (MTT) (Catalog No.: 11465007001) was used to assess cell viability. The MTT reagent was added to each well (final concentration of 0.5 mg / mL) for 3 hours at 37°C. Following the incubation, the supernatant was aspirated, and the formazan crystals were dissolved in DMSO (Make: Sigma-Aldrich, Catalog No.: D8418). Absorbance was measured at 570 nm using a Plate reader (Make: Thermo, Model: Varioskan LUX).

[0108] MTT Assay (Time-Dependent Analysis): For time-dependent cytotoxicity, the MTT assay was performed at different time points: 12, 24, 48, and 72 hours, post-transfection. The same procedure as described above for the concentration-dependent assay was followed, but with varying time points for data collection.

[0109] Results: The cytotoxicity of GBA GCase mRNA-loaded lipid nanoparticles was assessed using MTT assays under two conditions: over time and across increasing concentrations.

[0110] Concentration-dependent assay: MTT assay as depicted in Figure 4 showed a concentration-dependent decline in HEK293T cell viability following transfection with GBA GCase mRNA-LNPs. At lower concentrations (1-3 pg), cell viability remained above -90%, indicating good tolerability. However, higher concentrations (5 and 7 pg) resulted in a more pronounced reduction in viability (-85% and -78%, respectively). Statistical analysis confirmed significant differences between control and higher dose groups.

[0111] Time-dependent assay: Cell viability was also assessed over time following transfection with GBA GCase mRNA-LNPs. As apparent from Figure 5, viability remained high (-90-95%) at 12-24 h but showed a progressive decline at later time points, dropping to -85% at 48 h and -80% at 72 h. The results indicate a time-dependent cytotoxic effect associated with prolonged mRNA-LNP exposure.

[0112] Conclusion: These findings demonstrate that GBA GCase mRNA-LNPs are generally well tolerated by HEK293T cells at lower concentrations and earlier time points. However, higher doses and extended exposure times lead to a modest but significant reduction in cell viability. Together, the data suggests a balance between efficient protein expression and acceptable cytocompatibility, highlighting the importance of optimizing both dosage and exposure duration for mRNA-LNP applications.Example 6: Time-Dependent Analysis of GBA GCase Protein Expression Post Transfection with GBA GCase mRNA-LNP Formulation in HEK293T Cells

[0113] Cell Culture: HEK293T cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM, Gibco, Catalog No.: 10569) supplemented with 10% fetal bovine serum (FBS, Gibco, Catalog No.: 26140079) under standard conditions. Cells were transfected with 3 pg of in vitro synthesized GBA GCase mRNA encapsulated within lipid nanoparticles (LNPs). A blank LNP formulation served as a negative control. Transfection was performed by incubating cells with the GBA GCase mRNA-LNP formulation. Post-transfection, cells were harvested at 12, 24, 48, and 72 hours for downstream analysis.

[0114] ELISA quantification of expressed protein: GBA GCase protein levels in cell lysates were quantified using the Abeam ELISA kit (ab315298) according to the manufacturer’s protocol. Briefly, samples and standards were added to pre-coated plates, incubated, washed, and incubated with detection antibody and substrate. Absorbance was read at the 450 nm and sample concentrations interpolated from the standard curve. Values were normalized to total protein. Measurements were performed in technical duplicate for each biological replicate.

[0115] Result: ELISA quantification of GBA GCase protein expression in HEK293T cells transfected with GBA GCase mRNA-LNPs as shown in Figure 6 revealed a time-dependent increase in expression. Detectable levels were observed as early as 12 h, with a steady rise at 24 h and a peak at 48 h post-transfection. By 72 h, GBA GCase expression significantly declined compared to peak levels.

[0116] Conclusion: ELISA results confirmed that GBA GCase mRNA-LNPs successfully mediated intracellular delivery and translation of GBA GCase protein in HEK293T cells. Expression was sustained up to 48 h with maximum levels detected at this time point, followed by a decline at 72 h. This profile indicates efficient but transient protein production typical of mRNA therapeutics.Example 7: Time-Dependent Analysis of GBA GCase Enzyme activity Post Transfection with GBA GCase mRNA-LNP Formulation in HEK293T Cells

[0117] Glucocerebrosidase Activity Assay: The enzymatic activity of GBA GCase in HEK293T cells transfected with GBA GCase mRNA-LNPs was measured using the Glucocerebrosidase Activity Assay Kit (Abeam, Cat. No. ab273339), according to the manufacturer’s instructions. At 12, 24, 48, and 72 hours, post-transfection, cells were harvested, washed with cold PBS, and lysed in the provided lysis buffer on ice. Lysates were clarified by centrifugation at 12,000 x g for 10 min at 4 °C, and cell lysate were collected for analysis. Protein concentrations were determined using a BC A protein assay (Thermo Scientific, Cat. No.23225) to normalize enzyme activity across samples. For each reaction, equal amounts of protein lysate were incubated with the fluorogenic GBA GCase substrate (4-methylumbelliferyl-P-D-glucopyranoside) supplied in the kit. Reactions were carried out in black 96-well plates at 37 °C for 1 h in the presence and absence of the specific inhibitor (Conduritol B epoxide, CBE) to confirm assay specificity. Fluorescence was measured at Ex / Em = 365 / 445 nm using a microplate reader (Varioskan LUX). GBA GCase activity was calculated as relative fluorescence units (RFU) per pg of protein and expressed as fold-change compared to untreated control cells.

[0118] Result: The enzymatic activity of GBA GCase was evaluated in HEK293T cells transfected with GBA GCase mRNA-LNPs. From the graph provided in Figure 7 it can be observed that activity of GBA GCase enzyme was detectable at 12 h, increased further at 24 h, and reached maximum levels at 48 h post-transfection. By 72 h, enzymatic activity markedly declined compared to peak levels. These results confirm a strong correlation between the timedependent expression of GBA GCase protein and its functional activity, with 48 h representing the optimal expression window.

[0119] Conclusion: The enzymatic activity assay demonstrated that GBA GCase protein expressed following GBA GCase mRNA-LNP transfection was not only produced but also enzymatically functional. Peak activity was observed at 48 h, consistent with ELISA-based protein quantification. The decline at 72 h reflects the transient kinetics of mRNA expression, highlighting the necessity for time-optimized analysis when evaluating mRNA therapeutics.

Claims

I / We Claim:

1. A recombinant DNA molecule encoding P-glucocerebrosidase (GCase) enzyme, comprising a nucleic acid sequence selected from a group consisting of:a) a sequence having at least 80% identity to a sequence selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4,b) a sequence as set forth in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4, andc) a sequence complementary to (a) or (b).

2. The recombinant DNA molecule as claimed in claim 1, further comprising one or more non-coding regions and / or untranslated regions (UTRs).

3. The recombinant DNA molecule as claimed in claim 2, wherein the UTRs are positioned upstream (5' UTR) and / or downstream (3' UTR) of the nucleic acid sequence.

4. The recombinant DNA molecule as claimed in claim 3, wherein the 5' UTR comprises a sequence selected from SEQ ID NO: 5 (Hemoglobin Subunit Beta (HBB)), SEQ ID NO: 7 (Albumin), SEQ ID NO: 9 (Apolipoprotein), or SEQ ID NO: 11 (Hemoglobin subunit alpha 1).

5. The recombinant DNA molecule as claimed in claim 3, wherein the 3' UTR comprises a sequence selected from SEQ ID NO: 6 (Hemoglobin Subunit Beta (HBB)), SEQ ID NO: 8 (Albumin), or SEQ ID NO: 10 (Apolipoprotein), or SEQ ID NO: 12 (Hemoglobin subunit alpha 1).

6. A recombinant DNA construct comprising the recombinant DNA molecule as claimed in claims 1-5, operably linked to a heterologous promoter, preferably T7 promoter.

7. The recombinant DNA construct as claimed in claim 6, comprising a sequence selected from SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, or SEQ ID NO: 19.

8. A recombinant vector comprising the recombinant DNA construct as claimed in any one of claims 1-7.

9. The recombinant vector as claimed in claim 8, further comprising at least one origin of replication and one or more selectable markers.

10. A recombinant host cell comprising the recombinant DNA construct as claimed claim 6 or 7, or the recombinant vector as claimed in claim 8 or 9.

11. An mRNA molecule transcribed from the recombinant DNA molecule as claimed in any one of claims 1 to 5, the recombinant DNA construct as claimed in claim 6 or 7 or the recombinant vector as claimed in claim 8 or 9.

12. The mRNA molecule as claimed in claim 11, comprising one or more modified nucleotides.

13. The mRNAmolecule as claimed in claim 11 or 12, wherein the mRNAmolecule further comprises a 5’ cap structure and / or polyadenylated tail.

14. The mRNA molecule as claimed in any of the preceding claims, wherein the mRNA molecule is formulated in a delivery system.

15. A delivery system comprising the mRNA molecule as claimed in any one of claims 11 to 13.

16. The delivery system as claimed in claim 15, wherein the delivery system is selected from lipid nanoparticles, liposomes, polymeric nanoparticles, or viral vectors, preferably a lipid nanoparticle (LNP).

17. A process of preparing the mRNA molecule as claimed in claims 11 to 13, comprising amplifying the recombinant DNA molecule as claimed in claims 1 to 5 to obtain a plurality of DNA molecules, transcribing the plurality of DNA molecules to obtain a plurality of mRNA molecules, and optionally modifying the mRNA molecules.

18. The process as claimed in claim 17, wherein said amplifying the recombinant DNA molecule is by a method selected from Polymerase Chain Reaction (PCR), Loop- Mediated Isothermal Amplification (LAMP), Recombinase Polymerase Amplification (RPA), or by culturing the recombinant host cell as claimed in claim 10 in a suitable culture media.

19. The process as claimed in claim 17, further comprising a step of purifying the mRNA molecules.

20. A pharmaceutical composition comprising the mRNA molecule as claimed in any one of claims 11 to 14, and a pharmaceutically acceptable carrier.

21. A method of treating or managing symptoms of Gaucher disease in a subject, comprising administering a therapeutically effective amount of mRNA molecule as claimed in claims 11 to 14, the delivery system as claimed in claim 15 or 16 or the pharmaceutical composition as claimed in claim 20, to the subject.

22. The method as claimed in claim 17, wherein administering is via a route selected from intramuscular, subcutaneous, intradermal, intravenous, intranasal, or combinations thereof.

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