DESC:FORM 2
THE PATENTS ACT, 1970
(39 of 1970)
&
THE PATENTS RULES, 2003
COMPLETE SPECIFICATION
(Section 10 and Rule 13)

1. TITLE OF THE INVENTION:
MRNA BASED ENZYME PRECURSOR AND PREPARATION METHOD THEREOF

2. APPLICANT:
MICRO CRISPR Pvt. Ltd., an Indian Company, of the address Survey No: 1574, Muktanand Marg, Chala, Vapi-396191, Gujarat, India

3. The following specification particularly describes the invention and the manner in which it is to be performed:

FIELD OF INVENTION
[1] The present disclosure relates to an enzyme replacement therapy. More particularly, the present disclosure relates to an mRNA-based enzyme precursor for treating phenylketonuria.
BACKGROUND OF INVENTION
[2] Metabolic pathways in living organisms have complex biochemical processes to maintain cellular activities which are vital to sustain life. Few of the specific metabolic pathways inside the body are necessary processes which play primary functions to maintain daily life activities. Each pathway depends on pre-defined substrates and corresponding enzymes to ensure smooth functioning.
[3] Metabolic disorders like Inborn Error of Metabolism (IEMs) results in deficiency of a single enzyme activity in metabolic pathways. IEMs are inherited in an autosomal recessive manner. One of such rare inherited disorders is Phenylketonuria (PKU). Phenylketonuria (PKU) is caused by deficiency of the hepatic enzyme phenylalanine hydroxylase (PAH). Phenylalanine hydroxylase (PAH) is responsible for the conversion of phenylalanine (Phe) to tyrosine (Tyr) in the presence of the co-factor, tetrahydrobiopterin (BH4). Accumulation of Phe in blood and organs leads to high level of Phe in blood that is neurotoxic and lead to reduced cognitive development, may also lead to developmental delay and neurological impairment. This may lead to irreversible mental retardation, intellectual disability, seizures, behavioral, movement and psychiatric problems. In some patients it may also result in a musty smell and lighter skin. Newborns with poorly treated PKU may sometimes lead to heart problems, a small head and low birth weight. Also, presence of higher amount of Phe in the blood stream may get accumulated in brain causing detrimental effects on brain development and function.
[4] While there is no cure for PKU, some currently available treatments for PKU includes dietary restrictions, tetrahydrobiopterin (BH4) therapy, gene therapy and treatment using large neutral amino acids, Glycomacropeptides (GMP) and recombinant adeno-associated virus vector. However, these treatments have major issues leading to potential nutritional deficiencies, residual enzyme activity and genotoxicity.
[5] Thus, there arises a need to develop a new therapy to replace or supplement current dietary treatments and overcome the drawbacks associated with the conventional treatments.
SUMMARY OF INVENTION
[6] 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.
[7] In an exemplary embodiment, the present disclosure relates to a recombinant construct including a vector and a recombinant nucleic acid molecule. The vector including at least one promoter region. The recombinant nucleic acid molecule is encoded at least by SEQ ID No. 1. The recombinant nucleic acid molecule is disposed downstream of the at least one promoter region to enable transcription of the recombinant nucleic acid molecule by the promoter region to a plurality of messenger ribonucleic acid (mRNA) molecules encoded by SEQ ID No. 9.
[8] In another exemplary embodiment, the present disclosure relates to a transcript of a recombinant nucleic acid molecule as described above. The transcript including the messenger ribonucleic acid (mRNA) molecule encoded by SEQ ID No. 9. The mRNA molecule encodes for a phenylalanine hydroxylase (PAH) enzyme.
[9] In another exemplary embodiment, the present disclosure relates to an enzyme precursor including a composition of a plurality of mRNA molecules as described above.
[10] In yet another exemplary embodiment, the present disclosure relates to a method to prepare a precursor by ligating at least one recombinant nucleic acid molecule encoded at least by SEQ ID NO. 1 to a vector to obtain a plurality of recombinant circular constructs as described above. The plurality of recombinant circular constructs is digested using at least one restriction enzymes to obtain a plurality of recombinant linear constructs as described above. The recombinant linear constructs are transcribed to obtain a plurality of transcripts as described above.
BRIEF DESCRIPTION OF DRAWINGS
[11] 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.
[12] Fig. 1 depicts a method 100 to prepare an enzyme precursor, according to an embodiment of the present disclosure.
[13] Fig. 1a depicts a recombinant circular construct 10, according to an embodiment of the present disclosure.
[14] Fig. 1b depicts a recombinant linear construct 10a, according to an embodiment of the present disclosure.
[15] Figs. 2 - 6 depict experimental observations associated with the enzyme precursor, according to one or more exemplary embodiments of the present disclosure.
DETAILED DESCRIPTION OF THE DRAWINGS
[16] 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.
[17] 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.
[18] 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.
[19] 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.
[20] The present disclosure relates an enzyme precursor (or precursor) to treat and/or manage phenylketonuria (PKU).
[21] The precursor includes a composition containing at least a plurality of messenger RNA (mRNA) molecules that encodes one or more polypeptides and/or proteins of the phenylalanine hydroxylase (PAH) enzyme. The mRNAs are transcripts of a recombinant nucleic acid molecule encoding the one or more polypeptides and/or proteins of the PAH enzyme. The polypeptide(s) and/or protein(s) restores activity of PAH enzyme thereby managing symptoms of PKU in an individual receiving the precursor. The PAH enzyme, inside the body, catalyzes the conversion of phenylalanine (Phe) to tyrosine (Tyr).
[22] Additionally or optionally, a delivery system (or a carrier system) may be used to deliver the precursor. For example, the delivery system may be a plurality of lipid nanoparticles.
[23] 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 molecule 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.
[24] Now referring to the figures, Fig. 1 depicts an exemplary method 100 to prepare a precursor.
[25] The method 100 commences at step 101, where at least one recombinant nucleic acid molecule 1 is ligated to a vector to obtain a plurality of recombinant circular constructs 10 (as shown in Fig. 1a). The recombinant circular construct 10 is a substantially circular shaped polynucleotide molecule. The recombinant nucleic acid molecule 1 encodes for messenger RNA (mRNA) molecule that translates to one or more polypeptides and/or proteins of the phenylalanine hydroxylase (PAH) enzyme. The recombinant nucleic acid molecule 1 may be a single stranded DNA (ssDNA) or a double stranded DNA (dsDNA) encoded at least by SEQ ID NO. 1. In an exemplary embodiment, the recombinant nucleic acid molecule 1 has a dsDNA structure. In an exemplary embodiment, the recombinant nucleic acid molecule 1 is synthetically synthesized (from Genscript).
[26] The recombinant nucleic acid molecule 1 may optionally include non-coding regions (for example, untranslated regions (UTRs) and introns). In an exemplary embodiment, the recombinant nucleic acid molecule 1 does not include any non-coding regions. In an alternate embodiment, the recombinant nucleic acid molecule 1 is provided with a 3’ UTR and 5’ UTR. The UTRs of the recombinant nucleic acid molecule 1 are transcribed to the mRNA molecule but are not translated into the protein. The 5’ UTR is located upstream of the start codon of the mRNA molecule and the 3’ UTR is located downstream of the stop codon. The UTRs are important for translation Initiation, ribosome binding and stability of the mRNA molecule.
[27] The polypeptides and/or proteins obtained from the recombinant nucleic acid molecule 1 is the PAH enzyme.
[28] The vector may include at least one origin of replication region 11, at least one promoter region 13, one or more selectable markers 15, a plurality of restriction sites (i.e., pre-defined nucleotide sequences that are recognized by restriction enzymes), etc. The origin of replication region 11 helps the vector to replicate inside a host cell. Alternatively, the vector may include only the promoter region 13.
[29] The promoter region 13 may have a binding affinity to at least one RNA polymerase enzyme including at least one of T7 RNA polymerase, T3 RNA polymerase or SP6 RNA polymerase. The RNA polymerase enzymes help to produce mRNA molecules from the recombinant nucleic acid molecule 1 via transcription. The promoter region 13 may be at least one of T7 promoter encoded by SEQ ID No. 2 or SEQ ID No. 3, T3 promoter encoded by SEQ ID No. 4, or SP6 promoter encoded by SEQ ID No. 5, etc. The recombinant nucleic acid molecule 1 is disposed downstream of the at least one of the promoter region 13 to enable transcription of the recombinant nucleic acid molecule 1 by the promoter region 13. In an exemplary embodiment, the promoter region 13 includes T7 promoter encoded by SEQ ID No. 3.
[30] The selectable markers 15 may include resistance gene(s) of, for example, ampicillin encoded by SEQ ID No. 6, kanamycin encoded by SEQ ID No. 7, etc. In an exemplary embodiment, the vector includes resistance gene of kanamycin as the selectable marker 15. The selectable marker 15 may be disposed downstream of at least one second promoter region 13a that facilitates the expression of the resistance gene(s). The selectable marker 15 helps the host cell having the vector to resist the effects of the corresponding antibiotic.
[31] In an exemplary embodiment, the vector is pUC57 encoded by SEQ ID No. 8 (procured from Genescript) having a T7 promoter and kanamycin resistance genes.
[32] In an exemplary embodiment, two free ends of the recombinant nucleic acid molecule 1 is ligated to the (linearized) vector to obtain the plurality of recombinant circular constructs 10. The recombinant nucleic acid molecule 1 may be ligated with the vector using a ligase enzyme, for example, T4 DNA ligase (procured from New England Biolabs) by following manufacturer’s protocol. In an exemplary embodiment, the recombinant nucleic acid molecule 1 is ligated to the vector such that the promoter region 13 of the vector is disposed upstream of the recombinant nucleic acid molecule 1.
[33] Additionally or optionally, the recombinant nucleic acid molecule 1 and the vector may be digested with the help of one or more restriction enzymes to create sticky overhangs. The sticky overhangs allow the recombinant nucleic acid molecule 1 to correctly orient itself with respect to the vector. In an exemplary embodiment, the recombinant nucleic acid molecule 1 is digested with NotI (procured from New England Biolabs) by following manufacturer’s protocol. In another exemplary embodiment, the recombinant nucleic acid molecule 1 is digested with BspQ1 (procured from New England Biolabs) by following manufacturer’s protocol.
[34] At an optional step 103, the recombinant circular constructs 10 obtained from step 101 are amplified to increase their number. Increasing the number of recombinant circular constructs 10 is one of the factors to increase the yield of the mRNA molecules, i.e., the enzyme precursor.
[35] In an exemplary embodiment, the recombinant circular constructs 10 are amplified using natural replication mechanism of a competent host cell. The competent host cell may include Escherichia coli (E. coli) DH5a, E. coli DH1, E. coli C600 or strains thereof. In an exemplary embodiment, the host cell is E. coli DH5a.
[36] The recombinant circular constructs 10 are introduced inside the competent host cell via a technique selected from electroporation, heat shock, etc. In an exemplary embodiment, the recombinant circular constructs 10 are electroporated inside the competent host cells using an electroporator (procured from Thermo Scientific) by following manufacturer’s protocol to obtain a plurality of transformed host cells. The transformed host cells replicate the recombinant circular constructs 10 to amplify them, i.e., to increase the number of recombinant circular constructs 10 per transformed host cell.
[37] The transformed host cells are cultured at a pre-defined temperature and predefined rotations per minute (RPM) for at least a few hours, to increase their number. In an exemplary embodiment, the transformed host cells are cultured at 37 °C by shaking at 120 - 150 RPM inside a shaking incubator.
[38] The transformed host cells may be cultured in a pre-defined nutrient medium supplemented 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 15. In an exemplary embodiment, the nutrient medium is supplemented with 50 mg/mL of kanamycin (procured from Duschefa). Other functionally equivalent nutrient 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.
[39] 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.
[40] At step 103a, the plurality of recombinant circular constructs 10 are extracted from the transformed host cell(s). 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 techniques such as alkaline lysis. Other functionally equivalent technique to obtain the cell lysate is within the scope of the teachings of the present disclosure.
[41] 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 column-based purification (procured from Qiagen) by following manufacturer’s protocol.
[42] At step 105, the plurality of recombinant circular constructs 10 are digested to obtain a plurality of recombinant linear constructs 10a (as shown in Fig. 1b). Similar to the recombinant circular construct 10, the recombinant linear construct 10a includes the recombinant nucleic acid molecule 1, the origin of replication region 11, the promoter region 13, the selectable marker 15, the second promoter region 13a, the plurality of 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 the present disclosure.
[43] The recombinant circular constructs 10 may be digested using at least one restriction enzymes based on the vector used to create the recombinant circular constructs 10. The restriction enzyme is at least one of HindIII, KpnI, NotI, BamH1, EcoRI, and BspQI. The recombinant nucleic acid molecule 1 is disposed at an end of the recombinant linear construct 10a. And, the promoter region 13 of the vector is disposed upstream of the recombinant nucleic acid molecule 1 in the recombinant linear construct 10a.
[44] In an exemplary embodiment, the recombinant circular constructs 10 are digested using NotI (procured from New England Biolabs) by following the manufacturer’s protocol.
[45] At step 107, the recombinant linear constructs 10a are transcribed to obtain a plurality of transcripts. The transcript including the messenger RNA (mRNA) molecule of the recombinant nucleic acid molecule 1.
[46] In an exemplary embodiment, the recombinant linear constructs 10a are transcribed using an in vitro transcription technique. The recombinant linear construct 10a may be added to a buffer containing at least a pre-defined amount of at least one RNA polymerase and a plurality of nucleotide triphosphates (NTPs). The pre-defined amount of RNA polymerase ranges from 5000 U/mL to 20000 U/mL. In an exemplary 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 0.4 mM to 0.5 mM. The buffer may have a pre-defined pH ranging from 7.5 to 8.0. In an exemplary embodiment, the buffer is T7 polymerase reaction buffer (procured from New England Biolabs) having a pH of 8.0. The buffer along with the recombinant linear constructs 10a may be maintained at a pre-defined temperature ranging from 37 °C to 55 °C for a pre-defined time period ranging from 10 min to 180 min. In an exemplary embodiment, the buffer along with the recombinant linear constructs 10a is maintained at 37 °C for 120 mins. The RNA polymerase binds with the promoter region 13 of the recombinant linear construct 10a and transcribes the recombinant nucleic acid molecule 1 disposed downstream of the promoter region 13. The RNA polymerase polymerizes the NTPs to produce the mRNA molecules, and the sequence of the mRNA molecules correspond to the recombinant nucleic acid molecule 1. In an exemplary embodiment, the mRNA molecule is encoded by SEQ ID NO. 9. 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.
[47] Additionally or optionally, the mRNA molecules produced at step 107 includes at least one of one or more modified nucleotides, a 5’ cap, and a 3’ poly A tail. The modified nucleotide may be at least one of 2-thiouridine (s2U), pseudouridine (?), N1-methylpseudouridine (m1?), N6-methyladenosine (m6A) and 5-methylcytosine (m5C). The modified nucleotides help to improve stability and translation efficiency of the mRNA molecule.
[48] 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 Cap1 structure. The 5’ cap helps in efficient translation, stabilization, and transportation of mRNA molecules in eukaryotic cells.
[49] The 3’ 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 has a length ranging from 80 nucleotide to 150 nucleotide of adenosine. The poly A tail helps in maintaining stability of the mRNA molecule, prevention of degradation of the mRNA molecule and efficient translation of the mRNA molecule.
[50] At an optional step 109, the mRNA molecule obtained at the step 107 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 affinity chromatography by using POROS™ Oligo dT(25) GoPure™ Column (procured from Thermo Scientific) by following the manufacturer’s protocol.
[51] The mRNA molecule obtained from the method 100 is the precursor that may be administered to an individual to manage symptoms of and/or treat PKU. The composition containing the mRNA molecule(s) may be administered to an individual via any technique selected from intramuscular, subcutaneous, intravenous, or a combination thereof. Based on the toxicity and safety criteria, a route of administration may be decided for an individual.
[52] 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 LNPs are then injected within an individual. The LNPs may have an affinity towards hepatocytes, enabling targeted delivery of the mRNA molecules to the liver. The LNPs delivers the mRNA molecule within the liver cells of the individual where the mRNA molecules are translated to a plurality of polypeptides and/or proteins. The polypeptides and/or proteins catalyzes the conversion of phenylalanine (Phe) to tyrosine (Tyr) thereby managing symptoms and/or treating PKU in an individual. In other words, the mRNA molecule encodes for the phenylalanine hydroxylase (PAH) enzyme.
[53] An exemplary lipid nanoparticle is described in Indian Patent application number 202321061936, which is incorporated herein by reference and will form a part of this specification as if set forth herein in their entirety.
[54] 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.
[55] The precursor disclosed above will now be described with the help of the following examples.
[56] Example 1: Method to prepare the enzyme precursor of the present disclosure
[57] A recombinant nucleic acid molecule 1 encoded by SEQ ID NO. 1 was ligated to pUC57 vector using T4 ligase enzyme (procured from New England Biolabs) by following manufacturer’s protocol to obtain the recombinant circular construct 10. The recombinant circular construct 10 was then electroporated into competent E. coli DH5a cells using an electroporator (procured from Thermo Scientific) by following manufacturer’s protocol. The transformed E. coli cells were suspended in Luria-Bertani (LB) broth (procured from Himedia) having 1% kanamycin (procured from Duschefa) and incubated for 16 hours in a shaker incubator. The shaker incubator was maintained at a temperature of 37 °C and at 140 RPM. After 16 hours, the transformed E. coli cells were centrifuged at 6000 x g and lysed using alkaline lysis method. The cell lysate was subjected to column purification (procured from Qiagen) by following the manufacturer’s protocol to get the purified recombinant circular constructs 10. The purified recombinant circular construct 10 was then linearized using 5-10 U/µg the recombinant circular constructs 10 of NotI enzyme (procured from New England Biolabs) by following manufacturer’s protocol 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 recombinant linear constructs 10a suspended in the T7 polymerase reaction buffer was incubated at 37 °C for 120 minutes. The T7 polymerase reaction buffer was then subjected to column purification (procured from Thermo Scientific) by following manufacturer’s protocol to get purified mRNA molecules. 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. 9.
[58] Example 2: Administration of the precursor of the present disclosure to HEK293T cells
[59] A first group of 0.5 x 106 to 0.6 x 106 human embryonic kidney (HEK293T) cells (procured from American Type Culture Collection) were grown in Dulbecco's Modified Eagle Medium (DMEM) (procured from Gibco) supplemented with 10% (w/v) fetal bovine serum (FBS) (procured from Gibco) for 24 hours, at 37°C under 5% CO2. The HEK293T cells were transfected with different concentrations (0.5 µg, 1.0 µg, 1.5 µg, 2.0 µg, and 3.0 µg) of the mRNA molecule 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.
[60] A second group of 0.5 x 106 to 0.6 x 106 human embryonic kidney (HEK293T) cells (procured from American Type Culture Collection) were grown in Dulbecco's Modified Eagle Medium (DMEM) (procured from Gibco) supplemented with 10% (w/v) fetal bovine serum (FBS) (procured from Gibco) for 24 hours, at 37°C under 5% CO2. Different concentrations (0.5 µg, 1.0 µg, 1.5 µg, 2.0 µg, and 3.0 µg) of the mRNA molecules obtained from Example 1 above was encapsulated in lipid nano particles (LNPs) to obtain formulations of mRNA-LNPs (as described in Indian Patent Application No. 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.
[61] Example 3: Estimation of the level of expression of PAH enzyme in HEK293T cells via Western blot analysis
[62] 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 Biorad). The PVDF membrane was then blocked with 5% bovine serum albumin (BSA) (procured from MP Biomedicals) prepared in 1X tris-buffered saline with tween 20 (TBST) buffer.
[63] The membranes were incubated for 2 hours at room temperature with primary antibodies. The primary antibodies used was anti-PAH (procured from Invitrogen) at 1:4000 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 TBST and then again incubated for 2 hours at room temperature with horseradish peroxidase (HRP)-conjugated secondary antibodies. The secondary antibodies used was Goat Anti-Rabbit IgG (H + L)-HRP Conjugate (procured from Biorad) at 1:4000 dilution in 1% BSA and Goat Anti-Mouse IgG (H + L)-HRP Conjugate (procured from Biorad) at 1:4000 dilution in 1% BSA. The PAH protein and the ß-Actin 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).
[64] Fig. 2 depicts the western blot analysis of the Group A of cells and Fig. 3 depicts the western blot analysis of the Group B of cells. The PAH protein had an average molecular weight of 52 kD and the ß-Actin protein had an average molecular weight of 42 kD. The first lane in the figure depicts the control (i.e., untransfected cells) and the subsequent lane depicts the different concentrations (0.5 µg, 1.0 µg, 1.5 µg, 2.0 µg, and 3.0 µg) of the mRNA molecule used in Example 2 above. In Fig. 3, the second lane represents the LNP formulation without any mRNA molecule (i.e., negative control).
[65] It was confirmed from the western blot analysis that the mRNA molecule was introduced in the HEK293T cells and translated into PAH protein. The expression of the mRNA molecule (relative to the respective band intensity in the Fig. 2 and 3) increased with increase in the concentration of the mRNA molecule introduced inside the HEK293T cells.
[66] Example 4: Time dependent expression analysis of the mRNA molecule in HEK293T Cells and estimation of phenylalanine level in the media
[67] The Group B of cells transfected with the formulation of 3 µg of the mRNA molecule encapsulated in LNP (mRNA-LNP) and LNP without any mRNA molecule (i.e. negative control) were harvested after 24 hours, 48 hours, and 72 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. 4 depicts the western blot analysis. The analysis confirmed that the expression of the mRNA molecule, and simultaneously the level of PAH protein increases up to 48 hours.
[68] Along with the HEK293T cells, the media was collected after 24 hours, 48 hours, and 72 hours post-transfection. The media was used for phenylalanine level detection using the Phenylalanine Assay Kit (procured from Sigma Aldrich) by following the manufacturer’s protocol. The media was also used for estimating total protein using the bicinchoninic acid (BCA) reagent (procured from Merck) by following the manufacturer’s protocol. Thereafter, the level of phenylalanine detected was normalized with the amount of total protein and plotted as shown in Fig. 4a. The plot confirmed a significant reduction in phenylalanine levels at all time points in transfected HEK293T cells (***p < 0.001 at 24h and 48h, *p < 0.05 at 72h), confirming PAH-mediated phenylalanine metabolism. The data points in the plot are presented as mean ± standard deviation, with statistical significance indicated (*p < 0.05, ***p < 0.001).
[69] Example 5: Time dependent expression analysis of the mRNA molecule in HepG2 Cells, cell viability analysis and estimation of phenylalanine level in the media
[70] A group of 0.5 x 106 to 0.6 x 106 HepG2 cells (procured from ATCC) were grown in MEM-a media (procured from Gibco) supplemented with 10% (w/v) fetal bovine serum (FBS) (procured from Gibco) for 24 hours, at 37°C under 5% CO2. The HepG2 cells were transfected with a formulation of 3 µg of the mRNA molecule encapsulated in Lipid Nanoparticles (LNPs) formulation by incubating the cells with the mRNA-LNP formulation at 37 °C. A LNP formulation without any mRNA molecule was used as the negative control.
[71] Transfected HepG2 cells were harvested after 24 hours, 48 hours, and 72 hours post-transfection. The lysates of the harvested HepG2 cells were obtained and analyzed via western blot analysis as described in Example 3 above. Fig. 5 depicts the western blot analysis. The analysis confirmed that the expression of the mRNA molecule, and detection of the PAH protein up to 72 hours.
[72] Along with the HepG2 cells, the media was collected after 24 hours, 48 hours, and 72 hours post-transfection. The media was used for phenylalanine level detection as described in Example 4 above. The level of phenylalanine detected was normalized with the amount of total protein and plotted as shown in Fig. 5a. The plot confirmed a significant reduction in phenylalanine levels at all time points in transfected HEK293T cells (***p < 0.001), confirming PAH-mediated phenylalanine metabolism. The data points in the plot are presented as mean ± standard deviation, with statistical significance indicated (***p < 0.001).
[73] The PAH enzyme levels were detected in the lysates of the harvested HepG2 cells after 0 hour (control), 24 hours, 48 hours, and 72 hours post-transfection using the PAH ELISA Kit (procured from Abcam) by following manufacturer’s protocol. The PAH enzyme levels were normalized with the total protein amount for each sample. The PAH enzyme levels was plotted as unit ng of PAH protein per unit mg of total protein as shown in Fig. 5b. As shown in the plot of Fig. 5b, there was a significant increase in PAH expression at 24h, peaking at 48h, and decreasing at 72h. The data points in the plot are presented as mean ± standard deviation, with statistical significance indicated (*p < 0.05, **p < 0.01, ***p < 0.001).
[74] Cell viability of the HepG2 cells harvested after 0 hour, 24 hours, 48 hours, and 72 hours post-transfection was performed by Cell Proliferation Kit I (MTT) (procured from Roche) by following manufacturer’s protocol. The cell viability was plotted as shown in Fig. 5c. As shown in Fig. 5c, the viability remains above 80% at all time points, with a slight but statistically significant decrease at 72h compared to earlier time points (*p < 0.05, **p < 0.01). This suggests that transfection of the mRNA molecule does not significantly impact HepG2 cell viability over the observed time period. The data points in the plot are presented as mean ± standard deviation, with statistical significance indicated (*p < 0.05, **p < 0.01, ***p < 0.001).
[75] Example 6: Pharmacodynamics (PD) studies of the mRNA-LNP formulation in humanized Phenylketonuria (PKU) mice model
[76] The pharmacodynamics (PD) studies were conducted to evaluate the efficacy of the mRNA-LNP formulation in reducing phenylalanine levels in the plasma of a humanized PKU mice model (BTBR-Pahenu2/J mice). The PKU mice were tail bled, and the phenylalanine levels in the plasma (pre-bleed) were measured using the Phenylalanine Assay Kit (procured from Abcam) by following the manufacturer’ protocols. The formulation of the mRNA-LNPs were administered to the male (M) and female (F) homozygous PKU mice via intravenous (IV) and subcutaneous (SC) routes at doses of 0.5 mg/kg and 1.0 mg/kg. The negative controls used were homozygous PKU mice model (Sham) and heterozygous PKU mice model (Het) injected with blank/empty LNPs. The phenylalanine levels in the plasma were again measured after 24 hours using the Phenylalanine Assay Kit (procured from Abcam) by following the manufacturer’s protocol. A C57BL6 mice was used as the wild-type to compare the phenylalanine levels in the plasma.
[77] The phenylalanine levels (%) in the plasma (compared to Sham Empty LNP) were plotted as shown in Fig. 6. As shown in Fig. 6, the level of phenylalanine, after 24 hours of administration of the formulation of mRNA-LNP intravenously, were observed to be equivalent to that of the wild type mice, with levels significantly (p<0.05) lower than those in pre-bleed, Sham, and Het PKU mice. Whereas in case of the mice administered via subcutaneous route, the expression efficacy was low compared to intravenous by about 60%, suggesting limited efficacy of this route of administration at these doses. The data points in the plot are presented as mean ± standard deviation (n=3).
[78] 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 recombinant construct, comprising:
a. a vector including at least one promoter region (13); and
b. a recombinant nucleic acid molecule (1) encoded at least by SEQ ID No. 1, the recombinant nucleic acid molecule (1) is disposed downstream of the at least one promoter region (13) to enable transcription of the recombinant nucleic acid molecule (1) by the promoter region (13) to a plurality of messenger ribonucleic acid (mRNA) molecules encoded by SEQ ID No. 9.
2. The recombinant construct as claimed in claim 1, wherein the vector further includes at least one origin of replication region (11), one or more selectable markers (15), and a plurality of restriction sites.
3. The recombinant construct as claimed in claim 1, wherein the promoter region (13) is at least one of T7 promoter encoded by SEQ ID No. 2 or 3, T3 promoter encoded by SEQ ID No. 4, or SP6 promoter encoded by SEQ ID No. 5.
4. The recombinant construct as claimed in claim 2, wherein the one or more selectable markers (15) include resistance gene(s) of ampicillin encoded by SEQ ID No. 6, or kanamycin encoded by SEQ ID No. 7.
5. The recombinant construct as claimed in claim 2, wherein the one or more selectable markers (15) is disposed downstream of at least one second promoter region (13a) that facilitates the expression of a resistance gene.
6. The recombinant construct as claimed in claim 1, wherein the vector is pUC57 encoded by SEQ ID No. 8 having a T7 promoter and a kanamycin resistance gene.
7. The recombinant construct as claimed in claim 1, wherein recombinant nucleic acid molecule 1 may be a single stranded DNA (ssDNA) or a double stranded DNA (dsDNA).
8. The recombinant construct as claimed in any of the preceding claim 1-7, wherein the recombinant construct is a recombinant circular construct (10), the recombinant circular construct (10) is a substantially circular shaped polynucleotide molecule.
9. The recombinant construct as claimed in any of the preceding claim 1-7, wherein the recombinant construct is a recombinant linear construct (10a), the recombinant linear construct (10a) is a substantially linear shaped polynucleotide molecule.
10. A transcript of a recombinant nucleic acid molecule (1) as claimed in any of the preceding claims, the transcript comprising the messenger ribonucleic acid (mRNA) molecule encoded by SEQ ID No. 9, the mRNA molecule encodes for a phenylalanine hydroxylase (PAH) enzyme.
11. The transcript as claimed in claim 10, wherein the mRNA molecule includes one or more modified nucleotides, the modified nucleotide is at least one of 2-thiouridine (s2U), pseudouridine (?), N1-methylpseudouridine (m1?), N6-methyladenosine (m6A) and 5-methylcytosine (m5C).
12. The transcript as claimed in claim 10, wherein the mRNA molecule includes at least one of a 5’ cap, and a 3’ poly A tail.
13. An enzyme precursor comprising a composition of a plurality of mRNA molecules as claimed in any of the claims 10-12.
14. The enzyme precursor as claimed in claim 13, wherein the plurality of mRNA molecules is encapsulated within a plurality of lipid nanoparticles (LNPs).
15. A method (100) to prepare a precursor, comprising:
a. ligating at least one recombinant nucleic acid molecule (1) encoded at least by SEQ ID NO. 1 to a vector to obtain a plurality of recombinant circular constructs (10) as claimed claim 8;
b. digesting the plurality of recombinant circular constructs (10) using at least one restriction enzymes to obtain a plurality of recombinant linear constructs (10a) as claimed in claim 9; and
c. transcribing the recombinant linear constructs (10a) to obtain a plurality of transcripts as claimed in any of the claims 10-12.
16. The method (100) as claimed in claim 15, wherein after obtaining the plurality of recombinant circular constructs (10) at step a., the method (100) includes amplifying the plurality of recombinant circular constructs (10) to increase their number.
17. The method (100) as claimed in claim 15, wherein after obtaining the plurality of recombinant circular constructs (10) at step a., the method (100) includes
a. introducing the plurality of recombinant circular constructs (10) inside a competent host cell(s) to obtain a transformed host cell(s),
b. the transformed host cells are cultured in a pre-defined nutrient medium supplemented with at least one antibiotic at a pre-defined temperature for at least a few hours, to increase their number, and
c. extracting the plurality of recombinant circular constructs (10) from the transformed host cell(s).
18. The method (100) as claimed in claim 15, wherein after obtaining the plurality of transcripts, the method (100) includes purifying the mRNA molecules using a purification technique selected from one of bead-based techniques, cellulose based chromatography, and precipitation.
19. The method (100) as claimed in claim 15, wherein after obtaining the plurality of transcripts, the method (100) includes encapsulating the mRNA molecules within a plurality of lipid nanoparticles (LNPs).