[go: up one dir, main page]

WO2025126242A1 - Nucleic acid sequence(s) and construct(s) for treating methylmalonic acidemia and method(s) thereof - Google Patents

Nucleic acid sequence(s) and construct(s) for treating methylmalonic acidemia and method(s) thereof Download PDF

Info

Publication number
WO2025126242A1
WO2025126242A1 PCT/IN2024/052370 IN2024052370W WO2025126242A1 WO 2025126242 A1 WO2025126242 A1 WO 2025126242A1 IN 2024052370 W IN2024052370 W IN 2024052370W WO 2025126242 A1 WO2025126242 A1 WO 2025126242A1
Authority
WO
WIPO (PCT)
Prior art keywords
seq
mrna
utr
sequence
group
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
PCT/IN2024/052370
Other languages
French (fr)
Inventor
Aarti SEVILIMEDU
Rita RANI
Kishore PARSA
Kiranam Chatti
Srinivas ORUGANTI
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Dr Reddy's Institute Of Life Sciences
Original Assignee
Dr Reddy's Institute Of Life Sciences
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Dr Reddy's Institute Of Life Sciences filed Critical Dr Reddy's Institute Of Life Sciences
Publication of WO2025126242A1 publication Critical patent/WO2025126242A1/en
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/005—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
    • C12N2750/00011—Details
    • C12N2750/14011—Parvoviridae
    • C12N2750/14111—Dependovirus, e.g. adenoassociated viruses
    • C12N2750/14141—Use of virus, viral particle or viral elements as a vector
    • C12N2750/14143—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector

Definitions

  • the present disclosure relates to the technical field of biological therapeutics, in particular to nucleic acid therapeutics i.e. DNA and mRNA therapeutics, method(s) or application(s) thereof and a kit.
  • Methylmalonic acidemia refers to a group of inborn errors of metabolism caused by defects in multiple genes resulting in the accumulation of methyhnalonate or 5’- deoxyadenosylcobalamin.
  • the prevalence of treating Methylmalonic acidemia (MMA) is estimated to be between 1:25,000- 1:50,000 with detection rates of less than 2: 100,000.
  • Mutations in the gene MMUT (methyl malonyl-coenzyme A mutase) are the most common cause of this disorder, referred to as isolated MMA.
  • MMA continues to be a significant cause of morbidity and mortality because of the multisystem damage during the multiple metabolic crises faced by these patients.
  • liver transplantation is increasingly being used to alleviate recurrent metabolic strokes in patients with severe disease. While this supports the idea that addressing MMUT deficiency in the liver alone could be an effective therapy, LT is not an affordable or risk-free solution for all patients, and therefore there is need for efficacious disease modifying treatments with a better prognosis.
  • Replacement therapies in the form of protein are considered the closest to a “cure” for monogenic disorders.
  • ERT enzyme replacement therapies
  • MMUT is a mitochondrial enzyme
  • protein ERT is unsuitable.
  • IVTT mRNA In vitro transcribed messenger RNA (IVT mRNA) has always been considered an excellent option for protein replacement, as they do not need to be delivered to the nucleus, can be translated instantaneously. With the recent breakthroughs in LNP mediated delivery, mRNA candidates have progressed rapidly through preclinical as well as early clinical trials.
  • Adeno-associated virus Adeno-associated virus
  • the present disclosure provides an mRNA sequence comprising a CDS bearing at least about 90% identity to a sequence represented by SEQ ID No. 2.
  • an mRNA construct comprising the mRNA sequence as described above or a fragment thereof, a 5’ cap, a 5'UTR element, a 3'UTR element, a poly(A) sequence and optionally, a natural or synthetic IRES element and/or a circularizing element.
  • the present disclosure further envisages a DNA template suitable for generating the mRNA as described above, the said DNA template comprising a CDS having sequence bearing at least about 90% identity to a sequence represented by SEQ ID No. 15 or a fragment thereof, one or more elements selected from a group comprising an AT -rich region, a promoter, a 5' untranslated region (UTR), an enhancer region, a terminator region, a 3'UTR, a polyA signal, and a restriction site.
  • a DNA template suitable for generating the mRNA as described above comprising a CDS having sequence bearing at least about 90% identity to a sequence represented by SEQ ID No. 15 or a fragment thereof, one or more elements selected from a group comprising an AT -rich region, a promoter, a 5' untranslated region (UTR), an enhancer region, a terminator region, a 3'UTR, a polyA signal, and a restriction site.
  • the present disclosure further provides a recombinant AAV vector comprising the DNA template described above. Also envisaged herein is a lipid nanoparticle (LNP) comprising the mRNA construct as described above.
  • LNP lipid nanoparticle
  • the present disclosure further provides a method of treating and/or preventing MMA, said method comprising administering the mRNA construct, the mRNA-LNP or the recombinant AAV vector or the composition as described above to a subject in need thereof.
  • Figure 1A shows the Codon Adaptation Index (CAI) of the mRNA of the present disclosure [SEQ ID NO. 2] (in green) vis-a-vis the wild-type sequence (in blue).
  • CAI Codon Adaptation Index
  • Figure 3 shows the Construct design and optimization of in vitro transcription and translation.
  • 3A shows the design of the construct used to generate linear mRNA, according to an exemplary embodiment.
  • 3B is representative gel image of in vitro transcribed capped polyadenylated mRNA for a reporter gene.
  • 3C shows the translation of a fluorescent reporter accompanying the mRNA construct of the present disclosure after transfection into cells.
  • 3D shows translation of a chemiluminescent reporter made with uridine or pseudouridine accompanying the mRNA construct of the present disclosure after transfection into cells.
  • 3E shows translation of a fluorescent reporter accompanying the mRNA construct of the present disclosure injected into a zebrafish embryo at 24 hours post injection.
  • Figure 4 shows A. Details of the noncoding and coding elements in various MMUT mRNA constructs generated and tested in vitro B. Details of the type of UTR elements used C: Details of the CDS sequences used in the constructs.
  • WT Wild-type (native H.s. MMUT), L-CO/CO: Liver-Codon optimized.
  • D-F Comparison of MMUT protein levels in WT HEK-293T cells as is (E), MMUT knockout HEK 293T cells (KO), and KO cells transfected with the indicated constructs (numbers in red). Immunoblotting analysis using the MMUT antibody and GAPDH antibody (used as a loading control) performed on cell lysates.
  • G Details of the noncoding and coding elements in various MMUT mRNA constructs generated and tested in vitro
  • Figure 5 shows a measurement of the levels of methylmalonic acid (MMA) in total zebrafish larval lysates by LC-MS in samples of injected and un-injected larvae. The figure shows that accumulation of MMA is reduced in the mRNA injectants, indicating functional activity of the injected mRNA.
  • MMA methylmalonic acid
  • Figure 6 shows expression levels of MMUT examined by western blotting after AAV constructs encoding MMUT were transfected into HepG2 cells.
  • MUT Metal-malonyl CoA mutase
  • MMUT mitochondrial enzyme that catalyzes the isomerization of methyhnalonyl-CoA to succinyl- CoA. Mutations in the MMUT gene are a common cause of Methylmalonic acidemia (MMA).
  • MMA Methylmalonic acidemia
  • nucleic acid therapeutics refer to therapeutic substances, drugs or compositions with nucleic acids as their therapeutically active component, such as DNA or RNA of all forms.
  • RNA Malignant RNA refers to any polynucleotide that encodes a (at least one) polypeptide (a naturally-occurring, non-naturally-occurring, or modified polymer of amino acids) and can be translated to produce the encoded polypeptide in vitro, in vivo, in situ or ex vivo.
  • mRNA Genetic RNA
  • polynucleotide sequences set forth in the instant application will recite “T”s in a representative DNA sequence but where the sequence represents RNA (e.g., mRNA), the “T”s would be substituted for “U”s.
  • any of the RNA polynucleotides encoded by a DNA identified by a particular sequence identification number may also comprise the corresponding RNA (e.g., mRNA) sequence encoded by the DNA, where each “T” of the DNA sequence is substituted with “U ”
  • RNA in vitro transcription refers to a process wherein RNA is synthesized in a cell-free system (in vitro).
  • DNA particularly plasmid DNA
  • RNA may be obtained by DNA-dependent in vitro transcription of an appropriate DNA template.
  • the present disclosure provides an mRNA construct comprising a CDS bearing at least about 90% identity to the sequence represented by SEQ ID No. 2 or a fragment thereof and a 3’UTR selected from a group comprising sequences represented by SEQ ID NO. 9, SEQ ID NO. 10, SEQ ID NO. 11, SEQ ID NO. 12, SEQ ID NO. 13 and SEQ ID NO. 14.
  • the present disclosure provides an mRNA construct comprising a CDS bearing at least about 90% identity to the sequence represented by SEQ ID No. 2 or a fragment thereof, a 5’ terminal cap structure selected from a group comprising m7Gppp5'N (CapO), m7Gppp5'Nm (Capl), chemically synthesized cap analogs such as ARCA (3'-O-Me-m7G(5') ppp(5')G) or CleanCap capping reagents such as CleanCapAG or CleanCapM6 or other commercially available or novel cap reagents that perform the same function, a 5 ’ UTR selected from a group comprising sequences represented by SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO.
  • SEQ ID NO. 6 SEQ ID NO. 7 and SEQ ID NO. 8
  • a 3’UTR selected from a group comprising sequences represented by SEQ ID NO. 9, SEQ ID NO. 10, SEQ ID NO. 11, SEQ ID NO. 12, SEQ ID NO. 13 and SEQ ID NO. 14.
  • the polyA tail may be contiguous or bipartite or of any other suitable design.
  • the present disclosure provides an mRNA construct comprising a CDS bearing at least about 90% identity to the sequence represented by SEQ ID No. 2 or a fragment thereof and a polyA tail comprising about 60 As to about 120 As.
  • the present disclosure provides an mRNA construct comprising a CDS bearing at least about 90% identity to the sequence represented by SEQ ID No. 2 or a fragment thereof, a 5’ terminal cap structure selected from a group comprising m7Gppp5'N (CapO), m7Gppp5'Nm (Capl), chemically synthesized cap analogs such as ARCA (3'-O-Me-m7G(5') ppp(5')G) or CleanCap capping reagents such as CleanCapAG or CleanCapM6 or other commercially available or novel cap reagents that perform the same function and a polyA tail comprising about 60 As to about 120 As.
  • a 5’ terminal cap structure selected from a group comprising m7Gppp5'N (CapO), m7Gppp5'Nm (Capl), chemically synthesized cap analogs such as ARCA (3'-O-Me-m7G(5') ppp(5')G) or CleanCap capping reagent
  • the present disclosure provides an mRNA construct comprising a CDS bearing at least about 90% identity to the sequence represented by SEQ ID No. 2 or a fragment thereof, a 5’ UTR selected from a group comprising sequences represented by SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO. 5, SEQ ID NO. 6, SEQ ID NO. 7 and SEQ ID NO. 8 and a polyA tail comprising about 60 As to about 120 As.
  • the present disclosure provides an mRNA construct comprising a CDS bearing at least about 90% identity to the sequence represented by SEQ ID No. 2 or a fragment thereof, a 3 ’UTR selected from a group comprising sequences represented by SEQ ID NO. 9, SEQ ID NO. 10, SEQ ID NO. 11, SEQ ID NO. 12, SEQ ID NO. 13 and SEQ ID NO. 14 and a polyA tail comprising about 60 As to about 120 As.
  • the present disclosure provides an mRNA construct comprising a CDS encoding the MMUT protein or a fragment thereof, a 5 ’ terminal cap structure selected from a group comprising m7Gppp5'N (CapO), m7Gppp5'Nm (Capl), chemically synthesized cap analogs such as ARCA (3'-O-Me-m7G(5') ppp(5')G) or CleanCap capping reagents such as CleanCapAG or CleanCapM6 or other commercially available or novel cap reagents that perform the same function, a 5 ’ UTR selected from a group comprising sequences represented by SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO. 5, SEQ ID NO.
  • SEQ ID NO. 7 and SEQ ID NO. 8 a 3 ’UTR selected from a group comprising sequences represented by SEQ ID NO. 9, SEQ ID NO. 10, SEQ ID NO. 11, SEQ ID NO. 12, SEQ ID NO. 13 and SEQ ID NO. 14 and a polyA tail comprising about 60 As to about 120 As.
  • the present disclosure provides an mRNA construct comprising a CDS bearing at least about 90% identity to the sequence represented by SEQ ID No. 2 or a fragment thereof, a 5’ terminal cap structure selected from a group comprising m7Gppp5'N (CapO), m7Gppp5'Nm (Capl), chemically synthesized cap analogs such as ARCA (3'-O-Me-m7G(5') ppp(5')G) or CleanCap capping reagents such as CleanCapAG or CleanCapM6 or other commercially available or novel cap reagents that perform the same function, a 5 ’ UTR selected from a group comprising sequences represented by SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO.
  • SEQ ID NO. 6 SEQ ID NO. 7 and SEQ ID NO. 8
  • a 3 ’UTR selected from a group comprising sequences represented by SEQ ID NO. 9, SEQ ID NO. 10, SEQ ID NO. 11, SEQ ID NO. 12, SEQ ID NO. 13 and SEQ ID NO. 14 and a polyA tail comprising about 60 As to about 120 As.
  • the cDNA may be obtained by reverse transcription of mRNA or chemical synthesis.
  • the DNA template for in vitro RNA synthesis may also be obtained by gene synthesis.
  • the DNA template may be obtained by restriction digestion or via PCR.
  • the promoter is selected from a group comprising, T7, SP6, T3 and LP1 or HCR hAAT promoters.
  • the DNA template comprises an AT-rich region, a promoter selected from a group comprising HCRhAAT, T7, SP6, T3 and LP1 promoters, a 5' untranslated region (UTR) selected from a group comprising sequences represented by SEQ ID NO. 16, SEQ ID NO. 17, SEQ ID NO. 18, SEQ ID NO. 19, SEQ ID NO. 20 and SEQ ID NO. 21, SEQ ID NO. 33, SEQ ID NO. 34, SEQ ID NO. 35, SEQ ID NO. 36 and SEQ ID NO. 38, a terminator region, a CDS bearing at least about 90% identity to a sequence represented by SEQ ID No.
  • a promoter selected from a group comprising HCRhAAT, T7, SP6, T3 and LP1 promoters
  • UTR 5' untranslated region
  • SEQ ID NO. 16 selected from a group comprising sequences represented by SEQ ID NO. 16, SEQ ID NO. 17, SEQ ID NO. 18, SEQ ID NO. 19, SEQ ID NO
  • a 3'UTR selected from a group comprising sequences represented by SEQ ID NO. 22, SEQ ID NO. 23, SEQ ID NO. 24, SEQ ID NO. 25, SEQ ID NO. 26, SEQ ID NO. 27 and SEQ ID NO. 37
  • a polyA signal selected from a group comprising SV40 PolyA sequence, Synthetic PolyA site and Bovine growth hormone (bGH) PolyA site and a suitable restriction site to generate an mRNA construct without an overhang.
  • the DNA template comprises an AT-rich region, a promoter selected from a group comprising HCRhAAT, T7, SP6, T3 and LP1 promoters, a 5' untranslated region (UTR) selected from a group comprising sequences represented by SEQ ID NO. 16, SEQ ID NO. 17, SEQ ID NO. 18, SEQ ID NO. 19, SEQ ID NO. 20, SEQ ID NO. 21, SEQ ID NO. 33, SEQ ID NO. 34, SEQ ID NO. 35, SEQ ID NO. 36 and SEQ ID NO. 38, a CDS represented by SEQ ID No. 15 or SEQ ID No. 31 or a fragment thereof, a 3 'UTR selected from a group comprising sequences represented by SEQ ID NO.
  • a 3'UTR selected from a group comprising sequences represented by SEQ ID NO. 22, SEQ ID NO. 23, SEQ ID NO. 24, SEQ ID NO. 25, SEQ ID NO. 26, SEQ ID NO. 27 and SEQ ID NO. 37
  • a polyA signal selected from a group comprising SV40 PolyA sequence, Synthetic PolyA site and Bovine growth hormone (bGH) PolyA site and a suitable restriction site to generate an mRNA construct without an overhang.
  • the DNA template comprises an AT-rich region which includes but is not limited to TTAATT, a promoter selected from a group comprising HCR hAAT, T7, SP6, T3 and LP1 promoters, a 5' untranslated region (UTR) selected from a group comprising sequences represented by SEQ ID NO. 16, SEQ ID NO. 17, SEQ ID NO. 18, SEQ ID NO. 19, SEQ ID NO. 20 and SEQ ID NO. 21, SEQ ID NO. 33, SEQ ID NO. 34, SEQ ID NO. 35, SEQ ID NO. 36 and SEQ ID NO. 38, a terminator region, a CDS represented by SEQ ID No. 15 or SEQ ID No.
  • the DNA template comprises an AT-rich region which includes but is not limited to TTAATT, a promoter selected from a group comprising HCR hAAT, T7, SP6, T3 and LP1 promoters, a 5' untranslated region (UTR) selected from a group comprising sequences represented by SEQ ID NO. 16, SEQ ID NO. 17, SEQ ID NO. 18, SEQ ID NO. 19, SEQ ID NO. 20 and SEQ ID NO. 21, SEQ ID NO. 33, SEQ ID NO. 34, SEQ ID NO. 35, SEQ ID NO. 36 and SEQ ID NO. 38, a CDS represented by SEQ ID No. 15 or SEQ ID No.
  • a 3'UTR selected from a group comprising sequences represented by SEQ ID NO. 22, SEQ ID NO. 23, SEQ ID NO. 24, SEQ ID NO. 25, SEQ ID NO. 26, SEQ ID NO. 27 and SEQ ID NO. 37
  • a polyA signal selected from a group comprising SV40 PolyA sequence, Synthetic PolyA site and Bovine growth hormone (bGH) PolyA site and a suitable restriction site for linearization of the IVT template for mRNA synthesis by in vitro transcription, examples of which include but are not limited to a Type II restriction site such as that recognized by Type II enzymes such as Notl, EcoRI, or Type IIS enzymes such as BbsI and BstBl.
  • the DNA template comprises each of the aforesaid elements in the order as defined in the above embodiment.
  • the DNA template comprises a CDS bearing at least about 90% identity to a sequence represented by SEQ ID No. 15 or a fragment thereof along with a promoter represented by SEQ ID No. 32, one or more 5' untranslated regions selected from SEQ ID NO. 33, SEQ ID NO. 34, SEQ ID NO. 35 and SEQ ID NO. 38, one or more copies of 3' untranslated region represented by SEQ ID NO. 37 and polyA signal(s) selected from SEQ ID NO. 39 and SEQ ID NO. 40.
  • the DNA template comprises a suitable enzyme cleavage site between the different elements in the template as described above.
  • the DNA template is placed in the plasmid between two restriction sites.
  • the DNA template of the present disclosure may alternatively also be employed for gene therapy. Accordingly, in some embodiments, the DNA template of the present disclosure may be directly delivered to a subject in need thereof. In some embodiments, the direct delivery of the DNA template is facilitated by a viral vector.
  • the recombinant vector is a recombinant parvoviral vector. In some exemplary, non-limiting embodiments, the recombinant vector is a recombinant AAV (rAAV) vector.
  • the recombinant AAV vector may be of any serotype (e.g., comprise any AAV serotype genome and/or capsid protein), provided it is capable of infecting or transducing the target cells and/or tissues.
  • a recombinant AAV vector which retains only the replication and packaging signals of AAV, and which comprises a nucleic acid sequence encoding MMUT, or a derivative or functional fragment thereof.
  • an AAV vector comprising the nucleic acid sequence encoding MMUT sequence bearing at least about 90% identity to a sequence represented by SEQ ID No. 15 or a fragment thereof.
  • an AAV vector comprising the DNA template as described above comprising the nucleic acid sequence encoding MMUT sequence bearing at least about 90% identity to a sequence represented by SEQ ID No. 15 or a fragment thereof.
  • the AAV vector comprises the nucleic acid sequence encoding MMUT sequence bearing at least about 90% identity to a sequence represented by a CDS represented by SEQ ID No. 15 or SEQ ID No. 31 or a fragment thereof.
  • the present disclosure provides a recombinant AAV vector comprising the DNA template described above which comprises the nucleic acid sequence encoding MMUT sequence bearing at least about 90% identity to a sequence, preferably represented by SEQ ID No. 15 or SEQ ID No. 31 or a fragment thereof.
  • the present disclosure provides a recombinant AAV vector comprising a DNA template that comprises along with the CDS having sequence bearing at least about 90% identity to a sequence represented by SEQ ID No. 15 or a fragment thereof, one or more elements selected from a group comprising an AT-rich region, a promoter, a 5' untranslated region (UTR), an enhancer region, a terminator region, a 3'UTR, optionally, a polyA signal.
  • a recombinant AAV vector comprising a DNA template that comprises along with the CDS having sequence bearing at least about 90% identity to a sequence represented by SEQ ID No. 15 or a fragment thereof, one or more elements selected from a group comprising an AT-rich region, a promoter, a 5' untranslated region (UTR), an enhancer region, a terminator region, a 3'UTR, optionally, a polyA signal.
  • the present disclosure provides a recombinant AAV vector comprising a DNA template that comprises along with the CDS having sequence bearing at least about 90% identity to a sequence represented by SEQ ID No. 15 or a fragment thereof, three or more elements selected from a group comprising an AT-rich region, a promoter, a 5' untranslated region (UTR), an enhancer region, a terminator region, a 3'UTR, optionally, a polyA signal.
  • a recombinant AAV vector comprising a DNA template that comprises along with the CDS having sequence bearing at least about 90% identity to a sequence represented by SEQ ID No. 15 or a fragment thereof, three or more elements selected from a group comprising an AT-rich region, a promoter, a 5' untranslated region (UTR), an enhancer region, a terminator region, a 3'UTR, optionally, a polyA signal.
  • the present disclosure provides both mRNA and DNA based therapeutics by way of the mRNA construct as well as the DNA template that is capable of being directly delivered to a subject through recombinant AAV medicated delivery. While the recombinant AAV comprising the DNA template of the present disclosure is described in the above embodiments, the following embodiments further describe the mode of delivery of the mRNA construct of the present disclosure.
  • the host cell may be a prokaryotic cell or a eukaryotic cell.
  • the prokaryotic cell is selected from a group comprising bacterial cell, fungal cell, yeast cell, viral cell and protozoan cell.
  • the host cell is selected from a group comprising HEK293, HEK293T, Sf9, C12 and HeLa cell.
  • the mRNA construct of the present disclosure may be encapsulated in a lipid nanoparticle (LNP) or exosomes.
  • LNP lipid nanoparticle
  • lipid nanoparticle encapsulated mRNA construct comprising a CDS encoding the MMUT protein or a fragment thereof, a 5’ cap selected from a group comprising m7Gppp5'N (CapO), m7Gppp5'Nm (Capl), chemically synthesized cap analogs such as ARCA (3'-O-Me-m7G(5') ppp(5')G) or CleanCap capping reagents such as CleanCapAG or CleanCapM6 or other commercially available or novel cap reagents that perform the same function, a 5 'UTR, a 3 'UTR element and/or a poly(A) sequence .
  • mRNA-LNP lipid nanoparticle encapsulated mRNA construct
  • lipid nanoparticle encapsulated mRNA construct comprising a CDS encoding the MMUT protein or a fragment thereof, a 5’ cap, a 5'UTR element selected from a group comprising sequences represented by SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO. 5, SEQ ID NO. 6, SEQ ID NO. 7 and SEQ ID NO. 8, a 3'UTR element and/or a poly (A) sequence.
  • a lipid nanoparticle encapsulated mRNA construct comprising a CDS encoding the MMUT protein or a fragment thereof, a 5’ cap selected from a group comprising m7Gppp5'N (CapO), m7Gppp5'Nm (Capl), chemically synthesized cap analogs such as ARCA (3'-O-Me-m7G(5') ppp(5')G) or CleanCap capping reagents such as CleanCapAG or CleanCapM6 or other commercially available or novel cap reagents that perform the same function, a 5'UTR element selected from a group comprising sequences represented by SEQ ID NO. 3, SEQ ID NO.
  • a lipid nanoparticle encapsulated mRNA construct comprising a CDS bearing at least 90% identity to the sequence represented by SEQ ID No. 2 or a fragment thereof, a 5’ cap, a 5'UTR element selected from a group comprising sequences represented by SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO. 5, SEQ ID NO. 6, SEQ ID NO. 7 and SEQ ID NO. 8, a 3'UTR element selected from a group comprising sequences represented by SEQ ID NO. 9, SEQ ID NO. 10, SEQ ID NO. 11, SEQ ID NO. 12, SEQ ID NO. 13 and SEQ ID NO. 14 and/or a poly (A) sequence.
  • mRNA-LNP lipid nanoparticle encapsulated mRNA construct
  • a lipid nanoparticle encapsulated mRNA construct comprising a CDS represented by SEQ ID No. 2 or SEQ ID No. 29 or a fragment thereof, a 5’ cap selected from a group comprising m7Gppp5'N (CapO), m7Gppp5'Nm (Capl), chemically synthesized cap analogs such as ARCA (3'-O-Me-m7G(5') ppp(5')G) or CleanCap capping reagents such as CleanCapAG or CleanCapM6 or other commercially available or novel cap reagents that perform the same function, a 5'UTR element selected from a group comprising sequences represented by SEQ ID NO.
  • SEQ ID NO. 4 SEQ ID NO. 5, SEQ ID NO. 6, SEQ ID NO. 7 and SEQ ID NO. 8 a 3'UTR element selected from a group comprising sequences represented by SEQ ID NO. 9, SEQ ID NO. 10, SEQ ID NO. 11, SEQ ID NO. 12, SEQ ID NO. 13 and SEQ ID NO. 14, and a poly(A) sequence.
  • non-limiting examples of such pharmaceutically acceptable excipient(s) or carrier(s) include LNP components such as ionizable lipids, PEGylated lipid, cholesterol and its derivatives, structural lipids, and additional components such as triglycerides, mixed glycerides, surfactants and co-solvents.
  • LNP components such as ionizable lipids, PEGylated lipid, cholesterol and its derivatives, structural lipids, and additional components such as triglycerides, mixed glycerides, surfactants and co-solvents.
  • the subject is a mammal, including but not limited to human being.
  • the administration of the mRNA construct, the mRNA-LNP comprising the nucleotide encoding MMUT protein or fragment thereof or the recombinant AAV or the composition of the present disclosure to a subject in need thereof leads to higher expression of MMUT in the subject and therefore leads to reduction of MMA accumulation.
  • the present disclosure also relates to use of the mRNA sequence, the mRNA construct, the DNA template, the mRNA-LNP, the recombinant AAV or the composition of the present disclosure in treating and/or preventing MMA.
  • a kit comprising the mRNA sequence, the mRNA construct, the DNA template, the mRNA-LNP, the recombinant AAV or the composition, means for administration of the same and optionally, an instruction manual.
  • Example 1 Codon optimization of the sequence encoding MMUT
  • the codon optimized version (sample 3) showed significantly higher protein production in cells (Fig 4D). Further, it was also observed that the sample having first 8 amino acids of wild type and rest codon optimized sequence (sample 7), resulted in a significantly higher level of translation as compared to sample 6, where the codon-optimized nucleotides encoding the first 8 amino acids and rest wild type resulted in poor expression. This was contrary to expectation given the known influence of the first 1-8 amino acids on protein expression.
  • MMUT knockout zebrafish larvae generated in-house were used as the animal model for this experiment.
  • the zebra fish larvae were microinjected with mRNA candidate sample 3 listed in Table 2, at about 500pg/embryo in the appropriate buffer.
  • Five days post injection larvae from the un-injected and injected groups were harvested, along with age matched wild type larvae. Lysates were prepared and MMA levels were measured by LC-MS. Accumulation of MMA was observed in the un-injected F2, while a significant reduction in the same was observed in the group injected with the mRNA (Figure 5).
  • Example 4 AAV constructs comprising the MMUT nucleotide sequence of the present disclosure
  • a small panel of codon optimized MMUT AAV constructs was generated by sub-cloning a combination of different 5’ and 3’ UTR regulatory elements in the AAV vector.
  • the regulatory elements of the different constructs are shown in Table 3.
  • Codon optimized MMUT (CO- MMUT) AAV constructs were then transfected into HepG2 cells.
  • the expression levels of MMUT was examined by western blotting (Figure 6). As shown in the figure, the expression of MMUT protein was evident in the cells transfected with MUT-AAV constructs.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Engineering & Computer Science (AREA)
  • Chemical & Material Sciences (AREA)
  • Biotechnology (AREA)
  • Organic Chemistry (AREA)
  • Molecular Biology (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Health & Medical Sciences (AREA)
  • Biomedical Technology (AREA)
  • General Engineering & Computer Science (AREA)
  • Public Health (AREA)
  • Physics & Mathematics (AREA)
  • Biophysics (AREA)
  • Veterinary Medicine (AREA)
  • Plant Pathology (AREA)
  • Animal Behavior & Ethology (AREA)
  • Microbiology (AREA)
  • Epidemiology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Biochemistry (AREA)
  • Medicinal Chemistry (AREA)
  • Virology (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

The present disclosure provides a nucleic acid sequence encoding MMUT for the treatment of Methylmalonic acidemia (MMA). Said nucleic acid is codon optimized to improve various parameters and allows high expression in the cells it is expressed in. The present disclosure also provides nucleic acid construct(s) comprising the nucleic acid sequence encoding MMUT along with one or more regulatory elements. Modes of delivery of the said nucleic acid sequence include delivery by way of encapsulation in a lipid nanoparticle when the nucleic acid is an mRNA and by way of a recombinant AAV vector when the nucleic acid is a DNA sequence. The present disclosure also relates to a composition and kit comprising the nucleic acid sequence, constructs or delivery vehicles as well as their applications in treating MMA.

Description

“NUCLEIC ACID SEQUENCE(S) AND CONSTRUCT(S) FOR TREATING METHYLMALONIC ACIDEMIA AND METHOD(S) THEREOF”
TECHNICAL FIELD
The present disclosure relates to the technical field of biological therapeutics, in particular to nucleic acid therapeutics i.e. DNA and mRNA therapeutics, method(s) or application(s) thereof and a kit.
BACKGROUND OF THE DISCLOSURE
Methylmalonic acidemia refers to a group of inborn errors of metabolism caused by defects in multiple genes resulting in the accumulation of methyhnalonate or 5’- deoxyadenosylcobalamin. The prevalence of treating Methylmalonic acidemia (MMA) is estimated to be between 1:25,000- 1:50,000 with detection rates of less than 2: 100,000. Mutations in the gene MMUT (methyl malonyl-coenzyme A mutase) are the most common cause of this disorder, referred to as isolated MMA. Patients with this disease are unable to metabolize branched chain amino acids, odd chain fatty acid, gut-derived propionate, and cholesterol, and experience lethargy, vomiting, hypothermia, respiratory distress, severe ketoacidosis, and hyperammonemia during early childhood. If diagnosed early, patients can be treated with a specialized protein restricted diet and supplementary vitamin B12 to manage the symptoms, however most will continue to suffer severe metabolic instability requiring hospitalization. Intellectual deficits, renal failure, metabolic stroke and movement disorders are some of the secondary symptoms that develop with age in these patients.
MMA continues to be a significant cause of morbidity and mortality because of the multisystem damage during the multiple metabolic crises faced by these patients. Since most of the metabolic conversion of MMA occurs in the liver, liver transplantation (LT) is increasingly being used to alleviate recurrent metabolic strokes in patients with severe disease. While this supports the idea that addressing MMUT deficiency in the liver alone could be an effective therapy, LT is not an affordable or risk-free solution for all patients, and therefore there is need for efficacious disease modifying treatments with a better prognosis.
Replacement therapies in the form of protein (such as enzyme replacement therapies (ERT)), are considered the closest to a “cure” for monogenic disorders. However, since MMUT is a mitochondrial enzyme, protein ERT is unsuitable. In vitro transcribed messenger RNA (IVT mRNA) has always been considered an excellent option for protein replacement, as they do not need to be delivered to the nucleus, can be translated instantaneously. With the recent breakthroughs in LNP mediated delivery, mRNA candidates have progressed rapidly through preclinical as well as early clinical trials. mRNA cancer vaccines, replacement therapies for oncology, cardiology, hematology, endocrinology, pulmonary medicine and others are in late phase clinical trials and are likely to be approved soon given the safety and success of the mRNA vaccines for COVID-19. Additionally, delivering a therapeutic nucleic acid via Adeno-associated virus (AAV) vector has attracted considerable attention as a highly effective viral vector for gene therapy due to its low immunogenicity and ability to effectively transduce non-dividing cells. AAV has been shown to infect a variety of cell and tissue types, and significant progress has been made over the last decade to adapt this viral system for use in human gene therapy.
Therefore, the development of novel therapeutics for MMA patients, particularly in the form of nucleic acid therapeutics would be of great benefit. The present disclosure addresses this need.
SUMMARY OF THE DISCLOSURE
Addressing the aforesaid need in the art, the present disclosure provides an mRNA sequence comprising a CDS bearing at least about 90% identity to a sequence represented by SEQ ID No. 2.
Also provided herein is an mRNA construct comprising the mRNA sequence as described above or a fragment thereof, a 5’ cap, a 5'UTR element, a 3'UTR element, a poly(A) sequence and optionally, a natural or synthetic IRES element and/or a circularizing element.
The present disclosure further envisages a DNA template suitable for generating the mRNA as described above, the said DNA template comprising a CDS having sequence bearing at least about 90% identity to a sequence represented by SEQ ID No. 15 or a fragment thereof, one or more elements selected from a group comprising an AT -rich region, a promoter, a 5' untranslated region (UTR), an enhancer region, a terminator region, a 3'UTR, a polyA signal, and a restriction site.
The present disclosure further provides a recombinant AAV vector comprising the DNA template described above. Also envisaged herein is a lipid nanoparticle (LNP) comprising the mRNA construct as described above.
The present disclosure further provides composition comprising the mRNA construct, the mRNA-LNP or the recombinant AAV vector as described above 8 in combination with pharmaceutically acceptable excipient(s) or carrier(s).
The present disclosure further provides a method of treating and/or preventing MMA, said method comprising administering the mRNA construct, the mRNA-LNP or the recombinant AAV vector or the composition as described above to a subject in need thereof.
Also provided herein is a use of the mRNA sequence, the mRNA construct, the mRNA-LNP or the recombinant AAV vector or the composition as described above for treating and/or preventing MMA.
Further, the present disclosure envisages a kit comprising the mRNA sequence as claimed in any of claims 1 or 2, the mRNA construct as claimed in any of claims 3 or 4, the mRNA-LNP as claimed in claim 9 or the recombinant AAV vector as claimed in claim 8 or the composition as claimed in claim 10, means for administration of the same and optionally, an instruction manual.
BRIEF DESCRIPTION OF THE ACCOMPANYING FIGURES
In order that the disclosure may be readily understood and put into practical effect, reference will now be made to exemplary embodiments as illustrated with reference to the accompanying figures. The figures together with detailed description below, are incorporated in and form part of the specification, and serve to further illustrate the embodiments and explain various principles and advantages, in accordance with the present disclosure where:
Figure 1A shows the Codon Adaptation Index (CAI) of the mRNA of the present disclosure [SEQ ID NO. 2] (in green) vis-a-vis the wild-type sequence (in blue).
Figure IB is a graph showing the frequency of optimal codons in the mRNA of the present disclosure [SEQ ID NO. 2] (in green) vis-a-vis the wild-type sequence (in blue).
Figure 2 shows the GC content of the mRNA of the present disclosure [SEQ ID NO. 2] (in green) vis-a-vis the wild-type sequence (in blue).
Figure 3 shows the Construct design and optimization of in vitro transcription and translation. 3A shows the design of the construct used to generate linear mRNA, according to an exemplary embodiment. 3B is representative gel image of in vitro transcribed capped polyadenylated mRNA for a reporter gene. 3C shows the translation of a fluorescent reporter accompanying the mRNA construct of the present disclosure after transfection into cells. 3D shows translation of a chemiluminescent reporter made with uridine or pseudouridine accompanying the mRNA construct of the present disclosure after transfection into cells. 3E shows translation of a fluorescent reporter accompanying the mRNA construct of the present disclosure injected into a zebrafish embryo at 24 hours post injection.
Figure 4 shows A. Details of the noncoding and coding elements in various MMUT mRNA constructs generated and tested in vitro B. Details of the type of UTR elements used C: Details of the CDS sequences used in the constructs. WT: Wild-type (native H.s. MMUT), L-CO/CO: Liver-Codon optimized. D-F. Comparison of MMUT protein levels in WT HEK-293T cells as is (E), MMUT knockout HEK 293T cells (KO), and KO cells transfected with the indicated constructs (numbers in red). Immunoblotting analysis using the MMUT antibody and GAPDH antibody (used as a loading control) performed on cell lysates. G. Quantification of relative MMUT protein levels from three independent experiments, in cells transfected with equal amounts of the indicated mRNA. H. Measurement of the levels of methylmalonic acid (MMA) in cell lysates by LC-MS, in WT cells (no accumulation), Knockout cells (MMA detected), and KO cells transfected with mRNA 1 or 3 (complete reduction of MMA).
Figure 5 shows a measurement of the levels of methylmalonic acid (MMA) in total zebrafish larval lysates by LC-MS in samples of injected and un-injected larvae. The figure shows that accumulation of MMA is reduced in the mRNA injectants, indicating functional activity of the injected mRNA.
Figure 6 shows expression levels of MMUT examined by western blotting after AAV constructs encoding MMUT were transfected into HepG2 cells.
DETAILED DESCRIPTION OF THE DISCLOSURE
In view of the limitations discussed above in presently available methods for MMA treatment, the present disclosure provides polynucleotide sequences and constructs for effectively treating MMA. However, before describing the invention in greater detail, it is important to take note of the common terms and phrases that are employed throughout the present disclosure for better understanding of the technology provided herein.
Definitions The terms “Methyl-malonyl CoA mutase” abbreviated as “MUT” or “MMUT” refers to a mitochondrial enzyme that catalyzes the isomerization of methyhnalonyl-CoA to succinyl- CoA. Mutations in the MMUT gene are a common cause of Methylmalonic acidemia (MMA).
The term “nucleic acid therapeutics” refer to therapeutic substances, drugs or compositions with nucleic acids as their therapeutically active component, such as DNA or RNA of all forms.
“Messenger RNA” (mRNA) refers to any polynucleotide that encodes a (at least one) polypeptide (a naturally-occurring, non-naturally-occurring, or modified polymer of amino acids) and can be translated to produce the encoded polypeptide in vitro, in vivo, in situ or ex vivo. A skilled artisan will appreciate that except where otherwise noted, polynucleotide sequences set forth in the instant application will recite “T”s in a representative DNA sequence but where the sequence represents RNA (e.g., mRNA), the “T”s would be substituted for “U”s. Thus, any of the RNA polynucleotides encoded by a DNA identified by a particular sequence identification number may also comprise the corresponding RNA (e.g., mRNA) sequence encoded by the DNA, where each “T” of the DNA sequence is substituted with “U ”
A “5' untranslated region” (5'UTR) refers to a region of an mRNA that is directly upstream (i.e., 5') from the start codon (i.e., the first codon of an mRNA transcript translated by a ribosome) that does not encode a polypeptide.
A “3' untranslated region” (3'UTR) refers to a region of an mRNA that is directly downstream (i.e., 3') from the stop codon (i.e., the codon of an mRNA transcript that signals a termination of translation) that does not encode a polypeptide.
A “coding sequence” or “CDS” is a region of DNA or RNA whose sequence determines the sequence of amino acids in a protein.
A “polyA tail” is a region of mRNA that is downstream, e.g., directly downstream (i.e., 3'), from the 3' UTR that contains multiple, consecutive adenosine monophosphates. A polyA tail may contain 10 to 300 adenosine monophosphates. For example, a polyA tail may contain 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290 or 300 adenosine monophosphates. In some embodiments, a polyA tail contains 50 to 250 adenosine monophosphates. In a relevant biological setting (e.g., in cells, in vivo) the poly(A) tail functions to protect mRNA from enzymatic degradation, e.g., in the cytoplasm, and aids in transcription termination, export of the mRNA from the nucleus and translation. The term “mRNA construct” or “therapeutic mRNA” refers to a stabilized mRNA sequence, wherein the means of stabilization are envisaged in later embodiments of the present disclosure.
The term “DNA construct” or “DNA template” refers to an expression cassette, transcription of which yields the mRNA construct of the present disclosure. The DNA construct may be carried in a vector or plasmid. In the context of the present disclosure, the DNA template or DNA construct may serve one of the two purposes: generation of mRNA though in vitro transcription or direct delivery to a subject through an AAV vector as a mode of gene therapy.
The terms “RNA in vitro transcription”, “zft vitro transcription” or obvious variants thereof relate to a process wherein RNA is synthesized in a cell-free system (in vitro). DNA, particularly plasmid DNA, is used as template for the generation of RNA transcripts. RNA may be obtained by DNA-dependent in vitro transcription of an appropriate DNA template.
The term "AAV vector" or "AAV construct" or "AAV mRNA construct" or obvious variants thereof refers to a vector comprising some or all of the viral genes encoding a gene product, control sequences and viral packaging sequences along with a polynucleotide (encoding MMUT in the context of the present disclosure) to be delivered into a host cell, either in vivo, ex vivo or in vitro.
As used herein, the term ‘comprising’ when placed before the recitation of steps in a method means that the method encompasses one or more steps that are additional to those expressly recited, and that the additional one or more steps may be performed before, between, and/or after the recited steps. For example, a method comprising steps a, b, and c encompasses a method of steps a, b, x, and c, a method of steps a, b, c, and x, as well as a method of steps x, a, b, and c. Furthermore, the term “comprising” when placed before the recitation of steps in a method does not (although it may) require sequential performance of the listed steps, unless the content clearly dictates otherwise. For example, a method comprising steps a, b, and c encompasses, for example, a method of performing steps in the order of steps a, c, and b, the order of steps c, b, and a, and the order of steps c, a, and b, etc.
With respect to the use of substantially any plural and/or singular terms herein, those having skill in the art can translate from the plural to the singular and/or from the singular to the plural as is appropriate to the context and/or application. The various singular/plural permutations may be expressly set forth herein for sake of clarity. The suffix ‘(s)’ at the end of any term in the present disclosure envisages in scope both the singular and plural forms of said term. As used in this specification and the appended claims, the singular forms ‘a’, ‘an’ and ‘the’ includes both singular and plural references unless the content clearly dictates otherwise.
Numerical ranges stated in the form ‘from xto y’ include the values mentioned and those values that lie within the range of the respective measurement as known to the skilled person. If several preferred numerical ranges are stated in this form, of course, all the ranges formed by a combination of the different end points are also included.
The use of the expression ‘at least’ or ‘at least one’ suggests the use of one or more elements or ingredients or quantities, as the use may be in the embodiment of the disclosure to achieve one or more of the desired objects or results. As such, the terms “a” (or “an”), “one or more”, and “at least one” can be used interchangeably herein.
The terms ‘about’ or ‘approximately’ as used herein when referring to a measurable value such as a parameter, an amount, a temporal duration, and the like, are meant to encompass variations of and from the specified value, such as variations of +/-10% or less, +/-5% or less, +/-1% or less, and +/-0.1% or less of and from the specified value, insofar such variations are appropriate to perform in the disclosed invention. It is to be understood that the value to which the modifier ‘about’ or ‘approximately’ refers is itself also specifically, and preferably, disclosed.
As used herein, the terms ‘include’, ‘have’, ‘comprise’, ‘contain’ etc. or any form of said terms such as ‘having’, ‘including’, ‘containing’, ‘comprising’ or ‘comprises’ are inclusive and will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.
As regards the embodiments characterized in this specification, it is intended that each embodiment be read independently as well as in combination with another embodiment. For example, in case of an embodiment 1 reciting 3 alternatives A, B and C, an embodiment 2 reciting 3 alternatives D, E and F and an embodiment 3 reciting 3 alternatives G, H and I, it is to be understood that the specification unambiguously discloses embodiments corresponding to combinations A, D, G; A, D, H; A, D, I; A, E, G; A, E, H; A, E, I; A, F, G; A, F, H; A, F, I;
B, D, G; B, D, H; B, D, I; B, E, G; B, E, H; B, E, I; B, F, G; B, F, H; B, F, I; C, D, G; C, D, H;
C, D, I; C, E, G; C, E, H; C, E, I; C, F, G; C, F, H; C, F, I, unless specifically mentioned otherwise. Reference throughout this specification to “some embodiments”, “one embodiment”, “an embodiment”, “a preferred embodiment”, “a non-limiting embodiment” or “an exemplary embodiment” means that a particular feature, structure or characteristic described in connection with the embodiment may be included in at least one embodiment of the present disclosure. Thus, the appearances of the phrases “in some embodiments”, “in one embodiment”, “in an embodiment”, “a preferred embodiment”, “a non-limiting embodiment” or “an exemplary embodiment” in various places throughout this specification may not necessarily all refer to the same embodiment. It is appreciated that certain features of the disclosure, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the disclosure, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable sub-combination.
Throughout this specification, the term ‘a combination thereof, ‘combinations thereof or ‘any combination thereof or ‘any combinations thereof are used interchangeably and are intended to have the same meaning, as regularly known in the field of patent disclosures.
Disclosure
The present disclosure provides a therapeutic nucleic acid candidate for treating Methylmalonic acidemia (MMA). The therapeutic candidate is based on a codon optimized mRNA or gene therapy candidate for increasing the expression of Methyl-malonyl CoA mutase (MUT) in a subject, thereby decreasing the levels of Methylmalonic acidemia (MMA), leading to a phenotype rescue in the subject. Accordingly, the present disclosure envisages DNA and mRNA sequences, transcription and translation, respectively of which leads to increased expression of Methyl-malonyl CoA mutase (MUT).
More specifically, the present disclosure provides a messenger ribonucleic acid (mRNA) comprising a coding sequence (CDS) encoding the Methyl-malonyl CoA mutase (MUT) protein or a fragment thereof.
In some embodiments, the CDS encoding the MMUT protein or a fragment thereof is an engineered sequence. In some embodiments, the CDS encoding the MMUT protein or a fragment thereof is a codon optimized sequence. Codon optimization, in some embodiments, may be used to match codon frequencies in target and host organisms to ensure proper folding; bias GC content to increase mRNA stability or reduce secondary structures; maximize codon adaptation index; minimize tandem repeat codons or base runs that may impair gene construction or expression; customize transcriptional and translational control regions; insert or remove protein trafficking sequences; remove/add post translation modification sites in encoded protein (e.g., glycosylation sites); add, remove or shuffle protein domains; insert or delete restriction sites; modify ribosome binding sites and mRNA degradation sites; adjust translational rates to allow the various domains of the protein to fold properly; and/or reduce or eliminate problem secondary structures within the polynucleotide.
In a non-limiting embodiment, the CDS encoding the MMUT protein or a fragment thereof is codon optimized for mammalian expression. In an exemplary embodiment, the CDS encoding the MMUT protein or a fragment thereof is codon optimized for maximum expression in the mammalian liver.
Thus, in some embodiments, provided herein is a messenger ribonucleic acid (mRNA) sequence comprising a codon optimized CDS encoding the MMUT protein or a fragment thereof. In some embodiments, the messenger ribonucleic acid (mRNA) sequence shows high Codon Adaptation Index (CAI) of about 0.80 to about 0.99, preferably about 0.94.
In an exemplary, non-limiting embodiment, the present disclosure provides an mRNA sequence comprising a CDS bearing at least about 90% identity to a sequence represented by SEQ ID No. 2. In an exemplary, non-limiting embodiment, the present disclosure provides an mRNA sequence bearing at least about 90% identity to a sequence represented by SEQ ID No. 2.
In some embodiments, the present disclosure provides an mRNA sequence comprising a CDS bearing at least about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or about 100% identity to the sequence represented by SEQ ID No. 2. In some embodiments, at identities below 90%, the sequences of the present disclosure do not show the intended effect in terms of MMUT expression. In some embodiments, the present disclosure provides a nucleotide sequence represented by SEQ ID No. 29 which is a non-limiting example of a sequence bearing at least about 90% identity to SEQ ID No. 1.
In some embodiments, the mRNA CDS as defined above may comprise from about 0% to about 100% modified nucleotides. In some embodiments, the mRNA CDS as defined above may comprise 0-100% pseudouridine, Nl-methyl pseudouridine, 5 -methylcytidine, m6A or any other modified nucleotides.
In some embodiments, the above-described mRNA sequence of the present disclosure may further comprise, in addition to the CDS as defined above or a fragment thereof, a 5'UTR element, a 3'UTR element and a poly(A) sequence or polyA tail wherein said elements stabilize the mRNA.
In other words, in some embodiments, the present disclosure provides an mRNA construct comprising the CDS or mRNA sequence as defined above or a fragment thereof, a 5’ cap, a 5'UTR element, a 3'UTR element and a poly(A) sequence.
Accordingly, in some embodiments, the present disclosure provides an mRNA construct comprising a CDS encoding the MMUT protein or a fragment thereof, a 5’ cap, a 5'UTR element, a 3'UTR element and a poly(A) sequence.
In some embodiments, provided herein is an mRNA construct comprising a CDS bearing at least about 90% identity to the sequence represented by SEQ ID No. 2 or a fragment thereof, a 5’ cap, a 5'UTR element, a 3'UTR element and a poly(A) sequence.
In some embodiments, the 5 ’ terminal cap structure is selected from a group comprising of standard cap analogs m7Gppp5'N (CapO), m7Gppp5'Nm (Capl), chemically synthesized cap analogs such as ARCA (3'-O-Me-m7G(5') ppp(5')G) or CleanCap capping reagents such as CleanCapAG or CleanCapM6 or other commercially available or novel cap reagents that perform the same function. In some embodiments, the capping is performed co- transcriptionally. In some embodiments, the capping may be performed post-transcriptionally.
Thus, in some embodiments, the present disclosure provides an mRNA construct comprising a CDS encoding the MMUT protein or a fragment thereof and a 5 ’ terminal cap structure selected from a group comprising m7Gppp5'N (CapO), m7Gppp5'Nm (Capl), chemically synthesized cap analogs such as ARCA (3'-O-Me-m7G(5') ppp(5')G) or CleanCap capping reagents such as CleanCapAG or CleanCapM6 or other commercially available or novel cap reagents that perform the same function.
In some embodiments, provided herein is an mRNA construct comprising a CDS bearing at least about 90% identity to the sequence represented by SEQ ID No. 2 or a fragment thereof and a 5’ terminal cap structure selected from a group comprising m7Gppp5'N (CapO), m7Gppp5'Nm (Capl), chemically synthesized cap analogs such as ARCA (3'-O-Me-m7G(5') ppp(5')G) or CleanCap capping reagents such as CleanCapAG or CleanCapM6 or other commercially available or novel cap reagents that perform the same function.
In an exemplary embodiment, provided herein is an mRNA construct comprising a CDS bearing at least about 90% identity to the sequence represented by SEQ ID No. 2 or a fragment thereof, and a 5’ terminal cap structure selected from ARCA (3'-O-Me-m7G(5') ppp(5')G) and CleanCap capping reagents such as CleanCapAG or CleanCapM6.
In some embodiments, the mRNA construct may comprise one or more 5’ and 3’ UTR elements. In some embodiments, the mRNA construct may comprise one or more copies of the same 5’ and/or 3’ UTR elements. Accordingly, reference to ‘a’ UTR element in the above or below embodiments must not be interpreted as reference a single UTR element and it must be construed as a possibility of incorporating one or more 5’ and 3’ UTR elements.
In some embodiments, the 5’ UTR(s) is selected from a group comprising sequences represented by SEQ ID NO. 3 (bg), SEQ ID NO. 4 (mod), SEQ ID NO. 5 (ag), SEQ ID NO. 6 (cyba), SEQ ID NO. 7 (N7d) and SEQ ID NO. 8 (Pcbp) and others that act in a similar manner.
Thus, in some embodiments, the present disclosure provides an mRNA construct comprising a CDS encoding the MMUT protein or a fragment thereof and a 5’ UTR selected from a group comprising sequences represented by SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO. 5, SEQ ID NO. 6, SEQ ID NO. 7 and SEQ ID NO. 8.
In some embodiments, the present disclosure provides an mRNA construct comprising a CDS bearing at least about 90% identity to the sequence represented by SEQ ID No. 2 or a fragment thereof and a 5’ UTR selected from a group comprising sequences represented by SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO. 5, SEQ ID NO. 6, SEQ ID NO. 7 and SEQ ID NO. 8.
In some embodiments, the present disclosure provides an mRNA construct comprising a CDS bearing at least about 90% identity to the sequence represented by SEQ ID No. 2 or a fragment thereof, a 5’ terminal cap structure selected from a group comprising m7Gppp5'N (CapO), m7Gppp5'Nm (Capl), chemically synthesized cap analogs such as ARCA (3'-O-Me-m7G(5') ppp(5')G) or CleanCap capping reagents such as CleanCapAG or CleanCapM6 or other commercially available or novel cap reagents that perform the same function and a 5 ’ UTR selected from a group comprising sequences represented by SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO. 5, SEQ ID NO. 6, SEQ ID NO. 7 and SEQ ID NO. 8. In some embodiments, the 3’UTR(s) is selected from a group comprising sequences represented by SEQ ID NO. 9 (2bg), SEQ ID NO. 10 (AES), SEQ ID NO. 11 (ag), SEQ ID NO. 12 (cyba), SEQ ID NO. 13 (S27a) and SEQ ID NO. 14 (Hbaf).
Thus, in some embodiments, the present disclosure provides an mRNA construct comprising a CDS encoding the MMUT protein or a fragment thereof and a 3’UTR selected from a group comprising sequences represented by SEQ ID NO. 9, SEQ ID NO. 10, SEQ ID NO. 11, SEQ ID NO. 12, SEQ ID NO. 13 and SEQ ID NO. 14.
In some embodiments, the present disclosure provides an mRNA construct comprising a CDS bearing at least about 90% identity to the sequence represented by SEQ ID No. 2 or a fragment thereof and a 3’UTR selected from a group comprising sequences represented by SEQ ID NO. 9, SEQ ID NO. 10, SEQ ID NO. 11, SEQ ID NO. 12, SEQ ID NO. 13 and SEQ ID NO. 14.
In a non-limiting embodiment, some preferred combinations of 5 ’ and 3 ’ UTRs include SEQ ID No. 3 and SEQ ID No. 9, SEQ ID No. 4 and SEQ ID No. 9, SEQ ID No. 3 and SEQ ID No. 11, SEQ ID No. 6 and SEQ ID No. 11, SEQ ID No. 4 and SEQ ID No. 11, SEQ ID No. 5 and SEQ ID No. 10, SEQ ID No. 6 and SEQ ID No. 12, SEQ ID No. 7 and SEQ ID No.l 1, and SEQ ID No. 7 and SEQ ID No. 13.
Thus, in some embodiments, the present disclosure provides an mRNA construct comprising a CDS bearing at least about 90% identity to the sequence represented by SEQ ID No. 2 or a fragment thereof along with a combination of 5 ’ and 3 ’ UTRs selected from a group comprising SEQ ID No. 3 and SEQ ID No. 9, SEQ ID No. 4 and SEQ ID No. 9, SEQ ID No. 3 and SEQ ID No. 11, SEQ ID No. 6 and SEQ ID No. 11, SEQ ID No. 4 and SEQ ID No. 11, SEQ ID No. 5 and SEQ ID No. 10, SEQ ID No. 6 and SEQ ID No. 12, SEQ ID No. 7 and SEQ ID No.l l, and SEQ ID No. 7 and SEQ ID No. 13.
In some embodiments, the present disclosure provides an mRNA construct comprising a CDS bearing at least about 90% identity to the sequence represented by SEQ ID No. 2 or a fragment thereof, a 5’ terminal cap structure selected from a group comprising m7Gppp5'N (CapO), m7Gppp5'Nm (Capl), chemically synthesized cap analogs such as ARCA (3'-O-Me-m7G(5') ppp(5')G) or CleanCap capping reagents such as CleanCapAG or CleanCapM6 or other commercially available or novel cap reagents that perform the same function and a 3’UTR selected from a group comprising sequences represented by SEQ ID NO. 9, SEQ ID NO. 10, SEQ ID NO. 11, SEQ ID NO. 12, SEQ ID NO. 13 and SEQ ID NO. 14. In some embodiments, the present disclosure provides an mRNA construct comprising a CDS represented by SEQ ID No. 2 or SEQ ID No. 29 or a fragment thereof, a 5’ terminal cap structure selected from a group comprising m7Gppp5'N (CapO), m7Gppp5'Nm (Capl), chemically synthesized cap analogs such as ARCA (3'-O-Me-m7G(5') ppp(5')G) or CleanCap capping reagents such as CleanCapAG or CleanCapM6 or other commercially available or novel cap reagents that perform the same function and a 3’UTR selected from a group comprising sequences represented by SEQ ID NO. 9, SEQ ID NO. 10, SEQ ID NO. 11, SEQ ID NO. 12, SEQ ID NO. 13 and SEQ ID NO. 14.
In some embodiments, the present disclosure provides an mRNA construct comprising a CDS encoding the MMUT protein or a fragment thereof, a 5 ’ terminal cap structure selected from a group comprising m7Gppp5'N (CapO), m7Gppp5'Nm (Capl), chemically synthesized cap analogs such as ARCA (3'-O-Me-m7G(5') ppp(5')G) or CleanCap capping reagents such as CleanCapAG or CleanCapM6 or other commercially available or novel cap reagents that perform the same function, a 5’ UTR selected from a group comprising sequences represented by SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO. 5, SEQ ID NO. 6, SEQ ID NO. 7 and SEQ ID NO. 8, and a 3’UTR selected from a group comprising sequences represented by SEQ ID NO. 9, SEQ ID NO. 10, SEQ ID NO. 11, SEQ ID NO. 12, SEQ ID NO. 13 and SEQ ID NO. 14.
In some embodiments, the present disclosure provides an mRNA construct comprising a CDS bearing at least about 90% identity to the sequence represented by SEQ ID No. 2 or a fragment thereof, a 5’ terminal cap structure selected from a group comprising m7Gppp5'N (CapO), m7Gppp5'Nm (Capl), chemically synthesized cap analogs such as ARCA (3'-O-Me-m7G(5') ppp(5')G) or CleanCap capping reagents such as CleanCapAG or CleanCapM6 or other commercially available or novel cap reagents that perform the same function, a 5 ’ UTR selected from a group comprising sequences represented by SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO. 5, SEQ ID NO. 6, SEQ ID NO. 7 and SEQ ID NO. 8, and a 3’UTR selected from a group comprising sequences represented by SEQ ID NO. 9, SEQ ID NO. 10, SEQ ID NO. 11, SEQ ID NO. 12, SEQ ID NO. 13 and SEQ ID NO. 14.
In some embodiments, the present disclosure provides an mRNA construct comprising a CDS represented by SEQ ID No. 2 or SEQ ID No. 29 or a fragment thereof, a 5’ terminal cap structure selected from a group comprising m7Gppp5'N (CapO), m7Gppp5'Nm (Capl), chemically synthesized cap analogs such as ARCA (3'-O-Me-m7G(5') ppp(5')G) or CleanCap capping reagents such as CleanCapAG or CleanCapM6 or other commercially available or novel cap reagents that perform the same function, a 5 ’ UTR selected from a group comprising sequences represented by SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO. 5, SEQ ID NO. 6, SEQ ID NO. 7 and SEQ ID NO. 8, and a 3 ’UTR selected from a group comprising sequences represented by SEQ ID NO. 9, SEQ ID NO. 10, SEQ ID NO. 11, SEQ ID NO. 12, SEQ ID NO. 13 and SEQ ID NO. 14.
In some embodiments, the mRNA construct additionally comprises a polyA tail.
In some embodiments, the polyA tail comprises about 60 As to about 120 As.
In some embodiments, the polyA tail may be contiguous or bipartite or of any other suitable design.
Thus, in some embodiments, the present disclosure provides an mRNA construct comprising a CDS bearing at least about 90% identity to the sequence represented by SEQ ID No. 2 or a fragment thereof and a polyA tail comprising about 60 As to about 120 As.
In some embodiments, the present disclosure provides an mRNA construct comprising a CDS bearing at least about 90% identity to the sequence represented by SEQ ID No. 2 or a fragment thereof, a 5’ terminal cap structure selected from a group comprising m7Gppp5'N (CapO), m7Gppp5'Nm (Capl), chemically synthesized cap analogs such as ARCA (3'-O-Me-m7G(5') ppp(5')G) or CleanCap capping reagents such as CleanCapAG or CleanCapM6 or other commercially available or novel cap reagents that perform the same function and a polyA tail comprising about 60 As to about 120 As.
In some embodiments, the present disclosure provides an mRNA construct comprising a CDS bearing at least about 90% identity to the sequence represented by SEQ ID No. 2 or a fragment thereof, a 5’ UTR selected from a group comprising sequences represented by SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO. 5, SEQ ID NO. 6, SEQ ID NO. 7 and SEQ ID NO. 8 and a polyA tail comprising about 60 As to about 120 As.
Thus, in some embodiments, the present disclosure provides an mRNA construct comprising a CDS bearing at least about 90% identity to the sequence represented by SEQ ID No. 2 or a fragment thereof, a 3 ’UTR selected from a group comprising sequences represented by SEQ ID NO. 9, SEQ ID NO. 10, SEQ ID NO. 11, SEQ ID NO. 12, SEQ ID NO. 13 and SEQ ID NO. 14 and a polyA tail comprising about 60 As to about 120 As. In some embodiments, the present disclosure provides an mRNA construct comprising a CDS encoding the MMUT protein or a fragment thereof, a 5 ’ terminal cap structure selected from a group comprising m7Gppp5'N (CapO), m7Gppp5'Nm (Capl), chemically synthesized cap analogs such as ARCA (3'-O-Me-m7G(5') ppp(5')G) or CleanCap capping reagents such as CleanCapAG or CleanCapM6 or other commercially available or novel cap reagents that perform the same function, a 5 ’ UTR selected from a group comprising sequences represented by SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO. 5, SEQ ID NO. 6, SEQ ID NO. 7 and SEQ ID NO. 8, a 3 ’UTR selected from a group comprising sequences represented by SEQ ID NO. 9, SEQ ID NO. 10, SEQ ID NO. 11, SEQ ID NO. 12, SEQ ID NO. 13 and SEQ ID NO. 14 and a polyA tail comprising about 60 As to about 120 As.
In some embodiments, the present disclosure provides an mRNA construct comprising a CDS bearing at least about 90% identity to the sequence represented by SEQ ID No. 2 or a fragment thereof, a 5’ terminal cap structure selected from a group comprising m7Gppp5'N (CapO), m7Gppp5'Nm (Capl), chemically synthesized cap analogs such as ARCA (3'-O-Me-m7G(5') ppp(5')G) or CleanCap capping reagents such as CleanCapAG or CleanCapM6 or other commercially available or novel cap reagents that perform the same function, a 5 ’ UTR selected from a group comprising sequences represented by SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO. 5, SEQ ID NO. 6, SEQ ID NO. 7 and SEQ ID NO. 8, a 3 ’UTR selected from a group comprising sequences represented by SEQ ID NO. 9, SEQ ID NO. 10, SEQ ID NO. 11, SEQ ID NO. 12, SEQ ID NO. 13 and SEQ ID NO. 14 and a polyA tail comprising about 60 As to about 120 As.
In some embodiments, the present disclosure provides an mRNA construct comprising a CDS represented by SEQ ID No. 2 or SEQ ID No. 29 or a fragment thereof, a 5’ terminal cap structure selected from a group comprising m7Gppp5'N (CapO), m7Gppp5'Nm (Capl), chemically synthesized cap analogs such as ARCA (3'-O-Me-m7G(5') ppp(5')G) or CleanCap capping reagents such as CleanCapAG or CleanCapM6 or other commercially available or novel cap reagents that perform the same function, a 5 ’ UTR selected from a group comprising sequences represented by SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO. 5, SEQ ID NO. 6, SEQ ID NO. 7 and SEQ ID NO. 8, a 3 ’UTR selected from a group comprising sequences represented by SEQ ID NO. 9, SEQ ID NO. 10, SEQ ID NO. 11, SEQ ID NO. 12, SEQ ID NO. 13 and SEQ ID NO. 14 and a polyA tail comprising about 60 As to about 120 As. In some embodiments, the mRNA construct may additionally comprise a natural or synthetic IRES element and/or a circularizing element. In a non-limiting embodiment, the circularizing element may be obtained from Group I or Group II introns, T4 td intron or other viral sources, derived from intronic sequences from any RNAs known to generate circRNAs by regulation of the spliceosome or from any other published sources.
Thus, in some embodiments, the messenger ribonucleic acid (mRNA) construct of the present disclosure comprises a CDS encoding the MMUT protein or a fragment thereof and 5'UTR element(s), 3'UTR element(s) and apoly(A) sequence(s), and optionally, a natural or synthetic IRES element and/or a circularizing element as defined above.
In some embodiments, the messenger ribonucleic acid (mRNA) construct of the present disclosure comprises a CDS encoding the MMUT protein or a fragment thereof and a 5'UTR element, a 3'UTR element, a poly(A) sequence and a natural or synthetic IRES element and/or a circularizing element.
In an exemplary embodiment, the messenger ribonucleic acid (mRNA) construct of the present disclosure comprises a CDS bearing at least about 90% identity to the sequence represented by SEQ ID No. 2 or a fragment thereof and a 5'UTR element, a 3'UTR element, a poly(A) sequence and a natural or synthetic IRES element and/or a circularizing element as defined above, wherein the CDS optionally comprises from about 1% to about 100% modified nucleotides.
In some embodiments, the messenger ribonucleic acid (mRNA) construct of the present disclosure comprises sequence represented by SEQ ID No. 2 or SEQ ID No. 29 or a fragment thereof and a 5'UTR element, a 3'UTR element, a poly(A) sequence and a natural or synthetic IRES element and/or a circularizing element as defined above, wherein the CDS optionally comprises from about 1% to about 100% modified nucleotides.
In some embodiments, the mRNA construct as described in the above embodiments may be a circular or a linear construct.
The mRNA therapeutics manufacturing process is generally divided into two stages: preparation of DNA templates and preparation/purification of the mRNA produced from the DNA templates. In some embodiments, the mRNA is produced by in vitro transcription of the DNA template. To facilitate the production of the mRNA therapeutic of the present disclosure, further provided herein is a DNA template for production of the mRNA construct as described above.
In a non-limiting embodiment, DNA template for in vitro RNA transcription (IVT) may be obtained by cloning of a nucleic acid, in particular cDNA corresponding to the respective RNA to be in vitro transcribed and introducing it into an appropriate vector for in vitro transcription, for example into plasmid DNA. In some embodiments, the DNA template encodes a 5' untranslated region (UTR), includes a CDS flanked by start and stop codons, and encodes a 3'UTR and poly A tail. In some embodiments, the DNA template comprises an RNA polymerase promoter. The promoter for controlling in vitro transcription can be any promoter for any DNA-dependent RNA polymerase. In some embodiments, the DNA template further comprises a terminator region which serves to stop transcription. In some embodiments, the DNA template may additionally comprise an expression enhancing element.
The cDNA may be obtained by reverse transcription of mRNA or chemical synthesis. Alternatively, the DNA template for in vitro RNA synthesis may also be obtained by gene synthesis. In some embodiments, the DNA template may be obtained by restriction digestion or via PCR.
In some embodiments, cells, eg, bacterial cells such as E. coli, DH-5 alpha cells are transformed using the plasmid DNA template. In some embodiments, transformed cells are cultured to replicate plasmid DNA, then isolated and purified. The next step is in vitro transcription during which the DNA template is transcribed into mRNA. Without intending to be limited by theory, this enzymatic reaction uses elements of the natural transcription process, including RNA polymerase and nucleotide triphosphates.
Methods for in vitro transcription (IVT) are known in the art (see, e.g., Geall et al. (2013) Semin. Immunol. 25(2): 152-159; Brunelle et al. (2013) Methods Enzymol. 530: 101-14). Reagents used in said method typically include:
1) a DNA template with a promoter sequence that has a high binding affinity for its respective RNA polymerase such as bacteriophage-encoded RNA polymerases, wherein the DNA template may be linearized;
2) ribonucleoside triphosphates (NTPs) for the four bases (adenine, cytosine, guanine and uracil);
3) optionally a CAP analogue as defined above (e.g. m7G(5')ppp(5')G (m7G)); 4) a DNA-dependent RNA polymerase capable of binding to the promoter sequence within the linearized DNA template (e.g. T7, T3 or SP6 RNA polymerase);
5) optionally a ribonuclease (RNase) inhibitor to inactivate any contaminating RNase;
6) optionally a pyrophosphatase to degrade pyrophosphate, which may inhibit transcription;
7) MgCh, which supplies Mg2+ ions as a co-factor for the polymerase;
8) a buffer to maintain a suitable pH value, which can also contain antioxidants (e.g. DTT), and/or polyamines such as spermidine at optimal concentrations.
Accordingly, to facilitate production of the mRNA construct of the present disclosure, in some embodiments, further provided herein is a DNA template that is capable of generating the mRNA construct as defined above.
In some embodiments, the DNA template comprises regions and/or regulatory elements involved in pre -mRNA processing, transcription, mRNA stability, along with the nucleotide sequence as defined above.
In some embodiments, the DNA template comprises a 5' untranslated region (UTR), a promoter, a sequence encoding the MMUT protein or a fragment thereof and a 3 'UTR.
In some embodiments, the DNA template, optionally, further comprises enhancer and/or terminator regions. In some embodiments, the enhancer and terminator regions are each optionally present in the DNA template. When present, the enhancer and terminator regions are selected in accordance with the polymerase employed to regulate its activity. In some embodiments, the enhancer and terminator regions may not be incorporated into the DNA template as such and may be part of the plasmid into which they are subsequently introduced.
In some embodiments, the DNA template comprises a 5' untranslated region (UTR), a promoter, a sequence encoding the MMUT protein or a fragment thereof, a 3 'UTR and optionally, enhancer and/or terminator regions.
In some embodiments, the DNA template comprises a 5' untranslated region (UTR), a promoter, a sequence encoding the MMUT protein or a fragment thereof, a 3 'UTR and enhancer and/or terminator regions.
In some embodiments, the DNA template comprises a 5' untranslated region (UTR), a promoter, a sequence encoding the MMUT protein or a fragment thereof, a 3 'UTR and terminator regions. In some embodiments, the 5’ UTR in the DNA template may be preceded by regulatory elements such as but not limited to an AT-rich region, and suitable promoter(s).
In some embodiments, the AT-rich region can be any combination of A and T about 4-6 bases upstream of the promoter. A non-limiting example of this region includes but it not limited to TTAATT.
Thus, in some embodiments, the 5 ’ UTR in the DNA template may be preceded by regulatory elements such as but not limited to an AT-rich region, an example of which includes but is not limited to TTAATT or any combination of A and T about 4-6 bases upstream of the promoter the T7 promoter), and suitable promoter(s).
Taken together, in some embodiments, the DNA template comprises along with a CDS encoding the MMUT protein or a fragment thereof, one or more elements selected from a group comprising an AT -rich region, a promoter, a 5' untranslated region (UTR), an enhancer region a terminator region and a 3 'UTR region.
In some embodiments, the DNA template comprises along with a CDS encoding the MMUT protein or a fragment thereof, two or more elements selected from a group comprising an AT rich region, a promoter, a 5' untranslated region (UTR), an enhancer region, a terminator region and a 3 'UTR region.
In some embodiments, the DNA template comprises along with the CDS encoding the MMUT protein or a fragment thereof, three or more elements selected from a group comprising an AT rich region, a promoter, a 5' untranslated region (UTR), an enhancer region, a terminator region and a 3 'UTR region.
In some embodiments, the DNA template comprises along with the CDS encoding the MMUT protein or a fragment thereof, four or more elements selected from a group comprising an AT -rich region, a promoter, a 5' untranslated region (UTR), an enhancer region, a terminator region and a 3 'UTR region.
In some embodiments, the DNA template comprises along with the CDS encoding the MMUT protein or a fragment thereof, an AT -rich region, a promoter, a 5' untranslated region (UTR), a terminator region and a 3 'UTR region. In some embodiments, the DNA template comprises along with the CDS encoding the MMUT protein or a fragment thereof, an AT -rich region, a promoter, a 5' untranslated region (UTR) and a 3 'UTR region.
In some embodiments, the DNA template comprises along with the CDS encoding the MMUT protein or a fragment thereof, an AT -rich region, a promoter, a 5' untranslated region (UTR), an enhancer region, a terminator region and a 3'UTR.
The polyA sequence in the mRNA construct may be encoded by the template or added post- transcriptionally. Thus, in some embodiments, the DNA template may optionally further comprise a polyA signal.
Thus, in some embodiments, the DNA template comprises along with the CDS encoding the MMUT protein or a fragment thereof, an AT -rich region, a promoter, a 5' untranslated region (UTR), a terminator region, a 3'UTR, and optionally, a polyA signal.
In some embodiments, the DNA template comprises along with the CDS encoding the MMUT protein or a fragment thereof, an AT -rich region, a promoter, a 5' untranslated region (UTR), a terminator region, a 3'UTR, and a polyA signal.
In some embodiments, the DNA template comprises along with the CDS encoding the MMUT protein or a fragment thereof, an AT -rich region, a promoter, a 5' untranslated region (UTR), a 3'UTR, and a polyA signal.
In some embodiments, the DNA template comprises along with the CDS encoding the MMUT protein or a fragment thereof, an AT -rich region, a promoter, a 5' untranslated region (UTR), an enhancer region, a terminator region, a 3'UTR, and a polyA signal..
In some embodiments, the DNA template comprises along with the CDS encoding the MMUT protein or a fragment thereof, an AT -rich region, a promoter, a 5' untranslated region (UTR), an enhancer region, a terminator region, a 3'UTR, and a polyA signal.
In some embodiments, non-limiting examples of poly(A) signal sequence(s) employable in the present disclosure include SV40 PolyA sequence (SEQ ID No. 39), Synthetic PolyA site, Bovine growth hormone (bGH) PolyA site (SEQ ID No. 40), and other suitable poly(A) signal known to a person skilled in the art. In some embodiments, the poly(A) signal sequence(s) are selected from a group comprising SV40 PolyA sequence, Synthetic PolyA site and Bovine growth hormone (bGH) PolyA site.
In some embodiments, the DNA template may further comprise a suitable restriction site for linearization of the IVT template for mRNA synthesis by in vitro transcription. Examples of such restriction sites include but are not limited to a Type II restriction site such as that recognized by Type II enzymes such as Notl, EcoRI, or Type IIS enzymes such as BbsI and BstB 1.
Accordingly, in some embodiments, the DNA template comprises along with the CDS encoding the MMUT protein or a fragment thereof, one or more elements selected from a group comprising an AT -rich region, a promoter, a 5' untranslated region (UTR), an enhancer region, a terminator region, a 3 'UTR, optionally, a polyA signal, and a suitable restriction site to generate an mRNA construct without an overhang.
In some embodiments, the DNA template comprises along with the CDS encoding the MMUT protein or a fragment thereof, one or more elements selected from a group comprising an AT - rich region, a promoter, a 5' untranslated region (UTR), a terminator region, a 3 'UTR, a polyA signal, and a suitable restriction site to generate an mRNA construct without an overhang.
In some embodiments, the DNA template comprises along with the CDS encoding the MMUT protein or a fragment thereof, one or more elements selected from a group comprising an AT - rich region, a promoter, a 5' untranslated region (UTR), an enhancer region, a terminator region, a 3'UTR, a polyA signal, and a suitable restriction site to generate an mRNA construct without an overhang.
In some embodiments, the DNA template comprises along with the CDS encoding the MMUT protein or a fragment thereof, one or more elements selected from a group comprising an AT - rich region, a promoter, a 5' untranslated region (UTR), a terminator region, a 3'UTR, a polyA signal, and a suitable restriction site to generate an mRNA construct without an overhang.
In some embodiments, the DNA template comprises along with the CDS encoding the MMUT protein or a fragment thereof, one or more elements selected from a group comprising an AT - rich region, a promoter, a 5' untranslated region (UTR), a 3'UTR, a polyA signal, and a suitable restriction site to generate an mRNA construct without an overhang. In some embodiments, the DNA template comprises along with the CDS encoding the MMUT protein or a fragment thereof, one or more elements selected from a group comprising an AT - rich region, a promoter, a 5' untranslated region (UTR), a terminator region, a 3'UTR, a polyA signal, and a Type II restriction site to generate an mRNA construct without an overhang.
In some embodiments, the DNA template comprises along with the CDS encoding the MMUT protein or a fragment thereof, one or more elements selected from a group comprising an AT - rich region, a promoter, a 5' untranslated region (UTR), a 3'UTR, a polyA signal, and a Type II restriction site to generate an mRNA construct without an overhang.
In some embodiments, the CDS in the DNA template has a sequence bearing at least about 90% identity to a sequence represented by SEQ ID No. 15 or is a fragment thereof.
In some embodiments, the CDS has a sequence represented by SEQ ID No. 15 or SEQ ID No. 31 or a fragment thereof.
Accordingly, in some embodiments, the DNA template comprises along with the CDS having sequence bearing at least about 90% identity to a sequence represented by SEQ ID No. 15 or a fragment thereof, one or more elements selected from a group comprising an AT -rich region, a promoter, a 5' untranslated region (UTR), an enhancer region, a terminator region, a 3'UTR, optionally, a polyA signal, and a suitable restriction site to generate an mRNA construct without an overhang.
In some embodiments, the DNA template comprises along with the CDS having sequence bearing at least about 90% identity to a sequence represented by SEQ ID No. 15 or a fragment thereof, one or more elements selected from a group comprising an AT -rich region, a promoter, a 5' untranslated region (UTR), a terminator region, a 3'UTR, optionally, a polyA signal, and a suitable restriction site to generate an mRNA construct without an overhang.
In some embodiments, the DNA template comprises along with the CDS having sequence bearing at least about 90% identity to a sequence represented by SEQ ID No. 15 or a fragment thereof, one or more elements selected from a group comprising an AT -rich region, a promoter, a 5' untranslated region (UTR), an enhancer region, a terminator region, a 3'UTR, a polyA signal, and a suitable restriction site to generate an mRNA construct without an overhang.
In some embodiments, the DNA template comprises along with the CDS having sequence bearing at least about 90% identity to a sequence represented by SEQ ID No. 15 or a fragment thereof, one or more elements selected from a group comprising an AT -rich region, a promoter, a 5' untranslated region (UTR), a 3'UTR, a polyA signal, and a suitable restriction site to generate an mRNA construct without an overhang.
In some embodiments, the promoter may be any promoter capable of enabling adequate expression of the polynucleotide of the present disclosure. Examples of such promoters include but are not limited to T7, SP6, T3 and LP1 or HCR hAAT promoter.
In some embodiments, the promoter is selected from a group comprising, T7, SP6, T3 and LP1 or HCR hAAT promoters.
In a non-limiting exemplary embodiment, the promoter is an T7 promoter with an initiator.
In an exemplary embodiment, the promoter is an LP1 promoter with an initiator (SEQ ID No. 32). The LP1 promoter is interchangeably also referred as HCR hAAT.
In some embodiments, the 5 ’ UTR is selected from a group comprising sequences represented by SEQ ID NO. 16, SEQ ID NO. 17, SEQ ID NO. 18, SEQ ID NO. 19, SEQ ID NO. 20 and SEQ ID NO. 21, SEQ ID NO. 33, SEQ ID NO. 34, SEQ ID NO. 35, SEQ ID NO. 36 and SEQ ID NO. 38.
In some embodiments, the 3 ’UTR is selected from a group comprising sequences represented by SEQ ID NO. 22, SEQ ID NO. 23, SEQ ID NO. 24, SEQ ID NO. 25, SEQ ID NO. 26, SEQ ID NO. 27 and SEQ ID NO. 37.
In some embodiments, the DNA template comprises an AT-rich region, a promoter, a 5' untranslated region (UTR) selected from a group comprising sequences represented by SEQ ID NO. 16, SEQ ID NO. 17, SEQ ID NO. 18, SEQ ID NO. 19, SEQ ID NO. 20 and SEQ ID NO. 21, SEQ ID NO. 33, SEQ ID NO. 34, SEQ ID NO. 35, SEQ ID NO. 36 and SEQ ID NO. 38, a CDS encoding the MMUT protein or a fragment thereof, an enhancer region, a terminator region, a 3'UTR selected from a group comprising sequences represented by SEQ ID NO. 22, SEQ ID NO. 23, SEQ ID NO. 24, SEQ ID NO. 25, SEQ ID NO. 26, SEQ ID NO. 27 and SEQ ID NO. 37, optionally a polyA signal and a suitable restriction site to generate an mRNA construct without an overhang.
In some embodiments, the DNA template comprises an AT-rich region, a promoter, a 5' untranslated region (UTR) selected from a group comprising sequences represented by SEQ ID NO. 16, SEQ ID NO. 17, SEQ ID NO. 18, SEQ ID NO. 19, SEQ ID NO. 20 and SEQ ID NO. 21, SEQ ID NO. 33, SEQ ID NO. 34, SEQ ID NO. 35, SEQ ID NO. 36 and SEQ ID NO. 38, a terminator region, a CDS bearing at least about 90% identity to a sequence represented by SEQ ID No. 15 or a fragment thereof, a 3'UTR selected from a group comprising sequences represented by SEQ ID NO. 22, SEQ ID NO. 23, SEQ ID NO. 24, SEQ ID NO. 25, SEQ ID NO. 26, SEQ ID NO. 27 and SEQ ID NO. 37, optionally a polyA signal and a suitable restriction site to generate an mRNA construct without an overhang.
In some embodiments, the DNA template comprises an AT-rich region, a promoter, a 5' untranslated region (UTR) selected from a group comprising sequences represented by SEQ ID NO. 16, SEQ ID NO. 17, SEQ ID NO. 18, SEQ ID NO. 19, SEQ ID NO. 20 and SEQ ID NO. 21, SEQ ID NO. 33, SEQ ID NO. 34, SEQ ID NO. 35, SEQ ID NO. 36 and SEQ ID NO. 38, a terminator region, a CDS represented by SEQ ID No. 15 or SEQ ID No. 31, a 3'UTR selected from a group comprising sequences represented by SEQ ID NO. 22, SEQ ID NO. 23, SEQ ID NO. 24, SEQ ID NO. 25, SEQ ID NO. 26, SEQ ID NO. 27 and SEQ ID NO. 37, optionally a polyA signal and a suitable restriction site to generate an mRNA construct without an overhang.
In some embodiments, the DNA template comprises an AT-rich region, a promoter, a 5' untranslated region (UTR) selected from a group comprising sequences represented by SEQ ID NO. 16, SEQ ID NO. 17, SEQ ID NO. 18, SEQ ID NO. 19, SEQ ID NO. 20 and SEQ ID NO. 21, SEQ ID NO. 33, SEQ ID NO. 34, SEQ ID NO. 35, SEQ ID NO. 36 and SEQ ID NO. 38, a terminator region, a CDS bearing at least about 90% identity to a sequence represented by SEQ ID No. 15 or a fragment thereof, a 3'UTR selected from a group comprising sequences represented by SEQ ID NO. 22, SEQ ID NO. 23, SEQ ID NO. 24, SEQ ID NO. 25, SEQ ID NO. 26, SEQ ID NO. 27 and SEQ ID NO. 37, a polyA signal and a suitable restriction site to generate an mRNA construct without an overhang.
In some embodiments, the DNA template comprises an AT-rich region, a promoter, a 5' untranslated region (UTR) selected from a group comprising sequences represented by SEQ ID NO. 16, SEQ ID NO. 17, SEQ ID NO. 18, SEQ ID NO. 19, SEQ ID NO. 20 and SEQ ID NO. 21, SEQ ID NO. 33, SEQ ID NO. 34, SEQ ID NO. 35, SEQ ID NO. 36 and SEQ ID NO. 38, a terminator region, a CDS bearing at least about 90% identity to a sequence represented by SEQ ID No. 15 or a fragment thereof, a 3'UTR selected from a group comprising sequences represented by SEQ ID NO. 22, SEQ ID NO. 23, SEQ ID NO. 24, SEQ ID NO. 25, SEQ ID NO. 26, SEQ ID NO. 27 and SEQ ID NO. 37, a polyA signal and a Type II restriction site to generate an mRNA construct without an overhang.
In some embodiments, the DNA template comprises an AT-rich region, a promoter selected from a group comprising HCRhAAT, T7, SP6, T3 and LP1 promoters, a 5' untranslated region (UTR) selected from a group comprising sequences represented by SEQ ID NO. 16, SEQ ID NO. 17, SEQ ID NO. 18, SEQ ID NO. 19, SEQ ID NO. 20 and SEQ ID NO. 21, SEQ ID NO. 33, SEQ ID NO. 34, SEQ ID NO. 35, SEQ ID NO. 36 and SEQ ID NO. 38, a terminator region, a CDS represented by SEQ ID No. 15 or SEQ ID No. 31 or a fragment thereof, a 3 'UTR selected from a group comprising sequences represented by SEQ ID NO. 22, SEQ ID NO. 23, SEQ ID NO. 24, SEQ ID NO. 25, SEQ ID NO. 26, SEQ ID NO. 27 and SEQ ID NO. 37, a polyA signal and a suitable restriction site for linearization of the IVT template for mRNA synthesis by in vitro transcription, examples of which include but are not limited to a Type II restriction site such as that recognized by Type II enzymes such as Notl, EcoRI, or Type IIS enzymes such as BbsI and BstBl.
In some embodiments, the DNA template comprises an AT-rich region, a promoter selected from a group comprising HCRhAAT, T7, SP6, T3 and LP1 promoters, a 5' untranslated region (UTR) selected from a group comprising sequences represented by SEQ ID NO. 16, SEQ ID NO. 17, SEQ ID NO. 18, SEQ ID NO. 19, SEQ ID NO. 20 and SEQ ID NO. 21, SEQ ID NO. 33, SEQ ID NO. 34, SEQ ID NO. 35, SEQ ID NO. 36 and SEQ ID NO. 38, a terminator region, a CDS bearing at least about 90% identity to a sequence represented by SEQ ID No. 15 or a fragment thereof, a 3'UTR selected from a group comprising sequences represented by SEQ ID NO. 22, SEQ ID NO. 23, SEQ ID NO. 24, SEQ ID NO. 25, SEQ ID NO. 26, SEQ ID NO. 27 and SEQ ID NO. 37, optionally a polyA signal selected from a group comprising SV40 PolyA sequence, Synthetic PolyA site and Bovine growth hormone (bGH) PolyA site and a suitable restriction site to generate an mRNA construct without an overhang.
In some embodiments, the DNA template comprises an AT-rich region, a promoter selected from a group comprising HCRhAAT, T7, SP6, T3 and LP1 promoters, a 5' untranslated region (UTR) selected from a group comprising sequences represented by SEQ ID NO. 16, SEQ ID NO. 17, SEQ ID NO. 18, SEQ ID NO. 19, SEQ ID NO. 20 and SEQ ID NO. 21, SEQ ID NO. 33, SEQ ID NO. 34, SEQ ID NO. 35, SEQ ID NO. 36 and SEQ ID NO. 38, a terminator region, a CDS represented by SEQ ID No. 15 or SEQ ID No. 31 or a fragment thereof, a 3'UTR selected from a group comprising sequences represented by SEQ ID NO. 22, SEQ ID NO. 23, SEQ ID NO. 24, SEQ ID NO. 25, SEQ ID NO. 26, SEQ ID NO. 27 and SEQ ID NO. 37, apolyA signal selected from a group comprising SV40 PolyA sequence, Synthetic PolyA site and Bovine growth hormone (bGH) PolyA site and a suitable restriction site for linearization of the IVT template for mRNA synthesis by in vitro transcription, examples of which include but are not limited to a Type II restriction site such as that recognized by Type II enzymes such as Notl, EcoRI, or Type IIS enzymes such as BbsI and BstBl.
In some embodiments, the DNA template comprises an AT-rich region, a promoter selected from a group comprising HCRhAAT, T7, SP6, T3 and LP1 promoters, a 5' untranslated region (UTR) selected from a group comprising sequences represented by SEQ ID NO. 16, SEQ ID NO. 17, SEQ ID NO. 18, SEQ ID NO. 19, SEQ ID NO. 20, SEQ ID NO. 21, SEQ ID NO. 33, SEQ ID NO. 34, SEQ ID NO. 35, SEQ ID NO. 36 and SEQ ID NO. 38, a CDS represented by SEQ ID No. 15 or SEQ ID No. 31 or a fragment thereof, a 3 'UTR selected from a group comprising sequences represented by SEQ ID NO. 22, SEQ ID NO. 23, SEQ ID NO. 24, SEQ ID NO. 25, SEQ ID NO. 26, SEQ ID NO. 27 and SEQ ID NO. 37, apolyA signal selected from a group comprising SV40 PolyA sequence, Synthetic PolyA site and Bovine growth hormone (bGH) PolyA site and a suitable restriction site for linearization of the IVT template for mRNA synthesis by in vitro transcription, examples of which include but are not limited to a Type II restriction site such as that recognized by Type II enzymes such as Notl, EcoRI, or Type IIS enzymes such as BbsI and BstB 1.
In some embodiments, the DNA template comprises an AT-rich region that is any combination of A and T about 4-6 bases upstream of the promoter, a promoter selected from a group comprising HCR hAAT, T7, SP6, T3 and LP1 promoters, a 5' untranslated region (UTR) selected from a group comprising sequences represented by SEQ ID NO. 16, SEQ ID NO. 17, SEQ ID NO. 18, SEQ ID NO. 19, SEQ ID NO. 20 and SEQ ID NO. 21, SEQ ID NO. 33, SEQ ID NO. 34, SEQ ID NO. 35, SEQ ID NO. 36 and SEQ ID NO. 38, a terminator region, a CDS bearing at least about 90% identity to a sequence represented by SEQ ID No. 15 or a fragment thereof, a 3'UTR selected from a group comprising sequences represented by SEQ ID NO. 22, SEQ ID NO. 23, SEQ ID NO. 24, SEQ ID NO. 25, SEQ ID NO. 26, SEQ ID NO. 27 and SEQ ID NO. 37, optionally a polyA signal selected from a group comprising SV40 PolyA sequence, Synthetic PolyA site and Bovine growth hormone (bGH) PolyA site and a suitable restriction site to generate an mRNA construct without an overhang. In some embodiments, the DNA template comprises an AT-rich region which includes but is not limited to TTAATT, a promoter selected from a group comprising HCR hAAT, T7, SP6, T3 and LP1 promoters, a 5' untranslated region (UTR) selected from a group comprising sequences represented by SEQ ID NO. 16, SEQ ID NO. 17, SEQ ID NO. 18, SEQ ID NO. 19, SEQ ID NO. 20 and SEQ ID NO. 21, SEQ ID NO. 33, SEQ ID NO. 34, SEQ ID NO. 35, SEQ ID NO. 36 and SEQ ID NO. 38, a terminator region, a CDS represented by SEQ ID No. 15 or SEQ ID No. 31 or a fragment thereof, a 3'UTR selected from a group comprising sequences represented by SEQ ID NO. 22, SEQ ID NO. 23, SEQ ID NO. 24, SEQ ID NO. 25, SEQ ID NO. 26, SEQ ID NO. 27 and SEQ ID NO. 37, a polyA signal selected from a group comprising SV40 PolyA sequence, Synthetic PolyA site and Bovine growth hormone (bGH) PolyA site and a suitable restriction site for linearization of the IVT template for mRNA synthesis by in vitro transcription, examples of which include but are not limited to a Type II restriction site such as that recognized by Type II enzymes such as Notl, EcoRI, or Type IIS enzymes such as BbsI and BstBl.
In some embodiments, the DNA template comprises an AT-rich region which includes but is not limited to TTAATT, a promoter selected from a group comprising HCR hAAT, T7, SP6, T3 and LP1 promoters, a 5' untranslated region (UTR) selected from a group comprising sequences represented by SEQ ID NO. 16, SEQ ID NO. 17, SEQ ID NO. 18, SEQ ID NO. 19, SEQ ID NO. 20 and SEQ ID NO. 21, SEQ ID NO. 33, SEQ ID NO. 34, SEQ ID NO. 35, SEQ ID NO. 36 and SEQ ID NO. 38, a CDS represented by SEQ ID No. 15 or SEQ ID No. 31 or a fragment thereof, a 3'UTR selected from a group comprising sequences represented by SEQ ID NO. 22, SEQ ID NO. 23, SEQ ID NO. 24, SEQ ID NO. 25, SEQ ID NO. 26, SEQ ID NO. 27 and SEQ ID NO. 37, a polyA signal selected from a group comprising SV40 PolyA sequence, Synthetic PolyA site and Bovine growth hormone (bGH) PolyA site and a suitable restriction site for linearization of the IVT template for mRNA synthesis by in vitro transcription, examples of which include but are not limited to a Type II restriction site such as that recognized by Type II enzymes such as Notl, EcoRI, or Type IIS enzymes such as BbsI and BstBl.
In a non-limiting exemplary embodiment, the DNA template comprises each of the aforesaid elements in the order as defined in the above embodiment.
In an exemplary embodiment, the DNA template comprises a CDS bearing at least about 90% identity to a sequence represented by SEQ ID No. 15 or a fragment thereof along with a promoter represented by SEQ ID No. 32, one or more 5' untranslated regions selected from SEQ ID NO. 33, SEQ ID NO. 34, SEQ ID NO. 35 and SEQ ID NO. 38, one or more copies of 3' untranslated region represented by SEQ ID NO. 37 and polyA signal(s) selected from SEQ ID NO. 39 and SEQ ID NO. 40.
In some embodiments, the DNA template comprises a suitable enzyme cleavage site between the different elements in the template as described above.
In some embodiments, modification to the mRNA such as modified nucleotides, 5’ capping and Poly A tail to obtain the mRNA construct may be introduced during or after in vitro transcription for stability and efficient transduction of the mRNA in cells.
In some embodiments, the DNA template is contained in a plasmid. In some embodiments, the plasmid is a mammalian expression system plasmid that allows for codon optimization and mRNA generation using T7, SP6 or T3 polymerase.
In some embodiments, the DNA template is placed in the plasmid between two restriction sites.
A non-limiting representation of the DNA template is provided in Figure 3A.
In some embodiments, apart from mRNA generation, the DNA template of the present disclosure may alternatively also be employed for gene therapy. Accordingly, in some embodiments, the DNA template of the present disclosure may be directly delivered to a subject in need thereof. In some embodiments, the direct delivery of the DNA template is facilitated by a viral vector. In some embodiments, the recombinant vector is a recombinant parvoviral vector. In some exemplary, non-limiting embodiments, the recombinant vector is a recombinant AAV (rAAV) vector.
In some embodiments, the recombinant AAV vector may be of any serotype (e.g., comprise any AAV serotype genome and/or capsid protein), provided it is capable of infecting or transducing the target cells and/or tissues.
In a non-limiting embodiment, the recombinant AAV vector may be of any serotype selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10 and AAV 11 or recombinant serotypes such as Rec2 and Rec3.
Therefore, particularly encompassed by the present disclosure is a recombinant AAV vector which retains only the replication and packaging signals of AAV, and which comprises a nucleic acid sequence encoding MMUT, or a derivative or functional fragment thereof. Specifically, envisaged herein is an AAV vector comprising the nucleic acid sequence encoding MMUT sequence bearing at least about 90% identity to a sequence represented by SEQ ID No. 15 or a fragment thereof. Further envisaged herein is an AAV vector comprising the DNA template as described above comprising the nucleic acid sequence encoding MMUT sequence bearing at least about 90% identity to a sequence represented by SEQ ID No. 15 or a fragment thereof.
In some embodiments, the AAV vector comprises the nucleic acid sequence encoding MMUT sequence bearing at least about 90% identity to a sequence represented by a CDS represented by SEQ ID No. 15 or SEQ ID No. 31 or a fragment thereof.
In some embodiments, the present disclosure provides a recombinant AAV vector comprising the DNA template described above which comprises the nucleic acid sequence encoding MMUT sequence bearing at least about 90% identity to a sequence, preferably represented by SEQ ID No. 15 or SEQ ID No. 31 or a fragment thereof.
In some embodiments, the present disclosure provides a recombinant AAV vector comprising a DNA template that comprises along with the CDS having sequence bearing at least about 90% identity to a sequence represented by SEQ ID No. 15 or a fragment thereof, one or more elements selected from a group comprising an AT-rich region, a promoter, a 5' untranslated region (UTR), an enhancer region, a terminator region, a 3'UTR, optionally, a polyA signal.
In some embodiments, the present disclosure provides a recombinant AAV vector comprising a DNA template that comprises along with the CDS having sequence bearing at least about 90% identity to a sequence represented by SEQ ID No. 15 or a fragment thereof, two or more elements selected from a group comprising an AT-rich region, a promoter, a 5' untranslated region (UTR), an enhancer region, a terminator region, a 3'UTR, optionally, a polyA signal.
In some embodiments, the present disclosure provides a recombinant AAV vector comprising a DNA template that comprises along with the CDS having sequence bearing at least about 90% identity to a sequence represented by SEQ ID No. 15 or a fragment thereof, three or more elements selected from a group comprising an AT-rich region, a promoter, a 5' untranslated region (UTR), an enhancer region, a terminator region, a 3'UTR, optionally, a polyA signal.
In some embodiments, the present disclosure provides a recombinant AAV vector comprising a DNA template that comprises along with the CDS having sequence bearing at least about 90% identity to a sequence represented by SEQ ID No. 15 or a fragment thereof, four or more elements selected from a group comprising an AT-rich region, a promoter, a 5' untranslated region (UTR), an enhancer region, a terminator region, a 3'UTR, optionally, a polyA signal.
More preferably, the present disclosure provides a recombinant AAV vector comprising a DNA template that comprises along with the CDS having sequence bearing at least about 90% identity to a sequence represented by SEQ ID No. 15 or a fragment thereof, one or more elements selected from a group comprising a promoter, a 5' untranslated region (UTR), a 3'UTR and a polyA signal.
Taken together, the present disclosure provides both mRNA and DNA based therapeutics by way of the mRNA construct as well as the DNA template that is capable of being directly delivered to a subject through recombinant AAV medicated delivery. While the recombinant AAV comprising the DNA template of the present disclosure is described in the above embodiments, the following embodiments further describe the mode of delivery of the mRNA construct of the present disclosure.
The present disclosure also provides a host cell comprising the DNA template as described hereinabove.
In some embodiments, the host cell may be a prokaryotic cell or a eukaryotic cell.
In some embodiments, the prokaryotic cell is selected from a group comprising bacterial cell, fungal cell, yeast cell, viral cell and protozoan cell.
In some embodiments, the eukaryotic cell is selected from a group comprising an insect cell or a mammalian cell.
In some exemplary, non-limiting embodiments, the host cell is selected from a group comprising HEK293, HEK293T, Sf9, C12 and HeLa cell.
Although the subsequent embodiments focus on further aspects of the present disclosure such as a lipid nanoparticle comprising the mRNA construct of the present disclosure, a composition comprising the mRNA construct and a kit comprising the mRNA construct, the features of the mRNA construct as well as the DNA template, including the components as described by any of the embodiments above are applicable to the each of the said additional aspects of the present disclosure. For the sake of brevity, and to avoid repetition, each of those embodiments are not being reiterated here again with respect to the said aspects of the invention. However, each of the said embodiments completely fall within the purview of the said aspects of the invention.
Once purified and obtained after in-vitro transcription, to facilitate efficient delivery during administration, in some embodiments, the mRNA construct of the present disclosure may be encapsulated in a lipid nanoparticle (LNP) or exosomes.
Accordingly, in some embodiments, provided herein is a lipid nanoparticle encapsulated mRNA construct (mRNA-LNP) comprising a CDS encoding the MMUT protein, a 5’ cap, a 5'UTR element, a 3'UTR element and a poly(A) sequence.
In some embodiments, provided herein is a lipid nanoparticle encapsulated mRNA construct (mRNA-LNP) comprising a CDS encoding the MMUT protein or a fragment thereof, a 5’ cap selected from a group comprising m7Gppp5'N (CapO), m7Gppp5'Nm (Capl), chemically synthesized cap analogs such as ARCA (3'-O-Me-m7G(5') ppp(5')G) or CleanCap capping reagents such as CleanCapAG or CleanCapM6 or other commercially available or novel cap reagents that perform the same function, a 5 'UTR, a 3 'UTR element and/or a poly(A) sequence .
In some embodiments, provided herein is a lipid nanoparticle encapsulated mRNA construct (mRNA-LNP) comprising a CDS encoding the MMUT protein or a fragment thereof, a 5’ cap, a 5'UTR element selected from a group comprising sequences represented by SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO. 5, SEQ ID NO. 6, SEQ ID NO. 7 and SEQ ID NO. 8, a 3'UTR element and/or a poly (A) sequence.
In some embodiments, provided herein is a lipid nanoparticle encapsulated mRNA construct (mRNA-LNP) comprising a CDS encoding the MMUT protein or a fragment thereof, a 5’ cap, a 5'UTR element, a 3'UTR element selected from a group comprising sequences represented by SEQ ID NO. 9, SEQ ID NO. 10, SEQ ID NO. 11, SEQ ID NO. 12, SEQ ID NO. 13 and SEQ ID NO. 14 and/or a poly (A) sequence.
In some embodiments, provided herein is a lipid nanoparticle encapsulated mRNA construct (mRNA-LNP) comprising a CDS encoding the MMUT protein or a fragment thereof, a 5’ cap, a 5'UTR element selected from a group comprising sequences represented by SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO. 5, SEQ ID NO. 6, SEQ ID NO. 7 and SEQ ID NO. 8, a 3'UTR element selected from a group comprising sequences represented by SEQ ID NO. 9, SEQ ID NO. 10, SEQ ID NO. 11, SEQ ID NO. 12, SEQ ID NO. 13 and SEQ ID NO. 14, and a poly(A) sequence. In some embodiments, provided herein is a lipid nanoparticle encapsulated mRNA construct (mRNA-LNP) comprising a CDS encoding the MMUT protein or a fragment thereof, a 5’ cap selected from a group comprising m7Gppp5'N (CapO), m7Gppp5'Nm (Capl), chemically synthesized cap analogs such as ARCA (3'-O-Me-m7G(5') ppp(5')G) or CleanCap capping reagents such as CleanCapAG or CleanCapM6 or other commercially available or novel cap reagents that perform the same function, a 5'UTR element selected from a group comprising sequences represented by SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO. 5, SEQ ID NO. 6, SEQ ID NO. 7 and SEQ ID NO. 8, a 3'UTR element selected from a group comprising sequences represented by SEQ ID NO. 9, SEQ ID NO. 10, SEQ ID NO. 11, SEQ ID NO. 12, SEQ ID NO. 13 and SEQ ID NO. 14, and a poly(A) sequence.
In some embodiments, provided herein is a lipid nanoparticle encapsulated mRNA construct (mRNA-LNP) comprising a CDS bearing at least 90% identity to the sequence represented by SEQ ID No. 2 or a fragment thereof, a 5’ cap, a 5'UTR element selected from a group comprising sequences represented by SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO. 5, SEQ ID NO. 6, SEQ ID NO. 7 and SEQ ID NO. 8, a 3'UTR element selected from a group comprising sequences represented by SEQ ID NO. 9, SEQ ID NO. 10, SEQ ID NO. 11, SEQ ID NO. 12, SEQ ID NO. 13 and SEQ ID NO. 14 and/or a poly (A) sequence.
In some embodiments, provided herein is a lipid nanoparticle encapsulated mRNA construct (mRNA-LNP) comprising a CDS bearing at least 90% identity to the sequence represented by SEQ ID No. 2 or a fragment thereof, a 5’ cap selected from a group comprising m7Gppp5'N (CapO), m7Gppp5'Nm (Capl), chemically synthesized cap analogs such as ARCA (3'-O-Me- m7G(5') ppp(5')G) or CleanCap capping reagents such as CleanCapAG or CleanCapM6 or other commercially available or novel cap reagents that perform the same function, a 5'UTR element selected from a group comprising sequences represented by SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO. 5, SEQ ID NO. 6, SEQ ID NO. 7 and SEQ ID NO. 8, a 3'UTR element selected from a group comprising sequences represented by SEQ ID NO. 9, SEQ ID NO. 10, SEQ ID NO. 11, SEQ ID NO. 12, SEQ ID NO. 13 and SEQ ID NO. 14, and/or a poly(A) sequence.
In some embodiments, provided herein is a lipid nanoparticle encapsulated mRNA construct (mRNA-LNP) comprising a CDS represented by SEQ ID No. 2 or SEQ ID No. 29 or a fragment thereof, a 5’ cap selected from a group comprising m7Gppp5'N (CapO), m7Gppp5'Nm (Capl), chemically synthesized cap analogs such as ARCA (3'-O-Me-m7G(5') ppp(5')G) or CleanCap capping reagents such as CleanCapAG or CleanCapM6 or other commercially available or novel cap reagents that perform the same function, a 5'UTR element selected from a group comprising sequences represented by SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO. 5, SEQ ID NO. 6, SEQ ID NO. 7 and SEQ ID NO. 8 a 3'UTR element selected from a group comprising sequences represented by SEQ ID NO. 9, SEQ ID NO. 10, SEQ ID NO. 11, SEQ ID NO. 12, SEQ ID NO. 13 and SEQ ID NO. 14, and a poly(A) sequence.
In some non-limiting embodiments, the lipid or lipid nanoparticle is selected from a group comprising an ionizable amino lipid, a cationic lipid, a helper lipid, a sterol, steroid or steroid analogues, neutral lipid, phospholipid and a PEG lipid.
The present disclosure further provides a composition comprising the mRNA construct, mRNA-LNP or the recombinant AAV vector as described above in combination with pharmaceutically acceptable excipient(s) or carrier(s).
In some embodiments, non-limiting examples of such pharmaceutically acceptable excipient(s) or carrier(s) include LNP components such as ionizable lipids, PEGylated lipid, cholesterol and its derivatives, structural lipids, and additional components such as triglycerides, mixed glycerides, surfactants and co-solvents. Further provided herein is a method of treating and/or preventing MMA, said method comprising administering the mRNA construct, the mRNA- LNP comprising the nucleotide encoding MMUT protein or fragment thereof or the recombinant AAV or the composition of the present disclosure to a subject in need thereof.
In some embodiments, the subject is a mammal, including but not limited to human being.
In some embodiments, the administration may be carried out in a single dose or in multiple doses.
Without wishing to be limited by theory, the administration of the mRNA construct, the mRNA-LNP comprising the nucleotide encoding MMUT protein or fragment thereof or the recombinant AAV or the composition of the present disclosure to a subject in need thereof, leads to higher expression of MMUT in the subject and therefore leads to reduction of MMA accumulation.
The present disclosure also relates to use of the mRNA sequence, the mRNA construct, the DNA template, the mRNA-LNP, the recombinant AAV or the composition of the present disclosure in treating and/or preventing MMA. Further envisaged herein is a kit comprising the mRNA sequence, the mRNA construct, the DNA template, the mRNA-LNP, the recombinant AAV or the composition, means for administration of the same and optionally, an instruction manual.
Additional embodiments and features of the present disclosure will be apparent to one of ordinary skill in art based on the description provided herein. The embodiments herein provide various features and advantageous details thereof in the description. Descriptions of well- known/conventional methods and techniques are omitted so as to not unnecessarily obscure the embodiments herein.
Further, the disclosure herein provides for examples illustrating the above-described embodiments, and in order to illustrate the embodiments of the present disclosure certain aspects have been employed. The examples used herein for such illustration are intended merely to facilitate an understanding of ways in which the embodiments herein may be practiced and to further enable those of skill in the art to practice the embodiments herein. Accordingly, the following examples should not be construed as limiting the scope of the embodiments herein.
EXAMPLES
Example 1: Codon optimization of the sequence encoding MMUT
The mRNA polynucleotide sequence of the present disclosure (SEQ ID No. 2) encoding MMUT was obtained by in-vitro transcription and codon optimization to maximize expression in the human liver.
A comparison was carried out between SEQ ID NO. 2 and the wild type sequence encoding MMUT of SEQ ID NO. 1 for parameters of Codon Adaptation Index (CAI), Frequency of Optimal Codons (FOP) and GC content and the results shown in Figures 1-2.
As shown in Figure la, SEQ ID NO. 2 shows a high Codon Adaptation Index (CAI) of 0.94 (as shown in green) as compared to the wild type sequence which has a CAI of only 0.65 (shown in blue), which is considered low in terms of expression. Additionally, as shown in Figure lb, SEQ ID NO. 2 has a significantly higher frequency of optimal codons for the desired expression organism (human liver) as compared to the wild type sequence. Further, as shown in Figure 2, SEQ ID NO. 2 has a GC content of 54.86 which is significantly enhanced as compared to the wild-type sequence that has a GC content of only 45.69.
Example 2: construct design and in vitro testing
A series of vectors containing various combinations of 5’ UTR, 3’UTR and other elements as shown in Figure 3A were generated. The various combinations of 5’ UTR, 3 ’UTR tested are provided in the below table -
Table 1:
Figure imgf000036_0001
A reporter construct containing the coding sequences of nano luciferase and EGFP (nLuc-GFP) was cloned into each of the vectors in order to enable rapid testing. mRNA of the reporter was generated by in vitro transcription (IVT) using the mMESSAGE or HiScribe in vitro transcription kit following protocols provided by the manufacturer. Transcribed capped polyadenylated mRNA was purified using the MEGAClear column (Figure 3B). The purified mRNA was used for further analysis. The purified RNA was transfected into HEK293 cells using Jetmessenger RNA transfection reagent or Lipofectamine 3000. EGFP fluorescence and chemiluminescence measurements were made at about 24 hours post transfection and later timepoints as an indicator of translation efficacy. The chemiluminescence reporter used was made with uridine or pseudouridine.
Similarly, translation in vivo was assessed by EGFP fluorescence in zebrafish embryos injected with specific amounts of transcribed mRNA. Representative results for a specific vector (IVT4 - see Table 1) are shown in Figure 3C, D and E, each of which show translation of the fluorescent/chemiluminescent reporters in-vitro and in-vivo thus confirming expression of MMUT facilitated by the mRNA construct of the present disclosure.
Example: 3 Comparison of various MMUT mRNA constructs It is generally reported that the first 8-10 amino acids of coding sequences have a significant impact on the level of translation. In order to test this speculation, combinatorial constructs containing the first 8 amino acids from WT and the rest from codon optimized sequence of the present disclosure (SEQ ID No. 29), and vice versa (SEQ ID No. 28) were generated. A limited library of plasmids containing various combinations of 5’ and 3’UTRs, and 4 different mRNA encoding MMUT CDS were transfected into HEK293T cells. Cells were harvested for immune blotting about 24h after transfection. All of the constructs which were used as template for mRNA synthesis are listed in Table 2 and the protein expression levels are depicted in Figure 4A-C.
Table 2
Figure imgf000037_0001
Figure imgf000038_0001
In comparison with the endogenous WT MMUT (sample 5), the codon optimized version (sample 3) showed significantly higher protein production in cells (Fig 4D). Further, it was also observed that the sample having first 8 amino acids of wild type and rest codon optimized sequence (sample 7), resulted in a significantly higher level of translation as compared to sample 6, where the codon-optimized nucleotides encoding the first 8 amino acids and rest wild type resulted in poor expression. This was contrary to expectation given the known influence of the first 1-8 amino acids on protein expression.
Example 4: In vivo efficacy of the MMUT mRNA
The in vivo efficacy of the MMUT mRNA candidates listed in Table 2 above, was further tested in MMUT knockout zebrafish. MMUT knockout zebrafish larvae generated in-house were used as the animal model for this experiment. The zebra fish larvae were microinjected with mRNA candidate sample 3 listed in Table 2, at about 500pg/embryo in the appropriate buffer. Five days post injection, larvae from the un-injected and injected groups were harvested, along with age matched wild type larvae. Lysates were prepared and MMA levels were measured by LC-MS. Accumulation of MMA was observed in the un-injected F2, while a significant reduction in the same was observed in the group injected with the mRNA (Figure 5). These results indicated that the mRNA candidate of the present disclosure is effective in generation of a functional protein leading to a rescue of the phenotype (MMA levels).
Example 4: AAV constructs comprising the MMUT nucleotide sequence of the present disclosure
A small panel of codon optimized MMUT AAV constructs was generated by sub-cloning a combination of different 5’ and 3’ UTR regulatory elements in the AAV vector. The regulatory elements of the different constructs are shown in Table 3. Codon optimized MMUT (CO- MMUT) AAV constructs were then transfected into HepG2 cells. The expression levels of MMUT was examined by western blotting (Figure 6). As shown in the figure, the expression of MMUT protein was evident in the cells transfected with MUT-AAV constructs.
Table 3:
Figure imgf000039_0001
Foregoing description of the specific embodiments fully reveals the general nature of the embodiments herein that others can, by applying current knowledge, readily modify and/or adapt for various applications such specific embodiments without departing from the generic concept, and, therefore, such adaptations and modifications should and are intended to be comprehended within the meaning and range of equivalents of the disclosed embodiments. It is to be understood that the phraseology or terminology employed herein is for the purpose of description and not of limitation. Therefore, while the embodiments in this disclosure have been described in terms of preferred embodiments, those skilled in the art will recognize that the embodiments herein can be practiced with modification within the spirit and scope of the embodiments as described herein.
While considerable emphasis has been placed herein on the particular features of this disclosure, it will be appreciated that various modifications can be made, and that many changes can be made in the preferred embodiments without departing from the principles of the disclosure. These and other modifications in the nature of the disclosure or the preferred embodiments will be apparent to those skilled in the art from the disclosure herein, whereby it is to be distinctly understood that the foregoing descriptive matter is to be interpreted merely as illustrative of the disclosure and not as a limitation.
All references, articles, publications, general disclosures etc. cited herein are incorporated by reference in their entireties for all purposes. However, mention of any reference, article, publication etc. cited herein is not, and should not be taken as, an acknowledgment or any form of suggestion that they constitute valid prior art or form part of the common general knowledge in any country in the world.

Claims

WE CLAIM:
1. An mRNA sequence comprising a CDS bearing at least about 90% identity to a sequence represented by SEQ ID No. 2.
2. The mRNA sequence as claimed in claim 1, wherein the CDS has a sequence represented by SEQ ID No. 29.
3. An mRNA construct comprising the mRNA sequence as claimed in claim 1 or 2 or a fragment thereof, a 5’ cap, 5'UTR element(s), 3'UTR element(s), poly(A) sequence(s) and optionally, a natural or synthetic IRES element and/or a circularizing element.
4. The mRNA construct as claimed in claim 2, wherein the 5’ terminal cap structure is selected from a group comprising m7Gppp5'N (CapO), m7Gppp5'Nm (Capl), ARCA (3'-O-Me-m7G(5') ppp(5')G) or CleanCap capping reagents, CleanCapAG and CleanCapM6; wherein the 5’ UTR element(s) is selected from a group comprising sequences represented by SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO. 5, SEQ ID NO.
6, SEQ ID NO. 7 and SEQ ID NO. 8; wherein the 3 ’UTR element(s) is selected from a group comprising sequences represented by SEQ ID NO. 9, SEQ ID NO. 10, SEQ ID NO. 11, SEQ ID NO. 12, SEQ ID NO. 13 and SEQ ID NO. 14; wherein the polyA tail comprises about 60 As to about 120 As.
5. A DNA template suitable for generating the mRNA construct as claimed in any of claims 3-4, comprising a CDS having sequence bearing at least about 90% identity to a sequence represented by SEQ ID No. 15, one or more elements selected from a group comprising an AT -rich region, a promoter, 5' untranslated region(s) (UTR), an enhancer region, a terminator region, 3'UTR(s), polyA signal(s), and restriction site(s).
6. The DNA template as claimed in claim 5, wherein the CDS has a sequence represented by SEQ ID No. 31.
7. The DNA template as claimed in any of claims 5 or 6, wherein the promoter is selected from a group comprising T7, SP6, T3 and LP1 or HCR hAAT promoters; wherein the 5’ UTR(s) is selected from a group comprising sequences represented by SEQ ID NO.
16, SEQ ID NO. 17, SEQ ID NO. 18, SEQ ID NO. 19, SEQ ID NO. 20 and SEQ ID NO. 21, SEQ ID NO. 33, SEQ ID NO. 34, SEQ ID NO. 35, SEQ ID NO. 36 and SEQ ID NO. 38; wherein the 3’UTR(s) is selected from a group comprising sequences represented by SEQ ID NO. 22, SEQ ID NO. 23, SEQ ID NO. 24, SEQ ID NO. 25, SEQ ID NO. 26, SEQ ID NO. 27 and SEQ ID NO. 37; wherein the polyA signal(s) is selected from a group comprising SV40 PolyA sequence, Synthetic PolyA site and Bovine growth hormone (bGH) PolyA site; and/or wherein the restriction site(s) is a Type II restriction site.
8. A recombinant AAV vector comprising the DNA template as claimed in any of claims 5-7.
9. A lipid nanoparticle (LNP) comprising the mRNA construct as claimed in any of claims 3 or 4.
10. A composition comprising the mRNA construct as claimed in any of claims 3 or 4, the mRNA-LNP as claimed in claim 9 or the recombinant AAV vector as claimed in claim 8 in combination with pharmaceutically acceptable excipient(s) or carrier(s).
11. A method of treating and/or preventing MMA, said method comprising administering the mRNA construct as claimed in any of claims 3 or 4, the mRNA-LNP as claimed in claim 9 or the recombinant AAV vector as claimed in claim 8 or the composition as claimed in claim 10 to a subject in need thereof.
12. Use of the mRNA sequence as claimed in any of claims 1 or 2, the mRNA construct as claimed in any of claims 3 or 4, the mRNA-LNP as claimed in claim 9 or the recombinant AAV vector as claimed in claim 8 or the composition as claimed in claim 10 in treating and/or preventing MMA.
13. A kit comprising the mRNA sequence as claimed in any of claims 1 or 2, the mRNA construct as claimed in any of claims 3 or 4, the mRNA-LNP as claimed in claim 9 or the recombinant AAV vector as claimed in claim 8 or the composition as claimed in claim 10, means for administration of the same and optionally, an instruction manual.
PCT/IN2024/052370 2023-12-11 2024-12-11 Nucleic acid sequence(s) and construct(s) for treating methylmalonic acidemia and method(s) thereof Pending WO2025126242A1 (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
IN202341084213 2023-12-11
IN202341084213 2023-12-11

Publications (1)

Publication Number Publication Date
WO2025126242A1 true WO2025126242A1 (en) 2025-06-19

Family

ID=96056652

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/IN2024/052370 Pending WO2025126242A1 (en) 2023-12-11 2024-12-11 Nucleic acid sequence(s) and construct(s) for treating methylmalonic acidemia and method(s) thereof

Country Status (1)

Country Link
WO (1) WO2025126242A1 (en)

Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2013151666A2 (en) * 2012-04-02 2013-10-10 modeRNA Therapeutics Modified polynucleotides for the production of biologics and proteins associated with human disease
WO2017106799A1 (en) * 2015-12-17 2017-06-22 Modernatx, Inc. POLYNUCLEOTIDES ENCODING METHYLMALONYL-CoA MUTASE
WO2018231990A2 (en) * 2017-06-14 2018-12-20 Modernatx, Inc. Polynucleotides encoding methylmalonyl-coa mutase
WO2020056294A1 (en) * 2018-09-14 2020-03-19 Translate Bio, Inc. Composition and methods for treatment of methylmalonic acidemia
WO2022061002A1 (en) * 2020-09-18 2022-03-24 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Novel adeno-associated viral (aav) vectors to treat hereditary methylmalonic acidemia (mma) caused by methylmalonyl-coa mutase (mmut) deficiency
WO2022204369A1 (en) * 2021-03-24 2022-09-29 Modernatx, Inc. Polynucleotides encoding methylmalonyl-coa mutase for the treatment of methylmalonic acidemia
WO2023198930A1 (en) * 2022-04-14 2023-10-19 Genespire S.R.L. Lentiviral vector

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2013151666A2 (en) * 2012-04-02 2013-10-10 modeRNA Therapeutics Modified polynucleotides for the production of biologics and proteins associated with human disease
WO2017106799A1 (en) * 2015-12-17 2017-06-22 Modernatx, Inc. POLYNUCLEOTIDES ENCODING METHYLMALONYL-CoA MUTASE
WO2018231990A2 (en) * 2017-06-14 2018-12-20 Modernatx, Inc. Polynucleotides encoding methylmalonyl-coa mutase
WO2020056294A1 (en) * 2018-09-14 2020-03-19 Translate Bio, Inc. Composition and methods for treatment of methylmalonic acidemia
WO2022061002A1 (en) * 2020-09-18 2022-03-24 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Novel adeno-associated viral (aav) vectors to treat hereditary methylmalonic acidemia (mma) caused by methylmalonyl-coa mutase (mmut) deficiency
WO2022204369A1 (en) * 2021-03-24 2022-09-29 Modernatx, Inc. Polynucleotides encoding methylmalonyl-coa mutase for the treatment of methylmalonic acidemia
WO2023198930A1 (en) * 2022-04-14 2023-10-19 Genespire S.R.L. Lentiviral vector

Similar Documents

Publication Publication Date Title
JP7801093B2 (en) Enhancers for improving cell transfection and/or rAAV vector production
US12023386B2 (en) Composition for treatment of Crigler-Najjar syndrome
US12060567B2 (en) Engineered untranslated regions (UTR) for AAV production
US20240035047A1 (en) Manufacturing and use of recombinant aav vectors
WO2022187548A1 (en) Controlled expression of viral proteins
CA3115248A1 (en) Engineered nucleic acid constructs encoding aav production proteins
US20230383313A1 (en) Improved adeno-associated virus (aav) vector and uses therefor
JP2022505106A (en) Expression vector for large-scale production of rAAV in the baculovirus / Sf9 system
WO2020037161A1 (en) Combination therapy for spinal muscular atrophy
JP7525768B2 (en) Liver-specific inducible promoters and methods of use thereof
Yang et al. AAVone: A cost-effective, single-plasmid solution for efficient AAV production with reduced DNA impurities
WO2025126242A1 (en) Nucleic acid sequence(s) and construct(s) for treating methylmalonic acidemia and method(s) thereof
EP4045636A1 (en) Aav production strategy using a cell line expressing an inducible rep gene
WO2024196965A1 (en) Parvovirus compositions and related methods for gene therapy
US20250257367A1 (en) Gene therapy for gaucher disease
JP7799622B2 (en) Compositions and methods for reducing nuclease expression and off-target activity using promoters with low transcriptional activity
CN108103102B (en) An AAV1 virus-mediated skeletal muscle-specific PCK1 gene expression vector and use thereof
CN118879713B (en) Application of a new artificially designed NRG-1 in disease treatment
JP2024084843A (en) Method for enhancing gene expression by AAV vector
KR20250158061A (en) PAH-modulating systems and methods
WO2025106661A1 (en) Compositions with cardiac and skeletal musclespecific targeting motifs and uses thereof
WO2025102034A1 (en) Gene therapy for barth syndrome
WO2025054383A1 (en) Chemical stability of mrna
WO2024130070A2 (en) Recombinant aav capsids with cardiac- and skeletal muscle- specific targeting motifs and uses thereof
CN118434855A (en) PAH regulating compositions and methods

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 24903163

Country of ref document: EP

Kind code of ref document: A1