Compositions and methods for modulating antimicrobial responses

Abstract

This disclosure relates generally to compositions and methods for eliciting or enhancing the zinc toxicity antimicrobial response. More particularly, the present disclosure relates to compositions and methods for eliciting or enhancing the zinc toxicity response in innate immune cells in subjects with cystic fibrosis and methods related thereto.

Description

TITLE OF THE INVENTION“COMPOSITIONS AND METHODS FOR MODULATING ANTIMICROBIAL RESPONSES’’RELATED APPLICATIONS

[0001] This application claims priority to Australian Provisional Application No. 2024900187 entitled “Compositions And Methods For Modulating Antimicrobial Responses” filed 25 January 2024, the contents of which are incorporated herein by reference in their entirety.SEQUENCE LISTING

[0002] The present application contains a Sequence Listing which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety.FIELD OF THE INVENTION

[0003] This invention relates generally to compositions and methods for eliciting or enhancing the zinc toxicity antimicrobial response. More particularly, the present invention relates to compositions and methods for eliciting or enhancing the zinc toxicity response in innate immune cells in subjects with cystic fibrosis.BACKGROUND OF THE INVENTION

[0004] Cystic fibrosis (CF) is an autosomal recessive disorder caused by mutations in the cystic fibrosis transmembrane conductance regulator gene (CFTR). As many as 2,000 different mutations in CFTR have been reported, with approximately 150 of these known to cause disease (Ratjen et al., 2015). CFTR is a member of the ATP-binding cassette (ABC) family of proteins, functioning primarily as an apical anionic channel for chloride and bicarbonate ions (Watson et al., 2016). Mutations in CFTR can reduce the number of functional channels and / or interfere with the overall function of the protein, affecting the normal flow of chloride and bicarbonate ions and water into and out of cells (Ratjen et al., 2015). The most common CFTR mutation is the deletion of phenylalanine 508 (AF508), with this mutation occurring in approximately 70% of people with CF (pwCF) (Jih et al., 2011). The AF508 mutation results in destabilized CFTR protein folding due to altered stability of the nucleotide binding domain, leading to its proteasomal degradation and reduced cell surface residence time (Okiyoneda et al., 2010).

[0005] CFTR mutations result in mucus accumulation in the lungs, narrowing of the airway lumen, obstructive pulmonary disease, and bronchiectasis. This creates an environment conducive to inflammation and recurring infections caused by Pseudomonas aeruginosa, Staphylococcus aureus and other pathogens. P. aeruginosa infections in pwCF are associated with a decline in lung function, exaggerated lung inflammation, and increased mortality (Emerson et al., 2002; Gangell et al., 2011). Nontuberculous mycobacterium infections, especially those caused by Mycobacterium abscessus, are also common in patients with CFTR mutations, accounting for approximately 3-20% of infections in pwCF (Adjemian et al., 2014).

[0006] In addition to epithelial cells, CFTR is also expressed in immune cells, including macrophages, monocytes, neutrophils, and lymphocytes (13). Macrophages and neutrophils are key innate immune leukocytes that infiltrate compromised lungs of pwCF and contribute to CF pathology (Oz et al., 2022).

[0007] Macrophages from pwCF also exhibit defects that likely contribute to disease manifestations. These include increased secretion of proinflammatory cytokines such as IL-1 p, IL-6 and TNF (Kopp et al., 2012; Bruscia et al., 2011 ; Lara-Reyna et al., 2019), as well as impaired antimicrobial responses such as phagocytosis and phagolysosome acidification (Simonin-Le Jeune et al., 2013; Del Porto et al., 0211).

[0008] Despite the overall success of CFTR modulators in improving clinical outcomes, many pwCF, particularly those harbouring a G551 D mutation in CFTR, often report reemergence of symptoms after treatment cessation (Trimble et al., 2018). Furthermore, the prevalence of multi-antibiotic resistant bacterial pathogens presents a clear clinical need for the development of new strategies to combat persistent infections in pwCF.SUMMARY OF THE INVENTION

[0009] The present invention is predicated in part on the realization by the present inventors that mutations in the CFTR protein leading to CFTR dysfunction resulted in an impaired zinc toxicity antimicrobial response. Subjects with CFTR dysfunction were therefore unable to effectively defend against microbial infection. Accordingly, the inventors have derived compositions and methods for eliciting or enhancing the zinc toxicity antimicrobial response in a subject with CFTR dysfunction.

[0010] In one aspect, the present invention provides a method of treating or preventing a microbial infection in a subject with CFTR dysfunction, the method comprising administering to the subject an agent that elicits or enhances the zinc toxicity antimicrobial response of an innate immune cell of the subject. In some embodiments, the agent promotes SLC30A1 expression or SLC30A1 activity in the innate immune cell. In some alternative embodiments, the agent promotes SLC30A3 expression or SLC30A3 activity, or SLC30A8 expression or SLC30A8 activity in the innate immune cell.

[0011] In one aspect, the present invention provides a method of eliciting or enhancing a zinc toxicity antimicrobial response in an innate immune cell with CFTR dysfunction, the method comprising administering to the subject an agent that promotes SLC30A1 expression in the innate immune cell of the subject.

[0012] In some preferred embodiments, the agent promotes SLC30A1 expression in the innate immune cell.

[0013] Suitably, the agent does not promote the expression or activity of an SLC30A4 polypeptide.

[0014] In some embodiments, the CFTR dysfunction is caused by a heterozygous mutation or a homozygous mutation in the CFTR gene. In some embodiments, the CFTR mutation results in the deletion of the amino acid corresponding to phenylalanine 508 of the native human CFTR polypeptide. In someembodiments, the CFTR mutation results in a substitution of a glycine to an aspartic acid at the amino acid corresponding to position 551 of the native human CFTR polypeptide. In some embodiments, the CFTR dysfunction is caused by an RNA null allele of the CFTR gene. In some of the same embodiments and some other embodiments the CFTR dysfunction is caused by a genetic null allele of the CFTR gene).

[0015] In some embodiments the agent is a messenger RNA (mRNA) that comprises an open reading frame (ORF) that encodes a solute carrier 30 member A1 (SLC30A1) polypeptide. In some preferred embodiments, the SLC30A1 polypeptide has the amino acid sequence set forth in SEQ ID NO: 1 , or a biologically active fragment thereof.

[0016] In some embodiments, the innate immune cell of the subject has one or both of an impaired uptake of microbial cells, and an impaired clearance of microbial cells.

[0017] In some embodiments, the subject is diagnosed with cystic fibrosis or bronchiectasis.

[0018] In some embodiments, the innate immune cell is selected from a macrophage, neutrophil, monocyte, and dendritic cell. In some preferred embodiments, the innate immune cell is a macrophage.

[0019] In some embodiments, the agent is administered together with a treatment for a condition associated with dysfunctional CFTR (e.g., cystic fibrosis). Preferably, the treatment is a CFTR modulator. Suitable treatments of this type include, but are not limited to, TRIKAFTA (elexacaftor, ivacaftor, tezacaftor), KALYDECO (ivacaftor), lumacaftor, texacaftor, elexacaftor, vanzacaftor, VX-522, SYMDEKO (tezacaftor and ivacaftor), and ORKAMBI (lumacaftor and ivacaftor).

[0020] In yet another aspect, the invention provides messenger RNA (mRNA) for the restoration of a zinc toxicity antimicrobial response in a cell, the mRNA comprising an open reading frame (ORF) encoding a solute carrier 30 member A1 (SLC30A1) polypeptide and a 5' untranslated region (UTR). In some embodiments, the 5' untranslated region (UTR) comprises, consists, or consists essentially of a sequence set forth in SEQ ID NO: 13.

[0021] In some embodiments, the mRNA further comprises one or more of a 51cap, a 31UTR, and a poly(A) tail. Preferably, the mRNA further comprises a 51cap, a 31UTR, and a poly(A) tail. In some embodiments, the 3’ UTR comprises, consists, or consists essentially of the sequence set forth in SEQ ID NO: 12.

[0022] In some of the same embodiments and some other embodiments, the mRNA comprises one or both of an optimised codon and a chemical modification when compared to a corresponding mRNA that does not comprise the optimised codon and / or chemical modification. Preferably, the chemical modification and / or the optimised codon increases mRNA stability and / or mRNA translation in a mammalian cell when compared to a mRNA without the chemical modification and / or the optimised codon.

[0023] In some embodiments, the chemical modification is a nucleoside modification (e.g., a modified uracil or a modified cytosine).

[0024] In some embodiments, the optimised codon increases guanine (G) and / or cytosine (C) codon content.

[0025] In some embodiments, the mRNA encodes a SLC30A1 polypeptide that comprises an amino acid sequence set forth in SEQ ID NO: 1 , or having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 99%, at least 99.5%, at least 99.8% identity to a sequence set forth in SEQ ID NO: 1 , or a biologically active fragment thereof.

[0026] In yet another aspect, the invention provides a pharmaceutical composition comprising the mRNA as described above and / or as described herein, and a pharmaceutically acceptable carrier, diluent, or excipient.

[0027] In some embodiments, the pharmaceutical composition further comprises a treatment for a condition associated with dysfunctional CFTR (e.g., cystic fibrosis). Preferably, the treatment is a CFTR modulator. Suitable treatments of this type include, but are not limited to, TRIKAFTA (elexacaftor, ivacaftor, tezacaftor), KALYDECO (ivacaftor), lumacaftor, texacaftor, elexacaftor, vanzacaftor, VX-522, SYMDEKO (tezacaftor and ivacaftor), and ORKAMBI (lumacaftor and ivacaftor).BRIEF DESCRIPTION OF THE FIGURES

[0028] The following figures form part of the present specification and are included to further demonstrate certain aspects of the present disclosure. The disclosure may be better understood by reference to one or more of these figures in combination with the detailed description of specific embodiments presented herein.

[0029] Figure 1 provides graphical representation that CFTR is required for uptake and clearance of intracellular E. coli by human macrophages. (A) HMDM were differentiated with CSF-1 + 10 pM C172 or vehicle (DMSO) for 6 days, after which phagocytosis assays were performed using pHrodo E. coli and flow cytometry. Representative flow cytometry plot (left) and quantified flow cytometry data normalised to the baseline DMSO control in each experiment (right) (n = 4). (B-D) HMDM were differentiated with CSF-1 + 10 pM C172 (a CFTR inhibitor) or vehicle (DMSO) for 6 days, after which they were infected with EC958 (UPEC) (MOI 100) for the indicated time points. Intracellular bacterial loads were quantified using gentamicin exclusion assays. Data in (B) are normalised to the DMSO control for each experiment and data in (C-D) are presented relative to uptake for the relevant treatment in (B) (n = 6). (E) Schematic representation of infection assay protocol. (F-G) HMDM were infected with UPEC (MOI 100) for 1 hour after which C172 (10 pM) or vehicle (DMSO) was added to the cells for the indicated time points. Intracellular bacterial loads were quantified using gentamicin exclusion assays. Data are normalised to the DMSO control for each experiment (n = 7). (H-l) HMDM from healthy donors (HD) or people with cystic fibrosis (pwCF, or“CF”) were differentiated with CSF-1 for 6 days, then infected with UPEC (MOI 100) for the indicated time points. Intracellular bacterial loads were quantified using gentamicin exclusion assays. Data in (H) are normalised to the HD for each experiment and data in (I) are presented relative to bacterial uptake in (H) (n = 4). Data (mean+SEM) are combined from at least four independent experiments (each using different donors). Statistical significance was assessed by two-way ANOVA (A) followed by Sidak's multiple comparison test or Wilcoxon test (B-D, F-l) (ns - not significant, * p<0.05).

[0030] Figure 2 provides a graphical representation showing that CFTR is required for a zinc stress response in non-pathogenic E. coli within macrophages. (A) Non-pathogenic E. coli (strain MG1655)was grown in the presence of 500 pM ZnSO4 for 2 hours. Total RNA was extracted and zntA mRNA levels were assessed using qPCR. Data are representative of two independent experiments. (B) HMDM were differentiated with CSF-1 + 10 pM C172 or vehicle (DMSO) and infected with E. coli strain MG1655 (MOI 100) for the indicated time points, after which total RNA was extracted. Expression levels of E. coli zntA mRNA (relative to gapA) within HMDM versus bacterial alone were assessed using qPCR. Data are normalized to the 2-hour DMSO infection control (n = 4). (C) Total RNA was extracted from healthy or CF HMDM and infected with E. coli (MOI 100) for 8 hours. Expression levels of E. coli zntA mRNA (relative to gapA) within HMDM were assessed using qPCR (n = 5). Data (mean+SEM) are combined from at least four independent experiments (each using different donors). Statistical significance was assessed using two-way ANOVA followed by Sidak's multiple comparison test (B) or Mann- hitney test (C) (* p<0.05).

[0031] Figure 3 provides a graphical representation demonstrating that CFTR is required for zinc accumulation in human macrophages. (A) HMDM were differentiated with CSF-1 + 10 pM C172 or vehicle (DMSO) for 6 days, after which they were either stimulated with LPS (20 ng / mL) or infected with E. coli (MOI 100) for 24 hours. Relative intracellular zinc levels were determined by FluoZin-3AM staining and flow cytometry (left: DMSO control, right: C172). Representative flow cytometry plots and quantified flow cytometry data normalised to the baseline control in each experiment are depicted (n = 4). (B) Monocytes from healthy donors (HD) or pwCF (CF) were differentiated into HMDM with CSF-1 for 6 days, after which they were infected with E. coli (MOI 100) for 24 hours. Relative intracellular zinc levels were determined by FluoZin-3AM staining and flow cytometry. Data are normalised to the HD control in each experiment (n = 5). (C) Monocytes from healthy donors (HD) or pwCF (CF) were differentiated into HMDM with CSF-1 for 6 days, after which they were infected with E. co / / -mCherry (MOI 100) for 24 hours. Intracellular zinc- containing vesicles were visualised by immunofluorescence microscopy (FluoZin-3AM: green; E. coli- mCherry: white, DAPI: blue; scale bars: 10 pm), left. Number of zinc-containing puncta per cell were quantified, right (n = 4). (D) HMDM were infected with E. coli (MOI 100) for 1 hour afterwhich 10 pM C172 or vehicle (DMSO) was added to the cells for 24 hours. Relative intracellular zinc levels were determined by FluoZin-3AM staining and flow cytometry. Data are normalised to the control in each experiment (n = 6). (E) HMDM were differentiated with CSF-1 + 10 pM C172 or vehicle (DMSO) for 6 days, afterwhich they were infected with E. coli (MOI 100) for 24 hours. Absolute intracellular zinc levels were determined by ICP-OES. Data are normalised to the DMSO control in each experiment (n = 5). (F-G) Total RNA was extracted from HMDM differentiated + 10 pM C172 or vehicle (DMSO) for 6 days (n = 4) (F) or from matched healthy donors (HD) and pwCF (CF) (n = 5) (G), then infected with E. coli (MOI 100) for the indicated time points. Expression levels of SLC30A1 mRNA were assessed using qPCR. Data in (F) are normalized to the DMSO control for 24 hour post E. coli infection. Data (mean+SEM) are combined from at least four independent experiments (each using different donors). Statistical significance was assessed using non-parametric Kruskal Wallis test (A), Friedman's test (D) or two-way ANOVA (B-C, E-G) followed by Sidak's multiple comparison test (ns - not significant, * p<0.05, ** p<0.01 , **** p<0.0001).

[0032] Figure 4 provides graphical representation showing that CFTR is required for the macrophage zinc toxicity response, with ectopic SLC30A1 expression or zinc supplementation restoring host defence in macrophages with defective CFTR function. (A-B) HMDM were differentiated with CSF-1 +10 |JM C172 or vehicle (DMSO) for 6 days, after which they were infected with E. coli (blue bars, left-hand side) or E. coli AzntA (green bars, right-hand side) (MOI 100) for the indicated time points. Intracellular bacterial loads were quantified using gentamicin exclusion assays and expressed as percentage bacteria retrieved (B), relative to initial uptake (A) (n = 5). (C-D) Monocytes from healthy donors (HD) or pwCF (CF) were differentiated into HMDM with CSF-1 for 6 days, after which they were infected with E. coli (blue bars, left-hand side) or E. coli AzntA (green bars, right-hand side) (MOI 100) for the indicated time points. Intracellular bacterial loads were quantified using gentamicin exclusion assays and expressed as percentage bacteria retrieved (D) with respect to uptake (C) (n = 4). (E-F) PMA-differentiated THP-1 cells stably transduced with lentivirus expressing either empty vector (green bars, left-hand side) or SLC30A1_V5 (blue bars, right-hand side) were treated + doxycycline for 24 hours, then assessed for SLC30A1-V5 protein levels by anti-V5 immunoblots on cell lysates (E). Cells + 24 hour doxycycline treatment were infected with E. coli (MOI 100) for 8 hours, after which intracellular bacterial loads were assessed using gentamicin exclusion assays (n = 5) (F). The immunoblots in (E) are representative of three independent experiments. (G-H) HMDM were differentiated with CSF-1 + 10 pM C172 or vehicle (DMSO) for 6 days, after which they were treated with vehicle (grey bars, left-hand side) or 200 pM of ZnSO4 (turquoise bars, right-hand side) for 1 hour then infected with E. coli (G) or UPEC (H) (MOI 100) for 8 hours. Intracellular bacterial loads were quantified using gentamicin exclusion assays and expressed as percentage bacteria retrieved with respect to uptake (n = 5). Data are combined from at least four independent experiments (each using different donors) in (A-D) and (G-H) or five independent experiments in (F) and are expressed as mean+SEM. Statistical significance was assessed using two-way ANOVA followed by Tukey's multiple comparison test (ns - not significant, * p<0.05, ** p <0.01).

[0033] Figure 5 provides graphical representation showing that CFTR contributes to the antimicrobial zinc response against Pseudomonas aeruginosa in GM-CSF-derived macrophages. (A-C) HMDM were differentiated with GM-CSF + 10 pM C172 or vehicle (DMSO) for 6 d, afterwhich they were infected with Pseudomonas aeruginosa (PAM) (MOI 5) for the indicated time points. Intracellular bacterial loads were quantified using gentamicin exclusion assays. Data in (A) are normalised to the DMSO control for each experiment and data in (B-C) are presented relative to uptake for the relevant treatment in (A) (n = 6). (D-E) HMDM from healthy donors (HD) or pwCF (CF) were differentiated with GM-CSF for 6 days, after which they were infected with PA (MOI 5) for the indicated time points. Intracellular bacterial loads were quantified using gentamicin exclusion assays. Data in (D) are normalised to the healthy donor (HD) control for each experiment and data in (E) are presented relative to uptake for the relevant treatment in (D) (n = 5). (F) Schematic representation of infection assay used in (G-l). (G)-(l) GM-CSF-derived HMDM were infected with PAM (MOI 5) for 1 hour after which C172 (10 pM) was added to the cells for the indicated time points. Intracellular bacterial loads were quantified using gentamicin exclusion assays. Data are normalised to the DMSO control for each experiment (n = 5). (J) Monocytes from healthy donors (HD) or pwCF (CF) were differentiated into HMDM with GM-CSF for 6 days. CF monocytes were also differentiated + 3 pM Elexacaftor / 18 pM Tezacaftor / 1 pM Ivacaftor (ETI). Cells were then infected with PAM (MOI 5) for 24 hours afterwhich relative intracellular zinc levels were determined by FluoZin-3AM staining and flow cytometry. Data are normalised to the HD control for each experiment (n = 5). (J) Monocytes from pwCF(CF) were differentiated into HMDM with CSF-1 for 6 days + Elexacaftor 3 |jM / Tezacaftor 18 |jM / lvacaftor 1 |JM (ETI). In parallel, monocytes from healthy donors (HD) were differentiated with CSF-1 for use as a positive control for zinc vesicle formation. Cells were infected with E. co / / -mCherry (MOI 100) for 24 hours (EC), afterwhich intracellular zinc-containing vesicles were visualised by FluoZin-3AM staining / immunofluorescence microscopy and numbers of zinc-containing puncta per cell were quantified. Data (mean+SEM) are from 2 (healthy) or 3 (pwCF) donors. In these experiments, two CF donors (circles, squares) were analysed in parallel with one healthy donor positive control (circles). Data (mean+SEM) are combined from 2-6 independent experiments (each using different donors). (K-M) GM-CSF-derived HMDM from healthy donors (HD) or pwCF (CF), were treated with 200 pM of ZnSO4 for 1 hour prior to infection with PAM (MOI 5) for 20 minutes or 24 hours. Intracellular bacterial loads were quantified using gentamicin exclusion assays. Data in (L) are normalised to the healthy donor (HD) control for each experiment and data in (M) are presented relative to uptake for the relevant treatment in (L) (n = 5). Data (mean+SEM) are combined from at least four independent experiments (each using different donors). Statistical significance was assessed using Wilcoxon test (A-C and G-l), Mann-Whitney test (D-E) or two- way ANOVA (J-L) followed by Tukey's multiple comparison test (ns - not significant, * p<0.05, ** p <0.01 , *** p<0.001 , **** p<0.0001).

[0034] Figure 6 provides graphical representation showing that mRNA delivery of SLC30A1 reduces non-pathogenic E. coli loads in macrophages. THP1 cells were transfected with water (vehicle), 500 ng of GFP, SLC30A1-V5 (30A1), SLC30A4-V5 (30A4), or SLC30A1-V5 and SLC30A4-V5 combined (30A1+A4) mRNAs using the Neon NxT electroporation system. Transfected cells were then differentiated with 30 ng / mL PMA for 48 hours and then infected with MG1655 for 1 hour (MOI 100). After 1 hour, media was replaced with media containing 200 mg / mL gentamicin for 1 hour, followed by media containing 20 mg / mL gentamicin to exclude extracellular bacteria. At (A) 2 hours and (B) 24 hours post-infection, THP1 cells were lysed with 0.01% Triton X-100 and plated on LB agar. CFUs were calculated and graphed relative to the vehicle control. Data are mean + SEM combined from three independent experiments. (C) A schematic diagram of SLC30A1-V5_A (top) and SLC30A1-V5_B (bottom) mRNAs featuring different 31UTR regions. (D) HEK293 or (E) THP1 cells were transfected with water (vehicle), 2000 ng GFP, SLC30A1-V5_A (SEQ ID NO: 10) or SLC30A1-V5_B (SEQ ID NO: 11) mRNA molecules using the Neon NxT Electroporation system. (D) HEK293 cells were lysed 24 hours post-transfection, and protein expression of V5 (SLC30A1), GFP and tubulin (loading control) were assessed via immunoblotting. Data are representative blots of two independent experiments. (E) After transfection THP1 cells were differentiated with PMA for 24 hours, lysed and protein expression assessed via immunoblotting as in (D). Data are representative of one independent experiment.DETAILED DESCRIPTION OF THE INVENTION1. Definitions

[0035] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in thepractice or testing of the present invention, preferred methods and materials are described. For the purposes of the present invention, the following terms are defined below.

[0036] The articles “a” and “an” are used herein to refer to one or to more than one ( / .e. to at least one) of the grammatical object of the article. By way of example, “an element” means one element or more than one element.

[0037] The term “about” as used herein refers to the usual error range for the respective value readily known to the skilled person in this technical field. Reference to “about” a value or parameter herein includes (and describes) embodiments that are directed to that value or parameter perse.

[0038] The “amount” or “level” of a biomarker is a detectable level in a sample. These can be measured by methods known to one skilled in the art and also disclosed herein. The expression level or amount of biomarker assessed can be used to determine the response to treatment.

[0039] As used herein, “and / or” refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations when interpreted in the alternative (or).

[0040] As used herein, “CFTR” means cystic fibrosis transmembrane conductance regulator. This includes, but is not limited to, the wild-type human CFTR (UniProt Accession No. Q6KEI2) with the amino acid sequence as set forth in SEQ ID NO: 3. The term also includes any CFTR variants, or biologically active fragments of a native CFTR polypeptide sequence.

[0041] As used herein, mutations can refer to mutations in the CFTR gene or variants of the CFTR protein. A “CFTR gene mutation” refers to a mutation in the CFTR gene, and a “CFTR protein mutation” refers to a variation in the CFTR protein amino acid sequence. A generic defect or mutation (i.e. , a change in one or more nucleotides) in the CFTR gene in general results in a mutation (i.e., a change in one or more amino acids) in the CFTR protein translated from that gene, a frame shift (i.e., a change in the reading frame that governs how the CFTR gene is translated into the CFTR protein), or a splice mutation.

[0042] As used herein, the term “AF508”, “F508del” and the like refer to a mutant CFTR protein which is lacking the amino acid phenylalanine at position 508, or to a mutant CFTR gene which encodes for a CFTR protein lacking the amino acid phenylalanine at position 508.

[0043] The term “CFTR dysfunction”, also referred to as “CFTR ion channel dysfunction” (which terms are considered synonymous for the purposes of the invention and are therefore used interchangeably herein) means that the functional activity of CFTR is reduced as compared a corresponding wild-type CFTR. Insufficient numbers of functional CFTR ion channels at the epithelial cell surface, for example, as a consequence of mechanisms which cause reduced numbers of CFTR at the cell surface and / or insufficient ion channel activity in the population of CFTR that are present at the cell surface, results in the pathological state referred to as CFTR dysfunction. Typically, the reduced activity arises from a variation in the CFTR amino acid sequence which affects its activity and / or its cellular processing and delivery (trafficking) to the cell surface. Such defects may in many cases be due to mutations in the CFTR gene (i.e., due to an underlying genetic defect), but can also arise due to extrinsicfactors which may for example cause aberrant or impaired expression of the CFTR at the cell surface. Illustrative examples CFTR impairment that result in CFTR dysfunction include the presence of a premature stop codon in the CFTR mRNA transcript giving rise to truncated CFTR with reduced function and / or which are poorly transported to the cell membrane; impaired intracellular processing of full length CFTR translation products which interferes with the trafficking of CFTR to the cell membrane (e.g. misfolding, defective post-translational modification, inappropriate intracellular protein sorting, degradation prior to reaching the cell membrane); disordered ion channel regulation (e.g., poor activation by ATP or cAMP, reduced channel open time); reduced channel conductance; splicing defects in the transcription of CFTR mRNA or reduced CFTR mRNA transcription perse.

[0044] Throughout this specification, unless the context requires otherwise, the words “comprise”, “comprises” and “comprising” will be understood to imply the inclusion of a stated step or element or group of steps or elements but not the exclusion of any other step or element or group of steps or elements. Thus, use of the term “comprising” and the like indicates that the listed elements are required or mandatory, but that other elements are optional and may or may not be present. By “consisting of” is meant including, and limited to, whatever follows the phrase “consisting of”. Thus, the phrase “consisting of” indicates that the listed elements are required or mandatory, and that no other elements may be present. By “consisting essentially of is meant including any elements listed after the phrase, and limited to other elements that do not interfere with or contribute to the activity or action specified in the disclosure for the listed elements. Thus, the phrase “consisting essentially of indicates that the listed elements are required or mandatory, but that other elements are optional and may or may not be present depending upon whether or not they affect the activity or action of the listed elements.

[0045] The terms “correlated” and “associated” are used interchangeably herein to refer to the association between two measurements (or measured entities). The disclosure provides genetic and / or epigenetic variations, the level(s) of which are associated with disease diagnosis and / or prognosis and / or response to treatment.

[0046] The terms “decrease”, “reduced”, “reduction”, “inhibit”, “suppress”, “attenuate” and the like are all used herein to mean a decrease by a statistically significant amount. In some embodiments, these terms typically mean a decrease by at least 10% as compared to a reference level (e.g., the absence of a given treatment or agent) and can include, for example, a decrease by at least about 10%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or more. As used herein “reduction”, “suppression”, and “inhibition” does not necessitate a complete inhibition or reduction as compared to a reference level. “Complete inhibition” and the like is a 100% inhibition as compared to a reference level. A decrease can be preferably down to a level accepted as within the range of normal (e.g., for an individual without a given disorder).

[0047] The terms “increased”, “increase”, “enhance”, or “activate” are all used herein to mean an increase by a statistically significant amount. In some embodiments, the terms “increased”, “increase”, “enhance”, or “activate” can mean an increase of at least 10% as compared to a reference level (e.g., theabsence of a given treatment or agent) and can include, for example, of at least about 10% as compared to a reference level, for example an increase of at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or up to and including a 100% increase or any increase between 10-100% as compared to a reference level or at least about a 2-fold, or at least about a 3-fold, or at least about a 3-fold, or at least about a 4-fold, or at least about a 5-fold, or at least about a 10-fold increase, or any increase between 2-fold and 10-fold or greater as compared to a reference level. In the context of a marker or symptom, an “increase” is a statistically significant increase in such level.

[0048] As used herein, the term “lipid nanoparticle” or “LNP” shall be understood to refer to lipid- based particles having at least one dimension in the order of nanometers (e.g., 1-1 ,000 nm) and which typically comprises a mRNA described herein.

[0049] “Measuring" or “measurement” means assessing the presence, absence, quantity or amount (which can be an effective amount) of a given substance within a sample, including the derivation of qualitative or quantitative concentration levels of such substances, or otherwise evaluating the values or categorization of a subject's clinical parameters. Alternatively, the term “assaying,” “detecting" or “detection” may be used to refer to all measuring or measurement as described in this specification.

[0050] The terms “modification” or “modified” refer to modification of a mRNA of the invention with respect to A, G, U or C ribonucleotides. Generally, the modification refers to the coding region of the mRNA. However, the modification may also be introduced into the flanking regions and / or the terminal regions if the modification increases protein expression, function, thermal stability or structure.

[0051] As used herein, a “subject” means a human or animal. Usually the animal is a vertebrate such as a primate, rodent, domestic animal or game animal. Primates include chimpanzees, cynomolgus monkeys, spider monkeys, and macaques (e.g., Rhesus). Rodents include mice, rats, woodchucks, ferrets, rabbits, and hamsters. Domestic and game animals include cows, horses, pigs, deer, bison, buffalo, feline species (e.g., domestic cat), canine species (e.g., dog, fox, wolf), avian species (e.g., chicken, emu, ostrich), and fish (e.g., trout, catfish, and salmon). In some embodiments the subject is a mammal (e.g., a primate (e.g., a human)). The terms “individual”, “patient” and “subject” are used interchangeably herein.

[0052] Preferably the subject is a mammal. The mammal can be a human, non-human primate, mouse, rat, dog, cat, horse or cow, but is not limited to these examples. Mammals other than humans can be advantageously used as subjects that represent animal models of CFTR dysfunction (e.g., models of cystic fibrosis). A subject can be male or female.

[0053] As used herein, the terms “treat”, “treatment”, “treating” and the like, refer to therapeutic treatments, wherein the object is to reverse, alleviate, ameliorate, inhibit, slow down or stop the progression or severity of a condition associated with a disease or disorder (e.g., an inflammatory or autoimmune disorder). The term “treating” includes reducing or alleviating at least one adverse effect orsymptom of a condition, disease or disorder associated with an inflammatory or autoimmune disorder. Treatment is generally “effective” if one or more symptoms or clinical markers are reduced. Alternatively, treatment is “effective” if the progression of a disease is reduced or halted. That is, “treatment” includes not just the improvement of symptoms or markers, but also a cessation of, or at least slowing of, progress or worsening of symptoms compared to what would be expected in the absence of treatment. Beneficial or desired clinical results include, but are not limited to, alleviation of one or more symptom(s), diminishment of extent of disease, stabilized (i.e., not worsening) state of disease, delay or slowing of disease progression, amelioration, or palliation of the disease state, remission (whether partial or total), and / or decreased mortality, whether detectable or undetectable. The term “treatment” of a disease also includes providing relief from the symptoms or side-effects of the disease (including palliative treatment). A treatment need not cure a disorder (i.e., complete reversal or absence of disease) to be considered effective.

[0054] The phrases “zinc toxicity antimicrobial response”, “zinc toxicity response”, and variations thereof, refer to an innate immune defence mechanism utilised by innate immune cells (e.g., macrophages) to combat bacterial infection. The zinc toxicity antimicrobial response occurs from innate immune cell activation, which triggers the mobilization of zinc into vesicular-like structures (e.g., phagolysosomal compartments) that co-localize with foreign microorganisms. Such a response may, for example, culminate in the foreign microorganism producing a transcriptional response consistent with zinc poisoning. The zinc toxicity antimicrobial response can include any aspect of this response including, but not limited to, the transport of trafficking of zinc into cellular compartments (e.g., phagosomes), orthe uptake of foreign microorganisms into the innate immune cells (to bring the microorganisms into proximity with the zinc). It is known that the SLC30A1 protein is involved in the trafficking of zinc from the cytoplasm into the phagolysosomal compartments.

[0055] Each embodiment described herein is to be applied mutatis mutandis to each and every embodiment unless specifically stated otherwise.2. RNA therapeutics

[0056] The RNA therapeutic molecules of the present disclosure may comprise at least one agent that elicits or enhances the zinc toxicity antimicrobial response of an innate immune cell of the subject. For example, the agent may be a ribonucleic acid (RNA) comprising an ORF encoding an SLC30A1 polypeptide. In certain embodiments, the RNA is a messenger RNA (mRNA) comprising an ORF encoding an SLC30A1 polypeptide. In certain embodiments, the RNA (e.g., mRNA) further comprises at least one 5' UTR, 3' UTR, a poly(A) tail, and / or a 5' cap.2.1 Coding seguences / ooen reading frames (ORF)

[0057] In some embodiments, the ORF is translatable in a mammalian cell to express the human solute carrier family 30 member 1 (SLC30A1) protein having zinc transportation activity. In some embodiments, the ORF is translatable in a subject in vivo to express the human SLC30A1 protein having zinc transportation activity.

[0058] SLC30A1 is responsible for zinc ion transport and packaging in innate immune cells (e.g., macrophages). This function is known to be essential for the effective zinc toxicity antimicrobial response, and has a key role in phagocytosis. The full length wild-type human SLC30A1 protein has the UniProt accession no. A0A8J4Y5A2, and the following amino acid sequence:MGCWGRNRGRLLCMLALTFMFMVLEVVVSRVTSSLAMLSDSFHMLSDVLALVVALVAERFAR RTHATQKNTFGWIRAEVMGALVNAIFLTGLCFAILLEAIERFIEPHEMQQPLVVLGVGVAGLLVN VLGLCLFHHHSGFSQDSGHGHSHGGHGHGHGLPKGPRVKSTRPGSSDINVAPGEQGPDQEE TNTLVANTSNSNGLKLDPADPENPRSGDTVEVQVNGNLVREPDHMELEEDRAGQLNMRGVFL HVLGDALGSVIWVNALVFYFSWKGCSEGDFCVNPCFPDPCKAFVEIINSTHASVYEAGPCWVL YLDPTLCVVMVCILLYTTYPLLKESALILLQTVPKQIDIRNLIKELRNVEGVEEVHELHVWQLAGS RIIATAHIKCEDPTSYMEVAKTIKDVFHNHGIHATTIQPEFASVGSKSSVVPCELACRTQCALKQC CGTLPQAPSGKDAEKTPAVSISCLELSNNLEKKPRRTKAENIPAVVIEIKNMPNKQPESSL [SEQ ID NO: 1 ],

[0059] The full-length wild-type gene assembly of human SLC30A1 has GenBank accession no. NM_021194.3, with a coding sequence set forth in SEQ ID NO: 2. In some embodiments, the coding sequence (or ORF) of the present mRNA comprises the nucleic acid sequence set forth in SEQ ID NO: 2, or a nucleic acid sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to the sequence set forth in SEQ ID NO: 2. In some embodiments, the coding sequence (or ORF) of the present mRNA comprises the nucleic acid sequence set forth in SEQ ID NO: 8, or a nucleic acid sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to the sequence set forth in SEQ ID NO: 8. In some embodiments, the coding sequence (or ORF) of the present mRNA comprises the nucleic acid sequence set forth in SEQ ID NO: 9, or a nucleic acid sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to the sequence set forth in SEQ ID NO: 9. In some embodiments, the coding region of the mRNA molecule or the region that encodes a SLA30A1 polypeptide may comprise from about 100 to about 200, from about 200 to about 300, from about 300 to about 400, from about 400 to about 500, from about 500 to about 600, from about 600 to about 700, from about 700 to about 800, from about 800 to about 900, from about 900 to about 1000, from about 1000 to about 1200, from about 1200 to about 1400, or from about 1400 to about 1600. Preferably, the mRNA molecule is about 700 to about 1521 nucleotides or bases.

[0060] It is envisaged that a skilled person will be able to modify SLC30A1 gene known in the art to generate a mRNA of the invention containing a codon optimisation and a chemical modification. For example, the mRNA codon optimised to provide for enrichment of guanine (G) and cytosine (C) bases. It is known in the art the guanine and cytosine enrichment of nucleic acid constructs increase or restore protein expression, and enhance mRNA stability, thermal stability, and / or functionality.

[0061] In some embodiments, the present invention provides a SLC30A1 therapeutic composition, comprising a mRNA that comprises an open reading frame (ORF) encoding a SLC30A1 polypeptide. In some embodiments, the mRNA comprises a nucleic acid sequence that corresponds to at least a region of the SLC30A1 coding sequence as set forth in SEQ ID NO: 2. The SLC30A1 polypeptides described herein may have deletions or substitutions of different lengths relative to a wild-type SLC30A1 polypeptide. For example, the SLC30A1 polypeptide of the present invention may have an N-terminal or C- terminal truncation.

[0062] In some embodiments, the translatable mRNA sequence encoding the SLC30A1 protein may comprise a sequence immediately downstream of a coding region (i.e. , ORF) that creates a triple stop codon. A triple stop codon is a sequence of three consecutive stop codons. The triple stop codon can ensure total insulation of an expression cassette and may be incorporated to enhance the efficiency of translation. In some embodiments, the mRNA may comprise the sequence UAG, UGA, or UAA immediately downstream of an ORF described herein. The triple combination can be three of the same codons, three different codons, or any other permutation of the three stop codons.

[0063] In some alternative embodiments, the ORF is translatable in a mammalian cell to express the human SLC30A3 protein having zinc transportation activity. In some embodiments, the ORF is translatable in a subject in vivo to express the human SLC30A3 protein having zinc transportation activity.

[0064] SLC30A3 is responsible for zinc ion transport and packaging in innate immune cells (e.g., macrophages). This function is known to be implicated in the zinc toxicity antimicrobial response, and has a role in phagocytosis. The full length wild-type human SLC30A3 protein has the UniProt accession no. Q99726, and the following amino acid sequence:MEPSPAAGGLETTRLVSPRDRGGAGGSLRLKSLFTEPSEPLPEESKPVEMPFHHCHRDPLPPP GLTPERLHARRQLYAACAVCFVFMAGEVVGGYLAHSLAIMTDAAHLLADVGSMMGSLFSLWLS TRPATRTMTFGWHRSETLGALASVVSLWMVTGILLYLAFVRLLHSDYHIEGGAMLLTASIAVCAN LLMAFVLHQAGPPHSHGSRGAEYAPLEEGPEEPLPLGNTSVRAAFVHVLGDLLQSFGVLAASILI YFKPQYKAADPISTFLFSICALGSTAPTLRDVLRILMEGTPRNVGFEPVRDTLLSVPGVRATHEL HLWALTLTYHVASAHLAIDSTADPEAVLAEASSRLYSRFGFSSCTLQVEQYQPEMAQCLRCQE PPQA [SEQ ID NO: 6],

[0065] The full-length wild-type gene assembly of human SLC30A3 has GenBank accession no. NM_003459.5.

[0066] In some alternative embodiments, the ORF is translatable in a mammalian cell to express the human SLC30A8 protein having zinc transportation activity. In some embodiments, the ORF is translatable in a subject in vivo to express the human SLC30A8 protein having zinc transportation activity.

[0067] SLC30A8 is responsible for zinc ion transport and packaging in innate immune cells (e.g., macrophages). This function is known to be implicated in the zinc toxicity antimicrobial response, and has a role in phagocytosis. The full length wild-type human SLC30A8 protein has the UniProt accession no. Q8IWU4, and the following amino acid sequence:MEFLERTYLVNDKAAKMYAFTLESVELQQKPVNKDQCPRERPEELESGGMYHCHSGSKPTEK GANEYAYAKWKLCSASAICFIFMIAEVVGGHIAGSLAVVTDAAHLLIDLTSFLLSLFSLWLSSKPP SKRLTFGWHRAEILGALLSILCIWVVTGVLVYLACERLLYPDYQIQATVMIIVSSCAVAANIVLTVV LHQRCLGHNHKEVQANASVRAAFVHALGDLFQSISVLISALIIYFKPEYKIADPICTFIFSILVLASTI TILKDFSILLMEGVPKSLNYSGVKELILAVDGVLSVHSLHIWSLTMNQVILSAHVATAASRDSQVV RREIAKALSKSFTMHSLTIQMESPVDQDPDCLFCEDPCD [SEQ ID NO: 7],

[0068] The full-length wild-type gene assembly of human SLC30A8 has GenBank accession no. NM_173851.3.2.2 5' Cap

[0069] An mRNA 5' cap can provide resistance to nucleases found in most eukaryotic cells and promote translation efficiency. Several types of 5' caps are known. A 7-methylguanosine cap (also referred to as “m7G” or “Cap-0”), comprises a guanosine that is linked through a 5' — 5'-triphosphate bond to the first transcribed nucleotide.

[0070] A 5' cap is typically added to an RNA construct as follows: first, an RNA terminal phosphatase removes one of the terminal phosphate groups from the 5' nucleotide, leaving two terminal phosphates; guanosine triphosphate (GTP) is then added to the terminal phosphates via a guanylyl transferase, producing a 5 '5 '5 triphosphate linkage; and the 7-nitrogen of guanine is then methylated by a methyltransferase. Examples of cap structures include, but are not limited to, m7G(5')ppp(5')A, G(5')ppp(5')A, and G(5')ppp(5')G. Additional cap structures are described in U.S. Patent Publication Nos. US 2016 / 0032356 and US 2018 / 0125989, which are incorporated herein by reference.

[0071] 5'-capping of polynucleotides may be completed concomitantly during the in vitro transcription reaction using the following chemical RNA cap analogs to generate the 5'-guanosine cap structure according to manufacturer protocols: 3'-O-Me-m7G(5')ppp(5')G (the ARCA cap); G(5')ppp(5')A; G(5')ppp(5')G; m7G(5')ppp(5')A; m7G(5')ppp(5')G; m7G(5')ppp(5')(2'OMeA)pG; m7G(5')ppp(5')(2'OMeA)pU; m7G(5')ppp(5')(2'OMeG)pG (New England BioLabs, Ipswich, MA; TriLink Biotechnologies, San Diego, CA). 5'-capping of modified RNA may be completed post-transcriptionally using a vaccinia virus capping enzyme to generate the Cap 0 structure: m7G(5')ppp(5')G. Cap 1 structure may be generated using both vaccinia virus capping enzyme and a 2'-O methyltransferase to generate: m7G(5')ppp(5')G-2'-O-methyl. Cap 2 structure may be generated from the Cap 1 structure followed by the 2'-0-methylation of the 5'- antepenultimate nucleotide using a 2'-O methyl-transferase. Cap 3 structure may be generated from the Cap 2 structure followed by the 2'-0-methylation of the 5'-preantepenultimate nucleotide using a 2'-O methyl-transferase.

[0072] In some embodiments, the mRNA comprises a 5' cap selected from the group consisting of 3'-O-Me-m7G(5')ppp(5')G (the ARCA cap), G(5')ppp(5')A, G(5')ppp(5')G, m7G(5')ppp(5')A, m7G(5')ppp(5')G, m7G(5')ppp(5')(2'OMeA)pG, m7G(5')ppp(5')(2'OMeA)pU, and m7G(5')ppp(5')(2'OMeG)pG.2.3 Untranslated regions (UTR)

[0073] In some embodiments, the mRNA of the present invention includes a 5' and / or 3' untranslated region (UTR). UTRs are regions of a gene that are transcribed but not translated. In mRNA, the 5' UTR starts at the transcription start site and continues to the start codon but does not include the start codon. The 3' UTR starts immediately following the stop codon and continues until the transcriptional termination signal.

[0074] 5' UTR play a role in stability and translation initiation. They harbour signatures (e.g.,Kozak sequences) which are known to be involved in the process by which the ribosome initiates translation of many genes. Kozak sequences have the consensus sequences CCR(NG)CCAUGG, where Risa purine (adenine or guanine) three bases upstream of the start codon (AUG), which is followed by another “G”. 5' UTR have also been known to form secondary structures which are involved in elongation factor binding.

[0075] By engineering the features typically found in abundantly expressed genes of specific target cells or tissues, one can enhance the stability and protein production of mRNA of the invention. For example, introduction of 5' UTR of macrophage-expressed mRNA (such as CD44), leukocyte-expressed mRNA (such as CD45 and CD18), or myeloid cell mRNA (such as C / EBP, AML1 , G-CSF, GM-CSF, CD11 b, MSR, Fr-1 , i-NOS). Alternatively or in addition, the 5' UTR of lung epithelial cell-expressed mRNA could be introduced (e.g., SP-NB / C / D). In some embodiments, the 51UTR sequence and / orthe 31UTR sequence target the mRNA molecule to macrophages.

[0076] Other non-UTR sequences may be incorporated into the 5' UTR (or 3' UTR). For example, introns or portions of intron sequences may be incorporated into the flanking regions of the nucleic acid constructs of the invention. Incorporation of intronic sequences may increase protein production as well as mRNA levels.

[0077] In some embodiments, the mRNA disclosed herein may comprise a 5' UTR that includes one or more elements that positively impact on the stability or translation of mRNA. In some embodiments, a 5' UTR may be about 10 to 5,000 nucleotides in length. In some embodiments, a 5' UTR may be about 50 to 500 nucleotides in length. In some embodiments, the 5' UTR is at least about 10 nucleotides in length, about 20 nucleotides in length, about 30 nucleotides in length, about 40 nucleotides in length, about 50 nucleotides in length, about 100 nucleotides in length, about 150 nucleotides in length, about 200 nucleotides in length, about 250 nucleotides in length, about 300 nucleotides in length, about 350 nucleotides in length, about 400 nucleotides in length, about 450 nucleotides in length, about 500 nucleotides in length, about 550 nucleotides in length, about 600 nucleotides in length, about 650 nucleotides in length, about 700 nucleotides in length, about 750 nucleotides in length, about 800 nucleotides in length, about 850 nucleotides in length, about 900 nucleotides in length, about 950 nucleotides in length, about 1 ,000 nucleotides in length, about 1 ,500 nucleotides in length, about 2,000 nucleotides in length, about 2,500 nucleotides in length, about 3,000 nucleotides in length, about 3,500 nucleotides in length, about 4,000 nucleotides in length, about 4,500 nucleotides in length or about 5,000 nucleotides in length.

[0078] In some embodiments, the mRNA disclosed herein may comprise a 5' or 3' UTR that is derived from a gene distinct from the one encoded by the mRNA transcript (i.e. , the UTR is a heterologous UTR).

[0079] In certain embodiments, the 5' and / or 3' UTR sequences can be derived from mRNA which are stable (e.g., globin, actin, GAPDH, tubulin, histone, or citric acid cycle enzymes) to increase the stability of the mRNA. For example, a 5' UTR sequence may include a partial sequence of a CMV immediate-early 1 (IE1) gene, or a fragment thereof, to improve the nuclease resistance and / or improve the half-life of the mRNA. Also contemplated is the inclusion of a sequence encoding human growth hormone (hGH), or a fragment thereof, to the 3' end or untranslated region of the mRNA. Generally, these modifications improve the stability and / or pharmacokinetic properties (e.g., half-life) of the mRNA relative to their unmodified counterparts, and include, for example, modifications made to improve such mRNA resistance to in vivo nuclease digestion.

[0080] Exemplary 5' UTRs include a sequence derived from a CMV IE1 gene (U.S. Publication Nos. 2014 / 0206753 and 2015 / 0157565, each of which is incorporated herein by reference), or the sequence GGGAUCCUACC [SEQ ID NO: 3] (U.S. Publication No. 2016 / 0151409, incorporated herein by reference). In various embodiments, the 5' UTR may be derived from the 5' UTR of a TOP gene. TOP genes are typically characterized by the presence of a 5'-terminal oligopyrimidine (TOP) tract. Furthermore, most TOP genes are characterized by growth-associated translational regulation. However, TOP genes with a tissue specific translational regulation are also known. In some embodiments, the 5' UTR derived from the 5' UTR of a TOP gene lacks the 5' TOP motif (the oligopyrimidine tract) (e.g., U.S. Publication Nos. 2017 / 0029847, 2016 / 0304883, 2016 / 0235864, and 2016 / 0166710, each of which is incorporated herein by reference). In some embodiments, the 5' UTR is derived from a ribosomal protein Large 32 (L32) gene (U.S. Publication No. 2017 / 0029847, supra). In some embodiments, the 5' UTR is derived from the 5' UTR of an hydroxysteroid (17-b) dehydrogenase 4 gene (HSD17B4) (U.S. Publication No. 2016 / 0166710, supra). In certain embodiments, the 5' UTR is derived from the 5' UTR of an ATP5A1 gene (U.S. Publication No. 2016 / 0166710, supra). In some embodiments, an internal ribosome entry site (IRES) is used instead of a 5' UTR.

[0081] 3' UTRs are known to have stretches of adenosines and uridines embedded in them.These adenosine / uridine rich signatures are particularly prevalent in genes with high rates of turnover. Based on their sequence features and functional properties, the adenosine / uridine rich elements (AREs) can be separated into three classes: Class I AREs, which contain several dispersed copies of an AUUUA motif within uridine-rich regions. C-Myc and MyoD contain class I AREs. Class II AREs possess two or more overlapping UUAUUUA(U / A)(U / A) nonamers. Molecules containing this type of AREs include GM- CSF and TNF. Class III AREs are less well defined. These uridine rich regions do not contain an AUUUA motif. c-Jun and Myogenin are two well-studied examples of this class.

[0082] Most proteins binding to the AREs are known to destabilize the messenger, whereas members of the ELAV family, most notably HuR, have been documented to increase the stability of mRNA. HuR binds to AREs of all three classes. Therefore, it is envisaged that HuR specific binding sites may beengineered into the 3' UTR of nucleic acid molecules which will lead to HuR binding and thus, stabilization of the mRNA in vivo.

[0083] Introduction, removal or modification of 3' UTR adenosine / uridine-rich elements (AREs) can be used to modulate the stability of mRNA of the present invention. Although less preferable, when engineering specific mRNA, one or more copies of an ARE can be introduced to make mRNA of the invention less stable and thereby curtail translation and decrease production of the resultant protein. Likewise, AREs can be identified and removed or mutated to increase the intracellular stability and thus increase translation and production of the resultant protein. Transfection experiments can be conducted in relevant cell lines, using mRNA of the invention and protein production can be assayed at various time points post-transfection. For example, cells can be transfected with different ARE-engineering molecules and by using an ELISA kit to the relevant protein and assaying protein produced at 6 hour, 12 hour, 24 hour, 48 hour, and 7 days post-transfection.

[0084] In some embodiments, the mRNA disclosed herein may comprise a 3' UTR comprising one or more of a polyadenylation signal, a binding site for proteins that affect an mRNA's stability of location in a cell, or one or more binding sites for miRNAs. In some embodiments, a 3' UTR may be 50 to 5,000 nucleotides in length or longer. In some embodiments, a 3' UTR may be 50 to 1 ,000 nucleotides in length or longer. In some embodiments, the 3' UTR is at least about 50 nucleotides in length, about 100 nucleotides in length, about 150 nucleotides in length, about 200 nucleotides in length, about 250 nucleotides in length, about 300 nucleotides in length, about 350 nucleotides in length, about 400 nucleotides in length, about 450 nucleotides in length, about 500 nucleotides in length, about 550 nucleotides in length, about 600 nucleotides in length, about 650 nucleotides in length, about 700 nucleotides in length, about 750 nucleotides in length, about 800 nucleotides in length, about 850 nucleotides in length, about 900 nucleotides in length, about 950 nucleotides in length, about 1 ,000 nucleotides in length, about 1 ,500 nucleotides in length, about 2,000 nucleotides in length, about 2,500 nucleotides in length, about 3,000 nucleotides in length, about 3,500 nucleotides in length, about 4,000 nucleotides in length, about 4,500 nucleotides in length, or about 5,000 nucleotides in length.

[0085] A suitable non-limiting example of a 3' UTR that is useful in the invention is the sequence set forth in SEQ ID NO: 4. Other 3' UTRs are well known in the art, and could be readily utilized in compositions of the present invention.

[0086] In some embodiments, the 5' UTR comprises, consists, or consists essentially of a nucleic acid sequence set forth in SEQ ID NO: 3. In some embodiments, the 3' UTR comprises, consists, or consists essentially of a nucleic acid sequence set forth in SEQ ID NO: 4 and the 5' UTR comprises, consists, or consists essentially of a nucleic acid sequence set forth in SEQ ID NO: 5. Suitable 5' UTR and 3' UTR are described in further detail in International PCT Patent No. W02012 / 075040, incorporated herein by reference.

[0087] In some preferred embodiments, the 3' UTR comprises, consists, or consists essentially of a nucleic acid sequence set forth in SEQ ID NO: 12. In some embodiments of this type, the 5' UTR comprises, consists, or consists essentially of a nucleic acid sequence set forth in SEQ ID NO: 13.2.4 Polvadenylated (oolvtA)) tail

[0088] As used herein, the terms “poly(A) sequence,” “poly(A) tail,” and “poly(A) region” refer to a sequence of adenosine nucleotides at the 3' end of the mRNA molecule. The poly(A) tail may confer stability to the mRNA and protect it from exonuclease degradation. The poly(A) tail may enhance translation. In some embodiments, the poly(A) tail is essentially homopolymeric. For example, a poly(A) tail of 100 adenosine nucleotides may have essentially a length of 100 nucleotides. In certain embodiments, the poly(A) tail may be interrupted by at least one nucleotide different from an adenosine nucleotide (e.g., a nucleotide that is not an adenosine nucleotide). For example, a poly(A) tail of 100 adenosine nucleotides may have a length of more than 100 nucleotides (comprising 100 adenosine nucleotides and at least one nucleotide, or a stretch of nucleotides, that are different from an adenosine nucleotide).

[0089] The “poly(A) tail,” as used herein, typically relates to RNA. However, in the context of the disclosure, the term likewise relates to corresponding sequences in a DNA molecule (e.g., a “poly(T) sequence”).

[0090] The poly(A) tail may comprise about 10 to about 500 adenosine nucleotides, about 10 to about 200 adenosine nucleotides, about 40 to about 200 adenosine nucleotides, or about 40 to about 150 adenosine nucleotides. The length of the poly(A) tail may be at least about 10, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, or 500 adenosine nucleotides.

[0091] In certain embodiments, the poly(A) tail comprises the sequence:AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAGCAUAUGACUAAAAAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA [SEQ ID NO: 5],

[0092] In some embodiments where the nucleic acid is an RNA, the poly(A) tail of the nucleic acid is obtained from a DNA template during RNA in vitro transcription. In certain embodiments, the poly(A) tail is obtained in vitro by common methods of chemical synthesis without being transcribed from a DNA template. In various embodiments, poly(A) tails are generated by enzymatic polyadenylation of the RNA (after RNA in vitro transcription) using commercially available polyadenylation kits and corresponding protocols, or alternatively, by using immobilized poly(A)polymerases (e.g., using methods and means as described in International PCT Patent Publication No. WO2016 / 174271).

[0093] The nucleic acid may comprise a poly(A) tail obtained by enzymatic polyadenylation, wherein the majority of nucleic acid molecules comprise about 100 (+ / -20) to about 500 (+ / -50) or about 250 (+ / -20) adenosine nucleotides.

[0094] In some embodiments, the nucleic acid may comprise a poly(A) tail derived from a template DNA and may additionally comprise at least one additional poly(A) tail generated by enzymatic polyadenylation, e.g., as described in International PCT Patent No. WO2016 / 091391.

[0095] In certain embodiments, the nucleic acid comprises at least one polyadenylation signal. In various embodiments, the nucleic acid may comprise at least one poly(C) sequence. The term “poly(C) sequence,” as used herein, is intended to be a sequence of cytosine nucleotides of up to about 200cytosine nucleotides. In some embodiments, the poly(C) sequence comprises about 10 to about 200 cytosine nucleotides, about 10 to about 100 cytosine nucleotides, about 20 to about 70 cytosine nucleotides, about 20 to about 60 cytosine nucleotides, or about 10 to about 40 cytosine nucleotides. In some embodiments, the poly(C) sequence comprises about 30 cytosine nucleotides.

[0096] In some embodiments, the mRNA molecules of the present invention comprise the nucleic acid sequence set forth in SEQ ID NO: 11 . In similar embodiments, the mRNA molecules of the present invention may comprise a nucleic acid sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to the sequence set forth in SEQ ID NO: 11 .In some alternative embodiments, the mRNA molecules of the present invention comprise the nucleic acid sequence set forth in SEQ ID NO: 10. In some embodiments, the mRNA molecule may comprise a nucleic acid sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to the sequence set forth in SEQ ID NO: 10.2.5 Codon optimisation

[0097] The nucleotide sequence of a mRNA of the invention is preferably codon optimized. In performing codon optimisation, codon frequencies in target and host organisms are generally matched to ensure proper folding, bias GC content to increase mRNA stability or reduce secondary structures, 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, to adjust translational rates to allow the various domains of the protein to fold properly, or to reduce or eliminate problem secondary structures within the mRNA.

[0098] Codon composition is known to affect translation efficiency. Replacing rare codons with synonymous frequent codons improves translational yield because reuse of the same tRNA accelerates translation owing to amino-acylation of tRNAs in the vicinity of the ribosomes. Codon context (that is, neighbouring nucleotides and codons) also affects the translational elongation rate and translational efficiency. Similar to recombinant DNA-based approaches, codon-optimized in vitro transcribed (IVT) mRNAs have been successfully used. However, in some cases, there may be valid reasons to refrain from using optimized codons as understood by a skilled person. Some proteins require slow translation, which is ensured by rare codons, for their proper folding. It may also be beneficial for some IVT mRNA-encoded to maintain the original ORF.

[0099] In a preferred embodiment of the invention, codon optimization methods are useful to increase expression, structural stability, thermal stability or increased function of the encoded protein. Codon optimization tools, algorithms and services are known in the art, and non-limiting examples include services from GeneArt (Life Technologies), DNA2.0 (Menlo Park, CA) or other methods known in the art. However, specific strategies for codon optimization vary considerably based on underlying assumptionsabout which codon features are important to translation. One approach involves substituting in the most frequently used codon for all instances of a given amino acid. Another approach involves only replacing rare codons with more abundant synonymous codons. Still other approaches involve adjusting the codon usage frequency to match the natural frequencies in a host organism, or choosing codons based on cognate transfer RNA (tRNA) abundance. A skilled person will generally understand that codon optimization involves the replacement of a codon with an optimized codon that is synonymous with the replaced codon.

[0100] In one embodiment, the ORF sequence is optimized using optimization algorithms. In a preferred embodiment, codon optimisation is conducted by enriching a DNA template that will encode a mRNA of the invention for guanine (G) and cytosine (C) content. By way of an example regarding this method, G and C content is enriched by substituting codons with adenine (A) or uracil (U) at the third base with codons enriched in guanine (G) or cytosine (C). Using such an optimization methos, a skilled person will understand methods for determining suitable nucleotide substitutions based on codon options for each amino acid as outlined in Table 1 herein and known in the art. Non-limiting examples of sequences that may be codon optimised in accordance with the invention include SEQ ID NO: 2, being a wild-type mRNA sequence of SLC30A1 .TABLE 1EXEMPLARY CODON SELECTION FOR OPTIMISATION

[0101] In an embodiment of the invention, an codon optimized SLC30A1 sequence in accordance with the invention may comprise a sequence having at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5% at least 99.8% or 100% identity to a sequence set forth as SEQ ID NO: 2.

[0100] Whilst preferable, codon optimized mRNA need not be uniformly codon optimized along the entire length of the mRNA molecule. Different nucleotide modifications and / or backbone structures may exist at various positions in the nucleic acid. One of ordinary skill in the art will appreciate that the modification(s) may be located at any position(s) of a mRNA such that the polypeptide function, polypeptide expression, mRNA thermal stability or structure is preferably increased or improved. A modification may also be a 5' or 3' terminal modification. Further, the mRNA may contain at a minimum one and at maximum 100% optimized codons, or any intervening percentage, such as at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% optimized codons.

[0101] The identity of a polynucleotide or mRNA is determined by GAP (Needleman and Wunsch, 1970) analysis (GCG program) with a gap creation penalty=5, and a gap extension penalty=0.3. The query sequence is at least 900 nucleotides in length, and the GAP analysis aligns the two sequences over a region of at least 900 nucleotides. Preferably, the query sequence is at least 975 nucleotides in length, and the GAP analysis aligns the two sequences over a region of at least 975 nucleotides. Even more preferably, the query sequence is at least 1 ,050 nucleotides in length and the GAP analysis aligns the two sequences over a region of at least 1 ,050 nucleotides. Even more preferably, the GAP analysis aligns two sequences over their entire length.

[0102] With regard to the defined polynucleotides or mRNA, it will be appreciated that percentage identity figures higher than those provided above will encompass preferred embodiments. Thus, where applicable, in light of the minimum percentage identity figures, it is preferred that the mRNA comprises a sequence which is at least 50%, at least 60%, more preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, more preferably at least 99.1%, more preferably at least 99.2%, more preferably at least 99.3%, more preferably at least 99.4%, more preferably at least 99.5%, more preferably at least 99.6%, more preferably at least 99.7%, more preferably at least 99.8%, and even more preferably at least 99.9% identical to the relevant nominated sequence identifier.

[0103] When referring to polynucleotide or mRNA identity herein, it will be understood that a given sequence identity is in reference to the open reading frame sequence.

[0104] In a further embodiment, the present invention relates to polynucleotides or mRNA which are substantially identical or identical to those specifically described herein. As used herein, with reference to a mRNA or polynucleotide the term “substantially identical” means the substitution of one or a few (for example two, three, or four) nucleotides whilst maintaining activity of the native protein encoded by the polynucleotide. In addition, this term includes the addition or deletion of nucleotides which results in the increase or decrease in size of the encoded native protein by one or a few (for example, two, three, or four) amino acids whilst maintaining activity of the native protein encoded by the polynucleotide.2.6 Chemical modification

[0105] The mRNA disclosed herein may be modified or unmodified. In some embodiments, the mRNA may comprise at least one chemical modification. In some embodiments, the mRNA disclosed herein may contain one or more modifications that typically enhance RNA stability.

[0106] Exemplary modifications can include backbone modifications, sugar modifications, or base modifications. In some embodiments, the mRNA of the invention may be synthesized from naturally occurring nucleotides and / or nucleotide analogues (modified nucleotides) including, but not limited to, purines (adenine (A) and guanine (G)) or pyrimidines (thymine (T), cytosine (C), and uracil (U)). In certain embodiments, the disclosed mRNA may be synthesized from modified nucleotide analogues or derivatives of purines and pyrimidines, such as, e.g., 1-methyl-adenine, 2-methyl-adenine, 2-methylthio-N-6- isopentenyl-adenine, N6-methyl-adenine, N6-isopentenyladenine, 2-thio-cytosine, 3-methyl-cytosine, 4- acetyl-cytosine, 5-methyl-cytosine, 2,6-diaminopurine, 1-methyl-guanine, 2-methyl-guanine, 2,2-dimethyl- guanine, 7-methyl-guanine, inosine, 1-methyl-inosine, pseudouracil (5-uracil), dihydro-uracil, 2-thio-uracil, 4-thio-uracil, 5-carboxymethylaminomethyl-2-thio-uracil, 5-(carboxyhydroxymethyl)-uracil, 5-fluoro-uracil, 5- bromo-uracil, 5-carboxymethylaminomethyl-uracil, 5-methyl-2-thio-uracil, 5-methyl-uracil, Nuracil-5-oxy acetic acid methyl ester, 5-methylaminomethyl-uracil, 5-methoxyaminomethyl-2-thiouracil, 5'- methoxycarbonylmethyl-uracil, 5-methoxy-uracil, uracil-5-oxy acetic acid methyl ester, uracil-5-oxyacetic acid (v), 1-methyl-pseudouracil, queosine, p-D-mannosyl-queosine, phosphoramidates, phosphorothioates, peptide nucleotides, methyl phosphonates, 7-deazaguanosine, 5-methylcytosine, and inosine.

[0107] In some embodiments, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% of the uracil nucleotides in the mRNA are chemically modified.

[0108] In some embodiments, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% of the uracil nucleotides in the ORF are chemically modified.

[0109] The preparation of such analogues is described, for example, in U.S. Patent Nos. 4,373,071 , 4,401 ,796, 4,415,732, 4,458,066, 4,500,707, 4,668,777, 4,973,679, 5,047,524, 5,132,418, 5,153,319, 5,262,530, and 5,700,642.

[0110] The terms “modification” or “modified” refer to modification of a mRNA of the invention with respect to A, G, U or C ribonucleotides. Generally, the modification refers to the coding region of themRNA. However, the modification may also be introduced into the flanking regions and / or the terminal regions if the modification increases protein expression, function, thermal stability or structure.

[0111] Because chemical bonds will necessarily be broken and reformed to effect a structural modification, structural modifications are of a chemical nature and hence are chemical modifications. However, structural modifications will result in a different sequence of nucleotides. For example, the polynucleotide "ATCG" may be chemically modified to "AT -5meC-G". The same polynucleotide may be structurally modified from "ATCG" to "ATCCCG". Here, the dinucleotide "CC" has been inserted, resulting in a structural modification to the polynucleotide.

[0112] The mRNA or polynucleotides can include any useful modification, such as to the sugar, the nucleobase, or the internucleoside linkage (e.g. to a linking phosphate / to a phosphodiester linkage to the phosphodiester backbone). For example, the major groove of a polynucleotide, or the major groove face of a nucleobase may comprise one or more modifications. One or more atoms of a pyrimidine nucleobase (e.g. on the major groove face) may he replaced or substituted with optionally substituted amino, optionally substituted thiol, optionally substituted alkyl (e.g., methyl or ethyl), or halo (e.g., chloro or fluoro). In certain cases (e.g., one or more modifications) are present in each of the sugar and the internucleoside linkage. Modifications according to the present invention may be modifications of ribonucleic acids (RNAs) to deoxyribonucleic acids (DNAs), e.g., the substitution of the 2'OH of the ribofuranosyl ring to 2'H, threose nucleic acids (TNAs), glycol nucleic acids (GNAs), peptide nucleic acids (PNAs), locked nucleic acids (LNAs) or hybrids thereof). Additional modifications are known in the art.

[0113] In some embodiments the chemical modification increases mRNA stability and / or mRNA translation when compared to a mRNA without chemical modification. Preferably the modification is to a uracil (U) or a cytosine (C) but may be to any mRNA base including adenine (A), and guanine (G) if the modification increases mRNA stability and / or mRNA translation when compared to a mRNA without chemical modification. Suitable uracil modifications may include but are not limited to pseudouridine (ip), pyridin-4-one ribonucleoside, 5-aza-uridine, 6-aza-uridine, 2-thio-5-aza-uridine, 2-thio-uridine (s2U), 4-thio- uridine (s4U), 4-thiopseudouridine, 2-thio-pseudouridine, 5-hydroxy-uridine (ho5U), 5-aminoallyl-uridine, 5- halo-uridine (e.g., 5-iodo-uridine or 5-bromo-uridine), 3-methyluridine (m3U), 5-methoxy-uridine (mo5U), 5- methoxy-uridine triphosphate (mo5UTP), uridine 5-oxyacetic acid (cmo5U), uridine 5-oxyacetic acid methyl ester (mcmo5U), 5-carboxymethyluridine n(cm5U), 1-carboxymethyl-pseudouridine, 5- carboxyhydroxymethyl-uridine (chm5U), 5-carboxyhydroxymethyl-uridine methyl ester (mchm5U), 5- methoxycarbonylmethyl-uridine (mcm5U), 5-methoxycarbonylmethyl-2-thio-uridine (mcm5s2U), 5- aminomethyl-2-thio-uridine (nm5s2U), 5-methylaminomethyl-uridine (mnm5U), 5-methylaminomethyl-2-thio- uridine (mnm5s2U), 5-methylaminomethyl-2-seleno-uridine (mnm5se2U), 5-carbamoylmethyl-uridine (ncm5U), 5-carboxymethylaminomethyl-uridine (cmnm5U), 5-carboxymethylaminomethyl-2-thio-uridine (cmnm5s2U), 5-propynyluridine, 1-propynyl-pseudouridine, 5-taurinomethyl-uridine (tm5U), 1-taurinomethyl- pseudouridine, 5-taurinomethyl-2-thio-uridine (tm5s2U), 1-taurinomethyl-4-thio-pseudouridine, 5-methyl- uridine (m5U, i.e. , having the nucleobase deoxythymine), 1-methylpseudouridine (m1i ), 5-methyl-2- thiouridine (m5s2U), 1-methyl-4-thiopseudouridine (m1s4i ), 4-thio-1-methyl-pseudouridine, 3-methyl- pseudouridine (m3i ), 2-thio-1-methyl-pseudouridine, 1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-1 -deaza-pseudouridine, dihydrouridine (D), dihydropseudouridine, 5,6-dihydrourid ine, 5-methyldihydrouridine (m5D), 2-thio-dihydrouridine , 2-thio-dihydropseudouridine, 2-methoxy-uridine, 2-methoxy-4-thio-uridine, 4- methoxy-pseudouridine, 4-methoxy-2-thio-pseudouridine, N1-methyl-pseudouridine (also known as 1- methylpseudouridine (m1i ), 3-(3-amino-3-carboxypropyl)uridine (acp3U), 1-methyl-3-(3-amino-3 - carboxypropyl)pseudouridine (acp3i ), 5-(isopentenylaminomethyl)uridine (inm5U), 5- (isopentenylaminomethyl)-2-thio-uridine (inm5s2U), 4-thio-uridine, 2'-O-methyluridine (Um), 5,2'-O- dimethyl-uridine (m5Um), 2'-0-methyl-pseudouridine (i m), 2-thio-2'-0-methyl-uridine (s2Um), 5- methoxycarbonylmethyl-2'-0-methyl-uridine (mcm5Um), 5-carbamoylmethyl-2'-0-methyl -uridine (ncm5Um), 5-carboxymethylaminomethyl-2'-0-methyluridine (cmnm5Um), 3,2'-O-dimethyl-uridine (m3Um), 5-(isopentenylaminomethyl)-2'-0-methyl-uridine (inm5Um), 1 -thio-uridine, deoxythymidine, 2'-F-arauridine, 2'-F-urid ine, 2'-OH-ara-uridine, 5-(2-carbomethoxyvinyl) uridine, and 5-[3-(1-E-propenylamino)]uridine.

[0114] In some embodiments, the modified nucleobase is a modified cysteine. Suitable cytosine modifications may include but are not limited to 5-aza-cytidine, 6-aza-cytidine, pseudoisocytidine, 3- methyl-cytidine (m3C), N4-acetyl-cytidine (ac4C), 5-formylcytidine (f5C), N4-methyl-cytidine (m4C), 5- methyl-cytidine (m5C), 5-methyl-cytidine 5'-triphosphate (5mCTP), 5-halo-cytidine (e.g., 5-iodo-cytidine), 5- hydroxymethyl-cytidine (hm5C), 1-methyl-pseudoisocytidine, pyrrolo-cytidine, pyrrolo-pseudoisocytidine, 2- thio-cytidine (s2C), 2-thio-5-methyl-cytidine, 4-thio-pseudoisocytidine, 4-thio-1-methyl-pseudoisocytidine, 4- thio-1-methyl-1-deaza-pseudoisocytidine, 1-methyl-1-deaza-pseudoisocytidine, zebularine, 5- azazebularine, 5-methyl-zebularine, 5-aza-2-thio-zebularine, 2-thiozebularine, 2-methoxy-cytidine, 2- methoxy-5-methyl-cytidine, 4-methoxy-pseudoisocytidine, 4-methoxy-1 -methylpseudoisocytidine, lysidine (k2C) , a-thiocytidine, 2'-O-methyl-cytidine (Cm), 5,2'-O-dimethylcytidine (m5Cm), N4-acetyl-2'-O-methyl- cytidine (ac4Cm), N4,2'-O-dimethyl-cytidine (m4Cm), 5-formyl- 2'-O-methyl-cytidine (f5Cm), N4,N4,2'-O- trimethyl-cytidine (m42Cm), 1 -thio-cytidine, 2'-F-ara-cytidine, 2'-F-cytidine, and 2'-OH-ara-cytidine.

[0115] In some embodiments, the modified nucleobase is a modified adenine. Exemplary nucleobases and nucleosides having a modified adenine include 2-amino-purine, 2,6-diaminopurine, 2- amino-6-halo-purine (e.g., 2-amino-6-chloro-purine), 6-halo-purine (e.g., 6-chloro-purine), 2-amino-6- methyl-purine, 8-azido-adenosine, 7--adenine, 7-deaza-8-aza-adenine, 7-deaza-2-amino-purine, 7-deaza- 8-aza-2-amino-purine, 7-deaza-2,6-diaminopurine, 7-deaza-8-aza-2,6-diaminopurine, 1-methyl-adenosine (m1A), 2-methyl-adenine (m2A), N6-methyl-adenosine (m6A), 2-methylthio-N6-methyl-adenosine (ms2m6A), N6-isopentenyl-adenosine (i6A), 2-methylthio-N6-isopentenyl-adenosine (ms2i6A), N6-(cis- hydroxyisopentenyl)adenosine (io6A), 2-methylthio-N6-(cis-hydroxyisopentenyl)adenosine (ms2io6A), N6- glycinylcarbamoyl-adenosine (g6A), N6-threonylcarbamoyl-adenosine (t6A), N6-methyl-N6- threonylcarbamoyl-adenosine (m6t6A), 2-methylthio-N6-threonylcarbamoyl-adenosine (ms2g6A), N6,N6- dimethyl-adenosine (m62A), N6-hydroxynorvalylcarbamoyl-adenosine (hn6A), 2-methylthio-N6- hydroxynorvalylcarbamoyl-adenosine (ms2hn6A), N6-acetyl-adenosine (ac6A), 7-methyl-adenine, 2- methylthio-adenine, 2-methoxy-adenine, a-thio-adenosine, 2'-0-methyl-adenosine (Am), N6,2'-O-dimethyl- adenosine (m6Am), N6,N6,2'-0-trimethyl-adenosine (m62Am), 1 ,2'-0-dimethyl-adenosine (m1Am), 2'-O- ribosyladenosine (phosphate) (Ar(p)), 2-amino-N6-methyl-purine, 1-thio-adenosine, 8-azido-adenosine, 2'-F-ara-adenosine, 2'-F-adenosine, 2'-OH-ara-adenosine, and N6-(19-amino-pentaoxanonadecyl)- adenosine.

[0116] In some embodiments, the modified nucleobase is a modified guanine. Exemplary nucleobases and nucleosides having a modified guanine include inosine (I), 1-methyl-inosine (m1l), wyosine (imG), methylwyosine (mimG), 4-demethyl-wyosine (imG-14), isowyosine (imG2), wybutosine (yW), peroxywybutosine (02yW), hydroxywybutosine (OhyW), undermodified hydroxywybutosine (OhyW*),7-deaza-guanosine, queuosine (Q), epoxyqueuosine (oQ), galactosyl-queuosine (galQ), mannosyl- queuosine (manQ), 7-cyano-7-deaza-guanosine (preQo), 7-aminomethyl-7-deaza-guanosine (preQi), archaeosine (G+), 7-deaza-8-aza-guanosine, 6-thio-guanosine, 6-thio-7-deaza-guanosine, 6-thio-7-deaza-8-aza-guanosine, 7-methyl-guanosine (m7G), 6-thio-7-methyl-guanosine, 7-methyl-inosine, 6-methoxy- guanosine, 1-methyl-guanosine (m1G), N2-methyl-guanosine (m2G), N2,N2-dimethyl-guanosine (m22G), N2,7-dimethyl-guano sine (m2?G), N2,N2,7-dimethyl-guanosine (m227G), 8-oxo-guanosine, 7-methyl-8- oxo-guanosine, 1-methyl-6-thio-guanosine, N2-methyl-6-thio-guanosine, N2,N2-dimethyl-6-thio-guanosine, a-thio-guanosine, 2'-0-methyl-guanosine (Gm), N2-methyl-2'-0-methyl-guanosine (m2Gm), N2,N2- dimethyl-2'-0-methyl-guanosine (m22Gm), 1-methyl-2'-0-methyl-guanosine (m'Gm), N2,7-dimethyl-2'-O- methyl-guanosine (m27Gm), 2'-0-methyl-inosine (Im), 1 ,2'-0-dimethyl-inosine (m1lm), 2'-O- ribosylguanosine (phosphate) (Gr(p)), 1-thio-guanosine, 0(6)-methyl-guanosine, 2'-F-ara-guanosine, and 2'-F-guanosine.

[0117] Modified nucleic acids need not be uniformly chemically modified along the entire length of the molecule. Different nucleotide modifications and / or backbone structures may exist at various positions in the nucleic acid. One of ordinary skill in the art will appreciate that the modification(s) may be located at any position(s) of a mRNA such that the polypeptide function, polypeptide expression, mRNA thermal stability or structure is preferably increased or improved.

[0118] A modification may also be a 5' or 3' terminal modification. Further, the mRNA may contain at a minimum one and at maximum 100% chemical modifications, preferably nucleoside modifications, or any intervening percentage, such as at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% modified nucleotides.2.7 Methods of synthesis of mRNA

[0119] mRNA for use in accordance with the invention may be prepared according to any available technique including, but not limited to chemical synthesis, enzymatic synthesis, which is generally termed in vitro transcription (IVT), enzymatic or chemical cleavage of a longer precursor, etc.

[0120] Methods of synthesizing RNAs are known in the art (see, e.g., Gait, M.J., 1984; and Herdewijn, 2005; both of which are incorporated herein by reference).

[0121] In a preferred embodiment, a mRNA of the invention is prepared by IVT using methods known in the art. The machinery of the transfected cell is utilized for in vivo translation of the message to the corresponding protein, which is the therapeutic polypeptide suitable for the methods described herein. IVT mRNA is engineered to structurally resemble naturally occurring mature and processed mRNA in the cytoplasm of eukaryotic cells. Hence, the IVT mRNA is single-stranded, has a 5' cap and a 3' poly(A) tail.The open reading frame (ORF) encoding the protein of interest is marked by start and stop codons and is flanked by untranslated region (UTRs). The mRNA is generally synthesized in a cell-free system by IVT from a DNA template, such as a linearized plasmid or a PCR product. With the exception of the 5' cap, this DNA template encodes all the structural elements of a functional mRNA. IVT is performed with T7 or SP6 RNA polymerase in the presence of nucleotides and thereafter the mRNA is capped enzymatically. The template DNA is then digested by DNases and the mRNA is purified by conventionally used methods for isolating nucleic acids.2.8 Polynucleotides and Genes

[0122] The present invention relates to various polynucleotides encoding mRNA, particularly those used in in vitro transcription (IVT) for the generation of a mRNA of the invention. As used herein, a “polynucleotide” or “nucleic acid” or “nucleic acid molecule” means a polymer of nucleotides, which may be DNA or RNA or a combination thereof, and includes genomic DNA, mRNA, cRNA, and cDNA. A given polynucleotide may be of cellular, genomic, or synthetic origin, for example made on an automated synthesizer, and may be combined with carbohydrate, lipids, protein or other materials, labelled with fluorescent or other groups, or attached to a solid support to perform a particular activity defined herein, or comprise one or more modified nucleotides not found in nature. The polymer may be single-stranded, essentially double-stranded, or partly double-stranded. Base pairing as used herein refers to standard base pairing between nucleotides, including G:U base pairs. “Complementary” means two polynucleotides are capable of base pairing (hybridizing) along part of their lengths, or along the full length of one or both. The term “polynucleotide” is used interchangeably herein with the term “nucleic acid”. The polynucleotides or nucleic acid sequences of the present application may be deoxyribonucleic acid (DNA) sequences or ribonucleic acid (RNA) sequences and may include naturally occurring bases including adenine, guanine, cytosine, thymidine and uracil. The sequences may also contain modified bases. Examples of such modified bases include aza and deaza adenine, guanine, cytosine, thymidine and uracil; and xanthine and hypoxanthine. The nucleic acid can be either double stranded or single stranded, and represents the sense or antisense strand. Further, the term "nucleic acid" includes the complementary nucleic acid sequences.

[0123] The term “nucleic acid molecule” or its derivatives, as used herein, is intended to include unmodified DNA or RNA or modified DNA or RNA. For example, it may be useful for the nucleic acid molecules of the disclosure to be composed of single- and double-stranded DNA, DNA that is a mixture of single- and double-stranded regions, single- and double-stranded RNA, and RNA that is a mixture of single- and double stranded regions, hybrid molecules comprising DNA and RNA that may be single stranded or, more typically double-stranded or a mixture of single- and double-stranded regions. In addition, it may be useful for the nucleic acid molecules to be composed of triple stranded regions comprising RNA or DNA or both RNA and DNA. The nucleic acid molecules of the disclosure may also contain one or more modified bases or DNA or RNA backbones modified for stability or for other reasons. “Modified” bases include, for example, tritiated bases and unusual bases such as inosine. A variety of modifications can be made to DNA and RNA; thus “nucleic acid molecule” encompasses chemically,enzymatically , or metabolically modified forms. The term "polynucleotide" shall have a corresponding meaning.

[0124] Furthermore, the term "exogenous” in the context of a polynucleotide (nucleic acid) refers to the polynucleotide when present in a cell that does not naturally comprise the polynucleotide. The cell may be a cell which comprises a non-endogenous polynucleotide resulting in an altered amount of production of the encoded polypeptide, for example an exogenous polynucleotide which increases the expression of an endogenous polypeptide, or a cell which in its native state does not produce the polypeptide. Increased production of a polypeptide of the invention is also referred to herein as "overexpression”.

[0125] The present invention also relates to the use of oligonucleotides, for instance in methods of screening for a mRNA molecule of the invention. As used herein, "oligonucleotides” are polynucleotides up to 50 nucleotides in length. The minimum size of such oligonucleotides is the size required for the formation of a stable hybrid between an oligonucleotide and a complementary sequence on a nucleic acid molecule of the present invention. They can be RNA, DNA, or combinations or derivatives of either. Oligonucleotides are typically relatively short single stranded molecules of 10 to 30 nucleotides, commonly 15-25 nucleotides in length. When used as a guide for genome editing, probe or as a primer in an amplification reaction, the minimum size of such an oligonucleotide is the size required for the formation of a stable hybrid between the oligonucleotide and a complementary sequence on a target nucleic acid molecule. Preferably, the oligonucleotides are at least 15 nucleotides, more preferably at least 18 nucleotides, more preferably at least 19 nucleotides, more preferably at least 20 nucleotides, more preferably at least 22 nucleotides, even more preferably at least 25 nucleotides in length. Oligonucleotides of the present invention used as a probe are typically conjugated with a label such as a radioisotope, an enzyme, biotin, a fluorescent molecule or a chemiluminescent molecule.

[0126] As those skilled in the art would be aware, the sequence of the oligonucleotide primers described herein can be varied to some degree without effecting their usefulness for the methods of the invention. A "variant” of an oligonucleotide disclosed herein (also referred to herein as a "primer” or "probe” depending on its use) useful forthe methods of the invention includes molecules of varying sizes of, and / or are capable of hybridising to the genome close to that of, the specific oligonucleotide molecules defined herein. For example, variants may comprise additional nucleotides (such as 1 , 2, 3, 4, or more), or less nucleotides as long as they still hybridise to the target region. Furthermore, a few nucleotides may be substituted without influencing the ability of the oligonucleotide to hybridise the target region. In addition, variants may readily be designed which hybridise close (for example, but not limited to, within 50 nucleotides or within 100 nucleotides) to the region of the genome where the specific oligonucleotides defined herein hybridise.

[0127] The present invention includes oligonucleotides that can be used as, for example, guides for RNA-guided endonucleases, probes to identify nucleic acid molecules, or primers to produce nucleic acid molecules. Probes and / or primers can be used to clone homologues of the polynucleotides of the invention from other species. Furthermore, hybridization techniques known in the art can also be used to screen genomic or cDNA libraries for such homologues.

[0128] Polynucleotides and oligonucleotides of the present invention include those which hybridize under stringent conditions to one or more of the sequences disclosed herein. As used herein, stringent conditions are those that: (1) employ low ionic strength and high temperature for washing, for example, 0.015 M NaC1 / 0.0015 M sodium citrate / 0.1 % NaDodS04 at 50°C; (2) employ during hybridisation a denaturing agent such as formamide, for example, 50% (vol / vol) formamide with 0.1% bovine serum albumin, 0.1% Ficoll, 0.1% polyvinylpyrrolidone, 50 mM sodium phosphate buffer at pH 6.5 with 750 mM NaC1 , 75 mM sodium citrate at 42°C; or (3) employ 50% formamide, 5 x SSC (0.75 M NaC1 , 0.075 M sodium citrate), 50 mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5 x Denhardt's solution, sonicated salmon sperm DNA (50 g / ml), 0.1% SDS and 10% dextran sulfate at 42°C in 0.2 x SSC and 0.1% SDS.

[0129] Polynucleotides of the present invention may possess, when compared to naturally occurring molecules, one or more mutations which are deletions, insertions, or substitutions of nucleotide residues. Mutants can be either naturally occurring (that is to say, isolated from a natural source) or synthetic (for example, by performing site-directed mutagenesis on the nucleic acid). A variant of a polynucleotide of the invention includes molecules of varying sizes when compared to the reference polynucleotides defined herein. For example, variants may comprise additional nucleotides (such as 1 , 2, 3, 4, or more), or less nucleotides as long as they encode a functional protein. Furthermore, a few nucleotides may be substituted without influencing the integrity of the encoded protein. In addition, variants may include polynucleotides which encode the same polypeptide or amino acid sequence but which vary in nucleotide sequence by redundancy of the genetic code. The terms “polynucleotide variant” and “variant” also include naturally occurring allelic variants.2.9 Polypeptides

[0130] According to the present invention, a mRNA of the invention is designed to encode a polypeptide that is suitable for treating or preventing a microbial infection in subject with CFTR dysfunction, and preferably a SLC30A1 polypeptide. As used herein, the term “polypeptide” means a polymer of amino acid residues (natural or unnatural) linked together typically by peptide bonds. The term, as used herein, refers to proteins, polypeptides, and peptides of any size, structure, or function. In some instances the polypeptide encoded is smaller than about 50 amino acids and the polypeptide is then termed a peptide. If the polypeptide is a peptide, it will be at least about 2, 3, 4, or at least 5 amino acid residues long. Thus, polypeptides include gene products, naturally occurring polypeptides, synthetic polypeptides, homologs, orthologs, paralogs, fragments and other equivalents, and variants.

[0131] A “wildtype SLC301A polypeptide” refers to any polypeptide or protein encoded by a wildtype gene that is capable of having normal (level of function absent disease or disorder) biological activity when expressed in vivo. Such functionality can be tested by any means known to establish functionality of a protein. The term “mutant SLC30A1 gene” refers to any non — wildtype SLC30A1 sequence and may include SLC30A1 gene mutations.

[0132] In some embodiments, a codon optimised and / or chemically modified SLC30A1 mRNA of the invention may encode a polypeptide having a similar protein expression profile when compared to awild-type SLC30A1 mRNA counterpart. In another embodiment, a codon optimised and / or chemically modified SLC30A1 mRNA may encode a polypeptide that restores a protein insufficiency in cells in a subject with a deficient zinc toxicity anti-microbial response.

[0133] In another embodiment, a codon optimised and / or chemically modified SLC30A1 mRNA may encode a polypeptide that restores the zinc toxicity antimicrobial response in cells (e.g., macrophages) in a manner that is similar to a wild-type SLC30A1 mRNA counterpart.

[0134] Thus, codon optimisation and / or chemical modification of an mRNA of the invention can provide for protein function that is similar to that of the wild-type SLC30A1 protein counterpart.

[0135] The protein encoded by the codon optimised and / or chemically modified SLC30A1 mRNA is generally about 30 to about 60, from about 60 to about 100, from about 100 to about 130, from about 130 to about 160, from about 160 to about 200, from about 200 to about 230, from about 230 to about 260, from about 260 to about 300, from about 300 to about 330, from about 330 to about 400, from about 400 to about 460, from about 460 to about 530, from about 530 to about 600, from about 600 to about 660, from about 660 to about 800, from about 800 to about 930, from about 930 to about 1000, from about 1000 to about 1060, from about 1060 to about 1130, from about 1130 to about 1200, from about 1200 to about 1260, from about 1260 to about 1330, from about 1330 to about 1400 or more amino acids.

[0136] In an embodiment, the polypeptide encoded by a mRNA of the invention may have a function that is from at least about 60 to 65%, from at least about 65 to 70%, from at least about 70 to 75%, from at least about 75 to 80%, from at least about 80 to 85%, from at least about 85 to 90%, from at least about 90 to 95%, from at least about 95 to 100%, from at least about 105 to 110%, from at least about 110 to 115%, from at least about 115 to 120%, from at least about 120 to 125%, from at least about 125 to 130%, from at least about 130 to 135%, from at least about 135 to 140%, from at least about 145 to 150%, from at least about 155 to 160%, from at least about 160 to 165%, from at least about 165 to 170%, from at least about 170 to 175%, from at least about 175 to 180%, from at least about 180 to 185%, from at least about 185 to 190%, from at least about 190 to 195%, from at least about 195 to 200% or more compared to a corresponding wild-type polypeptide. Preferably, the polypeptide encoded by a mRNA of the invention has a function that is about the same as a wild-type counterpart. The corresponding wild-type polypeptide may be a sequence set forth in SEQ ID NO: 1 .

[0137] The SLC30A1 protein may be functionally characterized by its ability, when expressed in target cells, including those of the immune cells (e.g., macrophages), to accumulate zinc in vesicles, such as phagosomes. Methods for determining levels zinc in cellular compartments are known in the art and described herein including in the Examples.

[0138] “Substitutional variants” when referring to polypeptides are those that have at least one amino acid residue in a native or starting sequence removed and a different amino acid inserted in its place at the same position. The substitutions may be single, where only one amino acid in the molecule has been substituted, or they may be multiple, where two or more amino acids have been substituted in the same molecule.

[0139] As used herein the term “conservative amino acid substitution” refers to the substitution of an amino acid that is normally present in the sequence with a different amino acid of similar size, charge, or polarity. Examples of conservative substitutions include the substitution of a non-polar (hydrophobic) residue such as isoleucine, valine and leucine for another non-polar residue.

[0140] Likewise, examples of conservative substitutions include the substitution of one polar (hydrophilic) residue for another such as between arginine and lysine, between glutamine and asparagine, and between glycine and serine.

[0141] Additionally, the substitution of a basic residue such as lysine, arginine or histidine for another, or the substitution of one acidic residue such as aspartic acid or glutamic acid for another acidic residue are additional examples of conservative substitutions. Examples of non-conservative substitutions include the substitution of a non-polar (hydrophobic) amino acid residue such as isoleucine, valine, leucine, alanine, methionine for a polar (hydrophilic) residue such as cysteine, glutamine, glutamic acid or lysine and / or a polar residue for a non-polar residue.

[0142] As used herein the terms “termini” or “terminus” when referring to polypeptides refers to an extremity of a peptide or polypeptide. Such extremity is not limited only to the first or final site of the peptide or polypeptide but may include additional amino acids in the terminal regions. The polypeptide- based molecules of the present invention may be characterized as having both an N-terminus (terminated by an amino acid with a free amino group (NH2)) and a C-terminus (terminated by an amino acid with a free carboxyl group (CO OH)). Proteins of the invention are in some cases made up of multiple polypeptide chains brought together by disulfide bonds or by non-covalent forces (multimers, oligomers). These sorts of proteins will have multiple N- and C-termini. Alternatively, the termini of the polypeptides may be modified such that they begin or end, as the case may be, with a non-polypeptide based moiety such as an organic conjugate.

[0143] Once any of the features have been identified or defined as a desired component of a polypeptide to be encoded by the mRNA of the invention, any of several manipulations and / or modifications of these features may be performed by moving, swapping, inverting, deleting, randomizing or duplicating. Furthermore, it is understood that manipulation of features may result in the same outcome as a modification to the molecules of the invention. For example, a manipulation which involved deleting a domain would result in the alteration of the length of a molecule just as modification of a nucleic acid to encode less than a full-length molecule would.

[0144] Modifications and manipulations can be accomplished by methods known in the art such as, but not limited to, site directed mutagenesis. The resulting modified molecules may then be tested for activity using in vitro or in vivo assays such as those described herein or any other suitable screening assay known in the art.

[0145] As recognized by those skilled in the art, protein fragments, functional protein domains, and homologous proteins are also considered to be within the scope of polypeptides of interest of this invention. For example, provided herein is any protein fragment (meaning a polypeptide sequence at least one amino acid residue shorter than a reference polypeptide sequence but otherwise identical) of areference protein 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, or greater than 100 amino acids in length. In another example, any protein that includes a stretch of about 20, about 30, about 40, about 50, or about 100 amino acids which are about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, or about 100% identical to any of the sequences described herein can be utilized in accordance with the invention. In certain embodiments, a polypeptide to be utilized in accordance with the invention includes 2, 3, 4, 5, 6, 7, 8, 9, 10, or more mutations as shown in any of the sequences provided or referenced herein.

[0146] The term “identity” as known in the art, refers to a relationship between the sequences of two or more peptides, as determined by comparing the sequences. In the art, identity also means the degree of sequence relatedness between peptides, as determined by the number of matches between strings of two or more amino acid residues. Identity measures the percent of identical matches between the smaller of two or more sequences with gap alignments (if any) addressed by a particular mathematical model or computer program (i.e. , “algorithms”). Identity of related peptides can be readily calculated by known methods. Such methods include, but are not limited to, those described in Lesk (1988); Smith (1993); Griffin and Griffin, (1994); von Heinje (1987); Gribskov and Devereux (1991); and Carillo (1988).

[0147] In some embodiments, the polypeptide variant may have the same or a similar activity as the reference polypeptide. Alternatively, the variant may have an altered activity (e.g., increased or decreased) relative to a reference polypeptide. In preferred embodiments, the reference polypeptide may be that set forth in SEQ ID NO: 1 .

[0148] Generally, variants of a particular polynucleotide or polypeptide of the invention will have at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% but less than 100% sequence identity to that particular reference polynucleotide or polypeptide as determined by sequence alignment programs and parameters described herein and known to those skilled in the art. Such tools for alignment include those of the BLAST suite (Altschul (1997)) Other tools are described herein, specifically in the definition of “identity.”

[0149] Default parameters in the BLAST algorithm include, for example, an expect threshold of 10, Word size of 28, Match / Mismatch Scores 1 , -2, Gap costs Linear. Any filter can be applied as well as a selection for species specific repeats, e.g., Homo sapiens. The percentage identity of a polypeptide is determined by GAP (Needleman and Wunsch (1970)) analysis (GCG program) with a gap creation penalty=5, and a gap extension penalty=0.3. The query sequence is at least 100 amino acids in length, and the GAP analysis aligns the two sequences over a region of at least 100 amino acids. More preferably, the query sequence is at least 300 amino acids in length and the GAP analysis aligns the two sequences over a region of at least 300 amino acids. Alternatively, the query sequence is at least 500 amino acids in length and the GAP analysis aligns the two sequences over a region of at least 500 amino acids. Even more preferably, the GAP analysis aligns two sequences over their entire length of any amino acid sequence disclosed herein.

[0150] With regard to a defined polypeptide, it will be appreciated that percentage identity figures higher than those provided above will encompass preferred embodiments. Thus, where applicable, in lightof the minimum percentage identity figures, it is preferred that the polypeptide comprises an amino acid sequence which is preferably at least 50%, at least 60%, at least 70%, more preferably at least 75%, more preferably at least 76%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, more preferably at least 99.1%, more preferably at least 99.2%, more preferably at least 99.3%, more preferably at least 99.4%, more preferably at least 99.5%, more preferably at least 99.6%, more preferably at least 99.7%, more preferably at least 99.8%, and even more preferably at least 99.9% identical to the relevant nominated sequence identifier.2.10 Vectors

[0151] In one aspect, disclosed herein are vectors comprising the mRNA compositions disclosed herein. The RNA sequences encoding a protein of interest (e.g., mRNA encoding a SLC30A1 polypeptide) can be cloned into a number of types of vectors. For example, the nucleic acids can be cloned into a vector including, but not limited to, a plasmid, a phagemid, a phage derivative, an animal virus, and a cosmid. Vectors of particular interest can include expression vectors, replication vectors, probe generation vectors, sequencing vectors, and vectors optimized for in vitro transcription.

[0152] In certain embodiments, the vector can be used to express mRNA in a host cell. In various embodiments, the vector can be used as a template for IVT. The construction of optimally translated IVT mRNA suitable for therapeutic use is disclosed in detail in Sahin, et al. (2014) Nat. Rev. Drug Discov. 13, 759-780; Weissman (2015) Expert Rev. Vaccines 14, 265-281.

[0153] In some embodiments, the vectors disclosed herein can comprise at least the following, from 5' to 3': an RNA polymerase promoter; a polynucleotide sequence encoding a 5' UTR; a polynucleotide sequence encoding an ORF; a polynucleotide sequence encoding a 3' UTR; and a polynucleotide sequence encoding at least one RNA aptamer. In some embodiments, the vectors disclosed herein may comprise a polynucleotide sequence encoding a poly(A) sequence and / or a polyadenylation signal.

[0154] A variety of RNA polymerase promoters are known. In some embodiments, the promoter can be a T7 RNA polymerase promoter. Other useful promoters can include, but are not limited to, T3 and SP6 RNA polymerase promoters. Consensus nucleotide sequences for T7, T3, and SP6 promoters are known.

[0155] Also disclosed herein are host cells (e.g., mammalian cells, e.g., human cells) comprising the vectors or mRNA compositions disclosed herein.

[0156] Polynucleotides can be introduced into target cells using any of a number of different methods, for instance, commercially available methods which include, but are not limited to, electroporation (Amaxa Nucleofector-ll (Amaxa Biosystems, Cologne, Germany)), (ECM 830 (BTX) (Harvard Instruments, Boston, Mass.) orthe Gene Pulser II (BioRad, Denver, Colorado), Multiporator (Eppendorf, Hamburg, Germany), cationic liposome-mediated transfection using lipofection, polymerencapsulation, peptide mediated transfection, biolistic particle delivery systems such as “gene guns” (see, for example, Nishikawa, et al. (2001) Hum Gene Ther. 12(8):861 -70, or the TransIT-RNA transfection Kit (Mirus, Madison, Wl).

[0157] Chemical means for introducing a polynucleotide into a host cell include colloidal dispersion systems, such as macromolecule complexes, nanocapsules, microspheres, beads, and lipid- based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes. An exemplary colloidal system for use as a delivery vehicle in vitro and in vivo is a liposome (e.g., an artificial membrane vesicle). In preferred embodiments, the polynucleotides can be introduced into target cells using a lipid nanoparticle system, as described in more detail below.3. Pharmaceutical Compositions and Delivery Methods

[0158] In some embodiments, the mRNA of the invention may be formulated in a liposome, lipoplex or a lipid nanoparticle so as to increase stability of the mRNA.3.1 Liposome and lipoplex delivery

[0159] Liposomes are artificially-prepared vesicles which are primarily be composed of a lipid bilayer and may be used as a delivery vehicle for the administration of nutrients and pharmaceutical formulations. Liposomes can be of different sizes such as, but not limited to, a multilamellar vesicle (MLV) which may be hundreds of nanometers in diameter and may contain a series of concentric bilayers separated by narrow aqueous compartments, a small unicellular vesicle (SUV) which may be smaller than 50 nm in diameter, and a large unilamellar vesicle (LUV) which may be between 50 and 500 nm in diameter.

[0160] Liposomes and / or lipoplexes may include opsonins or ligands in order to improve the attachment of liposomes, lipoplex or lipid nanoparticles to unhealthy tissue or to activate events such as, but not limited to, endocytosis.3.2 Lipid nanoparticle delivery

[0161] In some embodiments, the mRNA of the invention is formulated a lipid nanoparticle. LNPs are composed primarily of cationic lipids along with other lipid ingredients. These typically include neutral phospholipid molecules belonging to the phosphatidylcholine (PC) class and sterols, such as cholesterol. Another common lipid ingredient is what is known as a PEGylated phospholipid-a polyethylene glycol (PEG) polymer covalently attached to the head-group of a phospholipid. In some embodiments, lipid nanoparticles (LNPs) are formulated in a composition for delivery of the mRNA to a desired target such as a cell, tissue, or organ. In some preferred embodiments, the LNPs are formulated for delivery of the mRNA to the lungs of a subject. In even more preferred embodiments, the LNPs are formulated for delivery of the mRNA to the macrophages of a subject. The LNPs of the invention may be any lipid composition For example, the LNP may be selected from, but not limited to, liposomes or vesicles in which an aqueous volume is encapsulated by amphipathic lipid bilayers, micelle-like lipid nanoparticles having a non-aqueous core, and solid lipid nanoparticles.

[0162] Whilst liposomes include one or more rings of lipid bilayer surrounding an aqueous pocket, not all lipid nanoparticles have a contiguous bilayer like liposomes. Instead, it is understood that some LNPs assume a micelle-like structure, encapsulating drug molecules in a non-aqueous core.

[0163] Where the use of lipid nanoparticles is contemplated, a lipid nanoparticle generally comprises a cationic lipid, a non-cationic lipid, a PEG lipid and a structural lipid. Suitable cationic lipids may include those described in the cationic lipid may be selected from, but not limited to, a cationic lipid described in International PCT Publication Nos. WO2012 / 040184, WO2011 / 153120, WO2011 / 149733, WO2011 / 090965, WO2011 / 043913, WO2011 / 022460, WO2012 / 061259, WO2012 / 054365, WO2012 / 044638, WO2010 / 080724, WO2010 / 21865, W02008 / 103276, WO2013 / 086373 and WO2013 / 086354, US Patent Nos. 7,893,302, 7,404,969, 8,283,333, and 8,466,122 and U.S. Patent Publication No. US2010 / 0036115, US2012 / 0202871 , US2013 / 0064894, US2013 / 0129785, US2013 / 0150625, 2US013 / 0178541 and US2013 / 0225836. Other suitable cationic lipids, non-cationic lipids, PEG lipids and structural lipids, and suitable ratios thereof include those disclosed in WO 2015 / 164674 and WO 2013 / 090648. For example, mRNA according to the invention may be formulated in a lipid nanoparticle at a 20:1 weight ratio of total lipid to modified mRNA.

[0164] Exemplary lipid nanoparticle compositions and methods of making same that are suitable for use with the present invention are described, for example, in Semple et al. (2010); Jayarama et al. (2012); and Maier et al. (2013). Alternatively, the LNP formulation may be formulated by the methods described in International PCT Publication Nos. WO 2011 / 127255 or WO 2008 / 103276.

[0165] Further, the particle size of the lipid nanoparticle may be increased and / or decreased. The change in particle size may be able to help counter biological reaction such as, but not limited to, inflammation or may increase the biological effect of the modified mRNA delivered to a given subject.

[0166] Lipid nanoparticles suitable for use in the present disclosure will be apparent to the skilled person and / or are described herein. The lipids can have an anionic, cationic or zwitterionic hydrophilic head group. In some embodiments, the lipid nanoparticle comprises a PEG-lipid, a sterol structural lipid, and / or a neutral lipid. In one example, the lipid nanoparticle further comprises a cationic lipid. In one example, the lipid nanoparticle does not comprise a cationic lipid. In one embodiment, the LNP comprises a PEG-lipid. For example, the PEG-lipid is selected from the group consisting of PEG-DSPE, PEG-c-DMG, PEG-DMG, PEG-DLPE, PEG-DMPE, PEGDPPC, a lipid and combinations thereof.

[0167] In some embodiments, the LNP comprises a structural lipid. For example, the structural lipid is selected from the group consisting of cholesterol fecosterol, sitosterol, campesterol, stigmasterol, brassicasterol, ergosterol, tomatidine, tomatine, ursolic acid and a-tocopherol, and combinations thereof.

[0168] In some embodiments, the LNP comprises a neutral lipid. Exemplary phospholipids (anionic or zwitterionic) for use in the present disclosure include, for example, phosphatidylethanolamines, phosphatidylcholines, phosphatidylserines, and phosphatidylglycerols. For example, the neutral lipid is selected from the group consisting of 1 ,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1 ,2-dioleoyl-sn- glycero-3-phosphoethanolamine (DOPE), 1 ,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1 ,2- dimyristoyl-sn-glycero-phosphocholine (DMPC), 1 ,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1 ,2-dipalmitoy l-sn-glycero-3-phosphocholine (DPPC), 1 ,2-diundecanoyl-sn-glycero-phosphocholine (DUPC), 1-palmitoyl-2-oleoyl- sn-glycero-3 -phosphocholine (POPC), 1 ,2-di-0-octadecenyl-sn-glycero-3- phosphocholine (18:0 Diether PC), 1-oleoyl-2-cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecylsn-glycero-3-phosphocholine (C16 Lyso PC), 1 ,2-dilinolenoyl-sn-glycero-3- phosphocholine, 1 ,2-diarachidonoyl-sn-glycero-3-phosphocholine, 1 ,2-didocosahexaenoyl-sn-glycero-3- phosphocholine, 1 ,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (ME 16.0 PE), 1 ,2-distearoyl-sn- glycero-3-phosphoethanolamine (DSPE), 1 ,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine, 1 ,2- dilinolenoyl-sn-glycero-3-phosphoethanolamine, 1 ,2-diarachidonoylsn-glycero-3-phosphoethanolamine, 1 ,2-didocosahexaenoyl-sn-glycero-3-phosphoethanolamine, 1 ,2-dioleoyl-sn-glycero-3-phospho-rac-(1- glycerol) sodium salt (DOPG), and sphingomyelin and combinations thereof.

[0169] In some embodiments, the LNP comprises a cationic lipid. Exemplary cationic lipids include, but are not limited to, dioleoyl trimethylammonium propane (DOTAP), 1 ,2-distearyloxy- N,Ndimethyl-3-aminopropane (DSDMA), 1 ,2-dioleyloxy-N,N-dimethyl-3-aminopropane (DODMA),1 ,2- dilinoleyloxy-N,N-dimethyl-3- aminopropane (DLinDMA), 1 ,2-dilinolenyloxy-N,N-dimethyl-3-aminopropane (DLenDMA), 2,5-bis((9Z,12Z)-octadeca-9,12,dien-1-yloxyl)benzyl-4-(dimethylamino)butnoate (LKY750). Exemplary zwitterionic lipids include, but are not limited to, acyl zwitterionic lipids and ether zwitterionic lipids, such as dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylcholine (DOPC) and dodecylphosphocholine. The lipids can be saturated or unsaturated.

[0170] By way of an illustrative example, in some embodiments the lipid nanoparticle comprises: (i) hyaluronic acid (HA) to target CD44 proteins overexpressed on the cell surface of macrophages; (ii) 1 ,2- distearoyl-sn-glycero-3-phosphoethanolamine-poly(ethylene glycol) (DSPE-PEG) to stabilise the nanoparticle system; and (Hi) a targeting moiety of mannose conjugated to DSPE-PEG on the mRNA nanoparticle surface to further dual target glycose transporters overexpressed on the surface of macrophages. Alveolar macrophages are the major cell type known to internalize HA for degradation in normal lung, and CD44 participates in this process (Hajj et al., 2019; and Wang et al., 2012).3.3 Polymeric microparticles

[0171] In one example, the pharmaceutical composition further comprises a polymeric microparticle.

[0172] The skilled person will be aware that various polymers can form microparticles to encapsulate or adsorb the mRNA agents of the present invention. It will be apparent that use of a substantially non-toxic polymer means that particles are safe, and the use of a biodegradable polymer means that the particles can be metabolised after delivery to avoid long-term persistence. Useful polymers are also sterilisable, to assist in the preparation of pharmaceutical grade formulations.

[0173] Exemplary non-toxic and biodegradable polymers include, but are not limited to, poly(a- hydroxy acids), polyhydroxy butyric acids, polylactones (including polycaprolactones), polydioxanones, polyvalerolactone, polyorthoesters, polyanhydrides, polycyanoacrylates, tyrosine-derived polycarbonates, polyvinyl-pyrrolidinones or polyester-amides, and combinations thereof.3.4 Oil-in-water emulsion

[0174] In embodiment, the pharmaceutical composition of the present disclosure further comprises an oil-in-water cationic emulsion.

[0175] Suitable oils for use in an oil-in-water emulsion will be apparent to the skilled person and / or are described herein. For example, the emulsion comprises one or more oils derived, for example, from an animal (e.g., fish) or a vegetable source (e.g., nuts, seeds, grains). The skilled person will recognise that biocompatible and biodegradable oils are preferentially used. Exemplary animal oils (i.e. , fish oils) include cod liver oil, shark liver oils, and whale oil. Exemplary vegetable oils include peanut oil, coconut oil, olive oil, soybean oil, jojoba oil, safflower oil, cottonseed oil, sunflower seed oil, sesame seed oil, corn oil.

[0176] In addition to the oil, the oil-in-water emulsion also comprises a cationic lipid to facilitate formation and stabilisation of the emulsion. Suitable cationic lipids will be apparent to the skilled person and / or are described herein. Exemplary cationic lipids include, but are not limited to: 1 ,2-dioleoyloxy-3- trimethylammonio-propane (DOTAP), 3beta [N-(N',N'-dimethylaminoethane)-carbamoyl] cholesterol (DC cholesterol), dimethyldioctadecyl-ammonium (DDA), 1 ,2-dimyristoy-3-trimethylammonium-propane (DMTAP), 1 ,2-dipalmitoyl-3-trimethylammonium-propane (DPTAP), and 1 ,2-distearoyl-3- trimethylammonium-propane (DSTAP).

[0177] In some embodiments, the oil-in-water emulsion also comprises a non-ionic surfactant and / or a zwitterionic surfactant. The skilled person will be aware of surfactants suitable for use in the present disclosure. Exemplary surfactants include, but are not limited to, the polyoxyethylene sorbitan esters surfactants (e.g., polysorbate 20 and polysorbate 80) and copolymers of ethylene oxide (EO), propylene oxide (PO), and / or butylene oxide (BO).3.5 Pharmaceutical compositions

[0178] RNA purified according to this disclosure can be useful as a component in pharmaceutical compositions, for example. These compositions will typically include RNA and a pharmaceutically acceptable carrier. A pharmaceutical composition of the present disclosure can also include one or more additional components such as a cystic fibrosis therapeutic.

[0179] A pharmaceutical composition of the present disclosure can also include a delivery system for the RNA, such as a liposome, an oil-in-water emulsion, or a microparticle. In some embodiments, the pharmaceutical composition comprises a lipid nanoparticle (LNP). In certain embodiments, the composition comprises an SLC30A1 polypeptide-encoding nucleic acid molecule encapsulated within a LNP.

[0180] To facilitate expression of mRNA in vivo, the nucleic acid lipid formulation delivery vehicles described herein can be combined with one or more additional nucleic acids, carriers, targeting ligands or stabilizing reagents, or in pharmacological compositions where it is mixed with suitable excipients. Techniques for formulation and administration of drugs may be found in “Remington's Pharmaceutical Sciences,” Mack Publishing Co., Easton, Pa., latest edition. Preferably, the nucleic acid lipid formulation is a SLC30A1 mRNA-lipid nanoparticle formulation as described herein. Preferably, the mRNA encodes a human SLC30A1 protein of SEQ ID NO: 1 , preferably formulated in a lipid delivery system or lipid carrierand preferably comprising pharmaceutically acceptable excipients. In some embodiments, the pharmaceutical composition further comprises pharmaceutically acceptable excipients. Pharmaceutical compositions disclosed herein preferably facilitate expression of SLC30A1 mRNA in vivo.

[0181] The lipid formulations and pharmaceutical compositions of the present disclosure may be administered and dosed in accordance with current medical practice, taking into account the clinical condition of the subject, the site and method of administration, the scheduling of administration, the subject's age, sex, body weight and other factors relevant to clinicians of ordinary skill in the art. The “effective amount” for the purposes herein may be determined by such relevant considerations as are known to those of ordinary skill in experimental clinical research, pharmacological, clinical and medical arts. In some embodiments, the amount administered is effective to achieve at least some stabilization, improvement or elimination of symptoms and other indicators as are selected as appropriate measures of disease progress, regression or improvement by those of skill in the art. For example, a suitable amount and dosing regimen is one that causes at least transient protein (e.g., enzyme) production.

[0182] The pharmaceutical compositions described herein can achieve expression of a SLC30A1 protein in an innate immune cell (e.g., a macrophage) of a subject. Suitable routes of administration include, for example, intratracheal, inhaled, or intranasal. In some embodiments, the administration results in delivery of the mRNA to a lung innate immune cell. In some embodiments, the administration shows a selectivity towards innate immune cells over other types of cells present in the lung and cells of the airways.

[0183] The pharmaceutical compositions disclosed herein can be formulated using one or more excipients to: (1) increase stability; (2) increase cell transfection; (3) permit a sustained or delayed release (e.g., from a depot formulation of the polynucleotide, primary construct, or mRNA); (4) alterthe biodistribution (e.g., target the polynucleotide, primary construct, or mRNA to specific tissues or cell types); (5) increase the translation of encoded protein in vivo\ and / or (6) alter the release profile of encoded protein in vivo.

[0184] Preferably, mRNAs and lipid formulations thereof may be administered in a local rather than systemic manner. Local delivery can be affected in various ways, depending on the tissue to be targeted. For example, aerosols containing compositions of the present disclosure can be inhaled (for nasal, tracheal, or bronchial delivery).

[0185] Pharmaceutical compositions may be administered to any desired tissue. In some embodiments, the SLC30A1 mRNA delivered by a lipid formulation or composition of the present disclosure is expressed in the tissue in which the lipid formulation and / or composition was administered. In some embodiments, the mRNA delivered is expressed in a tissue different from the tissue in which the lipid formulation and / or composition was administered. Example tissues in which delivered mRNA may be delivered and / or expressed include, but are not limited to the lung, trachea, and / or nasal passages.

[0186] The pharmaceutical compositions described herein may be prepared by any method known or hereafter developed in the art of pharmacology. In general, such preparatory methods include the step of associating the active ingredient (i.e. , nucleic acid) with an excipient and / or one or more otheraccessory ingredients. A pharmaceutical composition in accordance with the present disclosure may be prepared, packaged, and / or sold in bulk, as a single unit dose, and / or as a plurality of single unit doses.

[0187] Pharmaceutical compositions may additionally comprise a pharmaceutically acceptable excipient, which, as used herein, includes, but is not limited to, any and all solvents, dispersion media, diluents, or other liquid vehicles, dispersion or suspension aids, surface active agents, isotonic agents, thickening or emulsifying agents, preservatives, and the like, as suited to the particular dosage form desired.

[0188] In addition to traditional excipients such as any and all solvents, dispersion media, diluents, or other liquid vehicles, dispersion or suspension aids, surface active agents, isotonic agents, thickening or emulsifying agents, preservatives, excipients of the present disclosure can include, without limitation, liposomes, lipid nanoparticles, polymers, lipoplexes, core-shell nanoparticles, peptides, proteins, cells transfected with primary DNA construct, or mRNA (e.g., for transplantation into a subject), hyaluronidase, nanoparticle mimics and combinations thereof.

[0189] Accordingly, the formulations described herein can include one or more excipients, each in an amount that together increases the stability of the nucleic acid in the lipid formulation, increases cell transfection by the nucleic acid (e.g., mRNA), increases the expression of the encoded protein, and / or alters the release profile of the encoded protein. Further, the mRNA of the present disclosure may be formulated using self-assembled nucleic acid nanoparticles.

[0190] Various excipients for formulating pharmaceutical compositions and techniques for preparing the composition are known in the art (see Remington: The Science and Practice of Pharmacy, 21st Edition, A. R. Gennaro, Lippincott, Williams & Wilkins, Baltimore, Md., 2006; incorporated herein by reference in its entirety). The use of a conventional excipient medium may be contemplated within the scope of the embodiments of the present disclosure, except insofar as any conventional excipient medium may be incompatible with a substance or its derivatives, such as by producing any undesirable biological effect or otherwise interacting in a deleterious manner with any other component(s) of the pharmaceutical composition.

[0191] A dosage form of the composition of this disclosure can be solid, which can be reconstituted in a liquid prior to administration. The solid can be administered as a powder. In some embodiments, the pharmaceutical composition comprises a nucleic acid lipid formulation that has been lyophilized.

[0192] In a preferred embodiment, the dosage form of the pharmaceutical compositions described herein can be a liquid suspension of SLC30A1 mRNA lipid nanoparticles described herein. In some embodiments, the liquid suspension is in a buffered solution. In some embodiments, the buffered solution comprises a buffer selected from the group consisting of HEPES, MOPS, TES, and TRIS. In some embodiments, the buffer has a pH of about 7.4. In some preferred embodiments, the buffer is HEPES. In some further embodiments, the buffered solution further comprises a cryoprotectant. In some embodiments, the cryoprotectant is selected from a sugar and glycerol or a combination of a sugar and glycerol. In some embodiments, the sugar is a dimeric sugar. In some embodiments, the sugar is sucrose.In some preferred embodiments, the buffer comprises HEPES, sucrose, and glycerol at a pH of 7.4. In some embodiments, the suspension is frozen during storage and thawed priorto administration. In some embodiments, the suspension is frozen at a temperature below about -70 °C. In some embodiments, the suspension is diluted with sterile water prior to inhalable administration. In some embodiments, inhalable administration comprises diluting the suspension with about 1 volume to about 4 volumes of sterile water. In some embodiments, a lyophilized SLC30A1-mRNA lipid nanoparticle formulation can be resuspended in a buffer as described herein.

[0193] The compositions and methods of the disclosure may be administered to subjects by a variety of mucosal administration modes, including intranasal and / or intrapulmonary. In some aspects of this disclosure, the mucosal tissue layer includes an epithelial cell layer. The epithelial cell can be pulmonary, tracheal, bronchial, alveolar, nasal, and / or buccal. Compositions of this disclosure can be administered using conventional actuators such as mechanical spray devices, as well as pressurized, electrically activated, or othertypes of actuators.

[0194] The mRNA compositions of this disclosure may be administered in an aqueous solution as a nasal or pulmonary spray and may be dispensed in spray form by a variety of methods known to those skilled in the art. Pulmonary delivery of a composition of this disclosure is achieved by administering the composition in the form of drops, particles, or spray, which can be, for example, aerosolized, atomized, or nebulized. Particles of the composition, spray, or aerosol can be in either a liquid or solid form, for example, a lyophilized lipid formulation. Preferred systems for dispensing liquids as a nasal spray are disclosed in United States Patent No. 4,511 ,069. Such formulations may be conveniently prepared by dissolving compositions according to the present disclosure in water to produce an aqueous solution, and rendering said solution sterile. The formulations may be presented in multi-dose containers, for example in the sealed dispensing system disclosed in United States Patent No. 4,511 ,069. Other suitable nasal spray delivery systems have been described in TRANSDERMAL SYSTEMIC MEDICATION, Y. W. Chien ed., Elsevier Publishers, New York, 1985; and in United States Patent No. 4,778,810. Additional aerosol delivery forms may include, e.g., compressed air-, jet-, ultrasonic-, and piezoelectric nebulizers, which deliverthe SLC30A1 mRNA lipid formulation or suspended in a pharmaceutical solvent, e.g., water, ethanol, or mixtures thereof.

[0195] Nasal and pulmonary spray solutions of the present disclosure typically comprise the drug or drug to be delivered, optionally formulated with a surface-active agent, such as a non-ionic surfactant (e.g., polysorbate-80), and one or more buffers, provided that the inclusion of the surfactant does not disrupt the structure of the lipid formulation. In some embodiments of the present disclosure, the nasal spray solution further comprises a propellant. The pH of the nasal spray solution may be from pH 6.8 to 7.2. The pharmaceutical solvents employed can also be a slightly acidic aqueous buffer of pH 4-6. Other components may be added to enhance or maintain chemical stability, including preservatives, surfactants, dispersants, or gases.

[0196] In some embodiments, this disclosure provides a pharmaceutical product which includes a solution containing a composition of this disclosure and an actuator for a pulmonary, mucosal, or intranasal spray or aerosol.

[0197] A dosage form of the composition of this disclosure can be liquid, in the form of droplets or an emulsion, or in the form of an aerosol.

[0198] A dosage form of the composition of this disclosure can be solid, which can be reconstituted in a liquid prior to administration. The solid can be administered as a powder. The solid can be in the form of a capsule, tablet, or gel.

[0199] To formulate compositions for pulmonary delivery within the present disclosure, the SLC301A mRNA lipid formulation can be combined with various pharmaceutically acceptable additives, as well as a base or carrier for dispersion of the SLC30A1 mRNA lipid formulation(s). Examples of additives include pH control agents such as arginine, sodium hydroxide, glycine, hydrochloric acid, citric acid, and mixtures thereof. Other additives include local anesthetics (e.g., benzyl alcohol), isotonizing agents (e.g., sodium chloride, mannitol, sorbitol), adsorption inhibitors (e.g., Tween 80), solubility enhancing agents (e.g., cyclodextrins and derivatives thereof), stabilizers (e.g., serum albumin), and reducing agents (e.g., glutathione). When the composition for mucosal delivery is a liquid, the tonicity of the formulation, as measured with reference to the tonicity of 0.9% (w / v) physiological saline solution taken as unity, is typically adjusted to a value at which no substantial, irreversible tissue damage will be induced in the mucosa at the site of administration. Generally, the tonicity of the solution is adjusted to a value of 1 / 3 to 3, more typically 1 / 2 to 2, and most often 3 / 4 to 1 .7.

[0200] The SLC30A1 mRNA lipid formulation may be dispersed in a base or vehicle, which may comprise a hydrophilic compound having a capacity to disperse the CFTR mRNA lipid formulation and any desired additives. The base may be selected from a wide range of suitable carriers, including but not limited to, copolymers of polycarboxylic acids or salts thereof, carboxylic anhydrides (e.g., maleic anhydride) with other monomers (e.g., methyl(meth)acrylate, acrylic acid, etc.), hydrophilic vinyl polymers such as polyvinyl acetate, polyvinyl alcohol, polyvinylpyrrolidone, cellulose derivatives such as hydroxymethylcellulose, hydroxypropylcellulose, etc., and natural polymers such as chitosan, collagen, sodium alginate, gelatin, hyaluronic acid, and nontoxic metal salts thereof. Often, a biodegradable polymer is selected as a base or carrier, for example, polylactic acid, poly(lactic acid-glycolic acid) copolymer, polyhydroxybutyric acid, poly(hydroxybutyric acid-glycolic acid) copolymer, and mixtures thereof. Alternatively or in addition, synthetic fatty acid esters such as polyglycerin fatty acid esters, sucrose fatty acid esters, etc., can be employed as carriers. Hydrophilic polymers and other carriers can be used alone or in combination and enhanced structural integrity can be imparted to the carrier by partial crystallization, ionic bonding, crosslinking, and the like. The carrier can be provided in a variety of forms, including fluid or viscous solutions, gels, pastes, powders, microspheres, and films for direct application to the nasal mucosa. The use of a selected carrier in this context may result in promotion of absorption of the SLC30A1 mRNA lipid formulation.

[0201] The compositions of the invention may alternatively contain as pharmaceutically acceptable carriers substances as required to approximate physiological conditions, such as pH adjusting and buffering agents, tonicity adjusting agents, and wetting agents, for example, sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate, triethanolamine oleate, and mixtures thereof. For solid compositions, conventional nontoxic pharmaceutically acceptablecarriers can be used which include, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, talcum, cellulose, glucose, sucrose, magnesium carbonate, and the like.

[0202] In certain embodiments, the SLC30A1 mRNA lipid formulation may be administered in a time release formulation, for example in a composition which includes a slow-release polymer. The SLC30A1 mRNA lipid formulation can be prepared with carriers that will protect against rapid release, for example a controlled release vehicle such as a polymer, microencapsulated delivery system, or a bioadhesive gel. Prolonged delivery of the SLC30A1 mRNA lipid formulation, in various compositions of the disclosure can be brought about by including in the composition agents that delay absorption, for example, aluminium monostearate hydrogels and gelatin.

[0203] It has been demonstrated that nucleic acids can be delivered to the lungs by intratracheal administration of a liquid suspension of the nucleic acid composition and inhalation of an aerosol mist produced by a liquid nebulizer or the use of a dry powder apparatus such as that described in United States Patent No. 5,780,014, incorporated herein by reference.

[0204] In certain embodiments, the compositions of the disclosure may be formulated such that they may be aerosolized or otherwise delivered as a particulate liquid or solid prior to or upon administration to the subject. Such compositions may be administered with the assistance of one or more suitable devices for administering such solid or liquid particulate compositions (such as, e.g., an aerosolized aqueous solution or suspension) to generate particles that are easily respirable or inhalable by the subject. In some embodiments, such devices (e.g., a metered dose inhaler, jet-nebulizer, ultrasonic nebulizer, dry-powder-inhalers, propellant-based inhaler, or an insufflator) facilitate the administration of a predetermined mass, volume or dose of the compositions (e.g., about 0.5 mg / kg of mRNA per dose) to the subject. For example, in certain embodiments, the compositions of the disclosure are administered to a subject using a metered dose inhaler containing a suspension or solution comprising the composition and a suitable propellant. In certain embodiments, the compositions of the disclosure may be formulated as a particulate powder (e.g., respirable dry particles) intended for inhalation. In certain embodiments, compositions of the disclosure formulated as respirable particles are appropriately sized such that they may be respirable by the subject or delivered using a suitable device (e.g., a mean D50 or D90 particle size less than about 500 pm, 400 pm, 300 pm, 250 pm, 200 pm, 150 pm, 100 pm, 75 pm, 50 pm, 25 pm, 20 pm, 15 pm, 12.5 pm, 10 pm, 5 pm, 2.5 pm, or smaller). In yet other embodiments, the compositions of the disclosure are formulated to include one or more pulmonary surfactants (e.g., lamellar bodies). In some embodiments, the compositions of the disclosure are administered to a subject such that a concentration of at least 0.05 mg / kg, at least 0.1 mg / kg, at least 0.5 mg / kg, at least 1 mg / kg, at least 2 mg / kg, at least 3 mg / kg, at least 4 mg / kg, at least 5 mg / kg, at least 6 mg / kg, at least 7 mg / kg, at least 8 mg / kg, at least 9 mg / kg, at least 10 mg / kg, at least 15 mg / kg, at least 20 mg / kg, at least 25 mg / kg, at least 30 mg / kg, at least 35 mg / kg, at least 40 mg / kg, at least 45 mg / kg, at least 50 mg / kg, at least 55 mg / kg, at least 60 mg / kg, at least 65 mg / kg, at least 70 mg / kg, at least 75 mg / kg, at least 80 mg / kg, at least 85 mg / kg, at least 90 mg / kg, at least 95 mg / kg, or at least 100 mg / kg body weight is administered in a single dose. In some embodiments, the compositions of the disclosure are administered to a subject such that a totalamount of at least 0.1 mg, at least 0.5 mg, at least 1 mg, at least 2 mg, at least 3 mg, at least 4 mg, at least 5 mg, at least 6 mg, at least 7 mg, at least 8 mg, at least 9 mg, at least 10 mg, at least 15 mg, at least 20 mg, at least 25 mg, at least 30 mg, at least 35 mg, at least 40 mg, at least 45 mg, at least 50 mg, at least 55 mg, at least 60 mg, at least 65 mg, at least 70 mg, at least 75 mg, at least 80 mg, at least 85 mg, at least 90 mg, at least 95 mg or at least 100 mg mRNA is administered in one or more doses.

[0205] In some embodiments, a pharmaceutical composition is administered to a subject once per month. In some embodiments, a pharmaceutical composition of the present disclosure is administered to a subject twice per month. In some embodiments, a pharmaceutical composition of the present disclosure is administered to a subject three times per month. In some embodiments, a pharmaceutical composition of the present disclosure is administered to a subject four times per month.

[0206] According to the present disclosure, a therapeutically effective dose of the provided composition, when administered regularly, results in an increased SLC30A1 protein expression or activity level in a subject as compared to a baseline SLC30A1 protein expression or activity level before treatment. Typically, the SLC30A1 protein expression or activity level is measured in a biological sample obtained from the subject such as blood, plasma or serum, urine, or solid tissue extracts. The baseline level can be measured immediately before treatment. In some embodiments, administering a pharmaceutical composition described herein results in an increased SLC30A1 protein expression or activity level in a biological sample (e.g., plasma / serum or lung epithelial swab) by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% as compared to a baseline level before treatment. In some embodiments, administering the provided composition results in an increased SLC30A1 protein expression or activity level in a biological sample (e.g., plasma / serum or lung epithelial swab) by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% as compared to a baseline level before treatment for at least about 24 hours, at least about 48 hours, at least about 72 hours, at least about 4 days, at least about 5 days, at least about 6 days, at least about 7 days, at least about 8 days, at least about 9 days, at least about 10 days, at least about 11 days, at least about 12 days, at least about 13 days, at least about 14 days, or at least about 15 days.4. Combinations

[0207] The SLC30A1 mRNA, formulations thereof, or encoded SLC30A1 proteins described herein may be used in combination with one or more other therapeutic, prophylactic, diagnostic, or imaging agents. By “in combination with,” it is not intended to imply that the agents must be administered at the same time and / or formulated for delivery together, although these methods of delivery are within the scope of the present invention. Compositions can be administered concurrently with, prior to, or subsequent to, one or more other desired therapeutics or medical procedures. In general, each agent will be administered at a dose and / or on a time schedule determined forthat agent. Preferably, the methods of treatment of the present disclosure encompass the delivery of pharmaceutical, prophylactic, diagnostic, or imaging compositions in combination with agents that may improve their bioavailability, reduce and / or modify their metabolism, inhibit their excretion, and / or modify their distribution within the body. As a non- limiting example, mRNA encoding a SLC30A1 protein of SEQ ID NO: 1 may be used in combination with a pharmaceutical agent for the treatment of CFTR deficiency. The pharmaceutical agent includes, but is notlimited to one or more of: TRIKAFTA (elexacaftor, ivacaftor, tezacaftor), KALYDECO (ivacaftor), lumacaftor, texacaftor, elexacaftor, vanzacaftor, VX-522, SYMDEKO (tezacaftor and ivacaftor), and ORKAMBI (lumacaftor and ivacaftor), compositions and agents for airway clearance, antibiotics, antiinflammatory agents, bronchodilators, mucus thinners, etc. Multiple vitamins, calcium supplements or combined with a low protein / high caloric diet regimen. In general, it is expected that agents utilized in combination with the presently disclosed SLC30A1 mRNA and formulations thereof be utilized at levels that do not exceed the levels at which they are utilized individually. In some embodiments, the levels utilized in combination will be lower than those utilized individually. In one embodiment, the combinations, each or together may be administered according to the split dosing regimens as are known in the art.5. Methods of eliciting or enhancing zinc toxicity antimicrobial response

[0208] The CFTR protein functions as a channel across the membrane of cells that produce mucus, sweat, saliva, tears, and digestive enzymes. The channel transports negatively charged chloride ions into and out of cells. The transport of chloride ions assists control the movement of water in tissues, which is necessary for the production of thin, freely flowing mucus.

[0209] As described above and elsewhere herein, mutations in the CFTR protein typically culminate in CTFR dysfunction which results in an impaired zinc toxicity antimicrobial response in innate immune cells (including macrophages). Accordingly, the inventors have derived compositions and methods for eliciting or enhancing the zinc toxicity antimicrobial response in innate immune cells with CFTR dysfunction.

[0210] The compositions of the present disclosure can be used for eliciting or enhancing the zinc toxicity antimicrobial response in an innate immune cell with CFTR dysfunction. The eliciting or enhancing of zinc toxicity antimicrobial response includes both increasing phagocytic uptake and acidification. In some embodiments, the present invention provides methods for eliciting or enhancing the zinc toxicity antimicrobial response in an innate immune cell with CFTR dysfunction, the methods comprising administering to the innate immune cell an mRNA encoding a SLC30A1 protein as described herein or a pharmaceutical composition containing the mRNA. In some embodiments, the methods are performed in vivo. In alternative embodiments, the methods are performed in vitro.

[0211] Innate immune cells relevant to the present invention include those that express SLC30A1 and under normal conditions have a zinc toxicity antimicrobial response. Such innate immune cells include, but are not limited to, the group comprising macrophages, neutrophils, monocytes, and dendritic cells. In some preferred embodiments, the innate immune cell is a macrophage. In some more specific embodiments, the macrophage is an alveolar macrophage. Alternatively, the macrophage could be an interstitial macrophage. In some embodiments the macrophage could be present in a population of macrophages that include both alveolar macrophages and interstitial macrophages.

[0212] The mRNA or a pharmaceutical composition containing the mRNA may be administered directly to the lung environment of a subject with CFTR dysfunction. Various administration routes delivery to the lungs of a subject may be used. In some embodiments, an mRNA or a composition containing an mRNA described herein is administered by inhalation, nebulization or aerosolization. In variousembodiments, administration of the mRNA results in expression of SLC30A1 in one or more cells present in the lung environment of the subject (e.g., the innate immune cells residing in the lungs). In some of the same embodiments and some other embodiments, the administration of the mRNA results in expression of SLCA30A1 in the macrophages residing in the lungs.

[0213] In some embodiments, the method of eliciting or enhancing the zinc toxicity antimicrobial response in an innate immune cell is achieved by administering to the lung of a subject in need of treatment an mRNA comprising an open reading frame that encodes a SLC30A1 protein. A suitable SLC30A1 protein includes the full length wild-type human SLC30A1 protein sequence set forth in SEQ ID NO: 1 , or a biologically active fragment thereof. In some embodiments, the method of eliciting or enhancing the zinc toxicity antimicrobial response in an innate immune cell is achieved by administering to the lung of a subject in need of treatment an mRNA comprising an open reading frame which encodes an amino acid sequence at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 1 .

[0214] The mRNA or compositions of the present invention may be administered to a subject who is suffering from or susceptible to a condition associated with CFTR dysfunction. Examples of such conditions include cystic fibrosis and bronchiectasis. Cystic fibrosis is a genetic disorder characterized by mutations in the gene for CFTR.

[0215] In some embodiments, the present invention provides methods of treating and / or preventing a microbial infection in a subject with CFTR dysfunction. Such methods include the step of administering a mRNA or pharmaceutical composition as described above or elsewhere herein.

[0216] In some embodiments, the microbial infection is caused by a pathogenic microorganism. In some of the same embodiments and some other embodiments, the microbial infection is a persistent infection. In some embodiments the microbial infection is a bacterial infection, which includes but is not limited to Gram-negative and positive bacteria such as Pseudomonas (P. aeruginosa, P. paucimobilis, P. fluorescens, P. putida, and P. acidovorans) , methicillin-resistant Staphylococcus aureus (MRSA), streptococcus pneumoniae (Streptococcuspneumoniae), Escherichia coll, Klebsiella, Enterobacter, Serratia, Haemophilus, Yersinia pestis, Burkholderia pseudomallei, B. cepacia, B. gladioli, B. multibolans (B. multivorans), B. vietnamiensis, Mycobacterium tuberculosis, M. avium complex (MAC), M. avium, M. abscessus, M. intracellulare, M. kansasii, M. xenopi, M. marinum, M. ulcerans, M. fortuitum (M. fortuitum complex, M. fortuitum, orM. cheronei, Brevibacterium, Coryneform bacteria, Nocardia, Brevibacterium linens, Burkholderia cenocepecia, Methanosarcina mazei, Pantoea agglomerans, Pectobacterium atrosepticum, Pelagio variabilis, Streptomyces anulatus, and Streptomyces cinnamonensis. In some preferred embodiments, the infection is a Pseudomonas aeruginosa infection.6. Kits

[0217] In an aspect, the present invention provides a kit comprising one or more of the following: (i) a mRNA of the invention; and (ii) a pharmaceutical composition of the invention. In the case of a kit for therapeutic use, the kit can additionally comprise a pharmaceutically acceptable carrier, diluent or excipient.

[0218] Optionally, a kit of the invention is packaged with instructions for use in a method described herein according to any example.

[0219] In order that the invention may be readily understood and put into practical effect, particular preferred embodiments will now be described by way of the following non-limiting experimental examples.EXPERIMENTALEffect of CFTR inhibition on macrophage antimicrobial response.

[0220] To begin to generate a molecular understanding of how CFTR contributes to host defence in macrophages, the inventors assessed the effects of the selective and reversible thiazolidinone small molecule CFTR inhibitor 4-[[4-oxo-2-thioxo-3-[3-(trifluoromethyl)phenyl]-5-thiazolidinylidene]methyl]- benzoic acid (“C172”) on macrophage responses to E. coli. Initial experiments assessed responses of primary human macrophages that had been differentiated from monocytes in the presence of C172 (to mimic the long-term defect that is apparent in cystic fibrosis). C172 significantly reduced phagocytic uptake and / or acidification of fluorescently-labelled E. coli b primary human macrophages (Figure 1A). The inventors investigated this defect in phagocytosis using the UPEC ST131 reference strain EC958 (Petty et al., 2014). Uptake of UPEC, as assessed by intracellular bacterial loads at 20 min post infection (“p.i.”) (Figure 1 B), was also significantly reduced. Consequently, intracellular bacterial loads were also reduced at 2 hours and 8 hours p.i. (data not shown). However, when quantifying intracellular UPEC survival relative to initial uptake, bacterial loads were substantially increased at 2 hours and 8 hours p.i. (Figure 1C- D). This is consistent with previous observations using P. aeruginosa (Del Porto et al., 2011 ; and Barnaby et al., 2018) and suggests that macrophage antimicrobial responses engaged after the initial uptake of bacteria require CFTR.

[0221] To further examine this possibility and to avoid the complication of CFTR inhibition affecting phagocytosis, intramacrophage bacterial loads were next assessed when human monocyte- derived macrophages (HMDM) were first treated with C172 at 1 hour p.i. with UPEC (Figure 1 E). In these experiments, intracellular bacterial loads were again increased, particularly at the later time point of 8 hours p.i. (Figure 1 F-G), suggesting that CFTR contributes to antimicrobial responses in macrophages after bacterial uptake.

[0222] The inventors further validated these phenotypes using monocyte-derived macrophages from people with cystic fibrosis (pwCF) that harbour the AF508 mutation. Consistent with the CFTR inhibition data (Figure 1A-G), these macrophages were significantly impaired in both uptake and clearance of intracellular UPEC (Figure 1H-I). CFTR deficiency thus compromises at least two aspects of antimicrobial defence, phagocytosis and direct killing of bacteria.

[0223] Macrophages employ several inducible antimicrobial responses to combat persistent intracellular infections, including nutrient starvation and metal ion toxicity (Stocks et al., 2018). Of note, zinc toxicity has emerged as an important antimicrobial mechanism in macrophages, with this pathway being subverted by both UPEC and Salmonella (Kapetanovic et al., 2016; Stocks et al., 2019; and Stockset al., 2021). In E. coli and other Gram-negative bacteria, the transporter ZntA effluxes zinc when zinc concentrations reach cytotoxic levels, thus conferring zinc resistance (Beard et al., 1997; Rensing et al., 1997)). High levels of environmental zinc upregulate the expression of this transporter in UPEC (Figure 2A), with this effect being selective to zinc versus other metal ions (Stocks et al., 2019). The expression of this transporter is also elevated in E. coli within infected macrophages, consistent with engagement of the zinc toxicity response by these innate immune cells (Stocks et al., 2019).

[0224] Moreover, UPEC partly evades the macrophage zinc toxicity response, unlike the non- pathogenic E. coli K12 strain MG1655 (Stocks et al., 2019). To investigate if CFTR contributes to macrophage-mediated zinc toxicity against E. coli, the present inventors assessed zntA expression in the otherwise susceptible E. coli strain MG1655. In CFTR-inhibited macrophages, zntA mRNA levels were significantly lower than in vehicle-treated control macrophages (Figure 2B), suggesting that the zinc toxicity response was impaired. A similar phenotype was observed in macrophages from pwCF, where zntA mRNA levels of intramacrophage E. coli were lower than those in macrophages from healthy donors (Figure 2C).

[0225] Collectively, these data suggest that CFTR is required for optimal zinc trafficking in macrophages for deployment of the zinc toxicity response against intracellular bacteria.CFTR is required for bacteria-inducible zinc accumulation in human macrophages.

[0226] In response to TLR4 signalling or bacterial infection, macrophages accumulate intracellular zinc and mobilize this metal ion into vesicular structures that contain intracellular bacteria (Stocks et al., 2019; and Stocks et al., 2021).

[0227] Since analysis of zntA expression in E. coli within macrophages implicated CFTR in the zinc toxicity response (Figure 2), the inventors next sought to determine whether CFTR was required for E. co / / -induced zinc accumulation in macrophages. Treatment with either bacterial lipopolysaccharide (LPS) or E. coli for 24 hours increased levels of exchangeable zinc in macrophages, as detected by FluoZin-3 staining (Figure 3A, left panels), with this response being attenuated in CFTR-inhibited cells (Figure 3A, right panels). Similarly, the E. co / / -induced increase in FluoZin-3 staining did not occur in macrophages from pwCF (Figure 3B-C). To eliminate the possibility that the reduced intracellular zinc staining resulted from reduced uptake of bacteria (Figure 1A-B, H), CFTR was inhibited pharmacologically with C172 after bacterial uptake. Even in this setting where intracellular bacterial loads were increased (Figure 1 F-G), E. co / / -induced zinc accumulation was significantly reduced (Figure 3D). Total intracellular zinc levels were also quantified by ICP-OES to provide a more direct measure of zinc accumulation.

[0228] These experiments confirmed that infection with E. coli increased zinc levels within macrophages, with CFTR inhibition reducing this response (Figure 3E).

[0229] Zinc trafficking is controlled by the SLC39A / ZIP family of zinc importers and the SLC30A / ZNT family of zinc exporters. TLR ligation and E. coli infection upregulates SLC30A1 expression in human macrophages, with this transporter being functionally linked to zinc vesicle formation and the zinc toxicity response. For example, ectopic expression of SLC30A1 in human monocyte like THP-1 cells was sufficient to generate zinc-containing vesicles in macrophages in the absence of TLR activation (Stocks etal., 2021). The present inventors found that CFTR inhibition attenuated E. co / / -inducible SLC30A1 mRNA expression in human macrophages (Figure 3F). In contrast, there was a clear trend towards elevated TNF mRNA levels (data not shown), consistent with the hyperinflammatory phenotype that is observed in pwCF (Kopp et al., 2012; Bruscia et al., 2011 ; and Lara-Reyna et al., 2019). CFTR-dependent control of SLC30A1 mRNA expression was more evident when examining macrophages from pwCF. These data demonstrate that SLC30A1 mRNA expression is significantly reduced in macrophages from pwCF infected with E. coll for 8 hours, compared to healthy donor macrophages (Figure 3G). The coordinated actions of SLC39A and SLC30A zinc transporters are likely to enable zinc mobilisation for antimicrobial responses in macrophages. The requirement for CFTR in inducible SLC30A1 mRNA expression is therefore consistent with impaired zinc accumulation in CFTR-inhibited macrophages.CFTR is required for the zinc toxicity response in human macrophages.

[0230] Genetic deletion of zntA reduces the intracellular survival of E. coli within macrophages (Stocks et al., 2019). If CFTR is required for zinc-mediated antimicrobial defence, the reduction in intramacrophage survival of zntA E. coli should not occur in the absence of a functional CFTR. Indeed, although C172 impaired phagocytic uptake of both wild type and zntA E. coli as expected (Figure 4A), C172 prevented the ~two-fold reduction in intramacrophage survival of the zntA E. coli at 8 hours p.i. (Figure 4B). In fact, a clear trend for increased intracellular loads of zntA E. coli, compared to wild type E. coli, in C172-treated cells. Specifically, the recovery of zntA E. coli compared to wild type E. coli in DMSO vehicle-treated HMDM at 8 hours p.i. was 61%, compared to 156% recovered in C172-treated HMDM (means of five experiments). Similar findings were apparent in macrophages from pwCF. In these cells, initial uptake of both bacterial strains was again impaired compared to macrophages from healthy donors (Figure 4C). Furthermore, the ~two-fold reduction in intramacrophage survival of AznfA E. coli that was observed in macrophages from healthy donors did not occur in macrophages generated from pwCF (Figure 4D). Here, the recovery of zntA E. coli compared to wild type E. coli in macrophages from healthy donors at 8 hours p.i. was 43%, compared to 96% in macrophages from pwCF (means of four experiments). Collectively, these data confirm that macrophages without a functional CFTR protein fail to deploy the zinc toxicity response against intracellular E. coli.Overcoming defective zinc toxicity response in macrophages from pwCF.

[0231] The inventors next investigated the possibility that artificial amplification of the zinc toxicity response in CFTR-inhibited macrophages might be able to overcome the defect in bacterial killing. Ectopic expression of SLC30A1 in THP-1 cells using a doxycycline-inducible system (Figure 4E) reduced intracellular loads of E. coli in these cells at 8 hours p.i. (Figure 4F) and 24 hours p.i. (data not shown), whereas doxycycline did not affect bacterial loads in empty vector control cells at these time points. Interestingly, overexpressing SLC30A1 in CFTR-inhibited THP-1 cells eliminated the defect in antimicrobial responses of these macrophages, with these cells having similar intracellular bacterial loads to those with a functional CFTR.

[0232] Previous work by the inventors showed that exogenous zinc treatment facilitated intramacrophage clearance of Salmonella, which normally evades this pathway (Kapetanovic et al., 2016).- M -Given that SLC30A1 overexpression overcame the CFTR-mediated defect in the macrophage zinc toxicity response and that zinc supplementation of macrophages enhanced clearance of intracellular bacteria, the inventors predicted that exogenous zinc treatment of macrophages might also overcome this defect. Here we found that concentrations of zinc that did not inhibit the growth of either non-pathogenic E. coli or pathogenic EC958 (data not shown) significantly improved the ability of CFTR inhibited macrophages to reduce intracellular loads of both non-pathogenic and pathogenic E. coli (Figure 4G-H). Collectively, these data demonstrate a requirement for CFTR in the macrophage zinc toxicity response and suggest that artificial enhancement of this pathway may overcome defective innate immune defence in pwCF.CFTR is required for the antimicrobial zinc response against P. aeruginosa in GM-CSF-derived macrophages.

[0233] Having established a role for CFTR in the macrophage zinc toxicity response, the inventors next assessed the potential clinical significance of this using an in vitro model of lung infection. Alveolar macrophages are crucial in preserving lung function during pulmonary infections. These resident tissue macrophages of the lung rely on GM-CSF fortheir development and function. The inventors therefore assessed the zinc toxicity response in GM-CSF-derived human macrophages responding to P. aeruginosa, a common and significant bacterial pathogen in pwCF.

[0234] Consistent with the previous observations assessing E. coli uptake and killing by CSF-1- derived macrophages (Figure 1A-D), pharmacological inhibition of CFTR significantly impaired the ability of GM-CSF-derived macrophages to take up P. aeruginosa (Figure 5A) and to subsequently kill this pathogen (Figure 5B-C). Similarly, GM-CSF-derived macrophages from pwCF were defective in P. aeruginosa uptake and killing (Figure 5D-E). To uncouple the roles of CFTR in bacterial uptake versus the subsequent bacterial killing response, the inventors next pharmacologically inhibited CFTR after initial P. aeruginosa uptake (Figure 5F). In this setting, intracellular bacterial loads were elevated after CFTR inhibition, though some variability in macrophages from different donors was observed (Figure 5G-I).

[0235] Effects of zinc on P. aeruginosa growth are complex. Zinc promotes P. aeruginosa growth and biofilm formation, but higher concentrations are growth inhibitory (Marguettaz et al., 2014; Lee et al., 2014). Zinc also inhibits signalling pathways associated with P. aeruginosa virulence. The inventors therefore assessed effects of zinc in macrophage responses against P. aeruginosa infection. Here, the inventors observed a trend towards increased zinc accumulation in GM-CSF-derived macrophages from healthy donors after P. aeruginosa infection (Figure 5J), although the effect was not as pronounced as was observed after E. coli challenge in CSF-1-derived macrophages (Figure 3A-B). In contrast, P. aeruginosa did not increase the zinc content of GM-CSF-derived macrophages from pwCF, as assessed by FluoZin-3 staining (Figure 5J). The combination drug cocktail elexacaftor-tezacaftor-ivacaftor (ETI) corrects CFTR function and restores lung function in pwCF carrying the AF508 mutation (Ong et al., 2023). CFTR correction with ETI also reduces, but does not eliminate, infections caused by common bacterial pathogens such as P. aeruginosa in pwCF (Sheikh et al., 2023; Nichols et al., 2023). Here, the inventors demonstrate that ETI does not restore cellular zinc levels in GM-CSF-derived macrophages from pwCF after infection with P. aeruginosa (Figure 5J). Similarly, ETI does not restore zinc vesicle formation in CSF- 1-derived macrophages from pwCF, as assessed by FluoZin-3 staining and confocal microscopy (Figure5Ks). Nonetheless, while exogenous zinc treatment did not increase the capacity of macrophages from pwCF to take up bacteria (Figure 5L), it did significantly reduce P. aeruginosa loads within macrophages from both healthy donors and pwCF (Figure 5M).

[0236] Collectively, these data clearly show that CFTR is required for the macrophage zinc toxicity response and that zinc supplementation can overcome the defect in host defence that is apparent in macrophages from pwCF.SLC30A1 mRMA promotes clearance of intracellular bacteria by macrophages.

[0237] The present inventors next sought to determine the effects of manipulating the expression of specific zinc transporters in human macrophage-like PMA-differentiated THP1 cells. Strikingly, overexpression of SLC30A1-V5 or SLC30A1-V5 and SLC30A4-V5 together, but not SLC30A4-V5 alone, by mRNA delivery to human macrophage-like PMA-differentiated THP1 cells reduces the intracellular loads of E. coli in these cells at 24 hours post-infection, as assessed by colony forming unit assays (see, Figure 6B). These manipulations did not affect bacterial uptake, as assessed by colony forming unit assays at two hours post-infection (see, Figure 6A).

[0238] Varying mRNA design (Figure 6C) can enhance levels of SLC30A1-V5 protein expression in both HEK293 cells (Figure 6D) and human macrophage-like PMA-differentiated THP1 cells (see, Figure 6E). In particular, these data show that incorporation of the AES + mtRNRI 31UTR (SEQ ID NO: 11) results in higher expression of SLC30A1 than is observed for an alternative 31UTR (SEQ ID NO: 10) in both HEK293 (see, Figure 6D) and THP1 cells (see, Figure 6E).Materials & MethodsEthics statement

[0239] Human peripheral blood was collected from healthy donors and pwCF following informed consent, after which mononuclear cells were isolated. Monocytes from pwCF were either homozygous (12 out of 18) or heterozygous (6 out of 18) for the AF508 mutation (Table 2). All experiments that used these cells were approved by The University of Queensland Institutional Human Research Ethics Committee (2013 / HE001519, 2022 / HE002118 and 2020001275 / HREC / 20 / QCHQ / 64229).TABLE 2DONOR CHARACTERISTICSChemicals and Reagents

[0240] The CFTR inhibitor 4-[[4-oxo-2-thioxo-3-[3-(trifluoromethyl)phenyl]-5- thiazolidinylidene]methyl]-benzoic acid (C172, Cayman Chemical) was dissolved in DMSO and used at a final concentration of 10 pM. The Toll-like receptor (TLR)4 agonist lipopolysaccharide (LPS) from Salmonella enterica serotype Minnesota was purchased from Sigma Aldrich (Cat - L2137) and dissolved in RPMI media (Gibco). The concentrations used for the three drugs that constitute TRIKAFTA were elexacaftor 3 pM, tezacaftor 18 pM and ivacaftor 1 pM (ETI).Bacterial culture

[0241] Bacterial strains were cultured at 37 °C on solid or liquid Luria-Bertani (LB) medium. Non- pathogenic E. coll K12 strain MG1655, a zinc-sensitive zntA mutant of MG1655, MG1655_mCherry that constitutively expresses mCherry, a representative strain of the globally-disseminated multidrug-resistant ST131 clone isolated from the urine of a patient with a urinary tract infection (EC958), and a clinically- relevant reference strain of P. aeruginosa (PAM) (96), were used in this study. Overnight cultures of E. coll MG1655 and PA were grown under shaking conditions, while EC958 cultures were grown statically to induce type I fimbriae production, as previously described. Before infection, bacteria were washed twice and resuspended in macrophage infection medium (described below). Optical density at 600 nm (ODsoo) was measured and bacterial suspensions were diluted to ODsoo = 0.6, which corresponds to ~109cfu / mL for E. coll and P. aeruginosa. Bacterial growth + zinc sulphate (Sigma-Aldrich) was assessed by monitoring ODsoo using a POLARSTAR Omega plate reader (BMG Labtech) at 30 min intervals.Mammalian cell culture

[0242] CD14+ human monocytes derived from either healthy donors or pwCF were purified from buffy coats provided by the Australian Red Cross Blood Service or from pwCF, respectively. Monocytes were differentiated into HMDM by culturing for 7 days in IMDM (Gibco) containing 10% foetal calf serum (FCS, Gibco), 50 U / mL penicillin (Life Technologies), 50 mg / mL streptomycin (Life Technologies), 2 mM L- glutamine and recombinant human CSF-1 (150 ng / mL) (The University of Queensland Protein Expression Facility) or recombinant human GM-CSF (50 ng / mL) (Milteny Biotec). Where indicated, the CFTR inhibitor C172 (Cayman Chemical) was added daily to differentiating monocytes to mimic chronic CFTR deficiency, as occurs in cystic fibrosis. For experiments involving CFTR correctors / potentiators, ETI was added to monocytes on day 0, 3 and 7 of differentiation. ETI was also added to the cell culture media for the duration of the infection assays.

[0243] The doxycycline-inducible system for expression of the zinc transporter SLC30A1 with a C437 terminal V5 epitope tag in THP-1 cells has been described (Stocks et al., 2021). THP-1 cells lentivirally transduced with either empty vector (EV) or SLC30A1-V5 were cultured in RPMI-1640 media (Gibco) supplemented with 10% FCS (Gibco), 50 U / mL penicillin (Life Technologies), 50 mg / mL streptomycin (Life Technologies), 2 mM GLUTAMAX (Life Technologies), 1% HEPES (Thermo Fischer Scientific) and 1 % sodium pyruvate (Gibco). Cells were differentiated using phorbol 12-myristate 13- acetate (PMA) (30 ng / mL) (Sigma Aldrich) for 48 hours + C172. SLC30A1-V5 protein expression wasinduced by treating cells with 100 ng / mL doxycycline (Cat - D9891 , Sigma Aldrich) for 24 hours, as described below. For all infection assays (see below), cells were cultured in IMD supplemented with 10% FCS. All cells were cultured at 37 °C and 5% CO2, unless otherwise indicated.Quantification of phagocytic uptake of bacteria.

[0244] Phagocytic uptake of bacteria was assessed using pHrodo Green E. coli bioparticles (Thermo Fisher Scientific), as previously described (Das Gupta et al., 2023). Briefly, primary macrophages (5 x 105cells) were treated with 100 pg of pHRODO bacterial bioparticles for one hour, after which bacterial uptake was assessed by flow cytometry (Cytoflex, Beckman Coulter). Alternatively, primary macrophages were spin-fected with either E. coli MG1655, EC958 or PAM for 5 minutes at 500 g at 35 °C, using a multiplicity of infection (MOI) of 100 (for E. coli) or 5 (PAM). The cells were then rested for 5 minutes before removing extracellular bacteria with gentamicin (200 pg / mL) (Thermo Fisher Scientific). Bacterial uptake at 20 min post-p.i. was assessed by counting colony forming units (CFU), as described below.In vitro infection assays.

[0245] In vitro bacterial infections of primary macrophages and cell lines were carried out as previously described (Bokil et al., 2011). Briefly, 1-4 x 105cells were seeded overnight in antibiotic-free IMDM media. An MOI of 100 was used for both E. coli MG1655 and EC958, whereas P. aeruginosa infections were carried out using MOI of 5. At 1 hour p.i. , cells were washed and maintained in medium containing 200 pg / mL gentamicin to exclude any extracellular bacteria for 1 hour, after which cells were washed with media again and maintained in medium containing 20 pg / mL gentamicin (Thermo Fisher Scientific). At appropriate time points, cells were washed twice with PBS before being lysed in PBS containing 0.01% Triton X-100. Diluted lysates were plated onto LB agar and incubated overnight at 37°C. Numbers of colonies were counted to determine intracellular CFU. At all time points where the media was replaced during infection assays, 10 pM C172 or 200 pM ZnSO4 was re-introduced to the media for relevant treatment groups.Gene expression analyses.

[0246] Total RNA (bacterial and human) was extracted using RNA purification kits (Qiagen), as perthe manufacturer's instructions. Genomic DNA was removed using an on-column DNAse digestion (Qiagen). RNA was reverse transcribed to cDNA using SUPERSCRIPT III Reverse Transcriptase (Invitrogen) and oligo dT (for mammalian targets) or random hexamers (for bacterial genes). Levels of specific mRNAs were quantified by qPCR using SyBR Green-PCR mix (Invitrogen) in the Applied Biosystems Viia 7 RT-PCR system. Appropriate negative controls with no SUPERSCRIPT III Reverse T ranscriptase were included for all experiments. Data were expressed relative to the housekeeping gene hypoxanthine phosphoribosyltransferase (HPRT, human) or gapA (E. coli housekeeping gene) using the ACt method (Livak et al., 2001). Primers used for RT-qPCR are listed in Table 3.TABLE 3RT-QPCR PRIMERSEstimation of total cellular zinc levels by flow cytometry

[0247] The zinc content of human macrophages post-LPS stimulation or bacterial infection was indirectly assessed using staining with 5 pM FluoZin-3AM (Thermo-Fisher Scientific), as previously described (Kapetanovic et al., 2016). Briefly, primary macrophages (2 x 105cells) were eithertreated with LPS (20 ng / mL) or infected with indicated bacterial species for 24 hours, as described above. Posttreatment, cells were washed with PBS and stained for 30 minutes with FLUOZIN-3AM. The cells were washed again with PBS, and then harvested in ice-cold PBS containing 0.1 % sodium azide and 25 mM EDTA.

[0248] Flow cytometric analysis was performed using a CYTOFLEX flow cytometer (Beckman).Quantification of total cellular zinc levels by ICP-OES.

[0249] The zinc content of human macrophages was directly measured by inductively coupled plasma optical emission spectroscopy (ICP-OES), as previously described (Stocks et al., 2019). Briefly, 5 x 106HMDM were infected with MG1655 (MOI 100) for 24 hours, as described above. At 24 hours p.i., cells were washed twice with Hanks' balanced salt solution, lysed in 5 mL of lysis solution (0.1% sodium dodecyl sulfate in MilliQ water), and placed into pre-weighed 10 mL plastic tubes (School of Earth and Environmental Sciences, The University of Queensland, Australia). Triple distilled HNO3 was then added to acidify the sample to 2%. The sample was then made up to a total volume of 10 mL with MilliQ water. Samples were analysed using an Optima 8300 DV ICPOES spectrometer (Perkin Elmer, USA). Freshly prepared calibration standards were used to estimate intramacrophage zinc concentrations (with two or three spectral lines measured as a quality control). The overall concentration of zinc within a macrophage was approximated based on the zinc concentration within 1 million macrophages lysed in 1 mL of lysis buffer (as determined by ICP-OES).Confocal microscopy and zinc vesicle quantification.

[0250] 2 x 105cells were plated on coverslips in a 24 well plate in complete IMDM media and left to adhere overnight. Cells were infected using fluorescent E. coli MG1655_mCherry as described in figure legends before being washed twice with PBS and fixed with 4% PFA (Sigma-Aldrich) for 20 minutes. Cells were washed 3 times with PBS before being stained for intracellular zinc with 5 pM FluoZin-3AM and nuclear DNA with 1 pg / mL DAPI (Life Technologies). Coverslips were washed three times with PBS beforebeing mounted on slides using IM Biol mounting media (IMB) and slides were viewed using a Zeiss Axiovert 200 Upright Microscope stand with LSM 710 Meta Confocal Scanner and spectral detection with 63x magnification (Zeiss). Images were processed with FIJI (Imaged) and Zinc puncta were quantified by counting FLUOZIN-3AM maxima per cell (minimum 50 cells per condition) using the ‘Find maxima’ function.Immunoblotting.

[0251] Whole cell lysates were prepared in RIPA buffer, containing a cocktail of 1 x protease inhibitors (Roche) and 1 x PhosSTOP phosphatase inhibitors (Sigma-Aldrich). Immunoblotting was performed by electrophoresing equal amounts of protein through precast BOLT gels (Invitrogen), followed by turbo transfer onto nitrocellulose membranes at 25 V for 9 minutes (Bio-Rad Laboratories). Membranes were blocked using 5% BSA diluted in Tris-buffered saline containing 0.05% Tween-20 followed by probing with either anti-V5 (CAT: MCA1360, Bio-Rad Laboratories) or rhodamine-conjugated anti-tubulin (CAT: 12004165, Bio-Rad Laboratories). Proteins were visualised using Clarity ECL (Bio-Rad Laboratories) or through detection of fluorescent antibodies using a Chemidoc (Bio-Rad Laboratories). mRNA transfections.

[0252] THP1 or HEK293 cells were transfected with the indicated mRNAs using the Neon Transfection System (ThermoFisher Scientific). For each condition, 2 x 106cells were resuspended in 100 pL Buffer R from the Neon transfection system kit. mRNAs were diluted to indicated concentrations to a final volume of 10 pL in nuclease-free water and mixed with the cell suspension. Cells were transfected using the Neon transfection system and settings of voltage 1400 V, width 20 ms and 2 pulses. Cells were immediately plated on 12-well plates in media without antibiotics. Transfected THP1 cells were differentiated into macrophage-like cells by treating with 30 ng / mL PMA for 48 hours, then infected with E. co / / (K12 strain MG1655) at MOI 100 for 2 hours or 24 hours. Intracellular bacterial loads were counted as described above. HEK293 cells and PMA-differentiated THP1 cells were also lysed at 24 hours posttransfection and analysed for expression of V5, GFP and Tubulin (loading control) by immunoblotting.Statistical analyses.

[0253] Where statistical analyses were performed, data were combined from three or more independent experiments, with each experiment (n) designated by a different symbol. In the case of experiments on primary human macrophages, each experiment used cells from different donors and is represented by a different symbol. Statistical analyses were performed using Prism 9 software (GraphPad) with data combined from at least three independent experiments (taking averages from replicates within each experiment) and error bars indicating the standard error of the mean (SEM).

[0254] Statistical analyses were performed using non-parametric tests (Mann-Whitney t-test (unpaired), Wlcoxon t-test (paired), Kruskal-Wallis' or Friedman's test). For experiments analysing matched samples or data, repeated measures (RM) ANOVA was performed. For data sets with two or more variables, a two-way analysis of variance (ANOVA) was performed followed by Tukey's or Sidak's multiple comparison test. Statistical tests used for individual experiments are described in the figure legends. Differences with confidence values of 95% (P < 0.05) were considered statistically significant.REFERENCESAdjemian J. et al., Nontuberculous mycobacteria among patients with cystic fibrosis in the United States: screening practices and environmental risk. Am J Respir Crit Care Med. 190, 581-586 (2014).Barnaby R. et al., Lumacaftor (VX-809) restores the ability of CF macrophages to phagocytose and kill Pseudomonas aeruginosa. American Journal of Physiology-Lung Cellular and Molecular Physiology 314, L432-L438 (2018).Beard S.J. et al., Zinc(ll) tolerance in Escherichia coli K-12: evidence that the zntA gene (o732) encodes a cation transport ATPase. Mol Microbiol 25, 883-891 (1997).Bokil N.J. et al., Intramacrophage survival of uropathogenic Escherichia coli: Differences between diverse clinical isolates and between mouse and human macrophages. Immunobiology 216, 1164-1171 (2011).Bruscia E.M., et al., Abnormal Trafficking and Degradation of TLR4 Underlie the Elevated Inflammatory Response in Cystic Fibrosis. The Journal of Immunology 186, 6990-6998 (2011).Das Gupta K. et al., HDAC7 is an immunometabolic switch triaging danger signals for engagement of antimicrobial versus inflammatory responses in macrophages. Proc Natl Acad Sci USA 120, e2212813120 (2023).Del Porto P. et al., Dysfunctional CFTR alters the bactericidal activity of human macrophages against Pseudomonas aeruginosa. PLoS One 6, e19970 (2011).Gangell C. et al., Inflammatory responses to individual microorganisms in the lungs of children with cystic fibrosis. Clin Infect Dis 53, 425-432 (2011).Emerson J. et al., Pseudomonas aeruginosa and other predictors of mortality and morbidity in young children with cystic fibrosis. Pediatr Pulmonol. 582 34, 91-100 (2002).Hajj K.A. et al., Branched-tail lipid nanoparticles potently deliver mRNA in vivo due to enhanced ionization at endosomal pH. Small, 5, e1805097 (2019).Jih K.Y. et al., The most common cystic fibrosis-associated mutation destabilizes the dimeric state of the nucleotide-binding domains of CFTR. J Physiol 589, 2719-2731 (2011).Kapetanovic R. et al., Salmonella employs multiple mechanisms to subvert the TLR-inducible zinc mediated antimicrobial response of human macrophages. FASEB J 30, 1901-1912 (2016).Kopp B.T. et al., Exaggerated inflammatory responses mediated by Burkholderia cenocepacia in human macrophages derived from Cystic fibrosis patients. Biochemical and Biophysical Research Communications 424, 221-227 (2012).Lara-Reyna S. et al., Metabolic Reprograming of Cystic Fibrosis Macrophages via the IREIa Arm of the Unfolded Protein Response Results in Exacerbated Inflammation. Frontiers in Immunology 10, 10.3389 / fimmu.2019.01789 (2019).Lee J.H. et al., ZnO nanoparticles inhibit Pseudomonas aeruginosa biofilm formation and virulence factor production. Microbiol Res 169, 888-896 (2014).Livak, J.K., Schmittgen, T.D., Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods 25, 402-408 (2001).Marguerettaz, M. et al., Sputum containing zinc enhances carbapenem resistance, biofilm formation and virulence of Pseudomonas aeruginosa. Microb Pathog 77, 36-41 (2014).Nichols D.P. et al., Pharmacologic improvement of CFTR function rapidly decreases sputum pathogen density, but lung infections generally persist. J Clin Invest 133, e167957 (2023).Okiyoneda T. et al., Peripheral protein quality control removes unfolded CFTR from the plasma membrane. Science 329, 805-810 (2010).Ong, S., Ramsey, B. W., Cystic Fibrosis: A Review. Jama 329, 1859-1871 (2023).Petty et al., Global dissemination of a multidrug resistant Escherichia coli clone. Proc Natl Acad Sci U S A 111, 5694-5699 (2014).Oz H.H. et al., Recruited monocytes / macrophages drive pulmonary neutrophilic inflammation and irreversible lung tissue remodeling in cystic fibrosis. Cell Rep 41 , 111797 (2022).Ratjen F. et al., Cystic fibrosis. Nature Reviews Disease Primers 1 , 15010 (2015).Rensing C. et al., The zntA gene of Escherichia coli encodes a Zn(ll)-translocating P-type ATPase. Proc Natl Acad Sci U S A 94, 14326-14331 (1997).Sheikh S. et al., Impact of elexacaftor-tezacaftor-ivacaftor on bacterial colonization and inflammatory responses in cystic fibrosis. Pediatr Pulmonol 58, 825-833 (2023).Simonin-Le Jeune K. et al., Impaired Functions of Macrophage from Cystic Fibrosis Patients: CD11 b, TLR- 5 Decrease and sCD14, Inflammatory Cytokines Increase. PLOS ONE 8, e75667 (2013).Stocks C.J. et al., For when bacterial infections persist: Toll-like receptor-inducible direct antimicrobial pathways in macrophages. Journal of Leukocyte Biology 103, 35-51 (2018).Stocks C.J. et al., Uropathogenic Escherichia coliemploys both evasion and resistance to subvert innate immune-mediated zinc toxicity for dissemination. Proc Natl Acad Sci U S A 116, 6341-6350 (2019).Stocks C.J. et al., Frontline Science: LPS-inducible SLC30A1 drives human macrophage-mediated zinc toxicity against intracellular Escherichia coli. Journal of leukocyte biology 109, 287-297 (2021).Trimble A.T. et al., Ivacaftor withdrawal syndrome in cystic fibrosis patients with the G551 D mutation. Journal of Cystic Fibrosis 17, e13-e16 (2018).Wang et al., A combinatorial library of unsaturated lipidoids for efficient intracellular gene delivery. ACS Synth. Biol. 1 , 403-407 (2012).Watson M.J. et al., The Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Uses its C Terminus to Regulate the A2B Adenosine Receptor. Scientific Reports 6, 27390 (2016).

Claims

WHAT IS CLAIMED IS:

1. A method of treating or preventing a microbial infection in a subject with CFTR dysfunction, the method comprising administering to the subject an agent that elicits or enhances the zinc toxicity antimicrobial response of an innate immune cell of the subject.

2. The method of claim 1 , wherein the agent promotes SLC30A1 expression or SLC30A1 activity in the innate immune cell.

3. A method of eliciting or enhancing a zinc toxicity antimicrobial response in an innate immune cell with CFTR dysfunction, the method comprising administering to the subject an agent that promotes SLC30A1 expression or SLC30A1 activity in the innate immune cell.

4. The method of any one of claims 1 to 3, wherein the agent promotes SLC30A1 expression in the innate immune cell.

5. The method of any one of claims 1 to 4, wherein the CFTR deficiency is a heterozygous mutation or a homozygous mutation.

6. The method of any one of claims 1 to 5, wherein the agent is a messenger RNA (mRNA) that comprises an open reading frame (ORF) that encodes a solute carrier 30 member A1 (SLC30A1) polypeptide.

7. A method of eliciting or enhancing a zinc toxicity antimicrobial response in a subject with CFTR dysfunction, the method comprising administering to the subject an agent that promotes SLC30A1 expression or SLC30A1 activity in an innate immune cell of the subject.

8. The method of any one of claims 1 to 7, wherein the innate immune cell of the subject has one or both of: an impaired uptake of a microbial cells and an impaired clearance of microbial cells.

9. The method of any one of claims 1 to 8, wherein the subject is diagnosed with cystic fibrosis or bronchiectasis.

10. The method of any one of claims 1 to 9, wherein the innate immune cell is selected from a macrophage, neutrophil, monocyte, and dendritic cell.11 . The method of any one of claims 1 to 10, wherein the innate immune cell is a macrophage.

12. The method of any one of claims 1 to 11 , wherein the agent is administered together with a treatment for a condition associated with dysfunctional CFTR (e.g., cystic fibrosis).

13. The method of claim 12, wherein the treatment is selected from TRIKAFTA (elexacaftor, ivacaftor, tezacaftor), KALYDECO (ivacaftor), lumacaftor, texacaftor, elexacaftor, vanzacaftor, VX-522SYMDEKO (tezacaftor and ivacaftor), and ORKAMBI (lumacaftor and ivacaftor).

14. A messenger RNA (mRNA) for eliciting or enhancing a zinc toxicity antimicrobial response in an innate immune cell, the mRNA comprising an open reading frame (ORF) encoding a solute carrier 30 member A1 (SLC30A1) polypeptide and a 5' untranslated region (UTR).

15. The mRNA of claim 14, further comprising one or more of a 5’ cap, a 3’ UTR, and a poly(A) tail.

16. The mRNA of claim 14 or claim 15, further comprising one or both of an optimised codon and a chemical modification when compared to a corresponding mRNA that does not comprise the optimised codon and / or chemical modification.

17. The mRNA of claim 16, wherein the chemical modification and / orthe optimised codon increases mRNA stability and / or mRNA translation in a mammalian cell when compared to a mRNA without the chemical modification and / or the optimised codon.

18. The mRNA of claim 16 or claim 17, wherein the chemical modification is a nucleoside modification (e.g., a modified uracil or a modified cytosine).

19. The mRNA of any one of claims 16 to 18, wherein the optimised codon increases guanine (G) and / or cytosine (C) codon content.

20. The mRNA of any one of claims 16 to 19, wherein the mRNA encodes a SLC30A1 polypeptide that comprises an amino acid sequence set forth in SEQ ID NO: 1 , or having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 99%, at least 99.5%, at least 99.8% identity to a sequence set forth in SEQ ID NO: 1 , or a biologically active fragment thereof.21 . A pharmaceutical composition comprising the mRNA according to any one of claims 13 to 19, and a pharmaceutically acceptable carrier, diluent, or excipient.

22. The pharmaceutical composition of claim 21 , further comprising a treatment for a condition associated with dysfunctional CFTR (e.g., cystic fibrosis).

23. The pharmaceutical composition of claim 22, wherein the treatment is selected from TRIKAFTA (elexacaftor, ivacaftor, tezacaftor), KALYDECO (ivacaftor), lumacaftor, texacaftor, elexacaftor, vanzacaftor, VX-522, SYMDEKO (tezacaftor and ivacaftor), and ORKAMBI (lumacaftor and ivacaftor).

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