EP4695269A1

EP4695269A1 - Mucosal- and cell-membrane penetrating peptides and uses thereof

Info

Publication number: EP4695269A1
Application number: EP24789430.6A
Authority: EP
Inventors: Melissa SOTO; Debadyuti GHOSH
Original assignee: University of Texas System; University of Texas at Austin
Current assignee: University of Texas System; University of Texas at Austin
Priority date: 2023-04-11
Filing date: 2024-04-11
Publication date: 2026-02-18
Also published as: AU2024249750A1; WO2024215859A1

Abstract

Peptides which are capable of penetrating mucosal membranes or cell membranes are provided. In some aspects, functionalized peptide conjugates are provided. Compositions of peptide conjugates are disclosed, and methods of using such compositions are provided.

Description

DESCRIPTION

MUCOSAL- AND CELL-MEMBRANE PENETRATING PEPTIDES

AND USES THEREOF

REFERENCE TO A SEQUENCE LISTING

This application contains a Sequence Listing XML, which has been submitted electronically and is hereby incorporated by reference in its entirety. Said XML Sequence Listing, created on April 9, 2024, is named UTFBP1332WO.xml and is 52,961 bytes in size.

REFERENCE TO RELATED APPLICATIONS

[0001] The present application claims the priority benefit of United States provisional application number 63/495,481 filed April 11, 2023, the entire contents of which are incorporated herein by reference.

BACKGROUND OF THE INVENTION

Field of the Invention

[0003] The present disclosure relates generally to the fields of biochemistry, molecular biology, pharmaceutical formulation, and biologies. The present application relates to peptides, functionalized peptide conjugates, and related compositions which can be used to target delivery of therapeutic agents. More specifically, the present disclosure relates peptides that selectively target cells, such as lung cells, and conjugates, compositions, and methods of use thereof.

Description of Related Art

[0004] Patients with Cystic Fibrosis (CF) may have one of many known mutations in the gene encoding the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) protein, which often leads to abnormal and excessive build-up of mucus in the lungs. Lung related complications are often the main cause of death for CF patients. As such, lung targeted gene therapy shows promise for being a potential curative treatment for patients with CF. However, the hostile environment found in the lungs of CF patients, which includes hyper-concentrated mucus and immune cells, such as macrophages, greatly hinders locally delivered treatments from reaching intended target sites, i.e., bronchial epithelial cells, secretory cells, or basal cells. In CF patients, these delivery barriers are a primary reason why many gene therapies fail clinically. Existing efforts to overcome the mucus barrier have been focused on using hydrophilic, net- neutrally charged polymers (e.g., poly(ethylene) glycol) that have properties for improved diffusion and transport through mucus. While promising, these current formulations may possess potential immunogenicity upon initial or repeated administration or may have decreased cellular uptake due to steric hindrance issues. Furthermore, even when cellular uptake is possible, it is not always targeted or selective. For pulmonary gene delivery systems to be clinically translatable, it is essential to evaluate their targeting ability. Cell penetrating peptides (CPPs) have been studied to transport therapeutic cargo intracellularly, but their positively charged nature poses a problem for mucus diffusion.

[0005] Thus, gene delivery vehicles that can tackle the multiple barriers associated with gene delivery, including crossing the CF mucus barrier, and achieve targeted cellular uptake are sought. Also sought are delivery systems for shuttling therapeutics to a target site, such as lung cells, that are non-inflammatory, that enhance cell internalization, and that are easily incorporated into non-viral delivery systems where larger payloads can be delivered.

[0006] Research for this invention was supported by the Cystic Fibrosis Foundation under Grant No. GHOSH19XXO.

SUMMARY OF THE INVENTION

[0007] The presently disclosed peptides, conjugates, compositions, and methods are based, in part, on the discovery and identification of certain peptides that can be used to aid in prevention and/or treatment of a disease or disorder such as a disease or disorder of epithelial cells.

[0008] More particularly, the present application provides peptides and lipid nanoparticles that facilitate targeted delivery of cargo to primary human epithelial cells such as primary human bronchial epithelial cells (pHBECs), thereby providing a possible solution to the current obstacle of gene delivery to epithelial cells as part of treatment or prevention of disease or disorders including diseases or disorders associated with epithelial cells. Targeted delivery of therapies may provide benefits including, but not limited to, reduced systemic side effects, lower therapeutic dose compared to systemic delivery, or facilitation of alternative modes of administration of the therapy.

[0009] In one aspect, the present disclosure provides peptides of 9-15 amino acids, wherein the peptide comprises the sequence CX7C, wherein the peptide has a net positive charge of at least 1 at neutral pH and the peptide is capable of penetrating a mucosal membrane or cell membrane.

[0010] In some embodiments, the peptide has a net positive charge from about 1 to about 5. In further embodiments, the peptide has a net positive charge from about 1.5 to about 3. In some embodiments, the peptide has a net positive charge of about 1.9. In other embodiments, the peptide has a net positive charge of about 2.9. In still other embodiments, the peptide has a net positive charge of about 3.9.

[0011] In some embodiments, the peptide has a GRAVY score of from about -0.2 to about -4. In further embodiments, the peptide has a GRAVY score that is less than -1. In still further embodiments, the peptide has a GRAVY score that is less than -2. In yet further embodiments, the peptide has a GRAVY score that is less than -3.

[0012] In some embodiments, the peptide comprises at least 1 amino acid residue that is positively charged at neutral pH. In further embodiments, the peptide comprises at least 2 amino acid residues that are positively charged at neutral pH. In still further embodiments, the peptide comprises at least 3 amino acid residues that are positively charged at neutral pH. In yet further embodiments, the peptide comprises at least 4 amino acid residues that are positively charged at neutral pH.

[0013] In some embodiments, the peptide sequence comprises at least one serine residue. In further embodiments, the peptide sequence comprises one serine residue. In still further embodiments, the peptide sequence comprises two serine residues. In yet further embodiments, the peptide sequence comprises three serine residues. In even further embodiments, the peptide sequence comprises four serine residues.

[0014] In some embodiments, the peptide sequence comprises at least one proline residue. In further embodiments, the peptide sequence comprises one proline residue. In still further embodiments, the peptide sequence comprises two proline residues. In yet further embodiments, the peptide sequence comprises three proline residues. In even further embodiments, the peptide sequence comprises four proline residues.

[0015] In some embodiments, the peptide sequence comprises at least one lysine residue. In further embodiments, the peptide sequence comprises one lysine residue. In still further embodiments, the peptide sequence comprises two lysine residues. In yet further embodiments, the peptide sequence comprises three lysine residues. In even further embodiments, the peptide sequence comprises four lysine residues.

[0016] In some embodiments, the peptide sequence comprises at least one arginine residue. In further embodiments, the peptide sequence comprises one arginine residue. In still further embodiments, the peptide sequence comprises two arginine residues. In yet further embodiments, the peptide sequence comprises three arginine residues. In even further embodiments, the peptide sequence comprises four arginine residues.

[0017] In yet further embodiments, the peptide is capable of penetrating a mucosal membrane or cell membrane by direct penetration. In even further embodiments, the peptide is capable of penetrating a mucosal membrane or cell membrane by endocytosis.

[0018] In some embodiments, the cell membrane is a membrane of a human cell. In further embodiments, the cell membrane is a membrane of an epithelial cell. In still further embodiments, the epithelial cell membrane is a membrane of a lung epithelial cell. In another embodiment, the epithelial cell membrane is a membrane of a gastrointestinal epithelial cell. In another embodiment, the epithelial cell membrane is a membrane of a cervicovaginal epithelial cell. In another embodiment, the epithelial cell membrane is a membrane of a nasal epithelial cell. In another embodiment, the epithelial cell membrane is a membrane of a skin epithelial cell. In another embodiment, the epithelial cell membrane is a membrane of a hepatic epithelial cell. In another embodiment, the epithelial cell membrane is a membrane of a corneal epithelial cell.

[0019] In still further embodiments, the epithelial cell membrane is a membrane of a basal lung epithelial cell. In still further embodiments, the epithelial cell membrane is a membrane of a basal lung epithelial cell. In yet further embodiments, the lung epithelial cell membrane is a membrane of a primary cell. In even further embodiments, the primary cell membrane is a membrane of a primary human bronchial epithelial cell.

[0020] In some embodiments, the cell membrane is derived from a cell from a patient with a disease or disorder. In some embodiments, the disease or disorder is a lung disease or disorder. In some embodiments, the lung disease or disorder is chronic obstructive pulmonary disease. In another embodiment, the lung disease or disorder is respiratory syncytial virus. In another embodiment, the lung disease or disorder is influenza. In another embodiment, the lung disease is cytomegalovirus. In another embodiment, the lung disease is primary ciliary dyskinesia such as primary ciliary dyskinesia has a genetic mutation of the DNAI1 or DNAH5 genes. In another embodiment, the lung disease is alpha- 1 antitrypsin deficiency such as alpha- 1 antitrypsin deficiency has a mutation of the SERPINA1 gene. In another embodiment, the mutation of the SERPINA1 gene is E342K and V264E. In another embodiment, the lung disease is cystic fibrosis such as cystic fibrosis has a genetic mutation associated with cystic fibrosis. In some embodiments, the mutation is selected from the group consisting of AF5O8, G542X, N1303K, W1282X, E56K, G178R, S549R, K1060T, G1244E, P67L, E193K, G551D, A1067T, S1251N, R74W, L206W, G551S, G1069R, S1255P, D110E, R347H, D579G, R1070Q, D1270N, D110H, R352Q, S945L, R1070W, G1349D, R117C, A455E, S977F, F1074L, R117H, S549N, F1052V, and D1152H. In some embodiments, the mutation is selected from AF508, G542X, G551D, N1303K, and W1282X. In some embodiments, the genetic mutation is the AF508 mutation.

[0021] In another embodiment, the disease or disorder is a cervicovaginal disease or disorder. In some embodiments, the cervicovaginal disease or disorder is human immunodeficiency virus (HIV). In another embodiment, the cervicovaginal disease or disorder is human papillomavirus (HPV). In another embodiment, the cervicovaginal disease or disorder is a Mullerian anomaly. In another embodiment, the cervicovaginal disease or disorder is endometriosis.

[0022] In another embodiment, the disease or disorder is a disease or disorder of the nasal pathway. In another embodiment, the disease or disorder is a disease or disorder of the skin. In some embodiments, the skin disease or disorder is epidermolysis bullosa. In another embodiment, the skin disease or disorder is epidermolytic hyperkeratosis. In another embodiment, the skin disease or disorder is a polygenic skin disease. In some embodiments, the polygenic skin disease is systemic lupus, psoriasis, androgenic alopecia, atopic dermatitis, systemic sclerosis, vitiligo, alopecia areata, pemphigus vulgaris and foliaceus, or Sjogren’s syndrome.

[0023] In another embodiment, the disease or disorder is a gastrointestinal disease or disorder. In some embodiments, the gastrointestinal disease or disorder is oral carcinoma. In another embodiments, the gastrointestinal disease or disorder is Sjogren’s syndrome. In another embodiment, the gastrointestinal disease or disorder is colitis. In another embodiment, the gastrointestinal disease or disorder is inflammatory bowel disorder. In another embodiment, the disease or disorder is a corneal or ocular disease or disorder such as a retinal genetic disease. In some embodiments, the peptide selectively targets lung cells relative to macrophages.

[0024] In some embodiments, the peptide has a sequence identity of at least 95% of SEQ ID. 1-16. In further embodiments, the sequence identity is at least 98%.

[0025] In another aspect, the present disclosure provides conjugates comprising

(a) a peptide as provided above; and

(b) a hydrophobic group, wherein the hydrophobic group is covalently linked to the peptide.

[0026] In some embodiments, the hydrophobic group is a lipid. In further embodiments, the hydrophobic group is a fatty acid. In still further embodiments, the hydrophobic group is a long-chain fatty acid. In yet further embodiments, the fatty acid is an unsaturated fatty acid. In other embodiments, the fatty acid is a saturated fatty acid, such as myristic acid, palmitic acid, stearic acid, or arachidic acid. In some embodiments, the fatty acid is myristic acid. In some embodiments, the hydrophobic group and the peptide are covalently linked by an ester, an amide, a thioether, a carbamate, a carbonate, a urea, a thiocarbonate, a thiocarbamate, or a thiourea group.

[0027] In another aspect, the present disclosure provides compositions comprising:

(a) a peptide as provided above or a conjugate as provided above: and

(b) a lipid nanoparticle.

[0028] In some embodiments, the lipid nanoparticles comprise one or more different types of lipids. In some embodiments, the lipid nanoparticle comprises a cationic lipid. In further embodiments, the cationic lipid is an ionizable cationic lipid.

[0029] In some embodiments, the lipid nanoparticle comprises a phospholipid. In some embodiments, the lipid nanoparticle comprises a sterol, such as cholesterol. In some embodiments, the lipid nanoparticle comprises a polymer conjugated lipid, such as a polyethylene glycol conjugated lipid. In some embodiments, the lipid nanoparticle encapsulates a protein or a therapeutic agent. In some embodiments, the lipid nanoparticle encapsulates a protein. In other embodiments, the lipid nanoparticle encapsulates a therapeutic agent. In some embodiments, the therapeutic agent is a small molecule. In further embodiments, the therapeutic agent is a nucleic acid. In other embodiments, the therapeutic agent is a polypeptide or an antibody.

[0030] In another aspect, the present disclosure provides methods of treating a disease or disorder comprising administering to a patient in need thereof comprising administering to the patient a therapeutically effective amount of a composition as provided above.

[0031] In some embodiments, the disease or disorder is a lung disease or disorder. In some embodiments, the lung disease or disorder is chronic obstructive pulmonary disease. In another embodiment, the lung disease or disorder is respiratory syncytial virus. In another embodiment, the lung disease or disorder is influenza. In another embodiment, the lung disease is cytomegalovirus. In another embodiment, the lung disease is primary ciliary dyskinesia such as primary ciliary dyskinesia has a genetic mutation of the DNAI1 or DNAH5 genes. In another embodiment, the lung disease is alpha- 1 antitrypsin deficiency such as alpha- 1 antitrypsin deficiency has a mutation of the SERPINA1 gene. In another embodiment, the mutation of the SERPINA1 gene is E342K and V264E. In another embodiment, the lung disease is cystic fibrosis such as cystic fibrosis has a genetic mutation associated with cystic fibrosis. In some embodiments, the mutation is selected from the group consisting of AF5O8, G542X, N1303K, W1282X, E56K, G178R, S549R, K1060T, G1244E, P67L, E193K, G551D, A1067T, S1251N, R74W, L206W, G551S, G1069R, S1255P, D110E, R347H, D579G, R1070Q, D1270N, D110H, R352Q, S945L, R1070W, G1349D, R117C, A455E, S977F, F1074L, R117H, S549N, F1052V, and D1152H. In some embodiments, the mutation is selected from AF508, G542X, G551D, N1303K, and W1282X. In some embodiments, the genetic mutation is the AF508 mutation.

[0032] In another embodiment, the disease or disorder is a cervicovaginal disease or disorder. In some embodiments, the cervicovaginal disease or disorder is human immunodeficiency virus (HIV). In another embodiment, the cervicovaginal disease or disorder is human papillomavirus (HPV). In another embodiment, the cervicovaginal disease or disorder is a Mullerian anomaly. In another embodiment, the cervicovaginal disease or disorder is endometriosis.

[0033] In another embodiment, the disease or disorder is a disease or disorder of the nasal pathway. In another embodiment, the disease or disorder is a disease or disorder of the skin. In some embodiments, the skin disease or disorder is epidermolysis bullosa. In another embodiment, the skin disease or disorder is epidermolytic hyperkeratosis. In another embodiment, the skin disease or disorder is a polygenic skin disease. In some embodiments, the polygenic skin disease is systemic lupus, psoriasis, androgenic alopecia, atopic dermatitis, systemic sclerosis, vitiligo, alopecia areata, pemphigus vulgaris and foliaceus, or Sjogren’s syndrome.

[0034] In another embodiment, the disease or disorder is a gastrointestinal disease or disorder. In some embodiments, the gastrointestinal disease or disorder is oral carcinoma. In another embodiments, the gastrointestinal disease or disorder is Sjogren’s syndrome. In another embodiment, the gastrointestinal disease or disorder is colitis. In another embodiment, the gastrointestinal disease or disorder is inflammatory bowel disorder. In another embodiment, the disease or disorder is a corneal or ocular disease or disorder such as a retinal genetic disease.

[0035] In another aspect, the present disclosure provides methods of delivering a compound to an epithelial cell in a patient comprising administering to the patient a composition as provided above. In some embodiments, the compound is a nucleic acid. In some embodiment, the nucleic acid is a gene editing system.

[0036] In some embodiments, the epithelial cell is a lung epithelial cell. In another embodiment, the epithelial cell is a gastrointestinal epithelial cell. In another embodiment, the epithelial cell is a corneal or ocular epithelial cell. In another embodiment, the epithelial cell is a cervicovaginal epithelial cell. In another embodiment, the epithelial cell is a nasal epithelial cell. In another embodiment, the epithelial cell is a hepatic epithelial cell.

[0037] In another aspect, the present disclosure provides methods of selectively delivering a compound to organ cells in a patient comprising administering to the patient a composition as provided above. In some embodiments, the organ cells are skin cells, lung cells, liver cells, cornea cells, cervical cells, vaginal cells, nasal cells, or gastrointestinal cells. In some embodiments, the lung cells are bronchial epithelial cells. In some embodiments, the composition is administered via inhalation. In other embodiments, the composition is administered systemically.

[0038] Other objects, features, and advantages of the present disclosure will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating specific embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

[0039] The following drawings 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 drawings in combination with the detailed description of specific embodiments presented herein. Throughout the Figures described below, certain lipid nanoparticle formulations may be denoted by multiple names. See Table 7.

[0040] FIG. 1 illustrates the difference in the (right) presently described methods for identifying mucosal membrane penetrating or cell penetrating peptides compared to (left) those known in the art. The presently described methods are more clinically relevant, as the phage library is introduced to CF bronchial epithelial cells from human patients with the AF508 mutation.

[0041] FIG. 2 is an illustrative representation of a selection strategy to arrive at the peptides disclosed herein. At left, cells from a patient with CF with the AF508 mutation are collected. The cells from seven such patients were pooled and the CX7C phage library was introduced at ALL At right is illustrated the screening strategy using a cysteine constrained random 7-amino acid peptide T7 phage display library (CX7C) against differentiated primary human bronchial epithelial cells (pHBECs) through iterative, high-throughput screening for a total of five rounds. In some instances, iterative, high-throughput screening was carried out for a total of four rounds. Image made with BioRender.

[0042] FIG. 3 shows the enrichment over four rounds of a CX7C phage library for enhanced CF pHBEC uptake. C_out represents the concentration of phage collected after each round and Cm is the initial input phage concentration. Phage were added at 1000 viral genomes/cell and incubated for 16 hours at 37°C for round 1 and 1 hour at 37°C for round 4. WT = wild-type (peptide-less phage).

[0043] FIG. 4 provides, at left, the physicochemical properties of sequences that were selected after four rounds to undergo further cell-uptake validation studies. Both charge and grand average of hydropathicity (GRAVY) score at pH 7 were calculated as described previously (Leal 2020). At right: validation of selected clones for enhanced CF pHBEC uptake. Co represents the concentration of phage collected after each round and Cm is the initial input phage concentration, a; significant difference from all controls, b: significant difference from Naive library (phage library without selection) only. Phage were incubated against pHBECs at ALI for 1 hour at 37°C. Data was pooled from two separate experiments (n=6; Kruskal -Wallis test, uncorrected Dunn’s)

[0044] FIG. 5 shows the cell uptake data in pHBEC and MO non-polarized macrophage cells for phage displaying sequences identified after four iterative rounds of selection for mucus- and cell-penetrating properties, as described above and in the Examples section. MO (differentiated from THP-1 cells). % Phage uptake: [(plaque forming units (pfu)/pL collected x volume collected)/(input pfu/pL x volume input)] x 100 . * denotes p- value <0.05 and *** denotes p-value <0.0005. Phage were incubated against either pHBECs or M0 for 1 hour at 37°C. As can be seen, Clones 14 and 26 demonstrate significantly higher uptake in primary CF cells (pHBECs) than a human macrophage cell line (M0).

[0045] FIG. 6 illustrates the effect of scrambling the sequence of the mucosal membrane and cell penetrating peptides identified after four rounds of iterative selection as described in FIG. 6. C_out represents the concentration of phage collected after each round and Cm is the initial input phage concentration. * denotes p-value <0.05. Phage were incubated against pHBECs for 1 hour at 37°C. Sequence 9 (SEQ ID NO: 5) and sequence 26 (SEQ ID NO: 8) demonstrated improved uptake in primary CF cells versus their scrambled counterparts.

[0046] FIG. 7 shows the optimized enrichment of sequences after five rounds of iterative selection. C_out represents the concentration of phage collected after each round and G_n is the initial input phage concentration. Phage were added at 1000 viral genomes/cell (vg/cell) and incubated for 16 hours at 37°C for round 1 and 1 hour at 37°C for round 5. Data represents change in C_Out/Ci_n for three separate replicates from the first to last round of selection.

[0047] FIG. 8 shows the significant shift in weighted mean net charge and weighted mean hydropathicity of peptides identified after one round of selection and peptides identified after five rounds of selection. Peptides identified after five rounds of selection had a more positive net charge and a more negative GRAVY score, which corresponds with increased hydrophilicity, in comparison to the peptides identified after one round of selection. Each dot represents the weighted mean value for net charge or for GRAVY score from each replicate (n=30).

[0048] FIG. 9A-9D show the physicochemical properties of the top 30 most frequent peptide sequences from each replicate (n=3; total of 90 sequences analyzed per round). Peptide sequences were ranked 1-30 based on frequency. Graphed are the mean values for each ranked peptide sequence (n=3). FIG. 9A shows the net charge of the pooled top 30 (n=3) sequences. FIG. 9B shows the GRAVY score of the pooled top 30 (n=3 ) sequences. The physicochemical properties of these top 30 most frequent peptide sequences demonstrate that the optimized selection strategy contrast with previously published data in that more positively charged and hydrophilic peptide sequences were identified and are disclosed herein. (Leal et al., 2020). FIG. 9C shows multiple sequence alignment visual representation using Seq2Logo for the top 30 sequences of each replicate after 5 rounds of selection (Thomsen & Nielsen, 2012). FIG. 9D shows the consensus sequence obtained from earlier four-selection round panning experiments.

[0049] FIG. 10 shows enrichment of libraries according to earlier biopanning protocols that were tested en route to the optimized protocol; results for the optimized protocol are shown in FIG. 8.

[0050] FIG. 11 shows the enrichment of select peptide sequences after each round of selection in a five- sei ection round biopanning experiment. Enrichment data is shown for peptide-displaying clones that were identified to be present in the top 10 most abundant sequences across all three replicates. For each clone peptide sequence, the enrichment is provided for round 1 to round 5 (left to right). For each round of panning, the % of total sequences sampled represents the (average peptide frequency count/total # of sequences obtained from NGS data) x 100.

[0051] FIG. 12 shows the validation results for the most abundant peptides after five rounds of selection identified as described above and in the Examples section. Physicochemical data for the clones assayed in this figure are provided in Table 2. C_out represents the output concentration of phage collected from ALI cells after Ih and Cin is the initial input phage concentration. Controls include WT, a wild-type phage which lacks peptides on its capsid surface; Naive library, which is a mixture of randomized CX7C 7-mer peptide presenting phage clones; and CPS, a positive mucus-penetrating clone displaying the peptide CPSSSREKC (net charge 1 and GRAVY score of -1.2). One-way ANOVA (Kruskal- Wallis test; uncorrected Dunn’s); a,b p<0.05.

[0052] FIG. 13 shows the results of validation of clones identified from five-selection round biopanning (Clones A-F) in comparison to clones identified from four-selection round biopanning (clones 14 and 26) and controls (WT: peptide-less phage; NL: phage displaying randomized CX7C sequence; CPS: phage displaying known mucus penetrating peptide). Data analysis: One-way ANOVA (Kruskal-Wallis test; uncorrected Dunn's). Statistically significant compared to clones: #: A, F, WT, NL, CPS, 26 ##: F, WT, NL, CPS ###: WT, NL, CPS ####: NL. CPS denotes CPSSSREKC (SEQ ID NO: 17).

[0053] FIG. 14 shows the characterization of peptide-LNP compositions during optimization experiments. Nomenclature: Moderna_%peptide-lipid. The peptide-LNP comprising 6.25% peptide exhibited homogeneous size with a small polydispersity index.

[0054] FIG. 15 shows the mRNA transfection efficiency of peptide-LNP formulations with varying peptide percentage. Nomenclature: Modema_%peptide-lipid. At top, peptide content of 25% and 50% showed the lowest transfection efficiency. At bottom, the peptide- LNP comprising 6.25% peptide demonstrated the highest transfection efficiency and was selected for further use. GFP fluorescence was analyzed 24 hours post-transfection in HEK293T cells. 700 ng of GFP mRNA were dosed to HEK-293 cells using LNP formulations presently disclosed peptide-lipids content ranging from (top) 0% (Moderna formulation) to 50% (Modema_50) or (bottom) 0% (Moderna formulation) to 25% (Modema_25). After 24 hours, fluorescence was imaged by microscopy.

[0055] FIG. 16 shows the characterization data for control (Spikevax, CPS, PEG) LNPs and peptide-LNPs comprising presently disclosed peptides (Peptide C). The formulation of each LNP control and peptide-LNPs may be found in Table 6 (see also Table 7). “Peptide C” denotes an LNP formulation comprising a peptide with sequence CTSTRKKQC (SEQ ID NO: 11); “CPS” denotes an LNP formulation comprising a peptide with sequence CPSSSREKC (SEQ ID NO: 17). The left-most plot in FIG. 16 shows the dynamic light scattering results for size (in nm) and polydispersity index (PDI) (N=3). The plot in the center of FIG. 16 shows the zeta potentials of the LNP formulations. Zeta potentials were determined using the Zetasizer Nano (N=3) according to methods known in the art. The right-most plot of FIG. 16 shows the encapsulation efficiency of the LNP formulations. Encapsulation efficiencies were determined by modified Ribogreen assay (N=2) according to methods known in the art.

[0056] FIG. 17 provides evidence that peptide-LNPs comprising presently disclosed peptides can successfully deliver Nluc mRNA to HBECs. 450 ng Nluc mRNA was administered to the apical side of differentiated primary HBECs (N=4 per treatment group). Readout was collected at the 48hr timepoint. Following incubation, bioluminescence was measured by plate reader using methods known in the art. * denotes p- value <0.05, ** denotes p-value <0.01. Data analysis: one-way ANOVA (Tukey’s multiple comparisons; single pooled variance). The formulation of each LNP control and peptide-LNPs may be found in Table 6. CTS denotes CTSTRKKQC (SEQ ID NO: 11); CPS denotes CPSSSREKC (SEQ ID NO: 17). Formulation “Moderna” corresponds with formulation “Spikevax” as used elsewhere in this application; formulation “CTS” corresponds with formulation “Peptide C” as used elsewhere in this application (see Table 7).

[0057] FIG. 18 provides evidence that peptide-LNPs comprising presently-disclosed peptides have lower uptake by macrophages. THP-1 cells were seeded on 24- well plate and grown in 15 ng/mL PMA containing media for 48 hours, followed by 24 hours without PMA for differentiation. 450 ng Nluc mRNA was administered to the apical side of THP-1 derived macrophages. Readout was collected at the 48hr timepoint. Following incubation, bioluminescence was measured by plate reader using methods known in the art. * denotes p- value <0.05, ** denotes p-value <0.01, *** denotes p-value <0.005. Data analysis: one-way ANOVA (Tukey’s multiple comparisons; single pooled variance). The formulation of each LNP control and peptide-LNP may be found in Table 6. CTS denotes CTSTRKKQC (SEQ ID NO: 11); CPS denotes CPSSSREKC (SEQ ID NO: 17).

[0058] FIG. 19 shows the IVIS imaging at 24-hr post dose of (left image) control LNP (Moderna) and (right image) a peptide-LNP comprising a presently disclosed peptide (Moderna-CTS). CTS denotes LNP comprising CTSTRKKQC (SEQ ID NO: 11), formulated according to Table 6.

[0059] FIG. 20 shows additional IVIS imaging data at a 24hr post dose image of control (PBS, Moderna LNP, CPS peptide-LNP) and LNPs comprising presently disclosed peptide (CTS). 40 uL of either PBS or LNPs formulated at 20 ng/uL were delivered intratracheally to balb/c mice (6-8 weeks old, n=4). PBS sample contains no LNP and Moderna denotes a peptide-less LNP control. CTS denotes LNP comprising CTSTRKKQC (SEQ ID NO: 11); CPS denotes LNP comprising CPSSSREKC (SEQ ID NO: 17). The formulation of each LNP control and peptide-LNP may be found in Table 6.

[0060] FIG. 21 shows the quantification of the data presented in the imaging data of FIG. 20. Average Radiance (p/s/cm²/sr) was calculated for each lung (N=4). As for FIG. 20, PBS sample contains no LNP and Modema denotes a peptide-less LNP control. CTS denotes an LNP comprising peptide CTSTRKKQC (SEQ ID NO: 11); CPS denotes LNP comprising peptide CPSSSREKC (SEQ ID NO: 17). The formulation of each LNP control and peptide- LNP may be found in Table 6. See also Table 7. One-way ANOVA, multiple comparisons (test: Tukey) ** denotes p<0.005, *** denotes p<0.0005.

[0061] FIG. 22 shows the amino acid characteristics for the top performing identified peptides. At top is shown the hydropathic characteristics of clone C. At bottom is shown the hydropathic characteristics of clone 14. Analysis performed using www.pepcalc.com.

[0062] FIGS. 23A-23E provide evidence that LNPs comprising peptides disclosed herein are able to be incorporated into multiple classes of LNPs. FIGS. 23A-23C: LNP characterization for all LNP classes. (FIG. 23A) LNP sizes with PDI values, (FIG. 23B) encapsulation efficiencies, and (FIG. 23C) zeta potential measurements. (FIG. 23D) LNPs were incubated on top of Calu-3 ALI cells for 24 hours prior to determining the percentage of GFP positive cells and (FIG. 23E) live cells by flow cytometry. Ordinary one-way ANOVA. Tukey’s multiple comparison’s test with single pooled variance. *p<0.0332; **p<0.0021, ***p<0.0002, ****p<0.0001.

[0063] FIG. 24 shows data related to LNP GFP transfection in undifferentiated HBECs. Undifferentiated pHBECs were treated with either PBS, a peptide-less control LNP formulation (Spikevax) or Peptide C - an LNP composition comprising a presently disclosed peptide. After 24 hours, cells were collected and the percentage of GFP positive cells was determined with flow cytometry. (N=3).

[0064] FIGS. 25A-25C illustrate and show data related to intratracheal administration of Cre mRNA LNPs in vivo. (FIG. 25 A, FIG. 25B) Flow cytometry analysis of tdTomato + cells in various cell types in the lungs following intratracheal delivery of Cre mRNA to Ai9 mice. (FIG. 25 A) (N=3; mean + SD; Multiple unpaired t-tests (Holm-Sidak multiple comparisons test); *p <0.001) (FIG. 25B) (N=3; mean + SD); Unpaired t-test: *p = 0.0300). (FIG. 25C) Schematic of Cre recombinase gene editing. Created with BioRender.

[0065] FIGS. 26A-26C provide further insight into the role of peptide-lipids comprising a presently disclosed peptide and the mechanism of uptake of presently disclosed peptide- LNP compositions. (FIG. 26A) Schematic of competition assays described in Example 8. (FIG. 26B) For the competition experiments described, for example, in Example 8, fully differentiated pHBECs were treated with 50 pL of peptide-lipid conjugate comprising a presently disclosed peptide (1 mg/mL) for 10 minutes at 37°C prior to treating cells with 15 pL of a peptide-less NLuc LNP formulation (Spikevax/Modema; See Table 6 and Table 7) or an NLuc peptide-LNP composition of the present disclosure (Spikevax CTS/Moderna_CTS_6.25/CTS/See Tables 6 and 7), each formulated at an mRNA concentration of 40 ng/ pL. Following 48 hours of incubation at 37°C, bioluminescence was measured. (FIG. 26C) In a separate macropinocytosis inhibitor study, 50 pL of the macropinocytosis inhibitor EIPA (25 pM) was delivered, and cells were incubated for 30 minutes at 37°C prior to treatment of cells with 15 pL of Nluc peptide-LNP composition of the present disclosure (formulated at an mRNA concentration of 35 ng pL). Following 48 hours of incubation at 37°C, bioluminescence was measured. **p<0.0021; ****p<0.0001

[0066] FIG. 27 shows that the optimized biopanning procedure described in the Examples led to greater CX7C library enrichment.

BRIEF DESCRIPTION OF THE SEQUENCES LISTED

The sequences listed below provides amino acid sequences of biopanned peptides, some of which exhibit improved penetration of primary human bronchial epithelial cells.

SEQ ID NO: 1

>Clone 1

CSRSTASTC

SEQ ID NO: 2

>Clone 2

CPSPTRRKC

SEQ ID NO: 3

>Clone 3

CKKSSTRQC

SEQ ID NO: 4

>Clone 7

CGPSSKRKC

SEQ ID NO: 5

>Clone 9

CSKGPKQIC

SEQ ID NO: 6

>Clone 14

CERSSKSSC

SEQ ID NO: 7

>Clone 18

CDRQPRKRC

SEQ ID NO: 8 > Clone 26

CGGKSGGRC

SEQ ID NO: 9

> Clone A

CTPKRSRAC

SEQ ID NO: 10

> Clone B

CTRPTRSKC

SEQ ID NO: 11

> Clone C

CTSTRKKQC

SEQ ID NO: 12

> Clone D

CPAPRGKRC

SEQ ID NO: 13

> Clone E

CAPSKRNRC

SEQ ID NO: 14

>Clone F

CLSPTGKAC

SEQ ID NO: 15

>Clone G

CRSKPGRKC

SEQ ID NO: 16 >Clone H

CRKRPSSKC

SEQ ID NO: 17

>Control peptide (CPS) CPSSSREKC

DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

[0067] Provided herein are, in one embodiment, peptides for targeting cells, such as lung cells, tissues, or organs. In some embodiments, the peptides have mucus barrier or mucosal membrane penetrating or cell penetrating properties. In some embodiments, the presently disclosed peptides penetrate said membranes via direct penetration. Without wishing to be bound by any theory, it is believed that the presently disclosed peptides penetrate said membranes via endocytosis or a receptor-mediated mechanism. In some embodiments, the peptides disclosed herein may be attached, conjugated, or coupled with a wide variety of therapeutics.

[0068] Also disclosed herein are peptide- lipid nanoparticle compositions. The peptidelipid nanoparticle compositions (peptide-LNPs) disclosed herein may, in some embodiments, allow targeted delivery of cargo, including therapeutic compounds or cargo relevant for geneediting applications, to cells. The peptides or peptide-LNPs may have the benefit of having a small size, having cell targeting capabilities, having cell-membrane penetrating capabilities, having mucus barrier or mucosal membrane penetrating capabilities, or possessing low immunogenicity or low immunogenicity risk. The peptides disclosed herein may be used with a wide variety of therapeutic modalities to localize a therapeutic compound to an organ, tissue, or cell type in vivo or in vitro. The peptides or compositions disclosed herein may be used to improve transfection or gene delivery to an organ, tissue, or cell type. The present compositions in some embodiments have favorable uptake in cells, particularly in lung cells. In some embodiments, the present compositions have favorable uptake in epithelial cells. In some embodiments, the present compositions have favorable uptake in basal stem cells, and as such may be useful for the delivery of cargo for gene-editing applications. In some embodiments, the present compositions have enhanced uptake in target organs, tissues, or cells compared to macrophages, which provides an improvement over the known art. Methods of treating diseases or disorders using such compositions are also disclosed herein. Further details of these aspects and more are provided below.

I. Biopanning

[0052] Peptides as disclosed in the present application may be identified through the use of phage display libraries. Phage display libraries genetically engineered to display peptides on the surface of bacteriophage can be generated by inserting random oligonucleotides into cDNAs encoding a phage surface protein, generating collections of phage particles displaying unique peptides in many permutations. Phage display is a technique in which a phage library expresses, for example, a set of random peptide sequences of defined length, incorporated into a phage coat protein and peptide sequences that bind to a target molecule, cell, for example primary human bronchial epithelial cell, tissue, or organ were identified by incubating a phage display library with the target and selecting for bound peptides. Unbound phage is washed away and bound phage eluted and collected. The collected phage may be amplified and taken through further binding/amplification cycles to enrich the pool of peptides for those that selectively and/or specifically bind to or penetrate the target. In some embodiments, the phage is collected from whole cell lysate. In some embodiments, the lysed cells may first be centrifuged or spun down, and the phage displaying selected peptides is collected from the supernatant. Thus, in some embodiments, biopanning methods may select for internalized phage. The collected phage may be amplified and taken through 1 binding or penetration/amplification cycle, 2 binding or penetration/amplification cycles, 3 binding or penetration/amplification cycles, 4 binding or penetration/amplification cycles, 5 binding or penetration/amplification cycles, 6 binding or penetration/amplification cycles, 7 binding or penetration/amplification cycles, 8 binding or penetration/amplification cycles, 9 binding or penetration/amplification cycles, or 10 binding or penetration/amplification cycles. Selection pressure may be applied in round 1, in round 2, in round 3, in round 4, in round 5, in round 6, in round 7, in round 8, in round 9, in round 10, or any combination thereof. For example, selection pressure may be applied in round 1, in round 2, and in round 3. With each cycle, the proportion of phage in the pool that contains targeting peptides for the target of interest is enriched. After several cycles, individual phage clones may be characterized by DNA sequencing to identify the targeting peptide sequences (biopanning, see for example, US20050187161 as well as Pasqualini and Ruoslahti, Nature 380:364-66. 1996; Arap et al., Science 279:377-80, 1998).

[0053] In some embodiments, particular peptides capable of penetrating a mucosal membrane or a cell membrane are provided. Such peptides may have a length of 5 amino acid residues, 6 amino acid residues, 7 amino acid residues, 8 amino acid residues, 9 amino acid residues, 10 amino acid residues, 11 amino acid residues, 12 amino acid residues, 13 amino acid residues, 14 amino acid residues, 15 amino acid residues, 16 amino acid residues, 17 amino acid residues, 18 amino acid residues, 19 amino acid residues, 20 amino acid residues, or any range derivable therein. In some embodiments, the peptide may have a length of between 9 amino acid residues and 15 amino acid residues. [0054] Peptides may be characterized by the net charge of the peptide at pH 7. The net charge of a peptide according to the present disclosure may be positive or negative. In preferred embodiments, the net charge of a mucosal membrane- and/or cell membrane- penetrating peptide according to the present disclosure is positive. In some embodiments, the mucosal membrane- and/or cell membrane- penetrating peptide has a net positive charge from about 1 to about 5. In some embodiments, the mucosal membrane- and/or cell membrane- penetrating peptide has a net positive charge of about 1.0, about 1.5, about 2.0, about 2.5, about 3.0, about 3.5, about 4.0, about 4.5, about 5.0, or any range derivable therein. In some embodiments, the mucosal membrane- and/or cell membrane- penetrating peptide has a net positive charge from about 1.5 to about 3. In some embodiments, the mucosal membrane- and/or cell membranepenetrating peptide has a net positive charge of 1.9. In some embodiments, the mucosal membrane- and/or cell membrane- penetrating peptide has a net positive charge of 2.9. In some embodiments, the mucosal membrane- and/or cell membrane- penetrating peptide has a net positive charge of 3.9.

[0055] The mucosal membrane- and/or cell membrane- penetrating peptides described herein may comprise amino acid residues that are charged or neutral at neutral pH. For example, the peptides disclosed herein may comprise one or more independently selected amino acid residue(s) that are positively charged at neutral pH. Non-limiting examples of amino acid residues that are positively charged at neutral pH are arginine, lysine, and histidine. In some embodiments, the presently disclosed peptides comprise one amino acid residue that is positively charged at neutral pH. In some embodiments, the presently disclosed peptides comprise two amino acid residues that are positively charged at neutral pH. In some embodiments, the presently disclosed peptides comprise three amino acid residues that are positively charged at neutral pH. In some embodiments, the presently disclosed peptides comprise four amino acid residues that are positively charged at neutral pH.

[0056] Peptides and proteins may also be characterized by a grand average of hydropathicity index (GRAVY) score. Each amino acid has been assigned a hydropathic index on the basis of its hydrophobicity' and charge characteristics. They are: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine/cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine (-0.4); threonine (-0.7); serine (-0.8); tryptophan (-0.9); tyrosine (-1.3); proline (-1.6); histidine (-3.2); glutamate (-3.5); glutamine (-3.5); aspartate (-3.5); asparagine (-3.5); lysine (-3.9); and arginine (-4.5). The use of the hydropathic amino acid index in conferring interactive biological function on a protein is understood in the art (Kyte and Doolittle, J. Mol. Biol. 157:105-132, 1982). A GRAVY score is calculated as the sum of the hydropathy values for all the amino acids in a peptide or protein divided by the total number of residues. A negative GRAVY score indicates that the peptide is polar (hydrophilic) and a positive value indicates that the peptide is non-polar (hydrophobic). In some embodiments, a mucosal membrane- and/or cell membrane- penetrating peptide according to the present disclosure has a negative GRAVY score. In some embodiments, the GRAVY score of a mucosal membrane- and/or cell membrane- penetrating peptide according to the present disclosure is about -0.2, about -0.4, about -0.6, about -0.8, about -1.0, about -1.2, about -1.4, about -1.6, about -1.8, about -2.0, about -2.2, about -2.4, about -2.6, about -2.8, about -3.0, about -3.2, about -3.4, about -3.6, about -3.8, about -4.0, or any range derivable therein. In some embodiments, the present disclosure provides mucosal membrane- and/or cell membrane- penetrating peptides with GRAVY scores that are less than -1. In some embodiments, the present disclosure provides mucosal membrane- and/or cell membranepenetrating peptides with GRAVY scores that are less than -2. In some embodiments, the present disclosure provides mucosal membrane- and/or cell membrane- penetrating peptides with GRAVY scores that are less than -3. It is known that in certain instances, certain amino acids may be substituted for other amino acids having a similar hydropathic index or score and still retain a similar biological activity. In making changes based upon the hydropathic index, in certain embodiments the substitution of amino acids whose hydropathic indices are within ±2 is included, while in other embodiments amino acid substitutions that are within ±1 are included, and in yet other embodiments amino acid substitutions within ±0.5 are included.

[0057] In some embodiments, the presently disclosed mucosal membrane- and/or cell membrane- penetrating peptides comprise specific amino acid residues. For example, in some embodiments, the mucosal membrane- and/or cell membrane- penetrating peptides disclosed herein comprise at least one serine residue, at least one proline residue, or at least one arginine residue, or any combination thereof.

[0058] In some embodiments, the presently disclosed mucosal membrane- and/or cell membrane- penetrating peptides comprise at least one serine residue. The presently disclosed mucosal membrane- and/or cell membrane- penetrating peptides, in some embodiments, comprise one serine residue, two serine residues, three serine residues, four serine residues, or five serine residues. In some embodiments, the presently disclosed mucosal membrane- and/or cell membrane- penetrating peptides comprise one serine residue. In some embodiments, the presently disclosed mucosal membrane- and/or cell membrane- penetrating peptides comprise two serine residues. In some embodiments, the presently disclosed mucosal membrane- and/or cell membrane- penetrating peptides comprise three serine residues. In some embodiments, the presently disclosed mucosal membrane- and/or cell membrane- penetrating peptides comprise four serine residues.

[0059] In some embodiments, the presently disclosed mucosal membrane- and/or cell membrane- penetrating peptides comprise at least one proline residue. The presently disclosed mucosal membrane- and/or cell membrane- penetrating peptides, in some embodiments, comprise one proline residue, two proline residues, three proline residues, four proline residues, or five proline residues. In some embodiments, the presently disclosed mucosal membrane- and/or cell membrane- penetrating peptides comprise one proline residue. In some embodiments, the presently disclosed mucosal membrane- and/or cell membrane- penetrating peptides comprise two proline residues. In some embodiments, the presently disclosed mucosal membrane- and/or cell membrane- penetrating peptides comprise three proline residues. In some embodiments, the presently disclosed mucosal membrane- and/or cell membranepenetrating peptides comprise four proline residues.

[0060] In some embodiments, the presently disclosed mucosal membrane- and/or cell membrane- penetrating peptides comprise at least one arginine residue. The presently disclosed mucosal membrane- and/or cell membrane- penetrating peptides, in some embodiments, comprise one arginine residue, two arginine residues, three arginine residues, four arginine residues, or five arginine residues. In some embodiments, the presently disclosed mucosal membrane- and/or cell membrane- penetrating peptides comprise one arginine residue. In some embodiments, the presently disclosed mucosal membrane- and/or cell membrane- penetrating peptides comprise two arginine residues. In some embodiments, the presently disclosed mucosal membrane- and/or cell membrane- penetrating peptides comprise three arginine residues. In some embodiments, the presently disclosed mucosal membrane- and/or cell membrane- penetrating peptides comprise four arginine residues.

[0061] In some embodiments of the present invention, the disclosure provides a peptide comprising an amino acid sequence selected from the group consisting of Clone 1, Clone 2, Clone 3, Clone 7, Clone 9, Clone 14, Clone 18, Clone 26, Clone A, Clone B, Clone C, Clone D, Clone E, or Clone F (SEQ ID NOS: 1-16), wherein said peptide demonstrates mucosal membrane or cell membrane penetrating properties. In another embodiment of the present invention, a peptide according to the present disclosure comprises the amino acid sequence CTSTRKKQC (SEQ ID NO: 11). In another embodiment of the present invention, a peptide as disclosed herein comprises the amino acid sequence CERSSKSSC (SEQ ID NO: 6). Peptides comprising the sequences listed above may be used to form peptide conjugates, including peptide- lipid conjugates such as peptide-myristic acid conjugates, as described elsewhere in the present application. Peptides comprising the sequences listed above, functional equivalents thereof, or peptide conjugates of either the peptides or their functional equivalents may be incorporated into delivery systems for therapeutic cargo, such as lipid nanoparticles. Further details of the latter aspect are provided below. Thus, the present invention provides, through use of the sequences identified above or other peptides of the present disclosure, targeted delivery of therapeutic cargo, such as mRNA to lung cells.

[0062] The mucosal membrane- and cell membrane- penetrating peptides disclosed herein may penetrate the cell membrane in a variety of ways. In some embodiments, the peptides disclosed herein may penetrate the mucosal membrane or cell membrane by being transported via endocytosis. In some embodiments, the peptides disclosed herein may directly penetrate the plasma membrane. In some embodiments, the peptides disclosed herein exhibit uptake or penetration into certain types of cells, such as epithelial cells. In some embodiments, the peptides disclosed herein exhibit improved or selective uptake or penetration into certain types of cells, such as epithelial cells. In some embodiments, the peptides disclosed herein exhibit improved or selective uptake or penetration into certain types of cells, such as epithelial cells, in comparison to endothelial cells or immune cells. In some embodiments, the presently disclosed peptides exhibit uptake into basal epithelial cells.

[0063] Peptides disclosed herein may be functionally equivalent to any peptides described herein. Functionally equivalent mucosal membrane- and/or cell membranepenetrating peptides amino acid sequences include those with amino acid substitutions made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the residues involved. For example: nonpolar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, and methionine; polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine; positively charged (basic) amino acids include arginine, lysine, and histidine; and negatively charged (acidic) amino acids include aspartic acid and glutamic acid. The amino acid residues of peptides of the present disclosure may be altered to facilitate synthesis. For example, motifs or fragments present in the peptides disclosed herein may be incorporated into longer sequences which are more easily synthesized, isolated, or produced. As another non-limiting example, alteration of presently described peptides comprising L amino acids may be altered to the corresponding D amino acid form to minimize proteolytic degradation.

[0064] While the desired peptide amino acid sequences described can be chemically synthesized (see, e.g., “Proteins: Structures and Molecular Principles” (Creighton, ed., W. H. Freeman & Company, New York, N.Y., 1984)), large polypeptides sequences may advantageously be produced by recombinant DNA technology using techniques well-known in the art for expressing nucleic acids containing a nucleic acid sequence that encodes the desired peptide. Peptides disclosed herein may be synthesized using solid-phase peptide synthesis techniques that are known in the art (see e.g., Coin, et al., Nat Protoc 2, 3247-3256, 2007). Such methods can be used to construct expression vectors containing peptide encoding nucleotide sequences and appropriate transcriptional and translational control signals. These methods include, for example, in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination (see, e.g., “Molecular Cloning, A Laboratory Manual”, supra, and “Current Protocols in Molecular Biology”, supra). Alternatively, RNA and/or DNA encoding desired peptide encoding nucleotide sequences may be chemically synthesized using, for example, synthesizers (see, e.g., “Oligonucleotide Synthesis: A Practical Approach” (Gait, ed., IRL Press, Oxford, United Kingdom, 1984)).

[0065] A variety of host-expression vector systems may be utilized to express peptide encoding nucleotide sequences. When the desired peptide or polypeptide is soluble, or a soluble derivative, the peptide or polypeptide can be recovered from the host cell culture, i.e. , from the host cell in cases where the peptide or polypeptide is not secreted, and from the culture media in cases where the peptide or polypeptide is secreted by the host cell. However, suitable expression systems also encompass engineered host cells that express the desired polypeptide, peptide or functional equivalents anchored in the cell membrane. Purification or enrichment of the desired peptide from such expression systems can be accomplished using appropriate detergents and lipid micelles, and methods well-known to those skilled in the art. Furthermore, such engineered host cells themselves may be used in situations where it is desired not only to retain the structural and functional characteristics of the peptide, but to assess biological activity, e.g., in certain drug screening assays.

[0066] In certain applications, transient expression systems are desired. However, for long-term, high-yield production of recombinant proteins or peptides, stable expression is generally preferred. For example, cell lines that stably express the desired protein, polypeptide, peptide, or fusion protein may be engineered. Rather than using expression vectors that contain viral origins of replication, host cells can be transformed with DNA controlled by appropriate expression control elements (e.g., promoter, enhancer sequences, transcription terminators, polyadenylation sites, etc.), and a selectable marker. Following the introduction of the foreign DNA, engineered cells are allowed to grow for about 1-2 days in an enriched media, and then switched to a selective media. The selectable marker in the recombinant plasmid confers resistance to the selection and allows cells to stably integrate the plasmid into their chromosomes and grow to form foci, which in turn can be cloned and expanded into cell lines. This method may advantageously be used to engineer cell lines that express the desired gene products or portions thereof. Such engineered cell lines may be particularly useful in screening and evaluation of compounds that affect the endogenous activity of the desired protein, polypeptide or peptide.

[0067] A number of selection systems may be used, including, but not limited to, the herpes simplex virus thymidine kinase (Wigler et al. , Cell 11:223-232, 1977), hypoxanthine - guanine phosphoribosyltransferase (Szybalska and Szybalski, Proc. Natl. Acad. Sci. USA 48:2026-2034, 1962), and adenine phosphoribosyltransferase (Lowy et al. , Cell 22:817-823, 1980) genes, which can be employed in tk-, hgprt- or aprt- cells, respectively. Anti-metabolite resistance can also be used as the basis of selection for the following genes: dihydrofolate reductase (dhfr), which confers resistance to methotrexate (Wigler et al., Proc. Natl. Acad. Sci. USA 77:3567-3570, 1980, and O'Hare et al., Proc. Natl. Acad. Sci. USA 78:1527-1531, 1981); guanine phosphoribosyl transferase (gpt), which confers resistance to mycophenolic acid (Mulligan and Berg, Proc. Natl. Acad. Sci. USA 78:2072-2076, 1981); neomycin phosphotransferase (neo), which confers resistance to the aminoglycoside G-418 (Colbere- Garapin et al., J. Mol. Biol. 150:1-14, 1981); and hygromycin B phosphotransferase (hpt), which confers resistance to hygromycin (Santerre et al., Gene 30:147-156, 1984).

[0068] Host cells/expression systems that may be used for purpose of providing compositions to be used in the disclosed methods include, but are not limited to, microorganisms such as bacteria (e.g., E. coli, B. subtilis) transformed with a recombinant bacteriophage DNA, plasmid DNA, or cosmid DNA expression vector containing a desired peptide encoding nucleotide sequence; yeast (e.g., Saccharomyces cerevisiae, Pichia pastoris) transformed with a recombinant yeast expression vector containing a desired peptide encoding nucleotide sequence; insect cell systems infected with a recombinant virus expression vector (e.g., baculovirus) containing a desired peptide encoding nucleotide sequence; plant cell systems infected with a recombinant virus expression vector (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV), or transformed with a recombinant plasmid expression vector (e.g., Ti plasmid), containing a desired peptide encoding nucleotide sequence; or mammalian cell systems (e.g., COS, CHO, BHK, 293, 3T3) harboring a recombinant expression construct containing a desired peptide encoding nucleotide sequence and a promoter derived from the genome of mammalian cells (e.g., metallothionein promoter) or from mammalian viruses (e.g., the adenovirus late promoter, the vaccinia virus 7.5K promoter).

[0069] In bacterial systems, a number of different expression vectors may be advantageously selected depending upon the use intended for the desired gene product being expressed. For example, when a large quantity of such a protein is to be produced, such as for the generation of pharmaceutical compositions comprising a desired peptide, or for raising antibodies to the protein, vectors that direct the expression of high levels of fusion protein products that are readily purified may be desirable. Such vectors include, but are not limited to: the E. coli expression vector pUR278 (Ruther and Muller-Hill, EMBO J. 2: 1791-1794, 1983), in which a desired peptide encoding sequence may be ligated individually into the vector in frame with the lacZ coding region so that a fusion protein is produced; pIN vectors (Inouye and Inouye, Nucleic Acids Res. 13:3101-3110, 1985, and Van Heeke and Schuster, J. Biol. Chem. 264:5503-5509, 1989); and the like. pGEX vectors (GE Healthcare, Piscataway, N.J.) may also be used to express a desired peptide moiety as a fusion protein with glutathione S- transferase (GST). In general, such fusion proteins are soluble and can easily be purified from lysed cells by adsorption to glutathione-agarose beads, followed by elution in the presence of free glutathione. The pGEX vectors are designed to include thrombin or factor Xa protease cleavage sites so that the cloned desired peptide encoding gene product can be released from the GST moiety. Because, in some embodiments, D-amino acids are preferred, methods such as those described in, among others, Park, et al. , (Production of D-amino acid using whole cells of recombinant Escherichia coli with separately and coexpressed D-hydantoinase and N- carbamoylase. Biotechnol Prog. July-August; 16(4):564-70, 2000). [0070] In an exemplary insect system, Autographa californica nuclear polyhedrosis virus (AcNPV) is used as a vector to express a desired peptide encoding sequence. The virus grows in Spodoptera fnigiperda cells. A desired peptide encoding sequence may be cloned individually into a non-essential region (for example the polyhedrin gene) of the virus and placed under control of an AcNPV promoter (for example the polyhedrin promoter). Successful insertion of a desired peptide encoding sequence will result in inactivation of the polyhedrin gene and production of non-occluded recombinant virus (z.e., virus lacking the proteinaceous coat coded for by the polyhedrin gene). The recombinant viruses are then used to infect Spodoptera frugiperda cells in which the inserted polynucleotide is expressed (see, e.g., Smith et al., J. Virol. 46:584-593, 1983, and U.S. Pat. No. 4,215,051).

[0071] In mammalian host cells, a number of viral-based expression systems may be utilized. In cases where an adenovirus is used as an expression vector, a desired peptide encoding nucleotide sequence may be ligated to an adenovirus transcription/translation control complex, e.g., the late promoter and tripartite leader sequence. This chimeric sequence may then be inserted in the adenovirus genome by in vitro or in vivo recombination. Insertion in a non-essential region of the viral genome e.g., region El or E3) will result in a recombinant virus that is viable and capable of expressing desired peptide products in infected hosts (see, e.g., Logan and Shenk, Proc. Nall. Acad. Sci. USA 81:3655-3659, 1984). Specific initiation signals may also be required for efficient translation of inserted desired peptide encoding nucleotide sequences. These signals include the ATG initiation codon and adjacent sequences. In some cases, exogenous translational control signals, including, perhaps, the ATG initiation codon, may be provided. Furthermore, the initiation codon should be in phase with the reading frame of the desired peptide encoding coding sequence to ensure translation of the entire insert. These exogenous translational control signals and initiation codons can be of a variety of origins, both natural and synthetic. The efficiency of expression may be enhanced by the inclusion of appropriate transcription enhancer elements, transcription terminators, etc. (see, e.g., Nevins, CRC Crit. Rev. Biochem. 19:307-322, 1986).

[0072] In yeast, a number of vectors containing constitutive or inducible promoters may be used. For a review, see, e.g., “Current Protocols in Molecular Biology”, supra, Ch. 13, Bitter et al., Meth. Enzymol. 153:516-544, 1987, “DNA Cloning”, Vol. II, Ch. 3 (Glover, ed., IRL Press, Washington, D.C., 1986); Bitter, Meth. Enzymol. 152:673-684, 1987, “The Molecular Biology of the Yeast Saccharomyces: Life Cycle and Inheritance” (Strathem et al., eds., Cold Spring Harbor Press, Cold Spring Harbor, N.Y., 1981), and “The Molecular Biology of the Yeast Saccharomyces: Metabolism and Gene Expression” (Strathern et al., eds., Cold Spring Harbor Press, Cold Spring Harbor, N.Y., 1982).

[0073] In plants, a variety of different plant expression vectors can be used, and expression of a desired peptide encoding sequence may be driven by any of a number of promoters. For example, viral promoters such as the 35S RNA or 19S RNA promoters of CaMV (Brisson et al., Nature 310:511-514, 1984), or the coat protein promoter of TMV (Takamatsu et al., EMBO J. 6:307-311, 1987) may be used. Alternatively, plant promoters such as the promoter of the small subunit of RUBISCO (Coruzzi etal., EMBO J. 3: 1671-1679, 1984, and Broglie et al. , Science 224:838-843, 1984), or heat shock promoters, e.g., soybean hsp 17.5- E or hspl7.3-B (Gurley et al. , Mol. Cell. Biol. 6:559-565, 1986) may be used. These constructs can be introduced into plant cells using, for example, Ti plasmids, Ri plasmids, plant virus vectors, direct DNA transformation, microinjection, or electroporation. For reviews of such techniques, see, e.g., Weissbach and Weissbach, in “Methods in Plant Molecular Biology”, Section VIII (Schuler and Zielinski, eds., Academic Press, Inc., New York, N.Y., 1988), and “Plant Molecular Biology”, 2nd Ed., Ch. 7-9 (Grierson and Covey, eds., Blackie & Son, Ltd., Glasgow, Scotland, United Kingdom, 1988).

[0074] In addition, a host cell strain may be chosen that modulates the expression of the inserted desired peptide encoding sequence or modifies and processes the desired peptide encoding nucleic acid sequence in a desired fashion. Such modifications (e.g., glycosylation) and processing (e.g., cleavage) of protein products may affect certain functions of the protein. Different host cells have characteristic and specific mechanisms for post-translational processing and modification of proteins and peptides. Appropriate cell lines or host systems can be chosen to ensure the correct or desired modification and processing of the desired protein, polypeptide, or peptide expressed. To this end, eukaryotic host cells that possess the cellular machinery for desired processing of the primary transcript, and glycosylation and/or phosphorylation of desired peptide encoding nucleic acid sequence be used. Such mammalian host cells include, but are not limited to, Chinese hamster ovary (CHO), VERO, baby hamster kidney (BHK), HeLa, monkey kidney (COS), MDCK, 293, 3T3, WI38, human hepatocellular carcinoma (e.g., Hep G2), and U937 cells.

[0075] In some embodiments, the mucosal membrane- and/or cell membranepenetrating peptides are functionalized for further use, such as described in the section that follows. For example, the mucosal membrane- and/or cell membrane- penetrating peptides may be conjugated to a lipid, a nucleic acid (e.g., mRNA, siRNA), or a polymer. Any of these peptide conjugates may be incorporated into nonviral delivery systems, a non-limiting example of which is a lipid nanoparticle. For example, the mucosal membrane- and/or cell membranepenetrating peptides may be conjugated to a lipid, such as a fatty acid. Peptides of the present disclosure may, for example, be conjugated to a fatty acid with five or fewer carbons, a fatty acid with about 6 to about 12 carbons, a fatty acid with about 13 to about 21 carbons, or a fatty acid with about 22 or more carbons. The fatty acid may be saturated or unsaturated, branched or unbranched. In some embodiments, the fatty acid is saturated. Examples of saturated fatty acids that may be conjugated to peptides of the present disclosure are caprylic acid, capric acid, lauric acid, myristic acid, palmitic acid, stearic acid, arachidic acid, behenic acid, lignoceric acid, or cerotic acid. In some embodiments, mucosal membrane- and/or cell membranepenetrating peptides are conjugated to myristic acid.

IL Nanoparticle and nanoparticle compositions

[0076] In certain embodiments the presently disclosed compositions are used in the treatment of a disease or disorder. The disease or disorder may be a disease or disorder associated with the function or deregulation of an endothelium cells. These endothelium cells are located in the stomach or intestinal lining (i.e. the gastrointestinal tract), the nasal passages, the liver, the skin, the lungs, the reproductive organs, or the eyes. In particular, the present compositions may be used to treat a disease or disorder of the reproductive organs such as ovarian or cervical cancer, human immunodeficiency virus (HIV), a Mullerian anomaly, endometriosis, or an HPV infection. In some embodiments, the present compositions may be used to treat a disease or disorder of the gastrointestinal system such as oral carcinoma, Sjogren’ s syndrome, inflammatory bowel disease, or colitis. In other embodiments, the present compositions may be used to treat a disease or disorder of the corneal and ocular diseases or disorders such as retinal genetic disease. In other embodiments, the present composition may be used to treat lung or nasal diseases or disorders such as chronic obstructive pulmonary disease, respiratory syncytial virus (RSV), influenza, cytomegalovirus (CMV), cystic fibrosis, primary ciliary dyskinesia, or alpha- 1 antitrypsin deficiency. These diseases or disorders may be associated with one or more genetic mutations such as those described in Seixas et al. , Appl. Clin. Genet., 14:173-194, 2021, which is incorporated herein by reference. In other embodiments, the present composition may be used to treat skin diseases or disorders such as epidermolysis bullosa, epidermolytic hyperkeratosis, or a polygenic skin disease such as systemic lupus, psoriasis, androgenic alopecia, atopic dermatitis, systemic sclerosis, vitiligo, alopecia areata, pemphigus vulgaris and foliaceus, or Sjogren’s syndrome. In other embodiments, the present composition may be used to treat liver disease or disorders. In certain embodiments the presently disclosed compositions can be administered in combination with one or more additional compounds or agents (“additional active agents”) for the treatment, management, and/or prevention of the diseases or disorders. Such therapies can be administered to a patient at therapeutically effective doses to treat or ameliorate one or more of these diseases or disorders, or symptoms or disorders associated with one or more of these diseases or disorders. A therapeutically effective dose refers to that amount of the compound sufficient to result in any delay in onset, amelioration, or retardation of disease symptoms. The presently disclosed compositions in some embodiments comprise peptides described above and elsewhere in combination with a lipid nanoparticle, details of which are provided below.

[0077] As used herein, the term “nanoparticle” refers to any material having dimensions in the 1-1,000 nm range. In some embodiments, nanoparticles have dimensions in the 50-500 nm range. Nanoparticles used in the present embodiments include such nanoscale materials as a lipid-based nanoparticle, a superparamagnetic nanoparticle, a nanoshell, a semiconductor nanocrystal, a quantum dot, a polymer-based nanoparticle, a silicon-based nanoparticle, a silica-based nanoparticle, a metal-based nanoparticle, a fullerene and a nanotube (Ferrari, 2005). The conjugation of polypeptide or nucleic acids to nanoparticles provides structures with potential application for targeted delivery, controlled release, enhanced cellular uptake and intracellular trafficking, and molecular imaging of therapeutic peptides in vitro and in vivo (Stayton et al., 2000; Ballou et al.. 2004; Frangioni, 2003; Dubertret et al., 2002; Michalet et al., 2005; Dwarakanath et al., 2004.)

(1) Lipid nanoparticles (LNPs)

[0078] Lipid-based nanoparticles include lipid nanoparticles, liposomes, lipid preparations and lipid-based vesicles. As mentioned above, in some embodiments the present application provides compositions comprising lipid nanoparticles. Lipid-based nanoparticles may be positively charged, negatively charged or neutral. In preferred embodiments of the present disclosure, the lipid-based nanoparticles of the present disclosure comprise a cationic ionizable lipid. [0079] In specific aspects, a polypeptide or nucleic acids may be, for example, encapsulated in the aqueous interior of a lipid nanoparticle or liposome, interspersed within the lipid bilayer of a lipid nanoparticle or liposome, attached to a lipid nanoparticle or liposome via a linking molecule that is associated with both the lipid nanoparticle or liposome and the polypeptide/nucleic acid, entrapped in a lipid nanoparticle or liposome, complexed with a lipid nanoparticle or liposome, or the like.

[0080] The size of a lipid nanoparticle or liposome varies depending on the method of synthesis. Lipid nanoparticles or liposomes in the present disclosure can have a variety of sizes. In certain embodiments, the lipid nanoparticles are small, e.g., less than about 200 nm, about 190 nm, about 180 nm, about 170 nm, about 160 nm, about 150 nm, about 140 nm, about 130 nm, about 120 nm, about 110 nm, about 100 nm, about 90 nm, about 80 nm, about 70 nm, about 60 nm, or about 50 nm in external diameter. In general, prior to the incorporation of nucleic acid, a lipid nanoparticle or liposome for use according to the present embodiments comprises a size of about 50 to 250 nm or about 50 to about 150 nm. In other embodiments, the liposomes or lipid nanoparticles may have a larger diameter, such as about 300 nm, about 350 nm, about 400 nm, about 450 nm, about 500 nm, about 550 nm, about 600 nm, about 650 nm, about 700 nm, about 750 nm, about 800 nm, about 850 nm, about 900 nm, about 950 nm, about 1000 nm, or any range derivable therein. Such lipid nanoparticle or liposome formulations may also be defined by particle charge (zeta potential) and/or optical density (OD). For instance, a lipid nanoparticle or liposome formulation will typically comprise an OD400 of less than 0.45 prior to nucleic acid incorporation.

[0081] Lipid nanoparticles of the present disclosure have, in some embodiments, a zeta potential of about -20 mV, -19 mV, about -18 mV, about -17 mV, about -16 mV, -15 mV, about -14 mV, about -13 mV, about -12 mV, about -10 mV, about -9 mV, about -8 mV, about -7 mV, about -6 mV, about -5 mV, about -4 mV, about -3 mV, about -2 mV, about -1 mV, about 0 mV, about 1 mV, about 2 mV, about 3 mV, about 4 mV, about 5 mV, about 6 mV, about 7 mV, about 8 mV, about 9 mV, about 10 mV, or any range derivable therein. In some embodiments, the presently disclosed lipid nanoparticle compositions have a negative zeta potential. In some embodiments, the presently disclosed lipid nanoparticle compositions have a positive zeta potential. In some embodiments, the presently disclosed lipid nanoparticle compositions have a zeta potential of between 0 and about 1 mV, about 1 mV, about 2 mV, about 3 mV, about 4 mV, about 5 mV, about 6 mV, about 7 mV, about 8 mV, about 9 mV, about 10 mV, or any range derivable therein. In some embodiments, the zeta potential of the presently disclosed lipid nanoparticles is about 2 mV.

[0082] The lipid nanoparticle compositions provided by the present disclosure are shown, for example, above in the summary of the invention section and in the claims below. They may be made using the methods outlined in the Examples section. These methods can be further modified and optimized using the principles and techniques of organic chemistry as applied by a person skilled in the art. Such principles and techniques are taught, for example, in March’s Advanced Organic Chemistry: Reactions, Mechanisms, and Structure (2007), which is incorporated by reference herein.

[0083] Furthermore, in preparing such lipid nanoparticles or liposomes, any protocol described herein, or as would be known to one of ordinary skill in the art may be used. Additional non-limiting examples of preparing lipid nanoparticles or liposomes are described in US20240051789, WO2016144376, W02012170930, W002/100435A1, W003/015757A1, WO04029213A2, U.S. Application 2004/0208921 , U.S. Patents 4,728,578, 4,728,575, 4,737,323, 4,533,254, 4,162,282, 4,310,505, and 4,921,706; 5,030,453, 5,962,016, 6,680,068, International Applications PCT/US85/01161 and PCT/US89/05040; U.K. Patent Application GB 2193095 A; Mayer et al., 1986; Hope et al., 1985; Mayhew et al. 1987; and Mayhew et al., 1984, each incorporated herein by reference). A process of making liposomes is also described in W004/002453A1.

[0084] In certain embodiments, the lipid-based nanoparticle is a positive lipid nanoparticle or positive liposome. “Positive lipid nanoparticle”, “cationic lipid nanoparticle”, “positive liposomes”, or “cationic liposomes”, as used herein, are defined as liposomes having one or more lipid components that yield an essentially positive net charge (substantially positive). “Positive lipid nanoparticles” or “cationic lipid nanoparticles”, as used herein, are defined as lipid nanoparticles having one or more lipid components that yield an essentially positive net charge (substantially positive). By “essentially positive”, it is meant that overall, lipid components within a given population (e.g., a population of lipid nanoparticles or liposomes) include positive charges that are not canceled by an opposite charge of another component (z.e. , fewer than 10% of components are canceled by an opposite charge of another component, more preferably fewer than 5%, and most preferably fewer than 1%). In certain embodiments, positive lipid nanoparticles or positive liposomes of the present disclosure may include mostly lipids and/or phospholipids that are themselves positive under physiological conditions (i.e., at about pH 7). As used herein, the lipid components that yield an essentially positive net charge of the positive lipid nanoparticles or positive liposomes, that is, the lipids that are themselves positive under physiological conditions, may also be known as cationic ionizable lipids. In some embodiments, the cationic ionizable lipids may be neutral at physiological pH and positively charged in acidic pH or environments. The localized microenvironment surrounding cationic ionizable lipids may affect the protonation state of the cationic ionizable lipids and result in positively charged cationic ionizable lipids under conditions which would not otherwise be thought to result in positively charged cationic ionizable lipids. In some embodiments, the cationic ionizable lipid is an amino lipid. In some embodiments, the cationic ionizable lipids comprise a tertiary amine. In some embodiments, the alkyl groups attached to the tertiary amine may be independently substituted with functional groups, such as esters or hydroxy groups. In some embodiments, the cationic ionizable lipid is SM-102, MC3, or ALC-0315.

[0085] The cationic ionizable lipid component of lipid nanoparticle compositions of the present disclosure may be present in a variety of molar ratios with respect to the composition. In some embodiments of the present invention, the cationic ionizable lipid is present in a molar ratio with respect to the lipid nanoparticle composition of from about 0.2 to about 1.0. In some embodiments, the molar ratio of cationic ionizable lipid to lipid nanoparticle composition is from about 0.3 to about 0.7 or from about 0.4 to about 0.6. The molar ratio of cationic lipid to lipid nanoparticle composition may be about 0.2, about 0.25 about 0.3, about 0.35, about 0.4, about 0.45, about 0.5, about 0.55, about 0.6, about 0.65, about 0.7, about 0.75, about 0.8, about 0.85, about 0.9, about 0.95, or about 1.0, or any range derivable therein. In some embodiments, the molar ratio of cationic lipid to lipid nanoparticle composition is about 0.45.

[0086] The cationic ionizable lipids and other lipids of the present disclosure may contain one or more asymmetrically-substituted carbon or nitrogen atoms, and may be isolated in optically active or racemic form. Thus, all chiral, diastereomeric, racemic form, epimeric form, and all geometric isomeric forms of a chemical formula are intended, unless the specific stereochemistry or isomeric form is specifically indicated. Cationic ionizable lipids may occur as racemates and racemic mixtures, single enantiomers, diastereomeric mixtures and individual diastereomers. In some embodiments, a single diastereomer is obtained. The chiral centers of the cationic ionizable lipids of the present disclosure can have the S or the R configuration. Furthermore, it is contemplated that one or more of the cationic ionizable lipids may be present as constitutional isomers. In some embodiments, the compounds have the same formula but different connectivity.

[0087] Chemical formulas used to represent cationic ionizable lipids of the present disclosure will typically only show one of possibly several different tautomers. For example, many types of ketone groups are known to exist in equilibrium with corresponding enol groups. Similarly, many types of imine groups exist in equilibrium with enamine groups. Regardless of which tautomer is depicted for a given formula, and regardless of which one is most prevalent, all tautomers of a given chemical formula are intended.

[0088] The cationic ionizable lipids of the present disclosure may also have the advantage that they may be more efficacious than, be less toxic than, be longer acting than, be more potent than, produce fewer side effects than, be more easily absorbed than, more metabolically stable than, more lipophilic than, more hydrophilic than, and/or have a better pharmacokinetic profile (e.g., higher oral bioavailahility and/or lower clearance) than, and/or have other useful pharmacological, physical, or chemical properties over, compounds known in the prior art, whether for use in the indications stated herein or otherwise.

[0089] In addition, atoms making up the cationic ionizable lipids of the present disclosure are intended to include all isotopic forms of such atoms. Isotopes, as used herein, include those atoms having the same atomic number but different mass numbers. By way of general example and without limitation, isotopes of hydrogen include tritium and deuterium, and isotopes of carbon include ¹³C and ¹⁴C.

[0090] It should be recognized that the particular anion or cation forming a part of any salt form of a cationic ionizable lipids provided herein is not critical, so long as the salt, as a whole, is pharmacologically acceptable. Additional examples of pharmaceutically acceptable salts and their methods of preparation and use are presented in Handbook of Pharmaceutical Salts: Properties, and Use (2002), which is incorporated herein by reference.

(2) Lipids

[0091] In some aspects of the present disclosure, one or more additional types of lipids are mixed with the cationic ionizable lipids of the instant disclosure to create a nanoparticle composition. In some embodiments, the cationic ionizable lipids are mixed with 1, 2, 3, 4, or 5 different types of lipids. It is contemplated that the cationic ionizable lipids can be mixed with multiple different lipids of a single type.

[0092] In some embodiments, at least one of the additional lipids may be a steroid or a steroid derivative. In some embodiments, the additional lipid may be a PEG lipid. In some embodiments, the additional lipid may be a phospholipid. In some embodiments, the nanoparticle composition comprises a steroid or a steroid derivative, a PEG lipid, and a phospholipid, or any combination thereof. Additional details of the types of lipids that may be used to form the nanoparticle composition are provided in the sections that follow.

Steroids and Steroid Derivatives

[0093] In the lipid nanoparticle compounds of the present disclosure of the present disclosure, the cationic ionizable lipids (or compounds) are mixed with one or steroids or steroid derivatives and other components described below to form the nanoparticle composition. In some embodiments, the steroid or steroid derivative comprises any steroid or steroid derivative. As used herein, in some embodiments, the term “steroid” is a class of compounds with a four ring 17 carbon cyclic structure which can further comprises one or more substitutions including alkyl groups, alkoxy groups, hydroxy groups, oxo groups, acyl groups, or a double bond between two or more carbon atoms. In one aspect, the ring structure of a steroid comprises three fused cyclohexyl rings and a fused cyclopentyl ring as shown in the formula below:

[0094] In some embodiments, a steroid derivative comprises the ring structure above with one or more non-alkyl substitutions. In some embodiments, the steroid or steroid derivative is a sterol wherein the formula is further defined as:

[0095] Another steroid or steroid derivative is a cholestane or cholestane derivative. In a cholestane, the ring structure is further defined by the formula:

[0096] As described above, a cholestane derivative includes one or more non-alkyl substitution of the above ring system. The cholestane or cholestane derivative may be a cholestene or cholestene derivative or a sterol or a sterol derivative. The cholestane or cholestane derivative may be both a cholestere and a sterol or a derivative thereof. In preferred embodiments, the nanoparticle composition comprises cholesterol.

[0097] In some embodiments, the present composition comprises a molar ratio of the steroid or steroid derivative to the lipid nanoparticle composition of from about 0.05 to about 0.12 or from about 0.1 to about 0.6. The molar ratio may be from about 0.15 to about 0.5 such as a molar ratio of about such as a molar ratio of steroid or steroid derivative to lipid nanoparticle composition of about 0.34. In some embodiments, the molar ratio of steroid or steroid derivative to lipid nanoparticle composition is about 0.05, about 0.10, about 0.15, about 0.20, about 0.25, about 0.30, about 0.35, about 0.4, about 0.45, about 0.50, about 0.55, about 0.60, about 0.65, about 0.70, about 0.75, about 0.80, about 0.85, about 0.90, about 1.0, or about 1.2, or any range derivable therein.

PEG or PEGylated lipid

[0098] In the lipid nanoparticle compounds of the present disclosure of the present disclosure, the cationic ionizable lipids (or compounds) are mixed with one or more PEGylated lipids (or PEG lipid) and other components described above and below to form the nanoparticle composition. In some embodiments, the present disclosure comprises using any lipid to which a PEG group has been attached. In some embodiments, the PEG lipid is a diglyceride which also comprises a PEG chain attached to the glycerol group. In other embodiments, the PEG lipid is a compound which contains one or more C6-C24 long chain alkyl or alkenyl group or a C6-C24 fatty acid group attached to a linker group with a PEG chain. In some embodiments, the PEG-lipid has an advantage such as that it prevents aggregation or reduces uptake of the composition by immune cells. Some non-limiting examples of a PEG lipid includes a PEG modified phosphatidylethanolamine and phosphatidic acid, a PEG ceramide conjugated, PEG modified dialkylamines and PEG modified l,2-diacyloxypropan-3-amines, PEG modified diacylglycerols and dialkylglycerols. In some embodiments, the PEG lipid is PEG modified diastearoylphosphatidylethanolamine. In some embodiments, the PEG lipid comprises a PEG modified phospholipid, such as any of the lipids mentioned in the section that follows. In some embodiments, the PEG lipid is a PEG modified dimyristoyl phosphatidylethanolamine or a PEG modified myristoyl diglyceride.

[0099] In some embodiments, the PEG modification is measured by the molecular weight of PEG component of the lipid. In some embodiments, the PEG modification has a molecular weight from about 100 to about 5,000. In some embodiments, the molecular weight is from about 200 to about 500 or from about 1,200 to about 3,000. Some non- limiting examples of lipids that may be used in the present disclosure are taught by U.S. Patent 5,820,873, WO 2010/141069, or U.S. Patent 8,450,298, which is incorporated herein by reference.

[00100] In some embodiments, the present composition comprises a molar ratio of the PEG lipid to the lipid nanoparticle composition of from about 0.001 to about 0.04 or from about 0.005 to about 0.03. The molar ratio may be from about 0.01 to about 0.015. In some embodiments, the molar ratio of PEG lipid to the lipid nanoparticle composition may be about 0.01. In some embodiments, the ratio is about 0.001, about 0.005, about 0.006, about 0.007, about 0.008, about 0.009, about 0.01, about 0.011, about 0.012, about 0.013, about 0.014, about 0.015, about 0.02, about 0.025, about 0.03, about 0.035, to about 0.04 or any range derivable therein.

Phospholipids

[00101] In the lipid nanoparticle compounds of the present disclosure of the present disclosure, the cationic ionizable lipids (or compounds) are mixed with one or more phospholipids and other components described above and below to form the nanoparticle composition. The phospholipids may also be referred to herein as “helper lipids.” In some embodiments, compositions disclosed herein comprise a helper lipid which comprises a phosphate group. In some embodiments, more than one kind of phospholipid may be used to form the composition. In some embodiments, the phospholipid is a structure which contains one or two long chain C6-C24 alkyl or alkenyl groups, a glycerol or a sphingosine, one or two phosphate groups, and, optionally, a small organic molecule. In some embodiments, the small organic molecule is an amino acid, a sugar, or an amino substituted alkoxy group, such as choline or ethanolamine. In some embodiments, the phospholipid is a phosphatidylcholine. In some embodiments, the phospholipid is distearoylphosphatidylcholine, dioleoylphosphatidylycholine or dipalmitoylphosphatidylcholine. In some embodiments, the helper lipid may be neutral under physiological conditions (i.e., at about pH 7). In some embodiments, the helper lipid has an advantage such as that it improves the structure or enhances endosomal escape.

[00102] Phospholipids include, for example, phosphatidylcholines, phosphatidylglycerols, and phosphatidylethanolamines; because phosphatidylethanolamines and phosphatidyl cholines are non-charged under physiological conditions (i.e., at about pH 7), these compounds may be particularly useful for generating lipid nanoparticles or positive liposomes. In certain embodiments, the phospholipid DPPC is used to produce lipid nanoparticles or positive liposomes.

[00103] Phospholipids that may be components of compositions disclosed herein include glycerophospholipids and certain sphingolipids. Phospholipids include, but are not limited to, dioleoylphosphatidylycholine ("DOPC"), egg phosphatidylcholine ("EPC"), dilauryloylphosphatidylcholine ("DLPC"), dimyristoylphosphatidylcholine ("DMPC"), dipalmitoylphosphatidylcholine ("DPPC"), distearoylphosphatidylcholine ("DSPC"), 1- myristoyl-2-palmitoyl phosphatidylcholine ("MPPC"), l-palmitoyl-2-myristoyl phosphatidylcholine ("PMPC"), l-palmitoyl-2-stearoyl phosphatidylcholine ("PSPC"), 1- stearoyl-2-palmitoyl phosphatidylcholine ("SPPC"), dilauryloylphosphatidylglycerol ("DLPG"), dimyristoylphosphatidylglycerol ("DMPG"), dipalmitoylphosphatidylglycerol ("DPPG"), distearoylphosphatidylglycerol ("DSPG"), distearoyl sphingomyelin ("DSSP"), distearoylphophatidylethanolamine ("DSPE"), dioleoylphosphatidylglycerol ("DOPG"), dimyristoyl phosphatidic acid ("DMPA"), dipalmitoyl phosphatidic acid ("DPPA"), dimyristoyl phosphatidylethanolamine ("DMPE"), dipalmitoyl phosphatidylethanolamine ("DPPE"), dimyristoyl phosphatidylserine ("DMPS"), dipalmitoyl phosphatidylserine ("DPPS"), brain phosphatidylserine ("BPS"), brain sphingomyelin ("BSP"), dipalmitoyl sphingomyelin ("DPSP"), dimyristyl phosphatidylcholine ("DMPC"), 1 ,2-distearoyl-sn- glycero-3 -phosphocholine ("DAPC"), l,2-diarachidoyl-sn-glycero-3-phosphocholine

("DBPC"), l,2-dieicosenoyl-sn-glycero-3-phosphocholine ("DEPC"), dioleoylphosphatidylethanolamine ("DOPE"), palmitoyloeoyl phosphatidylcholine ("POPC"), palmitoyloeoyl phosphatidylethanolamine ("POPE"), lysophosphatidylcholine, lysophosphatidylethanolamine, and dilinoleoylphosphatidylcholine. [00104] In some embodiments, the present composition comprises a molar ratio of the phospholipid to the lipid nanoparticle composition from about 0.01 to about 0.5 or about 0.02 to about 0.4. The molar ratio may be from about 0.05 to about 0.3 such as a molar ratio of about 0.2. In some embodiments, the molar ratio of phospholipid to lipid nanoparticle composition is from about 0.01, about 0.03, about 0.05, about 0.07, about 0.09, about 0.1, about 0.12, about 0.14, about 0.16, about 0.18, about 0.2, about 0.22, about 0.24, about 0.26, about 0.28, about 0.3, about 0.35, to about 0.4, or any range derivable therein.

[00105] Phospholipids may be from natural or synthetic sources. However, phospholipids from natural sources, such as egg or soybean phosphatidylcholine, brain phosphatidic acid, brain or plant phosphatidylinositol, heart cardiolipin and plant or bacterial phosphatidylethanolamine are not used, in certain embodiments, as the primary phosphatide (i.e., constituting 50% or more of the total phosphatide composition) because this may result in instability and leakiness of the resulting lipid nanoparticles or liposomes.

Biologically active polynucleotides

[00106] Methods and composition of the embodiments concern biologically active polynucleotides. In some cases, these can comprise single stranded or double stranded RNA or DNA. It should be clear that the present disclosure is not limited to the specific nucleic acids disclosed herein. The present disclosure is not limited in scope to any particular source, sequence, or type of nucleic acid, however, as one of ordinary skill in the art could readily identify related homologs in various other sources of the nucleic acid including nucleic acids from non-human species (e.g., mouse, rat, rabbit, dog, monkey, gibbon, chimp, ape, baboon, cow, pig, horse, sheep, cat and other species). It is contemplated that the nucleic acid used in the present disclosure can comprises a sequence based upon a naturally-occurring sequence.

[00107] The amount of nucleic acid encapsulated by or located within the lipid nanoparticle may vary based on the intended use. The amount of nucleic acid may be calculated as a ratio with respect to the lipid nanoparticle composition (w/w) or to any of the individual components of the lipid nanoparticle composition (w/w). For example, the ratio of cationic ionizable lipid to nucleic acid may be about from about 50:1 (w/w), about 20: 1 (w/w), about 15: 1 (w/w), about 14:1 (w/w), about 13:1 (w/w), about 12:1 (w/w), about 11:1 (w/w), about 10: 1 (w/w), about 9:1 (w/w), about 8:1 (w/w), about 7: 1 (w/w), about 6: 1 (w/w), to about 5:1 (w/w), or any range derivable therein. In some embodiments, the ratio of cationic ionizable lipid to nucleic acid is about 11.33 (w/w). The length of the nucleic acid encapsulated by or located within the lipid nanoparticle may also vary based on the intended use. The length of the nucleic acid may be about 20 bp, about 50 bp, about 75 bp, about 100 bp, about 150 bp, about 200 bp, about 250 bp, about 300 bp, about 350 bp, about 400 bp, about 450 bp, about 500 bp, about 550 bp, about 600 bp, about 650 bp, about 700 bp, about 750 bp, about 800 bp, about 850 bp, about 900 bp, about 950 bp, about 1000 bp, or any range derivable therein. Longer nucleic acids are also contemplated, such as nucleic acids that are about 1500 bp, about 2000 bp, about 2500 bp, about 3000 bp, about 3500 bp, about 4000 bp, about 4500 bp, about 5000 bp, about 5500 bp, about 6000 bp, about 6500 bp, about 7000 bp, about 7500 bp, about 8000 bp, about 8500 bp, about 9000 bp, about 9500 bp, about 10,000 bp, or any range derivable therein.

[00108] In some aspects, the nucleic acid is a sequence which silences, is complimentary to, or replaces another sequence present in vivo. Sequences of 17 bases in length should occur only once in the human genome and, therefore, suffice to specify a unique target sequence. Although shorter oligomers are easier to make and increase in vivo accessibility, numerous other factors are involved in determining the specificity of hybridization. Both binding affinity and sequence specificity of an oligonucleotide to its complementary target increases with increasing length. It is contemplated that exemplary oligonucleotides of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 or more base pairs will be used, although others are contemplated. Longer polynucleotides encoding 250, 500, 1000, 1212, 1500, 2000, 2500, 3000 or longer are contemplated as well.

[00109] The nucleic acid used herein may be derived from genomic DNA, i.e., cloned directly from the genome of a particular organism. In preferred embodiments, however, the nucleic acid would comprise complementary DNA (cDNA). Also contemplated is a cDNA plus a natural intron or an intron derived from another gene; such engineered molecules are sometime referred to as "mini-genes." At a minimum, these and other nucleic acids of the present disclosure may be used as molecular weight standards in, for example, gel electrophoresis.

[00110] The term "cDNA" is intended to refer to DNA prepared using messenger RNA (mRNA) as template. The advantage of using a cDNA, as opposed to genomic DNA or DNA polymerized from a genomic, non- or partially-processed RNA template, is that the cDNA primarily contains coding sequences of the corresponding protein. There may be times when the full or partial genomic sequence is preferred, such as where the non-coding regions are required for optimal expression or where non-coding regions such as introns are to be targeted in an antisense strategy.

[00111] In some embodiments, the nucleic acid comprises one or more antisense segments which inhibits expression of a gene or gene product. Antisense methodology takes advantage of the fact that nucleic acids tend to pair with "complementary" sequences. By complementary, it is meant that polynucleotides are those which are capable of base-pairing according to the standard Watson-Crick complementarity rules. That is, the larger purines will base pair with the smaller pyrimidines to form combinations of guanine paired with cytosine (G:C) and adenine paired with either thymine (A:T) in the case of DNA, or adenine paired with uracil (A:U) in the case of RNA. Inclusion of less common bases such as inosine, 5- methylcytosine, 6-methyladenine, hypoxanthine and others in hybridizing sequences does not interfere with pairing.

[00112] Targeting double-stranded (ds) DNA with polynucleotides leads to triple-helix formation; targeting RNA will lead to double-helix formation. Antisense polynucleotides, when introduced into a target cell, specifically bind to their target polynucleotide and interfere with transcription, RNA processing, transport, translation and/or stability. Antisense RNA constructs, or DNA encoding such antisense RNA’s, may be employed to inhibit gene transcription or translation or both within a host cell, either in vitro or in vivo, such as within a host animal, including a human subject.

[00113] Antisense constructs may be designed to bind to the promoter and other control regions, exons, introns or even exon-intron boundaries of a gene. It is contemplated that the most effective antisense constructs will include regions complementary to intron/exon splice junctions. Thus, it is proposed that a preferred embodiment includes an antisense construct with complementarity to regions within 50-200 bases of an intron-exon splice junction. It has been observed that some exon sequences can be included in the construct without seriously affecting the target selectivity thereof. The amount of exonic material included will vary depending on the particular exon and intron sequences used. One can readily test whether too much exon DNA is included simply by testing the constructs in vitro to determine whether normal cellular function is affected or whether the expression of related genes having complementary sequences is affected. [00114] As stated above, "complementary" or "antisense" means polynucleotide sequences that are substantially complementary over their entire length and have very few base mismatches. For example, sequences of fifteen bases in length may be termed complementary when they have complementary nucleotides at thirteen or fourteen positions. Naturally, sequences which are completely complementary will be sequences which are entirely complementary throughout their entire length and have no base mismatches. Other sequences with lower degrees of homology also are contemplated. For example, an antisense construct which has limited regions of high homology, but also contains a non-homologous region (e.g., ribozyme; see below) could be designed. These molecules, though having less than 50% homology, would bind to target sequences under appropriate conditions.

[00115] It may be advantageous to combine portions of genomic DNA with cDNA or synthetic sequences to form a siRNA or to generate specific constructs. For example, where an intron is desired in the ultimate construct, a genomic clone will need to be used. The cDNA, siRNA, or a synthesized polynucleotide may provide more convenient restriction sites for the remaining portion of the construct and, therefore, would be used for the rest of the sequence. Other embodiments include dsRNA or ssRNA, which may be used to target genomic sequences or coding/non-coding transcripts.

[00116] In other embodiments, the nanoparticles may comprise a nucleic acid which comprises one or more expression vectors are used in a gene therapy. Expression requires that appropriate signals be provided in the vectors, and which include various regulatory elements, such as enhancers/promoters from both viral and mammalian sources that drive expression of the genes of interest in host cells. Elements designed to optimize messenger RNA stability and translatability in host cells also are defined. The conditions for the use of a number of dominant drug selection markers for establishing permanent, stable cell clones expressing the products are also provided, as is an element that links expression of the drug selection markers to expression of the polypeptide.

[00117] Throughout this application, the term "expression construct" is meant to include any type of genetic construct containing a nucleic acid coding for a gene product in which part or all of the nucleic acid encoding sequence is capable of being transcribed. The transcript may be translated into a protein, but it need not be. In certain embodiments, expression includes both transcription of a gene and translation of mRNA into a gene product. In other embodiments, expression only includes transcription of the nucleic acid encoding a gene of interest.

[00118] The term "vector" is used to refer to a carrier nucleic acid molecule into which a nucleic acid sequence can be inserted for introduction into a cell where it can be replicated. A nucleic acid sequence can be "exogenous," which means that it is foreign to the cell into which the vector is being introduced or that the sequence is homologous to a sequence in the cell but in a position within the host cell nucleic acid in which the sequence is ordinarily not found. Vectors include plasmids, cosmids, viruses (bacteriophage, animal viruses, and plant viruses), and artificial chromosomes (e.g., YACs). One of skill in the art would be well equipped to construct a vector through standard recombinant techniques, which are described in Sambrook et al. (1989) and Ausubel et al. (1994), both incorporated herein by reference.

[00119] The term "expression vector" refers to a vector containing a nucleic acid sequence coding for at least part of a gene product capable of being transcribed. In some cases, RNA molecules are then translated into a protein, polypeptide, or peptide. In other cases, these sequences are not translated, for example, in the production of antisense molecules or ribozymes. Expression vectors can contain a variety of "control sequences," which refer to nucleic acid sequences necessary for the transcription and possibly translation of an operably linked coding sequence in a particular host organism. In addition to control sequences that govern transcription and translation, vectors and expression vectors may contain nucleic acid sequences that serve other functions as well and are described infra. mRNA

[00120] In some aspects, the present compounds and compositions may be used in the delivery of an mRNA to a cell. Messenger RNA or mRNA are short RNA strands which transfer the genetic code from the DNA to the ribosomes so the mRNA may be translated into a therapeutic protein or peptide, or an antigen. The mRNAs described herein may be unprocessed or have undergone processing to add a poly(A) tail, be edited in vivo, or have a 5' cap added. The mRNA molecules may comprise a 5’ UTR or a 3’UTR. In some embodiments, the mRNA has an advantage of exhibiting reduced degradation, for example due to the presence of pseudouridine base or bases in the mRNA sequence. The present compositions are contemplated in the delivery of a variety of different mRNA including those which have not undergone processing or have been further processed. Additionally, these nucleic acids may be used therapeutically, used to produce an antibody in vivo, or in a vaccine formulation. mRNA molecules can provide a more direct method of expressing a polypeptide of interest in a target cell. However, such molecules are typically highly liable and rapidly degraded. In some aspects, LNP processing according to the embodiments can be used to substantially stabilize mRNA. In preferred aspects, mRNA is provided encapsulated in or in complex with LNPs.

[00121] As mentioned above, in some aspects a nucleic acid molecule of the embodiments encodes a therapeutic polypeptide. For example, the therapeutic protein may be a protein, such as an enzyme that is non-functional or disrupted in a particular disease state (e.g., CFTR in cystic fibrosis).

[00122] In further aspects, a polynucleotide of the embodiments encodes an antigen, such as an antigen from a pathogen or a cancer cell-associated antigen. For example, the cancer associated antigen can be CD 19, CD20, ROR1, CD22, carcinoembryonic antigen, alphafetoprotein, CA-125, 5T4, MUC-1 , epithelial tumor antigen, prostate-specific antigen, melanoma-associated antigen, mutated p53, mutated ras, HER2/Neu, folate binding protein, GD2, CD123, CD33, CD138, CD23, CD30 , CD56, c-Met, mesothelin, GD3, HERV-K, IL- HRalpha, kappa chain, lambda chain, CSPG4, ERBB2, EGFRvIII or VEGFR2. In some specific aspects the antigen is GP240, 5T4, HER1, CD-33, CD-38, VEGFR-1, VEGFR-2, CEA, FGFR3, IGFBP2, IGF-1R, BAFF-R, TACI, APRIL, Fnl4, ERBB2 or ERBB3

[00123] Antigens useful in the present disclosure may include those derived from viruses including, but not limited to, those from the family Arenaviridae (e.g., Lymphocytic choriomeningitis virus), Arterivirus (e.g., Equine arteritis virus), Astroviridae (Human astrovirus 1), Birnaviridae (e.g., Infectious pancreatic necrosis virus, Infectious bursal disease virus), Bunyaviridae (e.g., California encephalitis virus Group), Caliciviridae (e.g., Caliciviruses), Coronaviridae (e.g., Human coronaviruses 299E and OC43), Deltavirus (e.g., Hepatitis delta virus), Filoviridae (e.g., Marburg virus, Ebola virus), Flaviviridae (e.g., Yellow fever virus group, Hepatitis C virus), Hepadnaviridae (e.g., Hepatitis B virus), Herpesviridae (e.g., Epstein-Bar virus, Simplexvirus, Varicellovirus, Cytomegalovirus, Roseolovirus, Lymphocryptovirus, Rhadinovirus), Orthomyxoviridae (e.g., Influenzavirus A, B, and C), Papovaviridae (e.g., Papillomavirus), Paramyxoviridae (e.g., Paramyxovirus such as human parainfluenza virus 1, Morbillivirus such as Measles virus, Rubulavirus such as Mumps virus, Pneumovirus such as Human respiratory syncytial virus), Picornaviridae (e.g., Rhinovirus such as Human rhinovirus 1A, Hepatovirus such Human hepatitis A virus, Human poliovirus, Cardiovirus such as Encephalomyocarditis virus, Aphthovirus such as Foot-and-mouth disease virus O, Coxsackie virus), Poxyiridae (e.g., Orthopoxvirus such as Variola virus or monkey poxvirus), Reoviridae (e.g., Rotavirus such as Groups A-F rotaviruses), Retroviridae (Primate lentivirus group such as human immunodeficiency virus 1 and 2), Rhabdoviridae (e.g., rabies virus), Togaviridae (e.g., Rubivirus such as Rubella virus), Human T-cell leukemia virus, Murine leukemia virus, Vesicular stomatitis virus, Wart virus, Blue tongue virus, Sendai virus, Feline leukemia virus, Simian virus 40, Mouse mammary tumor virus, Dengue virus, HIV-1 and HIV-2, West Nile, H1N1, SARS, 1918 Influenza, Tick-borne encephalitis virus complex (Absettarov, Hanzalova, Hypr), Russian Spring-Summer encephalitis virus, Congo-Crimean Hemorrhagic Fever virus, Junin Virus, Kumlinge Virus, Marburg Virus, Machupo Virus, Kyasanur Forest Disease Virus, Lassa Virus, Omsk Hemorrhagic Fever Virus, FIV, SIV, Herpes simplex 1 and 2, Herpes Zoster, Human parvovirus (B19), Respiratory syncytial virus, Pox viruses (all types and serotypes), Coltivirus, Reoviruses — all types, and/or Rubivirus (rubella).

[00124] Antigens useful in the present disclosure may include those derived from bacteria including, but not limited to, Streptococcus agalactiae, Legionella pneumophilia, Streptococcus pyogenes, Escherichia coli, Neisseria gonorrhosae, Neisseria meningitidis, Pneumococcus, Hemophilis influenzae B, Treponema pallidum, Lyme disease spirochetes, Pseudomonas aeruginosa, Mycobacterium leprae, Brucella abortus, Mycobacterium tuberculosis, Plasmodium falciparum, Plasmodium vivax, Toxoplasma gondii, Trypanosoma rangeli, Trypanosoma cruzi, Trypanosoma rhodesiensei, Trypanosoma brucei, Schistosoma mansoni, Schistosoma japanicum, Babesia bovis, Elmeria tenella, Onchocerca volvulus, Leishmania tropica, Trichinella spiralis, Theileria parva, Taenia hydatigena, Taenia ovis, Taenia saginata, Echinococcus granulosus, Mesocestoides corti, Mycoplasma arthritidis, M. hyorhinis, M. orale, M. arginini, Acholeplasma laidlawii, M. salivarium, M. pneumoniae, Candida albicans, Cryptococcus neoformans, Histoplasma capsulatum, Coccidioides immitis, Blastomyces dermatitidis, Aspergillus fumigatus, Penicillium marneffei, Bacillus anthracis, Bartonella, Bordetella pertussis, Brucella — all serotypes, Chlamydia trachomatis, Chlamydia pneumoniae, Clostridium botulinum — anything from Clostridium serotypes, Haemophilus influenzae, Helicobacter pylori, Klebsiella — all serotypes, Legionella — all serotypes, Listeria, Mycobacterium — all serotypes, Mycoplasma — human and animal serotypes, Rickettsia — all serotypes, Shigella — all serotypes, Staphylococcus aureus, Streptococcus — S. pneumoniae, S. pyogenes, Vibrio cholera, Yersinia enterocolitica, and/or Yersinia pestis.

[00125] Antigens useful in the present disclosure may include those derived from parasites including, but not limited to, Ancylostomahuman hookworms, Leishmania — all strains, Microsporidium, Necator human hookworms, Onchocerca filarial worms, Plasmodium — all human strains and simian species, Toxoplasma — all strains, Trypanosoma — all serotypes, and/or Wuchereria bancrofti filarial worms. siRNA

[00126] As mentioned above, the present disclosure contemplates the use of one or more inhibitory nucleic acid for reducing expression and/or activation of a gene or gene product. Examples of an inhibitory nucleic acid include but are not limited to molecules targeted to an nucleic acid sequence, such as an siRNA (small interfering RNA), short hairpin RNA (shRNA), double- stranded RNA, an antisense oligonucleotide, a ribozyme and molecules targeted to a gene or gene product such as an aptamer.

[00127] An inhibitory nucleic acid may inhibit the transcription of a gene or prevent the translation of the gene transcript in a cell. An inhibitory nucleic acid may be from 16 to 1000 nucleotides long, and in certain embodiments from 18 to 100 nucleotides long.

[00128] Inhibitory nucleic acids are well known in the art. For example, siRNA, shRNA and double-stranded RNA have been described in U.S. Patents 6,506,559 and 6,573,099, as well as in U.S. Patent Publications 2003/0051263, 2003/0055020, 2004/0265839, 2002/0168707, 2003/0159161, and 2004/0064842, all of which are herein incorporated by reference in their entirety.

[00129] Since the discovery of RNAi by Fire and colleagues in 1998, the biochemical mechanisms have been rapidly characterized. Double stranded RNA (dsRNA) is cleaved by Dicer, which is an RNAase III family ribonuclease. This process yields siRNAs of ~21 nucleotides in length. These siRNAs are incorporated into a multiprotein RNA-induced silencing complex (RISC) that is guided to target mRNA. RISC cleaves the target mRNA in the middle of the complementary region. In mammalian cells, the related microRNAs (miRNAs) are found that are short RNA fragments (~22 nucleotides). miRNAs are generated after Dicer-mediated cleavage of longer (~70 nucleotide) precursors with imperfect hairpin RNA structures. The miRNA is incorporated into a miRNA-protein complex (miRNP), which leads to translational repression of target mRNA.

[00130] In designing a nucleic acid capable of generating an RNAi effect, there are several factors that need to be considered such as the nature of the siRNA, the durability of the silencing effect, and the choice of delivery system. To produce an RNAi effect, the siRNA that is introduced into the organism will typically contain exonic sequences. Furthermore, the RNAi process is homology dependent, so the sequences must be carefully selected so as to maximize gene specificity, while minimizing the possibility of cross-interference between homologous, but not gene-specific sequences. Particularly the siRNA exhibits greater than 80, 85, 90, 95, 98% or even 100% identity between the sequence of the siRNA and a portion of a EphA nucleotide sequence. Sequences less than about 80% identical to the target gene are substantially less effective. Thus, the greater identity between the siRNA and the gene to be inhibited, the less likely expression of unrelated genes will be affected.

[00131] In addition, the size of the siRNA is an important consideration. In some embodiments, the present disclosure relates to siRNA molecules that include at least about 19- 25 nucleotides, and are able to modulate gene expression. In the context of the present disclosure, the siRNA is particularly less than 500, 200, 100, 50, 25, or 20 nucleotides in length. In some embodiments, the siRNA is from about 25 nucleotides to about 35 nucleotides or from about 19 nucleotides to about 25 nucleotides in length.

[00132] To improve the effectiveness of siRNA-mediated gene silencing, guidelines for selection of target sites on mRNA have been developed for optimal design of siRNA (Soutschek et al., 2004; Wadhwa et al., 2004). These strategies may allow for rational approaches for selecting siRNA sequences to achieve maximal gene knockdown. To facilitate the entry of siRNA into cells and tissues, a variety of vectors including plasmids and viral vectors such as adenovirus, lentivirus, and retrovirus have been used (Wadhwa et al., 2004).

[00133] Within an inhibitory nucleic acid, the components of a nucleic acid need not be of the same type or homogenous throughout (e.g., an inhibitory nucleic acid may comprise a nucleotide and a nucleic acid or nucleotide analog). Typically, an inhibitory nucleic acid form a double-stranded structure; the double-stranded structure may result from two separate nucleic acids that are partially or completely complementary. In certain embodiments of the present disclosure, the inhibitory nucleic acid may comprise only a single nucleic acid (polynucleotide) or nucleic acid analog and form a double-stranded structure by complementing with itself (e.g., forming a hairpin loop). The double- stranded structure of the inhibitory nucleic acid may comprise 16-500 or more contiguous nucleobases, including all ranges derivable thereof. The inhibitory nucleic acid may comprise 17 to 35 contiguous nucleobases, more particularly 18 to 30 contiguous nucleobases, more particularly 19 to 25 nucleobases, more particularly 20 to 23 contiguous nucleobases, or 20 to 22 contiguous nucleobases, or 21 contiguous nucleobases that hybridize with a complementary nucleic acid (which may be another part of the same nucleic acid or a separate complementary nucleic acid) to form a double-stranded structure.

[00134] siRNA can be obtained from commercial sources, natural sources, or can be synthesized using any of a number of techniques well-known to those of ordinary skill in the art. For example, commercial sources of predesigned siRNA include Invitrogen’s StealthTM Select technology (Carlsbad, CA), Ambion®(Austin, TX), and Qiagen® (Valencia, CA). An inhibitory nucleic acid that can be applied in the compositions and methods of the present disclosure may be any nucleic acid sequence that has been found by any source to be a validated downregulator of the gene or gene product.

[00135] In some embodiments, the disclosure features an isolated siRNA molecule of at least 19 nucleotides, having at least one strand that is substantially complementary to at least ten but no more than thirty consecutive nucleotides of a nucleic acid that encodes a gene, and that reduces the expression of a gene or gene product. In one embodiment of the present disclosure, the siRNA molecule has at least one strand that is substantially complementary to at least ten but no more than thirty consecutive nucleotides of the mRNA that encodes a gene or a gene product.

[00136] In one embodiments, the siRNA molecule is at least 75, 80, 85, or 90% homologous, particularly at least 95%, 99%, or 100% similar or identical, or any percentages in between the foregoing (e.g., the disclosure contemplates 75% and greater, 80% and greater, 85% and greater, and so on, and said ranges are intended to include all whole numbers in between), to at least 10 contiguous nucleotides of any of the nucleic acid sequences encoding a target therapeutic protein.

[00137] The siRNA may also comprise an alteration of one or more nucleotides. Such alterations can include the addition of non-nucleotide material, such as to the end(s) of the 19 to 25 nucleotide RNA or internally (at one or more nucleotides of the RNA). In certain aspects, the RNA molecule contains a 3'-hydroxyl group. Nucleotides in the RNA molecules of the present disclosure can also comprise non-standard nucleotides, including non-naturally occurring nucleotides or deoxyribonucleotides. The double-stranded oligonucleotide may contain a modified backbone, for example, phosphorothioate, phosphorodithioate, or other modified backbones known in the art, or may contain non-natural internucleoside linkages. Additional modifications of siRNAs (e.g., 2'-O-methyl ribonucleotides, 2'-deoxy-2'-fluoro ribonucleotides, “universal base” nucleotides, 5-C-methyl nucleotides, one or more phosphorothioate internucleotide linkages, and inverted deoxyabasic residue incorporation) can be found in U.S. Publication 2004/0019001 and U.S. Patent 6,673,611 (each of which is incorporated by reference in its entirety). Collectively, all such altered nucleic acids or RNAs described above are referred to as modified siRNAs.

[00138] In one embodiment, siRNA is capable of decreasing the expression of a particular genetic product by at least 10%, at least 20%, at least 30%, or at least 40%, at least 50%, at least 60%, or at least 70%, at least 75%, at least 80%, at least 90%, at least 95% or more or any ranges in between the foregoing.

III. Pharmaceutical Formulations and Routes of Administration

[00139] In another aspect, for administration to a patient in need of such treatment, pharmaceutical formulations (also referred to as a pharmaceutical preparations, pharmaceutical compositions, pharmaceutical products, medicinal products, medicines, medications, or medicaments) comprise a therapeutically effective amount of a compound disclosed herein formulated with one or more excipients and/or drug carriers appropriate to the indicated route of administration. In some embodiments, the compounds disclosed herein are formulated in a manner amenable for the treatment of human and/or veterinary patients. In some embodiments, formulation comprises admixing or combining one or more of the compounds disclosed herein with one or more of the following excipients: lactose, sucrose, starch powder, cellulose esters of alkanoic acids, cellulose alkyl esters, talc, stearic acid, magnesium stearate, magnesium oxide, sodium and calcium salts of phosphoric and sulfuric acids, gelatin, acacia, sodium alginate, polyvinylpyrrolidone, and/or polyvinyl alcohol. In some embodiments, e.g., for oral administration, the pharmaceutical formulation may be tableted or encapsulated. In some embodiments, the compounds may be dissolved or slurried in water, polyethylene glycol, propylene glycol, ethanol, com oil, cottonseed oil, peanut oil, sesame oil, benzyl alcohol, sodium chloride, and/or various buffers. In some embodiments, the pharmaceutical formulations may be subjected to pharmaceutical operations, such as sterilization, and/or may contain drug carriers and/or excipients such as preservatives, stabilizers, wetting agents, emulsifiers, encapsulating agents such as lipids, dendrimers, polymers, proteins such as albumin, nucleic acids, and buffers.

[00140] Pharmaceutical formulations may be administered by a variety of methods, e.g., orally or by injection (e.g. subcutaneous, intravenous, and intraperitoneal). Depending on the route of administration, the compounds disclosed herein may be coated in a material to protect the compound from the action of acids and other natural conditions which may inactivate the compound. To administer the active compound by other than parenteral administration, it may be necessary to coat the compound with, or co-administer the compound with, a material to prevent its inactivation. In some embodiments, the active compound may be administered to a patient in an appropriate carrier, for example, liposomes, or a diluent. Pharmaceutically acceptable diluents include saline and aqueous buffer solutions. Liposomes include water-in-oil-in-water CGF emulsions as well as conventional liposomes.

[00141] The compounds disclosed herein may also be administered parenterally, intraperitoneally, intraspinally, or intracerebrally. Dispersions can be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations may contain a preservative to prevent the growth of microorganisms.

[00142] Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (such as, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, sodium chloride, or polyalcohols such as mannitol and sorbitol, in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate or gelatin.

[00143] The compounds disclosed herein can be administered orally, for example, with an inert diluent or an assimilable edible carrier. The compounds and other ingredients may also be enclosed in a hard or soft-shell gelatin capsule, compressed into tablets, or incorporated directly into the patient’s diet. For oral therapeutic administration, the compounds disclosed herein may be incorporated with excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like. The percentage of the therapeutic compound in the compositions and preparations may, of course, be varied. The amount of the therapeutic compound in such pharmaceutical formulations is such that a suitable dosage will be obtained.

[00144] The therapeutic compound may also be administered topically to the skin, eye, ear, or mucosal membranes. Administration of the therapeutic compound topically may include formulations of the compounds as a topical solution, lotion, cream, ointment, gel, foam, transdermal patch, or tincture. When the therapeutic compound is formulated for topical administration, the compound may be combined with one or more agents that increase the permeability of the compound through the tissue to which it is administered. In other embodiments, it is contemplated that the topical administration is administered to the eye. Such administration may be applied to the surface of the cornea, conjunctiva, or sclera. Without wishing to be bound by any theory, it is believed that administration to the surface of the eye allows the therapeutic compound to reach the posterior portion of the eye. Ophthalmic topical administration can be formulated as a solution, suspension, ointment, gel, or emulsion. Finally, topical administration may also include administration to the mucosa membranes such as the inside of the mouth. Such administration can be directly to a particular location within the mucosal membrane such as a tooth, a sore, or an ulcer. Alternatively, if local delivery to the lungs is desired the therapeutic compound may be administered by inhalation in a dry-powder or aerosol formulation.

[00145] In some embodiments, it may be advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the patients to be treated; each unit containing a predetermined quantity of therapeutic compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. In some embodiments, the specification for the dosage unit forms of the invention are dictated by and directly dependent on (a) the unique characteristics of the therapeutic compound and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the art of compounding such a therapeutic compound for the treatment of a selected condition in a patient. In some embodiments, active compounds are administered at a therapeutically effective dosage sufficient to treat a condition associated with a condition in a patient. For example, the efficacy of a compound can be evaluated in an animal model system that may be predictive of efficacy in treating the disease in a human or another animal.

[00146] In some embodiments, the effective dose range for the therapeutic compound can be extrapolated from effective doses determined in animal studies for a variety of different animals. In some embodiments, the human equivalent dose (HED) in mg/kg can be calculated in accordance with the following formula (see, e.g., Reagan-Shaw et al., FASEB J., 22(3): 659- 661, 2008, which is incorporated herein by reference):

HED (mg/kg) = Animal dose (mg/kg) x (Animal K_m/Human K_m)

[00147] Use of the Km factors in conversion results in HED values based on body surface area (BSA) rather than only on body mass. Km values for humans and various animals are well known. For example, the Km for an average 60 kg human (with a BSA of 1.6 m2) is 37, whereas a 20 kg child (BSA 0.8 m2) would have a Km of 25. Km for some relevant animal models are also well known, including: mice Km of 3 (given a weight of 0.02 kg and BSA of 0.007); hamster Km of 5 (given a weight of 0.08 kg and BSA of 0.02); rat Km of 6 (given a weight of 0.15 kg and BSA of 0.025) and monkey Km of 12 (given a weight of 3 kg and BSA of 0.24).

[00148] Precise amounts of the therapeutic composition depend on the judgment of the practitioner and are specific to each individual. Nonetheless, a calculated HED dose provides a general guide. Other factors affecting the dose include the physical and clinical state of the patient, the route of administration, the intended goal of treatment and the potency, stability and toxicity of the particular therapeutic formulation.

[00149] The actual dosage amount of a compound of the present disclosure or composition comprising a compound of the present disclosure administered to a patient may be determined by physical and physiological factors such as type of animal treated, age, sex, body weight, severity of condition, the type of disease being treated, previous or concurrent therapeutic interventions, idiopathy of the patient and on the route of administration. These factors may be determined by a skilled artisan. The practitioner responsible for administration will typically determine the concentration of active ingredient(s) in a composition and appropriate dose(s) for the individual patient. The dosage may be adjusted by the individual physician in the event of any complication.

[00150] In some embodiments, the therapeutically effective amount typically will vary from about 0.001 mg/kg to about 1000 mg/kg, from about 0.01 mg/kg to about 750 mg/kg, from about 100 mg/kg to about 500 mg/kg, from about 1 mg/kg to about 250 mg/kg, from about 10 mg/kg to about 150 mg/kg in one or more dose administrations daily, for one or several days (depending of course of the mode of administration and the factors discussed above). Other suitable dose ranges include 1 mg to 10,000 mg per day, 100 mg to 10,000 mg per day, 500 mg to 10,000 mg per day, and 500 mg to 1 ,000 mg per day. In some embodiments, the amount is less than 10,000 mg per day with a range of 750 mg to 9,000 mg per day.

[00151] In some embodiments, the amount of the active compound in the pharmaceutical formulation is from about 2 to about 75 weight percent. In some of these embodiments, the amount if from about 25 to about 60 weight percent.

[00152] Single or multiple doses of the agents are contemplated. Desired time intervals for delivery of multiple doses can be determined by one of ordinary skill in the art employing no more than routine experimentation. As an example, patients may be administered two doses daily at approximately 12-hour intervals. In some embodiments, the agent is administered once a day.

[00153] The agent(s) may be administered on a routine schedule. As used herein a routine schedule refers to a predetermined designated period of time. The routine schedule may encompass periods of time which are identical, or which differ in length, as long as the schedule is predetermined. For instance, the routine schedule may involve administration twice a day, every day, every two days, every three days, every four days, every five days, every six days, a weekly basis, a monthly basis or any set number of days or weeks there-between. Alternatively, the predetermined routine schedule may involve administration on a twice daily basis for the first week, followed by a daily basis for several months, etc. In other embodiments, the invention provides that the agent(s) may be taken orally and that the timing of which is or is not dependent upon food intake. Thus, for example, the agent can be taken every morning and/or every evening, regardless of when the patient has eaten or will eat.

IV. Definitions

[00154] In this disclosure, the use of the singular includes the plural, the use of the word “a” or “an” when used in conjunction with the term “comprising” in the claims and/or the specification may mean “one,” but it is also consistent with the meaning of “one or more,” “at least one,” and “one or more than one.” As used herein “another” may mean at least a second or more. As used herein, “or” means “and/or”, unless specifically stated otherwise.

[00155] As used in this specification and claim(s), the words “comprising” (and any form of comprising, such as “comprise” and “comprises”), “having” (and any form of having, such as “have” and “has”), “including” (and any form of including, such as “includes” and “include”), or “containing” (and any form of containing, such as “contains” and “contain”) are inclusive or open-ended and do not exclude additional, unrecited elements or method steps. For example, any method that “comprises,” “has” or “includes” one or more steps is not limited to possessing only those one or more steps and also covers other unlisted steps.

[00156] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described. All documents, or portions of documents, cited in this application, including, but not limited to, patents, patent applications, articles, books, and treatises, are hereby expressly incorporated herein by reference in their entirety for any purpose. In the event that one or more of the incorporated literature and similar materials defines a term in a manner that contradicts the definition of that term in this application, this application controls.

[00157] All the compounds of the present invention may in some embodiments be used for the prevention and treatment of one or more diseases or disorders discussed herein or otherwise. In some embodiments, one or more of the compounds characterized or exemplified herein as an intermediate, a metabolite, and/or prodrug, may nevertheless also be useful for the prevention and treatment of one or more diseases or disorders. As such unless explicitly stated to the contrary, all the compounds of the present invention are deemed “active compounds” and “therapeutic compounds” that are contemplated for use as active pharmaceutical ingredients (APIs). Actual suitability for human or veterinary use is typically determined using a combination of clinical trial protocols and regulatory procedures, such as those administered by the Food and Drug Administration (FDA). In the United States, the FDA is responsible for protecting the public health by assuring the safety, effectiveness, quality, and security of human and veterinary drugs, vaccines and other biological products, and medical devices.

[00158] In some embodiments, the compounds of the present invention have the advantage that they may be more efficacious than, be less toxic than, be longer acting than, be more potent than, produce fewer side effects than, be more easily absorbed than, more metabolically stable than, more lipophilic than, more hydrophilic than, and/or have a better pharmacokinetic profile (e.g., higher oral bioavailability and/or lower clearance) than, and/or have other useful pharmacological, physical, or chemical properties over, compounds known in the prior art, whether for use in the indications stated herein or otherwise.

[00159] In some embodiments, compounds of the present invention function as prodrugs or can be derivatized to function as prodrugs. Since prodrugs are known to enhance numerous desirable qualities of pharmaceuticals (e.g., solubility, bioavailability, manufacturing, etc.), the compounds employed in some methods of the invention may, if desired, be delivered in prodrug form. Thus, the invention contemplates prodrugs of compounds of the present invention as well as methods of delivering prodrugs. Prodrugs of the compounds employed in the invention may be prepared by modifying functional groups present in the compound in such a way that the modifications are cleaved, either in routine manipulation or in vivo, to the parent compound. Accordingly, prodrugs include, for example, compounds described herein in which a hydroxy, amino, or carboxy group is bonded to any group that, when the prodrug is administered to a patient, cleaves to form a hydroxy, amino, or carboxylic acid, respectively. For example, a compound comprising a hydroxy group may be administered as an ester that is converted by hydrolysis in vivo to the hydroxy compound. Nonlimiting examples of suitable esters that may be converted in vivo into hydroxy compounds include acetates, citrates, lactates, phosphates, tartrates, malonates, oxalates, salicylates, propionates, succinates, fumarates, maleates, methylene-bis-hydroxynaphthoate, gentisates, isethionates, di p toluoyl tartrates, methane-isulfonates, ethanesulfonates, benzenesulfonates, p toluenesulfonates, cyclohexyl- sulfamates, quinates, and esters of amino acids. Similarly, a compound comprising an amine group may be administered as an amide that is converted by hydrolysis in vivo to the amine compound.

[00160] In some embodiments, compounds of the present invention exist in salt or non-salt form. With regard to the salt form(s), in some embodiments the particular anion or cation forming a part of any salt form of a compound provided herein is not critical, so long as the salt, as a whole, is pharmacologically acceptable. Additional examples of pharmaceutically acceptable salts and their methods of preparation and use are presented in Handbook of Pharmaceutical Salts: Properties, and Use (2002), which is incorporated herein by reference.

[00161] It will be appreciated that many organic compounds can form complexes with solvents in which they are reacted or from which they are precipitated or crystallized. These complexes are known as “solvates.” Where the solvent is water, the complex is known as a “hydrate.” It will also be appreciated that many organic compounds can exist in more than one solid form, including crystalline and amorphous forms. All solid forms of the compounds provided herein, including any solvates thereof are within the scope of the present invention.

[00162] As used herein, the terms “drug”, “pharmaceutical”, “therapeutic agent”, and “therapeutically active agent” are used interchangeably to represent a compound which invokes a therapeutic or pharmacological effect in a human or animal and is used to treat a disease, disorder, or other condition. In some embodiments, these compounds have undergone and received regulatory approval for administration to a living creature.

[00163] An “active ingredient” (Al) or active pharmaceutical ingredient (API) (also referred to as an active compound, active substance, active agent, pharmaceutical agent, agent, biologically active molecule, or a therapeutic compound) is the ingredient in a pharmaceutical drug that is biologically active.

[00164] The term “effective,” as that term is used in the specification and/or claims, means adequate to accomplish a desired, expected, or intended result. “Effective amount,” “Therapeutically effective amount” or “pharmaceutically effective amount” when used in the context of treating a patient or subject with a compound means that amount of the compound which, when administered to the patient or subject, is sufficient to effect such treatment or prevention of the disease as those terms are defined below.

[00165] An “excipient” is a pharmaceutically acceptable substance formulated along with the active ingredient(s) of a medication, pharmaceutical composition, formulation, or drug delivery system. Excipients may be used, for example, to stabilize the composition, to bulk up the composition (thus often referred to as “bulking agents,” “fillers,” or “diluents” when used for this purpose), or to confer a therapeutic enhancement on the active ingredient in the final dosage form, such as facilitating drug absorption, reducing viscosity, or enhancing solubility. Excipients include pharmaceutically acceptable versions of antiadherents, binders, coatings, colors, disintegrants, flavors, glidants, lubricants, preservatives, sorbents, sweeteners, and vehicles. The main excipient that serves as a medium for conveying the active ingredient is usually called the vehicle. Excipients may also be used in the manufacturing process, for example, to aid in the handling of the active substance, such as by facilitating powder flowability or non-stick properties, in addition to aiding in vitro stability such as prevention of denaturation or aggregation over the expected shelf life. The suitability of an excipient will typically vary depending on the route of administration, the dosage form, the active ingredient, as well as other factors.

[00166] The term “hydrate” when used as a modifier to a compound means that the compound has less than one (e.g., hemihydrate), one (e.g., monohydrate), or more than one (e.g., dihydrate) water molecules associated with each compound molecule, such as in solid forms of the compound.

[00167] As used herein, the term “TC50” refers to an inhibitory dose which is 50% of the maximum response obtained. This quantitative measure indicates how much of a particular drug or other substance (inhibitor) is needed to inhibit a given biological, biochemical or chemical process (or component of a process, i.e. an enzyme, cell, cell receptor or microorganism) by half.

[00168] As used herein, the term “patient” or “subject” refers to a living mammalian organism, such as a human, monkey, cow, sheep, goat, dog, cat, mouse, rat, guinea pig, or transgenic species thereof. In certain embodiments, the patient or subject is a primate. Nonlimiting examples of human patients are adults, juveniles, infants and fetuses.

[00169] As generally used herein “pharmaceutically acceptable” refers to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues, organs, and/or bodily fluids of human beings and animals without excessive toxicity, irritation, allergic response, or other problems or complications commensurate with a reasonable benefit/risk ratio.

[00170] “Pharmaceutically acceptable salts” means salts of compounds disclosed herein which are pharmaceutically acceptable, as defined above, and which possess the desired pharmacological activity. Such salts include acid addition salts formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like; or with organic acids such as 1,2 ethanedisulfonic acid, 2 hydroxy ethanesulfonic acid, 2 naphthalenesulfonic acid, 3 phenylpropionic acid, 4,4' methylenebis(3 hydroxy 2 ene-1 carboxylic acid), 4 methylbicyclo[2.2.2]oct 2 ene-1 carboxylic acid, acetic acid, aliphatic mono- and dicarboxylic acids, aliphatic sulfuric acids, aromatic sulfuric acids, benzenesulfonic acid, benzoic acid, camphorsulfonic acid, carbonic acid, cinnamic acid, citric acid, cyclopentanepropionic acid, ethanesulfonic acid, fumaric acid, glucoheptonic acid, gluconic acid, glutamic acid, glycolic acid, heptanoic acid, hexanoic acid, hydroxynaphthoic acid, lactic acid, laurylsulfuric acid, maleic acid, malic acid, malonic acid, mandelic acid, methanesulfonic acid, muconic acid, o (4 hydroxybenzoyl)benzoic acid, oxalic acid, p chlorobenzenesulfonic acid, phenyl-substituted alkanoic acids, propionic acid, p toluenesulfonic acid, pyruvic acid, salicylic acid, stearic acid, succinic acid, tartaric acid, tertiarybutylacetic acid, trimethylacetic acid, and the like. Pharmaceutically acceptable salts also include base addition salts which may be formed when acidic protons present are capable of reacting with inorganic or organic bases. Acceptable inorganic bases include sodium hydroxide, sodium carbonate, potassium hydroxide, aluminum hydroxide and calcium hydroxide. Acceptable organic bases include ethanolamine, diethanolamine, triethanolamine, tromethamine, N methylglucamine and the like. It should be recognized that the particular anion or cation forming a part of any salt of this invention is not critical, so long as the salt, as a whole, is pharmacologically acceptable. Additional examples of pharmaceutically acceptable salts and their methods of preparation and use are presented in Handbook of Pharmaceutical Salts: Properties, and Use (P. H. Stahl & C. G. Wermuth eds., Verlag Helvetica Chimica Acta, 2002).

[00171] A “pharmaceutically acceptable carrier,” “drug carrier,” or simply “carrier” is a pharmaceutically acceptable substance formulated along with the active ingredient medication that is involved in carrying, delivering and/or transporting a chemical agent. Drug carriers may be used to improve the delivery and the effectiveness of drugs, including for example, controlled-release technology to modulate drug bioavailability, decrease drug metabolism, and/or reduce drug toxicity. Some drug carriers may increase the effectiveness of drug delivery to the specific target sites. Examples of carriers include: lipid nanoparticles, liposomes, microspheres (e.g., made of poly(lactic-co-glycolic) acid), albumin microspheres, synthetic polymers, nanofibers, protein-DNA complexes, protein conjugates, erythrocytes, virosomes, and dendrimers. [00172] A “pharmaceutical drug” (also referred to as a pharmaceutical, pharmaceutical preparation, pharmaceutical composition, pharmaceutical formulation, pharmaceutical product, medicinal product, medicine, medication, medicament, or simply a drug, agent, or preparation) is a composition used to diagnose, cure, treat, or prevent disease, which comprises an active pharmaceutical ingredient (API) (defined above) and optionally contains one or more inactive ingredients, which are also referred to as excipients (defined above).

[00173] The term “identity” refers to a relationship between the sequences of two or more polypeptide molecules or two or more nucleic acid molecules, as determined by aligning and comparing the sequences. “Percent identity” means the percent of identical residues between the amino acids or nucleotides in the compared molecules and is calculated based on the size of the smallest of the molecules being compared. For these calculations, gaps in alignments (if any) are preferably addressed by a particular mathematical model or computer program (i.e., an “algorithm”). Methods that can be used to calculate the identity of the aligned nucleic acids or polypeptides include those described in Computational Molecular Biology, (Lesk, A. M., ed.), 1988, New York: Oxford University’ Press; Biocomputing Informatics and Genome Projects, (Smith, D. W., ed.), 1993, New York: Academic Press; Computer Analysis of Sequence Data, Part I, (Griffin, A. M., and Griffin, H. G., eds.), 1994, New Jersey: Humana Press; von Heinje, G., 1987, Sequence Analysis in Molecular Biology, New York: Academic Press; Sequence Analysis Primer, (Gribskov, M. and Devereux, J., eds.), 1991, New York: M. Stockton Press; and Carillo et al., 1988, SIAM J. Applied Math. 48:1073.

[00174] In calculating percent identity, the sequences being compared are typically aligned in a way that gives the largest match between the sequences. One example of a computer program that can be used to determine percent identity is the GCG program package, which includes GAP (Devereux et al., 1984, Nucl. Acid Res. 12:387; Genetics Computer Group, University of Wisconsin, Madison, Wis.). The computer algorithm GAP is used to align the two polypeptides or polynucleotides for which the percent sequence identity is to be determined. The sequences are aligned for optimal matching of their respective amino acid or nucleotide (the “matched span”, as determined by the algorithm). A gap opening penalty (which is calculated as 3x the average diagonal, wherein the “average diagonal” is the average of the diagonal of the comparison matrix being used; the “diagonal” is the score or number assigned to each perfect amino acid match by the particular comparison matrix) and a gap extension penalty (which is usually 1/10 times the gap opening penalty), as well as a comparison matrix such as PAM 250 or BLOSUM 62 are used in conjunction with the algorithm. In certain embodiments, a standard comparison matrix (see, Dayhoff et al., 1978, Atlas of Protein Sequence and Structure 5:345-352 for the PAM 250 comparison matrix; Henikoff et al., 1992, Proc. Natl. Acad. Sci. U.S.A. 89:10915-10919 for the BLOSUM 62 comparison matrix) is also used by the algorithm.

[00175] Examples of parameters that can be employed in determining percent identity for polypeptides or nucleotide sequences using the GAP program can be found in Needleman etal., 1970, J. Mol. Biol. 48:443-453.

[00176] Certain alignment schemes for aligning two amino acid sequences may result in matching of only a short region of the two sequences, and this small aligned region may have very high sequence identity even though there is no significant relationship between the two full-length sequences. Accordingly, the selected alignment method (GAP program) can be adjusted if so desired to result in an alignment that spans at 50 or fewer of contiguous amino acids of the target peptide or polypeptide.

[00177] As used in this specification, the term “significant” (and any form of significant such as “significantly”) is not meant to imply statistical differences between two values but only to imply importance or the scope of difference of the parameter.

[00178] Throughout this application, the term “about” is used to indicate that a value includes the inherent variation of error for the device, the method being employed to determine the value, or the variation that exists among the study subjects or experimental studies. Unless another definition is applicable, the term “about” refers to ±10% of the indicated value.

[00179] As used herein, the term “substantially free of’ or “substantially free” in terms of a specified component, is used herein to mean that none of the specified component has been purposefully formulated into a composition and/or is present only as a contaminant or in trace amounts. The total amount of all containments, by-products, and other material is present in that composition in an amount less than 2%. The term “more substantially free of” or “more substantially free” is used to represent that the composition contains less than 1 % of the specific component. The term “essentially free of’ or “essentially free” contains less than 0.5% of the specific component. [00180] As used herein, “Treatment” or “treating” includes (1) inhibiting a disease in a subject or patient experiencing or displaying the pathology or symptomatology of the disease (e.g., arresting further development of the pathology and/or symptomatology), (2) ameliorating a disease in a subject or patient that is experiencing or displaying the pathology or symptomatology of the disease (e.g., reversing the pathology and/or symptomatology), and/or (3) effecting any measurable decrease in a disease or symptom thereof in a subject or patient that is experiencing or displaying the pathology or symptomatology of the disease.

[00181] As used herein, and unless otherwise indicated, the terms “prevent,” “preventing,” and “prevention” includes: (1) inhibiting the onset of a disease in a subject or patient which may be at risk and/or predisposed to the disease but does not yet experience or display any or all of the pathology or symptomatology of the disease, and/or (2) slowing the onset of the pathology or symptomatology of a disease in a subject or patient which may be at risk and/or predisposed to the disease but does not yet experience or display any or all of the pathology or symptomatology of the disease.

[00182] The term “unit dose” refers to a formulation of the compound or composition such that the formulation is prepared in a manner sufficient to provide a single therapeutically effective dose of the active ingredient to a patient in a single administration. Such unit dose formulations that may be used include but are not limited to a single tablet, capsule, or other oral formulations, a single vial with a syringeable liquid or other injectable formulations, or a dose that is formulated for administration in a single inhalation.

[00183] As used herein, a “targeting peptide” is a peptide comprising a contiguous sequence of amino acids, which is characterized by localization or penetration to an organ, tissue or cell type. Localization or penetration may be determined, for example, by methods disclosed below, wherein the putative targeting peptide sequence is incorporated into a protein that is displayed on the outer surface of a phage. Exposure of a library of such phage that have been genetically engineered to express a multitude of such targeting peptides of different amino acid sequence is followed by collection of one or more organs, tissues or cell types and identification of phage found in that organ, tissue or cell type. A phage expressing a targeting peptide sequence is considered to be selectively localized to or is considered to selectively target an organ, tissue or cell type if it exhibits greater binding or penetration in that organ, tissue or cell type compared to a control organ, tissue or cell type. Selective localization or selective targeting of the peptide includes but is not limited to increased uptake in the targeted organ, tissue, or cell type in comparison to a control organ, tissue, or cell type. Preferably, localization of a targeting peptide should result in a two-fold or higher enrichment of the phage in the target organ, tissue or cell type, compared to a control organ, tissue or cell type. Localization resulting in at least a three-fold, four-fold, five-fold, six-fold, seven-fold, eight-fold, nine-fold, ten-fold or higher enrichment in the target organ, tissue, or cell type compared to a control organ, tissue or cell type is more preferred. Alternatively, a phage expressing a targeting peptide sequence that exhibits localization preferably shows an increased enrichment in the target organ, tissue, or cell type when phage recovered from the target organ, tissue, or cell type are re-injected into a second amount of organ, tissue, or cell type for another round of screening. Further enrichment may be exhibited following a third round, a fourth round, or a fifth round of screening. Another alternative means to determine localization is that phage expressing the putative target peptide preferably exhibit a two-fold, more preferably a three-fold or higher enrichment in the target organ compared to control phage that express a non-specific peptide or that have not been genetically engineered to express any putative target peptides. “Targeting peptide” and “homing peptide” are used synonymously herein.

[00184] As used herein “phage display library” means a collection of phage that have been genetically engineered to express a set of putative targeting peptides on their outer surface. In preferred embodiments, DNA sequences encoding the putative targeting peptides are inserted in frame into a gene encoding a phage capsule protein. In other preferred embodiments, the putative targeting peptide sequences are in part random mixtures of all twenty amino acids and in part non-random. In certain preferred embodiments the putative targeting peptides of the phage display library exhibit one or more cysteine residues at fixed locations within the targeting peptide sequence. Cysteines may be used, for example, to create a cyclic peptide.

[00185] As used herein, the term “nanoparticle” has its customary and ordinary definition and refers to discrete particles which behave as a whole unit rather than as individual molecules within the particle. A nanoparticle may have a size from about 1 to about 10,000 nm with ultrafine nanoparticles having a size from 1 nm to 100 nm, fine particles having a size from 100 nm to 2,500 nm, and coarse particles having a size from 2,500 nm to 10,000 nm. In some embodiments, the nanoaggregates described herein may comprise a composition of multiple nanoparticles and have a size from about 10 nm to about 100 pm. [00186] Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the invention are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. Any numerical value, however, inherently contain certain errors necessarily resulting from the standard deviation found in their respective testing measurements and parameters.

V. Examples

[00187] To facilitate a better understanding of the present disclosure, the following examples of specific embodiments are given. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function well in the practice of the disclosure, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the disclosure. In no way should the following examples be read to limit or define the entire scope of the disclosure. Throughout the Examples provided below, certain lipid nanoparticle formulations may be denoted by multiple names. See Table 7.

Example 1: Biopanning

[00188] Bacteriophage (phage) display technology was used to screen and select for mucus-penetrating, cell-penetrating peptides using primary human bronchial epithelial cells (pHBECs) from cystic fibrosis (CF) patients containing the AF508 mutation that were cultured at air- liquid interface (ALI) (FIG. 1). At ALI, these cells produce mucus and reflect CF disease pathology, and as such, without being bound by theory, peptides selected under these conditions have both mucus penetration and cell penetration capabilities. Therefore, the methods described herein to identify mucus or cell penetrating peptides represent a more clinically relevant selection model compared to, for example, previously utilized mucus-only models. Peptide displaying phage that were taken up by primary cells were collected and their DNA was isolated and submitted for high throughput sequencing using Next Generation Sequencing. A custom python script was used to analyze thousands of sequences to identify peptide sequences that were responsible for the enrichment of specific phage that were able to overcome both mucus and cell transport. The unique sequences were identified and then further validated by cloning back into T7 phage. Validation studies, as discussed below and supported by the drawings, demonstrated that phage displaying these selected peptide sequences were able to better penetrate mucus producing primary cells when compared to phage displaying no peptides on its surface.

[00189] For the enrichment process, a cysteine constrained random 7-amino acid peptide T7 phage display library (CX7C) was repeatedly screened against mucus producing and well- differentiated pHBECs through iterative, high-throughput selection. In some embodiments, one library stock of T7 library was used as described below. In other embodiments, two stocks of T7 library were combined for use as described below. The pHBECs were pooled from seven different CF patients with the prevalent AF508 mutation and grown at ALI according to Pneumacult™ Ex-Plus and ALI media protocols (StemCell Technologies; Vancouver, BC). Cells were fully differentiated by 21- days post airlift (indicated by ciliation and mucus production which was observed by microscopy). In some embodiments, there were four iterative selection cycles. In other embodiments, there were five iterative selection cycles. After each round of selection, a portion of the collected phage internalized by the cells was quantified via standard double-layer plaque assay, and the remaining phage were amplified. DNA was isolated from amplified phage, prepared for next generation sequencing (NGS), and peptide sequence analysis was performed as previously reported. (Leal et al 2020; Mohanty et al 2019). Further details regarding the selection strategy described above are provided in Table 1 and are illustrated in FIG. 2. Certain peptide-displaying clones were selected for further validation in cell uptake studies, as described below.

Table 1: Selection details for enriching phage peptide-presenting library for mucus penetration and cellular uptake.

Round Incubation Wash Steps MOI (vg/cell)

1* 16 h Elution Buffer (EB) x 20 min 1000

-> 7 DPBS Wash steps

2* h EB x 20 min 4 DPBS x 20 min 1000

-> 6 additional DPBS washes

3* 1 h EB x 20 min DPBS x 20 min 1000

6 additional DPBS washes

4 1 h EB x 20 min -> DPBS x 20 min 1000

6 additional DPBS washes

5 1 h EB x 20 min 4 DPBS x 2 min 1000

6 additional DPBS washes

* Selection pressure was applied after round 1, round 2, and round 3. Four-round selection strategies according to the present disclosure follow, for example, the conditions identified for rounds 1 -4; Five-round selection strategies according to the present disclosure follow, for example, the conditions identified for rounds 1-5. DPBS= Dulbecco’s Phosphate-Buffered Saline. Example 2: Characterization and Validation - Peptides Identified After Four Selection Rounds

[00190] A phage library with four iterative selection rounds led to an average of —231- fold enrichment of the CX7C library across three replicates (FIG. 3). Eight clones were selected for validation of CF pHBEC uptake (FIG. 4), of which four clones demonstrated significantly enhanced CF pHBEC uptake compared to naive library (phage library without selection) and peptide-less phage controls and two further sequences demonstrated significantly enhanced CF pHBEC uptake in comparison to a naive library (phage library without selection) control. The selectivity of sequences that were demonstrated significantly enhanced CF pHBEC uptake in comparison to both controls were further assayed to determine selectivity for pHBECs in comparison to THP-1 macrophages. Clones 14 and 26 (FIG. 5) demonstrated significantly higher uptake in primary CF cells than non-polarized macrophages, MO, which are differentiated from THP-1 cell line. Next, the effect of scrambling the sequence of the sequences that were demonstrated to have significantly enhanced CF pHBEC uptake in comparison to both controls was investigated (FIG. 6). Clone 9 and Clone 26 were found to demonstrate improved uptake in primary CF cells versus their scrambled counterparts.

Example 3: Characterization and Validation - Peptides Identified After Five Selection Rounds

[00191] In a separate optimized enrichment process, five rounds of iterative selection led to an average of ~380-fold enrichment of the CX7C library across three replicates (FIG. 7). From high throughput sequencing results, the physicochemical properties of the top 30 most abundant peptides from each replicate of the five-round selection were further analyzed by calculating the net charge and hydropathicity after rounds 1 and 5. A significant difference in the weighted means of both net charge and hydropathicity was observed between the peptides of round 1 and round 5 (FIG. 8), further validating the selection process. The mean net charge of the peptides analyzed after five rounds of selection was higher than the mean net charge of the peptides analyzed after one round of selection (FIG. 9A).

[00192] The weighted mean GRAVY score of the peptides analyzed after five rounds of selection was more negative than the weighted mean GRAVY score of the peptides analyzed after one round of selection (FIG. 8B), corresponding with improved hydrophilicity. There is evidence known in the art that increased hydrophilicity is associated with improved mucus diffusion (Leal et al., 2020, Kumari et al., 2022). Therefore, the present application provides evidence that the peptides disclosed herein have, without being bound by theory, improved mucus diffusion. The Seq2Logo tool was used to visualize a multiple sequence alignment for the top 30 sequences of all replicates (N=3) after the 5th round and identify the amino acid frequency at each position of the 7-mer peptide sequence (Thomsen & Nielsen, 2012). The consensus sequence was observed to be rich in the basic and hydrophilic amino acids arginine and lysine, which further supported, without being bound, observed trends for net charge and GRAVY scores (FIG. 9C). Interestingly, this consensus differed slightly from earlier panning results, where less arginine and lysine were present (FIG. 9D).

[00193] It is worth noting that this enrichment was a significant improvement from the four-round selection strategy described above, which was 231 -fold after the final round of panning (FIG. 4). In the earlier panning strategy, four rounds of panning were completed and more variability was observed amongst the three replicates (FIG. 10). Furthermore, it was found that under the conditions used in Example 2, the addition of a fifth round did not improve enrichment or reproducibility (FIG. 10, top). Importantly, without being bound, a significant improvement in enrichment and reproducibility was observed amongst all replicates from initial panning methods (Table 1, FIG. 3). Without being bound by theory, the stringency in the selection process described in Example 3, in addition to optimized phage collection methods, was responsible for the improvement. Firstly, after each round, extensive wash steps were performed, to adequately remove bound phage from the apical surface of ALI cell cultures. The probability of collecting only internalized phage from pHBECS was increased by spinning down lysed cells and only collecting phage that remained in the lysate supernatant. Previously, phage collected from complete cell lysate (containing both cell debris and lysate supernatant) were quantified. The previous method was not optimal, without being bound by theory, since it increased the chances of enriching phage that are strongly bound to the cell surface (despite the multiple wash and elution steps). Lastly, internalized phage from all three replicates were pooled prior to each subsequent round. Before, internalized phage from each replicate were collected and amplified separately before being added onto pHBECs for the subsequent round. Surprisingly, pooling after each round resulted in improved reproducibility and enrichment profiles. Failure to pool even the last two rounds led to a decrease in total enrichment and noticeable differences in reproducibility (FIG. 10, bottom). [00194] Six sequences that were present in all three replicates after 5 rounds of selection (of the top 10 most abundant sequences among all three replicates) were identified, engineered into T7, and validated with ALI cell uptake assays (FIG. 11, FIG. 12). Sequence information for the six sequences, along with physicochemical data, including net charge and GRAVY score of each sequence, are presented in Table 2. Table 3 provides the top ten most abundant sequences from each of the three replicates that were analyzed to identify the six sequences as mentioned above. Each clone showed an increase in their abundance, i.e., percentage of total sequences identified from NGS analysis after each subsequent round (FIG. 11). The enrichment of relative frequency of individual clones ranged from ~7.7 (Clone B) to 13.7 (Clone C) from the first round to fifth round. Except for Clone F, net charges were 2.9 and GRAVY scores were less than -2.0. Clone F had a net charge and GRAVY score of 0.9 and -0.26, respectively (Table 2). Four clones demonstrated significantly higher uptake in primary CF HBEs at ALI versus an unselected CX7C library control. In addition, three of those four clones also demonstrated significantly higher uptake compared to a peptide-less “WT” phage control.

[00195] Clones identified after five selection rounds and clones identified after four selection rounds were validated and compared (FIG. 12, FIG. 13). Clone C (SEQ ID NO: 11) was found to be the most enriched in pHBECs, whereas clone 14 (SEQ ID NO: 6) was the second-most enriched in pHBECs in these validation studies. From the validation study of the six clones identified after five selection rounds (clones A-F of Table 2), clones B-E demonstrated a significant difference in uptake compared to an unselected naive library control (p<0.05) (FIG. 12). Additionally, Clones B, C, and E had significantly improved uptake compared to a peptide insertless “WT” control and an internal control sequence (denoted as “CPS”). Clone CPS was previously known to have improved diffusion through CF-like mucus (Leal et al., 2020. The best performing clone, Clone C, demonstrated a ~454-fold (p<0.0005), 56-fold (p<0.005), and 50-fold (p<0.005) improvement in uptake compared to Naive library, WT, and Clone CPS, respectively. To validate that Clone C outperformed top clones discovered after four rounds of selection (see Example 2), pHBEC uptake between Clone C and two top performing clones identified after four rounds of selection were compared and found a 2.3-fold and 17.2-fold improvement in uptake for clones that had been subjected to an additional round of selection (FIG. 13).

Table 2: T7 phage clones selected for validation and the physicochemical properties of their displayed peptides. Table 3: Top 10 clones from each replicate following optimized selection methods.

Example 4: Peptide-LNP Compositions

[00196] The present disclosure also provides for the formation of a peptide- mRNA lipid nanoparticle (LNP) system. After identification and validation of the clones after four and five selection rounds, the lead candidate peptide was conjugated with myristic acid and incorporated into formulated lipid nanoparticles (LNPs) encapsulating Nanoluciferase (NLuc) reporter mRNA using microfluidic mixing. Optimization of the formulation identified a lipid nanoparticle composition comprising 6.25% peptide as preferable for further use (FIG.

14 and FIG. 15). The LNP formulations used to determine the preferable peptide-lipid nanoparticle composition are provided in Table 4 and Table 5. Dynamic light scattering and a modified Ribogreen Assay were used to determine size and mRNA encapsulation efficiency, respectively (FIG. 16). Peptide-LNP compositions comprising a peptide as disclosed herein were homogeneous in size, and all formulations had diameter sizes below 80 nanometers (FIG.

16, left). The polydispersity index (PDI), which approximates the monodispersity of the formulations, was highest for the PEG formulation, while all other formulations had PDIs < 0.2 (FIG. 16, left). Next, encapsulation efficiencies (EEs) were characterized; all but the PEG

LNP formulation resulted in EEs > 90% (FIG. 16, right). Lastly, zeta potentials were measured for all four formulations. It was observed that an LNP composition comprising a presently disclosed peptide resulted in the most positive zeta potential at pH 7 (FIG. 16, center).

Table 4: Determination of highest peptide-lipid concentration

“Modema” is a peptideless control. Modema_25 and Moderna_50 are formulated with presently disclosed peptide C (also referenced herein as CTS). N/P

= nitrogen to phosphate ratio; CIL = cationic ionizable lipid; PEG = polyethylene glycol; DMG-PEG = l,2-dimyristoyl-rac-glycero-3-methoxypolyethylene

5 glycol-2000; DSPC = Distearoylphosphatidylcholine; Myr = myristoyl group.

Table 5: LNP formulations for optimizing peptide-LNP composition formulation

“Modema” is a peptideless control. Moderna_6.25, Modema_12.5, Modema_18.75, and Modema_25 are formulated with presently disclosed peptide C

(also referenced herein as CTS). N/P = nitrogen to phosphate ratio; CIL = cationic ionizable lipid; PEG = polyethylene glycol; DM G- PEG = 1,2-dimyristoyl- 5 rac-glycero-3-methoxypolyethylene glycol-2000; DSPC = Distearoylphosphatidylcholine; Myr = myristoyl group.

Example 5: Peptide-functionalized LNPs enhance mRNA expression

[00197] Peptide-functionalized and control LNPs (formulations shown in Table 6) were used to deliver Nluc to primary CF cells and THP-1 derived macrophages in order to demonstrate targeted delivery. Reporter expression was quantified via standardized luciferase assay.

[00198] First, NLuc-LNP compositions were delivered to differentiated pHBECs (i.e., cultured at ALI) and incubated for 48h before measuring bioluminescence. Peptide-LNP compositions disclosed herein demonstrated targeted uptake with significantly higher relative reporter expression in CF epithelia compared all other formulations, including a relevant peptide-less LNP control (Moderna’s LNP composition Spikevax with NLuc mRNA, see Table 7) and, surprisingly, a peptide-LNP composition comprising a previously known mucus-penetrating peptide (Modema_CPS_6.25 of Table 6) (FIG. 16). More specifically, a 10.5-fold and 4.5-fold increase in luminescence was observed from mRNA delivered by an LNP comprising a presently disclosed peptide compared to mRNA delivered by LNPs with additional PEG or a composition comprising a known mucus -penetrating peptide but no presently disclosed peptide, respectively (FIG. 17). Additionally, cells transfected with an LNP composition according to the present disclosure demonstrated 7.8- fold higher bioluminescence than Moderna’s Spikevax LNP (FIG. 17)

[00199] In some embodiments, the LNP compositions of the present disclosure demonstrate preferential transfection in primary HBECs. LNP transfection was measured in an alternative cell model, a THP-1 derived macrophage cell line. Using a similar incubation time (i.e., 48 hours) to the validation study on primary human bronchial epithelial cells (pHBECs) described above, administration of LNP compositions of the present disclosure resulted in significantly lower Nluc bioluminescence (1.7-fold) compared to administration of Moderna/Spikevax LNPs. Bioluminescence from cells transfected with LNP compositions of the present disclosure was slightly lower than for cells transfected with an LNP formulation comprising a known mucus-penetrating peptide (CPS) and slightly higher (but not statistically different) than the group treated with the PEG LNP formulation (FIG. 18).

Example 5: Peptide-functionalized LNPs enhance mRNA expression in vivo

[00200] In some embodiments, LNP compositions of the present disclosure enhance delivery and NLuc bioactivity in vivo in BALB/c mice. In order to investigate the delivery of peptide-LNP mRNA in vivo, peptide-LNP compositions were prepared with nanoLuc mRNA. The prepared control LNPs and peptide-LNPs comprising peptides as identified according to details provided above were dialyzed against lx PBS for four hours, then characterized by Ribogreen Assay to determine encapsulation efficiency. The LNPs and peptide-LNPs were delivered intratracheally to balb/c mice. The theoretical dose was 0.5 pg mRNA per mouse. At the 24 hour timepoint, each lung was harvested and separated into its five separate lobes (i.e. , left lung, right cranial lobe, right accessory lobe, right caudal lobe, and right middle lobe). Bioluminescence was confirmed via IVIS imaging (FIG. 19). A similar trend to in vitro ALI transfection study was observed; cells transfected with a presently disclosed LNP composition showed the highest bioluminescence, followed by the LNP formulation comprising a known mucus -penetrating peptide (CPS), the peptide-less Moderna/Spikevax LNP formulation and lastly PBS group (FIG. 21). Compared to the peptideless Moderna/Spikevax LNP formulations, presently disclosed peptide-LNP compositions demonstrated 4.4-fold higher bioluminescence. Notably, bioluminescence signal was distributed throughout all lobes for the group treated with presently disclosed peptide-LNP compositions (FIG. 20).

Table 6: Formulations of peptide-LNP and control LNPs

“CTS” denotes

CTSTRKKQC (Clone C, SEQ ID 11) ; “CPS” denotes CPSSSREKC (SEQ ID 17) . “Modema” is a peptideless control. Moderna_CTS_6.25 is

5 formulated with presently disclosed peptide C. Modema_PEG control is a peptideless control with increased PEG concentration (Huckaby et al, 2018). Moderna_CPS_6.25 is a peptide-LNP control comprising previously reported peptide (Leal et al., 2020). N/P = nitrogen to phosphate ration; CIL = cationic ionizable lipid; PEG = polyethylene glycol; DMG-PEG = l,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000; DSPC = Distcaroylphosphatidylcholinc; Myr = myristoyl group.

Table 7: Equivalent Nomenclature of Formulations described in the present disclosure

10 In the present disclosure, LNP compositions with the same formulation may be referenced herein by multiple names. Each row of the table provides alternative, equivalent names for the same formulation. The left column corresponds to the formulation as described in Table 6. Unless otherwise noted, “Modema” and “Spikevax” are generally interchangeable herein within the nomenclature of presently disclosed lipid nanoparticle formulations. For example, formulations labeled according to the template Formulation_%Peptide -lipid (see for example FIG. 14 and FIG. 15), “Moderna” and “Spikevax” are interchangeable. Therefore, the LNP composition labeled as “Moderna_6.25” of FIG. 15 may also equivalently be referenced herein as “Spikevax_6.25”, or vice-versa. “Peptide C” as used herein is equivalent to “Moderna_CTS_6.25”

15 (Table 1), “CTS” (for example, FIG. 17), and synonyms thereof as shown in the table above.

Example 6: Peptide-lipids can be incorporated into multiple classes of lipid nanoparticles and enhance transfection in an alternative ALI model

[00201] In some embodiments, presently disclosed peptide-LNP compositions may comprise a peptide selected from a variety of peptides according to the present disclosure, such as those identified according to panning protocols described above. Presently disclosed peptide-LNP compositions may comprise an LNP selected from a variety of LNP formulations known in the art. For example, a peptide of the present disclosure was incorporated into the three currently FDA approved LNP formulations (Onpattro, Comirnaty, and Spikevax LNPs) as well as a previously known formulation for nebulization termed Bl (Lewis el al., 2023). Size, polydispersity index (PDI), and zeta potential values were quantified through DLS (FIG 23A, FIG. 23C). These peptide-LNP compositions of the present disclosure were observed to have a size less than 150 nm and PDI less than or equal to 0.2. Presently disclosed LNP compositions resulted in a shift to more positive zeta potential values when compared to their respective LNP control formulation, which did not comprise a presently disclosed peptide. Lastly, the encapsulation efficiencies of all formulations were evaluated using a modified Quant- it™ RiboGreen RNA Assay. It was found that the encapsulation efficiencies of the peptide-LNP compositions described in this section were greater than 80% (FIG. 23B).

[00202] In some embodiments, presently disclosed peptide-LNP compositions enhance transfection efficiency for various LNP classes. An alternative ALI model with Calu-3 cells was used to determine if such enhanced transfection efficiency could be observed. At ALI, Calu-3 cells were observed to demonstrate lower autofluorescence, making this model more useful to complete flow cytometry studies with a GFP mRNA reporter. 1 pg of eGFP mRNA was delivered to ALI cells and after 24 hours, the percentage of cells expressing GFP was determined through flow cytometry. Delivery of the eGFP mRNA via presently disclosed peptide-LNP compositions significantly enhanced the percentage of cells expressing GFP for all four formulation classes without affecting cell viability compared to a PBS control (FIG. 23D, FIG. 23E). Given that the inclusion of presently disclosed peptides into the Spikevax/Modema formulation enhanced reporter bioactivity for both Calu-3 and pHBECs at ALI, transfection efficiency in submerged pHBECs was compared. Interestingly, the presently disclosed peptide-LNPs were not observed to significantly enhance transfection in submerged cells, which supports the notion, without being bound by theory, that these peptides were enriched specifically for differentiated cell uptake (FIG. 24). Example 7: Peptide-lipids can enhance gene editing in vivo

[00203] In some embodiments, peptide-LNP compositions of the present disclosure can be applied to gene editing in vivo. Ai9 mice, which contain a LoxP flanked STOP cassette that prevents expression of a TdTomato reporter gene downstream (Madisen el al. , 2010), were used to demonstrate this property of the presently disclosed peptide-LNP compositions. When Cre recombinase is introduced, it excises the STOP cassette and TdTomato is expressed. This conditional expression is seen as a proxy for gene editing since cells that are effectively edited will express TdTomato and fluoresce. Here, Cre recombinase mRNA (Cre mRNA) was intratracheally delivered using peptide-LNP compositions according to the present disclosure to Ai9 mice whose lungs were harvested for flow cytometry analysis 72 hours following peptide-LNP composition administration. For mice treated with peptide-LNP compositions of the present disclosure (Peptide C; FIG. 25; see also Table 6 and Table 7), significantly higher TdTomato bioactivity in epithelia was observed compared to PBS treated mice (FIG. 25A). Also, 6.37-fold higher expression in epithelia was observed upon administration of presently disclosed peptide-LNP compositions in comparison to immune cells (FIG. 25A). Non-targeted cell types (i.e., endothelia and immune cells) did not exhibit significantly higher tdTomato-i- cells than PBS treated mice (FIG. 25A). Following these results, delivery to basal cells was investigated. Statistically higher editing in basal cells following pulmonary delivery was observed for presently disclosed peptide-LNP compositions (Peptide C; see also Table 7) in comparison to a PBS treated control (FIG. 25B; p=0.03}.

Example 8: Peptide-lipid conjugate is crucial for mRNA bioactivity

[00204] A competition assay was performed by pre-incubating differentiated pHBECs with a peptide-lipid conjugate comprising a presently disclosed peptide prior to adding NLuc LNP compositions (either peptide-less control LNP or a presently disclosed peptide LNP composition). If a peptide-lipid undergoes receptor mediated endocytosis, it follows that adding the peptide-lipid in excess could potentially saturate cell surface receptors and hinder uptake of LNPs added thereafter, leading to a decrease in or lower observed NLuc bioactivity. A ~59.3-fold decrease in NLuc bioactivity was observed for cells that were pre-treated with peptide-lipid comprising a presently disclosed peptide followed by treatment with an NLuc peptide-LNP composition of the present disclosure (Spikevax CTS; FIG. 26B right; see also Table 7). For peptide-lipid pretreated cells that were subsequently administered a peptide-less LNP formulation, the decrease in NLuc bioactivity was markedly less at ~6.1-fold (Spikevax/Moderna; FIG. 26B left; see also see also Table 7).

Example 9: Macropinocytosis inhibitor does not affect transfection of LNPs comprising presently disclosed peptide, indicating that nonselective uptake is less likely

[00205] In some embodiments, presently disclosed peptide-LNP compositions comprising are beneficial in that they are not taken up in a non- selective manner. Differentiated pHBECs were treated with a macropinocytosis inhibitor, EIPA (5-[N-ethyl-N-isopropyl] amiloride), prior to transfecting with presently disclosed peptide-LNP compositions. EIPA is an established macropinocytosis blocker but does not inhibit receptor-mediated endocytosis (Kim et al..2018; Koivusalo et al. , 2010). If the presently disclosed peptide-LNP compositions behave as a targeting ligand and undergo receptor-mediated endocytosis, it follows that EIPA would not be expected to have a significant effect on uptake of presently disclosed peptide- LNP compositions. The inventors report no statistically significant decrease in Nluc bioactivity after EIPA treatment was observed (FIG. 26C), which is consistent with a selective receptor- mediated endocytosis mechanism of uptake for the peptide-LNPs compositions disclosed herein.

Example 10: Materials and Methods

Culturing and differentiation of primary human bronchial epithelial cells

[00206] For coating Transwell® inserts (0.4-micron, 6.5 mm diameter, Corning Product, catalog #3470), Human Placenta Collagen Type IV (Sigma, catalog #C7521) were prepared according to protocols established by University of North Carolina, Marisco Lung Institute (MLI) Core. A lOx stock solution (10 mg Collagen, 20 mL ddl LO, 50 pL concentrated acetic acid) was first prepared and was then incubated from 4-8 hours at 37°C to dissolve. The solution was then filter sterilized with a 0.2 pm syringe filter, aliquotted and stored at -20°C. Prior to coating, a lx solution was prepared with sterile cell culture grade water. Next, 100 pL of the solution was added to each insert, dried the plates containing inserts (with lids off) in a biosafety cabinet overnight and UV sterilized inserts for at least 30 minutes before use.

[00207] From MLI, primary human bronchial epithelial cells (pHBECs) were purchased from seven different cystic fibrosis (CF) patients homozygous for the AF508 mutation (patient demographics in Table 8). All cells used for experiments were passage 2 (P2) unless otherwise noted. All seven patients’ cells were pooled and seeded onto pre-coated inserts for differentiation at air-liquid interface (ALI) according to Pneumacult™ Ex-Plus (STEMCELL Technologies Inc., catalog #05040; supplemented with amphotericin B (0.25 pg/mL final concentration) (Thermo Fisher Scientific, catalog #BP264520), gentamicin (50 g/mL final concentration) (Sigma, catalog #G1397), and lx penicillin/streptomycin) and Pneumacult™ ALI media (STEMCELL Technologies Inc., catalog #05001 ; supplemented with lx penicillin/streptomycin) protocols. Briefly, P2 cells were first expanded on pre-coated Transwell® inserts until at least 80% confluency. Once confluency was reached, apical media was removed (i.e., cells were airlifted), and added Pneumacult™- ALI maintenance media to the basolateral side only. Cells were then maintained in Pneumacult™- ALI maintenance media until differentiation occurred (at least 21 days post airlift and confirmed by observation with mucus production and cilia movement).

[00208] For transfection studies using undifferentiated cells, patient 1 (Table 8) was seeded at a density of 1.5E5 cells/mL onto a 96-well plate (100 pL of cell suspension) and transfected cells 48 hours later.

Table 8: List of patient donors and their demographics for primary human bronchial epithelial cells used

All patients were homozygous for AF5O8 mutation.

Selection Strategy

[00209] T7 cysteine constrained heptapeptide (CX7C) phage libraries (Leal et al., 2020; Mohanty et al., 2019) previously developed using T7Select415-l cloning kit (Novagen, catalog #70015) were used to select against CF pHBECs to discover mucus and cell-penetrating peptides. Either one (for four-round selection protocol) or two (for our optimized five-round biopanning protocol) combined T7 libraries were diluted in DPBS (without Ca²⁺ and Mg²⁺) to a final concentration of 3.3E5 plaque forming units/pL or PFU/pL to obtain 1 ,000 viral genomes per cell (3.3E4 pHBECs were seeded per insert). 100 pL of phage solution was then added to the apical side of each Transwell® insert (N=3) and incubated for a given amount of time depending on the round of selection (Round 1 = 16 hours, Round 2 = 4h, Rounds 3- 5 =lh). Initial four-round biopanning selection consisted of four rounds, while optimized biopanning selection consisted of five total rounds. After incubation, apical and basolateral solutions were removed, and cells were washed with phage elution buffer (20 mM Tris-HCl pH 8, 100 mM NaCl, 6 mM MgSO4) and DPBS to remove any unbound/bound phage remaining. After wash steps, 200 pL of M-PER lysis buffer (Thermo Fisher Scientific Inc., catalog #78503) was added on top of cells and the cells were incubated for 5 minutes at 180 rotations per minute (RPM) on an orbital shaker to ensure cell lysis. Following lysis, cells were scraped (using a P200 pipette tip) and collected. In optimized five-round biopanning strategy, cells were further and spun down for 10 min at 14,000 x g to separate cell debris from internalized phage (present in supernatant). Collected phage from whole cell lysate (four-round selection) or supernatant only (in optimized biopanning) were then quantified and titered using standard double-layer plaque assay. In our optimized panning protocol, by titering only the supernatant, the presently described methods, without being bound by theory, increase the likelihood of only collecting internalized phage (that is, reduce the likelihood of collecting bound phage). In the initial four-round panning strategy, phage collected from each replicate after every round were amplified separately before use in the subsequent round. In the five- round optimized strategy, internalized phage from each replicate were amplified and then pooled before being used as input for the following round. This additional optimization step may, without being bound by theory, improve reproducibility in the selection process compared to screening strategies known in the art, which were known to have few clones present in all three replicates (Leal et al., 2020). Additionally, after sample collection, a portion of this sample was reserved for Next Generation Sequencing (NGS) and the remaining sample was amplified in E. coll (BL21) until lysis was observed (~2 hours), per manufacturer’s protocol, so input concentrations remained the same in the following rounds. As a control to identify parasitic sequences, (Liu etal., 2015) the naive library (unselected CX7C library) was amplified 5 subsequent times prior to NGS sample preparation.

Next Generation Sequencing Data Analysis

[00210] Following each round of selection, DNA from amplified phage was isolated, samples were prepared for NGS, and peptide sequence analysis was performed according to known methods (Leal et al., 2020; Mohanty et al., 2019). After obtaining a frequency count of all peptide sequences from each round of panning, and in order to properly select enriched CX7C sequences from the NGS data, sequences that were linear (i.e., did not contain the expected CX7C motif) or did not contain the correct flanking sequences surrounding the insert initially cloned into our library were omitted. The top 30 most abundant sequences after all five rounds of selection were analyzed to determine their physicochemical properties. Specifically, net charge and grand average of hydropathy score (GRAVY score) for each 7- mer sequences were calculated using in silico tools (https://pepcalc.com) and (http://www.gravy-calculator.de/index.php), respectively. Additionally, a multiple sequence alignment visual representation was created using Seq2Logo after combination of the top 30 sequences from each replicate (total of 90 sequences) after 5 rounds of selection (FIG. 22C) (Thomsen & Nielsen, 2012). Peptide sequences that were found in the top 10 most frequently occurring peptide sequences, in all three replicates, and absent from an amplified naive library control (amplified five times to correlate with the five rounds of panning) were selected for further validation studies. The enrichment for each clone selected for validation studies was evaluated by calculating the average (N=3) percentage of total unique sequences retrieved from NGS for each round of selection. Additionally, peptide sequences were analyzed using online tool, “Scanner and Reporter of Target-Unrelated Peptides” or SAROTUP to confirm sequences used for validation were not target- unrelated peptides (Huang et al., 2010; Huang et al., 2012; He et al., 2016).

Validation of Clones

[00211] After peptides were selected from NGS data for further validation, the selected peptide sequences were cloned back into T7 phage and verified through sanger sequencing according to known methods (Mohanty et al., 2019). Briefly, complementary pairs of oligonucleotides (i.e., sense and anti-sense oligos) were obtained for each peptide sequence through IDT (Table 9). Oligos were diluted in IDTE buffer, pH 8 (IDT) to a 100 pM stock solution and then to 10 pM before use. Oligos were annealed (5 pL of 10 pM sense and antisense oligos in a 50 pL reaction with ultrapure water at 95 °C for 3 minutes) then cooled to RT (25°C) at 0. 1°C/S (Ramp). After annealing, inserts were ligated to T7-Select 415-1 vector arms (Novagen (EMD Millipore), catalog #70015-3) using T4 DNA ligase (New England Biolabs, catalog #M0202L). Ligation reactions were then packaged using T7Select415-lb cloning kit (Novagen (EMD Millipore), catalog #70015-3) according to manufacturer’s instructions. Packaged clones were plated using a standard double-layer plaque assay with BL21 E. coli. After incubating clones overnight at 37°C, individual plaques were isolated and sequenced to confirm proper cloning. Once clones were validated, they were amplified in liquid BL21 E. coli culture prior to validation studies. To validate clones for enhanced CF pHBEC uptake, 3.3E7 PFU of each clone (in 100 pL DPBS) was incubated on the apical side of differentiated pHBECs for Ih at 37°C. Phages were collected and quantified in the same manner as their selection for either round 4 (in initial biopanning validation studies) or round 5 (for optimized biopanning validation studies). In order to evaluate the sequence specificity of the top clones identified from our initial four-round panning strategy, clones 1,9,14, and 26 were validated with their respective scramble controls using pHBECs cultured at ALL Scrambled peptide controls were designed using peptide nexus online scramble tool (https://peptidenexus.com/article/sequence-scrambler). Scrambled peptide sequences were cloned back into T7 phage as described earlier. Clones and scramble controls were incubated on top of pHBECs for 1 hour at 37°C. Cells were lysed and whole cell lysate was collected and tiered to quantify phage uptake.

[00212]

Table 9: Oligonucleotide sequences used for validation studies.

Sequences were optimized using GenSmart Codon Optimization Tool. Restriction Site (to be joined with 415-b vector arms that are precut); ((Inserted to make insert in reading frame)); Stop Codon; Cysteine. Note: Clone CPS was obtained from a previously made stock in our lab (Marson et al., 2016). Asn = Anti-sense.

Statistical analysis

[00213] All data analyses were performed with GraphPad Prism 10 (GraphPad Software, La Jolla, CA) at a significance level of p < 0.05 unless otherwise indicated.

Differentiation of THP-1 cells for macrophage uptake studies

[00214] For macrophage uptake studies, THP-1 cells (ATCC, catalog #TIB-202) were cultured according to ATCC’ s recommendations. Briefly, cells were maintained in RPMI- 1640 media (Sigma, catalog #R8758) supplemented with 10% Fetal Bovine Serum (FBS) and gentamicin (final concentration of 50 iig/mL). For differentiation into nonpolarized macrophages, cells were resuspended in phorbol 12-myristate 13-acetate (PMA) (Sigma, catalog #P8139) containing media (15 ng/mL) and seeded at a final density of 4E5 viable cells/mL. 0.5 mL of cell suspension were seeded per well for a 24-well plate. Cells were incubated at 37°C/5 % CO2 for 48 hours before exchanging media with PMA-free media and incubating cells for an additional 24 hours prior to use for experiments.

Polarization of Calu-3 Cell Culture

[00215] For uptake studies, Calu-3 cells (catalog # HTB-55, American Type Culture Collection) grown in minimal essential medium (MEM) (catalog # 11095-080) supplemented with 10% FBS and lx penicillin/streptomycin were used. After reaching 80% confluency, cells were passaged and seeded onto Transwell® inserts (0.4-micron, 6.5 mm diameter, Coming Product, catalog #3470) at a density of 3E5 cells/cm². Cells were expanded on inserts until confluent (3 days), and airlifted cells by removing apical media and continuing to culture by only replacing the basolateral media every 2-3 days. Studies were completed on cells two weeks post-ALI lift.

In vitro transcription

[00216] Nanoluciferase mRNA (NLuc) was prepared according to previously known methods (Lewis el al., 2023). Briefly, a template was created for NLuc mRNA encoding sequences for a T7 promoter, 5’ UTR, codon-optimized NLuc, and 3’ UTR. mRNA was synthesized using AmpliScribe™ T7- Flash Transcription Kit (Lucigen, catalog #ASF-3507) as described previously (Kauffman etal., 2015; Zeng et al., 2020). After purification with RNA Clean & Concentrator- 100 (Zymo, catalog #R1019), a capl structure was added using the Vaccinia Capping System (NEB, M2080S) and mRNA Cap 2’-O-methyltransferase (NEB, catalog #M0366S). A 3'-poly(A) tail (E. Coli Poly (A) Polymerase, NEB, catalog #M0276L) was then added. After polyadenylation, the mRNA was purified again and mRNA concentration was determined by Nanodrop 1000 (Thermo Fisher Scientific Inc.), and stored aliquots at -80 °C until use.

Lipid nanoparticle synthesis

[00217] To determine if peptides discovered through phage display could be incorporated into a nanoparticle system, cyclic peptides were conjugated to a myristic acid lipid tail (synthesized and N-terminally modified by LifeTein®). This peptide-lipid was used as a fifth component to Modema’s Spikevax lipid nanoparticle formulation using a Nanoassemblr™ benchtop instrument (Precision Nanosystems Inc., Vancouver, BC, Canada). Briefly, lipids (peptide-lipid, SM-102 (Echelon, catalog #N-1102), cholesterol (Sigma, catalog #C8667), distearoylphosphatidylcholine (DSPC) (Avanti, catalog #850365), and 1,2- dimyristoyl-rac-glycero-3-methoxypolyethylene (DMG-PEG) (Avanti, catalog #88015 IP)) were dissolved in molecular grade ethanol at a concentration of 10 mg/mL and reporter mRNA (NLuc) was dissolved in pre-chilled sodium citrate buffer (pH 4, 100 mM). Formulations were prepared at an aqueous: organic flow ratio of 3:1, flow rate of 4 mL/min, and at a volume of 500- 700 pL. After formulation, LNPs were dialyzed in lx phosphate buffered saline (PBS; pH 7.4) for 2 hours (for in vitro experiments) or 4 hours (for in vivo experiments) in 10K MWCO Slide-A-Lyzer dialysis cassettes (Thermo Fisher Scientific, catalog #87730). Lipid nanoparticle synthesis

[00218] For characterization, LNPs were prepared in lx PBS (pH 7.4) for size measurements (10-fold dilution) by dynamic light scattering (DLS) and ultrapure water for zeta potential measurements (10-fold dilution) using the Zetasizer Nano-ZS (Malvern Instruments MA, USA). To determine encapsulation efficiency percentages (EE %), a modified Quant-it™ RiboGreen RNA Assay Kit protocol (Thermo Fisher Scientific, Catalog # R11490) was used. Briefly, LNPs were diluted 100-fold in lx Tris-EDTA (TE) or 1% Triton® X- 100 (Thermo Fisher Scientific, catalog #BP151) to measure unencapsulated mRNA or total mRNA, respectively. Two low- range standard curves (one in TE and one in 1% Triton® were also prepared 100 pL of LNP sample or standard was added to a clear-bottom 96-well black plate (Coming, Catalog # 3631) in duplicate then all samples and standards were incubated at 37°C for 10 minutes. A 2,000-fold dilution of RiboGreen Reagent followed by 100 pL of the working solution was then added to each sample or standard. After 5 minutes, fluorescence was measured using a SpectraMax M3 plate reader (Molecular Devices) at excitation/emission of 480 nm/520 nm. EE% was calculated according to the following equation:

EE% = [1 -(unencapsulated mRNA/total mRNA)] * 100

Lipid nanoparticle cell uptake in vitro

[00219] To compare transfection of LNP formulations, differentiated CF pHBECs or THP-1 derived macrophages were incubated with 450 ng of mRNA (based off EE% values) for 48 hours. LNPs were diluted in lx PBS to a total volume of 125 pL. mRNA expression was measured by bioluminescence assay using Nano-Gio® Luciferase Assay System (Promega, Catalog # Ni l 10). Briefly, for pHBECs, cells were scraped from each treated well (remaining LNP solution left on top) and transferred to a 96-well white flat bottom plate (Corning, catalog #3912). For differentiated THP-1 cells, media was removed first, then added 50 pL of lx PBS (without Ca²⁺ or Mg²⁺) to cells before scraping and collecting (to keep apical volume consistent with pHBECs). Collected samples were incubated with a 1:1 ratio of Nano-Gio® Luciferase Assay Buffer to lyse the cells. Nano-Gio® substrate was added to each well (one group at a time) and luminescence was read 3 minutes later using a SpectraMax M3 plate reader (Molecular Devices). Lipid nanoparticle delivery in vivo

[00220] All procedures were performed in accordance and under approval by the University of Texas at Austin Institutional Animal Care and Use Committee. For intratracheal administration, Balb/c mice (Charles River, female, 6-8 weeks old) were first anesthetized under 2% isoflurane before delivering 40 pL of each LNP formulation in two separate instillations of 20 p L. Following 24 hours after administration of LNPs, mice were euthanized via carbon dioxide inhalation followed by cervical dislocation and immediately harvested lungs. Harvested lungs were briefly rinsed by dipping in lx PBS and separated each lung into five separate lobes (left, cranial, middle, accessory, and caudal). Lobes for each lung were then collected into a 1.5 mL microcentrifuge tube and incubated in 300 pL of Nano-Gio® substrate solution for 5 minutes, before proceeding directly to imaging using an IVIS Spectrum In Vivo imaging system. Bioluminescence (average radiance [p/s/cm²/srj calculated for area surrounding all five lobes) was measured using Living Image 4.3 software (PerkinElmer).

Analysis ofmRNA expression in vitro through flow cytometry

[00221] LNPs or lipofectamine messenger MAX (catalog # LMRNA003; Invitrogren, Thermo Fisher Scientific) (1 pg of mRNA or 75 pL of LNP solution) were added on top of Calu-3 cells cultured at ALL After 24 hours of treatment, apical media/LNP solution and detached cells were removed by adding 200 pL of 0.5% Trypsin (Catalog # 15400054, Gibco) on the apical side of the Transwells®, a 1 : 1 volume of Trypsin neutralization solution (Catalog # CC-5002, Lonza) was then added and cells were transferred to 96- well U-bottom plates. Next, cells were spun down at 350 x g for 6 minutes, and each sample was resuspended in 200 pL of FACS buffer (DPBS, 1% FBS, 1 mM EDTA). Prior to flow cytometry, 20 pL of lOx (4 pg/mL) propidium iodine was added to each well. Cells were compensated using GFP BrightComp eBeads™ (Catalog # A10514, Invitrogen, Thermo Fisher Scientific). Flow cytometry was performed using the Attune NxT Flow Cytometer.

[00222] To determine the percentage of cells transfected in submerged conditions, LNPs (10 pL) containing eGFP mRNA were delivered (at two separate doses (100 ng and 200 ng), to primary human bronchial epithelia cultured in submerged conditions (i.e., not differentiated into multiple cell types) and analyzed the percentage of GFP expressing cells via flow cytometry 24 hours later. Briefly, cells were trypsinized using the Animal Component Free Cell Dissociation Kit (Catalog #05426, Stemcell Technologies), spun down at 350g for 5.5 minutes and resuspended in 200 pL of FACS buffer. Prior to flow cytometry, 20 pL of lOx (4 pg/mL) propidium iodine was added to each well. Cells were compensated using GFP B rightComp eBeads™. Flow cytometry was performed using the Attune NxT Flow Cytometer.

In vivo gene editing study

[00223] For intratracheal administration of Cre mRNA, mice (B6.Cg- Gt(ROSA)26Sortm9(CAG-tdTomato)Hze/J (Ai9) mice (Catalog # 007909, The Jackson Laboratory)) were first anesthetized under 2% isoflurane before delivering 50 pL of each LNP formulation (0.5 mg/kg of Cre recombinase mRNA (Cat L7211, TriLink) in two separate instillations of 25 L. Following 72 hours after administration of LNPs, mice were euthanized via carbon dioxide inhalation followed by cervical dislocation and lungs were immediately harvested. Harvested lungs were briefly rinsed by dipping in lx PBS. To determine in vivo gene editing following local administration of presently disclosed peptide-LNP compositions, the harvested lungs were processed for flow cytometry as described previously (Wei et al., 2023; Lewis et al., 2023). Briefly, lungs were digested in 10 mL of digestion media (90 units/mL Collagenase Type I (Sigma, Catalog # SCR103), 50 units/mL DNAse I (Roche, Catalog # 11284932001), 60 units/mL Hyaluronidase (Sigma, Catalog # H3506) for 1 hour at 37°C with intermittent shaking. A single cell suspension was formed by pouring the digested contents over a 70-micron filter and quenched digestion with 10 mL of wash buffer (DMEM, 20% FBS, and 1% penicillin/streptomycin). The quenched solution was centrifuged at 300 x g for 10 minutes at 4°C, washed cells with 5 mL lx PBS, and lysed red blood cells by incubating cells in 5 mL ACK lysing buffer at RT for 3 minutes, prior to quenching with 10 mL of Quench buffer (90% lx PBS, 10% FBS). Cells were washed with wash buffer followed by an additional PBS wash, and cells were resuspended in lx PBS prior to staining for flow cytometry as previously described (Lewis et al., 2023). For staining the following antibodies were used: immune cells (CD45-Pacific Blue, BioLegend, Catalog # 103126), endothelial cells (CD31- AF488, BioLegend, Catalog #102414), epithelial cells (CD326-AF647, Catalog # 118212), and viability (Zombie NIR , BioLegend, Catalog # 77184). To determine the percentage of TdTomato % basal cells, cells were stained with CD271-APC (Invitrogen, Catalog # 17-9400- 42.) Flow cytometry was performed using the Attune NxT Flow Cytometer.

In vitro competition experiments

[00224] First, peptide-lipid conjugates were equilibrated at room temperature and diluted peptide-lipid stocks were prepared at a 1 mg/mL concentration in DPBS (Cat 21- 031-CV, Coming). A 25 pM stock of EIPA (Cat A3085, Sigma Aldrich) in lx PBS was also prepared. For competition experiments, either 50 pL of 1 mg/mL peptide-lipid, 25 pM EIPA,or lx PBS was added on top of cells (Cat 10010-023, Gibco™) (for cells treated with LNP only to keep volumes consistent across wells). Cells were either incubated with peptide-lipid conjugate for 10 minutes at 37°C or EIPA for 30 minutes at 37°C. Following this, cells were treated with 200 ng NLuc mRNA peptide-LNPs of the present disclosure and incubated at 37°C for 48 hours. Bioluminescence was measured using NanoGio Assay Buffer.

[0052] All of the compositions and methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this disclosure have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the disclosure. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved.

All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the disclosure as defined by the appended claims.

REFERENCES

The following references, to the extent that they provide exemplary procedural or other details supplementary to those set forth herein, are specifically incorporated herein by reference.

U.S. Pat. No. 4,215,051

Arap et al., Science 279:377-80, (1998).

Ausubel et al. (eds.), Curr. Prot. Molec. Biol., (2003).

Ballou et al., Bioconjugate Chem. 15( l):79-86, (2004).

Bitter et al., Meth. Enzymol. 153:516-544, (1987).

Bitter, Meth. Enzymol. 152:673-684, (1987).

Brisson et al., Nature 310:511-514, (1984).

Broglie et al., Science 224:838-843, (1984).

Cheng et al., Nature Nanotechnology. 15, 313-320 (2020).

Colbere-Garapin et al., J. Mol. Biol. 150:1-14, (1981).

Coin et al., Nat Protoc 2, 3247-3256, (2007).

Coruzzi et al., EMBO J. 3:1671-1679, (1984).

Devereux et al., Nucl. Acid Res. 12:387, (1984).

Dubertret et al., Science 298(5599): 1759- 1762, (2002).

Dwarakanath et al., Biochem Biophys Res. Commun. 325(3):739-743, (2004).

Ferrari, Curr. Opin .Chem. Biol. 9:343-346, (2005).

Frangioni et al., Curr. Opin. Chem. Bio. 7(5):626-634, (2003).

Gurley et al. , Mol. Cell. Biol. 6:559-565, (1986).

He et al., Nucleic Acids Res. 44(D1):D1127-32 (2016).

Henikoff et al. , Proc. Natl. Acad. Sci. U.S.A. 89:10915-10919, (1992).

Hope et al., Biochem Biophys Acta, 812(l):55-65, (1985).

Huang et al., J Biomed Biotechnol. 2010: 101932 (2010).

Huang et al., Nucleic Acids Res. 40, D271-7 (2012).

Huckaby J. . .Lai S, Advanced drug delivery reviews, 2018

Inouye & Inouye, Nucleic Acids Res. 13:3101-3110, (1985).

Kauffman et al., Nano Lett. 15(11):7300-6 (2015).

Kim et al., Cancer Discov. 8(7):866-83 (2018).

Kim et al., Science Advances. 7 eabf4398. doi.org/10.1126/sciadv.abf4398. (2021).

Kim et al., ACS Nano, doi.org/10.1021/acsnano.2c05647. (2022). Koivusalo et al. , J Cell Biol. 188(4):547-63 (2010).

Kulkami el al., ACS Nano. 12, 4787-4795 (2018).

Kulkami et al., Nanoscale. 11, 9023-9031 (2019).

Kumari et al., Mol Pharm. 19(5): 1309-24 (2022).

Leal et al., J Control Release. 322:457-69 (2020).

Lewis et al., Bioeng Transl Med. 8(6):el0580 (2023).

Liu et al., Bioconjug Chem. 26(8): 1811-7 (2015).

Logan & Shenk, Proc. Natl. Acad. Sci. USA 81:3655-3659, (1984).

Lowy et al., Cell 22:817-823, (1980).

Madisen et al., Nat Neurosci. 13(1): 133-40 (2010).

Marson et al., Lancet Respir Med. 4(8):e37-e8 (2016).

Mayer etal., Biochimica et Biophysica Acta 858: 161-168, (1986).

Mayhew et al., Experimental Cell Research 171(l):195-202, (1987).

Mayhew et al., Biochimica et Biophysica Acta (BBA) Biomembranes 775(2): 169-174, (1984).

Michalet et al., Science 307(5709):538-544, (2005).

Mohanty et al., Nanoscale. 11(38) : 17664-81 (2019).

Mulligan & Berg, Proc. Natl. Acad. Sci. USA 78:2072-2076, (1981).

Needleman et al., J. Mol. Biol. 48:443-453 (1970).

Nevins, CRC Crit. Rev. Biochem. 19:307-322, (1986).

O'Hare et al., Proc. Natl. Acad. Sci. USA 78:1527-1531, 1981.

Park, et al., Biotechnol Prog. 16(4):564-70, (2000).

Pasqualini & Ruoslahti, Nature 380:364-66, (1996).

Patel et al., Nature Communications. 11, 983 (2020).

Qiu et al., Proceedings of the National Academy of Sciences. 118, e2020401118 (2021).

Qiu et al., Proceedings of the National Academy of Sciences. 119, e2116271119 (2022).

Reagan-Shaw et al., FASEB J., 22(3):659-661, (2008).

Ruther & Miiller-Hill, EMBO J. 2: 1791-1794, (1983).

Sambrook et al., Molecular Cloning, 2^nd Edition (1989).

Santerre et al., Gene 30:147-156, (1984).

Seixas et al., Appl. Clin. Genet., 14:173-194, (2021).

Smith et al., J. Virol. 46:584-593, (1983).

Stayton et al., J. Control Release 65:23-220, (2000).

Szybalska & Szybalski, Proc. Natl. Acad. Sci. USA 48:2026-2034, (1962). Takamatsu et al., EMBO J. 6:307-311, (1987).

Thomsen & Nielsen, Nucleic Acids Res. 40:W281-7 (2012).

Van Heeke & Schuster, J. Biol. Chem. 264:5503-5509, (1989).

Wei et al., Nat Commun. 14(1):7322 (2023).

Wiglcr c/ al., Cell, 11:223-232, (1977).

Wigler c/ al., Proc. Natl. Acad. Sci. USA 77:3567-3570, (1980).

Zeng et al., Adv Mater. 32(40) :e2004452 (2020).

Zhang et al., Sci Adv 6, eabc2315 (2020a).

Zhang et al., Pharmaceutics 12, 1042 (2020b).

Claims

WHAT IS CLAIMED IS

1. A peptide of 9-15 amino acids, wherein the peptide comprises the sequence CX7C, wherein the peptide has a net positive charge of at least 1 at neutral pH and the peptide is capable of penetrating a mucosal membrane or cell membrane.

2. The peptide of claim 1, wherein the peptide has a net positive charge from about 1 to about 5.

3. The peptide of claim 2, wherein the peptide has a net positive charge from about 1.5 to about 3.

4. The peptide of claim 3, wherein the peptide has a net positive charge of about 1.9.

5. The peptide of claim 3, wherein the peptide has a net positive charge of about 2.9.

6. The peptide of claim 3, wherein the peptide has a net positive charge of about 3.9.

7. The peptide according to any one of claims 1-6, wherein the peptide has a GRAVY score of from about -0.2 to about -4.

8. The peptide according to claim 7, wherein the peptide has a GRAVY score that is less than - 1.

9. The peptide according to claim 7, wherein the peptide has a GRAVY score that is less than -2.

10. The peptide according to claim 7, wherein the peptide has a GRAVY score that is less than -3.

11. The peptide according to any one of claims 1-10, wherein the peptide comprises at least 1 amino acid residue that is positively charged at neutral pH.

12. The peptide of claim 11, wherein the peptide comprises at least 2 amino acid residues that are positively charged at neutral pH.

13. The peptide of either claim 11 or claim 12, wherein the peptide comprises at least 3 amino acid residues that are positively charged at neutral pH.

14. The peptide according to any one of claims 11-13, wherein the peptide comprises at least 4 amino acid residues that are positively charged at neutral pH.

15. The peptide according to any one of claims 1-14, wherein the peptide sequence comprises at least one serine residue.

16. The peptide of claim 15, wherein the peptide sequence comprises two serine residues.

17. The peptide of claim 15, wherein the peptide sequence comprises three serine residues.

18. The peptide of claim 15, wherein the peptide sequence comprises four serine residues.

19. The peptide according to any one of claims 1-18, wherein the peptide sequence comprises at least one proline residue.

20. The peptide of claim 19, wherein the peptide sequence comprises one proline residue.

21. The peptide of claim 19, wherein the peptide sequence comprises two proline residues.

22. The peptide of claim 19, wherein the peptide sequence comprises three proline residues.

23. The peptide of claim 19, wherein the peptide sequence comprises four proline residues.

24. The peptide according to any one of claims 1-23, wherein the peptide sequence comprises at least one lysine residue.

25. The peptide of claim 24, wherein the peptide sequence comprises two lysine residues.

26. The peptide according to any one of claims 1-25, wherein the peptide sequence comprises at least one arginine residue.

27. The peptide of claim 26, wherein the peptide sequence comprises one arginine residue.

28. The peptide of claim 26, wherein the peptide sequence comprises two arginine residues.

29. The peptide according to any one of claims 1-28, wherein the peptide is capable of penetrating a mucosal membrane or cell membrane by direct penetration.

30. The peptide according to any one of claims 1-28, wherein the peptide is capable of penetrating a mucosal membrane or cell membrane by endocytosis.

31 . The peptide according to any one of claims 1 -28, wherein the cell membrane is a membrane of a human cell.

32. The peptide of claim 31, wherein the cell membrane is a membrane of an epithelial cell.

33. The peptide of claim 32, wherein the epithelial cell membrane is a membrane of a gastrointestinal epithelial cell.

34. The peptide of claim 32, wherein the epithelial cell membrane is a membrane of a cervicovaginal epithelial cell.

35. The peptide of claim 32, wherein the epithelial cell membrane is a membrane of a nasal epithelial cell.

36. The peptide of claim 32, wherein the epithelial cell membrane is a membrane of a skin epithelial cell.

37. The peptide of claim 32, wherein the epithelial cell membrane is a membrane of a hepatic epithelial cell.

38. The peptide of claim 32, wherein the epithelial cell membrane is a membrane of a corneal epithelial cell.

39. The peptide of claim 32, wherein the epithelial cell membrane is a membrane of a lung epithelial cell.

40. The peptide of claim 39, wherein the epithelial cell membrane is a membrane of a basal lung epithelial cell.

41. The peptide of claim 39, wherein the lung epithelial cell membrane is a membrane of a primary cell.

42. The peptide of claim 41, wherein the primary cell membrane is a membrane of a primary human bronchial epithelial cell.

43. The peptide according to any one of claims 31-42, wherein the cell membrane is derived from a cell from a patient with a disease or disorder.

44. The peptide of claim 43, wherein the disease or disorder is a lung disease or disorder.

45. The peptide of claim 44, wherein the lung disease or disorder is chronic obstructive pulmonary disease.

46. The peptide of claim 44, wherein the lung disease or disorder is respiratory syncytial virus.

47. The peptide of claim 44, wherein the lung disease or disorder is influenza.

48. The peptide of claim 44, wherein the lung disease is cytomegalovirus.

49. The peptide of claim 44, wherein the lung disease is primary ciliary dyskinesia.

50. The peptide of claim 44, wherein the patient with primary ciliary dyskinesia has a genetic mutation of the DNAI1 or DNAH5 genes.

51. The peptide of claim 44, wherein the lung disease is alpha- 1 antitrypsin deficiency.

52. The peptide of claim 51, wherein the patient with alpha- 1 antitrypsin deficiency has a mutation of the SERPINA1 gene.

53. The peptide of claim 52, wherein the mutation of the SERPINA1 gene is E342K and V264E.

54. The peptide of claim 44, wherein the lung disease is cystic fibrosis.

55. The peptide of claim 54, wherein the patient with cystic fibrosis has a genetic mutation associated with cystic fibrosis.

56. The method of claim 55, wherein the mutation is selected from the group consisting of AF508, G542X, N1303K, W1282X, E56K, G178R, S549R, K1060T, G1244E, P67L, E193K, G551D, A1067T, S1251N, R74W, L206W, G551S, G1069R, S1255P, D110E, R347H, D579G, R1070Q, D1270N, D110H, R352Q, S945L, R1070W, G1349D, R117C, A455E, S977F, F1074L, R117H, S549N, F1052V, and D1152H.

57. The method of claim 56, wherein the mutation is selected from AF508, G542X, G55 ID, N1303K, and W1282X.

58. The peptide of claim 57, wherein the genetic mutation is the AF508 mutation.

59. The peptide of claim 43, wherein the disease or disorder is a cervicovaginal disease or disorder.

60. The peptide of claim 59, wherein the cervicovaginal disease or disorder is human immunodeficiency virus (HIV).

61. The peptide of claim 59, wherein the cervicovaginal disease or disorder is human papillomavirus (HPV).

62. The peptide of claim 59, wherein the cervicovaginal disease or disorder is a Mullerian anomaly.

63. The peptide of claim 59, wherein the cervicovaginal disease or disorder is endometriosis.

64. The peptide of claim 43, wherein the disease or disorder is a disease or disorder of the nasal pathway.

65. The peptide of claim 43, wherein the disease or disorder is a disease or disorder of the skin.

66. The peptide of claim 65, wherein the skin disease or disorder is epidermolysis bullosa.

67. The peptide of claim 65, wherein the skin disease or disorder is epidermolytic hyperkeratosis.

68. The peptide of 41.4, wherein the skin disease or disorder is a polygenic skin disease.

69. The peptide of claim 68, wherein the polygenic skin disease is systemic lupus, psoriasis, androgenic alopecia, atopic dermatitis, systemic sclerosis, vitiligo, alopecia areata, pemphigus vulgaris and foliaceus, or Sjogren’s syndrome.

70. The peptide of claim 43, wherein the disease or disorder is a gastrointestinal disease or disorder.

71. The peptide of claim 70, wherein the gastrointestinal disease or disorder is oral carcinoma.

72. The peptide of claim 70, wherein the gastrointestinal disease or disorder is Sjogren’s syndrome.

73. The peptide of claim 70, wherein the gastrointestinal disease or disorder is colitis.

74. The peptide of claim 70, wherein the gastrointestinal disease or disorder is inflammatory bowel disorder.

75. The peptide of claim 43, wherein the disease or disorder is a corneal or ocular disease or disorder.

76. The peptide of claim 43, wherein the corneal or ocular disease or disorder is a retinal genetic disease.

77. The peptide according to any one of claims 1-76, wherein the peptide selectively targets organ cells relative to macrophages.

78. The peptide of claim 77, wherein the organ cell is a skin cell, a lung cell, a liver cell, a cornea cell, a cervical cell, a vaginal cell, a nasal cell, or a gastrointestinal cell.

79. The peptide according to any one of claims 1-78, wherein the peptide has a sequence identity of at least 95% of SEQ ID. 1-16.

80. The peptide of claim 79, wherein the sequence identity is at least 98%.

81. A conjugate comprising

(a) a peptide according to any one of claims 1-80; and

82. The conjugate of claim 81, wherein the hydrophobic group is a lipid.

83. The conjugate according to either claim 81 or claim 82, wherein the hydrophobic group is a fatty acid.

84. The conjugate according to any one of claims 81-83, wherein the hydrophobic group is a long-chain fatty acid.

85. The conjugate of claim 84, wherein the fatty acid is an unsaturated fatty acid.

86. The conjugate of claim 84, wherein the fatty acid is a saturated fatty acid.

87. The conjugate of claim 86, wherein the fatty acid is myristic acid, palmitic acid, stearic acid, or arachidic acid.

88. The conjugate of claim 87, wherein the fatty acid is myristic acid.

89. The conjugate according to any one of claims 81-88, wherein the hydrophobic group and the peptide are covalently linked by an ester, an amide, a thioether, a carbamate, a carbonate, a urea, a thiocarbonate, a thiocarbamate, or a thiourea group.

90. A composition comprising:

(a) a peptide according to any one of claims 1-80 or a conjugate according to any one of claims 81-89; and

(b) a lipid nanoparticle.

91. The composition of claim 90, wherein the lipid nanoparticles comprise one or more different types of lipids.

92. The composition of either claim 90 or claim 91, wherein the lipid nanoparticle comprises a cationic lipid.

93. The composition of claim 92, wherein the cationic lipid is an ionizable cationic lipid.

94. The composition according to any one of claims 90-93, wherein the lipid nanoparticle comprises a phospholipid.

95. The composition according to any one of claims 90-94, wherein the lipid nanoparticle comprises a sterol.

96. The composition of claim 95, wherein the sterol is cholesterol.

97. The composition according to any one of claims 90-96, wherein the lipid nanoparticle comprises a polymer conjugated lipid.

98. The composition of claim 97, wherein the polymer conjugated lipid is a polyethylene glycol conjugated lipid.

99. The composition according to any one of claims 90-98, wherein the lipid nanoparticle encapsulates a protein or a therapeutic agent.

100. The composition of claim 99, wherein the lipid nanoparticle encapsulates a protein.

101. The composition of claim 99, wherein the lipid nanoparticle encapsulates a therapeutic agent.

102. The composition of claim 101, wherein the therapeutic agent is a small molecule.

103. The composition of claim 101, wherein the therapeutic agent is a nucleic acid.

104. The composition of claim 101, wherein the therapeutic agent is a polypeptide or an antibody.

105. A method of treating a disease or disorder comprising administering to a patient in need thereof comprising administering to the patent a therapeutically effective amount of a composition according to any one of claims 90-104.

106. The method of claim 105, wherein the disease or disorder is a lung disease or disorder.

107. The method of claim 106, wherein the lung disease or disorder is chronic obstructive pulmonary disease.

108. The method of claim 106, wherein the lung disease or disorder is respiratory syncytial virus (RSV).

109. The method of claim 106, wherein the lung disease or disorder is influenza.

110. The method of claim 106, wherein the lung disease is cytomegalovirus.

111. The method of claim 106, wherein the lung disease is primary ciliary dyskinesia.

1 12. The method of claim 1 1 1 , wherein the patient with primary ciliary dyskinesia has a genetic mutation of the DNAI1 or DNAH5 genes.

113. The method of claim 106, wherein the lung disease is alpha-1 antitrypsin deficiency.

114. The method of claim 113, wherein the patient with alpha- 1 antitrypsin deficiency has a mutation of the SERPINA1 gene.

115. The method of claim 114, wherein the mutation of the SERPINA1 gene is E342K and V264E.

116. The method of claim 106, wherein the lung disease is cystic fibrosis.

117. The method of claim 116, wherein the patient with cystic fibrosis has a genetic mutation associated with cystic fibrosis.

1 18. The method of claim 1 17, wherein the mutation is selected from the group consisting of AF508, G542X, N1303K, W1282X, E56K, G178R, S549R, K1060T, G1244E, P67L, E193K, G551D, A1067T, S 125 IN, R74W, L206W, G551S, G1069R, S1255P, D110E, R347H, D579G, R1070Q, D1270N, D110H, R352Q, S945L, R1070W, G1349D, R117C, A455E, S977F, F1074L, R117H, S549N, F1052V, and D1152H.

119. The method of claim 118, wherein the mutation is selected from AF508, G542X, G551D, N1303K, and W1282X.

120. The method of claim 119, wherein the genetic mutation is the AF508 mutation.

121. The method of claim 105, wherein the disease or disorder is a cervicovaginal disease or disorder.

122. The method of claim 121, wherein the cervicovaginal disease or disorder is human immunodeficiency virus (HIV).

123. The method of claim 121, wherein the cervicovaginal disease or disorder is human papillomavirus (HPV).

124. The method of claim 121, wherein the cervicovaginal disease or disorder is a Mullerian anomaly.

125. The method of claim 121, wherein the cervicovaginal disease or disorder is endometriosis.

126. The method of claim 105, wherein the disease or disorder is a disease or disorder of the nasal pathway.

127. The method of claim 105, wherein the disease or disorder is a disease or disorder of the skin.

128. The method of claim 127, wherein the skin disease or disorder is epidermolysis bullosa.

129. The method of claim 127, wherein the skin disease or disorder is epidermolytic hyperkeratosis.

130. The method of 69.56, wherein the skin disease or disorder is a polygenic skin disease.

131. The method of claim 130, wherein the polygenic skin disease is systemic lupus, psoriasis, androgenic alopecia, atopic dermatitis, systemic sclerosis, vitiligo, alopecia areata, pemphigus vulgaris and foliaceus, or Sjogren’s syndrome.

132. The method of claim 105, wherein the disease or disorder is a gastrointestinal disease or disorder.

133. The method of claim 132, wherein the gastrointestinal disease or disorder is oral carcinoma.

134. The method of claim 132, wherein the gastrointestinal disease or disorder is Sjogren’s syndrome.

135. The method of claim 132, wherein the gastrointestinal disease or disorder is colitis.

136. The method of claim 132, wherein the gastrointestinal disease or disorder is inflammatory bowel disorder.

137. The method of claim 105, wherein the disease or disorder is a corneal or ocular disease or disorder.

138. The method of claim 105, wherein the corneal or ocular disease or disorder is a retinal genetic disease.

139. A method of delivering a compound to an epithelial cell in a patient comprising administering to the patient a composition according to any one of claims 90-104.

140. The method of claim 139, wherein the epithelial cell is a lung epithelial cell.

141. The method of claim 139, wherein the epithelial cell is a gastrointestinal epithelial cell.

142. The method of claim 139, wherein the epithelial cell is a corneal or ocular epithelial cell.

143. The method of claim 139, wherein the epithelial cell is a cervicovaginal epithelial cell.

144. The method of claim 139, wherein the epithelial cell is a nasal epithelial cell.

145. The method of claim 139, wherein the epithelial cell is a hepatic epithelial cell.

146. The method according to any one of claims 1 9-145, wherein the compound is a nucleic acid.

147. The method of claim 146, wherein the nucleic acid is a gene editing system.

148. A method of selectively delivering a compound to organ cells in a patient comprising administering to the patient a composition according to any one of claims 90-104.

149. The method of claim 148, wherein the organ cells are skin cells, lung cells, liver cells, cornea cells, cervical cells, vaginal cells, nasal cells, or gastrointestinal cells.

150. The method of either claim 148 or claim 149, wherein the composition is administered via inhalation.

151. The method of either claim 148 or claim 149, wherein the composition is administered systemically.