Mucosal permeability and cell membrane permeability peptides and their use

Abstract

Peptides capable of permeating mucous membranes or cell membranes are provided. Functionally linked peptide conjugates are provided in some aspects. Compositions of peptide conjugates are disclosed, and methods of using such compositions are provided.

Description

[Technical Field]

[0001] Sequence listing reference This application includes an XML sequence listing file, submitted electronically and incorporated in its entirety by reference herein. The XML sequence listing file, created on 9 April 2024, is named UTFBP1332WO.xml and has a size of 52,961 bytes.

[0002] Reference to related applications This application claims priority under U.S. Provisional Patent Application No. 63 / 495,481, filed on 11 April 2023, the entire contents of which are incorporated herein by reference.

[0003] Field of Invention This disclosure generally relates to the fields of biochemistry, molecular biology, pharmaceutical formulations, and biologics. This application relates to peptides, functionalized peptide conjugates, and related compositions that can be used to target the delivery of therapeutic substances. More specifically, this disclosure relates to peptides that selectively target cells such as lung cells, as well as their conjugates, compositions, and methods of use. [Background technology]

[0004] Description of related fields Patients with cystic fibrosis (CF) may have one of many known mutations in the gene encoding the cystic fibrosis membrane conductance regulator (CFTR) protein, which often leads to abnormal and excessive mucus accumulation in the lungs. Lung-related complications are often the leading cause of death in CF patients. Therefore, lung-targeted gene therapy has the potential to be a curative treatment for patients with CF. However, the unfavorable environment found in the lungs of CF patients, including high concentrations of mucus and immune cells such as macrophages, significantly hinders locally delivered treatments from reaching their intended target sites, i.e., bronchial epithelial cells, secretory cells, or basal cells. In CF patients, these delivery barriers are the main cause of clinical failure in many gene therapies. Existing efforts to overcome the mucus barrier have focused on the use of hydrophilic, net-charge-neutral polymers (e.g., poly(ethylene) glycol) with properties to improve diffusion and transport through mucus. While these current formulations are promising, they may exhibit immunogenicity at the time of initial or repeated administration and may have reduced intracellular uptake due to steric hindrance. Furthermore, even when intracellular uptake is possible, it is not necessarily target-oriented or selective. Evaluating the targeting ability of lung gene delivery systems is essential for clinical deployment. Cell-permeable peptides (CPPs) are being studied for transporting therapeutic cargo into cells, but their positively charged nature poses a problem for mucolytic diffusion.

[0005] Therefore, there is a need for gene delivery vehicles that can overcome multiple barriers associated with gene delivery, including the passage of the CF mucus barrier, and that can achieve targeted intracellular uptake. There is also a need for delivery systems that are non-inflammatory, enhance intracellular translocation, and can be easily integrated into non-viral delivery systems capable of delivering larger payloads, for transporting therapeutic drugs to target sites such as lung cells.

[0006] This research was supported by the Cystic Fibrosis Foundation under grant number GHOSH19XX0. [Overview of the project]

[0007] The peptides, conjugates, compositions, and methods of this disclosure are, in part, based on the discovery and identification of certain peptides that may be used to assist in the prevention and / or treatment of diseases or disorders, such as epithelial cell diseases or disorders.

[0008] More specifically, the present invention provides peptide and lipid nanoparticles that facilitate targeted delivery of cargo to primary human epithelial cells, e.g., primary human bronchial epithelial cells (pHBEC), thereby providing a possible solution to the current challenges of gene delivery to epithelial cells as part of the treatment or prevention of diseases or disorders, e.g., diseases or disorders related to epithelial cells. Targeted delivery of therapies may offer advantages including, but are not limited to, reducing systemic side effects, reducing therapeutic doses compared to systemic delivery, or facilitating alternative methods of administration of therapies.

[0009] In one aspect, the present disclosure provides a peptide comprising 9 to 15 amino acids, comprising the sequence CX7C, having a net positive charge of at least 1 at neutral pH, and capable of permeating mucosa or cell membranes.

[0010] In some embodiments, the peptide has a net positive charge of about 1 to about 5. In further embodiments, the peptide has a net positive charge of 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 yet another embodiment, the peptide has a net positive charge of about 3.9.

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

[0012] In some embodiments, the peptide contains at least one amino acid residue that is positively charged at a neutral pH. In further embodiments, the peptide contains at least two amino acid residues that are positively charged at a neutral pH. In further embodiments, the peptide contains at least three amino acid residues that are positively charged at a neutral pH. In further embodiments, the peptide contains at least four amino acid residues that are positively charged at a neutral pH.

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

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

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

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

[0017] In a further embodiment, the peptide can permeate the mucous membrane or cell membrane by direct permeation. In a further embodiment, the peptide can permeate the mucous membrane or cell membrane by endocytosis.

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

[0019] In a further embodiment, the epithelial cell membrane is the membrane of a basal lung epithelial cell. In a further embodiment, the epithelial cell membrane is the membrane of a basal lung epithelial cell. In a further embodiment, the lung epithelial cell membrane is the membrane of a primary cell. In a further embodiment, the primary cell membrane is the membrane of a primary human bronchial epithelial cell.

[0020] In some embodiments, the cell membrane is derived from cells of a patient having 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 a respiratory multinuclear 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 dysfunction, for example, primary ciliary dysfunction having a gene mutation in the DNAI1 gene or the DNAH5 gene. In another embodiment, the lung disease is α1 antitrypsin deficiency, for example, α1 antitrypsin deficiency having a mutation in the SERPINA1 gene. In another embodiment, the SERPINA1 gene mutations are E342K and V264E. In another embodiment, the lung disease is cystic fibrosis, for example, cystic fibrosis having a gene mutation associated with cystic fibrosis. In some embodiments, the mutation is selected from the group consisting of ΔF508, 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 ΔF508, G542X, G551D, N1303K, and W1282X. In some embodiments, the gene mutation is the ΔF508 mutation.

[0021] In another aspect, the disease or disorder is a disease or disorder of the cervical vagina. In some aspects, the disease or disorder of the cervical vagina is human immunodeficiency virus (HIV). In another aspect, the disease or disorder of the cervical vagina is human papillomavirus (HPV). In another aspect, the disease or disorder of the cervical vagina is Müllerian duct anomaly. In another aspect, the disease or disorder of the cervical vagina is endometriosis.

[0022] In another aspect, the disease or disorder is a disease or disorder of the nasal pathway. In another aspect, the disease or disorder is a disease or disorder of the skin. In some aspects, the disease or disorder of the skin is epidermolysis bullosa. In another aspect, the disease or disorder of the skin is epidermolytic hyperkeratosis. In another aspect, the disease or disorder of the skin is a polygenic skin disease. In some aspects, the polygenic skin disease is systemic lupus, psoriasis, male pattern hair loss, atopic dermatitis, systemic sclerosis, vitiligo, alopecia areata, pemphigus vulgaris and pemphigus foliaceus, or Sjögren's syndrome.

[0023] In another aspect, the disease or disorder is a disease or disorder of the gastrointestinal tract. In some aspects, the disease or disorder of the gastrointestinal tract is oral cancer. In another aspect, the disease or disorder of the gastrointestinal tract is Sjögren's syndrome. In another aspect, the disease or disorder of the gastrointestinal tract is colitis. In another aspect, the disease or disorder of the gastrointestinal tract is inflammatory bowel disease. In another aspect, the disease or disorder is a disease or disorder of the cornea or eye, such as a hereditary disease of the retina. In some aspects, the peptide selectively targets lung cells compared to macrophages.

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

[0025] In another aspect, the present disclosure provides (a) the peptide and (b) a hydrophobic group to form a conjugate, wherein the hydrophobic group is covalently bonded to the peptide.

[0026] In some embodiments, the hydrophobic group is a lipid. In further embodiments, the hydrophobic group is a fatty acid. In further embodiments, the hydrophobic group is a long-chain fatty acid. In 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 bonded by an ester group, an amide group, a thioether group, a carbamate group, a carbonate group, a urea group, a thiocarbonate group, a thiocarbamate group, or a thiourea group.

[0027] In another aspect, the present disclosure provides (a) the peptide or the conjugate and (b) lipid nanoparticles to form a composition.

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

[0029] In some embodiments, the lipid nanoparticles contain phospholipids. In some embodiments, the lipid nanoparticles contain sterols, such as cholesterol. In some embodiments, the lipid nanoparticles contain lipids conjugated with a polymer, such as lipids conjugated with polyethylene glycol. In some embodiments, the lipid nanoparticles encapsulate a protein or therapeutic substance. In some embodiments, the lipid nanoparticles encapsulate a protein. In other embodiments, the lipid nanoparticles encapsulate a therapeutic substance. In some embodiments, the therapeutic substance is a low molecular weight substance. In further embodiments, the therapeutic substance is a nucleic acid. In other embodiments, the therapeutic substance is a polypeptide or an antibody.

[0030] In another aspect, the present disclosure provides a method for treating a disease or disorder, comprising the step of administering a therapeutically effective amount of the composition to a patient in need.

[0031] In some embodiments, the disease or disorder is a disease or disorder of the lung. In some embodiments, the disease or disorder of the lung is chronic obstructive pulmonary disease. In another embodiment, the disease or disorder of the lung is a polynuclear respiratory virus. In another embodiment, the disease or disorder of the lung is influenza. In another embodiment, the disease of the lung is cytomegalovirus. In another embodiment, the disease of the lung is primary ciliary dysfunction, for example, primary ciliary dysfunction having a gene mutation in the DNAI1 gene or the DNAH5 gene. In another embodiment, the disease of the lung is α1 antitrypsin deficiency, for example, α1 antitrypsin deficiency having a mutation in the SERPINA1 gene. In another embodiment, the mutations in the SERPINA1 gene are E342K and V264E. In another embodiment, the disease of the lung is cystic fibrosis, for example, cystic fibrosis having a gene mutation associated with cystic fibrosis. In some embodiments, the mutation is selected from the group consisting of ΔF508, 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 ΔF508, G542X, G551D, N1303K, and W1282X. In some embodiments, the gene mutation is the ΔF508 mutation.

[0032] In another embodiment, the disease or disorder is a disease or disorder of the cervix. In some embodiments, the disease or disorder of the cervix is ​​human immunodeficiency virus (HIV). In another embodiment, the disease or disorder of the cervix is ​​human papillomavirus (HPV). In another embodiment, the disease or disorder of the cervix is ​​Müllerian duct anomaly. In another embodiment, the disease or disorder of the cervix is ​​endometriosis.

[0033] In another embodiment, the disease or disorder is a disease or disorder of the nasal passages. In another embodiment, the disease or disorder is a disease or disorder of the skin. In some embodiments, the disease or disorder of the skin is epidermolysis bullosa. In another embodiment, the disease or disorder of the skin is epidermal lytic keratosis. In another embodiment, the disease or disorder of the skin is a polygenic skin disease. In some embodiments, the polygenic skin disease is systemic lupus, psoriasis, male pattern baldness, atopic dermatitis, systemic sclerosis, vitiligo, alopecia areata, pemphigus vulgaris and pemphigus foliaceus, or Sjögren'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 cancer. In another embodiment, the gastrointestinal disease or disorder is Sjögren's syndrome. In another embodiment, the gastrointestinal disease or disorder is colitis. In another embodiment, the gastrointestinal disease or disorder is inflammatory bowel disease. In another embodiment, the disease or disorder is a corneal or eye disease or disorder, such as a genetic disorder of the retina.

[0035] In another aspect, the present disclosure provides a method for delivering a compound to the epithelial cells of a patient, comprising the step of administering the composition to the patient. In some embodiments, the compound is a nucleic acid. In some embodiments, the nucleic acid is a gene editing system.

[0036] In some embodiments, the epithelial cells are lung epithelial cells. In another embodiment, the epithelial cells are gastrointestinal epithelial cells. In another embodiment, the epithelial cells are corneal or ocular epithelial cells. In another embodiment, the epithelial cells are cervical vaginal epithelial cells. In another embodiment, the epithelial cells are nasal epithelial cells. In another embodiment, the epithelial cells are hepatic epithelial cells.

[0037] In another aspect, the present disclosure provides a method for selectively delivering a compound to organ cells of a patient, comprising the step of administering the composition to the patient. In some embodiments, the organ cells are skin cells, lung cells, hepatocytes, corneal 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 by inhalation. In other embodiments, the composition is administered systemically.

[0038] Other purposes, features, and advantages of this disclosure will become apparent from the following detailed description. However, since various variations and modifications that fall within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description, it should be understood that the detailed description and specific examples are provided for illustrative purposes only, although they illustrate specific aspects of the invention. [Brief explanation of the drawing]

[0039] The following drawings form part of this specification and are included to further illustrate certain aspects of the disclosure. This disclosure can be better understood by referring to one or more of these drawings in conjunction with the detailed description of the specific embodiments presented herein. In the drawings described below, certain lipid nanoparticle formulations may be referred to by multiple names. See Table 7. [Figure 1]The difference between the method described herein (right) and methods known in the art (left) for identifying mucosal or cell-permeable peptides is illustrated. Because the phage library is introduced into CF bronchial epithelial cells derived from human patients with the ΔF508 mutation, the method described herein is more clinically appropriate. [Figure 2] This figure shows the selection strategies for reaching the peptides disclosed herein. On the left, patient-derived cells with CF containing the ΔF508 mutation are collected. Cells from seven such patients are pooled and the CX7C phage library is introduced in ALI. On the right, a screening strategy using the cysteine-restricted random 7-amino acid peptide T7 phage display library (CX7C) against differentiated primary human bronchial epithelial cells (pHBEC) is shown, consisting of a total of five iterative high-throughput screenings. In some cases, a total of four iterative high-throughput screenings were performed. Image created with BioRender. [Figure 3] This shows four enrichments of a CX7C phage library for enhanced uptake within CF pHBEC. Cout represents the phage concentration collected after each enrichment, and Cin is the initial input phage concentration. Phages were added with 1000 viral genomes / cell, incubated at 37°C for 16 hours for the first enrichment, and at 37°C for 1 hour for the fourth enrichment. WT = wild type (peptide-less phage). [Figure 4]Figure 4 (left) provides the physicochemical properties of selected sequences after four trials for further intracellular uptake validation studies. Both charge at pH 7 and the overall mean hydroxyl (GRAVY) score were calculated as previously described (Leal 2020). Right: Validation of selected clones for enhanced uptake within CF pHBEC. Cout represents the phage concentration collected after each trial, and Cin is the initial input phage concentration. a: Significant difference from all controls, b: Significant difference from untreated library (unselected phage library) only. Phages were incubated against pHBEC in ALI at 37°C for 1 hour. Data are pooled from two separate experiments (n=6; Kruskal-Wallis test, uncorrected Dunn). [Figure 5] As described above and in the Examples section, intracellular uptake data of phages exhibiting sequences identified by mucin and cell permeability characteristics after four repeated selections are shown in pHBEC and M0 unpolarized macrophage cells. M0 (differentiated from THP-1 cells). % phage uptake: [(collected plaque-forming units (pfu) / μL × collected volume) / (input pfu / μL × input volume)] × 100. * indicates p-value < 0.05, and *** indicates p-value < 0.0005. Phages were incubated in either pHBEC or M0 at 37°C for 1 hour. As shown, clones 14 and 26 show significantly higher uptake in primary CF cells (pHBEC) than in the human macrophage cell line (M0). [Figure 6] Figure 6 shows the effect of scrambling mucosal and cell-permeable peptide sequences identified after four repeated selections. Cout represents the phage concentration collected after each selection, and Cin is the initial input phage concentration. * indicates a p-value < 0.05. Phages were incubated in pHBEC at 37°C for 1 hour. Sequences 9 (SEQ ID NO: 5) and 26 (SEQ ID NO: 8) showed improved uptake in primary CF cells compared to the scrambled counterparts. [Figure 7]This shows the optimized enrichment of sequences after five repeated selections. Cout represents the phage concentration collected after each iteration, and Cin is the initial input phage concentration. Phages were added at 1000 viral genomes / cell (vg / cell), and incubated at 37°C for 16 hours for the first selection and at 37°C for 1 hour for the fifth selection. The data represent the change in Cout / Cin from the first to the final selection for three separate replicates. [Figure 8] This shows significant shifts in the weighted mean net charge and hydropathies between peptides identified after one selection and those identified after five selections. Peptides identified after five selections had higher net positive charge and lower negative GRAVY scores, corresponding to increased hydrophilicity, compared to peptides identified after one selection. Each dot represents the weighted mean net charge or GRAVY score from each replicate (n=30). [Figure 9] Figures 9A–9D show the physicochemical properties of the top 30 high-frequency peptide sequences from each replicate (n=3, a total of 90 sequences analyzed per iteration). The peptide sequences were ranked from 1st to 30th based on frequency. The average values ​​for each ranked peptide sequence (n=3) are graphed. Figure 9A shows the net charge of the pooled top 30 sequences (n=3). Figure 9B shows the GRAVY score of the pooled top 30 sequences (n=3). The physicochemical properties of these top 30 high-frequency peptide sequences demonstrate that the optimized selection strategy is in contrast to previously published data, in that more positively charged and hydrophilic peptide sequences were identified, as disclosed herein (Leal et al., 2020). Figure 9C shows a visual representation of the multiple sequence alignment using Seq2Logo for the top 30 sequences from each replicate after 5 selections (Thomsen & Nielsen, 2012). Figure 9D shows the consensus sequence obtained from the initial four-selection panning experiment. [Figure 10] The enrichment of the library by the initial biopanning protocol tested on the way to the optimized protocol is shown; the results of the optimized protocol are shown in Figure 8. [Figure 11] This shows the enrichment of selected peptide sequences after each selection run in a five-selection biopanning experiment. Enrichment data for clones presenting peptides identified as being present in the top 10 most abundant sequences across all three replicates is shown. For each clone peptide sequence, enrichments from the 1st to the 5th run are provided (from left to right). For each panning run, the percentage relative to all sampled sequences is expressed as (average peptide frequency / total number of sequences obtained from NGS data) × 100. [Figure 12] The validation results for the peptide most abundant after five selections, identified as described above and in the Examples section, are shown. Physicochemical data for the clones assayed in this figure are provided in Table 2. Cout represents the phage output concentration collected from ALI cells after 1 hour, and Cin is the initial input phage concentration. Controls include WT, which is wild-type phage lacking the capsid surface peptide; an untreated library, which is a mixture of phage clones presenting random CX7C 7mer peptides; and CPS, which is a positive mucus-permeable clone presenting the peptide CPSSSREKC (net charge 1 and GRAVY score -1.2). One-way ANOVA (Kruskal-Wallis test; uncorrected Dunn); a, bp < 0.05. [Figure 13] The validation results for clones identified from five-selection biopanning (clones A-F) compared to clones identified from four-selection biopanning (clones 14 and 26) and controls (WT: peptide-less phage; NL: phage presenting a random CX7C sequence; CPS: phage presenting a known mucopermeable peptide) are shown. Data analysis: One-way ANOVA (Kruskal-Wallis test; unadjusted Dunn). Statistically significant differences were observed compared to the following clones: #: A, F, WT, NL, CPS, 26, ##: F, WT, NL, CPS, ###: WT, NL, CPS, ####: NL. CPS is CPSSSREKC (SEQ ID NO: 17). [Figure 14]This report describes the characterization of peptide-LNP compositions in optimization experiments. Nomenclature: Moderna_%peptide-lipid. Peptide-LNPs containing 6.25% peptide exhibited a uniform size with a small polydispersity index. [Figure 15] This paper shows the mRNA transfection efficiency of peptide-LNP formulations with varying peptide content. Nomenclature: Moderna_%peptide-lipid. The 25% and 50% peptide content formulations at the top showed the lowest transfection efficiency. The peptide-LNP formulation containing 6.25% peptide at the bottom showed the highest transfection efficiency and was selected for further use. GFP fluorescence was analyzed 24 hours after transfection into HEK293T cells. 700 ng of GFP mRNA was administered to HEK-293 cells using LNP formulations with peptide-lipid content ranging from (top) 0% (Moderna formulation) to 50% (Moderna_50) or (bottom) 0% (Moderna formulation) to 25% (Moderna_25). Fluorescence was imaged by microscopy after 24 hours. [Figure 16] Characterization data for control (Spikevax, CPS, PEG) LNPs and peptide-LNPs, including the peptide of this disclosure (peptide C), are shown. Formulations for each LNP control and peptide-LNP can be found in Table 6 (see also Table 7). "Peptide C" refers to an LNP formulation containing the peptide having the sequence CTSTRKKQC (SEQ ID NO: 11); "CPS" refers to an LNP formulation containing the peptide having the sequence CPSSSREKC (SEQ ID NO: 17). The leftmost plot in Figure 16 shows the results of dynamic light scattering for size (nm) and polydispersity index (PDI) (N=3). The middle plot in Figure 16 shows the zeta potential of the LNP formulations. The zeta potential was determined using a Zetasizer Nano according to a method known in the art (N=3). The rightmost plot in Figure 16 shows the encapsulation efficiency of the LNP formulations. Encapsulation efficiency was determined by a modified Ribogreen assay according to a method known in the art (N=2). [Figure 17]This provides evidence that peptide-LNPs, including the peptides of this disclosure, can successfully deliver Nluc mRNA to HBECs. 450 ng of Nluc mRNA was administered to the apical side of differentiated primary HBECs (N=4 in each treatment group). Readings were collected at 48 hours. After incubation, bioluminescence was measured by a plate reader using methods known in the art. * indicates p-value < 0.05, ** indicates p-value < 0.01. Data analysis: One-way ANOVA (Tukey's multiple comparison; single pooled variance). Formulations of each LNP control and peptide-LNP can be found in Table 6. CTS is represented by CTSTRKKQC (SEQ ID NO: 11); CPS is represented by CPSSSREKC (SEQ ID NO: 17). Formulation "Moderna" corresponds to formulation "Spikevax" used elsewhere in this application. Formulation "CTS" corresponds to formulation "Peptide C" used elsewhere in this application (see Table 7). [Figure 18] This disclosure provides evidence that peptide-LNPs, including the peptides of this disclosure, have lower macrophage uptake. THP-1 cells were seeded in 24-well plates and grown for 48 hours in 15 ng / mL PMA-containing medium, then grown for 24 hours without PMA for differentiation. 450 ng of Nluc mRNA was administered to the apical side of THP-1-derived macrophages. Readings were collected at 48 hours. After incubation, bioluminescence was measured by a plate reader using methods known in the art. * indicates p-value < 0.05, ** indicates p-value < 0.01, and *** indicates p-value < 0.005. Data analysis: One-way ANOVA (Tukey's multiple comparison; single pooled variance). Formulations of each LNP control and peptide-LNP can be found in Table 6. CTS is shown as CTSTRKKQC (SEQ ID NO: 11); CPS is shown as CPSSSREKC (SEQ ID NO: 17). [Figure 19](Left image) shows IVIS imaging 24 hours after administration of a control LNP (Moderna) and (right image) shows IVIS imaging of a peptide-LNP (Moderna-CTS) containing the peptide of this disclosure. CTS represents an LNP containing CTSTRKKQC (SEQ ID NO: 11) formulated according to Table 6. [Figure 20] Additional IVIS imaging data 24 hours after administration of controls (PBS, Moderna LNP, CPS peptide-LNP) and LNPs containing the peptide of this disclosure (CTS) are shown. 40 μL of either PBS or LNPs formulated at 20 ng / uL was delivered intratracheally to balb / c mice (6-8 weeks old, n=4). PBS samples contained no LNPs, and Moderna represents a peptide-less LNP control. CTS represents LNPs containing CTSTRKKQC (SEQ ID NO: 11); CPS represents LNPs containing CPSSSREKC (SEQ ID NO: 17). Formulations of each LNP control and peptide-LNP can be found in Table 6. [Figure 21] The data presented in the imaging data in Figure 20 are quantified. Mean radiance (p / s / cm2 / sr) was calculated for each lung (N=4). Similar to Figure 20, the PBS sample does not contain LNPs, and Moderna represents a peptide-less LNP control. CTS represents an LNP containing the peptide CTSTRKKQC (SEQ ID NO: 11); CPS represents an LNP containing the peptide CPSSSREKC (SEQ ID NO: 17). Formulations of each LNP control and peptide-LNP can be found in Table 6. See also Table 7. One-way ANOVA, multiple comparisons (test: Tukey). ** indicates p<0.005, and *** indicates p<0.0005. [Figure 22] The amino acid characteristics of the best-identified peptide are shown. Above, the hydroxyl characteristics of clone C are shown. Below, the hydroxyl characteristics of clone 14 are shown. Analysis performed using www.pepcalc.com. [Figure 23]Figures 23A–23E provide evidence that LNPs containing the peptides disclosed herein can be incorporated into multiple classes of LNPs. Figures 23A–23C: LNP characterization for all LNP classes. (Figure 23A) LNP size and PDI value, (Figure 23B) encapsulation efficiency, and (Figure 23C) zeta potential measurement. After incubation of LNPs on Calu-3 ALI cells for 24 hours, the percentage of GFP-positive cells (Figure 23D) and (Figure 23E) viable cells was determined by flow cytometry. Standard one-way ANOVA. Tukey's multiple comparison test with single pooled variances. *p<0.0332; **p<0.0021, ***p<0.0002, ****p<0.0001. [Figure 24] Data on LNP GFP transfection in undifferentiated HBECs are presented. Undifferentiated HBECs were treated with either PBS, a peptide-less control LNP formulation (Spikevax), or peptide C (an LNP composition containing the peptides disclosed herein). After 24 hours, cells were collected and the percentage of GFP-positive cells was measured by flow cytometry (N=3). [Figure 25] Figures 25A-25C illustrate and show data on in vivo intratracheal administration of Cre mRNA LNP. (Figures 25A, 25B) Flow cytometry analysis of tdTomato+ cells in various cell types in the lungs after intratracheal delivery of Cre mRNA to Ai9 mice (Figure 25A) (N=3; mean + SD; unpaired multiple t-test (Holm-Sidak multiple comparison test); *p<0.001) (Figure 25B) (N=3; mean + SD); unpaired t-test: *p=0.0300). (Figure 25C) Schematic diagram of Cre recombinase gene editing. Created with BioRender. [Figure 26]Figures 26A–26C provide further insights into the role of peptide-lipids, including the peptides of this disclosure, and the mechanism of uptake of the peptide-LNP compositions of this disclosure. (Figure 26A) Schematic diagram of the competitive assay described in Example 8. (Figure 26B) For example, for the competitive experiment described in Example 8, fully differentiated pHBEC cells were treated with 50 μL of a peptide-lipid conjugate (1 mg / mL) containing the peptides of this disclosure for 10 minutes at 37°C, and then treated with 15 μL of either a peptide-less NLuc LNP formulation (Spikevax / Moderna; see Tables 6 and 7) or the NLuc peptide-LNP composition of this disclosure (Spikevax CTS / Moderna_CTS_6.25 / CTS / see Tables 6 and 7), each formulated at an mRNA concentration of 40 ng / μL. After incubation at 37°C for 48 hours, bioluminescence was measured. (Figure 26C) In a separate macropinocytosis inhibitor study, 50 μL of the macropinocytosis inhibitor EIPA (25 μM) was delivered, and cells were incubated at 37°C for 30 minutes. Cells were then treated with 15 μL of the Nluc peptide-LNP composition of this disclosure (formulated at an mRNA concentration of 35 ng μL). After 48 hours of incubation at 37°C, bioluminescence was measured. **p<0.0021;****p<0.0001. [Figure 27] This demonstrates that the optimized biopanning technique described in the examples resulted in greater enrichment of the CX7C library. [Modes for carrying out the invention]

[0040] A brief explanation of the enumerated arrays The sequences listed below provide amino acid sequences of biopanned peptides, some of which demonstrate improved permeability to primary human bronchial epithelial cells. TIFF2026515751000001.tif189128TIFF2026515751000002.tif21733TIFF2026515751000003.tif50128

[0041] Exemplary Description In one embodiment, peptides for targeting cells, tissues, or organs, such as lung cells, are provided herein. In some embodiments, the peptides have properties that allow them to penetrate mucous barriers or mucous membranes, or to penetrate cells. In some embodiments, the peptides of this disclosure penetrate the membranes via direct permeation. While we do not wish to be bound by any theory, the peptides of this disclosure are thought to penetrate the membranes via endocytosis or receptor-mediated mechanisms. In some embodiments, the peptides disclosed herein can be attached to, conjugated to, or coupled with a wide variety of therapeutic agents.

[0042] Peptide-lipid nanoparticle compositions are also disclosed herein. The peptide-lipid nanoparticle compositions (peptide-LNPs) disclosed herein may, in some embodiments, enable targeted delivery to cells of cargo, such as therapeutic compounds or cargo suitable for gene editing applications. Peptides or peptide-LNPs may have advantages such as small size, cell targeting ability, ability to penetrate cell membranes, ability to penetrate mucous barriers or mucous membranes, or low immunogenicity or low immunogenicity risk. The peptides disclosed herein can be used with a wide variety of therapeutic modalities to localize therapeutic compounds in vivo or in vitro to organs, tissues, or cell types. The peptides or compositions disclosed herein may be used to improve transfection or gene delivery to organs, tissues, or cell types. The compositions of the present invention, in some embodiments, have favorable uptake in cells, specifically lung cells. In some embodiments, the compositions of the present invention have favorable uptake in epithelial cells. In some embodiments, the compositions of the present invention have favorable uptake in basal stem cells and may therefore be useful for cargo delivery for gene editing applications. In some embodiments, the compositions of the present invention have enhanced uptake in target organs, tissues, or cells compared to macrophages, which provides an improvement over known techniques. Methods for treating diseases or disorders using such compositions are also disclosed herein. Further details of these aspects, etc., are provided below.

[0043] I. Biopanning The peptides disclosed herein can be identified by the use of a phage display library. A phage display library can be generated by genetically modifying bacteriophages to present peptides on their surface by inserting random oligonucleotides into cDNA encoding a phage surface protein, thereby generating a collection of phage particles that present many permutations of unique peptides. Phage display is a technique in which a set of random peptide sequences of a predetermined length, incorporated into a phage coat protein, is expressed in a phage library, for example. The phage display library is incubated with a target, and the bound peptides are selected to identify peptide sequences that bind to target molecules, cells, e.g., primary human bronchial epithelial cells, tissues, or organs. Unbound phages are washed away, and bound phages are eluted and collected. The collected phages can be amplified and further binding / amplification cycles can be performed to enrich the peptide pool for peptides that selectively and / or specifically bind to or permeate the target. In some embodiments, phages are collected from whole cell lysates. In some embodiments, the lysed cells may first be centrifuged or spun down, and phages presenting the selected peptide are collected from the supernatant. Thus, in some embodiments, the biopanning method can select internally migrated phages. The collected phages can be amplified, and one binding or permeabilization / amplification cycle, two binding or permeabilization / amplification cycles, three binding or permeabilization / amplification cycles, four binding or permeabilization / amplification cycles, five binding or permeabilization / amplification cycles, six binding or permeabilization / amplification cycles, seven binding or permeabilization / amplification cycles, eight binding or permeabilization / amplification cycles, nine binding or permeabilization / amplification cycles, or ten binding or permeabilization / amplification cycles can be performed. Selective pressure can be applied in the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth cycles, or any combination thereof. For example, selective pressure can be applied in the first, second, and third cycles.In each cycle, the proportion of phages containing the targeting peptide for the target of interest is enriched within the pool. After several cycles, individual phage clones can be characterized by DNA sequencing to identify the targeting peptide sequence (see biopanning, e.g., US20050187161 and Pasqualini and Ruoslahti, Nature 380:364-66, 1996; Arap et al., Science 279:377-80, 1998).

[0044] In some embodiments, specific peptides capable of permeating mucous membranes or cell membranes are provided. Such peptides may have lengths of 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 amino acid residues, or any range derivable therefrom. In some embodiments, the peptides may have lengths of 9 to 15 amino acid residues.

[0045] Peptides can be characterized by their net charge at pH 7. The net charge of peptides according to this disclosure may be positive or negative. In a preferred embodiment, the net charge of mucosal permeability and / or cell membrane permeability peptides according to this disclosure is positive. In some embodiments, the mucosal permeability and / or cell membrane permeability peptide has a net positive charge of about 1 to about 5. In some embodiments, the mucosal permeability and / or cell membrane permeability 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 of net positive charges that can be derived from them. In some embodiments, the mucosal permeability and / or cell membrane permeability peptide has a net positive charge of about 1.5 to about 3. In some embodiments, the mucosal permeability and / or cell membrane permeability peptide has a net positive charge of 1.9. In some embodiments, the mucosal permeable and / or cell membrane permeable peptide has a net positive charge of 2.9. In some embodiments, the mucosal permeable and / or cell membrane permeable peptide has a net positive charge of 3.9.

[0046] The mucosal permeable and / or cell membrane permeable peptides described herein may contain amino acid residues that are charged or neutral at neutral pH. For example, the peptides disclosed herein may contain one or more independently selected amino acid residues 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 peptides disclosed contain one amino acid residue that is positively charged at neutral pH. In some embodiments, the peptides disclosed contain two amino acid residues that are positively charged at neutral pH. In some embodiments, the peptides disclosed contain three amino acid residues that are positively charged at neutral pH. In some embodiments, the peptides disclosed contain four amino acid residues that are positively charged at neutral pH.

[0047] Peptides and proteins can also be characterized by their grand average of hydropathicity index (GRAVY) score. Each amino acid is assigned a hydropathic index based on its hydrophobic and charge characteristics. These are as follows: 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); glutamic acid (-3.5); glutamine (-3.5); aspartic acid (-3.5); asparagine (-3.5); lysine (-3.9); and arginine (-4.5). The use of hydroxy amino acid indices to confer interactive biological functions to proteins is well understood in the art (Kyte and Doolittle, J. Mol. Biol. 157:105-132, 1982). The GRAVY score is calculated by dividing the sum of the hydroxy values ​​of all amino acids in a peptide or protein 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 nonpolar (hydrophobic). In some embodiments, the mucosal permeable and / or cell membrane permeable peptides according to this disclosure have a negative GRAVY score. In some embodiments, the GRAVY scores of the mucosal permeability and / or cell membrane permeability peptides according to the present disclosure are approximately -0.2, approximately -0.4, approximately -0.6, approximately -0.8, approximately -1.0, approximately -1.2, approximately -1.4, approximately -1.6, approximately -1.8, approximately -2.0, approximately -2.2, approximately -2.4, approximately -2.6, approximately -2.8, approximately -3.0, approximately -3.2, approximately -3.4, approximately -3.6, approximately -3.8, approximately -4.0, or any range derivable therefrom. In some embodiments, the present disclosure provides mucosal permeability and / or cell membrane permeability peptides having a GRAVY score less than -1.In some embodiments, the Disclosure provides mucosal permeable and / or cell membrane permeable peptides having a GRAVY score of less than -2. In some embodiments, the Disclosure provides mucosal permeable and / or cell membrane permeable peptides having a GRAVY score of less than -3. It is known that in certain circumstances, certain amino acids may be substituted with other amino acids having similar hydroxyl indices or scores while retaining similar biological activity. Changes based on the hydroxyl indices include, in some embodiments, amino acid substitutions where the hydroxyl indices are within ±2, in other embodiments, amino acid substitutions where they are within ±1, and in yet another embodiment, amino acid substitutions where they are within ±0.5.

[0048] In some embodiments, the mucosal permeable and / or cell membrane permeable peptides of this disclosure include specific amino acid residues. For example, in some embodiments, the mucosal permeable and / or cell membrane permeable peptides disclosed herein include at least one serine residue, at least one proline residue, or at least one arginine residue, or any combination thereof.

[0049] In some embodiments, the mucosal permeable and / or cell membrane permeable peptides of this disclosure contain at least one serine residue. In some embodiments, the mucosal permeable and / or cell membrane permeable peptides of this disclosure contain one serine residue, two serine residues, three serine residues, four serine residues, or five serine residues. In some embodiments, the mucosal permeable and / or cell membrane permeable peptides of this disclosure contain one serine residue. In some embodiments, the mucosal permeable and / or cell membrane permeable peptides of this disclosure contain two serine residues. In some embodiments, the mucosal permeable and / or cell membrane permeable peptides of this disclosure contain three serine residues. In some embodiments, the mucosal permeable and / or cell membrane permeable peptides of this disclosure contain four serine residues.

[0050] In some embodiments, the mucosal permeable and / or cell membrane permeable peptides of this disclosure contain at least one proline residue. In some embodiments, the mucosal permeable and / or cell membrane permeable peptides of this disclosure contain one proline residue, two proline residues, three proline residues, four proline residues, or five proline residues. In some embodiments, the mucosal permeable and / or cell membrane permeable peptides of this disclosure contain one proline residue. In some embodiments, the mucosal permeable and / or cell membrane permeable peptides of this disclosure contain two proline residues. In some embodiments, the mucosal permeable and / or cell membrane permeable peptides of this disclosure contain three proline residues. In some embodiments, the mucosal permeable and / or cell membrane permeable peptides of this disclosure contain four proline residues.

[0051] In some embodiments, the mucosal permeable and / or cell membrane permeable peptides of this disclosure contain at least one arginine residue. In some embodiments, the mucosal permeable and / or cell membrane permeable peptides of this disclosure contain one arginine residue, two arginine residues, three arginine residues, four arginine residues, or five arginine residues. In some embodiments, the mucosal permeable and / or cell membrane permeable peptides of this disclosure contain one arginine residue. In some embodiments, the mucosal permeable and / or cell membrane permeable peptides of this disclosure contain two arginine residues. In some embodiments, the mucosal permeable and / or cell membrane permeable peptides of this disclosure contain three arginine residues. In some embodiments, the mucosal permeable and / or cell membrane permeable peptides of this disclosure contain four arginine residues.

[0052] In some aspects of the present invention, the disclosure provides peptides comprising an amino acid sequence (SEQ ID NO: 1-16) 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, and exhibiting mucosal or cell membrane permeability. In another aspect of the present invention, the peptide according to the disclosure comprises the amino acid sequence CTSTRKKQC (SEQ ID NO: 11). In another aspect of the present invention, the peptide disclosed herein comprises the amino acid sequence CERSSKSSC (SEQ ID NO: 6). Peptides comprising such sequences may be used to form peptide conjugates as described elsewhere in this application, e.g., peptide-lipid conjugates, e.g., peptide-myristic acid conjugates. Peptides comprising such sequences, their functional equivalents, or peptide conjugates of either such peptides or their functional equivalents may be incorporated into therapeutic cargo delivery systems, e.g., lipid nanoparticles. Further details of the latter aspect are provided below. Therefore, the present invention provides targeted delivery of therapeutic cargo, such as mRNA, to lung cells by using the sequence or other peptides of this disclosure.

[0053] The mucosal and cell membrane permeable peptides disclosed herein can permeate cell membranes in a variety of ways. In some embodiments, the peptides disclosed herein can permeate mucosa or cell membranes by transport via endocytosis. In some embodiments, the peptides disclosed herein can directly permeate cell membranes. In some embodiments, the peptides disclosed herein exhibit uptake or permeation into certain types of cells, e.g., epithelial cells. In some embodiments, the peptides disclosed herein exhibit improved or selective uptake or permeation into certain types of cells, e.g., epithelial cells. In some embodiments, the peptides disclosed herein exhibit improved or selective uptake or permeation into certain types of cells, e.g., epithelial cells, compared to endothelial cells or immune cells. In some embodiments, the peptides disclosed herein exhibit uptake into basal epithelial cells.

[0054] The peptides disclosed herein may be functionally equivalent to any peptide described herein. The amino acid sequences of functionally equivalent mucosal and / or cell membrane permeable peptides include those having amino acid substitutions based on the similarity of the polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or amphiphilicity of the residues they contain. 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 the peptides disclosed herein may be modified to facilitate synthesis. For example, motifs or fragments present in the peptides disclosed herein can be incorporated into longer sequences that are more easily synthesized, isolated, or constructed. As another non-restrictive example, to minimize proteolysis, modifications to peptides described herein that include L amino acids can be modified to the corresponding D amino acid type.

[0055] While the desired peptide amino acid sequences described may be chemically synthesized (see, e.g., "Proteins: Structures and Molecular Principles" (Creighton, ed., WH Freeman & Company, New York, NY, 1984)), larger polypeptide sequences may be advantageously prepared by recombinant DNA technology using techniques known in the art for expressing nucleic acids containing the nucleic acid sequence encoding the desired peptide. The peptides disclosed herein may be synthesized using solid-phase peptide synthesis techniques known in the art (see, e.g., Coin, et al., Nat Protoc 2, 3247-3256, 2007). Such methods may be used to construct expression vectors containing the nucleotide sequence encoding the peptide and appropriate transcriptional and translational regulatory signals. These methods include, for example, in vitro recombinant DNA techniques, synthesis techniques, and in vivo genetic recombination (see, e.g., "Molecular Cloning, A Laboratory Manual" (above) and "Current Protocols in Molecular Biology" (above)). Alternatively, RNA and / or DNA encoding the desired peptide and nucleotide sequence may be chemically synthesized, for example, using a synthesizer (see, for example, "Oligonucleotide Synthesis: A Practical Approach" (Gait, ed., IRL Press, Oxford, United Kingdom, 1984)).

[0056] A variety of host expression vector systems can be used to express nucleotide sequences encoding peptides. 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, if the peptide or polypeptide is not secreted, or from the culture medium if the peptide or polypeptide is secreted by the host cell. However, a suitable expression system also includes modified host cells that express the desired polypeptide, peptide, or functional equivalent immobilized on the cell membrane. Purification or concentration of the desired peptide from such an expression system can be achieved using appropriate surfactants and lipid micelles, as well as methods well known to those skilled in the art. Furthermore, such modified host cells themselves can be used, for example, in a particular drug screening assay, where it is desirable not only to preserve the structural and functional characteristics of the peptide but also to assess its biological activity.

[0057] In certain applications, transient expression systems are desirable. However, stable expression is generally preferred for long-term, high-yield production of recombinant proteins or peptides. For example, cell lines can be modified to stably express a desired protein, polypeptide, peptide, or fusion protein. Instead of using expression vectors containing viral replication origins, host cells can be transformed with DNA and selectable markers regulated by appropriate expression regulators (e.g., promoters, enhancer sequences, transcriptional terminators, polyadenylation sites, etc.). After introducing the foreign DNA, the modified cells are grown in nutrient-rich medium for about 1-2 days, and then switched to a selective medium. The selectable markers in the recombinant plasmid provide resistance to selection, allowing the cells to stably incorporate the plasmid into the chromosome, grow, and form a focus, which can then be cloned and expanded into a cell line. This method can be advantageously used to modify cell lines to express a desired gene product or a portion thereof. Such modified cell lines may be particularly useful in screening and evaluating compounds that affect the endogenous activity of a desired protein, polypeptide, or peptide.

[0058] Numerous select systems may be used, including, but not limited to, the herpes simplex virus thymidine kinase gene (Wigler et al., Cell 11:223-232, 1977), the hypoxanthine-guanine phosphoribosyltransferase gene (Szybalska and Szybalski, Proc. Natl. Acad. Sci. USA 48:2026-2034, 1962), and the adenine phosphoribosyltransferase gene (Lowy et al., Cell 22:817-823, 1980), which can be used in tk-, hgprt-, or aprt- cells, respectively. Metabolite resistance to the following genes can also be used as a basis for selection: dihydrofolate reductase (dhfr) conferring methotrexate resistance (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 phosphoribosyltransferase (gpt) conferring mycophenolate resistance (Mulligan and Berg, Proc. Natl. Acad. Sci. USA 78:2072-2076, 1981); neomycin phosphotransferase (neo) conferring aminoglycoside G-418 resistance (Colbere-Garapin et al.) al., J. Mol. Biol. 150:1-14, 1981); and hygromycin B phosphotransferase (hpt) that confers hygromycin resistance (Santerre et al., Gene 30:147-156, 1984).

[0059] Host cells / expression systems that may be used for the purpose of providing the compositions used in the disclosed methods include microorganisms transformed with recombinant bacteriophage DNA, plasmid DNA, or cosmid DNA expression vectors containing a nucleotide sequence encoding the desired peptide, such as bacteria (e.g., Escherichia coli, Bacillus subtilis); and yeast transformed with recombinant yeast expression vectors containing a nucleotide sequence encoding the desired peptide (e.g., Saccharomyces cerevisiae, Pichia pastris). This includes, but is not limited to, pastoris; insect cell lines infected with a recombinant viral expression vector (e.g., baculovirus) containing a nucleotide sequence encoding the desired peptide; plant cell lines infected with a recombinant viral expression vector (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) containing a nucleotide sequence encoding the desired peptide, or transformed with a recombinant plasmid expression vector (e.g., Ti plasmid); or mammalian cell lines (e.g., COS, CHO, BHK, 293, 3T3) holding a recombinant expression construct containing a nucleotide sequence encoding the desired peptide and a promoter derived from the genome of a mammalian cell (e.g., metallothionein promoter) or a promoter derived from a mammalian virus (e.g., adenovirus late promoter, vaccinia virus 7.5K promoter).

[0060] In bacterial systems, a number of different expression vectors can be advantageously selected depending on the intended use of the desired gene product to be expressed. For example, when it is necessary to produce large quantities of such proteins, such as for the production of pharmaceutical compositions containing a desired peptide or for the production of antibodies against a protein, a vector that directs the expression of a readily purified fusion protein product at a high level 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); and the pIN vector (Inouye and Inouye, Nucleic Acids Res.13:3101-3110, 1985 and Van Heeke and Schuster, J.Biol.Chem.264:5503-5509, 1989), in which the desired peptide coding sequence can be individually ligated into the vector in-frame with the lacZ coding region so that the fusion protein is produced. pGEX vectors (GE Healthcare, Piscataway, NJ) can also be used to express a desired peptide portion as a fusion protein with glutathione S-transferase (GST). Generally, such fusion proteins are soluble and can be readily purified from lysed cells by adsorption onto glutathione-agarose beads and subsequent elution in the presence of free glutathione. pGEX vectors are designed to include a thrombin or factor Xa protease cleavage site so that the product of the gene encoding the cloned desired peptide can be released from the GST portion. In some embodiments, D-amino acids are preferred, particularly methods such as those described in 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).

[0061] In exemplary insect systems, Autographa californica nuclear polyhedron disease virus (AcNPV) is used as a vector to express a sequence encoding a desired peptide. This virus grows in Spodoptera frugiperda cells. The sequence encoding the desired peptide can be individually cloned into a non-essential region of the virus (e.g., the polyhedrin gene) and placed under the regulation of the AcNPV promoter (e.g., the polyhedrin promoter). Successful insertion of the sequence encoding the desired peptide results in the inactivation of the polyhedrin gene and the generation of an unembedded recombinant virus (i.e., a virus lacking the protein coat encoded by the polyhedrin gene). The recombinant virus is then used to infect Spodoptera fulgiperda cells, where the inserted polynucleotide is expressed (see, for example, Smith et al., J. Virol. 46:584-593, 1983 and U.S. Patent No. 4,215,051).

[0062] In mammalian host cells, numerous virus-based expression systems can be utilized. When using adenoviruses as expression vectors, the nucleotide sequence encoding the desired peptide can be ligated to the adenovirus transcription / translation regulatory complex, e.g., the late promoter and trielemental reader sequence. This chimeric sequence can then be inserted into the adenovirus genome by in vitro or in vivo recombination. Insertion into a non-essential region of the viral genome (e.g., region E1 or E3) yields a recombinant virus that is viable and capable of expressing the desired peptide product in the infected host (see, e.g., Logan and Shenk, Proc. Natl. Acad. Sci. USA 81:3655-3659, 1984). Specific start signals may also be required for efficient translation of the inserted nucleotide sequence encoding the desired peptide. These signals include the ATG start codon and its adjacent sequences. In some cases, exogenous translational regulatory signals, possibly including the ATG start codon, may be provided. Furthermore, the start codon must be phase-matched with the reading frame of the coding sequence encoding the desired peptide to ensure translation of the entire insert. These exogenous translational regulatory signals and start codons can be of diverse origins, both natural and synthetic. Expression efficiency can be enhanced by including appropriate transcriptional enhancer elements, transcriptional terminators, etc. (see, e.g., Nevins, CRC Crit. Rev. Biochem. 19:307-322, 1986).

[0063] In yeast, a number of vectors containing constitutive or inducible promoters can be used. For an overview, see, for example, "Current Protocols in Molecular Biology" (mentioned above) Ch.13, Bitter et al., Meth.Enzymol.153:516-544, 1987; "DNA Cloning", Vol.II, Ch.3 (Glover, ed., IRL Press, Washington, DC, 1986); Bitter, Meth.Enzymol.152:673-684, 1987; "The Molecular Biology of the Yeast Saccharomyces: Life Cycle and Inheritance" (Strathern et al., eds., Cold Spring Harbor Press, Cold Spring Harbor, NY, 1981); and "The Molecular Biology of the Yeast Saccharomyces: Metabolism and Gene Expression" (Strathern et al., eds., Cold Spring Harbor Press, Cold Spring Harbor, NY, 1982).

[0064] In plants, a variety of different plant expression vectors can be used, and the expression of a sequence encoding a desired peptide can be driven by any of a number of promoters. For example, viral promoters, such as the 35S RNA promoter or 19S RNA promoter 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), can be used. Alternatively, plant promoters, such as the small subunit promoter of RUBISCO (Coruzzi et al., EMBO J.3:1671-1679, 1984 and Broglie et al., Science 224:838-843, 1984), or heat shock promoters, such as soybean hsp17.5-E or hsp17.3-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 viral vectors, direct DNA transformation, microinjection, or electroporation. For an overview of such techniques, see, for example, Weissbach and Weissbach, in "Methods in Plant Molecular Biology", Section VIII (Schuler and Zielinski, eds., Academic Press, Inc., New York, NY, 1988) and "Plant Molecular Biology", 2nd Ed., Ch.7-9 (Grierson and Covey, eds., Blackie & Son, Ltd., Glasgow, Scotland, United Kingdom, 1988).

[0065] Furthermore, a host cell line may be selected that modulates the expression of the sequence encoding the desired inserted peptide, or modifies and processes the nucleic acid sequence encoding the desired peptide in a desired manner. Such modification (e.g., glycosylation) and processing (e.g., cleavage) of a protein product may affect certain functions of the protein. Various host cells have characteristic specific mechanisms for post-translational processing and modification of proteins and peptides. To ensure the correct or desired modification and processing of the expressed desired protein, polypeptide, or peptide, an appropriate cell line or host system can be selected. For this purpose, eukaryotic host cells with cellular mechanisms for desired processing of the primary transcript of the nucleic acid sequence encoding the desired peptide, as well as glycosylation and / or phosphorylation, can be used. Such mammalian host cells include, but are not limited to, Chinese hamster ovary (CHO) cells, VERO cells, baby hamster kidney (BHK) cells, HeLa cells, monkey kidney (COS) cells, MDCK cells, 293 cells, 3T3 cells, WI38 cells, human hepatocellular carcinoma (e.g., Hep G2) cells, and U937 cells.

[0066] In some embodiments, mucosal permeable and / or cell membrane permeable peptides are functionalized for further use, as described in the following sections. For example, mucosal permeable and / or cell membrane permeable peptides may be conjugated to lipids, nucleic acids (e.g., mRNA, siRNA), or polymers. Any of these peptide conjugates may be incorporated into nonviral delivery systems, but a non-limiting example is lipid nanoparticles. For example, mucosal permeable and / or cell membrane permeable peptides may be conjugated to lipids, such as fatty acids. The peptides of this disclosure may be conjugated to fatty acids containing, for example, five or fewer carbon atoms, fatty acids containing about six to about twelve carbon atoms, fatty acids containing about thirteen to about twenty-one carbon atoms, or fatty acids containing about twenty-two or more carbon atoms. The fatty acids may be saturated or unsaturated, branched or unbranched. In some embodiments, the fatty acids are saturated. Examples of saturated fatty acids that can be conjugated to the peptides of this disclosure are caprylic acid, capric acid, lauric acid, myristic acid, palmitic acid, stearic acid, arachidic acid, behenic acid, lignoceric acid, or serotic acid. In some embodiments, mucosal permeable and / or cell membrane permeable peptides are conjugated with myristic acid.

[0067] II. Nanoparticles and Nanoparticle Compositions In certain embodiments, the compositions of this disclosure are used in the treatment of diseases or disorders. The diseases or disorders may be diseases or disorders related to the dysfunction or deregulation of endothelial cells. These endothelial cells are located in the lining of the stomach or intestines (i.e., the gastrointestinal tract), the nasal cavity, the liver, the skin, the lungs, the genitals, or the eyes. Specifically, the compositions of the present invention may be used to treat diseases or disorders of the genitals, such as ovarian or cervical cancer, human immunodeficiency virus (HIV), Müllerian anomalies, endometriosis, or HPV infection. In some embodiments, the compositions of the present invention may be used to treat diseases or disorders of the gastrointestinal system, such as oral cancer, Sjögren's syndrome, inflammatory bowel disease, or colitis. In other embodiments, the compositions of the present invention may be used to treat diseases or disorders of the cornea and eye, such as genetic disorders of the retina. In other embodiments, compositions of the present invention may be used to treat lung or nasal diseases or disorders, such as chronic obstructive pulmonary disease, polynuclear respiratory virus (RSV), influenza, cytomegalovirus (CMV), cystic fibrosis, primary ciliary dysfunction, or α1 antitrypsin deficiency. These diseases or disorders may be associated with one or more gene mutations, for example, those described in Seixas et al., Appl. Clin. Genet., 14:173-194, 2021, which are incorporated herein by reference. In other embodiments, compositions of the present invention may be used to treat skin diseases or disorders, such as epidermolysis bullosa, epidermal keratosis, or polygenic skin diseases, such as systemic lupus, psoriasis, male pattern baldness, atopic dermatitis, systemic sclerosis, vitiligo, alopecia areata, pemphigus vulgaris and pemphigus foliaceus, or Sjögren's syndrome. In other embodiments, compositions of the present invention may be used to treat liver diseases or disorders. In certain embodiments, the compositions of the Disclosure may be administered in combination with one or more additional compounds or agents ("additional activators") for the treatment, management, and / or prevention of a disease or disorder.Such treatments may be administered to a patient in a therapeutically effective dose to treat or improve 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 means an amount of the compound sufficient to delay, improve, or delay the onset of disease symptoms. In some embodiments, the compositions of this disclosure include the peptides described above and elsewhere in combination with lipid nanoparticles, details of which lipid nanoparticles are provided below.

[0068] As used herein, the term “nanoparticles” refers to any material having dimensions in the range of 1 to 1,000 nm. In some embodiments, nanoparticles have dimensions in the range of 50 to 500 nm. Nanoparticles used in embodiments of the present invention include nanoscale materials, such as lipid-based nanoparticles, superparamagnetic nanoparticles, nanoshells, semiconductor nanocrystals, quantum dots, polymer-based nanoparticles, silicon-based nanoparticles, silica-based nanoparticles, metal-based nanoparticles, fullerenes, and nanotubes (Ferrari, 2005). Conjugation of polypeptides or nucleic acids with nanoparticles provides structures that can be applied to targeted delivery, release control, enhanced intracellular uptake and transport of therapeutic peptides in vitro and in vivo, as well as molecular imaging (Stayton et al., 2000; Ballou et al., 2004; Frangioni, 2003; Dubertret et al., 2002; Michaelet et al., 2005; Dwarakanath et al., 2004).

[0069] (1) Lipid nanoparticles (LNPs) Lipid-based nanoparticles include lipid nanoparticles, liposomes, lipid preparations, and lipid-based vesicles. As described above, in some embodiments, the application provides compositions comprising lipid nanoparticles. Lipid-based nanoparticles may be positively charged, negatively charged, or neutral. In a preferred embodiment of the disclosure, the lipid-based nanoparticles of the disclosure comprise cationic ionized lipids.

[0070] In specific situations, polypeptides or nucleic acids can, for example, be encapsulated in the inner aqueous phase of lipid nanoparticles or liposomes, dispersed within the lipid bilayer of lipid nanoparticles or liposomes, attached to lipid nanoparticles or liposomes via linking molecules that associate with both lipid nanoparticles or liposomes and polypeptides / nucleic acids, captured by lipid nanoparticles or liposomes, or complexed with lipid nanoparticles or liposomes.

[0071] The size of lipid nanoparticles or liposomes varies depending on the synthesis method. Lipid nanoparticles or liposomes in this disclosure may have a variety of sizes. In certain embodiments, lipid nanoparticles are small, for example, with outer diameters of 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. Generally, lipid nanoparticles or liposomes for use according to embodiments of the present invention include sizes of about 50 to 250 nm, or about 50 to about 150 nm, before nucleic acid incorporation. In other embodiments, liposomes or lipid nanoparticles may have larger diameters, e.g., 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 from there. Such lipid nanoparticle or liposome formulations may also be defined by particle charge (zeta potential) and / or optical density (OD). For example, lipid nanoparticle or liposome formulations typically have an OD of less than 0.45 before nucleic acid incorporation. 400 Includes.

[0072] The lipid nanoparticles of the Disclosure, in some cases, have zeta potentials of approximately -20mV, -19mV, approximately -18mV, approximately -17mV, approximately -16mV, -15mV, approximately -14mV, approximately -13mV, approximately -12mV, approximately -10mV, approximately -9mV, approximately -8mV, approximately -7mV, approximately -6mV, approximately -5mV, approximately -4mV, approximately -3mV, approximately -2mV, approximately -1mV, approximately 0mV, approximately 1mV, approximately 2mV, approximately 3mV, approximately 4mV, approximately 5mV, approximately 6mV, approximately 7mV, approximately 8mV, approximately 9mV, approximately 10mV, or any range that can be derived from there. In some cases, the lipid nanoparticle compositions of the Disclosure have a negative zeta potential. In some cases, the lipid nanoparticle compositions of the Disclosure have a positive zeta potential. In some embodiments, the lipid nanoparticle compositions of the Disclosure have a zeta potential of 0 to 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 from there. In some embodiments, the zeta potential of the lipid nanoparticles of the Disclosure is about 2 mV.

[0073] The lipid nanoparticle compositions provided by this disclosure are, for example, shown in the summary section of the invention above and in the claims below. They can be prepared using the methods outlined in the examples section. These methods can be further modified and optimized using the principles and techniques of organic chemistry applicable to those 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 herein by reference.

[0074] Furthermore, any protocol described herein or known to those skilled in the art may be used in the preparation of such lipid nanoparticles or liposomes. Further non-limiting examples of the preparation of lipid nanoparticles or liposomes are incorporated herein by reference, respectively, US20240051789, WO2016144376, WO2012170930, WO02 / 100435A1, WO03 / 015757A1, WO04029213A2, U.S. Patent Application No. 2004 / 0208921, and U.S. Patent Application No. 4,728,578. This is described in patent applications Nos. 4,728,575, 4,737,323, 4,533,254, 4,162,282, 4,310,505, and 4,921,706; Nos. 5,030,453, 5,962,016, and 6,680,068, international applications PCT / US85 / 01161 and PCT / US89 / 05040; UK patent application GB 2193095 A; Mayer et al., 1986; Hope et al., 1985; Mayhew et al., 1987; and Mayhew et al., 1984. A method for preparing liposomes is also described in WO04 / 002453A1.

[0075] In certain embodiments, lipid-based nanoparticles are positive lipid nanoparticles or positive liposomes. “Positive lipid nanoparticles,” “cationic lipid nanoparticles,” “positive liposomes,” or “cationic liposomes” are defined as liposomes having one or more lipid components that generate an essentially positive (substantially positive) net charge, as used herein. “Essentially positive” means that the lipid components within a given population (e.g., a population of lipid nanoparticles or liposomes) contain a positive charge that, as a whole, cannot be offset by the opposite charges of other components (i.e., less than 10%, more preferably less than 5%, and most preferably less than 1%) of the components are offset by the opposite charges of other components. In certain embodiments, the positive lipid nanoparticles or positive liposomes of this disclosure may primarily consist of lipids and / or phospholipids that are positive themselves under physiological conditions (i.e., about pH 7). As used herein, lipid components that generate an essentially positive net charge in positive lipid nanoparticles or positive liposomes, i.e., lipids that are positive in themselves under physiological conditions, may also be known as cationic ionized lipids. In some embodiments, cationic ionized lipids may be neutral at physiological pH and positively charged at acidic pH or environments. The localized microenvironment surrounding the cationic ionized lipid can influence its protonation state, resulting in a positively charged cationic ionized lipid under conditions where, without this influence, a positively charged cationic ionized lipid would not be expected. In some embodiments, the cationic ionized lipid is an aminolipid. In some embodiments, the cationic ionized lipid contains a tertiary amine. In some embodiments, the alkyl group attached to the tertiary amine may be independently substituted with a functional group, such as an ester group or a hydroxyl group. In some embodiments, the cationic ionized lipid is SM-102, MC3, or ALC-0315.

[0076] The cationic ionized lipid component of the lipid nanoparticle composition of this disclosure may be present in a variety of molar ratios relative to the composition. In some embodiments of the present invention, the cationic ionized lipid is present in a molar ratio of about 0.2 to about 1.0 relative to the lipid nanoparticle composition. In some embodiments, the molar ratio of cationic ionized lipid to the lipid nanoparticle composition is about 0.3 to about 0.7, or about 0.4 to about 0.6. The molar ratio of cationic lipid to the 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 that can be derived from there. In some embodiments, the molar ratio of cationic lipid to the lipid nanoparticle composition is about 0.45.

[0077] The cationic ionized lipids and other lipids of this disclosure may contain one or more asymmetrically substituted carbon or nitrogen atoms and may be isolated as optically active or racemic compounds. Therefore, unless a specific stereochemistry or isomer is specifically indicated, all chiral compounds, diastereomers, racemic compounds, epimers, and all geometric isomers of a given chemical formula are intended. Cationic ionized lipids may exist as racemic compounds and racemic mixtures, single enantiomers, diastereomer mixtures, and individual diastereomers. In some embodiments, a single diastereomer is obtained. The chiral centers of the cationic ionized lipids of this disclosure may have an S configuration or an R configuration. Furthermore, it is intended that one or more of the cationic ionized lipids may exist as constituent isomers. In some embodiments, compounds may have the same formula but different bonding identities.

[0078] The chemical formulas used to represent cationic ionized lipids in this disclosure typically represent only one of several possible different tautomers. For example, many types of ketone groups are known to exist in equilibrium with their corresponding enol groups. Similarly, many types of imine groups exist in equilibrium with enamine groups. Regardless of which tautomers are illustrated for a given formula, and regardless of which is most dominant, all tautomers of a given chemical formula are intended.

[0079] The cationic ionized lipids of this disclosure, whether or not they are intended for use in the indications described herein, may also have advantages over compounds known in the prior art, such as being more effective, less toxic, longer-acting, potent, having fewer side effects, being more readily absorbed, metabolically stable, lipophilic, hydrophilic, and / or having a superior pharmacokinetic profile (e.g., high oral bioavailability and / or low clearance), and / or other useful pharmacological, physical, or chemical properties.

[0080] Furthermore, the atoms constituting the cationic ionized lipids of this disclosure shall include all isotopes of such atoms. Isotopes include atoms that have the same atomic number but different mass numbers, as used herein. As a general example, non-limitingly, isotopes of hydrogen include tritium and deuterium, and isotopes of carbon include 13 C and 14 C is included.

[0081] It should be recognized that the specific anions or cations that form part of any salt form of cationic ionized lipids provided herein are irrelevant as long as the salt as a whole is pharmacologically acceptable. Further examples of pharmacologically acceptable salts, as well as methods for their preparation and use, are presented in the Handbook of Pharmaceutical Salts: Properties, and Use (2002), incorporated herein by reference.

[0082] (2) Lipids In some aspects of this disclosure, one or more additional types of lipids are mixed with the cationic ionized lipids of this disclosure to prepare a nanoparticle composition. In some embodiments, the cationic ionized lipids are mixed with one, two, three, four, or five different types of lipids. It is intended that the cationic ionized lipids may be mixed with multiple different lipids of a single type.

[0083] In some embodiments, at least one of the additional lipids may be a steroid or a steroid derivative. In some embodiments, the additional lipids may be PEG lipids. In some embodiments, the additional lipids may be phospholipids. In some embodiments, the nanoparticle composition comprises a steroid or steroid derivative, PEG lipids, and phospholipids, or any combination thereof. Further details of the types of lipids that may be used to form the nanoparticle composition are provided in the following sections.

[0084] Pisces and piscesces In the lipid nanoparticle compounds of this disclosure, a cationic ionized lipid (or compound) is mixed with a steroid or steroid derivative and other components listed below to form a nanoparticle composition. In some embodiments, the steroid or steroid derivative includes any steroid or steroid derivative. As used herein, in some embodiments, the term “steroid” refers to a class of compounds having a 4-ring, 17-carbon ring structure, which may further include one or more substitutions, e.g., alkyl groups, alkoxy groups, hydroxyl groups, oxo groups, acyl groups, or double bonds between two or more carbon atoms. In one aspect, the ring structure of a steroid is given by the following formula: It contains three condensed cyclohexyl rings and one condensed cyclopentyl ring, as shown in TIFF2026515751000004.tif17128.

[0085] In some embodiments, the steroid derivative comprises the ring structure having one or more non-alkyl substitutions. In some embodiments, the steroid or steroid derivative is a sterol, whose formula is further as follows: It is defined as TIFF2026515751000005.tif19128.

[0086] Another steroid or steroid derivative is cholestane or a cholestane derivative. In cholestane, the ring structure is further defined by the following formula: Defined by TIFF2026515751000006.tif34128.

[0087] As described above, the cholestane derivative comprises one or more non-alkyl substitutions of the cyclic system. Cholestane or cholestane derivative may be cholestene or cholestene derivative, or sterol or sterol derivative. Cholestane or cholestane derivative may be both cholesterol and sterol or derivatives thereof. In a preferred embodiment, the nanoparticle composition contains cholesterol.

[0088] In some embodiments, the compositions of the present invention include a molar ratio of steroid or steroid derivative to lipid nanoparticle composition of about 0.05 to about 0.12, or about 0.1 to about 0.6. The molar ratio of steroid or steroid derivative to lipid nanoparticle composition may be about 0.15 to about 0.5, for example, 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 from there.

[0089] PEG or PEGylated lipids In the lipid nanoparticle compounds of this disclosure, a cationic ionized lipid (or compound) is mixed with one or more PEGylated lipids (or PEG lipids) and other components described above and below to form a nanoparticle composition. In some embodiments, this disclosure includes the use of any lipid to which a PEG group is attached. In some embodiments, the PEG lipid is a diglyceride that also includes a PEG chain attached to a glycerol group. In other embodiments, the PEG lipid is a compound containing one or more C6-C24 long-chain alkyl or alkenyl groups or C6-C24 fatty acid groups attached to a linker group along with a PEG chain. In some embodiments, the PEG lipid has advantages such as preventing aggregation or reducing the uptake of the composition by immune cells. Some non-limiting examples of PEG lipids include PEG-modified phosphatidylethanolamine and phosphatidic acid, PEG-modified dialkylamines conjugated with PEG ceramide, and PEG-modified 1,2-diacyloxypropane-3-amine, PEG-modified diacylglycerol and dialkylglycerol. In some embodiments, the PEG lipid is PEG-modified diastearoylphosphatidylethanolamine. In some embodiments, the PEG lipid includes any of the PEG-modified phospholipids, e.g., those mentioned in the following sections. In some embodiments, the PEG lipid is PEG-modified dimyristoylphosphatidylethanolamine or PEG-modified myristoyl diglyceride.

[0090] In some embodiments, PEG modification is measured by the molecular weight of the PEG component of the lipid. In some embodiments, the PEG modification has a molecular weight of about 100 to about 5,000. In some embodiments, the molecular weight is about 200 to about 500, or about 1,200 to about 3,000. Some non-limiting examples of lipids that may be used in this disclosure are taught by U.S. Patent No. 5,820,873, WO 2010 / 141069, or U.S. Patent No. 8,450,298, which are incorporated herein by reference.

[0091] In some embodiments, the compositions of the present invention include a molar ratio of PEG lipids to a lipid nanoparticle composition of about 0.001 to about 0.04, or about 0.005 to about 0.03. The molar ratio may be about 0.01 to about 0.015. In some embodiments, the molar ratio of PEG lipids to a lipid nanoparticle composition may be about 0.01. In some embodiments, the ratio may be 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 from there.

[0092] Phospholipids In the lipid nanoparticle compounds of this disclosure, a cationic ionized lipid (or compound) is mixed with one or more phospholipids and other components described above and below to form a nanoparticle composition. Phospholipids are also referred to herein as “helper lipids”. In some embodiments, the compositions disclosed herein include helper lipids containing phosphate groups. In some embodiments, multiple types of phospholipids may be used to form a composition. In some embodiments, the phospholipid has a structure containing one or two long-chain C6-C24 alkyl or alkenyl groups, glycerol or sphingosine, one or two phosphate groups, and optionally also containing organic low molecular weights. In some embodiments, the organic low molecular weight is an amino acid, sugar, or amino-substituted alkoxy group, such as choline or ethanolamine. In some embodiments, the phospholipid is phosphatidylcholine. In some embodiments, the phospholipid is distearoylphosphatidylcholine, dioleoylphosphatidylcholine, or dipalmitoylphosphatidylcholine. In some embodiments, helper lipids can be neutral under physiological conditions (i.e., at approximately pH 7). In some embodiments, helper lipids have advantages such as improving structure or enhancing endosomal escape.

[0093] Phospholipids include, for example, phosphatidylcholine, phosphatidylglycerol, and phosphatidylethanolamine. Since phosphatidylethanolamine and phosphatidylcholine are uncharged under physiological conditions (i.e., at approximately pH 7), these compounds may be particularly useful for generating lipid nanoparticles or positive liposomes. In certain embodiments, phospholipid DPPC is used to produce lipid nanoparticles or positive liposomes.

[0094] Phospholipids that may be components of the compositions disclosed herein include glycerophospholipids and certain sphingolipids. Phospholipids include dioleoylphosphatidyl lycorine ("DOPC"), egg phosphatidylcholine ("EPC"), dilauryloylphosphatidylcholine ("DLPC"), dimyristoylphosphatidylcholine ("DMPC"), dipalmitoylphosphatidylcholine ("DPPC"), distearoylphosphatidylcholine ("DSPC"), 1-myristoyl-2-palmitoylphosphatidylcholine ("MPPC"), and 1-palmitoyl 2-myristoylphosphatidylcholine ("PMPC"), 1-palmitoyl-2-stearoylphosphatidylcholine ("PSPC"), 1-stearoyl-2-palmitoylphosphatidylcholine ("SPPC"), dilauryloylphosphatidylglycerol ("DLPG"), dimyristoylphosphatidylglycerol ("DMPG"), dipalmitoylphosphatidylglycerol ("DPPG"), distearoylphosph Phosphate-dylglycerol ("DSPG"), distearoyl sphingomyelin ("DSSP"), distearoylphophatidylethanolamine ("DSPE"), dioleoylphosphatidylglycerol ("DOPG"), dimyristoylphosphatidic acid ("DMPA"), dipalmitoylphosphatidic acid ("DPPA"), dimyristoylphosphatidylethanolamine ("DMPE"), dipalmitoylphosphatidic acid Tanolamine ("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"), 1,2-diarachidoyl-sn-glycero-3-phosphocholine ("DBPC"), 1,This includes, but is not limited to, 2-dieicosenoyl-sn-glycero-3-phosphocholine ("DEPC"), dioleoylphosphatidylethanolamine ("DOPE"), palmitoyloeoylphosphatidylcholine ("POPC"), palmitoyloeoylphosphatidylethanolamine ("POPE"), lysophosphatidylcholine, lysophosphatidylethanolamine, and dilinoleoylphosphatidylcholine.

[0095] In some embodiments, the compositions of the present invention include a molar ratio of phospholipids to a lipid nanoparticle composition of about 0.01 to about 0.5, or about 0.02 to about 0.4. The molar ratio may be about 0.05 to about 0.3, for example, about 0.2. In some embodiments, the molar ratio of phospholipids to a lipid nanoparticle composition is 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 from there.

[0096] Phospholipids may originate from natural or synthetic sources. However, phospholipids of natural origin, such as phosphatidylcholine from eggs or soybeans, phosphatidic acid from brains, phosphatidylinositol from brains or plants, cardiolipin from hearts, and phosphatidylethanolamine from plants or bacteria, are not used as major phosphatides (i.e., constituting more than 50% of the total phosphatide composition) in certain embodiments because they may result in instability and leakability of the resulting lipid nanoparticles or liposomes.

[0097] Bioactive polynucleotides The methods and compositions of the above embodiments relate to bioactive polynucleotides. In some cases, these may include single-stranded or double-stranded RNA or DNA. It is clear that this disclosure is not limited to the specific nucleic acids disclosed herein. However, since those skilled in the art can readily identify relevant homologs in various other origins of nucleic acids, including nucleic acids from non-human species (e.g., mice, rats, rabbits, dogs, monkeys, gibbons, chimpanzees, apes, baboons, cattle, pigs, horses, sheep, cattle, and other species), this disclosure is not limited to any particular origin, sequence, or type of nucleic acid. The nucleic acids used in this disclosure are intended to include sequences based on naturally occurring sequences.

[0098] The amount of nucleic acid encapsulated by or located within lipid nanoparticles may vary depending on the intended use. The amount of nucleic acid can be calculated as a ratio (w / w) to the lipid nanoparticle composition or to any of the individual components of the lipid nanoparticle composition (w / w). For example, the ratio of cationic ionized lipid to nucleic acid may be 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 deriveable from these. In some embodiments, the ratio of cationic ionized lipids to nucleic acids is approximately 11.33 (w / w). The length of nucleic acids encapsulated by or located within lipid nanoparticles may also vary depending on the intended use. The length of nucleic acids may be approximately 20 bp, 50 bp, 75 bp, 100 bp, 150 bp, 200 bp, 250 bp, 300 bp, 350 bp, 400 bp, 450 bp, 500 bp, 550 bp, 600 bp, 650 bp, 700 bp, 750 bp, 800 bp, 850 bp, 900 bp, 950 bp, 1000 bp, or any range deriveable from these. Longer nucleic acids are also intended, for example, approximately 1500 bp, 2000 bp, 2500 bp, 3000 bp, 3500 bp, 4000 bp, 4500 bp, 5000 bp, 5500 bp, 6000 bp, 6500 bp, 7000 bp, 7500 bp, 8000 bp, 8500 bp, 9000 bp, 9500 bp, 10,000 bp, or any range that can be derived from these.

[0099] In some cases, nucleic acids are sequences that either repress, complement, or replace another sequence present in vivo. A 17-nucleotide sequence exists only once in the human genome and should therefore be sufficient to specify a unique target sequence. Shorter oligomers are easier to construct and increase in vivo accessibility, but many other factors are involved in determining the specificity of hybridization. Increasing length increases both the binding affinity and sequence specificity of the oligonucleotide to the complementary target. 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 base pairs or more are intended for use, but others are also intended. Longer polynucleotides encoding 250, 500, 1000, 1212, 1500, 2000, 2500, 3000, or more are also being considered.

[0100] The nucleic acids used herein may be derived from genomic DNA, i.e., directly cloned from the genome of a particular organism. However, in a preferred embodiment, the nucleic acids include complementary DNA (cDNA). cDNA containing native introns or introns derived from other genes is also intended, and such modified molecules are also called “minigenes.” At least these and other nucleic acids of this disclosure can be used, for example, as molecular weight standards in gel electrophoresis.

[0101] The term "cDNA" refers to DNA prepared using messenger RNA (mRNA) as a template. In contrast to genomic DNA, or DNA polymerized from unprocessed or partially processed genomic RNA templates, the advantage of using cDNA is that it primarily contains the coding sequence for the corresponding protein. In some cases, a complete or partial genomic sequence is preferable, such as when non-coding regions are required for optimal expression, or when non-coding regions, such as introns, are targeted in antisense strategies.

[0102] In some embodiments, nucleic acids contain one or more antisense segments that inhibit the expression of a gene or gene product. Antisense methodologies take advantage of the fact that nucleic acids tend to pair with "complementary" sequences. Complementarity means that polynucleotides can base-pair according to the standard Watson-Crick complementarity rules. That is, larger purines base-pair with smaller pyrimidines to form combinations such as guanine-cytosine (G:C), adenine-thymine (A:T) in the case of DNA, and adenine-uracil (A:U) in the case of RNA. Including uncommon bases, such as inosine, 5-methylcytosine, 6-methyladenine, hypoxanthine, etc., in the hybridizing sequence does not interfere with pairing.

[0103] Targeting double-stranded (ds) DNA, along with polynucleotides, induces triple helix formation; targeting RNA induces double helix formation. Antisense polynucleotides, when introduced into target cells, specifically bind to the target polynucleotide and interfere with transcription, RNA processing, transport, translation, and / or stability. Antisense RNA constructs, or DNA encoding such antisense RNA, can be used to inhibit the transcription or translation, or both, of genes in host cells, either in vitro or in vivo, in host animals, for example, including human subjects.

[0104] Antisense constructs can be designed to bind to gene promoters and other regulatory regions, exons, introns, or even exon-intron boundaries. The most effective antisense constructs are intended to contain regions complementary to intron / exon splice junctions. Therefore, a preferred embodiment is envisioned to contain an antisense construct complementary to a region within 50–200 base pairs of an intron-exon splice junction. It has been observed that several exon sequences can be included in constructs without significantly affecting their target selectivity. The amount of exon material included varies depending on the specific exon and intron sequences used. To determine whether normal cellular function is affected, or whether the expression of related genes with complementary sequences is affected, the presence of excess exon DNA can be easily tested simply by testing the construct in vitro.

[0105] As stated above, "complementary" or "antisense" refers to polynucleotide sequences that are substantially complementary throughout their entire length and have very few base mismatches. For example, a 15-base sequence can be called complementary if it has a complementary nucleotide at position 13 or 14. Naturally, a perfectly complementary sequence is one that is perfectly complementary throughout its entire length and has no base mismatches. Other sequences with a lower degree of homology are also considered. For example, antisense constructs (e.g., ribozymes; see below) can be designed that have limited high-homonymity regions and also contain non-homonymous regions. These molecules have less than 50% homology but will bind to target sequences under appropriate conditions.

[0106] It may be advantageous to combine a portion of genomic DNA with cDNA or a synthetic sequence to form siRNA or generate a specific construct. For example, if the presence of introns in the final construct is desirable, a genomic clone must be used. cDNA, siRNA, or synthetic polynucleotides can provide more convenient restriction sites for the remainder of the construct and are therefore 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.

[0107] In other embodiments, nanoparticles may comprise nucleic acids containing one or more expression vectors used in gene therapy. Expression requires that appropriate signals be provided to the vectors, which include various regulatory elements of both viral and mammalian origin, e.g., enhancers / promoters, that drive the expression of the gene of interest in host cells. Elements designed to optimize the stability and translatability of messenger RNA in host cells have also been identified. Conditions for using numerous dominant drug selection markers to establish persistent and stable cell clones expressing the product have also been provided, as have elements that associate the expression of drug selection markers with the expression of polypeptides.

[0108] Throughout this application, the term “expression construct” includes any type of gene construct containing a nucleic acid encoding a gene product, wherein part or all of the nucleic acid coding sequence can be transcribed. The transcript may, but not necessarily, be translated into a protein. In certain embodiments, expression includes both the transcription of a gene and the translation of mRNA into a gene product. In other embodiments, expression includes only the transcription of the nucleic acid encoding the gene of interest.

[0109] The term "vector" is used to refer to a carrier nucleic acid molecule into which a nucleic acid sequence can be inserted in order to introduce the nucleic acid sequence into a cell and allow it to replicate within the cell. A nucleic acid sequence can be "exogenous," meaning it is foreign to the cell into which the vector is introduced, or that the sequence is homologous to an intracellular sequence but located in a position within the host cell nucleic acid where the sequence is not normally found. Vectors include plasmids, cosmids, viruses (bacteriophages, animal viruses, and plant viruses), and artificial chromosomes (e.g., YACs). Those skilled in the art will know that vectors can be adequately constructed by standard recombination techniques described in Sambrook et al. (1989) and Ausubel et al. (1994), both of which are incorporated herein by reference.

[0110] The term “expression vector” refers to a vector containing nucleic acid sequences that encode at least a portion of a gene product that can be transcribed. In some cases, RNA molecules are then translated into proteins, polypeptides, or peptides. In other cases, for example, in the production of antisense molecules or ribozymes, these sequences are not translated. Expression vectors may contain a variety of “regulatory sequences,” which are nucleic acid sequences required for the transcription of functionally linked coding sequences in a particular host organism, and sometimes for translation as well. In addition to regulatory sequences that govern transcription and translation, vectors and expression vectors may also contain nucleic acid sequences that perform other functions, which are described below.

[0111] mRNA In several aspects, the compounds and compositions of the present invention can be used in the delivery of mRNA to cells. Messenger RNA, or mRNA, is a short RNA chain that transmits the genetic code from DNA to ribosomes so that the mRNA can be translated into therapeutic proteins or peptides, or antigens. The mRNAs described herein may be unprocessed or processed, with poly(A) tails added, edited in vivo, or 5' capped. The mRNA molecules may contain a 5'UTR or a 3'UTR. In some embodiments, the mRNA has the advantage of exhibiting reduced degradation, for example, due to the presence of pseudouridine bases in the mRNA sequence. The compositions of the present invention are intended for the delivery of a variety of different mRNAs, including unprocessed and further processed ones. Furthermore, these nucleic acids may be used therapeutically, for in vivo antibody production, or in vaccine formulations. mRNA molecules can provide a more direct method for expressing polypeptides of interest in target cells. However, such molecules are typically very unstable and rapidly degraded. In some aspects, LNP processing according to the above embodiment can be used to substantially stabilize mRNA. In preferred aspects, mRNA is provided encapsulated in LNPs or as a complex with LNPs.

[0112] As described above, in some aspects, the nucleic acid molecule of the above embodiment encodes a therapeutic polypeptide. For example, the therapeutic protein may be a protein that becomes non-functional or is destroyed in a particular disease condition, such as an enzyme (e.g., CFTR in cystic fibrosis).

[0113] In a further aspect, the polynucleotides of the above embodiment encode antigens, such as pathogen-derived antigens or cancer cell-associated antigens. For example, the cancer-associated antigens may be CD19, CD20, ROR1, CD22, carcinoembryonic antigen, α-fetoprotein, CA-125, 5T4, MUC-1, epithelial tumor antigen, prostate-specific antigen, melanoma-associated antigen, variant p53, variant ras, HER2 / Neu, folate-binding protein, GD2, CD123, CD33, CD138, CD23, CD30, CD56, c-Met, mesoserine, GD3, HERV-K, IL-11Rα, kappa chain, lambda chain, CSPG4, ERBB2, EGFRvIII, or VEGFR2. In some specific contexts, the antigens are GP240, 5T4, HER1, CD-33, CD-38, VEGFR-1, VEGFR-2, CEA, FGFR3, IGFBP2, IGF-1R, BAFF-R, TACI, APRIL, Fn14, ERBB2, or ERBB3.

[0114] Antigens useful in this disclosure include arenaviridae (e.g., lymphocytic choriomeningitis virus), arteriviridae (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., calicivirus), coronavirusidae (e.g., human coronavirus 299E and OC43), deltaviridae (e.g., hepatitis D virus), and filovirus. Viridae (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-Barr virus, simplex virus, varicella virus, cytomegalovirus, roseolovirus, lymphocyptovirus, razinovirus), Orthomyxoviridae (e.g., influenza viruses A, B, and C), Papovaviridae (e.g., papillomavirus), Paramyxoviridae (e.g., Paramyxoviruses (e.g., human parainfluenza virus 1, morbilliviruses (e.g., measles virus, rubraviruses (e.g., mumps virus, pneumovirus (e.g., human respiratory polynuclear virus)), Picornaviridae (e.g., rhinoviruses (e.g., human rhinovirus 1A, hepatoviruses (e.g., human hepatitis A virus, human poliovirus, cardioviruses (e.g., cardiomyitis virus, aftoviruses (e.g., foot-and-mouth disease virus O, coxsackievirus)), Poxviridae (e.g., orthopox virus) Viruses (e.g., smallpox virus or monkeypox virus), Reoviridae (e.g., rotavirus, e.g., groups A-F rotaviruses), Retroviridae (primate lentivirus group, e.g., human immunodeficiency virus 1 and 2), Rhabdoviridae (e.g., rabies virus), Togaviridae (e.g., rubivirus, e.g., rubella virus), human T-cell leukemia virus, mouse leukemia virus, vesicular stomatitis virus, wart virus, blue tongue virus, Sendai virus, feline leukemia virus, monkey virus 40, mouse mammary cancer virus,This may include, but is not limited to, those derived from viruses, including, dengue virus, HIV-1 and HIV-2, West Nile virus, 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 virus 1 and 2, herpes zoster, human parvovirus (B19), polynuclear respiratory virus, poxvirus (all types and serotypes), cortivirus, reovirus (all types), and / or rubivirus (rubella).

[0115] Antigens useful in this disclosure include Streptococcus agalactiae, Legionella pneumophila, Streptococcus pyogenes, Escherichia coli, Neisseria gonorrhosae, Neisseria meningitidis, Pneumococcus, Hemophilis influenzae B, Treponema pallidum, Lyme disease spirochetes, Pseudomonas aeruginosa, Mycobacterium leprae, Brucellella abortus, Mycobacterium tuberculosis, and Plasmodium falciparum. 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 parva), Taenia hydatigena, Taenia ovis, Taenia saginata, EchinococcusMycoplasma granulosus, Mesocestoides corti, Mycoplasma arthritidis, M. hyorhinis, M. orare, M. arginini, Acholeplasma laidlawii, M. salivarium, Mycoplasma pneumoniae, Candida albicans, Cryptococcus neoformans, Histoplasma capsulatum, Coccidioides immitis, Blastomyces dermatitidis, Aspergillus fumigatus * Helicobacter fumigatus*, *Penicillium marneffei*, *Bacillus anthracis*, *Bartonella*, *Bordetella pertussis*, *Brucella* (all serotypes), *Chlamydia trachomatis*, *Chlamydia pneumoniae*, *Clostridium botulinum* (any serotype derived from *Clostridium*), *Haemophilus influenzae*, *Helicobacter pylori* *This may include, but is not limited to, bacteria derived from bacteria such as *Streptococcus aureus*, *Streptococcus pneumoniae*, *Streptococcus pyogenes*, *Vibrio cholera*, *Yersinia enterocolitica*, and / or *Yersinia pestis*.

[0116] Antigens useful in this disclosure may include, but are not limited to, those derived from parasites such as Ancylostoma hominoides, Leishmania (all strains), Microsporidium, Necator hominoides, Onchocerca filarial worms, Plasmodium (all human and monkey strains), Toxoplasma (all strains), Trypanosoma (all serotypes), and / or Wuchereria bancrofti filarial worms.

[0117] siRNA As stated above, this disclosure envisions the use of one or more inhibitory nucleic acids to reduce the expression and / or activation of a gene or gene product. Examples of inhibitory nucleic acids include, but are not limited to, molecules targeted to nucleic acid sequences, such as siRNA (small interfering RNA), small hairpin RNA (shRNA), double-stranded RNA, antisense oligonucleotides, ribozymes, and molecules targeted to genes or gene products, such as aptamers.

[0118] Inhibitory nucleic acids can inhibit gene transcription in cells or prevent the translation of gene transcripts. Inhibitory nucleic acids can be 16 to 1000 nucleotides long, and in certain embodiments, 18 to 100 nucleotides long.

[0119] Inhibitory nucleic acids are well known in the art. For example, siRNA, shRNA, and double-stranded RNA are described in U.S. Patent Nos. 6,506,559 and 6,573,099, and U.S. Patent Publications 2003 / 0051263, 2003 / 0055020, 2004 / 0265839, 2002 / 0168707, 2003 / 0159161, and 2004 / 0064842, all of which are incorporated herein by reference in their entirety.

[0120] Since the discovery of RNAi by Fire et al. in 1998, its biochemical mechanism has been rapidly characterized. Double-stranded RNA (dsRNA) is cleaved by Dicer, an RNAase III family ribonuclease. This process yields siRNAs approximately 21 nucleotides long. These siRNAs are incorporated into a multiprotein RNA-induced silencing complex (RISC), which is induced to target mRNA. RISC cleaves the target mRNA in the middle of its complementary region. In mammalian cells, related microRNAs (miRNAs), which are short RNA fragments (approximately 22 nucleotides), have been found. miRNAs are generated after Dicer-mediated cleavage of a longer (approximately 70 nucleotides) precursor with an incomplete hairpin-shaped RNA structure. miRNAs are incorporated into a miRNA-protein complex (miRNP), which leads to translational repression of the target mRNA.

[0121] Designing nucleic acids capable of producing RNAi effects requires consideration of several factors, including the properties of the siRNA, the sustainability of the silencing effect, and the choice of delivery system. siRNAs introduced into organisms to produce RNAi effects typically contain exon sequences. Furthermore, because the RNAi process is homology-dependent, sequences must be carefully selected to maximize gene specificity while minimizing the possibility of cross-interference from homologous but non-gene-specific sequences. Specifically, siRNAs exhibit greater than 80%, 85%, 90%, 95%, 98%, or even 100% identity between the siRNA sequence and a portion of the EphA nucleotide sequence. Sequences with less than approximately 80% identity to the target gene have substantially low efficacy. Therefore, the greater the identity between the siRNA and the gene to be inhibited, the less likely it is that the expression of unrelated genes will be affected.

[0122] Furthermore, the size of the siRNA is also an important consideration. In some embodiments, the disclosure relates to siRNA molecules comprising at least about 19 to 25 nucleotides and capable of modulating gene expression. In relation to the disclosure, the siRNA is specifically less than 500, 200, 100, 50, 25, or 20 nucleotides in length. In some embodiments, the siRNA is about 25 to about 35 nucleotides in length, or about 19 to about 25 nucleotides in length.

[0123] To improve the effectiveness of siRNA-mediated gene silencing, guidelines for selecting target sites on mRNA for optimal siRNA design have been developed (Soutschek et al., 2004; Wadhwa et al., 2004). These strategies may enable a rational approach to selecting siRNA sequences to achieve maximum gene knockdown. To facilitate the entry of siRNA into cells and tissues, a variety of vectors are used, including plasmids and viral vectors, such as adenoviruses, lentiviruses, and retroviruses (Wadhwa et al., 2004).

[0124] The nucleic acid components within an inhibitory nucleic acid do not need to be of the same or homogeneous type overall (for example, an inhibitory nucleic acid may contain nucleotides and nucleic acids or nucleotide analogs). Typically, an inhibitory nucleic acid forms a double-stranded structure; this double-stranded structure may arise from two separate nucleic acids that are partially or completely complementary. In certain embodiments of this disclosure, an inhibitory nucleic acid may consist of only a single nucleic acid (polynucleotide) or nucleic acid analog, forming a double-stranded structure by complementary bonding (e.g., the formation of a hairpin loop). The double-stranded structure of an inhibitory nucleic acid may contain 16 to 500 or more consecutive nucleic acid bases (including the entire derivable range). Inhibitory nucleic acids may contain 17 to 35 consecutive nucleic acid bases, more specifically 18 to 30 consecutive nucleic acid bases, more specifically 19 to 25 consecutive nucleic acid bases, more specifically 20 to 23 consecutive nucleic acid bases, or 20 to 22 consecutive nucleic acid bases, or 21 consecutive nucleic acid bases, in order to form a double-stranded structure and hybridize with a complementary nucleic acid (which may be another part of the same nucleic acid or a separate complementary nucleic acid).

[0125] siRNA may be obtained from commercial sources, from natural sources, or synthesized using any of the many techniques well known to those skilled in the art. For example, commercial sources of pre-designed siRNA include Invitrogen's Stealth™ Select technology (Carlsbad, CA), Ambion® (Austin, TX), and Qiagen® (Valencia, CA). The inhibitory nucleic acid that may be applied in the compositions and methods of this disclosure may be any nucleic acid sequence that has been found by any source to be a validated down-regulator of a gene or gene product.

[0126] In some embodiments, the disclosure features isolated siRNA molecules of at least 19 nucleotides having at least one strand that is substantially complementary to at least 10 and up to 30 consecutive nucleotides of a nucleic acid encoding a gene, and that reduces the expression of the gene or gene product. In one embodiment of the disclosure, the siRNA molecule has at least one strand that is substantially complementary to at least 10 and up to 30 consecutive nucleotides of mRNA encoding a gene or gene product.

[0127] In one embodiment, an siRNA molecule is at least 75, 80, 85, or 90% homologous to at least 10 consecutive nucleotides of any nucleic acid sequence encoding a therapeutic target protein, specifically having at least 95%, 99%, or 100% similarity or identity, or any intermediate percentage therein (for example, the disclosure intends 75% or more, 80% or more, 85% or more, etc., and these ranges include all integers in between).

[0128] siRNA may include one or more nucleotide modifications. Such modifications may include, for example, the addition of non-nucleotide material (on one or more nucleotides of the RNA) to the terminal or interior of a 19-25 nucleotide RNA. In certain aspects, the RNA molecule contains a 3'-hydroxyl group. The nucleotides of the RNA molecule of this disclosure may also include non-standard nucleotides, such as nucleotides or deoxyribonucleotides that do not exist in nature. Double-stranded oligonucleotides may contain a modified skeleton, such as a phosphorothioate skeleton, a phosphorodithioate skeleton, or other modified skeletons known in the art, or they may contain non-natural nucleoside bonds. Further modifications of siRNA (e.g., incorporation of 2'-O-methylribonucleotides, 2'-deoxy-2'-fluororibonucleotides, "universal base" nucleotides, 5-C-methylnucleotides, internucleotide bonds of one or more phosphorothioate nucleotides, and inverted deoxyabasic residues) can be found in U.S. Patent Application Publication No. 2004 / 0019001 and U.S. Patent No. 6,673,611 (each of which is incorporated in whole by reference). Collectively, all such modified nucleic acids or RNAs are referred to as modified siRNAs.

[0129] In one embodiment, siRNA can reduce the expression of a particular gene 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 range in between.

[0130] III. Pharmaceutical preparations and routes of administration In another context, a pharmaceutical preparation (also called a pharmaceutical preparation, pharmaceutical composition, pharmaceutical product, medicinal product, drug, drug therapy, or medicinal product) for administration to a patient requiring such treatment comprises a therapeutically effective amount of one of the compounds disclosed herein, formulated with one or more excipients and / or drug carriers suitable for the indicated route of administration. In some embodiments, the compounds disclosed herein are formulated in a manner suitable for the treatment of human and / or animal patients. In some embodiments, the preparation comprises mixing 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 alkanates, cellulose alkyl esters, talc, stearic acid, magnesium stearate, magnesium oxide, sodium and calcium salts of phosphoric acid and sulfate, gelatin, gum arabic, sodium alginate, polyvinylpyrrolidone, and / or polyvinyl alcohol. In some embodiments, for example, for oral administration, the pharmaceutical preparation may be made into tablets or capsules. In some embodiments, the compound may be dissolved in water, polyethylene glycol, propylene glycol, ethanol, corn oil, cottonseed oil, peanut oil, sesame oil, benzyl alcohol, sodium chloride, and / or various buffers, or may be formed into a slurry. In some embodiments, the pharmaceutical formulation may undergo 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.

[0131] Pharmaceutical formulations can be administered by a variety of methods, such as orally or by injection (e.g., subcutaneously, intravenously, and intraperitoneally). Depending on the route of administration, the compounds disclosed herein may be coated with materials to protect them from the action of acids and other natural conditions that may inactivate them. For administration of active compounds by means other than parenteral administration, it may be necessary to coat the compound with a material to prevent inactivation or to administer the compound simultaneously with it. In some embodiments, active compounds may be administered to the patient by a suitable carrier, such as liposomes or diluents. Pharmaceutically acceptable diluents include physiological saline and buffered aqueous solutions. Liposomes include not only conventional liposomes but also water-in-oil-in-water CGF emulsions.

[0132] The compounds disclosed herein may be administered parenterally, intraperitoneally, intraspinally, or intracerebrally. Dispersions may be prepared with glycerol, liquid polyethylene glycol, and mixtures thereof, as well as oils. Under normal storage and use conditions, these preparations may contain preservatives to prevent microbial growth.

[0133] Pharmaceutical compositions suitable for injection include sterile aqueous solutions (if water-soluble) or dispersions, and sterile powders for the immediate preparation of sterile injectable solutions or dispersions. The carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), suitable mixtures thereof, and vegetable oils. Appropriate fluidity can be maintained, for example, by the use of coatings such as lecithin, maintaining the required particle size in the case of dispersion, and the use of surfactants. Prevention of microbial action can be achieved by various antimicrobial and antifungal agents, such as parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, etc. In many cases, it is preferable to include isotonic agents, such as sugars, sodium chloride, or polyhydric alcohols, such as mannitol and sorbitol, in the composition. Prolonged absorption of the injectable composition can be achieved by including absorption-delaying agents, such as aluminum monostearate or gelatin, in the composition.

[0134] The compounds disclosed herein may be administered orally, for example, by an inert diluent or an assimilated food carrier. The compounds and other components may be encapsulated in hard-shell or soft-shell gelatin capsules, compressed into tablets, or directly incorporated into the patient's diet. For therapeutic oral administration, the compounds disclosed herein may be incorporated with excipients and used in the form of ingestible tablets, buccal tablets, lozenges, capsules, elixirs, suspensions, syrups, wafers, etc. The proportion of the therapeutic compound in the composition and preparation may, of course, vary. The amount of the therapeutic compound in such a pharmaceutical preparation should be such that an appropriate dosage is obtained.

[0135] Therapeutic compounds may be administered topically to the skin, eyes, ears, or mucous membranes. Topical administration of therapeutic compounds may include formulations of the compound as a topical solution, lotion, cream, ointment, gel, foam, transdermal patch, or tincture. When a therapeutic compound is formulated for topical administration, it may be combined with one or more agents that increase the penetration of the compound into the tissue to which it is administered. In other embodiments, topical administration is intended to be administered to the eye. Such administration may be applied to the surface of the cornea, conjunctiva, or sclera. While we do not wish to be bound by any theory, administration to the surface of the eye is considered to allow the therapeutic compound to reach the back of the eye. Ophthalmic topical administration may be formulated as a solution, suspension, ointment, gel, or emulsion. Finally, topical administration may also include administration to mucous membranes, for example, the oral cavity. Such administration may be made directly to specific locations within the mucous membrane, for example, teeth, wounds, or ulcers. Alternatively, if topical delivery to the lungs is desired, the therapeutic compound may be administered by inhalation in the form of a dry powder or aerosol formulation.

[0136] In some embodiments, it may be advantageous to formulate parenteral compositions in dosage units for ease of administration and uniformity of dosage. A dosage unit, as used herein, refers to a physically distinct unit suitable as a unit dose for a patient being treated, each containing a predetermined amount of the therapeutic compound, calculated to produce a desired therapeutic effect, along with the necessary pharmaceutical carrier. In some embodiments, the details of the dosage unit forms of the present invention are indicated by and directly depend on (a) the unique characteristics of the therapeutic compound and the specific therapeutic effect to be achieved, and (b) the inherent limitations in the field of compounding such therapeutic compounds for the treatment of a selected condition in a patient. In some embodiments, the active compound is administered in a sufficiently therapeutically effective dose to treat a condition relevant to the patient's condition. For example, the efficacy of a compound can be evaluated in an animal model system, thereby allowing for prediction of its efficacy in treating a disease in humans or other animals.

[0137] In some embodiments, the effective dose range of a therapeutic compound can be extrapolated from the effective dose determined in animal experiments for a variety of different animals. In some embodiments, the human equivalent dose (HED) (mg / kg) is given by the following formula: HED (mg / kg) = Animal dose (mg / kg) × (Animal K m / Human K m ) It can be calculated according to (see, for example, Reagan-Shaw et al., FASEB J., 22(3):659-661, 2008, incorporated herein by reference).

[0138] The use of the Km coefficient in the conversion yields a HED value based not only on body weight but also on body surface area (BSA). The Km values ​​for humans and various animals are well known. For example, the Km of an average 60kg human (with a BSA of 1.6m2) is 37, while a 20kg child (BSA 0.8m2) has a Km of 25. The Km values ​​for several suitable animal models are also well known; for example, the Km of a mouse is 3 (for a body weight of 0.02kg and a BSA of 0.007), the Km of a hamster is 5 (for a body weight of 0.08kg and a BSA of 0.02), the Km of a rat is 6 (for a body weight of 0.15kg and a BSA of 0.025), and the Km of a monkey is 12 (for a body weight of 3kg and a BSA of 0.24).

[0139] The precise amount of therapeutic composition depends on the practitioner's judgment and is specific to each individual. Nevertheless, the calculated HED dose provides general guidance. Other factors that influence the dose include the patient's physical and clinical condition, the route of administration, the intended target of the treatment, and the potency, stability, and toxicity of the specific therapeutic formulation.

[0140] The actual dosage administered to a patient of the compounds of this disclosure, or compositions containing the compounds of this disclosure, may be determined by physical and physiological factors, such as the species, age, sex, weight, severity of the condition of the animal being treated, the type of disease being treated, previous or simultaneous therapeutic interventions, the patient's idiopathic nature, and the route of administration. These factors may be determined by those skilled in the art. The practitioner responsible for administration typically determines the concentration of the active ingredient in the composition and the appropriate dosage for the individual patient. The dosage may be adjusted by the individual physician if complications occur.

[0141] In some embodiments, the therapeutically effective dose, administered once or multiple times daily for one or several days, typically varies (depending, of course, on the mode of administration and the aforementioned factors) 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, and from about 10 mg / kg to about 150 mg / kg. Other appropriate 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 dose is in the range of less than 10,000 mg per day and from 750 mg to 9,000 mg per day.

[0142] In some embodiments, the amount of the active compound in the pharmaceutical formulation is about 2 to about 75 percent by weight. In some of these embodiments, the amount is about 25 to about 60 percent by weight.

[0143] The agent may be administered in single or multiple doses. Desired time intervals for the delivery of multiple doses can be determined by those skilled in the art using only routine experimental methods. For example, a patient may be administered two doses per day at approximately 12-hour intervals. In some embodiments, the agent is administered once daily.

[0144] The agent may be administered on a routine schedule. As used herein, a routine schedule means a designated period of time. A routine schedule may include periods of the same length or different lengths, as long as the schedule is planned. For example, a routine schedule may include administration twice a day, daily, every two days, every three days, every four days, every five days, every six days, weekly, monthly, or any number of days or weeks in between. Alternatively, a planned routine schedule may include administration twice a day for the first week, followed by daily administration for the following months. In other embodiments, the agent may be taken orally, and the timing of administration may or may not depend on food intake, as the present invention provides. Thus, for example, the agent may be taken every morning and / or every evening, regardless of when the patient has eaten or is scheduled to eat.

[0145] IV. Definition In this disclosure, the use of the singular includes the plural, and when used in conjunction with the term “including” in the claims and / or specification, the use of the word “one (a)” or “one (an)” may mean “one,” but also coincide with the meanings of “one or more,” “at least one,” and “one or more.” As used herein, “another” may mean at least a second or more. As used herein, “or” means “and / or” unless otherwise specified.

[0146] As used herein and in the claims, the words “comprising” (and any form of “comprising,” e.g., “comprise” and “comprises”), “having” (and any form of “having,” e.g., “have” and “has”), “including” (and any form of “including,” e.g., “includes” and “include”), or “containing” (and any form of “containing,” e.g., “contains” and “contain”) are comprehensive or non-exclusive and do not exclude additional undescribed elements or steps of the method. For example, a method that “includes,” “has,” or “contains” one or more steps is not limited to the inclusion of those one or more steps, but also covers other undescribed steps.

[0147] Section headings used herein are for organizational purposes only and should not be construed as limiting the subject matter described herein. All documents or parts of documents cited herein, including but not limited to patents, patent applications, articles, books, and professional works, are expressly incorporated herein by reference for any purpose. In the event of any conflict between the definition of a term in one or more of the incorporated documents and similar materials and the definition of such term in this application, this application shall prevail.

[0148] All compounds of the present invention may, in some embodiments, be used for the prevention and treatment of one or more diseases or disorders described herein or otherwise. In some embodiments, one or more of the compounds characterized or exemplified herein may also be useful as intermediates, metabolites, and / or prodrugs for the prevention and treatment of one or more diseases or disorders. Accordingly, unless expressly stated otherwise, all compounds of the present invention are considered “active compounds” and “therapeutic compounds” intended for use as active pharmaceutical ingredients (APIs). Actual suitability for use in humans or animals is typically determined using a combination of clinical trial protocols and regulatory procedures, such as those carried out by the Food and Drug Administration (FDA). In the United States, the FDA is responsible for protecting public health by ensuring the safety, efficacy, quality, and security of human and animal drugs, vaccines, and other biological products, as well as medical devices.

[0149] In some embodiments, the compounds of the present invention, whether or not they are intended for use in the indications described herein, also have advantages over compounds known in the prior art, such as being more effective, less toxic, longer-acting, potent, producing fewer side effects, being more readily absorbed, metabolically stable, lipophilic, hydrophilic, and / or having a superior pharmacokinetic profile (e.g., high oral bioavailability and / or low clearance) and / or other useful pharmacological, physical, or chemical properties.

[0150] In some embodiments, the compounds of the present invention may function as prodrugs or may be derivatized to function as prodrugs. Since prodrugs are known to enhance many desirable qualities of pharmaceuticals (e.g., solubility, bioavailability, manufacture, etc.), the compounds used in some methods of the present invention may, if desired, be delivered in the form of prodrugs. Accordingly, the present invention aims to provide prodrugs of the compounds of the present invention and methods for delivering prodrugs. Prodrugs of the compounds used in the present invention may be prepared by modifying functional groups present in the compound in a manner that is cleaved to form a parent compound, either by routine operations or in vivo. Accordingly, prodrugs include, for example, compounds described herein in which a hydroxyl group, an amino group, or a carboxyl group is bonded to any group that is cleaved to form a hydroxyl, amino, or carboxylic acid, respectively, when the prodrug is administered to a patient. For example, compounds containing a hydroxyl group may be administered as esters that are converted to a hydroxyl compound by hydrolysis in vivo. Non-limiting examples of suitable esters that can be converted to hydroxy compounds in vivo include acetate esters, citrate esters, lactate esters, phosphate esters, tartaric acid esters, malonic acid esters, oxalic acid esters, salicylic acid esters, propionic acid esters, succinic acid esters, fumarate esters, maleic acid esters, methylene-bis-hydroxynaphthate esters, gentisic acid esters, isethionate esters, di-p-toluoleoyl tartaric acid esters, methanesulfonic acid esters, ethanesulfonic acid esters, benzenesulfonic acid esters, p-toluenesulfonic acid esters, cyclohexyl sulfamic acid esters, quinic acid esters, and amino acid esters. Similarly, compounds containing an amine group can be administered as amides that are converted to amine compounds by hydrolysis in vivo.

[0151] In some embodiments, the compounds of the present invention exist in salt or non-salt forms. With respect to salt forms, in some embodiments, the specific anions or cations that form part of any salt form of the compounds provided herein are irrelevant, as long as the salt as a whole is pharmacodynamically acceptable. Further examples of pharmacodynamically acceptable salts, as well as methods for their preparation and use, are presented in the Handbook of Pharmaceutical Salts: Properties, and Use (2002), incorporated herein by reference.

[0152] It is understood that many organic compounds can react with them or form complexes with solvents that precipitate or crystallize them. These complexes are known as “solvates.” When the solvent is water, the complex is known as a “hydrate.” It is also understood that many organic compounds can exist in multiple solid forms, 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.

[0153] As used herein, the terms “drug,” “pharmaceutical,” “therapeutic substance,” and “therapeutically active agent” are interchangeable to describe compounds used to produce therapeutic or pharmacological effects in humans or animals and to treat diseases, disorders, or other conditions. In some embodiments, these compounds are approved by regulatory authorities for administration to living organisms.

[0154] An "active ingredient" (AI) or active pharmaceutical ingredient (API) (also called an active compound, active substance, activator, drug, agent, physiologically active molecule, or therapeutic compound) is a component of a pharmaceutical drug that possesses physiological activity.

[0155] The term “effective” means sufficient to achieve the desired, expected, or intended result, as used herein and / or in the claims. “Effective amount,” “therapeutably effective amount,” or “pharmaceutically effective amount” means, when used in relation to the treatment of a patient or subject with a compound, an amount of the compound sufficient to result in the treatment or prevention of a disease (these terms are defined below) when administered to a patient or subject.

[0156] Excipients are pharmaceutically acceptable substances that are formulated together with the active ingredient of a drug therapy, pharmaceutical composition, formulation, or drug delivery system. Excipients may be used, for example, to stabilize a composition, to increase the volume of a composition (and therefore often called "bulkers," "fillers," or "diluents" when used for this purpose), or to provide therapeutic enhancements to the active ingredient in the final dosage form, such as promoting drug absorption, reducing viscosity, or increasing solubility. Excipients include pharmaceutically acceptable versions of antifouling agents, binders, coatings, colorants, disintegrants, flavorings, flow aids, lubricants, preservatives, adsorbents, sweeteners, and media. The main excipient that functions as a medium for carrying the active ingredient is usually called the media. Excipients may also be used in the manufacturing process, for example, to assist in handling the active substance by promoting the flowability or non-stick properties of the powder, in addition to assisting in-vitro stability, such as preventing denaturation or aggregation over the expected storage period. The suitability of excipients typically varies depending on the route of administration, dosage form, active ingredient, and other factors.

[0157] When the term "hydrate" is used as a modifier for a compound, it means that the compound has, for example, fewer than one (e.g., hemihydrate), one (e.g., monohydrate), or more (e.g., dihydrate) water molecules associated with each compound molecule, for example, in the solid form of the compound.

[0158] As used herein, "IC 50The term "inhibitory dose" refers to the inhibitory dose that represents 50% of the maximum response obtained. This quantitative measure indicates the amount of a particular drug or other substance (inhibitor) required to half inhibit a given biological, biochemical, or chemical process (or component of the process, i.e., an enzyme, cell, cell receptor, or microorganism).

[0159] As used herein, the terms “patient” or “subject” refer to living mammalian organisms, such as humans, monkeys, cattle, sheep, goats, dogs, cats, mice, rats, guinea pigs, or their transgenic species. In certain embodiments, the patient or subject is a primate. Limited examples of human patients include adults, juveniles, infants, and fetuses.

[0160] As is commonly used herein, “pharmaceutically acceptable” means a compound, material, composition, and / or dosage form that, within the bounds of reasonable medical judgment, is suitable for use in contact with human and animal tissues, organs, and / or bodily fluids without excessive toxicity, irritation, allergic reaction, or other problems or complications, and is commensurate with a reasonable benefit / risk ratio.

[0161] "Pharmacologically acceptable salt" means a salt of a compound disclosed herein that is pharmaceutically acceptable (as defined above) and has the desired pharmacological activity. Such salts include inorganic acids, e.g., hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, etc.; or organic acids, e.g., 1,2-ethanedisulfonic acid, 2-hydroxyethanesulfonic acid, 2-naphthalenesulfonic acid, 3-phenylpropionic acid, 4,4'methylenebis(3-hydroxy2ene-1carboxylic acid), 4-methylbicyclo[2.2.2]octa2ene-1carboxylic acid, acetic acid, aliphatic monocarboxylic and dicarboxylic acids, aliphatic sulfuric acid, aromatic sulfuric acid, benzenesulfonic acid, benzoic acid, camphorsulfonic acid, carbonic acid, cinnamic acid, citric acid, cyclopentanepropionate This includes acid addition salts formed with benzoic acid, ethanesulfonic acid, fumaric acid, glucoheptonic acid, gluconic acid, glutamic acid, glycolic acid, heptanoic acid, hexanoic acid, hydroxynaphthic acid, lactic acid, lauryl sulfate, maleic acid, malic acid, malonic acid, mandelic acid, methanesulfonic acid, muconic acid, o(4-hydroxybenzoyl)benzoic acid, oxalic acid, p-chlorobenzenesulfonic acid, phenyl-substituted alkanic acid, propionic acid, p-toluenesulfonic acid, pyruvic acid, salicylic acid, stearic acid, succinic acid, tartaric acid, tert-butylacetic acid, trimethylacetic acid, etc. Pharmaceutically acceptable salts also include base addition salts that can be formed when the present acidic protons can react 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, and N-methylglucamine. It should be recognized that the specific anions or cations that form part of any salt of the present invention are not important, as long as the salt as a whole is pharmacokinetically acceptable. Further examples of pharmacokinetically acceptable salts, as well as methods for their preparation and use, are presented in Handbook of Pharmaceutical Salts: Properties, and Use (PHStahl & CGWermuth eds., Verlag Helvetica Chimica Acta, 2002).

[0162] A "pharmaceutically acceptable carrier," "drug carrier," or simply "carrier" is a pharmaceutically acceptable substance formulated with a drug therapy that involves the transport, delivery, and / or transport of a chemical agent. Drug carriers may be used to improve drug delivery and efficacy (e.g., controlled release technologies), to modulate drug bioavailability, to reduce drug metabolism, and / or to reduce drug toxicity. Some drug carriers may increase the effectiveness of drug delivery to specific target sites. Examples of carriers include lipid nanoparticles, liposomes, microspheres (e.g., made from lactate-glycolic acid copolymers), albumin microspheres, synthetic polymers, nanofibers, protein-DNA complexes, protein conjugates, erythrocytes, visomes, and dendrimers.

[0163] A "pharmaceutical drug" (also called a pharmaceutical, pharmaceutical preparation, pharmaceutical composition, pharmaceutical preparation, pharmaceutical product, pharmaceutical product, pharmaceutical, drug therapy, medicinal product, or simply a drug, preparation, or preparation) is a composition used for the diagnosis, treatment, management, or prevention of a disease, comprising an active pharmaceutical ingredient (API) (as defined above) and optionally containing one or more inactive ingredients, also called excipients (as defined above).

[0164] The term "identity" refers to the relationship between the sequences of two or more polypeptide molecules or two or more nucleic acid molecules, determined by aligning and comparing their sequences. "Percent identity" means the percentage of identical amino acid or nucleotide residues within the molecules being compared, and is calculated based on the size of the smallest of the molecules being compared. For these calculations, any gaps in the alignment (if any) are preferably addressed by a specific mathematical model or computer program (i.e., an "algorithm"). Methods that can be used to calculate the identity of aligned nucleic acids or polypeptides include those described in Computational Molecular Biology, (Lesk, AM, ed.), 1988, New York: Oxford University Press; Biocomputing Informatics and Genome Projects, (Smith, DW, ed.), 1993, New York: Academic Press; Computer Analysis of Sequence Data, Part I, (Griffin, AM, and Griffin, HG, 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.

[0165] In calculating percent identity, the sequences to be compared are typically aligned to maximize the match between them. An 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 GAP computer algorithm is used to align two polypeptides or polynucleotides whose percent sequence identity should be determined. Their sequences are aligned for the best possible matching of each amino acid or nucleotide (the "matched span" determined by the algorithm). The gap opening penalty and gap extension penalty (generally 1 / 10th of the gap opening penalty), as well as a comparison matrix, e.g., PAM 250 or BLOSUM 62, are used with the algorithm. In certain embodiments, standard comparison matrices are also used by the algorithm (see Dayhoff et al., 1978, Atlas of Protein Sequence and Structure 5:345-352 for the PAM 250 comparison matrix; see Henikoff et al., 1992, Proc.Natl.Acad.Sci.USA89:10915-10919 for the BLOSUM 62 comparison matrix).

[0166] Examples of parameters that can be used in determining the percentage identity of polypeptide or nucleotide sequences using the GAP program can be found in Needleman et al., 1970, J.Mol.Biol.48:443-453.

[0167] A particular alignment scheme for aligning two amino acid sequences may result in matching only short regions of the two sequences, and this aligned small region may have extremely high sequence identity even though there is no significant relationship between the two full-length sequences. Therefore, the selected alignment method (GAP program) may, if desired, be modified to result in alignment of 50 or fewer consecutive amino acids of the target peptide or target polypeptide.

[0168] As used herein, the term “significant” (and any form of significant, e.g., “significantly”) does not mean a statistical difference between two values, but merely the importance or range of the difference between parameters.

[0169] Throughout this application, the term “approximately” is used to indicate that the value includes variations in error inherent to the devices or methods used to determine the value, or variations that exist between subjects of study or between experimental studies. Unless otherwise defined, “approximately” means ±10% of the indicated value.

[0170] As used herein, the terms “substantially absent” or “substantially absent” are used with respect to a particular ingredient to mean that the particular ingredient is not intentionally formulated in the composition and / or is present only as an inclusion or in trace amounts. The total amount of all containment, by-products, and other materials is present in the composition at an amount of less than 2%. The terms “more substantially absent” or “more substantially absent” are used to indicate that the composition contains less than 1% of the particular ingredient. The terms “essentially absent” or “essentially absent” indicate that the composition contains less than 0.5% of the particular ingredient.

[0171] As used herein, “treatment” or “to treat” includes (1) inhibiting a disease in a subject or patient experiencing or presenting with the pathology or symptoms of the disease (e.g., preventing further development of the pathology and / or symptoms), (2) relieving a disease in a subject or patient experiencing or presenting with the pathology or symptoms of the disease (e.g., reversing the pathology and / or symptoms), and / or (3) resulting in a measurable reduction of the disease or its symptoms in a subject or patient experiencing or presenting with the pathology or symptoms of the disease.

[0172] As used herein, unless otherwise indicated, the terms “prevent,” “prevent,” and “prevent” include (1) inhibiting the onset of the disease in subjects or patients who may be at risk and / or predisposed to the disease but have not yet experienced or presented some or all of the pathologies or symptoms of the disease, and / or (2) delaying the onset of the pathologies or symptoms of the disease in subjects or patients who may be at risk and / or predisposed to the disease but have not yet experienced or presented some or all of the pathologies or symptoms of the disease.

[0173] The term "unit dose" refers to a formulation of a compound or composition that is prepared in a manner sufficient to deliver 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 formulation, a single vial containing an injectable liquid or other injectable formulation, or a dose formulated for administration by a single inhalation.

[0174] As used herein, “targeting peptide” means a peptide comprising a sequence of amino acids characterized by localization or permeation to an organ, tissue, or cell type. Localization or permeation may be determined by the methods disclosed below, for example, by incorporating the putative targeting peptide sequence into a protein presented on the outer surface of a phage. After exposure to a library of such phages genetically modified to express a large number of such targeting peptides of different amino acid sequences, one or more organs, tissues, or cell types are collected, and the phages found in those organs, tissues, or cell types are identified. A phage expressing a targeting peptide sequence is considered to selectively localize to or selectively target an organ, tissue, or cell type if it shows greater binding or permeation to that organ, tissue, or cell type compared to a control organ, tissue, or cell type. Selective localization or targeting of peptides includes, but is not limited to, increased uptake in the targeted organ, tissue, or cell type compared to a control organ, tissue, or cell type. Preferably, the localization of the targeting peptide should result in a 2-fold or greater enrichment of the phage in the target organ, tissue, or cell type compared to a control organ, tissue, or cell type. More preferably, the localization results in a 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, or greater enrichment in the target organ, tissue, or cell type compared to a control organ, tissue, or cell type. Alternatively, a phage expressing a localized targeting peptide sequence preferably shows increased enrichment in the target organ, tissue, or cell type when the phage recovered from the target organ, tissue, or cell type is reinjected into a second volume of the organ, tissue, or cell type for further screening. Further enrichment may be shown after the third, fourth, or fifth screening.Another alternative method for determining localization is that phages expressing the putative target peptide exhibit, preferably 2-fold, more preferably 3-fold, or greater enrichment in the target organ compared to control phages expressing a nonspecific peptide or control phages that have not been genetically modified to express the putative target peptide. "Targeting peptide" and "homing peptide" are used synonymously herein.

[0175] As used herein, “phage display library” means a collection of phages genetically modified to express a set of putative targeting peptides on their outer surface. In a preferred embodiment, the DNA sequence encoding the putative targeting peptide is inserted in-frame into the gene encoding the phage capsule protein. In another preferred embodiment, the putative targeting peptide sequence is partly a random mixture of all 20 amino acids and partly non-random. In a particular preferred embodiment, the putative targeting peptide of the phage display library exhibits one or more cysteine ​​residues at fixed positions within the targeting peptide sequence. Cysteine ​​may be used, for example, to construct a cyclic peptide.

[0176] As used herein, the term “nanoparticle” has its conventional and common definition and refers to individual particles that behave as a whole unit rather than as individual molecules within a particle. Nanoparticles may have a size of about 1 to about 10,000 nm, ultrafine nanoparticles have a size of 1 nm to 100 nm, fine particles have a size of 100 nm to 2,500 nm, and coarse particles have a size of 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 of about 10 nm to about 100 μm.

[0177] Although the numerical ranges and parameters representing the broad scope of this invention are approximate, the figures shown in the specific examples are reported as accurately as possible. However, any figures inherently contain a certain degree of error that inevitably arises from the standard deviations found in each test measurement and parameter. [Examples]

[0178] V. Examples To facilitate a better understanding of this disclosure, specific examples of embodiments are given below. Those skilled in the art will understand that the techniques disclosed in the following examples represent techniques that the inventors have found to work well in the implementation of this disclosure and may therefore be considered to constitute a preferred mode for the implementation of the invention. However, those skilled in the art will understand that, considering this disclosure, it is possible to obtain similar or analogous results without departing from the spirit and scope of this disclosure, even with many modifications to the specific embodiments disclosed. The following examples should not be construed as limiting or defining the entire scope of this disclosure. Throughout the examples provided below, certain lipid nanoparticle formulations may be referred to by multiple names. See Table 7.

[0179] Example 1: Biopanning Using primary human bronchial epithelial cells (pHBEC) derived from cystic fibrosis (CF) patients containing the ΔF508 mutation, cultured at the gas-liquid interface (ALI), bacteriophage (phage) display technology was used to screen and select mucus-permeable and cell-permeable peptides (Figure 1). In ALI, these cells produce mucus and reflect the pathology of CF disease, so the theory is not constrained, but peptides selected under these conditions possess both mucus-permeable and cell-permeable capabilities. Therefore, the methods described herein for identifying mucus-permeable or cell-permeable peptides represent a more clinically relevant selection model compared, for example, to previously utilized mucus-only models. Peptide-presenting phages taken up by primary cells were collected, their DNA isolated, and subjected to high-throughput sequencing using next-generation sequencing. Thousands of sequences were analyzed using a custom Python script to identify peptide sequences responsible for enriching specific phages capable of overcoming transport in both mucus and cells. Unique sequences were identified and then further validated by cloning them back into T7 phages. Validation studies, described below and supported by figures, demonstrated that phages presenting these selected peptide sequences were able to permeate more effectively into mucin-producing primary cells compared to phages that did not present peptides on their surface.

[0180] For the enrichment process, a cysteine-restricted random 7-amino acid peptide T7 phage display library (CX7C) was repeatedly screened against highly differentiated, mucus-producing pHBEC cells via repeated high-throughput selection. In some embodiments, one library stock of the T7 library was used as described below. In other embodiments, two stocks of the T7 library were combined and used as described below. pHBEC cells derived from seven different CF patients with the most common ΔF508 mutation were pooled and cultured in ALI according to the Pneumacult® Ex-Plus medium and ALI medium protocols (StemCell Technologies; Vancouver, BC). Cells were fully differentiated by 21 days after airlift (indicated by pili and mucus production observed by microscopy). In some embodiments, four repeated selection cycles were performed. In other embodiments, five repeated selection cycles were performed. After each selection cycle, phages that had migrated internally into the cells were collected, a portion of which were quantified via a standard two-layer plaque assay, and the remaining phages were amplified. DNA was isolated from the amplified phages, 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 are provided in Table 1 and illustrated in Figure 2. Clones presenting a specific peptide were selected for further validation in the intracellular uptake studies described below.

[0181] (Table 1) Details of selections for enriching phage peptide presentation libraries for mucus permeability and intracellular uptake TIFF2026515751000007.tif59143 * Selective pressure was applied after the first, second, and third selections. The four-selection strategy according to this disclosure follows, for example, the conditions shown for selections 1-4; the five-selection strategy according to this disclosure follows, for example, the conditions shown for selections 1-5. DPBS = Dulbecco's phosphate-buffered saline.

[0182] Example 2: Feature determination and validation - Peptides identified after 4 selections Phage libraries subjected to four repeated selections resulted in an average enrichment of the CX7C library of approximately 231 times across three replicates (Figure 3). Eight clones were selected to validate CF pHBEC uptake (Figure 4). Four of these clones showed significantly enhanced CF pHBEC uptake compared to the untreated library (unselected phage library) and peptide-less phage controls, and two additional sequences showed significantly enhanced CF pHBEC uptake compared to the untreated library (unselected phage library) control. The selectivity of sequences showing significantly enhanced CF pHBEC uptake compared to both controls was further assayed to determine their selectivity for pHBEC compared to THP-1 macrophages. Clones 14 and 26 (Figure 5) showed significantly higher uptake in primary CF cells than unpolarized macrophages M0 differentiated from the THP-1 cell line. Next, we investigated the effect of scrambling the sequences of the sequences that had been shown to significantly enhance uptake within CF pHBEC compared to both controls (Figure 6). Clones 9 and 26 were found to show improved uptake into primary CF cells compared to their scrambled counterparts.

[0183] Example 3: Characterization and Validation - Peptides identified after 5 selections In separate, optimized enrichment processes, five repeated selections resulted in an average enrichment of approximately 380-fold in the CX7C library across three replicates (Figure 7). High-throughput sequencing results allowed for further analysis of the physicochemical properties of the top 30 most abundant peptides from each of the five selected replicates by calculating net charge and hydropathies after the first and fifth selections. Significant differences were observed in the weighted average of both net charge and hydropathies between the first and fifth selected peptides (Figure 8), further validating this selection process. The average net charge of peptides analyzed after five selections was higher than the average net charge of peptides analyzed after one selection (Figure 9A).

[0184] The weighted average GRAVY score of peptides analyzed after five selections was significantly more negative than the weighted average GRAVY score of peptides analyzed after one selection (Figure 8B), which corresponded to an improvement in hydrophilicity. There is known evidence in the art that increased hydrophilicity is associated with improved mucolytic diffusion (Leal et al., 2020, Kumari et al., 2022). Therefore, this application provides evidence that the peptides disclosed herein improved mucolytic diffusion, although not constrained by theory. Using the Seq2Logo tool, the multiple sequence alignments of the top 30 sequences of all replicates (N=3) after five selections were visualized, and the amino acid frequencies at each position of the 7mer peptide sequence were identified (Thomsen & Nielsen, 2012). The consensus sequence was observed to be rich in arginine and lysine, which are basic and hydrophilic amino acids, which further supported the observed trend in net charge and GRAVY score, although not constrained by theory (Figure 9C). Interestingly, this consensus differed slightly from the initial panning results, which showed lower levels of arginine and lysine (Figure 9D).

[0185] It is noteworthy that this enrichment was a significant improvement from the aforementioned four-selection strategy, which was 231-fold after the final panning (Figure 4). In the initial panning strategy, four pannings were performed, and greater variability was observed between the three replicates (Figure 10). Furthermore, it was found that a fifth addition did not improve enrichment or reproducibility under the conditions used in Example 2 (Figure 10, top). Importantly, although not constrained, a significant improvement in enrichment and reproducibility from the initial panning method was observed in all replicates (Table 1, Figure 3). Although not constrained by theory, the stringency of the selection process described in Example 3, in addition to the optimized phage collection method, was the cause of the improvement. First, after each iteration, a thorough washing step was performed to adequately remove bound phages from the apical surface of the ALI cell culture. By spinning down the lysed cells and collecting only the phages remaining in the lysate supernatant, the probability of collecting only internally migrated phages from pHBECS was increased. Previously, phages collected from whole cell lysates (containing both cell fragments and lysate supernatant) were quantified. While not theoretically constrained, the previous method was not optimal because it increased the probability of enriching phages strongly bound to the cell surface (despite multiple washing and elution steps). Finally, internally migrated phages from all three replicates were pooled before each subsequent round. Previously, internally migrated phages from each replicate were collected separately, amplified, and then added to pHBEC for the next round. Surprisingly, pooling after each round improved reproducibility and enrichment profiles. Even without pooling the last two rounds, it led to a significant difference in overall enrichment and reproducibility (Figure 10, bottom).

[0186] Six sequences present in all three replicates after five selections (of the top 10 most abundant sequences in all three replicates) were identified, incorporated into T7 cells, and validated by the ALI intracellular uptake assay (Figures 11 and 12). Sequence information for the six sequences, along with physicochemical data including the net charge and GRAVY score for each sequence, is shown in Table 2. Table 3 provides the top 10 most abundant sequences from each of the three replicates analyzed to identify the six sequences, as described above. Each clone showed an increase in abundance, i.e., the proportion of all sequences identified from NGS analysis, after each subsequent selection (Figure 11). The relative frequency enrichment of individual clones from the 1st to the 5th selection ranged from approximately 7.7 (clone B) to 13.7 (clone C). Except for clone F, the net charge was 2.9 and the GRAVY score was less than -2.0. Clone F had a net charge of 0.9 and a GRAVY score of -0.26 (Table 2). Four clones showed significantly higher uptake into primary CF HBE in ALI compared to the unselected CX7C library control. Furthermore, three of these four clones also showed significantly higher uptake compared to the peptide-less "WT" phage control.

[0187] Clones identified after 5 selections and those identified after 4 selections were validated and compared (Figures 12 and 13). In these validation studies, clone C (SEQ ID NO: 11) was found to be the most enriched in pHBEC, and clone 14 (SEQ ID NO: 6) was the second most enriched in pHBEC. From validation studies of the six clones identified after 5 selections (clones A-F in Table 2), clones B-E showed a significant difference in uptake compared to the unselected, untreated library control (p<0.05) (Figure 12). Furthermore, clones B, C, and E had significantly improved uptake compared to the peptide insertless "WT" control and the internal control sequence (indicated as "CPS"). It was previously known that clone CPS has improved diffusion in CF-like mucus (Leal et al., 2020). Clone C, the best performing clone, showed approximately 454-fold (p<0.0005), 56-fold (p<0.005), and 50-fold (p<0.005) improvements in uptake compared to the untreated library, WT, and clone CPS, respectively. To validate that clone C was superior to the top clones found after four selections (see Example 2), we compared uptake in pHBEC between clone C and the two best performing clones identified after four selections. We found 2.3-fold and 17.2-fold improvements in uptake for the clones subjected to additional selections (Figure 13).

[0188] (Table 2) T7 phage clones selected for validation and the physicochemical properties of their presented peptides TIFF2026515751000008.tif181147

[0189] (Table 3) Top 10 clones from each replica after optimized selection method TIFF2026515751000009.tif216127TIFF2026515751000010.tif21646

[0190] Example 4: Peptide-LNP composition This disclosure also provides the formation of peptide-mRNA lipid nanoparticle (LNP) systems. Following clone identification and validation after quaternary and quinary selection, lead candidate peptides were conjugated with myristic acid and incorporated into formulated lipid nanoparticles (LNPs) encapsulating nanoluciferase (NLuc) reporter mRNA using microfluidic mixing. Through formulation optimization, a lipid nanoparticle composition containing 6.25% peptide was identified as preferred for further use (Figures 14 and 15). The LNP formulations used to determine the preferred peptide-lipid nanoparticle composition are provided in Tables 4 and 5. Size and mRNA encapsulation efficiency were determined using dynamic light scattering and modified Ribogreen assays, respectively (Figure 16). The peptide-LNP compositions containing the peptides disclosed herein exhibited uniform size, with all formulations having a diameter size of less than 80 nanometers (Figure 16, left). The polydispersity index (PDI), which approximates the monodispersity of the formulations, was highest for the PEG formulation, while all other formulations had a PDI of less than 0.2 (Figure 16, left). Next, encapsulation efficiency (EE) was characterized, and all formulations except the PEG LNP formulation yielded an EE of over 90% (Figure 16, right). Finally, the zeta potential was measured for all four formulations. The LNP composition containing the peptide of this disclosure was observed to yield the largest positive zeta potential at pH 7 (Figure 16, center).

[0191] (Table 4) Determination of the highest peptide-lipid concentration TIFF2026515751000011.tif21562 "Moderna" is a peptide-free control. Moderna_25 and Moderna_50 are formulated with peptide C (also referred to herein as CTS) of the present disclosure. N / P = ratio of nitrogen to phosphate; CIL = cationic ionized lipid; PEG = polyethylene glycol; DMG-PEG = 1,2-dimiristoyl-rac-glycero-3-methoxypolyethylene glycol-2000; DSPC = distearoylphosphatidylcholine; Myr = myristoyl group.

[0192] (Table 5) LNP formulations for optimizing peptide-LNP composition formulations TIFF2026515751000012.tif20189 "Moderna" is a peptide-free control. Moderna_6.25, Moderna_12.5, Moderna_18.75, and Moderna_25 are formulated with peptide C (also referred to herein as CTS) of the present disclosure. N / P = ratio of nitrogen to phosphate; CIL = cationic ionized lipid; PEG = polyethylene glycol; DMG-PEG = 1,2-dimiristoyl-rac-glycero-3-methoxypolyethylene glycol-2000; DSPC = distearoylphosphatidylcholine; Myr = myristoyl group.

[0193] Example 5: LNPs with peptide-functional groups enhance mRNA expression. To demonstrate targeted delivery, Nluc was delivered to primary CF cells and THP-1-derived macrophages using peptide-functionalized LNPs and control LNPs (formulations shown in Table 6). Reporter expression was quantified via a standardized luciferase assay.

[0194] First, the NLuc-LNP composition was delivered to differentiated (i.e., ALI-cultured) pHBEC cells, incubated for 48 hours, and then bioluminescence was measured. The peptide-LNP composition disclosed herein exhibited targeted uptake, and relative reporter expression in CF epithelium was significantly higher compared to all other formulations, for example, compared to a suitable peptide-less LNP control (Moderna's LNP composition Spikevax containing NLuc mRNA, see Table 7), and surprisingly, even compared to a conventionally known peptide-LNP composition containing a mucopermeable peptide (Moderna_CPS_6.25 in Table 6) (Figure 16). More specifically, an increase in luminescence was observed from mRNA delivered by the peptide-containing LNP of this disclosure, 10.5 times compared to mRNA delivered by an LNP containing an additional PEG, and 4.5 times compared to mRNA delivered by a composition containing a known mucopermeable peptide but without the peptide of this disclosure (Figure 17). Furthermore, cells transfected with the LNP composition according to this disclosure exhibited bioluminescence 7.8 times higher than that of Moderna's Spikevax LNP (Figure 17).

[0195] In some embodiments, the LNP compositions of this disclosure exhibit preferential transfection in primary HBECs. LNP transfection was measured in an alternative cell model, a THP-1-derived macrophage cell line. Using the same incubation time (i.e., 48 hours) as in the validation study in primary human bronchial epithelial cells (pHBECs) described above, administration of the LNP compositions of this disclosure resulted in significantly (1.7 times) lower Nluc bioluminescence compared to administration of Moderna / Spikevax LNP. Bioluminescence from cells transfected with the LNP compositions of this disclosure was slightly lower than that from cells transfected with known mucopermeable peptide (CPS)-containing LNP formulations and slightly higher than that from the group treated with PEG LNP formulations (but the difference was not statistically significant) (Figure 18).

[0196] Example 5: LNPs with peptide-functionalized groups enhance mRNA expression in vivo. In some embodiments, the LNP compositions of this disclosure enhance delivery and NLuc bioactivity in vivo in BALB / c mice. To investigate the in vivo delivery of peptide-LNP mRNA, peptide-LNP compositions containing nanoLuc mRNA were prepared. Prepared control LNPs and peptide-LNPs containing the peptides identified according to the details above were dialyzed against 1x PBS for 4 hours and then characterized by a Ribogreen assay to determine encapsulation efficiency. LNPs and peptide-LNPs were delivered intratracheally to balb / c mice. The theoretical dose was 0.5 μg mRNA per mouse. At 24 hours, each lung was harvested and separated into five separate lobes (i.e., left lung, right anterior lobe, right accessory lobe, right posterior lobe, and right middle lobe). Bioluminescence was confirmed via IVIS imaging (Figure 19). Similar trends were observed in in vitro ALI transfection studies; cells transfected with the LNP composition of this disclosure exhibited the highest bioluminescence, followed by LNP formulations containing known mucopermeable peptides (CPS), peptide-less Moderna / Spikevax LNP formulations, and finally the PBS group (Figure 21). Compared with the peptide-less Moderna / Spikevax LNP formulations, the peptide-LNP composition of this disclosure exhibited 4.4 times higher bioluminescence. Notably, in the group treated with the peptide-LNP composition of this disclosure, the bioluminescent signal was distributed across all leaves (Figure 20).

[0197] (Table 6) Formulations of peptide-LNPs and control LNPs In TIFF2026515751000013.tif21151, "CTS" refers to CTSTRKKQC (clone C, SEQ ID NO: 11); and "CPS" refers to CPSSSREKC (SEQ ID NO: 17). "Moderna" is a peptide-less control. Moderna_CTS_6.25 is formulated with peptide C of this disclosure. Moderna_PEG control is a peptide-less control with increased PEG concentration (Huckaby et al, 2018). Moderna_CPS_6.25 is a peptide-LNP control containing a previously reported peptide (Leal et al., 2020). N / P = ratio of nitrogen to phosphoric acid; CIL = cationic ionized lipid; PEG = polyethylene glycol; DMG-PEG = 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000; DSPC = distearoylphosphatidylcholine; Myr = myristoyl group.

[0198] (Table 7) Alternative names for the formulations described herein In this disclosure, LNP compositions of the same formulation may be referred to by multiple names herein. Each row in the table provides an alternative name for the same formulation. The left column corresponds to the formulations listed in Table 6. Unless otherwise indicated, “Moderna” and “Spikevax” are generally interchangeable in the nomenclature of lipid nanoparticle formulations of this disclosure herein. For example, formulations denoted according to template formulation_%peptide-lipid (see, e.g., Figures 14 and 15), “Moderna” and “Spikevax” are interchangeable. Thus, the LNP composition denoted as “Moderna_6.25” in Figure 15 may be referred to herein as synonymous with “Spikevax_6.25,” and vice versa. "Peptide C" is synonymous with "Moderna_CTS_6.25" (Table 1), "CTS" (e.g., Figure 17), and their synonyms shown in the table above, as used herein.

[0199] Example 6: Peptide-lipids can be incorporated into multiple classes of lipid nanoparticles, which can enhance transfection in alternative ALI models. In some embodiments, the peptide-LNP compositions of the Disclosure may include peptides selected from the diverse peptides of the Disclosure, for example, those identified according to the panning protocol described above. The peptide-LNP compositions of the Disclosure may include LNPs selected from a diverse range of LNP formulations known in the Art. For example, the peptides of the Disclosure were incorporated into three LNP formulations currently approved by the FDA (Onpattro, Comirnaty, and Spikevax LNP), as well as a conventionally known spray formulation called B1 (Lewis et al., 2023). Size, polydispersity index (PDI), and zeta potential values ​​were quantified by DLS (Figures 23A and 23C). These peptide-LNP compositions of the Disclosure were observed to have a size of less than 150 nm and a PDI of 0.2 or less. The LNP compositions of the Disclosure resulted in a greater positive zeta potential shift compared to each LNP control formulation that did not contain the peptides of the Disclosure. Finally, the encapsulation efficiency of all formulations was evaluated using the modified Quant-it® RiboGreen RNA assay. The encapsulation efficiency of the peptide-LNP compositions described in this section was found to be greater than 80% (Figure 23B).

[0200] In some embodiments, the peptide-LNP compositions of the present disclosure enhance the transfection efficiency of various LNP classes. To determine whether such enhanced transfection efficiency could be observed, an alternative ALI model with Calu-3 cells was used. In ALI, Calu-3 cells were observed to exhibit lower autofluorescence, and therefore this model was more useful for conducting flow cytometry studies with GFP mRNA reporters. 1 μg of eGFP mRNA was delivered to ALI cells, and after 24 hours, the percentage of cells expressing GFP was determined by flow cytometry. Delivery of eGFP mRNA via the peptide-LNP compositions of the present disclosure significantly enhanced the percentage of GFP-expressing cells for all four formulation classes compared to PBS controls, without affecting cell viability (Figures 23D, 23E). Since the inclusion of the peptides of the present disclosure in the Spikevax / Moderna formulation enhanced reporter bioactivity for both Calu-3 and pHBEC in ALI, transfection efficiency in in-liquid pHBEC was compared. Interestingly, the peptide-LNPs of this disclosure were observed not to significantly enhance transfection in fluid cells, which, while not theoretically bound, supports the idea that these peptides were specifically enriched by uptake into differentiated cells (Figure 24).

[0201] Example 7: Peptide-lipids can enhance in vivo gene editing. In some embodiments, the peptide-LNP compositions of the present disclosure may be applied to in vivo gene editing. To demonstrate this property of the peptide-LNP compositions of the present disclosure, we used Ai9 mice (Madisen et al., 2010) containing a STOP cassette flanked by LoxP that inhibits the expression of a downstream TdTomato reporter gene. Upon introduction of Cre recombinase, it excises the STOP cassette, and TdTomato is expressed. Since the cells to be effectively edited express TdTomato and fluoresce, this conditional expression is understood as a surrogate for gene editing. Here, Cre recombinase mRNA (Cre mRNA) was delivered intratracheally to Ai9 mice using the peptide-LNP compositions of the present disclosure, and the lungs were collected 72 hours after administration of the peptide-LNP composition for flow cytometry analysis. Mice treated with the peptide-LNP composition of this disclosure (peptide C; see also Figure 25; Tables 6 and 7) showed significantly higher TdTomato bioactivity in the epithelium compared to PBS-treated mice (Figure 25A). Furthermore, administration of the peptide-LNP composition of this disclosure resulted in a 6.37-fold higher expression in the epithelium compared to immune cells (Figure 25A). Non-target cell types (i.e., endothelial cells and immune cells) did not show significantly higher tdTomato+ cells than PBS-treated mice (Figure 25A). Following these results, delivery to basal cells was investigated. After lung delivery of the peptide-LNP composition of this disclosure (peptide C; see also Table 7), statistically higher levels of editing were observed in basal cells compared to PBS-treated controls (Figure 25B; p=0.03).

[0202] Example 8: Peptide-lipid conjugates are important for mRNA physiological activity. A competitive assay was performed by pre-incubating differentiated pHBEC cells with a peptide-lipid conjugate containing the peptides of this disclosure, followed by the addition of an NLuc LNP composition (either a peptide-less control LNP or the peptide-LNP composition of this disclosure). If peptide-lipids undergo receptor-mediated endocytosis, the addition of excess peptide-lipids may saturate cell surface receptors, hindering the uptake of subsequently added LNPs and resulting in a decrease or reduction in observed NLuc bioactivity. Cells pre-treated with peptide-lipids containing the peptides of this disclosure and subsequently treated with the NLuc peptide-LNP composition of this disclosure (Spikevax CTS; Figure 26B right; see also Table 7) showed an approximately 59.3-fold decrease in NLuc bioactivity. Cells pre-treated with peptide-lipids and subsequently administered with the peptide-less LNP formulation showed a significantly smaller decrease in NLuc bioactivity, approximately 6.1-fold (Spikevax / Moderna; Figure 26B left; see also Table 7).

[0203] Example 9: Macropinocytosis inhibitors do not affect the transfection of LNPs containing the peptides of this disclosure, and therefore the possibility of non-selective uptake is low. In some embodiments, the peptide-LNP compositions of this disclosure are beneficial in that they are not taken up non-selectively. Differentiated pHBECs were treated with the macropinocytosis inhibitor EIPA (5-[N-ethyl-N-isopropyl]amyloride) and then transfected with the peptide-LNP compositions of this disclosure. EIPA is an established macropinocytosis blocker, but it does not inhibit receptor-mediated endocytosis (Kim et al., 2018; Koivusalo et al., 2010). If the peptide-LNP compositions of this disclosure behave as targeting ligands and undergo receptor-mediated endocytosis, then EIPA would not be expected to have a significant effect on the uptake of the peptide-LNP compositions of this disclosure. We have reported that no statistically significant decrease in Nluc physiological activity was observed after EIPA treatment (Figure 26C), which is consistent with the selective receptor-mediated endocytosis mechanism of uptake of the peptide-LNP compositions disclosed herein.

[0204] Example 10: Materials and Methods Culture and differentiation of primary human bronchial epithelial cells To coat Transwell® inserts (0.4 micrometers, 6.5 mm diameter, Corning Product, catalog number 3470), human placental collagen type IV (Sigma, catalog number C7521) was prepared according to a protocol established by the Marisco Lung Institute (MLI) Core, University of North Carolina. First, a 10-fold stock solution (10 mg collagen, 20 mL ddH2O, 50 μL concentrated acetic acid) was prepared and then incubated at 37°C for 4–8 hours to dissolve. The solution was then filtered and sterilized through a 0.2 μm syringe filter, divided into equal parts, and stored at -20°C. Before coating, a 1-fold solution was prepared with cell culture-grade sterile water. Next, 100 μL of the solution was added to each insert, the plate containing the inserts was dried overnight in a biosafety cabinet (with the lid removed), and the inserts were UV sterilized for at least 30 minutes before use.

[0205] Primary human bronchial epithelial cells (pHBEC) derived from seven different cystic fibrosis (CF) patients who were homozygous for the ΔF508 mutation were purchased from MLI (see patient demographics in Table 8). Unless otherwise specified, all cells used in the experiment were second-generation (P2) cells. All cells from seven patients were pooled and seeded into pre-coated inserts for differentiation at the gas-liquid interface (ALI) according to the protocols for Pneumacult® Ex-Plus medium (STEMCELL Technologies Inc., catalog no. 05040; supplemented with amphotericin B (final concentration 0.25 μg / mL) (Thermo Fisher Scientific, catalog no. BP264520), gentamicin (final concentration 50 μg / mL) (Sigma, catalog no. G1397), and 1x penicillin / streptomycin) and Pneumacult® ALI medium (STEMCELL Technologies Inc., catalog no. 05001; supplemented with 1x penicillin / streptomycin). Briefly, P2 cells were first augmented in pre-coated Transwell® inserts to at least 80% confluence. Once confluence was reached, the apical medium was removed (i.e., the cells were airlifted), and Pneumacult®-ALI maintenance medium was added only to the basolateral side. The cells were then maintained in Pneumacult®-ALI maintenance medium until differentiation occurred (confirmed by observation of mucus production and ciliary movement for at least 21 days after airlifting).

[0206] For a transfection study using undifferentiated cells, patient 1 (Table 8) was seeded at a density of 1.5E5 cells / mL in a 96-well plate (in 100 μL of cell suspension) and transfected after 48 hours.

[0207] (Table 8) List of patient donors and their demographics for primary human bronchial epithelial cells used TIFF2026515751000015.tif56147 All patients were homozygous for the ΔF508 mutation.

[0208] Selection strategy Previously developed T7 cysteine-constrained heptapeptide (CX7C) phage libraries (Leal et al., 2020; Mohanty et al., 2019) using the T7Select415-1 cloning kit (Novagen, catalog number 70015) were selected against CF pHBECs to discover mucus- and cell-permeable peptides. One T7 library (for the 4-round selection protocol) or two combined T7 libraries (for the optimized 5-round biopanning protocol) were used in DPBS (Ca 2+ and Mg 2+The solution was diluted (without any additives) to obtain 1,000 viral genomes per cell, resulting in a final concentration of 3.3E5 plaque-forming units / μL (PFU / μL) (3.3E4 pHBEC was seeded per insert). Then, 100 μL of phage solution was added to the apical side of each Transwell® insert (N=3), and incubated for a predetermined time according to the selection trials (1st trial = 16 hours, 2nd trial = 4 hours, 3rd to 5th trials = 1 hour). The initial four biopanning selection trials consisted of four trials, and the optimized biopanning selection trials consisted of a total of five trials. After incubation, the apical and batholateral solutions were removed, and the cells were washed with phage elution buffer (20 mM Tris-HCl pH8, 100 mM NaCl, 6 mM MgSO4) and DPBS to remove any remaining unbound / bound phages. Following the washing step, 200 μL of M-PER lysis buffer (Thermo Fisher Scientific Inc., catalog no. 78503) was added to the cells, and the cells were incubated on an orbital shaker at 180 revolutions per minute (RPM) for 5 minutes to ensure cell lysis. After lysis, the cells were scraped and collected (using a P200 pipette tip). In the optimized 5-pass biopanning strategy, the cells were further spun down at 14,000 × g for 10 minutes to separate cell fragments from internally migrated phages (present in the supernatant). The phages collected from the entire cell lysate (4-pass selection) or the supernatant only (optimized biopanning) were then quantified and titrated using a standard two-layer plaque assay. The methods described herein are not theoretically bound, but in the optimized panning protocol, titrating only the supernatant increases the likelihood of collecting only internally migrated phages (i.e., reduces the likelihood of collecting bound phages). In the initial four-pass panning strategy, phages collected from each replicate after each pass were amplified separately and then used in the next pass. In the optimized five-pass strategy, phages that migrated internally from each replicate were amplified, then pooled, and then used as input for the next pass.This additional optimization step, while not theoretically constrained, may improve the reproducibility of the selection process compared to known screening strategies in the art, which were known to have very few clones present in all three replicates (Leal et al., 2020). Furthermore, after sample collection, a portion of the sample was saved for next-generation sequencing (NGS), and the remaining sample was amplified in E. coli (BL21) according to the manufacturer's protocol until lysis was observed (approximately 2 hours), so that the input concentration remained the same for subsequent amplified samples. As a control for identifying parasitic sequences (Liu et al., 2015), an untreated library (unselected CX7C library) was subsequently amplified five times before preparing the NGS sample.

[0209] Next-generation sequencing data analysis After each selection run, DNA was isolated from the amplified phages, samples were prepared for NGS, and peptide sequencing analysis was performed according to known methods (Leal et al., 2020; Mohanty et al., 2019). After obtaining frequency counts of all peptide sequences from each panning run, linear sequences (i.e., those not containing the expected CX7C motif) or sequences not containing the correct adjacent sequences around the insert initially cloned into the library were excluded to appropriately select enriched CX7C sequences from the NGS data. The top 30 most frequently present sequences after all five selection runs were analyzed to determine their physicochemical properties. Specifically, the net charge and overall mean hydropathy score (GRAVY score) of each 7mer sequence were calculated using in silico tools (https: / / pepcalc.com) and (http: / / www.gravy-calculator.de / index.php), respectively. Furthermore, after combining the top 30 sequences from each replicate (a total of 90 sequences) following five selections, a visual representation of the multiple sequence alignment was created using Seq2Logo (Figure 22C) (Thomsen & Nielsen, 2012). Peptide sequences found in the top 10 most abundant peptide sequences in all three replicates and not present in the amplified untreated library control (amplified five times to correlate with five pannings) were selected for further validation studies. The enrichment of each clone selected for validation studies was evaluated by calculating the mean (N=3) percentage of all unique sequences obtained from NGS for each selection. In addition, peptide sequences were analyzed using the online tool "Scanner and Reporter of Target-Unrelated Peptides" or SAROTUP to confirm that the sequences used for validation were not target-unrelated peptides (Huang et al., 2010; Huang et al., 2012; He et al., 2016).

[0210] Clone validation After selecting peptides from NGS data for further validation, the selected peptide sequences were cloned back into T7 phages and validated by Sanger sequencing using known methods (Mohanty et al., 2019). Briefly, complementary oligonucleotides (i.e., sense oligos and antisense oligos) were obtained for each peptide sequence by IDT (Table 9). The oligos were diluted in IDTE buffer, pH 8 (IDT) to prepare a 100 μM stock solution, which was then diluted to 10 μM before use. The oligos were annealed (5 μL of 10 μM sense oligos and antisense oligos in 50 μL of reaction mixture containing ultrapure water at 95°C for 3 minutes) and then cooled to RT (25°C) at a gradient of 0.1°C / S. After annealing, the insert was ligated to a T7-Select 415-1 vector arm (Novagen (EMD Millipore), catalog number 70015-3) using T4 DNA ligase (New England Biolabs, catalog number M0202L). The ligation reaction was then packaged using the T7Select415-1b cloning kit (Novagen (EMD Millipore), catalog number 70015-3) according to the manufacturer's instructions. The packaged clones were seeded using a standard two-layer plaque assay with BL21 E. coli. After incubation of the clones overnight at 37°C, individual plaques were isolated and sequenced to confirm proper cloning. Once the clones were validated, they were amplified in liquid BL21 E. coli cultures and then subjected to validation studies. To validate the uptake of clones into enhanced CF pHBEC, each clone of 3.3E7 PFU (in 100 μL DPBS) was incubated at 37°C for 1 hour on the apical side of differentiated pHBEC. Phages were collected and quantified as in either the fourth (initial biopanning validation study) or fifth (optimized biopanning validation study) selection.To evaluate the sequence specificity of the top clones identified from the initial four-pass panning strategy, clones 1, 9, 14, and 26 were validated with their respective scrambled controls using pHBEC cultured on ALI. Scrambled peptide controls were designed using the peptide nexus online scrambling tool (https: / / peptidenexus.com / article / sequence-scrambler). The scrambled peptide sequences were cloned back into the T7 phage as described above. Clones and scrambled controls were incubated on pHBEC at 37°C for 1 hour. Cells were lysed, and whole cell lysates were collected and tiered to quantify phage uptake.

[0211] (Table 9) Oligonucleotide sequences used in validation studies The TIFF2026515751000016.tif193145 and TIFF2026515751000017.tif71145 sequences were optimized using the GenSmart codon optimization tool. Restriction sites (joined to pre-cut 415-b vector arms); ((inserted to make the insert in-reading frame)); Stop codon Cysteine. Note: Clonal CPS was obtained from a stock previously prepared in our laboratory (Marson et al., 2016). Asn = antisense.

[0212] statistical analysis Unless otherwise indicated, all data analyses were performed using GraphPad Prism 10 (GraphPad Software, La Jolla, CA) with a significance level of p ≤ 0.05.

[0213] Differentiation of THP-1 cells for macrophage uptake studies For macrophage uptake studies, THP-1 cells (ATCC, catalog number TIB-202) were cultured according to ATCC's recommendations. Briefly, cells were maintained in RPMI-1640 medium (Sigma, catalog number R8758) supplemented with 10% fetal bovine serum (FBS) and gentamicin (final concentration 50 μg / mL). For differentiation into unpolarized macrophages, cells were resuspended in medium containing phorbol 12-myrisstart 13-acetate (PMA) (Sigma, catalog number P8139) (15 ng / mL) and seeded at a final density of 4E5 live cells / mL. 0.5 mL of cell suspension was seeded per well in a 24-well plate. After incubating the cells at 37°C / 5% CO2 for 48 hours, the medium was changed to PMA-free medium, and the cells were incubated for a further 24 hours before being used in experiments.

[0214] Polarization of Calu-3 cell cultures For uptake studies, Calu-3 cells (catalog number HTB-55, American Type Culture Collection) grown in minimal essential medium (MEM) (catalog number 11095-080) supplemented with 10% FBS and 1x penicillin / streptomycin were used. After reaching 80% confluence, the cells were passaged to 3E5 cells / cm². 2 Cells were seeded at a density onto Transwell® inserts (0.4 micrometers, 6.5 mm in diameter, Corning Product, catalog number 3470). Cells were grown on the inserts until confluence (3 days), then airlifted by removing the apical medium, and culture was continued by changing only the basolateral medium every 2-3 days. The study was conducted on cells 2 weeks after ALI lift.

[0215] In vitro transfer Nanoluciferase mRNA (NLuc) was prepared according to a conventionally known method (Lewis et al., 2023). Briefly, templates for NLuc mRNA encoding the T7 promoter, 5'UTR, codon-optimized NLuc, and 3'UTR sequences were constructed. The mRNA was synthesized using the AmpliScribe® T7-Flash Transcription Kit (Lucigen, catalog number ASF-3507) as previously described (Kauffman et al., 2015; Zeng et al., 2020). After purification with RNA Clean & Concentrator-100 (Zymo, catalog number R1019), a cap 1 structure was added using the Vaccinia Capping System (NEB, M2080S) and mRNA Cap 2'-O-methyltransferase (NEB, catalog number M0366S). Next, 3'-Poly(A)tail (E. coli poly(A) polymerase, NEB, catalog number M0276L) was added. After polyadenylation, the mRNA was purified again, the mRNA concentration was determined using Nanodrop 1000 (Thermo Fisher Scientific Inc.), and aliquots were stored at -80°C until use.

[0216] Lipid nanoparticle synthesis To determine whether peptides discovered by phage display could be incorporated into nanoparticle systems, cyclic peptides (synthesized with LifeTein® and N-terminally modified) were conjugated to myristic lipid tails. This peptide-lipid was used as a fifth component in the Moderna Spikevax lipid nanoparticle formulation using a Nanoassemblr® benchtop apparatus (Precision Nanosystems Inc., Vancouver, BC, Canada). In short, lipids (peptide-lipids, SM-102 (Echelon, catalog number N-1102), cholesterol (Sigma, catalog number C8667), distearoylphosphatidylcholine (DSPC) (Avanti, catalog number 850365), and 1,2-dimiristoyl-rac-glycero-3-methoxypolyethylene (DMG-PEG) (Avanti, catalog number 880151P)) were dissolved in molecular-grade ethanol at a concentration of 10 mg / mL, and reporter mRNA (NLuc) was dissolved in pre-cooled sodium citrate buffer (pH 4, 100 mM). The formulations were prepared at a water:organic flow ratio of 3:1, a flow rate of 4 mL / min, and a volume of 500-700 μL. After formulation, LNPs were dialyzed in 1x phosphate-buffered saline (PBS; pH 7.4) for 2 hours (in vitro) or 4 hours (in vivo) using a 10K MWCO Slide-A-Lyzer dialysis cassette (Thermo Fisher Scientific, catalog number 87730).

[0217] Lipid nanoparticle synthesis For characterization, LNPs were prepared in 1x PBS (pH 7.4) for size measurement by dynamic light scattering (DLS) (10-fold dilution), and in ultrapure water for zeta potential measurement using Zetasizer Nano-ZS (Malvern Instruments MA, USA) (10-fold dilution). To determine the encapsulation efficiency percentage (EE%), the Modified Quant-it® RiboGreen RNA assay kit protocol (Thermo Fisher Scientific, catalog number R11490) was used. Briefly, to measure unencapsulated mRNA or total mRNA, LNPs were diluted 100-fold with 1x Tris-EDTA (TE) or 1% Triton® X-100 (Thermo Fisher Scientific, catalog number BP151), respectively. Two low-pass standard curves (one for TE and one for 1% Triton®) were also prepared. 100 μL of LNP sample or standard material was added in duplicate to a clear-bottom 96-well black plate (Corning, catalog no. 3631), and all samples and standards were incubated at 37°C for 10 minutes. A 2,000-fold dilution of RiboGreen reagent, followed by 100 μL of working solution, was then added to each sample or standard material. After 5 minutes, fluorescence was measured using a SpectraMax M3 plate reader (Molecular Devices) at 480 nm / 520 nm excitation / emission. EE% was expressed by the following formula: EE% = [1 - (unencapsulated mRNA / total mRNA)] * 100 The calculation was performed according to the following method.

[0218] In vitro intracellular uptake of lipid nanoparticles To compare the transfection of LNP formulations, differentiated CF pHBEC or THP-1-derived macrophages were incubated with 450 ng of mRNA (based on EE% value) for 48 hours. LNP was diluted to a total volume of 125 μL in 1x PBS. mRNA expression was measured by bioluminescence assay using the Nano-Glo® luciferase assay system (Promega, catalog no. N1110). Briefly, for pHBEC, cells were scraped from each treated well (with residual LNP solution remaining on top) and transferred to a 96-well white flat-bottom plate (Corning, catalog no. 3912). For differentiated THP-1 cells, the medium was first removed, and then 50 μL of 1x PBS (Ca) was added (to match the apical volume with pHBEC). 2+ or Mg 2+ After adding (none) to the cells, the samples were scraped and collected. To lyse the cells, the collected samples were incubated with Nano-Glo® luciferase assay buffer in a 1:1 ratio. Nano-Glo® substrate was added to each well (one group at a time), and the luminescence was read after 3 minutes using a SpectraMax M3 plate reader (Molecular Devices).

[0219] In vivo delivery of lipid nanoparticles All procedures were carried out in accordance with and under the approval of 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 with 2% isoflurane, and then 40 μL of each LNP preparation was delivered by injecting 20 μL in two divided doses. 24 hours after LNP administration, the mice were euthanized by carbon dioxide inhalation, followed by cervical dislocation, and the lungs were immediately collected. The collected lungs were briefly rinsed by immersion in 1x PBS, and each lung was separated into five separate lobes (left lobe, anterior lobe, middle lobe, accessory lobe, and posterior lobe). The lobes of each lung were then collected in a 1.5 mL microcentrifuge tube and incubated in 300 μL of Nano-Glo® substrate solution for 5 minutes, after which imaging proceeded directly using the IVIS Spectrum in vivo imaging system. Bioluminescence (average brightness calculated for the area surrounding all five leaves [p / s / cm²]) 2 The sr value was measured using Living Image 4.3 software (PerkinElmer).

[0220] Analysis of in vitro mRNA expression by flow cytometry LNP or lipofectamine messenger MAX (catalog no. LMRNA003; Invitrogren, Thermo Fisher Scientific) (1 μg mRNA or 75 μL LNP solution) was added to Calu-3 cells cultured in ALI. After 24 hours of treatment, the apical side medium / LNP solution and detached cells were removed by adding 200 μL of 0.5% trypsin (catalog no. 15400054, Gibco) to the apical side of a Transwell® plate. Then, 1:1 volume of trypsin neutralization solution (catalog no. CC-5002, Lonza) was added and the cells were transferred to a 96-well U-bottom plate. Next, the cells were spun down at 350 × g for 6 minutes and each sample was resuspended in 200 μL of FACS buffer (DPBS, 1% FBS, 1 mM EDTA). Prior to flow cytometry, 20 μL of 10x (4 μg / mL) propidium iodide was added to each well. Cells were compensated using GFP BrightComp eBeads® (catalog number A10514, Invitrogen, Thermo Fisher Scientific). Flow cytometry was performed using an Attune NxT flow cytometer.

[0221] To determine the percentage of cells transfected under in-liquid conditions, LNPs (10 μL) containing eGFP mRNA were delivered (in two separate doses (100 ng and 200 ng)) to primary human bronchial epithelium cultured under in-liquid conditions (i.e., not differentiated into multiple cell types), and the percentage of GFP-expressing cells was analyzed via flow cytometry after 24 hours. Briefly, cells were trypsinized using the Animal Component Free Cell Dissociation Kit (catalog no. 05426, Stemcell Technologies), spun down at 350 g for 5.5 minutes, and resuspended in 200 μL of FACS buffer. Prior to flow cytometry, 20 μL of 10x (4 μg / mL) propidium iodide was added to each well. Cells were corrected using GFP BrightComp eBeads®. Flow cytometry was performed using an Attune NxT flow cytometer.

[0222] In vivo gene editing research For intratracheal administration of Cre mRNA, mice (B6.Cg-Gt(ROSA)26Sortm9(CAG-tdTomato)Hze / J(Ai9) mice (catalog number 007909, The Jackson Laboratory)) were first anesthetized with 2% isoflurane, and then 50 μL of each LNP preparation (0.5 mg / kg Cre recombinase mRNA (Cat L7211, TriLink)) was delivered in two divided doses of 25 μL each. 72 hours after LNP administration, the mice were euthanized by carbon dioxide inhalation, followed by cervical dislocation, and the lungs were immediately collected. The collected lungs were briefly rinsed by immersion in 1x PBS. To determine in vivo gene editing after local administration of the peptide-LNP composition of this disclosure, the collected lungs were processed for flow cytometry as previously described (Wei et al., 2023; Lewis et al.). (al., 2023). In short, 10 mL of digestion medium (90 units / mL collagenase type I (Sigma, catalog number SCR103), 50 units / mL DNAse Lungs were digested at 37°C for 1 hour with intermittent shaking using hyaluronidase (Sigma, catalog no. H3506) at 60 units / mL (Roche, catalog no. 11284932001). A single-cell suspension was formed by pouring the digested contents into a 70-micrometer filter, and the digestion reaction was stopped with 10 mL of washing buffer (DMEM, 20% FBS, and 1% penicillin / streptomycin). The stopped solution was centrifuged at 300 × g at 4°C for 10 minutes, the cells were washed with 5 mL of 1x PBS, and the erythrocytes were lysed by incubating the cells at RT for 3 minutes in 5 mL of ACK lysis buffer, followed by stopping the reaction with 10 mL of stop buffer (90% 1x PBS, 10% FBS). After washing the cells with washing buffer and then with PBS, the cells were resuspended in 1x PBS and stained for flow cytometry as previously described (Lewis et al., 2023). The following antibodies were used for staining.Immune cells (CD45-Pacific Blue, BioLegend, catalog number 103126), endothelial cells (CD31-AF488, BioLegend, catalog number 102414), epithelial cells (CD326-AF647, catalog number 118212), and viability (Zombie NIR, BioLegend, catalog number 77184). To determine the percentage of TdTomato% basal cells, cells were stained with CD271-APC (Invitrogen, catalog number 17-9400-42). Flow cytometry was performed using an Attune NxT flow cytometer.

[0223] In vitro competitive experiment First, the peptide-lipid conjugate was equilibrated at room temperature, and diluted peptide-lipid stocks were prepared at a concentration of 1 mg / mL in DPBS (Catalog No. 21-031-CV, Corning). A 25 μM stock of EIPA (Catalog No. A3085, Sigma Aldrich) in 1x PBS was also prepared. For competitive testing, 50 μL of either 1 mg / mL peptide-lipid, 25 μM EIPA, or 1x PBS (Catalog No. 10010-023, Gibco®) was added to the cells (to match the volume between wells, for cells treated with LNP only). Cells were incubated with the peptide-lipid conjugate at 37°C for 10 minutes or with EIPA at 37°C for 30 minutes. Subsequently, cells were treated with 200 ng of NLuc mRNA peptide-LNP of this disclosure and incubated at 37°C for 48 hours. Bioluminescence was measured using NanoGlo assay buffer.

[0224] All compositions and methods disclosed herein and described in the claims can be manufactured and carried out without excessive experimental methods, taking into consideration the present disclosure. While the compositions and methods of the present disclosure have been described in terms of preferred embodiments, it will be apparent to those skilled in the art that the methods and the steps or order of steps of the methods described herein can be varied without departing from the concepts, essence, and scope of the present disclosure. More specifically, it will be apparent that the same or similar results can be achieved by using certain chemically and physiologically relevant agents in place of the agents described herein. All such similar substitutions and modifications, apparent to those skilled in the art, are deemed to fall within the essence, scope, and concepts of the present disclosure as defined by the appended claims.

[0225] References The following references are incorporated herein by reference to the extent that they provide exemplary techniques or other details that supplement what is described herein. TIFF2026515751000018.tif191131TIFF2026515751000019.tif225137TIFF2026515751000020.tif57128

Claims

1. A peptide consisting of 9 to 15 amino acids, with sequence CX 7 A peptide comprising C, having a net positive charge of at least 1 at neutral pH, and capable of permeating mucous membranes or cell membranes.

2. The peptide according to claim 1, having a net positive charge of approximately 1 to approximately 5.

3. The peptide according to claim 2, having a net positive charge of approximately 1.5 to approximately 3.

4. The peptide according to claim 3, having a net positive charge of approximately 1.

9.

5. The peptide according to claim 3, having a net positive charge of approximately 2.

9.

6. The peptide according to claim 3, having a net positive charge of approximately 3.

9.

7. A peptide according to any one of claims 1 to 6, having a GRAVY score of approximately -0.2 to approximately -4.

8. The peptide according to claim 7, having a GRAVY score of less than -1.

9. The peptide according to claim 7, having a GRAVY score of less than -2.

10. The peptide according to claim 7, having a GRAVY score of less than -3.

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

12. The peptide according to claim 11, comprising at least two amino acid residues that are positively charged at a neutral pH.

13. The peptide according to claim 11 or claim 12, comprising at least three amino acid residues that are positively charged at a neutral pH.

14. The peptide according to any one of claims 11 to 13, comprising at least four amino acid residues that are positively charged at a neutral pH.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

29. A peptide according to any one of claims 1 to 28, which can permeate through mucous membranes or cell membranes by direct permeation.

30. A peptide according to any one of claims 1 to 28, which can permeate a mucous membrane or cell membrane by endocytosis.

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

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

33. The peptide according to claim 32, wherein the epithelial cell membrane is the membrane of gastrointestinal epithelial cells.

34. The peptide according to claim 32, wherein the epithelial cell membrane is the membrane of cervical vaginal epithelial cells.

35. The peptide according to claim 32, wherein the epithelial cell membrane is the membrane of nasal epithelial cells.

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

37. The peptide according to claim 32, wherein the epithelial cell membrane is the membrane of hepatic epithelial cells.

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

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

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

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

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

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

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

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

46. The peptide according to claim 44, wherein the lung disease or disorder is a respiratory multinuclear virus.

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

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

49. The peptide according to claim 44, wherein the lung disease is primary ciliary dysfunction.

50. The peptide according to claim 44, wherein the patient having primary ciliary dysfunction has a gene mutation in the DNAI1 gene or the DNAH5 gene.

51. The peptide according to claim 44, wherein the lung disease is α1 antitrypsin deficiency.

52. The peptide according to claim 51, wherein the patient having α1 antitrypsin deficiency has a mutation in the SERPINA1 gene.

53. The peptide according to claim 52, wherein the mutations in the SERPINA1 gene are E342K and V264E.

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

55. The peptide according to claim 54, wherein the patient having cystic fibrosis has a gene mutation associated with cystic fibrosis.

56. The aforementioned mutations are ΔF508, G542X, N1303K, W1282X, E56K, G178R, S549R, K1060T, G1244E, P67L, E193K, G551D, A1067T, S1251N, R74W, L206W, G551S, G1069R, S1255P, D110E, R3 The method according to claim 55, selected from the group consisting of 47H, D579G, R1070Q, D1270N, D110H, R352Q, S945L, R1070W, G1349D, R117C, A455E, S977F, F1074L, R117H, S549N, F1052V, and D1152H.

57. The method according to claim 56, wherein the mutation is selected from ΔF508, G542X, G551D, N1303K, and W1282X.

58. The peptide according to claim 57, wherein the gene mutation is a ΔF508 mutation.

59. The peptide according to claim 43, wherein the disease or disorder is a disease or disorder of the cervix.

60. The peptide according to claim 59, wherein the disease or disorder of the cervix is ​​caused by human immunodeficiency virus (HIV).

61. The peptide according to claim 59, wherein the disease or disorder of the cervix is ​​caused by human papillomavirus (HPV).

62. The peptide according to claim 59, wherein the disease or disorder of the cervical vagina is a Müllerian duct abnormality.

63. The peptide according to claim 59, wherein the disease or disorder of the cervix and vagina is endometriosis.

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

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

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

67. The peptide according to claim 65, wherein the skin disease or disorder is epidermal lytic keratosis.

68. Peptides of 41.4 in which the skin disease or disorder is a polygenic skin disease.

69. The peptide according to claim 68, wherein the polygenic skin disease is systemic lupus, psoriasis, male pattern baldness, atopic dermatitis, systemic sclerosis, vitiligo, alopecia areata, pemphigus vulgaris and pemphigus foliaceus, or Sjögren's syndrome.

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

71. The peptide according to claim 70, wherein the gastrointestinal disease or disorder is oral cancer.

72. The peptide according to claim 70, wherein the gastrointestinal disease or disorder is Sjögren's syndrome.

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

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

75. The peptide according to claim 43, wherein the disease or disorder is a disease or disorder of the cornea or eye.

76. The peptide according to claim 43, wherein the disease or disorder of the cornea or eye is a hereditary retinal disorder.

77. A peptide according to any one of claims 1 to 76, which selectively targets organ cells compared to macrophages.

78. The peptide according to claim 77, wherein the organ cells are skin cells, lung cells, liver cells, corneal cells, cervical cells, vaginal cells, nasal cells, or gastrointestinal cells.

79. The peptide according to any one of claims 1 to 78, having at least 95% sequence identity with respect to SEQ IDs 1 to 16.

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

81. (a) A peptide according to any one of claims 1 to 80, (b) Hydrophobic group and A conjugate comprising a peptide, wherein the hydrophobic group is covalently bonded to the peptide.

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

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

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

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

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

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

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

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

90. (a) A peptide according to any one of claims 1 to 80 or a conjugate according to any one of claims 81 to 89, (b) Lipid nanoparticles and A composition containing the following:

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

92. The composition according to claim 90 or claim 91, wherein the lipid nanoparticles include cationic lipids.

93. The composition according to claim 92, wherein the cationic lipid is an ionized cationic lipid.

94. The composition according to any one of claims 90 to 93, wherein the lipid nanoparticles contain phospholipids.

95. The composition according to any one of claims 90 to 94, wherein the lipid nanoparticles contain sterols.

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

97. The composition according to any one of claims 90 to 96, wherein the lipid nanoparticles include lipids conjugated with a polymer.

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

99. The composition according to any one of claims 90 to 98, wherein the lipid nanoparticles encapsulate a protein or a therapeutic substance.

100. The composition according to claim 99, wherein the lipid nanoparticles are encapsulated with proteins.

101. The composition according to claim 99, wherein the lipid nanoparticles encapsulate a therapeutic substance.

102. The composition according to claim 101, wherein the therapeutic substance is a low molecular weight substance.

103. The composition according to claim 101, wherein the therapeutic substance is nucleic acid.

104. The composition according to claim 101, wherein the therapeutic substance is a polypeptide or an antibody.

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

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

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

108. The method according to claim 106, wherein the lung disease or disorder is caused by a multinuclear respiratory virus (RSV).

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

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

111. The method according to claim 106, wherein the lung disease is primary ciliary dysfunction.

112. The method according to claim 111, wherein the patient having primary ciliary dysfunction has a gene mutation in the DNAI1 gene or the DNAH5 gene.

113. The method according to claim 106, wherein the lung disease is α1 antitrypsin deficiency.

114. The method according to claim 113, wherein the patient having α1 antitrypsin deficiency has a mutation in the SERPINA1 gene.

115. The method according to claim 114, wherein the mutations in the SERPINA1 gene are E342K and V264E.

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

117. The method according to claim 116, wherein the patient having cystic fibrosis has a gene mutation associated with cystic fibrosis.

118. The aforementioned mutations are ΔF508, G542X, N1303K, W1282X, E56K, G178R, S549R, K1060T, G1244E, P67L, E193K, G551D, A1067T, S1251N, R74W, L206W, G551S, G1069R, S1255P, D110E, R3 The method according to claim 117, selected from the group consisting of 47H, D579G, R1070Q, D1270N, D110H, R352Q, S945L, R1070W, G1349D, R117C, A455E, S977F, F1074L, R117H, S549N, F1052V, and D1152H.

119. The method according to claim 118, wherein the mutation is selected from ΔF508, G542X, G551D, N1303K, and W1282X.

120. The method according to claim 119, wherein the gene mutation is a ΔF508 mutation.

121. The method according to claim 105, wherein the disease or disorder is a disease or disorder of the cervix.

122. The method according to claim 121, wherein the disease or disorder of the cervix is ​​caused by human immunodeficiency virus (HIV).

123. The method according to claim 121, wherein the disease or disorder of the cervix is ​​caused by human papillomavirus (HPV).

124. The method according to claim 121, wherein the disease or disorder of the cervical vagina is a Müllerian duct anomaly.

125. The method according to claim 121, wherein the disease or disorder of the cervix and vagina is endometriosis.

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

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

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

129. The method according to claim 127, wherein the skin disease or disorder is epidermal lytic keratosis.

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

131. The method according to claim 130, wherein the polygenic skin disease is systemic lupus, psoriasis, male pattern baldness, atopic dermatitis, systemic sclerosis, vitiligo, alopecia areata, pemphigus vulgaris and pemphigus foliaceus, or Sjögren's syndrome.

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

133. The method according to claim 132, wherein the gastrointestinal disease or disorder is oral cancer.

134. The method according to claim 132, wherein the gastrointestinal disease or disorder is Sjögren's syndrome.

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

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

137. The method according to claim 105, wherein the disease or disorder is a disease or disorder of the cornea or eye.

138. The method according to claim 105, wherein the disease or disorder of the cornea or eye is a hereditary retinal disorder.

139. A method for delivering a compound to the epithelial cells of a patient, comprising the step of administering to the patient a composition according to any one of claims 90 to 104.

140. The method according to claim 139, wherein the epithelial cells are lung epithelial cells.

141. The method according to claim 139, wherein the epithelial cells are gastrointestinal epithelial cells.

142. The method according to claim 139, wherein the epithelial cells are corneal or ocular epithelial cells.

143. The method according to claim 139, wherein the epithelial cells are cervical vaginal epithelial cells.

144. The method according to claim 139, wherein the epithelial cells are nasal epithelial cells.

145. The method according to claim 139, wherein the epithelial cells are hepatic epithelial cells.

146. The method according to any one of claims 139 to 145, wherein the compound is a nucleic acid.

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

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

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

150. The method according to either claim 148 or claim 149, wherein the composition is administered by inhalation.

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

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