Chimeric fusogenic peptides and methods of use thereof

Abstract

The present invention relates to fusogenic peptide carrier molecules for delivery of therapeutic nucleic acid to cells and methods of use thereof for the treatment of a disease or disorder.

Description

Attorney Docket No.206678-0001-00WOCHIMERIC FUSOGENIC PEPTIDES AND METHODS OF USE THEREOFCROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U. S. Provisional Application No. 63 / 726,542, filed November 30, 2024, which is hereby incorporated by reference herein in its entirety.REFERENCE TO A “SEQUENCE LISTING” SUBMITTED AS AN XML FILE

[0002] The present application hereby incorporates by reference the entire contents of the sequence listing xml document named “206678-0001-00WO_SequenceListing.xml”. The xml file containing the Sequence Listing of the present application was created on November 28, 2025 and is 294,137 bytes in size.BACKGROUND OF THE INVENTION

[0003] Delivering therapeutic nucleic acids (DNA, mRNA, siRNA, etc.) into target cells safely and efficiently remains a major challenge. LNPs have emerged as promising delivery vehicles, for example in gene therapies for diseases like cystic fibrosis (CF). Conventional LNPs, however, typically enter cells via endocytosis and must rely on endosomal escape to release their cargo intracellularly. This process is notoriously inefficient - studies estimate only ~l-2% of internalized nucleic acid payloads successfully escape the endosomes into the cytosol. The rest remains trapped and is eventually degraded in lysosomes. Such poor cytosolic delivery greatly limits the bioavailability of LNP-encapsulated therapeuticsmdpi.com.

[0004] Furthermore, endosomal entrapment and escape can trigger unwanted immune responses and cellular stress. The rupture of endosomal membranes during escape may activate innate immune receptors (e.g. certain toll-like receptors like TLR9) and danger-associated pathways, leading to inflammation and cytotoxicity. Indeed, conventional LNP delivery is often accompanied by activation of immune sensors and cytokine release, partly due to exposure of the nucleic acid cargo to endosomal toll-like receptors and other intracellular surveillance mechanisms. These inflammatory responses can reduce tolerability and complicate repeated dosing of LNP -based therapies.

[0005] There remains a need in the art for improved methods for delivery of therapeutic nucleic acid molecules, including for delivery of gene therapy agents. The present invention addresses this need.SUMMARY OF THE INVENTION

[0006] The present invention relates to fusogenic peptide carrier molecules for delivery of therapeutic nucleic acid to cells and methods of use thereof for the treatment of a disease or disorder.

[0007] In one embodiment, the invention relates to a chimeric fusogenic peptide comprising at least one of a ectodomain and a transmembrane domain (TMD) of a first fusion-associated small transmembrane (FAST) protein linked to an endodomain of a second FAST protein. In one embodiment, the endodomain is an endodomain of a pl4, p 15, plO or p22 FAST protein.

[0008] In one embodiment, the endodomain is one of SEQ ID NO:259-281. In one embodiment, the peptide comprises at least one peptide of SEQ ID NO:248-258 linked to at least one peptide of SEQ ID NO:259-281. In one embodiment, the peptide comprises a sequence as set forth in SEQ ID NO: 1-242. In one embodiment, the peptide comprises a sequence as set forth in SEQ ID NO: 1-8.

[0009] In one embodiment, the invention relates to a composition comprising a chimeric fusogenic peptide comprising at least one of a ectodomain and a TMD of a first FAST protein linked to an endodomain of a second FAST protein. In one embodiment, the endodomain is an endodomain of a pl4, p 15, plO or p22 FAST protein. In one embodiment, the endodomain is one of SEQ ID NO:259-281. In one embodiment, the peptide comprises at least one peptide of SEQ ID NO:248-258 linked to at least one peptide of SEQ ID NO:259-281. In one embodiment, the peptide comprises a sequence as set forth in SEQ ID NO: 1-242. In one embodiment, the peptide comprises a sequence as set forth in SEQ ID NO: 1-8.

[0010] In one embodiment, the composition comprises a delivery vehicle comprising at least one therapeutic agent for the treatment of a disease or disorder. In one embodiment, the delivery vehicle is a liposome, a lipid nanoparticle, a polymeric nanoparticle, a polystyrene nanoparticle, or a micelle. In one embodiment, the delivery vehicle is a lipid nanoparticle.206678-0001-00WQ

[0011] In one embodiment, the therapeutic agent comprises a therapeutic nucleic acid molecule. In one embodiment, the therapeutic agent comprises a nucleic acid molecule encoding a therapeutic protein. In one embodiment, the therapeutic agent comprises a nucleic acid molecule encoding cystic fibrosis transmembrane conductance regulator (CFTR). In one embodiment, the nucleic acid molecule comprises a 5’ UTR comprising SEQ ID NO:243 and a 3’ UTR comprising SEQ ID NO:244. In one embodiment, the nucleic acid molecule comprises a 5’ UTR comprising SEQ ID NO:243, a coding sequence encoding SEQ ID NO:245, and a 3’ UTR comprising SEQ ID NO:244. In one embodiment, the coding sequence encoding SEQ ID NO:245 comprises SEQ ID NO:246. In one embodiment, the nucleic acid molecule comprises SEQ ID NO: 247.

[0012] In one embodiment, the delivery vehicle further comprises a targeting domain. In one embodiment, the targeting domain is an antibody.

[0013] In one embodiment, the invention relates to a method of treating a disease or disorder associated in a subject in need thereof, the method comprising administering a composition comprising a chimeric fusogenic peptide to the subject or to a transplantable cell for administration to the subject. In one embodiment, the chimeric fusogenic peptide comprises at least one of a ectodomain and a TMD of a first FAST protein linked to an endodomain of a second FAST protein. In one embodiment, the endodomain is an endodomain of a p!4, p 15, plO or p22 FAST protein. In one embodiment, the endodomain is one of SEQ ID NO:259-281. In one embodiment, the peptide comprises at least one peptide of SEQ ID NO:248-258 linked to at least one peptide of SEQ ID NO:259-281. In one embodiment, the peptide comprises a sequence as set forth in SEQ ID NO: 1-242. In one embodiment, the peptide comprises a sequence as set forth in SEQ ID NO: 1-8.

[0014] In one embodiment, the composition comprises a delivery vehicle comprising at least one therapeutic agent for the treatment of a disease or disorder. In one embodiment, the delivery vehicle is selected from the group consisting of a liposome, a lipid nanoparticle, a polymeric nanoparticle, a polystyrene nanoparticle, and a micelle. In one embodiment, the delivery vehicle is a lipid nanoparticle.

[0015] In one embodiment, the therapeutic agent comprises a therapeutic nucleic acid molecule. In one embodiment, the therapeutic agent comprises a nucleic acid molecule encoding a therapeutic protein.206678-0001-00WQ

[0016] In one embodiment, the disease or disorder is a monogenic disease, a metabolic disease, a condition benefiting from increased gene / hormone activity, enhanced metabolic signaling, or restored deficient pathways (a Boost condition), a condition benefiting from reduced gene / protein activity, suppressed lipogenesis, substrate reduction, or lowered pathological signaling (a Knockdown condition), a cosmetic, skin or hair condition, a condition associated with aging, a condition associated with mitochondrial function, an autoimmune disease, an immunodeficiency disease, an inflammatory disease, a neurological disease or disorder, an infectious disease or a cancer.

[0017] In one embodiment, the disease or disorder is cystic fibrosis (CF). In one embodiment, the therapeutic agent comprises a nucleic acid molecule encoding cystic fibrosis transmembrane conductance regulator (CFTR). In one embodiment, the nucleic acid molecule comprises a 5’ UTR comprising SEQ ID NO:243 and a 3’ UTR comprising SEQ ID NO:244. In one embodiment, the nucleic acid molecule comprises a 5’ UTR comprising SEQ ID NO:243, a coding sequence encoding SEQ ID NO:245, and a 3’ UTR comprising SEQ ID NO:244. In one embodiment, the coding sequence encoding SEQ ID NO:245 comprises SEQ ID NO:246. In one embodiment, the nucleic acid molecule comprises SEQ ID NO:247.

[0018] In one embodiment, the composition is administered by inhalation, intraocular delivery, intravitreal delivery, subretinal delivery, suprachoroidal delivery, subcutaneous delivery, intraperitoneal delivery, intramuscular delivery, intradermal delivery, intrasternal delivery, intratumoral delivery, intravenous delivery, intracerebroventricular injection, or kidney dialytic infusion.BRIEF DESCRIPTION OF THE DRAWINGS

[0019] The following detailed description of embodiments of the invention will be better understood when read in conjunction with the appended drawings. It should be understood that the invention is not limited to the precise arrangements and instrumentalities of the embodiments shown in the drawings.

[0020] Fig. 1 depicts images showing fusion of HEK293T cells transfected with a plasmid encoding fusogenic protein 1.18 1.8 at 27thhour (27H) following transfection. Images of lOx objective of the same cells, left: FITC, right: TRANS.

[0021] Fig. 2 depicts images showing fusion of HEK293T cells transfected with a plasmid encoding fusogenic protein 1.18 1.8 at 24thhour (24H) and 11H following transfection. Images of lOx objective of the same well location, left: 24H, right: 11H.

[0022] Fig. 3 depicts images showing fusion of HEK293T cells transfected with a plasmid encoding fusogenic protein 1.18 1.8 at 12H and 5H following transfection. Images of 40x objective of different wells, left: 12H, right: 5H.

[0023] Fig. 4 depicts images showing fusion of HEK293T cells transfected with a plasmid encoding fusogenic protein 1.18 1.8 at 11H and 9H following transfection. Images of lOx objective of the same well location, left: 11H, right: 9H.

[0024] Fig. 5 depicts images showing fusion of HEK293T cells transfected with a plasmid encoding fusogenic protein 1.18 1.8 at 7H and 5H following transfection. Images of lOx objective of the same well location, left: 7H, right: 5H.

[0025] Fig. 6 depicts images showing fusion of HEK293T cells transfected with a plasmid encoding fusogenic protein 1.3 3.1 at 27H following transfection. Images of lOx objective of the same cells, left: FITC, right: TRANS.

[0026] Fig. 7 depicts images showing fusion of HEK293T cells transfected with a plasmid encoding fusogenic protein 1.3 3.1 at 24H and 11H following transfection. Images of lOx objective of the same well location, left: 24H, right: 11H.

[0027] Fig. 8 depicts images showing fusion of HEK293T cells transfected with a plasmid encoding fusogenic protein 1.3 3.1 at 12H and 5H following transfection. Images of 40x objective of different wells, left: 12H, right: 5H.

[0028] Fig. 9 depicts images showing fusion of HEK293T cells transfected with a plasmid encoding fusogenic protein 1.3 3.1 at 11H and 9H following transfection. Images of 1 Ox objective of the same well location, left: 11H, right: 9H.

[0029] Fig. 10 depicts images showing fusion of HEK293T cells transfected with a plasmid encoding fusogenic protein 1.45 11.23 at 27thhour (27H) following transfection.Images of lOx objective of the same cells, left: FITC, right: TRANS.

[0030] Fig. 11 depicts images showing fusion of HEK293T cells transfected with a plasmid encoding fusogenic protein 1.45_11.23 at 24thhour (24H) and 11H following transfection. Images of lOx objective of the same well location, left: 24H, right: 11H.206678-0001-00WQ

[0031] Fig. 12 depicts images showing fusion of HEK293T cells transfected with a plasmid encoding fusogenic protein 1.45 11.23 at 12H and 5H following transfection. Images of 40x objective of different wells, left: 12H, right: 5H.

[0032] Fig. 13 depicts images showing fusion of HEK293T cells transfected with a plasmid encoding fusogenic protein 1.9 9.1 at 27thhour (27H) following transfection. Images of lOx objective of the same cells, left: FITC, right: TRANS.

[0033] Fig. 14 depicts images showing fusion of HEK293T cells transfected with a plasmid encoding fusogenic protein 1.9 9.1 at 24thhour (24H) and 11H following transfection. Images of lOx objective of the same well location, left: 24H, right: 11H.

[0034] Fig. 15 depicts images showing fusion of HEK293T cells transfected with a plasmid encoding fusogenic protein 1.9 9.1 at 12H and 5H following transfection. Images of 40x objective of different wells, left: 12H, right: 5H.

[0035] Fig. 16 depicts images showing fusion of HEK293T cells transfected with a plasmid encoding fusogenic protein 1.9 9.1 at 11H and 9H following transfection. Images of lOx objective of the same well location, left: 11H, right: 9H.

[0036] Fig. 17 depicts images showing fusion of HEK293T cells transfected with a plasmid encoding fusogenic protein 1.9 9.1 at 7H and 5H following transfection. Images of lOx objective of the same well location, left: 7H, right: 5H.

[0037] Fig. 18 depicts images showing fusion of HEK293T cells transfected with a plasmid encoding fusogenic protein 1.17_1.7 at 27thhour (27H) following transfection. Images of lOx objective of the same cells, left: FITC, right: TRANS.

[0038] Fig. 19 depicts images showing fusion of HEK293T cells transfected with a plasmid encoding fusogenic protein 1.17 1.7 at 24thhour (24H) and 11H following transfection. Images of lOx objective of the same well location, left: 24H, right: 11H.

[0039] Fig. 20 depicts images showing fusion of HEK293T cells transfected with a plasmid encoding fusogenic protein 1.17 1.7 at 12H and 5H following transfection. Images of 40x objective of different wells, left: 12H, right: 5H.

[0040] Fig. 21 depicts images showing fusion of HEK293T cells transfected with a plasmid encoding fusogenic protein 1.17 1.7 at 11H and 9H following transfection. Images of lOx objective of the same well location, left: 11H, right: 9H.206678-0001-00WQ

[0041] Fig. 22 depicts images showing fusion of HEK293T cells transfected with a plasmid encoding fusogenic protein 1.17 1.7 at 7H and 5H following transfection. Images of lOx objective of the same well location, left: 7H, right: 5H.

[0042] Fig. 23 depicts images showing fusion of HEK293T cells transfected with a plasmid encoding fusogenic protein 1.13 1.3 at 27thhour (27H) following transfection. Images of lOx objective of the same cells, left: FITC, right: TRANS.

[0043] Fig. 24 depicts images showing fusion of HEK293T cells transfected with a plasmid encoding fusogenic protein 1.13 1.3 at 24thhour (24H) and 11H following transfection. Images of lOx objective of the same well location, left: 24H, right: 11H.

[0044] Fig. 25 depicts images showing fusion of HEK293T cells transfected with a plasmid encoding fusogenic protein 1.13 1.3 at 12H and 5H following transfection. Images of 40x objective of different wells, left: 12H, right: 5H.

[0045] Fig. 26 depicts images showing fusion of HEK293T cells transfected with a plasmid encoding fusogenic protein 1.13 1.3 at 11H and 9H following transfection. Images of lOx objective of the same well location, left: 11H, right: 9H.

[0046] Fig. 27 depicts images showing fusion of HEK293T cells transfected with a plasmid encoding fusogenic protein 1.13 1.3 at 7H and 5H following transfection. Images of lOx objective of the same well location, left: 7H, right: 5H.

[0047] Fig. 28 depicts images showing fusion of HEK293T cells transfected with a plasmid encoding fusogenic protein 1,25_1.15 at 27thhour (27H) following transfection. Images of lOx objective of the same cells, left: FITC, right: TRANS.

[0048] Fig. 29 depicts images showing fusion of HEK293T cells transfected with a plasmid encoding fusogenic protein 1.25 1.15 at 24thhour (24H) and 11H following transfection. Images of lOx objective of the same well location, left: 24H, right: 11H.

[0049] Fig. 30 depicts images showing fusion of HEK293T cells transfected with a plasmid encoding fusogenic protein 1.25 1.15 at 12H and 5H following transfection. Images of 40x objective of different wells, left: 12H, right: 5H.

[0050] Fig. 31 depicts images showing fusion of HEK293T cells transfected with a plasmid encoding fusogenic protein 1.16 1.6 at 27thhour (27H) following transfection. Images of lOx objective of the same cells, left: FITC, right: TRANS.

[0051] Fig. 32 depicts images showing fusion of HEK293T cells transfected with a plasmid encoding fusogenic protein 1.16 1.6 at 24thhour (24H) and 11H following transfection. Images of lOx objective of the same well location, left: 24H, right: 11H.

[0052] Fig. 33 depicts images showing fusion of HEK293T cells transfected with a plasmid encoding wild-type fusogenic protein pl4 at 27thhour (27H) following transfection. Images of lOx objective of the same cells, left: FITC, right: TRANS.

[0053] Fig. 34 depicts images showing fusion of HEK293T cells transfected with a plasmid encoding eGFP at 27thhour (27H) following transfection. Images of lOx objective of the same cells, left: FITC, right: TRANS.

[0054] Fig. 35 depicts images showing fusion of HEK293T cells transfected with a plasmid encoding eGFP at 24thhour (24H) and 11H following transfection. Images of lOx objective of the same well location, left: 24H, right: 11H.

[0055] Fig. 36 depicts a diagram of the naming convention used for the chimeric fusion peptides.DETAILED DESCRIPTION

[0056] The present invention relates to cell-fusion (fusogenic) peptides and delivery vehicles comprising the fusogenic peptides for efficient cellular delivery and uptake of therapeutic agents. In some embodiments, the fusogenic peptide carrier molecule delivers full length, intact, functional DNA or RNA. In some embodiments, the fusogenic peptide carrier molecule delivers a nucleic acid molecule capable of producing functional protein.

[0057] In some embodiments, it also relates to methods of use of the compositions described herein for treating diseases or disorders in subjects including, but not limited to, monogenic diseases and disorders and cancers.Definitions

[0058] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0059] As used herein, each of the following terms has the meaning associated with it in this section.206678-0001-00WQ

[0060] The articles “a” and “an” are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, “an element” means one element or more than one element.

[0061] “About” as used herein when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass variations of ±20%, ±10%, ±5%, ±1%, or ±0.1% from the specified value, as such variations are appropriate to perform the disclosed methods.

[0062] A “disease” is a state of health of an animal wherein the animal cannot maintain homeostasis, and wherein if the disease is not ameliorated then the animal’s health continues to deteriorate. In contrast, a “disorder” in an animal is a state of health in which the animal is able to maintain homeostasis, but in which the animal’s state of health is less favorable than it would be in the absence of the disorder. Left untreated, a disorder does not necessarily cause a further decrease in the animal’s state of health.

[0063] An “effective amount” as used herein, means an amount which provides a therapeutic or prophylactic benefit.

[0064] The term “physiologically effective dosage” refers to an amount of an agent that produces a measurable biologic or physiologic effect in the recipient subject that is related to the activity of the agent(s). The physiologically effective dosage will vary depending on the compound, the age, weight, etc., of the subject being administered the agent, and the biologic or physiologic effect being measured.

[0065] “Encoding” refers to the inherent property of specific sequences of nucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA, to serve as templates for synthesis of other polymers and macromolecules in biological processes having either a defined sequence of nucleotides (i.e., rRNA, tRNA and mRNA) or a defined sequence of amino acids and the biological properties resulting therefrom. Thus, a gene encodes a protein if transcription and translation of mRNA corresponding to that gene produces the protein in a cell or other biological system. Both the coding strand, the nucleotide sequence of which is identical to the mRNA sequence and is usually provided in sequence listings, and the non-coding strand, used as the template for transcription of a gene or cDNA, can be referred to as encoding the protein or other product of that gene or cDNA.

[0066] “Homologous” refers to the sequence similarity or sequence identity between two polypeptides or between two nucleic acid molecules. When a position in both of the two compared sequences is occupied by the same base or amino acid monomer subunit, e.g., if a position in each of two DNA molecules is occupied by adenine, then the molecules are homologous at that position. The percent of homology between two sequences is a function of the number of matching or homologous positions shared by the two sequences divided by the number of positions compared X 100. For example, if 6 of 10 of the positions in two sequences are matched or homologous then the two sequences are 60% homologous. By way of example, the DNA sequences ATTGCC and TATGGC share 50% homology. Generally, a comparison is made when two sequences are aligned to give maximum homology.

[0067] “Isolated” means altered or removed from the natural state. For example, a nucleic acid or a peptide naturally present in a living animal is not “isolated,” but the same nucleic acid or peptide partially or completely separated from the coexisting materials of its natural state is “isolated.” An isolated nucleic acid or protein can exist in substantially purified form, or can exist in a non-native environment such as, for example, a host cell.

[0068] In the context of the present invention, the following abbreviations for the commonly occurring nucleosides (nucleobase bound to ribose or deoxyribose sugar via N-glycosidic linkage) are used. “A” refers to adenosine, “C” refers to cytidine, “G” refers to guanosine, “T” refers to thymidine, and “U” refers to uridine.

[0069] Unless otherwise specified, a “nucleotide sequence encoding an amino acid sequence” includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence. The phrase nucleotide sequence that encodes a protein or an RNA may also include introns to the extent that the nucleotide sequence encoding the protein may in some version contain an intron(s).

[0070] By the term “modulating,” as used herein, is meant mediating a detectable increase or decrease in the level of a response in a subject compared with the level of a response in the subject in the absence of a treatment or compound, and / or compared with the level of a response in an otherwise identical but untreated subject. The term encompasses perturbing and / or affecting a native signal or response thereby mediating a beneficial therapeutic response in a subject, preferably, a human.

[0071] Unless otherwise specified, a “nucleotide sequence encoding an amino acid sequence” includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence. Nucleotide sequences that encode proteins and RNA may include introns. In addition, the nucleotide sequence may contain modified nucleosides that are capable of being translated by translational machinery in a cell. For example, an mRNA where all of the uridines have been replaced with pseudouridine, 1 -methyl pseudouridine, or another modified nucleoside.

[0072] The terms “patient,” “subject,” “individual,” and the like are used interchangeably herein, and refer to any animal, or cells thereof whether in vitro or in situ, amenable to the methods described herein. In certain non-limiting embodiments, the patient, subject or individual is a human.

[0073] The term “polynucleotide” as used herein is defined as a chain of nucleotides. Furthermore, nucleic acids are polymers of nucleotides. Thus, nucleic acids and polynucleotides as used herein are interchangeable. One skilled in the art has the general knowledge that nucleic acids are polynucleotides, which can be hydrolyzed into the monomeric “nucleotides.” The monomeric nucleotides can be hydrolyzed into nucleosides. As used herein polynucleotides include, but are not limited to, all nucleic acid sequences which are obtained by any means available in the art, including, without limitation, recombinant means, i.e., the cloning of nucleic acid sequences from a recombinant library or a cell genome, using ordinary cloning technology and PCR™, and the like, and by synthetic means.

[0074] In certain instances, the polynucleotide or nucleic acid of the invention is a “nucleoside-modified nucleic acid,” which refers to a nucleic acid comprising at least one modified nucleoside. A “modified nucleoside” refers to a nucleoside with a modification. For example, over one hundred different nucleoside modifications have been identified in RNA (Rozenski, et al., 1999, The RNA Modification Database: 1999 update. Nucl Acids Res 27: 196-197).

[0075] As used herein, the terms “peptide,” “polypeptide,” and “protein” are used interchangeably, and refer to a compound comprised of amino acid residues covalently linked by peptide bonds. A protein or peptide must contain at least two amino acids, and no limitation is placed on the maximum number of amino acids that can comprise a protein’s or peptide’s sequence. Polypeptides include any peptide or protein comprising two or more amino acidsjoined to each other by peptide bonds. As used herein, the term refers to both short chains, which also commonly are referred to in the art as peptides, oligopeptides and oligomers, for example, and to longer chains, which generally are referred to in the art as proteins, of which there are many types. “Polypeptides” include, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, fusion proteins, among others. The polypeptides include natural peptides, recombinant peptides, synthetic peptides, or a combination thereof.

[0076] By the term “specifically binds,” as used herein with respect to an affinity ligand, in particular, an antibody, is meant an antibody which recognizes a specific antigen, but does not substantially recognize or bind other molecules in a sample. For example, an antibody that specifically binds to an antigen from one species may also bind to that antigen from one or more other species. But, such cross-species reactivity does not itself alter the classification of an antibody as specific. In another example, an antibody that specifically binds to an antigen may also bind to different allelic forms of the antigen. However, such cross reactivity does not itself alter the classification of an antibody as specific. In some instances, the terms “specific binding” or “specifically binding,” can be used in reference to the interaction of an antibody, a protein, or a peptide with a second chemical species, to mean that the interaction is dependent upon the presence of a particular structure (e.g., an antigenic determinant or epitope) on the chemical species; for example, an antibody recognizes and binds to a specific protein structure rather than to proteins generally. If an antibody is specific for epitope “A”, the presence of a molecule containing epitope A (or free, unlabeled A), in a reaction containing labeled “A” and the antibody, will reduce the amount of labeled A bound to the antibody.

[0077] The term “therapeutic” as used herein means a treatment and / or prophylaxis. A therapeutic effect is obtained by suppression, diminution, remission, or eradication of at least one sign or symptom of a disease or disorder.

[0078] The term “therapeutically effective amount” refers to the amount of the subject compound that will elicit the biological or medical response of a tissue, system, or subject that is being sought by the researcher, veterinarian, medical doctor or other clinician. The term “therapeutically effective amount” includes that amount of a compound that, when administered, is sufficient to prevent development of, or alleviate to some extent, one or more of the signs or symptoms of the disorder or disease being treated. The therapeutically effective amount will varydepending on the compound, the disease and its severity and the age, weight, etc., of the subject to be treated.

[0079] To “treat” a disease as the term is used herein, means to reduce the frequency or severity of at least one sign or symptom of a disease or disorder experienced by a subject.

[0080] The term “transfected” as used herein refers to a process by which exogenous nucleic acid is transferred or introduced into the host cell. A “transfected” cell is one which has been transfected with exogenous nucleic acid. The cell includes the primary subject cell and its progeny.

[0081] The phrase “under transcriptional control” or “operatively linked” as used herein means that the promoter is in the correct location and orientation in relation to a polynucleotide to control the initiation of transcription by RNA polymerase and expression of the polynucleotide.

[0082] A “vector” is a composition of matter which comprises an isolated nucleic acid and which can be used to deliver the isolated nucleic acid to the interior of a cell. Numerous vectors are known in the art including, but not limited to, linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids, and viruses. Thus, the term “vector” includes an autonomously replicating plasmid or a virus. The term should also be construed to include non-plasmid and non-viral compounds which facilitate transfer of nucleic acid into cells, such as, for example, polylysine compounds, liposomes, lipid nanoparticles, and the like.Examples of viral vectors include, but are not limited to, adenoviral vectors, adeno-associated virus vectors, retroviral vectors, and the like.

[0083] “Optional” or “optionally” means that the subsequently described event of circumstances may or may not occur, and that the description includes instances where said event or circumstance occurs and instances in which it does not.

[0084] Ranges: throughout this disclosure, various aspects of the invention can be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of the range.Fusogenic Peptide

[0085] In various embodiments of the invention, a therapeutic nucleic acid molecule is complexed to a fusogenic peptide to enhance cellular uptake. In one embodiment, the fusogenic peptide comprises a fusion-associated small transmembrane (FAST) protein, or a fragment or variant thereof. FAST proteins are a family of small nonstructural proteins occurring in limited genera of reoviruses, double-stranded RNA viruses that infect a wide variety of hosts. FAST proteins are generally under 200 residues in size and are expressed as single pass transmembrane proteins oriented with the N-terminus in the extracellular space and the C-terminus in the cytoplasm. They are post-translationally modified, either myristoylated or in some cases palmitoylated. The transmembrane (TM) domain functions as a reverse signal anchor, orienting the protein in the membrane. The extracellular portion of certain canonical FAST protein contains an N-terminal myristoylation site followed by a putative amphipathic helix termed the fusion peptide and the transmembrane domain. The intracellular portion contains a membrane-proximal region rich in basic residues (the polybasic region) and a proline-rich region.

[0086] The invention is based, in part on the development of fusogenic proteins, which have enhanced fusogenic ability, based on a chimeric recombinant of two or more domains of modified FAST proteins. In one embodiment, the chimeric fusogenic peptide comprises an ectodomain, transmembrane domain (TMD), or endodomain wherein at least one of the ectodomain, TMD, and endodomain is from a first FAST protein and at least one of the ectodomain, TMD, and endodomain is from a second FAST protein. In one embodiment, the chimeric fusogenic peptide comprises an ectodomain, transmembrane domain (TMD), or endodomain wherein the ectodomain, is from a first FAST protein, the TMD is from a second FAST protein, and the endodomain is from a third FAST protein.

[0087] In one embodiment, the chimeric fusogenic peptide comprises a fusion of an ectodomain and transmembrane domain (E+TMD) of Table 1 with an endodomain of Table 2. In one embodiment the chimeric fusogenic peptide comprises a fusion of any one of SEQ ID NO:248-258 with any one of SEQ ID NO:259-281.206678-0001-00WQ

[0088] Exemplary fusogenic peptides include, but are not limited to, those listed in SEQ ID NO: 1-242. In some embodiments, the fusogenic peptide comprises a sequence as set forth in SEQ ID NO: 1-242, or a fragment or variant thereof. In some embodiments, the fusogenic peptide comprises a sequence as set forth in SEQ ID NO: 1-8, or a fragment or variant thereof. In some embodiments, the fragment of SEQ ID NO: 1-242 comprises at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% of the full length sequence of SEQ ID NO: 1-242. In one embodiment, the fragment of SEQ ID NO: 1-242 retains the fusogenic property of the full length peptide. In some embodiments, the variant of SEQ ID NO: 1-242 comprises at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to the parental sequence of SEQ ID NO: 1-242. In one embodiment, the variant of SEQ ID NO: 1-242 retains the fusogenic property of the parental peptide.

[0089] In some embodiments, the fusogenic peptide comprises at least one D-amino acid. In some embodiments, the fusogenic peptide comprises a combination of D-amino acids and L-amino acids. In some embodiments, the fusogenic peptide comprises at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least or more than 90% D-amino acids. In some embodiments, the fusogenic peptide comprises only D-amino acids. In some embodiments, the fusogenic peptide comprises only L-amino acids.

[0090] In one embodiment, the peptide is between 10 and 300 amino acids in length. In one embodiment, the peptide is between 20 and 200 amino acids in length. In one embodiment, the peptide is between 50 and 100 amino acids in length.

[0091] In one aspect, the present invention provides a composition comprising a peptide comprising a polyarginine tract, or derivative thereof, as described herein. For example, in one embodiment, the peptide comprises a polyarginine tract comprising at least four arginine residues. In some instances, at least one of the arginine residues is a D-form stereoisomer. In some instances, at least one arginine residue is a D-form stereoisomer, and at least one arginine residue is a L-form stereoisomer. In some embodiments, the polyarginine tract comprises all D-form residues. In some embodiments, the polyarginine tract comprises all L-form residues. In some embodiments, the polyarginine tract comprises a combination of both D-form and L-form residues. In some embodiments, the polyarginine tract comprises only arginine residues. In some instances, the polyarginine tract is interrupted by at least one nonpolar residue. In some206678-0001-00WQinstances, the nonpolar residue is alanine. In certain instances, the alanine is D-alanine or L-alanine.

[0092] In certain embodiments, the peptides of the present invention further comprise conservative variants of the peptides herein described. As used herein, a “conservative variant” refers to alterations in the amino acid sequence that do not substantially and adversely affect the binding or association capacity of the peptide. A substitution, insertion or deletion is said to adversely affect the peptide when the altered sequence prevents, reduces, or disrupts a function or activity associated with the peptide. For example, the overall charge, structure or hydrophobic-hydrophilic properties of the peptide can be altered without adversely affecting an activity. Accordingly, the amino acid sequence can be altered, for example to render the peptide more hydrophobic or hydrophilic, without adversely affecting the activities of the peptide.

[0093] These variants, though possessing a slightly different amino acid sequence than those recited elsewhere herein, will still have the same or similar properties associated with any of the peptides discussed herein. Ordinarily, the conservative substitution variants, will have an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with any of the peptides discussed elsewhere herein.

[0094] The peptides of the present invention may be made using chemical methods. For example, peptides can be synthesized by solid phase techniques (Roberge J Y et al (1995) Science 269: 202-204), cleaved from the resin, and purified by preparative high performance liquid chromatography. Automated synthesis may be achieved, for example, using the ABI 431 A Peptide Synthesizer (Perkin Elmer) in accordance with the instructions provided by the manufacturer.

[0095] The peptides may alternatively be made by recombinant means or by cleavage from a longer polypeptide. The composition of a peptide may be confirmed by amino acid analysis or sequencing.

[0096] The variants of the polypeptides according to the present invention may be (i) one in which one or more of the amino acid residues are substituted with a conserved or nonconserved amino acid residue (for example, a conserved amino acid residue) and such substituted amino acid residue may or may not be one encoded by the genetic code, (ii) one in which there are one or more modified amino acid residues, e.g., residues that are modified by the attachmentof substituent groups, (iii) one in which the polypeptide is an alternative splice variant of the polypeptide of the present invention, (iv) fragments of the polypeptides and / or (v) one in which the polypeptide is fused with another polypeptide, such as a leader or secretory sequence or a sequence which is employed for purification (for example, His-tag) or for detection (for example, Sv5 epitope tag). The fragments include polypeptides generated via proteolytic cleavage (including multi-site proteolysis) of an original sequence. Variants may be post-translationally, or chemically modified. Such variants are deemed to be within the scope of those skilled in the art from the teaching herein.Peptide analogs

[0097] In one embodiment, the present invention relates to peptide analogs of the peptides of the invention appropriate for use with the invention and uses thereof. For example, in certain instances the invention provides peptides and peptide analogs or fragments of SEQ ID NO: 1-242 wherein the peptides, including peptides and analogs, fragments, and derivatives thereof, exhibit fusogenic properties.Fusion. Chimeric and Modified Polypeptides

[0098] A fusogenic peptide of the invention may be conjugated with other molecules, such as proteins, to prepare fusion proteins or chimeric peptides. This may be accomplished, for example, by the synthesis of N-terminal or C-terminal fusion proteins provided that the resulting fusion protein or chimeric peptide retains the functionality of the peptide of the invention.

[0099] A peptide or chimeric protein of the invention may be phosphorylated using conventional methods such as the method described in Reedijk et al. (The EMBO Journal 11(4):1365, 1992).

[0100] Cyclic derivatives of the peptides or chimeric proteins of the invention are also part of the present invention. Cyclization may allow the peptide or chimeric protein to assume a more favorable conformation for association with other molecules. Cyclization may be achieved using techniques known in the art. For example, disulfide bonds may be formed between two appropriately spaced components having free sulfhydryl groups, or an amide bond may be formed between an amino group of one component and a carboxyl group of another component. Cyclization may also be achieved using an azobenzene-containing amino acid as described byUlysse, L., et al., J. Am. Chem. Soc. 1995, 117, 8466-8467. The components that form the bonds may be N-termini of the peptide, C-termini of the peptide, side chains of amino acids within the peptide, non-amino acid components, or any combination thereof. In an embodiment of the invention, cyclic peptides may comprise a beta-turn in the right position. Beta-turns may be introduced into the peptides of the invention by adding the amino acids Pro-Gly at the right position.

[0101] It may be desirable to produce a cyclic peptide which is more flexible than the cyclic peptides containing peptide bond linkages as described above. A more flexible peptide may be prepared by introducing cysteines at the right and left position of the peptide and forming a disulfide bridge between the two cysteines. The two cysteines are arranged so as not to deform the beta-sheet and turn. The peptide is more flexible as a result of the length of the disulfide linkage and the smaller number of hydrogen bonds in the beta-sheet portion. The relative flexibility of a cyclic peptide can be determined by molecular dynamics simulations.

[0102] In some embodiments, the subject compositions are peptidomimetics of the peptides of the invention. Peptidomimetics are compounds based on, or derived from, peptides and proteins. The peptidomimetics of the present invention typically can be obtained by structural modification of a known peptide sequence using unnatural amino acids, conformational restraints, isosteric replacement, and the like. The subject peptidomimetics constitute the continuum of structural space between peptides and non-peptide synthetic structures; peptidomimetics may be useful, therefore, in delineating pharmacophores and in helping to translate peptides into nonpeptide compounds with the activity of the parent peptides.

[0103] The peptidomimetics of the invention may include unnatural amino acids formed by post-translational modification or by introducing unnatural amino acids during translation. A variety of approaches are available for introducing unnatural amino acids during protein translation. By way of example, special tRNAs, such as tRNAs which have suppressor properties, suppressor tRNAs, have been used in the process of site-directed non-native amino acid replacement (SNAAR). In SNAAR, a unique codon is required on the mRNA and the suppressor tRNA, acting to target a non-native amino acid to a unique site during the protein synthesis (described in W090 / 05785). However, the suppressor tRNA must not be recognizable by the aminoacyl tRNA synthetases present in the protein translation system. In certain cases, a non-native amino acid can be formed after the tRNA molecule is aminoacylated using chemicalreactions which specifically modify the native amino acid and do not significantly alter the functional activity of the aminoacylated tRNA. These reactions are referred to as post-aminoacylation modifications. For example, the epsilon-amino group of the lysine linked to its cognate tRNA (tRNALYS), could be modified with an amine specific photoaffinity label.

[0104] In some embodiments, the peptides of the invention may be modified to produce variants, analogs and fragments of the proteins which have an increased release rate of a complexed nucleic acid molecule, so long as the desired biological properties (i.e., the ability to bind to the nucleic acid molecule and promote its uptake by a cell) are retained. The peptides may be modified by using various genetic engineering or protein engineering techniques.

[0105] In one embodiment, a peptide of the invention is further modified. In one embodiment, a functional fragment of a peptide of the invention contains a further modification. In one example, a further modification of a peptide includes a modification at the N-terminus. In one embodiment a further modification comprises a modification at the C-terminus. In one embodiment a peptide of the invention is modified at both the N- and C-termini.

[0106] In one embodiment, a modification of a peptide of the invention is a chemical modification, conjugation to a synthetic or natural polymer, glycosylation, acetylation, methylation, phosphorylation, conjugation of a chemical linker, fatty acyl derivatization, or conjugation of a polysaccharide. In one embodiment, a polymer to be conjugated to a peptide on the invention is one of polyethylene glycol (PEG), polypropylene glycol (PPG), polysialic acid (PSA), XTENTM and hydroxyethyl starch (HES). Conventional PEGylation methodologies directed to monomeric proteins are well known in the art.

[0107] In one embodiment, a single polymer is conjugated to a single peptide of the invention. In one embodiment, multiple polymers are conjugated to a single peptide of the invention. In one embodiment, a population of peptides of the invention will contain a mixture of modified and unmodified peptides having 0 to more than 1 polymer conjugated to each peptide.

[0108] The peptides of the invention can be post-translationally modified. For example, post-translational modifications that fall within the scope of the present invention include signal peptide cleavage, glycosylation, acetylation, isoprenylation, proteolysis, myristoylation, protein folding and proteolytic processing, etc. Some modifications or processing events require introduction of additional biological machinery. For example, processing events, such as signal206678-0001-00WQpeptide cleavage and core glycosylation, are examined by adding canine microsomal membranes or Xenopus egg extracts (U. S. Pat. No. 6,103,489) to a standard translation reaction.Fusogenic Peptide-Based Nanoparticle

[0109] In some embodiments, the invention relates to fusogenic peptide-based nanoparticles for delivery of a therapeutic nucleic acid molecule to a cell. In some embodiments, the fusogenic peptide-based nanoparticle delivers a DNA or RNA molecule for expression of a therapeutic protein. In some embodiments, the therapeutic nucleic acid molecule is at least 1 kbp. In some embodiments, the therapeutic nucleic acid molecule is at least 3 kbp. In some embodiments, the therapeutic nucleic acid molecule is in vitro synthesized. In some embodiments, the therapeutic nucleic acid molecule produces RNA and / or protein.

[0110] In some embodiments, a therapeutic nucleic acid molecule is coated with fusogenic peptide using electrostatic interactions between the fusogenic peptide and the therapeutic nucleic acid molecule to fully complex with the therapeutic nucleic acid molecule within the fusogenic peptide-based nanoparticle. In one embodiment, the complexing is reversible, such that the fusogenic peptide can be disassociated from the therapeutic nucleic acid molecule following cellular uptake. In some embodiments, a positively charged fusogenic peptide interacts electrostatically with the negatively charged therapeutic nucleic acid molecule.

[0111] In various embodiments, the fusogenic peptide based nanoparticles have a mean diameter of from about 30 nm to about 500 nm, from about 40 nm to about 400 nm, from about 50 nm to about 300 nm, from about 60 nm to about 250 nm, from about 70 nm to about 200 nm, or from about 80 nm to about 150 nm. In some embodiments, the fusogenic peptide based nanoparticles are substantially non-toxic.Delivery Vehicle

[0112] In some embodiments, the delivery vehicle is a colloidal dispersion system, such as macromolecule complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes. An exemplary colloidal system for use as a delivery vehicle in vitro and in vivo is a liposome (e.g., an artificial membrane vesicle).206678-0001-00WQ

[0113] The use of lipid formulations is contemplated for the introduction of the at least one agent into a host cell (in vitro, ex vivo or in vivo). In another aspect, the at least one agent may be associated with a lipid. The at least one agent associated with a lipid may be encapsulated in the aqueous interior of a liposome, interspersed within the lipid bilayer of a liposome, attached to a liposome via a linking molecule that is associated with both the liposome and the oligonucleotide, entrapped in a liposome, complexed with a liposome, dispersed in a solution containing a lipid, mixed with a lipid, combined with a lipid, contained as a suspension in a lipid, contained or complexed with a micelle, or otherwise associated with a lipid. Lipid, lipid / nucleic acid or lipid / expression vector associated compositions are not limited to any particular structure in solution. For example, they may be present in a bilayer structure, as micelles, or with a “collapsed” structure. They may also simply be interspersed in a solution, possibly forming aggregates that are not uniform in size or shape. Lipids are fatty substances which may be naturally occurring or synthetic lipids. For example, lipids include the fatty droplets that naturally occur in the cytoplasm as well as the class of compounds which contain long-chain aliphatic hydrocarbons and their derivatives, such as fatty acids, alcohols, amines, amino alcohols, and aldehydes.

[0114] Lipids suitable for use can be obtained from commercial sources. For example, dimyristyl phosphatidylcholine (“DMPC”) can be obtained from Sigma, St. Louis, MO; dicetyl phosphate (“DCP”) can be obtained from K & K Laboratories (Plainview, NY); cholesterol (“Choi”) can be obtained from Calbiochem-Behring; dimyristyl phosphatidylglycerol (“DMPG”) and other lipids may be obtained from Avanti Polar Lipids, Inc. (Birmingham, AL). Stock solutions of lipids in chloroform or chloroform / methanol can be stored at about -20°C.Chloroform is used as the only solvent since it is more readily evaporated than methanol.“Liposome” is a generic term encompassing a variety of single and multilamellar lipid vehicles formed by the generation of enclosed lipid bilayers or aggregates. Liposomes can be characterized as having vesicular structures with a phospholipid bilayer membrane and an inner aqueous medium. Multilamellar liposomes have multiple lipid layers separated by aqueous medium. They form spontaneously when phospholipids are suspended in an excess of aqueous solution. The lipid components undergo self-rearrangement before the formation of closed structures and entrap water and dissolved solutes between the lipid bilayers (Ghosh et al., 1991 Glycobiology 5: 505-10). However, compositions that have different structures in solution than206678-0001-00WQthe normal vesicular structure are also encompassed. For example, the lipids may assume a micellar structure or merely exist as nonuniform aggregates of lipid molecules. Also contemplated are lipofectamine-agent complexes.

[0115] In one embodiment, delivery of the at least one agent comprises any suitable delivery method, including exemplary delivery methods described elsewhere herein. In certain embodiments, delivery of the at least one agent to a subject comprises mixing the at least one agent with a transfection reagent prior to the step of contacting. In another embodiment, a method of the present invention further comprises administering the at least one agent together with the transfection reagent. In another embodiment, the transfection reagent is a cationic lipid reagent.

[0116] In another embodiment, the transfection reagent is a lipid-based transfection reagent. In another embodiment, the transfection reagent is a protein-based transfection reagent. In another embodiment, the transfection reagent is a polyethyleneimine based transfection reagent. In another embodiment, the transfection reagent is calcium phosphate. In another embodiment, the transfection reagent is Lipofectin®, Lipofectamine®, or TransIT®. In another embodiment, the transfection reagent is any other transfection reagent known in the art.

[0117] In another embodiment, the transfection reagent forms a liposome. Liposomes, in another embodiment, increase intracellular stability, increase uptake efficiency and improve biological activity. In another embodiment, liposomes are hollow spherical vesicles composed of lipids arranged in a similar fashion as those lipids which make up the cell membrane. In some embodiments, the liposomes comprise an internal aqueous space for entrapping water-soluble compounds. In another embodiment, liposomes can deliver the at least one agent to cells in an active form.

[0118] In one embodiment, the composition comprises a lipid nanoparticle (LNP) and at least one agent.

[0119] The term “lipid nanoparticle” refers to a particle having at least one dimension on the order of nanometers (e.g., 1-1,000 nm) which includes one or more lipids. In some embodiments, lipid nanoparticles are included in a delivery vehicle comprising at least one agent as described herein. In some embodiments, such lipid nanoparticles comprise a cationic lipid and one or more excipient selected from neutral lipids, charged lipids, steroids and polymer conjugated lipids (e.g., a pegylated lipid). In some embodiments, the at least one agent is206678-0001-00WQencapsulated in the lipid portion of the lipid nanoparticle or an aqueous space enveloped by some or all of the lipid portion of the lipid nanoparticle, thereby protecting it from enzymatic degradation or other undesirable effects induced by the mechanisms of the host organism or cells e.g. an adverse immune response.

[0120] In various embodiments, the lipid nanoparticles have a mean diameter of from about 30 nm to about 150 nm, from about 40 nm to about 150 nm, from about 50 nm to about 150 nm, from about 60 nm to about 130 nm, from about 70 nm to about 110 nm, from about 70 nm to about 100 nm, from about 80 nm to about 100 nm, from about 90 nm to about 100 nm, from about 70 to about 90 nm, from about 80 nm to about 90 nm, from about 70 nm to about 80 nm, or about 30 nm, 35 nm, 40 nm, 45 nm, 50 nm, 55 nm, 60 nm, 65 nm, 70 nm, 75 nm, 80 nm, 85 nm, 90 nm, 95 nm, 100 nm, 105 nm, 110 nm, 115 nm, 120 nm, 125 nm, 130 nm, 135 nm, 140 nm, 145 nm, or 150 nm. In one embodiment, the lipid nanoparticles have a mean diameter of about 83 nm. In one embodiment, the lipid nanoparticles have a mean diameter of about 102 nm. In one embodiment, the lipid nanoparticles have a mean diameter of about 103 nm. In some embodiments, the lipid nanoparticles are substantially non-toxic. In certain embodiments, the at least one agent, when present in the lipid nanoparticles, is resistant in aqueous solution to degradation by intra- or intercellular enzymes.

[0121] The LNP may comprise any lipid capable of forming a particle to which the at least one agent is attached, or in which the at least one agent is encapsulated. The term “lipid” refers to a group of organic compounds that are derivatives of fatty acids (e.g., esters) and are generally characterized by being insoluble in water but soluble in many organic solvents. Lipids are usually divided in at least three classes: (1) “simple lipids” which include fats and oils as well as waxes; (2) “compound lipids” which include phospholipids and glycolipids; and (3) “derived lipids” such as steroids.

[0122] In one embodiment, the LNP comprises one or more cationic lipids, and one or more stabilizing lipids. Stabilizing lipids include neutral lipids and pegylated lipids.

[0123] In one embodiment, the LNP comprises a cationic lipid. As used herein, the term “ionizable cationic lipid” refers to a lipid that is cationic or becomes cationic (protonated) as the pH is lowered below the pK of the ionizable group of the lipid, but is progressively more neutral at higher pH values. At pH values below the pK, the lipid is then able to associate withnegatively charged nucleic acids. Tn certain embodiments, the cationic lipid comprises a zwitterionic lipid that assumes a positive charge on pH decrease.

[0124] In certain embodiments, the cationic lipid or ionizable cationic lipid comprises any of a number of lipid species which carry a net positive charge at a selective pH, such as physiological pH or becomes cationic (protonated) at a selective pH. Such lipids include, but are not limited to, N, N-dioleyl-N, N-dimethylammonium chloride (DODAC); N-(2,3-dioleyloxy)propyl)-N, N, N-trimethylammonium chloride (DOTMA); N, N-distearyl-N, N-dimethylammonium bromide (DDAB); N-(2,3-dioleoyloxy)propyl)-N, N, N-trimethylammonium chloride (DOTAP); 3-(N — (N', N'-dimethylaminoethane)-carbamoyl)cholesterol (DC-Chol), N-(l-(2,3-dioleoyloxy)propyl)-N-2-(sperminecarboxamido)ethyl)-N, N-dimethylammonium trifluoracetate (DOSPA), dioctadecylamidoglycyl carboxyspermine (DOGS), l,2-dioleoyl-3-dimethylammonium propane (DODAP), N, N-dimethyl-2,3-dioleoyloxy)propylamine (DODMA), and N-(l,2-dimyristyloxyprop-3-yl)-N, N-dimethyl-N-hydroxyethyl ammonium bromide (DMRIE), l,2-dilinoleyloxy-N, N-dimethylaminopropane (DLinDMA), N, N-dimethyl-2,3-bis(((9Z, 12Z, 15Z)-octadeca-9, 12, 15-trien-l -yl)oxy)propan-l -amine (DLenDMA), (6Z,9Z,28Z,3 lZ)-Heptatriaconta-6,9,28,31 -tetraen- 19-yl 4-(dimethylamino)butanoate (DLin-MC3-DMA), heptadecan-9-yl 8-((2-hydroxyethyl)(6-oxo-6-(undecyloxy)hexyl)amino)octanoate (SM-102), ((4-hydroxybutyl)azanediyl)bis(hexane-6,l-diyl) bis(2-hexyldecanoate) (ALC-0315), l,l'-((2-(4-(2-((2-(bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperazin-1 -yl)ethyl)azanediyl)bi s(dodecan-2-ol) (C 12-200), 3, 6-bi s [4- [bi s(2-hydroxydodecyl)amino]butyl]-2,5-piperazinedione (cKK-E12), and Ethylenediamine-based Cysteinyl Oleoyl Lipid (ECO). Additionally, a number of commercial preparations of cationic lipids are available which can be used in the present invention. These include, for example, LIPOFECTIN® (commercially available cationic liposomes comprising DOTMA and 1,2-dioleoyl-sn-3-phosphoethanolamine (DOPE), from GIBCO / BRL, Grand Island, N. Y.);LIPOFECT AMINE® (commercially available cationic liposomes comprising N-(l-(2,3-dioleyloxy)propyl)-N-(2-(sperminecarboxamido)ethyl)-N, N-dimethylammonium trifluoroacetate (DOSPA) and (DOPE), from GIBCO / BRL); and TRANSFECTAM® (commercially available cationic lipids comprising di octadecyl amidoglycyl carboxyspermine (DOGS) in ethanol from Promega Corp., Madison, Wis.).206678-0001-00WQ

[0125] In one embodiment, the cationic lipid is an amino lipid. Suitable amino lipids useful in the invention include those described in WO 2012 / 016184, incorporated herein by reference in its entirety. Representative amino lipids include, but are not limited to, 1,2-dilinol ey oxy-3 -(dimethylamino)acetoxypropane (DLin-DAC), 1,2-dilinoley oxy-3 -morpholinopropane (DLin-MA), l,2-dilinoleoyl-3 -dimethylaminopropane (DLinDAP), 1,2-dilinoleylthio-3-dimethylaminopropane (DLin-S-DMA), l-linoleoyl-2-linoleyloxy-3-dimethylaminopropane (DLin-2-DMAP), l,2-dilinoleyloxy-3 -trimethylaminopropane chloride salt (DLin-TMA Cl), l,2-dilinoleoyl-3 -trimethylaminopropane chloride salt (DLin-TAP. Cl), 1,2-dilinoleyloxy-3-(N-methylpiperazino)propane (DLin-MPZ), 3-(N, N-dilinoleylamino)-l,2-propanediol (DLinAP), 3-(N, N-dioleylamino)-l,2-propanediol (DOAP), l,2-dilinoleyloxo-3-(2-N, N-dimethylamino)ethoxypropane (DLin-EG-DMA), and 2,2-dilinoleyl-4-di m ethyl aminomethyl - [ 1, 3 ] -di oxol ane (DLi n-K-DM A).

[0126] In certain embodiments, the cationic lipid is present in the LNP in an amount from about 20 to about 95 mole percent. In one embodiment, the cationic lipid is present in the LNP in an amount from about 20 to about 70 mole percent. In one embodiment, the cationic lipid is present in the LNP in an amount from about 30 to about 60 mole percent. In one embodiment, the cationic lipid is present in the LNP in an amount of about 30 to about 50 mole percent.

[0127] In certain embodiments, the LNP comprises one or more additional lipids which stabilize the formation of particles during their formation.

[0128] Suitable stabilizing lipids include neutral lipids and anionic lipids.

[0129] The term “neutral lipid” refers to any one of a number of lipid species that exist in either an uncharged or neutral zwitterionic form at physiological pH. Representative neutral lipids include diacylphosphatidylcholines, diacylphosphatidylethanolamines, ceramides, sphingomyelins, dihydro sphingomyelins, cephalins, and cerebrosides.

[0130] Exemplary neutral lipids include, for example, distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoylphosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoyl-phosphatidylethanolamine (POPE) and dioleoyl-phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-l -carboxylate (DOPE-mal), dipalmitoyl phosphatidyl ethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoyl-206678-0001-00WQphosphatidylethanolamine (DSPE), 16-0-monomethyl PE, 16-O-dimethyl PE, 18-1 -trans PE, 1-stearioyl-2-oleoyl-phosphatidyethanol amine (SOPE), and l,2-dielaidoyl-sn-glycero-3-phophoethanol amine (transDOPE). In one embodiment, the neutral lipid is 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC).

[0131] In some embodiments, the LNPs comprise a neutral lipid selected from DSPC, DPPC, DMPC, DOPC, POPC, DOPE and SM. In certain embodiments, the neutral lipid is present in the LNP in an amount from about 20 to about 60 mole percent. In one embodiment, the neutral lipid is present in the LNP in an amount from about 25 to about 40 mole percent. In one embodiment, the neutral lipid is present in the LNP in an amount from about 25 to about 30 mole percent. In various embodiments, the molar ratio of the cationic lipid to the neutral lipid ranges from about 2: 1 to about 8:1.

[0132] In various embodiments, the LNPs further comprise a steroid or steroid analogue. In certain embodiments, the steroid or steroid analogue is cholesterol. In certain embodiments, the cholesterol is present in the LNP in an amount from about 10 to about 40 mole percent. In one embodiment, the cholesterol is present in the LNP in an amount from about 14 to about 30 mole percent. In some of these embodiments, the molar ratio of the cationic lipid to cholesterol ranges from about 2: 1 to 1:1.

[0133] The term “anionic lipid” refers to any lipid that is negatively charged at physiological pH. These lipids include phosphatidylglycerol, cardiolipin, diacylphosphatidylserine, diacylphosphatidic acid, N-dodecanoylphosphatidylethanolamines, N-succinylphosphatidylethanolamines, N-glutarylphosphatidylethanolamines, lysylphosphatidylglycerols, palmitoyloleyolphosphatidylglycerol (POPG), and other anionic modifying groups joined to neutral lipids.

[0134] In some embodiments, the LNPs comprise a polymer conjugated lipid. The term “polymer conjugated lipid” refers to a molecule comprising both a lipid portion and a polymer portion. An example of a polymer conjugated lipid is a pegylated lipid. The term “pegylated lipid” refers to a molecule comprising both a lipid portion and a polyethylene glycol portion. Pegylated lipids are known in the art and include 1 (monomethoxy polyethyleneglycol) 2,3 dimyristoylglycerol (PEG s- DMG) and the like.

[0135] In certain embodiments, the LNP comprises an additional, stabilizing -lipid which is a polyethylene glycol-lipid (pegylated lipid). Suitable polyethylene glycol-lipids include PEG-206678-0001-00WQmodified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramides (e g., PEG-CerC14 or PEG-CerC20), PEG-modified dialkylamines, PEG-modified diacylglycerols, PEG-modified dialkylglycerols. Representative polyethylene glycol-lipids include PEG-c-DOMG, PEG-c-DMA, and PEG-s-DMG. In one embodiment, the polyethylene glycol-lipid is N-[(methoxy poly(ethylene glycol)2000)carbamyl]-l,2-dimyristyloxlpropyl-3-amine (PEG-c-DMA). In one embodiment, the polyethylene glycol-lipid is PEG-c-DOMG). In other embodiments, the LNPs comprise a pegylated diacylglycerol (PEG-DAG) such as 1 (monomethoxy polyethyleneglycol) 2,3 dimyristoylglycerol (PEG-DMG), a pegylated phosphatidylethanoloamine (PEG-PE), a PEG succinate di acylglycerol (PEG-S-DAG) such as 4-O-(2’, 3 ’-di(tetradecanoyloxy)propyl-l-0-(co-methoxy(poly ethoxy )ethyl)butanedioate (PEG-S-DMG), a pegylated ceramide (PEG-cer), or a PEG dialkoxypropylcarbamate such as co-methoxy(polyethoxy)ethyl-N-(2,3-di(tetradecanoxy)propyl)carbamate or 2,3-di(tetradecanoxy)propyl-N-((D-methoxy(polyethoxy)ethyl)carbamate.

[0136] In certain embodiments, the pegylated lipid is present in the LNP in an amount from about 0 to about 20 mole percent. In one embodiment, the pegylated lipid is present in the LNP in an amount from about 0.1 to about 10 mole percent. In one embodiment, the pegylated lipid is present in the LNP in an amount from about 0.1 to about 5 mole percent. In various embodiments, the molar ratio of the cationic lipid to the pegylated lipid ranges from about 100:1 to about 25:1.

[0137] In certain embodiments, the LNP comprises one or more targeting moieties that targets the LNP to a cell or cell population. For example, in one embodiment, the targeting domain is a ligand which directs the LNP to a receptor found on a cell surface.

[0138] In certain embodiments, the LNP comprises one or more internalization domains. For example, in one embodiment, the LNP comprises one or more domains which bind to a cell to induce the internalization of the LNP. For example, in one embodiment, the one or more internalization domains bind to a receptor found on a cell surface to induce receptor-mediated uptake of the LNP. In certain embodiments, the LNP is capable of binding a biomolecule in vivo, where the LNP-bound biomolecule can then be recognized by a cell-surface receptor to induce internalization. For example, in one embodiment, the LNP binds systemic ApoE, which leads to the uptake of the LNP and associated cargo.206678-0001-00WQ

[0139] Exemplary LNPs and their manufacture are described in the art, for example in U. S. Patent Application Publication No. US20120276209, Semple et al., 2010, Nat Biotechnol., 28(2): 172-176; Akinc et al., 2010, Mol Ther., 18(7): 1357-1364; Basha et al., 2011, Mol Ther, 19(12): 2186-2200; Leung et al., 2012, J Phys Chem C Nanomater Interfaces, 116(34): 18440-18450; Lee et al., 2012, Int J Cancer., 131(5): E781-90; Belliveau et al., 2012, Mol Ther nucleic Acids, 1: e37; Jayaraman et al., 2012, Angew Chem Int Ed Engl., 51(34): 8529-8533; Mui et al., 2013, Mol Ther Nucleic Acids. 2, el39; Maier et al., 2013, Mol Ther., 21(8): 1570-1578; and Tam et al., 2013, Nanomedicine, 9(5): 665-74, each of which are incorporated by reference in their entirety.

[0140] In certain embodiments, at least one fusogenic peptide is present in the LNP in an amount from about 0.1 to about 10 mole percent. In one embodiment, the fusogenic peptide is present in the LNP in an amount from about 0.1 to about 5 mole percent. In various embodiments, the molar ratio of the cationic lipid to the fusogenic peptide ranges from about 100:1 to about 25:1.Fusogenic Lipid Nanoparticles

[0141] Inclusion of FAST proteins into LNPs has been shown to promote direct lipid-cell fusion events. The LNP-membrane fusion bypasses the endosomal pathway and allows for direct delivery of nanoparticle payloads to the cytoplasm. Avoidance of the endosomal compartment obviates the need for disrupting endosomal membranes, resulting in enhanced transfection efficiency, efficient payload release, reduced dose and less cytotoxicity to a patient. Therefore, in some embodiments, the invention relates to fusogenic lipid nanoparticles, and methods of use thereof for delivery of therapeutic agents.Agents

[0142] In one embodiment, the delivery vehicle comprises at least one agent. In some embodiments, the agent is a therapeutic agent, an imaging agent, diagnostic agent, a contrast agent, a labeling agent, or a detection agent. The agent may also include substances with biological activities which are not typically considered to be active ingredients, such as fragrances, sweeteners, flavorings and flavor enhancer agents, pH adjusting agents, effervescent206678-0001-00WQagents, emollients, bulking agents, soluble organic salts, permeabilizing agents, anti-oxidants, colorants or coloring agents, and the like.

[0143] In one embodiment, the delivery vehicle comprises at least one therapeutic agent. The present invention is not limited to any particular therapeutic agent, but rather encompasses any suitable therapeutic agent that can be included within the delivery vehicle. Exemplary therapeutic agents include, but are not limited to, anti-viral agents, anti-bacterial agents, antioxidant agents, thrombolytic agents, chemotherapeutic agents, anti-inflammatory agents, immunogenic agents, antiseptics, anesthetics, analgesics, pharmaceutical agents, small molecules, peptides, nucleic acids, and the like.Imaging Agents

[0144] In one embodiment, the delivery vehicle comprises an imaging agent. Imaging agents are materials that allow the delivery vehicle to be visualized after exposure to a cell or tissue. Visualization includes imaging for the naked eye, as well as imaging that requires detecting with instruments or detecting information not normally visible to the eye, and includes imaging that requires detecting of photons, sound or other energy quanta. Examples include stains, vital dyes, fluorescent markers, radioactive markers, enzymes or plasmid constructs encoding markers or enzymes. Many materials and methods for imaging and targeting that may be used in the delivery vehicle are provided in the Handbook of Targeted delivery of Imaging Agents, Torchilin, ed. (1995) CRC Press, Boca Raton, Fla.

[0145] Visualization based on molecular imaging typically involves detecting biological processes or biological molecules at a tissue, cell, or molecular level. Molecular imaging can be used to assess specific targets for gene therapies, cell-based therapies, and to visualize pathological conditions as a diagnostic or research tool. Imaging agents that are able to be delivered intracellularly are particularly useful because such agents can be used to assess intracellular activities or conditions. Imaging agents must reach their targets to be effective; thus, in some embodiments, an efficient uptake by cells is desirable. A rapid uptake may also be desirable to avoid the RES, see review in Allport and Weissleder, Experimental Hematology 1237-1246 (2001).

[0146] Further, imaging agents preferably should provide high signal to noise ratios so that they may be detected in small quantities, whether directly, or by effective amplificationtechniques that increase the signal associated with a particular target. Amplification strategies are reviewed in Allport and Weissleder, Experimental Hematology 1237-1246 (2001), and include, for example, avidin-biotin binding systems, trapping of converted ligands, probes that change physical behavior after being bound by a target, and taking advantage of relaxation rates.Examples of imaging technologies include magnetic resonance imaging, radionuclide imaging, computed tomography, ultrasound, and optical imaging.

[0147] Delivery vehicles as set forth herein may advantageously be used in various imaging technologies or strategies, for example by incorporating imaging agents into delivery vehicles. Many imaging techniques and strategies are known, e.g., see review in Allport and Weissleder, Experimental Hematology 1237-1246 (2001); such strategies may be adapted to use with delivery vehicles. Suitable imaging agents include, for example, fluorescent molecules, labeled antibodies, labeled avidimbiotin binding agents, colloidal metals (e.g., gold, silver), reporter enzymes (e.g., horseradish peroxidase), superparamagnetic transferrin, second reporter systems (e.g., tyrosinase), and paramagnetic chelates.

[0148] In some embodiments, the imaging agent is a magnetic resonance imaging contrast agent. Examples of magnetic resonance imaging contrast agents include, but are not limited to, l,4,7,10-tetraazacyclododecane-N, N', N" N'"-tetracetic acid (DOTA),di ethylenetriaminepentaacetic (DTP A), 1,4,7, 10-tetraazacy cl ododecane-N, N', N", N'"-tetraethylphosphorus (DOTEP), 1,4,7, 10-tetraazacyclododecane-N, N', N''-triacetic acid (DOTA) and derivatives thereof (see U. S. Pat. Nos. 5,188,816, 5,219,553, and 5,358,704). In some embodiments, the imaging agent is an X-Ray contrast agent. X-ray contrast agents already known in the art include a number of halogenated derivatives, especially iodinated derivatives, of 5-amino-isophthalic acid.Small molecule therapeutic agents

[0149] In various embodiments, the agent is a therapeutic agent. In various embodiments, the therapeutic agent is a small molecule. When the therapeutic agent is a small molecule, a small molecule may be obtained using standard methods known to the skilled artisan. Such methods include chemical organic synthesis or biological means. Biological means include purification from a biological source, recombinant synthesis and in vitro translation systems, using methods well known in the art. In one embodiment, a small molecule therapeutic agents206678-0001-00WQcomprises an organic molecule, inorganic molecule, biomolecule, synthetic molecule, and the like.

[0150] The small molecule and small molecule compounds described herein may be present as salts even if salts are not depicted, and it is understood that the invention embraces all salts and solvates of the therapeutic agents depicted here, as well as the non-salt and non-solvate form of the therapeutic agents, as is well understood by the skilled artisan. In some embodiments, the salts of the therapeutic agents of the invention are pharmaceutically acceptable salts.Nucleic acid therapeutic agents

[0151] In other related aspects, the therapeutic agent is an isolated nucleic acid. In certain embodiments, the isolated nucleic acid molecule is one of a DNA molecule or an RNA molecule. In certain embodiments, the isolated nucleic acid molecule is a cDNA, mRNA, siRNA, shRNA or miRNA molecule. In one embodiment, the isolated nucleic acid molecule encodes a therapeutic peptide such as cystic fibrosis transmembrane conductance regulator (CFTR). In some embodiments, the isolated nucleic acid is nanoplasmid comprising a CFTR coding sequence, or a fragment or variant thereof. In some embodiments, the isolated nucleic acid is CFTR mRNA, or a fragment or variant thereof. In one embodiment, the nucleic acid molecule encoding CFTR encodes SEQ ID NO:245. In one embodiment, the nucleic acid molecule encoding CFTR comprises SEQ ID NO:246 or a fragment or variant thereof. In one embodiment, the nucleic acid molecule encoding CFTR comprises a 5’ UTR sequence as set forth in SEQ ID NO:243 and a 3’ UTR sequence as set forth in SEQ ID NO:244. In one embodiment, the nucleic acid molecule encoding CFTR comprises SEQ ID NO:247.

[0152] The isolated nucleic acid may be further modified to increase its stability in vivo. Possible modifications include, but are not limited to, the addition of flanking sequences at the 5' and / or 3' ends; the use of phosphorothioate or 2' O-methyl rather than phosphodiester linkages in the backbone; and / or the inclusion of nontraditional bases such as inosine, queuosine, and wybutosine and the like, as well as acetyl- methyl-, thio- and other modified forms of adenine, cytidine, guanine, thymine, and uridine.

[0153] In one embodiment of the invention, an antisense nucleic acid sequence, which is expressed by a plasmid vector is used as a therapeutic agent to inhibit the expression of a targetprotein. Antisense molecules and their use for inhibiting gene expression are well known in the art (see, e.g., Cohen, 1989, In: Oligodeoxyribonucleotides, Antisense Inhibitors of Gene Expression, CRC Press). Antisense nucleic acids are DNA or RNA molecules that are complementary, as that term is defined elsewhere herein, to at least a portion of a specific mRNA molecule (Weintraub, 1990, Scientific American 262:40). In the cell, antisense nucleic acids hybridize to the corresponding mRNA, forming a double-stranded molecule thereby inhibiting the translation of genes.

[0154] The use of antisense methods to inhibit the translation of genes is known in the art, and is described, for example, in Marcus-Sakura (1988, Anal. Biochem. 172:289). Such antisense molecules may be provided to the cell via genetic expression using DNA encoding the antisense molecule as taught by Inoue, 1993, U. S. Patent No. 5,190,931.

[0155] Alternatively, antisense molecules of the invention may be made synthetically and then delivered to the cell. Antisense oligomers of between about 10 to about 30, and more preferably about 15 nucleotides, are preferred, since they are easily synthesized and introduced into a target cell. Synthetic antisense molecules contemplated by the invention include oligonucleotide derivatives known in the art which have improved biological activity compared to unmodified oligonucleotides (see U. S. Patent No. 5,023,243).

[0156] In one embodiment of the invention, a ribozyme is used as a therapeutic agent to inhibit expression of a target protein. Ribozymes useful for inhibiting the expression of a target molecule may be designed by incorporating target sequences into the basic ribozyme structure, which are complementary, for example, to the mRNA sequence encoding the target molecule. Ribozymes targeting the target molecule, may be synthesized using commercially available reagents (Applied Biosystems, Inc., Foster City, CA) or they may be genetically expressed from DNA encoding them.

[0157] In one embodiment, the therapeutic agent may comprise one or more components of a CRISPR-Cas system, where a guide RNA (gRNA) targeted to a gene encoding a target molecule, and a CRISPR-associated (Cas) peptide form a complex to induce mutations within the targeted gene. In one embodiment, the therapeutic agent comprises a gRNA or a nucleic acid molecule encoding a gRNA. In one embodiment, the therapeutic agent comprises a Cas peptide or a nucleic acid molecule encoding a Cas peptide.

[0158] In one embodiment, the agent comprises a miRNA or a mimic of a miRNA. In one embodiment, the agent comprises a nucleic acid molecule that encodes a miRNA or mimic of a miRNA.

[0159] MiRNAs are small non-coding RNA molecules that are capable of causing post-transcriptional silencing of specific genes in cells by the inhibition of translation or through degradation of the targeted mRNA. A miRNA can be completely complementary or can have a region of noncomplementarity with a target nucleic acid, consequently resulting in a "bulge" at the region of non-complementarity. A miRNA can inhibit gene expression by repressing translation, such as when the miRNA is not completely complementary to the target nucleic acid, or by causing target RNA degradation, which is believed to occur only when the miRNA binds its target with perfect complementarity. The disclosure also can include double-stranded precursors of miRNA. A miRNA or pri-miRNA can be 18- 100 nucleotides in length, or from 18-80 nucleotides in length. Mature miRNAs can have a length of 19-30 nucleotides, or 21-25 nucleotides, particularly 21, 22, 23, 24, or 25 nucleotides. MiRNA precursors typically have a length of about 70-100 nucleotides and have a hairpin conformation. miRNAs are generated in vivo from pre- miRNAs by the enzymes Dicer and Drosha, which specifically process long pre-miRNA into functional miRNA. The hairpin or mature microRNAs, or pri-microRNA agents featured in the disclosure can be synthesized in vivo by a cell-based system or in vitro by chemical synthesis.

[0160] In various embodiments, the agent comprises an oligonucleotide that comprises the nucleotide sequence of a disease-associated miRNA. In certain embodiments, the oligonucleotide comprises the nucleotide sequence of a disease-associated miRNA in a pre -microRNA, mature or hairpin form. In other embodiments, a combination of oligonucleotides comprising a sequence of one or more disease-associated miRNAs, any pre -miRNA, any fragment, or any combination thereof is envisioned.

[0161] MiRNAs can be synthesized to include a modification that imparts a desired characteristic. For example, the modification can improve stability, hybridization thermodynamics with a target nucleic acid, targeting to a particular tissue or cell -type, or cell permeability, e.g., by an endocytosis-dependent or -independent mechanism.

[0162] Modifications can also increase sequence specificity, and consequently decrease off-site targeting. Methods of synthesis and chemical modifications are described in greaterdetail below. If desired, miRNA molecules may be modified to stabilize the miRNAs against degradation, to enhance half-life, or to otherwise improve efficacy. Desirable modifications are described, for example, in U. S. Patent Publication Nos. 20070213292, 20060287260, 20060035254. 20060008822. and 2005028824, each of which is hereby incorporated by reference in its entirety. For increased nuclease resistance and / or binding affinity to the target, the single- stranded oligonucleotide agents featured in the disclosure can include 2'-O-methyl, 2'-fluorine, 2'-O-methoxy ethyl, 2'-O-aminopropyl, 2'-amino, and / or phosphorothioate linkages. Inclusion of locked nucleic acids (LNA), ethylene nucleic acids (ENA), e.g., 2'-4'-ethylene-bridged nucleic acids, and certain nucleotide modifications can also increase binding affinity to the target. The inclusion of pyranose sugars in the oligonucleotide backbone can also decrease endonucleolytic cleavage. An oligonucleotide can be further modified by including a 3' cationic group, or by inverting the nucleoside at the 3'-terminus with a 3 -3' linkage. In another alternative, the 3 '-terminus can be blocked with an aminoalkyl group. Other 3' conjugates can inhibit 3'-5' exonucleolytic cleavage. While not being bound by theory, a 3' may inhibit exonucleolytic cleavage by sterically blocking the exonuclease from binding to the 3' end of the oligonucleotide. Even small alkyl chains, aryl groups, or heterocyclic conjugates or modified sugars (D-ribose, deoxyribose, glucose etc.) can block 3'-5'-exonucleases.

[0163] In one embodiment, the miRNA includes a 2'-modified oligonucleotide containing oligodeoxynucleotide gaps with some or all intemucleotide linkages modified to phosphorothioates for nuclease resistance. The presence of methylphosphonate modifications increases the affinity of the oligonucleotide for its target RNA and thus reduces the IC50. This modification also increases the nuclease resistance of the modified oligonucleotide. It is understood that the methods and reagents of the present disclosure may be used in conjunction with any technologies that may be developed to enhance the stability or efficacy of an inhibitory nucleic acid molecule.

[0164] miRNA molecules include nucleotide oligomers containing modified backbones or non-natural internucleoside linkages. Oligomers having modified backbones include those that retain a phosphorus atom in the backbone and those that do not have a phosphorus atom in the backbone. For the purposes of this disclosure, modified oligonucleotides that do not have a phosphorus atom in their internucleoside backbone are also considered to be nucleotide oligomers. Nucleotide oligomers that have modified oligonucleotide backbones include, forexample, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotri esters, aminoalkyl-phosphotriesters, methyl and other alkyl phosphonates including 3 '-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriest- ers, and boranophosphates. Various salts, mixed salts and free acid forms are also included.

[0165] A miRNA described herein, which may be in the mature or hairpin form, may be provided as a naked oligonucleotide. In some cases, it may be desirable to utilize a formulation that aids in the delivery of a miRNA or other nucleotide oligomer to cells (see, e.g., U. S. Pat. Nos. 5,656,61 1, 5,753,613, 5,785,992, 6,120,798, 6,221,959, 6,346,613, and 6,353,055, each of which is hereby incorporated by reference).

[0166] In some examples, the miRNA composition is at least partially crystalline, uniformly crystalline, and / or anhydrous (e.g., less than 80, 50, 30, 20, or 10% water). In another example, the miRNA composition is in an aqueous phase, e.g., in a solution that includes water. The aqueous phase or the crystalline compositions can be incorporated into a delivery vehicle, e.g., a liposome (particularly for the aqueous phase), or a particle (e.g., a microparticle as can be appropriate for a crystalline composition). Generally, the miRNA composition is formulated in a manner that is compatible with the intended method of administration. A miRNA composition can be formulated in combination with another agent, e.g., another therapeutic agent or an agent that stabilizes an oligonucleotide agent, e.g., a protein that complexes with the oligonucleotide agent. Still other agents include chelators, e.g., EDTA (e.g., to remove divalent cations such as Mg), salts, and RNAse inhibitors (e.g., abroad specificity RNAse inhibitor). In one embodiment, the miRNA composition includes another miRNA, e.g., a second miRNA composition (e.g., a microRNA that is distinct from the first). Still other preparations can include at least three, five, ten, twenty, fifty, or a hundred or more different oligonucleotide species.

[0167] In certain embodiments, the composition comprises an oligonucleotide composition that mimics the activity of a miRNA. In certain embodiments, the composition comprises oligonucleotides having nucleobase identity to the nucleobase sequence of a miRNA, and are thus designed to mimic the activity of the miRNA. In certain embodiments, the oligonucleotide composition that mimics miRNA activity comprises a double-stranded RNA molecule which mimics the mature miRNA hairpins or processed miRNA duplexes.

[0168] In one embodiment, the oligonucleotide shares identity with endogenous miRNA or miRNA precursor nucleobase sequences. An oligonucleotide selected for inclusion in a composition of the present invention may be one of a number of lengths. Such an oligonucleotide can be from 7 to 100 linked nucleosides in length. For example, an oligonucleotide sharing nucleobase identity with a miRNA may be from 7 to 30 linked nucleosides in length. An oligonucleotide sharing identity with a miRNA precursor may be up to 100 linked nucleosides in length. In certain embodiments, an oligonucleotide comprises 7 to 30 linked nucleosides. In certain embodiments, an oligonucleotide comprises 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 28, 29, or 30 linked nucleotides. In certain embodiments, an oligonucleotide comprises 19 to 23 linked nucleosides. In certain embodiments, an oligonucleotide is from 40 up to 50, 60, 70, 80, 90, or 100 linked nucleosides in length.

[0169] In certain embodiments, an oligonucleotide has a sequence that has a certain identity to a miRNA or a precursor thereof. Nucleobase sequences of mature miRNAs and their corresponding stem-loop sequences described herein are the sequences found in miRBase, an online searchable database of miRNA sequences and annotation. Entries in the miRBase Sequence database represent a predicted hairpin portion of a miRNA transcript (the stem-loop), with information on the location and sequence of the mature miRNA sequence. The miRNA stem-loop sequences in the database are not strictly precursor miRNAs (pre-miRNAs), and may in some instances include the pre-miRNA and some flanking sequence from the presumed primary transcript. The miRNA nucleobase sequences described herein encompass any version of the miRNA, including the sequences described in Release 10.0 of the miRBase sequence database and sequences described in any earlier Release of the miRBase sequence database. A sequence database release may result in the re-naming of certain miRNAs. A sequence database release may result in a variation of a mature miRNA sequence. The compositions of the present invention encompass oligomeric compound comprising oligonucleotides having a certain identity to any nucleobase sequence version of a miRNAs described herein.

[0170] In certain embodiments, an oligonucleotide has a nucleobase sequence at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98% or 99% identical to the miRNA over a region of 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleobases. Accordingly, in certain embodiments the nucleobase sequence of an oligonucleotide may have one or more non-identical nucleobases with respect to the miRNA.206678-0001-00WQ

[0171] In certain embodiments, the composition comprises a nucleic acid molecule encoding a miRNA, precursor, mimic, or fragment thereof. For example, the composition may comprise a viral vector, plasmid, cosmid, or other expression vector suitable for expressing the miRNA, precursor, mimic, or fragment thereof in a desired mammalian cell or tissue.In vitro transcribed RNA

[0172] In one embodiment, the composition of the invention comprises in vitro transcribed (IVT) RNA. In one embodiment, the composition of the invention comprises in vitro transcribed (IVT) RNA encoding a therapeutic protein. In one embodiment, the composition of the invention comprises IVT RNA encoding a plurality of therapeutic proteins.

[0173] In one embodiment, an IVT RNA can be introduced to a cell as a form of transient transfection. The RNA is produced by in vitro transcription using a plasmid DNA template generated synthetically. DNA of interest from any source can be directly converted by PCR into a template for in vitro mRNA synthesis using appropriate primers and RNA polymerase. The source of the DNA can be, for example, genomic DNA, plasmid DNA, phage DNA, cDNA, synthetic DNA sequence or any other appropriate source of DNA. In one embodiment, the desired template for in vitro transcription is a therapeutic protein, as described elsewhere herein.

[0174] In one embodiment, the DNA to be used for PCR contains an open reading frame. The DNA can be from a naturally occurring DNA sequence from the genome of an organism. In one embodiment, the DNA is a full-length gene of interest of a portion of a gene. The gene can include some or all of the 5' and / or 3' untranslated regions (UTRs). The gene can include exons and introns. In one embodiment, the DNA to be used for PCR is a human gene. In another embodiment, the DNA to be used for PCR is a human gene including the 5' and 3' UTRs. In another embodiment, the DNA to be used for PCR is a gene from a pathogenic or commensal organism, including bacteria, viruses, parasites, and fungi. In another embodiment, the DNA to be used for PCR is from a pathogenic or commensal organism, including bacteria, viruses, parasites, and fungi, including the 5' and 3' UTRs. The DNA can alternatively be an artificial DNA sequence that is not normally expressed in a naturally occurring organism. An exemplary artificial DNA sequence is one that contains portions of genes that are ligated together to form anopen reading frame that encodes a fusion protein. The portions of DNA that are ligated together can be from a single organism or from more than one organism.

[0175] Genes that can be used as sources of DNA for PCR include genes that encode polypeptides that induce or enhance an adaptive immune response in an organism. Preferred genes are genes which are useful for a short-term treatment, or where there are safety concerns regarding dosage or the expressed gene.

[0176] In various embodiments, a plasmid is used to generate a template for in vitro transcription of an mRNA molecule which is encapsulated into a delivery vehicle (e g., an LNP) for delivery to a target cell.

[0177] Chemical structures with the ability to promote stability and / or translation efficiency may also be used. The RNA preferably has 5' and 3' UTRs. In one embodiment, the 5' UTR is between zero and 3000 nucleotides in length. The length of 5' and 3' UTR sequences to be added to the coding region can be altered by different methods, including, but not limited to, designing primers for PCR that anneal to different regions of the UTRs. Using this approach, one of ordinary skill in the art can modify the 5' and 3' UTR lengths required to achieve optimal translation efficiency following transfection of the transcribed RNA.

[0178] The 5' and 3' UTRs can be the naturally occurring, endogenous 5' and 3' UTRs for the gene of interest. Alternatively, UTR sequences that are not endogenous to the gene of interest can be added by incorporating the UTR sequences into the forward and reverse primers or by any other modifications of the template. The use of UTR sequences that are not endogenous to the gene of interest can be useful for modifying the stability and / or translation efficiency of the RNA. For example, it is known that AU-rich elements in 3' UTR sequences can decrease the stability of RNA. Therefore, 3' UTRs can be selected or designed to increase the stability of the transcribed RNA based on properties of UTRs that are well known in the art.

[0179] In one embodiment, the 5' UTR can contain the Kozak sequence of the endogenous gene. Alternatively, when a 5' UTR that is not endogenous to the gene of interest is being added by PCR as described above, a consensus Kozak sequence can be redesigned by adding the 5' UTR sequence. Kozak sequences can increase the efficiency of translation of some RNA transcripts, but does not appear to be required for all RNAs to enable efficient translation. The requirement for Kozak sequences for many RNAs is known in the art. In other embodiments the 5' UTR can be derived from an RNA virus whose RNA genome is stable in cells. In otherembodiments various nucleotide analogues can be used in the 3' or 5' UTR to impede exonuclease degradation of the RNA.

[0180] To enable synthesis of RNA from a DNA template without the need for gene cloning, a promoter of transcription should be attached to the DNA template upstream of the sequence to be transcribed. When a sequence that functions as a promoter for an RNA polymerase is added to the 5' end of the forward primer, the RNA polymerase promoter becomes incorporated into the PCR product upstream of the open reading frame that is to be transcribed. In one preferred embodiment, the promoter is a T7 RNA polymerase promoter, as described elsewhere herein. Other useful promoters include, but are not limited to, T3 and SP6 RNA polymerase promoters. Consensus nucleotide sequences for T7, T3 and SP6 promoters are known in the art.

[0181] In a preferred embodiment, the RNA has both a cap on the 5' end and a 3' poly(A) tail which determine ribosome binding, initiation of translation and stability mRNA in the cell. On a circular DNA template, for instance, plasmid DNA, RNA polymerase produces a long concatameric product which is not suitable for expression in eukaryotic cells. The transcription of plasmid DNA linearized at the end of the 3' UTR results in normal sized RNA which is effective in eukaryotic transfection when it is polyadenylated after transcription.

[0182] On a linear DNA template, phage T7 RNA polymerase can extend the 3' end of the transcript beyond the last base of the template (Schenborn and Mierendorf, Nuc Acids Res., 13:6223-36 (1985); Nacheva and Berzal-Herranz, Eur. J. Biochem., 270:1485-65 (2003).

[0183] The conventional method of integration of polyA / T stretches into a DNA template is molecular cloning. However polyA / T sequence integrated into plasmid DNA can cause plasmid instability, which can be ameliorated through the use of recombination incompetent bacterial cells for plasmid propagation.

[0184] Poly(A) tails of RNAs can be further extended following in vitro transcription with the use of a poly(A) polymerase, such as E. coli polyA polymerase (E-PAP) or yeast polyA polymerase. In one embodiment, increasing the length of a poly(A) tail from 100 nucleotides to between 300 and 400 nucleotides results in about a two-fold increase in the translation efficiency of the RNA. Additionally, the attachment of different chemical groups to the 3' end can increase RNA stability. Such attachment can contain modified / artificial nucleotides, aptamers and othercompounds. For example, ATP analogs can be incorporated into the poly(A) tail using poly(A) polymerase. ATP analogs can further increase the stability of the RNA.

[0185] 5' caps on also provide stability to RNA molecules. In a preferred embodiment, RNAs produced by the methods to include a 5' capl structure. Such capl structure can be generated using Vaccinia capping enzyme and 2 ’-O-methyl transferase enzymes (CellScript, Madison, WI). Alternatively, 5' cap is provided using techniques known in the art and described herein (Cougot, et al., Trends in Biochem. Sci., 29:436-444 (2001); Stepinski, et al., RNA, 7:1468-95 (2001); Elango, etal., Biochim. Biophys. Res. Commun., 330:958-966 (2005)).Polypeptide therapeutic agents

[0186] In other related aspects, the therapeutic agent includes an isolated peptide or a variant or fragment thereof. The variants of the polypeptide therapeutic agents may be (i) one in which one or more of the amino acid residues are substituted with a conserved or non-conserved amino acid residue (preferably a conserved amino acid residue) and such substituted amino acid residue may or may not be one encoded by the genetic code, (ii) one in which there are one or more modified amino acid residues, e.g., residues that are modified by the attachment of substituent groups, (iii) one in which the polypeptide is an alternative splice variant of the polypeptide of the present invention, (iv) fragments of the polypeptides and / or (v) one in which the polypeptide is fused with another polypeptide, such as a leader or secretory sequence or a sequence which is employed for purification (for example, His-tag) or for detection (for example, Sv5 epitope tag). The fragments include polypeptides generated via proteolytic cleavage (including multi-site proteolysis) of an original sequence. Variants may be post-translationally, or chemically modified. Such variants are deemed to be within the scope of those skilled in the art from the teaching herein.Targeting Domain

[0187] In various embodiments of the invention, the delivery vehicle is conjugated to a targeting domain. In one embodiment, the conjugation is a reversible conjugation, such that the delivery vehicle can be disassociated from the targeting domain upon exposure to certain conditions or chemical agents. In another embodiment, the conjugation is an irreversible206678-0001-00WQconjugation, such that under normal conditions the delivery vehicle does not dissociate from the targeting domain.

[0188] In some embodiments, the conjugation comprises a covalent bond between an activated polymer conjugated lipid and the targeting domain. The term “activated polymer conjugated lipid” refers to a molecule comprising a lipid portion and a polymer portion that has been activated via functionalization of a polymer conjugated lipid with a first coupling group. In one embodiment, the activated polymer conjugated lipid comprises a first coupling group capable of reacting with a second coupling group. In one embodiment, the activated polymer conjugated lipid is an activated pegylated lipid. In one embodiment, the first coupling group is bound to the lipid portion of the pegylated lipid. In another embodiment, the first coupling group is bound to the polyethylene glycol portion of the pegylated lipid. In one embodiment, the second functional group is covalently attached to the targeting domain.

[0189] The first coupling group and second coupling group can be any functional groups known to those of skill in the art to together form a covalent bond, for example under mild reaction conditions or physiological conditions. In some embodiments, the first coupling group or second coupling group are selected from the group consisting of maleimides, N-hydroxysuccinimide (NHS) esters, carbodiimides, hydrazide, pentafluorophenyl (PFP) esters, phosphines, hydroxymethyl phosphines, psoralen, imidoesters, pyridyl disulfide, isocyanates, vinyl sulfones, alpha-haloacetyls, aryl azides, acyl azides, alkyl azides, diazirines, benzophenone, epoxides, carbonates, anhydrides, sulfonyl chlorides, cyclooctyne, aldehydes, and sulfhydryl groups. In some embodiments, the first coupling group or second coupling group is selected from the group consisiting of free amines (-NH2), free sulfhydryl groups (-SH), free hydroxide groups (-OH), carboxylates, hydrazides, and alkoxyamines. In some embodiments, the first coupling group is a functional group that is reactive toward sulfhydryl groups, such as maleimide, pyridyl disulfide, or a haloacetyl. In one embodiment, the first coupling group is a maleimide.

[0190] In one embodiment, the second coupling group is a sulfhydryl group. The sulfhydryl group can be installed on the targeting domain using any method known to those of skill in the art. In one embodiment, the sulfhydryl group is present on a free cysteine residue. In one embodiment, the sulfhydryl group is revealed via reduction of a disulfide on the targeting domain, such as through reaction with 2-mercaptoethylamine. In one embodiment, the sulfhydryl206678-0001-00WQgroup is installed via a chemical reaction, such as the reaction between a free amine and 2-iminothilane or N-succinimidyl S-acetylthioacetate (SATA).

[0191] In some embodiments, the polymer conjugated lipid and targeting domain are functionalized with groups used in “click” chemistry. Bioorthogonal “click” chemistry comprises the reaction between a functional group with a 1,3-dipole, such as an azide, a nitrile oxide, a nitrone, an isocyanide, and the link, with an alkene or an alkyne dipolarophiles. Exemplary dipolarophiles include any strained cycloalkenes and cycloalkynes known to those of skill in the art, including, but not limited to, cyclooctynes, dibenzocyclooctynes, monofluorinated cyclcooctynes, difluorinated cyclooctynes, and biarylazacyclooctynone.

[0192] In some embodiments, the polymer conjugated lipid and targeting domain are functionalized with groups used in EDC / NHS (N-ethyl-N'-(3-(dimethylamino)propyl)carbodiimide / N-hydroxysuccinimide) crosslinking chemistry in which the intermediate molecule succinimidyl ester (NHS-ester) is used to immobilize biomolecules containing free primary amino groups via amide linkage.

[0193] In one embodiment, the composition comprises a targeting domain that directs the delivery vehicle to a target cell. The targeting domain may comprise a nucleic acid, peptide, antibody, small molecule, organic molecule, inorganic molecule, glycan, sugar, hormone, and the like that targets the particle to a site in particular need of the therapeutic agent. In certain embodiments, the particle comprises multivalent targeting, wherein the particle comprises multiple targeting mechanisms described herein. In certain embodiments, the targeting domain of the delivery vehicle specifically binds to a target associated with a site in need of an agent comprised within the delivery vehicle. For example, the targeting domain may be chosen to recognize a ligand that acts as a cell surface marker on target cells associated with a particular disease state. Such a target can be a protein, protein fragment, antigen, or other biomolecule that is associated with the targeted site. In some embodiments, the targeting domain is an affinity ligand which specifically binds to a target. In certain embodiments, the target (e.g. antigen) associated with a site in need of a treatment with an agent. In some embodiments, the targeting domain may be co-polymerized with the composition comprising the delivery vehicle. In some embodiments, the targeting domain may be covalently attached to the composition comprising the delivery vehicle, such as through a chemical reaction between the targeting domain and the composition comprising the delivery vehicle. In some embodiments, the targeting domain is an206678-0001-00WQadditive in the delivery vehicle. Targeting domains of the instant invention include, but are not limited to, antibodies, antibody fragments, proteins, peptides, and nucleic acids.Combinations

[0194] In one embodiment, the composition of the present invention comprises a combination of agents described herein. In one embodiment, the composition comprises a combination of at least one fusogenic nanoparticle and one or more additional therapeutic agent. In one embodiment, the composition comprises a combination of two or more fusogenic nanoparticles.

[0195] In certain embodiments, a composition comprising a combination of agents described herein has an additive effect, wherein the overall effect of the combination is approximately equal to the sum of the effects of each individual agent. In other embodiments, a composition comprising a combination of agents described herein has a synergistic effect, wherein the overall effect of the combination is greater than the sum of the effects of each individual agent.

[0196] A composition comprising a combination of agents comprises individual agents in any suitable ratio. For example, in one embodiment, the composition comprises a 1:1 ratio of two individual agents. However, the combination is not limited to any particular ratio. Rather any ratio that is shown to be effective is encompassed.

[0197] In one embodiment, two or more two or more compositions are formulated independently and then combined into a single composition prior to administration. In one embodiment, two or more two or more compositions are formulated independently and administered in combination temporally or as individual parts of a single treatment regime.Therapeutic Methods

[0198] In some embodiments, the invention provides methods for treatment or prevention of a disease or disorder. In some embodiments, the disease or disorder is associated with mitochondrial dysfunction. In some embodiments, the disease or disorder is a genetic disease or disorder resulting from a mutation in a gene.

[0199] In some embodiments, the invention provides methods for delivery of a nucleic acid molecule to a cell of a subject having a disease or disorder, wherein the nucleic acid206678-0001-00WQmolecule is therapeutic for the disease or disorder or produces an expression product that is therapeutic for the disease or disorder. In some embodiments the compositions are added to patient derived cells for the treatment of a disease or disorder. In some embodiments the patient derived cells are transplantable cells (e.g., hemopoietic stem cells (HSCs), T-cells, fibroblasts, cardiac cells, retinal pigment epithelial cells, or any other transplantable cell).

[0200] Exemplary diseases or disorders that can be treated using the methods of the invention include, but are not limited to, monogenic diseases, metabolic diseases, conditions benefiting from increased gene / hormone activity, enhanced metabolic signaling, or restored deficient pathways (Boost conditions), conditions benefiting from reduced gene / protein activity, suppressed lipogenesis, substrate reduction, or lowered pathological signaling (Knockdown conditions), cosmetic or skin conditions, conditions associated with aging, conditions associated with mitochondrial function, autoimmune diseases, immunodeficiency diseases, inflammatory diseases, neurological diseases or disorder, infectious diseases and cancer.

[0201] Exemplary monogenic diseases include, but are not limited to, cystic fibrosis, severe combined immunodeficiency, lysosomal storage diseases (e.g, Gaucher, Hurler, Hunter, Fabry, Neimann-Pick, Tay-Sach, etc.), sickle cell anemia, thalassemia Primary Ciliary Dyskinesia (PCD), Recessive Dystrophic Epidermolysis Bullosa (RDEB), Lamellar Ichthyosis (LI) of Autosomal Recessive Congenital Ichthyosis (ARCI) Family, Cystic fibrosis, Duchenne muscular dystrophy, Becker muscular dystrophy, spinal muscular atrophy, hemophilia A, hemophilia B, sickle cell disease, beta-thalassemia, alpha-thalassemia, Tay-Sachs disease, Sandhoff disease, Gaucher disease, Fabry disease, Pompe disease, Niemann-Pick disease, metachromatic leukodystrophy, Krabbe disease, adrenoleukodystrophy, mucopolysaccharidoses (MPS I- VII), Rett syndrome, Angelman syndrome, Huntington’s disease, Friedreich’s ataxia, ALS (genetic forms), Charcot-Mari e-Tooth disease, Leber congenital amaurosis, retinitis pigmentosa, Stargardt disease, congenital blindness from RPE65 defects, primary immunodeficiencies (SCID variants, ADA-SCID, X-linked SCID), Wiskott-Aldrich syndrome, chronic granulomatous disease, Fanconi anemia, alpha-1 antitrypsin deficiency, Wilson disease, hemochromatosis, urea cycle disorders, OTC deficiency, familial hypercholesterolemia (LDLR, PCSK9 variants), LPL deficiency, congenital leptin deficiency, congenital hypothyroidism (genetic forms), glycogen storage diseases (types I-IX), galactosemia, maple syrup urine disease,phenylketonuria, homocystinuria, mitochondrial DNA depletion syndromes (nuclear-encoded), and congenital hearing loss due to single-gene defects.

[0202] Exemplary cancers include, but are not limited to, breast cancer, prostate cancer, lung cancer (NSCLC, SCLC), colorectal cancer, pancreatic cancer, gastric cancer, esophageal cancer, liver cancer (HCC), cholangiocarcinoma, kidney cancer (RCC), bladder cancer, ovarian cancer, cervical cancer, endometrial cancer, testicular cancer, thyroid cancer, head and neck cancers, skin cancers (melanoma, basal cell, squamous cell), bone cancers (osteosarcoma, Ewing sarcoma), brain cancers (gliomas, glioblastoma, astrocytoma), neuroblastoma, sarcomas (soft-tissue and others), lymphoma (Hodgkin and non-Hodgkin), leukemia (ALL, AML, CLL, CML), multiple myeloma, mesothelioma, uveal melanoma, and Merkel cell carcinoma.

[0203] Exemplary metabolic diseases include, but are not limited to, GLP-1 against, cholesterol psk-9 inhibitors, obesity (general, monogenic, polygenic), metabolic syndrome, type 2 diabetes mellitus, insulin resistance, nonalcoholic fatty liver disease (NAFLD), nonalcoholic steatohepatitis (NASH), dyslipidemia, familial hypercholesterolemia, hypertriglyceridemia, mitochondrial diabetes (MIDD), lipodystrophy (generalized and partial), Prader-Willi syndrome (metabolic aspects), Bardet-Biedl syndrome, Alstrbm syndrome, glycogen storage diseases (types I-IX), galactosemia, fructose intolerance, maple syrup urine disease, phenylketonuria, tyrosinemia, homocystinuria, urea cycle disorders, organic acidemias (propionic, methylmalonic, isovaleric), medium-chain acyl-CoA dehydrogenase deficiency (MCAD) and other fatty acid oxidation defects, primary carnitine deficiency, CPT1 / 2 deficiencies, peroxisomal disorders (Zellweger spectrum), Wilson disease (copper metabolism), hemochromatosis (iron overload), a-1 antitrypsin deficiency (metabolic liver disease), congenital leptin deficiency, congenital leptin receptor deficiency, MC4R-related obesity, PCSK9-related hypercholesterolemia, LPL deficiency, APOC2 deficiency, ABCA1 deficiency (Tangier disease), Niemann-Pick type C (cholesterol trafficking), Gaucher disease, Fabry disease, Pompe disease, mucopolysaccharidoses (metabolic lysosomal disorders), thyroid hormone metabolism disorders (hypothyroidism, hyperthyroidism genetic variants), adrenal metabolic dysregulation (CAH forms), and rare monogenic forms of hypoglycemia and hyperglycemia such as congenital hyperinsulinism or MODY types 1-14.

[0204] Exemplary Boost conditions include, but are not limited to, obesity (certain subtypes with impaired satiety signaling), metabolic syndrome with low adiponectin or low206678-0001-00WQmitochondrial activity, type 2 diabetes requiring enhanced incretin action (GLP-1 pathway boost), insulin resistance driven by deficient anabolic signaling, NAFLD / NASH with underactive fatty-acid oxidation, mitochondrial diabetes (MIDD), generalized and partial lipodystrophy (adipokine and mitochondrial boosting), Prader-Willi syndrome (hypometabolic aspects), Bardet-Biedl syndrome, Alstrbm syndrome, fatty acid oxidation defects (MCAD and related FAO deficiencies), primary carnitine deficiency, CPT1 / 2 deficiencies, peroxisomal disorders (Zellweger spectrum), select glycogen storage diseases needing increased metabolic flux (some GSD III / IX contexts), urea cycle disorders requiring enhanced enzyme expression, congenital leptin deficiency, leptin receptor deficiency, MC4R-related obesity, certain thyroid hormone deficiency states, adrenal insufficiency variants, rare monogenic hypoglycemia syndromes needing upregulated counter-regulatory pathways, and metabolic diseases benefiting from increased mitochondrial biogenesis (PGC-la), thermogenesis (UCP1), and incretin signaling (GLP-1 pathway reinforcement).

[0205] Exemplary Knockdown conditions include, but are not limited to, obesity driven by excess orexigenic signaling (e.g., AGRP overactivity), atherosclerosis, dyslipidemia, familial hypercholesterolemia (PCSK9 knockdown), hypertriglyceridemia (APOC3, ANGPTL3 / 4 pathways), NAFLD / NASH driven by overactive lipogenesis (SREBPlc, FASN, ACC), LPL deficiency (pathway suppression of triglyceride production), APOC2 and ABCA1 deficiency contexts where substrate reduction mitigates burden, Wilson disease (copper accumulation pathways), hemochromatosis (iron regulatory excess), a-1 antitrypsin deficiency (misfolded-protein stress pathways), Niemann-Pick type C, Gaucher, Fabry, Pompe, mucopolysaccharidoses (lysosomal substrate-reduction strategies), maple syrup urine disease, phenylketonuria, tyrosinemia, homocystinuria, organic acidemias (propionic, methylmalonic, isovaleric), galactosemia, hereditary fructose intolerance, glycogen storage diseases that overproduce or accumulate toxic intermediates (notably GSD I), congenital hyperinsulinism (suppression of excessive insulin secretion), MODY forms with excessive hepatic glucose output (e.g., GCK or HNF-driven), and metabolic inflammatory states in which reducing diabetogenic cytokines improves insulin sensitivity.

[0206] In some embodiments, compositions for treating a condition associated with aging include, but are not limited to, compositions for increasing one or more transcription-factor programs that enhance longevity by increasing youthful gene expression, restoringmitochondrial / metabolic function, improving chromatin state, or reversing epigenetic age.Exemplary transcription-factors that enhance longevity include, but are not limited to, Yamanaka factors: OSKM (full set, mainly research use), OSK (OCT4, SOX2, KLF4), enhanced OSK variants (OSK + LIN28A / B, OSK + ESRRB / ESRRA, OSK + SALL4, OSK + PRDM14), metabolic / mitochondrial youth factors (F0X01 / 3, PGC-la / p, NRF1 / NRF2, TFAM), circadian rejuvenation TFs (BMAL1, CLOCK), chromatin and DNA-stability TFs (NANOG partial, TCF3 / E47, MEIS1), tissue-specific partial-reprogramming sets (NEUR0D1 / ASCL1 for neural, MY0D / PAX7 for muscle, HNF4A / FOXA2 for liver, GATA4 / TBX5 / MEF2C for cardiac), senescence-suppression TFs (F0XM1, controlled KLF4, regulated E2F variants), and pro-youth secretome regulators (Klotho activators, adiponectin-promoting TFs, NF-KB suppressors via TF modulation). These combinations aim for partial, cyclic, or tissue-restricted expression that resets epigenetic age while maintaining cell identity.

[0207] In some embodiments, compositions for treating a condition associated with aging include, but are not limited to, compositions for decreasing transcriptional programs or genesuppression targets intended to reduce pro-aging pathways, senescence signals, or harmful transcriptional outputs. Exemplary knockdown targets for treatment of conditions associated with aging include, but are not limited to suppression of c-MYC for safer reprogramming boundaries, knockdown of NF-KB-driven inflammatory transcription, reduction of AP-l / SASP-enhancing TFs (JUN / FOS), inhibition of TGF-p / SMAD-mediated fibrotic and pro-aging transcription, attenuation of pl6 and p21 senescence-locking circuits (in tightly controlled contexts), suppression of GATA4 in chronic SASP-associated states, downregulation of proaging mitochondrial stress TF outputs (ATF4 / ATF5 under maladaptive UPRmt), partial inhibition of FOXA1 / 2 in age-related lineage-drift scenarios, and targeted reduction of RUNX family hyperactivation in chronic inflammation. These knockdown strategies seek to minimize inflammatory, fibrotic, senescent, or dedifferentiation-driving transcriptional programs that accumulate with aging.

[0208] Exemplary immune dysregulation conditions or diseases associated with immunodeficiencies include, but are not limited to, primary immunodeficiencies (CVID, SCID subtypes, IgA deficiency), chronic granulomatous disease (defective NADPH oxidase), leukocyte adhesion deficiency, complement deficiencies (C2, C3, C4, C5-C9), severe neutropenia or neutrophil dysfunction syndromes, chronic mucocutaneous candidiasis, WHIM206678-0001-00WQsyndrome, hyper-IgM syndrome, hyper-IgE syndrome (STAT3 loss-of-function), antibody production defects (AID, UNG deficiencies), viral susceptibility disorders (TLR3, IRF7, IFNAR defects), chronic or recurrent bacterial infections due to impaired phagocytosis, certain autoimmune conditions requiring increased tolerance pathways (IL-10, TGF-P, FOXP3 / Treg boosting in autoimmunity broadly), immune exhaustion states requiring enhanced cytotoxicity (e.g., chronic viral infections), impaired vaccine responses, and mast cell activation disorders.

[0209] Exemplary inflammatory or autoinfl ammatory diseases include, but are not limited to, systemic lupus erythematosus (SLE; suppression of type I interferon pathway, TLR7 / 9 overactivation), mast cell activation syndrome (MCAS; reduction of mast-cell hyperreactivity pathways including KIT overactivation), rheumatoid arthritis, psoriatic arthritis, ankylosing spondylitis, multiple sclerosis (Thl / Thl7 overactivity), type 1 diabetes (autoimmune 3-cell destruction), celiac disease, inflammatory bowel disease (Crohn’s, ulcerative colitis), Sjogren’s syndrome, systemic sclerosis, mixed connective tissue disease, autoimmune thyroiditis, myasthenia gravis, autoimmune cytopenias, eosinophilic disorders (eosinophilic esophagitis, hypereosinophilic syndrome), severe asthma and allergic diseases (IgE pathway, IL-4 / IL-5 / IL-13 axis), chronic urticaria, hyperactive complement disorders (C3 gain-of-function, factor H deficiency contexts), autoinflammatory syndromes (NLRP3 inflammasome hyperactivation), cytokine storm syndromes (IL-6, IL-1, TNF-a overproduction), vasculitides driven by abnormal immune activation, and graft-versus-host disease.

[0210] For skin rejuvenation, in certain embodiments the composition increases expression or activity of COL1A1, COL3A1, ELN, HAS2, PPARG and CEBPA and / or growth factors such as FGF7 and IGF1 to promote collagen, elastin, hyaluronan and dermal adipose production, while reducing or modulating LOX / LOXL family members, selected MMPs and pro-inflammatory mediators (e.g., IL1B, TNF) to enable remodeling of aged or cross-linked collagen and scar tissue. For hair rejuvenation, in certain embodiments the composition increases or activates WNT pathway effectors (e.g., WNT7A, WNT10A, CTNNB1), KITLG and melanogenesis factors (MITF, TYR, TYRP1), VEGFA and follicular-supportive FGFs and antioxidant defenses (e.g., SOD1, GPX1), and decreases or inhibits negative regulators including DKK1, SFRP1, TGF-P 1 / 2 and FGF5 to promote pigmentation, anagen entry, follicle vascularization, and increased hair density and quality.206678-0001-00WQ

[0211] In some embodiments, compositions for treating a condition associated with mitochondrial dysfunction include, but are not limited to, compositions for mitochondria function enhancement. Exemplary targets for enhancing mitochondria function include, but are not limited to, PGC-la / 0, NRF1, NRF2 / GABP, TFAM, SIRT1 / 3 / 6 / 7, AMPK-LKB1-FOXO axis, PINKl / Parkin mitophagy, OPA1 / MFN1 / MFN2 fusion regulators, DRP1 / MFF fission regulators, POLG / TWNK mtDNA maintenance, NRF2 antioxidant pathways, PPARa / 8 metabolic regulators, COQ / ETC nuclear subunits, mt ribosomal genes, PRDM16 / UCP1 thermogenic regulators, and mitochondrial proteostasis regulators (LONP1, CLPP, HSP60 / 10).

[0212] Exemplary infectious diseases include, but are not limited to, diseases due to infection by HIV-1, Hepatitis B (HBV), Hepatitis C (HCV), Influenza A / B, SARS-CoV-2, RSV, CMV, EBV, HSV-1 / 2, VZV, Dengue, Zika, Chikungunya, West Nile virus, Nipah virus, Hendra virus, Lassa virus, Ebola, Marburg, Monkeypox, Mycobacterium tuberculosis, Streptococcus pneumoniae, Neisseria meningitidis, Haemophilus influenzae type b, Bordetella pertussis, Clostridioides difficile, Bacillus anthracis, Corynebacterium diphtheriae, Clostridium botulinum (toxin-oriented antibody strategies), Plasmodium species (malaria), Trypanosoma cruzi, Leishmania species, Toxoplasma gondii, Candida auris, Aspergillus fumigatus, Cryptococcus neoformans, Histoplasma species, and Coccidioides species, and transmissible spongiform encephalopathies (TSEs), such as Creutzfeldt-Jakob disease (CJD).

[0213] To practice the methods of the invention; the skilled artisan would understand, based on the disclosure provided herein, how to formulate and administer the appropriate composition to a subject. The present invention is not limited to any particular method of administration or treatment regimen.

[0214] The invention encompasses delivery of a delivery vehicle, comprising at least one therapeutic nucleic acid molecule, wherein the delivery vehicle is complexed to a fusogenic peptide or molecule to increase cellular uptake of the delivery vehicle, wherein cellular uptake and release of the therapeutic nucleic acid molecule provides a beneficial or therapeutic effect. In one embodiment, the therapeutic agent is a known or established therapeutic for the disease being treated. In one embodiment, the therapeutic agent boosts the level or expression of a protein or gene product that is under-expressed or absent in a subject having a disease or disorder. In one embodiment, the therapeutic agent decreases the level or expression of a protein or gene product that is over-expressed or present in a subject having a disease or disorder.

[0215] It will be appreciated by one of skill in the art, when armed with the present disclosure including the methods detailed herein, that the invention is not limited to treatment of diseases or disorders that are already established. Particularly, the disease or disorder need not have manifested to the point of detriment to the subject; indeed, the disease or disorder need not be detected in a subject before treatment is administered. That is, significant signs or symptoms of diseases or disorders do not have to occur before the present invention may provide benefit. Therefore, the present invention includes a method for preventing diseases or disorders, in that a composition, as discussed previously elsewhere herein, can be administered to a subject prior to the onset of diseases or disorders, thereby preventing diseases or disorders.

[0216] One of skill in the art, when armed with the disclosure herein, would appreciate that the prevention of a disease or disorder, encompasses administering to a subject a composition as a preventative measure against the development of, or progression of, a disease or disorder.

[0217] One of skill in the art will appreciate that the compositions of the invention can be administered singly or in any combination. Further, the compositions of the invention can be administered singly or in any combination in a temporal sense, in that they may be administered concurrently, or before, and / or after each other. One of ordinary skill in the art will appreciate, based on the disclosure provided herein, that the compositions of the invention can be used to prevent or to treat a disease or disorder, and that a composition can be used alone or in any combination with another composition to affect a therapeutic result. In various embodiments, any of the compositions of the invention described herein can be administered alone or in combination with other modulators of other molecules associated with diseases or disorders.

[0218] In one embodiment, the invention includes a method comprising administering a combination of compositions described herein. In certain embodiments, the method has an additive effect, wherein the overall effect of administering a combination of compositions is approximately equal to the sum of the effects of administering each individual inhibitor. In other embodiments, the method has a synergistic effect, wherein the overall effect of administering a combination of compositions is greater than the sum of the effects of administering each individual composition.

[0219] The method comprises administering a combination of composition in any suitable ratio. For example, in one embodiment, the method comprises administering twoindividual compositions at a 1:1 ratio. However, the method is not limited to any particular ratio. Rather any ratio that is shown to be effective is encompassed.Pharmaceutical Compositions

[0220] The formulations of the pharmaceutical compositions described herein may be prepared by any method known or hereafter developed in the art of pharmacology. In general, such preparatory methods include the step of bringing the active ingredient into association with a carrier or one or more other accessory ingredients, and then, if necessary or desirable, shaping or packaging the product into a desired single- or multi-dose unit.

[0221] Although the description of pharmaceutical compositions provided herein are principally directed to pharmaceutical compositions which are suitable for ethical administration to humans, it will be understood by the skilled artisan that such compositions are generally suitable for administration to animals of all sorts. Modification of pharmaceutical compositions suitable for administration to humans in order to render the compositions suitable for administration to various animals is well understood, and the ordinarily skilled veterinary pharmacologist can design and perform such modification with merely ordinary, if any, experimentation. Subjects to which administration of the pharmaceutical compositions of the invention is contemplated include, but are not limited to, humans and other primates, mammals including commercially relevant mammals such as non-human primates, cattle, pigs, horses, sheep, cats, and dogs.

[0222] Pharmaceutical compositions that are useful in the methods of the invention may be prepared, packaged, or sold in formulations suitable for ophthalmic, oral, rectal, vaginal, parenteral, topical, pulmonary, intranasal, buccal, intravenous, intracerebroventricular, intradermal, intramuscular, or another route of administration. Other contemplated formulations include projected nanoparticles, liposomal preparations, resealed erythrocytes containing the active ingredient, and immunogenic-based formulations.

[0223] In certain embodiments, the composition of the invention is administered by inhalation. In certain embodiments, the invention is conveniently delivered from an insufflator, nebulizer or a pressurized pack or other convenient means of delivering an aerosol spray.Pressurized packs may comprise a suitable propellant such as dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In thecase of a pressurized aerosol, the dosage unit may be determined by providing a valve to deliver a metered amount.

[0224] A pharmaceutical composition of the invention may be prepared, packaged, or sold in bulk, as a single unit dose, or as a plurality of single unit doses. As used herein, a “unit dose” is discrete amount of the pharmaceutical composition comprising a predetermined amount of the active ingredient. The amount of the active ingredient is generally equal to the dosage of the active ingredient which would be administered to a subject or a convenient fraction of such a dosage such as, for example, one-half or one-third of such a dosage.

[0225] The relative amounts of the active ingredient, the pharmaceutically acceptable carrier, and any additional ingredients in a pharmaceutical composition of the invention will vary, depending upon the identity, size, and condition of the subject treated and further depending upon the route by which the composition is to be administered. By way of example, the composition may comprise between 0.1% and 100% (w / w) active ingredient.

[0226] In addition to the active ingredient, a pharmaceutical composition of the invention may further comprise one or more additional pharmaceutically active agents.

[0227] In addition to the active ingredient, a pharmaceutical composition of the invention may further comprise one or more additional adjuvants. Exemplary adjuvants include, but are not limited to, aluminum-based adjuvant and monophosphoryl lipid A.

[0228] Controlled- or sustained-release formulations of a pharmaceutical composition of the invention may be made using conventional technology.

[0229] As used herein, “parenteral administration” of a pharmaceutical composition includes any route of administration characterized by physical breaching of a tissue of a subject and administration of the pharmaceutical composition through the breach in the tissue. Parenteral administration thus includes, but is not limited to, administration of a pharmaceutical composition by injection of the composition, by application of the composition through a surgical incision, by application of the composition through a tissue-penetrating non-surgical wound, and the like. In particular, parenteral administration is contemplated to include, but is not limited to, inhalation, intraocular, intravitreal, subretinal, suprachoroidal, subcutaneous, intraperitoneal, intramuscular, intradermal, intrasternal, intratumoral, intravenous, intracerebroventricular injections and kidney dialytic infusion techniques.

[0230] Formulations of a pharmaceutical composition suitable for parenteral administration comprise the active ingredient combined with a pharmaceutically acceptable carrier, such as sterile water or sterile isotonic saline. Such formulations may be prepared, packaged, or sold in a form suitable for bolus administration or for continuous administration. Injectable formulations may be prepared, packaged, or sold in unit dosage form, such as in ampules or in multi-dose containers containing a preservative. Formulations for parenteral administration include, but are not limited to, suspensions, solutions, emulsions in oily or aqueous vehicles, pastes, and implantable sustained-release or biodegradable formulations. Such formulations may further comprise one or more additional ingredients including, but not limited to, suspending, stabilizing, or dispersing agents. In one embodiment of a formulation for parenteral administration, the active ingredient is provided in dry (i.e., powder or granular) form for reconstitution with a suitable vehicle (e g., sterile pyrogen-free water) prior to parenteral administration of the reconstituted composition.

[0231] The pharmaceutical compositions may be prepared, packaged, or sold in the form of a sterile injectable aqueous or oily suspension or solution. This suspension or solution may be formulated according to the known art, and may comprise, in addition to the active ingredient, additional ingredients such as dispersing agents, wetting agents, or suspending agents described herein. Such sterile injectable formulations may be prepared using a non-toxicparenterally-acceptable diluent or solvent, such as water or 1,3-butane diol, for example. Other acceptable diluents and solvents include, but are not limited to, Ringer's solution, isotonic sodium chloride solution, and fixed oils such as synthetic mono- or di-glycerides. Other parentally-administrable formulations which are useful include those which comprise the active ingredient in microcrystalline form, in a liposomal preparation, or as a component of biodegradable polymer systems. Compositions for sustained release or implantation may comprise pharmaceutically acceptable polymeric or hydrophobic materials such as an emulsion, an ion exchange resin, a sparingly soluble polymer, or a sparingly soluble salt.

[0232] A pharmaceutical composition of the invention may be prepared, packaged, or sold in a formulation suitable for pulmonary administration via the buccal cavity. Such a formulation may comprise dry particles which comprise the active ingredient and which have a diameter in the range from about 0.5 to about 7 nanometers, and preferably from about 1 to about 6 nanometers. Such compositions are conveniently in the form of dry powders for administrationusing a device comprising a dry powder reservoir to which a stream of propellant may be directed to disperse the powder or using a self-propelling solvent / powder-dispensing container such as a device comprising the active ingredient dissolved or suspended in a low-boiling propellant in a sealed container. Preferably, such powders comprise particles wherein at least 98% of the particles by weight have a diameter greater than 0.5 nanometers and at least 95% of the particles by number have a diameter less than 7 nanometers. More preferably, at least 95% of the particles by weight have a diameter greater than 1 nanometer and at least 90% of the particles by number have a diameter less than 6 nanometers. Dry powder compositions preferably include a solid fine powder diluent such as sugar and are conveniently provided in a unit dose form.

[0233] Low boiling propellants generally include liquid propellants having a boiling point of below 65°F at atmospheric pressure. Generally the propellant may constitute 50 to 99.9% (w / w) of the composition, and the active ingredient may constitute 0.1 to 20% (w / w) of the composition. The propellant may further comprise additional ingredients such as a liquid non-ionic or solid anionic surfactant or a solid diluent (preferably having a particle size of the same order as particles comprising the active ingredient).

[0234] As used herein, “additional ingredients” include, but are not limited to, one or more of the following: excipients; surface active agents; dispersing agents; inert diluents; granulating and disintegrating agents; binding agents; lubricating agents; sweetening agents; flavoring agents; coloring agents; preservatives; physiologically degradable compositions such as gelatin; aqueous vehicles and solvents; oily vehicles and solvents; suspending agents; dispersing or wetting agents; emulsifying agents, demulcents; buffers; salts; thickening agents; fillers; emulsifying agents; antioxidants; antibiotics; antifungal agents; stabilizing agents; and pharmaceutically acceptable polymeric or hydrophobic materials. Other “additional ingredients” which may be included in the pharmaceutical compositions of the invention are known in the art and described, for example in Remington's Pharmaceutical Sciences (1985, Genaro, ed., Mack Publishing Co., Easton, PA), which is incorporated herein by reference.EXPERIMENTAL EXAMPLES

[0235] The invention is further described in detail by reference to the following experimental examples. These examples are provided for purposes of illustration only, and are not intended to be limiting unless otherwise specified. Thus, the invention should in no way be206678-0001-00WQconstrued as being limited to the following examples, but rather should be construed to encompass any and all variations which become evident as a result of the teaching provided herein.

[0236] Without further description, it is believed that one of ordinary skill in the art can, using the preceding description and the following illustrative examples, make and utilize the present invention and practice the claimed methods. The following working examples therefore are not to be construed as limiting in any way for the remainder of the disclosure.Example 1: Engineering Chimeric Fusogens

[0237] Fusogenic proteins were designed as chimeric constructs derived from the FAST protein family, rationally designed and screened to maximize fusion activity while minimizing any unnecessary elements. In embodiments of the invention, specific ectodomain, transmembrane domain (TMD), and endodomain (cytoplasmic tail) segments from different FAST proteins are recombined to create synthetic fusion proteins. This invention discloses a library of chimeric FAST variants, where the N-terminal ectodomain of one FAST protein was fused to the transmembrane region of another, or the ectodomain+ transmembrane domains (E+TMD) of one combined with the C-terminal tail of another, and so on. Exemplary source proteins for these chimeras include pl4 and p 15 FAST proteins (from reptilian and baboon reoviruses), as well as other homologs such as plO (from avian reovirus) and p22 (from aquareovirus), among others (Table 1 and Table 2). These particular FAST proteins were chosen to sample a diverse range of fusion peptide structures and cytosolic tail motifs.Table 1: Ecto + Transmembraine Domain (E+TMD) SequencesE+TMD No. SEQIDNO: Sequence1 248 MGSGPSNFVNHAPGEAIVTGLEKGADKVAGTISHTIWEVIAGLVALLTFLAFGFWLF2 252 MGNGPSNFVNHSPAEAILTGLDKNTHSITSTFTNGIKELLVGLLVLILFFVAAGAACWYW 3 249 MGSGPSNFVNHASGEAIISGLSDQTNRLGSLLSQNVYNIIYFFVIGGLILSAGYGLYKYC 4 253 MGNGPSNFVNHSPAETIISGLDKGADKVAGTVSNTIWEVIAGLVALLTFLAFGFWLF5 254 MGNGPSNFVNHSPAETIISGLDKGADKVAGTVSNTIWEWGGVLLLLFLIAVGFGLY6 255 MGSGPSNFVNHASGEAIISGLSDQTNRLGSLLSQNIWEVIAGLVALLTFLAFGFWLF 7 256 MGSGPSNFVNHASGEAIISGLSDQTNRLGSLLSQNVYNIIYFFVIGGLILSAGYGLY8 257 MGNGPSNFVNHSPAETIISGLDKGADKVAGTVSNTVYNIIYFFVIGGLILSAGYGLY9 250 MGSGPSNFVNHASGEAIISGLSDQTNRLGSLLSQNIWEWGGVLLLLFLIAVGFGLY 10 258 MLRMPPGSCNGATAVFGNVHCQAAQNTAGGDLQATSSIIAYWPYLAAGGGFLLIVIIFALLYCC11 251 MADGACNHATSIFGAVYCQISQNIAHGNIDSYTSWTSYLPPILGGGFGLIVLLVLWLIVYCC206678-0001-00WQTable 2: Endodomain SequencesEndodomain Endodomain SEQ ID NO: SequenceSource No.1 260 KYLQKRRERRRQLTEFQKRYLRNSYRLSEIQRPISQHEYEDPYEPPSRRKPPPPPY STYVNIDNVSAI2 266 KYCQKKRDKARQLTEFQRRYLRNSYRLSDLHPTAVPPVYEDPYEPPVRKKAPPPP YNTYVNINDVSSFN3 262 RYAKGRREKKRELTEFQKRFLRNSYRLSQRRSLTQSPDYEEPTEYGITKPLPPPPYA TYINI4 267 YWRKRRAKRRRLTKYQIRFLNDFSRSARLNQTPIPVASKRETWPQKTRSVSPPPYT ANYVNVNDYDEAESAPTFRRY5 268 YCKYKQRRAKNLTRQLSRELIDLNRKIEHISGGKIPATKPSAPRYTPPCYKEPIYNEV CEGGFYGNCpl4 Endodomain6 265 KYLQKRRERRRQLTEFQKRYLRNSYRLSDLHPTAVPPVYEDPYEPPVRKKAPPPPY NTYVNINDVSSFN7 261 KYLQKRRERRRQLTEFQKRYLRNSYRLSQRRSLTQSPDYEEPTEYGITKPLPPPPY ATYINI8 259 KYLQKRRERRRQLTEFQKRYLRNSYRLSDLHPTAVPPVYEDPTEYGITKPLPPPPY ATYINI9 269 KYLQKRRERRRQLTEFQKRYLRSYRLSQRRSLTQSPDYEEEPYEPPSRRKPPPPPP PPPYSTYVNIDNVSAI10 270 RYLKEKRDRKRELSEYQRRFLRDSYRLSEVHKPISLHEYEDPYEPPSRKKAPPPPY STYVNIDNVSAI11 271 HSGRIPSISRRLDVLRDTRSASEYKVRSNRNPKSRVRRVSISDSSDSSSLSDLELS RHRSHPLAHSFRPESYSQRPHSPSQAQSSIILPLVPVHSRTSLDDGVIRSQPSRY QGPHQQFEDWLQQAHLLRPGDVSRDTNPFR12 272 HSGRFPGLSRRLDVLGGSGSTPKHSLRSHRHPKPRVHRVSFSDSSDSSDISDLE LPRHGSHPLAHSFRPEVDRHRPRPSTQVQQTSFIPLVPLRSGSSLDDGIVRSQPS RDSRPHEQFEDWLQQAHLLRPGRVSGSTNPFT13 273 HSGRIPSLSRRLDVLGSQGSAPKHTLRSHRIPKPRVRRLSFSDSSDSSGISDLELP GHRSHPLAHPFRPEVDRNRPRPATPVQSSNLIPLIHLRDGTGLEDGWCTQPPR DPLPHHQFEDWLQQAHLLRPGEVSRNTNPFL14 274 HSGRLPSISRRLDVLRDSRSTPEHKVRRDRFTKPRIHRPRLSSTSDSSDLTDLELS WDGFDPLAYPHWSAVNSPRARPAPSVHSQGLIPLVTLQPRAGLDNGWHSQPS RSPRPHQRFEDWLQQAHLLRPGDVPRDTNPFRRPYPRRDTEH15 264 YTGRLPKPRCLDVWPTTRTPPEYDVSPTVTSQPNRGYASVHASRYTRTGTDMELL QLGSANMADLGLCSKTGSASPPAYQPINAAQSPPPTYGSDDDAYGSGRLR p22 Endodomain 16 275 HSGRFPGISRRLDVLGSQGSTPKHTLRRHRFTKSRIHRPRFSDTSDSSDISDLELP WDRFHPLAHPFRSEVNRQRPRSASPAQSQNFLPLVHVHDRASLDNGVIHTQPS RYPGPHERFEDWLQQAHLLRPGDVSRNTNPFR17 276 HSGRIPGISRRLDVLRDQGSAPEHKVRRNRHPKSRIRRPRISDTSDSSSISDLELP RHRFHPLAHSFRPEVDRQRPHSASPAQSTNFLPLVHVHDRASLDDGVIRTQPSR YQGPHEQFEDWLQQAHLLRPGDVSRNTNPFR18 277 HSGRFPGISRRLDVLGSQGSTPKHTLRRHRFTKSRIHRPRFSDTSDSSDISDLELP WDRFHPLAHPFRSEVNRQRPRSASPAQSQNFLPLVHVHDRASLDNGVIHTQPS RYPGPHERFEDWLQQAHLLRPGDVSRNTNPFRRPYPRRDTEH19 278 HSGRIPGISRRLDVLRDQGSAPEHKVRRNRHPKSRIRRPRISDTSDSSSISDLELP RHRFHPLAHSFRPEVDRQRARPAPSVHSTGFLPLVHVHDRASLDDGVIRTQPSR YQGPHEQFEDWLQQAHLLRPGDVSRNTNPFR20 279 YTGRLPKPRCLDVWPTTRTPPEYDVSPTVTSQPNRGYASVHASRYTRTGTDMELL QLGSANMADLGLCSKTGSASPPAYQPINAAQSPPPTYGSDDDAYGSGRLRSQP SRYQGPHQQFEDWLQQAHLLRPGDVSRDTNPFRpl4 Endodomain 22 280 KAKVKADAARSVFHRELVALSSGKHNAMAPPYDV23 263 NHSKILSAVKATDSAVTTLLRDVAPANPDPVQWp!5 Endodomain 29 281 KLLQWYNRKSKNKKRKEQIREQIELGLLSYGAGVASLPLLNVIAHNPGSVISATPIYKGPCTGVPNSRLLQITSGTAEENTRILNHDGRNPDGSINV

[0238] Chimeric constructs were designed combining each of the E+TMD constructs of Table 1 with each of the Endodomain constructs of Table 2. The chimeric fusion proteins were provided designations depending on the combination of E+TMD and Endodomain. For example, for a fusion construct named ##_1.8 the construct is a fusion of E+TMD domain No. 1 and Endodomain No. 8 (Fig. 36).

[0239] Each chimeric construct was expressed and evaluated for fusogenic activity in vitro. A cell-cell fusion assay (syncytium formation assay) was used as a proxy for fusion activity: cells expressing the candidate fusogen were co-cultured with indicator cells, and the formation of multinucleated syncytia was quantified. Many of the engineered chimeras successfully induced cell-cell fusion, confirming that they are functional fusogens. Notably, several chimeric variants outperformed the native FAST proteins in fusion efficiency. For example, peptide 1.18 1.8 exhibited a markedly higher fusion rate than wild-type. This particular variant induced large syncytia in cell culture, indicating an enhanced ability to drive membrane merger.

[0240] After multiple rounds of screening, the results demonstrate that strategic motif substitutions and domain shuffling among FAST proteins can produce a synthetic fusogen with greater activity than naturally occurring ones. Importantly, some non-essential motifs present in native FAST proteins may be removed in the chimeras without much loss of function.Experiments of Chimeric FAST Proteins

[0241] Plasmid DNAs (pDNAs) encoding variants of fusogenic proteins (fusogen) using pTwist cDNA 3.1 backbone by Twist Biosciences were introduced into HEK293T cells using Lipofectamine 3000, with each well in the 24-well plate. Additionally, Efla-eGFP Nanoplasmid DNA by Aldevron was co-formulated with fusogen pDNA at two different dosing levels. In the first experiment 1 (Exp#l), pDNAs encoding fusogens was administered at 500 ng per well and the eGFP pDNA was administered at 100 ng per well. In the second and third experiments (Exp#2 and Exp#3), the doses for both plasmid DNAs encoding fusogen and eGFP were administered at 500 ng per well each where eGFP dose was increased to allow for better visualization of cell fusion.

[0242] Cells were imaged using the M7000 EVOS microscope system from Thermo Fisher. Images were captured using the eGFP fdter at 10X objective and at 40X objective on the scope respectively. The images were collected individually and merge images were also created to allow full dynamic visualization of the transfection morphology changes. Exemplary images for a subset of fusion proteins are provided.Fusion of cells expressing Fusogenic Protein 1.18 1.8.

[0243] Fusogenic Protein 1.18 1.8 demonstrated high fusion activity as shown in Fig. 1-5. Fig. 1 Left (Fig. IL) shows large eGFP diffusion due to cell fusion while Fig. 1 Right (Fig. 1R) is light microscopy of the same cells, both at 27th hour (27H) using lOx objective. Fig. 2 shows 24H vs 11H hour cell morphology of the same well location. Large areas of diffused eGFP were observed as well as cell clearing Fig. 2L, as compared to Fig. 2R 13 hours earlier. After cell-cell fusion, the cell membranes start to disintegrate, causing cell death and clearing. Therefore, the difference between Fig. 2L and Fig. 2R is a key ranking parameter, the larger the cell clearing area is, the more potent the fusogenic activity is. Fig. 3 shows two separate experiments at 12H and 5H at 40x objective. Large patches of cell membrane fusion and disintegration, cell death and clearing happened at 12H, and syncytia formation took place as early as 5H. Fig. 4 shows 11H and 9H of the same well location, where syncytia formation is visible. Fig. 5 shows 7H and 5H of the same well location, where syncytia formation was visible as early as 5H.Fusion of cells expressing Fusogenic Protein 1.3 3.1.

[0244] Fusogenic Protein 1.3 3.1 demonstrated high fusion activity as shown in Fig. 6-9. Fig. 6L shows large eGFP diffusion due to cell fusion while Fig. R is light microscopy of the same cells, both at 27H using lOx objective. Fig. 7 shows 24H vs 11H hour cell morphorlogy of the same well location. Large areas of diffused eGFP were observed as well as cell clearing Fig.7L, as compared to Fig. 7R 13 hours earlier. Fig. 8 shows two separate experiments at 12H and 5H at 40x objective. Large patches of cell membrane fusion and disintegration, cell death and clearing happened at 12H, and syncytia formation took place as early as 5H. Fig. 9 shows 11H and 9H of the same well location, where syncytia formation is visible.Fusion of cells expressing Fusogenic Protein 1.45 11.23.

[0245] Fusogenic Protein 1.45 11.23 demonstrated moderate fusion activity as shown in Fig. 10-12. Fig. 10L shows moderate eGFP diffusion due to cell fusion while Fig. R is light206678-0001-00WQmicroscopy of the same cells, both at 27H using lOx objective. Fig. 11 shows 24H vs 11H hour cell morphology of the same well location. Moderate areas of diffused eGFP were observed as well as cell clearing Fig. 1 IL, as compared to Fig. 11R 13 hours earlier. Fig. 12 shows two separate experiments at 12H and 5H at 40x objective. Large patches of cell membrane fusion and disintegration, cell death and clearing happened at 12H, and syncytia formation took place as early as 5H.

[0246] The method to interpret and rank the fusogenic activities are similar for the other figures. Fig. 13-32 showed various levels of fusogenic activities where 1.17_1.7, 1.13 1.3 showed high level of fusogenic activities while 1.9 9.1, 1.25_1.15, 1.16 1.6 showed moderate level of fusogenic activity.

[0247] As controls, pl4 has low level of fusogenic activity (Fig. 33) while eGFP alone indicates no activity (Fig. 34-35).Example 2: Development of fusogenic LNPs.

[0248] The present invention provides a new class of LNP-based delivery vehicles that fuse directly with target cell membranes to release their cargo. By incorporating engineered chimeric FAST proteins, these fusogenic LNPs overcome the historical limitations of nanoparticle delivery - namely, endosomal trapping and immunotoxicity. The minimal fusogens devised here combine the best features of viral fusion proteins (efficient membrane merger) with enhanced fusogenic potency.

[0249] All modifications, variations, and combinations of the compositions and methods described above that would be apparent to one skilled in the art are intended to be encompassed within the scope of this invention. The examples and embodiments are illustrative and not limiting, demonstrating the principles and advantages of fusogen-enhanced LNP delivery.

[0250] Such a platform technology is particularly well suited for diseases requiring longterm or durable protein expression. Key areas of application include: genetic disorders such as cystic fibrosis (CF) and Duchenne muscular dystrophy, infectious diseases that require durable production of protective antibodies or therapeutic cytokines, oncology that requires sustained expression of immune-modulating proteins, such as checkpoint inhibitors, antibodies or engineered cytokines, and anti-aging therapies that boost certain protein / peptide expressions that have declined with aging, contributing the aging process.

[0251] Nanoparticles were designed comprising the fusogenic peptides. Experiments were designed to demonstrate the efficacy of delivery of therapeutic agents via the fusogenic nanoparticles.

[0252] The lead formulation integrates several lipids with a fusogenic protein to achieve efficient syncytium formation at the plasma membrane. It is well suited for all routes of administration including but not limited to IV, IM and nebulization. Exemplary LNP formulations are provided in Table 3. Additionally, lipid formulation ratio can be optimized for each route of administration, balancing various constraints and requirements, such as nebulization to target lung epithelial cells.Table 3: Lipid Nanoparticle FormulationsMolarComponent RationaleRatio (%)Ionizable Primary ionizable lipid for DNA encapsulation and reductive 30-50Lipids release in cytoplasm.Promotes membrane destabilization and fusion, aiding DOPE 15-30syncytium formation.Stabilizes nanoparticle structure during nebulization and Cholesterol 15-30enhances bilayer integrity.Enhances colloidal stability and facilitates penetration through PEG 0-5lung mucus.Fusogenic Optimized for direct syncytium formation, minimizing protein- 0.1-10Protein to-lipid ratio to maintain particle integrity.Ionizable Lipids

[0253] Ionizable lipids provide positive charge at low PH condition to complex with and compact the gene delivery payload such as nucleic acids (e.g., plasmid DNA, messenger RNA, or proteins and peptides, or small molecules). At physiological condition, DODAP is neutral therefore avoiding the potential toxicity typically seen in cationic lipids that remain positively charged even in the physiological condition. Notable examples of ionizable lipids include DODAP, DLin-MC3-DMA, SM-102, and ALC-0315, C12-200, cKK-E12, DODMA, and Ethylenediamine-based Cysteinyl Oleoyl Lipid (ECO).Cholesterol:

[0254] Cholesterol strengthens the lipid bilayer, improving LNP resilience under shear forces and droplet formation during nebulization. A cholesterol content of 20-30% ensures balance between bilayer rigidity and flexibility, preventing particle disruption. Cholesterol can be left out of the formulation if nebulization is not required or if liver targeting is not required.PEG:

[0255] PEG such as DSPE-PEG2000 enhances mucosal penetration, overcoming the protective mucus barrier in the lungs, and maintaining LNP dispersibility.Formulation

[0256] There are several different formulation methods. An example method includes the following steps:1. Organic Phase Preparation:• Dissolve lipids (DODAP, DOPE, cholesterol, and DSPE-PEG2000) in ethanol or another organic solvent at concentrations optimized for microfluidic mixing.• Add the fusogenic protein to the organic phase to integrate it into the lipid bilayer. 2. Aqueous Phase Preparation:• Prepare an acetate or Tris-HCl with sucrose buffer at pH 4.0-5, containing the plasmid DNA (e.g., encoding CFTR for CF therapy).• DNA concentration: Typically 0.5-1 mg / mL in the aqueous phase.3. Microfluidic Mixing:• Use a microfluidic device (e.g., NanoAssemblr Ignite from Cytiva, Sunscreen or Sunshine from Unchain Labs) to achieve precise particle size and monodispersity. • Parameters:• Flow Rate Ratio (Aqueous: Organic): 3:1.• Total Flow Rate: ~12 mL / min.• Lipid-to-DNA ratio (w / w): 12:1 -24:1.4. Post-Processing:• Dialysis: Dialyze the resulting LNP suspension against PBS (or another physiological buffer) using an 8,000 MWCO membrane for 2-4 hours to remove residual ethanol.• Concentration: Concentrate LNPs using ultrafiltration (e.g., 100 kDa ultrafiltration devices).• Sterilization: Filter the final formulation through a 0.2 pm sterile filter.Fusogen-Enhanced LNPs

[0257] The top-performing chimeric fusogens are incorporated into LNP formulations containing payloads (e.g. a luciferase-encoding mRNA or plasmid). These fusogen-functionalized LNPs are applied to target cells in vitro, and their delivery efficiency is measured in comparison to conventional LNPs lacking a fusogen. The fusogenic LNPs consistently achieved much higher levels of reporter expression, indicating successful direct cytosolic delivery. In some experiments, nearly the entire cell population expressed the reporter after treatment with a fusogen-LNP, whereas LNPs without fusogen produced only sparse expression due to the endosomal barrier.

[0258] Incorporating a FAST fusogen into an LNP dramatically increases intracellular delivery of nucleic acids. Adding a FAST protein to a lipid nanoparticle formulation greatly boosted mRNA and plasmid DNA expression in target cells, compared to a control LNP without the fusogen. This result demonstrates the efficacy of direct fusion-mediated delivery, as the fusogen-enhanced LNP (right) bypasses endosomal trapping and releases its genetic cargo directly into the cytosol, leading to high gene expression.

[0259] The fusogen-enhanced LNPs offer substantial advantages over conventional LNP delivery systems.

[0260] High Delivery Efficiency: Direct fusion with the plasma membrane releases the full dose of cargo into the cytosol. This circumvents the endosomal escape bottleneck where <2% of material might otherwise reach the cytosol. The improved efficiency means therapeutic effect can be achieved at lower doses.

[0261] Bypass of Endosomal Pathway: Since the feLNP avoids endosomal uptake, it avoids exposure to degradative enzymes and low pH. There is no need for the nanoparticle to disrupt an endosome from within. Consequently, cellular health is better preserved (no endosomal rupture damage), and the risk of triggering endosomal toll-like receptors orinflammasome pathways is minimized. In other words, the delivery is less immunostimulatory and less likely to cause inflammation.

[0262] Lower Toxicity and Improved Safety: Because more of the payload is utilized and little is wasted in endosomes, there is less immune sensing and clearance, a smaller quantity of nanoparticle can achieve the desired effect. This can reduce dose-dependent toxicities. Moreover, the fusion mechanism itself is relatively gentle to cells (transient membrane fusion as opposed to particle uptake and endosome bursting).

[0263] Reduced Immunogenicity: The fusogenic proteins used are small and derived from non -pathogenic virus elements, and they operate by facilitating membrane fusion rather than by creating pores via endosomal membrane rupture. The feLNP does not heavily engage the innate immune system - for instance, it does not leave behind DNA or RNA in endosomes where nucleic acid-sensing TLRs reside. A fusogen-LNP therefore elicits a more muted immune response. This feature allows for repeated administration of the therapy, a key requirement in treating chronic conditions.

[0264] Broad Therapeutic Applicability: This delivery strategy is platform-agnostic with respect to the therapeutic cargo. It can improve the intracellular delivery of messenger RNAs, DNA (plasmid, linear, doggybone, or other forms of DNA), gene editing plasmids, siRNA or antisense oligos, protein therapeutics (by encoding them in mRNA), etc. The technology is particularly beneficial for diseases that require gene delivery to cells that are hard to transfect or where only partial delivery was achieved before. For example, in cystic fibrosis, airway epithelial cells need efficient delivery of functional CFTR gene copies; a fusogen-LNP could greatly enhance transfection of these cells, potentially improving therapeutic outcomes.Likewise, muscle cells, neurons, skin, and other targets that are sensitive or require repeated dosing (such as in muscular dystrophy or neurodegenerative diseases) could be treated more effectively.Example 3: Delivery of Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Protein

[0265] Cystic fibrosis (CF) is one of the most prevalent genetic diseases in the United States (US). CF arises from mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, which encodes a protein critical for the proper function of severalepithelial tissues. The absence of functional CFTR protein disrupts the barrier function of airway epithelial cells, leading to chronic infections, airway remodeling, and progressive lung failure. While Trikafta, a breakthrough prescription therapy, has significantly improved the quality of life and lifespan for approximately 90% of CF patients, critical gaps remain for the remaining 10%. These gaps are due to the following factors:Specific CFTR Variants: Certain mutations do not respond to modulator therapies.Side Effects: Many patients experience intolerable reactions to existing treatments. Access Limitations: Geographic and economic barriers prevent universal access to care.

[0266] Gene therapy provides a transformative approach to address these unmet needs by directly targeting the lungs, the primary organ responsible for morbidity and mortality in CF patients. By introducing functional copies of the CFTR gene, gene therapy has the potential to modify the course of CF disease and provide long-term, effective treatment for all patients, including those who cannot benefit from current small-molecule therapies. Beyond CF, gene therapy holds immense potential to treat a broad range of diseases, including genetic disorders, certain cancers, neurological conditions, and acquired diseases such as cardiovascular or infectious diseases, by addressing the underlying molecular or cellular defects driving these conditions.

[0267] Experiments have been designed to demonstrate that the novel non-viral fusogenically enhanced lipid nanoparticles (feLNP) can effectively delivery nucleic acid-based drug substances encoding therapeutic proteins, polypeptides, peptides to treat a wide range of diseases including but not limited to the human wild-type Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) protein.

[0268] The drug substance encoding CFTR is delivered via nebulization to lungs of CF patients. The target patient population could be 100% of the CF patients since the drug substance allows for the expression full-length wild-type CFTR protein. Specifically, the initial goal is to develop, optimize, and express full-length nanoplasmid DNA-encoded CFTR gene constructs expressing wild-type CFTR protein and to use a novel non-viral enhanced lipid nanoparticle (feLNP) to deliver to hBE cells grown at air-liquid interface (ALI) for therapeutically effective and sustained expression. The feLNP delivery method circumvents major limitations in the LNP field.

[0269] The turnkey technology platform is designed to enable the safe and effective delivery and sustained expression of therapeutic transgenes encoded by DNA or RNA, targeting diseases including but not limited to CF. This platform integrates three key components:

[0270] Highly optimized DNA or RNA constructs for optimized transgene expression. Initially, nanoplasmid DNA construct (npDNA) is engineered to contain the full-length CFTR coding region with various optimizations, built on Aldevron’s nanoplasmid backbone.

[0271] A nanoplasmid backbone from Aldevron that minimizes immunogenicity and overhead. The nanoplasmid backbone, which incorporates several cutting-edge features. CpG-depletion minimizes immunogenicity for safer, more efficient in vivo applications. Its compact size (500 bp bacterial region) reduces payload overhead. The antibiotic-free RNA-OUT selection system eliminates traditional antibiotic-resistance genes, simplifying regulatory compliance. The Nanoplasmid has been used in several phase 1 / 2 clinical trials.

[0272] A Fusogenically Enhanced Lipid Nanoparticle (feLNP) that uses direct lipid-cell fusion-based entry directly into the cytoplasm and bypasses endosomal entry and achieves superior delivery efficiency. This innovative delivery approach bypasses the need for endosomal escape, achieving higher transfection efficiency and reducing toxicity and immunogenicity risks associated with immune activation.Nebulize npDNA / feLNP nanoparticles

[0273] feLNP particles are aerosolized using a vibrating mesh nebulizer. npDNA / feLNP nanoparticles, nanoplasmid DNA (npDNA) containing the sequence-optimized coding region for CFTR followed by two tandem 2A “self-cleaving” peptides and eGFP is obtained from Aldevron. npDNA is then encapsulated into different feLNP formulations using microfluidic process equipment such as benchtop Nanoassmblr (Cytiva).

[0274] Formulations with particles sizes < 200 nm and encapsulation efficiencies of > 60 % is aerosolized. Single-use Aerogen Solo nebulizers are used for delivery of lx PBS to confirm the specified flow rate range. In a chemical hood, 500 mL of CFTR-eGFP npDNA / feLNP formulations at 1 mg / mL and 0.5 mg / mL is aerosolized through a Aerogen Solo nebulizer. Time for nebulization of the entire content is recorded and flow rate calculated. Nebulized material is collected in a 50 mL conical tube. Particle size and encapsulation efficiency post-nebulization ismeasured and compared to values pre-nebulization. npDNA / feLNP formulations showing values within 20 % range is tested in cells.

[0275] npDNA / feLNPs that retain their particles sizes and encapsulation efficiencies within 20% are identified when comparing pre- and post- nebulization values and are selected for further development.Aerosolized DNA / feLNP formulations efficiently transfect CFF-16HBEge CFTR G542X cells.

[0276] Immortalized human bronchial epithelial cells that have been gene edited at the endogenous CFTR locus (CFF-16HBEge CFTR G542X cells (CFFT lab, see LOS) grown in HTS transwell-24 plates and maintained at ALI) were contacted with 200 mL of the aerosolized CFTR-eGFP npDNA / feLNPs formulations using a Aerogen Solo nebulizer (Aerogen). Six transwells are nebulized with each formulation. Cells are washed with lx PBS at 4 h posttransfection to remove any material remaining in the media. At 48 h post-transfection two replicates for each sample are processed for one of the following assays: a) cell lysate for western-blot (WB); b) total RNA extraction for RT-qPCR; c) basal media collection for LDH assay; and d) eGFP signal for transfection efficiency. Cells is lysed in NETN buffer (10 mM Tris-HCl, pH 7.5, 150mMNaCl, 5 mMEDTA, 1% [v / v] Triton X-100, IX Protease Inhibitor Cocktail). Proteins is separated on Tris-Acetate gels (Thermo Scientific) and transferred to Immobilon-P PVDF membranes (Millipore-Sigma). Membranes is probed with anti-CFTR antibody-596 (CFF / UNC, Chapel Hill) and anti-eGFP (Invitrogen) antibodies. Vinculin is used as internal reference (anti -vinculin, Cell Signaling). Signal is developed with ECL Western Blotting Substrate. Densitometry of bands is performed, CFTR and eGFP signals is normalized to Vinculin signal to assess the relative expression of CFTR and eGFP for each sample. The CFTR protein undergoes a series of glycosylation steps during its synthesis. In fact, for proper CFTR protein folding glycosylation is a pre-requisite. In western-blot assays, two main bands are usually observed for CFTR, bands B and C. Band B has a lower molecular weight than band C corresponding to the “core glycosylated” and the “fully glycosylated” forms, respectively. Fully glycosylated forms suggest that CFTR if properly folded. Therefore, ratio of normalized CFTR bands B (core-glycosylated) and C (complex-glycosylated) is calculated to assess the relative expression and maturation of CFTR for each sample (C / C+B). Total RNA extraction isperformed with Trizol (Thermo Scientific) and RT-qPCR is performed with Luna Universal One-step RT-qPCR kit (New England Biolabs). GAPDH, ACTB, HPRT1, TUBA1A and Vinculin is tested as housekeeping genes for RT-qPCR normalization. geNorm software (https: / / genorm.cmgg.be / ) is used to assess most stable housekeeping gene. The two most stable genes is used as reference for RT-qPCR studies using CFF-16HBEge CFTR G542X cells grown at ALI. Cytotoxicity is measured with LDH-Glo Cytotoxicity assay kit (Promega) from the media collected from the samples. eGFP signal is assessed using a fluorescence microscope to obtain a semi -quantitative value for transfection efficiency. As reference for all experiments described in this step, we use four npDNAs: 1) Identical composition as described in Figure 1; 2) with almost identical composition as 1), except that the designed UTRs are removed (FIG. 2); 3) Same as 1) except change the promoter from CAG to hPGK; 4) Same as 1) except change the promoter from CAG to EFl A.

[0277] To facilitate the assessment of transfection efficiency, the sequence for a reporter gene, eGFP, was inserted in the same DNA construct as CFTR. Both genes were transcribed as a single mRNA separated by a sequence coding for two tandem 2A “self-cleaving” peptides (P2A and T2A). Although, there are a few limitations associated with usage of 2A peptides, this system remains widely used in molecular biology as it allows co-expression of multiple proteins from a single mRNA. Here, the presence of eGFP provides fast and simple visualization and evaluation of transfection efficiency without interfering with the protein of interest. The eGFP coding region was followed by a stop codon and the 3’ UTR.

[0278] npDNA / feLNP formulations that successfully transfect CFF-16HBEge CFTR G542X cells after nebulization are identified by detecting CFTR mRNA and CFTR protein at 48 h post-dosing. The initial target is to have at least 10-20% of cells transfected based on eGFP signal. This step also establishes a preliminary rank of the formulations based on CFTR protein expression and transfection efficiency.Durable expression of wild-type CFTR protein in aerosolized primary CF-patient derived human bronchial epithelial cells (hBE G542X).

[0279] The durability of CFTR protein expression after cells have been nebulized with CFTR-eGFP npDNA / feLNP formulations. A time-course is preformed after nebulization with the formulations selected from Step 1. Forthat the cell culture model is changed to primaryhuman bronchial epithelial cells derived from CF-patients (CFFT lab), since the immortalized cells do not growth arrest after confluency. Primary hBEs is maintained at ALI for 5 weeks to allow cells to fully-differentiate and then is treated with the selected formulations as described above. Briefly, 200 mb of CFTR-eGFP npDNA / feLNP formulations (1 mg / mL) is nebulized onto hBEs with a Aerogen Solo nebulizer. For each condition, six inserts are dosed. Cells are washed with lx PBS at 4 h post-transfection to remove any material remaining in the media. At days 1, 2, 3, 4, 7, 14, 20, 30, 45 and 60 post-transfection two replicates for each npDNA / feLNP is processed for one of the following assays: a) cell lysate for western-blot (WB); b) total RNA extraction for RT-qPCR; c) basal media collection for LDH assay; and d) eGFP signal for transfection efficiency. Cell lysis, WB, total RNA extraction, RT-qPCR, LDH assay and eGFP fluorescence is assessed as described in Specific Step 2 at each of the time-points. An important difference relates to the LDH assay, since cell culture media needs to be replaced / refreshed three times per week when maintaining cells at ALI. Hence, we collect and freeze the media from all exchanges but initially test only the media at 4 h post-dosing (to be used as reference) and the media on the pre-established time-point (1, 2, 3, 4, 7, 14, 20, 30, 45 and 60 post-dosing). In addition to LDH assay, visual inspection of cell culture health is assessed immediately before each time-point processing and during maintenance of the cultures; “leakage” of media is expected if treatment is cytotoxic. When performing WB and RT-PCR experiments the “day 1” sample is used as an internal reference for all protein gels and qPCR plates.Screen for top npDNA / feLNP constructs

[0280] This comprehensive system offers transformative advantages in the field of gene therapy, combining the long-term benefits of DNA-based therapeutics with the enhanced delivery capabilities of fusogenic lipid nanoparticles. By targeting unmet needs in Cystic Fibrosis (CF) therapy, particularly for patients unresponsive to small-molecule modulators, fusogenic feLNPs offer a scalable, safe, effective and durable therapy solution for addressing one of the most challenging genetic diseases. The reduction in toxicity and enhanced delivery properties make this approach broadly applicable to other genetic diseases as well.Higher Transfection Efficiency

[0281] The feLNP system's direct lipid-cell fusion-based entry results in significantly higher cellular uptake compared to traditional LNPs relying on endosomal escape. The smallsize of the nanoplasmid backbone also facilitates efficient intracellular delivery, enhancing transgene expression efficiency.Reduce toxicity and immunogenicity

[0282] feLNP’s direct lipid-cell fusion-based entry avoids toxicity and immunogenicity by avoiding immune responses typically associated with nucleic acid delivery via endosomal pathways. The elimination of antibiotic resistance genes from the nanoplasmid further enhances safety. Features such as CpG-depletion also contribute to minimized potential immune responses.Extended Therapeutic Half-Life

[0283] Durability of expression from npDNA is on the order of months.Dose Reduction Potential

[0284] The combination of longer duration of effect and higher transfection efficiency reduces the need for frequent dosing, which may further lower the risk of cumulative toxicity and improve patient compliance.Dose repeatedly

[0285] Due to the lack of immunogenicity, repeat dosing potentially has the additive effect on transgene expression.Refrigerator Stable Storage

[0286] Both npDNA and feLNP formulations should be stable for at least months if not longer.Manufacture scale and economically:

[0287] In contrast to the high complexity and high cost to manufacture viral gene therapy or RNA / LNP products, both npDNA and feLNPs can be integrated in an established GMP manufacturing process more economically and scalably.

[0288] Such a platform technology is particularly well suited for diseases requiring longterm or durable protein expression. Key areas of application include: genetic disorders such as cystic fibrosis (CF) and Duchenne muscular dystrophy, infectious diseases that require durable production of protective antibodies or therapeutic cytokines, oncology that requires sustained expression of immune-modulating proteins, such as checkpoint inhibitors, antibodies orengineered cytokines, and anti-aging therapies that boost certain protein / peptide expressions that have declined with aging, contributing the aging process.Example 4: SequencesTable 4: Sequences of Lead Chimeric Fusion PeptidesPeptide SEQ ID NO Sequence1.18 1.8 SEQ ID NO:1 MGSGPSNFVNHAPGEAIVTGLEKGADKVA GTISHTIWEVIAGLVALLTFLAFGFWLFKYL QKRRERRRQLTEFQKRYLRNSYRLSDLHPT AVPPVYEDPTEYGITKPLPPPPYATYINI1.3 3.1 SEQ ID NO: 2 MGSGPSNFVNHASGEAIISGLSDQTNRLGSL LSQNVYNIIYFFVIGGLILSAGYGLYKYCKY LQKRRERRRQLTEFQKRYLRNSYRLSEIQRP ISQHEYEDPYEPPSRRKPPPPPYSTYVNIDNV SAI1.17 1.7 SEQ ID NO: 3 MGSGPSNFVNHAPGEAIVTGLEKGADKVA GTISHTIWEVIAGLVALLTFLAFGFWLFKYL QKRRERRRQLTEFQKRYLRNSYRLSQRRSL TQ SPD YEEPTEYGITKPLPPPPYATYINI1.9 9.1 SEQ ID NO: 4 MGSGPSNFVNHASGEAIISGLSDQTNRLGSL LSQNIWEVVGGVLLLLFLIAVGFGLYKYLQ KRRERRRQLTEFQKRYLRNSYRLSEIQRPIS QHEYEDPYEPPSRRKPPPPPYSTYVNIDNVSAl1.13 1.3 SEQ ID NO: 5 MGSGPSNFVNHAPGEAIVTGLEKGADKVA GTISHTIWEVIAGLVALLTFLAFGFWLFRYA KGRREKKRELTEFQKRFLRNSYRLSQRRSL TQ SPD YEEPTEYGITKPLPPPPYATYINI1.45 11.23 SEQ ID NO: 6 MADGACNHATSIFGAVYCQISQNIAHGNID SYTSWTSYLPPILGGGFGLIVLLVLVVLIVY CCNHSKILSAVKATDSAVTTLLRDVAPANP DPVQVV1.25_1.15 SEQ ID NO: 7 MGSGPSNFVNHAPGEAIVTGLEKGADKVA GTISHTIWEVIAGLVALLTFLAFGFWLFYTG RLPKPRCLDVWPTTRTPPEYDVSPTVTSQPN RGYASVHASRYTRTGTDMELLQLGSANMA DLGLC SKTGS ASPP A YQPINAAQ SPPPT YGS DDDAYGSGRLR1.16 1.6 SEQ ID NO: 8 MGSGPSNFVNHAPGEAIVTGLEKGADKVA GT1SHT1WEV1AGLVALLTFLAFGFWLFKYL QKRRERRRQLTEFQKRYLRNSYRLSDLHPT AVPPVYEDPYEPPVRKKAPPPPYNTYVNINDVSSFN206678-0001-00WQTable 5: Additional Fusogenic PeptidesPeptide SEQ ID NO 1.35_2.22 SEQ ID NO:36 1.1 1.1 SEQ ID NO:9 1.36_3.22 SEQ ID NO:37 1.2_2.1 SEQ ID NO: 10 1.37_4.22 SEQ ID NO:38 1.4_4.1 SEQ ID NO: 11 1.38_5.22 SEQ ID NO:39 1.5_5.1 SEQ ID NO: 12 1.39_6.22 SEQ ID NO:40 1.6 6.1 SEQ ID NO: 13 1.40_7.22 SEQ ID NO:41 1.7_7.1 SEQ ID NO: 14 1.41_8.22 SEQ ID NO:42 1.8 8.1 SEQ ID NO: 15 1.42-9.22 SEQ ID NO:43 1.10 10.1 SEQ ID NO: 16 1.43_10.22 SEQ ID NO:44 1.11 11.1 SEQ ID NO: 17 1.44-11.22 SEQ ID NO:45 1.12 1.2 SEQ ID NO: 18 2.1_2.2 SEQ ID NO:46 1.14 1.4 SEQ ID NO: 19 2.2_2.3 SEQ ID NO:47 1.15 1.5 SEQ ID NO:20 2.3_2.5 SEQ ID NO:48 1.19 1.9 SEQ ID NO:21 2.4_2.6 SEQ ID NO:49 1.20 1.10 SEQ ID NO: 22 2.5_2.7 SEQ ID NO:50 1.21 1.11 SEQ ID NO: 23 2.6_2.8 SEQ ID NO:51 1.22 1.12 SEQ ID NO: 24 2.7_2.9 SEQ ID NO: 52 1.23 1.13 SEQ ID NO:25 2.8-2.10 SEQ ID NO:53 1.24 1.14 SEQ ID NO: 26 2.9-2.11 SEQ ID NO: 54 1.26 1.16 SEQ ID NO:27 2.10_2.12 SEQ ID NO:55 1.27 1.17 SEQ ID NO: 28 2.11-2.13 SEQ ID NO:56 1.28 1.18 SEQ ID NO: 29 2.12 2.14 SEQ ID NO:57 1.29 1.19 SEQ ID NO:30 2.13_2.15 SEQ ID NO:58 1.30 1.20 SEQ ID NO:31 2.14_2.16 SEQ ID NO:59 1.31J.22 SEQ ID NO:32 2.15_2.17 SEQ ID NO:60 1.32 1.23 SEQ ID NO:33 2.16_2.18 SEQ ID NO:61 1.33_2.4 SEQ ID NO:34 2.17_2.19 SEQ ID NO: 62 1.34 3.5 SEQ ID NO:35 2.18-2.20 SEQ ID NO: 63206678-0001-00WQ2.19_2.23 SEQ ID NO: 64 2.49_4.12 SEQ ID NO: 94 2.20_3.2 SEQ ID NO: 65 2.50_4.13 SEQ ID NO:95 2.21_3.3 SEQ ID NO:66 2.51_4.14 SEQ ID NO:96 2.22_3.4 SEQ ID NO: 67 2.52_4.15 SEQ ID NO: 97 2.23_3.6 SEQ ID NO: 68 2.53_4.16 SEQ ID NO:98 2.24_3.7 SEQ ID NO: 69 2.54_4.17 SEQ ID NO: 99 2.25_3.8 SEQ ID NO: 70 2.55_4.18 SEQ ID NO: 100 2.26_3.9 SEQ ID NO:71 2.56_4.19 SEQ ID NO:101 2.27_3.10 SEQ ID NO: 72 2.57_4.20 SEQ ID NO: 102 2.28_3.11 SEQ ID NO:73 2.58_4.23 SEQ ID NO: 103 2.29_3.12 SEQ ID NO: 74 2.59_5.2 SEQ ID NO: 104 2.30_3.13 SEQ ID NO: 75 2.60_5.3 SEQ ID NO: 105 2.31 3.14 SEQ ID NO: 76 2.61_5.4 SEQ ID NO: 106 2.32_3.15 SEQ ID NO: 77 2.62_5.5 SEQ ID NO: 107 2.33 3.16 SEQ ID NO:78 2.63_5.6 SEQ ID NO: 108 2.34_3.17 SEQ ID NO: 79 2.64_5.7 SEQ ID NO: 109 2.35 3.18 SEQ ID NO: 80 2.65_5.8 SEQ ID NO: 110 2.36_3.19 SEQ ID NO: 81 2.66_5.9 SEQ ID NO:111 2.37 3.20 SEQ ID NO: 82 2.67 5.10 SEQ ID NO: 112 2.38_3.23 SEQ ID NO: 83 2.68 5.11 SEQ ID NO: 113 2.39_4.2 SEQ ID NO: 84 2.69_5.12 SEQ ID NO: 114 2.40_4.3 SEQ ID NO: 85 2.70_5.13 SEQ ID NO:115 2.41_4.4 SEQ ID NO: 86 2.71_5.14 SEQ ID NO: 116 2.42_4.5 SEQ ID NO: 87 2.72_5.15 SEQ ID NO: 117 2.43_4.6 SEQ ID NO: 88 2.73_5.16 SEQ ID NO:118 2.44_4.7 SEQ ID NO: 89 2.74_5.17 SEQ ID NO: 119 2.45_4.8 SEQ ID NO: 90 2.75_5.18 SEQ ID NO: 120 2.46_4.9 SEQ ID NO:91 2.76_5.19 SEQ ID NO: 121 2.47_4.10 SEQ ID NO:92 2.77_5.20 SEQ ID NO: 122 2.48_4.11 SEQ ID NO: 93 2.78_5.23 SEQ ID NO: 123206678-0001-00WQ2.79_6.2 SEQ ID NO: 124 2.109_7.12 SEQ ID NO:154 2.80_6.3 SEQ ID NO: 125 2.110 7.13 SEQ ID NO:155 2.81_6.4 SEQ ID NO: 126 2.111 7.14 SEQ ID NO: 156 2.82_6.5 SEQ ID NO: 127 2.112 7.15 SEQ ID NO:157 2.83_6.6 SEQ ID NO: 128 2.113_7.16 SEQ ID NO: 158 2.84_6.7 SEQ ID NO: 129 2.114 7.17 SEQ ID NO:159 2.85_6.8 SEQ ID NO: 130 2.115 7.18 SEQ ID NO:160 2.86_6.9 SEQ ID NO: 131 2.116 7.19 SEQ ID NO:161 2.87_6.10 SEQ ID NO: 132 2.117_7.20 SEQ ID NO:162 2.88_6.11 SEQ ID NO: 133 2.118 7.23 SEQ ID NO: 163 2.89_6.12 SEQ ID NO: 134 2.119 8.2 SEQ ID NO:164 2.90_6.13 SEQ ID NO: 135 2.120_8.3 SEQ ID NO: 165 2.91 6.14 SEQ ID NO: 136 2.121_8.4 SEQ ID NO:166 2.92_6.15 SEQ ID NO: 137 2.122_8.5 SEQ ID NO:167 2.93_6.16 SEQ ID NO: 138 2.123_8.6 SEQ ID NO:168 2.94_6.17 SEQ ID NO: 139 2.124_8.7 SEQ ID NO:169 2.95_6.18 SEQ ID NO: 140 2.125_8.8 SEQ ID NO: 170 2.96_6.19 SEQ ID NO: 140 2.126_8.9 SEQ ID NO:171 2.97 6.20 SEQ ID NO: 142 2.127 8.10 SEQ ID NO: 172 2.98_6.23 SEQ ID NO: 143 2.128 8.11 SEQ ID NO:173 2.99_7.2 SEQ ID NO: 144 2.129_8.12 SEQ ID NO:174 2.100_7.3 SEQ ID NO: 145 2.130 8.13 SEQ ID NO:175 2.101_7.4 SEQ ID NO: 146 2.131 8.14 SEQ ID NO:176 2.102_7.5 SEQ ID NO: 147 2.132 8.15 SEQ ID NO: 177 2.103_7.6 SEQ ID NO: 148 2.133 8.16 SEQ ID NO:178 2.104_7.7 SEQ ID NO: 149 2.134 8.17 SEQ ID NO: 179 2.105_7.8 SEQ ID NO: 150 2.135 8.18 SEQ ID NO:180 2.106_7.9 SEQ ID NO: 151 2.136_8.19 SEQ ID NO:181 2.107_7.10 SEQ ID NO: 152 2.137_8.20 SEQ ID NO: 182 2.108 7.11 SEQ ID NO: 153 2.138_8.23 SEQ ID NO:183206678-0001-00WQ2.139_9.2 SEQ ID NO: 184 2.169 10.12 SEQ ID NO:214 2.140_9.3 SEQ ID NO: 185 2.170_10.13 SEQ ID NO:215 2.141_9.4 SEQ ID NO: 186 2.171_10.14 SEQ ID NO:216 2.142-9.5 SEQ ID NO: 187 2.172_10.15 SEQ ID NO:217 2.143-9.6 SEQ ID NO: 188 2.173_10.16 SEQ ID NO:218 2.144-9.7 SEQ ID NO: 189 2.174_10.17 SEQ ID NO:219 2.145-9.8 SEQ ID NO: 190 2.175_10.18 SEQ ID NO:220 2.146-9.9 SEQ ID NO: 191 2.176 10.19 SEQ ID NO:221 2.147_9.10 SEQ ID NO: 192 2.177_10.20 SEQ ID NO:222 2.148-9.11 SEQ ID NO: 193 2.178_10.23 SEQ ID NO:223 2.149_9.12 SEQ ID NO: 194 2.179-11.2 SEQ ID NO: 224 2.150_9.13 SEQ ID NO: 195 2.180-11.3 SEQ ID NO:225 2.151_9.14 SEQ ID NO: 196 2.181-11.4 SEQ ID NO: 226 2.152_9.15 SEQ ID NO: 197 2.182-11.5 SEQ ID NO:227 2.153_9.16 SEQ ID NO: 198 2.183_11.6 SEQ ID NO:228 2.154-9.17 SEQ ID NO: 199 2.184-11.7 SEQ ID NO:229 2.155_9.18 SEQ ID NO: 200 2.185-11.8 SEQ ID NO:230 2.156_9.19 SEQ ID NO:201 2.186-11.9 SEQ ID NO:231 2.157 9.20 SEQ ID NO: 202 2.187 11.10 SEQ ID NO:232 2.158-9.23 SEQ ID NO:203 2.188-11.11 SEQ ID NO:233 2.159_10.2 SEQ ID NO: 204 2.189-11.12 SEQ ID NO:234 2.160_10.3 SEQ ID NO:205 2.190_11.13 SEQ ID NO:235 2.161_10.4 SEQ ID NO: 206 2.191-11.14 SEQ ID NO:236 2.162-10.5 SEQ ID NO: 207 2.192-11.15 SEQ ID NO:237 2.163_10.6 SEQ ID NO:208 2.193-11.16 SEQ ID NO:238 2.164_10.7 SEQ ID NO: 209 2.194-11.17 SEQ ID NO:239 2.165_10.8 SEQ ID NO:210 2.195-11.18 SEQ ID NO: 240 2.166_10.9 SEQ ID NO:211 2.196-11.19 SEQ ID NO:241 2.167_10.10 SEQ ID NO:212 2.197-11.20 SEQ ID NO:2422.168-10.11 SEQ ID NO:213Attorney Docket No.206678-0001-00WO5’ UTR (SEQ ID NO: 243)CAGTTGGACACTTGAAGTACAGACCTGAAGCCACC3’ UTR hHBB (human beta-globin) (SEQ ID NO:244) GCTCGCTTTCTTGCTGTCCAATTTCTATTAAAGGTTCCTTTGTTCCCTAAGTCCAA CTACTAAACTGGGGGATATTATGAAGGGCCTTGAGCATCTGGATTCTGCCTAATA AAAAACATTTATTTTCATTGCAGCTCGCTTTCTTGCTGTCCAATTTCTATTAAAGG TTCCTTTGTTCCCTAAGTCCAACTACTAAACTGGGGGATATTATGAAGGGCCTTG AGCATCTGGATTCTGCCTAATAAAAAACATTTATTTTCATTGCACFTR Amino Acid Sequence (SEQ ID NO:245)CFTR nucleotide sequence (no UTRs) (SEQ ID NO:246)CFTR nucleotide sequence (with UTRs) (SEQ ID NO:247)

[0289] The disclosures of each and every patent, patent application, and publication cited herein are hereby incorporated herein by reference in their entirety. While this invention has been disclosed with reference to specific embodiments, it is apparent that other embodiments and variations of this invention may be devised by others skilled in the art without departing from the true spirit and scope of the invention. The appended claims are intended to be constmed to include all such embodiments and equivalent variations.

Claims

206678-0001-00WQCLAIMSWhat is claimed is:

1. A chimeric fusogenic peptide comprising at least one of a ectodomain and a transmembrane domain (TMD) of a first fusion-associated small transmembrane (FAST) protein linked to an endodomain of a second FAST protein.

2. The chimeric fusogenic peptide of claim 1, wherein the endodomain is an endodomain of apl4, pl5, plO or p22 FAST protein.

3. The chimeric fusogenic peptide of claim 2, wherein the endodomain is selected from the group consisting of SEQ ID NO:259-281.

4. The chimeric fusogenic peptide of claim 3, wherein the peptide comprises at least one peptide of SEQ ID NO:248-258 linked to at least one peptide of SEQ ID NO:259-281.

5. The chimeric fusogenic peptide of claim 3, wherein the peptide comprises a sequence as set forth in SEQ ID NO: 1-242.

6. The chimeric fusogenic peptide of claim 3, wherein the peptide comprises a sequence as set forth in SEQ ID NO: 1-8.

7. A composition comprising a chimeric fusogenic peptide of claim 1.

8. The composition of claim 7 comprising a delivery vehicle comprising at least one therapeutic agent for the treatment of a disease or disorder.

9. The composition of claim 8, wherein the delivery vehicle is selected from the group consisting of a liposome, a lipid nanoparticle, a polymeric nanoparticle, a polystyrene nanoparticle, and a micelle.

10. The composition of claim 9, wherein the delivery vehicle is a lipid nanoparticle.

11. The composition of claim 8, wherein the therapeutic agent comprises a nucleic acid molecule encoding a therapeutic protein.

12. The composition of claim 11, wherein the therapeutic agent comprises a nucleic acid molecule encoding cystic fibrosis transmembrane conductance regulator (CFTR), wherein the nucleic acid molecule comprises a 5’ UTR comprising SEQ ID NO:243 and a 3’ UTR comprising SEQ ID NO: 244.

13. The composition of claim 8, wherein the delivery vehicle further comprises a targeting domain.

14. The composition of claim 13, wherein the targeting domain is an antibody.

15. A method of treating a disease or disorder associated in a subject in need thereof, the method comprising administering a composition of claim 7 to the subject or to a transplantable cell for administration to the subject.

16. The method of claim 15, wherein the composition comprises a chimeric fusogenic peptide comprising an amino acid sequence as set forth in SEQ ID NO: 1-8.

17. The method of claim 15, comprising administering a delivery vehicle comprising at least one therapeutic agent for the treatment of a disease or disorder.

18. The method of claim 17, wherein the delivery vehicle is selected from the group consisting of a liposome, a lipid nanoparticle, a polymeric nanoparticle, a polystyrene nanoparticle, and a micelle.

19. The method of claim 18, wherein the delivery vehicle is a lipid nanoparticle.

20. The method of claim 17, wherein the therapeutic agent comprises a nucleic acid molecule encoding a therapeutic protein.206678-0001-00WQ21. The method of claim 15, wherein the disease or disorder is selected from the group consisting of monogenic diseases, metabolic diseases, conditions benefiting from increased gene / hormone activity, enhanced metabolic signaling, or restored deficient pathways (Boost conditions), conditions benefiting from reduced gene / protein activity, suppressed lipogenesis, substrate reduction, or lowered pathological signaling (Knockdown conditions), cosmetic or skin conditions, conditions associated with aging, conditions associated with mitochondrial function, autoimmune diseases, immunodeficiency diseases, inflammatory diseases, neurological diseases or disorder, infectious diseases and cancer.

22. The method of claim 15, wherein the disease or disorder is cystic fibrosis (CF).

23. The method of claim 22, wherein the therapeutic agent comprises a nucleic acid molecule encoding cystic fibrosis transmembrane conductance regulator (CFTR).

24. The method of claim 22, wherein the therapeutic agent comprises a nucleic acid molecule encoding SEQ ID NO:245, wherein the coding sequence is operably linked to a 5’ UTR comprising SEQ ID NO:243 and a 3’ UTR comprising SEQ ID NO:244.

25. The method of claim 15, wherein the composition is administered by a delivery route selected from the group consisting of inhalation, intraocular, intravitreal, subretinal, suprachoroidal, subcutaneous, intraperitoneal, intramuscular, intradermal, intrasternal, intratumoral, intravenous, intracerebroventricular injections, and kidney dialytic infusion.

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