EP4731186A2 - Methods and compositions for treating epithelial diseases - Google Patents

Methods and compositions for treating epithelial diseases

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Publication number
EP4731186A2
EP4731186A2 EP24826734.6A EP24826734A EP4731186A2 EP 4731186 A2 EP4731186 A2 EP 4731186A2 EP 24826734 A EP24826734 A EP 24826734A EP 4731186 A2 EP4731186 A2 EP 4731186A2
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EP
European Patent Office
Prior art keywords
lipid nanoparticle
seq
nucleic acid
acid sequence
lipid
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
EP24826734.6A
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German (de)
French (fr)
Inventor
Paul Ayoub
Donald B. Kohn
Brigitte GOMPERTS
Steven J. Jonas
Ruth FOLEY
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
University of California
University of California Berkeley
University of California San Diego UCSD
Original Assignee
University of California
University of California Berkeley
University of California San Diego UCSD
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Publication date
Application filed by University of California, University of California Berkeley, University of California San Diego UCSD filed Critical University of California
Publication of EP4731186A2 publication Critical patent/EP4731186A2/en
Pending legal-status Critical Current

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    • A61K47/6929—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle
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    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
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    • C12N2800/00—Nucleic acids vectors
    • C12N2800/22—Vectors comprising a coding region that has been codon optimised for expression in a respective host

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Abstract

Described herein is a delivery system and method that can be used to deliver therapeutic compositions to epithelial cells. Provided are lipid nanoparticles comprising an ionizable lipid, a sterol, a phospholipid, and a lipid conjugated to PEG. Also described are novel molecules that can be used for gene editing. Methods include methods for delivering a therapeutic cargo to the airway or epithelium of a subject and/or a method for treating an airway or gastrointestinal disorder in a subject, the method comprising administering: i) a first lipid nanoparticle comprising a surfactant; and ii) a second lipid nanoparticle comprising a therapeutic cargo. Further methods describe a method for delivering a therapeutic cargo to the airway or epithelium of a subject and/or a method for treating an airway or gastrointestinal disorder in a subject, the method comprising administering: a lipid nanoparticle comprising a therapeutic cargo, wherein the lipid nanoparticle comprises a surfactant.

Description

METHODS AND COMPOSITIONS FOR TREATING EPITHELIAL DISEASES
[0001] This application claims priority of U.S. Provisional Patent Application No. 63/522,979, filed June 23, 2023; U.S. Provisional Patent Application No. 63/546,597, filed October 31, 2023; U.S. Provisional Patent Application No. 63/627,977, filed February 1, 2024; and U.S. Provisional Patent Application No. 63/643,016, filed May 6, 2024, all of which are hereby incorporated by reference in their entirety.
Sequence Listing
[0002] The application contains a Sequence Listing in compliance with ST.26 format and is hereby incorporated by reference in its entirety. Said Sequence Listing, created on June 21, 2024 is named UCLAP0184WO.xml and is 31,974 bytes in size.
1. Field of the Invention
[0003] The present invention relates generally to the field of medicine. In particular, embodiments are directed to treatment of airway disorders.
2. Description of Related Art
[0004] Several obstacles have precluded the successful realization and clinical translation of gene therapies for epithelial diseases such as cystic fibrosis (CF). One significant hurdle for these interventions is that effective and durable correction of the disease phenotype in CF (for example) hinges on the ability to deliver nucleic acids and/or other gene-editing biomolecules to airway basal stem/progenitor cell (ABSC) populations of the airway epithelium. Accessing and targeting ABSCs directly via inhaled delivery of therapeutic biomolecules, although ideal for treating CF, is particularly challenging and limited by failure of existing delivery approaches to overcome physical (e.g., thick mucus, epithelial tight junctions) barriers imposed by the CF airway. While strategies exist to encapsulate cargoes capable of gene-editing into particle-based nanocarriers, these approaches have yet to demonstrate modification of the airway epithelium by accessing and preferentially targeting ABSCs.
SUMMARY OF THE INVENTION
[0005] The current disclosure provides for delivery systems and methods that can be used to deliver therapeutic compositions to epithelial cells. Provided are lipid nanoparticles comprising an ionizable lipid, a sterol, a phospholipid, and a lipid conjugated to PEG. Also described is a lipid nanoparticle comprising: an ionizable lipid, sitosterol, DOPE, and/or DMG- PEG2000. Also described is a small guide RNA (sgRNA) comprising the nucleotide sequence of one of SEQ ID NOS: 1-5 or a RNA with a sequence that is at least 90% identical to one of SEQ ID NOS: 1-5. The small guide RNA (sgRNA) may comprise a nucleotide sequence having or having at least 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence identity, or any derivable range therein, to one of SEQ ID NOS: 1-5. Also provided is a donor DNA molecule comprising a CFTR transgene or mutation correction cassette and at least one homology arm. The disclosure describes a donor DNA molecule comprising a transgene or mutation correction cassette and at least one homology arm, wherein the homology arm is fewer than 150 nucleotides and comprises at least 90% homology to an endogenous genomic sequence and wherein the DNA is modified with 6-12 carbons at the 5’ terminus of the DNA molecule. The homology arm may be, may be at least, or may be at most 20, 21, 22, 23, 24,
25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49,
50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74,
75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99,
100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, or 150 nucleotides in length, or any range derivable therein.
[0006] The disclosure also describes a cDNA encoding a sgRNA or donor DNA of the disclosure. Also included are expression vectors comprising a cDNA of the disclosure and cells comprising an expression vector, cDNA, sgRNA, and/or donor DNA of the disclosure. Compositions include those comprising a lipid nanoparticle, an expression vector, cDNA, sgRNA, donor DNA, or cell of the disclosure.
[0007] The disclosure also describes methods for delivering a therapeutic cargo to the airway or epithelium of a subject and/or a method for treating an airway or gastrointestinal disorder in a subject, the method comprising administering: i) a first lipid nanoparticle comprising a surfactant; and ii) a second lipid nanoparticle comprising a therapeutic cargo. Further methods describe a method for delivering a therapeutic cargo to the airway or epithelium of a subject and/or a method for treating an airway or gastrointestinal disorder in a subject, the method comprising administering: a lipid nanoparticle comprising a therapeutic cargo, wherein the lipid nanoparticle comprises a surfactant. Also described is a method for delivering a therapeutic cargo to the airway or epithelium of a subject or for treating an airway or gastrointestinal disorder in a subject, the method comprising administering a lipid nanoparticle, expression vector, cDNA, sgRNA, donor DNA, and/or composition of the disclosure to the subject. [0008] Also provided is a lipid nanoparticle comprising an expression vector, cDNA, sgRNA, and/or donor DNA of the disclosure. The lipid nanoparticle may further comprise a Cas protein or a nucleic acid encoding a Cas protein.
[0009] The sterol may comprise or exclude a sitosterol. The lipid nanoparticle, first lipid nanoparticle, and/or second lipid nanoparticle may comprise or exclude one or more of the ionizable lipids: 9-Heptadecanyl 8-{(2-hydroxyethyl)[6-oxo-6-
(undecyloxy)hexyl] amino] octanoate (SM-102); 7-[(2-Hydroxyethyl)[8-(nonyloxy)-8- oxooctyl]amino]heptyl 2-octyldecanoate (Lipid 5); 9-[4-(dimethylamino)-l-oxobutoxy]- heptadecanedioic acid, l,17-di-(dec-3-yn-l-yl) ester Di(dec-3-yn-l-yl)9-((4- (dimethylamino)butanoyl)oxy)heptadecanedioate (Lipid A6); 1 , 1 '-[[2-[4-[2-[[2-[bis(2- hydroxydodecyl)amino]ethyl](2-hydroxydodecyl)amino]ethyl]-l- piperazinyl]ethyl]imino]bis-2-dodecanol (C 12-200); l,2-dioleoyl-3 -trimethylammonium - propane chloride (DOTAP); and 4,7,10,13,16-Pentaazanonadecanedioic acid, 4, 10, 16-tris[3- [2-[2-methyl-3-(octylthio)-l-oxopropoxy]ethoxy]-3-oxopropyl]-, l,19-bis[2-[2-methyl-3- (octylthio)-l-oxopropo (5A2-SC8). The lipid nanoparticle, first lipid nanoparticle, and/or second lipid nanoparticle may comprise or exclude one or more of the sterols: 30-[N-(N’N, N’-dimethylaminoethane)-carbamoyl]cholesterol (DC-cholesterol), cholesterol, and P- sitosterol. The lipid nanoparticle, first lipid nanoparticle, and/or second lipid nanoparticle may comprise or exclude one or more of the lipids conjugated to PEG: l,2-Dioleoyl-sn-glycero-3- phosphoethanolamine-N-[methoxy(poly ethylene glycol)- 1000] (DOPE-PEG 1000), 1,2- dioleoyl-sn-glycero-3-phosphoethanolamine-N-[amino(poly ethylene glycol)-2000] (DOPE- PEG2000), 1,2 distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(poly-ethylene glycol)-2000] (DSPE-PEG2000), l,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-
[carboxy(polyethylene glycol)-1000 (DSPE-PEG1000); and l,2-Dimyristoyl-rac-glycero-3- [methoxy(poly-ethylene glycol)-2000] (DMG-PEG2000). The lipid nanoparticle, first lipid nanoparticle, and/or second lipid nanoparticle may comprise or exclude the phospholipid dioleoylphosphatidylethanolamine (DOPE).
[0010] The lipid nanoparticle, first lipid nanoparticle, and/or second lipid nanoparticle may comprise molar ratios or weight ratios of 40-60 ionizable lipid; 25-50 sterol; 5-15 phospholipid; and 0.5-5 lipid conjugated to PEG. The lipid nanoparticle, first lipid nanoparticle, and/or second lipid nanoparticle may comprise, comprise at least, or comprise at most a molar or weight ratio of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, or 80 ionizable lipid, or any derivable range therein. The lipid nanoparticle, first lipid nanoparticle, and/or second lipid nanoparticle may comprise, comprise at least, or comprise at most a molar or weight ratio of 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,
28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52,
53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77,
78, 79, or 80% sterol, or any derivable range therein. The lipid nanoparticle, first lipid nanoparticle, and/or second lipid nanoparticle may comprise, comprise at least, or comprise at most a molar or weight ratio of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, or 80% phospholipid, or any derivable range therein. The lipid nanoparticle, first lipid nanoparticle, and/or second lipid nanoparticle may comprise, comprise at least, or comprise at most a molar or weight ratio of 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5, 5.1, 5.2,
5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4,
7.5, 7.6, 7.7, 7.8, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6,
9.7, 9.8, 9.9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20% lipid conjugated to PEG, or any derivable range therein. The lipid nanoparticle, first lipid nanoparticle, and/or second lipid nanoparticle may comprise a molar or weight ratio of 50% ionizable lipid. The lipid nanoparticle, first lipid nanoparticle, and/or second lipid nanoparticle may comprise a molar or weight ratio of 38.5% sterol. The lipid nanoparticle, first lipid nanoparticle, and/or second lipid nanoparticle may comprise a molar or weight ratio of 10% phospholipid. The lipid nanoparticle, first lipid nanoparticle, and/or second lipid nanoparticle may comprise a molar or weight ratio of 1.5% lipid conjugated to PEG.
[0011] The percent by weight or volume of ionizable lipid, sterol, phospholipid, lipid conjugated to PEG, and/or surfactant may be, be at least, or be at most 1, 2, 3, 4, 5, 6, 7, 8, 9,
10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,
35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59,
60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84,
85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%, or any derivable range therein. [0012] The lipid nanoparticle may comprise or further comprise or exclude an ionizable lipid, sitosterol, DOPE, and/or DMG-PEG2000. The lipid nanoparticle may comprise 20-60 mole or weight percent of ionizable lipid. The ionizable lipid may comprise or exclude SM- 102. The lipid nanoparticle may comprise, comprise at least, or comprise at most 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37,
38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62,
63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87,
88, 89, or 90 mole or weight percent (or any derivable range therein) of ionizable lipid. The lipid nanoparticle may comprise 50 mole or weight percent of SM-102. The lipid nanoparticle may comprise 25-55 mole or weight percent of sitosterol. The lipid nanoparticle may comprise 38.5 mole or weight percent of Sitosterol. The lipid nanoparticle may comprise, comprise at least, or comprise at most 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,
28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52,
53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77,
78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, or 90 mole or weight percent (or any derivable range therein) of sitosterol or sterol. The lipid nanoparticle may comprise 5-15 mole or weight percent DOPE or phospholipid. The lipid nanoparticle may comprise, comprise at least, or comprise at most 2, 3, 4, 5, 6, 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 mole or weight percent (or any derivable range therein) of DOPE or phospholipid. The lipid nanoparticle may comprise 10 mole or weight percent of DOPE or phospholipid. The lipid nanoparticle may comprise 0.5-3 weight or mole percent DMG- PEG2000 or lipid conjugated to PEG. The lipid nanoparticle may comprise, comprise at least, or comprise at most 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, or 10 mole or weight percent (or any derivable range therein of DMG-PEG2000 or lipid conjugated to PEG. The lipid nanoparticle may comprise 1.5 mole or weight percent DMG-PEG2000 or lipid conjugated to PEG.
[0013] The lipid nanoparticle may comprises DMG-PEG2000 and DSPE-PEG1000. The lipid nanoparticle may comprise 0.6% DMG-PEG2000 and 0.9% DSPE-PEG1000. The weight or molar ratio of DMG-PEG2000 to DSPE-PEG1000 may be 2:3. The PEG of the lipid conjugated to PEG may comprise a functional end group at the PEG terminus. The functional end group may comprise -SH, -orthopyridyl-disulfide, -maleimide, -transcyclooctene, and/or - dibenzocyclooctyne. [0014] The lipid nanoparticle, first lipid nanoparticle, second lipid nanoparticle, and/or composition may be nebulized/aerosolized. The lipid nanoparticle, first lipid nanoparticle, second lipid nanoparticle, and/or composition may be aerolized. The lipid nanoparticle, first lipid nanoparticle, and/or second lipid nanoparticle may be 100-300 nm in size and/or diameter. The lipid nanoparticle, first lipid nanoparticle, and/or second lipid nanoparticle may be 100- 200 nm in size and/or diameter. The size or diameter may be an average size/diameter of a lipid nanoparticle composition. The lipid nanoparticle, first lipid nanoparticle, and/or second lipid nanoparticle may be, be at least, or be at most 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128,
129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147,
148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166,
167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185,
186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204,
205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223,
224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242,
243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261,
262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280,
281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299,
300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318,
319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337,
338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356,
357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375,
376, 377, 378, 379, 380, 381, 382, 383, 384, 385, 386, 387, 388, 389, 390, 391, 392, 393, 394,
395, 396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413,
414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428, 429, 430, 431, 432,
433, 434, 435, 436, 437, 438, 439, 440, 441, 442, 443, 444, 445, 446, 447, 448, 449, 450, 451,
452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470,
471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 484, 485, 486, 487, 488, 489,
490, 491, 492, 493, 494, 495, 496, 497, 498, 499, or 500 nm in size and/or diameter, or any derivable range therein. Any one or more of these may be excluded in an an aspect described herein. [0015] The lipid nanoparticle, first lipid nanoparticle, or second lipid nanoparticle may comprise a surfactant. The surfactant may be covalently bound to the lipid nanoparticle or first lipid nanoparticle. The surfactant may be non-covalently bound to the lipid nanoparticle or first lipid nanoparticle. The surfactant may comprise or exclude an amphiphilic surfactant. The surfactant may comprise or exclude polidocanol (PDOC). The surfactant may comprise or exclude lysophosphatidylcholine (LPC). The surfactant may comprise or exclude a mixture of surfactants, such as a mixture of PDOC and LPC, or a surfactant disclosed herein. The lipid nanoparticle, first lipid nanoparticle, and/or second lipid nanoparticle may exclude PDOC, LPC, or a surfactant described herein. The lipid nanoparticle or first lipid nanoparticle may exclude a nucleic acid, therapeutic cargo, and/or small molecule. The lipid nanoparticle or first lipid nanoparticle may consist or consist essentially of SM-102, Beta-sitosterol, DOPE, DMG- PEG2000, and a surfactant. The lipid nanoparticle or first lipid nanoparticle may comprise, consist, or consist essentially of an ionizable lipid, a sterol, a phospholipid, a lipid conjugated to PEG, and a surfactant. The lipid nanoparticle or first lipid nanoparticle may comprise 10-40 wt % surfactant. The lipid nanoparticle or first lipid nanoparticle may comprise, comprise at least, or comprise at most 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, or 80 wt % surfactant.
[0016] The lipid nanoparticle or composition may comprise a DNA-PK inhibitor and/or a DNA Polymerase Theta (POLO) inhibitor. The nanoparticle may comprise AZD7648 and/or ART558. The lipid nanoparticle or composition may comprise a Rho kinase (ROCK) inhibitor. Exemplary ROCK inhibitors include the compounds: Y27632, Fasudil, Ripasudil, Netarsudil, RKL1447, GSK429286A, and Y30141. The lipid nanoparticle or composition may exclude one or more of a ROCK inhibibitor, DNA-PK inhibitor, and DNA Polymersae Theta inhibitor. [0017] The lipid nanoparticle or second lipid nanoparticle may comprise or further comprise a therapeutic cargo. The lipid nanoparticle or second lipid nanoparticle may consist or consist essentially of SM-102, Beta-sitosterol, DOPE, DMG-PEG2000, and a therapeutic cargo. The lipid nanoparticle or second lipid nanoparticle may comprise, consist, or consist essentially of an ionizable lipid, a sterol, a phospholipid, a lipid conjugated to PEG, and a therapeutic cargo. The lipid nanoparticle, second lipid nanoparticle, and/or composition of the disclosure may comprise a Cas protein or a nucleic acid encoding a Cas protein. The lipid nanoparticle, second lipid nanoparticle, and/or composition of the disclosure may comprise a RNA or DNA encoding a Cas protein. The lipid nanoparticle, second lipid nanoparticle, and/or composition of the disclosure may comprise a guide RNA or a DNA encoding a guide RNA. The lipid nanoparticle, second lipid nanoparticle, and/or composition of the disclosure may comprise a donor DNA or exclude a donor DNA.
[0018] The therapeutic cargo may comprise a nucleic acid, tRNA, small molecule, antibody, and/or polypeptide. The therapeutic cargo may exclude one or more of a nucleic acid, tRNA, small molecule, antibody, and/or polypeptide. The nucleic acid may be a DNA, RNA, or cDNA. The nucleic acid may comprise one or more of a small interfering RNA (siRNA), short hairpin RNA (shRNA), double-stranded RNA, an antisense oligonucleotide, and a ribozyme. The Cas protein may comprise a Cas9 protein. The Cas9 protein may comprise the amino acid sequence of SEQ ID NO: 6, an amino acid sequence of a fragment of SEQ ID NO: 6, an amino acid sequence with at least 80% sequence identity to SEQ ID NO:6, or an amino acid sequence with at least 80% sequence identity to a fragment of SEQ ID NO:6. The Cas9 protein may comprise an amino acid sequence with or with at least 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%, or any derivable range therein, sequence identity to SEQ ID NO:6. The guide RNA may comprise one of SEQ ID NOS: 1-5 or a RNA with a sequence that is at least 90% identical to one of SEQ ID NOS: 1-5. The guide RNA may exclude one or more of SEQ ID NOS: 1-5. The donor DNA may be ssDNA or dsDNA. The therapeutic cargo or composition of the disclosure may comprise or consist of or exclude a guide RNA, a Cas protein or a nucleic acid encoding a Cas protein, and/or a donor DNA.
[0019] The lipid nanoparticle may comprise a ratio of the sgRNA to the Cas RNA or DNA of 0.5-3 sgRNA to 0.5-3 Cas RNA or DNA w/w. The ratio of sgRNA to the Cas (RNA or
DNA) may be from, from at least, or from at most 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1,
1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2,
3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5, 5.1, 5.2, 5.3, 5.4,
5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6,
7.7, 7.8, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8,
9.9, 10 (or any derivabe Irange therein) sgRNA to, to at least, or to at most 0.1, 0.2, 0.3, 0.4,
0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6,
2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8,
4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1,
7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9, 9.1, 9.2, 9.3,
9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10 (or any derivabe Irange therein) Cas w/w, or any derivable range therein. The ratio of the sgRNA to the Cas RNA or DNA may be 1.2: 1 w/w. The ratio of the sgRNA to the Cas RNA or DNA may be 1.6: 1 w/w. The ratio of the sgRNA to the Cas RNA or DNA may be 1.2-1.6 sgRNA to 1 Cas RNA or Cas DNA.
[0020] The lipid nanoparticle may comprise a ratio of the donor DNA to the Cas RNA or DNA of 1-5 donor DNA to 0.5-3 Cas RNA or DNA w/w. The ratio of donor DNA to the Cas (RNA or DNA) may be from, from at least, or from at most 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1,
3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5, 5.1, 5.2, 5.3,
5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5,
7.6, 7.7, 7.8, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7,
9.8, 9.9, 10 (or any derivabe Irange therein) donor DNA to, to at least, or to at most 0.1, 0.2,
0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4,
2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6,
4.7, 4.8, 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8,
6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9, 9.1,
9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10 (or any derivabe Irange therein) Cas w/w, or any derivable range therein. The ratio of the donor DNA to the Cas RNA or DNA may be 3 : 1 w/w. The ratio of the donor DNA to the Cas RNA or DNA may be 4: 1 w/w.
[0021] The nitrogen-to-phosphate ratio (N/P) may be 5-13. The N/P may be, be at least, or be at most 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2,
4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4,
6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6,
8.7, 8.8, 8.9, 9, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10, 10.1, 10.2, 10.3, 10.4, 10.5, 10.6,
10.7, 10.8, 10.9, 11, 11.1, 11.2, 11.3, 11.4, 11.5, 11.6, 11.7, 11.8, 11.9, 12, 12.1, 12.2, 12.3,
12.4, 12.5, 12.6, 12.7, 12.8, 12.9, 13, 13.1, 13.2, 13.3, 13.4, 13.5, 13.6, 13.7, 13.8, 13.9, 14,
14.1, 14.2, 14.3, 14.4, 14.5, 14.6, 14.7, 14.8, 14.9, 15, 15.1, 15.2, 15.3, 15.4, 15.5, 15.6, 15.7,
15.8, 15.9, 16, 16.1, 16.2, 16.3, 16.4, 16.5, 16.6, 16.7, 16.8, 16.9, 17, 17.1, 17.2, 17.3, 17.4,
17.5, 17.6, 17.7, 17.8, 17.9, 18, 18.1, 18.2, 18.3, 18.4, 18.5, 18.6, 18.7, 18.8, 18.9, 19, 19.1,
19.2, 19.3, 19.4, 19.5, 19.6, 19.7, 19.8, 19.9, 20, 20.1, 20.2, 20.3, 20.4, 20.5, 20.6, 20.7, 20.8,
20.9, 21, 21.1, 21.2, 21.3, 21.4, 21.5, 21.6, 21.7, 21.8, 21.9, 22, 22.1, 22.2, 22.3, 22.4, 22.5,
22.6, 22.7, 22.8, 22.9, 23, 23.1, 23.2, 23.3, 23.4, 23.5, 23.6, 23.7, 23.8, 23.9, 24, 24.1, 24.2,
24.3, 24.4, 24.5, 24.6, 24.7, 24.8, 24.9, 25, 25.1, 25.2, 25.3, 25.4, 25.5, 25.6, 25.7, 25.8, 25.9, or 30, or any derivable range therein. The N/P refers to the ratio of moles of the amine groups of cationic polymers (such as those from the polymers of the liposome) to phosphate polymers, such as nucleic acid polymers. The N/P may be 9 or greater and/or 13 or fewer. The N/P may be 11.
[0022] The weight of the Cas RNA or Cas DNA in the lipid nanoparticle may be 50-100 ng. The weight of the Cas RNA or Cas DNA in the lipid nanoparticle may be 40-200ng. The weight of the Cas RNA or Cas DNA in the lipid nanoparticle may be 100-200ng. The weight of the donor DNA in the lipid nanoparticle may be 50-100 ng. The weight of the donor DNA in the lipid nanoparticle may be 40-200ng. The weight of the donor DNA in the lipid nanoparticle may be 100-200ng. The weight of the sgRNA or DNA encoding the sgRNA in the lipid nanoparticle may be 50-100 ng. The weight of the sgRNA or DNA encoding the sgRNA in the lipid nanoparticle may be 40-200ng. The weight of the sgRNA or DNA encoding the sgRNA in the lipid nanoparticle may be 100-200ng. The weight of the donor DNA in the lipid nanoparticle may be 50 ng. The weight of the donor DNA in the lipid nanoparticle may be 75 ng. The weight of the donor DNA in the lipid nanoparticle may be 100 ng. The weight of the sgRNA or DNA encoding the sgRNA in the lipid nanoparticle may be 50 ng. The weight of the sgRNA or DNA encoding the sgRNA in the lipid nanoparticle may be 75 ng. The weight of the sgRNA or DNA encoding the sgRNA in the lipid nanoparticle may be 100 ng. The weight of the Cas RNA or Cas DNA in the lipid nanoparticle may be 50 ng. The weight of the Cas RNA or Cas DNA in the lipid nanoparticle may be 75 ng. The weight of the Cas RNA or Cas DNA in the lipid nanoparticle may be 100 ng. The total weight of all the nucleic acid moleucles in the nanoparticle may be 50-100 ng. The total weight of all the nucleic acid moleucles in the nanoparticle may be 40-200ng. The total weight of all the nucleic acid moleucles in the nanoparticle may be 100-200ng. The total weight of all the nucleic acid moleucles in the nanoparticle may be 50 ng. The total weight of all the nucleic acid moleucles in the nanoparticle may be 75 ng. The total weight of all the nucleic acid moleucles in the nanoparticle may be 100 ng.
[0023] The weight of the Cas RNA, Cas DNA, sgRNA, DNA encoding a sgRNA, donor DNA and/or all nucleic acid molecules in the nanoparticle may be, be at least, or be at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103,
104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122,
123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141,
142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, , 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179,, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198,, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217,, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236,, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255,, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274,, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293,, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312,, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331,, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350,, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369,, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, 383, 384, 385, 386, 387, 388,, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 407,, 409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426,, 428, 429, 430, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442, 443, 444, 445,, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464,, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483,, 485, 486, 487, 488, 489, 490, 491, 492, 493, 494, 495, 496, 497, 498, 499, 500, 501, 502,, 504, 505, 506, 507, 508, 509, 510, 511, 512, 513, 514, 515, 516, 517, 518, 519, 520, 521,, 523, 524, 525, 526, 527, 528, 529, 530, 531, 532, 533, 534, 535, 536, 537, 538, 539, 540,, 542, 543, 544, 545, 546, 547, 548, 549, 550, 551, 552, 553, 554, 555, 556, 557, 558, 559,, 561, 562, 563, 564, 565, 566, 567, 568, 569, 570, 571, 572, 573, 574, 575, 576, 577, 578,, 580, 581, 582, 583, 584, 585, 586, 587, 588, 589, 590, 591, 592, 593, 594, 595, 596, 597,, 599, 600, 601, 602, 603, 604, 605, 606, 607, 608, 609, 610, 611, 612, 613, 614, 615, 616,, 618, 619, 620, 621, 622, 623, 624, 625, 626, 627, 628, 629, 630, 631, 632, 633, 634, 635,, 637, 638, 639, 640, 641, 642, 643, 644, 645, 646, 647, 648, 649, 650, 651, 652, 653, 654,, 656, 657, 658, 659, 660, 661, 662, 663, 664, 665, 666, 667, 668, 669, 670, 671, 672, 673,, 675, 676, 677, 678, 679, 680, 681, 682, 683, 684, 685, 686, 687, 688, 689, 690, 691, 692,, 694, 695, 696, 697, 698, 699, 700, 701, 702, 703, 704, 705, 706, 707, 708, 709, 710, 711,, 713, 714, 715, 716, 717, 718, 719, 720, 721, 722, 723, 724, 725, 726, 727, 728, 729, 730,, 732, 733, 734, 735, 736, 737, 738, 739, 740, 741, 742, 743, 744, 745, 746, 747, 748, 749,, 751, 752, 753, 754, 755, 756, 757, 758, 759, 760, 761, 762, 763, 764, 765, 766, 767, 768,, 770, 771, 772, 773, 774, 775, 776, 777, 778, 779, 780, 781, 782, 783, 784, 785, 786, 787,, 789, 790, 791, 792, 793, 794, 795, 796, 797, 798, 799, 800, 801, 802, 803, 804, 805, 806, 807, 808, 809, 810, 811, 812, 813, 814, 815, 816, 817, 818, 819, 820, 821, 822, 823, 824, 825,
826, 827, 828, 829, 830, 831, 832, 833, 834, 835, 836, 837, 838, 839, 840, 841, 842, 843, 844,
845, 846, 847, 848, 849, 850, 851, 852, 853, 854, 855, 856, 857, 858, 859, 860, 861, 862, 863,
864, 865, 866, 867, 868, 869, 870, 871, 872, 873, 874, 875, 876, 877, 878, 879, 880, 881, 882,
883, 884, 885, 886, 887, 888, 889, 890, 891, 892, 893, 894, 895, 896, 897, 898, 899, 900, 901,
902, 903, 904, 905, 906, 907, 908, 909, 910, 911, 912, 913, 914, 915, 916, 917, 918, 919, 920,
921, 922, 923, 924, 925, 926, 927, 928, 929, 930, 931, 932, 933, 934, 935, 936, 937, 938, 939,
940, 941, 942, 943, 944, 945, 946, 947, 948, 949, 950, 951, 952, 953, 954, 955, 956, 957, 958,
959, 960, 961, 962, 963, 964, 965, 966, 967, 968, 969, 970, 971, 972, 973, 974, 975, 976, 977,
978, 979, 980, 981, 982, 983, 984, 985, 986, 987, 988, 989, 990, 991, 992, 993, 994, 995, 996,
997, 998, 999, or 1000 (or any derivable range therein) picograms, nanograms, or micrograms [0024] The DNA-PK inhibitor, DNA Polymerase Theta (POLO) inhibitor, sgRNA, donor DNA, therapeutic cargo, Cas, or other elements recited herein as part of the nanoparticle may be further defined as being encapsulated within the nanoparticle. It is also contemplated that the DNA-PK inhibitor, DNA Polymerase Theta (POLO) inhibitor, sgRNA, donor DNA, therapeutic cargo, Cas, or other elements recited herein may be in a composition with the nanoparticles and are not encapsulated within the nanoparticle. The DNA-PK inhibitor, DNA Polymerase Theta (POLO) inhibitor, sgRNA, donor DNA, therapeutic cargo, and/or Cas may be in a composition wherein at least or at most 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,
19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43,
44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68,
69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93,
94, 95, 96, 97, 98, 99, 100 percent (or any derivable range therein) of the DNA-PK inhibitor, DNA Polymerase Theta (POLO) inhibitor, sgRNA, donor DNA, therapeutic cargo, and/or Cas is encapsulated in the lipid nanoparticle.
[0025] The composition may comprise or exclude a second lipid nanoparticle, wherein the second lipid nanoparticle is selected from a lipid nanoparticle of the disclosure. The composition may comprise i) a lipid nanoparticle comprising a sgRNA of the disclosure and a Cas protein or a nucleic acid encoding a Cas protein; and ii) a second lipid nanoparticle comprising a donor DNA of the disclosure. The composition may comprise a carrier. The carrier may comprise ethylene glycol-bis(P-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA). The carrier may comprise 1-10 mM EGTA. The carrier may comprise, comprise at least, or comprise at most 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7,
7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9, 9.1, 9.2,
9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10, 10.1, 10.2, 10.3, 10.4, 10.5, 10.6, 10.7, 10.8, 10.9, 11, 11.1,
11.2, 11.3, 11.4, 11.5, 11.6, 11.7, 11.8, 11.9, 12, 12.1, 12.2, 12.3, 12.4, 12.5, 12.6, 12.7, 12.8,
12.9, 13, 13.1, 13.2, 13.3, 13.4, 13.5, 13.6, 13.7, 13.8, 13.9, 14, 14.1, 14.2, 14.3, 14.4, 14.5,
14.6, 14.7, 14.8, 14.9, 15, 15.1, 15.2, 15.3, 15.4, 15.5, 15.6, 15.7, 15.8, 15.9, 16, 16.1, 16.2,
16.3, 16.4, 16.5, 16.6, 16.7, 16.8, 16.9, 17, 17.1, 17.2, 17.3, 17.4, 17.5, 17.6, 17.7, 17.8, 17.9,
18, 18.1, 18.2, 18.3, 18.4, 18.5, 18.6, 18.7, 18.8, 18.9, 19, 19.1, 19.2, 19.3, 19.4, 19.5, 19.6,
19.7, 19.8, 19.9, 20, 20.1, 20.2, 20.3, 20.4, 20.5, 20.6, 20.7, 20.8, 20.9, 21, 21.1, 21.2, 21.3,
21.4, 21.5, 21.6, 21.7, 21.8, 21.9, 22, 22.1, 22.2, 22.3, 22.4, 22.5, 22.6, 22.7, 22.8, 22.9, 23,
23.1, 23.2, 23.3, 23.4, 23.5, 23.6, 23.7, 23.8, 23.9, 24, 24.1, 24.2, 24.3, 24.4, 24.5, 24.6, 24.7,
24.8, 24.9, 25, 25.1, 25.2, 25.3, 25.4, 25.5, 25.6, 25.7, 25.8, 25.9, or 30 mM, nM, M, or pM (or any derivable range therein) of EGTA. The carrier may comprise 6mM EGTA. The carrier may comprise or further comprise a buffer. The buffer may comprise HEPES. The carrier may comprise, comprise at least, or comprise at most 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,
19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43,
44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68,
69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93,
94, 95, 96, 97, 98, 99, or 100 mM, nM, M, or pM (or any derivable range therein) of buffer. The composition may comprise 0.5-5 ng/pL therapeutic cargo suspended in a carrier. The composition may comprise, comprise at least, or comprise at most 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8,
2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3,
7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, or 10 ng, pg, pg, mg, or g therapeutic cargo (or any derivable range therein). [0026] The donor DNA may be linear. The donor DNA may be circular. The donor DNA may comprise or exclude a transgene or mutation correction cassette and at least one homology arm that is 5’ or 3’ proximal to the transgene or cassette. The donor DNA may comprise two homology arms and wherein one homology arm is 5’ proximal to a transgene or a mutation correction cassette and at least one homology arm that is 5’ or 3’ proximal to the transgene or cassette, wherein the mutation correction cassette edits at least one pathogenic mutation in an endogenous gene. The transgene or endogenous gene may comprise or exclude CFTR, a dynein gene, DNAI1, DNAH5, GATA4, NR2F2, ZFPM2, WT1, a surfactant gene, surfactant protein C, surfactant protein B, alphal -antitrypsin gene, SMAD4, CHD7, Trisomy 18, APC, LKB1, MLH1, MSH2, MSH6, PMS2, EPCAM, N0D2, ATG16L1, IL23R, IRGM, HFE, SMAD4, or BMPR1 A, or a cDNA thereof. The transgene or endogenous gene may comprise a CFTR gene or cDNA. The donor DNA may comprise two homology arms and wherein one homology arm is 5’ proximal to the transgene or a mutation correction cassette and at least one homology arm that is 5’ or 3’ proximal to the transgene or cassette, wherein the mutation correction cassette edits at least one pathogenic mutation in an endogenous gene. A first region is 5’-proximal to a second region when the first region is attached to the 5’ terminus of the second region. There may be further intervening nucleotides between the first and second regions or intervening nucleotides may be excluded. Thus, the regions need not be immediately adjacent, unless specifically specified as not having intervening nucleotides.
[0027] The donor DNA may comprise a transgene and wherein the transgene is at least 3000 DNA bases. The transgene may be fewer than 8000 DNA bases. The transgene may comprse at least 4000 DNA bases. The transgene may comprise or comprise at least or comprise at most 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100,
2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000, 3100, 3200, 3300, 3400, 3500, 3600,
3700, 3800, 3900, 4000, 4100, 4200, 4300, 4400, 4500, 4600, 4700, 4800, 4900, 5000, 5100,
5200, 5300, 5400, 5500, 5600, 5700, 5800, 5900, 6000, 6100, 6200, 6300, 6400, 6500, 6600,
6700, 6800, 6900, 7000, 8000, 9000, 10000, 11000, or 12000 bases, or any range derivable therein. The donor DNA may comprise at least 1700 kDa. The donor DNA may be, be at least, or be at most 300, 305, 310, 315, 320, 325, 330, 335, 340, 345, 350, 355, 360, 365, 370, 375, 380, 385, 390, 395, 400, 405, 410, 415, 420, 425, 430, 435, 440, 445, 450, 455, 460, 465, 470,
475, 480, 485, 490, 495, 500, 505, 510, 515, 520, 525, 530, 535, 540, 545, 550, 555, 560, 565,
570, 575, 580, 585, 590, 595, 600, 605, 610, 615, 620, 625, 630, 635, 640, 645, 650, 655, 660,
665, 670, 675, 680, 685, 690, 695, 700, 705, 710, 715, 720, 725, 730, 735, 740, 745, 750, 755,
760, 765, 770, 775, 780, 785, 790, 795, 800, 805, 810, 815, 820, 825, 830, 835, 840, 845, 850,
855, 860, 865, 870, 875, 880, 885, 890, 895, 900, 905, 910, 915, 920, 925, 930, 935, 940, 945,
950, 955, 960, 965, 970, 975, 980, 985, 990, 995, 1000, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000, 3500, 4000, 4500, 5000, 6000, 7000, 8000, 9000, 10000, 11000, 12000, 13000, 14000, 15000, 16000, 17000, 18000, 19000, or 20000 kDa (or any derivable range therein) in molecular mass. The donor DNA may comprise a nucleic acid that encodes for a protein that is at least 1700 kDa. The donor DNA may comprise a nucleic acid that encodes for a protein that is, is at least, or is at most 300, 305, 310, 315, 320, 325, 330, 335, 340, 345, 350, 355, 360, 365, 370, 375, 380, 385, 390, 395, 400, 405, 410, 415, 420, 425, 430, 435, 440, 445, 450, 455, 460, 465, 470,
475, 480, 485, 490, 495, 500, 505, 510, 515, 520, 525, 530, 535, 540, 545, 550, 555, 560, 565,
570, 575, 580, 585, 590, 595, 600, 605, 610, 615, 620, 625, 630, 635, 640, 645, 650, 655, 660,
665, 670, 675, 680, 685, 690, 695, 700, 705, 710, 715, 720, 725, 730, 735, 740, 745, 750, 755,
760, 765, 770, 775, 780, 785, 790, 795, 800, 805, 810, 815, 820, 825, 830, 835, 840, 845, 850,
855, 860, 865, 870, 875, 880, 885, 890, 895, 900, 905, 910, 915, 920, 925, 930, 935, 940, 945,
950, 955, 960, 965, 970, 975, 980, 985, 990, 995, 1000, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000, 3500, 4000, 4500, 5000, 6000, 7000, 8000, 9000, 10000, 11000, 12000, 13000, 14000, 15000, 16000, 17000, 18000, 19000, or 20000 kDa (or any derivable range therein) in molecular mass. [0028] The term “3 ’-proximal” is similarly defined in that a first region is 3 ’-proximal to a second region when the first region is attached to the 3’ terminus of the second region. Similarly, there may be further intervening nucleotides between the first and second regions unless stated otherwise.
[0029] The 5’ proximal, 3’ proximal, or both 5’ and 3’ proximal homology arm may be fewer than 150 nucleotides and be at least 90% homologous to an endogenous genomic sequence. The 5’ proximal, 3’ proximal, or both 5’ and 3’ proximal homology arm may be fewer than 150 nucleotides and comprise at least 90% sequence identity to an endogenous genomic sequence of the same length of the homology arm. The 5’ proximal, 3’ proximal, or both 5’ and 3’ proximal homology arm may be or be fewer than 20, 21, 22, 23, 24, 25, 26, 27,
28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52,
53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77,
78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101,
102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120,
121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139,
140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158,
159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177,
178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196,
197, 198, 199, or 200 nucleotides, or any derivable range therein. The 5’ proximal, 3’ proximal, or both 5’ and 3’ proximal homology arm may have or have at least 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%, or any deriviable range therein, of homology or sequence identity to an endogenous genomic sequence. The endogenous genomic sequence may be one of the same length as the homology arm. The donor DNA may be modified. The modification may comprise a modification of 6-12 Carbons at the 5’ terminus of the DNA molecule. The modification may comprise or consist of 6 carbons at the 5’ terminus of the DNA molecule.
[0030] A donor DNA may include additional elements that enhances expression of the gene. The donor DNA may include a posttranscriptional regulatory element. The posttranscriptional regulatory element may comprise SEQ ID NO: 11, an amino acid sequence comprising a fragment of SEQ ID NO: 11, an amino acid sequence with at least 80% sequence identity to SEQ ID NO: 11, or an amino acid sequence with at least 80% sequence identity to a fragment of SEQ ID NO: 11. The posttranscriptional regulatory element may comprise an amino acid sequence with or with at least 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%, or any derivable range therein, sequence identity to SEQ ID NO: 11.
[0031] The donor DNA may comprise a polyadenylation signal. The polyadenylation signal may comprise the amino acid sequence of SEQ ID NO: 12, an amino acid sequence comprising a fragment of SEQ ID NO: 12, an amino acid sequence with at least 80% sequence identity to SEQ ID NO: 12, or an amino acid sequence with at least 80% sequence identity to a fragment of SEQ ID NO: 12. The polyadenylation signal may comprise an amino acid sequence with or with at least 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%, or any derivable range therein, sequence identity to SEQ ID NO: 12.
[0032] The donor DNA may comprise a nucleic acid encoding for the amino acid sequence of SEQ ID NO: 14, an amino acid sequence of a fragment of SEQ ID NO: 14, an amino acid sequence with at least 80% sequence identity to SEQ ID NO: 14, or an amino acid sequence with at least 80% sequence identity to a fragment of SEQ ID NO: 14. The donor DNA may comprise a nucleic acid encoding or an amino acid sequence with or with at least 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%, or any derivable range therein, sequence identity to SEQ ID NO: 14.
[0033] The donor DNA may comprise the nucleic acid sequence of SEQ ID NO: 13, a nucleic acid that is a fragment of SEQ ID NO: 13, a nucleic acid sequence with at least 80% sequence identity to the nucleic acid sequence of SEQ ID NO: 13, or a nucleic acid sequence with at least 80% sequence identity to a fragment of the nucleic acid sequence of SEQ ID NO: 13. The donor DNA may comprise a nucleic acid sequence with or with at least 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%, or any derivable range therein, sequence identity to SEQ ID NO: 13.
[0034] The 5’ proximal homology arm may comprise the nucleic acid sequence of SEQ ID NO: 15 or 16, a nucleic acid that is a fragment of SEQ ID NO: 15 or 16, a nucleic acid sequence with at least 80% sequence identity to the nucleic acid sequence of SEQ ID NO: 15 or 16, or a nucleic acid sequence with at least 80% sequence identity to a fragment of the nucleic acid sequence of SEQ ID NO: 15 or 16. The 5’ proximal homology arm may comprise a nucleic acid sequence with or with at least 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%, or any derivable range therein, sequence identity to SEQ ID NO: 15 or 16.
[0035] The 3’ proximal homology arm may comprise the nucleic acid sequence of SEQ ID NO: 17, a nucleic acid that is a fragment of SEQ ID NO: 17, a nucleic acid sequence with at least 80% sequence identity to the nucleic acid sequence of SEQ ID NO: 17, or a nucleic acid sequence with at least 80% sequence identity to a fragment of the nucleic acid sequence of SEQ ID NO: 17. The 3’ proximal homology arm may comprise a nucleic acid sequence with or with at least 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%, or any derivable range therein, sequence identity to SEQ ID NO: 17.
[0036] The donor DNA may comprise the nucleic acid sequence of SEQ ID NO: 18, a nucleic acid that is a fragment of SEQ ID NO: 18, a nucleic acid sequence with at least 80% sequence identity to the nucleic acid sequence of SEQ ID NO: 18, or a nucleic acid sequence with at least 80% sequence identity to a fragment of the nucleic acid sequence of SEQ ID NO: 18. The donor DNA may comprise a nucleic acid sequence with or with at least 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%, or any derivable range therein, sequence identity to SEQ ID NO: 18.
[0037] The sgRNA may comprise or consists of the nucleotide sequence of one of SEQ ID NOS: 1-5. The sgRNA may be single-stranded. The sgRNA may be double stranded.
[0038] A pathogenic mutation refers to a mutation in a gene that contributes to the pathogenesis of a disease. Correcting such mutation may restore the endogenous function of the protein encoded by the gene and treat the disease. The mutation correction cassette refers to a donor DNA that, instead of an entire transgene, comprises a fragment of a gene that corresponds to a pathogenic mutation in the genome of the subject. The donor DNA may comprise a DNA sequence that corrects the pathogenic mutation in the DNA to encode for a protein with the wild-type amino acid or a protein with a non-pathogenic amino acid.
[0039] The gene editing may include or exclude homology directed repair. The gene editing may include or exclude non-homologous end joining. The gene editing achieved by the compositions, methods, and embodiments of the disclosure may be, be at least, or be at most, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2,
2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4,
4.5, 4.6, 4.7, 4.8, 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6,
6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8,
8.9, 9, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10, 10.1, 10.2, 10.3, 10.4, 10.5, 10.6, 10.7, 10.8,
10.9, 11, 11.1, 11.2, 11.3, 11.4, 11.5, 11.6, 11.7, 11.8, 11.9, 12, 12.1, 12.2, 12.3, 12.4, 12.5,
12.6, 12.7, 12.8, 12.9, 13, 13.1, 13.2, 13.3, 13.4, 13.5, 13.6, 13.7, 13.8, 13.9, 14, 14.1, 14.2,
14.3, 14.4, 14.5, 14.6, 14.7, 14.8, 14.9, 15, 15.1, 15.2, 15.3, 15.4, 15.5, 15.6, 15.7, 15.8, 15.9,
16, 16.1, 16.2, 16.3, 16.4, 16.5, 16.6, 16.7, 16.8, 16.9, 17, 17.1, 17.2, 17.3, 17.4, 17.5, 17.6,
17.7, 17.8, 17.9, 18, 18.1, 18.2, 18.3, 18.4, 18.5, 18.6, 18.7, 18.8, 18.9, 19, 19.1, 19.2, 19.3,
19.4, 19.5, 19.6, 19.7, 19.8, 19.9, 20, 20.1, 20.2, 20.3, 20.4, 20.5, 20.6, 20.7, 20.8, 20.9, 21,
21.1, 21.2, 21.3, 21.4, 21.5, 21.6, 21.7, 21.8, 21.9, 22, 22.1, 22.2, 22.3, 22.4, 22.5, 22.6, 22.7,
22.8, 22.9, 23, 23.1, 23.2, 23.3, 23.4, 23.5, 23.6, 23.7, 23.8, 23.9, 24, 24.1, 24.2, 24.3, 24.4,
24.5, 24.6, 24.7, 24.8, 24.9, 25, 25.1, 25.2, 25.3, 25.4, 25.5, 25.6, 25.7, 25.8, 25.9, 26, 26.1,
26.2, 26.3, 26.4, 26.5, 26.6, 26.7, 26.8, 26.9, 27, 27.1, 27.2, 27.3, 27.4, 27.5, 27.6, 27.7, 27.8,
27.9, 28, 28.1, 28.2, 28.3, 28.4, 28.5, 28.6, 28.7, 28.8, 28.9, 29, 29.1, 29.2, 29.3, 29.4, 29.5,
29.6, 29.7, 29.8, 29.9, 30, 30.1, 30.2, 30.3, 30.4, 30.5, 30.6, 30.7, 30.8, 30.9, 31, 31.1, 31.2,
31.3, 31.4, 31.5, 31.6, 31.7, 31.8, 31.9, 32, 32.1, 32.2, 32.3, 32.4, 32.5, 32.6, 32.7, 32.8, 32.9, 33, 33.1, 33.2, 33.3, 33.4, 33.5, 33.6, 33.7, 33.8, 33.9, 34, 34.1, 34.2, 34.3, 34.4, 34.5, 34.6,
34.7, 34.8, 34.9, 35, 35.1, 35.2, 35.3, 35.4, 35.5, 35.6, 35.7, 35.8, 35.9, 36, 36.1, 36.2, 36.3,
36.4, 36.5, 36.6, 36.7, 36.8, 36.9, 37, 37.1, 37.2, 37.3, 37.4, 37.5, 37.6, 37.7, 37.8, 37.9, 38,
38.1, 38.2, 38.3, 38.4, 38.5, 38.6, 38.7, 38.8, 38.9, 39, 39.1, 39.2, 39.3, 39.4, 39.5, 39.6, 39.7,
39.8, 39.9, 40, 40.1, 40.2, 40.3, 40.4, 40.5, 40.6, 40.7, 40.8, 40.9, 41, 41.1, 41.2, 41.3, 41.4,
41.5, 41.6, 41.7, 41.8, 41.9, 42, 42.1, 42.2, 42.3, 42.4, 42.5, 42.6, 42.7, 42.8, 42.9, 43, 43.1,
43.2, 43.3, 43.4, 43.5, 43.6, 43.7, 43.8, 43.9, 44, 44.1, 44.2, 44.3, 44.4, 44.5, 44.6, 44.7, 44.8,
44.9, 45, 45.1, 45.2, 45.3, 45.4, 45.5, 45.6, 45.7, 45.8, 45.9, 46, 46.1, 46.2, 46.3, 46.4, 46.5,
46.6, 46.7, 46.8, 46.9, 47, 47.1, 47.2, 47.3, 47.4, 47.5, 47.6, 47.7, 47.8, 47.9, 48, 48.1, 48.2,
48.3, 48.4, 48.5, 48.6, 48.7, 48.8, 48.9, 49, 49.1, 49.2, 49.3, 49.4, 49.5, 49.6, 49.7, 49.8, 49.9, 50, 50.1, 50.2, 50.3, 50.4, 50.5, 50.6, 50.7, 50.8, 50.9, 51, 51.1, 51.2, 51.3, 51.4, 51.5, 51.6, 51.7, 51.8, 51.9, 52, 52.1, 52.2, 52.3, 52.4, 52.5, 52.6, 52.7, 52.8, 52.9, 53, 53.1, 53.2, 53.3, 53.4, 53.5, 53.6, 53.7, 53.8, 53.9, 54, 54.1, 54.2, 54.3, 54.4, 54.5, 54.6, 54.7, 54.8, 54.9, 55, 55.1, 55.2, 55.3, 55.5, 55.5, 55.6, 55.7, 55.8, 55.9, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 % (or any derivable range therein) of gene editing/gene correct and/or of restoration of gene fuction.
[0040] The airway or gastrointestinal disorder may comprise or exclude an epithelial disorder, COVID, a respiratory virus, a pulmonary fibrosis disorder, or cystic fibrosis. The airway or gastrointestinal disorder may comprise cystic fibrosis. The treatment may exclude treatment of a virus.
[0041] The subject may be administered the first lipid nanoparticle before, concurrently with, and/or after the second lipid nanoparticle. The subject may be administered the first lipid nanoparticle before the second lipid nanoparticle. The subject may be administered the second lipid nanoparticle before, concurrently with, and/or after the first lipid nanoparticle. The subject may be administered the second lipid nanoparticle after the first lipid nanoparticle. The first lipid nanoparticle may be administered at least 3 hours before the second lipid nanoparticle. The first lipid nanoparticle may be administered at, administered at least, or administered at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 hours, days, or weeks (or any derivable range therein) before or after the administration of the second lipid nanoparticle. The first and second lipid nanoparticle may be administered within 1 day of each other. The first and second lipid nanoparticle may be administered within, within at least, or within at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,
24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48,
49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73,
74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98,
99, or 100 hours, days, or weeks (or any derivable range therein) of each other.
[0042] The dose of the lipid nanoparticle, first lipid nanoparticle, and/or second lipid nanoparticle may be, be at least, or be at most .1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129,
130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148,
149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167,
168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186,
187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205,
206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224,
225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243,
244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262,
263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281,
282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300,
301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319,
320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338,
339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357,
358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376,
377, 378, 379, 380, 381, 382, 383, 384, 385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395,
396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414,
415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428, 429, 430, 431, 432, 433,
434, 435, 436, 437, 438, 439, 440, 441, 442, 443, 444, 445, 446, 447, 448, 449, 450, 451, 452,
453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471,
472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 484, 485, 486, 487, 488, 489, 490,
491, 492, 493, 494, 495, 496, 497, 498, 499, or 500 pg, ng, pg, mg, g, pg/kg, ng/kg, pg/kg, mg/kg, or g/kg, or any derivable range therein.
[0043] The subject may be one that has or has been diagnosed with a disease. The disease may be an airway disease or a gut disease. The therapeutic cargo may comprise a CRISPR/Cas system that corrects a pathogenic mutation associated with the disease. The pathogenic mutation may be one that is known in the art and/or determined to be present in cells from the subject. The mutation correction cassette may edit an edogenous gene that comprises a diseasecausing mutation. Example of disorders and corresponding endogenous genes that may be edited/replaced with a transgene are provide in the table below:
[0044] The term “gene” refers to a nucleic acid encoding for a functional protein. The gene may include or exclude introns. The gene may be a transgene. The nucleic acid may be further defined as a cDNA.
[0045] The term “subject” and “patient” may be used interchangeably and may refer to a human subject. The subject may be defined as a mammalian subject. The subject may also be a mouse, rat, pig, horse, non-human primate, cat, dog, cow, and the like.
[0046] Throughout this application, the term “about” is used according to its plain and ordinary meaning in the area of cell and molecular biology to indicate that a value includes the standard deviation of error for the device or method being employed to determine the value.
[0047] The use of the word “a” or “an” when used in conjunction with the term “comprising” may mean “one,” but it is also consistent with the meaning of “one or more,” “at least one,” and “one or more than one.”
[0048] As used herein, the terms “or” and “and/or” are utilized to describe multiple components in combination or exclusive of one another. For example, “x, y, and/or z” can refer to “x” alone, “y” alone, “z” alone, “x, y, and z,” “(x and y) or z,” “x or (y and z),” or “x or y or z.” It is specifically contemplated that x, y, or z may be specifically excluded from an embodiment or aspect.
[0049] The words “comprising” (and any form of comprising, such as “comprise” and “comprises”), “having” (and any form of having, such as “have” and “has”), “including” (and any form of including, such as “includes” and “include”), “characterized by” (and any form of including, such as “characterized as”), or “containing” (and any form of containing, such as “contains” and “contain”) are inclusive or open-ended and do not exclude additional, unrecited elements or method steps.
[0050] The compositions and methods for their use can “comprise,” “consist essentially of,” or “consist of’ any of the ingredients or steps disclosed throughout the specification. The phrase “consisting of’ excludes any element, step, or ingredient not specified. The phrase “consisting essentially of’ limits the scope of described subject matter to the specified materials or steps and those that do not materially affect its basic and novel characteristics. It is contemplated that embodiments and asp ectsde scribed in the context of the term “comprising” may also be implemented in the context of the term “consisting of’ or “consisting essentially of.”
[0051] Any method in the context of a therapeutic, diagnostic, or physiologic purpose or effect may also be described in “use” claim language such as “Use of’ any compound, composition, or agent discussed herein for achieving or implementing a described therapeutic, diagnostic, or physiologic purpose or effect.
[0052] Use of the one or more sequences or compositions may be employed based on any of the methods described herein. Other embodiments and aspects are discussed throughout this application. Any embodiment discussed with respect to one aspect of the disclosure applies to other aspects of the disclosure as well and vice versa. Furthermore, any composition of the invention may be used in any method of the invention, and any method of the invention may be used to produce or to utilize any composition of the invention. Aspects of an embodiment set forth in the Examples are also embodiments that may be implemented in the context of embodiments discussed elsewhere in a different Example or elsewhere in the application, such as in the Summary of Invention, Detailed Description of the Embodiments, Claims, and description of Figure Legends.
[0053] Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating specific embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description. BRIEF DESCRIPTION OF THE DRAWINGS
[0054] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.
[0055] FIG. 1. Schematic outlining strategies for nebulized delivery of lipid nanoparticles (LNPs) engineered to enable inhalable gene therapy strategies for treating inherited diseases of the airway.
[0056] FIG. 2. Schematic illustrating the workflow for preparing lipid mixtures for the synthesis of epithelial-penetrating nanoparticles (epNPs) and CRISPR-nanoparticles (crNPs).
[0057] FIG. 3A-3D. (A) Schematic illustrating the microfluidic mixing methodology to synthesize epithelial-penetrating nanoparticles (NPs). (B) Summary size and poly dispersity data collected via dynamic light scattering (DLS) for epNPs containing increasing mole % of either lysophosphatydilcholine (LPC) or polidocanol (PDOC). (C) Size distribution of epNPs containing LPC and (D) PDOC as determined by DLS analysis.
[0058] FIG. 4A-4C. (A) Schematic illustrating mixing methodologies that leverage rapid pipetting or commercial microfluidic platforms such as the NanoAssemblr Spark (Precision NanoSystems) that combine lipid constituents with messenger RNA (mRNA) constructs to synthesize CRISPR-nanoparticles (crNPs).
[0059] FIG. 5A-5C. (A) Schematic illustrating aerosol-based delivery of the invented CRISPR-nanoparticles (crNPs) to airway basal stem cells (ABSCs) cultured on an air-liquid interface (ALI). (B & C) Confocal micrographs displaying green fluorescent protein (GFP) expression in ABSCs cultured on an ALI. Cells were counterstained for keratin 5 (magenta, AlexaFluor 594) and with a nuclear dye (blue, DAPI) after treatment with LNPs loaded with GFP-encoding mRNA. The ABSC cultures shown were exposed to the nebulized LNP aerosols for 2.5 min.
[0060] FIG. 6A-6B. Fig. 6A: (Left, upper) Fluorescence microscope image of immortalized human bronchial epithelial cells (16HBE14o-) expressing green fluorescent protein (GFP) and counter-stained with a nuclear dye (DAPI) and (left, lower) flow cytometry data reporting viability and GFP expression in 16HBE14o- cells 24 h after treatment with CRISPR-nanoparticles (crNPs) composed of ionizable lipids Lipid A6, Lipid 5 or SM-102, either cholesterol (chol) or 30-[N-(N’N, N’-dimethylaminoethane)-carbamoyl]cholesterol (DC-chol), and either poly(ethylene glycol)-mysristoyl diglyceride (DPEG) or PEG dioleoylphosphatidylethanolamine (PPEG). These crLNPs were loaded with messenger RNA (mRNA) constructs encoding GFP. Scale bar = 100 pm. (Right, upper) Confocal micrographs of air-liquid interface (ALI) cultures 4 and 24 h post-treatment with polidocanol-decorated lipid nanoparticles (PDOC-epLNPs). Cells were counterstained for basal stem cell (AB SC) marker keratin 5 (red, AlexaFluor 594) and with a nuclear dye (blue, DAPI). Increased K5 fluorescence indicates exposure of ABSCs. Scale bars = 100 pm. (Right, lower) Cutting efficiencies as measured by tracking of insertion-deletions by decomposition (TIDE) at the cystic fibrosis transmembrane conductance regulator (CFTR) locus in 16HBE14o- cells 72 h after treatment with crNPs loaded with mRNA transcripts encoding Cas9 along with a guide RNA targeting CFTR. FIG. 6B: GFP expression in LNP -treated primary human bronchial epithelial cells (HBEs).
[0061] FIG. 7 Donor Comparison of dsDNA against plasmid and nanoplasmid.
[0062] FIG. 8. Donor Comparison HA Sizes and Chem Mod.
[0063] FIG. 9 AmC6 Donor compared to other dsDNA modifications.
[0064] FIG. 10. AmC6 Integration (by HDR/MMEJ) after NHEJ Inhibition (using AZD 7468 small molecule).
[0065] FIG. 11. HBE vs T84 sgRNA Editing Data at CFTR locus.
[0066] FIG. 12. Liposome synthesis (further described in Example 3).
[0067] FIG. 13A-13D. LNP synthesis optimization for larger CRISPR/Cas9 mRNA cargoes using a microfluidic mixing device.
[0068] FIG. 14. (Left) Flow cytometry analysis of blue fluorescent protein (BFP)- expressing T84 cells 72 h post-treatment with (right) LNPs carrying Cas9 constructs to edit the BFP locus.
[0069] FIG. 15. PDOC Incorporation into LNPs. Cationic LNPs synthesized with increasing wt % PDOC exhibit decreasing surface charge. Successful fabrication of small (>200 nm), monodisperse LNPs containing up to 40 wt % PDOC. Surface charge measurements suggest LNP-polidocanol (PDOC) interactions and small (<200nm), monodisperse particles up to 40 wt% PDOC.
[0070] FIG. 16A-16D Flow cytometry and live cell imaging analysis of CFTRexpressing T84 cells treated with plasmid-lipoplexes of varying composition.
[0071] FIG. 17A-17B Fluorescence imaging and flow cytometry confirm lipoplex- mediated delivery of GFP expression plasmids in CFTR-expressing T84 cells.
[0072] FIG. 18. Validation of LNP platform.
[0073] FIG. 19. Optimizing Synthetic Protocols for Larger Cargoes. [0074] FIG. 20. Optimizing Synthetic Protocols for Larger Cargoes.
[0075] FIG. 21A-21G. (A) Schematic depicting workflow for LNP -mediated editing, expansion, and downstream functional analysis of 16HBE-G542x cells. (B) ddPCR data reflecting site-specific integration of a codon-optimized CFTR cassette within the 5’UTR of the endogenous CFTR gene and (C) MTS assay data depicting relative cell survival in 16HBE- G542x- cells 24 hrs after treatment with LNPs at various dosages. (D) Western blot data depicting protein expression in cultures expanded from edited 16HBE-G542x cells. (E) Ussing chamber traces depicting CFTR-dep endent Cl- ion current in CF mutant 16HBEgeG542x cells, wild type 16HBE14o- cells, and 16HBEgeG542x cells bulk-edited to express codon-optimized CFTR. (F) Maximum Cl- ion current inhibitable by CFTR inhibitor 172 (CFTRinh-172) as measured via Ussing chamber. (E) LNPs containing dsDNA exhibit greater heterogeneity compared to LNPs containing only RNA.
[0076] FIG. 22. Synthesis and culture of LNPs.
[0077] FIG. 23. Size distribution of crLNPs.
[0078] FIG. 24A-24B. Screening and characterization of crLNP formulations.
[0079] FIG. 25. LNPs achieve site-specific integration of CFTR. crLNPs were packaged with Cas9 mRNA, mCitrine (left) or CFTR (right) double-stranded DNA (dsDNA) donor cassettes, and guide RNAs (gRNA) targeting the CFTR 5’UTR. Integration at the CFTR locus in 16HBE14o- and 16HBEG542x cells was measured by digital droplet PCR (ddPCR).
[0080] FIG. 26. Cystic fibrosis (CF) is caused by mutations within the cystic fibrosis transmembrane conductance regulator (CFTR) gene, resulting in formation of thick airway mucus & decreased mucociliary clearance. The inventors sought to establish inhalable gene therapy solutions for correcting any CFTR mutation in airway basal stem cells (ABSCs). Do do this, they plan to: 1) configure lipid nanoparticle (LNP) carriers to package CRISPR/Cas9 gene-editing reagents to form CRISPR-LNPs (crLNPs); 2) establish parameters for EGTA- based permeabilization of epithelia to enable gene delivery to ABSCs; 3) deliver LNP- packaged therapeutic cargoes to ABSCs via aerosol.
[0081] FIG. 27. ABSCs were grown in air-liquid interface (ALI) cultures to recapitulate the structure of the airway epithelium.
[0082] FIG. 28. crLNPs synthesized with the Precision NanoSystems Spark form uniform size distributions regardless of cargo type.
[0083] FIG. 29A-29G. (A) Schematic illustrating the blue fluorescent protein (BFP)- expressing 16HBE14o- reporter line used as a tool to assess capabilities of LNPs to deliver functional gene editing cargos including mRNA constructs encoding Cas9, single guide RNA (sgRNA), and a single-stranded oligonucleotide (ssODN) donor, and to screen cargo component ratios. Loss of blue fluorescence indicates non-homologous end-joining (NHEJ), and expression of green fluorescent protein (GFP) indicates homology-directed repair (HDR). (B) Flow cytometry data depicting rates of NHEJ and HDR in BFP-expressing 16HBE14o- cells 5 days after treatment with LNPs loaded with mRNA transcripts encoding Cas9, sgRNA, and an ssODN targeting the BFP gene. The ratio of ssODN to mRNA was held constant at 3 : 1 w/w, with the ratio of sgRNA to mRNA titrated from 0.8: 1 w/w to 2: 1 w/w. (C) Flow cytometry data depicting rates of NHEJ and HDR in BFP-expressing 16HBE14o- cells 5 days after treatment with LNPs loaded with mRNA transcripts encoding Cas9, sgRNA, and an ssODN targeting the BFP gene. The ratio of sgRNA to mRNA was held constant at 1.2:1 w/w, with the ratio of ssODN to mRNA titrated from 2: 1 w/w to 4: 1 w/w. (D) Fluorescence microscopy image displaying GFP expression indicating HDR in BFP-expressing 16HBE14o- cells 5 days after treatment with LNPs loaded with mRNA transcripts encoding Cas9, sgRNA, and an ssODN targeting the BFP gene at a mRNA: sgRNA: ssODN ratio of 1 :1.2:3 w/w/w. Cell nuclei were counterstained with DAPI. (E) Dynamic light scattering (DLS) data depicting the size distributions of LNPs loaded with various ssODN:mRNA w/w ratios. (F-G) DLS data depicting the Z-average diameter and encapsulation efficiencies as measured via the RiboGreen assay (ThermoFisher) of LNPs loaded with various ssODN:mRNA w/w ratios.
[0084] FIG. 30A-30F. (A) Schematic defining comparison between lipid nanoparticles (LNPs) packaged with either mRNA encoding Cas9 (Cas9-mRNA) together with a singleguide RNA (sgRNA) and/or a double-stranded DNA (dsDNA) donor cassette. Particle mixtures separating the dsDNA into a distinct LNP are referred to as sepLNPs, and formulations that combine all three gene-editing cargoes into a single formulation are referred to as togLNPs. (B) Dynamic light scattering (DLS) data depicting the Z-average diameter of sepLNPs and togLNPs and encapsulation efficiencies as measured via the RiboGreen assay (ThermoFisher). (C) Digital droplet PCR (ddPCR) data reflecting site-specific integration of an mCitrine reporter cassette within the 5’ untranslated region (UTR) of the endogenous cystic fibrosis transmembrane conductance regulator (CFTR) gene in 16HBE14o- cells 48 hrs after treatment with sepLNPs or togLNPs. (D) Fluorescence microscopy images depicting 16HBE14o- cells 48 hrs after treatment with sepLNPs or togLNPs. Cell nuclei were counterstained with DAPI. Scale bars represent 200 pm. (E) ddPCR data reflecting site-specific integration of a codon-optimized CFTR cassette within the 5 ’UTR of the endogenous CFTR gene and (F) tetrazolium (MTS) assay data depicting relative cell survival in 16HBE-G542x- cells 48 hrs after treatment with LNPs formulated with various dsDNA: Cas9-mRNA w/w and nitrogen-to-phosphate (N/P) ratios. Cells were incubated overnight with LNPs either with or without drugs AZD7648 and ART558.
[0085] FIG. 31. Cell survival (left) and integration efficiency (left) in 16HBEge-G542x cells 48 h after exposure to LNPs packaged with Cas9 mRNA, dsDNA encoding CFTR, and gRNA targeting the CFTR 5’UTR. LNPs comprised various cargo ratios (top) and N/P ratios (bottom). Drugs added include AZD7648 and ART558.
[0086] FIG. 32. Treatment of ALIs with LNPs loaded with mRNA transcripts encoding GFP suspended in hypotonic EGTA permeabilize the epithelia and enable transfection of airway cells. Scale bars represent 100 pm.
[0087] FIG. 33A-33F. (A) Schematic depicting stepwise selection of lipid nanoparticle (LNP) constituents for optimized delivery of mRNA to a human bronchial epithelial cell line (16HBE14O-). (B) Representative transmission electron microscopy (TEM) image depicting optimized LNPs of uniform size and morphology. (C) Schematic outlining workflow for synthesis of LNPs using a commercial bifurcating toroidal mixer and screening for LNP- mediated delivery of reporter mRNA constructs encoding GFP to 16HBE14o- cells, quantified using flow cytometry. (D) Fluorescence microscope image of 16HBE14o- cells 24 h after treatment with LNPs carrying mRNA constructs encoding GFP. Cell nuclei were counterstained with DAPI.
[0088] FIG. 34A-34C. (A) Cryogenic transmission electron microscopy (cryo-TEM) images depicting LNPs containing Cas9 mRNA and single guide RNA (sgRNA) (top), doublestranded DNA (dsDNA) encoding CFTR (middle), or Cas9 mRNA, sgRNA, and CFTR dsDNA (bottom). Scale bar represent 200 nm. (B) Dynamic light scattering (DLS) data portraying the size distribution of LNPs containing Cas9 mRNA and sgRNA only, CFTR dsDNA only, or Cas9 mRNA, sgRNA, and CFTR dsDNA. (C) DLS and RiboGreen assay data depicting the Z- ave diameter (first bar in each group of 3 bars, left y-axis), poly dispersity (Pdl) (second bar in each group of 3 bars, right y-axis), and encapsulation efficiency (EE) (third bar in each group of 3 bars, right y-axis) of LNPs containing Cas9 mRNA and sgRNA only, CFTR dsDNA only, or Cas9 mRNA, sgRNA, and CFTR dsDNA.
[0089] FIG. 35A-35C. (A) Dynamic light scattering (DLS) and encapsulation data depicting the size distributions (left) and diameter, polydispersity (Pdl), and encapsulation efficiencies (right) of LNPs packaged with GFP mRNA and containing either Lipid 5, SM-102, or Lipid A6. (B) DLS and encapsulation data depicting the size distributions (left) and diameter, Pdl, and encapsulation efficiencies (right) of LNPs packaged with GFP mRNA and containing either sitosterol, cholesterol, or DC-cholesterol. (C) DLS and encapsulation data depicting the size distributions (left) and diameter, Pdl and encapsulation efficiencies (right) of LNPs packaged with GFP mRNA and containing either DMG-PEG200, DOPE-PEG2000, or DSPE-PEG2000.
[0090] FIG. 36. Viability and editing of BFP-16HBE14o- cells treated with LNPs loaded with Cas9 mRNA, sgRNA targeting the BFP locus, and ssODNs configured to convert BFP to GFP, at various sgRNA:mRNA w/w ratios (top) or ssODN:mRNA ratios (bottom). Cells were stained with 7-AAD viability dye and live cells were quantified via flow cytometry. “Experiment 1 a” and “Experiment 2a” refer to the editing data reported in the text. “Experiment lb” and “Experiment 2b” are biological replicates of the data reported in the text.
[0091] FIG. 37. Analysis of insertion-deletions producing allelic disruption of the 5’UTR within the endogenous cystic fibrosis transmembrane conductance regulator (CFTR) gene. Mock samples were treated with unloaded LNPs containing no nucleic acid. “RNA only” LNPs were loaded with Cas9 mRNA and guide RNA (gRNA) only. “DNA only” LNPs were loaded with a double-stranded DNA (dsDNA) donor cassette encoding CFTR. Analysis was conducted using Synthego’s online ICE tool.
[0092] FIG. 38. Chloride ion flux traces of 16HBE samples grown on SnapWell inserts and assayed in an Ussing chamber.
DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS
[0093] Cystic fibrosis (CF) is a disorder arising from mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene that leads to chronic lung infections and respiratory failure. Diseases like CF are appealing targets for gene therapies, but existing gene editing approaches that enable the site-specific correction of disease-causing defects rely on large double-stranded DNA (dsDNA) constructs that are inefficient to package and deliver to targeted airway basal stem cells (ABSCs) lining the CF airway. To address this challenge, the inventors designed and tested a gene therapy strategy that applies lipid nanoparticle (LNP) vehicles that facilitate transport of CFTR-directed CRISPR/Cas9 gene editing tools to ABSCs. The inventors have shown that hypotonic EGTA carrier solutions clear a path to ABSCs in air liquid interface (ALI) culture models of the human airway for LNPs loaded with green fluorescent protein (GFP)-encoding mRNA cargoes. Confocal microscopy analysis of these ALI cultures exhibit expression of the GFP reporter. The capabilities of LNPs packaged with CRISPR/Cas9 cargoes configured to direct editing within the 5’ untranslated region of the endogenous CFTR locus were tested using a human bronchial epithelial cell line (16HBE14o- ). Packaging these gene editing reagents into a single LNP resulted in ca. 14% integration compared to ca. 10% when the dsDNA donor was packaged in a separate nanoparticle. These studies informed experiments applying the LNPs for correction of CFTR in a CF mutant cell line (16HBEge-G542x). LNPs carrying a CFTR dsDNA correction template achieve up to 3.5% integration. These nanotechnology-enabled tools overcome limitations in the cargocarrying capacity of existing viral vector and nanoparticle delivery vehicles. This work sets a new precedent for packaging and delivering large dsDNA templates in nanocarriers, which can be leveraged to accelerate the clinical deployment of gene therapies for CF and other genetic diseases.
[0094] Methods described herein enable: 1) localized perturbation of the apical epithelium to better access airway basal stem/progenitor cell (AB SC) populations and 2) efficient delivery of biomolecules to enable long-term gene correction of these progenitor cells (FIG. 1). Methods include a two-step nanoparticle platform that facilitates access and delivery of gene editing reagents to ABSCs, and is robust when aerosolized.
I. Lipid Nanoparticles
[0095] In some embodiments, a lipid nanoparticle is a liposomes, an exosomes, lipid preparations, or another lipid nanoparticle, such as a lipid-based vesicle (e.g., a DOTAP:cholesterol vesicle). Lipid-based nanoparticles may be positively charged, negatively charged or neutral.
[0096] A “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 may be characterized as having vesicular structures with a bilayer membrane, generally comprising a phospholipid, and an inner medium that generally comprises an aqueous composition. Liposomes provided herein include unilamellar liposomes, multilamellar liposomes, and multivesicular liposomes. Liposomes provided herein may be positively charged, negatively charged, or neutrally charged. In certain embodiments, the liposomes are neutral in charge.
[0097] A multilamellar liposome has multiple lipid layers separated by aqueous medium. Such liposomes form spontaneously when lipids comprising 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. Lipophilic molecules or molecules with lipophilic regions may also dissolve in or associate with the lipid bilayer.
[0098] In specific aspects, a polypeptide, a nucleic acid, or a small molecule drug may be, for example, 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 polypeptide/nucleic acid, entrapped in a liposome, complexed with a liposome, or the like.
[0099] A liposome used according to the present embodiments can be made by different methods, as would be known to one of ordinary skill in the art. For example, a phospholipid, such as for example the neutral phospholipid dioleoylphosphatidylcholine (DOPC), is dissolved in tert-butanol. The lipid(s) is then mixed with a polypeptide, nucleic acid, and/or other component(s). Tween 20 is added to the lipid mixture such that Tween 20 is about 5% of the composition's weight. Excess tert-butanol is added to this mixture such that the volume of tert-butanol is at least 95%. The mixture is vortexed, frozen in a dry ice/acetone bath and lyophilized overnight. The lyophilized preparation is stored at -20 C and can be used up to three months. When required the lyophilized liposomes are reconstituted in 0.9% saline.
[0100] Alternatively, a liposome can be prepared by mixing lipids in a solvent in a container, e.g., a glass, pear-shaped flask. The container should have a volume ten-times greater than the volume of the expected suspension of liposomes. Using a rotary evaporator, the solvent is removed at approximately 40 C under negative pressure. The solvent normally is removed within about 5 min to 2 h, depending on the desired volume of the liposomes. The composition can be dried further in a desiccator under vacuum. The dried lipids generally are discarded after about 1 week because of a tendency to deteriorate with time.
[0101] Dried lipids can be hydrated at approximately 25-50 mM phospholipid in sterile, pyrogen-free water by shaking until all the lipid film is resuspended. The aqueous liposomes can be then separated into aliquots, each placed in a vial, lyophilized and sealed under vacuum. [0102] The dried lipids or lyophilized liposomes prepared as described above may be dehydrated and reconstituted in a solution of a protein or peptide and diluted to an appropriate concentration with a suitable solvent, e.g., DPBS. The mixture is then vigorously shaken in a vortex mixer. Unencapsulated additional materials, such as agents including but not limited to hormones, drugs, nucleic acid constructs and the like, are removed by centrifugation at 29,000 g and the liposomal pellets washed. The washed liposomes are resuspended at an appropriate total phospholipid concentration, e.g., about 50-200 mM. The amount of additional material or active agent encapsulated can be determined in accordance with standard methods. After determination of the amount of additional material or active agent encapsulated in the liposome preparation, the liposomes may be diluted to appropriate concentrations and stored at 4 C until use. A pharmaceutical composition comprising the liposomes will usually include a sterile, pharmaceutically acceptable carrier or diluent, such as water or saline solution. [0103] Additional liposomes which may be useful with the present embodiments include cationic liposomes, for example, as described in W002/100435A1, U.S Patent 5,962,016, U.S. Application 2004/0208921, W003/015757A1, WO04029213A2, U.S. Patent 5,030,453, and U.S. Patent 6,680,068, all of which are hereby incorporated by reference in their entirety without disclaimer.
[0104] In preparing such liposomes, any protocol described herein, or as would be known to one of ordinary skill in the art may be used. Additional non-limiting examples of preparing liposomes are described in U.S. Patents 4,728,578, 4,728,575, 4,737,323, 4,533,254, 4,162,282, 4,310,505, and 4,921,706; W01986/000238 and WO 1990/004943, each incorporated herein by reference.
[0105] In certain embodiments, the lipid based nanoparticle is a neutral liposome (e.g., a DOPC liposome). “Neutral liposomes” or “non-charged liposomes”, as used herein, are defined as liposomes having one or more lipid components that yield an essentially-neutral, net charge (substantially non-charged). By “essentially neutral” or “essentially non-charged”, it is meant that few, if any, lipid components within a given population (e.g., a population of liposomes) include a charge that is not canceled by an opposite charge of another component (i.e., fewer than 10% of components include a non-canceled charge, more preferably fewer than 5%, and most preferably fewer than 1%). In certain embodiments, neutral liposomes may include mostly lipids and/or phospholipids that are themselves neutral under physiological conditions (i.e., at about pH 7).
[0106] Liposomes and/or lipid-based nanoparticles of the present embodiments may comprise or exclude a phospholipid. In certain embodiments, a single kind of phospholipid may be used in the creation of liposomes (e.g., a neutral phospholipid, such as DOPC, may be used to generate neutral liposomes). In other embodiments, more than one kind of phospholipid may be used to create liposomes. Phospholipids may be from natural or synthetic sources. Phospholipids may include or exclude, for example, phosphatidylcholines, phosphatidylglycerols, and phosphatidylethanolamines; because phosphatidylethanolamines and phosphatidyl cholines are non-charged under physiological conditions (i.e., at about pH 7), these compounds may be particularly useful for generating neutral liposomes. In certain embodiments, the phospholipid DOPC is used to produce non-charged liposomes. In certain embodiments, a lipid that is not a phospholipid (e.g., a cholesterol) may be used
[0107] Phospholipids useful for incorporation into a lipid nanoparticle may include or exclude glycerophospholipids and certain sphingolipids. Phospholipids include, but are not limited to, dioleoylphosphatidylycholine ("DOPC"), egg phosphatidylcholine ("EPC"), dilauryloylphosphatidylcholine ("DLPC"), dimyristoylphosphatidylcholine ("DMPC"), dipalmitoylphosphatidylcholine ("DPPC"), distearoylphosphatidylcholine ("DSPC"), 1- myristoyl-2-palmitoyl phosphatidylcholine ("MPPC"), l-palmitoyl-2-myristoyl phosphatidylcholine ("PMPC"), l-palmitoyl-2-stearoyl phosphatidylcholine ("PSPC"), 1- stearoyl-2-palmitoyl phosphatidylcholine ("SPPC"), dilauryloylphosphatidylglycerol ("DLPG"), dimyristoylphosphatidylglycerol ("DMPG"), dipalmitoylphosphatidylglycerol ("DPPG"), distearoylphosphatidylglycerol ("DSPG"), distearoyl sphingomyelin ("DSSP"), distearoylphophatidylethanolamine ("DSPE"), dioleoylphosphatidylglycerol ("DOPG"), dimyristoyl phosphatidic acid ("DMPA"), dipalmitoyl phosphatidic acid ("DPPA"), dimyristoyl phosphatidylethanolamine ("DMPE"), dipalmitoyl phosphatidylethanolamine ("DPPE"), dimyristoyl phosphatidylserine ("DMPS"), dipalmitoyl phosphatidylserine ("DPPS"), brain phosphatidylserine ("BPS"), brain sphingomyelin ("BSP"), dipalmitoyl sphingomyelin ("DPSP"), dimyristyl phosphatidylcholine ("DMPC"), 1,2-distearoyl-sn- glycero-3 -phosphocholine ("DAPC "), 1 ,2-diarachidoyl-sn-glycero-3 -phosphocholine
("DBPC "), 1 ,2-dieicosenoyl-sn-glycero-3 -phosphocholine ("DEPC"), dioleoylphosphatidylethanolamine ("DOPE"), palmitoyloeoyl phosphatidylcholine ("POPC"), palmitoyloeoyl phosphatidylethanolamine ("POPE"), lysophosphatidylcholine, lysophosphatidylethanolamine, and dilinoleoylphosphatidylcholine.
[0108] In addition, other lipids such as steroids, cholesterol, aliphatic amines such as long- chained aliphatic amines and carboxylic acids, long chained sulfates and phosphates, diacetyl phosphate, butylated hydroxytoluene, tocopherols, retinols, and isoprenoid compounds can be can be incorporated into the lipid nanoparticle or can be excluded from the nanoparticle.
[0109] The lipid nanoparticle, first lipid nanoparticle, and/or second lipid nanoparticle may comprise, comprise at least, or comprise at most a molar or weight ratio of 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% of a phospholipid and/or lipid described herein. II. Therapeutic cargo
[0110] The term “therapeutic agent” or “therapeutic cargo” refers to any chemical moiety that is a biologically, physiologically, or pharmacologically active substance that acts locally or systemically in a subject. Examples of therapeutic agents, also referred to as “drugs”, are described in well-known literature references such as the Merck Index, the Physicians Desk Reference, and The Pharmacological Basis of Therapeutics, and they include, without limitation, medicaments; vitamins; mineral supplements; substances used for the treatment, prevention, diagnosis, cure or mitigation of a disease or illness; substances which affect the structure or function of the body; or pro-drugs, which become biologically active or more active after they have been placed in a physiological environment.
[OHl] In some embodiments, therapeutic agents or diagnostic agents may be loaded to the lipid nanoparticle for delivery to a subject, such as by electroporation or other method known in the art. The therapeutic agents may be a therapeutic nucleic acid, a protein or antibody fragment, or a small molecule.
[0112] The therapeutic cargo may comprise a nucleic acid that can be administered to a subject for the purpose of treating or preventing a disease. The nucleic acid is one which is known or suspected to be of benefit in the treatment of a disease or health-related condition in a subject.
[0113] Therapeutic benefit may arise, for example, as a result of alteration of expression of a particular gene or genes by the nucleic acid. Alteration of expression of a particular gene or genes may be inhibition or augmentation of expression of a particular gene (e.g., via miRNA). In certain embodiments, the therapeutic nucleic acid encodes one or more proteins or polypeptides that can be applied in the treatment or prevention of a disease or health-related condition in a subject (i.e., via mRNA). The terms “protein” and “polypeptide” are used interchangeably herein. Both terms refer to an amino acid sequence comprising two or more amino acid residues.
[0114] Any nucleic acid known to those of ordinary skill in the art that is known or suspected to be of benefit in the treatment or prevention of a disease or health-related condition is contemplated in certain aspects as a therapeutic nucleic acid. The phrase “nucleic acid sequence encoding,” as set forth throughout this application, refers to a nucleic acid which directs the expression of a specific protein or peptide. The nucleic acid sequences include both the DNA strand sequence that is transcribed into RNA and the RNA sequence that is translated into protein. In some embodiments, the nucleic acid includes a therapeutic gene. The term “gene” is used to refer to a nucleic acid sequence that encodes a functional protein, polypeptide, or peptide-encoding unit.
[0115] As will be understood by those in the art, the term “therapeutic nucleic acid” includes genomic sequences, cDNA sequences, and smaller engineered gene segments that express, or may be adapted to express, proteins, polypeptides, domains, peptides, fusion proteins, and mutants. The nucleic acid may comprise a contiguous nucleic acid sequence of about 5 to about 12000 or more nucleotides, nucleosides, or base pairs.
[0116] Encompassed within the definition of “nucleic acid” is a “biologically functional equivalent” of a nucleic acid that has proved to be of benefit in the treatment or prevention of a disease or health-related condition. Accordingly, sequences that have about 70% to about 99% homology to a known nucleic acid are contemplated in certain aspects.
B. CRISPR/Cas Systems
[0117] In general, “CRISPR system” refers collectively to transcripts and other elements involved in the expression of or directing the activity of CRISPR-associated (“Cas”) genes, including sequences encoding a Cas gene, a tracr (trans-activating CRISPR) sequence (e.g. tracrRNA or an active partial tracrRNA), a tracr-mate sequence (encompassing a “direct repeat” and a tracrRNA-processed partial direct repeat in the context of an endogenous CRISPR system), a guide sequence (also referred to as a “spacer” in the context of an endogenous CRISPR system), and/or other sequences and transcripts from a CRISPR locus.
[0118] The CRISPR/Cas nuclease or CRISPR/Cas nuclease system can include a noncoding RNA molecule (guide) RNA, which sequence-specifically binds to DNA, and a Cas protein (e.g., Cas9), with nuclease functionality (e.g., two nuclease domains). One or more elements of a CRISPR system can derive from a type I, type II, or type III CRISPR system, e.g., derived from a particular organism comprising an endogenous CRISPR system, such as Streptococcus pyogenes.
[0119] In some aspects, a Cas nuclease and gRNA (including a fusion of crRNA specific for the target sequence and fixed tracrRNA) are introduced into the cell. In general, target sites at the 5' end of the gRNA target the Cas nuclease to the target site, e.g., the gene, using complementary base pairing. The target site may be selected based on its location immediately 5' of a protospacer adjacent motif (PAM) sequence, such as typically NGG, or NAG. In this respect, the gRNA is targeted to the desired sequence by modifying the first 20, 19, 18, 17, 16, 15, 14, 14, 12, 11, or 10 nucleotides of the guide RNA to correspond to the target DNA sequence. In general, a CRISPR system is characterized by elements that promote the formation of a CRISPR complex at the site of a target sequence. Typically, "target sequence" generally refers to a sequence to which a guide sequence is designed to have complementarity, where hybridization between the target sequence and a guide sequence promotes the formation of a CRISPR complex. Full complementarity is not necessarily required, provided there is sufficient complementarity to cause hybridization and promote formation of a CRISPR complex.
[0120] The CRISPR system can induce double stranded breaks (DSBs) at the target site, followed by disruptions as discussed herein. In other embodiments, Cas9 variants, deemed "nickases," are used to nick a single strand at the target site. Paired nickases can be used, e.g., to improve specificity, each directed by a pair of different gRNAs targeting sequences such that upon introduction of the nicks simultaneously, a 5' overhang is introduced. In other embodiments, catalytically inactive Cas9 is fused to a heterologous effector domain such as a transcriptional repressor or activator, to affect gene expression.
[0121] The target sequence may comprise any polynucleotide, such as DNA or RNA polynucleotides. The target sequence may be located in the nucleus or cytoplasm of the cell, such as within an organelle of the cell. Generally, a sequence or template that may be used for recombination into the targeted locus comprising the target sequences is referred to as an "editing template" or "editing polynucleotide" or "editing sequence". In some aspects, an exogenous template polynucleotide may be referred to as an editing template. In some aspects, the recombination is homologous recombination.
[0122] Typically, in the context of an endogenous CRISPR system, formation of the CRISPR complex (comprising the guide sequence hybridized to the target sequence and complexed with one or more Cas proteins) results in cleavage of one or both strands in or near (e.g. within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50, or more base pairs from) the target sequence. The tracr sequence, which may comprise or consist of all or a portion of a wild-type tracr sequence (e.g. about or more than about 20, 26, 32, 45, 48, 54, 63, 67, 85, or more nucleotides of a wildtype tracr sequence), may also form part of the CRISPR complex, such as by hybridization along at least a portion of the tracr sequence to all or a portion of a tracr mate sequence that is operably linked to the guide sequence. The tracr sequence has sufficient complementarity to a tracr mate sequence to hybridize and participate in formation of the CRISPR complex, such as at least 50%, 60%, 70%, 80%, 90%, 95% or 99% of sequence complementarity along the length of the tracr mate sequence when optimally aligned.
[0123] One or more vectors driving expression of one or more elements of the CRISPR system can be introduced into the cell such that expression of the elements of the CRISPR system direct formation of the CRISPR complex at one or more target sites. Components can also be delivered to cells as proteins and/or RNA. For example, a Cas enzyme, a guide sequence linked to a tracr-mate sequence, and a tracr sequence could each be operably linked to separate regulatory elements on separate vectors. Alternatively, two or more of the elements expressed from the same or different regulatory elements, may be combined in a single vector, with one or more additional vectors providing any components of the CRISPR system not included in the first vector. The vector may comprise one or more insertion sites, such as a restriction endonuclease recognition sequence (also referred to as a “cloning site”). In some embodiments, one or more insertion sites are located upstream and/or downstream of one or more sequence elements of one or more vectors. When multiple different guide sequences are used, a single expression construct may be used to target CRISPR activity to multiple different, corresponding target sequences within a cell.
[0124] A vector may comprise a regulatory element operably linked to an enzyme-coding sequence encoding the CRISPR enzyme, such as a Cas protein. Non-limiting examples of Cas proteins include Casl, CaslB, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csnl and Csxl2), CaslO, Csyl, Csy2, Csy3, Csel, Cse2, Cscl, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmrl, Cmr3, Cmr4, Cmr5, Cmr6, Csbl, Csb2, Csb3, Csxl7, Csxl4, CsxlO, Csxl6, CsaX, Csx3, Csxl, Csxl5, Csfl, Csf2, Csf3, Csf4, homologs thereof, or modified versions thereof. These enzymes are known; for example, the amino acid sequence of S. pyogenes Cas9 protein may be found in the SwissProt database under accession number Q99ZW2.
[0125] The CRISPR enzyme can be Cas9 (e.g., from S. pyogenes or S. pneumonia). The CRISPR enzyme can direct cleavage of one or both strands at the location of a target sequence, such as within the target sequence and/or within the complement of the target sequence. The vector can encode a CRISPR enzyme that is mutated with respect to a corresponding wild-type enzyme such that the mutated CRISPR enzyme lacks the ability to cleave one or both strands of a target polynucleotide containing a target sequence. For example, an aspartate-to-alanine substitution (D10A) in the RuvC I catalytic domain of Cas9 from S. pyogenes converts Cas9 from a nuclease that cleaves both strands to a nickase (cleaves a single strand). In some embodiments, a Cas9 nickase may be used in combination with guide sequence(s), e.g., two guide sequences, which target respectively sense and antisense strands of the DNA target. This combination allows both strands to be nicked and used to induce NHEJ or HDR.
[0126] In some embodiments, an enzyme coding sequence encoding the CRISPR enzyme is codon optimized for expression in particular cells, such as eukaryotic cells. The eukaryotic cells may be those of or derived from a particular organism, such as a mammal, including but not limited to human, mouse, rat, rabbit, dog, or non-human primate. In general, codon optimization refers to a process of modifying a nucleic acid sequence for enhanced expression in the host cells of interest by replacing at least one codon of the native sequence with codons that are more frequently or most frequently used in the genes of that host cell while maintaining the native amino acid sequence. Various species exhibit particular bias for certain codons of a particular amino acid. Codon bias (differences in codon usage between organisms) often correlates with the efficiency of translation of messenger RNA (mRNA), which is in turn believed to be dependent on, among other things, the properties of the codons being translated and the availability of particular transfer RNA (tRNA) molecules. The predominance of selected tRNAs in a cell is generally a reflection of the codons used most frequently in peptide synthesis. Accordingly, genes can be tailored for optimal gene expression in a given organism based on codon optimization.
[0127] In general, a guide sequence is any polynucleotide sequence having sufficient complementarity with a target polynucleotide sequence to hybridize with the target sequence and direct sequence-specific binding of the CRISPR complex to the target sequence. In some embodiments, the degree of complementarity between a guide sequence and its corresponding target sequence, when optimally aligned using a suitable alignment algorithm, is about or more than about 50%, 60%, 75%, 80%, 85%, 90%, 95%, 97.5%, 99%, or more.
[0128] Optimal alignment may be determined with the use of any suitable algorithm for aligning sequences, non-limiting example of which include the Smith-Waterman algorithm, the Needleman-Wunsch algorithm, algorithms based on the Burrows- Wheel er Transform (e.g. the Burrows Wheeler Aligner), Clustal W, Clustal X, BLAT, Novoalign (Novocraft Technologies, ELAND (Illumina, San Diego, Calif.), SOAP (available at soap.genomics.org.cn), and Maq (available at maq.sourceforge.net).
[0129] The CRISPR enzyme may be part of a fusion protein comprising one or more heterologous protein domains. A CRISPR enzyme fusion protein may comprise any additional protein sequence, and optionally a linker sequence between any two domains. Examples of protein domains that may be fused to a CRISPR enzyme include, without limitation, epitope tags, reporter gene sequences, and protein domains having one or more of the following activities: methylase activity, demethylase activity, transcription activation activity, transcription repression activity, transcription release factor activity, histone modification activity, RNA cleavage activity and nucleic acid binding activity. Non-limiting examples of epitope tags include histidine (His) tags, V5 tags, FLAG tags, influenza hemagglutinin (HA) tags, Myc tags, VSV-G tags, and thioredoxin (Trx) tags. Examples of reporter genes include, but are not limited to, glutathione- 5- transferase (GST), horseradish peroxidase (HRP), chloramphenicol acetyltransferase (CAT) beta galactosidase, beta-glucuronidase, luciferase, green fluorescent protein (GFP), HcRed, DsRed, cyan fluorescent protein (CFP), yellow fluorescent protein (YFP), and autofluorescent proteins including blue fluorescent protein (BFP). A CRISPR enzyme may be fused to a gene sequence encoding a protein or a fragment of a protein that bind DNA molecules or bind other cellular molecules, including but not limited to maltose binding protein (MBP), S-tag, Lex A DNA binding domain (DBD) fusions, GAL4A DNA binding domain fusions, and herpes simplex virus (HSV) BP 16 protein fusions. Additional domains that may form part of a fusion protein comprising a CRISPR enzyme are described in US 20110059502, incorporated herein by reference.
[0130] The Cas protein may be a Cas protein of SEQ ID NO:6: NAMDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDS GETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKK HERHPIFGNIVDEVAYHEI<YPTIYHLRI<I<LVDSTDI<ADLRLIYLALAHMII<FRGHFLI EGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQ LPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQ YADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQL PEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRK QRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNS RFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIE CFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIE
ERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFA NRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDE LVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVEN TQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTR SDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKA GFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQF YI<VREINNYHHAHDAYLNAVVGTALII<I<YPI<LESEFVYGDYI<VYDVRI<MIAI<SEQ EIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRK VLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYS VLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPK YSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQ LFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLT NLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGDGSPKKK RI<VEDPI<I<I<RI<VD (SEQ ID NO: 6).
C. Nucleic Acid Inhibitors
[0131] Inhibitory nucleic acids or any ways of inhibiting gene expression are known in the art are contemplated in certain embodiments. Examples of an inhibitory nucleic acid include but are not limited to siRNA (small interfering RNA), short hairpin RNA (shRNA), double-stranded RNA, an antisense oligonucleotide, a ribozyme, and a nucleic acid encoding thereof. An inhibitory nucleic acid may inhibit the transcription of a gene or prevent the translation of a gene transcript in a cell. An inhibitory nucleic acid may be from 16 to 1000 nucleotides long, and in certain embodiments from 18 to 100 nucleotides long. The nucleic acid may have nucleotides of at least or at most 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 40, 50, 60, 70, 80, 90 or any range derivable therefrom.
[0132] As used herein, “isolated” means altered or removed from the natural state through human intervention. For example, an siRNA naturally present in a living animal is not “isolated,” but a synthetic siRNA, or an siRNA partially or completely separated from the coexisting materials of its natural state is “isolated.” An isolated siRNA can exist in substantially purified form, or can exist in a non-native environment such as, for example, a cell into which the siRNA has been delivered.
[0133] Antisense oligonucleotide (ASO) therapeutic agents are single stranded nucleic acid therapeutics, typically about 16 to 30 nucleotides in length, and are complementary to a target nucleic acid sequence in the target cell, either in culture or in an organism.
[0134] In some embodiments, the agent is a single-stranded antisense RNA molecule, a single-stranded antisense DNA molecule, or a single-stranded antisense polynucleotide comprising both DNA and RNA. In a particular embodiment, the antisense molecule is an ASO comprising both DNA and RNA. An antisense molecule is complementary to a sequence within the target mRNA, e.g., a STAT3 mRNA. Antisense molecules can inhibit translation in a stoichiometric manner by base pairing to the mRNA and physically obstructing the translation machinery. The antisense molecule may have at least or at most 15-30 nucleotides that are complementary to the target mRNA. For example, the antisense molecule may have a sequence of at least or at most 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25, or any range or value derivable therein, contiguous nucleotides that are complementary to the target mRNA.
[0135] In some embodiments, the ASO comprises at least or at most 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleotides, or any range or value derivable therein. Any of these values may be used to define a range for the number of nucleotides in the ASO. For example, the ASO may comprise, comprise at least or, or comprise at most 8-50, 15- 30, or 20-25 nucleotides. In some embodiments, the ASO consists of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39,
40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleotides, or any range or value derivable therein. Any of these values may be used to define a range for the number of nucleotides in the ASO. For example, the ASO may consist of 8-50, 15-30, or 20-25 nucleotides.
[0136] In one aspect of the disclosure, the agent is a single-stranded antisense nucleic acid molecule (ASO). Antisense oligonucleotides (ASOs) are synthetic molecules and, in some embodiments, comprise between 18-21 nucleotides in length and are complementary to the mRNA sequence of the target gene. ASOs bind cognate mRNA sequences through sequencespecific hybridization resulting in cleavage or disablement of the mRNA and inhibition of the expression of the target gene.
[0137] siRNA (e.g., siNA) are well known in the art. For example, siRNA and doublestranded RNA have been described in U.S. Pat. Nos. 6,506,559 and 6,573,099, as well as in U.S. Patent Applications 2003/0051263, 2003/0055020, 2004/0265839, 2002/0168707, 2003/0159161, and 2004/0064842, all of which are herein incorporated by reference in their entirety.
[0138] Within a siRNA, the components of a nucleic acid need not be of the same type or homogenous throughout (e.g., a siRNA may comprise a nucleotide and a nucleic acid or nucleotide analog). Typically, siRNA form a double-stranded structure; the double-stranded structure may result from two separate nucleic acids that are partially or completely complementary. In certain embodiments of the present disclosure, the siRNA may comprise only a single nucleic acid (polynucleotide) or nucleic acid analog and form a double-stranded structure by complementing with itself (e.g., forming a hairpin loop). The double-stranded structure of the siRNA may comprise, comprise at least, or comprise at most 16, 20, 25, 30, 35, 40, 45, 50, 60, 65, 70, 75, 80, 85, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500 or more contiguous nucleobases, including all ranges and values therein. The siRNA may comprise 17 to 35 contiguous nucleobases, 18 to 30 contiguous nucleobases, 19 to 25 nucleobases, 20 to 23 contiguous nucleobases, 20 to 22 contiguous nucleobases, or 21 contiguous nucleobases that hybridize with a complementary nucleic acid (which may be another part of the same nucleic acid or a separate complementary nucleic acid) to form a double-stranded structure. [0139] Agents of the present disclosure useful for practicing the methods of the present disclosure include, but are not limited to siRNAs. Typically, introduction of double-stranded RNA (dsRNA), which may alternatively be referred to herein as small interfering RNA (siRNA), induces potent and specific gene silencing, a phenomenon called RNA interference or RNAi. RNA interference has been referred to as “cosuppression,” “post-transcriptional gene silencing,” “sense suppression,” and “quelling.” RNAi is an attractive biotechnological tool because it provides a means for knocking out the activity of specific genes.
[0140] In designing RNAi there are several factors that need to be considered, such as the nature of the siRNA, the durability of the silencing effect, and the choice of delivery system. To produce an RNAi effect, the siRNA that is introduced into the organism will typically contain exonic sequences. Furthermore, the RNAi process is homology dependent, so the sequences must be carefully selected so as to maximize gene specificity, while minimizing the possibility of cross-interference between homologous, but not gene-specific sequences. Preferably the siRNA exhibits or exhibits greater than 80%, 85%, 90%, 95%, 98%, or even 100% identity, or any range or value derivable therein, between the sequence of the siRNA and the gene to be inhibited. Sequences less than about 80% identical to the target gene are substantially less effective. Thus, the greater homology between the siRNA and the gene to be inhibited, the less likely expression of unrelated genes will be affected.
[0141] In addition, the size of the siRNA is an important consideration. In some embodiments, the present disclosure relates to siRNA molecules that include, include at least, or include at most 19-25 nucleotides, or any range or value derivable therein, and are able to modulate gene expression. In the context of the present disclosure, the siRNA is, in some embodiments, less than 500, 200, 100, 50, or 25 nucleotides in length. In some embodiments, the siRNA is from about 19 nucleotides to about 25 nucleotides in length.
[0142] A target gene generally means a polynucleotide comprising a region that encodes a polypeptide, or a polynucleotide region that regulates replication, transcription, or translation or other processes important to expression of the polypeptide, or a polynucleotide comprising both a region that encodes a polypeptide and a region operably linked thereto that regulates expression. Any gene being expressed in a cell can be targeted. Preferably, a target gene is one involved in or associated with the progression of cellular activities important to disease or of particular interest as a research object.
[0143] siRNA can be obtained from commercial sources, natural sources, or can be synthesized using any of a number of techniques well-known to those of ordinary skill in the art. For example, one commercial source of predesigned siRNA is Ambion®, Austin, Tex. Another is Qiagen® (Valencia, Calif.). An inhibitory nucleic acid that can be applied in the compositions and methods of the present disclosure may be any nucleic acid sequence that has been found by any source to be a validated downregulator of a protein of interest. Without undue experimentation and using the disclosure of this disclosure, it is understood that additional siRNAs can be designed and used to practice the methods of the disclosure.
[0144] The siRNA may also comprise an alteration of one or more nucleotides. Such alterations can include the addition of non-nucleotide material, such as to the end(s) of the 19 to 25 nucleotide RNA or internally (at one or more nucleotides of the RNA). In certain aspects, the RNA molecule contains a 3 '-hydroxyl group. Nucleotides in the RNA molecules of the present disclosure can also comprise non-standard nucleotides, including non-naturally occurring nucleotides or deoxyribonucleotides. The double-stranded oligonucleotide may contain a modified backbone, for example, phosphorothioate, phosphorodithioate, or other modified backbones known in the art, or may contain non-natural internucleoside linkages. Additional modifications of siRNAs (e.g., 2'-O-methyl ribonucleotides, 2 '-deoxy-2 '-fluoro ribonucleotides, “universal base” nucleotides, 5-C-methyl nucleotides, one or more phosphorothioate intemucleotide linkages, and inverted deoxyabasic residue incorporation) can be found in U.S. Application Publication 2004/0019001 and U.S. Pat. No. 6,673,611 (each of which is incorporated by reference in its entirety). Collectively, all such altered nucleic acids or RNAs described above are referred to as modified siRNAs.
[0145] In some embodiments, the nucleic acid inhibitor is comprises a modification, such as a chemical modification or a modified base. In some embodiments, one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19. 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 (or any derivable range therein) of the nucleotide positions in one or both strands of an siRNA molecule are modified. Modifications include nucleic acid sugar modifications, base modifications, backbone (internucleotide linkage) modifications, non-nucleotide modifications, and/or any combination thereof. In certain instances, purine and pyrimidine nucleotides are differentially modified. For example, purine and pyrimidine nucleotides can be differentially modified at the 2'-sugar position (i.e., at least one purine has a different modification from at least one pyrimidine in the same or different strand at the 2'-sugar position). In other instances, at least one modified nucleotide is a 2'- deoxy-2 '-fluoro nucleotide, a 2'-deoxy nucleotide, or a 2'-O-alkyl nucleotide. In certain embodiments, the siRNA molecule has 3' overhangs of one, two, three, or four nucleotide(s) on one or both of the strands. In other embodiments, the siRNA lacks overhangs (i.e., has blunt ends). The overhangs can be modified or unmodified. Examples of modified nucleotides in the overhangs include, but are not limited to, 2'-O-alkyl nucleotides, 2 '-deoxy -2 '-fluoro nucleotides, or 2'-deoxy nucleotides. The overhang nucleotides in the antisense strand can comprise nucleotides that are complementary to nucleotides in the target sequence. Likewise, the overhangs in the sense stand can comprise nucleotides that are in the target sequence. In certain instances, the siRNA molecules have two 3 ' overhang nucleotides on the antisense stand that are 2'-O-alkyl nucleotides and two 3' overhang nucleotides on the sense stand that are 2'- deoxy nucleotides.
[0146] Particularly, an inhibitory nucleic acid may be capable of decreasing the expression of a protein or mRNA by at least 10%, 20%, 30%, or 40%, more particularly by at least 50%, 60%, or 70%, and most particularly by at least 75%, 80%, 90%, 95% or more or any range or value in between the foregoing.
[0147] In further embodiments, there are synthetic nucleic acids that are inhibitors of a gene. An inhibitor may be between 17 to 25 nucleotides in length and comprises a 5’ to 3’ sequence that is at least 90% complementary to the 5’ to 3’ sequence of a mature mRNA. In certain embodiments, an inhibitor molecule is 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length, or any range derivable therein. Moreover, an inhibitor molecule has a sequence (from 5’ to 3’) that is or is at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 99.1, 99.2, 99.3, 99.4, 99.5, 99.6, 99.7, 99.8, 99.9 or 100% complementary, or any range derivable therein, to the 5’ to 3’ sequence of a mature mRNA, particularly a mature, naturally occurring mRNA. One of skill in the art could use a portion of the probe sequence that is complementary to the sequence of a mature mRNA as the sequence for an mRNA inhibitor. Moreover, that portion of the probe sequence can be altered so that it is still 90% complementary to the sequence of a mature mRNA.
D. Modifications of Nucleic Acids
[0148] In certain embodiments, the nucleic acid of the disclosure may be modified. A “modified nucleic acid” refers to a molecule in which one or more of the components of the nucleic acid, namely sugars, bases, and phosphate moi eties, are different from that which occur in nature, for example, different from that which occurs in the human body. Several modifications to nucleic acids are described in the art. These modifications may improve properties such as resistance to nucleases, permeability across biological membranes, solubility, stability, or modulation of pharmacokinetic and pharmacodynamics properties while maintaining specificity to the target mRNA. For example, the modifications on the nucleotides can include, but are not limited to, LNA, HNA, CeNA, 2'-methoxy ethyl, 2'-O-alkyl, 2'-O-allyl, 2'-C-allyl, 2'-fluoro, 2'-deoxy, 2'-hydroxyl, and combinations thereof. It is contemplated that one or more of these modifications may be excluded in an embodiment.
[0149] Patents directed to antisense nucleic acids, chemical modifications, and therapeutic uses are provided, for example, in U.S. Pat. No. 5,898,031 related to chemically modified RNA-containing therapeutic compounds, and U.S. Pat. No. 6,107,094 related methods of using these compounds as therapeutic agent. U.S. Pat. No. 7,432,250 related to methods of treating patients by administering single-stranded chemically modified RNA-like compounds; and U.S. Pat. No. 7,432,249 related to pharmaceutical compositions containing single-stranded chemically modified RNA-like compounds. U.S. Pat. No. 7,629,321 is related to methods of cleaving target mRNA using a single-stranded oligonucleotide having a plurality RNA nucleosides and at least one chemical modification. Each of the patents listed in this paragraph are incorporated herein by reference in their entirety.
1. Modified Bases
[0150] Therapeutic nucleic acid may include natural (i.e. A, G, U, C, or T) or modified (e.g. 7-deazaguanosine, inosine, etc.) bases. Modification of bases includes the incorporation of modified bases (or modified nucleoside or modified nucleotides) that are variations of standard bases, sugars and/or phosphate backbone chemical structures occurring in ribonucleic (i.e., A, C, G and U) and deoxyribonucleic (i.e., A, C, G and T) acids. Included or excluded within this scope are, for example: Gm (2'-methoxyguanylic acid), Am (2'-methoxyadenylic acid), Cf (2'-fluorocytidylic acid), Uf (2'-fluorouridylic acid), Ar (riboadenylic acid). The aptamers may also include cytosine or any cytosine-related base including 5-methylcytosine, 4-acetylcytosine, 3 -methylcytosine, 5 -hydroxymethyl cytosine, 2-thiocytosine, 5-halocytosine (e.g., 5-fluorocytosine, 5 -bromocytosine, 5-chlorocytosine, and 5-iodocytosine), 5-propynyl cytosine, 6-azocytosine, 5-trifluoromethylcytosine, N4,N4-ethanocytosine, phenoxazine cytidine, phenothiazine cytidine, carbazole cytidine or pyridoindole cytidine. The aptamer may further include guanine or any guanine-related base including 6-methylguanine, 1- methylguanine, 2,2-dimethylguanine, 2-methylguanine, 7-methylguanine, 2-propylguanine, 6- propylguanine, 8-haloguanine (e.g., 8-fluoroguanine, 8-bromoguanine, 8-chloroguanine, and 8-iodoguanine), 8-aminoguanine, 8-sulfhydrylguanine, 8-thioalkylguanine, 8- hydroxylguanine, 7-methylguanine, 8-azaguanine, 7-deazaguanine or 3 -deazaguanine. The aptamer may still further include adenine or any adenine-related base including 6- methyladenine, N6-isopentenyladenine, N6-methyladenine, 1 -methyladenine, 2- methyladenine, 2-methylthio-N6-isopentenyladenine, 8-haloadenine (e.g., 8-fluoroadenine, 8- bromoadenine, 8-chloroadenine, and 8-iodoadenine), 8-aminoadenine, 8-sulfhydryladenine, 8- thioalkyladenine, 8-hydroxyladenine, 7-methyladenine, 2-haloadenine (e.g., 2-fluoroadenine, 2-bromoadenine, 2-chloroadenine, and 2-iodoadenine), 2-aminoadenine, 8-azaadenine, 7- deazaadenine or 3 -deazaadenine. Also included are uracil or any uracil-related base including 5-halouracil (e.g., 5 -fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil), 5- (carboxyhydroxylmethyl)uracil, 5-carboxymethylaminomethyl-2-thiouracil, 5- carboxymethylaminomethyluracil, dihydrouracil, 1 -methylpseudouracil, 5- methoxyaminomethyl-2-thiouracil, 5 '-methoxy carbonylmethyluracil, 5-methoxyuracil, 5- methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid, pseudouracil, 5-methyl-2-thiouracil, 2-thiouracil, 3-(3- amino-3-N-2-carboxypropyl)uracil, 5-methylaminomethyluracil, 5-propynyl uracil, 6- azouracil, or 4-thiouracil.
[0151] The nucleic acids of the disclosure may include or exclude modified base variants. The modified base variant may include or exclude 4-acetylcytidine, 5- (carboxyhydroxylmethyl)uridine, 2'-methoxy cytidine, 5-carboxymethylaminomethyl-2- thioridine, 5-carboxymethylaminomethyluridine, dihydrouridine, 2'-O-methylpseudouridine, b-D-galactosylqueosine, inosine, N6-isopentenyladenosine, 1 -methyladenosine, 1- methylpseudouridine, 1 -methylguanosine, 1 -methylinosine, 2,2-dimethylguanosine, 2- methyladenosine, 2-methylguanosine, 3 -methylcytidine, 5-methylcytidine, N6- methyladenosine, 7-methylguanosine, 5-methylaminomethyluridine, 5 -methoxy aminomethyl- 2-thiouridine, b-D-mannosylqueosine, 5-methoxycarbonylmethyluridine, 5-methoxyuridine, 2-methylthio-N6-isopentenyladenosine, N-((9-b-D-ribofuranosyl-2-methylthiopurine-6- yl)carbamoyl)threonine, N-((9-b-D-ribofuranosylpurine-6-yl)N-methyl-carbamoyl)threonine, urdine-5-oxyacetic acid methylester, uridine-5-oxyacetic acid (v), wybutoxosine, pseudouridine, queosine, 2-thiocytidine, 5-methyl-2-thiouridine, 2-thiouridine, 4-thiouridine, 5-methyluridine, N-((9-b-D-ribofuranosylpurine-6-yl)carbamoyl)threonine, 2'-O-methyl-5- methyluridine, 2'-O-methyluridine, and wybutosine, and 3-(3-amino-3-carboxypropyl)uridine. [0152] Also included are the modified nucleobases described in U.S. Pat. Nos. 3,687,808, 3,687,808, 4,845,205, 5,130,302, 5,134,066, 5,175,273, 5,367,066, 5,432,272, 5,457,187,
5,459,255, 5,484,908, 5,502,177, 5,525,711, 5,552,540, 5,587,469, 5,594,121, 5,596,091,
5,614,617, 5,645,985, 5,830,653, 5,763,588, 6,005,096, and 5,681,941, each of which is incorporated herein by reference in its entirety. 2. Modified Sugars
[0153] Modified sugar moieties for use in nucleic acids are well known in the art and are described for example in U.S. Pat. No. 9,045,754 which is incorporated by reference herein in its entirety. Modified sugars can be used to alter, typically increase, the affinity of the nucleic acid for its target and/or increase nuclease resistance. For example, in some embodiments, the binding affinity of the nucleic acids to their target can be increased by incorporating substituent groups in the nucleoside subunits of the nucleic acids. In some embodiments, the substituent groups are T substituent groups, substituent groups located at the 2' position of the pentofuranosyl sugar moieties of the nucleoside subunits of the nucleic acids. Substituent groups include or exclude fluoro, alkoxy, amino-alkoxy, allyloxy, imidazolylalkoxy and polyethylene glycol. Alkoxy and aminoalkoxy groups generally include lower alkyl groups, particularly C1-C9 alkyl. In a particular embodiment, the 2' substituent group is 2'-O-methyl. Polyethylene glycols are of the structure (O — CH2 — CH2)n — O-alkyl. In a particular embodiment, the substituent is a polyethylene glycol substituent of the formula ( — O — CH2 — CH2)n — O-alkyl, wherein n=l and alkyl=CH3. This modification has been shown to increase both affinity of an oligonucleotide for its target and nuclease resistance of an oligonucleotide. See U.S. Pat. No. 7,629,321 cited above. A further particularly useful 2 '-substituent group for increasing the binding affinity is the 2 '-fluoro group.
[0154] Examples of modified nucleoside and nucleotide sugar backbone variants known in the art include, without limitation, those having, e.g., 2' ribosyl substituents such as F, SH, SCH3, OCN, Cl, Br, CN, CF3, OCF3, SOCH3, SO2, CH3, ONO2, NO2, N3, NH2, OCH2CH2OCH3, O(CH2)2ON(CH3)2, OCH2OCH2N(CH3)2, O(Cl-10 alkyl), O(C2-10 alkenyl), O(C2-10 alkynyl), S(Cl-10 alkyl), S(C2-10 alkenyl), S(C2-10 alkynyl), NH(Cl-10 alkyl), NH(C2-10 alkenyl), NH(C2-10 alkynyl), and O-alkyl-O-alkyl. Desirable 2' ribosyl substituents include 2'-methoxy (2'-OCH3), 2 '-aminopropoxy (2' OCH2CH2CH2NH2), 2'-O- allyl (2'-CH2— CH=CH2), 2'-O-allyl (2'-O— CH2— CH=CH2), 2'-amino (2'-NH2), and 2'- fluoro (2'-F). The 2 '-substituent may be in the arabino (up) position or ribo (down) position. One or more of these variants may be excluded from embodiments of the disclosure.
[0155] Another class of modified nucleic acids known in the art and that may be utilized in the nucleic acids of the disclosure contain alkyl modifications at the 2' position of the ribose moiety. These nucleic acids were developed to improve the binding affinity and hybridization stability with target mRNA, and to increase the nuclease resistance of the nucleic acids. In this category, the most commonly used nucleic acids are 2'-O-Methyl (2'-0ME) and 2'-O- Methoxyethyl (2'-M0E) nucleic acids. Nucleic acids with this type of modification are incapable of activating RNAse H. Therefore, to induce RNAse H activation, chimeric nucleic acids have been developed in which a central gap region consisting of a phosphorothioate deoxyribose core is flanked with nuclease resistant arms such as 2'-0ME or 2'-M0E that possess greater nuclease resistance. A “gapmer” is produced as a result, in which RNAse H can sit in the central gap and activate target specific mRNA degradation, while the arms prevent the nucleic acid degradation. Nucleic acids in this category may possess higher affinity for mRNA, show better tissue uptake, and have increased resistance to nucleases, longer in vivo half life, and lesser toxicity, as compared to the modified nucleic acids of the first class.
[0156] A further class of nucleic acids known in the art and that may be utilized in the nucleic acids of the disclosure contain modifications of the furanose ring along with modifications of the phosphate linkage, the ribose moiety, or the nucleotides. These modifications were designed to improve the nuclease stability, target affinity and pharmacokinetic profiles of the nucleic acids. Common examples of third category of nucleic acids are Locked nucleic acid (LNA), Peptide nucleic acid (PNA) and Morpholino phosphoroamidates (MF) nucleic acids in this category are more stable in biological fluids because of their high resistance to degradation by nucleases and peptidases. They also exhibit a strong hybridization affinity with the mRNA. Further, PNAs recognize double stranded DNA, and are able to modulate gene expression or induce mutation by strand invasion of chromosomal duplex DNA. Nucleic acids in this category also do not activate RNAse H and rely on sterically hindering the ribosomal machinery to cause translational arrest. They do not bind to serum proteins as they are uncharged. Lack of charge reduces the odds of non-specific interactions but increases the rate of clearance from the body. Their electrostatically neutral backbones may reduce solubility and make uptake more difficult.
[0157] A representative list of preferred modified sugars includes but is not limited to bicyclic modified sugars (BNA's), including methyleneoxy (4'-CH2 — 0-2') BNA and ethyleneoxy (4'-(CH2)2 — 0-2' bridge) BNA; substituted sugars, especially 2 '-substituted sugars having a 2'-F, 2'-OCH3 or a 2'-O(CH2)2 — 0CH3 substituent group; and 4'-thio modified sugars. Sugars can also be replaced with sugar mimetic groups among others. Methods for the preparations of modified sugars are well known to those skilled in the art. Some representative patents and publications that teach the preparation of such modified sugars include, but are not limited to, U.S. Pat. Nos. 4,981,957; 5,118,800; 5,319,080; 5,359,044; 5,393,878; 5,446,137; 5,466,786; 5,514,785; 5,519,134; 5,567,811; 5,576,427; 5,591,722; 5,597,909; 5,610,300; 5,627,053; 5,639,873; 5,646,265; 5,658,873; 5,670,633; 5,792,747; 5,700,920; 6,531,584; and 6,600,032; and WO 2005/121371.
3. Modified Internucleotide Linkages
[0158] Nucleic acid therapeutics may further comprise at least one phosphorothioate or methylphosphonate internucleotide linkage. The phosphorothioate or methylphosphonate internucleotide linkage modification may occur on any nucleotide of the sense strand or antisense strand or both (in nucleic acid therapeutics including a sense strand) in any position of the strand. For instance, the internucleotide linkage modification may occur on every nucleotide on the sense strand or antisense strand; each internucleotide linkage modification may occur in an alternating pattern on the sense strand or antisense strand; or the sense strand or antisense strand may contain both intemucleotide linkage modifications in an alternating pattern. The alternating pattern of the internucleotide linkage modification on the sense strand may be the same or different from the antisense strand, and the alternating pattern of the internucleotide linkage modification on the sense strand may have a shift relative to the alternating pattern of the internucleotide linkage modification on the antisense strand.
[0159] In certain embodiments, the nucleic acids of the disclosure comprise or exclude one or more nucleoside subunits connected by phosphorus linkages including phosphodiester, phosphorothioate, 3 '(or -5 ')deoxy-3 '-(or -5')thio-phosphorothioate, phosphorodithioate, phosphoroselenates, 3 '-(or -5')deoxy phosphinates, borano phosphates, 3 '-(or 5'-)amino phosphoramidates, hydrogen phosphonates, borano phosphate esters, phosphoramidates, alkyl or aryl phosphonates and phosphotriester phosphorus linkages. In some embodiments, the nucleic acids of the disclosure comprise nucleoside subunits connected by carbonate, carbamate, silyl, sulfur, sulfonate, sulfonamide, formacetal, thioformacetyl, oxime, methyleneimino, methylenemethylimino, methylenehydrazo, methylenedimethylhydrazo and methyleneoxymethylimino linkages.
[0160] For example, one class of modified nucleic acids described in the art and that may be utilized in the nucleic acids of the disclosure are those that have one of the non-bridging oxygen atoms in the phosphate group of the nucleic acids replaced with either a sulfur group (phosphorothioates), a methyl group (methyl phosphonates) or an amine group (phosphoramidates). These nucleic acids have greater resistance to nucleases and longer plasma half life as compared with phosphodiester oligonucleotides. They are capable of activating RNAse H, carry negative charges which facilitate their delivery to cells, and have suitable pharmacokinetics. Among these modifications, phosphorothioate modifications are used most widely. For example, Vitravene, an FDA approved ASO drug, and most of the other ASO drugs in clinical trials are phosphorothioate ASOs.
[0161] In addition, the bases in nucleotide may be joined by a linkage other than a phosphodiester bond, so long as it does not interfere with hybridization. Thus, inhibitory nucleic acids may be peptide nucleic acids in which the constituent bases are joined by peptide bonds rather than phosphodiester linkages. The inhibitory nucleic acids may be prepared by converting the RNA to cDNA using known methods (see, e.g., Ausubel et. al., Current Protocols in Molecular Biology Wiley 1999). The inhibitory nucleic acids can also be cRNA (see, e.g., Park et. al., (2004) Biochem. Biophys. Res. Commun. 325(4): 1346-52).
III. Delivery of the CRISPR System
[0162] In some aspects, a nucleic acid encoding the CRISPR-Cas9 targeting molecule, complex, or combination, is administered or introduced to the cell. In some aspects, the system may already be present in the cell, or within liposome nanoparticles in cell. The nucleic acid typically is administered in the form of an expression vector, such as a viral expression vector. In some aspects, the expression vector is a retroviral expression vector, an adenoviral expression vector, a DNA plasmid expression vector, or an AAV expression vector. In some aspects, one or more polynucleotides encoding the disruption molecule or complex, such as the DNA-targeting molecule, is delivered to the cell. In some aspects, the delivery is by delivery of one or more vectors, one or more transcripts thereof, and/or one or more proteins transcribed therefrom, is delivered to the cell.
[0163] In some embodiments, the polypeptides are synthesized in situ in the cell as a result of the introduction of polynucleotides encoding the polypeptides into the cell. In some aspects, the polypeptides could be produced outside the cell and then introduced thereto. Methods for introducing a polynucleotide construct into animal cells are known and include, as non-limiting examples stable transformation methods wherein the polynucleotide construct is integrated into the genome of the cell, transient transformation methods wherein the polynucleotide construct is not integrated into the genome of the cell, and virus mediated methods. In some embodiments, the polynucleotides may be introduced into the cell by for example, recombinant viral vectors (e.g. retroviruses, adenoviruses), liposome and the like. For example, in some aspects, transient transformation methods include microinjection, electroporation, or particle bombardment. In some embodiments, the polynucleotides may be included in vectors, more particularly plasmids or virus, in view of being expressed in the cells.
[0164] In some embodiments, viral and non-viral based gene transfer methods can be used to introduce nucleic acids in mammalian cells or target tissues. Such methods can be used to administer nucleic acids encoding components of a CRISPR system to cells in culture, or in a host organism. Non-viral vector delivery systems include DNA plasmids, RNA (e.g. a transcript of a vector described herein), naked nucleic acid, and nucleic acid complexed with a delivery vehicle, such as a liposome. Viral vector delivery systems include DNA and RNA viruses, which have either episomal or integrated genomes after delivery to the cell. For a review of gene therapy procedures, see Anderson, 1992; Nabel & Feigner, 1993; Mitani & Caskey, 1993; Dillon, 1993; Miller, 1992; Van Brunt, 1988; Vigne, 1995; Kremer & Perricaudet, 1995; Haddada et al., 1995; and Yu et al., 1994.
[0165] Methods of non-viral delivery of nucleic acids include exosomes, lipofection, nucleofection, microinjection, biolistics, virosomes, liposomes, nanoparticles, lipid nanoparticles, immunoliposomes, polycation or lipidmucleic acid conjugates, naked DNA, artificial virions, and agent-enhanced uptake of DNA. Lipofection is described in (e.g., U.S. Pat. Nos. 5,049,386, 4,946,787; and 4,897,355) and lipofection reagents are sold commercially (e.g., Transfectam™ and Lipofectin™). Cationic and neutral lipids that are suitable for efficient receptor-recognition lipofection of polynucleotides include those of Feigner, WO 91117424; WO 91116024. Delivery can be to cells (e.g. in vitro or ex vivo administration) or target tissues (e.g. in vivo administration).
[0166] In some embodiments, delivery is via the use of RNA or DNA viral based systems for the delivery of nucleic acids. Viral vectors in some aspects may be administered directly to patients (in vivo) or they can be used to treat cells in vitro or ex vivo, and then administered to patients. Viral-based systems in some embodiments include retroviral, lentivirus, adenoviral, adeno-associated and herpes simplex virus vectors for gene transfer.
[0167] In some aspects, a reporter gene which includes but is not limited to glutathione- 5-transferase (GST), horseradish peroxidase (HRP), chloramphenicol acetyltransferase (CAT) beta-galactosidase, beta-glucuronidase, luciferase, green fluorescent protein (GFP), HcRed, DsRed, cyan fluorescent protein (CFP), yellow fluorescent protein (YFP), and autofluore scent proteins including blue fluorescent protein (BFP), may be introduced into the cell to encode a gene product which serves as a marker by which to measure the alteration or modification of expression of the gene product. In a further embodiment, the DNA molecule encoding the gene product may be introduced into the cell via a vector. In some embodiments, the gene product is luciferase.
[0168] As will be appreciated by one of skill in the art, prior or subsequent to loading with cargo, the present nanoparticles may be further altered by inclusion of a targeting moiety to enhance the utility thereof as a vehicle for delivery of cargo. In this regard, nanoparticles may be engineered to incorporate an entity that specifically targets a particular cell to tissue type. This target-specific entity, e.g. peptide having affinity for a receptor or ligand on the target cell or tissue, may be integrated within the exosomal membrane, for example, by fusion to an exosomal membrane marker using methods well-established in the art.
IV. Surfactants
[0169] As disclosed elsewhere herein, in some embodiments, the nanoparticle composition comprises a surfactant. In some embodiments, the nanoparticle composition does not comprises a surfactant. In several embodiments, the surfactant is a pharmaceutically acceptable surfactant. In several embodiments, the surfactant comprises or exclude one or more of a polyoxyethylene sorbitan esters (e.g., polysorbates/tweens, including polysorbate 80, polysorbate 20, etc.), cremophor (e.g., a non-ionic solubilizer and emulsifier that is made by reacting ethylene oxide with castor oil), propylene oxide-modified polymethylsiloxane, dodecyl betaine, lauramidopropyl betaine, cocoamido-2-hydroxypropyl sulfobetaine, sodium stearate (or other stearate salts), polyoxyethylene alcohol, lecithins, mono- and diglycerides of fatty acids (MDG), acetic acid esters of MDG, lactic acid esters of MDG, citric acid esters of MDG, mono- and diacetyl tartaric acid esters of MDG, sucrose esters of fatty acids, polyglycerol esters of fatty acids (e.g., polyglycerol esters), lysophoshatidylcholine, polidocanol, polyglycerol polyricinoleate, propane- 1,2-diol esters of fatty acids, propylene glycol esters, sodium stearoyl-2-lactylate, calcium stearoyl-2-lactylate, sorbitan fatty acid esters, quillaja extract surfactant, yucca extract surfactant, saponins, silicone emulsifiers, sorbitan trioleate, soya lecithin, dioctyl sodium sulfosuccinate, dioctyl sodium sulfonate, polyoxyethylene, hydrogenated castor oil, sucrose fatty acid ester, or combinations of any of the foregoing. Natural or synthetic surfactants can be used, including polyethylene glycol and dextrans, such as cyclodextran.
[0170] Surfactants can include or exclude anionic surfactants such as sulfate, sulfonate, and phosphate, carboxylate derivatives. Anionic surfactants contain anionic functional groups at their head. Anionic surfactants include alkyl sulfates such as ammonium lauryl sulfate, sodium lauryl sulfate (sodium dodecyl sulfate, SLS, or SDS), and the related alkyl-ether sulfates sodium laureth sulfate (sodium lauryl ether sulfate or SLES), and sodium myreth sulfate. Anionic surfactants also include docusate (dioctyl sodium sulfosuccinate), perfluorooctanesulfonate (PFOS), perfluorobutanesulfonate, alkyl-aryl ether phosphates, and alkyl ether phosphates. Also include are carboxylates such as carboxylate salts (soaps), such as sodium stearate. Anionic surfactants also incoude sodium lauroyl sarcosinate and carboxylatebased fluorosurfactants such as perfluorononanoate, perfluorooctanoate (PFOA or PFO). [0171] Cationic surfactants may include or exclude octenidine dihydrochloride and ammonium salts such as cetrimonium bromide (CTAB), cetylpyridinium chloride (CPC), benzalkonium chloride (BAC), benzethonium chloride (BZT), dimethyldioctadecylammonium chloride, and dioctadecyldimethylammonium bromide (DODAB). Cationic surfactants also include
[0172] Zwitterionic surfactants have both cationic and anionic centers attached to the same molecule. The cationic part may be based on primary, secondary, or tertiary amines or quaternary ammonium cations. The anionic part can be more variable and include or exclude sulfonates, as in the sultaines CHAPS (3-[(3-cholamidopropyl)dimethylammonio]-l- propanesulfonate) and cocamidopropyl hydroxysultaine. Betaines such as cocamidopropyl betaine have a carboxylate with the ammonium. The zwitterionic surfactant may have a phosphate anion with an amine or ammonium, such as the phospholipids phosphatidylserine, phosphatidylethanolamine, phosphatidylcholine, and sphingomyelins. Zwitterionic surfactants include lauryldimethylamine oxide and myristamine oxide.
[0173] Other surfactants include or exclude ethoxylates, fatty alcohol ethoxylates, narrowrange ethoxylate, octaethylene glycol monododecyl ether, pentaethylene glycol monododecyl ether, alkylphenol ethoxylates (APEs or APEOs), nonoxynols, Triton X-100, fatty acid ethoxylates, ethoxylated fatty esters and oils, ethoxylated amines and/or fatty acid amides, polyethoxylated tallow amine, cocamide monoethanolamine, cocamide diethanolamine, terminally blocked ethoxylates, poloxamers, fatty acid esters of polyhydroxy compounds, fatty acid esters of glycerol, glycerol monostearate, glycerol monolaurate, fatty acid esters of sorbitol, sorbitan monolaurate, sorbitan monostearate, sorbitan tristearate, tweens such as Tween 20, Tween 40, Tween 60, and Tween 80, fatty acid esters of sucrose, alkyl polyglucosides, decyl glucoside, lauryl glucoside, and octyl glucoside.
[0174] The one or more surfactants may be present in the nanoparticle composition (collectively or individually) at a dry wt. % of equal to or less than about: 0%, 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, or ranges including and/or spanning the aforementioned values. Surfactants can include cationic, anionic, non-ionic, and zwitterionic surfactants. The one or more surfactants (collectively or individually) may be present in the composition at a wet wt. % of equal to or less than about: 0%, 0.1%, 0.5%, 1.0%, 2.5%, 4%, 5%, 6%, 7.5%, 10%, 12.5%, 15%, 17.5%, or ranges including and/or spanning the aforementioned values. The one or more surfactants (collectively or individually) may be present in the composition at a wet w/v of equal to or less than about: 0 mg/mL, 0.1 mg/mL, 0.5 mg/mL, 1.0 mg/mL, 2.5 mg/mL, 4 mg/mL, 5 mg/mL, 6 mg/mL, 7.5 mg/mL, 10 mg/mL, 12.5 mg/mL, 15 mg/mL, 17.5 mg/mL, or ranges including and/or spanning the aforementioned values. In several embodiments, the surfactant exceeds requirements of the United States Pharmacopeia (is USP grade) and/or is National Formulary (NF) grade.
V. Proteinaceous Compositions
[0175] The polypeptides or polynucleotides of the disclosure may include, may include at least, or may include at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 or more variant amino acids or nucleic acid substitutions or be at least 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% similar, identical, or homologous with at least, or at most 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,
25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49,
50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74,
75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99,
100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118,
119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137,
138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156,
157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175,
176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194,
195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213,
214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232,
233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 300,
400, 500, 550, 1000 or more contiguous amino acids or nucleic acids, or any range derivable therein, of SEQ ID NOs: 1-20.
[0176] The polypeptides or polynucleotides of the disclosure may include, may include at least, or may include at most 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47,
48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72,
73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97,
98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116,
117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135,
136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154,
155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192
193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211
212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230
231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249
250, 300, 400, 500, 550, 1000 or more contiguous amino acids, or any range derivable therein of SEQ ID NO: 1-20.
[0177] A polypeptide of the disclosure may comprise amino acids 1 to 2, 3, 4, 5, 6, 7, 8 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106
107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125,
126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144,
145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163,
164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182,
183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201,
202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220,
221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239,
240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258,
259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277,
278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296,
297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315,
316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334,
335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353,
354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372,
373, 374, 375, 376, 377, 378, 379, 380, 381, 382, 383, 384, 385, 386, 387, 388, 389, 390, 391,
392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410,
411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428, 429,
430, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442, 443, 444, 445, 446, 447, 448,
449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467,
468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 484, 485, 486,
487, 488, 489, 490, 491, 492, 493, 494, 495, 496, 497, 498, 499, 500, 501, 502, 503, 504, 505,
506, 507, 508, 509, 510, 511, 512, 513, 514, 515, 516, 517, 518, 519, 520, 521, 522, 523, 524,
525, 526, 527, 528, 529, 530, 531, 532, 533, 534 535, 536, 537, 538, 539, 540, 541, 542, 543, 544, 545, 546, 547, 548, 549, 550, 551, 552, 553, 554, 555, 556, 557, 558, 559, 560, 561, 562,
563, 564, 565, 566, 567, 568, 569, 570, 571, 572, 573, 574, 575, 576, 577, 578, 579, 580, 581,
582, 583, 584, 585, 586, 587, 588, 589, 590, 591, 592, 593, 594, 595, 596, 597, 598, 599, 600,
601, 602, 603, 604, 605, 606, 607, 608, 609, 610, 611, 612, 613, 614, or 615 (or any derivable range therein) of SEQ ID NOs: 1-20.
[0178] A polypeptide of the disclosure may comprise, comprise at least, or comprise at most 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,
28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90^ 91, 92, 93, 94 , 95, 96, 97, 98 , 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, 383, 384, 385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428, 429, 430, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442, 443, 444, 445, 446, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 484, 485, 486, 487, 488, 489, 490, 491, 492, 493, 494, 495, 496, 497, 498, 499, 500, 501, 502, 503, 504, 505, 506, 507, 508, 509, 510, 511, 512, 513, 514, 515, 516, 517, 518, 519, 520, 521, 522, 523, 524, 525, 526, 527, 528, 529, 530, 531, 532, 533, 534, 535, 536, 537, 538, 539, 540, 541, 542, 543, 544, 545, 546, 547, 548, 549, 550, 551, 552, 553, 554, 555, 556, 557, 558, 559, 560, 561, 562, 563, 564, 565, 566, 567, 568, 569, 570, 571, 572, 573, 574, 575, 576,
577, 578, 579, 580, 581, 582, 583, 584, 585, 586, 587, 588, 589, 590, 591, 592, 593, 594, 595,
596, 597, 598, 599, 600, 601, 602, 603, 604, 605, 606, 607, 608, 609, 610, 611, 612, 613, 614, or 615 (or any derivable range therein) contiguous amino acids of SEQ ID NOs: 1-20.
[0179] The polypeptides of the disclosure may comprise, may comprise at least, or may comprise at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,
24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48,
49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73,
74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98,
99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136,
137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155,
156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174,
175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193,
194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212,
213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231,
232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250,
251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269,
270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288,
289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307,
308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326,
327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345,
346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364,
365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, 383,
384, 385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402,
403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421,
422, 423, 424, 425, 426, 427, 428, 429, 430, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440,
441, 442, 443, 444, 445, 446, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459,
460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478,
479, 480, 481, 482, 483, 484, 485, 486, 487, 488, 489, 490, 491, 492, 493, 494, 495, 496, 497,
498, 499, 500, 501, 502, 503, 504, 505, 506, 507, 508, 509, 510, 511, 512, 513, 514, 515, 516,
517, 518, 519, 520, 521, 522, 523, 524, 525, 526, 527, 528, 529, 530, 531, 532, 533, 534, 535,
536, 537, 538, 539, 540, 541, 542, 543, 544, 545, 546, 547, 548, 549, 550, 551, 552, 553, 554,
555, 556, 557, 558, 559, 560, 561, 562, 563, 564, 565, 566, 567, 568, 569, 570, 571, 572, 573, 574, 575, 576, 577, 578, 579, 580, 581, 582, 583, 584, 585, 586, 587, 588, 589, 590, 591, 592, 593, 594, 595, 596, 597, 598, 599, 600, 601, 602, 603, 604, 605, 606, 607, 608, 609, 610, 611, 612, 613, 614, or 615 (or any derivable range therein) contiguous amino acids of SEQ ID NOs: l-20 that are at least, at most, or exactly 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% similar, identical, or homologous with any one of SEQ ID NOS: 1-20.
[0180] A polypeptide of the disclosure may have, have at least, or have at most 60%, 61%,
62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% (or any range derivable therein) sequence identity or homology with one of SEQ ID NOS: 1-20.
[0181] The disclosure includes a nucleic acid molecule or polypeptide starting at position
1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,
29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53,
54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78,
79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102,
103, 104, 105, 106, 107, 108, 109, 110, 111, 112 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378 379, 380, 381, 382, 383, 384, 385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397 398, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416 417, 418, 419, 420, 421, 422, 423, 424, 425, 6, 427, 428, 429, 430, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442, 443, 444,5, 446, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463,4, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482,3, 484, 485, 486, 487, 488, 489, 490, 491, 492, 493, 494, 495, 496, 497, 498, 499, 500, 501,2, 503, 504, 505, 506, 507, 508, 509, 510, 511, 512, 513, 514, 515, 516, 517, 518, 519, 520,1, 522, 523, 524, 525, 526, 527, 528, 529, 530, 531, 532, 533, 534, 535, 536, 537, 538, 539,0, 541, 542, 543, 544, 545, 546, 547, 548, 549, 550, 551, 552, 553, 554, 555, 556, 557, 558,9, 560, 561, 562, 563, 564, 565, 566, 567, 568, 569, 570, 571, 572, 573, 574, 575, 576, 577,8, 579, 580, 581, 582, 583, 584, 585, 586, 587, 588, 589, 590, 591, 592, 593, 594, 595, 596,7, 598, 599, 600, 601, 602, 603, 604, 605, 606, 607, 608, 609, 610, 611, 612, 613, 614, or5 of any of SEQ ID NOS: 1-20 and comprising 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41,, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66,, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91,, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112,3, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131,2, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150,1, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169,0, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188,9, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207,8, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226,7, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245,6, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264,5, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283,4, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302,3, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321,2, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340,1, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359,0, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378,9, 380, 381, 382, 383, 384, 385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397,8, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416,7, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428, 429, 430, 431, 432, 433, 434, 435,6, 437, 438, 439, 440, 441, 442, 443, 444, 445, 446, 447, 448, 449, 450, 451, 452, 453, 454,5, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 484, 485, 486, 487, 488, 489, 490, 491, 492,
493, 494, 495, 496, 497, 498, 499, 500, 501, 502, 503, 504, 505, 506, 507, 508, 509, 510, 511,
512, 513, 514, 515, 516, 517, 518, 519, 520, 521, 522, 523, 524, 525, 526, 527, 528, 529, 530,
531, 532, 533, 534, 535, 536, 537, 538, 539, 540, 541, 542, 543, 544, 545, 546, 547, 548, 549,
550, 551, 552, 553, 554, 555, 556, 557, 558, 559, 560, 561, 562, 563, 564, 565, 566, 567, 568,
569, 570, 571, 572, 573, 574, 575, 576, 577, 578, 579, 580, 581, 582, 583, 584, 585, 586, 587,
588, 589, 590, 591, 592, 593, 594, 595, 596, 597, 598, 599, 600, 601, 602, 603, 604, 605, 606,
607, 608, 609, 610, 611, 612, 613, 614, or 615 contiguous nucleotides or amino acids of any of SEQ ID NOS: 1-20.
[0182] The polypeptides and nucleic acids of the disclosure may include, may include at least, or may inclu at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 1' ), 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, , 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 55, 57, 58, 59, 60, 61, 62, 63, 64, 65, , 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, , 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 , 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119 , 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134,
135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, 383, 384, 385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428, 429, 430, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442, 443, 444, 445, 446, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 484, 485, 486, 487, 488, 489, 490, 491, 492, 493, 494, 495
496, 497, 498, 499, 500, 501, 502, 503, 504, 505, 506, 507, 508, 509, 510, 511, 512, 513, 514
515, 516, 517, 518, 519, 520, 521, 522, 523, 524, 525, 526, 527, 528, 529, 530, 531, 532, 533
534, 535, 536, 537, 538, 539, 540, 541, 542, 543, 544, 545, 546, 547, 548, 549, 550, 551, 552
553, 554, 555, 556, 557, 558, 559, 560, 561, 562, 563, 564, 565, 566, 567, 568, 569, 570, 571
572, 573, 574, 575, 576, 577, 578, 579, 580, 581, 582, 583, 584, 585, 586, 587, 588, 589, 590
591, 592, 593, 594, 595, 596, 597, 598, 599, 600, 601, 602, 603, 604, 605, 606, 607, 608, 609
610, 611, 612, 613, 614, or 615 substitutions (or any range derivable therein).
[0183] The substitution may be at amino acid position or nucleic acid position 1, 2, 3, 4 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31
32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69. 70. 71. 72. 73. 74. 75. 76. 77. 78. 79. 80. 81 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93 , 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, 383, 384, 385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428, 429, 430, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442, 443, 444, 445, 446, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 484, 485, 486, 487, 488, 489, 490, 491, 492, 493, 494, 495, 496, 497, 498, 499, 500, 501, 502, 503, 504, 505, 506, 507, 508, 509, 510, 511, 512, 513, 514, 515, 516, 517, 518, 519, 520, 521, 522,
523, 524, 525, 526, 527, 528, 529, 530, 531, 532, 533, 534, 535, 536, 537, 538, 539, 540, 541,
542, 543, 544, 545, 546, 547, 548, 549, 550, 551, 552, 553, 554, 555, 556, 557, 558, 559, 560,
561, 562, 563, 564, 565, 566, 567, 568, 569, 570, 571, 572, 573, 574, 575, 576, 577, 578, 579,
580, 581, 582, 583, 584, 585, 586, 587, 588, 589, 590, 591, 592, 593, 594, 595, 596, 597, 598,
599, 600, 601, 602, 603, 604, 605, 606, 607, 608, 609, 610, 611, 612, 613, 614, or 615 of one of SEQ ID NO: 1-20. One or more of these substitutions may be specifically excluded from an aspect.
[0184] The amino acid at position 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43,
44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68,
69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93,
94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113,
114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132,
133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151,
152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170,
171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189,
190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208,
209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227,
228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246,
247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265,
266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284,
285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303,
304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322,
323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341,
342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360,
361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379,
380, 381, 382, 383, 384, 385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398,
399, or 400 of the peptide or polypeptide of one of SEQ ID NOS: 1-20 may be substituted with an alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, or valine.
[0185] Peptides, polypeptides, and proteins of the disclosure may have, may have at least, or may have at most 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity to any one of SEQ ID NOS: 1-20 and may includes a fragment or segment starting at amino acid 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37,
38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62,
63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87,
88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109,
110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128,
129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147,
148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166,
167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185,
186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, or 200 (or any range derivable therein) and ending at amino acid 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,
23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47,
48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72,
73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97,
98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135,
136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154,
155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173,
174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192,
193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, or 205 (or any range derivable therein).
[0186] Substitutional variants typically contain the exchange of one amino acid for another at one or more sites within the protein, and may be designed to modulate one or more properties of the polypeptide, with or without the loss of other functions or properties. Substitutions may be conservative, that is, one amino acid is replaced with one of similar shape and charge. Conservative substitutions are well known in the art and include, for example, the changes of: alanine to serine; arginine to lysine; asparagine to glutamine or histidine; aspartate to glutamate; cysteine to serine; glutamine to asparagine; glutamate to aspartate; glycine to proline; histidine to asparagine or glutamine; isoleucine to leucine or valine; leucine to valine or isoleucine; lysine to arginine; methionine to leucine or isoleucine; phenylalanine to tyrosine, leucine or methionine; serine to threonine; threonine to serine; tryptophan to tyrosine; tyrosine to tryptophan or phenylalanine; and valine to isoleucine or leucine. Alternatively, substitutions may be non-conservative such that a function or activity of the polypeptide is affected. Non- conservative changes typically involve substituting a residue with one that is chemically dissimilar, such as a polar or charged amino acid for a nonpolar or uncharged amino acid, and vice versa. One or more of these substitutions may be specifically excluded from an aspect.
[0187] Proteins may be recombinant, or synthesized in vitro. Alternatively, a nonrecombinant or recombinant protein may be isolated from bacteria. It is also contemplated that bacteria containing such a variant may be implemented in compositions and methods. Consequently, a protein need not be isolated.
[0188] The term “functionally equivalent codon” is used herein to refer to codons that encode the same amino acid, such as the six codons for arginine or serine, and also refers to codons that encode biologically equivalent amino acids.
[0189] It also will be understood that amino acid and nucleic acid sequences may include additional residues, such as additional N- or C-terminal amino acids, or 5' or 3' sequences, respectively, and yet still be essentially as set forth in one of the sequences disclosed herein, so long as the sequence meets the criteria set forth above, including the maintenance of biological protein activity where protein expression is concerned. The addition of terminal sequences particularly applies to nucleic acid sequences that may, for example, include various noncoding sequences flanking either of the 5' or 3' portions of the coding region.
[0190] The following is a discussion based upon changing of the amino acids of a protein to create an equivalent, or even an improved, second-generation molecule. For example, certain amino acids may be substituted for other amino acids in a protein structure without appreciable loss of interactive binding capacity. Structures such as, for example, an enzymatic catalytic domain or interaction components may have amino acid substituted to maintain such function. Since it is the interactive capacity and nature of a protein that defines that protein’s biological functional activity, certain amino acid substitutions can be made in a protein sequence, and in its underlying DNA coding sequence, and nevertheless produce a protein with like properties. It is thus contemplated by the inventors that various changes may be made in the DNA sequences of genes without appreciable loss of their biological utility or activity.
[0191] In other aspects, alteration of the function of a polypeptide is intended by introducing one or more substitutions. For example, certain amino acids may be substituted for other amino acids in a protein structure with the intent to modify the interactive binding capacity of interaction components. Structures such as, for example, protein interaction domains, nucleic acid interaction domains, and catalytic sites may have amino acids substituted to alter such function. Since it is the interactive capacity and nature of a protein that defines that protein’s biological functional activity, certain amino acid substitutions can be made in a protein sequence, and in its underlying DNA coding sequence, and nevertheless produce a protein with different properties. It is thus contemplated by the inventors that various changes may be made in the DNA sequences of genes with appreciable alteration of their biological utility or activity.
[0192] In making such changes, the hydropathic index of amino acids may be considered. The importance of the hydropathic amino acid index in conferring interactive biologic function on a protein is generally understood in the art (Kyte and Doolittle, 1982). It is accepted that the relative hydropathic character of the amino acid contributes to the secondary structure of the resultant protein, which in turn defines the interaction of the protein with other molecules, for example, enzymes, substrates, receptors, DNA, antibodies, antigens, and the like.
[0193] It also is understood in the art that the substitution of like amino acids can be made effectively on the basis of hydrophilicity. U.S. Patent 4,554,101, incorporated herein by reference, states that the greatest local average hydrophilicity of a protein, as governed by the hydrophilicity of its adjacent amino acids, correlates with a biological property of the protein. It is understood that an amino acid can be substituted for another having a similar hydrophilicity value and still produce a biologically equivalent and immunologically equivalent protein.
[0194] As outlined above, amino acid substitutions generally are based on the relative similarity of the amino acid side-chain substituents, for example, their hydrophobicity, hydrophilicity, charge, size, and the like. Exemplary substitutions that take into consideration the various foregoing characteristics are well known and include: arginine and lysine; glutamate and aspartate; serine and threonine; glutamine and asparagine; and valine, leucine and isoleucine.
[0195] In specific aspects, all or part of proteins described herein can also be synthesized in solution or on a solid support in accordance with conventional techniques. Various automatic synthesizers are commercially available and can be used in accordance with known protocols. See, for example, Stewart and Young, (1984); Tam et al., (1983); Merrifield, (1986); and Barany and Merrifield (1979), each incorporated herein by reference. Alternatively, recombinant DNA technology may be employed wherein a nucleotide sequence that encodes a peptide or polypeptide is inserted into an expression vector, transformed or transfected into an appropriate host cell and cultivated under conditions suitable for expression.
[0196] One aspect includes the use of gene transfer to cells, including microorganisms, for the production and/or presentation of proteins. The gene for the protein of interest may be transferred into appropriate host cells followed by culture of cells under the appropriate conditions. A nucleic acid encoding virtually any polypeptide may be employed. The generation of recombinant expression vectors, and the elements included therein, are discussed herein. Alternatively, the protein to be produced may be an endogenous protein normally synthesized by the cell used for protein production.
VI. Nucleic Acids
[0197] In certain aspects, the current disclosure concerns recombinant polynucleotides encoding the polypeptides of the disclosure. Therefore, certain aspects relate to nucleotides encoding for polypeptides, chimeric polypeptides, or multimeric polypeptides of the disclosure.
[0198] As used in this application, the term “polynucleotide” refers to a nucleic acid molecule that either is recombinant or has been isolated free of total genomic nucleic acid. Included within the term “polynucleotide” are oligonucleotides (nucleic acids of 100 residues or less in length), recombinant vectors, including, for example, plasmids, cosmids, phage, viruses, and the like. Polynucleotides include, in certain aspects, regulatory sequences, isolated substantially away from their naturally occurring genes or protein encoding sequences. Polynucleotides may be single-stranded (coding or antisense) or double-stranded, and may be RNA, DNA (genomic, cDNA or synthetic), analogs thereof, or a combination thereof. Additional coding or non-coding sequences may, but need not, be present within a polynucleotide.
[0199] In this respect, the term “gene,” “polynucleotide,” or “nucleic acid” is used to refer to a nucleic acid that encodes a protein, polypeptide, or peptide (including any sequences required for proper transcription, post-translational modification, or localization). As will be understood by those in the art, this term encompasses genomic sequences, expression cassettes, cDNA sequences, and smaller engineered nucleic acid segments that express, or may be adapted to express, proteins, polypeptides, domains, peptides, fusion proteins, and mutants. A nucleic acid encoding all or part of a polypeptide may contain a contiguous nucleic acid sequence of: 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 441, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 910, 920, 930, 940, 950, 960, 970, 980, 990, 1000, 1010, 1020, 1030, 1040, 1050, 1060, 1070, 1080, 1090, 1095, 1100, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 9000, 10000, or more nucleotides, nucleosides, or base pairs (or any range derivable therein), including all values and ranges there between, of a polynucleotide encoding one or more amino acid sequence described or referenced herein. It also is contemplated that a particular polypeptide may be encoded by nucleic acids containing variations having slightly different nucleic acid sequences but, nonetheless, encode the same or substantially similar protein.
[0200] In particular aspects, the invention concerns isolated nucleic acid segments and recombinant vectors incorporating nucleic acid sequences that encode a polypeptide or peptide of the disclosure. The term “recombinant” may be used in conjunction with a polynucleotide or polypeptide and generally refers to a polypeptide or polynucleotide produced and/or manipulated in vitro or that is a replication product of such a molecule.
[0201] In other aspects, the invention concerns isolated nucleic acid segments and recombinant vectors incorporating nucleic acid sequences that encode a polypeptide or peptide of the disclosure.
[0202] The nucleic acid segments used in the current disclosure can be combined with other nucleic acid sequences, such as promoters, polyadenylation signals, additional restriction enzyme sites, multiple cloning sites, other coding segments, and the like, such that their overall length may vary considerably. It is therefore contemplated that a nucleic acid fragment of almost any length may be employed, with the total length preferably being limited by the ease of preparation and use in the intended recombinant nucleic acid protocol. In some cases, a nucleic acid sequence may encode a polypeptide sequence with additional heterologous coding sequences, for example to allow for purification of the polypeptide, transport, secretion, post- translational modification, or for therapeutic benefits such as targeting or efficacy. As discussed above, a tag or other heterologous polypeptide may be added to the modified polypeptide-encoding sequence, wherein “heterologous” refers to a polypeptide that is not the same as the modified polypeptide.
[0203] In certain aspects, the current disclosure provides polynucleotide variants having substantial identity to the sequences disclosed herein; those comprising at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or higher sequence identity, including all values and ranges there between, compared to a polynucleotide sequence of this disclosure using the methods described herein (e.g., BLAST analysis using standard parameters).
[0204] The disclosure also contemplates the use of polynucleotides which are complementary to all the above described polynucleotides.
A. Vectors
[0205] Polypeptides of the disclosure may be encoded by a nucleic acid molecule comprised in a vector. The term “vector” is used to refer to a carrier nucleic acid molecule into which a heterologous nucleic acid sequence can be inserted for introduction into a cell where it can be replicated and expressed. A nucleic acid sequence can be “heterologous,” which means that it is in a context foreign to the cell in which the vector is being introduced or to the nucleic acid in which is incorporated, which includes a sequence homologous to a sequence in the cell or nucleic acid but in a position within the host cell or nucleic acid where it is ordinarily not found. Vectors include DNAs, RNAs, plasmids, cosmids, viruses (bacteriophage, animal viruses, and plant viruses), and artificial chromosomes (e.g., YACs). One of skill in the art would be well equipped to construct a vector through standard recombinant techniques (for example Sambrook et al., 2001; Ausubel et al., 1996, both incorporated herein by reference). In addition to encoding a polypeptide of the disclosure, the vector can encode other polypeptide sequences such as a one or more other bacterial peptide, a tag, or an immunogenicity enhancing peptide. Useful vectors encoding such fusion proteins include pIN vectors (Inouye et al., 1985), vectors encoding a stretch of histidines, and pGEX vectors, for use in generating glutathione S-transferase (GST) soluble fusion proteins for later purification and separation or cleavage.
[0206] The term “expression vector” refers to a vector containing a nucleic acid sequence coding for at least part of a gene product capable of being transcribed. In some cases, RNA molecules are then translated into a protein, polypeptide, or peptide. Expression vectors can contain a variety of “control sequences,” which refer to nucleic acid sequences necessary for the transcription and possibly translation of an operably linked coding sequence in a particular host organism. In addition to control sequences that govern transcription and translation, vectors and expression vectors may contain nucleic acid sequences that serve other functions as well and are described herein.
B. Promoters and Enhancers
[0207] A “promoter” is a control sequence. The promoter is typically a region of a nucleic acid sequence at which initiation and rate of transcription are controlled. It may contain genetic elements at which regulatory proteins and molecules may bind such as RNA polymerase and other transcription factors. The phrases “operatively positioned,” “operatively linked,” “under control,” and “under transcriptional control” mean that a promoter is in a correct functional location and/or orientation in relation to a nucleic acid sequence to control transcriptional initiation and expression of that sequence. A promoter may or may not be used in conjunction with an “enhancer,” which refers to a cis-acting regulatory sequence involved in the transcriptional activation of a nucleic acid sequence. [0208] Naturally, it may be important to employ a promoter and/or enhancer that effectively directs the expression of the DNA segment in the cell type or organism chosen for expression. Those of skill in the art of molecular biology generally know the use of promoters, enhancers, and cell type combinations for protein expression (see Sambrook et al., 2001, incorporated herein by reference). The promoters employed may be constitutive, tissuespecific, or inducible and in certain aspects may direct high level expression of the introduced DNA segment under specified conditions, such as large-scale production of recombinant proteins or peptides.
[0209] The particular promoter that is employed to control the expression of peptide or protein encoding polynucleotide of the invention is not believed to be critical, so long as it is capable of expressing the polynucleotide in a targeted cell, preferably a bacterial cell. Where a human cell is targeted, it is preferable to position the polynucleotide coding region adjacent to and under the control of a promoter that is capable of being expressed in a human cell. Generally speaking, such a promoter might include either a bacterial, human or viral promoter.
C. Initiation Signals and Internal Ribosome Binding Sites (IRES)
[0210] A specific initiation signal also may be required for efficient translation of coding sequences. These signals include the ATG initiation codon or adjacent sequences. Exogenous translational control signals, including the ATG initiation codon, may need to be provided. One of ordinary skill in the art would readily be capable of determining this and providing the necessary signals.
[0211] In certain aspects of the invention, the use of internal ribosome entry sites (IRES) elements are used to create multigene, or polycistronic, messages. IRES elements are able to bypass the ribosome scanning model of 5’ methylated Cap dependent translation and begin translation at internal sites (Pelletier and Sonenberg, 1988; Macejak and Sarnow, 1991). IRES elements can be linked to heterologous open reading frames. Multiple open reading frames can be transcribed together, each separated by an IRES, creating polycistronic messages. Multiple genes can be efficiently expressed using a single promoter/enhancer to transcribe a single message (see U.S. Patents 5,925,565 and 5,935,819, herein incorporated by reference).
D. Selectable and Screenable Markers
[0212] In certain aspects of the invention, cells containing a nucleic acid construct of the current disclosure may be identified in vitro or in vivo by encoding a screenable or selectable marker in the expression vector. When transcribed and translated, a marker confers an identifiable change to the cell permitting easy identification of cells containing the expression vector. Generally, a selectable marker is one that confers a property that allows for selection. A positive selectable marker is one in which the presence of the marker allows for its selection, while a negative selectable marker is one in which its presence prevents its selection. An example of a positive selectable marker is a drug resistance marker.
E. Host Cells
[0213] As used herein, the terms “cell,” “cell line,” and “cell culture” may be used interchangeably. All of these terms also include their progeny, which is any and all subsequent generations. It is understood that all progeny may not be identical due to deliberate or inadvertent mutations. In the context of expressing a heterologous nucleic acid sequence, “host cell” refers to a prokaryotic or eukaryotic cell, and it includes any transformable organism that is capable of replicating a vector or expressing a heterologous gene encoded by a vector. A host cell can, and has been, used as a recipient for vectors or viruses. A host cell may be “transfected” or “transformed,” which refers to a process by which exogenous nucleic acid, such as a recombinant protein-encoding sequence, is transferred or introduced into the host cell. A transformed cell includes the primary subject cell and its progeny.
[0214] Host cells may be derived from prokaryotes or eukaryotes, including bacteria, yeast cells, insect cells, and mammalian cells for replication of the vector or expression of part or all of the nucleic acid sequence(s). Numerous cell lines and cultures are available for use as a host cell, and they can be obtained through the American Type Culture Collection (ATCC), which is an organization that serves as an archive for living cultures and genetic materials (www.atcc.org).
F. Expression Systems
[0215] Numerous expression systems exist that comprise at least a part or all of the compositions discussed above. Prokaryote- and/or eukaryote-based systems can be employed for use with the present invention to produce nucleic acid sequences, or their cognate polypeptides, proteins and peptides. Many such systems are commercially and widely available.
[0216] The insect cell/baculovirus system can produce a high level of protein expression of a heterologous nucleic acid segment, such as described in U.S. Patents 5,871,986, 4,879,236, both herein incorporated by reference, and which can be bought, for example, under the name MAXBAC® 2.0 from INVITROGEN® and BACPACK™ BACULOVIRUS EXPRESSION SYSTEM FROM CLONTECH®.
[0217] In addition to the disclosed expression systems of the invention, other examples of expression systems include STRATAGENE®’s COMPLETE CONTROL Inducible Mammalian Expression System, which involves a synthetic ecdysone-inducible receptor, or its pET Expression System, an E. coli expression system. Another example of an inducible expression system is available from INVITROGEN®, which carries the T-REX™ (tetracycline-regulated expression) System, an inducible mammalian expression system that uses the full-length CMV promoter. INVITROGEN® also provides a yeast expression system called the Pichia methanolica Expression System, which is designed for high-level production of recombinant proteins in the methyl otrophic yeast Pichia methanolica. One of skill in the art would know how to express a vector, such as an expression construct, to produce a nucleic acid sequence or its cognate polypeptide, protein, or peptide.
VII. Pharmaceutical compositions
[0218] In certain aspects, the compositions or agents for use in the methods are suitably contained in a pharmaceutically acceptable carrier. The carrier is non-toxic, biocompatible and is selected so as not to detrimentally affect the biological activity of the agent. The agents in some aspects of the invention may be formulated into preparations for local delivery (i.e. to a specific location of the body, such as skeletal muscle or other tissue) or systemic delivery, in solid, semi-solid, gel, liquid or gaseous forms such as tablets, capsules, powders, granules, ointments, solutions, depositories, inhalants and injections allowing for oral, parenteral or surgical administration. Certain aspects of the invention also contemplate local administration of the compositions by coating medical devices and the like.
[0219] Suitable carriers for parenteral delivery via injectable, infusion or irrigation and topical delivery include distilled water, physiological phosphate-buffered saline, normal or lactated Ringer's solutions, dextrose solution, Hank's solution, or propanediol. In addition, sterile, fixed oils may be employed as a solvent or suspending medium. For this purpose any biocompatible oil may be employed including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid find use in the preparation of injectables. The carrier and agent may be compounded as a liquid, suspension, polymerizable or non-polymerizable gel, paste or salve.
[0220] The carrier may also comprise a delivery vehicle to sustain (i.e., extend, delay or regulate) the delivery of the agent(s) or to enhance the delivery, uptake, stability or pharmacokinetics of the therapeutic agent(s). Such a delivery vehicle may include, by way of non-limiting examples, microparticles, microspheres, nanospheres or nanoparticles composed of proteins, liposomes, carbohydrates, synthetic organic compounds, inorganic compounds, polymeric or copolymeric hydrogels and polymeric micelles.
[0221] In certain aspects, the actual dosage amount of a composition administered to a patient or subject can be determined by physical and physiological factors such as body weight, severity of condition, the type of disease being treated, previous or concurrent therapeutic interventions, idiopathy of the patient and on the route of administration. The practitioner responsible for administration will, in any event, determine the concentration of active ingredient(s) in a composition and appropriate dose(s) for the individual subject.
[0222] In certain embodiments, pharmaceutical compositions may comprise, for example, at least about 0.1% of an active agent, such as lipid nanoparticle or a related lipid nanovesicle loaded with therapeutic cargo or diagnostic agents. In other embodiments, the active agent may comprise between about 2% to about 75% of the weight of the unit, or between about 25% to about 60%, for example, and any range derivable therein. In other non-limiting examples, a dose may also comprise from about 1 microgram/kg/body weight, about 5 microgram/kg/body weight, about 10 microgram/kg/body weight, about 50 microgram/kg/body weight, about 100 microgram/kg/body weight, about 200 microgram/kg/body weight, about 350 microgram/kg/body weight, about 500 microgram/kg/body weight, about 1 milligram/kg/body weight, about 5 milligram/kg/body weight, about 10 milligram/kg/body weight, about 50 milligram/kg/body weight, about 100 milligram/kg/body weight, about 200 milligram/kg/body weight, about 350 milligram/kg/body weight, about 500 milligram/kg/body weight, to about 1000 mg/kg/body weight or more per administration, and any range derivable therein. In nonlimiting examples of a derivable range from the numbers listed herein, a range of about 5 microgram/kg/body weight to about 100 mg/kg/body weight, about 5 microgram/kg/body weight to about 500 milligram/kg/body weight, etc., can be administered.
[0223] Solutions of pharmaceutical compositions can be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose. Dispersions also can be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.
[0224] In certain aspects, the pharmaceutical compositions are advantageously administered in the form of injectable compositions either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid prior to injection may also be prepared. These preparations also may be emulsified. A typical composition for such purpose comprises a pharmaceutically acceptable carrier. For instance, the composition may contain less, than, equal to, or more than 10 mg, 25 mg, 50 mg or up to about 100 mg of human serum albumin per milliliter of phosphate buffered saline. Other pharmaceutically acceptable carriers include aqueous solutions, non-toxic excipients, including salts, preservatives, buffers and the like. [0225] Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oil and injectable organic esters such as ethyloleate. Aqueous carriers include water, alcoholic/aqueous solutions, saline solutions, parenteral vehicles such as sodium chloride, Ringer's dextrose, etc. Intravenous vehicles include fluid and nutrient replenishers. Preservatives include antimicrobial agents, anti-fungal agents, anti-oxidants, chelating agents and inert gases. The pH and exact concentration of the various components the pharmaceutical composition are adjusted according to well-known parameters.
[0226] Additional formulations are suitable for oral administration. Oral formulations include such typical excipients as, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate and the like. The compositions take the form of solutions, suspensions, tablets, pills, capsules, sustained release formulations or powders.
[0227] In further aspects, the pharmaceutical compositions may include classic pharmaceutical preparations. Administration of pharmaceutical compositions according to certain aspects may be via any common route so long as the target tissue is available via that route. This may include oral, nasal, buccal, rectal, vaginal or topical. Administration may be by orthotopic, intradermal, subcutaneous, intramuscular, intraperitoneal or intravenous injection. Such compositions would normally be administered as pharmaceutically acceptable compositions that include physiologically acceptable carriers, buffers or other excipients. For treatment of conditions of the lungs, aerosol delivery can be used. Volume of the aerosol is between about 0.01 ml and 0.5 ml.
[0228] An effective amount of the pharmaceutical composition is determined based on the intended goal. The term “unit dose” or “dosage” refers to physically discrete units suitable for use in a subject, each unit containing a predetermined-quantity of the pharmaceutical composition calculated to produce the desired responses discussed above in association with its administration, i.e., the appropriate route and treatment regimen. The quantity to be administered, both according to number of treatments and unit dose, depends on the protection or effect desired.
[0229] Precise amounts of the pharmaceutical composition also depend on the judgment of the practitioner and are peculiar to each individual. Factors affecting the dose include the physical and clinical state of the patient, the route of administration, the intended goal of treatment (e.g., alleviation of symptoms versus cure) and the potency, stability and toxicity of the particular therapeutic substance. VIII. Sequences
IX. Kits
[0230] Some embodiments concern kits, such as diagnostic and therapeutic kits, as well as kits for preparing and/or delivering lipid nanoparticles. For example, a kit may comprise one or more pharmaceutical compositions and/or lipid nanoparticles as described herein and optionally instructions for their use. Kits may also comprise one or more devices for accomplishing administration of such compositions. For example, a subject kit may comprise a pharmaceutical composition and catheter for accomplishing direct administration of the composition to a patient having or at risk for a demyelination disorder. In other embodiments, a subject kit may comprise pre-filled ampoules of isolated lipid nanoparticles, optionally formulated as a pharmaceutical, or lyophilized, for use with a delivery device. [0231] Kits may comprise a container with a label. Suitable containers include, for example, bottles, vials, and test tubes. The containers may be formed from a variety of materials such as glass or plastic. The container may hold a composition which includes an antibody that is effective for therapeutic or non-therapeutic applications, such as described above. The label on the container may indicate that the composition is used for a specific therapy or non- therapeutic application, and may also indicate directions for either in vivo or in vitro use, such as those described above. In some embodiments, kits will comprise the container described above and one or more other containers comprising materials desirable from a commercial and user standpoint, including buffers, diluents, filters, needles, syringes, and package inserts with instructions for use.
X. Examples
[0232] The following examples are included to demonstrate preferred embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.
EXAMPLE 1: Nanoparticle Toolkit to Enable Choreographed Epithelial Disruption and Gene Editing of Epithelial Stem Cells
A. Nanoparticle Formulation
[0233] Epithelial -Penetrating Nanoparticles: Epithelial-penetrating nanoparticles (epNPs) are designed to penetrate pathological airway mucus and transiently disrupt the apical epithelia to expose the stem cells. Current embodiments of epNPs are comprised of a lipid mix containing molar ratios of 50% l,2-dioleoyl-3 -trimethylammonium -propane chloride (DOTAP), 38.5% 3 -[N-(N’N, N’-dimethylaminoethane)-carbamoyl]cholesterol (DC- cholesterol), 10% dioleoylphosphatidylethanolamine (DOPE), and 1.5% DOPE-poly(ethylene glycol)- 1000 (PE-PEG1000). The identities and respective ratios of these lipids may be varied to tune their ability to disrupt different types of epithelia under various environmental conditions. Further, and critical to the novelty of this invention, this base mixture of structural lipids is then doped with a surfactant moiety (e.g., either polidocanol (PDOC) or lysophosphatidylcholine (LPC)). Individual lipids are dissolved in chloroform and combined in the above ratios in a glass vial or borosilicate test tube (FIG. 2). In the case of LPC, this surfactant is likewise dissolved in chloroform and added to the lipid mix. PDOC, which is insoluble in chloroform, is added to the lipid mix later, In aqueous solution, as described below. The chloroform is then evaporated from the lipid mix under a gentle stream of air, and trace remaining amounts are removed by placing the lipid film under vacuum for 4-24 h (FIG. 2).
[0234] Airway Stem Cell-Targeting Nanoparticles: The second component of AB SC delivery platform includes a nanoparticle that carries a therapeutic cargo. The inventors used CRISPR-nanoparticles (crNPs) to package and deliver gene-editing cargoes to the stem cells of the airway. These lipid nanoparticles are formulated using lipid mixes comprised of molar ratios of 50% ionizable lipid SM-102, 38.5% DC-cholesterol, 10% DOPE, and 1.5% PE- PEG1000. The identities and respective ratios of these lipids may be varied to optimize their affinity for airway stem cells and ability to package and deliver gene editing biomolecules. Individual lipids are dissolved in chloroform and combined in the above ratios in a glass vial or borosilicate test tube (FIG. 2). Chloroform is then evaporated from the lipid mix under a gentle stream of air, and remaining trace amounts of solvent are removed by placing the lipid film under vacuum for 4-24 hrs (FIG. 2).
B. Nanoparticle Fabrication
[0235] Epithelial -Penetrating Nanoparticles: Liposomal structures are formed by dissolving the lipid mixture outlined above in ethanol to the concentration of 10 mg/mL, then flowing this solution through a microfluidic flow-focusing device (Fluigent) at a rate of 10 pL/min while an aqueous phase of phosphate-buffered saline (PBS) and PDOC (where applicable) is co-flowed at a rate of 180 pL/min (FIG. 3). The output is collected and typically contains uniform epNPs of 50-60 nm as measured by dynamic light scattering (DLS) analysis (FIG. 3).
[0236] Airway Stem Cell-Targeting Nanoparticles (with CRISPR/Cas9 system— crNPs): Lipid nanoparticle structures are formed via a rapid dilution and mixing method based on Mitchell et al. and Sweigert et al. (1, 2) by dissolving the lipid mixture outlined above in ethanol to a concentration of 65 mM, then rapidly mixing this solution with a 10 mM citric acid buffer solution of messenger RNA (mRNA), single guide RNA (sgRNA), and single-stranded oligonucleotide donor (ssODN) DNA sequence in an aqueous:organic volume ratio of 2: 1 and a nitrogen-to-phosphate ratio of 7. Rapid mixing may be achieved either by rapid pipetting or by microfluidic mixing using a commercial system (Precision NanoSystems Inc.) or laboratory-designed microfluidic devices (FIG. 4)(1). crNPs are then diluted 4: 1 in PBS for characterization and are typically uniform and 100-200 nm in size as measured by dynamic light scattering (DLS) and transmission electron microscopy (TEM) analysis (FIG. 4). C. Nanoparticle Usage In Vitro
[0237] Suspensions containing epNPs are diluted to a concentration of 10 pg lipid/mL in PBS, and crLNPs are diluted in PBS to a concentration of 1 ng/pL mRNA. In tests, both epNPs and crNPs are applied to the apical surface of airway epithelia cultured in air-liquid interface (ALI) models and may be administered in suspension or as aerosols generated via vibrating mesh nebulizers (e.g., Aeroneb, Kent Scientific). The inventors demonstrated that crLNPs loaded with mRNA transcripts encoding green fluorescent protein (GFP) administered as aerosols to ABSCs cultured in ALI readily transfect ABSCs (FIG. 5). When administered to immortalized human bronchial epithelial (16HBEol4-) cells in suspension, these same crNPs achieve transfection efficiencies of up to ca. 90% with minimal toxicity as measured by flow cytometry (FIG. 6). Further, the inventors observed that crNPs loaded with mRNA transcripts encoding Cas9 along with a sgRNA targeting the CFTR locus achieve site-specific nonhom ologus end joining (NHEJ)-mediated cutting efficiencies of ca. 10% as measured by tracking of insertion-deletions by decomposition (TIDE) (FIG. 6). The inventors observed that PDOC-incorporating epNPs deposited onto ALI cultures show faster airway repair compared to ALIs exposed to PDOC alone (FIG. 6). Dosage (i.e., administered volume of suspension or duration of aerosol exposure) and timing between epNP treatment and crNP treatment may vary depending on the application.
D. Clinical Translation
[0238] The inventors sought to provide inhalable gene therapies, with CF as an initial disease target. Intended clinical use will involve one-time nebulized administration of 1) epLNPs to clear the physical barriers to accessing airway stem cells, followed by nebulized administration of 2) crLNPs to correct the dysfunctional CFTR gene that causes CF and provide long-term or permanent relief of respiratory symptoms of the disease. This two-step strategy may be tailored for a broad spectrum of applications. Specifically, this method is designed to address physical barriers that present challenges in topically accessing stem cells residing in any epithelia (e.g., gastrointestinal epithelium) and can be adapted to deliver multiple types of cargo (e.g., siRNA, tRNA, small molecules, proteins). Accordingly, while the example is directed to the treatment of CF, the methodologies and techniques may apply more broadly and are expected to function for other genetic and non-genetic diseases of the airway and other epithelia. Example 2: CHEMICALLY MODIFIED DSDNA FOR DECREASED TOXICITY AND INCREASED HOMOLOGY DIRECTED REPAIR
[0239] One common approach utilizes the CRISPR/Cas9 system to correct multiple mutated sites in a gene with the site-specific insertion of the corrected target gene’s cDNA. By utilizing site specific cDNA insertion downstream of the endogenous promoter, the corrected cDNA template can be transcribed from the endogenous promoter to correct all downstream mutations. CRISPR/Cas9-mediated site-specific gene editing can be a critical gene editing strategy for diseases with multiple known disease causing mutations (e.g. Cystic Fibrosis, XLA, WAS, X-CGD, XLP, XHIM, etc.). Increased efficiency by means of homology directed repair (after a CRISPR/Cas9-induced double stranded break) requires homology arms of length >500bp; however, the delivery of the entire gene cassette downstream of the endogenous promoter with homology arms of such size can elicit safety concerns within gene editing strategies. Specifically, homology arms of large length (e.g. 100bp+) may include promoter sequence of the target gene, thereby increasing safety risks upon integration into an off-target location. As a result, the inventors utilized the generation of short-homology arm dsDNA donors with chemically modified ” ends to not only increase the safety of integration, but also increase integration capacity and decrease toxicity over standard dsDNA donors. To generate the donors, plasmid DNA of the donor template is amplified with a forward and reverse primer oligo that each contain ” Amino Modifier C6 or C12 modifications (termed AmC6 or AmC12). The resulting PCR product is purified using silica- or carboxyl-coated paramagnetic bead isolation. The inventors tested dsDNA donors with homology arms of various sizes and demonstrated that donors with as little as 50bp of homology arm length could integrate into the target locus by means of homology directed repair, microhomology -mediated end joining, or homology independent targeted integration. To test the effects of the ” AmC6 chemically modified dsDNA donors, the inventors attempted targeted integration into the ” untranslated region (UTR) of the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Specifically, the inventors designed and analyzed various sgRNAs within the 5’ UTR of the CFTR gene for site-specific insertion of the ” AmC6 donor cassette for regulation by the endogenous CFTR promoter. The sequences of each sgRNA are as follows: GGGUCCCUGCUAGGGCCGUC (SEQ ID NO:1); GGUCCCUGCUAGGGCCGUCU (SEQ ID NO:2); UGAGCCCAGACGGCCCUAGC (SEQ ID NO:3); GCAUGGUCUCUCGGGCGCUG (SEQ ID NO:4); GCGCCCGAGAGACCAUGCAG (SEQ ID NO:5). The primers to amplify the AmC6 donors for integration into the CFTR cassette were as follows: AGACAACGCTGGCCTTTTC (SEQ ID NOTO); GTCTTTGGCATTAGGAGCTT (SEQ ID NO: 7); GTTTTCTGAGCACTTACTATATGC (SEQ ID NO:8); GTGGCTTCTTCTGTCCTCCA (SEQ ID NO:9). ” AmC6 dsDNA donors were generated by amplifying a reporter85assettee with 5Obp or 5OObp flanking homology arms around the CFTR target cut site (cut site using sgRNA GCATGGTCTCTCGGGCGCTG (SEQ ID NO:4)). PCR products were purified using AmPureXP magnetic bead purification. The dsDNA donors were tested in a pilot study in various DNA concentrations (0.5 pg, I g, 2pg, 3pg, 5pg, lOpg) for their effect on toxicity and integration. To assess sgRNA cutting efficiency and CFTR gene correction, the inventors utilized T84 epithelial cells (derived from a human colon carcinoma) and human bronchial epithelial cells (16HBE 14o-). These cell lines, that endogenously expresses CFTR, serve as a representative model for CFTR gene repair. For these studies, the inventors transfected T84 or HBE cells with a sgRNA-Cas9 ribonucleoprotein (RNP) complex using the Lonza 4D- Nucleofector using programs DS-138 or CM-137, respectively. 120 pmol of Cas9 and 100 pmol of sgRNA were delivered to the cells. The inventors quantified site-specific reporter cassette integration via in-out digital-droplet (dd) PCR, using Taqman probes designed to the amplicon cassette. The dsDNA donor faired the best in both integration and toxicity when compared to plasmid and nanoplasmid donors. By capping the 50 bp HA dsDNA donor with a 5’ end AmC6 chemical modification, the inventors observed greater than 2-fold reduction in toxicity and maintained equivalent integration efficiency compared to a model plasmid donor with 500bp homology arms. AmC6 modified 50bp donors displayed a 2-fold increase in integration over their non-modified 50bp dsDNA counterpart. Furthermore, AmC6 modified 500 bp HA dsDNA donors displayed a two-fold increase in integration efficiency over 500bp homology arm plasmid donors. Integration by the ” AmC6 donors demonstrated equivalent increases to integration and decreases to toxicity in both T84 cells and HBE cells. Upon assessment of integration fidelity by sequencing of the target locus, ” AmC6 dsDNA donors integrated both through homology directed repair mechanisms and homology -independent targeted integration. FIGS. 7-11 exemplify the data of this example.
Example 3: Lipid nanoparticle aerosols to enable gene therapies for cystic fibrosis.
[0240] Cystic fibrosis (CF) is caused by mutations within the cystic fibrosis transmembrane conductance regulator (CFTR) gene, resulting in formation of thick airway mucus and decreased mucociliary clearance.
[0241] The inventors sought to establish inhalable gene therapy solutions for correcting any CFTR mutation in airway basal stem cells (ABSCs). To accomplish this, they configured lipid nanoparticle (LNP) carriers to package CRISPR/Cas9 gene-editing reagents. These were then delivered to ABSCs via aerosol. The methods also incorporate functionalities to circumnavigate thick mucus layer and epithelium present within the CF airway.
[0242] Liposomes were synthesized via thin-film extrusion and complexed to plasmid or mRNA constructs encoding GFP. ABSCs cultured at ALI (air-liquid interface) were treated with aerosolized lipoplexes carrying reporter cargo (FIG. 12). As exemplified in the data of FIGS. 13-17, aerosolized LNPs offer a versatile inhalable solution for delivery of gene-editing reagents to ABSCs. Incorporation of CRISPR/Cas9 machinery into LNPs paves the way for CF-directed gene therapies.
Example 4: Lipid Nanoparticle Aerosols to Enable Gene Therapies for Cystic Fibrosis [0243] In cystic fibrosis (CF), mutations within the cystic fibrosis transmembrane conductance regulator (CFTR) gene lead to dehydration of airway mucus and decreased antimicrobial function via impaired mucociliary clearance. Because CF is a monogenic disorder, it is a promising candidate for therapies that employ gene editing technologies to provide temporary or even curative symptom relief by correcting CF-associated mutations directly in airway basal stem cells (ABSCs). However, this stem cell population lies below the thick mucus layer lining the airway of CF patients, which represents a considerable physical barrier to the effective delivery of biomolecules used genome engineering. To enable gene therapies capable of delivering gene editing reagents to ABSCs via inhalable aerosolized suspensions, the inventors are developing and testing lipid nanoparticles (LNPs) configured to circumvent the mucus layer and access ABSCs. They leveraged microfluidic mixing strategies to screen LNP compositions rapidly and identified optimal LNP formulations comprised of di oleoyl-3 -trimethylammonium propane (DOTAP), di oleoyl-3 -trimethylammonium propane (DOPE), and 3-P-[N-(”,’'-dimethylaminoethane) carbamoyl] cholesterol (DC-Chol). Dynamic light scattering measurements and transmission electron micrographs confirm that synthesized LNPs are on average <200 nm with a narrow size distribution. Delivery efficiency of LNPs loaded with a model green fluorescent protein (GFP)-expressing plasmid cargo was evaluated in CFTR-expressing T84 cells as well as to air-liquid interface (ALI) monolayer cultures of ABSCs via aerosolized suspensions. Up to 40% GFP-positive cells are observed based on flow cytometry and confocal microscopy -based analyses. Formulations with a nitrogen to phosphate (N/P) ratio of 2: 1 produced the highest transfection efficiencies. Comparable transfection in both T84 cells and ALI cultures was also observed from LNPs packaged with eGFP-encoding mRNA constructs. These studies inform the present evaluation of the delivery performance of LNPs laden with mRNA packages encoding for Cas9 ribonucleoprotein complexes designed for disruption of CFTR. Altogether, insights from the development of this LNP platform may be applied to inform the design and rapid clinical translation of stem cell-based gene therapies directed at CF and other diseases of the airway
[0244] Current approaches to nebulized gene therapies have leveraged either viral-based gene editing or non-viral gene delivery platforms. Transient expression and small effect sizes limit prospects of lipid vectors and viral vectors pose problems with immunogenicity and inefficient transduction across epithelium to access progenitor cells of airway. True gene correction of airway basal stem cells beneath differentiated mucociliary epithelium remains on the frontiers of CF gene therapy. The inventors aim to address this unmet challenge by leveraging the modular design and amenability of lipid nanoparticles to decoration with molecules that may be used to disrupt the apical epithelium, enabling access to the selfrenewing populations of cells in the airway through inhaled aerosol-based delivery (FIG. 1).
[0245] To verify capability to produce LNPs capable of delivering nucleic acid cargos to CF-relevant cells, the inventors used liposome complexation and microfluidic encapsulation methods to deliver reporter constructs to model CFTR-expressing T84 cell line and ABSCs. Liposomes containing DOTAP, DOPE, and lipid-anchored cholesterol were found to effectively complex and deliver reporter cargoes. Dynamic light scattering data shown in the center of FIG. 18 provide information about particles size, and these particles tend to be homogenous and roughly 130 nm in size (-40% transfection in T84s).
[0246] Using Aeroneb vibrating mesh nebulizers, the inventors were able to induce comparable GFP expression with aerosolized eGFP-mRNA lipoplexes in airway basal stem cells before mucociliary differentiation. While these results suggest that a good foundation for a clinical therapy may exist, obstacles include the delivery of large cargoes capable of gene editing, including Cas9-mRNA constructs and even ribonucleoprotein complexes, and effectively crossing the epithelium to access ABSCs directly.
[0247] The inventors’ synthetic methods accommodate larger constructs, and they have been able to reproducibly synthesize monodisperse LNPs around 200 nm in size, coencapsulating Cas9 mRNA with a guide RNA (FIG. 13 and 19). LNPs encapsulating RNPs have tended to be larger but remain monodisperse. It is expected that the size of the LNP may be reduced to 100-200 nm. As a model system to probe delivery and gene-editing capabilities of the particles, the inventors used a T84 cell line transduced to stably express blue fluorescent protein. Homology-directed repair inducing a 3 -nucleotide mutation within the target gene results in green, rather than blue, fluorescence (FIG. 20). Loss of fluorescence altogether indicates cutting and non-homologous end-joining at the target locus. Before introducing the added complexity of incorporating a donor to induce HDR, the inventors focused solely on cutting induced by delivery of a Cas9 construct and guide RNA only. While cutting efficiencies are roughly 5-10% compared to the untreated control, they show promise for a variety of cargoes as the inventors optimize the synthetic methods (FIG. 20).
[0248] The second objective is to overcome the significant physical barriers to accessing ABSCS for permanent edits. Just as is seen in-vivo, ABSCs capable of self-renewal, differentiation, and propagation of permanent gene edits are difficult to access given their location beneath a thick, sticky layer of mucus and a mucociliary epithelium rendered largely impermeable by tight junctions. To address this barrier, the inventors utilized polidocanol. Polidocanol is a surfactant capable of disrupting cell-cell junctions that help form the tight epithelial barrier to luminal cells of the airway. This molecule is routinely used to induce transient injury to the epithelia and access ABSCs in mice. It is hypothesized that, due to its amphiphilic structure, this molecule may participate in the spontaneous self-assembly of LNPs and confer epithelial-penetrating capabilities to the particles.
[0249] While polidocanol may increase access to the ABSCs, direct administration of this compound to the airway is not feasible in a clinical setting. The inventors therefore focused efforts towards developing synthetic protocols which may effectively incorporate polidocanol into the LNPs in such a way to temper the harshness of its detergent action.
[0250] Zeta potential measurements, which give information about particle surface charge, decrease with increasing weight percent added polidocanol, which the inventors hypothesize may be due either to replacement of cationic lipids at the surface of the particles with uncharged polidocanol molecules, or due to ion shielding by polidocanol at the shear plane of the particles. Dynamic light scattering measurements confirm production of small and uniform particles containing up to 40 weight percent polidocanol regardless of whether the particles are formed using thin-film liposome extrusion or whether the polidocanol is incorporated into the aqueous or ethanol phase during microfluidic- synthesis of LNPs (FIG. 15).
[0251] Pilot data comparing the injury and repair of the ALIs after treatment with naked polidocanol as opposed to polidocanol-LNPs shows promising results. While polidocanol- LNPs induce a similar level of epithelial injury, exposing the stem cells (which you can see here indicated with a Keratin 5 marker), epithelial repair was shown to occur much faster in ALIs treated with polidocanol-LNPs (FIG. 6A, upper right panel). The brief transience of LNP- induced epithelial injury holds promise for maintaining the health and proliferative capabilities of the stem cells, and the inventors hypothesize that this will result in faster repair and more efficient gene-editing of these populations. [0252] To summarize, the inventors induced expression of reporter cargoes in ABSCs with
LNP-laden aerosols. These LNPs are being further engineered to induce gene editing in a model cell line, and have induced transient injury to full ALI cultures with polidocanol-LNPs. Objectives moving forward will largely involve tuning lipid composition and microfluidic mixing parameters for improved cutting efficiencies and eventual HDR in the BFP-T84 model. In parallel, the inventors aim to do the same type of optimization for eGFP mRNA and polidocanol co-encapsulation, followed by dosage titration for efficient transfection of ABSCs in full mucociliary epithelia. The inventors will then apply these techniquest to coencapsulate polidocanol with CRISPR-based cargoes to induce gene editing. Finally, the inventors propose investigation of the impact of this sort of coencapsulation on transfection efficiencies and HDR. This work may lead to new tools for enabling clinically relevant, streamlined, and permanent gene therapy solutions for treating cystic fibrosis.
Example 5: A lipid nanoparticle toolkit to correct cystic fibrosis in airway stem cells
[0253] Cystic fibrosis (CF) is caused by mutations within the cystic fibrosis transmembrane conductance regulator (CFTR) gene, resulting in formation of thick airway mucus & decreased mucociliary clearance. The inventors are developing a inhalable gene therapy solution for correcting any CFTR mutation in airway basal stem cells (ABSCs). To achieve this, they are configuring lipid nanoparticle (LNP) carriers to package CRISPR/Cas9 gene-editing reagents to form CRISPR-LNPs (crLNPs), designing liposomes that incorporate the surfactant polidocanol (PDOC) to form epithelial-penetrating LNPs (epLNPs), and delivering LNP -packaged therapeutic cargoes to ABSCs via aerosol (FIG. 1).
[0254] LNPs were synthesized via microfluidic mixing. ABSCs were grown in air-liquid interface (ALI) cultures to recapitulate the structure of the airway epithelium. 16HBE14o- cells ere used as an immortalized model cell line (FIG. 22). crLNPs synthesized with the Precision NanoSystems Spark form uniform size distributions regardless of cargo type (FIG. 23). Various crLNP formulations were screened for their gene-editing capabilities using a reporter system. Editing at the blue fluorescence protein (BFP) locus in reporter 16HBE14o- cells (left) was measured by flow cytometry to rapidly screen LNP composition & dosage (FIG. 24). crLNPs were packaged with Cas9 mRNA, mCitrine (left) or CFTR (right) double-stranded DNA (dsDNA) donor cassettes, and guide RNAs (gRNA) targeting the CFTR 5’UTR. Integration at the CFTR locus in 16HBE14oand 16HBEG542x cells was measured by digital droplet PCR (ddPCR) (FIG. 25). Confocal micrographs displaying GFP expression in ABSCs cultured at ALI 72 h after treatment with LNP-laden aerosols (FIG. 5C). Treatment of ALIs with epLNPs containing PDOC induce similar levels of injury and faster repair of the epithelium as compared to PDOC alone. Scale bars represent 100 pm (FIG. 6).
[0255] In conclusion, particle 1 epLNP formulations demonstrate epithelial modulation & ABSC exposure with minimal injury, particle 2 crLNP formulations deliver a variety of cargoes in suspension & aerosol, and achieve site-specific CFTR editing in airway cells, and incorporation of CRISPR/Cas9 machinery into aerosolizable LNPs paves the way for inhalable CF-directed gene therapies.
Example 6: Cargo-agnostic lipid nanoparticles to correct cystic fibrosis mutations in airway stem cells.
[0256] Cystic fibrosis (CF) is a monogenic disorder arising from mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene that leads to chronic lung infections & respiratory failure. Genetic diseases like CF are appealing targets for gene therapies, but mutation-agnostic gene correction strategies rely on genomic integration of large double-stranded DNA (dsDNA) cassettes that are difficult to package and deliver to target cells. The inventors report the design and testing of a gene therapy strategy that utilizes lipid nanoparticles (LNPs) configured to transport CRISPR/Cas9 payloads designed for site-specific insertion of CFTR in in vitro models of the human airway.
[0257] LNP formulations were synthesized via microfluidic mixing and characterized with dynamic light scattering. The inventors systematically screened LNPs with varying ratios of mRNA encoding Cas9, single guide RNAs (sgRNAs), and donor DNA templates to optimize gene editing in a human bronchial epithelial cell line (16HBE14o-). 16HBE14o- cells stably expressing blue fluorescent protein (BFP) were applied to evaluate editing at the BFP locus via flow cytometry. In this model, non-homologous end joining (NHEJ)-mediated repair at the locus results in loss of fluorescence whereas homology-directed repair (HDR) can be detected following successful integration of donors designed to convert BFP to green fluorescent protein (GFP). Next, they applied HDR donors designed to insert a mCitrine reporter within the endogenous 5’ untranslated region of CFTR in 16HBE14o- cells to assess whether dsDNA cassettes should be co-encapsulated with Cas9-mRNA and sgRNA in a single LNP or packaged separately. The dsDNA: Cas9-mRNA and nitrogen-to-phosphate (N/P) ratios were systematically varied to improve the integration of a CFTR-encoding cassette in 16HBEge- G542x CF mutant cells. Integration of the reporter mCitrine and CFTR-correction cassettes was measured with digital droplet PCR. Successfully edited 16HBEge-G542x colonies were transferred to TransWell inserts and evaluated in an Ussing chamber to validate restoration of CFTR function. [0258] In the presence of an HDR donor, ca. 90% NHEJ-mediated repair and up to 35% HDR were observed in 16HBE14o-BFP cells treated with LNPs packaged with BFP editing cargoes. LNPs with a sgRNA:Cas9-mRNA w/w ratio of 1.2: 1 achieved the highest overall editing efficiencies. Encapsulation of dsDNA donor cassettes with Cas9-mRNA and sgRNA in a single LNP yielded ca. 14% integration of the mCitrine reporter in 16HBE14o- cells compared to ca. 10% when the donor was packaged separately. In 16HBEge-G542x cells, LNPs with a dsDNA: Cas9 mRNA w/w ratio of 3:1 demonstrated up to 3.5% integration, compared to <2% integration for 2: 1 and 4: 1 w/w ratios. Increasing the N/P of the LNPs to 9 further improved CFTR dsDNA integration. Initial validation of CFTR dsDNA donors electroporated in 16HBEge-G542x cells restored CFTR-dependent Cl- current to ca. 80% of wild-type, confirming functional correction. As shown in FIG. 21A-D, LNPs achieved up to 3.5% editing, restoring CFTR protein expression to wild-type levels. FIG. 21E-G shows that Bulk-edited populations of 16HBEgeG542x cells exhibit rescue of the CF phenotype, exhibiting 160% recovery of non-CF wild-type Cl-current as measured via Ussing chamber (FIG. 21E-F) and that LNPs containing dsDNA exhibit greater heterogeneity compared to LNPs containing only RNA (FIG. 21G).
[0259] The inventors demonstrate a LNP platform capable of transporting large gene editing biomolecules to airway epithelial cells that can achieve mutation-agnostic, site-specific correction of CFTR in a CF-deficient cell line. These nanotechnologies offer solutions to overcome limitations in the cargo-carrying capacity of existing gene delivery vectors. This work sets a new precedent for packaging and delivering large dsDNA templates with LNPs that is poised to accelerate progress toward definitive cures for CF and other genetic diseases. [0260] The data and drawings relating to this example is shown in FIGS. 21 and 26-34. LNP formulations deliver a variety of cargoes in suspension & aerosol, and achieve sitespecific CFTR editing in airway cells. Hypotonic EGTA permeabilizes airway epithelia to enable gene delivery. Incorporation of CRISPR/Cas9 machinery into LNPs paves the way for inhalable CF-directed gene therapies.
Example 7: Lipid Nanoparticles for the Delivery of CRISPR/Cas9 Machinery to Enable Site-Specific Integration of CFTR and Mutation-Agnostic Disease Rescue
[0261] Lipid nanoparticles are under rapid and comprehensive development for the packaging and intracellular delivery of nucleic acid-based payloads configured for gene editing. Here, the inventors report the design and testing of lipid nanoparticles (LNPs) engineered to transport CRISPR/Cas9 payloads, including a linear double-stranded DNA (dsDNA) donor template, designed for site-specific insertion of the cystic fibrosis transmembrane conductance regulator (CFTR) gene to correct cystic fibrosis (CF) in diseased human airway epithelial cells. Nanoparticle formulations were systematically screened with varying ratios of Cas9-encoding mRNA, single guide RNAs (sgRNAs), and donor DNA templates to facilitate gene editing in a human bronchial epithelial cell line (16HBE14o-). Bulk populations of 16HBEge-G542x cells edited via LNP delivery of codon-optimized CFTR cDNA cassettes to achieve 3 - 3.5% integration exhibited CFTR-dependent Cl- current to ca. 160% of values measured in normal (non-CF) 16HBE14o- cells. Western blot analyses confirmed greater CFTR protein expression in these bulk edited populations compared to 16HBE14o- cells. Altogether, this versatile LNP platform adds new capabilities for transporting large gene editing machinery to airway epithelial cells for genomic integration of entire genes, paving the way for therapeutic solutions that achieve site-specific correction of any CF-causing mutation.
[0262] The clustered regularly interspersed short palindromic repeat-associated protein 9 (CRISPR/Cas9) technology and its rapidly advancing derivatives including base editors, prime editors, and CRISPR-associated transposases (CASTs) are poised to offer groundbreaking treatment options to patients suffering from genetic disorders. These tools promise long-lived correction of the underlying genetic causes of patient pathology by manipulating the patient genome to knock out pathogenic genes, correct disease-causing mutations, or insert healthy copies of genes. Successful implementation of these tools to edit a cell’s genome necessitates the intracellular delivery of all necessary biomolecular components (i.e. nucleases, guide RNAs, and donor templates). Intracellular delivery is relatively straightforward in vitro, and generally involves the implementation of well-established and effective tools such as electroporation or viral transduction. However, these tools are impractical and sometimes impossible for use in vivo, as electroporation requires the removal of cells from the patient’s body, and viral transduction is fraught with packaging limitations, immunogenicity, and the massive expense of producing good manufacturing practice (GMP)-grade vectors. Translation of exciting and powerful gene editing systems to the clinic will depend largely on the development of platforms for in vivo intracellular delivery of these systems’ multiple required biomolecular components. Lipid nanoparticles (LNPs) are one platform under intense exploration for gene delivery applications following the widespread success of the Pfizer- BioNTech and Modema COVID-19 vaccines, which leverage LNPs to deliver mRNA encoding the COVID-19 spike protein. Prior to the COVID-19 pandemic, LNPs had already demonstrated utility for delivering siRNA. Since then, the lipids used in traditional LNP formulations have been largely replaced by materials generated through the systematic synthesis and screening of vast libraries of novel lipids and lipid-like compounds. These new lipids demonstrate superior mRNA encapsulation and delivery profiles and enhanced tissuetargeting tunability compared to early lipids optimized for hepatic siRNA delivery.
[0263] The application of new LNP formulations toward delivering genes and geneediting components is becoming increasingly common as interest rises in alternatives to viral vectors. In particular, LNP -based gene delivery strategies for targeting cystic fibrosis (CF) are under rapid development in both industry and academia. Cystic fibrosis (CF) is an especially appealing target for gene therapies, as it stems from mutations in a single gene: the cystic fibrosis transmembrane conductance regulator (CFTR) gene. This disease of the epithelia results in impaired ion transport across the epithelium, resulting in dehydrated, sticky mucus and impaired mucociliary clearance. Patients suffer from chronic respiratory infections and eventual respiratory failure. While many patients can be managed with pharmaceuticals that augment the function of the CFTR protein, approximately 10% of patients have mutations that are not eligible for treatment with modulators. For these patients, gene therapies promise definitive treatment. The delivery of mRNA transcripts encoding functional CFTR represents an exciting advancement in the field and a promising option for modulator-resistant CF patients, but the transient nature of mRNA means that these patients will require frequent repeated dosing. This poses accessibility and sustainability issues and raises questions about whether immune responses to LNP components will present a clinical challenge, as they have in other clinical trials. Gene editing platforms, including CRISPR/Cas9, offer a more durable solution. Permanent, robust gene editing of airway stem cells (ASCs) to express functional CFTR would result in long-lived alleviation of respiratory symptoms for virtually 100% of CF patients, limited in durability by only the rate of stem cell turnover.
[0264] However, many CRISPR-based systems (e.g. Cas9-mediated insertion; CRISPR- associated transposases or CASTs; CRISPR- associated recombinases/integrases etc.) rely on large DNA donor cassettes templates for gene editing.7, 8 These systems offer a few distinct advantages over mutation-specific correction using ssODN donor templates. First, insertion of long sequences or entire genes offers a mutation-agnostic platform that does not require patientcustomization, ultimately reducing cost and scalability of the therapy. Second, insertion of entire gene offers the flexibility to utilize codon-optimized and/or gain-of-function variants of the donor cassette, increasing protein expression and/or function and potentially overcoming limitations to delivery and editing efficiencies.9- 13 However, the packaging capacity of synthetic nanocarriers like LNPs is still poorly understood, and LNPs have not yet been applied for the delivery of linear dsDNA. Here, the inventors report the design and validation of a LNP platform that encapsulates in one formulation an mRNA transcript encoding Cas9, guide RNAs targeting CFTR, and for the first time ever reported, a codon-optimized dsDNA donor template encoding the entirety of the 5kb CFTR gene. The engineered formulations achieved integration efficiencies high enough to result in complete rescue of the CF phenotype, restoring CF-mutant cells to chloride transport rates and CFTR expression levels to values well above non-CF control cells. The inventors believe that the work serves as proof-of-concept that LNPs can successfully deliver very large dsDNA transcripts and offer an exciting delivery modality for gene editing tools that have not yet been applied in conjunction with this nanocarrier platform.
A. Results
1. Selection of lipid nanoparticle constituents
[0265] Traditional LNP formulations comprise an ionizable lipid, a helper phospholipid, a sterol, and a lipid conjugated to poly(ethylene glycol) (PEG). The exact identities and molar ratios of each of these constituents must be designed according to application, since LNP selfassembly depends on the nature of the encapsulated cargo, and the biology of LNP uptake and endosomal trafficking varies according to cell type.14, 15 With this in mind, the inventors aimed to establish an LNP formula suitable for nucleic acid delivery to a disease-relevant bronchial epithelial cell line. To do this, the inventors began with a base formula synthesizing LNPs, at a classic molar composition of 50% ionizable lipid, 38.5% sterol, 10% helper lipid, 1.5 % lipid-PEG, and 50% ionizable lipid. The initial LNP formula comprised 3B-[N-(N',N'- dimethylaminoethane)-carbamoyl]cholesterol (DC-cholesterol), l,2-dioleoyl-sn-glycero-3- phosphoethanolamine (DOPE), DOPE-PEGIOOO, and SM-102. The inventors systematically substituted various lipid compounds into this base formula to screen for their ability to encapsulate and deliver reporter mRNA cargoes encoding green fluorescent protein (GFP) to 16HBE14o- cells.
[0266] The inventors first tested commercially available ionizable lipids containing degradable ester groups including Lipid 5 and Lipid A6 against SM-102 as candidate LNP components. Each of these ionizable lipids were compared for their ability to form small, uniform LNPs with high (>80%) encapsulation efficiencies and optimal delivery capabilities. All ionizable lipids were found to produce LNPs with similar sizes and encapsulation efficiencies (Supplementary Information S.1). LNPs containing SM-102 were found to achieve transfection efficiencies 7-8% higher than LNPs containing Lipid 5 (p = 0.0003) or Lipid A6 (p = 0.0007) (FIG. 33E). All LNPs preserved 85 - 95% viability across the three ionizable lipids (n.s.) (FIG. 33F). SM-102 was thus selected as the ionizable lipid of choice as the inventors proceeded with engineering the LNP composition for bronchial epithelial cells. [0267] The inventors further investigated whether delivery of mRNA-based cargoes could be enhanced by substituting DC-cholesterol with either cholesterol or B-sitosterol. The inventors found particles containing DC-cholesterol to tend toward higher encapsulation efficiencies and smaller sizes (FIG. 35); however, LNPs containing B-sitosterol were found to achieve transfection efficiencies ca. 5-10% higher than particles containing cholesterol (n.s.) and 30 - 40% higher than particles containing DC-Cholesterol (p < 0.0001).
[0268] Finally, the inventors probed the impact of the structure of the PEG-lipid comprising the LNPs on particle size, encapsulation efficiencies, and transfection capabilities. The inventors directly comparing encapsulation and delivery of mRNA constructs encoding GFP in LNPs containing either DOPE-PEG2000, l,2-Dimyristoyl-rac-glycero-3- [methoxy(poly-ethylene glycol)-2000] (DMG-PEG2000) or 1,2 distearoyl-sn-glycero-3- phosphoethanolamine-N-[amino(poly-ethylene glycol)-2000] (DSPE-PEG2000). Regardless of PEG-lipid, the inventors found all LNPs to exhibit similar sizes and encapsulation efficiencies (FIG. 35). LNPs containing DMG-PEG2000 achieved 4-5% greater transfection efficiency compared to LNPs containing DOPE-PEG2000 (p < 0.0001), and ca. 90% greater transfection efficiency compared to LNPs containing DSPE-PEG2000 (p < 0.0001) (FIG. 33E). While LNPs containing DOPE-PEG2000 preserved 16% greater cell viability compared to DSPE-PEG2000 (p = 0.0364), there was no significant difference in viability between LNPs containing DOPE-PEG2000 and DMG-PEG2000 (FIG. 33F). LNPs comprised entirely of optimized constituents achieve transfection efficiencies of 95% (FIG. 33E) and preserved >80% cell viability (FIG. 33F).
2. Screening ratios of gene-editing cargoes
[0269] While the inventors were encouraged by the capabilities of the LNP formulation to efficiently deliver homogenous model GFP transcripts to 16HBE14o- cells, LNP-mediated gene editing relies on the delivery of multiple cargoes at once. Specifically, the HDR-mediated gene editing approach requires the intracellular delivery of an mRNA transcript encoding a Cas9 endonuclease, a single guide RNA (sgRNA), and a donor DNA template. The inventors designed a set of experiments to rapidly screen the impact of relative ratios of these components on the gene-editing capabilities of the LNPs. For these studies, the inventors generated a 16HBE14o- model reporter line stably expressing blue fluorescent protein (BFP) according to a protocol from Corn and colleagues (FIG. 29A). (18) LNPs were loaded with Cas9 mRNA, a sgRNA targeting the BFP locus, and a single-stranded oligonucleotide (ssODN) donor designed to introduce a three-nucleotide mutation (targeting H66) to convert BFP to GFP. The inventors systematically screened various ratios of mRNA, sgRNA, and ssODN to determine the conditions that maximize HDR-mediated repair. Initially, the ssODN:mRNA ratio was held fixed at 3 : 1 w/w and the sgRNA:mRNA ratio was titrated from 0.8: 1 w/w to 2: 1 w/w. For all ratios, LNPs induced 10 - 20% HDR and 25 - 40% overall editing, with no significant differences reproducibly observed between any sgRNA:mRNA ratios (FIG. 29B). Viability for all conditions was maintained at ca. 80 - 90% (FIG. 35). Biological replicates of this experiment show 4-6% greater incidence of HDR for LNPs loaded at a sgRNA:mRNA ratio of 1.2:1 w/w as compared to 0.8: 1 w/w (p = 0.0002) and 2: 1 w/w (p = 0.0101), with no significant difference compared to 1.6: 1 w/w (FIG. 36). The inventors therefore selected a sgRNA:mRNA ratio of 1.2: 1 w/w for use in subsequent experiments.
[0270] Next, the inventors similarly titrated the ssODN:mRNA ratio from 2 to 4 w/w while maintaining the sgRNA:mRNA ratio constant at 1.2: 1 w/w. For all ratios, LNPs induced 30 - 35% HDR and 90 - 95% overall editing, with no significant differences reproducibly observed between any ssODN:mRNA ratios (FIG. 29C). Viability for all conditions was maintained at ca. 80 - 95% (FIG. 36). Biological replicates of this experiment show 3% greater incidence of HDR for LNPs loaded at a ssODN:mRNA ratio of 3: 1 w/w as compared to 2: 1 w/w (p = 0.0031), with no significant difference compared to 4: 1 w/w (FIG. 36). The inventors accordingly selected a ssODN:mRNA ratio of 3: 1 w/w for use in subsequent experiments.
3. Optimizing LNP formulation to accommodate double-stranded DNA donor cassettes
[0271] Extremely encouraged by the editing data in the BFP to GFP model, the inventors asked whether the same LNP formula could be applied toward delivering double-stranded DNA (dsDNA) donor templates for whole-genes. As a reporter for the disease target, the inventors aimed to integrate an mdtrine reporter construct at the 5’UTR of the endogenous CFTR gene. The inventors anticipated that differences in physicochemical properties of dsDNA compared to ssODNs would require re-optimization of LNP constituent ratios. First, the inventors probed whether delivery of cargoes separately or encapsulated within one LNP formulation can achieve higher integration efficiencies. The optimized lipid formula was combined at a nitrogen/phosphate (N/P) ratio of 7 with either mRNA encoding Cas9 together with a sgRNA and/or a dsDNA donor cassette. Particle mixtures separating the dsDNA into a distinct LNP are referred to as sepLNPs, and formulations that combine all three gene-editing cargoes into a single formulation are referred to as togLNPs (FIG. 30 A). Cargo encapsulation efficiencies were found to be ca. 80% for both togLNPs and RNA-sepLNPs, and lower (62.6%) for DNA-sepLNPs (FIG. 30B). Similarly, both togLNPs and RNA-sepLNPs exhibited small (ca. 150 nm) diameters and low (ca. 0.2) poly dispersity indices, whereas DNA-sepLNPs were slightly larger (ca. 200 nm) with a Pdl of 0.34 (FIG. 30B). The inventors found that, when coadministered with drugs AZD7648 and ART558, togLNPs achieved integration efficiencies of ca. 14%, compared to ca. 10% achieved by sepLNPs (p = 0.0082) (FIG. 30C,D). When administered without drug, togLNPs achieved 36% greater integration efficiencies than sepLNPs (p = 0.0452) (FIG. 30).
[0272] Excitingly, these integration efficiencies of mdtrine were comparable to previously reported results of Cas9 ribonucleoprotein complex (RNP) electroporation experiments. The inventors were therefore inspired to proceed by replacing the mdtrine reporter construct with a codon-optimized CFTR dsDNA construct, which was shown previously to achieve up to 90% restoration of wild-type CFZR-dependent ion current at low (2-3%) levels of integration in 16HBE-G542x cells. The CFTR construct is nearly triple the size of the mCitrine reporter, and the inventors accordingly anticipated that formulaic optimization would again be required. The inventors began by reevaluating the appropriate dsDNA:mRNA ratio, assessing 2: 1, 3: 1, and 4:1 w/w. The inventors found a dsDNA:mRNA ratio of 3: 1 to achieve greater (up to 3.5%) integration compared to ratios of 2: 1 or 4:1, which produced integration efficiencies of <2% (n.s.). The inventors found no differences in cell viability across conditions, with all conditions maintaining 70 - 90% survival relative to the untreated control. The inventors therefore proceeded with the dsDNA:mRNA ratio of 3 : 1 w/w for further experiments. Similarly, the inventors titrated the N/P ratio from 5 to 9. When administered with AZD7648 and ART558. the inventors found LNPs exhibiting a N/P of 9 to achieve significantly higher integration efficiencies compared to N/Ps of 5 or 9 (stats), with comparable relative cell survival across all conditions (stats). To ensure the inventors captured the optimal N/P ratio within the ranges the inventors tested, the inventors further explored N/Ps of 11 and 13. The inventors found no statistically siginifcant differences in integration efficiencies between N/Ps of 9, 11, and 13, but found a N/P of 11 to confer a small increase in integration efficiency. Further, cell survival in samples treated with N/P 9 formulations was significantly lower than samples treated with N/P 13 formulations, and lower (although not significantly so) than samples treated with N/P 11 formulations. Because cells treated with N/P 11 formulations exhibited higher integration efficiencies than N/P 13 formulations and higher viabilities than N/P 9 formulations, the inventors selected a N/P of 11 for subsequent dosage optimization and functional assays. 4. LNP-mediated site-specific integration restores CFTR function in a G542x-mutant human bronchial epithelial cell line and in a F508del- mutant human bronchial epithelial cell line
[0273] As the final step in optimizing the platform for the editing of the model 16HBE- G542x cell line, the inventors titrated the dose of LNPs from 50 ng mRNA to 100 ng mRNA. All conditions achieved ca. 3% integration. The 50 ng condition achieved the highest integration efficiencies with a mean of 3.3% and a maximum of 3.5%, and no significant differences in relative cell survival. The inventors next sought to evaluate whether these seemingly modest editing efficiencies could confer any degree of CFTR production and function. To do this, the inventors expanded cells from the 50 ng treatment condition to conduct Western blot and Ussing chamber analyses. LNP-edited populations of 16HBEgeG542x cells exhibited inhibitable CFTR-dependent chloride currents at 160% of the values measured in non-CF 16HBE14o- cells (p = 0.0045). The inventors hypothesized that the large currents measured in populations exhibiting at most 3.5% gene correction were due to high CFTR expression levels resulting from the use of a codon-optimized CFTR donor cassette. To verify this, the inventors performed a Western blot, which confirmed ca. 1.6x protein expression in edited 16HBEgeG542x populations compared to the non-CF 16HBE14o- control. The inventors repeated these studies in CFBE41o- cells which are patient-derived and homozygous for the common CF-causing F508del mutation. To the inventors’ knowledge, this is the first report of linear dsDNA cassettes of this size encapsulated and delivered intracellularly to achieve phenotypic rescue via an LNP platform.
5. LNPs are capable of encapsulating large dsDNA constructs
[0274] Historically, traditional LNPs optimized for the delivery of RNA have not been explored for their ability to encapsulate and deliver larger dsDNA constructs. The success of the in-vitro assays inspired us to explore the morphology of LNPs containing either Cas9 mRNA and sgRNA only, dsDNA encoding CFTR only, or Cas9 mRNA, sgRNA, and dsDNA encoding CFTR all together in a single formulation. All three formulations primarily contained particles ca. 30 - 100 nm with either round or polyhedral morphology and an electron-dense multilamellar core. The inventors considered these findings corroborative of the editing data, assuming that integration of the CFTR donor construct depends on successful encapsulation of all nucleic acid payloads into LNPs of endocytosable size. However, very large (>200 nm) particles with irregular morphology, empty unilamellar liposomes, and large cylindrical and sheet-like structures were also observed in the samples containing dsDNA. This heterogeneity is consistent with broader size distributions measured in dsDNA-containing samples via DLS. Qualitatively, these secondary structures were observed to a greater degree in the samples containing only dsDNA compared to samples containing both RNA and DNA. The inventors intuited that lipids sequestered in these secondary structures are not free to interact with nucleic acid payloads, perhaps explaining the lower encapsulation efficiencies in dsDNA-containing LNPs measured via the RiboGreen assay. Still, the cryo-TEM images verify the formation of multilamellar particles loaded with nucleic acid in all samples, confirming that large linear dsDNA constructs may be encapsulated within LNPs.
6. LNPs are capable of encapsulating large dsDNA constructs
[0275] Historically, traditional LNPs engineered for the delivery of RNA have not been explored for their ability to encapsulate and deliver larger dsDNA constructs. The success of the in-vitro assays inspired the inventors to explore the morphology of LNPs containing either Cas9 mRNA and sgRNA only, dsDNA encoding CFTR only, or Cas9 mRNA, sgRNA, and dsDNA encoding CFTR all together in a single formulation. All three formulations primarily contained particles ca. 30 - 100 nm with either round or polyhedral morphology and an electron-dense multilamellar core. The inventors considered these findings corroborative of the editing data, assuming that integration of the CFTR donor construct depends on successful encapsulation of all nucleic acid payloads into LNPs of endocytosable size. However, very large (>200 nm) particles with irregular morphology, empty unilamellar liposomes, and large cylindrical and sheet-like structures were also observed in the samples containing dsDNA. This heterogeneity is consistent with broader size distributions measured in dsDNA-containing samples via DLS. Qualitatively, these secondary structures were observed to a greater degree in the samples containing only dsDNA compared to samples containing both RNA and DNA. The inventors intuited that lipids sequestered in these secondary structures are not free to interact with nucleic acid payloads, perhaps explaining the lower encapsulation efficiencies in dsDNA-containing LNPs measured via the RiboGreen assay. Still, the cryo-TEM images verify the formation of multilamellar particles loaded with nucleic acid in all samples, confirming that large linear dsDNA constructs may be encapsulated within LNPs.
B. Discussion
[0276] Lipid nanoparticles have demonstrated remarkable versatility and widespread clinical success as a platform for siRNA therapeutics and mRNA vaccines, in addition to rapid preclinical development as delivery system for gene editing tools. These nanocarriers are amenable to encapsulating and delivering RNAs of multiple sizes and types, plasmid DNA, and RNPs.19-22 However, to date, it has remained unclear whether large linear dsDNA cassettes are amenable to encapsulation and delivery by LNPs. In this study, the inventors aimed to address two key questions: first, can LNPs fabricated from commercially available reagents encapsulate and deliver linear dsDNA cargoes? Second, can LNPs be used to deliver all components of the CRISPR/Cas9 system to bronchial epithelial cells to achieve site-specific integration of the large (>5kb) CFTR gene and permanently restore ion channel function in airway epithelia?
[0277] To probe the former, we used DLS, encapsulation assays, and cryo-TEM to compare our dsDNA-containing formulations to formulations containing only RNA. Our data suggest that identical lipid formulations can encapsulate RNA cargoes and linear dsDNA cargoes alike. However, morphological changes (including the formation of large, irregular LNPs, empty liposomes, and sheet-like structures) in our dsDNA-containing LNPs suggest that cargo encapsulation and particle formation appear to be less efficient in these formulations. We speculate this may result from altered thermodynamics of self-assembly, perhaps due to the greater size and/or rigidity of the dsDNA donor compared to single-stranded RNA molecules, or because of steric hindrance preventing electrostatic interactions between the ionizable lipids and the dsDNA phosphate backbone. Molecular dynamics simulations may be useful to deconvolute the mechanistic differences in self-assembly of LNPs containing RNA versus dsDNA. Interestingly, samples containing both dsDNA and RNA tended to exhibit intermediate encapsulation efficiencies, sizes, polydispersities, and morphological heterogeneity. It is unclear whether this is because the presence of RNA augments the selfassembly process to stabilize LNPs containing both RNA and dsDNA, or if it is because the RNA and dsDNA partition into separate small, uniform RNA-containing LNPs, and larger, heterogenous DNA-containing LNPs. More specialized analytical techniques, such as methods developed by Li et al. based on the multi-laser cylindrical illumination confocal spectroscopy technique (CICS), are required to elucidate the payload copy number per particle and answer these questions.23
[0278] This work demonstrates the feasibility of using LNPs to deliver large, linear dsDNA constructs intracellularly. The inventors further hypothesized that, if they could encapsulate all necessary components for Cas9-mediated insertion of CFTR into an LNP optimized for delivery to bronchial epithelial cells, then LNPs could be used as an alternative to viral vectors previously relied upon for genomic integration of donor sequences. While the integration efficiencies are modest, the high (50 - 60%) rate of insertion-deletions introduced by NHEJ at the cut site in samples edited with both mCitrine and CFTR donor constructs suggest that uptake and endosomal escape, which are classically understood to be the primary bottlenecks to intracellular delivery of nucleic acids, are not the limiting factors for our platform. It is instead possible that donor trafficking to the nucleus is inefficient due either to the lack of nuclear localization signal (NLS) or to degradation by the cells’ immune machinery. Foreign double-stranded DNA is known to be immunogenic to cells;24, 25 however, it is worth noting that cell survival and proliferation observed in this study were not significantly impacted by LNP -mediated delivery of dsDNA. This is especially exciting because electroporation of RNPs together with identical dsDNA constructs encoding mCitrine and CFTR into 16HBE cells has been previously reported to negatively impact cell survival, suggesting that cytotoxicity of large dsDNA donors may be mitigated by the use of alternative delivery platforms such as LNPs. Further studies are required to elucidate the mechanisms of toxicity and immunogenicity of dsDNA when delivered via LNP compared to electroporation. Alternatively, Cas9 expression and nuclear localization may peak at a different time point than nuclear localization of the donor. Further experiments may elucidate the kinetics of the expression and nuclear localization of each component and enable enhanced HDR efficiencies by informing well-timed delivery of the donor.
[0279] It is also probable that HDR is limited by the internal biology of the cell. This repair process is cell-cycle dependent, occurring primarily in S/G2 phases, and in competition with NHEJ and microhomology-mediated end-joining (MMEJ).
[0280] This is the first time LNP-mediated integration of an entire gene has been reported, and our data establish a new benchmark for the cargo-carrying capacity of lipid nanoparticles. While there is ample precedent that LNPs can carry large mRNA transcripts for proteins including Cas9, CFTR, CAR constructs, and base and prime editors, 17, 29-31 dsDNA molecules are approximately double the molecular weight of their mRNA counterparts. Here, the inventors demonstrate the encapsulation and functional delivery of an entire gene with homology arms, 5.5 kb in length and weighing in at approximately 3400 kDa. To the inventors’ knowledge, this is the first time a nucleic acid LNP payload of this size has been reported . This data offer a proof-of-concept that LNPs can mediate the functional delivery of large dsDNA constructs, indicating that such an effort may be a worthwhile endeavor for researchers in the lipid nanoparticle field.
[0281] This work has vast implications. Genetic disorders such as CF that arise from any one of thousands of disease-causing mutations may theoretically be treated with CRISPR/Cas9 approaches that leverage ssODNs, but this strategy would require customized design and validation of sgRNAs and ssODNs for each individual mutation, presenting cost and scalability issues. Gene addition strategies, including the CRISPR/Cas9 approach described in this paper in addition to CAST approaches currently under rapid development for similar applications, offer virtually universal gene correction tools for monogenic diseases like CF. One massive barrier to the implementation of these groundbreaking technologies is the challenge of delivery, and we demonstrate here that LNPs offer an exciting and elegant platform to deliver the multiple components required for site-specific whole-gene insertion.
C. Methods
1. Chemicals
[0282] SM-102, Lipid 5, Lipid A6, and DMG-PEG2000 were purchased from Echelon
Biosciences. Cholesterol, DC-cholesterol, B-sitosterol, and DOPE-PEG2000 were purchased from Avanti Polar Lipids. DSPE-PEG2000 was purchased from Nanosoft Polymers. Chloroform and ethanol were purchased from the UCLA Chemistry & Biochemistry Store Room. TritonTM X-100 was purchased from Millipore Sigma. Quant-iTTM Ribogreen® RNA reagent and rRNA standards were purchased from Thermo Scientific. CellTiter 96® AQueous One Solution Cell Proliferation Assay was purchased from Promega. EGFP mRNA and Cas9 mRNA were purchased from TriLink Biotechnologies. Single guide RNAs were obtained from Synthego. Phosphate-buffered saline (PBS) was purchased from Thermo Scientific. Citric acid buffer (50 mM, pH 5.35) was made by combining 8.55 g sodium citrate dihydrate (Fisher) and 4.02 g citric acid (Fisher) per 1 L diethyl pyrocarbonate (DEPC)-treated water (Thermo Scientific), and adjusting the pH with HC1 or NaOH (Sigma-Aldrich). Citric acid buffer (50 mM, pH 4) was made by combining 4.96 g sodium citrate dihydrate and 6.36 g citric acid per 1 L DEPC-treated water and adjusting the pH with HC1 or NaOH.
2. Nanoparticle formulation, synthesis, and characterization
[0283] LNPs containing ionizable lipid (SM-102, Lipid 5, or Lipid A6): sterol (DC- cholesterol, cholesterol, or B-sitosterol) :DOPE:PEG-lipid (DOPE-PEG 1000, DOPE-PEG2000, DSPE-PEG2000, or DMG-PEG2000) at molar ratios of 50:38.5: 10: 1.5 were formulated using NanoAssemblr Spark (Precision NanoSystems). A lipid mix concentration of 65 mM was used. NanoAssemblr Spark functions at a constant flow rate and 3 : 1 ratio of aqueous phase to organic phase. LNPs were diluted in PBS and hydrodynamic size and Pdl of the particles was measured using DLS on a Malvern Zetasizer Nano. To determine nucleic acid encapsulation efficiency, LNPs or PBS blanks were diluted in tris-EDTA (TE) buffer to achieve a concentration of 2 - 10 ng/pL nucleic acid per well. These samples were aliquoted and diluted 1 : 1 in TE buffer (to measure unencapsulated nucleic acid) or TE buffer with 2% Triton-X-100 (to measure total nucleic acid). Quant-iT RiboGreen reagent (to measure total nucleic acid) or PicoGreen reagent (to measure dsDNA ONLY) was added and fluorescence signal was quantified with a Varioskan LUX multimode microplate reader (Thermo Scientific). Encapsulation efficiency was calculated as follows:
[0284] 2.5 pL undiluted LNPs were deposited on a glow-discharged 200 mesh Cu grid with thin carbon film supported by holey carbon substrate (QuantiFoil). Grids were blotted for 2 s at 22 °C then plunged into liquid ethane using a manual plunger. Images were taken using a K2 camera (Gatan) on a TF20 microscope (FEZ) operated at 200 kV.
3. Cell Culture
[0285] 16HBE14o- cells were obtained from Millipore Sigma. 16HBE14o-BFP cells were generated by transducing 16HBE14o- cells with a lenti virus carrying a blue fluorescent protein (BFP) reporter cassette driven by the EFla promoter based on a protocol described by Dr. Corn and colleagues.18 Cells were single-cell sorted into clonal populations via fluorescence activated cell sorting (FACS). Clones were expanded and their VCN was measured via ddPCR. A colony with a VCN of 1.1 was selected for use in experiments. 16HBEge-G542x cells were obtained via a materials transfer agreement between the Regents of the University of California, Los Angeles and Dr. Hillary Valley at the Cystic Fibrosis Foundation (CFF). All cell lines were cultured in Minimum Essential Medium Eagle (Sigma) with 10% fetal bovine serum (FBS) (Millipore Sigma), 1% penicillin/streptomycin (Fisher Scientific), and 2 mM L- glutamine (Millipore Sigma) (complete medium referred to as E10). Cells were grown on tissue culture flasks and well plates (Corning) coated with a matrix of bovine serum albumin (Millipore Sigma), fibronectin from human plasma (Millipore Sigma), and collagen type I (Millipore Sigma). Cells were passaged by lifting the cells via trypsinization with trypsin- EDTA 0.25% (Thermo Fisher Scientific) and split at ratios between 1 :4 and 1 : 10.
4. Transfection
[0286] 16HBE cells cultured for 10 or fewer passages were plated in clear-bottom 96 well plates at 20,000 cells/well. For GFP experiments, cells were plated in 100 pL E10 and incubated overnight before replacing medium with 100 pL fresh E10 immediately prior to transfection. For gene editing experiments, cells were plated in 100 pL OptiMEM (Thermo Fisher) with or without AZD-7648 and ART-558 and incubated for 18 h before replacing medium with 100 pL E10 4 h prior to transfection. For LNP experiments including CFTR donor cassettes, 10 uM rho-kinase inhibitor Y-27632 was added to the cell culture medium 24 h prior to transfection, and cells were cultured with Y-27632 until harvest. Nanoparticles were added directly to the medium and incubated with the cells overnight before replacing medium with 200 uL E10 the next day. Primary HBE cells on TransWell inserts were incubated with CHIR and 10 pM Y-27632 for 24 h prior to transfection. LNPs were added to the apical chamber 24 h prior to air lifting the cultures.
5. Flow Cytometry
[0287] 16HBE14o- and 16HBE14o-BFP were plated in 96-well plates at 20,000 cells/well and treated with LNPs carrying either GFP mRNA or Cas9 mRNA, sgRNA-BFP, and ssODN- BFP. For GFP delivery experiments, cells were harvested the following day via trypsinization and resuspended in PBS containing 5% FBS. For BFP to GFP editing experiments, cells were harvested 5 days after transfection via trypsinization and resuspended in PBS containing 5% FBS. Cells were stained for viability using 7AAD dye (Thermo Fisher). Analysis was performed on a BD LSRII flow cytometer equipped with 355, 405, 488, 561, and 633 nm lasers and running BD FACS Diva v.8.0.1 software. Forward scatter and side scatter gating was used to exclude debris. Doublets were excluded based on forward scatter width and heigh gating. GFP was detected through a 525/50 bandpass filter, BFP was detected through a 450/50 bandpass filter, and 7AAD was detected through a 710/50 bandpass filter.
6. Immunostaining and Confocal Microscopy
[0288] Complete fixation and immunostaining protocols can be found in Supplementary Information. 16HBEs on TransWell membranes were excised from the supporting inserts with a scalpel and placed in Eppendorf tubes. Cells were fixed in ice-cold methanol for 30 min and rehydrated in PBS + 1% DMSO + 0.1% Triton X 100 (PBT). Cells were washed 3x with PBT, 3x with DI water, then permeabilized with cold acetone for 7 min. Cells were again washed 3x with DI water, 3x with PBT, and then blocked using Dako (Agligent) for 2 h at RT. Primary antibodies were diluted to 0.05 pg/pL in Dako and incubated with cells at 4oC overnight. Cells were rinsed 4x with PBT, and secondary antibodies were diluted to 0.04 pg/uL in Dako with 0.02 pg/uL DAPI and incubated with cells for 60 min at RT. A full list of antibodies is provided in Supplementary Information. Cells were rinsed 4x with PBT, clarified with glycerol, and membranes were carefully sandwiched between a coverslip and glass slide in a droplet of ProLong Gold AntiFade with DAPI (Thermo Fisher). Cells were visualized using a Leica SP8- STED/FLIM/FCS laser scanning confocal microscope (Leica Microsystems, Wetzlar, Germany). 7. DNA Extraction and Analysis
[0289] Cells were harvested for DNA extraction 48 - 72 h after transfection via trypsinization or cell scraping. Genomic DNA was extracted from cell pellets for all ddPCR and sequencing analyses using the GeneJet Genomic DNA Purification Kit (Thermo Fisher).
8. Ussing Chamber Analysis
[0290] 16HBEge-G542x cells were seeded on 12 mm polyester SnapWell inserts (Corning) coated in collagen type IV from human placenta (Millipore Sigma) at a density of 500,000 cells/well, and cultured in E10 in basal and apical chambers for at least 7 days prior to Ussing assays. Assays were performed using an EasyMount Ussing chamber (Physiologic Instruments) at 37°C in HEPES buffered solutions with an imposed chloride gradient across the epithelia (full buffer recipes in Supplementary Information) for 16HBEge-G542x samples. [0291] Electrode tips (Physiologic instruments) were partially filled with 3% LB Agar (Thermo Fisher) in 3M KC1 and backfilled with 3M KC1.
[0292] Cells grown on SnapWell inserts were mounted in the chambers and allowed to equilibrate for 10 - 15 min before recording baseline short-circuit current (Isc) for 10 min. At 10 minute intervals, 10 pM amiloride, 10 pM forskolin, 1 pM VX-770, and 20 pM CFTR- Inhl72 were added sequentially to the basal and apical chambers. At 10 min after the initial dose of CFTR-Inhl72, a second dose of 20 pM CFTR-Inhl72 was added to the basal and apical chambers to extinguish any remaining current, and 5 minutes later 100 pM adenosine triphosphate (ATP) was added to the basal and apical chambers. Isc was recorded for an additional 10 minutes before ending the experiment.
9. Western Blot
[0293] For immunoblots, cells were lysed in RIPA Lysis and Extraction Buffer (Cat: 89901; ThermoFisher Scientific; Grand Island, NY) with added HALT protease inhibitor (Cat: 87786; ThermoFisher Scientific; Grand Island, NY) at a 1 x concentration following the manufacturer's protocols. Lysate concentrations were determined using the Pierce BCA protein assay (Cat: 23227; ThermoFisher Scientific, Grand Island, NY) following the manufacturer's protocol. Samples were treated for sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with NuPAGE LDS Sample Buffer (Cat: NP0007; ThermoFisher Scientific; Grand Island, NY) and NuPAGE Sample Reducing Agent (Cat: NP0009; ThermoFisher Scientific; Grand Island, NY), each to a 1 x concentration. Lysates were diluted to contain 50pg of total protein for immunoblot gel loading to keep the total amount of protein loaded per lane constant to allow for valid loading controls. Note, CFTR protein becomes insoluble if the sample is heated above 60°C and will not enter stacking gel. As such, the samples were denatured at 37°C for 20 minutes prior to loading into the stacking gel. Wild-type cells 16HBE14o- were used as a control to indicate the relative expression levels of CFTR protein. CFTR levels were detected using Ab 596 (obtained via MTA from J. Riordan, UNC;) at 1 : 1000 in 5% milk in TBST.33 Protein quantification was assessed through densitometry via the ImageJ software. CFTR protein levels were normalized to the actin protein levels after quantification.
10. Double stranded DNA donor synthesis and amplification
[0294] Double-stranded DNA (dsDNA) donor fragments were synthesized as gBlocks by Integrated DNA Technologies (Coralville, IA). These gBlocks were subsequently cloned into plasmids using the TOPO Zero Blunt Cloning Kit (Thermo Fisher Scientific, Catalog #K2800J10). The dsDNA donors were then amplified by PCR from these plasmids with Platinum SuperFi II DNA Polymerase using the manufacturer's protocol (ThermoFisher; Catalog #12369010). Amplification utilized oligonucleotide primers "GTCTTTGGCATTAGGAGCTT" (SEQ ID NO:7) and "AGACAACGCTGGCCTTTTC" (SEQ ID NO: 10) that were modified at the 5' end with AmC6, also supplied by Integrated DNA Technologies (Coralville, IA). The PCR products were then purified using SPRI paramagnetic bead-based purification (complete protocol in Supplementary Information).
11. Integration analysis by ddPCR:
[0295] Genomic DNA was extracted from edited cells for integration site analysis using the Invitrogen PureLink Genomic DNA Kit (Cat: KI 82002; Thermo fisher Scientific) or QuickExtract DNA Extraction Solution (LGC Biosearch Technologies; #QE09050) and quantified using the NanoDrop system (Cat: ND-2000; Thermo Fisher Scientific). DNA samples were then analyzed by droplet digital PCR to measure integration rates. Two sets of primers were duplexed, each with their own fluorescent probe (FAM/HEX). To measure integration rates of the mCitrine reporter cassette, one primer was complementary to a CFTR gene sequence upstream from the left homology arm of the donor. The second primer bound to the mCitrine reporter cassette, to allow for specific measurement of the integrated mCitrine transgene distinct from the endogenous CFTRE gene. To measure integration rates of the CFTR donor cassette, one primer was bound to the bovine growth hormone polyA to allow for specific measurement of the integrated CFTR donor. The second primer was complementary to a CFTR gene sequence upstream from the right homology arm of the donor cassette. The F AM-conjugated nucleotide probe with a quencher also bound to the minus-strand DNA near the second primer. A reference primer/probe set was also delivered to recognize the SDC4 gene on chromosome 20. One microliter of Dral endonuclease (Cat: R0129S; New England Biolabs) was added to the reaction mixture (an enzyme that does not disrupt the experimental or reference amplicons) to reduce background. Each sample was digested at 37°C for 1 h before droplet generation with the Bio-Rad QX200 Droplet Generator (Cat: 186-4002; Bio-Rad, Hercules, CA). The prepared samples were then assayed via the QX200 Bio-Rad Droplet Reader on the “Absolute” measurement setting (Cat: 186-4003; Bio-Rad).
12. Measuring allelic disruption:
[0296] To measure allelic disruption of the reporter gene cassete, a 728 bp amplicon that encompasses CFTR 5’UTR and exon 1 was amplified from genomic DNA taken from edited cells (primer sequences: “AAAGCCGCTAGAGCAAATTT,” (SEQ ID NO: 19) “TGTTGGCTGAATTCAGTCAA” (SEQ ID NO:20)). The resulting amplicon was Sanger sequenced and uploaded for analysis via the Synthego ICE web tool (Synthego Corporation, Menlo Park, CA).
D. Supplemental
1. Lonza Nucleofector Electroporation Protocol
1. CalcpLate number of cells needed for total experiments
2. Count cells/mL
3. Obtain mL needed for total cells needed
4. Spin cells in 50 mL conical at 94g or 15 mins
While waiting,
1. Set up eppendorf tubes (1 per sample/well)
2. Aliquot reagents into Eppendorf tubes a. i.e. 3 ug of Cas9 mRNA + 2 ug sgRNA; store on ice
3. Make SF media (0.818 SF + 0.181 Supplement) - enough for 20 pL/tube + 2 extra
4. Obtain electroporation cuvette and holder a. Blue holder and well strip
5. After 15 mins, Aspirate supernatant
6. Resuspend cells in SF media (200,000 cells/20 pL)
7. Transfer 20 pL of cells to Eppendorf tubes containing editing reagents (protein/mRNA/ plasmid)
8. Pipette to mix, transfer everything to cuvette. NO BUBBLES a. Push past the first stop to get all 20 pL + extra volume
9. Start electroporation a. Choose X unit b. Cuvette well holder c. Click number of wells d. Choose cell type e. Press start
10. Rest cells for 10 mins at RT
11. Add 400 pL E10 media into wells of new plate
After 10 mins.
12. Add 80 pL of E10 into each well
13. Transfer total volume (100 pL) to well containing 400 pL media
14. Mix gently by shaking plate
15. Incubate at 37C
2. Whole-mount immunofluorescent immunostaining
Day 1
1- Rehydration with PBT (PBS +1% DMSO + 0.1% triton lOOx so 500ml + 5ml +500ul)/methanol
75% methanol/ 25% PBT (7.5ml/2.5ml) 5min
50% methanol/ 50% PBT (5.5ml/5.5ml) 5 min
25% methanol/75% PBT (2.5ml/7.5ml) 5 min
Make sure that the membranes are suspended in the eppendorf at each step
The liquids are removed with a lOOOul pipette and are deposited with a pasteurette.
2- Wash with PBT 3x5 min
3- Wash with DI Water 3 min
4- Permeabilization with cold acetone for 7 min under a hood
5- Wash with DI Water 3 min
6- Wash with PBT 3x 5min
7- Blocking with PBT/BSA 10% or other blocking solution 2h at RT
8- Add primary antibody diluted in blocking solution (approximatively 200 pl/ tube) (1/200)
9- 4°C overnight
Day 2 10- Wash with PBT 4*5min
11- Add secondary antibody and DAPI diluted in blocking solution or in PBT (l/5000e) (protect from light the tubes)
12- Wash with PBT 4*5min (cover from light between washes)
13 - Clarification with glycerol from a series of PBT/glycerol washes of 20 min each at RT
- 75% PBT/ 25% Glycerol (7.5ml/2.5ml) 2-3 HOURS
- 50% PBT/ 50% Glycerol (5.5ml/5.5ml) 2-3 HOURS
- 25% PBT/75% Glycerol (2.5ml/7.5ml) OVERNIGHT
• You can leave them overnight in glycerol or directly mount them
Day 3
14- Prepare slides and coverslip and weight
15- Add a few drops of mounting fluid
16- Use clean forceps retrieve the membrane and remove excess glycerol by using a kimtech
17- Deposit the membrane with cells upwards (check under the microscope if necessary)
18- Add drop of mounting media over the membrane and put the coverslip (no bubble)
19- Use weights to flatten the slides
20- Let dry
3. Cell Culture
Medium (Store complete medium at
Coating Solution (Make fresh)
[0297] Coat flasks with the coating solution: 1ml of the solution for a T-25 flask, 2ml for a T-75. Distribute the solution evenly across the surface, making sure the entire surface is wetted by the solution and leave for 2-3 hours at 37°C. After incubation, thoroughly remove liquid. Do not reuse this solution. Do not rinse the containers. The coated flasks can be stored at 4°C for several months. 4. Ussing Chamber Buffer Recipes
For gradient assay:
Note: All IM solutions unless noted (even for pH). Recipes all for IL total volume
5. Double stranded DNA Bead Purification
[0298] To begin the PCR purification process, divide the PCR reaction mixture into 500pL portions. Vortex the beads, then add an equal volume of l.Ox SPRI paramagnetic bead-based technology (Ampure XP; Beckman Coulter; # A63881) to the PCR reaction. Mix thoroughly by pipetting up and down 10 times. Allow the mixture to sit for 5 minutes before placing it on a magnet until the liquid clarifies. Remove the supernatant carefully without disturbing the bead pellet. While keeping the tube on the magnetic plate, wash the beads with at least ImL of 70% ethanol, ensuring the beads are covered but the pellet remains undisturbed on the magnet. Allow the ethanol to incubate for at least 10-30 seconds at room temperature before removing the supernatant. Repeat this washing step with another ImL of 70% ethanol. After removing all ethanol, rehydrate the beads by adding 150pL of l/10th TE, mixing thoroughly with pipetting, and then incubating for 5 minutes. Return the tube to the magnet and wait until the solution is clear to extract the supernatant. Add another 150pL of l/10th TE, mix, incubate, and then extract the supernatant to combine with the previous extract. Assess the DNA concentration using a nanodrop. Add 30pL of CutSmart and 7pL of Dpnl, incubating at 37°C for an hour. For a second round of purification, use 340pL of SPRI paramagnetic beads without dividing the sample, elute the DNA in 80pL of pure TE, and perform a nanodrop measurement.
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* * *
[0299] All of the methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.
REFERENCES
The following references and the references cited herein, to the extent that they provide additional emboidments and/or exemplary procedural or other details supplementary to those set forth herein, are specifically incorporated herein by reference.
1. Shepherd, S. J.; Warzecha, C. C.; Yadavali, S.; El-Mayta, R.; Alameh, M.-G.; Wang, L.; Weissman, D.; Wilson, J. M.; Issadore, D.; Mitchell, M. J., Scalable mRNA and siRNA lipid nanoparticle production using a parallelized microfluidic device. Nano Letters 2021, 21 (13), 5671-5680.
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4. Krishna S Ghanta, Zexiang Chen, Aamir Mir, Gregoriy A Dokshin, Pranathi M Krishnamurthy, Yeonsoo Yoon, Judith Gallant, Ping Xu, Xiao-Ou Zhang, Ahmet Rasit Ozturk, Masahiro Shin, Feston Idrizi, Pengpeng Liu, Hassan Gneid, Alireza Edraki, Nathan D Lawson, Jaime A Rivera-Perez, Erik J Sontheimer, Jonathan K Watts, Craig C Mello (2021) 5'- Modifications improve potency and efficacy of DNA donors for precision genome editing eLife 10:e72216
5. Kanca O, Zirin J, Garcia-Marques J, Knight SM, Yang-Zhou D, Amador G, Chung H, Zuo Z, Ma L, He Y, Lin WW, Fang Y, Ge M, Yamamoto S, Schulze KL, Hu Y, Spradling AC, Mohr SE, Perrimon N, Bellen HJ. An efficient CRISPR-based strategy to insert small and large fragments of DNA using short homology arms. Elife. 2019 Nov l;8:e51539. doi: 10.7554/eLife.51539. PMID: 31674908; PMCID: PMC6855806.

Claims

1. A lipid nanoparticle comprising one or more lipids and a sterol, wherein the one or more lipids comprise one or more of an ionizable lipid, a phospholipid, and a lipid conjugated to PEG.
2. The lipid nanoparticle of claim 1, wherein the sterol comprises a sitosterol.
3. The lipid nanoparticle of claim 1 or 2, wherein the lipid nanoparticle comprises: i) one or more of the ionizable lipids: 9-Heptadecanyl 8-{(2-hydroxyethyl)[6-oxo-6-
(undecyloxy)hexyl] amino} octanoate (SM-102); 7-[(2-Hydroxyethyl)[8-(nonyloxy)-8- oxooctyl]amino]heptyl 2-octyldecanoate (Lipid 5); 9-[4-(dimethylamino)-l-oxobutoxy]- heptadecanedioic acid, l,17-di-(dec-3-yn-l-yl) ester Di(dec-3-yn-l-yl)9-((4- (dimethylamino)butanoyl)oxy)heptadecanedioate (Lipid A6); l,l'-[[2-[4-[2-[[2-[bis(2- hydroxydodecyl)amino]ethyl](2-hydroxydodecyl)amino]ethyl]-l- piperazinyl]ethyl]imino]bis-2-dodecanol (C 12-200); l,2-dioleoyl-3 -trimethylammonium - propane chloride (DOTAP); and 4,7,10,13,16-Pentaazanonadecanedioic acid, 4, 10, 16-tris[3- [2-[2-methyl-3-(octylthio)-l-oxopropoxy]ethoxy]-3-oxopropyl]-, l,19-bis[2-[2-methyl-3- (octylthio)-l-oxopropo (5A2-SC8); and/or ii) one or more of the sterols: 3 -[N-(N’N, N’-dimethylaminoethane)- carbamoyl]cholesterol (DC-cholesterol), cholesterol, and 0-sitosterol, and lithochoic acid; and/or iii) one or more of the lipids conjugated to PEG: l,2-Dioleoyl-sn-glycero-3- phosphoethanolamine-N-[methoxy(poly ethylene glycol)- 1000] (DOPE-PEG 1000), 1,2- dioleoyl-sn-glycero-3-phosphoethanolamine-N-[amino(poly ethylene glycol)-2000] (DOPE- PEG2000), 1,2 distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(poly-ethylene glycol)-2000] (DSPE-PEG2000), l,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-
[carboxy(polyethylene glycol)-1000 (DSPE-PEG1000); and l,2-Dimyristoyl-rac-glycero-3- [methoxy(poly-ethylene glycol)-2000] (DMG-PEG2000); and/or iv) the phospholipid dioleoylphosphatidylethanolamine (DOPE).
4. The lipid nanoparticle of any one of claims 1-3, wherein the nanoparticle comprises molar ratios of 40-60 ionizable lipid; 25-50 sterol; 5-15 phospholipid; and 0.5-5 lipid conjugated to PEG.
5. The lipid nanoparticle of claim 4, wherein the nanoparticle comprises molar ratios of 50 ionizable lipid to 38.5 sterol to 10 phospholipid to 1.5 lipid conjugated to PEG.
6. The lipid nanoparticle of any one of claims 1-5, wherein the ionizable lipid comprises
SM-102.
7. The lipid nanoparticle of any one of claims 5-6, wherein the lipid nanoparticle comprises 20-60 mole percent of ionizable lipid, 25-55 mole percent of sterol, 5-15 mole percent phospholipid, and/or 0.5-3 mole percent lipid conjugated to PEG.
8. The lipid nanoparticle of any one of claims 1-7, wherein the lipid nanoparticle comprises DMG-PEG2000 and DSPE-PEG1000.
9. The lipid nanoparticle of claim 8, wherein the lipid nanoparticle comprises 0.6% DMG- PEG2000 and 0.9% DSPE-PEG1000 or wherein the weight or molar ratio of DMG-PEG2000 to DSPE-PEG1000 is 2:3.
10. The lipid nanoparticle of claim 7, wherein the lipid nanoparticle comprises 50 mole percent of SM-102, 38.5 mole percent of beta-sitosterol, 10% DOPE, and/or 1.5% DMG- PEG2000.
11. The lipid nanoparticle of any one of claims 1-10, wherein the PEG of the lipid conjugated to PEG comprises a functional end group at the PEG terminus.
12. The lipid nanoparticle of claim 11, wherein the functional end group comprises -SH, - orthopyridyl-disulfide, -maleimide, -transcyclooctene, and/or -dibenzocyclooctyne.
13. The lipid nanoparticle of any one of claims 1-12, wherein the lipid nanoparticle is nebulized.
14. The lipid nanoparticle of any one of claims 1-13, wherein the lipid nanoparticle is 100- 200 nm in size.
15. The lipid nanoparticle of any one of claims 1-14, wherein the nanoparticle comprises a surfactant.
16. The lipid nanoparticle of claim 15, wherein the surfactant is covalently or non- covalently bound to the lipid nanoparticle.
17. The lipid nanoparticle of claim 15 or 16, wherein the surfactant comprises an amphiphilic surfactant.
18. The lipid nanoparticle of claim 17, wherein the surfactant comprises polidocanol (PDOC) or lysophosphatidylcholine (LPC).
19. The lipid nanoparticle of any one of claims 1-18, wherein the lipid nanoparticle excludes a nucleic acid, therapeutic cargo, and/or small molecule.
20. The lipid nanoparticle of any one of claims 1-19, wherein the lipid nanoparticle consists or consists essentially of SM-102, beta-sitosterol, DOPE, DMG-PEG2000, and a surfactant.
21. The lipid nanoparticle of any one of claims 1-20, wherein the lipid nanoparticle comprises, consists, or consists essentially of an ionizable lipid, a sterol, a phospholipid, a lipid conjugated to PEG, and a surfactant.
22. The lipid nanoparticle of any one of claims 15-21, wherein the lipid nanoparticle comprises 10-40 wt % surfactant.
23. The lipid nanoparticle of any one of claims 1-22, wherein the lipid nanoparticle comprises a therapeutic cargo.
24. The lipid nanoparticle of any one of claims 1-23, wherein the lipid nanoparticle consists or consists essentially of SM-102, beta-sitosterol, DOPE, DMG-PEG2000, and a therapeutic cargo.
25. The lipid nanoparticle of any one of claims 1-3 or 13-14, wherein the lipid nanoparticle comprises, consists, or consists essentially of an ionizable lipid, a sterol, a phospholipid, a lipid conjugated to PEG, and a therapeutic cargo.
26. The lipid nanoparticle of any one of claims 23-25, wherein the therapeutic cargo comprises a nucleic acid, tRNA, small molecule, antibody, and/or polypeptide.
27. The lipid nanoparticle of claim 26, wherein the lipid nanoparticle comprises a DNA- PK inhibitor and/or a DNA Polymerase Theta (POLO) inhibitor.
28. The lipid nanoparticle of claim 27, wherein the lipid nanoparticle comprises AZD7648 and/or ART558.
29. The lipid nanoparticle of any one of claims 26-28, wherein the nucleic acid comprises one or more of a small interfering RNA (siRNA), short hairpin RNA (shRNA), double-stranded RNA, an antisense oligonucleotide, and a ribozyme.
30. The lipid nanoparticle of any one of claims 23-29, wherein the lipid nanoparticle comprises a Cas protein or a nucleic acid encoding a Cas protein.
31. The lipid nanoparticle of claim 30, wherein the lipid nanoparticle comprises a RNA or DNA encoding a Cas protein.
32. The lipid nanoparticle of claim 30 or 31, wherein the Cas protein comprises a Cas9 protein.
33. The lipid nanoparticle of claim 32, wherein the Cas9 protein comprises the amino acid sequence of SEQ ID NO: 6, an amino acid sequence of a fragment of SEQ ID NO: 6, an amino acid sequence with at least 80% sequence identity to SEQ ID NO:6, or an amino acid sequence with at least 80% sequence identity to a fragment of SEQ ID NO:6.
34. The lipid nanoparticle of any one of claims 23-33, wherein the lipid nanoparticle comprises a small guide RNA or a DNA encoding a small guide RNA.
35. The lipid nanoparticle of claim 34, wherein the small guide RNA comprises one of SEQ ID NOS: 1-5 or a RNA with a sequence that is at least 90% identical to one of SEQ ID NOS: 1- 5.
36. The lipid nanoparticle of any one of claims 23-34, wherein the lipid nanoparticle comprises a donor DNA.
37. The lipid nanoparticle of any one of claims 34-36, wherein the ratio of the sgRNA to the Cas RNA or DNA is 0.5-3 sgRNA to 0.5-3 Cas RNA or DNA w/w.
38. The lipid nanoparticle of any one of claims 34-37, wherein the ratio of the sgRNA to the Cas RNA or DNA is 1.2: 1 or 1.6:1.
39. The lipid nanoparticle of any one of claims 34-38, wherein the ratio of the sgRNA to the Cas RNA or DNA is 1.2-1.6 sgRNA to 1 Cas RNA or Cas DNA.
40. The lipid nanoparticle of any one of claims 36-39, wherein the ratio of the donor DNA to the Cas RNA or DNA is 1-5 donor DNA to 0.5-3 Cas RNA or DNA w/w.
41. The lipid nanoparticle of any one of claims 36-40, wherein the ratio of the donor DNA to the Cas RNA or DNA is 3 : 1 or 4: 1.
42. The lipid nanoparticle of any one of claims 1-41, wherein the nitrogen-to-phosphate ratio (N/P) is 5-13.
43. The lipid nanoparticle of claim 42, wherein the N/P is 9 or greater and/or 13 or fewer.
44. The lipid nanoparticle of claim 43, wherein the N/P is 11.
45. The lipid nanoparticle of any one of claims 36-44, wherein the donor DNA is ssDNA or dsDNA.
46. The lipid nanoparticle of any one of claims 36-43, wherein the donor DNA is linear.
47. The lipid nanoparticle of any one of claims 36-46, wherein the donor DNA comprises a transgene or a mutation correction cassette and at least one homology arm that is 5’ or 3’ proximal to the transgene or cassette, wherein the mutation correction cassette edits at least one pathogenic mutation in an endogenous gene.
48. The lipid nanoparticle of claim claim 47, wherein the donor DNA comprises a transgene and wherein the transgene is at least 3000 DNA bases.
49. The lipid nanoparticle of claim claim 47 or 48, wherein the donor DNA is at least 1700 kDa.
50. The lipid nanoparticle of any one of claims 47-49, wherein the transgene is fewer than 8000 DNA bases.
51. The lipid nanoparticle of any one of claims 47-50, wherein the transgene or endogenous gene is CFTR, a dynein gene, DNAI1, DNAH5, GATA4, NR2F2, ZFPM2, WT1, a surfactant gene, surfactant protein C, surfactant protein B, alphal -antitrypsin gene, SMAD4, CHD7, Trisomy 18, APC, LKB1, MLH1, MSH2, MSH6, PMS2, EPC AM, N0D2, ATG16L1, IL23R, IRGM, HFE, SMAD4, or BMPRIA.
52. The lipid nanoparticle of claim 51, wherein the transgene or endogenous gene comprises a CFTR gene.
53. The lipid nanoparticle of claim 52, wherein the donor DNA comprises a nucleic acid encoding for the amino acid sequence of SEQ ID NO: 14, an amino acid sequence of a fragment of SEQ ID NO: 14, an amino acid sequence with at least 80% sequence identity to SEQ ID NO: 14, or an amino acid sequence with at least 80% sequence identity to a fragment of SEQ ID NO: 14.
54. The lipid nanoparticle of claim 52-53, wherein the donor DNA comprises the nucleic acid sequence of SEQ ID NO: 13, a nucleic acid that is a fragment of SEQ ID NO: 13, a nucleic acid sequence with at least 80% sequence identity to the nucleic acid sequence of SEQ ID NO: 13, or a nucleic acid sequence with at least 80% sequence identity to a fragment of the nucleic acid sequence of SEQ ID NO: 13.
55. The lipid nanoparticle of any one of claims 52-54, wherein the 5’ proximal homology arm comprises the nucleic acid sequence of SEQ ID NO: 15 or 16, a nucleic acid that is a fragment of SEQ ID NO: 15 or 16, a nucleic acid sequence with at least 80% sequence identity to the nucleic acid sequence of SEQ ID NO: 15 or 16, or a nucleic acid sequence with at least 80% sequence identity to a fragment of the nucleic acid sequence of SEQ ID NO: 15 or 16.
56. The lipid nanoparticle of any one of claims 52-55, wherein the 3’ proximal homology arm comprises the nucleic acid sequence of SEQ ID NO: 17, a nucleic acid that is a fragment of SEQ ID NO: 17, a nucleic acid sequence with at least 80% sequence identity to the nucleic acid sequence of SEQ ID NO: 17, or a nucleic acid sequence with at least 80% sequence identity to a fragment of the nucleic acid sequence of SEQ ID NO: 17.
57. The lipid nanoparticle of any one of claims 52-56, wherein the donor DNA comprises the nucleic acid sequence of SEQ ID NO: 18, a nucleic acid that is a fragment of SEQ ID NO: 18, a nucleic acid sequence with at least 80% sequence identity to the nucleic acid sequence of SEQ ID NO: 18, or a nucleic acid sequence with at least 80% sequence identity to a fragment of the nucleic acid sequence of SEQ ID NO: 18.
58. The lipid nanoparticle of any one of claims 47-57, wherein the donor DNA comprises two homology arms and wherein one homology arm is 5’ proximal to the transgene or cassette and one homology arm is 3’ proximal to the transgene or cassette.
59. The lipid nanoparticle of any one of claims 47-58, wherein the 5’ proximal, 3’ proximal, or both 5’ and 3’ proximal homology arm is fewer than 150 nucleotides and is at least 90% homologous to an endogenous genomic sequence.
60. The lipid nanoparticle of any one of claims 47-59, wherein the 5’ proximal, 3’ proximal, or both 5’ and 3’ proximal homology arm is fewer than 150 nucleotides and comprises at least 90% sequence identity to an endogenous genomic sequence of the same length of the homology arm.
61. The lipid nanoparticle of any one of claims 36-60, wherein the donor DNA comprises a posttranscriptional regulatory element.
62. The lipid nanoparticle of claim 61, wherein the posttranscriptional regulatory element comprises SEQ ID NO: 11, an amino acid sequence of a fragment of SEQ ID NO: 11, an amino acid sequence with at least 80% sequence identity to SEQ ID NO: 11, or an amino acid sequence with at least 80% sequence identity to a fragment of SEQ ID NO: 11.
63. The lipid nanoparticle of any one of claims 36-62, wherein the donor DNA comprises a polyadenylation signal.
64. The lipid nanoparticle of claim 63, wherein the polyadenylation signal comprises SEQ ID NO: 12, an amino acid sequence of a fragment of SEQ ID NO: 12, an amino acid sequence with at least 80% sequence identity to SEQ ID NO: 12, or an amino acid sequence with at least 80% sequence identity to a fragment of SEQ ID NO: 12.
65. The lipid nanoparticle of any one of claims 36-64, wherein the donor DNA comprises one or more modifications.
66. The lipid nanoparticle of claim 65, wherein the modification comprises a modification of 6-12 Carbons at the 5’ terminus of the DNA molecule.
67. The lipid nanoparticle of claim 66, wherein the modification comprises or consists of 6 carbons at the 5’ terminus of the DNA molecule.
68. A small guide RNA (sgRNA) comprising the nucleotide sequence of one of SEQ ID NOS: 1-5 or a RNA with a sequence that is at least 90% identical to one of SEQ ID NOS: 1-5.
69. The sgRNA of claim 68, wherein the RNA sequence consists of the nucleotide sequence of one of SEQ ID NOS: 1-5.
70. The sgRNA of claim 68 or 69, wherein the sgRNA comprises one or more modifications.
71. The sgRNA of any one of claims 68-70, wherein the sgRNA is single-stranded.
72. A donor DNA molecule comprising a CFTR transgene and at least one homology arm.
73. A donor DNA molecule comprising a transgene or mutation correction cassette and at least one homology arm, wherein the homology arm is fewer than 150 nucleotides and comprises at least 90% homology to an endogenous genomic sequence and wherein the DNA is modified with 6-12 carbons at the 5’ terminus of the DNA molecule; and wherein the mutation correction cassette edits at least one pathogenic mutation in an endogenous gene.
74. The donor DNA molecule of claim 73, wherein the donor DNA comprises a transgene and wherein the transgene is at least 3000 DNA bases.
75. The donor DNA molecule of claim 73 or 74, wherein the donor DNA is at least 1700 kDa.
76. The donor DNA of any one of claims 73-74, wherein the transgene is fewer than 8000 DNA bases.
77. The donor DNA of any one of claims 73-76, wherein the transgene or endogenous gene comprises CFTR.
78. The donor DNA of any one of claims 72-77, wherein the donor DNA comprises a nucleic acid encoding for the amino acid sequence of SEQ ID NO: 14, an amino acid sequence of a fragment of SEQ ID NO: 14, an amino acid sequence with at least 80% sequence identity to SEQ ID NO: 14, or an amino acid sequence with at least 80% sequence identity to a fragment of SEQ ID NO: 14.
79. The donor DNA of any one of claims 72-78, wherein the donor DNA comprises the nucleic acid sequence of SEQ ID NO: 13, a nucleic acid that is a fragment of SEQ ID NO: 13, a nucleic acid sequence with at least 80% sequence identity to the nucleic acid sequence of SEQ ID NO: 13, or a nucleic acid sequence with at least 80% sequence identity to a fragment of the nucleic acid sequence of SEQ ID NO: 13.
80. The donor DNA of any one of claims 72-78, wherein the 5’ proximal homology arm comprises the nucleic acid sequence of SEQ ID NO: 15 or 16, a nucleic acid that is a fragment of SEQ ID NO: 15 or 16, a nucleic acid sequence with at least 80% sequence identity to the nucleic acid sequence of SEQ ID NO: 15 or 16, or a nucleic acid sequence with at least 80% sequence identity to a fragment of the nucleic acid sequence of SEQ ID NO: 15 or 16.
81. The donor DNA of any one of claims 72-79, wherein the 3’ proximal homology arm comprises the nucleic acid sequence of SEQ ID NO: 17, a nucleic acid that is a fragment of SEQ ID NO: 17, a nucleic acid sequence with at least 80% sequence identity to the nucleic acid sequence of SEQ ID NO: 17, or a nucleic acid sequence with at least 80% sequence identity to a fragment of the nucleic acid sequence of SEQ ID NO: 17.
82. The donor DNA of any one of claims 72-81, wherein the donor DNA comprises the nucleic acid sequence of SEQ ID NO: 18, a nucleic acid that is a fragment of SEQ ID NO: 18, a nucleic acid sequence with at least 80% sequence identity to the nucleic acid sequence of SEQ ID NO: 18, or a nucleic acid sequence with at least 80% sequence identity to a fragment of the nucleic acid sequence of SEQ ID NO: 18.
83. The donor DNA of any one of claims 72-82, wherein the donor DNA comprises two homology arms and wherein one homology arm is 5’ proximal to the transgene or cassette and one homology arm is 3’ proximal to the transgene or cassette.
84. The donor DNA of any one of claims 72-83, wherein the 5’ proximal, 3’ proximal, or both 5’ and 3’ proximal homology arm is fewer than 150 nucleotides and is at least 90% homologous to an endogenous genomic sequence.
85. The donor DNA of any one of claims 72-84, wherein the 5’ proximal, 3’ proximal, or both 5’ and 3’ proximal homology arm is fewer than 150 nucleotides and comprises at least 90% sequence identity to an endogenous genomic sequence of the same length of the homology arm.
86. The donor DNA of any one of claims 72-85, wherein the donor DNA comprises a posttranscriptional regulatory element.
87. The donor DNA of claim 86, wherein the posttranscriptional regulatory element comprises SEQ ID NO: 11, an amino acid sequence of a fragment of SEQ ID NO: 11, an amino acid sequence with at least 80% sequence identity to SEQ ID NO: 11, or an amino acid sequence with at least 80% sequence identity to a fragment of SEQ ID NO: 11.
88. The donor DNA of any one of claims 72-87, wherein the donor DNA comprises a polyadenylation signal.
89. The donor DNA of claim 88, wherein the polyadenylation signal comprises SEQ ID NO: 12, an amino acid sequence of a fragment of SEQ ID NO: 12, an amino acid sequence with at least 80% sequence identity to SEQ ID NO: 12, or an amino acid sequence with at least 80% sequence identity to a fragment of SEQ ID NO: 12.
90. The donor DNA of any one of claims 72-89, wherein the donor DNA comprises one or more modifications.
91. The donor DNA of claim 90, wherein the modification comprises a modification of 6- 12 Carbons at the 5’ terminus of the DNA molecule.
92. The donor DNA of claim 91, wherein the modification comprises or consists of 6 carbons at the 5’ terminus of the DNA molecule.
93. A cDNA encoding the sgRNA of any one of claims 68-71.
94. A cDNA encoding the donor DNA of any one of claims 72-92.
95. An expression vector comprising the cDNA of claim 93 and/or the cDNA of claim 94.
96. A cell comprising the expression vector of claim 95, the cDNA of claim 93, the cDNA of claim 94, a sgRNA of any one of claims 68-71, and/or the donor DNA of any one of claims 72-92.
97. A lipid nanoparticle comprising the expression vector of claim 95, the cDNA of claim 93, the cDNA of claim 94, a sgRNA of any one of claims 68-71, and/or the donor DNA of any one of claims 72-92.
98. The lipid nanoparticle of claim 97, wherein the lipid nanoparticle further comprises a Cas protein or a nucleic acid encoding a Cas protein.
99. The lipid nanoparticle of claim 97 or 98, wherein the donor DNA, sgRNA, and/or Cas nucleic acid comprises one or more modifications..
100. The lipid nanoparticle of any one of claims 97-99, wherein the lipid nanoparticle comprises a sgRNA of any one of claims 68-71, and a Cas protein or a nucleic acid encoding a Cas protein.
101. The lipid nanoparticle of claim 98 or 100, wherein the lipid nanoparticle comprises a RNA or DNA encoding a Cas protein.
102. The lipid nanoparticle of any one of claims 98-101, wherein the Cas protein comprises a Cas9 protein.
103. The lipid nanoparticle of claim 102, wherein the Cas9 protein comprises the amino acid sequence of SEQ ID NO: 6, an amino acid sequence of a fragment of SEQ ID NO: 6, an amino acid sequence with at least 80% sequence identity to SEQ ID NO:6, or an amino acid sequence with at least 80% sequence identity to a fragment of SEQ ID NO:6.
104. The lipid nanoparticle of any one of claims 100-103, wherein the ratio of the sgRNA to the Cas RNA or Cas DNA is 0.5-3 sgRNA to 0.5-3 Cas RNA or Cas DNA w/w.
105. The lipid nanoparticle of any one of claims 100-104, wherein the ratio of the sgRNA to the Cas RNA or DNA is 1.2: 1 or 1.6:1.
106. The lipid nanoparticle of any one of claims 100-105, wherein the ratio of the sgRNA to the Cas RNA or DNA is 1.2-1.6 sgRNA to 1 Cas RNA or Cas DNA.
107. The lipid nanoparticle of any one of claims 97-106, wherein the ratio of the donor DNA to the Cas RNA or DNA is 1-5 donor DNA to 0.5-3 Cas RNA or DNA w/w.
108. The lipid nanoparticle of any one of claims 97-107, wherein the ratio of the donor DNA to the Cas RNA or DNA is 3 : 1 or 4: 1.
109. The lipid nanoparticle of any one of claims 97-108, wherein the nitrogen-to-phosphate ratio (N/P) is 5-13.
110. The lipid nanoparticle of claim 109, wherein the N/P is 9 or greater and/or 13 or fewer.
111. The lipid nanoparticle of claim 109, wherein the N/P is 11.
112. The lipid nanoparticle of any one of claims 101-111, wherein the weight of the cas9
RNA or cas9 DNA, donor DNA, and/or sgRNA is 50-100 ng.
113. The lipid nanoparticle of any one of claims 97-112, wherein the lipid nanoparticle comprises one or more lipids and a sterol, wherein the one or more lipids comprise one or more of: an ionizable lipid, a phospholipid, and a lipid conjugated to PEG.
114. The lipid nanoparticle of claim 113, wherein the sterol comprises a sitosterol.
115. The lipid nanoparticle of claim 113 or 114, wherein the lipid nanoparticle comprises: i) one or more of the ionizable lipids: 9-Heptadecanyl 8-{(2-hydroxyethyl)[6-oxo-6-
(undecyloxy)hexyl] amino} octanoate (SM-102); 7-[(2-Hydroxyethyl)[8-(nonyloxy)-8- oxooctyl]amino]heptyl 2-octyldecanoate (Lipid 5); 9-[4-(dimethylamino)-l-oxobutoxy]- heptadecanedioic acid, l,17-di-(dec-3-yn-l-yl) ester Di(dec-3-yn-l-yl)9-((4- (dimethylamino)butanoyl)oxy)heptadecanedioate (Lipid A6); l,l'-[[2-[4-[2-[[2-[bis(2- hydroxydodecyl)amino]ethyl](2-hydroxydodecyl)amino]ethyl]-l- piperazinyl]ethyl]imino]bis-2-dodecanol (C 12-200); l,2-dioleoyl-3 -trimethylammonium - propane chloride (DOTAP); and 4,7,10,13,16-Pentaazanonadecanedioic acid, 4, 10, 16-tris[3- [2-[2-methyl-3-(octylthio)-l-oxopropoxy]ethoxy]-3-oxopropyl]-, l,19-bis[2-[2-methyl-3- (octylthio)-l-oxopropo (5A2-SC8); and/or ii) one or more of the sterols: 3 -[N-(N’N, N’-dimethylaminoethane)- carbamoyl]cholesterol (DC-cholesterol), cholesterol, 0-sitosterol, and lithochoic acid; and/or iii) one or more of the lipids conjugated to PEG: l,2-Dioleoyl-sn-glycero-3- phosphoethanolamine-N-[methoxy(poly ethylene glycol)- 1000] (DOPE-PEG 1000), 1,2- dioleoyl-sn-glycero-3-phosphoethanolamine-N-[amino(poly ethylene glycol)-2000] (DOPE- PEG2000), 1,2 distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(poly-ethylene glycol)-2000] (DSPE-PEG2000), l,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-
[carboxy(polyethylene glycol)-1000 (DSPE-PEG1000); and l,2-Dimyristoyl-rac-glycero-3- [methoxy(poly-ethylene glycol)-2000] (DMG-PEG2000); and/or iv) the phospholipid dioleoylphosphatidylethanolamine (DOPE).
116. The lipid nanoparticle of any one of claims 113-115, wherein the nanoparticle comprises molar ratios of 40-60 ionizable lipid; 25-50 sterol; 5-15 phospholipid; and 0.5-5 lipid conjugated to PEG.
117. The lipid nanoparticle of claim 116, wherein the nanoparticle comprises molar ratios of 50 ionizable lipid; 38.5 sterol; 10 phospholipid; and 1.5 lipid conjugated to PEG.
118. The lipid nanoparticle of claim 114, wherein the nanoparticle comprises an ionizable lipid, sitosterol, DOPE, and/or DMG-PEG2000.
119. The lipid nanoparticle of claim 118, wherein the ionizable lipid comprises SM-102.
120. The lipid nanoparticle of claim 118 or 119, wherein the lipid nanoparticle comprises 20-60 mole percent of ionizable lipid, 25-55 mole percent of sterol, 5-15 mole percent phospholipid, and/or 0.5-3 mole percent lipid conjugated to PEG.
121. The lipid nanoparticle of claim 120, wherein the lipid nanoparticle comprises 50 mole percent of SM-102, 38.5 mole percent of Sitosterol, 10% DOPE, and/or 1.5% DMG-PEG2000.
122. The lipid nanoparticle of any one of claims 113-121, wherein the lipid nanoparticle comprises DMG-PEG2000 and DSPE-PEG1000.
123. The lipid nanoparticle of claim 122, wherein the lipid nanoparticle comprises 0.6% DMG-PEG2000 and 0.9% DSPE-PEG1000 or wherein the weight or molar ratio of DMG- PEG2000 to DSPE-PEG1000 is 2:3.
124. The lipid nanoparticle of any one of claims 113-123, wherein the PEG of the lipid conjugated to PEG comprises a functional end group at the PEG terminus.
125. The lipid nanoparticle of claim 113-123, wherein the functional end group comprises - SH, -orthopyridyl-disulfide, -maleimide, -transcyclooctene, and/or -dibenzocyclooctyne.
126. The lipid nanoparticle of any one of claims 97-125, wherein the lipid nanoparticle is nebulized or in an aerosol.
127. The lipid nanoparticle of any one of claims 97-126, wherein the nanoparticle comprises a surfactant.
128. The lipid nanoparticle of claim 127, wherein the surfactant is covalently or non- covalently bound to the lipid nanoparticle.
129. The lipid nanoparticle of claim 127 or 128, wherein the surfactant comprises an amphiphilic surfactant.
130. The lipid nanoparticle of claim 129, wherein the surfactant comprises polidocanol (PDOC) or lysophosphatidylcholine (LPC).
131. The lipid nanoparticle of any one of claims 127-130, wherein the lipid nanoparticle comprises 10-40 wt % surfactant.
132. The lipid nanoparticle of any one of claims 97-131, wherein the nanoparticle comprises a DNA-PK inhibitor and/or a DNA Polymerase Theta (POLO) inhibitor.
133. The lipid nanoparticle of claim 132, wherein the nanoparticle comprises AZD7648 and/or ART558.
134. The lipid nanoparticle of any one of claims 97-133, wherein the lipid nanoparticle is 100-200 nm in size.
135. A composition comprising the lipid nanoparticle of any one of claims 1-67 or 97-134, expression vector of claim 95, the cDNA of claim 93, the cDNA of claim 94, a sgRNA of any one of claims 68-71, and/or the donor DNA of any one of claims 72-92.
136. The composition of claim 135, wherein the composition comprises a DNA-PK inhibitor and/or a DNA Polymerase Theta (POLO) inhibitor.
137. The composition of claim 136, wherein the composition comprises AZD7648 and/or ART558.
138. The composition of any one of claims 135-137, wherein the composition further comprises a Cas protein or a nucleic acid encoding a Cas protein.
139. The composition of any one of claims 135-138, wherein the donor DNA, sgRNA, and/or Cas nucleic acid comprises one or more modifications.
140. The composition of any one of claims 135-139, wherein the composition comprises a sgRNA of any one of claims 68-71, and a Cas protein or a nucleic acid encoding a Cas protein.
141. The composition of claim 138 or 140, wherein the composition comprises a RNA or DNA encoding a Cas protein.
142. The composition of any one of claims 138-141, wherein the Cas protein comprises a Cas9 protein.
143. The composition of claim 142, wherein the Cas9 protein comprises the amino acid sequence of SEQ ID NO: 6, an amino acid sequence of a fragment of SEQ ID NO: 6, an amino acid sequence with at least 80% sequence identity to SEQ ID NO:6, or an amino acid sequence with at least 80% sequence identity to a fragment of SEQ ID NO:6.
144. The composition of any one of claims 138-143, wherein the composition further comprises the donor DNA of any one of claims 72-92.
145. The composition of any one of claims 135-144, wherein the composition comprises the lipid nanoparticle of any one of claims 1-67 or 97-134 and a second lipid nanoparticle, wherein the second lipid nanoparticle is selected from the lipid nanoparticle of any one of claims 1-67 or 97-134.
146. The composition of claim 145, wherein the composition comprises i) a lipid nanoparticle comprising a sgRNA of any one of claims 68-71 and a Cas protein or a nucleic acid encoding a Cas protein; and ii) a second lipid nanoparticle comprising a donor DNA of any one of claims 72-92.
147. The composition of any one of claims 135-146, wherein the composition comprises a carrier.
148. The composition of claim 147, wherein the carrier comprises ethylene glycol-bis(P- aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA).
149. The composition of claim 148, wherein the carrier comprises 1-10 mM EGTA.
150. The composition of claim 149, wherein the carrier comprises 6 mM EGTA.
151. The composition of any one of claims 147-150, wherein the carrier comprises a buffer.
152. The composition of claim 151, wherein the carrier comprises HEPES buffer.
153. The composition of any one of claims 135-152, wherein the composition comprises 0.5-5 ng/pL therapeutic cargo suspended in a carrier.
154. A method for delivering a therapeutic cargo to the airway or epithelium of a subject or for treating an airway or gastrointestinal disorder in a subject, the method comprising administering the lipid nanoparticle of any one of claims 1-67 or 97-134, expression vector of claim 95, the cDNA of claim 93, the cDNA of claim 94, a sgRNA of any one of claims 68-71, the donor DNA of any one of claims 72-92; and/or the composition of any one of claims 135- 154 to the subject.
155. A method for delivering a therapeutic cargo to the airway or epithelium of a subject, the method comprising administering to the subject: i) a first lipid nanoparticle comprising a surfactant; and ii) at least one second lipid nanoparticle comprising a therapeutic cargo.
156. A method for treating an airway or gastrointestinal disorder in a subject, the method comprising administering to the subject: i) a first lipid nanoparticle comprising a surfactant; and ii) at least one second lipid nanoparticle comprising a therapeutic cargo.
157. The method of claim 154 or 156, wherein the airway or gastrointestinal disorder comprises an epithelial disorder, COVID, a respiratory virus, a pulmonary fibrosis disorder, cystic fibrosis, primary ciliary dyskinesia, congenital diaphragmatic hernia, interstitial lung disease, alphal -antitrypsin deficiency, hemorrhagic telangiectasia, trachea-esophageal fistula, familial adenomatous polyposis, Peutz-Jerghers syndrome, Lynch syndrome, inflammatory bowel disease, hemochromatosis, polyposis syndrome, or juvenile polyposis syndrome.
158. The method of claims 157, wherein the airway or gastrointestinal disorder comprises cystic fibrosis.
159. The method of any one of claims 154-158, wherein the surfactant of the first lipid nanoparticle is covalently or non-covalently bound to the lipid nanoparticle.
160. The method of any one of claims 154-159, wherein the surfactant of the first lipid nanoparticle comprises an amphiphilic surfactant.
161. The method of any one of claims 154-160, wherein the surfactant of the first lipid nanoparticle comprises polidocanol (PDOC) or lysophosphatidylcholine (LPC).
162. The method of any one of claims 154-161, wherein the first lipid nanoparticle excludes a nucleic acid, therapeutic cargo, and/or small molecule.
163. The method of any one of claims 154-162, wherein the first lipid nanoparticle consists or consists essentially of SM-102, beta-sitosterol, DOPE, DMG-PEG2000, and a surfactant.
164. The method of any one of claims 154-163, wherein the first lipid nanoparticle comprises, consists, or consists essentially of an ionizable lipid, a sterol, a phospholipid, a lipid conjugated to PEG, and a surfactant.
165. The method of any one of claims 154-164, wherein the first lipid nanoparticle comprises 10-40 wt % surfactant.
166. The method of any one of claims 154-165, wherein the second lipid nanoparticle comprises a therapeutic cargo.
167. The method of any one of claims 154-166, wherein the second lipid nanoparticle consists or consists essentially of SM-102, beta-sitosterol, DOPE, DMG-PEG2000, and a therapeutic cargo.
168. The method of any one of claims 154-167, wherein the second lipid nanoparticle comprises, consists, or consists essentially of an ionizable lipid, a sterol, a phospholipid, a lipid conjugated to PEG, and a therapeutic cargo.
169. The method of any one of claims 164-168, wherein the sterol comprises a sitosterol.
170. The method of any one of claims 164-169, wherein the ionizable lipid comprises one or more of: 9-Heptadecanyl 8-{(2-hydroxyethyl)[6-oxo-6-
(undecyloxy)hexyl] amino} octanoate (SM-102); 7-[(2-Hydroxyethyl)[8-(nonyloxy)-8- oxooctyl]amino]heptyl 2-octyldecanoate (Lipid 5); 9-[4-(dimethylamino)-l-oxobutoxy]- heptadecanedioic acid, l,17-di-(dec-3-yn-l-yl) ester Di(dec-3-yn-l-yl)9-((4- (dimethylamino)butanoyl)oxy)heptadecanedioate (Lipid A6); l,l'-[[2-[4-[2-[[2-[bis(2- hydroxydodecyl)amino]ethyl](2-hydroxydodecyl)amino]ethyl]-l- piperazinyl]ethyl]imino]bis-2-dodecanol (C 12-200); l,2-dioleoyl-3 -trimethylammonium - propane chloride (DOTAP); and 4,7,10,13,16-Pentaazanonadecanedioic acid, 4, 10, 16-tris[3- [2-[2-methyl-3-(octylthio)-l-oxopropoxy]ethoxy]-3-oxopropyl]-, l,19-bis[2-[2-methyl-3- (octylthio)-l-oxopropo (5A2-SC8).
171. The method of any one of claims 164-170, wherein the sterol comprises one or more of DC-cholesterol, cholesterol, P-sitosterol, and lithochoic acid.
172. The method of any one of claims 164-171, wherein the lipid conjugated to PEG comprises one or more of l,2-Dioleoyl-sn-glycero-3-phosphoethanolamine-N- [methoxy(poly ethylene glycol)- 1000] (DOPE-PEGIOOO), l,2-dioleoyl-sn-glycero-3- phosphoethanolamine-N-[amino(poly ethylene glycol)-2000] (DOPE-PEG2000), 1,2 distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(poly-ethylene glycol)-2000] (DSPE- PEG2000), l,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[carboxy(polyethylene glycol)-1000 (DSPE-PEG1000); and l,2-Dimyristoyl-rac-glycero-3-[methoxy(poly-ethylene glycol)-2000] (DMG-PEG2000).
173. The method of any one of claims 164-172, wherein the phospholipid comprises dioleoylphosphatidylethanolamine (DOPE).
174. The method of any one of claims 164-173, wherein the nanoparticle comprises molar ratios of 40-60 ionizable lipid; 25-50 sterol; 5-15 phospholipid; and 0.5-5 lipid conjugated to PEG.
175. The method of claim 174, wherein the nanoparticle comprises molar ratios of 50 ionizable lipid; 38.5 sterol; 10 phospholipid; and 1.5 lipid conjugated to PEG.
176. The method of any one of claims 166-175, wherein the therapeutic cargo comprises a nucleic acid, tRNA, small molecule, antibody, and/or polypeptide.
177. The method of claim 176, wherein the nucleic acid comprises one or more of a small interfering RNA (siRNA), short hairpin RNA (shRNA), double-stranded RNA, an antisense oligonucleotide, and a ribozyme.
178. The method of any one of claims 154-177, wherein the therapeutic cargo comprises a guide RNA, a Cas protein or a nucleic acid encoding a Cas protein, and/or a donor DNA.
179. The method of any one of claims 155-178, wherein the second lipid nanoparticle comprises a Cas protein or a nucleic acid encoding a Cas protein.
180. The method of claim 178 or 179, wherein the second lipid nanoparticle comprises a RNA or DNA encoding a Cas protein.
181. The method of any one of claims 178-180, wherein the Cas protein comprises a Cas9 protein.
182. The method of any one of claims 178-181, wherein the donor DNA, sgRNA, and/or Cas nucleic acid comprises one or more modifications.
183. The method of claim 181 or 182, wherein the Cas9 protein comprises the amino acid sequence of SEQ ID NO: 6, an amino acid sequence of a fragment of SEQ ID NO: 6, an amino acid sequence with at least 80% sequence identity to SEQ ID NO:6, or an amino acid sequence with at least 80% sequence identity to a fragment of SEQ ID NO:6.
184. The method of any one of claims 154-183, wherein the second lipid nanoparticle comprises a small guide RNA (sgRNA) or a DNA encoding a sgRNA.
185. The method of any one of claims 178-184, wherein the sgRNA comprises one of SEQ ID NOS: 1-5 or a RNA with a sequence that is at least 90% identical to one of SEQ ID NOS: 1- 5.
186. The method of any one of claims 154-185, wherein the second lipid nanoparticle comprises a donor DNA.
187. The method of any one of claims 178-186, wherein the donor DNA is ssDNA or dsDNA.
188. The method of any one of claims 178-187, wherein the donor DNA is linear.
189. The method of any one of claims 178-188, wherein the ratio of the sgRNA to the Cas RNA or Cas DNA is 0.5-3 sgRNA to 0.5-3 Cas RNA or Cas DNA w/w.
190. The method of any one of claims 178-189, wherein the ratio of the sgRNA to the Cas RNA or DNA is 1.2: 1 or 1.6: 1.
191. The method of any one of claims 178-190, wherein the ratio of the sgRNA to the Cas RNA or DNA is 1.2-1.6 sgRNA to 1 Cas RNA or Cas DNA.
192. The method of any one of claims 178-191, wherein the ratio of the donor DNA to the Cas RNA or DNA is 1-5 donor DNA to 0.5-3 Cas RNA or DNA w/w.
193. The method of any one of claims 178-192, wherein the ratio of the donor DNA to the Cas RNA or DNA is 3 : 1 or 4: 1.
194. The method of any one of claims 178-193, wherein the nitrogen-to-phosphate ratio (N/P) is 5-13.
195. The method of claim 194, wherein the N/P is 9 or greater and/or 13 or fewer.
196. The method of claim 195, wherein the N/P is 11.
197. The method of any one of claims 178-196, wherein the weight of the cas9 RNA or cas9 DNA, donor DNA, and/or sgRNA is 50-100 ng.
198. The method of any one of claims 178-197, wherein the donor DNA comprises a transgene or a mutation correction cassette and at least one homology arm that is 5’ or 3’ proximal to the transgene or cassette, wherein the mutation correction cassette edits at least one pathogenic mutation in an endogenous gene.
199. The method of claim 198, wherein the donor DNA comprises a transgene and wherein the transgene is at least 3000 DNA bases.
200. The method of claim 198 or 199, wherein the donor DNA is at least 1700 kDa.
201. The method of any one of claims 198-200, wherein the transgene is fewer than 8000 DNA bases.
202. The method of any one of claims 198-201, wherein the transgene or endogenous gene comprises CFTR, a dynein gene, DNAI1, DNAH5, GATA4, NR2F2, ZFPM2, WT1, a surfactant gene, surfactant protein C, surfactant protein B, alphal -antitrypsin gene, SMAD4, CHD7, Trisomy 18, APC, LKB1, MLH1, MSH2, MSH6, PMS2, EPCAM, N0D2, ATG16L1, IL23R, IRGM, HFE, SMAD4, or BMPRIA.
203. The method of claim 202, wherein the transgene or endogenous gene comprises a CFTR gene.
204. The method of claim 203, wherein the donor DNA comprises a nucleic acid encoding for the amino acid sequence of SEQ ID NO: 14, an amino acid sequence of a fragment of SEQ ID NO: 14, an amino acid sequence with at least 80% sequence identity to SEQ ID NO: 14, or an amino acid sequence with at least 80% sequence identity to a fragment of SEQ ID NO: 14.
205. The method of claim 203 or 204, wherein the donor DNA comprises the nucleic acid sequence of SEQ ID NO: 13, a nucleic acid that is a fragment of SEQ ID NO: 13, a nucleic acid sequence with at least 80% sequence identity to the nucleic acid sequence of SEQ ID NO: 13, or a nucleic acid sequence with at least 80% sequence identity to a fragment of the nucleic acid sequence of SEQ ID NO: 13.
206. The method of any one of claims 203-205, wherein the 5’ proximal homology arm comprises the nucleic acid sequence of SEQ ID NO: 15 or 16, a nucleic acid that is a fragment of SEQ ID NO: 15 or 16, a nucleic acid sequence with at least 80% sequence identity to the nucleic acid sequence of SEQ ID NO: 15 or 16, or a nucleic acid sequence with at least 80% sequence identity to a fragment of the nucleic acid sequence of SEQ ID NO: 15 or 16.
207. The method of any one of claims 203-206, wherein the 3’ proximal homology arm comprises the nucleic acid sequence of SEQ ID NO: 17, a nucleic acid that is a fragment of SEQ ID NO: 17, a nucleic acid sequence with at least 80% sequence identity to the nucleic acid sequence of SEQ ID NO: 17, or a nucleic acid sequence with at least 80% sequence identity to a fragment of the nucleic acid sequence of SEQ ID NO: 17.
208. The method of any one of claims 203-207, wherein the donor DNA comprises the nucleic acid sequence of SEQ ID NO: 18, a nucleic acid that is a fragment of SEQ ID NO: 18, a nucleic acid sequence with at least 80% sequence identity to the nucleic acid sequence of SEQ ID NO: 18, or a nucleic acid sequence with at least 80% sequence identity to a fragment of the nucleic acid sequence of SEQ ID NO: 18.
209. The method of any one of claims 154-208, wherein the subject has or wherein the airway or gastrointestinal disorder comprises cystic fibrosis and the transgene or endogenous gene comprises CFTR.
210. The method of any one of claims 154-208, wherein the subject has or wherein the airway or gastrointestinal disorder comprises primary ciliary dyskinesia and the transgene or endogenous gene comprises a cilia related gene.
211. The method of claim 210, wherein the cilia related gene comprises a dynein gene, DNAI1, and/or DNAH5.
212. The method of any one of claims 154-208, wherein the subject has or wherein the airway or gastrointestinal disorder comprises congenital diaphragmatic hernia and the transgene or endogenous gene comprises GATA4, NR2F2, ZFPM2, or WT1.
213. The method of any one of claims 154-208, wherein the subject has or wherein the airway or gastrointestinal disorder comprises interstitial lung disease and the transgene or endogenous gene comprises a surfactant gene.
214. The method of claim 213, wherein the surfactant gene comprises surfactant protein C and/or surfactant protein B.
215. The method of claim 213 or 214, wherein the interstitial lung disease comprises childhood interstitial lung disease.
216. The method of any one of claims 154-208, wherein the subject has or wherein the airway or gastrointestinal disorder comprises alpha 1 -antitrypsin deficiency and the transgene or endogenous gene comprises alphal- antitrypsin gene.
217. The method of any one of claims 154-208, wherein the subject has or wherein the airway or gastrointestinal disorder comprises hemorrhagic telangiectasia and the transgene or endogenous gene comprises SMAD4.
218. The method of claim 217, wherein the hemorrhagic telangiectasia comprises hereditary hemorrhagic telangiectasia.
219. The method of any one of claims 154-208, wherein the subject has or wherein the airway or gastrointestinal disorder comprises trachea-esophageal fistula and the transgene or endogenous gene comprises CHD7 or Trisomy 18.
220. The method of any one of claims 154-208, wherein the subject has or wherein the airway or gastrointestinal disorder comprises familial adenomatous polyposis and the transgene or endogenous gene comprises APC.
221. The method of any one of claims 154-208, wherein the subject has or wherein the airway or gastrointestinal disorder comprises Peutz-Jerghers syndrome and the transgene or endogenous gene comprises LKB1.
222. The method of any one of claims 154-208, wherein the subject has or wherein the airway or gastrointestinal disorder comprises Lynch syndrome and the transgene or endogenous gene comprises MLH1, MSH2, MSH6, PMS2, and/or EPCAM.
223. The method of any one of claims 154-208, wherein the subject has or wherein the airway or gastrointestinal disorder comprises hemochromatosis and the transgene or endogenous gene comprises HFE.
224. The method of claim 223, wherein the hemochromatosis comprises hereditary hemochromatosis.
225. The method of any one of claims 154-208, wherein the subject has or wherein the airway or gastrointestinal disorder comprises inflammatory bowel disease and the transgene or endogenous gene comprises N0D2, ATG16L1, IL23R, and/or IRGM.
226. The method of any one of claims 154-208, wherein the subject has or wherein the airway or gastrointestinal disorder comprises polyposis syndrome and the transgene or endogenous gene comprises SMAD4 and/or BMPR1 A.
227. The method of claim 226, wherein polyposis syndrome comprises juvenile polyposis syndrome.
228. The method of any one of claims 198-227, wherein the donor DNA comprises two homology arms and wherein one homology arm is 5’ proximal to the transgene or cassette and one homology arm is 3’ proximal to the transgene or cassette.
229. The method of any one of claims 198-228, wherein the 5’ proximal, 3’ proximal, or both 5’ and 3’ proximal homology arm is fewer than 150 nucleotides and is at least 90% homologous to an endogenous genomic sequence.
230. The method of any one of claims 198-229, wherein the 5’ proximal, 3’ proximal, or both 5’ and 3’ proximal homology arm is fewer than 150 nucleotides and comprises at least 90% sequence identity to an endogenous genomic sequence of the same length of the homology arm.
231. The method of any one of claims 178-229, wherein the donor DNA comprises a posttranscriptional regulatory element.
232. The method of claim 231 , wherein the posttranscriptional regulatory element comprises SEQ ID NO: 11, an amino acid sequence of a fragment of SEQ ID NO: 11, an amino acid sequence with at least 80% sequence identity to SEQ ID NO: 11, or an amino acid sequence with at least 80% sequence identity to a fragment of SEQ ID NO: 11.
233. The method of any one of claims 178-232, wherein the donor DNA comprises a polyadenylation signal.
234. The method of claim 233, wherein the polyadenylation signal comprises SEQ ID NO: 12, an amino acid sequence of a fragment of SEQ ID NO: 12, an amino acid sequence with at least 80% sequence identity to SEQ ID NO: 12, or an amino acid sequence with at least 80% sequence identity to a fragment of SEQ ID NO: 12.
235. The method of any one of claims 178-234, wherein the donor DNA is modified.
236. The method of claim 235, wherein the modification comprises a modification of 6-12 Carbons at the 5’ terminus of the DNA molecule.
237. The method of claim 236, wherein the modification comprises or consists of 6 carbons at the 5’ terminus of the DNA molecule.
238. The method of any one of claims 155-237, wherein the first and/or second lipid nanoparticle comprises: SM-102; beta-sitosterol; DOPE; and DMG-PEG2000.
239. The method of claim 238, wherein the first and/or second lipid nanoparticle comprises molar ratios of 40-60 SM-102; 25-50 beta-sitosterol; 5-15 DOPE; and 0.5-5 DMG-PEG2000.
240. The method of claim 239, wherein the first and/or second lipid nanoparticle comprises molar ratios of 50 SM-102; 38.5 beta-sitosterol; 10 DOPE; and 1.5 DMG-PEG2000.
241. The method of any one of claims 163-240, wherein the PEG of the lipid conjugated to PEG comprises a functional end group at the PEG terminus.
242. The method of claim 163-241, wherein the functional end group comprises -SH, - orthopyridyl-disulfide, -maleimide, -transcyclooctene, and/or -dibenzocyclooctyne.
243. The method of any one of claims 155-242, wherein the first and/or second lipid nanoparticle is nebulized.
244. The method of any one of claims 155-243, wherein the lipid nanoparticle is 100-200 nm in size.
245. The method of any one of claims 155-244, wherein the subject is administered the first lipid nanoparticle before, concurrently with, and/or after the second lipid nanoparticle.
246. The method of any one of claims 155-244, wherein the subject is administered the second lipid nanoparticle before, concurrently with, and/or after the first lipid nanoparticle.
247. The method of any one of claims 155-246, wherein the subject is administered the first lipid nanoparticle before the administration of the second lipid nanoparticle.
248. The method of any one of claims 245-247, wherein the first lipid nanoparticle is administered at least 3 hours before the second lipid nanoparticle.
249. The method of any one of claims 155-248, wherein the first and second lipid nanoparticle are administered within 1 day of each other.
250. A method for delivering a therapeutic cargo to the airway or epithelium of a subject, the method comprising administering: a lipid nanoparticle comprising a therapeutic cargo, wherein the lipid nanoparticle comprises a surfactant.
251. A method for treating an airway or gastrointestinal disorder in a subject, the method comprising administering: a lipid nanoparticle comprising a therapeutic cargo, wherein the lipid nanoparticle comprises a surfactant.
252. The method of claim 251, wherein the airway or gastrointestinal disorder comprises an epithelial disorder, CO VID, a respiratory virus, a pulmonary fibrosis disorder, cystic fibrosis, primary ciliary dyskinesia, congenital diaphragmatic hernia, interstitial lung disease, alphal- antitrypsin deficiency, hemorrhagic telangiectasia, trachea-esophageal fistula, familial adenomatous polyposis, Peutz-Jerghers syndrome, Lynch syndrome, inflammatory bowel disease, hemochromatosis, polyposis syndrome, or juvenile polyposis syndrome.
253. The method of claims 252, wherein the airway or gastrointestinal disorder comprises cystic fibrosis.
254. The method of any one of claims 250-253, wherein the surfactant is covalently or non- covalently bound to the lipid nanoparticle.
255. The method of any one of claims 250-254, wherein the surfactant comprises an amphiphilic surfactant.
256. The method of any one of claims 250-255, wherein the surfactant comprises polidocanol (PDOC) or lysophosphatidylcholine (LPC).
257. The method of any one of claims 250-255, wherein the lipid nanoparticle comprises 10- 40 wt % surfactant.
258. The method of any one of claims 250-257, wherein the therapeutic cargo comprises a nucleic acid, tRNA, small molecule, antibody, and/or polypeptide.
259. The method of claim 258, wherein the nucleic acid comprises one or more of a small interfering RNA (siRNA), short hairpin RNA (shRNA), double-stranded RNA, an antisense oligonucleotide, and a ribozyme.
260. The method of any one of claims 250-259, wherein the therapeutic cargo comprises a guide RNA, a Cas protein or a nucleic acid encoding a Cas protein, and/or a donor DNA.
261. The method of any one of claims 250-260, wherein the lipid nanoparticle comprises a Cas protein or a nucleic acid encoding a Cas protein.
262. The method of claim 260 or 261, wherein the lipid nanoparticle comprises a RNA or DNA encoding a Cas protein.
263. The method of any one of claims 260-262, wherein the Cas protein comprises a Cas9 protein.
264. The method of any one of claims 260-263, wherein the donor DNA, sgRNA, and/or Cas nucleic acid comprises one or more modifications.
265. The method of claim 263 or 264, wherein the Cas9 protein comprises the amino acid sequence of SEQ ID NO: 6, an amino acid sequence of a fragment of SEQ ID NO: 6, an amino acid sequence with at least 80% sequence identity to SEQ ID NO:6, or an amino acid sequence with at least 80% sequence identity to a fragment of SEQ ID NO:6.
266. The method of any one of claims 250-263, wherein the lipid nanoparticle comprises a guide RNA or a DNA encoding a guide RNA.
267. The method of any one of claims 260-266, wherein the guide RNA comprises one of SEQ ID NOS: 1-5 or a RNA with a sequence that is at least 90% identical to one of SEQ ID NOS: l-5.
268. The method of any one of claims 250-267, wherein the lipid nanoparticle comprises a donor DNA.
269. The method of any one of claims 260-268, wherein the donor DNA is ssDNA or dsDNA.
270. The method of any one of claims 250-269, wherein the donor DNA is linear.
271. The method of any one of claims 260-270, wherein the donor DNA comprises a transgene or a mutation correction cassette and at least one homology arm that is 5’ or 3’ proximal to the transgene or cassette, wherein the mutation correction cassette edits at least one pathogenic mutation in an endogenous gene.
272. The method of claim claim 271, wherein the donor DNA comprises a transgene and wherein the transgene is at least 3000 DNA bases.
273. The method of claim claim 271 or 272, wherein the donor DNA is at least 1700 kDa.
274. The method of any one of claims 271-273, wherein the transgene is fewer than 8000 DNA bases.
275. The method of any one of claims 271-274, wherein the transgene or endogenous gene comprises CFTR, a dynein gene, DNAI1, DNAH5, GATA4, NR2F2, ZFPM2, WT1, a surfactant gene, surfactant protein C, surfactant protein B, alphal -antitrypsin gene, SMAD4, CHD7, Trisomy 18, APC, LKB1, MLH1, MSH2, MSH6, PMS2, EPCAM, N0D2, ATG16L1, IL23R, IRGM, HFE, SMAD4, or BMPRIA.
276. The method of claim 275, wherein the transgene or endogenous gene comprises a CFTR gene.
277. The method of claim 276, wherein the donor DNA comprises a nucleic acid encoding for the amino acid sequence of SEQ ID NO: 14, an amino acid sequence of a fragment of SEQ ID NO: 14, an amino acid sequence with at least 80% sequence identity to SEQ ID NO: 14, or an amino acid sequence with at least 80% sequence identity to a fragment of SEQ ID NO: 14.
278. The method of claim 276 or 277, wherein the donor DNA comprises the nucleic acid sequence of SEQ ID NO: 13, a nucleic acid that is a fragment of SEQ ID NO: 13, a nucleic acid sequence with at least 80% sequence identity to the nucleic acid sequence of SEQ ID NO: 13, or a nucleic acid sequence with at least 80% sequence identity to a fragment of the nucleic acid sequence of SEQ ID NO: 13.
279. The method of any one of claims 276-278, wherein the 5’ proximal homology arm comprises the nucleic acid sequence of SEQ ID NO: 15 or 16, a nucleic acid that is a fragment of SEQ ID NO: 15 or 16, a nucleic acid sequence with at least 80% sequence identity to the nucleic acid sequence of SEQ ID NO: 15 or 16, or a nucleic acid sequence with at least 80% sequence identity to a fragment of the nucleic acid sequence of SEQ ID NO: 15 or 16.
280. The method of any one of claims 276-279, wherein the 3’ proximal homology arm comprises the nucleic acid sequence of SEQ ID NO: 17, a nucleic acid that is a fragment of SEQ ID NO: 17, a nucleic acid sequence with at least 80% sequence identity to the nucleic acid sequence of SEQ ID NO: 17, or a nucleic acid sequence with at least 80% sequence identity to a fragment of the nucleic acid sequence of SEQ ID NO: 17.
281. The method of any one of claims 276-279, wherein the donor DNA comprises the nucleic acid sequence of SEQ ID NO: 18, a nucleic acid that is a fragment of SEQ ID NO: 18, a nucleic acid sequence with at least 80% sequence identity to the nucleic acid sequence of SEQ ID NO: 18, or a nucleic acid sequence with at least 80% sequence identity to a fragment of the nucleic acid sequence of SEQ ID NO: 18.
282. The method of any one of claims 271-281, wherein the donor DNA comprises two homology arms and wherein one homology arm is 5’ proximal to the transgene or cassette and one homology arm is 3’ proximal to the transgene or cassette.
283. The method of any one of claims 271-282, wherein the 5’ proximal, 3’ proximal, or both 5’ and 3’ proximal homology arm is fewer than 150 nucleotides and is at least 90% homologous to an endogenous genomic sequence.
284. The method of any one of claims 271-283, wherein the 5’ proximal, 3’ proximal, or both 5’ and 3’ proximal homology arm is fewer than 150 nucleotides and comprises at least 90% sequence identity to an endogenous genomic sequence of the same length of the homology arm.
285. The method of any one of claims 260-284, wherein the donor DNA comprises a posttranscriptional regulatory element.
286. The method of claim 285, wherein the posttranscriptional regulatory element comprises SEQ ID NO: 11, an amino acid sequence of a fragment of SEQ ID NO: 11, an amino acid sequence with at least 80% sequence identity to SEQ ID NO: 11, or an amino acid sequence with at least 80% sequence identity to a fragment of SEQ ID NO: 11.
287. The method of any one of claims 260-286, wherein the donor DNA comprises a polyadenylation signal.
288. The method of claim 287, wherein the polyadenylation signal comprises SEQ ID NO: 12, an amino acid sequence of a fragment of SEQ ID NO: 12, an amino acid sequence with at least 80% sequence identity to SEQ ID NO: 12, or an amino acid sequence with at least 80% sequence identity to a fragment of SEQ ID NO: 12.
289. The method of any one of claims 260-288, wherein the donor DNA is modified.
290. The method of claim 289, wherein the modification comprises a modification of 6-12 Carbons at the 5’ terminus of the DNA molecule.
291. The method of claim 290, wherein the modification comprises or consists of 6 carbons at the 5’ terminus of the DNA molecule.
292. The method of any one of claims 250-291, wherein the lipid nanoparticle comprises: 1 SM-102; Beta-sitosterol; dioleoylphosphatidylethanolamine (DOPE); and DMG-PEG2000.
293. The method of claim 292, wherein the lipid nanoparticle comprises molar ratios of 40- 60 SM-102; 25-50 Beta-sitosterol; 5-15 DOPE; and 0.5-5 DMG-PEG2000.
294. The method of claim 293, wherein the lipid nanoparticle comprises molar ratios of 50% SM-102; 38.5 Beta-sitosterol; 10 DOPE; and 1.5 PE-PEG1000.
295. The method of any one of claims 292-294, wherein the PEG of the lipid conjugated to PEG comprises a functional end group at the PEG terminus.
296. The method of claim 292-295, wherein the functional end group comprises -SH, - orthopyridyl-disulfide, -maleimide, -transcyclooctene, and/or -dibenzocyclooctyne.
297. The method of any one of claims 250-296, wherein the lipid nanoparticle is nebulized.
298. The method of any one of claims 154-297, wherein the subject is a human.
-MO-
299. A lipid nanoparticle comprising 50 mole percent of SM-102, 38.5 mole percent of betasitosterol, 10 mole percent DOPE, and 1.5 mole percent DMG-PEG2000.
300. A method for treating cystic fibrosis in a subject, the method comprising administering to the subject: i) a first lipid nanoparticle comprising SM-102, beta-sitosterol, DOPE, and DMG- PEG2000, and a surfactant; wherein the ratio of SM-102: beta-sitosterol :DOPE: DMG- PEG2000 is 50:38.5: 10: 1.5 and the surfactant comprises PDOC or LPC; and ii) a second lipid nanoparticle comprising SM-102, beta-sitosterol, DOPE, and DMG- PEG2000, and a therapeutic cargo; wherein the ratio of SM-102: beta-sitosterol :DOPE: DMG- PEG2000 is 50:38.5: 10: 1.5; wherein the therapeutic cargo comprises a guide RNA, a mRNA encoding a Cas protein, and a donor DNA; wherein the donor DNA encodes a CFTR transgene; and wherein the first lipid nanoparticle is administered prior to the second lipid nanoparticle to disrupt the the apical epithelia.
EP24826734.6A 2023-06-23 2024-06-21 Methods and compositions for treating epithelial diseases Pending EP4731186A2 (en)

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