Lipid nanoparticles for topical delivery

Abstract

The instant disclosure relates to lipid particles that harbor cationic lipids, the particles found to be capable of delivering associated cargoes - particularly nucleic acid cargoes when formulated as nucleic acid-lipid particles - intracellularly to skin tissue cells when administered topically to a subject. The instant disclosure provides compositions comprising such lipid particles, optionally in association with a therapeutic agent (e.g., a therapeutic mRNA and / or nucleic acid controller system), as well as methods and kits for delivering a lipid particle-associated therapeutic agent and / or for treating or preventing a disease or disorder, e.g., a skin disease or disorder, in a subject, using one or more lipid particle compositions provided herein.

Description

[0001] LIPID NANOPARTICLES FOR TOPICAL DELIVERY

[0002] CROSS-REFERENCE TO RELATED APPLICATION

[0003] The present application is related to and claims priority under 35 U.S.C. § 119(e) to U.S. provisional patent application No. 63 / 655,260, entitled “LIPID NANOPARTICLES FOR TOPICAL DELIVERY,” filed June 3, 2024. The entire content of the aforementioned patent application is incorporated herein by this reference.

[0004] FIELD OF THE INVENTION

[0005] The current disclosure relates to lipid-based compositions and methods useful for administering therapies, especially nucleic acid-based therapies. In particular, the disclosure relates to lipid particles, having cationic lipids, capable of delivery of a cargo (e.g., nucleic acids) to a subject, including for treatment or prevention of diseases or disorders of a subject, such as skin diseases or disorders.

[0006] BACKGROUND OF THE INVENTION

[0007] Alopecia areata is a common autoimmune skin disease, in which hair is lost from the head or all areas of the body. Also known as spot baldness, it results in a few bald spots on the scalp, each the size of a coin. As many as 6.8 million people are affected in the U.S., of all ages, of both sexes, and of all ethnic groups. In a few cases, all the hair on the scalp is lost or all body hair is lost, and the loss is permanent. It can cause psychological distress because of self-image issues, even though a subject is otherwise generally healthy. Alopecia in general has different classifications (i.e., alopecia areata, androgenetic alopecia, central centrifugal cicatricial alopecia, chemotherapy induced alopecia, lichen planopilaris, telogen effluvium, traction alopecia, and trichotillomania); but in each case, it does not affect life expectancy and is not contagious. Alopecia areata is a systemic autoimmune disorder in which the body attacks its own anagen hair follicles and suppresses or stops hair growth. T cell lymphocytes cluster around affected follicles, causing inflammation and subsequent hair loss. Alopecia areata shares genetic risk with other autoimmune diseases, including rheumatoid arthritis, type 1 diabetes, and celiac disease. Treatment assessment is very difficult, and spontaneous remission is unpredictable. None of the existing therapeutic options are curative or preventative, and a major challenge in the field remains to develop therapeutic agents that effectively treat alopecia areata as well as other skin diseases. Nucleic acid therapies offer tremendous potential for treatment of diseases at the level of individual, targeted genes. However, safe and effective delivery systems are essential for realizing the full promise of nucleic acid therapeutics. Non-specific delivery of nucleic acid therapeutics to all organs and tissues can often result in off-site (non-targeted and / or off-target) effects and toxicity. Delivery of nucleic acid therapeutics preferentially to an organ, tissue and / or cell type of interest in which a specific action is desirable is a continuing goal for drag delivery and delivery of nucleic acid-based agents in particular. There is therefore a previously unmet need in the art for vehicles that are capable of achieving safe and effective delivery of nucleic acid cargoes to target tissue(s). In particular, because topical administration is an attractive route for gene therapy, particularly for treatment of diseases or disorders of the skin, there is also a specific need in the art for such vehicles capable of delivering nucleic acid cargoes to skin tissues, as well as cell types within the skin.

[0008] BRIEF SUMMARY OF THE INVENTION

[0009] The instant disclosure is based, at least in part, upon identification of lipid-based nanoparticle compositions and formulations capable of topical delivery of a cargo moiety (e.g., a nucleic acid cargo). In particular, the instant disclosure relates to as series of independent lipid particles formed using distinct obligate cationic lipid or ionizable lipid components selected from a set of twelve such lipids, with the series of lipid particles evaluated for effective delivery of nucleic acid cargoes intracellularly to skin, including sub-populations of skin cells (e.g., dermal papilla cells, hair follicle cells, etc.). Certain of the instantly disclosed formulations achieved robust intracellular delivery of nucleic acid cargoes to skin tissues, including selective delivery to dermal papilla cells and / or hair follicle cells. The instant disclosure therefore provides a set of twelve distinct lipid particles possessing varying obligate cationic lipids or ionizable lipids, among other structural components (e.g., phospholipid(s), non-cationic lipid(s), and conjugated lipid(s) that inhibit particle aggregation), a selection of which demonstrated delivery of nucleic acid cargoes selectively to skin tissue cells upon topical administration to a subject, under the range of conditions currently tested. While the current lipid particles were initially evaluated via topical administration and assessed for effective nucleic acid cargo delivery to specific types of skin tissue cells (i.e., dermal papilla cells, hair follicle cells), it is expressly contemplated that these lipid particles could be employed for administration of nucleic acid cargo(es) via other routes of o administration, including parenteral administration (e.g., intravenous injection, intrathecal injection, etc.), and / or could be used to deliver non-nucleic acid cargo(es) via any such currently contemplated route of administration. Exemplary contemplated nucleic acid cargo(es) for the lipid particles of the instant disclosure include, e.g., nucleic acid modulating controllers, therapeutic mRNAs, RNA interference (RNAi) and antisense agents (siRNAs, miRNAs, etc.), among others known in the art.

[0010] In one aspect, the instant disclosure provides a lipid particle for topical delivery of a cargo to a subject, the lipid particle including a cationic lipid present at about 20 mol % to about 70 mol % (or optionally at about 20 mol % to about 50 mol %) of total lipid in the lipid particle; a phospholipid present at about 4 mol % to about 30 mol % of the total lipid in the lipid particle; a non-cationic lipid present at about 10 mol % to about 75 mol % of the total lipid present in the lipid particle; and a conjugated lipid that inhibits aggregation of particles present at 0.01 mol % to about 3 mol % of the total lipid present in the lipid particle.

[0011] In one embodiment, the cationic lipid is an obligate cationic lipid. In a related embodiment, the obligate cationic lipid is Didodecyldimethylammonium bromide (DDAB), N -( 4 - carboxy benzy l)-N,N -dimethy l-2,3-bis(oleoy loxyjpropan- 1 -aminium (DOB AQ), 1 ,2-dioleoyl-3 - trimethylammonium-propane (DOTAP), 3B-[N-(N',N'-dimethylaminoethane)- carbamoyljcholesterol (DOTMA), ethyl phosphatidylcholine (EPC), or N,N,N-trimethyl-D- erythro-sphingosine (Trimethyl Sphingosine). Optionally, the obligate cationic lipid is EPC, DOTMA or DDAB. hi a related embodiment, the obligate cationic lipid is EPC.

[0012] In another embodiment, the obligate cationic lipid has the structure of compound (I): where Ri and R? are each optionally (un)substituted alkyl or alkenyl groups having a hydrocarbon chain length between 10 and 24 carbon atoms, optionally where Ri and R2 are circularized.

[0013] In one embodiment, the cationic lipid is an ionizable lipid. In a related embodiment, the ionizable lipid is (((((disulfanediylbis(ethane-2,l-diyl))bis(piperidine-l,4-diyl))bis(ethane-2,l- diyl))bis(oxy))bis(2-oxoethane-2, 1 -diyl))bis(4, 1 -phenylene) dioleate (SS-OP), dimethylaminoethane)-carbamoyl]cholesterol (DC-Cholesterol), l,2-dioleyloxy-N,N-dimethyl-3- aminopropane (DODMA), N,N-dimethyl-D-erythro-sphingosine (Dimethyl Sphingosine),

[0014] Heptadecan-9-yl 8 - { (2-hydroxyethyl)[6-oxo-6-(undecyloxy)hexy I] amino } octanoate equivalent

[0015] (SM-102), or l ,2-Dioleoyl-3-dimethylammonium-propane (DODAP).

[0016] In some embodiments, the ionizable lipid is a disulfide (SS) cleavable lipid. Optionally, the disulfide (SS) cleavable lipid is SS-OC, SS-OP or SS-EC. hi certain embodiments, the cationic lipid is present at about 25 mol % to about 65 mol % of the total lipid present in the lipid particle. Optionally, the cationic lipid is present at about 30 mol % to about 60 mol % of the total lipid present in the lipid particle. Optionally, the cationic lipid is present at about 35 mol % to about 50 mol % of the total lipid present in the lipid particle. Optionally, the cationic lipid is present at about 45 mol % of the total lipid present in the lipid particle.

[0017] In some embodiments, the phospholipid is distearoyl-sn-glycero-phosphoethanolamine, distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoyl-phosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoylphosphatidylethanolamine (POPE), dioleoyl-phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane- 1 -carboxylate (DOPE- mal), dipalmitoyl phosphatidyl ethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoyl -phosphatidyl -ethanolamine (DSPE), monomethyl -phosphatidylethanolamine (such as 16- O-monomethyl PE), dimethyl-phosphatidylethanolamine (such as 16-O-dimethyl PE), 18-1- trans PE, l-stearoyl-2-oleoyl-phosphatidyethanolamine (SOPE), hydrogenated soy phosphatidylcholine (HSPC), ethyl phosphatidylcholine (EPC), dioleoylphosphatidylserine (DOPS), sphingomyelin (SM), dimyristoyl phosphatidylcholine (DMPC), dimyristoyl phosphatidylglycerol (DMPG), distearoylphosphatidylglycerol (DSPG), dierucoylphosphatidylcholine (DEPC), palmitoyloleyolphosphatidylglycerol (POPG), dielaidoyl - phosphatidylethanolamine (DEPE), 1,2- dilauroyl-sn-glycero-3 -pho sphoethanolamine (DLPE); l,2-diphytanoyl-sn-glycero-3- phosphoethanolamine (DPHyPE); lecithin, phosphatidylethanolamine, lysolecithin, lysophosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, sphingomyelin, egg sphingomyelin (ESM), cephalin, cardiolipin, phosphatidicacid, cerebrosides, dicetylphosphate, lysophosphatidylcholine, or dilinoleoylphosphatidylcholine, or mixtures thereof. In a related embodiment, the phospholipid is DOPC, DSPC or DOPE. Optionally, the phospholipid is DSPC.

[0018] In certain embodiments, the phospholipid is present at about 5 mol % to about 20 mol % of the total lipid present in the lipid particle. Optionally, the phospholipid is present at about 7 mol % to about 15 mol % of the total lipid present in the lipid particle. Optionally, the phospholipid is present at about 8 mol % to about 12 mol % of the total lipid present in the lipid particle. Optionally, the phospholipid is present at about 9 mol % to about 11 mol % of the total lipid present in the lipid particle. Optionally, the phospholipid is present at about 10 mol % of the total lipid present in the lipid particle.

[0019] In one embodiment, the non-cationic lipid is cholesterol, ^-sitosterol, or derivative(s) thereof.

[0020] In some embodiments, the non-cationic lipid is present at about 20 mol % to about 65 mol % of the total lipid present in the lipid particle. Optionally, the non-cationic lipid is present at about 25 mol % to about 60 mol % of the total lipid present in the lipid particle. Optionally, the noncationic lipid is present at about 30 mol % to about 55 mol % of the total lipid present in the lipid particle. Optionally, the non-cationic lipid is present at about 35 mol % to about 50 mol % of the total lipid present in the lipid particle. Optionally, the non-cationic lipid is present at about 40 mol % to about 45 mol % of the total lipid present in the lipid particle. Optionally, the non-cationic lipid is present at about 43.5 mol % of the total lipid present in the lipid particle.

[0021] In another embodiment, the conjugated lipid that inhibits aggregation of particles includes or is a polyethyleneglycol (PEG)-lipid conjugate. Optionally, the PEG of the PEG-lipid conjugate has an average molecular weight of from 550 daltons to 3000 daltons. Optionally, the PEG-lipid conjugate is a PEGsooo-lipid conjugate. Optionally, the PEG2ooo-lipid conjugate includes one or more of l,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG2000), 1,2- Dipalmitoyl-rac-glycero-3-methylpolyoxyethylene (DPG-PEG2000), and 1 ,2-distearoyl-rac- glycero-3 -methoxypolyethylene glycol-2000 (DSG-PEG2000). Optionally, the PEGiooo-lipid conjugate is l,2-Dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG2o<x)).

[0022] In certain embodiments, the conjugated lipid that inhibits aggregation of particles is present at about 0.05 mol % to about 2.8 mol % of the total lipid present in the lipid particle. Optionally, the conjugated lipid that inhibits aggregation of particles is present at about 0.1 mol % to about 2.5 mol % of the total lipid present in the lipid particle. Optionally, the conjugated lipid that inhibits aggregation of particles is present at about 0.5 mol % to about 2.0 mol % of the total lipid present in the lipid particle. Optionally, the conjugated lipid that inhibits aggregation of particles is present at about 1.2 mol % to about 1.8 mol % of the total lipid present in the lipid particle. Optionally, the conjugated lipid that inhibits aggregation of particles is present at about 1.5 mol % of the total lipid present in the lipid particle.

[0023] In some embodiments, the lipid particle includes a cationic lipid at about 45 mol % of the total lipid present in the lipid particle, phospholipid at about 10 mol % of the total lipid present in the lipid particle, cholesterol at about 43.5 mol % of the total lipid present in the lipid particle, and PEG-lipid at about 1 .5 mol % of the total lipid present in the lipid particle.

[0024] In one embodiment, the lipid particle further includes a nucleic acid cargo. Optionally, the nucleic acid cargo is a mRNA.

[0025] Another aspect of the instant disclosure provides a lipid particle for topical delivery of a cargo to a subject, the lipid particle including (((((disulfanediylbis(ethane-2,l-diyl))bis(piperidine- 1 ,4-diyl))bis(ethane-2, 1 -diyl))bis(oxy))bis(2-oxoethane-2, 1 - diy 1) )bi s(4 , 1 -phenylene) dioleate

[0026] (SS-OP) at about 20 mol % to 70 mol % of the total lipid in the lipid particle; a phospholipid at about 4 mol % to 30 mol % of the total lipid in the lipid particle; cholesterol, P-sitosterol or derivative(s) thereof at about 10 mol % to about 75 mol % of the total lipid present in the lipid particle; and a conjugated lipid that inhibits aggregation of particles at 0.01 mol % to about 3 mol % of the total lipid present in the lipid particle.

[0027] In certain embodiments, the SS-OP is present at about 45 mol % of the total lipid in the lipid particle; 1,2-distearoyl-sn-glycero-phosphocholine (DSPC) is present at about 10 mol % of the total lipid in the lipid particle; cholesterol is present at about 43.5 mol % of the total lipid in the lipid particle, and / or l,2-dimyristoyl-rac-glycero-3-methoxypoly ethylene glycol-2000 (DMG- PEG2k) is present at about 1.5 mol % of the total lipid in the lipid particle.

[0028] An additional aspect of the instant disclosure provides a lipid particle for topical delivery of a cargo to a subject, the lipid particle including ethyl phosphatidylcholine (EPC) at about 20 mol % to 70 mol % of the total lipid in the lipid particle; a phospholipid at about 4 mol % to 30 mol % of the total lipid in the lipid particle; cholesterol, p-sitosterol or derivative(s) thereof at about 10 mol % to about 75 mol % of the total lipid present in the lipid particle; and a conjugated lipid that inhibits aggregation of particles at 0.01 mol % to about 3 mol % of the total lipid present in the lipid particle.

[0029] In some embodiments, the EPC is present at about 45 mol % of the total lipid in the lipid particle; 1,2-distearoyl-sn-glycero-phosphocholine (DSPC) is present at about 10 mol % of the total lipid in the lipid particle; cholesterol is present at about 43.5 mol % of the total lipid in the lipid particle, and / or l ,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG- PEG2k) is present at about 1.5 mol % of the total lipid in the lipid particle.

[0030] A further aspect of the instant disclosure provides a lipid particle for topical delivery of a cargo to a subject, the lipid particle including 3B-[N-(N',N'-dimethylaminoethane)- carbamoyl]cholesterol (DOTMA) at about 20 mol % to 70 mol % of the total lipid in the lipid particle; a phospholipid at about 4 mol % to 30 mol % of the total lipid in the lipid particle; cholesterol, P-sitosterol or derivative(s) thereof at about 10 mol % to about 75 mol % of the total lipid present in the lipid particle; and a conjugated lipid that inhibits aggregation of particles at 0.01 mol % to about 3 mol % of the total lipid present in the lipid particle.

[0031] In one embodiment, the DOTMA is present at about 45 mol % of the total lipid in the lipid particle; 1,2-distearoyl-sn-glycero-phosphocholine (DSPC) is present at about 10 mol % of the total lipid in the lipid particle; cholesterol is present at about 43.5 mol % of the total lipid in the lipid particle, and / or l,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG- PEG2k) is present at about 1.5 mol % of the total lipid in the lipid particle.

[0032] Another aspect of the instant disclosure provides a lipid particle for topical delivery of a cargo to a subject, the lipid particle including 3B-[N-(N',N‘-dimethylaminoethan.e)- carbamoyl] cholesterol (DC-Cholesterol) at about 20 mol % to 70 mol % of the total lipid in the lipid particle; a phospholipid at about 4 mol % to 30 mol % of the total lipid in the lipid particle; cholesterol, p-sitosterol or derivative(s) thereof at about 10 mol % to about 75 mol % of the total lipid present in the lipid particle; and a conjugated lipid that inhibits aggregation of particles at 0.01 mol % to about 3 mol % of the total lipid present in the lipid particle.

[0033] In certain embodiments, the DC-Cholesterol is present at about 45 mol % of the total lipid in the lipid particle; 1,2-distearoyl-sn-glycero-phosphocholine (DSPC) is present at about 10 mol % of the total lipid in the lipid particle; cholesterol is present at about 43.5 mol % of the total lipid in the lipid particle, and / or l ,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG2k) is present at about 1.5 mol % of the total lipid in the lipid particle. An additional aspect of the instant disclosure provides a lipid particle for topical delivery of a cargo to a subject, the lipid particle including l,2-dioleyloxy~N,N-dimethyl-3- aminopropane (DODMA) at about 20 mol % to 70 mol % of the total lipid in the lipid particle; a phospholipid at about 4 mol % to 30 mol % of the total lipid in the lipid particle; cholesterol, [1- sitosterol or derivative(s) thereof at about 10 mol % to about 75 mol % of the total lipid present in the lipid particle; and a conjugated lipid that inhibits aggregation of particles at 0.01 mol % to about 3 mol % of the total lipid present in the lipid particle. hi some embodiments, the DODMA is present at about 45 mol % of the total lipid in the lipid particle; 1 ,2-distearoyl-sn-glycero-phosphocholine (DSPC) is present at about 10 mol % of the total lipid in the lipid particle; cholesterol is present at about 43.5 mol % of the total lipid in the lipid particle, and / or l,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG- PEG2k) is present at about 1.5 mol % of the total lipid in the lipid particle.

[0034] A further aspect of the instant disclosure provides a lipid particle for topical delivery of a cargo to a subject, the lipid particle including N,N-dimethyl-D-erythro-sphingosine (Dimethyl Sphingosine) at about 20 mol % to 70 mol % of the total lipid in the lipid particle; a phospholipid at about 4 mol % to 30 mol % of the total lipid in the lipid particle; cholesterol, p-sitosterol or derivative(s) thereof at about 10 mol % to about 75 mol % of the total lipid present in the lipid particle; and a conjugated lipid that inhibits aggregation of particles at 0.01 mol % to about 3 mol % of the total lipid present in the lipid particle.

[0035] In one embodiment, the Dimethyl Sphingosine is present at about 45 mol % of the total lipid in the lipid particle; 1 ,2-distearoyl-sn-glycero-phosphocholine (DSPC) is present at about 10 mol % of the total lipid in the lipid particle; cholesterol is present at about 43.5 mol % of the total lipid in the lipid particle, and / or l,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG2k) is present at about 1.5 mol % of the total lipid in the lipid particle.

[0036] An additional aspect of the instant disclosure provides a lipid particle for topical delivery of a cargo to a subject, the lipid particle including Didodecyldimethylammonium bromide (DDAB) at about 20 mol % to 70 mol % of the total lipid in the lipid particle; a phospholipid at about 4 mol % to 30 mol % of the total lipid in the lipid particle; cholesterol, p-sitosterol or derivative(s) thereof at about 10 mol % to about 75 mol % of the total lipid present in the lipid particle; and a conjugated lipid that inhibits aggregation of particles at 0.01 mol % to about 3 mol % of the total lipid present in the lipid particle. In some embodiments, the DDAB is present at about 45 mol % of the total lipid in the lipid particle; 1,2-distearoyl-sn-glycero-phosphocholine (DSPC) is present at about 10 mol % of the total lipid in the lipid particle; cholesterol is present at about 43.5 mol % of the total lipid in the lipid particle, and / or 1 ,2-dimyristoyl-rac-glycero-3 -methoxypolyethylene glycol-2000 (DMG- PEG2k) is present at about 1.5 mol % of the total lipid in the lipid particle.

[0037] In one embodiment, the cargo is a nucleic acid. Optionally, the nucleic acid cargo is a mRNA. hi certain embodiments, the nucleic acid cargo includes a synthetic or naturally occurring RNA or DNA, or derivatives thereof. Optionally, the nucleic acid cargo is a modified RNA. Optionally, the modified RNA is a modified mRNA, a modified antisense oligonucleotide, and / or a modified siRNA. Optionally, the modified mRNA encodes a nucleic acid modulating controller.

[0038] In some embodiments, the nucleic acid cargo includes one or more of the following modifications: 2'-O-methyl modified nucleotides, a nucleotide having a 5'-phosphorothioate group, a terminal nucleotide linked to a cholesteryl derivative, a 2 '-deoxy-2 '-fluoro modified nucleotide, a 5'-methoxy-modified nucleotide (e.g., 5 '-methoxyuridine), a 2'-deoxy-modified nucleotide, a locked nucleotide, an abasic nucleotide, a 2'-amino-modified nucleotide, a 2'-alkyl- modified nucleotide, a morpholino nucleotide, a phosphoramidate, a non-natural base comprising nucleotide; intemucleoside linkages or backbones including phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates including 3'-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates including 3 '-amino phosphoramidate and aminoalky Iphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, and / or boranophosphates having normal 3'-5' linkages, 2'-5' linked analogs of these, and / or those having inverted polarity wherein the adjacent pairs of nucleoside units are linked 3'-5' to 5'-3 ' or 2'-5' to 5'-2'.

[0039] A further aspect of the instant disclosure provides a composition that includes the lipid particle of the instant disclosure, where the composition is fonnulated for topical application to the skin of the subject. Optionally, the composition is formulated for application to the scalp of the subject.

[0040] Another aspect of the instant disclosure provides a pharmaceutical composition that includes a lipid particle of the disclosure and a pharmaceutically acceptable carrier. An additional aspect of the instant disclosure provides a topical formulation that includes a lipid particle, composition and'or a pharmaceutical composition of the disclosure.

[0041] In some embodiments, the lipid particle, composition, pharmaceutical composition, or topical formulation is administered to a subject for treatment of a skin or inflammatory disease or disorder. Optionally, the skin or inflammatory disease or disorder is alopecia, psoriasis, atopic dermatitis, scleroderma, eczema, rosacea, seborrheic dermatitis, melanoma, solar keratosis, ichthyosis, Grover's disease, common warts, keratoacanthoma, and / or seborrheic keratosis.

[0042] A further aspect of the instant disclosure provides a method for delivering a cargo to a dermal tissue cell of a subject, the method involving administering the lipid particle, composition, pharmaceutical composition, or topical formulation disclosed herein to the subject.

[0043] Another aspect of the instant disclosure provides a method for treating or preventing a disease or disorder in a subject, the method involving administering the lipid particle, composition, pharmaceutical composition, or topical formulation disclosed herein to the subject.

[0044] In one embodiment, the disease or disorder is an epidermal disease or disorder, a joint disease or disorder, and'or an inflammatory disease or disorder.

[0045] In some embodiments, the epidermal disease or disorder is alopecia, psoriasis, atopic dermatitis, scleroderma, eczema, rosacea, seborrheic dermatitis, melanoma, solar keratosis, ichthyosis, Grover's disease, common warts, keratoacanthoma and / or seborrheic keratosis. Optionally, the alopecia is alopecia areata, androgenetic alopecia, central centrifugal cicatricial, chemotherapy induced alopecia, frontal fibrosing alopecia, lichen planopilaris, telogen effluvium, and / or traction alopecia.

[0046] In another embodiment, the joint disease or disorder is rheumatoid arthritis, psoriatic arthritis, gout, tendinitis, bursitis, Carpal Tunnel Syndrome, and / or osteoarthritis.

[0047] In some embodiments, the inflammatory disease or disorder is osteoarthritis, rheumatoid arthritis, arthritis, osteoarthritis, gout, spondyloarthropathies, ankylosing spondylitis, caustic gout, non-arthritic rheumatism, bursitis, hay fever, suppurative inflammation, neuropathic joint disease, hypertrophic osteoarthritis, multisized hemorrhoids, and / or systemic lupus erythematosus (SLE).

[0048] In embodiments, the lipid particle, composition or pharmaceutical composition is administered via topical, transdermal, or intradermal routes of administration. Alternatively, the lipid particle, composition or pharmaceutical composition can be administered by intravenous injection, intratracheal injection, intra-articular injection, subcutaneous injection, intradermal injection and / or intramuscular injection.

[0049] Another aspect of the instant disclosure provides a method for delivering a cargo intracellularly to a skin cell of a subject, the method involving contacting the skin of the subject with a lipid particle, composition, pharmaceutical composition or topical formulation of the disclosure having a cargo, thereby delivering the cargo intracellularly to the skin cell of the subject. In one embodiment, the lipid particle, composition, pharmaceutical composition or topical formulation of the instant disclosure is applied to the scalp of the subject.

[0050] In some embodiments, the cargo is a nucleic acid cargo. Optionally, the lipid particle delivers the nucleic acid cargo intracellularly to the skin cell of the subject. In a related embodiment, the nucleic acid cargo is expressed in the cytosol of the skin cell of the subject and / or the nucleic acid cargo is delivered to the nucleus of the skin cell of the subject.

[0051] In one embodiment, the skin cell of the subject is an epidermal tissue cell, a dermal tissue cell, and / or a subcutaneous tissue cell. Optionally, the epidermal tissue cell is a keratinocyte (optionally a stratum corneum cell, a stratum granulosum cell, a stratum spinulosum cell, and / or a stratum basale cell), a melanocyte, a Langerhans cell, and / or a Merkel cell. Optionally, the dermal tissue cell is a mast cell, a vascular smooth muscle cell, a specialized muscle cell, a fibroblast, a hair follicle cell (e.g., a dermal papilla cell and / or a dermal sheath cell), an immune cell, and / or a leukocyte (e.g., a neutrophil, a T or B lymphocyte, an eosinophil and / or a monocyte). Optionally, the subcutaneous tissue cell is a fat cell, a nerve cell, and / or a vascular cell.

[0052] In one embodiment, the skin cell of the subject is a hair follicle cell. Optionally, the hair follicle cell is a dermal papilla cell.

[0053] Definitions

[0054] Unless specifically stated or obvious from context, as used herein, the term “about” is understood as within a range of normal tolerance in the art, for example within 2 standard deviations of the mean. “About” can be understood as within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the stated value.

[0055] In certain embodiments, the term "approximately" or "about" refers to a range of values that fall within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1 %, or less in either direction (greater than or less than) of the stated reference value unless otherwise stated or otherwise evident from the context (except where such number would exceed 100% of a possible value).

[0056] Unless otherwise clear from context, all numerical values provided herein are modified by the term “about."

[0057] The term “lipid” refers to a group of organic compounds that include, but are not limited to, esters of fatty acids and are characterized by being insoluble in water, but soluble in many organic solvents. They are usually divided into at least three classes: (1) “simple lipids” which include fats and oils as well as waxes; (2) “compound lipids” which include phospholipids and glycolipids; (3) “derived lipids” such as steroids.

[0058] As used herein, the term “cationic lipid” refers to any lipid species that carries a net positive charge at a selected pH. The pH may be a physiologically relevant pH. For example and without limitation, the pH may be between about 3.0 and 8.0, e.g., the pH may be about 3.0, the pH may be about 3.5, the pH may be about 4.0, the pH may be about 4.5, the pH may be about 5.0, the pH may be about 5.5, the pH may be about 6.0, the pH may be about 6.5, the pH may be about 7.0, the pH may be about 7.5, and / or the pH may be about 8.0, including all pH values between a pH of about 3.0 and a pH of about 8.0. A cationic lipid may have a head group that is always positively charged in aqueous solution (an “obligate cationic lipid”). For example and without limitation, an obligate cationic lipid may have a quaternary amine as a head group. Alternatively, a cationic lipid may have a head group that accepts a proton in solution such that the lipid exists predominantly as a cation below its pKa and predominantly as a neutral moiety above its pKa, e.g., it may have a pH-titratable amino head group (e.g., for an “ionizable lipid”, as defined infra). For example and without limitation, an ionizable lipid may have a primary, secondary, or tertiary amine as a head group, (e.g., an alkylamino or dialkylamino head group). In some embodiments, the ionizable lipids comprise: a protonatable tertiary amine (e.g., pH-titratable) head group; C18 hydrocarbon chains e.g., alkyl, alkenyl, or alkynyl chains, wherein each hydrocarbon chain independently has 0 to 3 (e.g., 0, 1, 2, or 3) double bonds; and ether, ester, or ketal linkages between the head group and hydrocarbon chains.

[0059] Examples of obligate cationic lipids include, but are not limited to, Dimethyldioctadecylammonium, Bromide Salt (DDAB), N-(4-carboxybenzyl)-N,N-dimethyl- 2,3-bis(oleoyloxy) propan- 1 -aminium (DOBAQ), l,2-dioleoyl-3-trimethylammonium-propane or 18:1 TAP, a di-chain or gemini, cationic lipid (DOTAP), l,2-di-O-octadecenyl-3- trimethyl ammonium propane, chloride salt (DOTMA), ethyl phosphatidylcholine (EPC), and trimethyl sphingosine.

[0060] As used herein, the term "ionizable lipid" or "ionizable cationic lipid" refers to a lipid that becomes cationic (protonated) as the pH is lowered below the pKa of the ionizable group of the lipid but is progressively more neutral at higher pH values. When a component of a lipid-nucleic acid particle, at pH values below the pKa, the lipid is then able to associate with negatively charged polynucleic acids. Certain examples of such ionizable lipids include lipids and salts thereof having one, two, three, or more fatty acid or fatty hydrocarbon chains and a pH-titratable amino head group (e.g, an alkylamino or dialkylamino head group). Exemplary ionizable lipids include, without limitation, l,2-Dioleoyl-3-dimethylammonium-propane (DODAP), 9-Heptadecanyl 8- {(2-hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl] amino (octanoate (SM-102), disulfanediylbis(ethane-2, 1 -diyl)bis(piperidine- 1 ,4-diyl)bis(ethane-2, 1 -diyl)bis(oxy)bis(2- oxoethane-2, l-diyl)bis(4,l -phenylene) dioleate (SS-OP), Dimethyl Sphingosine, 3-(N—(N',N'- dimethylaminoethane)-carbamoyl)cholesterol (DC-Cholesterol), C 12-200; N4-Cholesteryl- Spemiine HC1 Salt (GL67); Nl-[2-((lS)-l-[(3-aminopropyl)amino]-4-[di(3-amino- propyl)amino]butylcarboxamido)ethyl]-3,4-di[oleyloxy]-benzamide (MVL5); 1,2-distearyloxy- N,N-dimethyl-3-aminopropane (DSDMA); 1 ,2-dioleyloxy-N,N-dimethyl-3-aminopropane (DODMA); l,2-dilinoleyloxy-N,N-dimethyl-3-aminopropane (DLinDMA); 1,2-dilinolenyloxy- N,N-dimethyl-3-aminopropane (DLenDMA); 1 ,2-di-Y-linolenyloxy-N,N-dimethylaminopropane (y-DLenDMA); l,2-dilinoleyloxy-keto-N,N-dimethyl-3 -aminopropane (DLinK-DMA); 1,2- dilinoleyl-4-(2-dimethylaminoethyl)-[l ,3]-dioxolane (DLinKC2-DMA) (also known as DLin- C2K-DMA, XTC2, and C2K); 2,2-dilinoleyl-4-(3-dimethylaminopropyl)[l,3]-dioxolane (DLin- K-C3-DMA); 2,2-dilinoleyl-4-(4-dimethylaminobutyl)[l,3]-dioxolane (DLin-K-C4-DMA); 1,2- dilinolenyloxy-4-(2-dimethylaminoethyl)- [1,3] -dioxolane (y-DLen-C2K-DMA); 1 ,2-di-v- linolenyloxy-4-(2-dimethylaminoethyl)-[l,3]-dioxolane (y-DLen-C2K-DMA); dilinoleylmethyl- 3-dimethylaminopropionate (DLin-M-C2-DMA) (also known as MC2); (6Z,9Z,28Z,31Z)- heptatriaconta-6,9,28,31-tetraen- 19-yl 4-(dimethylamino) butanoate (DLin-M-C3-DMA) (also known as MC3); 3-(dilinoleylmethoxy)-N,N-dimethylpropan-l-amine (DLin-MP-DMA) (also known as 1-B11); 2-({8-[(3[J)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z,12Z)- octadeca-9,12-dien-l-yloxy]propan-l -amine (Octyl-CLinDMA); (2R) 2-({8-[(3[3)-cholest-5-en- 3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-l-yloxy]propan-l-amine (R- Octyl-CLinDMA); (2S) 2-({8-[(3p)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z,12Z)- octadeca-9,12-dien-l-yloxy]propan-l -amine (S-Octyl-CLinDMA); (2S)-l-{7-[(3p)-cholest-5-en- 3-yIoxy]heptyloxy}-3-[(4Z)-dec-4-en-l-yloxy]-7V, N -dimethylpropan-2-amine; (2R)-l-{4-[(3p)- cholest-5-en-3-yloxy]butoxy}-3-[(4Z)-dec-4-en-l-yloxy]-N,N-dimethylpropan-2-amine; 1-[(2R)- 1 -{4-[(3P)-cholest-5-en-3-yloxy]butoxy}-3-(octyloxy)propan-2-yl]guanidine; l-[(2R)-l-{7- [(3P)-cholest-5-en-3-yloxy]heptyloxy}-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9, 12-dien-l- yloxy]propan-2-amine; l-[(2R)-l-{4-[(3p)-cholest-5-en-3-yloxy]butoxy}-N,N-dimethyl-3- [(9Z,12Z)-octadeca-9,12-dien-l-yloxy]propan-2-amine; (2S)-l-({6-[(3p))-cholest-5-en-3- yloxy]hexyl } oxy)-N ,N -dimethyl-3 - [(9Z)-octadec-9-en- 1 -yloxy]propan-2-amine; (3 P)-3 - [6-

[0061] {[(2S)-3-[(9Z)-octadec-9-en-l -yloxyl]-2-(pyrrolidin-l-yl)propyl]oxy}hexy])oxy]cholest-5-ene; (2R)-l-{4-[(3p)-cholest-5-en-3-yloxy]butoxy}-3-(octyloxy)propan-2-amine; (2R)-l-({8-[(3p)- cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-(pentyloxy)propan-2-amine; (2R)-l-({8-[(3P)- cholest-5-en-3-yloxy]octyl}oxy)-3-(heptyloxy)-N,N-dimethylpropan-2-amine; (2R)-l-({8-[(3P)- cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(2Z)-pent-2-en-1.-yloxy]propan-2-amine;

[0062] (2S)-l-butoxy-3-({8-[(3P)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethylpropan-2-amine; (2S- l-({8-[(3P)-cholest-5-en-3-yloxy]octyd}oxy)-3-[2,2,3,3,4,4,5,5,6,6,7,7,8,8,9,9- hexadecafluorononyl)oxy]-N,N-dimethylpropan-2-amine; 2-amino-2- {[(9Z, 12Z)-octadeca-9, 12- dien-1 -yloxy]methyl}propane-l ,3-ciiol ; 2-amino-3-({9-[(3p, 8^,9^, 14^,17^,20^)-cholest-5-en-3- yloxy]nonyl} oxy)-2- { [(9Z , 12Z)-octadeca-9, 12-dien- 1 -yloxy]methyl } propan- 1 -ol; 2-ammo-3 -

[0063] ( {6-[(3p,8^,9^, 14^, 17^.20^)-cholest-5-en-3-yloxy]hexyl} oxy)-2- { [(9Z)-octadec-9-en- 1 - yloxy]methyl}propan-l-ol; (20Z,23Z)-N,N-dimethylnonacosa-20,23-dien-l 0-amine; (17Z,20Z)- N,N-dimethylhexacosa-17,20-dien-9-amine; (16Z,19Z)-N,N-dimethylpentacosa-16,19-dien-8- amine; ( 13Z, 16Z)-N,N-dimethyldocosa- 13,16-dien-5-amine; ( 12Z, 15Z)-N,N-dimethylhenicosa- 12, 15-dien-4-amine; ( 14Z, 17Z)-N,N-dimethyltricosa- 14, 17-dien-6-amine; ( 15Z, 18Z)-N,N- dimethyltetracosa- 15,18-dien-7-amine; ( 18Z,21 Z)-N,N-dimethylheptacosa- 18,21 -dien- 10-amine; (15Z,18Z)-N,N-dimethyltetracosa-15,18-dien-5-amine; (14Z,17Z)-N,N-dimethyltricosa-14,l 7- dien-4-amine; ( 19Z,22Z)-N,N-dimethyloctacosa- 19,22-dien-9-amine; ( 18Z,21 Z)-N,N- dimethylheptacosa-18,21-dien-8-amine; (17Z,20Z)-N,N-dimethylhexacosa-17,20-dien-7-amine; ( 16Z, 19Z)-N,N-dimethylpentacosa- 16, 19-dien-6-amine; (22Z,25Z)-N,N-dimethylhentriaconta- 22,25-dien-l 0-amine; (21Z,24Z)-N,N-dimethyltriaconta-21,24-dien-9-amine; (18Z)-N,N- dimethylheptacos- 18-en- 10-amine; ( 17Z)-N,N-dimethylhexacos- 17-en-9-amine; ( 19Z,22Z)-N,N - dimethyl octacosa- 19,22-dien-7-amine; N,N-dimethylheptacosan-l 0-amine; (20Z,23Z)-N-ethyl- N-methylnonacosa-20,23-dien-l 0-amine; 1-[(1 1Z,14Z)-l-nonylicosa-ll,14-dien-l- yl]pyrrolidine; (20Z)-N,N-dimethylheptacos-20-en-l 0-amine; (15Z)-N,N-dimethylheptacos-15- en- 10-amine; ( 14Z)-N,N-dimethylnonacos- 14-en- 10-amine; ( 17Z)-N,N-dimethylnonacos- 17-en- 10-amine; (24Z)-N,N-dimethyltritriacont-24-en-10-amine; (20Z)-N,N-dimethylnonacos-20-en- 10-amine; (22Z)-N,N-dimethylhentriacont-22-en-l 0-amine; (16Z)-N,N-dimethylpentacos-16-en- 8-amine; (12Z, 15Z)-N,N-dimethyl-2-nonylhenicosa- 12,15-dien- 1 -amine; ( 13Z, 16Z)-N,N- dimethyl~3 -nonyldocosa- 13,16-dien- 1 -amine; N,N-dimethyl- 1 -[( 1 S,2R)-2- octylcyclopropyl]heptadecan-8-amine; 1 -[( 1 S,2R)-2-hexylcyclopropyl]-N,N- dimethylnonadecan-10-amine; N,N-dimethyl-l-[(l S,2R)-2-octylcyclopropyl]nonadecan-l 0- amine; N,N-dimethyl-21 -[( 1 S,2R)-2-octylcyclopropyl]henicosan- 10-amine; N,N-dimethyl- 1 - [(lS,2S)-2-{[(lR,2R)-2-pentylcyclopropyl]methyl}cyclopropyl]nonadecan-10-amine; N,N- dimethyl- 1 -[( 1 S,2R)-2-octylcyclopropyl]hexadecan-8-amine; N,N-dimethyl- 1 -[( 1 R,2S)-2- undecylcyclopropyl]tetradecan-5-amine; N,N-dimethyl-3-{7-[(lS,2R)-2- octylcyclopropyljheptyl} dodecan- 1 -amine; 1 -[(1 R,2S)-2-heptylcyclopropyl]-N,N- dimethyloctadecan-9-amine; 1 -[(1 S,2R)-2-decylcyclopropyl]-N,N-dimethylpentadecan-6-amine; N,N-dimethyl-l-[(lS,2R)-2-octylcyclopropyl]pentadecan-8-amine; (11E,2OZ,23Z)-N,N- dimethylnonacosa-11 ,20,23-trien-10-amine; 2,2-dilinoleyl-5-dimethylaminomethyl-[l ,3]- dioxane (DLin-K6-DMA), 2,2-dilinoleyl-4-N-methylpepiazino-[l,3]-dioxolane (DLin-K-MPZ), 2,2-dioleoyl-4-dimethylaminomethyl-[ 1 ,3]-dioxolane (DO-K-DMA), 2,2-distearoyl-4- dimethylaminomethyl-[l,3]-dioxolane (DS-K-DMA), 2,2-dilinoleyl-4-N-morpholino-[l,3]- dioxolane (DLin-K-MA), 2,2-Dilinoleyl-4-trimethylamino-[l,3]-dioxolane chloride (DLin-K- TMA.C1), 2,2-dilinoleyl-4,5-bis(dimethylaminomethyl)-[l,3]-dioxolane (DLin-K2-DMA), 2,2- dilinoleyl-4-methylpiperzine-[l,3]-dioxolane (D-Lin-K — N-methylpiperzine), DLen-C2K-DMA, y-DLen-C2K-DMA, DPan-C2K-DMA, DPan-C3K-DMA, DLen-C2K-DMA, y-DLen-C2K- DMA, DPan-C2K-DMA, TLinDMA, C2-TLinDMA, C3-TLinDMA, l,2-di-y-linolenyloxy-N,N- dimethylaminopropane (y-DLenDMA), l,2-dilinoleyloxy-(N,N-dimethyl)-butyl-4-amine (C2- DLinDMA), l,2-dilinoleoyloxy-(N,N-dimethyl)-butyl-4-amine (C2-DLinDAP), CP-LenMC3, CP-y-LenMC3, CP-MC3, CP-DLen-C2K-DMA, CP-yDLen-C2K-DMA, CP-C2K-DMA, CP- DODMA, CP-DPetroDMA, CP-DLinDMA, CP-DLenDMA, CP-yDLenDMA, 1,2- dioeylcarbamoyloxy-3 -dimethylaminopropane (DO-C-DAP), l,2-dimyristoleoyl-3- dimethyl aminopropane (DMDAP), l ,2-dioleoyl-3-trimethylaminopropane chloride (DOTAP.C1), 1 ,2-dilinoleylcarbamoyloxy-3-dimethylaminopropane (DLin-C-DAP), 1 ,2-dilinoleyoxy-3- (dimethylamino)acetoxypropane (DLin-DAC), 1,2-dilinoley oxy-3 -morpholinopropane (DLin- MA), l,2-dilinoleoyl-3 -dimethylaminopropane (DLinDAP), l,2-dilinoleylthio-3- dimethyl aminopropane (DLin-S-DMA), l-linoleoyl-2-linoleyloxy-3-dimethylaminopropane (DLin-2-DMAP), l,2-dilinoleyloxy-3-trimethylaminopropane chloride salt (DLin-TMA.Cl), 1,2- dilinoleoyl-3-trimethylaminopropane chloride salt (DLin-TAP.Cl), l,2-dilinoleyloxy-3-(N- methylpiperazino)propane (DLin-MPZ), 3-(N,N-dilinoleylamino)-l,2-propanediol (DLinAP), 3- (N,N-dioleylamino)-l ,2-propanedio (DOAP), l,2-dilinoleyloxo-3-(2-N,N- dimethylaminojethoxypropane (DLin-EG-DMA), 3-dimethylamino-2-(cholest-5-en-3-beta- oxybutan-4-oxy)- 1 -(cis,cis-9, 12-octadecadienoxy)propane (CLinDMA), 2-[5'-(cholest-5-en-3- beta-oxy)-3'-oxapentoxy)-3-dimethy-l-(cis,cis-9',l-2'-octadecadienoxy)propane (CpLinDMA), N,N-dimethyl-3,4-dioleyloxybenzylamine (DMOBA), 1 ,2-N,N'-dioleylcarbamyl-3- dimethyl aminopropane (DOcarbDAP), and l,2-N,N'-dilinoleylcarbamyl-3- dimethylaminopropane (DLincarbDAP); as well as pharmaceutically acceptable salts thereof, and stereoisomers of any of the foregoing.

[0064] As used herein, the term "non-cationic lipid" refers to any uncharged, anionic, or zwitterionic lipid. At physiological pH, such lipids include, for example, diacylphosphatidylcholine, diacylphosphatidylethanolamine, ceramide, sphingomyelin, cephalin, cholesterol, cerebrosides, diacylglycerols, phosphatidylglycerols, cardiolipins, diacylphosphatidylserines, diacylphosphatidic acids, N-dodecanoyl phosphatidylethanolamines, N-succinyl phosphatidylethanolamines, N-glutarylphosphatidylethanolamines, lysylphosphatidylglycerols, palmitoyloleyolphosphatidylglycerol (POPG), and other anionic modifying groups joined to neutral lipids. In some embodiments, the non-cationic lipid used in the instant disclosure is l,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1 ,2-Distearoyl-sn- glycero-3 -phosphocholine ( DSPC), and 'or 1 ,2-Dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), hi embodiments, the non-cationic lipid is cholesterol (CHE) and / or [l-sitosterol.

[0065] In some embodiments, the non-cationic lipid present in the lipid particles comprises or consists of a mixture of one or more phospholipids and cholesterol or a derivative thereof. Exemplary zwitterionic non-cationic lipids include the following phospholipids. 16:0- 18:0 PC, also known as l-palmitoyl-2-stearoyl-sn-glycero-3-phosphocholine. has the following structure:

[0066] 16:0 / 16: 1 (9Z)-PC, also known as 1 -(1 -enyl-palmitoyl)-2-palmitoleoyl-sn-glycero-3- phosphocholine, has the following structure:

[0067] 16:0-18:2 PC, also known as l-palmitoyl-2-linoleoyl-sn-glycero-3-phosphocholine, has the following structure:

[0068] 18:0-18: 1(9Z)-PC, also known as l-stearoyl-2-ok‘oyl-sn-glycero-3-phosphocholine, has the following structure:

[0069] 18:0-18:2(92, 12Z)-PC, also known as l-Octadecanyl-2-(9Z,12Z-octadecadienoyl)-sn-glycero-3- phosphocholine, has the following structure:

[0070] 18:1-18 :2(9Z, 12Z)-PC, also known as 1 -(9Z, 12Z-octadecadienoy l)-2-(9Z-octadecenoyl)-glycero- 3-phosphocholine, has the following structure:

[0071] In some embodiments, the non-cationic lipid present in the lipid particles comprises or consists of a mixture of one or more phospholipids and cholesterol or a derivative thereof.

[0072] In certain embodiments, a lipid composition of the disclosure can include lipids such as "neutral lipids", "helper lipids", and / or "stealth lipids".

[0073] "Neutral lipids" suitable for use in a lipid composition of the disclosure include, for example, a variety of neutral, uncharged or zwitterionic lipids. In some embodiments, neutral lipids disclosed herein may include, for example, diacylphosphatidylcholine, diacylphosphatidylethanolamine, ceramide, sphingomyelin, cephalin, cholesterol, cerebrosides and diacylglycerols. Other examples of neutral phospholipids suitable for use in the present disclosure include, but are not limited to, distearoylphosphatidylcholine (DSPC), pohsphocholine (DOPC), dimyristoylphosphatidylcholine (DMPC), phosphatidylethanolamine (PE), egg phosphatidylcholine (EPC), palmitoyloleoyl phosphatidylcholine (POPC), dioleoyl phosphatidylethanolamine (DOPE), dilinoleoylphosphatidylcholine distearoylphosphatidylethanolamine (DSPE), dimyristoyl phosphatidylethanolamine (DMPE), dipalmitoyl phosphatidylethanolamine (DP PE), palmitoyloleoyl phosphatidylethanolamine (POPE), lysophosphatidylethanolamine and combinations thereof. In one embodiment, the neutral phospholipid may be selected from the group consisting of dioleoyl phosphatidylethanolamine (DOPE). In another embodiment, the neutral phospholipid may be distearoylphosphatidylcholine (DSPC). Without wishing to be bound by theory, neutral lipids have been described to function to stabilize and improve processing of the LNPs.

[0074] "Helper lipids" are lipids that enhance transfection (e.g., transfection of the nanoparticle including the biologically active agent). Without wishing to be bound by theory, the mechanism by which the helper lipid enhances transfection includes enhancing particle stability. In certain embodiments, the helper lipid enhances membrane fusogenicity. Helper lipids include the abovereferenced "neutral lipids", including but not limited to include, but are not limited to, distearoylphosphatidylcholine (DSPC), pohsphocholine (DOPC), dimyristoylphosphatidylcholine (DMPC), phosphatidylethanolamine (PE), egg phosphatidylcholine (EPC), palmitoyloleoyl phosphatidylcholine (POPC), dioleoyl phosphatidylethanolamine (DOPE), dilinoleoylphosphatidylcholine distearoylphosphatidylethanolamine (DSPE), dimyristoyl phosphatidylethanolamine (DMPE), dipalmitoyl phosphatidylethanolamine (DPPE), palmitoyloleoyl phosphatidylethanolamine (POPE), lysophosphatidylethanolamine and combinations thereof, as well as steroids, and sterols. Helper lipids suitable for use in the present disclosure include, but are not limited to, neutral lipids, cholesterol, and PEG-chol esterol. In one embodiment, the helper lipid may be cholesterol. In one embodiment, the helper lipid may be PEG- cholesterol.

[0075] "Stealth lipids" are lipids that alter the length of time the nanoparticles can exist in vivo (e.g., in the blood). Without wishing to be bound by theory, stealth lipids may assist in the formulation process by, for example, reducing particle aggregation and controlling particle size. Stealth lipids used herein may modulate pharmacokinetic properties of the LNP. Stealth lipids suitable for use in a lipid composition of the disclosure include, but are not limited to, stealth lipids having a hydrophilic head group linked to a lipid moiety. Stealth lipids suitable for use in a lipid composition of the present disclosure and information about the biochemistry of such lipids can be found in Romberg et al, Pharmaceutical Research, Vol. 25, No. 1 , 2008, pg. 55-71 and Hoekstra et al, Biochimica et Biophysica Acta 1660 (2004) 41 -52. In one embodiment, the hydrophilic head group of stealth lipid comprises a polymer moiety selected from polymers based on PEG (sometimes referred to as polyethylene oxide)), poly(oxazoline), poly(vinyl alcohol), poly(glycerol), poly(N- vinylpyrrolidone), polyaminoacids and poly N-(2- hydroxypropyl)methacrylamide. Stealth lipids may comprise a lipid moiety. In some embodiments, the lipid moiety of the stealth lipid may be derived from diacyl glycerol or diacylglycamide, including those comprising a dialkylglycerol or dialkylglycamide group having alkyl chain length independently comprising from about C4 to about C40 saturated or unsaturated carbon atoms, wherein the chain may comprise one or more functional groups such as, for example, an amide or ester. The dialkylglycerol or dialkylglycamide group can further comprise one or more substituted alkyl groups. In some embodiments, stealth lipids may comprise a-Methoxy-co-(3- oxopropoxy), polyoxyethylene (Methoxy PEG, Aldehyde), PEG2k-DMG, PEG2k-DSG, PEG2k- DSPE, PEG2K-DOPE, PEG5k-DOPE, Methoxy PEG aldehyde 20k, PEG2K-Cholesterol, and the like.

[0076] The term "‘lipid nanoparticle (LNP)” as used herein refers to different types of compositions of nano-scale particles, wherein the particles comprising lipids function as carriers across cell membranes and biological barriers and deliver compounds to targeted cells and tissues of humans and other organisms. As used herein, “lipid nanoparticles” of the instant disclosure may further comprise additional lipids and other components. Other lipids may be included for a variety of purposes, such as to prevent lipid oxidation or to attach ligands onto the lipid nanoparticle surface. Any of a number of lipids may be present in lipid nanoparticles of the present disclosure, including amphipathic, neutral, cationic, and anionic lipids. Such lipids can be used alone or in combination, and can also include bilayer stabilizing components such as polyamide oligomers (see, e.g., U.S. Pat. No. 6,320,017), peptides, proteins, detergents, lipid-derivatives, such as PEG coupled to phosphatidylethanolamine and PEG conjugated to ceramides (see, e.g., U.S. Pat. No. 5,885,613).

[0077] As used herein, a “PEG” conjugated lipid that inhibits aggregation of particles refers to one or more of a polyethyleneglycol (PEG)-lipid conjugate, a polyamide (ATTA)-lipid conjugate, and a mixture thereof. In one aspect, the PEG-lipid conjugate is one or more of a PEG- dialkyloxypropyl (DAA), a PEG-diacyl glycerol (DAG), a PEG-phospholipid, a PEG-ceramide, and a mixture thereof. In one aspect, the PEG-DAG conjugate is one or more of a PEG- dilauroylglycerol (C12), a PEG-dimyristoylglycerol (CM), a PEG-dipalmitoylglycerol (CM), and a PEG-distearoylglycerol (C is). In one aspect, the PEG-DAA conjugate is one or more of a PEG- di lauryloxypropyl (Cj 2), a PEG-dimyristyl oxypropyl (Cta), a PEG-dipalmityloxypropyl (Ci 6), and a PEG-di stearyloxypropyl (Cis). In some embodiments, PEG is 2-dimyristoyl-rac-glycero-3- methoxypolyethylene glycol-2000 (PEG-DMG), l,2-dipalmitoyl-rac-glycero-3- methylpolyoxyethylene glycol-2000 (PEG-DPG), and / or 1,2-di stearoyl -rac-glycero-3- methoxypolyethylene glycol-2000 (PEG-DSG).

[0078] The term “N / P ratio” as used herein refers to the (N)itrogen-to-(P)hosphate molar ratio between the cationic amino lipid and negatively charged phosphate groups of the nucleic acid.

[0079] The “polydispersity index” or “PDF’ as used herein is a measure of the heterogeneity of a sample based on size. Polydispersity can occur due to size distribution in a sample or agglomeration or aggregation of the sample during isolation or analysis.

[0080] The “zeta potential” or “surface charge” as used herein refers to the degree of electrostatic repulsion between adjacent, similarly charged particles in a dispersion. For molecules and particles that are small enough, a high zeta potential will confer stability, i.e., the solution or dispersion will resist aggregation.

[0081] As used herein, the term nucleic acid “cargo” refers to the intended nucleic acid for delivery to the cell or tissue (in embodiments, a therapeutic nucleic acid for delivery to the cell or tissue).

[0082] As used herein, the term “nucleic acid-lipid nanoparticle” refers to lipid nanoparticles as described above that associate with or encapsulate one or more nucleic acids to deliver one or more nucleic acid cargoes to a tissue.

[0083] As used herein, “encapsulated” can refer to a nucleic acid-lipid nanoparticle formulation that provides a nucleic acid with full encapsulation, partial encapsulation, association by ionic or van der Waals forces, or all of the aforementioned. In one embodiment, the nucleic acid is fully encapsulated in the nucleic acid-lipid nanoparticle.

[0084] As used herein, “nucleic acid” refers to a synthetic or naturally occurring RNA or DNA, or derivatives thereof. In one embodiment, a cargo and / or agent of the instant disclosure is a nucleic acid, such as a double-stranded RNA (dsRNA). In one embodiment, the nucleic acid or nucleic acid cargo is a single-stranded DNA or RNA, or double-stranded DNA or RNA, or DNA-RNA hybrid. For example, a double-stranded DNA can be a structural gene, a gene including control and termination regions, or a self-replicating system such as a viral or plasmid DNA. A doublestranded RNA can be, e.g., a dsRNA or another RNA interference reagent. A single-stranded nucleic acid can be, e.g., an mRNA, an antisense oligonucleotide, ribozyme, a microRNA, or triplex-forming oligonucleotide. In certain embodiments, the nucleic acid or nucleic acid cargo may comprise a modified RNA, wherein the modified RNA is one or more of a modified mRNA, a modified antisense oligonucleotide and a modified siRNA, In some embodiments, a nucleic acid cargo of the instant disclosure includes or is a modified mRNA that encodes a nucleic acid modulating controller.

[0085] As used herein, the term “modified nucleic acid” refers to any non-natural nucleic acid, including but not limited to those selected from the group comprising 2'-O-methyl modified nucleotides, a nucleotide comprising a 5'-phosphorothioate group, a terminal nucleotide linked to a cholesteryl derivative, a 2'-deoxy-2'-fluoro modified nucleotide, a 5'-methoxy-modified nucleotide (e.g., 5'-methoxyuridine), a 2'-deoxy-modified nucleotide, a locked nucleotide, an abasic nucleotide, a 2'-amino-modified nucleotide, a 2'-alkyl-modified nucleotide, a morpholino nucleotide, a phosphoramidate, a non-natural base comprising nucleotide; intemucleoside linkages or backbones including phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates including 3'- alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates including 3'- amino phosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, and boranophosphates having nonnal 3'-5' linkages, 2'-5' linked analogs of these, and those having inverted polarity wherein the adjacent pairs of nucleoside units are linked 3 '-5' to 5'-3' or 2'-5 ' to 5'-2'.

[0086] As used herein, the term “nucleic acid modulating controller” refers to a mRNA that encodes for protein controller components, though reference to “nucleic acid modulating controller” can also refer to the mRNA-expressed protein controller components themselves. In certain embodiments, the mRNA-encoded protein controller components include Zinc-Finger proteins (ZFPs) or other forms of DNA or RNA binding domains (DBDs or RBDs) that are associated with (and optionally tethered to) one or more epigenetic regulators or nucleases (the epigenetic regulators or nucleases are generally referred to as effectors, effector domains, or effector moieties). Without wishing to be bound by theory, an advantage of a nucleic acid modulating controller as described herein is that it provides durable gene programming only at the confluence of (I) where the nucleic acid modulating controller-encoding mRNA is expressed, (2) where nucleic acid binding of the ZFP or other nucleic acid binding domain occurs and (3) where the associated effector domain is able to exert activity (i.e. where the effector domain is capable of changing the epigenomic state (e.g., in the instance of an epigenomic controller)).

[0087] As used herein, the term “effector moiety” or “effector domain” refers to a domain that is capable of altering the expression of a target gene when localized to an appropriate site in a cell, e.g., in the nucleus of a cell. In some embodiments, an effector moiety recruits components of the transcription machinery. In some embodiments, an effector moiety inhibits recruitment of components of transcription factors or expression repressing factors. In some embodiments, an effector moiety comprises an epigenetic modifying moiety (e.g., epigenetically modifies a target DNA sequence). Specific examples of effector moieties include, without limitation, effectors capable of binding Krueppel-associated box (KRAB) domains (KRAB is a domain of around 75 amino acids that is found in the N-terminal part of about one third of eukaryotic Krueppel-type C2H2 zinc finger proteins (ZFPs)) and the engineered prokaryotic DNA methyltransferase MQ1, among others.

[0088] As used herein, “epigenetic modifying moiety” refers to a domain that alters: i) the structure, e.g., two-dimensional structure, of chromatin; and / or ii) an epigenetic marker (e.g., one or more of DNA methylation, histone methylation, histone acetylation, histone sumoylation, histone phosphorylation, and RNA-associated silencing), when the epigenetic modifying moiety is appropriately localized to a nucleic acid (e.g., by a targeting moiety). In some embodiments, an epigenetic modifying moiety comprises an enzyme, or a functional fragment or variant thereof, that affects (e.g., increases or decreases the level of) one or more epigenetic markers. In some embodiments, an epigenetic modifying moiety comprises a DNA methyltransferase, a histone methyltransferase, CREB-binding protein (CBP), or a functional fragment of any thereof.

[0089] As used herein, the term “expression control sequence” refers to a nucleic acid sequence that increases or decreases transcription of a gene, and includes (but is not limited to) a promoter and an enhancer. An “enhancing sequence” refers to a subtype of expression control sequence and increases the likelihood of gene transcription. A “silencing or repressor sequence” refers to a subtype of expression control sequence and decreases the likelihood of gene transcription.

[0090] As used herein, the term “expression repressor” refers to an agent or entity with one or more functionalities that decreases expression of a target gene in a cell and that specifically binds to a DNA sequence (e.g., a DNA sequence associated with a target gene or a transcription control element operably linked to a target gene). In certain embodiments, an expression repressor comprises at least one targeting moiety and optionally one effector moiety.

[0091] As used herein, the term “targeting moiety” means an agent or entity that specifically targets, e.g., binds, a genomic sequence element (e.g., an expression control sequence or anchor sequence; promoter, enhancer or CTCF site). In some embodiments, the genomic sequence element is proximal to and / or operably linked to a target gene (e.g., MYC).

[0092] As used herein, the term "skin" or “skin tissue” describes the outer covering of a mammalian form including, without limitation, the epidermis, dermis, and subcutaneous tissues of a mammal. Skin tissue as used herein can also include other components such as, without limitation, hair follicles and sweat glands. Exemplary epidermal cell types include, without limitation, keratinocytes (optionally stratum comeum cells, stratum granulosum cells, stratum spinulosum cells, and / or stratum basale cells), melanocytes, Langerhans cells, and / or a Merkel cells. Exemplary dermal tissue cell types include, without limitation, mast cells, vascular smooth muscle cells, specialized muscle cells, fibroblasts, hair follicle cells (e.g., dermal papilla cells and / or dermal sheath cells), immune cells, and / or leukocytes (e.g., neutrophils, T or B lymphocytes, eosinophils and or monocytes). Exemplary subcutaneous tissue cell types include, without limitation, fat cells, nerve cells, and / or vascular cells. "Skin tissue" may refer to any cell or population of cells within the organ of the skin including but not limited to, vascular cells, endothelial cells, parenchymal cells, non-parenchymal cells, fibroblasts, mesenchymal cells, immune cells, cancer cells, and skin-derived stem / progenitor cells. In certain embodiments, a lipid particle of the instant disclosure targets skin tissue. In some other embodiments, a lipid particle of the instant disclosure may target other cell types or tissues including but not limited to cell types accessible via mucus membrane, vascular tissue cells, brain cells, nerve cells, eye cells, pharynx cells, and / or larynx cells, among other cell types or tissues.

[0093] As used herein, “localization” refers to the position of a lipid, peptide, or other component of a lipid particle of the instant disclosure, within an organism and / or tissue. In some embodiments, localization can be detectible in individual cells. In some embodiments a label can be used for detecting localization, e.g., a fluorescent label, optionally a fluorescently labeled lipid, optionally Cy7. In some embodiments, the label of the lipid nanoparticle may be a quantum dot, or the lipid detectible by stimulated Raman scattering. In other embodiments, the label is any fluorophore known in the art, i.e., with excitation and emission in the ultraviolet, visible, or infrared spectra. In some embodiments the localization is detected or further corroborated by immunohistochemistry or immunofluorescence.

[0094] As used herein, the term “activity” refers to any detectable effect that is mediated by a component or composition of the instant disclosure. In embodiments, “activity” as used herein, can refer to a measurable (whether directly or by proxy) effect, e.g., of a cargo of the instant lipid particles of the disclosure. Examples of activity include, without limitation, the intracellular expression and resulting effect(s) of a nucleic acid cargo (e.g., a mRNA, a CRISPR / Cas system, a RNAi agent, a nucleic acid modulating controller, etc.), which can optionally be measured at a cellular, tissue, organ and / or organismal level.

[0095] As used herein, “multidosing” refers to two or more doses of a lipid nanoparticle formulation given as part of a therapeutic regimen to a subject.

[0096] As used herein, the term “skin disease or disorder” may include, without limitation, a disease or disorder such as alopecia, including alopecia areata, androgenic alopecia, anagen effluvium, telogen effluvium, or scarring alopecia. In certain embodiments, the "skin disease or disorder" is a dermatologic condition, including those that might predispose a subject to a greater risk of developing skin cancer, such as actinic keratosi s, xeroderma pigmentosum, or albinism, for example. In some embodiments, the term "skin disease or disorder" includes a wide variety of other dennatologic conditions, including but not limited to bruising or senile purpura, bums, age spots, sun spots, scars including keloids, eye bags, xerosis, ichthyosis, keratoderma, dermatofibroma, dermatitis, acne, neurodermatitis, dermatitis herpetiformis, vitiligo, vasculitis, pemphigus, bullous pemphigoid, hyperkeratosis, eczema, psoriasis, rosacea, pityriasis rosea, warts; bacterial, viral, fungal, or other infections can also be potentially treated using the lipid particles, formulations, pharmaceutical compositions, and methods as disclosed herein. In some embodiments, the dermatologic condition to be treated could be a manifestation of a systemic disease, such as an autoimmune disease such as systemic lupus erythematosus (SI.. E), scleroderma, or rheumatoid arthritis, for example. The condition could also be an epidermal disease or disorder, such as - without limitation - psoriasis, atopic dermatitis, scleroderma, eczema, rosacea, seborrheic dermatitis, melanoma, solar keratosis, ichthyosis, Grover's disease, common warts, keratoacanthoma andfor seborrhoeic keratosis.

[0097] As used herein, the term "subject" includes humans and mammals (e.g., mice, rats, pigs, cats, dogs, and horses). In many embodiments, subjects are mammals, particularly primates, especially humans. In some embodiments, subjects are livestock such as cattle, sheep, goats, cows, swine, and the like; poultry such as chickens, ducks, geese, turkeys, and the like; and domesticated animals particularly pets such as dogs and cats. In some embodiments (e.g., particularly in research contexts) subject mammals will be, for example, rodents (e.g., mice, rats, hamsters), rabbits, primates, or swine such as inbred pigs and the like.

[0098] As used herein, “administration” to a subject may include topical administration, parenteral administration, optionally for intravenous injection, inhalation, intravenous, intra-arterial, intratracheal, or involve direct injection into a tissue.

[0099] The term "treating" includes the administration of compositions to prevent or delay the onset of the symptoms, complications, or biochemical indicia of a disease (e.g., cancer, including, e.g., tumor formation, growth and / or metastasis), alleviating the symptoms or arresting or inhibiting further development of the disease, condition, or disorder. Treatment may be prophylactic (to prevent or delay the onset of the disease, or to prevent the manifestation of clinical or subclinical symptoms thereof) or therapeutic suppression or alleviation of symptoms after the manifestation of the disease.

[0100] As used herein, a “pharmaceutical composition” comprises a pharmacologically effective amount of a lipid particle, optionally a nucleic-acid lipid nanoparticle (NLNP) and a pharmaceutically acceptable earner. As used herein, “pharmacologically effective amount,” “therapeutically effective amount” or simply “effective amount” refers to that amount of nucleic acid effective to produce the intended pharmacological, therapeutic or preventive result. For example, if a given clinical treatment is considered effective when there is at least a 25% reduction in a measurable parameter associated with a disease or disorder, a therapeutically effective amount of a drug for the treatment of that disease or disorder is the amount necessary to induce at least a 25% reduction in that parameter.

[0101] The term “pharmaceutically acceptable carrier” refers to a carrier for administration of a therapeutic agent. Such carriers include, but are not limited to, saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof.

[0102] Unless specifically stated or obvious from context, as used herein, the term "or" is understood to be inclusive. Unless specifically stated or obvious from context, as used herein, the terms "a", "an", and "the" are understood to be singular or plural. Ranges can be expressed herein as from “about” one particular value, and / or to “about” another particular value. When such a range is expressed, another aspect includes from the one particular value and / or to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent “about,” it is understood that the particular value forms another aspect. It is further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint. It is also understood that there are a number of values disclosed herein, and that each value is also herein disclosed as “about” that particular value in addition to the value itself. It is also understood that throughout the application, data are provided in a number of different formats and that this data represent endpoints and starting points and ranges for any combination of the data points. For example, if a particular data point “10” and a particular data point “15” are disclosed, it is understood that greater than, greater than or equal to, less than, less than or equal to, and equal to 10 and 15 are considered disclosed as well as between 10 and 15. It is also understood that each unit between two particular units are also disclosed. For example, if 10 and 15 are disclosed, then 11, 12, 13, and 14 are also disclosed.

[0103] Ranges provided herein are understood to be shorthand for all of the values within the range. For example, a range of 1 to 50 is understood to include any number, combination of numbers, or sub-range from the group consisting 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 as well as all intervening decimal values between the aforementioned integers such as, for example, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, and 1.9. With respect to sub-ranges, “nested sub-ranges” that extend from either end point of the range are specifically contemplated. For example, a nested sub-range of an exemplary range of 1 to 50 may comprise 1 to 10, 1 to 20, 1 to 30, and 1 to 40 in one direction, or 50 to 40, 50 to 30, 50 to 20, and 50 to 10 in the other direction.

[0104] The transitional term “comprising,” which is synonymous with “including,” is inclusive or open-ended and does not exclude additional, unrecited elements or method steps. By contrast, the transitional phrase “consisting of’ excludes any element, step, or ingredient not specified in the claim. The transitional phrase “consisting essentially of’ limits the scope of a claim to the specified materials or steps “and those that do not materially affect the basic and novel characteristic(s)” of the claimed invention. The embodiments set forth below and recited in the claims can be understood in view of the above definitions.

[0105] Other features and advantages of the disclosure will be apparent from the following description of the preferred embodiments thereof, and from the claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, suitable methods and materials are described below. All published foreign patents and patent applications cited herein are incorporated herein by reference. All other published references, documents, manuscripts and scientific literature cited herein are incorporated herein by reference. In the case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

[0106] BRIEF DESCRIPTION OF THE DRAWINGS

[0107] The following detailed description, given by way of example, but not intended to limit the disclosure solely to the specific embodiments described, may best be understood in conjunction with the accompanying drawings, in which:

[0108] FIG. 1 shows cargo delivery results for seven LNP types disclosed herein that transfected successfully into dermal papilla cells of swine hair follicles. The dermal papilla cell regions are marked in red, and the treatment effect was demonstrated by staining firefly luciferase (FLuc) reporter protein in brown.

[0109] FIG. 2 shows cargo delivery results for five LNP types disclosed herein that were not observed to transfect into dermal papilla cells of swine hair follicles, at least under the currently tested conditions. Dermal papilla cell regions are marked in red, and the treatment effect (or here, absence thereof) was demonstrated by staining FLuc reporter protein in brown.

[0110] DETAILED DESCRIPTION OF THE INVENTION

[0111] The instant disclosure provides, at least in part, lipid particle compositions, formulations and associated methods, for delivery of lipid particle-associated molecular cargoes to the cells of a subject. In certain aspects, nucleic acid-lipid nanoparticles are provided that deliver associated nucleic acid cargoes to the skin of a subject, with delivery occurring to various types of tissue within the skin of a subject. In particular, the particles of the instant disclosure include cationic lipids (e.g„ obligate cationic lipids or ionizable lipids), as well as a non-cationic “helper” lipid, a structural lipid (e.g., cholesterol, ^-sitosterol or derivatives thereof) and / or a stabilizer / anti- aggregation lipid (e.g., PEG-lipid).

[0112] Nucleic acid therapy has well-known, tremendous potential to treat diseases at the gene level. However, safe and effective delivery systems are essential for nucleic acid therapeutics. To be effective, a nucleic acid cargo delivery system needs to facilitate a nucleic acid cargo reaching the intracellular space of a target cell (including, e.g., cytosol and / or nuclear compartments of the target cell type).

[0113] LNPs used for the delivery of nucleic acids to cells have typically been composed of four main components. An ionizable lipid or an obligate cationic lipid for mRNA encapsulation, amphipathic helper phospholipids for increased efficacy, cholesterol for structural stability and polyethylene glycol (PEG)-lipids for steric stability. Conventionally, effective intracellular delivery materials have relied on an optimal balance of ionizable amines to bind and release RNAs (pKa between 6.0 and 6.5) and nanoparticle-stabilizing hydrophobicity. Thus, there has been a significant focus on developing ionizable lipids, which have been proven to be highly effective delivery platforms for liver and hepatocytes. However, changing the chemical structure of the ionizable lipid to achieve different pKa values and generating libraries, although validated, is a time consuming, investment heavy and labor-intensive exercise.

[0114] Accessing the intracellular environment of skin cells in particular has been a challenge, as the skin's exposure to the environment has likely applied selective pressure against skin tissue cells being readily penetrated by exogenous nucleic acid agents. There is therefore a well-recognized need for systems and formulations that are capable of delivering payloads (including nucleic acid cargoes, as well as other cargoes) to the intracellular environment of skin cells, as well as to other cell types.

[0115] Certain aspects of the instant disclosure are based, at least in part, upon the surprising discovery that nucleic acid-lipid particles formulated with specific types of obligate cationic lipid or ionizable lipid, but not with other types of obligate cationic lipid or ionizable lipid, could robustly deliver a nucleic acid cargo (mRNA as exemplified herein, though a wide range of cargoes are contemplated) to the cytosol of hair follicle cells of the skin, particularly to dermal papilla cells of the skin, when administered topically. The exemplified nucleic acid-lipid particles shown to deliver cargo to dermal papilla cells effectively were specifically formulated to include: the phospholipid 1,2-distearoyl-sn-glycero-phosphocholine (DSPC) at about 10 mol % of the total lipid in the nucleic acid-lipid particle; the non-cationic lipid cholesterol at about 43.5 mol % of the total lipid in the nucleic acid-lipid particle; and the PEGylated lipid 1 ,2-dimyristoyl-rac-glycero- 3 -methoxypolyethylene glycol-2000 (DMG-PEG2k) at about 1.5 mol % of the total lipid in the nucleic acid-lipid particle, together with any one of the following obligate cationic lipids or ionizable lipids present at about 45 mol % of the total lipid in the nucleic acid-lipid particle: obligate cationic lipids ethyl phosphatidylcholine (EPC), 3B-[N-(N',N'-dimethylaminoethane)- carbamoyl]choiesterol (DOTMA), or Didodecyldimethylammonium bromide (DDAB), or ionizable lipids (((((disulfanediylbis(ethane-2,l-diyl))bis(piperidine-l,4-diyl))bis(ethane-2,l- diyl))bis(oxy))bis(2-oxoethane-2,l-diyl))bis(4,l -phenylene) dioleate (SS-OP), 3B-[N-(N’,N'- dimethylaminoethane)-carbamoyl]cholesterol (DC-Cholesterol), l,2-dioleyloxy-N,N-dimethyl-3- aminopropane (DODMA), or N,N-dimethyl-D-erythro-sphingosine (Dimethyl Sphingosine).

[0116] The instant disclosure has therefore specifically identified a panel of obligate cationic lipids or ionizable lipids individually capable of facilitating delivery of nucleic acid cargoes to skin tissue (including sub-populations of skin tissue cells, such as hair follicle cells, e.g., dermal papilla cells) when included in lipid particle formulations. The instant disclosure provides, without limitation, features and benefits of skin-specific delivery of nucleic acid cargoes / therapeutics via use of obligate cationic lipid particles or ionizable lipid sparticles as described herein.

[0117] Although the lipid particles of the instant disclosure have been identified to deliver cargoes to the cytosol of skin tissue cells, in some aspects, delivery to other regions and / or organs, such as joint and / or inflammation sites, as well as mucus membranes and / or oral administration, with the lipid particles of the instant disclosure or variations thereof, is also contemplated. In addition, administration of the particles of the instant disclosure via intravenous injection, inhalation, or non-intravenous injection is also expressly contemplated. Without limitation, a nucleic acid-lipid particle, pharmaceutical composition, topical solution or lotion, or inj ectate of the instant disclosure can be administered to a subject via a non-intravenous route, e.g., by topical application, intratracheal injection, intra-articular injection, subcutaneous injection, intradermal injection and / or intramuscular injection. Various expressly contemplated components of certain compositions and methods of the instant disclosure are considered in additional detail below.

[0118] Obligate Cationic Lipid- or Ionizable Lipid-Based Lipid Nanoparticle Compositions

[0119] In certain embodiments, the LNPs of the instant disclosure feature a series of distinct obligate cationic lipids or ionizable lipids, with exemplified LNPs having one of the following twelve obligate cationic lipids or ionizable lipids present at about 45 mol % of the total lipid in the LNP, each independently formulated with 1,2-distearoyl-sn-glycero-phosphocholine (DSPC) at about 10 mol % of the total lipid in the LNP; cholesterol at about 43.5 mol % of the total lipid in the LNP, and l,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG2k) at about 1.5 mol % of the total lipid in the LNP.

[0120] Obligate Cationic Lipids

[0121] EPC

[0122] A range of forms of the obligate cationic lipid EPC are commercially available. The instant Examples have employed ethyl phosphatidylcholine, 18: 1 EPC (Cl Salt), also known as 1,2- dioleoyl-sn-glycero-3-ethylphosphocholine (chloride salt), which has the following structure:

[0123] 18:0 EPC (Cl Salt), also known as l^-distearoyl-sn-gjycero-S-ethsdphosphocholine (chloride salt), has the followina structure:

[0124] 16:0 EPC (Cl Salt), also known as i,2-dipalmitoyl-sn-glycero"3"ethy1phosphocholine (chloride salt), has the following structure:

[0125] 14:0 EPC (Cl Salt), also known as l,2-dimyristoyl-sn-glycero-3-ethylphosphocholine (chloride salt), has the following structure:

[0126] 12:0 EPC (Cl Salt), also known as l,2-dilauroyl-sn-glycero-3-ethylphosphocholine (chloride salt), has the following structure:

[0127] 14:1 EPC (Tf Salt), also known as l,2-dimyristoleoyl-sn-glycero-3-ethylphosphocholine (Tf salt), has the following structure:

[0128] 16:0-18:1 EPC (Cl Salt), also known as l-palmitoyl-2-oleoyl-sn-glycero-3-ethylphosphocholine (chloride salt), has the following structure:

[0129] 18:1 EPC (Cl Salt), also known as l,2-dioleoyl-sn-glycero-3 -ethylphosphocholine (chloride salt), has the following structure:

[0130] In certain embodiments, the instant disclosure contemplates using an EPC-related compound having the structure of compound (1): wherein Ri and R2 are each optionally (un)substituted alkyl or alkenyl groups comprising between 10 and 24 carbon atoms.

[0131] DOTMA

[0132] 'The obligate cationic lipid 3B-[N-(N‘,N'-dimethylaminoethane)-carbamoyl]cholesterol (DOTMA) is a tetra-methylated DOTA analogue that can be used as a non-viral vector for gene therapy. DOTMA induces a positive charge on liposomes and thus promotes efficient liposome* cell membrane interaction.

[0133] The structure of DOTMA (C42H84CINO2) is shown below: DDAB

[0134] The obligate cationic lipid didodecykiimethylammonium bromide (DDAB) is a doublechain cationic surfactant that is widely used for an efficient deliver)' system into mammalian cells. It has been previously used as a lipid for gene delivery, similar to DOTAB and DOTMA.

[0135] The structure of DDAB ([CH3(CH2)ii]2N(CH3)2(Br)) is shown below:

[0136] DOTAP

[0137] The obligate cationic lipid l,2-dioleoyl-3-trimethylammonium-propane (DOTAP) or 18:1

[0138] TAP possesses a quaternary amine. The structure of DOTAP (C42H8oN04+) is shown below:

[0139] DOBAQ

[0140] N"(4-carboxybenzyl)~N,N-dimethyl~2,3"bis(oleoyloxy)propaii" 1 -aminium (DOBAQ) as employed herein is is an obligate cationic lipid. It has been used in the art to form lipid nanoparticles that can deliver DNA or mRNA.

[0141] The structure of DOBAQ (C49H83NO6) is shown below:

[0142] Trimethyl Sphingosine

[0143] The obligate cationic lipid N,N,N-trimethyl-D-erythro-sphingosine (Trimethyl

[0144] Sphingosine) (C21H44NO2) has the following structure:

[0145] Ionizable Lipids

[0146] SS-OP

[0147] The ionizable lipid (((((disulfanediylbis(ethane-2,l-diyl))bis(piperidine-l ,4- diyl))bis(ethane-2, 1 -diyl))bis(oxy))bis(2-oxoethane-2, 1 -diy l))bis(4, 1 -phenylene) dioleate (SS- OP) is a component of Coatsome (NOF America Corp.). The structure of SS-OP is shown below:

[0148] In certain embodiments, the instant disclosure also contemplates use of SS-OC and / or SS-EC, which have the following structures:

[0149] SS-OC:

[0150] SS-EC:5: . Other disulfide lipid components of Coatsome have also been described, possessing the following genus structure: including SS-M (RCO- = myristoyl; -O-X- = SS-E (RCO- = a-D-

[0151] DC-Cholesterol

[0152] The ionizable lipid 3B-[N“(N',N'“dimethylaminoethane)-carbamoyl]cholesterol (DC- Cholesterol), has been characterized as an effective transfection agent and a liposome reagent. It is believed to impart positive surface charge to lipid particles, including microbubbles for ultrasound based transfection of clustered regularly interspaced short palindromic repeats (CRISPRs).

[0153] The structure of DC cholesterol (C32H56N2O2) is shown below:

[0154] DODMA l,2-dioleyloxy-N,N-dimethyT3-ammopropane (DODMA) is an ionizable lipid that is considered a non-viral earner for gene therapy. It has been used for the preparation of liposomes and lipid nanoparticles that need a neutral or low zeta potential. DODMA is effective at encapsulating nucleic acids due to its ability to temporarily reduce pH. The structure of DODMA (C41H81NO2) is shown below:

[0155] Dimethyl Sphingosine

[0156] The ionizable lipid N,N-dimethyl-D-erythro-sphingosine (Dimethyl Sphingosine) is an inhibitor of sphingosine kinase. It is a natural metabolite of sphingosine in some cancer cells and tissues. Dimethyl sphingosine induces apoptosis, but it is not an inhibitor of protein kinase C. Recently, it has been identified as an inducer of pain in a rat model of chronic pain, having properties similar to capsaicin. Dimethyl sphingosine has been used in the preparation of positively charged liposomes for DNA transfection.

[0157] The structure of dimethyl sphingosine (C20H41NO2) is shown below:

[0158] SM-102

[0159] The ionizable lipid heptadecan-9-yl 8-{(2-hydroxyethyl)[6-oxo-6- (undecyloxy)hexyl] amino (octanoate equivalent (SM-102) is a synthetic lipid used with other lipids to form lipid nanoparticles in drug delivery. It holds a neutral charge at physiological pH but is positively charged within the nanoparticle. SM-102-containing lipid nanoparticles have been used to deliver mRNA-based vaccines.

[0160] The structure of SM-102 (C44H87NO5) is shown below:

[0161] DODAP

[0162] The ionizable lipid l,2-Dioleoyl-3-dimethylammonium-propane (DODAP) possesses low cytotoxicity and high transfection efficiency. It is neutral at physiological pH, but acquires a positive charge inside the endosome due to the protonation of free amines when pH is lower than its pKa (<7). Without being bound by theory, the electrostatic interactions between DODAP and naturally occurring anionic lipids in endosomal membranes trigger the release of nucleic acid. These interactions promote membrane lytic non-bilayer structures to enable the intracellular delivery of nucleic acid.

[0163] The structure of DODAP (CnH / vNO.-;) is shown below:

[0164] In certain embodiments of the lipid particles of the instant disclosure, and in related methods of the instant disclosure, at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, or between about 10% and about 50% (molar basis), or between about 20% and about 70% (molar basis), or between about 30% and 50% (molar basis), or between about 35% and about 49% (molar basis), or between about 40% and about 48% (molar basis), or about 45%> (molar basis), of the total lipids present in a lipid nanoparticle of the disclosure are an obligate cationic lipid or an ionizable lipid (e.g., one of the twelve abovedescribed obligate cationic lipids or ionizable lipids). In some embodiments, the lipid particles of the instant disclosure include cholesterol, [3-sitosterol, and / or derivatives thereof at a level of at least about 0.1%, at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, between about 10% and about 75%, between about 20% and about 65%, between about 30% and about 55%, between about 40% and about 45%, and / or at about 43.5% (molar basis) of the total lipid present in the lipid particle. In certain embodiments, at least about 0.1%, at least about 3%, at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, at least about 10%, at least about 20%, at least about 40%, between about 3% and about 46%, between about 5% and about 30%, between about 6% and about 25%, between about 7% and about 20%, between about 8% and about 15%, between about 9% and about 11%, or about 10% (molar basis) of the total lipid in a lipid particle of the disclosure is a phospholipid, e.g., DSPC. In some embodiments of the lipid particles of the instant disclosure, and in related methods of the instant disclosure, the particle includes a conjugated lipid that inhibits aggregation of particles present at 0.01 rnol% to about 3 mol%, at about 0.5 mol% to about 2.5 niol%, at about 1.0 mol% to about 2.0 mol%, at about 1.3 mol% to about 1.7 mol%, at about 1.4 mol% to about 1.6 mol%, at about 1.5 mol % of the total lipid present. Examples of such conjugated lipids include, without limitation, polyethyleneglycol (PEG)-lipid conjugates, e.g., PEG2ooo-lipid conjugates, e.g., l,2-dimyristoyl-rac-glycero-3 -methoxypolyethylene glycol-2000 (DMG-PEG2k) and 1 ,2- distearoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DSG-PEG2k).

[0165] Lipid nanoparticles of any size may be used according to the instant disclosure, hi certain embodiments of the instant disclosure, lipid nanoparticles have a size ranging from about 0.02 microns to about 0.4 microns, between about 0.05 and about 0.2 microns, or between 0.07 and 0.12 microns in diameter. In some embodiments, the lipid-based compositions described herein typically have a mean diameter of from about 30 nm to about 250 nm, from about 40 nm to about 200 nm, from about 50 nm to about 150 nm, from about 60 nm to about 130 nm, from about 70 nm to about 110 nm, or from about 70 to about 90 nm. in some embodiments, the lipid-based compositions disclosed herein have a lipid:therapeutic agent (e.g., lipidmucleic acid) ratio (mass / mass ratio) of from about 1:1 to about 1000:1, from about 1:1 to about 500:1, from about 2:1 to about 250:1, from about 3:1 to about 200:1, from about 5:1 to about 150:1, from about 5:1 to about 100:1, from about 5 : 1 to about 50:1, from about 5 : 1 to about 25:1, from about 5 : 1 to about 20:1, from about 5:1 to about 10:1, or from about 6: 1 to about 9: 1. Alternatively, the lipid-based compositions disclosed herein have a lipid:therapeutic agent (e.g., lipidmucleic acid) ratio (mole / mole ratio) of from about 1 : 1 to about 30: 1, from about 2: 1 to about 20: 1 , from about 2: 1 to about 15: 1 , from about 3: 1 to about 10:1, from about 4:1 to about 9: 1 , from about 5:1 to about 8: l , or from about 6: 1 to about 8:1.

[0166] In some embodiments that employ PEG-conjugated lipids, the PEG-conjugated lipid is one or more of a polyethyleneglycol (PEG)-lipid conjugate, a polyamide (ATTA)-lipid conjugate, and a mixture thereof. In one aspect, the PEG-lipid conjugate is one or more of a PEG- dialkyloxypropyl (DAA), a PEG -di acylglycerol (DAG), a PEG-phospholipid, a PEG-ceramide, and a mixture thereof. In one aspect, the PEG-DAG conjugate is one or more of a PEG- dilauroylglycerol (C12), a PEG-dimyristoylglycerol (C14), a PEG-dipalmitoylglycerol (Ci6), and a PEG-di stearoyl glycerol (Cis). In one aspect, the PEG-DAA conjugate is one or more of a PEG- di lauryl oxypropyl (Cj 2), a PEG-dimyristyloxypropyl (CM), a PEG-dipalmityloxypropyl (Cj6), and a PEG-di stearyloxypropyl (Cis). In some embodiments, PEG is 2-dimyristoyl-rac-glycero-3- methoxypolyethylene glycol-2000 (PEG-DMG) and / or l ,2-distearoyl-rac-glycero-3- methoxypolyethylene glycol-2000 (PEG-DSG).

[0167] In some embodiments, amphipathic lipids are included in particles of the instant disclosure. Amphipathic lipids may refer to any suitable material, wherein the hydrophobic portion of the lipid material orients into a hydrophobic phase, while the hydrophilic portion orients toward the aqueous phase. Such compounds include, but are not limited to, phospholipids, aminolipids, and sphingolipids. Representative phospholipids include sphingomyelin, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoyloleoyl phosphatdylcholine, lysophosphatidylcholine, lysophosphatidylethanolamine, dipalmitoylphosphatidylcholine, dioleoylphosphatidylcholine, distearoylphosphatidylcholine, or dilinoleoylphosphatidylcholine. Other phosphorus-lacking compounds, such as sphingolipids, glycosphingolipid families, diacylglycerols, and 0-acyloxyacids, can also be used. Additionally, such amphipathic lipids can be readily mixed with other lipids, such as triglycerides and sterols.

[0168] Also suitable for inclusion in the lipid particles of the instant disclosure are programmable fusion lipid formulations. Such formulations have little tendency to fuse with cell membranes and deliver their cargo until a given signal event occurs. This allows the lipid formulation to distribute more evenly after injection into an organism or disease site before it starts fusing with cells. The signal event can be, for example, a change in pH, temperature, ionic environment, or time. In the latter case, a fusion delaying or “cloaking” component, such as an ATTA-lipid conjugate or a PEG- lipid conjugate, can simply exchange out of the lipid nanoparticle membrane over time. By the time the formulation is suitably distributed in the body, it has lost sufficient cloaking agent so as to be fusogenic. With other signal events, it is desirable to choose a signal that is associated with the disease site or target cell, such as increased temperature at a site of inflammation.

[0169] In certain embodiments, it can be desirable to target the lipid particles of this disclosure further, using targeting moieties that are specific to a cell type or tissue. Targeting of lipid nanoparticles using a variety of targeting moieties, such as ligands, cell surface receptors, glycoproteins, vitamins (e.g., riboflavin) and monoclonal antibodies, has been previously described (see, e.g., U.S. Pat. Nos. 4,957,773 and 4,603,044). The targeting moieties can comprise the entire protein or fragments thereof.

[0170] Targeting mechanisms generally require that the targeting agents be positioned on the surface of the lipid nanoparticle in such a manner that the target moiety is available for interaction with the target, for example, a cell surface receptor. A variety of different targeting agents and methods are known and available in the art, including those described, e.g., in Sapra, P. and Allen, T M, Prog. Lipid Res. 42(5):439-62 (2003); and Abra, R M et al., J. Lipid nanoparticle Res. 12:1- 3, (2002).

[0171] Standard methods for coupling target agents can be used. For example, phosphatidylethanolamine, which can be activated for attachment of target agents, or derivatized lipophilic compounds, such as lipid-derivatized bleomycin, can be used. Antibody-targeted lipid nanoparticles can be constructed using, for instance, lipid nanoparticles that incorporate protein A (see, Renneisen, et al., J. Bio. Chem., 265:16337-16342 (1990) and Leonetti, et al., Proc. Natl. Acad. Sci. (USA), 87:2448-2451 (1990). Other examples of antibody conjugation are disclosed in U.S. Pat. No. 6,027,726, the teachings of which are incorporated herein by reference. Examples of targeting moieties can also include other proteins, specific to cellular components, including antigens associated with neoplasms or tumors. Proteins used as targeting moieties can be attached to the lipid nanoparticles via covalent bonds (see, Heath, Covalent Attachment of Proteins to Lipid nanoparticles, 149 Methods in Enzymology 111-119 (Academic Press, Inc. 1987)). Other targeting methods include the biotin-avidin system.

[0172] A variety of methods for preparing lipid nanoparticles are known in the art, including e.g., those described in Szoka, et al., Ann. Rev. Biophys. Bioeng., 9A61 (1980); U.S. Pat. Nos. 4,186,183, 4,217,344, 4,235,871, 4,261,975, 4,485,054, 4,501,728, 4,774,085, 4,837,028, 4,946,787; PCT Publication No. WO 91 / 17424; Deamer and Bangham, Biochim. Biophys. Acta, 443:629-634 (1976); Fraley, et al., Proc. Natl. Acad. Sci. USA, 76:3348-3352 (1979); Hope, et al., Biochim. Biophys. Acta, 812:55-65 (1985); Mayer, et al., Biochim. Biophys. Acta, 858: 161- 168 (1986); Williams, et al., Proc. Natl. Acad. Sci., 85:242-246 (1988): Lipid nanoparticles, Marc J. Ostro, ed., Marcel Dekker, Inc., New York, 1983, Chapter 1; Hope, et al., Chem. Phys. Lip., 40:89 (1986); and Lipid nanoparticles: A Practical Approach, Torchilin, V. P. et aL, ed., Oxford University Press (2003), and references cited therein. Suitable methods include, but are not limited to, sonication, extrusion, high pressure / homogenization, microfluidization, detergent dialysis, calcium-induced fusion of small lipid nanoparticle vesicles, and ether-infusion methods, all of which are well known in the art.

[0173] In some embodiments of the instant disclosure, LNPs were prepared using a microfluidic mixing process. Briefly, lipid stocks of obligate cationic lipid (e.g., DDAB, DOBAQ, DOTAP, DOTMA, EPC, trimethyl sphingosine) or ionizable lipid (e.g., DODAP, SM-102, SS-OP, DODMA, dimethyl sphingosine, DC-Cholesterol), DSPC, CHE and PEG-DMG were prepared in ethanol at 20 mg / ml concentration (it is noted that lipid stocks of 20 mg / ml to 80 mg / ml can readily be used, e.g., for animal studies). Lipids were mixed together for the given compositions in ethanol with a final lipid concentration of 6.5-8.5 mg / mL for in vitro studies, or a final lipid concentration of 15-120 mg / mL was used for animal studies, with the mRNA cargo in the aqueous phase at a concentration of 0.25-2 mg / ml. The mixing of two phases and LNP preparation was performed using a 2:1 or 3:1 aqueous to organic volume ratio, and at an 8 or 12 ml / min flow rate in a microfluidic chip with staggered herringbone structure. Resulting LNPs were subjected to purification and buffer exchange by tangential flow filtration (TFF) against molecular biology grade water. Alternatively, resulting LNPs were subjected to dialysis against molecular biology grade water using a membrane with a MWCO range between 8-300 kDa. Characterization parameters of the formulations are summarized below in Table 1. Precise control of the characterization parameters enabled the preparation of obligate cationic lipid-based LNPs or ionizable lipid-based LNPs in the size range of 55-110 nm for eleven of the twelve distinctly exemplified obligate cationic lipid or ionizable lipid particles (DDAB lipid particles being the outlier at about 230 nm in diameter), surface charge (Zeta values) between 4 and -7 mV, and PDI below 0.22. For preparation of Firefly Luciferase (FLuc)-loaded lipid particles exemplified herein, FLuc mRNA at 0.25 mg / mL in running buffer pH 4.5 (Citrate or Malate) and ethanol stock solution of the lipids listed in Table 1 at 20-100 mg / mL were used to produce the lipid nanoparticle formulations. Briefly, the aqueous phase containing the drug substance (mRNA) was mixed with the ethanol phase containing the lipids using a microfluidic mixer at 2: 1 volume ratio (RNA:lipid) and 8 mL / min flow. The product was purified for 3h under dialysis (MW 100 KDa) against 3L of running buffer for removal of residual ethanol (final EtOH concentration lower than 0.2% v / v). Buffer exchange was performed overnight, by replacing the running buffer with 3L of Tris Acetate or HEPES buffer pH 7-7.5. The end-product was further concentrated to 0.5 mg of mRNA / mL by tangential flow filtration (MicroKros column, mPES, 300-750K) and sterile filtered through 0.22 pm Polyethersulfone (PES) membrane. Particle size, polydispersity index, and zeta potential were measured on a light scattering instrument.

[0174] Lipid particles prepared according to methods as disclosed herein and as known in the art can in certain embodiments be stored for substantial periods of time prior to drug loading and administration to a patient. For example, lipid nanoparticles can be dehydrated, stored, and subsequently rehydrated and loaded with one or more active agents, prior to administration. Lipid nanoparticles may also be dehydrated after being loaded with one or more active agents. Dehydration can be accomplished by a variety of methods available in the art, including the dehydration and lyophilization procedures described, e.g., in U.S. Pat. Nos. 4,880,635, 5,578,320, 5,837,279, 5,922,350, 4,857,319, 5,376,380, 5,817,334, 6,355,267, and 6,475,517. In one embodiment, lipid nanoparticles are dehydrated using standard freeze-drying apparatus, i.e., they are dehydrated under low pressure conditions. Also, the lipid nanoparticles can be frozen, e.g., in liquid nitrogen, prior to dehydration. Sugars can be added to the LNP environment, e.g., to the buffer containing the lipid nanoparticles, prior to dehydration, thereby promoting the integrity of the lipid nanoparticle during dehydration. See, e.g., U.S. Pat. No. 5,077,056 or 5,736,155.

[0175] Lipid nanoparticles may be sterilized by conventional methods at any point during their preparation, including, e.g., after sizing or after generating a pH gradient.

[0176] Cargo-Loaded Lipid Particle Compositions

[0177] In various embodiments, lipid particles of the instant disclosure may be used for many different applications, including the delivery of an active agent to a cell, tissue, organ or subject. For example, lipid nanoparticles of the instant disclosure may be used to deliver a therapeutic agent systemically via the bloodstream or to deliver a cosmetic agent to the skin. Accordingly, lipid nanoparticles of the instant disclosure and one or more active agents as cargo(es) are included in the instant disclosure.

[0178] Lipid Particle Cargoes

[0179] The instant disclosure describes lipid nanoparticles (i.e., a lipid nanoparticle comprising an obligate cationic lipid or an ionizable lipid, a phospholipid, a non-cationic lipid, and a conjugated lipid that inhibits aggregation of particles) in combination with an active agent as a cargo. Active agents, as used herein, include any molecule or compound capable of exerting a desired effect on a cell, tissue, organ, or subject. Such effects may be biological, physiological, or cosmetic, for example. Active agents may be any type of molecule or compound, including e.g., nucleic acids, such as single- or double-stranded polynucleotides, plasmids, antisense RNA, RNA interference agents, including, e.g., DNA-DNA hybrids, DNA-RNA hybrids, RNA-DNA hybrids, RNA-RNA hybrids, short interfering RNAs (siRNA), micro RNAs (mRNA) and short hairpin RNAs (shRNAs); peptides and polypeptides, including, e.g., antibodies, such as, e.g., polyclonal antibodies, monoclonal antibodies, antibody fragments; humanized antibodies, recombinant antibodies, recombinant human antibodies, and Primatized™ antibodies, cytokines, growth factors, apoptotic factors, differentiation-inducing factors, cell surface receptors and their ligands; hormones; and small molecules, including small organic molecules or compounds.

[0180] Nucleic acids associated with or encapsulated by LNPs may contain modifications including but not limited to those selected from the following group: 2'-O-methyl modified nucleotides, a nucleotide comprising a 5'-phosphorothioate group, a terminal nucleotide linked to a cholesteryl derivative, a 2 '-deoxy-2 '-fluoro modified nucleotide, a 5'-methoxy-modified nucleotide (e.g., 5 '-methoxyuridine), a 2'-deoxy-modified nucleotide, a locked nucleotide, an abasic nucleotide, a 2'-amino-modified nucleotide, a 2'-alkyl-modified nucleotide, a morpholino nucleotide, a phosphoramidate, a non-natural base comprising nucleotide; internucleoside linkages or backbones including phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates including 3'- alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates including 3'- amino phosphoramidate and aminoalky Iphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotri esters, and boranophosphates having normal 3'-5' linkages, 2'-5' linked analogs of these, and those having inverted polarity wherein the adjacent pairs of nucleoside units are linked 3'-5' to 5'-3' or 2'-5' to 5'-2.'

[0181] In certain embodiments, the active agent is a mRNA or a vector capable expressing a mRNA in a cell.

[0182] In embodiments, the active agent is a CRISPR / Cas system. Optionally, a LNP of the instant disclosure can be formulated to include, e.g., both a guide strand (gRNA) and a Cas enzyme as cargoes, thereby providing a self-contained delivery' vehicle capable of effecting and controlling CRIS PR-mediated targeting of a gene in a target cell.

[0183] In certain featured embodiments, the active agent is a nucleic acid modulating controller (e.g., a mRNA that encodes protein controller components, as described above).

[0184] In some embodiments, the active agent is a therapeutic agent, or a salt or derivative thereof. Therapeutic agent derivatives may be therapeutically active themselves or they may be prodrugs, which become active upon further modification. Thus, in one embodiment, a therapeutic agent derivative retains some or all of the therapeutic activity as compared to the unmodified agent, while in another embodiment, a therapeutic agent derivative lacks therapeutic activity.

[0185] In certain embodiments, the LNP formulations of the instant disclosure are contemplated to include a nucleic acid payload, optionally in combination with a further active agent, optionally a therapeutic agent. Lipid particles of the instant disclosure can be administered with a variety of non-nucleic acid agents, optionally alone within lipid particles, or in combination with nucleic acid-lipid particles of the instant disclosure. Exemplary non-nucleic acid agents for topical delivery include, without limitation, topical anesthetic compounds, topical antibiotic agents, topical corticosteroids, decongestants, steroids, chemotherapeutics, unguents and / or salves for wound, bum, rash, abrasion or other topical injury treatment, among other active agents. Further examples of active agents for topical delivery with the lipid particles and / or nucleic acid-lipid particles of the instant disclosure include, e.g., anti-inflammatory compounds, narcotics, depressants, antidepressants, stimulants, hallucinogens, analgesics, antibiotics, birth control medication, antipyretics, vasodilators, anti-angiogenics, cytovascular agents, signal transduction inhibitors, vasoconstrictors, hormones, and anti-itch agents, etc.

[0186] In certain embodiments, the active agent is an oncology drug, which may also be referred to as an anti-tumor drug, an anti-cancer drug, a tumor drug, an antineoplastic agent, or the like. Examples of oncology drugs that may be used according to the instant disclosure include, but are not limited to, dacarbazine (DTIC), temozolomide, nab-paclitaxel, paclitaxel, cisplatin, carboplatin, adriamycin, alkeran, allopurinol, altretamine, amifostine, anastrozole, araC, arsenic trioxide, azathioprine, bexarotene, biCNU, bleomycin, busulfan intravenous, busulfan oral, capecitabine (Xeloda), carmustine, CCNU, celecoxib, chlorambucil, cladribine, cyclosporin A, cytarabine, cytosine arabinoside, daunorubicin, cytoxan, daunorubicin, dexamethasone, dexrazoxane, dodetaxel, doxorubicin, doxorubicin, epinibicin, estramustine, etoposide phosphate, etoposide and VP- 16, exemestane, FK506, fludarabine, fluorouracil, 5-FU, gemcitabine (Gemzar), gemtuzumab-ozogamicin, goserelin acetate, hydrea, hydroxyurea, idarubicin, ifosfamide, imatinib mesylate, interferon, irinotecan (Camptostar, CPT-111), letrozole, leucovorin, leustatin, leuprolide, levamisole, litretinoin, megastrol, melphalan, L-PAM, mesna, methotrexate, methoxsalen, mithramycin, mitomycin, mitoxantrone, nitrogen mustard, pamidronate, Pegademase, pentostatin, porfimer sodium, prednisone, rituxan, streptozocin, STI-571, tamoxifen, taxotere, temozolamide, teniposide, VM-26, topotecan (Hycamtin), toremifene, tretinoin, ATRA, valrubicin, velban, vinblastine, vincristine, VP 16, and vinorelbine. Other examples of oncology drugs that may be used according to the instant disclosure are ellipticin and ellipticin analogs or derivatives, epothilones, intracellular kinase inhibitors and camptothecins.

[0187] While LNP compositions of the instant disclosure generally comprise a single active agent, in certain embodiments, they may comprise more than one active agent.

[0188] The instant disclosure also provides lipid nanoparticles and variations thereof in kit form. The kit may comprise a ready-made formulation or a formulation that requires mixing before administration. The kit will typically comprise a container that is compartmentalized for holding the various elements of the kit. The kit will contain the nucleic acid-lipid particle s of the instant disclosure or the components thereof, in hydrated or dehydrated form, with instructions for their rehydration and administration. In particular embodiments, a kit comprises at least one compartment containing a lipid nanoparticle of the instant disclosure that is loaded with an active agent. In another embodiment, a kit comprises at least two compartments, one containing a lipid nanoparticle of the instant disclosure and the other containing an active agent. Of course, it is understood that any of these kits may comprise additional compartments, e.g., a compartment comprising a buffer, such as those described in U.S. Patent Publication No. 2004-0228909-Al. Kits of the instant disclosure, which comprise lipid nanoparticles comprising an obligate cationic lipid or an ionizable lipid disclosed herein (e.g., obligate cationic lipids such as DOTAP, DOBAQ, EPC, DOTMA, DDAB or trimethyl sphingosine, or ionizable lipids such as SS-OP, DC- Cholesterol, DODMA, Dimethyl Sphingosine, SM-102, or DDAB), may also contain other features of the kits described in U.S. Patent Publication No. 2004-0228909 Al. Further, the kit may contain drug-loaded lipid nanoparticles in one compartment and empty lipid nanoparticles in a second compartment. Alternatively, the kit may contain a lipid nanoparticle of the instant disclosure, an active agent to be loaded into the lipid nanoparticle of the instant disclosure in a second compartment, and an empty lipid nanoparticle in a third compartment.

[0189] In a particular embodiment, a kit of the instant disclosure includes a lipid nanoparticle having an obligate cationic lipid or an ionizable lipid as disclosed herein, as well as a phospholipid, a non-cationic lipid and a conjugated lipid that inhibits aggregation of particles, optionally where the obligate cationic lipid or the ionizable lipid constitutes about 35-50% (molar basis) of total lipids present in the lipid nanoparticle, and optionally where a therapeutic compound is encapsulated in the lipid nanoparticle. Efficacy of Lipid Particle-Mediated Cargo Delivery

[0190] In certain embodiments, the instant disclosure is based, at least in part, upon observation of effective delivery of nucleic acid cargo to hair follicle cells of the dermis using lipid nanoparticles having certain obligate cationic lipids or ionizable lipids. Such nucleic acid-lipid particles were identified as highly effective at delivering active nucleic acid cargoes into cells of the skin (e.g., hair follicle cells, e.g., dermal papilla cells). In some embodiments, the efficacy of localization of a lipid particle may be described as the fold difference (increase or decrease) in localization of the nucleic acid-lipid particle to a particular tissue and / or population of cells within a tissue of the subject relative to that of one or more other tissues and / or other populations of cells of the subject. The efficacy of activity as a further component in assessing delivery may be described as the fold difference (increase or decrease) in activity of the active agent, e.g., a nucleic acid cargo or other compound, within a particular type of cells of a particular tissue of the subject, relative to that observed in surrounding cells of a different type within the tissue(s) of the subject. For example, delivery of nucleic acid cargo to dermal papilla cells may be compared to surrounding, non-dermal papilla cells of the skin, when assessing the specificity of delivery efficacy of the nucleic acid-lipid particles of the instant disclosure. In some embodiments, the fold difference may therefore be detected at the cellular level, or can be detected by appropriate proxy for events occurring at the cellular level. In some embodiments, the cell of the tissue affected is a hair follicle cell and / or dermal papilla cell of the skin; however, delivery to other types of cells (including not only skin cells, but also cell types such as endothelial cells, fibroblasts, mesenchymal cells, immune cells, cancer cells, astrocytes, vascular endothelial cells, stem / progenitor cells, etc. can also be assessed for cargo delivery efficacy of the lipid particles of the instant disclosure. In some embodiments, the fold-difference in effect / activity may be detected at a sub-cellular level, i.e., where activity is detectible in the nuclei of targeted cells.

[0191] To determine the efficacy of localization of the LNP and / or the activity of the active agent encapsulated by the lipid particle, assays may be performed according to the characteristics of the labeled or detected molecule of interest and / or the characteristics of the active agent, hi embodiments of the instant disclosure, a fluorescently labeled lipid can also be used to determine LNP localization. In other embodiments, a labeled peptide, or other component of a lipid particle may be used. In some embodiments, the localization is detectible in individual cells. In some embodiments the label is a fluorescent label, i.e., a fluorescently labeled lipid such as Cy7. In other embodiments the label of the lipid nanoparticle may be a quantum dot, or the lipid detectible by stimulated Raman scattering. In other embodiments the label is any fluorophore known in the art, i.e. with excitation and emission in the ultraviolet, visible, or infrared spectra. In some embodiments the localization is detected or further corroborated by immunohistochemistry or immunofluorescence methods.

[0192] In certain embodiments, the active agent in the lipid particle is a nucleic acid. In other embodiments, the active agent in the lipid particle is a small molecule or other compound. In some embodiments, the active agent in the lipid particle is a mRNA. In illustrative embodiments, the localized expression of a reporter mRNA, i.e., firefly luciferase served as an indication of intracellular delivery efficacy for an mRNA as the active agent / cargo. In other embodiments, the mRNA may encode Cre enzyme, green fluorescent protein, red fluorescent protein, yellow fluorescent protein or blue fluorescent protein. Or in therapeutic embodiments, the mRNA may encode for a protein for therapeutic intracellular expression in LNP-targeted cells of a subject (including delivery and expression of nucleic acid modulatory controllers), optionally where intracellular levels of delivered mRNA or encoded protein can be detected by methods known in the art, as appropriate for the therapeutic mRNA that is delivered. In other embodiments, a reporter mRNA encodes a cell surface marker, such as a Lyt2 cell surface marker. In still other embodiments, the reporter can be a j3-galactosidase, a-lactamase, an alkaline phosphatase or a horseradish peroxidase. In other embodiments, the reporter mRNA encodes a negative selection marker, such as thymidine kinase (tk), HRPT or APRT. In some embodiments, immunohistochemistry or immunofluorescence is used to detect or corroborate activity of the reporter mRNA.

[0193] In certain embodiments, the effectiveness of a lipid particle of the instant disclosure in delivering a cargo is assessed based upon the levels of activity observed for the cargo (active agent) intracellularly within a lipid particle-targeted tissue. Such effects can be identified as folddifferences in activity, as compared to an appropriate control formulation and / or tissue, e.g., the delivery efficacy of a LNP with nucleic acid cargo may be described as the fold difference (increase or decrease) in activity of the nucleic acid cargo in cells of a targeted tissue, i.e., to skin tissue and / or one or more cell types within a tissue, of a subject relative to one or more other cell types and / or other tissues of the subject. Thus, for certain nucleic acid cargoes, delivery efficacy of an LNP formulation can be identified as a LNP that achieves, e.g., two-fold greater intracellular activity of the nucleic acid payload in targeted tissue cells than in non-targeted tissue cells, or relative to a LNP formulation that does not include the nucleic acid cargo. Optionally, an effective LNP formulation for delivery of a nucleic acid cargo can be described as one that achieves at least about a three-fold greater, optionally about a four- fold greater, optionally about a five-fold greater, optionally about a six-fold greater, optionally about a seven-fold greater, optionally about an eightfold greater, optionally about a nine-fold greater, optionally about a ten-fold greater, optionally about a twenty- fold greater, optionally about a 50-fold greater, optionally about a 100-fold greater, etc. intracellular activity of the nucleic acid payload in targeted tissue cells than in non-targeted tissue cells, or relative to a LNP formulation that does not include the nucleic acid cargo

[0194] In other embodiments, lipid particles can be employed to deliver a RNAi agent (e.g., a siRNA) to a tissue, i.e., a skin tissue. For siRNA or other RNAi agents, delivery and activity efficacy measurements can employ, for example, target-specific PCR to detect transcript levels, immunosorbent or other immunological methods to detect target protein levels, and / or Flow Cytometry (FACS) (Testoni et al., Blood 1996, 87:3822.). In some embodiments, a siRNA may be active in cells of the skin tissue and / or cell type within the skin tissue of the subject at a level that is at least two-fold higher than in surrounding / other cell types of the skin tissue, or of other non-targeted tissues, such as heart, spleen, liver, ovary, pancreas, kidney and / or other non-skin organ or tissue of the subject. In some embodiments, a siRNA may be active in cells and / or a cell type of the skin tissue of the subject at a level that is at least three-fold higher than in surrounding / different cell types of the skin or of other non-targeted tissues. In some embodiments, the siRNA may be active in cells and / or a cell type of the skin tissue of the subject at a level at least four-fold higher, in some embodiments, the siRNA may be active in cells and / or a cell type of the skin tissue of the subject at a level at least five-fold higher, in some embodiments the siRNA may be active in cells and / or a cell type of the skin tissue of the subject at a level at least six-fold higher, in some embodiments at least seven-fold higher, in some embodiments at least eight-fold higher, in some embodiments at least nine-fold higher, in some embodiments at least ten-fold higher, in some embodiments at least eleven-fold higher, in some embodiments at least twelvefold higher, in some embodiments at least thirteen-fold higher, in some embodiments at least fourteen-fold higher, in some embodiments at least fifteen-fold higher, in some embodiments at least twenty-fold higher, etc., than activity of the siRNA in surrounding / different cell types of the skin or of other non-targeted tissues of the subject. In related embodiments, a lipid particle that delivers a RNAi cargo preferentially to a cell type of the skin (e.g., dermal papilla cells) may exhibit, e.g., greater than 20% reduction in target transcript and / or protein levels in targeted cells of skin tissue, as compared to non-targeted cell types of skin tissue, or as compared to cells of nontargeted tissue(s), or as compared to some other appropriate control (e.g., levels of target transcript in untreated skin tissue cells). Optionally, a lipid particle that delivers a RNAi cargo preferentially to a cell type of the skin may exhibit, e.g., more than 30% reduction, more than 40% reduction, more than 50% reduction, more than 60% reduction, more than 70% reduction, more than 80% reduction, more than 90% reduction, more than 95% reduction, more than 97% reduction, more than 97% reduction, more than 98% reduction or more than 99% reduction in target transcript and / or protein levels in targeted cells of skin tissue, as compared to non-targeted cells of the skin tissue, or of non-targeted tissue, or as compared to some other appropriate control (e.g., levels of target transcript in untreated skin tissue cells).

[0195] In some embodiments, lipid particles of the instant disclosure can be used to deliver a CRISPR-Cas9 system to a tissue, i.e., to skin tissue. CRISPR-Cas9 delivery and activity efficacy measurements may require, for example, PCR to detect Cas9, the genomic structures of targeted regions and / or target transcript levels, immunosorbent or other immunological methods to detect Cas9 or knock-in, knock-out, or other modifications of target proteins, and / or Flow Cytometry (FACS) (Testoni et al., Blood 1996, 87:3822.). In some embodiments, CRISPR-Cas9-mediated effects may be identified in hair follicle cells (e.g., dermal papilla cells) of the skin tissue of the subject at a level that is at least two-fold higher than in surrounding non-hair follicle skin cells and / or other non-skin tissue cells of the subject. In some embodiments, CRISPR-Cas9-mediated effects may be identified in hair follicle cells (e.g., dermal papilla cells) of the skin tissue of the subject at a level that is at least three-fold higher than in surrounding non-hair follicle skin cells and / or other non-skin tissue cells of the subject. In some embodiments, CRISPR-Cas9-mediated effects may be identified in skin cells at a level at least four-fold higher in the hair follicle cells, in some embodiments, CRISP R-Cas9-mediated effects may be identified in skin cells at a level at least five-fold higher in the hair follicle cells, in some embodiments, CRISPR-Cas9-mediated effects may be identified in skin cells at a level at least six-fold higher, in some embodiments, CRISPR-Cas9-mediated effects may be identified in skin cells at least seven-fold higher, in some embodiments at least eight-fold higher, in some embodiments at least nine-fold higher, in some embodiments at least ten-fold higher, in some embodiments at least eleven-fold higher, in some embodiments at least twelve-fold higher, in some embodiments at least thirteen-fold higher, in some embodiments at least fourteen-fold higher, in some embodiments at least fifteen-fold higher, in some embodiments at least twenty-fold higher in hair follicle cells, than CRISPR-Cas9- mediated effects in surrounding non-hair follicle skin cells and / or other non-skin tissue cells of the subject.

[0196] In other embodiments, lipid particles of the disclosure may deliver a mRNA or other nucleic acid cargo to a tissue, i.e., a skin tissue (including a sub-population of skin cells, e.g., hair follicle cells, e.g., dermal papilla cells), where expression and possibly activity occurs in the nucleus. For example, Cre recombinase enzyme can be used as a reporter for nuclear activity of an active agent. The Cre recombinase enzyme requires translocation of the encoded protein to the nucleus and thus can serve as a reporter of nuclear translocation. The Cre recombinase catalyzes site-specific recombination of DNA between loxP sites. Upon Cre recombinase activity expression, due to loxP recombination, reporter fluorescent proteins are expressed. In one embodiment, a mouse line can use a Cre reporter loxP-flanked STOP cassette preventing transcription of a CAG promoter-driven red fluorescent protein variant (tdTomato), inserted into the Gt(ROSA)26Sor locus. Such mice can be contacted with mCre-loaded LNPs of the instant disclosure, with effective nuclear delivery of the mCre cargo signaled via observation of tdTomato fluorescence in the nuclei of skin cells, reflecting delivery and expression of the Cre enzyme, nuclear translocation of the Cre enzyme, and subsequently, Cre -mediated recombination of the tdTomato promoter.

[0197] In other embodiments, lipid particles of the instant disclosure may deliver small molecules or other compounds to a tissue, i.e., to skin tissue. The efficacy of localization or activity of small molecules may be determined by a number of in vivo imaging methods (e.g., PET / CT), mass spectrometry, as well as immunohistochemistry and immunofluorescence of target effects.

[0198] In certain embodiments, a lipid particle that is formulated for skin deliver}' refers to a lipid particle that exhibits preferential localization and intracellular delivery (based upon assessment of intracellular activity either directly or by proxy) of a cargo to dermal cells, as compared to one or more other cell types of skin, or to cells of one or more other tissues of a subject. For example, a lipid particle for skin delivery is one capable of inducing at least two-fold greater activity of a cargo (e.g., a nucleic acid cargo, e.g., a mRNA, a CRISPR / Cas system, a nucleic acid modulating controller, etc.) in skin cells of a subject, than in other tissues of the subject. Such effects in skin cells of a subject can be evaluated within one or more cell types of the skin, as described elsewhere herein. In certain embodiments, a lipid particle for skin delivery and / or for dermal papilla cell delivery is one capable of inducing at least three-fold greater, at least four-fold greater, at least five-fold greater, at least six-fold greater, at least seven-fold greater, at least eight-fold greater, at least nine-fold greater, at least ten-fold greater, at least fifteen-fold greater, at least twenty-fold greater, at least thirty-fold greater, at least 40-fold greater, at least 50-fold greater, at least 60-fold greater, at least 70-fold greater, at least 80-fold greater, at least 90-fold greater, at least 100-fold greater, at least 1000-fold greater, etc. activity of a cargo (e.g., a nucleic acid cargo, e.g., a mRNA, a CRISPR / Cas system, a nucleic acid modulating controller, etc.) in skin cells and / or dermal papilla cells of a subject, than in other tissues and / or than other skin cell types of the subject.

[0199] LNP-Mediated Cargo Delivery

[0200] The lipid particle compositions disclosed herein can be used for a variety of purposes, including the delivery of an active agent or therapeutic agent or compound to a subject or patient in need thereof. Subjects include both humans and non-human animals. In certain embodiments, subjects are mammals. In other embodiments, subjects are one or more particular species or breed, including, e.g., humans, mice, rats, dogs, cats, cows, pigs, sheep, or birds.

[0201] Thus, the instant disclosure also provides methods of treatment for a variety of diseases and disorders, as well as methods intended to provide a cosmetic benefit.

[0202] Methods of Treatment

[0203] In certain embodiments, the lipid particle compositions of the instant disclosure can be used to treat or prevent an epidermal disease or disorder, including but not limited to alopecia, atopic dermatitis, scleroderma, eczema, rosacea, sebomheic dermatitis, melanoma, solar keratosis, ichthyosis, Grover's disease, common warts, keratoacanthoma, and seborrheic keratosis. Optionally, the alopecia is alopecia areata, androgenetic alopecia, central centrifugal cicatricial, chemotherapy induced alopecia, frontal fibrosing alopecia, lichen planopilaris, telogen effluvium, and / or traction alopecia.

[0204] In some embodiments, the lipid particle compositions of the instant disclosure can be used to treat or prevent a joint disease or disorder, including but not limited to rheumatoid arthritis, psoriatic arthritis, gout, tendinitis, bursitis, Carpal Tunnel Syndrome and / or osteoarthritis. In certain embodiments, the lipid particle compositions of the instant disclosure can be used to treat or prevent an inflammatory disease or disorder, including but not limited to osteoarthritis, rheumatoid arthritis, arthritis, osteoarthritis, gout, spondyloarthropathies, ankylosing spondylitis, caustic gout, non-arthritic rheumatism, bursitis, hay fever, suppurative inflammation, neuropathic joint disease, hypertrophic osteoarthritis, multisized hemorrhoids, and / or systemic lupus erythematosus (SLE).

[0205] In some embodiments, administration of the nucleic acid-lipid particle or pharmaceutical composition can be performed to treat or prevent a dermatologic condition that could predispose a patient to a greater risk of developing skin cancer, including one or more dermatologic conditions such as actinic keratosis, xeroderma pigmentosum, or albinism. A wide variety of other dermatologic conditions, including but not limited to bruising or senile purpura, bums, age spots, sun spots, scars including keloids, eye bags, xerosis, ichthyosis, keratoderma, dermatofibroma, dermatitis, acne, neurodermatitis, dermatitis herpetiformis, vitiligo, vasculitis, pemphigus, bullous pemphigoid, hyperkeratosis, eczema, psoriasis, rosacea, pityriasis rosea, warts; bacterial, viral, fungal, or other infections can also potentially be treated using the systems, preparations, and methods as disclosed herein. In some embodiments, the dermatologic condition to be treated could be a manifestation of a systemic disease, such as an autoimmune disease such as systemic lupus erythematosus, scleroderma, or rheumatoid arthritis, for example, the condition could be an epidermal disease or disorder (e.g., psoriasis, atopic dermatitis, scleroderma, eczema, rosacea, seborrheic dermatitis, melanoma, solar keratosis, ichthyosis, Grover's disease, common warts, keratoacanthoma and / or seborrhoeic keratosis); a joint disease or disorder such as rheumatoid arthritis, psoriatic arthritis, gout, tendinitis, bursitis, Carpal Tunnel Syndrome and / or osteoarthritis; an inflammatory disease or disorder (e.g., allergic rhinitis, osteoarthritis, rheumatoid arthritis, infectious arthritis, post-infectious arthritis, tuberculous arthritis, arthritis, osteoarthritis, gout, spondyloarthropathies, ankylosing spondylitis, arthritis associated with vasculitis syndrome, nodular polyarteritis nervosa, irritable vasculitis, rugenic granulomatosis, rheumatoid polyposis myalgia, arthritis cell arteritis, calcium polycystic arthropathy, caustic gout, non-arthritic rheumatism, bursitis, hay fever, suppurative inflammation (e.g., tennis elbow), neuropathic joint disease, hemarthrosic, Henoch-Schlein purpura, hypertrophic osteoarthritis, multisized hemorrhoids, scoliosis, hemochromatosis, hyperlipoproteinemia, hypogammaglobulinemia, COPD, acute respiratory distress syndrome, acute lung injury, broncho-pulmonary dysplasia and / or systemic lupus erythematosus (SLE)); and an epidermal disease or disorder (e.g., psoriasis, atopic dermatitis, scleroderma, eczema, rosacea, seborrheic dermatitis, melanoma, solar keratosis, ichthyosis, Grover's disease, common warts, keratoacanthoma and / or seborrhoeic keratosis).

[0206] In further embodiments, the lipid particle compositions of the instant disclosure may be used to treat any of a wide variety of diseases or disorders, including, but not limited to, inflammatory diseases, cardiovascular diseases, nervous system diseases, tumors, demyelinating diseases, digestive system diseases, endocrine system diseases, reproductive system diseases, hemic and lymphatic diseases, immunological diseases, mental disorders, muscoloskeletal diseases, neurological diseases, neuromuscular diseases, metabolic diseases, sexually transmitted diseases, skin and connective tissue diseases, urological diseases, and infections.

[0207] The lipid particle compositions of the instant disclosure may be administered as first line treatments or as secondary treatments. In addition, where cancer is treated, the lipid particle compositions may be administered as a primary chemotherapeutic treatment or as adjuvant or neoadjuvant chemotherapy. For example, treatments of relapsed, indolent, transformed, and aggressive forms of melanoma may be administered following at least one course of a primary anti-cancer treatment, such as chemotherapy and / or radiation therapy.

[0208] The lipid particles and / or pharmaceutical compositions disclosed herein may be formed using techniques know in the art such as, for example, continuous mixing in which the process of continuously introducing lipid and buffer solutions into a mixing area causes a continuous dilution of the lipid solution with the buffer solution, which has the effect of producing a lipid vesicle almost immediately upon mixing. By mixing an aqueous solution comprising a therapeutic agent with an organic lipid solution, the organic lipid solution may undergo a continuous stepwise dilution in the presence of the buffer solution to produce a therapeutic agent-lipid particle. Such particles may have a size of from about 30 nm to about 250 nm, from about 40 nm to about 200 nm, from about 50 nm to about 150 nm, from about 60 nm to about 130 nm, from about 70 nm to about 1 10 nm, from about 70 nm to about 100 nm, from about 80 nm to about 100 nm, from about 90 nm to about 100 nm, from about 70 to about 90 nm, from about 80 nm to about 90 nm, from about 70 nm to about 80 nm, less than about 120 nm, 110 nm, 100 nm, 90 nm, or 80 nm, or about 30 nm, 35 nm, 40 nm, 45 nm, 50 nm, 55 nm, 60 nm, 65 nm, 70 nm, 75 nm, 80 nm, 85 nm, 90 nm, 95 nm, 100 nm, 105 nm, 110 nm, 115 nm, 120 nm, 125 nm, 130 nm, 135 nm, 140 nm, 145 nm, 150 nm, 155 nm, 160 nm, 165 nm, 170 nm, 175 nm, 180 nm, 185 nm, 190 nm, 195 nm, 200 nm, 205 nm, 210 nm, 215 nm, 220 nm, 225 nm, 230 nm, 235 nm, 240 nm, 245 nm, or 250 nm, or any intermediate value or sub-range therein. Once formed, the particles do not aggregate. According to the techniques herein, the particles may be sized to achieve a uniform particle size.

[0209] It is also contemplated within the scope of the disclosure that such particles may be prepared by a direct dilution process (e.g, forming a lipid vesicle solution and directly introducing it into a container having a controlled amount of dilution buffer) such as is described in U.S. Patent Publication No. 20070042031, the disclosure of which is herein incorporated by reference in its entirety for all purposes. The particles formed using the direct dilution processes typically have a size of from about 30 nm to about 250 nm, from about 40 nm to about 200 nm, from about 50 nm to about 150 nm, from about 60 nm to about 130 nm, from about 70 nm to about 110 nm, from about 70 nm to about 100 nm, from about 80 nm to about 100 nm, from about 90 nm to about 100 nm, from about 70 to about 90 nm, from about 80 nm to about 90 nm, from about 70 nm to about 80 nm, less than about 120 nm, 1 10 nm, 100 nm, 90 nm, or 80 nm, or about 30 nm, 35 nm, 40 nm, 45 nm, 50 nm, 55 nm, 60 nm, 65 nm, 70 nm, 75 nm, 80 nm, 85 nm, 90 nm, 95 nm, 100 nm, 105 nm, 110 nm, 115 nm, 120 nm, 125 nm, 130 nm, 135 nm, 140 nm, 145 nm, 150 nm, 155 nm, 160 nm, 165 nm, 170 nm, 175 nm, 180 nm, 185 nm, 190 nm, 195 nm, 200 nm, 205 nm, 210 nm, 215 nm, 220 nm, 225 nm, 230 nm, 235 nm, 240 nm, 245 nm, or 250 nm, or any intermediate value or sub-range therein. Once formed, the particles do not aggregate. According to the techniques herein, the particles may be sized to achieve a uniform particle size.

[0210] Administration ofLNP Compositions

[0211] Lipid particle compositions of the instant disclosure are primarily administered topically, i.e., are applied to body surfaces such as the skin or mucous membranes to treat ailments via a large range of classes including creams, foams, gels, lotions, and ointments. In certain embodiments, topical administration can be epicutaneous, meaning application directly to the skin. Topical administrati on may also be inhalational, and / or applied to the surface of tissues other than the skin, such as administration to the eye / conjunctiva, administration to the ear, and / or administration to the surface of a tooth.

[0212] In some embodiments, other modes of administration are contemplated, including without limitation, parenteral, intravenous, systemic, local, oral, intratumoral, intramuscular, subcutaneous, intraperitoneal, inhalation, or any such method of delivery. In one embodiment, the compositions are administered topically to the skin tissue of a subject. Alternatively, the compositions are administered parenterally, i.e., intraarticularly, intravenously, intraperitoneally, subcutaneously, or intramuscularly. In a specific embodiment, the lipid particle compositions disclosed herein are administered by dermal application and / or dermal patch. For example, in one embodiment, a patient is given dermal patch of the lipid nanoparticle-encapsulated active agent via skin application over, e.g., 5-10 minutes, 15-20 minutes, 30 minutes, 60 minutes, 90 minutes, 6 hours, 12 hours, 24 hours, two days, four days, a week, or over a multi- week period. Administrations can also be performed periodically, e.g., once every 1, 3, 5, 7, 10, 14, 21, or 28 days or longer, preferably once every 7-21 days, and preferably once every 7 or 14 days.

[0213] Lipid particle compositions of the instant disclosure may be formulated as pharmaceutical compositions suitable for delivery to a subject. The pharmaceutical compositions of the instant disclosure will often further comprise one or more buffers (e.g., neutral buffered saline or phosphate buffered saline), carbohydrates (e.g., glucose, mannose, sucrose, dextrose or dextrans), mannitol, proteins, polypeptides or amino acids such as glycine, antioxidants, bacteriostats, chelating agents such as EDTA or glutathione, adjuvants (e.g., aluminum hydroxide), solutes that render the formulation isotonic, hypotonic or weakly hypertonic with the blood of a recipient, suspending agents, thickening agents and / 'or preservatives. Alternatively, compositions of the instant disclosure may be formulated as a lyophilizate.

[0214] The concentration of drug and lipid nanoparticles in the pharmaceutical formulations can vary widely, i.e., from less than about 0.05%, usually at or at least about 2-5% to as much as 10 to 30% by weight and will be selected depend upon the particular drug used, the disease state being treated and the judgment of the clinician taking. Further, the concentration of drug and lipid nanoparticles will also take into consideration the fluid volume administered, the osmolality of the administered solution, and the tolerability of the drug and lipid nanoparticles. In some instances, it may be preferable to use a lower drug or lipid nanoparticle concentration to reduce the incidence or severity of any administration-related side effects that might occur.

[0215] Suitable formulations for use in the instant disclosure can be found, e.g., va Remington's Pharmaceutical Sciences, Mack Publishing Company, Philadelphia, Pa., 17thEd. (1985). Topical compositions can comprise a solution of the lipid nanoparticles suspended in an acceptable carrier, such as an aqueous carrier. Any of a variety of aqueous carriers can be used, e.g., water, buffered water, 0.4% saline, 0.9% isotonic saline, 0.3% glycine, 5% dextrose, and the like, and may include glycoproteins or other components for enhanced stability, such as albumin, lipoprotein, globulin, etc. Often, normal buffered saline (135-150 mM NaCl) or 5% dextrose will be used. These compositions can be sterilized by conventional sterilization techniques, such as filtration. The resulting aqueous solutions may be packaged for use or filtered under aseptic conditions and lyophilized, the lyophilized preparation being combined with a sterile aqueous solution prior to administration. The compositions may also contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions, such as pH adjusting and buffering agents, tonicity adjusting agents and the like, for example, sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, etc. Additionally, the composition may include lipid-protective agents, which protect lipids against free-radical and lipid-peroxidative damages on storage. Lipophilic free-radical quenchers, such as u-tocopherol and water-soluble iron-specific chelators, such as fen'ioxamine, are suitable.

[0216] The amount of active agent administered per dose is selected to be above the minimal therapeutic dose but below a toxic dose. The choice of amount per dose will depend on a number of factors, such as the medical history of the patient, the use of other therapies, and the nature of the disease. In addition, the amount of active agent administered may be adjusted throughout treatment, depending on the patient's response to treatment and the presence or severity of any treatment-associated side effects. In certain embodiments, the dosage of LNP composition or the frequency of administration is approximately the same as the dosage and schedule of treatment with the corresponding free active agent. However, it is understood that the dosage may be higher or more frequently administered as compared to free drug treatment, particularly where the LNP composition exhibits reduced toxicity. It is also understood that the dosage may be lower or less frequently administered as compared to free drug treatment, particularly where the LNP composition exhibits increased efficacy as compared to the free drug. Exemplary dosages and treatment for a variety of chemotherapy compounds (free drug) are known and available to those skilled in the art and are described in, e.g., Physician's Cancer Chemotherapy Drug Manual, E. Chu and V. Devita (Jones and Bartlett, 2002).

[0217] Patients typically will receive one or more courses of such treatment, and potentially many, depending on the response of the patient to the treatment. In single agent regimens, total courses of treatment are determined by the patient and physician based on observed responses and toxicity.

[0218] Combination Therapies In certain embodiments, LNP compositions of the instant disclosure can be administered in combination with one or more additional compounds or therapies, such as surgery, radiation treatment, chemotherapy, or other active agents, including any of those described above. LNP compositions may be administered in combination with a second active agent for a variety of reasons, including increased efficacy or to reduce undesirable side effects. The LNP composition may be administered prior to, subsequent to, or simultaneously with the additional treatment. Furthermore, where a LNP composition of the instant disclosure (which comprises a first active agent) is administered in combination with a second active agent, the second active agent may be administered as a free drug, as an independent LNP formulation, or as a component of the LNP composition comprising the first drug. In certain embodiments, multiple active agents are loaded into the same lipid nanoparticles, hr other embodiments, lipid nanoparticles comprising an active agent are used in combination with one or more free drugs. In particular embodiments, LNP compositions comprising an active agent are formed individually and subsequently combined with other compounds for a single co-administration. Alternatively, certain therapies are administered sequentially in a predetermined order. Accordingly, LNP compositions of the instant disclosure may comprise one or more active agents.

[0219] Other combination therapies known to those of skill in the art can be used in conjunction with the methods of the instant disclosure.

[0220] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

[0221] Reference will now be made in detail to exemplary embodiments of the disclosure. While the disclosure will be described in conjunction with the exemplary embodiments, it will be understood that it is not intended to limit the disclosure to those embodiments. To the contrary, it is intended to cover alternatives, modifications, and equivalents as may be included within the spirit and scope of the disclosure as defined by the appended claims. Standard techniques well known in the art or the techniques specifically described below were utilized.

[0222] EXAMPLES

[0223] Example 1: Production Method for Lipid Nanoparticle (LNP) Formulations Using a Microfluidic Approach

[0224] Formulations of LNPs were prepared using a cationic lipid, non-cationic lipids (e.g., as helper lipids), cholesterol (e.g., as structural lipids) and conjugated lipids (e.g., PEG-lipids) for encapsulating oligonucleotides as cargo. A reporter gene, firefly Luciferase (FLuc) mRNA in aqueous buffer pH 4.5 and ethanol stock solution of the lipids listed in Table 1 were used to produce twelve distinct LNP formulations. Briefly, the aqueous phase containing the drug substance (mRNA) was mixed with the ethanol phase containing the lipids using a microfluidic mixer. The product was purified by overnight dialysis (MW 100 KDa) against 3L of water for injection for removal of residual ethanol. The LNPs were characterized after dialysis and filtration through Polyethersulfone (PES) membrane filter. Particle size and zeta potential were measured on a light scattering instrument.

[0225] Example 2: Obligate Cationic Lipid- or Ionizable Lipid-Containing Lipid Nanoparticles Robustly Delivered a mRNA Cargo to Dermal Papilla Cells

[0226] A panel of twelve distinct lipid nanoparticles (LNPs) harboring different cationic lipids was assessed for delivery of a mRNA cargo to dermal papilla cells. For each cationic lipid listed in Table 1, a different LNP was formulated and topically administered to Yorkshire pigs, with reporter dosed at 0.3-0.5 mg of mRNA / mL. Full thickness skin biopsies were collected 6 hours after administration and were analyzed by immunohistochemistry to image Firefly Luciferase protein. The Firefly Luciferase reporter protein was detected in dermal papilla cells in each of SS- OP, ethyl phosphatidylcholine (EPC), 3B-[N-(N,,N,-dimethyiaminoethaiie)- carbamoyl]cholesterol (DOTMA), 3B-[N-(N^N'-dimetiliyiaininoethane)- carbamoyl]cholesterol (DC-Cholesterol), 1 ,2-dioleyloxy-N,N-dimethyl-3- aminopropane (DODMA), N,N-dimethyl-D-erythro-sphingosine (Dimethyl Sphingosine), Didodecyldimethylammonium bromide (DDAB)-containing LNPs, respectively (FIG. 1). In contrast, SM-102, l,2-dioleoyl-3-trimethylammonium-propane (DOTAP), l,2-Dioleoyl-3- dimethylammonium-propane (DODAP), N“(4*carboxybenzyl)"N,N-dimethyl*2,3“ bis(oleoyloxy)propan- 1 -aminium (DOBAQ), and N,N,N-trimrthyl-D-erythro-sphingosine (Trimethyl Sphingosinej-containing LNPs were respectively not identified to deliver the Firefly Luciferase reporter protein to dermal papilla cells, at least under conditions and for the LNP formulation ranges tested (FIG. 2). Accordingly, LNP formulations respectively containing any one of SS-OP, EPC, DOTMA, DC-Cholesterol, DODMA, Dimethyl Sphingosine, or DDAB present at 45% of total lipid, further including DSPC at 10% of total lipid, cholesterol at 43.5% of total lipid and DMG-PEG2000 at 1.5% of total lipid, were prioritized for further development for skin-directed delivery of nucleic acid cargoes.

[0227] Table I. LNP Compositions Tested in a Swine Model (American Yorkshire Pig)

[0228] All patents and publications mentioned in the specification are indicative of the levels of skill of those skilled in the art to which the disclosure pertains. All references cited in this disclosure are incorporated by reference to the same extent as if each reference had been incorporated by reference in its entirety individually.

[0229] One skilled in the art would readily appreciate that the present disclosure is well adapted to carry out the objects and obtain the ends and advantages mentioned, as well as those inherent therein. The methods and compositions described herein as presently representative of preferred embodiments are exemplary and are not intended as limitations on the scope of the disclosure. Changes therein and other uses will occur to those skilled in the art, which are encompassed within the spirit of the disclosure, are defined by the scope of the claims.

[0230] In addition, where features or aspects of the disclosure are described in terms of Markush groups or other grouping of alternatives, those skilled in the art will recognize that the disclosure is also thereby described in terms of any individual member or subgroup of members of the Markush group or other group.

[0231] The use of the terms "a" and "an” and "the” and similar referents in the context of describing the disclosure (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The terms "comprising" and "including" are to be construed as open-ended terms (i.e., meaning "including, but not limited to,") unless otherwise noted. Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein.

[0232] All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., "such as") provided herein, is intended merely to better illuminate the disclosure and does not pose a limitation on the scope of the disclosure unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the disclosure.

[0233] Embodiments of this disclosure are described herein, including the best mode known to the inventors for carrying out the disclosed invention. Variations of those embodiments may become apparent to those of ordinary skill in the art upon reading the foregoing description.

[0234] The disclosure illustratively described herein suitably can be practiced in the absence of any element or elements, limitation or limitations that are not specifically disclosed herein. Thus, for example, in each instance herein any of the terms "comprising", "consisting essentially of', and "consisting of' may be replaced with either of the other two terms. The terms and expressions which have been employed are used as terms of description and not of limitation, and there is no intention that in the use of such terms and expressions of excluding any equival ents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention claimed. Thus, it should be understood that although the present disclosure provides preferred embodiments, optional features, modification and variation of the concepts herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this disclosure as defined by the description and the appended claims.

[0235] It will be readily apparent to one skilled in the art that varying substitutions and modifications can be made to the invention disclosed herein without departing from the scope and spirit of the invention. Thus, such additional embodiments are within the scope of the present disclosure and the following claims. The present disclosure teaches one skilled in the art to test various combinations and / or substitutions of chemical modifications described herein toward generating conjugates possessing improved contrast, diagnostic and / or imaging activity. Therefore, the specific embodiments described herein are not limiting and one skilled in the art can readily appreciate that specific combinations of the modifications described herein can be tested without undue experimentation toward identifying conjugates possessing improved contrast, diagnostic and / or imaging activity.

[0236] The inventors expect skilled artisans to employ such variations as appropriate, and the inventors intend for the disclosure to be practiced otherwise than as specifically described herein. Accordingly, this disclosure includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the disclosure unless otherwise indicated herein or otherwise clearly contradicted by context. Those skilled in the art will recognize or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the disclosure described herein. Such equivalents are intended to be encompassed by the following claims.

Claims

We Claim:

1. A lipid particle for topical delivery of a cargo to a subject, the lipid particle comprising: a cationic lipid comprising from about 20 mol % to about 50 mol % of total lipid in the lipid particle; a phospholipid comprising from about 4 mol % to about 30 mol % of the total lipid in the lipid particle; a non-cationic lipid comprising from about 10 mol % to about 75 mol % of the total lipid present in the lipid particle; and a conjugated lipid that inhibits aggregation of particles comprising from 0.01 mol % to about 3 mol % of the total lipid present in the lipid particle.

2. The lipid particle of claim 1, wherein the cationic lipid is an obligate cationic lipid.

3. The lipid particle of claim 2, wherein the obligate cationic lipid is selected from the group consisting of Didodecyldimethylammonium bromide (DDAB), N-(4-carboxybenzyl)-N,N- dimethyl-2,3-bis(oleoyloxy)propan-l-aminium (DOBAQ), l,2-dioleoyl-3-trimethylammonium- propane (DOTAP), 3B-[N-(N’,N'-dimethyiammoethane)-carbamoyl]cholesterol (DOTMA), ethyl phosphatidylcholine {EPC), and N,N,N-trimethyl-D-erythro-sphingosine (Trimethyl Sphingosine).

4. The lipid particle of claim 2, wherein the obligate cationic lipid is selected from the group consisting of EPC, DOTMA, and DDAB, optionally wherein the obligate cationic lipid is EPC.

5. The lipid particle of claim 2, wherein the obligate cationic lipid has the structure of compound (1):wherein Ri and R2 are each optionally (un)substituted alkyl or alkenyl groups comprising between 10 and 24 carbon atoms.

6. The lipid particle of claim 1 , wherein the cationic lipid is an ionizable lipid.

7. The lipid particle of claim 6, wherein the ionizable lipid is selected from the group consisting of (((((disulfanediylbis(ethane-2,l-diyl))bis(piperidine-l,4-diyl))bis(ethane-2,l- diyl))bis(oxy))bis(2-oxoethane-2,l-diyl))bis(4,l -phenylene) dioleate (SS-OP), 3B-[N-(N’,N'- dimethylaminoethane)-carbamoyl]cholesterol (DC-Cholesterol), l,2-dioleyloxy-N,N-dimethyl-3- aminopropane (DODMA), N,N-dimethyl-D-erythro-sphingosine (Dimethyl Sphingosine), Heptadecan-9-yl 8- {(2-hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino} octanoate equivalent (SM-102), and l,2-Dioleoyl-3-dimethylammonium-propane (DODAP).

8. The lipid particle of claim 6, wherein the ionizable lipid is a disulfide (SS) cleavable lipid, optionally wherein the disulfide (SS) cleavable lipid is selected from the group consisting of SS- OC, SS-OP and SS-EC.

9. The lipid particle of any one of the preceding claims, wherein the cationic lipid is present at about 25 mol % to about 65 mol % of the total lipid present in the lipid particle, optionally wherein the cationic lipid is present at about 30 mol % to about 60 mol % of the total lipid present in the lipid particle, optionally wherein the cationic lipid is present at about 35 mol % to about 50 mol % of the total lipid present in the lipid particle, optionally wherein the cationic lipid is present at about 45 mol % of the total lipid present in the lipid particle.

10. The lipid particle of any one of the preceding claims, wherein the phospholipid is selected from the group consisting of distearoyl-sn-glycero-phosphoethanolamine, distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoyl-phosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoylphosphatidylethanolamine (POPE), dioleoyl-phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane- 1 -carboxylate (DOPE- mal), dipalmitoyl phosphatidyl ethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoyl -phosphatidyl -ethanolamine (DSPE), monomethyl -phosphatidylethanolamine (such as 16- O-monomethyl PE), dimethyl-phosphatidylethanolamine (such as 16-O-dimethyl PE), 18-1- trans PE, l-stearoyl-2-oleoyl-phosphatidyethanolamine (SOPE), hydrogenated soy phosphatidylcholine (HSPC), ethyl phosphatidylcholine (EPC), dioleoylphosphatidylserine(DOPS), sphingomyelin (SM), dimyristoyl phosphatidylcholine (DMPC), dimyristoyl phosphatidylglycerol (DMPG), distearoylphosphatidylglycerol (DSPG), dierucoylphosphatidylcholine (DEPC), palmitoyloleyolphosphatidylglycerol (POPG), dielaidoyl - phosphatidylethanolamine (DEPE), 1,2- dilauroyl-sn-glycero-3 -pho sphoethanolamine (DLPE);I,2-diphytanoyl-sn-glycero-3- phosphoethanolamine (DPHyPE); lecithin, phosphatidylethanolamine, lysolecithin, lysophosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, sphingomyelin, egg sphingomyelin (ESM), cephalin, cardiolipin, phosphatidicacid, cerebrosides, dicetylphosphate, lysophosphatidylcholine, dilinoleoylphosphatidylcholine, and mixtures thereof, optionally wherein the phospholipid is selected from the group consisting of DOPC, DSPC and DOPE, optionally wherein the phospholipid is DSPC.I I. The lipid particle of any one of the preceding claims, wherein the phospholipid is present at about 5 mol % to about 20 mol % of the total lipid present in the lipid particle, optionally wherein the phospholipid is present at about 7 mol % to about 15 mol % of the total lipid present in the lipid particle, optionally wherein the phospholipid is present at about 8 mol % to about 12 mol % of the total lipid present in the lipid particle, optionally wherein the phospholipid is present at about 9 mol % to about 11 mol % of the total lipid present in the lipid particle, optionally wherein the phospholipid is present at about 10 mol % of the total lipid present in the lipid particle.

12. The lipid particle of any one of the preceding claims, wherein the non-cationic lipid is selected from the group consisting of cholesterol, p-sitosterol and a derivative thereof.

13. The lipid particle of any one of the preceding claims, wherein the non-cationic lipid is present at about 20 mol % to about 65 mol % of the total lipid present in the lipid particle, optionally wherein the non-cationic lipid is present at about 25 mol % to about 60 mol % of the total lipid present in the lipid particle, optionally wherein the non-cationic lipid is present at about 30 mol % to about 55 mol % of the total lipid present in the lipid particle, optionally wherein the non-cationic lipid is present at about 35 mol % to about 50 mol % of the total lipid present in the lipid particle, optionally wherein the non-cationic lipid is present at about 40 mol % to about 45 mol % of the total lipid present in the lipid particle, optionally wherein the non-cationic lipid is present at about 43.5 mol % of the total lipid present in the lipid particle.

14. The lipid particle of any one of the preceding claims, wherein the conjugated lipid that inhibits aggregation of particles comprises a polyethyleneglycol (PEG)-lipid conjugate, optionally wherein the PEG of the PEG-lipid conjugate has an average molecular weight of from 550 daltons to 3000 daltons, optionally wherein the PEG-lipid conjugate is a PEGaooo-lipid conjugate, optionally wherein the PEGzooo-lipid conjugate comprises one or more of 1,2-dimyristoyl-rac- glycero-3 -methoxypolyethylene glycol-2000 (DMG-PEG2000), l,2-Dipalmitoyl-rac-glycero-3- methylpolyoxyethylene (DPG-PEG2000), and l,2-distearoyl-rac-glycero-3 -methoxypoly ethylene glycol-2000 (DSG-PEG2000), optionally wherein the PEG2ooo-lipid conjugate is 1,2-Dimyristoyl- rac-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG2000).

15. The lipid particle of any one of the preceding claims, wherein the conjugated lipid that inhibits aggregation of particles is present at about 0.05 mol % to about 2.8 mol % of the total lipid present in the lipid particle, optionally wherein the conjugated lipid that inhibits aggregation of particles is present at about 0.1 mol % to about 2.5 mol % of the total lipid present in the lipid particle, optionally wherein the conjugated lipid that inhibits aggregation of particles is present at about 0.5 mol % to about 2.0 mol % of the total lipid present in the lipid particle, optionally wherein the conjugated lipid that inhibits aggregation of particles is present at about 1.2 mol % to about 1.8 mol % of the total lipid present in the lipid particle, optionally wherein the conjugated lipid that inhibits aggregation of particles is present at about 1.5 mol % of the total lipid present in the lipid particle.

16. The lipid particle of any one of the preceding claims comprising cationic lipid at about 45 mol % of the total lipid present in the lipid particle, a phospholipid at about 10 mol % of the total lipid present in the lipid particle, cholesterol at about 43.5 mol % of the total lipid present in the lipid particle, and a PEG-lipid at about 1.5 mol % of the total lipid present in the lipid particle.

17. The lipid particle of any one of the preceding claims, further comprising nucleic acid.

18. The lipid particle of claim 17, wherein the nucleic acid is mRNA.

19. A lipid particle for topical delivery of a cargo to a subject, the lipid particle comprising:(((((disulfanediylbis(ethane-2,l -diyl))bis(piperidine-l,4-diyl))bis(ethane-2, 1 - diyl))bis(oxy))bis(2-oxoethane-2,l-diyl))bis(4,l -phenylene) dioleate (SS-OP) at about 20 mol % to 70 mol % of the total lipid in the lipid particle; a phospholipid comprising from about 4 mol % to 30 mol % of the total lipid in the lipid particle; cholesterol, [3-sitosterol or derivatives thereof from about 10 mol % to about 75 mol % of the total lipid present in the lipid particle; and a conjugated lipid that inhibits aggregation of particles comprising from 0.01 mol % to about 3 mol % of the total lipid present in the lipid particle.

20. The lipid particle of claim 19, wherein: the SS-OP is present at about 45 mol % of the total lipid in the lipid particle; 1,2-distearoyl-sn-glycero-phosphocholine (DSPC) is present at about 10 mol % of the total lipid in the lipid particle; cholesterol is present at about 43.5 mol % of the total lipid in the lipid particle, and / or l ,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG2k) is present at about 1.5 mol % of the total lipid in the lipid particle.

21. A lipid particle for topical delivery of a cargo to a subject, the lipid particle comprising: ethyl phosphatidylcholine (EPC) at about 20 mol % to 70 mol % of the total lipid in the lipid particle; a phospholipid comprising from about 4 mol % to 30 mol % of the total lipid in the lipid particle; cholesterol, [3-sitosterol or derivatives thereof from about 10 mol % to about 75 mol % of the total lipid present in the lipid particle; and a conjugated lipid that inhibits aggregation of particles comprising from 0.01 mol % to about 3 mol % of the total lipid present in the lipid particle.

22. The lipid particle of claim 21, wherein: the EPC is present at about 45 mol % of the total lipid in the lipid particle; 1,2-distearoyl-sn-glycero-phosphocholine (DSPC) is present at about 10 mol % of the total lipid in the lipid particle; cholesterol is present at about 43.5 mol % of the total lipid in the lipid particle, and / or l,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG2k) is present at about 1 .5 mol % of the total lipid in the lipid particle.

23. A lipid particle for topical delivery of a cargo to a subject, the lipid particle comprising: 3B-[N-(N',N'-dimethylaminoethane)-carbamoyl]cholesterol (DOTMA) at about 20 mol % to 70 mol % of the total lipid in the lipid particle; a phospholipid comprising from about 4 mol % to 30 mol % of the total lipid in the lipid particle; cholesterol, [3-sitosterol or derivatives thereof from about 10 mol % to about 75 mol % of the total lipid present in the lipid particle; and a conjugated lipid that inhibits aggregation of particles comprising from 0.01 mol % to about 3 mol % of the total lipid present in the lipid particle.

24. The lipid particle of claim 23, wherein: the DOTMA is present at about 45 mol % of the total lipid in the lipid particle; 1,2-distearoyl-sn-glycero-phosphocholine (DSPC) is present at about 10 mol % of the total lipid in the lipid particle; cholesterol is present at about 43.5 mol % of the total lipid in the lipid particle, and / or l ,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG2k) is present at about 1.5 mol % of the total lipid in the lipid particle.

25. A lipid particle for topical delivery of a cargo to a subject, the lipid particle comprising: 3B-[N-(N’,N'-dimethylamiiioethane)-carbamoyl]cholesterol (DC-Cholesterol) at about 20 mol % to 70 mol % of the total lipid in the lipid particle; a phospholipid comprising from about 4 mol % to 30 mol % of the total lipid in the lipid particle; cholesterol, ^-sitosterol or derivatives thereof from about 10 mol % to about 75 mol % of the total lipid present in the lipid particle; and a conjugated lipid that inhibits aggregation of particles comprising from 0.01 mol % to about 3 mol % of the total lipid present in the lipid particle.

26. The lipid particle of claim 25, wherein: the DC-Cholesterol is present at about 45 mol % of the total lipid in the lipid particle; 1,2-distearoyl-sn-glycero-phosphocholine (DSPC) is present at about 10 mol % of the total lipid in the lipid particle; cholesterol is present at about 43.5 mol % of the total lipid in the lipid particle, and / or l,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG2k) is present at about 1.5 mol % of the total lipid in the lipid particle.

27. A lipid particle for topical delivery of a cargo to a subject, the lipid particle comprising:1 ^“dioleyloxy-NjN-dimethyl-S-aminopropane (DODMA) at about 20 mol % to 70 mol % of the total lipid in the lipid particle; a phospholipid comprising from about 4 mol % to 30 mol % of the total lipid in the lipid particle; cholesterol, ^-sitosterol or derivatives thereof from about 10 mol % to about 75 mol % of the total lipid present in the lipid particle; and a conjugated lipid that inhibits aggregation of particles comprising from 0.01 mol % to about 3 mol % of the total lipid present in the lipid particle.

28. The lipid particle of claim 27, wherein: the DODMA is present at about 45 mol % of the total lipid in the lipid particle; 1,2-distearoyl-sn-glycero-phosphocholine (DSPC) is present at about 10 mol % of the total lipid in the lipid particle; cholesterol is present at about 43.5 mol % of the total lipid in the lipid particle, and / or l,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG2k) is present at about 1.5 mol % of the total lipid in the lipid particle.

29. A lipid particle for topical delivery of a cargo to a subject, the lipid particle comprising: N,N-dimethyl-D-erythro-sphingosine (Dimethyl Sphingosine) at about 20 mol % to 70 mol% of the total lipid in the lipid particle; a phospholipid comprising from about 4 mol % to 30 mol % of the total lipid in the -lipid particle; cholesterol, ^-sitosterol or derivatives thereof from about 10 mol % to about 75 mol % of the total lipid present in the lipid particle; and a conjugated lipid that inhibits aggregation of particles comprising from 0.01 mol % to about 3 mol % of the total lipid present in the lipid particle.

30. The lipid particle of claim 29, wherein: the Dimethyl Sphingosine is present at about 45 mol % of the total lipid in the lipid particle; 1,2-distearoyl-sn-glycero-phosphocholine (DSPC) is present at about 10 mol % of the total lipid in the lipid particle; cholesterol is present at about 43.5 mol % of the total lipid in the lipid particle, and / or l,2-dimyristoyl-rac-glycero-3- methoxypoly ethylene glycol-2000 (DMG-PEG2k) is present at about 1.5 mol % of the total lipid in the lipid particle.

31. A lipid particle for topical delivery of a cargo to a subject, the lipid particle comprising:Didodecyldimethylammonium bromide (DDAB) at about 20 mol % to 70 mol % of the total lipid in the lipid particle; a phospholipid comprising from about 4 mol % to 30 mol % of the total lipid in the lipid particle; cholesterol, ^-sitosterol or derivatives thereof from about 10 mol % to about 75 mol % of the total lipid present in the lipid particle; and a conjugated lipid that inhibits aggregation of particles comprising from 0.01 mol % to about 3 mol % of the total lipid present in the lipid particle.

32. The lipid particle of claim 31 , wherein: the DDA B is present at about 45 mol % of the total lipid in the lipid particle; 1,2-distearoyl-sn-glycero-phosphocholine (DSPC) is present at about 10 mol % of the total lipid in the lipid particle; cholesterol is present at about 43.5 mol % of the total lipid in the lipid particle, and / or l,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG2k) is present at about 1 .5 mol % of the total lipid in the lipid particle.

33. The lipid particle of any one of claims 19-32, wherein the cargo is a nucleic acid, optionally wherein the nucleic acid is mRNA.

34. The lipid particle of claim 33, wherein the nucleic acid cargo comprises a synthetic or naturally occurring RNA or DNA, or derivatives thereof, optionally wherein the nucleic acid cargo is a modified RNA, optionally wherein the modified RNA is selected from the group consisting of a modified mRNA, a modified antisense oligonucleotide and a modified siRNA, optionally wherein the modified mRNA encodes a nucleic acid modulating controller.

35. The lipid particle of claim 33, wherein the nucleic acid cargo comprises one or more modifications selected from the group consisting of 2'-O-methyl modified nucleotides, a nucleotide comprising a 5'-phosphorothioate group, a terminal nucleotide linked to a cholesteryl derivative, a 2 '-deoxy-2'- fluoro modified nucleotide, a 5'-methoxy-modified nucleotide (e.g., 5'- methoxyuridine), a 2 '-deoxy -modified nucleotide, a locked nucleotide, an abasic nucleotide, a 2'- amino-modified nucleotide, a 2'-alkyl-modified nucleotide, a morpholino nucleotide, a phosphoramidate, a non-natural base comprising nucleotide; intemucleoside linkages or backbones including phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates including 3'-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates including 3'- amino phosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, and boranophosphates having normal 3'- 5 ' linkages, 2 '-5' linked analogs of these, and those having inverted polarity wherein the adjacent pairs of nucleoside units are linked 3'-5' to 5 '-3' or 2 -5' to 5'-2'.

36. A composition comprising the lipid particle of any one of the preceding claims, wherein the composition is formulated for topical application to the skin of the subject, optionally wherein the composition is formulated for application to the scalp of the subject.

37. A pharmaceutical composition comprising the lipid particle of any one of claims 1 -35 or the composition of claim 36, and a pharmaceutically acceptable carrier.

38. A topical formulation comprising the pharmaceutical composition of claim 37.

39. The lipid particle, composition, pharmaceutical composition, or topical formulation of any one of the preceding claims, wherein the lipid particle, composition, pharmaceutical composition, or topical formulation is administered to a subject for treatment of a skin or inflammatory disease or disorder, optionally wherein the skin or inflammatory disease or disorder is selected from the group consisting of alopecia, psoriasis, atopic dermatitis, scleroderma, eczema, rosacea, seborrheic dermatitis, melanoma, solar keratosis, ichthyosis, Grover's disease, common warts, keratoacanthoma and seborrheic keratosis.

40. A method for delivering a cargo to a demial tissue cell of a subject, the method comprising administering the lipid particle, composition, pharmaceutical composition, or topical formulation of any one of the preceding claims with the cargo to the subject.

41. A method for treating or preventing a disease or disorder in a subject, the method comprising administering the lipid particle, composition, pharmaceutical composition, or topical formulation of any one of claims 1-39 to the subject.

42. The method of claim 41, wherein the disease or disorder is selected from the group consisting of an epidermal disease or disorder, a joint disease or disorder, and an inflammatory disease or disorder.

43. The method of claim 42, wherein the epidermal disease or disorder is selected from the group consisting of alopecia, psoriasis, atopic dermatitis, scleroderma, eczema, rosacea, seborrheic dermatitis, melanoma, solar keratosis, ichthyosis, Grover's disease, common warts, keratoacanthoma and seborrheic keratosis, optionally wherein the alopecia is selected from the group consisting of alopecia areata, androgenetic alopecia, central centrifugal cicatricial, chemotherapy induced alopecia, frontal fibrosing alopecia, lichen planopilaris, telogen effluvium, and traction alopecia.

44. The method of claim 42, wherein the joint disease or disorder is selected from the group consisting of rheumatoid arthritis, psoriatic arthritis, gout, tendinitis, bursitis, Carpal Tunnel Syndrome and osteoarthritis.

45. The method of claim 42, wherein the inflammatory disease or disorder is selected from the group consisting of osteoarthritis, rheumatoid arthritis, arthritis, osteoarthritis, gout, spondyloarthropathies, ankylosing spondylitis, caustic gout, non-arthritic rheumatism, bursitis, hay fever, suppurative inflammation, neuropathic joint disease, hypertrophic osteoarthritis, multisized hemorrhoids, and systemic lupus erythematosus (SLE).

46. A method for delivering a cargo intracellularly to a skin cell of a subject, the method comprising contacting the skin of the subject with a lipid particle of any one of claims 1 -35 having the cargo, composition of claim 36 having the cargo, pharmaceutical composition of claim 37 having the cargo, or a topical formulation of claim 38 having the cargo, thereby delivering the cargo intracellularly to the skin cell of the subject.

47. The method of claim 46, wherein the cargo is a nucleic acid cargo, optionally wherein the nucleic acid cargo is delivered intracellularly to the skin cell of the subject, optionally wherein the nucleic acid cargo is expressed in the cytosol of the skin cell of the subject and / or wherein the nucleic acid cargo is delivered to the nucleus of the skin cell of the subject.

48. The method of claim 46, wherein the skin cell of the subject is selected from the group consisting of an epidermal tissue cell, a dermal tissue cell and a subcutaneous tissue cell, optionally wherein the epidermal tissue cell is selected from the group consisting of a keratinocyte (optionally a stratum corneum cell, a stratum granulosum cell, a stratum spinulosum cell, and / or a stratumbasale cell), a melanocyte, a Langerhans cell and a Merkel cell; the dermal tissue cell is selected from the group consisting of a mast cell, a vascular smooth muscle cell, a specialized muscle cell, a fibroblast, a hair follicle cell (e.g., a dermal papilla cell and / or a dermal sheath cell), an immune cell, and / or a leukocyte (e.g., a neutrophil, a T or B lymphocyte, an eosinophil and / or a monocyte); and / or a subcutaneous tissue cell selected from group consisting of a fat cell, a nerve cell and / or a vascular cell.

49. The method of claim 46, wherein the skin cell of the subject is a hair follicle cell, optionally wherein the hair follicle cell is a dermal papilla cell.

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