Lipid NANO delivery particles for targeted transport of nucleic acids and methods of making and using the same

Abstract

Compositions comprising lipid nano delivery particles for the targeted transport of nucleic acids as well as methods of manufacturing and using lipid nano delivery particles in the targeted transport of nucleic acids once administered to patients.

Description

LIPID NANO DELIVERY PARTICLES FOR TARGETED TRANSPORT OF NUCLEIC ACIDS AND METHODS OF MAKING AND USING THE SAMEREFERENCE TO SEQUENCE LISTING

[0001] The instant application contains a Sequence Listing which has been submitted electronically in ST.26 XML format and is hereby incorporated by reference in its entirety. Said ST.26 XML Sequence Listing was created on April 4, 2025, is named 30664_WO.xml and is 10 kilobytes in size.FIELD OF THE INVENTION

[0002] The present disclosure relates generally to the field of medicine. More particularly the present disclosure relates to compositions comprising lipid nano delivery particles (LNDPs) for the targeted transport of nucleic acids as well as methods of manufacturing and using LNDPs in the targeted transport of nucleic acids once administered to patients. The LNDPs of the present disclosure aide in the treatment of diseases, including in some embodiments diseases of the central nervous system (CNS).BACKGROUND

[0003] Nucleic acids as a class of therapeutics are a promising field. However, a major challenge for realizing such therapeutic promise remains packaging and delivery technology that provides stability and protection for the therapeutic nucleic acids, as well as targeted transport, or delivery, to a desired area / therapeutic target once administered to the patient. For central nervous system (CNS) diseases, therapeutic nucleic acids face the additional challenge of traversing the blood brain barrier (BBB) to enter the CNS once administered to the patient.

[0004] Proposals for packaging and targeted transport of therapeutic nucleic acids have included linking the nucleic acid to moieties such as antibodies, antibody fragments and peptides that specifically bind a therapeutic target. For example, conjugating a therapeutic nucleic acid to a peptide (e.g., RGD peptides or TAT peptides) or an antibody or a fragment thereof e.g., antibody-oligonucleotide conjugate (AOC)), where the peptide or antibody binds to a therapeutic target such as a cell surface receptor, have been suggested for targeted transport. For CNS diseases one such target of interest is the transferrin receptor (TfR); conjugating therapeutic nucleic acids to antibodies, or fragments thereof, which specificallybind TfR has been proposed for targeted transport. See, e.g., McQuiad el al. (2021) PLoS ONE 16:e0252341 ; and Inti. Patent Application Publication Nos. WO 2020 / 144233 and 2022 / 026555). While such options may offer selectively in targeted transport, they do not address packaging and protection of the therapeutic nucleic acid.

[0005] Other proposals for packaging and targeted transport of therapeutic nucleic acids includes forms of nano delivery particles such as exosomes, liposomes, lipid nanoparticles and the like. While these approaches may aid in delivering therapeutic nucleic acid agents across the cell membrane they may, however, lack cellular specificity and may induce toxicity associated with broad exposure to therapeutic agents. Furthermore, to date these technologies continue to present significant challenges associated with manufacturing, encapsulation of nucleic acid, and protection and stability for the therapeutic nucleic acid, as well as pharmacokinetic issues once administered.

[0006] Thus, there remains a need for enhancements for the packaging and targeted transport of therapeutic nucleic acids that overcomes one or more of the challenges described above. Such enhancements must be able to deliver therapeutic nucleic acids to selective targets once administered with increased efficiency and stability. It is also desirable that such enhancements not be more invasive than the therapeutic molecule and not be attendant upon unacceptable immunogenicity, toxicity and off-target issues, and demonstrate acceptable pharmacokinetic properties. Such enhancements should also allow for improved manufacturing and should not interfere with the properties of the therapeutic nucleic acid necessary for treating the disease indication. In addition, for diseases of the CNS, such enhancements must improve targeting of the BBB and, in some instances enable peripheral systemic delivery of the therapeutic to overcome the need to invasively deliver the therapeutic directly into the CNS.

[0007] The present disclosure provides lipid nano delivery particles (LNDPs) for packaging and enhanced targeted transport of therapeutic nucleic acids. Embodiments of the LNDPs disclosed herein are also useful in the treatment of CNS diseases and targeting a BBB target for enhanced delivery of therapeutic nucleic acids into the CNS.BRIEF SUMMARY

[0008] Embodiments of the present disclosure comprise a LNDP comprising an outer lipid membrane comprised of phospholipids; and an inner core, the inner core being interior to the outer lipid membrane, wherein a plurality of the phospholipids of the outer lipid membrane are conjugated to a hydrophilic polymer (such as polyethylene glycol (PEG) molecule), andwherein a plurality of the phospholipids of the outer lipid membrane conjugated to a hydrophilic polymer comprise a targeting moiety conjugated to the hydrophilic polymer.

[0009] According to some embodiments of the present disclosure, the LNDP is selected from the group consisting of an enveloped nanocomplex (eNCP), a lipid nanoparticle (LNP), a liposome, a nanocapsule (NC), a nanocrystal (NCR), a polymer lipid nanoparticle (PLNP), a nanoemulsion (NE) and a proteolipid vehicle (PLV).

[0010] According to particular embodiments the inner core is one of oily or liquid. According to some embodiments, the inner core is solid.

[0011] Embodiments of LNDP of the present disclosure further comprising an inner lipid membrane comprising phospholipids, where the inner lipid membrane is interior to the outer lipid membrane.

[0012] Additionally, embodiments of the LNDPs of the present disclosure include a targeting moiety being one of a peptide, antibody, Fab, scFv, VHH, or organic compound. In some specific embodiments, the targeting moiety specifically binds TfR. In even further embodiments where the targeting moiety specifically binds TfR, it has an affinity of between 0.1 and 500 nanomolar (nM). In some further embodiments, the targeting moiety specifically binds a peripheral cell target. In a specific embodiment, the peripheral cell may be a T lymphocyte.

[0013] According to some embodiments of the LNDPs of the present disclosure, the hydrophilic polymer has a molecular weight of between 1 and 10 kiloDalton (kDa). In some embodiments, the hydrophilic polymer has a molecular weight of between 1 and 5 kDa. In some embodiments, the hydrophilic polymer has a molecular weight of between 1 and 3 kDa. In some embodiments, the hydrophilic polymer has a molecular weight of approximately 2.5 kDa. According to some embodiments, the hydrophilic polymer are conjugated to the plurality of phospholipids of the outer lipid membrane by a maleimide (that is, by a maleimide reaction). According to embodiments the hydrophilic polymer is one of a polyethylene glycol (PEG) molecule, a zwitterionic polymer or OEGMA. According to more specific embodiments the hydrophilic polymer is a PEG molecule.

[0014] According to some embodiments of the LNDPs of the present disclosure, the targeting moieties are conjugated to the plurality of hydrophilic polymers by a maleimide. According to specific embodiments the hydrophilic polymer is a PEG molecule.

[0015] According to some embodiments of the LNDPs of the present disclosure, between approximately 1 to approximately 5 percent (%) of total molar particle composition (that is,approximately 1-5% by mole percentage) is the plurality of the phospholipids of the outer lipid membrane having a hydrophilic polymer conjugated thereto. According to some embodiments between approximately 1 to approximately 2.5 % of total molar particle composition is the plurality of the phospholipids of the outer lipid membrane having a hydrophilic polymer conjugated thereto. According to specific embodiments the hydrophilic polymer is a PEG molecule.

[0016] According to some embodiments between approximately 5 % to approximately 20 % of the hydrophilic polymers conjugated to phospholipids of the outer lipid membrane have a targeting moiety conjugated thereto. According to some embodiments approximately 10 % of the hydrophilic polymers conjugated to phospholipids of the outer lipid membrane have a targeting moiety conjugated thereto. According to specific embodiments the hydrophilic polymer is a PEG molecule.

[0017] Further, embodiments of the present disclosure include pharmaceutical compositions comprising LNDPs of the present disclosure.

[0018] Additionally, embodiments of the present disclosure include methods of targeted transport of a therapeutic nucleic acid to a patient. Such methods comprise encapsulating a therapeutic nucleic acid within the inner core of a LNDP of the present disclosure and administering the LNDP comprising the therapeutic nucleic acid to a patient. According to some such embodiments, the therapeutic nucleic acid comprises one or a combination of any of mRNA, guide RNAs (gRNAs), microRNA (miRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), interfering RNA (iRNA), antisense oligonucleotide (ASO), Dicer substrate interfering RNA (DsiRNA), miRNA, short hairpin RNA (shRNA), short interfering RNA (siRNA), short activating RNAs (saRNAs), suppressor tRNAs (sup-tRNAs), circularized RNA (circRNA), and activating RNA (aRNA). Additionally, according to some embodiments, the step of administering comprises any of enteral, parenteral, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, subcutaneous, intraventricular, transdermal, interdermal, rectal, intravaginal, intraperitoneal, topical, mucosal, nasal, buccal, sublingual, and intracarotid.BRIEF DESCRIPTION OF THE DRAWINGS

[0019] FIG. 1 is a representative illustration of a LNDP of the present disclosure.

[0020] FIG. 2 is an illustrative exemplification of direct conjugation of a Targeting Moiety Fab to a PEG molecule conjugated to a phospholipid of the outer lipid membrane and shown here with a maleimide reactive group.

[0021] FIG. 3 is an illustrative exemplification of post-nano particle (NP) insertion of a phospholipid having a PEG molecule and a PEG molecule with a targeting moiety linked thereto already conjugated thereto into the phospholipid outer membrane.

[0022] FIG. 4 provides pharmacokinetic assessment results of exemplified LNDPs of the present disclosure of Example 1 A.

[0023] FIG. 5 provides results demonstrating targeted delivery of exemplified LNDPs of the present disclosure of Example 1A to non-central nervous systems.

[0024] FIG. 6 provides results demonstrating targeted delivery of exemplified LNDPs of the present disclosure of Example 1 to the BBB and demonstrates exemplified therapeutic nucleic acid activity.

[0025] FIG. 7 provides pharmacokinetic assessment results of exemplified LNDPs of the present disclosure of Example 2.

[0026] FIG. 8 provides assessment results of cellular level distribution at the brain of exemplified LNDPs of the present disclosure of Example 2.

[0027] FIG. 9 provides results demonstrating targeted delivery of exemplified LNDPs of the present disclosure of Example 3 to the brain.

[0028] FIG. 10 is a plot demonstrating that siRNA delivery is enhanced in primary human T-cells by encapsulation in an LDNP decorated with anti-CD7 fab fragment.DETAILED DESCRIPTION

[0029] Lipid nano delivery particle (LNDP), as used herein, refers to a nano particle (NP) composition having a phospholipid outer (i.e., surface) membrane encapsulating an inner core. LNDPs of the present disclosure are capable of encapsulating (i.e., packaging), either fully or partially, a nucleic acid within the inner core. In some embodiments, LNDPs of the present disclosure may comprise a phospholipid bilayer, such as a liposome, or may comprise a single phospholipid outer membrane such as a lipid nanoparticle (LNP). Additionally, LNDPs of the present disclosure may comprise a solid core such as a LNP, a liquid core such as a nanoemulsion (NE), or an oily core such as a nanocapsule (NC), or combinations thereof. Examples of NPs comprising LNDPs of the present disclosure include, by way of example,LNPs, NEs, NCs, liposomes, lipoids, lipoplexes, proteolipid vehicles (PLVs), polymers, dendrimers, enveloped nanocomplex (eNCPs) and nanocrystals (NCRs).

[0030] LNDPs of the present disclosure include hydrophilic polymers, such as PEG molecules, linked or conjugated to a plurality of phospholipids comprising the outer (i.e., surface) membrane. Hydrophilic polymers, as used herein, are oxygen containing hydrophile polymers which possess an affinity for water such as being capable of interacting with water, including PEG molecules, zwitterionic polymers and OEGMA. Hydrophilic polymers linked to a phospholipid of the present disclosure may be, independently, straight, branched, and aromatic in nature and be approximately 1-10 kDa in molecule weight. According to some particular embodiments, molecular weight of the PEG molecules linked to respective phospholipids, of the present disclosure may be approximately l-4.5kDa. According to embodiments, the plurality of phospholipids comprising hydrophilic polymers linked thereto may comprise approximately 1% to approximately 5% of the total molar percent of outer lipid membrane phospholipids. In some particular embodiments, the total molar percent of phospholipids comprising hydrophilic polymers linked thereto is approximately 1.5%. In even more particular embodiments the hydrophilic polymers are PEG molecules.

[0031] LNDPs of the present disclosure also include a targeting moiety linked or conjugated to a plurality of the hydrophilic polymers, for example PEG molecules, linked to phospholipids comprising the outer (i.e., surface) membrane. According to embodiments of the LNDPs of the present disclosure, a targeting moiety is linked or conjugated to approximately 10% to approximately 20% of the PEG molecules linked to phospholipids. According to even more particular embodiments, a targeting moiety is linked or conjugated to approximately 10% the PEG molecules linked to phospholipids.

[0032] As used herein, a targeting moiety refers to a chemical moiety (e.g., an antibody or fragment thereof such as a scFV, VHH or Fab, or the like, a peptide, a receptor, etc.) that selectively binds a therapeutic target such as a cell surface receptor. A targeting moiety facilitates delivery of the LNDP to a particular cell through such selective binding to the therapeutic target such as a r cell surface receptor and therefore aides in the internalization of the packaged therapeutic nucleic acid into the cell. According to some embodiments useful in aiding in the treatment of CNS diseases, targeting moieties are specific for BBB targets.

[0033] According to some embodiments, the LNDP as described herein may be used to deliver a nucleic acid to an immune cell. In a specific embodiment, the immune cell is a T lymphocyte (or “T cell”). In such an embodiment, the nucleic acid delivered may encode oneor more proteins to be expressed by the T cell, and / or may serve to edit the genetic material of the T cell such that the T cell expresses one or more proteins. In an embodiment, the nucleic acid delivered by the LNDP serves to encode a chimeric antigen receptor (CAR), thereby making the T cell a CAR-T cell. The LNDP may deliver the nucleic acid cargo ex vivo or in vivo. The targeting moiety provided on or conjugated to the outer surface of the LNDP for such an application may target a molecule expressed on the surface of a T cell, such as CD5, CD7, or CD8, among others (see Billingsley et al., “In Vivo mRNA CAR T Cell Engineering via Targeted Ionizable Lipid Nanoparticles with Extrahepatic Tropism.” Small 2024, 20, 2304378).

[0034] The term “specific for”, “specifically binds” or “specifically targets”, as used interchangeably herein in relation to a targeting moiety, refers to the ability of the targeting moiety to bind and / or associate with a particular target molecule, such as a protein or receptor, over background levels or non-target levels. In some embodiments, specific for may be demonstrated by affinity (Kd) measures for the target over non-targets.

[0035] The term “conjugated” or “linked”, as used interchangeably herein, refers to a first molecule or compound, for example a NP, being associated, attached, connected or otherwise joined to a second molecule or compound, for example a hydrophilic polymer such as a PEG molecule (or for example, a PEG molecule having a targeting moiety linked thereto). Conjugation of a PEG molecule (or PEG molecule having a targeting moiety linked thereto) can be by directed conjugation (e.g., with a reactive group such as a thiol-based reactive group such as a maleimide, see FIG. 2 for an exemplification) or via post-NP insertion of a phospholipid having a PEG molecule (or a PEG molecule with a targeting moiety linked thereto, see FIG. 3 for an exemplification) already conjugated thereto into the phospholipid outer membrane. It is understood it is possible to have a combination of post-NP insertion of a phospholipid having a PEG molecule and directed conjugation of a targeting moiety.

[0036] As used herein, “linker” refers to an atom, group of atoms, molecule or compound, such as an amino acid or group of amino acids, comprising at least one bond that attaches or links a molecule or compound (e.g., a NP) to another molecule or compound e.g., a PEG molecule or a PEG molecule having a targeting moiety linked thereto). A linker can be “labile” or “cleavable,” meaning the linker can be cleaved (e.g., by acidic pH or enzyme). Alternatively, a linker can be “stable” or “non-cleavable,” meaning the linker cannot be cleaved in physiological conditions. Linkers, according to the present disclosure, may also include hydrolysable and / or enzymatically degradable linkers. Linkers may also attach multipletherapeutic compositions to a multispecific binding molecule of the present disclosure. Additionally, examples of linkers comprise thiol-based reactive groups (e.g., maleimides, sulfhydryls, haloacetyls, thiosulfonates, disulfide), amine-based groups (e.g., N- hydroxysuccimide esters, imidoesters, etc) and aldehyde-reactive groups (e.g., hydrazides and alkoxyamines), and carbohydrate-based groups (e.g., native Fc glycan, engineered glycans etc.).

[0037] As used herein, therapeutic nucleic acid refers to a nucleic acid molecule used for the treatment of a disease. Examples of therapeutic nucleic acids include editing nucleic acids, including for example, DNA, mRNA, guide RNAs (gRNAs), microRNA (miRNA), transfer RNA (tRNA) and ribosomal RNA (rRNA). Additionally, therapeutic nucleic acids as referred to herein include interfering RNA (iRNA), antisense oligonucleotide (ASO), Dicer substrate interfering RNA (DsiRNA), miRNA, short hairpin RNA (shRNA), short interfering RNA (siRNA), short activating RNAs (saRNAs), suppressor tRNAs (sup-tRNAs), circularized RNA (circRNA) and small interfering RNA (siRNA). In addition, therapeutic nucleic acids of the present disclosure may include activating RNA (aRNA). As used herein, a therapeutic nucleic acid may be a single strand or double strand nucleic acid that has a therapeutic application (i.e. , application in diagnosing and / or treating a disease, disorder or condition). Therapeutic nucleic acids of the present disclosure may contain one or more modified nucleotide residues or linkages.

[0038] As used herein, “nucleic acid’’ means a polymer of nucleotides. Although it may comprise any type of nucleotide units, the term generally applies to nucleotide polymers of DNA, cDNA and / or RNA. Polynucleotide is used to include single- stranded (ss) nucleic acids, double-stranded (ds) nucleic acids, and DNA, cDNA and / or RNA made from nucleotide or nucleoside analogues that may be identified by their sequences, which are generally presented in the 5' to 3' direction (as the coding strand), where the 5' and 3' indicate the linkages formed between the 5' hydroxyl group of one nucleotide and the 3'-hydroxyl group of the next nucleotide. For a coding strand presented in the 5'-3' direction, its complement (or non-coding strand) is the strand that hybridizes to that sequence according to Watson-Crick base pairing. Thus, as used herein, the complement of a nucleic acid such as a polynucleotide is the same as the “reverse complement” and describes the nucleic acid that in its natural form, would be based paired with the nucleic acid in question.

[0039] A therapeutic nucleic acid of the present disclosure may be presented within a vector. As used herein, “vector” means a nucleic acid molecule capable of transporting another nucleicacid sequence (or multiple nucleic acid sequences) to which it has been ligated into a host cell or genome. One type of vector is a “plasmid,’- which refers to a circular DNA loop, typically double-stranded (ds), into which additional DNA segments may be ligated. Another type of vector is a viral vector, wherein additional DNA segments may be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g. , bacterial vectors having a bacterial origin of replication). Moreover, certain vectors are capable of directing the expression of genes (e.g., genes encoding an exogenous peptide or protein of interest) to which they are operatively linked when combined with appropriate control sequences such as promoter and operator sequences and replication initiation sites. Such vectors are commonly referred to as “expression vectors” and may also include a multiple cloning site for insertion of the gene encoding the protein of interest. Alternatively, the gene encoding the peptide or protein of interest may be introduced by site- directed mutagenesis techniques such as Kunkel mutagenesis. See, e.g. , Handa et al., Rapid and Reliable Site-Directed Mutagenesis Using Kunkel’s Approach, Methods in Molecular Biology, vol 182: In Vitro: Mutagenesis Protocols, 2nd Ed.).

[0040] According to some embodiments, a vector may include a “viral vector”, which as used herein refers to a vector that is derived from a naturally occurring or modified virus, especially a recombinant AAV (“rAAV”) vector or a Baculovirus vector (e.g., Autographa californica nuclear polyhedrosis (AcNPV) vector)). A “recombinant adeno-associated virus,” “recombinant AAV” and “rAAV” are synthetic polynucleotide vectors comprising one or more heterologous sequences (i.e., nucleic acid sequence not of an AAV origin) that are flanked by at least one AAV ITR sequence. Such rAAV vectors can be replicated and packaged into infectious viral particles when present in a host cell that has been infected with a suitable helper virus (or that is expressing suitable helper functions) that expresses AAV rep and cap gene products (i.e., AAV Rep and Cap proteins).

[0041] As used herein, “engineered,” with respect nucleic acid sequence means having an altered nucleotide sequence obtained from genetic engineering techniques. Genetic engineering techniques include, but are not limited to, PCR and DNA cloning technologies; transfection, transformation and other gene transfer technologies; homologous recombination; site-directed mutagenesis; and gene fusion. For example, “engineered” may refer to a change, such as an addition, deletion and / or substitution of a nucleic acid residue or amino acid residue with respect to a given wild-type nucleotide or amino acid sequence.

[0042] As used herein, “sequence identity,” in the context of two nucleotide sequences or two amino acid sequences, means that residues in the two sequences that are the same when aligned for maximum correspondence over a specified comparison window.

[0043] As used interchangeably herein, the term “patient,” “subject,” and “individual,” refers to a human. In certain embodiments, the patient is further characterized with a CNS disease, disorder, or condition (for example, a CNS neurodegenerative disorder). In some embodiments, the patient may be further characterized as being at risk of developing a CNS disorder, disease, or condition.

[0044] As used herein, “treat,” “to treat,” “treatment” or “treating” means an act of providing care to an individual in need thereof, for example, by administering a therapeutic agent to the individual for purposes of improving the health and / or well-being of the individual with respect to an existing a disease, disorder or condition, or to prevent or decrease the likelihood of the occurrence of a disease, disorder or condition. Treating also can involve reducing the frequency or severity of at least one sign, symptom or contributing factor of a disease, disorder or condition experienced by the individual.

[0045] As used herein, “administer,” “administering,” “administration” and the like mean providing a substance (e.g., a lipid nano delivery particle or pharmaceutical composition thereof provided herein) to an individual in a manner that is pharmacologically useful (e.g., to treat a disease, disorder or condition in the individual). Administration may be by any route, including enteral (e.g., oral), parenteral, intravenous, intramuscular, intra-arterial, intramedullary, intrathecal, subcutaneous, intraventricular, transdermal, interdermal, rectal, intravaginal, intraperitoneal, topical (as by powders, ointments, creams and / or drops), mucosal, nasal, buccal, sublingual; by intratracheal instillation, bronchial instillation and / or inhalation; and / or as an oral spray, nasal spray, and / or aerosol. Specifically contemplated routes are IV injection (e.g., systemic intravenous injection), IC (e.g., vascular intracarotid injection), SC injection, ICV injection, ICM injection, IPM injection, IT injection or a combination thereof.

[0046] As used herein, “effective amount” means an amount, concentration or dose of a therapeutic agent, or a pharmaceutical composition thereof, upon single or multiple dose administration to an individual in need thereof, provides a desired effect in such an individual under diagnosis or treatment (i.e., may produce a clinically measurable difference in a condition of the individual). An effective amount can be readily determined by one of skill in the art by using known techniques and by observing results obtained under analogous circumstances. In determining the effective amount for an individual, a number of factors areconsidered, including, but not limited to, the species of mammal, its size, age and general health, the specific disease, disorder or condition involved, the degree of or involvement or the severity of the disease, disorder or condition, the response of the individual, the therapeutic agent administered, the mode of administration, the bioavailability characteristics of the preparation administered, the dose regimen selected, the use of concomitant medication, and other relevant circumstances.

[0047] As used herein, “pharmaceutical composition” means a composition or therapeutic agent, mixed with at least one pharmaceutically acceptable chemical component, such as, but not limited to carriers, stabilizers, diluents, dispersing agents, suspending agents, thickening agents, excipients and the like.

[0048] As used herein, “pharmaceutically acceptable,” when referring to a material such as a carrier or diluent, means that it does not abrogate the biological activity or properties of a therapeutic agent and is relatively non-toxic (i.e. , the material may be administered to an individual without causing undesirable biological effects or interacting in a deleterious manner with any of the components of the composition in which it is contained.

[0049] As used herein, “pharmaceutically acceptable carrier” means a pharmaceutically acceptable material, composition or carrier, such as a liquid or solid filler, stabilizer, dispersing agent, suspending agent, diluent, excipient, thickening agent, solvent or encapsulating material, involved in carrying or transporting a therapeutic agent within or to an individual such that it may perform its intended function. Additional ingredients that may be included in the pharmaceutical compositions used in the practice of the invention are known in the art and described, for example in Remington’s Pharmaceutical Sciences, 21stEdition, University of the Sciences in Philadelphia, PA (2006).

[0050] According to some embodiments of the present disclosure, the LNDPs disclosed herein are also useful in the treatment of CNS diseases, targeting a BBB target for enhanced delivery of therapeutic nucleic acids into the CNS. As used herein, “CNS disease, disorder or condition” or “disease of the CNS” means a disease, disorder or condition that affects the brain (e.g., frontal lobe, occipital lobe, parietal lobe and / or temporal lobe including regions such as, for example, the amygdala, basal ganglia, Broca’s area, cerebellum, corpus callosum, hypothalamus, medulla oblongata and thalamus), brain stem, glial cells (e.g., astrocytes, ependymal cells, microglia and oligodendrocytes), neurons and / or spinal cord. Examples of CNS diseases, disorders or conditions include, but are not limited to, attention deficit hyperactivity disorder (ADHD), autism, Alzheimer’s disease (AD), cancer, epilepsy,frontotemporal dementia (FTD), Huntington’s disease (HD), infection / inflammation, migraine, multiple sclerosis (MS), Parkinson’s disease (PD), seizure and stroke. In some instances, use of the LNDP for the treatment of CNS diseases may include administration via direct (i.e., central) injection into the CNS of the individual, which can be an ICV injection, an ICM injection, an IPM injection, an IT injection or a combination thereof. In other instances, administering can be via a peripheral injection including IV and SC.

[0051] A BBB target, as referred to herein, is a membrane receptor presented on the apical side of the endothelial BBB and include, but are not limited to, TfR, Low Density Lipoprotein Receptor-related Protein 1 (LRP1), LRP2 or megalin / GP33O, LRP3, LRP5, LRP6, LRP8, insulin-like growth factor receptor 1 (IGF1R), CD98 heavy chain (SLC3A2), large amino acid transporter small subunit 1 (LAT-1), insulin receptor, low-density lipoprotein (LDL) receptor, CDC50A and the like. As used herein, TfR includes both isoforms, TfRl and TfR2. Of particular interest herein is TfRl, exemplary nucleotide sequences thereof which can be found in NCBI Ref. Seq. Nos. NM_003234.4 (human), NM_011638.4 (mouse), NM_001257303.1 (non-human primate), XM_003310190.3 (non-human primate) and NM_022712.1 (rat). Likewise, exemplary amino acid sequences for TfRl monomers can be found in NCBI Ref. Seq. Nos. NP_003225.2 (human; SEQ ID NO:1), NP_035768.1 (mouse), NP_001244232.1 (non-human primate), XP_003310238.1 (non-human primate) and NP_073203.1 (rat). One of skill in the art, however, understands that additional examples of TfRl sequences are readily available using publicly available databases such as, for example, GenBank and UniProt.

[0052] Additionally, reference to an element by the indefinite article “a” or “an” does not exclude the possibility that more than one element is present, unless the context clearly requires that there be one and only one element. The indefinite article “a” or “an” thus usually means “at least one”.

[0053] As used interchangeably herein, “about” and / or “approximately”, means within a statistically meaningful range of a value or values such as, for example, a stated concentration, length, molecular weight, pH, sequence similarity, time frame, temperature, volume, etc. Such a value or range can be within an order of magnitude typically within 20%, more typically within 10%, and even more typically within 5% of a given value or range. The allowable variation encompassed by “about” and / or “approximately” will depend upon the particular system under study, and can be readily appreciated by one of skill in the art.Components of targeted LNDPs

[0054] Lipid nanoparticles useful for delivering nucleic acid cargo have been known for over a decade and are composed of several different classes of lipid molecules which imbue the nanoparticle with its properties. The categories of lipid molecules useful in making a targeted LNDP as described in the present disclosure include, but are not limited to, the following.Ionizable lipids

[0055] Ionizable lipids generally have pKa values which render the lipid neutral at physiological pH (such as in the bloodstream or after the LNDP passes through the cell membrane), but these lipids become positively charged as the pH drops (such as when associated with an endosome) in order to facilitate endosomal escape of the nucleic acid cargo. Generally, the pKa of such lipids is between 6 and 7. Ionizable lipids which may be included in LNDPs as described herein include, but are not limited to, 9Z,12Z-octadecadienoic acid, 3- [4,4-bis(octyloxy)-l-oxobutoxy]-2-[[[[3-(diethylamino)propoxy]carbonyl]oxy]methyl]propyl ester (LP-01); 4-(dimethylamino)-butanoic acid, (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31- tetraen- 19-yl 4-(dimethylamino)butanoate (D-Lin-MC3-DMA or MC3); and 9-heptadecanyl 8-((2-hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino)octanoate (SM-102), among others. The ionizable lipid is generally 35% to 60% of the LNDP (molar ratio).Structural lipids

[0056] Structural lipids assist in maintaining the structure and rigidity of the LNDP. Generally, the structural lipid is cholesterol, or a cholesterol analog. The structural lipid is generally 0% to 60% of the LNDP (molar ratio).Phospholipids (“helper” lipids)

[0057] Phospholipids contribute a degree of fluidity to the LNDP while maintaining particle integrity. Phospholipids which may be used in LNDPs as described herein include, but are not limited to, distearoylphosphatidylcholine (DSPC) and l,2-distearoyl-sn-glycero-3- phosphoethanolamine-poly(ethylene glycol) (DSPE). The phospholipid is generally 0% to 30% of the LNDP (molar ratio).Polymer-conjugated lipids

[0058] Polymer-conjugated lipids, which in embodiments include hydrophilic polymers, are useful for modulating particle size and charge. Several polymers have been described herein. In one embodiment, the polymer is a polyethylene glycol (PEG) polymer having a molecular weight of about 1 to 10 kilodaltons. In some embodiments, the lipid component of a polymer- conjugated lipid is a phospholipid. In such embodiments, the phospholipid of the polymer- conjugated lipid may be the same as the phospholipid which is deemed a “helper lipid” above.In other embodiments, the phospholipid of the polymer-conjugated lipid may be the different from the phospholipid which is deemed a “helper lipid” above. In other embodiments, the lipid component of a polymer-conjugated lipid is not a phospholipid. In specific embodiments, the polymer conjugated lipid may be l,2-distearoyl-sn-glycero-3-phosphoethanolamine- poly(ethylene glycol) (DSPE-PEG); l,2-dimyristoyl-sn-glycero-3-methoxypolyethylene glycol (DMG-PEG); or 2- [(polyethylene glycol)-2000]-N,N-ditetradecylacetamide (ALC- 0159), among others. The polymer-conjugated lipid is generally 1% to 5% of the LNDP (molar ratio).Targeting lipids

[0059] Targeting lipids, as described herein, are lipids conjugated to a targeting moiety, such as an antibody or portion thereof, a peptide, a sugar, or the like. In a specific embodiment, the targeting lipid also includes a polymer. The LNDP may be generated with a precursor (such as with a maleimide moiety) and the targeting moiety added after the LNDP is purified. Specific targeting lipids are described herein. The targeting lipid is generally present in an amount of 5% to 20% relative to the polymer-conjugated lipid, or 0.05% to 1% of the LNDP overall (molar ratio).

[0060] Exemplary LNDP compositions are provided in Table 1.Table 1: Exemplary LNDP compositions (all values mol% of total LNDP composition)EXAMPLES Example 1AExemplified Lipid Nano Delivery Particles

[0061] Exemplified LNDPs of the present disclosure comprise an outer lipid membrane comprised of phospholipids wherein a plurality of the phospholipids are conjugated to a PEG molecule. Exemplified embodiments the LNDP core composition comprises ionizable lipid (for example, 9Z,12Z-octadecadienoic acid, 3-[4,4-bis(octyloxy)-l-oxobutoxy]-2-[[[[3- (diethylamino)propoxy]carbonyl]oxy]methyl]propyl ester (LP-01) at 45 mol%, cholesterol present at 43.5 mol%, and distearoylphosphatidylcholine (DSPC) present at 9 mol%).

[0062] According to particular exemplified embodiments, lipids of the outer lipid membrane are independently conjugated to PEG, for example l,2-distearoyl-sn-glycero-3- phosphoethanolamine-poly(ethylene glycol) (DSPE-PEG; PEG length: 500-4000Da), or 1,2- dimyristoyl-sn-glycero-3-methoxypolyethylene glycol (DMG-PEG; PEG length 500-3400Da), and these PEG-lipids can be present in this example at a concentration of about 2.25% by mol. % of the particles. According to particular exemplified embodiments, approximately 2% by mol. % of PEG-lipids of the outer lipid membrane are, independently, DMG-PEG (PEG MW approximately 2kDa) and approximately 0.25% mol. % of phospholipids of the outer lipid membrane are, independently, DSPE, and conjugated to a PEG of approximately 2kDa to yield DSPE-PEG. The DSPE-PEG is further conjugated to a ligand, exemplified by a Fab targeting transferrin receptor. According to the exemplified embodiment, the Fab comprises an engineered cysteine at residue 124 of the heavy chain constant region. The exemplified Fab comprises the following amino acid sequences:Heavy chain:QSLEESGGDLVKPEGSLTLTCTASGFSFSGSYWICWVRQAPGKGLEWIGCIYSTS GGRTYYASWVKGRFTISKTSSTTVTLQMTSLTAADTATYFCARGDDSISDAYFDL WGPGTLVTVSSASTKGPCVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSG ALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVE PKSCDKTH (SEQ ID NO: 1);Light chain:ALDMTQTASPVSAAVGGTVTINCQSSQSVYNNNRLAWYQQKPGQPPKLLIYDAS TLASGVPSRFKGSGSGTQFTLTISGVQSDDSATYYCQGTYFSSGWSWAFGGGTEV VVKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGE C (SEQ ID NO: 2).

[0063] Additionally, according to exemplified embodiments the LNDP inner core composition comprises a hydrophobic core and includes a nucleic acid (e.g., a therapeutic nucleic acid) therein. Further exemplified embodiments include an oily inner core. Exemplified embodiments include an siRNA nucleic acid. Additional exemplified embodiments include an mRNA encapsulated therein.Encapsulation of Therapeutic Nucleic Acids

[0064] Encapsulation of nucleic acids according to the present disclosure may be accomplished substantially as set forth below. Briefly, by way of exemplification, mRNA coding for Cre protein, is encapsulated with exemplified LNDPs of the present disclosure according to the following: a. Lipids are combined in 200-proof ethanol in the specific ratios of 2.25mol% DSPE-PEG (2kDa), 0.25mol% DSPE-PEG-Maleimide (2kDa), 45 mol% LP-01 (9Z,12Z- octadecadienoic acid, 3 - [4,4-bis(octyloxy)- 1 -oxobutoxy ]-2- [ [ [ [3-(diethylamino)propoxy]carbonyl]oxy]methyl]propyl ester), 43.5mol% cholesterol, and 9mol% DSPC (distearoylphosphatidylcholine). b. If the lipids do not fully dissolve, process is repeated. c. Prepare the RNA solution in water and mix with sodium citrate buffer, d. Mix aqueous and lipid solutions using microfluidic channels. The following exemplified parameters may be used: flow rate volume ratio to 3:1 (RNA: lipid), total flow rate 12 mL / min. e. Buffer exchange is performed by preparing PD-10 columns to exchange ethanol and citrate buffer to 0.1 M PBS.Conjugation of a Targeting Moiety to the Lipid Nano Delivery Particles

[0065] Exemplified LNDPs of the present disclosure may have a targeting moiety conjugated thereto substantially as described herein. Fabs, functionalized with a redoxed thiol, are presented to the LNDPs at a molar ratio of 4:1 (maleimide-PEG-DSPE:fab fragment) for 4 hours. Isotype lipid nano particle controls may be made in the same manner but with nontargeting analogs of the same fab or other structure. Bare lipid nanoparticle controls may also be made in the same manner, but with functionalized lipid-PEG molecules replaced bynon-functionalized lipid-PEG molecules (e.g., no reactive group associated with the PEG molecule or other hydrophilic polymer).Pharmacokinetic (Pk) Assessment

[0066] Pharmacokinetics of exemplified LNDPs of the present disclosure may be assessed according to the following. Briefly, female C57BL / 6 mice at 8 weeks are dosed with 1 mg / kg of (i) LNDPs of the present disclosure comprising a transferrin receptor-targeting moiety with encapsulated Cy3-labeled siRNA, or (ii) PBS, or (iii) control isotype lipid nano particles or (iv) bare lipid nano particle. Levels of the respective molecules are determined by fluorescent imaging of the bulk tissue and flow cytometry of homogenized tissue at 4 and 18 hours, respectively. Following procedures substantially as described herein, a peak of approximately 65-95% of endothelial cells are positive for the fluorescent Cy3-labeled siRNA after around between 4-48hrs. Clearance from blood circulation is demonstrated by 48 hours. Results are provided in FIG. 4 and suggest enhanced PK over bare and isotype lipid nano particle controls.Targeted Delivery - Peripheral

[0067] Targeting of non-CNS organs by exemplified LNDPs of the present disclosure may be demonstrated according to the following. Briefly, female C57BL / 6 mice at 8 weeks are dosed via IV with 2 mg / kg of (I) LNDPs of the present disclosure comprising a transferrin receptor-targeting moiety with encapsulated Alexafluor647 labeled siRNA, or (ii) PBS, or (iii) control isotype lipid nano particles or (iv) bare lipid nano particle in two separate studies. Mice are sacrificed four hours after dosing and levels of the respective molecules are determined at brain, heart, spleen, lings, kidney, gastric and liver tissue. Results are provided in FIG. 5 and suggest enhanced targeting of exemplified LNDPs of the present disclosure over isotype and bare lipid nano particle controls.Targeted Delivery - Blood Brain Barrier

[0068] Targeting of the blood-brain barrier, and therapeutic nucleic acid activity, by exemplified LNDPs of the present disclosure may be demonstrated according to the following. Briefly, female C57BL / 6 mice at 8 weeks are dosed via IV with 2 mg / kg of (i) LNDPs of the present disclosure comprising a transferrin receptor-targeting moiety with encapsulated Cy3- siRNA or Cre mRNA, or (ii) PBS, or (III) control isotype lipid nano particle or (IV) bare lipid nano particles in two separate studies. Mice are sacrificed four hours after dosing and levelsof the respective molecules are determined at brain, and Cre3 mRNA activity are determined. Results are provided in FIG. 6 and suggest enhanced targeting of exemplified LNDPs of the present disclosure over isotype and bare lipid nano particle controls as well as therapeutic nucleic acid activity.Example IBTargeted Delivery - Enhanced in vitro Activity

[0069] Targeting-enriched therapeutic nucleic acid activity, by exemplified LNDPs of the present disclosure may be demonstrated according to the following and may be accomplished substantially as set forth in the method provided in Example 1A. Briefly, N2A and BV2 cells were cultured at 5 x 103cells / well and treated at doses from 0 to 100 nM siRNA and incubated for 24 hours. The siRNA was formulated as (i) LNDPs of the present disclosure comprising a transferrin receptor- targeting moiety, or (ii) bare lipid nano particles, or (III) control isotype lipid nano particle, or (IV) PBS. HPRT expression was measured by Taqman qPCR and normalized against the expression of Actin-b. Results are provided in Table 2 normalized to the PBS-treated group and suggest enhanced targeting of exemplified LNDPs of the present disclosure over isotype and bare lipid nano particle controls as well as therapeutic nucleic acid activity. LNDPs with TfR-binding fab delivering the HPRT siRNA resulted in enriched delivery to BV2 cells (a murine microglial line) and N2A cells (a murine neuroblastoma line) over undecorated and LNDPs decorated with isotype control fab.Table 2: Reduced HPRT expression from HPRT expression from HPRT siRNA delivered by TfR-binding fab decorated in human TfR ELISAExample 1CIn vivo Brain Enrichment

[0070] LNDPs with TfR-binding fab delivering Alexafluor647-labeled HPRT siRNA (encapsulation of which was achieved substantially as set forth in the method provided in Example 1 A) resulting in enriched delivery to the brain over undecorated and LNDPs decorated with isotype control fab. Delivery is quantified by ex vivo invital imaging (IVIS) as total flux (photons / second, p / s). Results are shown in Table 3.Table 3: Enhanced delivery of Alexafhior647-labeled HPRT siRNA from delivery by TfR-binding fab-decorated LNDP in C57BL / 6 miceExample 2Exemplified Lipid Nano Delivery Particles

[0071] Exemplified LNDPs of the present disclosure comprise an outer lipid membrane comprised of phospholipids wherein a plurality of the phospholipids are conjugated to a PEG molecule. Exemplified embodiments of the LNDP core composition comprises ionizable lipid (for example, 4-(dimethylamino)-butanoic acid, (6Z,9Z,28Z,31Z)-Heptatriaconta-6,9,28,31-tetraen- 19-yl 4-(dimethylamino)butanoate (D-Lin-MC3-DMA) at 50 mol%, cholesterol present at 37.5 mol%, and distearoylphosphatidylcholine (DSPC) present at 10 mol%). Additional exemplified embodiments of the LNDP core composition comprises ionizable lipid (for example, 9Z,12Z-octadecadienoic acid, 3-[4,4-bis(octyloxy)-l-oxobutoxy]-2-[[[[3- (diethylamino)propoxy]carbonyl]oxy]methyl]propyl ester (LP-01) at 45 mol%, cholesterol present at 43.5 mol%, and distearoylphosphatidylcholine (DSPC) present at 9 mol%).

[0072] According to particular exemplified embodiments, lipids at the outer lipid membrane comprise, independently, DMG-PEG of approximately 2kDa at approximately 2.5% of total lipid in LNP. According to some embodiments, between 2.5-25% of the total PEG-lipids may be replaced by DSPE-PEG-glucose of approximately 2kDa.

[0073] Additionally, according to exemplified embodiments the LNDP inner core composition comprises a hydrophobic core and includes a nucleic acid (e.g., a therapeutic nucleic acid) therein. Further exemplified embodiments include an oily inner core. Exemplified embodiments include an siRNA nucleic acid. Additional exemplified embodiments include an mRNA encapsulated therein.Encapsulation of Therapeutic Nucleic Acids

[0074] Encapsulation of nucleic acids according to the present disclosure may be accomplished substantially as set forth in the method provided in Example 1A.Pharmacokinetic (Pk) Assessment

[0075] Pharmacokinetics of exemplified LNDPs of the present disclosure may be assessed according to the following processes. Briefly, male B ALB / c mice at 8 weeks are put on 24 hour fasting before dosing via tail vein injection with 2mg / kg of (i) LNDPs of the present disclosure comprising a GLUT 1 -targeting moiety, with encapsulated Alexafluor647-labeled siRNA, (ii) PBS, or (iii) bare lipid nano particles. Thirty minutes following sample injection, 200 uL of 20% glucose solution in PBS is injected through intraperitoneal administration. Levels of the respective molecules are determined by fluorescent imaging of the bulk tissue at 4-6 hours. Results are provided in FIG. 7 and demonstrate that glucose decorated exemplified LNDPs with siRNA cargo show enhanced localization to brain.Therapeutic Nucleic Acids Accumulation at Cellular Level

[0076] Cellular level distribution of exemplified LNDPs at the brain may be assessed according to the following. Briefly, female C57BL / 6J mice aged 8-10 weeks are put on 24 hour fasting before dosing via tail vein (IV) injection or intracarotid (IC) injection with 2mg / kg of (i) exemplified LNDP carrying Cy3-labeled siRNA or (ii) PBS. Thirty minutes after sample injection, 200 uL of 20% glucose solution in PBS was injected through intraperitoneal administration. Levels of the respective molecules at the brain cellular level are determined by flow cytometry 24 h after injection. Result are provided in FIG. 8 and demonstrate that glucose decorated exemplified LNDP of the present invention with siRNA cargo shows penetration of the BBB.Example 3Exemplified Lipid Nano Delivery Particles

[0077] Exemplified LNDPs of the present disclosure comprise an outer lipid membrane comprised of phospholipids wherein a plurality of the phospholipids are conjugated to a PEG molecule. Exemplified embodiments the LNDP core composition comprises ionizable lipid (for example, 9Z,12Z-octadecadienoic acid, 3-[4,4-bis(octyloxy)-l-oxobutoxy]-2-[[[[3- (diethylamino)propoxy]carbonyl]oxy]methyl]propyl ester (LP-01) at 45 mol%, cholesterol present at 43.5 mol%, and distearoylphosphatidylcholine (DSPC) present at 9 mol%).

[0078] According to particular exemplified embodiments, lipids of the outer lipid membrane are independently polymer- lipid conjugates such as l,2-distearoyl-sn-glycero-3- phosphoethanolamine-poly(ethylene glycol) (DSPE-PEG; PEG length: 500-4000Da), or 1,2- dimyristoyl-sn-glycero-3-methoxypolyethylene glycol (DMG-PEG; PEG length 500-3400Da), and can be present in this example at a concentration of about 2.25% by mol. % of the particles. According to particular exemplified embodiments, approximately 2% by mol. % of the LNDP are lipids of the outer lipid membrane and are, independently, DMG conjugated to PEG of approximately 2kDa, and approximately 0.25% mol. % of are, independently, DSPE conjugated to PEG of approximately 2kDa, to yield DSPE-PEG. The DSPE-PEG is further conjugated to a ligand, exemplified by peptides targeting human transferrin receptor. According to exemplified embodiments, the peptides comprise a target binding region containing approximately 7-20 amino acid sequences and a peptide or PEG (approximately 88- 700Da) linker attached to the c-terminal of the binding region. The linker region comprisesdibenzocyclooctyne (DBCO) or tetrazine. The exemplified peptides comprise the following amino acid sequences:T7 peptide: HAIYPRH (SEQ ID NO: 6) (European Journal of Biochemistry, 2001, 268(7), 2004-2012.); andLilly proprietary peptide: amino acid sequence (not shown).

[0079] Additionally, according to exemplified embodiments the LNDP inner core composition comprises a hydrophobic core and includes a nucleic acid (e.g., a therapeutic nucleic acid) therein. Further exemplified embodiments include an oily inner core. Exemplified embodiments include a siRNA nucleic acid. Additional exemplified embodiments include an mRNA encapsulated therein.Encapsulation of Therapeutic Nucleic Acids

[0080] Encapsulation of nucleic acids according to the present disclosure may be accomplished substantially as set forth in the method provided in Example 1A.Conjugation of a Targeting Moiety to the Lipid Nano Delivery Particles

[0081] Exemplified LNDPs of the present disclosure may have a targeting moiety conjugated thereto substantially as described herein. Peptides, functionalized with a DBCO, are presented to the LNDPs at a molar ratio of 2:1 (Azide-PEG-DSPE:peptides) for 40 hours.Targeted Delivery - Brain

[0082] Targeted delivery to the brain of exemplified LNDPs of the present disclosure may be assessed according to the following. Briefly, female C57BL / 6NTac-Tfrctm7330.1_B- F1 l(TFRC*)Tac at 19 weeks are dosed via IV with 2 mg / kg of (i) LNDPs of the present disclosure comprising a transferrin receptor targeting moiety with encapsulated Alexafluor647- labeled siRNA, (ii) PBS, (III) bare lipid nano particles, or (iv) LNDP comprising a T7 protein. Mice are sacrificed four hours after dosing and levels of the respective molecules are determined at brain, and Cre3 mRNA activity are determined. Results are provided in FIG. 9 and suggest enhanced targeting of exemplified LNDPs of the present disclosure over isotype and bare lipid nano particle controls as well as therapeutic nucleic acid activity. Four hours later, levels of respective molecules are determined by fluorescent imaging. Following procedures as described herein, a 6-fold increase in brain total fluorescent signal is observedfor Lilly proprietary peptide targeted nano delivery particles compared to non-targeted nano delivery particles. Results are shown in FIG. 9.Example 4In vitro delivery to T cells

[0083] Targeted delivery to T cells of exemplified LNDPs of the present disclosure may be assessed according to the following. A CD7 targeting moiety, in this case a fab fragment, may be accomplished substantially as set forth in the method provided in Example 1A. Briefly, cultured primary human T cells are dosed with either a dose range of (i) LNDPs of the present disclosure comprising a CD7 targeting moiety with encapsulated HPRT siRNA, (ii) PBS, (iii) bare lipid nano particles, or (iv) cholesterol-labeled HPRT siRNA. Cells were incubated for 24 hours after dosing and levels of gene expression silencing were quantified by qPCR, and HPRT mRNA activity are determined. Results are provided in FIG. 10 and suggest enhanced targeting of exemplified LNDPs of the present disclosure over bare lipid nano particle and lipid-siRNA controls as well as successful nucleic acid delivery.SEQUENCE LISTING

[0084] SEQ ID NO. 1 : amino acid sequence of exemplified heavy chain Fab targeting TfR.QSLEESGGDLVKPEGSLTLTCTASGFSFSGSYWICWVRQAPGKGLEWIGCIYSTSGGR TYYASWVKGRFTISKTSSTTVTLQMTSLTAADTATYFCARGDDSISDAYFDLWGPGT LVTVSSASTKGPCVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVH TFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTH

[0085] SEQ ID NO. 2: amino acid sequence of exemplified light chain Fab targeting TfR. ALDMTQTASPVSAAVGGTVTINCQSSQSVYNNNRLAWYQQKPGQPPKLLIYDASTL ASGVPSRFKGSGSGTQFTLTISGVQSDDSATYYCQGTYFSSGWSWAFGGGTEVVVKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVT EQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0086] SEQ ID NO. 3: Cre mRNA SequenceAUGGGCACCCCCAAGAAGAAGCGGAAGGUGCGGACCGACCUGAACAGCAACCU GCUGACCGUGCACCAGAACCUGCCCGCCCUGCCCGUGGACGCCACCAGCGACG AGGUGCGGAAGAACCUGAUGGACAUGUUCCGGGACCGGCAGGCCUUCAGCGAG CACACCUGGAAGAUGCUGCUGAGCGUGUGCCGGAGCUGGGCCGCCUGGUGCAA GCUGAACAACCGGAAGUGGUUCCCCGCCGAGCCCGAGGACGUGCGGGACUACC UGCUGUACCUGCAGGCCCGGGGCCUGGCCGUGAAGACCAUCCAGCAGCACCUG GGCCAGCUGAACAUGCUGCACCGGCGGAGCGGCCUGCCCCGGCCCAGCGACAG CAACGCCGUGAGCCUGGUGAUGCGGCGGAUCCGGAAGGAGAACGUGGACGCCG GCGAGCGGGCCAAGCAGGCCCUGGCCUUCGAGCGGACCGACUUCGACCAGGUG CGGAGCCUGAUGGAGAACAGCGACCGGUGCCAGGACAUCCGGAACCUGGCCUU CCUGGGCAUCGCCUACAACACCCUGCUGCGGAUCGCCGAGAUCGCCCGGAUCC GGGUGAAGGACAUCAGCCGGACCGACGGCGGCCGGAUGCUGAUCCACAUCGGC CGGACCAAGACCCUGGUGAGCACCGCCGGCGUGGAGAAGGCCCUGAGCCUGGG CGUGACCAAGCUGGUGGAGCGGUGGAUCAGCGUGAGCGGCGUGGCCGACGACC CCAACAACUACCUGUUCUGCCGGGUGCGGAAGAACGGCGUGGCCGCCCCCAGC GCCACCAGCCAGCUGAGCACCCGGGCCCUGGAGGGCAUCUUCGAGGCCACCCA CCGGCUGAUCUACGGCGCCAAGGACGACAGCGGCCAGCGGUACCUGGCCUGGA GCGGCCACAGCGCCCGGGUGGGCGCCGCCCGGGACAUGGCCCGGGCCGGCGUG AGCAUCCCCGAGAUCAUGCAGGCCGGCGGCUGGACCAACGUGAACAUCGUGAUGAACUACAUCCGGAACCUGGACAGCGAGACCGGCGCCAUGGUGCGGCUGCUGG AGGACGGCGACUGA

[0087] SEQ ID NO. 4: HPRT siRNA Sense StrandUCCUAUGACUGUAGAUUUUAU

[0088] SEQ ID NO. 5: HPRT siRNA Antisense StrandAUAAAAUCUACAGUCAUAGGAAU

[0089] SEQ ID NO. 6: T7 PeptideHAIYPRH

[0090] SEQ ID NO. 7: amino acid sequence of exemplified heavy chain Fab targetingCD7.EVQLVESGGGLVKPGGSPRLSCAAYGVTFSNAWMSWVRQAPGKGLEWVGRIKSKT DGGTTDYAAPVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCTIEAVAGHFDYWG QGTLVTVSSASTKGPCVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTS GVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDK TH

[0091] SEQ ID NO. 8: amino acid sequence of exemplified light chain Fab targeting CD7. IQMTQSPSTLSASVGDRVTITCRASQSISSWLAWYQQKPGKAPKLLIYKASSLESGVP SRFSGSGSGTEFTLTISSLQPDDFATYYCQQYNNYSPTFGQGTKVEIKRTVAAPSVFIF PPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYS LSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

Claims

CLAIMSWe claim:

1. A lipid nano delivery particle comprising: an outer lipid membrane comprising phospholipids; and an inner core, the inner core being interior to the outer lipid membrane, wherein a plurality of the phospholipids of the outer lipid membrane are conjugated to a hydrophilic polymer and a targeting moiety.

2. The lipid nano delivery particle of claim 1 , wherein the hydrophilic polymer is selected from a PEG molecule, a zwitterionic polymer or OEGMA.

3. The lipid nano delivery particle of claim 1 or 2, wherein the hydrophilic polymer is a PEG molecule.

4. The lipid nano delivery particle of any one of claims 1-3, wherein the lipid nano delivery particle is selected from the group consisting of an enveloped nanocomplex (eNCP), a lipid nanoparticle (LNP), a liposome, a nanocapsule (NC), a nanocrystal (NCR), a polymer lipid nanoparticle (PLNP), a nanoemulsion (NE) and a proteolipid vehicle (PLV).

5. The lipid nano delivery particle according to any one of claims 1-4, wherein the inner core is oily or liquid.

6. The lipid nano delivery particle according to any one of claims 1-4, wherein the inner core is solid.

7. The lipid nano delivery particle according to any one of claims 1-6 further comprising an inner lipid membrane comprising phospholipids, wherein the inner lipid membrane is interior to the outer lipid membrane.

8. The lipid nano delivery particle according to any one of claims 1-7, wherein the targeting moiety is a peptide, antibody, Fab, scFv, VHH, or organic compound.

9. The lipid nano delivery particle according to any one of claims 1-8, wherein the targeting moiety specifically binds transferrin receptor (TfR).

10. The lipid nano delivery particle of claim 9, wherein the targeting moiety that specifically binds TfR has an affinity of between 0.1-500 nanomolar.

11. The lipid nano delivery particle according to any one of claims 1-8, wherein the targeting moiety specifically binds a target on a peripheral cell.

12. The lipid nano delivery particle according to claim 11 , wherein the peripheral cell is a T cell.

13. The lipid nano delivery particle according to any one of claims 1-12, wherein the hydrophilic polymer has a molecular weight selected from the group consisting of: between 1 and 10 kDa; between 1 and 5 kDa; and between 1 and 3.5 kDa.

14. The lipid nano delivery particle according to any one of claims 1-12, wherein the hydrophilic polymer has a molecular weight of approximately 2.5 kDa.

15. The lipid nano delivery particle according to any one of claims 1-14, wherein the hydrophilic polymer is a PEG molecule.

16. The lipid nano delivery particle according to any of claims 1-15, wherein the targeting moiety is conjugated to a hydrophilic polymer by a maleimide reaction.

17. The lipid nano delivery particle according to any of claims 1-15, wherein the targeting moiety is conjugated to a hydrophilic polymer by an azide / dibenzocyclooctyne (DBCO) reaction or by an N-Hydroxysuccinimide (NHS) / amine reaction.

18. The lipid nano delivery particle according to any one of claims 1-17, wherein between approximately 1 percent to approximately 5 percent mole percentage of the lipid nano delivery particle is the plurality of the phospholipids of the outer lipid membrane having a hydrophilic polymer conjugated thereto.

19. The lipid nano delivery particle according to any one of claims 1-18, wherein between approximately 1 percent to approximately 2.5 percent mole percentage of the lipid nano delivery particle is the plurality of the phospholipids of the outer lipid membrane having a hydrophilic polymer conjugated thereto.

20. The lipid nano delivery particle according to any one of claims 1-19, wherein between approximately 5 percent to approximately 20 percent of the hydrophilic polymers conjugated to phospholipids of the outer lipid membrane have a targeting moiety conjugated thereto.

21. The lipid nano delivery particle according to any one of claims 1-20, wherein approximately 10 percent of the hydrophilic polymers conjugated to phospholipids of the outer lipid membrane have a targeting moiety conjugated thereto.

22. The lipid nano delivery particle according to any one of claims 1-21, wherein targeting moieties are conjugated to the hydrophilic polymers via a cleavable linker.

23. The lipid nano delivery particle according to any one of claims 1-22, wherein the inner core comprises a therapeutic nucleic acid .

24. The lipid nano delivery particle of claim 23, wherein the therapeutic nucleic acid comprises one or a combination of any of mRNA, guide RNAs (gRNAs), microRNA (miRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), interfering RNA (iRNA), antisense oligonucleotide (ASO), Dicer substrate interfering RNA (DsiRNA), miRNA, short hairpin RNA (shRNA), short interfering RNA (siRNA), short activating RNAs (saRNAs), suppressor tRNAs (sup-tRNAs), circularized RNA (circRNA), and activating RNA (aRNA).

25. A method of targeted transport of a therapeutic nucleic acid to a patient, said method comprising: encapsulating a therapeutic nucleic acid within the inner core of a lipid nano delivery particle of any one of claims 1-22; and administering the lipid nano delivery particle comprising the therapeutic nucleic acid to a patient.

26. The method of claim 25, wherein the therapeutic nucleic acid comprises one or a combination of any of mRNA, guide RNAs (gRNAs), microRNA (miRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), interfering RNA (iRNA), antisense oligonucleotide (ASO), Dicer substrate interfering RNA (DsiRNA), miRNA, short hairpin RNA (shRNA), short interfering RNA (siRNA), short activating RNAs (saRNAs), suppressor tRNAs (sup-tRNAs), circularized RNA (circRNA), and activating RNA (aRNA).

27. The method of any one of claims 25-26, wherein said step of administering comprises any of enteral, parenteral, intravenous, intramuscular, intra-arterial, intramedullary, intrathecal, intraocular, subcutaneous, intraventricular, transdermal, interdermal, rectal, intravaginal, intraperitoneal, topical, mucosal, nasal, buccal, sublingual, and intracarotid.

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