DESC:FORM 2
THE PATENTS ACT, 1970
(39 of 1970)
&
THE PATENTS RULES, 2003
COMPLETE SPECIFICATION
(Section 10 and Rule 13)

1. TITLE OF THE INVENTION:
DELIVERY SYSTEM FOR CRISPR CAS COMPONENTS

2. APPLICANT:
MICRO CRISPR Pvt. Ltd., an Indian company of the address Survey No: 1574, Muktanand Marg, Chala, Vapi-396191, Gujarat, India.

The following specification particularly describes the invention and the manner in which it is to be performed:

FIELD OF INVENTION
[1] The present disclosure relates to a particle and a delivery system formed thereof. More particularly, the present disclosure relates to a nanoparticle-based delivery system for CRISPR-Cas components.
BACKGROUND OF INVENTION
[2] In vivo techniques for gene editing within the cells offer a significant potential for addressing a wide range of medical conditions, including genetic, viral, bacterial, autoimmune, cancer, aging-related, and inflammatory diseases. In recent years, drug delivery systems used to deliver gene-editing agents for treatment of various diseases/conditions include a carrier like a micelle or a nanoparticle that protects the gene editing agent from degradation and allows it to be delivered at the desired site of treatment.
[3] Lipids and nanoparticles made using lipids, are potent to carry and deliver gene-editing agents to the desired site of treatment. For example, lipid nanoparticles are used to encapsulate and deliver complex nucleic acid molecules, proteins, and other entities of biological origin.
[4] Accordingly, the lipid nanoparticles are being studied as an alternative to conventionally available delivery systems, like vaccines. Gene-editing agents are encapsulated into the lipid nanoparticles and administered to individuals for therapeutic effect. The compositions of the lipid nanoparticles influence the characteristic properties (such as, targeting ability (or affinity) of the delivery system, amount of gene-editing agent being encapsulated, particle size, etc.) of the delivery system formed.
[5] However, conventionally available delivery systems (made of lipid nanoparticles) are incompetent as they prematurely degrade exposing the gene-editing agents to vulnerable environments, inefficient translation of the mRNAs (an exemplary gene-editing agent) due to inefficient release of the mRNA into the host cell, incompatibility with the membranes of the host cell leading to inefficient cellular uptake. The aforesaid challenges with the conventional lipid nanoparticles encapsulating gene-editing agents lead to limited therapeutic efficacy and often result in toxicity to host cells/tissues.
[6] Further, specially in cases where conventional nanoparticles are used to encapsulate nucleic acids (like DNA, RNA), the nucleic acid may go through nicking, degradation, or structural changes during long-term storage or freeze/thaw cycles leading to partial or total loss of therapeutic effectiveness. Due to the aforesaid shortcomings, the shelf-life of such conventional nanoparticle is very less.
[7] Thus, there is a need for a delivery system that overcomes the problems associated with the conventional delivery systems.
SUMMARY OF INVENTION
[8] Particular embodiments of the present disclosure are described herein below with reference to the accompanying drawings; however, it is to be understood that the disclosed embodiments are mere examples of the disclosure, which may be embodied in various forms. Well-known functions or constructions are not described in detail to avoid obscuring the present disclosure in unnecessary detail. Therefore, specific structural and functional details disclosed herein are not to be interpreted as limiting, but merely as a basis for the claims and as a representative basis for teaching one skilled in the art to variously employ the present disclosure in virtually any appropriately detailed structure.
[9] In an embodiment, the present disclosure relates to a particle including at least one bi-layer outer membrane, a plurality of vacuoles and at least one payload. The bi-layer outer membrane defining a lumen including an outer layer and an inner layer. The outer layer defines a hydrophobic portion and a hydrophilic portion. The outer layer including at least a plurality of cationic lipids, a plurality of neutral lipids, a plurality of helper lipid, and a plurality of polyethers. The inner layer is disposed between the outer layer and the lumen. The inner layer defines a hydrophobic portion and a hydrophilic portion. The inner layer including at least the plurality of cationic lipids, the plurality of neutral lipids, and the plurality of helper lipids. The vacuoles are disposed within the lumen defined by the bi-layer outer membrane. Each of the vacuole includes a membrane layer defining a lumen. The membrane layer including at least the plurality of cationic lipids, the plurality of neutral lipids, and the plurality of helper lipids. The payload is encapsulated within the lumen of the at least one vacuole. The payload includes at least one mRNA and at least one gRNA in a ratio by weight of 1:1, 1:3, 1:7, 1:10, 3:1, 7:1, or 10:1. The mRNA encodes for at least one of an endonuclease, and a base editor nuclease. The gRNA includes at least one crRNA having binding specificity to a portion a genomic DNA of a host cell.
BRIEF DESCRIPTION OF DRAWINGS
[10] The summary above, as well as the following detailed description of illustrative embodiments, is better understood when read in conjunction with the apportioned drawings. For the purpose of illustrating the present disclosure, exemplary constructions of the disclosure are shown in the drawings. However, the disclosure is not limited to specific methods and instrumentality disclosed herein. Moreover, those in the art will understand that the drawings are not to scale.
[11] Fig. 1 depicts a particle 100 of a delivery system according to an embodiment of the present disclosure.
[12] Fig. 2 depicts a method 200 to prepare the particle 100 of the delivery system according to an embodiment of the present disclosure.
[13] Fig. 2a depicts an assembly 300 for preparing the particle 100 of the delivery system according to an embodiment of the present disclosure.
[14] Figs. 3-25 depict experimental data related to the particle 100 according to one or more embodiments of the present disclosure.
DETAILED DESCRIPTION OF THE ACCOMPANYING DRAWINGS
[15] Prior to describing the invention in detail, definitions of certain words or phrases used throughout this patent document will be defined: the terms "include" and "comprise", as well as derivatives thereof, mean inclusion without limitation; the term "or" is inclusive, meaning and/or; the phrases "coupled with" and "associated therewith", as well as derivatives thereof, may mean to include, be included within, interconnect with, contain, be contained within, connect to or with, couple to or with, be communicable with, cooperate with, interleave, juxtapose, be proximate to, be bound to or with, have a property of, or the like. Definitions of certain words and phrases are provided throughout this patent document, and those of ordinary skill in the art will understand that such definitions apply in many, if not most, instances to prior as well as future uses of such defined words and phrases.
[16] Reference throughout this specification to “one embodiment,” “an embodiment,” or similar language means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment. Thus, appearances of the phrases “in one embodiment,” “in an embodiment,” and similar language throughout this specification may, but do not necessarily, all refer to the same embodiment, but mean “one or more but not all embodiments” unless expressly specified otherwise. The terms “including,” “comprising,” “having,” and variations thereof mean “including but not limited to” unless expressly specified otherwise. An enumerated listing of items does not imply that any or all of the items are mutually exclusive and/or mutually inclusive, unless expressly specified otherwise. The terms “a,” “an,” and “the” also refer to “one or more” unless expressly specified otherwise.
[17] Although the operations of exemplary embodiments of the disclosed method may be described in a particular, sequential order for convenient presentation, it should be understood that the disclosed embodiments can encompass an order of operations other than the particular, sequential order disclosed. For example, operations described sequentially may in some cases be rearranged or performed concurrently. Further, descriptions and disclosures provided in association with one particular embodiment are not limited to that embodiment, and may be applied to any embodiment disclosed herein. Moreover, for the sake of simplicity, the attached figures may not show the various ways in which the disclosed system, method, and apparatus can be used in combination with other systems, methods, and apparatuses.
[18] Furthermore, the described features, advantages, and characteristics of the embodiments may be combined in any suitable manner. One skilled in the relevant art will recognize that the embodiments may be practiced without one or more of the specific features or advantages of a particular embodiment. In other instances, additional features and advantages may be recognized in certain embodiments that may not be present in all embodiments. These features and advantages of the embodiments will become more fully apparent from the following description and apportioned claims, or may be learned by the practice of embodiments as set forth hereinafter.
[19] The present disclosure discloses a nanoparticle-based delivery system (or system) including a plurality of particles to deliver gene-editing agents (for example, CRISPR-Cas components) and a method to prepare the system. In an exemplary embodiment, the system of the present disclosure is used to deliver a messenger ribonucleic acid (mRNA) and guide ribonucleic acid (gRNA) to a host cell. The host cell may be a mammalian cell including, but not limited to, a liver cell, liver stem cells, liver sinusoidal endothelial cells (LSECs), Kupffer cells, hepatic stellate cells, and liver tumor cells. In an exemplary embodiment, the host cell is a liver stem cell. In another exemplary embodiment, the host cell is a cell expressing ApoE-binding receptors. The combination of the mRNA and the gRNA (mRNA+gRNA) may edit one or more gene of a host cell to treat various conditions like allergies, autoimmune disease, infectious diseases, cancers, rare diseases, genetic disorders, etc. In an exemplary embodiment, the system of the present disclosure is used to deliver the mRNA+gRNA to treat conditions like but not limited to tuberculosis, hypertension, leukemia.
[20] The particles of the system of the present disclosure are non-toxic and biodegradable, thus, do not accumulate to toxic levels inside the body when administered in therapeutically effective doses. The particles (a compositions thereof) do not trigger any innate immune response that would result in significant adverse effect when administered in therapeutically effective doses.
[21] The system of the present disclosure blocks the degradation of mRNA+gRNA in the plasma and facilitate efficient cellular uptake of nucleotides (such as mRNA+gRNA).
[22] The system of the present disclosure includes a plurality of particles. Each particle of the system includes a mixture of components arranged in a pre-defined arrangement. The pre-defined arrangement may be in any suitable shape/order to encapsulate and deliver a pre-defined concentration of a payload. The components include one or more lipids, at least one helper lipid (or structural lipid), at least one polyether (or stealth lipid), and at least one nucleic acid stabilizer in a pre-defined concentration. The pre-defined concentration of the components influences, without limitation, the targeting ability (or affinity) of the system towards a pre-defined host cell, amount/length of a payload being encapsulated in each particle, particle size, etc. The one or more lipids may be selected from one or more cationic lipids (or ionizable lipids), one or more neutral lipids, etc.
[23] The system may include a plurality of particle having a pre-defined particle size (i.e., median diameter) ranging from 10 nm – 250 nm. The particle size of the particles is measured by a dynamic light scattering (DLS) technique, for example, using a Zetasizer Ultra (Malvern, USA). In an exemplary embodiment, the particles of the system include a unimodal particle size distribution thus providing homogenous encapsulation and dosage. The pre-defined particle size of the system allows the particles to efficiently drain to the lymph nodes after being administered (for example, subcutaneously). In an exemplary embodiment, the pre-defined particle size of the system prevents digestion of the particles by the macrophages and the particles are preferentially taken up by the dendritic cells. The physicochemical properties of the particles may be modified to enhance selectivity for particular bodily targets. For example, the particle size can be adjusted based on the fenestration sizes of different organs.
[24] The particles of the system may have a polydispersity index (PDI) ranging from 0.005 to 0.75. Alternatively, the PDI of the particles of the system may range from 0 to 0.08.
[25] The particles of the system may have a pre-defined zeta potential ranging from -60 mV to +60 mV. The said zeta potential of the system provides stability to the system during long term storage by preventing aggregation of the particles due to like-charge repulsion.
[26] Each particle of the system may encapsulate a pre-defined amount of payload having a pre-defined size (or length). The payload includes at least one messenger RNA (mRNA), at least one guide RNA (gRNA), or a combination thereof. The mRNA and the gRNA may together be encapsulated in the particle at a pre-defined ratio (by weight or mole) with respect to each other. The ratio between the gRNA and the mRNA may be one of 50:1, 40:1, 30:1, 20:1, 18:1, 16:1, 14:1, 12:1, 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, or 1:10 by weight or mole. In an exemplary embodiment, the ratio between the gRNA and the mRNA is 1:1 by weight. In another exemplary embodiment, the ratio between the gRNA and the mRNA is 1:1 by mole.
[27] The amount of payload encapsulated by the particles may be represented as a ratio by weight between the particle and the payload. The amount of payload (i.e., the mRNA and the gRNA) in the system may be determined using absorption spectroscopy techniques, such as ultraviolet-visible spectroscopy. The ratio by weight between the particle and the payload is at least one of 5:1, 6:1, 10:1, 20:1, or 40:1. The encapsulation efficiency (EE) of the mRNA and the gRNA in the particles of the system is at least 80%.
[28] The mRNA may encode for at least one of an endonuclease, a base editor nuclease, etc. The endonuclease may be at least one of a Cas9 nuclease, a FnCas9 nuclease, a Cpf nuclease, etc. The endonuclease may introduce a single stranded break (SSB) or a double stranded break (DSB) in a polynucleotide sequence (for example, genomic deoxyribonucleic acid or gDNA). The base editor nuclease may be at least one of a cytosine base editor (CBE), an adenine base editor (ABE), etc. In an exemplary embodiment, the mRNA encodes for Cas9 endonuclease derived from Streptococcus pyogenes.
[29] The mRNA may include one or more untranslated regions (UTRs) disposed at a 3’ end or a 5’ end of the mRNA. The UTR may be derived from human a- and ß-globin, human albumin, human HSD17B4, human eukaryotic elongation factor 1a, orthopoxvirus or cytomegalovirus. The UTR helps in mRNA metabolism.
[30] Additionally or optionally, the mRNA may include a 3’ tail, and a 5’ cap. The 5’ cap includes for example, m7G(5')ppp(5')N which may be a cap-0 (without 2'OMe), cap-1 (with 2'OMe at nucleotide N), or cap-2 (with 2'OMe at both nucleotide N and N+1). The 5’ cap helps to regulate nuclear export, prevent exonuclease degradation, promote translation, and aid in 5' proximal intron excision. The 3’ tail includes a poly(A) tail having 40 to 300 adenosine (A) residues, 40 to 100 adenosine (A) residues, 100 to 300 adenosine (A) residues, or 50 to 200 adenosine (A) residues. The 3’ tail may include modifications, such as phosphorothioate linkages or altered nucleobases, to prevent degradation by exonucleases. Alternatively, the 3’ tail includes a 3’ cap including modified or non-natural nucleobases or synthetic moieties.
[31] The gRNA may depend on a gene to be edited of a host cell. In other words, the gRNA varies based on the condition to be treated. Based on the sequence of the gRNA, the binding specificity of the gRNA to for example, a portion/gene of the genomic DNA of the host cell changes. In an exemplary embodiment, the gRNA encodes for a transthyretin (TTR) gene related to a condition called transthyretin amyloidosis (ATTR). In another exemplary embodiment, the gRNA encodes for proprotein convertase subtilisin/kexin type 9 (PCSK9) gene which provides a protein that regulates cholesterol levels in blood. The gRNA helps to control the specificity of proteins translated from the mRNA. For example, the gRNA helps to control the specificity of the endonuclease when the mRNA encodes for an endonuclease like the Cas9 endonuclease.
[32] In an embodiment, the gRNA includes a CRISPR RNA (crRNA) and trans-activating RNA (tracrRNA). In another embodiment, a gRNA includes a crRNA and another gRNA includes the tracrRNA forming a double guide RNA (dgRNA). The crRNA having binding specificity to for example, a portion/gene of the genomic DNA of the host cell. The tracrRNA is configured to couple the gRNA to the endonuclease (or any other protein) encoded by the mRNA.
[33] Similar to the gRNA, the host cell may vary based on the condition to be treated. In an exemplary embodiment, the host cells are liver cells.
[34] The mRNA and the gRNA are polynucleotides, i.e., they include a plurality of standard nucleotides from a group of A, G, C, and U residues. The sequence of polynucleotides may be codon-optimized depending upon the host cell to which the system is delivered. At least one of the nucleotides of at least one of the mRNA and the gRNA is chemically modified. The chemical modifications of the nucleotides include, without limitation, pseudouridine, 1-methyl-pseudouridine, and 5-methyl-cytidine, changes to the phosphate backbone by replacing or altering one or both of the non-linking phosphate oxygens or linking phosphate oxygens; changes to the ribose sugar by altering the 2' hydroxyl group, replacing the phosphate group with “dephospho” linkers, modification or replacement of a naturally occurring nucleobase with a non-canonical nucleobase, modifications to the ribose-phosphate backbone; alterations to the 3' or 5' end of the oligonucleotide by removing, modifying, or replacing terminal phosphate groups or attaching moieties, caps, or linkers, and modifications to the sugar.. The chemical modifications improve the stability, expression, and immunogenicity of the mRNA and the gRNA.
[35] After encapsulation of the mRNA, each particle of the system may have a pre-defined ratio between the nitrogen on the lipids (i.e., the positively charged amine groups of the cationic lipid) to (negatively charged) phosphate groups on the mRNAs/gRNAs. The said pre-defined ratio is also called as N/P ratio that ranges from 0.5 to 30 for the particle of the present disclosure. In an exemplary embodiment, the N/P ratio of the particles in the system is 5. The N/P ratio defines the encapsulation efficiency of the particles. The encapsulation efficiency of the particles is around 90%. Such high encapsulation efficiency ensures protection of the payload (i.e., the mRNA and/or the gRNA) with enhanced transfection efficiency and minimal loss of activity post administration.
[36] The cationic lipid may include at least one of DODMA or lipid-T (1,2-dioleyloxy-3-dimethylamino-propane, having formula I), DODAP ((Z)-3-(Dimethylamino)propane-1,2-diyl dioleate, having formula II), DOTAP (1,2-Dioleoyloxy-3-(trimethylammonium)propane, having formula III), DHA-1 or lipid-D (N-[(2-Hydroxyethyl)oxyethyl]azanediyl)bis(hexane-6,1-diyl) bis(2-hexyldecanoate), having formula IV), DHA-2 (di(heptadecan-9-yl) 8,8'-((4-hydroxybutyl)azanediyl)dioctanoate, having formula V), DHA-6 or lipid-H (6-((6-((3-heptylundecanoyl)oxy)hexyl)(2-(2-hydroxyethoxy)ethyl)amino)hexyl 3-hexylundecanoate, having formula VI), D-Lin-MC3-DMA ((6Z,9Z,28Z,31Z)-Heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoate, not shown), DLin-KC2-DMA (2-[2,2-bis[(9Z,12Z)-octadeca-9,12-dienyl]-1,3-dioxolan-4-yl]-N,N-dimethylethanamine, not shown), cKK - E12 (3,6-bis(4-(bis(2-hydroxydodecyl)amino)butyl)piperazine-2,5-dione, not shown), C12 – 200 (1,1'-[[2-[4-[2-[[2-[bis(2-hydroxydodecyl)amino]ethyl](2-hydroxydodecyl)amino]ethyl]-1-piperazinyl]ethyl]imino]bis-2-dodecanol, not shown), HUO – 2 (undecyl 6-((7-(((heptadecan-9-yloxy)carbonyl)oxy)heptyl)(2-(2-hydroxyethoxy)ethyl)amino)hexanoate, not shown), etc. and a pharmaceutically acceptable salt or solvate thereof. The concentration of the cationic lipid in the system (and/or for each particle) may range from 20 mol% to 80 mol%. Alternatively, the concentration of the cationic lipid in the system (and/or for each particle) may range from 40 mol% to 60 mol%. Alternatively, the concentration of the cationic lipid in the system (and/or for each particle) may range from 50 mol% to 60 mol%. In an exemplary embodiment, the cationic lipids help to encapsulate negatively charged nucleic acid (such as the mRNAs and the gRNAs) into the particles to form a core shell structure and/or a vacuole in vacuole system. The cationic lipids may include one or more nitrogen atoms At least one of the one or more nitrogen atoms of the cationic lipids may be ionized. The amount of nitrogen atoms that are ionized compared to the all the nitrogen atoms of the cationic lipids is 10 mol %, 20 mol %, 30 mol %, 40 mol %, 50 mol %, 60 mol %, 70 mol %, 80 mol %, 90 mol %, 95 mol %, or 99 mol %. The intrinsic pKa value of the cationic lipids may range from 4.5 to 6.8, thereby effectively targeting both liver as well as tumors as required. In an exemplary embodiment, the pKa value of the cationic lipids is 6.1. The cationic lipids are biodegradable in vivo and exhibit low toxicity, even up to 20 mg/kg or higher doses. The cationic lipids (and the particles itself) have a clearance rate of at least 50% from the plasma within 8, 10, 12, 24, or 48 hours of administration. The higher clearance rate provided by the cationic lipids reduces long term accumulation of the particles in circulation and within the tissues, thereby making the particle safe with minimal to none adverse effects.

(Formula I)
(Formula II)
(Formula III)

(Formula IV)

(Formula V)

(Formula VI)

[37] The structural formula of the cationic lipids may include at least one of Formula CI, Formula CII, Formula CIa, Formula CIIb, Formula CIII, Formula CIV and pharmaceutically acceptable salt or solvate thereof.
(Formula CI)

[38] For Formula CI, each of R¹ and R² are at least one of C3-C30 alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl, C3-C10 cycloalkyl, —C0-C10 alkylene-L-R6, or —C2-C10 alkenylene-L-R6. These groups may be substituted or unsubstituted. For Formula CI, each of X, Y, and Z are at least one of functional groups including, but not limited to, —C(-O)NR4—, —NR4C(-O)—, —C(-O)O—, —OC(-O)—, —OC(-O)O—, —NR4C(-O)O—, —OC(-O)NR4—, —NR4C(-O)NR4—, —NR4C(-NR4)NR4—, sulfur-containing groups (e.g., —C(-S)NR4—, —NR4C(-S)—), and other specified linkages or bonds. For Formula C1, L includes similar functional groups as X, Y, and Z, with additional moieties like —O—N-CR4— or —CR4=N—O—, and the like. For Formula CI, R³ is at least one of —C0-C10 alkylene-NR7R8, —C0-C10 alkylene-heterocycloalkyl, or —C0-C10 alkylene-heterocycloaryl, with all components optionally substituted or unsubstituted. For Formula CI, R4 is at least one of hydrogen or a C1-C6 alkyl group that is substituted or unsubstituted. For Formula CI, R5: is at least one of hydrogen or a substituted or unsubstituted C1-C6 alkyl group. For Formula CI, R6 is at least one of C3-C30 alkyl, alkenyl, alkynyl, or their cyclic counterparts, which may be substituted or unsubstituted. For Formula CI, Cy: is at least one of substituted or unsubstituted cycloalkyl, heterocycloalkyl, aryl, or heteroaryl groups. For Formula CI, each of R7 and R8 are at least one of hydrogen, a C1-C6 alkyl group (substituted or unsubstituted), or together with the attached nitrogen form a C2-C6 heterocyclic group. For Formula CI, each of n, m, and q are at least one of 0, 1, 2, 3, 4, or 5. These definitions specify the structural diversity and substitution patterns for the cationic lipid having Formula CI.
(Formula CII)

[39] For Formula CII, R¹ is at least one of hydrogen, C3-C30 alkyl, alkenyl, or alkynyl; C3-C30 heteroalkyl, heteroalkenyl, or heteroalkynyl; C3-C10 cycloalkyl; —C0-C10 alkylene-L-R6; or —C2-C10 alkenylene-L-R6. Each of these groups, including alkyl, alkylene, alkenyl, alkenylene, alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl, and cycloalkyl, may be substituted or unsubstituted. For Formula CII, R² is at least one of hydrogen, C3-C30 alkyl, alkenyl, or alkynyl; C3-C30 heteroalkyl, heteroalkenyl, or heteroalkynyl; C3-C10 cycloalkyl; —C0-C10 alkylene-L-R6; or —C2-C10 alkenylene-L-R6, with each group being independently substituted or unsubstituted. For Formula CII, each of X, Y, and Z are at least one linkers, including, but not limited to, —C(=O)NR4—, —NR4C(=O)—, —C(=O)O—, —OC(=O)—, and many other functional groups or bonds as specified. L is independently chosen from linkers like —C(=O)NR4—, —NR4C(=O)—, —C(=O)O—, —OC(=O)—, and additional linkages, including bonds, substituted or unsubstituted forms, as listed. For Formula CII, R³ is at least one of —C0-C10 alkylene-NR7R8, —C0-C10 alkylene-heterocycloalkyl, or —C0-C10 alkylene-heterocycloaryl, where the alkylene, heterocycloalkyl, and heterocycloaryl groups may be substituted or unsubstituted. For Formula CII, R4 is at least one of hydrogen, substituted or unsubstituted C1-C16 alkyl, or heteroalkyl. For Formula CII, R5 is at least one of hydrogen or a substituted/unsubstituted —C0-C10 alkylene-L-R4 group. For Formula CII, R6 is at least one of hydrogen, C3-C30 alkyl, alkenyl, or alkynyl; and Cy-C3-30 alkyl, alkenyl, or alkynyl, with all being substituted or unsubstituted. For Formula CII, Cy is at least one of substituted or unsubstituted cycloalkyl, heterocycloalkyl, aryl, or heteroaryl. For Formula CII, each of R7 and R8 are at least one of hydrogen, substituted or unsubstituted C1-C16 alkyl, or heteroalkyl; or together with the attached nitrogen, form a substituted or unsubstituted C2-C6 heterocyclic group. For Formula CII, each of R?, R¹°, and R¹¹ are at least one of hydrogen, substituted/unsubstituted C1-C16 alkylene or heteroalkylene groups. For Formula CII, each of n, m, and q are at least one of 0, 1, 2, 3, 4, or 5. These definitions specify the structural diversity and substitution patterns for the cationic lipid having Formula CII.
(Formula CIa)

[40] For Formula CIa, each of R¹ and R² is at least one of C3-C30 alkyl, C3-C30 alkenyl, C3-C30 alkynyl, C3-C30 heteroalkyl, C3-C30 heteroalkenyl, C3-C30 heteroalkynyl, C3-C10 cycloalkyl, —C0-C10 alkylene-L-R6, or —C2-C10 alkenylene-L-R6, where each alkyl, alkylene, alkenyl, alkenylene, alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl, and cycloalkyl may be substituted or unsubstituted. For Formula CIa, each of X, Y, and Z are at least one linkers including, but not limited to, —C(-O)NR4—, —NR4C(-O)—, —C(-O)O—, —OC(-O)—, —NR4C(-O)O—, —OC(-S)—, —NR4C(-S)—, and many others, as well as simple groups like —O—, —S—, —C1-C10 alkylene-O—, or a bond, where the alkylene may be substituted or unsubstituted. For Formula CIa, L is at least one of —C(-O)NR4—, —NR4C(-O)—, —OC(-S)—, —NR4C(-S)—, —SC(-O)—, —C(-S)NR4—, —SC(-S)—, and others, or may also be —O—, —S—, —C1-C10 alkylene-O—, —C1-C10 alkylene-C(-O)O—, or a bond, where the alkylene may be substituted or unsubstituted. For Formula CIa, R³ is at least one of —C0-C10 alkylene-NR7R8 or —C0-C10 alkylene-heterocycloalkyl, where the alkylene and heterocycloalkyl may each be substituted or unsubstituted. For Formula CIa, R4 is at least one of hydrogen or C1-C6 alkyl, which may be substituted or unsubstituted. For Formula CIa, R6 is at least one of C3-C30 alkyl, alkenyl, or alkynyl, as well as Cy-C3-C30 alkyl, alkenyl, or alkynyl, with all groups optionally substituted. For Formula CIa, Cy is at least one of substituted or unsubstituted cycloalkyl, heterocycloalkyl, aryl, or heteroaryl. For Formula CIa, each of R7 and R8 are at least one of hydrogen or C1-C6 alkyl, which may be substituted or unsubstituted, or R7 and R8 together with the nitrogen to which they are attached can form a substituted or unsubstituted C2-C6 heterocyclic ring. These definitions specify the structural diversity and substitution patterns for the cationic lipid having Formula CIa.
(Formula CIIb)

[41] For Formula CIIb, each of X and Y are at least one of —OC(-O)—, —OC(-O)O—, —OC(-O)NR4—, —C(-S)O—, —OC(-S)—, —OC(-S)O—, —NR4C(-S)O—, —OC(-S)NR4—, —SC(-O)—, —OC(-O)S—, —SC(-S)O—, —O—, —C1-C10 alkylene-O—, or a bond, where the alkylene may be substituted or unsubstituted. For Formula CIIb, each of R¹ and R² are at least one of hydrogen, C1-C30 alkyl, C1-C30 heteroalkyl, or Cy-C3-C30 alkyl, with each alkyl or heteroalkyl being substituted or unsubstituted. For Formula CIIb, R4 is at least one of hydrogen or C1-C6 alkyl, either substituted or unsubstituted. For Formula CIIb, Cy is at least one of substituted or unsubstituted cycloalkyl, heterocycloalkyl, aryl, or heteroaryl. For Formula CIIb, each of R7 and R8 are at least one of hydrogen or C1-C6 alkyl, which can be substituted or unsubstituted, or together with the attached nitrogen, R7 and R8 form a substituted or unsubstituted C2-C6 heterocyclic ring. For Formula CIIb, A is at least one of —O—, —CH2—, —S—, or —NR¹²—. For Formula CIIb, R¹² is at least one of hydrogen, C1-C10 alkyl, or C1-C10 heteroalkyl, with the alkyl or heteroalkyl being substituted or unsubstituted. For Formula CIIb, Cy is at least one of substituted or unsubstituted cycloalkyl, heterocycloalkyl, aryl, or heteroaryl. For Formula CIIb, p is at least one of 1, 2, 3, 4, 5, or 6. For Formula CIIb, R6 is at least one of C3-C30 alkyl, C3-C30 alkenyl, C3-C30 alkynyl, Cy-C3-C30 alkyl, Cy-C3-C30 alkenyl, or Cy-C3-C30 alkynyl, where each alkyl, alkenyl, or alkynyl may be substituted or unsubstituted. These definitions specify the structural diversity and substitution patterns for the cationic lipid having Formula CIIb.
(Formula CIII)

[42] For Formula CIII, each of R¹ and R² are at least one of C3-C30 alkyl, C3-C30 alkenyl, C3-C30 alkynyl, C3-C30 heteroalkyl, C3-C30 heteroalkenyl, C3-C30 heteroalkynyl, C3-C10 cycloalkyl, —C0-C10 alkylene-L-R6, or —C1-C10 alkenylene-L-R6, where each alkyl, alkylene, alkenyl, alkenylene, alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl, and cycloalkyl can be substituted or unsubstituted. For Formula CIII, each of X, Y, and Z are at least one of —C(-O)NR4—, —NR4C(-O)—, —C(-O)O—, —OC(-O)—, —OC(-O)O—, —NR4C(-O)O—, —OC(-O)NR4—, —NR4C(-O)NR4—, —NR4C(-NR4)NR4—, —C(-S)NR4—, —NR4C(-S)—, —C(-S)O—, —OC(-S)—, —OC(-S)O—, —NR4C(-S)O—, —OC(-S)NR4—, —NR4C(-S)NR4—, —C(-O)S—, —SC(-O)—, —OC(-O)S—, —NR4C(-O)S—, —SC(-O)NR4—, —C(-S)S—, —SC(-S)—, —SC(-S)O—, —NR4C(-S)S—, —SC(-S)NR4—, —O—, —S—, or a bond. For Formula CIII, L is at least one of —C(-O)NR4—, —NR4C(-O)—, —C(-O)O—, —OC(-O)—, —OC(-O)O—, —NR4C(-O)O—, —OC(-O)NR4—, —NR4C(-O)NR4—, —NR4C(-NR4)NR4—, —C(-S)NR4—, —NR4C(-S)—, —C(-S)O—, —OC(-S)—, —OC(-S)O—, —NR4C(-S)O—, —OC(-S)NR4—, —NR4C(-S)NR4—, —C(-O)S—, —SC(-O)—, —OC(-O)S—, —NR4C(-O)S—, —SC(-O)NR4—, —C(-S)S—, —SC(-S)—, —SC(-S)O—, —NR4C(-S)S—, —SC(-S)NR4—, —O—N-CR4—, —CR4-N—O—, —O—, —S—, —C1-C10 alkylene-O—, —C1-C10 alkylene-C(-O)O—, —C1-C10 alkylene-OC(-O)—, or a bond, where the alkylene may be substituted or unsubstituted. For Formula CIII, R³ is at least one of —C0-C10 alkylene-NR7R8, —C0-C10 alkylene-heterocycloalkyl, or —C0-C10 alkylene-heterocycloaryl, with the alkylene, heterocycloalkyl, and heterocycloaryl being independently substituted or unsubstituted. For Formula CIII, R4 is at least one of hydrogen or C1-C6 alkyl, either substituted or unsubstituted. For Formula CIII, R6 is at least one of C3-C30 alkyl, C3-C30 alkenyl, C3-C30 alkynyl, Cy-C3-C30 alkyl, Cy-C3-C30 alkenyl, or Cy-C3-C30 alkynyl, with the alkyl, alkenyl, and alkynyl being independently substituted or unsubstituted. For Formula CIII, Cy is at least one of substituted or unsubstituted cycloalkyl, heterocycloalkyl, aryl, or heteroaryl. For Formula CIII, each of R7 and R8 are at least one of hydrogen or C1-C6 alkyl, substituted or unsubstituted, or together with the nitrogen they form a substituted or unsubstituted C2-C6 heterocyclic ring. For Formula CIII, each of a and b are at least one of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. For Formula CIII, m is at least one of 0, 1, 2, 3, 4, or 5. These definitions specify the structural diversity and substitution patterns for the cationic lipid having Formula CIII.
(Formula CIV)

[43] For Formula CIV, each of R¹ and R² are at least on of hydrogen, C3-C30 alkyl, C3-C30 alkenyl, C3-C30 alkynyl, C3-C30 heteroalkyl, C3-C30 heteroalkenyl, C3-C30 heteroalkynyl, C3-C10 cycloalkyl, —C0-C10 alkylene-L-R6, or —C1-C10 alkenylene-L-R6, where each alkyl, alkylene, alkenyl, alkenylene, alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl, and cycloalkyl can be substituted or unsubstituted. For Formula CIV, each of X, Y, and Z are at least one of —C(-O)NR4—, —C(-O)—, —NR4C(-O)—, —C(-O)O—, —OC(-O)—, —OC(-O)O—, —NR4C(-O)O—, —OC(-O)NR4—, —NR4C(-O)NR4—, —NR4C(-NR4)NR4—, —C(-S)NR4—, —NR4C(-S)—, —C(-S)O—, —OC(-S)—, —OC(-S)O—, —NR4C(-S)O—, —OC(-S)NR4—, —NR4C(-S)NR4—, —C(-O)S—, —SC(-O)—, —OC(-O)S—, —NR4C(-O)S—, —SC(-O)NR4—, —C(-S)S—, —SC(-S)—, —SC(-S)O—, —NR4C(-S)S—, —SC(-S)NR4—, —O—, —S—, or a bond. Each L is independently selected from: —C(-O)NR4—, —NR4C(-O)—, —C(-O)O—, —OC(-O)—, —OC(-O)O—, —NR4C(-O)O—, —OC(-O)NR4—, —NR4C(-O)NR4—, —NR4C(-NR4)NR4—, —C(-S)NR4—, —NR4C(-S)—, —C(-S)O—, —OC(-S)—, —OC(-S)O—, —NR4C(-S)O—, —OC(-S)NR4—, —NR4C(-S)NR4—, —C(-O)S—, —SC(-O)—, —OC(-O)S—, —NR4C(-O)S—, —SC(-O)NR4—, —C(-S)S—, —SC(-S)—, —SC(-S)O—, —NR4C(-S)S—, —SC(-S)NR4—, —O—N-CR4—, —CR4-N—O—, —O—, —S—, —C1-C10 alkylene-O—, —C1-C10 alkylene-C(-O)O—, —C1-C10 alkylene-OC(-O)—, or a bond, where the alkylene can be substituted or unsubstituted. For Formula CIV, R³ is at least one of hydrogen, C1-C6 alkyl, —C0-C10 alkylene-NR7R8, —C0-C10 alkylene-heterocycloalkyl, or —C0-C10 alkylene-heterocycloaryl, where the alkyl, alkylene, heterocycloalkyl, and heterocycloaryl can be independently substituted or unsubstituted. For Formula CIV, R4 is at least one of hydrogen or C1-C6 alkyl, either substituted or unsubstituted. For Formula CIV, R6 is at least one of C3-C30 alkyl, C3-C30 alkenyl, C3-C30 alkynyl, Cy-C3-C30 alkyl, Cy-C3-C30 alkenyl, or Cy-C3-C30 alkynyl, with the alkyl, alkenyl, and alkynyl being independently substituted or unsubstituted. For Formula CIV, Cy is at least one of substituted or unsubstituted cycloalkyl, heterocycloalkyl, aryl, or heteroaryl. For Formula CIV, each of R7 and R8 are at least one of hydrogen or C1-C6 alkyl, either substituted or unsubstituted, or together with the nitrogen to which they are attached forms a substituted or unsubstituted C2-C6 heterocyclic ring. For Formula CIV, each of a and b is at least one of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. For Formula CIV, m is at least one of 0, 1, 2, 3, 4, or 5. These definitions specify the structural diversity and substitution patterns for the cationic lipid having Formula CIV.
[44] In an embodiment, the cationic lipid is symmetric, which leads to fewer metabolites and make the molecule achiral. The symmetric, achiral cationic lipid contributes to the formation of smaller and/or more compact particles compared to asymmetric cationic lipids, which enhances efficient delivery to the liver in mammalian subjects, especially when the particles are smaller in size. In an embodiment, the cationic lipid includes more hydrolysable bonds, resulting in faster metabolic clearance of the particle made of the said cationic lipid.
[45] In an exemplary embodiment, the cationic lipid (for example, DOTAP) below 50% increases the affinity of the particles of the system towards the liver cells.
[46] In another exemplary embodiment, the cationic lipid (for example, DOTAP) over 50% increases the affinity of the particles of the system towards lungs and/or spleen, as the particles acquired a net positive charge.
[47] In an exemplary embodiment, the cationic lipid (for example, DOTAP) in low-concentrations produced a larger particle size (for example 190?±?8.4 nm), compared to particles made with a high concentration of DOTAP (for example 140?±?3.8 nm).
[48] In an exemplary embodiment, the cationic lipid (for example, DOTAP) has low toxicity. Further, DOTAP has shorter lipid chains, thus reducing the particle size of the particles. DOTAP also provides extremely positive zeta potential of approximately?+?40 mV to?+?60 mV to the particles.
[49] The neutral lipid may include at least one of DOPE (3-{[(2-Aminoethoxy)(hydroxy)phosphoryl]oxy}-2-[(octadec-9-enoyl)oxy]propyl octadec-9-enoate, having formula VII), and DSPC (1,2-Distearoyl-sn-glycero-3-phosphocholine, having formula VIII), DOPS (1,2-Dioleoyl-sn-glycero-3-phospho-L-serine, having formula IX), DOPC (1,2-Dioleoyl-sn-Glycero-3-Phosphocholine, having formula X), DPPC (1,2-Dipalmitoyl-rac-glycero-3-phosphocholine, having formula XI), DMPC (dimyristoylphosphatidylcholine, having formula XII), DAPC (1,2-distearoyl-sn-glycero-3-phosphocholine, having formula XIII), DLPC (dilauryloylphosphatidylcholine, having formula XIV), EPC (egg phosphatidylcholine, having formula XV), MPPC (1-myristoyl-2-palmitoyl phosphatidylcholine, having formula XVI), POPC (1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, not shown), resorcinol (5-heptadecylbenzene-1,3-diol, not shown), PLPC (phosphatidylcholine, not shown), PE (phosphatidylethanolamine, not shown), PMPC (1-palmitoyl-2-myristoyl phosphatidylcholine, not shown), PSPC (1-palmitoyl-2-stearoyl phosphatidylcholine, not shown), DBPC (1,2-diarachidoyl-sn-glycero-3-phosphocholine, not shown), SPPC (1-stearoyl-2-palmitoyl phosphatidylcholine, not shown), DEPC (1,2-dieicosenoyl-sn-glycero-3-phosphocholine, not shown), lysophosphatidylcholine (not shown), dilinoleoylphosphatidylcholine (not shown), DSPE (distearoylphosphatidylethanolamine, not shown), DMPE (dimyristoyl phosphatidylethanolamine, not shown), DPPE (dipalmitoyl phosphatidylethanolamine, not shown), POPE (palmitoyloleoyl phosphatidylethanolamine, not shown), 18:0 Diether PC (1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine, not shown), OChemsPC (1-oleoyl-2-cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine, not shown), C16 Lyso PC (1-hexadecyl-sn-glycero-3-phosphocholine, not shown), DUPC (1,2-diundecanoyl-sn-glycero-phosphocholine, not shown), SOPE (1-stearoyl-2-oleoyl-phosphatidylethanolamine, not shown), DOPG (1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt, not shown) lysophosphatidylethanolamine (not shown), PC (phosphatidylcholine, not shown), phosphatidylethanolamine amine, glycerophospholipids, sphingophospholipids, Guriserohosuhono, sphingolipid phosphono lipids, natural lecithins (not shown), hydrogenated phospholipids, 16-0-Monome Le PE, 16-0-dimethyl PE, 18-1-trans PE, plasmalogen, phosphatidate, phosphatidylserine, phosphatidic acid, phosphatidylglycerol, phosphatidylinositol, POPG (palmitoyl oleoyl phosphatidylglycerol, not shown) lysophosphatidylcholine, sphingomyelin, ceramide phosphoethanolamine, ceramide phosphoglycerol, ceramide phosphoglycerophosphoric acid, egg yolk lecithin, soybean lecithin, hydrogenated soybean phosphatidylcholine, etc. and a pharmaceutically acceptable salt or solvate thereof. The concentration of the neutral lipid in the system (and/or for each particle) may range from 1 mol% to 20 mol%. Alternatively, the concentration of the neutral lipid may range from 2 mol% to 25 mol%. Alternatively, the concentration of the neutral lipid may range from 5 mol% to 10 mol%. The neutral lipid imparts transfection efficiency by facilitating membrane fusion between the particles of the system and the cell membrane of the host cell. The neutral lipids maintain the charge balance of the particles in order to prevent their aggregation, thereby increasing their stability.

(Formula VII)
(Formula VIII)
(Formula IX)
(Formula X)
(Formula XI)
(Formula XII)
(Formula XIII)
(Formula XIV)
(Formula XV)
(Formula XVI)

[50] The neutral lipids (for example, DOPE and DSPC) are used as structural lipids. Due to amphipathic nature of the neutral lipids, they readily form concentric bi-layers. In an exemplary embodiment, the two unsaturated fatty acid chains of DOPE attain a hexagonal shape once it is exposed to the endosomal pH of the host cell thereby destabilizing the endosomal membrane and releasing the mRNA within the host cell. In another exemplary embodiment, the DSPC attains a cylindrical geometry and forms a lamellar structure that stabilizes the particle core. Both, DOPE and DSPC contributes to structural and functional properties of the particles of the system.
[51] In an exemplary embodiment, DOPE imparts higher positive charge in acidic environment. Due to the higher positive charge and negative charge of the mRNAs/gRNAs, the encapsulation efficiency of the particle is enhanced, i.e., a greater number of mRNAs/gRNAs are encapsulated in a single particle of the system. Effectively, the amount of delivery system required to deliver a pre-defined amount of mRNA and/or gRNA decreases significantly. This not only translates to cheaper cost of production, but also minimizes the amount of particle constituents being introduced inside the body.
[52] The helper lipid may include at least one of a steroid, sterol, and alkyl resorcinol, cholesterol, etc. and derivatives thereof. The steroids may be one of a 5-heptadecylresorcinol, cholesterol hemisuccinate, fecosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatidine, tomatine, ursolic acid, alpha-tocopherol, prednisolone, dexamethasone, prednisone, or hydrocortisone, cholesterol having formula XVII, formula XVIII, formula XIX, formula XX and derivatives thereof. Alternatively, the helper lipids include polar cholesterol analogues including, but not limited to, 5a-cholestanol, 5ß-coprostanol, cholesteryl-(2'-hydroxy)-ethyl ether, cholesteryl-(4'-hydroxy)-butyl ether, or 6-ketocholestanol. Alternatively, the helper lipids include non-polar cholesterol analogues including, but not limited to, 5a-cholestane, cholestenone, 5a-cholestanone, 5ß-cholestanone, or cholesteryl decanoate. In an exemplary embodiment, the helper lipid of the system may include a cholesterol (having formula XVII, XVIII, XIX). The concentration of the helper lipid in the system (and/or for each particle) may range from 10 mol% to 50 mol%. Alternatively, the concentration of the helper lipid may range from 40 mol% to 60 mol%. Alternatively, the concentration of the helper lipid may range from 20 mol% to 40 mol%. The helper lipid helps in reducing the amount of surface-bound protein on the particles and improves their circulation half-life. The circulation half-life is defined as the time period in which the amount of particles of the system in circulation becomes half after being administered to the patient. The helper lipid pulls the neutral lipids towards a liquid-ordered phase. The helper lipid improves the transfection efficiency, especially for particles delivering biologically active agnets by increasing the stability of the particles. The helper lipids help to enhance membrane fusogenicity.

(Formula XVII)
(Formula XVIII)
(Formula XIX)
(Formula XX)

[53] In an exemplary embodiment, when the helper lipids are combined with neutral lipids having low gel-liquid crystalline phase transitions (Tm), the helper lipid helps in formation of a liquid-ordered phase having decreased membrane fluidity and increased bi-layer thickness.
[54] In another exemplary embodiment, when the helper lipids are combined with neutral lipids having high Tm, the helper lipid boosts membrane fluidity and narrows the bi-layer thickness.
[55] Although the particles of the present disclosure are described with the example of the cholesterol (having formula XVII), use of other functionally equivalent helper lipid in place of the cholesterol (having formula XVII) is within the scope of the teachings of the present disclosure.
[56] The polyether may include a hydrophilic head group attached to a lipid moiety. The hydrophilic head group of the polyether may include a polymer moiety including, but not limited to, PEG (poly(ethylene glycol)), poly(oxazoline), PVA poly(vinyl alcohol), poly(glycerol), poly(N-vinylpyrrolidone), polyamino acids, poly[N-(2-hydroxypropyl)methacrylamide], etc. and derivatives thereof. The lipid moiety of the polyether may be one of diacylglycerol or diacylglycamide, etc. and derivatives thereof, optionally having a dialkylglycerol group or a dialkylglycamide group with alkyl chains (either saturated or unsaturated) having 4 to 40 carbon atoms. The alkyl chains of the lipid moiety may include one or more functional groups including, but not limited to, amides or esters. Additionally or optionally, the dialkylglycerol group or the dialkylglycamide group of the lipid moiety may include one or more substituted alkyl groups.
[57] The polyether may include a PEG moiety conjugated to the stealth lipid having an empirical formula XXI. The PEG moiety includes at least one of polyethylene glycol, polyalkylene ether polymer, or copolymers like PEG-polyurethane, PEG-polypropylene, etc. and derivatives thereof. The PEG moiety may be one of a linear or branched polymer of ethylene glycol, ethylene oxide or derivatives thereof. The number of ethylene glycol units in the polyether may be at least one of 1, 2, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 120, or 150. Alternatively, the PEG moiety may be a PEG monopolymer or a copolymer including, but not limited to, PEG-polyurethane, PEG-polypropylene. The PEG moiety may either be unsubstituted or substituted with groups including, but not limited to, an alkyl group, an alkoxy group, an acyl group, a hydroxy group, an aryl groups or a combination thereof. The molecular weight of the PEG moiety may range from 200 daltons to 5000 daltons. Alternatively, the molecular weight of the PEG moiety may range from 500 daltons to 3000 daltons. Alternatively, the molecular weight of the PEG moiety may range from 750 daltons to 2500 daltons.

(Formula XXI)
[58] In the above formula XXI of the PEG moiety, ‘n’ represents the average degree of polymerization. For example, for PEG-2000 having molecular of 2000 daltons (approx.), ‘n’ is equal to 45 subunits. The degree of polymerization ‘n’ of the PEG moiety may range from 15 subunits to 200 subunits.
[59] In the above formula XIII of the PEG moiety, ‘R’ may be one of hydrogen (H), substituted alkyl groups, or unsubstituted alkyl groups. In an exemplary embodiment, the ‘R’ is a methyl group (an exemplary unsubstituted alkyl group).
[60] The polyether is at least one of a PEG-lipid conjugate, such as PEG attached to dialkyloxypropyls (e.g., PEG-DAA conjugates), PEG linked to diacylglycerols (e.g., PEG-DAG conjugates), PEG bonded to cholesterol, PEG connected to phosphatidylethanolamines, or PEG conjugated to ceramides, cationic PEG-lipids, polyoxazoline (POZ)-lipid conjugates, polyamide oligomers (e.g., ATTA-lipid conjugates), etc. The polyether may include at least one of DMG-PEG 2000 (Azane;[3-(2-methoxyethoxy)-2-tetradecanoyloxypropyl] tetradecanoate, having formula XXII), DSPE-PEG 5000 (1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-5000], having formula XXIII), DSG PEG 2000 (1,2-distearoyl-rac-glycero-3-methylpolyoxyethylene 2000, having formula XXIV), DMPE PEG 2000 (1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-([methoxy(polyethylene glycol)-2000], not shown), PEG-DPPC, PEG-DLPE, PEG-c-DOMG, PEG-dilauroylglycerol, PEG-dipalmitoylglycerol, PEG-dipalmitoylglycamide, PEG-disterylglycamide, PEG-cholesterol, PEG-DMB (3,4-ditetradecoxylbenzyl-[omega]-methyl-poly(ethylene glycol) ether), 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000]), etc. and derivatives thereof. The concentration of the polyether in the system (and/or for each particle) may range from 0.1 mol% to 6 mol%. Alternatively, the concentration of the polyether in the system (and/or for each particle) may range from 1.0 mol% to 3.5 mol%. The polydispersity index of the polyether is less than 2. The polyether prevents adsorption of the serum protein(s), thus, inhibiting uptake of the particles by the mononuclear phagocyte system (MPS). Polyether further ensures particle stability by preventing particle aggregation during preparation of the system and its storage thereby controlling particle size. Polyether also affects factors such as encapsulation efficiency, in-vivo distribution of particles, transfection efficiency and immune response. Polyether also increases the circulation half-life of the particles of the system. The polyether improves the pharmacokinetic properties of the particles.

(Formula XXII)

(Formula XXIII)
(Formula XXIV)
[61] Additionally or optionally, the polyether’s may contain one or more terminal functional groups, such as amine or maleimide, which can be used to conjugate other molecules that improve cellular targeting and uptake of the particles, improve storage stability and circulation half-life of the particles.
[62] The nucleic acid stabilizer is at least one of polyethylene glycol (PEG 200, PEG 400, PEG 600, PEG 1000, PEG 3350, PEG 4000, PEG 8000, PEG 10000, PEG 20000), cetrimonium bromide or cetrimonium chloride, cetrimonium bromide (also known as cetyltrimethylammonium bromide or CTAB), low molecular weight chitosan (50,000 to 190,000 Da or lower than 50,000 Da), etc.
[63] The nucleic acid stabilizer may optionally include a cryoprotectant including at least one of polyols (e.g., propylene glycol, glycerol, ethylene glycol, or diethylene glycol), nondetergent sulfobetaines (e.g., NDSB-201), osmolytes (e.g., L-proline or trimethylamine N-oxide dihydrate), water-soluble polymers (e.g., polyethylene glycol, polyvinylpyrrolidone, or block polymers of polyethylene glycol and polypropylene glycol), organic solvents (e.g., dimethyl sulfoxide or ethanol), sugars (e.g., sucrose, trehalose, or glucose), salts (e.g., lithium acetate, sodium chloride, or magnesium acetate), etc. The concentration of the nucleic acid stabilizer in the system (and/or for each particle) may range from 0.001% (w/w) to 50% (w/w). Alternatively, the concentration of the nucleic acid stabilizer in the system (and/or for each particle) may range from 0.001% (w/w) to 20% (w/w). Alternatively, the concentration of the nucleic acid stabilizer in the system (and/or for each particle) may range from 0.001% (w/w) to 5% (w/w). The nucleic acid stabilizer prevents degradation of the cationic lipids and the payload (i.e., the mRNA and the gRNA) thereby preserving their structural integrity over long period of time. The aforesaid improvement in stability helps to extend the shelf-life of the system and makes it resilient to multiple freeze-thaw cycles.
[64] Additionally or optionally, the particles of the system may include at least one stabilizer. The stabilizer is be at least one of ethylenediaminetetraacetic acid (EDTA), citrate, vitamin E isomers, polyphenols, etc. and a pharmaceutically acceptable salt or solvate thereof. The concentration of the stabilizer in the system (and/or for each particle) may range from 1 mol% to 100 mol%. In an exemplary embodiment, the concentration of the stabilizer is 20 mol%.
[65] Additionally or optionally, the particles of the system may include at least one surfactants. The surfactant is at least one of 2-acrylamido-2-methylpropane sulfonic acid, ammonium lauryl sulfate, ammonium perfluorononanoate, docusate, disodium cocoamphodiacetate, magnesium laureth sulfate, perfluorobutanesulfonic acid, perfluorononanoic acid, perfluorooctanesulfonic acid, perfluorooctanoic acid, potassium lauryl sulfate, sodium alkyl sulfate, sodium dodecyl sulfate, sodium dodecylbenzenesulfonate, sodium laurate, sodium laureth sulfate, sodium lauroyl sarcosinate, sodium myreth sulfate, sodium nonanoyloxybenzenesulfonate, sodium pareth sulfate, sodium stearate, and sulfolipid. In some embodiments, the surfactants are cationic, such as behentrimonium chloride, benzalkonium chloride, benzethonium chloride, benzododecinium bromide, bronidox, carbethopendecinium bromide, cetalkonium chloride, cetrimonium bromide, cetrimonium chloride, cetylpyridinium chloride, didecyldimethylammonium chloride, dimethyldioctadecylammonium bromide, dimethyldioctadecylammonium chloride, domiphen bromide, lauryl methyl gluceth-10 hydroxypropyl dimonium chloride, octenidine dihydrochloride, olaflur, n-oleyl-1,3-propanediamine, pahutoxin, stearalkonium chloride, tetramethylammonium hydroxide, and thonzonium bromide. In other cases, the surfactants are zwitterionic, such as cocamidopropyl betaine, cocamidopropyl hydroxysultaine, dipalmitoylphosphatidylcholine, egg lecithin, hydroxysultaine, lecithin, myristamine oxide, peptitergents, sodium lauroamphoacetate, alkyl polyglycoside, cetomacrogol 1000, cetostearyl alcohol, cetyl alcohol, cocamide DEA, cocamide MEA, decyl glucoside, decyl polyglucose, glycerol monostearate, Igepal CA-630, isoceteth-20, lauryl glucoside, maltosides, monolaurin, mycosubtilin, narrow-range ethoxylate, Nonidet P-40, nonoxynol-9, nonoxynols, NP-40, octaethylene glycol monododecyl ether, N-octyl beta-D-thioglucopyranoside, octyl glucoside, oleyl alcohol, PEG-10 sunflower glycerides, pentaethylene glycol monododecyl ether, polidocanol, poloxamers (e.g., poloxamer 188 and poloxamer 407), polyethoxylated tallow amine, polyglycerol polyricinoleate, polysorbates (e.g., polysorbate 20, polysorbate 40, polysorbate 60, or polysorbate 80), sorbitan, sorbitan monolaurate, sorbitan monostearate, sorbitan tristearate, stearyl alcohol, surfactin, Triton X-100, lauryl alcohol, tridecyl alcohol, myristyl alcohol, pentadecyl alcohol, cetyl alcohol, palmitoleyl alcohol, heptadecyl alcohol, stearyl alcohol, oleyl alcohol, nonadecyl alcohol, arachidyl alcohol, oleyl alcohol, stearyl alcohol, etc. The concentration of the surfactants in the particle (and the system) ranges from 0.5 mol% to 20 mol%. Alternatively, the concentration of the surfactants in the particle (and the system) ranges from 1 mol% to 10 mol%. Alternatively, the concentration of the surfactants in the particle (and the system) ranges from 3 mol% to 8 mol%. The surfactants prevent aggregation of the particles thereby, increasing their stability.
[66] Additionally or optionally, the particles of the system may include at least one permeability enhancers. The permeability enhancer is at least one of glucose, polysaccharides like glycogen, polyamines, polyethers, polyamides, polyesters, polycarbamates, polyureas, polycarbonates, polystyrenes, polyimides, polysulfones, polyurethanes, polyacetylenes, polyethylenes, polyethyleneimines, polyisocyanates, polyacrylates, polymethacrylates, polyacrylonitriles, polyarylates, poly(caprolactone) (PCL), ethylene vinyl acetate (EVA), poly(lactic acid) (PLA), poly(L-lactic acid) (PLLA), poly(glycolic acid) (PGA), poly(lactic acid-co-glycolic acid) (PLGA), poly(D,L-lactide) (PDLA), poly(D,L-lactide-co-caprolactone), poly(D,L-lactide-co-caprolactone-co-glycolide), poly(L-lactide-co-glycolic acid) (PLLGA), poly(D,L-lactide-co-PEO-co-D,L-lactide), poly(D,L-lactide-co-PPO-co-D,L-lactide), polyalkyl cyanoacrylate, polyurethane, poly-L-lysine (PLL), hydroxypropyl methacrylate (HPMA), polyethyleneglycol (PEG), poly-L-glutamic acid, poly(hydroxy acids), polyanhydrides, polyorthoesters, poly(ester amides), polyamides, poly(ester ethers), polycarbonates, polyalkylenes (e.g., polyethylene and polypropylene), polyalkylene glycols (e.g., poly(ethylene glycol), PEO), polyalkylene terephthalates (e.g., poly(ethylene terephthalate)), polyvinyl alcohols (PVA), polyvinyl ethers, polyvinyl esters (e.g., poly(vinyl acetate)), polyvinyl halides (e.g., poly(vinyl chloride)), polyvinylpyrrolidone (PVP), polysiloxanes, polystyrene, polyurethanes, derivatized celluloses (e.g., alkyl celluloses, hydroxyalkyl celluloses), polymers of acrylic acids (e.g., PMMA), polydioxanone, polyhydroxyalkanoates, polypropylene fumarate, polyoxymethylene, poloxamers, poloxamines, poly(ortho)esters, poly(butyric acid), poly(valeric acid), poly(lactide-co-caprolactone), trimethylene carbonate, poly(N-acryloylmorpholine), poly(2-methyl-2-oxazoline), poly(2-ethyl-2-oxazoline), polyglycerol and their derivatives or analogs. The permeability enhancers improve permeation of the particles of the present disclosure across biological membranes, for example, cell membrane of the host cells.
[67] In an exemplary embodiment, the system includes 40.4 mol% DODMA, 18.6 mol% DOPE, 35.5 mol% cholesterol, and 4.5 mol% DMG-PEG 2000. Each particle of the system encapsulated at least one mRNA having a length of 800 bp to 4000 bp. The system is administered for treatment of a rare metabolic disorder such as Gaucher’s diseases, by dosing in the range of 0.1 mg – 2.0 mg per kg weight of the patient as decided by the physician.
[68] In an alternate embodiment, the system includes 43.6 mol% DODAP, 12.6 mol% DOPE, 40.2 mol% cholesterol, and 3.4 mol% DSPE-PEG 5000. Each particle of the system encapsulated at least one mRNA having a length of 400 bp to 2000 bp. The system is administered for treatment of Polyketoneuria by dosing in the range of 0.1 mg – 2.0 mg per kg weight of the patient as decided by the physician.
[69] In yet another embodiment, the system includes 46 mol% DOTAP, 7.5 mol% DSPC, 8.5 mol% DOPE, 35.5 mol% cholesterol, and 2.5 mol% DMG-PEG 2000. Each particle of the system encapsulated at least one mRNA having a length of 1900 bp to 4500 bp and a GC content ranging from 45% to 65%. The system is administered to inhibit TNF-a within a dose range of 0.1 mg – 2.0 mg per kg weight of the patient as decided by the physician.
[70] Now referring to the figures, Fig. 1 depicts a cross-sectional view of a particle 100 of the system in accordance with an exemplary embodiment of the present disclosure. The particle 100 of the system includes at least one bi-layer outer membrane 110 (i.e., a unilamellar structure) and a plurality of vacuoles 130 disposed within a lumen 110a defined by the bi-layer outer membrane 110.
[71] The bi-layer outer membrane 110 (and the particle 100) may include a pre-defined shape including but not limited to spherical/microspheres, oval, elliptical, corona-like, etc. In an exemplary embodiment, as shown in Fig. 1, the bi-layer outer membrane 110 is spherical shaped.
[72] Although the particle 100 of the present disclosure is described with examples of one bi-layer outer membrane 110 (i.e., unilamellar structure), the particle 100 may be provided with more than one bi-layer (i.e., a multilamellar structure) and the same is within the scope of the teachings of the present disclosure.
[73] The bi-layer outer membrane 110 may include an outer layer 111 and an inner layer 113. The inner layer 113 is disposed between the outer layer 111 and the lumen 110a. Each of the outer layer 111 and the inner layer 113 include a hydrophobic portion and a hydrophilic portion. The hydrophobic portions of the outer layer 111 and the inner layer 113 are disposed adjacent to each other, i.e., hydrophobic portions of the outer layer 111 and the inner layer 113 are disposed between the respective hydrophilic portions of the outer layer 111 and the inner layer 113.
[74] The outer layer 111 includes at least a plurality of cationic lipids 101, a plurality of neutral lipids 103, a plurality of helper lipid 105, and a plurality of polyethers 107. In an exemplary embodiment, as shown in Fig. 1, the cationic lipids 101 and the neutral lipids 103 are disposed alternatively. The helper lipid 105 and the polyethers 107 are disposed intermittently between the cationic and neutral lipids 101, 103, as a group together.
[75] The inner layer 113 includes at least the plurality of cationic lipids 101, the plurality of neutral lipids 103, and the plurality of helper lipid 105. In an exemplary embodiment, as shown in Fig. 1, the cationic lipids 101 and the neutral lipids 103 are disposed alternatively. The helper lipid 105 are disposed intermittently between the cationic and neutral lipids 101, 103.
[76] The vacuoles 130 includes a membrane layer 131 defining a lumen 130a. The membrane layer 131 (and the vacuole 130) may include a pre-defined shape including but not limited to spherical, oval, elliptical, etc. In an exemplary embodiment, as shown in Fig. 1, the membrane layer 131 is spherical shaped.
[77] The membrane layer 131 includes at least the plurality of cationic lipids 101, the plurality of neutral lipids 103, and the plurality of helper lipid 105. In an exemplary embodiment, as shown in Fig. 1, the cationic lipids 101 and the neutral lipids 103 are disposed alternatively. The helper lipid 105 are disposed intermittently between the cationic and neutral lipids 101, 103.
[78] The membrane layer 131 includes a hydrophobic portion and a hydrophilic portion. The hydrophilic portion of the membrane layer 131 is disposed towards the lumen 130a of the vacuole 130.
[79] At least one payload 150 having a pre-defined size (or length) may be encapsulated within the lumen 130a of the vacuoles 130. The payload 150 includes at least one messenger ribonucleic acid molecule (or mRNA), and at least one guide ribonucleic acid molecule (or gRNA). The mRNA and the gRNA may be encapsulated in a ratio by weight (or mole) of 1:1, 1:3, 1:7, 1:10, 3:1, 7:1, or 10:1. In an exemplary embodiment, each vacuole 130 encapsulates a single mRNA molecule. In another exemplary embodiment, each vacuole 130 encapsulates a single gRNA molecule. The length of the mRNA and the gRNA molecules encapsulated by the vacuole 130 may range from 1kb to 20kb or a combination thereof. The mRNA molecules may either be modified or unmodified. The modified mRNA molecules may include at least one of altered nucleotides, modified sugar-phosphate backbones, modified 5' and/or 3' untranslated regions (UTRs), proving a 5’ cap, providing a 3’ tail, etc. In an exemplary embodiment, the 5’ capping is provided by adding a guanosine triphosphate (GTP) at the 5’ end of the mRNA using a guanylyl transferase enzyme and adding a 2'-O-methyl to the 7th nitrogen of the guanine using methyltransferase enzyme. The 5’ cap may be one of m7GpppNp-RNA, m7GpppNmp-RNA, and m7GpppNmpNmp-RNA (where ‘m’ represents the 2'-O-methyl group). The 5’ cap is crucial for protecting the mRNA molecules from degradation by nucleases usually present in most eukaryotic cells. The 3’ tail includes a poly-A tail that protects the mRNA molecule from exonuclease activity.
[80] In an exemplary embodiment, the mRNA/gRNA molecules constituting the payload 150 is first synthesized using in vitro transcription (IVT) technique before it is being encapsulated in the particles 100. For example, in IVT technique, a linear or circular deoxynucleotide acid (DNA) template having a promoter is incubated at a pre-defined temperature for a predefined time period along with a plurality of additives. The pre-defined temperature and the pre-defined time period vary depending upon the mRNA molecule and application thereof. The additives include, without limitation, ribonucleotide triphosphates (rNTPs), a buffer (including dithiothreitol and magnesium ions), an RNA polymerase enzyme (for example T3, T7, or SP6 RNA polymerase), a DNase 1 enzyme, a pyrophosphatase enzyme, and an RNAse inhibitor.
[81] Other functionally equivalent techniques to synthesize the mRNA/gRNA molecules constituting the payload 150 are within the scope of the teachings of the present disclosure.
[82] As an example, the gRNA molecules may be synthesized corresponding to at least one of, but not limited to, spinal motor neuron 1 (SMN1) gene, alpha-galactosidase (GLA) gene, argininosuccinate synthetase 1 (ASS1) gene, ornithine transcarbamylase (OTC) gene, Factor IX (FIX) gene, phenylalanine hydroxylase (PAH) gene, erythropoietin (EPO) gene, cystic fibrosis transmembrane conductance receptor (CFTR) gene, firefly luciferase (fLUC) gene, etc. to manage conditions related to the respective gene.
[83] Fig. 2 depicts an exemplary method 200 to prepare the particles 100 of the system of the present disclosure. The method commences at step 201 by preparing a first mixture. The first mixture includes at least one cationic lipid 101, at least one neutral lipid 103, at least one helper lipid 105 and at least one polyether 107 dissolved in at least one first solvent. The amount of cationic lipid 101 in the first mixture ranges from 30 mol% to 60 mol%. The amount of neutral lipid 103 in the first mixture ranges from 1 mol% to 20 mol%. The amount of helper lipid 105 in the first mixture ranges from 30 mol% to 60 mol%. The amount of polyether 107 in the first mixture ranges from 1 mol% to 10 mol%. The first solvent may include an organic solvent, selected from alcohols, such as methanol, ethanol, isopropyl alcohol, dimethylsulfoxide (DMSO), chloroform, diethyl ether, cyclohexane, tetrahydrofuran etc. or a combination and/or dilutions thereof. In an exemplary embodiment, the first mixture includes 40.4 mol% DODMA, 18.6 mol% DOPE, 35.5 mol% cholesterol, and 4.5 mol% DMG-PEG 2000 dissolved in 15 mL of ethanol. In an alternate embodiment, the first mixture includes 43.6 mol% DODAP, 12.6 mol% DOPE, 40.2 mol% cholesterol, and 3.4 mol% DSPE-PEG 5000 dissolved in 15 mL of ethanol. In yet another embodiment, the first mixture includes 46 mol% DOTAP, 7.5 mol% DSPC, 8.5 mol% DOPE, 35.5 mol% cholesterol, and 2.5 mol% DMG-PEG 2000 dissolved in 15 mL of 100% ethanol. The cationic lipids 101 helps to encapsulate the mRNA and in endosomal escape via membrane disruption and proton sponge effect. For example, the cationic lipid 101 is protonated in the acidic environment of the cellular endosome thus, attracting counter ions and water. This leads to swelling and subsequent rupture of the endosome, thereby facilitating the release of the payload 150 in the cytoplasm of the cell. The neutral lipids 103 and the helper lipids 105 improves stability of the particles 100.
[84] At step 203, a second mixture is prepared. The second mixture includes a pre-defined amount of the payload 150, a pre-defined amount of at least one buffering agent (or salts thereof) dissolved in at least one second solvent. The amount of payload 150 ranges from 0.01 mg/mL to 1 mg/mL that may be directly added to the second mixture. Alternatively, a stock solution of the payload 150 may be prepared and diluted (for example with the buffering agent or the like) before being added to the second mixture. The amount of the payload 150 in the stock solution ranges from 0.02 mg/mL to 5 mg/mL. The payload 150 may be at least one of a mRNA, gRNA. The amount of buffering agent may range from 0.1 mM to 100 mM. The buffering agent (or salts thereof) includes at least one of, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), ammonium sulfate, sodium bicarbonate, sodium acetate, sodium chloride, sodium citrate, potassium phosphate, sodium phosphate, etc. The second solvent may include an aqueous solvent, selected from distilled water, buffers (like phosphate buffer saline (PBS), Tris buffer, citrate buffer), saline solution, or a combination thereof. In an exemplary embodiment, the second mixture includes 2.1 mg of mRNA and 2.1 mg of gRNA (i.e., the payload 150 in a ratio of 1:1 by weight), 25 mM of sodium acetate, and 85 mM of sodium chloride is dissolved in 45 mL of RNAse free water having its pH adjusted to 5.0. In another embodiment, 3.5 mg of mRNA (i.e., the payload 150) is dissolved in 45 mL of 10 mM to 50 mM citrate buffer having a pH ranging from 2.5 to 4.5. In yet another embodiment, 3.5 mg of gRNA (i.e., the payload 150) is dissolved in 45 mL of 20mM sodium acetate buffer having a pH of 5.0.
[85] The second mixture may have an acidic pH significantly lower than the apparent pKa value of the cationic lipids 101. The buffering agent helps to maintain the pH of the second mixture which is less than 6.5, and preferably more than 3. In an exemplary embodiment, the acidic pH provides a net positive charge on the cationic lipids 101, thereby, enhancing the encapsulation efficiency of the negatively charged mRNA into the particles 100.
[86] Additionally or optionally, the payload 150 may be purified before adding the payload 150 to the second mixture. The payload 150 may be purified using techniques including, but not limited to, dialysis, filtration, centrifugation, or a combination thereof. Purifying the payload 150 helps to remove unwanted impurities such as enzymes and other additives used during the synthesis of the mRNA/gRNA molecules (i.e., the payload 150).
[87] Additionally or optionally, the nucleic acid stabilizer is added to the second solution in a pre-defined amount. The amount of nucleic acid stabilizer added to the second solution ranges from 0.001% (w/w) to 50% (w/w). In an exemplary embodiment, 1% polyethylene glycol is added to the second solution as the nucleic acid stabilizer. he nucleic acid stabilizer prevents degradation of the cationic lipids 101 and the payload 150 (i.e., the mRNA and the gRNA) thereby preserving their structural integrity over long period of time. The aforesaid improvement in stability helps to extend the shelf-life of the system and makes it resilient to multiple freeze-thaw cycles.
[88] Alternatively, a pre-defined amount of cationic lipid 101 is added to the second solution to result in a bi-phase system. In other words, an amount of cationic lipid 101 from the total amount of cationic lipid 101 that is to be dissolved in the first solution is added to the second solution instead of the first solution. The cationic lipid 101 may bind with the mRNA/gRNA molecules in the second solution, thereby, allowing the mRNA/gRNA molecules to easily interact with the constituents of the first solution in the subsequent steps of the method 200. The nitrogen of the cationic lipid 101 neutralizes the phosphates of the payload 150 by 0.5% to 90% depending upon the N/P ratio.
[89] At step 205a, a primary emulsion is prepared by mixing the first and second mixture at a pre-defined ratio ranging from 1:1 to 1:10. In an exemplary embodiment, the first mixture and the second mixture is mixed in the ratio of 1:3. The first mixture and the second mixture is mixed using at least one of stirring, microfluidic mixing, T-mixing, cross-mixing, high-energy mixers (for example, T-junction, confined impinging jets, vortex mixers), etc. The second mixture is subjected to stirring at a pre-defined speed ranging from 600 rpm to 2000 rpm. In an exemplary embodiment, the second mixture is stirred at 1200rpm using a magnetic stirrer. In an alternate embodiment, the second mixture is stirred at 800rpm using a magnetic stirrer. While the second mixture is stirred, the first mixture is injected into the second mixture to form the primary emulsion.
[90] At step 205b, an emulsion is prepared by preparing a secondary emulsion from the primary emulsion obtained from step 205a. The secondary emulsion is prepared by subjecting the primary emulsion to a high-pressure homogenization technique. The primary emulsion is subjected to a pre-defined temperature, pressure, and flow rate. The temperature ranges from 3°C to 10°C. The pressure ranges from 10000 psi to 22000 psi. The flow rate ranges from 4 mL/min to 20 mL/min. The high-pressure homogenization technique helps to reduce the particle size of the particles 100 with the help of pressure and microfluidic channels. In an exemplary embodiment, the high-pressure homogenization technique reduces the particle size of the particles 100 from 800 nm – 1000 nm to 50 nm – 150 nm.
[91] In an exemplary embodiment, the primary emulsion is subjected to a pressure of 16300psi at 4°C inside a LM20 microfluidizer to obtain the secondary emulsion. The primary emulsion is fed at a flow rate of 6mL/min to the microfluidizer.
[92] In an alternate embodiment, the primary emulsion is subjected to a pressure of 19200psi at 4°C inside a LM20 microfluidizer to obtain the secondary emulsion. The primary emulsion is fed at a flow rate of 6mL/min to the microfluidizer.
[93] In yet another embodiment, the primary emulsion is subjected to a pressure of 17500psi at 4°C inside a LM20 microfluidizer to obtain the secondary emulsion. The primary emulsion is fed at a flow rate of 6mL/min to the microfluidizer.
[94] At an alternative step 207 (instead of steps 205a and 205b), an emulsion is prepared by an assembly 300 as shown in Fig. 2a. In an exemplary embodiment, the assembly 300 is a Tee mixer assembly. The assembly 300 includes a first T-tube 310 having a first inlet 311, a second inlet 313, and an outlet 315. The first T-tube 310 may have a pre-defined diameter ranging from 0.762 mm to 12.7 mm. The diameter of the first T-tube 310 may be uniform or may be tapered. In an exemplary embodiment, the diameter of the first T-tube 310 is 1.524 mm. The first T-tube 310 helps to mix the first mixture and the second mixture to prepare the emulsion having the particles 100. Although the first T-tube 310 is depicted with exemplary tubular geometry, other functionally equivalent geometry of the first T-tube 310 is within the scope of the teachings of the present disclosure.
[95] The first inlet 311 and the second inlet 313 are co-aligned. The first inlet 311 and the second inlet 313 may each have a diameter ranging from 0.762 mm to 12.7 mm. The diameters of the first inlet 311 and the second inlet 313 may either be different or same. In an exemplary embodiment, the diameter of the first inlet 311 and the second inlet 313 is 0.75 mm and 13.5 mm, respectively. The first T-tube 310 may define a length between the first inlet 311 and the second inlet 313 ranging from 20 mm to 50 mm. In an exemplary embodiment, the length between the first inlet 311 and the second inlet 313 is 25 mm. The first inlet 311 is configured to receive the first mixture and the second inlet 313 is configured to receive the second mixture at a pre-defined flow rate ratio. The predefined flow rate ratio between the first mixture and the second mixture ranges from 1:1 to 1:10. In an exemplary embodiment, the first mixture is fed to the first inlet 311 of the first T-tube 310 at 10 mL/min and the second mixture is fed to the second inlet 313 of the first T-tube 310 at 30 mL/min.
[96] Additionally or optionally, one or more flow meters 350 may be provided at the first inlet 311 and/or the second inlet 313 to observe and control the flow rate ratio between the first mixture and the second mixture, respectively.
[97] In an exemplary embodiment, as shown in Fig. 2a, the first mixture is fed to the first inlet 311 of the first T-tube 310 via a first syringe 311a. And, the second mixture is fed to the second inlet 313 of the first T-tube 310 via a second syringe 313a. Although the feeding of the first mixture and the second mixture into the first T-tube 310 is described with the examples of the syringes, other functionally equivalent apparatus/technique to feed the first mixture and the second mixture into the first T-tube 310 is within the scope of the teachings of the present disclosure.
[98] The outlet 315 is perpendicular to the first inlet 311 and the second inlet 313. Alternatively, the outlet 315 may define an acute angle with at least one of the first inlet 311 and the second inlet 313. The outlet 315 may have a pre-defined diameter ranging from 0.762 mm to 12.7 mm. In an exemplary embodiment, the diameter of the outlet 315 is 1.524 mm. The outlet 315 may protrude away from the co-aligned first inlet 311 and the second inlet 313, thereby defining a length ranging from 5 mm to 50 mm. In an exemplary embodiment, the outlet 315 protrudes away by 15 mm. The outlet 315 is configured to expel the emulsion (containing the particles 100) that is formed inside the first T-tube 310 via the mixing of the first mixture and the second mixture.
[99] Additionally or optionally, as shown in Fig. 2a, the emulsion obtained from the first T-tube 310 is diluted with the help of a second T-tube 330. The second T-tube 330 may be structurally similar to the first T-tube 310. Similar to the first T-tube 310, the second T-tube 330 includes a first inlet 331, a second inlet 333 and an outlet 335.
[100] The first inlet 331 is configured to receive the emulsion that is expelled by the outlet 115 of the first T-tube 310 at a pre-defined flow rate corresponding to a total flow rate of the first mixture and the second mixture. In an exemplary embodiment, the total flow rate is 40 mL/min.
[101] The second inlet 333 is configured to receive a third solvent used to dilute the particles 100. The third solvent may be at least one of buffer solution, such as HEPES (2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid), Tris buffer, Phosphate buffer, etc. In an exemplary embodiment, the third solvent is to dilute the ethanol content to 12.5% ethanol. The third solvent may be fed into the second inlet 333 at a pre-defined flow rate based on the total flow rate of the first mixture and the second mixture and/or based on the dilution required. In an exemplary embodiment, the third solvent is fed into the second inlet 333 at a flow rate of 40 mL/min using a third syringe 333a. Although the feeding of the buffer into the second T-tube 330 is described with the examples of the syringes, other functionally equivalent apparatus/technique to feed the buffer into the second T-tube 330 is within the scope of the teachings of the present disclosure.
[102] The outlet 335 of the of the second T-tube 330 is configured to expel the diluted emulsion. A collection tube 370 or the like is used to collect the diluted emulsion from the outlet 335 of the second T-tube 330.
[103] At step 209, the emulsion obtained either from step 205b or step 207 is purified to obtain the particles 100 of the system. The emulsion may be purified using at least one of dialysis, diafiltration, tangential flow filtration, chromatography (for example, using a desalting column like a PD10 column), etc. The purification step also helps bring the pH of the system including the particles 100 to neutral or physiological pH.
[104] In an exemplary embodiment, the emulsion is subjected to dialysis using a Tangential Flow Filtration (TFF) assembly against a buffer. In an exemplary embodiment, the emulsion is dialyzed against 10 mM to 50mM phosphate buffer having a pH of 7.4 to remove any residual solvent (for example, ethanol) and to provide buffer exchange. In an alternate embodiment, the emulsion is dialyzed against 10 mM to 50 mM Tris-HCl buffer having a pH of 7.4. In yet another embodiment, the emulsion is dialyzed against 10 mM to 50 mM HEPES buffer (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) having a pH of 7.4.
[105] At an optional step 211, the system including the particles 100 is sterilized by a pre-defined technique to remove any microbes or spores (if present). The pre-defined technique includes at least one of filtration, terminal sterilization, etc.
[106] In an exemplary embodiment, the system including the particles 100 is subjected to filtration using a Millipore filter (having a pore size of 0.22 µ).
[107] The system including at least the particles 100 obtained from step 211 may be suspended in buffer including, but not limited to, phosphate buffer (PBS), Tris buffer, citrate buffer, or a combination thereof to obtain a composition having at least two of the particles 100. The buffer may include at least one salt including, but not limited to, a pre-defined amount of Tris and a pre-defined amount of sodium chloride (NaCl). The amount of Tris ranges from 40 mM to 60 mM. The amount of NaCl ranges from 40 mM to 50 mM.
[108] At yet another optional step 213, the system including the particles 100 are processed for preservation and storage using a pre-defined technique to preserve the integrity of the lipids (i.e., to prevent the lipids from oxidizing). The composition may be stored as suspensions, emulsions, or lyophilized powders. The composition may be stored at either room temperature, at 2°C to 8°C, at 0°C to -80°C, or at -20°C to -80°C basis the duration of storage. If frozen, the composition may be thawed before use either at room temperature, at 25°C, or inside an ice bath or the like.
[109] In an exemplary embodiment, the at least one cryoprotectant (an exemplary nucleic acid stabilizer) is added to the composition and optionally lyophilized. The cryoprotectant may include at least one of trehalose, sucrose (ß-D-Fructofuranosyl a-D-glucopyranoside, having formula XXIV), mannitol ((2R,3R,4R,5R)-Hexane-1,2,3,4,5,6-hexol, having formula XXV), sorbitol (D-Glucitol, having formula XXVI), alpha-methyl D-glucose ether (Alpha-D-glucopyranoside, having formula XXVII), cyclodextrin (ß-Cyclodextrin, having formula XXVIII), lactose (ß-D-Galactopyranosyl-(1-4)-D-glucose not shown), glycerol, dimethylsulfoxide (DMSO), and ethylene glycol, with or without amino acids, like lysine (l-lysine, having formula XXIX), glycine, and additional protein such as bovine serum albumin (BSA), human serum albumin (HSA), etc. and derivatives thereof. The amount of cryoprotectant being added to the system may be up to 10% (w/v). In an exemplary embodiment, the cryoprotectant used to lyophilize the system includes 10% (w/v) sucrose (formula IX) with 0.5% PEG, 0.6% (w/v) HSA and 0.3% (w/v) glycine as adjuvants. In an alternate embodiment, the cryoprotectant used to lyophilize the composition includes 10% (w/v) sucrose with 0.5% PEG (w/v), and 0.3% (w/v) glycine as adjuvants.

(Formula XXIV)
(Formula XXV)
(Formula XXVI)
(Formula XXVII)
(Formula XXVIII)
(Formula XXIX)

[110] In an exemplary embodiment, the composition includes the particles 100 suspended in 5% (w/v) sucrose and 45 mM NaCl in a 50 mM Tris buffer. The amount of salt, buffer, and cryoprotectant in the composition is adjusted to maintain the osmolarity of the composition between 250 mOsm/L and 350 mOsm/L.
[111] The composition including the particles 100 are diluted as required and administered to the patient systemically as injections or infusions via parenteral routes (intravenous, subcutaneous, intramuscular, intradermal), intraperitoneal, intrathecal, intracardiac, intraventricular, intraurethral, intrasternal, intracranial, subretinal, intravitreal, intra-anterior chamber, intramuscular, intrasynovial, intraarterial routes to bloodstream, tissues, muscles, and/or internal organs. The composition may be injected using needles, microneedles, needle-free injector, or the like. The composition may be diluted in a buffer including, but not limited to, HEPES (2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid), TBS (tris buffer saline), PBS (phosphate buffer saline), etc. The pharmaceutically acceptable buffer helps to maintain the pH that ranges from 6.8 to 8.2.
[112] Alternatively, the composition including the particles 100 are administered locally to the patient by topical application. The particles 100 of the composition, before their administration to the patient, may behave as a dispersed phase of an emulsion, an internal phase of a suspension, or a micelle(s).
[113] Additionally or optionally, one or more excipients are added to the composition before it is administered. The excipients help in release of the payload 150 from the particles 100 and/or act as diluents, etc. The selection of excipients is influenced by factors like the intended method of administration, their impact on solubility and stability, and the characteristics of the dosage form, etc.
[114] In an exemplary embodiment, parenteral formulations of the composition are generally either aqueous or oily solutions or suspensions. For aqueous formulations of the composition, the excipients may include sugars (such as glucose, mannitol, or sorbitol), salts, carbohydrates, and buffering agents (typically maintaining a pH between 3 and 9). For some uses, formulations of the composition might be better suited as sterile non-aqueous solutions or in a dried form, which can be reconstituted with a suitable vehicle like sterile, pyrogen-free water (WFI).
[115] In an exemplary embodiment, the particles 100 of the present disclosure are preferentially absorbed by the hepatocytes, i.e., the liver cells. The particles 100 specifically bind with apolipoproteins, including apolipoprotein E (ApoE), which are proteins in plasma that play a crucial role in regulating lipid transport across the body (and cell membranes). Specifically, as an example, the ApoE interacts with cell surface heparin sulfate proteoglycans (an exemplary an ApoE receptor) in the liver during the uptake of the ApoE bound to the particles 100.
[116] The present disclosure will now be explained with the help of the following examples:
[117] Example 1 (Present disclosure): Preparing the particles 100 of the present disclosure
[118] 45 mol% (12.7 mM) of DHA-1 (cationic lipid 101), 10 mol% (2.62 mM) DSPC (neutral lipid 103), 41.5 mol% (11.6 mM) cholesterol (helper lipid 105), and 2.5 mol% (0.623 mM) PEG2k-DMG (polyether 107) were dissolved in 100% ethanol to prepare the first mixture. The second mixture was prepared by dissolving RNA molecules (payload 150) in 20 mM acetate buffer (pH 4.5) at 0.45 mg/mL. The payload 150 was one of mRNA, gRNA, or mRNA+gRNA.
[119] One part of the first mixture was mixed with three parts of the second mixture by T-mixing using a 0.2-inch ID Tee (T-tube) and syringe pump at differential flow rates to obtain the particles 100. The particles 100 were then collected and diluted in Tris buffer saline (TBS) at 1:1 ratio. The diluted particles 100 were then exchanged with 100 times volume of TBS overnight at 4°C, under gentle stirring using 10 kDa slide-a-lyzer G2 dialysis cassette. After dialysis, the particles 100 were filtered using 0.2 µ sterile filter and stored at 2-8°C. The N/P ratio of the particles 100 were 5.
[120] Repeating the above steps, the particles 100 with the following composition were prepared:
Particle 100 samples Cationic lipid 101 Neutral lipid 103 Helper lipid 105 Polyether 107 Payload 150
LNP003 45 mol% DHA-1 10 mol% DSPC 41.5 mol% cholesterol 2.5 mol% DMG-PEG2k 0.45 mg/mL eGFP
mRNA
LNP012 45 mol% DHA-1 10 mol% DSPC 41.5 mol% Cholesterol 2.5 mol% DSPE-PEG2k 0.45 mg/mL eGFP
mRNA
LNP017 45 mol% DHA-1 10 mol% DSPC 41.5 mol% Cholesterol 2.5 mol% C14-PEG2K 0.45 mg/mL eGFP
mRNA
LNP018 45 mol% DHA-2 10 mol% DSPC 41.5 mol% Cholesterol 2.5 mol% DMG-PEG2k 0.45 mg/mL eGFP mRNA
LNP023 45 mol% DHA-1 10 mol% DSPC 41.5 mol% Cholesterol 2.5 mol% DMG-PEG2k 0.45 mg/mL fLUC mRNA
LNP026 45 mol% DHA-1 10 mol% DSPC 41.5 mol% Cholesterol 2.5 mol% DSPE-PEG2k 0.45 mg/mL fLUC mRNA
LNP028 45 mol% DHA-1 10 mol% DSPC 41.5 mol% Cholesterol 2.5 mol% C14-PEG2K 0.45 mg/mL fLUC mRNA
LNP036 45 mol% DHA-2 10 mol% DSPC 41.5 mol% Cholesterol 2.5 mol% DMG-PEG2k 0.45 mg/mL fLUC mRNA
LNP041 45 mol% DHA-1 10 mol% DSPC 41.5 mol% Cholesterol 2.5 mol% DMG-PEG2k 0.45 mg/mL eGFP mRNA
LNP045 45 mol% DHA-1 10 mol% DSPC 41.5 mol% Cholesterol 2.5 mol% DMG-PEG2k 0.45 mg/mL fLUC mRNA
LNP062 45 mol% DHA-1 10 mol% DSPC 41.5 mol% Cholesterol 2.5 mol% DMG-PEG2k 0.45 mg/mL eGFP mRNA
LNP084 45 mol% DHA-1 10 mol% DSPC 41.5 mol% Cholesterol 2.5 mol% DMG-PEG2k 0.45 mg/mL AB4IH mRNA
LNP091 45 mol% DHA-1 10 mol% DSPC 41.5 mol% Cholesterol 2.5 mol% DMG-PEG2k 0.45 mg/mL AB6IH mRNA
LNP106 45 mol% DHA-1 10 mol% DSPC 41.5 mol% Cholesterol 2.5 mol% DMG-PEG2k 0.45 mg/mL Cas9 mRNA
LNP138 45 mol% DHA-1 10 mol% DSPC 41.5 mol% Cholesterol 2.5 mol% DMG-PEG2k 0.45 mg/mL fLUC mRNA
LNP141 46 mol% DOTAP 7.5 mol% DOPE + 8.5 mol% DSPC 35 mol% Cholesterol 3 mol% DMG-PEG2k 0.45 mg/mL eGFP mRNA
LNP144 43.8 mol% DODAP 12.6 mol% DOPE 40.2 mol% Cholesterol 3.4 mol% DMG-PEG2k 0.45 mg/mL eGFP mRNA
LNP153 46.5 mol% DOTAP 11 mol% DSPC 40 mol% Cholesterol 2.5 mol% DMG-PEG2k 0.45 mg/mL eGFP mRNA
LNP165 43.5 mol% DOTAP 12.5 mol% DSPC 42 mol% Cholesterol 2 mol% DMG-PEG2k 0.45 mg/mL eGFP mRNA
LNP171 41.4 mol% DOTAP 10.6 mol% DOPE 46.2 mol% Cholesterol 1.8 mol% DMG-PEG2k 0.45 mg/mL eGFP mRNA
LNP179 46 mol% DOTMA 12 mol% DOPE 39.5 mol% Cholesterol 2.5 mol% C14-PEG2K 0.45 mg/mL eGFP mRNA
LNP186 28 mol% DOTMA 20 mol% DOTAP + 10 mol% DOPE 40.5 mol% Cholesterol 1.5 mol% DMG-PEG2k 0.45 mg/mL eGFP mRNA
LNP195 43.8 mol% DODAP 12.6 mol% DOPE 40.2 mol% Cholesterol 3.4 mol% DMG-PEG2k 0.45 mg/mL eGFP mRNA
LNP202 43.5 mol% DOTAP 12.5 mol% DOPE 41.5 mol% Cholesterol 2.5 mol% DMG-PEG2k 0.45 mg/mL eGFP mRNA
(Table 1)
[121] Example 2 (Present disclosure): Characterization of the particle 100 samples obtained in Example 1 above
[122] The particle 100 samples were assessed for average particle size (Z-average diameter, which represents the intensity-based average particle size), polydispersity (PDI), and concentration of the payload 150, and encapsulation efficiency (EE). The average particle size and PDI were determined using dynamic light scattering (DLS) with a Malvern Zetasizer DLS instrument. Prior to measurement, the particle 100 samples were diluted 20-fold in Tris buffer saline (TBS).
[123] A fluorescence-based assay was employed to measure total concentration of the payload 150 and encapsulation efficiency. The particle 100 samples were diluted 75-fold with 1× Tris-EDTA (TE) buffer to fall within the linear range of the RiboGreen™ dye. 50 µL of the diluted particle 100 samples was mixed with 50 µL of either 1× TE buffer or 1× TE buffer containing 0.2% Triton X-100, in duplicate. The particle 100 samples were incubated at 37°C for 10 minutes to allow Triton X-100 to fully disrupt the particles 100, thereby exposing the payload 150 (mRNA) for interaction with the RiboGreen™ dye. For the standard curve, an RNA solution used in the preparation of the particle 100 samples was used. Diluted RiboGreen™ dye (100 µL of 100× in 1× TE buffer) was then added to each of the particle 100 samples and incubated for 10 minutes at room temperature, shielded from light. A Victore Nivo Multimodal Microplate Reader was used to measure the particle 100 samples with excitation and emission wavelengths set to 480 nm and 530 nm, respectively. The encapsulation efficiency (% EE) was calculated using the following equation: [(1 – Fluoresence525 nm / Fluoresence525 nm +triton) x 100]. Fluoresence525 nm represents the fluorescence measured without triton at 525 nm and Fluoresence525 nm +triton represents the fluorescence measured with triton at 525 nm. Total payload 150 concentration was determined using a liner standard curve and average fluorescence reading from Fluoresence525 nm +triton.
Particle 100 samples Avg. particle size (nm) PDI EE (%)
LNP003 81.06 0.09 91
LNP012 94.45 0.14 78
LNP017 93.82 0.24 82
LNP018 85.72 0.13 88
LNP023 65.59 0.09 92
LNP026 96 0.20 86
LNP028 90.48 0.30 83
LNP036 76.49 0.07 89
LNP041 80.53 0.06 93
LNP045 69.98 0.09 91
LNP062 70.26 0.07 92
LNP084 85.65 0.08 94
LNP091 87.13 0.04 92
LNP106 80.53 0.06 94
LNP138 71.12 0.05 95
LNP141 108.1 0.24 75
LNP144 84.48 0.15 84
LNP153 123.1 0.26 76
LNP165 93.17 0.13 78
LNP171 90.64 0.26 82
LNP179 57.9 0.19 71
LNP186 110.8 0.25 82
LNP195 78.02 0.17 75
LNP202 109.4 0.25 79
(Table 2)
[124] Example 3 (Present disclosure): In vivo delivery of the particle 100 samples obtained in Example 1 above
[125] Balb/c female mice, ranging from 7 – 10 weeks of age were used in each study. Animals were weighed and grouped according to their weights in order to prepare specific dose for each group. The particle 100 samples were injected via intravenous injection into lateral vein in a volume of 0.2 mL per animal (approximately 10 mL per kg). The animals were also observed for any adverse events till 24 hours, and were euthanized at various time intervals by exsanguinations via cardiac puncture under isoflurane anesthesia. Blood was collected into serum separator tubes for measurement of various liver parameters and other body function tests. The organs were isolated, preserved in formalin and sent for histopathological examination to check for any signs of organ toxicities.
[126] The results for histopathological examination displayed no significant changes in tissue distribution, indicating the absence of any significant organ toxicities for all the tested particle 100 samples. The liver function parameters such as ALT (alanine aminotransferase), AST (aspartate aminotransferase) and total bilirubin were well within the acceptable ranges, i.e. ALT – 31 u/L, AST – 109 u/L and total bilirubin – 13 mg/dL.
[127] Example 4 (Present disclosure): In vitro delivery of particle 100 samples (LNP003) obtained in Example 1 above
[128] The particle 100 sample encapsulating the mRNA encoding eGFP (payload 150) were delivered to human embryonic kidney cells (HEK293) or human hepatocyte cells (HepG2), with total amounts of eGFP mRNA ranging from 100ng to 500ng per well. The particle 100 samples were incubated with each cell for 24 hours and eGFP expression was measured using a Fluorescence-activated cell sorting (FACS) Lyric flow cytometer.
[129] As observed in Figs. 3 (depicting data for HEK293T cells) and 4 (depicting data for HepG2 cells), the eGFP expression was observed for the particle 100 sample at different doses in HEK293T and HepG2 cells along with its cell viability, which shows successful delivery and expression of the particles 100 within the cells.
[130] Example 5 (Present disclosure): In vivo delivery of particle 100 (LNP045) samples obtained in Example 1 above
[131] The particle 100 samples encapsulating the mRNA encoding firefly luciferase (fLUC) (payload 150) were tested for mRNA delivery in vivo in animals. The animals were injected with the particle 100 samples at dose of 125 µg/kg and 250 µg/kg. The animals were imaged for their bioluminescence at time interval 3 hours, 6 hours and 24 hours. The blood was collected from the animals after 24 hours and was euthanized, and serum luciferase expression was measured using a Pierce firefly luciferase glow assay kit.
[132] As shown in Fig. 5, dose dependent fLUC expression was measured as flux photons per seconds for group of animals n=5 as compared to TBS (control).
[133] Example 6 (Present disclosure): Effect of pH on particle size, PDI
[134] Fig. 6 depicts a graph demonstrating the effect (while preparing the particles 100) of increase in pH of the buffer (sodium citrate buffer) on particles size and polydispersity. The direct effect on particle size after pH 4, due to its reduced electrostatic stabilization of particles 100, as the cationic lipids 101 lose their charge, combined with the effects of citrate on lipid interactions and possible aggregation.
[135] Fig. 7 depicts a graph demonstrating the effect (while preparing the particles 100) of increase in pH of the buffer (sodium acetate buffer) on particle size and polydispersity. The direct effect on particle size, as the pH and pKa of the buffer are almost same, there is a balance between ionization of cationic lipid 101, leading to the balance between protonation and deprotonation which may result in a more stable system where particles 100 are less likely to aggregate, resulting in a smaller average particle size.
[136] Example 7 (Present disclosure): Effect of pH on EE
[137] Fig. 8 depicts a graph demonstrating the encapsulation of mRNA measured by Ribogreen assay, encapsulated using sodium citrate buffer of varied pH in increasing order. The graph indicates that as the pH of buffer increases, the encapsulation improves, this can be due to the fact that, as the pH increases the protonation of cationic lipid 101 reduces and the packaging around payload 150 tightens, leading to reduced leakage and increased encapsulation.
[138] Fig. 9 depicts a graph demonstrating the encapsulation of mRNA measured by Ribogreen assay, encapsulated using sodium acetate buffer of varied pH in increasing order. The graph indicates that as the pH of buffer increases, the encapsulation increases, the lipids in the particles 100, particularly cationic lipids 101, may be partially protonated. This partial protonation creates an optimal charge density on the lipid molecules that promotes strong electrostatic interactions with the encapsulated payload 150 (especially if the payload 150 is negatively charged, like nucleic acids). This enhanced interaction can lead to more efficient encapsulation.
[139] Example 8 (Present disclosure): Effect of molarity of buffer on particle size, PDI and EE
[140] Fig. 10 depicts a graph demonstrating the effect (while preparing the particles 100) of buffer molarity on particle size and polydispersity. This represents, that low molarity of buffer below 20 mM was giving high particle size and heterogenous particles 100 due to reduced ionic strength, inadequate pH control, compromised electrostatic and steric stabilization, and potential osmotic imbalance. These factors can cause aggregation, swelling, and instability in the bi-layer outer membrane 110, leading to an overall increase in the size of the particles 100.
[141] Fig. 11 depicts a graph demonstrating the effect (while preparing the particles 100) of buffer molarity on encapsulation efficiency of the mRNA (payload 150) into the particles 100, as low molarity can weaken the electrostatic interactions between the mRNA (payload 150) and the lipids, disrupt the formation and integrity of the particles 100, and lead to potential leakage or aggregation.
[142] Example 9 (Present disclosure): Effect of dialysis
[143] Fig. 12 depicts a graph demonstrating the particle size and polydispersity of lipid particles 100 at increasing pH and buffer exchange, as the particles 100 reach towards neutralization, cationic lipid 101 gets deprotonated and attains the steady state, which tend to decreased fusion and aggregation between the particles 100, that reduces the particle size and forms a uniformly distributed system, though there is slight increase in the particle size as that of before dialysis due to increased membrane fluidity and formation of bi-layer outer membrane 110.
[144] Fig. 13 depicts a graph of demonstrating encapsulation efficiency of mRNA (payload 150) within the particles 100. As the dialysis process progresses, the cationic lipid 101 gets deprotonated and there is balance between hydrophilic and hydrophobic components of the particle 100 that leads to tightly packed particles 100, wherein the mRNA (payload 150) is encapsulated at maximum amount due to increased interaction between mRNA-lipid components.
[145] Example 10 (Present disclosure): Effect of cryoprotectant during dialysis
[146] Fig. 14 depicts a graph demonstrating the effect of dialysis buffer with or without cryoprotectant on particle size and polydispersity, showing that the particles 100 with Tris as dialysis buffer along with sucrose 20% is able to retain the original particle size and able to withstand the freeze thaw of particles 100 upto six cycles without affecting the size and polydispersity at a significant level.
[147] Fig. 15 depicts a graph demonstrating the dialysis activity with different buffers alongwith cryoprotectant does not have a significant effect on the encapsulation efficiency, though on exposure to freeze thaw cycles. The mRNA (payload 150) concentration tends to reduce in comparison to Tris.
[148] Example 11 (Present disclosure): Effect of polyether 107
[149] Fig. 16 depicts a graph demonstrating the effect of polyether 107 concentration on particle size and polydispersity of the particles 100. The increase in polyether 107 concentration reduces the particle size and PDI up to a certain extent, afterwards the size increases due to increased osmotic pressure that leads to more water absorption inside the particle 100 core, as well as the increased PEG concentration leads to extended PEG chain on surface that gives increase hydrodynamic diameter by DLS.
[150] Fig. 17 depicts a graph demonstrating the effect of polyether 107 concentration on encapsulation of mRNA (payload 150) within the particle 100 core, due to the fact that PEG imparts hydrophilicity to the particles 100, the encapsulation increases with increased concentration.
[151] Example 12 (Present disclosure): Effect of technique to prepare the primary emulsion
[152] Fig. 18 depicts a graph demonstrating the effect of method of preparing the primary emulsion on particle size and polydispersity index of the particles 100.
[153] Fig. 19 depicts a graph demonstrating the effect of method of preparing the primary emulsion on encapsulation of the mRNA (payload 150) within the particle 100 core, the methods such as t – mixing, microfluidic mixing and post hoc loading showed significant encapsulation efficiency.
[154] Example 13 (Present disclosure): Effect of technique to prepare the primary emulsion
[155] Fig. 20 depicts a graph demonstrating the effect of aqueous (second mixture) to organic (first mixture) flow rate ratio on particle size and polydispersity index. As the ratio of aqueous layer increases the particle size decreases and the homogeneity between particles 100 also increase as this is facilitated by promoting faster nucleation, improved dispersion, reduced lipid aggregation, and more controlled precipitation of lipids.
[156] Fig. 21 depicts a graph demonstrating the effect of aqueous (second mixture) to organic (first mixture) flow rate ratio on encapsulation efficiency as this environment allows for more effective interactions between the mRNA (payload 150) and lipids, resulting in higher encapsulation efficiency. The combination of optimized mixing, improved stability, and efficient nucleation contributes to the successful encapsulation of mRNA within the particles 100 when the aqueous phase (second mixture) is increased relative to the organic phase (first mixture).
[157] Example 14 (Present disclosure): Effect of cryoprotectant
[158] Fig. 22 depicts a graph demonstrating the effect of varied concentration of cryoprotectant and different cryprotectants on particle size and polydispersity of the particles 100 in either liquid or lyophilized state. The effect of sucrose and trehalose is shown here which impacts particle size and homogeneity of particles 100.
[159] Fig. 23 depicts a graph demonstrating effect of addition of sucrose in varied concentration and additives for lyophilization on particle size and polydispersity index. The graph represents the size taken just after dialysis with addition of given sucrose concentration before lyophilization.
[160] Example 15 (Present disclosure): Effect of technique to prepare the primary emulsion
[161] Fig. 24 depicts a graph demonstrating the particles size and polydispersity of the particles 100 after lyophilization/reconstitution in solvent such as WFI. The particle size tends to increase post lyophilization and reconstitution, though it imparts the stability and ability to store the particles 100 at refrigerated conditions.
[162] Example 16 (Present disclosure): Effect of ratio between mRNA and gRNA on encapsulation efficiency
[163] Fig. 25 depicts a graph demonstrating of encapsulation efficiency of different ratios (by weight) of mRNA and gRNA (the payloads 150) within the particles 100 of the present disclosure. The composition of the particle 100 used to determine encapsulation efficiency is provided as LNP106 described in Example 1 above.
[164] The scope of the invention is only limited by the appended patent claims. More generally, those skilled in the art will readily appreciate that all parameters, dimensions, materials, and configurations described herein are meant to be exemplary and that the actual parameters, dimensions, materials, and/or configurations will depend upon the specific application or applications for which the teachings of the present invention is/are used. ,CLAIMS:WE CLAIM,
1. A particle (100) comprising:
a. at least one bi-layer outer membrane (110) defining a lumen (110a), the bi-layer outer membrane (110) including:
i. an outer layer (111) defining a hydrophobic portion and a hydrophilic portion, the outer layer (111) including at least a plurality of cationic lipids (101), a plurality of neutral lipids (103), a plurality of helper lipid (105), and a plurality of polyethers (107), and
ii. an inner layer (113) disposed between the outer layer (111) and the lumen (110a), the inner layer (113) defining a hydrophobic portion and a hydrophilic portion, the inner layer (113) including at least the plurality of cationic lipids (101), the plurality of neutral lipids (103), and the plurality of helper lipids (105);
b. a plurality of vacuoles (130) disposed within the lumen (110a), each of the vacuole (130) includes a membrane layer (131) defining a lumen (130a), the membrane layer (131) including at least the plurality of cationic lipids (101), the plurality of neutral lipids (103), and the plurality of helper lipids (105);
c. at least one payload (150) encapsulated within the lumen (130a) of the at least one vacuole (130), the payload (150) includes at least one mRNA and at least one gRNA in a ratio by weight of 1:1, 1:3, 1:7, 1:10, 3:1, 7:1, or 10:1, the mRNA encodes for at least one of an endonuclease, and a base editor nuclease, the gRNA includes at least one crRNA having binding specificity to a portion a genomic DNA of a host cell.
2. The particle (100) as claimed in claim 1, wherein the bi-layer outer membrane (110) and the particle (100) include a pre-defined shape including at least one of spherical/microspheres, oval, elliptical, or corona-like.
3. The particle (100) as claimed in claim 1, wherein the hydrophobic portions of the outer layer (111) and the inner layer (113) of the bi-layer outer membrane (110) are disposed adjacent to each other.
4. The particle (100) as claimed in claim 1, wherein the respective cationic lipids (101) and the respective neutral lipids (103) of the outer layer (111), the inner layer (113) and the membrane layer (131) are disposed alternatively.
5. The particle (100) as claimed in claim 1, wherein the helper lipid (105) and the polyethers (107) of the outer layer (111) are disposed intermittently between the cationic lipids (101) and the neutral lipids (103) as a group together.
6. The particle (100) as claimed in claim 1, wherein the respective helper lipid (105) of the inner layer (113) and the membrane layer (131) are disposed intermittently between the respective cationic lipids (101) and the respective neutral lipids (103).
7. The particle (100) as claimed in claim 1, wherein the membrane layer (131) includes a hydrophobic portion and a hydrophilic portion disposed towards the lumen (130a) of the vacuole (130).
8. The particle (100) as claimed in claim 1, wherein the gRNA of the payload (150) includes a tracrRNA configured to couple the gRNA to the endonuclease encoded by the mRNA.
9. The particle (100) as claimed in claim 1, wherein the mRNA encodes for at least one of a Cas9 nuclease, a FnCas9 nuclease, a Cpf nuclease, a cytosine base editor (CBE), and an adenine base editor (ABE).
10. The particle (100) as claimed in claim 1, wherein the particle (100) includes at least one nucleic acid stabilizer in the concentration ranging from 0.001% (w/w) to 50% (w/w).
11. The particle (100) as claimed in claim 10, wherein the nucleic acid stabilizer is at least one of polyethylene glycol, cetrimonium bromide, cetrimonium chloride, cetrimonium bromide, low molecular weight chitosan, polyols, nondetergent sulfobetaines, osmolytes, water-soluble polymers, organic solvents, sugars, and salts.
12. The particle (100) as claimed in claim 1, wherein the concentration of the cationic lipids (101) in the particles (100) ranges from 20 mol% to 80 mol%.
13. The particle (100) as claimed in claim 12, wherein the cationic lipid (101) is at least one of DODMA or lipid-T (1,2-dioleyloxy-3-dimethylamino-propane, having formula I), DODAP ((Z)-3-(Dimethylamino)propane-1,2-diyl dioleate, having formula II), DOTAP (1,2-Dioleoyloxy-3-(trimethylammonium)propane, having formula III), DHA-1 or lipid-D (N-[(2-Hydroxyethyl)oxyethyl]azanediyl)bis(hexane-6,1-diyl) bis(2-hexyldecanoate), having formula IV), DHA-2 (di(heptadecan-9-yl) 8,8'-((4-hydroxybutyl)azanediyl)dioctanoate, having formula V), DHA-6 or lipid-H (6-((6-((3-heptylundecanoyl)oxy)hexyl)(2-(2-hydroxyethoxy)ethyl)amino)hexyl 3-hexylundecanoate, having formula VI), D-Lin-MC3-DMA ((6Z,9Z,28Z,31Z)-Heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoate), DLin-KC2-DMA (2-[2,2-bis[(9Z,12Z)-octadeca-9,12-dienyl]-1,3-dioxolan-4-yl]-N,N-dimethylethanamine), cKK - E12 (3,6-bis(4-(bis(2-hydroxydodecyl)amino)butyl)piperazine-2,5-dione), C12 – 200 (1,1'-[[2-[4-[2-[[2-[bis(2-hydroxydodecyl)amino]ethyl](2-hydroxydodecyl)amino]ethyl]-1-piperazinyl]ethyl]imino]bis-2-dodecanol), HUO – 2 (undecyl 6-((7-(((heptadecan-9-yloxy)carbonyl)oxy)heptyl)(2-(2-hydroxyethoxy)ethyl)amino)hexanoate) and a pharmaceutically acceptable salt or solvate thereof.
14. The particle (100) as claimed in claim 1, wherein the concentration of the neutral lipids (103) in the particles (100) ranges from 2 mol% to 25 mol%.
15. The particle (100) as claimed in claim 14, wherein the neutral lipid (103) is at least one of DOPE (3-{[(2-Aminoethoxy)(hydroxy)phosphoryl]oxy}-2-[(octadec-9-enoyl)oxy]propyl octadec-9-enoate, having formula VII), and DSPC (1,2-Distearoyl-sn-glycero-3-phosphocholine, having formula VIII), DOPS (1,2-Dioleoyl-sn-glycero-3-phospho-L-serine, having formula IX), DOPC (1,2-Dioleoyl-sn-Glycero-3-Phosphocholine, having formula X), DPPC (1,2-Dipalmitoyl-rac-glycero-3-phosphocholine, having formula XI), DMPC (dimyristoylphosphatidylcholine, having formula XII), DAPC (1,2-distearoyl-sn-glycero-3-phosphocholine, having formula XIII), DLPC (dilauryloylphosphatidylcholine, having formula XIV), EPC (egg phosphatidylcholine, having formula XV), MPPC (1-myristoyl-2-palmitoyl phosphatidylcholine, having formula XVI), POPC (1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine), resorcinol (5-heptadecylbenzene-1,3-diol), PLPC (phosphatidylcholine), PE (phosphatidylethanolamine), PMPC (1-palmitoyl-2-myristoyl phosphatidylcholine), PSPC (1-palmitoyl-2-stearoyl phosphatidylcholine), DBPC (1,2-diarachidoyl-sn-glycero-3-phosphocholine), SPPC (1-stearoyl-2-palmitoyl phosphatidylcholine), DEPC (1,2-dieicosenoyl-sn-glycero-3-phosphocholine), lysophosphatidylcholine, dilinoleoylphosphatidylcholine, DSPE (distearoylphosphatidylethanolamine), DMPE (dimyristoyl phosphatidylethanolamine), DPPE (dipalmitoyl phosphatidylethanolamine), POPE (palmitoyloleoyl phosphatidylethanolamine), 18:0 Diether PC (1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine), OChemsPC (1-oleoyl-2-cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine), C16 Lyso PC (1-hexadecyl-sn-glycero-3-phosphocholine), DUPC (1,2-diundecanoyl-sn-glycero-phosphocholine), SOPE (1-stearoyl-2-oleoyl-phosphatidylethanolamine), DOPG (1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt) lysophosphatidylethanolamine, PC (phosphatidylcholine), phosphatidylethanolamine amine, glycerophospholipids, sphingophospholipids, Guriserohosuhono, sphingolipid phosphono lipids, natural lecithins, hydrogenated phospholipids, 16-0-Monome Le PE, 16-0-dimethyl PE, 18-1-trans PE, plasmalogen, phosphatidate, phosphatidylserine, phosphatidic acid, phosphatidylglycerol, phosphatidylinositol, POPG (palmitoyl oleoyl phosphatidylglycerol) lysophosphatidylcholine, sphingomyelin, ceramide phosphoethanolamine, ceramide phosphoglycerol, ceramide phosphoglycerophosphoric acid, egg yolk lecithin, soybean lecithin, hydrogenated soybean phosphatidylcholine and a pharmaceutically acceptable salt or solvate thereof.
16. The particle (100) as claimed in claim 1, wherein the concentration of the helper lipids (105) in the particles (100) ranges from 40 mol% to 60 mol%.
17. The particle (100) as claimed in claim 16, wherein the helper lipid (105) is at least one of a sterol, and alkyl resorcinol, cholesterol (having formula XVII, formula XVIII, formula XIX, formula XX), 5-heptadecylresorcinol, cholesterol hemisuccinate, fecosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatidine, tomatine, ursolic acid, alpha-tocopherol, prednisolone, dexamethasone, prednisone, and hydrocortisone.
18. The particle (100) as claimed in claim 1, wherein the concentration of the polyethers (107) in the particles (100) ranges from 0.1 mol% to 6 mol%.
19. The particle (100) as claimed in claim 18, wherein the polyether (107) is at least one of DMG-PEG 2000 (Azane;[3-(2-methoxyethoxy)-2-tetradecanoyloxypropyl] tetradecanoate, having formula XXII), DSPE-PEG 5000 (1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-5000], having formula XXIII), DSG PEG 2000 (1,2-distearoyl-rac-glycero-3-methylpolyoxyethylene 2000, having formula XXIV), DMPE PEG 2000 (1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-([methoxy(polyethylene glycol)-2000]), PEG-DPPC, PEG-DLPE, PEG-c-DOMG, PEG-dilauroylglycerol, PEG-dipalmitoylglycerol, PEG-dipalmitoylglycamide, PEG-disterylglycamide, PEG-cholesterol, PEG-DMB (3,4-ditetradecoxylbenzyl-[omega]-methyl-poly(ethylene glycol) ether), 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000]) and derivatives thereof.