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WO2023192624A2 - Co-administration d'acides nucléiques de charge utile et de promotion - Google Patents

Co-administration d'acides nucléiques de charge utile et de promotion Download PDF

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Publication number
WO2023192624A2
WO2023192624A2 PCT/US2023/017174 US2023017174W WO2023192624A2 WO 2023192624 A2 WO2023192624 A2 WO 2023192624A2 US 2023017174 W US2023017174 W US 2023017174W WO 2023192624 A2 WO2023192624 A2 WO 2023192624A2
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Prior art keywords
nucleic acid
oligonucleotide
promoting
loaded
payload
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WO2023192624A3 (fr
WO2023192624A8 (fr
Inventor
Ozan ALKAN
Wei Zhong LEONG
Yumi KAWAMURA
Dean Zi Yang LEE
Kimberly Moyan BURNETT
Ronne YEO
Donald David HAUT JR.
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Carmine Therapeutics Pte Ltd
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Carmine Therapeutics Pte Ltd
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Publication of WO2023192624A3 publication Critical patent/WO2023192624A3/fr
Publication of WO2023192624A8 publication Critical patent/WO2023192624A8/fr
Anticipated expiration legal-status Critical
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0641—Erythrocytes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/14—Blood; Artificial blood
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/0008—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition
    • A61K48/0025—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition wherein the non-active part clearly interacts with the delivered nucleic acid
    • A61K48/0041—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition wherein the non-active part clearly interacts with the delivered nucleic acid the non-active part being polymeric
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C12N15/88—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation using microencapsulation, e.g. using amphiphile liposome vesicle

Definitions

  • Nucleic acids offer exciting therapeutic possibilities, including the possibility of treating otherwise untreatable diseases. Nucleic acids can be delivered to a system to accomplish a wide range of biological effects. However, efforts to develop nucleic acid therapies often face challenges including, for example, poor transgene expression and immune response to the nucleic acid. These challenges can limit the effectiveness and therapeutic potential of nucleic acid delivery. SUMMARY [0002] Among other things, the present disclosure provides an insight that one or more aspects of nucleic acid delivery and/or administration are sometimes inhibited by activity of one or more biological pathways endogenous to the host or cell to which the nucleic acid is administered.
  • the present disclosure demonstrates that administration of a second nucleic acid, and specifically of a promoting oligonucleotide as described herein, can improve or otherwise promote certain features of nucleic acid administration (e.g., of administration of a payload nucleic acid as described herein).
  • administration of a promoting oligonucleotide as described herein can enhance or increase one or more features of expression and/or activity of a payload nucleic acid.
  • administration of a promoting oligonucleotide as described herein can inhibit or reduce one or more features of an undesirable effect of or response to such payload nucleic acid.
  • a promoting oligonucleotide is or comprises DNA, RNA, or non-natural residues. In some embodiments, a promoting oligonucleotide is or comprises double-stranded and/or single-stranded portions. A promoting oligonucleotide may be self-complementary. In some embodiments, a promoting oligonucleotide is or comprises a nucleic acid sequence of particular length (e.g., as measured by base pairs or nucleotides). In some embodiments, a promoting oligonucleotide does not encode an expression product.
  • a promoting oligonucleotide increases expression and/or activity of a payload nucleic acid in a cell to which it is administered or delivered.
  • a promoting oligonucleotide increases the number of copies of payload nucleic acid delivered to a system (e.g., a cell, tissue, or organism).
  • a promoting oligonucleotide increases the number of cells that receive delivery of a payload nucleic acid.
  • a promoting oligonucleotide increases the amount of expression product expressed per copy of payload nucleic acid.
  • a promoting oligonucleotide decreases the amount of payload nucleic acid (e.g., or of a product it encodes) degraded upon delivery to a system.
  • a promoting oligonucleotide interacts with (e.g., hybridizes with) an endogenous factor of a cell to which it (and/or the payload nucleic acid) is administered or delivered.
  • a useful promoting nucleic acid may, upon binding to an endogenous factor of a cell, absorb and/or neutralize the biologic effects of the endogenous factor such that its endogenous functions are lessened.
  • a promoting oligonucleotide inhibits or neutralizes an immune response (e.g., to a payload nucleic acid).
  • a promoting oligonucleotide acts as a decoy, lure, trap, bait, mimic, squelch, and/or sink to an endogenous factor of a cell.
  • the present disclosure provides technologies (e.g., by administration of one or more promoting oligonucleotides as described herein) for suppressing and/or inhibiting one or more biological pathway, and demonstrates beneficial impact in certain nucleic acid administration contexts.
  • a biological pathway may be a nucleic acid sensing pathway.
  • such a biological pathway may be an inflammatory signaling pathway.
  • a promoting oligonucleotide suppresses and/or inhibits a specific effector of a biological pathway.
  • a promoting oligonucleotide inhibits multiple effectors of the same or different biological pathways.
  • a promoting oligonucleotide inhibits or neutralizes a transcription factor. In some embodiments, a promoting oligonucleotide inhibits or neutralizes NF- ⁇ B. In some embodiments, a promoting oligonucleotide inhibits or neutralizes TLR9. In some embodiments, a promoting oligonucleotide inhibits or neutralizes cGAS. In some embodiments, a promoting oligonucleotide inhibits or neutralizes RIG-I. [0010] In some embodiments, delivery vehicles are used to deliver cargo nucleic acid(s) as described herein.
  • cargo nucleic acids e.g., a promoting oligonucleotide
  • a delivery vehicle e.g., the present disclosure
  • EVs e.g., RBCEVs
  • a promoting oligonucleotide improves or increases delivery (e.g., in delivery vehicle loading) of cargo nucleic acid(s).
  • one or more promoting oligonucleotides are loaded into a delivery vehicle with one more payload nucleic acids.
  • one or more promoting oligonucleotides are loaded into a delivery vehicle, and one or more payload nucleic acids are loaded into a further delivery vehicle.
  • a population of delivery vehicles co-loaded with one or more promoting oligonucleotides and one or more payload nucleic acids are administered.
  • a population of delivery vehicles loaded with one or more promoting oligonucleotides are administered prior to administration of a population of delivery vehicles loaded with one or more payload nucleic acids.
  • a promoting oligonucleotide is useful for therapeutic purposes.
  • a promoting oligonucleotide is useful for therapeutic nucleic acid delivery.
  • a promoting oligonucleotide is useful for gene therapy. In some embodiments, a promoting oligonucleotide is useful for delivery of a replacement gene for a genetic disorder. In some embodiments, a promoting oligonucleotide is useful for delivery of a gene editing system. In some embodiments, a promoting oligonucleotide is useful for delivery of an antibody or antigen-binding fragment. In some embodiments, a promoting oligonucleotide is useful for delivery of a vaccine or epitope.
  • the present disclosure provides certain promoting oligonucleotides, compositions including them (e.g., associated with delivery vehicles) and/or methods of making and/or using the foregoing or related materials.
  • the present disclosure provides a population of delivery vehicles loaded with at least two cargo nucleic acids, wherein at least one nucleic acid is a payload nucleic and at least one nucleic acid is a promoting oligonucleotide, wherein the promoting oligonucleotide is associated with: (a) increased level of an expression product of the payload nucleic acid; and/or (b) decreased immune response associated with administration or delivery of the payload nucleic acid and/or a pharmaceutical composition thereof.
  • the present disclosure provides methods of manufacturing, characterizing, and/or using such populations, including for example, in methods of suppressing an immune response following administration of a payload nucleic acid in a human subject comprising administration of a promoting oligonucleotide.
  • the present disclosure provides methods comprising a step of administering to a subject who has received or will receive a payload nucleic acid a composition comprising a promoting oligonucleotide associated with a delivery vehicle.
  • the present disclosure provides methods comprising a step of co-loading a payload nucleic acid and a promoting oligonucleotide into a delivery vehicle by electroporation.
  • the present disclosure provides methods comprising a step of co-loading a payload nucleic acid and a promoting oligonucleotide into a delivery vehicle by transfection. In some embodiments, the present disclosure provides methods comprising a step of administering a payload nucleic acid to a subject to whom a composition comprising a promoting oligonucleotide associated with a delivery vehicle has been administered. [0019] In some embodiments, the present disclosure provides technologies for characterizing populations of particulars as descried herein, and compositions characterized by certain features as described herein.
  • the present disclosure provides compositions comprising a population of delivery vehicles co-loaded with a payload nucleic acid and a promoting oligonucleotide, which population is characterized in that, when administered to a mammalian subject, the composition achieves an elevated level of expression, and/or a longer period of expression, of the payload nucleic acid as compared with that achieved by comparable reference composition lacking the promoting oligonucleotide.
  • a population of delivery vehicles co-loaded with a payload nucleic acid and a promoting oligonucleotide which population is characterized in that, when administered to a mammalian subject, the composition achieves an elevated level of expression, and/or a longer period of expression, of the payload nucleic acid as compared with that achieved by comparable reference composition lacking the promoting oligonucleotide.
  • Huh-7 (Panels A-C), HepG2 (Panels D-F), and THP-1 cells (Panels G-I) were transfected with RBCEVs loaded with DNA plasmid alone (EV- NP) or co-loaded with DNA plasmid and NF- ⁇ B decoy (ODN), scrambled (SCD), phosphorothioate-modified NF- ⁇ B decoy (ODN-PS), or phosphorothioate-modified scrambled (SCD-PS) oligonucleotides at increasing dosages from 12.5 to 100 pmol.
  • EV- NP DNA plasmid alone
  • ODN NF- ⁇ B decoy
  • SCD scrambled
  • SCD-PS phosphorothioate-modified NF- ⁇ B decoy
  • SCD-PS phosphorothioate-modified scrambled
  • Luminescence of HiBiT-tagged FIX protein was measured from day 1 to day 49 after administration of RBCEVs loaded with DNA plasmid alone (0) or co-loaded with DNA plasmid and NF- ⁇ B decoy (ODN) or scrambled (SCD) oligonucleotides at 25pmol (low dose; ODN-25 and SCD-25) or 100 pmol (high dose; ODN-100 and SCD-100).
  • ODN NF- ⁇ B decoy
  • SCD scrambled
  • Trastuzumab was measured in the serum of mice from day 1 to day 49 after administration of RBCEVs loaded with DNA plasmid alone (0) or co- loaded with DNA plasmid and NF- ⁇ B decoy (ODN) oligonucleotides at 4 mg/kg and 6 mg/kg dose.
  • ODN NF- ⁇ B decoy
  • Figure 4 RBCEVs co-loaded with DNA vector and NF- ⁇ B decoy oligonucleotides can reduce systemic IFNa and IFNb in vivo.
  • BL/6 mice were injected with RBCEVs loaded with DNA plasmid alone (EV-NP) or co-loaded with DNA plasmid and NF- ⁇ B decoy (ODN) or scrambled (SCD) oligonucleotides at 100 pmol (ODN-100 and SCD-100) by intravenous tail- vein injection. Naked DNA plasmid was injected via hydrodynamic injection (HDI). Blood was drawn at different timepoints and quantified for mouse interferon alpha (IFNa; Panel A) and interferon beta (IFNb; Panel B) levels by ELISA.
  • Figure 5 Comparison of oligonucleotide designs co-loaded in RBCEVs in vitro.
  • HepG2 (Panels A-C) and Huh7 (Panels D-F) cells were transfected with RBCEVs co-loaded with a CMV-copGFP plasmid and an oligonucleotide of different design.
  • GFP expression Panels A, D
  • mean fluorescence intensity MFI; Panels B, E
  • RBCEVs were co-loaded with plasmid DNA and bait oligonucleotides from Table 2 or NF- ⁇ B decoy oligonucleotide. Plasmid and oligonucleotide loading efficiencies were measured with agarose gel electrophoresis of DNA extracted from loaded RBCEVs. [0027] Figure 7. RBCEVs co-loaded with a bait oligonucleotide can improve transgene expression in vitro. Huh7 cells were transfected with RBCEVs co-loaded with plasmid and bait oligonucleotide or NF- ⁇ B decoy oligonucleotide.
  • FIX-Hibit protein expression (Panel A) and EGFP expression (Panel B) were measured at 24 hours after transfection.
  • Figure 8 RBCEVs co-loaded with a double-stranded or a single-stranded oligonucleotide can increase transgene expression in vitro.
  • Huh7 (Panel A) and HepG2 (Panel B) cells were transfected with RBCEVS co-loaded with a DNA plasmid that expresses hFIX-HiBit luciferase reporter construct and a double-stranded oligonucleotide (scrambled, NF- ⁇ B decoy) or a single-stranded oligonucleotide. Luminescence was measured 24 hours later with NANO-GLO ® HiBiT assay. [0029] Figure 9. RBCEVs co-loaded with single-stranded oligonucleotides can modulate gene expression associate with immune response in vitro.
  • THP-1 cells were transfected with RBCEVs co-loaded with a DNA plasmid that expresses hFIX-HiBit luciferase reporter construct and a double-stranded oligonucleotide (scrambled, NF- ⁇ B decoy) or a single- stranded oligonucleotide. Gene expression was measured 6 hours later by quantitative PCR (qPCR). Taqman target specific probes for IFNb1 (Panel A), IL6 (Panel B), CXCL10 (Panel C) and CCL2 (Panel D) were used for target genes. Taqman target specific probes for GAPDH was used for normalization of cDNA input.
  • FIG. 10 Pre-treatment with RBCEVs loaded with double-stranded oligonucleotides can increase transgene expression of subsequently delivered DNA plasmid.
  • HepG2 (Panels A-C) and Huh-7 (Panels D-F) cells were transfected with RBCEVs loaded with NF- ⁇ B decoy (ODN) or scrambled (SCD) oligonucleotides at 25 pmol or 50 pmol doses. After 24 hours, cells that were pre-conditioned by ODN or SCD or neither (no pre- treat) were subsequently transfected with RBCEVs loaded with DNA plasmid (CMV-copGFP) alone.
  • ODN NF- ⁇ B decoy
  • SCD scrambled
  • Pre-treatment with RBCEVs loaded with double-stranded oligonucleotides can further increase transgene expression of subsequently delivered DNA plasmid co-loaded with NF- ⁇ B decoy (ODN) oligonucleotides.
  • ODN NF- ⁇ B decoy
  • HepG2 cells were transfected with RBCEVs loaded with NF- ⁇ B decoy (ODN) or scrambled (SCD) oligonucleotides at increasing dosages from 25 to 50 pmol.
  • Administration typically refers to the administration of a composition to a subject or system (e.g., that is or comprises one or more cells, tissues, organisms, etc.), for example to achieve delivery of an agent that is, is included in, or is otherwise delivered by, the composition.
  • Affinity As is known in the art, “affinity” is a measure of the tightness with which two or more binding partners associate with one another. Those skilled in the art are aware of a variety of assays that can be used to assess affinity, and will furthermore be aware of appropriate controls for such assays. In some embodiments, affinity is assessed in a quantitative assay.
  • affinity is assessed over a plurality of concentrations (e.g., of one binding partner at a time). In some embodiments, affinity is assessed in the presence of one or more potential competitor entities (e.g., that might be present in a relevant – e.g., physiological – setting). In some embodiments, affinity is assessed relative to a reference (e.g., that has a known affinity above a particular threshold [a “positive control” reference] or that has a known affinity below a particular threshold [ a “negative control” reference”]. In some embodiments, affinity may be assessed relative to a contemporaneous reference; in some embodiments, affinity may be assessed relative to a historical reference. Typically, when affinity is assessed relative to a reference, it is assessed under comparable conditions.
  • an analog refers to a substance that shares one or more particular structural features, elements, components, or moieties with a reference substance. Typically, an “analog” shows significant structural similarity with the reference substance, for example sharing a core or consensus structure, but also differs in certain discrete ways.
  • an analog is a substance that can be generated from the reference substance, e.g., by chemical manipulation of the reference substance. In some embodiments, an analog is a substance that can be generated through performance of a synthetic process substantially similar to (e.g., sharing a plurality of steps with) one that generates the reference substance.
  • an analog is or can be generated through performance of a synthetic process different from that used to generate the reference substance.
  • Two events or entities are “associated” with one another, as that term is used herein, if the presence, level, degree, type and/or form of one is correlated with that of the other.
  • a particular entity e.g., cargo nucleic acid
  • a biological event e.g., expression or activity of a polypeptide encoded by a payload nucleic acid, level of cytokine indicative of an inflammatory response, level of expression of a gene regulated by an inflammation-associated regulator, cell viability, etc.
  • a biological event e.g., expression or activity of a polypeptide encoded by a payload nucleic acid, level of cytokine indicative of an inflammatory response, level of expression of a gene regulated by an inflammation-associated regulator, cell viability, etc.
  • two or more entities are physically “associated” with one another if they interact, directly or indirectly, so that they are and/or remain in physical proximity with one another.
  • two or more entities that are physically associated with one another are covalently linked to one another; in some embodiments, two or more entities that are physically associated with one another are not covalently linked to one another but are non-covalently associated, for example by means of hydrogen bonds, van der Waals interaction, hydrophobic interactions, magnetism, and combinations thereof.
  • Binding typically refers to a non-covalent association between or among two or more entities. “Direct” binding involves physical contact between entities or moieties; indirect binding involves physical interaction by way of physical contact with one or more intermediate entities.
  • Binding between two or more entities can typically be assessed in any of a variety of contexts – including where interacting entities or moieties are studied in isolation or in the context of more complex systems (e.g., while covalently or otherwise associated with a carrier entity and/or in a biological system or cell). Binding between two entities may be considered “specific” if, under the conditions assessed, the relevant entities are more likely to associate with one another than with other available binding partners.
  • Comparable refers to two or more agents, entities, situations, sets of conditions, etc., that may not be identical to one another but that are sufficiently similar to permit comparison therebetween so that one skilled in the art will appreciate that conclusions may reasonably be drawn based on differences or similarities observed.
  • comparable sets of conditions, circumstances, individuals, or populations are characterized by a plurality of substantially identical features and one or a small number of varied features.
  • Those of ordinary skill in the art will understand, in context, what degree of identity is required in any given circumstance for two or more such agents, entities, situations, sets of conditions, etc to be considered comparable.
  • sets of circumstances, individuals, or populations are comparable to one another when characterized by a sufficient number and type of substantially identical features to warrant a reasonable conclusion that differences in results obtained or phenomena observed under or with different sets of circumstances, individuals, or populations are caused by or indicative of the variation in those features that are varied.
  • Cargo Nucleic Acid refers to a nucleic acid that is administered or otherwise delivered to a subject or system of interest (e.g., that is or comprises one or more cells, tissues, organisms, etc).
  • a cargo nucleic acid is present in and/or delivered from an extracellular vesicle (EV, e.g., a red blood cell extracellular vesicle, RBCEV).
  • EV extracellular vesicle
  • a cargo nucleic acid is or comprises a payload nucleic acid.
  • a cargo nucleic acid is or comprises a promoting oligonucleotide.
  • more than one cargo nucleic acid is administered or otherwise delivered to the same subject or system in accordance with the present disclosure.
  • at least one payload nucleic acid and at least one promoting oligonucleotide are administered or otherwise delivered to the same subject or system in accordance with the present disclosure, in some embodiments as cargo within the same EV (e.g., RBCEV), in some embodiments as separate cargos within different EVs (e.g., RBCEVs) or otherwise separately.
  • the term “corresponding to” refers to a relationship between two or more entities.
  • corresponding to may be used to designate the position/identity of a structural element in a compound or composition relative to another compound or composition (e.g., to an appropriate reference compound or composition).
  • a monomeric residue in a polymer e.g., an amino acid residue in a polypeptide or a nucleic acid residue in a polynucleotide
  • corresponding to a residue in an appropriate reference polymer.
  • residues in a polypeptide are often designated using a canonical numbering system based on a reference related polypeptide, so that an amino acid "corresponding to" a residue at position 190, for example, need not actually be the 190 th amino acid in a particular amino acid chain but rather corresponds to the residue found at 190 in the reference polypeptide; those of ordinary skill in the art readily appreciate how to identify "corresponding" amino acids.
  • sequence alignment strategies including software programs such as, for example, BLAST, CS-BLAST, CUSASW++, DIAMOND, FASTA, GGSEARCH/GLSEARCH, Genoogle, HMMER, HHpred/HHsearch, IDF, Infernal, KLAST, USEARCH, parasail, PSI-BLAST, PSI-Search, ScalaBLAST, Sequilab, SAM, SSEARCH, SWAPHI, SWAPHI-LS, SWIMM, or SWIPE that can be utilized, for example, to identify “corresponding” residues in polypeptides and/or nucleic acids in accordance with the present disclosure.
  • software programs such as, for example, BLAST, CS-BLAST, CUSASW++, DIAMOND, FASTA, GGSEARCH/GLSEARCH, Genoogle, HMMER, HHpred/HHsearch, IDF, Infernal, KLAST, USEARCH, parasail, PSI-BLAST, PSI-Search, Scala
  • the term “corresponding to” may be used to describe an event or entity that shares a relevant similarity with another event or entity (e.g., an appropriate reference event or entity).
  • a gene or protein in one organism may be described as “corresponding to” a gene or protein from another organism in order to indicate, in some embodiments, that it plays an analogous role or performs an analogous function and/or that it shows a particular degree of sequence identity or homology, or shares a particular characteristic sequence element.
  • Delivery vehicle refers to an agent that complexes or otherwise interacts with nucleic acid for the purpose of delivering said nucleic acid to a system.
  • Delivery vehicles may stabilize nucleic acid in otherwise harsh conditions (e.g., a bloodstream after in vivo administration). Delivery vehicles may allow for nucleic acid to pass through the plasma membrane of a cell (i.e., be delivered to a cell). Furthermore, delivery vehicles may provide cell-type or tissue-type specificity in delivering of a nucleic acid. Delivery vehicles may be, for example, polyplexes, nanoconjugates, micelles, vesicles, nanocapsules, dendrimers, or nanoparticles (NPs).
  • NPs nanoparticles
  • Dosing regimen As used herein, the term “designed” refers to an agent (i) whose structure is or was selected by the hand of man; (ii) that is produced by a process requiring the hand of man; and/or (iii) that is distinct from natural substances and other known agents.
  • Dosing regimen may be used to refer to a set of unit doses (typically more than one) that are administered individually to a subject, typically separated by periods of time. In some embodiments, a given therapeutic agent has a recommended dosing regimen, which may involve one or more doses.
  • a dosing regimen comprises a plurality of doses each of which is separated in time from other doses. In some embodiments, individual doses are separated from one another by a time period of the same length; in some embodiments, a dosing regimen comprises a plurality of doses and at least two different time periods separating individual doses. In some embodiments, all doses within a dosing regimen are of the same unit dose amount. In some embodiments, different doses within a dosing regimen are of different amounts. In some embodiments, a dosing regimen comprises a first dose in a first dose amount, followed by one or more additional doses in a second dose amount different from the first dose amount.
  • a dosing regimen comprises a first dose in a first dose amount, followed by one or more additional doses in a second dose amount same as the first dose amount.
  • a dosing regimen is correlated with a desired or beneficial outcome when administered across a relevant population (i.e., is a therapeutic dosing regimen).
  • Engineered In general, the term “engineered” refers to the aspect of having been manipulated by the hand of man.
  • a polynucleotide is considered to be “engineered” when two or more sequences that are not linked together in that order in nature are manipulated by the hand of man to be directly linked to one another in the engineered polynucleotide and/or when a particular residue in a polynucleotide is non- naturally occurring and/or is caused through action of the hand of man to be linked with an entity or moiety with which it is not linked in nature.
  • a cell or organism is considered to be “engineered” if it has been subjected to a manipulation, so that its genetic, epigenetic, and/or phenotypic identity is altered relative to an appropriate reference cell such as otherwise identical cell that has not been so manipulated.
  • such a manipulation is or comprises a genetic manipulation, so that its genetic information is altered (e.g., new genetic material not previously present has been introduced, for example by transformation, mating, somatic hybridization, transfection, transduction, or other mechanism, or previously present genetic material is altered or removed, for example by substitution or deletion mutation, or by mating protocols).
  • an engineered cell is one that has been manipulated so that it contains and/or expresses a particular agent of interest (e.g., a protein, a nucleic acid, and/or a particular form thereof) in an altered amount and/or according to altered timing relative to such an appropriate reference cell.
  • a gene product can be a transcript.
  • a gene product can be a polypeptide.
  • expression of a nucleic acid sequence involves one or more of the following: (1) production of an RNA template from a DNA sequence (e.g., by transcription); (2) processing of an RNA transcript (e.g., by splicing, editing, etc); (3) translation of an RNA into a polypeptide or protein; and/or (4) post- translational modification of a polypeptide or protein.
  • homology refers to overall relatedness between polymeric molecules, e.g., between nucleic acid molecules (e.g., DNA molecules and/or RNA molecules) and/or between polypeptide molecules.
  • polymeric molecules are considered to be “substantially homologous” to one another if their sequences are at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% homologous, meaning that identical or homologous residues are present in corresponding positions of both molecules.
  • Calculation of percent homology of two nucleic acid or polypeptide sequences can be performed by aligning two sequences for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second sequences for optimal alignment and non-identical sequences can be disregarded for comparison purposes).
  • a length of a sequence aligned for comparison purposes is at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or substantially 100% of length of a reference sequence; residues at corresponding positions are then compared.
  • residues at corresponding positions are then compared.
  • Percent homology between two sequences is a function of the number of homologous positions shared by the two sequences being compared, taking into account the number of gaps, and the length of each gap, which needs to be introduced for optimal alignment of the two sequences. Comparison of sequences and determination of percent homology between two sequences can be accomplished using a mathematical algorithm.
  • percent homology between two nucleotide sequences can be determined using the algorithm of Meyers and Miller (CABIOS, 1989, 4: 11-17, which is herein incorporated by reference in its entirety), which has been incorporated into the ALIGN program (version 2.0).
  • “Improved,” “increased” or “reduced” indicate values that are relative to a comparable reference measurement. For example, in some embodiments, an assessed value achieved with an agent of interest may be “improved” relative to that obtained with a comparable reference agent.
  • an assessed value achieved in a subject or system of interest may be “improved” relative to that obtained in the same subject or system under different conditions (e.g., prior to or after an event such as administration of an agent of interest), or in a different, comparable subject (e.g., in a comparable subject or system that differs from the subject or system of interest in presence of one or more indicators of a particular disease, disorder or condition of interest, or in prior exposure to a condition or agent, etc).
  • comparative terms refer to statistically relevant differences (e.g., that are of a prevalence and/or magnitude sufficient to achieve statistical relevance).
  • Indicative Marker refers to an entity or moiety whose presence or level is a characteristic of a particular state or event. In some embodiments, presence or level of a particular indicative marker may be characteristic of presence or stage of a disease, disorder, or condition. To give but one example, in some embodiments, the term refers to a gene expression product that is characteristic of an immune effect or response, inflammation, cytokine release, etc.
  • a presence or level of a particular indicative marker correlates with activity (or activity level) of a particular signaling pathway, for example that may be characteristic of a particular pathway of inflammation.
  • the statistical significance of the presence or absence of an indicative marker may vary depending upon the particular marker.
  • detection of an indicative marker is highly specific in that it reflects a high probability that a biological event or process has occurred. Such specificity may come at the cost of sensitivity (i.e., a negative result may occur even if the biological event or process has occurred and would be expected to result in the indicative marker being present at a certain level). Conversely, indicative markers with a high degree of sensitivity may be less specific that those with lower sensitivity.
  • Nanoparticle refers to a discrete entity of small size, e.g., typically having a longest dimension that is shorter than about 1000 nanometers (nm) and often is shorter than 500 nm, or even 100 nm or less. In many embodiments, a nanoparticle may be characterized by a longest dimension between about 1 nm and about 100 nm, or between about 1 ⁇ m and about 500 nm, or between about 1 nm and 1000 nm.
  • a population of microparticles is characterized by an average size (e.g., longest dimension) that is below about 1000 nm, about 500 nm, about 100 nm, about 50 nm, about 40 nm, about 30 nm, about 20 nm, or about 10 nm and often above about 1 nm.
  • a microparticle may be substantially spherical (e.g., so that its longest dimension may be its diameter).
  • a nanoparticle has a diameter of less than 100 nm as defined by the National Institutes of Health.
  • nanoparticles are micelles in that they comprise an enclosed compartment, separated from the bulk solution by a micellar membrane, typically comprised of amphiphilic entities which surround and enclose a space or compartment (e.g., to define a lumen).
  • a micellar membrane is comprised of at least one polymer, such as for example a biocompatible and/or biodegradable polymer.
  • Nanoparticle composition refers to a composition that contains at least one nanoparticle and at least one additional agent or ingredient.
  • a nanoparticle composition contains a substantially uniform collection of nanoparticles as described herein.
  • nucleic acid refers to a polymer of at least three nucleotides.
  • a nucleic acid comprises DNA.
  • RNA RNA
  • a nucleic acid is single-stranded.
  • a nucleic acid is double-stranded.
  • a nucleic acid comprises both single- and double-stranded portions.
  • a nucleic acid comprises a backbone that comprises one or more phosphodiester linkages.
  • a nucleic acid comprises a backbone that comprises both phosphodiester and non-phosphodiester linkages.
  • a nucleic acid may comprise a backbone that comprises one or more phosphorothioate or 5'- N-phosphoramidite linkages and/or one or more peptide bonds, e.g., as in a “peptide nucleic acid”.
  • a nucleic acid comprises one or more, or all, natural residues (e.g., adenine, cytosine, deoxyadenosine, deoxycytidine, deoxyguanosine, deoxythymidine, guanine, thymine, uracil).
  • a nucleic acid comprises on or more, or all, non-natural residues.
  • a non-natural residue comprises a nucleoside analog (e.g., 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolo-pyrimidine, 3 -methyl adenosine, 5-methylcytidine, C-5 propynyl-cytidine, C-5 propynyl-uridine, 2-aminoadenosine, C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5-propynyl-uridine, C5 -propynyl-cytidine, C5-methylcytidine, 2-aminoadenosine, 7- deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, 0(6)-methylguanine, 2-thiocytidine, methylated bases, intercalated bases, and combinations thereof).
  • a non-natural residue comprises one or more modified sugars (e.g., 2'- fluororibose, ribose, 2'-deoxyribose, arabinose, and hexose) as compared to those in natural residues.
  • a nucleic acid has a nucleotide sequence that encodes a functional gene product such as an RNA or polypeptide.
  • a nucleic acid has a nucleotide sequence that comprises one or more introns.
  • a nucleic acid may be prepared by isolation from a natural source, enzymatic synthesis (e.g., by polymerization based on a complementary template, e.g., in vivo or in vitro, reproduction in a recombinant cell or system, or chemical synthesis.
  • enzymatic synthesis e.g., by polymerization based on a complementary template, e.g., in vivo or in vitro, reproduction in a recombinant cell or system, or chemical synthesis.
  • a nucleic acid is at least 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000 or more residues long.
  • Payload Nucleic Acid refers to a nucleic acid that is administered or otherwise delivered to a subject or system of interest (e.g., that is or comprises one or more cells, tissues, organisms, etc.) that results in or is intended to achieve a particular biological result.
  • a payload nucleic acid encodes an expression product (e.g., a transcript or polypeptide) that achieves or is intended to achieve the relevant result.
  • a payload nucleic acid wholly or partly makes up a cargo nucleic acid.
  • a payload nucleic acid is present in and/or delivered from an extracellular vesicle (EV, e.g., a red blood cell extracellular vesicle, RBCEV).
  • EV extracellular vesicle
  • RBCEV red blood cell extracellular vesicle
  • at least one payload nucleic acid and at least one promoting oligonucleotide are administered or otherwise delivered to the same subject or system in accordance with the present disclosure, in some embodiments as cargo within the same EV (e.g., RBCEV), in some embodiments as separate cargos within different EVs (e.g., RBCEVs) or otherwise separately.
  • composition refers to an active agent, formulated together with one or more pharmaceutically acceptable carriers.
  • active agent is present in unit dose amount appropriate for administration in a therapeutic regimen that shows a statistically significant probability of achieving a predetermined therapeutic effect when administered to a relevant population.
  • compositions may be specially formulated for administration in solid or liquid form, including those adapted for the following: oral administration, for example, drenches (aqueous or non-aqueous solutions or suspensions), tablets, e.g., those targeted for buccal, sublingual, and systemic absorption, boluses, powders, granules, pastes for application to the tongue; parenteral administration, for example, by subcutaneous, intramuscular, intravenous or epidural injection as, for example, a sterile solution or suspension, or sustained-release formulation; topical application, for example, as a cream, ointment, or a controlled-release patch or spray applied to the skin, lungs, or oral cavity; intravaginally or intrarectally, for example, as a pessary, cream, or foam; sublingually; ocularly; transdermally; or nasally, pulmonary, and to other mucosal surfaces [0053] Promoting Oligonucleotide: As used herein, the
  • a promoting oligonucleotide wholly or partly makes up a cargo nucleic acid.
  • a promoting oligonucleotide is present in and/or delivered from an extracellular vesicle (EV, e.g., a red blood cell extracellular vesicle, RBCEV).
  • EV extracellular vesicle
  • RBCEV red blood cell extracellular vesicle
  • at least one promoting oligonucleotide and at least one payload nucleic acid are administered or otherwise delivered to the same subject or system in accordance with the present disclosure, in some embodiments as cargo within the same EV (e.g., RBCEV), in some embodiments as separate cargos within different EVs (e.g., RBCEVs) or otherwise separately.
  • reference describes a standard or control relative to which a comparison is performed.
  • an agent, animal, individual, population, sample, sequence or value of interest is compared with a reference or control agent, animal, individual, population, sample, sequence or value.
  • a reference or control is tested and/or determined substantially simultaneously with the testing or determination of interest.
  • a reference or control is a historical reference or control, optionally embodied in a tangible medium.
  • a reference or control is determined or characterized under comparable conditions or circumstances to those under assessment.
  • the term “specific”, with reference to an agent having an activity, is understood by those skilled in the art to mean that the agent discriminates between potential target entities or states. For example, and in some embodiments, an agent is said to bind “specifically” to its target if it binds preferentially with that target in the presence of one or more competing alternative targets. In many embodiments, specific interaction is dependent upon the presence of a particular structural feature of the target entity (e.g., an epitope, a cleft, a binding site). It is to be understood that specificity need not be absolute.
  • specificity may be evaluated relative to that of the binding agent for one or more other potential target entities (e.g., competitors). In some embodiments, specificity is evaluated relative to that of a reference specific binding agent. In some embodiments specificity is evaluated relative to that of a reference non- specific binding agent. In some embodiments, the agent or entity does not detectably bind to the competing alternative target under conditions of binding to its target entity. In some embodiments, binding agent binds with higher on-rate, lower off-rate, increased affinity, decreased dissociation, and/or increased stability to its target entity as compared with the competing alternative target(s).
  • therapeutically effective amount means an amount of a substance (e.g., a therapeutic agent, composition, and/or formulation) that elicits a desired biological response when administered as part of a therapeutic regimen.
  • a therapeutically effective amount of a substance is an amount that is sufficient, when administered to a subject suffering from or susceptible to a disease, disorder, and/or condition, to treat, diagnose, reduce the risk of developing the disease, and/or delay the onset of the disease, disorder, and/or condition.
  • the effective amount of a substance may vary depending on such factors as the desired biological endpoint, the substance to be delivered, the target cell or tissue, etc.
  • the effective amount of compound in a formulation to treat a disease, disorder, and/or condition is the amount that alleviates, ameliorates, relieves, inhibits, delays onset of, reduces severity of and/or reduces incidence of one or more symptoms or features of the disease, disorder, and/or condition.
  • a therapeutically effective amount is administered in a single dose; in some embodiments, multiple unit doses are required to deliver a therapeutically effective amount.
  • Unit dose The expression “unit dose” as used herein refers to an amount administered as a single dose and/or in a physically discrete unit of a pharmaceutical composition. In many embodiments, a unit dose contains a predetermined quantity of an active agent.
  • a unit dose contains an entire single dose of the agent. In some embodiments, more than one unit dose is administered to achieve a total single dose. In some embodiments, administration of multiple unit doses is required, or expected to be required, in order to achieve an intended effect.
  • a unit dose may be, for example, a volume of liquid (e.g., an acceptable carrier) containing a predetermined quantity of one or more therapeutic agents, a predetermined amount of one or more therapeutic agents in solid form, a sustained release formulation or drug delivery device containing a predetermined amount of one or more therapeutic agents, etc. It will be appreciated that a unit dose may be present in a formulation that includes any of a variety of components in addition to the therapeutic agent(s).
  • acceptable carriers e.g., pharmaceutically acceptable carriers
  • diluents e.g., pharmaceutically acceptable carriers
  • stabilizers e.g., buffers, preservatives, etc.
  • a total appropriate daily dosage of a particular therapeutic agent may comprise a portion, or a plurality, of unit doses, and may be decided, for example, by the attending physician within the scope of sound medical judgment.
  • the specific effective dose level for any particular subject or organism may depend upon a variety of factors including the disorder being treated and the severity of the disorder; activity of specific active compound employed; specific composition employed; age, body weight, general health, sex and diet of the subject; time of administration, and rate of excretion of the specific active compound employed; duration of the treatment; drugs and/or additional therapies used in combination or coincidental with specific compound(s) employed, and like factors well known in the medical arts.
  • the present disclosure provides technologies relating to administration of a nucleic acid of interest (e.g., a payload nucleic acid as described herein), and specifically relating to administration of a second nucleic acid – e.g., a promoting oligonucleotide as described herein, to a subject who has received or will receive such a nucleic acid of interest.
  • a nucleic acid of interest e.g., a payload nucleic acid as described herein
  • a second nucleic acid – e.g., a promoting oligonucleotide as described herein e.g., a promoting oligonucleotide as described herein
  • provided technologies can be particularly useful in the context of gene therapy applications (e.g., wherein a nucleic acid of interest, e.g., a payload nucleic acid, is or provides gene therapy).
  • a nucleic acid of interest e.g., a payload nucleic acid
  • the present disclosure teaches that such administration of a promoting oligonucleotide can provide unexpected benefits including, for example, potential enhancement or increase in one or more features of expression and/or activity of a payload nucleic acid, potential inhibition or reduction in one or more features of an undesirable effect of or response to such payload nucleic acid, and/or potential improvement or increase in delivery (e.g., in delivery vehicle loading) of cargo nucleic acid(s).
  • nucleic acid delivery may be used to treat/or otherwise manage various diseases, disorders, or indications. Nucleic acid delivery may be more effective at treating and/or otherwise managing certain diseases, disorders, or indications as compared to other therapeutic classes (e.g., small molecules).
  • nucleic acids i.e., of exogenous nucleic acids
  • a delivered nucleic acid e.g., or of a product it encodes
  • Various reports have described the challenges that may arise when delivering nucleic acids to a system (e.g., that is or comprises cells, tissues, and/or organisms) of interest (see, for example, Elsabahy, et al., "Non-viral nucleic acid delivery: key challenges and future directions.” Current drug delivery 8.3 (2011), which is hereby incorporated by reference in its entirety).
  • a particular challenge that can be encountered in various contexts of nucleic acid delivery may be associated with loading of a nucleic acid of interest into a delivery vehicle of interest in sufficient amount and/or with sufficient stability to achieve introduction of the nucleic acid to a system of interest.
  • Another challenge associated with nucleic acid delivery may relate to achieving sufficient level, expression, or activity of the nucleic acid of interest upon delivery.
  • Another challenge that can be encountered in various contexts of nucleic acid delivery may be associated with specificity of delivery (e.g., to tissue-type, cell- type, subcellular site, etc.).
  • Factors influencing the specificity of delivery may include, for example, the selected delivery vehicle, selected route of administration, bioavailability of the delivery vehicle and/or nucleic acid, extracellular degradation of the delivery vehicle and/or nucleic acid, intracellular processing of the delivery vehicle and/or nucleic acid, persistence of the nucleic acid following delivery, or combinations thereof.
  • the selected delivery vehicle selected route of administration
  • bioavailability of the delivery vehicle and/or nucleic acid extracellular degradation of the delivery vehicle and/or nucleic acid
  • intracellular processing of the delivery vehicle and/or nucleic acid intracellular processing of the delivery vehicle and/or nucleic acid
  • persistence of the nucleic acid following delivery or combinations thereof.
  • nucleic acid following delivery or combinations thereof.
  • neutralizing antibodies may hinder the delivery of a delivery vehicle and/or nucleic acid to its intended site (e.g., a cell or tissue type) upon administration to an organism of interest.
  • intracellular processes may be activated upon delivery of a nucleic acid of interest to a system of interest. Activation of such intracellular processes may result in, for example, degradation of the nucleic acid of interest, poor expression of the nucleic acid of interest, inflammation, local and/or systemic toxicities, or combinations thereof.
  • teachings of the present disclosure relate to avoiding and/or limiting challenges associated with nucleic acid delivery. In some embodiments, the present disclosure relates to avoiding and/or limiting challenges associated with achieving sufficient level, expression or activity of a delivered nucleic acid.
  • the present disclosure relates to avoiding and/or limiting challenges associated with one or more undesirable effects associated with such delivery. In some embodiments, the present disclosure relates to avoiding and/or limiting challenges associated with both (1) achieving sufficient level, expression or activity of a delivered nucleic acid and (2) one or more undesirable effects associated with such delivery. [0067]
  • the present disclosure teaches that administration of a second nucleic acid, and specifically of a promoting oligonucleotide as described herein, can avoid and/or limit challenges associated with nucleic acid delivery. Teachings of the present disclosure are particularly relevant to non-viral vectors and/or systems used for nucleic acid delivery. II.
  • nucleic Acid sensing and inflammatory immune responses can negatively impact the intended effect(s) of nucleic acid delivery.
  • the present disclosure demonstrates that administration of a second nucleic acid, and specifically of a promoting oligonucleotide as described herein, can lessen the negative impacts of nucleic acid sensing and inflammatory immune responses on nucleic acid delivery.
  • inflammatory immune responses and innate immune responses refer to essentially the same set of processes, and thus can be used interchangeably.
  • nucleic acid sensing refers to a cellular process in which (a) damage to endogenous DNA, and/or (b) aberrantly localized DNA and/or RNA is detected and/or sensed within a cell (see, for example, Nastasi et al., "DNA damage response and immune defense” International Journal of Molecular Sciences 21.20 (2020) incorporated herein by reference).
  • Teachings of the present disclosure relate to sensing of nucleic acids following administration (i.e, exogenous nucleic acid). Nucleic acid may be aberrantly localized for several reasons including, but not limited to, cellular dysfunction, pathogen infection, or exogenous nucleic acid delivery to a cell.
  • Nucleic acid sensing of exogenous nucleic acid that is aberrantly localized. Nucleic acid sensing is typically sequence-independent. [0071] Nucleic acid sensing involves multiple pathways and proteins endogenous to a cell. Pattern recognition receptors (PRRs) detect molecular patterns associated with pathogen infection (e.g., pathogen-associated molecular patterns (PAMPS)) or damage (e.g., damage- associated molecular patterns (DAMPS)).
  • PAMPS pathogen-associated molecular patterns
  • DAMPS damage- associated molecular patterns
  • PRRs include, but are not limited to, toll-like receptors (TLRs), retinoic acid-inducible gene-I (RIG-I)-like receptors (RLRs), Nod- like receptors (NLRs), absent in melanoma 2 (AIM2)-like receptors (ALRs), C-type lectin receptors (CLRs), and intracellular DNA sensors such as cyclic GMP-AMP synthase (cGAS).
  • TLRs toll-like receptors
  • RLRs retinoic acid-inducible gene-I
  • NLRs Nod- like receptors
  • AIM2 absent in melanoma 2
  • AIM2 AIM2
  • CLRs C-type lectin receptors
  • intracellular DNA sensors such as cyclic GMP-AMP synthase (cGAS).
  • cGAS cyclic GMP-AMP synthase
  • Teachings of the present disclosure relate to PRR detection of DAMPs, specifically DNA and/or
  • Intracellular DNA sensors may include, but are not limited to, cyclic GMP-AMP synthase (cGAS), toll-like receptors (TLRs), DExD/H-box helicase 41 (DDX41), and interferon- ⁇ (IFN ⁇ )- inducible protein 16 (IFI16).
  • cGAS cyclic GMP-AMP synthase
  • TLRs toll-like receptors
  • DDX41 DExD/H-box helicase 41
  • IFI16 interferon- ⁇ - inducible protein 16
  • RIG-I-like receptors RLRs
  • RLRs are intracellular PRRs that detect single-stranded and/or double-stranded RNA.
  • RLRs Retinoic acid-inducible gene I
  • MDA5 melanoma differentiation associated gene 5
  • LGP2 laboratory of genetics and physiology 2
  • RLRs are typically characterized by a central DEAD box helicase/ATPase domain and a C-terminal regulatory domain (CTD).
  • CCD C-terminal regulatory domain
  • RLRs may be activated by specific RNA motifs, such as, for example, 5’ end-di/triphosphorylated (5’-pp/5’-ppp) RNA sequences rich in poly-U or poly-UC tracts, high-molecular weight viral RNAs, or synthetic double- stranded RNA poly(I:C).
  • RIG-I Upon binding of aberrantly localized RNA, RIG-I interacts with mitochondrial antiviral signaling protein (MAVS) on the surface of mitochondria. Downstream signaling from this interaction between RIG-I and MAVS may involve TANK binding kinase 1-(TBK1), interferon regulatory factors (IRFs) 3 and 7, and/or nuclear factor-kappa B (NF- ⁇ B) signaling to upregulate proinflammatory genes (see, for example, Zevini et al., "Crosstalk between cytoplasmic RIG-I and STING sensing pathways.” Trends in immunology 38.3 (2017), hereby incorporated in its entirety by reference).
  • IRFs interferon regulatory factors
  • NF- ⁇ B nuclear factor-kappa B
  • Innate immune signaling from intracellular DNA and/or RNA sensors may signal via effectors including, but not limited to, mitochondrial antiviral signaling protein (MAVS), stimulator of IFN genes (STING), MYD88, serine/threonine kinases, and/or transcription factors (e.g., NF- ⁇ B).
  • Innate immune signaling through these pathways can result in innate immunity activation, inflammation, and/or recruitment of leukocytes to the region.
  • innate immune signaling can lead to the production of inflammatory molecules, including but not limited to, type I interferons (IFNs), type III IFNs, inflammasomes, pro-inflammatory cytokines, and/or pro- inflammatory chemokines.
  • the cGAS-STING signaling axis is a major cytosolic sensing pathway of single- stranded and double-stranded DNA (e.g., exogenous nucleic acid).
  • Various reports have described the signaling pathway and effects of the cGAS-STING signaling axis (see, for example, Gao, Menghui, et al. "cGAS/STING: novel perspectives of the classic pathway.” Molecular Biomedicine 1.1 (2020)).
  • This axis comprises cGAS sensing of aberrantly localized DNA, subsequent production of secondary messenger cGAMP by cGAS, and downstream activation of STING by the secondary messenger cGAMP. STING may also directly sense aberrantly localized DNA.
  • STING can induce multiple innate immune responses.
  • STING may signal through TANK binding kinase 1-(TBK1) to activate transcription factors (e.g., interferon regulatory factor 3 (IRF3), interferon regulatory factor 7 (IRF7), signal transducer and activator of transcription 6 (STAT6), etc.).
  • transcription factors e.g., interferon regulatory factor 3 (IRF3), interferon regulatory factor 7 (IRF7), signal transducer and activator of transcription 6 (STAT6), etc.
  • IRF3 interferon regulatory factor 3
  • IRF7 interferon regulatory factor 7
  • STAT6 signal transducer and activator of transcription 6
  • STING may also activate the NF- ⁇ B transcription factor and the NF- ⁇ B signaling pathway.
  • STING signaling can ultimately result in inflammatory cytokine and/or chemokine expression and release (e.g., type I IFNs, IL6, CXCL10, CCL2, CCL20, etc.).
  • cytokines and/or chemokines can be used as an indicative marker of inflammatory immune responses upon administration of nucleic acids.
  • the expression and/or release of cytokines and/or chemokines can be measured to ascertain the magnitude or type of immune response following administration of a nucleic acid of interest.
  • the TLR9 signaling axis is an endolysosomal sensing pathway of unmethylated CpG motifs. Upon activation, TLR9 signals through TLR adaptor myeloid differentiation primary response gene 88 (MyD88), which in turn activates transcription factors such as NF- ⁇ B and IRF7.
  • MyD88 myeloid differentiation primary response gene 88
  • TNF tumor necrosis factor
  • IL interleukin
  • IL-6 interleukin-6
  • type I IFNs type I IFNs.
  • Other TLRs are known in the art to sense nucleic acids in certain cell compartments (e.g., endosomes) such as TLR3, TLR7, TLR8, and TLR13. Activation of different TLR effectors can result in the same type I IFN gene transcription mediated by the same or different effector and adaptor proteins.
  • inflammatory immune response can present a challenge to nucleic acid delivery. Indeed, inflammation is a concerning toxicity associated with nucleic acid delivery in vivo and can limit the therapeutic potential of nucleic acid delivery.
  • nucleic acid agents e.g., to cargo nucleic acids such as payload nucleic acids and/or promoting oligonucleotides as described herein.
  • a nucleic acid agent comprises DNA.
  • a nucleic acid agent comprises RNA.
  • a nucleic acid agent is single- stranded.
  • a nucleic acid agent is double-stranded.
  • a nucleic acid comprises both single- and double-stranded portions.
  • a strand of a nucleic acid agent comprises self-complementary element(s) such that one or more double-stranded structures can form by self- hybridization within the strand.
  • a nucleic acid comprises a backbone that comprises one or more phosphodiester linkages.
  • a nucleic acid comprises a backbone that comprises both phosphodiester and non-phosphodiester linkages.
  • a nucleic acid may comprise a backbone that comprises one or more phosphorothioate or 5'-N-phosphoramidite linkages and/or one or more peptide bonds, e.g., as in a “peptide nucleic acid”.
  • a nucleic acid comprises one or more, or all, natural residues (e.g., adenine, cytosine, deoxyadenosine, deoxycytidine, deoxyguanosine, deoxythymidine, guanine, thymine, uracil). In some embodiments, a nucleic acid comprises on or more, or all, non-natural residues.
  • natural residues e.g., adenine, cytosine, deoxyadenosine, deoxycytidine, deoxyguanosine, deoxythymidine, guanine, thymine, uracil.
  • a non-natural residue comprises a nucleoside analog (e.g., 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolo-pyrimidine, 3 -methyl adenosine, 5-methylcytidine, C-5 propynyl-cytidine, C-5 propynyl-uridine, 2- aminoadenosine, C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5-propynyl-uridine, C5 -propynyl-cytidine, C5-methylcytidine, 2-aminoadenosine, 7-deazaadenosine, 7- deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, 0(6)-methylguanine, 2-thiocytidine, methylated bases, intercalated bases, and combinations thereof), an intercalator (e.g.,
  • a non-natural residue comprises one or more modified sugars (e.g., 2'-fluororibose, ribose, 2'-deoxyribose, 2’-amino (2’-NH), 2’-O-methyl (2’-OMe), arabinose, and hexose) as compared to those in natural residues.
  • modified sugars e.g., 2'-fluororibose, ribose, 2'-deoxyribose, 2’-amino (2’-NH), 2’-O-methyl (2’-OMe), arabinose, and hexose
  • a non-natural residue comprises one or more modified bases (e.g., 5- position pyrimidine modifications, 8-position purine modifications, modifications at exocyclic amines, substitution of 4-thiouridine, substitution of 5-bromo- or 5-iodo-uracil, backbone modifications, methylations, unusual base-pairing combinations such as the isobases isocytidine and isoguanidine) as compared to those in natural residues.
  • a non-natural residue comprises one or more 3’ and 5’ modifications (e.g., capping) as compared to those in natural residues.
  • any of the hydroxyl groups ordinarily present in a sugar may be replaced by a phosphonate group or a phosphate group; protected by standard protecting groups; or activated to prepare additional linkages to additional nucleotides or to a solid support.
  • the 5' and 3' terminal OH groups can be phosphorylated or substituted with amines, organic capping group moieties of from about 1 to about 20 carbon atoms, or organic capping group moieties of from about 1 to about 20 polyethylene glycol (PEG) polymers or other hydrophilic or hydrophobic biological or synthetic polymers.
  • Nucleic acids may be of variant types, such as locked nucleic acid (LNA), peptide nucleic acid (PNA), or gapmer.
  • a nucleic acid has a nucleotide sequence that encodes a functional gene product such as an RNA or polypeptide.
  • a nucleic acid has a nucleotide sequence that comprises one or more introns.
  • a nucleic acid may be prepared by isolation from a natural source, enzymatic synthesis (e.g., by polymerization based on a complementary template, e.g., in vivo or in vitro, reproduction in a recombinant cell or system, or chemical synthesis.
  • a nucleic acid is at least 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000 or more residues long.
  • Nucleic acid agents generally, can be super-coiled or not super-coiled.
  • Nucleic acid agents generally, can be chromosomal or non-chromosomal. Nucleic acid agents may be linear or circular. Nucleic acid agents may be conjugated to, or complexed with, other molecules (e.g., carriers, stabilizers, histones, lipophilic agent, etc.). IV. Cargo Nucleic Acids [0087] As described herein, a cargo nucleic acid is a nucleic acid that is administered or otherwise delivered to a subject or system of interest (e.g., that is or comprises one or more cells, tissues, organisms, etc.). [0088] In some embodiments, a cargo nucleic acid is present in and/or delivered from a delivery vehicle.
  • a cargo nucleic acid is present in and/or delivered from an extracellular vesicle (EV, e.g., an RBCEV).
  • EV extracellular vesicle
  • one or more copies of an identical cargo nucleic acid is present in and/or delivered from an extracellular vesicle (EV, e.g., an RBCEV).
  • two or more non-identical cargo nucleic acids are present in and/or delivered from the same extracellular vesicle (EV, e.g., an RBCEV).
  • cargo nucleic acids may be non-identical for a various reasons (e.g., sequence, strandedness; length, chemical composition and/or modification, etc.).
  • a cargo nucleic acid is or comprises a payload nucleic acid.
  • a cargo nucleic acid is or comprises a promoting oligonucleotide.
  • more than one cargo nucleic acid is administered or otherwise delivered to the same subject or system in accordance with the present disclosure.
  • at least one payload nucleic acid and at least one promoting oligonucleotide are administered or otherwise delivered to the same subject or system in accordance with the present disclosure, in some embodiments as cargo within the same EV (e.g., RBCEV), in some embodiments as separate cargos within different EVs (e.g., RBCEVs) or otherwise separately.
  • a payload nucleic acid is a nucleic acid that is administered or otherwise delivered to a subject or system of interest (e.g., that is or comprises one or more cells, tissues, organisms, etc.) that results in or is intended to achieve a particular biological result.
  • a payload nucleic acid encodes an expression product (e.g., a transcript or polypeptide) that achieves or is intended to achieve the relevant result.
  • teachings of the present disclosure relate to payload nucleic acids that are not intended for use with viral vectors.
  • a payload nucleic acid does not comprise ITR sequences.
  • a payload nucleic acid may be delivered to at least one cell type or tissue within a subject or system of interest.
  • a payload nucleic acid expresses or is intended to express an expression product within the cell type or tissue in which it was delivered.
  • a payload nucleic acid expresses or is intended to express an expression product which is subsequently secreted and/or released from the cell type or tissue in which it was delivered.
  • a payload nucleic acid is therapeutic to a subject or system of interest in which the payload nucleic acid was administered.
  • a payload nucleic acid is therapeutic to one or more cell types or tissues in which the payload nucleic acid was delivered. In some embodiments, a payload nucleic acid is therapeutic to one or more cell types or tissues other than in which the payload nucleic acid was delivered. [0094] In some embodiments, a payload nucleic acid is or comprises DNA that encodes an expression product. In some embodiments, a payload nucleic acid that is or comprises DNA has a maximum size of 30,000 kb.
  • a payload nucleic acid that is or comprises DNA may have a size of about 30,000, 25,000, 20,000, 15,000, 10,000, 9,000, 8,000, 7,000, 6,000, 5,000, 4,000, 3,000, 2,000, 1,000 or less kb.
  • a payload nucleic acid is or comprises RNA that encodes an expression an expression product.
  • a payload nucleic acid that is or comprises RNA has a maximum size of 2,000 kb.
  • a payload nucleic acid that is or comprises RNA may have a size of about 2,000, 1,500, 1,000, 900, 800, 700, 600, 500, 400, 300, 200, 100 or less kb.
  • a payload nucleic acid is or comprises a DNA plasmid, an RNA plasmid, a circular DNA, a linear double-stranded DNA, a DNA minicircle, a dumbbell- shaped DNA minimal vector, a doggy bone vector, a closed-end linear DNA vector, a nicked linear DNA vector, an RNA minicircle, a small interfering RNA (siRNA), a messenger RNA (mRNA), a guide RNA (gRNA), a prime editing guide RNA (peg RNA), a CRISPR RNA (crRNA), a trans-activating CRISPR RNA (tracrRNA), a circular RNA, a microRNA (miRNA), a primary miRNA (pri-miRNA), a precursor miRNA (pre-miRNA), a piwi-interacting RNA (piRNA), a transfer RNA (tRNA), a long noncoding RNA (lncRNA), an antisense oligon
  • siRNA messenger RNA
  • a payload nucleic acid is or comprises a minicircle.
  • Minicircles are circular replicons around 4 kbp.
  • a minicircle is or comprises DNA.
  • a minicircle is or comprises RNA.
  • a minicircle is double-stranded or comprises double-stranded regions.
  • a minicircle is synthetically derived.
  • a minicircle does not comprise an origin of replication and therefore does not replicate within a cell.
  • a minicircle is or comprises a reporter gene. Minicircles are known to those of ordinary skill in the art (e.g.
  • a payload nucleic acid is or comprises a dumbbell-shaped DNA minimal vector.
  • a dumbbell-shaped DNA minimal vector is or comprises a DNA oligonucleotide with a secondary structure comprising one or more hairpins. Dumbbell- shaped DNA minimal vectors are described, for example, in Yu et al (Nucleic Acids Research 2015: 43(18): e120), Jiang et al (Molecular Therapy 2016: 24(9): 1581-1591) and Zanta et al (PNAS 1999: 96: 91-96), each incorporated herein by reference in its entirety.
  • a payload nucleic acid is or comprises a doggy bone vector. In some embodiments, a payload nucleic acid is or comprises a closed-end linear DNA vector. In some embodiments, a payload nucleic acid is or comprises a nicked linear DNA vector. [0099] In some embodiments, a payload nucleic acid is or comprises a plasmid. In some embodiments, a plasmid is able to replicate independently in a cell. In some embodiments, a plasmid comprises an origin of replication sequence. In some embodiments, a plasmid is a nanoplasmid. [0100] In some embodiments, a payload nucleic acid is or comprises RNA.
  • a payload nucleic acid is or comprises therapeutic RNA.
  • a payload nucleic acid is or comprises RNA that encodes an expression product (e.g., one or more polypeptides or antigen-binding molecules).
  • a payload nucleic acid is or comprises RNA that comprises a sequence complementary to a nucleic acid sequence endogenous to a cell in which the payload nucleic acid is delivered.
  • a payload nucleic acid is or comprises RNA that is useful in methods of gene silencing or downregulating gene expression. [0101]
  • a payload nucleic acid is antisense to an endogenous nucleic acid sequence within a cell.
  • an antisense nucleic acid is single or double-stranded.
  • an antisense nucleic acid comprises double- stranded RNA (dsRNA) or partially double-stranded RNA that is complementary to a target nucleic acid sequence.
  • dsRNA double-stranded RNA
  • a double-stranded RNA molecule is formed by the complementary pairing between a first RNA portion and a second RNA portion within an antisense nucleic acid.
  • the length of an RNA sequence i.e. one portion
  • the length of an RNA sequence is within a range of about 18-24 nucleotides.
  • a complementary first RNA portion and a second RNA portion form a “stem” of a hairpin structure.
  • the two portions can be joined by a linking sequence, which may form the “loop” in the hairpin structure.
  • the linking sequence can vary in length and may be, for example, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more nucleotides in length. Suitable linking sequences are known in the art.
  • an antisense nucleic acid hybridizes to a corresponding DNA sequence within a cell.
  • An antisense nucleic acid may hybridize to a corresponding mRNA within a cell, forming a double-stranded molecule.
  • An antisense nucleic acid may interfere or otherwise disrupt translation of a complementary mRNA, as translation of double- stranded mRNA does not occur. Antisense inhibition of translation is known in the art (see, e.g., Marcus-Sakura, Anal. Biochem.1988, 172:289).
  • an antisense nucleic acid hybridizes to a corresponding micro RNA (miRNA).
  • miRNA micro RNA
  • an antisense nucleic acid inhibits the function of a miRNA and/or prevents the miRNA from post-transcriptionally regulating gene expression.
  • an antisense nucleic acid functions to upregulate expression of one or more genes that are otherwise downregulated by a miRNA. In some embodiments, an antisense nucleic acid functions to downregulate expression of target genes.
  • an antisense nucleic acid include, but are not limited to, small interfering RNA (siRNA; including derivatives or pre-cursors, such as nucleotide analogs), short hairpin RNA (shRNA), micro RNA (miRNA), saRNA (small activating RNA), small nucleolar RNA (snoRNA) or derivatives or pre-cursors, long non-coding RNA (lncRNA), or single stranded molecules such as chimeric ASO or gapmers.
  • siRNA small interfering RNA
  • shRNA short hairpin RNA
  • miRNA micro RNA
  • saRNA small activating RNA
  • snoRNA small nucleolar RNA
  • lncRNA long non-coding RNA
  • an antisense nucleic acid stimulates RNA interference (RNAi) or other cellular degradation mechanisms (e.g., RNase degradation).
  • a payload nucleic acid is or comprises a siRNA.
  • a "siRNA,” “small interfering RNA,” “small RNA,” or “RNAi” as provided herein, refers to a nucleic acid that forms a double-stranded RNA, which double-stranded RNA has the ability to reduce or inhibit expression of a gene or target gene when expressed in the same cell as the gene or target gene.
  • Complementary portions of RNA that hybridize to form double-stranded RNA may have substantially or completely complementary sequences.
  • a siRNA has a sequence that is substantially or completely complementary to a target gene sequence. In some embodiments, a siRNA has a length within a range of about 15-50 nucleotides (e.g., each complementary sequence of double-stranded siRNA is about 15-50 nucleotides in length and the double-stranded siRNA is about 15-50 base pairs in length).
  • a siRNA may have a length within a range of 20-30 nucleotides, 20-25 nucleotides, or 24-29 nucleotides (e.g., 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length).
  • RNAi and siRNA are described in, for example, Dana et al., Int J Biomed Sci.2017; 13(2): 48–57, herein incorporated by reference in its entirety.
  • Suitable siRNA molecules for use in the methods of the present invention may be designed by schemes known in the art (see, for example, Elbashire et al., Nature, 2001 411:494-8; Amarzguioui et al., Biochem. Biophys. Res. Commun.2004316(4):1050-8; and Reynolds et al., Nat. Biotech.2004, 22(3):326-30).
  • siRNA molecules are designed and/or found from commercial vendors.
  • a potential siRNA candidate may be checked for possible complementation and/or interaction with other nucleic acid sequences or polymorphisms using a BLAST alignment program (see, for example, the National Library of Medicine website).
  • a number of siRNAs are generated and screened to obtain a potential candidate (see, for example, U.S. Pat. No.7,078,196).
  • a siRNA is expressed from a vector and/or produced chemically or synthetically. Synthetic RNAi may be obtained from commercial sources, for example, Invitrogen (Carlsbad, California).
  • RNAi vectors may be obtained from commercial sources, for example, Invitrogen.
  • a payload nucleic acid is or comprises a miRNA.
  • miRNA is used in accordance with its ordinary meaning and refers to a small non-coding RNA molecule capable of post-transcriptionally regulating gene expression.
  • a miRNA is a nucleic acid that has substantial or complete identity to a target gene.
  • a miRNA inhibits gene expression by interacting with a complementary cellular mRNA thereby interfering with the expression of the complementary mRNA.
  • a miRNA has a length within a range of about 15-50 nucleotides.
  • a miRNA comprises a stem-loop and/or hairpin structure.
  • a miRNA is synthetic or recombinant.
  • a miRNA is associated with cancer.
  • a miRNA is miR-125b.
  • a payload nucleic acid is or comprises an expression vector or expression cassette sequence.
  • expression vector or expression cassette sequence refer to a nucleic acid molecule used to express exogenous nucleic acid within a cell. Suitable expression vectors and expression cassettes are known in the art.
  • Expression vectors may comprise elements that facilitate the expression of one or more nucleic acid sequences in a target system (e.g. cell, tissue, organism, etc.).
  • a target system e.g. cell, tissue, organism, etc.
  • an expression vector comprises a promoter sequence operably linked to the nucleotide sequence encoding the nucleic acid sequence to be expressed.
  • an expression vector comprises a termination codon.
  • an expression vector comprises expression enhancers. Suitable promoters, termination codons, and enhancers may be used and are known in the art.
  • a payload nucleic acid is or comprises a plurality of expression vectors encoding for different peptides or proteins.
  • a payload nucleic acid is or comprises a first expression vector encoding a first protein of a protein complex and a further expression vector encoding a further protein of the protein complex.
  • the further protein may be non- identical to the first protein.
  • a payload nucleic acid is or comprises a first expression vector encoding a first domain of a protein and a further expression vector encoding a further domain of the protein.
  • a payload nucleic acid is or comprises a first expression vector encoding a first segment of a protein and a further expression vector encoding a further segment of the protein.
  • a payload nucleic acid expresses or is intended to express an expression product that is endogenous to the subject or system of interest in which the payload nucleic acid is administered.
  • a payload nucleic expresses or is intended to express a functional gene, or fragment thereof, to replace and/or supplement a gene that is otherwise not fully functional.
  • a payload nucleic acid encodes human factor IX (FIX).
  • a payload nucleic acid expresses or is intended to express an expression product that is exogenous to the subject or system of interest in which the payload nucleic acid is administered.
  • a payload nucleic acid is or comprises a transgene.
  • a payload nucleic acid encodes and/or expresses (or is the complement of a nucleic acid that encodes or expresses) an antibody, an antibody gene therapy system, and/or an antigen-binding molecule.
  • An antibody gene therapy system refers to a system in which nucleic acids encoding an antibody of interest is delivered to cells wherein said cells produce and secrete the encoded antibody.
  • a payload nucleic acid encodes and/or expresses (or is the complement of a nucleic acid that encodes or expresses) one or more components of an antibody gene therapy system.
  • an antibody gene therapy system is encoded by the same nucleic acid molecule or separate nucleic acid molecules.
  • an antibody gene therapy system is encoded by one or more DNA molecules.
  • an antibody gene therapy system is encoded by one or more plasmids.
  • an antibody gene therapy system is encoded by one or more expression vectors.
  • an antibody gene therapy system is encoded by one or more mRNA molecules.
  • an antibody gene therapy system is encoded by one or more minicircles.
  • an antibody gene therapy system is encoded by one or more dumbbell- shaped DNA minimal vectors.
  • An antigen-binding molecule refers to a molecule which is capable of binding to a target antigen.
  • An antigen-binding molecule may be a monoclonal antibody, a polyclonal antibody, a monospecific antibody, a multispecific antibody (e.g., a bispecific antibody), or an antibody fragment (e.g., Fv, scFv, Fab, scFab, F(ab’)2, Fab2, diabody, triabody, scFv-Fc, minibody, single domain antibody (e.g., VhH), etc.), as long as it displays binding to the relevant target molecule(s).
  • an antibody, or fragment thereof, or antigen-binding molecule is human, humanized, murine, camelid, chimeric, or from another suitable source.
  • an antibody, or fragment thereof, or antigen-binding molecule is humanized.
  • Methods of humanizing antibodies may involve the fusing of variable domains of rodent origin to constant domains of human origin such that the resultant antibody retains the antigenic specificity of the rodent parented antibody, for example, as described in Morrison et al (1984) Proc. Natl. Acad. Sd. USA 81, 6851-6855.
  • Monoclonal antibodies refer to a homogenous population of antibodies that specifically bind a single epitope on an antigen.
  • Suitable monoclonal antibodies to selected antigens may be prepared by known techniques, for example, those disclosed in Köhler, G.; Milstein, C. (1975) "Continuous cultures of fused cells secreting antibody of predefined specificity”. Nature 256 (5517): 495; Siegel DL (2002). "Recombinant monoclonal antibody technology”;. Schmitz U, Versmold A, Kaufmann P, Frank HG (2000) "Phage display: a molecular tool for the generation of antibodies--a review”. Placenta.21 Suppl A: S106–12; Helen E.
  • Polyclonal antibodies refer to a heterologous population of antibodies that bind different epitopes on a single antigen.
  • polyclonal antibodies are monospecific. Suitable polyclonal antibodies can be prepared using methods known in the art.
  • a payload nucleic acid encodes and/or expresses (or is the complement of a nucleic acid that encodes or expresses) a heavy chain or light chain of an antibody.
  • a payload nucleic acid encodes and/or expresses (or is the complement of a nucleic acid that encodes or expresses) a heavy chain of an antibody, and a further payload nucleic acid encodes and/or expresses (or is the complement of a nucleic acid that encodes or expresses) a light chain of an antibody, and when the at least two payloads are delivered in the same cell, cell type, or tissue an antibody is formed.
  • An antibody fragment refers to a fragment or shortened sequence of an antibody which retains binding to relevant target molecule(s). Antigenic specificity is conferred by variable domains and is independent of constant domains.
  • Molecules that possess antigen-binding properties include, but are not limited to, Fab-like molecules (Better et al. (1988) Science 240, 1041); Fv molecules (Skerra et al. (1988) Science 240, 1038); single- chain Fv (ScFv) molecules where the VH and VL partner domains are linked via a flexible oligopeptide (Bird et al. (1988) Science 242, 423; Huston et al. (1988) Proc. Natl. Acad. Sd. USA 85, 5879) and single domain antibodies (dAbs) comprising isolated V domains (Ward et al. (1989) Nature 341, 544).
  • a single-chain variable fragment refers to molecules wherein the heavy chain variable domain (VH) and light chain variable domain (VL) are covalently linked (e.g., by a peptide or a flexible oligopeptide).
  • VH heavy chain variable domain
  • VL light chain variable domain
  • sdAb single domain antibody
  • scAb single chain antibody
  • VH and VL partner domains e.g., by a peptide or a flexible oligoeptide
  • a payload nucleic acid may encode and/or express (or is the complement of a nucleic acid that encodes or expresses) 3F8, 8H9, Abagovomab, Abciximab (ReoPro), Abituzumab, Abrezekimab, Abrilumab, Actoxumab, Adalimumab (Humira), Adecatumumab, Aducanumab, Afasevikumab, Afelimomab, Alacizumab pegol, Alemtuzumab (Lemtrada), Alirocumab (Praluent), Altumomab pentetate (Hybri-ceaker), Amatuximab, Amivantamab, Anatumomab mafenatox, Andecaliximab, Anetumab ravtansine, Anifrolumab, Ansuvimab (Ebanga), Anrukinzum
  • An antigen-binding molecule may be a derivative of any of the abovementioned antibodies.
  • a payload nucleic acid encodes and/or expresses (or is the complement of a nucleic acid that encodes or expresses) one or more components of a gene editing system.
  • CRISPR is an abbreviation of Clustered Regularly Interspaced Short Palindromic Repeats. CRISPR comprises segments of DNA containing short, repetitive base sequences in a palindromic repeat (wherein the sequence of nucleotides is the same in both directions). Each repetition is followed by short segments of spacer DNA from previous integration of foreign DNA from a virus or plasmid.
  • CRISPR-associated genes Small clusters of Cas (CRISPR- associated) genes are located next to CRISPR sequences. RNA harboring the spacer sequence helps Cas (CRISPR-associated) proteins recognize and cut foreign pathogenic DNA. Other RNA-guided Cas proteins cut foreign RNA.
  • An embodiment of the CRISPR/Cas system, CRISPR/Cas9 has been modified to edit genomes. By delivering the Cas9 nuclease and a synthetic guide RNA (gRNA) into a cell, the cell's genome can be cut at a desired location, allowing existing genes to be removed and/or new ones added. CRISPR/Cas systems fall into two classes. Class 1 systems use a complex of multiple Cas proteins to degrade foreign nucleic acids.
  • Class 2 systems use a single large Cas protein for the same purpose.
  • Class 1 is divided into types I, III, and IV; class 2 is divided into types II, V, and VI.
  • CRISPR genome editing uses a type II CRISPR system. he complement of a nucleic acid that encodes or expresses) one or more components of a CRISPR/Cas gene editing system.
  • a payload nucleic acid recognizes a particular target sequence.
  • a payload nucleic acid is or comprises a guide RNA (gRNA).
  • a guide RNA comprises a CRISPR RNA (crRNA) and a trans-activating CRISPR RNA (tracrRNA).
  • crRNA may comprise a sequence that binds and/or identifies a host DNA sequence and a region that binds to tracrRNA to form an active complex.
  • a gRNA combines both crRNA and tracrRNA thereby encoding an active complex.
  • a gRNA may comprises multiple crRNAs and/or multiple tracrRNAs.
  • a gRNA is designed to bind and/or otherwise identify a sequence or gene of interest.
  • a gRNA targets a sequence or gene of interest for cleavage.
  • a template DNA sequence is included.
  • a template DNA sequence is utilized in either non-homologous end joining (NHEJ) or homology directed repair (HDR).
  • NHEJ non-homologous end joining
  • HDR homology directed repair
  • a payload nucleic acid encodes and/or expresses (or is the complement of a nucleic acid that encodes or expresses) a nuclease.
  • a payload nucleic acid encodes and/or expresses (or is the complement of a nucleic acid that encodes or expresses) a Cas nuclease.
  • Cas nuclease may refer to any Cas protein (e.g., Cas 9, Cas12, etc.).
  • nuclease may refer to any protein that functions to modify nucleic acid (e.g., single strand nicking, double strand breaking, DNA binding, etc.).
  • a nuclease recognizes a DNA site and allows for site-specific DNA editing.
  • a nuclease is modified.
  • a nuclease is fused to a reverse transcriptase.
  • a nuclease is catalytically inactive.
  • a nuclease is fused to a transcription factor.
  • a modified nuclease may be useful, for example, in a prime editing system or in systems to regulate transcription.
  • a payload nucleic acid encodes and/or expresses (or is the complement of a nucleic acid that encodes or expresses) at least a gRNA and a nuclease. In some embodiments, a payload nucleic acid encodes and/or expresses (or is the complement of a nucleic acid that encodes or expresses) at least a gRNA and a nuclease on a plasmid. In some embodiments, a gRNA and a nuclease are encoded on a single plasmid. In some embodiments, a gRNA and a nuclease are encoded on separate plasmids.
  • a payload nucleic acid is or comprises a DNA repair template.
  • a DNA repair template is or comprises a linear double- stranded DNA.
  • a DNA repair template is a plasmid.
  • a DNA repair template is present on the same nucleic acid which encodes a gRNA and/or nuclease.
  • a DNA repair template is present on a separate nucleic acid from the nucleic acid which encodes a gRNA and/or a nuclease.
  • CRISPR/Cas9 and related systems are reviewed, for example, in Nakade et al., Bioengineered (2017) 8(3):265-273, which is hereby incorporated by reference in its entirety.
  • These systems comprise an endonuclease (e.g., Cas9, Cpf1, etc.) and a single-guide RNA (sgRNA) molecule.
  • sgRNA single-guide RNA
  • a sgRNA can be engineered to target endonuclease activity to nucleic acid sequences of interest.
  • a payload nucleic acid encodes and/or expresses (or is the complement of a nucleic acid that encodes or expresses) one or more components of a gene editing system other than a CRISPR/Cas gene editing system (e.g., zinc finger nucleases (ZFNs) or transcription activator-like effector nucleases (TALENs)).
  • a gene editing system specifically targets a miRNA.
  • a gene editing system specifically targets miR-125b.
  • a gene editing system employs targeted gene editing using a site-specific nuclease (SSN).
  • Enzymes capable of creating site-specific double strand breaks may be engineered to introduce DSBs to target nucleic acid sequence(s) of interest.
  • DSBs may be repaired by error-prone non-homologous end-joining (NHEJ), in which the two ends of the break are rejoined, often with insertion or deletion of nucleotides.
  • NHEJ error-prone non-homologous end-joining
  • DSBs may be repaired by homology-directed repair (HDR), in which a DNA template with ends homologous to the break site is supplied and introduced at the site of the DSB.
  • HDR homology-directed repair
  • SSNs capable of being engineered to generate target nucleic acid sequence-specific DSBs include ZFNs, TALENs and clustered regularly interspaced palindromic repeats/CRISPR-associated-9 (CRISPR/Cas9) systems.
  • ZFN systems are reviewed, for example, in Umov et al., Nat Rev Genet. (2010) 11(9):636-46, which is hereby incorporated by reference in its entirety.
  • ZFNs comprise a programmable Zinc Finger DNA-binding domain and a DNA-cleaving domain (e.g. a FokI endonuclease domain).
  • the DNA-binding domain may be identified by screening a Zinc Finger array capable of binding to the target nucleic acid sequence.
  • ZFNs work in pairs as the endonuclease (e.g., FokI) functions as a dimer.
  • a ZFN system comprises two monomers with unique DNA recognition sites in the target genome with proper orientation (i.e. on opposite DNA strands) and spacing to allow the endonuclease to function.
  • a payload nucleic acid encodes and/or expresses (or is the complement of a nucleic acid that encodes or expresses) one or more components of a ZFN gene editing system.
  • a ZFN gene editing system comprises a ZFN pair having two polypeptide monomers.
  • a ZFN gene editing system is encoded by the same nucleic acid molecule or separate nucleic acid molecules. In some embodiments, a ZFN gene editing system is encoded by one or more DNA molecules. In some embodiments, a ZFN gene editing system is encoded by one or more plasmids. In some embodiments, a ZFN gene editing system is encoded by one or more expression vectors. In some embodiments, a ZFN gene editing system is encoded by one or more mRNA molecules. In some embodiments, a ZFN gene editing system is encoded by one or more minicircles. In some embodiments, a ZFN gene editing system is encoded by one or more dumbbell-shaped DNA minimal vectors.
  • two payload nucleic acids comprise a first nucleic acid molecule that encodes first monomer of a ZFN pair and a further nucleic acid molecule that encodes a second monomer of a ZFN pair.
  • the nucleic acids may comprise an expression cassette such that the ZFN monomers are expressed within a target cell.
  • the expressed ZFN monomers may bind to their respective DNA recognition sites and allow dimerization of endonuclease.
  • the endonuclease may function to introduce a DSB into the DNA.
  • TALENs comprise a programmable DNA-binding TALE domain and a DNA-cleaving domain (e.g., a FokI endonuclease domain).
  • TALEs comprise repeat domains consisting of repeats of 33-39 amino acids, which are identical except for two residues at positions 12 and 13 of each repeat which are repeat variable di-residues (RVDs).
  • Each RVD determines binding of the repeat to a nucleotide in the target DNA sequence according to the following relationship: “HD” binds to C, “NI” binds to A, “NG” binds to T and “NN” or “NK” binds to G (see, for example, Moscou and Bogdanove, Science (2009) 326(5959):1501 which is hereby incorporated by reference in its entirety).
  • TALENs work in pairs as the endonuclease (e.g., FokI) functions as a dimer.
  • a TALEN system comprises two monomers with unique DNA recognition sites in the target genome with proper orientation (i.e., on opposite DNA strands) and spacing to allow the endonuclease to function.
  • a payload nucleic acid encodes and/or expresses (or is the complement of a nucleic acid that encodes or expresses) one or more components of a TALEN gene editing system.
  • a TALEN gene editing system comprises a TALEN pair having two polypeptide monomers.
  • a TALEN gene editing system is encoded by the same nucleic acid molecule or separate nucleic acid molecules.
  • a TALEN gene editing system is encoded by one or more DNA molecules.
  • a TALEN gene editing system is encoded by one or more plasmids.
  • a TALEN gene editing system is encoded by one or more expression vectors. In some embodiments, a TALEN gene editing system is encoded by one or more mRNA molecules. In some embodiments, a TALEN gene editing system is encoded by one or more minicircles. In some embodiments, a TALEN gene editing system is encoded by one or more dumbbell-shaped DNA minimal vectors. [0143] In some embodiments, two payload nucleic acids comprise a first nucleic acid molecule that encodes first monomer of a TALEN pair and a further nucleic acid molecule that encodes a second monomer of a TALEN pair.
  • the nucleic acids may comprise an expression cassette such that the TALEN monomers are expressed within a target cell.
  • the expressed ZFN monomers may bind to their respective DNA recognition sites and allow dimerization of endonuclease.
  • the endonuclease may function to introduce a DSB into the DNA.
  • a payload nucleic acid encodes and/or expresses (or is the complement of a nucleic acid that encodes or expresses) a vaccine.
  • a payload nucleic acid encodes and/or expresses (or is the complement of a nucleic acid that encodes or expresses) an epitope sequence.
  • a payload nucleic acid encodes and/or expresses (or is the complement of a nucleic acid that encodes or expresses) a vaccine to cancer.
  • Cancer vaccines involve displaying a tumor-specific antigen or a tumor-associated antigen to a subject’s immune system such that the immune system is able to more effectively recognize cancerous cells. Cancer vaccines are reviewed, for example, in Vergati, Matteo, et al. "Strategies for cancer vaccine development.” Journal of Biomedicine and Biotechnology (2010), which is hereby incorporated by reference.
  • One of ordinary skill in the art will be able to select a tumor-specific antigen or tumor-associated antigen for any particular cancer type using methods known in the art.
  • a payload nucleic acid encodes and/or expresses (or is the complement of a nucleic acid that encodes or expresses) a tumor-specific antigen. In some embodiments, a payload nucleic acid encodes and/or expresses (or is the complement of a nucleic acid that encodes or expresses) a tumor-associated antigen.
  • a cancer vaccine is encoded by one or more DNA molecules. In some embodiments, a cancer vaccine is encoded by one or more plasmids. In some embodiments, a cancer vaccine is encoded by one or more expression vectors. In some embodiments, a cancer vaccine is encoded by one or more mRNA molecules.
  • a cancer vaccine is encoded by one or more minicircles. In some embodiments, a cancer vaccine is encoded by one or more dumbbell-shaped DNA minimal vectors. [0147] In some embodiments, a payload nucleic acid encodes and/or expresses (or is the complement of a nucleic acid that encodes or expresses) a vaccine to a pathogen. In some embodiments, a payload nucleic acid encodes and/or expresses (or is the complement of a nucleic acid that encodes or expresses) a vaccine to a bacteria.
  • a payload nucleic acid encodes and/or expresses (or is the complement of a nucleic acid that encodes or expresses) a vaccine to a virus.
  • Pathogen vaccines involve displaying a pathogen-specific antigen to a subject’s immune system such that the immune system is able to more effectively recognize foreign pathogens.
  • a pathogen vaccine is encoded by one or more DNA molecules.
  • a pathogen vaccine is encoded by one or more plasmids.
  • a pathogen vaccine is encoded by one or more expression vectors.
  • a pathogen vaccine is encoded by one or more mRNA molecules. In some embodiments, a pathogen vaccine is encoded by one or more minicircles. In some embodiments, a pathogen vaccine is encoded by one or more dumbbell-shaped DNA minimal vectors. [0149] In some embodiments, a payload nucleic acid is diagnostic. In some embodiments, a payload nucleic acid encodes and/or expresses (or is the complement of a nucleic acid that encodes or expresses) a reporter gene and/or a molecule that is detectable.
  • a promoting oligonucleotide is a nucleic acid whose presence is associated with (a) increased level and/or activity of an expression product of a payload; and/or (b) decreased inflammatory and/or otherwise undesirable effect or response (e.g., immune effect or response) associated with administration or delivery of a payload nucleic acid.
  • a promoting oligonucleotide is or comprises double- stranded DNA (dsDNA).
  • dsDNA promoting oligonucleotide is or comprises two DNA strands.
  • a dsDNA promoting oligonucleotide has a length within a range of 5-200 base pairs. In some embodiments, a dsDNA promoting oligonucleotide has a length of 5, 10, 15, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, or 200 base pairs. In some embodiments, a dsDNA promoting oligonucleotide has a length of at least 5 base pairs. In some embodiments, a dsDNA promoting oligonucleotide has a length of at most 40 base pairs.
  • a promoting oligonucleotide is or comprises single-stranded DNA (ssDNA).
  • An ssDNA promoting oligonucleotide may or may not comprise self- complementary regions.
  • an ssDNA promoting oligonucleotide comprises one or more stem-loop structures.
  • an ssDNA promoting oligonucleotide comprises two stem-loop structures (e.g., a ribbon shaped promoting oligonucleotide).
  • an ssDNA promoting oligonucleotide has a length within a range of 5-100 nucleotides.
  • an ssDNA promoting oligonucleotide has a length of 5, 10, 15, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, or 200 nucleotides. In some embodiments, an ssDNA promoting oligonucleotide has a length of at least 5 nucleotides. In some embodiments, an ssDNA promoting oligonucleotide has a length of at most 40 nucleotides. [0153] In some embodiments, a promoting oligonucleotide is or comprises a single RNA strand. An RNA promoting oligonucleotide may or may not comprise self-complementary regions.
  • an RNA promoting oligonucleotide has a length within a range of 5-100 nucleotides. In some embodiments, an RNA promoting oligonucleotide has a length of 5, 10, 15, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, or 200 nucleotides. In some embodiments, an RNA promoting oligonucleotide has a length of at least 5 nucleotides. In some embodiments, an RNA promoting oligonucleotide has a length of at most 40 nucleotides. [0154] In some embodiments, a promoting oligonucleotide comprises chemically modified nucleic acid.
  • Chemical modifications may relate to, for example, a nucleotide, a sugar, a base, or a bond of or within a promoting oligonucleotide.
  • a promoting oligonucleotide comprises at least one phosphorothioate-modified bond.
  • every nucleotide bond of a promoting oligonucleotide is a phosphorothioate-modified bond.
  • at most 50% of the nucleotide bonds of the promoting oligonucleotide are phosphorothioate-bonds.
  • the nucleotide bonds that are phosphorothioate-bonds of the promoting oligonucleotide are at the 5’ and 3’ ends of the nucleic acid sequence.
  • phosphorothioate-modified bonds are incorporated into a promoting oligonucleotide to control the oligonucleotide’s in vivo half-life (e.g., rate of degradation in a cell, tissue, organism, etc.).
  • the ratio of phosphorothioate-modified bonds to unmodified bonds in a promoting oligonucleotide is used to control the in vivo half-life.
  • a promoting oligonucleotide’s in vivo half- life By modifying a promoting oligonucleotide’s in vivo half- life, the duration of the oligonucleotide’s effects may be controlled. In some embodiments, a promoting oligonucleotide’s in vivo half-life is decreased. In some embodiments, a promoting oligonucleotide’s in vivo half-life is decreased to minimize constitutive inhibition (e.g., of NF- ⁇ B). In some embodiments, a promoting oligonucleotide’s in vivo half-life is increased.
  • a promoting oligonucleotide’s in vivo half-life is increased to lessen the quantity of oligonucleotide that is required to achieve a biologic effect.
  • a promoting oligonucleotide comprises one or more spacer molecules.
  • a spacer molecule comprises a linker used to cap the ends of dsDNA and DNA duplexes, such as, for example, hexaethylene glycol.
  • a promoting oligonucleotide does not encode for an expression product.
  • a promoting oligonucleotide can avoid and/or limit one or more challenges associated with nucleic acid delivery (e.g., a payload nucleic acid).
  • a promoting oligonucleotide increases the amount of nucleic acid loaded into a delivery vehicle, especially when the promoting oligonucleotide is co-loaded with a payload nucleic acid in an RBCEV.
  • a promoting oligonucleotide can increase the level, expression or activity of a delivered nucleic acid (e.g., or of a product it encodes).
  • a promoting oligonucleotide increases the number of copies of payload nucleic acid delivered to a system (e.g., a cell, tissue, or organism). In some embodiments, a promoting oligonucleotide increases the number of cells that receive delivery of a payload nucleic acid. In some embodiments, a promoting oligonucleotide increases the amount of expression product expressed per copy of payload nucleic acid. In some embodiments, a promoting oligonucleotide decreases the amount of payload nucleic acid (e.g., or of a product it encodes) degraded upon delivery to a system.
  • a system e.g., a cell, tissue, or organism.
  • a promoting oligonucleotide can decrease inflammatory and/or otherwise undesirable effect or response (e.g., immune effect or response) associated with administration or delivery of a payload nucleic acid.
  • administration of a promoting oligonucleotide decreases expression and/or release of indicative marker(s) of inflammatory and/or otherwise undesirable effect or response (e.g., immune effect or response) associated with administration or delivery of a payload nucleic acid.
  • administration of a promoting oligonucleotide decreases cytokine expression and/or release associated with administration or delivery of a payload nucleic acid.
  • administration of a promoting oligonucleotide decreases type I IFN (e.g., IFNa, IFNb, etc.), IL6, CXCL10, and/or CCL2 expression and/or release associated with administration or delivery of a payload nucleic acid.
  • a promoting oligonucleotide interacts with a factor endogenous to a cell in which the promoting oligonucleotide has been delivered in order to effect decreased inflammatory and/or otherwise undesirable effect or response (e.g., immune effect or response) associated with administration or delivery of a payload nucleic acid.
  • a promoting oligonucleotide interacts with a factor endogenous to a cell that typically functions to bind nucleic acid. In some embodiments, a promoting oligonucleotide interacts with a transcription factor. In some embodiments, a promoting oligonucleotide interacts with an RNA-binding protein. In some embodiments, a promoting oligonucleotide interacts with any factor that can be bound by an aptamer. [0162] In some embodiments, a promoting oligonucleotide prevents and/or inhibits an endogenous factor of a cell from interacting with a payload nucleic acid.
  • This prevention and/or inhibition of interaction between an endogenous factor of a cell and a payload nucleic acid by a promoting oligonucleotide may be through direct means (e.g., a promoting oligonucleotide interacting with a factor such that it is unable to interact with a payload nucleic acid) or through indirect means (e.g., a promoting oligonucleotide interacting with a factor that regulates the function or activity of a further factor which might otherwise interact with a payload nucleic acid).
  • a promoting oligonucleotide acts as a decoy, lure, trap, bait, mimic, squelch, and/or sink to a factor endogenous to a cell in which the promoting oligonucleotide has been delivered (i.e., acts to absorb and/or neutralize the biologic effects of an endogenous factor such that its endogenous functions are lessened).
  • a promoting oligonucleotide may be or comprise a decoy to a transcription factor; such a decoy could interact with a target transcription factor upon delivery to a cell and decrease the transcription factor’s binding to target DNA sequences within the cell’s nucleus.
  • a promoting oligonucleotide is or comprises a decoy to an effector of a nucleic acid sensing pathway. In some embodiments, a promoting oligonucleotide is or comprises a decoy to an effector of the cGAS-STING signaling axis. In some embodiments, a promoting oligonucleotide is or comprises a decoy to an effector of the TLR9 signaling axis. In some embodiments, a promoting oligonucleotide is or comprises a decoy to an effector of an inflammatory and/or innate immune pathway.
  • a promoting oligonucleotide is or comprises an NF- ⁇ B decoy. In some embodiments, a promoting oligonucleotide is or comprises a decoy to DNA-dependent protein kinase (DNA-PK) and/or poly (ADP-ribose) polymerase (PARP). In some embodiments, a promoting oligonucleotide is or comprises a RIG-I decoy.
  • a delivery vehicle is an agent that complexes or otherwise interacts with nucleic acid for the purpose of delivering said nucleic acid to a system (e.g., a cell, tissue, or organism).
  • Delivery vehicles may stabilize nucleic acid in otherwise harsh conditions (e.g., a bloodstream after in vivo administration). Delivery vehicles may allow for nucleic acid to pass through the plasma membrane of a cell (i.e., be delivered to a cell). Furthermore, delivery vehicles may provide cell-type or tissue-type specificity in delivering of a nucleic acid. [0166] Teachings of the present disclosure relate to non-viral vectors and/or systems used for nucleic acid delivery.
  • the insights that the present disclosure provides are not limited to a particular delivery vehicle species.
  • a delivery vehicle is or comprises a polymer.
  • a delivery vehicle is or comprises cationic molecules.
  • a delivery vehicle is or comprises linear polycations.
  • a delivery vehicle is or comprises proton sponges.
  • a delivery vehicle is or comprises amphiphilic polycations.
  • a delivery vehicle is or comprises polyethylene glycol (PEG).
  • a delivery vehicle is or comprises polyethylenimine (PEI).
  • PEI polyethylenimine
  • a delivery vehicle is or comprises a biodegradable polymer.
  • a biodegradable polymer may be natural or synthetic.
  • a delivery vehicle is or comprises poly(lactic acid) (PLA), poly(glycolic acid) (PGA), or poly(lactic-co-glycolic acid) (PLGA).
  • a delivery vehicle is chemically modified (e.g., PEGylated).
  • a delivery vehicle is or comprises lipid nanoparticles (LNPs).
  • a delivery vehicle is or comprises extracellular vesicles.
  • a delivery vehicle is or comprises red blood cell derived extracellular vesicles.
  • Extracellular Vesicles EVs
  • an extracellular vesicle EV is a lipid-bound vesicle-like structure.
  • EVs have a membrane.
  • EVs have a membrane that is a double layer membrane (e.g., a lipid bilayer).
  • EVs have a membrane that originates from a cell.
  • EVs have a membrane that originates from the plasma membrane of a cell.
  • the term extracellular vesicle encompasses exosomes, microvesicles, membrane microparticles, ectosomes, blebs or apoptotic bodies.
  • an EV is classified as an exosome, microvesicle, membrane microparticle, ectosome, bleb or apoptotic body based on the origin of formation.
  • EVs are substantially transparent.
  • EVs are substantially spherical.
  • an EV has a diameter within a range of 50 to 1000 nm.
  • an EV has a diameter within a range of 50 to 750 nm. In some embodiments, an EV has a diameter within a range of 50 to 500 nm. In some embodiments, an EV has a diameter within a range of 50 to 300 nm. In some embodiments, an EV has a diameter within a range of 50 to 200 nm. In some embodiments, an EV has a diameter within a range of 50 to 150 nm. In some embodiments, an EV has a diameter within a range of 100 to 1000 nm. In some embodiments, an EV has a diameter within a range of 100 to 750 nm.
  • an EV has a diameter within a range of 100 to 500 nm. In some embodiments, an EV has a diameter within a range of 100 to 300 nm. In some embodiments, an EV has a diameter within a range of 100 to 200 nm. In some embodiments, an EV has a diameter of at least 100 nm. In some embodiments, an EV has a diameter of at most 300 nm.
  • a population of EVs (e.g., as present in a composition, pharmaceutical composition, medicament, preparation or otherwise) will comprise EVs with a range of diameters.
  • the median diameter of EVs within a population is 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, or 300 nm ( ⁇ 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 nm).
  • the mean diameter of EVs within a population is 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, or 300 nm ( ⁇ 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 nm).
  • a population of EVs may comprise at least 10, 100, 1000, 10 4 , 10 5 , 10 6 , 10 7 , 10 8 , 10 9 , 10 10 , 10 11 , 10 12 , 10 13 or 10 14 EVs.
  • a population of EVs may comprise at least 10, 100, 1000, 10 4 , 10 5 , 10 6 , 10 7 , 10 8 , 10 9 , 10 10 , 10 11 , 10 12 , 10 13 or 10 14 EVs per mL of carrier.
  • an extracellular vesicle is derived from a cell into the extracellular environment.
  • An extracellular vesicle may be derived from various cell types under both pathological and physiological conditions.
  • EVs have a similar composition to the cell from which it is derived from.
  • an EV is produced from outward budding and fission of cellular membrane.
  • An EV may be produced via a natural process or a chemically-induced or enhance process.
  • EVs are derived from cells that are contacted with a vesicle- inducing agent.
  • a vesicle-inducing agent may be calcium ionophore, lysophosphatidic acid (LPA), or phorbol-12-myristat-13-acetate (PMA).
  • EVs are derived from human cells, or cells of human origin. In some embodiments, EVs are derived from cells that are not modified (e.g., transduced, transfected, infected, or otherwise modified). In some embodiments, EVs are derived from cells that are ex vivo.
  • EVS are derived from hematopoietic cells.
  • EVs are derived from immune cells.
  • EVs may be derived from red blood cells, white blood cells, cancer cells, stem cells, dendritic cells, macrophages, or other cell types.
  • Characterization [0180]
  • an EV is a microvesicle or membrane microparticle produced via chemical induction.
  • a microvesicle or membrane microparticle is shed from the plasma membrane of a cell and does not originate from the endosomal system.
  • an EV selected for loading with cargo nucleic acid is not an exosome.
  • an EV selected for loading with cargo nucleic acid is not an ectosome. In some embodiments, an EV selected for loading with cargo nucleic acid is not a bleb. In some embodiments, an EV selected for loading with cargo nucleic acid is not an apoptotic body.
  • Red Blood Cell Derived Extracellular Vesicles [0182] In some embodiments, EVs are derived from red blood cells. In some embodiments, EVs are red blood cell derived extracellular vesicles (RBCEVs). In some embodiments, EVs are derived from red blood cells ex vivo from a blood draw from a subject.
  • Red blood cells e.g, erythrocytes
  • Red blood cells are enucleated.
  • Red blood cells are characterized in that they do not contain DNA or they contain substantially no DNA.
  • Red blood cells may contain miRNAs or other RNAs.
  • Red blood cells do not contain oncogenic DNA or oncogenic DNA mutations.
  • Red blood cells lack cellular organelles, such as endosomes and endoplasmic reticulum. Red blood cells cannot produce exosomes.
  • RBCEVs contain less nucleic acid than EVs that have been derived from other cell types.
  • RBCEVs do not contain nucleic acid (e.g., DNA) that was present in the cells from which they were derived.
  • RBCEVs are non-exosomal EVs.
  • RBCEVs comprise hemoglobin, stomatin, and/or flotilin-2.
  • RBCEVs are red in color.
  • RBCEVs exhibit a domed (i.e., concave) surface, or “cup shape” when viewed under transmission electron microscopes.
  • RBCEVs comprise cell surface CD235a.
  • an RBCEV has a diameter within a range of 50 to 1000 nm. In some embodiments, an RBCEV has a diameter within a range of 50 to 750 nm.
  • an RBCEV has a diameter within a range of 50 to 500 nm. In some embodiments, an RBCEV has a diameter within a range of 50 to 300 nm. In some embodiments, an RBCEV has a diameter within a range of 50 to 200 nm. In some embodiments, an RBCEV has a diameter within a range of 50 to 150 nm. In some embodiments, an RBCEV has a diameter within a range of 100 to 1000 nm. In some embodiments, an RBCEV has a diameter within a range of 100 to 750 nm. In some embodiments, an RBCEV has a diameter within a range of 100 to 500 nm.
  • an RBCEV has a diameter within a range of 100 to 300 nm. In some embodiments, an RBCEV has a diameter within a range of 100 to 200 nm. In some embodiments, an RBCEV has a diameter of at least 100 nm. In some embodiments, an RBCEV has a diameter of at most 300 nm. [0187] A population of RBCEVs (e.g., as present in a composition, pharmaceutical composition, medicament, preparation or otherwise) will comprise RBCEVs with a range of diameters.
  • the median diameter of RBCEVs within a population is 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, or 300 nm ( ⁇ 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 nm).
  • the mean diameter of RBCEVs within a population is 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, or 300 nm ( ⁇ 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 nm).
  • a population of RBCEVs may comprise at least 10, 100, 1000, 10 4 , 10 5 , 10 6 , 10 7 , 10 8 , 10 9 , 10 10 , 10 11 , 10 12 , 10 13 or 10 14 RBCEVs.
  • a population of RBCEVs may comprise at least 10, 100, 1000, 10 4 , 10 5 , 10 6 , 10 7 , 10 8 , 10 9 , 10 10 , 10 11 , 10 12 , 10 13 or 10 14 RBCEVs per mL of carrier.
  • RBCEVs are derived from a human or animal blood sample.
  • RBCEVs are derived from red blood cells derived from primary cells or immobilized red blood cell lines.
  • RBCEVs are derived from blood cells type matched to the subject that is to be treated.
  • RBCEVs are derived from blood cells of Group A, Group B, Group AB, or Group O blood.
  • RBCEVs are derived from blood cells of Group O blood.
  • blood is any blood type.
  • blood is rhesus positive or rhesus negative.
  • blood is Group O and/or rhesus negative, such as Type O-.
  • blood has been determined to be free from disease or disorder.
  • blood has been determined to be free from HIV, HBV, HCV, syphilis, sickle cell anemia, SARS-CoV2, and/or malaria.
  • RBCEVs are derived from a blood sample obtained from a subject that is to be treated. In some embodiments, RBCEVs are autologous.
  • RBCEVs are derived from a blood sample obtained from a subject other than one that is to be treated. In some embodiments, RBCEVs are allogenic. [0192] In some embodiments, RBCEVs are isolated from a sample of red blood cells. Protocols for obtaining EVs from red blood cells are known in the art, for example in Danesh et al. (2014) Blood.2014 Jan 30; 123(5): 687–696. Methods useful for obtaining RBCEVs may include steps of providing or obtaining a sample comprising red blood cells, inducing the red blood cells to produce EVs, and isolating the EVs. A sample may be a whole blood sample.
  • Red blood cells in a sample may be separated from other components of a whole blood sample (e.g., white blood cells or plasma). Red blood cells may be concentrated (e.g., by centrifugation). A blood sample may be subjected to leukocyte reduction. [0193] Cells other than red blood cells may have been removed from the sample, such that the cellular component of the sample is entirely or substantially only red blood cells.
  • EVs are induced from red blood cells by contacting the cells with a vesicle-inducing agent.
  • a vesicle-inducing agent is calcium ionophore, lysophosphatidic acid (LPA), or phorbol-12-myristat-13-acetate (PMA).
  • a vesicle-inducing agent is about 10 nM calcium ionophore.
  • RBCEVs are isolated from red blood cells and other components of a sample and/or mixture. In some embodiments, RBCEVs are isolated by centrifugation (with or without ultracentrifugation), precipitation, filtration (e.g., tangential flow filtration), or chromatography.
  • red blood cells are separated from a whole blood sample which contains white blood cells and plasma by low speed centrifugation and leukodepletion filters. In some embodiments, a red blood cell sample comprises no other cell types (e.g., white blood cells).
  • red blood cells are diluted in buffer (e.g., PBS) prior to contacting with a vesicle-inducing agent.
  • red blood cells are contacted with a vesicle-inducing agent overnight, or for at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12 or more than 12 hours.
  • red blood cells are contacted with a vesicle-inducing agent at a plurality of time points.
  • RBCEVs are isolated by subjecting a sample to low speed centrifugation and/or passing a sample through an about 0.45 ⁇ m syringe filter.
  • RBCEVs are concentrated by ultracentrifugation. In some embodiments, RBCEVs are concentrated by ultracentrifugation at a speed of 10,000 x g, 15,000 x g, 20,000 x g, 25,000 x g, 30,000 x g, 40,000 x g, 50,000 x g, 60,000 x g, 70,000 x g, 80,000 x g, 90,000 x g or 100,000 x g. In some embodiments, RBCEVs are concentrated by ultracentrifugation at a speed within a range of 10,000 x g and 50,000 x g.
  • RBCEVs are concentrated by ultracentrifugation at a speed of about 15,000 x g. In some embodiments, RBCEVs are concentrated by ultracentrifugation for at least 10 minutes, at least 20 minutes, at least 30 minutes, at least 40 minutes, at least 50 minutes or at least one hour. [0196] In some embodiments, concentrated RBCEVs are suspended in cold PBS. In some embodiments, concentrated RBCEVs are layered on a sucrose cushion. In some embodiments, a sucrose cushion comprises frozen 60% sucrose.
  • RBCEVs layered on a sucrose cushion are subjected to ultracentrifugation at 100,000 x g for at least 1 hour, at least 2 hours, at least 3 hours, at least 4 hours, at least 5 hours, at least 6 hours, at least 7 hours, at least 8 hours, at least 9 hours, at least 10 hours, at least 11 hours, at least 12 hours, at least 13 hours, at least 14 hours, at least 15 hours, at least 16 hours, at least 17 hours, at least 18 hours or longer.
  • RBCEVs layered on a sucrose cushion are subjected to ultracentrifugation at 100,000 x g for about 16 hours. RBCEVs may then be obtained by collecting the red layer above the sucrose cushion.
  • loading of an EV with a cargo nucleic acid refers to associating the EV and the cargo nucleic acid in stable or semi-stable form such that the EV is useful as a carrier of the cargo nucleic acid (e.g., allowing its delivery to cells).
  • cargo nucleic acids are loaded such that they are present in the lumen of the EV. In some embodiments, cargo nucleic acids are attached to, adhered to, inserted through, or complexed with the external surface (e.g., the membrane) of the EV. In some embodiments, cargo nucleic acids are loaded such that there are nucleic acids present in the lumen of the EV and there are nucleic acids attached to, adhered to, inserted through, or complexed with the external surface (e.g., the membrane) of the EV. [0199] In some embodiments, at least one copy of a single cargo nucleic acid is loaded into EVs.
  • At least one copy each of two different cargo nucleic acids are loaded into EVs.
  • EVs are loaded with a first cargo nucleic acid, followed by loading of a second cargo nucleic acid.
  • EVs are loaded first with a payload nucleic acid followed by loading of a promoting oligonucleotide.
  • EVs are loaded first with a promoting oligonucleotide followed by loading of a payload nucleic acid.
  • EVs are loaded with two cargo nucleic acids simultaneously.
  • EVs are loaded simultaneously with a promoting oligonucleotide and a payload nucleic acid.
  • methods of EV loading comprise contacting cargo nucleic acid with transfection reagent.
  • cargo nucleic acid and transfection reagent are brought together under suitable conditions and for suitable time to allow for EV loading to occur.
  • transfection reagents comprise cationic reagents such as cationic lipid reagents.
  • Transfection reagents may be Lipofectamine TM 3000 TM (ThermoFisher), Turbofect TM (ThermoFisher), Lipofectamine TM MessengerMAX TM (ThermoFisher), Exofect TM (System Biosciences), Linear Polyethylenimine Hydrochlorides (e.g., having an average molecular weight of 25,000 Da or 40,000Da, such as PEIMax TM (Polysciences, Inc.) and jetPEI® (Polyplus transfection)), polybrene or protamine sulfate (see, for example, Delville et al.
  • Lipofectamine TM 3000 TM ThermoFisher
  • Turbofect TM ThermoFisher
  • Lipofectamine TM MessengerMAX TM ThermoFisher
  • Exofect TM System Biosciences
  • Linear Polyethylenimine Hydrochlorides e.g., having an average molecular weight of 25,000
  • loading of cargo nucleic acids into EVs does not comprise viral delivery methods.
  • loading of cargo nucleic acids into EVs does not comprise a viral vector (e.g., an adenoviral vector, adeno-associated vector, lentiviral vector, retroviral vector, etc.).
  • a viral vector e.g., an adenoviral vector, adeno-associated vector, lentiviral vector, retroviral vector, etc.
  • methods of EV loading comprise a step of preparing the cargo nucleic acid to be loaded.
  • the preparation step comprises contacting the nucleic acid to be loaded into EVs with transfection reagent under conditions suitable for the formation of a complex between the transfection reagent and the nucleic acid.
  • the nucleic acid and transfection reagent may form a complex (e.g., DNA:PEIMax complex).
  • the preparation step comprises concentration or dilution of the nucleic acid.
  • the preparation step comprises addition of buffers or other reagents or media (e.g., Opti-MEM reduced serum media (Gibco)).
  • the nucleic acid and transfection reagent are contacted for at least 1 minute, at least 2 minutes, at least 3 minutes, at least 4 minutes, at least 5 minutes, at least 6 minutes, at least 7 minutes, at least 8 minutes, at least 9 minutes, at least 10 minutes, at least 11 minutes, at least 12 minutes, at least 13 minutes, at least 14 minutes, at least 15 minutes, at least 16 minutes, at least 17 minutes, at least 18 minutes, at least 19 minutes, at least 20 minutes, or more than 20 minutes.
  • the preparation step comprises combining a nucleic acid:transfection reagent complex with a further nucleic acid:transfection reagent complex wherein the nucleic acids are non-identical.
  • nucleic acid:transfection reagent complexes contain identical nucleic acids. In some embodiments, nucleic acid:transfection reagent complexes contain non-identical nucleic acids in particular ratios. In some embodiments, two non- identical nucleic acid:transfection reagent complexes are combined.
  • the transfection reagent of multiple complexes may or may not be identical. Non-identical nucleic acids may be present in complexes at equimolar amounts (i.e., at an equimolar ratio). Non- identical nucleic acids may not be present in complexes at equimolar amounts (i.e., at an equimolar ratio).
  • the ratio may refer to the amount of a first nucleic acid in relation a further nucleic acid present in a mixture, wherein the first nucleic acid and further nucleic acid are to be contacted with EVs simultaneously.
  • the ratio may refer to the amount of a first nucleic acid in relation a further nucleic acid present in a mixture, wherein the first nucleic acid and further nucleic acid are to be contacted with EVs in separate steps.
  • the first nucleic acid to be loaded and the further nucleic acid to be loaded may be present at a ratio of about 400:1, 300:1, 250:1, 200:1, 150:1, 100:1, 75:1, 50:1, 25:1, 20:1, 15:1, 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, 1:10, 1:15, 1:20, 1:25, 1:50, 1:75, 1:100, 1:150, 1:200, 1:250, 1:300, 1:400, 1:500.
  • the first nucleic acid to be loaded and the further nucleic acid to be loaded may be present at a ratio of about 100:1, 75:1, 50:1, 25:1, 20:1, 15:1, 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, 1:10, 1:15, 1:20, 1:25, 1:50, 1:75, 1:100, 1:150, 1:200, 1:250, 1:300, 1:400, 1:500.
  • the first nucleic acid to be loaded and the further nucleic acid to be loaded may be present at a ratio of about 25:1, 20:1, 15:1, 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, 1:10, 1:15, 1:20, 1:25.
  • the first nucleic acid to be loaded and the further nucleic acid to be loaded may be present at a ratio of 1:1.
  • the first nucleic acid to be loaded and the further nucleic acid to be loaded may be present at a ratio of between 100:1-1:100, 75:1-1:75, 50:1-1:50, 25:1-1:25, 20:1-1:20, 15:1- 1:15, 10:1-1:10, 9:1-1:9, 8:1-1:8, 7:1-1:7, 6:1-1:6, 5:1-1:5, 4:1-1:4, 3:1-1:3, 2:1-1:2, or about 1:1.
  • the first, second and third nucleic acids may be present in a ratio of about 1:1:2, 1:1:3, 1:1:4, 1:1:5, 1:1:6, 1:1:7, 1:1:8, 1:1:9, 1:1:10, 1:2:1, 1:3:1, 1:4:1, 1:5:1, 1:6:1, 1:7:1, 1:8:1, 1:9:1, 1:10:1, 2:1:1, 3:1:1, 4:1:1, 5:1:1, 6:1:1, 7:1:1, 8:1:1, 9:1:1, 10:1:1, 1:2:2, 1:3:3.1:4:4, 1:5:5, 1:6:6, 1:7:7.1:8:8: 1:9:9, 1:10:10, 1:2:3, 1:2:4, 1:3:6, 1:4:8, 1:5:10, 2:4:6, 2:8:4 or other ratio.
  • the length of a nucleic acid to be loaded will influence the ratio.
  • a nucleic acid with longer length will be loaded at a greater ratio than a nucleic acid with less length.
  • the relative structure of a nucleic acid to be loaded will influence the ratio.
  • a more compact nucleic acid structure e.g., a DNA plasmid
  • a less compact nucleic acid structure e.g., a linear DNA
  • the strandedness (e.g. single or double) of a nucleic acid will influence the ratio.
  • a single-stranded nucleic acid will be loaded at a greater ratio than a double-stranded nucleic acid. In some embodiments, a single-stranded nucleic acid will be loaded at a doubled ratio than a double-stranded nucleic acid. The ratio maybe adjusted from 1:1 to 2:1 where the first nucleic acid is a single-stranded nucleic acid and the further nucleic acid is a double-stranded nucleic acid.
  • Loading EVs with Cargo Nucleic Acids [0208] In some embodiments, methods of EV loading comprise a step of loading the EVs with cargo nucleic acid.
  • prepared nucleic acid:transfection reagent complexes are contacted with the EVs that are to be loaded. In some embodiments, contacting with the EVs is performed subsequently to the contacting of the nucleic acid to be loaded with the transfection reagent. In some embodiments, the nucleic acid:transfection reagent complexes are contacted with a composition comprising a plurality of EVs. In some embodiments, the nucleic acid:transfection reagent complexes and EVs to be loaded are incubated for sufficient time and under appropriate conditions to allow the EV to be loaded with the one or more nucleic acid:transfection reagent complexes.
  • the nucleic acid:transfection reagent complexes are internalized into the EV. In some embodiments, the nucleic acid:transfection reagent complexes are loaded onto the surface of the EVs (e.g., onto the membranes of the EVs). [0209] In some embodiments, EVs are isolated, washed, and/or concentrated after the step of loading with cargo nucleic acid. In some embodiments, loaded EVs are washed with phosphate buffered saline (PBS). In some embodiments, the washing step is repeated 1, 2, 3, 4, 5, 6, or more times. [0210] In some embodiments, methods of EV loading comprise a temporary or semi- permanent increase in permeability of the membrane of the EVs.
  • PBS phosphate buffered saline
  • Suitable methods to temporarily or semi-permanently increase permeability of the EV membranes are, for example, electroporation, sonication, ultrasound, lipofection or hypotonic dialysis as described in PCT/SG2018/050596 which is herein incorporated by reference in its entirety.
  • loaded EVs are treated to increase the permeability of the membranes of the EVs.
  • the loaded EVs are chilled prior to treatment to increase the permeability of the membranes of the EVs.
  • treatment of the EVs to increase the permeability of the membranes of the EVs further involves one or more buffers (e.g., PBS).
  • buffers e.g., PBS
  • EVs are further contacted with nucleic acid:transfection reagent complexes after previous contact with nucleic acid:transfection reagent complexes.
  • the further nucleic acid:transfection reagent complexes comprise a nucleic acid which is non- identical to the nucleic acid loaded in the previous loading step.
  • the further loading step is conducted under the same or different time and the same or different conditions as used in the previous loading step.
  • a washing step may be performed after a first loading step and/or subsequent loading steps following the first loading step.
  • Treatment to increase the permeability of the membranes of the EVs may be performed after a first loading step and/or subsequent loading steps following the first loading step.
  • Electroporation or electropermeabilization, is a microbiology technique in which an electrical field is applied to cells in order to increase the permeability of the cell membrane, allowing, for example, chemicals, drugs or DNA to be introduced into the cell.
  • EVs are induced to encapsulate cargo nucleic acids by electroporation.
  • electroporation involves passing thousands of volts across a distance of one to two millimeters of suspended cells in an electroporation cuvette (1.0-1.5 kV, 250- 750V/cm).
  • electroporation is a multi-step process with distinct phases.
  • a first phase comprises application of a short electrical pulse.
  • voltage settings for a fist phase would be within the range of 300-400 mV for less than 1 millisecond across the membrane.
  • Application of the potential may charge the membrane like a capacitor through the migration of ions from the surrounding solution. There may be a rapid localized rearrangement in lipid morphology once the critical field is achieved.
  • the resulting structure may not be electrically conductive but may lead to the rapid creation of a conductive pore.
  • the conductive pores may heal by resealing the bilayer or expand and eventually rupture.
  • EVs are subjected to electroporation at between about 25 and 300 V or between about 50 and 250 V.
  • EVs are loaded with cargo nucleic acid by sonication.
  • Sonication is the act of applying sound energy to agitate particles in a sample. Ultrasonic frequencies (>20 kHz) may be used, leading to the process also being known as ultrasonification or ultra-sonification. Sonication may be applied using an ultrasonic bath or an ultrasonic probe, also known as a sonicator.
  • EVs are loaded with cargo nucleic acid by ultrasound. Ultrasound is known to disrupt cell membranes and thereby load cells with molecules. Sound waves with frequencies from 20 kHz up to several gigahertz may be applied to EVs.
  • EVs are loaded with cargo nucleic acid by lipofection.
  • Lipofection or liposome transfection, is a technique used to deliver nucleic acid into a cell by means of liposomes. Liposomes are vesicles that readily merge with phospholipid bilayers as liposomes are made of phospholipid bilayer.
  • nucleic acids are loaded at an equimolar ratio when they are of similar size. In some embodiments, nucleic acids are loaded at an equimolar ratio when they are plasmids.
  • methods of EV loading comprise removing nucleic acid not contained within the lumen of EVs.
  • EVs are contacted with DNAse to remove nucleic acid not contained within the lumen of EVs. In some embodiments, EVs are contacted with heparin to dissociate nucleic acid or nucleic acid:transfection reagent complexes. VIII. Pharmaceutical Compositions Characterization [0219] Compositions comprising EVs are described herein. A composition comprising EVs comprises one or more EVs. [0220] In some embodiments, a composition comprising EVs is a pharmaceutical composition. In some embodiments, a composition comprising EVs is a pharmaceutical composition further comprising a pharmaceutically acceptable carrier.
  • compositions may be formulated for administration by a particular route (e.g., intravenous, intratumoral, intraperitoneal, intradermal, subcutaneous, intranasal or other administration route).
  • a composition comprising EVs further comprises a buffer solution.
  • a composition comprising EVs further comprises a preservative compound
  • a composition comprises an amount of EV particles within a range of 10 6 to 10 15 particles per mL.
  • a composition may comprise an amount of EV particles at least 10 5 particles per mL, at least 10 6 particles per mL, at least at least 10 7 particles per mL, at least 10 8 particles per mL, at least 10 9 particles per mL, at least 10 10 particles per mL, at least 10 11 particles per mL, at least 10 12 particles per mL, at least 10 13 particles per mL, at least 10 14 particles per mL, or at least 10 15 particles per mL.
  • a composition comprising EVs contains EVs that have substantially homologous dimensions. Refer to VI. Extracellular Vesicles (EVs), Populations for a description of embodiments of EV dimensions when present in a composition.
  • a composition comprises EVs that are loaded with the same, or substantially the same, cargo nucleic acid. In some embodiments, a composition comprises EVs that are loaded with different cargo nucleic acid. In some embodiments, a composition comprises two EV populations that are loaded with different cargo nucleic acid. In some embodiments, a composition comprises two EV populations that are loaded with different cargo nucleic acid in an equal or an unequal ratio within the composition. In some embodiments, a composition comprises three EV populations, wherein at least on population is loaded with different cargo nucleic acid. In some embodiments, a composition comprises three EV populations, wherein at least on population is loaded with different cargo nucleic acid, in an equal or an unequal ratio within the composition.
  • a proportion of EVs contain cargo nucleic acid when present in a composition.
  • at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 97% of EVs contain cargo nucleic acid when present in a composition.
  • Administration As described herein, a composition comprising EVs may be used to deliver two or more nucleic acids to a cell, wherein one nucleic acid is a promoting oligonucleotide.
  • a composition comprising EVs loaded with at least two non-identical nucleic acids delivers said nucleic acids simultaneously, wherein one nucleic acid is a promoting oligonucleotide.
  • EVs loaded with two non-identical cargo nucleic acids affords superior expression as compared to encoding the peptides on a single nucleic acid (e.g., a bicistronic vector), as well as delivering said cargo nucleic acids separately in different EVs, wherein one nucleic acid is a promoting oligonucleotide.
  • a composition comprising EVs is used to deliver cargo nucleic acid to at least one target cell.
  • a target cell is a cancer cell or tumor cell. In some embodiments, a target cell is an immune cell. In some embodiments, a target cell comprises at least one genetic mutation. In some embodiments, a target cell comprises a gene that is dysfunctional or otherwise not functioning properly. In some embodiments, a target cell comprises a loss of function mutation. In some embodiments, a target cell comprises a gain of function mutation. In some embodiments, a target cell comprises a mutation that results in overexpression or underexpression of a protein. [0228] In some embodiments, a composition comprising EVs contacts a target cell.
  • the target cell and the composition comprising EVs are contacted for a sufficient time under sufficient conditions suitable for the target cell to uptake the EVs.
  • delivering cargo nucleic acid to a target cell is performed in vitro or ex vivo. In some embodiments, delivering cargo nucleic acid to a target cell is performed in vivo. In some embodiments, delivering cargo nucleic acid to a target cell is performed on at least one cell that has isolated from a subject (e.g., a human subject). In some embodiments, delivering cargo nucleic acid to a target cell is performed on at least one cell that is present in a subject (e.g., a human subject).
  • a composition comprising EVs is incubated with a target cell.
  • the terms “incubating”, “incubation”, “incubate” are used to refer to contacting target cell(s) and EV(s) loaded with cargo nucleic acid together at suitable temperature and for suitable time such that the EV(s) are taken up (i.e., assimilated, incorporated or taken in) by the target cell(s).
  • incubating target cell(s) involves culturing the cells, or populations thereof, in vitro or ex vivo in cell culture medium comprising EVs loaded with cargo nucleic acid.
  • incubation is performed at a temperature close to body temperature of a mammal (e.g., a human). Incubation may be performed at a temperature of at least 35.0 ° C, at least 35.5 ° C, at least 36.0 ° C, at least 36.1 ° C, at least 36.2 ° C, at least 36.3 ° C, at least 36.4 ° C, at least 36.5 ° C, at least 36.6 ° C, at least 36.7 ° C, at least 36.8 ° C, at least 36.9 ° C, at least 37.0 ° C, at least 37.1 ° C, at least 37.2 ° C, at least 37.3 ° C, at least 37.4 ° C, and/or at least 37.5 ° C.
  • incubation is performed at two or more temperatures (e.g., as above). In some embodiments, incubation is performed at a single temperature. In some embodiments, incubation is performed at human body temperature. In some embodiments, incubation is performed at at least 37.0 ° C. In some embodiments, incubation may be repeated on same cells. [0231] In some embodiments, incubation comprises controlling CO 2 level of the cell culture. Controlling CO2 level of the cell culture may control the pH of the incubated mixture. In some embodiments, CO2 level of the incubating mixture is maintained at or close to the CO 2 level of blood (e.g., mammalian blood).
  • blood e.g., mammalian blood
  • Incubation may be performed at one or more of at least 4.0%, at least 4.1%, at least 4.2%, at least 4.3%, at least 4.4%, at least 4.5%, at least 4.6%, at least 4.7%, at least 4.8%, at least 4.9%, at least 5.0%, at least 5.1%, at least 5.2%, at least 5.3%, at least 5.4%, at least 5.5%, at least 5.6%, at least 5.7%, at least 5.8%, at least 5.9% and/or at least 6.0% CO 2 .
  • Incubation may be performed at one or more of at least 30mmHg, at least 31mmHg, at least 32mmHg, at least 33mmHg, at least 34mmHg, at least 35mmHg, at least 36mmHg, at least 37mmHg, at least 38mmHg, at least 39mmHg, at least 40mmHg, at least 41mmHg, at least 42mmHg, at least 43mmHg, at least 44mmHg, and/or at least 45mmHg CO 2 .
  • incubation is performed at a level at least 5% CO 2 . In some embodiments, incubation is performed at a level about 5% CO 2 .
  • incubation is performed at a level of 5% CO2. In some embodiments, incubation is performed at a level at least 38mmHg CO 2 . In some embodiments, incubation is performed at a level about 38mmHg CO 2 . In some embodiments, incubation is performed at a level of 38mmHg CO2. In some embodiments, incubation is performed in a humidified environment (e.g., in a humidified incubator). [0233] In some embodiments, incubation is performed for a length of time such that EVs are taken up by target cells. Incubation may be performed, at a combination of temperature and CO 2 level as above, for one of 12, 24, 36, 48, 60, or 72 hours.
  • Incubation may be performed for at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, at least 30, at least 31, at least 32, at least 33, at least 34, at least 35, at least 36, at least 37, at least 38, at least 39, at least 40, at least 41, at least 42, at least 43, at least 44, at least 45, at least 46, at least 47, at least 48, at least 49, at least 50, at least 51, at least 52, at least 53, at least 54, at least 55, at least 56, at least 57, at least 58, at least 59, at least 60, at least 61, at least 62, at least 63, at least 64, at least 65, at least 66, at least 67, at least 68, at least 69, at least 70, at least 71, or at least 72 hours.
  • incubation is performed for at least 36 or at least 48 hours. In some embodiments, incubation is performed for 48 hours. In some embodiments, target cells are washed one or more times after incubation. In some embodiments, target cells are washed to remove any non-assimilated EVs. In some embodiments, target cells are washed using PBS and centrifugation (e.g., at 4 ° C). [0235] The methods described herein may comprise an incubation step comprising any combination of temperature, CO 2 level, and/or time (e.g., as described above). In some embodiments, incubation is performed at 37 ° C at 5% CO 2 for 48 hours.
  • Incubation may be performed in any suitable medium (e.g., a cell culture medium). In some embodiments, incubation comprises agitating the mixture for some or all of the incubation time.
  • IX. Methods of Treatment and Uses of EVs [0237] EVs, as described herein, may be useful in methods of treatment. [0238] In some embodiments, a composition comprising EVs is useful in methods of treatment that are known to benefit from administration of nucleic acid. For example, a composition comprising EVs may be useful for delivering a functional gene, or fragment thereof, to replace and/or supplement a gene that is otherwise not fully functional.
  • a composition comprising EVs is useful in methods of treatment that are known to benefit from administration of multiple nucleic acids.
  • a composition comprising EVs may be useful for delivering a gene editing system or a vectorized antibody.
  • a composition comprising EVs may be useful in methods of treatment for a genetic disease, an inflammatory disease, a cancer, an autoimmune disorder, a cardiovascular disease, or a gastrointestinal disease.
  • a genetic disorder treated with a composition comprising EVs may be thalassemia, sickle cell anemia, a genetic metabolic disorder, a muscular dystrophy, a neuromuscular dystrophy, or a retinopathy.
  • a composition comprising EVs may be useful in methods of treatment for a disorder of the liver, bone marrow, lung, spleen, brain, pancreas, stomach, or intestine.
  • the target cell depends on the disease or disorder that is to be treated.
  • the target cell may be a breast cancer cell, a colorectal cancer cell, a lung cancer cell, a kidney cancer cell or the like.
  • a subject treated with a composition comprising EVs has an inflammatory disease.
  • a subject treated with a composition comprising EVs has cancer.
  • a subject treated with a composition comprising EVs has an autoimmune disease.
  • a subject treated with a composition comprising EVs has a cardiovascular disease. In some embodiments, a subject treated with a composition comprising EVs has a gastrointestinal disease. In some embodiments, a subject treated with a composition comprising EVs ha a genetic disease. In some embodiments, a subject treated with a composition comprising EVs has a monogenic disease. In some embodiments, a subject treated with a composition comprising EVs has a polygenic disease. [0244] In some embodiments, a composition comprising EVs is used for the treatment of cancer. A composition comprising EVs may be useful for inhibiting the growth, proliferation, or survival of cancerous cells.
  • a composition comprising EVs is used for the treatment of liquid or blood cancer (e.g., leukemia, lymphoma, or myeloma).
  • a composition comprising EVs may be administered, or formulated for administration, by a number of routes, including but not limited to systemic, intratumoral, intraperitoneal, parenteral, intravenous, intra-arterial, intradermal, subcutaneous, intramuscular, oral and/or nasal administration.
  • a composition comprising EVs is formulated in liquid or solid form.
  • a liquid formulation is administered by injection to a specific region of the subject or via a specific route of administration.
  • Administration of a composition comprising EVs may be in a “therapeutically effective amount”, this being sufficient to show benefit to the subject.
  • the amount administered, the rate at which it is administered, and the time-course of administration may depend on the nature and severity of the disease that is to be treated. Prescriptions of treatment (e.g., decisions on dosage) may be within the responsibility of general practitioners and other medical doctors. Prescriptions of treatment may be depend on the disease and/or condition that is to be treated, the condition of the individual subject, the site of delivery, the route of administration, and/or other factors. Examples of the techniques and protocols mentioned above may be found in Remington’s Pharmaceutical Sciences, 20th Edition, 2000, pub. Lippincott, Williams & Wilkins.
  • a composition comprising EVs is administered alone. In some embodiments, a composition comprising EVs is administered in combination with at least one other treatment. A compositions comprising EVs may be administered simultaneously or sequentially when administered in combination with at least one other treatment. [0248] In some embodiments, a composition comprising EVs is administered to an animal. In some embodiments, a composition comprising EVs is administered to a mammal. In some embodiments, a composition comprising EVs is administered to a non-human mammal. In some embodiments, a composition comprising EVs is administered to a human. In some embodiments, a composition comprising EVs is administered to a male or female human.
  • a composition comprising EVs is administered to a human that is a patient. In some embodiments, a composition comprising EVs is administered to non-human animal for veterinary purposes. [0249] In some embodiments, a composition comprising EVs is used to deliver cargo nucleic acid to a target cell in vitro. In some embodiments, a composition comprising EVs is used to deliver a labeling molecule or a plasmid to a target cell in vitro.
  • EXEMPLIFICATION Example 1 RBCEVs co-loaded with double-stranded oligonucleotides can increase transgene expression and suppress immune response(s) to the transgene [0250]
  • the present Example demonstrates that co-loading of RBCEVs with double-stranded oligonucleotides can increase transgene expression when delivered in vitro or in vivo while also suppressing the immune response to the transgene. Specifically, transgene expression and inflammatory cytokines were assessed after co-loaded RBCEVs were delivered to multiple mouse strains and multiple human cell lines of different tissue origin.
  • Exogenous DNA found in the cytoplasm can activate the innate immune response by DNA-activated signaling pathways such as the cGAS-STING pathway, resulting in the production of pro-inflammatory cytokines.
  • the present invention proposes the co-loading of decoy oligonucleotides with red blood cell derived extracellular vesicles (RBCEVs) to enhance the activity of the delivered nucleic acid payload in vitro and in vivo, relative to the corresponding RBCEVs loaded with payload alone.
  • RBCEVs co-loaded with decoy oligonucleotides can act to mitigate cellular innate immune response by reducing the induction of Type I interferons in vivo.
  • the term “decoy oligonucleotide” represents a class of oligonucleotides that can block the transcriptional activity of immune response and signaling-related transcription factors such as NF- ⁇ B, as an effective strategy to counter the surveillance of the cGAS-STING pathway. Oligonucleotides containing the NF- ⁇ B consensus sequence blocks the binding of the NF- ⁇ B to the promoter regions of its target genes, resulting in the inhibition of downstream genes and the release of inflammatory cytokines. Materials and Methods Purification and quantification of EVs from human RBCs [0253] Whole blood samples were obtained through Alternative Research, Inc from healthy donors with informed consent.
  • RBCs were separated from plasma and white blood cells by using centrifugation and leukodepletion filters (Terumo Japan). Isolated RBCs were diluted in PBS and treated with 10 mM calcium ionophore (Sigma-Aldrich) overnight. To purify EVs, RBCs and cell debris were removed by centrifugation at 600 ⁇ g for 60 min, 1,600 ⁇ g for 30 min, and 4,000 ⁇ g for 30 minutes at 4°C. EVs were pelleted at 15,000 ⁇ g for 180 minutes at 4 °C, resuspended in PBS and passed through a 0.45 ⁇ m filter.
  • EVs were washed with 4 diavolumes of PBS and concentrated by tangential flow filtration (Pall Minimate). Purified EVs were stored at ⁇ 80 °C. EVs were quantified by assessing their hemoglobin content using the Hemoglobin Assay Kit (Abcam). Plasmids [0254] DNA plasmids (CMV-copGFP, LSP-FIX-HiBit, CAG-LC, CAG-HC) were custom synthesized. The full-length heavy chain (HC) and light chain (LC) of trastuzumab were obtained from the drug bank database (Accession Number: DB00072).
  • Oligonucleotides [0255] All oligonucleotide designs were synthesized by IDT and purified by either standard desalting or by HPLC. [0256] Decoy to the NF- ⁇ B consensus sequence (ODN) – a 22-bp synthetic oligonucleotide containing the 11-bp NF- ⁇ B binding sequence: GGGGACTTTTCC (De Stefano et al., "A decoy oligonucleotide to NF- ⁇ B delivered through inhalable particles prevents LPS-induced rat airway inflammation.” American Journal of Respiratory Cell and Molecular Biology 49.2 (2013)).
  • Scrambled NF- ⁇ B consensus sequence (SCD) – a scrambled sequence based on the 22-bp ODN, generated using the program from Stothard P (2000) The Sequence Manipulation Suite: JavaScript programs for analyzing and formatting protein and DNA sequences. Biotechniques 28:1102-1104.
  • a scrambled sequence, as described herein, contains the same residues but in a different order as compared to a reference sequence (e.g., NF- ⁇ B decoy oligonucleotide).
  • a scrambled sequences changes the function and/or structure of an oligonucleotide. In some cases, a scrambled sequences does not change the function and/or structure of an oligonucleotide. 5’-ATCTGGCGGTCCAGTTAGAGCC-3’ 3’-TAGACCGCCAGGTCAATCTCGG-5’ Nucleic acid loading of RBCEVs [0258] 1 ⁇ g of DNA was added to 20 ⁇ g of washed RBCEVs and 7 ⁇ l of a chemical-based transfection reagent in Opti-MEM (ThermoFisher) and mixed gently. The reaction was incubated at room temperature for 1 hour with gentle rotation.
  • Opti-MEM ThermoFisher
  • loaded RBCEVs were pelleted at 15,000 ⁇ g and washed with PBS.
  • plasmids and oligonucleotides were first mixed with RBCEVs before the chemical-based transfection reagent was added. After the addition of the chemical-based transfection reagent, the reaction was incubated at room temperature for 1 hour with gentle rotation. Loaded RBCEVs were pelleted at 15,000 ⁇ g and washed with PBS. DNA was quantified by gel densitometry.
  • Loading efficiency was calculated based on starting amount of nucleic acid added to the loading reaction and final amount of nucleic acid recovered after the loading reaction.
  • Cell lines and in vitro transfection [0259] HepG2 cells were purchased from ATCC, and Huh-7 cells were purchased from Elabscience. Cells were cultured in DMEM containing 10% FBS and 1% Penicillin- Streptomycin. THP-1 cells were purchased from ATCC and cultured in RPMI 1640 media containing 10% FBS and 1% Penicillin-Streptomycin. All cell lines were maintained at 37 °C in a 5% CO2 incubator. [0260] For in vitro transfection, 50,000 cells were seeded in each well of 24-well plate and incubated overnight.
  • Flow cytometry of cells was performed using the CytoFLEX S Flow Cytometer (Beckman Coulter) and analyzed using FlowJo V10 (FlowJo, USA). Cells were initially gated based on FSC-A vs. SSC-A to exclude the debris and dead cells (low FSC-A). Cells were further gated based on FSC-width vs. FSC-height, to exclude doublets and aggregates. Using untreated cells as controls, GFP-positive cells were gated in the FITC channel and PI-stained cells were gated in the PE channel. Percentage of GFP and/or PI-stained cells were assessed.
  • Nano-Glo HiBiT Lytic Detection System to quantify HiBiT-tagged protein expression .
  • samples were diluted 100-fold using PBS.
  • 100 ⁇ l of the diluted samples were mixed with an equal volume of Nano-Glo HiBiT Lytic Reagent (Promega), consisting of Nano-Glo HiBiT Lytic Buffer, Nano-Glo HiBiT Lytic Substrate and LgBiT protein. This mixture was incubated for 10 minutes at room temperature in the dark. The luminescence was measured using a Tecan M200 microplate reader with an integration time of 1000ms.
  • trastuzumab HerceptinTM, Genentech
  • the level of trastuzumab in cell culture supernatant was determined by the anti- HER2 ELISA kit (ab237645, Abcam) following the manufacturer’s protocol. This detection method was chosen because a positive signal only occurs in the presence of an intact whole antibody that includes both heavy and light chains.
  • Statistical analysis [0264] The results are expressed as mean ⁇ standard deviation (SD). Data were analyzed by Student’s t-test, one-way/two-way ANOVA, or Pearson’s correlation. The difference between means was considered statistically significant at p ⁇ 0.05.
  • RBCEVs co-loaded with a double-stranded decoy oligonucleotide increases transgene expression in Huh-7, HepG2, THP-1 cell lines
  • RBCEVs were loaded with a DNA plasmid (CMV-copGFP), or simultaneously loaded with a DNA plasmid and NF- ⁇ B decoy (ODN), scrambled (SCD), phosphorothioate-modified NF- ⁇ B decoy (ODN-PS), or phosphorothioate-modified scrambled (SCD-PS) oligonucleotides at increasing dosages from 12.5 to 100 pmol.
  • the loading efficiency of RBCEVs was measured post-loading.
  • Loaded RBCEVs were transfected to 50,000 Huh-7, HepG2, or THP- 1 cells on a 24-well plate format at an equimass amount of the DNA plasmid at 200ng, 400ng, and 500ng respectively. 48 hours after transfection, cells were harvested and analyzed by flow cytometry for GFP expression and cell viability using propidium iodide (PI) staining. Using untreated cells as controls, GFP-positive cells were gated in the FITC channel and PI-positive cells were gated in the PE channel. [0266] Results obtained are presented in Figure 1.
  • Increased expression of GFP was observed in cells that were transfected with RBCEVs that were co-loaded with DNA plasmid and ODN or SCD oligonucleotides.
  • Co-loaded RBCEVs increased transgene expression in some cases by roughly a factor of 2, 3, 4, 5, or 6.
  • Cells that were treated with RBCEVs co- loaded with a single DNA plasmid and ODN showed increased transfection efficiency and expression of the DNA payload in cells.
  • the increase in transfection efficiency and transgene expression was observed to be dose-dependent. Specifically, in each case, transgene expression showed greater enhancement after administration of higher oligonucleotide dose. Pre-treatment with oligonucleotides did not negatively impact cell viability.
  • RBCEVs co-loaded with a double-stranded decoy oligonucleotide enhances transgene expression in BL/6 and SCID mice
  • RBCEVs were loaded with a DNA plasmid that expresses hFIX-HiBit under the LSP promoter (hFIX-HiBit) or co-loaded with DNA plasmid and NF- ⁇ B decoy (ODN) or scrambled (SCD) oligonucleotides.
  • ODN NF- ⁇ B decoy
  • SCD scrambled
  • High dose NF- ⁇ B decoy oligonucleotide treatment resulted in increased HiBit expression at all time-points measured from day 1 up to day 49 as compared to RBCEVs loaded with DNA plasmid alone.
  • RBCEVs were co-loaded with 3 components: a DNA plasmid that expresses the light chain of monoclonal antibody trastuzumab (HERCEPTINTM, Genentech), a DNA plasmid that expresses the trastuzumab heavy chain, and NF- ⁇ B decoy oligonucleotides.
  • Co-loaded RBCEVs increased trastuzumab expression in some cases by roughly a factor of 2, 3, 4, 5, or 6.
  • RBCEVs co-loaded with a double-stranded decoy oligonucleotide suppresses the release of type I interferons (IFN) after IV injection
  • IFN type I interferons
  • Type I IFN release was assayed as a measurement of immune response so as to assess the immunological effect of decoy oligonucleotides or scrambled oligonucleotides co-loaded into RBCEVs.
  • Type I IFN The secretion of Type I IFN was quantified after the intravenous tail-vein injection of RBCEVs co-loaded with DNA plasmid and NF- ⁇ B decoy (ODN) or scrambled (SCD) oligonucleotides in BL/6 mice. Blood was drawn at 0, 6, and 24 hours post-injection of loaded RBCEVs. Type I IFN induction was quantified from mouse serum using the InvivoGen mouse IFN-beta bioluminescent ELISA kit and mouse IFN-alpha bioluminescent ELISA kit. [0272] Results obtained are presented in Figure 4. Administration of RBCEVs loaded with plasmid payload alone induced Type I IFN in vivo.
  • RBCEVs co-loaded with different double-stranded decoy oligonucleotides also increases transgene expression in Huh-7 and HepG2 cell lines [0273]
  • Different double-stranded oligonucleotides were utilized to assess the effect on transgene expression in vitro with RBCEVs.
  • Table 1 shows the sequences of double- stranded, NF- ⁇ B decoy oligonucleotides designed and tested with RBCEVs.
  • RBCEVs were loaded with a DNA plasmid that expresses copGFP under the CMV promoter (CMV-copGFP) or co-loaded with DNA plasmid along with 100 pmol of annealed NF- ⁇ B decoy oligonucleotide.
  • CMV-copGFP CMV promoter
  • Loaded RBCEVs were transfected to 50,000 HepG2 and Huh-7 cells on a 24-well plate format at an equimass amount of the DNA plasmid at 400ng and 200ng respectively.24 and 48 hours after transfection, cells were harvested and analyzed by flow cytometry for GFP expression and cell viability using propidium iodide (PI) staining. [0274] Results obtained are presented in Figure 5. Cells that were treated with RBCEVs co- loaded with a DNA plasmid and an oligonucleotide design showed increased transfection efficiency and expression of the DNA payload in HepG2 and Huh7 cells as compared to RBCEVs loaded with DNA plasmid alone.
  • oligonucleotide designs achieved some level of increase of enhancement than its corresponding group lacking an oligonucleotide without reducing cell viability.
  • Co-loaded RBCEVs increased transgene expression in some cases by roughly a factor of 2, 3, 4, 5, or 6.
  • Transgene expression in cells transfected with co-loaded RBCEVs varied with each oligonucleotide design, depending on the composition, length, and structure of the design. For instance, increased phosphorothioate-bonds in the oligonucleotides from end-modifications to full length modifications decreased transfection efficiency. Reducing the length of dsDNA oligonucleotide design from 22 base pairs to 11 base pairs reduced the transfection efficiency of the DNA plasmid.
  • Ribbon shaped oligonucleotide designs outperformed all other designs, which could be due to longer half-life from reduced nuclease degradation of open-ended oligonucleotides.
  • Table 1 Exemplary representative decoy oligonucleotide designs co-loaded with RBCEVs. Highlighted in bold is the 11 base pair NF- ⁇ B binding sequence, asterisks represent phosphorothioate-bond modifications.
  • RBCEVs co-loaded with double-stranded bait oligonucleotides increases transgene expression in Huh-7, HepG2, THP-1 cell lines [0275]
  • RBCEVs were loaded with two DNA plasmids, one which encodes eGFP and one which encodes FIX-HiBit luciferase reporter construct, or simultaneously loaded with the two plasmids and a self-complementary single-stranded (e.g., comprises one or more double-stranded portions and stem-loop structures) bait oligonucleotide as laid out in Table 2 at increasing dosages from 1 to 2 ⁇ g by chemical-based transfection reagent.
  • the bait oligonucleotides in Table 2 are short interfering DNA molecules (siDNA) that mimic either double-strand breaks (Dbait) or single-strand breaks (Pbait).
  • siDNA short interfering DNA molecules
  • Such oligonucleotides are known to promote DNA-dependent protein kinase (DNA-PK) and/or poly (ADP-ribose) polymerase (PARP) activation (see, for example, Croset, et al., "Inhibition of DNA damage repair by artificial activation of PARP with siDNA.” Nucleic acids research 41.15 (2013)).
  • the loading efficiency of RBCEVs was measured post-loading ( Figure 6).
  • Loaded RBCEVs were transfected to 100,000 Huh-7 cells at an equimass amount of the loaded RBCEVs at 200ng. 24 hours after transfection, supernatant was harvested and Nano-Glo® HiBiT assay was performed to evaluate the transgene expression, and cells were harvested and analyzed by flow cytometry for GFP expression. [0276] Results obtained are presented in Figure 7. As can be seen, several RBCEV compositions loaded with a bait oligonucleotide increased expression of both transgenes. Specifically, Pbait32, Pbait32L, Dbait8H, and Dbait32Hc increased both eGFP and FIX-HiBit expression at various dosages.
  • Example 2 RBCEVs co-loaded with single-stranded oligonucleotides can increase transgene expression and suppress immune response to the transgene
  • the present Example demonstrates that co-loading of RBCEVs with single-stranded oligonucleotides can increase transgene expression when delivered in vitro while also suppressing the immune response to the transgene. Specifically, transgene expression and inflammatory cytokine expression were assessed after co-loaded RBCEVs were delivered to multiple human cell lines of different tissue origin.
  • RBCEVs were loaded with a DNA plasmid that expresses hFIX-HiBit luciferase reporter construct or co-loaded with a DNA plasmid that expresses hFIX-HiBit luciferase reporter construct and a double-stranded oligonucleotide (scrambled, NF- ⁇ B decoy) or a single-stranded oligonucleotide.
  • Table 3 shows the sequences of single-stranded, NF- ⁇ B decoy oligonucleotides designed and tested with RBCEVs. 100,000 of Huh7 and HepG2 cells were transfected with the loaded RBCEVs containing 200ng of DNA plasmid.
  • Results obtained are presented in Figure 8. Multiple different designs of single- stranded oligonucleotide increased transgene expression as compared to RBCEVs loaded without oligonucleotide. Specifically, RBCEVs loaded with a single-stranded oligonucleotide increased FIX-Hibit expression and/or activity in multiple cell lines when co- loaded with FIX-HiBit construct.
  • RBCEVs were loaded with a DNA plasmid that expresses hFIX-HiBit luciferase reporter construct or co-loaded with a DNA plasmid that expresses hFIX-HiBit luciferase reporter and double-stranded oligonucleotide (scrambled, NF- ⁇ B decoy) or single-stranded oligonucleotide.
  • Table 3 shows the sequences of single-stranded, NF- ⁇ B decoy oligonucleotides designed and tested with RBCEVs.
  • Pre-treatment with oligonucleotide-loaded RBCEVs can increase expression of subsequently-delivered transgene
  • the present Example demonstrates that pre-treatment with oligonucleotide-loaded RBCEVs can enhance expression of a transgene subsequently delivered by RBCEVs.
  • the present example describes transgene expression in two different human liver cell lines (Huh-7 and HepG2) after RBCEV-mediated delivery with and without pre- treatment with RBCEVs loaded with certain double-stranded oligonucleotides.
  • RBCEVs were loaded with DNA plasmid (CMV-copGFP), NF- ⁇ B decoy (ODN) oligonucleotides or NF- ⁇ B scrambled (SCD) oligonucleotides.
  • Cells were pre-conditioned with ODN or SCD oligonucleotides;
  • RBCEVs loaded with ODN or SCD were transfected to 50,000 Huh-7 and HepG2 cells on a 24-well plate format at oligonucleotide amounts of 25pmol (low dose) and 50pmol (high dose).
  • RBCEVs were separately loaded with NF- ⁇ B decoy (ODN) oligonucleotides, NF- ⁇ B scrambled (SCD) oligonucleotides, or simultaneously loaded with a DNA plasmid (CMV-copGFP) and NF- ⁇ B decoy (ODN) oligonucleotides.
  • Cells were pre- conditioned with ODN or SCD oligonucleotides; RBCEVs loaded with ODN or SCD were transfected to 50,000 HepG2 cells on a 24-well plate format at oligonucleotide amounts of 25pmol (low dose) and 50pmol (high dose).
  • Example 4 Exemplary double-stranded decoy oligonucleotide designs
  • the present Example provides exemplary designs of double-stranded decoy oligonucleotides (e.g., promoting oligonucleotides as described herein). These decoy oligonucleotides are designed to target endogenous proteins as described herein (e.g., endogenous proteins associated with nucleic acid sensing and/or inflammatory immune response).
  • decoy oligonucleotides are designed to target NF- ⁇ B, IRF3, IRF7, STAT3, or interferon-sensitive response element (ISRE).
  • Table 4 Exemplary representative double-stranded oligonucleotide designs. Highlighted in bold are the 11 binding sequences to the respective targets.
  • Nucleic acids of mammalian origin can act as endogenous ligands for Toll-like receptors and may promote systemic lupus erythematosus.
  • the Journal of experimental medicine, 202(8), pp.1131-1139. Bezzerri, V., Borgatti, M., Nicolis, E., Lampronti, I., Dechecchi, M.C., Mancini, I., Rizzotti, P., Gambari, R. and Cabrini, G., 2008.
  • Transcription factor oligodeoxynucleotides to NF- ⁇ B inhibit transcription of IL-8 in bronchial cells.
  • a decoy oligonucleotide to NF- ⁇ B delivered through inhalable particles prevents LPS-induced rat airway inflammation.
  • a nontoxic transduction enhancer enables highly efficient lentiviral transduction of primary murine T cells and hematopoietic stem cells. Molecular Therapy-Methods & Clinical Development, 10, pp.341-347.

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Abstract

L'invention concerne des compositions et des procédés d'utilisation concernant la délivrance d'acides nucléiques. L'invention concerne également des oligonucléotides de promotion qui améliorent, ou favorisent d'une autre manière, certaines caractéristiques de délivrance et/ou d'administration d'acides nucléiques. Dans certains modes de réalisation, les oligonucléotides de promotion améliorent ou augmentent une ou plusieurs caractéristiques d'expression et/ou d'activité d'un acide nucléique de charge utile. Dans certains modes de réalisation, les oligonucléotides de promotion inhibent ou réduisent une ou plusieurs caractéristiques d'un effet indésirable ou d'une réponse à un tel acide nucléique de charge utile. Dans certains modes de réalisation, les oligonucléotides de promotion améliorent ou augmentent la délivrance (p. ex. dans la délivrance de la charge excipient) d'acide(s) nucléique(s) de cargo.
PCT/US2023/017174 2022-03-31 2023-03-31 Co-administration d'acides nucléiques de charge utile et de promotion Ceased WO2023192624A2 (fr)

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US20220002730A1 (en) * 2018-08-24 2022-01-06 City Of Hope Oligonucleotide inhibitors of nuclear factor kappa-light-chain-enhancer of activated b cells and the uses thereof
US11970718B2 (en) * 2020-01-13 2024-04-30 Carmine Therapeutics Pte. Ltd. Nucleic acid loaded extracellular vesicles

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