WO2024229254A2 - Administration cellulaire d'agents thérapeutiques à l'aide de vésicules fusogènes - Google Patents

Administration cellulaire d'agents thérapeutiques à l'aide de vésicules fusogènes Download PDF

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WO2024229254A2
WO2024229254A2 PCT/US2024/027464 US2024027464W WO2024229254A2 WO 2024229254 A2 WO2024229254 A2 WO 2024229254A2 US 2024027464 W US2024027464 W US 2024027464W WO 2024229254 A2 WO2024229254 A2 WO 2024229254A2
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cell
cells
protein
fact
proteins
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WO2024229254A3 (fr
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Tim LUETKENS
Aneesh KARATT VELLATT
Andrea MARTOS ESTEBAN
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University of Maryland Baltimore
University of Maryland College Park
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University of Maryland College Park
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/10—Cellular immunotherapy characterised by the cell type used
    • A61K40/11—T-cells, e.g. tumour infiltrating lymphocytes [TIL] or regulatory T [Treg] cells; Lymphokine-activated killer [LAK] cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/30—Cellular immunotherapy characterised by the recombinant expression of specific molecules in the cells of the immune system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/40—Cellular immunotherapy characterised by antigens that are targeted or presented by cells of the immune system
    • A61K40/41—Vertebrate antigens
    • A61K40/42—Cancer antigens
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • 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
    • C12N5/0636—T lymphocytes
    • 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
    • A61K35/17—Lymphocytes; B-cells; T-cells; Natural killer cells; Interferon-activated or cytokine-activated lymphocytes
    • 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
    • C12N2510/00—Genetically modified cells
    • 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
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/10022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • 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
    • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
    • C12N2760/00011—Details
    • C12N2760/20011—Rhabdoviridae
    • C12N2760/20211—Vesiculovirus, e.g. vesicular stomatitis Indiana virus
    • C12N2760/20222—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes

Definitions

  • Biovesicles represent a range of extracellular vesicles (EV) including exosomes, microvesicles and virus like particles (VLPs) with the ability to encapsulate and be released as cargo-containing vesicles.
  • EV extracellular vesicles
  • VLPs virus like particles
  • High levels of BVs are tolerated by the human host [29] as these are naturally generated in the body and can deliver a wide variety of cargo such as small molecules, proteins, mRNAs and microRNAs.
  • EV- and BV-based therapeutics has also proven to be challenging especially for the treatment of chronic diseases.
  • VSV-G vesicular stomatitis virus
  • the present invention is directed to overcoming these hurdles and other important goals in the development of effective means for intracellular delivery of cargo, such as therapeutic molecules, to target cells.
  • the present invention is broadly directed to engineered cells that serve as a source of biovesicles (BVs) that can deliver cargo molecules, such as therapeutic molecules, to other cells in vivo or in vitro.
  • cargo molecules are shuttled to target cells within cell-derived fusogenic BVs, generated by the engineered source cell administered in vivo.
  • engineered cells serve as a source for production or manufacture of the cell-derived fusogenic BVs that can then be used in subsequent therapeutic administration.
  • Encapsulated cargo molecules include, but are not limited to, protein therapeutics as well as mRNA that encodes protein therapeutics that will be translated in target cells.
  • This invention can be applied to therapeutic applications (such as diseased cells or healthy cells acting as carriers) using therapeutic molecules as the cargo molecules and/or non-therapeutic applications (such as cell-based assays) using non- therapeutic molecules as the cargo molecules.
  • fusogenic BV-producing engineered cells will persist in patients over extended periods of time, acting as a constant source of the respective protein therapeutic, for example.
  • Such fusogenic BV-producing adoptive cell therapy combines durability of a cell therapy with the functional flexibility of EVs to deliver in v vo-produced payloads from engineered source cells to target receiving cells.
  • the FACT platform for the delivery of genetically encoded therapeutics by engineered cells represents a paradigm-shifting technology, potentially allowing for the treatment of many diseases and conditions.
  • BVs enables delivery of cargos, such as drugs, to intracellular, transmembrane, and secreted targets.
  • fusogenic BVs have the capacity to incorporate cargo molecules from a source cell, bud from the cell membrane of the source cell to the extracellular space and, upon fusing to the target cell membrane, release their cargo to the cytosol of the target cell.
  • in vivo generated fusogenic BVs comprising one or more therapeutic molecule(s) of interest as the cargo, such as a protein, can be used as protein replacement therapy to substitute absent or dysfunctional proteins or to deliver a protein or peptide therapeutic to modulate aberrant cellular pathways.
  • the present invention is directed to engineering FACT (fusogenic biovesicle-producing adoptive cell therapy) cells comprising (i) a polynucleotide sequence encoding one or more fusogens and/or proteins or peptides with cell penetrating properties, (ii) a polynucleotide sequence serving as or encoding one or more cargo molecules, and (iii) optionally a polynucleotide sequence encoding one or more gag-like proteins.
  • FACT fusogenic biovesicle-producing adoptive cell therapy
  • Each of the noted polynucleotide sequences can, independently of the other polynucleotide sequences, be a genomic (chromosomal) or non-genomic sequence.
  • Non-genomic sequences are extra-chromosomal sequences such as those of a plasmid or virus present in the FACT cells.
  • Suitable cargo molecules include, but are not limited to, DNA molecules, RNA molecules, peptides and proteins.
  • Exemplary peptides and proteins include, but are not limited to, enzymes such as gene-editing enzymes, including base editors, prime editors, TALE nucleases, and CRISPR/Cas, zine-finger nucleases.
  • Additional exemplary DNA molecules, RNA molecules, peptides and proteins are those reducing RNA and protein levels in target cells, including, but not limited to, small -interfering RNA, short-hairpin RNA, and proteins and peptides containing domains targeting molecules for proteasomal degradation.
  • Additional exemplary DNA molecules, RNA molecules, peptides and proteins include those correcting aberrant protein function, for example by altering protein conformation or trafficking, or providing wildtype protein function.
  • the FACT cells of the invention produce and release fusogenic BVs comprising the one or more cargo molecules and the optional one or more gag-like proteins.
  • the cargo molecules are therapeutic molecules.
  • the invention is directed to fusogenic BVs comprising (i) one or more fusogens and/or proteins with cell penetrating properties, (ii) one or more cargo molecules, and (iii) optionally one or more gag-like proteins.
  • the fusogenic BVs themselves are constructed of (i) the fusogens or proteins with cell penetrating properties, and (ii) the gag-like proteins, when present, to form a structure.
  • the cargo molecules are present inside of the structure and thus carried within the fusogenic BVs.
  • the cargo molecules are therapeutic molecules. Suitable therapeutic molecules include, but are not limited to, DNA molecules, RNA molecules, peptides and proteins.
  • Exemplary peptides and proteins include, but are not limited to, enzymes such as gene-editing enzymes, including base editors, prime editors, TALE nucleases, and CRISPR/Cas, zinc-finger nucleases.
  • Additional exemplary DNA molecules, RNA molecules, peptides and proteins are those reducing RNA and protein levels in target cells, including, but not limited to, small-interfering RNA, short-hairpin RNA, and proteins and peptides containing domains targeting molecules for proteasomal degradation.
  • Additional exemplary DNA molecules, RNA molecules, peptides and proteins include those correcting aberrant protein function, for example by altering protein conformation or trafficking, or providing wildtype protein function. Such genetically-encoded therapeutics can be used in the treatment of selected diseases and conditions.
  • the invention is directed to methods for delivering one or more cargo molecules to a target cell, comprising culturing a target cell with a fusogenic BV of the present invention under conditions promoting delivery of a cargo molecule from the fusogenic BV to the target cell. Such delivery is via binding of the fusogenic BVs to the target cell and release of the cargo molecules into the target cell.
  • the cargo molecules are therapeutic molecules.
  • the therapeutic molecules carried by the fusogenic BVs may be, but are not limited to, DNA molecules, RNA molecules, peptides or proteins that are therapeutic for a selected disease or condition.
  • Exemplary peptides and proteins include, but are not limited to, enzymes such as gene-editing enzymes, including base editors, prime editors, TALE nucleases, and CRISPR/Cas, zine-finger nucleases.
  • Additional exemplary DNA molecules, RNA molecules, peptides and proteins are those reducing RNA and protein levels in target cells, including, but not limited to, small-interfering RNA, short-hairpin RNA, and proteins and peptides containing domains targeting molecules for proteasomal degradation.
  • Additional exemplary DNA molecules, RNA molecules, peptides and proteins include those correcting aberrant protein function, for example by altering protein conformation or trafficking, or providing wildtype protein function.
  • the invention is directed to methods for delivering one or more cargo molecules to a target cell, comprising culturing a target cell with a FACT cell of the present invention under conditions promoting delivery of a cargo molecule from the FACT cell to the target cell.
  • Such delivery is via fusogenic BVs that are released from the FACT cells and that subsequently bind to the target cell.
  • the cargo molecules are therapeutic molecules.
  • the therapeutic molecules carried by the fusogenic BVs may be, but are not limited to, DNA molecules, RNA molecules, peptides or proteins that are therapeutic for a selected disease or condition.
  • Exemplary peptides and proteins include, but are not limited to, enzymes such as geneediting enzymes, including base editors, prime editors, TALE nucleases, and CRISPR/Cas, zinc- finger nucleases.
  • Additional exemplary DNA molecules, RNA molecules, peptides and proteins are those reducing RNA and protein levels in target cells, including, but not limited to, small-interfering RNA, short-hairpin RNA, and proteins and peptides containing domains targeting molecules for proteasomal degradation.
  • Additional exemplary DNA molecules, RNA molecules, peptides and proteins include those correcting aberrant protein function, for example by altering protein conformation or trafficking, or providing wildtype protein function.
  • the invention is directed to methods for delivering one or more cargo molecules to a target cell of a subject, comprising administering to a subject in need thereof a population of FACT cells of the present invention.
  • FACT cells Upon administration of the FACT cells to the subject, fusogenic BVs are released from the FACT cells and subsequently bind to target cells of the subject. Cargo molecules are then released into the target cells.
  • cargo molecules encompassed by the fusogenic BVs are DNA molecules, RNA molecules, peptides or proteins.
  • Exemplary peptides and proteins include, but are not limited to, enzymes such as gene-editing enzymes, including base editors, prime editors, TALE nucleases, and CRISPR/Cas, zinc-finger nucleases.
  • Additional exemplary DNA molecules, RNA molecules, peptides and proteins are those reducing RNA and protein levels in target cells, including, but not limited to, small-interfering RNA, short-hairpin RNA, and proteins and peptides containing domains targeting molecules for proteasomal degradation.
  • Additional exemplary DNA molecules, RNA molecules, peptides and proteins include those correcting aberrant protein function, for example by altering protein conformation or trafficking, or providing wildtype protein function.
  • the invention is directed to methods for treating a disease or condition in a subject, comprising administering to a subject in need thereof a population of FACT cells of the present invention.
  • FACT cells Upon administration of the FACT cells to the subject, fusogenic BVs are released from the FACT cells and subsequently bind to target cells of the subject.
  • Therapeutic molecules are then released into the target cells.
  • therapeutic molecules encompassed by the fusogenic BVs are DNA molecules, RNA molecules, peptides or proteins that are therapeutic for the disease or condition.
  • Exemplary peptides and proteins include, but are not limited to, enzymes such as gene-editing enzymes, including base editors, prime editors, TALE nucleases, and CRISPR/Cas, zinc-finger nucleases.
  • Additional exemplary DNA molecules, RNA molecules, peptides and proteins are those reducing RNA and protein levels in target cells, including, but not limited to, small-interfering RNA, short-hairpin RNA, and proteins and peptides containing domains targeting molecules for proteasomal degradation.
  • Additional exemplary DNA molecules, RNA molecules, peptides and proteins include those correcting aberrant protein function, for example by altering protein conformation or trafficking, or providing wildtype protein function.
  • the invention is directed to methods for delivering fusogenic BVs carrying therapeutic molecules to a target cell, comprising culturing a target cell with a population of fusogenic BVs carrying therapeutic molecules under conditions promoting fusion of fusogenic BVs to the target cell.
  • therapeutic molecules carried by the fusogenic BVs are DNA molecules, RNA molecules, peptides or proteins that are therapeutic for a selected disease or condition.
  • Exemplary peptides and proteins include, but are not limited to, enzymes such as gene-editing enzymes, including base editors, prime editors, TALE nucleases, and CRISPR/Cas, zinc-finger nucleases.
  • Additional exemplary DNA molecules, RNA molecules, peptides and proteins are those reducing RNA and protein levels in target cells, including, but not limited to, small-interfering RNA, short-hairpin RNA, and proteins and peptides containing domains targeting molecules for proteasomal degradation. Additional exemplary DNA molecules, RNA molecules, peptides and proteins include those correcting aberrant protein function, for example by altering protein conformation or trafficking, or providing wildtype protein function.
  • the invention is directed to methods for treating a disease or condition in a subject, comprising administering to a subject in need thereof a population of fusogenic BVs carrying therapeutic molecules, wherein the therapeutic molecules are DNA molecules, RNA molecules, peptides or proteins that are therapeutic for a selected disease or condition.
  • Exemplary peptides and proteins include, but are not limited to, enzymes such as gene-editing enzymes, including base editors, prime editors, TALE nucleases, and CRISPR/Cas, zinc-finger nucleases.
  • Additional exemplary DNA molecules, RNA molecules, peptides and proteins are those reducing RNA and protein levels in target cells, including, but not limited to, small-interfering RNA, short-hairpin RNA, and proteins and peptides containing domains targeting molecules for proteasomal degradation. Additional exemplary DNA molecules, RNA molecules, peptides and proteins include those correcting aberrant protein function, for example by altering protein conformation or trafficking, or providing wildtype protein function.
  • the invention is directed to methods for producing fusogenic BVs comprising culturing FACT cells of the present invention under conditions promoting production and release of fusogenic BVs from the FACT cells.
  • the fusogen or protein with cell penetrating properties may be encoded in a viral genome, in the human genome, or the genome of a non-human mammal.
  • the fusogen or protein with cell penetrating properties may be a viral fusogen, for example VSV-G, mammalian syncytins such as syncytin A (SynA), including human SynA (hSynA), human endogenous retrovirus K (HERV-K) envelope, or functional variants thereof.
  • Such functional variants retain the activity of the protein upon which they are based.
  • the biovesicles (BVs) comprising the fusogens and/or proteins with cell penetrating properties may be fully human BVs.
  • the BVs may comprise one or more elements that are not fully human in origin.
  • the BVs may comprise non-human mammalian fusogens with reduced propensity for immunogenicity in humans, or fusogens that have been humanized, i.e. engineered to be less immunogenic (e.g. mouse SynA) to suit in vivo or chronic dosing.
  • the fusogenic BVs of the invention may be defined as poorly immunogenic in a subject, such as a human.
  • the fusogenic BVs of the invention may be fully human fusogenic BVs.
  • the fusogenic BVs of the invention may be described as fusogenic BVs having low or poor immunogenicity. Such low or poor immunogenicity refers to the immunogenicity of the fusogenic BVs when administered to a subject, such as a human.
  • the FACT cell may be a T cell, a monocyte, a megakaryocyte, a neuron, an epithelial cell, such as lung epithelial cells, or a stem cell.
  • the subject may be suffering from a disease or condition such as, but not limited to, a chronic neurodegenerative disease, such as amyotrophic lateral sclerosis, Alzheimer’s disease, or cancer.
  • a chronic neurodegenerative disease such as amyotrophic lateral sclerosis, Alzheimer’s disease, or cancer.
  • the gag-like protein may be encoded in the human genome or the genome of a non-human mammal.
  • the gag-like protein may be a viral gag protein, for example human Arc (hArc) or PEG10, or functional variants thereof.
  • FIG. 1 Transfer of genetically-encoded payload by engineered FACT cell to target cells.
  • A Schema of experiment set up using a luciferase complementation assay. FACT cells engineered to express the C-terminal fragment of NanoLuc were transfected with different fusogen expression constructs and then co-cultured with target cells expressing the N-terminal NanoLuc fragment. Payload transfer was determined by luminescence following NanoLuc complementation.
  • B Luminescence signal from co-cultures containing genetically engineered 293T-based FACT cells expressing the C-terminal NanoLuc fragment and a fusogen as well SW480 cells expressing the N- terminal NanoLuc fragment.
  • FIG. 2 Payload transfer by fusogenic biovesicles expressing the HERV-K envelope.
  • FIG. 3 Effect of fusogen expression on cell manufacturing.
  • A mCherry fluorescence (top) and bright-field (bottom) microscopy images of 293T cells transiently transfected with an mCherry payload plasmid and one of the indicated fusogens or a GFP expression plasmid as a negative control at 100-fold magnification.
  • White arrows indicate syncytia.
  • B Number of 293 T cells 48 h after transfection with the indicated fusogens or a GFP expression plasmid as a negative control as determined by automated cell counter.
  • FIG. 4 Contribution of gag-like proteins on payload transfer by fusogenic biovesicles.
  • A Schema of co-culture experiment using FACT cells transiently transfected with payload, hSYNA, and gag-like protein PEG10 or hArc. mCherry Payload transfer to CellTrace dye Far Redpositive target cells was determined by flow cytometry.
  • B Mean fluorescence intensity (MFI) of mCherry in target cells following co-culture with FACT cells transfected with mCherry, hSYNA and the indicated gag-like proteins as determined by flow cytometry.
  • MFI Mean fluorescence intensity
  • FIG. 5 Expression of fluorescent reporter proteins co-expressed with fusogen and payload in Jurkat cells engineered using Sleeping Beauty transposase as determined by flow cytometry. The percentage of double positive cells is indicated in each panel.
  • FIG. 6. Viability of Jurkat cells engineered to express the indicated fusogens as determined by trypan blue staining and automated cell counting.
  • FIG. 7. Transfer of C-terminal luciferase fragment payload by Jurkat cells engineered to express the indicated fusogens. Engineered Jurkat cells and SW480 cells expressing the N-terminal luciferase fragment were co-cultured at an effector-target ratio of 5: 1 for 24h before addition of furimazine. Luminescence was determine using a Spark multi-mode plate reader (Tecan).
  • FIG. 8 Transfer of C-terminal luciferase fragment payload using conditioned supernatants collected after 24h from Jurkat cells engineered to express the indicated fusogens. Conditioned supernatants and SW480 cells expressing the N-terminal luciferase fragment were incubated for 24h before addition of furimazine. Luminescence was determined using a Spark multimode plate reader (Tecan).
  • FIG. 9. Validation of hSYNA expression in engineered human cells.
  • HSYNA-HA expression as determined by western blot after probing with an anti-HA, anti-TDP-43 or an anti-J3- actin (ACTB) antibody in 293T cells transiently transfected with hSYNA-HA expression construct.
  • FIG. 10. Cell-to-cell delivery of a gene-editing enzyme using hSYNA.
  • A Schema of construct used to detect Cre activity in target cells in the absence of Cre (left) and after Cre- mediated excision (right). Black triangles indicate LoxP sites.
  • FIG. 11 Delivery of gene-editing enzyme using hSYNA-based fusogenic extracellular vesicles. Conversion of EpCAM+ SW480 cells to eGFP following Cre-mediated excision of LoxP cassette after 48 h incubation with supernatants from FACTcre cells and tamoxifen as determined by flow cytometry.
  • FIG. 12 Degradation of mutant TDP-43 using FACT cells.
  • 293T cells stably engineered to express wildtype TDP-43 (TDP-43wt) or mutant TDP-43 (TDP-43ci73s ci75s) fused to GFP under control of a tetracycline-inducible promoter were incubated with 293T cells expressing hSYNA alone or together with a TDP-43 -specific single-chain variable fragment (3B12A (SEQ ID NO: 7) or Vh7Vk9 (SEQ ID NO: 6)).
  • TDP-43wt wildtype TDP-43
  • TDP-43ci73s ci75s mutant TDP-43 fused to GFP under control of a tetracycline-inducible promoter
  • FIG. 13 Base editor-mediated correction of mutation using FACT technology.
  • FIG. 14 Construct Map. pSBbi-RP-3B12A-hSYNA (FACT open-reading frame; SEQ ID NO: 16)).
  • FIG. 15 Construct Map. pSBbi-RP-Vh7Vk9-hSYNA (FACT open-reading frame; SEQ ID NO: 17)).
  • FIG. 16 Construct Map. pTwist-CMV-neo-hSYNA (polynucleotide sequence set forth in SEQ ID NO: 18)).
  • “about” refers to a numeric value, including, for example, whole numbers, fractions, and percentages, whether or not explicitly indicated.
  • the term “about” generally refers to a range of numerical values (e.g., +/- 5-10% of the recited value) that one of ordinary skill in the art would consider equivalent to the recited value (e.g., having the same function or result). In some instances, the term “about” may include numerical values that are rounded to the nearest significant figure.
  • HVLPs human virus-like particles
  • gag-like protein Arc is able to form capsids and shuttles mRNA between neurons as well as human embryonic kidney cells overexpressing Arc [1,2].
  • Arc-based HVLPs are pseudotyped via endogenous fusogens present in these cells or whether Arc-based capsids are able to fuse with target cells and deliver their payload without a fusogen. This is important because tropism of retroviruses is to a large extent determined by fusogens and pseudotyping may augment transduction efficiency and increase tissue specificity.
  • BVs fusogenic biovesicles
  • FACT fusogenic biovesicle-producing adoptive cell therapy
  • This platform is highly flexible, permitting the targeting of various cell types, including neurons, immune cells, and epithelial cells, and allowing delivery of a wide range of genetically-encoded cargo molecules.
  • gag-like proteins such as human Arc and PEG10
  • FACT cells a genetically-encoded cargo molecules carried by FACT cells to target cells for expression therein and, when the cargo molecules are therapeutic molecules, the concomitant treatment of diseases and conditions, including various currently incurable diseases.
  • the FACT approach is made possible by the use of genetically engineered cells expressing fusogenic proteins or peptides that are poorly immunogenic in the subject to which they are administered.
  • the genetically engineered cells express fully human or humanised or deimmunised fusogenic proteins or peptides, such as those contained in the human genome, that allow the stable production of fully human fusogenic biovesicles or fusogenic BVs having low or poor immunogenicity that can be used to shuttle custom, genetically-encoded therapeutic payloads into neighbouring cells (Fig. 1).
  • fusogenic proteins are naturally and spontaneously expressed in different healthy human tissues, they may carry limited immunogenicity which may allow long-term persistence of engineered cells expressing these proteins.
  • Suitable proteins and peptides include those from closely-related mammals.
  • the FACT platform is based on the production of cells (herein “FACT cells”) that produce the elements required for delivery of therapeutic molecules to target cells.
  • FACT cells include (i) fusogens or proteins with cell penetrating properties, and in some cases (ii) gag-like proteins.
  • the FACT cells of the invention encode (i) one or more fusogens or proteins with cell penetrating properties, (ii) one or more genetically encoded cargo molecules, such as therapeutic molecules, and optionally (iii) one or more gag-like proteins.
  • fusogenic proteins or peptides that are “poorly immunogenic” are proteins and peptides that either do not induce an immune response or induce an immune response with undetectable or mild symptoms in a subject that allows cells expressing the fusogenic proteins or peptides to persist in the subject rather than being targeted by the immune system for immediate eradication.
  • fusogenic BVs having “low or poor immunogenicity” are fusogenic BVs that either do not induce an immune response or induce an immune response with undetectable or mild symptoms in a subject that allows the fusogenic BVs to persist in the subject rather than being targeted by the immune system for immediate eradication.
  • Fusogens also known as fusion proteins, are proteins that allow membranes to fuse by overcoming the repulsion between membranes. Viruses use fusogens to efficiently merge with their target cells and deliver their payload. Viruses encode their own fusogens, such as the vesicular stomatitis virus (VSV) G fusion protein, which has broad tropism and is widely used in the production of recombinant viruses for therapeutic applications.
  • VSV vesicular stomatitis virus
  • viral envelope proteins including fusogens that are expressed on the surface of host cells prior to viral budding may serve as a key target of antibody-mediated immunity. Therefore, careful thought is required in selecting suitable fusogens for use in the embodiments of the present invention.
  • Fusogens that may be used in the FACT cells of the present invention include endogenous human or other mammalian retroviral fusogens, such as human endogenous retrovirus K (HERV-K), syncytin A (SynA) (e.g. human SynA), baboon retroviral envelope glycoprotein (BaEV), and the endogenous retroviral envelope protein EnVP(b)l . These proteins are likely to exhibit reduced propensity for immunogenicity [26], Commonly used non-human or viral fusogens such as fusogens from vesicular stomatitis virus (VSV) may be used as a tool for optimizing different components of FACT system or as a control.
  • VSV vesicular stomatitis virus
  • fusogens from one species may be used in the preparation of BVs from another species.
  • suitable fusogens may be non-human fusogens that have been humanized or deimmunised, i.e. engineered to be less immunogenic in a human.
  • Suitable fusogens include those from closely-related mammals.
  • other proteins and peptides having cell penetrating properties may be used in the embodiments of the invention, either in place of traditional fusogens, such as those mentioned above, or in addition to these fusogens.
  • Suitable cell-penetrating proteins and peptides encompassed within the scope of the invention include, but are not limited to:
  • RALA WEARLARALARALARHLARALARALRACEA; SEQ ID NO: 8;
  • PF14 (Stearyl-AGYLLGKLLXXLAAAALXXLL, where X is ornithine; SEQ ID NO: 9);
  • GALA WEAALAEALAEALAEHLAEALAEALEALAA; SEQ ID NO: 12;
  • LAH4-L1 (KKALLAHALHLLALLALHLAHALKKA; SEQ ID NO: 13);
  • KL4 (KLLLLKLLLLKLLLLKLLLLKLLLLLLKLLLLK; SEQ ID NO: 14);
  • OligoArgAib (RRXRRXRRXRRXRRXRRX, where X is a-aminoisobutyric acid (Aib); SEQ ID NO: 15)
  • proteins can be engineered to have cell penetrating properties.
  • Such engineered proteins are encompassed within the scope of the invention.
  • cell-permeable proteins may be engineered by genetically grafting a short cell-penetrating peptide (CPP) to an exposed loop of a protein of interest.
  • CPP cell-penetrating peptide
  • Functional variants of the fusogens and proteins with cell penetrating properties defined above may also be used in the various aspects and embodiments of the invention. These functional variants will retain the activity of the protein upon which they are based, but have at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with the protein upon which they are based.
  • the FACT cells can be engineered to either transiently or stably express the fusogens.
  • transient expression actual mRNA or recombinant proteins, or any plasmids containing promoters active in eukaryotic cells, such as CMV, EF1A, PGK, and SFFV may be used.
  • transposon-based plasmids together with a transposase, or adeno/retroviral transfer plasmids using the same type of promoters or promoters contained within the long-terminal repeats of the viral vectors may be used.
  • Suitable means for preparing FACT cells expressing the proteins include, but are not limited to, calcium phosphate, lipofection, nucleofection, electroporation, adeno-/retroviral transduction.
  • the same plasmid/vector can encode both the gag proteins and the fusogens, when gag proteins are present.
  • the fusogens and proteins having cell penetrating properties can be genomic (chromosomal) or non-genomic sequences.
  • Non-genomic sequences are extra- chromosomal sequences such as those of a plasmid or virus present in the FACT cells.
  • the present invention also encompassed fusogenic BVs comprising (i) one or more fusogens or proteins with cell penetrating properties, (ii) one or more cargo molecules, and (iii) optionally one or more gag-like proteins.
  • fusogenic BVs themselves are constructed of (i) the fusogens or proteins with cell penetrating properties, and (ii) the gag-like proteins, when present, to form a structure.
  • the cargo molecules are present inside of the structure and thus carried within the fusogenic BVs.
  • the fusogenic BVs may be fully human fusogenic BVs.
  • the BVs may comprise one or more elements that are not fully human in origin.
  • the BVs may comprise non-human mammalian fusogens with reduced propensity for immunogenicity in humans, or fusogens that have been humanized, i.e. engineered to be less immunogenic (e.g. mouse SynA) to suit in vivo or chronic dosing.
  • the fusogenic BVs of the invention may be described as fully human fusogenic BVs.
  • the fusogenic BVs of the invention may be described as fusogenic BVs having low or poor immunogenicity.
  • Such low or poor immunogenicity refers to the immunogenicity of the fusogenic BVs when administered to a subject, such as a human.
  • the term “fully human” refers to peptides and proteins that are encoded by the human (Homo sapiens) genome (e.g. human SynA), and biovesicles that only comprise and/or contain peptides, proteins and other molecules that are encoded by the human genome, derived from the human body, or produced by the human body.
  • human Homo sapiens
  • biovesicles that only comprise and/or contain peptides, proteins and other molecules that are encoded by the human genome, derived from the human body, or produced by the human body.
  • the fusogenic biovesicles produced by the FACT cells of the invention shuttle cargo molecules to target (receiving) cells.
  • the cargo molecules will typically be therapeutic molecules.
  • the cargo molecules may also have other functions, such as a signalling function if the cargo molecule is used in the context of a cell-based assay system.
  • the FACT cells can be engineered to either transiently or stably express the genetically encoded cargo molecules, such as DNA molecules, RNA molecules, peptides or proteins.
  • Exemplary peptides and proteins include, but are not limited to, enzymes such as gene-editing enzymes, including base editors, prime editors, TALE nucleases, and CRISPR/Cas, zine-finger nucleases.
  • Base editors are engineered proteins that are able to alter individual nucleotides at defined positions in a cell’s genome without introducing double-strand breaks. BEs include, but are not limited to, cytosine and adenine base editors.
  • Additional exemplary DNA molecules, RNA molecules, peptides and proteins are those reducing RNA and protein levels in target cells, including, but not limited to, small-interfering RNA, short-hairpin RNA, and proteins and peptides containing domains targeting molecules for proteasomal degradation. Additional exemplary DNA molecules, RNA molecules, peptides and proteins include those correcting aberrant protein function, for example by altering protein conformation or trafficking, or providing wildtype protein function. [0075] For transient expression, DNA, RNA, recombinant peptides or recombinant proteins, or any plasmids containing promoters active in eukaryotic cells, such as CMV, EF1A, PGK, SFFV, U6 may be used.
  • transposon-based plasmids together with a transposase, or adeno/retroviral transfer plasmids using the same type of promoters or promoters contained within the long-terminal repeats of the viral vectors may be used.
  • a suitable plasmid for both transient and stable expression of cargo molecules is, for example, pSBBi-RP, a Sleeping Beauty plasmid.
  • Suitable means for preparing FACT cells expressing the cargo molecules include calcium phosphate, lipofection, nucleofection, electroporation, adeno-/retroviral transduction.
  • the identity of the cargo molecules that makes up the payload can vary widely and is not limited to DNA, RNA, peptides or specific proteins themselves.
  • gag-like proteins that may be expressed by the FACT cells of the invention are retroviral gag-like proteins that are endogenous proteins encoded by the human genome or genome of other mammals.
  • PEG10 is a suitable gag-like protein for use in the FACT cells of the invention.
  • PEG10 contains not only a gag-like sequence consisting of a capsid and nucleocapsid polypeptide but also a partial pol segment, likely encoding a protease (PR) and reverse transcriptase (RT).
  • PR and RT domains may be deleted from the PEG10 ORF. It has been shown that the RT domain is not required for efficient cargo transport.
  • the human Arc gene also encodes a gag-like capsid protein that may be used to increase cargo transfer by fusogenic BVs.
  • gag-like proteins that can be expressed by FACT cells include, but are not limited to, Pnmal, Pnma3, Pnma5, Pnma6a, Pnma8, Asprvl, Moapl, Zcchcl2, and Rtll.
  • Functional variants of the gag-like proteins defined above may also be used in the various aspects and embodiments of the invention. These functional variants will retain the activity of the protein upon which they are based, but have at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with the protein upon which they are based.
  • the FACT cells can be engineered to either transiently or stably express the gag-like proteins.
  • actual mRNA or recombinant proteins, or any plasmids containing promoters active in eukaryotic cells, such as CMV, EF1A, PGK, SFFV may be used.
  • transposon-based plasmids together with a transposase, or adeno/retroviral transfer plasmids using the same type of promoters or promoters contained within the long-terminal repeats of the viral vectors may be used.
  • Suitable means for preparing FACT cells expressing the proteins include calcium phosphate, lipofection, nucleofection, electroporation, adeno-/retroviral transduction.
  • the FACT cells of the invention may use any cell type that will produce fusogenic biovesicles that can carry selected cargo molecules, such as proteins or RNA.
  • Suitable cell types include, but are not limited to, cells of the immune system, such as T cells, natural killer cells and monocytes; megakaryocytes; neurons; epithelial cells, such as lung epithelial cells; and stem cells.
  • An exemplary cell type is T cells.
  • the FACT cells of the invention can be used, inter alia, in the treatment of certain diseases and conditions.
  • diseases include, but are not limited to, chronic neurodegenerative diseases, such as amyotrophic lateral sclerosis and Alzheimer’s disease, cancer, mitochondrial diseases, lysosomal deficiencies, and genetic diseases resulting from loss of heterozygosity.
  • a population of FACT cells can be formulated for administration to a subject, such as a human subject, as a pharmaceutical composition.
  • pharmaceutical compositions will comprise one or more populations of FACT cells and a suitable carrier or excipient.
  • the fusogenic BVs of the invention can also be formulated for administration to a subject, such as a human subject, as a pharmaceutical composition.
  • a pharmaceutical composition will comprise one or more populations of fusogenic BVs and a suitable carrier or excipient.
  • compositions of the present invention may be formulated, for example, for oral, sublingual, intranasal, intraocular, rectal, transdermal, mucosal, pulmonary, topical or parenteral administration.
  • Parenteral modes of administration include without limitation, intradermal, subcutaneous (s.c., s.q., sub-Q, Hypo), intramuscular (i.m.), intravenous (i.v.), intraperitoneal (i.p ), intra-arterial, intramedulary, intracardiac, intra-articular (joint), intrasynovial (joint fluid area), intracranial, intraspinal, and intrathecal (spinal fluids). Any known device useful for parenteral injection or infusion of pharmaceutical compositions can be used to effect such administration.
  • the dosage may be administered all at once, such as with an oral formulation in a capsule or liquid, or slowly over a period of time, such as with an intramuscular or intravenous administration.
  • Administration frequencies for the pharmaceutical compositions of the present invention include 4, 3, 2 or once daily, every other day, every third day, every fourth day, every fifth day, every sixth day, once weekly, every eight days, every nine days, every ten days, bi-weekly, monthly, and bi-monthly. The duration of treatment will be based on the condition being treated and will be best determined by the attending physician.
  • the present invention includes methods for utilizing the fusogenic BVs and FACT cells defined herein.
  • the invention includes methods for delivering one or more cargo molecules to a target cell, comprising culturing a target cell with a fusogenic BV of the present invention under conditions promoting delivery of a cargo molecule from the fusogenic BV to the target cell.
  • Such delivery is via binding of the fusogenic BVs to the target cell and release of the cargo molecules into the target cell.
  • the invention also includes methods for delivering one or more cargo molecules to a target cell, comprising culturing a target cell with a FACT cell of the present invention under conditions promoting delivery of a cargo molecule from the FACT cell to the target cell. Such delivery is via fusogenic BVs that are released from the FACT cells and that subsequently bind to the target cell.
  • the invention further includes methods for delivering one or more cargo molecules to a target cell of a subject, comprising administering to a subject in need thereof a population of FACT cells of the present invention.
  • FACT cells Upon administration of the FACT cells to the subject, fusogenic BVs are released from the FACT cells and subsequently bind to target cells of the subject. Cargo molecules are then released into the target cells.
  • the invention includes methods for treating a disease or condition in a subject, comprising administering to a subject in need thereof a population of FACT cells of the present invention.
  • a population of FACT cells of the present invention Upon administration of the FACT cells to the subject, fusogenic BVs are released from the FACT cells and subsequently bind to target cells of the subject. Therapeutic molecules are then released into the target cells.
  • the invention includes methods for delivering fusogenic BVs carrying therapeutic molecules to a target cell, comprising culturing a target cell with a population of fusogenic BVs carrying therapeutic molecules under conditions promoting fusion of fusogenic BVs to the target cell.
  • the invention also includes methods for treating a disease or condition in a subject, comprising administering to a subject in need thereof a population of fusogenic BVs carrying therapeutic molecules, wherein the therapeutic molecules are RNA molecules, peptides or proteins that are therapeutic for a selected disease or condition.
  • the invention further includes methods for producing fusogenic BVs comprising culturing FACT cells of the present invention under conditions promoting production and release of fusogenic BVs from the FACT cells.
  • the cargo molecules may be therapeutic molecules.
  • the cargo molecules may be, but are not limited to, RNA molecules, peptides or proteins as defined above.
  • the therapeutic molecules may be, but are not limited to, RNA molecules, peptides or proteins as defined above that are therapeutic for a selected disease or condition
  • the subject is any vertebrate animal including, but not limited to, human, non-human primate, bird, horse, cow, goat, sheep, a companion animal, such as a dog, cat or rodent, or other mammal.
  • Example 1 Cells producing fully human BVs are an effective approach for the intercellular transfer of genetically-encoded cargo
  • Persistent delivery of genetically-encoded therapeutics using cells engineered to produce fusogenic biovesicles comprising endogenous human fusogens may be an effective approach to treating disease.
  • a new assay was developed to measure the cytosolic delivery of a genetically -encoded payload using a luciferase complementation system (Fig. 1A).
  • a luciferase-complementation assay was developed using a previously described split NanoLuc pair [32], with the C-terminal luciferase fragment stably expressed in the FACT cells and the N-terminal fragment stably expressed in the target cells.
  • an N-terminal NanoLuc (nLuc) overexpressing colorectal cancer cell line SW480 was generated via lentiviral transduction using the LeGO system [35] followed by fluorescence-activated cell sorting on a FACSaria cell sorter (BD).
  • BD FACSaria cell sorter
  • 5xl0 5 FACT cells were transiently transfected with the human fusogen syncytin A (hSYNA, SEQ ID NO: 1) using the construct shown in Fig. 16 or the established fusogen vesicular stomatitis virus G (VSV-G) envelope (control) in a 24-well plate following the manufacturer’s instructions.
  • FACT and target cells were washed twice with 1 ml PBS and cLuc-expressing FACT cells transfected with fusogen constructs were subsequently co-cultured with nLuc-expressing SW480 cells at an effector-target ratio of 5:1.
  • cells were transferred to a black non-binding plate (Greiner) and furimazine substrate (Promega) was added according to the manufacturer’s instructions.
  • Luminescence signal was read on a Spark multi-mode plate reader (Tecan) after a 5 min incubation at 37°C.
  • Example 2 Fully human fusogenic biovesicles can be generated using the HERV-K envelope
  • HERV-K human endogenous retrovirus K
  • HERV-K is the most recently identified endogenized human retrovirus [36] and its envelope protein has previously been suggested to have potentially broad tropism [37], It was therefore hypothesized that it may represent a promising alternative to syncytins in the context of the engineering of fusogenic biovesicles.
  • FACT cells were generated expressing mCherry as the payload and HERV-K (SEQ ID NO 2) as the fusogen. Specifically, 70-80% confluent 293T cells were transiently transfected in T75 flasks with an mCherry expression plasmid and an HERV-K expression plasmid, both in the pcDNA3.4 backbone, using Lipofectamine 2000. After 24 h, the 293T cells were washed with PBS and fresh complete culture medium was added.
  • the supernatants were harvested and filtered using a 0.45 pm polyethersulfone filter (MilliporeSigma) and the filtered supernatants were concentrated 10-fold using commercially available Retro-X concentrator solution (Takara).
  • Retro-X concentrator solution Takara
  • wells of a 24-well tissue culture plate were coated with 10 pg/ml retronectin (Takara), the wells were blocked with 2% bovine serum albumin (BSA), and concentrated supernatants were immobilized on retronectin-coated plates by centrifugation according to the manufacturer’s instructions.
  • BSA bovine serum albumin
  • 5xlO 5 untransfected 293T cells were added to wells coated with fusogenic vesicles and incubated for 24 h at 37°C/5% CO2. After 24 h, cells were harvested by trypsinization, filtered through a 70 pm cell strainer (BD), and analyzed on an LSR II flow cytometer (BD).
  • HERV-K envelope proteins substantially increased transfer of the mCherry payload to target cells (Fig. 2), demonstrating that the HERV-K envelope represents an alternative to syncytins for the generation of fusogenic vesicles as a standalone drug or when delivered by engineered FACT cells.
  • Fusogenic proteins are able to overcome the repulsion between plasma cell membranes and actively facilitate the fusion of multiple cells or a cell with a fusogenic biovesicle.
  • the functionality of FACT cells relies on the expression of individual fusogenic proteins and it was hypothesized that expression of some fusogens may cause fusion not only by the fusogenic vesicles to target cells but also between the engineered FACT cells and can lead to reduced FACT cell viability and production efficiency.
  • 293T cells were plated at 5xl0 5 cells/well in a 24-well plate and transiently transfected with expression constructs for hSYNA, mouse syncytin A (mSYNA, SEQ ID NO: 3), HERV-K, VSV-G (control) or GFP (green fluorescent protein) as a negative control together with an mCherry payload plasmid using Lipofectamine 2000 according to the manufacturer’s instructions. After 24 h, the cells were analyzed by fluorescence and bright field microscopy, and the remaining cells were counted following trypsinization using an automated cell counter (ThermoFisher).
  • Example 4 Addition of gag-like proteins to fusogenic biovesicles can enhance cargo transfer
  • fusogenic proteins are sufficient for the transfer of payloads, it may be possible to increase the transfer efficiency of fusogenic biovesicles in some cases.
  • Conventional retroviruses use gag proteins to specifically load and encapsulate their viral payloads. Therefore, experiments were also conducted to analyze whether addition of fully human gag-like proteins, such as Paternally Expressed- 10 (PEG10) and Activity Regulated Cytoskeleton Associated Protein (hArc) encoded in the human genome are able to further increase payload transfer efficiency using the human fusogen hSYNA.
  • PEG10 Paternally Expressed- 10
  • hArc Activity Regulated Cytoskeleton Associated Protein
  • FACT cells were washed twice with 1 ml PBS and untransfected 293T target cells were stained with CellTrace dye Far Red (ThermoFisher). FACT cells were co-cultured with 5xlO 5 target cells at an effector-target ratio of 5: 1 for 24 h at 37°C/5% CO2. After 24 h, cells were harvested by trypsinization, filtered through a 70 pm cell strainer (BD), and analyzed on an LSR II flow cytometer (BD). Target cells were identified by CellTrace dye Far red positivity.
  • T cells can provide an ideal vehicle for the delivery of therapeutic payloads, due to their long-term persistence and demonstrated clinical safety.
  • T cells can be engineered to overexpress fusogenic biovesicles (BVs) that can transfer payloads to relevant target cells.
  • BVs fusogenic biovesicles
  • the potential impact of fusogen expression on T cell viability was also unknown.
  • Fig. 5 a transposase-based gene transfer to express the C-terminal NanoLuc fragment together with human Arc and the fusogens VSV-G or human syncytin A (using the construct shown in Fig. 16).
  • Proteins were transferred to a nitrocellulose membrane using an iBlot 2 device.
  • the membrane was probed with an anti-HA and a [Lactin (ACTB) antibody and protein levels were visualized by chemiluminescence.
  • ACTB a [Lactin (ACTB) antibody
  • protein levels were visualized by chemiluminescence.
  • high levels of hSYNA-HA were detected (Fig. 9).
  • Example 7 Cell-to-cell delivery of a gene-editing enzyme using hSYNA
  • colorectal EpCAM-positive SW480 cells were engineered using lentivirus to carry a LoxP-flanked DsRed fluorescent reporter including a stop codon followed by an eGFP reporter (SW480-LoxP-DsRed-eGFP) using the pLV-CMV-LoxP- DsRed-LoxP-eGFP plasmid. Following Cre-mediated excision of the LoxP-flanked cassette, SW480 cells switch from DsRed to eGFP.
  • FACT cells were engineered by lipofection to express hSYNA as well as tamoxifen-inducible Cre using the pcDNA3.1-CMV-CFP;UBC-Cre25nt plasmid. FACTc- cells were co-cultured with SW480-LoxP-DsRed-eGFP cells in the presence of 3uM tamoxifen for
  • SW480-LoxP-DsRed-eGFP cells to eGFP (Fig. 10B), with the top panel showing all cells and the bottom panel only showing the EpCAM-positive target cells.
  • Example 8 Generation of fusogenic extracellular vesicles capable of delivering of gene-editing enzymes
  • Extracellular vesicles are a promising tool for the delivery of therapeutic payloads, especially gene-editing enzymes, due to their high biocompatibility and low immunogenicity.
  • the most commonly used protein to generate fusogenic EVs is the vesicular stomatitis virus G (VSVG) envelope protein. While VSVG is a highly efficient fusogen with broad tropism, as a large viral protein it elicits potent immune responses limiting efficacy and potentially preventing repeat infusions.
  • VSVG vesicular stomatitis virus G
  • FACT cells are able to efficiently deliver gene-editing enzymes when co-cultured directly with target cells.
  • FACT cells could serve as the source of fusogenic EVs, an alternative method of delivering therapeutic payloads. Therefore, supernatants were collected from FACT&e cells engineered using different fusogens. Supernatants were added to SW480-LoxP- DsRed-eGFP cells and incubated for 48h in the presence of tamoxifen. Conversion of the cells to eGFP was determined by flow cytometry as described above. It was found that supernatants harvested from FACTcre cells were able to induce eGFP expression in SW480 cells, indicating that FACT cells generate fusogenic EVs (Fig. 11).
  • TDP-43 a key protein found in neurofibrils in patients with amyotrophic lateral sclerosis (ALS)
  • ALS amyotrophic lateral sclerosis
  • 293T cells were engineered by lentiviral transduction to express either wildtype TDP-43 or TDP-43C173S/C175S fused to GFP under control of a tetracycline-inducible promoter.
  • 293T cells were engineered to stably express hSYNA and one of two single-chain variable fragments (clones 3B12A and Vh7Vk9) targeting aggregated TDP-43 [21, 24] (see Figs. 14 and 15).
  • FACT cells and TDP-43-GFP- expressing target cells were co-cultured for 24h before addition of tetracycline. After 24h, target 293T cells were purified by fluorescence activated cell sorting.
  • FACT cells were lysed in RIPA buffer and analyzed by western blot using antibodies against GFP antibody and ACTB.
  • FACT cells showed efficient reduction in total TDP-43 in target cells as determined by western blot using FACT cells engineered to deliver the TDP-43-specific scFv Vh7Vk9 (SEQ ID NO: 6) (Fig. 12). These data indicate that FACT cells are able to cause the degradation of specific proteins in target cells.
  • Example 10 FACT-mediated delivery of a genome editor
  • BE Base editors
  • FACT FACT can serve as a technology for the delivery of BEs
  • SW480 target cells expressing mutated eGFPL202 were generated by lentiviral transduction using the plenti- CMV-mCherry-T2A-GFPL202 plasmid.
  • FACT cells were engineered by lipofection to express hSYNA, an adenine BE using the A3Ai-Cas9n-UGI-NLS construct, as well as a guide RNA targeting the L202 mutation in eGFP and co-cultured target cells with these cells for 48h.
  • Increased GFP expression in SW480- eGFPL202 target cells was observed after co-culture with FACT cells by flow cytometry after staining with an anti-EpCAM antibody as described above (Fig. 13). This finding demonstrates that FACT technology is able to deliver functional BEs together with guide RNA to correct mutations on the DNA level, an application with high therapeutic potential.

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Abstract

L'invention concerne des cellules modifiées qui servent de véhicules pour délivrer des molécules cargo génétiquement codées, telles que des molécules thérapeutiques, à d'autres cellules in vivo. En particulier, des biovésicules fusogènes ayant un potentiel d'immunogénicité réduit, exprimant des fusogènes faiblement immunogènes (entièrement humains, humanisés ou désimmunisés) renfermant les molécules cargo sont produits par les cellules modifiées. Les cellules modifiées peuvent être utilisées dans des méthodes de traitement de maladies et d'états pathologiques tels que la sclérose latérale amyotrophique, la maladie d'Alzheimer et le cancer.
PCT/US2024/027464 2023-05-02 2024-05-02 Administration cellulaire d'agents thérapeutiques à l'aide de vésicules fusogènes Ceased WO2024229254A2 (fr)

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