Engineered lipid bilayer particles displaying sirpa

Abstract

The present disclosure provides methods and compositions for targeting engineered lipid bilayer particles, such as secreted extracellular vesicles, and cargos included therein to recipient cells by displaying one or more signal regulatory protein alpha (SIRPa) on surfaces of the lipid bilayer particles.

Description

ENGINEERED LIPID BILAYER PARTICLES DISPLAYING SIRPACross-Reference to Related Applications

[0001] The present application claims priority to and the benefit of U.S. Provisional Patent Application No. 63 / 599,153 filed on November 15, 2023, the entirety of which is incorporated herein by reference.Background

[0002] The burgeoning field of nucleic acid therapeutics has garnered much attention, among other things because of its potential to treat a variety of diseases, disorders, or conditions that may not be readily addressable by other modalities. However, as noted in a recent review, “[w]hile nucleic acid therapeutics can expand the array of treatable diseases, their broader use is limited by multiple delivery challenges” (Gupta et al., “Nucleic acid delivery for therapeutic applications” in Adv . Drug Delivery Reviews 178: 113834, 2021).Summary

[0003] The present disclosure provides a remarkable new technology for achieving delivery of a cargo (e.g., a nucleic acid cargo) to a recipient cell or cell population using engineered lipid bilayer particles.

[0004] Among other things, the present disclosure provides an insight that presence of SIRPa on surfaces of lipid bilayer particles can confer surprisingly improved delivery attributes to such particles, for example achieving more efficient delivery and / or delivery to particular cell types. Without wishing to be bound by any particular theory, it is proposed that SIRPa confers its function by binding to a target (e.g., present on a recipient cell), mediating internalization, and / or potentially catalyzing fusogen-mediated fusion between lipid bilayer particles and cellular membranes.

[0005] The present disclosure identifies the source of one or more problems associated with many conventional strategies for payload delivery (e.g., nucleic acid payload delivery) to recipient cell(s) of interest, including specifically with technologies intended to achieve in vivo delivery. Among other things, the present disclosure identifies the source of a particular problem associated with conventional strategies that utilize a viral fusogen (e.g., VSV-G), or a variant thereof, to achieve payload delivery; the present disclosuredemonstrates that expressing SIRPa on the surfaces of lipid bilayer particles, solves such identified problem(s) and / or otherwise achieves particularly beneficial results (e.g., particularly precise and / or efficient payload delivery).

[0006] Among other things, the present disclosure provides engineered lipid bilayer particles, and preparations thereof, whose surfaces include a SIRPa as described herein. In some embodiments, the present disclosure provides engineered lipid bilayer particles, and preparations thereof, whose surfaces display a SIRPa and optionally further display a fusogen entity polypeptide and / or an affinity entity polypeptide. In some embodiments, such provided particles and / or preparations are characterized by particular payload delivery attributes. In some embodiments, such provided particles and / or preparations achieve payload delivery (e.g., specific payload delivery) to particular cell(s) or cell population(s) of interest; in some embodiments such delivery is in vivo. In some embodiments, such provided particles and / or preparations achieve enhanced payload delivery (e.g., specific payload delivery) to particular cell(s) or cell population(s) of interest, comparable to particles that do not express SIRPa on their surfaces.

[0007] In some embodiments, provided technology is useful for delivery of viral vectors (e.g., viral capsid, nucleocapsid, lentivirus cores, adeno-associated virus particles), and / or virus-like particles. Alternatively, or additionally, in some embodiments, provided technologies are useful for delivery of non-viral vectors (e.g., nucleic acid payloads that are not packaged within a protein core or capsid structure).

[0008] Certain particularly useful applications of provided technologies include, for example, CAR T cell therapy, for example in the manufacture of CAR T cells for oncology treatment, immune system disorders, and other applications.

[0009] Among other things, the present disclosure identifies challenges with in vivo gene delivery to cells (e.g., T cells), in particular in vivo delivery of a cargo (e.g., payload) for specifically and efficiently targeting T cells. In vitro delivery of a cargo (e.g., payload) to T cells in a specific and efficient fashion is partially met by some conventional methods, but there remains unmet need for specific in vivo delivery and non-toxic in vivo and in vitro delivery, as well as more efficient in vivo and in vitro delivery.

[0010] Among other things, in some embodiments, the present disclosure provides technologies (e.g., systems, engineered lipid bilayer parties, production cells, method ofmanufacturing and delivery) that mediate fusion of an engineered lipid bilayer particle that displays SIRPa to a recipient cell (e.g., to deliver a cargo).

[0011] Certain particularly useful applications of provided technologies include, for example, CAR T cell therapy, for example in the manufacture of CAR T cells for oncology treatment and other applications.

[0012] Among other things, in some embodiments, the present disclosure provides technologies that enhance delivery of a particular cargo (e.g., payload) and / or delivery to a particular recipient cell or cell population.

[0013] In some embodiments, the present disclosure achieves specificity and / or efficiency of payload delivery through combined activity of a SIRPa and a fusogen entity polypeptide and / or an affinity entity polypeptide. In some embodiments, provided technologies achieve delivery that shows greater specificity and / or efficiency when compared with a particular reference; in some embodiments, such reference may be a sufficiently comparable system including one or the other of the fusogen entity polypeptide and the affinity entity polypeptide, but not both. In many embodiments, an appropriate reference may be a sufficiently comparable system including the fusogen entity polypeptide but not the affinity entity polypeptide.Brief Description of the Drawing

[0014] The Drawing includes the following Figures:

[0015] Figure 1 shows an overview of the plasmids used in Example 1.

[0016] Figures 2A-2F: Figures 2A, 2B, and 2C show transduction of Jurkat T cells with exemplary lipid bilayer particles comprising a lentivirus core together with no fusogen entity (Figure 2A) or either wild type (wt) VSV-G (Figure 2B) or mutated (mut) VSV-G (Figure 2C) as a fusogen entity. Five doses of 1 pl, 2.5 pl, 10 pl, 25 pl and 100 pl lipid bilayer particles were tested. Figures 2D, 2E, and 2F show transduction of Jurkat T cells evaluated under similar conditions Figures 2A-2C, respectively, but with doses reported in viral genomes (quantified by qPCR) added per recipient Jurkat T cell for no fusogen entity (Figure 2D) and either VSV-Gwt (Figure 2E) or VSV-Gmut (Figure 2F).

[0017] Figures 3A-3G show transduction of HEK293FT cells with lipid bilayer particles. In each case, the lipid bilayer particles were formulated such that their surfaces include neithera fusogen nor SIRPa (Figure 3A), SIRPa only (Figure 3D), a fusogen only (Figures 3B and 3C), or combinations of each fusogen with SIRPa (Figures 3E and 3F). Figure 3G shows transduction of HEK293FT cells evaluated under similar conditions to Figures 3A- 3F, but in this case with doses reported in viral genomes (quantified by qPCR) added per recipient HEK293FT cell.

[0018] Figure 4 shows an overview of the plasmids used in Example 2.

[0019] Figures 5A-5B show transduction of activated primary human T cells showing CD4+ activation (Figure 5A) or CD8+ activation (Figure 5B) with lipid bilayer particles with different surface modifications.

[0020] Figure 6 shows transduction of CD4+ activated primary human T cells using VSV- Gmut alone and expressed along with SIRPa and / or an affinity reagent anti-CD5scFv or anti-CD3scFv.

[0021] Figures 7A-7B are graphs depicting functional titer based on transduction of different cell types (x-axis: HEK29FT cells; y-axis: Lenti-X HEK293T cells) with exemplary engineered lipid bilayer particles as described herein, assessed by flow cytometry. Data points are shaded based on the percentage of SIRPa plasmid DNA relative to total plasma DNA (Figure 7A) or based on the exemplary lentiviral packaging system used (Figure 7B). Points represent the mean of calculated functional titer from data points within the linear transduction range from two biologic replicates. Error bars represent the standard error of the mean. A bold line depicts the fit of a linear regression through the data points, with the R squared, slope, and y-intercept of the model fit shown in the upper right comer.

[0022] Figures 7C-7D are graphs depicting percent transduction of HEK293FT cells (Figure 7C) or Lenti-X HEK293T cells (Figure 7D) with different viral dosages (pL) of an exemplary composition of engineered lipid bilayer particles comprising a fusogen (VSV-G) and SIRPa compared to a composition comprising a fusogen (VSV-G) without SIRPa. Percent transduction was assessed by flow cytometry. Points represent the mean of two biologic replicates. Error bars represent the standard error of the mean.

[0023] Figure 8A is a bar graph depicting functional titer of Jurkat T cells transduced with compositions of engineered lipid bilayer particles comprising a fusogen only (VSV-G) or afusogen and SIRPa. Bars represent the mean of calculated functional titer data points within the linear transduction range, and error bars represent the standard error of the mean.

[0024] Figure 8B is a graph depicting percent transduction of Jurkat T cells with different viral dosages (pL) of an exemplary composition of engineered lipid bilayer particles comprising a fusogen (VSV-G) and SIRPa compared to a composition comprising a fusogen (VSV-G) without SIRPa, as assessed by flow cytometry.

[0025] Figures 9A-9B are schematics depicting exemplary plasmid systems used to produce engineered lipid bilayer particles as described in the Examples.Definitions

[0026] In this application, unless otherwise clear from context, (i) the term “a” may be understood to mean “at least one”; (ii) the term “or” may be understood to mean “and / or”; (iii) the terms “comprising” and “including” may be understood to encompass itemized components or steps whether presented by themselves or together with one or more additional components or steps; and (iv) the terms “about” and “approximately” may be understood to permit standard variation as would be understood by those of ordinary skill in the art; and (v) where ranges are provided, endpoints are included.

[0027] 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. In some embodiments, 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 or that has a known affinity below a particular threshold. 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.

[0028] Affinity entity polypeptide. As used herein the term “affinity entity polypeptide” refers to a polypeptide that binds to a target on a recipient cell, or on populations thereof. In some embodiments, an affinity entity polypeptide binds preferentially to its recipient cells as compared with other cells within a system that includes the recipient cells. In some embodiments, an affinity entity polypeptide binds to a particular target ligand on recipient cell surface(s). In some embodiments, an affinity entity polypeptide shows specific binding to its target ligand relative to one or more other entities on the surface of its recipient cells. Alternatively, or additionally, in some embodiments, an affinity entity polypeptide shows preferential binding to its target ligand relative to one or more (or all) entities present on surfaces of non- recipient cell(s) (e.g., non- recipient cell(s) that may be present in a system that includes recipient cells). In some embodiments, an affinity entity polypeptide is characterized by the ability to bind to a target on the surface of a recipient cell during the fusion and / or delivery process (e.g., cargo delivery). In some embodiments, an affinity entity polypeptide is characterized by faster on rate of binding kinetics compared to a negative control reference. In some embodiments, an affinity entity polypeptide is characterized by a slower off rate of binding kinetics to a target compared to a negative control reference. In some embodiments, an affinity entity polypeptide is characterized by the ability to induce a conformational change in a target that slows the rate of dissociation. In some embodiments, an affinity entity polypeptide is characterized by the ability to include a functional change in the target (e.g., cross linking a receptor to induce or suppress signaling). In some embodiments, an affinity entity polypeptide is characterized by the ability to induce internalization of a target.

[0029] Affinity moiety . In general, the term “affinity moiety” is used herein to refer to a moiety that binds to a target ligand of interest as described herein. In many embodiments, an affinity moiety of interest is one that binds specifically with its target in that it discriminates its target from other potential binding partners in a particular interaction context. In general, an affinity moiety may be or comprise a moiety of any chemical class (e.g., polymer, non-polymer, small molecule, polypeptide, carbohydrate, lipid, nucleic acid, etc). In some embodiments, an affinity moiety is a single chemical entity. In some embodiments, an affinity moiety is a complex of two or more discrete chemical entities associated with one another under relevant conditions by non-covalent interactions. For example, those skilled in the art will appreciate that in some embodiments, an affinitymoiety may comprise a “generic” binding moiety (e.g., one of biotin / avidin / streptavidin and / or a class-specific antibody) and a “specific” binding moiety (e.g., an antibody or aptamers with a particular molecular target) that is linked to the partner of the generic biding moiety. In some embodiments, such an approach can permit modular assembly of multiple affinity moieties through linkage of different specific binding moieties with the same generic binding moiety partner. In some embodiments, affinity moieties are or comprise polypeptides (including, e.g., antibodies or antibody fragments). In some embodiments, affinity moieties are or comprise small molecules. In some embodiments, affinity moieties are or comprise nucleic acids. In some embodiments, affinity moieties are aptamers. In some embodiments, affinity moieties are polymers; in some embodiments, affinity moieties are not polymers. In some embodiments, affinity moieties are non-polymeric in that they lack polymeric moieties. In some embodiments, affinity moieties are or comprise carbohydrates. In some embodiments, affinity moieties are or comprise peptidomimetics. In some embodiments, affinity moieties are or comprise scaffold proteins. In some embodiments, affinity moieties are or comprise mimeotopes. In some embodiments, affinity moieties are or comprise stapled peptides. In certain embodiments, affinity moieties are or comprise nucleic acids, such as DNA or RNA.

[0030] Characteristic sequence element: As used herein, the phrase “characteristic sequence element” refers to a sequence element found in a polymer (e.g., in a polypeptide or nucleic acid) that represents a characteristic portion of that polymer. In some embodiments, presence of a characteristic sequence element correlates with presence or level of a particular activity or property of the polymer. In some embodiments, presence (or absence) of a characteristic sequence element defines a particular polymer as a member (or not a member) of a particular family or group of such polymers. A characteristic sequence element typically comprises at least two monomers (e.g., amino acids or nucleotides). In some embodiments, a characteristic sequence element includes at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50, or more monomers (e.g., contiguously linked monomers). In some embodiments, a characteristic sequence element includes at least first and second stretches of contiguous monomers spaced apart by one or more spacer regions whose length may or may not vary across polymers that share the sequence element.

[0031] Comparable: As used herein, the term “comparable” refers to two or more agents, entities, situations, sets of conditions, etc., that may not be identical to one another but thatare sufficiently similar to permit comparison there between so that one skilled in the art will appreciate that conclusions may reasonably be drawn based on differences or similarities observed. In some embodiments, 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. For example, those of ordinary skill in the art will appreciate that 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.

[0032] Corresponding to. As used herein, the term “corresponding to” refers to a relationship between two or more entities. For example, the term “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). For example, in some embodiments, a monomeric residue in a polymer (e.g., an amino acid residue in a polypeptide or a nucleic acid residue in a polynucleotide) may be identified as “corresponding to” a residue in an appropriate reference polymer. For example, those of ordinary skill will appreciate that, for purposes of simplicity, 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 190thamino 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. For example, those skilled in the art will be aware of various sequence alignment strategies, including software programs such as, for example, BLAST, CS-BLAST, CUSASW++, DIAMOND, FASTA, GGSEARCH / GL SEARCH, 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 inpolypeptides and / or nucleic acids in accordance with the present disclosure. Those of skill in the art will also appreciate that, in some instances, 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). To give but one example, 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.

[0033] Engineered: In general, the term “engineered” refers to the aspect of having been designed, produced, and / or manipulated by the hand of man. For example, a polynucleotide is considered to be “engineered” when two or more sequences that are not linked together in that order in nature are designed or otherwise caused 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. For example, in some embodiments described and / or utilized herein, an engineered polynucleotide comprises a regulatory sequence that is found in nature in operative association with a first coding sequence but not in operative association with a second coding sequence, is linked by the hand of man so that it is operatively associated with the second coding sequence. Comparably, in some embodiments a polypeptide may be considered to be “engineered” if encoded by or expressed from an engineered polynucleotide, and / or if produced other than natural expression in a cell. Analogously, 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. In some embodiments, the 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). In some embodiments, 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 / oraccording to altered timing relative to such an appropriate reference cell. As is common practice and is understood by those in the art, progeny of an engineered polynucleotide or cell are typically still referred to as “engineered” even though the actual manipulation was performed on a prior entity.

[0034] Engineered lipid bilayer particle. As used herein refers to a lipid bilayer particle engineered as described herein. For example, in some embodiments, a lipid bilayer particle may be considered to be “engineered” if it is synthetically produced, i.e., not produced by a cell. Alternatively, or additionally, in some embodiments, a lipid bilayer particle may be considered to be “engineered’ if it is produced by an engineered production cell. In some embodiments, an engineered lipid bilayer particle is produced by a production cell engineered to have both a fusogen entity polypeptide and an affinity entity polypeptide on its surface. In some such embodiments, an engineered production cell differs from an appropriate reference cell in that it has been engineered to express a fusogen entity polypeptide, an affinity entity polypeptide, or both, or to express one or both at a different level (e.g., an elevated level) such that lipid bilayer particles (e.g., exosomes) produced (e.g., released) by such engineered production cell bind to a recipient cell, or population of cells, with significantly greater affinity and / or specificity than do comparable particles produced (e.g., released) by the reference cell.

[0035] Functional: As used herein, the term “functional” is used to refer to a form or fragment of an entity that exhibits a particular property and / or activity.

[0036] Fusogen entity polypeptide: The term “fusogen entity polypeptide” as used herein refers to a polypeptide that (a) mediates fusion between lipid bilayers; and (b) shares at least one characteristic sequence element with and / or shows an overall degree of identity with a reference fusogen entity polypeptide (and / or with a portion [e.g., a fragment] thereof, particularly with a portion that is or comprises a characteristic sequence thereof): in many embodiments, a fusogen entity polypeptide (a) mediates fusion between lipid bilayers; and (bl) shares at least one characteristic sequence element with a reference fusogen entity polypeptide (or portion thereof) and (b2) shows an overall degree of sequence identity with such reference fusogen entity polypeptide (or portion thereof). Table 3 presents amino acid sequence(s) of certain known fusogen entity polypeptides (e.g., reference fusogen entity polypeptides). Table 9 presents certain sequences that may, in some embodiments, be considered characteristic sequences of a fusogen entity polypeptide relevant to the presentdisclosure. In some embodiments, a fusogen entity polypeptide shows at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, or 100% sequence identity with a reference fusogen entity polypeptide (or portion thereof).

[0037] In vitro'. The term “in vitro” as used herein refers to events that occur in an artificial environment, e.g., in a test tube or reaction vessel, in cell culture, etc., rather than within a multi-cellular organism.

[0038] In vivo: as used herein refers to events that occur within a multi-cellular organism, such as a human and a non-human animal. In the context of cell-based systems, the term may be used to refer to events that occur within a living cell (as opposed to, for example, in vitro systems).

[0039] Linker: as used herein, is used to refer to that portion of a multi -element agent that connects different elements to one another. For example, those of ordinary skill in the art appreciate that a polypeptide whose structure includes two or more functional or organizational domains often includes a stretch of amino acids between such domains that links them to one another. In some embodiments, a polypeptide comprising a linker element has an overall structure of the general form S1-L-S2, wherein SI and S2 may be the same or different and represent two domains associated with one another by the linker. In some embodiments, a polypeptide linker is at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 or more amino acids in length. In some embodiments, a linker is characterized in that it tends not to adopt a rigid three-dimensional structure, but rather provides flexibility to the polypeptide. In some embodiments, a linker is characterized in that it adopt a rigid three-dimensional structure and provides a stability to the polypeptide. A variety of different linker elements that can appropriately be used when engineering polypeptides (e.g., fusion polypeptides) known in the art (see e.g., Holliger, P., et al. (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448; Poljak, R. J., et al. (1994) Structure 2: 1 121- 1123).

[0040] Polypeptide: As used herein refers to a polymeric chain of amino acids. In some embodiments, a polypeptide has an amino acid sequence that occurs in nature. In some embodiments, a polypeptide has an amino acid sequence that does not occur in nature. In some embodiments, a polypeptide has an amino acid sequence that is engineered in that it isdesigned and / or produced through action of the hand of man. In some embodiments, a polypeptide may comprise or consist of natural amino acids, non-natural amino acids, or both. In some embodiments, a polypeptide may comprise or consist of only natural amino acids or only non-natural amino acids. In some embodiments, a polypeptide may comprise D-amino acids, L-amino acids, or both. In some embodiments, a polypeptide may comprise only D-amino acids. In some embodiments, a polypeptide may comprise only L-amino acids. In some embodiments, a polypeptide may include one or more pendant groups or other modifications, e.g., modifying or attached to one or more amino acid side chains, at the polypeptide’s N-terminus, at the polypeptide’s C-terminus, or any combination thereof. In some embodiments, such pendant groups or modifications may be selected from the group consisting of acetylation, amidation, lipidation, methylation, pegylation, etc., including combinations thereof. In some embodiments, a polypeptide may be cyclic, and / or may comprise a cyclic portion. In some embodiments, a polypeptide is not cyclic and / or does not comprise any cyclic portion. In some embodiments, a polypeptide is linear. In some embodiments, a polypeptide may be or comprise a stapled polypeptide. In some embodiments, the term “polypeptide” may be appended to a name of a reference polypeptide, activity, or structure; in such instances it is used herein to refer to polypeptides that share the relevant activity or structure and thus can be considered to be members of the same class or family of polypeptides. For each such class, the present specification provides and / or those skilled in the art will be aware of exemplary polypeptides within the class whose amino acid sequences and / or functions are known; in some embodiments, such exemplary polypeptides are reference polypeptides for the polypeptide class or family. In some embodiments, a member of a polypeptide class or family shows significant sequence homology or identity with, shares a common sequence motif (e.g., a characteristic sequence element) with, and / or shares a common activity (in some embodiments at a comparable level or within a designated range) with a reference polypeptide of the class; in some embodiments with all polypeptides within the class). For example, in some embodiments, a member polypeptide shows an overall degree of sequence homology or identity with a reference polypeptide that is at least about 30-40%, and is often greater than about 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more and / or includes at least one region (e.g., a conserved region that may in some embodiments be or comprise a characteristic sequence element) that shows very high sequence identity, often greater than 90% or even 95%, 96%, 97%, 98%, or 99%. Such a conserved region usuallyencompasses at least 3-4 and often up to 20 or more amino acids; in some embodiments, a conserved region encompasses at least one stretch of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more contiguous amino acids. In some embodiments, a relevant polypeptide may comprise or consist of a fragment of a parent polypeptide. In some embodiments, a useful polypeptide as may comprise or consist of a plurality of fragments, each of which is found in the same parent polypeptide in a different spatial arrangement relative to one another than is found in the polypeptide of interest (e.g., fragments that are directly linked in the parent may be spatially separated in the polypeptide of interest or vice versa, and / or fragments may be present in a different order in the polypeptide of interest than in the parent), so that the polypeptide of interest is a derivative of its parent polypeptide.

[0041] Specificity . As is known in the art, “specificity” is a measure of the ability of a particular ligand to distinguish its binding partner from other potential alternative binding partners. In many embodiments, specificity is assessed in a context in which one or more such potential alternative binding partners is also present (e.g., in a competitive binding context), and specificity reflects a preference for a particular target binding partner as compared with one or more such other potential alternative binding partners present in such context. In some embodiments, such preference is observed as a ratio of equilibrium dissociation constants (Kd) wherein the Kd associated with the specific interaction is smaller (indicating more preference for the bound state) than the Kd associated with non-specific binding; such ratios of Kd non-specific to Kd non-specific may be 2, 3, 4, 5, 6, 7, 8, 9, 10- fold or more.

[0042] Wild-type: As used herein, the term “wild-type” has its art-understood meaning that refers to an entity having a structure and / or activity as found in nature in a “normal” (as contrasted with mutant, diseased, altered, etc.) state or context. Those of ordinary skill in the art will appreciate that wild-type genes and polypeptides often exist in multiple different forms (e.g., alleles).Detailed Description of Certain Embodiments

[0043] The present disclosure provides technologies (e.g., engineered lipid bilayer particles, systems, engineered production cells, methods of manufacturing and methods of delivering a cargo) utilizing a fusogen entity polypeptide and an affinity entity polypeptide. Inparticular, the present disclosure provides technologies for delivering a cargo from bilayer lipid particles into cells - e.g., in vivo and / or in vitro.Engineered Lipid Bilayer Particles

[0044] Engineered lipid bilayer particles according to the present disclosure display at least one signal regulatory protein alpha (SIRPa). In some embodiments, engineered lipid bilayer particles are characterized by the presence of their surface of SIRPa and a fusogen entity polypeptide. In some embodiments, engineered lipid bilayer particles are characterized by the presence of their surface of SIRPa and an affinity entity polypeptide. In some embodiments, engineered lipid bilayer particles are characterized by the presence of their surface of SIRPa, a fusogen entity polypeptide and an affinity entity polypeptide.

[0045] The present disclosure provides a population of engineered lipid bilayer particles that display one or more SIRPa. In some embodiments, a population of engineered lipid bilayer particles display SIRPa and further display a fusogen entity polypeptide, an affinity entity polypeptide, or a combination thereof.

[0046] In some embodiments, particles of a population of engineered lipid bilayer particles display one or more SIRPa. In some embodiments, particles of a population of engineered lipid bilayer particles display one or more SIRPa and one or more fusogen entity polypeptides. In some embodiments, particles of a population of engineered lipid bilayer particles display one or more SIRPa and one or more affinity entity polypeptides. In some embodiments, particles of a population of engineered lipid bilayer particles display one or more SIRPa, one or more fusogen entity polypeptides, and one or more affinity entity polypeptides.

[0047] Without wishing to be bound by any particular theory, it is proposed that SIRPa, when displayed on the surface of an engineered lipid bilayer particle, binds to a target on a recipient cell and mediates internalization between the particle and the recipient cell membrane. In some embodiments, SIRPa alone mediates fusion between an engineered lipid bilayer particle and a recipient cell (see Example 1, Figures 2A and 2D). In some embodiments, SIRPa enhances the specificity of delivery of engineered lipid bilayer particles to a recipient cell. In some embodiments, SIRPa reduces engineered lipid bilayer particle delivery to non-recipient cells while maintaining delivery to recipient cells. In someembodiments, SIRPa enables delivery to a recipient cell by complementing a fusogen entity polypeptide (e.g., where the fusogen entity polypeptide lacks specificity).

[0048] Provided engineered lipid bilayer particles may be suitable for therapeutic applications.

[0049] In some embodiments, engineered lipid bilayer particles are produced by one or more engineered production cells as described herein so that the engineered lipid bilayer particles display at least one SIRPa. In some embodiments, engineered lipid bilayer particles are produced by one or more engineered production cells as described herein so that the engineered lipid bilayer particles display at least one SIRPa, and further display a fusogen entity polypeptide, an affinity entity polypeptide, or a combination thereof.

[0050] In some embodiments, a particle of a population of engineered lipid bilayer particles are characterized in that the engineered lipid bilayer particles are smaller than a eukaryotic cell. In some embodiments, a population of engineered lipid bilayer particles is characterized in that the average diameter of the engineered lipid bilayer particles is at the most 1000 nm, such as at the most 800 nm, such as at the most 300, such as at the most 100 nm. In some embodiments, a population of engineered lipid bilayer particles is characterized in that the average diameter of the engineered lipid bilayer particles is at least 30 nm, such as at least 50 nm, such as at least 80 nm, such as at least 100 nm, such as at least 150 nm, such as at least 200 nm, such as at least 250 nm, such as at least 300 nm. In some embodiments, a population of engineered lipid bilayer particles is characterized in that the average diameter of the engineered lipid bilayer particles is about 10 nm to about 1000 nm, such as about 30 nm to about 800 nm, such as about 50 nm and about 500 nm.

[0051] In some embodiments, entry of an engineered lipid bilayer particles into a recipient cell depends on the entry environment. An entry may be dependent on pH, temperature, buffer, temperature, radiation (e.g., nuclear, ultraviolet, visual, etc.), electric signal, magnetic field, induced by proximity, mechanical tension, stress, etc. or a combination hereof.

[0052] In some embodiments, an engineered lipid bilayer particle displays at least 5 copies of a SIRPa, such as at least 10 copies, such as at least 50 copies, such as at least 100 copies, such as at least 200 copies, such as at least 300 copies, such as at least 400 copies, such as at least 500 copies of a SIRPa.

[0053] In some embodiments, an engineered lipid bilayer particle displays at least 5 copies of a fusogen entity polypeptide, such as at least 10 copies, such as at least 50 copies, such as at least 100 copies, such as at least 200 copies, such as at least 300 copies, such as at least 400 copies, such as at least 500 copies of a fusogen entity polypeptide.

[0054] In some embodiments, an engineered lipid bilayer particle displays at least 10 copies of an affinity entity polypeptide, such as at least 50 copies, such as at least 100 copies, such as at least 200 copies, such as at least 300 copies, such as at least 400 copies, such as at least 500 copies of an affinity entity polypeptide.

[0055] In some embodiments, engineered lipid bilayer particles are cell-derived membrane particles (CDMPs). In some embodiments, CDMPs are selected from extracellular vesicles, virus particles, virus-like particles (VLPs), apoptotic bodies, platelet-like particles, and combinations thereof. In some embodiments, extracellular vesicles are exosomes, microvesicles, and combinations thereof.

[0056] In some embodiments, a CDMP is an extracellular vesicle, which can be selected from an exosome or a microvesicle. In some embodiments, a CDMP can be a virus particle, a virus-like particles (VLP), an apoptotic body, and a platelet-like particle. In some embodiments, the CDMP can be a hybrid particle generated by mixing a cell-derived particle or vesicle (e.g., a particle or vesicle that blebbed or budded from a cell) and a synthetic vesicle. In some embodiments, an extracellular vesicle is a modified extracellular vesicle. In some embodiments, a modified extracellular vesicle is prepared by adding lipids and / or molecules (e.g., polypeptides etc.) to an extracellular vesicle and thereby produce the modified extracellular vesicle. In some embodiments, the added lipids modify the properties of the extracellular vesicle lipid bilayer.

[0057] In some embodiments, engineered lipid particles are lentiviral vectors.

[0058] In some embodiment, a lentiviral vector comprises one or more molecules selected from the group consisting of structural protein, enzymatic protein, and viral genomic RNA components that, when self-assembled and delivered as cargo within a lipid bilayer particle to the cytoplasm of a recipient cell, confer transgene expression in recipient cells. In some embodiments, lentiviral vectors comprise one or more of gag, pol, and rev gene products and two copies of a single-stranded RNA genome derived from HIV.

[0059] In some embodiments, engineered lipid particles are synthetic lipid nanoparticles or liposomes (e.g., such as immunosomes, virosomes, or a combination thereof).SIRPa

[0060] Signal regulatory protein alpha (SIRPa) is a regulatory membrane glycoprotein from SIRP family expressed mainly by myeloid cells and also by stem cells or neurons. In some embodiments, SIRPa is expressed on the surface of an engineered lipid bilayer particle as provided herein.

[0061] Exemplary amino acid sequences of SIRPa and portions thereof are provided in Table 1. Exemplary nucleic acid sequences encoding SIRPa, and portions thereof are provided in Table 2.Table 1 - Example SIRPa amino acid sequencesTable 2 - Example SIRPa nucleic acid sequences

[0062] In some embodiments, a SIRPa comprises a secretory signal. In some embodiments, a SIRPa comprises an amino acid sequence according to SEQ ID NO: 2. In some embodiments, a SIRPa comprises an extraparticle portion. In some embodiments, a SIRPa comprises an amino acid sequence according to SEQ ID NO: 3. In some embodiments, a SIRPa comprises a lipid bilayer association portion such as a transparticle portion. In some embodiments, a SIRPa comprises an amino acid sequence according to SEQ ID NO: 4. In some embodiments, a SIRPa comprises an intraparticle portion. In some embodiments, a SIRPa comprises an amino acid sequence according to SEQ ID NO: 5.

[0063] In some embodiments, a SIRPa comprises an amino acid sequence that is at least 75% identical, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 1.

[0064] Provided herein are also nucleotide sequences encoding a SIRPa. In some embodiments, a nucleotide sequence encoding a SIRPa comprises a nucleic acid sequence of SEQ ID NO: 7. In some embodiments, a nucleotide sequence encoding a SIRPa comprises a nucleic acid sequence of SEQ ID NO: 8. In some embodiments, a nucleotide sequence encoding a SIRPa comprises a nucleic acid sequence of SEQ ID NO: 9. In some embodiments, a nucleotide sequence encoding a SIRPa comprises a nucleic acid sequence of SEQ ID NO: 10.

[0065] In some embodiments, a nucleotide sequence encoding a SIRPa comprises a nucleic acid sequence that is at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the nucleic acid sequence of SEQ ID NO: 6.

[0066] In some embodiments, SIRPa is overexpressed on the surface of an engineered lipid bilayer particle. In some embodiments, SIRPa is overexpressed on the surface of an engineered lipid bilayer particle relative to a basal level of SIRPa found on a lipid bilayer particle that is not engineered to express SIRPa.

[0067] In some embodiments, at least 5 copies of a SIRPa, such as at least 10 copies, such as at least 50 copies, such as at least 100 copies, such as at least 200 copies, such as at least 300 copies, such as at least 400 copies, such as at least 500 copies of a SIRPa are present on the surface of an engineered lipid bilayer particle. In some embodiments, SIRPa is the only polypeptide overexpressed on the surface of an engineered lipid bilayer particle.

[0068] Example 1 included herein documents usefulness of SIRPa alone (no fusogen entity polypeptide). Engineered lipid bilayer particles displaying SIRPa yield transduction of Jurkat T cells (Figures 2A and D).

[0069] In some embodiments, a SIRPa for use in accordance with the present disclosure is characterized in that it mediates fusion between two lipid bilayers (i.e., fusion activity).

[0070] In some embodiments, engineered bilayer particles displaying SIRPa as described herein and / or compositions that include them, may be characterized by one or more features or attributes (e.g., one or more physical and / or functional parameters) that, for example, may make them particularly useful in context(s) of interest (e.g., in therapeutic context(s) and / or in diagnostic and / or research contexts).

[0071] In some embodiments, a provided SIRPa is characterized in that, when it is expressed by an engineered production cell (e.g., via administration of a system or composition that includes or encodes it), it displays at an acceptable level on engineered lipid bilayer particles that are produced by such engineered production cell. Those skilled in the art will be aware of what level might be “acceptable” in a given context and / or in a particular assay.Fusogen Entity Polypeptide

[0072] In some embodiments, engineered lipid bilayer particles are characterized by presence of their surfaces of at least one SIRPa and at least one fusogen entity polypeptide.

[0073] Fusogen entity polypeptides as described herein mediate cell entry of an engineered lipid bilayer particle displaying such a fusogen entity polypeptide (alone or in combination with SIRPa). In some embodiments, a fusogen entity polypeptide is characterized by its ability to mediate fusion between lipid bilayers. In some embodiments, a fusogen entity polypeptide mediates transduction of a recipient cell. In some embodiments, a fusogen entity polypeptide binds to a target on a recipient cell.

[0074] Without wishing to be bound by a particular theory, it is proposed that the unique combination of SIRPa and a fusogen entity polypeptide provides targeted entry of engineered lipid bilayer particles into specific recipient cells with high efficiency. SIRPa may enhance specific delivery of engineered lipid bilayer particles displaying such SIRPa on their surfaces compared to engineered lipid bilayer particles that do not display SIRPa on their surfaces (See Examples 2, 4, and 5 and Figures 2, 3, 5, 7, and 8).

[0075] In some embodiments, technologies according to the present disclosure comprise a fusogen entity polypeptide. In some embodiments, technologies according to the present disclosure comprise at least one fusogen entity polypeptide. In some embodiments, technologies according to the present disclosure comprise one or more fusogen entity polypeptides.

[0076] In some embodiments, a fusogen entity polypeptide is a wild-type polypeptide. In some embodiments, a fusogen entity polypeptide is native to a particular production cell. In some embodiments, a fusogen entity polypeptide is an engineered polypeptide. In some embodiments, a fusogen entity polypeptide (e.g., an engineered fusogen entity polypeptide) is a variant of a wild-type polypeptide and / or of a native polypeptide.

[0077] In some embodiments, a fusogen entity polypeptide comprises a fusogen secretory signal. In some embodiments, a fusogen entity polypeptide comprises a fusogen moiety. In some embodiments, a fusogen entity polypeptide comprises a membrane association portion such as a transmembrane portion. In some embodiments, a fusogen entity polypeptide comprises an intraparticle portion.

[0078] In some embodiments, a fusogen entity polypeptide consists of or comprises a fusogen moiety and a membrane association portion such as a transmembrane portion.

[0079] In some embodiments, the order from the N-terminal to the C-terminal of a fusion entity polypeptide is as follows: a fusogen secretory signal, a fusogen moiety, a fusogen transmembrane portion, and / or a fusogen intraparticle portion.

[0080] In some embodiments, a fusogen entity polypeptide, or a fusogen moiety thereof, has an amino acid sequence that includes a characteristic sequence element and / or shares an overall degree of sequence identity with a reference fusogen entity polypeptide (e.g., a wild type fusogen entity polypeptide, and / or a fusogen entity polypeptide whose amino acid sequence are included in Table 3). In some embodiments, a fusogen entity polypeptide is a variant of such a reference fusogen entity polypeptide. In some embodiments, a fusogen entity polypeptide includes one or more modifications, such as glycosylation, lipidation, phosphorylation, etc.

[0081] In some embodiments, a fusogen entity polypeptide is a constitutive fusogen entity polypeptide in that its fusogenic activity does not depend on a particular stimulus or condition.

[0082] In some embodiments, a fusogen entity polypeptide is a conditional fusogen entity polypeptide in that its fusogenic activity is dependent upon or triggered by a particular stimulus or condition (e.g., pH, temperature, buffer radiation (e.g., nuclear, ultraviolet, visual, etc.), electric signal, magnetic field, induced by proximity, mechanical tension, stress, etc., or a combination thereof).

[0083] Exemplary amino acid sequences of fusogen entity polypeptides and portions thereof are provided in Table 3 and exemplary nucleic acid sequences encoding fusogen entity polypeptides or portions thereof are provided in Table 4.Table 3: Exemplary Amino acid sequencesTable 4: Exemplary Nucleic acid sequences

[0084] In some embodiments, a fusogen entity polypeptide comprises a secretory signal, e.g., that is functional in mammalian cells. In some embodiments, a utilized secretory signal is a heterologous secretory signal. In some embodiments, a heterologous secretory signal comprises or consists of a non-human secretory signal. In some embodiments, a heterologous secretory signal comprises or consists of a viral secretory signal. In some embodiments, a secretory signal comprises or consists of a VSV-G secretory signal. In some embodiments, a secretory signal is characterized by a length of about 10 to 30 amino acids. In some embodiments, a secretory signal is positioned at the N-terminus of a fusogen entity polypeptide described herein. In some embodiments, a secretory signal preferably allows transport of a fusogen entity polypeptide with which it is associated into a defined cellular compartment, preferably a cell surface, endoplasmic reticulum (ER), endosomal-lysosomal compartment and / or Golgi apparatus (e.g., glycosylate the fusogen entity polypeptide).

[0085] In some embodiments, a secretory signal is one listed in Table 3, or a secretory signal having 1, 2, 3, 4, or 5 amino acid differences relative thereto. In some embodiments, a fusogen entity polypeptide comprises an amino acid sequence according to SEQ ID NO: 11.

[0086] In some embodiments, a fusogen entity polypeptide comprises a fusogen moiety. Fusogen moieties as described herein mediate cell entry of an engineered lipid bilayer particle displaying a fusogen entity polypeptide comprising a fusogen moiety. In some embodiments, a fusogen moiety mediates transduction of a recipient cell. Fusogen moieties cover moieties or functional fragments thereof that are characterized in that they promote fusion lipid bilayers.

[0087] In some embodiments, a fusogen moiety is displayed on the surface of an engineered lipid bilayer particle. It may be displayed in a way that promotes fusion of the lipid bilayer of the engineered lipid bilayer particle with the lipid bilayer of a recipient cell.

[0088] In some embodiments, a fusogen moiety targets a specific epitope on recipient cells such that binding of this target epitope enables or enhances uptake, fusion and / or functional delivery of a cargo contained within the engineered lipid bilayer particle. In some embodiments, a target epitope may be expressed on all cells (e.g., a universal feature of the cell surface), or on a subset of cells, or on cells that occupy a subset of possible states (e.g., activated T cells versus resting T cells).

[0089] In some embodiments a fusion moiety mediates fusion between the engineered lipid bilayer particle and the target cell in a manner that does not require target epitope binding by the fusogen.

[0090] In some embodiments, a fusogen moiety is a viral fusogen moiety. In some embodiments, a fusogen moiety is an enveloped vial fusogen moiety. In some embodiments, an enveloped fusogen moiety fusogen is a fusogen moiety selected from the group consisting of a polypeptide from vesicular stomatitis virus, Measles virus, Sindbis virus, Tupaia paramyxovirus, Nipah virus, Chandipura virus, Rabies virus, Lymphocytic choriomeningitis virus, Mokola virus, Ross River virus, Ross River virus, Semliki Forest virus, Venezuelan equine encephalitis virus, Ebola virus, Marburg virus, Lassa virus, Avian leukosis virus, Jaagsiekte sheep retrovirus, Moloney Murine leukemia virus, Gibbon ape leukemia virus, Feline endogenous retrovirus (RD114), Human T-lymphotropic virus 1, Human foamy virus, Maedi-visna virus, SARS-CoV, SARS-CoV-2, Sendai virus,Respiratory syncytia virus, Human parainfluenza virus type 3, Human parainfluenza virus type 4, Hepatitis C virus, Hepatitis C virus, Influenza virus, Fowl plague virus, Autographa californica multiple nucleopolyhedro virus, Baboon endogenous retrovirus, Cocal virus, Japanese encephalitis virus, Dengue virus, Zika virus, West Nile virus, Yellow fever virus, Tick-borne encephalitis virus, Herpes simplex virus 1, Hendra virus, Newcastle disease virus, Epstein Barr virus, Bourbon virus, Varicella-zoster virus, Severe fever with thrombocytopenia virus, Hantavirus, Vaccinia virus, Simian immunodeficiency virus, Human immunodeficiency virus, Junin virus, Machupo virus, Bas-Congo virus, La Crosse virus, Human cytomegalovirus, Human cytomegalovirus, Thogoto virus, and Dhori virus

[0091] In some embodiments, a fusogen moiety is a non-viral fusogen moiety.

[0092] In some embodiments, a non-viral fusogen moiety is an endogenous protein. In some embodiments, a non-viral fusogen moiety is myomeger.

[0093] In some embodiments, a fusogen moiety is a heterologous fusogen moiety (e.g., to SIRPa).

[0094] In some embodiments, a fusogen moiety is positioned at the N-terminus of a fusogen entity polypeptide. In some embodiments, a fusogen moiety is positioned between a fusogen secretory signal and a fusogen transmembrane portion of a fusogen entity polypeptide.

[0095] In some embodiments, a fusogen moiety is or comprises a fragment of a wild type VSV-G. In some embodiments, a wild type VSV-G fusogen moiety has an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid SEQ ID NO: 15. In some embodiments, a wild type VSV-G fusogen moiety has an amino acid sequence that is identical to the amino acid SEQ ID NO: 15.

[0096] In some embodiments, a fusogen moiety is or comprises a fragment of a mutated VSV-G. In some embodiments, a mutated VSV-G fusogen moiety has an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid SEQ ID NO: 17. In some embodiments, a wild type VSV-G fusogen moiety has an amino acid sequence that is identical to the amino acid SEQ ID NO: 17.

[0097] Exemplary amino acid sequences of certain fusogen entity polypeptides or fragments thereof are provided in Table 1 and Table 3 and exemplary nucleic acid sequences encoding certain fusogen entity polypeptides are provided in Table 2 and Table 4.

[0098] In some embodiments, a fusogen entity polypeptide comprises a membrane association portion, such as a transmembrane portion. In some embodiments, a membrane association portion is positioned at the C-terminus of a fusogen entity polypeptide. In some embodiments, a membrane association portion is characterized by a length of about 10 amino acids to about 300 amino acids. In some embodiments, a membrane association portion is a heterologous membrane association portion. In some embodiments, a membrane association portion is a transmembrane portion.

[0099] In some embodiments, a membrane association portion is a viral membrane association portion. In some embodiments, a membrane association portion is a viral envelope membrane association portion, such as an envelope transmembrane association portion. In some embodiments, a membrane association portion is a non-viral membrane association portion, such as a non-viral transmembrane portion. In some embodiments, a membrane association portion is an engineered non-natural membrane association portion.

[0100] Membrane association portions, such as transmembrane portions are known in the art. In some embodiments, a membrane association portion comprises or is a transmembrane domain of Hemagglutinin (HA) of Influenza virus, Env of HIV- 1, equine infectious anaemia virus (EIAV), murine leukaemia virus (MLV), mouse mammary tumor virus, G protein of vesicular stomatitis virus (VSV-G), Rabies virus, Platelet-derived growth factor receptor (PDGFR), or a seven transmembrane domain receptor.

[0101] In some embodiments, a transmembrane domain comprises a platelet-derived growth factor receptor (PDGFR) transmembrane domain of SEQ ID NO: 25. In some embodiments, a PDGFR transmembrane domain is encoded by a nucleotide sequence according to SEQ ID NO: 26.

[0102] In some embodiments, a membrane association portion is or comprises a wild type VSV-G transmembrane portion or a fragment thereof. In some embodiments, a wild type VSV-G fusogen transmembrane portion has an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid SEQ ID NO: 12. In some embodiments, a wild type VSV-Gfusogen transmembrane portion has an amino acid sequence that is identical to the amino acid SEQ ID NO: 12.

[0103] An exemplary amino acid sequence of a membrane association portion is provided in Table 3 and an exemplary nucleic acid sequence encoding a membrane association portion is provided in Table 4.

[0104] In some embodiments, a fusogen entity polypeptide described herein comprises a fusogen intraparticle portion.

[0105] In some embodiments, a fusogen intracellular portion is or comprises a wild type VSV-G intracellular portion or a fragment thereof. In some embodiments, a wild type VSV- G fusogen intracellular portion has an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid SEQ ID NO: 9. In some embodiments, a wild type VSV-G fusogen intracellular portion has an amino acid sequence that is identical to the amino acid SEQ ID NO: 9.

[0106] An exemplary amino acid sequence of a fusogen intracellular portion is provided in Table 3 and an exemplary nucleic acid sequence encoding a fusogen intracellular portion is provided in Table 4.

[0107] In some embodiments, a fusogen entity polypeptide is or comprises a measles virus polypeptide, such as a measles virus glycoprotein. In some embodiments, a fusogen entity polypeptide is or comprises a measles virus glycoprotein H and / or F.

[0108] In some embodiments, a fusogen entity polypeptide is or comprises a sindbis virus polypeptide, such as a sindbis virus glycoprotein. In some embodiments, a fusogen entity polypeptide is or comprises a sindbis virus glycoprotein El and / or E2.

[0109] In some embodiments, a fusogen entity polypeptide is or comprises a tupaia paramyxovirus polypeptide, such as a tupaia paramyxovirus glycoprotein. In some embodiments, a fusogen entity polypeptide is or comprises a tupaia paramyxovirus glycoprotein H and / or F.

[0110] In some embodiments, a fusogen entity polypeptide is or comprises a nipah virus polypeptide, such as a nipah virus glycoprotein. In some embodiments, a fusogen entity polypeptide is or comprises a nipah virus glycoprotein G and / or F.[oni] In some embodiments, a fusogen entity polypeptide is or comprises a chandipura virus polypeptide, such as a chandipura virus glycoprotein. In some embodiments, a fusogen entity polypeptide is or comprises a chandipura virus glycoprotein G.

[0112] In some embodiments, a fusogen entity polypeptide is or comprises a rabies virus polypeptide, such as a rabies virus glycoprotein. In some embodiments, a fusogen entity polypeptide is or comprises a rabies virus glycoprotein G.

[0113] In some embodiments, a fusogen entity polypeptide is or comprises a lymphocytic choriomeningitis virus polypeptide, such as a lymphocytic choriomeningitis virus glycoprotein. In some embodiments, a fusogen entity polypeptide is or comprises a lymphocytic choriomeningitis virus glycoprotein GP-1 and / or GP-2.

[0114] In some embodiments, a fusogen entity polypeptide is or comprises a mokola virus polypeptide, such as a mokola virus glycoprotein. In some embodiments, a fusogen entity polypeptide is or comprises a mokola virus glycoprotein G.

[0115] In some embodiments, a fusogen entity polypeptide is or comprises a ross river virus polypeptide, such as a ross river virus glycoprotein. In some embodiments, a fusogen entity polypeptide is or comprises a ross river virus glycoprotein El and / or E2.

[0116] In some embodiments, a fusogen entity polypeptide is or comprises a semliki forest virus polypeptide, such as a semliki forest virus glycoprotein. In some embodiments, a fusogen entity polypeptide is or comprises a semliki forest virus glycoprotein El and / or E2.

[0117] In some embodiments, a fusogen entity polypeptide is or comprises a Venezuelan equine encephalitis virus polypeptide, such as a Venezuelan equine encephalitis virus glycoprotein. In some embodiments, a fusogen entity polypeptide is or comprises a Venezuelan equine encephalitis virus glycoprotein El and / or E2.

[0118] In some embodiments, a fusogen entity polypeptide is or comprises an ebola virus polypeptide, such as an ebola virus glycoprotein. In some embodiments, a fusogen entity polypeptide is or comprises an ebola virus glycoprotein GP.

[0119] In some embodiments, a fusogen entity polypeptide is or comprises a marburg virus polypeptide, such as a marburg virus glycoprotein. In some embodiments, a fusogen entity polypeptide is or comprises a marburg virus glycoprotein GP.

[0120] In some embodiments, a fusogen entity polypeptide is or comprises a lassa virus polypeptide, such as a lassa virus glycoprotein. In some embodiments, a fusogen entity polypeptide is or comprises a lassa virus glycoprotein GPC.

[0121] In some embodiments, a fusogen entity polypeptide is or comprises an avian leucosis virus polypeptide, such as an avian leucosis virus glycoprotein. In some embodiments, a fusogen entity polypeptide is or comprises an avian leucosis virus envelope glycoprotein.

[0122] In some embodiments, a fusogen entity polypeptide is or comprises a jaagsiekte sheep virus polypeptide, such as a jaagsiekte sheep virus glycoprotein. In some embodiments, a fusogen entity polypeptide is or comprises a jaagsiekte sheep virus envelope glycoprotein.

[0123] In some embodiments, a fusogen entity polypeptide is or comprises a moloney murine leukemia virus polypeptide, such as a moloney murine leukemia virus glycoprotein. In some embodiments, a fusogen entity polypeptide is or comprises a moloney murine leukemia virus envelope glycoprotein.

[0124] In some embodiments, a fusogen entity polypeptide is or comprises a gibbon ape leukemia virus polypeptide, such as a gibbon ape leukemia virus glycoprotein. In some embodiments, a fusogen entity polypeptide is or comprises a gibbon ape leukemia virus envelope glycoprotein.

[0125] In some embodiments, a fusogen entity polypeptide is or comprises a feline endogenous retrovirus polypeptide, such as a feline endogenous retrovirus glycoprotein. In some embodiments, a fusogen entity polypeptide is or comprises a RD114 glycoprotein.

[0126] In some embodiments, a fusogen entity polypeptide is or comprises a human T- lymphocyte virus 1 polypeptide, such as a human T-lymphocyte virus 1 glycoprotein. In some embodiments, a fusogen entity polypeptide is or comprises a human T-lymphocyte virus 1 glycoprotein SU and / or TM. In some embodiments, a fusogen entity polypeptide is or comprises a human T-lymphocyte virus 1 glycoprotein gp46.

[0127] In some embodiments, a fusogen entity polypeptide is or comprises a human foamy virus polypeptide, such as a human foamy virus glycoprotein. In some embodiments, a fusogen entity polypeptide is or comprises a human foamy virus envelope glycoprotein.

[0128] In some embodiments, a fusogen entity polypeptide is or comprises a maedi-visna virus polypeptide, such as a maedi-visna virus glycoprotein. In some embodiments, a fusogen entity polypeptide is or comprises a maedi-visna virus envelope glycoprotein.

[0129] In some embodiments, a fusogen entity polypeptide is or comprises a SARS-CoV polypeptide (e.g., SARS-CoV 2), such as a SARS-CoV glycoprotein. In some embodiments, a fusogen entity polypeptide is or comprises a SARS-CoV spike glycoprotein (S).

[0130] In some embodiments, a fusogen entity polypeptide is or comprises a sendai virus polypeptide, such as a sendai virus glycoprotein. In some embodiments, a fusogen entity polypeptide is or comprises a sendai virus glycoprotein HN and / or F.

[0131] In some embodiments, a fusogen entity polypeptide is or comprises a respiratory syncytia virus polypeptide, such as a respiratory syncytia virus glycoprotein. In some embodiments, a fusogen entity polypeptide is or comprises a respiratory syncytia virus glycoprotein G and / or F.

[0132] In some embodiments, a fusogen entity polypeptide is or comprises a human parainfluenza virus type 3 and / or type 4 polypeptide, such as a respiratory human parainfluenza virus type 3 and / or type 4 glycoprotein. In some embodiments, a fusogen entity polypeptide is or comprises a human parainfluenza virus type 3 and / or type 4 glycoprotein HN and / or F.

[0133] In some embodiments, a fusogen entity polypeptide is or comprises a hepatitis C virus polypeptide, such as a hepatitis C virus glycoprotein. In some embodiments, a fusogen entity polypeptide is or comprises a hepatitis C virus glycoprotein El and / or E2.

[0134] In some embodiments, a fusogen entity polypeptide is or comprises an influenza virus polypeptide, such as an influenza virus glycoprotein. In some embodiments, a fusogen entity polypeptide is or comprises an influenza virus glycoprotein HA and / or NA.

[0135] In some embodiments, a fusogen entity polypeptide is or comprises a fowl plague virus polypeptide, such as a fowl plague virus glycoprotein. In some embodiments, a fusogen entity polypeptide is or comprises a fowl plague virus glycoprotein HA.

[0136] In some embodiments, a fusogen entity polypeptide is or comprises an autographa californica multiple nucleopolyhedro virus polypeptide, such an autographa californica multiple nucleopolyhedro virus glycoprotein. In some embodiments, a fusogen entitypolypeptide is or comprises an autographa californica multiple nucleopolyhedro virus glycoprotein gp64.

[0137] In some embodiments, a fusogen entity polypeptide is or comprises a baboon endogenous retrovirus polypeptide, such a baboon endogenous retrovirus glycoprotein. In some embodiments, a fusogen entity polypeptide is or comprises a baboon endogenous retrovirus glycoprotein envelope.

[0138] In some embodiments, a fusogen entity polypeptide is or comprises a cocal virus polypeptide, such a cocal virus glycoprotein (G).

[0139] In some embodiments, a fusogen entity polypeptide is or comprises a Japanese encephalitis virus polypeptide, such a Japanese encephalitis virus glycoprotein. In some embodiments, a fusogen entity polypeptide is or comprises a Japanese encephalitis virus glycoprotein E.

[0140] In some embodiments, a fusogen entity polypeptide is or comprises a dengue virus polypeptide, such a dengue virus glycoprotein. In some embodiments, a fusogen entity polypeptide is or comprises a dengue virus glycoprotein E.

[0141] In some embodiments, a fusogen entity polypeptide is or comprises a zika virus polypeptide, such a zika virus glycoprotein. In some embodiments, a fusogen entity polypeptide is or comprises a zika virus glycoprotein E.

[0142] In some embodiments, a fusogen entity polypeptide is or comprises a west nile virus polypeptide, such a west nile virus glycoprotein. In some embodiments, a fusogen entity polypeptide is or comprises a west nile virus glycoprotein E.

[0143] In some embodiments, a fusogen entity polypeptide is or comprises a yellow fever virus polypeptide, such a yellow fever virus glycoprotein. In some embodiments, a fusogen entity polypeptide is or comprises a yellow fever virus glycoprotein E.

[0144] In some embodiments, a fusogen entity polypeptide is or comprises a tick-borne encephalitis virus polypeptide, such a tick-borne encephalitis virus glycoprotein. In some embodiments, a fusogen entity polypeptide is or comprises a tick-borne encephalitis virus glycoprotein E.

[0145] In some embodiments, a fusogen entity polypeptide is or comprises a herpes simplex virus polypeptide, such a herpes simplex virus glycoprotein. In some embodiments, afusogen entity polypeptide is or comprises a herpes simplex virus glycoprotein HSV-1 gB, HSV-1 gH, HSV-1 gL, and / or HSV-1 gD.

[0146] In some embodiments, a fusogen entity polypeptide is or comprises a hendra virus polypeptide, such a hendra virus glycoprotein. In some embodiments, a fusogen entity polypeptide is or comprises a hendra virus glycoprotein G and / or F.

[0147] In some embodiments, a fusogen entity polypeptide is or comprises a Newcastle disease virus polypeptide, such a Newcastle disease virus glycoprotein. In some embodiments, a fusogen entity polypeptide is or comprises a Newcastle disease virus glycoprotein Fl and / or F2.

[0148] In some embodiments, a fusogen entity polypeptide is or comprises an Epstein Banvirus polypeptide, such an Epstein Barr virus glycoprotein. In some embodiments, a fusogen entity polypeptide is or comprises an Epstein Barr virus glycoprotein gB, gH and / or gL. In some embodiments, a fusogen entity polypeptide is or comprises an Epstein Barr virus glycoprotein gp42.

[0149] In some embodiments, a fusogen entity polypeptide is or comprises a bourbon virus polypeptide, such a bourbon virus glycoprotein. In some embodiments, a fusogen entity polypeptide is or comprises a bourbon virus glycoprotein Gp.

[0150] In some embodiments, a fusogen entity polypeptide is or comprises a varicella-zoster virus polypeptide, such a varicella-zoster virus glycoprotein. In some embodiments, a fusogen entity polypeptide is or comprises a varicella-zoster virus glycoprotein gB, gH, gE, and / or gL.

[0151] In some embodiments, a fusogen entity polypeptide is or comprises a severe fever with thrombocytopenia virus polypeptide, such a severe fever with thrombocytopenia virus glycoprotein. In some embodiments, a fusogen entity polypeptide is or comprises a severe fever with thrombocytopenia virus glycoprotein gB, gH, and / or gL.

[0152] In some embodiments, a fusogen entity polypeptide is or comprises a hantavirus polypeptide, such a hantavirus glycoprotein. In some embodiments, a fusogen entity polypeptide is or comprises a hantavirus glycoprotein Gn and / or Gc.

[0153] In some embodiments, a fusogen entity polypeptide is or comprises a vaccinia virus polypeptide, such a vaccinia virus glycoprotein. In some embodiments, a fusogen entitypolypeptide is or comprises a vaccinia virus glycoprotein A28, A21, A16, F9, G9, G3, H2, J5, L5, LI, A33, A34, B5, and / or 03.

[0154] In some embodiments, a fusogen entity polypeptide is or comprises a simian immunedeficient virus polypeptide, such a simian immunedeficient virus glycoprotein. In some embodiments, a fusogen entity polypeptide is or comprises a simian immunedeficient virus Env glycoprotein.

[0155] In some embodiments, a fusogen entity polypeptide is or comprises a human immunedeficient virus (e.g., HIV-1) polypeptide, such a human immunedeficient virus glycoprotein. In some embodiments, a fusogen entity polypeptide is or comprises a human immunedeficient virus Env glycoprotein.

[0156] In some embodiments, a fusogen entity polypeptide is or comprises a junin virus polypeptide, such a junin virus glycoprotein. In some embodiments, a fusogen entity polypeptide is or comprises a junin virus glycoprotein complex GPC.

[0157] In some embodiments, a fusogen entity polypeptide is or comprises a machupo virus polypeptide, such a machupo virus glycoprotein. In some embodiments, a fusogen entity polypeptide is or comprises a machupo virus glycoprotein complex GPC.

[0158] In some embodiments, a fusogen entity polypeptide is or comprises a bas-congo virus polypeptide, such a bas-congo virus glycoprotein. In some embodiments, a fusogen entity polypeptide is or comprises a bas-congo virus glycoprotein G.

[0159] In some embodiments, a fusogen entity polypeptide is or comprises a la crosse virus polypeptide, such a la crosse virus glycoprotein. In some embodiments, a fusogen entity polypeptide is or comprises a la crosse virus glycoprotein Gc and / or Gn.

[0160] In some embodiments, a fusogen entity polypeptide is or comprises a human cytomegalovirus polypeptide, such a human cytomegalovirus glycoprotein. In some embodiments, a fusogen entity polypeptide is or comprises a human cytomegalovirus glycoprotein gH, gL, gO, UL128, UL130, and / or UL131 A.

[0161] In some embodiments, a fusogen entity polypeptide is or comprises a thogoto virus polypeptide, such a thogoto virus glycoprotein. In some embodiments, a fusogen entity polypeptide is or comprises a thogoto virus glycoprotein Gp.

[0162] In some embodiments, a fusogen entity polypeptide is or comprises a dhori virus polypeptide, such a dhori virus glycoprotein. In some embodiments, a fusogen entity polypeptide is or comprises a dhori virus glycoprotein Gp.

[0163] In some embodiments, a fusogen entity polypeptide comprises a non-viral fusogen moiety.

[0164] In some embodiments, a fusogen entity polypeptide comprises a human fusogen moiety.

[0165] In some embodiments, a fusogen entity polypeptide is or comprises a human fusogen moiety selected from the group consisting of Syncytin-1, Syncytin-2, CD9, CD81, myomarker, CD200 (OX-2G), DC-STAMP, OC-STAMP, E-Cadherin (CADH1), Cadherin 11 (CAD11), matrix meralloproteinase-9, zonula occludens-1 (ZO-1), myomerger, Annexin Al, Annexin A5, CD44, P2X purinoceptor 7, IZUMO1, Juno, StartD7, receptor-like 1, associated protein 4, CD63, connexin 43 (Cx43), CD36, MFR, tumor-associated member 1, GLPRl-like protein 1, associated protein 43, ERVV-1, ERVV-2, ERVH48-1, ERVMER34- 1, ERV3-1, and ERVK13-l.

[0166] In some embodiments, a fusogen entity polypeptide is or comprises a human fusogen moiety selected from the group consisting of Syncytin-1, Syncytin-2, CD9, CD81, myomarker, CD200 (OX-2G), DC-STAMP, OC-STAMP, E-Cadherin (CADH1), Cadherin 11 (CAD11), matrix meralloproteinase-9, zonula occludens-1 (ZO-1), myomerger, Annexin Al, Annexin A5, CD44, P2X purinoceptor 7, IZUMO1, Juno, StartD7, receptor-like 1, associated protein 4, CD63, connexin 43 (Cx43), CD36, MFR, tumor-associated member 1, GLPRl-like protein 1, and associated protein 43.

[0167] In some embodiments, a fusogen entity polypeptide mediates fusion between lipid bilayers when displayed alone or co-displayed with SIRPa on the surface of an engineered lipid bilayer particle.

[0168] Fusogen entity polypeptides provided by and / or utilized in accordance with the present disclosure are characterized by an ability to mediate fusion between lipid bilayers (e.g., fusion between an engineered lipid bilayer particle and a lipid bilayer recipient cell). In some embodiments, a fusogen entity polypeptide mediates fusion between lipid bilayers when displayed alone (e.g., without SIRPa and / or an affinity entity polypeptide) on the surface of an engineered lipid bilayer particle. In some embodiments, a fusogen entitypolypeptide mediates fusion between lipid bilayers when displayed with its natural partner fusogen entity polypeptide (e.g., Nipah F and G envelope proteins) on the surface of an engineered lipid bilayer particle.

[0169] In some embodiments, a fusogen entity polypeptide demonstrates greater fusion activity to a recipient cell when co-displayed with SIRPa on an engineered lipid bilayer particle when SIRPa is bound to its corresponding target ligand on the surface of the recipient cell, compared to fusion activity to a recipient cell that does not express a corresponding SIRPa target ligand.

[0170] Those skilled in the art will be aware of a variety of assays and technologies that may be useful to assess the ability of a fusogen entity polypeptide to mediate fusion between lipid bilayers. In some embodiments, fusion activity is quantified via a functional delivery assay in which a readout is generated only when the engineered lipid bilayer particle fuses with a recipient cell membrane. For example, if an engineered lipid bilayer particle is engineered to contain the core of a lentivirus vector, rate of functional delivery can be quantified by the number of recipient cells that express a lentivirus-encoded reporter gene after being exposed to such engineered lipid bilayer particles. Rate of functional delivery can thus provide a metric of fusion activity. Other non-limiting examples include complementation assays (e.g., delivery of half of a split reporter protein that that only functional delivery leads to reconstitution and generation of a signal that can be measured by fluorescence or luminescence-based assays). Another non-limiting example is delivery of an enzyme that acts upon DNA (e.g., Cre or a Cas9-sgRNA complex) such that modifications of the recipient cell DNA can be evaluated and quantified.

[0171] In some embodiments, the ability of a fusogen entity polypeptide to mediate fusion between two lipid bilayers may be assessed in a complex context - e.g., in the context of an engineered lipid bilayer particle displaying a SIRPa and a fusogen entity polypeptide (or extract or other fraction thereof, such as for example a population of engineered lipid bilayer particles, particle extract or fraction thereof). In some embodiments, a fusogen entity polypeptide is considered to have sufficient activity to be used as fusogen if it mediates functional delivery that is distinguishable from background in a given assay. In some embodiments, a fusogen entity polypeptide is considered to have sufficient activity to be used as fusogen if delivers a cargo to at least 10%, at least 15%, at least 20%, at least 25%,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% of recipient cells.

[0172] Examples included herein (e.g., Examples 1, 2, 4, and 5) document usefulness of SIRPa alone or in combination with a fusogen entity polypeptide as described herein. For instance, Example 1 demonstrates that SIRPa when co-displayed with a fusogen entity polypeptide on the surface of an engineered lipid bilayer particle showed good transduction of Jurkat T cells (Figures 2B and 2E) and HEK293FT cells (Figures 3E and 3F)

[0173] In many embodiments, the ability of a fusogen entity polypeptide to mediate fusion between lipid bilayers is assessed with respect to functional delivery / transduction of a cargo to a recipient cell.

[0174] In some embodiments, a fusogen entity polypeptide for use in accordance with the present disclosure is characterized in that it mediates fusion between two lipid bilayers (i.e., fusion activity). In some embodiments, fusogen entity polypeptide fusion activity is comparable to a reference fusogen entity polypeptide (e.g., a VSV-G or variant thereof). In some embodiments, SIRPa is co-displayed with a fusogen entity polypeptide that combined increase transduction of engineered lipid bilayer particles to recipient cells.

[0175] As noted herein, in some embodiments, a fusogen entity polypeptide may show sufficient structural and functional similarity to a known reference fusogen entity polypeptide (e.g., to a VSV-G whose mature amino acid sequence is set forth in SEQ ID NO: 14 or SEQ ID NO: 16) that one skilled in the art will appreciate its proper classification as an “fusogen entity polypeptide” as described herein, but may nonetheless include certain amino acid sequence difference(s) and / or other modifications (e.g., attachment of pendant groups such as glycans, PEG moieties, etc., fusion with tag or other functional peptides - while preferably still preserving attribute(s) and / or benefit(s) as described herein relative to such reference fusogen entity polypeptide.

[0176] In some embodiments, fusogen entity polypeptides as described herein, and / or compositions that include them, may be characterized by one or more features or attributes (e.g., one or more physical and / or functional parameters) that, for example, may make them particularly useful in context(s) of interest (e.g., in therapeutic context(s) and / or in diagnostic and / or research contexts).

[0177] Relevant physical parameters may include, for example, size and / or stability. Relevant functional parameters may include, for example, ability to mediate fusion and / or selectivity, toxicity, pharmacokinetic and / or pharmacodynamics performance, etc.

[0178] In some embodiments, a provided fusogen entity polypeptide is characterized in that, when it is expressed by an engineered production cell (e.g., via administration of a system or composition that includes or encodes it), it displays at an acceptable level on engineered lipid bilayer particles that are produced by such engineered production cell. Those skilled in the art will be aware of what level might be “acceptable” in a given context and / or in a particular assay.

[0179] In some embodiments, a fusogen entity polypeptide is found in a virus selected from the group consisting of vesicular stomatitis virus, Measles virus, Sindbis virus, Tupaia paramyxovirus, Nipah virus, Chandipura virus, Rabies virus, Lymphocytic choriomeningitis virus, Mokola virus, Ross River virus, Ross River virus, Semliki Forest virus, Venezuelan equine encephalitis virus, Ebola virus, Marburg virus, Lassa virus, Avian leukosis virus, Jaagsiekte sheep retrovirus, Moloney Murine leukemia virus, Gibbon ape leukemia virus, Feline endogenous retrovirus (RD114), Human T-lympho tropic virus 1, Human foamy virus, Maedi-visna virus, SARS-CoV, SARS-CoV-2, Sendai virus, Respiratory syncytia virus, Human parainfluenza virus type 3, Human parainfluenza virus type 4, Hepatitis C virus, Hepatitis C virus, Influenza virus, Fowl plague virus, Autographa californica multiple nucleopolyhedro virus, Baboon endogenous retrovirus, Cocal virus, Japanese encephalitis virus, Dengue virus, Zika virus, West Nile virus, Yellow fever virus, Tick-bome encephalitis virus, Herpes simplex virus 1, Hendra virus, Newcastle disease virus, Epstein Barr virus, Bourbon virus, Varicella-zoster virus, Severe fever with thrombocytopenia virus, Hantavirus, Vaccinia virus, Simian immunodeficiency virus, Human immunodeficiency virus, Junin virus, Machupo virus, Bas-Congo virus, La Crosse virus, Human cytomegalovirus, Human cytomegalovirus, Thogoto virus, and Dhori virus.

[0180] In some embodiments, a fusogen entity polypeptide is selected from a lentiviral glycoprotein or a glycoprotein selected from vesicular stomatitis glycoprotein (VSV-G), measles virus glycoprotein H, measles virus glycoprotein F, rabies virus glycoprotein (RVG), gibbon ape leukemia virus glycoprotein (GaLV), ampho tropic murine leukemia virus glycoprotein (MLV-A), feline endogenous virus (RD114) glycoprotein, fowl plaguevirus (FPV) glycoprotein, Ebola virus (EboV) glycoprotein, vesicular stomatitis virus (VSV) glycoprotein, and lymphocytic choriomeningitis virus (LCMV) glycoprotein.

[0181] In some embodiments, a fusogen entity polypeptide is selected from the group consisting of VSV-G, Cocal glycoprotein, RD114 / TR glycoprotein, Nippa glycoprotein, Measels glycoprotein, and variants thereof.

[0182] In some embodiments, a fusogen entity polypeptide is a VSV-G.

[0183] In some embodiments, a fusogen entity polypeptide is a Cocal glycoprotein.

[0184] In some embodiments, a VSV-G is a wild type VSV-G. In some embodiments, a wild type VSV-G has an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid SEQ ID NO: 14. In some embodiments, a wild type VSV-G has an amino acid sequence that is identical to the amino acid SEQ ID NO: 14.

[0185] In some embodiments, a VSV-G is a mutated VSV-G. In some embodiments, a VSV-G comprises one or more mutations that ablate binding of the mutated VSV-G to its cognate receptor relative to a comparable wild type VSV-G that does not comprise the one or more mutations. In some embodiments, one or more mutations are one more amino acid substitutions. In some embodiments, one or more amino acid substitutions comprise K47Q, R354A and combination thereof, wherein the numbering is with reference to SEQ ID NO: 11. In some embodiments, a mutated VSV-G has an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid SEQ ID NO: 16. In some embodiments, a mutated VSV-G has an amino acid sequence that is identical to the amino acid SEQ ID NO: 16.

[0186] In some embodiments, a fusogen entity polypeptide comprises one or more fragments of a SIRPa. In some embodiments, a fusogen entity polypeptide comprises a SIRPa secretory signal or a portion thereof. In some embodiments, a fusogen entity polypeptide comprises an amino acid sequence according to SEQ ID NO: 2. In some embodiments, a fusogen entity polypeptide comprises a SIRPa extraparticle portion or a portion thereof. In some embodiments, a fusogen entity polypeptide comprises an amino acid sequence according to SEQ ID NO: 3. In some embodiments, a fusogen entity polypeptide comprises a SIRPa lipid bilayer association portion such as a transparticle portion or portion thereof. In some embodiments, a fusogen entity polypeptide comprises anamino acid sequence according to SEQ ID NO: 4. In some embodiments, a fusogen entity polypeptide comprises a SIRPa intraparticle portion or portion thereof. In some embodiments, a fusogen entity polypeptide comprises an amino acid sequence according to SEQ ID NO: 5

[0187] Exemplary amino acid sequences of certain fusogen entity polypeptides are provided in Table 1 and Table 3 and exemplary nucleic acid sequences encoding certain fusogen entity polypeptides are provided in Table 2 and Table 4.Affinity Entity Polypeptides

[0188] Without wishing to be bound by a particular theory, it is proposed that the unique combination of a SIRPa and an affinity entity polypeptide provides targeted entry and fusion of engineered lipid bilayer particles into specific recipient cells with high efficiency (See Example 2, Figure 6).

[0189] The present disclosure provides affinity entity polypeptides that binds to a specific target on a recipient cell. In some embodiments, engineered lipid bilayer particles can be useful in mediating specific binding of an engineered lipid bilayer particle to a target on a particular recipient cell or a particular subset of such recipient cells (e.g., provide specificity to technologies described herein). In some embodiments, an affinity entity polypeptide mediates binding of an engineered lipid bilayer particle to a recipient cell of interest. In some embodiments, an affinity polypeptide alone does not promote cell entry and transduction. Affinity entity polypeptides provided herein are useful when designing binding to a specific type of recipient cells.

[0190] In some embodiments, an affinity entity polypeptide binds to a specific target ligand hereby limiting binding to recipient cells expressing such specific target ligand. In some embodiments, an affinity entity polypeptide is selected from the group consisting of an antibody, a Fab, a Fab', a F(ab')2, a Fd, a scFv, a single-chain antibody, a disulfide-linked Fvs (sdFv), an affinibody, a DARPIN, a nanobody, a variable lymphocyte receptor (VLR), and a camelid antibody. In some embodiments, when an affinity entity polypeptide is codisplayed on an engineered lipid bilayer particle with a SIRPa and optionally a fusogen entity polypeptide, binding of the affinity entity polypeptide to at target on the surface of the recipient cell promotes fusion of particle with the recipient cell. Without wishing to be bound by a particular theory, it is proposed that the unique combination of SIRPa and anaffinity entity polypeptide provides synergistic binding and / or entry of engineered lipid bilayer particles into specific recipient cells. In some embodiments, when SIRPa and an affinity entity polypeptide are co-displayed together they bind synergistically to recipient cell targets.

[0191] In some embodiments, the present disclosure provides and / or utilizes an affinity entity polypeptide. In some embodiments, the present disclosure provides and / or utilizes a nucleotide sequence that encodes an affinity entity polypeptide or fragments thereof.

[0192] In some embodiments, technologies (e.g., a system, engineered lipid bilayer particle and engineered production cells) according to the present disclosure comprise an affinity entity polypeptide. In some embodiments, technologies according to the present disclosure comprise at least one affinity entity polypeptide. In some embodiments, technologies according to the present disclosure comprise one or more affinity entity polypeptides.

[0193] In some embodiments, an affinity entity polypeptide comprises a secretory signal. In some embodiments, an affinity entity polypeptide comprises a FLAG tag. In some embodiments, an affinity entity polypeptide does not comprise a FLAG tag, e.g., when the affinity entity polypeptide is a native polypeptide. In some embodiments, an affinity entity polypeptide comprises an affinity moiety. In some embodiments, an affinity entity polypeptide comprises a linker. In some embodiments, an affinity entity polypeptide does not comprise a linker e.g., when the affinity entity polypeptide is a native polypeptide. In some embodiments, an affinity entity polypeptide comprises a membrane association portion. In some embodiments, an affinity entity polypeptide comprises an intraparticle portion.

[0194] In some embodiments, an affinity entity polypeptide consists or comprises of an affinity moiety and a membrane association portion.

[0195] In some embodiments, an affinity entity polypeptide is an engineered polypeptide. In some embodiments, the order from the N-terminal to the C-terminal of an affinity entity polypeptide is as follows: a secretory signal, an affinity moiety, a linker, and / or a membrane association portion. In some embodiments, the order from the N-terminal to the C-terminal of an entity polypeptide is as follows: a secretory signal, a FLAG tag, an affinity moiety, a linker, and / or a membrane association portion.

[0196] In some embodiments, a fiisogen entity polypeptide is a wild type fiisogen entity polypeptide. In some embodiments, a fusogen entity polypeptide is a native fusogen entity polypeptide. In some embodiments, a fiisogen entity polypeptide is native to a particular production cell. In some embodiments, an affinity entity polypeptide is an engineered polypeptide. In some embodiments, an affinity entity polypeptide (e.g., an engineered fusogen entity polypeptide) is a variant of a wild-type polypeptide and / or of a native polypeptide.

[0197] In some embodiments, the order from the N-terminal to the C-terminal of an affinity entity polypeptide is as follows: a secretory signal, an affinity moiety, a membrane association portion, and / or an intraparticle portion.

[0198] In some embodiments, an affinity entity polypeptide, or an affinity moiety thereof, has an amino acid sequence that includes a characteristic sequence element and / or shares an overall degree of sequence identity with a reference affinity entity polypeptide (e.g., a wildtype affinity entity polypeptide, and / or an affinity entity polypeptide whose amino acid sequence is included in Table 11). In some embodiments, an affinity entity polypeptide is a variant of such a reference affinity entity polypeptide. In some embodiments, an affinity entity polypeptide includes one or more modifications, such as glycosylation, lipidation, phosphorylation, etc.

[0199] In some embodiments, an affinity entity polypeptide comprises a secretory signal, e.g., that is functional in mammalian cells. In some embodiments, a utilized secretory signal is a heterologous secretory signal. In some embodiments, a heterologous secretory signal comprises or consists of a non-human secretory signal. In some embodiments, a heterologous secretory signal comprises or consists of a viral secretory signal. In some embodiments, a secretory signal comprises or consists of a VSV-G secretory signal. In some embodiments, a secretory signal is characterized by a length of about 10 to 30 amino acids. In some embodiments, a secretory signal is positioned at the N-terminus of an affinity entity polypeptide described herein. In some embodiments, a secretory signal preferably allows transport of an affinity entity polypeptide with which it is associated into a defined cellular compartment, preferably a cell surface, endoplasmic reticulum (ER), endosomal-lysosomal compartment and / or Golgi apparatus (e.g., glycosylate the affinity entity polypeptide).

[0200] In some embodiments, an affinity entity polypeptide comprises a FLAG tag. In some embodiments, a utilized FLAG tag is a heterologous secretory tag.

[0201] In some embodiments, an affinity entity polypeptide comprises an affinity moiety. In some embodiments, an affinity moiety binds to the surface of a recipient cell (e.g., a target ligand on the surface of a recipient cell). In some embodiments, an affinity moiety binds a specific recipient cell target ligand. A recipient cell may express a number of unique targeting ligands on the cell surface and an affinity moiety can be designed to target one or more such target ligands. In some embodiments, a target ligand is an epitope, a receptor, a protein, a carbohydrate, a lipid, or combinations thereof or conformational states thereof.

[0202] In some embodiments, an affinity moiety is a heterologous affinity moiety. In some embodiments, an affinity moiety is positioned at the N-terminus of an affinity entity polypeptide. In some embodiments, an affinity moiety is displayed on the surface of an engineered lipid bilayer particle. It may be displayed in a way that promotes binding of the lipid bilayer of the engineered lipid bilayer particle with a target ligand on the surface of a recipient cell.

[0203] In some embodiments, an affinity moiety is characterized in that it binds to a particular target ligand (e.g., an epitope on a recipient cell surface). In some embodiments, an affinity moiety is characterized in that it preferentially binds a particular recipient cell target ligand compared to another cell ligand. In some embodiments, an affinity entity moiety binds to a biologic target and changes its conformation thereby drive activity, activate the target, or a combination thereof. In some embodiments, an affinity entity moiety binds to a target that is exposed in the particle uptake and / or fusion process (e.g., endosome).

[0204] In some embodiments, an affinity moiety specifically binds to the surface of an immune cell. In some embodiments, an affinity moiety is an immune polypeptide. In some embodiments, an affinity moiety is selected from the group consisting of an antibody, a Fab, a Fab', a F(ab')2, a Fd, a scFv, a single-chain antibody, a disulfide-linked Fvs (sdFv), an affinibody, a DARPIN, a nanobody, a variable lymphocyte receptor (VLR), and a camelid antibody.

[0205] In some embodiments, an affinity moiety specifically binds to the surface of an immune cell (e.g., a T cell, such as a CD4+ and / or CD8+ T cell). In some embodiments, an affinity moiety is characterized in that it binds to a recipient cell expressing CD5, CD2, CD3, CD47, or a combination thereof.

[0206] In some embodiments, an affinity moiety binds to CD2. In some embodiments, an affinity moiety comprises or is an anti-CD2 scFv or a fragment thereof. In some embodiments, an affinity moiety comprises an amino acid according to SEQ ID NO: 27 or an amino acid sequence that is at least 80%, such as at least 85%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to that of SEQ ID NO: 27.

[0207] In some embodiments, an affinity moiety binds to CD3. In some embodiments, an affinity moiety comprises or is an anti-CD3 scFv or a fragment thereof. In some embodiments, an affinity moiety comprises an amino acid according to SEQ ID NO: 28 or an amino acid sequence that is at least 80%, such as at least 85%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to that of SEQ ID NO: 28.

[0208] In some embodiments, an affinity moiety binds to CD5. In some embodiments, an affinity moiety comprises or is an anti-CD5 VLR or a fragment thereof. In some embodiments, an affinity moiety comprises an amino acid according to SEQ ID NO: 29 or an amino acid sequence that is at least 80%, such as at least 85%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to that of SEQ ID NO: 29.

[0209] In some embodiments, an affinity moiety binds to CD5. In some embodiments, an affinity moiety comprises or is an anti-CD5 scFv or a fragment thereof. In some embodiments, an affinity moiety comprises an amino acid according to SEQ ID NO: 30 or an amino acid sequence that is at least 80%, such as at least 85%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to that of SEQ ID NO: 30.

[0210] Exemplary amino acid sequences of certain affinity moieties are provided in Table 5 and exemplary nucleic acid sequences encoding affinity moieties are provided in Table 6.Table 5: Exemplary Amino acid SequencesTable 6: Exemplary Nucleic Acid Sequences

[0211] In some embodiments, an affinity entity polypeptide described herein includes one or more linkers. In some embodiments, an affinity moiety and a membrane association portion are directly linked or indirectly linked.

[0212] In some embodiments, an affinity moiety and a membrane association portion are directly linked or indirectly linked.

[0213] In some embodiments, an affinity linker is or comprises 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acids. In some embodiments, an affinity linker is or comprises no more than about 30, 25, 20, 15, 10 or fewer amino acids. An affinity linker can include any amino acid sequence and is not limited to any particular amino acids. In some embodiments, an affinity linker comprises one or more glycine (G) amino acids. In some embodiments, an affinitylinker comprises one or more serine (S) amino acids. In some embodiments, a linker is or comprises GGGS. A linker may improve spacing and flexibility. In some embodiments, a linker is a structured element (e.g., a hinge derived from an immunoglobulin family protein, or an alpha helix, or combinations thereof).

[0214] In some embodiments, an affinity linker is one listed in Table 7, or a linker having 1, 2, 3, 4, or 5 amino acid differences relative thereto.

[0215] An exemplary amino acid sequence of a linker is provided in Table 7 and exemplary nucleic acid sequences encoding a linker is provided in Table 8.Table 7: Exemplary Amino acid SequencesTable 8: Exemplary Nucleic Acid Sequences

[0216] In some embodiments, an affinity entity polypeptide comprises a membrane association portion, such as a transmembrane portion. In some embodiments, a membrane association portion is positioned at the C-terminus of an affinity entity polypeptide. In some embodiments, a membrane association portion is characterized by a length of about 15 amino acids to about 25 amino acids. In some embodiments, a membrane association portion is a heterologous a membrane association portion. In some embodiments, a membrane association portion is a transmembrane portion.

[0217] Membrane association portions such as transmembrane portions are known in the art. In some embodiments, a membrane association portion comprises or is a transmembrane domain of Hemagglutinin (HA) of Influenza virus, Env of HIV- 1, equine infectious anaemia virus (EIAV), murine leukaemia virus (MLV), mouse mammary tumor virus, G protein ofvesicular stomatitis virus (VSV-G), Rabies virus, or a seven transmembrane domain receptor.

[0218] In some embodiments, a membrane association portion allows that an affinity moiety is displayed on the surface of a lipid bilayer particle. In some embodiments, a membrane association portion positions an affinity moiety on the surface of a lipid bilayer particle such that it can bind to a recipient cell surface epitope.

[0219] In some embodiments, a membrane association portion comprises or consists of a non-human transmembrane region. In some embodiments, a membrane association portion comprises or consists of a viral transmembrane region. In some embodiments, a membrane association portion comprises or consists of a transmembrane portion of PDGFR. In some embodiments, a transmembrane domain comprises a platelet-derived growth factor receptor (PDGFR) transmembrane domain of SEQ ID NO: 25. In some embodiments, a PDGFR transmembrane domain is encoded by a nucleotide sequence according to SEQ ID NO: 26.

[0220] In some embodiments, a membrane association portion comprises an amino acid according to SEQ ID NO: 29 or an amino acid sequence that is at least 80%, such as at least 85%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to that of SEQ ID NO: 29.

[0221] In some embodiments, a membrane association portion comprises an amino acid according to SEQ ID NO: 30 or an amino acid sequence that is at least 80%, such as at least 85%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to that of SEQ ID NO: 30.

[0222] Exemplary amino acid sequences of certain membrane association portions are provided in Table 9 and exemplary nucleic acid sequences encoding membrane association portions are provided in Table 10.Table 9: Exemplary Amino acid SequencesTable 10: Exemplary Nucleic Acid Sequences

[0223] Affinity entity polypeptides provided by and / or utilized in accordance with the present disclosure are characterized by an ability to bind to specific target ligands expressed on the surface of a recipient cell (e.g., mediate binding between an engineered lipid bilayer particle and a lipid bilayer recipient cell).

[0224] In some embodiments, an affinity entity polypeptide is a folded protein. In some embodiments, an affinity entity polypeptide is displayed on the surface of a bilayer lipid particle. In some embodiments, lipid bilayer particles display at least 5, at least 10, at least 15, at least 20, at least 50, at least 100, at least 200, at least 300, at least 400, at least 500 copies of the membrane polypeptide

[0225] In some embodiments, binding of an affinity entity polypeptide to its target triggers signaling in the recipient cell. In some embodiments, bindings of an affinity entity polypeptide to its target triggers internalization of the target. In some embodiments, an affinity entity polypeptide binds it target at a high valency and this mode of binding induced signaling and / or internalization of its target into the recipient cell.

[0226] In many embodiments, an affinity entity polypeptide according to the present disclosure mediates binding between lipid bilayers.

[0227] An important distinction between a naturally evolved viral envelope protein and the engineered envelope functions that are of use for vector engineering derives from a distinction between viral replication and packaging of viral vectors. A virus typicallyreplicates in one or several cell types, meaning that a virus is typically produced in the same type of cell that it can infect. In order to infect a cell, a virus binds a target on the cell surface and then mediates fusion to gain entry to the cell’s machinery. However, premature fusion of a virus to the virus-producing cell surface would render the virus ineffective at infecting a new cell (and reproducing). Thus, viruses evolve mechanisms to suppress fusion during virus biogenesis, leading to innovations such as low pH-induced exposure of fusogenic domains (viruses do not experience low pH during budding, but only later after endosomal uptake in recipient cells). In contrast, viral vectors can be produced in cells that lack the targets of the envelope proteins being expressed and loaded onto viral vectors. This provides additional options as to which proteins and mechanisms we can employ when producing viral vectors (compared to what one might find in nature when examining natural viral proteins and mechanisms).

[0228] In some embodiments, an affinity entity polypeptide specifically binds to the surface of an immune cell (e.g., a T cell, such as a CD4+ and / or CD8+ T cell).

[0229] In some embodiments, an affinity entity polypeptide is characterized in that it binds to a recipient cell expressing CD2, CD3, CD5, or a combination thereof.

[0230] In some embodiments, an affinity entity polypeptide is selected from the group consisting of anti-CD2 scFv, anti-CD scFv, anti-CD5 scFv, anti-CD5 VLR, or combination thereof.

[0231] In some embodiments, an affinity entity polypeptide comprises an amino acid according to SEQ ID NO: 37 or an amino acid sequence that is at least 80%, such as at least 85%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to that of SEQ ID NO: 37.

[0232] In some embodiments, an affinity entity polypeptide comprises an amino acid according to SEQ ID NO: 38 or an amino acid sequence that is at least 80%, such as at least 85%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to that of SEQ ID NO: 38.

[0233] In some embodiments, an affinity entity polypeptide comprises an amino acid according to SEQ ID NO: 39 or an amino acid sequence that is at least 80%, such as at least85%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to that of SEQ ID NO: 39.

[0234] In some embodiments, an affinity entity polypeptide comprises an amino acid according to SEQ ID NO: 40 or an amino acid sequence that is at least 80%, such as at least 85%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to that of SEQ ID NO: 40.

[0235] Exemplary amino acid sequences of certain affinity entity polypeptides are provided in Table 11 below and exemplary nucleic acid sequences of certain affinity entities are provided in Table 12.Table 11: Exemplary Affinity Amino Acid SequencesTable 12: Exemplary Affinity Nucleic Acid SequencesPolynucleotides

[0236] In some embodiments, the present disclosure provides and / or utilizes polynucleotides that encode and / or regulate expression of one or more SIRPa. In some embodiments, the present disclosure provides and / or utilizes polynucleotides that encode and / or regulate expression of one or more fusogen entity polypeptides and / or affinity entity polypeptides as described herein.

[0237] In some embodiments, polynucleotides provided by and / or utilized in accordance with the present disclosure may have a nucleotide sequence that is codon optimized, e.g., for expression in a particular production cell (e.g., engineered production cell) of interest.

[0238] Those skilled in the art are familiar with preferred codons for use in various different cell types of interest. In general, as is understood, codon optimization typically involves selecting a nucleic acid sequence for optimal expression in a host cell of interest by utilizing codons that are more frequently or most frequently used in genes of that host cell while maintaining native desired amino acid sequence.

[0239] Various species exhibit particular preference for certain codons that encode a particular amino acid. Without wishing to be bound by any particular theory, codon preference (differences in codon usage between organisms) often correlates with mRNA translation efficiency in the relevant host cell, which is in turn believed to be dependent on, among other things, the properties of codons being translated and / or availability of particular transfer RNAs (tRNAs). The predominance of particular tRNAs in a cell may generally be a reflection of the codons used most frequently in polypeptide synthesis in that cell. Accordingly, genes may be tailored for optimal gene expression in a given cell type (e.g., organism) through codon optimization. Codon usage tables are available, for example, at the "Codon Usage Database" available at www.kazusa.orjp / codon / and these tables may be adapted in a number of ways. Computer algorithms for codon optimizing a particular sequence for expression in a particular subject or its cells, such as Gene Forge (Aptagen; Jacobus, PA), are also available.

[0240] In some embodiments, a polynucleotide as comprises a nucleic acid sequence that encodes a SIRPa as provided herein above. In some embodiments, a polynucleotide comprises a nucleic acid sequence according to any one of SEQ ID NO: 6-10 or an nucleic acid sequence that is at least 80%, such as at least 85%, such as at least 90%, such as at least91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to any one of SEQ ID NO: 6-10.

[0241] In some embodiments, a polynucleotide comprises a nucleic acid sequence that encodes a fusogen entity polypeptide or a fragment thereof as provided herein above.

[0242] In some embodiments, a polynucleotide comprises a nucleic acid sequence according to SEQ ID NO: 18 or an nucleic acid sequence that is at least 80%, such as at least 85%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to that of SEQ ID NO: 18.

[0243] In some embodiments, a polynucleotide comprises a nucleic acid sequence according to SEQ ID NO: 19 or an nucleic acid sequence that is at least 80%, such as at least 85%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to that of SEQ ID NO: 19.

[0244] In some embodiments, a polynucleotide comprises a nucleic acid sequence according to SEQ ID NO: 20 or an nucleic acid sequence that is at least 80%, such as at least 85%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to that of SEQ ID NO: 20.

[0245] In some embodiments, a polynucleotide comprises a nucleic acid sequence according to SEQ ID NO: 21 or SEQ ID NO: 23 or an nucleic acid sequence that is at least 80%, such as at least 85%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to that of SEQ ID NO: 21 or SEQ ID NO: 23.

[0246] In some embodiments, a polynucleotide comprises a nucleic acid sequence according to SEQ ID NO: 22 or SEQ ID NO: 24 or an nucleic acid sequence that is at least 80%, such as at least 85%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least97%, such as at least 98%, such as at least 99%, such as 100% identical to that of SEQ ID NO: 22 or SEQ ID NO: 24.

[0247] In some embodiments, a polynucleotide comprises a nucleic acid sequence that encodes an affinity entity polypeptide or a fragment thereof as provided herein above.

[0248] In some embodiments, a polynucleotide comprises a nucleic acid sequence according to any one of SEQ ID NO:31-34, or an nucleic acid sequence that is at least 80%, such as at least 85%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to any one of SEQ ID NO: 31-34.

[0249] In some embodiments, a polynucleotide comprises a nucleic acid sequence according to SEQ ID NO:36, or an nucleic acid sequence that is at least 80%, such as at least 85%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to SEQ ID NO: 36.

[0250] In some embodiments, a polynucleotide comprises a nucleic acid sequence according to SEQ ID NO:26, or an nucleic acid sequence that is at least 80%, such as at least 85%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to SEQ ID NO: 26.

[0251] In some embodiments, a polynucleotide comprises a nucleic acid sequence according to any one of SEQ ID NO:41-44, or an nucleic acid sequence that is at least 80%, such as at least 85%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to any one of SEQ ID NO: 41-44.

[0252] In some embodiments, a polynucleotide provided by and / or utilized in accordance with the present disclosure may be or comprise RNA. In some embodiments, a polynucleotide provided by and / or utilized in accordance with the present disclosure may be or comprise DNA. In some embodiments, a polynucleotide provided by and / or utilized in accordance with the present disclosure may be or comprise single-stranded DNA. In someembodiments, a polynucleotide provided by and / or utilized in accordance with the present disclosure may be or comprise double-stranded DNA.

[0253] In some embodiments, the present disclosure provides a system comprising a polynucleotide encoding SIRPa. In some embodiments, a system comprising a polynucleotide encoding SIRPa as provided herein further comprises a polynucleotide encoding fusogen entity polypeptide, and / or a polynucleotide encoding an affinity entity polypeptide.

[0254] In some embodiments, provided polynucleotides may be incorporated into one or more molecules (e.g., vectors or plasmids). In some embodiments, the polynucleotides are part of a single polynucleotide molecule (e.g., vector or plasmid). In some embodiments, polynucleotides are separate polynucleotide molecules (i.e., each polynucleotide is expressed on different vectors or plasmids). In some embodiments, at least one of the polynucleotides are circular. In some embodiments, a polyribonucleotide comprising a nucleic acid encoding SIRPa is circular. In some embodiments, a polyribonucleotide comprising a nucleic acid encoding SIRPa is linear.

[0255] In some embodiments, a polynucleotide comprising a nucleic acid sequence encoding SIRPa is stably introduced by an expression cassette into a production cell (e.g., using lentivirus, transposons, integrates, recombinases, homology-directed repair, etc.). In some embodiments, a polynucleotide comprising a nucleic acid sequence encoding SIRPa is constitutively expressed in a production cell. In some embodiments, a polynucleotide comprising a nucleic acid sequence encoding SIRPa is inducible expressed in a production cell.

[0256] In some embodiments, such molecules (e.g., vectors) may include one or more expression control elements (e.g., one or more of promoters, transcriptional regulator binding sites such as enhancers or repressor sites, transcriptional terminators, splice donor and / or acceptor sites, translation start sites, polyA tails, etc.), or a complement thereof. Alternatively, or additionally, in some embodiments, such molecule (e.g., vector) may include a replication site such as an origin of replication or a primer landing site, etc., or a complement thereof In some embodiments, a polypeptide includes expression control elements sufficient to direct production of the encoded polypeptide in a production cell (e.g., an engineered production cell). In some embodiments, an expression element is a promoter. In some embodiments, a promoter is a small molecule-inducible promoter.

[0257] In some embodiments, a molecule (e.g., vector) may include an insertion site, such as an integration site or a polycloning site (e.g., that may include recognition sequences for multiple restriction enzymes) so that the molecule (e.g., vector) is adapted to receive heterologous (e.g., “payload”) nucleic acid(s), e.g., such as a provided nucleic acid encoding a fusion entity polypeptide and / or an affinity entity polypeptide, and, in some embodiments to permit, facilitate, or achieve expression and / or replication or other duplication thereof.

[0258] In some embodiments, a molecule (e.g., vector) may include one or more detection and / or one or more selection markers, as is known in the art, e.g., to facilitate identification of cells that have received the molecule (e.g., vector).

[0259] Those skilled in the art are aware of a variety of vector systems, including vector systems particularly useful and / or adapted for introduction into certain cells (e.g., microbial, such as bacterial or yeast, cells, insect cells, mammalian cells, etc.), and / or for expression (e.g., of heterologous nucleic acids) therein. For example, those skilled in the art are aware of a variety of plasmids, phage, viral vectors, etc., useful for delivering payload nucleic acids (e.g., encoding a fusion entity polypeptide and an affinity entity polypeptide as described herein) into particular cell(s) of interest in vitro and / or in vivo, and / or for expressing such payload nucleic acids in such cells.

[0260] In some particular embodiments, a provided nucleic acid encoding a fusion entity polypeptide and an affinity entity polypeptide as described herein is incorporated into a plasmid, for example, useful for introduction into and / or expression by a mammalian cell (e.g., a CHO cell, a BHK cell, a HEK293 cell, an NS0 cell, an HSC, a MSC etc.).

[0261] Provided systems may be stored and / or delivered using appropriate methods known in the art, e.g., delivery in one or more containers, such as one or more tubes. In some embodiments, two or more polynucleotides are disposed in separate containers (e.g., tubes). In some embodiments, one or more polynucleotides are disposed in the same container (e.g., tubes).

[0262] Systems as described herein are suitable for transfecting production cells so that production cells (e.g., engineered production cells) display the fusogen entity polypeptide and / or the affinity entity polypeptide. In some embodiments, provided systems deliver a polynucleotide as described herein and / or a further polynucleotide as described herein. Insome embodiments, systems as described herein are within an engineered production cell (e.g., delivered in an engineered production cell).Cargo

[0263] The present disclosure may, in some embodiments, provide technologies that comprises or deliver a cargo. In some embodiments, a lipid bilayer particle or a population of lipid bilayer particles comprises a cargo. In some embodiments, disclosed technologies comprise one or more cargos, such as multiple cargos (e.g., a first cargo, a second cargo, etc. or a combination thereof). A cargo described herein can of any chemical class, e.g., polypeptides, nucleic acids, saccharides, lipids, small entities, and combinations thereof. In some embodiments, a cargo is selected from the group consisting of plasmids, mRNA, miRNA, antisense oligonucleotide (ASO) and oligodeoxynucleotide (ODN).

[0264] In some embodiments, a cargo comprises a nucleic acid payload.

[0265] In some embodiments, a cargo comprises a polypeptide payload.

[0266] In some embodiments, a cargo is selected from the group consisting of nucleases, transcriptional regulators, based editors and enzymes.

[0267] In some embodiments, a cargo is or comprises viral particles (e.g., a lentivirus core, a nucleocapsid, or variants thereof). In some embodiments, a cargo is or comprises non-viral particles.

[0268] In some embodiments, a cargo comprises a nucleic acid payload. In some embodiments, a nucleic acid payload is integrated in the genome of the engineered production cell. In some embodiments, a nucleic acid payload is integrated in the genome of the engineered production cell. In some embodiments, a nucleic acid payload is expressed from a plasmid.

[0269] A nucleic acid payload may after transduction of the engineered lipid bilayer particle with a recipient cell be expressed by the recipient cell. In some embodiments, a recipient cell is a T cell. In some embodiments, a nucleic acid payload encodes a CAR-T.

[0270] In some embodiments, a cargo comprises or is a nucleic acid cargo. In some embodiments, a nucleic acid cargo is a synthetic nucleic acid cargo. In some embodiments, a nucleic acid cargo entity comprises chemically modifies nucleotides. Without wishing to be bound by a particular theory, a synthetic nucleic acid cargo and / or a nucleic acid cargocomprising chemically modifies nucleotides provides stability to the nucleic acid cargo. This may be particular useful for guide RNA (e.g., for use in a Cas gRNA complex).

[0271] In some embodiments, a synthetic nucleic acid cargo comprises or is an ASO or chemically modified RNA.

[0272] In some embodiments, a cargo may be a nucleic acid (e.g., DNA or RNA) or another molecule. Nucleic acid cargo entities can include, but are not limited to, DNA encoding a polypeptide of interest, mRNA, siRNA, shRNA, miRNA, an antisense oligonucleotide, and combinations thereof. Other potential cargo entities include, but are not limited to, viral and non-viral vectors (e.g., nucleocapsids) that are expressed inside a cell (or can be delivered to the cytosol of a cell), and ribonucleoprotein complexes, such as CRISPR-type entities and endogenous complexes such as DICER or RISC bound to natural or synthetic RNA such as miRNA, shRNA, etc.).

[0273] In some embodiment’s, a cargo is an intraparticle macromolecular assembly (e.g., lentiviral cores, AAV particles, other viral cores, VLP cores, and subunits thereof).

[0274] In some embodiments, a cargo is a nucleocapsid. In some embodiments, a nucleocapsid is a viral nucleocapsid. In some embodiments, a nucleocapsid is a recombinant viral nucleocapsid. In some embodiments, a nucleocapsid comprises cargo nucleic acids and cargo polypeptides.

[0275] In some embodiments, a cargo is or encodes an AAV nucleocapsid, or a LVV nucleocapsid, or fragments thereof.

[0276] In some embodiments, a cargo comprises or is a polypeptide cargo. In some embodiments, a polypeptide cargo comprises or is a cytosolic cargo molecule, wherein the cytosolic cargo may be a peptide, polypeptide, or protein of interest to be delivered to a recipient cell, such as an enzyme, a therapeutic agent (e.g., an antibody, inhibitor, an agonist, and an antagonist), or a fluorescent protein. In some embodiments, a cytosolic polypeptide cargo can be selected from any one or more of base editors, prime editors, TALENs, ZFNs, kinases, kinase inhibitors, activators or inhibitors of receptor-signaling, intrabodies, chromatin-modifying synthetic transcription factors, natural transcription factors, and mutant forms thereof. In some embodiments, a cytosolic polypeptide cargo is a CRISPR enzyme, e.g., a Type II CRISPR enzyme. In some embodiments, the CRISPR enzyme catalyzes DNA cleavage. In some embodiments, the CRISPR enzyme catalyzesRNA cleavage. In some embodiments, the CRISPR enzyme is a Cas9 protein (e.g., a naturally occurring bacterial Cas9 as well as any chimeras, mutants, homologs or orthologs). Non-limiting examples of Cas proteins include Casl, CaslB, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csnl and Csxl2), CaslO, Csyl, Csy2, Csy3, Csel, Cse2, Cscl, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmrl, Cmr3, Cmr4, Cmr5, Cmr6, Csbl, Csb2, Csb3, Csxl7, Csxl4, CsxlO, Csxl6, CsaX, Csx3, Csxl, Csxl5, Csfl, Csf2, Csf3, Csf4, homologues thereof, or modified variants thereof.Engineered Production cells

[0277] The present disclosure provides a population of engineered production cells engineered to express a polynucleotide encoding a SIRPa, such that lipid bilayer particles formed by the engineered production cells are characterized by presence on their surface at least a part of the SIRPa.

[0278] In some embodiments, a population of engineered production cells is engineered to express a polynucleotide encoding a SIRPa and a fusogen entity polypeptide, such that lipid bilayer particles formed by the engineered production cells are characterized by presence on their surface at least a part of the SIRPa and the fusogen entity polypeptide.

[0279] In some embodiments, a population of engineered production cells is engineered to express a polynucleotide encoding a SIRPa and an affinity entity polypeptide, such that lipid bilayer particles formed by the engineered production cells are characterized by presence on their surface at least a part of the SIRPa and the affinity entity polypeptide.

[0280] In some embodiments, a population of engineered production cells is engineered to express a polynucleotide encoding a SIRPa, a fusogen entity polypeptide and an affinity entity polypeptide, such that lipid bilayer particles formed by the engineered production cells are characterized by presence on their surface at least a part of the SIRPa, the fusogen entity polypeptide, and the affinity entity polypeptide.

[0281] In some embodiments, an engineered production cell engineered to express a polynucleotide encoding a SIRPa and optionally engineered to express a polynucleotide encoding a fusogen entity polypeptide; and / or a polynucleotide encoding an affinity entity polypeptide may comprise 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 othermechanism, or previously present genetic material is altered or removed, for example by substitution or deletion mutation, or by mating protocols). In some embodiments, an engineered production cell has been manipulated so that it contains and / or expresses a SIRPa, and optionally a fusogen entity polypeptide and / or an affinity entity polypeptide in an altered amount and / or according to altered timing relative to such an appropriate reference cell that has not be engineered so.

[0282] In some embodiments, a polynucleotide provided herein is integrated in the genome of the engineered cell. In some embodiments, an integration is a single copy integration per genomic loci. In some embodiments, a polynucleotide provided herein is expressed from a plasmid.

[0283] In some embodiments, engineered production cells are mammalian cells. In some embodiments, mammalian cells are human cells. Suitable mammalian cells include, but are not limited to, HEK293, HEK293FT, PER.C6, mesenchymal stem cells, megakaryocytes, iPSCs, T cells, erythrocytes and erythropoetic precursors, and iPSC-derived version of any of the preceding cells.

[0284] In some embodiments, engineered lipid bilayer particles are formed by budding from the cellular membrane of the cell. In some embodiments, a cellular membrane is a plasma membrane. In some embodiments, mammalian cells are HEK cells.

[0285] In some embodiments, engineered production cells are further engineered to express a cargo as described herein.Recipient cells

[0286] Among other things, the present disclosure demonstrates surprising effectiveness of provided technologies when applied to recipient cells, e.g., to particular recipient cells of interest, or to population(s) thereof. For example, the present disclosure documents surprising specificity and / or efficiency of cargo delivery recipient cells, or populations thereof, of interest, using engineered lipid bilayer particle, such as cell-derived membrane particles (CDMPs) as described herein. Using technologies of the present disclosure lipid bilayer particles deliver a cargo to a recipient cell in an efficient and specific manner. In some embodiments, provided technologies delivery a cargo when employed ex vivo. In some embodiments, provided technologies achieve, e.g., in vivo delivery of a cargo to a recipient cell.

[0287] In some embodiments, a recipient cell expresses a target ligand (e.g., a cell surface epitope). In some embodiments, a recipient cell expresses a particular target ligand or a specific combination of target ligands. In some embodiments, a recipient cell is CD5+, CD3+, CD2+, or a combination thereof. In some embodiments, a recipient cell is an immune cell. In some embodiments, an immune cell is a lymphocyte. In some embodiments, a lymphocyte is a T cell. In some embodiments, a T cell is an activated T cell. In some embodiments, a T cell is CD4+ and / or CD8+.

[0288] In some embodiments, a SIRPa provided herein binds a target ligand on the surface of a recipient cell. In some embodiments, a target ligand (e.g., target epitope) is expressed on all cells (a universal feature of the cell surface), or on a subset of cells, or on cells that occupy a subset of possible states (e.g., activated T cells versus resting T cells).

[0289] A recipient cell may express a number of unique targeting ligands on the cell surface and a targeting chimeric polypeptide can be designed to target one or more such target ligands. In some embodiments, a target ligand is on the surface of a recipient cell. In some embodiments, a target ligand is an epitope, receptor, protein, carbohydrate, lipid, or particular combinations or conformational states thereof.Exemplification

[0290] The following examples exemplify to one of ordinary skill in the art how to make and use certain compositions and methods described herein, and / or to confirm or otherwise provide representative illustration of certain findings provided by the present disclosure and are not intended to limit the scope of the present disclosure.Example 1: Functional delivery to Jurkat T cells and HEK293FT cells.

[0291] The present Example demonstrates the ability of lipid bilayer particles comprising a lentivirus core, a SIRPa entity, and, optionally, a fusogen entity to deliver cargo to Jurkat T cells and HEK293FT cells.

[0292] Exemplary particles were designed to display.1) SIRPa2) SIRPa combined with VSVGwt; or3) SIRPa combined with VSVGmut.

[0293] Figure 1 shows plasmid constructs used in Example 1. A first plasmid encodes the lentiviral helper genes: gag, poL rev, and tat in which expression is driven by an exemplary cytomegalovirus (CMV) early enhancer, chicken beta-actin promoter, and a chimeric chicken beta-actin and rabbit beta-globin intron (pCAG). A second plasmid encodes the lentiviral backbone including a 5’ long terminal repeat (LTR) and 3’ LTR in which the U3 promoter sequence has been disrupted by deletion to yield a self-inactivating lentiviral vector. The lentiviral payload is driven by the human elongation factor la promoter (phEFla) and comprises the mNeonGreen fluorescent protein. The 3’ portion of the lentiviral genome includes a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) to stabilize both lentiviral genomic RNA and payload encoding mRNA driven from this vector. A third plasmid encodes a fusogen (driven by an exemplary CMV enhancer and promoter and a human beta-globin intron); fusogen genes included in this vector include the wild-type vesicular stomatitis virus glycoprotein, VSVG (VSVGwt), or a mutant version of VSVG that cannot bind its cognate receptor (VSVGmut), or a non-coding sequence (blank). A fourth plasmid encodes SIRPa (driven by an exemplary pCMV enhancer and promoter).

[0294] LentiX (lentivirus producing cells) were plated and transfected with one or more plasmids via calcium-phosphate precipitation depending on the above study design. Cell media was changed appropriately. Equal genome copies of virus to T cells and HEK293FT cells were applied. The produced lentiviruses were harvested, and physical titer was determined by qPCR. Jurkat T cells (Figure 2) or HEK293FT cells (Figure 3) were next transduced with 5 doses of unconcentrated (logarithmically spaced) virus. Flow-based fluorescent readout was performed two days after transduction and qPCR for particle titer in parallel. Viral genomes per cell were derived from qPCR titer results (no correction for contaminating plasmid) with 40,000 cells plated per well.Lipid bilayer particles displaying only SIRPa without any V SVG delivery to Jurkat T cells.

[0295] Lipid bilayer particles expressing neither SIRPa nor a fusogen entity (e.g., VSVGwt or VSVGmut) do not infect Jurkat T cells (Figure 2A and 2D). However, lipid bilayer particles expressing SIRPa alone do transduce Jurkat T cells at rates up to approximately 14% at lOOul viral load (Figure 2A and 2D).Lipid bilayer particles with VSVGwt delivery to Jurkat T cells.

[0296] Lipid bilayer particles displaying the VSVGwt fusogen alone yield good transduction of Jurkat T cells (Figure 2B and Figure 2E). At a viral load greater than 10 pl, lipid bilayer particles displaying VSVGwt transduce 75-100% of Jurkat T cells (Figure 2B). However, lipid bilayer particles which display both VSVGwt and SIRPa show enhanced transduction at nearly all tested doses compared to VSVGwt alone, with approximately twofold increase in transduction at lul and 2.5ul doses.Lipid bilayer particles with VSVGmut delivery to Jurkat T cells.

[0297] Lipid bilayer particles displaying the binding-incompetent fusogen VSVGmut alone do not effectively transduce Jurkat T cells (Figure 2C and Figure 2F). By contrast, coexpression of VSVGmut with SIRPa rescues infectivity with VSVGmut alone, and lipid bilayer particles expressing both VSVGmut and SIRPa show high transduction at every viral dose tested and in a dose-dependent manner.Lipid bilayer particles with various VSVG delivery to HEK293FT cells.

[0298] Lipid bilayer particles without SIRPa or any fusogen (e.g., VSVGwt or VSVGmut) do not infect HEK293FT cells (Figure 3A). Particles with VSVGwt alone (Figure 3B) infect somewhat more efficiently than do those with VSVGmut alone (Figure 3C). In HEK293FT cells, co-expression of SIRPa with VSVGwt yields increased transduction compared to VSVGwt alone, particularly at lower viral loads, where transduction increased approximately five-fold (Figure 3E versus Figure 3B). A similar pattern is observed with co-expression of SIRPa and VSVGmut (Figure 3F versus Figure 3C), whereby SIRPa enhances transduction compared to VSVGmut alone. Infection plotted against viral dose (in viral genomes per cell) derived from qPCR titer results are shown in Figure 3G, showing improved transduction when SIRPa was displayed on the lipid bilayer particles in combination with VSVGwt or VSVGmut, compared to lipid bilayer particles only displaying VSVGwt or VSVGmut.Summary: Example 1

[0299] Lipid bilayer particles co-expressing SIRPa and a VSVG fusogen show enhanced cargo delivery to Jurkat T cells compared to lipid bilayer particles expressing a VSVG fusogen alone. Lipid bilayer particles displaying SIRPa along with VSVGwt are capable of transducing Jurkat T cells at a rate approximately twice that of reference lipid bilayerparticles displaying VSVGwt alone, which have been previously established as high performing (Milone & O’Doherty. Leukemia 32, 1529-1541 (2018)). The co-expression of SIRPa and the binding-incompetent VSVGmut enabled delivery of the fluorophore cargo to Jurkat T cells, which does not occur in the absence of SIRPa.Example 2: Functional delivery to primary human T cells.

[0300] The present Example demonstrates the ability of lipid bilayer particles comprising a lentivirus core, a SIRPa entity, and, optionally, a fusogen entity to deliver cargo to primary human T cells.

[0301] The following combinations were tested:1) No surface modification2) VSVGwt only3) VSVGmut only4) SIRPa only5) anti-CD3 scFv only6) anti-CD5 scFv only7) VSVGwt + SIRPa8) VSVGmut + SIRPa9) SIRPa + anti-CD3 scFv10) SIRPa + anti-CD5 scFv

[0302] Figure 4 shows an overview of the plasmids used in this Example. A first plasmid encodes the lentiviral helper genes: gag,pol, rev, and tat in which expression is driven by an exemplary cytomegalovirus (CMV) early enhancer, chicken beta-actin promoter, and a chimeric chicken beta-actin and rabbit beta-globin intron (pCAG). A second plasmid encodes the lentiviral backbone including a 5’ long terminal repeat (LTR) and 3’ LTR in which the U3 promoter sequence has been disrupted by deletion to yield a self-inactivating lentiviral vector. The lentiviral payload is driven by the human elongation factor la promoter (phEFla) and comprises the miRFP720 fluorescent protein. The 3’ portion of the lentiviral genome includes a woodchuck hepatitis virus post-transcriptional regulatoryelement (WPRE) to stabilize both lentiviral genomic RNA and payload encoding mRNA driven from this vector. A third plasmid encodes a fusogen (driven by an exemplary CMV enhancer and promoter and a human beta-globin intron); fusogen genes included in this vector include the wild-type vesicular stomatitis virus glycoprotein, VSV-G (VSVGwt), or a mutant version of VSV-G that cannot bind its cognate receptor (VSVGmut). A fourth plasmid encodes SIRPa (driven by an exemplary pCMV enhancer and promoter).

[0303] LentiX (lentivirus producing cells) were plated and transfected with one or more plasmids via calcium-phosphate precipitation according to the above study design. Cell media was changed appropriately. The produced lipid bilayer particles were harvested. Physical titer was determined by qPCR. Flow-based fluorescent readout was performed three days after transduction, and concurrent staining to identity T cell identity as CD4+ and / or CD8+ was also performed. For the experiment in Figure 6 used in this Example, cells were incubated with lipid bilayer particles for 3 d prior transgene fluorescence analysis via flow cytometry. The experiment was performed in biological triplicate or greater, and error bars represent the standard error of the mean.Lipid bilayer particles with VSVGwt delivery to primary human T cells.

[0304] Lipid bilayer particles displaying VSVGwt alone yield dose-dependent delivery of cargo to activated T cells (Figure 5A and Figure 5B). However, lipid bilayer particles displaying both SIRPa and VSVGwt show enhanced transduction compared to those particles displaying VSVGwt alone for both CD4+ (Figure 5A) and CD8+ (Figure 5B) activated human primary T cells.Lipid bilayer particles with VSVGmut to primary human T cells.

[0305] Lipid bilayer particles displaying binding-incompetent VSVGmut alone or in combination with SIRPa do not substantially transduce CD4+ activated (Figure 5A) or CD8+ activated (Figure 5B) primary human T cells. However, lipid bilayer particles that display all three of SIRPa, VSVGmut, and an additional affinity entity polypeptide such as anti-CD5 scFv or anti-CD3 scFv do demonstrate enhanced delivery of cargo compared to lipid bilayer particles that do not display SIRPa but do display both VSVGmut and an additional affinity entity polypeptide (Figure 6).Summary: Example 2

[0306] Lipid bilayer particles which co-express SIRPa and the VSVGwt fusogen demonstrate enhanced delivery of cargo to both CD4+ and CD8+ primary human T cells compared to those expressing the VSVGwt fusogen alone. Although lipid bilayer particles co-expressing SIRPa and the binding-incompetent VSVGmut fusogen do not substantially infect primary human T cells, the addition of an affinity entity polypeptide such as anti-CD5 scFv or anti-CD3 scFV rescues infectivity compared to the combination of VSVGmut and either anti-CD5 scFv or anti-CD3 scFv without SIRPa.Example 3: Methods and Materials

[0307] This Example describes exemplary methods for generating and testing of engineered lipid bilayer particles displaying SIRPa as described herein.Plasmid Preparation

[0308] Exemplary plasmids were prepared for transfection using either ZymoPURE preparation kits (mini, midi, or maxi) or were purchased directly from a DNA supplier as transfection-grade DNA in TE buffer. For preparation of exemplary SIRPa-encoding plasmids, other affinity reagent-encoding plasmids, and packaging plasmids (plasmids encoding gag / pol / rev / tat, gag / pol, rev, VSV-G), transformed, clonal bacterial cell lines were propagated to various culture sizes and grown for 12-16 h shaking at 37°C in LB containing 100 pg / mL ampicillin (midiprep- 50-100 mL culture per prep grown in vented conical tubes or baffled shake flasks; maxiprep- 200-300 mL culture per prep grown in baffled shake flasks). For preparation of lentiviral transfer vectors, transformed, clonal, stable bacterial cells were propagated to various culture sizes and grown for 16-24 h shaking at 30°C in LB containing 100 pg / mL ampicillin (midiprep- 100-150 mL culture per prep grown in vented conical tubes or baffled shake flasks; maxiprep- 200-300 mL culture per prep grown in baffled shake flasks). For all plasmid types, an endotoxin removal step (part of ZymoPURE kits) was performed for midipreps and maxipreps. All preps were validated by whole plasmid sequencing before use.Cell Culture

[0309] HEK293T Lenti-X cells and HEK293FT cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 1% penicillinstreptomycin, and 4 mM additional L-glutamine. Jurkat T cells were cultured in RPMIsupplemented with 10% FBS, and 1% pen-strep. All cells were maintained at 37°C with 5% CO2.Lentivirus Production

[0310] Lentiviruses were produced in adherent Lenti-X cells seeded at a density of 3.2e5 cells / mL in a volume of 0.5 mL DMEM in 24-well TC-treated plates 24 hr prior to transfection. Cells were transfected with 0.4 pg total DNA. For generation of exemplary 3rdgeneration lentiviral production plasmids, plasmid mixes comprised 28-63% transfer vector, 17-37% gag / pol, 8-20% rev, 5-11% VSV-G, and 0-22% SIRPa-encoding plasmid. For generation of exemplary 2ndgeneration lentiviral production plasmids, plasmid mixes comprised 28-58% transfer vector, 30-45% gag / pol / rev / tat, 5-12% VSV-G, and 0-22% SIRPa-encoding plasmid. The total DNA was always held constant. Plasmids and PEI MAX transfection reagent were diluted in Opti-MEM and mixed in equal volumes at a 4: 1 mass ratio of PEI to DNA and complexed for 24 min prior to transfection. Medium was changed 12-16 h later. At least 28 h post media change, LV was harvested from the conditioned medium and centrifuged at 1000 g for 10 min at 4°C, then aliquoted and stored at -80°C until use.Transduction of HEK293-derived cells

[0311] HEK293FT or Lenti-X HEK293T cells were plated at the time of transduction in PBS-moated, flat bottom, TC-treated 96-well plates in a volume of 100 uL per well (le4 cells / well). Crude lentiviral supernatant was diluted 5-fold seven times in complete DMEM to generate an eight-point dilution curve. Cells were transduced by adding 100 uL / well of exemplary lentivirus (crude and each dilution) and were incubated for 3 days at 37°C.

[0312] To analyze transduction by flow cytometry, cells were lifted from the 96-well plates by treatment with TrypLE at room temperature for 5-6 minutes before quenching with complete DMEM. Cells were then transferred to round bottom 96 well plates, spun down at 150 x g for 5 min, and resuspended in FACS buffer (PBS pH 7.4 with 0.05% BSA and 2 mM EDTA) + DRAQ7 viability dye (diluted lOOOx) after media removal by flicking. Flow cytometry was performed on a Novocyte Penteon using the 488 nm laser for mNeonGreen (525 / 45 filter), the 637 nm laser for DRAQ7 (725 / 40 filter). Approximately 3,000 live cells were collected per sample for analysis. Data were analyzed using FlowJo vlO. Briefly, cells were identified using an FSC-A vs SSC-A plot and gated for singlets using an FSC-A vsFSC-H plot. Live cells were identified using a DRAQ7 negative gate drawn on a non- DRAQ7-stained sample, and transduced cells were identified by drawing an mNeonGreen positive gate on a non-transduced sample.Transduction of Jurkat T cells

[0313] Jurkat T cells were plated at the time of transduction in PBS-moated, flat bottom, TC-treated 96-well plates in a volume of 100 uL per well (3e4 cells / well). Crude lentiviral supernatant was diluted 5-fold five times in RPMI to generate a six-point dilution curve. Cells were transduced by adding 100 uL / well of lentivirus (crude and each dilution) and were incubated for 3 days at 37°C.

[0314] To analyze transduction by flow cytometry, cells were transferred to round bottom 96 well plates, spun down at 400 x g for 4 min, and resuspended in FACS buffer (PBS pH 7.4 with 0.05% BSA and 2 mM EDTA) + DRAQ7 viability dye (diluted lOOOx) after media removal by flicking. Flow cytometry was performed on a Novocyte Penteon using the 488 nm laser for mNeonGreen (525 / 45 filter), the 637 nm laser for DRAQ7 (725 / 40 filter). Approximately 3,000 live cells were collected per sample for analysis. Data were analyzed using FlowJo vlO. Briefly, cells were identified using an FSC-A vs SSC-A plot and gated for singlets using an FSC-A vs FSC-H plot. Live cells were identified using a DRAQ7 negative gate drawn on a non-DRAQ7-stained sample, and exemplary transduced cells were identified by drawing an mNeonGreen positive gate on a non-transduced sample.Functional titer

[0315] The functional titer for lentiviral vectors (LV) is a measure of the concentration of infectious virus particles capable of transducing target cells. It is typically expressed in transducing units per milliliter (TU / mL). This metric quantifies the concentration of viral particles that can successfully deliver genetic material into a specific cell type per virus volume. Functional titer for the exemplary lentiviral vectors described herein was calculated by first calculating multiplicity of infection (MOI) using the Poisson distribution shown in the equation below.Next, for samples with MOI in the linear range (between 2 and 25% transduced), functional titer was calculated using the equation below.TU MOI x # cells plated mL mL virusExample 4: Functional delivery of cargo to Jurkat T cells using exemplary lipid bilayer particles.

[0316] This example documents the ability of exemplary lipid bilayer particles comprising a lentivirus core, a SIRPa entity, and a fusogen to deliver cargo to recipient Jurkat T cells.

[0317] Exemplary lipid bilayer particles were designed to display a fusogen (VSV-G) only or a fusogen (VSV-G) and SIRPa. Lipid bilayer particles were produced by transfecting adherent Lenti-X cells with an exemplary 3rd generation lentiviral production system as described in Example 3. The system employed plasmids as shown in Figure 9B. Transduction in recipient Jurkat T cells was assessed with flow cytometry as described in Example 3.

[0318] Enhanced cargo delivery was observed in recipient Jurkat T cells at a range of viral doses tested using lipid bilayer particles expressing a VSV-G fusogen and SIRPa, as compared to lipid bilayer particles expressing a VSV-G fusogen only (Figure 8B). As in HEK-derived recipient cells, lipid bilayer particles expressing VSV-G+SIRPa reach higher transduction efficiencies in Jurkat T cells at each dose evaluated, as evidenced by a leftward shift in the dose-response curve for VSV-G + SIRPa compared to VSV-G only (Figure 8B). Lipid bilayer particles expressing both a VSV-G fusogen and SIRPa demonstrated approximately 5-fold transduction compared to the VSV-G-only control composition, as assessed by functional titer (Figure 8A).

[0319] Lipid bilayer particles co-expressing SIRPa and a fusogen showed enhanced cargo delivery to Jurkat T cells compared to lipid bilayer particles expressing a fusogen alone. Lipid bilayer particles displaying SIRPa and a fusogen were capable of transducing Jurkat T cells at a rate approximately 5-fold better than that of reference lipid bilayer particles displaying a fusogen alone, which had been previously established as high performing (Milone & O’Doherty. Leukemia 32, 1529-1541 (2018)).

[0320] Taken together, the results of this Example confirm particular surprising and beneficial attributes of lipid bilayer particles expressing a fusogen and SIRPa, specifically for delivering cargo to various recipient cells, as described herein.Example 5: Functional delivery of cargo to HEK293-derived cells with exemplary lipid bilayer particles.

[0321] This Example further confirms the ability of lipid bilayer particles comprising a lentivirus core, a SIRPa entity, and a fusogen to deliver cargo to HEK293 -derived recipient T cells.

[0322] Exemplary lipid bilayer particles were engineered to display a fusogen (VSV-G) only or a fusogen (VSV-G) and SIRPa using 2ndand 3rdgeneration lentiviral production systems as described in Example 3 and shown in Figures 9A and Figure 9B. Lipid bilayer particles formulated with SIRPa were produced with various plasmid ratios employing varying amounts of SIRPa-encoding plasmid per mix to assess the robustness of improved transduction to varying production conditions. The amount of SIRPa-encoding plasmid ranged from 0 to 22% of total plasmid DNA Transduction in recipient cells was assessed with flow cytometry as described in Example 3. Figure 7A and Figure 7B include the same data points and linear regression model fit, replotted with different markers indicating either the fraction of SIRPa plasmid DNA (Figure 7A) or the production system (Figure 7B) used to generate the particles. The VSV-G + SIRPa composition that generated the highest functional titer in both cell lines (generated with the 3rdgeneration lentiviral packaging system) was selected from the dataset and used to generate full dose response curves for comparison with the control composition (VSV-G fusogen only).

[0323] Lipid bilayer particles engineered to express a VSV-G fusogen and SIRPa demonstrated improved transduction to both HEK293FT and Lenti-X HEK293T cells, as compared to those particles expressing a VSV-G fusogen alone (Figure 7A). Lipid bilayer particles created using a SIRPa-encoding plasmid which made up greater than 2% of the total plasmid DNA demonstrated the highest degree of transduction (Figure 7A). Functional titer values for the two different HEK-derived cell types tested were highly correlated (Figure 7A; R2=0.8671).

[0324] As shown in Figure 7B, enhanced delivery with compositions of lipid bilayer particles expressing a VSV-G fusogen and SIRPa, as compared to a composition of particles comprising expression of a VSV-G fusogen only was achieved with both 2ndand 3rdgeneration lentiviral production systems tested. The best-performing composition expressing both a VSV-G fusogen and SIRPa was the same for both cell recipient lines and demonstrated improved delivery 12-fold for HEK293FT cells and 52-fold for Lenti-X HEK293T cells compared to the VSV-G-only control composition.

[0325] The full viral dose response curves for the best-performing VSV-G+SIRPa composition versus control VSV-G only are shown in Figure 7C (HEK293FT recipient cells) and Figure 7D (Lenti-X HEK293T cells). Both compositions show similar dosedependent increases in transduction with viral volume with delivery to the two recipient cell types tested, with peaks at nearly 100% transduction. However, the compositions containing SIRPa reached higher transduction efficiencies at each dose evaluated, as evidenced by a leftward shift in the dose-response curve for VSV-G + SIRPa compared to VSV-G only in experiments with both recipient cell types (Figure 7C and Figure 7D).

[0326] In summary, enhanced transduction with lipid bilayer particles expressing SIRPa in combination with a fusogen was similar and highly correlated between two different HEK293 -derived cell lines. The exemplified lipid bilayer particles expressing a fusogen and SIRPa were capable of transducing HEK293 -derived cells at a rate approximately 12-52- fold compared to reference lipid bilayer particles displaying a VSV-G fusogen alone, which had been previously established as high performing (Milone & O’Doherty. Leukemia 32, 1529-1541 (2018)).

[0327] Taken together, the results of this Example confirm findings in Example 1, and further document the robustness of the effect of SIRPa in combination with a fusogen for enhancing delivery of cargo to recipient cells across varying lipid bilayer particle production conditions, including the use of different lipid bilayer particle production systems.Certain Embodiments

[0328] Embodiment 1. A population of engineered lipid bilayer particles characterized by presence on their surfaces of at least one Signal Regulatory Protein Alpha (SIRPa).

[0329] Embodiment 2. The population of embodiment 1, wherein the SIRPa comprises an amino acid sequence that is at least 75% identical to SEQ ID NO: 1.

[0330] Embodiment 3. The population of embodiment 1 or 2, wherein the SIRPa is overexpressed on the surfaces of the engineered lipid bilayer particles.

[0331] Embodiment 4. The population of any one of embodiments 1 to 3, wherein the SIRPa is overexpressed on the surface of an engineered lipid bilayer particle relative to a basal level of SIRPa found on a lipid bilayer particle that is not engineered to express SIRPa.

[0332] Embodiment 5. The population of any one of embodiments 1 to 4, wherein each lipid bilayer particle displays at least 10 copies of SIRPa.

[0333] Embodiment 6. The population of embodiment 5, wherein each lipid bilayer particle displays at least 100 copies of SIRPa.

[0334] Embodiment 7. The population of embodiment 6, wherein each lipid bilayer particle displays at least 500 copies of SIRPa.

[0335] Embodiment 8. The population of any one of embodiments 1-7, wherein the SIRPa is the only polypeptide overexpressed on the surfaces of the engineered lipid bilayer particles.

[0336] Embodiment 9. The population of any one of embodiments 1-8, wherein the SIRPa binds to a target present on a recipient cell.

[0337] Embodiment 10. The population of embodiment 9, wherein the SIRPa binds to CD47.

[0338] Embodiment 11. The population of any one of embodiments 1-10, wherein the SIRPa mediates internalization of the particles to the recipient cell.

[0339] Embodiment 12. The population of any one of embodiments 1-11, wherein the particles are synthetic particles or cell-derived membrane particles (CDMPs).

[0340] Embodiment 13. The population of embodiment 12, wherein the CDMPs are extracellular vesicles, virus particles, virus-like particles (VLPs), apoptotic bodies, plateletlike particles, or combinations thereof.

[0341] Embodiment 14. The population of embodiment 13, wherein the extracellular vesicles are exosomes, microvesicles, or combinations thereof.

[0342] Embodiment 15. The population of any one of embodiments 1-11, wherein the particles are lentiviral vectors.

[0343] Embodiment 16. The population of any one of embodiments 1-11, wherein the particles are synthetic lipid nanoparticles or liposomes.

[0344] Embodiment 17. The population of any one of embodiments 1-16, wherein the particles are further characterized by the presence on their surfaces of a fusogen entity polypeptide.

[0345] Embodiment 18. The population of embodiment 17, wherein the fusogen entity polypeptide comprises: a fusogen moiety; and a membrane association portion.

[0346] Embodiment 19. The population of embodiment 18, wherein the fusogen moiety and the membrane association portion are directly linked or indirectly linked via a linker.

[0347] Embodiment 20. The population of embodiment 19, wherein the fusogen entity polypeptide is heterologous to the SIRPa.

[0348] Embodiment 21. The population of any one of embodiments 18 to 20, wherein the fusogen moiety is characterized by an ability to promote fusion between lipid bilayers.

[0349] Embodiment 22. The population of any one of embodiments 17 to 21, wherein the fusogen entity polypeptide is a viral fusogen entity polypeptide or derivative thereof.

[0350] Embodiment 23. The population of any one of embodiments 17 to 21, wherein the fusogen entity polypeptide is a non-viral fusogen entity polypeptide or derivative thereof.

[0351] Embodiment 24. The population of embodiment 22, wherein the viral fusogen entity polypeptide is a polypeptide from vesicular stomatitis virus, Measles virus, Sindbis virus, Tupaia paramyxovirus, Nipah virus, Chandipura virus, Rabies virus, Lymphocytic choriomeningitis virus, Mokola virus, Ross River virus, Ross River virus, Semliki Forestvirus, Venezuelan equine encephalitis virus, Ebola virus, Marburg virus, Lassa virus, Avian leukosis virus, Jaagsiekte sheep retrovirus, Moloney Murine leukemia virus, Gibbon ape leukemia virus, Feline endogenous retrovirus (RD114), Human T-lymphotropic virus 1, Human foamy virus, Maedi-visna virus, SARS-CoV, SARS-CoV-2, Sendai virus, Respiratory syncytia virus, Human parainfluenza virus type 3, Human parainfluenza virus type 4, Hepatitis C virus, Hepatitis C virus, Influenza virus, Fowl plague virus, Autographa californica multiple nucleopolyhedro virus, Baboon endogenous retrovirus, Cocal virus, Japanese encephalitis virus, Dengue virus, Zika virus, West Nile virus, Yellow fever virus, Tick-borne encephalitis virus, Herpes simplex virus 1, Hendra virus, Newcastle disease virus, Epstein Barr virus, Bourbon virus, Varicella-zoster virus, Severe fever with thrombocytopenia virus, Hantavirus, Vaccinia virus, Simian immunodeficiency virus, Human immunodeficiency virus, Junin virus, Machupo virus, Bas-Congo virus, La Crosse virus, Human cytomegalovirus, Human cytomegalovirus, Thogoto virus, or Dhori virus.

[0352] Embodiment 25. The population of embodiment 22, wherein the viral fusogen entity polypeptide is a lentiviral glycoprotein or a glycoprotein selected from a group consisting of vesicular stomatitis glycoprotein (VSV-G), measles virus glycoprotein H, measles virus glycoprotein F, rabies virus glycoprotein (RVG), gibbon ape leukemia virus glycoprotein (GaLV), amphotropic murine leukemia virus glycoprotein (MLV-A), feline endogenous virus (RD114) glycoprotein, fowl plague virus (FPV) glycoprotein, Ebola virus (EboV) glycoprotein, vesicular stomatitis virus (VSV) glycoprotein, and lymphocytic choriomeningitis virus (LCMV) glycoprotein.

[0353] Embodiment 26. The population of embodiment 25, wherein VSV-G comprises an amino acid sequence that is at least 80% identical to that of SEQ ID NO: 14.

[0354] Embodiment 27. The population of embodiment 25, wherein a VSV-G comprises an amino acid sequence that is at least 80% identical to that of SEQ ID NO: 16.

[0355] Embodiment 28. The population of any one of embodiments 17 to 27, wherein the fusogen entity polypeptide enables fusion of the particles to a recipient cell.

[0356] Embodiment 29. The population of any one of embodiments 17 to 28, wherein the SIRPa catalyzes fusogen entity polypeptide mediated fusion between the engineered lipid bilayer particles and a recipient cell membrane.

[0357] Embodiment 30. The population of any one of embodiments 1 to 29, wherein the particles are further characterized by the presence on their surfaces of an affinity entity polypeptide.

[0358] Embodiment 31. The population of embodiment 30, wherein the affinity entity polypeptide is heterologous to the SIRPa.

[0359] Embodiment 32. The population of embodiment 30, wherein the fusogen entity polypeptide and the affinity entity polypeptide are heterologous to one another.

[0360] Embodiment 33. The population of any one of embodiments 30 to 33, wherein the affinity entity polypeptide comprises: an affinity moiety; and a membrane association portion.

[0361] Embodiment 34. The population of embodiment 33, wherein the affinity moiety and the membrane association portion are directly linked or indirectly linked via a linker.

[0362] Embodiment 35. The population of embodiment 33 or 34, wherein the membrane association portion is a viral transmembrane portion or derivative thereof.

[0363] Embodiment 36. The population of embodiment 33 or 34, wherein the membrane association portion is a non-viral transmembrane portion.

[0364] Embodiment 37. The population of any one of embodiments 33 to 36, wherein the affinity moiety specifically binds to a surface of a recipient cell.

[0365] Embodiment 38. The population of any one of embodiments 33 to 37, wherein the affinity moiety is selected from the group consisting of an antibody, a Fab, a Fab', a F(ab')2, a Fd, a scFv, a single-chain antibody, a disulfide-linked Fvs (sdFv), an affinibody, a DARPIN, a nanobody, a variable lymphocyte receptor (VLR), and a camelid antibody.

[0366] Embodiment 39. The population of any one of embodiments 33 to 38, wherein the affinity entity polypeptide is selected from the group consisting of anti-CD5 VLR, anti- CD2 scFv, anti-CD3 scFv, and anti-CD5 scFv.

[0367] Embodiment 40. The population of embodiment 39, wherein the anti-CD2 scFv comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 37.

[0368] Embodiment 41. The population of embodiment 39, wherein the anti-CD3 scFv comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 38.

[0369] Embodiment 42. The population of embodiment 39, wherein the anti-CD5 scFv comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 39.

[0370] Embodiment 43. The population of embodiment 39, wherein the anti-CD5 VLR comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 40.

[0371] Embodiment 44. The population of any one of embodiments 33 to 38, wherein the fusogen entity polypeptide is a viral fusion polypeptide and the affinity entity polypeptide specifically binds to surfaces of immune cells.

[0372] Embodiment 45. The population of any one of embodiments 33 to 38, wherein the fusion entity polypeptide is a VSV-G and the affinity entity polypeptide is an anti-CD2 scFv, anti-CD3 scFv or anti-CD5 scFv.

[0373] Embodiment 46. The population of any one of embodiments 33 to 38, wherein the fusion entity polypeptide is a VSV-G and the affinity entity polypeptide is an anti-CD5 VLR.

[0374] Embodiment 47. The population of any one of embodiments 1 to 46, wherein the particles further comprise a cargo.

[0375] Embodiment 48. The population of embodiment 47, wherein the cargo comprises a nucleic acid payload.

[0376] Embodiment 49. The population of embodiment 47, wherein the cargo comprises a polypeptide cargo payload.

[0377] Embodiment 50. The population of embodiment 47, wherein the cargo comprises a viral payload.

[0378] Embodiment 51. The population of embodiment 47, wherein the cargo comprises adeno-associated virus particles.

[0379] Embodiment 52. The population of embodiment 47, wherein the cargo comprises a non-viral payload.

[0380] Embodiment 53. The population of embodiment 52, wherein the non-viral payload includes ASO, RNA, mRNA, DNA, and siRNA gene delivery vector.

[0381] Embodiment 54. The population of any one of embodiments 1 to 53, wherein the SIRPa alone mediates cargo delivery to a recipient cell.

[0382] Embodiment 55. The population of any one of embodiments 1 to 54, wherein particles expressing SIRPa on their surfaces have enhanced cargo delivery to recipient cells compared to particles that do not express SIRPa on their surfaces.

[0383] Embodiment 56. The population of any one of embodiments 1 to 55, wherein the population is prepared from cells engineered to express SIRPa.

[0384] Embodiment 57. The population of embodiment 56, wherein the population is prepared from cells engineered to further express one or both of the fusogen entity polypeptide and the affinity entity polypeptide.

[0385] Embodiment 58. The population of embodiment 56 or 57, wherein the cells are mammalian cells.

[0386] Embodiment 59. The population of embodiment 58, wherein the lipid bilayer particles are formed by budding from a membrane from the cell.

[0387] Embodiment 60. The population of embodiment 59, wherein the membrane is a plasma membrane.

[0388] Embodiment 61. A system comprising.(1) a first polynucleotide encoding a Signal Regulatory Protein Alpha (SIRPa); and(2a) a second polynucleotide encoding a fusogen entity polypeptide; (2b) a third polynucleotide encoding an affinity entity polypeptide; or (2c) a combination thereof.

[0389] Embodiment 62. A population of engineered production cells engineered to express. a polynucleotide encoding a Signal Regulatory Protein Alpha (SIRPa), such that lipid bilayer particles formed by the engineered production cells are characterized by presence on their surface of at least one SIRPa.

[0390] Embodiment 63. A method of manufacturing a population of lipid bilayer particles, the method comprising a step of: isolating lipid bilayer particles produced by engineered production cells engineered to express.a Signal Regulatory Protein Alpha (SIRPa), such that the lipid bilayer particles contain the SIRPa on their surfaces.

[0391] Embodiment 64. The method of embodiment 63, wherein the lipid bilayer particles comprise a cargo.

[0392] Embodiment 65. A method of delivering a cargo to a recipient cell, the method comprising a step of: contacting the recipient cell with a population of lipid bilayer particles prepared from an engineered production cell, which engineered production cell was engineered to express. a Signal Regulatory Protein Alpha (SIRPa), such that the lipid bilayer particles contain the SIRPa on their surfaces.

[0393] Embodiment 66. The method of embodiment 65, wherein the recipient cell is CD2+, CD3+, CD5+, or a combination thereof.

[0394] Embodiment 67. The method of embodiment 65 or 66, wherein the recipient cell is an immune cell.

[0395] Embodiment 68. The method of embodiment 67, wherein the immune cell is a T cell.

[0396] Embodiment 69. The method of embodiment 68, wherein the T cell is an activated T cell.

[0397] Embodiment 70. The method of embodiment 67, wherein the T cell is CD4+ and / or CD8+.Equivalents

[0398] Those skilled in the art will recognize or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. The scope of the present invention is not intended to be limited to the above Description, but rather is as set forth in the following claims:

Claims

ClaimsWe claim:

1. A population of engineered lipid bilayer particles characterized by presence on their surfaces of at least one Signal Regulatory Protein Alpha (SIRPa).

2. The population of claim 1, wherein the SIRPa comprises an amino acid sequence that is at least 75% identical to SEQ ID NO: 1.

3. The population of claim 1 or 2, wherein the SIRPa is overexpressed on the surfaces of the engineered lipid bilayer particles.

4. The population of any one of claims 1 to 3, wherein the SIRPa is overexpressed on the surface of an engineered lipid bilayer particle relative to a basal level of SIRPa found on a lipid bilayer particle that is not engineered to express SIRPa.

5. The population of any one of claims 1 to 4, wherein each lipid bilayer particle displays at least 10 copies of SIRPa, at least 100 copies of SIRPa, or at least 500 copies of SIRPa.

6. The population of any one of claims 1-5, wherein the SIRPa binds to a target present on a recipient cell.

7. The population of claim 6, wherein the SIRPa binds to CD47.

8. The population of any one of claims 1-7, wherein the SIRPa mediates internalization of the particles to the recipient cell.

9. The population of any one of claims 1-8, wherein the particles are synthetic particles or cell-derived membrane particles (CDMPs).

10. The population of claim 9, wherein the CDMPs are extracellular vesicles, virus particles, virus-like particles (VLPs), apoptotic bodies, platelet-like particles, or combinations thereof.

11. The population of claim 10, wherein the extracellular vesicles are exosomes, microvesicles, or combinations thereof.

12. The population of any one of claims 1-8, wherein the particles are lentiviral vectors.

13. The population of any one of claims 1-8, wherein the particles are synthetic lipid nanoparticles or liposomes.

14. The population of any one of claims 1-13, wherein the particles are further characterized by the presence on their surfaces of a fusogen entity polypeptide.

15. The population of claim 14, wherein the fusogen entity polypeptide comprises: a fusogen moiety; and a membrane association portion.

16. The population of claim 15, wherein the fusogen entity polypeptide is heterologous to the SIRPa.

17. The population of claim 15 or 16, wherein the fusogen moiety is characterized by an ability to promote fusion between lipid bilayers.

18. The population of any one of claims 14 to 17, wherein the fusogen entity polypeptide is a viral fusogen entity polypeptide or derivative thereof.

19. The population of any one of claims 14 to 17, wherein the fusogen entity polypeptide is a non-viral fusogen entity polypeptide or derivative thereof.

20. The population of claim 18, wherein the viral fusogen entity polypeptide is a polypeptide from vesicular stomatitis virus, Measles virus, Sindbis virus, Tupaia paramyxovirus, Nipah virus, Chandipura virus, Rabies virus, Lymphocytic choriomeningitis virus, Mokola virus, Ross River virus, Ross River virus, Semliki Forest virus, Venezuelan equine encephalitis virus, Ebola virus, Marburg virus, Lassa virus, Avian leukosis virus, Jaagsiekte sheep retrovirus, Moloney Murine leukemia virus, Gibbon ape leukemia virus, Feline endogenous retrovirus (RD114), Human T-lymphotropic virus 1, Human foamy virus, Maedi-visna virus, SARS-CoV, SARS-CoV-2, Sendai virus, Respiratory syncytia virus, Human parainfluenza virus type 3, Human parainfluenza virus type 4, Hepatitis C virus, Hepatitis C virus, Influenza virus, Fowl plague virus, Autographa californica multiplenucleopolyhedro virus, Baboon endogenous retrovirus, Cocal virus, Japanese encephalitis virus, Dengue virus, Zika virus, West Nile virus, Yellow fever virus, Tick-borne encephalitis virus, Herpes simplex virus 1, Hendra virus, Newcastle disease virus, Epstein Barr virus, Bourbon virus, Varicella-zoster virus, Severe fever with thrombocytopenia virus, Hantavirus, Vaccinia virus, Simian immunodeficiency virus, Human immunodeficiency virus, Junin virus, Machupo virus, Bas-Congo virus, La Crosse virus, Human cytomegalovirus, Human cytomegalovirus, Thogoto virus, or Dhori virus.

21. The population of claim 18, wherein the viral fusogen entity polypeptide is a lentiviral glycoprotein or a glycoprotein selected from a group consisting of vesicular stomatitis glycoprotein (VSV-G), measles virus glycoprotein H, measles virus glycoprotein F, rabies virus glycoprotein (RVG), gibbon ape leukemia virus glycoprotein (GaLV), amphotropic murine leukemia virus glycoprotein (MLV-A), feline endogenous virus (RD114) glycoprotein, fowl plague virus (FPV) glycoprotein, Ebola virus (EboV) glycoprotein, and lymphocytic choriomeningitis virus (LCMV) glycoprotein.

22. The population of claim 21, wherein VSV-G comprises an amino acid sequence that is at least 80% identical to that of SEQ ID NO: 14 or SEQ ID NO: 16.

23. The population of any one of claims 14 to 22, wherein the fusogen entity polypeptide enables fusion of the particles to a recipient cell.

24. The population of any one of claims 14 to 23, wherein the SIRPa catalyzes fusogen entity polypeptide mediated fusion between the engineered lipid bilayer particles and a recipient cell membrane.

25. The population of any one of claims 1 to 24, wherein the particles are further characterized by the presence on their surfaces of an affinity entity polypeptide.

26. The population of claim 25, wherein the affinity entity polypeptide comprises: an affinity moiety; and a membrane association portion.

27. The population of claim 26, wherein the membrane association portion is a viral transmembrane portion or derivative thereof.

28. The population of claim 26, wherein the membrane association portion is a non-viral transmembrane portion.

29. The population of any one of claims 26 to 28, wherein the affinity moiety specifically binds to a surface of a recipient cell.

30. The population of any one of claims 26 to 29, wherein the affinity moiety is selected from the group consisting of an antibody, a Fab, a Fab', a F(ab')2, a Fd, a scFv, a single-chain antibody, a disulfide-linked Fvs (sdFv), an affinibody, a DARPIN, a nanobody, a variable lymphocyte receptor (VLR), and a camelid antibody.

31. The population of any one of claims 26 to 30, wherein the affinity entity polypeptide is selected from the group consisting of anti-CD5 VLR, anti-CD2 scFv, anti- CD3 scFv, and anti-CD5 scFv.

32. The population of claim 31, wherein the anti-CD2 scFv comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 37.

33. The population of claim 31, wherein the anti-CD3 scFv comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 38.

34. The population of claim 31, wherein the anti-CD5 scFv comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 39 or SEQ ID NO: 40.

35. The population of any one of claims 26 to 30, wherein the fusogen entity polypeptide is a viral fusion polypeptide and the affinity entity polypeptide specifically binds to surfaces of immune cells.

36. The population of any one of claims 26 to 30, wherein the fusion entity polypeptide is a VSV-G, and the affinity entity polypeptide is an anti-CD2 scFv, anti-CD3 scFv or anti-CD5 scFv.

37. The population of any one of claims 26 to 30, wherein the fusion entity polypeptide is a VSV-G, and the affinity entity polypeptide is an anti-CD5 VLR.

38. The population of any one of claims 1 to 37, wherein the particles further comprise a cargo.

39. The population of claim 38, wherein the cargo comprises a nucleic acid payload, a polypeptide payload, a viral payload, a non-viral payload, or a combination thereof.

40. The population of claim 39, wherein the viral payload comprises adeno- associated virus particles.

41. The population of claim 39, wherein the non-viral payload includes ASO, RNA, mRNA, DNA, and siRNA gene delivery vector.

42. The population of any one of claims 1 to 41, wherein particles expressing SIRPa on their surfaces have enhanced cargo delivery to recipient cells compared to particles that do not express SIRPa on their surfaces.

43. The population of any one of claims 1 to 42, wherein the population is prepared from cells engineered to express SIRPa.

44. The population of claim 43, wherein the population is prepared from cells engineered to further express one or both of the fusogen entity polypeptide and the affinity entity polypeptide.

45. The population of claims 43 or 44, wherein the cells are mammalian cells.

46. The population of claim 45, wherein the lipid bilayer particles are formed by budding from a membrane from the cell.

47. A system comprising(1) a first polynucleotide encoding a Signal Regulatory Protein Alpha (SIRPa); and(2a) a second polynucleotide encoding a fusogen entity polypeptide; (2b) a third polynucleotide encoding an affinity entity polypeptide; or (2c) a combination thereof.

48. A population of engineered production cells engineered to express a polynucleotide encoding a Signal Regulatory Protein Alpha (SIRPa),such that lipid bilayer particles formed by the engineered production cells are characterized by presence on their surface of at least one SIRPa.

49. A method of manufacturing a population of lipid bilayer particles, the method comprising a step of: isolating lipid bilayer particles produced by engineered production cells engineered to express: a Signal Regulatory Protein Alpha (SIRPa), such that the lipid bilayer particles contain the SIRPa on their surfaces.

50. The method of claim 49, wherein the lipid bilayer particles comprise a cargo.

51. A method of delivering a cargo to a recipient cell, the method comprising a step of: contacting the recipient cell with a population of lipid bilayer particles prepared from an engineered production cell, which engineered production cell was engineered to express: a Signal Regulatory Protein Alpha (SIRPa), such that the lipid bilayer particles contain the SIRPa on their surfaces.

52. The method of claim 51, wherein the recipient cell is CD2+, CD3+, CD5+, or a combination thereof.

53. The method of claim 51, wherein the recipient cell is a T cell, optionally wherein the T cell is an activated T cell.

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