Fusosome compositions and uses thereof

A fusosome comprising:

• a) a lipid bilayer comprising a fusogen;
• b) a nucleic acid that comprises or encodes:
• (i) a positive target cell-specific regulatory element operatively linked to a nucleic acid encoding an exogenous agent, wherein the positive tissue-specific regulatory element increases expression of the exogenous agent in a target cell or tissue relative to an otherwise similar fusosome lacking the positive tissue-specific regulatory element;
• and
• (ii) a non-target cell-specific regulatory element operatively linked to the nucleic acid encoding the exogenous agent, wherein the non-target cell-specific regulatory element decreases expression of the exogenous agent in a non-target cell or tissue relative to an otherwise similar fusosome lacking the non-target cell-specific regulatory element.

The fusosome of any of the preceding claims, wherein one or more of:

• i) the fusosome fuses at a higher rate with a target cell than with a non-target cell, optionally wherein the higher rate is by at least at least 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 20-fold, 50-fold, or 100-fold;
• ii) the fusosome fuses at a higher rate with a target cell than with another fusosome, optionally wherein the higher rate is by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 20-fold, 50-fold, or 100-fold;
• iii) the fusosome fuses with target cells at a rate such that the exogenous agent in the fusosome is delivered to at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%, of target cells after 24, 48, or 72 hours;
• iv) the fusosome delivers the nucleic acid to a target cell at a higher rate than to a non-target cell, optionally wherein the higher rate is by at least at least 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 20-fold, 50-fold, or 100-fold;
• v) the fusosome delivers the nucleic acid to a target cell at a higher rate than to another fusosome, optionally wherein the higher rate is by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 20-fold, 50-fold, or 100-fold;
• or
• vi) the fusosome delivers the nucleic acid to a target cell at a rate such that the exogenous agent in the fusosome is delivered to at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%, of target cells after 24, 48, or 72 hours.

The fusosome of any of the preceding claims, wherein, when the fusosome is administered to a subject, one or more of:

• i) less than 10%, 5%, 4%, 3%, 2%, or 1% of the exogenous agent detectably present in the subject is in non-target cells;
• ii) at least 90%, 95%, 96%, 97%, 98%, or 99% of the cells of the subject that detectably comprise the exogenous agent, are target cells, optionally wherein the target cells are of a single cell type, optionally wherein the target cells are T cells;
• iii) less than 1,000,000, 500,000, 200,000, 100,000, 50,000, 20,000, or 10,000 cells of the cells of the subject that detectably comprise the exogenous agent are non-target cells;
• iv) average levels of the exogenous agent in all target cells in the subject are at least 100-fold, 200-fold, 500-fold, or 1,000-fold higher than average levels of the exogenous agent in all non-target cells in the subject;
• or
• v) the exogenous agent is not detectable in any non-target cell in the subject.

The fusosome of any of the preceding claims, wherein the fusosome further comprises:

• i) a first exogenous or overexpressed immunosuppressive protein on the lipid bilayer and a second exogenous or overexpressed immunosuppressive protein on the lipid bilayer;
• ii) a first exogenous or overexpressed immunosuppressive protein on the lipid bilayer and a second immunostimulatory protein that is absent or present at reduced levels, optionally wherein the reduced level is by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%, compared to a fusosome generated from an otherwise similar, unmodified source cell;
• or
• iii) a first immunostimulatory protein that is absent or present at reduced levels, optionally wherein the reduced level by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%, compared to a fusosome generated from an otherwise similar, unmodified source cell and a second immunostimulatory protein that is absent or present at reduced levels, optionally wherein the reduced level is by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%, compared to a fusosome generated from an otherwise similar, unmodified source cell.

A fusosome comprising:

• a) a lipid bilayer comprising a fusogen, and
• b) an exogenous agent or a nucleic acid encoding an exogenous agent;
• and
• c) one or more of:
• i) a first exogenous or overexpressed immunosuppressive protein on the lipid bilayer and a second exogenous or overexpressed immunosuppressive protein on the lipid bilayer;
• ii) a first exogenous or overexpressed immunosuppressive protein on the lipid bilayer and a second immunostimulatory protein that is absent or present at reduced levels, optionally wherein the reduced level is by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%, compared to a fusosome generated from an otherwise similar, unmodified source cell;
• or
• iii) a first immunostimulatory protein that is absent or present at reduced levels, optionally wherein the reduced level is reduced by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%, compared to a fusosome generated from an otherwise similar, unmodified source cell and a second immunostimulatory protein that is absent or present at reduced levels, optionally wherein the reduced level is reduced by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%, compared to a fusosome generated from an otherwise similar, unmodified source cell;
• wherein, when administered to a subject, optionally wherein the subject is a human subject or a mouse, one or more of:
• i) the fusosome does not produce a detectable antibody response, or antibodies against the fusosome are present at a level of less than 10%, 5%, 4%, 3%, 2%, or 1% above a background level;
• ii) the fusosome does not produce a detectable cellular immune response, or a cellular immune response against the fusosome is present at a level of less than 10%, 5%, 4%, 3%, 2%, or 1% above a background level;
• iii) the fusosome does not produce a detectable innate immune response, or the innate immune response against the fusosome is present at a level of less than 10%, 5%, 4%, 3%, 2%, or 1% above a background level;
• iv) less than 10%, 5%, 4%, 3%, 2%, or 1% of fusosomes are inactivated by serum;
• v) a target cell that has received the exogenous agent from the fusosome does not produce a detectable antibody response, or antibodies against the target cell are present at a level of less than 10%, 5%, 4%, 3%, 2%, or 1% above a background level;
• or
• vi) a target cell that has received the exogenous agent from the fusosome does not produce a detectable cellular immune response, or a cellular response against the target cell is present at a level of less than 10%, 5%, 4%, 3%, 2%, or 1% above a background level.

A fusosome comprising:

• a) a lipid bilayer comprising a fusogen, wherein the fusogen comprises a domain of at least 100 amino acids in length having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to a wild-type paramyxovirus fusogen set forth in any one of SEQ ID NOS:
• 1-132;
• b) a nucleic acid encoding an exogenous agent;
• and
• c) an exogenous or overexpressed CD47 or a complement regulatory protein, or a combination thereof, on the lipid bilayer.

A fusosome comprising:

• a) a lipid bilayer comprising a fusogen, wherein the fusogen comprises a domain of at least 100 amino acids in length having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to a wild-type paramyxovirus fusogen set forth in any one of SEQ ID NOS:
• 1-132, and
• b) a nucleic acid encoding an exogenous agent;
• and
• c) MHC I, optionally wherein the MHC I is HLA-A, HLA-B, or HLA-C, or MHC II, optionally wherein MHC II is HLA-DP, HLA-DM, HLA-DOA, HLA-DOB, HLA-DQ, or HLA-DR, that is absent or present at reduced levels, optionally wherein the reduced level is, reduced by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%, compared to a fusosome generated from an otherwise similar, unmodified source cell.

A fusosome comprising:

• a) a lipid bilayer comprising a fusogen, wherein the fusogen comprises a domain of at least 100 amino acids in length having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to a wild-type paramyxovirus fusogen set forth in any one of SEQ ID NOS:
• 1-132;
• and
• b) a nucleic acid encoding an exogenous agent;
• and
• c) one or both of an exogenous or overexpressed immunosuppressive protein or an immunostimulatory protein that is absent or present at reduced levels, optionally wherein the reduced level is reduced by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%, compared to a fusosome generated from an otherwise similar, unmodified source cell.

A fusosome comprising:

• a) a lipid bilayer comprising a fusogen, wherein the fusogen comprises a domain of at least 100 amino acids in length having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to a wild-type paramyxovirus fusogen set forth in any one of SEQ ID NOS:
• 1-132;
• and
• b) a nucleic acid encoding an exogenous agent, wherein the nucleic acid comprises one or more insulator elements.

A source cell for producing a fusosome, comprising:

• a) a nucleic acid;
• b) structural proteins that can package the nucleic acid, wherein at least one structural protein comprises a fusogen that binds a fusogen receptor;
• and
• c) a fusogen receptor that is absent or present at reduced levels (e.g., reduced by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%) compared to an otherwise similar, unmodified source cell.