Lipid particles containing truncated baboon endogenous retrovirus (BaEV) envelope glycoproteins and related methods and uses - Patents.com

Abstract

Provided herein are lipid particles, e.g., lentiviral particles, incorporating or pseudotyped with a truncated Baboon endogenous retrovirus (BaEV) envelope glycoprotein comprising a cytoplasmic tail with a partial inhibitory R peptide that is shorter than the full-length wild-type BaEV inhibitory R peptide. Also provided herein are polynucleotides encoding the truncated BaEV envelope glycoproteins, and producer cells for preparing the lipid particles, e.g., lentiviral particles, comprising the truncated BaEV envelope glycoproteins, and methods for preparing and using the lipid particles, e.g., lentiviral particles.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 63 / 194,880, filed May 28, 2021, entitled "LIPID PARTICLES COMPRISING TRUNCATED BABOON ENDOGENOUS RETROVIRUS (BaEV) ENVELOPE GLYCOPROTEINS AND RELATED METHODS AND USES," the contents of which are incorporated by reference in their entirety herein for all purposes.

[0002] INCORPORATION BY REFERENCE TO SEQUENCE LISTING This application is filed with a Sequence Listing in electronic format. The Sequence Listing is provided in a file entitled "18615_2004440_SEQLIST", created on May 27, 2022, and is 138,255 bytes in size. The information in the electronic format of this Sequence Listing is incorporated herein by reference in its entirety.

[0003] Field The present disclosure relates to lipid particles, e.g., lentiviral particles, incorporating or pseudotyped with a truncated Baboon endogenous retrovirus (BaEV) envelope glycoprotein that includes a cytoplasmic tail with a partial inhibitory R peptide that is shorter than the full-length wild-type BaEV inhibitory R peptide. The present disclosure also provides polynucleotides encoding the truncated BaEV envelope glycoproteins, and producer cells for preparing lipid particles, e.g., lentiviral particles, that include the truncated BaEV envelope glycoproteins, as well as methods for preparing and using lipid particles, e.g., lentiviral particles. Summary of the Invention

[0004] overview Lipid particles, including virus-like particles and viral vectors, such as lentiviral particles, are commonly used to deliver exogenous agents to cells. For various particles, such as lentiviral vector particles, the host range can be modified by pseudotyping with heterologous envelope proteins. Efficient preparation and production of particles containing specific heterologous pseudotype envelope proteins may not always be efficient, such as due to the effect of the envelope proteins on the low titer of lentiviral vector particles produced. There is a need for improved lipid particles, including virus-like particles and viral vectors, that can be produced in desired cells with higher titers and efficient transduction efficiency. The disclosure provided addresses this need.

[0005] Provided herein are Baboon endogenous retrovirus (BaEV) envelope glycoprotein pseudotyped lentiviral particles comprising a truncated BaEV envelope glycoprotein that includes a cytoplasmic tail with a partial fusion-inhibitory R peptide compared to the inhibitory R peptide of wild-type BaEV envelope glycoprotein, wherein the partial fusion-inhibitory R peptide includes at least one amino-terminal amino acid, but less than the full-length, inhibitory R peptide of the wild-type BaEv envelope glycoprotein.

[0006] Also provided herein are Baboon endogenous retrovirus (BaEV) envelope glycoprotein pseudotyped lentiviral particles comprising a truncated BaEV envelope glycoprotein comprising a cytoplasmic tail with a partial fusion-inhibitory R peptide compared to a wild-type BaEV envelope glycoprotein, wherein the cytoplasmic tail is 25 amino acids in length and comprises an inhibitory R peptide of 8 consecutive amino-terminal acids (R+8) of the full-length inhibitory R peptide of the wild-type BaEV envelope glycoprotein.

[0007] In some of any of the provided embodiments, the lentiviral particles are replication-deficient. In some of any of the provided embodiments, the lentiviral particles provided are prepared by a method comprising transducing a producer cell with packaging plasmids encoding Gag-pol, Rev, Tat, and truncated BaEV envelope glycoproteins.

[0008] In some of any of the embodiments provided, the lentiviral particle further comprises a viral nucleic acid. In some of any of the embodiments, the viral nucleic acid comprises one or more (e.g., all) of the following nucleic acid sequences: 5'LTR (e.g., includes U5 and lacks a functional U3 domain), Psi packaging element (Psi), central polypurine tract (cPPT) / central termination sequence (CTS) (e.g., DNA flap), polyA tail sequence, post-transcriptional regulatory element (e.g., WPRE), Rev response element (RRE), and 3'LTR (e.g., includes U5 and lacks a functional U3). In some of any of the embodiments, the lentiviral particle does not have viral genomic DNA.

[0009] Provided herein is a lipid particle comprising a baboon endogenous retrovirus (BaEV) envelope glycoprotein, the lipid particle comprising: (a) a lipid bilayer surrounding a lumen; and (b) a truncated baboon endogenous retrovirus (BaEV) envelope glycoprotein comprising a cytoplasmic tail with a partial fusion-inhibitory R peptide compared to an inhibitory R peptide of a wild-type truncated BaEV envelope glycoprotein, where the partial fusion-inhibitory R peptide comprises at least one contiguous amino-terminal amino acid, but less than the full length, of the inhibitory R peptide of the wild-type BaEV envelope glycoprotein, and the envelope glycoprotein is embedded in the lipid bilayer.

[0010] Provided herein is a lipid particle comprising a Baboon endogenous retrovirus (BaEV) envelope glycoprotein, comprising (a) a lipid bilayer surrounding a lumen, and (b) a truncated Baboon endogenous retrovirus (BaEV) envelope glycoprotein comprising a cytoplasmic tail with a partial fusion-inhibitory R peptide compared to the inhibitory R peptide of a wild-type truncated BaEV envelope glycoprotein, wherein the cytoplasmic tail is 25 amino acids in length and comprises an inhibitory R peptide of 8 consecutive amino-terminal acids (R+8) of the full-length inhibitory R peptide of the wild-type BaEV envelope glycoprotein, and wherein the envelope glycoprotein is embedded in the lipid bilayer.

[0011] In some of any of the provided embodiments, the lipid bilayer is derived from the membrane of a host cell used to generate the retroviral or retrovirus-like particle. In some of any of the embodiments, the host cell is selected from the group consisting of CHO cells, BHK cells, MDCK cells, C3H 10T1 / 2 cells, FLY cells, Psi-2 cells, BOSC 23 cells, PA317 cells, WEHI cells, COS cells, BSC 1 cells, BSC 40 cells, BMT 10 cells, VERO cells, W138 cells, MRC5 cells, A549 cells, HT1080 cells, 293 cells, 293T cells, B-50 cells, 3T3 cells, NIH3T3 cells, HepG2 cells, Saos-2 cells, Huh7 cells, HeLa cells, W163 cells, 211 cells, and 211A cells.

[0012] In some of any of the embodiments provided, the lipid bilayer is or includes one or more other viral components other than BaEV envelope glycoprotein. In some of any of the embodiments provided, the one or more viral components are derived from a retrovirus. In some of any of the embodiments provided, the retrovirus is a lentivirus or lentivirus-like particle. In some of any of the embodiments provided, the truncated BaEV glycoprotein includes (i) a glycoprotein 70 (g70) subunit or a biologically active portion thereof, and (ii) a portion of a glycoprotein p20E (p20E) subunit that includes a cytoplasmic tail with a partial inhibitory R peptide. In some of any of the embodiments, the glycoprotein 70 (g70) subunit or a biologically active portion thereof, and the portion of a glycoprotein p20E (p20E) subunit are linked via an intersubunit disulfide bond. In some of any of the embodiments, the BaEV glycoprotein binds to the ASCT-2 or ASCT-1 receptor.

[0013] In some of any of the provided embodiments, the glycoprotein 70 (g70) subunit, or a biologically active portion thereof, has an amino acid sequence set forth in SEQ ID NO:25, or a sequence that is at least 80% or about 80%, at least 81% or about 81%, at least 82% or about 82%, at least 83% or about 83%, 84% or about 84%, at least 85% or about 85%, at least 86% or about 86%, or at least 87% or about 87% of the amino acid sequence set forth in SEQ ID NO:25. The present invention includes sequences exhibiting at least 7% or about 87%, at least 88% or about 88%, or at least 89% or about 89%, at least 90% or about 90%, at least 91% or about 91%, at least 92% or about 92%, at least 93% or about 93%, at least 94% or about 94%, at least 95% or about 95%, 96% or about 96%, at least 97% or about 97%, at least 98% or about 98%, or at least 99% or about 99% sequence identity. In some of any of the provided embodiments, the portion of the glycoprotein p20E (p20E) subunit is SEQ ID NO:26, or a sequence similar to SEQ ID NO:26, at least 80% or about 80%, at least 81% or about 81%, at least 82% or about 82%, at least 83% or about 83%, 84% or about 84%, at least 85% or about 85%, at least 86% or about 86%, or at least 87% or about 87%, at least 88% or about 88%, or a sequence similar to SEQ ID NO:26. %, or at least or about 89%, at least 90% or about 90%, at least 91% or about 91%, at least 92% or about 92%, at least 93% or about 93%, at least 94% or about 94%, at least 95% or about 95%, 96% or about 96%, at least 97% or about 97%, at least 98% or about 98%, or at least 99% or about 99% sequence identity to the R peptide, and comprises a partial inhibitory R peptide.

[0014] In some of any of the provided embodiments, the truncated BaEV envelope glycoprotein is truncated relative to SEQ ID NO:24 and lacks up to 16 contiguous amino acids from the C-terminal cytoplasmic tail of SEQ ID NO:24. In some of any of the provided embodiments, the truncated BaEV envelope glycoprotein is truncated relative to SEQ ID NO:24 and lacks 8-14 contiguous amino acids from the C-terminal cytoplasmic tail of SEQ ID NO:24, optionally lacking 8-13 contiguous amino acids from the C-terminal cytoplasmic tail of SEQ ID NO:24.

[0015] In some of any of the provided embodiments, the partial fusion-inhibiting R peptide is set forth as amino acids 1-9 of SEQ ID NO:22, with an optional cytoplasmic tail set forth in SEQ ID NO:14 (R+9). In some of any of the provided embodiments, the truncated BaEV envelope glycoprotein is set forth in SEQ ID NO:37. In some of any of the provided embodiments, the partial fusion-inhibiting R peptide is set forth as amino acids 1-8 of SEQ ID NO:22, with an optional cytoplasmic tail set forth in SEQ ID NO:13 (R+8).

[0016] In some of any of the provided embodiments, the truncated BaEV envelope glycoprotein is set forth in SEQ ID NO: 36. In some of any of the provided embodiments, the partial fusion-inhibiting R peptide is set forth as amino acids 1-7 of SEQ ID NO: 22, with an optional cytoplasmic tail set forth in SEQ ID NO: 12 (R+7). In some of any of the provided embodiments, the truncated BaEV envelope glycoprotein is set forth in SEQ ID NO: 35. In some of any of the provided embodiments, the partial fusion-inhibiting R peptide is set forth as amino acids 1-6 of SEQ ID NO: 22, with an optional cytoplasmic tail set forth in SEQ ID NO: 11 (R+6). In some of any of the provided embodiments, the truncated BaEV envelope glycoprotein is set forth in SEQ ID NO: 34. In some of any of the provided embodiments, the partial fusion-inhibiting R peptide is set forth as amino acids 1-5 of SEQ ID NO: 22, with an optional cytoplasmic tail set forth in SEQ ID NO: 10 (R+5). In some of any of the provided embodiments, the truncated BaEV envelope glycoprotein is set forth in SEQ ID NO: 33. In some of any of the provided embodiments, the partial fusion-inhibiting R peptide is set forth as amino acids 1-4 of SEQ ID NO: 22, with an optional cytoplasmic tail set forth in SEQ ID NO: 9 (R+4). In some of any of the provided embodiments, the truncated BaEV envelope glycoprotein is set forth in SEQ ID NO: 32. In some of any of the provided embodiments, the partial fusion-inhibiting R peptide is set forth as amino acids 1-3 of SEQ ID NO: 22, with an optional cytoplasmic tail set forth in SEQ ID NO: 8 (R+3). In some of any of the provided embodiments, the truncated BaEV envelope glycoprotein is set forth in SEQ ID NO: 31.

[0017] In some of any of the embodiments provided, the particle further comprises an exogenous agent. In some of any of the embodiments provided, the exogenous agent is present in the lumen. In some of any of the embodiments provided, the exogenous agent is a protein or a nucleic acid, and optionally, the nucleic acid is DNA or RNA. In some of any of the embodiments provided, the particle is produced as a preparation with increased titer compared to a reference particle preparation, which is produced in the same manner, but has the incorporation of BaEV envelope glycoprotein with a cytoplasmic tail with a full-length R peptide or an R peptide portion that is 10 amino acids long or more.

[0018] In some of any of the provided embodiments, the titer is increased by 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, 30-fold or more, or more than 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, 30-fold or more, optionally by 5-fold or more, or about 5-fold or more, or by more than 5-fold or more. In some of any of the provided embodiments, the titer in the target cells, optionally HEK293 cells, after transduction is increased by 3×10 6 TU / mL or more, 4×10 6 TU / mL or more, 5×10 6 TU / mL or more, 6×10 6 TU / mL or more, 7×10 6 TU / mL or more, 8×10 6 TU / mL or more, 9×10 6 TU / mL or more, 1×10 7 TU / mL or greater, or 1.2 x 10 7 In some of the embodiments provided, the truncated BaEV envelope glycoprotein is at least about (0.001, 0.002, 0.005, 0.01, 0.02, 0.05, 0.1, 0.2, or 0.5) truncated BaEV envelope glycoprotein / nm 2 is present on the surface of the particle at a density of

[0019] Provided herein is a truncated BaEV envelope glycoprotein comprising a cytoplasmic tail with a partial fusion-inhibiting R peptide compared to a wild-type BaEV envelope glycoprotein, wherein the partial fusion-inhibiting R peptide comprises at least one contiguous amino-terminal amino acid, but less than the full-length inhibitory R peptide amino acid, of the wild-type BaEV envelope glycoprotein.

[0020] In some of any of the provided embodiments, the truncated BaEV glycoprotein comprises (i) a glycoprotein 70 (g70) subunit, or a biologically active portion thereof, and (ii) a portion of a glycoprotein p20E (p20E) subunit that includes a cytoplasmic tail with a partial inhibitory R peptide. In some of any of the embodiments, the glycoprotein 70 (g70) subunit, or a biologically active portion thereof, and the portion of the glycoprotein p20E (p20E) subunit are linked via an intersubunit disulfide bond.

[0021] In some of any of the provided embodiments, the BaEV glycoprotein binds to the ASCT-2 or ASCT-1 receptor.

[0022] In some of any of the provided embodiments, the glycoprotein 70 (g70) subunit, or a biologically active portion thereof, has an amino acid sequence set forth in SEQ ID NO:25, or a sequence that is at least 80% or about 80%, at least 81% or about 81%, at least 82% or about 82%, at least 83% or about 83%, 84% or about 84%, at least 85% or about 85%, at least 86% or about 86%, or at least 87% or about 87% of the amino acid sequence set forth in SEQ ID NO:25. The present invention includes sequences exhibiting at least 7% or about 87%, at least 88% or about 88%, or at least 89% or about 89%, at least 90% or about 90%, at least 91% or about 91%, at least 92% or about 92%, at least 93% or about 93%, at least 94% or about 94%, at least 95% or about 95%, 96% or about 96%, at least 97% or about 97%, at least 98% or about 98%, or at least 99% or about 99% sequence identity. In some of any of the provided embodiments, the portion of the glycoprotein p20E (p20E) subunit is SEQ ID NO:26, or a sequence similar to SEQ ID NO:26, at least 80% or about 80%, at least 81% or about 81%, at least 82% or about 82%, at least 83% or about 83%, 84% or about 84%, at least 85% or about 85%, at least 86% or about 86%, or at least 87% or about 87%, at least 88% or about 88%, or a sequence similar to SEQ ID NO:26. %, or at least or about 89%, at least 90% or about 90%, at least 91% or about 91%, at least 92% or about 92%, at least 93% or about 93%, at least 94% or about 94%, at least 95% or about 95%, 96% or about 96%, at least 97% or about 97%, at least 98% or about 98%, or at least 99% or about 99% sequence identity to the R peptide, and comprises a partial inhibitory R peptide.

[0023] In some of any of the provided embodiments, the truncated BaEV envelope glycoprotein is truncated relative to SEQ ID NO:24 and lacks up to 16 contiguous amino acids from the C-terminal cytoplasmic tail of SEQ ID NO:24. In some of any of the provided embodiments, the truncated BaEV envelope glycoprotein is truncated relative to SEQ ID NO:24 and lacks 8-14 contiguous amino acids from the C-terminal cytoplasmic tail of SEQ ID NO:24, and optionally lacks 8-13 contiguous amino acids from the C-terminal cytoplasmic tail of SEQ ID NO:24. In some of any of the provided embodiments, the partial fusion-inhibiting R peptide is set forth as amino acids 1-9 of SEQ ID NO:22, optionally with the cytoplasmic tail set forth in SEQ ID NO:14 (R+9). In some of any of the provided embodiments, the truncated BaEV envelope glycoprotein is set forth in SEQ ID NO:37. In some of any of the provided embodiments, the partial fusion-inhibiting R peptide is set forth as amino acids 1-8 of SEQ ID NO:22, optionally with the cytoplasmic tail set forth in SEQ ID NO:13 (R+8). In some of any of the provided embodiments, the truncated BaEV envelope glycoprotein is set forth in SEQ ID NO: 36. In some of any of the provided embodiments, the partial fusion-inhibiting R peptide is set forth as amino acids 1-7 of SEQ ID NO: 22, with an optional cytoplasmic tail set forth in SEQ ID NO: 12 (R+7). In some of any of the provided embodiments, the truncated BaEV envelope glycoprotein is set forth in SEQ ID NO: 35. In some of any of the provided embodiments, the partial fusion-inhibiting R peptide is set forth as amino acids 1-6 of SEQ ID NO: 22, with an optional cytoplasmic tail set forth in SEQ ID NO: 11 (R+6). In some of any of the provided embodiments, the truncated BaEV envelope glycoprotein is set forth in SEQ ID NO: 34. In some of any of the provided embodiments, the partial fusion-inhibiting R peptide is set forth as amino acids 1-5 of SEQ ID NO: 22, with an optional cytoplasmic tail set forth in SEQ ID NO: 10 (R+5). In some of any of the provided embodiments, the truncated BaEV envelope glycoprotein is set forth in SEQ ID NO:33.In some of any of the provided embodiments, the partial fusion-inhibiting R peptide is set forth as amino acids 1-4 of SEQ ID NO:22, with an optional cytoplasmic tail set forth in SEQ ID NO:9 (R+4). In some of any of the provided embodiments, the truncated BaEV envelope glycoprotein is set forth in SEQ ID NO:32. In some of any of the provided embodiments, the partial fusion-inhibiting R peptide is set forth as amino acids 1-3 of SEQ ID NO:22, with an optional cytoplasmic tail set forth in SEQ ID NO:8 (R+3). In some of any of the provided embodiments, the truncated BaEV envelope glycoprotein is set forth in SEQ ID NO:31.

[0024] Provided herein is a polynucleotide comprising a nucleic acid encoding a truncated BaEV envelope glycoprotein of any of the provided embodiments. In some of any of the provided embodiments, the polynucleotide is codon optimized.

[0025] In some of any of the embodiments provided, the polynucleotide comprises at least one promoter operably linked to control the expression of the nucleic acid. In some of any of the embodiments provided, the promoter is a constitutive promoter. In some of any of the embodiments provided, the promoter is an inducible promoter.

[0026] Provided herein is a vector comprising any of the polynucleotides provided herein.Provided herein is a plasmid comprising any of the polynucleotides provided herein.In some of any of the embodiments provided herein, the plasmid further comprises one or more nucleic acids encoding proteins for lentivirus production.

[0027] Provided herein is a cell comprising any of the provided polynucleotides, vectors, or plasmids. In some of any of the provided embodiments, the cell is a producer cell for producing lentiviral particles.

[0028] Provided herein is a producer cell comprising (i) a viral nucleic acid and (ii) a nucleic acid encoding a truncated BaEV envelope glycoprotein according to any of claims 43 to 64, optionally wherein the viral nucleic acid is a lentiviral nucleic acid. In some of any of the provided embodiments, the producer cell is selected from the group consisting of CHO cells, BHK cells, MDCK cells, C3H 10T1 / 2 cells, FLY cells, Psi-2 cells, BOSC 23 cells, PA317 cells, WEHI cells, COS cells, BSC 1 cells, BSC 40 cells, BMT 10 cells, VERO cells, W138 cells, MRC5 cells, A549 cells, HT1080 cells, 293 cells, 293T cells, B-50 cells, 3T3 cells, NIH3T3 cells, HepG2 cells, Saos-2 cells, Huh7 cells, HeLa cells, W163 cells, 211 cells, and 211A cells. In some of any of the embodiments provided, the producer cells comprise 293T cells.

[0029] In some of any of the provided embodiments, the viral nucleic acid is deficient in one or more genes involved in viral replication. In some of any of the provided embodiments, the viral nucleic acid comprises a nucleic acid encoding a viral packaging protein selected from one or more of Gag, Pol, Rev, and Tat. In some of any of the embodiments, the viral nucleic acid comprises one or more (e.g., all) of the following nucleic acid sequences: 5'LTR (e.g., including U5 and lacking a functional U3 domain), Psi packaging element (psi), central polypurine tract (cPPT) / central termination sequence (CTS) (e.g., DNA flap), polyA tail sequence, post-transcriptional control element (e.g., WPRE), Rev response element (RRE), and 3'LTR (e.g., including U5 and lacking a functional U3).

[0030] Provided herein are methods for producing lipid particles comprising a truncated BaEV glycoprotein, comprising: a) introducing any of the provided polynucleotides, any of the provided vectors, or any of the provided plasmids into a source cell; b) culturing the cell under conditions capable of producing lipid particles; and c) isolating, enriching, or purifying the lipid particles from the cell, thereby producing the lipid particles.

[0031] In some of any of the provided embodiments, the source cell is a mammalian cell. In some of any of the provided embodiments, the source cell is a producer cell and the lipid particle is a viral or viral-like particle, optionally a retroviral or retroviral-like particle, and optionally a lentiviral or lentiviral-like particle.

[0032] Provided herein are methods for producing pseudotyped lentiviral particles, the methods comprising: a) providing a producer cell comprising lentiviral nucleic acid and any of the provided polynucleotides, or any of the provided nucleic acids encoding truncated BaEV envelope glycoproteins; b) culturing the cell under conditions capable of producing lentiviral particles; and c) isolating, concentrating, or purifying the lentiviral particles from the producer cell, thereby producing pseudotyped lentiviral particles.

[0033] In some of any of the provided embodiments, the producer cell is selected from the group consisting of CHO cells, BHK cells, MDCK cells, C3H 10T1 / 2 cells, FLY cells, Psi-2 cells, BOSC 23 cells, PA317 cells, WEHI cells, COS cells, BSC 1 cells, BSC 40 cells, BMT 10 cells, VERO cells, W138 cells, MRC5 cells, A549 cells, HT1080 cells, 293 cells, 293T cells, B-50 cells, 3T3 cells, NIH3T3 cells, HepG2 cells, Saos-2 cells, Huh7 cells, HeLa cells, W163 cells, 211 cells, and 211A cells. In some of any of the provided embodiments, the producer cell comprises a 293T cell. In some of any of the provided embodiments, the method produces a lentiviral preparation having increased titer compared to a reference lentiviral particle preparation produced similarly but pseudotyped with a BaEV envelope glycoprotein having a cytoplasmic tail with a full-length R peptide or an R peptide portion that is 10 amino acids or more in length.

[0034] In some of any of the provided embodiments, the titer is increased by 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, 30-fold or more, or more than 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, 30-fold or more, optionally by 5-fold or more, or about 5-fold or more, or by more than 5-fold or more. In some of any of the provided embodiments, the method provides a method for increasing the titer in transduced target cells, optionally HEK293 cells, by 3×10 6 TU / mL or more, 4×10 6 TU / mL or more, 5×10 6 TU / mL or more, 6×10 6 TU / mL or more, 7×10 6 TU / mL or more, 8×10 6 TU / mL or more, 9×10 6 TU / mL or more, 1×10 7 TU / mL or greater, or 1.2 x 10 7In some of any of the provided embodiments, the method produces lentiviral preparations that are TU / mL or greater. In some of any of the provided embodiments, the method results in reduced syncytia formation of producer cells compared to a similar method but for producing a reference lentiviral particle preparation pseudotyped with a BaEV envelope glycoprotein having a cytoplasmic tail with no R peptide (Rless) or an R peptide that is 3 consecutive amino-terminal amino acids or less in length compared to the wild-type BaEV envelope glycoprotein R peptide. In some of any of the provided embodiments, the method produces lentiviral preparations that produce high titers (e.g., 4×10 6 >5×10 TU / mL 6 >6×10 TU / mL 6 >7×10 TU / mL 6 >8 x 10 TU / mL 6 >9×10 TU / mL 6 >1×10 TU / mL 7 > TU / mL or 1.2 × 10 7 Generate lentiviral preparations with a cytotoxicity of 0.1% TU / mL or greater, resulting in minimal syncytia formation of producer cells during the production process.

[0035] Provided herein are lipid particles produced by any of the methods provided.Also provided herein are lentiviral particles produced by any of the methods provided.

[0036] Provided herein is a lipid particle comprising any of the truncated BaEV envelope glycoproteins provided. Provided herein is a composition comprising any of the plurality of lentiviral particles provided. Provided herein is a composition comprising any of the plurality of lipid particles provided.

[0037] In some of any of the provided embodiments, the composition further comprises a pharma- ceutically acceptable excipient.

[0038] Provided herein are methods of transducing a cell, the methods comprising contacting a cell with any of the lentiviral particles provided or any of the compositions provided. In some of any of the provided embodiments, the lipid particle or lentiviral vector comprises an exogenous agent, and the exogenous agent is introduced into the cell by transduction.

[0039] Provided herein are methods of delivering an exogenous agent into a cell, the methods comprising contacting a cell with any of the provided lentiviral particles, any of the provided lipid particles, or any of the provided compositions.

[0040] In some of any of the embodiments provided, the contacting is in vitro or ex vivo. In some of any of the embodiments provided, the contacting is in vivo in a subject.

[0041] Provided herein are methods of delivering an exogenous agent to cells of a subject, the methods comprising administering to the subject any of the lentiviral particles, lipid particles, or compositions provided.

[0042] In some of any of the provided embodiments, the cells are hematopoietic cells. In some of any of the provided embodiments, the cells are selected from the group consisting of myeloid-lymphoid balanced hematopoietic cells, myeloid-biased hematopoietic cells, lymphoid-biased hematopoietic cells, platelet-biased hematopoietic cells, platelet-myeloid-biased hematopoietic cells, long-term repopulating hematopoietic cells, intermediate-term repopulating hematopoietic cells, or short-term repopulating hematopoietic cells. In some of any of the provided embodiments, the cells are selected from monocytes, macrophages, neutrophils, basophils, eosinophils, erythrocytes, megakaryocytes, and platelets. In some of any of the provided embodiments, the cell is selected from a T cell, a B cell, a natural killer (NK) cell, and an innate lymphoid cell. In some of any of the provided embodiments, the cell is a hematopoietic stem cell (HSC). In some of any of the provided embodiments, the subject has undergone a hematopoietic stem cell transplant.

[0043] In some of any of the provided embodiments, the exogenous agent is a protein or a nucleic acid, and optionally the nucleic acid is DNA or RNA. In some of any of the provided embodiments, the exogenous agent is or codes for a therapeutic substance for treating a disease or condition in a subject. In some of any of the provided embodiments, the exogenous agent is or codes for a membrane protein, optionally a chimeric antigen receptor, for targeting an antigen associated with a disease or condition in a subject. In some of any of the provided embodiments, the exogenous agent is for use in gene therapy to correct a genetic defect in a subject, or to replace a missing or deleted gene. In some of any of the provided embodiments, the subject is a human subject. [Brief description of the drawings]

[0044] [Figure 1] 1 shows viral titers of lentiviral vector particles produced from producer cells transfected with packaging plasmids and various truncated BaEV envelope glycoproteins containing inhibitory R peptides of different lengths. [Diagram 2] FIG. 1 shows syncytium formation of producer cells during production of lentiviral vector particles by transfection of producer cells with packaging plasmids and various truncated BaEV envelope glycoproteins containing inhibitory R peptides of different lengths. [Diagram 3] FIG. 1 shows viral titers of an exemplary lentivector preparation pseudotyped with a truncated BaEV envelope glycoprotein containing a partial inhibitory R peptide having eight consecutive amino-terminal amino acids of the R peptide (R+8) of the wild-type BaEV envelope protein after production from producer cells transfected with a packaging plasmid and different concentrations of a plasmid encoding the R+8 truncated BaEV envelope glycoprotein. [Figure 4]The percentage of GFP+ cells 8 days after transduction with BaEV+8, BaEVTR, no BaEVR, or VSV-G pseudotyped vectors is shown. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0045] Detailed Description Provided herein is a lipid particle, e.g., a lentiviral particle, pseudotyped with a truncated Baboon endogenous retrovirus (BaEV) envelope glycoprotein that comprises a cytoplasmic tail with a partial fusion-inhibiting R peptide compared to a full-length wild-type BaEV envelope glycoprotein. Also provided herein is a lipid particle, e.g., a lentiviral particle, pseudotyped with a truncated Baboon endogenous retrovirus (BaEV) envelope glycoprotein that comprises (i) a glycoprotein 70 (g70) subunit or a biologically active portion thereof, and (ii) a glycoprotein p20E (p20E) subunit that comprises a partial fusion-inhibiting R peptide. In certain embodiments, the R peptide comprises at least three consecutive amino-terminal amino acids of the inhibitory R peptide. In certain embodiments, the partial fusion-inhibiting R peptide can mediate fusion of the truncated BaEV envelope glycoprotein to a desired target cell. In some embodiments, the truncated BaEV envelope glycoprotein is embedded in the lipid particle. The lipid particle may be a non-viral particle, a viral particle, or a virus-like particle (VLP).

[0046] The embodiments provided are based on BaEV envelope glycoproteins that exhibit transduction properties that are particularly suitable for gene transfer into hematopoietic cells, including hematopoietic stem cells (HSCs) and resting T and B cells. This provides a delivery vector for delivering therapeutic genes to HSCs that can differentiate into all hematopoietic lineages for gene replacement or correction in the context of gene therapy. Furthermore, efficient gene transfer into resting or quiescent T and B lymphocytes for gene therapy or immunotherapy also has many advantages in conjunction with ex vivo and in vivo methods of viral particle delivery.

[0047] BaEV envelope glycoprotein shares structural and functional features in common with envelope glycoproteins of other retroviruses, including an extracellular domain, a transmembrane domain, and a cytoplasmic tail domain. BaEV envelope glycoprotein is synthesized as an N-glycosylated inactive precursor and processed into two subunits, the surface unit protein or gp70, and the transmembrane protein p20E, by host cell furin or furin-like proteases in the Golgi. The cleavage site between gp70 and p20E requires a minimal sequence [KR]-X-[KR]-R (X is any amino acid), and gp70 and p20E remain associated in a labile interaction that may involve disulfide bonds. gp70 is believed to bind the virus to host cells by binding to a receptor, which triggers the refolding of p20E and promotes fusion with the host cell membrane. p20E functions as a class I viral fusion protein and shares common structural and functional features with many families of fusion proteins (e.g., HIV-1 gp41 or influenza virus hemagglutinin [HA]). Under native conditions, BaEV envelope glycoprotein also undergoes a second cleavage in the cytoplasmic domain or cytoplasmic tail of p20E. The C-terminal 17 amino acids of the cytoplasmic tail, the fusion-inhibitory R peptide, contains the tyrosine endocytosis signal YXXL and is removed as a result of proteolytic cleavage by the viral protease. Removal of the R peptide is associated with enhanced membrane fusogenicity of the envelope glycoprotein (Beneviste et al. (1974) Nature 248:17-20; Todaro et al. (1974) Cell 2:55-61; Aguilar et al. (2003) Journal of Virology 77(2):1281-1291).

[0048] Although the mechanism by which R-peptide cleavage enhances membrane fusion is not fully understood, the fusion efficiency of BaEV envelope glycoprotein can be improved by modifying the cleavage of the R-peptide in the absence of viral proteases. Mutations in BaEV that completely cleave the 17 amino acid R-peptide have been identified (US9249426, Bernardin et al. (2019) Blood. 3(3):461-475). Furthermore, although the homology between R-peptides is low, such as only 33% between murine leukemia virus (MuLV) and gibbon ape leukemia virus (GaLV), R-peptides are interchangeable (Christodoulopoulos et al. (2001) J. Viral 75(4):4129-4138). Several such studies have identified variant BaEV envelope glycoprotein cytoplasmic tails replaced by MuLV (US9249426, Bernardin et al. (2019) Blood. 3(3):461-475).

[0049] However, truncation of the full-length R peptide from the cytoplasmic tail renders the envelope glycoprotein more fusogenic and induces extensive syncytia formation (Olsen et al. (1999) Journal of Virology 8975-8981; Ragheb et al. (1994) J Virol. 68:3220-3231; Rein et al (1994) J Virol 1773-1781). Various envelope glycoproteins, including those that form MuLV and BaEV, show a greater ability to induce syncytia in cell-cell fusion assays in the absence of the R peptide compared to the full-length R peptide across a range of cell lines (Aguilar (2003) J. Virol. 77(2):1281-1291). Furthermore, syncytium formation during lentiviral vector production has been shown to result in low titers, even under conditions that attempt to optimize higher titer protocols (Bauler et al. (2019) Molecular Therapy vol. 17; Noguchi et al. (2020) ASGCT 23 rdAnnual Meeting. Poster #988).

[0050] Particles incorporating BaEV lacking wild-type R peptide have been generated, while hyperfusogenic mutants with low syncytia formation are found herein to result from specific tail truncations of BaEV envelope glycoprotein.Furthermore, it is found herein that mutant BaEV envelope glycoproteins result in improved titers of lentivirus preparations when specific amino acid truncations of R peptide are expressed.For example, it is found that BaEV envelope glycoproteins containing 8 amino acids of R peptide result in 5-fold higher titers than BaEV envelope glycoproteins expressing 10 amino acids of R peptide.

[0051] The provided lipid particles, such as lentiviral particles, incorporate a truncated BaEV envelope glycoprotein that includes a cytoplasmic tail with a partial fusion-inhibitory R peptide compared to the inhibitory R peptide of the full-length wild-type BaEV envelope glycoprotein. The partial fusion-inhibitory peptide can include 1, 2, 3, 4, 5, 6, 7, 8, or 9 consecutive amino-terminal amino acids of the inhibitory R peptide. In some embodiments, the provided lipid particles, such as lentiviral vectors, exhibit high titers after production by producer cells, especially compared to full-length BaEV envelope glycoproteins that include a complete inhibitory R peptide. Furthermore, the provided lipid particles, such as lentiviral vectors, exhibit lower membrane fusion activity, especially compared to mutant BaEV envelope glycoproteins that lack a complete R peptide (no R), resulting in reduced or minimal syncytium formation in producer cells.

[0052] Also provided is a particle comprising a truncated Baboon endogenous retrovirus (BaEV) envelope glycoprotein that comprises a cytoplasmic tail with a partial fusion-inhibiting R peptide compared to the wild-type BaEV envelope glycoprotein. The particle can include a lipid particle, a viral particle, a virus-like particle (VLP), a non-viral particle, or a synthetic particle, or any of the particles described herein.

[0053] Also provided are particles that additionally contain one or more exogenous agents, for example, for delivering diagnostic or therapeutic agents to cells, including after in vivo administration to a subject. In some embodiments, the exogenous agent may be a transgene, such as a nucleic acid encoding a desired protein, or may be a heterologous protein. In some aspects, the transgene may be used in conjunction with gene therapy, such as to replace a gene in a cell where the gene is missing or defective. In some embodiments, the exogenous agent may code for a protein that is desired to be delivered to a target cell, or is the desired protein. In some embodiments, the exogenous agent codes for or is a chimeric antigen receptor (CAR).

[0054] In some embodiments, the exogenous agent is or codes for a factor associated with gene editing. In some embodiments, the exogenous agent is or codes for a factor associated with base editing and / or prime editing (i.e., target-primed reverse transcription (TPRT)). In some embodiments, the exogenous agent codes for or is a nuclease used in gene editing methods or the like. In some embodiments, the nuclease is a zinc finger nuclease (ZFN), a transcription activator-like effector nuclease (TALEN), or a CRISPR-associated protein nuclease (Cas). In some embodiments, the exogenous agent is or codes for a transposase and / or a recombinase. In some embodiments, the exogenous agent is or codes for a DNA polymerase, an RNA polymerase, or a reverse transcriptase.

[0055] Also provided herein are methods and uses of the truncated BaEV envelope glycoproteins and particles, such as in diagnostic and therapeutic methods. Also provided are methods for modifying, preparing, and producing the polynucleotides, glycoproteins, and particles, as well as kits and devices containing, using, making, and administering the particles.

[0056] All publications, including patent documents, scientific articles, and databases, referred to in this application are incorporated by reference in their entirety for all purposes to the same extent as if each individual publication was individually disclosed by reference. To the extent that a definition set forth herein is contrary to or inconsistent with a definition set forth in a patent, application, published application, or other publication incorporated herein by reference, the definition set forth herein takes precedence over the definition incorporated herein by reference.

[0057] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0058] I. Definition Unless otherwise defined, all terms, notations, and other technical and scientific terms or terminology of the art used herein are intended to have the same meaning as commonly understood by one of ordinary skill in the art to which the claimed subject matter pertains. In some cases, terms having a commonly understood meaning have been defined herein for clarity and / or ease of reference, and the inclusion of such definitions herein should not necessarily be construed as representing a substantial difference from the meaning commonly understood in the art.

[0059] As used herein, the articles "a" and "an" refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element.

[0060] As used herein, the term "about" will be understood by those of ordinary skill in the art and will vary to some extent in the context in which it is used. As used herein, "about," when referring to a measurable value, such as an amount, a time duration, etc., encompasses a variation of ±20% or ±10%, more preferably ±5%, even more preferably ±1%, and even more preferably ±0.1% from the specified value, as such variations are appropriate for carrying out the disclosed methods.

[0061] As used herein, "lipid particle" refers to any biological or synthetic particle that contains a bilayer of amphiphilic lipids surrounding a lumen or cavity. Generally, the particle does not contain a nucleus. Examples of lipid particles include solid particles, such as nanoparticles, virus-derived particles, or cell-derived particles. Such lipid particles include, but are not limited to, virus-based particles, such as virus-like particles or virus vectors (e.g., lentiviral vectors), exosomes, enucleated cells, various vesicles, such as microvesicles, membrane vesicles, extracellular membrane vesicles, cell membrane vesicles, giant cell membrane vesicles, apoptotic bodies, mitoparticles, pyrenocytes, or lysosomes. In some embodiments, the lipid particle can be a fusosome. In some embodiments, the lipid particle is not a platelet.

[0062] The term "virus-based particle" can be any type of lipid particle derived from a virus or viral proteins, such as viral vector particles and virus-like particles.

[0063] The terms "viral vector particle" and "viral vector" are used interchangeably herein. A viral vector particle can be any type of lipid particle that contains one or more viral structural proteins in addition to at least one nonstructural viral genomic component or functional fragment thereof (i.e., polymerase, integrase, protease, or other nonstructural component).

[0064] The term "virus-like particle" or VLP can be any type of particle characterized by at least one viral structural protein and lacking viral genetic material.

[0065] As used herein, "fusosome" refers to a particle that contains a bilayer of amphipathic lipids surrounding a lumen or cavity, and a fusogen that interacts with the amphipathic lipid bilayer. In some embodiments, the fusosome contains an exogenous agent. In some embodiments, the exogenous agent is a nucleic acid (e.g., DNA or RNA), a peptide, or a protein. In some embodiments, the fusosome is a membrane-encapsulated preparation. In some embodiments, the fusosome is derived from a source cell.

[0066] As used herein, a "fusosome composition" refers to a composition that includes one or more fusosomes.

[0067] As used herein, "fusogen" refers to an agent or molecule that creates an interaction between two membranes, including a membrane-enclosed lumen. In embodiments, the fusogen promotes membrane fusion. In other embodiments, the fusogen creates a connection, for example, a pore between two membranes or lumen (e.g., the lumen of a retroviral vector and the cytoplasm of a target cell).

[0068] As used herein, "retroviral nucleic acid" refers to a nucleic acid that contains at least the minimum sequence requirements for packaging into a retrovirus or retroviral vector, alone or in combination with a helper cell, helper virus, or helper plasmid. In some embodiments, the retroviral nucleic acid comprises one or more of 5'LTR (e.g., to facilitate integration), U3 (e.g., to activate viral genome RNA transcription), R (e.g., Tat binding region), U5, 3'LTR (e.g., to facilitate integration), packaging site (e.g., psi (Ψ)), RRE (e.g., to bind Rev and facilitate nuclear transport). Retroviral nucleic acid may comprise RNA (e.g., as part of the virion) or DNA (e.g., as introduced into a source cell or after reverse transcription in a recipient cell). In some embodiments, the retroviral nucleic acid is packaged using a helper cell, helper virus, or helper plasmid that comprises one or more (e.g., all) of gag, pol, and env.

[0069] As used herein, with respect to peptide, polypeptide, or antibody sequences, "percent (%) amino acid sequence identity" and "homology" are defined as the percentage of amino acid residues in a candidate sequence that are identical to amino acid residues in a particular peptide or polypeptide sequence, without considering any conservative substitutions as part of the sequence identity, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percentage of sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be accomplished in a variety of ways that are within the skill of the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, or MEGALIGN™ (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms required to achieve maximum alignment over the entire length of the sequences being compared.

[0070] Amino acid substitutions can include, but are not limited to, the replacement of one amino acid with another in a polypeptide. Exemplary substitutions are shown in Table 1. Amino acid substitutions can be introduced into an antibody of interest and the product screened for the maintenance / improvement of a desired activity, e.g., binding.

[0071] (Table 1) TIFF2024521811000001.tif156165

[0072] Amino acids can be classified according to common side chain properties: (1) Hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile; (2) Neutral hydrophilic: Cys, Ser, Thr, Asn, Gln; (3) Acidic: Asp, Glu; (4) Basic: His, Lys, Arg; (5) Residues that affect chain orientation: Gly, Pro; (6) Aromatic: Trp, Tyr, Phe.

[0073] Non-conservative substitutions involve exchanging a member of one of these classes for another class.

[0074] The term "corresponding to" in reference to a protein position, e.g., a statement that a nucleotide or amino acid position "corresponds to" a nucleotide or amino acid position in a disclosed sequence, such as those shown in the sequence listing, refers to a nucleotide or amino acid position identified in an alignment with the disclosed sequence based on structural sequence alignment or using a standard alignment algorithm such as the GAP algorithm. For example, corresponding residues of similar sequences (e.g., fragments or species variants) can be determined by alignment with a reference sequence by structural alignment methods. By aligning sequences, one skilled in the art can identify corresponding residues, for example, using conserved and identical amino acid residues as guides.

[0075] The term "isolated" as used herein refers to a molecule that is separated from at least some of the components that are typically found or produced in nature. For example, a polypeptide is referred to as "isolated" when it is separated from at least some of the components of the cell that produced it. If the polypeptide is secreted by a cell after expression, physically separating the supernatant containing the polypeptide from the cell that produced it is considered to "isolate" the polypeptide. Similarly, a polynucleotide is referred to as "isolated" when it is not part of a larger polynucleotide that is typically found in nature (e.g., in the case of a DNA polynucleotide, genomic DNA or mitochondrial DNA, etc.), or, for example, in the case of an RNA polynucleotide, when it is separated from at least some of the components of the cell that produced it. Thus, a DNA polynucleotide contained in a vector within a host cell can be referred to as "isolated".

[0076] The term "effective amount" as used herein means an amount of a pharmaceutical composition sufficient to significantly and favorably modify the symptoms and / or condition being treated (e.g., provide a favorable clinical response). The effective amount of active ingredient for use in a pharmaceutical composition will vary according to the particular condition being treated, the severity of the condition, the duration of treatment, the nature of the combination therapy, the particular active ingredient being used, the particular pharma- ceutically acceptable excipients and / or carriers being used, and similar factors, according to the knowledge and expertise of the attending physician.

[0077] "Exogenous agent" as used herein with respect to particles refers to an agent that is not contained in or encoded by the corresponding wild-type virus or fusogen produced from the corresponding wild-type source cell. In some embodiments, the exogenous agent is not naturally occurring, e.g., a protein or nucleic acid with a sequence that is altered (e.g., by insertion, deletion, or substitution) relative to the naturally occurring protein. In some embodiments, the exogenous agent is not naturally occurring in the source cell. In some embodiments, the exogenous agent is naturally occurring in the source cell but exogenous to the virus. In some embodiments, the exogenous agent is not naturally occurring in the recipient cell. In some embodiments, the exogenous agent is naturally occurring in the recipient cell but not at the desired level or at the desired time. In some embodiments, the exogenous agent comprises an RNA or a protein.

[0078] As used herein, "promoter" refers to a cis-regulatory DNA sequence that drives the transcription of a gene when operably linked to a gene coding sequence. A promoter may contain a transcription factor binding site. In some embodiments, a promoter acts in cooperation with one or more enhancers distal to the gene.

[0079] As used herein, a composition refers to any mixture of two or more products, substances, or compounds, including cells. It may be a solution, suspension, liquid, powder, paste, aqueous, non-aqueous, or any combination thereof.

[0080] As used herein, the term "pharmacologically acceptable" refers to a substance, such as a carrier or diluent, that does not abrogate the biological activity or properties of the compound and is relatively non-toxic, i.e., the substance may be administered to an individual without causing undesired biological effects or interacting in a deleterious manner with any of the components of the composition in which it is contained.

[0081] As used herein, the term "pharmaceutical composition" refers to a mixture of at least one compound of the present invention with other chemical components, such as carriers, stabilizers, diluents, dispersants, suspending agents, thickeners, and / or excipients. The pharmaceutical composition facilitates administration of the compound to an organism. Multiple techniques exist in the art for administering compounds, including, but not limited to, intravenous, oral, aerosol, parenteral, ocular, pulmonary, and topical administration.

[0082] "Disease" or "disorder" as used herein refers to a condition for which treatment is necessary and / or desirable.

[0083] As used herein, the terms "treat", "treating" or "treatment" refer to improving a disease or disorder, e.g., delaying or stopping the onset of a disease or disorder, or alleviating at least one of its clinical symptoms. For purposes of this disclosure, improving a disease or disorder can include obtaining beneficial or desired clinical results, including, but not limited to, any one or more of the following: alleviating one or more symptoms, attenuating the extent of the disease, preventing or delaying the spread of the disease (e.g., metastasis, e.g., to the lungs or lymph nodes), preventing or delaying the recurrence of the disease, delaying or slowing the progression of the disease, improving the disease state, inhibiting the disease or disease progression, inhibiting or slowing the disease or its progression, halting its progression, and remission (whether partial or total).

[0084] The terms "individual" and "subject" are used interchangeably herein to refer to animals, e.g., mammals. The term patient includes human and veterinary subjects. In some embodiments, methods of treating mammals are provided, including, but not limited to, humans, rodents, monkeys, cats, dogs, horses, cows, pigs, sheep, goats, mammalian laboratory animals, mammalian farm animals, mammalian sport animals, and mammalian pets. The subject may be male or female and of any suitable age, including infants, juveniles, adolescents, adults, and geriatric subjects. In some examples, "individual" or "subject" refers to an individual or subject in need of treatment for a disease or disorder. In some embodiments, the subject receiving treatment may be a patient, which indicates the fact that the subject has a disorder relevant to the treatment or has been identified as being at sufficient risk of suffering from the disorder. In certain embodiments, the subject is a human, e.g., a human patient.

[0085] II. Truncated BaEV Fusogens Provided herein is a truncated Baboon endogenous retrovirus (BaEV) envelope glycoprotein that can be incorporated or incorporated into lipid particles, for example, into viral particles, including lentivirus particles or lentivirus-like particles.For example, provided herein is a lentivirus particle that is pseudotyped with any of the truncated BaEV envelope glycoproteins provided.Also provided herein is a polynucleotide that encodes the truncated BaEV envelope glycoprotein.

[0086] The wild-type BaEV envelope glycoprotein is a retroviral envelope protein that includes a C-terminal cytoplasmic tail (e.g., corresponding to amino acids 512-545 of SEQ ID NO:4, or SEQ ID NO:24), a transmembrane domain (e.g., corresponding to amino acids 489-511 of SEQ ID NO:3, or SEQ ID NO:24), and an extracellular domain (e.g., corresponding to amino acids 1-488 of SEQ ID NO:2, or SEQ ID NO:24). Maturation of the precursor protein in the Golgi, which requires the minimal sequence [KR]-X-[KR]-R (where X is any amino acid), results in two subunits: a surface unit protein, gp70, and a transmembrane protein, p20E. The surface unit protein, gp70 (e.g., corresponding to amino acids 1-358 of SEQ ID NO:25, or SEQ ID NO:24) and the transmembrane protein, p20E (e.g., corresponding to amino acids 359-545 of SEQ ID NO:26, SEQ ID NO:27, or SEQ ID NO:24) remain associated in an unstable interaction that may involve disulfide bonds. In wild-type BaEV envelope glycoprotein, membrane fusion is controlled by a short 17 amino acid sequence called the fusion-inhibitory R peptide (e.g., as shown in SEQ ID NO: 22), which is located at the C-terminus of the cytoplasmic tail domain. The fusion-inhibitory R peptide carries a tyrosine endocytosis signal YXXL, and its cleavage by the viral protease is thought to enhance membrane fusion activation through molecular rearrangements in the transmembrane domain and extracellular region of the envelope glycoprotein (Salamango et al (2015) Journal of virology 89 (24): 12492-12500).

[0087] In wild-type BaEV envelope glycoprotein, gp70 mediates receptor binding to ASCT-2 and ASCT-1 receptors on host cells. In some embodiments, the glycoprotein 70 (g70) subunit or a biologically active portion thereof binds to ASCT-2 and ASCT-1 receptors. In wild-type BaEV envelope glycoprotein, p20E functions as a class I viral fusion protein. It is believed that interaction of the gp70 subunit with the host cell membrane triggers the refolding of p20E, activating membrane fusion potential by unmasking the fusion peptide.

[0088] In some embodiments, the truncated BaEV envelope glycoprotein comprises a cytoplasmic tail with a partial fusion-inhibitory R peptide compared to the wild-type BaEV envelope glycoprotein, where the R peptide comprises a contiguous portion of the inhibitory R peptide but lacks the full-length R peptide of the wild-type BaEV envelope glycoprotein. In some embodiments, the truncated BaEV envelope glycoprotein has a cytoplasmic tail consisting of a partial inhibitory R peptide compared to the wild-type BaEV envelope glycoprotein, having at least one, at least two, or at least three contiguous amino-terminal amino acids of the inhibitory R peptide, but shorter than the full-length R peptide. In some embodiments, the truncated BaEV envelope glycoprotein has a cytoplasmic tail having a partial inhibitory R peptide consisting of 1 to 16 contiguous amino-terminal amino acids of the inhibitory R peptide of wild-type BaEV envelope glycoprotein, e.g., consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 12, 14, 15, or 16 amino-terminal amino acids of the inhibitory R peptide of wild-type BaEV envelope glycoprotein.

[0089] In certain embodiments, the partial fusion-inhibiting R peptide comprises 1-9 contiguous amino terminal amino acids (but less than the full length) of the inhibitory R peptide of the wild-type BaEV envelope glycoprotein. In certain embodiments, the partial fusion-inhibiting R peptide comprises 2-9 contiguous amino terminal amino acids (but less than the full length) of the inhibitory R peptide of the wild-type BaEV envelope glycoprotein. In certain embodiments, the partial fusion-inhibiting R peptide comprises 3-9 contiguous amino terminal amino acids (but less than the full length) of the inhibitory R peptide of the wild-type BaEV envelope glycoprotein. In certain embodiments, the partial fusion-inhibiting R peptide comprises 4-9 contiguous amino terminal amino acids (but less than the full length) of the inhibitory R peptide of the wild-type BaEV envelope glycoprotein. In certain embodiments, the partial fusion-inhibiting R peptide comprises 5-9 contiguous amino terminal amino acids (but less than the full length) of the inhibitory R peptide of the wild-type BaEV envelope glycoprotein. In certain embodiments, the partial fusion-inhibiting R peptide comprises 6-9 contiguous amino-terminal amino acids (but less than the full length) of the inhibitory R peptide of the wild-type BaEV envelope glycoprotein. In certain embodiments, the partial fusion-inhibiting R peptide comprises 7-9 contiguous amino-terminal amino acids (but less than the full length) of the inhibitory R peptide of the wild-type BaEV envelope glycoprotein. In certain embodiments, the partial fusion-inhibiting R peptide comprises 8-9 contiguous amino-terminal amino acids (but less than the full length) of the inhibitory R peptide of the wild-type BaEV envelope glycoprotein. In certain embodiments, the partial fusion-inhibiting R peptide comprises 9 contiguous amino-terminal amino acids (but less than the full length) of the inhibitory R peptide of the wild-type BaEV envelope glycoprotein.

[0090] In some embodiments, the truncated BaEV envelope glycoprotein comprises a cytoplasmic tail that is shorter than the full-length cytoplasmic tail of the truncated BaEV envelope glycoprotein due to the inclusion of a partial inhibitory R peptide. In some embodiments, the truncated BaEV envelope glycoprotein comprises a portion of the full-length cytoplasmic tail shown in SEQ ID NO:4. In some embodiments, the truncated BaEV envelope glycoprotein comprises 18-33 contiguous amino terminal amino acids of SEQ ID NO:4, e.g., 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 contiguous amino terminal amino acids of SEQ ID NO:4. In some embodiments, the cytoplasmic tail of the truncated BaEV envelope is 18-33 amino acids in length, e.g., 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 amino acids in length.

[0091] In certain embodiments, the truncated BaEV envelope glycoprotein comprises a portion of the sequence set forth in either SEQ ID NO:23 or 24, which is a functionally active variant or biologically active portion thereof that retains membrane fusion activity. In some embodiments, the truncated BaEV envelope glycoprotein lacks up to 16 contiguous amino acids from the C-terminal cytoplasmic tail of SEQ ID NO:23 or SEQ ID NO:24. In some embodiments, the truncated BaEV envelope glycoprotein lacks 1-16 contiguous amino acids of the C-terminal cytoplasmic tail of SEQ ID NO:23 or SEQ ID NO:24, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 contiguous amino acids of the C-terminal cytoplasmic tail of SEQ ID NO:23 or SEQ ID NO:24. In certain embodiments, the truncated BaEV envelope glycoprotein lacks up to 8-14 contiguous amino acids from the C-terminal cytoplasmic tail of SEQ ID NO:23 or SEQ ID NO:24. In some embodiments, the truncated BaEV envelope glycoprotein lacks up to 8-13 contiguous amino acids from the C-terminal cytoplasmic tail of SEQ ID NO:23 or SEQ ID NO:24.

[0092] In some embodiments, provided truncated baboon endogenous retrovirus (BaEV) envelope glycoproteins, such as when incorporated into lipid particles (e.g., lentiviral particles), consist of two chains comprising (i) the glycoprotein 70 (g70) subunit, or a biologically active portion thereof, and (ii) a portion of the glycoprotein p20E (p20E) subunit that includes a cytoplasmic domain with a partial fusion-inhibitory R peptide as described.

[0093] In certain embodiments, the truncated BaEV envelope glycoprotein gp70 subunit has a sequence as set forth in SEQ ID NO:25, or a sequence that is at least or about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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 9 ... % or about 88%, or at least 89% or about 89%, at least 90% or about 90%, at least 91% or about 91%, at least 92% or about 92%, at least 93% or about 93%, at least 94% or about 94%, at least 95% or about 95%, 96% or about 96%, at least 97% or about 97%, at least 98% or about 98%, or at least 99% or about 99% sequence identity thereto.

[0094] In certain embodiments, the portion of the truncated BaEV envelope glycoprotein p20E subunit has the sequence set forth in SEQ ID NO:26, or a functional or biologically active variant thereof that retains membrane fusion activity, and includes a partial inhibitory R peptide. In some embodiments, a functional or biologically active variant thereof of the p20E subunit comprises a sequence exhibiting at least or about 80%, at least or about 81%, at least 82% or about 82%, at least 83% or about 83%, 84% or about 84%, at least 85% or about 85%, at least 86% or about 86%, or at least 87% or about 87%, at least 88% or about 88%, or at least 89% or about 89%, at least 90% or about 90%, at least 91% or about 91%, at least 92% or about 92%, at least 93% or about 93%, at least 94% or about 94%, at least 95% or about 95%, 96% or about 96%, at least 97% or about 97%, at least 98% or about 98%, or at least 99% or about 99% sequence identity to SEQ ID NO:26, and comprises a partial inhibitory R peptide. In some embodiments, the portion of the p20E subunit has a sequence set forth in any of SEQ ID NOs:44-60.

[0095] In certain embodiments, the truncated BaEV envelope glycoproteins provided exhibit membrane fusion activity mediated by a partial fusion-inhibiting R peptide, such as when incorporated into lipid particles (e.g., lentiviral particles). In some embodiments, the truncated BaEV envelope glycoproteins provided comprising a partial fusion-inhibiting R peptide retain the membrane fusion activity of wild-type BaEV envelope glycoproteins. The membrane fusion activity includes the ability of the truncated BaEV envelope glycoproteins to promote or facilitate the fusion of two membrane lumens and cytoplasm of a target cell, e.g., cells that contain surface receptors or molecules that are recognized or bound by the truncated BaEV envelope protein. In some embodiments, the truncated BaEV envelope glycoproteins provided herein bind to the neutral amino acid transporter receptor ASCT-2 or ASCT-1. The ASCT-2 and ASCT-1 receptors share approximately 57% sequence identity, are differentially expressed in cells (e.g., T and B cells), and are transporters of overlapping, but not identical, sets of neutral amino acids (Colmartino et al. (2019) Frontiers in Immunology 10(2873):1-7; Girard-Gagnepain et al (2014) Blood 124:1221-1231; Levy et al. (2016) Journal of Thrombosis and Haemostasis 14:2478-2492).

[0096] References to retention of membrane fusion activity include from 10% to greater than or equal to about 150% of the binding level or degree of binding of the corresponding wild-type BaEV envelope glycoprotein, as set forth in SEQ ID NO: 24. In some embodiments, the membrane fusion activity of the truncated BaEV envelope glycoprotein is between 50% and 125% of the binding level or degree of binding of the corresponding wild-type BaEV envelope glycoprotein, as set forth in SEQ ID NO: 24. In some embodiments, the membrane fusion activity of the truncated BaEV envelope glycoprotein is between 80% and 120% of the binding level or degree of binding of the corresponding wild-type BaEV envelope glycoprotein, as set forth in SEQ ID NO: 24.

[0097] In some embodiments, the membrane fusion activity of the truncated BaEV envelope glycoprotein is lower than the membrane fusion activity of a BaEV envelope glycoprotein lacking the complete inhibitory R peptide (R-less). For example, in some embodiments, the membrane fusion activity of the truncated BaEV envelope glycoprotein is lower than the membrane fusion activity of an R-less BaEV envelope glycoprotein having a 17 amino acid truncation in the distal C-terminal portion of the wild-type BaEV envelope glycoprotein corresponding to amino acids 529-545 of SEQ ID NO:24. In some embodiments, the R-less BaEV envelope glycoprotein has the amino acid sequence shown in SEQ ID NO:28. In some embodiments, the truncated BaEV envelope glycoproteins provided exhibit 10% to 90% of the membrane fusion activity of the R-less BaEV envelope glycoprotein, e.g., 90% or about 90%, 85% or about 85%, 80% or about 80%, 75% or about 75%, 70% or about 70%, 60% or about 60%, 50% or about 50%, 40% or about 40%, 30% or about 30%, 20% or about 20%, or 10% or less of the membrane fusion activity of the R-less BaEV envelope glycoprotein, e.g., as set forth in SEQ ID NO: 28. In some embodiments, the reduced membrane fusion activity avoids high levels of cell fusion during production of the lipid particles, e.g., lentiviral particles, provided. For example, incorporation of the truncated BaEV envelope glycoprotein during a method for producing lipid particles, e.g., lentiviral particles, avoids or reduces high levels of syncytia formation of producer cells during production of the particles.

[0098] In certain embodiments, the truncated BaEV envelope glycoprotein comprises a partial fusion-inhibitory R peptide (e.g., R+9) that comprises 9 consecutive amino-terminal amino acids of the wild-type inhibitory R peptide. In certain embodiments, the truncated BaEV envelope glycoprotein comprises a partial fusion-inhibitory R peptide set forth as amino acids 1-9 of SEQ ID NO:22. In some embodiments, the BaEV envelope glycoprotein has a cytoplasmic tail set forth in SEQ ID NO:14. In some embodiments, the truncated BaEV envelope glycoprotein has a sequence that exhibits at least 85%, at least 90%, or at least 95% sequence identity to the sequence set forth in SEQ ID NO:37, e.g., a cytoplasmic domain that has a 9 amino acid partial inhibitor peptide set forth as amino acids 1-9 of SEQ ID NO:22. In some embodiments, the truncated BaEV envelope glycoprotein has a sequence set forth in SEQ ID NO:37. In some embodiments, the truncated BaEV envelope glycoprotein is in a two-chain form comprising gp70 or a biologically active portion thereof (e.g., as set forth in SEQ ID NO:25) and a portion of the p20E subunit comprising the cytoplasmic domain having a partial inhibitory polypeptide. In some embodiments, the truncated BaEV envelope glycoprotein is in a two-chain form comprising the gp70 subunit as set forth in SEQ ID NO:25 and a portion of the p20E subunit as set forth in SEQ ID NO:53. In some embodiments, the gp70 subunit is s and the portion of the p20E is linked via an intersubunit disulfide bond.

[0099] In certain embodiments, the truncated BaEV envelope glycoprotein comprises a partial fusion-inhibitory R peptide (e.g., R+8) that comprises 8 consecutive amino-terminal amino acids of the wild-type inhibitory R peptide. In certain embodiments, the truncated BaEV envelope glycoprotein comprises a partial fusion-inhibitory R peptide set forth as amino acids 1-8 of SEQ ID NO:22. In some embodiments, the BaEV envelope glycoprotein has a cytoplasmic tail set forth in SEQ ID NO:13. In some embodiments, the truncated BaEV envelope glycoprotein has a sequence that exhibits at least 85%, at least 90%, or at least 95% sequence identity to the sequence set forth in SEQ ID NO:36, e.g., a cytoplasmic domain that has a partial inhibitor peptide of 8 amino acids set forth as amino acids 1-8 of SEQ ID NO:22. In some embodiments, the truncated BaEV envelope glycoprotein has a sequence set forth in SEQ ID NO:36. In some embodiments, the truncated BaEV envelope glycoprotein is in a two-chain form comprising gp70 or a biologically active portion thereof (e.g., as set forth in SEQ ID NO:25) and a portion of the p20E subunit comprising the cytoplasmic domain having a partial inhibitory polypeptide. In some embodiments, the truncated BaEV envelope glycoprotein is in a two-chain form comprising the gp70 subunit as set forth in SEQ ID NO:25 and a portion of the p20E subunit as set forth in SEQ ID NO:52. In some embodiments, the gp70 subunit is s and the portion of the p20E is linked via an intersubunit disulfide bond.

[0100] In certain embodiments, the truncated BaEV envelope glycoprotein comprises a partial fusion-inhibitory R peptide (e.g., R+7) that comprises 7 consecutive amino-terminal amino acids of the wild-type inhibitory R peptide. In certain embodiments, the truncated BaEV envelope glycoprotein comprises a partial fusion-inhibitory R peptide set forth as amino acids 1-7 of SEQ ID NO:22. In some embodiments, the BaEV envelope glycoprotein has a cytoplasmic tail set forth in SEQ ID NO:12. In some embodiments, the truncated BaEV envelope glycoprotein has a sequence that exhibits at least 85%, at least 90%, or at least 95% sequence identity to the sequence set forth in SEQ ID NO:35, e.g., a cytoplasmic domain that has a partial inhibitor peptide of 7 amino acids set forth as amino acids 1-7 of SEQ ID NO:22. In some embodiments, the truncated BaEV envelope glycoprotein has a sequence set forth in SEQ ID NO:35. In some embodiments, the truncated BaEV envelope glycoprotein is in a two-chain form comprising gp70 or a biologically active portion thereof (e.g., as set forth in SEQ ID NO:25) and a portion of the p20E subunit comprising the cytoplasmic domain having a partial inhibitory polypeptide. In some embodiments, the truncated BaEV envelope glycoprotein is in a two-chain form comprising the gp70 subunit as set forth in SEQ ID NO:25 and a portion of the p20E subunit as set forth in SEQ ID NO:51. In some embodiments, the gp70 subunit is s and the portion of the p20E is linked via an intersubunit disulfide bond.

[0101] In certain embodiments, the truncated BaEV envelope glycoprotein comprises a partial fusion-inhibitory R peptide (e.g., R+6) that comprises six consecutive amino-terminal amino acids of the wild-type inhibitory R peptide. In certain embodiments, the truncated BaEV envelope glycoprotein comprises a partial fusion-inhibitory R peptide set forth as amino acids 1-6 of SEQ ID NO:22. In some embodiments, the BaEV envelope glycoprotein has a cytoplasmic tail set forth in SEQ ID NO:11. In some embodiments, the truncated BaEV envelope glycoprotein has a sequence that exhibits at least 85%, at least 90%, or at least 95% sequence identity to the sequence set forth in SEQ ID NO:34, e.g., a cytoplasmic domain that has a partial inhibitor peptide of six amino acids set forth as amino acids 1-6 of SEQ ID NO:22. In some embodiments, the truncated BaEV envelope glycoprotein has a sequence set forth in SEQ ID NO:34. In some embodiments, the truncated BaEV envelope glycoprotein is in a two-chain form comprising gp70 or a biologically active portion thereof (e.g., as set forth in SEQ ID NO:25) and a portion of the p20E subunit comprising the cytoplasmic domain having a partial inhibitory polypeptide. In some embodiments, the truncated BaEV envelope glycoprotein is in a two-chain form comprising the gp70 subunit as set forth in SEQ ID NO:25 and a portion of the p20E subunit as set forth in SEQ ID NO:50. In some embodiments, the gp70 subunit is s and the portion of the p20E is linked via an intersubunit disulfide bond.

[0102] In certain embodiments, the truncated BaEV envelope glycoprotein comprises a partial fusion-inhibitory R peptide (e.g., R+5) that comprises five consecutive amino-terminal amino acids of the wild-type inhibitory R peptide. In certain embodiments, the truncated BaEV envelope glycoprotein comprises a partial fusion-inhibitory R peptide set forth as amino acids 1-5 of SEQ ID NO:22. In some embodiments, the BaEV envelope glycoprotein has a cytoplasmic tail set forth in SEQ ID NO:10. In some embodiments, the truncated BaEV envelope glycoprotein has a sequence that exhibits at least 85%, at least 90%, or at least 95% sequence identity to the sequence set forth in SEQ ID NO:33, e.g., a cytoplasmic domain that has a partial inhibitor peptide of five amino acids set forth as amino acids 1-5 of SEQ ID NO:22. In some embodiments, the truncated BaEV envelope glycoprotein has a sequence set forth in SEQ ID NO:33. In some embodiments, the truncated BaEV envelope glycoprotein is in a two-chain form comprising gp70 or a biologically active portion thereof (e.g., as set forth in SEQ ID NO:25) and a portion of the p20E subunit comprising the cytoplasmic domain having a partial inhibitory polypeptide. In some embodiments, the truncated BaEV envelope glycoprotein is in a two-chain form comprising the gp70 subunit as set forth in SEQ ID NO:25 and a portion of the p20E subunit as set forth in SEQ ID NO:49. In some embodiments, the gp70 subunit is s and the portion of the p20E is linked via an intersubunit disulfide bond.

[0103] In certain embodiments, the truncated BaEV envelope glycoprotein comprises a partial fusion-inhibitory R peptide (e.g., R+4) that comprises the four consecutive amino-terminal amino acids of the wild-type inhibitory R peptide. In certain embodiments, the truncated BaEV envelope glycoprotein comprises a partial fusion-inhibitory R peptide set forth as amino acids 1-4 of SEQ ID NO:22. In some embodiments, the BaEV envelope glycoprotein has a cytoplasmic tail set forth in SEQ ID NO:9. In some embodiments, the truncated BaEV envelope glycoprotein has a sequence that exhibits at least 85%, at least 90%, or at least 95% sequence identity to the sequence set forth in SEQ ID NO:32, e.g., a cytoplasmic domain that has a partial inhibitor peptide of four amino acids set forth as amino acids 1-4 of SEQ ID NO:22. In some embodiments, the truncated BaEV envelope glycoprotein has a sequence set forth in SEQ ID NO:32. In some embodiments, the truncated BaEV envelope glycoprotein is in a two-chain form comprising gp70 or a biologically active portion thereof (e.g., as set forth in SEQ ID NO:25) and a portion of the p20E subunit comprising the cytoplasmic domain having a partial inhibitory polypeptide. In some embodiments, the truncated BaEV envelope glycoprotein is in a two-chain form comprising the gp70 subunit as set forth in SEQ ID NO:25 and a portion of the p20E subunit as set forth in SEQ ID NO:48. In some embodiments, the gp70 subunit is s and the portion of the p20E is linked via an intersubunit disulfide bond.

[0104] In certain embodiments, the truncated BaEV envelope glycoprotein comprises a partial fusion-inhibitory R peptide (e.g., R+3) that comprises three consecutive amino-terminal amino acids of the wild-type inhibitory R peptide. In certain embodiments, the truncated BaEV envelope glycoprotein comprises a partial fusion-inhibitory R peptide set forth as amino acids 1-3 of SEQ ID NO:22. In some embodiments, the BaEV envelope glycoprotein has a cytoplasmic tail set forth in SEQ ID NO:8. In some embodiments, the truncated BaEV envelope glycoprotein has a sequence that exhibits at least 85%, at least 90%, or at least 95% sequence identity to the sequence set forth in SEQ ID NO:31, e.g., a cytoplasmic domain that has a partial inhibitor peptide of three amino acids set forth as amino acids 1-3 of SEQ ID NO:22. In some embodiments, the truncated BaEV envelope glycoprotein has a sequence set forth in SEQ ID NO:31. In some embodiments, the truncated BaEV envelope glycoprotein is in a two-chain form comprising gp70 or a biologically active portion thereof (e.g., as set forth in SEQ ID NO:25) and a portion of the p20E subunit comprising the cytoplasmic domain having a partial inhibitory polypeptide. In some embodiments, the truncated BaEV envelope glycoprotein is in a two-chain form comprising the gp70 subunit as set forth in SEQ ID NO:25 and a portion of the p20E subunit as set forth in SEQ ID NO:47. In some embodiments, the gp70 subunit is s and the portion of the p20E is linked via an intersubunit disulfide bond.

[0105] In certain embodiments, the truncated BaEV envelope glycoprotein comprises a partial fusion-inhibitory R peptide (e.g., R+2) that comprises two consecutive amino-terminal amino acids of the wild-type inhibitory R peptide. In certain embodiments, the truncated BaEV envelope glycoprotein comprises a partial fusion-inhibitory R peptide set forth as amino acids 1-2 of SEQ ID NO:22. In some embodiments, the BaEV envelope glycoprotein has a cytoplasmic tail set forth in SEQ ID NO:7. In some embodiments, the truncated BaEV envelope glycoprotein has a sequence that exhibits at least 85%, at least 90%, or at least 95% sequence identity to the sequence set forth in SEQ ID NO:30, e.g., a cytoplasmic domain that has a partial inhibitor peptide of two amino acids set forth as amino acids 1-1 of SEQ ID NO:22. In some embodiments, the truncated BaEV envelope glycoprotein has a sequence set forth in SEQ ID NO:30. In some embodiments, the truncated BaEV envelope glycoprotein is in a two-chain form comprising gp70 or a biologically active portion thereof (e.g., as set forth in SEQ ID NO:25) and a portion of the p20E subunit comprising the cytoplasmic domain having a partial inhibitory polypeptide. In some embodiments, the truncated BaEV envelope glycoprotein is in a two-chain form comprising the gp70 subunit as set forth in SEQ ID NO:25 and a portion of the p20E subunit as set forth in SEQ ID NO:46. In some embodiments, the gp70 subunit is s and the portion of the p20E is linked via an intersubunit disulfide bond.

[0106] In certain embodiments, the truncated BaEV envelope glycoprotein comprises a partial fusion-inhibitory R peptide (e.g., R+1) that comprises one consecutive amino-terminal amino acid of the wild-type inhibitory R peptide. In certain embodiments, the truncated BaEV envelope glycoprotein comprises a partial fusion-inhibitory R peptide as shown as amino acid 1 of SEQ ID NO:22. In some embodiments, the BaEV envelope glycoprotein has a cytoplasmic tail as shown in SEQ ID NO:6. In some embodiments, the truncated BaEV envelope glycoprotein has a sequence that exhibits at least 85%, at least 90%, or at least 95% sequence identity to the sequence shown in SEQ ID NO:29, e.g., a cytoplasmic domain with a partial inhibitory peptide of one amino acid as shown as amino acid of SEQ ID NO:22. In some embodiments, the truncated BaEV envelope glycoprotein has a sequence as shown in SEQ ID NO:29. In some embodiments, the truncated BaEV envelope glycoprotein is a two-chain form that comprises a portion of the p20E subunit that includes gp70 or a biologically active portion thereof (e.g., as shown in SEQ ID NO:25) and a cytoplasmic domain with a partial inhibitory polypeptide. In some embodiments, the truncated BaEV envelope glycoprotein is in a two-chain form comprising the gp70 subunit as set forth in SEQ ID NO: 25 and a portion of the p20E subunit as set forth in SEQ ID NO: 45. In some embodiments, the gp70 subunit and the portion of the p20E are linked via an intersubunit disulfide bond.

[0107] A. Polynucleotides Provided herein is a polynucleotide comprising a nucleic acid sequence encoding a truncated BaEV envelope glycoprotein comprising a cytoplasmic tail with a partial fusion-inhibiting R peptide compared to the wild-type BaEV envelope glycoprotein described herein. The polynucleotide may comprise a sequence of nucleotides encoding any of the truncated BaEV envelope glycoproteins described above.

[0108] In some embodiments, the polynucleotide encodes a truncated BaEV envelope glycoprotein that includes a cytoplasmic tail with a partial fusion-inhibitory R peptide that includes 9 consecutive amino-terminal amino acids (e.g., R+9) of the wild-type inhibitory R peptide. In some embodiments, the polynucleotide encodes a truncated BaEV envelope glycoprotein having a sequence of amino acids that exhibits at least 85%, at least 90%, or at least 95% sequence identity to SEQ ID NO:37, e.g., a cytoplasmic domain with a partial inhibitory peptide of 9 amino acids shown as amino acids 1-9 of SEQ ID NO:22. In some embodiments, the polynucleotide encodes a truncated BaEV envelope glycoprotein shown in SEQ ID NO:37.

[0109] In some embodiments, the polynucleotide encodes a truncated BaEV envelope glycoprotein that includes a cytoplasmic tail with a partial fusion-inhibitory R peptide that includes 8 consecutive amino-terminal amino acids (e.g., R+8) of the wild-type inhibitory R peptide. In some embodiments, the polynucleotide encodes a truncated BaEV envelope glycoprotein having a sequence of amino acids that exhibits at least 85%, at least 90%, or at least 95% sequence identity to SEQ ID NO:36, e.g., a cytoplasmic domain with a partial inhibitory peptide of 8 amino acids shown as amino acids 1-8 of SEQ ID NO:22. In some embodiments, the polynucleotide encodes a truncated BaEV envelope glycoprotein shown in SEQ ID NO:36.

[0110] In some embodiments, the polynucleotide encodes a truncated BaEV envelope glycoprotein that includes a cytoplasmic tail with a partial fusion-inhibitory R peptide that includes seven consecutive amino-terminal amino acids (e.g., R+7) of the wild-type inhibitory R peptide. In some embodiments, the polynucleotide encodes a truncated BaEV envelope glycoprotein having a sequence of amino acids that exhibits at least 85%, at least 90%, or at least 95% sequence identity to SEQ ID NO:35, e.g., a cytoplasmic domain with a partial inhibitory peptide of seven amino acids shown as amino acids 1-7 of SEQ ID NO:22. In some embodiments, the polynucleotide encodes a truncated BaEV envelope glycoprotein shown in SEQ ID NO:35.

[0111] In some embodiments, the polynucleotide encodes a truncated BaEV envelope glycoprotein that includes a cytoplasmic tail with a partial fusion-inhibitory R peptide that includes six consecutive amino-terminal amino acids (e.g., R+6) of the wild-type inhibitory R peptide. In some embodiments, the polynucleotide encodes a truncated BaEV envelope glycoprotein having a sequence of amino acids that exhibits at least 85%, at least 90%, or at least 95% sequence identity to SEQ ID NO:34, e.g., a cytoplasmic domain with a partial inhibitory peptide of six amino acids shown as amino acids 1-6 of SEQ ID NO:22. In some embodiments, the polynucleotide encodes a truncated BaEV envelope glycoprotein shown in SEQ ID NO:34.

[0112] In some embodiments, the polynucleotide encodes a truncated BaEV envelope glycoprotein that includes a cytoplasmic tail with a partial fusion-inhibitory R peptide that includes five consecutive amino-terminal amino acids (e.g., R+5) of the wild-type inhibitory R peptide. In some embodiments, the polynucleotide encodes a truncated BaEV envelope glycoprotein having a sequence of amino acids that exhibits at least 85%, at least 90%, or at least 95% sequence identity to SEQ ID NO:33, e.g., a cytoplasmic domain with a partial inhibitory peptide of five amino acids shown as amino acids 1-5 of SEQ ID NO:22. In some embodiments, the polynucleotide encodes a truncated BaEV envelope glycoprotein shown in SEQ ID NO:33.

[0113] In some embodiments, the polynucleotide encodes a truncated BaEV envelope glycoprotein that includes a cytoplasmic tail with a partial fusion-inhibitory R peptide that includes four consecutive amino-terminal amino acids (e.g., R+4) of the wild-type inhibitory R peptide. In some embodiments, the polynucleotide encodes a truncated BaEV envelope glycoprotein having a sequence of amino acids that exhibits at least 85%, at least 90%, or at least 95% sequence identity to SEQ ID NO:32, e.g., a cytoplasmic domain with a partial inhibitory peptide of four amino acids shown as amino acids 1-4 of SEQ ID NO:22. In some embodiments, the polynucleotide encodes a truncated BaEV envelope glycoprotein shown in SEQ ID NO:32.

[0114] In some embodiments, the polynucleotide encodes a truncated BaEV envelope glycoprotein that includes a cytoplasmic tail with a partial fusion-inhibitory R peptide that includes three consecutive amino-terminal amino acids (e.g., R+3) of the wild-type inhibitory R peptide. In some embodiments, the polynucleotide encodes a truncated BaEV envelope glycoprotein having a sequence of amino acids that exhibits at least 85%, at least 90%, or at least 95% sequence identity to SEQ ID NO:31, including a cytoplasmic domain with a partial inhibitory peptide of three amino acids, e.g., as shown as amino acids 1-3 of SEQ ID NO:22. In some embodiments, the polynucleotide encodes a truncated BaEV envelope glycoprotein as shown in SEQ ID NO:31.

[0115] In some embodiments, the polynucleotide encodes a truncated BaEV envelope glycoprotein that includes a cytoplasmic tail with a partial fusion-inhibitory R peptide that includes two consecutive amino-terminal amino acids (e.g., R+2) of the wild-type inhibitory R peptide. In some embodiments, the polynucleotide encodes a truncated BaEV envelope glycoprotein having a sequence of amino acids that exhibits at least 85%, at least 90%, or at least 95% sequence identity to SEQ ID NO:30, including a cytoplasmic domain with a partial inhibitory peptide of two amino acids, e.g., shown as amino acids 1-2 of SEQ ID NO:22. In some embodiments, the polynucleotide encodes a truncated BaEV envelope glycoprotein as shown in SEQ ID NO:30.

[0116] In some embodiments, the polynucleotide encodes a truncated BaEV envelope glycoprotein that includes a cytoplasmic tail with a partial fusion-inhibiting R peptide that includes one consecutive amino-terminal amino acid (e.g., R+1) of the wild-type inhibitory R peptide. In some embodiments, the polynucleotide encodes a truncated BaEV envelope glycoprotein having a sequence of amino acids that exhibits at least 85%, at least 90%, or at least 95% sequence identity to SEQ ID NO:29, including a cytoplasmic domain with a partial inhibitory peptide of one amino acid, e.g., shown as amino acid 1 of SEQ ID NO:22. In some embodiments, the polynucleotide encodes a truncated BaEV envelope glycoprotein as shown in SEQ ID NO:29.

[0117] In some embodiments, the polynucleotides can be synthetic nucleic acids.Also provided are expression vectors containing any of the provided polynucleotides.

[0118] In some of the embodiments, expression of natural or synthetic nucleic acids is generally achieved by operably linking a nucleic acid encoding a gene of interest to a promoter and incorporating the construct into an expression vector. In some embodiments, the vector may be suitable for replication and integration in eukaryotes. In some embodiments, the cloning vector contains transcription and translation terminators, initiation sequences, and promoters useful for expression of the desired nucleic acid sequence. In some of the embodiments, the plasmid contains a promoter suitable for expression in a cell.

[0119] In some embodiments, the polynucleotide comprises at least one promoter operably linked to control the expression of the truncated BaEV envelope glycoprotein. For the expression of the truncated BaEV envelope glycoprotein, at least one module in each promoter functions to position the start site for RNA synthesis. The most well-known example of this is the TATA box, but in some promoters that lack a TATA box, such as the promoter for the mammalian terminal deoxynucleotidyl transferase gene and the promoter for the SV40 gene, another element overlapping the start site itself helps to anchor the point of initiation.

[0120] In some embodiments, additional promoter elements, e.g., enhancers, control the frequency of transcription initiation. In some embodiments, the additional promoter elements are located in the region 30-110 bp upstream of the start site, although multiple promoters have recently been shown to contain functional elements downstream of the start site as well. In some embodiments, the spacing between promoter elements is frequently flexible, so that promoter function is maintained when elements are inverted or moved relative to one another. In some embodiments, in the thymidine kinase (tk) promoter, the spacing between promoter elements can be increased to 50 bp apart before activity begins to decline. In some embodiments, depending on the promoter, individual elements can function either cooperatively or independently to activate transcription.

[0121] A promoter may be one that is naturally associated with a gene or polynucleotide sequence, as may be obtained by isolating the 5' non-coding sequences located upstream of the coding segment and / or exon. Such a promoter may be referred to as "endogenous". Similarly, an enhancer may be one that is naturally associated with a polynucleotide sequence located either downstream or upstream of that sequence. Alternatively, certain advantages are obtained by placing a coding polynucleotide segment under the control of a recombinant or heterologous promoter, which refers to a promoter that is not normally associated with a polynucleotide sequence in its natural environment. A recombinant or heterologous enhancer also refers to an enhancer that is not normally associated with a polynucleotide sequence in its natural environment. Such promoters or enhancers may include promoters or enhancers of other genes, and promoters or enhancers isolated from any other prokaryotic, viral, or eukaryotic cell, and "natural", i.e., promoters or enhancers that contain different elements of different transcriptional control regions and / or mutations that alter expression. In addition to producing promoter and enhancer nucleic acid sequences synthetically, the sequences can be produced using nucleic acid amplification techniques, including recombinant cloning and / or PCR, in combination with the compositions disclosed herein (U.S. Pat. Nos. 4,683,202 and 5,928,906).

[0122] In some embodiments, a suitable promoter is an immediate early cytomegalovirus (CMV) promoter sequence. In some embodiments, the promoter sequence is a strong constitutive promoter sequence capable of inducing high levels of expression of any polynucleotide sequence operably linked thereto. In some embodiments, a suitable promoter is elongation growth factor-la (EF-la). In some embodiments, other constitutive promoter sequences may also be used, including, but not limited to, the simian virus 40 (SV40) early promoter, mouse mammary tumor virus (MMTV), human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, MoMuLV promoter, avian leukemia virus promoter, Epstein-Barr virus immediate early promoter, Rous sarcoma virus promoter, and human gene promoters, such as, but not limited to, actin promoter, myosin promoter, hemoglobin promoter, and creatine kinase promoter.

[0123] In some embodiments, the promoter is an inducible promoter. In some embodiments, an inducible promoter provides a molecular switch capable of turning on expression of an operably linked polynucleotide sequence when such expression is desired, or turning off expression when expression is not desired. In some embodiments, inducible promoters include metallothionine promoters, glucocorticoid promoters, progesterone promoters, and tetracycline promoters.

[0124] In some embodiments, an exogenously controlled inducible promoter can be used to regulate the expression of the truncated BaEV envelope glycoprotein. For example, radiation-, heat-, and / or drug-inducible promoters can be used, for example, to selectively induce transgene expression in targeted regions. In such embodiments, the location, duration, and level of transgene expression can be regulated by administration of an exogenous source of induction.

[0125] In some embodiments, a drug-inducible promoter is used to regulate the expression of the truncated BaEV envelope glycoprotein. For example, in some cases, the promoter, enhancer, or transactivator comprises a Lac operator sequence, a tetracycline operator sequence, a galactose operator sequence, a doxycycline operator sequence, a rapamycin operator sequence, a tamoxifen operator sequence, or a hormone-responsive operator sequence, or an analog thereof. In some examples, the inducible promoter comprises a tetracycline response element (TRE). In some embodiments, the inducible promoter comprises an estrogen response element (ERE) that can activate gene expression in the presence of tamoxifen. In some examples, a drug-inducible element, such as a TRE, can be combined with a selected promoter to enhance transcription in the presence of a drug, such as doxycycline. In some embodiments, the drug-inducible promoter is a small molecule inducible promoter.

[0126] Any of the provided polynucleotides can be modified to remove CpG motifs and / or codon optimized for translation in a particular species, such as human, canine, feline, equine, ovine, bovine, etc. In some embodiments, the polynucleotides are optimized for human codon usage (i.e., human codon optimized). In some embodiments, the polynucleotides are modified to remove CpG motifs. In other embodiments, the provided polynucleotides are modified to remove CpG motifs and codon optimized, e.g., human codon optimized. Methods for codon optimization and CpG motif detection and modification are well known. In general, polynucleotide optimization improves transgene expression, increases transgene stability, and preserves the amino acid sequence of the encoded polypeptide.

[0127] To assess the expression of truncated envelope glycoproteins, the expression vector introduced into the cells may also contain either a selection marker gene or a reporter gene, or both, to facilitate identification and selection of expressing particles, e.g., viral particles. In other embodiments, the selection marker may be carried on a separate piece of DNA and used in a co-transfection procedure. Both the selection marker and the reporter gene may be flanked by appropriate regulatory sequences to enable expression in the host cell. Useful selection markers are known in the art and include, for example, antibiotic resistance genes, e.g., neo.

[0128] Reporter genes are used to identify potentially transfected cells and to evaluate the functionality of regulatory sequences. Reporter genes that code for easily assayable proteins are well known. In general, reporter genes are genes that code for proteins that are not present in or expressed by recipient organisms or tissues, and whose expression is manifested by some easily detectable property, such as enzymatic activity. The expression of the reporter gene is assayed at a suitable time point after DNA is introduced into the recipient cells.

[0129] Suitable reporter genes may include genes encoding luciferase, β-galactosidase, chloramphenicol acetyltransferase, secreted alkaline phosphatase, or green fluorescent protein genes (see, e.g., Ui-Tei et al., 2000, FEBS Lett. 479:79-82). Suitable expression systems are well known and may be prepared using well known techniques or obtained commercially. Internal deletion constructs may be generated using unique internal restriction enzyme sites or by partial digestion of non-unique restriction enzyme sites. The constructs may then be transfected into cells that exhibit high levels of desired polynucleotide and / or polypeptide expression. Typically, the construct with the smallest 5' flanking region that exhibits the highest level of expression of the reporter gene is identified as the promoter. Such promoter regions may be linked to reporter genes and used to evaluate agents for their ability to modulate promoter-induced transcription.

[0130] III. Lipid Particles and Methods for Producing Lipid Particles Provided herein are particles that include a lipid bilayer, a lumen surrounded by the lipid bilayer, and a truncated BaEV envelope protein such as those described, where the truncated BaEV envelope protein is embedded within the lipid bilayer. In some embodiments, the provided lipid particles preferentially target hematopoietic cells (e.g., T cells) mediated by the tropism of the truncated BaEV envelope protein. In some embodiments, the lipid particles may further include an exogenous agent (e.g., a therapeutic agent) for delivery to cells. In some embodiments, the lipid particles are introduced into cells of a subject. Also provided are methods of delivering any of the provided lipid particles to cells.

[0131] In some embodiments, the lipid particles provided exhibit membrane fusion activity mediated by truncated BaEV envelope protein, which promotes the merging or fusion of the two lumens of the lipid particle with the target cell membrane. Thus, the lipid particles provided are fusosomes. In some embodiments, the fusosomes comprise a naturally occurring bilayer of amphipathic lipids with truncated BaEV envelope protein as a fusogen. In some embodiments, the fusosomes comprise (a) a lipid bilayer, (b) a lumen (e.g., including the cytosol) surrounded by the lipid bilayer, and (c) a fusogen that is exogenous or overexpressed compared to the source cell. In some embodiments, the truncated BaEV envelope protein is disposed in the lipid bilayer. In some embodiments, the fusosomes comprise several different types of lipids, e.g., amphipathic lipids such as phospholipids. In some embodiments, the fusosomes comprise a lipid bilayer as the outermost surface.

[0132] In some embodiments, the lipid particle comprises a naturally occurring bilayer of amphipathic lipids surrounding a lumen or cavity. In some embodiments, the lipid particle comprises a lipid bilayer as the outermost surface. In some embodiments, the lipid bilayer surrounds a lumen. In some embodiments, the lumen is aqueous. In some embodiments, the lumen is in contact with the interior hydrophilic head groups of the lipid bilayer. In some embodiments, the lumen is the cytosol. In some embodiments, the cytosol contains cellular components present in the source cell. In some embodiments, the cytosol does not contain components present in the source cell. In some embodiments, the lumen is a cavity. In some embodiments, the lumen contains an aqueous environment. In some embodiments, the lumen does not contain an aqueous environment.

[0133] In some embodiments, the lipid particle can be a viral particle, a virus-like particle, a nanoparticle, a vesicle, an exosome, a dendrimer, a lentivirus, a viral vector, an enucleated cell, a microvesicle, a membrane vesicle, an extracellular membrane vesicle, a plasma membrane vesicle, a giant plasma membrane vesicle, an apoptotic body, a mitoparticle, a pyrenocyte, a lysosome, another membrane-enclosed vesicle, or a lentiviral vector, a virus-based particle, a virus-like particle (VLP) or a cell-based particle.

[0134] In some aspects, the lipid bilayer is obtained from a source cell during the process for making the lipid-containing particles. Exemplary methods for making lipid-containing particles are described herein. In some embodiments, the lipid bilayer includes membrane components of the host cell from which the lipid bilayer is derived, e.g., phospholipids, membrane proteins, etc. In some embodiments, the lipid bilayer includes cytosol, including components found in the cell from which the vehicle is derived, e.g., solutes, proteins, nucleic acids, etc., but not all of the components of the cell, e.g., they lack a nucleus. In some embodiments, the lipid bilayer is considered exosome-like. The lipid bilayer can vary in size, and in some instances has a diameter ranging from 30 to 300 nm, such as 30 to 150 nm, including 40 to 100 nm.

[0135] In certain embodiments, the lipid particle is of viral origin. In some embodiments, the lipid particle can be a virus-based particle, such as a viral vector particle (e.g., a lentiviral vector particle) or a virus-like particle (e.g., a lentiviral-like particle). In some embodiments, the lipid bilayer is a viral envelope. In some embodiments, the viral envelope is obtained from a host cell. In some embodiments, the viral envelope is obtained by a viral capsid from the plasma membrane of a source cell. In some embodiments, the lipid bilayer is obtained from a membrane other than the plasma membrane of a host cell. In some embodiments, the viral envelope lipid bilayer is embedded with viral proteins, including viral glycoproteins.

[0136] In certain embodiments, the lipid particle is not of viral origin. In some embodiments, the lipid particle may be a nanoparticle, a vesicle, an exosome, a dendrimer, an enucleated cell, a microvesicle, a membrane vesicle, an extracellular membrane vesicle, a plasma membrane vesicle, a giant plasma membrane vesicle, an apoptotic body, a mitoparticle, a pyrenocyte, a lysosome, another membrane-enclosed vesicle, or a cell-derived particle.

[0137] In some embodiments, the lipid bilayer includes membrane components of the host cell from which the lipid bilayer is derived, e.g., phospholipids, membrane proteins, etc. In some embodiments, the lipid bilayer includes cytosol, including components found in the cell from which the vehicle is derived, e.g., solutes, proteins, nucleic acids, etc., but not all of the components of the cell, e.g., they lack a nucleus. In some embodiments, the lipid bilayer is considered exosome-like. The lipid bilayer can vary in size, and in some instances has a diameter ranging from 30 to 300 nm, such as 30 to 150 nm, including 40 to 100 nm.

[0138] In other aspects, the lipid bilayer comprises a synthetic lipid complex. In some embodiments, the synthetic lipid complex is a liposome. In some embodiments, the lipid bilayer is a vesicular structure characterized by a phospholipid bilayer membrane and an inner aqueous medium. In some embodiments, the lipid bilayer has multiple lipid layers separated by aqueous medium. In some embodiments, the lipid bilayer forms spontaneously when phospholipids are suspended in an excess of aqueous solution. In some instances, the lipid components undergo self-rearrangement prior to the formation of a juxtaposed structure, trapping water and dissolved solutes between the lipid bilayers.

[0139] In some embodiments, the lipid particles include several different types of lipids. In some embodiments, the lipids are amphipathic lipids. In some embodiments, the amphipathic lipids are phospholipids. In some embodiments, the phospholipids include phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, and phosphatidylserine. In some embodiments, the lipids include phospholipids such as phosphocholine and phosphoinositol. In some embodiments, the lipids include DMPC, DOPC, and DSPC.

[0140] In certain embodiments, an exogenous agent, such as a polynucleotide or a polypeptide, is encapsulated within the lumen of the lipid particle. The lipid particle embodiments provided herein may have various properties that facilitate the delivery of a payload, such as a desired transgene or exogenous agent, to a target cell. The exogenous agent may be a polynucleotide or a polypeptide. In some embodiments, the lipid particles provided herein are administered to a subject, such as a mammal, such as a human. In such embodiments, the subject may be at risk of, have symptoms of, or have been diagnosed with or identified as having a particular disease or condition. In one embodiment, the subject has cancer. In one embodiment, the subject has an infectious disease. In some embodiments, the lipid particle includes a nucleic acid sequence (polynucleotide) encoding an exogenous agent, or a polypeptide exogenous agent, for treating a disease or condition.

[0141] The lipid particles may include spherical particles or may include elongated or irregularly shaped particles.

[0142] In some embodiments, the composition of particles can be assessed for one or more properties related to the size of the particles, including diameter, the range of variation above and below the mean (average) or median of that diameter, the coefficient of variation, the polydispersity index, or other measure of the size of the particles in the composition. A variety of methods can be used to characterize particles, including, but not limited to, laser diffraction, dynamic light scattering (DLS, also known as photon correlation spectroscopy), or image analysis, such as microscopy or automated image analysis.

[0143] In some embodiments, lipid particles are provided with a particle diameter or average (mean) diameter in the composition of less than about 3 μm, less than about 2 μm, less than about 1 μm, less than about 900 nm, less than about 800 nm, less than about 700 nm, less than about 600 nm, less than about 500 nm, less than about 400 nm, less than about 300 nm, less than about 200 nm, less than about 150 nm, less than about 100 nm, less than about 50 nm, or less than about 20 nm.In some embodiments, lipid particles are provided with a particle diameter or average (mean) diameter in the composition of less than about 400 nm.In another embodiment, lipid particles are provided with a particle diameter or average (mean) diameter in the composition of less than about 150 nm. In some embodiments, the lipid particles have a diameter or average (mean) diameter of the particles in the composition of from 2 μm or about 2 μm, from 1 μm or about 1 μm, from 1 μm or about 1 μm, from 900 nm or about 900 nm, from 900 nm or about 900 nm, from 800 nm or about 800 nm, from 800 nm or about 800 nm, from 700 nm or about 700 nm, from 700 nm or about 700 nm, from 600 nm or about 600 nm, from 600 nm or about 600 nm, 00 nm or about 500 nm, 500 nm or about 500 nm to 400 nm or about 400 nm, 400 nm or about 400 nm to 300 nm or about 300 nm, 300 nm or about 300 nm to 200 nm or about 200 nm, 200 nm or about 200 nm to 100 nm or about 100 nm, 100 nm or about 100 nm to 50 nm or about 50 nm, or 20 nm or about 20 nm to 50 nm or about 50 nm.

[0144] In some embodiments, the particles in the composition have a median diameter of from at or about 10 nm, at or about 1000 nM, at or about 25 nm, at or about 500 nm, at or about 40 nm, at or about 300 nm, at or about 50 nm, at or about 250 nm, at or about 60 nm, at or about 225 nm, at or about 70 nm, at or about 200 nm, at or about 80 nm, at or about 175 nm, or at or about 90 nm, at or about 150 nm.

[0145] In some embodiments, 90% of the lipid particles in the composition are within 50% of the lipid particle's median diameter. In some embodiments, 90% of the lipid particles in the composition are within 25% of the lipid particle's median diameter. In some embodiments, 90% of the lipid particles in the composition are within 20% of the lipid particle's median diameter. In some embodiments, 90% of the lipid particles in the composition are within 15% of the lipid particle's median diameter. In some embodiments, 90% of the lipid particles in the composition are within 10% of the lipid particle's median diameter.

[0146] In some embodiments, 75% of the lipid particles in the composition fall within ±2 or ±1 St Dev standard deviation (St Dev) of the mean diameter of the lipid particles. In some embodiments, 80% of the lipid particles in the composition fall within ±2 St Dev or ±1 St Dev of the mean diameter of the lipid particles. In some embodiments, 85% of the lipid particles in the composition fall within ±2 St Dev or ±1 St Dev of the mean diameter of the lipid particles. In some embodiments, 90% of the lipid particles in the composition fall within ±2 St Dev or ±1 St Dev of the mean diameter of the lipid particles. In some embodiments, 95% of the lipid particles in the composition fall within ±2 St Dev or ±1 St Dev of the mean diameter of the lipid particles.

[0147] In some embodiments, the lipid particles have an average hydrodynamic radius of about 100 nm to about 2 micrometers, as measured, for example, by DLS. In some embodiments, the lipid particles have an average hydrodynamic radius of about 2 μm to about 1 μm to about 1 μm to about 900 nm to about 800 nm to about 700 nm to about 600 nm to about 600 nm to about 500 nm or less. is about 500 nm, 500 nm or about 500 nm to 400 nm, 400 nm or about 400 nm to 300 nm or about 300 nm, 300 nm or about 300 nm to 200 nm or about 200 nm, 200 nm or about 200 nm to 100 nm or about 100 nm, 100 nm or about 100 nm to 50 nm or about 50 nm, or 20 nm or about 20 nm to 50 nm or about 50 nm.

[0148] In some embodiments, the lipid particles have a mean geometric radius of about 100 nm to about 2 micrometers, e.g., as measured by multi-angle light scattering. In some embodiments, the lipid particles have a mean geometric radius of about 2 μm to about 1 μm to about 1 μm to about 900 nm to about 900 nm to about 800 nm to about 800 nm to about 700 nm to about 700 nm to about 600 nm to about 600 nm to about 500 nm or less. is about 500 nm, 500 nm or about 500 nm to 400 nm, 400 nm or about 400 nm to 300 nm or about 300 nm, 300 nm or about 300 nm to 200 nm or about 200 nm, 200 nm or about 200 nm to 100 nm or about 100 nm, 100 nm or about 100 nm to 50 nm or about 50 nm, or 20 nm or about 20 nm to 50 nm or about 50 nm.

[0149] In some embodiments, the coefficient of variation (COV) of the composition of the lipid particles (i.e., the standard deviation divided by the mean) is less than or about 30%, less than or about 25%, less than or about 20%, less than or about 15%, less than or about 10%, or less than or about 5%.

[0150] In some embodiments, the lipid particle compositions provided herein are characterized by a polydispersity index, which is a measure of the size distribution of the particles and can have values ​​between 1 (maximum dispersion) and 0 (all particles are the same size). In some embodiments, the lipid particle compositions provided herein have a polydispersity index of 0.05 or about 0.05 to 0.7 or about 0.7, 0.05 or about 0.05 to 0.6 or about 0.6, 0.05 or about 0.05 to 0.5 or about 0.5, 0.05 or about 0.05 to 0.4 or about 0.4, 0.05 or about 0.05 to 0.3 or about 0.3, 0.05 or about 0.05 to 0.2 or about 0.2, 0.05 or about 0.05 to 0.5 or about 0.5 ... or from about 0.05, 0.1 or about 0.1, 0.05 or about 0.05, 0.1 or about 0.1, 0.1 or about 0.1 to, 0.7 or about 0.7, 0.1 or about 0.1 to, 0.6 or about 0.6, 0.1 or about 0.1 to, 0.5 or about 0.5, 0.1 or about 0.1 to, 0.4 or about 0.4, 0.1 or about 0.1 to, 0.3 or about 0.3, 0.1 or about 0.1 to, 0.2 or about 0.2, 0.2 or about 0.2 to, 0.7 or about 0.7, 0.2 or about 0.2 to, 0.6 or about 0.6, 0.2 or about 0.2 to, 0.5 or about 0.5, 0.2 or about 0.2 to, 0.4 or about 0.4, 0.2 or about 0.2 to, 0.3 or about 0.3, 0.3 or about 0.3 to, 0.7 or about 0.7, 0.3 or about 0.3 to, 0.6 or about 0.6, 0.3 or about 0.3 to, 0.5 or has a polydispersity index of at or about 0.5, 0.3 or about 0.3, 0.4 or about 0.4, 0.4 or about 0.4, 0.7 or about 0.7, 0.4 or about 0.4, 0.6 or about 0.6, 0.4 or about 0.4, 0.5 or about 0.5, 0.5 or about 0.5, 0.7 or about 0.7, 0.5 or about 0.5, 0.6 or about 0.6, or 0.6 or about 0.6, 0.7 or about 0.7.In some embodiments, the polydispersity index is less than or equal to 0.05, less than or equal to 0.1, less than or equal to 0.15, less than or equal to 0.15, less than or equal to 0.2, less than or equal to 0.25, less than or equal to 0.3, less than or equal to 0.3, less than or equal to 0.4, less than or equal to 0.5, less than or equal to 0.6, or less than or equal to 0.6. A variety of lipid particles are known, any of which can be produced in accordance with the embodiments provided. Non-limiting examples of lipid particles include those described in International Published PCT Application Nos. WO2017 / 095946; WO2017 / 095944; WO2017 / 095940; WO2019 / 157319; WO2018 / 208728; WO2019 / 113512; WO2019 / 161281; WO2020 / 102578; WO2019 / 222403; WO2020 / 014209; WO2020 / 102485; WO2020 / 102499; WO2020 / 102503; WO2013 / 148327; WO2017 / 182585; WO2011 / 058052; or WO2017 / 068077, each of which is incorporated by reference in its entirety.

[0151] Exemplary characteristics of provided lipid particles are described in the following subsections.

[0152] A. Virus-Based Particles Provided herein are virus-derived virus-based particles, including those derived from retroviruses or lentiviruses, that contain a truncated BaEV envelope protein as described in section I. In some embodiments, the amphipathic lipid bilayer of the lipid particle is or comprises a viral envelope. In some embodiments, the bilayer of the lipid particle of amphipathic lipid is or comprises a lipid derived from a producer cell. In some embodiments, the viral envelope may comprise a fusogen, e.g., a fusogen or pseudotype fusogen that is endogenous to the virus. In some embodiments, the lumen or cavity of the lipid particle comprises a viral nucleic acid, e.g., a retroviral nucleic acid, e.g., a lentiviral nucleic acid. In some embodiments, the viral nucleic acid may be a viral genome. In some embodiments, the lipid particle further comprises one or more viral nonstructural proteins, e.g., in its lumen or cavity. In some embodiments, the virus-based particle is or comprises a virus-like particle (VLP). In some embodiments, the VLP does not comprise viral genetic material. In some embodiments, the virus-based particle does not comprise a viral protein, such as a viral structural protein, or a viral nucleic acid derived from a virus.

[0153] Biological methods for introducing exogenous agents into host cells include the use of DNA and RNA vectors. DNA and RNA vectors can also be used to house and deliver polynucleotides and polypeptides. Viral vectors and virus-like particles, particularly retroviral vectors, are the most widely used methods for inserting genes into mammalian cells, e.g., human cells. Other viral vectors and virus-like particles can be derived from lentiviruses, poxviruses, herpes simplex virus I, adenoviruses, and adeno-associated viruses, etc. See, e.g., U.S. Patent Nos. 5,350,674 and 5,585,362. Methods for producing vectors and / or cells containing exogenous acids are well known in the art. See, e.g., Sambrook et al., 2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York.

[0154] In some embodiments, the viral or virus-like particle bilayer of amphipathic lipids is or comprises lipids derived from an infected host cell. In some embodiments, the lipid bilayer is a viral envelope. In some embodiments, the viral or virus-like particle envelope is obtained from a host cell. In some embodiments, the viral or virus-like particle envelope is obtained by a viral capsid from the plasma membrane of a source cell. In some embodiments, the lipid bilayer is obtained from a membrane other than the plasma membrane of a host cell. In some embodiments, the viral or virus-like particle envelope lipid bilayer has viral proteins embedded therein, including viral glycoproteins.

[0155] In some embodiments, one or more transducing units of a viral or viral-like particle, e.g., a retroviral or retroviral-like particle, are administered to the subject. In some embodiments, at least 1, 10, 100, 1000, 10 4 , 10 5 , 106 , 10 7 , 10 8 , 10 9 , 10 10 , 10 11 , 10 12 , 10 13 , or 10 14 The subject is administered at least 1, 10, 100, 1000, 10 transducing units per ml of blood. 4 , 10 5 , 10 6 , 10 7 , 10 8 , 10 9 , 10 10 , 10 11 , 10 12 , 10 13 , or 10 14 The transducing units / target cells are administered to the subject.

[0156] 1. Viral Vector Particles In some embodiments, the lipid particle is or comprises a virus or a viral vector, for example a retrovirus or a retroviral vector, for example a lentivirus or a lentiviral vector.In some embodiments, the virus or the viral vector is recombinant.For example, the viral particle may be referred to as a recombinant virus or a recombinant viral vector, which are used interchangeably.In some embodiments, the lipid particle is a recombinant lentiviral vector particle.

[0157] In some embodiments, the lipid particle comprises a lipid bilayer comprising a retroviral vector comprising an envelope. For example, in some embodiments, the amphipathic lipid bilayer is or comprises a viral envelope. The viral envelope can comprise a fusogen, e.g., a fusogen endogenous to the virus, e.g., a truncated BaEV fusogen or a pseudotype fusogen. In some embodiments, the lumen or cavity of the viral vector comprises a viral nucleic acid, e.g., a retroviral nucleic acid, e.g., a lentiviral nucleic acid. The viral nucleic acid can be a viral genome. In some embodiments, the viral vector can further comprise one or more viral nonstructural proteins, e.g., in its lumen or cavity. In some embodiments, the viral-based vector particle is a lentivirus. In some embodiments, the lentiviral vector particle is a human immunodeficiency virus-1 (HIV-1).

[0158] In some aspects, viral vector particles are limited in the number of polynucleotides that can be packaged. In some embodiments, the nucleotides encoding the packaged polypeptide can be modified to retain functional activity with fewer nucleotides in the coding region than the nucleotides encoding the wild-type peptide. Such modifications can include truncations or other deletions. In some embodiments, multiple polypeptides can be expressed from the same promoter to result in a fusion polypeptide. In some embodiments, the size of the packaged insert (i.e., the viral genome or a portion thereof, or the heterologous polynucleotide as described) can be 500-1000, 1000-2000, 2000-3000, 3000-4000, 4000-5000, 5000-6000, 6000-7000, or 7000-8000 nucleotides in length. In some embodiments, the insert can be more than 8000 nucleotides in length, for example, 9000, 10000, or 11000 nucleotides in length.

[0159] In some embodiments, the viral vector particle, e.g., retroviral vector particle, comprises one or more of gag polyprotein, polymerase (e.g., pol), integrase (e.g., functional or non-functional variants), protease, and fusogen. In some embodiments, the lipid particle further comprises rev. In some embodiments, one or more of the aforementioned proteins are encoded in the retroviral genome (i.e., the insert described above), and in some embodiments, one or more of the aforementioned proteins are provided in trans, e.g., by a helper cell, helper virus, or helper plasmid. In some embodiments, the lipid particle nucleic acid (e.g., retroviral nucleic acid) comprises one or more of the following nucleic acid sequences: 5'LTR (e.g., comprising U5 and lacking a functional U3 domain), Psi packaging element (Psi), a central polypurine tract (cPPT) promoter operably linked to a payload gene, a payload gene (optionally comprising an intron before the open reading frame), a polyA tail sequence, a WPRE, and a 3'LTR (e.g., comprising U5 and lacking a functional U3). In some embodiments, the lipid particle nucleic acid further comprises a retroviral cis-acting RNA packaging element and a cPPT / CTS element. In some embodiments, the lipid particle nucleic acid further comprises one or more insulator elements. In some embodiments, the recognition site is located between the polyA tail sequence and the WPRE.

[0160] In some embodiments, the lipid particle comprises a supramolecular complex formed by viral proteins that self-assemble into a capsid. In some embodiments, the lipid particle is a viral particle derived from a viral capsid. In some embodiments, the lipid particle is a viral particle derived from a viral nucleocapsid. In some embodiments, the lipid particle comprises a nucleocapsid derivative that retains the property of packaging nucleic acid.

[0161] In some embodiments, the lipid particle packages nucleic acid from host cell that carries one or more viral nucleic acids (e.g., retroviral nucleic acids) during the expression process.In some embodiments, the nucleic acid does not code for any gene involved in viral replication.In certain embodiments, the lipid particle is, for example, a viral-based particle that is replication-deficient, for example, a retroviral particle such as a lentiviral particle.

[0162] In some cases, the lipid particle is a viral particle that is morphologically indistinguishable from a wild-type infectious virus. In some embodiments, the viral particle presents the entire viral proteome as an antigen. In some embodiments, the viral particle presents only a portion of the proteome as an antigen.

[0163] In some embodiments, the retroviral nucleic acid comprises one or more (e.g., all) of a 5' promoter (e.g., for controlling expression of the entire packaged RNA), a 5' LTR (e.g., comprising a U5 containing R (polyadenylation tail signal) and / or a primer activation signal), a primer binding site, a Psi packaging signal, an RRE element for nuclear export, a promoter directly upstream of the transgene to control transgene expression, a transgene (or other exogenous agent element), a polypurine tract, and a 3' LTR (e.g., comprising a mutated U3, R, and U5). In some embodiments, the retroviral nucleic acid further comprises one or more of a cPPT, a WPRE, and / or an insulator element.

[0164] Retroviruses usually replicate by reverse transcription of their genomic RNA into a linear double-stranded DNA copy, and then covalently integrate their genomic DNA into the host genome.Exemplary retroviruses suitable for use in certain embodiments include, but are not limited to, Moloney murine leukemia virus (M-MuLV), Moloney murine sarcoma virus (MoMSV), Harvey murine sarcoma virus (HaMuSV), mouse mammary tumor virus (MuMTV), gibbon ape leukemia virus (GaLV), feline leukemia virus (FLV), spuma virus, friend murine leukemia virus, murine stem cell virus (MSCV), and Rous sarcoma virus (RSV), and lentivirus.

[0165] In some embodiments, the retrovirus is a gamma retrovirus. In some embodiments, the retrovirus is an epsilon retrovirus. In some embodiments, the retrovirus is an alpha retrovirus. In some embodiments, the retrovirus is a beta retrovirus. In some embodiments, the retrovirus is a delta retrovirus. In some embodiments, the retrovirus is a lentivirus. In some embodiments, the retrovirus is a spuma retrovirus. In some embodiments, the retrovirus is an endogenous retrovirus.

[0166] Exemplary lentiviruses include, but are not limited to, HIV (human immunodeficiency virus; including HIV type 1 and HIV type 2); Visna-Maedi virus (VMV); Caprine arthritis-encephalitis virus (CAEV); Equine infectious anemia virus (EIAV); Feline immunodeficiency virus (FIV); Bovine immunodeficiency virus (BIV); and Simian immunodeficiency virus (SIV). In some embodiments, an HIV-based vector backbone (i.e., HIV cis-acting sequence elements) is used.

[0167] Viral vectors may include nucleic acid molecules (e.g., transfer plasmids) that generally include virus-derived nucleic acid elements that facilitate the transfer or integration of nucleic acid molecules (e.g., including nucleic acids encoding exogenous agents) into the genome of a cell or into viral particles that mediate nucleic acid transfer. Viral vector particles may typically include various viral components and sometimes host cell components in addition to the nucleic acid. Viral vectors may include viruses or viral particles that are capable of transferring nucleic acids into cells (e.g., nucleic acids encoding exogenous agents) or transferred nucleic acids (e.g., as naked DNA). Viral vectors and transfer plasmids may include structural and / or functional genetic elements that are primarily derived from viruses. Retroviral vectors may include viral vectors or plasmids that include structural and functional genetic elements, or portions thereof, that are primarily derived from retroviruses. Lentiviral vectors may include viral vectors or plasmids that contain structural and / or functional genetic elements, or portions thereof, that include LTRs that are primarily derived from lentiviruses.

[0168] In embodiments, lentiviral vectors (e.g., lentiviral expression vectors) can comprise lentiviral transfer plasmids (e.g., as naked DNA) or infectious lentiviral particles. It should be understood that with respect to elements such as cloning sites, promoters, regulatory elements, heterologous nucleic acids, etc., the sequences of these elements can be present in RNA form in lentiviral particles and in DNA plasmids in DNA form.

[0169] In some vectors described herein, at least a portion of one or more protein coding regions that contribute to or are essential for replication may be absent compared to the corresponding wild-type virus. This renders the viral vector replication-deficient. In some embodiments, the vector is capable of transducing a target non-dividing host cell and / or integrating its genome into the host genome.

[0170] The structure of wild-type retroviral genomes often includes a 5' long terminal repeat (LTR) and a 3' LTR between or within which are located packaging signals to allow the genome to be packaged, primer binding sites, integration sites to allow integration into the host cell genome, and gag, pol, and env genes that code for packaging components that facilitate the assembly of viral particles. More complex retroviruses have additional features, such as the rev and RRE sequences in HIV, which allow efficient export of RNA transcripts of the integrated provirus from the nucleus to the cytoplasm of the infected target cell. In proviruses, viral genes are flanked on both ends by regions called long terminal repeats (LTRs). LTRs are involved in the integration and transcription of the provirus. LTRs may also function as enhancer-promoter sequences to control the expression of viral genes. Encapsidation of retroviral RNA occurs through the psi sequence located at the 5' end of the viral genome.

[0171] The LTRs themselves are usually similar (e.g., identical) sequences that can be divided into three elements called U3, R, and U5. U3 is derived from a sequence unique to the 3' end of the RNA. R is derived from repeated sequences at both ends of the RNA, and U5 is derived from a sequence unique to the 5' end of the RNA. The sizes of the three elements can vary greatly between different retroviruses.

[0172] For the viral genome, the site of transcription initiation is usually the boundary between U3 and R in one LTR, and the site of poly(A) addition (termination) is the boundary between R and U5 in the other LTR. U3 contains most of the transcriptional control elements of the provirus, including the promoter and multiple enhancer sequences responsive to cellular and, in some cases, viral transcriptional activator proteins. Some retroviruses contain any one or more of the following genes that code for proteins involved in the control of gene expression: tot, rev, tax, and rex. Regarding the structural genes gag, pol, and env themselves, gag codes for the internal structural protein of the virus. The gag protein is proteolytically processed into the mature proteins MA (matrix), CA (capsid), and NC (nucleocapsid). The pol gene codes for reverse transcriptase (RT), which contains the DNA polymerase, associated RNase H, and integrase (IN), which mediate genome replication. The env gene encodes the surface (SU) glycoprotein and the transmembrane (TM) protein of the virion, which form a complex that specifically interacts with cellular receptor proteins, facilitating infection, for example, by fusion of the viral membrane with the cellular membrane.

[0173] Replication-defective retroviral vector genomes gag, pol, and env may be absent or may not be functional. The R regions at both ends of the RNA are generally repeated sequences. U5 and U3 represent sequences unique to the 5' and 3' ends of the RNA genome, respectively.

[0174] Retroviruses may also contain additional genes that code for proteins other than gag, pol, and env. Examples of additional genes include one or more of (in HIV), vif, vpr, vpx, vpu, tat, rev, and nef. EIAV has an additional gene, S2 (among others). The proteins encoded by the additional genes perform various functions, some of which may be redundant of functions provided by cellular proteins. In EIAV, for example, tat acts as a transcriptional activator of the viral LTR (Derse and Newbold 1993 Virology 194:530-6; Maury et al. 1994 Virology 200:632-42). It binds to a stable stem-loop RNA secondary structure called TAR. Rev regulates and modulates the expression of viral genes through the rev response element (RRE) (Martarano et al. 1994 J. Virol. 68:3102-11). The mechanism of action of these two proteins appears to be largely similar to that of the analogous mechanisms in primate viruses.In addition, an EIAV protein, Ttm, has been identified, which is encoded by the first exon of tat spliced ​​into the env coding sequence at the start of the transmembrane protein.

[0175] In addition to protease, reverse transcriptase, and integrase, non-primate lentiviruses contain a fourth pol gene product that encodes a dUTPase, which may play a role in the ability of these lentiviruses to infect certain non-dividing or slowly dividing cell types.

[0176] In embodiments, a recombinant lentiviral vector (RLV) is a vector that carries sufficient retroviral genetic information to allow the RNA genome to be packaged into a viral particle capable of infecting a target cell in the presence of packaging components. Infection of a target cell may include reverse transcription and integration into the target cell genome. RLVs typically carry non-viral coding sequences that are delivered to the target cell by the vector, such as nucleic acids encoding exogenous agents described herein. In embodiments, RLVs are incapable of independent replication to generate infectious retroviral particles in the target cell. Typically, RLVs lack functional gag-pol and / or env genes and / or other genes involved in replication. Vectors may be configured as split-intron vectors, for example, as described in PCT Patent Application No. WO99 / 15683, which is incorporated herein by reference in its entirety.

[0177] In some embodiments, lentiviral vectors comprise a minimal viral genome, e.g., viral vectors have been engineered to remove non-essential elements and retain essential elements to provide the functionality required to infect, transduce, and deliver a nucleotide sequence of interest to a target host cell, e.g., as described in WO 98 / 17815, which is incorporated by reference in its entirety.

[0178] A minimal lentiviral genome may, for example, comprise (5')R-U5-one or more first nucleotide sequences-U3-R(3'). However, the plasmid vector used to produce the lentiviral genome in the source cell may also comprise a transcriptional regulatory control sequence operably linked to the lentiviral genome to induce transcription of the genome in the source cell. These regulatory sequences may comprise the natural sequence associated with the transcribed retroviral sequence, e.g., the 5'U3 region, or they may comprise a heterologous promoter, e.g., another viral promoter, e.g., the CMV promoter. Some lentiviral genomes contain additional sequences to facilitate efficient virus production. For example, in the case of HIV, rev and RRE sequences may be included. Alternatively or in combination, codon optimization may be used, e.g., the gene encoding the exogenous agent may be codon optimized, e.g., as described in WO01 / 79518, which is incorporated herein by reference in its entirety. Alternative sequences that perform similar or the same function as the rev / RRE system may also be used. For example, a functional analogue of the rev / RRE system is found in the Mason-Pfizer monkey virus, which contains an RRE-type sequence in its genome known as a CTE that is thought to interact with factors in infected cells. The cellular factors can be thought of as rev analogues. Thus, the CTE can be used as a substitute for the rev / RRE system. Furthermore, the Rex protein of HTLV-I can functionally replace the Rev protein of HIV-I. Rev and Rex have similar effects on the IRE-BP.

[0179] In some embodiments, the retroviral nucleic acid (e.g., a lentiviral nucleic acid, e.g., a primate or non-primate lentiviral nucleic acid) (1) has a gag gene deleted, where the gag deletion removes one or more nucleotides downstream of about nucleotide 350 or 354 of the gag coding sequence, (2) has one or more accessory genes not present in the retroviral nucleic acid, (3) lacks a tat gene but includes a leader sequence between the end of the 5'LTR and the ATG of gag, and (4) is a combination of (1), (2), and (3). In embodiments, a lentiviral vector includes all of the features of (1) and (2) and (3). This strategy is described in more detail in WO99 / 32646, which is incorporated herein by reference in its entirety.

[0180] In some embodiments, primate lentiviral minimal systems do not require any of the additional HIV / SIV genes vif, vpr, vpx, vpu, tat, rev, and nef for either vector production or transduction of dividing and non-dividing cells, hi some embodiments, EIAV minimal vector systems do not require S2 for either vector production or transduction of dividing and non-dividing cells.

[0181] The deletion of additional genes may allow vectors to be produced without genes associated with disease in lentivirus (e.g., HIV) infection. In particular, tat is associated with disease. Secondly, the deletion of additional genes allows the vector to package more heterologous DNA. Thirdly, genes with unknown functions such as S2 may not be included, thus reducing the risk of causing unwanted effects. Examples of minimal lentivirus vectors are disclosed in WO99 / 32646 and WO98 / 17815.

[0182] In some embodiments, the retroviral nucleic acid is defective in at least tat and S2 (in the case of an EIAV vector system), and optionally also defective in vif, vpr, vpx, vpu, and nef, hi some embodiments, the retroviral nucleic acid is defective in rev, RRE, or both.

[0183] In some embodiments, the retroviral nucleic acid comprises vpx. Vpx polypeptide binds to and induces the degradation of SAMHD1 restriction factor, which degrades free dNTPs in cytoplasm. Thus, the concentration of free dNTPs in cytoplasm increases as Vpx degrades SAMHD1 and reverse transcription activity increases, thus promoting the reverse transcription and integration of retroviral genome into target cell genome.

[0184] Different cells differ in their frequency of use of certain codons. This codon bias corresponds to the bias in the relative abundance of certain tRNAs in cell types. It is possible to increase expression by modifying codons in sequences to adjust them to match the relative abundance of corresponding tRNAs. Similarly, it is possible to decrease expression by deliberately selecting codons whose corresponding tRNAs are known to be rare in certain cell types. Thus, a further degree of translational control is possible. Additional description of codon optimization is described, for example, in WO99 / 41397, which is incorporated herein by reference in its entirety.

[0185] Many viruses, including HIV and other lentiviruses, use a large number of rare codons and by changing these to correspond to commonly used mammalian codons, expression of the packaging components in mammalian producer cells can be increased.

[0186] In some embodiments, codon optimization has several other advantages. In some embodiments, modifications in those sequences may reduce or eliminate RNA instability sequences (INS) from the nucleotide sequences encoding the packaging components. At the same time, the amino acid sequences coding sequences for the packaging components are retained so that the viral components encoded by the sequences remain the same, or at least similar enough, so that the function of the packaging components is not compromised. In some embodiments, codon optimization also overcomes the Rev / RRE requirement for export, making the optimized sequences Rev-independent. In some embodiments, codon optimization also reduces homologous recombination between different constructs within the vector system (e.g., between overlapping regions in the gag-pol and env open reading frames). In some embodiments, codon optimization results in increased viral titers and / or improved safety.

[0187] In some embodiments, only the codons associated with the INS are codon optimized, hi other embodiments, the sequence is codon optimized in its entirety, except for the sequence encompassing the frameshift site of gag-pol.

[0188] The gag-pol gene contains two overlapping reading frames that code for gag-pol proteins. Expression of both proteins is dependent on a frameshift during translation. This frameshift occurs as a result of ribosome "slippage" during translation. This slippage is thought to be caused, at least in part, by a ribosome-stalling RNA secondary structure. Such a secondary structure is present downstream of the frameshift site in the gag-pol gene. In the case of HIV, the overlapping region extends from nucleotide 1222 downstream of the start of gag (nucleotide 1 is A of the gag ATG) to the end of gag (nt 1503). Therefore, it is preferred that the 281 bp fragment spanning the frameshift site and the overlapping region of the two reading frames is not codon-optimized. In some embodiments, retention of this fragment allows for more efficient expression of the gag-pol proteins. In the case of EIAV, the start of the overlap is at nt 1262 (nucleotide 1 is A of the gag ATG). The end of the overlap is at nt 1461. The wild-type sequence can be retained from nt 1156 to 1465 to ensure that the frameshift site and the gag-pol overlap are preserved.

[0189] In some embodiments, derivation from optimal codon usage may be made and conservative amino acid changes may be introduced into the gag-pol protein, for example to provide convenient restriction enzyme sites.

[0190] In some embodiments, codon optimization is based on codons with low codon usage in mammalian systems. The third and sometimes the second and third bases may be altered.

[0191] It will be appreciated that in some embodiments, due to the degenerate nature of the genetic code, a large number of gag-pol sequences can be achieved by one skilled in the art. Also, many retrovirus variants have been described that can be used as a starting point to generate codon-optimized gag-pol sequences. The genome of lentiviruses can be very diverse. For example, there are many quasi-species of HIV-1 that still function. This is also true for EIAV. These variants may be used to enhance certain parts of the transduction process. Examples of HIV-I variants can be found in the HIV database maintained by Los Alamos National Laboratory. Details of EIAV clones can be found in the NCBI database maintained by the National Institutes of Health.

[0192] It is within the level of ordinary skill in the art to empirically determine the appropriate codon optimization of a viral sequence. The strategy for codon-optimized sequences, including gag-pol, can be used with any retrovirus, such as EIAV, FIV, BIV, CAEV, VMR, SIV, HIV-I and HIV-2. This method can also be used to increase expression of genes from HTLV-I, HTLV-2, HFV, HSRV, and human endogenous retroviruses (HERVs), MLV, and other retroviruses.

[0193] In embodiments, the retroviral vector comprises a packaging signal that comprises 255-360 nucleotides of gag in vectors that still retain the env sequence, or about 40 nucleotides of gag in certain combinations of splice donor mutations, gag and env deletions. In some embodiments, the retroviral vector comprises a gag sequence that includes one or more deletions, for example, the gag sequence comprises about 360 nucleotides derivable from the N-terminus.

[0194] In some embodiments, the retroviral vector, helper cell, helper virus, or helper plasmid may include retroviral structural and accessory proteins, such as gag, pol, env, tat, rev, vif, vpr, vpu, vpx, or nef proteins or other retroviral proteins. In some embodiments, the retroviral proteins are from the same retrovirus. In some embodiments, the retroviral proteins are from multiple retroviruses, such as 2, 3, 4, or more retroviruses.

[0195] In some embodiments, the gag and pol coding sequences are typically organized in native lentiviruses as the Gag-Pol precursor. The gag sequence encodes the 55 kD Gag precursor protein, also called p55. p55 is cleaved by a virally encoded protease (the product of the pol gene) during the process of maturation into four smaller proteins designated MA (matrix [p17]), CA (capsid [p24]), NC (nucleocapsid [p9]), and p6. The pol precursor protein is cleaved from Gag by the virally encoded protease and further digested to separate the protease (p10), RT (p50), RNase H (p15), and integrase (p31) activities.

[0196] In some embodiments, the lentiviral vector is integration-deficient. In some embodiments, pol is integrase-deficient, such as by encoding due to a mutation in the integrase gene. For example, the pol coding sequence may include an inactivating mutation in integrase, such as one or more mutations of amino acids involved in catalytic activity, i.e., one or more of aspartic acid 64, aspartic acid 116, and / or glutamic acid 152. In some embodiments, the integrase mutation is a D64V mutation. In some embodiments, the mutation in integrase allows packaging of viral RNA into the lentivirus. In some embodiments, the mutation in integrase allows packaging of viral proteins into the lentivirus. In some embodiments, the mutation in integrase reduces the likelihood of insertional mutagenesis. In some embodiments, the mutation in integrase reduces the likelihood of generating replication-competent recombinants (RCR) (Wanisch et al. 2009. Mol Ther. 1798:1316-1332). In some embodiments, native Gag-Pol sequences can be used in helper vectors (e.g., helper plasmids or helper viruses) or can be modified. These modifications include chimeric Gag-Pol, where the Gag and Pol sequences are derived from different viruses (e.g., different species, subspecies, strains, clades, etc.) and / or the sequences have been modified to improve transcription and / or translation and / or to reduce recombination.

[0197] In some embodiments, the retroviral nucleic acid comprises a polynucleotide encoding a 150-250 (e.g., 168) nucleotide portion of a gag protein, which (i) contains a mutant INS1 inhibitory sequence that reduces restriction of RNA export from the nucleus relative to wild-type INS1, (ii) contains a two nucleotide insertion that results in a frameshift and premature termination, and / or (iii) does not contain the INS2, INS3, and INS4 inhibitory sequences of gag.

[0198] In some embodiments, the vectors described herein are hybrid vectors that contain both retroviral (e.g., lentiviral) and non-lentiviral viral sequences. In some embodiments, the hybrid vectors contain retroviral, e.g., lentiviral, sequences for reverse transcription, replication, integration, and / or packaging.

[0199] According to certain specific embodiments, most or all of the backbone sequence of the viral vector is derived from a lentivirus, such as HIV-I. However, it should be understood that many different sources of retroviral and / or lentiviral sequences can be used or combined, providing numerous substitutions and modifications in a given lentiviral sequence without compromising the ability of the transfer vector to perform the functions described herein. A variety of lentiviral vectors are described in Naldini et al., (1996a, 1996b, and 1998), Zufferey et al., (1997), Dull et al., 1998, U.S. Patent Nos. 6,013,516, and 5,994,136, many of which can be adapted to produce retroviral nucleic acid.

[0200] At each end of the provirus, a long terminal repeat (LTR) is usually found. LTRs usually contain domains located at the ends of retroviral nucleic acids that are direct repeats in their natural sequence context and include U3, R, and U5 regions. LTRs generally promote retroviral gene expression (e.g., promotion, initiation, and polyadenylation of gene transcripts) and viral replication. LTRs may contain multiple regulatory signals, including transcriptional control elements, polyadenylation signals, and sequences for replication and integration of the viral genome. Viral LTRs are usually divided into three regions, called U3, R, and U5. The U3 region usually contains enhancer and promoter elements. The U5 region is usually a sequence between the primer binding site and the R region and may contain polyadenylation sequences. The R (repeat) region may flank the U3 and U5 regions. LTRs usually consist of U3, R, and U5 regions and may occur at both the 5' and 3' ends of the viral genome. In some embodiments, the 5'LTR is flanked by sequences for reverse transcription of the genome (tRNA primer binding site) and for efficient packaging of viral RNA into particles (psi site).

[0201] In some embodiments, the packaging signal may comprise a sequence located within the retroviral genome that mediates the insertion of viral RNA into the viral capsid or particle.See, for example, Clever et al., 1995. J. of Virology, Vol. 69, No. 4; pp. 2101-2109.Some retroviral vectors use a minimal packaging signal (psi[Y] sequence) for the encapsidation of the viral genome.

[0202] In various embodiments, the retroviral nucleic acid comprises a modified 5'LTR and / or 3'LTR. One or both of the LTRs may contain one or more modifications, including, but not limited to, one or more deletions, insertions, or substitutions. Modifications of the 3'LTR are often made to improve the safety of lentiviral or retroviral systems by rendering the virus replication-deficient, e.g., a virus that cannot replicate completely and efficiently such that infectious virions are not produced (e.g., replication-deficient lentiviral progeny).

[0203] In some embodiments, the vector is a self-inactivating (SIN) vector, e.g., a replication-deficient vector, e.g., a retroviral or lentiviral vector, in which the right (3') LTR enhancer-promoter region, known as the U3 region, has been modified (e.g., by deletion or substitution) to prevent viral transcription beyond the first round of viral replication. In some aspects, provided herein are replication-incompetent (also referred to herein as replication-deficient) vector particles that cannot participate in replication (i.e., no viral vector particles are produced from the transduced cell) in the absence of packaging cells. In some aspects, this is because the right (3') LTR U3 region can be used as a template for the left (5') LTR U3 region during viral replication, and thus viral replication is inhibited by the deletion of the U3 enhancer-promoter. In embodiments, the 3'LTR is modified such that the U5 region is removed, modified, or replaced, e.g., with an exogenous poly(A) sequence. The 3'LTR, the 5'LTR, or both the 3' and 5' LTRs may be modified LTRs. Other modifications to viral vectors, ie retroviral or lentiviral vectors, to render the vector incapable of replication are known in the art.

[0204] In some embodiments, the U3 region of the 5'LTR is replaced with a heterologous promoter to drive transcription of the viral genome during the production of viral particles. Examples of heterologous promoters that can be used include, for example, the viral Simian Virus 40 (SV40) (e.g., early or late) promoter, the cytomegalovirus (CMV) (e.g., immediate early) promoter, the Moloney Murine Leukemia Virus (MoMLV) promoter, the Rous Sarcoma Virus (RSV) promoter, and the Herpes Simplex Virus (HSV) (thymidine kinase) promoter. In some embodiments, the promoter can drive high levels of transcription in a Tat-independent manner. In certain embodiments, the heterologous promoter has the added advantage of controlling the manner in which the viral genome is transcribed. For example, the heterologous promoter can be inducible, such that transcription of all or part of the viral genome occurs only in the presence of an inducer. An inducer includes, but is not limited to, one or more compounds, or physiological conditions, such as temperature or pH, at which the host cells are cultured.

[0205] In some embodiments, the viral vector comprises a TAR (transactivation response) element, for example located in the R region of lentivirus (e.g., HIV) LTR. This element interacts with the transactivator (tat) gene element of lentivirus to enhance viral replication. However, this element is not required in some embodiments, for example, where the U3 region of 5'LTR is replaced by a heterologous promoter.

[0206] The R region, e.g., the region within a retroviral LTR that begins at the start of the capping group (i.e., the start of transcription) and ends just before the start of the polyA tract, may be flanked by U3 and U5 regions. The R region plays a role in the process of reverse transcription in the transfer of nascent DNA from one end of the genome to the other.

[0207] Retroviral nucleic acid may also include a FLAP element, for example, a nucleic acid whose sequence includes the central polypurine tract and central termination sequence (cPPT and CTS) of a retrovirus, for example, HIV-I or HIV-2. Suitable FLAP elements are described in U.S. Patent No. 6,682,907 and Zennou, et al., 2000, Cell, 101:173, which are incorporated herein by reference in their entirety. During the reverse transcription of HIV-I, the central initiation of the positive strand DNA in the central polypurine tract (cPPT) and the central termination in the central termination sequence (CTS) can result in the formation of a triple-stranded DNA structure: the HIV-I central DNA flap. In some embodiments, the retroviral or lentiviral vector backbone includes one or more FLAP elements upstream or downstream of the gene encoding the exogenous agent. For example, in some embodiments, the transfer plasmid includes a FLAP element, for example, a FLAP element derived from or isolated from HIV-L.

[0208] In embodiments, retroviral or lentiviral nucleic acid comprises one or more transport elements, such as cis-acting post-transcriptional regulatory elements that regulate the transport of RNA transcripts from the nucleus to the cytoplasm of cells. Examples of RNA transport elements include, but are not limited to, the human immunodeficiency virus (HIV) rev response element (RRE) (see, for example, Cullen et al., 1991. J.Virol.65:1053; and Cullen et al., 1991. Cell 58:423), and the hepatitis B virus post-transcriptional regulatory element (HPRE), which are incorporated herein by reference in their entirety. In general, RNA transport elements are located within the 3'UTR of genes and can be inserted as one or more copies.

[0209] In some embodiments, expression of a heterologous sequence (e.g., a nucleic acid encoding an exogenous agent) in a viral vector is increased by incorporating one or more, e.g., all, of a posttranscriptional regulatory element, a polyadenylation site, and a transcription termination signal into the vector. A variety of posttranscriptional regulatory elements, such as the woodchuck hepatitis virus posttranscriptional regulatory element (WPRE; Zufferey et al., 1999, J. Virol., 73:2886), the posttranscriptional regulatory element (HPRE) present in Hepatitis B virus (Huang et al., Mol. Cell. Biol., 5:3864), and the like (Liu et al., 1995, Genes Dev., 9:1766), each of which is incorporated herein by reference in its entirety, can increase expression of a heterologous nucleic acid in a protein. In some embodiments, the retroviral nucleic acid described herein comprises a posttranscriptional regulatory element, e.g., a WPRE or a HPRE.

[0210] In some embodiments, the retroviral nucleic acids described herein lack or do not include a post-transcriptional regulatory element, e.g., a WPRE or HPRE.

[0211] For example, elements directing termination and polyadenylation of heterologous nucleic acid transcripts may be included to increase expression of exogenous agents. Transcription termination signals may be found downstream of polyadenylation signals. In some embodiments, vectors contain polyadenylation sequences 3' of the polynucleotide encoding the exogenous agent. PolyA sites may contain DNA sequences directing both termination and polyadenylation of the nascent RNA transcript by RNA polymerase II. Polyadenylation sequences may promote mRNA stability and contribute to improved translation efficiency by adding a polyA tail to the 3' end of the coding sequence. Illustrative examples of polyA signals that can be used in retroviral nucleic acids include AATAAA, ATTAAA, AGTAAA, bovine growth hormone polyA sequence (BGHpA), rabbit beta globin polyA sequence (rPgpA), or another suitable heterologous or endogenous polyA sequence.

[0212] In some embodiments, the retroviral or lentiviral vector further comprises one or more insulator sequences, such as those described herein.

[0213] In various embodiments, the vector comprises a promoter operably linked to a polynucleotide encoding an exogenous agent. The vector may have one or more LTRs, any of which may comprise one or more modifications, such as one or more nucleotide substitutions, additions, or deletions. The vector may further comprise one or more accessory elements for increasing transduction efficiency (e.g., cPPT / FLAP), one or more accessory elements for increasing viral packaging (e.g., Psi(Y) packaging signal, RRE), and / or other elements for increasing expression of the exogenous gene (e.g., poly(A) sequence), and may optionally comprise a WPRE or HPRE.

[0214] In some embodiments, the lentiviral nucleic acid includes, e.g., from 5' to 3', one or more, e.g., all of: a promoter (e.g., CMV), an R sequence (e.g., including TAR), a U5 sequence (e.g., for integration), a PBS sequence (e.g., for reverse transcription), a DIS sequence (e.g., for genome dimerization), a psi packaging signal, a partial gag sequence, an RRE sequence (e.g., for nuclear export), a cPPT sequence (e.g., for nuclear import), a promoter for driving expression of an exogenous agent, a gene encoding an exogenous agent, a WPRE sequence (e.g., for efficient transgene expression), a PPT sequence (e.g., for reverse transcription), an R sequence (e.g., for polyadenylation and termination), and a U5 signal (e.g., for integration).

[0215] 2. Virus-like vector particles In some embodiments, the virus-based particle is a virus-like lipid particle (VLP) derived from a virus. In some embodiments, the viral envelope may contain fusogens, such as fusogens or pseudotype fusogens that are endogenous to the virus. VLPs include those derived from retroviruses or lentiviruses. VLPs mimic the natural virion structure but lack the viral genomic information required for independent replication in a host cell. Thus, in some aspects, the VLP is non-infectious. In certain embodiments, the VLP does not contain a viral genome. In some embodiments, the amphipathic lipid bilayer of the VLP is or comprises a viral envelope. In some embodiments, the bilayer of the amphipathic lipid targeted lipid particle is or comprises a lipid derived from a cell. In some embodiments, the VLP comprises at least one structural protein derived from a virus. In most cases, this protein forms a proteinaceous capsid. In some cases, the capsid is also covered by a lipid bilayer derived from the cell that released the assembled VLP (e.g., VLPs containing human immunodeficiency virus structural proteins such as GAGs). In some embodiments, the VLP further comprises a targeting moiety as an envelope protein within the lipid bilayer.

[0216] In some embodiments, the vector vehicle particle comprises a supramolecular complex formed by viral proteins that self-assemble into a capsid. In some embodiments, the vector vehicle particle is a virus-like particle derived from a viral capsid protein. In some embodiments, the vector vehicle particle is a virus-like particle derived from a viral nucleocapsid protein. In some embodiments, the vector vehicle particle comprises a nucleocapsid-derived protein that retains the property of packaging nucleic acid. In some embodiments, the virus-based particle, e.g., the virus-like particle, comprises only viral structural glycoproteins among proteins derived from the viral genome. In some embodiments, the vector vehicle particle does not comprise a viral genome.

[0217] In some embodiments, the vector vehicle particle packages a nucleic acid derived from the host cell during the expression process, for example, a nucleic acid encoding an exogenous agent. In some embodiments, the nucleic acid does not encode any genes involved in viral replication. In certain embodiments, the vector vehicle particle is, for example, a virus-like particle that is replication-deficient, for example, a retrovirus-like particle, such as a lentivirus-like particle.

[0218] In some embodiments, the vector vehicle particle is a virus-like particle that contains sequences that are absent or missing viral RNA, which may result in the removal or elimination of viral RNA from the sequence. In some embodiments, this may be accomplished by using an endogenous packaging signal binding site on gag. In some embodiments, the endogenous packaging signal binding site is on pol. In some embodiments, the RNA to be delivered contains a cognate packaging signal. In some embodiments, a heterologous binding domain (heterologous to gag) located on the RNA to be delivered and a cognate binding site located on gag or pol may be used to ensure packaging of the RNA to be delivered. In some embodiments, the heterologous sequence may be non-viral, or it may be viral, in which case it may be derived from a different virus. In some embodiments, the vector particle may be used to deliver a therapeutic RNA, in which case functional integrase and / or reverse transcriptase are not required. In some embodiments, the vector particle may also be used to deliver a therapeutic gene of interest, in which case pol is typically included.

[0219] In some embodiments, the VLP comprises a supramolecular complex formed by viral proteins that self-assemble into a capsid. In some embodiments, the VLP is derived from a viral capsid. In some embodiments, the VLP is derived from a viral nucleocapsid. In some embodiments, the VLP is derived from a nucleocapsid and retains the property of packaging nucleic acid. In some embodiments, the VLP comprises only viral structural glycoproteins. In some embodiments, the VLP does not comprise a viral genome.

[0220] 3. Methods for generating virus-based particles Large-scale production of viral particles is often useful to achieve a desired viral titer. Viral particles can be produced by transfecting a transfer vector into a packaging cell line that contains viral structural and / or accessory genes, such as the gag, pol, env, tat, rev, vif, vpr, vpu, vpx, or nef genes or other retroviral genes.

[0221] In some embodiments, viral vector particles can be produced in multiple cell culture systems, including bacteria, mammalian cell lines, insect cell lines, yeast, and plant cells. Exemplary methods for making viral vector particles are described.

[0222] In some embodiments, elements for the production of a viral vector, i.e., a recombinant viral vector such as a replication-incompetent lentiviral vector, are contained in a packaging cell line or are present on a packaging vector. In some embodiments, a viral vector can include packaging elements rev, gag, and pol that are delivered to a packaging cell line via one or more packaging vectors.

[0223] In embodiments, the packaging vector is an expression vector or a viral vector that lacks a packaging signal and contains a polynucleotide encoding one, two, three, four or more viral structural and / or accessory genes. Typically, the packaging vector is contained in a packaging cell and introduced into the cell via transfection, transduction, or infection. A retroviral, e.g., lentiviral, transfer vector can be introduced into the packaging cell line via transfection, transduction, or infection to generate a source cell or cell line. The packaging vector can be introduced into a human cell or cell line by standard methods, including, for example, calcium phosphate transfection, lipofection, or electroporation. In some embodiments, the packaging vector is introduced into the cell together with a dominant selection marker, e.g., neomycin, hygromycin, puromycin, blasticidin, zeocin, thymidine kinase, DHFR, Gln synthetase, or ADA, followed by selection in the presence of the appropriate drug to isolate clones. The selectable marker gene can be physically linked by the packaging vector, for example to a gene encoded by an IRES or a self-cleaving viral peptide, In some embodiments, the packaging vector is a packaging plasmid.

[0224] Producer cell lines (also called packaging cell lines) include cell lines that do not contain a packaging signal but stably or transiently express viral structural proteins and replicative enzymes (e.g., gag, pol, and env) that are capable of packaging viral particles. Any suitable cell line can be used, e.g., mammalian cells, e.g., human cells. Suitable cell lines that can be used include, for example, CHO cells, BHK cells, MDCK cells, C3H 10T1 / 2 cells, FLY cells, Psi-2 cells, BOSC 23 cells, PA317 cells, WEHI cells, COS cells, BSC 1 cells, BSC 40 cells, BMT 10 cells, VERO cells, W138 cells, MRC5 cells, A549 cells, HT1080 cells, 293 cells, 293T cells, B-50 cells, 3T3 cells, NIH3T3 cells, HepG2 cells, Saos-2 cells, Huh7 cells, HeLa cells, W163 cells, 211 cells, and 211A cells. In embodiments, the packaging cells are 293 cells, 293T cells, or A549 cells.

[0225] In some embodiments, producer cells (i.e., source cell lines) include packaging cell lines and cell lines capable of generating recombinant retroviral particles that contain a transfer vector construct that includes a packaging signal. Methods for preparing virus stock solutions are described, for example, by Y. Soneoka et al. (1995) Nucl. Acids Res. 23:628-633 and NRL Andau et al. (1992) J. Virol. 66:5110-5113, which are incorporated herein by reference. Infectious virus particles can be recovered from packaging cells, for example, by cell lysis or by harvesting the cell culture supernatant. Optionally, the recovered virus particles can be concentrated or purified.

[0226] In some embodiments, the source cell comprises one or more plasmids (i.e., packaging plasmids) encoding viral structural proteins and replicative enzymes (e.g., gag, pol, and env) capable of packaging viral particles. In some embodiments, the sequences encoding at least two of the gag, pol, and env precursors are on the same plasmid. In some embodiments, the sequences encoding the gag, pol, and env precursors are on different plasmids. In some embodiments, the sequences encoding the gag, pol, and env precursors have the same expression signal, e.g., promoter. In some embodiments, the sequences encoding the gag, pol, and env precursors have different expression signals, e.g., different promoters. In some embodiments, the expression of the gag, pol, and env precursors is inducible. In some embodiments, the plasmids encoding the viral structural proteins and replicative enzymes are transfected at the same time or at different times. In some embodiments, the plasmids encoding the viral structural proteins and replicative enzymes are transfected at the same time or at different times as the packaging vector.

[0227] In some embodiments, the source cell line contains one or more stably integrated viral structural genes. In some embodiments, expression of the stably integrated viral structural genes is inducible.

[0228] In some embodiments, expression of a viral structural gene is regulated at the transcriptional level. In some embodiments, expression of a viral structural gene is regulated at the translational level. In some embodiments, expression of a viral structural gene is regulated at the post-translational level.

[0229] In some embodiments, expression of viral structural genes is regulated by a tetracycline (Tet)-dependent system, in which the Tet-regulated transcriptional repressor (Tet-R) binds to DNA sequences contained in the promoter and represses transcription by steric hindrance (Yao et al., 1998; Jones et al., 2005). Addition of doxycycline (dox) releases Tet-R, allowing transcription. Several other suitable transcriptionally regulated promoters, transcription factors, and small molecule inducers are suitable for regulating transcription of viral structural genes.

[0230] In some embodiments, the third generation lentiviral components, human immunodeficiency virus type 1 (HIV) Rev, Gag / Pol, and envelope, under the control of a Tet regulated promoter and linked to an antibiotic resistance cassette, are separately integrated into the genome of a source cell, in some embodiments, the source cell has only one copy of each of the Rev, Gag / Pol, and envelope proteins integrated into its genome.

[0231] In some embodiments, a nucleic acid encoding an exogenous agent (e.g., a retroviral nucleic acid encoding an exogenous agent) is also integrated into the source cell genome. In some embodiments, the nucleic acid encoding the exogenous agent is maintained episomally. In some embodiments, the nucleic acid encoding the exogenous agent is transfected into the source cell with Rev, Gag / Pol, and envelope proteins stably integrated into the genome. See, e.g., Milani et al. EMBO Molecular Medicine, 2017, incorporated herein by reference in its entirety.

[0232] In some embodiments, the retroviral nucleic acid described herein is incapable of undergoing reverse transcription. Such nucleic acid, in embodiments, can transiently express an exogenous agent. The retrovirus or VLP may comprise a non-functional reverse transcriptase protein or may not comprise a reverse transcriptase protein. In embodiments, the retroviral nucleic acid comprises a non-functional primer binding site (PBS) and / or att site. In embodiments, one or more viral accessory genes, including rev, tat, vif, nef, vpr, vpu, vpx, and S2 or functional equivalents thereof, are non-functional or absent in the retroviral nucleic acid. In embodiments, one or more accessory genes selected from S2, rev, and tat are non-functional or absent in the retroviral nucleic acid.

[0233] Typically, modern retroviral vector systems include (1) a viral genome with cis-acting vector sequences for transcription, reverse transcription, integration, translation, and packaging of viral RNA into viral particles, and (2) a producer cell line expressing the trans-acting retroviral gene sequences (e.g., gag, pol, and env) necessary for the production of viral particles. Complete separation of the cis-acting and trans-acting vector sequences renders the virus unable to sustain replication for multiple cycles of infection. The generation of live virus can be avoided by several strategies, for example, by minimizing overlap between the cis-acting and trans-acting sequences to avoid recombination.

[0234] As described in Section III.A.2, virus-like particles (VLPs) that contain sequences that are free or deficient in viral RNA can be the result of removing or eliminating viral RNA from the sequence. Similar to the viral vector particles disclosed in Section III.A.1, VLPs contain an outer viral envelope made from a lipid bilayer of a host cell (i.e., a producer cell or a source cell) and at least one viral structural protein. In some embodiments, a viral structural protein refers to any viral protein or fragment thereof that contributes to the structure of the viral core or capsid.

[0235] Generally, for viral vector particles as described in section III.A.1, only expression of gag precursor protein mediates vector assembly and release. In some aspects, gag protein or fragments thereof have been shown to assemble into structures similar to the viral core. In one embodiment, this can be achieved by using endogenous packaging signal binding sites on gag. Alternatively, the endogenous packaging signal binding sites are on pol. In this embodiment, the delivered RNA contains the cognate packaging signal. In another embodiment, a heterologous binding domain (heterologous to gag) located on the delivered RNA and a cognate binding site located on gag or pol can be used to ensure packaging of the delivered RNA. The heterologous sequence can be non-viral or viral, in which case it can be derived from a different virus. VLPs can be used to deliver therapeutic RNA, in which case functional integrase and / or reverse transcriptase are not required. These VLPs can also be used to deliver therapeutic genes of interest, in which case pol is usually included.

[0236] In an embodiment, gag-pol is modified and packaging signal is replaced with corresponding packaging signal.In this embodiment, particle can package RNA with new packaging signal.The advantage of this approach is that it can package RNA sequence that lacks viral sequence, for example RNAi.

[0237] An alternative approach is to rely on overexpression of the RNA to be packaged.In one embodiment, the RNA to be packaged is overexpressed in the absence of the RNA that contains packaging signal.This can result in significant levels of therapeutic RNA being packaged, which is sufficient to transduce cells and produce biological effects.

[0238] In some embodiments, the polynucleotide comprises a nucleotide sequence encoding a viral gag protein or a retroviral gag and pol protein, wherein the gag protein or the pol protein comprises a heterologous RNA-binding domain capable of recognizing a corresponding sequence in an RNA sequence to facilitate packaging of the RNA sequence into a viral vector particle, In some embodiments, the heterologous RNA-binding domain comprises an RNA-binding domain derived from a bacteriophage coat protein, a Rev protein, a protein of the U1 small nuclear ribonucleoprotein particle, a Nova protein, a TF111A protein, a TIS11 protein, a trp RNA-binding attenuation protein (TRAP), or a pseudouridine synthase.

[0239] In some embodiments, the assembly of a viral-based vector vehicle particle (i.e., a VLP) is initiated by binding of the core protein to a unique encapsidation sequence (e.g., a UTR with a stem-loop structure) within the viral genome. In some embodiments, the interaction of the core with the encapsidation sequence promotes oligomerization.

[0240] In some embodiments, a source cell for VLP production contains one or more plasmids (i.e., packaging plasmids) encoding viral structural proteins (e.g., gag, pol) capable of packaging viral particles. In some embodiments, the sequences encoding at least two of the gag and pol precursors are on the same plasmid. In some embodiments, the sequences encoding the gag and pol precursors are on different plasmids. In some embodiments, the sequences encoding the gag and pol precursors have the same expression signal, e.g., promoter. In some embodiments, the sequences encoding the gag and pol precursors have different expression signals, e.g., different promoters. In some embodiments, expression of the gag and pol precursors is inducible.

[0241] In some embodiments, the formation of VLPs or any virus-based particle as described above in Section III can be detected by any suitable technique known in the art, including, for example, electron microscopy, dynamic light scattering, selective chromatographic separation and / or density gradient centrifugation.

[0242] B. Cell-Based Particles In some embodiments, the lipid particles are cell-based particles that include naturally occurring membranes. In some embodiments, the naturally occurring membranes include membrane vesicles prepared from cells or tissues. In some embodiments, the cell-based particles include vesicles that can be obtained from cells. In some embodiments, the cell-based particles include microvesicles, exosomes, membrane inclusions, apoptotic bodies (from apoptotic cells), particles (e.g., can be derived from platelets), ectosomes (e.g., can be derived from neutrophils and monocytes in serum), prostatosomes (can be obtained from prostate cancer cells), or cardiosomes (can be derived from cardiac cells).

[0243] In some embodiments, the source cells are endothelial cells, fibroblasts, blood cells (e.g., macrophages, neutrophils, granulocytes, leukocytes), stem cells (e.g., mesenchymal stem cells, umbilical cord stem cells, bone marrow stem cells, hematopoietic stem cells, induced pluripotent stem cells, e.g., induced pluripotent stem cells derived from a subject's cells), embryonic stem cells (e.g., stem cells from embryonic yolk sac, placenta, umbilical cord, fetal skin, adolescent skin, blood, bone marrow, adipose tissue, erythropoietic tissue, hematopoietic tissue), myoblasts, parenchymal cells (e.g., hepatocytes), alveolar cells. , neuronal (e.g., retinal neuronal) precursor cells (e.g., retinal precursor cells, myeloblasts, myeloid precursor cells, thymocytes, meiocytes, megakaryoblasts, promegakaryoblasts, melanoblasts, lymphoblasts, myeloid precursor cells, normoblasts, or hemangioblasts), progenitor cells (e.g., cardiac progenitor cells, satellite cells, radial glial cells, bone marrow stromal cells, pancreatic progenitor cells, endothelial progenitor cells, blast cells), or immortalized cells (e.g., HeEa, HEK293, HFF-1, MRC-5, WI-38, IMR 90, IMR 91, PER.C6, HT-1080, or BJ cells). In some embodiments, the source cells are other than 293 cells, HEK cells, human endothelial cells, or human epithelial cells, monocytes, macrophages, dendritic cells, or stem cells.

[0244] In some embodiments, the cell-based particles have a density of <1, 1-1.1, 1.05-1.15, 1.1-1.2, 1.15-1.25, 1.2-1.3, 1.25-1.35, or >1.35 g / ml. In some embodiments, the vector vehicle particle composition comprises less than 0.01%, 0.05%, 0.1%, 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 4%, 5%, or 10% of source cells based on protein mass, or less than 0.01%, 0.05%, 0.1%, 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 4%, 5%, or 10% of cells with functional nuclei.

[0245] In embodiments, the cell-based particles have a size that is less than about 0.01%, 0.05%, 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% of the size of the source cells, or the population of vector vehicle particles has an average size that is less than about 0.01%, 0.05%, 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% of the average size of the source cells.

[0246] In some embodiments, the cell-based particles are extracellular vesicles, e.g., cell-based vesicles that include a membrane that encloses an internal space and has a smaller diameter than the cell from which they originate. In embodiments, the extracellular vesicles have a diameter between 20 nm and 1000 nm. In embodiments, the cell-based particles are apoptotic bodies, cell fragments, vesicles obtained from cells by direct or indirect manipulation, vesiculated organelles, and vesicles produced by viable cells (e.g., by direct cell membrane budding or fusion of late endosomes with the cell membrane). In embodiments, the extracellular vesicles are derived from living or dead organisms, transplanted tissues or organs, or cultured cells.

[0247] In embodiments, the cell-based particles are nanovesicles, e.g., small (e.g., 20-250 nm in diameter, or 30-150 nm in diameter) cell-derived vesicles that contain a membrane enclosing an interior space and that are generated from the cell by direct or indirect manipulation. The production of nanovesicles may, in some instances, result in the destruction of the source cell. Nanovesicles may contain lipids or fatty acids and polypeptides.

[0248] In embodiments, the cell-based particle is an exosome. In embodiments, exosomes are small (e.g., 20-300 nm in diameter, or 40-200 nm in diameter) cell-derived vesicles that contain a membrane that encloses an internal space and is generated from the cell by direct cell membrane budding or by fusion of a late endosome with the cell membrane. In embodiments, the generation of exosomes does not result in the destruction of the source cell. In embodiments, exosomes contain lipids or fatty acids and polypeptides. Exemplary exosomes and other membrane inclusions are also described in WO / 2017 / 161010, WO / 2016 / 077639, US20160168572, US20150290343, and US20070298118, each of which is incorporated herein by reference in its entirety.

[0249] In some embodiments, the cell-based particles are microvesicles. In some embodiments, the microvesicles have a diameter of about 100 nm to about 2000 nm.

[0250] In some embodiments, the cell-based particle is a cell ghost. In some embodiments, the vesicle is a cell membrane vesicle, for example a giant cell membrane vesicle.

[0251] In some embodiments, the cell-based particles are derived from source cells that have genetic modifications that increase expression of immunomodulatory factors, such as immunosuppressive factors. In some embodiments, the immunosuppressive factors are on the outer surface of the cells. In some embodiments, the immunosuppressive factors are incorporated into the outer surface of the vector vehicle particles. In some embodiments, the vector vehicle particles include immunomodulatory factors bound to the surface of the solid particles by covalent or non-covalent bonds.

[0252] In some embodiments, the cell-based particles are generated by inducing budding of exosomes, microvesicles, membrane vesicles, extracellular membrane vesicles, plasma membrane vesicles, giant plasma membrane vesicles, apoptotic bodies, mitoparticles, pyrenocytes, lysosomes, or other membrane-enclosed vesicles.

[0253] In some embodiments, cell-based particles are generated by inducing cell enucleation. Enucleation can be performed using assays such as genetic, chemical (e.g., using actinomycin D, see Bayona-Bafaluy et al., "A chemical enucleation method for the transfer of mitochondrial DNA to ρ° cells" Nucleic Acids Res. 2003 Aug 15; 31(16): e98), mechanical (e.g., squeezing or aspiration, see Lee et al., "A comparative study on the efficiency of two enucleation methods in pig somatic cell nuclear transfer: effects of the squeezing and the aspiration methods" Anim Biotechnol. 2008; 19(2): 71-9), or a combination thereof.

[0254] In some embodiments, the cell-based particles are generated by inducing cell fragmentation. In some embodiments, cell fragmentation can be performed using the following methods, including but not limited to chemical methods, mechanical methods (e.g., centrifugation (e.g., ultracentrifugation, or density centrifugation), freeze-thawing, or sonication), or a combination thereof.

[0255] In some embodiments, the source cells used to generate the cell-based particles are not available for testing after the vector vehicle particles are generated.

[0256] In some embodiments, the characteristics of the cell-based particles are described by comparison to a reference cell. In some embodiments, the reference cell is a source cell. In some embodiments, the reference cell is a HeLa, HEK293, HFF-1, MRC-5, WI-38, IMR90, IMR91, PER.C6, HT-1080, or BJ cell. In some embodiments, the characteristics of the population of vector vehicle particles are described by comparison to a population of reference cells, for example, a population of source cells, or a population of HeLa, HEK293, HFF-1, MRC-5, WI-38, IMR 90, IMR 91, PER.C6, HT-1080, or BJ cell.

[0257] C. Exogenous agents In some embodiments, the lipid particles described herein or pharmaceutical compositions comprising same contain an exogenous agent. In some embodiments, the lipid particles described herein or pharmaceutical compositions comprising same contain a nucleic acid encoding an exogenous agent. In some embodiments, the lipid particles contain an exogenous agent. In some embodiments, the lipid particles contain a nucleic acid encoding an exogenous agent. Reference to the coding sequence of a nucleic acid encoding an exogenous agent is also referred to herein as a payload gene. In some embodiments, the exogenous agent or a nucleic acid encoding an exogenous agent is present in the lumen of the lipid particle.

[0258] In some embodiments, the exogenous agent is a protein or a nucleic acid (e.g., DNA, a chromosome (e.g., a human artificial chromosome), an RNA, e.g., an mRNA or an miRNA). In some embodiments, the exogenous agent is a protein. In some embodiments, the exogenous agent is a nucleic acid (e.g., DNA, a chromosome (e.g., a human artificial chromosome), an RNA, e.g., an mRNA or an miRNA). In some embodiments, the exogenous agent comprises or encodes a membrane protein. In some embodiments, the exogenous agent comprises or encodes a therapeutic agent. In some embodiments, the therapeutic agent is selected from one or more of a protein, e.g., an enzyme, a transmembrane protein, a receptor, or an antibody, a nucleic acid, e.g., DNA, a chromosome (e.g., a human artificial chromosome), an RNA, an mRNA, an siRNA, or an miRNA, or a small molecule.

[0259] In some embodiments, the lipid particle or pharmaceutical composition delivers at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% of the exogenous agent (e.g., an exogenous agent that includes or encodes a therapeutic agent) contained in the lipid particle to the target cell. In some embodiments, the lipid particle, e.g., fusosome, that contacts, e.g., fuses with the target cell delivers, on average, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% of the exogenous agent (e.g., an exogenous agent that includes or encodes a therapeutic agent) contained in the lipid particle, e.g., fusosome, that contacts, e.g., fuses with the target cell to the target cell. In some embodiments, the lipid particle composition delivers at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% of an exogenous agent included in the lipid particle composition (e.g., an exogenous agent that includes or encodes a therapeutic agent) to the target tissue.

[0260] In some embodiments, the exogenous agent is not naturally expressed in the cell from which the lipid particle is derived. In some embodiments, the exogenous agent is naturally expressed in the cell from which the lipid particle is derived. In some embodiments, the exogenous agent is loaded into the lipid particle via expression in the cell from which the lipid particle is derived (e.g., expression from DNA or mRNA introduced via transfection, transduction, or electroporation). In some embodiments, the exogenous agent is expressed from DNA integrated into the genome or maintained episomally. In some embodiments, the expression of the exogenous agent is constitutive. In some embodiments, the expression of the exogenous agent is induced. In some embodiments, the expression of the exogenous agent is induced immediately before generating the lipid particle. In some embodiments, the expression of the exogenous agent is induced simultaneously with the expression of the fusogen.

[0261] In some embodiments, the exogenous agent is loaded into the lipid particles via electroporation into the lipid particles themselves or into the cells from which the lipid particles are derived. In some embodiments, the exogenous agent is loaded into the lipid particles via transfection (e.g., of DNA or mRNA encoding the exogenous agent) into the lipid particles themselves or into the cells from which the lipid particles are derived.

[0262] In some embodiments, the exogenous agent may include one or more nucleic acid sequences, one or more polypeptides, a combination of nucleic acid sequences and / or polypeptides, one or more organelles, and any combination thereof. In some embodiments, the exogenous agent may include one or more cellular components. In some embodiments, the exogenous agent may include one or more cytoplasmic and / or nuclear components.

[0263] In some embodiments, the lipid particle contains an exogenous factor that is a nucleic acid or contains a nucleic acid that encodes an exogenous agent. In some embodiments, the nucleic acid is operably linked to a "positive target cell specific regulatory element" (or positive TCSRE). In some embodiments, the positive TCSRE is a functional nucleic acid sequence. In some embodiments, the positive TCSRE comprises a promoter or enhancer. In some embodiments, the TCSRE is a nucleic acid sequence that increases the level of the exogenous agent in the target cell. In some embodiments, the positive target cell specific regulatory element comprises a T cell specific promoter, a T cell specific enhancer, a T cell specific splice site, a T cell specific site that extends the half-life of an RNA or protein, a T cell specific mRNA export promoting site, a T cell specific translation enhancing site, or a T cell specific post-translational modification site. In some embodiments, the T cell specific promoter is a promoter described in the Immgen consortium, which is incorporated herein by reference in its entirety, for example, the T cell specific promoter is an IL2RA (CD25), LRRC32, FOXP3, or IKZF2 promoter. In some embodiments, the T cell specific promoter or enhancer is a promoter or enhancer described in Schmidl et a, Blood. 2014 Apr 24; 123(17): e68-78., which is incorporated herein by reference in its entirety. In some embodiments, the T cell specific promoter is a transcriptionally active fragment of any of the foregoing. In some embodiments, the T cell specific promoter is a variant having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to any of the foregoing.

[0264] In some embodiments, the lipid particle contains an exogenous agent that is a nucleic acid or contains a nucleic acid that encodes an exogenous agent. In some embodiments, the nucleic acid is operably linked to a "negative target cell specific regulatory element" (or negative TCSRE). In some embodiments, the negative TCSRE is a functional nucleic acid sequence. In some embodiments, the negative TCSRE is a miRNA recognition site that causes inhibition degradation of the lipid particle in non-target cells. In some embodiments, the exogenous agent is operably linked to a "non-target cell specific regulatory element" (i.e., NTCSRE). In some embodiments, the NTCSRE comprises a nucleic acid sequence that reduces the level of the exogenous agent in non-target cells compared to in target cells. In some embodiments, the NTCSRE comprises a non-target cell specific miRNA recognition sequence, a non-target cell specific protease recognition site, a non-target cell specific ubiquitin ligase site, a non-target cell specific transcriptional repression site, or a non-target cell specific epigenetic repression site. In some embodiments, the NTCSRE comprises a tissue-specific miRNA recognition sequence, a tissue-specific protease recognition site, a tissue-specific ubiquitin ligase site, a tissue-specific transcriptional repression site, or a tissue-specific epigenetic repression site. In some embodiments, the NTCSRE comprises a non-target cell-specific miRNA recognition sequence, a non-target cell-specific protease recognition site, a non-target cell-specific ubiquitin ligase site, a non-target cell-specific transcriptional repression site, or a non-target cell-specific epigenetic repression site. In some embodiments, the NTCSRE comprises a non-target cell-specific miRNA recognition sequence, to which one or more of miR31, miR363, or miR29c can bind. In some embodiments, the NTCSRE is located or encoded within a transcribed region that encodes an exogenous agent, and optionally, the RNA produced by the transcribed region comprises a miRNA recognition sequence within a UTR or coding region.

[0265] 1. Nucleic acids In some embodiments, the exogenous agent may include nucleic acid. For example, the exogenous agent may include RNA for improving the expression of endogenous protein, or siRNA or miRNA for inhibiting the protein expression of endogenous protein. For example, endogenous protein may regulate the structure or function in target cell. In some embodiments, the exogenous agent may include nucleic acid that encodes a modified protein that regulates the structure or function in target cell. In some embodiments, the exogenous agent is a nucleic acid that targets a transcription activator that regulates the structure or function in target cell.

[0266] In some embodiments, the lipid particles described herein comprise nucleic acid, such as RNA or DNA. In some embodiments, the nucleic acid is, comprises, or consists of one or more natural nucleic acid residues. In some embodiments, the nucleic acid is, comprises, or consists of one or more nucleic acid analogs. In some embodiments, the nucleic acid has a nucleotide sequence that encodes a functional gene product, such as RNA or protein. In some embodiments, the nucleic acid comprises one or more introns. In some embodiments, the nucleic acid is prepared by one or more of isolation from a natural source, enzymatic synthesis by polymerization based on a complementary template (in vivo or in vitro), replication in a recombinant cell or system, and chemical synthesis. In some embodiments, the nucleic acid is at least 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 20, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000 residues in length, or more. In some embodiments, the nucleic acid is partially or entirely single-stranded, and in some embodiments, the nucleic acid is partially or entirely double-stranded. In some embodiments, the nucleic acid has a nucleotide sequence that includes at least one element that encodes a polypeptide or is the complement of a sequence that encodes a polypeptide. The nucleic acid may include variants that have, for example, at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 99% overall sequence identity with the reference nucleic acid. In some embodiments, the variant nucleic acid does not share at least one characteristic sequence element with the reference nucleic acid. In some embodiments, the variant nucleic acid shares one or more of the biological activities of the reference nucleic acid. In some embodiments, the nucleic acid variant has a nucleic acid sequence identical to the reference nucleic acid sequence except for a small number of sequence modifications at certain positions.In some embodiments, less than about 20%, about 15%, about 10%, about 9%, about 8%, about 7%, about 6%, about 5%, about 4%, about 3%, or about 2% of the residues in the variant are substituted, inserted, or deleted compared to the reference. In some embodiments, the variant nucleic acid comprises about 10, about 9, about 8, about 7, about 6, about 5, about 4, about 3, about 2, or about 1 substituted residues compared to the reference. In some embodiments, the variant nucleic acid comprises a very small number (e.g., less than about 5, about 4, about 3, about 2, or about 1) of substituted, inserted, or deleted functional residues involved in a particular biological activity compared to the reference. In some embodiments, the variant nucleic acid comprises no more than about 15, about 12, about 9, about 3, or about 1 additions or deletions compared to the reference, and in some embodiments no additions or deletions. In some embodiments, the variant nucleic acid contains less than about 27, about 24, about 21, about 18, about 15, about 12, about 9, about 6, about 3, or less than about 9, about 6, about 3, or about 2 additions or deletions compared to the reference.

[0267] In some embodiments, the exogenous agent may be a nucleic acid, e.g., DNA, nDNA (nuclear DNA), mtDNA (mitochondrial DNA), protein-coding DNA, gene, operon, chromosome, genome, transposon, retrotransposon, viral genome, intron, exon, modified DNA, mRNA (messenger RNA), tRNA (transfer RNA), modified RNA, microRNA, siRNA (small interfering RNA), tmRNA (transfer messenger RNA), rRNA (ribosomal RNA), mtRNA (mitochondrial RNA), snRNA (small nuclear RNA), small nucleolar RNA (snoRNA), SmY RNA (mRNA trans-splicing RNA), gRNA (guide RNA), TERC (telomerase RNA component), aRNA (antisense RNA), cis-NAT (cis-natural antisense transcript), CRISPR. Examples of such nucleic acids include RNA (crRNA), IncRNA (long non-coding RNA), piRNA (piwi interacting RNA), shRNA (short hairpin RNA), tasiRNA (trans-acting siRNA), eRNA (enhancer RNA), satellite RNA, pcRNA (protein coding RNA), dsRNA (double stranded RNA), RNAi (interfering RNA), circRNA (circular RNA), reprogramming RNA, aptamers, and any combination thereof. In some embodiments, the nucleic acid is a wild type nucleic acid. In some embodiments, the protein is a mutant nucleic acid. In some embodiments, the nucleic acid is a fusion or chimera of multiple nucleic acid sequences.

[0268] In embodiments, the nucleic acid encodes one or more (e.g., two or more) inhibitory RNA molecules directed against one or more RNA targets. The inhibitory RNA molecules can be, for example, miRNAs or shRNAs. In some embodiments, the inhibitory molecules can be precursors of miRNAs, such as Pri-miRNAs or Pre-miRNAs, or precursors of shRNAs. In some embodiments, the inhibitory molecules can be artificially derived miRNAs or shRNAs. In other embodiments, the inhibitory RNA molecules can be dsRNAs (transcribed or artificially introduced) that are processed into siRNAs, or siRNAs themselves. In some embodiments, the inhibitory RNA molecules can be miRNAs or shRNAs that have a sequence not found in nature, have at least one functional segment not found in nature, or have a combination of functional segments not found in nature. In exemplary embodiments, at least one or all of the inhibitory RNA molecules are miR-l55. In some embodiments, the retroviral vectors described herein encode two or more inhibitory RNA molecules directed against one or more RNA targets. In some embodiments, the two or more inhibitory RNA molecules can be directed against different targets. In other embodiments, two or more inhibitory RNA molecules are directed against the same target. In some embodiments, the exogenous agent comprises an shRNA. An shRNA (short hairpin RNA) may comprise a double-stranded structure formed by a single self-complementary RNA strand. The shRNA construct may comprise a nucleotide sequence identical to any portion of the coding or non-coding sequence of the target gene. RNA sequences containing insertions, deletions, and single point mutations to the target sequence may also be used. More than 90% sequence identity, or even 100% sequence identity between the inhibitory RNA and the portion of the target gene may be used. In certain embodiments, the length of the duplex-forming portion of the shRNA is at least 20, 21, or 22 nucleotides long, which corresponds to the size of the RNA product generated, for example, by Dicer-dependent cleavage. In certain embodiments, the shRNA construct is at least 25, 50, 100, 200, 300, or 400 bases long.In certain embodiments, the shRNA construct is 400-800 bases long. The shRNA construct is highly tolerant to variations in loop sequence and loop size. In embodiments, the retroviral vector encoding the siRNA, miRNA, shRNA, or ribozyme includes one or more regulatory sequences, such as a strong constitutive pol III, e.g., human U6 snRNA promoter, mouse U6 snRNA promoter, human and mouse Hl RNA promoters, and human tRNA-val promoter, or a strong constitutive pol II promoter.

[0269] 2. Polypeptides In some embodiments, the lipid particle contains a nucleic acid encoding a protein exogenous agent (also referred to as an "exogenous agent-encoding payload gene"). In some embodiments, the lipid particle described herein comprises an exogenous agent that is or comprises a protein.

[0270] In some embodiments, a protein may include moieties other than amino acids (e.g., may be a glycoprotein, proteoglycan, etc.) and / or may be processed or modified. In some embodiments, a protein may include multiple polypeptide chains, for example, linked by one or more disulfide bonds or associated by other means.

[0271] In some embodiments, the protein may include L-amino acids, D-amino acids, or both, and may include any of a variety of amino acid modifications or analogs. In some embodiments, the protein may include natural amino acids, unnatural amino acids, synthetic amino acids, and combinations thereof. In some embodiments, the protein is an antibody, an antibody fragment, a biologically active portion thereof, and / or a characteristic portion thereof. In some embodiments, the polypeptide may include variants thereof having, for example, at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 99% overall sequence identity with the reference polypeptide. In some embodiments, the variant polypeptide does not share at least one characteristic sequence element with the reference polypeptide. In some embodiments, the variant polypeptide shares one or more of the biological activities of the reference polypeptide. In some embodiments, the polypeptide variant has an amino acid sequence identical to the reference amino acid sequence except for a small number of sequence modifications at certain positions. In some embodiments, less than about 20%, about 15%, about 10%, about 9%, about 8%, about 7%, about 6%, about 5%, about 4%, about 3%, or about 2% of the residues in the variant are substituted, inserted, or deleted compared to the reference. In some embodiments, the variant polypeptide comprises about 10, about 9, about 8, about 7, about 6, about 5, about 4, about 3, about 2, or about 1 substituted residues compared to the reference. In some embodiments, the variant polypeptide comprises a very small number (e.g., less than about 5, about 4, about 3, about 2, or about 1) of substitutions, insertions, or deletions that are responsible for a particular biological activity compared to the reference. In some embodiments, the variant polypeptide comprises no more than about 5, about 4, about 3, about 2, or about 1 additions or deletions compared to the reference, and in some embodiments no additions or deletions. In some embodiments, the variant polypeptide contains fewer than about 25, about 20, about 19, about 18, about 17, about 16, about 15, about 14, about 13, about 10, about 9, about 8, about 7, about 6, typically fewer than about 5, about 4, about 3, or about 2 additions or deletions compared to the reference.

[0272] In some embodiments, proteins include polypeptides, such as enzymes, structural polypeptides, signaling polypeptides, regulatory polypeptides, transport polypeptides, sensory polypeptides, motor polypeptides, defensive polypeptides, conservation polypeptides, transcription factors, antibodies, cytokines, hormones, catabolic polypeptides, anabolic polypeptides, proteolytic polypeptides, metabolic polypeptides, kinases, transferases, hydrolases, lyases, isomerases, ligases, enzyme regulator polypeptides, protein-binding polypeptides, lipid-binding polypeptides, membrane fusion polypeptides, cell differentiation polypeptides, epigenetic polypeptides, cell death polypeptides, nuclear transport polypeptides, nucleic acid binding polypeptides, reprogramming polypeptides, DNA editing polypeptides, DNA repair polypeptides, DNA recombination polypeptides, transposase polypeptides, DNA integration polypeptides, targeting endonucleases (e.g., zinc finger nucleases, transcription activator-like nucleases (TALENs), cas9 and its homologs), recombinases, transposases, DNA polymerases, RNA polymerases, reverse transcriptases, and any combination thereof.

[0273] In some embodiments, the protein targets a protein within a cell for degradation. In some embodiments, the protein targets a protein within a cell for degradation by localizing the protein to the proteasome. In some embodiments, the protein is a wild-type protein. In some embodiments, the protein is a mutant protein.

[0274] Exemplary protein exogenous agents are described in the following subsections. In some embodiments, the lipid particles provided herein can include any of such exogenous agents. In certain embodiments, the lipid particles contain a nucleic acid encoding any of such exogenous agents.

[0275] A. Cytoplasmic proteins In some embodiments, the exogenous agent comprises a cytoplasmic protein, e.g., a protein that is produced in the recipient cell and localized in the recipient's cytoplasm. In some embodiments, the exogenous agent comprises a secreted protein, e.g., a protein that is produced and secreted by the recipient cell. In some embodiments, the exogenous agent comprises a nuclear protein, e.g., a protein that is produced in the recipient cell and imported into the nucleus of the recipient cell. In some embodiments, the exogenous agent comprises an organelle protein (e.g., a mitochondrial protein), e.g., a protein that is produced in the recipient cell and imported into an organelle (e.g., a mitochondria) of the recipient cell. In some embodiments, the protein is a wild-type protein or a mutant protein. In some embodiments, the protein is a fusion protein or a chimeric protein.

[0276] b. Membrane proteins In some embodiments, the exogenous agent comprises a membrane protein. In some embodiments, the membrane protein comprises a chimeric antigen receptor (CAR), a T cell receptor, an integrin, an ion channel, a pore-forming protein, a Toll-like receptor, an interleukin receptor, a cell adhesion protein, or a transport protein.

[0277] 1) Chimeric antigen receptor (CAR) In some embodiments, the payload gene described herein encodes a chimeric antigen receptor (CAR) comprising an antigen binding domain. In some embodiments, the exogenous agent described herein comprises a chimeric antigen receptor (CAR) comprising an antigen binding domain. In some embodiments, the payload comprises or comprises a chimeric antigen receptor (CAR) comprising an antigen binding domain. In some embodiments, the CAR is or comprises a first generation CAR comprising an antigen binding domain, a transmembrane domain, and a signaling domain (e.g., one, two, or three signaling domains). In some embodiments, the CAR comprises a third generation CAR comprising an antigen binding domain, a transmembrane domain, and at least three signaling domains. In some embodiments, a fourth generation CAR comprises an antigen binding domain, a transmembrane domain, three or four signaling domains, and a domain that induces expression of a cytokine gene upon successful signaling of the CAR. In some embodiments, the antigen binding domain is or comprises an scFv or Fab.

[0278] In some embodiments, the antigen binding domain targets an antigen characteristic of a cell type, hi some embodiments, the antigen binding domain targets an antigen characteristic of a neoplastic cell. In some embodiments, the antigen specific to the neoplastic cell is a cell surface receptor, an ion channel-linked receptor, an enzyme-linked receptor, a G protein-coupled receptor, a receptor tyrosine kinase, a tyrosine kinase-associated receptor, a receptor-like tyrosine phosphatase, a receptor serine / threonine kinase, a receptor guanylyl cyclase, a histidine kinase-associated receptor, an epidermal growth factor receptor (EGFR) (including ErbB1 / EGFR, ErbB2 / HER2, ErbB3 / HER3, and ErbB4 / HER4), a fibroblast growth factor receptor (FGFR) (including FGF1, FGF2, FGF3, FGF4, FGF5, FGF6, FGF7, FGF18, and FGF21), a vascular endothelial growth factor receptor (VEGFR) (including VEGF-A, VEGF-B, VEGF-C, VEGF -D, and PIGF), RET receptors and the Eph receptor family (including EphA1, EphA2, EphA3, EphA4, EphA5, EphA6, EphA7, EphA8, EphA9, EphA10, EphB1, EphB2, EphB3, EphB4, and EphB6), CXCR1, CXCR2, CXCR3, CXCR4, CXCR6, CCR1, CCR2, CCR3, CCR4, C ... CR3, CCR4, CCR5, CCR6, CCR8, CFTR, CIC-1, CIC-2, CIC-4, CIC-5, CIC-7, CIC-Ka, CIC-Kb, bestrophin, TMEM16A, GABA receptor, glycine receptor, ABC transporter, NAV1.1, NAV1.2, NAV1.3, NAV1.4, NAV1.5, NAV1.6, NAV1.7, NAV1.8, NAV1.9, sphingosine-1-phosphate receptor (S1P1R), NMDA channel, transmembrane protein, multispanning transmembrane protein, T cell receptor motif; T cell α chain; T cell β chain; T cell γ chain; T cell δ chain; CCR7; CD3; CD4; CD5; CD7; CD8; CD11b; CD11c; CD16; CD19; CD20; CD21; CD22; CD25; CD28; CD34; CD35; CD40; CD45RA; CD45RO; CD52; CD56; CD62L;CD68;CD80;CD95;CD117;CD127;CD133;CD137(4-1BB);CD163;F4 / 80;IL-4Ra;Sca-1;CTLA-4;GITR;GARP;LAP;Granzyme B;LFA-1;Transferrin receptor;NKp46, perforin, CD4+;Th1;Th2;Th17;Th40;Th22;Th9;Tfh, canonical Treg, FoxP3+;Tr1;Th3;Treg17;T. RE G;CDCP1, NT5E, EpCAM, CEA, gpA33, mucin, TAG-72, carbonic anhydrase IX, PSMA, folate binding protein, gangliosides (e.g., CD2, CD3, GM2), Lewis-gamma 2, VEGF, VEGFR1 / 2 / 3, αVβ3, α5β1, ErbB1 / EGFR, ErbB1 / HER2, ErB3, c-MET, IGF1R, EphA3, TRAIL-R1, TRAIL-R2, RANKL, FAP, tenascin, P DL-1, BAFF, HDAC, ABL, FLT3, KIT, MET, RET, IL-1β, ALK, RANKL, mTOR, CTLA-4, IL-6, IL-6R, JAK3, BRAF, PTCH, Smoothened, PIGF, ANPEP , TIMP1, PLAUR, PTPRJ, LTBR, ​​or ANTXR1, folate receptor α (FRa), ERBB2 (Her2 / neu), EphA2, IL-13Ra2, epidermal growth factor receptor (EGFR), mesothelin, TSHR, CD19, CD123, CD22, CD30, CD171, CS-1, CLL-1, CD33, EGFRvIII, GD2, GD3, BCMA, MUC16 (CA125), L1CAM, LeY, MSLN, IL13Rα1, L1-CAM, Tn Ag, prostate-specific membrane antigen (PSMA), ROR1, FLT3, FAP, TAG72, CD38, CD44v6, CEA, EPCAM, B7H3, KIT, interleukin-11 receptor a (IL-11Ra), PSCA, PRSS21, VEGFR2, Lewis Y, CD24, platelet-derived growth factor receptor-β (PDGFR-β), SSEA-4, CD20, MUC1, NCAM, prostase, PAP, ELF2M, ephrin B2, IGF-1 receptor, CAIX, LMP2, gplOO, bcr-a bl, tyrosinase, fucosyl GM1, sLe, GM3, TGS5, HMWMAA, o-acetyl-GD2, folate receptor β, TEM1 / CD248, TEM7R, CLDN6, GPRC5D, CXORF61, CD97, CD179a, ALK, polysialic acid, PLACl, GloboH, NY-BR-1, UPK2, HAVCR1, ADRB3, PANX3, GPR20, LY6K, OR51E2, TARP, WT1, NY-ESO-1, LAGE-la, MAGE-A1, legumain, HPVE6, E7, ETV6-AML, sperm protein 17, XAGE1, Tie2, MAD-CT-1, MAD-CT-2, major histocompatibility complex class I-related gene protein (MR1), urokinase-type plasminogen activator receptor (uPAR), Fos-related antigen 1, p53, p53 mutant, prostein, survivin, telomerase, PCTA-1 / galectin 8, MelanA / MART1, Ras mutant, hTERT, sarcoma translocation breakpoint, ML-IAP, ERG (TMPRSS2 ETS fusion gene), NA17, PAX3, androgen receptor, cyclin B1, MYCN, RhoC, TRP-2, CYPIB I, BORIS, SART3, PAX5, OY-TES1, LCK, AKAP-4, SSX2, RAGE-1, human telomerase reverse transcriptase, RU1, RU2, intestinal carboxylesterase, mut The antigens are selected from hsp70-2, CD79a, CD79b, CD72, LAIR1, FCAR, LILRA2, CD300LF, CLEC12A, BST2, EMR2, LY75, GPC3, FCRL5, IGLL1, neoantigens, CD133, CD15, CD184, CD24, CD56, CD26, CD29, CD44, HLA-A, HLA-B, HLA-C (HLA-A,B,C), CD49f, CD151, CD340, CD200, tkrA, trkB, or trkC, or antigen fragments or portions thereof.

[0279] In some embodiments, the antigen binding domain targets an antigen characteristic of T cells, hi some embodiments, the antigen characteristic of T cells is selected from a cell surface receptor, a membrane transport protein (e.g., an active or passive transport protein, e.g., an ion channel protein, a pore forming protein, etc.), a transmembrane receptor, a membrane enzyme, and / or a cell adhesion protein characteristic of T cells.In some embodiments, the antigen characteristic of a T cell is selected from the group consisting of G protein-coupled receptors, receptor tyrosine kinases, tyrosine kinase-associated receptors, receptor-like tyrosine phosphatases, receptor serine / threonine kinases, receptor guanylate cyclases, histidine kinase-associated receptors, AKT1, AKT2, AKT3, ATF2, BCL10, CALM1, CD3D (CD3δ), CD3E (CD3ε), CD3G (CD3γ), CD4, CD8, CD28, CD45, CD80 (B7-1), CD86(B7-2), CD247(CD3ζ), CTLA4(CD152), ELK1, ERK1(MAPK3), ERK2, FOS, FYN, GRAP2(GADS), GRB2, HLA-DRA, HLA-DRB1, HLA-DRB3, H LA-DRB4, HLA-DRB5, HRAS, IKBKA(CHUK), IKBKB, IKBKE, IKBKG(NEMO), IL2, ITPR1, ITK, JUN, KRAS2, LAT, LCK, MAP2K1(MEK1), MAP2K2( MEK2), MAP2K3(MKK3), MAP2K4(MKK4), MAP2K6(MKK6), MAP2K7(MKK7), MAP3K1(MEKK1), MAP3K3, MAP3K4, MAP3K5, MAP3K8, MAP3K14(N IK), MAPK8 (JNK1), MAPK9 (JNK2), MAPK10 (JNK3), MAPK11 (p38β), MAPK12 (p38γ), MAPK13 (p38δ), MAPK14 (p38α), NCK, NFAT1, NFAT2, N It can be FKB1, NFKB2, NFKBIA, NRAS, PAK1, PAK2, PAK3, PAK4, PIK3C2B, PIK3C3 (VPS34), PIK3CA, PIK3CB, PIK3CD, PIK3R1, PKCA, PKCB, PKCM, PKCQ, PLCY1, PRF1 (perforin), PTEN, RAC1, RAF1, RELA, SDF1, SHP2, SLP76, SOS, SRC, TBK1, TCRA, TEC, TRAF6, VAV1, VAV2, or ZAP70.

[0280] In some embodiments, the antigen binding domain targets an antigen characteristic of a disorder. In some embodiments, the antigen binding domain targets an antigen characteristic of an autoimmune disorder or an inflammatory disorder. In some embodiments, the autoimmune disorder or an inflammatory disorder is selected from the group consisting of chronic graft-versus-host disease (GVHD), lupus, arthritis, immune complex glomerulonephritis, Goodpasture's disease, uveitis, hepatitis, systemic sclerosis or scleroderma, type I diabetes, multiple sclerosis, cold agglutinin disease, pemphigus vulgaris, Graves' disease, autoimmune hemolytic anemia, hemophilia A, primary Sjogren's syndrome, thrombotic thrombocytopenic purpura, neuromyelitis optica, Evans' syndrome, IgM-mediated neuropathy, and the like. thrombocytopenia, cryoglobulinemia, dermatomyositis, idiopathic thrombocytopenia, ankylosing spondylitis, bullous pemphigoid, acquired angioedema, chronic urticaria, antiphospholipid demyelinating polyneuropathy, and autoimmune thrombocytopenia or neutropenia or pure red cell aplasia, while non-limiting examples of exemplary alloimmune diseases include sensitization to foreign antigens such as may occur with hematopoietic or solid organ transplants, allosensitization (see, e.g., Blazar et al., 2015, Am. J. Transplant, 15(4):931-41) or xenosensitization due to blood transfusions, pregnancy with fetal allosensitization, alloimmune thrombocytopenia of the newborn, hemolytic disease of the newborn, enzyme or protein replacement therapy, blood products, and replacement of inherited or acquired deficiency disorders treated with gene therapy. In some embodiments, the antigen characteristic of an autoimmune or inflammatory disorder is selected from a cell surface receptor, an ion channel-linked receptor, an enzyme-linked receptor, a G protein-coupled receptor, a receptor tyrosine kinase, a tyrosine kinase-associated receptor, a receptor-like tyrosine phosphatase, a receptor serine / threonine kinase, a receptor guanylyl cyclase, or a histidine kinase-associated receptor.In some embodiments, the antigen binding domain of the CAR binds to ligands expressed on B cells, plasma cells, plasmablasts, CD10, CD19, CD20, CD22, CD24, CD27, CD38, CD45R, CD138, CD319, BCMA, CD28, TNF, interferon receptor, GM-CSF, ZAP-70, LFA-1, CD3γ, CD5, or CD2. See US2003 / 0077249, WO2017 / 058753, WO2017 / 058850, the contents of which are incorporated herein by reference.

[0281] In some embodiments, the antigen binding domain targets an antigen characteristic of senescent cells, such as urokinase-type plasminogen activator receptor (uPAR). In some embodiments, the CARs may be used to treat or prevent disorders characterized by the abnormal accumulation of senescent cells, such as liver and lung fibrosis, atherosclerosis, diabetes, and osteoarthritis.

[0282] In some embodiments, the antigen binding domain targets an antigen characteristic of an infectious disease, in some embodiments, the infectious disease is selected from HIV, Hepatitis B virus, Hepatitis C virus, human herpes virus, human herpes virus 8 (HHV-8, Kaposi's sarcoma associated herpes virus (KSHV)), human T-lymphotropic virus-1 (HTLV-1), Merkel cell polyoma virus (MCV), Simian virus 40 (SV40), Epstein-Barr virus, CMV, human papilloma virus. In some embodiments, the infectious disease specific antigen is selected from a cell surface receptor, an ion channel-linked receptor, an enzyme-linked receptor, a G protein-coupled receptor, a receptor tyrosine kinase, a tyrosine kinase-associated receptor, a receptor-like tyrosine phosphatase, a receptor serine / threonine kinase, a receptor guanylyl cyclase, a histidine kinase-associated receptor, an HIV Env, gpl20, or a CD4-inducible epitope on HIV-1 Env.

[0283] In some embodiments, the transmembrane domain of the CAR comprises at least the transmembrane region of the alpha, beta, or zeta chain of the T cell receptor, CD28, CD3e, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, or a functional variant thereof. In some embodiments, the transmembrane domain comprises at least the transmembrane region of CD8α, CD8β, 4-1BB / CD137, CD28, CD34, CD4, FcεRIγ, CD16, OX40 / CD134, CD3ζ, CD3ε, CD3γ, CD3δ, TCRα, TCRβ, TCRζ, CD32, CD64, CD64, CD45, CD5, CD9, CD22, CD37, CD80, CD86, CD40, CD40L / CD154, VEGFR2, FAS, and FGFR2B, or a functional variant thereof.

[0284] These CARs are B7-1 / CD80, B7-2 / CD86, B7-H1 / PD-L1, and B7-H 2. B7-H3, B7-H4, B7-H6, B7-H7, BTLA / CD272, CD28, CTLA-4, Gi24 / VISTA / B7-H5、ICOS / CD278、PD-1、PD-L2 / B7-DC、PDCD6)、4-1BB / TNFSF9 / CD137、4-1BBッガド / TNFSF9、BAFF / BLyS / TNFSF13B BAFF R / TNFRSF13C, CD27 / TNFRSF7, CD27 factor / TNFSF7, CD30 / TNFRSF8, CD30 factor / TNFSF8. CD40 / TNFRSF5, CD40 / TNFSF5, CD40, DR3 / TNFRSF25, GITR / TNFRSF18, GI TR factor / TNFSF18, HVEM / TNFRSF14, LIGHT / TNFSF14, glycoprotein-α / TNF-β, OX40 / TNFRSF 4. OX40リンド / TNFSF4, RELT / TNFRSF19L, TACI / TNFRSF13B, TL1A / TNFSF15, TNF-α, TNF RII / TNFRSF1B) 2B4 / CD244 / SLAMF4 BLAME / SLAMF8 CD2 CD2F-10 / SLAMF9 CD48 / SLAMF2 CD58 / LFA-3 CD84 / SLAMF5 CD229 / SLAM F3, CRACC / SLAMF7, NTB-A / SLAMF6, SLAM / CD150), CD2, CD7, CD53, CD82 / Kai-1, CD90 / Thy1, CD96, CD160, CD200, CD300a / LMIR1, HLA Class I, HLA-DR, Ikaros, Linkα4 / CD49d 4β1, ligandα4β7 / LPAM-1, LAG-3, TCL1A, TCL1B CRTAM, DAP12, CC-1 / CLEC7A, DPPIV / CD26, EphB6, TIM-1 / KIM-1 / HAVCR, TIM-4, TSLP, TSLPand at least one signaling domain selected from one or more of R, lymphocyte function-associated antigen-1 (LFA-1), NKG2C, CD3 zeta domain, immunoreceptor tyrosine-based activation motif (ITAM), CD27, CD28, 4-1BB, CD134 / OX40, CD30, CD40, PD-1, ICOS, a ligand that specifically binds lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, CD83, or functional fragments thereof.

[0285] In some embodiments, the CAR comprises a CD3 zeta domain or an immunoreceptor tyrosine-based activation motif (ITAM), or a functional variant thereof. In some embodiments, the CAR comprises (i) a CD3 zeta domain or an immunoreceptor tyrosine-based activation motif (ITAM), or a functional variant thereof, and (ii) a CD28 domain or a 4-1BB domain, or a functional variant thereof. In some embodiments, the CAR comprises (i) a CD3 zeta domain or an immunoreceptor tyrosine-based activation motif (ITAM), or a functional variant thereof, (ii) a CD28 domain, or a functional variant thereof, and (iii) a 4-1BB domain, or a CD134 domain, or a functional variant thereof. In some embodiments, the CAR comprises (i) a CD3 zeta domain or an immunoreceptor tyrosine-based activation motif (ITAM), or a functional variant thereof, (ii) a CD28 domain, or a functional variant thereof, and / or (iii) a 4-1BB domain, or a CD134 domain, or a functional variant thereof. In some embodiments, the CAR comprises (i) a CD3 zeta domain, or an immunoreceptor tyrosine-based activation motif (ITAM), or a functional variant thereof, (ii) a CD28 domain, or a functional variant thereof, (iii) a 4-1BB domain, or a CD134 domain, or a functional variant thereof, and (iv) a cytokine or costimulatory ligand transgene.

[0286] In some embodiments, the CAR further comprises one or more spacers, for example, the spacer is a first spacer between the antigen-binding domain and the transmembrane domain. In some embodiments, the first spacer comprises at least a portion of an immunoglobulin constant region or a variant or modified version thereof. In some embodiments, the spacer is a second spacer between the transmembrane domain and the signaling domain. In some embodiments, the second spacer is an oligopeptide, for example, the oligopeptide comprises a glycine-serine doublet.

[0287] In some embodiments, the exogenous agent is or comprises a CAR, e.g., a first generation CAR or a nucleic acid encoding a first generation CAR. In some embodiments, the first generation CAR comprises an antigen binding domain, a transmembrane domain, and a signaling domain. In some embodiments, the signaling domain mediates downstream signaling during T cell activation.

[0288] In some embodiments, the exogenous agent is or comprises a second generation CAR or a nucleic acid encoding a second generation CAR. In some embodiments, the second generation CAR comprises an antigen binding domain, a transmembrane domain, and two signaling domains. In some embodiments, the signaling domain mediates downstream signaling during T cell activation. In some embodiments, the signaling domain is a costimulatory domain. In some embodiments, the costimulatory domain enhances cytokine production, CAR T cell proliferation, and / or CAR T cell persistence during T cell activation.

[0289] In some embodiments, the exogenous agent is or comprises a third generation CAR or a nucleic acid encoding a third generation CAR. In some embodiments, the third generation CAR comprises an antigen binding domain, a transmembrane domain, and at least three signaling domains. In some embodiments, the signaling domain mediates downstream signaling during T cell activation. In some embodiments, the signaling domain is a costimulatory domain. In some embodiments, the costimulatory domain enhances cytokine production, CAR T cell proliferation, and / or CAR T cell persistence during T cell activation. In some embodiments, the third generation CAR comprises at least two costimulatory domains. In some embodiments, the at least two costimulatory domains are not the same.

[0290] In some embodiments, the exogenous is or comprises a fourth generation CAR or a nucleic acid encoding a fourth generation CAR. In some embodiments, the fourth generation CAR comprises an antigen binding domain, a transmembrane domain, and at least two, three, or four signaling domains. In some embodiments, the signaling domain mediates downstream signaling during T cell activation. In some embodiments, the signaling domain is a costimulatory domain. In some embodiments, the costimulatory domain enhances cytokine production, CAR T cell proliferation, and / or CAR T cell persistence during T cell activation.

[0291] In some embodiments, the first, second, third, or fourth generation CAR further comprises a domain that induces expression of a cytokine gene upon successful signaling of the CAR. In some embodiments, the cytokine gene is endogenous or exogenous to the target cell comprising the CAR that comprises a domain that induces expression of a cytokine gene upon successful signaling of the CAR. In some embodiments, the cytokine gene encodes a pro-inflammatory cytokine. In some embodiments, the cytokine gene encodes IL-1, IL-2, IL-9, IL-12, IL-18, TNF, or IFN-γ, or a functional fragment thereof. In some embodiments, the domain that induces expression of a cytokine gene upon successful signaling of the CAR is or comprises a transcription factor, or a functional domain or fragment thereof. In some embodiments, the domain that induces expression of a cytokine gene upon successful signaling of the CAR is or comprises a transcription factor, or a functional domain or fragment thereof. In some embodiments, the transcription factor, or a functional domain or fragment thereof, is or comprises a nuclear factor of activated T cells (NFAT), NF-kB, or a functional domain or fragment thereof. See, e.g., Zhang. C. et al., Engineering CAR-T cells. Biomarker Research. 5:22(2017); WO2016126608; Sha, H. et al. Chimaeric antigen receptor T-cell therapy for tumour immunotherapy. Bioscience Reports Jan 27, 2017, 37(1).

[0292] In some embodiments, the antigen binding domain of the CAR is or comprises an antibody or antigen binding portion thereof. In some embodiments, the antigen binding domain of the CAR is or comprises an scFv or Fab. In some embodiments, the antigen binding domain of the CAR is or comprises a T cell alpha chain antibody, a T cell beta chain antibody, a T cell gamma chain antibody, a T cell delta chain antibody, a CCR7 antibody, a CD3 antibody, a CD4 antibody, a CD5 antibody, a CD7 antibody, a CD8 antibody, a CD11b antibody, a CD11c antibody, a CD16 antibody, a CD19 antibody, a CD20 antibody, a CD21 antibody, a CD22 antibody, a CD25 antibody, a CD28 antibody, a CD34 antibody, a CD35 antibody, a CD40 antibody, a CD45RA antibody, a CD45RO antibody, a CD52 antibody, a CD56 antibody, a CD62L antibody, a CD68 antibody, a CD80 antibody, a CD95 antibody, a CD117 antibody, a CD127 antibody, a CD133 antibody, a CD137(4-1 BB) antibody, CD163 antibody, F4 / 80 antibody, IL-4Ra antibody, Sca-1 antibody, CTLA-4 antibody, GITR antibody, GARP antibody, LAP antibody, granzyme B antibody, LFA-1 antibody, MR1 antibody, uPAR antibody, or transferrin receptor antibody scFv or Fab fragments.

[0293] In some embodiments, the antigen binding domain binds to a cell surface antigen of a cell. In some embodiments, the cell surface antigen is characteristic of one cell type. In some embodiments, the cell surface antigen is characteristic of multiple cell types.

[0294] In some embodiments, the antigen binding domain of the CAR binds to a cell surface antigen characteristic of T cells. In some embodiments, the antigen characteristic of T cells can be a cell surface receptor, a membrane transport protein (e.g., active or passive transport proteins, such as, e.g., ion channel proteins, membrane pore forming proteins, etc.), a transmembrane receptor, a membrane enzyme, and / or a cell adhesion protein characteristic of T cells. In some embodiments, the antigen characteristic of T cells can be a G protein-coupled receptor, a receptor tyrosine kinase, a tyrosine kinase-associated receptor, a receptor-like tyrosine phosphatase, a receptor serine / threonine kinase, a receptor guanylyl cyclase, or a histidine kinase-associated receptor.

[0295] In some embodiments, the antigen characteristic of a T cell can be a T cell receptor. In some embodiments, the T cell receptor is AKT1; AKT2; AKT3; ATF2; BCL10; CALM1; CD3D (CD3δ); CD3E (CD3ε); CD3G (CD3γ); CD4; CD8; CD28; CD45; CD80 (B7-1); CD86 (B7-2); CD247 (CD3ζ); CTLA4 (CD152); ELK1; ERK1 (MAPK3); ERK2; FOS; FYN; GRAP2 (GADS); G RB2;HLA-DRA;HLA-DRB1;HLA-DRB3;HLA-DRB4;HLA-DRB5;HRAS;IKBKA(CHUK);IKBKB;IKBKE;IKBKG(NEMO);IL2;ITP R1;ITK;JUN;KRAS2;LAT;LCK;MAP2K1(MEK1);MAP2K2(MEK2);MAP2K3(MKK3);MAP2K4(MKK4);MAP2K6(MKK6);MAP2K7( MKK7);MAP3K1(MEKK1);MAP3K3;MAP3K4;MAP3K5;MAP3K8;MAP3K14(NIK);MAPK8(JNK1);MAPK9(JNK2);MAPK10(JNK3 );MAPK11(p38β);MAPK12(p38γ);MAPK13(p38δ);MAPK14(p38α);NCK;NFAT1;NFAT2;NFKB1;NFKB2;NFKBIA;NRAS;PAK 1; PAK2; PAK3; PAK4; PIK3C2B; PIK3C3 (VPS34); PIK3CA; PIK3CB; PIK3CD; PIK3R1; PKCA; PKCB; PKCM; PKCQ; PLCY1; PRF1 (perforin); PTEN; RAC1; RAF1; RELA; SDF1; SHP2; SLP76; SOS; SRC; TBK1; TCRA; TEC; TRAF6; VAV1; VAV2; or ZAP70.

[0296] In some embodiments, the CAR comprises a signaling domain that is a costimulatory domain. In some embodiments, the CAR comprises a second costimulatory domain. In some embodiments, the CAR comprises at least two costimulatory domains. In some embodiments, the CAR comprises at least three costimulatory domains. In some embodiments, the CAR comprises a costimulatory domain selected from one or more of CD27, CD28, 4-1BB, CD134 / OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, CD83 specific binding ligand.

[0297] In some embodiments, the CAR comprises a CD3 zeta domain or an immunoreceptor tyrosine-based activation motif (ITAM), or a functional variant thereof. In some embodiments, the CAR comprises (i) a CD3 zeta domain or an immunoreceptor tyrosine-based activation motif (ITAM), or a functional variant thereof, and (ii) a CD28 domain or a 4-1BB domain, or a functional variant thereof. In some embodiments, the CAR comprises (i) a CD3 zeta domain or an immunoreceptor tyrosine-based activation motif (ITAM), or a functional variant thereof, (ii) a CD28 domain, or a functional variant thereof, and (iii) a 4-1BB domain, or a CD134 domain, or a functional variant thereof. In some embodiments, the CAR comprises (i) a CD3 zeta domain or an immunoreceptor tyrosine-based activation motif (ITAM), or a functional variant thereof, (ii) a CD28 domain, or a functional variant thereof, and / or (iii) a 4-1BB domain, or a CD134 domain, or a functional variant thereof. In some embodiments, the CAR comprises (i) a CD3 zeta domain, or an immunoreceptor tyrosine-based activation motif (ITAM), or a functional variant thereof, (ii) a CD28 domain, or a functional variant thereof, (iii) a 4-1BB domain, or a CD134 domain, or a functional variant thereof, and (iv) a cytokine or costimulatory ligand transgene.

[0298] In certain embodiments, the intracellular signaling domain comprises the transmembrane and signaling domains of CD28 linked to the intracellular domain of CD3 (e.g., CD3ζ). In some embodiments, the intracellular signaling domain comprises a chimeric CD28 and CD137 (4-1BB, TNFRSF9) costimulatory domain linked to the CD3ζ intracellular domain.

[0299] In some embodiments, a CAR includes one or more, e.g., two or more, costimulatory domains and an activation domain, e.g., a primary activation domain, in the cytoplasmic portion. Exemplary CARs include the intracellular components of CD3zeta, CD28, and 4-1BB.

[0300] In some embodiments, the intracellular signaling domain comprises the signaling domain of 4-1BB and an intracellular component of the signaling domain of CD3ζ, hi some embodiments, the intracellular signaling domain comprises the signaling domain of CD28 and an intracellular component of the signaling domain of CD3ζ.

[0301] In some embodiments, a CAR comprises an extracellular antigen binding domain (e.g., an antibody or antibody fragment, e.g., scFv) that binds to an antigen (e.g., a tumor antigen), a spacer (e.g., including a hinge domain, such as any described herein), a transmembrane domain (e.g., any described herein), and an intracellular signaling domain (e.g., any intracellular signaling domain, e.g., a primary signaling domain or a costimulatory signaling domain described herein). In some embodiments, the intracellular signaling domain is or comprises a primary cytoplasmic signaling domain. In some embodiments, the intracellular signaling domain further comprises an intracellular signaling domain of a costimulatory molecule (e.g., a costimulatory domain). Examples of exemplary components of a CAR are set forth in Table 2. In provided aspects, the sequence of each component in a CAR can include any combination listed in Table 2.

[0302] Table 2: CAR components and exemplary sequences TIFF2024521811000002.tif196165

[0303] In some embodiments, the CAR further comprises one or more spacers, for example, the spacer is a first spacer between the antigen-binding domain and the transmembrane domain. In some embodiments, the first spacer comprises at least a portion of an immunoglobulin constant region or a variant or modified version thereof. In some embodiments, the spacer is a second spacer between the transmembrane domain and the signaling domain. In some embodiments, the second spacer is an oligopeptide, for example, the oligopeptide comprises a glycine-serine doublet.

[0304] In addition to the CARs described herein, various chimeric antigen receptors and nucleotide sequences encoding same are known in the art and would be suitable for fusomal delivery and reprogramming of target cells in vivo and in vitro as described herein. See, e.g., WO2013040557; WO2012079000; WO2016030414; Smith T, et al., Nature Nanotechnology. 2017. DOI: 10.1038 / NNANO.2017.57, the disclosures of which are incorporated herein by reference.

[0305] In some embodiments, lipid particles containing a CAR or a nucleic acid encoding a CAR (e.g., DNA, gDNA, cDNA, RNA, pre-MRNA, mRNA, miRNA, siRNA, etc.) are delivered to a target cell. In some embodiments, the target cell is an effector cell, e.g., a cell of the immune system, that expresses one or more Fc receptors and mediates one or more effector functions. In some embodiments, the target cell may include, but is not limited to, one or more of monocytes, macrophages, neutrophils, dendritic cells, eosinophils, mast cells, platelets, large granular lymphocytes, Langerhans cells, natural killer (NK) cells, T lymphocytes (e.g., T cells), γδ T cells, B lymphocytes (e.g., B cells), and may be derived from any organism, including, but not limited to, human, mouse, rat, rabbit, and monkey.

[0306] 3. Gene editing agents (e.g., nuclease enzymes) In some embodiments, the exogenous agent is related to gene editing technology. For example, any of the various agents related to gene editing technology for delivering gene editing mechanisms to cells can be included as exogenous agents. In some embodiments, gene editing technology can include systems including nucleases, integrases, transposases, and recombinases. In some embodiments, gene editing technology can be used to knock out or knock down genes. In some embodiments, gene editing technology can be used to knock in or integrate DNA into genomic regions. In some embodiments, the exogenous agent mediates double-strand breaks (DSBs), including those related to non-homologous end joining (NHEJ) or homology-directed repair (HDR). In some embodiments, the exogenous agent does not mediate DSBs. In some embodiments, the exogenous agent can be used for DNA-based editing or prime editing. In some embodiments, the exogenous agent can be used for Programmable Addition via Site-specific Targeting Element (PASTE).

[0307] In some embodiments, the exogenous agent is a nuclease for use in gene editing methods. In some embodiments, the nuclease is a zinc finger nuclease (ZFN), a transcription activator-like effector nuclease (TALEN), or a CRISPR-associated protein nuclease (Cas). In some embodiments, the Cas is Cas9 from Streptococcus pyogenes. In some embodiments, the Cas is Cas12a (also known as cpf1) from Prevotella or Francisella bacteria. In some embodiments, the Cas is Cas12b from Bacillus, optionally Bacillus hisashii.

[0308] In some embodiments, delivery of the nuclease is by a provided vector that encodes the nuclease (e.g., Cas).

[0309] In some embodiments, the provided viral vector particles contain a nuclease protein and deliver the nuclease protein directly to the target cell. Methods for delivering the nuclease protein include, for example, those described in Cai et al. Elife, 2014, 3: e01911 and International Patent Publication No. WO2017068077. For example, the provided viral vector particles contain one or more Cas proteins, such as Cas9. In some embodiments, the nuclease protein (e.g., Cas, such as Cas9) is modified as a chimeric nuclease protein with a viral structural protein (e.g., GAG) for packaging in the viral vector particle (e.g., lentiviral vector particle). For example, a fusion of a chimeric Cas9 protein and a structural GAG protein can be packaged in the lentiviral vector particle. In some embodiments, the fusion protein is a cleavable fusion protein between (i) a viral structural protein (e.g., GAG) and (ii) a nuclease protein (e.g., Cas protein, such as Cas9).

[0310] In some embodiments, the Cas is wild-type Cas9, which is capable of site-specific cleavage of double-stranded DNA, resulting in activation of the double-strand break (DSB) repair machinery. DSBs can be repaired by the cellular non-homologous end joining (NHEJ) pathway (Overballe-Petersen et al., 2013, Proc Natl Acad Sci USA, Vol. 110:19860-19865), which generates insertions and / or deletions (indels) and disrupts the target locus. Alternatively, if a donor template with homology to the target locus is provided, the DSB may be repaired by the homology-directed repair (HDR) pathway, allowing for the creation of precise replacement mutations (Overballe-Petersen et al., 2013, Proc Natl Acad Sci USA, Vol. 110: 19860-19865; Gong et al., 2005, Nat. Struct Mol Biol, Vol. 12: 304-312). In some embodiments, the Cas is a mutant form known as Cas9 D10A that only has nickase activity. This means that Cas9D10A only cleaves one DNA strand and does not activate NHEJ. Instead, when a homologous repair template is provided, DNA repair is exclusively via the high-fidelity HDR pathway, resulting in reduced indel mutations (Cong et al., 2013, Science, Vol. 339: 819-823; Jinek et al., 2012, Science, Vol. 337: 816-821; Qi et al., 2013 Cell, Vol. 152: 1173-1183). Cas9D10A is even more attractive in terms of target specificity when the locus is targeted by a paired Cas9 complex designed to generate adjacent DNA nicks (Ran et al., 2013, Cell, Vol. 154: 1380-1389). In some embodiments, Cas is a nuclease-deficient Cas9 (Qi et al., 2013 Cell, Vol. 152: 1173-1183). For example, the H840A mutation in the HNH domain and the D10A mutation in the RuvC domain inactivate cleavage activity but do not interfere with DNA binding.Therefore, this variant can be used to sequence-specifically target any region of the genome without cleavage. Alternatively, by fusing with various effector domains, dCas9 can be used as a gene silencing or activation tool. In addition, by conjugating the guide RNA or Cas9 protein with a fluorophore or fluorescent protein, it can be used as a visualization tool.

[0311] In certain embodiments, the nuclease is a Cas nuclease, such as Cas9. In some embodiments, CRISPR / Cas delivery can be used to introduce single point mutations (deletions or insertions) in specific target genes via a single gRNA. Alternatively, pairs of gRNA-directed Cas9 nucleases can be used to induce larger deletions and genomic rearrangements, such as inversions and translocations. In some embodiments, the dCas9 version of the CRISPR / Cas9 system can be used to target protein domains for transcriptional regulation, epigenetic modifications, and microscopic visualization of specific genomic loci.

[0312] In some embodiments, the provided viral particles (e.g., lentiviral particles) containing Cas nuclease (e.g., Cas9) further comprise or are further complexed with one or more CRISPR-Cas system guide RNAs for targeting desired target genes. In some embodiments, the CRISPR guide RNAs are efficiently packaged in the CAS-containing viral particles. In some embodiments, the provided viral particles (e.g., lentiviral particles) further comprise or are further complexed with targeting nucleic acids.

[0313] In some embodiments, the exogenous agent is for use in targeted primed reverse transcription (TPRT), or "prime editing." In some embodiments, prime editing mediates targeted insertions, deletions, possible 12 base pair base changes, and combinations thereof in human cells without the need for DSBs or donor DNA templates.

[0314] Prime editing is a genome editing method that uses a nucleic acid programmable DNA binding protein ("napDNAbp") that works in conjunction with a polymerase (i.e., provided in the form of a fusion protein or in trans with napDNAbp) to directly write new genetic information at a designated DNA site; the prime editing system is programmed with a prime editing (PE) guide RNA ("PEgRNA") that both specifies the target site and synthesizes and templates the desired edit in the form of a replacement DNA strand with an extension (either DNA or RNA) designed on the guide RNA (e.g., at the 5' or 3' end, or in an internal portion of the guide RNA). The replacement strand, containing the desired edit (e.g., a single nucleobase substitution), shares the same sequence as the endogenous strand of the target site to be edited (except that it contains the desired edit). Through DNA repair and / or replication mechanisms, the endogenous strand of the target site is replaced by the newly synthesized replacement strand containing the desired edit. In some cases, prime editing may be considered a "search and replace" genome editing technique because it searches for and identifies the desired target site to be edited, and simultaneously encodes a replacement strand containing the desired edit that is installed in the endogenous DNA strand at the corresponding target site. For example, prime editing can be adapted to perform precise CRISPR / Cas-based genome editing to bypass double-strand breaks. In some embodiments, the exogenous agent is or encodes a Cas protein-reverse transcriptase fusion or related system to target a specific DNA sequence with a guide RNA, generate a single-stranded nick at the target site, and use the nicked DNA as a primer to reverse transcribe a modified reverse transcriptase template integrated with the guide RNA.

[0315] In some embodiments, the exogenous agent is or codes for a primer editor that is a reverse transcriptase or any DNA polymerase known in the art.Thus, in one aspect, the primer editor can include Cas9 (or equivalent napDNAbp) that is programmed to target a DNA sequence by associating with a special guide RNA (i.e., PEgRNA) that includes a spacer sequence that anneals to a complementary protospacer in the target DNA.Such methods include those disclosed in Anzalone et al., (https: / / doi.org / 10.1038 / s41586-019-1711-4), or PCT Publication No. WO2020191248, WO2021226558, or WO2022067130, which are incorporated herein by reference in their entirety.

[0316] In some embodiments, the exogenous agent is for use in Programmable Addition via Site-specific Targeting Element (PASTE). In some aspects, PASTE is a platform that directs genomic insertion via a CRISPR-Cas9 nickase fused to both a reverse transcriptase and a serine integrase. As described in Ioannidi et al. (doi:https: / / doi.org / 10.1101 / 2021.11.01.466786), PASTE does not generate double-stranded breaks but allows for integration of sequences up to about 36 kb. In some embodiments, the serine integrase can be any known in the art. In some embodiments, the serine integrase has sufficient orthogonality such that PASTE can be used for multiplex gene integration to simultaneously integrate at least two different genes at at least two genomic loci. In some embodiments, PASTE has editing efficiency comparable to or better than homologous recombination repair or non-homologous end joining-based integration, is active in non-dividing cells, and has fewer detectable off-target events. In some embodiments, the exogenous agent is associated with base editing. Base editors (BEs) are typically fusions of a Cas ("CRISPR-associated") domain and a nucleobase-modifying domain (e.g., natural or evolved deaminases such as APOBEC1 ("apolipoprotein B mRNA editing enzyme, catalytic polypeptide 1"), CDA ("cytidine deaminase"), and AID ("activation-induced cytidine deaminase")) domains). In some cases, base editors can also include proteins or domains that modify the cell's DNA repair process to increase the efficiency and / or stability of the resulting single nucleotide changes.

[0317] In some embodiments, currently available base editors include a cytidine base editor (e.g., BE4) that converts targeted C·G to T·A, and an adenine base editor (e.g., ABE7.10) that converts targeted A·T to G·C. In some embodiments, Cas9-targeted deamination was first demonstrated in the context of a base editing (BE) system designed to induce base changes without introducing double-stranded DNA breaks. Additionally, rat deaminase APOBEC1 (rAPOBEC1) fused to deactivated Cas9 (dCas9) was successfully used to convert a cytidine to a thymidine upstream of the PAM of an sgRNA. In some embodiments, this initial BE system was optimized by altering dCas9 to the "nickase" Cas9 D10A, which nicks the strand opposite the deaminated cytidine. Without wishing to be bound by theory, it is predicted that this initiates long-patch base excision repair (BER). In this case, the deaminated strand is preferentially used as a template for repair to generate a U:A base pair, which is then converted to a T:A during DNA replication.

[0318] In some embodiments, the exogenous agent is or encodes a base editor (e.g., a nucleobase editor). In some embodiments, the exogenous agent is a nucleobase editor comprising a first DNA-binding protein domain that is catalytically inactive, a domain with base-editing activity, and a second DNA-binding protein domain with nickase activity, where the DNA-binding protein domains are expressed on a single fusion protein or separately (e.g., on separate expression vectors). In some embodiments, the base editor is a fusion protein comprising a domain with base-editing activity (e.g., cytidine deaminase or adenosine deaminase) and two nucleic acid programmable DNA-binding protein domains (napDNAbp), where the first domain comprises nickase activity, the second napDNAbp is catalytically inactive, and at least two napDNAbp are linked by a linker. In some embodiments, the base editor is a fusion protein that includes a DNA domain of CRISPR-Cas (e.g., Cas9) with nickase activity (nCas; nCas9), a catalytically inactive domain of a CRISPR-Cas protein (e.g., Cas9) with nucleic acid programmable DNA binding activity (dCas; e.g., dCas9), and a deaminase domain, where dCas is linked to nCas by a linker, and dCas is directly adjacent to the deaminase domain. In some embodiments, the base editor is an adenine to thymine, or "ATBE" (or thymine to adenine, or "TABE") converting base editor. Exemplary base editors and base editing systems include those described in Patent Publications US20220127622, ​​US20210079366, US20200248169, US20210093667, US20210071163, WO2020181202, WO2021158921, WO2019126709, WO2020181178, WO2020181195, WO2020214842, and WO2020181193, which are incorporated by reference in their entireties.

[0319] In some embodiments, the exogenous agent is, or encodes, one or more polypeptides having an activity selected from the group consisting of a nuclease activity (e.g., a programmable nuclease activity), a nickase activity (e.g., a programmable nickase activity), a homing activity (e.g., a programmable DNA binding activity), a nucleic acid polymerase activity (e.g., a DNA polymerase or RNA polymerase activity), an integrase activity, a recombinase activity, or a base editing activity (e.g., a cytidine deaminase or adenosine deaminase activity).

[0320] 4. Small molecules In some embodiments, the exogenous agent is a small molecule, e.g., an ion (e.g., Ca 2+ , C1-, Fe 2+ ), carbohydrates, lipids, reactive oxygen species, reactive nitrogen species, isoprenoids, signaling molecules, heme, polypeptide cofactors, electron withdrawing compounds, electron donating compounds, metabolites, ligands, and any combination thereof. In some embodiments, the small molecule is an agent that interacts with a target in a cell. In some embodiments, the small molecule targets a protein contained in a cell for degradation. In some embodiments, the small molecule targets a protein contained in a cell for degradation by localizing the protein to the proteasome. In some embodiments, the small molecule is a proteolytic targeting chimeric molecule (PROTAC).

[0321] In some embodiments, exogenous agents include mixtures of proteins, nucleic acids, or metabolites, such as multiple polypeptides, multiple nucleic acids, multiple small molecules, combinations of nucleic acids, polypeptides, and small molecules, ribonucleoprotein complexes (e.g., Cas9-gRNA complexes), multiple transcription factors, multiple epigenetic factors, reprogramming factors (e.g., Oct4, Sox2, cMyc, and Klf4), multiple regulatory RNAs, and any combination thereof.

[0322] D. Exemplary Features In some embodiments, the truncated BaEV envelope glycoprotein is increased in expression on the particle surface compared to a reference particle comprising a BaEV lacking all amino acid residues in the R peptide (e.g., BaEV as set forth in SEQ ID NO: 24). In some embodiments, the expression is increased by 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 125%, 150%, 200%, 300%, 400%, 500% or more, or by more than 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 125%, 150%, 200%, 300%, 400%, 500% or more, compared to a reference particle comprising a BaEV lacking all amino acid residues in the R peptide (e.g., BaEV as set forth in SEQ ID NO: 24). In some embodiments, the expression is increased by 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, 30-fold, or more, preferably 10-fold or more, or about 10-fold or more, or more than 10-fold, compared to a reference particle containing BaEV lacking all amino acid residues of the R peptide (e.g., BaEV as set forth in SEQ ID NO: 24). In some embodiments, expression can be assayed in vitro using flow cytometry, e.g., FACs. In some embodiments, expression can be expressed as the number or density of BaEV envelope glycoproteins, e.g., truncated BaEV envelope glycoproteins, on the surface of the lipid particle. In some embodiments, expression can be expressed as the mean fluorescence intensity (MFI) of surface expression of a truncated BaEV envelope glycoprotein, e.g., a truncated BaEV envelope glycoprotein, on the surface of a lipid particle. In some embodiments, expression can be shown as the percentage of lipid particles (e.g., lentiviral vectors) in a population that are surface positive for a BaEV envelope glycoprotein, e.g., a truncated BaEV envelope glycoprotein.

[0323] In some embodiments, in a population of lipid particles (e.g., lentiviral vectors), more than 50% or about 50% of the lipid particles are surface positive for truncated BaEV envelope glycoprotein. For example, in a population of lipid particles (e.g., lentiviral vectors) provided, more than 55% or about 55%, more than 60%, more than 65%, more than 70%, more than 75%, or about 75% of the cells in the population are surface positive for truncated BaEV envelope glycoprotein.

[0324] In some embodiments, the titer of the lipid particles after introduction into a target cell (e.g., a transduced cell), such as by transduction, is increased compared to the titer of a reference lipid particle (e.g., a reference lentiviral vector) incorporating a BaEV (e.g., a BaEV as set forth in SEQ ID NO: 24) lacking all amino acid residues of an R peptide, into the same target cell. In some examples, the titer is increased by 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, 30-fold, or more, or by more than 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, 30-fold, or more, compared to the titer of a reference lipid particle (e.g., a reference lentiviral vector), such as a reference lipid particle containing a BaEV (e.g., a BaEV as set forth in SEQ ID NO: 24) lacking all amino acid residues of an R peptide. In some of the optional embodiments, the titer in the target cells after transduction is 1×10 6 Transducing units (TU) / mL or greater, 2 x 10 6 TU / mL or more, 3×10 6 TU / mL or more, 4×10 6 TU / mL or more, 5×10 6 TU / mL or more, 6×10 6 TU / mL or more, 7×10 6 TU / mL or more, 8×10 6 TU / mL or more, 9×10 6 TU / mL or greater, or 1×10 7 ≥ TU / mL.

[0325] In certain embodiments, the truncated BaEV envelope glycoproteins are at least about (0.001, 0.002, 0.005, 0.01, 0.02, 0.05, 0.1, 0.2, or 0.5) truncated BaEV envelope glycoproteins / nm 2 is present on the surface of the particle at a density of

[0326] In some embodiments, provided lipid particles are preferentially targeted to target cells relative to non-target cells, hi some embodiments, provided lipid particles include an exogenous agent in a lumen or cavity, and such lipid particles exhibit preferential delivery of the exogenous agent to target cells relative to non-target cells.

[0327] As used herein, "target cell" refers to a type of cell that is specifically targeted by BaEV envelope glycoprotein-containing lipid particles. In embodiments, the target cell is a hematopoietic cell, such as a cell that is surface positive for CD34 (CD34+ cell).

[0328] As used herein, "non-target cell" refers to a type of cell that is not desirable to target lipid particles, for example, to deliver exogenous agents. In some embodiments, non-target cell is a non-hematopoietic cell. In some embodiments, non-target cell is a cell that is surface negative for CD34 (CD34- cell).

[0329] In some embodiments, lipid particles comprising truncated BaEV envelope glycoproteins, such as BaEV glycoprotein pseudotyped lentiviral particles, can target cells ex vivo. In some embodiments, lipid particles comprising truncated BaEV envelope glycoproteins, such as BaEV glycoprotein pseudotyped lentiviral particles, can target cells in vivo. In some embodiments, lipid particles comprising truncated BaEV envelope glycoproteins, such as BaEV glycoprotein pseudotyped lentiviral particles, can target cells in vivo after fixation.

[0330] In some embodiments, truncated BaEV envelope glycoproteins exhibit similar tropism and cell targeting as VSV-G. Thus, in some embodiments, lipid particles comprising a truncated Baboon endogenous retrovirus (BaEV) envelope glycoprotein, e.g., BaEV glycoprotein pseudotyped lentiviral particles, can be used in place of VSV-G.

[0331] In some embodiments, the target cells are positive for cell surface expression of ASCT1 and / or ASCT2.

[0332] In some embodiments, the target cell is a hematopoietic cell. Reference to "hematopoietic cell" includes blood cells derived from both the myeloid and lymphoid lineages. In particular, the term "hematopoietic cell" includes both undifferentiated or less differentiated cells, such as hematopoietic stem and progenitor cells, and differentiated cells, such as T lymphocytes, B lymphocytes, or dendritic cells. In some embodiments, the hematopoietic cell is a hematopoietic stem cell, a CD34+ progenitor cell, particularly peripheral blood CD34+ cells, an immediate early progenitor CD34+ cell, a B cell CD19+ progenitor cell, a bone marrow progenitor CD13+ cell, a T lymphocyte, a B lymphocyte, a monocyte, a dendritic cell, a cancer B cell, particularly a B cell chronic lymphocytic leukemia (BCLL) cell and a marginal zone lymphoma (MZL) B cell, and a thymic cell.

[0333] As is known to those of skill in the art, many hematopoietic cells are generated from bone marrow hematopoietic stem cells.

[0334] In some embodiments, the hematopoietic cells are hematopoietic stem cells (HSCs), which are cells capable of replenishing all blood cell types and self-renewing. Hematopoietic stem cells may be specifically defined as cells that maintain robustly detectable levels of myeloid, T, and B cells (usually >1% of peripheral blood cells) for 16 weeks when the depleted hematopoietic system is injected into the circulation of recipient mice (Schroeder (2010) Cell Stem Cell 6:203-207).

[0335] In some embodiments, the hematopoietic cells are "CD34+ progenitor cells," which are a heterogeneous cell population that includes HSCs, pluripotent stem cells, and a subpopulation of cells at an early stage of lineage commitment. CD34+ progenitor cells continually migrate to and from the bone marrow in normal adult animals. They can differentiate to generate all hematopoietic cell lineages found in the circulation. In some embodiments, the hematopoietic cells are immediate precursor CD34+ cells, a subgroup of CD34+ progenitor cells enriched from HSCs.

[0336] In some embodiments, hematopoietic cells include "peripheral blood CD34+ cells," which are CD34+ cells present in blood.

[0337] In some embodiments, the hematopoietic cells are B cell CD19+ progenitor cells, which are a population of B lineage cells that express cell surface CD10, CD34, and CD19.

[0338] In some embodiments, the hematopoietic cells are myeloid progenitor CD13+ cells, a population of myeloid cells that express cell surface CD34 and CD34, and in some cases also CD33.

[0339] In some embodiments, the cells are selected from the group consisting of hematopoietic cells with no myeloid-lymphoid bias, myeloid-biased hematopoietic cells, lymphoid-biased hematopoietic cells, platelet-biased hematopoietic cells, long-term repopulating hematopoietic cells, intermediate-term repopulating hematopoietic cells, or short-term repopulating hematopoietic cells. In some embodiments, the cells are selected from monocytes, macrophages, neutrophils, basophils, eosinophils, erythrocytes, megakaryocytes, and platelets. In some embodiments, the cells are selected from T cells, B cells, natural killer (NK) cells, and innate lymphoid cells.

[0340] In some embodiments, the hematopoietic cells are T cells. In some embodiments, the T cells are naive T cells. In some embodiments, the T cells are memory T cells.

[0341] In some embodiments, the hematopoietic cells are B cells. In some embodiments, the target cells are resting B cells, such as naive or memory B cells. In some embodiments, the target cells are cancer B cells, such as B cell chronic lymphocytic leukemia (BCLL) cells or marginal zone lymphoma (MZL) B cells.

[0342] In some embodiments, the target cell is a thymocyte. In some embodiments, the target cell is a natural killer (NK) cell. In some embodiments, the thymocyte expresses CD4 or CD8. In some embodiments, the thymocyte does not express CD4 or CD8. In some embodiments, the natural killer (NK) cell is a cell that expresses CD56.

[0343] IV. Pharmaceutical Compositions and Methods for Manufacturing the Same Also provided are compositions (including pharmaceutical compositions and formulations) containing the lipid particles herein that contain the truncated BaEV envelope glycoproteins or polynucleotides encoding the truncated BaEV envelope glycoproteins. The pharmaceutical compositions may include any of the truncated BaEV-containing lipid particles described.

[0344] The present disclosure also provides, in some aspects, a pharmaceutical composition comprising a composition described herein and a pharma- ceutically acceptable carrier.

[0345] The term "pharmaceutical formulation" refers to a preparation that is in a form such that the biological activity of the active ingredients contained therein is effective and does not contain any additional components that are unacceptably toxic to a subject to which the formulation will be administered.

[0346] "Pharmaceutically acceptable carrier" refers to an ingredient in a pharmaceutical formulation, other than an active ingredient, that is non-toxic to a subject. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives.

[0347] In some embodiments, the choice of carrier is determined in part by the particular lipid particle and / or the method of administration. Thus, there are a variety of suitable formulations. For example, the pharmaceutical composition can include a preservative. Suitable preservatives can include, for example, methylparaben, propylparaben, sodium benzoate, and benzalkonium chloride. In some embodiments, a mixture of two or more preservatives is used. The preservative or mixtures thereof are typically present in an amount of about 0.0001% to about 2% by weight of the total composition. Carriers are described, for example, in Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980). Pharmaceutically acceptable carriers are generally nontoxic to recipients at the dosages and concentrations employed and include, but are not limited to, buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl, or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; serum albumin; Examples of suitable surfactants include, but are not limited to, proteins such as amine, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., Zn-protein complexes); and / or non-ionic surfactants such as polyethylene glycol (PEG).

[0348] In some embodiments, the lipid particles meet Good Manufacturing Practice (GMP). In some embodiments, the lipid particles are made according to Good Manufacturing Practice (GMP). In some embodiments, the lipid particles have pathogen levels below a predefined reference value, e.g., are substantially free of pathogens. In some embodiments, the lipid particles have contaminant levels below a predefined reference value, e.g., are substantially free of contaminants. In some embodiments, the lipid particles have low immunogenicity.

[0349] In some embodiments, the formulation of pharmaceutical composition described herein can be prepared by any method known or hereafter developed in the field of pharmacology.In some embodiments, the preparation method includes combining active ingredient with carrier or one or more other auxiliary ingredients, and then, if necessary or desired, shaping or packaging the product into desired single or multiple dosage units.

[0350] In some embodiments, as used herein, a "unit dose" is a discrete amount of a pharmaceutical composition containing a predetermined amount of an active ingredient. In some embodiments, the amount of active ingredient is generally equal to the dose of the active ingredient that would be administered to a subject, or a convenient fraction of such a dose, such as one-half or one-third of such a dose. In some embodiments, the unit dosage form can be a single daily dose or multiple daily doses (e.g., about 1-4 or more times per day). In some embodiments, when multiple daily doses are used, the unit dosage form can be the same or different for each dose.

[0351] In some embodiments, the lipid particle containing the truncated BaEV envelope glycoprotein is a viral vector or a virus-like particle (e.g., Section II). In some embodiments, the compositions provided herein can be formulated in dose units of genome copies (GC). Suitable methods for determining GC have been described, including, for example, qPCR or digital droplet PCR (ddPCR) as described in M. Lock et al, Hu Gene Therapy Methods, Hum Gene Ther Methods 25(2):115-25.2014, which is incorporated herein by reference. In some embodiments, the dosage of the viral vector or virus-like particle is about 10 4 ~about 10 10 In some embodiments, the dosage of the viral vector or virus-like particle is about 10 9 ~about 10 15 In some embodiments, the dosage of the viral vector or virus-like particle is about 10 5 ~about 10 9 In some embodiments, the dosage of the viral vector or virus-like particle is about 10 6 ~about 10 9 In some embodiments, the dosage of the viral vector or virus-like particle is about 10 12 ~about 10 14 GC units (inclusive). In some embodiments, the dosage is 1.0×10 9 GC units, 5.0 × 10 9 GC units, 1.0 × 10 10 GC units, 5.0 × 10 10 GC units, 1.0 × 10 11 GC units, 5.0 × 10 11 GC units, 1.0 × 10 12 GC units, 5.0 × 10 12 GC units, or 1.0 x 10 13 GC units, 5.0 × 10 13 GC units, 1.0 × 10 14GC units, 5.0 × 10 14 GC units, or 1.0 x 10 15 It is in GC units.

[0352] In some embodiments, the dosage of the viral vector or virus-like particle is about 10 4 ~about 10 10 In some embodiments, the dosage of the viral vector or virus-like particle is about 10 9 ~about 10 15 In some embodiments, the viral vector or virus-like particle dosage is 10 5 ~about 10 9 In some embodiments, the dosage of the viral vector or virus-like particle is about 10 infectious units. 6 ~about 10 9 In some embodiments, the dosage of the viral vector or virus-like particle is about 10 infectious units. 12 ~about 10 14 In some embodiments, the dose is 1.0×10 infectious units (inclusive). 9 Infectious units, 5.0 x 10 9 Infectious units, 1.0×10 10 Infectious units, 5.0 x 10 10 Infectious units, 1.0×10 11 Infectious units, 5.0 x 10 11 Infectious units, 1.0×10 12 Infectious units, 5.0 x 10 12 Infectious units, or 1.0 × 10 13 Infectious units, 5.0 x 10 13 Infectious units, 1.0×10 14 Infectious units, 5.0 x 10 14 Infectious units, or 1.0 × 10 15Infectious unit. The available techniques for quantifying infectious unit are routine in the art and include determining the number of viral particles, fluorescence microscopy, and titration by plaque assay. For example, the number of adenovirus particles can be determined by measuring the absorbance at A260. Similarly, infectious unit can be determined by quantitative immunofluorescence or plaque assay of vector-specific protein using monoclonal antibody.

[0353] In some embodiments, methods for calculating infectious units include plaque assays, in which a titer of virus is grown on a cell monolayer and the number of plaques counted after several days to weeks. For example, infectious titers are determined by plaque assays, such as assays that evaluate cytopathic effect (CPE). In some embodiments, the CPE assay is performed by serially diluting the virus on a monolayer of cells, such as HFF cells, covered with agarose. After a period of incubation to achieve cytopathic effect (e.g., about 3-28 days, typically 7-10 days), the cells can be fixed and cell-free foci visualized as plaques can be determined. In some embodiments, infectious units are calculated by the endpoint dilution (TCID), which determines the dilution of virus that infects 50% of the cell culture. 50 ) method and therefore generally the titer can be determined within a particular range, such as 1 log.

[0354] In some embodiments, the dosage of the viral vector or virus-like particle is about 10 4 ~about 10 10 plaque forming units (pfu), inclusive. In some embodiments, the dosage of the viral vector or virus-like particle is about 10 9 ~about 10 15 In some embodiments, the dose of the viral vector or virus-like particle is 10 5 ~about 10 9 In some embodiments, the dose of the viral vector or virus-like particle is about 106 ~about 10 9 In some embodiments, the dose of the viral vector or virus-like particle is about 10 12 ~about 10 14 In some embodiments, the dose is 1.0×10 pfu inclusive. 9 p.f.u., 5.0 × 10 9 p.f.u., 1.0 × 10 10 p.f.u., 5.0 × 10 10 p.f.u., 1.0 × 10 11 p.f.u., 5.0 × 10 11 p.f.u., 1.0 × 10 12 p.f.u., 5.0 × 10 12 pfu, or 1.0 × 10 13 p.f.u., 5.0 × 10 13 p.f.u., 1.0 × 10 14 p.f.u., 5.0 × 10 14 pfu, or 1.0 × 10 15 It is pfu.

[0355] In some embodiments, subjects receive a single injection. In some embodiments, administration can be repeated at daily / weekly / monthly intervals indefinitely and / or until efficacy of treatment is established. As described herein, efficacy of treatment can be determined by assessing symptoms and clinical parameters as described herein and / or by detecting a desired response.

[0356] The exact amount of vehicle-providing lipid particles required will vary from subject to subject, depending on the species, age, weight, and general condition of the subject, the particular polynucleic acid, polypeptide, or vector used, its method of administration, etc. Given the teachings herein, appropriate amounts can be determined by one of ordinary skill in the art using only routine experimentation.

[0357] The composition in some embodiments is provided as a sterile liquid formulation, such as an isotonic aqueous solution, suspension, emulsion, dispersion, or viscous composition, and in some aspects may be buffered to a selected pH. Liquid formulations are usually easier to prepare than gels, other viscous compositions, and solid compositions. In addition, liquid compositions are somewhat more convenient to administer, especially by injection. On the other hand, viscous compositions can be formulated within an appropriate viscosity range to provide longer contact time with specific tissues. The liquid or viscous composition may include a carrier, which may be a solvent or dispersion medium, such as water, saline, phosphate buffered saline, polyol (e.g., glycerol, propylene glycol, liquid polyethylene glycol), and suitable mixtures thereof.

[0358] Sterile injectable solutions can be prepared by incorporating lipid particles in a solvent such as a mixture with a suitable carrier, diluent, or excipient, such as sterile water, physiological saline, glucose, dextrose, etc. The composition can also be lyophilized. The composition can contain auxiliary substances such as wetting agents, dispersing agents, or emulsifying agents (e.g., methylcellulose), pH buffering agents, gelling or viscosity enhancing additives, preservatives, flavoring agents, coloring agents, etc., depending on the route of administration and the preparation desired. In some embodiments, standard texts can be consulted to prepare suitable preparations.

[0359] Injectables may be prepared in conventional forms, either as liquid solutions or suspensions, in solid forms suitable for solution or suspension in liquid prior to injection, or as emulsions. As used herein, "parenteral administration" includes intradermal, intranasal, subcutaneous, intramuscular, intraperitoneal, intravenous, and intratracheal routes, as well as slow-release or sustained-release systems such that a constant dosage is maintained.

[0360] Various additives that enhance the stability and sterility of the composition can be added, including antimicrobial preservatives, antioxidants, chelating agents, and buffers. Prevention of the action of microorganisms can be ensured by various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, etc. Prolonged absorption of the injectable pharmaceutical form can be brought about by the use of agents delaying absorption, such as aluminum monostearate and gelatin.

[0361] Sustained-release preparations may be prepared. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, e.g., films, or microcapsules.

[0362] In some embodiments, the vehicle formulation may include a cryoprotectant. As used herein, the term "cryoprotectant" refers to one or more agents that, when combined with a given substance, help reduce or eliminate damage to that substance that occurs during freezing. In some embodiments, a cryoprotectant is combined with a vector vehicle to stabilize the vector vehicle during freezing. In some aspects, cryopreservation of RNA at -20°C to -80°C may be advantageous for long-term (e.g., 36 months) stability of polynucleotides. In some embodiments, the RNA species is mRNA. In some embodiments, a cryoprotectant is included in the vehicle formulation to stabilize the polynucleotide through freeze / thaw cycles and under cryopreservation conditions. Cryoprotectants of the provided embodiments include, but are not limited to, sucrose, trehalose, lactose, glycerol, dextrose, raffinose, and / or mannitol. Trehalose is listed by the Food and Drug Administration as Generally Regarded as Safe (GRAS) and is commonly used in commercial pharmaceutical formulations.

[0363] Formulations to be used for in vivo administration are generally sterile. Sterility can be readily accomplished, for example, by filtration through sterile filtration membranes.

[0364] V. Methods of Use and Therapeutic Applications In some embodiments, lipid particles (e.g., lentiviral particles) containing the truncated BaEV envelope glycoprotein provided herein are used to deliver exogenous agents to target cells. Exogenous agents can be proteins, nucleic acids such as DNA or RNA (e.g., mRNA), or small molecules. Exemplary exogenous agents that can be included in the non-cellular particles for delivery are described herein. Some methods provided herein include delivering exogenous agents to target cells. In some embodiments, the exogenous agents are agents that are completely heterologous or are not produced or normally expressed by target cells.

[0365] In some embodiments, the delivery is performed by transduction of the provided lentiviral vector particles into the target cells. Thus, also provided herein is a method of transducing a target cell with the provided lentiviral vector particles pseudotyped with a truncated BaEV envelope glycoprotein. The method comprises contacting a hematopoietic cell with the pseudotyped viral vector particles as defined above under conditions that result in transduction of the hematopoietic cell by the pseudotyped viral vector particles. In some embodiments, transduction by the viral vector particles (e.g., lentiviral vector particles) first delivers the biological material to the membrane or cytoplasm of the target cell upon binding to the target cell. After delivery, the biological material can migrate to other compartments of the cell. In some embodiments, the transduction mediates the integration of the exogenous gene expressed by the particle into the genome of the cell. Conditions for achieving transduction of target cells are well known to those skilled in the art and generally comprise incubating the cells to be transduced, such as by culturing in a flask, plate, or dish, in some cases in the presence of a transduction adjuvant (e.g., retronectin). In some embodiments, target cells can be pre-stimulated or activated, such as with a cytokine cocktail or other stimulant to stimulate or activate target cells. In some embodiments, viral vector particles are incubated with target cells at an MOI of 1, 5, 10, or 100, or any value between any of the above. In some embodiments, incubation is performed in serum-free medium.

[0366] In some embodiments, the target cells are hematopoietic cells. In some embodiments, the hematopoietic cells are blood cells, for example, of myeloid or lymphoid origin. In particular, the hematopoietic cells can be undifferentiated or less differentiated cells, such as hematopoietic stem and progenitor cells, or differentiated cells, such as T lymphocytes, B lymphocytes, or dendritic cells. In some embodiments, the hematopoietic cells are selected from the group consisting of hematopoietic stem cells, CD34+ progenitor cells, particularly peripheral blood CD34+ cells, very early progenitor CD34+ cells, B cell CD19+ progenitor cells, myeloid progenitor CD13+ cells, T lymphocytes, B lymphocytes, monocytes, dendritic cells, cancer B cells, particularly B cell chronic lymphocytic leukemia (BCLL) cells and marginal zone lymphoma (MZL) B cells, and thymic cells.

[0367] In some embodiments, the target cells are hematopoietic stem cells (HSCs). HSCs are stem cells that replenish all types of blood cells and self-renew. Hematopoietic stem cells may be specifically defined as cells that maintain robust detectable levels of bone marrow cells, T cells, and B cells (usually >1% of peripheral blood cells) for 16 weeks when the depleted hematopoietic system is injected into the circulation of recipient mice (Schroeder (2010) Cell Stem Cell 6:203-207).

[0368] In some embodiments, the target cells are CD34+ progenitor cells. In some aspects, CD34+ progenitor cells are a heterogeneous cell population that includes subpopulations of HSCs, pluripotent stem cells, and cells at an early stage of lineage commitment. CD34+ progenitor cells continuously migrate to and from the bone marrow in normal adult animals. They can differentiate to generate all hematopoietic cell lineages found in the circulation.

[0369] In some embodiments, the target cell is a T cell. In some embodiments, the T cell is a resting T cell or a resting T cell. In some embodiments, the T cell is a naive T cell or a memory T cell. In some embodiments, the T cell is not activated before delivery of the lipid particle, including before transduction with the provided lentiviral vector particles. Thus, in aspects of the provided methods, the T cell is not activated by a T cell stimulant, such as an anti-CD3 / anti-CD28 antibody reagent (e.g., Dynabeads), before transduction with the provided truncated BaEV envelope glycoprotein pseudotyped viral vector particles (e.g., lentiviral vector particles). The T cell may be a CD4+ T cell, a CD8+ T cell, or a subset thereof.

[0370] In some embodiments, the target cell is a B cell. In some embodiments, the B cell is a resting B cell, such as a naive B cell or a memory B cell. In some embodiments, the B cell is a cancer B cell, such as a B cell chronic lymphocytic leukemia (BCLL) cell or a marginal zone lymphoma (MZL) B cell.

[0371] In some embodiments, delivery of an exogenous agent to a target cell can provide a therapeutic effect for treating a disease or condition in a subject. The therapeutic effect can be by targeting, regulating, or modifying an antigen or protein present or expressed in the target cell that is associated with or involved in the disease or condition. The therapeutic effect can also be by providing an exogenous agent, where the exogenous agent is a protein (or a nucleic acid encoding a protein, e.g., an mRNA encoding a protein) that is absent, mutant, or has a lower level compared to wild type in the target cell. In some embodiments, the target cell is derived from a subject with a genetic disease, e.g., a monogenic disease, e.g., a monogenic intracellular protein disease.

[0372] In some embodiments, the target cells are derived from a subject with a hematopoietic disease or disorder. In some embodiments, the hematopoietic disorder may result from a blood disease, particularly a disease involving hematopoietic cells. In some embodiments, the hematopoietic disorder is a monogenic hematopoietic disease, such as due to a mutation in a single gene. In some embodiments, the hematopoietic disorder is a myelodysplasia, aplastic anemia, Fanconi anemia, paroxysmal nocturnal hemoglobinuria, sickle cell disease, Diamond-Blackfan anemia, Shwachman-Diamond disorder, Kostmann syndrome, chronic granulomatous disease, adrenoleukodystrophy, leukocyte adhesion deficiency, hemophilia, thalassemia, β-thalassemia, leukemia, e.g., acute lymphocytic leukemia (ALL), acute myeloid (myeloid) leukemia (AML), adult lymphoblastic leukemia, chronic lymphocytic ... The primary comorbidities are chronic lymphocytic leukemia (CLL), B-cell chronic lymphocytic leukemia (B-CLL), chronic myelogenous leukemia (CML), juvenile chronic myelogenous leukemia (CML), juvenile myelomonocytic leukemia (JMML), severe combined immunodeficiency (SCID), X-linked severe combined immunodeficiency, Wiskott-Aldrich syndrome (WAS), adenosine deaminase (ADA) deficiency, chronic granulomatous disease, Chediak-Higashi syndrome, Hodgkin's lymphoma, non-Hodgkin's lymphoma (NHL), or AIDS.

[0373] In some embodiments, the target cells are from a subject with an autoimmune disease. In some embodiments, the autoimmune disease is acute disseminated encephalomyelitis, acute hemorrhagic leukoencephalitis, Addison's disease, agammaglobulinemia, alopecia areata, amyotrophic lateral sclerosis, ankylosing spondylitis, antiphospholipid syndrome, antisynthetase syndrome, atopic allergy, autoimmune aplastic anemia, autoimmune cardiomyopathy, autoimmune enteropathy, autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune inner ear disease, autoimmune lymphoproliferative syndrome, autoimmune peripheral neuropathy, autoimmune pancreatitis, autoimmune polyendocrine syndrome, autoimmune progesterone dermatitis, autoimmune Thrombocytopenic purpura, autoimmune urticaria, autoimmune uveitis, Barot's disease, Barot's concentric sclerosis, Behçet's disease, Berger's disease, Bickerstaff encephalitis, Blau syndrome, bullous pemphigoid, cancer, Castleman's disease, celiac disease, chronic inflammatory demyelinating polyneuropathy, chronic relapsing multifocal osteomyelitis, Churg-Strauss syndrome, cicatricial pemphigoid, Cogan's syndrome, cold agglutinin disease, complement component 2 deficiency, cerebral arteritis, CREST syndrome, Crohn's disease, Cushing's syndrome, cutaneous leukocytoclastic vasculitis, Dego's disease, Dercum's disease, dermatitis herpetiformis, dermatomyelitis inflammation, type 1 diabetes mellitus, diffuse cutaneous systemic sclerosis, Dressler syndrome, discoid lupus erythematosus, eczema, enthesitis-associated arthritis, eosinophilic fasciitis, eosinophilic gastroenteritis, epidermolysis bullosa acquisita, erythema nodosum, essential mixed cryoglobulinemia, Evan's syndrome, fibrodysplasia ossificans progressiva, fibrosing alveolitis, gastritis, gastrointestinal pemphigoid, giant cell arteritis, glomerulonephritis, Goodpasture's syndrome, Graves' disease, Guillain-Barré syndrome (GBS), Hashimoto's encephalitis, Hashimoto's thyroiditis, hemolytic anemia, Henoch-Schönlein purpura, herpes gestationis, hypogammaglobulinemia inflammatory demyelinating disease, idiopathic pulmonary fibrosis, idiopathic thrombocytopenic purpura, IgA nephropathy, inclusion body myositis, inflammatory demyelinating polyneuropathy, interstitial cystitis, juvenile idiopathic arthritis, juvenile rheumatoid arthritis, Kawasaki disease, Lambert-Eaton myasthenic syndrome, leukocytoclastic vasculitis, lichen planus, lichen sclerosus, linear immunoglobulin A disease (LAD), Lou Gehrig's disease, lupoid hepatitis, lupus erythematosus, Majeed syndrome, Meniere's disease, microscopic polyangiitis, Miller-Fisher syndrome, mixed connective tissue disease, morphea, Mucha-Habermann disease, multiple sclerosis,Myasthenia gravis, myositis, optic nerve pyelitis, neuromyotonia, ocular cicatricial pemphigoid, opsoclonus-myoclonus syndrome, thyroiditis, relapsing rheumatism, paraneoplastic cerebellar degeneration, paroxysmal nocturnal hemoglobinuria (PNH), Parry-Romberg syndrome, Parsonage-Turner syndrome, pars planitis, pemphigus, pemphigus vulgaris, pernicious anemia, perivenous encephalomyelitis, POEMS syndrome, polyarteritis nodosa, polymyalgia rheumatica, polymyositis, primary biliary cirrhosis, primary sclerosing cholangitis, progressive inflammatory neuropathy, psoriasis, psoriatic arthritis, pyoderma gangrenosum, pure red cell aplasia, Rasmussen syndrome The following conditions may be considered: encephalitis, Raynaud's phenomenon, relapsing polychondritis, Reiter's syndrome, restless legs syndrome, retroperitoneal fibrosis, rheumatoid arthritis, rheumatic fever, sarcoidosis, Schmidt's syndrome, Schnitzler's syndrome, scleritis, scleroderma, Sjögren's syndrome, spondyloarthropathy, Still's disease, stiff-person syndrome, subacute bacterial endocarditis, Susac's syndrome, Sweet's syndrome, Sydenham's chorea, sympathetic ophthalmia, Takayasu's arteritis, temporal arteritis, Tolosa-Hunt syndrome, transverse myelitis, ulcerative colitis, undifferentiated connective tissue disease, undifferentiated spondyloarthropathy, vasculitis, vitiligo, or Wegener's granulomatosis.

[0374] In some embodiments, the target cell is derived from a subject having cancer. In some embodiments, the cancer is leukemia. In some embodiments, the leukemia is B-CLL, CML, or T-cell-based leukemia, such as ALT. In some embodiments, the cancer is melanoma.

[0375] In some embodiments, the target cells are derived from a subject with a demyelinating disease of the central nervous system.

[0376] The lipid particles described herein, such as lentiviral vectors, or pharmaceutical compositions comprising the same can be administered to a subject, such as a mammal, such as a human.In such embodiments, the subject may be at risk of, have symptoms of, or have been diagnosed with or identified as having a particular disease or condition (e.g., a disease or condition described herein).In some embodiments, the disease or condition may be treated by delivering an exogenous agent contained in the lipid particles administered to target cells of the subject.

[0377] In some embodiments, the present disclosure provides, in certain aspects, methods of administering a lipid particle composition to a subject (e.g., a human subject), comprising administering to the subject a provided lipid particle composition comprising a plurality of lipid particles as described herein, thereby administering the lipid particle composition to the subject.

[0378] In some embodiments, the present disclosure provides a method of delivering a lipid particle composition to a target cell, comprising, in certain aspects, contacting the target cell with a provided lipid particle composition comprising a plurality of lipid particles as described herein, thereby delivering the lipid particle composition to the target cell. In some embodiments, the contacting is performed by administering the provided lipid particles to a subject, thereby delivering the lipid particles to the target cell present in the subject.

[0379] In some embodiments, the present disclosure provides a method of delivering an exogenous agent, e.g., a therapeutic substance (e.g., a polypeptide, a nucleic acid, a metabolite, an organelle, or an intracellular structure) to a subject or cell, comprising, in certain aspects, administering to a subject a plurality of lipid particles described herein, or a pharmaceutical composition described herein, wherein the lipid particle composition is administered in an amount and / or at a time such that the therapeutic substance is delivered. Exemplary exogenous agents that may be included in lipid particles herein for delivery to a subject are described in Section III.D.

[0380] In some embodiments, the present disclosure provides a method for delivering an exogenous agent, such as a therapeutic agent (e.g., a polypeptide, a nucleic acid, a metabolite, an organelle, or an intracellular structure) to a target cell, comprising, in certain aspects, contacting the target cell with a plurality of lipid particles described herein or a pharmaceutical composition described herein, wherein the lipid particle composition is contacted with the target cell under conditions such that the therapeutic agent is delivered. Exemplary exogenous agents that can be included in the lipid particles herein for delivery to a subject are described in Section III.D. In some embodiments, the contacting is performed by administering the lipid particles provided to a subject, where the therapeutic agent (e.g., an exogenous agent) included in the lipid particles is delivered to a target cell present in the subject.

[0381] In some embodiments, delivery of exogenous agents by administration of lipid particle compositions described herein may modify cellular protein expression levels. In certain embodiments, the administered composition induces upregulation (through expression in a cell, delivery in a cell, or induction in a cell) of one or more exogenous agent cargoes (e.g., polypeptides or mRNAs) that provide substantially absent or reduced functional activity in the cells to which the polypeptides are delivered. In some embodiments, the missing functional activity may be enzymatic, structural, or regulatory in nature. In some embodiments, the administered composition induces upregulation of one or more polypeptides that increase (e.g., synergistically) functional activity present but substantially missing in the cells in which the polypeptides are upregulated. In some of any of the embodiments, the administered composition induces downregulation (through expression in a cell, delivery in a cell, or induction in a cell) of one or more cargoes (e.g., polypeptides, siRNAs, or miRNAs) that suppress functional activity present or upregulated in the cells to which the polypeptides, siRNAs, or miRNAs are delivered. In some of any of the embodiments, the upregulated functional activity may be enzymatic, structural, or regulatory in nature. In some embodiments, the administered composition induces downregulation of one or more polypeptides that are present in the cells in which the polypeptide is downregulated or that reduce (e.g., synergistically) the upregulated functional activity. In some embodiments, the administered composition induces upregulation of certain functional activities and downregulation of other functional activities.

[0382] In some of the optional embodiments, the lipid particle composition (e.g., those comprising mitochondria or DNA) mediates an effect on the target cell that lasts for at least 1, 2, 3, 4, 5, 6, or 7 days, 2, 3, or 4 weeks, or 1, 2, 3, 6, or 12 months. In some embodiments (e.g., where the lipid particle composition comprises an exogenous protein), the effect lasts for less than 1, 2, 3, 4, 5, 6, or 7 days, 2, 3, or 4 weeks, or 1, 2, 3, 6, or 12 months.

[0383] In some embodiments, the lipid particle further comprises a second exogenous agent, or the method further comprises delivering said second exogenous agent, said second exogenous agent comprises or codes for a second cell surface ligand or antibody that binds to a cell surface receptor, and optionally further comprises or codes for one or more additional cell surface ligands or antibodies that bind to cell surface receptors (e.g., 1, 2, 3, 4, 5, 10, 20, 50, or more). In some embodiments, the first exogenous agent and the second exogenous agent form a complex, and optionally, the complex further comprises one or more additional cell surface ligands. In some embodiments, the exogenous agent comprises or codes for a cell surface receptor, e.g., an exogenous cell surface receptor. In some embodiments, the lipid particle further comprises a second exogenous agent, or the method further comprises delivering said second exogenous agent, wherein said second exogenous agent comprises or encodes a second cell surface receptor, and optionally further comprises or encodes one or more additional cell surface receptors (e.g., 1, 2, 3, 4, 5, 10, 20, 50, or more cell surface receptors).

[0384] In some embodiments, the lipid particles can deliver (e.g., deliver) one or more cell surface receptors to a target cell (e.g., an immune cell). Similarly, in some embodiments, the methods herein include delivering one or more cell surface receptors to a target cell. In some embodiments, the first exogenous agent and the second exogenous agent form a complex, and optionally, the complex further comprises one or more additional cell surface receptors. In some embodiments, the exogenous agent comprises or encodes an antigen or an antigen-presenting protein.

[0385] In some embodiments, the lipid particles can cause (e.g., cause) a target cell to secrete a protein, e.g., a therapeutic protein. In some embodiments, the lipid particles can deliver (e.g., deliver) a secreted exogenous agent, e.g., a secreted protein, to a target site (e.g., an extracellular space), e.g., by delivering a nucleic acid (e.g., mRNA) encoding the protein to the target cell under conditions that allow the target cell to produce and secrete the protein. Similarly, in some embodiments, the methods herein include delivering a secreted exogenous agent as described herein. In embodiments, the secreted protein includes a protein therapeutic, e.g., an antibody molecule, a cytokine, or an enzyme. In embodiments, the secreted protein includes an autocrine signaling molecule or a paracrine signaling molecule. In embodiments, the secreted exogenous agent includes a secretory granule.

[0386] In some embodiments, the lipid particles can secrete (e.g., secrete) an exogenous agent, such as a protein. In some embodiments, the exogenous agent, such as a secreted agent, is delivered to a target site in a subject. In some embodiments, the exogenous agent is a protein that cannot be or is difficult to produce recombinantly. In some embodiments, the lipid particles that secrete proteins are derived from source cells selected from MSCs or chondrocytes.

[0387] In some embodiments, the lipid particles can reprogram (e.g., reprogram) target cells (e.g., immune cells) by delivering an exogenous agent selected from, for example, a transcription factor, a nucleic acid encoding a transcription factor, an mRNA, or a plurality of said exogenous agents. Similarly, in some embodiments, the methods herein include reprogramming a target cell. In embodiments, the reprogramming includes inducing exhausted T cells to acquire one or more characteristics of non-exhausted T cells, e.g., killer T cells. In some embodiments, the exogenous agent includes an antigen. In some embodiments, the lipid particles include a first exogenous agent including an antigen and a second exogenous agent including an antigen-presenting protein.

[0388] In some embodiments, the lipid particles can modify the target tumor cells, for example by delivering an exogenous agent (protein or nucleic acid) or a nucleic acid encoding an exogenous agent. Similarly, in some embodiments, the methods herein include modifying the target tumor cells. In embodiments, the lipid particles deliver mRNA encoding an immunostimulatory ligand, an antigen-presenting protein, a tumor suppressor protein, or a proapoptotic protein. In some embodiments, the lipid particles deliver miRNA that can reduce the level of an immunosuppressive ligand, a mitogenic signal, or a growth factor in the target cell.

[0389] In some embodiments, the lipid particles deliver immunomodulatory, eg, immunostimulatory, exogenous agents.

[0390] In some embodiments, the lipid particles can induce (e.g., induce) antigen presentation in target cells, for example, by delivering an exogenous agent, including an antigen or a nucleic acid encoding an antigen. Similarly, in some embodiments, the methods herein include presenting an antigen on a target cell. In some embodiments, the lipid particles promote regeneration in a target tissue. Similarly, in some embodiments, the methods herein include promoting regeneration in a target tissue.

[0391] In some embodiments, the lipid particles can deliver (e.g., deliver) nucleic acid to a target cell, e.g., for gene therapy, e.g., to stably modify the genome of the target cell. Similarly, in some embodiments, the methods herein include delivering nucleic acid to a target cell. In some embodiments, the target cell has an enzyme deficiency, e.g., includes a mutation in an enzyme that results in reduced activity (e.g., inactivity) of the enzyme.

[0392] In some embodiments, the lipid particles can deliver (e.g., deliver) reagents that mediate sequence-specific modifications to DNA (e.g., Cas9, ZFN, or TALEN) in target cells. Similarly, in some embodiments, the methods herein include delivering reagents to target cells. In embodiments, the target cells are CNS cells.

[0393] In some embodiments, the lipid particles are capable of delivering (eg, delivering) nucleic acids to target cells, for example, to transiently alter gene expression in the target cells.

[0394] In some embodiments, the lipid particles can deliver (e.g., deliver) proteins to target cells, for example, to transiently rescue a protein defect. Similarly, in some embodiments, the methods herein include delivering proteins to target cells. In embodiments, the protein is a membrane protein (e.g., a membrane transporter protein), a cytoplasmic protein (e.g., an enzyme), or a secreted protein (e.g., an immunosuppressant protein).

[0395] In some embodiments, the lipid particles are capable of intracellular molecular delivery, for example, delivering a protein exogenous agent to a target cell. Similarly, in some embodiments, the methods herein include delivering a molecule to an intracellular region of a target cell. In some embodiments, the protein exogenous agent is an inhibitor. In some embodiments, the protein exogenous agent comprises a nanobody, an scFv, a camelid antibody, a peptide, a macrocycle, or a small molecule.

[0396] In some embodiments, the lipid particle comprises one or more cell surface ligands (e.g., 1, 2, 3, 4, 5, 10, 20, 50, or more cell surface ligands) on its membrane, and the cell surface ligands are presented by the lipid particle to the target cell. Similarly, in some embodiments, the methods herein comprise presenting one or more cell surface ligands to the target cell. In some embodiments, the lipid particle with the cell surface ligand is derived from a source cell selected from a neutrophil (e.g., the target cell is a tumor-infiltrating lymphocyte), a dendritic cell (e.g., the target cell is a naive T cell), or a neutrophil (e.g., the target is a tumor cell or a virus-infected cell). In some embodiments, the lipid particle comprises a membrane complex, e.g., a complex comprising at least 2, 3, 4, or 5 proteins, e.g., homodimers, heterodimers, homotrimers, heterotrimers, homotetramers, or heterotetramers. In some embodiments, the lipid particles include an antibody, e.g., a toxic antibody, and the lipid particles can deliver the antibody to a target site, e.g., by homing to the target site. In some embodiments, the source cell is a NK cell or a neutrophil.

[0397] In some embodiments, the methods herein include causing ligand presentation on the surface of a target cell by presenting a cell surface ligand on the lipid particle. In some embodiments, the lipid particle can cause cell death of the target cell. In some embodiments, the lipid particle is derived from a NK source cell. In some embodiments, the lipid particle or the target cell is capable of phagocytosis (e.g., of a pathogen). Similarly, in some embodiments, the methods herein include causing phagocytosis. In some embodiments, the lipid particle senses and responds to its local environment. In some embodiments, the lipid particle can sense the level of a metabolite, an interleukin, or an antigen.

[0398] In embodiments, the lipid particles are capable of chemotaxis, extravasation, or one or more metabolic activities. In embodiments, the metabolic activities are selected from kyneurinin, gluconeogenesis, prostaglandin fatty acid oxidation, adenosine metabolism, urea cycle, and thermogenic respiration. In some embodiments, the source cells are neutrophils and the lipid particles are capable of homing to the site of injury. In some embodiments, the source cells are macrophages and the lipid particles are capable of phagocytosis. In some embodiments, the source cells are brown adipose tissue cells and the lipid particles are capable of lipolysis.

[0399] In some embodiments, the lipid particle comprises multiple exogenous agents (e.g., at least 2, 3, 4, 5, 10, 20, or 50 exogenous agents) or nucleic acids encoding multiple exogenous agents (e.g., capable of being delivered to a target cell). In embodiments, the lipid particle comprises an inhibitory nucleic acid (e.g., siRNA or miRNA) and mRNA.

[0400] In some embodiments, the lipid particle comprises a membrane protein or a nucleic acid encoding a membrane protein (e.g., capable of being delivered to a target cell). In embodiments, the lipid particle is capable of reprogramming or transdifferentiating a target cell, e.g., the lipid particle comprises one or more agents that induce reprogramming or transdifferentiation of a target cell.

[0401] VI. Illustrative Embodiments The following embodiments are provided: 1. A Baboon endogenous retrovirus (BaEV) envelope glycoprotein pseudotyped lentiviral particle comprising a truncated BaEV envelope glycoprotein that includes a cytoplasmic tail with a partial fusion-inhibitory R peptide compared to the inhibitory R peptide of wild-type BaEV envelope glycoprotein, wherein the partial fusion-inhibitory R peptide comprises at least one amino terminal amino acid but less than the full-length inhibitory R peptide of the wild-type BaEV envelope glycoprotein. 2. A Baboon endogenous retrovirus (BaEV) envelope glycoprotein pseudotyped lentiviral particle comprising a truncated BaEV envelope glycoprotein comprising a cytoplasmic tail with a partial fusion-inhibitory R peptide compared to a wild-type BaEV envelope glycoprotein, wherein the cytoplasmic tail is 25 amino acids in length a...

Claims

**Claim 1** A BaEV envelope glycoprotein pseudotyped lentiviral particle comprising a truncated BaEV envelope glycoprotein having a cytoplasmic tail with a partial fusion inhibitory R peptide compared to the inhibitory R peptide of the wild-type baboon endogenous retrovirus (BaEV) envelope glycoprotein, wherein the partial fusion inhibitory R peptide comprises an inhibitory R peptide that is at least one amino-terminal amino acid of the wild-type BaEv envelope glycoprotein but less than full length, said BaEV envelope glycoprotein pseudotyped lentiviral particle. **Claim 2** A BaEV envelope glycoprotein pseudotyped lentiviral particle comprising a truncated BaEV envelope glycoprotein having a cytoplasmic tail with a partial fusion inhibitory R peptide compared to the wild-type baboon endogenous retrovirus (BaEV) envelope glycoprotein, wherein the cytoplasmic tail is 25 amino acids in length and comprises an inhibitory R peptide of 8 consecutive amino-terminal acids of the full-length inhibitory R peptide of the wild-type BaEV envelope glycoprotein (R+8), said BaEV envelope glycoprotein pseudotyped lentiviral particle. **Claim 3** The lentiviral particle according to claim 1 or 2, further comprising a viral nucleic acid, said viral nucleic acid comprising one or more of the following nucleic acid sequences: 5' LTR, Psi packaging element (Psi), central polypurine tract (cPPT) / central termination sequence (CTS), poly A tail sequence, post-transcriptional regulatory element, Rev response element (RRE), and 3' LTR. **Claim 4** A lipid particle comprising a truncated baboon endogenous retrovirus (BaEV) envelope glycoprotein, (a) a lipid bilayer surrounding the lumen, and (b) a truncated BaEV envelope glycoprotein having a cytoplasmic tail with a partial fusion inhibitory R peptide compared to the inhibitory R peptide of the wild-type BaEV envelope glycoprotein, wherein the partial fusion inhibitory R peptide comprises an inhibitory R peptide that is at least one consecutive amino-terminal amino acid of the inhibitory R peptide of the wild-type BaEV envelope glycoprotein but less than full length, and the envelope glycoprotein is embedded in the lipid bilayer, said truncated BaEV envelope glycoprotein comprising said lipid particle. **Claim 5** A lipid particle comprising a truncated baboon endogenous retrovirus (BaEV) envelope glycoprotein, (a) a lipid bilayer surrounding the lumen, and (b) a truncated BaEV envelope glycoprotein comprising a cytoplasmic tail with a partial fusion inhibitory R peptide compared to the inhibitory R peptide of the wild-type BaEV envelope glycoprotein, said cytoplasmic tail being 25 amino acids in length and comprising the inhibitory R peptide (R+8) of 8 consecutive amino-terminal acids of the full-length inhibitory R peptide of the wild-type BaEV envelope glycoprotein, and the envelope glycoprotein being embedded in said lipid bilayer, said truncated BaEV envelope glycoprotein comprising said lipid particle. **Claim 6** Said lipid bilayer is derived from the membrane of a host cell used to generate retroviral particles or retrovirus-like particles, Said host cell is selected from the group consisting of CHO cells, BHK cells, MDCK cells, C3H 10T1 / 2 cells, FLY cells, Psi-2 cells, BOSC 23 cells, PA317 cells, WEHI cells, COS cells, BSC 1 cells, BSC 40 cells, BMT 10 cells, VERO cells, W138 cells, MRC5 cells, A549 cells, HT1080 cells, 293 cells, 293T cells, B-50 cells, 3T3 cells, NIH3T3 cells, HepG2 cells, Saos-2 cells, Huh7 cells, HeLa cells, W163 cells, 211 cells, and 211A cells, the lipid particle according to claim 4 or claim 5. **Claim 7** Said lipid bilayer is one or more other viral components other than said BaEV envelope glycoprotein or comprises the same, the lipid particle according to claim 4 or 5. **Claim 8** Said one or more viral components are derived from a retrovirus, the lipid particle according to claim 7. **Claim 9** Said retrovirus is a lentiviral particle or a lentivirus-like particle, the lipid particle according to claim 8. **Claim 10** (a) Said partial fusion inhibitory R peptide is shown as amino acids 1-8 of SEQ ID NO: 22; (b) Said partial fusion inhibitory R peptide is shown as amino acids 1-7 of SEQ ID NO: 22; (c) Said partial fusion inhibitory R peptide is shown as amino acids 1-6 of SEQ ID NO: 22; (d) Said partial fusion inhibitory R peptide is shown as amino acids 1-5 of SEQ ID NO: 22; (e) the partial fusion inhibitory R peptide is shown as amino acids 1 to 4 of SEQ ID NO: 22; or (f) the partial fusion inhibitory R peptide is shown as amino acids 1 to 3 of SEQ ID NO: 22, The lentiviral particle or lipid particle according to any one of claims 1, 2, 4, or 5.

11. (a) the partial fusion inhibitory R peptide is shown as amino acids 1 to 8 of SEQ ID NO: 22, and the cytoplasmic tail is shown as SEQ ID NO: 13 (R + 8); (b) the partial fusion inhibitory R peptide is shown as amino acids 1 to 7 of SEQ ID NO: 22, and the cytoplasmic tail is shown as SEQ ID NO: 12 (R + 7); (c) the partial fusion inhibitory R peptide is shown as amino acids 1 to 6 of SEQ ID NO: 22, and the cytoplasmic tail is shown as SEQ ID NO: 11 (R + 6); (d) the partial fusion inhibitory R peptide is shown as amino acids 1 to 5 of SEQ ID NO: 22, and the cytoplasmic tail is shown as SEQ ID NO: 10 (R + 5); (e) the partial fusion inhibitory R peptide is shown as amino acids 1 to 4 of SEQ ID NO: 22, and the cytoplasmic tail is shown as SEQ ID NO: 9 (R + 4); or (f) the partial fusion inhibitory R peptide is shown as amino acids 1 to 3 of SEQ ID NO: 22, and the cytoplasmic tail is shown as SEQ ID NO: 8 (R + 3); The lentiviral particle or lipid particle according to any one of claims 1, 2, 4, or 5.

12. The truncated BaEV envelope glycoprotein is (a) shown as SEQ ID NO: 36; (b) shown as SEQ ID NO: 35; (c) shown as SEQ ID NO: 34; (d) shown as SEQ ID NO: 33; (e) shown as SEQ ID NO: 32; or (f) shown as SEQ ID NO: 31, The lentiviral particle or lipid particle according to any one of claims 1, 2, 4, or 5.

13. The lentiviral particle or lipid particle according to any one of claims 1, 2, 4, or 5, further comprising an exogenous acting factor.

14. The titer is increased by 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, 30-fold, or more, or increases above 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, 30-fold, or more. The lentiviral particle or lipid particle according to any one of claims 1, 2, 4, or 5.

15. A truncated BaEV envelope glycoprotein comprising a cytoplasmic tail with a partial fusion inhibitory R peptide compared to the wild-type BaEV envelope glycoprotein, wherein the partial fusion inhibitory R peptide comprises an inhibitory R peptide that is at least one consecutive amino-terminal amino acid of the wild-type BaEV envelope glycoprotein but less than full length. The truncated BaEV envelope glycoprotein.

16. (a) The partial fusion inhibitory R peptide is shown as amino acids 1 to 8 of SEQ ID NO: 22; (b) The partial fusion inhibitory R peptide is shown as amino acids 1 to 7 of SEQ ID NO: 22; (c) The partial fusion inhibitory R peptide is shown as amino acids 1 to 6 of SEQ ID NO: 22; (d) The partial fusion inhibitory R peptide is shown as amino acids 1 to 5 of SEQ ID NO: 22; (e) The partial fusion inhibitory R peptide is shown as amino acids 1 to 4 of SEQ ID NO: 22; or (f) The partial fusion inhibitory R peptide is shown as amino acids 1 to 3 of SEQ ID NO: 22, The truncated BaEV envelope glycoprotein according to claim 15.

17. (a) The partial fusion inhibitory R peptide is shown as amino acids 1 to 8 of SEQ ID NO: 22, and the cytoplasmic tail is shown as SEQ ID NO: 13 (R + 8); (b) The partial fusion inhibitory R peptide is shown as amino acids 1 to 7 of SEQ ID NO: 22, and the cytoplasmic tail is shown as SEQ ID NO: 12 (R + 7); (c) The partial fusion inhibitory R peptide is shown as amino acids 1 to 6 of SEQ ID NO: 22, and the cytoplasmic tail is shown as SEQ ID NO: 11 (R + 6); (d) The partial fusion inhibitory R peptide is shown as amino acids 1 to 5 of SEQ ID NO: 22, and the cytoplasmic tail is shown as SEQ ID NO: 10 (R + 5); (e) The partial fusion inhibitory R peptide is shown as amino acids 1 to 4 of SEQ ID NO: 22, and the cytoplasmic tail is shown in SEQ ID NO: 9 (R + 4); or (f) The partial fusion inhibitory R peptide is shown as amino acids 1 to 3 of SEQ ID NO: 22, and the cytoplasmic tail is shown in SEQ ID NO: 8 (R + 3); The truncated BaEV envelope glycoprotein according to claim 15.

18. (a) Shown in SEQ ID NO: 36; (b) Shown in SEQ ID NO: 35; (c) Shown in SEQ ID NO: 34; (d) Shown in SEQ ID NO: 33; (e) Shown in SEQ ID NO: 32; or (f) Shown in SEQ ID NO: 31, The truncated BaEV envelope glycoprotein according to claim 15.

19. A polynucleotide comprising a nucleic acid encoding the truncated BaEV envelope glycoprotein according to any one of claims 15 to 18.

20. A vector comprising the polynucleotide according to claim 19.

21. A plasmid comprising the polynucleotide according to claim 19.

22. The plasmid according to claim 21, further comprising one or more nucleic acids encoding proteins for lentivirus production.

23. The polynucleotide according to claim 19, the vector according to claim 20, or the plasmid according to claim 21 or claim 22 Comprising a cell.

24. A producer cell comprising (i) viral nucleic acid and (ii) a nucleic acid encoding the truncated BaEV envelope glycoprotein according to any one of claims 15 to 18.

25. A method for producing lipid particles comprising a truncated BaEV glycoprotein, comprising a) introducing the polynucleotide according to claim 19, the vector according to claim 20, or the plasmid according to claim 21 or claim 22 into a source cell, b) culturing the cell under conditions that allow production of lipid particles, and c) separating, concentrating, or purifying the lipid particles from the cell, thereby producing the lipid particles The production method comprising the above steps.

26. A method for producing pseudotyped lentiviral particles, a) providing a producer cell comprising a lentiviral nucleic acid and a nucleic acid encoding a truncated BaEV envelope glycoprotein according to any one of claims 15 to 18 or a polynucleotide according to claim 19; b) culturing said cell under conditions that allow the production of lentiviral particles, and c) separating, concentrating, or purifying said lentiviral particles from said producer cell, thereby producing said pseudotyped lentiviral particles comprising said production method.

27. A composition comprising a plurality of lentiviral particles according to any one of claims 1 to 3, or 10 to 14; or a lipid particle according to any one of claims 4 to 14.

28. A method for transducing a cell, comprising contacting the cell with a lentiviral particle according to any one of claims 1 to 3, or 10 to 14, or a composition according to claim 27.

29. An in vitro method for delivering an exogenous agent into a cell, comprising contacting the cell with a lentiviral particle according to any one of claims 1 to 3 and 10 to 14 or a lipid particle according to any one of claims 4 to 14, or a composition according to claim 27.

30. A lentiviral particle according to any one of claims 1 to 3 and 10 to 14, or a lipid particle according to any one of claims 4 to 14, or a composition according to claim 27 for use in a method for delivering an exogenous agent to a cell of a subject: wherein said method comprises administering said lentiviral particle, said lipid particle, or said composition to said subject.

31. Use of a lentiviral particle according to any one of claims 1 to 3 and 10 to 14, or a lipid particle according to any one of claims 4 to 14, or a composition according to claim 27 in the delivery of an exogenous agent to a cell of a subject.

32. A lentiviral particle according to any one of claims 1 to 3 and 10 to 14, or a lipid particle according to any one of claims 4 to 14, or a composition according to claim 27, a method according to any one of claims 28 to 30, or use according to claim 31, wherein said cell is a hematopoietic cell.

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