Virus-like particles with programmable targeting and methods of using same for delivery to cells - Patents.com

Abstract

Described herein are programmable targeting virus-like particles (ptVLPs) comprising a membrane comprising a phospholipid bilayer with one or more wild-type or mutant / truncated viral glycoproteins on the exterior. The viral envelope glycoproteins may optionally be fused directly to a targeting domain (e.g., a targeting peptide, a single-chain variable fragment (scFv), a nanobody, a fibronectin type 3 domain (FN3), an arginylglycylaspartic acid motif (RGD), a single variable domain / nanobody (VHH) on the heavy chain, a variable domain of a novel antigen receptor (VNAR), a darpin, or other targeting ligand) and / or in combination with a membrane-tethered targeting domain fusion protein. A biomolecular cargo (preferably fused to a membrane recruitment domain, e.g., a pleckstrin homology domain) may be located in the core of the ptVLP. Preferably, the ptVLP does not contain proteins from any human endogenous or exogenous viral gag, pro, pol, or other viral proteins present inside the envelope particle.

Description

[Technical Field]

[0001] Priority claim This application claims the benefit of U.S. Provisional Application No. 63 / 425,894, filed November 16, 2022, the entire contents of which are incorporated herein by reference.

[0002] Federally funded research or development This invention was made with government support under Grant No. GM118158 awarded by the National Institutes of Health. The government has certain rights in this invention.

[0003] Described herein are programmable targeting virus-like particles (ptVLPs) that include a membrane comprising a phospholipid bilayer with one or more wild-type or mutant / truncated virus-derived envelope glycoproteins on the exterior, and a targeting domain (e.g., a targeting peptide, a single-chain variable fragment (scFv), a nanobody, a fibronectin type 3 domain (FN3), an arginylglycylaspartic acid motif (RGD), a single variable domain / nanobody (VHH) on the heavy chain, a variable domain of a novel antigen receptor (VNAR), a darpin, or other targeting ligand). The targeting domain may optionally be fused directly to the virus-derived envelope glycoprotein (e.g., terminally or internally) and / or may be present in combination with the envelope glycoprotein as a separate membrane-tethered targeting domain fusion protein. Optionally, a biomolecule cargo is located on the interior side of the membrane in the core of the ptVLP. [Background technology]

[0004] The ability to deliver cargo directly to a specific cell type is useful in a number of situations, particularly in the delivery of cargo including therapeutic gene editing agents. Summary of the Invention

[0005] Described herein are programmable targeting virus-like particles (ptVLPs) comprising a membrane comprising a phospholipid bilayer with one or more wild-type or mutant / truncated viral glycoproteins on the exterior. The viral envelope glycoproteins may optionally be fused directly to a targeting domain (e.g., a targeting peptide, a single-chain variable fragment (scFv), a nanobody, a fibronectin type 3 domain (FN3), an arginylglycylaspartic acid motif (RGD), a single variable domain / nanobody (VHH) on the heavy chain, a variable domain of a novel antigen receptor (VNAR), a darpin, or other targeting ligand) and / or in combination with a membrane-tethered targeting domain fusion protein. A biomolecular cargo (preferably fused to a membrane recruitment domain, e.g., a pleckstrin homology domain) may be located in the core of the ptVLP. Preferably, the ptVLP may or may not include any human endogenous retroviral (HERV) proteins other than env, e.g., gag, pol, or pro (as long as the cargo does not include viral proteins). Exogenous virus-derived gag, pol, or pro refers to any gag, pro, pol, gag-pol, gag-pro-pol, and / or pol proteins, or any other proteins expressed from gag, pro, or pol from any virus introduced into the cell.

[0006] Provided herein are fusion proteins comprising programmable targeting glycoproteins or envelope proteins (ptENVs), including viral glycoproteins or envelope proteins fused to a targeting domain, optionally located at the C-terminus, N-terminus, or inserted immediately after the signal sequence of the glycoprotein or envelope protein. Also provided are fusion proteins comprising membrane-tethered targeting domains, including targeting domains fused to transmembrane domains. In some embodiments, the targeting domain comprises a targeting peptide, e.g., as shown in Table A. In some embodiments, the targeting domain comprises a single-chain variable fragment (scFv), a nanobody, a fibronectin type 3 domain (FN3), an arginylglycylaspartic acid motif (RGD), a single variable domain / nanobody (VHH) on the heavy chain, a variable domain of a novel antigen receptor (VNAR), a darpin, or other targeting ligand. A linker may be present between any or all of the portions of the fusion protein.

[0007] In some embodiments, the targeting domain binds to human CD19, CD4, CD34, ASGR1, TfR1, HER2, CD25, CTLA-4, HB-EGF, ACE2, aryl hydrocarbon receptor (AhR), keratin 5 (KRT5), KRT13, fibronectin (FN1), amyloid precursor protein (APP), neurotrophin receptor (p75NTR), Thy-1 / CD90, EpCAM, and / or CFTR.

[0008] In some embodiments, the signal sequence comprises MKCLLYLAFLFIGVNCK (SEQ ID NO: 1), or a secretory signal sequence derived from VSVG (e.g., MKCLLYLAFLFIGVNC, SEQ ID NO: 2), or another signal sequence known in the art or described herein.

[0009] In some embodiments, the ptENV fusion protein comprises a sequence that is at least 95% identical to a sequence described herein, e.g., the ptENV comprises a targeting domain in addition to a glycoprotein or envelope protein of Table 1.

[0010] Further provided herein are nucleic acid sequences encoding the fusion proteins described herein, as well as vectors comprising the nucleic acid sequences optionally operably linked to a promoter for expression of the fusion proteins, and host cells (e.g., production cells) comprising the nucleic acid sequences, and optionally expressing the fusion proteins.

[0011] Also provided herein is a virus-like particle (VLP) comprising a fusion protein described herein, optionally with cargo disposed in the core of the VLP, optionally fused to a phospholipid bilayer recruitment domain.

[0012] Further provided is a programmable tropism virus-like particle (ptVLP) comprising: (a) a membrane comprising a phospholipid bilayer; and (b) a fusion protein comprising a ptENV described herein, or a fusion protein comprising a glycoprotein or envelope protein (optionally listed in Table 1) and a membrane-tethered targeting domain described herein; and (c) optionally, a cargo disposed in the core of the ptVLP, optionally fused to the phospholipid bilayer recruitment domain, wherein the ptVLP optionally does not comprise exogenous gag, pro, and / or pol proteins.

[0013] In some embodiments, the cargo is a therapeutic or diagnostic protein, and / or a nucleic acid encoding a therapeutic or diagnostic protein, and / or a chemical, optionally a small molecule therapeutic or diagnostic agent. In some embodiments, the cargo is a gene editing or epigenetic modulation reagent. In some embodiments, the gene editing or epigenetic modulation reagent comprises a zinc finger (ZF), a transcription activator-like effector (TALE), and / or a CRISPR-Cas protein, variant, or fusion thereof; a nucleic acid encoding a zinc finger (ZF), a transcription activator-like effector (TALE), and / or a CRISPR-Cas protein, variant, or fusion thereof; a guide RNA and / or crRNA; or a ribonucleoprotein complex (RNP) comprising a CRISPR-Cas protein, variant, or fusion thereof, and / or optionally a guide RNA and / or crRNA.

[0014] In some embodiments, the cargo is selected from proteins listed in Tables 2, 3, 4 and 5, or is at least 95% identical to a sequence set forth herein, eg, in Tables 2, 3, 4 or 5.

[0015] In some embodiments, the cargo comprises a CRISPR-Cas protein and the ptVLP further comprises one or more guide RNAs and / or crRNAs that bind to and guide the CRISPR-Cas protein to a target nucleic acid sequence.

[0016] In some embodiments, the cargo comprises a fusion to a phospholipid bilayer recruitment domain, preferably as shown in Table 6, or a phospholipid bilayer recruitment domain that is at least 95% identical to a sequence set forth in Table 6 herein.

[0017] Further provided herein are methods for delivering cargo to a target cell, optionally a cell in vivo or in vitro, by contacting the cell with a VLP or ptVLP comprising the cargo as described herein.

[0018] Further provided herein is a method of producing a cargo-containing VLP or ptVLP by: (i) providing a cell that expresses a fusion protein described herein, e.g., ptENV, or a glycoprotein or envelope protein (optionally listed in Table 1) and a separate membrane-tethered targeting domain described herein; and optionally also expresses cargo, optionally not expressing exogenous gag, pro, or pol proteins; and maintaining the cell under conditions such that the VLP or ptVLP is produced.

[0019] In some embodiments, the method includes recovering, and optionally purifying and / or concentrating, the VLPs or ptVLPs produced.

[0020] Further provided herein are cells that express (i) a ptENV fusion protein described herein, or (ii) a fusion protein comprising a glycoprotein or envelope protein (optionally listed in Table 1) and a membrane-tethered targeting domain described in part (ii) of the specification; and optionally a cargo optionally fused to a phospholipid bilayer recruitment domain, wherein optionally the cell does not express exogenous gag, pro, and / or pol proteins. In some embodiments, the cell is a primary or stable human cell line, e.g., a human embryonic kidney (HEK) 293 cell or a HEK293 T cell.

[0021] In some embodiments, the cargo is a therapeutic or diagnostic protein, and / or a nucleic acid encoding a therapeutic or diagnostic protein, and / or a small molecule, optionally a therapeutic or diagnostic small molecule. In some embodiments, the cargo is a gene editing or epigenetic modulation reagent. In some embodiments, the gene editing or epigenetic modulation reagent comprises a zinc finger (ZF), a transcription activator-like effector (TALE), and / or a CRISPR-Cas protein, variant, or fusion thereof; a nucleic acid encoding a zinc finger (ZF), a transcription activator-like effector (TALE), and / or a CRISPR-Cas protein, variant, or fusion thereof; a guide RNA and / or crRNA; or a ribonucleoprotein complex (RNP) comprising a CRISPR-Cas protein, variant, or fusion thereof, and optionally a guide RNA and / or crRNA.

[0022] In some embodiments, the cargo reagent is selected from the proteins listed in Tables 2, 3, 4 and 5, or is at least 95% identical to a sequence set forth herein, eg, in Tables 2, 3, 4 or 5.

[0023] In some embodiments, the cargo reagent comprises a CRISPR-Cas protein, variant, or fusion thereof, and the ptVLP further comprises one or more guide RNAs and / or crRNAs that bind to and guide the CRISPR-based genome editing or regulatory protein to the target sequence.

[0024] In some embodiments, the cargo comprises a fusion to a phospholipid bilayer recruitment domain, preferably as shown in Table 6, or a phospholipid bilayer recruitment domain that is at least 95% identical to a sequence set forth in Table 6 herein.

[0025] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention belongs. Methods and materials used in the present invention are described herein. Other suitable methods and materials known in the art can also be used. Materials, methods and examples are illustrative only and are not intended to be limiting. All publications, patent applications, patents, sequences, database entries and other references mentioned herein are incorporated herein by reference in their entirety. In case of conflict, the present specification, including definitions, will control.

[0026] Other features and advantages of the invention will be apparent from the following detailed description and drawings, and from the claims. [Brief explanation of the drawings]

[0027] [Figure 1-1] Figure 1 is an exemplary diagram of ptVLP DNA expression constructs (A and C) that can be transfected into production cells, and particle structures (B and D). The scFv as a membrane-bound targeting moiety shown in this figure is exemplary only and is not intended to be limiting. [Figure 1-2] (Continued) Figure 1 is an exemplary diagram of ptVLP DNA expression constructs (A and C) that can be transfected into production cells, and particle structures (B and D). The scFv as a membrane-bound targeting moiety shown in this figure is exemplary only and is not intended to be limiting. [Figure 2]HEK293T cells transfected with either a mock control plasmid or a CD19-encoding plasmid were treated with ptVLPs containing the human AKT pleckstrin homology domain fused to SpCas9 and a guide RNA (gRNA) targeting VEGF site 3. The ptVLPs were pseudotyped with either an ectodomain-truncated VSVG (VSVG-421, in which VSVG amino acids 421-511 are preceded by a signal sequence; see Table 1) or a mutated version of the VSVG envelope protein (VSVG mut), and further contained a membrane-tethered anti-CD19 scFV. Gene modification (y-axis) was measured by targeted amplicon sequencing of the intended VEGF site 3 on-target site. [Figure 3]

[0023] Figure 1 shows exemplary gene modification efficiencies induced in cells treated with eVLPs containing various PH-Cas9 / sgRNA (VEGFs3.1-targeting) RNP cargoes. HEK293T cells were treated with these VSVG-pseudotyped eVLPs, and gene modification efficiencies (y-axis) were determined in these cells by targeted amplicon sequencing of the VEGFs3.1 on-target site. PKD protein kinase D1 (PRKD1) DAPP dual adaptor for phosphotyrosine and 3-phosphoinositide-1 (DAPP-1) FAPP4 phosphoadapter protein (FAPP) OSBP oxysterol-binding protein (OSBP) SWAP70 switch-associated protein 70 (SWAP70) GRP cytohesin 3 (CYTH3, formerly GRP1) BTK Bruton's tyrosine kinase (Btk) PHLPP pleckstrin homology domain leucine-rich repeat protein phosphatase (PHLPP) AKT AKT serine / threonine kinase 1 (AKT1) PLC phospholipase C delta 1 (PLCδ1) [Figure 4]

[0033] Figure 1 shows exemplary gene modification efficiencies induced with eVLPs containing various mutant PH-Cas9 / sgRNA (RNF2-targeting) RNP cargoes. Primary T cells were treated with eVLPs pseudotyped with VSVG or VSVG+BaeVTRless, and gene modification efficiencies (y-axis) were determined in the cells by targeted amplicon sequencing of the RNF2 on-target site. DETAILED DESCRIPTION OF THE INVENTION

[0028] Therapeutic proteins and nucleic acids hold great promise, and delivery of proteins and nucleic acids to specific cell types is of great interest, especially because it offers the potential for reduced side effects. For example, genome editing reagents, such as zinc finger nucleases (ZFNs), or RNA-guided enzymatically active / inactive DNA-binding proteins, such as Cas9, have rapidly improved in terms of specificity and the types of edits that can be performed, but the hurdle of safety in in vivo delivery remains a significant challenge for gene editing and epigenetic editing therapies.

[0029] Virus-like particles (VLPs) have been used to deliver mRNA and protein cargo into the cytosol of cells. 2、3、25~30 VLPs have emerged as an alternative delivery method to retroviral or lentiviral particles. VLPs can be engineered to lack the ability to integrate retroviral DNA and to package and deliver protein / RNP / DNA combinations. However, most VLPs currently known to deliver genome editing reagents, including recently designed VLPs, utilize HIV or other virus-derived gag or gag-pol protein fusions and viral proteases to generate retrovirus-like particles. 25~27、29、30 Some VLPs contain an RNA-guided nuclease (RGN), which is required to further package and express guide RNA from lentiviral DNA transcripts. 27Some VLPs require viral proteases to form functional particles and release their genome-editing cargo. 25~27、29 This viral protease recognizes and cleaves multiple amino acid motifs, which can cause damage to the protein cargo or potentially to other endogenous proteins in the target recipient cell, which can be harmful or create challenges for therapeutic applications. Most published VLP formats for delivering genome editing proteins or RNPs to date exhibit low in vitro and in vivo gene modification efficiencies due to low packaging and transduction efficiencies. 25~27 The complex viral genomes utilized in these VLP components have multiple reading frames and utilize RNA splicing, which can result in incorrect fusion protein products being delivered. 25~27、29、30 The presence of reverse transcriptase, integrase, capsid, and viral envelope proteins in these VLPs makes them less than ideal for many therapeutic applications due to immunogenicity and off-target concerns. Furthermore, most retroviral particles, e.g., lentiviral particles, are pseudotyped with VSVG, and almost all described VLPs delivering genome editing reagents to date contain and rely on VSVG. 2、3、25~30 .

[0030] Various embodiments of programmable targeting virus-like particles (ptVLPs) are described herein that can be used for cell-type or tissue-specific delivery of cargo, including genome editing reagents. The ptVLPs contain a targeting moiety that is either incorporated into a glycoprotein (e.g., a sequence shown below) or is separate (e.g., on the outer surface of the particle but membrane-tethered (e.g., by linkage to a transmembrane or integral membrane protein, GPI anchor, or other membrane anchor)).

[0031] This specification describes methods and compositions for producing, purifying, and administering ptVLPs for in vitro and in vivo applications, such as genome editing, epigenome regulation, transcriptome editing, and proteome regulation. The desired editing or other regulatory outcome in target recipient cells depends on the therapeutic context and requires different gene editing or other cargo to be delivered. Streptococcus pyogenes Cas9 (SpCas9) and Acidaminococcus species Cas12a (AsCas12a) are two commonly used RNA-guided enzymes for editing that utilize NHEJ-mediated repair of DNA double-strand breaks (DSBs) induced by their nucleases to introduce stop codons, or insertions / deletions (indels), or homology-directed repair (HDR) of DSBs with exogenous DNA donor templates encoding desired genetic changes (e.g., precise point mutations or insertions). Cas9 deaminase fusions, also known as base editors, are the current standard for precise editing of single nucleotides without double-stranded DNA breaks.

[0032] Phospholipid bilayer recruitment domain Conventional VLPs engineered to encapsulate and deliver protein-based cargo generally fuse the cargo to an INT or GAG polyprotein. 25~27、29、30、39、40 After transient transfection of production plasmid DNA constructs encoding these proteins and viral envelope (ENV) proteins, the protein fusions are translated in the cytosol of conventional VLP-producing cell lines, the gag matrix is ​​acetylated and recruited to the plasma membrane, and the VLPs are budded from the membrane into the extracellular space, thereby encapsulating the gag fusions within the VLPs.

[0033] In contrast, in some embodiments, proteins can be packaged into ptVLPs by fusing a phospholipid bilayer recruitment domain from a selected human protein to the protein-based cargo (e.g., as described in WO 2022 / 020800 or shown in Table 6).

[0034] One such human protein-derived phospholipid bilayer recruitment domain used for this purpose is the human pleckstrin homology (PH) domain, which interacts with phosphatidylinositol lipids and proteins in biological membranes, such as PIP2, PIP3, the βγ subunits of GPCRs, and PKCs. 41、42 Alternatively, the human Arc protein may be fused to a protein-based cargo to recruit the cargo to the cytosolic side of the phospholipid bilayer. 43These human protein-derived phospholipid bilayer recruitment domains, or variants thereof (e.g., as shown in Table 6), can be fused to the N-terminus or C-terminus of a protein-based cargo by a polypeptide linker of variable length, regardless of the location of one or more nuclear localization sequences (NLSs) within the cargo. Preferably, the linker between the protein-based cargo and the phospholipid bilayer recruitment domain is a polypeptide linker of 5-20, e.g., 8-12, e.g., 10 amino acids in length, consisting primarily of glycine and serine. The human protein-derived phospholipid bilayer recruitment domain localizes the cargo to the cytosolic surface of the phospholipid bilayer, and this protein cargo is packaged into ptVLPs that further contain and use envelope glycoproteins to induce budding of the particle from the producing cell into the extracellular space. These human protein-derived domains and human proteins can promote localization of cargo to the cytosolic surface of the plasma membrane in ptVLP-producing cells, and also enable localization of cargo to the nucleus of ptVLP-transduced cells without the use of exogenous retroviral gag / pol, or chemical and / or light-based dimerization systems. For example, Cas9 can be delivered as cargo into particles much more efficiently by fusion to a phospholipid bilayer recruitment domain than without fusion to a phospholipid bilayer recruitment domain.

[0035] Targeted Domains To alter the tropism of VLPs, VLPs (e.g., ptVLPs) are provided herein that include a targeting domain that binds to an antigen on a target cell. Numerous such antigens are known in the art. Exemplary antigens include CD19 70 , asialoglycoprotein receptor 1 (ASGR1) 71 , transferrin receptor (TfR) 72 , HER2 73 , CD34 74 , CD4 75 , CD25 76 , CTLA-4 77 , HB-EGF 78 , ACE279 , aryl hydrocarbon receptor (AhR) 80 , keratin 5 (KRT5) 81 , keratin 17 (KRT17) 82 , keratin 14 (KRT14) 83 , keratin 13 (KRT13) 84 , neural cell adhesion molecule L1 85 , fibronectin (FN1) 86、87、88 , amyloid precursor protein (APP) 89 , programmed cell death protein 1 (PD-1) 90、91 , neurotrophin receptor (p75NTR) 92 , Thy-1 / CD90 93 , EpCAM 94 , and / or CFTR 95 Includes:

[0036] The ptVLP targeting domain can include a targeting peptide, a single chain variable fragment (scFv), a nanobody, a fibronectin type 3 domain (FN3), an arginylglycylaspartic acid motif (RGD), a single variable domain on the heavy chain / nanobody (VHH), a variable domain of a novel antigen receptor (VNAR), a darpin, or other targeting ligand that binds to an antigen on the target cell. 47~53 .

[0037] Targeting domains can also include, for example, the peptides shown in Table A.

[0038] The targeting domain can be inserted into the sequence of the envelope protein so that it is displayed on the surface of the ptVLP described herein, or it can exist as a separate molecule anchored on the outside of the ptVLP membrane.Thus, the present invention provides a fusion protein comprising (i) a targeting domain and an envelope glycoprotein, or (ii) a targeting domain and a membrane anchor, and a nucleic acid encoding the fusion protein.In some embodiments, the targeting domain is inserted between the signal sequence and the transmembrane domain of the ENV protein, and optionally replaces part or most of the N-terminus of the ENV, including the RBD.

[0039] The membrane anchor can be any transmembrane (TM) domain, e.g., platelet-derived growth factor receptor (PDGFR). 96 , CD9 97 , CD63 97 , CD81 97 , CD86, Notch 70 , CD28 98 , CD8 99 , or CD4 100 Generally, membrane-tethered targeting domain fusion proteins comprise, from N- to C-terminus, the following: secretory signal sequence-optional linker-targeting domain-optional linker-transmembrane domain (see, e.g., Figure 1). Preferably, the optional linker between the three domains is a polypeptide linker consisting primarily of glycines and serine, 5-20, e.g., 8-12, e.g., 10 amino acids in length.

[0040] [Table 1]

[0041] signal sequence Preferably, the membrane-tethered targeting domain and ptENV contain an N-terminal signal sequence, and the native signal sequence can be used or replaced with a heterologous signal sequence. Exemplary signal sequences include those from the VSV-G protein, such as MKCLLYLAFLFIGVNCK (SEQ ID NO: 1), and / or any other secretory signal sequence from VSVG (e.g., MKCLLYLAFLFIGVNC, SEQ ID NO: 2), or their homologs, or those from transmembrane proteins and / or synthetic / engineered signal sequences. A number of secretory signal sequence peptides, including human signal sequences, are known in the art, examples of which are shown in Table B (Table adapted from novoprolabs.com / support / articles / commonly-used-leader-peptide-sequences-forefficient-secretion-of-a-recombinant-protein-expressed-in-mammalian-cells-201804211337.html).

[0042] [Table 2]

[0043] In some embodiments, an alternative signal sequence is used to promote secretion, e.g., as described in Table 5 of U.S. Pat. No. 1,099,3967; von Heijne, J. Mol. Biol. 1985 Jul. 5;184(1)99-105; Kober et al., Biotechnol. Bioeng. 2013;110 1164-1173; Tsuchiya et al., Nucleic Acids Research Supplement No. 3 261 -262 (2003).

[0044] Generally, signal peptides are cleaved by signal peptidases after the nascent protein is inserted into the membrane as part of the cell's intrinsic secretory pathway processing.

[0045] ptVLP-mediated delivery of DNA, protein, and RNA The ptVLPs described herein can package and deliver biomolecular cargoes. ptVLP. "Cargo" refers to any payload that can be delivered, including chemicals, such as small molecule compounds, and combinations of DNA and RNPs, RNPs, DNA and protein combinations, or proteins, including combinations thereof, as well as biomolecules, including viruses and their parts, for example, for therapeutic or diagnostic applications, or for genome editing, epigenetic regulation, and / or transcriptome regulation applications. In this regard, RNA includes, for example, single guide RNA (sgRNA), clustered regularly interspaced short palindromic repeats (CRISPR) RNA (crRNA), and / or mRNA encoding the cargo. Other exemplary nucleic acids may include specialized single-stranded and / or double-stranded DNA molecules (e.g., plasmids, minicircles, linear closed-end DNA, AAV DNA, episomes, bacteriophage DNA, homology-directed repair templates, etc.), single-stranded and / or double-stranded RNA molecules (e.g., single guide RNAs, prime-editing guide RNAs, crRNAs, tracrRNAs, messenger RNAs, transfer RNAs, long non-coding RNAs, circular RNAs, RNA replicons, circular or linear splicing RNAs, microRNAs, small interfering RNAs, short hairpin RNAs, piwi-interacting RNAs, toehold switch RNAs, RNAs to which an RNA-binding protein can bind, bacteriophage RNAs, or internal ribosome entry site-containing RNAs). Combinations of the foregoing cargoes (e.g., AAV particles, and / or ribonucleoprotein (RNP) complexes containing RNA and protein, e.g., guide RNA / CRISPR Cas protein complexes) may also be included.

[0046] As used herein, "small molecule" refers to a small organic or inorganic molecule with a molecular weight of less than about 3,000 Daltons. Generally, small molecules useful in the present invention have a molecular weight of less than 3,000 Daltons (Da). Small molecules can be, for example, at least about 100 Da to about 3,000 Da (e.g., between about 100 and about 3,000 Da, between about 100 and about 2500 Da, between about 100 and about 2,000 Da, between about 100 and about 1,750 Da, between about 100 and about 1,500 Da, between about 100 and about 1,250 Da, between about 100 and about 1,000 Da, between about 100 and about 750 Da, between about 100 and about 500 Da, between about 200 and about 1500, between about 500 and about 1000, between about 300 and about 1000 Da, or between about 100 and about 250 Da).

[0047] In some embodiments, the cargo is limited by the diameter of the particle, which, for example, in some embodiments, can range from 30 nm to 500 nm.

[0048] In some embodiments, for example, when ptVLPs are produced via transient transfection of a production cell line, the cargo may comprise a combination of DNA and RNA. DNA transfected into cells has size-dependent mobility, with a fraction of the transfected DNA remaining in the cytosol, while another fraction of the transfected DNA will localize to the nucleus. 44~46 A fraction of the transfected DNA in the nucleus expresses the components encoded on those plasmids necessary to generate ptVLPs, while another fraction in the cytosol / near the plasma membrane is packaged and delivered within ptVLPs. See, e.g., Figures 1-4 of WO 2022 / 020800.

[0049] Cargos developed for genome or gene editing applications further include CRISPR-Cas nucleases, as well as fusions and variants thereof, such as prime editors and base editors. Nucleases include ZFNs and transcription activator-like effector nucleases (TALENs) having a FokI or AcuI nuclease domain; and CRISPR Cas proteins or functional derivatives thereof (e.g., as shown in Table 2) (ZFNs are described, e.g., in U.S. Patent Application Publication Nos. 20030232410; 20050208489; 20050026157; 20050064474; 20060188987; 20060063231; and WO 07 / 014275). (TALENs are described, e.g., in U.S. Patent Application Publication Nos. 9393257; and WO 2014134412). (CRISPR Cas proteins are described, e.g., in U.S. Patent Application Publication Nos. 9393257; and WO 2014134412). Cas proteins are described, for example, in U.S. Patent Publication No. 8,697,359; U.S. Patent Application Publication No. 20180208976; and International Publication Nos. 2014093661; 2017184786. 34~36 The base editor can be any CRISPR-based nuclease, e.g., any of those shown in Table 2, that contain a deaminase domain embedded internally using a polypeptide linker of variable length, with or without C-terminal fusion to one or more uracil glycosylase inhibitors (UGIs), or that are fused at the N-terminus to a nucleotide deaminase or nucleoside deaminase, or functional derivatives thereof (e.g., as shown in Table 3). and PCT Publication Nos. 20150166982; 20180312825; U.S. Patent No. 10,113,163; and WO 2015089406; 2018218188; 2017070632; 2018027078; 2018165629; and WO 2018 / 218166). 37、38Additionally, prime editors are also compatible with mVLP delivery methods (prime editors are described, for example, in Anzalone et al., Nature. 2019 Dec;576(7785)149-157). Prime editors can be delivered, for example, as a fusion of a Cas nickase to a reverse transcriptase or as a separate component (see, for example, Grunewald et al., Nat Biotechnol. 2022 Sep 26. doi 10.1038 / s41587-022-01473-1; and Liu et al., Nat Biotechnol. 2022 Sep;40(9)1388-1393).

[0050] Cargos designed for epigenetic regulation include CRISPR Cas proteins, zinc fingers (ZFs), and TALEs fused to epigenetic / epigenetic regulators, or combinations of epigenetic / epigenetic regulators or their functional derivatives, linked by one or more polypeptide linkers of variable length. Exemplary epigenetic regulators include CRISPR-Cas proteins (e.g., nickases, or catalytically inactive Cas) fused to DNA methylases, histone acetyltransferases, and deacetylases, and transcriptional activators or repressors (see, e.g., Tables 2 and 4). Examples include, for example, transcriptional repressors (e.g., KRAB, ERD, SID, and others, e.g., amino acids 473-530 of the ets2 repressor factor (ERF) repressor domain (ERD), amino acids 1-97 of the KRAB domain of KOX1, amino acids 1-36 of the Mad mSIN3-interacting domain (SID); Beerli et al., PNAS USA 9514628-14633 (1998)), or silencers, e.g., heterochromatin protein 1 (HP1, also known as swi6), e.g., HP1α or HP1β; proteins or peptides that can recruit long non-coding RNAs (lncRNAs) fused to fixed RNA-binding sequences, such as those bound by MS2 coat protein, endoribonuclease Csy4, or lambda N protein; enzymes that modify the methylation status of DNA (e.g., DNA methyltransferases (DNMTs) or TET proteins); or enzymes that modify histone subunits (e.g., histone acetyltransferases (HATs), histone deacetylases (HDACs), histone methyltransferases (e.g., methylation of lysine or arginine residues), or histone demethylases (e.g., demethylation of lysine or arginine residues)). In some embodiments, the sequence of the cargo is at least 95% identical to a sequence described herein.

[0051] The sgRNA can be complexed with the genome editing reagent during the packaging process for co-delivery within the ptVLP. Additionally, the linear or circular RNA encoding the cargo or edit to be introduced by the prime editor can be co-packaged with the genome editing reagent fused to an RNA-binding protein, such as MS2, PP7, COM, or TAR hairpin-binding protein (TBP) or human SLBP. Cargoes designed for transcriptome editing include a CRISPR Cas protein or any functional derivative thereof (e.g., as shown in Table 5), or a CRISPR Cas protein or any functional derivative thereof (e.g., as shown in Table 5) fused to a nucleotide or nucleoside deaminase (e.g., as shown in Table 3) via one or more variable-length polypeptide linkers.

[0052] Cargo can also comprise any therapeutically or diagnostically useful protein, DNA, RNP, or the combination of DNA, protein and / or RNP.See, for example, International Publication No. 2014005219; US Patent No. 10137206; US Patent Application Publication No. 20180339166; US Patent No. 5892020; European Patent No. 2134841; International Publication No. 2007020965.For example, the cargo that encodes or consists of nuclease, or base editor protein, or RNP, or their derivatives, can be delivered to retinal cells to correct the spline site defect that causes Leber congenital amaurosis type 10. In the mammalian inner ear, ptVLP delivery of base editing reagents or HDR-promoting cargoes to sensory cells, such as cochlear supporting cells and hair cells, with the aim of editing β-catenin (editing β-catenin Ser33 to Tyr, Pro, or Cys) to improve β-catenin stabilization can promote regression of hearing loss.

[0053] In another application, ptVLP delivery of RNA editing or proteome-perturbing reagents can cause a transient reduction in cellular levels of one or more specific proteins of interest (potentially at the systemic level, in a specific organ, or in a specific subset of cells, such as a tumor), which can create a therapeutic window during which a second drug can be administered (which second drug is more effective in the absence or presence of low levels of the protein of interest). For example, ptVLP delivery of RNA editing or proteome-perturbing reagents can induce targeted degradation of MAPK and PI3K / AKT proteins and associated mRNAs in vemurafenib / dabrafenib-resistant, BRAF-driven tumor cells, which can provide an opportunity for administration of vemurafenib / dabrafenib because it temporarily abolishes BRAF inhibitor resistance (the resistance mechanism based on the MAPK / PI3K / AKT pathway is temporarily downregulated by the ptVLP cargo). This example is particularly relevant when combined with ptVLPs that are antigen-inducing and therefore specific to tumor cells. Alternatively, the transient reduction in cellular levels of a particular protein of interest may itself have therapeutic utility.

[0054] In some embodiments, ptVLPs can be used to deliver factors, including, for example, the Yamanaka factors Oct3 / 4, Sox2, Klf4, and c-Myc, to cells such as human or mouse fibroblasts, for the purpose of generating induced pluripotent stem cells or to deliver factors that induce forward differentiation or transdifferentiation into specific cell types.

[0055] In some embodiments, the ptVLP may deliver a dominant negative form of a protein to elicit a therapeutic effect.

[0056] Antigen-specific (e.g., tumor antigen-specific) ptVLPs can be targeted to cancer cells to deliver the pro-apoptotic proteins BIM, BID, PUMA, NOXA, BAD, BIK, BAX, BAK, and / or HRK to induce apoptosis of the cancer cells. Tumor antigens are known in the art.

[0057] Ninety percent of patients with pancreatic cancer present with unresectable disease. Approximately 30% of patients with unresectable pancreatic tumors die from local disease progression; therefore, it is desirable to treat locally advanced pancreatic tumors with ablative radiation; however, the intestinal tract cannot tolerate the high doses of radiation required to cause tumor ablation. Selective radioprotection of the intestinal tract would allow ablative radiotherapy of pancreatic tumors while minimizing damage to the surrounding gastrointestinal tract. Toward this end, ptVLPs can carry dCas9 fused to the transcriptional repressor KRAB and a guide RNA targeting EGLN. EGLN inhibition has been shown to significantly reduce the gastrointestinal toxicity of ablative radiation treatments, resulting in selective radioprotection of the gastrointestinal tract but not the pancreatic tumor. 54 Such fusion proteins, ptVLPs, and methods of making and using such fusion proteins, ptVLPs, are provided herein.

[0058] Unbound steroid receptors reside in the cytosol. After ligand binding, these receptors translocate to the nucleus and initiate transcription of response genes. ptVLPs can deliver single-chain variable fragment (scFv) antibodies into the cytosol of cells that bind to and destroy cytosolic steroid receptors. For example, scFvs can bind to the glucocorticoid receptor and prevent its binding to dexamethasone, which can prevent transcription of response genes, such as metallothionein 1E, which has been associated with tumorigenesis. 55 .

[0059] ptVLPs can be adapted for treatments involving targeted destruction of proteins. For example, ptVLPs can be utilized to target and destroy proteins in the cytosol of cells by delivering antibodies / scFvs to the cytosol of cells. Traditionally, delivery of antibodies across the plasma membrane to the cytosol of cells is notoriously difficult and inefficient. This mode of protein inhibition is similar to how targeted small molecules bind to and destroy proteins in the cytosol, and can be useful in treating a wide variety of diseases. 56~58 Included herein are such fusion proteins, ptVLPs, and methods of making and using such fusion proteins, ptVLPs.

[0060] Furthermore, targeting of small molecules is limited to proteins of a certain size that contain binding pockets associated with catalytic functions or protein-protein interactions. This limitation does not prevent scFvs, because scFvs can be generated that bind to many different parts of a protein to disrupt catalysis and interactions with other proteins. For example, the RAS oncoprotein has been implicated across numerous cancer subtypes, and RAS is one of the most frequently observed oncogenes in cancer. For example, the International Cancer Genome Consortium found that KRAS was mutated in 95% of its pancreatic adenocarcinoma samples. RAS isoforms are known to activate various pathways that are dysregulated in human cancers, such as the PI3K and MAPK pathways. Despite the abnormal role RAS plays in cancer, no effective, pharmacological, direct or indirect small molecule inhibitors of RAS have been developed or approved for clinical use. One strategy for targeting RAS could be ptVLPs, which can specifically deliver scFvs that bind to multiple RAS isoforms and disrupt their function to cancer cells. 56~58 .

[0061] Composition, production, purification, and applications of ptVLPs ptVLPs can be produced from a producer cell line that is transiently transfected with at least one plasmid or stably expresses a construct that is integrated into the genomic DNA of the producer cell line.Thus, in some embodiments, the ptVLPs described herein can be produced by integrating all of the production DNA constructs into the genomic DNA of the producer cell line, and can package protein-based cargo.Once a cell line (e.g., a producer line) is created, protein-delivery ptVLPs can be produced constitutively or inducibly.

[0062] Alternatively, some or all of the components for producing ptVLPs may be transiently expressed. In some embodiments, for ptVLPs, a single plasmid is used in transfection that includes one or more transmembrane envelope glycoproteins (e.g., with or without the specified mutations / truncations and / or targeting domain fusions described herein) (e.g., unmodified envelopes are shown in Table 1), or transmembrane envelope glycoproteins containing membrane-tethered targeting domains in trans, with or without the specified mutations / truncations, cargo (e.g., therapeutic proteins or gene editing reagents, such as zinc fingers, transcription activator-like effectors (TALEs), and / or CRISPR-based genome editing / regulatory proteins and RNPs, as found in Tables 2, 3, 4, and 5), with or without fusion to a plasma membrane recruitment domain (e.g., shown in Table 6), and, if necessary, at least one guide RNA encoding sequence.

[0063] In some embodiments, transient transfections use two to three plasmids, which may include the following (any two or more components listed here may be combined in a single plasmid): 1. A plasmid containing a sequence encoding a cargo, e.g., a therapeutic protein or genome editing reagent, with or without fusion to a plasma membrane recruitment domain. 2. A plasmid containing one or more target envelope glycoproteins containing the specified mutations / truncations and / or targeting domain fusions (e.g., unmodified envelopes are listed in Table 1). 3. If the genome editing reagent in Plasmid 1 requires one or more guide RNAs, a plasmid containing one or more guide RNAs appropriate for the genome editing reagent in Plasmid 1. Additionally, three, four, or more plasmids may be used in transient transfections. These four or more plasmids may include the following (any two or more components may be combined in a single plasmid): 1. A plasmid containing a sequence encoding a cargo, e.g., a therapeutic protein or genome editing reagent, with or without fusion to a plasma membrane recruitment domain. 2. A plasmid containing one or more envelope glycoproteins containing the specified mutations / truncations (e.g., listed in Table 1). 3. For example, if the envelope glycoprotein does not contain a targeting domain, a plasmid containing one or more membrane-tethered targeting domains (e.g., a targeting peptide, scFv, nanobody, FN3, RGD, VHH, VNAR, darpin or other targeting ligand) (although in some embodiments, two or more different targeting domains are used in the ENV and / or as separate membrane-tethered targeting domains). 4. If the genome editing reagent in Plasmid 1 requires one or more guide RNAs, a plasmid containing one or more guide RNAs appropriate for the genome editing reagent in Plasmid 1. If delivery of certain DNA molecules other than plasmids is desired, the transfection can be performed using double-stranded linear closed-end DNA, episomes, minicircles, double-stranded oligonucleotides, and / or other specialized / modified DNA, RNA, AAV, adenovirus, anellovirus, or peptide nucleic acid (PNA) molecules. Alternatively, for ptVLPs, producer cell lines can be generated that stably express one or more of the constructs (1 to 3) described in the transfection procedure above.

[0064] In some embodiments, methods for producing ptVLPs can include using cells that have been engineered to express any exogenous proteins other than the target viral envelope protein containing the targeting domain fusion, or the viral envelope containing the relevant targeting domain in trans, with or without the specified mutations / truncations (e.g., as shown in Table 1), and, if desired, the plasma membrane recruitment domain (e.g., as shown in Table 6), or cells that do not express cargo. In this embodiment, the "empty" particles produced can be loaded with cargo and / or small molecules by incubation of purified particles mixed with cargo, nucleofection, lipid, polymer, or CaCl transfection, sonication, freeze-thawing, and / or heat shock. In all embodiments, the producing cells do not express any exogenous gag protein. This type of loading allows the cargo to be unmodified by fusion to the plasma membrane recruitment domain, a significant improvement over previous VLP technology.

[0065] Plasmids or other types of specialized DNA molecules known in the art or described herein may also preferably include other elements that drive expression or translation of the encoded sequence, such as a promoter sequence; an enhancer sequence, e.g., in a 5' untranslated region (UTR) or 3' UTR; a polyadenylation site; an IRES; a 2A peptide; an insulator sequence; or another sequence that increases or controls expression (e.g., an inducible promoter element).

[0066] Suitable producer cell lines may include primary or stable human cell lines that are resistant to the effects of transfection reagents and fusogenic effects caused by viral glycoproteins. Examples of suitable cell lines include human embryonic kidney (HEK) 293 cells, HEK293 T / 17 SF cells, kidney-derived Phoenix-AMPHO cells, and placenta-derived BeWo cells. For example, such cells may be selected for their ability to grow as adherent or suspension cells. In some embodiments, producer cells may be cultured in classic DMEM under serum, serum-free, or exosome-free serum conditions. ptVLPs may be produced from patient-derived cells (autologous ptVLPs) and other FDA-approved cell lines (allogeneic ptVLPs), as long as the cells can be transfected with DNA constructs encoding the ptVLP-producing components described above by various techniques known in the art.

[0067] Furthermore, if desired, two or more genome editing reagents encoded in the polynucleic acid construct can be included in the transfection. The DNA construct can be designed to overexpress proteins in the production cell line. For example, the plasmid backbone used in transfection can be known to those skilled in the art, such as a pCDNA3 backbone that uses a CMV promoter for RNA polymerase II transcription or a U6 promoter for RNA polymerase III transcription. Various techniques known in the art can be used to introduce polynucleic acids into production cells. Such techniques include chemically promoted transfection using compounds such as calcium phosphate, cationic lipids, and cationic polymers; liposome-mediated transfection, such as cationic liposomes like LIPOFECTAMINE (LIPOFECTAMINE 2000 or 3000, and TransIT-X2); polyethyleneimine; and non-chemical methods such as electroporation, particle bombardment, or microinjection.

[0068] Human producer cell lines that stably express the necessary ptVLP components constitutively and / or inducibly can be used for the production of ptVLPs. ptVLPs can be produced from patient-derived cells (autologous ptVLPs) and other FDA-approved cell lines (allogeneic ptVLPs), as long as the cells have been converted into stable cell lines that express the ptVLP components.

[0069] The production cells themselves are also provided herein.

[0070] Production of cargo-loaded ptVLPs and compositions Preferably, ptVLPs are harvested from the cell culture medium supernatant 36-48 hours after transfection, or when the ptVLPs are at maximum concentration in the producer cell medium (the producer cells release particles into the medium, and at some point the particle concentration in the medium becomes optimal for particle harvesting). The supernatant can be purified by any method known in the art, such as centrifugation, ultracentrifugation, sedimentation, ultrafiltration, tangential flow filtration, and / or chromatography. In some embodiments, the supernatant is first filtered, for example, through a 0.45 μm pore size polyvinylidene fluoride hydrophilic membrane (Millipore's Millex-HV) or a 0.8 μm pore size mixed cellulose ester hydrophilic membrane (Millipore's Millex-AA), to remove particles, for example, larger than 1 μm. After filtration, the supernatant can be further purified and concentrated using, for example, ultracentrifugation, e.g., at 80,000-100,000 x g for 1-2 hours at a temperature between 1°C and 5°C, or at 8,000-15,000 g for 10-16 hours at a temperature between 1°C and 5°C. After this centrifugation step, the ptVLPs are concentrated in the form of a centrifugate (pellet), which can be resuspended to a desired concentration, mixed with a transduction-enhancing reagent, buffer-exchanged, or used as is. In some embodiments, the ptVLP-containing supernatant can be filtered, precipitated, centrifuged, and resuspended into a concentrated solution. To precipitate the particles, for example, polyethylene glycol (PEG), e.g., PEG 8000, or antibody-bead conjugates that bind to ptVLP surface proteins or membrane components can be used. The purified particles are stable and can be stored at 4°C for up to one week or at -80°C for years without any apparent loss of activity.

[0071] Preferably, the ptVLPs are resuspended or subjected to buffer exchange, and the particles are suspended in a suitable carrier. In some embodiments, buffer exchange can be performed by ultrafiltration (e.g., Vivaspin 500 MWCO 100,000, Sartorius). An exemplary suitable carrier for ptVLPs that can be used for in vitro applications would preferably be a cell culture medium appropriate for the cells to be transduced by the ptVLPs. Transduction-enhancing reagents that can be mixed into purified and concentrated ptVLP solutions for in vitro applications include those known to those skilled in the art (e.g., Vectofusin-1, Miltenyi Biotec; Polybrene, Millipore; Retrone, Takara; Protamine sulfate, Sigma, etc.). After applying the ptVLPs in a suitable carrier to the cells to be transduced, the transduction efficiency can be further increased by centrifugation. Preferably, the plate containing the ptVLPs applied to the cells can be centrifuged at a speed of 1,150 g at room temperature for 30 minutes. After centrifugation, the cells are returned to a suitable cell culture incubator (eg, a humidified incubator at 37° C. and 5% CO 2 ).

[0072] Suitable carriers for ptVLPs to be administered to mammals, particularly humans, will preferably be pharmaceutically acceptable compositions. A "pharmaceutically acceptable composition" refers to a non-toxic semi-solid, liquid, or aerosolized filler, diluent, encapsulating material, colloidal suspension, or any type of formulation auxiliary. Preferably, the composition is suitable for injection. They may be, in particular, sterile isotonic saline solutions (monosodium phosphate or disodium phosphate, sodium chloride, potassium chloride, calcium chloride, or magnesium chloride and similar solutions, or mixtures of such salts), or dry compositions, particularly lyophilized compositions, that can be made into an injectable solution upon addition of sterile water or sterile saline, as the case may be. Other suitable dosage forms would be aerosolized particles for administration by intranasal inhalation or endotracheal intubation.

[0073] Suitable dosage forms for injection include sterile aqueous solutions or suspensions. The solutions or suspensions may contain additives that are compatible with the ptVLP and do not interfere with the entry of the ptVLP into target cells. In either case, the dosage form must be sterile and fluid enough to be administered by syringe. The dosage form must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi. A suitable solution is a buffer solution, such as phosphate-buffered saline.

[0074] Methods for preparing suitable pharmaceutical compositions are known in the art, see, for example, Remington: The Science and Practice of Pharmacy, 21st ed., 2005; and Drugs and the Pharmaceutical Sciences: a Series of Textbooks and Monographs (Dekker, NY) series. For example, solutions or suspensions used for parenteral, intradermal, or subcutaneous administration may contain the following components: a sterile diluent, such as water for injection, saline solution, fixed oils, polyethylene glycol, glycerin, propylene glycol, or other synthetic solvents; an antibacterial agent, such as benzyl alcohol or methylparaben; an antioxidant, such as ascorbic acid or sodium bisulfite; a chelating agent, such as ethylenediaminetetraacetic acid; a buffer, such as acetate, citrate, or phosphate; and a tonicity adjuster, such as sodium chloride or glucose. pH can be adjusted using an acid or base, such as hydrochloric acid or sodium hydroxide. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.

[0075] Pharmaceutical compositions suitable for injection may include sterile, aqueous solutions (where water soluble) or dispersions, and sterile powders for the extemporaneous preparation of sterile, injectable solutions or dispersions. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL™ (BASF, Parsippany, NJ), or phosphate-buffered saline (PBS). In all cases, the composition must be sterile and fluid to the extent that easy syringability exists. The composition must be stable under the conditions of manufacture and storage and preserved against the contaminating action of microorganisms, such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, etc. In many cases, it is preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol and sorbitol, and sodium chloride in the composition. Prolonged absorption of injectable compositions can be achieved by including in the composition an agent that delays absorption, for example, aluminum monostearate and gelatin.

[0076] Sterile injectable solution can be prepared by incorporating active compound in the required amount in a suitable solvent that contains one or a combination of ingredients listed above, and then optionally sterilize by filtration.Generally, dispersion is prepared by incorporating active compound into a sterile vehicle that contains a basic dispersion medium and other necessary ingredients from those listed above.For sterile powder for preparing sterile injectable solution, the preferred method of preparation is vacuum drying and freeze-drying, which produces powder of active ingredient with any desired additional ingredients from the solution that has been previously sterile-filtered.

[0077] Compositions comprising cargo-loaded ptVLPs described herein can be included in a container, pack, or dispenser together with instructions for administration.

[0078] [Table 3]

[0079] [Table 4-1]

[0080] [Table 4-2]

[0081] [Table 4-3]

[0082] [Table 4-4]

[0083] [Table 4-5]

[0084] [Table 4-6]

[0085] [Table 5]

[0086] [Table 6]

[0087] [Table 7]

[0088] [Table 8]

[0089] [Table 9]

[0090] Exemplary Sequences In some embodiments, the protein or nucleic acid sequences used in the compositions or methods described herein are at least 80%, 85%, 90%, 95%, 97%, 98%, or 99% identical to the sequences described herein. To determine the percent identity of two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps may be introduced into one or both of the first and second amino acid or nucleic acid sequences for optimal alignment, and non-homologous sequences may not be considered for comparison purposes). In a preferred embodiment, the length of the reference sequence aligned for comparison purposes is at least 80%, and in some embodiments, at least 90% or 100% of the reference sequence. The amino acid residues or nucleotides at corresponding amino acid or nucleotide positions are then compared. If a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, the molecules are identical at that position (as used herein, amino acid or nucleic acid "identity" is equivalent to amino acid or nucleic acid "homology"). The percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps that need to be introduced for optimal alignment of the two sequences, and the length of each gap.

[0091] The comparison of sequences and the determination of percent identity between two sequences can be achieved using a mathematical algorithm. For example, the percent identity between two amino acid sequences can be determined using the algorithm of Needleman and Wunsch ((1970) J. Mol. Biol. 48444-453), which is incorporated into the GAP program in the GCG software package (available from the World Wide Web at gcg.com), and uses default parameters, such as a Blossum62 scoring matrix, a gap penalty of 12, a gap extension penalty of 4, and a frameshift gap penalty of 5.

[0092] Prime editor spCas9 H840A-MMLV reverse transcriptase (delta RNase H domain)

[0093] [ka]

[0094] Rat (Rattus norvegicus) and synthetic APOBEC1-XTEN L8-nspCas9-UGI-SV40 NLS

[0095] [ka]

[0096] Human (Homo sapiens) AID

[0097] [ka]

[0098] A human (Homo sapiens) AIDv soluble variant lacking the N-terminal RNA-binding domain

[0099] [ka]

[0100] A soluble variant of human (Homo sapiens) AIDv lacking the N-terminal RNA-binding domain and the C-terminal poorly structured region

[0101] [ka]

[0102] Rat (Rattus norvegicus) APOBEC1

[0103] [ka]

[0104] Mouse (Mus musculus) APOBEC3

[0105] [ka]

[0106] Mouse (Mus musculus) APOBEC3 catalytic domain

[0107] [ka]

[0108] Human (Homo sapiens) APOBEC3A

[0109] [ka]

[0110] Human (Homo sapiens) APOBEC3G

[0111] [ka]

[0112] Human (Homo sapiens) APOBEC3G catalytic domain

[0113] [ka]

[0114] Human (Homo sapiens) APOBEC3H

[0115] [ka]

[0116] Human (Homo sapiens) APOBEC3F

[0117] [ka]

[0118] Human (Homo sapiens) APOBEC3F catalytic domain

[0119] [ka]

[0120] Escherichia coli TadA

[0121] [ka]

[0122] Human (Homo sapiens) Adar1

[0123] [ka]

[0124] Human (Homo sapiens) Adar2

[0125] [ka]

[0126] Streptococcus pyogenes Cas9 bisecting NLS

[0127] [ka]

[0128] Staphylococcus aureus Cas9

[0129] [ka]

[0130] Campylobacter jejuni Cas9

[0131] [ka]

[0132] Neisseria meningitidis Cas9

[0133] [ka]

[0134] Acidaminococcus species Cas12a

[0135] [ka]

[0136] Lachnospiraceae bacterium Cas12a

[0137] [ka]

[0138] Leptotrichia shahii Cas13a

[0139] [ka]

[0140] Leptotrichia wadei Cas13a

[0141] [ka]

[0142] The pleckstrin homology domain of human ARNO

[0143] [ka]

[0144] Pleckstrin homology domain of human ARNO R279C

[0145] [ka]

[0146] FYVE domain of human EEA1 DNEVQNCMACGKGFSVTVRRHHCRQCGNIFCAECSAKNALTPSSKKPVRVCDACFNDLQ (SEQ ID NO: 43)

[0147] FYVE domain of human EEA1 R1375L

[0148] DNEVQNCMACGKGFSVTVRRHHCLQCGNIFCAECSAKNALTPSSKKPVRVCDACFNDLQ (SEQ ID NO: 44)

[0149] PX domain of p40phox (NCF4)

[0150] [ka]

[0151] p40 phox PX domain of (NCF4)R58L

[0152] [ka]

[0153] The pleckstrin homology domain of human (Homo sapiens) DAPP1

[0154] [ka]

[0155] The pleckstrin homology domain of human (Homo sapiens) GRP1 (CYTH3)

[0156] [ka]

[0157] The pleckstrin homology domain of human (Homo sapiens) GRP1 (CYTH3) R284C

[0158] [ka]

[0159] Pleckstrin homology domain of human OSBP1

[0160] [ka]

[0161] Pleckstrin homology domain of human OSBP1 R108E

[0162] [ka]

[0163] The pleckstrin homology domain of human Btk1

[0164] [ka]

[0165] Pleckstrin homology domain of human Btk1 R28C

[0166] [ka]

[0167] The pleckstrin homology domain of human FAPP1 MEGVLYKWTNYLTGWQPRWFVLDNGILSYYDSQDDVCKGSKGSIKMAVCEIKVHSADNTRMELIIPGEQHFYMKAVNAAERQRWLVALGSSKACLTDT (SEQ ID NO: 54)

[0168] The pleckstrin homology domain of human CERT PVERCGVLSKWTNYIHGWQDRWVVLKNNALSYYKSEDETEYGCRGSICLSKAVITPHDFDECRFDISVNDSVWYLRAQDPDHRQQWIDAIEQHKT (SEQ ID NO: 55)

[0169] The pleckstrin homology domain of human PHLPP1

[0170] [ka]

[0171] The pleckstrin homology domain of human SWAP70 MDVLKQGYMMKKGHRRKNWTERWFVLKPNIISYYVSEDLKDKKGDILLDENCCVESLPDKDGKKCLFLVKCFDKTFEISASDKKKKQEWIQAIHSTIH (SEQ ID NO: 57)

[0172] Pleckstrin homology domain of human SWAP70 R223E and R224E MDVLKQGYMMKKGHEEKNWTERWFVLKPNIISYYVSEDLKDKKGDILLDENCCVESLPDKDGKKCLFLVKCFDKTFEISASDKKKKQEWIQAIHSTIH (SEQ ID NO: 58)

[0173] The pleckstrin homology domain of human MAPKAP1

[0174] [ka]

[0175] Pleckstrin homology domain of human PKD

[0176] [ka]

[0177] Pleckstrin homology domain of human Son of Sevenless homolog 2

[0178] [ka]

[0179] The pleckstrin homology domain of human dynamin

[0180] [ka]

[0181] Pleckstrin homology domain of human BCR

[0182] [ka]

[0183] Pleckstrin homology domain of human DBS

[0184] [ka]

[0185] The pleckstrin homology domain of human (Homo sapiens) phospholipase C delta 1 (hPLC delta 1)

[0186] [ka]

[0187] The pleckstrin homology domain of human (Homo sapiens) phospholipase C delta 1 (hPLC delta 1) R40L

[0188] [ka]

[0189] The pleckstrin homology domain of human (Homo sapiens) Akt1 (hAkt)

[0190] [ka]

[0191] The pleckstrin homology domain of human (Homo sapiens) Akt1 (hAkt) E17K

[0192] [ka]

[0193] The pleckstrin homology domain of human (Homo sapiens) Akt1 (hAkt) K14R

[0194] [ka]

[0195] The pleckstrin homology domain of human (Homo sapiens) Akt1 (hAkt) K8R

[0196] [ka]

[0197] The pleckstrin homology domain of human (Homo sapiens) Akt1 (hAkt) T72A

[0198] [ka]

[0199] The pleckstrin homology domain of human (Homo sapiens) Akt1 (hAkt) T92A

[0200] [ka]

[0201] The pleckstrin homology domain of human (Homo sapiens) Akt1 (hAkt) R25C

[0202] [ka]

[0203] The pleckstrin homology domain of human (Homo sapiens) Akt1 (hAkt) T34D

[0204] [ka]

[0205] The pleckstrin homology domain of human (Homo sapiens) Akt1 (hAkt) T34F

[0206] [ka]

[0207] The pleckstrin homology domain of human (Homo sapiens) Akt1 (hAkt) T34L

[0208] [ka]

[0209] The pleckstrin homology domain of human (Homo sapiens) Akt1 (hAkt) T81Y

[0210] [ka]

[0211] Pleckstrin homology domains of human (Homo sapiens) Akt1 (hAkt) K142A, H143A, and R144A

[0212] [ka]

[0213] The pleckstrin homology domain of human (Homo sapiens) Akt1 (hAkt) T101C

[0214] [ka]

[0215] The pleckstrin homology domain of human (Homo sapiens) PDPK1 (hPDPK1)

[0216] [ka]

[0217] MS2 (RNA-binding protein)

[0218] [ka]

[0219] COM (RNA-binding protein) MKSIRCKNCNKLLFKADSFDHIEIRCPRCKRHIIMLNACEHPTEKHCGKREKITHSDETVRY (SEQ ID NO: 82)

[0220] PP7 (RNA-binding protein)

[0221] [ka]

[0222] TBP (RNA-binding protein)

[0223] [ka]

[0224] Human SLBP (RNA-binding protein) MADFETDESVLMRRQKQINYGKNTIAYDRYIKEVPRHLRQPGIHPKTPNKFKKYSRRSWDQQIKLWKVALHFWD (SEQ ID NO: 85)

[0225] Herpes simplex virus (HSV) type 1 VP16 transcription activation domain PTDALDDFDLDMLPADALDDFDLDMLPADALDDFDLDM (SEQ ID NO: 86)

[0226] Herpes simplex virus (HSV) type 1 and synthetic VP64 GRADALDDFDLDMLGSDALDDFDLDMLGSDALDDFDLDMLGSDALDDFDLDML (SEQ ID NO: 87)

[0227] Human (Homo sapiens) P65

[0228] [ka]

[0229] Kaposi's sarcoma-associated herpesvirus transactivator RTA

[0230] [ka]

[0231] Human (Homo sapiens) KRAB MDAKSLTAWSRTLVTFKDVFVDFTREEWKLLDTAQQIVYRNVMLENYKNLVSLGYQLTKPDVILRLEKGEEP (SEQ ID NO: 90)

[0232] Human (Homo sapiens) MeCP2

[0233] [ka]

[0234] Human (Homo sapiens) Tet1

[0235] [ka]

[0236] Human (Homo sapiens) Dnmt3a

[0237] [ka]

[0238] Vesicular stomatitis virus glycoprotein (VSVG) WT

[0239] [ka]

[0240] Vesicular stomatitis virus glycoprotein (VSVG) (K47A)

[0241] [ka]

[0242] Vesicular stomatitis virus glycoprotein (VSVG)(K47E)

[0243] [ka]

[0244] Vesicular stomatitis virus glycoprotein (VSVG) (K47G)

[0245] [ka]

[0246] Vesicular stomatitis virus glycoprotein (VSVG) (K47Q)

[0247] [ka]

[0248] Vesicular stomatitis virus glycoprotein (VSVG) (K47W)

[0249] [ka]

[0250] Vesicular stomatitis virus glycoprotein (VSVG) (K47A) (R354A)

[0251] [ka]

[0252] Vesicular stomatitis virus glycoprotein (VSVG) (K47E) (R354A)

[0253] [ka]

[0254] Vesicular stomatitis virus glycoprotein (VSVG) (K47G) (R354A)

[0255] [ka]

[0256] Vesicular stomatitis virus glycoprotein (VSVG) (K47Q) (R354A)

[0257] [ka]

[0258] Vesicular stomatitis virus glycoprotein (VSVG) (K47W) (R354A)

[0259] [ka]

[0260] Targeting domain-VSVG fusion site

[0261] [ka]

[0262] Vesicular stomatitis virus glycoprotein (VSVG truncated) (F421 truncated)

[0263] [ka]

[0264] Vesicular stomatitis virus glycoprotein (VSVG truncated) (F440 truncated) MKCLLYLAFLFIGVNCKFFGDTGLSKNPIELVEGWFSSWKSSIASFFFIIGLIIGLFLVLRVGIHLCIKLKHTKKRQIYTDIEMNRLGK (SEQ ID NO: 107)

[0265] Vesicular stomatitis virus glycoprotein (VSVG truncated) (F448 truncated) MKCLLYLAFLFIGVNCKKNPIELVEGWFSSWKSSIASFFFIIGLIIGLFLVLRVGIHLCIKLKHTKKRQIYTDIEMNRLGK (SEQ ID NO: 108)

[0266] Targeting domain-VSVG truncation fusion 421

[0267] [ka]

[0268] Targeting domain-VSVG truncation fusion 440

[0269] [ka]

[0270] Targeting domain-VSVG truncation fusion 448

[0271] [ka]

[0272] Amphotropic murine leukemia virus glycoprotein (AMLVG)WT

[0273] [ka]

[0274] Amphotropic murine leukemia virus glycoprotein (AMLVG)(D86K) receptor-binding domain mutant

[0275] [ka]

[0276] Amphotropic murine leukemia virus glycoprotein (AMLVG) (R domain deleted)

[0277] [ka]

[0278] Amphotropic murine leukemia virus glycoprotein (AMLVG) (furin-cleaved mutant)

[0279] [ka]

[0280] Amphotropic murine leukemia virus glycoprotein (AMLVG)(L640A)

[0281] [ka]

[0282] Amphotropic murine leukemia virus glycoprotein (AMLVG)(Y644A) endocytosis signal mutant

[0283] [ka]

[0284] Targeting domain-AMLVG fusion site

[0285] [ka]

[0286] 10A1 murine leukemia virus glycoprotein (10A1MLVG)WT

[0287] [ka]

[0288] 10A1 murine leukemia virus glycoprotein (10A1MLVG)(D86K) receptor-binding domain mutant

[0289] [ka]

[0290] 10A1 murine leukemia virus glycoprotein (10A1MLVG) (R domain deleted)

[0291] [ka]

[0292] 10A1 murine leukemia virus glycoprotein (10A1MLVG) (furin truncation mutant)

[0293] [ka]

[0294] 10A1 murine leukemia virus glycoprotein (10A1MLVG) (L631A)

[0295] [ka]

[0296] 10A1 murine leukemia virus glycoprotein (10A1MLVG)(Y635A) endocytosis signal mutant

[0297] [ka]

[0298] Targeting domain-10A1MLVG fusion site

[0299] [ka]

[0300] Influenza A (FPV) / Rostock / 1934, H7 subtype virus hemagglutinin WT

[0301] [ka]

[0302] Influenza A (FPV) / Rostock / 1934, H7 subtype virus hemagglutinin (Y106F) (E199Q) (G237K)

[0303] [ka]

[0304] Influenza A (FPV) / Rostock / 1934, H7 subtype virus hemagglutinin (furin cleavage mutation)

[0305] [ka]

[0306] Influenza A / Puerto Rico / 8 / 34, N1 subtype, neuraminidase WT

[0307] [ka]

[0308] Influenza A / Puerto Rico / 8 / 34, N1 subtype neuraminidase (T55A)

[0309] [ka]

[0310] Sindbis virus glycoprotein (SINVG) WT

[0311] [ka]

[0312] Sindbis virus glycoprotein (SINVG) triple mutant, E3 (61-64del), E2 (68SLEQ71 to 68AAAA71), E2 (159KE160 to 159AA160)

[0313] [ka]

[0314] Sindbis virus glycoprotein (SINVG) triple mutant containing an HA tag, E3 (61-64del), E2 (68SLEQ71 to 68AAAA71), and E2 (159KE160 to 159AA160)

[0315] [ka]

[0316] Sindbis virus glycoprotein (SINVG) triple mutant containing the targeting domain fusion site: E3(61-64del), E2(68SLEQ71 to 68AAAA71), and E2(159KE160 to 159AA160).

[0317] [ka]

[0318] Sindbis virus glycoprotein (SINVG) triple mutant version 2, E3 (61-64del), E2 (68SLEQ71 to 68AAAA71), and E2 (159KE160 to 159AA160)

[0319] [ka]

[0320] Sindbis virus glycoprotein (SINVG) triple mutant version 2 containing an HA tag, E3 (61-64del), E2 (68SLEQ71 to 68AAAA71), and E2 (159KE160 to 159AA160).

[0321] [ka]

[0322] Sindbis virus glycoprotein (SINVG) triple mutant version 2 containing the targeting domain fusion site, E3 (61-64del), E2 (68SLEQ71 to 68AAAA71), and E2 (159KE160 to 159AA160).

[0323] [ka]

[0324] Sindbis virus glycoprotein (SINVG) quadruple mutant, E3 (61-64del), E2 (68SLEQ71 to 68AAAA71), E2 (159KE160 to 159AA160), E1 (250AK251 to 250SG251)

[0325] [ka]

[0326] Sindbis virus glycoprotein (SINVG) quadruple mutant containing an HA tag: E3 (61-64del), E2 (68SLEQ71 to 68AAAA71), E2 (159KE160 to 159AA160), and E1 (250AK251 to 250SG251).

[0327] [ka]

[0328] Sindbis virus glycoprotein (SINVG) quadruple mutant containing targeting domain fusion sites: E3 (61-64del), E2 (68SLEQ71 to 68AAAA71), E2 (159KE160 to 159AA160), and E1 (250AK251 to 250SG251).

[0329] [ka]

[0330] Sindbis virus glycoprotein (SINVG) quadruple mutant version 2, E3 (61-64del), E2 (68SLEQ71 to 68AAAA71), E2 (159KE160 to 159AA160), and E1 (250AK251 to 250SG251)

[0331] [ka]

[0332] Sindbis virus glycoprotein (SINVG) quadruple mutant version 2 containing an HA tag, E3 (61-64del), E2 (68SLEQ71 to 68AAAA71), E2 (159KE160 to 159AA160), and E1 (250AK251 to 250SG251).

[0333] [ka]

[0334] Sindbis virus glycoprotein (SINVG) quadruple mutant version 2 containing targeting domain fusion sites: E3 (61-64del), E2 (68SLEQ71 to 68AAAA71), E2 (159KE160 to 159AA160), and E1 (250AK251 to 250SG251).

[0335] [ka]

[0336] Measles virus hemagglutinin (MeV H) WT

[0337] [ka]

[0338] Measles virus hemagglutinin (MeV H) delta 18

[0339] [ka]

[0340] Measles virus hemagglutinin (MeV H) delta18 double mutant (Y463A)(R515A)

[0341] [ka]

[0342] Measles virus hemagglutinin (MeV H) delta18 double mutant (Y463A)(R515A) containing a targeting domain fusion site

[0343] [ka]

[0344] Measles virus hemagglutinin (MeV H) delta18 quadruple mutant (Y463A) (R515A) (530SF531 to 530LS531)

[0345] [ka]

[0346] Measles virus hemagglutinin (MeV H) delta18 quadruple mutant (Y463A) (R515A) (530SF531 to 530LS531) containing a targeting domain fusion site

[0347] [ka]

[0348] Measles virus hemagglutinin (MeV H) delta 19

[0349] [ka]

[0350] Measles virus hemagglutinin (MeV H) delta19 double mutant (Y463A)(R515A)

[0351] [ka]

[0352] Measles virus hemagglutinin (MeV H) delta19 double mutant (Y463A)(R515A) containing a targeting domain fusion site

[0353] [ka]

[0354] Measles virus hemagglutinin (MeV H) delta19 quadruple mutant (Y463A) (R515A) (530SF531 to 530LS531)

[0355] [ka]

[0356] Measles virus hemagglutinin (MeV H) delta19 quadruple mutant (Y463A) (R515A) (530SF531 to 530LS531) containing a targeting domain fusion site

[0357] [ka]

[0358] Measles virus hemagglutinin (MeV H) delta 24AAAA

[0359] [ka]

[0360] Measles virus hemagglutinin (MeV H) delta 24AAAA double mutant (Y463A) (R515A)

[0361] [ka]

[0362] Measles virus hemagglutinin (MeV H) delta 24AAAA double mutant (Y463A)(R515A) containing a targeting domain fusion site

[0363] [ka]

[0364] Measles virus hemagglutinin (MeV H) delta 24AAAA quadruple mutant (Y463A) (R515A) (530SF531 to 530LS531)

[0365] [ka]

[0366] Measles virus hemagglutinin (MeV H) delta 24AAAA quadruple mutant (Y463A) (R515A) (530SF531 to 530LS531) containing a targeting domain fusion site

[0367] [ka]

[0368] Measles virus fusion (MeV F) Delta 24

[0369] [ka]

[0370] Measles virus fusion (MeV F) delta 24 (T461I) hyperfusogenic mutant

[0371] [ka]

[0372] Measles virus fusion (MeV F) Delta 30

[0373] [ka]

[0374] Measles virus fusion (MeV F) delta 30 (T461I) highly fusogenic mutant

[0375] [ka]

[0376] Tupaia Paramyxovirus Hemagglutinin (TPMV H) WT

[0377] [ka]

[0378] Tupaia Paramyxovirus Hemagglutinin (TPMV H) Delta 32

[0379] [ka]

[0380] Tupaia Paramyxovirus Hemagglutinin (TPMV H) Delta 32 Contains a Targeting Domain Fusion Site

[0381] [ka]

[0382] Tupaia Paramyxovirus Hemagglutinin (TPMV H) Delta 80

[0383] [ka]

[0384] Tupaia Paramyxovirus Hemagglutinin (TPMV H) Delta 80 Contains a Targeting Domain Fusion Site

[0385] [ka]

[0386] Tupaia Paramyxovirus Fusion (TPMV F) WT

[0387] [ka]

[0388] Tupaia Paramyxovirus Fusion (TPMV F) Delta 32

[0389] [ka]

[0390] Canine distemper virus hemagglutinin (CDV H) WT

[0391] [ka]

[0392] Canine distemper virus hemagglutinin (CDV H) WT containing the targeting domain fusion site

[0393] [ka]

[0394] Canine distemper virus hemagglutinin (CDV H) delta 18

[0395] [ka]

[0396] Canine distemper virus hemagglutinin (CDV H) delta 18 containing a targeting domain fusion site

[0397] [ka]

[0398] Canine distemper virus hemagglutinin (CDV H) delta 19

[0399] [ka]

[0400] Canine distemper virus hemagglutinin (CDV H) delta 19 containing a targeting domain fusion site

[0401] [ka]

[0402] Canine distemper virus fusion (CDV F) WT

[0403] [ka]

[0404] Canine distemper virus fusion (CDV F), a highly fusogenic mutation from T to I

[0405] [ka]

[0406] Canine distemper virus fusion (CDV F) Delta 24

[0407] [ka]

[0408] Canine distemper virus fusion (CDV F) delta 24, a highly fusogenic mutation from T to I

[0409] [ka]

[0410] Canine distemper virus fusion (CDV F) delta 30

[0411] [ka]

[0412] Canine distemper virus fusion (CDV F) delta 30, a highly fusogenic mutation from T to I

[0413] [ka]

[0414] Canine distemper virus fusion (CDV F) WT, mini-signal sequence d107

[0415] [ka]

[0416] Canine distemper virus fusion (CDV F) WT, mini-signal sequence d107, and T to I mutation for high membrane fusion activity

[0417] [ka]

[0418] Canine distemper virus fusion (CDV F), mini-signal sequence d107, delta24

[0419] [ka]

[0420] Canine distemper virus fusion (CDV F), mini-signal sequence d107, delta 24, and highly fusogenic mutation from T to I

[0421] [ka]

[0422] Canine distemper virus fusion (CDV F), mini-signal sequence d107, delta30

[0423] [ka]

[0424] Canine distemper virus fusion (CDV F), mini-signal sequence d107, delta30, and highly fusogenic mutation from T to I

[0425] [ka]

[0426] Nipah virus glycoprotein (NiVG) WT

[0427] [ka]

[0428] Nipah virus glycoprotein (NiVG) WT containing the targeting domain fusion site

[0429] [ka]

[0430] Nipah virus glycoprotein (NiVG) delta33

[0431] [ka]

[0432] Nipah virus glycoprotein (NiVG) delta33 containing a targeting domain fusion site

[0433] [ka]

[0434] Nipah virus glycoprotein (NiVG) delta34

[0435] [ka]

[0436] Nipah virus glycoprotein (NiVG) delta34 containing a targeting domain fusion site

[0437] [ka]

[0438] Nipah virus fusion WT

[0439] [ka]

[0440] Nipah virus fusion Delta 22

[0441] [ka]

[0442] Nipah virus fusion delta 25

[0443] [ka]

[0444] Nipah virus glycoprotein (NiVG) (E501A)

[0445] [ka]

[0446] Nipah virus glycoprotein (NiVG) (W504A)

[0447] [ka]

[0448] Nipah virus glycoprotein (NiVG) (Q530A)

[0449] [ka]

[0450] Nipah virus glycoprotein (NiVG) (E533A)

[0451] [ka]

[0452] Cocal virus glycoprotein (CVG) WT

[0453] [ka]

[0454] Cocal virus glycoprotein (CVG) (K64Q)

[0455] [ka]

[0456] Cocal virus glycoprotein (CVG) (R371A)

[0457] [ka]

[0458] Cocal virus glycoprotein (CVG) (K64Q) (R371A)

[0459] [ka]

[0460] Targeting domain fusion site to transmembrane PDGFR anchor

[0461] [ka]

[0462] Targeting domain fusion site to transmembrane CD9 anchor

[0463] [ka]

[0464] Targeting domain fusion site for transmembrane CD28 anchor

[0465] [ka]

[0466] Targeting domain fusion site to transmembrane CD8 anchor

[0467] [ka]

[0468] Targeting domain fusion site for transmembrane CD4 anchor

[0469] [ka]

[0470] Targeting domain fusion site for transmembrane CD63 anchor

[0471] [ka]

[0472] Targeting domain fusion site for transmembrane CD81 anchor

[0473] [ka]

[0474] Targeting domain fusion site for transmembrane CD86 anchor

[0475] [ka]

[0476] Targeting domain fusion site for transmembrane Notch anchor

[0477] [ka] [Example]

[0478] The invention is further described in the following examples, which do not limit the scope of the invention, as claimed.

[0479] [Example 1] Programmed, tropic virus-like particles deliver gene-editing cargo to target cells method ptVLP particles were produced in HEK293T cells by transfecting the plasmids into the cells using polyethyleneimine (PEI). PEI is a 25 kD linear polyethyleneimine (Polysciences, #23966-2). To make a stock "PEI MAX" solution, 1 g of PEI was added to 1 L of endotoxin-free dH2O that had been preheated to approximately 80°C and cooled to room temperature. The mixture was neutralized to pH 7.1 by adding 10 N NaOH and filter-sterilized through 0.22 μm polyethersulfone (PES). The PEI MAX solution was stored at -20°C.

[0480] HEK293T cells were split to reach 70%-90% confluence at the time of transfection and cultured in DMEM medium with 10% FBS. A cargo-encoding plasmid vector, e.g., a CMV promoter driving expression of a fusion protein containing the hPLCδ1 PH domain linked to a codon-optimized Cas9, was co-transfected with a plasmid encoding a U6 promoter driving expression of the Cas9 sgRNA, a plasmid encoding a membrane-tethered targeting moiety, and a mutant VSV-G envelope plasmid. The transfection reaction was assembled in low-serum medium (Opti-MEM; GIBCO #31985-070). To produce ptVLP particles on a 10 cm plate, 7.5 μg of PH-Cas9 expression plasmid, 7.5 μg of sgRNA expression plasmid, and 5 μg of programmed targeting ENV expression plasmid were mixed in 1 mL of Opti-MEM, followed by the addition of 27.5 μl of PEI MAX. After 20–30 min of incubation at room temperature, the transfection reaction was dispensed dropwise onto HEK293T cells.

[0481] ptVLPs were harvested 48–72 hours after transfection. To do so, the ptVLP supernatant was filtered using a 0.45 μm PVDF or acetylcellulose or a 0.8 μm PES membrane filter and transferred to polypropylene Beckman ultracentrifuge tubes (Beckman Coulter #326823) for use in an SW28 rotor. Each ultracentrifuge tube was filled with the ptVLP-containing supernatant from three 10 cm plates to a final volume of approximately 35–37.5 ml. The ptVLP supernatant was ultracentrifuged at approximately 100,000 x g or 25,000 rpm at 4°C for 2 hours. After ultracentrifugation, the supernatant was decanted, and the ptVLP pellet was resuspended in DMEM 10% FBS medium so that it was approximately 1,000-fold more concentrated than before ultracentrifugation. The ptVLPs were added dropwise to cells seeded in 24-well plates 24 hours before transduction. If necessary, polybrene (5-10 μg / mL in cell culture medium; Sigma-Aldrich #TR-1003-G) was supplemented to enhance transduction efficiency. If necessary, Vectofusin-1 (10 μg / mL in cell culture medium; Miltenyi Biotec #130-111-163) was supplemented to enhance transduction efficiency. If necessary, the 24-well plate was centrifuged at 1,150 x g for 30 minutes at room temperature immediately after the addition of ptVLPs to enhance transduction efficiency.

[0482] Example 1.1 ptVLPs (shown in Figures 1A and 1C) were produced by transient plasmid transfection of HEK293T cells as described above. These ptVLPs (Figures 1B and 1D) were purified and concentrated 100-fold by filtration and PEG precipitation and applied to HEK293T cells expressing or lacking CD19 for a 48-hour incubation period. The HEK293T cells were then harvested and genomic DNA extracted. To quantify the frequency of gene modification / editing, targeted amplicon sequencing of the genomic site targeted by the VLP cargo was performed using the extracted genomic DNA (Figure 2). The results showed that transduction efficiency (measured by gene editing of the target site (VEGFs3)) was significantly increased in cells expressing the target antigen CD19 compared to cells lacking CD19 expression.

[0483] [Example 1.2] Figures 3 and 4 show that different phospholipid bilayer recruitment domains can deliver cargo in the previously described eVLPs (WO 2022 / 020800). For Figure 3, eVLPs were produced by transient transfection of HEK293T cells, purified and concentrated 100-fold by filtration and PEG precipitation, and normalized based on total Cas9 in the particles as determined by ELISA before transduction into HEK293T cells. The same pmol of Cas9 was applied to each well to ensure comparability between different PH domains. The frequency of gene modification / editing induced at the endogenous VEGF target site was determined by targeted amplicon sequencing (Figure 3). The eVLPs were pseudotyped with VSVG. These results demonstrate that fusion of various PH domains and mutant PH domains to cargo can mediate variable cargo delivery efficiency and, in turn, variable frequencies of targeted gene modification in target recipient cells.

[0484] With reference to Figure 4, different mutant PH-Cas9 fusions (and Cas9 lacking fusion to the PH domain) were packaged into eVLPs (prepared as described in WO 2022 / 020800), purified and concentrated 100-fold by PEG precipitation, and normalized based on total Cas9 in the particles as determined by ELISA. 5 pmol of Cas9 was added to 15,000 primary T cells per well. The frequency of gene modification / editing induced at the endogenous RNF2 target site was determined by targeted amplicon sequencing (Figure 4). The eVLPs were pseudotyped with VSVG or a combination of VSVG and BaEVTRless. These results demonstrated that fusion of various PH domains and mutant PH domains to cargo resulted in variable cargo delivery efficiencies and variable frequencies of targeted gene modification in target recipient cells. Furthermore, different pseudotype combinations also affected delivery efficiency. References

[0485] [Table 10-1]

[0486] [Table 10-2]

[0487] [Table 10-3]

[0488] [Table 10-4]

[0489] [Table 10-5]

[0490] [Table 10-6]

[0491] [Table 10-7]

[0492] [Table 10-8]

[0493] [Table 10-9]

[0494] [Table 10-10]

[0495] Other embodiments While the present invention has been described in connection with its detailed description, it should be understood that the foregoing description is intended to be illustrative and not limiting of the scope of the invention, which is defined by the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims

1. (i) a programmable targeting glycoprotein or envelope protein (ptENV) comprising a viral-derived glycoprotein or envelope protein, optionally fused to a targeting domain located at the C-terminus, N-terminus, or inserted immediately after the signal sequence of the glycoprotein or envelope protein; or (ii) A membrane-tethered targeting domain, optionally comprising a targeting domain fused to a transmembrane domain, including a targeting peptide, a single chain variable fragment (scFv), a nanobody, a fibronectin type 3 domain (FN3), an arginylglycylaspartic acid motif (RGD), a single variable domain on the heavy chain / nanobody (VHH), a variable domain of a novel antigen receptor (VNAR), a darpin, or other targeting ligand. A fusion protein comprising:

2. 2. The fusion protein of claim 1, wherein the targeting domain binds to human CD19, CD4, CD34, ASGR1, TfR1, HER2, CD25, CTLA-4, HB-EGF, ACE2, aryl hydrocarbon receptor (AhR), keratin 5 (KRT5), KRT13, fibronectin (FN1), amyloid precursor protein (APP), neurotrophin receptor (p75NTR), Thy-1 / CD90, EpCAM, and / or CFTR.

3. 3. The fusion protein of claim 1, wherein the signal sequence comprises MKCLLYLAFLFIGVNCK (SEQ ID NO: 1), or a secretory signal sequence derived from VSVG (optionally MKCLLYLAFLFIGVNC, SEQ ID NO: 2).

4. 4. The fusion protein of any one of claims 1 to 3, comprising a sequence that is at least 95% identical to a sequence described herein, and optionally, wherein ptENV comprises a targeting domain in addition to a glycoprotein or envelope protein of Table 1.

5. A nucleic acid sequence encoding the fusion protein of any one of claims 1 to 4.

6. A vector comprising the nucleic acid sequence of claim 5, optionally operably linked to a promoter for the expression of a fusion protein of any one of claims 1 to 4.

7. A host cell comprising a nucleic acid sequence according to claim 5, optionally expressing a fusion protein according to any one of claims 1 to 4.

8. 5. A virus-like particle (VLP) comprising a fusion protein according to any one of claims 1 to 4, optionally comprising a cargo located in the core of the VLP, optionally fused to a phospholipid bilayer recruitment domain.

9. (a) a membrane comprising a phospholipid bilayer; and (b) a fusion protein comprising the ptENV according to part (i) of claims 1 to 4, or a fusion protein comprising a glycoprotein or an envelope protein (optionally listed in Table 1) and a membrane-tethered targeting domain according to part (ii) of claims 1 to 4; and (c) optionally a cargo located in the core of the ptVLP, optionally fused to a phospholipid bilayer recruitment domain; A programmable tropism virus-like particle (ptVLP) comprising:

10. The VLP of claim 8 or the ptVLP of claim 9, wherein the cargo is a therapeutic or diagnostic protein, and / or a nucleic acid encoding a therapeutic or diagnostic protein, and / or a chemical, optionally a small molecule therapeutic or diagnostic agent.

11. The VLP of claim 8 or the ptVLP of claim 9, wherein the cargo is a gene editing or epigenetic modulation reagent.

12. 10. The VLP of claim 8 or the ptVLP of claim 9, wherein the gene editing or epigenetic modulation reagent comprises a zinc finger (ZF), a transcription activator-like effector (TALE), and / or a CRISPR-Cas protein, variant, or fusion thereof; a nucleic acid encoding a zinc finger (ZF), a transcription activator-like effector (TALE), and / or a CRISPR-Cas protein, variant, or fusion thereof; a guide RNA and / or crRNA; or a ribonucleoprotein complex (RNP) comprising a CRISPR-Cas protein, variant, or fusion thereof, and / or optionally a guide RNA and / or crRNA.

13. 13. The VLP or ptVLP of claim 12, wherein the cargo is selected from the proteins listed in Tables 2, 3, 4 and 5, or is at least 95% identical to a sequence set forth herein, optionally in Tables 2, 3, 4 and 5.

14. 13. The VLP or ptVLP of claim 12, wherein the cargo comprises a CRISPR-Cas protein, and the ptVLP further comprises one or more guide RNAs and / or crRNAs that bind to the CRISPR-Cas protein and guide the CRISPR-Cas protein to a target nucleic acid sequence.

15. 15. The VLP or ptVLP of any one of claims 9 to 14, wherein the cargo comprises a fusion to a phospholipid bilayer recruitment domain, preferably as shown in Table 6, or a phospholipid bilayer recruitment domain that is at least 95% identical to a sequence set out in Table 6 herein.

16. A method for delivering cargo to a target cell, optionally a cell in vivo or in vitro, comprising contacting said cell with a VLP or ptVLP comprising said cargo according to any one of claims 8 to 15.

17. 1. A method for producing cargo-containing VLPs or ptVLPs, comprising: (i) expressing a fusion protein comprising the ptENV according to part (i) of any one of claims 1 to 4, or (ii) a glycoprotein or envelope protein (optionally listed in Table 1) and a membrane-tethered targeting domain according to part (ii) of any one of claims 1 to 4; and Providing cells that optionally also express cargo, and optionally do not express exogenous gag, pro, or pol proteins; and maintaining said cells under conditions such that said VLPs or ptVLPs are produced; A method comprising:

18. 18. The method of claim 17, further comprising recovering, and optionally purifying and / or concentrating, the VLPs or ptVLPs produced.

19. 18. The method of claim 17, wherein the cargo is a therapeutic or diagnostic protein, and / or a nucleic acid encoding a therapeutic or diagnostic protein, and / or a small molecule, optionally a therapeutic or diagnostic small molecule.

20. 18. The method of claim 17, wherein the cargo is a gene editing or epigenetic modulation reagent.

21. 18. The method of claim 17, wherein the gene editing or epigenetic modulation reagent comprises a zinc finger (ZF), a transcription activator-like effector (TALE), and / or a CRISPR-Cas protein, variant, or fusion thereof; a nucleic acid encoding a zinc finger (ZF), a transcription activator-like effector (TALE), and / or a CRISPR-Cas protein, variant, or fusion thereof; a guide RNA and / or crRNA; or a ribonucleoprotein complex (RNP) comprising a CRISPR-Cas protein, variant, or fusion thereof, and optionally a guide RNA and / or crRNA.

22. 22. The method of claim 21, wherein the cargo reagent is selected from the proteins listed in Tables 2, 3, 4 and 5, or is at least 95% identical to a sequence set forth herein, optionally in Tables 2, 3, 4 and 5.

23. 22. The method of claim 21, wherein the cargo reagent comprises a CRISPR-Cas protein, variant, or fusion thereof, and the ptVLP further comprises one or more guide RNAs and / or crRNAs that bind to the CRISPR-based genome editing or regulatory protein and guide the CRISPR-based genome editing or regulatory protein to a target sequence.

24. 24. The method of any one of claims 17 to 23, wherein the cargo comprises a fusion to a phospholipid bilayer recruitment domain, preferably as shown in Table 6, or a phospholipid bilayer recruitment domain that is at least 95% identical to a sequence set out in Table 6 herein.

25. 1. A cell expressing (i) a fusion protein comprising the ptENV according to part (i) of any one of claims 1 to 4, or (ii) a fusion protein comprising a glycoprotein or envelope protein (optionally listed in Table 1) and a membrane-tethered targeting domain according to part (ii) of any one of claims 1 to 4; and optionally a cargo optionally fused to a phospholipid bilayer recruitment domain, Optionally, cells that do not express exogenous gag, pro and / or pol proteins.

26. 26. The cell of claim 25, wherein the cargo is a therapeutic or diagnostic protein, and / or a nucleic acid encoding a therapeutic or diagnostic protein, and / or a small molecule, optionally a therapeutic or diagnostic small molecule.

27. 26. The cell of claim 25, wherein the cargo is a gene editing or epigenetic modulation reagent.

28. 26. The cell of claim 25, wherein the gene editing or epigenetic modulation reagent comprises a zinc finger (ZF), a transcription activator-like effector (TALE), and / or a CRISPR-Cas protein, variant, or fusion thereof; a nucleic acid encoding a zinc finger (ZF), a transcription activator-like effector (TALE), and / or a CRISPR-Cas protein, variant, or fusion thereof; a guide RNA and / or a crRNA; or a ribonucleoprotein complex (RNP) comprising a CRISPR-Cas protein, variant, or fusion thereof, and optionally a guide RNA.

29. 29. The cell of claim 28, wherein the cargo reagent is selected from the proteins listed in Tables 2, 3, 4 and 5, or is at least 95% identical to a sequence set forth herein, optionally in Tables 2, 3, 4 and 5.

30. 29. The cell of claim 28, wherein the gene editing or epigenetic modulation reagent comprises a CRISPR-Cas protein, and the ptVLP further comprises one or more guide RNAs and / or crRNAs that bind to the CRISPR-Cas protein and guide the CRISPR-Cas protein to a target sequence.

31. 31. The cell of any one of claims 25 to 30, wherein the cargo comprises a fusion to a phospholipid bilayer recruitment domain, preferably as shown in Table 6, or a phospholipid bilayer recruitment domain that is at least 95% identical to a sequence set out in Table 6 herein.

32. The cell of any one of claims 25 to 31, wherein the cell is a primary or stable human cell line.

33. 33. The cell of claim 32, which is a human embryonic kidney (HEK) 293 cell or a HEK293 T cell.

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