EP4684007A1 - Improved yield of paramyxovirus-like particles through use of altered viral matrix proteins - Google Patents

Abstract

Provided are compositions and methods for introducing proteins into cells. The compositions and methods relate to introducing a foreign protein as an engineered component of a paramyxovirus virus like particle (VLP). The compositions and methods pertain to modified VLPs that contain a contiguous recombinant polypeptide comprising i) all or a segment of a C-terminal domain of a paramyxovirus nucleocapsid protein and ii) a polypeptide sequence of a distinct protein that is an enzyme such as a recombinase. The VLPs include at least one altered M protein, which increased yield of the VLPs by cells that produce the VLPs.

Description

IMPROVED YIELD OF PARAMYXOVIRUS-LIKE PARTICLES THROUGH USE OF ALTERED VIRAL MATRIX PROTEINS

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. provisional patent application no. 63/454.242, filed March 23, 2023, the disclosure of which is incorporated herein by reference.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

This invention was made with government support under Hatch Act Project No. PEN04771 awarded by the United States Department of Agriculture. The Government has certain rights in the invention.

SEQUENCE LISTING

The instant application contains a sequence listing which has been submitted electronically in xml format and is hereby incorporated by reference in its entirety. Said .xml copy, created March 22, 2024. is titled ”PSU-2023-5592.xmr and is 5.714 bytes in size.

BACKGROUND OF THE DISCLOSURE

There is an ongoing and unmet need for compositions and methods that are useful for protein delivery to a variety of cell types for a variety of purposes. The present disclosure pertains to this need.

SUMMARY

The present disclosure provides compositions and methods for introducing proteins into cells. The compositions and methods relate to introducing a foreign protein as an engineered component of a paramyxovirus virus like particle (VLP). The compositions and methods pertain to modified VLPs that contain a contiguous recombinant polypeptide comprising i) all or a segment of a C-terminal domain of a paramyxovirus nucleocapsid protein and li) a polypeptide sequence of a distinct protein. The VLPs include an M protein comprising a one or a combination of described mutations which permits, among other properties, increased yield of modified VLPs from producer cells, relative to producer cells that do not use the M protein that contains the same mutations. BRIEF DESCRIPTION OF THE FIGURES

Figures 1A and IB: Paramyxovirus particle-based cargo delivery. Virus particle binding to a cell receptor on a target cell plasma membrane allows the fusion of viral membrane to the host cell plasma membrane, which facilitates the direct release of cargo such as viral genome (A) and foreign cargo protein (B) into the cell interiors.

Figure 2: General procedure for VLP packaging and delivery of cargo proteins. Four different plasmids expressing matrix protein, attachment protein, fusion protein, and cargo-NP fused protein were transfected into VLP producer HEK 293T cells. VLPs released into the culture supernatants were purified by using sucrose gradients. VLP production and cargo incorporation efficiency was tested using immunoblotting. Cargo-loaded purified particles were then incubated with target cells and post-delivery analysis was carried out using fluorescence microscopy and flow cytometry.

Figures 3A-3C: Packaging of Cre recombinase into delivery capable PIV5 VLPs. (A) C-terminal amino acid sequences of paramyxovirus NP/N proteins (top panel), illustration of Cre recombinase protein appended with residues derived from PIV5 NP protein (bottom panel). (B) Purified Cre-loaded VLPs were detected by immunoblotting. Cre-NP20 was efficiently packaged into VLPs, while unmodified Cre w as not. (C) Total amount of Cre packaged into VLPs was calculated as the amount of Cre detected in VLP fractions and normalized to the value obtained with Cre. (n = 3). *P < 0.05, Welch’s Ltest. The sequences on Figure 3 (A) are C-terminal ends: QNAAAGAPIHTDDLNAALGDLDI (SEQ ID NO: 1) PIV5; EHGNTFPNNPNQNAQSQVGDWDE (SEQ ID NO:2) Mumps; GILEEQGSDTDTPRVYNDRDLLD (SEQ ID NO:3) Measles; and (SEQ ID SEKKNNQDLKPAQNDLDFVRADV (SEQ ID NO: 4) Nipah.

Figures 4A and 4B: Delivery of Cre recombinase into target cell nuclei using PIV5 VLPs. (A) Cre-loaded PIV5 VLPs having only the attachment glycoprotein HN (-F VLPs) and VLPs having both HN and F glycoproteins (+F VLPs) were produced and purified as described in Fig. 2. Purified VLPs were detected by immunoblotting. (B) Purified VLPs were incubated with reporter cells (switches green fluorescence to red fluorescence upon Cre- induced recombination) and visualized using a fluorescence microscope. Fusion-capable VLPs were successful in delivering active Cre to target cell nuclei. VLPs produced with no F protein were unable to deliver Cre to target cells.

Figures 5A-5C: Dose-response for delivery of Cre recombinase using PIV5 VLPs. Cre-loaded fusion-capable PIV5 VLPs were produced and purified as described above. An increasing amount of VLPs were incubated with reporter cells and visualized using (A) fluorescence microscopy and (B, C) flow cytometry to measure red fluorescence signal. 90% of the reporter cells switched to red fluorescence at the highest dose of VLPs.

Figure 6: Manipulation of multiple paramyxoviruses to allow protein delivery. 293T cells were transfected to produce Cre-loaded Nipah VLPs. Purified VLPs were incubated with the reporter cells and visualized using a fluorescence microscope. Nipah VLPs were successful in delivering active Cre cargo to the nuclei of the target cells.

Figure 7A-7D: G188D alteration to PIV5 M protein enhances VLP production and cargo delivery. Cre-loaded PIV5 VLPs were produced and purified as above using either wild-type M or an altered M protein with the single amino acid substitution G188D. (A) Purified VLPs were detected by immunoblotting. (B) VLP production efficiencies (yields) were calculated based on M band intensities observed in panel A, normalized to those obtained with wild-type M. (C) Total amount of Cre packaged into VLPs was calculated based on Cre band intensities observed in panel A, normalized to those obtained with wild-type M. (D) VLPs were incubated with reporter cells and Cre delivery was visualized using a fluorescence microscope (i.e., RFP signal intensity). Cre delivery was significantly improved when G188D M protein was used in place of the wild-type M protein. Error bars indicate standard deviations (n = 3). * P < 0.05, Welch’s t-test.

Figure 8: G188D, W235L, I278M, and G357W substitutions to M protein each have the ability to enhance the production of delivery-capable cargo-loaded PIV5 VLPs. 293T cells were transfected to produce PIV5 M protein variants as indicated, together with HN, F, and Cre-NP20 proteins for VLP production. 1 /250th of the resulting crude VLPs (each harvested from one 10-cm dish of producer cells) were incubated for 48 h with Cre reporter cells so that delivery of the Cre cargo could be evaluated based on conversion of green fluorescence signal to red. Of the five M substitutions that were tested, four of them (G188D, W235L, I278M, and G357W) led to enhanced delivery of the Cre cargo as compared to the wildtype M protein. On the other hand, F184L substitution led to delivery of Cre cargo that was not noticeably changed as compared to the wildtype M protein.

Figures 9A-9D. Second-site M mutant protein increases VLP quantity. (A) Based on the Cre signal detected in VLP fractions in figure 4, VLPs were normalized and fractionated on SDS gels, and proteins were detected by immunoblotting. (B, C, and D) Normalized amounts of Cre loaded VLPs were incubated with Cre reporter cells. GFP and RFP fluorescence signals were visualized using a fluorescence microscope (B) or using a flow cytometer (C and D). lx dose = 1/40 of crude VLPs from a 10-cm dish of producer 293T cells, and delivered to a single well of target cells on a 24-well plate. DESCRIPTION

Unless defined otherwise herein, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains.

The disclosures of U.S. Provisional Application No. 63/146,430, filed February 5, 2021, U.S. Patent No. 10,316,295, U.S. patent 11.339,375, U.S. Patent Application No. 17/542.337 filed December 2, 2021. published as US patent publication no. 2022-0162567, U.S. provisional patent application no. 62/268,921, filed December 17, 2015, and PCT publication WO 2017/106822, are incorporated herein by reference.

The present disclosure relates to modified virus-like particles of paramyxoviruses, compositions comprising them, and methods of using them for delivery of any particular protein of interest to any of a variety of cells. The cells include but are not necessarily limited to mammalian cells. Generally, the disclosure involves introducing into a cell a foreign protein as an engineered component of a paramyxovirus virus like particle (VLP). The compositions, methods and kits accordingly pertain to modified VLPs that contain a contiguous recombinant polypeptide comprising i) all or a segment of a C-terminal domain of a paramyxovirus nucleocapsid protein and ii) a polypeptide sequence of a distinct protein. Paramyxovirus nucleocapsid proteins are referred to as the "N" protein, but it will be recognized that in certain cases the nucleocapsid protein can be what may be referred to in the art for any particular paramyxovirus as the "NP" protein. The N protein can have an amino acid sequence that is the same as a segment of any naturally occurring N protein, or the sequence of the N protein can be modified, such as to provide for enhanced function. The type of paramyxovirus N protein C-terminal sequence that is used or is modified for use in examples of this disclosure is not particularly limited.

In non-limiting examples, the N protein C-terminal sequence that is used and/or modified is from PIV5, hPIV2, Nipah virus, Hendra virus, mumps virus (MuV), measles virus (MeV), Newcastle disease virus (NDV), Sendai virus (SeV), respiratory syncytial virus (RSV), and human metapneumovirus (hMPV). Non-limiting examples of the disclosure are provided using PIV5 VLPs, Nipah VLPs, and mumps VLPs. In certain examples, C-terminal segment of the N protein that is present in a fusion with a distinct protein is at least 10 amino acids in length, and can be from 10-120 amino acids in length, and the fusion protein may comprise one or more C-terminal segments, and may comprise any suitable linker or linkers. In certain examples, the C-terminal segment of the N protein can comprise a DLD or DWD amino acid motif. In certain examples, the disclosure includes complexes that comprise non- covalent associations of paramyxovirus M protein, and a fusion protein of this disclosure, wherein the fusion protein comprises a foreign protein and a paramyxovirus N protein C- terminal sequence. Such complexes may be present in VLPs of this disclosure. In examples, non-covalent associations of cells and modified VLPs are provided. In examples, non- covalent associations of VLPs of this disclosure that are formed between the VLPs and sialic acid are provided. In examples, non-covalent associations of VLPs of this disclosure with Ephrin B2. Ephrin B3. SLAM and/or Nectin4 receptors are provided. In certain examples, complexes comprising antibodies and VLPs of this disclosure are provided. Expression vectors encoding the fusion proteins are provided, as are cells that contain such expression vectors. Methods of making the VLPs are included, as are isolated and/or purified VLP preparations, wherein the VLPs have been separated from cells, including but not necessarily limited to VLP producer cells.

It will be recognized that the VLPs can be made by producing any one or any combination of VLP components recombinantly, i.e., by expression from an expression vector. In examples, the VLPs are produced using one or more expression vectors in cells, wherein the cells express at least: paramyxovirus M, N or NP, F and Attachment proteins, non-limiting examples or which are described herein. Paramyxoviruses are responsible for a wide range of diseases that affect both humans and animals. Paramyxovirus pathogens include measles virus, mumps virus, human respiratory syncytial virus, and the zoonotic paramyxoviruses Nipah virus and Hendra virus. Infectivity of paramyxovirus particles depends on matrix-nucleocapsid protein interactions which enable efficient packaging of encapsidated viral RNA genomes into budding virions.

Paramyxovirus infections are spread via particles, which bud from plasma membranes of infected cells. Formation of these particles is driven by the viral matrix (M) proteins which can self-assemble to form ordered yet flexible arrays that likely play key roles in generating the membrane curvature required for budding. M proteins also organize the particle assembly process by interacting with the viral glycoproteins via their cytoplasmic tails, and also with the viral ribonucleoprotein (vRNP) complexes via the nucleocapsid (N or NP) proteins. These interactions bring together and concentrate all of the viral structural components onto specific sites underlying infected cell plasma membranes, enabling infectious virions to subsequently bud from these locations.

For many paramyxoviruses, expression of M protein in the absence of any other viral components is sufficient to induce the assembly and release of virus-like particles (VLPs) from transfected cells. M proteins of Sendai virus, measles virus, Nipah virus, Hendra virus, Newcastle disease virus, and human parainfluenza virus 1 are all capable of directing VLP production and release from transfected cells when expressed alone. In these cases, additional viral components including the viral glycoproteins and the nucleocapsid-like structures that form upon expression of paramyxovirus N/NP proteins can be efficiently packaged into the VLPs if they are co-expressed along with the M proteins (Harrison MS, Sakaguchi T, Schmitt AP. 2010. Paramyxovirus assembly and budding: building particles that transmit infections. Int. J. Biochem. Cell. Biol. 42: 1416-1429.) For other paramyxoviruses, including mumps virus (Li M, Schmitt PT, Li Z, McCrory TS, He B, Schmitt AP. 2009. Mumps virus matrix, fusion, and nucleocapsid proteins cooperate for efficient production of virus-like particles. J. Virol. 83:7261-7272) and parainfluenza virus 5 (PIV5) (Schmitt AP, Leser GP, Waning DL. Lamb RA. 2002. Requirements for budding of paramyxovirus simian virus 5 virus-like particles. J. Virol. 76:3952-3964), the viral M proteins do not induce significant VLP production when expressed alone in transfected cells. In these cases, co-expression of M proteins together with viral glycoproteins and NP proteins is necessary for VLP production to occur.

This disclosure demonstrates that VLPs that contain modified M proteins can increase viral yield from producer cells. As such, VLPs that comprise at least one mutation in paramyxovirus M protein are provided. The at least one mutation increases yield of the VLPs by producer cells that are modified to produce the VLPs, relative to yield of VLPs produced by cells that are modified to produce the VLPs but do not comprise the at least one M protein mutation, or comprise an M protein mutation that does not increase yield, as illustrated by the description and accompanying figures. The VLPs further may comprise a contiguous recombinant polypeptide comprising i) all or a segment of a C-terminal domain of a paramyxovirus nucleocapsid (N) protein and ii) a polypeptide sequence of a distinct protein. In examples the disclosure provides VLPs comprising one or more mutations of Parainfluenza virus 5 strain W3A proteins, including but not necessarily limited to the membrane (M) protein.

The amino acid sequence of Parainfluenza virus 5 strain W3 M protein is: MPSISIPADPTNPRQSIKAFPIVINSDGGEKGRLVKQLRTTYLNDLDTHEPLVTFINTYG FIYEQDRGNTIVGEDQLGKKREAVTAAMVTLGCGPNLPSLGNVLGQLREFQVTVRK TSSKAEEMVFEIVKYPRIFRGHTLIQKGLVCVSAEKFVKSPGKIQSGMDYLFIPTFLSV TYCPAAIKFQVPGPMLKMRSRYTQSLQLELMIRILCKPDSPLMKVHTPDKEGRGCLV SVWLHVCNIFKSGNKNGSEWQEYWMRKCANMQLEVSIADMWGPTIIIHARGHIPKS AKLFFGKGGWSCHPLHEVVPSVTKTLWSVGCEITKAKAIIQESSISLLVETTDIISPKV KISSKHRRFGKSNWGLFKKTKSLPNLTELE (SEQ ID N0:5). Other than the M protein, other amino acid sequences corresponding to Parainfluenza virus 5 strain W3 proteins are available under GenBank ID AF052755. 1, from which all of the nucleotide and amino acid sequences are incorporated herein by reference as they exist in the GenBank database as of the filing date of this application. The disclosure includes all paramyxovirus proteins that have at least 80% sequence identity to the Parainfluenza virus 5 strain W3 proteins. In examples, at least one mutation is in a Parainfluenza virus 5 strain W3A M protein. In examples, at least one mutation to an M protein is at residues G188, W235, 1278, or G357. In examples, the at least one mutation is G188D, I278M, W235L, I278M, or G357W. Combinations of mutations are included. The disclosure includes corresponding changes in different paramyxovirus proteins that can be identified by those skilled in the art. such as by using amino acid sequence alignments.

The disclosure includes culturing modified cells to produce VLPs that comprise a described mutation, wherein the modified cells produce a higher yield of VLPs, relative to cells that produce the VLPs that do not comprise the same M protein mutation. The disclosure includes separating VLPs from the modified cells that produce VLPs that comprise the mutation in the M protein. The disclosure includes contacting cells with the VLPs such that the VLPs enter the cells. Demonstrations of increased yields, and using the modified VLPs to transport a variety of cargo are presented in the Figures.

With respect to changes to the amino acid sequence of the Parainfluenza virus 5 strain W3A M protein, Table 1 provides a summary of results obtained by comparing VLP yield using non-mutated M protein (wild type M protein) and multiple mutations of the M protein to analyze and compare the effects on VLP yield. In particular, Table 1 includes a relative comparison of yield of wild type VLPs, VLPs containing mutated M protein but with no heterologous cargo appended to the C-terminal domain of a paramyxovirus nucleocapsid (N) protein (Viral NP cargo/left column of Table 1), and ii) a with Cre as a representative cargo added to the C-terminal domain of the N protein (Cre-loaded VLPs/right column) in the nonmutated M protein (first row of Table 1) and the indicated M protein mutations in the columns of Table 1.

As can be seen from Table 1, 21 M protein mutations were produced and compared to wild type (non-mutated) M protein in terms of wild ty pe and Cre-loaded VLPs. Cre is used to illustrate an example and is not meant to limit the type of protein that can be added to the C- terminal domain of the N protein. Of the tested proteins, seven mutants produced yields that were comparable to VLPs with wild type M protein. Three mutants induced moderately higher yields and 11 induced substantially higher yields. All seven mutated proteins that failed to increase the yield of wild type VLPs also failed to induce the yield of function Cre-loaded VLPs. For three of these, the yield of functional Cre-loaded VLPs was similar to that induced by VLPs comprising wild type M protein. For the other four, the yield of functional Cre-loaded VLPs was reduced to a level that was lower than VLP production obtained with VLPs containing wild type M protein. Of the 11 mutations that induced higher yields of wild type M protein containing VLPs, only four were able to increase the yield of functional Cre-loaded VLPs, namely those with mutations of the M protein at residues G188, W235, 1278, and G357. Thus, the disclosure unexpectedly reveals that (1) it is possible to increase the yield of wild type VLPs via mutations to the viral M protein, and (2) of these yield-increasing M protein mutants, only a subset of selected mutations can increase the yield in a way that maintains a functional cargo, illustrated using Cre recombinase as heterologous cargo appended to the C-terminal domain of the N protein.

In addition to the data summarized in Table 1, the unexpected nature of only certain mutations increasing yield of functional Cre-loaded VLPs is also illustrated in Figure 8. As an example, Figure 8 includes results using an M protein with the F184L mutation. For a change in fluorescence, the sequence of events as represented by Figure 8 is as follows: (1) Cre-loaded VLPs are generated in producer cells, (2) the VLPs are incubated with reporter cells, (3) the VLPs bind to the reporter cells and fuse the VLP membranes with the reporter cell plasma membranes, thereby allowing transfer of the Cre cargo into the reporter cells as depicted in Figs. 1 A-1B, and (4) if functional, Cre induces recombination at a specific site in the reporter cell DNA, triggering production of RFP and halting production of green fluorescent protein (GFP). However, as shown in Figure 8, the F184L M protein failed to increase the yield of functional Cre-loaded VLPs despite its clear ability (shown in Table 1) to improve the yield of wild-type VLPs that have no Cre cargo appendage, but did not increase the yield of VLPs that do include the Cre cargo. Therefore, the present disclosure demonstrates that increasing yield of wild ty pe VLPs with one type of M protein mutation does not enable a prediction that the same mutation would increase yield of VLPs with a cargo protein added to the C-terminus of the N protein.

In certain implementations the disclosure comprises compositions comprising the VLPs, which may be provided as pharmaceutical compositions. In examples the disclosure comprises administering the VLPs and/or compositions comprising them to cells, and/or to individuals in need thereof. The administration results in a foreign protein that is present in a fusion protein of the VLPs being introduced to the cell. Methods of screening for anti-viral compounds are provided. These methods generally comprise determining whether one or more test agents can inhibit one or more steps of viral infection and/or reproduction by mixing modified VLPs of this disclosure, cells and test agents and determining w hether or not the test agents inhibit any of the one or more steps. Also provided are kits. The kits can comprise an expression vector encoding a segment of a C-terminal domain of a paramyxovirus nucleocapsid protein in proximity to a cloning site configured so that a polynucleotide encoding a distinct polypeptide can be introduced into the cloning site. This configuration results in the capability of the expression vector to express the segment of the C -terminal domain and the distinct polypeptide in a contiguous fusion protein, which may be incorporated into VLPs. The kits can comprise at least one additional expression vector encoding at least one additional VLP component, wherein the at least one additional component is selected from a viral matrix protein, a viral attachment glycoprotein, and a viral fusion glycoprotein. Or a single expression vector can be adapted to express more than one of these proteins.

The t pe of a foreign protein that is modified as described herein to be present in a C- terminal paramyxovirus N protein segment is not particularly limited. In examples, the foreign protein can comprise or consist of a functional protein or fragment thereof. In certain examples the foreign protein is selected from enzymes, receptor ligands, transcriptional factors, grow th factors, antibodies or antigen-binding fragments thereof including singlechain antibody fragments and Fabs, peptide or protein immunogens that can be used for stimulating an immune response (i.e., a vaccine), protein-based chemotherapeutic agents, and toxins. In certain examples, the foreign protein comprises insulin, a growth hormone or a grow th hormone releasing factor, a platelet derived growth factor, an epidermal growth factor, any insulin-like growth factor, a clotting factor, superoxide dismutase and other antioxidant enzymes, any interferon, any interleukin, a lymphotoxin, and the like. In examples the foreign protein comprises a protein-based toxin, such as enzymatically active toxins which include but are not limited to diphtheria A chain, nonbinding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha sarcin, Aleurites fordii proteins, dianthin proteins, and Phytolaca americana proteins (PAPI, PAPII, and PAP-S). It is expected that the length of the foreign protein will not be particularly limited. In a non-limiting example, the protein is not larger in mass than the N-encapsidated viral genome that is part of a wild type paramyxovirus virion. In examples, the fusion protein is modified to include, for example, an intracellular trafficking signal, including but not necessarily limited to a nuclear transport signal. In certain implementations, such as when using a nuclear transport signal, the disclosure encompasses using a nuclear import inhibiting drug, suitable examples of which are known in the art and include, for instance, importazole, wherein the drug is used in producer cells during VLP production. This approach is expected to prevent the nuclear localization signalbearing foreign protein from trafficking to the nucleus, where it may be subsequently unavailable to package into VLPs at the plasma membrane. But when the VLPs are delivered to cells in the absence of the nuclear localization inhibitor, the nuclear localization-bearing foreign protein would be able to travel to the nucleus and exert its function.

As discussed above, in one aspect the foreign protein comprises any protein, including any enzyme. In non-limiting examples the enzyme can comprise a nuclease or a nickase (and thus may include a nuclear localization signal). In examples the nuclease comprises a bacterial CRISPR (clustered regularly interspaced short palindromic repeats) nuclease, including but not necessarily limited to a CRISPR Cas enzyme. In an example, the Cas9 enzyme has the amino acid sequence of a Cas9 encoded by Streptococcus pyogenes, which are well known in the art. In examples, CRISPR enzyme is CRISPR enzyme that is distinct from S. pyogenes Cas9, which are also well known in the art.

In examples, the Cas enzyme is a Staph, aureus Cas9. Alternatively, the enzyme is a Cpfl enzyme. In examples, the foreign protein is an enzymatically inactive Cas protein, such as nuclease dead Cas9. In examples, the Cas protein comprises a Cas protein from at least one of the following CRISPR systems: ty pe I, type II, type III, I- A, I-B, I-D, or I-F.

In examples, the foreign protein may be a deaminases or any other base-altering enzymes currently referred to as so-called “base editors.” Base editors can convert one DNA base to another. In non-limiting examples, cytidine base editors promote C>T conversions and adenine base editors promote A>G conversions. The described proteins can be used to make genetic modifications without inducing double stranded DNA breaks.

In examples, the foreign protein may be an integrase or a transposase.

In another example, transcription activator-like effector nucleases (TALENs) can be included in the recombinant proteins. As know n in the art, TALENs are artificial restriction enzymes generated by fusing a TAL effector DNA binding domain to a DNA cleavage domain and can be adapted for use in examples of this disclosure. In yet another approach, zinc-finger nucleases (ZFNs) can be used. Thus, in examples, the disclosure comprises introducing an enzyme into a cell, wherein the enzyme has nuclease and/or nickase activity, and is the foreign component of a recombinant peptide that also comprises the segment of a paramyxovirus N protein as described herein. In an example, the foreign protein may be a polymerase, including but not limited to an RNA polymerase, or a reverse transcriptase.

Any cell type that is susceptible to infection by a paramyxovirus described herein can be modified by having any such enzyme introduced to it via the VLPs of this disclosure, and accordingly genetic material in the cell can be edited. The editing can comprise blunt end or sticky end cleavage. The editing can involve by homologous or non-homologous end-joining (NHEJ). The editing can involve insertions, deletions or other mutations, and can be used to make homozy gous or heterozygous mutations, and thus is suitable for a wide variety of purposes, including but not limited to making knock-out and knock-in mutations.

In examples, the cells into which the modified VLPs, (regardless of the nature of their cargo), can comprise animal cells, including mammalian cells. In examples the cells are totipotent, pluripotent, multipotent, or oligopotent stem cells. In examples, the cells are hematopoietic stem cells. In examples, the cells are leukocytes. In examples, the leukocytes are of a myeloid or lymphoid lineage. In examples, the cells are embryonic stem cells, or adult stem cells. In examples, the cells are epidermal stem cells or epithelial stem cells. In examples, the cells are differentiated cells when the VLPs are introduced. In examples, the cells are human, or are non-human animal cells. In examples, the cells are used to generate cell lines, and/or transgenic non-human animals.

In examples an effective amount of VLPs are administered to cells in an individual in need thereof. In examples, an effective amount is an amount of described VLPs that reduces one or more signs or symptoms of a disease and/or reduces the severity of the disease. An effective amount may also inhibit or prevent the onset of a disease or a disease relapse. A precise dosage of VLPs can be selected by the individual physician in view of the patient to be treated. Dosage and administration can be adjusted to provide sufficient levels of VLPs to maintain the desired effect. Additional factors that may be taken into account include the severity and type of the disease state, age, weight, and gender of the patient, desired duration of treatment, method of administration, time and frequency of administration, drug combination(s), reaction sensitivities, and/or tolerance/response to therapy, and the type of cargo that is included with the VLPs.

In examples, the disclosure includes obtaining cells from an individual, modifying the cells ex vivo or in vivo using any suitable CRISPR system that operates with the nuclease that is part of a recombinant protein described herein. Progeny of such cells are included. Such cells may be reintroducing to the individuals for prophylaxis and/or therapy of a condition, disease or disorder, or to treat an injury⁷. The disclosure comprises administering nucleases suitable for CRISPR-based gene editing, and may further comprise introducing any other agents that are involved in CRISPR-based DNA editing, such as any suitable guide RNA and/or tracrRNA. In certain examples, the guide RNA is provided to and/or is expressed by producer cells along with the other components, for example via a plasmid. The Cas9-guide RNA complex will then be packaged into the VLPs. Kits comprising all or some of such reagents and VLPs of this disclosure are included. It will be recognized that the disclosure encompasses fusion proteins wherein the foreign protein is intended to be introduced into a cell or cell population. Individual cells and cell populations that are complexed with the VLPs, and/or into which a fusion protein of this disclosure has been introduced, are included in the disclosure.

The disclosure in certain implementations can exclude fusions of a C-terminus of a paramyxovirus N protein segment solely with polypeptide sequences that are used for purposes such as protein separation or for visualization of the fusion protein. Accordingly in certain examples the fusion proteins of this disclosure do not comprise a C-terminus of a paramyxovirus N protein fused solely with an affinity' tag for use in protein purification, examples of which include but are not necessarily limited to chitin binding protein (CBP), maltose binding protein (MBP), glutathione-S-transferase (GST) and a poly(His) tag. Likewise, in certain examples, the disclosure can exclude fusions of a C-terminus of a paramyxovirus N protein with proteins that are used alone as detectable labels, including but not limited to green fluorescent protein. In examples, the disclosure can exclude fusion of an N protein from one type of paramyxovirus from a segment of any other paramyxovirus protein, and a configuration where a paramyxovirus N protein is rearranged so that its N protein is modified to provide a new N terminus using a segment of the N protein.

In other examples, the disclosure excludes fusion solely with polyanionic amino acids, such as found in a FLAG-tag, and can exclude fusion solely with epitope tags, which are short peptide sequences for producing high-affinity antibodies, examples of which include but are not limited V5-tag, Myc-tag, and HA-tag. Thus, examples of this disclosure comprise VLPs wherein the foreign protein component of the fusion does not consist only of one of the foregoing polypeptides. In particular, the foreign proteins are present in the fusion protein and are distinct from polypeptide sequences that are used for protein separation, or for visualization of the fusion protein, or as intracellular trafficking signals. How ever, it should be recognized that polypeptide sequences that are used for protein separation, or for visualization of the fusion protein, or as intracellular trafficking signals, or for any other purpose, may be present so long as the foreign protein is also present in the N protein fusion.

EXAMPLES

The Examples of this disclosure are provided on the accompanying Figures, which demonstrate the following.

The results presented in the panels of Figures 1A-1B demonstrate paramyxovirus particle-based cargo delivery. The results presented in the panels of Figure 2 demonstrate a non-limiting example of a procedure for VLP packaging and delivery’ of cargo proteins.

The results presented in the panels of Figures 3A-3C demonstrate packaging of the representative protein Cre recombinase into delivery capable PIV5 VLPs.

The results presented in the panels of Figures 4A-4B demonstrate delivery of Cre recombinase into target cell nuclei using PIV5 VLPs.

The results presented in the panels of Figures 5A-5C show a dose-response for delivery of Cre recombinase using PIV5 VLPs.

The results presented in the panels of Figure 6 demonstrate manipulation of multiple paramyxoviruses to allow protein delivery.

The results presented in the panels of Figures 7A-7D demonstrate a representative M protein mutation, namely a G188D alteration to PIV5 M protein, enhances VLP production, which improves cargo delivery'.

The results presented in the panels of Figure 8 demonstrate that G188D, W235L, I278M. and G357W substitutions to M protein each have the ability’ to enhance the production of delivery-capable cargo-loaded PIV5 VLPs.

The results presented in the panels of Figures 9A-9D demonstrate that second-site M mutant protein increases VLP quantity.

While the invention has been particularly shown and described with reference to specific examples, it should be understood by those having skill in the art that various changes in form and detail may be made therein yvithout departing from the spirit and scope of the present invention as disclosed herein.

Claims

What is claimed is:

1 . Paramyxovirus virus like particles (VLPs) comprising at least one mutation in a paramyxovirus M protein, wherein the at least one mutation increases yield of the VLPs by cells that are modified to produce the VLPs, relative to yield of VLPs produced by cells that are modified to produce the VLPs but do not comprise the same one or more M protein mutations, and wherein the VLPs further comprise a contiguous recombinant polypeptide comprising i) all or a segment of a C-terminal domain of a paramyxovirus nucleocapsid (N) protein and ii) a polypeptide sequence of a distinct protein that is not encoded by an unmodified paramyxovirus genome.

2. The VLPs of claim 1, wherein the at least one mutation is in a Parainfluenza virus 5 strain W3A M protein.

3. The VLPs of claim 2, wherein the at least one mutation is present at amino acid residue 188, 235, 278, or 357.

4. The VLPs of claim 3, wherein the at least one mutation is G188D, I278M, W235L, I278M. or G357W.

5. A method comprising culturing modified cells to produce VLPs that comprise a mutation as in any one of claims 1-4, wherein the modified cells produce a higher yield of VLPs, relative to cells that produce the VLPs that do not comprise the at least one mutation.

6. The method of claim 5, further comprising separating VLPs from the modified cells that produce VLPs that comprise the mutation in the M protein.

7. A method comprising contacting cells with VLPs of any one of claims 1-4, such that the VLPs enter the cells.