SARS-cov-2 virus-like particles for mRNA delivery

Abstract

Provided are engineered SARS-CoV-2 virus-like particles (VLPs) and methods of making and using the VLPs for mRNA delivery. A 3 plasmid (3P) VLP system displays ~7-fold higher viral entry efficiency compared to VLPs formed by co-transfection with 4 plasmids. A 2 plasmid (2P) VLP system is provided where one vector carries the viral surface glycoprotein and the second carries the remaining SARS-CoV-2 structural proteins and reporter gene. The VLPs can carry up to four transgenes, including functional Cas9 mRNA for genome editing. Gene editing of specific target cell types is performed modifying VLP tropism. Successful mRNA delivery to mouse lungs shows that the SARS-CoV-2 VLPs can overcome natural biological barriers to enable pulmonary gene delivery.

Description

[0001] Attorney Docket No.: 011520.01999

[0002] SARS-CoV-2 Virus-Like Particles for mRNA Delivery CROSS REFERENCE TO RELATED APPLICATIONS

[0003] This application claims the benefit of priority to U.S. provisional application no. 63 / 754,897, filed February 6, 2025, the entire disclosure of which is incorporated herein by reference.

[0004] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

[0005] This invention was made with government support under grant number TR001412 awarded by the National Institutes of Health. The government has certain rights in the invention.

[0006] SEQUENCE LISTING

[0007] The instant application contains a Sequence Listing which is submitted electronically in XML file format (the “Sequence Listing XML”). The Sequence Listing XML is named “011520.01861.xml,” was created on February 3, 2026, and is 12,377 bytes in size. The Sequence Listing XML is herein incorporated by reference in its entirety.

[0008] FIELD

[0009] This disclosure relates generally to nucleic acid delivery and more specifically use of Coronavirus virus-like particles (VLP) as a means of such delivery.

[0010] RELATED INFORMATION COVID-19 (Coronavirus disease 2019) is caused by the severe acute respiratory syndrome coronavirus 2 or SARS-CoV-2 (SARS2). A number of experimental platforms have been developed to study the biological features of SARS2 and to develop related countermeasures. One of these involves the development of non-replicative virus-like particles or SARS2 VLPs that resemble the authentic virus as they are self-assembled by coexpression of the four SARS2 structural proteins in cells (1). These structural proteins include the viral spike (S), nucleocapsid (N), membrane (M) and envelope (E) proteins. VLPs with fewer SARS2 structural proteins may be formulated, but these often result in less infectious agents (2). SARS2 VLPs are mostly used to advance vaccine development research as these particles are non-replicative, exhibit low toxicity, are more stable than subunit vaccines, and can promote development of immunological response against multiple viral proteins (3-5). While the earlier SARS2 VLP formulations did not carry a packaging sequence to encapsulate mRNA (6-8), Syed etal. demonstrated the feasibility of achieving this byincluding a SARS2 cis-acting RNA packaging sequence that engages the SARS2 N protein (9). The authors showed that tagging a reporter gene to a cis-acting segment of the SARS2 genome called “T20”, which encodes for the nonstructural proteins 15 and 16 (nucleotides: 20,080-22,222), enables the formation of a ribonucleoprotein complex with N protein that is then packaged into the VLP. In addition to T20, even a smaller segment called “PS9” (nucleotides: 20,080-21,171) allowed similar encapsulation of a reporter gene into the VLP. Such viral particles have been useful in basic science studies that quantify the effect of spike mutations (10) and glycosylation on viral infectivity (11).

[0011] Besides vaccine development and fundamental biomedical investigations, there is interest in testing VLPs as a platform for gene delivery to heterologous cells (12,13). Unlike 25 nm non-enveloped adeno-associated virus (AAV) with a -5 kb genome, VLPs formulated using SARS2 proteins could potentially carry larger mRNA due to their large 100-120 nm physical size and -29.9 kb genome (14,15). Such VLPs would be non-integrative as opposed to lentivirus, and they may potentially result in lower immunogenicity compared to adenovirus that promote T-cell mediated cytotoxicity (16). Compared to non-viral delivery strategies like polyplexes, lipoplexes and lipid nanoparticle formulations which display predilection to liver or spleen (17,18), the tropism of these viral mimetics may be tuned by modifying their surface glycoproteins. In systemic circulation, virions like SARS2 exhibit natural organotropism that is different from synthetic nanoparticles (19). Banskota et al. reported the engineering of the Friend murine leukemia virus VLPs for base editor targeting or Cas9 ribonucleoprotein (RNP) delivery in vitro and in vivo (20). Segel et al. describe a mammalian retrovirus-like protein PEG10 which enables the packaging of cargo mRNA, forming VLPs that can be pseudotyped with vesicular stomatitis virus G-protein (VSV-G) for mRNA delivery into cells (21). Despite these developments, there is an ongoing need for improved compositions and methods for producing VLPs and using them to deliver payloads. The present disclosure is pertinent to this need.

[0012] BRIEF SUMMARY

[0013] The present disclosure demonstrates tuning the tropism and packaging capacity of VLPs formulated using SARS2 structural proteins as representative examples of Coronaviridae proteins. The disclosure includes extensive molecular engineering to reduce the number of plasmids used to synthesize the VLPs from four in a previously described “4P” system (10,11), to three (“3P” system) and then down to two (“2P” system). The disclosure includes a nomenclature that is related to data on the structural proteins, viral glycoproteinand reporter encapsulated in the VLPs, with Luc-PS9 being the default reporter, and as a representative but not limiting payload. Examples of the described nomenclature in 3P SARS2 EGFP-PS9 VLP which denotes a 3 plasmid VLP containing a SARS-CoV-2 spike glycoprotein and EGFP reporter that is linked to PS9. The extensive biomolecular engineering described herein resulted in VLPs that are both easier to synthesize and display improved entry properties relative to previously available approaches. Transcomplementation with various viral glycoproteins enabled modification of viral tropism. Additionally, mRNA encoding for up to four different proteins could be packaged in a single VLP. In a representative and non-limiting example, gene editing was enabled by packaging the S. Pyrogenes Cas9 mRNA in the particles. In a non-limiting proof of principal, the pulmonary SARS2 virions can be readily aerosolized (22). The disclosure therefore demonstrates that VLPs may be used for pulmonary gene delivery. Thus, the disclosure describes a streamlined platform for the robust production of SARS2 VLPs, for use in payload delivery applications, among other uses.

[0014] BRIEF DESCRPTION OF THE FIGURES

[0015] Figure 1. 3P SARS2 VLPs are superior to 4P SARS2 VLPs A. 4P and 3P SARS2 Luc-PS9 VLPs were produced by co-transfection of either 4 or 3 plasmids into 293T producer cells. These plasmids encode for the viral structural proteins, along with a luciferase reporter coupled to PS9 packaging signal (Luc-PS9). B. Luciferase assay showed ~7-fold higher luminescence intensity in 293T-hACE2 cells upon using 3P versus 4P VLPs. C. VLP concentrate (10 pL for M and E, 2 pL for S2 and N) was loaded in each lane. Western blot analysis suggests incorporation of all SARS-CoV-2 structural components in VLPs, with 3P SARS2 Luc-PS9 VLPs displaying more intense protein bands compared to 4P VLPs. D. Higher Luc-PS9 transcript levels were observed using real-time PCR (RT-PCR) in the case of 3P SARS2 Luc-PS9 VLPs. E. Cryo-transmission electron microscopy (Cryo-TEM) images of 3P SARS2 Luc-PS9 VLPs showed spherical -lOOnrn sized VLPs with double-layered membrane structures. F. 3P SARS2 EGFP-PS9 VLPs produced with EGFP reporter efficiently infected 293T-hACE2 and A549-hACE2-TMPRSS2 cells. Fluorescence images were acquired 24 h post-infection. G. Flow cytometry VLP entry time-course showed peak fluorescence for 3P SARS2 EGFP-PS9 VLPs at 24 h followed by decrease at larger times. This was observed both for 293T-hACE2 and A549-hACE2-TMPRSS2 cells. Abbreviations: ‘aa’: amino acid; ‘nt’: nucleotide; ‘MFI’: mean fluorescence intensity. Data are Mean + STD. *P < 0.05, **P < 0.01, ***P < 0.001, **** < 0.0001, NS: not significant.Figure 2. Tuning VLP viral tropism by altering viral glycoprotein. A. Different types of VLPs were produced using the 3P system by varying the viral glycoprotein (VSV-G, SARS2 spike, SARS spike or MERS spike) and reporter genes (luciferase or EGFP). B. 1 pg of the viral glycoprotein was used to create various 3P Luc-PS9 VLPs and these were used to infect five cell types: wild-type 293 T (293 T), 293T-hACE2, A549-hACE2-TMPRSS2, Calu-3 and 293T-DPP4. SARS2 and SARS spike VLPs displayed similar tropism and entered only human ACE2 expressing cells (293T-hACE2, A549-hACE2-TMPRSS2, Calu-3), with SARS exhibiting higher luminescence intensity compared to SARS2. MERS VLPs infected Calu-3 cell at low level and efficiently entered 293T-DPP4 cells. VSV-G VLPs entered all cell types.

[0016] C. 3P EGFP-PS9 VLPs were produced with 4 pg VSV-G, 1 pg SARS2 spike or 1 pg MERS spike plasmid. VSV-G VLPs entered all cell types. SARS2 VLPs only entered ACE2 cells. MERS VLPs only entered DPP4 cells. Methods provides detailed steps for VLP production. Data are Mean

[0017]

[0018] < 0.05, **P < 0.01, ***P < 0.001, ****p < 0.0001, NS: not significant.

[0019] Figure 3. Streamlining VLP technology using a 2 plasmid (2P) system. A. Four constructs were developed with two independent promoters driving expression of reporter gene and SARS2 structural proteins. The promoters were separated by insulator and terminator sequences to minimize promoter interference: synthetic polyA (‘spa); a G-rich sequence from P-actin (Tactb); chicken hypersensitive site 4 (cHS4), and a synthetic MAR sequence 8 (sMAR8) at the end of the E protein. B-C. Each of these constructs was transfected into 293T cells along with spike plasmid to produce four different 2P SARS2 Luc-PS9 VLPs. 2P.2 VLPs displayed highest luminescence intensity (panel B). Its signal was comparable to 4P VLP but lower than 3P VLP (panel C). D. Western blots of SARS2 structural proteins showed different patterns of protein expression for different 2P plasmids.

[0020] 2P.2 SARS2 Luc-PS9 VLPs displayed more intense protein bands compared to 2P.3 and 2P.4, but this was lower than 2P.1. E. 2P.2.EGFP VLPs were created by replacing the luciferase reporter with EGFP. VLP entry of 2P.2.EGFP SARS2 VLPs into A549-hACE2-TMPRSS2 and 293T-hACE2 cells was measured using flow cytometry. Data are Mean + STD. ***P < 0.001, ****P < 0.0001, NS: not significant.

[0021] Figure 4. Delivery of four transgenes using 3P SARS2 VLPs. A. Schematic of six PS9 constructs with successive addition of four different reporter genes: EGFP, dTomato, TagBFP and / or luciferase. These constructs were employed to develop 3P VLPs for transgene delivery. B. Fluorescence and luminescence signal for multiple biological replicates using different 3P constructs (same amount of VLP in each case). The relationbetween payload size and viral entry was measured based on reporter intensity (upper panels) and % fluorescent cells (lower panels). While % of infected cells decreased gradually upon increasing mRNA package size, the decrease in fluorescence intensity was more dramatic. Data are Mean + STD. ** < 0.01, ***P < 0.001, ****P < 0.0001, NS: not significant.

[0022] Figure 5. Mechanisms regulating VLP function and efficacy. A. VLPs with either EGFP reporter (downstream of CMV promoter) or luciferase reporter (downstream of CMV or IRES) were produced using the 3P system. Various parameters were measured as illustrated. B-C. The individual panels in these plots from left to right present: i) reporter signal intensity in producer cells; ii) N protein ELISA levels for individual VLP preparations; iii) ddPCR quantitation of PS9 mRNA levels in VLPs; iv) reporter signal in target cells following VLP infection; and v) western blot of VLP structural proteins. EGFP fluorescence data are presented in panel B and luciferase reporter data in panel C. The data showed that VLPs with similar structural compositions and mRNA copy numbers were produced for all payloads. However, increasing payload size progressively decreased reporter signal in target cells. Data are Mean + STD. *** < 0.001, ****p < 0.0001, NS: not significant.

[0023] Figure 6. VLPs deliver functional Cas9 mRNA into target cells to achieve gene editing. A. 3P Cas9-P2A-dTo-T20 VLPs (dTo: dTomato) were used for gene editing studies, with surface glycoprotein encoding for either VSV-G or SARS2 spike. B. General workflow of gene editing study performed in panels C-E. sgRNAs were transfected into cells on day -1 using plasmids carrying BFP reporter. VLP carrying spCas9 mRNA was introduced into cells on day 0. Tropism of the VLP depends on surface glycoprotein. Gene editing efficiency was quantified on day 6. Editing efficiency quantified % of BFP(+) population that turned either EGFP(-) (panels C, E) or lost ACE2 expression based on anti-human ACE2 binding (panel D). C. sgRNAs targeting EGFP were introduced into 293 T-hACE2 -EGFP and spCas9 mRNA was delivered using 3P SARS2 Cas9-P2A-dTo-T20 VLPs. D. sgRNAs against hACE2 were introduced to knockout the receptor in 293T-hACE2 cells using 3P SARS2 Cas9-P2A-dTo-T20 VLPs. E. sgRNAs targeting EGFP were introduced in 293T-EGFP cells, with genome editing being performed using 3P Cas9-P2A-dTo-T20 VLPs bearing either VSV-G or SARS2 spike. In all panels, the target gene (EGFP or hACE2) was knocked out in 20-35% of cells expressing sgRNA. Higher VLP amount resulted in greater editing. F. 293T-hACE2 stably express sgRNAs against SLC35A1 were infected with 3P SARS2 Cas9-P2A-dTo-T20 VLPs (1.885 pg / pL N protein equivalent) or without SARS2 spike (1.385 pg / pL N protein equivalent). Gene editing efficiency was evaluated based on increase in fluorescent peanut agglutinin lectin (PNA) binding to cells. >70% gene editing was observed upon using3P VLPs to edit endogenous genes. Volume of VLP used in each assay is specified in individual panels. Data are Mean + STD. *** < 0.001, ****P < 0.0001, NS: not significant.

[0024] Figure 7. In vivo pulmonary gene delivery using VLPs. A. VLPs bearing either VSV-G or mouse-adapted SARS2 spike (‘maSARS2’) were instilled into mice at time=0. 24 h post-instillation, luciferase activity was measured in tissue extracts from left lung, right lung or trachea . Protein concentration in lysate was used to normalize luminescence signal, with untreated / mock values being set to 1.0 for all in vivo studies. B. VSV-G VLPs were instilled via either oropharyngeal aspiration (100 pL, o.p.a.) or intranasal (50 pL, i.n.) routes, o.p.a. resulted in VLP administration to mouse lung. C. Q493K and N501 Y mutations were introduced into SARS2 spike to generate maSARS2 spike. 3P VLPs with VSV-G, SARS2, maSARS2 and no spike were produced with N protein equivalents of 1.859, 1.149, 1.334 and 2.024 pg / pL, respectively. 50 pL of VLP was used to infect three target cell types: 293T, 293T-hACE2, and 293T-mACE2. VSV-G VLPs infected all three cell types, SARS2 spike only infected 293T-hACE2 (human ACE2), whereas maSARS2 spike was permissive to both 293T-hACE2 and 293T-mACE2. VSV-G luminescence was set to 1.0 in this panel. D. 100 pL of VSV-G VLPs or maSARS2 VLPs, both 0.820 pg / pL N protein equivalent, were instilled via o.p.a. into mice. Whole lung tissue was harvested. Mice without VLPs served as negative control. Both VSV-G and maSARS2 VLPs enabled luciferase signal in mouse lung with VSV-G being more efficient. Data are Mean + STD. N = 5-6 for each mouse treatment group. < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, NS: not significant.

[0025] Figure 8. VLP system requires a specific spike plasmid mass for efficient production (Related to Figure 1). A-B. SARS2 Luc VLPs were produced for 4P VLPs (panel A) and 3P VLPs (panel B) in 150mm petri dishes, keeping the total plasmid mass constant at 50 pg / plate in all cases, while varying the proportion of spike plasmid from 0-4 pg. VLP entry properties was evaluated by measuring luminescence in recipient 293 T-hACE2 cells. Highest entry peaked upon using 1 pg spike plasmid for both systems. C.

[0026] Dynamic light scattering (DLS) quantified particle size distribution of 3P SARS2 Luc-PS9 VLPs. VLPs with spike sized at -146 nm, slightly higher than that of the VLPs without spike (-125 nm). D. Representative flow cytometry histograms for data presented in Figure 1G (main Figure). Appreciable EGFP expression was observed in a vast majority of the cells, in these studies that measured 3P SARS2 EGFP-PS9 VLP entry into either 293T-hACE2 or A549-hACE2-TMPRSS2 cells. Data are Mean ± STD. **P < 0.01, ***P < 0.001, ****P < 0.0001, NS: not significant.Figure 9. Turning viral tropism (Related to Figure 2). A. Stable isogenic 293T-DPP4 cells were established and DPP4 expression level was measured using Alexa Fluor 488 conjugated anti-DPP4 antibody using flow cytometry. B. VLP entry data shown in Figure 2B (main Figure) were also collected upon increasing detection sensitivity in the luminescence plate reader. The data show that VSV-G VLPs have strong entry efficiency, although it is lower than SARS2 or SARS VLPs for 293T-hACE2 and A549-hACE2-TMPRSS2 cells. C-D. VSV-G and MERS spike plasmid mass was varied during production of 3P Luc-PS9 VLPs bearing these glycoproteins in 150mm petri dishes. VLPs produced using 4 pg VSV-G or 2 pg MERS spike plasmid caused maximal infection of target cells. E. Representative flow cytometry histograms for Figure 2C (main Figure) , showing that VLP tropism can be varied by changing viral glycoprotein. Data are Mean + STD.

[0027]

[0028] < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, NS: not significant.

[0029] Figure 10. Characterization of 2P VLPs (Related to Figure 3). A. The NeoR / KanR resistance gene in pcDNA3.1 N-T2A-M-IRES-E plasmid, which is driven by the SV40 promoter was replaced by Luc-PS9. This new ‘2P’ construct is called CMV-NME SV40-LucPS9. The proportion of this plasmid and SARS2 spike plasmid was varied during the production of SARS2 Luc-PS9 VLPs. Luminescence intensity in 293T-hACE2 cells was low for these particles in comparison to 3P VLPs, regardless of the VLP production condition. B.

[0030] All four 2P SARS2 Luc-PS9 VLPs entered 293T-hACE2 cells in a spike dependent manner, albeit with different efficiencies. C. A restriction enzyme site Pact, embedded in the insulator / terminator region of 2P, was digested to linearize 2P.1, 2P.2, 2P.3 and 2P.4 during the VLP production step. Such cutting, which presumably eliminates any promoter interference, failed to improve the entry infectivity of the resulting VLPs. D. Dynamic light scattering (DLS) showing the particle size distribution of 2P.2 SARS2 Luc-PS9 VLPs. VLPs with spike (-146 nm) was sized slightly larger than the VLPs without spike (-125 nm). E.

[0031] 2P.2 VLP production was optimal upon using 1 pg plasmid mass. F. Representative flow cytometry histogram data for Figure 3E (main Figure) showing appreciable VLP entry into hACE2 bearing cells when using 2P VLPs. Data are Mean + STD. **P < 0.01,

[0032]

[0033] 0.0001.

[0034] Figure 11. Effect of payload size on delivery (Related to Figure 4). Representative flow cytometry histograms for experiment in Figure 4B that quantified EGFP, dTomato and TagBFP fluorescence in 293T-hACE2 cells following addition of 3P SARS2 VLPs with different mRNA payloads. Data are presented for ‘mock’ and ‘no spike VLPs’ to illustratebackground fluorescence and spike-dependent VLP entry. Vertical line separates background signal from signal due to spike dependent VLP entry.

[0035] Figure 12. Strategies to enhance VLP efficacy (Related to Figure 4). A. Plasmid amounts were varied during VLP production to determine optimal stoichiometry needed to generate 3P VLPs. These studies were performed using const. 6 as payload. Amounts of spike, N-T2A-M-IRES-E and const. 6 plasmid in each case are presented. Increasing const. 6 plasmid amount to 40 pg increased viral entry by -25% compared to the standard stoichiometry that used equal amounts of N-T2A-M-IRES-E and const 6, both at 25 pg. Other conditions tested decreased EGFP signal. B. A duplicate PS9 packaging signal was appended at the end of the original PS9, resulting in an EGFP-PS9-PS9 construct. C. This construct failed to enhance viral entry measured based on fluorescence intensity for the 3P VLP system. Data are Mean + STD. ** < 0.01, ***P < 0.001, ****P < 0.0001.

[0036] Figure 13. Characterization of VLPs using ddPCR and ELISA (related to Figure 5). A. Representative ddPCR plot showing positive and negative population, when using probe directed against the PS9 sequence of the package. B. N protein ELISA standard curve for quantification of VLP concentration. VLP concentration was determined based on equivalent N protein amount incorporated into the particle.

[0037] Figure 14. T20 is a more efficient packaging sequence compared to PS9 in the 3P VLP system (Related to Figure 6). A. 3P SARS2 Cas9-P2A-dTo VLPs (dTo: dTomato) were produced when the packaging signal was either PS9 or T20. B. VLP entry was measured in 293T-hACE2 cells based on the dTomato signal from either the 3P SARS2 Cas9-P2A-dTo-PS9 VLPs (left) or 3P SARS2 Cas9-P2A-dTo-T20 VLPs (right). VLP entry measured based on both % of cells that are dTomato positive and MFI was higher for 3P SARS2 Cas9-P2A-dTo-T20 VLPs compared to Cas9-P2A-dTo-PS9 VLPs across a range of VLP titers. C. Cas9-P2A-dTo-T20 was replaced by luciferase reporter in the above assay. Luminescence signal was -2.5 times higher for 3P SARS2 Luc-T20 VLPs compared to 3P SARS2 Luc-PS9 VLPs. Due to these observations, T20 was used as packaging sequence in Figure 6 and 7 (main Figure). Data are Mean + STD. **** < 0.0001.

[0038] Figure 15. Gene editing of EGFP and human ACE2 (hACE2) (Related to Fig. 6). A. Stable isogenic 293 T-hACE2 -EGFP (left panel) and 293T-EGFP (right panel) cells were created for the spCas9 editing studies, by transduction using lentivirus carrying EGFP transgene. Both cell types expressed high levels of EGFP as assessed using flow cytometry.

[0039] B. Representative dot plot of hACE2 gene editing in 293T-hACE2 cells by SARS2 VLPs in Figure 6D (main manuscript). C. Genomic DNA was extracted from edited 293T-hACE2cells in panel B studies. The edited region was PCR amplified and then sequenced using 150 bp paired end Illumina next-generation sequencing (NGS). Editing efficiency was quantified based on the site of editing in single reads (site 1, 2 or both sites) and this was normalized based on the total amplicon reads. D. Representative dot plot of gene editing of EGFP by VSV-G pseudotyped SARS-CoV-2 VLPs in Figure 6E (main manuscript). E. Histogram shows dTomato fluorescence intensity measured upon infection of 3P SARS2 Cas9-P2A-dTo-T20 VLPs into 293T-hACE2 cells at 24h and 6 days post-infection. A majority of cells were dTomato positive at 24h. Signal was absent 6 days post-infection, indicating the transient mRNA delivery into target cells.

[0040] Figure 16. Knocking out SLC35A1 in 293T-hACE2 cells augments peanut agglutinin lectin (PNA) binding (Related to Figure 6). A. 293T cells showed enhanced PNA binding following sialidase treatment. B. 293T-hACE2 cells stably express sgRNAs against SLC35A1 (denoted as ‘293T-hACE2-SLC35Al sgRNA’ cells) were generated by transducing 293T-hACE2 cells with a pool of two VSV-G pseudotyped lentivirus each carrying an sgRNA targeting SLC35A1. Transduced cells were sorted based on BFP fluorescence (see vector schematic). C. 293T-hACE2-SLC35Al sgRNA cells were transfected with Cas9-P2A-dTo-T20 plasmid. Editing efficiency [=100 x Top right / (Top right + Top left)] was -65% in Cas9-P2A-dTomato positive cells at day 6, based on increased PNA binding. Representative cytometry plots are presented.

[0041] Figure 17. 293T mouse ACE2 (‘293T-mACE2’) cells (Related to Figure 7). 293T-mACE2 cells were produced by transducing VSV-G pseudotyped lentivirus carrying mouse ACE2 transgene into wild-type 293T cells. Mouse-ACE2 expressing cells were sorted using FACS and expression level quantified using anti-mouse ACE2 monoclonal antibody in flow cytometry run.

[0042] DETAILED DESCRIPTION

[0043] Unless defined otherwise, 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 invention belongs.

[0044] Every numerical range given throughout this specification includes its upper and lower values, as well as every narrower numerical range that falls within it, as if such narrower numerical ranges were all expressly written herein.

[0045] As used in the specification and the appended claims, the singular forms “a” “and” and “the" include plural referents unless the context clearly dictates otherwise. Ranges maybe expressed herein as from “about” one particular value, and / or to “about” another particular value. When such a range is expressed, another example includes from the one particular value and / or to the other particular value. Similarly, when values are expressed as approximations, by the use of the antecedent “about” it will be understood that the particular value forms another example. The term “about” in relation to a numerical value encompasses variations of + / - 10%, + / - 5%, or + / - 1%.

[0046] This disclosure includes proteins described or referenced herein and all nucleotide sequences encoding the amino acid sequences. Polynucleotide and amino acid sequences having from 80-99% similarity, inclusive, and including and all numbers and ranges of numbers there between, with the sequences provided here are included in the invention.

[0047] The disclosure includes all described polynucleotides, proteins, VLPs, methods of making the VLPs, and methods of using the VLPs.

[0048] Any described polynucleotide (e.g., the described plasmids) may be used for transient transfection, or one or all of the polynucleotides may be integrated into one or more chromosomes. The disclosure therefore includes stable cell lines for use as VLP producing cells.

[0049] In examples, the VLPs comprise at least one payload. In examples, more than one payload can be included. The additional payloads may be provided in tandem or separated. In examples, 1, 2, 3, 4, or more payloads are included.

[0050] The disclosure includes the following examples.

[0051] Based on the description herein and the accompanying figures, the present disclosure includes the following examples.

[0052] A method for producing virus-like particles (VLPs) derived from a coronavirus by introducing into a plurality of eukaryotic cells a combination of polynucleotides comprising or consisting of first, second, and optionally a third plasmid, wherein:

[0053] i) the first plasmid comprises a promoter operably linked to a sequence encoding a coronavirus nucleoprotein (“N” protein), a coronavirus membrane protein (“M” protein) and a coronavirus small membrane protein (“E” protein);

[0054] ii) the second plasmid comprises a promoter operably linked to a sequence encoding a protein that can bind to a target on the surface of a cell (a cell targeting protein), and wherein the cell targeting protein is optionally a spike protein (“S” protein);

[0055] iii) the third plasmid comprises a promoter operably linked to a coronavirus packaging signal and a sequence encoding a desired payload; andiv) allowing expression of the N, M, E, the cell targeting protein, the payload, and the packaging signal within the cells such that the VLPs are formed and comprise the N, M, and E, proteins, the packaging signal, and the payload; and

[0056] v) separating the VLPs of iv) from the cells to provide an isolated VLP preparation.

[0057] The disclosure includes all described ratios of plasmids used in generating the VLPs. In examples, the tropism of the VLPs is tunable by changing the sequence encoding the cell targeting protein such that the VLPs bind to and deliver the payload comprised by the VLPs into cells to which the cell targeting protein binds.

[0058] In examples, the N, M, and E proteins may be encoded by a single RNA and the N, M, and E proteins are separated from one another by polynucleotide segments that comprise an internal ribosome entry site (IRES), a self-cleaving peptide sequence, or a combination thereof.

[0059] In examples, the promoter of the first, second, or third plasmids are the same, or at least two of the promoters are different promoters.

[0060] In examples, the N, M, or E proteins, are present in a fusion protein.

[0061] In examples, the payload comprises a) an RNA polynucleotide that does not encode a protein, or b) an RNA polynucleotide that encodes a protein that is translated and is present as a component of the VLP, or a combination of a) and b).

[0062] In examples, entry of the VLPs into cells to which the cell targeting protein binds is greater relative to entry of VLPs produced using four plasmids, wherein a first plasmid encodes a coronavirus E protein, a second plasmid encodes a coronavirus S protein, a third plasmid encodes a coronavirus M protein, and a fourth plasmid encodes a coronavirus N protein.

[0063] The amino acid sequences of the described coronavirus proteins are known in the art. In non-limiting examples, the described VLPs can include proteins that identical to or are at least 80-99% identical to amino acid sequences available in GenBank accession numbers NC_045512, MZ208926, MZ571142, OMOl 1974, and MZ020420, from which the amino acid sequences are incorporated herein as they exist in the GenBank database on the filing date of this application. The disclosure includes all DNA and RNA sequences that encode the proteins. In examples, the described proteins are obtained from any Betacoronavirus. In examples, the proteins are obtained from a human coronavirus, a feline coronavirus, a bovine coronavirus, a porcine coronavirus, or a bat coronavirus.The disclosure includes isolated VLP preparation produced according to described method. In an example, the disclosure provides a method for producing the described VLPs derived from a coronavirus by introducing into a plurality of eukaryotic cells a combination of polynucleotides consisting of first and second polynucleotides, which optionally may be plasmids. In this example, the first plasmid comprises a promoter operably linked to a sequence comprising a payload and a coronavirus packaging signal, and a second promoter operably linked to a sequence encoding a coronavirus nucleoprotein N protein, and a coronavirus membrane M protein, and a coronavirus small membrane protein E. The second polynucleotide comprises a promoter operably linked to a sequence encoding a protein that can bind to a target on the surface of a cell (a cell targeting protein). The cell targeting protein is optionally a spike protein (“S” protein), and a sequence encoding a payload. The method includes allowing expression of the N, M, E, the cell targeting protein, the payload, and the packaging signal within the cells such that the VLPs are formed and comprise the N, M, and E, proteins, the packaging signal, and the payload. The method provides for separating the VLPs from the cells to provide an isolated VLP preparation. As described herein, the tropism of the VLPs is tunable by changing the sequence encoding the cell targeting protein such that the VLPs bind to and deliver the payload comprised by the VLPs into cells to which the cell targeting protein binds.

[0064] The disclosure includes a method comprising administering to cells a composition comprising an isolated VLP preparation as described herein such that the VLPs enter the cells and wherein the payload affects at least one property of the cells. The method may have a therapeutic or prophylactic effect. The payload may be encoded by a polynucleotide within the VLP or may be a component of the VLP itself.

[0065] The payload provided as a component of the VLP is not particularly limited. In examples, the payload is a protein, or a functional polynucleotide. In examples, the payload is foreign to the recipient cell, i.e., the cell did not include the payload prior to being contacted with the VLPs.

[0066] The described proteins may be modified to include those that produce a detectable signal, or for protein purification purposes, such as by including a Sumo or poly-histidine tag. In examples, any one or combination of the described N, M, or E proteins may be provided as components of a fusion protein. The payload proteins when provided as fusion proteins with the N, M, or E proteins, may also include ribosomal skipping sequences, protease recognition sequences, linker sequences, or any combination thereof.Any payload used in this disclosure may be modified to include a localization signal, such as a nuclear localization signal or a signal to localize the payload to any other organelle, or a secretion signal to promote secretion of the payload.

[0067] In examples, the payload is any one or a combination of enzymes, receptor ligands, transcriptional factors, growth factors, antibodies or antigen-binding fragments thereof including single-chain 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 examples, the payload comprises a hormone, a growth factor, a clotting factor, or a cytokine.

[0068] For gene editing applications, the VLPs may be configured such that they express or include or are used with a nuclease, a nickase, or enzymatically inactivated variants thereof. In examples, any CRISPR nuclease may be used. CRISPR nucleases are known in the art and include but are not necessarily limited to CRISPR effectors Cas9 (Type II) and Cast 2 (Type V) CRISPR proteins. Cas3 proteins may also be used. In examples, the described VLPs may be configured to include components for use in prime-editing, or may be configured to facilitate retron-based editing. In examples, the VLPs may include TALONS or zinc fingers for producing genetic modifications. In an example, a Cas9 enzyme that is included in a described VLP has the amino acid sequence of a Cas9 encoded by Streptococcus pyogenes. In an example, the enzyme is a Cpfl enzyme. Any cell type that is susceptible to infection by Coronavirus 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 and may produce indels, or insertion of a repair template. 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 homozygous 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 described VLPs may comprise all or some components used for prime editing, such as a prime editor protein composing a fusion protein that includes a Cas9 nickase. The VLP may be configured to include and / or encoding a prime editing guide RNA (pegRNA). In an example, the VLP may include components for retron editing, such as a reverse transcriptase, a non-coding RNA, and any suitable nickase.

[0069] Contacting cells with described VLPs can be performed using any suitable approach. The disclosure includes using the described VLPs in vivo and ex vivo.In examples, the described VLPs are considered suitable for use in any eukaryotic cells that are capable of receiving and internalizing the VLPs. In examples, eukaryotic cells that are modified using a VLP preparation of this disclosure are totipotent, pluripotent, multipotent, or oligopotent stem cells when the modification is made. In examples, the cells are neural 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 cancer cells, or cancer stem cells. In examples, the cells are differentiated cells when the modification is made. In examples, the cells are mammalian cells. In examples, the cells are human, or non-human animal cells.

[0070] In certain approaches, compositions of this disclosure, including the described VLPs, are used for treatment of condition or disorder in an individual in need thereof. The term “treatment” as used herein refers to alleviation of one or more symptoms or features associated with the presence of the particular condition or suspected condition being treated. Treatment does not necessarily mean complete cure or remission, nor does it preclude recurrence or relapses. Treatment can be effected over a short term, over a medium term, or can be a long-term treatment, such as, within the context of a maintenance therapy. Treatment can be continuous or intermittent.

[0071] In examples, a composition comprising VLPs is administered to an individual in a therapeutically effective amount. In examples, a therapeutically effective amount of a composition of this disclosure is used. The term “therapeutically effective amount” as used herein refers to an amount of the VLPs sufficient to achieve, in a single or multiple doses, the intended purpose of treatment. Appropriate effective amounts can be determined by one of ordinary skill in the art informed by the instant disclosure using routine experimentation. For example, a therapeutically effective amount, e.g., a VLP dose, can be estimated initially either in cell culture assays or in animal models. An animal model can also be used to determine a suitable VLP concentration range, and route of administration. Such information can then be used to determine useful doses and routes for administration in humans, or to non-human animals. A precise dosage can be selected by in view of the patient to be treated. Dosage and administration can be adjusted to provide sufficient levels of VLPs to achieve a desired effect, such as a modification in a threshold number of cells. Additional factors which may be taken into account, such as in the case of gene editing, include the particular gene or other genetic element involved, the type of condition, the age, weight and gender of thepatient, desired duration of treatment, method of administration, time and frequency of administration, drug combination(s), reaction sensitivities, and tolerance / response to therapy.

[0072] Administration of formulations comprising VLPs as described herein can be performed using any suitable route of administration, including but not limited to parenteral, intraperitoneal, intrapulmonary, oral, and intra-tumoral. Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal, and subcutaneous administration. In examples, a VLP preparation is configured to be delivered as aerosol, and thus may be administered by oral or nasal inhalation. The VLP preparation may be provided as a powder, and may be lyophilized, such as for reconstitution.

[0073] In examples, the disclosure provides an article of manufacture, which may comprise a kit. In examples, the article of manufacture may comprise one or more described cloning vectors, e.g., the described plasmids. The one or more cloning vectors may encode any one or combination of proteins and polynucleotides described herein. The cloning vectors may be adapted to include, for example, a multiple cloning site (MCS), into which a sequence encoding any protein or polynucleotide, such as any desired targeting RNA and / or payload, may be introduced. An article of manufacture may include one or more sealed containers that contain any of the aforementioned components, and may further comprise packaging and / or printed material. The printed material may provide information on the contents of the article, and may provide instructions or other indication of how the contents of the article may be used. In an example, the printed material provides an indication of a disease or disorder that is to be treated using the contents of the article.

[0074] In examples, a VLP / RNP may comprise modified polynucleotides, which may include phosphate backbone modifications, and modified nucleotides, such as nucleotide analogs.

[0075] As will be recognized from the description above and the figures, and the following Examples, this disclosure demonstrates, among other aspects, that SARS-CoV-2 VLP tropism could be varied by modifying surface glycoproteins. Payloads up to ~5kb were packaged, including Streptococcus pyogenes Cas9 for genome editing. The disclosure demonstrates the VLPs could cross physiological barriers to enable mRNA delivery to mouse lungs. The disclosure demonstrates Cas9 mRNA delivery using VLPs. sgRNA was delivered separately using an independent plasmid vector or using lentivirus.

[0076] The disclosure includes providing sgRNA and Cas9, or other proteins and RNAs, that are introduced into VLPs using two plasmids, both optionally with 3’ PS9 or T20 packaging signals. It is expected that co-transfection of these constructs along with other 3P plasmids will enable co-packaging of both mRNA in a single VLP. The disclosure includes using Cas9or other proteins fused with viral structural proteins to co-package Cas9 / sgRNA RNPs or other RNPs in the delivery vehicle. The disclosure includes aptamer / aptamer binding protein (ABP) pairing approaches to co-package VLP structural proteins that are fused with ABP complexed with sgRNA that are 3 ’-modified with the reciprocal aptamer sequences.

[0077] The following Examples are intended to illustrate but not limit the disclosure.

[0078] EXAMPLES

[0079] MATERIALS AND METHODS

[0080] Biochemicals: All monoclonal antibodies (mAbs) were from mouse unless otherwise stated. These include anti-SARS-CoV-2 nucleocapsid protein mAb 1035111 (IgG2b ,Cat#: MAB10474), anti-SARS-CoV-2 spike S2 subunit mAb 1034617 (IgG2a, Cat#: MAB10557), Alexa Fluor 647 conjugated anti-human ACE2 mAb 535919 (IgG2a, Cat#: FAB9332R) and rabbit anti-mouse ACE2 mAb 28181 (IgG, Cat#: FAB34372G), all from R&D Systems (Minneapolis, MN). Rabbit anti-SARS-CoV-2 envelope protein polyclonal antibody (pAb) (Cat#: 74698), anti-SARS-CoV-2 membrane protein mAb E5A8A (IgGl, Cat#: 15333), HRP conjugated horse anti-mouse IgG pAb (Cat#: 7076, RRID: AB 330924) and HRP conjugated goat anti-rabbit IgG pAb (Cat#: 7074, RRID: AB 2099233) were purchased from Cell Signaling (Danvers, MA). FITC conjugated anti-human CD26 / DPP4 IgG2a mAb BA5b was from Biolegend (Cat#: 302704). All other biochemicals were from Sigma Chemicals (St. Louis, MO), Gold Biotechnology (Olivette, MO) or ThermoFisher (Waltham, MA) unless otherwise mentioned.

[0081] Cell culture: Human embryonic kidney 293T Lenti-X cells (‘293T’) (Cat#: 632180) were purchased from Clontech / Takara Bio (Mountain View, CA). Stable 293T-human ACE2 (‘293T-hACE2’) cells were from Scripps Research, Jupiter, FL. A549 lung carcinoma overexpressing human ACE2 and TMPRSS2 (‘ A549-hACE2-TMPRSS2 cells’) (Cat#: a549-hace2tpsa) were purchased from Invivogen (San Diego, CA). Calu-3 human airway epithelial cells (Cat#: HTB-55) were from ATCC (Manassas, VA). 293T-DPP4 cells were produced by transducing lentivirus packaged with Dipeptidyl peptidase-4 (DPP4) gene into 293T cells. 293T-hACE2-EGFP cells and 293T-EGFP cells were made by transducing lentivirus packaged with EGFP gene into 293T-hACE2 and 293T cells, respectively. Isogenic clones were selected for the 293T-DPP4 and EGFP bearing cells using FACS sorting, and these were scaled up for downstream studies. 293T-mouse ACE2 (‘293T-mACE2’) cells wereproduced by transducing lentivirus packaged with mouse ACE2 gene into 293T cells. 293T-hACE2-SLC35Al sgRNA cells were produced by transducing 293T-hACE2 with a pool of two VSV-G pseudotyped lentivirus each carrying an sgRNA targeting SLC35A1. These cells were bulk sorted and scaled up prior to use in functional studies. All cells were cultured using Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10 % fetal bovine serum (FBS), 1 % Antibiotic- Antimycotic and 1 % GlutaMAX supplement. Cultures of Calu-3 were additionally supplemented with 1 % non-essential amino acids. 293T-hACE2, 293T-mACE2, 293T-hACE2-SLC35Al sgRNA and 293T-DPP4 cultures additionally contained 1 pg / mL puromycin for selection. A549-hACE2-TMPRSS2 culture media contained 100 pg / mL normocin, 0.5 pg / mL puromycin and 300pg / mL hygromycin based on manufacturer’s instructions. All cell culture was performed in incubators maintained at 37 °C, humidified, 5 % CO2 environment.

[0082] Molecular biology: The parent spike plasmid was as previously described (11). The original plasmid containing the full-length SARS-CoV-2 spike protein with C-terminal FLAG-tag was (23), was site-directed modified to introduce a D614G mutation. The pcDNA3.1 SARS-CoV-2 N containing R203M mutation (RRID: Addgene_l 77952), pcDNA3.1 SARS-CoV-2 M-IRES-E (RRID: Addgene_177938), pcDNA3.1 Luc-PS9 (RRID: Addgene_l 77942), pcDNA3.1 Luc-T20 (RRID: Addgene_l 77941), and pcDNA3.1 GFP-PS9 (RRID: Addgene_ 177944) plasmids were as described (9). The pcDNA3.1 N-T2A-M-IRES-E plasmid was made by inserting the N-T2A fragment upstream of M-IRES-E in pcDNA3.1 SARS-CoV-2 M-IRES-E. The pMD2.G which encodes for VSV-G glycoprotein RRID:

[0083] Addgene_12259) The pCDNA3.3 MERSD12 S plasmid encoding the MERS WT spike protein with a 12-amino acid deletion on the C-terminal tail is RRID: Addgene l 70448). pcDNA3.1 SARS S plasmid encoding the 2002 SARS spike protein was RRID:

[0084] Addgene_145031). The pLEX307-DPP4-puro plasmid encodes the DPP4 / CD26 was RRID: Addgene l 58451. The pscALPSpuro-MmACE2 encoding for mouse ACE2 was a RRID: Addgene l 58087. The lentiviral dual-promoter (LVDP) vector was as described (24). A family of four derivative plasmids were made based on this, including LVDP 2P.1, LVDP 2P.2, LVDP 2P.3 and LVDP 2P.4 each containing different promoter configurations. The LVDP 2P.2.EGFP was produced by replacing the luciferase gene in LVDP 2P.2 with EGFP. The pcDNA3.1 CMV-NME SV40-LucPS9 plasmid was made by replacing the NeoR / KanR gene of pcDNA3.1 SARS-CoV-2 N-T2A-M-IRES-E with the Luc-PS9 gene cassette. The const.2, const.3, const.4, const.5 and const.6 constructs were produced by successive addition of dTomato, TagBFP, and Luciferase reporter genes into the pcDNA3.1 GFP-PS9 plasmid(denoted const.1). These reporter genes were linked using P2A, T2A or encephalomyocarditis virus (EMCV) IRES sequences. The pcDNA3.1 GFP-PS9-PS9 plasmid was cloned by inserting a second PS9 packaging sequence to the end of PS9 in pcDNA3.1 GFP-PS9. sgRNA targeting EGFP, SLC35A1 and human- ACE2 (hACE2) were cloned into the original pKLV-U6gRNA(BbsI)-PGKpuro2ABFP as in RRID:

[0085] Addgene_50946. The pcDNA3.1 Cas9-P2A-dTomato-PS9 and pcDNA3.1 Cas9-P2A-dTomato-T20 plasmids were cloned by replacing the luciferase reporter gene with the Cas9-P2A-dTomato fragment in pcDNA3.1 Luc-PS9 and pcDNA3.1 Luc-T20, respectively. The lentiviral vector pLKO.1 TRC-EGFP was made by replacing the DsRed gene in pLKO.1 TRC-DsRed plasmid with EGFP gene (25). Constructs were typically produced using the NEB HiFi DNA Assembly kit, and verified using either Sanger and / or Oxford Nanopore whole plasmid sequencing.

[0086] Transfection: 293T cells and variants of this cell line were transfected using either the calcium phosphate method (26) or Lipofectamine 2000 reagent following manufacturer’s instructions. Such transfections were used to produce both lentivirus and VLPs, and for the expression of sgRNAs in 293T-hACE2, 293T-hACE2-EGFP and 293T-EGFP cells. Briefly, cells were plated in either cell culture treated 6-well plates or 150 mm petri dishes one day prior to transfection. The next day (day 1), when cell density reached -70% confluence, 2 pg DNA was used to transfect each well in a 6-well plate, while -50 pg DNA was used for each 150 mm petri dish. In all cases, 6-8 h post-transfection, media was switched to fresh Opti-MEM.

[0087] Virus-like particle (VLP) production: To produce VLPs, 15-20 million 293T cells were seeded in 150 mm petri dishes on day 0, in order to reach -70% confluence overnight. -50 pg of plasmid encoding for structural proteins was used in all experiments, unless stated otherwise. Thus, when producing VLPs using the 4 plasmid (‘4P’) VLP system, 8.25 pg M-IRES-E, 16.75 pg N, 25 pg Luc-PS9 and 1 pg spike plasmid were co-transfected on day 1. In the case of the 3 plasmid (‘3P’) VLP system, cells were co-transfected using 25 pg N-T2A-M-IRES-E, 25 pg Luc-PS9 and 1 pg spike plasmids. Similarly, 2 plasmid (‘2P’) VLPs were produced by co-transfecting 293T cells using 50 pg of one of the 2P plasmids, along with 1 pg spike plasmid. In some studies that titrated spike plasmid amounts, spike plasmid fraction was varied while preserving the mass ratio of the remaining constructs encoding for viral structural proteins and Luc / EGFP reporters such that a total of 50 pg plasmid was used for each 150 mm petri dish. In instances where plasmids were linearized prior to transfection for VLP production, 1 pL plasmid DNA (-2 pg / pL), 2 pL rCutSmart buffer (New EnglandBioLabs, Cat#: R0532S), 1 pL restriction enzyme and 16 pL nuclease free water were added into a 0.2 mL PCR tube. The reagents were mixed and incubated at 37 °C for 1 h prior to validation of cut by gel electrophoresis. This product was then also used for cell transfection and VLP production. The above ‘standard’ method for used in all studies unless otherwise stated.

[0088] In examples, 6-8 h post-transfection, the cell culture medium was switched to 20 mL fresh Opti-MEM. 48 h thereafter, the cell culture supernatant was harvested, centrifuged at 4000 g for 5 min. to remove any floating cells, and filtered through 0.45 pm polyethersulfone (PES) membrane to remove cell debris. In most cases, 20 mL of the clarified supernatant was added into polycarbonate centrifuge bottles (Beckman Coulter, Indianapolis, IN). A 4” long stainless-steel needle was then used to inject 20% (g / mL) sucrose solution at 1 / 10thsample volume as cushion to the bottom of the tube. Subsequently, the bottles were ultra-centrifuged at 150,000 g for 2.5 h at 4°C using a Type 70 Ti rotor in an Optima XE Ultracentrifuge (Beckman Coulter). The opaque VLP pellet obtained was resuspended in 200 L PBS (phosphate buffered saline, 1 mM KH2PO4, 155 mM NaCl, 3 mM Na2HPO4). When using a larger Type 45 Ti rotor, the above reagent volumes were proportionally scaled. Following this, the VLP sample (100 x concentrate) was vortexed thoroughly and spun down in a bench-top centrifuge at 13,000 g for 2 min. to remove any remaining debris. The supernatant was transferred into a new microcentrifuge tube. The VLP sample prepared in this manner was either used directly for downstream application or stored at -80 °C for future studies. In addition to the ultracentrifugation method above, in one biological repeat we compared the efficacy of 4P vs. 3P VLPs by simply concentrating the 20mL clarified supernatant 100-fold using a PES protein concentrator (100 kDa cutoff, ThermoFisher).

[0089] Pseudovirus production and engineering of stable cell line: VSV-Gpseudotyped-lentivirus was produced using the 3rdgeneration lentivirus system (27). 293T producer cells were plated in 150 mm petri dish on day 0 to reach -70% confluence the next day. The cells were co-transfected with 18.9 pg pLKO.l TRC-EGFP, 21.4 pg psPAX2 and 5.8 pg VSV-G plasmids. 6-8 h post-transfection, media was switched to 20 mL fresh Opti-MEM. The first virus batch was collected at 18-20 h and stored at 4 °C. Fresh Opti-MEM containing 10 mM sodium butyrate (Sigma- Aldrich) was then added and second virus batch was collected 16-20 h thereafter. Both batches were pooled and centrifuged at 2,000 g for 2 min. to remove floating cells, filtered through 0.45 pm PES membrane to remove cell debris, and then ultracentrifuged at 50,000 g for 2 h at 4 °C to obtain the viral pellet. The pellet was resuspended in 100 pL Opti-MEM, aliquoted and stored at -80 °C for further use.Luminescence assay to assess VLPs infectivity: The target cells were trypsinized and resuspended at 107cells / mL. In a 1.5 mL microcentrifuge tube, 50 pL VLP preparation (default volume, unless otherwise specified) was mixed with 80,000 cells (8 pL of stock), along with the addition of 8 pg / mL polybrene. After incubation at room temperature (RT) for 25 min. with periodic flicking, the cells were transferred into 96-well plates with additional 150 pL fresh DMEM media. Following overnight culture to allow for reporter expression, the cells were washed with 200 pL PBS buffer and lysed using 50 pL / well cell lysis buffer (Gold Biotechnology) at RT on a shaker for 20 min. In this time, fresh 2* TMCA buffer was made by mixing 20 pL MgCh (from 500 mM MgCh 100* stock), 20 pL Coenzyme A (from 25 mM 100* stock), 20 pL ATP (from 15 mM 100* stock), 500 pL Tris-HCl, pH = 7.8 (from 400 mM 4 stock) and additional 440 pL cell culture water to bring up the volume to 1 mL. Following cell lysis, 50 pL 2 TMCA was added into a 96-well white plate with round bottom followed by 50 pL cell lysate. 1 pL D-Luciferin (from 15 mg / mL D-Luciferin 100 / stock) was then added and resulting luminescence was immediately quantified using either a BioTek Synergy4 plate reader (Santa Clara, CA) or a GloMax luminescence microplate reader (Promega, Madison, WI). Studies with mouse tissue were performed identically only using 50 pL mouse lysates produced using tissue lysis buffer (Gold Biotechnology).

[0090] Flow cytometry measurement of receptor expression and VLP entry: Fluorescent reporter expression in VLP transduced cells was measured using methods identical to the luminescence assay above, only using a BD Fortessa X-20 flow cytometer (San Diego, CA) to quantify fluorescence signal in live cells. The cytometer was also used to quantify receptor expression on various cells. In both cases, cells were trypsinized from tissue culture flasks or 6-well plates, and resuspended in HEPES buffer (110 mM NaCl, 10 mM KC1, 2 mM MgCh, 10 mM Glucose, 30 mM HEPES, pH = 7.2-7.3) at 107 / mL. 20 pL cells were added into a 1.5 mL microcentrifuge tube along with 0.5-5 pg / mL fluorescent antibodies if necessary for 15-20 min. on ice. After washing using HEPES buffer, the cells were resuspended at 2xlO6 / mL and analyzed using the cytometer. Mock transduced cells were used as negative control for the VLP entry studies. Both % cells with more than baseline EGFP signal and mean fluorescence intensity (MFI) of all cells are reported.

[0091] Fluorescence microscopy: A 4-well glass chamber slide (Nest Scientific, NJ) was coated with 500 pL of 16 pg / mL matrigel dissolved in PBS for 1 h at 37 °C. The slide was washed once with 500 pL PBS and stored at 4 °C prior to use. 50,000293T-hACE2 or A549-hACE2-TMPRSS2 cells were incubated with 50 pL VLP for 25 min. at RT before beingtransferred into the chamber slides along with 500 pL fresh DMEM. Following overnight culture, the cells were washed once with 500 pL PBS and mounted using a few drops of ProLong glass antifade. Fluorescence images were collected using a Zeiss AxioObserver microscope (10X / 0.25 NA objective).

[0092] Western blot: VLP samples were denatured in SDS-DTT blue loading buffer (Cell Signaling) by heating at 98 °C for 5-10 min. 10 pL concentrated VLP sample for anti-M and anti-E or 2 pL sample for anti-S2 and anti-N were resolved using a 12 % Tris-glycine gel. Following transfer onto a nitrocellulose membrane using a Trans-Blot Turbo Transfer System (Biorad, Hercules, CA), the membranes were blocked for 1 h at RT using TBST (100 mM sodium chloride, 20 mM Tris-HCl, 0.1 % Tween-20) containing 5 % non-fat milk. Primary antibody was then added at manufacturer recommended concentrations in TBST solution containing 2 % non-fat milk at 4 °C overnight. The next day, the membrane was washed using TBST four times with each wash lasting 5 min. at RT. The membrane was then treated with HRP conjugated secondary antibody for 1 h at RT at manufacturer recommended concentrations. The membrane was then washed again and developed using the SuperSignal chemiluminescence substrate (ThermoFisher), and imaged using a ChemiDoc Imaging System (Biorad).

[0093] Cryo-Transmission electron microscopy (Cryo-TEM): Cryo-TEM images were acquired according to protocols published previously (28). Briefly, the VLP specimen were prepared using thin-film plunge freezing in a FEI Vitrobot. The vitrified specimen was mounted onto a Gatan 626. DH cryo-holder and transferred into

[0094] a FEI Tecnai F20 TEM. Cryo-TEM images were obtained using low-dose mode.

[0095] Real-time PCR (RT-PCR): The RT-PCR protocol was adapted from previous work (29). To this end, the VLP sample was mixed at 1 : 1 ratio with 2* RNA lysis buffer containing: 2 % Triton X-100, 50 mM KC1, 100 mM Tris-HCl [pH 7.4], 40 % glycerol and 0.4 U / pL of Superase in RNase inhibitor (ThermoFisher). After 10 min. incubation at RT, the lysed sample was diluted 1:5,000 in nuclease free water, resulting in 1:10,000 final dilution. Quantitative RT-PCR was performed using the SuperScript™ III Platinum™ SYBR™ Green One-Step qRT-PCR Kit (ThermoFisher). Here, a 50 pL reaction mixture was prepared in 0.2 mL PCR tube containing 10 pL diluted VLP lysate, 1 pL SuperScript® III RT / Platinum® Taq Mix (includes RNaseOUT™), 25 pL 2* SYBR® Green Reaction Mix, 1 pL forward primer (5’-CCAGGAGTCAAATGGAAATTGAT (SEQ ID NO:1), 0.2 pM final concentration), 1 pL reverse primer (5’-CGATATGTTCGAAGGCATAGCC (SEQ ID NO:2), 0.2 pM final concentration) and 12 pL nuclease free water. PCR was performed usinga C1000 Touch Thermal Cycler (Biorad) for 93-nucleotide product amplification. The cycling program was: hold 50 °C for 3 min; hold 95 °C for 5 min; 40 cycles of: 95 °C for 15 seconds, 60 °C for 30 sec. The CFX maestro software (Biorad) was utilized to monitor SYBR Green signal. As relative mRNA expression level was compared between different VLP samples, no internal control was used. Three identical wells were tested in each run to account for technical replicates, and three independent runs were performed to account for biological replicates. The threshold was determined using auto mode in the software. Relative mRNA expression quantified the amount of RNA in 3P VLPs vs. 4P VLPs based on measured ACt values: 2ACt(ACt = Ct,4PVLPs - Ct,3PVLPs).

[0096] Droplet digital PCR (ddPCR): VLP lysate was prepared using 1 : 1 volumetric ratio with 2X RNA lysis buffer as described in the previous section. The lysed sample was then diluted to 1:1,000,000 (final dilution) in nuclease free water. The primer / probe mix directed against the viral PS9 sequence, containing forward primer: 5’-AGACAGTGGTTGCCTACG-3’(SEQ ID NO:3), reverse primer: 5’-CAGTTGCACAATCACCAATCA-3’ (SEQ ID NO:4) and probe: 5’-AGATCTGAATCGACAAGCAGCGTACC-3’ (SEQ ID NO:5) produced by IDT (Coralville, IA) was resuspended in nuclease free water at a 20 x concentration. The One-Step RT-ddPCR Advanced Kit for Probes (Biorad) was used to prepare a 20 pL reaction in a high-profile 96-well PCR plate (Biorad). The reaction mixture contained 5 pL diluted VLP sample, 5 pL supermix, 2 pL reverse transcriptase, 1 pL 300mM DTT, 1 pL 20 x primer / probe mix (final lxconcentration at 500nM primer, 250nM probe) and 6 pL nuclease-free water. The plate was placed into the Automated Droplet Generator in a QX200 AutoDG Droplet Digital PCR System. Up to -20,000 droplets in each reaction were created using the default settings. Subsequently, the plate was foil-sealed and placed into a C1000 Touch Thermal Cycler (Biorad) for reverse-transcription and amplification. The cycling program was: 60 min. at 50 °C for cDNA synthesis, 10 min. at 95 °C for pre-denaturation, 40 cycles of: 30 sec at 95 °C for denaturation, 60 sec at 60 °C for annealing / extension, and 10 min. at the end at 98 °C for enzyme deactivation. Droplets were then read using the QX200 Droplet Reader and the data were analyzed using the Biorad QuantaSoft software 1.7. Droplets containing the viral RNA contributes to the positive population whereas those devoid of the viral RNA belong to the negative population. mRNA copy number per unit volume was calculated by the software, using default settings.

[0097] SARS-CoV-2 nucleocapsid (N) protein ELISA assay: The VLP sample was lysed over 10 min. at RT using 1 : 1 volume ratio with 2* lysis buffer containing: 2 % Triton X-100, 50 mM KC1, 100 mM Tris-HCl [pH 7.4], 40 % glycerol and 2* Halt protease inhibitor(ThermoFisher). The lysate was further diluted in nuclease free water to obtain 1 : 1,000,000 final VLP dilution. The LEGEND MAX™ SARS-CoV-2 Nucleocapsid Protein ELISA Kit (Biolegend) was used to quantify the N protein equivalent of the VLP samples, using calibration standard and instructions provided by the manufacturer. Absorbance at 450nm was measured and VLP titer was calculated based on the standard curve.

[0098] Dynamic Light Scattering (DLS): VLP samples were diluted 1:100 in PBS buffer.

[0099] 200 pL of this diluted sample was added into disposable plastic cuvettes and placed in a Zetasizer Ultra instrument (Malvern Panalytical, United Kingdom). In the ZS Xplorer software 3.0, materials was set to ‘liposomes’, dispersant was ‘water’, data processing was ‘general purpose’ and angle of detection was set to ‘back scatter’. All other parameters remained default setting. Particle diameter (in nm) was estimated by the software based on the measured scattering intensity (%).

[0100] CRISPR-Cas9 VLP editing assay: In some studies, gene editing was performed on 293T-EGFP, 293T-hACE2 and 293T-hACE2-EGFP cells. sgRNA against the target gene were designed using the CRISPRscan software (30), and cloned into pKLV-U6gRNA(BbsI)-PGKpuro2ABFP. These include 5’-GGCGAGGGCGATGCCACCTA-3’ (SEQ ID NO:6), 5’-GAGCTGGACGGCGACGTAAA-3’ (SEQ ID NO: 7), and 5’-GAGAGTGATCCCGGCGGCGG-3’(SEQ ID NO: 8) against EGFP 5’-TGGTATAGACTGCAGCCATC-3’ (SEQ ID NO: 9) and 5’-ttctgtgatacacacggctg-3’(SEQ ID NO: 10) against SLC35AP, and 5’-tgtcatttcagAATAATGCT-3’ (SEQ ID NO: 11) and 5’-CACTTGCCCAAATGTATCCA-3’ (SEQ ID NO: 12) against hACE2. All sgRNA were simultaneously used on the target cells by either co-transfection with multiple plasmids or applying pooled lentivirus when stable cells were established. To this end, cells were transfected with pooled plasmids when targeting EGFP or human- ACE2 (hACE2) 24-48 h prior to addition of Cas9 VLPs. 293T cells stably expressing sgRNA against SLC35A1 were used when targeting endogenous gene. To introduce Cas9, 3P VLPs were prepared carrying either Cas9-P2A-dTo-PS9 (dTo: dTomato) or Cas9-P2A-dTo-T20, with SARS-CoV-2 spike or VSV-G viral glycoprotein as 100 x concentrates. These VLPs were introduced into target cells in 1.5 mL microcentrifuge tubes by incubating 80,000 cells in 8 pL volume with varying amounts of VLPs in the presence of 8 pg / mL polybrene for 25 min. at RT. Transduced cells were then transferred into 6-well plates with the addition of 5 mL fresh DMEM media per well. Cells were cultured for 6 days with fresh media being added every 48 h. On day 6, gene editing efficiency was quantified using flow cytometer. Additionally, in studies that monitored human ACE2 editing, the cellular genomic DNA was purified from individualtreatments using the PureLink genomic DNA isolation kit (ThermoFisher). The region surrounding the hACE2 editing target site was PCR amplified using forward primer: 5’-tcaagcaatgccattccaacttc-3’ and reverse primer: 5’-CTGAGAGCACTGAAGACCCAT-3’) (SEQ ID NO: 13). Standard Nextera Index primers were then added using a second overlapping PCR and the product was subjected to 150bp paired-end sequencing on a MiSeq instrument (Illumina, San Diego, CA). Sequencing results were demultiplexed and editing efficiency was analyzed using a custom python script that quantified indels at the edit site. % editing quantifies the number of reads with indels / total number of reads.

[0101] Animal study: All animal experiments were approved by the University at Buffalo Institutional Animal Care and Use Committee and by the University Biosafety Committee (Buffalo, NY). CD-I mice (weight=20~21 g, equal number of both sexes) were purchased from Charles River Laboratory. 3P VSV-G Luc-T20 and 3P maSARS2 Luc-T20 VLPs were produced after 300-fold concentration. In studies with maSARS2, VLP amount was further adjusted based on N protein equivalent values. Mice were anesthetized in chamber using 3.0 % isoflurane in 100 % O2 at 1.5 L / min. until unresponsive. Equal numbers of mice were left untreated (negative control), treated with VLPs via intranasal (i.n.) route or subjected to VLP instillation via oropharyngeal aspiration (o.p.a.). In the case of i.n., mice were grasped behind the neck and held vertically with nose up, and a total of 50 pL VLP solution was alternatively applied on both nares as the animal inhaled. In the case of o.p.a., mice were hung by their front teeth with a suture on a 60° incline board. The tongue was grasped with gauze sponge and pulled straight out. With the chest being squeezed, a first 50 pL VLP dose was deposited into the back of oropharynx and then the chest was released. Following the recovery of regular breathing, additional 50 pL VLP was similarly instilled. Mice were sacrificed at 24 h. The left lung, right lung, and trachea tissue of each mouse were obtained and put in tared 1.5 mL “Navy bullet” tubes (Next Advance, Troy, NY) separately. Enough ice-cold tissue lysis buffer (Gold Biotechnology) with Halt protease inhibitor cocktail (ThermoFisher) was added to the individual tube to yield a ‘tissue + fluid’ total weight of 900 mg. Subsequently, the “Navy bullet” tubes were put in a Bullet Blender Storm 24 (Next Advance) in cold room and run at level-8 for 5 min twice. Homogenous supernatant was collected after spinning samples at 20,000 g for 30 min. at 4 °C. Samples were stored at -80 °C, prior to performing the luciferase assay as described previously, and measuring total protein concentration using the BCA Protein Assay kit (ThermoFisher). Luciferase signals reported in this manuscript were normalized based on BCA based protein concentration measurements. These measurements did not vary by more than 20 % among the different samples.Statistics: All data are presented as mean ± standard deviation for multiple biological replicates. Dual comparisons were performed using the Student’s two-tailed t-test. Multiple comparisons were performed using ANOVA followed by the Tukey post-test. E- value < 0.05 was considered to be statistically significant. Number of repeats are presented using discrete points in individual plots. Western blots are representative of multiple repeats. All samples in VLP infectivity assays were paired in order to account for biological variability.

[0102] EXAMPLES

[0103] Establishing a ‘3 plasmid’ SARS2 VLP system with improved viral entry properties: As the co-transfection of four plasmids into single cells could result in a heterogeneous VLP population due to cell-to-cell variability in exogenous DNA expression, we sought to reduce the number of plasmids required for such particle production. A number of combinations were tested, with the final product containing the N protein linked via a T2A self-cleaving peptide upstream of the M-IRES-E gene cassette (IRES sequence from encephalomyocarditis virus) (Figure 1A). Co-transfection of this plasmid, along with plasmid encoding for SARS2 spike and luciferase reporter linked to the PS9 packaging sequence resulted in functional 3P SARS2 Luc-PS9 VLPs capable of expressing reporter mRNA in recipient cells. As the number of plasmids used for VLP generation was reduced from four to three, this new system is referred to herein as the “3P system”.

[0104] Spike plasmid transfection amounts were varied in 150 mm petri dishes to optimize functional VLP production (Figure 8A, IB). Optimal 4P and 3P VLPs were formed upon using 1 pg spike plasmid along with 50 pg of plasmid encoding for other viral components. Thus, the stoichiometry of viral proteins in host cells dictated the efficacy of the produced VLPs. 3P VLPs not only resulted in a simpler VLP production workflow, but also displayed ~7-fold higher luminescence intensity using 293T-hACE2 target cells compared to 4P VLPs (Figure IB). VLP entry was strictly spike-dependent, as VLPs without spike did not induce luminescence signal in target cells. Western blot analysis showed the presence of all structural proteins for both the 4P and 3P VLPs: spike subunit 2 (S2, ~95kDa), nucleocapsid (N, ~46kDa), membrane (M, ~25kDa), and envelope (E, ~10kDa) (Figure 1C). 3P VLPs displayed more intense bands suggesting more uniform expression of structural proteins in producer cells. Real-time quantitative PCR analysis of VLP lysates suggests ~3-fold higher expression of the PS9 transcript in the 3P VLPs, compared to 4P VLPs (Figure ID). Cryotransmission electron microscopy (Cryo-TEM) showed that the 3P VLPs were uniform, 80-100 nm sized with prominent double layered membranes (Figure IE). Dynamic lightscattering showed that 3P SARS2 Luc-PS9 VLPs that carried the spike protein were slightly larger (-146 nm) compared to VLPs lacking spike -125 nm (Figure 8C).

[0105] EGFP-PS9 was introduced in place of Luc-PS9 to quantify infection in single cells using microscopy and flow cytometry. In microscopy studies, 3P SARS2 EGFP-PS9 VLPs infected considerable numbers of A549-hACE2-TMPRSS2 and 293T-hACE2 cells, both in a spike-dependent manner (Figure IF). Using flow cytometry for quantitation, we noted that -52% of A549-hACE2-TMPRSS2 and -96% of 293T-hACE2 cells were EGFP positive 24 h post-infection (Figure 1G). Fluorescence signal persisted up to 72 h, with 46% of A549-hACE2-TMPRSS2 cells, and 94% of 293T-hACE2 cells remaining EGFP positive. However, a decrease in the measured signal intensity was observed at greater times in the cytometry histogram (Figure 1G, Figure 8D), likely due to the degradation of EGFP mRNA and EGFP protein in recipient cells along with cell division which splits the signal among daughter cells. Overall, 3P VLPs were developed that both simplified SARS2 VLP usage, and improved efficacy compared to previously used 4P VLPs.

[0106] Tuning SARS2 VLP tropism by switching viral glycoprotein: We determined if viral tropism could be tuned by altering the surface glycoprotein. In addition to SARS2 spike, this was tested using the vesicular stomatitis virus G glycoprotein (VSV-G), and also betacoronavirus spike from the 2002 SARS and 2012 MERS (middle east respiratory syndrome) virus (Figure 2A). In this context, VSV-G binds low-density lipoprotein receptors and phosphatidylserine that are ubiquitously present on many mammalian cell types (31), and MERS spike recognizes dipeptidyl peptidase 4 (DPP4) as host cell receptor (32). To enable studies of MERS VLPs, isogenic HEK293T clones expressing DPP4 (‘293T-DPP4’) were produced (Figure 16A) and Calu-3 was used as a lung epithelial cell model that is permissive to SARS, SARS2 and MERS spike (33,34).

[0107] Initial studies were conducted using the 3P platform with Luc-PS9 reporter (Figure 2B). Here, the 3P SARS2 Luc-PS9 VLPs and 3P SARS Luc-PS9 VLPs exhibited strict specificity for cells expressing human ACE2, including 293T-hACE2, A549-hACE2-TMPRSS2 and Calu-3. The SARS VLPs displayed 1-3-fold higher infectivity for these cells compared to SARS2 VLPs. 3P MERS Luc-PS9 VLPs only infected 293T-DPP4 and Calu-3 cells. The 3P VSV-G Luc-PS9 VLPs infected all target cell types, albeit at lower levels compared to other VLPs. This was clear using both the standard sensitivity of the luminescence plate reader and upon increased detector sensitivity (Figure 8B). In the absence of spike, the VLPs failed to infect any of the cell types and the measured signal was similar to mock infection control. Titration studies were performed with VSV-G and MERS spikeVLPs to determine optimal conditions for VLP production (Figure 8C, 8D). This revealed that the 3P VSV-G and MERS VLPs required greater plasmid mass of 4 pg and 2 pg, respectively, for improved production as opposed to 3P SARS2 VLPs which only required 1 pg plasmid (Figure 8B).

[0108] Next, we evaluated the percentage of cells infected by these VLPs carrying the EGFP reporter (Figure 2C). Here, 3P SARS2 EGFP-PS9 VLPs exclusively infected ACE2 expressing cells, while 3P MERS EGFP-PS9 VLPs only infected DPP4 cells. 3P VSV-G EGFP-PS9 VLPs infected all cell types at >90% efficiency (Figure 2C), although the measured MFI was lower compared to 3P SARS2 EGFP-PS9 VLPs (Figure 8E). Without intending to be constrained by any particular theory, it is considered the differences observed in measured intensity across cell types for different VLPs, may be due to differences in the affinity of the VLP glycoprotein receptor for their ligand on host cells, and the expression levels of these ligands. This may cause variation in the number of EGFP mRNA copies and extent of measured fluorescence across infected cell types, even though most cells are infected. Overall, VLP tropism could be tuned to target specific recipient cell types. This aspect of the disclosure can be exploited for the directed mRNA delivery to specific target cells as further discussed herein.

[0109] SARS2 VLP system could be streamlined into a 2 plasmid (2P) system: We determined if the number of plasmids could be further reduced from three to two, as this would further simplify downstream applications. Thus, all structural and reporter genes were expressed using a single vector, with a second vector being used for trans-complementation of the desired viral glycoprotein. To test this concept, initially, we replaced the NeoR / KanR resistance gene located downstream of the SV40 promoter in pcDNA3.1 CMV N-T2A-M-IRES-E with Luc-PS9 (Figure 10A). Titration experiments were performed by changing the ratio of this plasmid and the SARS2 spike plasmid over a wide range. The resulting VLPs infected 293T-hACE2 cells at levels above the no spike control, but the measured luminescence was low compared to the 3P SARS2 Luc-PS9 VLPs. Without intending to be constrained by any particular interpretation, it is considered that this could be due to the low activity of the SV40 promoter or promoter interference within the single construct.

[0110] To address the above limitations, we modified a previously developed lentiviral dual promoter vector (LVDP), which can independently express two proteins driven by different promoters (Figure 3A) (24). This vector contains a series of insulator and terminator elements to minimize interference between the two promoters. Four different constructs (2P.1-2P.4) were developed, with PGK, EF-lot or CMV promoter driving either the SARS2structural proteins N, M and E, or the Luc-PS9 reporter. All 2P SARS2 Luc-PS9 VLPs were produced and VLP entry was evaluated using 293T-hACE2 recipient cells (Figure 3B). Here, the 2P.2 SARS2 Luc-PS9 VLPs showed high reporter signal comparable to 4P SARS2 Luc-PS9 VLPs, but this was lower than the 3P VLPs (Figure 3C). VLP entry for all 2P VLPs was strictly spike-dependent (Figure 10B). To rule out the possibility that promoter interference still exists in this system, we used the restriction enzyme Pad to linearize the vector between the two gene cassettes. Enzymatic digestion was confirmed using agarose gel electrophoresis of 2P vectors (Figure IOC). However, this treatment did not increase VLP entry function, as VLPs formed using the cut plasmids displayed -50% reduced signal in 293T-hACE2 cells compared to that of the uncut plasmids (Figure 10C). Upon analyzing SARS2 structural proteins using western blots, 2P.2 VLPs demonstrated stronger band intensities compared to 2P.3 VLPs and 2P.4 VLPs, but the measured signal was lower compared to the less infectious 2P.1 VLPs to some extent (Figure 3D). The 2P.2 VLPs were sized similarly to the 3P VLPs based on DLS, with the spike bearing 2P.2 SARS2 Luc-PS9 VLPs being -146 nm in size compared to -125 nm for the same VLPs without spike (Figure 10D). In studies that titrated the spike plasmid mass, similar to the 3P VLPs, 1 pg spike plasmid mass was sufficient for maximal 2P.2 VLP infectivity (Figure 10E). These data indicated that the concentrations of different structural proteins and amount of Luc-PS9 mRNA may be regulated by swapping the promoters, and this is at least in part determinant of VLP function.

[0111] To study single cell infection and perform time-course studies, the luciferase reporter in the 2P.2 plasmid was replaced by EGFP to create 2P.2 SARS2 EGFP-PS9 VLPs (Figure 3E). Here, similar to studies with the 3P VLPs (Figure 1G), reporter signal was maximum at 24 h, decreasing thereafter. 2P.2 SARS2 EGFP-PS9 VLPs infected 92% of the 293T-hACE2 and 45% of the A549-hACE2-TMPRSS2 cells at 24 h (Figure 3E, Figure 9F). Overall, these experiments resulted in the 2P VLP system, with a simplified workflow and reduced number of reagents. These reagents reduce the workload for experimentalists as illustrated in an independent publication (35).

[0112] SARS2 VLPs can package at least 5 kb mRNA and four transgenes: We investigated the packaging ability of the SARS2 VLPs in terms of the maximum mRNA size and number of gene payloads that can be delivered into target cells. To this end, a panel of six constructs were created with four reporter genes, EGFP, dTomato, TagBFP and luciferase (Figure 4A). These reporters were linked to a single CMV promoter either via T2A or P2A self-cleaving peptides, or IRES sequence. Thus, 3P SARS2 VLPs with payloads ranging from 900-4700 nt complexed with the PS9 packaging sequence were evaluated for viral entry into293T-hACE2 cells. Flow cytometry measured fluorescence reporter signal and a plate reader was used for luminescence measurement (Figure 4B). All viral entry was spike dependent. While ~85 % of the cells carried the first EGFP reporter in the smallest const.1, this was reduced to 50 % upon using the largest const.6 (Figure 4B, Figure 11). EGFP mean fluorescence intensity also decreased with increasing payload size. Similar observations were made upon following other reporters including dTomato, TagBFP and luciferase.

[0113] Several additional attempts were made to vary mRNA packaging efficiency while keeping spike plasmid constant, in order to enhance transduction into 293T-hACE2 target cells. In one aspect, we varied the concentration of the structural protein encoding plasmid and const. 6 reporter plasmid. This at most resulted in -25% signal improvement upon increasing reporter plasmid amount from 25 pg to 40 pg (Figure 12A). In a second case, we inserted a second PS9 sequence either at the 3 ’-end of the mRNA payload to generate the 3P SARS2 EGFP-PS9-PS9 VLP (Figure 12B), or at the 5 ’-end while varying the open reading frame to account for premature translation initiation. While the former manipulation resulted in % transduction comparable to the original 3P SARS2 EGFP-PS9 VLP, reporter signal intensity was reduced by -80% (Figure 12C). Surprisingly, the latter modification resulted in complete absence of reporter signal in recipient 293T-hACE2 cells. Overall, SARS2 VLPs were generated to carry payloads of -4.7 kb size, with a single promoter driving up to 4 transgenes.

[0114] Changing payload size does not affect VLP synthesis: To investigate the mechanism(s) contributing to the decreasing reporter signal with increasing payload size, we systematically compared the impact of four different PS9 coupled payloads on reporter expression in producer cells, the VLP composition including the amount of packaged RNA using ddPCR, and corresponding reporter expression in target cells (Figure 5A, Figure 13A).

[0115] For such work, N protein amounts in each VLP preparation was also monitored using ELISA as this allows quantification of VLP amounts in ‘N protein equivalent’ units (Figure 13B). The four VLPs include const.1, 3P SARS2 Luc-PS9 VLPs, const.4 and const.6, ranging in size from 903-4698 nt. The analysis revealed several notable findings: i) The mechanism regulating EGFP expression in target cells is different from luciferase. This is because EGFP is always the first reporter with its expression being controlled by CMV in all constructs. Luciferase expression, on the other hand, was more highly expressed in Luc-PS9 due to the CMV promoter and it was lower for constructs (#4, #6) containing the ribosome entry IRES (Figure 5B-5C, 1st column). These observations are consistent with previous work showing low efficiency of IRES dependent translation compared to the first Cap-dependent promoteractivity (36). ii) Virus payload size did not impact either VLP physical particle numbers (measured using N protein ELISA, 2ndcolumn) or amount of mRNA encapsulated in virus (measured using ddPCR, 3rdcolumn), iii) Western blots showed that RNA payload did not affect VLP structural proteins (5thcolumn), iv) Whereas EGFP signal did not vary with construct size in producer cells, it was reduced in the target cells. Thus, the mRNA may undergo 5 ’-truncation or other impairments during VLP assembly upon increasing payload size. Overall, while the VLPs are well formed independent of construct size, the number of functional mRNA is reduced upon increasing construct size.

[0116] SARS2 VLPs can be used to deliver functional Cas9 mRNA for genome editing:

[0117] We determined if SARS2 VLPs could be used for gene editing. Thus, we created 3P SARS2 VLPs that carried either Cas9-P2A-dTomato-PS9 mRNA or Cas9-P2A-dTomato-T20 mRNA (Figure 14A). Titration studies measured VLP entry into 293T-hACE2 cells for both constructs. While VLP-dose dependent entry was observed in both cases, T20 was a superior packaging signal compared to PS9 (Figure 14B). In luciferase-based reporter assays, also, 3P SARS2 Luc-T20 VLPs resulted in -2.8-fold higher entry signal in 293T-hACE2 cells compared to 3P SARS2 Luc-PS9 VLPs (Figure 14C). Thus, we use T20 as the packaging signal in downstream studies.

[0118] To determine if Cas9 mRNA delivered using the VLPs can mediate gene editing, two isogenic clones stably expressing high levels of EGFP were created using either 293T-hACE2 (‘293T-hACE2-EGFP’, Figure 15A, left panel) or wild-type 293T cells (‘293T-EGFP’, Figure 15A, right panel). These cells were used for Cas9 editing assays using VLPs that contained Cas9-P2A-dTomato-T20 mRNA with either the SARS2 spike or VSV-G glycoprotein (Figure 6A). The ability of these VLPs to edit different cell types and genes was assessed (Figure 6B). In one experiment, 293 T-hACE2 -EGFP cells were transfected with single guide RNAs (sgRNAs) against EGFP gene (Figure 6C). The transfected cells expressed blue fluorescence due to the presence of BFP reporter in the sgRNA vector (37). The next day, the 3P SARS2 Cas9-P2A-dTomato-T20 VLPs were applied. Editing efficiency was quantified 6 days later by assessing EGFP levels in BFP positive cells. Here, -20-30% gene editing of BFP positive cells was observed upon using VLPs bearing SARS2 spike, but not VLPs produced in the absence of spike. In other negative controls, cells lacking sgRNA and mock infected cells without VLPs failed to contain gene edits.

[0119] We examined if the above methodology can be extended to ‘self-inactivate’ the human ACE2 receptor (Figure 6D, Figure 15B). Thus, sgRNAs against human ACE2 was delivered using the same BFP reporter vector, this time, into non-fluorescent 293T-hACE2cells. Here, also, 15-25% knockout of cell surface ACE2 was observed at day 6 upon using 3P SARS2 Cas9-P2A-dTomato-T20 VLPs, but not upon using VLPs lacking SARS2 spike, cells lacking sgRNA against ACE2 or mock infected cells. To rule out any possible artifact, besides cytometry, gene editing was also confirmed using Illumina next-generation sequencing by performing amplicon-sequencing of the editing sites on the human ACE2 gene (Figure 15C). The results quantitatively agree with the cytometry results based on the % reads with gene edits, after accounting for the fraction of cells that are BFP negative.

[0120] Gene knockouts could be targeted to specific cell types by tuning VLP tropism. To demonstrate this, two types of VLPs were created carrying the VSV-G (3P VSV-G Cas9-P2A-dTomato-T20 VLPs) and SARS2 spike glycoprotein (3P SARS2 Cas9-P2A-dTomato-T20 VLPs). Both VLPs were applied to 293T-EGFP that lack the human ACE2 receptor, but transiently expressed sgRNAs against EGFP using the above BFP reporter vector (Figure 6E, Figure 15D). As anticipated, while the VSV-G VLPs could mediate -35% gene editing based on the measured decrease in EGFP fluorescent cells at day 6, editing was absent upon using SARS2 VLPs, in the absence of sgRNAs and in mock infection controls. Notably, while dTomato signal was observed in recipient cells at 24h, this signal was absent at day 6, confirming that payload delivery using SARS2 VLPs was transient and non-integrative (Figure 15E).

[0121] We determined if endogenous genes could be knocked out using this approach, in addition to reporter genes in above studies. To test this, we knocked out SLC35A1 (CMP-sialic acid transporter) as absence of cell-surface sialic acid exposes galactose terminated glycans that can be readily detected using lectins like PNA (Peanut agglutinin) (38)( Figure 16A). Thus, 293T-hACE2 cells stably expressing sgRNAs against SLC35A 1 were established, using co-expressed BFP as a selection marker (Figure 16B). Infection of 3P SASRS2 Cas9-P2A-dTomato-T20 VLPs into these ‘293T-hACE2-SLC35Al sgRNA’ cells resulted in >70% gene editing when using 1.885 pg / pL N protein equivalent VLPs, while this further increased to -85% upon doubling VLP volume. In comparison, transfection of Cas9-P2A-dTomato-T20 plasmid into these cells resulted in -65% gene editing (Figure 16C). In negative control no spike VLP (1.385 pg / pL N protein equivalent) failed to edit the cells. Overall, SARS2 VLPs can carry functional 5 kb Cas9 mRNA for gene editing in a target cell type-specific manner.

[0122] SARS2 VLPs could be used for in vivo delivery into mouse lung: As SARS2 is a pulmonary virus, we determined if these VLPs can overcome physiological barriers in the mouse lung to enable mRNA delivery (Figure 7A). In one study, we produced 3P VSV-GLuc-T20 VLPs and instilled them into the mouse lung via either oropharyngeal aspiration (100 pL, o.p.a.) or intranasal (50 pL, i.n.) routes. A third group without VLPs served as negative control. All mice were sacrificed at 24 h, lungs (left and right) and trachea were harvested, and luciferase activity was measured in tissue lysates. Here, o.p.a. allowed higher VSV-G VLP delivery to the lungs (Figure 7B).

[0123] Next, to enable spike dependent VLP delivery, mouse adapted SARS2 spike (‘maSARS2 spike’) was developed by introducing Q493K / N501 Y mutations into the natural spike as it does not bind mouse ACE2 (39) (Figure 7C). 293T cells stably expressing mouse ACE2 receptor (‘293T-mACE2’) were produced (Figure 17), and indeed these were permissive to 3P maSARS2 Luc-T20 VLPs (Figure 6C). In contrast, 293T-hACE2 cells were infected by both 3P SARS2 VLPs and 3P maSARS2 VLPs. As anticipated, the VSV-G VLPs infected all cell types. Next, equal N protein equivalents of 3P Luc-T20 VLPs bearing either VSV-G or maSARS2 were instilled into mouse via o.p.a route (Figure 7D). Following this, we noted higher delivery of luciferase reporter in mouse lung using VSV-G VLPs compared to maSARS2 spike VLPs, likely due to more ubiquitous expression of the lipoprotein receptor compared to mouse ACE2 (31). Thus spike- ACE2 dependent in vivo mRNA delivery was possible in lungs.

[0124] DISCUSSION OF EXAMPLES

[0125] Without intending to be constrained by any particular theory, it is considered this disclosure advanced SARS-CoV-2 VLP technology by analysis of factors related to: i) the design of plasmids necessary for efficient production of these particles, ii) methods to tune the tropism of the VLPs, iii) optimizing the packaging capacity and characterizing the particle formation of the VLPs, iv) gene editing applications and v) in vivo pulmonary delivery. In one aspect, the study presents approaches to simplify the production of SARS-CoV-2 VLPs, in order to produce more uniform particles. Decreasing the number of plasmids from four (4P system) to three (3P system) led to a less complex VLP synthesis process and at the same time markedly increased reporter signal by ~7 fold. Further decreasing the number of plasmids to two (2P system) resulted in VLPs with reporter signal comparable to the 4P VLPs. As 2P VLP efficiency was lower than that of 3P VLP, and as the composition and reporter signal varied with the utilized plasmid promoter, it is considered that the concentrations of SARS2 structural proteins in producer cells impacts VLP function. Despite the lower efficacy, the 2P system drastically simplified experimental workflows during biological investigations (35). Spike concentration in a narrow range influenced optimal VLPproduction. Whereas only 1 / 50 of the total plasmid in the transfection mix encoded for SARS2 spike during the production of optimal SARS2 VLPs, this fraction was 1 / 25 for MERS VLPs and 1 / 10 when producing VSV-G pseudotyped VLPs.

[0126] VLP tropism could be tuned by swapping the SARS2 spike protein with spike from the 2002 SARS virus, the 2012 MERS virus and the VSV-G glycoprotein. This allowed selective VLP delivery into permissive target cells, relying solely on the presence of the host cell receptor on the cell surface. Indeed, similar efforts have been made for targeted delivery of lipid or polymeric nanoparticles, for example by conjugating a sialic acid binding ‘GALA’ peptide onto EGFP-mRNA polyplexes for engaging dendritic cells (40), or surface-decorated exosomes with E3 aptamer for siRNA delivery to prostate cancer cells (41). While success has been demonstrated ex vivo, clinical translation is complicated in part due to high technical demand needed to make such particles (17,18,42). Additionally, a protein corona commonly coats nanoparticles in complex biological milieu resulting in either loss of nanoparticle targeting ability in vivo and / or redirection to the liver (43). Nevertheless, efforts continue in this area to promote non-hepatic mRNA delivery either by non-covalent attachment of antibodies (44), or surface modifications to modify the protein corona (45). In contrast to these synthetic methods, viral tropism has evolved with high efficiency in nature. In addition to targeting a given organ, these VLP particles can be designed to engage specific cell types. Exploiting this using the SARS2 VLP platform as novel strategies for targeted gene and protein delivery is encompassed by the disclosure.

[0127] Increasing payload size from ~0.9kb to ~5kb decreased reporter signal by -50-90%. Mechanistic investigations suggest that increasing payload size does not affect VLP synthesis or RNA incorporation into these particles but may affect protein production in target cells. Nevertheless, packaging of functional S. pyrogenes Cas9 (~5kb) was possible. Among the packaging sequences, we consistently observed that T20 was superior to PS9. This observation is different compared to the findings reported by Syed et al (9), perhaps due to the use of different plasmid constructs. The disclosure includes packaging signal design that accounts for multiple segments of the viral genome which may synergically promote mRNA package size and viral assembly efficiency.

[0128] Despite the low Cas9-dTomato reporter signal intensity, SARS2 VLPs encapsulating S. pyrogenes Cas9 mRNA (~5kb insert) were remarkably proficient at performing genome editing. Reporter genes expressed in cells at variable levels could be knocked out by >30% in the infected cells, while this fraction increased to >70% when targeting the endogenous gene SLC35A1 using efficient sgRNAs. The level of editing is comparable to the lentivirus-likebio-nanoparticle (LVLP) system reported by Lu et al (47). Varying the sgRNA specificity allowed editing of different gene targets and modification of VLP tropism enabled editing in different cell types. Cas9 mRNA delivered using VLPs is only transiently expressed, making it susceptible to degradation by cellular RNases. The short half-life of mRNA both reduces potential off-target editing and minimizes the chance of deleterious gene integration (48). Unlike DNA payloads, the nuclear translocation and transcription is not needed with mRNA, making protein expression and drug delivery more straightforward (49).

[0129] mRNA reporters could be delivered to mouse lung using VLPs bearing both VSV-G and maSARS2 spike glycoproteins. Thus, the particles can mediate pulmonary gene delivery, overcoming barriers including the lung cilia, mucus and humoral immunity (50,51). While an aspect of this disclosure is related to pulmonary delivery due to the natural tropism of the virus, the specific target cell type may be varied by tuning VLP tropism. Levels of mucosal antibodies against spike is low even in CO VID-19 immunized individuals (51) making this an attractive vehicle for mRNA delivery. Additionally, while the described luminescence were made using whole tissue lysates, it is likely that the measured signal would be amplified if these same measurements were made using lung epithelial cells dissociated from the larger organ. The demonstration that mRNA can be delivered to lungs supports non-integrative gene therapy to the lung both in the context of rare pulmonary genetic disorders like cystic fibrosis and lymphangioleiomyomatosis, and more common disorders like chronic obstructive pulmonary disease (53,54).

[0130] Overall, the disclosure presents advances in the SARS2 VLP system both for basic science virology studies and mRNA delivery applications. It demonstrates the packaging of multiple genes in a single vector, the ability to perform genome editing and in vivo gene delivery to the lungs. By exploiting the natural tropism and size features of the SARS-CoV-2 virion, these VLPs provide new applications that are not possible using conventional targeted drug delivery approaches.

[0131] Other examples of the disclosure will be apparent to those skilled in the art from consideration of the specification and practice of the disclosure disclosed herein. It is intended that the specification and examples be considered as exemplary only.REFERENCES - This reference listing is not an indication that any reference is material to patentability.

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Claims

1. What is claimed is:

1. A method for producing virus-like particles (VLPs) derived from a coronavirus by introducing into a plurality of eukaryotic cells a combination of polynucleotides consisting of first, second and third plasmids, wherein:i) the first plasmid comprises a promoter operably linked to a sequence encoding a coronavirus nucleoprotein (“N” protein), a coronavirus membrane protein (“M” protein) and a coronavirus small membrane protein (“E” protein);ii) the second plasmid comprises a promoter operably linked to a sequence encoding a protein that can bind to a target on the surface of a cell (a cell targeting protein), and wherein the cell targeting protein is optionally a spike protein (“S” protein);iii) the third plasmid comprises a promoter operably linked to a coronavirus packaging signal and a sequence encoding a desired payload; andiv) allowing expression of the N, M, E, the cell targeting protein, the payload, and the packaging signal within the cells such that the VLPs are formed and comprise the N, M, and E, proteins, the packaging signal, and the payload; andv) separating the VLPs of iv) from the cells to provide an isolated VLP preparation.

2. The method of claim 1, wherein tropism of the VLPs is tunable by changing the sequence encoding the cell targeting protein such that the VLPs bind to and deliver the payload comprised by the VLPs into cells to which the cell targeting protein binds.

3. The method of claim 1, wherein the N, M, and E proteins are encoded by a single RNA and wherein the N, M, and E proteins are separated from one another by polynucleotide segments that comprise an internal ribosome entry site (IRES), a self-cleaving peptide sequence, or a combination thereof.

4. The method of claim 3, wherein the promoter of the first, second, or third plasmids are the same, or wherein at least two of the promoters are different promoters.

5. The method of claim 2, wherein the N, M, or E proteins are present in a fusion protein.

6. The method of any one of claims 1-5, wherein the payload comprises a) an RNA polynucleotide that does not encode a protein, or wherein the payload comprises b) an RNApolynucleotide that encodes a protein that is translated and is present as a component of the VLP, or a combination of a) and b).

7. The method of claim 6, wherein entry of the VLPs into cells to which the cell targeting protein binds is greater relative to entry of VLPs produced using four plasmids, wherein the four plasmids comprise: a first plasmid encodes that a coronavirus E protein, a second plasmid that encodes a coronavirus S protein, a third plasmid that encodes a coronavirus M protein, and a fourth plasmid that encodes a coronavirus N protein.

8. An isolated VLP preparation produced according to any one of claims 1-5.

9. A method for producing virus-like particles (VLPs) derived from a coronavirus by introducing into a plurality of eukaryotic cells a combination of polynucleotides consisting of first and second polynucleotides that are optionally plasmids, wherein:i) the first polynucleotide comprises a promoter operably linked to a sequence comprising or encoding a payload and a coronavirus packaging signal, and a second promoter operably linked to a sequence encoding a coronavirus nucleoprotein (“N” protein), and a coronavirus membrane protein (“M” protein), and a coronavirus small membrane protein (“E” protein);ii) the second polynucleotide comprises a promoter operably linked to a sequence encoding a protein that can bind to a target on the surface of a cell (a cell targeting protein) wherein the cell targeting protein is optionally a spike protein (“S” protein)iii) allowing expression of the N, M, E, the cell targeting protein, the payload, and the packaging signal within the cells such that the VLPs are formed and comprise the N, M, and E, proteins, the packaging signal, and the payload; andiv) separating the VLPs of iv) from the cells to provide an isolated VLP preparation.

10. The method of claim 9, wherein tropism of the VLPs is tunable by changing the sequence encoding the cell targeting protein such that the VLPs bind to and deliver the payload comprised by the VLPs into cells to which the cell targeting protein binds.

11. The method of claim 9 or claim 10, wherein the N, M, and E proteins are encoded by a single RNA and wherein the N, M, and E proteins are separated from one another by polynucleotide segments that comprise an internal ribosome entry site (IRES), a self-cleaving peptide sequence, or a combination thereof.

12. The method of any one of claims 11, wherein the promoter of the first or second plasmids are the same, or wherein the promoters are different promoters.

13. The method of any one of claim 11, wherein the N, M, or E proteins, are present in a fusion protein.

14. The method of claim 11, wherein the payload comprises a) an RNA polynucleotide that does not encode a protein, or wherein the payload comprises b) an RNA polynucleotide that encodes a protein that is translated and is present as a component of the VLP, or a combination of a) and b).

15. An isolated VLP preparation produced according to claim 9.

16. A method comprising administering to cells a composition comprising an isolated VLP preparation of claim 8 or 15 such that the VLPs enter the cells and wherein the payload affects at least one property of the cells.

17. The method of claim 16, wherein the cells are present in an individual that is a mammal.

18. The method of claim 17, wherein the mammal is a human.

19. The method of claim 17, wherein the payload has a therapeutic effect.

20. The method of claim 17, wherein the payload has a prophylactic effect.

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