Cell strain for producing engineered virus-like particles, method thereof and engineered virus-like particles

Abstract

The invention provides a cell strain for producing engineered viroid particles, a method thereof and the engineered viroid particles, the cell strain is named as SZCV-Con28 and preserved in Guangdong Microbial Culture Collection Center on October 15, 2025, the preservation number is GDMCC No: 67101, and the preservation number is GDMCC No: 67101. A plurality of corresponding cell factories for producing the engineered viroid particles are established by cell lines obtained by introducing different target gRNA elements into the cell strain, so that the corresponding engineered viroid particles can be continuously produced in batches. According to the invention, large-scale preparation can be realized through cell expanding culture, the capacity limitation is overcome, the production process flow is simplified, and the consumption of production raw materials is reduced.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biotechnology, in particular to a cell strain for producing engineered virus-like particles, a method thereof and the engineered virus-like particles. BACKGROUND

[0002] Gene editing technologies such as CRISPR-Cas9 and Base Editor have made significant progress in scientific research and clinical applications, but there are still some limitations in effectively delivering these editing tools to cells. For example, traditional viral vectors such as adeno-associated virus (AAV) have limitations in delivery capacity and potential safety risks, lentivirus and retrovirus delivery of base editing DNA has the risk of random integration of genes and high off-target, and non-viral vectors such as lipid nanoparticles (LNP) have the problem of targeted delivery. In view of this, researchers have developed a new delivery tool based on viral vector derivatives, which fuses RNA binding proteins or target proteins with viral structural proteins (including different sites in the retroviral gag polyprotein). During the capsid self-assembly process, mRNA containing binding protein affinity elements or target proteins cut by viral enzymes can be directed into the virion to form virus-like particles (VLPs), which can deliver genes into cells and exert gene editing effects. Because gene editors are proteins, they have a low half-life in cells, which can effectively avoid off-target effects. Compared with the cell damage of CRISPR electroporation and the risk of persistent expression of retroviruses, engineered virus-like particles (eVLPs) have the advantages of high efficiency and safety in the form of transient delivery of ribonucleoprotein (RNP).

[0003] However, the existing eVLP production system relies on a four-plasmid transient 293T cell production system, which has serious industrialization obstacles: poor process stability, large raw material consumption, high scale-up cost, and repeated detection of host residues for clinical-grade quality control. Therefore, it is necessary to develop an innovative process solution with low cost, high stability, and suitable for large-scale production. SUMMARY

[0004] To solve the above problems, to solve the above problems, the present application provides a cell strain for producing engineered virus-like particles, which is named SZCV-Clon28 Homo sapiens and was preserved in the Guangdong Microbial Culture Collection Center on October 15, 2025, with the preservation number: GDMCC No: 67101.

[0005] In one embodiment, a method for preparing the cell strain of claim is provided, the method comprising: step 1: constructing a BaEV retrovirus stable packaging cell line, comprising transferring the structural protein gag-pol protein required for constructing the retrovirus into a HEK293T cell line or a cell line derived therefrom to obtain a corresponding cell line containing the structural protein gag-pol protein; based on the cell line containing the structural protein gag-pol protein, constructing a HEK293T cell line or a cell line derived therefrom which simultaneously knocks out ASCT-1 and ASCT-2 receptor proteins to obtain a corresponding cell line which knocks out ASCT-1 and ASCT-2 receptor proteins; transferring the BaEV envelope into the above-mentioned cell line which knocks out ASCT-1 and ASCT-2 receptor proteins, thereby obtaining the BaEV retrovirus stable packaging cell line; Step 2: transfecting the plasmid of Gag-Cas9 into the BaEV retrovirus stable packaging cell line obtained in step 1 to obtain a cell strain capable of producing engineered viroid particles containing BaEV envelope glycoprotein, gag-pol protein and Cas9 protein.

[0006] In one embodiment, in step 2, a monoclonal cell strain is selected by drug screening and limiting dilution method, the genomic DNA thereof is extracted and the Cas9 gene / Pol gene ratio and the Pol gene / inner reference gene ratio are calculated by detecting the copy number of Cas9 gene and Pol gene molecules in the cell genome, which are used for screening the cell line in step 2.

[0007] In one embodiment, the Cas9 gene / Pol gene ratio is between 0.4 and 1.2.

[0008] In one embodiment, the inner reference gene is RPPH1, and the Pol gene / inner reference gene ratio is >0.7.

[0009] In one embodiment, the above-mentioned cell strain is provided for use in producing engineered viroid particles.

[0010] In one embodiment, an engineered viroid particle is provided, which is prepared by the above-mentioned cell strain.

[0011] Traditional engineered virus-like particles (eVLP) are prepared by using four plasmids to co-transfect HEK293T cells. Because of the limited half-life of plasmid expression in cells, eVLPs are usually collected within 72 hours after transfection, and the production capacity of one preparation is limited. The cost of plasmid and transfection reagent used in large-scale preparation is extremely high, and the production is unstable between batches, which limits the large-scale production of eVLP. In view of this, the application creatively constructs a cell line integrated with essential elements of eVLP, and then establishes a cell line factory for various eVLPs by introducing different target gRNA elements. The constructed cell line can continuously and stably produce specific eVLPs, and has the following advantages: 1) no longer needs to be packaged by plasmid transfection, but can be prepared on a large scale by cell expansion, overcoming the capacity limitation and simplifying the production process; 2) reduces the consumption of raw materials (plasmid, transfection reagent, etc.), avoids quality control detection of plasmid residues after vector preparation, and effectively reduces the production cost; 3) the stable cell line for preparing eVLP can ensure the high stability of the vector between different batches, avoiding the risk of large difference between batches and possible preparation failure in traditional eVLP preparation, and the repeated quality and function verification of each batch in traditional preparation is tedious and costly; 4) increases the flexibility of production. After screening the cell line capable of producing engineered virus-like particles containing BaEV envelope glycoprotein, gag-pol protein and Cas9 protein, different target gRNA elements can be introduced into the above cell line to establish a variety of corresponding cell lines for producing engineered virus-like particles, thereby increasing the flexibility and convenience of batch production. BRIEF DESCRIPTION OF DRAWINGS

[0012] In order to more clearly illustrate the technical solutions in the embodiments of the present application, the drawings needed in the embodiments will be briefly introduced. Obviously, the drawings in the following description are only some embodiments described in the present application, and other drawings can be obtained by those skilled in the art without creative labor.

[0013] Figure 1 Detection of three envelope eVLPs targeting AAVS1 site 293T / NK92 cell genome editing efficiency result graph; Figure 2 TRAV-eVLP with Baev envelope knockout efficiency result graph for Jurkat cells; Figure 3 pCMV-MMLVgag-3xNES-Cas9-PolyA-hPGK-BSD plasmid design graph; Figure 4 qPCR method for detecting gene copy number ratio result graph; Figure 5Figure of flow cytometry detection of gene knockout results of different clone strains of eVLP vectors; Figure 6 Figure of CD7 knockout results of Jurkat cells after transduction by different eVLP vectors; Figure 7 Figure of proliferation results of Jurkat cells after transduction by different eVLP vectors. DETAILED DESCRIPTION

[0014] In order to make the technical field of the art better understand the technical solutions in the present application, the present application will be further described below in conjunction with examples. Obviously, the described examples are only a part of the examples of the present application, but not all the examples. Based on the examples in the present application, all other examples obtained by the person skilled in the art without making creative efforts shall fall within the scope of protection of the present application.

[0015] Example 1: Construction of BaEV retrovirus stable packaging cell line

[0016] 1. Resuscitate 293T and NK92 cells and inoculate 293T eVLP vectors are usually composed of four functional modules: Gag-Pol, Gag-Cas9, envelope protein (optional VSVG, GALV, Amphotropic, Ecotropic or Baev, etc.) and targeting gRNA plasmid. In order to establish a sustainable and stable toxin-producing cell factory, all the above elements except the targeting gRNA need to be integrated into the same host cell, and then eVLP can be produced by transfecting the gRNA plasmid alone. Since the subsequent research is suitable for immune cell (T / NK) treatment, we prefer Baev envelope, but whether the mechanism of action of eVLP envelope protein is consistent with that of RV has not been studied. Subsequently, we prepared three kinds of AAVS1 (adeno-associated virus integration site 1) eVLPs with VSVG, GALV and Baev envelopes, and performed targeting knockout on 293T and NK92 cells, respectively. The experimental steps are as follows:

[0017] 2. Four-plasmid transient transfection of 293T to produce eVLP targeting AAVS1 site Resuscitate 293T cells, and the culture medium is DMEM containing 10% FBS and 1% P / S. Resuscitate NK92 cells, and the culture medium is NK92 special culture medium (Pronosis: CM-0530). Place in a 5% CO2, 37°C incubator for culture. When the confluence of 293T cells reaches 80%, perform subculture. When the culture medium of NK92 cells turns yellow and the cell density exceeds 2x10 6 Subculture after that. Use after subculturing for 3 times. Take 3.5x10 6293T cells were seeded into 10 cm cell culture dishes for subsequent eVLP production.

[0018] When the density of 293T cells in a 10 cm culture dish reached 75%-85%, it was used for transfection, without replacing the culture medium, 1.1 μg VSVG / BaEV / Galv envelope plasmid, 3.375 μg MMLV-Gag-Pol structure plasmid, 1.125 μg Gag-Cas9 plasmid, 4.4 μg AAVS1-gRNA plasmid were mixed into 500 μl serum-free medium, 30 μl of transfection reagent lipomaster2000 (Vazyme: TL-201) was added into 500 μl serum-free medium, then the plasmid mixture was added dropwise into the transfection reagent mixture, inverted and mixed for 2 times, and then placed for 5 min. Then the reagent was evenly added to the 293T cells, slowly mixed in a cross shape, and placed in a 5% CO2, 37℃ incubator overnight. After 16 h, the fresh culture medium was replaced, and the cell supernatant was collected after 48 h, filtered with a 0.44 μm filter, aliquoted 2.2 ml / tube, and stored at -80℃.

[0019] 3. eVLP targeting HEK293 and NK92 cell AAVS1 site Retronectin diluted with PBS was used to coat a 12-well plate, which was placed at 4℃ overnight; the next day, the coating solution was discarded, and PBS was used to rinse once, then 1 ml of eVLP carrier supernatant was added, centrifuged at 2000 rpm for 1 h at 32℃. After completion, the supernatant was discarded, and a certain amount of cells (293T: 4.4×10 5 / 1.12×10 5 ; NK92: 4×10 5 ) were mixed with 1 ml of fresh eVLP supernatant, and transferred to the corresponding well plate, centrifuged at 2000 rpm for 1 h at 32℃. After 16 h in a 5% CO2, 37℃ incubator, the fresh corresponding culture medium was replaced, and the cells were collected after 72 h and genomic DNA was extracted.

[0020] 4. T7 Endonuclease I enzyme digestion to verify the targeting efficiency T7E1 endonuclease can recognize the mismatched base of more than 1 bp and cut at this site. The targeting efficiency of gene editing can be verified by this mechanism. The primer pair is usually designed to be about 200 bp away from the target site, and the other one is about 400 bp away from the target site. In this way, it can be distinguished directly by agarose gel electrophoresis. Using this primer pair, 100 ng of genomic DNA was used as a template for PCR, and 30 cycles were run. The PCR product was purified and recovered. Then 200 ng of product template and control template were taken respectively, and the T7E1 reaction solution (except T7E1, all other components) was configured, pre-denatured at 95°C for 5 min, gradually cooled to 85°C at 2°C per second, then cooled to 25°C at 0.1°C per second, and then kept at 4°C for cutting. After annealing, add 1 μl of T7E1 mixture, incubate at 37°C for 15 min, use 2% agarose gel for electrophoresis, and image observation, where the target sample cuts out 400 bp and 200 bp bands, and the control sample is not cut, indicating that the sample is edited successfully. Then the approximate editing efficiency value is obtained by gray scale analysis (ImageJ) software analysis.

[0021] The T7E1 enzyme cutting method verifies that eVLP indeed has the mechanism that different envelope proteins have delivery preferences for different target cells. In Figure 1 , as shown in Figure 1 A and 1B, VSVG envelope can effectively knock out 293T (28.4% - 44.8%), but the knock-out efficiency of NK92 AAVS1 site is 0, and Baev envelope is opposite, which can efficiently knock out the genome of NK92 cell line (60.1%), and the GALV vector has a low knock-out efficiency of NK92 (about 10.8%), and the eVLP of Baev envelope can efficiently edit NK cells; as shown in Figure 2 , we also prepared eVLP of Baev envelope targeting TRAC site and performed transduction knock-out on Jurkat cells. Flow cytometry detection found that the expression of TCRaP was significantly reduced by 50%, indicating that eVLP of Baev envelope can also efficiently edit T cells. Given that this study is mainly applied to immune cell therapy, we subsequently selected Baev envelope for subsequent cell line construction.

[0022] We have previously used CRISPR technology to knock out ASCT1 / ASCT2 in HEK293 cells, and constructed a Baev envelope stable expression retrovirus production cell line WT64 (Patent authorized announcement number: CN119020418B; Patent publication number: CN 119020419A; Patent publication number: CN118956766A; Patent publication number: CN 119020417A), to construct a BaEV retrovirus stable packaging cell line, including transferring the structural protein gag-pol protein required for constructing the retrovirus preparation into the HEK293T cell line or its derivative cell line, to obtain the corresponding cell line containing the structural protein gag-pol protein; on the basis of the cell line containing the structural protein gag-pol protein, a HEK293T cell line or its derivative cell line is constructed which simultaneously knocks out ASCT-1 and ASCT-2 receptor proteins, to obtain a cell line that knocks out ASCT-1 and ASCT-2 receptor proteins; the BaEV envelope is transferred into the above-mentioned cell line that knocks out ASCT-1 and ASCT-2 receptor proteins, thereby obtaining the BaEV retrovirus stable packaging cell line; this cell line already contains two key components, Gag-Pol and Baev envelope, and is free of external elements interference. Therefore, only by stably introducing Gag-Cas9 components can all eVLP components except gRNA be completed in this cell line, and the upgrade of the stable production of virus-like particle platform is completed.

[0023] Example two pCMV-MMLVgag-3xNES-Cas9-PolyA -hPGK-BSD plasmid design, transfection and drug screening 1. pCMV-MMLVgag-3xNES-Cas9-PolyA -hPGK-BSD plasmid construction

[0024] The Gag-Cas9 initial plasmid is derived from pCMV-MMLVgag-3xNES-Cas9 (Addgene Plasmid #181752), which we use as a backbone, and by seamless cloning method, we add hPGK promoter and Blasticidin resistance gene elements after Cas9-PolyA to construct pCMV-MMLVgag-3xNES-Cas9-PolyA -hPGK-BSD, and the plasmid map is as follows Figure 3 .

[0025] 2. Plasmid transfection of WT64 cell line The culture solution used for the WT64 cell line is DMEM with 10% FBS and 1% P / S, and is used after being recovered and subcultured for 3 times. 4E5 cells are inoculated into a 6-well plate, and after the confluence reaches 80%, lipomaster 2000 is used for plasmid transfection (see Example 1 for the method), and after 16 hours, the fresh culture solution is replaced.

[0026] 3. Drug screening and limiting dilution separation of monoclonal cells After 48 hours of transfection, the DMEM resistance screening medium containing 10 μg / mL Blasticidin is replaced, and during the period when the cell confluence exceeds 90%, the subculture is carried out, the resistance screening medium is used for culture, and after 14 days, the limiting dilution method (1 cell / well) is used to separate the monoclonal cell strain. The first week of monoclonal cells uses 7.5 μg / mL Blasticidin selection medium for culture, and then the selection medium containing 10 μg / mL Blasticidin is replaced for culture. Only cell strains with single cells are selected, and after the cells grow to more than 50% range, the cells are digested and transferred to a 6-well plate, and the culture solution is replaced with 7.5 μg / mL Blasticidin selection medium.

[0027] Example Three: Screening of Monoclonal Cell Strains for Producing eVLP Carriers One. qPCR detection of Pol and Cas9 gene copy number

[0028] The present application selects 42 monoclonal strains by drug screening and limiting dilution method, extracts their genomic DNA, and detects the copy number of Cas9 and Pol molecules in the cell genome by qPCR, and selects the qualified clone strain in combination with the cell growth and proliferation state.

[0029] 1. Verification of primer eligibility First, we selected multiple pairs of Pol and Cas9 primers for qPCR using the corresponding plasmid as the template, analyzed the melting curve of the primer pairs, and determined the specificity of the primers. Finally, one pair of primers was selected for subsequent identification. The primer sequences are as follows: Pol-GC-new-F: 5'-TGAGTATCGGCTACATGAGACCT-3' (SEQ ID NO. 1); Pol-GC-new-R: 5'-CATGCCCCCGGTTTCCG-3' (SEQ ID NO. 2); Cas9-F: 5'-CAGATTCGCCTGGATGACCA-3' (SEQ ID NO. 3); Cas9-R: 5'-ATCCGCTCGATGAAGCTCTG-3' (SEQ ID NO. 4).

[0030] 2. gag / pol / cas9 fragment standard construction We constructed the gene fragment containing the primer into a plasmid, and set the enzyme digestion site EcoRI. After enzyme digestion, we obtained a 1310 bp fragment and a 2112 bp fragment. The 1310 bp fragment contains all the gene fragments we need to detect. After agarose gel electrophoresis, we purified and recovered it as a linear standard.

[0031] 3. Absolute quantification of Pol, Cas9, RPPH (internal reference) gene copy number using qPCR We diluted the linear standard from 1 x 10 8 copies to 10 3 copies in 10-fold gradient. We added 0 copies as a negative control to establish a standard curve. Then, we diluted each sample genome to 40 ng / μl, and added 1 μl of sample (40 ng) to the SYBR Green reaction system. After vortexing for 15 s and centrifuging at 400 g for 2 min, we detected it on the machine. After exporting the results, we plotted the standard curve of each detected gene to generate a function. The standard curve: c is the copies concentration; Y = log (1 / c, 2); X = Cq. We made a standard curve. We substituted the sample Cq value into the standard curve to obtain Y value, then the sample copies concentration = power (2, -Y). We calculated the absolute copy number of each gene fragment in the genome, and then calculated the Cas9 / Pol ratio and the Pol / RPPH ratio for subsequent single clone screening.

[0032] The ratio of Gag-Cas9 and Gag-Pol gene copy number can significantly improve the efficiency of gene editing within a certain range, but simply increasing the proportion of Cas9 plasmid cannot produce efficient eVLP. Based on this, we selected 2, 6, 12, 14, 16, 17, 22, 27, 28, 32, 33, and 39 of the 12 clones whose Gag-Cas9 and Gag-Pol copy number ratio were within the optimized range (Cas9 / Pol = 0.4-1.2, Pol / RPPH > 0.7) for the next round of screening. Clone 47 was discarded due to slow proliferation, and clone 14 was supplemented (Table 1; Figure 4 ). These clones have higher expression of Pol and Cas9 elements and can produce higher titer eVLP.

[0033] Table 1. Cas9 / Pol and Pol / RPPH copy number detection results

[0034] II. Functional verification of eVLP vectors produced by single clone cell strains Since Jurkat cells have high expression of CD7 molecules on the cell surface, the knock-out efficiency can be evaluated by directly detecting the expression level of CD7 by flow cytometry. This method has the advantages of being intuitive and accurate, so in this patent, we further screened the effect of eVLP production by Clone 28 cells by knocking out CD7 from Jurkat cells.

[0035] 1. Production of eVLP vectors using cells of each monoclonal strain Each clone was inoculated into 6x10 5 When the cell confluence reached more than 80%, CD7 gRNA plasmid was transfected, and after 16 hours, the fresh culture medium was replaced. After 48 hours, the cell supernatant was collected, filtered through a 0.44 μm filter, and CD7 eVLP vectors were obtained.

[0036] 2. Screening of high-efficiency production monoclonal strains by eVLP supernatant targeting knock-out of Jurkat cells These supernatants were used to transduce Jurkat cells for CD7 gene targeting knock-out (see Example 1 for method), and flow cytometry was used to evaluate the CD7 gene knock-out efficiency mediated by CD7 eVLP produced by each monoclonal strain (Table 2; Figure 5 ). The experimental results showed that the knock-out efficiency of Jurkat cells treated with CD7 eVLP produced by Clone-28 cells reached 93.16% (Table 2). Based on this excellent performance, this cell strain can be used as the starting cell strain for producing eVLP vectors of Baev envelope protein and spCas9 nuclease.

[0037] Table 2. Efficiency of eVLP vector produced by different monoclonal strains in knocking out Jurkat cells

[0038] Example Four: Comparison of Clone 28 cell strain and BaEV RV cell line transient production of eVLP Stable production of eVLP cell strain to prepare eVLP: The cells were expanded in T225 culture bottles, and when the cell confluence reached 100%, the DMEM complete culture medium containing 1 mM sodium butyrate was replaced, and the cells were incubated at 32°C. After 24 hours, H1 was collected, and fresh culture medium was replaced. After 48 hours, H2 was collected. The eVLP supernatant of each batch was filtered through a 0.44 μm filter, and then it could be used for storage. It was stored at -80°C.

[0039] The patent compares two eVLP production methods: one is based on the stable expression of BaEV envelope glycoprotein and gag-pol gene in the stable packaging cell line WT64 constructed by Shenzhen Cell Valley, and the experiment prepares an eVLP vector by transfecting gag-Cas9 plasmid and CD7 gRNA plasmid; the other is to package eVLP by transfecting CD7 gRNA plasmid on Clone 28 cell line, and the experiment uses the two vectors to transduce Jurkat cells for efficacy verification (see Example 1 for specific methods), and the results show that the CD7 gene knockout efficiency mediated by the eVLP vector produced by the Clone 28 cell line can reach 76.9% ( Figure 6 ), and the proliferation rate of Jurkat cells after transduction is comparable to that of the eVLP vector prepared by the BaEV stable cell line (Table 3; Figure 7 ).

[0040] Table 3 Cell proliferation after eVLP vector knockout of Jurkat cells produced by different methods

[0041] It should be understood that the disclosed application is not limited only to the specific methods, protocols and substances described, as these can vary. It should also be understood that the terminology used herein is for the purpose of describing only particular embodiments and is not intended to limit the scope of the present application, which is limited only by the appended claims.

Claims

1. A cell line for producing engineered virus-like particles, characterized in that, The cell line was named SZCV-Clon28 and was deposited at the Guangdong Provincial Center for Microbial Culture Collection on October 15, 2025, with accession number GDMCC No: 67101.

2. The method for preparing the cell line according to claim 1, characterized in that, The method includes: Step 1: Constructing a stable packaging cell line for BaEV retrovirus, including transferring the structural protein gag-pol protein required for retrovirus preparation into the HEK293T cell line or its derivative cell line to obtain a cell line containing the structural protein gag-pol protein; based on the cell line containing the structural protein gag-pol protein, constructing a HEK293T cell line or its derivative cell line that simultaneously knocks out ASCT-1 and ASCT-2 receptor proteins to obtain a cell line that knocks out ASCT-1 and ASCT-2 receptor proteins; transferring the BaEV envelope into the above-mentioned cell line that knocks out ASCT-1 and ASCT-2 receptor proteins to obtain the stable packaging cell line for BaEV retrovirus; Step 2: Transfect the stable packaging cell line of BaEV retrovirus obtained in Step 1 with the Gag-Cas9 plasmid, and screen for cell lines that can produce engineered virus-like particles containing BaEV envelope glycoprotein, gag-pol protein and Cas9 protein.

3. The method according to claim 2, characterized in that, In step 2, monoclonal cell lines are selected by drug screening and limiting dilution method, their genomic DNA is extracted, and the copy number of Cas9 gene and Pol gene molecules in the cell genome is detected. The Cas9 gene / Pol gene ratio and the Pol gene / internal reference gene ratio are calculated and used to screen for the cell lines in step 2.

4. The method according to claim 3, characterized in that, The Cas9 gene / Pol gene ratio is between 0.4 and 1.

2.

5. The method according to claim 3, characterized in that, The internal reference gene is RPPH, and the ratio of Pol gene to internal reference gene is > 0.

7.

6. The application of the cell line according to claim 1 in the production of engineered virus-like particles.

7. Engineered virus-like particles, prepared using the cell line described in claim 1.

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