A method for producing engineered virus-like particles using stable cell lines and the engineered virus-like particles thereof.

Abstract

This invention provides a method for producing engineered virus-like particles using stable cell lines, and the engineered virus-like particles thereof. The method comprises the following steps: constructing a stable BaEV retrovirus packaging cell line; transfecting the BaEV retrovirus packaging cell line obtained in step 1 with a Gag-Cas9 plasmid; constructing a self-inactivating delivery plasmid for a retrovirus packaging system; and selecting a stable cell line capable of producing engineered virus-like particles. This allows for the production of engineered virus-like particles through stable cell lines. This invention innovatively develops a method for preparing eVLP stable cell lines. This method overcomes existing production bottlenecks, providing a stable, efficient, and economical gene-editing vector solution for research and clinical applications, and promoting further development in the field of gene therapy.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and more specifically to a method for producing engineered virus-like particles through stable cell lines and the engineered virus-like particles thereof. Background Technology

[0002] Gene editing technologies such as CRISPR-Cas9 and base editors have made significant progress in scientific research and clinical applications, but there are still some limitations to effectively delivering these editing tools into cells. For example, traditional viral vectors like adeno-associated virus (AAV) have delivery capacity limitations and potential safety risks; lentiviruses and retroviruses face random gene integration and a high risk of off-target effects when delivering base-edited DNA; and non-viral vectors such as lipid nanoparticles (LNPs) present challenges in targeted delivery. In light of this, researchers have developed a novel delivery tool based on viral vectors. This tool fuses RNA-binding proteins or target proteins with viral structural proteins (including at different sites on retroviral gag polymers). During capsid self-assembly, mRNA containing affinity elements of the binding protein or the target protein cleaved by viral enzymes can be directed into viral particles, forming virus-like particles (VLPs). These VLPs exert their gene-editing effects after being delivered into cells. Because the gene editor is a protein, it has a short half-life in cells, meaning that gene editing is only effective within that half-life, effectively avoiding off-target effects. Compared to the cell damage and persistent retrovirus expression risks associated with CRISPR electroporation, engineered viral particles (eVLPs) offer the advantages of both high efficiency and safety by delivering ribonucleoproteins (RNPs) transiently.

[0003] However, existing eVLP production systems relying on four plasmids for transient transduction of 293T cells face significant industrialization obstacles: poor process stability, high raw material consumption, and exorbitant large-scale costs. Furthermore, clinical-grade quality control requires repeated testing for host residues. Therefore, there is a need to develop innovative process solutions that are low-cost, highly stable, and suitable for large-scale production. Summary of the Invention

[0004] To address the aforementioned problems, this invention provides a method for producing engineered virus-like particles using stable cell lines. The method includes the following steps: Step 1: Constructing a stable BaEV retrovirus packaging cell line, comprising: transferring the structural protein gag-pol required for retrovirus preparation into a HEK293T cell line or a derived cell line to obtain a cell line containing the structural protein gag-pol; based on the cell line containing the structural protein gag-pol, constructing a HEK293T cell line or a derived cell line that simultaneously knocks out ASCT-1 and ASCT-2 receptor proteins to obtain a cell line with ASCT-1 and ASCT-2 receptor proteins knocked out; transferring the BaEV envelope into the aforementioned cell line with ASCT-1 and ASCT-2 receptor proteins knocked out, thereby obtaining the stable BaEV retrovirus packaging cell line.

[0005] Step 2: Transfect the stable packaging cell line of BaEV retrovirus obtained in Step 1 with the Gag-Cas9 plasmid, and screen to obtain cell lines that can produce engineered virus-like particles containing BaEV envelope glycoprotein, gag-pol protein and Cas9 protein.

[0006] Step 3: Construct a self-inactivating delivery plasmid for a retroviral packaging system, which includes gRNA elements targeting different targets; transfect the self-inactivating delivery plasmid into 293T cells or Ampho 293 cells, and then use the transfected cells to produce self-inactivating retroviruses targeting different targets;

[0007] Step 4: Use the retrovirus obtained in Step 3 to transduce the cell line obtained in Step 2, and screen it to obtain a stable cell line that produces engineered virus-like particles, thereby producing engineered virus-like particles through the stable cell line.

[0008] In one embodiment, the self-inactivating delivery plasmid in step 3 includes a modified LTR structure comprising a 5'LTR and a ΔU3-modified 3'LTR, such that after the viral genome integrates into the host cell, the ΔU3-modified 3'LTR transfers to the 5' end, causing promoter inactivation and thereby blocking the regeneration of the complete 5'LTR-3'LTR structure, preventing the delivery plasmid from being repackaged.

[0009] In one embodiment, the self-inactivating delivery plasmid in step 3 includes an SV40 promoter-driven GFP reporter system, which is used to monitor viral transduction efficiency in real time and verify the reliability of the self-inactivating system.

[0010] In one embodiment, the self-inactivating delivery plasmid in step 3 is co-transfected with retroviral structural proteins and envelope proteins into Ampho 293T cells, or Ampho 293 cells are transfected with the self-inactivating delivery plasmid to assemble self-inactivating retroviral particles with different targeting properties, suitable for transduction needs of various cells.

[0011] In one embodiment, 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 to calculate the Cas9 gene / Pol gene ratio and the Pol gene / internal reference gene ratio, which are used to screen and obtain the cell lines in step 2.

[0012] In one embodiment, the Cas9 gene / Pol gene ratio is between 0.4 and 1.2.

[0013] In one embodiment, the internal reference gene is RPPH, and the ratio of Pol gene to internal reference gene is > 0.7.

[0014] In one embodiment, an engineered virus-like particle is provided, which is prepared by the above method.

[0015] In one embodiment, a method for preparing cell lines for the sustainable, mass production of engineered virus-like particles is provided, the method comprising the following steps:

[0016] Step 1: Constructing a stable BaEV retrovirus packaging cell line, including transferring the structural protein gag-pol 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; based on the cell line containing the structural protein gag-pol, constructing a HEK293T cell line or its derivative cell line that simultaneously knocks out the ASCT-1 and ASCT-2 receptor proteins to obtain a cell line that knocks out the ASCT-1 and ASCT-2 receptor proteins; transferring the BaEV envelope into the above-mentioned cell line that knocks out the ASCT-1 and ASCT-2 receptor proteins to obtain the stable BaEV retrovirus packaging cell line;

[0017] Step 2: Transfect the stable packaging cell line of BaEV retrovirus obtained in Step 1 with the Gag-Cas9 plasmid, and screen to obtain cell lines that can produce engineered virus-like particles containing BaEV envelope glycoprotein, gag-pol protein and Cas9 protein.

[0018] Step 3: Construct a self-inactivating delivery plasmid for a retroviral packaging system, which includes gRNA elements targeting different targets; transfect the self-inactivating delivery plasmid into 293T cells, and then use the transfected 293T cells to produce self-inactivating retroviruses targeting different targets.

[0019] Step 4: Use the retrovirus obtained in Step 3 to transduce the cell line obtained in Step 2, and screen from it to obtain a stable cell line that produces engineered virus-like particles.

[0020] In one embodiment, the present invention provides a cell line for the sustainable mass production of engineered virus-like particles, which is prepared by the method described above.

[0021] Traditional engineered viral-like particles (eVLPs) are prepared by co-transfecting HEK293T cells with four plasmids. Because the expression half-life of the plasmids in the cells is limited, eVLPs are usually only suitable for collection within 72 hours after transfection. The production capacity of a single preparation is limited, and the cost of plasmids and transfection reagents used for large-scale preparation is extremely high. The production is unstable between batches, which limits the large-scale production of eVLPs.

[0022] In the field of viral vector production, stable cell lines combined with continuous perfusion culture have become the mainstream approach for large-scale production of retroviruses (RVs). This process constructs stable virus-producing cell lines through genetic engineering, avoiding the cumbersome steps of multi-plasmid co-transfection and reducing production costs by more than 30%. Furthermore, when combined with purification techniques such as tangential flow ultrafiltration, titers up to 10 can be obtained. 6 -10 7 The high-quality virus at TU / mL fully meets the large-scale demand for cell therapies such as CAR-T (10¹¹–10¹² TU per patient).

[0023] This invention addresses the key technological bottlenecks currently faced by eVLP vectors in large-scale production and clinical translation by innovatively developing a production platform based on engineered stable cell lines. This platform achieves high efficiency, safety, and scalability in vector production by constructing cell lines capable of continuously expressing all functional components of eVLP. Specifically, the constructed cell lines stably express four core components: (1) diverse envelope proteins (VSVG, BaEV, GALV, Ecotrophic, Amphotrophic, etc.); (2) MMLV-gag-pol retroviral structural and functional elements; (3) MMLV-gag-cas9 (or other gene editor) fusion proteins; and (4) targeted gRNA elements. This integrated design not only ensures the coordinated expression of all components required for vector assembly but also overcomes the problems of large batch variations and high production costs associated with traditional multi-plasmid transient transfection processes through a stable expression system, providing a reliable vector production solution for the clinical translation of gene editing therapies.

[0024] This protocol first constructed the basic production cell line Clone 28: using pre-constructed Baev WT64 cells (containing the Baev envelope and gag-pol elements) as a chassis, it was obtained through transfection with the gag-cas9-PGK-BSD plasmid, drug screening, and single-clone sorting. This cell line stably expresses the spCas9 editor and viral structural proteins, and its high-efficiency production capacity was confirmed by qPCR and functional validation. To obtain target specificity, a self-inactivating retroviral vector was further introduced to deliver gRNA: promoter activity was eliminated through 3'LTR mutation, ensuring that gRNA expression is driven only by the U6 promoter. Simultaneously, an SV40-GFP reporter system was integrated for dual monitoring—indicating both gRNA expression levels and verifying the absence of LTR contamination in the eVLP product (GFP negativity being the acceptance criterion).

[0025] This invention creatively constructs a cell line integrating essential elements of eVLP and a self-inactivating delivery plasmid for a retroviral packaging system. The self-inactivating delivery plasmid includes gRNA elements targeting different targets. The self-inactivating delivery plasmid is transfected into 293T cells, and then the transfected 293T cells produce self-inactivating retroviruses targeting different targets. The above cell line is transduced using retroviruses, and stable cell lines that produce engineered virus-like particles are screened from them. Engineered virus-like particles are then produced through stable cell lines, and the constructed cell line can sustainably and stably produce specific eVLPs. The advantages of this invention are: 1) It eliminates the need for plasmid transfection for packaging production, enabling large-scale preparation solely through cell culture, overcoming production capacity limitations and simplifying the production process; 2) It reduces the consumption of raw materials (plasmids, transfection reagents, etc.), avoids quality control testing for plasmid residues after vector preparation, and effectively reduces production costs; 3) Stable cell line preparation of eVLP ensures high stability of the vector between different batches, avoiding the risks of large batch-to-batch differences and potential preparation failures in traditional eVLP preparation, as well as the cumbersome and costly process of repeatedly verifying the vector quality and function for each batch in traditional preparation; 4) It increases production flexibility. After screening cell lines capable of producing engineered virus-like particles containing BaEV envelope glycoprotein, gag-pol protein, and Cas9 protein, various corresponding cell lines for producing engineered virus-like particles can be established by introducing different target gRNA elements into these cell lines, thereby increasing the flexibility and convenience of batch production.

[0026] This invention innovatively develops a method for preparing stable eVLP cell lines. This method breaks through existing production bottlenecks and provides a stable, efficient, and economical gene editing vector solution for scientific research and clinical practice, thus promoting the further development of the gene therapy field. Attached Figure Description

[0027] To more clearly illustrate the technical solutions in the embodiments of this application, the accompanying drawings used in the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are only some embodiments recorded in this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0028] Figure 1 Figure showing the genome editing efficiency of 293T / NK92 cells targeting the AAVS1 site with three types of enveloped eVLPs;

[0029] Figure 2 This is a graph showing the knockout efficiency of TRAV-eVLP in Jurkat cells using the Baev capsule.

[0030] Figure 3This is the design diagram of the pCMV-MMLVgag-3xNES-Cas9-PolyA-hPGK-BSD plasmid;

[0031] Figure 4 This is a graph showing the results of gene copy number ratio detection using the qPCR method;

[0032] Figure 5 This is a graph showing the results of gene knockout detection of different clones of the eVLP vector by flow cytometry.

[0033] Figure 6 This is a map of the SIN-CD7 gRNA plasmid;

[0034] Figure 7 This is a graph showing the positive rate of SIN-CD7 gRNA transduction.

[0035] Figure 8 This is a graph showing the results of gradient knockout of Jurkat cells using eVLP. Figure 8 Figure A shows the results of staining with CD7-PE flow cytometry antibody to analyze changes in CD7 knockout efficiency. Figure 8 B in the figure represents plotting the virus knockout efficiency curve;

[0036] Figure 9 This is a diagram showing the verification results of LTR-U6-gRNA-SV40-GFP-LTR nucleic acid molecules in stably produced eVLPs;

[0037] Figure 10 yes Figure 5 A comparison of the efficiency, stability, and T cell survival of transient and stable transgenic eVLP knockout. Figure 10 Figures A, B, and C in the figure show the comparison results of knockout efficiency, stability, and T cell survival rate, respectively.

[0038] Figure 11 This image shows the screening and validation results of monoclonal cell lines produced using the CD7 eVLP vector. Figure 11 Figure A shows the CD7 knockout efficiency of eVLP harvested from 49 candidate clones after infection with Jurkat cells. Figure 11 Figure B shows the CD7 knockout efficiency of primary T cells infected with eVLPs harvested from 12 selected clones. Figure 11 The C-value in the figure compares the cell viability after CD7 knockout. Detailed Implementation

[0039] To enable those skilled in the art to better understand the technical solutions in this application, the present invention will be further described below with reference to embodiments. Obviously, the described embodiments are only some embodiments of this application, and not all embodiments. Based on the embodiments in this application, all other embodiments obtained by those skilled in the art without creative effort should fall within the scope of protection of this application.

[0040] Example 1: Construction of a stable BaEV retrovirus packaging cell line

[0041] eVLP vectors typically consist of four functional modules: Gag-Pol, Gag-Cas9, envelope proteins (such as VSVG, GALV, Amphotropic, Ecotropic, or Baev), and a targeting gRNA plasmid. To establish a sustainable and stable cell factory for toxin production, all of these components, except the targeting gRNA, must be integrated into the same host cell. Subsequently, eVLPs can be produced by transfecting the gRNA plasmid alone. Given that future research is applicable to immune cell (T / NK) therapy, the Baev envelope is preferred; however, whether the mechanism of action of eVLP is consistent with that of the RV envelope protein remains to be investigated. Subsequently, three envelopes (VSVG, GALV, and Baev) of AAVS1 (adeno-associated virus integration site 1) eVLPs were prepared and knocked out in 293T and NK92 cells, respectively. The experimental procedures are as follows.

[0042] 1. Resuscitate 293T and NK92 cells and seed 293T cells.

[0043] 293T cells were resuscitated in DMEM containing 10% FBS and 1% P / S; NK92 cells were resuscitated in NK92-specific medium (Pronosai: CM-0530) and cultured in a 5% CO2 incubator at 37°C. 293T cells were passaged when they reached 80% confluence, and NK92 cells were passaged when the medium turned yellow and the cell density exceeded 2 × 10⁶ cells / cells. 6 Afterwards, it is propagated, and used after 3 generations. Take 3.5 × 10⁻⁶. 6 293T cells were seeded into 10 cm cell culture dishes for subsequent eVLP production.

[0044] 2. Transient 293T quadrature plasmid rotation produces eVLPs targeting the AAVS1 site.

[0045] When the density of 293T cells in a 10cm culture dish reaches 75%-85%, it is ready for transfection. Without changing the culture medium, mix 1.1μg of VSVG / BaEV / Galv envelope plasmid, 3.375μg of MMLV-Gag-Pol structural plasmid, 1.125μg of Gag-Cas9 plasmid, and 4.4μg of AAVS1-gRNA plasmid and add them to 500μl of serum-free medium. Add 30μl of transfection reagent Lipomaster 2000 (Vazyme: TL-201) to 500μl of serum-free medium and mix. Then, dropwise add the plasmid mixture to the transfection reagent mixture, invert and mix twice, and let stand for 5 min. The reagent was then added evenly to the 293T cells and slowly mixed in a cross shape. The cells were then incubated overnight in a 5% CO2, 37°C incubator. After 16 hours, the culture medium was replaced with fresh medium. After 48 hours, the cell supernatant was collected, filtered through a 0.44μm filter, dispensed into 2.2ml tubes, and stored at -80°C.

[0046] 3. eVLP targeting the AAVS1 site in HEK293 and NK92 cells

[0047] Retronectin diluted with PBS was used to coat 12-well plates and incubated overnight at 4°C. The next day, the coating solution was discarded, the plates were rinsed once with PBS, and 1 ml of eVLP vector supernatant was added. The plates were centrifuged at 2000 rpm for 1 h at 32°C. After centrifugation, the supernatant was discarded, and a quantitative sample of cells (293T: 4.4 × 10⁻⁶) was collected. 5 / 1.12×10 5 NK92: 4×10 5 Mix 1 ml of fresh eVLP supernatant and transfer to the corresponding well plate. Centrifuge at 2000 rpm for 1 h at 32°C. Incubate at 37°C with 5% CO2 for 16 h, then replace with fresh culture medium. Collect cells and extract genomic DNA after 72 h.

[0048] 4. T7 Endonuclease I digestion was used to verify the targeting efficiency.

[0049] The T7E1 endonuclease recognizes mismatched bases of 1 bp or more and cleaves at that site. The targeting efficiency of gene editing can be verified through this mechanism. Primer pairs are typically designed with one primer approximately 200 bp from the target site and the other approximately 400 bp from the target site, allowing for direct differentiation via agarose gel electrophoresis. Using this primer pair, PCR was performed with 100 ng of genomic DNA as a template, running for 30 cycles, and the PCR product was purified and recovered. Then, 200 ng of the product template and control template were used to prepare a T7E1 reaction solution (containing all components except T7E1). The solution was pre-denatured at 95°C for 5 min, gradually cooled to 85°C at 2°C per second, then gradually cooled to 25°C at 0.1°C per second, and finally held at 4°C until cleavage. After annealing, add 1 μl of T7E1 and mix well. Incubate at 37°C for 15 min. Perform electrophoresis on a 2% agarose gel and observe the images. The targeted sample showed bands of 400 bp and 200 bp, while the control sample was not cut, indicating successful editing. Subsequently, the approximate editing efficiency was obtained by analyzing the grayscale using ImageJ software.

[0050] Verification using the T7E1 restriction enzyme method confirmed that eVLP does indeed exhibit a mechanism where different envelope proteins have delivery preferences for different target cells. Figure 1 In, such as Figure 1 As shown in A and B, the VSVG envelope effectively knocked out 293T (28.4% - 44.8%), but the knockout efficiency of the NK92 AAVS1 site was 0. The Baev envelope, conversely, efficiently knocked out the NK92 cell line genome (60.1%), while the GALV vector had a lower NK92 knockout efficiency (around 10.8%). Baev-encapsulated eVLPs can efficiently perform gene editing in NK cells; Figure 2 As shown, eVLP targeting the TRAC site of the Baev capsule was prepared and transduced and knocked out on Jurkat cells. Flow cytometry analysis showed that the expression of TCRαβ was significantly reduced by 50%, indicating that the eVLP of the Baev capsule can also efficiently edit T cells. Since this study is mainly applied to immunotherapy, the Baev capsule was subsequently selected for the construction of subsequent cell lines.

[0051] Previously, CRISPR technology was used to knock out ASCT1 / ASCT2 in HEK293 cells, constructing the WT64 retroviral production cell line that stably expresses the Baev envelope (Patent authorization announcement number: CN119020418B; Patent publication number: CN 119020419A; Patent publication number: CN118956766A; Patent publication number: CN). (119020417A) Constructing a stable BaEV retrovirus packaging cell line includes: transferring the structural protein gag-pol, 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; based on the cell line containing the structural protein gag-pol, constructing a HEK293T cell line or its derivative cell line that simultaneously knocks out the ASCT-1 and ASCT-2 receptor proteins to obtain a cell line that knocks out the ASCT-1 and ASCT-2 receptor proteins; transferring the BaEV envelope into the above-mentioned cell line that knocks out the ASCT-1 and ASCT-2 receptor proteins to obtain the stable BaEV retrovirus packaging cell line; this cell line already contains the two key components, Gag-Pol and BaEV envelope, and is free from interference from foreign elements. Therefore, only by stably introducing the Gag-Cas9 component is it possible to complete all eVLP components except gRNA in this cell line, thus upgrading the platform for stable production of virus-like particles.

[0052] Example 2: Design, transfection, and drug screening of pCMV-MMLVgag-3xNES-Cas9-PolyA-hPGK-BSD plasmid.

[0053] 1. Construction of pCMV-MMLVgag-3xNES-Cas9-PolyA-hPGK-BSD plasmid

[0054] The initial Gag-Cas9 plasmid was derived from pCMV-MMLVgag-3xNES-Cas9 (Addgene Plasmid #181752). Using this as a backbone, an hPGK promoter and a plasticidin resistance gene element were added to the Cas9-PolyA line using a seamless cloning method to construct pCMV-MMLVgag-3xNES-Cas9-PolyA-hPGK-BSD. The plasmid map is shown below. Figure 3 .

[0055] 2. Plasmid transfection of WT64 cell line

[0056] The WT64 cell line was cultured in DMEM with 10% FBS and 1% P / S, and was used after three passages following resuscitation. 4E5 cells were seeded into 6-well plates, and plasmid transfection was performed using Lipomaster 2000 after 80% confluence (see Example 1). The culture medium was replaced with fresh medium after 16 hours.

[0057] 3. Drug screening and limiting dilution to isolate monoclonal cells

[0058] 48 hours after transfection, the medium was replaced with DMEM containing 10 μg / mL Blasticidin for resistance selection. During this period, when the cell confluence exceeded 90%, the cells were passaged and cultured in resistance selection medium. After 14 days, monoclonal cell lines were isolated using the limiting dilution method (1 cell / well). Monoclonal cells were cultured in 7.5 μg / mL Blasticidin selection medium for the first week, and then the medium was replaced with 10 μg / mL Blasticidin selection medium. Cell lines with only single cell lines were selected. When the cells grew to more than 50% confluence, the cells were digested and transferred to 6-well plates, and the culture medium was replaced with 7.5 μg / mL Blasticidin selection medium.

[0059] Example 3: Screening of monoclonal cell lines for producing eVLP vectors

[0060] I. qPCR detection of Pol and Cas9 gene copy numbers

[0061] This invention selects 42 monoclonal strains through 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. At the same time, combined with the cell growth and proliferation status, clones that meet the criteria are screened.

[0062] 1. Primer qualification verification

[0063] First, using the corresponding plasmids as templates, multiple Pol and Cas9 primer pairs were selected for qPCR. The melting curves of the primer pairs were analyzed to determine their specificity. Finally, one primer pair from each pair was selected for further 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).

[0064] 2. Construction of standard samples of gag / pol / cas9 fragments

[0065] The gene fragment containing the primers was constructed into a plasmid, and the EcoRI restriction site was set. After digestion, the plasmid yielded a 1310 bp fragment and a 2112 bp fragment. The 1310 bp fragment contained all the gene fragments to be detected. The fragments were purified and recovered by agarose gel electrophoresis and used as linear standards.

[0066] 3. Absolute quantification of Pol, Cas9, and RPPH (internal control) genome copy numbers was performed using qPCR.

[0067] With linear standard products from 1×10 8 Starting with copy number, perform a 10-fold serial dilution to 10. 3 A negative control (0 point) was added to establish a standard curve. Subsequently, the genome of each sample was diluted to 40 ng / μl, and 1 μl of sample (40 ng) was added to the SYBR Green reaction system. The mixture was vortexed for 15 seconds, centrifuged at 400g for 2 minutes, and then analyzed. After exporting the results, standard curves for each detected gene were plotted, generating functions were used. Standard curve: c is the copy concentration; Y = log(1 / c, 2); X = Cq. The sample Cq value was substituted into the standard curve to obtain the Y value; therefore, the sample copy concentration = power(2, -Y). The absolute copy number of each gene fragment in the genome was calculated, followed by the Cas9 / Pol ratio and the Pol / RPPH ratio, for subsequent monoclonal screening.

[0068] A certain range of Gag-Cas9 to Gag-Pol gene copy number ratios can significantly improve gene editing efficiency, rather than simply increasing the proportion of Cas9 plasmid to produce efficient eVLPs. Based on this, 12 clones (2, 6, 12, 14, 16, 17, 22, 27, 28, 32, 33, and 39) with Gag-Cas9 to Gag-Pol copy number ratios within the optimized range (Cas9 / Pol = 0.4-1.2, Pol / RPPH > 0.7) were selected for the next round of screening. Among them, clone 47 was discarded due to slow proliferation, and clone 14 was added (as shown in Table 1). Figure 4 These clones exhibit high expression of Pol and Cas9 elements and produce high titers of eVLP.

[0069] Table 1. Cas9 / Pol and Pol / RPPH copy number detection results

[0070]

[0071] II. Functional validation of eVLP vectors produced by monoclonal cell lines

[0072] Since Jurkat cells highly express CD7 molecules on their surface, their knockout efficiency can be assessed by directly detecting CD7 expression levels using flow cytometry. This method is intuitive and accurate; therefore, knocking out CD7 in Jurkat cells using eVLP can be used to further screen monoclonal lines for eVLP production.

[0073] 1. Production of eVLP vectors using various monoclonal cell lines

[0074] The clones were inoculated at 6 × 10⁶ cells / year. 5 Cells were placed in 6-well plates. When the cell confluence reached more than 80%, they were transfected with CD7 gRNA plasmid. After 16 hours, the culture medium was replaced with fresh medium. After 48 hours, the cell supernatant was collected and filtered through a 0.44 μm filter to obtain the CD7 eVLP vector.

[0075] 2. Targeting and knocking out Jurkat cells with eVLP supernatant to screen for efficient production of monoclonal lines

[0076] Jurkat cells were transduced with these supernatants for CD7 gene knockout (see Example 1). The CD7 gene knockout efficiency mediated by CD7 eVLP produced by each monoclonal line was evaluated by flow cytometry (as shown in Table 2). Figure 5 Experimental results showed that Jurkat cells treated with CD7 eVLP generated from the Clon-28 cell line achieved a knockout efficiency of 93.16% (as shown in Table 2). Based on this excellent performance, this cell line can be used as a starting cell line for the production of eVLP vectors for Baev envelope proteins and spCas9 nuclease.

[0077] Table 2. Efficiency of different monoclonal lines in producing eVLP vector knockout Jurkat cells

[0078]

[0079] Example 4: Preparation of SIN-CD7 gRNA Retrovirus

[0080] I. Design of SIN-CD7 gRNA plasmids (gRNA targeting the CD7 gene delivered via a self-inactivating viral vector)

[0081] This invention provides a multifunctional self-inactivating delivery plasmid for retroviral packaging systems. When this plasmid is co-transfected with retroviral structural proteins (MMLV-gag-pol) and envelope proteins (such as VSVG, Ecotrophic, Amphotrophic, GALV, Baev, etc.) into 293T cells, it can assemble into self-inactivating retroviral particles with different targeting properties, suitable for the transduction needs of various cells.

[0082] like Figure 6 As shown, the main functional elements of this plasmid include: (1) RSV promoter: responsible for driving the transcription of the 5'LTR-3'LTR element, ensuring the generation of RNA required for viral packaging; (2) Modified LTR structure: containing a 5'LTR and a 3'LTR modified by ΔU3. This design allows the 3'LTR (ΔU3) to transfer to the 5' end after the viral genome integrates into the host cell, which leads to promoter inactivation, thereby blocking the regeneration of the complete 5'LTR-3'LTR structure and effectively preventing the delivered plasmid from being repackaged; (3) Primer binding site: providing a primer binding site for the reverse transcription process; (4) MMLVΨ packaging signal: specifically binding to gag protein, mediating the assembly of viral particles; (5) CD7 gRNA (5'-ggagcaggtgatgttgacgg-3'): specifically guides the gene editor to the target site of CD7 gene exon 2; (6) SV40 promoter-driven GFP reporter system: used to monitor viral transduction efficiency in real time and verify the reliability of the self-inactivation system; (7) plasmid replication origin site (ori) and ampicillin resistance gene (AmpR): support plasmid replication and screening in Escherichia coli.

[0083] II. Preparation of SIN-CD7 gRNA Ampho-encapsulated retroviruses

[0084] 1. Resuscitate and inoculate the Ampho293 cell line.

[0085] Ampho293 cells were resuscitated and cultured in DMEM containing 10% FBS and 1% P / S at 37°C with 5% CO2. Cells were passaged after reaching 80% confluence, and used after three passages. 3.5 × 10⁶ cells were collected. 6 Cells were seeded into 10 cm cell culture dishes for subsequent retrovirus production.

[0086] 2. Preparation of retroviruses by plasmid transfection

[0087] When the Ampho293 cell density in a 10cm culture dish reaches 75%-85%, it is ready for transfection. Without changing the culture medium, add 15μg of SIN-CD7 gRNA plasmid to 500μl of serum-free medium. Add 45μl of transfection reagent Lipomaster 2000 (Vazyme: TL-201) to the same 500μl medium and mix. Then, dropwise add the plasmid mixture to the transfection reagent mixture, invert twice to mix, and let stand for 5 min. Next, evenly add the reagent to the Ampho293 cells, slowly mixing in a crosswise motion. Incubate overnight at 37°C with 5% CO2. After 16 h, replace with fresh culture medium. After 48 h, collect the cell supernatant, filter through a 0.44μm filter, aliquot into 3.3ml tubes, and store at -80°C.

[0088] Example 5: Construction of the SIN-CD7-eVLP cell line

[0089] I. Clon28 cell line resuscitation and seeding

[0090] Clon28 cell lines were revived and cultured in DMEM containing 10% FBS and 1% P / S at 37°C with 5% CO2. Cells were passaged after reaching 80% confluence, and used after three passages.

[0091] II. Transduction of Clon28 cell lines using SIN-CD7 gRNA Ampho retrovirus.

[0092] Retronectin diluted with PBS was coated onto 12-well plates and incubated overnight at 4°C. The next day, the coating solution was discarded, the plates were rinsed once with PBS, and 1 ml of SIN-CD7 gRNA retroviral supernatant was added. The plates were centrifuged at 32°C for 1 hour at 2000 rpm. After centrifugation, the supernatant was discarded, and 2 × 10⁻⁶ cells were collected. 5 Clone28 cells were inoculated with 1 ml of fresh viral supernatant into the corresponding wells of a plate and transduced once by centrifugation at 2000 rpm for 1 h at 32°C. The cells were then incubated in a 5% CO2 incubator at 37°C for 2 h. The supernatant was then discarded, and 1 ml of fresh viral supernatant was added again for a second transduction by centrifugation. After the second transduction, the cells were incubated for 2 h, followed by replacement with fresh DMEM complete medium.

[0093] III. Flow cytometry detection of gRNA positive expression rate

[0094] On the third day after viral transduction, cells were collected for positivity rate detection. After digestion and counting, 2 × 10⁶ cells were extracted. 5Cells were washed with PBS and then resuspended in 300 μl of cold PBS. Flow cytometry was used to detect the expression of the reporter gene GFP, which represents the proportion of gRNA expressed in the cell population. To ensure transduction quality, flow cytometry was used again on day 10 post-transduction to detect GFP expression. Figure 7 The results showed that the GFP positivity rate was greater than 70% in all cases, and the GFP positivity rate gradually increased over time, ensuring that gRNA could be stably expressed in cells.

[0095] Example 6: SIN-CD7-eVLP cell line produces and concentrates toxin; Jurkat gradient transduction verifies knockout efficiency.

[0096] I. Establishment of SIN-CD7-eVLP Toxin Production Process

[0097] This protocol involves expanding the SIN-CD7-eVLP cell line into T225 flasks. Once cell confluence reaches 100%, replace the supernatant with 40 ml of 1 mM sodium butyrate solution and incubate at 32°C. After 24 hours, collect the H1 virus supernatant, and replenish with another 40 ml of fresh 1 mM sodium butyrate solution. Collect the H2 virus supernatant after 48 hours, and so on up to H4. Add 4×PEG 8K virus concentrate to the collected virus supernatant, mix vigorously for 1 min, and incubate overnight at 4°C. The next day, centrifuge at 1500g for 20 min to precipitate the virus, discard the supernatant, resuspend the virus pellet in cold PBS, and aliquot. This concentrates the virus 20-30 times.

[0098] II. Validation of Jurakt Knockout Using Gradient Dilution of eVLP Vectors

[0099] First, coat 12-well plates with Retronectin diluted in PBS and incubate overnight at 4°C. The next day, discard the coating solution, rinse once with PBS, and then prepare serially diluted eVLP (0.5 μl, 1 μl, 5 μl, 10 μl, 25 μl) in Jurkat cell culture medium (1 ml each). Transfer each solution to the coated wells and centrifuge at 2000 rpm for 1 h at 32°C. After centrifugation, discard the supernatant and collect 2 × 10⁻⁶ cells / well. 5 Jurkat cells were added to serially diluted eVLP (0.5 μl, 1 μl, 5 μl, 10 μl, 25 μl) fresh viroid supernatant and seeded into corresponding wells of a plate. Transduction was performed by centrifugation at 2000 rpm for 1 h at 32 °C. Cells were then incubated in a 5% CO2 incubator at 37 °C for 2 h, followed by replacement with fresh Jurkat complete medium. After 72 h, cells were collected and stained with CD7-PE flow cytometry antibody to analyze changes in CD7 knockout efficiency (Table 3). Figure 8 (A) Plot the virus knockout efficiency curve ( Figure 8(B in the text). It can be seen that the eVLP knockout effect is positively correlated with the eVLP concentration, and the knockout efficiency of 25 μl vector is 75.92%, proving that this cell line can produce highly efficient eVLP vector.

[0100] Table 3. Efficiency of Jurkat cell knockout after concentrated eVLP gradient dilution

[0101]

[0102] Example 7: Verifying nucleic acid molecules without LTR elements in eVLP

[0103] The designed gRNA delivery system employs a self-inactivating plasmid vector. Its core innovation lies in ensuring, through a modified LTR structure, that the gRNA, after integration into the genome of a production cell line containing the MMLV-gag-pol element, will not be repackaged into a replicating retrovirus. The key safety verification mechanism of this system is based on the following principle: if the LTR-U6-gRNA-SV40-GFP-LTR nucleic acid structure is accidentally introduced into the eVLP, the SV40 promoter will drive GFP expression after transduction into target cells.

[0104] In this example, 25 μl of eVLP vector was used to knock out Jurkat cells (method described in Example 6). Analysis was then performed using CD7-PE flow cytometry with antibody staining and without antibody staining. The results showed that the CD7 knockout efficiency reached 88% (CD7-PE IgG detection), while the GFP positivity rate was only 0.28% (no IgG), significantly lower than the positive threshold. Figure 9 This result confirms the following important conclusions: (1) the LTR-related nucleic acid contamination rate in the produced eVLP vector is less than 0.3%; (2) the self-inactivation design effectively blocks the possibility of repackaging gRNA elements; and (3) the vector system meets the safety standard of "no risk of genome integration". This safety verification system provides a reliable quality control standard for the clinical translation of gene editing therapy.

[0105] Example 8: Comparison of knockout efficiency, stability, and proliferation activity of eVLP vectors produced by different methods

[0106] This study evaluated the differences in gene editing efficiency and stability between a stable eVLP system and a traditional four-plasmid transient transfection system (transient eVLP) through systematic functional comparison experiments.

[0107] Different. Primary T cells were used as the target cell model in the experiment. The knockout efficiency of the CD7 gene was quantitatively analyzed by flow cytometry (method described in Example 6). Cell viability and cell number were detected at different time points (as shown in Table 4). Figure 10The results showed that: (1) Comparison of editing efficiency: The knockout efficiency of the stable production system was 80%, which was slightly lower than the 92.9% of the instantaneous production system. Figure 10 (2) Stability assessment: Flow cytometry analysis on day 8 and day 15 after transduction showed that the editing efficiency of both systems remained stable (instantaneous: 95.4%~92.9%; stable: 81.5%~80%). Figure 10 Results B and C in the figure show the cell proliferation and viability curves of CD7 knockout T cells after transiently produced eVLP versus stably produced eVLP. The study indicates that the viability (above 80%, approximately 10% lower than the highest viability values ​​of other groups) and proliferation were slightly reduced in the stably produced knockout group before day 8. After day 8, cell viability recovered and was no different from other groups (less than 3%). These data confirm that although the stably produced system has a slightly lower absolute editing efficiency, it still has high and stable knockout efficiency with no significant toxicity. Given its better process stability and reproducibility, it better meets the requirements for clinical-grade production.

[0108] Table 4. Knockout efficiency, stability, and proliferation rate of eVLP knockout T cells after transient and stable transformation.

[0109]

[0110] Example 9: Isolation and screening of monoclonal cell lines suitable for large-scale industrial production of eVLPs

[0111] To achieve stable and reproducible production of CD7 eVLP on an industrial scale, systematic monoclonalization and functional verification of its production cell lines were carried out.

[0112] I. Limiting dilution method for isolating monoclonal cells

[0113] SIN-CD7 eVLP cell lines were collected and prepared with DMEM complete medium at a concentration of 1 cell / 100 μl / well. The cells were seeded into 96-well plates, and monoclonal cell lines were observed and labeled. GFP-positive cell lines were selected under a fluorescence microscope. A total of 49 monoclonal cell lines were selected.

[0114] II. Transferring monoclonal cells to 6-well plates for eVLP production

[0115] When the cells reached 50% confluence, they were transferred to 6-well plates. When they reached 80% confluence, 6 × 10⁶ cells were harvested. 5Cells were seeded into new 6-well plates for eVLP production. Once cells reached 100% confluence, the medium was replaced with DMEM containing 1 mM sodium butyrate (2.2 mL / well), and the plates were incubated at 32 °C and 5% CO2 for 24 h. The supernatant (H1) was collected. After adding an equal volume of fresh medium, the plates were cultured for another 48 h, and the supernatant (H2) was collected. Both supernatants were centrifuged at 3000 g for 15 min to remove cell debris, yielding high-purity eVLPs.

[0116] III. Verification of knockout efficiency using eVLP knockout Jurkat cells produced by each monoclonal cell line.

[0117] To verify the knockout efficiency and cytotoxicity of eVLP, a horizontal centrifugation transduction strategy was used to infect Jurkat cells or primary T cells activated after 72 h. Fresh culture medium was replaced 2 h after infection, and CD7 knockout efficiency and cell viability were quantitatively detected by flow cytometry after 72 h, thereby finally identifying the optimal production clone (see Example 6 for the method). Figure 11 Figure A shows the CD7 knockout efficiency of eVLP harvested from 49 candidate clones after infection of Jurkat cells, from which 12 preferred single clones were selected. Subsequently... Figure 11 Figure B shows the CD7 knockout efficiency of primary T cells infected with eVLPs harvested from 12 selected clones. Figure 11 The C-values ​​in the study compared the cell viability after CD7 knockout. Based on the above results, 10A3 was determined to be the optimal production clone, which can then be used for large-scale industrial production of CD7 eVLP vectors.

[0118] 1. Monoclonal cell expansion culture and GMP-grade production

[0119] The selected monoclonal cells were expanded using GMP-level production processes until the cells reached 100% confluence. The supernatants of H1 and H2 cells were collected separately and then filtered through a 0.44 μm filter membrane to remove cell debris.

[0120] 2. Gradient knockout curve and biotiter calculation

[0121] Subsequently, the supernatants of H1 and H2 were diluted five times (1 / 3, 1 / 6, 1 / 12, 1 / 24, 1 / 48), and the original solution was added to Jurkat cells for transduction knockout (see Example 6). Finally, the gradient knockout efficiency was detected, and the biotiter of eVLP was calculated. The formula is: biotiter = knockout efficiency * initial cell number / eVLP volume (TU / ml). The results are shown in Table 5.

[0122] Table 5. Knockout efficiency and biotiter of Jurkat cells using GMP-grade eVLP vector gradient knockout.

[0123]

[0124] It should be understood that the disclosed invention is not limited to the specific methods, schemes, and substances described, as these are all subject to variation. It should also be understood that the terminology used herein is for the purpose of describing specific embodiments only and is not intended to limit the scope of the invention, which is limited only by the appended claims.

Claims

1. A method for the production of engineered viroid particles by a stable cell line, characterized in that, The method comprises the following steps: Step 1: Constructing a BaEV retrovirus stable packaging cell line, comprising transferring the structural protein gag-pol protein required for constructing a retrovirus preparation into a HEK293T cell line or a cell line derived therefrom to obtain a corresponding cell line containing the structural protein gag-pol protein; on the basis of the cell line containing the structural protein gag-pol protein, constructing a HEK293T cell line or a cell line derived therefrom in which ASCT-1 and ASCT-2 receptor proteins are simultaneously knocked out to obtain a corresponding cell line in which ASCT-1 and ASCT-2 receptor proteins are knocked out; transferring the BaEV envelope into the above-mentioned cell line in which ASCT-1 and ASCT-2 receptor proteins are knocked out, thereby obtaining the BaEV retrovirus stable packaging cell line; Step 2: Transfecting the BaEV retrovirus stable packaging cell line obtained in step 1 with a Gag-Cas9-containing plasmid to obtain a cell line capable of producing engineered viroid particles containing a BaEV envelope glycoprotein, a gag-pol protein and a Cas9 protein, wherein the Gag-Cas9-containing plasmid is a pCMV-MMLVgag-3xNES-Cas9-PolyA-hPGK-BSD prepared by adding a hPGK promoter and a BSD resistance gene element to the rear of Cas9-PolyA in a pCMV-MMLVgag-3xNES-Cas9 as a backbone through a seamless cloning method; Step 3: Constructing a self-inactivating delivery plasmid for a retrovirus packaging system, wherein the self-inactivating delivery plasmid comprises gRNA elements targeting different target points; the self-inactivating delivery plasmid is transfected into 293T cells or Ampho 293 cells, and then a self-inactivating retrovirus targeting different target points is produced by the transfected cells; Step 4: Transducing the cell line obtained in step 2 with the retrovirus obtained in step 3 to screen a stable cell line capable of producing engineered viroid particles therefrom, so that the stable cell line can be used to produce engineered viroid particles; In step 2, a single clone cell strain is selected by drug screening and limiting dilution method, genomic DNA is extracted therefrom, and the copy number of Cas9 gene and Pol gene molecules in the cell genome is detected to calculate the ratio of Cas9 gene / Pol gene and the ratio of Pol gene / inner reference gene, which are used for screening the cell line in step 2, wherein the ratio of Cas9 gene / Pol gene is between 0.4 and 1.2, and the ratio of Pol gene / inner reference gene is > 0.7, and the inner reference gene is RPPH1; The self-inactivating delivery plasmid in step 3 comprises a modified LTR structure, which comprises a 5'LTR and a 3'LTR modified by ΔU3, so that after the viral genome is integrated into the host cell, the transfer of the 3'LTR modified by ΔU3 to the 5' end causes the promoter to be inactivated, thereby blocking the reformation of the complete 5'LTR-3'LTR structure and preventing the delivery plasmid from being repackaged.

2. The method of claim 1, wherein, The self-inactivating delivery plasmid in step 3 includes an SV40 promoter-driven GFP reporter system for real-time monitoring of viral transduction efficiency while verifying the reliability of the self-inactivating system.

3. The method of claim 1, wherein, The self-inactivating delivery plasmid in step 3 is co-transfected with retroviral structural proteins and envelope proteins into Ampho 293T cells, or transfected into Ampho 293 cells using the self-inactivating delivery plasmid, to assemble self-inactivating retroviral particles with different targeting properties suitable for the transduction needs of various cells.

4. A method for preparing a cell line for sustainable batch production of engineered viroid particles, comprising the following steps: Step 1: Constructing a BaEV retrovirus stable packaging cell line, including transferring the structural protein gag-pol protein required for constructing a retrovirus preparation into a HEK293T cell line or a cell line derived therefrom to obtain a corresponding cell line containing the structural protein gag-pol protein; on the basis of the cell line containing the structural protein gag-pol protein, constructing a HEK293T cell line or a cell line derived therefrom that simultaneously knocks out ASCT-1 and ASCT-2 receptor proteins to obtain a corresponding 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, thereby obtaining the BaEV retrovirus stable packaging cell line; Step 2: Transfecting the BaEV retrovirus stable packaging cell line obtained in step 1 with a Gag-Cas9-containing plasmid to obtain a cell line that can produce engineered viroid particles containing BaEV envelope glycoprotein, gag-pol protein, and Cas9 protein, wherein the Gag-Cas9-containing plasmid is a pCMV-MMLVgag-3xNES-Cas9-PolyA-hPGK-BSD prepared by adding a hPGK promoter and a BSD resistance gene element after Cas9-PolyA using a seamless cloning method with pCMV-MMLVgag-3xNES-Cas9 as the backbone; Step 3: Constructing a self-inactivating delivery plasmid for a retrovirus packaging system, which includes gRNA elements targeting different target sites; transfecting 293T cells or Ampho 293 cells with the self-inactivating delivery plasmid, and then producing self-inactivating retroviruses targeting different target sites through the transfected cells; Step 4: Transducing the cell line obtained in step 2 with the retroviruses obtained in step 3 to screen a stable cell line that can produce engineered viroid particles therefrom; In step 2, the single clone cell strain is selected by drug screening and limited dilution method, the genomic DNA is extracted, and the copy number of Cas9 gene and Pol gene molecules in the cell genome is detected, the ratio of Cas9 gene / Pol gene and the ratio of Pol gene / inner reference gene are calculated, and the cell line obtained in step 2 is screened, the ratio of Cas9 gene / Pol gene is between 0.4-1.2, and the inner reference gene is RPPH, and the ratio of Pol gene / inner reference gene is >0.7; The self-inactivation delivery plasmid in step 3 includes a modified LTR structure, which contains a 5' LTR and a 3' LTR modified by ΔU3, so that after the viral genome is integrated into the host cell, the transfer of the 3' LTR modified by ΔU3 to the 5' end will cause the promoter to be inactivated, thereby blocking the reformation of the complete 5' LTR-3' LTR structure and preventing the delivery plasmid from being repackaged.

5. The method of claim 4, wherein, The self-inactivation delivery plasmid in step 3 includes an SV40 promoter-driven GFP reporter system, which is used to monitor the virus transduction efficiency in real time and verify the reliability of the self-inactivation system.

6. The method of claim 4, wherein, The self-inactivation delivery plasmid in step 3 is co-transfected with retroviral structural proteins and envelope proteins into 293T cells to assemble into self-inactivation retroviral particles with different targeting properties, which are suitable for the transduction needs of various cells.

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