A modified cell line for efficient packaging of viral particles

Abstract

This invention relates to a modified cell line with one or two ASCT1 and ASCT2 genes knocked out, as well as related preparation methods and applications.

Description

Technical Field

[0001] This invention relates to a modified cell line for improving the encapsulation of viral particles. Prior Technology

[0002] Lentiviral delivery systems are essential tools for molecular biologists to introduce foreign genetic instructions into any desired cell type. The modularity of lentiviral encapsulation systems makes them easy to manipulate and highly flexible, suitable for a variety of purposes. Lentiviral tropism can be altered by changing the pseudotype coat, thereby endowing lentiviruses with the ability to infect different cell types. BaEVRless pseudotyped lentiviruses have been reported to effectively transduce primary human natural killer cells, primary human B cells, and hematopoietic stem cells. However, the formation of HEK293T syncytia during BaEVRless lentiviral encapsulation has been reported, often leading to encapsulated cell death and lower functional viral titer. The lower viral titer of BaEVRless viruses limits NK cell research and CAR-NK cell manufacturing. Therefore, a solution to this problem is needed in this technology. Summary of the Invention

[0003] In one embodiment, this disclosure provides a modified cell line in which one or both ASCT1 and ASCT2 genes are knocked out. In one embodiment, the ASCT2 gene is knocked out. In some other embodiments, the ASCT1 gene is further knocked out. Some examples of gene knockout include "ASCT2" or "ASCT2 and ASCT1".

[0004] In another embodiment, this disclosure provides a method for encapsulating high-valence retroviral particles without forming syncytia, comprising knocking out the ASCT2 gene from a cell line to obtain a modified cell line and transfecting the modified cell line with a retroviral particle containing BaEV protein. In another embodiment, the ASCT1 gene is further knocked out. Some examples of gene knockout include "ASCT2" or "ASCT2 and ASCT1".

[0005] In one embodiment, the IQGAP-1 gene is further knocked out in the modified cell line described herein.

[0006] In some embodiments, the modified cell line is an ASCT1 / ASCT2 double knockout cell line, an ASCT1 / IQGAP-1 double knockout cell line, an ASCT2 / IQGAP-1 double knockout cell line, or an ASCT1 / ASCT2 / IQGAP-1 triple knockout cell line.

[0007] In one embodiment, the cell line disclosed herein is a production cell line. In some embodiments, the cell line is a human fetal kidney 293 cell line (HEK293T cell line), an insect cell line, a TE 671 cell line, or an HT1080 cell line.

[0008] In one embodiment, the modified cell line disclosed herein is transfected with retroviral particles containing baboon endogenous retrovirus (BaEV) protein. In some embodiments, the BaEV protein is the full-length BaEV protein (BaWT) or the BaEV protein lacking the terminal R peptide (BaEVRless).

[0009] In some embodiments, the retroviral particles are lentiviral particles, gamma-retroviral particles, or alpha-retroviral particles. Examples of retroviral particles include (but are not limited to) murine leukosis virus (MLV) particles, avian leukosis virus (ALV) particles, respiratory syncytial virus (RSV) particles, Mason-Pfizer monkey virus (MPMV) particles, lentiviral particles, or spumavirus particles. In some embodiments, the lentiviral particles are: human immunodeficiency virus (HIV) particles, such as HIV-1 or HIV-2 particles; simian immunodeficiency virus (SIV) particles; feline immunodeficiency virus (FIV) particles; equine infectious anemia virus (EIAV) particles and caprine arthritis encephalitis virus (CAEV) particles; and spumavirus vector particles, such as human foamy virus (HFV) vector particles. In another embodiment, this disclosure provides retroviral particles generated by a method as described herein for encapsulating high-valence retroviral particles without forming syncytia.

[0010] In another embodiment, this disclosure provides a method for generating modified NK cells, comprising transducing NK cells with retroviral particles as described herein. Simple Explanation of the Diagram

[0011] Figures 1(A) through 1(E) show BaEVRless-induced cell fusion during lentivirus production. [(] [A] [)] An overview of lentivirus synthesis via transfection of encapsulated and transplastinated plasmids in HEK293T cells. Lentiviral tropism is determined by the mantle gene. [(] [B] [)] The transduction efficiency of BaEVRless versus VSV-G pseudotyped lentivirus (BaEVRless-LV versus VSVG-LV) on primary NK cells from two donors at different infection folds (MOI) was determined using flow cytometry with turboGFP expression. [(] [C] [)] Viral titers of VSVG-LV and BaEVRless-LV produced in parental HEK293T cells, and BaEVRless-LV from our optimized platform. Data are presented as mean ± standard deviation (SD) of three independent experiments (n=3). ns, not significant; *, p≤0.05. [D] Microscopic images of parental HEK293T cells transfected with VSV-G plasmids during lentiviral synthesis. [E]) Images of HEK293T cells transfected with BaEVRless plasmids under the same treatment. The scale bar in the 1× image is 500 μm, and the scale bar in the magnified image is 100 μm.

[0012] Figures 2(A) to 2(J) show that CRISPR knockout of the ASCT2 gene reduces BaEVRless-induced cell fusion. (A) Model of BaEVRless-induced cell fusion. BaEVRless binds to ASCT1 and ASCT2 receptors on adjacent HEK293T cells, resulting in membrane fusion, cell death, and low BaEVRless-LV titer. (B) sgRNA sequences of CRISPR knockout (KO) of ASCT1 and ASCT2 genes in HEK293T cells. Insertion-deletion (indel) % and KO score were determined by Sanger sequencing and ICE analysis. (C) Immunomodulatory spots of ASCT1 in four single ASCT1-KO colonies relative to parental HEK293T cells, with GAPDH as an internal reference. (D) Immunomodulatory spots of ASCT2 in four single ASCT2-KO colonies. (E) Microscopic image of ASCT1-KO colony 3 48 hours after transfection with BaEVRless plastids. (F) Microscopic image of ASCT2-KO line 8 under the same treatment. Scale bar is 500 μm in 1× image and 100 μm in magnified image. (G) Unpurified BaEVRless-LV titer from parental HEK293T, ASCT1-KO, and ASCT2-KO cells at 24 and 48 hours post-transfection. (H) Cell proliferation rate of parental HEK293T and ASCT2-KO line 8 under standard culture conditions, determined by cell counting. (I) Cell respiration rate of parental HEK293T and ASCT2-KO line 8 under standard culture conditions, determined by WST-1 analysis. (J) Unpurified BaEVRless-LV titer with increasing concentrations of inositol hexaphosphate (IP6) added during BaEVRless-LV synthesis. Data are shown as mean ± SD of three independent experiments (n=3). ns, not significant; *, p≤0.05; **, p≤0.01; ****, p≤0.0001.

[0013] Figures 3(A) through 3(H) show robust encapsulation of large transgenes in BaEVRless-LV using an optimized synthesis platform. (A) Lentiviral transcripts encoding turbogfp, TBX21, and the EGFR-CAR chimeric antigen receptor (EGFR-CAR) sequence. The transgenes are expressed by the PGK promoter. 5' LTR, ψ, RRE, cPPT, WPRE, and 3' LTR are essential lentiviral elements. The EGFR-CAR and TBX21 sequences are co-expressed with turbogfp and egfp, respectively, for transduction efficiency measurement by flow cytometry. (B) BaEVRless-LV valence encoding EGFR-CAR and TBX21 is determined in HEK293T cells based on turboGFP and EGFP co-expression, respectively. (C) TBX21 is transduced in primary NK cells from two donors, with efficiency estimated by EGFP co-expression on days 4, 7, and 14 post-transduction. (D) Transduction of EGFR-CAR into primary NK cells from two donors, with efficiency estimated by turboGFP co-expression on days 4, 7, and 14 post-transduction. (E) Mean fluorescence intensity (MFI) of turboGFP. (F) Correlation between turboGFP and HA tag expression in EGFR-CAR-transduced NK cells. (G) In vitro cytotoxicity of EGFR-CAR-expressing NK cells against U-87 MG glioblastoma cells compared to untreated NK cells (NTC). Analysis was performed on days 7 and 14 post-transduction. (H) Cytotoxicity of donor 2 cells. Data are presented as mean ± SD of triplicate experiments (n=3). ns, not significant; *, p≤0.05; **, p≤0.01; ***, p≤0.001.

[0014] Figures 4(A) through 4(E) show the kinome CRISPR KO screening in NK cells using BaEVRless-LV. (A) Workflow of kinome CRISPR KO screening using the SLICE method. A kinome CRISPR KO library containing 6204 sgRNAs targeting 763 kinase genes was encapsulated in BaEVRless-LV for transduction of primary NK cells. Cas9 protein was electroporated into NK cells to generate a unique KO cell population by targeting KO kinase genes. Changes in KO cell distribution and corresponding sgRNA characteristics were determined by next-generation sequencing (NGS) and bioinformatics analysis. (B) NGS verification of the number of sgRNAs in the plastome library and recovered from transduced NK cells. (C) Volcano plot showing the log2 fold change (LFC) and confidence level of enriched and depleted kinase genes from kinome KO screening. All values ​​were calculated from two donor cells as biological replicates. (D) Distribution of LFC values ​​for 6094 target sgRNAs and 100 non-target sgRNAs in the kinase somatic library. (E) LFC values ​​of all eight sgRNAs targeting four enriched genes (red lines) and four depleted genes (blue lines) in surviving NK cells.

[0015] Figures 5(A) through 5(E) show the optimization of BaEVRless-LV purification and NK cell transduction. (A) Workflow for purifying BaEVRless-LV by sucrose pad centrifugation or Lenti-X precipitation. (B) BaEVRless-LV recovery rates for the two purification methods. Data are shown as mean ± SD of four independent experiments (n=4). (C) NK cell viability 72 hours after transduction with BaEVRless-LV from the two purification methods. (D) Transduction efficiency of BaEVRless-LV encoding the turbogfp gene at an MOI of 0.3 under different spinfection and polybrene treatments. (E) NK cell viability transduced with BaEVRless-LV under different spinfection and polybrene treatments. Data in C, D, and E are shown as mean ± SD of three donor cells (n=3). ns, not significant; *, p≤0.05; **, p≤0.01; ***, p≤0.001. Implementation

[0016] The terms used in this specification generally have their usual meaning in the context of this invention and in the specific context in which they are used. Certain terms used to describe this invention are discussed below or elsewhere in this specification to provide additional guidance to those skilled in the art regarding the description of the invention. For convenience, certain terms may be highlighted, for example, using italics and / or quotation marks. The use of highlighting does not affect the scope and meaning of the terms; terms have the same scope and meaning in the same context, regardless of whether they are highlighted. It should be understood that the same thing can be expressed in more than one way. Therefore, alternative language and synonyms may be used for any or more of the terms discussed herein, without any particular meaning, whether the terms are elaborated or discussed in detail herein. Synonyms for certain terms are provided. The description of one or more synonyms does not preclude the use of other synonyms. The use of any instance of any term discussed herein, including examples of any of those terms, anywhere in this specification is illustrative only and does not limit the scope and meaning of the invention or any of the illustrative terms. Similarly, the invention is not limited to the various embodiments given in this specification.

[0017] It should be noted that, as used in this specification and the accompanying claims, unless the context clearly indicates otherwise, the singular forms “a”, “an” and “the” include the plural references.

[0018] As used in this article, the term "pseudotyped viral vector" refers to a viral vector that contains foreign viral mantle glycoproteins.

[0019] As used in this article, the term "baboon endogenous retrovirus" or "BaEV" refers to a type C retrovirus present in multiple proviral replicas in baboon DNA.

[0020] As used herein, the term "BaEV mantle glycoprotein" refers to the wild-type form of BaEV mantle glycoprotein or a mutant of the wild-type BaEV mantle glycoprotein.

[0021] As used herein, the term "transfection" refers to the uptake of a foreign nucleic acid molecule by a cell. Cells are "transfected" when a foreign nucleic acid has been introduced into the cell membrane. Several transfection techniques are commonly known in this field. See, for example, Graham et al. (1973) Virology, 52: 456; Sambrook et al. (1989) Molecular Cloning, a laboratory manual, Cold Spring Harbor lab. (1981) Gene 13: 197. These techniques can be used to introduce one or more foreign nucleic acid molecules into suitable host cells.

[0022] As used herein, the term "carrier particle" refers to any particle that readily displays chimeric mantle glycoproteins or modified BaEV mantle glycoproteins on a surface and reversibly binds to biomaterials.

[0023] Genetic modification of primary natural killer (PNK) cells has been a major challenge in PNK research and application. BaEVRless pseudoformed lentiviral vectors can effectively transduce PNK cells, thus showing great promise in overcoming this challenge. However, due to the known formation of syncytia in encapsulated cell lines, the yield of BaEVRless pseudoformed lentiviruses is usually low. Two BaEVRless lentivirus production protocols have been reported in the literature; one protocol describes using lysine-coated culture trays to delay the detachment of encapsulated cells after syncytia formation (J Virol Methods. 2023 Apr; 314: 114689), and the other protocol uses suspended HEK293T-derived cells to encapsulate the BaEVRless lentiviral vector to prevent syncytia formation (Mol Ther Methods Clin Dev. 2019 Nov 26; 17: 58-68). However, the formation of syncytia of attached HEK293T during BaEVRless lentivirus encapsulation remains unresolved.

[0024] This disclosure reveals that premature binding of the mantle protein to its receptor in the encapsulated cell line leads to the syncytial effect. This disclosure specifically knocks out one or both ASCT1 and ASCT2 genes in the cell line, and this knockout cell line can effectively encapsulate high-valence vectors (such as BaEVRless lentiviral vectors) without forming syncytials. Specifically, the cell line is an ASCT1 / ASCT2 / IQGAP-1 triple knockout cell line. Furthermore, these ASCT1 knockout cell lines, ASCT2 knockout cell lines, ASCT1 / ASCT2 double knockout cell lines, ASCT2 / IQGAP-1 double knockout cell lines, and ASCT1 / ASCT2 / IQGAP-1 triple knockout cell lines show no abnormal defects in cell morphology, proliferation, or metabolism. It further demonstrates that this encapsulation platform can effectively accommodate gene delivery vehicles of different sizes (e.g., chimeric antigen receptors, dCas9).

[0025] Therefore, this disclosure provides a modified cell line in which one or both ASCT1 and ASCT2 genes are disrupted; IQGAP-1 is further disrupted, depending on the situation. The resulting modified cell line is able to encapsulate high-titer BaEVRless lentivirus without forming syncytia. In one embodiment, the cell line is a human fetal kidney 293 cell line (HEK293T cell line).

[0026] SLC1A5 (referred to as ASCT2) and SLC1A4 (referred to as ASCT1) are neutral amino acid transport proteins belonging to the SLC1 family and located in the plasma membrane of several body regions (Mariafrancesca Scalise et al., Front Cell Dev Biol. 2018; 6: 96; published online on September 4, 2018. doi: 10.3389 / fcell.2018.00096). ASCT2 is an acronym for Alanine, Serine, Cysteine ​​Transporter 2, even though the preferred receptor is the conditionally essential amino acid glutamic acid, while cysteine ​​is a regulator rather than a receptor. ASCT2 and ASCT1 have been reported as major receptors for BaEVRless glycoproteins.

[0027] This disclosure discloses that ASCT2 and / or ASCT1, and, where appropriate, IQGAP-1 knockout cells, can improve the titer of viral vector particles (such as BaEVRless lentiviruses) by preventing syncytial formation during viral encapsulation. CRISPR-Cas9 ribonucleoprotein electroporation is one example of a method for specifically knocking out the ASCT2 gene in cells. This disclosure successfully generated one or both ASCT1 and ASCT2 knockout cell lines that increased the titer of BaEVRless lentiviruses by 5 to 10 times.

[0028] The viral vector particle is preferably a retroviral vector particle. More preferably, the retroviral vector particle is selected from the group consisting of: viral vector particles, including murine leukosis virus (MLV), avian leukosis virus (ALV), respiratory syncytial virus (RSV), or mason-Fischer monkey virus (MPMV) vector particles; lentiviral vector particles, such as human immunodeficiency virus (HIV) (e.g., HIV-1 or HIV-2), simian immunodeficiency virus (SIV), feline immunodeficiency virus (FIV), equine infectious anemia virus (EIAV), and caprine arthritis encephalitis virus (CAEV) vector particles; and foamy virus vector particles, such as human foamy virus (HFV) vector particles.

[0029] Any cell compatible with the expression of the lentiviral Gag and Pol genes, or any cell that can be engineered to support that expression, may be used in this disclosure. For example, production cells such as HEK293T cells and insect cells (especially for HIV-derived vectors), TE 671 and HT1080 cells (especially for MLV-derived vectors) may be used.

[0030] NK cell therapy is an emerging field with enormous potential. The allogeneic transfer capability of NK cells will significantly reduce the cost of cancer cell therapy. Effective methods of manipulating NK cells will benefit the research and development of NK cell therapy. Therefore, one or two ASCT1 and ASCT2 knockout HEK293T cell lines capable of producing high-titer BaEVRless lentiviruses can provide an effective platform for manipulating NK cells at a reduced cost.

[0031] In summary, this disclosure elucidates the challenges posed by BaEVRless in lentiviral synthesis and addresses these challenges through ASCT2 KO, IP6 supplementation, and optimized purification protocols. We demonstrate that despite initial hurdles in viral titer and syncytial formation, BaEVRless-LV outperforms VSVG-LV in transducing primary human NK cells. Through CRISPR-Cas9 genomic editing, we identified ASCT2 as a key mediator of BaEVRless-induced syncytial formation, highlighting its role in BaEVRless-LV production. Furthermore, our study demonstrates BaEVRless-LV's robust encapsulation of large transgenic genes and its utility in CAR-NK cell manufacturing and CRISPR KO screening in primary NK cells. To the best of our knowledge, this first successful demonstration of CRISPR gene screening in primary human NK cells paves the way for future whole-genome CRISPR screening and therapeutic development. Our platform also enables CRISPR screening in cells requiring BaEVRless-LV transduction, such as primary B cells and HSPCs. Overall, this disclosure establishes BaEVRless-LV as a valuable tool for gene manipulation in primary immune cells, with broad implications for cell therapy and basic research. [Example] []

[0032] Materials and Methods

[0033] [Reagents and Antibodies] []

[0034] Unless otherwise specified, all chemicals were purchased from Merck and cell culture reagents from Thermo Fisher Scientific. The following antibodies were used for the immunoblotting assay: anti-GAPDH (#60004-1-Ig, ProteinTech), anti-ASCT1 (#8442, Cell Signaling Technology), anti-ASCT2 (#8057, Cell Signaling Technology), anti-rabbit IgG (#7074, Cell Signaling Technology), and anti-mouse IgG (#7076, Cell Signaling Technology). Unless otherwise specified, the following antibodies were used for flow cytometry analysis and were purchased from BioLegend: APC anti-CD3 (#317317), PE anti-CD56 (#362507), PE anti-CD137L (also known as 4-1BB ligand, #311503), Alexa Fluor 647 anti-IL-21 (#513005), anti-HA antigenic determinant tag (#901513), and Alexa Fluor 647 anti-mouse IgG (Invitrogen #A21235).

[0035] [Cell Culture] []

[0036] HEK293T, K562, and U-87 MG cell lines were purchased from the American Type Culture Collection (ATCC) and maintained according to the ATCC protocol. In short, HEK293T and U-87 MG were maintained in complete DMEM medium containing high-glucose DMEM (HyClone) supplemented with 10% heat-inactivated fetal bovine serum (FBS), 25 mM HEPES, 1×GlutaMAX, and 1×Penicillin-Strep. K562 cells were maintained in RPMI-1640 (ATCC modified version) supplemented with 12.5% ​​heat-inactivated FBS, 25 mM HEPES, 1×GlutaMAX, and 1×Pen-Strep. Genetically modified K562 cells were used as feed cells to stimulate the in vitro proliferation of primary NK cells. Modified K562 cells were generated via VSVG-LV transduction using cells expressing 4-1BBL and membrane-bound IL21 (mIL-21). K562-fed cells were expanded in RPMI medium containing 2 µg / ml puromycin to maintain transgenic gene expression, followed by irradiation at a dose of 100 Gy to terminate cell proliferation. Irradiated K562 cells were then washed and immediately used for NK stimulation or cryopreserved in liquid nitrogen at 3 × 10⁶ cells / vial for future use. For routine subculture, HEK293T and U-87 MG cells were dissociated using trypsin-EDTA solution. Dissociated cells were diluted in fresh medium at ratios of 1:4 to 1:8. Cell density and viability during routine subculture were determined by trypan blue staining using a Countess II cell counter (Thermo Fisher Scientific). All cell lines were maintained in an incubator at 37°C with 5% CO2, and mycoplasma contamination was routinely checked using the EZ-PCR detection and analysis kit (Biological Industries).

[0037] [By means] [Cas9] [] [RNP] [Electroporation performed] [, ASCT , ] [] [KO]

[0038] Cas9 protein and single guide RNA (sgRNA) were prepared as described in the literature (Lin, S., V. Nguyen and S. Lin. 2022. Preparation of Cas9 Ribonucleoproteins for Genome Editing. Bio Protoc. 12: e4420.). The oligonucleotides used as templates for sgRNA synthesis are listed in Table S1. Benchling website was used (…). [www] [.] [benchling] [.] Using CRISPR design tools on [com], sgRNAs targeting ASCT1 and ASCT2 were designed. Cas9 RNP electroporation was performed using the Lonza 4D Nucleofector, as described in the literature and modified (Huang, R.-S., M.-C. Lai, H.-A. Shih, and S. Lin. 2021. A robust platform for expansion and genome editing of primary human natural killer cells. J. Exp. Med. 218: e20201529). In short, the electroporation reaction consisted of 20 μL of SF buffer (Lonza) containing 2 × 10⁵ HEK293T cells and 2 μL of 20 μM Cas9 RNP (equivalent to a final concentration of 40 pmol). The cell mixture was then loaded into 16-well nuclear transfection strips (Lonza) and electroporated using a pulse code DS150. Immediately after electroporation, 100 μL of pre-warmed complete RPMI 1640 medium was added to each well to allow cells to recover in a 37°C incubator for 15 minutes. Subsequently, the cells were transferred to 24-well culture dishes filled with 1 mL of pre-warmed complete DMEM medium. Gene editing analysis was performed 72 hours after electroporation.

[0039] [Through Sanger's ordination and] [ICE] [conduct] [, ASCT , ] [] [KO] [analyze] []

[0040] Using Sanger ordination and ICE analysis with preset parameters ( [https] [: / / ] [ice] [.] [synthego] [.] [com]) to determine ASCT KO efficiency. In short, edited HEK293T cells were collected by trypsin dissociation, precipitated by centrifugation at 300×g for 5 minutes, and washed once with DPBS. Following the manufacturer's instructions, the cell pellet was dissolved in QuickExtraction solution (Lucigen) at 65°C for 15 minutes, at 98°C for 5 minutes, and at 4°C for 10 minutes to extract genomic DNA. PCR amplification of the target locus was performed using 100 nanograms of genomic DNA using the KAPA HiFi HotStart PCR kit (Roche), and primer sets were prepared according to known techniques. After verification by DNA gel electrophoresis, the PCR products were purified using the QIAquick PCR purification kit (Qiagen), dissociated in H2O, and Sanger sequenced at the Institute of Biomedical Sciences, Academia Sinica. The results were obtained using the Inference of CRISPR Edits tool (ICE) on the Synthego website with preset settings ( [https] [: / / ] [www] [.] [synthego] [.] [com] [ / ] [products] [ / ] [bioinformatics] [ / ] [crispr] [-] [analysis]) determines insertions or missing values.

[0041] [Immune Ink Dot Method] []

[0042] ASCT1 and ASCT2 expression were detected by immunoblotting. KO HEK293T cell pellet was dissolved in 50 μL of SDS-PAGE containing 80 mM Tris (pH 6.8), 2% β-mercaptoethanol, 4% SDS, 15% glycerol, and 0.01% Orange G. The cell lysate was heated at 95°C for 5 min and resolved by electrophoresis on a 10% SDS-PAGE gel at 100 V for 1.5 h. Proteins were transferred from SDS-PAGE to a 0.2 μm, Millipore PVDF membrane using Trans-Blot SD semi-dry transfer (Bio-Rad) at 80 mA for 1.5 h in Tris-glycine transfer buffer (Bio-Rad). The membrane was incubated at room temperature for 1 h in TBST containing 5% (w / v) skim milk (BD Biosciences). After blocking, the membrane was incubated overnight at 4°C with the primary antibody in TBST containing 5% skim milk. The membrane was washed three times with TBST and incubated with the secondary antibody in TBST containing 0.5% BSA at room temperature for 1 hour. The membrane was washed three times with TBST. The target protein was visualized using Western Lightning PLUS-ECL or the Ultra kit (Perkin Elmer). Images were acquired using Azure Biosystems C300 or Thermo Fisher Scientific iBright FL1000 and processed using ImageJ.

[0043] [Flow cytometry analysis technology and] [FACS] []

[0044] Flow cytometry was performed on the CytoFLEX (Beckman Coulter) system at the Institute of Biological Chemistry, Academia Sinica, a core facility for flow cytometry analysis. NK cells were collected by centrifugation at 500×g for 5 minutes and washed once with 1 mL of ice-cold flow buffer (DPBS supplemented with 2% FBS, 25 mM HEPES, and 0.5 mM EDTA). For surface protein detection, cells were stained with antibody solution at the manufacturer's recommended ratio on ice in the dark for 15 minutes. After staining, cells were washed with 1 mL of flow buffer, precipitated at 500×g for 5 minutes, resuspended in 200 μL of flow buffer, and transferred to 5 mL Falcon polystyrene tubes (Corning) with cell filter snap caps. Modified K562-fed cells were similarly prepared to monitor the performance of 4-1BBL and mIL-21. Single-cell sorting of ASCT1-KO and ASCT2-KO HEK293T cells was performed using the FACSAria III (BD Biosciences) at the Flow Cytometry Core Facility of the Institute of Biomedical Sciences, Academia Sinica. The separated cells were collected in 96-well plates at 200 μL of complete DMEM medium, one cell per well, and expanded using standard culture methods. The elimination of ASCT1 and ASCT2 protein expression in the expanded cells was verified by immunoblotting, and the cells were subsequently stored in liquid nitrogen. All data were analyzed using FlowJo (BD Biosciences) and CytExpert (Beckman Coulter) software.

[0045] [Construction] [BaEVRless] [Cycloplegic reticulum] []

[0046] The encapsulated plasmid pCMV.deltaR8.91 and the VSV-G mantle plasmid pMD2.G were obtained from the C6 RNAi Core Facility of Academia Sinica. BaEVRless mantle plasmids were generated by replacing the VSV-G gene in the pMD2.G plasmid with the previously reported BaEVRless sequence (Girard-Gagnepain, A., F. Amirache, C. Costa, C. Lévy, C. Frecha, F. Fusil, D. Nègre, D. Lavillette, F.-L. Cosset and E. Verhoeyen. 2014. Baboon envelope pseudotyped LVs outperform VSV-G-LVs for gene transfer into early-cytokine-stimulated and resting HSCs. Blood. 124: 1221-1231). PCR primers are listed in Table S2. In short, the BaEVRless gene was codon-optimized and synthesized from IDT DNA into a gBlock fragment. The BaEVRless sequence was amplified from gBlock using primers 1 and 2 with the KAPA HiFi HotStart PCR kit. The pMD2.G vector backbone, lacking the VSV-G gene, was amplified using primers 3 and 4. The PCR products were analyzed by DNA gel electrophoresis, extracted using the QIAquick PCR purification kit, and ligated using NEBuilder DNA Assembly (NEB) according to the manufacturer's protocol. The plastids were transformed and maintained in *E. coli* Stbl3 strain (Thermo Fisher Scientific) and purified using the Qiagen plastid extraction kit. The BaEVRless plastids were validated by Sanger sequencing at the DNA Sequencing Core Facility of the Institute of Biomedical Sciences, Academia Sinica. The BaEVRless plastid map (pMD2.G_BaEVRless) is attached as a Genbank file in the supplementary information.

[0047] [Constructing lentiviral transfer plasmids] []

[0048] Using the same method as with BaEVRless plasmids, lentiviral transfer plasmids encoding different transgenes were constructed into pHR vectors (modified from Addgene #79125) via NEBuilder HiFi DNA assembly (NEB). PCR reactions were performed using a KAPA HiFi HotStart PCR kit, and primers were prepared according to known techniques. In short, to prepare pHR_turboGFP plasmids, primers 5 and 6 were used to amplify the turbogfp gene from pmaxGFP plasmids (Lonza), and this gene was ligated to the pHR vector amplified by primers 7 and 8. To prepare pHR_TBX21_P2A_EGFP plasmids, primers 9 and 10 were used to amplify the TBX21 gene from NK cell cDNA, and this gene was ligated to the pHR vector amplified by primers 11 and 12. The EGFR-CAR expression cartridge contains the following sequences: turbogfp gene, T2A, CSF2RA message peptide, anti-EGFR single-stranded variable fragment (scFv) from panitumumab, IgG hinge, CD8 transmembrane domain, 4-1BB co-stimulatory domain, and CD3σ activation domain. The CAR cartridge is synthesized from IDT DNA into a gBlock fragment, amplified by primers 13 and 14, and ligated into a pHR vector amplified by primers 15 and 16. All plastids were verified by Sanger sequencing. Plastid maps are attached in the supplementary materials as GenBank archives.

[0049] [Lentinus production] []

[0050] Maintain HEK293T and ASCTKO cells as described above. Subculture cells every 2 to 3 days when they reach 80% to 90% confluence. To ensure consistent and high lentiviral titer, it is recommended not to use cells that have been passaged more than 30 times. On day 0, seed 8.5–9.0 × 10⁶ cells in 10 mL of lentiviral encapsulation medium (Opti-MEM medium supplemented with 5% heat-inactivated FBS, 1×GlutaMAX, 1× Sodium pyruvate, and 1×MEM non-essential amino acid solution) in 10 cm culture dishes. Approximately 16 to 18 hours post-inoculation, transfect cells using Lipofectamine 3000 with 8 µg of transfer plasmids, 7 µg of encapsulated plasmids (pCMV deltaR8.91), and 3 µg of mantle plasmids (pMD2.G or pMD2.G_BaEVRless) according to the manufacturer's instructions. Six hours after transfection, the culture medium was removed, and the medium was replenished with fresh lentivirus-encapsulated medium supplemented with IP6 (also known as phytic acid). A 30 mM IP6 stock solution was prepared as follows: IP6 powder (Merck) was dissolved in DPBS, the pH was adjusted to 7, filtered, sterilized, and stored at 4°C. IP6 is based on... [picture] Add the lentivirus at the concentration indicated in [2J] or the optimal concentration of 150 µM. 48 to 50 hours post-transfection, collect the lentivirus-containing medium in a 15 mL conical tube and centrifuge at 650 × g for 5 minutes to remove cell debris. Pass the supernatant through a low-protein-binding 0.45 µm syringe filter (Millipore). Lentiviral virus was purified using two different methods for comparison. In the first method, lentivirus was precipitated from the supernatant using Lenti-X Concentrating Reagent (Takara) according to the manufacturer's protocol. In the second method, lentivirus was precipitated by centrifugation at 4500 × g for 20 hours in a 20% sucrose pad at 4°C. In both methods, the lentivirus precipitate was resuspended in basal NK MACS or Opti-MEM at 1 / 50 the volume of the cell culture supernatant. The purified lentivirus was aliquoted, flash-frozen in liquid nitrogen, and stored at -80°C.

[0051] With the exception of the kinase CRISPR sgRNA library, the lentiviral vectors in this study carried the gfp reporter gene for detection. Functional force valence of lentivirus was determined by transduction of HEK293T cells and by flow cytometric analysis techniques based on GFP phenometric measurement of transduction efficiency. Briefly, 5×104 HEK293T cells were inoculated in 24-well culture dishes in 500 μL of complete DMEM medium containing 8 μg / mL condensed amine. Different amounts of lentivirus in the range of 0.1 to 5 μL were added to transduce HEK293T cells for 2 days. Gene expression was determined by flow cytometry techniques, and the functional force price was calculated based on samples producing 10% to 30% transduction efficiency as follows: Functional force valence (TU / mL) = (5 × 104 cells × transduction efficiency × 103) / amount of virus added (μL).

[0052] Determining the lentiviral force valence of the CRISPR sgRNA library based on puromycin resistance. HEK293T cells were transduced with different amounts of lentivirus, and those cells were treated with 2 μg / mL puromycin on day 2 post-transduction. On day 4, 100% of untransduced cells should be killed by puromycin. The transduction efficiency was determined based on the number of viable cells in the transduced group determined by means of trypanosomatid blue staining compared with the number of cells in the untransduced group without puromycin treatment. The self-generating 30% transduction efficiency of the sample was calculated to calculate the functional force price.

[0053] Imaging analysis of BaEVRless-induced syncytium

[0054] Parental, ASCT1KO (colonial strain No. 3) and ASCT2KO (Colonial strain No. 8) HEK293T cells were transfected by lipostaining amine with lentiviral enveloped plastids encoding VSV - G or BaEVRless mantle genes and transfer plastids encoding turbogfp genes, as described above. No IP6 added. Forty-eight hours after transfection, bright-field and green fluorescence images of parental cells and KO colonizing lines were taken on an Olympus CKX41 inverted microscope and were treated with ImageJ version 1.53A.

[0055] Ex vivo expansion of primary NK cells

[0056] CD3-negative enriched and cryopreserved human peripheral blood NK cells were purchased from Lonza and HemaCare. All experiments involving human cells were performed in accordance with human experimental guidelines approved by the Institutional Review Board on Biomedical Science Research, Academia Sinica. On day 0, the cryopreserved NK cells were thawed in a 37°C water bath with gentle agitation until no ice was visible in the tubes. The NK cells were gently transferred to 15 mL Falcon tubes containing 8 mL of 4°C RPMI 1640 (ATCC modified) medium supplemented with 10 U / mL deoxyribonuclease I (Merck). The frozen vials were rinsed with 1 mL of RPMI medium to transfer the remaining NK cells to the 15 mL tubes. After centrifugation at 400×g for 10 minutes, the cell pellet was resuspended in 10 mL of pre-warmed complete NK MACS medium containing NK MACS medium (Miltenyi), 5% EliteGro-Adv human platelet lysate (EliteCell), 1% GlutaMAX, and 1% Pen-Strep. This medium was also supplemented with 10 U / mL deoxyribonuclease I and 2 ng / mL recombinant IL-15 (Peprotech). The cell suspension was transferred to T25 culture flasks (Corning) and maintained in an incubator at 37°C with 5% CO2. On day 1, NK cells were collected from the culture flasks, and cell density was determined to establish feed-dependent expansion. In T75 culture flasks (Corning), approximately 5 × 10⁶ NK cells were co-cultured with 1 × 10⁷ modified and irradiated K562 feed cells in 40 mL of complete NK MACS medium supplemented with 100 U / mL recombinant IL-2 (Peprotech). The final NK cell density was 1.25 × 10⁵ cells / mL in 40 mL of culture medium. On days 3 and 5, NK cells were pelleted by centrifugation at 300 × g for 5 minutes. Twenty mL of supernatant was carefully removed from the top layer and replaced with 20 mL of fresh complete NK MACS medium containing 100 U / mL IL-2. The NK cells were gently resuspended and transferred back to the culture flask. On day 7, NK cells were collected by centrifugation at 300 × g for 5 minutes, washed once with DPBS (Corning), and cryopreserved at 3 × 10⁶ NK cells per flask in CryoStor (Sigma Aldrich) for future use. The remaining NK cells were restimulated at a 1:1 ratio with K562 and maintained using the same method described above.Primary NK cells were maintained at a concentration below 3 × 10⁶ cells / mL and subcultured every 2 to 3 days by dilution in fresh complete NK MACS medium. The purity of the primary NK cells was analyzed by flow cytometry based on CD3 and CD56 expression.

[0057] [NK] [Cell transduction] []

[0058] Seven to ten days after expansion, 5 × 10⁴ primary NK cells were suspended in 100 µL of fresh, complete NK MACS medium per well in a 96-well flat-bottom culture dish. Lentiviral cells were added at the indicated MOI. Agglutamine (also known as hexamethylene bromide) was purchased from Merck, dissolved in H₂O to a concentration of 8 mg / mL, and sterilized by passing through a 0.2 µm filter. Agglutamine solutions were aliquoted and stored at -80°C to prevent repeated freezing and thawing. Agglutamine was added to the medium. Infection was performed by centrifugation at 1200 × g for 90 minutes at 32°C. Cells were then resuspended by gentle aspiration and incubated overnight at 37°C. One day after transduction, cells were washed and resuspended in 100 µL of fresh, complete NK MACS medium. Flow cytometry was used to analyze transduced cells to determine transduction efficiency at specified time points.

[0059] [Respiratory Analysis] []

[0060] Cellular respiration was determined using the manufacturer's protocol via WST-1 analysis (Abcam). In short, parental cells and ASCT2KO HEK293T cells were seeded at 1 × 10⁴ cells per well in 1 mL of complete DMEM medium in 12-well culture dishes, with three wells for each cell type. After one day of incubation, 100 µL of medium was removed from each well, and 100 µL of WST-1 reagent was gently added to the cell culture to avoid cell interference. The culture dish was gently agitated to ensure uniform mixing of the WST-1 reagent and medium, followed by incubation at 37°C for 2 hours. After incubation, 100 µL of culture supernatant was transferred to a clear 96-well culture dish, and absorbance was measured at 440 nm and 650 nm (reference) using an Infinite M1000 Pro microdispenser (Tecan).

[0061] [CAR] [-] [NK] [Cellular Cytotoxicity Analysis] []

[0062] Cytotoxicity analysis based on Calcein-AM was described as follows: Huang, R.-S., M.-C. Lai, H.-A. Shih, and S. Lin. 2021. A robust platform for expansion and genome editing of primary human natural killer cells. J. Exp. Med. 218: e20201529. U-87 MG cells were dissociated with trypsin (Gibco), neutralized with culture medium, and precipitated at 200×g for 3 min. Cells were washed once with DPBS and adjusted to 1×10⁶ cells / mL in 1 mL of DPBS containing 10 µM Calcein-AM (BioLegend) for staining at 37°C for 30 min. Cells were then washed three times with DPBS and resuspended in RPMI-1640 (ATCC modified) to 1×10⁵ cells / mL. CAR-NK cells were pelleted at 90×g for 10 min and resuspended in RPMI-1640 (ATCC modified) to 4×10⁵ cells / mL. One hundred μL of CAR-NK cells were added to each well of a 96-well round-bottom culture dish. Serial dilutions were performed at different ratios of CAR-NK cells to U-87 MG cells. One hundred μL of stained U-87 MG cell suspension was added to each well to initiate cytotoxicity analysis. The 96-well culture dish was centrifuged at 120×g for 3 min to initiate contact between NK cells and U-87 MG cells. The cell mixture was incubated at 37°C for 4 h. Spontaneous release of calcein-AM from U-87 MG cells was measured in the absence of CAR-NK cells. Maximum release was determined by completely dissolving U-87 MG cells in RPMI-1640 (ATCC modified) containing 2% Triton-X100. After co-culturing, the culture dish was centrifuged at 120×g for 3 minutes, and 100 µL of supernatant was transferred to 96-well Opti-plates (PerkinElmer). The 488 / 520 value was recorded using an Infinite M1000 pro (Tecan). The following equation was used for cytotoxicity calculation:

[0063] [use] [SLICE] [Methods for kinaseosome analysis] [CRISPR] [Removal Filtering] []

[0064] The kinaseosome CRISPR knockout screening was performed in primary NK cells using the sgRNA lentiviral infection with Cas9 protein electroporation (SLICE) method developed for primary T cells. The human kinosomal CRISPR knockout library was obtained from Addgene (catalog number 1000000082) and prepared as described in the literature (Doench, J.G., N. Fusi, M. Sullender, M. Hegde, E.W. Vaimberg, K.F. Donovan, I. Smith, Z. Tothova, C. Wilen, R. Orchard, H.W. Virgin, J. Listgarten and D.E. Root. 2016. Optimized sgRNA design to maximize activity and minimize off-target effects of CRISPR-Cas9. Nat. Biotechnol. 34: 184-191). The kinosomal CRISPR sgRNA library was encapsulated in BaEVRless-LV cells of ASCT2KO cells using our standard protocol. As described above, viral titer was determined in HEK293T cells by puromycin selection. Approximately 1 × 10⁷ feeder-expanded NK cells were transduced using BaEVRless-LV at an MOI of 0.3. Recombinant Cas9 protein was prepared as detailed in (Lin, S., V. Nguyen and S. Lin. 2022. Preparation of Cas9 Ribonucleoproteins for Genome Editing. Bio Protoc. 12: e4420). Twenty-four hours post-transduction, Cas9 was electroporated into NK cells using the Lonza 4D nuclear transfection system with pulse code CM137 in a 100 µL electroporation sample well. The reaction mixture contained 400 pmol of Cas9 protein and 1 × 10⁷ transduced NK cells in 100 µL of P3 solution (Lonza). Immediately after electroporation, 500 µL of pre-warmed complete NK MACS medium was added directly to the sample well to restore NK cells at 37°C for 15 minutes.Next, NK cells were transferred to T25 culture flasks at a density of 1 × 10⁶ cells / mL and cultured at 37°C. After 24 hours, NK cells were collected by centrifugation at 300 × g for 5 minutes and resuspended in complete NK MACS medium supplemented with 1 µg / mL puromycin to select sgRNA-transduced cells for 48 hours. Following this, NK cells were collected by centrifugation at 300 × g for 5 minutes, washed once with DPBS, and resuspended in puromycin-free complete NK MACS medium. NK cells were cultured for 7 days after puromycin selection, with fresh medium added every 2 to 3 days to maintain a NK cell density of 5 × 10⁵ to 2 × 10⁶ cells / mL. The cell suspension was aliquoted into multiple culture flasks as needed. The total cell count was maintained at a minimum of 3 × 10⁶ cells to preserve intact sgRNA expression. On day 21, we collected 3 × 10⁶ cells (equivalent to 1000 × coverage) for genomic DNA extraction and NGS to determine the sgRNA sequence in the NK cell population.

[0065] [Preparation of genomic DNA] [DNA] [For use] [NGS] []

[0066] Genome DNA from NK cells was purified using the Quick DNA Miniprep Plus kit (Zymo Research) according to the manufacturer's instructions. The sgRNA sequence from the genome DNA was amplified and barcoded using a two-step PCR reaction as described in the literature (Shifrut, E., J. Carnevale, V. Tobin, T. L. Roth, J. M. Woo, C. T. Bui, P. J. Li, M. E. Diolaiti, A. Ashworth and A. Marson. 2018. Genome-wide CRISPR Screens in Primary Human T Cells Reveal Key Regulators of Immune Function. Cell. 175: 1958-1971). PCR primers were prepared using known techniques. The reaction mixture consisted of 50 µL of NEBNext high-fidelity 2×PCR master mix (NEB), 4 µg of genomic DNA, 2.5 µL each of 10 µM NG-Lib-Fwd and NGS-Lib-KO-Rev primers, and water, totaling 100 µL. The thermal cycler was set to 98 °C for 3 min, followed by 98 °C for 10 s, 66 °C for 10 s, and 72 °C for 25 s, for 23 cycles; and a final extension at 72 °C for 2 min. PCR amplicones were purified by DNA gel electrophoresis and extracted using a Zymoclean gel DNA recovery kit (Zymo Research). Amplicon concentration and size were determined using Qubit DNA quantification (Thermo Fisher Scientific) and a fragment analyzer (Agilent). Dual-barcode transducers were added using a Nextera XT indexing kit v2 (Illumina). The molar concentrations of amplicones from each sample were normalized and then sequenced on a NextSeq 500 / 550 instrument (Illumina). The same procedure was also used to perform PCR amplification and sequencing of the purified kinaseosome CRISPR knockout library plasmids to verify the integrity of the sgRNA library.

[0067] [Regarding the merger] [CRISPR] [Analysis of the screening process] []

[0068] The MAGeCK software was used to identify enriched and depleted genes in the kinaseosome screening. Read count tables for each sgRNA were determined from the original fastq sequencing archive using the default MAGeCK "Count" module. During count table construction, sgRNAs with offset designs were extracted using the "-trim-5 48,49,50,51,52" parameter. To obtain gene enrichment, the robust rank aggregation (RRA) algorithm was executed using the MAGeCK "Test" module with default settings. Size factors were calculated using non-targeted control sgRNAs from each library for standardization between experiments. The distribution of individual sgRNAs from the top-ranked genes was visually examined using the MAGeCKFlute R suite with default settings. The list of genes from the MAGeCK analysis is shown in Table S5. The original NGS data archive is available in the NCBI Sequence Read Archive (PRJNA1078135).

[0069] [Statistical Analysis] []

[0070] Except for the screening experiment, all data were collected from three independent experiments to determine the mean ± standard deviation, as shown. The unpaired Welch's unequal variance t-test was used to detect significant differences between the two groups. A p-value ≤ 0.05 was considered statistically significant. Statistical analysis was performed using GraphPad Prism 9.

[0071] [Example] [1] [] [BaEVRless] [Induces cell fusion and disrupts lentivirus synthesis]

[0072] To understand the challenges posed by BaEVRless, we will briefly review lentiviral vector synthesis in HEK293T and related cell lines. The process begins with the transfection of lentiviral plasmids containing genes for Gag, Tat, Pol, Rev, and pseudomantle proteins, and transfer plasmids encoding the desired transfection genes (Figure 1A). The expression of these viral proteins and lentiviral RNA transcripts initiates lentiviral assembly. Subsequently, these viruses are secreted from HEK293T cells into culture medium, which is then collected for virus purification.

[0073] Pseudomantle proteins can be modified by altering the mantle genes within the plastid to change lentiviral tropism. During expression, mantle proteins are localized to the cell membrane and displayed on the surface of HEK293T cells. During viral particle release, a portion of the HEK293T cell membrane becomes the lentiviral outer membrane. For example, the VSV-G mantle protein on the outer membrane of VSV-G-LV interacts with LDLR on the target cell, causing VSV-G-LV internalization and the release of lentiviral RNA transcripts encoding transfection genes. Similarly, BaEVRless proteins bind to ASCT1 and ASCT2 receptors on NK cells to facilitate viral entry.

[0074] We transduced NK cells using two types of lentiviruses encoding the turboGFP gene and assessed transduction efficiency by measuring turboGFP expression using flow cytometry. BaEVRless-LV demonstrated significantly higher efficiency than VSVG-LV in transducing primary human NK cells (Figure 1B). Even at an infection fold change (MOI) as high as 50, VSVG-LV maintained a stable transduction efficiency of 20% to 40%, while BaEVRless-LV achieved 40% to 50% efficiency at an MOI of 1 and remained stable at approximately 80% at an MOI of 10. Our results confirm that BaEVRless-LV outperforms VSVG-LV in NK cell transduction efficiency across all MOI ranges.

[0075] However, BaEVRless-LV synthesis presents unique challenges not observed in VSVG-LV. When encapsulated in HEK293T cells using the same protocol, the viral titer of BaEVRless-LV was approximately 100-fold lower than that of VSVG-LV (Figure 1C). Furthermore, extensive syncytial formation and the production of large sheets of confluent cells were observed within 48 hours of transfection with BaEVRless-encapsulated plastids. [picture] [1D] (relative to Figure 1E). These fused cells subsequently undergo cell death and detach from the culture dish, resulting in excessive cell debris and complicating lentiviral purification.

[0076] [Example] [2] [] [ASCT2] [Mediation] [BaEVRless] [Induced syncytial formation] []

[0077] We hypothesize that BaEVRless binds to the ASCT2 receptor on neighboring HEK293T cells, triggering membrane fusion (Figure 2A). This process may also involve the helper receptor ASCT1 (Marin, M., C. S. Tailor, A. Nouri, and D. Kabat. 2000. Sodium-Dependent Neutral Amino Acid Transporter Type 1 Is an Auxiliary Receptor for Baboon Endogenous Retrovirus. J. Virol. 74: 8085-8093). To test this, we used CRISPR-Cas9 genome editing to knock out the ASCT1 and ASCT2 genes. We designed four single guide RNAs (sgRNAs) targeting exon 1 of the ASCT1 and ASCT2 genes and independently electroporated HEK293T cells using four Cas9 RNPs. Three days after electroporation, Sanger sequencing analysis was used to determine the insertion-deletion percentage (insertion or deletion %) and KO score at the target site (Figure 2B). ICE analysis revealed that Cas9 RNPs 1 and 3 exhibited higher insertion or deletion % and KO scores, indicating higher ASCT1 and ASCT2 KO efficiencies, respectively. Editing via Cas9 RNP 2 resulted in a nine-base frame deletion that did not disrupt ASCT1 expression, leading to a low KO score.

[0078] We performed fluorescence-activated cell sorting (FACS) on cells edited with Cas9 RNP 1 and 3. We isolated four single colonies from each KO cell line and analyzed ASCT1 or ASCT2 expression using immunoblotting. Most KO colonies showed reduced ASCT expression (Figs. 2C and 2D). Specifically, ASCT1KO colony 3 and ASCT2KO colony 8 showed almost complete elimination of ASCT1 and ASCT2 expression, respectively. Subsequently, we encapsulated colonies 3 and 8 with BaEVRless-LV and monitored the cells 48 hours after BaEVRless plastid transfection. Although syncytial formation was still observed in ASCT1KO colony 3, the effect was less pronounced compared to the parent HEK293T (Fig. 2E vs. Fig. 1E). ASCT1KO cells formed small, confluent, and attached cell communities, which eventually detached from the culture tray after 72 hours of culture. In contrast, ASCT2KO line 8 exhibited perfectly healthy cell morphology (Fig. 2F). ASCT2KO cells remained firmly attached to the culture dish, and no syncytiotrophoblasts were observed. Our results clearly indicate that ASCT2 is the main mediator of BaEVRless-induced syncytiotrophoblasts, while ASCT1 may play a supporting role in this process.

[0079] [Example] [3] [] [, ASCT2 , ] [Genes] [CRISPR] [Increase] [BaEVRless] [-] [LV] [Power Price] []

[0080] We aimed to determine whether inhibiting BaEVRless-induced syncytial formation increased BaEVRless-LV synthesis. We encapsulated BaEVRless-LV in parental and ASCT2KO cells, sampled the culture medium at 24 and 48 hours, and measured the titer of the unpurified virus. Of interest, we observed an eight-fold increase in viral titer in ASCT2KO cells (Fig. 2G), indicating that reducing BaEVRless-induced syncytial formation increased BaEVRless-LV production. Furthermore, ASCT2-KO cells exhibited the same normal proliferation and respiration rates as the parental HEK293T cells (Figs. 2H and 2I). Because ASCT2-KO cells can be maintained and efficiently transfected like parental cells, these KO cells can be seamlessly integrated into standard attach-type cell-based lentivirus production systems without modification to the cell culture system or transfection procedure.

[0081] [Example] [4] [Supplementing with inositol hexaphosphate will increase] [BaEVRless] [-] [LV] [Power Price] []

[0082] To assess whether supplementation with inositol hexaphosphate (IP6) during BaEVRless-LV production could enhance viral titer, we added various concentrations of IP6 to ASCT2-KO cells during viral encapsulation. We observed a dose-dependent increase in BaEVRless-LV titer with IP6 supplementation (Figure 2J). At a concentration of 150 μM, IP6 further tripled the viral titer. These results suggest that supplementation with IP6 (a non-toxic and naturally occurring metabolite) may enhance BaEVRless-LV production.

[0083] [Example] [5] [] [BaEVRless] [-] [LV] Optimization of purification and transduction methods is crucial. []

[0084] We compared two common lentiviral purification methods: sucrose-blended centrifugation and precipitation using Lenti-X concentrate (a commercially available polyethylene glycol-based solution). ASCT2-KO cells were transfected with BaEVRless-LV-encapsulated plasmids and turboGFP transfer plasmids. Three days later, the culture medium containing BaEVRless-LV was collected and purified using both methods. Viral titer before and after purification was quantified by transducing HEK293T cells and measuring turboGFP expression using flow cytometry. Sucrose-blended centrifugation recovered 30% BaEVRless-LV from the unpurified medium, while Lenti-X precipitation achieved a recovery rate of >90%. Notably, insoluble precipitate was observed after sucrose-blended centrifugation, resulting in a significant loss of viral yield. We also evaluated NK cell viability after transduction as an indicator of viral purity. Both purification methods showed similar cell viability in the range of 70% to 100%, indicating similar viral purity. Ultimately, the combination of ASCT2KO cells, IP6 supplementation, and optimized purification regimen increased the valence of BaEVRless-LV by 50-fold, reaching approximately 5 × 10⁸ TU / mL, which is close to the valence of VSVG-LV (Figure 1C).

[0085] Next, we evaluated lentiviral transduction using BaEVRless-LV encoding turbogfp purified by Lenti-X, focusing on amine supplementation and centrifugal infection. Transduction was performed on primary NK cells from three donors, with the MOI reduced to 0.3 for analytical sensitivity. The cationic polymer amine (hexammonium bromide) was added to enhance lentiviral transduction by neutralizing the charge repulsion between viral particles and the cell surface. We tested three amine concentrations (2, 4, and 8 µg / mL) and observed a dose-dependent increase in transduction efficiency, but higher amine doses also decreased cell viability. Centrifugal infection, involving centrifuging the NK cell and BaEVRless-LV mixture at 1200×g for 90 min to enhance cell-virus contact, did not significantly improve transduction but rather impaired cell viability. Therefore, we chose condition 3 (4 µg / mL amine, without centrifugal infection) for all downstream experiments because it achieved the optimal balance between enhanced transduction and cell viability. Figure 5 shows the optimization of BaEVRless-LV purification and NK cell transduction.

[0086] [Example] [6] [] [, ASCT2 , ] [] [KO] [Cells can robustly encapsulate large transgenic genes] []

[0087] Building upon the results of turbogfp, we further encapsulated two larger transgenic genes: the EGFR-CAR sequence and the TBX21 gene, which encodes the T-bet transcription factor crucial for NK cell development. Co-expression of turbogfp or egfp with EGFR-CAR and TBX21 simplified transduction analysis using flow cytometry (Figure 3A). The BaEVRless-LV valence of EGFR-CAR (2147 nt) and TBX21 (2400 nt) was comparable to that of turbogfp (717 nt), indicating robust encapsulation of large genes (Figure 3B). However, compared to turbogfp, the transduction and expression efficiencies of EGFR-CAR and TBX21 were lower. The TBX1 transduction efficiency was 30%–50% at an MOI of 1, increasing to 60%–80% at an MOI of 10 (Figure 3C). In contrast, turbogfp efficiency reached 70% at an MOI of 1 and stabilized at 80% at an MOI of 10 (Figure 1B). These results suggest that large transgenic genes may have lower transduction or expression efficiency in NK cells.

[0088] A similar decrease in EGFR-CAR transduction was observed. We transduced two different donor cells at low MOIs of 2 and 5, and monitored EGFR-CAR performance using turboGFP fluorescence on days 4, 7, and 14 post-transduction. Transduction efficiency, measured as the percentage of turboGFP+ cells, varied between donor cells (Fig. 3D). Furthermore, the percentage of turboGFP+ cells and the fluorescence intensity of turboGFP decreased over time (Fig. 3E). To ensure that turboGFP is a good indicator of EGFR-CAR performance, we also stained the HA affinity antigenic determinant within the CAR protein with an anti-HA antibody to detect EGFR-CAR protein displayed on the surface of NK cells. At different MOIs, the percentages of HA+ and turboGFP+ cells were highly similar in both donor cell types, confirming the correlation between turboGFP and EGFR-CAR performance.

[0089] Next, we evaluated the in vitro cytotoxicity of EGFR-CAR NK cells against the EGFR+ glioblastoma cell line U-87 MG. EGFR-CAR NK cells showed higher cytotoxicity than untransduced control NK cells (Figs. 3G and 3H), indicating that EGFR-CAR is functional. There was no difference in cytotoxicity between MOIs 2 and 5 for EGFR-CAR NK cells. Donor 1 CAR NK cells showed high cytotoxicity on day 7, but activity decreased significantly on day 14, possibly due to reduced CAR expression. In contrast, the cytotoxicity of Donor 2 CAR NK cells remained relatively consistent on days 7 and 14, indicating donor-specific differences in CAR-mediated cytotoxicity.

[0090] [Example] [7] [] [BaEVRless] [-] [LV] [The robust packaging enabled it to be able to be used in the first generation] [NK] [Occurs within the cell] [CRISPR] [] [KO] [filter] []

[0091] We expanded our research to include CRISPR KO screening in primary NK cells to validate the effectiveness of our BaEVRless-LV synthesis platform (Figure 4A). We adapted a SLICE editing protocol developed for human primary T cells and reused it to screen for kinase genes crucial for primary NK cell proliferation. We selected the Brunello CRISPR kinosomal library, which contains 6194 sgRNAs targeting 763 kinase genes in the human genome. The kinosomal sgRNA library was encapsulated in BaEVRless-LV using our optimized platform. To maintain 1000-fold sgRNA coverage of the kinosomal library, we transduced 2 × 10⁷ NK cells with BaEVRless-LV at an MOI of 0.3 and electroporated the cells with Cas9 protein one day later. The confluent KO population was selected by puromycin treatment and then cultured for 10 days. Subsequently, we amplified sgRNA cartridges in a surviving NK cell population for next-generation sequencing (NGS) to identify genes essential for NK cell proliferation.

[0092] In two independent experiments using two donor cells, the encapsulation and transduction of the kinosomal sgRNA library were highly efficient. Following BaEVRless-LV transduction and puromycin selection, we analyzed NK cells and detected almost all sgRNAs from the kinosomal library in both donor cells (Fig. 4B). The depletion of sgRNAs targeting essential genes was the expected result of successful CRISPR KO screening. Indeed, we observed depletion of sgRNAs targeting several important cell cycle kinase genes, such as CDK6, TRRAP, and TP53RK (a negative regulator of apoptosis in response to mitotic stress) (Figs. 4C and 4D). We also observed depletion of JAK3 (a downstream kinase in the IL-2 signaling pathway essential for NK cell survival), indicating successful screening.

[0093] TW202540399A_113143869_SEQL.xml

Claims

1. A modified cell line in which the IQGAP-1 and ASCT2 genes are knocked out.

2. The modified cell line of request item 1, wherein the cell line is a virus production cell line.

3. The modified cell line of request 1, wherein the cell line is a human fetal kidney 293 cell line (HEK293T cell line), an insect cell line, a TE 671 cell line, or an HT1080 cell line.

4. The modified cell line of claim 1, which has been transfected with plastids to produce retroviral particles containing BaEV protein.

5. The modified cell line of claim 4, wherein the retroviral particle is a lentiviral particle, a γ-retroviral particle, or an α-retroviral particle, or a combination thereof.

6. The modified cell line of claim 4, wherein the retroviral particle is a murine leukosis virus (MLV) particle, an avian leukosis virus (ALV) particle, a respiratory fusion virus (RSV) particle, a Mason-Pfizer monkey virus (MPMV) particle, a lentivirus particle, or a spumavirus particle, or a combination thereof.

7. The modified cell line of claim 6, wherein the lentiviral particle is a human immunodeficiency virus (HIV) particle, a simian immunodeficiency virus (SIV) particle, a feline immunodeficiency virus (FIV) particle, an equine infectious anemia virus (EIAV) particle, or a caprine arthritis encephalitis virus (CAEV) particle, or a combination thereof.

8. The modified cell line of claim 6, wherein the foam virus particles are human foamy virus (HFV) particles.

9. A modified cell line as claimed in any of claims 4 to 8, wherein the BaEV protein is a full-length BaEV protein (BaWT) or a BaEV protein lacking the terminal R peptide (BaEVRless).

10. The modified cell line of request 1, wherein the ASCT1 gene is further removed.

11. A method for encapsulating high-valence retroviral particles without forming syncytia, comprising knocking out the IQGAP-1 and ASCT2 genes of cells to obtain modified cells, and transfecting the modified cells with plastids for generating retroviral particles containing BaEV protein.

12. The method of claim 11, wherein the cell is a production cell line.

13. The method of claim 11, wherein the cell is a human fetal kidney 293 cell line (HEK293T cell line), an insect cell line, a TE 671 cell line, or an HT1080 cell line.

14. The method of claim 11, wherein the retroviral particle is a murine leukosis virus (MLV) particle, an avian leukosis virus (ALV) particle, a respiratory syncytial virus (RSV) particle, a Mason-Fischer monkey virus (MPMV) particle, a lentivirus particle, or a foam virus particle, or a combination thereof.

15. The method of claim 14, wherein the lentiviral particle is a human immunodeficiency virus (HIV) particle, a simian immunodeficiency virus (SIV) particle, a feline immunodeficiency virus (FIV) particle, an equine infectious anemia virus (EIAV) particle, or a caprine arthritis encephalitis virus (CAEV) particle.

16. The method of claim 14, wherein the foam virus particle is a human foam virus (HFV) particle.

17. The method of any one of claims 11 to 16, wherein the BaEV protein is a full-length BaEV protein (BaWT) or a BaEV protein lacking the terminal R peptide (BaEVRless).

18. The method of claim 11, wherein the ASCT1 gene of the cell is further knocked out.

Images