WO2023083224A1

WO2023083224A1 - The construction of a new virus vector packaging cell line of high productivity

Info

Publication number: WO2023083224A1
Application number: PCT/CN2022/130947
Authority: WO
Inventors: Shumin Zhou
Original assignee: Shanghai Sixth People's Hospital
Priority date: 2021-11-09
Filing date: 2022-11-09
Publication date: 2023-05-19

Abstract

Provided are a host cell that is modified to over-express FNDC5 or irisin and a method of preparing a viral vector comprising: 1) modifying a host cell to over-express FNDC5 or irisin; and 2) co-transfecting the host cell with a transfer plasmid and packaging plasmids. The host cell and the method provided herein can be used to prepare lentiviral vectors on a large scale.

Description

THE CONSTRUCTION OF A NEW VIRUS VECTOR PACKAGING CELL LINE OF HIGH PRODUCTIVITY

CROSS-REFERENCE TO RELATED APPLICATION

This application claims priority form PCT patent application PCT/CN2021/129604, filed Nov. 9, 2021, the content of which is incorporated herein by reference in its entirety

DEPOSIT INFORMATION

Under the terms of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purpose of Patent Procedure, the KX293T-3 cell strain was deposited on Oct. 20, 2021, with China Center for Type Culture Collection (CCTCC, Wuhan University, Wuhan, China) and was assigned deposit number CCTCC NO: C2021292.

SEQUENCE LISTING

This application includes a Sequence Listing submitted electronically as an xml file named “P11304-PCT. xml” , created on Nov. 9, 2022 with a size of about 8 kb. The Sequence Listing is incorporated by reference herein.

BACKGROUND

Lentivirus vectors have been used extensively for cellular therapy and gene therapy (PMID: 9305836) . The advantage of lentivirus vectors is that they can transduce wide range of cell types, both dividing cells, and silent cells as well. The lentivirus vector system now in use is developed from human immunodeficiency virus (HIV) , which makes it an ideal vehicle to transfer foreign genes into T cells which is the natural host of lentivirus and usually hard to be transfected. For this reason, lentivirus vector is the dominant tool for making CAR-T cells (PMID: 27189167) , a powerful immunotherapy against tumor.

CAR-T cells (Chimeric antigen receptor T cells) therapy is now a super-star within the tumor-related therapies (PMID: 33292660) . Generally speaking, CAR-T cells are T cells genetically engineered to produce an artificial chimeric antigen receptor (CAR) which combine both antigen-binding and T cell activating functions in a single molecule (PMID: 3122749) . These CARs make T cells gain new ability to bind a target protein (usually tumor-specific antigen) and is quite customized for immunotherapy (PMID: 23550147) . As the key step to make CAR-T cells, the engineered CARs should be transfected into T cells using a lentiviral (LV) vector (PMID: 27189167) . For the large number of T cells (～10 ^11-12) and high MOI (～5-10) used in the production of CAR T cells, massive amounts of lentiviral vectors (～10 ¹³) should be prepared just for a single patient treatment.

The lentivirus vectors, now are generated in packaging cells by transient co-transfection of three or four plasmids. The three-plasmids system including: (i) the packaging construct plasmid encoding Gag-Pol, Tat, Nef, Vpr, Vpu, Vif proteins, and HIV long terminal repeats (LTRs) , packaging signal, Rev response element; (ii) a plasmid encoding vesicular stomatitis virus G (VSV-G) protein; and (iii) a foreign promoter driving the transgene (PMID: 9294208, PMID: 8876144, PMID: 8602510) . For the four-plasmids system, the packaging signal and Rev response element is spun off from the packaging construct plasmid. In most conditions, recombinant lentiviruses could be generated from the titers of 10 ⁵ TU/ml to 10 ⁶ TU/ml according to the length of insert genes when packaged in 293T cells in a 10cm dish. It also could be concentrated by centrifugation to get higher titers (PMID: 9354796, PMID: 9733856) . Although the workflow of large-scale production of lentivirus is very mature now, it is still quite expensive to obtain a large number of lentiviral vectors, especially when applied to the scale like CAR-T therapy.

As mentioned above, companies usually need 200L stirred-tank bioreactors to produce lentivirus vectors in order to prepare enough dose for only few patients per batch. This situation makes the price of CAR-T therapy now available too high for ordinary patients to afford (https: //www. pharmacytimes. com/view/medically-integrated-pharmacies-oral-oncolytic-agents-grow-in-importance-during-covid-19) . Therefore, it is an urgent task to establish a more efficient lentivirus production platform to produce enough lentivirus vectors in smaller bioreactors, which will reduce the cost of CAR T treatment finally. And there’s no doubt that the packaging cell line has the top priority.

SUMMARY

In one aspect, the present disclosure provides a host cell that is modified to over-express FNDC5 or irisin.

In some embodiments, the modification comprises introducing into the host cell an exogenous nucleic acid molecule encoding FNDC5 or irisin.

In some embodiments, the expression level of FNDC5 or irisin in the host cell is higher than the expression level of FNDC5 or irisin in the host cell before the modification, preferably by at least 5 times.

In some embodiments, the host cell is a mammalian cell, preferably a human cell, more preferably a 293T cell.

In some embodiments, the exogenous nucleic acid molecule is a DNA or RNA molecule.

In some embodiments, the FNDC5 comprises an amino acid sequence shown in SEQ ID NO: 1 or a functional variant thereof; the irisin comprises an amino acid sequence shown in SEQ ID NO: 2 or a functional variant thereof.

In some embodiments, the host cell is a human embryonic kidney cell KX293T with a deposit number of CCTCC NO: C2021292.

In another aspect, the present disclosure provides use of the host cell as a viral vector packaging cell line.

In some embodiments, the viral vector packaging cell line is a lentiviral vector packaging cell line.

In another aspect, the present disclosure provides a method of preparing a viral vector comprising:

1) modifying a host cell to over-express FNDC5 or irisin; and

2) co-transfecting the host cell with a transfer plasmid and packaging plasmids.

In some embodiments, the expression level of FNDC5 or irisin in the host cell is higher than the expression level of FNDC5 or irisin in the host cell before the modification by at least 5 times.

In another aspect, the present disclosure provides a method of preparing a viral vector comprising co-transfecting the host cell with a transfer plasmid and packaging plasmids, wherein the transfer plasmid comprises a sequence encoding FNDC5 or irisin.

1) co-transfecting a host cell with a transfer plasmid and packaging plasmids in a medium; and

2) adding to the medium FNDC5 or irisin prior to, concurrent with, or after step 1) .

In some embodiments, the FNDC5 comprises the amino acid sequence shown in SEQ ID NO: 1 or a functional variant thereof; the irisin comprises the amino acid sequence shown in SEQ ID NO: 2 or a functional variant thereof.

In another aspect, the present disclosure provides a method of preparing a viral vector comprising co-transfecting a host cell with a transfer plasmid and packaging plasmids, wherein the host cell is a human embryonic kidney cell KX293T with a deposit number of CCTCC NO: C2021292.

In some embodiments, the viral vector is a lentiviral vector.

In some embodiments, the transfer plasmid comprises a sequence encoding a chimeric antigen receptor (CAR) .

In another aspect, the present disclosure provides a method of a method of preparing a CAR-T cell comprising:

1) preparing a viral vector by any one of the methods described above; and

2) transfecting a T cell with the viral vector.

In another aspect, the present disclosure provides a method of preparing a protein with a host cell comprising:

1) modifying the host cell to over-express FNDC5 or irisin;

2) modifying the host cell to express the protein; and

3) optionally, isolating or purifying the protein from the host cell.

In some embodiments, the modification in step 1) comprises introducing into the host cell a first exogenous nucleic acid molecule encoding FNDC5 or irisin, and the modification in step 2) comprises introducing into the host cell a second exogenous nucleic acid molecule encoding the protein.

In some embodiments, the protein is an enzyme, such as FUT8, or a cytokine, such as VEGF.

In another aspect, the present disclosure provides a method of preparing exosomes with a host cell comprising:

1) modifying the host cell to over-express FNDC5 or irisin;

2) culturing the host cell and optionally, isolating or purifying the exosomes from the host cell.

In some embodiments, the modification in step 1) comprises introducing into the host cell an exogenous nucleic acid molecule encoding FNDC5 or irisin.

The host cell and the method provided herein can be used to prepare lentiviral vectors, recombinant proteins or exosomes on a large scale.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1. The construction and expression of pLVX-EGFP-IRES-puro-irisin. A. The CDS with restriction enzyme sites of human irisin was cloned from the total RNA of 293T cells and inserted into the according site in the MCS of the pLVX-EGFP-IRES--puro empty vector. B. The qPCR and western-blot assays were employed to detect the irisin expression at mRNA and protein levels in 293T cells transiently transfected with pLVX-EGFP-IRES-puro-irisin plasmid.

Figure 2. The workflow of the construction of 293T-irisin. The pLVX-EGFP-IRES-puro-irisin plasmid was co-transfected into 293T cells with the packaging plasmids. The lentivirus vectors containing human irisin was harvested 48 hours posttransduction. After concentrated and titrated, the lentivirus particles were added into prepared 293T cells at the MOI=5.0. The cells were selectively cultured in the medium containing 1ug/ml puromycin 48 hours after infection. The positive cells remained after a 7 days selective culturing.

Figure 3. The characterization of 293T-irisin and KX293T cells. A. The fluorescent and bright field pictures of 293T-irisin and KX293T cells. B, C. The PCR and western-blot assays were employed to detect the irisin expression at mRNA and protein levels in 293T-irisin and KX293T cells. D. The titration assays of lentivirus particles were performed using the supernatant of 293T-irisin and 293T cells at 48 hours posttransduction. The mean value of three independent assays were calculated and the comparation of the titers of lentivirus from 293T-irisin and 293T cells at the same harvest time points were also performed.

Figure 4. The analysis of the KX293T series cell lines. A, and B. The PCR and western-blot assays were employed to detect the irisin expression at mRNA and protein levels in 293T-irisin and KX293T-1/2/3 cells. C. The fluorescent and bright field pictures of 293T-irisin and KX293T-1/2/3 cells. D. The titration assays of lentivirus particles were performed using the supernatant of KX293T-1/2/3 and 293T cells at 48 hours posttransduction. The mean value of three independent assays were calculated and the comparation of the titers of lentivirus from KX293T-1/2/3 and 293T cells were also performed.

Figure 5. The characterization of the KX293T cell line. A. The lentivirus particles were harvested from the supernatant of KX293T and

293T cells

48, 72 and 96 hours posttransduction. Then the lentivirus samples were titrated as described in material and method 1.7. The mean value of three independent assays were calculated and the comparation of the titers of lentivirus from KX293T and 293T cells at the same harvest time points were also performed. B. The representative picture of the titration of the lentivirus particle harvested 48 posttransduction. C. The concentration and diameter of the extracellular particles in the of lentivirus samples of interest were analyzed using a high-resolution nanoflow cytometer as described in material and method 1.10. The total events of the diameter ranged from 80 to 120nm were calculated.

Figure 6. Characterization of the lentivirus particles produced by KX293T and 293T cells. High-dividing and low/non-dividing cells were employed to perform the transfection assays. The representative fluorescent pictures of a high-dividing cell (HOS/MNNG) and low/non-dividing cells (HACC) 48 hours post-infection with lentivirus produced by KX293T and 293T cells at a same MOI (MOI=10) .

Figure 7. Analysis of foreign proteins expression in KX293T and 293T cells. Plasmids containing cDNA of FUT8 (A) and VEGF165 (B) were transiently transfected into KX293T and 293T cells. After 48 hours, the cells were harvested and homogenized, and target foreign proteins were detected by Western-Blot.

Figure 8. Determination of exosomes produced by KX293T and 293T cells. About 3 X 10 ⁶ KX293T and 293T cells were seeded in 10cm dishes. The supernatants were harvested 24h, and 48h post seeding. Prepared samples by ultracentrifuge containing exosomes were measured by nanoflow (A) , and the calculation and normalization were performed (B) .

DETAILED DESCRIPTION

Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by a person of ordinary skill in the art. Any methods, devices and materials similar or equivalent to those described herein can be used in the practice of the present invention. The following definitions are provided to facilitate understanding of certain terms used herein and are not meant to limit the scope of the present disclosure.

The articles “a” and “an” are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, “a cell" means one cell or more than one cell.

A “host cell, ” as used herein, refers to an in vivo or in vitro eukaryotic cell, or a cell from a multicellular organism (e.g., a cell line) cultured as a unicellular entity, which can be, or have been, used as recipients for a nucleic acid (e.g., an exogenous nucleic acid) , and include the progeny of the original cell which has been genetically modified by the nucleic acid.

“Modification” or “genetic modification” of a host cell, as used herein, refers to a purposeful modification of the genome of the host cell or the introduction of an exogenous nucleic acid into the host cell so as to express a gene of interest or increase its expression level.

A host cell “over-expressing” a peptide (e.g., irisin) , as used herein, refers to a host cell that expresses the peptide at a level higher than the baseline level. For example, after modification, the peptide concentration detected in the host cell is at least 5 times higher than the baseline concentration (i.e., the peptide concentration in the host cell before the modification) . In some cases, the expression level of the peptide can be determined indirectly by detecting the amount or concentration of mRNA encoding the polypeptide in the host cell. There are some ways to achieve this modification, for example, by introducing an exogenous gene encoding the peptide into the host cell, replacing the promoter of the gene encoding the peptide in the host cell with a strong promoter, or increasing copy number of the gene encoding the peptide (e.g., gene doubling) .

An "exogenous nucleic acid molecule" refers to a nucleic acid molecule that is independent of the inherent nucleic acids of the host cell. Its sequence is usually different from or not included in the host genome, but in some cases, for example, for the purpose of increasing the expression of a certain protein or peptide of the host cell, it can also be a part of the host genome or encoding the same protein or peptide. An exogenous nucleic acid molecule can also comprise operably linked nucleotide sequences such as a promoter.

A "vector" , as used herein, refers to a nucleic acid molecule capable of transporting a foreign nucleic acid molecule into a host cell. The foreign nucleic acid molecule is linked to the vector nucleic acid molecule by a recombinant technique, such as ligation or recombination. This allows the foreign nucleic acid molecule to be multiplied, selected, further manipulated or expressed in a host cell or organism. A vector can be a plasmid, phage, transposon, cosmid, chromosome, or virus. Vectors capable of directing the expression of expressible foreign nucleic acids to which they are operatively linked are commonly referred to "expression vectors. "

A "viral vector" , as used herein, refers to a recombinant viral vector particle that is able to accomplish transformation of a target host cell with a nucleotide sequence of interest, such as an adenovirus (ADV) vector, an adeno-associated virus (AAV) vector or a retroviral vector. Preferably, the viral vector is a retroviral vector, such as a lentiviral vector. A retroviral vector may be composed of a RNA, Pol protein (for reverse transcription of the RNA following infection) , Gag protein (structural protein present in the nucleocapsid) , and an envelope protein. The RNA of the retroviral vector is usually a recombinant RNA genome, e.g., contains an RNA sequence exogenous to the native retroviral genome and/or is defective in an endogenous retroviral sequence (e.g., is defective in pol, gag, and/or env, and is normally defective in all three genes) .

"Lentiviral vector" is a viral vector (e.g., in the form of a virus particle) derived from lentivirus, such as HIV-1. It is able to efficiently introduce target genes into primary cells or cell lines of animals or humans. Lentiviral vector-mediated gene expression is continuous and stable, as the inserted gene might integrate into the host cell genome and divides with the division of the cell genome. In addition, lentivirus has a wide range of hosts, including dividing and non-dividing cells. It is especially suitable for host cells with low plasmid vector transfection efficiency.

A "packaging cell line" , as used herein, refers to a cell line such as HEK 293 that is able to produce viral vectors (e.g., retroviral vectors, such as lentiviral vectors) . The packaging cell line may produce the viral vectors transiently or stably, depending on the kind of preparation of the cell line. Preferentially, the packaging cell line is a human packaging cell line. If the packaging cell line stably produces (generates) the viral vector particles, it may also be called as a "producer cell line" .

A "lentiviral vector packaging cell line" refers to a cell line used for the production of lentiviral vectors. In the process of preparing lentiviral vectors, packaging cell line such as 293T is co-transfected with a transfer plasmid (carrying the target gene) and one or more (2, 3, or 4) lentiviral packaging plasmids, and lentiviral vectors are subsequently separated from the cell culture supernatant by centrifugation or other means.

A "transfer plasmid" , as used herein, refers to a plasmid carrying a sequence of interest (e.g., CAR coding sequence) flanked by long terminal repeat (LTR) sequences, which facilitates integration of the transfer plasmid sequences into the host genome. Typically, it is the sequences between and including the LTRs that is integrated into the host genome upon viral transduction. For safety reasons, transfer plasmids are all replication incompetent and may contain an additional deletion in the 3'LTR, rendering the virus “self-inactivating” (SIN) after integration.

"Packing plasmids" , as used herein, refers to one or more plasmids that encode auxiliary proteins such as Rev, Gag and Pol that are needed to transcribe and package RNA into the recombinant viral vectors. As used herein, packaging plasmids may also include envelope plasmid, such as VSV-G-encoding plasmid.

"Functional variant" of a peptide (e.g., irisin) , as used herein, refers to a peptide or protein that differs from a reference/parental peptide (e.g., a wild-type peptide) by substitutions (such as, conservative amino acid substitutions) , deletions, and/or insertions at small number of amino acid residues while basically maintaining the original function of the reference/parental peptide (e.g., activity of irisin in enhancing the production of lentivirus vector in 293T cells) . The number of different amino acid residues between the variant and parental protein may be one or more, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, or more amino acid residues. For example, a functional variant of irisin may share at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or even at least about 99%, or more, amino acid sequence identity with the amino acid sequence of irisin. As used herein, "sequence identity" between two polypeptide sequences indicates the percentage of amino acids that are identical between the sequences. The amino acid sequence identity of polypeptides can be determined conventionally using known computer programs such as Bestfit, FASTA, or BLAST.

The term “chimeric antigen receptor (CAR) ” refers to a fusion protein which, in their usual format, graft the specificity of an antibody or an antigen-binding fragment thereof to the effector function of a T cell. Once the CARs are expressed in a T cell, the CARs modified T cell (i.e., CAR-T cell) acquires some properties, such as antigen specific recognition and antitumor reactivity. CARs are expressed as transmembrane proteins, including at least an antigen-specific binding domain, a transmembrane region, and a signaling cytoplasmic domain (e.g., a CD3ζ chain) . The antigen-specific binding site is usually a monoclonal antibody-derived single chain variable fragment (scFv) consisting of a heavy chain variable region and a light chain variable region joined by a flexible linker.

We accidentally discovered that 293T cells over-expressing human irisin could produce lentivirus vectors of higher titer compared to those in original 293T in the same conditions. Thus, we constructed a 293T cell line stably expressing human irisin proteins, and cherrypicked a monoclonal cell line of high productivity, which was termed as KX293T. The titer of the lentivirus vectors produced in KX293T was 5.22-fold higher than those form 293T cells. In addition, we also tested the abilities of producing recombined proteins and exosomes of KX293T. As a result, it was showed that the ability of KX293T cells to express recombined enzyme (FUT8) and cytokine (VEGF) was 5.48 and 3.81 times higher than the original 293T cells, respectively. Last but not the least, the ability of KX293T cells to produce exosomes was also 1.97-fold higher than that of original 293T cells (8.7 x 10 ¹⁰ vs 4.4 x 10 ¹⁰) . We believe this KX293T packaging cell line will facilitate the production of CAR-T cells, recombined proteins and exosomes, which benefit tumor patients in the future.

Example

1. Material and methods

1.1 Plasmid construction.

In order to construct a lentivirus vector that overexpressing human irisin peptide. The nucleotide sequence of the CDS of human FNDC5/irisin variant1 mRNA (NM_001171941.3) from 1-429 was amplified by PCR using the cDNA of HEK293T cells (short for 293T in the follow manuscript) . The amplicon with Xbal I and BamH I restriction endonuclease site in its 5’ and 3’ terminal, respectively. Then the amplicon was digested by Xbal I and BamH I, followed by inserted into the according site of a pre-digested lentivirus vector pLVX-EGFP-IRES-puro (Addgene, #128652) . Thus, the irisin-overexpressing lentivirus vector pLVX-EGFP -IRES-puro-irisin was successfully constructed and ready for transfection.

1.2 Cell culturing.

The human HEK293T cells were acquired from American Type Culture Collection (ATCC) . All cells used in this patent were cultured in Dulbecco’s modified Eagle’s medium (Merck, D6429) containing 10%fetal bovine serum (Gibco, 10099141C) . The cells were passaged when the confluence reached 80-90%.

1.3 Transient transfection.

In order to analyze whether the lentivirus vector pLVX-EGFP-IRES-puro-irisin could successfully expressed the target irisin peptide, transient transfection was performed on 293T cells. Generally, 1*10 ⁶ 293T cell line were placed in a well of 6-well plate before the day of transfection. When the cell confluence reached 80-90%, 4ug pLVX-EGFP-IRES-puro-irisin plasmid was mixed with 6ul Lipofectamine 3000 reagent (ThermoFisher, L3000015) and added onto the 293T cells. The culturing medium was changed on the next day to minimize the cytotoxicity of transfection reagents. The qPCR and western-blot analysis were performed 36 and 48 hours psottransduction, respectively.

1.4 RT-qPCR analysis.

For RNA analysis, the total RNA of about 1*10 ⁶ cells of detected were isolated using a total RNA isolation kit I (Omega, R6834-01) . Then 1ug total RNA was reverse-transcripted using a cDNA synthesis kit (ThermoFisher, K1612) . Quantitative real-time PCR (RT-qPCR) assays were carried out on QuantStudio ^TM 7 Flex Real-Time PCR System (ThermoFisher, 4485701) with FastStart Universal Probe Master (Merck, 4913949001) . The 2-ΔΔCt approach was used to calculate the fold change of the irisin mRNA expression in target cells and beta-actin worked as an internal control. All primers were shown in Table. 1.

Table 1 primer sequences used for RT-PCR analysis

1.5 Western blotting analysis.

To analyze the protein levels of human irisin in target cells. Cells were lysed using RIPA cell lysis buffer (ThermoFisher, 89900) with cocktail protease inhibitor (Roche, 11697498001) for 20 min on ice, and proteins were denatured by boiling for 10 min in a SDS loading buffer (ThermoFisher, AM8546G) . Twenty micrograms of the denatured proteins were separated on a 12.5%polyacrylamide gel containing SDS and blotted onto a 0.22um PVDF membrane (Merck, 3010040001) . After blocking with 5%nonfat milk in PBS–0.2%Tween 20 for 60 min, the membranes were incubated with rabbit polyclonal antibody anti-irisin (ThermoFisher, PA5-81569) or with mouse monoclonal antibody anti-beta actin (ThermoFisher, 8H10D10) and then with goat anti-mouse immunoglobulin horseradish peroxidase (ThermoFisher, #G-21040) or donkey anti-rabbit horseradish peroxidase (ThermoFisher, #SA1-200) , respectively. The protein bands were detected in a ChemiDoc Gel Imaging System (Bio-Rad, 1708370) with an ECL substrate kit (ThermoFisher, 32106) .

1.6 Pseudovirus production.

To generate the pseudovirus particles, we followed the procedure of the handbook of LentiSuite Basic Kit (SBI, Cat#LV340A-1) . On Day 1, about 3 x 10 ⁶ 293T cells were placed in a fresh 100mm plate in 10 mL of antibiotic-free DMEM medium (DMEM+10%FBS+Glu) and cultured overnight. On Day 2, the cells should be 70%to 85%confluent at day of transfection. Then, add 0.6 mL of serum-free DMEM media into a 1.5 mL Eppendorf tube, followed by adding 2 μg of shuttle plasmid and 20 μL pPACKH1-plasmid mix into the tube and mix by pipetting gently. Meanwhile, adding 40 μL of Lipofectamine 3000 reagent (ThermoFisher, L3000015) into another 1.5 mL tube containing 0.6 mL of serum-free DMEM media, and pipetting gently. Mixing the liquids in these two tubes by drop-wise and let the mixture sit at room temperature for 20 min. Adding the mixture drop-wise to the cell culturing plate, and swirl to disperse evenly throughout, and incubate the plates in a 37℃, 5%CO ₂ incubator. On Day 4, the supernatant containing pseudovirus was collected into a 50-mL sterile, capped conical centrifuge tubes after 48 hours past transfection, followed by centrifuged at 400 x g for 5 minutes at room temperature to eliminate suspended cells. Transferring the viral supernatant into new fresh tubes and centrifuged at 3000 x g for 10 minutes to pellet cell debris. The supernatant was then passed through a 0.22 um filter to sterilize. Add PEG-it at a final volume of 1: 5. Example: 2 mL of PEG-it should be added to 8 mL of viral supernatant, invert 10 times to mix well. Keep everything cold from this point onwards. Store virus supernatant containing PEG-it at 4℃ overnight. On Day 5, harvest PEG-it precipitated virus by centrifuging at 4℃ at 1500 x g for 30 min. Aspirate off the supernatant and resuspend the milky-white pellet in a small volume (1/100 to 1/1000 of original volume) using cold sterile PBS or cold DMEM. 2. Freeze virus aliquots at -80℃.

1.7 Titer determination.

To evaluate the titer of the pseudovirus particle produced, 293T cells were placed in a 96 well plate at the density of 1*10 ⁵ cell/well. The pseudovirus suspension were serial diluted from 10 ^-1 ～ 10 ^-10 and added onto 293T cells, respectively. Titers were scored 48 h posttransduction by dividing the number of GFP-positive foci by the dilution factor using an inverted fluorescence microscope (Leica, DMi8) .

1.8 Stable cell line establishment.

To generate the 293T cells stably expressing human irisin protein. About 1 x 10 ⁶ 293T cells were firstly placed in a 6-well plate in 1mL medium and cultured overnight. Then, the media were replaced with fresh ones and pseudovirus concentrates were added into the medium at the MOI=5.0. The cells were then cultured in a selective medium containing 1ug/ml puromycin 48 hours posttransduction. Replacing the selective culturing medium every 2-3 days for 7-10 days until all the surviving cells expressing GFP. We termed this cell line as 293T-irisin.

1.9 Monoclone cell line establishment.

In order to get a more homogeneous cell line, two round limiting dilutions were performed using 96-well plates. Generally, 293T-irisin cells were suspended using 0.25%trypsin and diluted to a density of 5-10 cells/ml. Mixing the cell suspension evenly, and fill each well of one 96 well plate with 100 uL medium containing cells. Check the plate the next day and eliminate the wells containing more than one cells. For the cells of high virus productivities are usually of high proliferation abilities, top 5 sub-cell lines of highest proliferation were selected in the first-round dilution. Repeat this procedure again. Finally, the sub-cell line with highest proliferation were obtained and termed as KX293T.

1.10 Lentivirus production efficiency determination.

A high-resolution nanoflow cytometer was employed to measure the concentration and diameter of the extracellular particles of interest as previously described (PMID: 29300458) . Initially, standard polystyrene nanoparticles (diameter: 200 nm, concentration: 1.58 x 10 ⁸/mL) were loaded to the nanoflow cytometer to measure the standard side scatter intensity (SSI) . Next, the SSI of the lentivirus vectors sample was evaluated by the nanoflow cytometer. Finally, the particle concentration of extracellular particles of interest was calculated accordingly. For size distribution, a standard curve was created using four different sizes of standard silica nanoparticles (diameter: 68, 91, 113, 155 nm) in the high-resolution nanoflow cytometer. Next, the diluted samples containing lentivirus vectors was loaded and the size distribution was generated by the standard cure.

1.11 Exosomes production efficiency determination.

For particle concentration detection, a standard nanoparticle with a diameter of 200 nm and a concentration of 1.58×10 ⁸/mL was used for quantification. Next, the samples containing exosomes were measured by nanoflow (NanoFCM Inc) . Finally, particle concentration of samples was calculated via the recorded particle number of the samples and the standard nanoparticles.

2. RESULTS

2.1 Recombined human irisin expression.

In order to elevate the expression level of human irisin in 293T cells, an over-expression lentivirus vector containing the CDS of human irisin was constructed as described in material and method 1.1 (Figure 1A. ) . Then, a transient transfection using the constructed plasmid: pLVX-EGFP-IRES-puro-irisin and according empty vector (pLVX-EGFP-IRES-puro) was performed in 293T cell line to analyze whether the lentivirus vector we constructed could over-express human irisin peptide. To validate the expression of human irisin in its mRNA and protein levels, the total mRNA and protein were extracted 36 and 48 hours posttransdoction, respectively. As a result, the mRNA levels and protein level in the 293T cells transfected with pLVX-EGFP-IRES-puro-irisin is 16967.25 and 12.38 folds higher than those in the cells with empty vector (Figure 1B. ) . Thus, we concluded that the constructed lentivirus vector successfully expressed recombined human irisin protein and ready for pseudovirus production.

2.2 293T-irisin stable cell line.

Encouraged by the results mentioned above, we embarked on the establishment of a stable 293T cell line that over-expressing recombined human irisin using lentivirus. To generate a pseudotyped lentivirus containing human irisin mRNA, we transiently co-transfecting 293T cells with the recombined plasmid pLVX-EGFP-IRES-puro-irisin, and the packaging plasmids that were previously described (PMID: 8876144) . The cell supernatant containing lentivirus particle was collected, followed by concentrated by centrifugation. After titrated, the pseudovirus particle was added into pre-placed 293T cells at the MOI=5. Since the pLVX-EGFP-IRES-puro-irisin packaging construct that also expresses the puromycin resistance gene, the 293T cells infected with pseudovirus particle were selectively cultured in the selective medium for 7 days as described above. Then the cells remained were supposed to be the positive ones that successfully transduced with lentivirus which over-expressing human irisin, which was termed as 293T-irisin (Figure 2. ) .

To further confirm the expression of irisin in the stable cell lines constructed, the 293T-irisin cells were also firstly check under an inverted fluorescence microscope (Figure 3A. ) and then performed the RT-qPCR and Western-Blotting analysis to check the expression of human irisin at both mRNA and protein levels (Figure 3B and C. ) . As a results, at least 86.7%of the 293T cells that transfected turned green. The mRNA and protein levels of irisin were 667.83 and 5.66 folds higher than those in the original 293T cells.

To analyze whether the overexpressing of irisin in 293T cell did increase the titer of lentivirus vectors, a lentivirus shuttle plasmid containing GFP only (pCDH-CMV-MCS-EF1-copGFP-T2A-Puro, SBI) were employed and co-transfected with packing plasmids into 293T-irisin and 293T cells at a same condition, simultaneously as described in material and methods 1.6. After 48 hours posttransfection, the supernatant of 293T-irisin and 293T cells were collected, followed by titration as described in material and methods 1.7. As shown in Figure 3D., the titer of the lentivirus producing form 293T-irisin cell is 4.80 X 10 ⁶ TU/ml, whereas those form 293T cell were 2.15 X 10 ⁶ TU/ml. Thus, the titers of lentivirus vectors from 293T-irisin were as 2.25 folds higher as those form 293T cell. This conformed that irisin-overexpression up-regulates the lentivirus vector production.

2.3 KX293T cell line.

In order to get a purer and more stable packaging cell lines over-expressing irisin, we started to make a monoclonal cell line by two round limiting dilutions. In each round limiting dilution, the top 5 fastest growth clones were picked. The final 25 subclones of high growth rates were got in the second round and the top 3 sub-cell lines of highest growth rate was cherrypicked and named as KX293T-1, KX293T-2 and KX293T-3. Then these KX293T cells was performed qPCR and Western-blot analysis to check the expression of human irisin at both mRNA and protein levels (Figure 4A and B. ) . As a results, the mRNA expression levels of irisin in KX293T-1, KX293T-2 and KX293T-3 cells were 39281.55, 23668.61 and 69663.98 folds higher than those in the 293T-irisin cells, respectively. Accordingly, the protein levels of irisin in KX293T-1, KX293T-2 and KX293T-3 cells were 23.53, 17.88 and 36.34 folds higher than those in the 293T-irisin cells. This result indicated that the constructed monocloned sub-cell line expressed irisin in higher levels than that in 293T-irisin cell in both mRNA and protein levels.

To compare the titration of lentivirus vectors from the KX293T-1/2/3 packaging cell line with those from original 293T cell line, the same shuttle plasmid (pCDH-CMV-MCS-EF1-copGFP-T2A-Puro, SBI) transfected in 293T-irisin were employed and performed titration as described in material and methods 1.6 and 1.7. As shown in Figure 4D., the titer of the lentivirus producing form KX293T-1/2/3 cell is 5.87 X 10 ⁶, 4.83 X 10 ⁶ and 1.18 X 10 ⁶ TU/ml, respectively. Thus, the titers of lentivirus vectors from KX293T-1/2/3 cell were as 2.75, 2.26 and 5.625 folds higher as those form 293T cell, respectively. Thus, we picked the KX293T-3 sub-cell lines as the representative of KX293T cell series and termed it as KX293T.

In order to analyze whether the KX293T cell could continually produce lentivirus vector at a high level, a long-time packaging assays were performed from 48 to 96 hours posttransduction. As shown in Figure 5A., the titer of the lentivirus producing form KX293T cell is 1.16 X 10 ⁷ TU/ml, 5.83 X 10 ⁶ TU/ml and 3.17 X 10 ⁶ TU/ml, whereas those form 293T cell were 2.23 X 10 ⁶ TU/ml, 1.80 X 10 ⁶ TU/ml and 1.63 X 10 ⁶ TU/ml at the

timepoint

48, 72 and 96 posttransfection. Thus, the titers of lentivirus vectors from KX293T were as 5.22, 3.24 and 1.94 folds high as those form 293T cell. Representative pictures of the titration of the lentivirus particles harvested 48 posttransduction were showed in Figure 5B.

We have deposited the KX293T-3 cell strain, and the deposit information is as follows:

applicant: Shanghai Jiaotong University affiliated sixth people’s hospital;

date of deposit: October 20, 2021;

name of the culture: Stablely transfected human embryonic kidney cells KX293T;

deposit number: CCTCC NO: C2021292.

2.4 Background analysis.

Furthermore, to analyze whether the lentivirus of high titer produced by KX293T cell will lead to high background which may increase the burden of downstream purification, a high-resolution nanoflow cytometer was employed to measure the particle concentration and particle diameter of the supernatant samples containing lentivirus vectors as previously described in material and method 1.10. Since, the diameter of lentivirus ranges from 80-120nm, we calculated the number of the particles in this range of the supernatant samples from KX293T and 293T cells. As shown in Figure 5C., the number of the particles of interest in the supernatants from KX293T and 293T cells were 2.8 X 10 ⁹ and 2.2 X 10 ⁹ events/ml. This means the elevated production of lentivirus in KX293T cells doesn’t increase the background accordingly.

2.5 Characterization of lentivirus vectors obtained from KX293T cell line.

To test whether the transduction of lentivirus vectors from KX293T cell line as efficient as that from 293T cell line, high-dividing and low/non-dividing cells were employed to perform the transfection assays. In this assay, an osteosarcoma cell line, HOS/MNNG cells which represented high-dividing cells and a primary human articular chondrocyte (HACC) which represented non/low-dividing ones were infected with lentivirus vectors form KX293T and 293T cells at a same MOI (MOI=10) . The positive cells with GFP expressed were examined under an inverted fluorescence Microscope. As a result, the lentivirus vector generated by the KX293T packaging cell line was as efficient as 293T cell line at transducing both non-dividing/low-dividing cells and proliferating cells (Figure 6. ) .

2.6 The analysis of foreign proteins expression using KX293T cell line.

Enzymes and cytokines are two main kinds of recombined proteins used in clinic and industry widely, and 293T is a commonly used host cell for recombined protein production. Thus, whether the KX293T cell line can express foreign proteins more efficiently than 293T is an interesting question. We tested the two proteins expression in KX293T and original 293T. FUT8 (～66KD, representing enzymes) and VEGF165 (～28KD, representing cytokines) were expressed in KX293T and 293T cell using transient transfection with according plasmids. As results, it was found the expression of FUT8 and VEGF165 protein in KX293T were 5.48 and 3.81 folds higher than those in original 293T, respectively (Figure 7A and 7B) . This means KX293T can produce not only lentivirus, but also foreign proteins of high efficiency as well.

2.7 The determination of exosomes produced by KX293T cell line.

Exosomes are membrane-bound extracellular vehicles (EVs) , ranged from 30-150nm. Currently, exosomes are being recognized as potential therapeutics as they have the ability to elicit potent cellular responses in vitro and in vivo. As the reason that exosomes from 293T was commonly used as standards for calculation and vehicles for drug delivery, we wonder whether KX293T can produce more exosomes than original 293T. Herein, we check the concentrations of exosomes from KX293T and 293T at the time points 24h, and 48h after seeding. As results, it was found the expression of exosomes released in 24h and 48h after seeding from KX293T were 1.98 and 2.73 folds higher than those from original 293T, respectively (Figure 8A and 8B) . This means KX293T can also produce more exosomes than original 293T.

3. DISCUSSION

Lentivirus vectors have the ability to deliver and to maintain long-term expression of transgenes in a broad range of cells, and is the dominant tool to generate CAR-T cells. To date, these lentivirus vectors have been produced by transient co-transfections of three-plasmid into packaging cells (PMID: 9354796, PMID: 8602510) . Undoubtedly, the packaging cells is at the key position of the whole lentivirus production workflow. Therefore, it is important to develop a cell line that can reproducibly generate high-titer lentivirus vectors in amounts that facility lentivirus-based therapies.

Irisin, the N-terminal peptide of a type I membrane protein that translated by fibronectin type III domain-containing protein 5 (FNDC5) gene, can be released into blood (PMID: 22237023, PMID: 32353410) . Blood irisin levels are increased in mice and humans after exercise. Irisin is highly conservative and its amino acid sequence is identical between humans and mice. Currently, FNDC5/Irisin was mostly studied in human metabolism bioprocess, including inducing browning of white fat (PMID: 22566556, PMID: 27504012) , improving insulin resistance (PMID: 27173461) , improving cognitive function (PMID: 32396989) and regulating bone metabolism (PMID: 26374841) . However, no study has been published about irisin functioning on lentivirus production.

Interestingly, we found that secondary transduction of 293T-irisin cells with the lentivirus vector results in a significant increase in producing lentivirus vector titers (data not shown) . The uncloned populations of 293T-irisin cells have showed an ability of packaging high-titer pseudotyped lentivirus vectors in a 10ml dish (data not shown) . Therefore, we further cherrypicked an individual clones of highest proliferation by two-round limitation dilutions, and named it as KX293T. As a result, the lentivirus vector titer packaged by KX293T could be 5.22 times higher than that from 293T cells at the harvest time 48 hours posttransduction. Moreover, the elevated production of lentivirus vectors from KX293T didn’t increase the background of extracellular secretion accordingly.

In summary, we report here the establishment of a lentivirus vector packaging cell line stably over-expressing human recombined irisin peptide, which reproducibly generates high-titer (up to 5.22 folds than 293T cell) pseudotyped lentivirus vectors. These vectors have a similar spectrum as the vectors produced by 293T cell. Vectors produced by the new packaging cell lines efficiently transduced both fast-growing cells and low/non growing cells in vitro. The new packaging cell line allows large scale production of lentivirus vectors and therefore will facilitate CAR-T therapy and human gene therapy efforts.

Listed below are some amino acid sequences mentioned herein.

amino acid sequence of FNDC5 (SEQ ID NO: 1) :

amino acid sequence of irisin (SEQ ID NO: 2) :

Claims

A host cell that is modified to over-express FNDC5 or irisin.
The host cell of claim 1, wherein the modification comprises introducing into the host cell an exogenous nucleic acid molecule encoding FNDC5 or irisin.
The host cell of claim 1 or 2, wherein the expression level of FNDC5 or irisin in the host cell is higher than the expression level of FNDC5 or irisin in the host cell before the modification, preferably by at least 5 times.
The host cell of any one of claims 1-3, wherein the host cell is a mammalian cell, preferably a human cell, more preferably a 293T cell.
The host cell of any one of claims 1-4, wherein the exogenous nucleic acid molecule is a DNA or RNA molecule.
The host cell of any one of claims 1-5, wherein the FNDC5 comprises the amino acid sequence shown in SEQ ID NO: 1 or a functional variant thereof; the irisin comprises the amino acid sequence shown in SEQ ID NO: 2 or a functional variant thereof.
The host cell of any one of claims 1-6, wherein the host cell is a human embryonic kidney cell KX293T with a deposit number of CCTCC NO: C2021292.
Use of the host cell of any one of claims 1-7 as a viral vector packaging cell line.
The use of claim 8, wherein the viral vector packaging cell line is a lentiviral vector packaging cell line.
A method of preparing a viral vector comprising:

1) modifying a host cell to over-express FNDC5 or irisin; and

2) co-transfecting the host cell with a transfer plasmid and packaging plasmids.
The method of claim 10, wherein the modification comprises introducing into the host cell an exogenous nucleic acid molecule encoding FNDC5 or irisin.
The method of claim 10 or 11, wherein the expression level of FNDC5 or irisin in the host cell is higher than the expression level of FNDC5 or irisin in the host cell before the modification by at least 5 times.
The method of any one of claims 10-12, wherein the exogenous nucleic acid molecule is a DNA or RNA molecule.
A method of preparing a viral vector comprising co-transfecting the host cell with a transfer plasmid and packaging plasmids, wherein the transfer plasmid comprises a sequence encoding FNDC5 or irisin.
A method of preparing a viral vector comprising:

1) co-transfecting a host cell with a transfer plasmid and packaging plasmids in a medium; and

2) adding to the medium FNDC5 or irisin prior to, concurrent with, or after step 1) .
The method of any one of claims 10-15, wherein the host cell is a mammalian cell, preferably a human cell, more preferably a 293T cell.
The method of any one of claims 10-16, wherein the FNDC5 comprises the amino acid sequence shown in SEQ ID NO: 1 or a functional variant thereof; the irisin comprises the amino acid sequence shown in SEQ ID NO: 2 or a functional variant thereof.
A method of preparing a viral vector comprising co-transfecting a host cell with a transfer plasmid and packaging plasmids, wherein the host cell is a human embryonic kidney cell KX293T with a deposit number of CCTCC NO: C2021292.
The method of any one of claims 10-18, wherein the viral vector is a lentiviral vector.
The method of any one of claims 10-19, wherein the transfer plasmid comprises a sequence encoding a chimeric antigen receptor (CAR) .
A method of preparing a CAR-T cell comprising:

1) preparing a viral vector by a method of any one of claims 10-20; and

2) transfecting a T cell with the viral vector.
A method of preparing a protein with a host cell comprising:

1) modifying the host cell to over-express FNDC5 or irisin;

2) modifying the host cell to express the protein; and

3) optionally, isolating or purifying the protein from the host cell.
The method of claim 22, wherein the modification in step 1) comprises introducing into the host cell a first exogenous nucleic acid molecule encoding FNDC5 or irisin, and the modification in step 2) comprises introducing into the host cell a second exogenous nucleic acid molecule encoding the protein.
The method of claim 22 or 23, wherein the protein is an enzyme, such as FUT8, or a cytokine, such as VEGF.
A method of preparing exosomes with a host cell comprising:

1) modifying the host cell to over-express FNDC5 or irisin;

2) culturing the host cell and optionally, isolating or purifying the exosomes from the host cell.
The method of claim 25, wherein the modification in step 1) comprises introducing into the host cell an exogenous nucleic acid molecule encoding FNDC5 or irisin.
The method of any one of claims 22-26, wherein the expression level of FNDC5 or irisin in the host cell is higher than the expression level of FNDC5 or irisin in the host cell before the modification by at least 5 times.
The method of any one of claims 22-27, wherein the host cell is a mammalian cell, preferably a human cell, more preferably a 293T cell.
The method of any one of claims 22-28, wherein the FNDC5 comprises the amino acid sequence shown in SEQ ID NO: 1 or a functional variant thereof; the irisin comprises the amino acid sequence shown in SEQ ID NO: 2 or a functional variant thereof.
The method of any one of claims 22-29, wherein the host cell is a human embryonic kidney cell KX293T with a deposit number of CCTCC NO: C2021292.