Characteristics of Transfection Reagents that Achieve High Production of Recombinant Adeno-Associated Virus Vectors

Abstract

Recombinant adeno-associated virus (rAAV) vectors are among the most effective for gene therapy. A significant advancement in rAAV vector production is developing the triple-plasmid transfection method, which remains the most widely used technique. In this study, we used Expi293F^TM (Expi293F) and Viral Production Cells 2.0 (VPC2.0 cells) to evaluate various transfection reagents, comparing transgene protein expression levels and intracellular plasmid copy numbers to optimize rAAV production. Our findings indicated that the effectiveness of transfection reagents in promoting higher rAAV production was cell-dependent and that rAAV productivity correlated more with plasmid levels in the cell nucleus than with transgene protein expression levels. Confocal laser microscopy revealed that in cells transfected with the high-yield transfection reagent, a large amount of free plasmid DNA entered the nucleus, whereas the transfection reagents themselves did not. These results provide new insights into the intracellular mechanisms underlying efficient rAAV vector production. Furthermore, identifying transfection reagents that facilitate nuclear plasmid delivery will aid in the selection of optimal reagents for high-yield AAV production.

Keywords

adeno-associated virus transfection reagent intracellular plasmid copies genome titer nuclear localization

INTRODUCTION

Gene therapy has advanced significantly, with recombinant adeno-associated virus (rAAV) vectors playing a key role owing to their low immunogenicity, high transduction efficiency, broad tissue specificity, and long-term transgene expression.^1–3

Efficient rAAV vector production is crucial for gene therapy, requiring a distinct approach owing to the complexity of the process and unique productivity challenges compared with simpler protein expression systems.^4,5 Significant advancements in rAAV vector production have been achieved with the development of the triple-plasmid transfection method, which has since become the predominant technique in the field.^6–8 This involves introducing three plasmids—one with the transgene flanked by inverted terminal repeats (ITRs) and two containing AAV and adenoviral elements—into cells. Viral elements transcribed from the two plasmids facilitate replication and encapsidation of the ITR-flanked genome, producing rAAV particles.^9–12

Although various techniques exist for introducing plasmid DNA (pDNA) into mammalian cells, common transfection methods for rAAV production include calcium phosphate coprecipitation, polycations such as polyethyleneimine (PEI), and cationic lipids. Although calcium phosphate coprecipitation of pDNA is a simple method, it is difficult to provide a stable supply on an industrial scale because of its dependence on serum and various factors such as reagent concentration, pH, and temperature.^4,13 Cationic polymers and cationic lipids are frequently used for rAAV production in suspended cells. PEI, a cationic polymer, was first used to deliver DNA to cultured cells in 1995.¹⁴ It forms polyplexes with DNA, which are taken up by cells and reach the nucleus. Many studies have been conducted on transfection events using PEI,^15–18 and it is known that PEI is nonbiodegradable and moderately cytotoxic.^4,13 Various types of PEI exist, such as branched and linear,¹⁹ and other cationic polymers used for transfection have also become more diverse.^20,21 Cationic lipids were first reported in 1987²² and enable efficient, biocompatible, and nontoxic gene transfer.^23–26 These cationic lipid-based reagents are expensive, making their use in AAV production challenging.⁴ However, in a recent study, Guan et al. reported that employing cationic lipids such as DOPE and DOTAP makes it possible to produce transfection reagents more cost-effectively.²⁶

Among these transfection reagents, the key factors responsible for increased rAAV production remain unknown. In this study, we evaluated different transfection reagents to determine the conditions required for higher rAAV production.

MATERIALS AND METHODS

Cells and culture medium

Expi293F^TM Expi293F cells (Thermo Fisher Scientific, Waltham, MA) and Viral Production Cells 2.0 (VPC2.0 cells) (Thermo Fisher Scientific) were used as host cells in the present study. Expi293F cells were maintained in Expi293 expression medium (Thermo Fisher Scientific), and VPC2.0 cells were maintained in viral production medium (Thermo Fisher Scientific) at 37°C in 8% CO₂ incubator.

Triple-plasmid transfection

The cells were transfected with three plasmid vectors, pRC2-mi342, pHelper, and pAAV-ZsGreen1 (Takara Bio, Shiga, Japan), at a ratio of 1:1:1 (w/w) using the supplier-recommended protocol for each transfection reagent. The details of each transfection reagent and protocol are summarized in Table 1. At 72 h post-transfection or during time-course sampling, 1.1 mL of AAV-MAX lysis buffer (Thermo Fisher Scientific) was added to 10 mL of culture medium. The flasks were then incubated at 37°C for 2 h. One milliliter of the cell lysate was transferred to a 1.5-mL tube and centrifuged at 4°C at 13,000 g for 10 min. The supernatant containing the crude AAV particles was transferred to a new tube, and the sample was stored at −80°C until use. Additionally, for confocal laser microscopy, pAAV-LacZ was used instead of the pAAV-ZsGreen1 plasmid because the fluorescence of ZsGreen1 was not required.

Table 1.

Comparison of Transfection Reagents

TF Reagents	ExpiFectamine^a	AAV-MAX^b	VirusGEN AAV Kit(virusGEN+)	TransIT-VirusGEN(virusGEN-)	PEIpro	FectoVIR-AAV
Supplier(Country)	Thermo Fisher Scientific(Waltham, MA, US)		Mirus Bio(Madison, WI, US)		Polyplus(Illkirch, France)
Component	Cationic Lipids	Cationic Lipids and Peptide	Cationic Lipids and Polymer	Cationic Lipids and Polymer	Cationic Polymer	Cationic Polymer
Cell density (× 10⁶ cells/mL)	3.0	3.0	2.0	2.0	2.0	2.5
Medium (mL)	25	30	25	25	25	30
Amount of pDNA/Buffer	25 µg DNA in 1.5 mL buffer and 80 µL in 1.4 mL buffer	45 µg in 3 mL buffer/(180 µL transfection reagent + 90 µL booster)	50 µg/75 µL in 2.5 mL buffer	50 µg/75 µL in 2.5 mL buffer	50 µg in 1.25 mL buffer and 50 µL in 1.25 mL buffer	75 µg/75 µL in 2.5 mL buffer
Complexation Buffer	Opti-MEM	Viral-Plex Complexation Buffer	VirusGEN AAV Complex Formation Solution and Enhancer	Opti-MEM	Opti-MEM	Opti-MEM
Incubate (min)	10–20	20–30	15–60	15–60	15	30

This reagent requires that the enhancer (Enhancer 1: 150 µL, Enhancer 2: 1.5 mL) be added to the culture medium between 18 and 22 h after transfection.

This reagent requires the addition of an enhancer (300 µL) to the culture medium at the time of cell seeding prior to transfection.

Cell analysis and sorting using a flow cytometer

Cell analysis and sorting were performed using a cell sorter SH800S (Sony Biotechnology, San Jose, CA, USA). ZsGreen1-positive cells were detected using a 488 nm fluorescein isothiocyanate laser (Ex/Em: 493/505 nm).²⁷ Apoptotic cells were identified using Annexin V-Alexa Fluor 647 conjugate (Thermo Fisher Scientific, Ex/Em: 650/665) and detected with a 638 nm Cy5 laser. Cutoff values for ZsGreen1 and Annexin V positivity were determined by flow cytometer (FCM) using untransfected²⁸ or annexin-treated normal cells (Supplementary Fig. S1), respectively, and percentages of cells above the cutoffs were calculated. The transfected cells were sorted, each 2.0 × 10⁶ cells, using a 15-mL tube in an “ultra purity” mode. The cells were washed in 1× phosphate-buffered saline (PBS), and the samples were stored at −80°C until use.

Droplet digital polymerase chain reaction for rAAV genome titer and pAAV-ZsGreen1 plasmid copies

The protocol used to titer rAAV genomes has been described previously.²⁸ Briefly, 2 µL of each rAAV solution was added to 17 µL of a DNase I solution and incubated at 37°C for 30 min. DNase I was inactivated by adding 1 µL of 0.5 M ethylenediaminetetraacetic acid (pH 8.0), followed by incubation at 25°C for 5 min. The sample was then heated at 95°C for 10 min to denature the AAV capsid. This sample was used as the pretreated sample for droplet digital polymerase chain reaction (ddPCR). After proper dilution of the pretreated sample with 0.05% Pluronic F68 in Tris-EDTA solution, 1 µL of the rAAV sample was mixed with 19 µL of the reaction mixture with ddPCR Supermix for Probe (No dUTP) following the instructions from Bio-Rad (Hercules, CA). After droplet generation, ddPCR was performed using the C1000 Touch Thermal Cycler (Bio-Rad). The droplets were analyzed using a QX200 Droplet Reader and QuantaSoft (v1.7.4.0917; Bio-Rad). Copy numbers were calculated based on a droplet volume of 0.85 nL and expressed as copies/µL. Vector genome (vg) titers were then converted to vg/mL by incorporating dilution factors. For flow-sorted samples or intracellular plasmid quantification, calculations were based on known cell counts to calculate copies/cell. ddPCR primers and probes targeted the ZsGreen1 gene for rAAV genomes and plasmid-specific backbone sequences for pAAV-ZsGreen1. All oligonucleotides were custom-synthesized and HPLC-purified by Eurofins Genomics (Tokyo, Japan), with sequences described previously.²⁸

Cytoplasmic and nuclear localization of plasmid using transfection reagents

Expi293F and VPC2.0 cells were transfected with appropriate transfection reagents. At each time point after transfection, 3 mL of each culture medium was sampled and washed with PBS. From that, 2.0 × 10⁶ cells were used for the isolation of the cytoplasmic and nuclear fractions using NE-PER^TM Nuclear and Cytoplasmic Extraction Reagent (Thermo Fisher Scientific). The procedure was performed according to the manufacturer’s instructions.

Nuclear localization of labeled pDNA/transfection reagent polyplex using confocal laser microscopy

The pAAV-LacZ plasmid (Takara Bio) was directly labeled with Cy5 using the Label IT® Tracker™ Intracellular Nucleic Acid Localization Kit (Mirus Bio, Madison, WI). Transfection reagents, PEIpro, FectoVIR-AAV, and AAV-MAX, were labeled with amine-reactive dye (Oregon Green^TM 488 carboxylic acid, succinimidyl ester, and 5-isomer [Thermo Fisher Scientific] in dimethyl sulfoxide at 10 mg/mL) as previously described.²⁹ Triple transfection was performed on the preculture of VPC2.0 cells in a 125 mL flask using an appropriate amount of labeled each transfection reagent and three plasmids, including the labeled pAAV-LacZ instead of pAAV-ZsGreen1. At 24 h post-transfection, 2 mL of each culture medium was collected, and viable cells were counted, fixed with 1 mL of 4% paraformaldehyde per 1 × 10⁶ cells, and incubated at room temperature for 10 min. The cells were washed twice with PBS, nuclear-stained with Hoechst 33342 (Thermo Fisher Scientific), washed again with PBS, placed in glass-bottomed Petri dishes, and observed under a confocal laser scanning microscope (FV1000; Evident Corporation, Shinjuku, Tokyo, Japan). 4',6-diamidino-2-phenylindole, Alexa Fluor 488, and Cy5 dye’s channels were employed for visualization of cellular nucleus (stained with Hoechst33342, Ex/Em: 350/461), Oregon Green^TM 488-labeled transfection reagent (Ex/Em: 496/524), and Cy5-labeled pDNA (Ex/Em: 651/670), respectively. All experiments were carried out in duplicate.

Statistical analysis

Statistical differences between datasets were assessed using Student’s t-test, with a p-value of <0.05 considered statistically significant. Correlation coefficient values between 0.7 and 1.0 indicated a strong positive linear relationship, reflecting a clear linear trend.

RESULTS

rAAV2 production when using different transfection reagents

Initially, we compared various transfection reagents using Expi293F and VPC2.0 cells, both derived from Human Embryonic Kidney cells 293 (HEK293) cells. The transfection reagents used in this study were ExpiFectamine, AAV-MAX, TransIT-VirusGEN transfection reagent, VirusGEN AAV kit, PEIpro, and FectoVIR-AAV according to the manufacturer’s instructions (Table 1). Each cell line was transfected with the triple plasmid using the optimized protocol for each transfection reagent and cultured for 72 h to produce rAAV2. Then, rAAV2 was harvested from a portion of the cells using a lysis buffer, and the remaining transfected cells were collected using an FCM, which was used to determine the percentage of ZsGreen1-positive cells simultaneously. The genome titer was calculated using ddPCR, using the harvested rAAV2, and the intracellular plasmid levels, using the pAAV-ZsGreen1 plasmid backbone as a target, were determined using ddPCR using cells collected using the FCM. Comparing different transfection reagents in Expi293F cells, the highest rAAV2 genome titers were obtained using FectoVIR-AAV (Fig. 1A). The ExpiFectamine transfection reagent, optimized for Expi293F cells for protein production, was found to be unsuitable for AAV production. In contrast, in VPC2.0 cells, which are thought to be the same HEK293-derived line as the Expi293F cells, the highest rAAV2 genome titers were obtained using FectoVIR-AAV and AAV-MAX (Fig. 1B). The AAV-MAX transfection reagent was sold as a kit together with VPC2.0 cells for rAAV production and could produce high titers only with VPC2.0 cells but had no effect on Expi293F cells. This suggests cell-specific compatibility between the transfection reagent and cell line. The percentages of ZsGreen1 protein-positive cells were similar between the two cell lines, Expi293F and VPC2.0 cells (Fig. 1C, D, line graphs), whereas the intracellular plasmid copy numbers, as well as the rAAV2 genome titers, were higher in VPC2.0 than in Expi293F. Furthermore, previous studies have shown that rAAV2 genome titers correlate with the amount of intracellular plasmid rather than the amount of transgene protein expressed.²⁸ These observations prompted speculation that intracellular plasmid levels may be important in promoting high-level AAV production.

Figure 1.

Evaluation of the performance of Expi293F and VPC2.0 cells with various transfection reagents. Each transfection protocol used its own recommended protocol. Expifect: Expifectamine 293 transfection kit, AAV-MAX: AAV-MAX transfection kit, virusGEN+: VirusGEN AAV Transfection Kit, virusGEN−: TransIT-VirusGEN transfection reagent, FectoVIR: FectoVIR-AAV. (A) Comparison of rAAV2 genome titer values in Expi293F cells using different transfection reagents. (B) Comparison of rAAV2 genome titer values in VPC2.0 cells using different transfection reagents. (C) Comparison of ZsGreen1 protein positivity by FCM and pAAV-ZsGreen1 plasmid amount per cell in Expi293F cells using different transfection reagents by FCM and ddPCR. (D) Comparison of ZsGreen1 protein positivity and pAAV-ZsGreen1 plasmid amount per cell in VPC2.0 cells using different transfection reagents. This experiment was conducted independently in triplicate. FCM, flow cytometer; ddPCR, droplet digital polymerase chain reaction; rAAV2, recombinant adeno-associated virus 2.

Cytoplasmic and nuclear localization of plasmid using transfection reagents

To assess the relationship between intracellular plasmid copy number and rAAV2 productivity, we isolated the cytoplasmic and nuclear fractions from transfected cells using low- and high-production reagents and quantified pAAV-ZsGreen1 plasmid copies via ddPCR. In Expi293F cells transfected with low-yield PEIpro, plasmids were distributed in both the cytoplasm and nucleus within 4 h post-transfection, with a stable proportion over time (Fig. 2A). In contrast, when employing the high-yield transfection reagent FectoVIR-AAV in Expi293F cells, minimal plasmid presence was observed in the cytoplasmic and nuclear fractions 4 h after transfection. However, by day 2, an increased quantity of plasmids was detected in the cytoplasmic and nuclear fractions compared with that with PEIpro (Fig. 2A). Similar results were obtained in VPC2.0 cells, especially when using AAV-MAX reagent, a high-productivity transfection reagent in VPC2.0 cells, the behavior was similar to that with FectoVIR-AAV (Fig. 2B). These findings suggested that the early arrival of DNA into the nucleus by transfection reagents did not necessarily lead to high AAV production.

Figure 2.

Cytoplasmic and nuclear localization of plasmid using various transfection reagents. The “cyto.” indicates cytoplasmic localization, and “nuc.” indicates the nuclear localization. (A) Comparison of pAAV-ZsGreen1 plasmid copy numbers in Expi293F cells using PEIpro or FectoVIR-AAV transfection reagent. (B) Comparison of pAAV-ZsGreen1 plasmid copy numbers in VPC2.0 cells using PEIpro, AAV-MAX, or FectoVIR-AAV transfection reagent. This experiment was independently performed three times.

Cytoplasmic or nuclear localization of plasmid correlation with AAV genomic titer

Using the values from Figure 2, we numerically compared the number of plasmid copies in the cytoplasmic and nuclear fractions relative to the rAAV2 genome titers. rAAV2 genome titer values exhibited a stronger correlation with nuclear localization than with cytoplasmic (Fig. 3A). Furthermore, the correlation between rAAV2 genome titer values and plasmid copies was notably higher using AAV-MAX or FectoVIR-AAV transfection reagents than using PEIpro (comparison of Figs. 3A–C). Based on this, we suspected that the interaction between the transfection reagent and the plasmid DNA may be different only when PEIpro was used in this study. As a next step, we sought to visualize the intracellular localization of the plasmid and the transfection reagent by confocal laser microscopy.

Figure 3.

Correlation between the copy number of pAAV-ZsGreen1 plasmid backbone and rAAV2 genome titer in the cytoplasmic or nuclear localization in VPC2.0 cells. (A) PEIpro transfection reagent, (B) AAV-MAX transfection kit, and (C) Fecto-VIR-AAV. The “titer” indicates rAAV genome titer, “cyto.” shows the amount of plasmid in the cytoplasm, and “nuc.” shows the amount of plasmid in the cell nucleus.

Nuclear localization of labeled pDNA/transfection reagent polyplex using confocal laser microscopy

To investigate the behavior of the plasmid and transfection reagent in the cell nucleus, the pAAV-LacZ plasmid (labeled with Cy5) was triple-transfected with various transfection reagents (labeled with Oregon Green^TM 488), along with the pHelper and pRC2 plasmids in VPC2.0. We previously showed a positive correlation between intracellular plasmid copy number and genomic titer, and that the ratio of the three plasmids remains proportional.²⁸ Thus, only the cisplasmid was labeled for analysis in this study. The cells were harvested 24 h after transfection, and the nuclei were stained with Hoechst33342 and observed under a confocal laser microscope. Using PEIpro, we consistently observed significantly larger pDNA clumps within the cytoplasm (Fig. 4, top: plasmid). In contrast, when FectoVIR-AAV or AAV-MAX were used, fewer large clumps of pDNA and finer pDNA particles were observed in the nuclei (Fig. 4, arrowheads). By comparing the behavior of the transfection reagents, we found that FectoVIR-AAV or AAV-MAX did not enter the nucleus and remained in the cytoplasm, unlike PEIpro (Fig. 4, white arrows). These results indicated that although the compositions of the transfection reagents FectoVIR-AAV and AAV-MAX have not been disclosed, we hypothesized that they may enable the “complete” release of plasmid DNA (pDNA) in the cytoplasm of host cells, thereby facilitating the delivery of free pDNA into the nucleus.

Figure 4.

Localization observation of pDNA and transfection reagents after 24 h post-transfection using confocal laser microscopy. The red color shows pAAV-LacZ plasmids labeled with Cy5, the green color shows the transfection reagents with Oregon Green^TM 488, and the blue color shows cell nuclei stained with Hoechst 33342. The arrowhead indicates the pAAV-LacZ present in the nucleus, and the white arrow indicates the behavior of the transfection reagent within the nucleus at that time.

Assessment of transfection reagent toxicity

Polyplexes such as PEI have been reported to be more cytotoxic than lipoplexes.^13,24,30 Indeed, increasing the amount of PEI increased the rate of apoptosis and reduced cell viability and AAV productivity (Supplementary Fig. S2). However, when comparing different transfection reagents, the highest cell viability and the lowest rate of apoptosis were observed (Supplementary Figs. S3 and Figs. S4) with PEIpro compared with lipid-based AAV-MAX. Furthermore, it was observed that the higher the cytotoxicity, the higher the genome titer (Supplementary Fig. S3B).

Flow cytometry analysis of the cells after transfection revealed that, particularly when using the FectoVIR-AAV reagent, a novel cell population (Supplementary Fig. S5, group b) emerged in addition to the typical cell population (Supplementary Fig. S5, group a) 72 h post-transfection. The new cell populations in group b were found to be apoptotic and contained many atypical cells (Supplementary Fig. S6). However, when the rAAV2 productivity of each cell group was investigated using ddPCR, we found that normal and apoptotic cells produced similar levels of rAAV2 (Supplementary Fig. S7). These results suggest that, while the link between cytotoxicity and genome titer during rAAV production is unclear, moderate cytotoxicity does not hinder high rAAV production. During rAAV production, rAAV tends to accumulate inside the cells, particularly serotype 2. In fact, the production of AAV5, which is secreted extracellularly,³¹ showed a more than 9% higher viable cell rate and over 3% fewer apoptotic cells compared with AAV2 (data not shown). Therefore, these apoptotic events may result from rAAV production and not just from the transfection reagent. Unlike antibody production using Chinese hamster ovary cells cultured over extended periods, rAAV is typically harvested 3 days post-transfection; therefore, reagent toxicity is unlikely to be a major concern during this period.

DISCUSSION

In this study, we compared transfection reagents and examined their mechanisms with optimize rAAV2 production. While many studies have evaluated nucleic acid delivery, most focus on small interfering RNA uptake.^32–37 Among the few addressing AAV production, Guan et al. compared adherent and suspension cells and showed that cationic liposomes yielded the highest titers in suspension HEK293F cells.²⁶ Building on this, we compared reagents in the same suspension cell type and monitored transfection events in greater detail.

This study offers key insights into optimizing rAAV production by comparing transfection reagents in Expi293F and VPC2.0 cells. AAV-MAX showed cell-specific effects, greatly increasing titers in VPC2.0 but not in Expi293F cells (Fig. 1A, B). While prior work noted cell- and reagent-specific transfection outcomes,³³ our results show that even closely related HEK293-derived cells respond differently, underscoring the importance of cell line-specific reagent selection.

Moreover, this study revealed that rAAV2 production correlated with high intracellular plasmid copy numbers, not with transgene protein expression. ZsGreen1-positive cell rates were similar between Expi293F and VPC2.0 cells at 24 and 48 h post-transfection (data not shown), and high protein expression did not equate to high rAAV2 yield (Figs. 1 and 3). While transfection efficiency is often judged by protein output,^38,39 our findings—and previous work²⁸—suggest that rAAV2 production involves distinct mechanisms, as seen in the low yield from high protein-expressing Expi293F cells.

Further analysis showed that rAAV genome titers correlated more strongly with nuclear, rather than cytoplasmic, plasmid copy numbers (Fig. 3), and these relationships did not change with varying amounts of transfection reagent (data not shown). The transfected nucleic acid DNA is then transported into the host cell and transcribed into mRNA. Therefore, it is crucial for foreign pDNA to reliably reach the cell nucleus.^37,40 In the case of rAAV, in addition to transcription, there is a significant event where the AAV genome is replicated in the nucleus by Rep proteins, making the transport of pDNA into the nucleus even more important.¹²

Although nuclear plasmid levels correlate with rAAV titers, they are unreliable as titer markers. With PEIpro, plasmids were already present within the nucleus 4 h after transfection (Fig. 2), possibly overestimating productivity. At 24 h post-transfection, FectoVIR-AAV had only 1.2-fold more nuclear plasmids than PEIpro but a 5.5-fold higher genome titer. By 48 h, the plasmid-to-titer ratio became similar (3-fold or more). This difference was likely due to the different behaviors of the plasmids in the cell nucleus 24 h after transfection. With PEIpro, large pDNA clumps were seen in the cytoplasm 24 h post-transfection (Fig. 4). In Figure 2, when PEIpro was used, the results indicated that pDNA appeared to be localized in the nucleus as early as 4 hours after transfection; however, it may actually be accumulating at the nuclear envelope rather than within the nucleus. Similar to findings by Haraguchi et al.,⁴¹ lipid-wrapped pDNA may fuse with the nuclear membrane, though such structures were minimal with FectoVIR-AAV and AAV-MAX. While PEIpro-DNA complexes were not clearly nuclear, PEIpro itself was detected, consistent with Godbey et al.²⁹

With high-yield reagents such as FectoVIR-AAV and AAV-MAX, the reagents were absent from the nucleus, while small DNA particles were present (Fig. 4), suggesting free nuclear DNA enables efficient replication by Rep proteins. Both FectoVIR-AAV and PEIpro are cationic polymers; however, the sequences and conformation differences likely explain their performance variation.

This study emphasizes the importance of transporting free pDNA into the nucleus to achieve high rAAV production. Transfection reagents, such as FectoVIR-AAV or AAV-MAX, excel in this process, making them superior choices. Future research should focus on optimizing transfection reagents and elucidating the mechanisms underlying their efficacy in enhancing gene therapy.

AUTHORS’ CONTRIBUTIONS

K.M.-K. performed experiments and wrote the article. K.U. conceived the experiments and secured the funding. E.I.-K., Y.Y., N.H., and K.Y. supervised the study.

Footnotes

ACKNOWLEDGMENT

The authors would like to thank Editage () for editing and reviewing this article for English language.

FUNDING INFORMATION

This study was supported by the Japan Agency for Medical Research and Development under Grant Number 18ae0201001h0001r.

AUTHOR DISCLOSURE

The authors declare no competing interests.

Supplemental Material

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