what is the ratio of cesium chloride to dmso for the cancer protocol
Mol Ther Methods Clin Dev. 2020 Jun 12; 17: 612–621.
Protocol Optimization for the Production of the Non-Cytotoxic JΔNI5 HSV Vector Scarce in Expression of Immediately Early Genes
Seiji Kuroda
1Department of Biochemistry and Molecular Biology, Japan Medical School, Tokyo, Japan
3Section of Surgery, Nippon Medical School Musashikosugi Hospital, Kawasaki, Nihon
fourDepartment of Gastrointestinal and Hepato-Biliary-Pancreatic Surgery, Japan Medical School, Tokyo, Nippon
Yoshitaka Miyagawa
1Department of Biochemistry and Molecular Biological science, Nippon Medical Schoolhouse, Tokyo, Japan
Yuriko Sato
aneDepartment of Biochemistry and Molecular Biological science, Japan Medical School, Tokyo, Japan
Motoko Yamamoto
aneDepartment of Biochemistry and Molecular Biology, Nippon Medical School, Tokyo, Japan
Kumi Adachi
iDepartment of Biochemistry and Molecular Biological science, Nippon Medical Schoolhouse, Tokyo, Japan
Hiromi Kinoh
1Section of Biochemistry and Molecular Biology, Nippon Medical School, Tokyo, Nihon
William F. Goins
2Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, Pittsburgh, PA 15219, USA
Justus B. Cohen
2Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, Pittsburgh, PA 15219, USA
Joseph C. Glorioso
2Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, Pittsburgh, PA 15219, USA
Nobuhiko Taniai
threeSection of Surgery, Japan Medical School Musashikosugi Hospital, Kawasaki, Nippon
Hiroshi Yoshida
4Department of Gastrointestinal and Hepato-Biliary-Pancreatic Surgery, Nippon Medical School, Tokyo, Japan
Takashi Okada
5Partition of Molecular and Medical Genetics, Center for Cistron and Cell Therapy, The Institute of Medical Science, The Academy of Tokyo, Tokyo, Japan
Received 2020 February nineteen; Accepted 2020 Mar 12.
- Supplementary Materials
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Document S1. Figures S1 and S2
GUID: C511F1FF-2F5D-4C70-846C-02287D880BC1
Certificate S2. Article plus Supplemental Information
GUID: ADB9487F-90F8-4FC7-B69D-C767DB7A88DF
Abstruse
Non-toxic herpes simplex virus (HSV) vectors tin can be generated by functional deletion of all immediate-early (IE) genes, providing a benign vehicle with potential for gene therapy. However, deletion of multiple IE genes raises manufacturing concerns and thus limits clinical application of these vectors. To address this effect, nosotros previously developed a novel production cell line, chosen U2OS-ICP4/27, by lentiviral transduction of human osteosarcoma U2OS cells with two essential HSV IE genes, ICP4 and ICP27. To optimize the procedure of vector manufacturing on this platform, nosotros evaluated several cell culture parameters of U2OS-ICP4/27 for high-titer and -quality production of non-toxic HSV vectors, revealing that the yields and functionality of these vectors can be significantly influenced past culturing weather. We also found that several chemical compounds can enhance the replication of non-toxic HSV vectors and their release from producer cells into the supernatants. Notably, the vector produced by our optimized protocol displayed a profoundly improved vector yield and quality and showed elevated transgene expression in cultures of master dorsal root ganglion neurons. Taken together, our optimized production arroyo emerges equally a relevant protocol for high-yield and high-quality training of not-toxic HSV-based gene therapy vectors.
Graphical Abstract
Introduction
The potential of engineered herpes simplex virus (HSV) vectors every bit factor transfer vehicles has been studied for years and these vectors are now emerging as promising cistron therapy and oncolytic agents.1, 2, three, 4, 5, 6 A major drawback of the HSV vector system as a gene transfer tool has been its toxicity for a multifariousness of jail cell types, due mainly to cell-bicycle arrest and apoptosis induced by viral gene products.7 Several groups have attempted to reduce the cytopathic effects of the HSV vector past genetic manipulation. Originally, recombinant HSV vectors had unmarried immediate-early (IE) genes deleted, such as ICP4, which encodes a major transcription factor, or ICP27, which encodes an essential mail service-transcriptional regulator,8, 9, 10, eleven, 12 to block lytic replication. All the same, these mutants still exhibited cytotoxic furnishings on many cell types in vitro.xiii, 14, 15 Subsequently, double and multiple IE gene-deletion mutants in diverse combinations were developed to further reduce vector cytotoxicity and their safe was evaluated.8,12,16 About of these recombinants still showed residual cytotoxic effects and their use as gene therapy vectors has therefore been limited. To eliminate these barriers, nosotros recently developed a genetically engineered not-cytotoxic HSV vector by ablation of all IE gene expression,17 causing heterochromatinization of the viral genome upon entry into the prison cell and shut-downwardly of all toxic viral cistron expression; only the latency-associated transcript (LAT) locus remains transcriptionally active, allowing the expression of therapeutic genes.17 Nosotros have demonstrated that big and multiple transgene expression cassettes can be inserted into the HSV vector genome and expressed under unlike regulatory sequences.17,18 The engineered non-toxic HSV has successfully provided robust transgene expression without vector-mediated cytotoxicity in rat encephalon, indicating that the vector can be used for transduction in vivo.19 Thus, this not-toxic HSV vector platform can exist expected to become a new rubber and useful tool for efficient gene therapy.
Since non-toxic HSV vectors lack the ability to express whatever IE gene products, they cannot replicate in normal cells. Therefore, to produce the highly defective virus particle, we generated a specific producer jail cell line, U2OS-ICP4/27, by lentiviral transduction of human osteosarcoma U2OS cells with two essential HSV IE genes, ICP4 and ICP27.17 Because U2OS cells naturally complement the growth-promoting activity of the toxic IE protein ICP0,20 our IE gene-disabled HSV vectors could be propagated substantially more than efficiently on U2OS-ICP4/27 cells than on traditional ICP4/ICP27-complementing (eastward.g., Vero-based 7B) cells.8,17
In the present report, we evaluated unlike jail cell-civilisation parameters in order to optimize the production of a prototype non-toxic HSV vector, JΔNI5,17 on U2OS-ICP4/27 cells. We demonstrate that modification of each of the culture parameters significantly affected the physical vector yield and its biological activity. Additionally, several chemical agents, including histone deacetylase (HDAC) and bromodomain and extra-terminal (BET) inhibitors, were tested for their ability to raise the replication of non-toxic HSV. Furthermore, nosotros examined two other chemicals, sodium chloride and cesium chloride, for their ability to increase the release of the HSV vector from its U2OS-ICP4/27 producer cells. Finally, we examined the differences in the results obtained from the conventional production method compared with an optimized protocol using the elements with the highest impact on virus yield.
Results
Optimization of Culture Weather condition for JΔNI5 Vector Production
We commencement determined the optimal multiplicity of infection (MOI) for efficient production of JΔNI5 vector, a not-toxic HSV vector previously described past one of our labs.17 The JΔNI5 genome carries an mCherry expression cassette equally a transgene in place of the terminal ICP4 locus. In previous reports, MOIs of 10−two to 10−4 were considered suitable for recombinant HSV vector production.21 In our system, the physical titers at these MOIs increased rapidly and reached peak values of 1.66–1.79 × 1010 genome copies (gc)/mL at vii–8 days post infection (dpi) (Figure 1A). At input MOIs of 10−five or x−half dozen, the titers peaked at 9 or ten dpi, respectively, with the highest titer (2.1 × ten10 gc/mL) achieved by MOI = 10−5 at 9 dpi. Likewise, the height biological titers (in plaque-forming units [PFU]/mL) also depended on the input MOI (Figure 1B). The highest biological titer (2.9 × 106 PFU/mL) was once more achieved using MOI = 10−5 at 9 dpi. The gc/PFU ratio was as well examined to judge the quality of the virus (Effigy 1C). The minimum gc/PFU ratio was achieved at four–6 dpi, except for MOI = 10−vi (viii dpi), and a modest increase was more often than not observed at later time points. The gc/PFU ratio was non significantly unlike amid the conditions at 9 dpi. Together, these results indicated that an input MOI of 10−5 was optimal, establishing it as the standard input for optimization of other culture parameters. To verify that the gc/PFU ratio represented an accurate measure of the functionality of the vector preparation, we infected fetal rat dorsal root ganglion (rDRG) neurons in culture with 5,000 gc/prison cell of the 9 dpi MOI = 10−ii and MOI = 10−5 and the transduction efficiencies were examined 4 d subsequently. As expected, the HSV vector produced by MOI = x−five showed a more than widespread mCherry expression than to the vector produced by MOI = ten−2 (Figure oneD). This result was confirmed by quantitative reverse transcriptase PCR (qRT-PCR) measurement of mCherry mRNA levels at 7 dpi (Figure 1E). The mRNA level of mCherry from the HSV vector produced past MOI = x−5 at nine dpi was approximately 2.7-fold college than that of the vector produced by MOI = 10−2 at ix dpi (Figure 1E). These observations validated the gc/PFU ratio as an informative measure of the quality of our vector stocks. We likewise produced a closely related vector, JΔNI8, which was derived from JΔNI5 by deletion of the virion host shutoff (vhs) cistron,22 and compared its yields with those of JΔNI5 over time at the optimal input MOI (10−5; Figure S1). Both vectors grew at a comparable rate in U2OS-ICP4/27 cells.
JΔNI5 Vector Growth at Dissimilar MOIs
Confluent U2OS-ICP4/27 cells in T225 flasks were infected with JΔNI5 vector at ten−2–10−half-dozen plaque formation unit of measurement (PFU)/jail cell. (A and B) Physical titers in gc/mL (A) and biological titers in PFU/mL (B) in the supernatants were measured daily past quantitative real-time PCR and standard plaque assay, respectively. (C) gc/PFU ratios were calculated based on the results of (A) and (B). (D and E) Cultures of rat primary dorsal root ganglia (rDRGs) were infected with JΔNI5 vector preparations (five,000 gc/prison cell) produced at MOIs of 10−2 or ten−5. At 4 d mail-infection (dpi), vector-mediated mCherry fluorescence was photographed (D) and relative mCherry mRNA levels were measured by qRT-PCR with normalization to 18S rRNA (E). Group comparisons were performed with Student's t test or one-way ANOVA with post hoc Dunnett's multiple comparison test. Differences between an input MOI of 10−5 and ten−2 were statistically highly significant (∗p < 0.05). Quantitative data are presented as means ± SD (n = three).
To explore whether the cell culture atmospheric condition influence the yield of JΔNI5 virus, we tested variations in pH, temperature, glucose, and serum concentration23 (Effigy two). A media pH of 7.5–8.0 resulted in the highest vector yield, whereas pH = 6 significantly inhibited virus product (Figure iiA). Virus growth at 33°C or 35°C resulted in the highest virus titers, reached at nine dpi (Figure 2B), while significantly lower yields were obtained at higher temperatures (37°C–39°C). To examine the effect of glucose concentration, nosotros first recorded the changes in its concentration in the media during vector product (Effigy S2A). The glucose concentration decreased over time in our system, consequent with previous observations.xx Daily supplementation of glucose then enabled us to maintain the glucose concentration throughout the menstruation of vector production (Figure S2B). To make up one's mind the glucose requirement during virus growth, we propagated JΔNI5 virus under naturally decreasing levels of glucose (group A), at a abiding glucose concentration due to daily supplementation (grouping B), or in the absence of glucose in the culture medium (grouping C). The supplementation of glucose did not influence virus growth, whereas its absence clearly inhibited viral replication (Effigy 2C). To examine whether serum concentration affects virus production, nosotros tested iii dissimilar serum concentrations (0%, five%, and x%). As shown in Figure iiD, no significant differences in virus growth kinetics or yield were observed, suggesting that serum can be eliminated from the culture medium in our organisation.
Effects of Cell Civilisation Weather condition on JΔNI5 Vector Product
Confluent U2OS-ICP4/27 cells in T75 flasks were infected with JΔNI5 virus at an MOI of ten−5 PFU/cell for 2 h. (A–D) The infected cells were then incubated in media at various pH values (A), temperatures (B), glucose concentrations (C), or serum concentrations (D). Supernatant titers in gc/mL were determined daily by quantitative real-time PCR. Group comparisons were performed with one-way ANOVA with post hoc Dunnett'south multiple comparison test. Statistically significant differences betwixt pH 7.5 and pH six are indicated by asterisks (∗p < 0.05). Differences between 33°C and 37°C were statistically highly meaning (∗p < 0.05). Statistically pregnant differences between 4.v m/50 and 0 thousand/L glucose are indicated (∗p < 0.05). Grouping A, glucose levels decreasing from the initial 4.v g/L concentration; grouping B, glucose levels maintained at ~4.v g/L; group C, glucose-free media. Data are presented as means ± SD (n = iii).
Effect of Frequency of Media Drove on JΔNI5 Vector Production
Next, we asked whether the frequency of culture media drove could bear on virus production. Four media collection protocols were tested (Effigy 3A). In each protocol, whole culture supernatant was nerveless on the days indicated by circles in the figure and replaced with fresh medium, namely, culture supernatant was collected iv times at four, half-dozen, 8, and ten dpi in protocol (a), and 3 times at half-dozen, viii, and 10 dpi in protocol (b). Alternatively, we harvested culture supernatant 2 times at half-dozen and 10 dpi in protocol (c) and only in one case at 10 dpi in protocol (d). The virus growth curves (Figures threeB and 3C) and accompanying changes in gc/PFU ratios (Figure 3D) were very similar between the protocols, even protocol (a) involving multiple media replacements. Total virus yields and gc/PFU ratios are listed in Table 1. The total gc of protocols (a) and (b) were higher relative to protocol (d) and the total PFU of protocol (b) was approximately 2-fold higher than that of protocol (d). In addition, the total gc/PFU ratio of protocol (b) was superior to that of all other groups. Together, these results indicated that frequent media drove can amend vector yield and quality.
Effect of Media Substitution Frequency on JΔNI5 Vector Production
(A) Timeline of media changes in four experimental groups (a–d). Circles in the diagram stand for complete media changes. (B and C) Confluent U2OS-ICP4/27 cells in T75 flasks were infected with JΔNI5 virus at an MOI of 10−5 PFU/cell and viral titers in the media were measured daily by quantitative real-time PCR (B) and standard plaque assay (C). (D) gc/PFU ratios were calculated from the results of (B) and (C). Information are presented as means ± SD (n = 3).
Table 1
Summary of Vector Yields Using Different Media Replacement Protocols (n = 3)
| (a) | (b) | (c) | (d) | |
|---|---|---|---|---|
| Total gc | seven.viii ± 0.91 × 10ten | 6.0 ± 0.24 × ten10 | 4.nine ± 0.44 × 1010 | 4.9 ± 0.10 × 1010 |
| Full PFU | iv.3 ± 1.5 × 107 | 6.0 ± 1.vii × 107∗ | 3.2 ± 0.75 × xvii | 2.nine ± 0.93 × 10vii |
| Total gc/PFU | 1,816 ± 607 | 990 ± 371 | 1520 ± 267 | one,665 ± 506 |
Enhancement of Non-toxic HSV Vector Production by Treatment with Chemical Agents
It has been shown that HDAC inhibitors and BET motif inhibitors enhance oncolytic HSV replication in tumor cells but not normal cells.24, 25, 26, 27, 28 Therefore, nosotros evaluated whether these agents tin can likewise promote non-toxic HSV replication in U2OS-ICP4/27 cells, which are derived from an osteosarcoma tumor cell line. Based on previous reports, we selected the nine agents listed in Table 2 for initial analysis. We infected U2OS-ICP4/27 cells with JΔNI5, treated the cells with the indicated agents at different doses, and measured the virus titers at 6 dpi. For accurate comparison to negative controls, experiments were divided into ii groups based on the solvent (Table ii; Figures fourA and 4B). All HDAC inhibitors showed dose-related increases in vector yields compared with the control while BET inhibitor JQ1 did non enhance JΔNI5 virus production. In detail, meaning increases in vector yields were observed with Valproic acid (VPA, 10 mM), Belinostat (BEL, ten μM), sodium butyrate (NaB, 0.1 mM), and suberoylanilide hydroxamic acid (SAHA, 0.one μM). Considering SAHA showed the highest vector yield among these agents, we focused on SAHA for further experiments. Previous reports take suggested that pre-treatment of the cells with HDAC inhibitors followed by infection in the absence of inhibitor ("pre-treatment") is more effective in enhancing virus replication than in the continued presence of inhibitor ("co-treatment").28, 29, thirty We therefore compared these two atmospheric condition in our system. As illustrated in Figure 5A, SAHA removal prior to infection resulted in more efficient viral spread. Furthermore, the vector yield was slightly college using this protocol compared to co-handling (Figure vB). Nosotros also explored whether concentrations of SAHA between 0.ane and one μM analyzed in Effigy ivB would increase vector yield further (Figures 5C and 5D). Both the physical and the biological titer reached a meridian at 0.5 μM SAHA (i.82-fold compared to no-SAHA control, p < 0.05), while the gc/PFU ratio was similar to that of the command (Table 3), indicating that SAHA tin increase the yield of not-toxic HSV vector without reducing its quality.
Table 2
Chemic Agents Used in This Report
| Inhibitor | Solvent | Concentration | |
|---|---|---|---|
| HDACa | VPA (Valproic acrid) | dH2O | 100~0.1 mM |
| BEL (Belinostat) | dH2O | 100~0.1 μM | |
| NaB (Sodium butyrate) | dH2O | ane~0.01 mM | |
| APHA8 (APHA chemical compound 8) | DMSO | 50~0.5 μM | |
| PAN (Panobinostat) | DMSO | 100~1 nM | |
| SAHA (Suberoylanilide hydroxamic acid, Vorinostat) | DMSO | one~0.01 μM | |
| TSA (Trichostatin A) | DMSO | 250~2.five nM | |
| BETb | JQ1 | DMSO | 300~3 nM |
Effect of Chemical Agents on JΔNI5 Vector Production
Confluent U2OS-ICP4/27 cells in 24-well civilisation dishes were treated with HDAC or BET inhibitors for 2 h prior to infection with 10−v viral PFU/cell. At vi dpi, supernatants were harvested and viral vector titers adamant by quantitative real-time PCR. (A and B) Agents were analyzed in 2 groups according to their solvent, dH2O (A) or dimethyl sulfoxide (DMSO) (B); controls were the respective solvents alone. Group comparisons were performed with one-style ANOVA with post hoc Dunnett'south multiple comparison test. (∗p < 0.05). Data are presented as means ± SD (due north = 3).
Comparison of Viral Yields under Different SAHA Treatment Conditions
Confluent U2OS-ICP4/27 cells in T225 flasks were treated with 0.5 μM SAHA for 2 h. Cells in the "co-handling" group were then infected with JΔNI5 virus. Cells in the "pre-treatment" group were washed with PBS and fresh medium without SAHA was added prior to viral infection. All infections were performed with 10−5 viral PFU/cell. (A and B) Fluorescence was recorded at seven dpi (A) and the titers of supernatant samples were analyzed by quantitative real-fourth dimension PCR at 9 dpi (B). (C and D) Confluent U2OS-ICP4/27 cells in T225 flasks were treated with the indicated concentrations of SAHA for two h and infected with JΔNI5 vector in SAHA-free media. Supernatant samples were collected at 9 dpi and viral titers determined by quantitative real-time PCR (C) and standard plaque assay (D). Differences between pairs were analyzed by Educatee's t test for (B), and group comparisons (C and D) were performed with Student'south t examination or one-way ANOVA with post hoc Dunnett's multiple comparing test. Data are presented every bit the ways ± SD (n = three; ∗p < 0.05, ∗∗p < 0.01 compared to solvent).
Table 3
gc/PFU Yields at Different SAHA Concentrations (due north = 3)
| Concentration (μM) | 0 | 0.1 | 0.25 | 0.5 | 1.0 |
|---|---|---|---|---|---|
| gc/PFU | 1,695 ± 398 | ane,580 ± 491 | one,799 ± 542 | 1,474 ± 185 | 1,906 ± 39 |
Induction of Non-toxic HSV Vector Release from Producer Cells by Salt Treatment
Viral vectors tin be recovered from the surface of producer cells by treatment with sodium chloride31,32 or cesium chloride.33 Hence, we next evaluated whether these salts can function to promote the release of trapped virus also in our organisation. Culture supernatants were harvested at 9 dpi and fresh media with or without the indicated salt was added to the cells (Figures 6A and 6B). Consistent with previous results, both sodium chloride and cesium chloride induced the release of the JΔNI5 virus from its producer cells. The physical and biological titers peaked at 0.25 M sodium chloride handling with a 37-fold increase in gc compared to the control (p < 0.05). The released virus was infectious and superior in gc/PFU ratio to the control (Table four), showing that both salts can better the recovery of active virus as well in our system.
Induction of JΔNI5 Virus Release from Producer Cells by Salts
Confluent U2OS-ICP4/27 cells in T75 flasks were infected with JΔNI5 virus at ten−5 PFU/jail cell. At ix dpi, supernatants were replaced with media supplemented with NaCl or CsCl at the indicated concentrations or media alone (control). (A and B) Cultures were then incubated at 33°C for six h and supernatants were nerveless and titered by quantitative existent-time PCR (A) and standard plaque assay (B). Group comparisons were performed with one-mode ANOVA with post hoc Dunnett's multiple comparison test. Data are presented as means ± SD (n = three; ∗p < 0.05 compared to command).
Table 4
gc/PFU Ratios of Viruses Released by Different Salt Concentrations (north = 3)
| Concentration (G) | gc/PFU | |
|---|---|---|
| NaCl | 0.1 | 1,462 ± 392 |
| 0.25 | 1,841 ± 480 | |
| 0.5 | one,443 ± 358 | |
| 1.0 | one,945 ± 29 | |
| CsCl | 0.1 | 2,343 ± 1133 |
| 0.25 | 1,303 ± 296 | |
| 0.5 | ane,251 ± 478 | |
| 1.0 | ane,052 ± 457 | |
| Control | – | 4,227 ± 1545 |
Comparison of Optimal and Conventional Production Procedures
We combined the optimal parameters established above for infection and civilization conditions and virus harvest to maximize virus yield and quality in what we refer to as the optimal protocol (Table v). To evaluate its performance, we performed JΔNI5 vector production in parallel using the optimal protocol and the conventional vector production method (Table 5, conventional protocol) and the vector yields and quality were compared (Table vi). The vector yield reached past the optimal protocol was higher than that accomplished by the conventional protocol (approximately 2.8-fold in gc titer, 3.8-fold in PFU titer). Furthermore, the gc/PFU ratio obtained using the optimal protocol was superior to that achieved with the conventional protocol (approximately 1.3-fold). To confirm the superior quality of the optimal protocol vector stock, we compared the transduction efficiencies of equal gc of the 2 preparations in rDRG cultures (Effigy vii). Equally expected, rDRG cultures infected with the optimal-protocol virus displayed more robust transgene expression than cultures infected with the conventional-protocol virus (Figure 7A). Lastly, we compared intracellular viral DNA levels and transgene mRNA levels between cultures infected with either virus stock. Total cellular DNA and mRNA were harvested at 4 dpi and analyzed by quantitative real-time PCR for the viral gD gene (Figure 7B) and qRT-PCR for the mCherry transgene (Figure 7C), respectively, equally previously described.17 The results demonstrated that both virus entry and transgene expression were increased in cultures infected with the optimal-protocol virus compared with cultures infected with conventional-protocol virus, confirming that the optimized protocol improved not only the yield, but also the specific activity of our virus stocks.
Table 5
Summary of Optimal and Conventional JΔNI5 Vector Production Methods
| Optimal Protocol | Conventional Protocol | |
|---|---|---|
| MOI | E-five | E-iv |
| Temperature | 33°C | 33°C |
| Serum | 0% | 0% |
| Chemical agent | SAHA (0.v μM, pre 2 h) | none |
| Vector release | NaCl (0.25 M, 2 h) | none |
| Medium collection | days 6, 8, 9 | day 9 |
Tabular array 6
Summary of Results from JΔNI5 Vector Production Runs using the Conventional and the Optimal Protocol (northward = 3)
| Total gc | Total PFU | gc/PFU | |
|---|---|---|---|
| Conventional protocol | 6.eight ± 0.35 × xx | 4.vii ± 2.three × ten7 | ane,827 ± 730 |
| Optimal protocol | 1.9 ± 0.17 × 1011 | i.8 ± 0.99 × x8 | i,408 ± 710 |
rDRG Transduction past JΔNI5 Viruses Produced by Optimal and Conventional Protocols
(A) rDRGs were infected with each JΔNI5 virus preparation at 5,000 gc/prison cell and mCherry fluorescence was photographed at 4 dpi. (B and C) Relative amounts of viral genomes (B) and mCherry mRNA (C) in rDRGs at iv dpi as determined past quantitative real-time PCR and qRT-PCR, respectively. Intracellular viral genome numbers and mCherry mRNA levels were normalized to cellular 18S rRNA genes and RNA levels, respectively. Differences between pairs were analyzed by Educatee'south t test. Information are presented equally means ± SD (north = three; ∗∗p < 0.01).
Discussion
Non-toxic HSV vectors are harmless high-chapters vehicles for therapeutic gene delivery, but they replicate extremely inefficiently in traditional complementing cell lines because of the functional deletion of all IE genes, which severely limits their clinical applicability. To accost these manufacturing concerns, nosotros previously generated a novel producer cell line, U2OS-ICP4/27,17 and the experiments described in this newspaper were aimed at optimizing the weather for loftier-yield and high-quality production of stocks of our non-toxic HSV vectors by these cells.
Our study demonstrates that several infection and cell culture parameters significantly affect the yield and quality of JΔNI5 virus preparations (Figures ane and two). A previous report showed that an MOI of 0.02 was the most suitable for replication-defective HSV vector production.20 However, here we establish that the optimal MOI for JΔNI5 vector production by our ICP4/ICP27-complementing U2OS cell line is 10−five. The difference is most likely related to the nature of the producer cells; in the by, complementing producer cells were typically based on Vero cells, which intrinsically restrict HSV infection and spread and exercise not support significant replication of ICP0 null HSV mutants.34, 35, 36 In contrast, U2OS cells are highly permissive for HSV infection37,38 and inherently complement ICP0 naught HSV mutants.20 These backdrop appear to favor the utilize of a very low MOI for the production of our ICP0 cypher JΔNI5 vector and virtually probable of other similarly defective vectors. As shown here, JΔNI5 infection at higher MOIs provided less robust virus replication (Effigy 1A) and lower maximum PFU titers, resulting in higher gc/PFU ratios (Figures 1B and 1C), suggesting a relative increase in lacking, non-infectious particle formation. The optimal cell culture parameters, including temperature, pH, and serum and glucose concentrations during JΔNI5 vector production were too different from those reported in other systems.21,39 Hence, our results demonstrate the value of varying the input MOI and prison cell-culture parameters when using a not-standard virus production protocol.
We determined the gc/PFU ratios of viruses produced under varying weather condition (Figures oneC and threeD; Tables 1, 3, 4, and 6) because these ratios reverberate the proportion of functional HSV particles in vector stocks; the college the ratio, the lower the pct of agile particles. Indeed, JΔNI5 virus preparations exhibiting a lower gc/PFU ratio showed higher transduction efficiencies in rDRG cultures than preparations with a higher gc/PFU ratio (Figures 1D, 1E, and seven). The presence of excess defective particles in vector stocks can crusade adverse events in gene therapy, including undesired immune responses.forty,41 Thus, monitoring of the gc/PFU ratio is essential for the establishment of optimal procedures to generate high-quality HSV stocks.
We evaluated the effect of HDAC inhibitors on virus production since these agents have been reported to enhance the replication of oncolytic HSV in cancer cells.24,25,28 Consequent with previous inquiry, most HDAC inhibitors tested in this study increased virus yield in our organisation (Figures iv and 5). In improver, cell conditioning with the HDAC inhibitor SAHA prior to infection followed by virus growth in SAHA-free media ("pre-treatment") was more constructive than infection and growth in the connected presence of the drug ("co-handling") (Figure v). Previous studies have shown that HDAC inhibitor pretreatment, but not cotreatment, promotes cistron expression of HSV and cell-cycle regulators such as p16 and p21, whereas it inhibits expression of interferon responsive genes including STAT1 and PKR.24 Thus, our results revealed that HDAC inhibitor-mediated modifications of gene expression contribute to the molecular mechanisms for high yield non-toxic HSV production. Chiefly, SAHA handling had no discernible impact on the gc/PFU ratio of the product (Table 3), indicating that the drug is capable of increasing vector yield without reducing vector functionality. Because SAHA is a simplified structural analogs of TSA and suitable for mass-production42 and SAHA has already been used to treat cutaneous T cell lymphoma in clinical practice and its safety has been confirmed,43 its use in the product of non-toxic HSV factor therapy vectors for potential future patient trials should non cause safe or regulatory concerns. However, the utilise of SAHA in large-scale vector production runs will require careful aligning of its concentration since a doubling of the optimal dose in our pocket-size runs was counter-productive (Figures vC and 5D).
Efficient viral vector release from producer cells simplifies vector purification and minimizes contagion of the product with cellular impurities. Sodium chloride,31,32 cesium chloride,33 heparin,44 and dextran sulfate44,45 accept all been used previously to promote virus disengagement from producer cells and membrane debris. Here, we documented productive recovery of JΔNI5 virus from our U2OS-based producer cells using either sodium chloride or cesium chloride treatment and determined the concentration of both salts for optimal vector release in this system (Figure six). Moreover, our results revealed that those handling weather condition did not adversely touch on the specific infectious activity of the production (Table 4).
In conclusion, we adult an efficient method for the production of our novel class of non-toxic HSV vectors by the optimization of infection parameters, including the MOI and the composition and exchange frequency of the culture media, and achieved an important comeback in the quality and yield of our paradigm of this class of vectors. In addition, we demonstrated that judicious use of HDAC inhibitors and sodium chloride increase the recovery of active virus. These results will contribute to the institution of a cost-effective and reproducible protocol for efficient product of highly defective, non-toxic HSV vectors for pre-clinical and clinical studies besides every bit in vitro transduction applications.
Materials and Methods
Cells and Viruses
U2OS-ICP4/27 cells were as described previously18 and were grown in DMEM (Thermo Fisher) with ten% (vol/vol) fetal bovine serum (FBS, Thermo Fisher), penicillin-streptomycin (P/S, Sigma), puromycin (1 μg/mL), and blasticidin (5 μg/mL). All animal care and use procedures were carried out in understanding with the Fauna Experiments Ethical Review Committee, and approved by the President of Nippon Medical School (Blessing number 28-037). Fetal rat dorsal root ganglia (rDRGs) were microdissected from day 21 rat embryos, dissociated with 5 mg/mL collagenase A (Roche), ane mg/mL Dispase II (Roche) in PBS for thirty min at 37°C with abiding shaking, and then treated with 0.125% Trypsin/EDTA (Sigma) for 30 min at 37°C with abiding shaking. Afterwards dissociation, rDRGs were washed twice with DMEM/F12 (Thermo Fisher) and plated on poly-D-lysine/laminin-coated coverslips (BD Biosciences) at five × 10four cells per well in 24-well dishes in 500 μL of rDRG culture medium (Neurobasal-A medium, Thermo Fisher) with 2% B-27 supplement (Thermo Fisher), P/South, 50 ng/mL nerve growth cistron-7S (Sigma), and ii ng/mL GDNF (R&D Systems). At one d post-plating, rDRGs were treated with arabinofuranoside hydrochloride (Sigma) in the above media for vii d to remove dividing cells. Cells were then incubated with fresh rDRG culture media equally above. HSV transduction was performed vii d after plating. JΔNI5 and JΔNI8 vectors were amplified and titered on U2OS-ICP4/27 cells as described.eighteen,23
Virus Growth Curves
When using T225 flasks, triplicate flasks of two × ten7 U2OS-ICP4/27 cells were infected with JΔNI vector at a MOI (in PFU/cell) indicated in the respective figure legends for 2 h at 37°C and 5% COtwo prior to incubation at 33°C and 5% CO2. For culture parameter optimization, triplicate T75 flasks of 6.7 × 106 U2OS-ICP4/27 cells were used. The supernatants were collected daily for virus titration. Viral titers were determined past quantitative real-fourth dimension PCR for the gD gene or standard plaque assay on U2OS-ICP4/27 cells. For optimization of the glucose concentration, cells were grown in D-MEM (high glucose) with L-glutamine and phenol red (044-29765, Wako) or D-MEM (no glucose) with L-glutamine and phenol red (042-32255, Wako). In order to continue the concentration of glucose at ~four.v g/Fifty, D(+)-glucose (049-31165, Wako) was added to the culture media. For consecration of vector release, cells were infected every bit described above and supernatants were harvested 9 d later. Fresh media supplemented with NaCl or CsCl to dissimilar final concentrations were added to the cultures and incubation was connected for six h at room temperature. Supernatants were and then nerveless and their physical and biological viral titers determined.
Chemical Agents
HDAC and BET inhibitors and their commercial sources were every bit follows: APHA (three-[4-aroyl-1H-2-pyrrolyl]-N-hydroxypropenamide) compound 8 (A2478, Sigma), Belinostat (CS-0453, ChemScene), NaB (B5887, Sigma), Panobinostat (PAN; CS-0267, ChemScene), SAHA (Vorinostat; 10009929, Cayman Chemic), Trichostatin A (TSA; T-8552, Sigma), VPA (P4543, Sigma), and JQ-1 (2070-i,5, BioVision). Stock solutions of VPA, BEL, and NaB were prepared in dH2O and the remaining stock solutions (APHA8, PAN, SAHA, TSA, and JQ-1) were prepared in dimethyl sulfoxide (DMSO; 472301, Sigma). Sodium chloride (191-01665) and cesium chloride (037-19685) were purchased from Wako. For evaluation of SAHA treatment conditions, confluent U2OS-ICP4/27 cells in T225 flasks were treated with 0.5 μM SAHA for two h prior to viral infection. Cells were then infected with JΔNI5 virus at 10−five (co-treatment group) or supernatants were replaced with SAHA-free media prior to infection at the same MOI (pre-treatment group). Fine-tuning of the SAHA concentration was performed in the pre-treatment protocol.
qRT-PCR and Genomic Quantitative Real-Time PCR
rDRGs were seeded in 15 wells of a 24-well tissue civilization dish and infected with JΔNI5 virus at v,000 gc/cell. At four dpi, cells were harvested and pooled in groups of five, genomic DNA and total RNA were extracted from each group by RNeasy Mini kit (QIAGEN), and RNA was contrary transcribed by SuperScript IV Reverse Transcriptase (Thermo Fisher). Genomic DNA and cDNA were analyzed by quantitative existent-time PCR in triplicate using the 7500 Fast Existent-Time PCR Organisation (Practical Biosystems). Results were normalized to cellular 18S rRNA genes or RNA. PCR primers used in this study were as described.18
Statistical Analyses
All values are presented as the mean ± SD. Differences betwixt pairs were analyzed past Educatee's t examination or one-way analysis of variance (ANOVA) with post hoc Dunnett's multiple comparison exam using Microsoft Excel 14.seven.7 or IBM SPSS statistics version 25.0. p values below 0.05 (p < 0.05) were considered statistically meaning.
Author Contributions
S.G. and Y.M. conceived and designed the research. S.1000., Y.1000., G.A., Chiliad.Y., Y.S., and H.M. performed the experiments. Southward.M. and Y.One thousand. analyzed the data. S.K. and Y.M. wrote the manuscript. Y.Chiliad., W.F.1000., J.B.C., J.C.G., and T.O. supervised the enquiry. All authors reviewed and edited the manuscript.
Conflicts of Interest
Y.Thousand., J.B.C., and J.C.G. are co-inventors of intellectual property licensed to Coda Biotherapeutics, and J.B.C. and J.C.G. are co-inventors of intellectual property licensed to Oncorus. J.C.Grand. is a founder and consultant of Coda Biotherapuetics and Oncorus. W.F.K. is a consultant of Oncorus.
Acknowledgments
We are grateful to Taro Tomono (Tsukuba Academy) for virus product, Makoto Sukegawa (Nippon Medical School) for prison cell culturing, and Guillermo Posadas-Herrera (Nippon Medical School) for comments on the manuscript. This research was supported by grants to Y.Thousand. and Due south.K. from Japan Society for the Promotion of Scientific discipline (JSPS) Grant-in-Aid for Scientific Research JP16K08249 and Japan Medical Schoolhouse Grant-in-Aid for Medical Research.
Footnotes
Supplemental Information
Document S1. Figures S1 and S2:
Document S2. Article plus Supplemental Information:
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