Abstract
We evaluated the use of the Product Enhanced Reverse Transcriptase (PERT) assay as a means of detecting virus in retroviral vectors products pseudotyped with Gibbon Ape Leukemia Virus (GALV) and Vesicular Stomatitis Virus G (VSVG) envelopes. PERT provides greater standardization than the S+/L− assay which has been used extensively in virus detection. A challenge is that PERT will also detect residual retroviral vectors as vector particles contain reverse transcriptase. Vector products were cultured for 3 weeks on HEK293 cells to amplify any potential virus. In addition, vector supernatant and end-of-production cells were spiked with GALV to evaluate for inhibition by the test article. Results of PERT and the S+/L− assay were compared. PERT and S+/L− assays were both effective in detecting virus. Vector supernatants were negative at the end of 3 weeks of culture by PERT for both GAVL and VSVG pseudotyped vector. In contrast, end-of-production cells were positive by PERT due to persistent vector producing cells. A one-week culture of cell-free media obtained at the 3 weeks timepoint allowed distinction of virus-free test articles from those with virus. The PERT assay is suitable for detecting replication competent retrovirus in vector products pseudotyped with GALV and VSVG envelopes.
Keywords: Replication competent retrovirus, gene therapy, safety, product enhanced reverse transcriptase assay
1. Introduction
Retroviral vectors based on murine leukemia viruses were the first vectors used in humans(Blaese et al., 1995; Rosenberg et al., 1990) and are now approved by regulatory agencies for use in cancer and other diseases.(Exothera, 2021; FDA, 2023) This virus is amenable to the generation of packaging cell lines, which provided efficient and consistent vector production,(Miller, 1990) but early cell lines were prone to recombination leading to replication competent retroviruses (RCR).(Muenchau et al., 1990) Evaluations of RCR in mice and non-human primates were shown to cause malignancy that resembled the cancer seen in mice infected with the parent murine leukemia virus.(Cornetta et al., 1993; Donahue et al., 1992)
The demonstrated pathogenicity of RCR in animal models,(Cornetta et al., 1993; Donahue et al., 1992) and the association of retroviral vectors with insertional oncogenesis in humans,(Wu and Dunbar, 2011) has led regulatory bodies to require extensive screening of vector production for RCR. Testing is required at the manufacturing stage (vector products and end-of-production cells), transduced cell products (ex., transduced hematopoietic stem/progenitor cells and CAR-T cells), and in treated patients.(Cornetta et al., 2023) The FDA Guidance states an expectation that RCR assays for vector and cell products be cultured for at least 3 weeks of culture in order to permit amplification of low titer virus.(FDA, 2020) Our laboratory has used the PG-4 S+/L− (sarcoma positive, leukemia negative) cell line to detect RCR at the end of the 3 week culture.(Bassin et al., 1982) This cell line contains a murine sarcoma virus genome that leads to cell transformation in the presence of a murine leukemia virus.
While our laboratory has found the S+/L− assay to be quantitative and reliable,(Chen, Reeves, and Cornetta, 2001) it does require highly skilled technicians to identify foci and adds one week of cell culture time to the assay. In this manuscript, we compared the potential of the Product Enhanced Reverse Transcriptase (PERT) Assay(Sastry et al., 2005) to replace S+/L− assay as an indicator of RCR. PERT utilizes qPCR technology to quantify the level of reverse transcriptase (RT) activity in a test article. In this paper, we document the assay is sensitive and suitable for replacing the S+/L− assay, but it does not eliminate the need for further cell culture after the 3-week amplification phase for certain test articles.
2. Material and methods.
2.1. Cell Line and Positive Control Preparation.
HEK293 (American Type Culture Collection ATCC, Manassas, VA, catalog CRL-1573) and Mink GALV SEATO cells (a kind gift of M. Eiden and C. Wilson, National Institutes of Health, Bethesda, MD) were maintained in D10 medium (Dulbecco’s modified Eagle medium (Invitrogen, Carlsbad, CA) with 10 % fetal bovine serum [FBS; Hyclone, Logan, UT], 2 mM L-glutamine [Invitrogen], 1 mM Sodium Pyruvate, and 100 units/ml penicillin and 100 μg/ml streptomycin [Invitrogen]). PG4 cells (ATCC, CRL-2032) were maintained in McCoys 5A media (Thermo Fisher Scientific, Waltham, MA, USA) with 10% fetal bovine serum and 100 units/ml penicillin and 100 μg/ml streptomycin. All cell lines were maintained at 37° C and 5% CO2. The neomycin resistance gene expressing vector G1PLII generated by a stable clone of PG13 cells.(Hanazono et al., 1999) The RT value of the vector supernatant was 2.14 × 107, and the PG13 clone had previously been found to be negative for RCR. After vector was collected, EOP cells were trypsinized and then stored in 10% DMSO at < −70 C°.
2.2. PERT Assay.
The assay was based on the method previously described.(Sastry et al., 2005) Samples were mixed with an equal volume of disruption buffer consisting of 40 mM of Tris-Buffer pH 7.5 (Invitrogen, Carlsbad, CA, USA), 50 mM of KCL (Invitrogen), 20 mM of dithiothreitol (DDT) (VWR, Radnor, PA, USA), and 0.2% of Nonidot-P40 (G-BioSciences, St. Louis, MO, USA). A standard curve of ten-fold serial dilutions of purified HIV reverse transcriptase (HIV-RT) (Worthington Biomedical, Lakewood, NJ, USA) is prepared for each assay. Each sample was tested in triplicate on a 96-well PCR plate (Applied Biosystems, Waltham, MA, USA). In each well, 5 μL of samples were mixed with 20 μL of master mix. Master mix per reaction consists of 5.3 μL of Diethylpyrocarbonate-water (Invitrogen), 0.5 μL of 22.5 μM PERT-F, 0.5 μL of 22.5 μM PERT-R, 0.5 μL of 10 μM probe, 0.25 μL of 40 U/μL RNasin (Sigma-Aldrich, St. Louis, MO, USA), 0.45 μL of MS2 RNA (Sigma-Aldrich), and 12.5 μL of TaqMan 2X Universal PCR Master Mix, (Thermo Fisher Scientific). The positive control contains 5×106 molecules in 20 μL of master mix. Media and no template controls serve as background and negative controls. PERT reactions contain forward primer (PERT-F, 5’-TCCTGCTCAACTTCCTGTCGA-3’), reverse primer (PERT-R, 5’-CACAGGTCAAACCTCCTAGGAATG-3’), and probe (5’-FAMCGAGACGCTACCATGGCTATCGCTGTAG-TAMRA-3’) and analyzed in the Applied Biosystems™ 7500 Real-Time PCR System. The reaction was programmed as follows: 1 cycle of step 1 (48°C for 30 min.), 1 cycle of step 2 (95°C for 10 min.), and 40 cycles of step 3 (95°C for 15 sec. followed by 60°C for 1 min.).
2.3. GALV RCR Amplification Phase
On Day −1 of the amplification phase, 2 × 106 HEK293 cells were placed into a 25 cm2 flasks intended to test the negative control, positive control, and supernatants. For flasks intended to evaluate EOP cells, the flasks receive 2 × 106 HEK293 and 4 × 105 EOP cells. After an overnight incubation, media was removed from each flask and replaced with 1 ml of D10 media (the 3 negative controls and the EOP cell flasks), 1 mL of G1PLII vector, or 1 mL of GALV/SEATO virus at the TCID50 (5 positive control flasks). To evaluate potential inhibition by vector (inhibition control), HEK293 cultures were inoculated with 1 mL of G1PLII vector spiked with GALV/SEATO virus at 5 times or 50 times the TCID50. To test for inhibition by EOP cells, flasks with HEK293 and EOP cell received GALV/SEATO at 5 times and 50 times the TCID50. All cultures received Polybrene® (8 μg/mL) and where cultured for 4 to 6 hours after which media was removed and replaced with D10 media. Cultures were maintained in log phase growth for 3 weeks with a minimum of 5 passages. Media was harvested at the end of the 3 weeks, passaged through a 0.45 micron filter and mixed 1:1 with PERT lysis buffer. A portion of the remaining media was frozen and stored at < −70 C°. Media was also used to inoculate flasks in the S+/L− Indicator Phase or HEK293 Indicator Phase.
2.4. VSV-G RCR Amplification Phase
The assay was set using the same method as described above. As the test article was larger in size, larger culture flasks were utilized. To maintain sensitivity, the vector supernatant ratio should be approximately 1 mL per 105 HEK293 cells plated, and 1 EOP cells per 5 HEK293 cells plated.
2.5. S+/L− Indicator Phase
On Day −1, PG-4 cells were plated in 6 well dishes with 4 mL of McCoy’s 5A media and 1 × 105 cells per well. On Day 0, the media was removed from the wells and for each test article and control, 3 wells were evaluated; two wells containing 1 mL of undiluted amplification phase media and 1 well containing 1 mL of amplification media diluted 1:100. Polybrene® (8 μg/mL) is added to all control and test articles. Cultures were incubated from 4–6 hours and the media replaced with 4 mL of McCoy media. On day 2, the medium was removed and replaced. When wells were confluent (Day 4–6) they were inspected independently by two technicians for foci using an inverted microscope. For an assay to be acceptable, negative controls must be without foci, and one or more wells of the positive controls plated at the TCID50 must contain foci. A test article is considered positive for RCR if one or more foci are detected.
2.6. HEK293 Indicator Phase
On day −1, 2 × 106 naïve HEK293 cells were plated in 25 cm2 flasks. On Day 0, media was replaced with 1 ml of fresh D10 (negative control) or cell-free media from amplification phase cultures. All cultures received 8 μg/mL of Polybrene® and cultured for 4 to 6 hours after which media was replaced with D10 media. The indicator phase cultures were passaged at least twice over a minimum of 7 days at which time media was collected, filtered (0.45 μL) and analyzed by PERT. Reserve samples were stored at < −70 C°. Each assay evaluated triplicate flasks (except for the 50 X TCID50 cultures where a single culture was evaluated).
3. Results
3.1. PERT Values in Cell-Free and Cell-Conditioned Media
Our current assay for detecting RCR uses a two-stage approach (Figure 1A). The initial Amplification Phase exposes HEK293 cells to test articles then maintains cells in log phase growth for 3 weeks in compliance with current US FDA Guidance.(FDA, 2020) We previously reported that HEK293 cells are highly susceptible to infection by the GALV/SEATO virus and will amplify the virus to high titer.(Chen et al., 2001) The second phase of the assay is the Indicator Phase, in which cell-free media from confluent Amplification Phase cultures are tested for RCR in the S+/L− assay.
Figure 1.
Description of the Replication Competent Retrovirus assay. A. Our current assay subjects HEK293 cells are exposed to test articles then passaged for 3 weeks (Amplification Phase). Media from confluent cultures are then filtered (0.45 micron) and evaluated in the S+/L− assay. B. The modified assay tests filtered amplification phase media by Product Enhanced Reverse Transcriptase assay directly and after one week of passage on naïve HEK293 cells (Indicator Phase).
To determine the potential of PERT replacing the S+/L− assay, we began by assessing the lower limits of detection of virus in the PERT assay. Figure 2A illustrates the PERT activity in various media. Fetal bovine serum is associated with higher RT activity, but the overall contribution is low, with a RT value of 1.4. Figure 2B compares RT values for serial dilutions of GALV/SEATO virus in serum containing media, with and without correction for background media RT. The curves are superimposable up until the 1× 105 dilution, where RT activity in media impacts the detection of GALV/SEATO. At the 1 × 106 and 1 × 107 dilutions, the RT value is within the range of D10 media alone. The GALV/SEATO stock used in Figure 2 has a TCID50 of 1 × 105 (calculated titer of 7 × 104 infectious units, IU). Figure 2C compares the RT activity and the estimated number of infectious units at each of the dilutions tested. At the 1 ×106 and 1 ×107 dilutions, the estimated number of infectious GALV/SEATO virus particles is less than 1.
Figure 2.
Reverse Transcriptase activity in media spiked with serial dilutions of GALV SEATO virus stock. A. Media with or without serum. B DMEM media with 10% fetal bovine serum spiked with serial dilution of GALV/SEATO virus. C. Comparison of PERT values and estimated Infectious Units per PERT reaction.
To determine the role cells may play in background RT activity, cell-conditioned medium were evaluated at the end of 3 weeks of culture. Table 1 shows the results from 126 negative control cultures from 42 RCR assays. The media had a higher baseline and range of RT values than the media alone. Based on this data, an RT value of 10 was set as a tentative cut off value for test articles to be considered positive.
Table 1.
Number of Reverse Transcriptase Molecules estimated per reaction for negative control assays at the end of 3-week amplification phase.
| Total replicates | 126 |
| Mean of replicates | 0.9698 |
| Max replicate value | 8.7862 |
| Min replicate value | 0.1165 |
| Median replicates | 0.5537 |
| standard deviation (SD) | 1.2669 |
| Mean + 2SD | 3.5036 |
PERT was then used to assess RT in Amplification Phase positive control cultures (inoculated with GALV/SEATO at the TCID50 and passaged for 3 weeks). Shown in Figure 3A are RT values for 35 positive control cultures from 7 assays. All controls were positive for RCR, and samples that had lower FFU by S+/L− also had lower RT values by PERT. Most positive control samples had PERT values 4 to 5 logs higher than the proposed positive cut-off value of 10 RT.
Figure 3.
Assay Control PERT Analysis at the end of the Amplification Phase. A. Negative Control Cultures B. Positive control cultures infected with GALV/SEATO virus at the TCID50.
3.2. PERT Evaluation of Test Articles
To evaluate the level of RT activity in cultures exposed to test articles, the G1PLII vector was assessed in a modified RCR assay. (Figure 1B). The evaluation included negative controls (media alone), G1PLII vector supernatant and end-of-production (EOP) cells, and positive controls inoculated with GALV/SEATO at the TCID50. To evaluate potential inhibition by vector or EOP cells, cultures of G1PLII supernatant and EOP cells were inoculated with GALV/SEATO at 5 or 50 times (5X or 50X) the TCID50. The experiment was performed three times.
The mean RT values were less than 10 for media, negative controls, and G1PLII vector in both the amplification and indicator phases (Figure 4A). EOP cells were positive in the amplification phase, but RT values were less than 10 in the indicator phase. This is consistent with continued vector production by EOP cells during the amplification phase with subsequent loss of vector through integration and dilution during passage in the Indicator Phase. In contrast, G1PLII vector and EOP cells spiked with GALV/SEATO were positive in both the amplification and indicator phase (Figure 4B). Figure 4C evaluates the range of PERT values in the indicator phase and supports our hypothesis that a value of 10 may be an appropriate cutoff for determining whether a sample is RCR negative.
Figure 4.
Reverse Transcriptase Values for in Assays Evaluating the G1PII Vector. A and B and C. A. The mean and standard deviation for RT testing in negative controls and G1PLII test articles. Mean of 9 samples. AP = amplification phase, IP = Indicator Phase. B. RT values for Positive Control and Inhibition Controls for supernatant and EOP cells. IP = Indicator Phase samples evaluated G1PLII test articles spiked with 5 or 50 times the GALV/SEATO TCID50. Values represent the mean of 9 samples except for the 50 times TCID50 which represents mean of 3 samples. C. The minimum and maximum value is provided for each Indicator Phase sample tested.
3.3. PERT for Detecting RCR in VSVG-Pseudotyped Vectors
While the GALV envelope has been used extensively in clinical trials, investigators are exploring alternative pseudotypes, one of which is the Vesicular Stomatitis Virus G protein (VSV-G). In seeking to adapt our current RCR assay, the first challenge was assessing the ability of VSV-G pseudotyped vector to transduce HEK293 cells. We prepared a GFP expressing retroviral vectors pseudotyped with the VSV-G or GALV envelopes by transient transfection and transduced HEK293 and PG-4 cells. Vectors were normalized by RT activity (5.26 × 107 RT/mL) and were diluted 1:5 and 1:20 before exposing cells to the vector. Higher levels of gene transfer were noted with the VSV-G pseudotyped vector compared to GALV (Figure 5). Due, in part, to the fusogenic nature of the VSV-G envelope, we assess cell toxicity of different vector preparations with RT values ranging from 1 × 106 to 4 × 107. When cell counts were compared 10 days after vector exposure, there was no statistical difference in cell number, but a trend starting at 3.4 × 107 suggested inhibition of growth may occur at higher concentrations (data not shown).
Figure 5.
Transduction of HEK293 and GP4 cells with VSV-G and GALV pseudotyped retroviral vectors. Retroviral vectors pseudotyped with VSV-G and GALV envelopes were standardized by PERT before transducing HEK293 (A and B) and PG4 (C and D) cells. The vectors were diluted at 1:5 (A and C) or 1:20 (B and D) with culture media. Mock groups were transduction using culture media without vectors. Cells were harvested 3 days post-transduction and analyzed by flow cytometry for GFP expression. The results shown were representative of three independent experiments.
To evaluate RCR detection in a clinical vector product, we evaluated the 8R-70CAR (IL-8 receptor-linked CD70CAR) vector. The vector was generated by transient transfection and 560 mL was diluted 1:1 to 1120 mL with D10 media to a RT value of 4.1 × 106. In the RCR assay the vector supernatant was divided among 13 300-cm2 flasks and 1 × 108 EOP cells were divided among 24 300-cm2 flasks (vector volume and EOP cell number to HEK cell number using previously established to maintain assay sensitivity).(Chen et al., 2001) As the S+/L− assay has been our gold standard, Amplification Phase media was evaluated and was negative for RCR in the negative control, vector and EOP cell cultures (0/3, 0/13, and 0/24 cultures positive, respectively). Inhibition, and positive controls were positive (5/5 and 3/3 positive respectively).
As a preliminary evaluation of PERT as an alternative to S+/L−, Amplification Phase media were assessed. The results were similar to that seen with GALV vector with RT values above 10 in Amplification Phase EOP, although the values were not as high as that seen with G1PLII (Table 1). This is not unexpected as the PG13/G1PLII packaging cells are stable producer cell lines that can continue to produce vector during the Amplification Phase. The 8R-70CAR vector was generated by transient transfection in HEK293T cells and production of vector will decrease over time. As noted in Table 2, persistence of EOP cells was confirmed by evaluating media for SV40 large T antigen DNA at the end of the Amplification Phase (the SV40 large T antigen is what distinguishes HEK293 from HEK293T cells).
Table 2.
Comparison of Amplification Phase Samples analyzed by PERT to corresponding Indicator Phase samples analyzed by S+/L−.
| PERT RT value Amplification Phase | S+/L− Results Indicator Phase | |
|---|---|---|
| Negative Control #1 | 0.578 | Negative |
| Negative Control #2 | 0.604 | Negative |
| Negative Control #3 | 0.482 | Negative |
| Positive Control #1 | 5.43 × 105 | Positive |
| Positive Control #2 | 4.84 × 105 | Positive |
| Positive Control. #3 | 5.14 × 105 | Positive |
| EOP cells #1 | 2.62 × 103 | Negative |
| EOP cells #12 | 5.76 × 103 | Negative |
| EOP cells #24 | 6.03 × 103 | Negative |
| Supernatant #1 | 13.256 | Negative |
| Supernatant #7 | 1.216 | Negative |
| Supernatant #13 | 1.103 | Negative |
| Inhibition Control EOP cells #1 | 1.43 × 106 | Positive |
| Inhibition Control EOP cells #2 | 1.56 × 106 | Positive |
| Inhibition Control EOP cells #3 | 1.27 × 106 | Positive |
| Inhibition Control Supernatant #1 | 6.62 × 105 | Positive |
| Inhibition Control Supernatant #2 | 6.07 × 105 | Positive |
| Inhibition Control Supernatant #3 | 5.81 × 105 | Positive |
One finding of note was that Supernatant Flask #1, had a PERT value of 13.3 at the end of the amplification phase but was negative in the S+/L− assay. The sample was retested and PERT value remained above 10; repeat S+/L− testing remained negative.
4. Discussion
We have previously developed PERT for the detection of replication competent lentivirus (RCL) in an assay that complies with current FDA regulations.(Cornetta et al., 2018) PERT appears to be suitable as an indicator assay when testing vector supernatant for RCR. PERT can be utilized to test EOP cells if the cell-free amplification phase media is cultured on naïve HEK293 cells for a week prior to PERT analysis.
Both the S+/L− assay and PERT have advantages and disadvantages. S+/L− is a functional assay that requires replication competent virus, as such false positives are rare. The disadvantage is the requirement for a week of cell culture and experienced technicians to identify foci. The advantages of PERT include standardizable PCR technology, and the ability to obtain same-day results. Moreover, PERT can identify a wide variety of viruses expressing reverse transcriptase, while S+/L− assays are generally limited to a subset of gammaretroviruses. The downside is the inability to distinguish between RCR and replication-defective retroviral vectors.
Determining the RCR assay limit of detection is critical given that one RCR is above the acceptability criteria for a vector or cell product. Serum-containing media and cell-conditioned media had low level RT activity, likely due to cellular elements and impurities in molecular reagents.(Pyra, Boni, and Schupbach, 1994; Su et al., 2019) Of the 27 samples from Amplification Phase supernatants and Indicator Phase supernatant and EOP cells, at least one value was above 2 + the standard deviation of the mean. Evaluation of minimum and maximum values for GALV vector testing (Figure 3) shows that some samples were as high as 6.59 and one VSV-G pseudotyped vector sample had a value of 13.4. Our evaluation suggests that a value of > 10 RT raises the possibility of an RCR but this requires further monitoring as different serum lots, vector manufacturing methods, vector titers, and/or EOP cell types could impact the background level of RT activity. Given that most positive cultures had RT values greater than 10,000, test articles with RT values between 10 and 100 should be evaluated in the S+/L− assay before calling the sample positive for RCR.
There are two important considerations in determining an appropriate positive control for an RCR assay. Ideally, an assay positive control would be identical to the virus that the assay hopes to detect. Unfortunately, an RCR assay is expected to detect a recombinant virus whose structure cannot be predicted with certainty. Therefore, the assay positive control aims to confirm that adequate conditions existed for RCR infection and expansion. Secondly, the selection of an RCR positive control must balance function with the biosafety risk for lab personnel. For retroviruses, the gammaretroviral LTR present in most vectors is known to cause cancer in non-human primates and is implicated in hematologic malignancies in humans.
For our GALV RCR assay, we use the wild-type GALV/SEATO virus as the positive control. While GALV does cause disease in non-human primates, GALV infection has not been reported in humans. Generating a GALV pseudotyped RCR with gammaretroviral LTRs is likely to increase the biosafety risk and would not guarantee improved detection of an RCR. The biosafety concerns for generating a VSV-G pseudotyped RCR appear to be even greater. Like GALV/SEATO, VSV is zoonotic and could facilitate cross-species transmission. An important difference is VSV does infect humans.(Liu et al., 2021; Quiroz et al., 1988) Therefore, the biosafety risk of generating VSV-G pseudotyped RCR with gammaretroviral LTR appears even greater. As the GALV/SEATO virus meets the criteria for high infectivity and expansion in culture and lacks the biosafety issues associated with a VSV-G RCR, we have utilized this virus as the positive control in both assays.
Determining the amount of vector product to be tested can be challenging. The US FDA Guidance (FDA, 2020) states “In all cases, we recommend testing at least 5% of the total supernatant by amplification on a permissive cell line.” The Guidance then goes on to say “However, this recommendation may not be applicable to all current clinical manufacturing practices.” It then states, “Therefore, we now recommend that sufficient supernatant be tested to ensure a 95% probability of detection of RCR if present at a concentration of 1 RCR/dose equivalent.” While the Guidance does provide suggestions on how to calculate the amount of material needed to test (in the Guidance Appendix), the formula does not consider the concentration of vector. High titer vectors can significantly decrease the sensitivity of RCR detection due to receptor interference (i.e. vector particles overwhelming receptors and preventing RCR infection). When investigators wish to utilize the 1 RCR/dose equivalent option, we suggest performing a qualification study, with vector supernatant of similar titer to the clinical vector product, to ensure the RCR assay meets the expectations described in the Guidance. At this time, most investigators continue to request testing utilizing the 5% volume option.
In summary, PERT is an alternative to the S+/L− assay for RCR detection. When testing supernatant, PERT might allow direct testing after the 3-week Amplification Phase, which would shorten the assay time and cost. When testing EOP cells, the Amplification Phase media is still likely to contain vector particles that result in a false positive test. Passaging the media for another week appears to dilute particles to a level below detection by PERT.
Table 3.
Quantitative PCR analysis of amplification phase media for the presence of SV40 T antigen DNA as a surrogate for detecting HEK293T (EOP) End-of-Production Cells. The # designation indicates the replicate flask tested.
| Test Article Name | Test Article (SV40 T antigen copies per reaction) | Test Article Spiked with 100 Copies SV40 plasmid (SV40 T antigen copies per reaction) |
|---|---|---|
| Water | Undetected | Not tested |
| Negative Control #1 | Undetected | 135.2 |
| EOP cells #1 | 126.145 | 229.549 |
| EOP cells #12 | 181.185 | 311.643 |
| EOP cells #24 | 227.548 | 367.154 |
| Supernatant #1 | Undetected | 133.421 |
| Supernatant #7 | Undetected | 116.877 |
| Supernatant #13 | Undetected | 114.256 |
Highlights.
Retroviral vectors have been associated with cancers
Ensuring retroviral vectors are free of replication competent viruses is required by regulatory bodies
Distinguishing retroviruses from retroviral vectors requires biologic assays
Performance enhanced reverse transcriptase assays has similar sensitivity and fosters standardization in detecting replication competent retroviruses
Acknowledgements
This project has been funded in part with Federal funds from the National Heart, Lung, and Blood Institute, National Institutes of Health, Department of Health and Human Services, under Contract No. 75N92019D00018 to K.C. and from the Department of Defense W81XWH-20-1-0726 to J.H. The contents of this manuscript are the views of the authors alone. We thank Kyle Miller for his assistance with flow cytometric experiments. Figure 1 was created with BioRender.
Abbreviations:
- RCL
replication competent retroviruses
- RT
reverse transcriptase
- PERT
Product Enhanced Reverse Transcriptase
- GALV
Gibbon Ape Leukemia Virus
- VSV-G
Vesicular Stomatitis Virus G virus
- CAR
chimeric antigen receptor
Footnotes
Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Declaration of Competing Interest
Indiana University has licensed Replication Competent Lentiviral testing technology to Charles River Laboratories and Genezen Labs, of which K.C. and T-Y. L. receive a portion of the fees.
The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:
Indiana University has licensed Replication Competent Lentiviral testing technology to Charles River Laboratories and Genezen Labs, of which K.C. and T-Y. L. receive a portion of the fees. Note: we have not licensed replication competent retroviral testing assays, such as the one in this paper.
Data availability statement
The data presented in this paper is available from the corresponding author upon reasonable request.
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Data Availability Statement
The data presented in this paper is available from the corresponding author upon reasonable request.