Development of a Sensitive Assay for Detection of Replication-Competent Recombinant Lentivirus in Large-Scale HIV-Based Vector Preparations

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1 METHOD doi: /s (03) Development of a Sensitive Assay for Detection of Replication-Competent Recombinant Lentivirus in Large-Scale HIV-Based Vector Preparations Paul Escarpe, Nathalie Zayek, Peggy Chin, Flavia Borellini, Romain Zufferey, Gabor Veres, and Veronique Kiermer* Cell Genesys, Inc., 500 Forbes Boulevard, South San Francisco, California 94080, USA *To whom correspondence and reprint requests should be addressed. Fax: (650) Veronique.Kiermer@cellgenesys.com. Lentiviral vectors have demonstrated great potential as gene therapy vectors mediating efficient ex vivo and in vivo gene delivery and long-term transgene expression in both dividing and nondividing cells. However, for clinical studies it must be demonstrated that lentiviral vector preparations are safe and not contaminated by replication-competent recombinants related to the parental pathogenic virus. Here we describe a sensitive assay for the detection of replicationcompetent lentiviruses (RCL) in large-scale preparations of HIV-based lentiviral vectors. This RCL assay for lentiviral vectors is based on the principles used for retroviral vectors, using a highly permissive cell line, C , for RCL amplification and an appropriate positive control virus to establish the assay sensitivity. The assay is capable of detecting 1 RCL infectious unit in a background of transducing units of vector in a single test culture. Statistically representative samples from large-scale lentiviral vector productions were assayed using multiple test cultures for each lot. Overall, a total of transducing units of vector from 10 independent 14-liter production lots were screened and no RCL was detected. We propose to implement this assay as a release testing for clinical-grade lentiviral vector preparations intended for gene therapy clinical trials. Key Words: gene therapy, lentiviral vectors, safety, virus replication, biological assay INTRODUCTION Lentiviral vectors have the potential to be extremely valuable tools for human gene therapy. They share the advantages of the retroviral vectors derived from murine oncoretroviruses: a large cloning capacity, the lack of a requirement for viral gene expression, and the capacity to integrate into the target cell genome for stable transduction. In addition, they are highly efficient for in vivo gene delivery and they overcome a major limitation of the retroviral vectors by efficiently transducing nonmitotic cells such as neurons, endothelial cells, hepatocytes, and hematopoietic stem cells [1 6]. HIV-1-based vectors are also less sensitive to gene silencing during cell differentiation [7], another important advantage for achieving therapeutic effect when targeting stem cells. Despite the strong rationale for their utilization in human gene therapy and extensive preclinical data, concerns regarding their safety have hampered their testing in clinical trials. Lentiviral vectors are not able to replicate; however, a principal safety concern is the possibility that the parental pathogenic virus or a related replication-competent recombinant might be reconstituted. In an early study with retroviral vectors derived from murine leukemia virus, 3 of 10 rhesus monkeys that were administered ex vivo-transduced stem cells developed fatal lymphomas etiologically associated with the presence of high doses of contaminant replication-competent retroviruses (RCR) in the vector preparation [8]. This experiment, conducted with rudimentary vectors in severely immune-suppressed animals, was the precedent-setting study that demonstrated the risk of replication-competent recombinants and led to the development of improved vector systems and RCR detection assays. Extensive study of the pathogenesis of lentiviruses, in particular HIV-1, has facilitated the development of successive generations of lentiviral vectors with an increasing number of built-in safety features designed to prevent the reconstitution of a replication-competent lentivirus (RCL). In the current generation of HIV-derived lentiviral vectors, known as the third generation [9], the HIV acces /03 $30.00

2 doi: /s (03) METHOD sory genes vif, vpu, vpr, nef, and tat have been deleted from the packaging constructs [10], the necessary vector components are expressed from four distinct plasmids, and the overlapping regions between these units have been minimized to limit the possibility of homologous recombination. In addition, with the self-inactivating lentiviral vectors, the repair of the vector promoter would also be required for a recombinant to be able to replicate [11]. Although these built-in safety features make the generation of an RCL very unlikely, a sensitive test for RCL detection in large-scale vector productions is necessary before clinical trials with lentiviral vectors can be envisioned. Sensitive assays for the detection of RCR have been developed and consist of the amplification of RCR by culturing vector-containing supernatant on a permissive cell line followed by a sensitive detection method [12 14]. A similar assay was described by Farson and colleagues for lentiviral vectors and used to test small amounts of vector supernatants [15]. The identification of a control virus that represents the probable replication characteristics of an RCL is one of the challenges presented by the adaptation of RCR assays for lentiviral vectors. An RCR standard, representing the most likely recombinant to be generated by the Moloney murine leukemia virus amphotropic vector system, is available and used as a positive control in RCR assays [16]. The equivalent RCL standard is difficult to develop because the lentiviral vector safety features, which are designed precisely to avoid recombination, render the nature of an RCL extremely difficult to predict. As a best approximation of an RCL, Farson et al. [15] used a virus derived from HIV, R8.71, multiply attenuated by inactivation of the four accessory genes vif, vpr, vpu, and nef, and phenotypically pseudotyped by VSV G, allowing its first round of infection to proceed through the VSV envelope and the subsequent infections to occur through the HIV envelope. This phenotypic pseudotyping allows one to approximate a potential RCL better while avoiding the generation of an HIV strain genetically pseudotyped with VSV G and the biosafety concerns that such a virus would pose. We have used this phenotypically pseudotyped virus as an RCL standard to develop an RCL detection assay with a sensitivity and volume adapted for the testing of large-scale productions of HIV-based lentiviral vector. Here we describe the development of this sensitive RCL assay and the testing of large-scale preparations of the third-generation lentiviral vectors. RESULTS RCL Amplification Cell Line The choice of an amplification cell line able to amplify any potential RCL efficiently is a determining factor of the RCL assay sensitivity. We compared the C line used by Farson et al. with five other lymphocytic cell lines TABLE 1: Choice of a permissive cell line for RCL amplification 50% infectious dose of virus (fg p24) R8.71-VSV G R8.71 R8 Cells C , a CEM-SS , ,000 b MT ,000 c 100 PM ,000 1,000 H ,000 1,000 SupT ,000 10,000 Primary PBMC , Doses of virus tested (fg p24) , ,000 Infections of various cell lines by 10-fold serial dilutions of each virus were performed as described under Material and Methods. The range of virus doses tested varied according to the virus and is indicated (in fg p24). Ten replicates were tested for each dilution of virus. The dose of each virus (expressed in fg of p24) necessary to infect 50% of the cultures productively is shown. a In two independent experiments, the 10- and 100-fg doses gave respectively four and eight productively infected cultures of 10. b The 100-fg dose did not lead to any productive infection, whereas the 1000-fg dose gave 100% productive infections. c Doses expressed as indicate that at this highest tested dose, none of the replicates became productively infected for their capacity to support the replication of the RCL standard, R8.71 phenotypically pseudotyped with VSV G [15]. We compared the infectivity of this positive control with the nonpseudotyped version of R8.71, using only the HIV envelope, and the parental clone R8, representing wild-type HIV. This comparison was done by determining the 50% tissue culture infectious dose (TCID 50 ) of each virus in the different cell lines. As shown in Table 1, the TCID 50 for the RCL positive control (R8.71-VSV G) was the lowest in C , indicating that C was the most permissive cell line to the RCL positive control, in agreement with results by Farson et al. [15]. Remarkably, these cells were similarly permissive to the RCL positive control and wild-type HIV. The presence of VSV G on viral particles during the first round of infection resulted in an approximately 100-fold reduction in the TCID 50 for at least three cell lines, including C , demonstrating their susceptibility to VSV G-pseudotyped viruses. In addition, the C cells grow in suspension culture at high cell densities, they stably express the HIV receptor CD4 and coreceptor CXCR4, and show a constant infectivity with the RCL control over time in culture (data not shown). These characteristics make the C well suited to the amplification of RCL in large-scale testing. RCL Assay Design We modeled the RCL assay design, in a scalable format, on the RCR assays previously described [13 15] and it is 333

3 METHOD doi: /s (03) FIG. 1. RCL assay principle. The RCL-permissive cell line C is exposed to the vector sample for 7 days to maximize the chances of RCL infection and amplification. The culture is then diluted regularly for six passages to allow RCL amplification. Samples of culture supernatant are collected at each passage and assayed for viral replication by measure of the p24 capsid protein concentration. by introducing the equivalent of 1 infectious unit of the RCL positive control in preparations of vector. In a typical experiment, six identical vector samples were spiked with the minimal infectious dose of positive control virus (1 TCID 50 ) and tested by the RCL assay. As a control, the amplification of the same dose of positive control virus was similarly monitored in six cultures, in the absence of vector. In the absence of interference, we expected 50% of the cultures to become productively infected by the positive control virus, regardless of the presence of vector. A representative experiment is shown in Fig. 2. In four of illustrated in Fig. 1. We exposed the permissive cells C to the test sample of lentiviral vector and cultivated them for six passages to allow amplification of a potential RCL. To maximize the chances for an RCL to infect and amplify in C cells, we inoculated the cultures at a high cell density (10 6 cells/ml) and initially maintained them in the presence of the vector samples for 7 days. To ensure cell viability during this prolonged infection period, the culture was minimally diluted by addition of medium but no portion of the culture was discarded. After the 7-day infection, we diluted onefourth of the cell suspension four times every 3 or 4 days for a total of six passages. The RCL detection was done by ELISA determination of the HIV p24 protein concentration in culture supernatant. Since the lentiviral vectors are packaged with the HIV capsid, the original p24 concentration in the assay culture supernatant depends upon the vector sample concentration. However, in the absence of RCL, this concentration should decrease over time as the cells undergo multiple passages. To monitor that decay, we collected samples of culture supernatant at each passage. This assay design can be scaled up to accommodate relatively large sample volumes (up to 50 ml in static cultures). To determine whether 1 infectious unit of RCL can be efficiently amplified and detected by this assay, we measured the RCL positive control TCID 50, previously determined in C (Table 1), following the initial culture schedule of this assay, in culture volumes ranging from 100 l to 50 ml. The minimal dose of the RCL positive control virus required to establish productive infection was unaffected by the culture format, confirming the scalability of the assay. RCL Assay Sensitivity The presence of replication-defective vector could possibly interfere with the replication of an RCL. To address this issue we performed spike and recovery experiments FIG. 2. Sensitivity of the RCL assay. (A) Spike and recovery of the positive control. Six 30-ml unpurified vector samples, each containing transducing units of lentiviral vector (i.e., vector particles or 930 ng p24), were spiked with 1 TCID 50 (10 fg p24) of the RCL positive control and assayed for RCL. The p24 concentration of culture supernatant samples collected at each passage is shown. (B) Amplification of the positive control in the absence of vector. Six RCL assay cultures were set up without vector samples, inoculated with 1 TCID 50 of RCL positive control, and processed as in A. (C) Assay of the vector sample in the absence of positive control. Three 30-ml vector samples, identical to those tested in A but not spiked, were assayed for RCL as in A. 334

4 doi: /s (03) METHOD TABLE 2: Sensitivity and interference study by spike and recovery of the positive control virus Number of cultures becoming productively infected (of 6) Sample Culture volume (ml) Vector particles per culture a Vector TU per culture M.o.i. of vector (TU/cell) Presence of vector Bulk vector supernatant Absence of vector Purified vector b 3 b Total productively infected cultures 17/42 22/42 For each experiment, RCL assays were conducted on six vector samples and on six mock samples (medium equivalent) spiked with 1 TCID 50 of the positive control virus. Viral infection was monitored by p24 concentration as described for the RCL assay and productive infection was determined after 28 days. a Calculated on the basis of the p24 concentration assuming that 1 fg p24 represents 12 viral particles (see Material and Methods). b Transducing units estimated according to a ratio of 1 transducing unit per 1000 viral particles. the six cultures inoculated with the spiked vector samples, the p24 concentration in the supernatant decayed gradually over time and decreased below the limit of detection of the ELISA (3 pg/ml) after 28 days (Fig. 2A). In the other two cultures, however, the p24 concentration rebounded after 15 days and increased, indicating a productive infection. This result is consistent with the 50% productive infection rate expected after inoculation of 1 TCID 50 of virus. Similarly, five of the six cultures infected with 1 TCID 50 of the positive control in the absence of vector became productively infected (Fig. 2B). The difference between the numbers of cultures becoming infected in the presence and absence of vector is most likely attributable to statistical variability given the small number of replicates. This experiment also included three cultures exposed to the same vector-containing supernatant but not spiked with the positive control. The p24 concentration in these cultures decayed gradually over the course of the experiment and no viral replication was detected (Fig. 2C). We analyzed the two test cultures of vector spiked with the positive control that became productively infected further to confirm that the virus replicating in these cultures was indeed the positive control and not a replication-competent recombinant. Surface expression of the HIV envelope on the cells at the end of the assay was demonstrated by flow cytometry (data not shown). In addition, we infected HeLa-CD4-LTR- Gal cells with culture supernatants collected at the end of the assay. Only the supernatants from the two productively infected cultures scored positive on the HIV indicator cells, demonstrating the presence of the Tat transactivator in the replicating virus (data not shown). Since neither the HIV envelope nor the HIV Tat transactivator is part of the vector production system, the presence of these two HIV components confirms that the replicating virus is the positive control and not a recombinant originating from the vector production. To determine the sensitivity of the assay, we performed similar spike and recovery of 1 TCID 50 of the RCL positive control virus using increasing amounts of vector, in the purified or bulk unpurified form. The results of seven representative experiments are summarized in Table 2. Despite some statistical variability due to the limited number of replicates, cultures became productively infected by the positive control in all experiments, consistently with the 50% infection rate expected following the inoculation of 1 TCID 50 of control virus. Most importantly, the number of cultures becoming productively infected in the presence and absence of vector was generally comparable, indicating no limitation of sensitivity by the vector at the concentrations tested. Overall, of the 42 cultures inoculated with the positive control in the presence of vector, 17 became productively infected (40%), while 22 of 42 cultures inoculated in the absence of vector became productively infected (52%). The difference between the overall percentage of cultures becoming productively infected in the presence and absence of vector was not statistically significant (see Materials and Methods for statistical analysis). From this series of experiments we concluded that the RCL assay could detect 1 infectious unit of replication-competent lentivirus in as much as transducing units of unpurified vector 335

5 METHOD doi: /s (03) or transducing units of purified vector per culture. Analysis of Lentiviral Vector Lots We used the RCL assay to analyze large-scale vector production lots. To test a statistically representative sample of each vector lot, we assayed multiple test units concurrently. Shown in Fig. 3A are the p24 concentration curves for a representative testing of unpurified supernatant from a 14-liter production lot. The total test sample (460 ml) represented 3% of the large-scale production lot. The p24 concentration in the test culture supernatant decreased gradually and no sign of active replication was detected even after 43 days of culture. Remarkably, since the initial p24 concentration was high (587 ng/ml) it took 10 passages of the culture for the p24 concentration to become undetectable. However, in a spike and recovery experiment run in parallel, productive infection by 1 TCID 50 of the positive control virus was clearly detectable after the fourth passage with a p24 concentration fold higher than in the test article (Fig. 3B). We have tested 10 large-scale production lots and no RCL has been detected. The 10 independent production runs were vectors with various transgenes. We conducted the tests using both bulk vector supernatants and purified vector products. To achieve the demonstrated assay sensitivity, we set up the RCL assays according to the following criteria. For testing bulk vector supernatant samples, each test unit was a 30- to 32-ml culture containing less than transducing units of vector. For the purified vector samples, each test unit was a 50-ml culture containing less than transducing units of vector. The amplification cultures were stopped after six passages (28 days). The testing data are summarized in Table 3. Overall, we have screened approximately transducing units of vector from different large-scale productions without detecting any RCL. Complementary End-Point Analysis In addition to the p24 ELISA, we have designed a complementary end-point analysis targeting a second viral function, the pseudotyping envelope VSV G. Reasoning that the propagation of a virus using the VSV G envelope during the amplification culture would lead to an increase in the number of cells containing the VSV G sequence, we designed a quantitative PCR assay to measure VSV G copies in C We designed this detection method to screen 1 g of genomic DNA, or approximately cells, per PCR, and we assessed its sensitivity in two ways. First, we spiked various amounts of the VSV G plasmid pmd.g [9] into human genomic DNA and demonstrated that the limit of detection of the method was approximately 5 VSV G copies per test reaction ( cell equivalents). Second, we used this PCR to quantify the VSV G sequence in cell preparations composed of C mixed in various proportions with a cell clone, FIG. 3. RCL testing of a large-scale vector production lot. (A) Test results. One 14-liter production lot (LPR25) was tested for RCL. The vector concentration in this lot was transducing units/ml (587 ng p24/ml). A 460-ml sample of the bulk unpurified lot of vector was split into ml parts, each containing TU of vector. Fourteen replicate RCL assays were performed with 32-ml cultures. The p24 concentrations in samples collected at each passage of the cultures are shown. (B) Representative spike/recovery controls. As a control for the testing, six samples identical to the 32-ml test samples were spiked with 1 TCID 50 of the positive control virus and assayed for RCL. One of six cultures became productively infected. The growth kinetics of the virus in this culture is shown by the open squares. The filled squares stand for a representative culture that did not become productively infected. (C) Representative positive control. Six additional cultures were set up similarly without vector sample and inoculated with 1 TCID 50 of the positive control virus. In this case also, one of the six cultures became productively infected with the positive control; the growth kinetics of the positive control in that culture is shown by the open triangle. The filled triangles stand for a representative culture that did not become productively infected. 52G, stably transfected with a VSV G plasmid and containing approximately 7 VSV G gene copies. The assay appeared to be able to detect down to 1 VSV G-containing 336

6 doi: /s (03) METHOD Production lot identification Volume of production lot a (ml) TABLE 3: RCL testing of large-scale production lots Nature of RCL sample b Test sample volume (ml) Vector particles in test sample Vector transducing units in test sample RCL LPR 15 13,800 Bulk None detected LPR21 8,400 Bulk None detected LPR22 13,800 Bulk None detected LPR24 13,800 Bulk None detected LPR25 13,800 Bulk None detected LPR27 13,800 Bulk None detected LPR28 13,800 (60) Purified None detected LPR26 13,800 (50) Purified None detected LPR 38 13,800 (90) Purified None detected LPR 42 55,460 (419) Purified None detected a Numbers in parentheses indicate the final volume after purification. b Bulk indicates that the sample was collected from the unpurified vector-containing supernatant, purified indicates that the vector was tested after purification cell in a mixed population of cells (Fig. 4A). When less than 1 52G cell was present per reaction, a background level of 1 to 10 copies of VSV G per reaction was observed, potentially due to nonspecific priming of the PCR in the presence of large quantities of genomic DNA. Because of this noise in the PCR, we considered 10 copies as the threshold for detection of the VSV G sequence. We used this PCR to quantify VSV G sequences in C cells harvested at different times during the amplification phase in RCL assays performed on three vector lots (Figs. 4B, 4C, and 4D). Similar to what we observed for the p24 measurement, the RCL assay cultures contained an initial level of VSV G sequence, depending on the original vector concentration. This was most likely due to the presence of residual plasmid DNA originating from the transient transfection procedure of vector production. This initial level decreased gradually to reach the limit of detection of the PCR within the 35 days of the assay in most of the cultures tested. Overall, we have screened 15 cultures inoculated by a total of transducing units (TU) of vector from three independent vector lots for VSV G propagation. No increase in the VSV G concentration was observed in any of the cultures, indicating that no VSV G propagation caused by the replication of a recombinant virus occurred in the cultures tested. DISCUSSION Lentiviral vectors are potentially the gene delivery system of choice for some gene therapy applications that currently lack adequate vectors. However, their clinical use may be envisioned only after it has been demonstrated that clinical lentiviral vector preparations do not contain replication-competent recombinants with the pathogenic potential of the parental virus. Here we describe the development of a sensitive assay for the detection of RCL in large-scale preparations of HIV-based lentiviral vectors. This assay allows the detection of 1 RCL infectious unit in samples containing as much as transducing units of vector. This high sensitivity is conferred by the efficient amplification of RCL following culture on a particularly permissive cell line, C The selection of a highly permissive cell line and the analysis of the assay sensitivity were made possible by the use of a positive control virus designed to match the probable replication characteristics of an RCL in a preparation of second- or thirdgeneration lentiviral vector [15]. Since this phenotypically pseudotyped virus is severely attenuated in primary cells and used in small amounts as an experimental control, the RCL testing may be conducted under the same biosafety conditions used for routine procedures involving small amounts of wild-type HIV, that is, an adapted Biosafety Level 2 facility with special practices and safety equipment for Biosafety Level 3 [17]. C cells, which were known to be permissive for vif-defective HIV mutants [15,18], proved to be the most permissive to the multiply attenuated virus. These cells are infected with HTLV-I, but do not produce the virus. Two of the three HTLV-I proviruses in C have major deletions encompassing most of the gag and env regions. The absence of genome expression from the fulllength provirus is due to a mutation that introduced a premature termination codon in the rex ORF, leading to a truncated and nonfunctional version of the HTLV-I Rex protein [19]. The HTLV-I Tax protein, however, is expressed in C The Tax transactivator is known to be able to synergize with HIV-1 Tat to increase HIV-1 337

7 METHOD doi: /s (03) FIG. 4. (A) Sensitivity of the VSV G PCR detection method. A cell clone, 52G, containing approximately 7 copies of the VSV G gene was mixed with C cells in proportions ranging from 10 to % 52G cells. Genomic DNA was extracted from the mixed-cell populations and analyzed by quantitative PCR. PCRs were performed in triplicate with 1 g of DNA per reaction (the equivalent of cells). The PCR readout expressed in VSV G copies per reaction, or cells, is plotted against the number of VSV G-containing cells per reaction. The average of six independent DNA extractions is shown. The threshold of detection was determined to be 10 copies per reaction and is indicated on the graph. (B, C, and D) Detection of VSV G-containing cells in three RCL assays. During the testing of three large-scale vector lots described in Table 3, C cells from some of the RCL test cultures were collected at each passage. VSV G sequence in genomic DNA extracted from these cells was quantified by PCR. The average of three PCRs per sample is shown and expressed as VSV G copies per PCR ( cells). Each curve represents the evolution of VSV G levels over time in one test culture. The assays were set up with the following amounts of vector per culture: (B) vector lot LPR38, TU (2.8 g p24), (C) vector lot LPR28, TU (18.9 g p24), (C) vector lot LPR42, TU (10.4 g p24). transcription [20,21]. This mechanism may be, in part, responsible for the complementation of the replication defect of an attenuated HIV-1 strain. The presence of Tax, however, is not likely to be sufficient to confer the high level of permissiveness observed in the C cells since Tax is also expressed in MT-4 cells [22], which are less permissive to R8.71 infection. In the spike and recovery experiments performed to determine the sensitivity of the RCL assay, we observed that the positive control replication was slightly delayed in the presence of unpurified vector-containing supernatant (Figs. 2A and 2B), indicating some degree of interference between vector and RCL positive control. Since the vectors used in this study were self-inactivating, this interference should not take place at the level of gene expression or packaging but is likely to occur at an early stage of infection such as entry [23]. We have shown that the RCL positive control virus is sufficiently stable during the RCL assay to be capable of infecting the C cells 24 h after inoculation of the cultures (Escarpe and Kiermer, unpublished data). Most likely, a delayed infection due to interference with vector particles or impurities in the vector sample could be responsible for the delayed kinetics. In a similar spike and recovery experiment using a comparable amount of purified vector, the positive control kinetics were delayed in only two of the four productively infected cultures (data not shown), indicating that the events causing interference may be less frequent with purified material than with crude production supernatant. Nevertheless, once initiated, the infection with the positive control virus proceeds at the same rate in the presence or absence of vector and the degree of interference observed does not affect the sensitivity of the assay. Even at the highest vector dose tested in spike/recovery, the minimal infectious dose of RCL positive control was amplified and detected. This indicates that the sensitivity of the assay could actually be higher than 1 RCL in TU of vector. However, in this experimental format the multiplicity of infection is approximately 5 vector TU/cell. At a significantly higher m.o.i., only part of the vector can be expected to infect the amplification cells, and this would jeopardize the sensitivity and reproducibility of the assay. To increase the capacity of a single test culture even more, the cell number and culture volume 338

8 doi: /s (03) METHOD should be increased proportionally to the vector sample, and this would require a switch to a different culture device. The p24 ELISA, although not the most sensitive detection method, is highly reliable for monitoring virus replication. We demonstrated that by following the p24 concentration in the test culture supernatant, the amplification of 1 TCID 50 of the RCL positive control becomes clearly detectable in 21 days. In cultures not infected by the RCL positive control, the initial p24 levels, corresponding to the sample vector particles, decreased gradually to become undetectable. However, with higher vector concentrations in the sample, it takes longer for the p24 levels to decrease below the limit of detection of the ELISA. While assaying vector-containing supernatant at a p24 concentration of approximately 600 ng/ml, we have observed that clearance of the vector-associated p24 can take up to 43 days or 10 culture passages. However, the difference in p24 concentrations between cultures exposed to the vector and the vector containing a spike of replication-competent virus is at least 10,000-fold after the sixth passage, and there is then no ambiguity about the assay outcome. Therefore, we have defined this time point (six passages, 28 days) as the assay end point in our systematic testing of vector production lots. The p24 ELISA is suitable for detection of all amplified RCL originating from a recombination between the components of the vector system. However, in the less likely case of a recombination between the VSV G envelope and a gag pol function from another virus, such as an endogenous human retrovirus potentially present in the vectorproducer cells, the p24 detection method would not be suitable. To broaden the detection range of this RCL assay, we used a quantitative PCR to detect the propagation of the VSV G sequence caused by the replication of a VSV G-pseudotyped RCL in the test culture. This quantitative PCR could be implemented as a complementary endpoint analysis to the p24 ELISA. However, the appropriate positive control to qualify this assay, an RCL propagating with the VSV G envelope, is not available. The VSV G PCR end-point should therefore remain a complementary informative assay but should not replace the p24 ELISA. In addition, it has to be noted that sensitive PCR-based methods, such as this VSV G detection assay, are impacted by the presence of contaminating viral sequences, most likely originating from the transient transfection procedure used for vector production. These residual contaminants create a background in the detection test. Using extremely sensitive PCR-based methods as end-point analyses may become more appropriate if more purified vector preparations become available, with improvements to the purification procedure or use of stable producer cells. The RCL assay in its current design can accommodate 50-ml samples of vector preparation per culture and it is capable of detecting 1 RCL in a total of up to transducing units of vector. Moreover, the RCL assay described here can accommodate both unpurified vectorcontaining supernatant and purified vector preparations, which allows flexibility in the testing strategy. By assaying multiple samples we have tested up to 465 ml of unpurified vector supernatant from a 14-liter production. Remarkably, if one assumes that the distribution of potential RCLs in vector-containing supernatants follows a Poisson distribution, one can calculate that testing 460 ml without finding an RCL indicates with 99% confidence that there is less than 1 RCL per 100 ml of supernatant [12]. For reference, the Food and Drug Administration recommends, for retroviral vector-based gene therapy protocols, a testing protocol capable of demonstrating, at a 95% confidence level, that there is less than 1 RCR per 100 ml of vector supernatant used as clinical material. Similarly, the largest test sample assayed from a single purified vector preparation was TU of vector. According to the same statistical model, the absence of RCL in this sample indicated with 99.5% confidence that there is less than 1 RCL per 10 9 TU of vector. Given the transducing efficiency of lentiviral vectors, preclinical studies indicate that a clinical dose for an ex vivo gene therapy application would be in the range of 10 9 TU of vector [3,5,24,25]. Therefore, the capacity of this RCL assay should be sufficient to test vector preparations intended for gene therapy applications. MATERIALS AND METHODS Virus and vector production. Virus stocks of clones R8.71 and R8 were prepared by transient transfection of plasmid into 293T cells by the calcium phosphate precipitation method. Briefly, T cells were seeded in a 10-cm-diameter dish 24 h prior to transfection in 10 ml of Iscove s modified Dulbecco s medium (JRH Biosciences) containing 10% fetal bovine serum (HyClone) and 1% penicillin streptomycin (GIBCO BRL), in a 5% CO 2 incubator at 37 C. The culture medium was changed 4 h prior to transfection. Twenty micrograms of the pr8 [26] or pr8.71 [15] plasmid DNA was used per transfection. The medium was replaced after 16 to 18 h and then collected after an additional 24 h. The conditioned medium was filtered through a m cellulose acetate membrane (Nalgene), aliquoted, and stored at 80 C. Determination of virus concentration was performed by p24 antigen capture ELISA (NEN). The positive control virus stock, R8.71 transiently VSV G pseudotyped, was prepared in a similar manner by cotransfecting 20 g of the pr8.71 plasmid and 15 g of the pmd.g envelope plasmid [9]. Among the combinations tested, a 20:15 ratio of pr8.71 to pmd.g yielded virus with the highest infectivity. The positive control virus was diluted with complete culture medium and stored in small aliquots at 80 C. The large-scale lentiviral vector preparations were produced in 10- chamber cell factory units (Nalgene Nunc) by calcium phosphate-based transient transfection using the third-generation lentiviral vector and helper plasmids [9]. Vector titers were determined by transduction of HeLa cells and quantification of transduced cells by flow cytometry for GFP expression or by quantitative PCR as previously described [9,27]. Cell culture. C , MT4, CEM-SS, PM1, H9, and SupT1 cells were obtained from the National Institutes of Health AIDS Research and Reference Reagent Program and maintained in RPMI 1640 medium (JRH Biosciences) containing 10% fetal bovine serum and 1% penicillin streptomycin at 37 C with 5% CO 2. For the RCL assay, C cells were adapted to culture in Iscove s modified Dulbecco s medium (JRH Bio- 339

9 METHOD doi: /s (03) science) containing 10% fetal bovine serum, 1% penicillin streptomycin, and6mml-glutamine. Human peripheral blood mononuclear cells (PBMCs) were obtained from healthy donors (Stanford Blood Bank, Stanford, CA) by centrifugation on Ficoll Hypaque density gradients. PBMCs were activated with 5 g/ml PHA (Sigma) and 20 U/ml recombinant human IL-2 (Sigma) for 3 days in RPMI medium containing 20% fetal bovine serum and 1% penicillin streptomycin at 37 C with 5% CO 2 prior to viral infection. p24 ELISA. Measurement of p24 concentration was performed using the HIV-1 p24 ELISA kit (Perkin Elmer Life Science Products) according to the manufacturer s recommendations. A standard curve was generated for each assay using twofold serial dilutions of the kit standard ranging from 100 to 3 pg/ml. Supplemented culture medium was used as a diluent and blank. Vector particle concentrations were calculated on the basis of the p24 concentration. According to the stoichiometry of 2000 p24 molecules (molecular mass 24 kda) per viral particle [28 30], 1 fg p24 corresponds to 12 viral particles. VSV G quantitative PCR. Taqman PCR was performed on the ABI Prism 7700 thermocycler, using the Universal PCR master mix (Roche) and the following primers and probe at 10 M each: Fwd primer 5 -CGAGATG- GCTGATAAGGATCTC-3, Rev primer 5 -ATTGATTATGGTGAAAGCAG- GAC-3, and probe 6FAM-TGCTGCAGCCAGATTCCCTGAATG. The PCRs were incubated at 50 C for 2 min and at 95 C for 10 min and then submitted to 40 cycles of 15 s at 95 C and 1 min at 60 C. Amplification plots were analyzed with the Sequence Detection System 1.7 software (ABI). Samples for PCR were prepared by extracting genomic DNA from cell pellets of cells using the Puregene kit (Gentra Systems). DNA concentration was normalized by UV spectrophotometry and 1 gofdna was used per PCR (equivalent to cells). The standard curves for VSV G quantification were made by serial dilutions of VSV G-containing plasmid in a background of human genomic DNA (Clontech). TCID 50 determination. For the selection of an RCL-permissive cell line, infections with 10-fold serial dilutions of each virus were performed in a final volume of 200 l at a cell density of cells/ml in the presence of 2 g/ml Polybrene. After 24 h, the Polybrene was diluted 10-fold by the addition of fresh medium. The cells were cultivated by 5-fold dilutions in fresh medium every 3 or 4 days and assayed for productive infection after 14 days by ELISA measurement of p24 concentration. To determine more precisely the TCID 50 of the positive control virus in the RCL assay, infections of C cells, at a concentration of cells/ml, were performed in 96-well flat-bottom plates (Falcon) in a 100- l volume. Serial dilutions of the virus were tested ranging typically from 0.1 fg to 10 pg p24/well, in 10 replicates each. The cultures were diluted by addition of 50 l of fresh medium after 3 and 7 days. Ten days after the initiation of infection, cultures were analyzed for virus production by p24 antigen capture ELISA. A portion of each culture was diluted fourfold and analyzed 4 days later for confirmation. The cultures productively infected by the virus were counted and the TCID 50 for each virus was calculated by the Reed Muench method [31]. The average TCID 50 obtained corresponded to 9 fg p24 equivalent of virus. This value of the RCL positive control TCID 50 was confirmed using a smaller dilution range in culture formats of 1, 30, and 50 ml. RCL assay. Cultures for the RCL assay were set up as follows. C cells were pelleted and resuspended at a concentration of cells/ml. For testing of purified vector, the cells were resuspended in fresh medium and vector was subsequently added. For testing of the bulk supernatant vector, the cells were resuspended directly in the vector sample to avoid dilution. Cultures set up to monitor positive control amplification in the absence of vector were resuspended in fresh medium; the control virus was then added to the culture. The culture volume was modified during assay development to accommodate large samples of vector. The final standard assay was conducted in 50-ml static cultures in T-175 cm 2 flasks (Falcon) for purified vector samples and 30-ml cultures for bulk vector supernatant samples. Three days after inoculation, half the volume of fresh medium was added to the cultures for feeding. After an additional 4-day incubation, one-fourth of the cell suspension was transferred to a new culture flask and diluted fourfold with fresh medium. Every 3 4 days, the cultures were similarly diluted according to a 1:4 ratio. At each dilution, samples of the culture supernatants were collected, clarified by low-speed centrifugation, and stored at 80 C until assayed by p24 antigen capture ELISA. 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