Retroviral Producer Research Articles

BTK mediated apoptosis, a possible mechanism for failure to generate high titer retroviral producer clones

BTK mediated apoptosis, a possible mechanism for failure to generate high titer retroviral producer clones

BTK mediated apoptosis, a possible mechanism for failure to generate high titer retroviral producer clones.

It has been shown previously that mutations in the cytoplasmic protein kinase, Bruton's tyrosine kinase (BTK) lead to X-linked agammaglobulinemia, an inherited primary immunodeficiency, thus making it a potential candidate for gene therapy. Producer cell lines using retroviral BTK constructs were generated and retroviral titers determined. Southern blot analysis was performed to check for pro-viral integrity in the respective clones. Furthermore, cotransfection of green fluorescent protein (GFP) with BTK expression plasmids was used in HeLa cells to establish and characterize the role of BTK in apoptosis. Following the attempt to generate retroviral producer clones by conventional methods, we observed that the BTK gene is deleted from neomycin-resistant high titer clones. We show that BTK mediated apoptosis in GP#E86 and HeLa cells. Furthermore, membrane targeting and kinase activity are required for this effect. In addition, BTK induced apoptosis could be inhibited by using a specific inhibitor for p38 mitogen-activated protein kinase (MAPK), SB203580. Failure to generate retroviral producer clones may be caused by the induction of apoptosis mediated by the therapeutic gene product.

Modeling retrovirus production for gene therapy. 1. Determination Of optimal bioreaction mode and harvest strategy.

Although retroviruses are a promising tool for gene therapy, there are two major problems limiting the establishment of viable industrial processes: retrovirus stability and low final yield in the supernatant. This fact emphasizes the need for an effective process optimization, not only at a genetic level but also at a bioprocess engineering level. In part 1 of this paper a mathematical model was developed to optimize the bioreaction yield by determining the best retrovirus harvest strategy in perfusion cultures. PA317 cells producing recombinant retroviruses were used to develop and test this model. Cell culture was performed in stirred tanks using porous supports. The parameters of the proposed model were experimentally determined for batch and perfusion cultures at 32 and 37 degrees C both with and without additives to enhance production; the model was then validated. This model allowed the determination of the optimal values of all operational variables included: batch and perfusion duration and perfusion rate. The highest productivity (2682 virus cm(-)(3) h(-)(1)) was obtained under the following conditions: batch at 37 degrees C for 53 h followed by perfusion at 32 degrees C for 23 h with a perfusion rate of 0.107 h(-)(1). This value was 3.5-fold higher than the best result obtained in batch cultures for the same conditions of titer and quality. A sensitivity analysis of the parameters showed that the parameters that affect most the final productivity depend on the bioreaction phase: cell growth in batch culture and production and product degradation in perfusion culture. In part 2 of this paper, this model is extended to the first step of downstream processing, and the addition of further steps to the process is discussed in order to achieve global process optimization.

DNA methylation of helper virus increases genetic instability of retroviral vector producer cells.

Retroviral vector producer cells (VPC) have been considered genetically stable. A clonal cell population exhibiting a uniform vector integration pattern is used for sustained vector production. Here, we observed that the vector copy number is increased and varied in a population of established LTKOSN.2 VPC. Among five subclones of LTKOSN.2 VPC, the vector copy number ranged from 1 to approximately 29 copies per cell. A vector superinfection experiment and Northern blot analysis demonstrated that suppression of helper virus gene expression decreased Env-receptor interference and allowed increased superinfection. The titer production was tightly associated with helper virus gene expression and varied between 0 and 2.2 x 10(5) CFU/ml in these subclones. In one analyzed subclone, the number of integrated vectors increased from one copy per cell to nine copies per cell during a 31-day period. Vector titer was reduced from 1.5 x 10(5) CFU to an undetectable level. To understand the mechanism involved, helper virus and vectors were examined for DNA methylation status by methylation-sensitive restriction enzyme digestion. We demonstrated that DNA methylation of helper virus 5' long terminal repeat occurred in approximately 2% of the VPC population per day and correlated closely with inactivation of helper virus gene expression. In contrast, retroviral vectors did not exhibit significant methylation and maintained consistent transcription activity. Treatment with 5-azacytidine, a methylation inhibitor, partially reversed the helper virus DNA methylation and restored a portion of vector production. The preference for methylation of helper virus sequences over vector sequences may have important implications for host-virus interaction. Designing a helper virus to overcome cellular DNA methylation may therefore improve vector production. The maintenance of increased viral envelope-receptor interference might also prevent replication-competent retrovirus formation.

Extended analysis of the in vitro tropism of porcine endogenous retrovirus.

We previously reported that mitogenic activation of porcine peripheral blood mononuclear cells resulted in production of porcine endogenous retrovirus(es) (PERV[s]) capable of productively infecting human cells (C. Wilson et al., J. Virol. 72:3082-3087, 1998). We now extend that analysis to show that additional passage of isolated virus, named here PERV-NIH, through a human cell line yielded a viral population with a higher titer of infectious virus on human cells than the initial isolate. We show that in a single additional passage on a human cell line, the increase in infectivity for human cells is accounted for by selection against variants carrying pig-tropic envelope sequences (PERV-C) as well as by enrichment for replication-competent genomes. Sequence analysis of the envelope cDNA present in virions demonstrated that the envelope sequence of PERV-NIH is related to but distinct from previously reported PERV envelopes. The in vitro host range of PERV was studied in human primary cells and cell lines, as well as in cell lines from nonhuman primate and other species. This analysis reveals three patterns of susceptibility to infection among these host cells: (i) cells are resistant to infection in our assay; (ii) cells are infected by virus, as viral RNA is detected in the supernatant by reverse transcription-PCR, but the cells are not permissive to productive replication and spread; and (iii) cells are permissive to low-level productive replication. Certain cell lines were permissive for efficient productive infection and spread. These results may prove useful in designing appropriate animal models to assess the in vivo infectivity properties of PERV.

Characterization of Recombination Events Leading to the Production of an Ecotropic Replication-Competent Retrovirus in a GP+envAM12-Derived Producer Cell Line

Replication-competent retrovirus (RCR) was identified in a GP+envAM12-derived producer cell, containing the MFG-S-Neo retroviral vector, using a marker rescue assay. Studies were undertaken to determine the origin and structure of this RCR. Receptor interference assays demonstrated that the virus was pseudotyped with an ecotropic envelope. Molecular analysis demonstrated the presence of a MoMLV ecotropic env recombinant where the neomycin resistance gene of the MFG-S-Neo vector was replaced by MoMLV ecotropic env. Additional recombinants linking the retroviral pol gene to neo and the neo gene to MoMLV env were also identified. A full-length MoMLV retroviral genome was detected by nested PCR in the contaminated amphotropic producer cells and in cells infected with its supernatant. Unexpectedly, this was also present in the GP+E86 packaging cells together with a previously undescribed envelope construct possessing a full 5′ and 3′ LTR, although these cells were consistently negative for the presence of RCR. These anomalies in the GP+E86 packaging cell line result in increased homology with the MFG-S-Neo vector, leading to an increased risk for the production of RCR. Our findings point to a need for increased vigilance when using these packaging lines to generate replication-defective retrovirus.

T-Ag inhibits implantation by EC cell derived embryoid bodies.

When introduced into EC cells of a blastocyst, polyomavirus (Py) T-Ag results in mice mosaic for T-Ag but otherwise essentially normal. It had been reported that SV40 T-Ag does not inhibit differentiation of F9 EC cells, but did inhibit endogenous retrovirus (ERV) production. We therefore sought to determine if Py T-Ag had any affect on EC derived embryoid body implantation onto mouse placenta. F9 EC cells were selected for T-Ag maintenance. Like the SV40 transformed cells, we show that these Py T-Ag selected EC cells no longer express IAP transcripts following differentiation into embryoid bodies. Normal and Py T-Ag selected F9 cells were differentiated into embryoid bodies then implanted into pseudopregnant mice. We observe, that normal F9 derived embryoid bodies underwent the initial stages of implantation whereas the Py T-Ag selected embryoid bodied did not implant. The implications of this observation with respect to trophectoderm and ERV function are discussed. We examine the idea that ERVs may be a required element for normal embryo implantation.

Retroviral Vector Biosafety: Lessons from Sheep.

The safety of retroviral-based systems and the possible transmission of replication-competent virus to patients is a major concern associated with using retroviral vectors for gene therapy. While much effort has been put into the design of safe retroviral production methods and effective in vitro monitoring assays, there is little data evaluating the risks resulting from retroviral vector instability at post-transduction stages especially following in vivo gene delivery. Here, we briefly describe and discuss our observations in an in vivo experimental model based on the inoculation of retroviral vector-transduced tumor cells in sheep. Our data indicates that the in vivo generation of mosaic viruses is a dynamic process and that virus variants, generated by retroviral vector-mediated recombination, may be stored and persist in infected individuals prior to selection at the level of replication. Recombination may not only restore essential viral functions or provide selective advantages in a changing environment but also reestablish or enhance the pathogenic potential of the particular virus undergoing recombination. These observations in sheep break new ground in our understanding of how retroviral vectors may have an impact on the course of a preestablished disease or reactivate dormant or endogenous viruses. The in vivo aspects of vector stability raise important biosafety issues for the future development of safe retroviral vector-based gene therapy.

Retrovirus-mediated transfer of human alpha-galactosidase A gene to human CD34+ hematopoietic progenitor cells.

Fabry disease, caused by a deficiency of lysosomal enzyme alpha-galactosidase A (alpha-gal A), is one of the inherited disorders potentially treatable by gene transfer to hematopoietic stem cells. In this study, a high-titer amphotropic retroviral producer cell line, MFG-alpha-gal A, was established. CD34+ cells from normal umbilical cord blood were transduced by centrifugal enhancement. The alpha-gal A activity in transduced cells increased 3.6-fold above the activity in nontransduced cells. Transduction efficiency measured by PCR for the integrated alpha-gal A cDNA in CFU-GM colonies was in the range of 42-88% (average, 63%). The expression of functional enzyme in TFI erythroleukemia was sustained for as long as cells remained in culture (84 days) and for 28 days in LTC-IC cultures of CD34+ cells. The ability of the transduced CD34+ cells to secrete the enzyme and to correct enzyme-deficient Fabry fibroblasts was assessed by cocultivation of these cells. The enzyme was secreted into the medium from transduced CD34+ cells and taken up by Fabry fibroblasts through mannose 6-phosphate receptors. These findings suggest that genetically corrected hematopoietic stem/progenitor cells can be an enzymatic source for neighboring enzyme-deficient cells, and can potentially be useful for gene therapy of Fabry disease.

Characterization of the P140K, PVP(138-140)MLK, and G156A O6-methylguanine-DNA methyltransferase mutants: implications for drug resistance gene therapy.

The G156A O6-alkylguanine-DNA alkyltransferase (AGT) mutant protein, encoded by the G156A O6-methylguanine-DNA methyltransferase gene (MGMT), is resistant to O6-benzylguanine (BG) inactivation and, after transduction into hematopoietic progenitors, transmits remarkable resistance to BG and BCNU. As a result, a clinical trial, in which the MGMT gene is transduced into CD34+ cells of patients with cancer, has been approved. A newly identified AGT mutation, P140K, generates dramatically increased BG resistance relative to G156A, and suggests that gene transfer of P140K may confer improved hematopoietic cell protection. To address this hypothesis, we measured BG + BCNU and BG + TMZ resistance in G156A, P140K, or P138M/V139L/P140K (MLK) MGMT-transduced K562 cells. In addition, we performed a detailed characterization of individual properties including BG resistance, activity, and protein stability of these mutants in human hematopoetic K562 cells and E86 retroviral producer cells. In K562 cell extracts, the MLK and P140K mutants retained full activity at doses up to 1 mM BG, while G156A had a BG ED50 of 15 microM, compared with 0.1 microM for wtAGT. In the absence of BG, the G156A protein possessed a 56% reduction in specific O6-methyltransferase activity compared with wtAGT. MLK, P140K, and wtAGT all possessed similar specific activities, although the O6-methyl repair rate of all mutants was reduced 4- to 13-fold relative to wtAGT. The wtAGT, MLK, and P140K proteins were stable, with half-lives of greater than 18 hr. In contrast, only 20% of the G156A protein was stable after 12 hr in cycloheximide and, interestingly, the remaining protein appeared to retain most of the activity present in non-cycloheximide-treated cells. Differences in BG resistance, activity, and stability between P140K, MLK, and G156A suggest that P140K may be the optimal mutant for drug resistance gene transfer. However, hematopoietic K562 cells transduced with MFG-G156A, P140K, or MLK had similar degrees of BG and BCNU as well as BG and TMZ resistance when treated with concentrations of BG (< or =25 microM) achieved in clinical trials, suggesting similar efficacy in many in vivo applications.

Retroviral marking of acute myelogenous leukemia progenitors that initiate long-term culture and growth in immunodeficient mice

Rare primitive progenitors among the malignant cells from most patients with AML include AML long-term culture-initiating cells (AML LTC-IC) and NOD/SCID mouse leukemia-initiating cells (NOD/SL-IC). To evaluate the feasibility of genetic modification of these progenitors for gene marking and/or gene therapy strategies, cells from patients with newly-diagnosed AML were cocultured with retroviral producer cells and then placed in colony (AML-CFC) assays, LTC, and injected intravenously into NOD/SCID mice. Southern blotting demonstrated transfer of the neo r gene to 30% to 80% of leukemic blasts when cells were cultured for 48 hours in the presence of IL-3 and steel factor (SF) prior to 48-hour coculture with viral producers. Three of six retrovirally-infected AML samples showed both engraftment in NOD/SCID mice and the presence of the neo r transgene in mouse tissues 8–15 weeks after injection of transduced cells. Thirteen weeks after injection of one of these samples, >80% of cells from mouse bone marrow were the progeny of two retrovirally-transduced AML progenitors. Four of the remaining five samples showed markedly reduced ability to engraft in mice after retroviral infection. Subsequent experiments demonstrated that the loss of engraftment potential took place within 24 hours of culture initiation in the absence of retroviral producers and regardless of the cytokines present. Interestingly, the majority of AML-CFC or AML LTC-IC survived the 24-hour culture period. A retroviral vector containing the murine cell surface marker heat stable antigen (HSA), which allows purification of transduced cells on immunomagnetic columns, was used to obtain an enriched population of gene-modified AML cells following an infection protocol that eliminated the 48 hours of prestimulation in IL-3 and SF and reduced coculture with viral producers to 10–36 hours. These modifications failed to improve engraftment of the infected cells. In addition, in these experiments more than 10 hours of cocultivation with viral producer cells was necessary to achieve gene transfer and expression in AML LTC-IC. These data demonstrate that although retroviral-mediated gene transfer can be achieved to AML progenitors, including NOD/SL-IC, improved culture conditions will be required before substantial numbers of such transduced primitive progenitors can be obtained. In addition, the difference in the ability of AML LTC-IC and NOD/SL-IC to survive ex vivo suggests that these assays may detect different populations of cells or that changes are induced in vitro in primitive cells which can only be detected in the mouse assay.

Molecular characterization of the glycoproteins from two warm water rhabdoviruses: snakehead rhabdovirus (SHRV) and rhabdovirus of penaeid shrimp (RPS)/spring viremia of carp virus (SVCV)

We have determined the complete coding sequences for the glycoprotein (G) genes from two rhabdoviruses that infect warm water aquatic animals, the snakehead rhabdovirus (SHRV) and rhabdovirus of penaeid shrimp (RPS). Surprisingly, the G nucleotide sequence from RPS, a virus which has been isolated from diseased shrimp in Hawaii on numerous occasions, was over 99% identical to the G nucleotide sequence from spring viremia of carp virus (SVCV), a fish virus from Europe and Asia. This is the first report of SVCV isolation outside of Europe and Asia, and it is also the first report of SVCV infecting a non-vertebrate species. The G gene from SHRV was most closely related to the G genes from the three Novirhabdoviruses, viral hemorrhagic septicemia virus (VHSV), infectious hematopoietic necrosis virus (IHNV), and hirame rhabdovirus (HIRRV), with 47, 37, and 36% amino acid identity, respectively. In addition, a phylogenetic analysis using the amino acid sequence from rhabdovirus G genes indicated that SHRV should be classified within the Novirhabdovirus genus. Finally, the SHRV-G gene was successfully expressed in mammalian cells under the control of the cytomegalovirus (CMV) promoter, establishing that it can potentially be used in the production of pseudotyped retroviruses designed to infect fish.

Retroviral-mediated transfer and expression of the multidrug resistance protein 1 gene (MRP1) protect human hematopoietic cells from antineoplastic drugs.

Multidrug resistance protein (MRP1) is a member of the ATP-binding cassette (ABC) transmembrane transporter superfamily that confers multidrug resistance. The transfer and expression of the MRP1 gene in human hematopoietic stem cells may be a useful alternative to multidrug resistance (MDR1) gene transfer for protection from the myelosuppressive effects of chemotherapy in cancer patients. We constructed a gibbon ape leukemia virus packaging cell line (PG13) using the human MRP1 cDNA in a Moloney murine leukemia virus (MoMuLV) backbone containing a modified LTR. This PG13-based cell line, designated MRP1-PG13, produces retroviral vectors bearing the MRP1 gene at a titer of 1.7x10(5) viral particles/ml. Transduction of the human leukemic cell line K562 showed that viral MRP1-PG13 supernatants routinely transfer the MRP1 gene to approximately 35% of target K562 cells, of which at least one third are capable of proliferating in the presence of otherwise toxic concentrations of etoposide. Southern blot analyses indicated that most clones had only one proviral integration. Northern blot analysis of expanded K562 clones showed the presence of a major full-length approximately 8-kb MRP1 transcript as well as a minor approximately 6-kb transcript in all clones. Flow cytometric analysis of the producer cells and clones of transduced K562 cells demonstrated significantly increased MRP1 expression in these cells (approximately 30-fold increase). Human bone marrow mononuclear cells and CD34+ cells were also transduced with MRP1-PG13 supernatants on fibronectin-coated culture flasks in the presence of SCF, IL-3, and IL-6. PCR analysis of individual hematopoietic colonies in methylcellulose cultures demonstrated proviral DNA in approximately 10% of unselected human hematopoietic progenitor cells cultured from nonsorted mononuclear cell samples and in up to approximately 75% of progenitors when CD34-enriched cell populations were targeted. To assess functional MRP1 gene expression, normal human hematopoietic progenitors and K562 cells were cultured in methylcellulose assays containing vincristine or etoposide. All transduced samples gave rise to approximately 10% drug-resistant colonies, which were shown to be provirus-positive by PCR. Our studies document the development of an amphotropic MRP1 retroviral vector producer cell line and pave the way for large animal and preclinical studies of chemoprotection by MRP1 gene transfer.

One step screening of retroviral producer clones by real time quantitative PCR.

Recombinant retroviruses are obtained from either stably or transiently transfected retrovirus producer cells. In the case of stably producing lines, a large number of clones must be screened in order to select the one with the highest titre. The multi-step selection of high titre producing clones is time consuming and expensive. We have taken advantage of retroviral endogenous reverse transcription to develop a quantitative PCR assay on crude supernatant from producing clones. We used Taqman PCR technology, which, by using fluorescence measurement at each cycle of amplification, allows PCR product quantification. Fluorescence results from specific degradation of a probe oligonucleotide by the Taq polymerase 3'-5' exonuclease activity. Primers and probe sequences were chosen to anneal to the viral strong stop species, which is the first DNA molecule synthesised during reverse transcription. The protocol consists of a single real time PCR, using as template filtered viral supernatant without any other pre-treatment. We show that the primers and probe described allow quantitation of serially diluted plasmid to as few as 15 plasmid molecules. We then test 200 GFP-expressing retroviral-producing clones either by FACS analysis of infected cells or by using the quantitative PCR. We confirm that the Taqman protocol allows the detection of virus in supernatant and selection of high titre clones. Furthermore, we can determine infectious titre by quantitative PCR on genomic DNA from infected cells, using an additional set of primers and probe to albumin to normalise for the genomic copy number. We demonstrate that real time quantitative PCR can be used as a powerful and reliable single step, high throughput screen for high titre retroviral producer clones.

Gelsolin gene therapy by retrovirus producer cells for human bladder cancer in nude mice.

Gelsolin, a regulator of the actin cytoskeleton, has been shown previously to act as a tumor suppressor in vitro and in vivo when introduced into certain cancer cell lines. To investigate the in vivo efficacy of gene therapy with the gelsolin gene, we inoculated nude mice with human urinary bladder cancer cells (UMUC-2 or DAB-1) and tested the effects of adding either retroviral DNA constructs containing gelsolin cDNA or retrovirus producer cells that produce the same retroviral constructs at high levels. The addition of retroviral gelsolin cDNA constructs did not inhibit tumor growth; however, this form of gene therapy, in which retrovirus producer cells were introduced, resulted in marked and reproducible tumor growth inhibition and prolonged survival time in the majority of animals tested. These findings demonstrate the potential for treating human urinary bladder carcinomas with the gelsolin gene.

Distribution of retroviral vectors and vector producer cells using two routes of administration in rats.

The clinical use of retroviral vector producer cells (VPCs) to deliver retroviral vectors efficiently to target cells has been investigated as a method to increase efficiency of gene delivery, presumably as a result of continued vector production in vivo. Studies were conducted in rats to evaluate the distribution of vector to distal organs and tissues as measured by transduction. Rats were treated with two doses of VPCs using two routes of administration: (1) subcutaneous injection, chosen to maximize both the dose and exposure of animals, thereby enabling identification of potential target organs under worst-case conditions; and (2) direct injection into brain parenchyma, chosen to mimic the intended clinical route of administration and provide an estimate of risk to patients receiving this therapy. Twelve organs or tissues were collected 7 days after administration of VPCs and analyzed by PCR for the presence of vector and vector producer cell sequences. Vector was detected most frequently at the site of injection by either route of administration. Less frequently, vector was detected in draining lymph nodes at the higher dose only using either route of injection. Single specimens of lung and contralateral skin were positive for vector following subcutaneous administration only. Vector was detected in gonadal tissue from a single low-dose male following subcutaneous administration, but this finding was not reproduced in any high-dose male or any males injected intracerebrally. In contrast, VPCs were detected only at the site of administration. The frequency of detection of VPCs 7 days after administration was higher when rats were injected by the intracerebral route. Based on these studies, gene transfer to distal organs or gonadal tissue following intracerebral administration of VPCs is not considered to be a risk to patients undergoing retroviral vector gene therapy for the treatment of brain cancer (glioblastoma multiforme; GBM).

Retrovirus producer cells encoding antisense VEGF prolong survival of rats with intracranial GS9L gliomas

With increasing size tumors are continually dependent on a functional blood vessel system to guarantee the supply with oxygen and nutrients. Vascular endothelial growth factor (VEGF) is a key mediator not only of developmental but also of hypoxia-mediated and tumor-induced angiogenesis. Gene therapy using antisense VEGF with the aim to inhibit tumor angiogenesis may be a successful strategy for the treatment of highly vascular and invasive malignant gliomas. We investigated whether retrovirus producer cells encoding antisense VEGF can be used for in vivo gene transfer. The full length mouse VEGF164 cDNA was cloned in a sense and antisense direction into the retroviral expression vector pLEN. pLEN–VEGF (sense) and pLEN–FGEV (antisense) expression vectors were used to transfect the packaging cell line GP+E86 and to establish ecotropic virus producer cell lines. GP+E86:LEN–FGEV (#5) cells showed high expression of antisense VEGF mRNA, whereas GP+E86:LEN–VEGF (#8) showed high expression of sense VEGF mRNA and active VEGF protein. Co-implantation of GS-9L cells with retrovirus producing cells containing the antisense VEGF construct into the brains of syngeneic rats showed a statistically significant inhibition of tumor growth and prolongation of survival time, while co-implantation of retrovirus producer cells containing the sense VEGF expression vector resulted in an increasing tumor growth and reduced survival time of the rats compared to control animals. Histological analysis of the tumors co-implanted with GP+E86:LEN–FGEV (#5) cells showed the suppression of angiogenesis, high degree of necrosis and no evidence of a significant immune response. Expression of antisense VEGF mRNA in these tumors was confirmed by in situ hybridization analysis. This is the first report demonstrating the potential utility of virus producer cells as in vivo gene transfer vehicles for antisense VEGF gene therapy of malignant gliomas.

Kinetics of retrovirus production and decay

There has been only limited success in using recombinant retroviruses to transfer genes for the purposes of human gene therapy, in part because the average number of genes delivered to the target cells (transduction efficiency) is often too low to achieve the desired therapeutic effect [Miller, AD. 1990. Blood 76:271–278; Mulligan RC. 1993. Science 260:926–932; Orkin SH, Mo- tulsky AG. 1995. Report and recommendations of the panel to assess the NIH investment in research on gene therapy. Bethesda, MD: National Institutes of Health.]. One strategy to improve transduction efficiency is to focus on understanding and improving the processes used to produce recombinant retroviruses. In this report, we characterized the dynamics of retrovirus production and decay in batch cultures of virus producer cells using a simple mathematical model, a recombinant retrovirus encoding the Escherichia coli lacZ gene, and quantitative assays for virus activity and number. We found that the rate at which recombinant retroviruses spontaneously lose their activity (decay) is a strong function of temperature, decreasing roughly 2-fold for every 5°C reduction in temperature, whereas the rate at which retroviruses are produced is only weakly affected by temperature, decreasing about 10% for every 5°C reduction in temperature. In addition, we developed a simple mathematical model of virus production and decay that predicted that the virus titer in batch cultures of virus producer cells would reach a maximum steady-state at a rate that is inversely proportional to the virus decay rate and to a level that is proportional to the ratio of the virus production rate to the virus decay rate. Consistent with the model, we observed that the steady-state levels of virus titer increased more than 3-fold when the cell culture temperature was reduced from 37 to 28°C. Despite their higher titers, virus stocks produced at 28°C, when used in undiluted form so as to mimic human gene transfer protocols, did not transduce substantially more cells than virus stocks produced at 37°C. The implications of our findings on the production of retroviruses for use in human gene therapy protocols are discussed. © 1999 John Wiley & Sons, Inc. Biotechnol Bioeng 63: 654–662, 1999.

Kinetics of retrovirus production and decay

There has been only limited success in using recombinant retroviruses to transfer genes for the purposes of human gene therapy, in part because the average number of genes delivered to the target cells (transduction efficiency) is often too low to achieve the desired therapeutic effect [Miller, AD. 1990. Blood 76:271-278; Mulligan RC. 1993. Science 260:926-932; Orkin SH, Motulsky AG. 1995. Report and recommendations of the panel to assess the NIH investment in research on gene therapy. Bethesda, MD: National Institutes of Health.]. One strategy to improve transduction efficiency is to focus on understanding and improving the processes used to produce recombinant retroviruses. In this report, we characterized the dynamics of retrovirus production and decay in batch cultures of virus producer cells using a simple mathematical model, a recombinant retrovirus encoding the Escherichia coli lacZ gene, and quantitative assays for virus activity and number. We found that the rate at which recombinant retroviruses spontaneously lose their activity (decay) is a strong function of temperature, decreasing roughly 2-fold for every 5 degrees C reduction in temperature, whereas the rate at which retroviruses are produced is only weakly affected by temperature, decreasing about 10% for every 5 degrees C reduction in temperature. In addition, we developed a simple mathematical model of virus production and decay that predicted that the virus titer in batch cultures of virus producer cells would reach a maximum steady-state at a rate that is inversely proportional to the virus decay rate and to a level that is proportional to the ratio of the virus production rate to the virus decay rate. Consistent with the model, we observed that the steady-state levels of virus titer increased more than 3-fold when the cell culture temperature was reduced from 37 to 28 degrees C. Despite their higher titers, virus stocks produced at 28 degrees C, when used in undiluted form so as to mimic human gene transfer protocols, did not transduce substantially more cells than virus stocks produced at 37 degrees C. The implications of our findings on the production of retroviruses for use in human gene therapy protocols are discussed.

Retrovirus-mediated transfer of anti-MDR1 hammerhead ribozymes into multidrug-resistant human leukemia cells: screening for effective target sites.

One of the underlying mechanisms of multidrug resistance (MDR) is cellular over-production of P-glycoprotein (P-gp), which acts as a drug efflux pump. P-gp is encoded by a small group of related genes termed MDR; only MDR1 is known to confer drug resistance. To overcome P-gp-mediated drug resistance, we have developed two anti-MDR1 hammerhead ribozymes driven by the beta-actin promoter. Upon transduction of the ribozymes into MDR cells, vincristine resistance was decreased. These two ribozymes were constructed, which showed different cleavage activities. In this study, to determine suitable target sites for the anti-MDR1 ribozyme, the exon 1b-intron 1 boundary, the translation-initiation site, the intron 1-exon 2 boundary and the exon 2-intron 2 boundary, codons 179 and 196 of the MDR1 gene were selected as candidates. To improve the ribozyme activity, a retroviral vector containing RNA polymerase III promoter was used. Stable retrovirus producer cells were generated by transfecting the retroviral vector plasmids carrying the ribozyme into the packaging cell line. Retroviral vector transduction of human leukemia cell lines expressing MDR1 was accomplished by co-culturing these with virus producer cells. Stably transduced cells were selected by G418 and pooled to determine the efficacy of each ribozyme. These ribozyme-transduced cells became vincristine-sensitive concomitant with the decreases in MDR1 expression, P-gp amount and drug efflux pump function. Among the ribozymes tested, the anti-MDR1 ribozyme against the translation-initiation site exhibited the strongest efficacy. This retrovirus-mediated transfer of anti-MDR1 ribozyme may be applicable to the treatment of MDR cells as a specific means to reverse resistance.