Replication Competent Lentivirus Research Articles

Generation of lentivirus vectors using recombinant baculoviruses

In spite of advances in conventional four-plasmid transient transfection methods and development of inducible stable production cell lines, production of replication-defective lentiviral vectors in clinical scale has been challenging. Baculovirus technology offers an alternative to scalable virus production as a result of fast and easy production of baculoviruses, efficient transduction of mammalian cells and safety of the baculoviruses. As a first step toward scalable lentiviral production system, we have constructed four recombinant baculoviruses: the BAC-transfer virus expresses green fluorescent protein (GFP) as a transgene and BAC-gag-pol, BAC-vesicular stomatitis virus glycoprotein G and BAC-rev express all elements required for a safe lentivirus vector generation. Following 293T cell transduction with recombinant baculoviruses functional lentiviruses were produced. Different baculovirus concentrations, mediums and transduction times were used to find optimal conditions for lentivirus production. The unconcentrated lentiviral titers in cell culture mediums were on average 2.5 x 10(6) TU ml(-1), which are comparable to titers of the lentiviruses produced by conventional four-plasmid methods. Lentiviruses produced by baculovirus method transduced HeLa cells and showed sustained GFP expression. No evidence of the formation of replication competent lentiviruses was detected by p24 enzyme-linked immunosorbent assay. Our results show that baculoviruses are an attractive alternative for the production of lentiviruses in mammalian cells.

Retroviral infections in sheep and the associated diseases

Retroviruses are RNA viruses widely distributed in most vertebrate and in many non-vertebrate species. Among the retrovirus family, small ruminant lentiviruses (SRLV), Jaagsiekte sheep retrovirus (JSRV) and enzootic nasal tumor virus (ENTV) infect small ruminants. JSRV and ENTV are oncogenic retroviruses responsible for pulmonary and nasal adenocarcinomas, respectively, which can lead to the death of the animals in few weeks or months. SRLV are non-oncogenic and replication-competent lentiviruses, related to human immunodeficiency virus (HIV). SRLV are responsible for slowly evolving progressive degenerative diseases leading to the death of the host after few months or years. These viruses are transmitted through the colostrum or milk and aerosolization of infectious particles. This review focuses on the biology of the small ruminant retrovirus and on the main mechanisms of disease induction.

Gene delivery by lentivirus vectors

The capacity to efficiently transduce nondividing cells, shuttle large genetic payloads, and maintain stable long-term transgene expression are attributes that have brought lentiviral vectors to the forefront of gene delivery vehicles for research and therapeutic applications in a clinical setting. Our discussion initiates with advances in lentiviral vector development and how these sophisticated lentiviral vectors reflect improvements in safety, regarding the prevention of replication competent lentiviruses (RCLs), vector mobilization, and insertional mutagenesis. Additionally, we describe conventional molecular regulatory systems to manage gene expression levels in a spatial and temporal fashion in the context of a lentiviral vector. State of the art technology for lentiviral vector production by transient transfection and packaging cell lines are explicitly presented with current practices used for concentration, purification, titering, and determining the safety of a vector stock. We summarize lentiviral vector applications that have received a great deal of attention in recent years including the generation of transgenic animals and the stable delivery of RNA interference molecules. Concluding remarks address some of the successes in preclinical animals, and the recent transition of lentiviral vectors to human clinical trials as therapy for a variety of infectious and genetic diseases.

Design of a trans protease lentiviral packaging system that produces high titer virus

BackgroundThe structural and enzymatic proteins of the human immunodeficiency virus (HIV) are initially generated as two long polyproteins encoded from overlapping reading frames, one producing the structural proteins (Gag) and the second producing both structural and enzymatic proteins (Gag-Pol). The Gag to Gag-Pol ratio is critical for the proper assembly and maturation of viral particles. To minimize the risk of producing a replication competent lentivirus (RCL), we developed a "super-split" lentiviral packaging system in which Gag was separated from Pol with minimal loss of transducibility by supplying protease (PR) in trans independently of both Gag and Pol.ResultsIn developing this "super-split" packaging system, we incorporated several new safety features that include removing the Gag/Gag-Pol frameshift, splitting the Gag, PR, and reverse transcriptase/integrase (RT/IN) functions onto separate plasmids, and greatly reducing the nucleotide sequence overlap between vector and Gag and between Gag and Pol. As part of the construction of this novel system, we used a truncated form of the accessory protein Vpr, which binds the P6 region of Gag, as a vehicle to deliver both PR and RT/IN as fusion proteins to the site of viral assembly and budding. We also replaced wt PR with a slightly less active T26S PR mutant in an effort to prevent premature processing and cytoxicity associated with wt PR. This novel "super-split" packaging system yielded lentiviral titers comparable to those generated by conventional lentiviral packaging where Gag-Pol is supplied intact (1.0 × 106 TU/ml, unconcentrated).ConclusionHere, we were able to create a true "split-function" lentiviral packaging system that has the potential to be used for gene therapy applications. This novel system incorporates many new safety features while maintaining high titers. In addition, because PR is supplied in trans, this unique system may also provide opportunities to examine viral protein processing and maturation.

A trans-lentiviral packaging cell line for high-titer conditional self-inactivating HIV-1 vectors

Lentiviral vector safety has been the impetus underlying the progress in packaging cell line development. The prospects of generating replication-competent lentiviruses (RCLs) and the potential for vector mobilization continue to be the driving force for the advancement of packaging cell lines. We have exploited the trans-lentiviral packaging system to develop the SODk3 packaging cell line for the generation of conditional self-inactivating (cSIN) vectors. Separating the gag–pol genome into two distinct expression cassettes (gag–pro and vpr–RT–IN) may reduce the potential for RCL formation, while concurrently employing cSIN vectors supports retention of the SIN phenotype in target cells and alleviates technical constraints associated with generating producer cell lines. Through development of the SODk3 packaging cell line we determined that the ratio of Gag/Pol in vector particles may be used as an indicator for packaging cell clones that yield high vector titers. Conditional SIN vector titers (1 × 107 TU/ml) were augmented through clonal selection. Distinct producer cell clones revealed a parallel between vector titer and transgene expression levels. We exploited this observation to demonstrate that incorporation of an internal ribosome entry site between the GFP marker and a relevant transgene affords efficient selection of high-titer producer cell lines. Furthermore, cSIN vectors generated from SODk3 packaging cells imparted efficient transduction of primary human fibroblasts, an indication of the future applicability of the SODk3 packaging cell line.

755. Phase II Clinical Trial Demonstrates the Safety and Tolerability of Multiple Doses of Autologous CD4+ T Cells Transduced with VRX496, a Lentiviral Vector Delivering Anti-HIV Antisense in Patients Failing 1 or More HAART Treatment

Gene therapy for HIV-1 infection has been proposed as an alternative to antiretroviral drug regimens due to emerging drug resistance and toxicity that raises concerns about HAART as a long term therapy. We have previously reported the successful completion of our Phase I clinical trial testing the safety and tolerability of a single dose of autologous HIV infected CD4+ T cells transduced with a lentiviral vector delivery system expressing a 937-base antisense gene against the HIV envelope (VRX496) for use in T cell therapy for HIV/AIDS. These results have lead us to initiate a Phase II clinical trial to evaluate the safety, tolerability, and biological activity of repeated infusions (4 or 8 doses) of autologous VRX496 transduced T cells. The study will enroll up to 40 male and female HIV-positive subjects in up to 8 centers. Subjects will be 18 years of age and over who have failed or are intolerant to at least one triple combination of antiretroviral drugs. Subjects will have, a viral load between 5,000 and 200,000 copies/ml and a CD4+ count of |[ge]|150. Additionally, subjects must have a Karnofsky Performance score of 80 or higher, have no evidence of active opportunistic infection, congestive heart failure, hemodynamic instability, bleeding diathesis and no contraindications for leukapheresis. Patients will receive repeated doses by i.v. of |[sim]|1|[ast]|1010 genetically modified autologous T-cells, every two weeks. The primary safety objectives will include incidence of adverse events, changes in viral load, changes in CD4+ T cells, changes in TCR v beta repertoire, and tests for replication competent lentivirus (RCL). Tissue trafficking of gene modified cells will also be monitored in GALT tissue. The first part of the study is evaluating the safety and tolerability of multiple dosing using a repeat dosing design. All patients in the 4 dose cohort have received their infusions as scheduled and have reached the 3 month post-infusion visit, and one patient in the 8 dose cohort has received all of the doses as scheduled. To date, all of the doses have been well tolerated and preliminary results suggest multiple infusions are safe, with no SAE's due to the product, no changes in hematology or chemistry laboratory evaluations, and no detection of VSVG DNA or RCL. In the second part, we are determining an optimal dosing regimen for future confirmatory trials. To date 18 patients have been enrolled and 11 have started receiving infusions. The data generated from the current clinical trial demonstrates the clinical utility of lentiviral vector technology as an alternative for treatment of HIV infection.

A replication competent lentivirus (RCL) assay for equine infectious anaemia virus (EIAV)-based lentiviral vectors

Lentiviral vectors are being developed to satisfy a wide range of currently unmet medical needs. Vectors destined for clinical evaluation have been rendered multiply defective by deletion of all viral coding sequences and nonessential cis-acting sequences from the transfer genome. The viral envelope and accessory proteins are excluded from the production system. The vectors are produced from separate expression plasmids that are designed to minimize the potential for homologous recombination. These features ensure that the regeneration of the starting virus is impossible. It is a regulatory requirement to confirm the absence of any replication competent virus, so we describe here the development and validation of a replication competent lentivirus (RCL) assay for equine infectious anaemia virus (EIAV)-based vectors. The assay is based on the guidelines developed for testing retroviral vectors, and uses the F-PERT (fluorescent-product enhanced reverse transcriptase) assay to test for the presence of a transmissible reverse transcriptase. We have empirically modelled the replication kinetics of an EIAV-like entity in human cells and devised an amplification protocol by comparison with a replication competent MLV. The RCL assay has been validated at the 20 litre manufacturing scale, during which no RCL was detected. The assay is theoretically applicable to any lentiviral vector and pseudotype combination.

Product-Enhanced Reverse Transcriptase Assay for Replication-Competent Retrovirus and Lentivirus Detection

The product-enhanced reverse transcriptase (PERT) assay has been used to detect reverse transcriptase (RT) activity associated with retroviruses. Although the PERT assay has been proposed as a method for detection of replication-competent retrovirus (RCR) and lentivirus (RCL), it has not been rigorously compared with existing methods for RCR and RCL detection. We have assessed the PERT assay for detection of RCL and RCR that may contaminate lentiviral and retroviral vectors and compared it with published methods for RCL (p24gag ELISA/gag PCR) and RCR (S+/L-) detection. Our results suggest that the PERT assay is as sensitive as p24gag ELISA and gag PCR for detection of replication-competent HIV-1 in an RCL detection assay. Comparison of detection of replication-competent retroviruses, GALV and RD114, by extended S+/L- and PERT assays indicates that both assays can detect 1 IU of each virus. Our findings suggest that the PERT assay can be used for RCL and RCR testing of a variety of retroviral vectors regardless of the structure, sequence, and envelope of the vectors.

1075. Phase I Clinical Trial Demonstrates Safety and Feasibility of Autologous Cellular Therapy with Lentiviral Modified CD4 T Cells Expressing Anti-HIV Antisense in Patients with HAART Resistant HIV-1 Infection

Top of pageAbstract Emerging drug resistance raises concerns about HAART as a long-term therapy. We have developed an HIV-1-based lentiviral vector expressing a 937-base antisense gene against the HIV envelope for autologous T cell therapy for HIV/AIDS. The vector introduces no new sequences to an HIV-infected individual. In preclinical studies, HIV replication was inhibited over 95%, and up to 4 logs, in primary CD4 T lymphocytes isolated from HIV-infected patients. For evaluation of safety and tolerability of this therapy, a Phase I open-label non-randomized clinical trial has been carried out. Five patients who have failed at least two HAART regimens due to intolerance or resistance, and have CD4 counts above 150 cells/mm3 and a viral load greater than 5000 copies/ml, have been serially enrolled for a 6-month study with 21 day, 3, 6, 9, and 12-month monitoring points. Each patient was given a single i.v. dose of |[sim]| 1 |[times]| 1010 genetically-modified autologous T cells, and then monitored for viral load, CD4 count, replication competent lentivirus (RCL), outgrowth of vector-modified cells, immunological parameters, and adverse events. Serious adverse events (SAE) were defined in part by a sustained 30% increase in viral load, or sustained 0.5 log decrease in CD4 count within 3 weeks post dose. All five patients have been dosed, and have completed the critical 3-week safety timepoint. No SAEs related to the product have occurred, and all RCL tests have been negative, including a biological RCL assay performed on apheresed patient PBMCs at 6 months. Patients 1-3 have reached their 1 year follow-up and all three have demonstrated clinically significant (>0.5 log) reductions in their viral loads at 1 year at 0.78 log, 1.61 log, and 0.57 log, respectively. Furthermore, patients 2 and 3 have had a 20% and 30% increase in CD4 counts, respectively, while patient 1 has maintained a steady CD4 count. Patient 4 will reach his 9-month follow up in March, and Patient 5 has just completed his 3-month follow up. Circulating vector-modified cells was detected in patient 2 at one year, to month 9 in patients 1 and 3, and they remain detectable in patients 4 and 5. To date, 211 total integration sites from the transduced clinical products of all 5 patients have been analyzed; initial results indicate that VRX496 preferentially integrates into transcription units and disfavors integration into alphoid repeats, a pattern that is comparable to and indistinguishable from wild type HIV. These data support the safety and feasibility of lentiviral vector therapy in the setting of ex vivo cellular therapy.

Report from the Lentivirus Vector Working Group: Issues for Developing Assays and Reference Materials for Detecting Replication-Competent Lentivirus in Production Lots of Lentivirus Vectors

Report from the Lentivirus Vector Working Group: Issues for Developing Assays and Reference Materials for Detecting Replication-Competent Lentivirus in Production Lots of Lentivirus Vectors

Generation of a packaging cell line for prolonged large‐scale production of high‐titer HIV‐1‐based lentiviral vector

A stable packaging cell line facilitates large-scale lentivirus vector manufacture. However, it has been difficult to produce clinical-scale HIV-1-based lentiviral vectors using a packaging cell line, in part due to toxicity of packaging genes, and gene silencing that occurs during the long culture period necessary for sequential addition of packaging constructs. To avoid these problems, we developed a three-level cascade gene regulation system designed to remove tetracycline transactivator (tTA) from cytomegalovirus immediate early promoter (CMV)-controlled expression to reduce cytotoxicity from constitutive expression of tTA and leaky expression of packaging genes. We also performed a one-step integration of the three packaging plasmids to shorten the culture time for clonal selection. Although leaky expression of p24 and vector production still occurred despite the three-level regulation system, little cytotoxicity was observed and producer cells could be expanded for large-scale production. Producer cells yielded remarkably stable vector production over a period greater than 11 days with the highest titer 3.5 x 10(7) transducing units (TU)/ml and p24 300 ng/ml, yielding 2.2 x 10(11) TU and 1.8 milligram (mg) p24 from one cell factory. No replication-competent lentivirus (RCL) was detected. Long-term analysis demonstrated that, although the cells are genetically stable, partial gene silencing occurs after 2-3 months in culture; however, the one-step construct integration allowed prolonged vector production before significant gene silencing. Concentrated vector resulted in 90% transduction in CD4+ lymphocytes at 20 TU per cell. CD34+ progenitor cells were transduced at 41-46% efficiency, and long-term initiating culture (LTC-IC) was transduced at 45-51%. These results demonstrate for the first time HIV-1-based lentiviral vector production on the large scale using a packaging cell line.

Regulatory Considerations for Novel Gene Therapy Products: A Review of the Process Leading to the First Clinical Lentiviral Vector

This review is intended to exemplify the roles and responsibilities of the two agencies under the Department of Health and Human Services, the National Institutes of Health and the Food and Drug Administration, that have oversight for human gene transfer clinical protocols, as seen through our experience of bringing a first-in-its-class lentiviral vector to clinical trials. In response to the changing circumstances in gene therapy research between 1999 and 2002, the concerns of these agencies regarding gene therapy have been evolving. This review provides an overview of the major safety concerns regarding insertional oncogenesis, the generation of a replication- competent lentivirus (RCL), and vector mobilization thought to be related to lentiviral vectors, which had to be addressed during the regulatory review process before initiating the clinical trial. Specific monitoring assays to address these concerns were established to test for RCL generation, vector mobilization, persistence of vector-modified cells, and abnormal clonal expansion of modified cells. We hope to provide a basic understanding and appreciation of the regulatory process and major safety concerns, toward providing useful insight to those presently embarking on the development of clinical application of lentiviral vectors.

A Synthetic Rev-Independent Bovine Immunodeficiency Virus-Based Packaging Construct

Replication competent lentivirus (RCL) has been the major safety concern associated with applications of lentivirus-based gene transfer systems for human gene therapy. Minimization and elimination of overlaps between the packaging and the transfer vector constructs are expected to reduce the potential to generate RCL. We previously developed second- and third-generation bovine immunodeficiency virus (BIV)-based gene transfer systems. However, some sequence homologies between the vector and gag/pol packaging constructs remained. In order to minimize the sequence homologies, we recoded gag/pol with codon usage optimized for expression in human cells in this report. Expression of the recoded gag/pol was Rev/RRE independent. Thus, RRE was eliminated from the packaging construct, thereby removing a 312 bp block of homology. In addition, recoding gag/pol minimized overall homologies between the packaging and transfer vector constructs. Vectors generated by the recoded packaging construct with a four plasmid system had titers greater than 1 × 106 transducing units per milliliter, equivalent to those of the earlier generation systems. The vectors were functional in vitro and efficiently transduced rat pigment epithelial cells in vivo. Generation of the synthetic packaging construct provides further advances to the safety of lentiviral vectors for clinical applications.

A synthetic Rev-independent bovine immunodeficiency virus-based packaging construct.

Replication competent lentivirus (RCL) has been the major safety concern associated with applications of lentivirus-based gene transfer systems for human gene therapy. Minimization and elimination of overlaps between the packaging and the transfer vector constructs are expected to reduce the potential to generate RCL. We previously developed second- and third-generation bovine immunodeficiency virus (BIV)-based gene transfer systems. However, some sequence homologies between the vector and gag/pol packaging constructs remained. In order to minimize the sequence homologies, we recoded gag/pol with codon usage optimized for expression in human cells in this report. Expression of the recoded gag/pol was Rev/RRE independent. Thus, RRE was eliminated from the packaging construct, thereby removing a 312 bp block of homology. In addition, recoding gag/pol minimized overall homologies between the packaging and transfer vector constructs. Vectors generated by the recoded packaging construct with a four plasmid system had titers greater than 1 x 10(6) transducing units per milliliter, equivalent to those of the earlier generation systems. The vectors were functional in vitro and efficiently transduced rat pigment epithelial cells in vivo. Generation of the synthetic packaging construct provides further advances to the safety of lentiviral vectors for clinical applications.

Safe two-plasmid production for the first clinical lentivirus vector that achieves >99% transduction in primary cells using a one-step protocol.

We report the design of a unique two-plasmid production system for the first lentiviral vector to be evaluated in humans, VRX496. VRX496 is an optimized VSV-G pseudotyped vector derived from HIV-1 that expresses antisense to the HIV envelope gene. We found that a two-plasmid approach to production resulted in higher vector production titers when compared with a three-plasmid approach, which is particularly important for vector production at the large scale. Therefore, we carefully designed a single packaging construct, VIRPAC, for safety by reducing its homology with VRX496 and by insertion of functionally validated genetic elements designed to reduce the risk of generation of a replication-competent lentivirus (RCL). A native cis-acting ribozyme is used to prevent read through into the envelope gene from the upstream gag-pol genes in the packaging vector, thus preventing RNAs containing gag-pol and env together for comparable safety to a three-plasmid system. We demonstrate that there is no significant in vivo vector mobilization using a primary SCID-hu mouse transplantation model, which correlates with the presence of an anti-HIV payload and suggests that inclusion of antisense may be a useful tool to restrict mobilization in other vector constructs. Gene transfer is achieved using a one-step transduction procedure that is simple and clinically translatable, which reaches stable transduction efficiencies of >99% in CD4+ T lymphocytes within 3 days of culture initiation.

82. Adaptation of a Lentiviral Vector Producer Cell Line to Reduced-Serum, Suspension Culture for Large Scale Production

The ability of lentiviral-based vectors to integrate into non-dividing cells such as neuronal cells has been exploited to correct a number of disease models. Using vectors based on the equine infectious anaemia virus (EIAV) we have previously reported the correction of an animal model of Parkinson's disease (Azzouz et al. J Neurosci. 2002 Dec 1;22(23):10302–12). To use such vectors in the clinic each manufactured lot must be tested and demonstrated to be free of replication competent lentivirus (RCL). We have designed the vectors in such a way to limit the possibility of homologous recombination taking place; the vector genome contains only 824 nt of EIAV sequence with no coding regions; all the accessory genes have been removed entirely from the system; the gag/pol sequence has been codon optimised and a heterologous envelope is used (VSV-G). However, there is still the formal possibility of some form of recombination leading to the generation of an RCL. We have therefore developed an RCL assay based on the property common to all members of the Retroviradae family, namely the presence of reverse transcriptase (RT). RT activity can be detected by a sensitive assay: Product Enhanced Reverse Transcriptase (PERT). The test article is passaged on human cells to amplify any replicating entity and then a PERT assay is carried out on the supernatant. A typical set of assay results will be presented.

1003. HIV-1-Derived Lentivirus Vector-Based Antisense Gene Therapy: Towards an Alternative Treatment for HIV/AIDS

Despite the significant advancement in HIV/AIDS treatment with highly active antiretroviral therapy (HAART), HIV/AIDS remains incurable. Limitations of HAART include expense, disabling complications, and a stringent requirement for compliance. In the United States, where HAART has become the standard of care, approximately 20% of newly transmitted virus is already resistant to at least one antiretroviral drug. In anticipation of expanded HAART in Africa, there is concern regarding rapid emergence of drug resistant strains of HIV that brings into question HAART as a long-term solution. We have developed an alternative therapy for HIV/AIDS therapy using an HIV-1-based lentiviral vector expressing a 937-base antisense gene against the HIV envelope, VRX496, for autologous T cell therapy. T cells are transduced with vector in the presence of co-stimulatory CD3/CD28 beads, and subsequently expanded in culture. Preclinical studies demonstrated the feasibility of this approach in normal CD4+ T cells and inCD4+ T cells isolated from 20 early and late stage patients, as defined by viral loads. An efficient gene transfer procedure was established, which demonstrated stable transfer rates of 94% +/- 5%. Antisense inhibited HIV in cultures by >93% over mock transduced controls, regardless of patient status or the tropism of the infecting virus. A Phase I open label non-randomized clinical trial was then initiated to investigate the safety and tolerability of autologous T cells modified with VRX496. In this study, 5 HAART resistant patients having CD4 T cells counts between 200-500 cells/mm3 are serially enrolled in the study that includes 3-week, and 3 and 6-month monitoring points. Each patient is given approximately 1 1010 modified autologous CD4 T cells in a single dose. Patients are monitored for viral load, CD4 count, emergence of any replication competent lentivirus (RCL), immunological parameters, and clinical indications. Adverse events are defined in part by a sustained 0.5 log increase in viral load, or sustained 0.5 log decrease in CD4 count within 3 weeks post dose. To date, 3 subjects have been infused, and the first and second subjects have completed the 6 and 3-month monitoring points, respectively. No adverse events have been noted, and all RCL assays have been negative. CD4 counts remained steady and engraftment of VRX496-modified cells was observed for at least 6 months in the first patient and 3 months in the second patient. At dosing, subject 1 had an average viral load of 188,500 and subject 2 had an average viral load of 40,428. After three and six months post dosing, subjects 1 and 2 respectively, had viral loads below baseline. This interim analysis supports the promise of lentiviral vectors for late stage HIV infection, and that VRX496 could become a viable treatment modality for individuals infected with HIV/AIDS.

67. Bovine Immunodeficiency Virus Based Lentiviral Vector with Improved Safety Features

Replication competent lentivirus (RCL) has been the major safety concern associated with applications of lentivirus based gene transfer systems for human gene therapy. Minimization and elimination of overlaps between the packaging and the transfer vector constructs are expected to reduce the potential to generate RCL. We previously developed second and third generation Bovine Immunodeficiency Virus (BIV) based gene transfer systems (M. Matukonis, M. Li, R. P. Molina, B. Paszkiet, M. Kaleko, and T. Luo, 2002, Hum. Gene Ther. 13,1293–1303). However, some sequence homologies between the vector and gag/pol packaging constructs remained. We now describe a fourth generation packaging construct in which the gag/pol was recoded with codon usage optimized for expression in human cells. Expression of the recoded gag/pol was Rev/RRE independent. Thus, RRE was eliminated from the packaging construct, thereby removing a 312 bp block of homology. In addition, recoding gag/pol minimized overall homologies between the packaging and transfer vector constructs. The longest homologous sequence between the packaging construct and the transfer vector construct is only 8 bps. Moreover, recoding resulted in a 10-fold improvement in Gag/Pol expression levels. Vectors generated by the recoded packaging construct with a four plasmid system had titers greater than 1 × 106 transducing units per milliliter, equivalent to those of the earlier generation systems. The vectors were functional in vitro and efficiently transduced rat pigment epithelial cells in vivo. Generation of the synthetic packaging construct provides further advances to the safety of lentiviral vectors for clinical applications.

69. Closing-in on the titer gap between transient transfection and packaging cell lines

Lentiviral vectors are a potent gene delivery system that show promise for hematopoietic and neuronal cell applications. However, several safety concerns exist regarding their use. These include the possibility of a replication-competent lentivirus (RCL) arising, which is a particular concern when the parental virus is a human pathogen such as HIV-1. In addition, in common with other integrating vectors, lentiviral vectors have the potential to cause insertional mutagenesis. This can occur directly by gene disruption or read-through transcription from the vector cassette, or indirectly through the action at a distance effects of vector enhancer/promoter elements.

Certification assays for HIV-1-based vectors: frequent passage of gag sequences without evidence of replication-competent viruses

A principal concern regarding the safety of HIV-1-based vectors is replication-competent lentivirus (RCL). We have developed two PCR assays for detecting RCL; the first detects recombination between gag regions in the transfer vector and the packaging construct (sensitivity of detection ∼10–100 copies of target sequence). The second assay uses real-time PCR to detect vesicular stomatitis virus glycoprotein (VSVG) envelope DNA (sensitivity ∼5–50 VSVG sequences). In an attempt to amplify any RCL, test vectors were used to transduce C8166 and 293 cells, which were then screened weekly for 3 weeks. Psi–gag recombinants were routinely detected (20 of 21 analyses) in four transductions using the RRL-CMV-GFP vector. In contrast, VSVG sequences were detected only once in 21 analyses. Interestingly, p24 levels (as measured by ELISA) were occasionally detectable after 3 weeks of culture. To determine if a true RCL was present, 21-day cell-free medium was used to transduce naïve cells. No evidence of psi–gag or VSVG transfer was detected, indicating that the recombination events were insufficient to reconstitute a true RCL. These findings have important implications for the design and safety of HIV-1-based vectors intended for clinical applications.