Multiple platforms of a HIV-2 derived lentiviral vector for expanded utility

Human immunodeficiency virus type 1 (HIV-1) based gene transfer systems are gaining in popularity due to their ability to transduce terminally differentiated and non-dividing cells. Oncoretroviral vectors based on Moloney murine leukemia virus (MoMLV), on the other hand, can only transduce dividing cells. The reasons for increased ability of lentivirus vectors to transduce such cells has been attributed to several of the viral proteins (integrase, matrix and Vpr) that are purported to be involved in the nuclear import of the pre-integration complex (PIC). Nuclear import is also augmented by a unique triple stranded DNA region created during reverse transcription of the incoming viral RNA in the target cell (discussed in chapter 3). This chapter deals with the rationale behind the design of human immunodeficiency virus type 1 (HIV-1) based packaging systems with an emphasis on some recent advances in the field for the creation of safe and efficient HIV-1 based vectors. The review covers trans-acting proteins and cis-sequences required for the deployment of HIV-1 vectors for gene transfer. This is a rapidly advancing field that with further refinements may soon allow the utilization of HIV-1 based and/or other lentivirus vectors in a clinical setting.

Self-Inactivating Lentiviral Vectors with U3 and U5 Modifications

A retrovirus‐based gene transfer system consists of two components: the transfer vector, which harbours a foreign gene linked to elements needed for retroviral replication, and the packaging cell, which supplies the necessary retroviral proteins for transfer of the vector through a single round of viral replication. A hallmark of retroviral replication is the stable maintenance of the transferred gene(s) during cell division. The first type of transfer vectors used in gene therapy trials were derived from mouse retroviruses and referred to as retroviral vectors. Lentiviral vectors are derived from human immunodeficiency virus type 1 and exhibit distinct features that provide advantages in some settings. Retroviral and lentiviral vectors are being used in clinical trials for several diseases, including monogenic diseases and cancer. The clinical protocols include gene transfer in vivo as well as ex vivo , the latter being followed by installation of the genetically modified cells into the body. Key Concepts: The retroviral replication machinery leads to stable maintenance of a transferred gene. The stable integration of retroviral vectors into a chromosome of the target cell may have adverse effects on neighbouring genes. Retroviral gene transfer does not require the expression of retroviral genes in the target cell. The term retroviral vector is used for a vector based on murine leukaemia virus. The term lentiviral vector is used for a vector based on human immunodeficiency virus type 1. Retroviral vectors allow gene transfer only to dividing cells, whereas lentiviral vectors target dividing as well as nondividing cells. In clinical protocols using retroviral or lentiviral vectors, gene transfer may take place ex vivo or in vivo . The selectivity of retroviral vectors for dividing cells has been exploited to target tumour cells.

Chimeric antigen receptor (CAR) T cell immunotherapy has been a major advancement in cancer therapeutics. Reprogramming of T cells is achieved by using gammaretroviral or lentiviral vectors, which may interfere with human immunodeficiency virus type 1 (HIV-1) nucleic acid amplification testing (NAAT). Here, we describe three clinical scenarios in which CAR T cell immunotherapy interfered with HIV-1 testing, including (i) routine infectious disease screening prior to stem cell transplantation in a 16-year-old female with B cell acute lymphoblastic leukemia, post CAR T cell treatment; (ii) routine infectious disease screening prior to second CAR T cell collection in a 65-year-old male with diffuse large B cell lymphoma who failed initial CAR T cell treatment; and (iii) routine infectious risk assessment following an occupational health exposure from a 58-year-old male with multiple myeloma, who received CAR T cell treatment. In each case, patients initially tested negative by the "fourth-generation" HIV-1 screening enzyme immunoassay (targeting the p24 antigen and anti-HIV-1 antibodies), but positive by the Roche Cobas AmpliPrep/Cobas TaqMan HIV-1 test v2.0 (targeting gag and the long terminal repeat [LTR]). These samples subsequently retested negative using the Abbott m2000 RealTime HIV-1 assay, which targets the integrase gene. These results indicated that cross-reactions between lentiviral vectors and LTR genomes targeted in the HIV-1 NAAT caused the HIV-1 NAAT false-positive results.

Lentiviral Vector Design for Multiple shRNA Expression and Durable HIV-1 Inhibition

Stable genetic modification of human embryonic stem cells by lentiviral vectors.

The goal of this study was to design improved regulatable lentivirus vector systems. The aim was to design tetracycline (tet)-regulatable lentivirus vectors based on the Tet-on system displaying low background expression in the absence of the doxycycline (DOX) inducer and high transgene expression levels in the presence of DOX. We constructed a binary lentivirus vector system that is composed of a self-inactivating (SIN) lentivirus vector bearing inducible first- or second-generation tet-responsive promoter elements (TREs) driving expression of a transgene and a second lentivirus vector encoding a reverse tetracycline-controlled transactivator (rtTA) that activates transgene expression from the TRE in the presence of DOX. We evaluated a number of different rtTAs and found rtTA2S-M2 to induce the highest levels of transgene expression. Regulated transgene expression was stable in human breast carcinoma cells implanted into nude mice for up to 11 weeks. In an attempt to minimize background expression levels, the chicken beta-globin cHS4 insulator element was cloned into the 3' long terminal repeat (LTR) of the transgene transfer vector. The cHS4 insulator element reduced background expression but expression levels following DOX addition were lower than those observed with vectors lacking an insulator sequence. In a second strategy, vectors bearing second-generation TREs harboring repositioned tetracycline operator elements were used. Such vectors displayed greatly reduced leakiness in the absence of DOX and induced transgene expression levels were up to 522-fold above those seen in the absence of DOX. Inducible lentivirus vectors bearing insulators or second-generation TREs will likely prove useful for applications demanding the lowest levels of background expression.

In this report, we evaluated the efficiency of stable gene transfer into established CD8(+) human tumor antigen-specific cytotoxic T cell (CTL) lines and peripheral blood lymphocytes (PBL) by oncoretroviral and lentiviral vectors. In the oncoretroviral vector, the green fluorescent protein (GFP) reporter gene was regulated by the murine stem cell virus (MSCV) promoter. In three human immunodeficiency virus type 1 (HIV-1)-based lentiviral vectors, the GFP transgene was regulated by either a chimeric MSCV/HIV-1 promoter, or cellular promoters from human housekeeping genes PGK and EF1 alpha. We found that several lines of proliferating tumor-specific CTL were poorly (=2%) transduced by the oncoretroviral vector that transduced Jurkat T cell line efficiently (=80%). In contrast, three lentiviral vectors transduced 38-63% of these proliferating CTL. More interestingly, all lentiviral vectors packaged without the HIV-1 accessory proteins transduced human bulk PBL and purified CD4(+) and CD8(+) lymphocyte subsets without prior stimulation. Detailed analysis indicated that the lentiviral vectors containing the EF1 alpha or PGK ubiquitous promoter can transduce unstimulated PBL and achieve low-level transgene expression in the absence of any T-cell activation. However, T-cell activation subsequent to the transduction of unstimulated PBL is required for high-level transgene expression. Transduced PBL expressing transgene delivered by the lentiviral vectors still preserved resting and naïve cell phenotypes. Taken together, prior T cell stimulation and HIV-1 accessory proteins are dispensable for lentivirus-mediated gene transfer into resting naïve and memory T lymphocytes. These results will have significant implications for the study of T-cell biology and for the improvement of clinical gene therapies of acquired immune deficiency syndrome (AIDS) and cancer.

Retroviral vectors provide the means for gene transfer with long-term expression. The lentivirus subgroup of retroviruses, such as human immunodeficiency virus type 1 (HIV-1) and type 2 (HIV-2), possesses a number of regulatory and accessory genes and other special elements. These features can be exploited to design vectors for transducing non-dividing as well as dividing cells with the potential for regulated transgene expression. Encapsidation of the transgene RNA in lentiviral vectors is determined by the leader sequence-based multipartite packaging signal. Embedded in the packaging signal is a major splice donor site that, this study shows, is not by itself essential for transgene expression or encapsidation. We designed HIV-2 vectors that contained all the sequence elements thought to be necessary and sufficient for vector RNA encapsidation. Unexpectedly, despite abundant expression, only a small fraction of the transgene RNA was encapsidated and the titre of the vector was low. Redesign of the vector with a mutant splice donor resulted in increased vector RNA encapsidation and yielded vectors with high titre. Inefficient encapsidation by the conventionally designed vector was not due to suboptimal Rev responsive element (RRE)-Rev function. Varying the length of RRE in the vector did not change vector RNA encapsidation, nor did the introduction of a synthetic intron into the mutant vector. The vector RNA with the intact splice donor may have been excessively spliced, decreasing the amount of packageable RNA. A titre of 10(5) transducing units (TU)/ml was readily obtained for vectors with the neo or GFP transgene, and the vector could be concentrated to a titre of 1-5x10(7) TU/ml.

Packaging systems based on human immunodeficiency virus type 1 (HIV-1) can be used to transfer genes (both ex vivo and in vivo in experimental animals), with high efficiency into a wide variety of cell types including nondividing and terminally differentiated cells such as muscle and neuronal cells (1–6). In this chapter, I begin with an overview of HIV-1 structure, genetics, and replication cycle, emphasizing those features essential for the creation of packaging systems based on HIV-1. Next, I describe the plasmid constructs for preparation of HIV-1 vector stocks, including some recent advances in the design of helper and gene transfer vectors. Finally, a detailed protocol for generation and testing of vectors is provided. Although HIV-1 is used as a prototype for other lentiviruses and there are many similarities between different viruses, there are also significant differences. The extensive knowledge base of HIV-1-based gene transfer systems can be readily adapted to develop gene transfer vectors based onother lentiviruses such as feline immunodeficiency virus (FIV; see Chapter 23). These other lentivirus vectors are likely to share many of the advantages of HIV-1 vectors. Because of space limitations, comparisons with other lentiviruses that highlight similarities or differences are not given. I recommend the excellent treatise on retroviruses (6a) edited by J.M. Coffin, S.H. Hughes, and H.E. Varmus (CSHL,1997) for this purpose and to support many of the statements made in this introduction.KeywordsLong Terminal RepeatPackaging SystemPackaging PlasmidGene Transfer VectorConstitutive Transport ElementThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Hematopoietic stem cells (HSCs) are clonogenic cells capable of both selfrenewal and multilineage differentiation. An efficient method for gene transfer into HSCs is required for exploring HSC biology as well as for gene therapy of hematopoietic disorders. Retroviral vectors have been the most widely used vectors for gene transfer to HSCs. However, retroviral vectors require cell division for integration, limiting their use for gene transfer into HSCs that are exclusively quiescent. Although prestimulation of HSCs with cytokines can enhance gene-transfer efficiency, exposure to cytokines also stimulates HSCs to differentiate, resulting in the reduction of long-term repopulating capacity (8-15). In contrast, lentiviral vectors based on the human immunodeficiency virus type 1 (HIV-1) can efficiently transduce human CD34+ cells without cytokine prestimulation and long-term multilineage expression of the transgene is detected in nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice after transplantation (16-22). Murine HSCs can also be easily transduced with lentiviral vectors without cytokine prestimulation (23-26). In this chapter, the protocols for transduction of human CD34+ cells that contain HSCs are described. The procedures include the isolation of human CD34+ cells, preparation of lentiviral vectors, transduction of human CD34+ cells, and in vivo analysis of transduced HSCs using NOD/SCID mice.

Heterologous lentiviral vectors (LVs) represent a way to address safety concerns in the field of gene therapy by decreasing the possibility of genetic recombination between vector and packaging constructs and the generation of replication-competent viruses. Using described LVs based on human immunodeficiency virus type 1 (HIV-1) and simian immunodeficiency virus MAC251 (SIV(MAC251)), we asked whether heterologous virion particles in which trans-acting factors belonged to HIV-1 and cis elements belonged to SIV(MAC251) (HIV-siv) would behave as parental homologous vectors in all cell types. To our surprise, we found that although the heterologous HIV-siv vector was as infectious as its homologous counterpart in most human cells, it was defective in the transduction of dendritic cells (DCs) and, to a lesser extent, macrophages. In DCs, the main postentry defect was observed in the formation of two-long-terminal-repeat circles, despite the fact that full-length proviral DNA was being synthesized and was associated with the nucleus. Taken together, our data suggest that heterologous HIV-siv vectors display a cell-dependent infectivity defect, most probably at a post-nuclear entry migration step. As homologous HIV and SIV vectors do transduce DCs, we believe that these results underscore the importance of a conserved interaction between cis elements and trans-acting viral factors that is lost or suboptimal in heterologous vectors and essential only in the transduction of certain cell types. For gene therapy purposes, these findings indicate that the cellular tropism of LVs can be modulated not only through the use of distinct envelope proteins or tissue-specific promoters but also through the specific combinatorial use of packaging and transfer vector constructs.

Virus Vectors for use in the Central Nervous System: Lentiviral Vectors

Adeno-associated Virus-mediated, Mifepristone-regulated Transgene Expression in the Brain

Lentiviral vectors derived from human immunodeficiency virus type 1 (HIV-1) show great promise as gene carriers for future gene therapy. Insertion of a fragment containing the central polypurine tract (cPPT) in HIV-1 vector constructs is known to enhance transduction efficiency drastically, reportedly by facilitating the nuclear import of HIV-1 cDNA through a central DNA flap. We have studied the impact of the cPPT on the kinetics of HIV-1 vector transduction by real-time PCR. The kinetics of total HIV-1 DNA, two-long-terminal-repeat (2-LTR) circles, and, by an Alu-PCR, integrated proviral DNA were monitored. About 6 to 12 h after transduction, the total HIV-1 DNA reached a maximum level, followed by a steep decrease. The 2-LTR circles peaked after 24 to 48 h and were diluted upon cell division. Integration of HIV-1 DNA was first detected at 12 h postinfection. When HIV-1 vectors that contained the cPPT were used, DNA synthesis was similar but a threefold higher amount of 2-LTR circles was detected, confirming the impact on nuclear import. Moreover, a 10-fold increase in the amount of integrated DNA was observed in the presence of the cPPT. Only in the absence of the cPPT was a saturation in 2-LTR circle formation seen at a high multiplicity of infection, suggesting a role for the cPPT in overcoming a barrier to the nuclear import of HIV-1 DNA. A major effect of the central DNA flap on the juxtaposition of both LTRs is unlikely, since transduction with HIV-1 vectors containing ectopic cPPT fragments resulted in increased amounts of 2-LTR circles as well as integrated DNA. Inhibitors of transduction by cPPT-containing HIV vectors were also studied by real-time PCR. The reverse transcriptase inhibitor azidothymidine (AZT) and the nonnucleoside reverse transcriptase inhibitor alpha-APA clearly inhibited viral DNA synthesis, whereas integrase inhibitors such as the diketo acid L-708,906 and the pyranodipyrimidine V-165 specifically inhibited integration.