Lentiviral and MLV based retroviral vectors for ex vivo and in vivo gene transfer

Retroviral vectors have become an important tool for gene transfer in vitro and in vivo. Classical Moloney murine leukemia virus (MLV) based retroviral vectors have been used for over 20 years to transfer genes into dividing cells. Cell lines for production of retroviral vectors have become commonly available and modifications in retroviral vector design and use of envelope proteins have made the production of high titer, helper-free, infectious virus stocks relatively easy. More recently, lentiviral vectors, another class of retroviruses, have been modified for in vitro and in vivo gene transfer. The ability of lentiviral vectors to transduce non-dividing cells has made them especially attractive for in vivo gene transfer into differentiated, non-dividing tissues. Several improvements in helper plasmids and vectors have made lentivirus a safe vector system for ex vivo and in vivo gene transfer. This review will briefly summarize the background of these vector systems and provide some common protocols available for the preparation of MLV based retroviral vectors and HIV-1 based lentiviral vectors.     

Retroviral vectors for vaccine development: induction of HIV-1-specific humoral and cellular immune responses in rhesus macaques using a novel MLV(HIV-1) pseudotype vector

Retroviral vectors have yet not been tested for their potential as vaccines despite their frequent utilization in gene therapy allowing for highly efficient gene transfer into a number of cell types and their suitability for large-scale production in biotechnology. To investigate MLV-based vectors suitability for inducing immune response against HIV-1-antigens, we generated a MLV(HIV-1) pseudotype vector enabling CD4-specific transduction of HIV-1 genes env, vpu, tat and rev originating from the pathogenic SHIV-89.6P. Functional expression of the lentiviral genes in packaging cells, human and rhesus CD4+ target cells was demonstrated by various assays. Following highly efficient ex vivo transduction, up to 3.4x10(7) autologous, transfer vector-positive rhesus peripheral blood mononuclear cells (rhPBMCs) were re-inoculated into a rhesus macaque. Five weeks after the initial inoculation HIV-1 Env-specific antibodies were detected using ELISA. ELIspot-assay revealed the induction of a HIV-1 Rev and Env-specific CTL-response 7.5 weeks after immunization. Thus, these novel MLV(HIV-1) vectors facilitate efficient transduction and subsequent expression of HIV-1-genes in CD4-positive host cells. Induction of both humoral and cellular HIV-1-specific immune responses in vivo confirmed their potential as an effective HIV-1 vaccine to be further studied in SHIV/rhesus macaque model of lentivirus infection.     

Lentivirus vectors using human and simian immunodeficiency virus elements

Lentivirus vectors based on human immunodeficiency virus (HIV) type 1 (HIV-1) constitute a recent development in the field of gene therapy. A key property of HIV-1-derived vectors is their ability to infect nondividing cells. Although high-titer HIV-1-derived vectors have been produced, concerns regarding safety still exist. Safety concerns arise mainly from the possibility of recombination between transfer and packaging vectors, which may give rise to replication-competent viruses with pathogenic potential. We describe a novel lentivirus vector which is based on HIV, simian immunodeficiency virus (SIV), and vesicular stomatitis virus (VSV) and which we refer to as HIV/SIVpack/G. In this system, an HIV-1-derived genome is encapsidated by SIVmac core particles. These core particles are pseudotyped with VSV glycoprotein G. Because the nucleotide homology between HIV-1 and SIVmac is low, the likelihood of recombination between vector elements should be reduced. In addition, the packaging construct (SIVpack) for this lentivirus system was derived from SIVmac1A11, a nonvirulent SIV strain. Thus, the potential for pathogenicity with this vector system is minimal. The transduction ability of HIV/SIVpack/G was demonstrated with immortalized human lymphocytes, human primary macrophages, human bone marrow-derived CD34(+) cells, and primary mouse neurons. To our knowledge, these experiments constitute the first demonstration that the HIV-1-derived genome can be packaged by an SIVmac capsid. We demonstrate that the lentivirus vector described here recapitulates the biological properties of HIV-1-derived vectors, although with increased potential for safety in humans.     

Marking and gene expression by a lentivirus vector in transplanted human and nonhuman primate CD34(+) cells

Recently, gene delivery vectors based on human immunodeficiency virus (HIV) have been developed as an alternative mode of gene delivery. These vectors have a number of advantages, particularly in regard to the ability to infect cells which are not actively dividing. However, the use of vectors based on human immunodeficiency virus raises a number of issues, not the least of which is safety; therefore, further characterization of marking and gene expression in different hematopoietic lineages in primate animal model systems is desirable. We use two animal model systems for gene therapy to test the efficiency of transduction and marking, as well as the safety of these vectors. The first utilizes the rhesus animal model for cytokine-mobilized autologous peripheral blood CD34(+) cell transplantation. The second uses the SCID-human (SCID-hu) thymus/liver chimeric graft animal model useful specifically for human T-lymphoid progenitor cell reconstitution. In the rhesus macaques, detectable levels of vector were observed in granulocytes, lymphocytes, monocytes, and, in one animal with the highest levels of marking, erythrocytes and platelets. In transplanted SCID-hu mice, we directly compared marking and gene expression of the lentivirus vector and a murine leukemia virus-derived vector in thymocytes. Marking was observed at comparable levels, but the lentivirus vector bearing an internal cytomegalovirus promoter expressed less efficiently than did the murine retroviral vector expressed from its own long terminal repeats. In assays for infectious HIV type 1 (HIV-1), no replication-competent HIV-1 was detected in either animal model system. Thus, these results indicate that while lentivirus vectors have no apparent deleterious effects and may have advantages over murine retroviral vectors, further study of the requirements for optimal use are warranted.     

Theoretical design of a gene therapy to prevent AIDS but not human immunodeficiency virus type 1 infection

Recent reports confirm that, due to the presence of long-lived, latently infected cell populations, eradication of human immunodeficiency virus type 1 (HIV-1) from infected patients by using antiretroviral drugs will be exceedingly difficult. An alternative to virus eradication may be to use gene therapy to induce a pseudo-latent state in virus-producing cells, thus transforming HIV-1 into a lifelong, but manageable, virus. Conditionally replicating HIV-1 (crHIV-1) gene therapy vectors provide an avenue for subduing HIV-1 expression in infected cells (by creating a parasite, crHIV-1, of the parasite HIV-1), potentially reducing the HIV-1 set point and delaying AIDS onset. Development of crHIV-1 vectors has proceeded in vitro, but the requirements for a crHIV-1 vector to proliferate and persist in vivo have not been explored. We expand a widely accepted mathematical model of HIV-1 in vivo dynamics to include a crHIV-1 gene therapy virus and derive a simple criterion for designing crHIV-1 viruses that will persist in vivo. The model introduces only two new parameters-HIV-1 inhibition and crHIV-1 production-and both can be experimentally engineered and controlled. Analysis demonstrates that crHIV-1 gene therapy can indefinitely reduce HIV-1 set point to levels comparable to those achieved with highly active antiretroviral therapy, provided crHIV-1 production is more efficient than HIV-1. Paradoxically, highly efficient therapeutic inhibition of HIV-1 was found to be disadvantageous. Thus, the field may benefit by shifting the search for more potent antiviral genes toward engineering optimized therapy viruses that package ultra-efficiently while downregulating viral production moderately.     

Human immunodeficiency virus type 1 escapes from RNA interference-mediated inhibition

Short-term assays have suggested that RNA interference (RNAi) may be a powerful new method for intracellular immunization against human immunodeficiency virus type 1 (HIV-1) infection. However, RNAi has not yet been shown to protect cells against HIV-1 in long-term virus replication assays. We stably introduced vectors expressing small interfering RNAs (siRNAs) directed against the HIV-1 genome into human T cells by retroviral transduction. We report here that an siRNA directed against the viral Nef gene (siRNA-Nef) confers resistance to HIV-1 replication. This block in replication is not absolute, and HIV-1 escape variants that were no longer inhibited by siRNA-Nef appeared after several weeks of culture. These RNAi-resistant viruses contained nucleotide substitutions or deletions in the Nef gene that modified or deleted the siRNA-Nef target sequence. These results demonstrate that efficient inhibition of HIV-1 replication through RNAi is possible in stably transduced cells. Therefore, RNAi could become a realistic gene therapy approach with which to overcome the devastating effect of HIV-1 on the immune system. However, as is known for antiviral drug therapy against HIV-1, antiviral approaches involving RNAi should be used in a combined fashion to prevent the emergence of resistant viruses.     

Induction of anti-HIV-1 immune responses by retroviral vectors.

Retroviral vectors encoding HIV-1 proteins, in particular, the envelope from HIV-1 IIIB, have been constructed and used to generate infectious vector particles. Murine cells transduced with these vectors express HIV proteins. Vector-transduced cells, when injected into syngeneic BALB/c mice, induce potent CD8+, class I MHC-restricted cytotoxic T-lymphocyte responses and elicit the production of neutralizing antibody specific for HIV-1. The induction of similar responses in primates may provide the basis for considering the use of these vectors as immunostimulants in humans. The retroviral vectors or vector-transduced cells would probably be first employed as an immunotherapeutic for HIV-infected individuals.     

Efficient gene transfer mediated by HIV-1-based defective lentivector and inhibition of HIV-1 replication

Lentiviral vectors have drawn considerable attention recently and show great promise to become important delivery vehicles for future gene transfer manipulation. In the present study we have optimized a protocol for preparation of human immunodeficiency virus type-1 (HIV-1)-based defective lentiviral vectors (DLV) and characterized these vectors in terms of their transduction of different cells. Transient co-transfection of 293T packaging cells with DNA plasmids encoding lentiviral vector constituents resulted in production of high-titer DLV (0.5–1.2 × 10⁷IU/mL), which can be further concentrated over 100-fold through a single step ultracentrifugation. These vectors were capable of transducing a variety of cells from both primate and non-primate sources and high transduction efficiency was achieved using concentrated vectors. Assessment of potential generation of RCV revealed no detection of infection by infectious particles in DLV-transduced CEM, SupT-1 and MT-2 cells. Long-term culture of transduced cells showed a stable expression of transgenes without apparent alteration in cellular morphology and growth kinetics. Vector mobilization to untransduced cells mediated by wild-type HIV-1 infection was confirmed in this test. Challenge of transduced human T-lymphocytes with wild-type HIV-1 showed these cells are totally resistant to the viral infection. Considering the effective gene transfer and stable gene expression, safety and anti-HIV activity, these DLV vectors warrant further exploration for their potential use as a gene transfer vehicle in the development of gene therapy protocols. Foundation items: National Institute of Health (S11 NS43499); RCMI (G12RR/AI03061, USA.)     

Evaluation of Tat-encoding bicistronic human immunodeficiency virus type 1 gene transfer vectors in primary canine bone marrow mononuclear cells

Tat-encoding human immunodeficiency virus type 1 (HIV-1) gene transfer vectors were evaluated in primary canine bone marrow mononuclear cells. Tat vectors provided higher levels of gene expression than vectors with internal promoters. The HIV-1 vector was also more efficient than Moloney murine leukemia virus (MoMLV) vectors for transduction of canine bone marrow mononuclear cells in vitro. Transplantation experiments in dogs with transduced autologous marrow cells confirmed the superiority of HIV-1 vectors over MoMLV vectors for gene transfer into canine bone marrow cells. Tat vectors may be useful not only for providing high levels of therapeutic gene expression in hematopoietic cells but also for study of the biological effects of Tat in those tissues in the canine model.     

Hepatocyte-specific gene expression from integrated lentiviral vectors

BACKGROUND: For many applications, efficient gene therapy will require long-term, organ-specific therapeutic gene expression. Lentiviral vectors based on HIV-1 are promising gene delivery vehicles due to their ability to integrate transgenes into non-dividing cells. Many experimental vectors express transgenes under the control of the cytomegalovirus (CMV) immediate-early gene promoter. Although this promoter directs strong gene expression in vitro, it may be shut off rapidly in vivo. This study explores the potential of HIV-1-based vectors to transduce hepatocytes and compares gene expression from different promoters in integrated vectors. METHODS: HIV-1-based vector plasmids expressing the green fluorescent protein (GFP) under the control of the CMV promoter, the alpha-1 antitrypsin gene promoter or promoters derived from the hepatitis B virus (HBV) genome were used to compare expression in transfected and transduced cell lines. RESULTS: Hepatocyte cell lines differed strikingly in their transfectability. Transduction with replication-deficient HIV-1-based vector particles incorporating the different promoter elements was uniformly effective in hepatocyte and non-hepatocyte lines. However, in hepatocytes, only the CMV, alpha-1 antitrypsin and HBV core but not HBV surface promoters were able to produce GFP expression. Addition of the HBV enhancer 2 element improved the transducing ability of the HBV surface promoter and suppressed expression in non-hepatocytes increasing specificity for hepatocytes. CONCLUSIONS: Integrated lentiviral vectors can be used to direct transgene expression in liver cells both promiscuously and specifically. Promoters derived from the alpha-1 antitrypsin gene or HBV are alternatives to the CMV promoter. Inclusion of the HBV enhancer 2 permits strong liver-specific gene expression in vitro.     

Evolutionary Analysis of Human Immunodeficiency Virus Type 1 Therapies Based on Conditionally Replicating Vectors

Efforts to reduce the viral load of human immunodeficiency virus type 1 (HIV-1) during long-term treatment are challenged by the evolution of anti-viral resistance mutants. Recent studies have shown that gene therapy approaches based on conditionally replicating vectors (CRVs) could have many advantages over anti-viral drugs and other approaches to therapy, potentially including the ability to circumvent the problem of evolved resistance. However, research to date has not explored the evolutionary consequences of long-term treatment of HIV-1 infections with conditionally replicating vectors. In this study, we analyze a computational model of the within-host co-evolutionary dynamics of HIV-1 and conditionally replicating vectors, using the recently proposed ‘therapeutic interfering particle’ as an example. The model keeps track of the stochastic process of viral mutation, and the deterministic population dynamics of T cells as well as different strains of CRV and HIV-1 particles. We show that early in the co-infection, mutant HIV-1 genotypes that escape suppression by CRV therapy appear; this is similar to the dynamics observed in drug treatments and other gene therapies. In contrast to other treatments, however, the CRV population is able to evolve and catch up with the dominant HIV-1 escape mutant and persist long-term in most cases. On evolutionary grounds, gene therapies based on CRVs appear to be a promising tool for long-term treatment of HIV-1. Our model allows us to propose design principles to optimize the efficacy of this class of gene therapies. In addition, because of the analogy between CRVs and naturally-occurring defective interfering particles, our results also shed light on the co-evolutionary dynamics of wild-type viruses and their defective interfering particles during natural infections.     

Tailored HIV-1 Vectors for Genetic Modification of Primary Human Dendritic Cells and Monocytes

Monocyte-derived dendritic cells (MDDCs) play a key role in the regulation of the immune system and are the target of numerous gene therapy applications. The genetic modification of MDDCs is possible with human immunodeficiency virus type 1 (HIV-1)-derived lentiviral vectors (LVs) but requires high viral doses to bypass their natural resistance to viral infection, and this in turn affects their physiological properties. To date, a single viral protein is able to counter this restrictive phenotype, Vpx, a protein derived from members of the HIV-2/simian immunodeficiency virus SM lineage that counters at least two restriction factors present in myeloid cells. By tagging Vpx with a short heterologous membrane-targeting domain, we have obtained HIV-1 LVs incorporating high levels of this protein (HIV-1-Src-Vpx). These vectors efficiently transduce differentiated MDDCs and monocytes either as previously purified populations or as populations within unsorted peripheral blood mononuclear cells (PBMCs). In addition, these vectors can be efficiently pseudotyped with receptor-specific envelopes, further restricting their cellular tropism almost uniquely to MDDCs. Compared to conventional HIV-1 LVs, these novel vectors allow for an efficient genetic modification of MDDCs and, more importantly, do not cause their maturation or affect their survival, which are unwanted side effects of the transduction process. This study describes HIV-1-Src-Vpx LVs as a novel potent tool for the genetic modification of differentiated MDDCs and of circulating monocyte precursors with strong potential for a wide range of gene therapy applications.     

Advances in lentiviral vectors: a patent review

Lentiviral vectors are at the forefront of gene delivery systems for research and clinical applications. These vectors have the ability to efficiently transduce nondividing and dividing cells, to insert large genetic segment in the host chromatin, and to sustain stable long-term transgene expression. Most of lentiviral vectors systems in use are derived from HIV-1. Numerous modifications in the basic HIV structure have been made to ensure safety and to promote efficiency to vectors. Lentiviral vectors can be pseudotyped with distinct viral envelopes that influence vector tropism and transduction efficiency. Moreover, these vectors can be used to reprogram cells and generate induced pluripotent stem cells. This review aims to show the patents that resulted in improved safety and efficacy of lentiviral vector with important implications for clinical trials.     

Lentivirus-mediated RNA interference of DC-SIGN expression inhibits human immunodeficiency virus transmission from dendritic cells to T cells

In the early events of human immunodeficiency virus type 1 (HIV-1) infection, immature dendritic cells (DCs) expressing the DC-specific intercellular adhesion molecule 3-grabbing nonintegrin (DC-SIGN) receptor capture small amounts of HIV-1 on mucosal surfaces and spread viral infection to CD4(+) T cells in lymph nodes (22, 34, 45). RNA interference has emerged as a powerful tool to gain insight into gene function. For this purpose, lentiviral vectors that express short hairpin RNA (shRNA) for the delivery of small interfering RNA (siRNA) into mammalian cells represent a powerful tool to achieve stable gene silencing. In order to interfere with DC-SIGN function, we developed shRNA-expressing lentiviral vectors capable of conditionally suppressing DC-SIGN expression. Selectivity of inhibition of human DC-SIGN and L-SIGN and chimpanzee and rhesus macaque DC-SIGN was obtained by using distinct siRNAs. Suppression of DC-SIGN expression inhibited the attachment of the gp120 envelope glycoprotein of HIV-1 to DC-SIGN transfectants, as well as transfer of HIV-1 to target cells in trans. Furthermore, shRNA-expressing lentiviral vectors were capable of efficiently suppressing DC-SIGN expression in primary human DCs. DC-SIGN-negative DCs were unable to enhance transfer of HIV-1 infectivity to T cells in trans, demonstrating an essential role for the DC-SIGN receptor in transferring infectious viral particles from DCs to T cells. The present system should have broad applications for studying the function of DC-SIGN in the pathogenesis of HIV as well as other pathogens also recognized by this receptor.     

化学趋化因子受体在HIV感染中的作用及其在AIDS治疗中的应用价值

化学趋化因子受体作为协同受体,为人免疫缺陷病毒（ＨＩＶ－１）进入细胞所必需。其中ＣＸＣＲ４被亲Ｔ细胞的病毒株利用,而ＣＣＲ５被亲巨噬细胞的病毒株利用,它们是大多数病毒株利用的协同受体。协同受体和ＣＤ４一起形成复合受体,ｇｐ１２０与之结合后发生构象改变,使ｇｐ４１暴露出来,引起膜的融合。ＨＩＶ协同受体发现为治疗艾滋病开辟了新的途径。利用趋化因子拮抗剂、单克隆抗体和天然配体封闭趋化因子受体可阻止ＨＩＶ     

Packaging of human immunodeficiency virus type 1 RNA requires cis-acting sequences outside the 5' leader region.

cis elements required for the encapsidation of human immunodeficiency virus type 1 (HIV-1) RNA have been investigated by using a replication-competent helper virus to package a series of HIV-1-based vectors which had been stably transfected into human CD4 T-cell lines. A previously identified packaging signal in the 5' leader region was not sufficient for the encapsidation of small vectors containing heterologous genes. In contrast, vectors containing additional gag and env sequences were packaged with high efficiency and transduced into CD4-expressing target cells with titers exceeding 10(4) CFU/ml. The presence of gag sequences did not enhance vector packaging efficiency. A 1.1-kb env gene fragment encompassing the Rev-responsive element was absolutely required for the expression and encapsidation of vectors containing cis-acting repressive sequences and appeared also to contain an important packaging signal. Vectors as small as 2.6 kb were successfully packaged in this system. The presence of abundant, packageable vector RNA did not appear to interfere with encapsidation of the wild-type HIV-1 genome, suggesting that HIV-1 RNA packaging capacity is not saturated during acute infection.     

人免疫缺陷病毒1型TAT突变蛋白及反义TAT RNA对TAT反式激活作用的抑制

Ｔａｔ是人免疫缺陷病毒（ＨＩＶ）基因组编码的反式激活因子,突变分析表明它含有几个重要的功能域。为寻找控制ＨＩＶ复制的途径,构建了以ＨＩＶ－１ＬＴＲ（－１５８－＋８０）为启动子的Ｔａｔ ｃＤＮＡ全长反义表达质粒ｐＡＳ－Ｔａｔ,并用已经构建的ＨＩＶ ＬＴＲ－１５８到＋８０为启动子,具有不同突变点的突变Ｔａｔ基因表达质粒,以荧光酶基因为报告基因,共转染Ｊｕｒｋａｔ细胞,结果发现无论是反义Ｔａｔ表达质粒还