Optimized human CYP4B1 in combination with the alkylator prodrug 4-ipomeanol serves as a novel suicide gene system for adoptive T-cell therapies.

Engineering autologous or allogeneic T cells to express a suicide gene can control potential toxicity in adoptive T-cell therapies. We recently reported the development of a novel human suicide gene system that is based on an orphan human cytochrome P450 enzyme, CYP4B1, and the naturally occurring alkylator prodrug 4-ipomeanol. The goal of this study was to systematically develop a clinically applicable self-inactivating lentiviral vector for efficient co-expression of CYP4B1 as an ER-located protein with two distinct types of cell surface proteins, either MACS selection genes for donor lymphocyte infusions after allogeneic stem cell transplantation or chimeric antigen receptors for retargeting primary T cells. The U3 region of the myeloproliferative sarcoma virus in combination with the T2A site was found to drive high-level expression of our CYP4B1 mutant with truncated CD34 or CD271 as MACS suitable selection markers. This lentiviral vector backbone was also well suited for co-expression of CYP4B1 with a codon-optimized CD19 chimeric antigen receptor (CAR) construct. Finally, 4-ipomeanol efficiently induced apoptosis in primary T cells that co-express mutant CYP4B1 and the divergently located MACS selection and CAR genes. In conclusion, we here developed a clinically suited lentiviral vector that supports high-level co-expression of cell surface proteins with a potent novel human suicide gene.

Chemogenetic ON and OFF switches for RNA virus replication.

Therapeutic application of RNA viruses as oncolytic agents or gene vectors requires a tight control of virus activity if toxicity is a concern. Here we present a regulator switch for RNA viruses using a conditional protease approach, in which the function of at least one viral protein essential for transcription and replication is linked to autocatalytical, exogenous human immunodeficiency virus (HIV) protease activity. Virus activity can be en- or disabled by various HIV protease inhibitors. Incorporating the HIV protease dimer in the genome of vesicular stomatitis virus (VSV) into the open reading frame of either the P- or L-protein resulted in an ON switch. Here, virus activity depends on co-application of protease inhibitor in a dose-dependent manner. Conversely, an N-terminal VSV polymerase tag with the HIV protease dimer constitutes an OFF switch, as application of protease inhibitor stops virus activity. This technology may also be applicable to other potentially therapeutic RNA viruses.

Foamy combinatorial anti-HIV vectors with MGMTP140K potently inhibit HIV-1 and SHIV replication and mediate selection in vivo.

Highly active antiretroviral therapy has greatly reduced the morbidity and mortality from human immunodeficiency virus (HIV) infection, but AIDS continues to be a serious health problem worldwide. Despite enormous efforts to develop a vaccine, there is still no cure, and alternative approaches including gene therapy should be explored. In this study we developed and compared combinatorial foamy virus (FV) anti-HIV vectors that also express a mutant methylguanine methyltransferase (MGMTP140K) transgene to increase the percentage of gene-modified cells after transplantation. These FV vectors inhibit replication of HIV-1 and also the simian immunodeficiency virus/HIV-1 (SHIV) chimera that can be used in monkey AIDS gene therapy studies. We identified a combinatorial FV vector that expresses 3 anti-HIV transgenes and inhibits viral replication by over 4 logs in a viral challenge assay. This FV anti-HIV vector expresses an HIV fusion inhibitor and two short hairpin RNAs (shRNAs) targeted to HIV-1 tat and rev, and can be produced at high titer (3.8 x 10(7) transducing units ml(-1)) using improved helper plasmids suitable for clinical use. Using a competitive repopulation assay, we show that human CD34(+) cells transduced with this combinatorial FV vector efficiently engraft in a mouse xenotransplantation model, and that the percentage of transduced repopulating cells can be increased after transplantation.

Antiviral gene therapy.

This chapter describes the major gene therapeutic approaches for viral infections. The vast majority of published approaches target severe chronic viral infections such as hepatitis B or C and HIV infection. Two basic gene therapy strategies are introduced here. The first involves the expression of a protein or an RNA that inhibits viral replication by targeting crucial steps of the viral life cycle or by interfering with a cellular factor required for virus replication. The major limitation of this approach is that primary levels of gene modification have generally not been sufficient to reduce the availability of target cells permissive for virus replication to a level that significantly decreases overall viral load. Thus, investigators have banked on the expectation that gene-protected cells have a sufficient selective advantage to accumulate and gain prevalence over time, a prediction that so far could not be confirmed in clinical trials. In vivo levels of gene modification can be improved, however, by introducing an additional selectable marker. In addition, a secreted antiviral gene product that exerts a bystander effect could significantly reduce overall virus replication despite relatively low levels of gene modification. In addition to these direct antiviral approaches, several strategies have been developed that employ or aim to enhance host immune responses. The innate immune response has been enhanced, for example, by the in vivo expression of interferons. Alternatively, T cells can be grafted with recombinant receptors to boost adaptive virus-specific immunity. These approaches are especially promising for chronic virus infection, where natural immune responses are evidently not sufficient to effectively control virus replication.

Efficient entry inhibition of human and nonhuman primate immunodeficiency virus by cell surface-expressed gp41-derived peptides.

Membrane-anchored C-peptides (for example, maC46) derived from human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein gp41 effectively inhibit HIV-1 entry in cell lines and primary human CD4+ cells in vitro. Here we evaluated this gene therapy approach in animal models of AIDS. We adapted the HIV gp41-derived maC46 vector construct for use in rhesus monkeys. Simian immunodeficiency virus (SIV and SHIV) sequence-adapted maC46 peptides, and the original HIV-1-derived maC46 expressed on the surface of established cell lines blocked entry of HIV-1, SIVmac251 and SHIV89.6P. Furthermore, primary rhesus monkey CD4+ T cells expressing HIV sequence-based maC46 peptides were also protected from SIV entry. Depletion of CD8+ T cells from PBMCs enhanced the yield of maC46-transduced CD4+ T cells. Supplementation with interleukin-2 (IL-2) increased transduction efficiency, whereas IL-7 and/or IL-15 provided no additional benefit. Phenotypic analysis showed that maC46-transduced and expanded cells were predominantly central memory CD4+ T cells that expressed low levels of CCR5 and slightly elevated levels of CD62L, beta7-integrin and CXCR4. These findings show that maC46-based cell surface-expressed peptides can efficiently inhibit primate immunodeficiency virus infection, and therefore serve as the basis for evaluation of this gene therapy approach in an animal model for AIDS.

Germinal centre CD4+ T cells are an important site of HIV replication in vivo.

OBJECTIVE: CD4+ T cells are the main target for HIV. However, the highest HIV antigen concentration in infected subjects accumulates on the cell surface of follicular dendritic cells in the germinal centres of the lymphoid tissue. Germinal centres contain a T-helper cell subset which expresses CD57 molecules. Here we analysed virus replication and viral load in CD57+CD4+ germinal centre T cells and in the CD4+ T cells found mostly outside germinal centres (CD57-CD4+). METHODS: Peripheral blood mononuclear cells and lymph-node cells were prepared, stained for CD4 and CD57 and purified by FACS. Defined cell numbers of CD4+CD57+ cells and CD4+CD57- cells were sorted directly into polymerase chain reaction (PCR) tubes by FACS, equipped with an automated cell deposition unit and analysed by PCR to detect proviral DNA. Based on Poisson distribution, the expected level of infection was calculated. Viral replication was determined by amplifying double-spliced, single-spliced, and full-length transcripts of HIV using serially diluted cDNA of the FACS-sorted cells. RESULTS: An up to 10-fold higher frequency of infected cells was found in the CD57+CD4+ germinal centre T cells compared with CD57-CD4+ T cells. Furthermore, active viral replication was detected almost exclusively in the CD57+CD4+ T cells. CONCLUSIONS: The CD57+CD4+ germinal centre T cells are one of the sites of HIV infection and replication that may play a pivotal role in the pathogenesis of HIV infection.

Lack of superinfection interference in retroviral vector producer cells.

Vesicular stomatitis virus G protein (VSV-G)-pseudotyped retroviral vectors have become more feasible for clinical gene transfer protocols since stable tetracycline (tet)-regulated packaging cell lines have become available. Here, we analyzed superinfection interference in VSV-G-pseudotyped and classic amphotropic packaging cell lines. No superinfection interference was observed in VSV-G-pseudotyped packaging cell lines. Thus, integrated retroviral vector genomes accumulated during culture. Similar results were obtained with the amphotropic packaging cells, but to a lesser degree. In addition, VSV-G packaging cells were susceptible to infection with vector particles devoid of envelope proteins, which are produced by these cells in high titers when VSV-G expression is suppressed by tetracycline. For both packaging systems, superinfection could be blocked by azidothymidine (AZT). With regard to safety, this study suggests that in clinical protocols amphotropic producer clones should be tested for superinfection interference and VSV-G packaging cells should always be cultured in the presence of AZT.

Human Kupffer cells infected with HIV-1 in vivo.

Blood monocytes as well as cells of the macrophage-phagocytic system in several tissues are targets for HIV-1 in vivo and in vitro. However, the data on HIV-1 infection of liver macrophages/Kupffer cells (KCs), which make up the main pool of fixed-tissue macrophages, is controversial. We therefore studied HIV-1 infection of KCs in vivo. Blood and liver tissue was obtained from seven AIDS patients shortly after death. Liver tissue was minced before processing. Cell suspensions were further purified by density gradient centrifugation, stained with anti-CD14 (blood monocytes) or anti-macrophage 25F9 (KCs), and separated by fluorescence-activated cell sorting (FACS). These highly purified cell populations were then analyzed for HIV-1 proviral DNA by the polymerase chain reaction (PCR). By performing PCR on FACS-purified cell populations, we could show that both KCs and peripheral blood monocytes were HIV-1-infected in 3 of 7 patients. KCs harbored HIV-1 proviral DNA only if peripheral blood monocytes were infected. These data show that KCs of the liver are infected with HIV-1 in vivo.

Antibodies to p24 antigen do not specifically detect HIV-infected lymphocytes in AIDS patients.

A flow cytometric assay (FCA), which detects the p24 antigen in HIV-infected cell lines and in peripheral blood mononuclear cells (PBMC) of AIDS patients, has been described in several studies. However, the results presented here clearly show that this p24-FCA, although useful for the analysis of HIV infection of cells in vitro, does not specifically detect HIV-infected PBMC from patients. Isotype control antibodies also stained PBMC from HIV-infected patients to a greater degree than the PBMC from healthy controls. Furthermore, the CD4-negative lymphocytes, which are generally not infected with HIV, were also found to stain with anti-p24. Finally, no enrichment of HIV-infected cells was found in the FACS-purified CD4+p24+ lymphocytes, compared to the CD4+p24- cell fraction. The p24-FCA, therefore, was not useful for determining the percentage of infected PBMCs from HIV-infected individuals.

Human immunodeficiency virus (HIV)-specific antibodies, neutralizing activity and antibody-dependent cellular cytotoxicity (ADCC) in the cerebrospinal fluid of HIV-infected patients.

We assessed the capacity of cerebrospinal fluids (CSF) and sera from human immunodeficiency virus (HIV)-seropositive patients to neutralize HIV and to mediate specific antibody-dependent lysis of HIV-infected target cells. A local HIV-specific intrathecal antibody synthesis was found in all stages of HIV infection regardless of neurological manifestations. Virus-neutralizing antibodies could not be detected in the CSF of patients with primary encephalitis or polyneuropathy. Cytotoxic antibodies mediating HIV-specific antibody-dependent cellular cytotoxicity (ADCC) were demonstrable in the CSF of most patients without evidence of central nervous system (CNS) involvement, but only in 43% of cases with HIV encephalitis. In some cases, the exclusive detection of ADCC activity in either the CSF or serum compartment suggested the presence of non-identical target antigens in the CSF and serum of the same patient. Further studies are needed to clarify the significance of these findings for the manifestation of CNS involvement in HIV infection.

Detection of DNA in single cells using an automated cell deposition unit and PCR.

A highly effective single-cell PCR method using a fluorescence-activated cell sorting (FACS)-based automated cell deposition unit (ACDU) that sorts single cells directly into PCR tubes was developed. To evaluate the sensitivity of this method, single ACH-2 cells (containing one HIV-1 genome per cell) were sorted, and 220 out of 228 samples (96.5%) were HIV DNA-positive by PCR. Furthermore, the number of samples accidentally containing more than one cell was determined by sorting single cells from a mixture of human cytomegalovirus (HCMV)-infected fibroblasts and ACH-2 cells. Multiplex nested PCR (nPCR) was then performed, detecting HCMV and HIV DNA simultaneously. From 66 sorted cells, 2 (3%) were double-positive for HIV and HCMV, 31 (47%) for HCMV alone, 30 (45.5%) for HIV alone and 3 (4.5%) were PCR-negative. The ACDU was then programmed to sort defined numbers of cells into PCR tubes. This is similar to classic dilution assays in that it allows the determination of the percentage of cells that was positive for a specific DNA. The accuracy of multiple cell deposition by the ACDU was evaluated by determining the percentage of HIV-positive cells in defined mixtures of ACH-2 and uninfected H9 cells. Infection rates determined by the ACDU correlated well with the rates expected from the given dilutions.

Amphotropic and VSV-G-pseudotyped retroviral vectors transduce human hematopoietic progenitor cells with similar efficiency.

One restriction of retroviral gene transfer into hematopoietic stem cells is the low level of amphotropic virus receptor. In the present study, we examined whether retroviral vectors pseudotyped with the G-protein of vesicular stomatitis virus (VSV) can overcome this restriction. Human progenitor cells purified by magnetic beads and cell sorting were transduced with an amphotropic or VSV-G-pseudotyped retroviral vector containing the truncated human nerve growth factor receptor as a marker gene. Cells were prestimulated with flt-3 ligand, stem cell factor, and interleukin-3 and transduced on fibronectin. Marker gene expression was analyzed by flow cytometry. Transduction efficiencies of amphotropic and VSV-G-pseudotyped virus for CD34+ cells did not differ significantly. Gene transfer into CD34+CD38- cells, which are enriched in more immature progenitors, was not restricted and transfer efficiencies for this subset were also similar for both pseudotypes. The addition of fibronectin improved gene transfer with the amphotropic vector considerably (5- to 19.3-fold, mean 12.6), while the effect on the VSV-G-pseudotype was far less pronounced (1- to 3.9-fold, mean 2.1, P = 0.04). In conclusion, high levels of gene transfer to human hematopoietic progenitors were achieved with an optimized transduction protocol, and transduction efficiencies could not be improved further by the use of VSV-G-pseudotypes.

Infection of blood and bone marrow cells with the human cytomegalovirus in vivo.

The human cytomegalovirus (HCMV) is a major pathogen in immunocompromised patients. Both, primary infection and reactivation of latent virus can cause disease. Peripheral blood leukocytes (PBL) most likely play an important role in viral persistence and dissemination of infection. However, an open question has been whether HCMV actively replicates in PBL in vivo and whether the progenitor cells in the bone marrow are also infected. Previous studies on this issue are controversial. Here we summarize data on the tropism of HCMV for mature leukocyte populations as well as bone marrow progenitor cells during HCMV viremia. All cell populations were highly purified by a fluorescence activated cell sorter (FACS) and analyzed by PCR for the presence of viral genomic DNA. Moreover, mature leukocyte populations were investigated for mRNA expression of regulatory and viral structural proteins. We could show, that HCMV DNA was detected most frequently in granulocytes and monocytes as well as in CD34+ progenitor cells of immunosuppressed patients. Viral mRNA expression was found in granulocytes, monocytes, and lymphocyte fractions. In contrast, no HCMV DNA was found in healthy, seropositive individuals.

T-cell receptor diversity prevents T-cell lymphoma development.

Mature T-cell lymphomas (MTCLs) have an extremely poor prognosis and are much less frequent than immature T-cell leukemias. This suggests that malignant outgrowth of mature T lymphocytes is well controlled. Indeed, in a previous study we found that mature T cells are resistant to transformation with known T-cell oncogenes. Here, however, we observed that T-cell receptor (TCR) mono-/oligoclonal mature T cells from TCR transgenic (tg) mice (OT-I, P14) expressing the oncogenes NPM/ALK or ΔTrkA readily developed MTCLs in T-cell-deficient recipients. Analysis of cell surface markers largely ruled out that TCR tg lymphomas were derived from T-cell precursors. Furthermore, cotransplanted non-modified TCR polyclonal T cells suppressed malignant outgrowth of oncogene expressing TCR tg T lymphocytes. A dominant role of an anti-leukemic immune response or Tregs in the control of MTCLs seems unlikely as naïve T cells derived from oncogene expressing stem cells, which should be tolerant to leukemic antigens, as well as purified CD4 and CD8 were resistant to transformation. However, our results are in line with a model in which homeostatic mechanisms that stabilize the diversity of the normal T-cell repertoire, for example, clonal competition, also control the outgrowth of potentially malignant T-cell clones. This study introduces a new innate mechanism of lymphoma control.

Woodchuck hepatitis virus post-transcriptional regulatory element deleted from X protein and promoter sequences enhances retroviral vector titer and expression.

Introduction of the post-transcriptional regulatory element (PRE) of woodchuck hepatitis virus (WHV) into the 3' untranslated region of retroviral and lentiviral gene transfer vectors enhances both titer and transgene expression. Optimal use of the PRE is often necessary to obtain vectors with sufficient performance for therapeutic applications. The enhancing activity of the PRE depends on the precise configuration of its sequence and the context of the vector and cell into which it is introduced. However, data obtained in the context of WHV-associated hepatocellular carcinomas suggests that the PRE might potentially contribute to tumorigenesis, especially if encoding a truncated version of the WHV X protein. Oncogenic side effects of lentiviral vectors containing the PRE have reinforced these safety concerns, although a causal role of the PRE remained unproven. Here, we demonstrate that PRE mutants can be generated that are devoid of X protein open reading frames (ORFs) as well as other ORFs exceeding 25 amino acids, without significant loss of RNA enhancement activity. Furthermore, the X protein promoter could be deleted without compromising the enhancement of vector titers and transgene expression. Such a modified PRE sequence appears useful for future vector design.

Towards hematopoietic stem cell-mediated protection against infection with human immunodeficiency virus.

The failure of pharmacological approaches to cure infection with the human immunodeficiency virus (HIV) has renewed the interest in gene-based therapies. Among the various strategies that are currently explored, the blockade of HIV entry into susceptible T cells and macrophages promises to be the most powerful intervention. For long-term protection of both of these lineages, genetic modification of hematopoietic stem cells (HSCs) would be required. Here, we tested whether HSCs and their progeny can be modified to express therapeutic levels of M87o, a gammaretroviral vector encoding an artificial transmembrane molecule that blocks fusion-mediated uptake of HIV. In serial murine bone marrow transplantations, efficient and multilineage expression of M87o was observed for more than 1 year (range 37-75% of mononuclear cells), without signs of toxicity related to the transmembrane molecule. To allow enrichment of M87o-modified HSCs after transplant, we constructed vectors coexpressing the P140K mutant of O(6)-methylguanine-DNA-methyltransferase (MGMT-P140K). This clinically relevant selection marker mediates a survival advantage in HSCs if exposed to combinations of methylguanine-methyltransferase (MGMT) inhibitors and alkylating agents. A bicistronic vector mediated sufficient expression of both M87o and MGMT to confer a selective survival advantage in the presence of HIV and alkylating agents, respectively. These data encourage further investigations in large animal models and clinical trials.

Suppression of HIV-1 infection in primary CD4 T cells transduced with a self-inactivating lentiviral vector encoding a membrane expressed gp41-derived fusion inhibitor.

Peptidomimetics of HIV-1 gp41 sequences required for membrane fusion are potent inhibitors of HIV-1 entry. We hypothesize that expression of a membrane-bound gp41-derived fusion inhibitor will confer HIV-1 resistance to primary CD4 T cells. Efficient gene delivery and stable expression of a membrane-bound gp41-derived fusion inhibitor to primary CD4 T cells was accomplished using a self-inactivating lentiviral vector. A potent antiviral effect was observed when transduced CD4 T cells were challenged with a highly virulent CXCR4-tropic strain of HIV-1. Production of soluble p24 in the supernatant was inhibited 100-fold, and cytopathic effects were evident early in non-transduced cells and absent in transduced cells. Expression of the gp41 sequences was not detrimental to CD4 cells as transduced CD4 T cells exhibited a population doubling time that was equivalent to T cells transduced with a control vector. Results from this study support the rationale to use this lentiviral vector targeted at HIV entry as a potential gene therapy for HIV infection.

Leukocyte differentiation antigens in the bone marrow of patients with HIV-related disease.

To understand at what level hematopoiesis is impaired in AIDS patients, we quantitated bone marrow cells of the myeloid and lymphoid lineages in their different stages of differentiation. The bone marrow aspirates of 20 HIV-infected patients (18 with CDC IV) and 11 controls were analyzed by flow cytometry, using a panel of monoclonal antibodies. The mean percentages of CD34+ and CD10+ hematopoietic precursor cells were not significantly altered in the HIV-infected patients. The values of these patients however scattered in a much wider range than in the control group, some patients showing extremely high values. Patients with high levels of CD34+ and CD10+ precursor cells in the bone marrow had lower blood leukocyte counts and lower blood and bone marrow CD4 cell numbers than patients with a low percentage of CD10+ and CD34+ cells (p < 0.01). The percentages of the CD34+ cell subsets studied (CD34+CD33+, CD34+CD14+, CD34+CD15+) were not altered. Bone marrow CD4+ lymphocytes, especially those of CD45RA-phenotype, were reduced, while the CD8+ cells (especially HLA-DR+/CD45RA+ cells) were increased. These results show that the hematologic disorders in AIDS are not simply caused by an altered number or composition of bone marrow precursor cells. We postulate that hematopoiesis is inhibited due to an imbalance in the bone marrow T lymphocyte populations.

Delayed formation of defective interfering particles in vesicular stomatitis virus-infected cells: kinetic studies of viral protein and RNA synthesis during autointerference.

The time course of defective interfering (DI) particle and B particle release from vesicular stomatitis virus-infected BHK-21 cells was studied at different multiplicities of defective and infective particles. Particle release was progressively delayed in cells infected with an increasing DI-to-B particle ratio. The delayed particle release during interference was found to be connected with a reduced but prolonged synthesis of viral proteins, a slower accumulation of viral proteins, and a delayed shutoff of cellular protein synthesis. The relative synthesis of M and G proteins was reduced during interference, whereas the relative synthesis of N and NS proteins was increased. On the level of genomic RNA replication, we found that DI RNA was replicated more slowly during interference than the standard genomic RNA was during acute infection. The ratio of DI particles to B particles which were released increased throughout the infectious cycle. At a given time in the infectious cycle, this ratio was independent of the multiplicity of infecting DI and B particles. On the basis of the kinetic studies, we argue that cells infected with higher amounts of DI particles compared with B particles synthesize a higher DI-to-B particle ratio and release these progeny particles later than cells infected with a low DI-to-B particle ratio.