Development of molecular genetic interventions for HIV infection.

This unit includes a set of protocols for the ex vivo transfer of genes into CD4+ T cells, to be used in the initial evaluation of genes protecting against HIV infection in gene therapy protocols. The describes isolating and expanding CD4+ T cells from the patient. The cells are then transduced by either retroviral transduction or particle-mediated gene transfer and reinfused into the patient. To monitor the effectiveness of gene transfer, genomic DNA is prepared from the patient's cells. Detection of vector DNA by PCR analysis of the patient's genomic DNA following gene transfer is also described in detail.

Generation of retroviral vector for clinical studies using transient transfection.

Transient transfection of 293T cells was utilized to produce high-titer murine recombinant retroviral vectors for clinical studies. This system was initially optimized by gene transfer using different retroviral envelope proteins into activated human CD4+ T lymphocytes in vitro. Higher titer and infectivity were obtained than with stable murine producer lines; titers of 0.3-1 x 10(7) infectious units per milliliter for vectors encoding the green fluorescent protein (GFP) were achieved. Virions pseudotyped with envelope proteins from gibbon ape leukemia virus or amphotropic murine leukemia virus resulted in gene transfer of > or = 50% in CD4+ human T lymphocytes with this marker. Gene transfer of Rev M10 with this vector conferred resistance to HIV infection compared with negative controls in the absence of drug selection. Thus, the efficiency of transduction achieved under these conditions obviated the need to include selection to detect biologic effects in T cells. Finally, a protocol for the production of large-scale supernatants using transient transfection was optimized up to titers of 1.9 x 10(7) IU/ml. These packaging cells can be used to generate high-titer virus in sufficient quantities for clinical studies and will facilitate the rapid, cost-effective generation of improved retroviral, lentiviral, or other viral vectors for human gene therapy.

Gene transfer into human umbilical cord blood-derived CD34+ cells by particle-mediated gene transfer.

Delivery of genes into hematopoietic progenitor cells offers an attractive means for the introduction of corrective or protective genes into cells of both the myeloid and lymphoid lineage. Previously, investigators have often used murine retroviral vectors for gene delivery which require cells to be cycling for efficient delivery. We describe a nonviral method of gene delivery using particle-mediated gene transfer to obviate many disadvantages of viral vectors related to safety, production costs and the need for cell cycle proliferation. Using a CMV-CAT reporter plasmid, we show transfection of highly purified CD34+ cells isolated from umbilical cord blood. Effective gene transfer was shown in unstimulated and in growth-stimulated cells. Following transfection with a neomycin resistance gene, differentiation into cells of the myeloid lineage was observed, assayed by CFU-GM in the presence of G-418. Both unstimulated and stimulated cells gave rise to CFU-GM in the presence of G-418, indicating that stable expression of the neomycin resistance gene was maintained in early progenitors. These results demonstrate that particle-mediated gene transfer into human hematopoietic cells from umbilical cord blood can be achieved without affecting their CFU-GM differentiation potential. This gene transfer method offers an alternative approach to gene therapy studies involving human hematopoietic progenitor cells.

HIV transcriptional activation by the accessory protein, VPR, is mediated by the p300 co-activator.

The accessory protein, Vpr, is a virion-associated protein that is required for HIV-1 replication in macrophages and regulates viral gene expression in T cells. Vpr causes arrest of cell cycle progression at G2/M, presumably through its effect on cyclin B1.Cdc2 activity. Here, we show that the ability of Vpr to activate HIV transcription correlates with its ability to induce G2/M growth arrest, and this effect is mediated by the p300 transcriptional co-activator, which promotes cooperative interactions between the Rel A subunit of NF-kappaB and cyclin B1.Cdc2. Vpr cooperates with p300, which regulates NF-kappaB and the basal transcriptional machinery, to increase HIV gene expression. Similar effects are seen in the absence of Vpr with a kinase-deficient Cdc2, and overexpression of p300 increases levels of HIV Vpr+ replication. Taken together, these data suggest that p300, through its interactions with NF-kappaB, basal transcriptional components, and Cdks, is modulated by Vpr and regulates HIV replication. The regulation of p300 by Vpr provides a mechanism to enhance viral replication in proliferating cells after growth arrest by increasing viral transcription.

Enhanced T cell engraftment after retroviral delivery of an antiviral gene in HIV-infected individuals.

Intracellular expression of gene products that inhibit viral replication have the potential to complement current antiviral approaches to the treatment of AIDS. We previously have shown that a mutant inhibitory form of an essential viral protein, Rev M10, prolongs the survival of T cells transduced with a nonviral vector in HIV-infected individuals. Because these gene-modified cells were not observed in patients beyond 8 weeks, efforts were made to improve the duration of engraftment. In this study, we used retroviral vector delivery of Rev M10 to CD4(+) cells and analyzed relevant immune responses in a pilot study of three HIV-seropositive patients. DNA and RNA PCR analyses revealed that cells transduced with Rev M10 retroviral vectors survived and expressed the recombinant gene for significantly longer time periods than those transduced with a negative control vector in all three patients. Immune responses were not detected either to Rev M10 or to Moloney murine leukemia virus gp70 envelope protein. Rev M10-transduced cells were detected for an average of 6 months after retroviral gene transfer compared with approximately 3 weeks for the previously reported nonviral vector delivery. These findings suggest that retroviral delivery of an antiviral gene may potentially contribute to immune reconstitution in AIDS and could provide a more effective vector to prolong survival of CD4(+) cells in HIV infection.

Cell and viral regulatory elements enhance the expression and function of a human immunodeficiency virus inhibitory gene.

Regulated expression of recombinant genes in CD4+ cells is an important objective for gene therapy of AIDS, as these cells represent the principal target for viral replication of human immunodeficiency virus (HIV). We report here that specific combinations of CD4 cell-specific and viral regulatory elements can enhance expression of an antiviral gene product. Different viral regulatory elements were incorporated into a previously reported CD4 locus control region to increase the expression of reporter genes in T and monocytic cell lines. The CD4-specific regulatory elements were included to enhance expression in CD4 cells, and viral regulatory regions, including the cytomegalovirus immediate-early (CMV IE) upstream enhancer, which contains the kappa B and Ap1 regulatory elements and a Tat-responsive element of the HIV type 1 long terminal repeat, were used to increase gene expression and modulate its activity in response to viral infection. In transient transfection assays, this vector was 100- to 1,000-fold more active than the original CD4 regulatory elements alone. Expression of an inhibitory form of the Rev protein, Rev M10, was more effective than previously described vectors and protected against productive viral replication in CD4+ peripheral blood mononuclear cells. The combination of CD4 lineage-specific and viral regulatory elements will facilitate the development of more effective antiviral genetic strategies for AIDS.

RANTES inhibits HIV-1 replication in human peripheral blood monocytes and alveolar macrophages.

Infection with human immunodeficiency virus (HIV)-1 most often leads to the development of acquired immune deficiency syndrome, which may manifest with opportunistic infections, many of which occur in the lung. Mononuclear phagocytes infected by HIV-1, being relatively resistant to its cytopathic effects, potentially act as a reservoir for the virus. The alveolar macrophage (AM), a differentiated lung tissue macrophage, is readily infected by HIV-1, after which the virus becomes relatively dormant. C-C chemokines, secreted by CD8 T lymphocytes and other cells, are known to suppress HIV replication in lymphocytes. In view of this observation, and the relative increase in CD8+ T lymphocytes during HIV-1 disease, particularly in the lung, we hypothesized that C-C chemokines might play a key role in suppressing HIV-1 replication in AM. We examined the effect of the C-C chemokines macrophage inflammatory protein (MIP)-1 alpha, MIP-1 beta, and regulated on activation normal T cell expressed and secreted (RANTES) singly and in combination on HIV-1 replication in peripheral blood monocytes (PBM) and AM infected in vitro. Our findings indicate that RANTES suppresses HIV-1 replication, as measured by reverse transcriptase activity, in PBM (41.3 +/- 15.2% of control, n = 3, P < 0.05) and AM (30.3 +/- 7.8% of control, n = 3, P < 0.05) in a dose-dependent manner. The other C-C chemokines had no significant effect singly (MIP-1 alpha PBM: 64.8 +/- 21.9%; AM: 115.0 +/- 2.4% of control; MIP-1 beta PBM: 68 +/- 19.6; AM: 63.3 +/- 26.2% of control) but modestly decreased HIV replication when incubated in addition to RANTES (24.5 +/- 6.5% of control). These observations suggest that RANTES plays a key role in modulating HIV-1 replication in mononuclear phagocytes in the blood and lung, and this may have therapeutic implications for prevention and/or treatment of HIV disease.

Inhibition of recombinant human immunodeficiency virus type 1 replication by a site-specific recombinase.

Current molecular genetic strategies to inhibit productive human immunodeficiency virus type 1 (HIV-1) replication have involved the generation of gene products which provide intracellular inhibition of essential virally encoded proteins or RNA structures. A molecular strategy to excise proviral DNA from HIV-1-infected cells and render these cells virus free would provide an attractive direct antiviral strategy, providing a mechanism to remove viral genes from infected cells. The potential of such a molecular genetic intervention was examined by using the Cre-loxP recombination system. A recombinant HIV-1 clone, designated HIV(lox), that contains loxP within a nonessential U3 region of the long terminal repeats was synthesized. The loxP motif was maintained during replication of HIV(lox) in CEM cells, as demonstrated by reverse transcriptase PCR analyses of genomic RNA isolated from virions. Two different types of HIV-1-permissive cells, CEM cells and 293 cells expressing the CD4 glycoprotein, were transformed with a Cre expression vector which was shown to encode Cre DNA binding and recombinase activities. HIV(lox) infection of CEM or CD4+ 293 cells expressing Cre resulted in a substantial reduction in virus replication compared to control cells, and evidence for the presence of the expected excision product was found. Site-specific excision of HIV-1 can therefore be achieved by using this model system with acute infection. These studies represent one step toward the development of a novel antiviral strategy for the treatment of AIDS.

Distinct domains of IkappaB-alpha inhibit human immunodeficiency virus type 1 replication through NF-kappaB and Rev.

Among the regulators of human immunodeficiency virus (HIV) replication is the cellular transcription factor NF-kappaB, whose activity is regulated through inhibition by IkappaB family members. We have shown previously that I kappaB-alpha inhibits HIV type 1 (HIV-1) replication, and unexpectedly, IkappaB-alpha was found both to suppress HIV-1 transcription and to inhibit Rev function. The relative contributions and specificities of these mechanisms to HIV replication were unknown. Here, we report that the region of IkappaB-alpha which blocks Rev function is separable from that required for inhibition of NF-kappaB. Molecular mutagenesis revealed that the N terminus of IkappaB-alpha is required for inhibition of Rev function, whereas mutants lacking the N terminus retained the ability to inhibit NF-kappaB function. Interestingly, the nuclear export sequence of IkappaB-alpha was not required for inhibition of Rev or NF-kappaB function in mammalian transfection assays. Conversely, the C terminus of IkappaB-alpha was not required for the inhibition of Rev, while deletion of this region resulted in a loss of NF-kappaB inhibition. Another IkappaB family member with a distinct amino-terminal sequence, IkappaB-beta, inhibited NF-kappaB but not Rev function. These studies indicate that the inhibition of Rev by IkappaB-alpha is independent of NF-kappaB. Mutants defective in inhibition of either Rev or NF-kappaB retained the ability to inhibit HIV-1 replication, suggesting that both functions may contribute to the inhibition of HIV replication by I kappaB-alpha.

The inhibition of pro-apoptotic ICE-like proteases enhances HIV replication.

Accelerated programmed cell death, or apoptosis, contributes to the CD4+ T-cell depletion characteristic of infection by human immunodeficiency virus (HIV). It has therefore been proposed that limiting apoptosis may represent a therapeutic modality for HIV infection. We found, however, that T leukemia cells or peripheral blood mononuclear cells (PBMCs) exposed to HIV-1 underwent enhanced viral replication in the presence of the cell death inhibitor, N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (z-AVD-fmk). Furthermore, z-VAD-fmk, which targets the pro-apoptotic interleukin-1 beta-converting enzyme (ICE)-like proteases, stimulated endogenous virus production in activated PBMCs derived from HIV-1-infected asymptomatic individuals. These findings suggest that programmed cell death may serve as a beneficial host mechanism to limit HIV spread and that strategies to inhibit it may have deleterious consequences for the infected host.

Propagation of a human herpesvirus from AIDS-associated Kaposi's sarcoma.

BACKGROUND: Although unique DNA sequences related to gammaherpesviruses have been found in Kaposi's sarcoma lesions, it is uncertain whether this DNA encodes a virus that is able to reproduce. METHODS: We isolated and propagated a filterable agent whose DNA sequences were found to be identical to those of the Kaposi's sarcoma-associated herpesvirus (KSHV). We obtained early-passage spindle cells from skin lesions of patients with the acquired immunodeficiency syndrome (AIDS) who had Kaposi's sarcoma and cultured them with cells of the human embryonal-kidney epithelial-cell line 293. We characterized the virus according to its effects on cellular morphology and viral replication and its appearance on electron microscopy. RESULTS: KSHV was cytotoxic to 293 cells and was detected by the polymerase chain reaction (PCR) in infected cells but not uninfected ones. Cytotoxicity and positive PCR signals were consistently maintained with viral titers of 1 million per milliliter, for about 20 serial infections of 293 cells. The viral copy number was relatively low (1 to 10 copies per cell). Viral replication was confirmed by Southern blot analysis of DNA isolated from the enriched nuclear fraction of infected cells and by a semiquantitative PCR using dilutions of the lysates of infected cells to detect the 233-bp viral DNA fragment originally described in association with Kaposi's lesions. Electron microscopy revealed herpesvirus-like particles in about 1 percent of cells from infected cultures, as compared with none in cells from uninfected cultures. CONCLUSIONS: A herpesvirus with DNA sequences identical to those of KSHV can be propagated from skin lesions of patients with AIDS-associated Kaposi's sarcoma.

Expression of a protective gene-prolongs survival of T cells in human immunodeficiency virus-infected patients.

The resistance of acquired immunodeficiency syndrome (AIDS) to traditional drug therapy has prompted a search for alternative treatments for this disease. One potential approach is to provide genetic resistance to viral replication to prolong latency. This strategy requires the definition of effective antiviral genes that extend the survival of T cells in human immunodeficiency virus (HIV)-infected individuals. We report the results of a human study designed to determine whether a genetic intervention can prolong the survival of T cells in HIV-infected individuals. Gene transfer was performed in enriched CD4+ cells with plasmid expression vectors encoding an inhibitory Rev protein, Rev M10, or a deletion mutant control, deltaRev M10, delivered by gold microparticles. Autologous cells separately transfected with each of the vectors were returned to each patient, and toxicity, gene expression, and survival of genetically modified cells were assessed. Cells that expressed Rev M10 were more resistant to HIV infection than those with deltaRev M10 in vitro. In HIV-infected subjects, Rev M10-transduced cells showed preferential survival compared to deltaRev M10 controls. Rev M10 can therefore act as a specific intracellular inhibitor that can prolong T-cell survival in HIV-1-infected individuals and potentially serve as a molecular genetic intervention which can contribute to the treatment of AIDS.

Genetic modification of human peripheral blood lymphocytes with a transdominant negative form of Rev: safety and toxicity.

A transdominant mutant form of the rev gene, M10, confers resistance to infection by the human immunodeficiency virus (HIV) in vitro and is currently under investigation as a potential intervention in acquired immunodeficiency syndrome (AIDS). In this report, we examine three issues relevant to the safety of autologous transfer of human T cells genetically modified with Rev M10. First, the potential for malignant transformation was assessed in vitro using interleukin-2 (IL-2) dependence and fibroblast transformation assays, and tumorigenicity was evaluated in severe combined immunodeficient (SCID) mice. Possible toxicity was evaluated by pathologic analysis following adoptive transfer of genetically modified human T cells into SCID mice. Second, methods were developed that permit T cell activation required for gene transfer but do not allow replication of endogenous HIV. Third, T cell function was evaluated in peripheral blood lymphocytes (PBL) of HIV-seropositive donors transduced with Rev M10 and compared to a negative control mutant, delta Rev M10. By all criteria, no oncogenicity or toxicity was observed. Human T cells transduced with these vectors did not grow in the absence of IL-2 in vitro, and no tumors were observed following transplantation of genetically modified human cells into recipient SCID mice. Histopathological analysis of heart, lung, liver, spleen, and kidney of animals 1-21 weeks following adoptive transfer of gene-modified human T cells revealed no significant abnormalities. Additionally, no differences were observed in the pattern of cytokine secretion in enriched human PBL expressing Rev M10 compared to delta Rev M10. (ABSTRACT TRUNCATED AT 250 WORDS)

Regulation of human retroviral latency by the NF-kappa B/I kappa B family: inhibition of human immunodeficiency virus replication by I kappa B through a Rev-dependent mechanism.

The cellular transcription factor NF-kappa B stimulates human immunodeficiency virus type 1 (HIV-1) transcriptional initiation, but its role in the retroviral life cycle has not been fully defined. In this report, we show that I kappa B alpha acts as a cellular inhibitor of human retroviral replication through a discrete mechanism, independent of its effect on HIV transcription. I kappa B alpha inhibited HIV replication and gp160 expression by negatively regulating Rev function, most likely acting through a cellular factor involved in Rev transactivation. A similar effect was observed with human T leukemia virus I, in which I kappa B alpha inhibited Rex function. In contrast, no effect was observed on the replication of a DNA virus, adenovirus type 5. The NF-kappa B/I kappa B regulatory pathway therefore modulates human retroviral replication by regulating a program of cellular gene expression required for several steps in the viral life cycle, including not only viral transcription but also RNA export. This interaction between cellular and viral gene products suggests that NF-kappa B plays a broader role in the regulation of human retroviral replication, providing a previously unrecognized link between two important regulators of HIV gene expression and common NF-kappa B-dependent programs of gene expression used by human retroviruses.

Nonviral and viral delivery of a human immunodeficiency virus protective gene into primary human T cells.

Because AIDS has been refractory to traditional pharmacologic interventions, alternative approaches have been developed. Although the introduction of specific antiviral genes into T leukemia cells can provide relative resistance to human immunodeficiency virus (HIV) replication, the testing of such genes against primary viral isolates in human CD4+ lymphocytes has been limited, and safety questions remain regarding gene delivery into cells from HIV-infected patients. In this report, we evaluate the efficacy of a transdominant mutant protein, Rev M10, against cloned and primary HIV isolates in human peripheral blood lymphocytes and describe different methods of gene transfer into peripheral blood lymphocytes from HIV-infected individuals. We show that gold microparticles can mediate stable Rev M10 gene transfer into these cells. Introduction of Rev M10 by these techniques conferred resistance to HIV infection in vitro to cloned and clinical isolates. Nonviral delivery of HIV protective genes will facilitate the development of gene therapy for AIDS and the analysis of viral and cellular gene expression in human T lymphocytes.

Regulated expression of a dominant negative form of Rev improves resistance to HIV replication in T cells.

Infection by the human immunodeficiency virus (HIV) has remained refractory to treatment, and molecular genetic interventions have been developed for the treatment of the acquired immunodeficiency syndrome (AIDS). Previous studies have focused on the development of gene products which inhibit productive HIV replication, including transdominant proteins, RNA decoys and ribozymes. In this report, we show that appropriate expression vectors which optimize production and regulated synthesis of a transdominant mutant form of Rev improve its antiviral effect. The combination of a strong constitutive enhancer, a Tat activation response (TAR) regulatory element and transdominant Rev take advantage of three aspects of early viral gene expression to confer increased resistance to HIV replication. This vector may be useful, alone or in combination with other antiviral genes, to provide gene therapy for AIDS.

Mammalian mRNAs encoding protein closely related to ubiquitin-conjugating enzyme encoded by yeast DNA repair gene RAD6.

A clone of about 1 kb has been isolated from a human brain cDNA library. The clone possesses a 151 amino acid open reading frame that exhibits 72% amino acid identity with the E2 ubiquitin-conjugating enzyme encoded by the RAD6 gene of Saccharomyces cerevisiae. A 90% amino acid identity was observed in a central sequence surrounding a cysteine, which most likely contributes the sulfhydryl group involved in the formation of the ubiquitin-E2 thiolester linkage. Northern hybridization analyses have identified a poly(A)-containing mRNA of about 1 kb encoding the E2-like sequence in human CEM lymphoblastoid and HeLa cells, Novikoff rat hepatoma cells and S49 mouse leukemia cells. Southern hybridization analyses indicate the presence of a single gene encoding this sequence in both human cell lines, but of two or more related genes in the rodent cell lines.

Mycoplasma contamination alters 2'-deoxyadenosine metabolism in deoxycoformycin-treated mouse leukemia cells.

Deoxycoformycin-treated P388 and L1210 mouse leukemia cells salvage 2'-deoxyadenosine from the medium only inefficiently, because deoxyadenosine deamination is blocked and its phosphorylation is limited by feedback controls. Mycoplasma contamination at a level that had no significant effect on the growth of the cells increased the salvage of deoxyadenosine greater than 10 fold over a 90 min period of incubation at 37 degrees C, but in this case deoxyadenosine was mainly incorporated into ribonucleotides and RNA via adenine formed from deoxyadenosine by mycoplasma adenosine phosphorylase. Deoxyadenosine was an efficient substrate for this enzyme, in contrast to 2',3'-dideoxyadenosine which was not phosphorolyzed. Mycoplasma infection was confirmed by the presence of uracil phosphoribosyltransferase activity and by culture isolation. The contaminant has been identified as Mycoplasma orale. Mycoplasma infection had no effect on the deamination and phosphorylation of deoxyadenosine and adenosine, on the salvage of hypoxanthine and adenine, or on the degradation of dAMP and dATP by the cells or on their acid and alkaline phosphatase activities.

Mobility of nucleoside transporter of human erythrocytes differs greatly when loaded with different nucleosides.

Time courses of transmembrane equilibration of 2-chloroadenosine, 2'-deoxyadenosine, 3'-deoxyadenosine, cytidine and 2'-deoxycytidine were measured by rapid kinetic techniques in human erythrocytes under equilibrium exchange and zero-trans conditions. The kinetic parameters for transport were computed by fitting appropriate integrated rate equations to the data pooled for seven concentrations and compared to the kinetic parameters for uridine, adenosine, thymidine and formycin B transport determined previously for human erythrocytes under comparable experimental conditions. The transport of all nucleosides conformed to the simple carrier model and was directionally symmetric. The Michaelis-Menten constants for equilibrium exchange (Kee) ranged from 22 microM for 2-chloroadenosine to about 4 mM for cytidine and the maximum velocities (Vee) differed in a similar manner, so that the first-order rate constants (Vee/Kee) were similar for all nucleosides. The kinetic parameters for 2'-deoxyadenosine transport were similar to those for adenosine transport, whereas the lack of the 3'-OH group greatly reduced the affinity of 3'-deoxyadenosine (cordycepin) for the carrier. 2', 3'-Dideoxynucleosides were transported less than 1% as efficiently as 2'- and 3'-deoxynucleosides. Thus, the 2'- and 3'-OH groups play an important role in nucleoside transport. The mobility of the carrier when loaded with pyrimidine nucleosides (reflected by Vee) was 5-10-times greater than that of the empty carrier, whereas the mobility of the adenosine-loaded or 2'-deoxyadenosine-loaded carrier was about equal to that of the empty carrier. Loading the carrier with 2-chloroadenosine or 3'-deoxyadenosine actually decreased its mobility. Thus, the differential mobility of the loaded and empty carrier differs greatly with the nucleoside substrate. The mobility of the loaded carrier as well as Kee increased with a decrease in lipid solubility of the nucleoside substrate, but the relationship was complex.