Biomedical subjects
G J Nabel
Publications and source records attributed to G J Nabel.
Cancer gene therapy: present status and future directions.
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Challenges and opportunities for development of an AIDS vaccine.
Among the devastating consequences of AIDS has been its epidemic spread in the developing world. The disease has caused unprecedented suffering, debilitation, loss of life and disruption of family, social and economic stability. Because of the considerable expense and logistical difficulty in providing antiviral drugs to populations infected with the human immunodeficiency virus throughout the world, the biomedical community is looking towards vaccines to help solve this compelling problem.
Coexpression of guanylate kinase with thymidine kinase enhances prodrug cell killing in vitro and suppresses vascular smooth muscle cell proliferation in vivo.
Herpes simplex virus-thymidine kinase (HSV-TK) phosphorylates the prodrugs ganciclovir (GCV) and acyclovir (ACV), leading to disruption of DNA synthesis and inhibition of cell proliferation. HSV-TK vectors have been successfully employed in cardiovascular and cancer gene therapy. Activation of GCV and ACV, after an initial phosphorylation step by the viral thymidine kinase, is carried out by guanylate kinase. We reasoned that coexpression of guanylate kinase (GK) with HSV-TK would augment phosphorylation of GCV or ACV, leading to increased cell killing. To test this hypothesis, a vector expressing TK with GK (TKciteGK) was developed and tested on vascular smooth muscle cells (vsmcs) in vitro and in vivo. Compared to HSV-TK vectors, killing of vascular cells transduced with TKciteGK and exposed to GCV was significantly increased (P = 0.03). The TKciteGK construct was evaluated with three promoters: CMV, EF1alpha, and SM22alpha. TKciteGK expression driven by a CMV promoter induced cell killing more effectively than SM22alpha or EF1alpha promoters in primary vsmcs. Based upon these in vitro findings, TKciteGK vectors with a CMV promoter were tested in two animal models of cardiovascular disease: balloon angioplasty and stent deployment in pig arteries. Following vascular injury, expression of CMV-TKciteGK with GCV significantly reduced vsmc proliferation and intimal lesion formation compared to control vectors with GCV. In the angioplasty model, there was an 80% reduction in intima-to-media area ratio (P = 0.0002). These findings were paralleled in a stent model with 66% reduction in intimal lesions (P = 0.006). Coexpression of GK with TK increases cell killing and permits administration of GCV at lower doses. These modifications in TKciteGK vectors and GCV showed enhanced efficacy at lower prodrug doses, leading to improved safety for cardiovascular gene therapy.
Injection of human herpesvirus-8 in human skin engrafted on SCID mice induces Kaposi's sarcoma-like lesions.
Kaposi's sarcoma-associated herpesvirus (KSHV) or human herpesvirus 8 (HHV-8) has been implicated in the development of Kaposi's sarcoma (KS) and several B-cell lymphoproliferative diseases. Serologic and molecular genetic association data has implicated HHV-8 as the causal agent of KS, but its role in the development of KS lesions is not understood. To examine the etiology of KS, HHV-8 was injected into normal human skin transplanted onto SCID mice. Injection of HHV-8 induced lesion formation that is morphologically and phenotypically consistent with KS, including the presence of angiogenesis and spindle-shaped cells latently infected with HHV-8. These findings suggest that HHV-8 is indeed the etiologic agent of KS, and that the virus plays an important role in initiation of this disease.
Vaccines for the prevention of HIV-1 disease.
Clinical investigation in humans and experimental lentivirus infection in nonhuman primates have advanced our understanding of immune responses that control HIV-1 disease. Recently, immunization approaches in macaques have shown that the immune response can control viremia and improve clinical outcome. When such vaccine strategies are formulated to be similarly immunogenic in humans, they could form the basis for the development of candidate AIDS vaccines that would prevent infection, suppress progression to disease or reduce HIV-1 transmission in humans.
Extracellular matrix interacts with soluble CD95L: retention and enhancement of cytotoxicity.
Fas ligand (CD95L) is synthesized both on the cell surface membrane and in a soluble form. Although CD95L contributes to immune privilege in the cornea and testis, the functions of these alternatively processed proteins are not well understood. Some reports suggest that the cytotoxicity of soluble CD95L is insignificant, whereas others show potent responses in vivo, including hepatocyte apoptosis that causes liver failure. We show here that extracellular matrix proteins interact with soluble CD95L and potentiate its pro-apoptotic activity. The cytotoxicity of supernatants from CD95L-expressing cells was increased by incubation on tissue culture plates coated with these matrix proteins; this effect was mediated by trimeric soluble CD95L. With the use of immunoprecipitation, it was found that CD95L binds directly to fibronectin. In addition, immunohistochemical analysis of the cornea revealed that soluble CD95L binds primarily to extracellular matrix. The retention of soluble CD95L on extracellular matrices is likely to play an important role in the development of peripheral tolerance in immune-privileged sites.
Heme oxygenase-1 protects against vascular constriction and proliferation.
Heme oxygenase (HO-1, encoded by Hmox1) is an inducible protein activated in systemic inflammatory conditions by oxidant stress. Vascular injury is characterized by a local reparative process with inflammatory components, indicating a potential protective role for HO-1 in arterial wound repair. Here we report that HO-1 directly reduces vasoconstriction and inhibits cell proliferation during vascular injury. Expression of HO-1 in arteries stimulated vascular relaxation, mediated by guanylate cyclase and cGMP, independent of nitric oxide. The unexpected effects of HO-1 on vascular smooth muscle cell growth were mediated by cell-cycle arrest involving p21Cip1. HO-1 reduced the proliferative response to vascular injury in vivo; expression of HO-1 in pig arteries inhibited lesion formation and Hmox1-/- mice produced hyperplastic arteries compared with controls. Induction of the HO-1 pathway moderates the severity of vascular injury by at least two adaptive mechanisms independent of nitric oxide, and is a potential therapeutic target for diseases of the vasculature.
Human immunodeficiency virus type 1-specific immunity after genetic immunization is enhanced by modification of Gag and Pol expression.
Immunity to human immunodeficiency virus virion-like structures or a polyprotein has been examined after DNA immunization with Rev-independent expression vectors. A Gag-Pol fusion protein stimulated cytotoxic T lymphocyte and antibody responses to Gag and Pol, while a Gag-Pol pseudoparticle did not elicit substantial Pol responses. This fusion protein may be useful for AIDS vaccines.
The Gordon Wilson Lecture: viruses and human disease.
In many ways, Ebola virus infection provides a model for understanding the toxicity of viruses and their causal role in human disease. The highly aggressive course of Ebola virus infection provides a model for understanding the molecular mechanisms of viral cytotoxicity. In addition, the use of animal models and definition of immune correlates, which lead to protection, may provide lessons that are applicable to other viral infections. Perhaps the greatest challenge facing biomedical science today is the containment of the human immunodeficiency virus, the causative agent of AIDS. In many ways the critical obstacles to the development of a vaccine for HIV are similar to those observed with Ebola virus infection. Because the reservoir of infection is not known and human-to-human spread has been documented, vaccines may provide the best opportunity to contain and limit the spread of infection worldwide. Similar to Ebola virus, there are few convincing examples of immune resistance of HIV infection. In addition, it has been difficult to identify broadly neutralizing antibodies that can prevent infection in vitro or in vivo. In defining immune correlates, relevant animal models, and mechanisms of cytotoxicity, it is hoped that similar efforts may lead to effective vaccines for other infectious diseases. In this way, Ebola virus infection provides a useful paradigm for understanding the genetic determinants of viral disease and in facilitating the development of treatments and prevention of viral infections.
Development of a preventive vaccine for Ebola virus infection in primates.
Outbreaks of haemorrhagic fever caused by the Ebola virus are associated with high mortality rates that are a distinguishing feature of this human pathogen. The highest lethality is associated with the Zaire subtype, one of four strains identified to date. Its rapid progression allows little opportunity to develop natural immunity, and there is currently no effective anti-viral therapy. Therefore, vaccination offers a promising intervention to prevent infection and limit spread. Here we describe a highly effective vaccine strategy for Ebola virus infection in non-human primates. A combination of DNA immunization and boosting with adenoviral vectors that encode viral proteins generated cellular and humoral immunity in cynomolgus macaques. Challenge with a lethal dose of the highly pathogenic, wild-type, 1976 Mayinga strain of Ebola Zaire virus resulted in uniform infection in controls, who progressed to a moribund state and death in less than one week. In contrast, all vaccinated animals were asymptomatic for more than six months, with no detectable virus after the initial challenge. These findings demonstrate that it is possible to develop a preventive vaccine against Ebola virus infection in primates.
Regulation of beta -catenin transformation by the p300 transcriptional coactivator.
The beta-catenin protein plays a critical role in embryonic development and mature tissue homeostasis through its effects on E-cadherin-mediated cell adhesion and Wnt-dependent signal transduction. In colon and other cancers, mutations of beta-catenin or the adenomatous polyposis coli (APC) tumor suppressor appear to stabilize beta-catenin and enhance its interaction with T cell factor (TCF) or lymphoid enhancer factor (Lef) transcription factors. At present, a complete picture of the means by which beta-catenin's interactions with TCF/Lef proteins contribute to neoplastic transformation is lacking. We report that the transcriptional coactivator p300 interacts with beta-catenin in vitro and in vivo and is critical for beta-catenin-mediated neoplastic transformation. p300 synergistically activates beta-catenin/TCF transcription, and their biochemical association requires the CH1 domain of p300 and a region of beta-catenin that includes its NH(2)-terminal transactivation domain and the first two armadillo repeats. Lowering of cellular p300 levels by using a ribozyme directed against p300 reduced TCF transcriptional activity and inhibited the neoplastic growth properties of a beta-catenin-transformed rat epithelial cell line and a human colon carcinoma line with a beta-catenin mutation. These findings demonstrate a critical role for p300 in beta-catenin/TCF transcription and in cancers arising from defects in beta-catenin regulation.
Antibodies and resistance to natural HIV infection.
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Differential effects of the cyclin-dependent kinase inhibitors p27(Kip1), p21(Cip1), and p16(Ink4) on vascular smooth muscle cell proliferation.
BACKGROUND: The cyclin-dependent kinase inhibitors (CKIs) have different patterns of expression in vascular diseases. The Kip/Cip CKIs, p27(Kip1) and p21(Cip1), are upregulated during arterial repair and negatively regulate the growth of vascular smooth muscle cells (VSMCs). In contrast, the Ink CKI, p16(Ink4), is not expressed in vascular lesions. We hypothesized that a variation in the inactivation of cdk2 and cdk4 during the G(1) phase of the cell cycle by p27(Kip1), p21(Cip1), and p16(Ink4) leads to different effects on VSMC growth in vitro and in vivo. METHODS AND RESULTS: The expression of p27(Kip1) and p21(Cip1) in serum-stimulated VSMCs inactivated cdk2 and cdk4, leading to G(1) growth arrest. p16(Ink4) inhibited cdk4, but not cdk2, kinase activity, producing partial inhibition of VSMC growth in vitro. In an in vivo model of vascular injury, overexpression of p27(Kip1) reduced intimal VSMC proliferation by 52% (P<0.01) and the intima/media area ratio by 51% (P<0.005) after vascular injury and gene transfer to pig arteries, when compared with control arteries. p16(Ink4) was a weak inhibitor of intimal VSMC proliferation in injured arteries (P=NS), and it did not significantly reduce intima/media area ratios (P=NS), which is consistent with its minor effects on VSMC growth in vitro. CONCLUSIONS: p27(Kip1) and p21(Cip1) are potent inhibitors of VSMC growth compared with p16(Ink4) because of their different molecular mechanisms of cyclin-dependent kinase inhibition in the G(1) phase of the cell cycle. These findings have important implications for our understanding of the pathophysiology of vascular proliferative diseases and for the development of molecular therapies.
Human gene marker/therapy clinical protocols.
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Ebola virus secretory glycoprotein (sGP) diminishes Fc gamma RIIIB-to-CR3 proximity on neutrophils.
Previous studies have shown that Ebola virus' secretory glycoprotein (sGP) binds to Fc gamma RIIIB (CD16b) and inhibits L-selectin shedding. In this study, we test the hypothesis that sGP interferes with the physical linkage between CR3 and Fc gamma RIIIB. Neutrophils were stained with rhodamine-conjugated anti-CD16b mAb (which does not inhibit sGP binding) and fluorescein-conjugated anti-CR3 mAb reagents and then incubated in media with or without sGP. Physical proximity between fluorochrome-labeled CR3 and Fc gamma RIIIB on individual cells was measured by resonance energy transfer (RET) imaging, quantitative RET microfluorometry, and single-cell imaging spectrophotometry. Cells incubated with control supernatants displayed a significant RET signal, indicative of physical proximity (<7 nm) between CR3 and Fc gamma RIIIB. In contrast, cells exposed to sGP showed a significant reduction in the CR3-Fc gamma RIIIB RET signal using these methods. Interestingly, colocalization and cocapping of CR3 and Fc gamma RIIIB were not affected, suggesting that the proximity of these two receptors is reduced without triggering dissociation. Thus, sGP alters the physical linkage between Fc gamma RIIIB and CR3.
Restricted expression of an adenoviral vector encoding Fas ligand (CD95L) enhances safety for cancer gene therapy.
Gene transfer of Fas ligand (CD95L) using adenoviral vectors has been shown to generate apoptotic responses and potent inflammatory reactions that can be used to induce the regression of malignancies in vivo, but these vectors also cause significant hepatotoxicity that may limit their clinical utility. Here we describe an adenoviral vector encoding CD95L with restricted gene expression that reduces its toxicity in vivo. Preclinical efficacy and gene expression studies of lineage-restricted CD95L adenoviral vectors were performed. To enhance its cytotoxicity and reduce potential systemic effects, a noncleavable CD95L was made by deleting a segment containing the cleavage site (CD95L deltaQP). Higher CD95L expression of this mutant was observed on the tumor cell surface, together with a reduction in the release of soluble CD95L. This CD95L cleavage mutant was then expressed under control of a smooth muscle-specific promoter, SM22apha, and analyzed for its ability to suppress the growth of tumors of smooth muscle origin in vivo. Growth of human leiomyosarcomas but not gliomas was inhibited after ADV gene transfer into tumor-bearing immunodeficient mice. In contrast to viral promoters, in which mortality was uniformly seen after injection of 10(12) particles, no significant hepatic injury or systemic toxicity was observed in mice, and the maximum tolerated dose was increased > or = 10- to 100-fold. These findings suggest that restricted specificity of adenoviral CD95L gene expression enhances the safety of this approach for cancer gene therapy.
How much longer will tumour cells fool the immune system?
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