Evaluation of the safety and brain-related tissues distribution characteristics of TAT-HaFGF via intranasal administration.

Disabilities triggered by neurodegeneration mainly result in mortality in the elderly, and patients with neurodegenerative disease also display deficits in olfactory function. Therefore drug distribution to the brain through intranasal administration has become one of the most difficult challenges in the treatment of central nervous system (CNS) diseases. TAT-human acidic fibroblast growth factor (HaFGF) is a new fused protein retaining the neuroprotective activities of HaFGF, and is a promising prospect in the treatment of neurodegenerative diseases. TAT (a cell-penetrating peptide) contains a high relative abundance of positively charged amino acids such as lysine and arginine, which have a powerful attraction to the negatively charge on the nasal epithelial membrane. The present study focused on the evaluation of the safety and absorption characteristics of TAT-HaFGF following intranasal administration. After TAT-HaFGF intranasal administration (100, 300, 600 µg/kg) for 5 weeks, hematoxylin-eosin (HE) staining showed no pathology in any of the investigated tissues and organs. The expression of olfactory marker protein (OMP) was observed with immunohistochemical staining, which showed no altered expression in the sensory neurons of the nasal epithelium. Nasal ciliotoxicity studies carried out using an in situ palate model and optical microscope showed that TAT-HaFGF had no nasal ciliotoxicity. The distribution of the TAT-HaFGF following intranasal administration was assessed using a radioisotopic tracing method. Radioactivity was observed in the brain after 15 min. This became stronger at 30 min and weaker at 1 h. All of the results confirmed the in vivo safety of TAT-HaFGF via intranasal administration.

Comparative study on transduction and toxicity of protein transduction domains.

BACKGROUND AND PURPOSE: Protein transduction domains (PTDs), such as Tat, antennapedia homeoprotein (Antp), Rev and VP22, have been extensively utilized for intracellular delivery of biologically active macromolecules in vitro and in vivo. There is little known, however, about the relative transduction efficacy, cytotoxicity and internalization mechanism of individual PTDs. EXPERIMENTAL APPROACH: We examined the cargo delivery efficacies of four major PTDs (Tat, Antp, Rev and VP22) and evaluated their toxicities and cell internalizing pathways in various cell lines. KEY RESULTS: The relative order of the transduction efficacy of these PTDs conjugated to fluorescein was Rev>Antp>Tat>VP22, independent of cell type (HeLa, HaCaT, A431, Jurkat, MOLT-4 and HL60 cells). Antp produced significant toxicity in HeLa and Jurkat cells, and Rev produced significant toxicity in Jurkat cells. Flow cytometric analysis demonstrated that the uptake of PTD-fluorescein conjugate was dose-dependently inhibited by methyl-beta-cyclodextrin, cytochalasin D and amiloride, indicating that all four PTDs were internalized by the macropinocytotic pathway. Accordingly, in cells co-treated with 'Tat-fused' endosome-disruptive HA2 peptides (HA2-Tat) and independent PTD-fluorescent protein conjugates, fluorescence spread throughout the cytosol, indicating that all four PTDs were internalized into the same vesicles as Tat. CONCLUSIONS AND IMPLICATIONS: These findings suggest that macropinocytosis-dependent internalization is a crucial step in PTD-mediated molecular transduction. From the viewpoint of developing effective and safe protein transduction technology, although Tat was the most versatile carrier among the peptides studied, PTDs should be selected based on their individual characteristics.

Proliferative activity of extracellular HIV-1 Tat protein in human epithelial cells: expression profile of pathogenetically relevant genes.

BACKGROUND: Tat is being tested as a component of HIV vaccines. Tat activity has been mainly investigated on cells of lymphoid/hematopoietic lineages. HIV-1, however, is known to infect many different cells of both solid organs and mucosal surfaces. The activity of two-exon (aa 1-101) and synthetic (aa 1-86) Tat was studied on mammary and amniotic epithelial cells cultured under low serum conditions. RESULTS: small concentrations of Tat (100 ng/ml) stimulated cell proliferation. Tat antibodies neutralized the mitogenic Tat activity. Changes of gene expression in Tat-treated cells were evaluated by RT-PCR and gene-array methods. Within 4 hours of treatment, exposure to Tat is followed by up-regulation of some cell cycle-associated genes (transcription factors, cyclin/cdk complexes, genes of apoptotic pathways) and of genes relevant to HIV pathogenesis [chemokine receptors (CXCR4, CCR3), chemotactic cytokines (SDF-1, RANTES, SCYC1, SCYE1), IL6 family cytokines, inflammatory cytokines, factors of the TGF-beta family (TGFb, BMP-1, BMP-2)]. Up-regulation of anti-inflammatory cytokines (IL-10, IL-19, IL-20), a hallmark of other persistent viral infections, was a remarkable feature of Tat-treated epithelial cell lines. CONCLUSION: extracellular Tat is mitogenic for mammary and amniotic epithelial cells and stimulates the expression of genes of pathogenetic interest in HIV infection. These effects may favor virus replication and may facilitate the mother-to-child transmission of virus.

Novel biocompatible anionic polymeric microspheres for the delivery of the HIV-1 Tat protein for vaccine application.

Two novel classes of biocompatible core-shell anionic microspheres, composed of an inner hard insoluble core, either made of poly(styrene) (PS) or poly(methyl methacrylate) (PMMA), and a soft outer tentacular shell made of long soluble negatively charged arms derived from the steric stabilizer, hemisuccinated poly(vinyl alcohol) or Eudragit L100/55, respectively, were prepared by dispersion polymerization and characterized. Five types of these novel microspheres, two made of poly(styrene) and hemisuccinated poly(vinyl alcohol) (A4 and A7), and three made of poly(methyl methacrylate) and Eudragit L100/55 (1D, 1E, H1D), differing for chemical composition, size, and surface charge density were analyzed for the delivery of the HIV-1 Tat protein for vaccine applications. All microspheres reversibly adsorbed the native biologically active HIV-1 Tat protein preventing Tat from oxidation and maintaining its biological activity, therefore increasing the shelf-life of the Tat protein vaccine. The microspheres efficiently delivered Tat intracellularly, and were not toxic in vitro nor in mice, even after multiple administrations. These results indicate that these novel microparticles are safe and represent a promising delivery system for vaccination with Tat, as well as for other subunit vaccines, particularly when a native protein conformation is required.

Minimization of chronic plasma viremia in rhesus macaques immunized with synthetic HIV-1 Tat peptides and infected with a chimeric simian/human immunodeficiency virus (SHIV33).

HIV-1 Tat protein activates resting cells, rendering them permissive for viral replication. Replication of HIV-1 in vitro is enhanced by intercellular passage of Tat protein and inhibited by anti-Tat antibodies. Tat dependence of HIV-1 replication in vivo during acute, chronic asymptomatic and AIDS stages of infection was assessed by comparisons of plasma viremia in Tat-immunized or control monkeys challenged with SHIV(33) or SHIV(33A). Chronic plasma viremia became undetectable or minimized in Tat-immunized asymptomatic SHIV(33)-infected monkeys (p<0.008) while the high viral loads of acute infection or SHIV(33A)-induced simian AIDS were unaffected by Tat immunization. Active or passive immunotherapies targeting Tat provide potential approaches to controlling chronic HIV-1 viremia and preventing AIDS.

Safety and immunogenicity of HIV-1 Tat toxoid in immunocompromised HIV-1-infected patients.

OBJECTIVES: To antagonize the deleterious effects of the HIV-1 toxin extracellular Tat on uninfected immune cells, we developed a new strategy of anti-HIV-1 vaccine using an inactivated but immunogenic Tat (Tat toxoid). Tat toxoid has been assayed for safety and immunogenicity in seropositive patients. METHOD: The phase I vaccine clinical trial testing Tat toxoid preparation in Seppic Isa 51 oil adjuvant was performed on 14 HIV-1-infected asymptomatic although biologically immunocompromised individuals (500-200 CD4+ cells/mm3). RESULTS: Following as many as 8 injections, no clinical defects were observed. All patients exhibited an antibody (Ab) response to Tat, and some had cell-mediated immunity (CMI) as evaluated by skin test in vivo and T-cell proliferation in vitro. CONCLUSION: These results provide initial evidence of safety and potency of Tat toxoid vaccination in HIV-1-infected individuals.