Eleni Papanikolaou

Antiviral properties of isoborneol, a potent inhibitor of herpes simplex virus type 1

Isoborneol, a monoterpene and a component of several plant essential oils, showed dual viricidal activity against herpes simplex virus 1 (HSV-1). First, it inactivated HSV-1 by almost 4 log10 values within 30 min of exposure, and second, isoborneol at a concentration of 0.06% completely inhibited viral replication, without affecting viral adsorption. Isoborneol did not exhibit significant cytotoxicity at concentrations ranging between 0.016% and 0.08% when tested against human and monkey cell lines. Isoborneol specifically inhibited glycosylation of viral polypeptides based on the following data: (1) the mature fully glycosylated forms of two viral glycoproteins gB and gD were not detected when the virus was replicated in the presence of isoborneol, (2) no major changes were observed in the glycosylation pattern of cellular polypeptides between untreated and isoborneol treated Vero cells, (3) isoborneol did not affect the glycosylation of gB produced from a copy of the gB gene resident in the cellular genome, and (4) other monoterpenes such as 1,8-cineole and borneol, a stereoisomer of isoborneol, did not inhibit HSV-1 glycosylation.

Therapeutic levels of fetal hemoglobin in erythroid progeny of β-thalassemic CD34+ cells after lentiviral vector-mediated gene transfer.

β-Thalassemia major results from severely reduced or absent expression of the β-chain of adult hemoglobin (α₂β₂;HbA). Increased levels of fetal hemoglobin (α₂γ₂;HbF), such as occurs with hereditary persistence of HbF, ameliorate the severity of β-thalassemia, raising the potential for genetic therapy directed at enhancing HbF. We used an in vitro model of human erythropoiesis to assay for enhanced production of HbF after gene delivery into CD34(+) cells obtained from mobilized peripheral blood of normal adults or steady-state bone marrow from patients with β-thalassemia major. Lentiviral vectors encoding (1) a human γ-globin gene with or without an insulator, (2) a synthetic zinc-finger transcription factor designed to interact with the γ-globin gene promoters, or (3) a short-hairpin RNA targeting the γ-globin gene repressor, BCL11A, were tested. Erythroid progeny of normal CD34(+) cells demonstrated levels of HbF up to 21% per vector copy. For β-thalassemic CD34(+) cells, similar gene transfer efficiencies achieved HbF production ranging from 45% to 60%, resulting in up to a 3-fold increase in the total cellular Hb content. These observations suggest that both lentiviral-mediated γ-globin gene addition and genetic reactivation of endogenous γ-globin genes have potential to provide therapeutic HbF levels to patients with β-globin deficiency.

Identification and characterization of the gene products of open reading frame U86/87 of human herpesvirus 6.

The human herpesvirus 6 (HHV-6) immediate early-A locus (IE-A) locates in the position analogous to the human cytomegalovirus (HCMV) major IE (MIE) locus that is well-known to play critical roles in viral infection. Similarly to HCMV MIE, HHV-6 IE-A consists of two genetic units, IE1 and IE2, corresponding to open reading frames U90-U89 and U90-U86/87, respectively. However, the HHV-6 IE-A locus exhibits limited sequence homology with the HCMV MIE locus. In this study, to characterize HHV-6 IE2 gene products, polyclonal antibodies against four domains of the U86/87 open reading frame were generated by immunization of rabbits with bacterially-expressed proteins. Three polypeptides derived from the U86/87 region with apparent molecular masses of 100, 85 and 55 kD were detected in HHV-6-infected cells 3 days after infection, while IE1 polypeptides with apparent molecular mass greater than 170 kD were detectable as early as 8 h. Mapping of the IE2 gene products with the antibodies suggests differential splicing and alternative translation initiation in the IE2 genetic unit. The IE2 products show a mixed cytoplasmic and nuclear localization pattern. In addition, the 437 amino acid carboxyl-terminus domain bound to a DNA fragment containing the putative IE-A promoter. These results suggest that HHV-6 IE2 plays a critical role in transcriptional regulation and viral growth as does HCMV IE2, although it is likely that HHV-6 IE2 has expression kinetics different from HCMV IE2.

Characterization and comparative performance of lentiviral vector preparations concentrated by either one-step ultrafiltration or ultracentrifugation

Gene therapy utilizing lentiviral vectors (LVs) constitutes a real therapeutic alternative for many inherited monogenic diseases. Therefore, the generation of functional vectors using fast, non-laborious and cost-effective strategies is imperative. Among the available concentration methods for VSV-G pseudotyped lentiviruses to achieve high therapeutic titers, ultracentrifugation represents the most common approach. However, the procedure requires special handling and access to special instrumentation, it is time-consuming, and most importantly, it is cost-ineffective due to the high maintenance expenses and consumables of the ultracentrifuge apparatus. Here we describe an improved protocol in which vector stocks are prepared by transient transfection using standard cell culture media and are then concentrated by ultrafiltration, resulting in functional vector titers of up to 6×10(9) transducing units per millilitre (TU/ml) without the involvement of any purification step. Although ultrafiltration per se for concentrating viruses is not a new procedure, our work displays one major novelty; we characterized the nature and the constituents of the viral batches produced by ultrafiltration using peptide mass fingerprint analysis. We also determined the viral functional titer by employing flow cytometry and evaluated the actual viral particle size and concentration in real time by using laser-based nanoparticle tracking analysis based on Brownian motion. Vectors generated by this production method are contained in intact virions and when tested to transduce in vitro either murine total bone marrow or human CD34(+) hematopoietic stem cells, resulted in equal transduction efficiency and reduced toxicity, compared to lentiviral vectors produced using standard ultracentrifugation-based methods. The data from this study can eventually lead to the improvement of protocols and technical modifications for the clinical trials for gene therapy.

The New Self-Inactivating Lentiviral Vector for Thalassemia Gene Therapy Combining Two HPFH Activating Elements Corrects Human Thalassemic Hematopoietic Stem Cells

To address how low titer, variable expression, and gene silencing affect gene therapy vectors for hemoglobinopathies, in a previous study we successfully used the HPFH (hereditary persistence of fetal hemoglobin)-2 enhancer in a series of oncoretroviral vectors. On the basis of these data, we generated a novel insulated self-inactivating (SIN) lentiviral vector, termed GGHI, carrying the (A)γ-globin gene with the -117 HPFH point mutation and the HPFH-2 enhancer and exhibiting a pancellular pattern of (A)γ-globin gene expression in MEL-585 clones. To assess the eventual clinical feasibility of this vector, GGHI was tested on CD34(+) hematopoietic stem cells from nonmobilized peripheral blood or bone marrow from 20 patients with β-thalassemia. Our results show that GGHI increased the production of γ-globin by 32.9% as measured by high-performance liquid chromatography (p=0.001), with a mean vector copy number per cell of 1.1 and a mean transduction efficiency of 40.3%. Transduced populations also exhibited a lower rate of apoptosis and resulted in improvement of erythropoiesis with a higher percentage of orthochromatic erythroblasts. This is the first report of a locus control region (LCR)-free SIN insulated lentiviral vector that can be used to efficiently produce the anticipated therapeutic levels of γ-globin protein in the erythroid progeny of primary human thalassemic hematopoietic stem cells in vitro.

Major challenges for gene therapy of thalassemia and sickle cell disease.

Gene therapy utilizing retroviral vectors is being postulated as a real therapeutic alternative for many hemopoietic inherited diseases, such as β-thalassemia or sickle cell disease. A major limitation of current vectors is their inability to achieve efficient gene transfer into quiescent cells, such as human CD34+ cells that reside in the Go phase of the cell cycle and are highly enriched in hemopoietic stem cells. For that reason, lentiviral vectors (LVs) were proven to be more efficient than oncoretroviral vectors. Additional problems of these vectors are a) the low titers observed due to regulatory elements of the β-globin locus, used for the improvement of the transgenes expression b) the eventual silencing of the transgene and c) the toxicity posed on CD34+ cells due to the usage of VSV-G as an envelope protein. These facts hamper their application for gene therapy of hematopoietic cells. Thus, the major current drawbacks of the field affecting therapeutic efficacy, include 1) insufficient transduction efficiency of the target hemopoietic stem cells, 2) inconsistent expression of the transgene, 3) putative aberrant expression near integration sites raising safety issues and 4) lack of long term expression of the transgene exhibiting eventual silencing. This review presents the current status of globin gene therapy for the hemoglobin disorders, reviews the recent results and discusses how the knowledge gained from these trials can be used to develop a safe and effective gene therapy approach for the treatment of β-thalassemia and SCD.

Cell Cycle Status of CD34+ Hemopoietic Stem Cells Determines Lentiviral Integration in Actively Transcribed and Development-related Genes

Gene therapy utilizing lentiviral-vectors (LVs) is postulated as a dynamic therapeutic alternative for monogenic diseases. However, retroviral gene transfer may cause insertional mutagenesis. Although, such risks had been originally estimated as extremely low, several reports of leukemias or clonal dominance, have led to a re-evaluation of the mechanisms operating in insertional mutagenesis. Therefore, unraveling the mechanism of retroviral integration is mandatory toward safer gene therapy applications. In the present study, we undertook an experimental approach which enabled direct correlation of the cell cycle stage of the target cell with the integration profile of LVs. CD34+ cells arrested at different stages of cell cycle, were transduced with a GFP-LV. LAM-PCR was employed for integration site detection, followed by microarray analysis to correlate transcribed genes with integration sites. The results indicate that ~10% of integration events occurred in actively transcribed genes and that the cell cycle stage of target cells affects integration pattern. Specifically, use of thymine promoted a safer profile, since it significantly reduced integration within cell cycle-related genes, while we observed increased possibility for integration into genes related to development, and decreased possibility for integration within cell cycle and cancer-related genes, when transduction occurs during mitosis.Gene therapy utilizing lentiviral-vectors (LVs) is postulated as a dynamic therapeutic alternative for monogenic diseases. However, retroviral gene transfer may cause insertional mutagenesis. Although, such risks had been originally estimated as extremely low, several reports of leukemias or clonal dominance, have led to a re-evaluation of the mechanisms operating in insertional mutagenesis. Therefore, unraveling the mechanism of retroviral integration is mandatory toward safer gene therapy applications. In the present study, we undertook an experimental approach which enabled direct correlation of the cell cycle stage of the target cell with the integration profile of LVs. CD34(+) cells arrested at different stages of cell cycle, were transduced with a GFP-LV. LAM-PCR was employed for integration site detection, followed by microarray analysis to correlate transcribed genes with integration sites. The results indicate that ~10% of integration events occurred in actively transcribed genes and that the cell cycle stage of target cells affects integration pattern. Specifically, use of thymine promoted a safer profile, since it significantly reduced integration within cell cycle-related genes, while we observed increased possibility for integration into genes related to development, and decreased possibility for integration within cell cycle and cancer-related genes, when transduction occurs during mitosis.

The Ongoing Challenge of Hematopoietic Stem Cell-Based Gene Therapy for β-Thalassemia

β-thalassemia is characterized by reduced or absence of β-globin production, resulting in anemia. Current therapies include blood transfusion combined with iron chelation. BM transplantation, although curative, is restricted by the matched donor limitation. Gene therapy, on the other hand, is promising, and its success lies primarily on designing efficient globin vectors that can effectively and stably transduce HSCs. The major breakthrough in β-thalassemia gene therapy occurred a decade ago with the development of globin LVs. Since then, researchers focused on designing efficient and safe vectors, which can successfully deliver the therapeutic transgene, demonstrating no insertional mutagenesis. Furthermore, as human HSCs have intrinsic barriers to HIV-1 infection, attention is drawn towards their ex vivo manipulation, aiming to achieve higher yield of genetically modified HSCs. This paper presents the current status of gene therapy for β-thalassemia, its success and limitations, and the novel promising strategies available involving the therapeutic role of HSCs.

A novel BaEVRless-pseudotyped γ-globin lentiviral vector drives high and stable HbF expression and improves thalassemic erythropoiesis in vitro

It has previously been demonstrated that the self-inactivating γ-globin lentiviral vector GGHI can significantly increase fetal hemoglobin (HbF) in erythroid cells from thalassemia patients and thus improve the disease phenotype in vitro. In the present study, the GGHI vector was improved further by incorporating novel enhancer elements and also pseudotyping it with the baboon endogenous virus envelope glycoprotein BaEVRless, which efficiently and specifically targets human CD34+ cells. We evaluated the hypothesis that the newly constructed vector designated as GGHI-mB-3D would increase hCD34+ cell tropism and thus transduction efficiency at low multiplicity of infection, leading to increased transgene expression. High and stable HbF expression was demonstrated in thalassemic cells for the resulting GGHI-mB-3D/BaEVRless vector, exhibiting increased transduction efficiency compared to the original GGHI-mB-3D/VSVG vector, with a concomitant 91% mean HbF increase at a mean vector copy number per cell of 0.86 and a mean transduction efficiency of 56.4%. Transduced populations also exhibited a trend toward late erythroid, orthochromatic differentiation and reduced apoptosis, a further indication of successful gene therapy treatment. Monitoring expression of ATG5, a key link between autophagy and apoptosis, it was established that this correction correlates with a reduction of enhanced autophagy activation, a typical feature of thalassemic polychromatophilic normoblasts. This work provides novel mechanistic insights into gene therapy-mediated correction of erythropoiesis and demonstrates the beneficial role of BaEVRless envelope glycoprotein compared to VSVG pseudotyping and of the novel GGHI-mB-3D/BaEVRless lentiviral vector for enhanced thalassemia gene therapy.

Pediculosis in a paediatric greek population: regressions of socioeconomic factors

Background: Pediculosis in humans and especially children is a very common dermatological disorder caused by the parasites Pediculus humanus capitis, Pediculus humanus humanus and/or Pediculus phthirus pubis. Aim: The aim of this study was to further investigate the relations of studied socioeconomic variables towards the understanding of pediculosis dynamics in the Greek population. Materials and methods: A total of 435 parents or parent couples were investigated with respect to their socioeconomic background and the prevalence of pediculosis in their children. Linear regression has been applied in order to identify further patterns of pediculosis dynamics. Results: Regression analysis revealed distinct dynamics with respect to several socioeconomic factors such as education and income. Discussion: It appeared that income and education are influential with respect to the prevalence of pediculosis, while parents’ gender appears also of great importance since mothers are the determining factor in pediculosis prevalence. Correspondence to: George I. Lambrou, First Department of Pediatrics, Choremeio Research Laboratory, University of Athens, Athens-Goudi 11527, Greece, Tel: +00302107467427; Fax: +00302107467427; E-mail: glamprou@med.uoa.gr