The Suicide Gene Therapy Challenge: How to Improve a Successful Gene Therapy Approach

The transfer of a suicide gene into donor lymphocytes to control alloreactivity in the context of allogeneic hematopoietic stem cell transplantation (allo-HSCT) represents the widest clinical application of T-cell based gene transfer, as shown by more than 100 patients treated worldwide to date, several phase I–II studies completed, and a registrative phase III study, sponsored by a biotech firm, about to begin. In this mini-review, we will summarize the clinical results obtained to date, and attempt to identify the steps envisaged to optimize the suicide gene therapy approach

A CRIATIVIDADE E OS BLOQUEIOS MENTAIS - ESTUDO DE CASO: AS PERCEPÇÕES DOS COLABORADORES DA ENCOPEL PAPELARIA SOBRE ASPECTOS DA CRIATIVIDADE

The Covid 19 pandemic has brought many socioeconomic changes in the world. To adapt to these changes, there has been a growing search for the adoption of digital services and innovations, accelerating the technological revolution. Unleashing an urgent demand for professionals with high skills and competencies, including in terms of creativity. However, there are mental blocks and other factors that create misperceptions about creativity and hinder its development. A project was carried out, with action research methodology, with the employees of the company Encopel Papelaria, which sought to analyze whether the participants considered themselves creative or not. If they were aware of the existence of mental blocks to creativity and if, in self-analysis, they identified in themselves the occurrence of some blockage. Through the case study, collection, and analysis of quality-quantitative data, the article aims to demonstrate the perceptions of employees about creativity and factors that can hinder the development of this skill. Data analysis revealed that almost 80% of the participants were unaware of mental blocks and almost all of them identified in themselves the incidence of at least one mental block. This may indicate that this, perhaps, is the reason why almost half of them did not consider themselves creative. That is, they had some blockage or factor that interferes in the perception of aspects of creativity, as well as in its development. Through theoretical bibliographic analyses, the article aims to describe the concepts of creativity and the factors related to its development and importance, especially at present.La pandemia de Covid 19 trajo muchos cambios socioeconómicos en el mundo, Con el fin de adaptarse a estos cambios, hubo una creciente búsqueda de la adopción de servicios e innovaciones digitales, acelerando la revolución tecnológica. Desencadenar una demanda urgente de profesionales con altas habilidades y competencias, incluso en la cuestión de la creatividad. Pero hay bloqueos mentales y otros factores que crean percepciones erróneas sobre la creatividad y dificultan su desarrollo. Se llevó a cabo un proyecto, con metodología de investigación-acción, con empleados de la empresa Encopel Papelaria que buscaba analizar si los participantes se consideraban creativos o no. Si eran conscientes de la existencia de los bloqueos mentales de la creatividad y si, en el autoanálisis, identificaban en sí mismos la ocurrencia de algún bloqueo. A través del estudio de caso, la recopilación y el análisis de datos cualitativos-cuantitativos, el artículo tiene como objetivo demostrar las percepciones de los empleados sobre la creatividad y los factores que pueden perjudicar el desarrollo de esta capacidad. El análisis de los datos reveló que casi el 80% de los participantes desconocían los bloqueos mentales y casi todos identificaron la incidencia de al menos un bloqueo mental. Lo que se puede notar es que esta puede ser la razón por la cual casi la mitad de ellos no se consideraban creativos. Es decir, tenían algún bloqueo o factor que interfiere en la percepción de aspectos de la creatividad, así como en su desarrollo. A través de análisis teóricos bibliográficos.A pandemia de Covid 19 trouxe muitas mudanças socioeconômicas no mundo, A fim de se adequar às essas mudanças, notou-se uma crescente busca da adoção de serviços digitais e inovações, acelerando a revolução tecnológica. Desencadeando uma demanda urgente por profissionais com alta habilidades e competências, inclusive no quesito criatividade. Porém existem bloqueios mentais, e outros fatores que criam percepções equivocadas sobre a criatividade e dificultam o seu desenvolvimento. Foi realizado um projeto, com metodologia pesquisa-ação, com os colaboradores da empresa Encopel Papelaria que buscou analisar se os participantes se consideravam criativos ou não. Se tinham consciência da existência dos bloqueios mentais da criatividade e se, em auto análise, identificavam em si a ocorrência de algum bloqueio. Através do estudo de caso, coleta e análises de dados quali-quantitativos, o artigo visa demonstrar as percepções dos colaboradores sobre a criatividade e fatores que podem prejudicar o desenvolvimento dessa habilidade. A análise dos dados revelou que quase 80% dos participantes desconheciam os bloqueios mentais e quase todos eles identificaram em si a incidência de, pelo menos, um bloqueio mental. O que pode denotar que essa, talvez, seja a razão pela qual quase metade deles não se consideravam criativos. Ou seja, possuíam algum bloqueio ou fator que interfere na percepção de aspectos da criatividade, bem como no seu desenvolvimento. Através das análises teóricas bibliográficas, o artigo visa descrever os conceitos de criatividade e os fatores relacionados ao seu desenvolvimento e importância, principalmente no momento atual

A CRIATIVIDADE E OS BLOQUEIOS MENTAIS - ESTUDO DE CASO: AS PERCEPÇÕES DOS COLABORADORES DA ENCOPEL PAPELARIA SOBRE ASPECTOS DA CRIATIVIDADE

The Covid 19 pandemic has brought many socioeconomic changes in the world. To adapt to these changes, there has been a growing search for the adoption of digital services and innovations, accelerating the technological revolution. Unleashing an urgent demand for professionals with high skills and competencies, including in terms of creativity. However, there are mental blocks and other factors that create misperceptions about creativity and hinder its development. A project was carried out, with action research methodology, with the employees of the company Encopel Papelaria, which sought to analyze whether the participants considered themselves creative or not. If they were aware of the existence of mental blocks to creativity and if, in self-analysis, they identified in themselves the occurrence of some blockage. Through the case study, collection, and analysis of quality-quantitative data, the article aims to demonstrate the perceptions of employees about creativity and factors that can hinder the development of this skill. Data analysis revealed that almost 80% of the participants were unaware of mental blocks and almost all of them identified in themselves the incidence of at least one mental block. This may indicate that this, perhaps, is the reason why almost half of them did not consider themselves creative. That is, they had some blockage or factor that interferes in the perception of aspects of creativity, as well as in its development. Through theoretical bibliographic analyses, the article aims to describe the concepts of creativity and the factors related to its development and importance, especially at present.La pandemia de Covid 19 trajo muchos cambios socioeconómicos en el mundo, Con el fin de adaptarse a estos cambios, hubo una creciente búsqueda de la adopción de servicios e innovaciones digitales, acelerando la revolución tecnológica. Desencadenar una demanda urgente de profesionales con altas habilidades y competencias, incluso en la cuestión de la creatividad. Pero hay bloqueos mentales y otros factores que crean percepciones erróneas sobre la creatividad y dificultan su desarrollo. Se llevó a cabo un proyecto, con metodología de investigación-acción, con empleados de la empresa Encopel Papelaria que buscaba analizar si los participantes se consideraban creativos o no. Si eran conscientes de la existencia de los bloqueos mentales de la creatividad y si, en el autoanálisis, identificaban en sí mismos la ocurrencia de algún bloqueo. A través del estudio de caso, la recopilación y el análisis de datos cualitativos-cuantitativos, el artículo tiene como objetivo demostrar las percepciones de los empleados sobre la creatividad y los factores que pueden perjudicar el desarrollo de esta capacidad. El análisis de los datos reveló que casi el 80% de los participantes desconocían los bloqueos mentales y casi todos identificaron la incidencia de al menos un bloqueo mental. Lo que se puede notar es que esta puede ser la razón por la cual casi la mitad de ellos no se consideraban creativos. Es decir, tenían algún bloqueo o factor que interfiere en la percepción de aspectos de la creatividad, así como en su desarrollo. A través de análisis teóricos bibliográficos.A pandemia de Covid 19 trouxe muitas mudanças socioeconômicas no mundo, A fim de se adequar às essas mudanças, notou-se uma crescente busca da adoção de serviços digitais e inovações, acelerando a revolução tecnológica. Desencadeando uma demanda urgente por profissionais com alta habilidades e competências, inclusive no quesito criatividade. Porém existem bloqueios mentais, e outros fatores que criam percepções equivocadas sobre a criatividade e dificultam o seu desenvolvimento. Foi realizado um projeto, com metodologia pesquisa-ação, com os colaboradores da empresa Encopel Papelaria que buscou analisar se os participantes se consideravam criativos ou não. Se tinham consciência da existência dos bloqueios mentais da criatividade e se, em auto análise, identificavam em si a ocorrência de algum bloqueio. Através do estudo de caso, coleta e análises de dados quali-quantitativos, o artigo visa demonstrar as percepções dos colaboradores sobre a criatividade e fatores que podem prejudicar o desenvolvimento dessa habilidade. A análise dos dados revelou que quase 80% dos participantes desconheciam os bloqueios mentais e quase todos eles identificaram em si a incidência de, pelo menos, um bloqueio mental. O que pode denotar que essa, talvez, seja a razão pela qual quase metade deles não se consideravam criativos. Ou seja, possuíam algum bloqueio ou fator que interfere na percepção de aspectos da criatividade, bem como no seu desenvolvimento. Através das análises teóricas bibliográficas, o artigo visa descrever os conceitos de criatividade e os fatores relacionados ao seu desenvolvimento e importância, principalmente no momento atual

Absence of Metabolic Cross-correction in Tay-Sachs Cells: IMPLICATIONS FOR GENE THERAPY

We have investigated the ability of a receptor-mediated gene transfer strategy (cross-correction) to restore ganglioside metabolism in fibroblasts from Tay-Sachs (TS) patients in vitro. TS disease is a GM2 gangliosidosis attributed to the deficiency of the lysosomal enzyme beta-hexosaminidase A (HexA) (beta-N-acetylhexosaminidase, EC ). The hypothesis is that transduced cells overexpressing and secreting large amounts of the enzyme would lead to a measurable activity in defective cells via a secretion-recapture mechanism. We transduced NIH3T3 murine fibroblasts with the LalphaHexTN retroviral vector carrying the cDNA encoding for the human Hex alpha-subunit. The Hex activity in the medium from transduced cells was approximately 10-fold higher (up to 75 milliunits) than observed in non-transduced cells. TS cells were cultured for 72 h in the presence of the cell medium derived from the transduced NIH3T3 cells, and they were analyzed for the presence and catalytic activity of the enzyme. Although TS cells were able to efficiently uptake a large amount of the soluble enzyme, the enzyme failed to reach the lysosomes in a sufficient quantity to hydrolyze the GM2 ganglioside to GM3 ganglioside. Thus, our results showed that delivery of the therapeutic HexA was not sufficient to correct the phenotype of TS cells

Coronaviridae and SARS-associated Coronavirus Strain HSR1

During the recent severe acute respiratory (SARS) outbreak, the etiologic agent was identified as a new coronavirus (CoV). We have isolated a SARS-associated CoV (SARS-CoV) strain by injecting Vero cells with a sputum specimen from an Italian patient affected by a severe pneumonia; the patient traveled from Vietnam to Italy in March 2003. Ultrastructural analysis of infected Vero cells showed the virions within cell vesicles and around the cell membrane. The full-length viral genome sequence was similar to those derived from the Hong-Kong Hotel M isolate. By using both real-time reverse transcription–polymerase chain reaction TaqMan assay and an infectivity plaque assay, we determined that approximately 360 viral genomes were required to generate a PFU. In addition, heparin (100 μg/mL) inhibited infection of Vero cells by 50%. Overall, the molecular and biologic characteristics of the strain HSR1 provide evidence that SARS-CoV forms a fourth genetic coronavirus group with distinct genomic and biologic features

512 the cytokine release syndrome crucially contributes to the anti leukemic effects of cd44v6 car t cells

Background: Despite the remarkable clinical results of CD19 CAR-T cells in B-cell leukemias, their long-term efficacy is limited by the emergence of CD19-loss escape variants. Moreover, whether the cytokine release syndrome (CRS) is necessary for durable remissions is a matter of debate. Currently available xenograft models in NSG mice are not suited for studying the antitumor effects of CAR-T cells beyond 3-4 weeks, because of xenograft-versus-host disease (X-GVHD). Moreover, since NSG mice lack functional myeloid cells, the CRS does not develop. Aim: To verify whether the CRS contributes to the antileukemic effects of CAR in an innovative xenotolerant mouse model.Results: NSG mice triple transgenic for human IL-3, GM-CSF and SCF (NSG-3GS) were sub-lethally irradiated and injected intra-liver with human HSCs soon after birth, enabling an accelerated and better balanced lympho-hematopoietic reconstitution compared with NSG mice. Reconstituting human T cells were single CD4+/CD8+ T cells, representing all memory sub-populations. After ex vivo isolation and activation with CD3/CD28-beads and IL-7/IL-15, NSG-3GS T cells were transduced with a CD44v6 CAR, retaining an early-differentiated (stem-cell/central-memory) phenotype and full antitumor functionality against acute myeloid leukemia (AML). NSG-3GS-derived CD44v6 CAR T cells were subsequently infused in tumor-bearing secondary recipients previously humanized with autologous HSCs. CAR-T cells persisted in vivo for at least 6 months and mediated durable leukemia remissions (P<0.001 vs controls) in the absence of X-GVHD. Tumor clearance associated with an acute malaise syndrome, characterized by high fevers and a surge in human IL-6 levels, which was lethal in 30% of the mice. Differently from CD19 CAR-T cells, the CRS by CD44v6 CAR-T cells was significantly anticipated (3 vs 8 days), coinciding with human CD44v6+ monocyte depletion. In humanized mice, previous myeloid-cell depletion by clodronate administration completely prevented this syndrome, but associated with late leukemia relapses. Conversely, mice developing the CRS entered a state of durable and profound remission, as demonstrated by prolonged observation times and secondary transplantation. Conclusions: By using an innovative xenotolerant mouse model, we have demonstrated that the CRS is needed for sustained antileukemic effects by CD44v6 CAR-T cells

Lentiviral vector-mediated gene transfer in T cells from Wiskott-Aldrich syndrome patients leads to functional correction

Wiskott–Aldrich syndrome (WAS) is an X-linked primary immunodeficiency with a median survival below the age of 20 due to infections, severe hemorrhage, and lymphomas. Transplantation of hematopoietic stem cells from HLA-identical sibling donors is a resolutive treatment, but is available for a minority of patients. Transplantation of genetically corrected autologous hematopoietic stem cells or T cells could represent an alternative treatment applicable to all patients. We investigated whether WAS gene transfer with MMLV-based oncoretroviral and HIV-based lentiviral vectors could restore normal functions of patients' T cells. T cells transduced either with lentiviral vectors expressing the WAS protein (WASP) from the ubiquitous PGK promoter or the tissue-specific WASP promoter or with an oncoretroviral vector expressing WASP from the LTR, reached normal levels of WASP with correction of functional defects, including proliferation, IL-2 production, and lipid raft upregulation. Lentiviral vectors transduced T cells from WAS patients at higher rates, compared to oncoretroviral vectors, and efficiently transduced both activated and naive WAS T cells. Furthermore, a selective growth advantage of T cells corrected with the lentiviral vectors was demonstrated. The observation that lentiviral vector-mediated gene transfer results in correction of T cell defects in vitro supports their application for gene therapy in WAS patients

130 purification of large scale mrna encoding zfn nucleases by dhplc technology

A novel strategy of targeted gene correction of the interleukin-2 receptor common gamma chain (IL2RG) gene for the treatment of X-linked Severe Combined Immunodeficiency (SCID-X1) is achieved by the combination of a pair of IL2RG-specific Zinc Finger Nucleases (ZFN) and the correct-gene template DNA delivered by integration-defective lentiviral vector (IDLV).The transient expression of the ZFN pair targeting the disease-causing gene is obtained by the electroporation of the two corresponding mRNAs, produced by in vitro transcription starting from plasmid DNA template. A major limitation of the mRNA transcribed in vitro is the presence of residual contaminants such as short RNAs and double stranded (ds)RNAs that may affect the function and spectrophotometric quantification of the product hampering therefore the delivery of high quality and precise amount of mRNA to target cells. Moreover, dsRNA contaminants represent a possible risk in terms of immunogenicity of the product, leading to activation of unwanted innate immune response with consequent reduction/abrogation of mRNA translation as well as potential alteration of the properties of the transfected cells. To improve nuclease expression while decreasing cellular innate response to mRNA transfection we combined different strategies: (i) inclusion of UTRs and polyA tails in the DNA template used for mRNA production; (ii) use of modified nucleotides during mRNA production and (iii) purification of the mRNAs by dHPLC with a reverse phase column made of non-porous matrix consisting of polystyrene-divinylbenzene copolymer beads alkylated with C-18 chains (Transgenomic, LTD.). In particular, the purification of in vitro transcribed mRNAs by means of dHPLC has been shown to strongly improve the translation of mRNA and significantly reduce the contaminant presence thus preventing innate immunity and eventually increasing modified cells persistence in vivo. We have developed feasible and reproducible, small and large scale mRNA production and downstream purification processes of the ZFN pairs obtaining accurate RNA quantification and reduced risk of immunogenicity. The full process achieved a 60% yield, loading with a 500µg RNA for each run with a single clean chromatographic peak. Furthermore, the level of residual organic solvent (i.e. Acetonitrile) used in the purification process is compatible with that applicable into clinic. The highly translatable non-immunogenic dHPLC-purified mRNA can be delivered without toxicity and represents a powerful and safe tool for the application of gene therapy protocols

Molecular and Immunological Characterization of Staphylococcus aureus in Pediatric Atopic Dermatitis: Implications for Prophylaxis and Clinical Management

S. aureus represents a critical cofactor in atopic dermatitis (AD). In this paper, the prevalence of S. aureus infection/colonization was evaluated in 117 children as well as in their cohabitants, in order to assess the value of S. aureus characterization in predicting disease onset and severity and in providing indications for prophylaxis. Results showed that children with AD as well as their cohabitants had a significantly greater incidence of S. aureus infection/colonization as compared to controls. The genetic characterization showed a virtual identity of the bacteria strains collected at different sites of the patients with those found in the cohabitants, suggesting both a direct transmission between the nasal reservoir and the lesions in the same atopic subject and a risk for reinfection within family cohabitants. These data stress the need of preliminary laboratory assessment and posttherapy control in both AD patients and their close contacts for effective S. aureus eradication