Use Of Lentiviral Vectors Research Articles

Vectors in stealth mode

Gene Therapy Gene therapy using adeno-associated virus (AAV) vectors has shown safety and efficacy in hemophilia. However, AAV vectors have limitations hindering their efficacy in some patients. The use of lentiviral vectors (LVs) has been explored as a possible alternative; however, preclinical data reported low transduction efficacy, possibly owing to fast clearance by phagocytes. Milani et al. developed a shielded LV able to escape phagocytosis by expressing the phagocytosis inhibitor CD47 on its surface. Upon intravenous administration in monkeys, the LVs showed high transduction efficacy without signs of toxicity. Thus, LV-mediated gene therapy might be an effective strategy for treating hemophilia. Sci. Transl. Med. 11 , eaav7325 (2019).

Genetic Modification of Human Primary Keratinocytes by Lentiviral Vectors.

Keratinocytes are hard to transfect. Viral vectors are a good alternative to genetically modify primary keratinocytes. A classical method is the use of retroviral vectors by co-culture of keratinocytes with virus-producer cells. This method is efficient in high-calcium conditions with feeder cells. However, sometimes co-culture is not possible and is more laborious as producer cells need to be replaced by feeder cells. Our solution is the use of lentiviral vectors, far more efficient as supernatant on keratinocytes. In this chapter we describe improved detailed protocols for stable genetic modification of human primary keratinocytes of the skin or head and neck, in both low- and high-calcium conditions by lentiviral vectors.

Multiple Inhibitory Factors Act in the Late Phase of HIV-1 Replication: a Systematic Review of the Literature.

The use of lentiviral vectors for therapeutic purposes has shown promising results in clinical trials. The ability to produce a clinical-grade vector at high yields remains a critical issue. One possible obstacle could be cellular factors known to inhibit human immunodeficiency virus (HIV). To date, five HIV restriction factors have been identified, although it is likely that more factors are involved in the complex HIV-cell interaction. Inhibitory factors that have an adverse effect but do not abolish virus production are much less well described. Therefore, a gap exists in the knowledge of inhibitory factors acting late in the HIV life cycle (from transcription to infection of a new cell), which are relevant to the lentiviral vector production process. The objective was to review the HIV literature to identify cellular factors previously implicated as inhibitors of the late stages of lentivirus production. A search for publications was conducted on MEDLINE via the PubMed interface, using the keyword sequence "HIV restriction factor" or "HIV restriction" or "inhibit HIV" or "repress HIV" or "restrict HIV" or "suppress HIV" or "block HIV," with a publication date up to 31 December 2016. Cited papers from the identified records were investigated, and additional database searches were performed. A total of 260 candidate inhibitory factors were identified. These factors have been identified in the literature as having a negative impact on HIV replication. This study identified hundreds of candidate inhibitory factors for which the impact of modulating their expression in lentiviral vector production could be beneficial.

Optimized lentiviral transduction of human amniotic mesenchymal stromal cells

Mesenchymal stromal cells are excellent candidates for regenerative medicine since they are multipotent, easy to isolate, can be expanded to obtain clinically relevant numbers and are immunoprivileged. Stable genetic modification with viral vectors can improve mesenchymal stromal cell function and enhance their therapeutic potential. However, standard viral vectors achieve sub-optimal transduction efficiency with a single infection. On the other hand, multiple transduction cycles or antibiotic-based selection methods may alter the stem cell phenotype. We hypothesized that the use of lentiviral vectors containing specific regulatory sequences may result in improved transduction efficiency. Thus, we compared two types of third generation lentiviral vectors, one of which, the pLenti7.3 vector, contains the optimized sequences for Polypurine Tract and Woodchuck Post-transcriptional Regulatory Element. We demonstrated that with the pLenti7.3 it is possible to efficiently transduce human mesenchymal stromal cells with a single transduction cycle. Additionally, we successfully showed that by using the pLenti7.3 vector it is possible to efficiently over-express different growth factors, particularly relevant for cardiac protection and differentiation, in human mesenchymal stromal cells.

In Vitro Erythroid Differentiation and Lentiviral Knockdown in Human CD34+ Cells from Umbilical Cord Blood.

Human umbilical cord blood is a rich source of hematopoietic stem and progenitor cells. CD34+ cells in umbilical cord blood are more primitive than those in peripheral blood or bone marrow, and can proliferate at a high rate and differentiate into multiple cell types. In this protocol, a dependable method is described for the isolation of fetal CD34+ cells from umbilical cord blood and expanding these cells in culture. The cells can then be in vitro differentiated along an erythroid pathway, while simultaneously performing knockdown of a gene of choice. The use of lentiviral vectors that express small hairpin RNA (shRNA) is an efficient method to downregulate genes. Flow cytometric analyses are used to enrich for erythroid cells. Using these methods, one can generate in vitro differentiated cells to use for quantitative reverse transcriptase PCR and other purposes.

Measles virus envelope pseudotyped lentiviral vectors transduce quiescent human HSCs at an efficiency without precedent

AbstractHematopoietic stem cell (HSC)–based gene therapy trials are now moving toward the use of lentiviral vectors (LVs) with success. However, one challenge in the field remains: efficient transduction of HSCs without compromising their stem cell potential. Here we showed that measles virus glycoprotein–displaying LVs (hemagglutinin and fusion protein LVs [H/F-LVs]) were capable of transducing 100% of early-acting cytokine-stimulated human CD34+ (hCD34+) progenitor cells upon a single application. Strikingly, these H/F-LVs also allowed transduction of up to 70% of nonstimulated quiescent hCD34+ cells, whereas conventional vesicular stomatitis virus G (VSV-G)–LVs reached 5% at the most with H/F-LV entry occurring exclusively through the CD46 complement receptor. Importantly, reconstitution of NOD/SCIDγc−/− (NSG) mice with H/F-LV transduced prestimulated or resting hCD34+ cells confirmed these high transduction levels in all myeloid and lymphoid lineages. Remarkably, for resting CD34+ cells, secondary recipients exhibited increasing transduction levels of up to 100%, emphasizing that H/F-LVs efficiently gene-marked HSCs in the resting state. Because H/F-LVs promoted ex vivo gene modification of minimally manipulated CD34+ progenitors that maintained stemness, we assessed their applicability in Fanconi anemia, a bone marrow (BM) failure with chromosomal fragility. Notably, only H/F-LVs efficiently gene-corrected minimally stimulated hCD34+ cells in unfractionated BM from these patients. These H/F-LVs improved HSC gene delivery in the absence of cytokine stimulation while maintaining their stem cell potential. Thus, H/F-LVs will facilitate future clinical applications requiring HSC gene modification, including BM failure syndromes, for which treatment has been very challenging up to now.

CRISPR/Cas9-Mediated Knockin Application in Cell Therapy: A Non-viral Procedure for Bystander Treatment of Glioma in Mice.

The use of non-viral procedures, together with CRISPR/Cas9 genome-editing technology, allows the insertion of single-copy therapeutic genes at pre-determined genomic sites, overcoming safety limitations resulting from random gene insertions of viral vectors with potential for genome damage. In this study, we demonstrate that combination of non-viral gene delivery and CRISPR/Cas9-mediated knockin via homology-directed repair can replace the use of viral vectors for the generation of genetically modified therapeutic cells. We custom-modified human adipose mesenchymal stem cells (hAMSCs), using electroporation as a transfection method and CRISPR/Cas9-mediated knockin for the introduction and stable expression of a 3 kb DNA fragment including the eGFP (selectable marker) and a variant of the herpes simplex virus 1 thymidine kinase genes (therapeutic gene), under the control of the human elongation factor 1 alpha promoter in exon 5 of the endogenous thymidine kinase 2 gene. Using a U87 glioma model in SCID mice, we show that the therapeutic capacity of the new CRISPR/Cas9-engineered hAMSCs is equivalent to that of therapeutic hAMSCs generated by introduction of the same therapeutic gene by transduction with a lentiviral vector previously published by our group. This strategy should be of general use to other applications requiring genetic modification of therapeutic cells.

Abstract 4561: Non-integrative lentiviral particles for immunotherapy: RNA delivery to drive tumoral antigen presentation in a safe and efficient way

Abstract Safe and efficient cancer therapies using adoptive transfer of engineered cells are very promising but very challenging approaches. Opportunities to improve gene transfer into primary cells involve a better design of the vectors used. Such improvements must lead to an increase of the efficiency of gene expression, the cell phenotype preservation and the increase of the gene number delivered. The use of lentiviral vectors has largely increased in clinical protocols over the past few years but safety concerns have slowed down this progression. Actually, the permanent genetic modification remains a focus of significant regulatory oversight. On another side, mRNA delivery is a versatile, flexible, and safe mean for protein therapies, but existing techniques, such as chemical or electroporation-based transfection protocols, are known to induce cell toxicity and phenotype modifications of the target cells. Here, we present an innovative tool, named LentiFlash, allowing RNA delivery into target cells without any genomic scar. This tool combines RNA delivery, providing the best expression system to get efficient and transient expression, and lentiviral delivery which exhibits the best efficiency of entry into primary and stem cells. The RNA encapsidation is mediated via an RNA/protein interaction using properties of the MS2 bacteriophage. This new vector breaks with all existing systems. The resulting lentiviral particle is able to efficiently deliver non-viral coding or non-coding RNA into the cytoplasm of any cell type. These LentiFlash particles, are able to transduce delicate primary cells, such as dendritic cells, T cells and hematopoietic stem cells. They show an efficient, fast and transient expression of protein and RNA, such as antigenic peptides or genome editing tools, with no cell phenotype modification. Here we show a proof of concept on primary murine dendritic cells transduction with LentiFlash particles, successfully used to deliver several tumoral antigens. As a result, these dendritic cells are able to present the antigens to the immune system which in turn can efficiently fight a tumor development into wild type mice. This new delivery system gives the opportunity for multiple antigens co-expression to enhance immune responses, avoiding tumor relapse. Such multiple co-expressions into immune cells are expected to mimic the innate and adaptive immune responses. This transient RNA delivery mediated by LentiFlash is a powerful tool to induce an efficient gene delivery. The possibility to express multiple genes at once in the target cells is an attractive therapeutic perspective. The absence of any viral sequence avoids any integration event, an important safety consideration for human use. Finally, another advantage of the LentiFlash system is its ability to utilize lentiviral production platforms already validated in clinical settings. Citation Format: Pascale Bouillé, Christine Duthoit, Nicolas Martin, Lucille Lamouroux, Jean-Christophe Pages. Non-integrative lentiviral particles for immunotherapy: RNA delivery to drive tumoral antigen presentation in a safe and efficient way [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4561. doi:10.1158/1538-7445.AM2017-4561

Gene Therapy in Fanconi Anemia: A Matter of Time, Safety and Gene Transfer Tool Efficiency.

Fanconi anemia (FA) is a rare genetic syndrome characterized by progressive marrow failure. Gene therapy by infusion of FA-corrected autologous hematopoietic stem cells (HSCs) may offer a potential cure since it is a monogenetic disease with mutations in the FANC genes, coding for DNA repair enzymes [1]. However, the collection of hCD34+-cells in FA patients implies particular challenges because of the reduced numbers of progenitor cells present in their bone marrow (BM) [2] or mobilized peripheral blood [3-5]. In addition, the FA genetic defect fragilizes the HSCs [6]. These particular features might explain why the first clinical trials using murine leukemia virus derived retroviral vectors conducted for FA failed to show engraftment of corrected cells. The gene therapy field is now moving towards the use of lentiviral vectors (LVs) evidenced by recent succesful clinical trials for the treatment of patients suffering from adrenoleukodystrophy (ALD) [7], β-thalassemia [8], metachromatic leukodystrophy [9] and Wiskott-Aldrich syndrome [10]. LV trials for X-linked severe combined immunodificiency and Fanconi anemia (FA) defects were recently initiated [11, 12]. Fifteen years of preclinical studies using different FA mouse models and in vitro research allowed us to find the weak points in the in vitro culture and transduction conditions, which most probably led to the initial failure of FA HSC gene therapy. In this review, we will focus on the different obstacles, unique to FA gene therapy, and how they have been overcome through the development of optimized protocols for FA HSC culture and transduction and the engineering of new gene transfer tools for FA HSCs. These combined advances in the field hopefully will allow the correction of the FA hematological defect in the near future.

Synthetic biology and occupational risk

ABSTRACTSynthetic biology is an emerging interdisciplinary field of biotechnology that involves applying the principles of engineering and chemical design to biological systems. Biosafety professionals have done an excellent job in addressing research laboratory safety as synthetic biology and gene editing have emerged from the larger field of biotechnology. Despite these efforts, risks posed by synthetic biology are of increasing concern as research procedures scale up to industrial processes in the larger bioeconomy. A greater number and variety of workers will be exposed to commercial synthetic biology risks in the future, including risks to a variety of workers from the use of lentiviral vectors as gene transfer devices. There is a need to review and enhance current protection measures in the field of synthetic biology, whether in experimental laboratories where new advances are being researched, in health care settings where treatments using viral vectors as gene delivery systems are increasingly being used, or in the industrial bioeconomy. Enhanced worker protection measures should include increased injury and illness surveillance of the synthetic biology workforce; proactive risk assessment and management of synthetic biology products; research on the relative effectiveness of extrinsic and intrinsic biocontainment methods; specific safety guidance for synthetic biology industrial processes; determination of appropriate medical mitigation measures for lentiviral vector exposure incidents; and greater awareness and involvement in synthetic biology safety by the general occupational safety and health community as well as by government occupational safety and health research and regulatory agencies.

Ex vivo Generation of Genetically Modified Macrophages from Human Induced Pluripotent Stem Cells

Background: Pluripotent stem cells, including induced pluripotent stem cells (iPSCs), have the capacity to differentiate towards all three germ layers and have been highlighted as an attractive cell source for the field of regenerative medicine. Thus, stable expression of therapeutic transgenes in iPSCs, as well as thereof derived progeny of hematopoietic lineage, may lay the foundation for innovative cell replacement therapies. Methods: We have utilized human iPSC lines genetically modified by lentiviral vector technology or targeted integration of reporter genes to evaluate transgene expression during hematopoietic specification and differentiation towards macrophages. Results: Use of lentiviral vectors equipped with an ubiquitous chromatin opening element (CBX3-UCOE) as well as zinc finger nuclease-mediated targeting of an expression cassette into the human adeno-associated virus integration site 1 (AAVS1) safe harbor resulted in stable transgene expression in iPSCs. When iPSCs were differentiated along the myeloid pathway into macrophages, both strategies yielded sustained transgene expression during the hematopoietic specification process including mature CD14+ and CD11b+ macrophages. Conclusion: Combination of human iPSC technology with either lentiviral vector technology or designer nuclease-based genome editing allows for the generation of transgenic iPSC-derived macrophages with stable transgene expression which may be useful for novel cell and gene replacement therapies.

Improvement ofDe NovoCholesterol Biosynthesis Efficiently Promotes the Production of Human Immunodeficiency Virus Type 1-Derived Lentiviral Vectors

The use of lentiviral vectors (LVs) for gene transfer in research, technological, or clinical applications requires the production of large amounts of vector. Mass production of clinical-grade LVs remains a challenge and limits certain perspectives for therapeutic use. Some improvements in LV production protocols have been possible by acting on multiple steps of the production process. The addition of animal-derived cholesterol to the culture medium of producer cells is known to increase the infectivity of LVs. To avoid the use of this animal-derived product in clinical settings, an alternative approach is to increase de novo the production of cholesterol by overexpressing a crucial cholesterogenic enzyme, namely, 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGCR). This project evaluates the impact of such an approach on the production, infectivity, and stability of LVs. We demonstrated that the overexpression of human HMGCR isoform 1 (hHMGCR1) in LV producer cells efficiently increased de novo cholesterol biosynthesis and enhanced by 2- to 3-fold the physical and infectious titers of LVs. We also observed that LVs produced in hHMGCR1-overexpressing cells were comparable in stability to LVs produced under classical conditions and were capable of transducing human CD34+ hematopoietic stem/progenitor cells efficiently. Interestingly, we also showed that LV production in the absence of fetal calf serum (FCS) but under hHMGCR1-overexpressing conditions allowed a viral production yield comparable to that achieved under classical conditions in high FCS content, leading the way to the establishment of new LV production protocols on adherent cells without serum.

Optimal Lentivirus Production and Cell Culture Conditions Necessary to Successfully Transduce Primary Human Bronchial Epithelial Cells.

In vitro culture of primary human bronchial epithelial (HBE) cells using air-liquid interface conditions provides a useful model to study the processes of airway cell differentiation and function. In the past few years, the use of lentiviral vectors for transgene delivery became common practice. While there are reports of transduction of fully differentiated airway epithelial cells with certain non-HIV pseudo-typed lentiviruses, the overall transduction efficiency is usually less than 15%. The protocol presented here provides a reliable and efficient method to produce lentiviruses and to transduce primary human bronchial epithelial cells. Using undifferentiated bronchial epithelial cells, transduction in bronchial epithelial growth media, while the cells attach, with a multiplicity of infection factor of 4 provides efficiencies close to 100%. This protocol describes, step-by-step, the preparation and concentration of high-titer lentiviral vectors and the transduction process. It discusses the experiments that determined the optimal culture conditions to achieve highly efficient transductions of primary human bronchial epithelial cells.

Finding Expandable Induced Cardiovascular Progenitor Cells.

Expandable Cardiovascular Progenitor Cells Reprogrammed From Fibroblasts Zhang et al Cell Stem Cell . 2016;18:368–381. Lineage Reprogramming of Fibroblasts Into Proliferative Induced Cardiac Progenitor Cells by Defined Factors Lalit et al Cell Stem Cell . 2016;18:354–367. Cardiovascular progenitor cells (CPCs) are a promising cell source for cardiac regenerative therapy because of their ability for self-renewal and differentiation into various cardiovascular cell types beneficial for myocardial repair: cardiomyocytes, smooth muscle cells, and endothelial cells. Previous evaluations of exogenously derived CPCs have focused mainly on their capacity for trilineage differentiation rather than self-renewal because of the lack of an effective protocol to maintain and expand CPCs long-term in culture. In a recent issue of Cell Stem Cell , 2 groups of investigators independently reported their success in isolating, maintaining, and expanding mouse CPCs in culture for >18 to 20 passages (1010- to 1015-fold expansion), with faithful preservation of progenitor phenotype and ability for trilineage-restricted differentiation in both cell culture and mouse models of myocardial infarction (MI). This commentary will discuss the unique findings of these 2 studies, highlight the strengths and weaknesses of each CPC derivation/expansion technique, and propose additional steps necessary to accelerate their clinical translation. The past 2 decades have witnessed some of the most dramatic developments in the field of cardiac regeneration. Central to numerous controversies is the debate on whether certain stem cell types can differentiate into functional cardiomyocytes at a clinically relevant efficiency after implantation for repair of MI. Bone marrow–derived hematopoietic stem cells1,2 and c-kit+ resident cardiac stem cells3 are 2 notable cell types at the center of contention. Although many adult stem cells can still confer modest therapeutic benefits via paracrine effects, the search for a bona fide progenitor cell type that can generate a significant …

Lentiviral Vectors for the Engineering of Implantable Cells Secreting Recombinant Antibodies.

The implantation of genetically modified cells is considered for the chronic delivery of therapeutic recombinant proteins in vivo. In the context of gene therapy, the genetic engineering of cells faces two main challenges. First, it is critical to generate expandable cell sources, which can maintain stable high productivity of the recombinant protein of interest over time, both in culture and after transplantation. In addition, gene transfer techniques need to be developed to engineer cells synthetizing complex polypeptides, such as recombinant monoclonal antibodies, to broaden the range of potential therapeutic applications. Here, we provide a workflow for the use of lentiviral vectors as a flexible tool to generate antibody-producing cells. In particular, lentiviral vectors can be used to genetically engineer the cell types compatible with encapsulation devices protecting the implanted cells from the host immune system. Detailed methods are provided for the design and production of lentiviral vectors, optimization of cell transduction, as well as for the quantification and quality control of the produced recombinant antibody.

Stable Delivery of CCR5-Directed shRNA into Human Primary Peripheral Blood Mononuclear Cells and Hematopoietic Stem/Progenitor Cells via a Lentiviral Vector.

RNAi is a powerful tool to achieve suppression of a specific gene expression and therefore it has tremendous potential for gene therapy applications. A number of vector systems have been developed to express short-hairpin RNAs (shRNAs) to produce siRNAs within mammalian T-cells, primary hematopoietic stem/progenitor cells (HSPC), human peripheral blood mononuclear cells, and in animal model systems. Among these, vectors based on lentivirus backbones have significantly transformed our ability to transfer shRNAs into nondividing cells, such as HSPC, resulting in high transduction efficiencies. However, delivery and long-term expression of shRNAs should be carefully optimized for efficient knock down of target gene without causing cytotoxicity in mammalian cells. Here, we describe our protocols for the development of shRNA against a major HIV co-receptor/chemokine receptor CCR5 and the use of lentiviral vectors for stable shRNA delivery and expression in primary human PBMC and HSPC.

B-108 Specific HIV integration sites are linked to clonal expansion and persistence of infected cells

HIV integrates its DNA into many sites in the host genome; thus integration sites can be used as markers to identify clonally expanded cells. We identified the integration sites in PBMCs and CD4+ T cells from patients and used these data to show that there is extensive clonal expansion of infected cells. We have started to determine the sequences of viral DNAs in clonally expanded cells; these sequences are being used to study the structure and expression of the proviruses present in expanded cells. We have identified >2500 integration sites in PBMCs and CD4+ cells from infected individuals on combination anti-retroviral therapy (cART). About 40% of the integrations were in clonally expanded cells. In one patient, more than 50% of the infected cells were from a single clone; some of the expanded clones persisted for more than 10 years. There were multiple independent integrations in the same orientation as the gene in 2 introns of the MKL2 and BACH2 genes; many of these integrations were in clonally expanded cells. Both BACH2 and MKL2 are involved in regulating the growth of cells. DNA rearrangements involving these genes have been found in human tumors. There was no evidence for integration in one orientation, or in specific introns, in either of these genes in large libraries prepared by infecting stimulated or unstimulated PBMCs, CD34+ cells, or HeLa cells that were infected in culture. There were, in patients, multiple independent integrations in a number of other growth related genes, some of which were associated with clonal expansion of the infected cells. These data show that HIV integration at certain sites can play a critical role in the expansion and persistence of HIV infected cells. In one case, we showed that the provirus in an expanded clone was responsible for producing the majority of the virus that was present in the blood of a patient on cART, showing that, in this patient, immune surveillance was not sufficient to prevent clonally expanded cells from producing virions. Additional experiments showed that this virus is replication competent, demonstrating that clonally expanded cells can carry replication competent proviruses that produce infectious virus in patients. Thus, proviruses in clonally expanded cells can be part of the reservoir that gives rise to replicating virus when therapy is interrupted. Our findings have important implications for the development and maintenance of the viral reservoir, for designing and implementing strategies to eliminate persistent HIV infection, for the use of lentiviral vectors for gene therapy in human patients, and, possibly, for the origin of some HIV-related malignancies.

SNAIL transcription factor increases the motility and invasive capacity of prostate cancer cells.

The incidence and mortality rates of prostate cancer (PCa) are increasing, and PCa is almost the second-leading cause of cancer-associated mortality in men. During tumor progression, epithelial cells decrease the number of adhesion molecules, change their polarity and position, rearrange their cytoskeleton and increase their migratory and invasive capacities. These changes are known under the concept of epithelial-mesenchymal transition (EMT). EMT is characterized by an upregulation of certain transcription factors, including SNAIL1, which represses genes that are characteristic of an epithelial phenotype, including E-cadherin, and indirectly increase the expression levels of genes, which are associated with the mesenchymal phenotype. It has been suggested that the transcription factor, SNAIL1, decreases the proliferation and increases the migratory and invasive capacities of PCa cell lines. The present study was performed using LNCaP and PC3 cell lines, in which the expression levels of SNAIL1 were increased or silenced through the use of lentiviral vectors. The expression levels of EMT markers were quantified using reverse transcription-quantitative polymerase chain reaction and western blot analysis. In addition, cell survival was analyzed using an MTS assay; cell proliferation was examined using an antibody targeting Ki-67; migration on plates with 8 µm pores to allow the passage of cells; and invasiveness was analyzed using a membrane chamber covered in dried basement membrane matrix solution. The levels of apoptosis were determined using a Caspase 3/7 assay containing a substrate modified by caspases 3 and 7. The results demonstrated that the overexpression and silencing of SNAIL1 decreased cell proliferation and survival. However, the overexpression of SNAIL1 decreased apoptosis, compared with cells with the SNAIL1-silenced cells, in which cell apoptosis increased. The migration and invasive capacities increased in the cells overexpressing SNAIL1, and decreased when SNAIL1 was silenced. In conclusion, PCa cells overexpressing SNAIL1 exhibited characteristics of an EMT phenotype, whereas the silencing of the SNAIL1 transcriptional repressor promoted an epithelial-like phenotype, with decreased migration and invasion, characteristic of mesenchymal cells.

Transgenic mouse offspring generated by ROSI.

The production of transgenic animals is an important tool for experimental and applied biology. Over the years, many approaches for the production of transgenic animals have been tried, including pronuclear microinjection, sperm-mediated gene transfer, transfection of male germ cells, somatic cell nuclear transfer and the use of lentiviral vectors. In the present study, we developed a new transgene delivery approach, and we report for the first time the production of transgenic animals by co-injection of DNA and round spermatid nuclei into non-fertilized mouse oocytes (ROSI). The transgene used was a construct containing the human CMV immediate early promoter and the enhanced GFP gene. With this procedure, 12% of the live offspring we obtained carried the transgene. This efficiency of transgenic production by ROSI was similar to the efficiency by pronuclear injection or intracytoplasmic injection of male gamete nuclei (ICSI). However, ICSI required fewer embryos to produce the same number of transgenic animals. The expression of Egfp mRNA and fluorescence of EGFP were found in the majority of the organs examined in 4 transgenic lines generated by ROSI. Tissue morphology and transgene expression were not distinguishable between transgenic animals produced by ROSI or pronuclear injection. Furthermore, our results are of particular interest because they indicate that the transgene incorporation mediated by intracytoplasmic injection of male gamete nuclei is not an exclusive property of mature sperm cell nuclei with compact chromatin but it can be accomplished with immature sperm cell nuclei with decondensed chromatin as well. The present study also provides alternative procedures for transgene delivery into embryos or reconstituted oocytes.

Advanced T-Cell Engineering for Cancer Therapy (ATECT).

Human Gene Therapy Clinical DevelopmentVol. 26, No. 2 European Commission Project SummariesOpen AccessAdvanced T-Cell Engineering for Cancer Therapy (ATECT)Published Online:18 Jun 2015https://doi.org/10.1089/humc.2015.2520AboutSectionsPDF/EPUB ToolsPermissionsDownload CitationsTrack CitationsAdd to favorites Back To Publication ShareShare onFacebookTwitterLinked InRedditEmail Contract No.: 602239; EU contribution: € 5,931,151; Total costs: € 7,780,342.20; Starting date: 01/12/2013; Duration: 60 monthsBackground and ObjectivesT-effector cells (T-cells) are immune cells whose major role is surveillance for and destruction of virally infected cells. T-cells can be readily isolated from peripheral blood of patients or normal donors and cultured in vitro. By ex vivo manipulation, T-cells that recognize a specific cancer antigen can be selected and administered as a therapeutic. This therapy, known as adoptive immunotherapy, is an established method for treating a small group of human cancers, but is limited by an inability to generate T-cells specific for a wider range of cancer antigens.More recently, gene therapy technology has provided a radically different strategy: by introducing a coding sequence for a chimeric antigen receptor (CAR) or a native T-cell receptor (TCR) using an integrating vector, we can rapidly confer any specificity to a large T-cell population in vitro. We and others have tested such “positive” engineering in T-cells as a cancer therapeutic with clear and, in some cases, dramatic results.Despite the considerable advance afforded us by genetic engineering, considerable barriers still exist, holding back the full potential of adoptive immunotherapy with engineered T-cells.At present, a bespoke autologous therapeutic product must be produced for each patient. We believe that this limits practicality and scalability, restricting CARs to an experimental therapy in few specialized centers worldwide. Further, a lag time of weeks is needed for T-cell product generation. Production may fail or may be qualitatively different between patients. Recent success with B-cell malignancy represents the “low-hanging fruits” of CAR therapy. The problem of targeting other antigens, recruiting sufficient T-cells to sites of disease, and a hostile microenvironment are considerable barriers to function in broader range of human cancers, in particular solid tumors.Recent genome engineering methods now give us an additional powerful T-cell engineering strategy to use alongside integrating vectors: Custom endonucleases targeting arbitrary sequences can result in inactivation of any desired genes—“negative” gene therapy. The transcription activator-like effector nuclease (TALEN) technology is emerging as the most robust and effective technology platform for genomic engineering. We propose to develop advanced T-cell gene therapy with a combination of “positive” and “negative” engineering strategies, which we believe will revolutionize cellular therapy. Not only can we now engineer T-cells to have any specificity, but also we can render them resistant to hostile microenvironment and immunosuppressive agents and confer new properties. In addition, this combination of engineering strategies opens up the possibility of generating universal or “off-the-shelf” T-cells that will facilitate effective commercialization of T-cell therapy.The core aim of this consortium is to test advanced T-cell therapy in early clinical studies and continue to develop it in the laboratory. We will first establish the feasibility and safety of the approach in a simple phase I study of TCR-edited, CD19 CAR donor T-cells in allogeneic hematopoietic stem cell transplantation. We will build on this study to a second phase I study, testing a more elaborate strategy to develop universal CAR T-cells that can be mass-produced and given “off the shelf.” In addition, we will perform developmental and preclinical work, which we believe is necessary to expand the scope of engineered T-cells to more challenging malignancies by exploiting gene-editing technology.Approach and MethodologyWe will implement clinical studies of CAR T-cells in patients with relapsed/refractory B-cell cancers. In addition to the use of lentiviral vectors to modify the T-cells, T-cell products will be subjected to genome editing to prevent allogeneic effects. In addition to this, more basic research and development will be applied to exploiting this concept of combining lentiviral engineering with genome editing for enhanced adoptive immunotherapy.Main FindingsNone reported yet.Expected OutcomeTwo complementary clinical studies of CD19-directed CAR therapy that together explore the potential and challenges for “off-the-shelf” CAR T-cell therapy. Development of new approaches to enhance adoptive immunotherapy by genome editing of T-cell products.Major PublicationsNone reported yet.© European Union 2015. Published by Mary Ann Liebert, Inc.FiguresReferencesRelatedDetails Volume 26Issue 2Jun 2015 InformationCopyright 2015, Mary Ann Liebert, Inc.To cite this article:Advanced T-Cell Engineering for Cancer Therapy (ATECT).Human Gene Therapy Clinical Development.Jun 2015.78-78.http://doi.org/10.1089/humc.2015.2520creative commons licensePublished in Volume: 26 Issue 2: June 18, 2015PDF download