Download or read book Engineering Lentiviral Vectors for Gene Therapy and Development of Live Cell Arrays for Dynamic Gene Expression Profiling written by Jun Tian and published by . This book was released on 2010 with total page 140 pages. Available in PDF, EPUB and Kindle. Book excerpt: With their advantages including wide tropism, high efficiency in gene transfer to both dividing and non-dividing cells, and stable and long-term expression of transgenes, lentiviral vectors have been attractively used for genes therapies and widely used for basic biomedical researches where gene transfer is required. As expression of multiple genes from the same target cell is required in such applications, this research work focused on designing novel multicistronic lentiviral vectors to develop gene therapy of diabetes through regulated insulin delivery from skin cells and live cell arrays for analyzing gene expression in a high-throughput and real-time manner.^Specifically, first, lentiviral vectors were engineered to produce a fusion protein between the furin cleavable proinsulin gene and the self-dimerization mutant of FK506-binding protein to yield bioactive insulin in keratinocytes that could be released following exogenous administration of a small organic molecule, rapamycin. The engineered keratinocytes retained normal morphology and grew similar to lentiviral-treated control cells. Epidermal keratinocytes in culture and in stratified bioengineered epidermis released insulin within 30 min after addition of rapamycin, and secretion slowed or stopped within 2-3 hours after removal of the inducer. When the cells were implanted into athymic mice that were rendered diabetic with streptozotocin, insulin was detected in the plasma within 1 hr after addition of rapamycin. Concomitantly, glucose decreased to normal levels even in diabetic animals suffering severe hyperglycemia. Repeated rapamycin administration yielded similar results.^These experiments provide proof-of-concept that insulin released from the skin in a regulatable manner can be an effective treatment for diabetic patients. Second, a lentiviral vector carrying two transcriptional units was designed to reach independent and high level dual-gene expression. The two transcriptional units were separated by polyadenylation, terminator and insulator sequences in order to eliminate promoter interference existing between the transcriptional units. With this design, the expression level of both genes was as high as that yielded from lentiviral vectors containing only a single transcriptional unit. Similar results were observed with several promoters and cell types including epidermal keratinocytes, bone marrow mesenchymal stem cells and hair follicle stem cells.^Notably, this research work also demonstrated quantitative dynamic monitoring of gene expression in primary cells with no need for selection protocols suggesting that this optimized lentivirus may be useful in high-throughput gene expression profiling studies. Last, using the novel double-promoter lentiviral vector scalable live-cell microarrays were developed to measure gene expression dynamics in a real-time and high-throughput manner. To this end, dual-promoter lentiviral vectors were prepared harboring a transcriptional regulatory element encoding for green fluorescence protein to monitor cell activation in response to exogenous stimuli and a constitutive promoter driving red fluorescence protein for internal signal normalization.^Lentivirus preparations were immobilized in a microarray format and after transduction on the array surface target cells were treated with cytokines and interrogated in real-time using automated fluorescence microscopy, providing rich dynamic information over a period of several days. Data normalization by red fluorescence intensity eliminated errors due to spot-to-spot variability in transduction efficiency or changes in cell proliferation upon cytokine treatment. These results suggest that the LVA can monitor gene expression in real-time and high-throughput manner thereby providing a useful tool for quantitatively measuring gene expression dynamics and deciphering gene regulatory networks.
Download or read book Lentivirus Gene Engineering Protocols written by Maurizio Federico and published by Springer Science & Business Media. This book was released on 2008-02-03 with total page 310 pages. Available in PDF, EPUB and Kindle. Book excerpt: Cell gene engineering is emerging as a field with outstanding impact, not only in medicine/biology, but also, and perhaps most importantly, in agriculture and in all those food sciences involved in the fight against world hunger. Lentivirus vector-based technologies represent the last frontier in the development of powerful and reliable methods for both in vitro and in vivo gene transfer in eukaryotic animal cells. Although the design of lentivirus vectors is closely reminiscent of those already successfully applied to the construction of oncoretroviral vectors, some unique features, e.g., the ef- ciency in transducing both postmitotic and stem cells, render the use of lentivirus vectors invaluable. It has been a great pleasure to edit Lentivirus Gene Engineering Pro- cols, owing in part to the high level of enthusiasm that the authors dem- strated in contributing to this book. The fact that so many outstanding scientists engaged in lentivirus vector research have provided articles renders it so- thing more than a technical handbook. In addition to detailed descriptions of the most innovative methodologies, the reader may find very informative ov- views concerning both theoretical and practical aspects of the origin and the development of diverse lentivirus vector types. This, in my opinion, rep- sents a unique added value of this volume, which should help our work resist the passage of time, to which books such as this are particularly sensitive.
Download or read book Lentiviral Vectors and Gene Therapy written by David Escors and published by Springer Science & Business Media. This book was released on 2012-03-22 with total page 110 pages. Available in PDF, EPUB and Kindle. Book excerpt: Gene therapy was conceived during the early and mid part of the 20th century. At first, it was considered a revolutionary biomedical procedure, which could potentially cure any disease for which the molecular bases were understood. Since then, gene therapy has gone through many stages and has evolved from a nearly unrealistic perspective to a real life application. Clinical efficacy in humans was demonstrated at the beginning of this century after its successful application in small-scale clinical trials to cure severe immunodeficiency in children. However, their successes were overshadowed some time later by the occurrence of vector-related leukaemia in a number of treated children. It is in this context that lentiviral vectors have appeared, with improved efficiency and, possibly, increased biosafety. Very recently, the first clinical trials with lentivectors have been carried out with some success. This Brief firstly defines gene therapy, and places lentivectors within this fascinating therapeutic strategy. Then follows a comprehensive description of the development of retroviral and lentiviral vectors and how to specifically target distinct cell types and tissues. The authors also discuss the application of lentivector gene therapy for the treatment of cancer and autoimmune diseases, ending with the application of lentivectors in human gene therapy clinical trials.
Download or read book Lentiviral Vectors written by Didier Trono and published by Springer Science & Business Media. This book was released on 2012-12-06 with total page 261 pages. Available in PDF, EPUB and Kindle. Book excerpt: For the first time a compilation of chapters that depict the biological bases underlying the development of lentiviral vectors, the techniques involved in the manufacture of this new gene delivery tool, and its most promising applications.
Download or read book Lentiviral Vector Systems for Gene Transfer written by Gary L. Buchschacher and published by Taylor & Francis US. This book was released on 2003-01-31 with total page 210 pages. Available in PDF, EPUB and Kindle. Book excerpt: The human immunodeficiency viruses (HIVs), in particular HIV-1, are the causative agent responsible for the current worldwide epidemic of acquired immunodeficiency syndrome (AIDS). A major effort has thus been underway over the past two decades to understand and control this pathogen. During this time, an enormous knowledge base has accumulated regarding the role of viral factors in the HIV-1 life cycle, and the interaction of HIV-1 with the host cell is becoming increasingly understood. Certain features of HIV, for example its ability to infect non-dividing cells, are being exploited in the development of novel gene therapy vehicles. This volume provides an overview of the current information regarding the HIV replication cycle and will serve as an introduction to subsequent chapters that address specific aspects of lentiviral-based gene therapy.
Download or read book Viral Vectors for Gene Therapy written by Curtis A. Machida and published by Springer Science & Business Media. This book was released on 2008-02-02 with total page 591 pages. Available in PDF, EPUB and Kindle. Book excerpt: Viral Vectors for Gene Therapy: Methods and Protocols consists of 30 ch- ters detailing the use of herpes viruses, adenoviruses, adeno-associated viruses, simple and complex retroviruses, including lentiviruses, and other virus systems for vector development and gene transfer. Chapter cont- butions provide perspective in the use of viral vectors for applications in the brain and in the central nervous system. Viral Vectors for Gene Therapy: Methods and Protocols contains step-by-step methods for successful rep- cation of experimental procedures, and should prove useful for both experienced investigators and newcomers in the field, including those beginning graduate study or undergoing postdoctoral training. The “Notes” section contained in each chapter provides valuable troublesho- ing guides to help develop working protocols for your laboratory. With Viral Vectors for Gene Therapy: Methods and Protocols, it has been my intent to develop a comprehensive collection of modern molecular methods for the construction, development, and use of viral vectors for gene transfer and gene therapy. I would like to thank the many chapter authors for their contributions. They are all experts in various aspects of viral vectors, and I appreciate their efforts and hard work in developing comprehensive chapters. As editor, it has been a privilege to preview the development of Viral Vectors for Gene Therapy: Methods and Protocols, and to acquire insight into the various methodological approaches from the many different contri- tors.
Download or read book Gene Transfer Vectors for Clinical Application written by and published by Academic Press. This book was released on 2012-12-31 with total page 450 pages. Available in PDF, EPUB and Kindle. Book excerpt: This volume of Methods in Enzymology looks at Gene Transfer Vectors for Clinical Application. The chapters provide an invaluable resource for academics, researchers and students alike. With an international board of authors, this volume covers such topics as General principles of retrovirus vector design, Chronic granulomatous disease (CGD), Gene therapy for blindness, and Retrovirus genetic strategy and vector design. Chapters provide an invaluable resource for academics, researchers and students alike International board of authors This volume covers such topics as general principles of retrovirus vector design, chronic granulomatous disease (CGD), gene therapy for blindness, and retrovirus genetic strategy and vector design
Download or read book Development and Application of Non integrating Lentiviral Vectors for Gene Therapy written by L. F. S. Apolonia and published by . This book was released on 2009 with total page pages. Available in PDF, EPUB and Kindle. Book excerpt: Lentiviruses stably integrate their genome into the host genome. Although this feature can be advantageous for long term transgene expression, it also has the potential to cause mutagenesis and cell transformation. To address this problem, thereby improving the safety of lentiviral gene therapy, non-integrating lentiviral vectors (NILVs) were developed. NILVs were generated by mutating the cis-acting sequences that interact with integrase (att sites) or by mutating specific residues in integrase in different domains (catalysis - D64V, strand transfer - Q148A, K264R, K266R, K273R, DNA or chromatin binding - N120L, W235E). Relevant mutations were then combined in order to improve the safety of these vectors. It was shown that all mutant vectors were efficiently produced and mutations did not affect infectivity. In contrast to dividing cells, differentiated muscle cells infected with NILVs show stable transgene expression over time without degradation of episomal viral DNA. The vectors were also tested in vivo by intramuscular injection in neonate mice. Transgene expression from muscle cells was maintained for 8 months using both integrating and NILVs. The vectors were then tested in a haemophilia B disease model. It was shown that plasma levels of FIX produced by muscle cells infected with integrating lentiviral vectors were above the therapeutic threshold. However, expression from NILVs was lower. This was studied in detail and it was found that integrating lentiviral vectors are transcriptionally more active than NILVs. A comparison of expression levels revealed that integrated lentivectors express more transgene protein per vector copy than NILVs and AAV vectors, but both episomal vectors display similar levels of transgene expression per vector copy. In conclusion, NILVs have the potential to be used as tools for prolonged transgene expression in non-dividing muscle cells or transient expression in dividing cells. However, vectors may need to be optimised if high expression levels are required.
Download or read book Gene Delivery to Mammalian Cells written by William C. Heiser and published by Springer Science & Business Media. This book was released on 2008-02-02 with total page 561 pages. Available in PDF, EPUB and Kindle. Book excerpt: The efficiency of delivering DNA into mammalian cells has increased t- mendously since DEAE dextran was first shown to be capable of enhancing transfer of RNA into mammalian cells in culture. Not only have other chemical methods been developed and refined, but also very efficient physical and viral delivery methods have been established. The technique of introducing DNA into cells has developed from transfecting tissue culture cells to delivering DNA to specific cell types and organs in vivo. Moreover, two important areas of biology—assessment of gene function and gene therapy—require succe- ful DNA delivery to cells, driving the practical need to increase the efficiency and efficacy of gene transfer both in vitro and in vivo. TM These two volumes of the Methods in Molecular Biology series, Gene Del- ery to Mammalian Cells, are designed as a compendium of those techniques that have proven most useful in the expanding field of gene transfer in mammalian cells. It is intended that these volumes will provide a thorough background on chemical, physical, and viral methods of gene delivery, a synopsis of the myriad techniques currently available to introduce genes into mammalian cells, as well as a practical guide on how to accomplish this. It is my expectation that it will be useful to the novice in the field as well as to the scientist with expertise in gene delivery.
Download or read book Approaches to Blocking the Immune Response to Gene Transfer with Viral Vectors written by Katherine High and published by Frontiers E-books. This book was released on with total page 96 pages. Available in PDF, EPUB and Kindle. Book excerpt: Viral vectors are superior tools for gene therapy and as a genetic vaccine platform because viruses have evolved to efficiently infect and transfer their genomes to cells. Several impressive successes in viral vector-based gene therapies have been reported in humans, including restoration of vision in patients with Leber’s congenital amaurosis by retinal gene transfer and cures for severe immune deficiencies by gene transfer to hematopoietic stem cells. However, the mammalian immune system has evolved in parallel to fend off invading pathogens such as viruses. Innate and antigen-specific adaptive immune responses against viral vectors and therapeutic transgene products pose serious hurdles for successful gene therapy. Pre-existing immunity in humans, resulting from prior exposure to the parent virus that forms the basis for the gene transfer vehicle may be derived from, often prevents efficient gene transfer. This problem also reduces our ability to use certain vectors for genetic vaccination or in anti-cancer therapy. For these reasons, the gene transfer community has been extensively studying the mechanisms of immune responses against viral vectors and has started to develop strategies and protocols to block or circumvent such responses. Choice, design and engineering of a vector as well as the route of administration/target tissue can be optimized/ altered to minimize immune responses or evade pre-existing immunity. Immune suppression and modulation strategies are being developed in order to minimize inflammation, prevent antibody or T cell responses against vectors, and to promote tolerance to therapeutic gene products. Combinations of these approaches will likely facilitate clinical applications of gene therapy for many target diseases and also aid in vaccine development.
Download or read book Next Generation Lentiviral Vectors for the Gene Therapy of Hemoglobinopathies written by Richard Morgan and published by . This book was released on 2019 with total page 224 pages. Available in PDF, EPUB and Kindle. Book excerpt: We set out to improve upon current -globin expressing lentiviral vectors (LV) designs by rationally reengineering human genomic sequences through the removal and/or addition of known elements. Although this approach generated improved LV designs, it is a low-throughput model for LV development, where large sections of DNA containing roughly-defined elements are simply inserted and/or removed from LVs and tested against a litany of criteria until a combination of well-performing elements are found (typically occurring over a span of years). To speed up LV development, we concurrently developed an advanced LV-engineering technology that can be broadly applied to the design of any gene therapy LV that requires high-level and lineage specific expression. Our Lentiviral Vector based, Massively Parallel Reporter Assay (LV-MPRA) allowed us to map the precise boundaries of lineage specific enhancers within large genomic regions (>16kb). The enhancer maps generated guided the assembly of novel enhancer combinations that when placed in a globin expression LV conferred robust transgene expression within a minimal DNA footprint. While our original "intelligent" design approach generated LV designs with improved performance (when compared to antecedent clinical LVs), vectors designed using the LV-MPRA based enhancer mapping approach possessed similar improvements in performance and were generated in only a fraction of the time (~3 months vs ~3 years). Moreover, LVs generated by either approach corrected the "Townes" mouse model of Sickle Cell Disease. The yields of this research offer improved "next generation" LVs for the gene therapy of hemoglobinopathies.
Download or read book Vector Targeting for Therapeutic Gene Delivery written by David T. Curiel and published by Wiley-Liss. This book was released on 2002-08-26 with total page 744 pages. Available in PDF, EPUB and Kindle. Book excerpt: -Cancer Biology and Therapy.
Download or read book Engineering Gene Delivery Vectors Through Genetic and Conjugational Modifications written by Gary Ka Leong Lee and published by . This book was released on 2004 with total page 408 pages. Available in PDF, EPUB and Kindle. Book excerpt:
Download or read book Engineering Cellular Resistance to HIV 1 Infection in Vitro Using Therapeutic Lentiviral Vector written by Fadi Kandarian and published by . This book was released on 2019 with total page 26 pages. Available in PDF, EPUB and Kindle. Book excerpt: Hematopoietic stem cells genetically modified to express anti-HIV genes can repopulate the immune system, replacing HIV target cells such as CD4+ T lymphocytes and macrophages. If such cells express therapeutic genes that halt early steps in the viral life cycle, HIV replication can be suppressed prior to reverse- transcription and integration. The HIV host restriction factor, huTrimCyp, was recently developed and does not occur naturally in humans. It is believed that huTrimCyp inhibits HIV shortly after viral entry by binding to capsid proteins and sequestering them for degradation. My data indicates th a t T-cell lines transduced with a lentivirus vector expressing huTrimCyp showed no short-term or long- term cytotoxicity, as well as exhibited a stable gene expression for a period greater than six weeks in vitro. Furthermore, transduced T-cell lines showed an inhibition of R5- tropic HIV strain greater than 5 f o l d when compared to cells expressing ZsGreen alone as determined by flow analysis of HIV-reporter infected MOLT4 T-cell line. I have performed in-vitro experiments in CD34+ hematopoietic stem cells in order to determine the feasibility of pursuing huTrimCyp as an anti-HIV gene therapy candidate in vivo. I have shown that huTrimCyp is capable of a fivefold reduction in supernatant p24 levels in CD34+ derived macrophages when infected with dual tropic HIV-1 (89.6) and a threefold reduction when infected with R5-tropic HIV-1 (NFNS-X), compared to macrophages expressing my control vector.
Download or read book Process Development of Lentiviral Vector Expression Purification and Formulation for Gene Therapy Applications written by S. M. Nilsson and published by . This book was released on 2016 with total page pages. Available in PDF, EPUB and Kindle. Book excerpt:
Download or read book Lentiviral Vector Systems for Gene Transfer written by Gary L. Buchschacher and published by Eurekah.Com Incorporated. This book was released on 2003 with total page 195 pages. Available in PDF, EPUB and Kindle. Book excerpt:
Download or read book Process Development of Lentiviral Vector Expression Purification and Formulation for Gene Therapy Applications written by Sara Margareta Nilsson and published by . This book was released on 2016 with total page 0 pages. Available in PDF, EPUB and Kindle. Book excerpt:
