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Clinical trials over the last 15 years have demonstrated that cell and gene therapies for cancer, monogenic and infectious disease are feasible and can lead to long-term benefit for patients. However, these trials have been limited to proof-of-principle and were conducted on modest numbers of patients or over long periods of time. In order for these studies to move towards standard practice and commercialization, scalable technologies for the isolation, ex vivo manipulation and delivery of these cells to patients must be developed. Additionally, regulatory strategies and clinical protocols for the collection, creation and delivery of cell products must be generated. In this article we review recent progress in hematopoietic cell and gene therapy, describe some of the current issues facing the field and discuss clinical, technical and regulatory approaches used to navigate the road to product development. 相似文献
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Lentiviral vectors are more efficient at transducing quiescent hematopoietic stem cells than murine retroviral vectors. This characteristic is due to multiple karyophilic components of the lentiviral vector pre-integration complex. Lentiviral vectors are also able to carry more complex payloads than murine retroviral vectors, making it possible to deliver expression cassettes that direct either constitutive or targeted expression in various hematopoietic stem cell progeny. 相似文献
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Production of adenovirus vector for gene therapy 总被引:20,自引:0,他引:20
The field of gene therapy is rapidly expanding with a major focus on the treatment of cancer. Replication-defective adenoviruses are vectors of choice for delivering corrective genes into human cells. Major efforts are directed to design new generations of adenoviral vectors that feature reduced immunogenicity and improved targeting ability. However, the production of adenoviral vectors for gene therapy applications faces a number of challenges that limit the availability of high quality material at the early stages of research and development in the gene therapy field. Moreover, very few papers have been published on the subject and information on large-scale production methods are only available through specialized conference proceedings. This review outlines the problems associated with mass production of adenovirus vectors and describes research efforts by a number of groups who have contributed to optimize production methods. Better understanding of the adenovirus infection and replication kinetics as well as better understanding of complementing cell line physiology and metabolism greatly contributed to improving vector titers and volumetric productivity at higher cell densities. Also, the critical aspect of viral vector quantitation is discussed. 相似文献
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Recent discoveries on human and non-human stem cells have prompted the development of several studies aimed at the translation of laboratory evidences into novel clinical procedures collectively known as ‘cellular therapy’.In this regard, a number of features specifically related to the clinical setting require stringent evaluation, including, but not limited to: ethical appropriateness; donor and recipient informed consent; autologous versus allogeneic use; media and devices for cell collection, processing, characterization, storage and distribution; donor and recipient adverse events registration and management; risk-to-benefit and cost analysis; outcome analysis; production sites accreditation and management; regulatory oversight.This article describes recent national and international developments related to the distribution of cells and tissues for clinical use. Moreover, an example is reported of the implementation of a cellular therapy production site compliant with good manufacturing practices (GMPs) in a large European University hospital. 相似文献
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Highly efficient and sustained gene transfer in adult neurons with a lentivirus vector. 总被引:30,自引:1,他引:30
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The identification of monogenic and complex genes responsible for neurological disorders requires new approaches for delivering therapeutic protein genes to significant numbers of cells in the central nervous system. A lentivirus-based vector capable of infecting dividing and quiescent cells was investigated in vivo by injecting highly concentrated viral vector stock into the striatum and hippocampus of adult rats. Control brains were injected with a Moloney murine leukemia virus, adenovirus, or adeno-associated virus vector. The volumes of the areas containing transduced cells and the transduced-cell densities were stereologically determined to provide a basis for comparison among different viral vectors and variants of the viral vector stocks. The efficiency of infection by the lentivirus vector was improved by deoxynucleoside triphosphate pretreatment of the vector and was reduced following mutation of integrase and the Vpr-matrix protein complex involved in the nuclear translocation of the preintegration complex. The lentivirus vector system was able to efficiently and stably infect quiescent cells in the primary injection site with transgene expression for over 6 months. Triple labeling showed that 88.7% of striatal cells transduced by the lentivirus vector were terminally differentiated neurons. 相似文献
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Overview of vector design for mammalian gene expression 总被引:4,自引:0,他引:4
Kaufman RJ 《Molecular biotechnology》2000,16(2):151-160
The expression of cloned genes in mammalian cells is a basic tool for understanding gene expression, protein structure, and
function, and biological regulatory mechanisms. The level of protein expression from heterologous genes introduced into mammlaian
cells depends upon multiple factors including DNA copy number, efficiency of transportation, mRNA processing, mRNA transport,
mRNA stability, and translational efficiency, and protein processing, transport, and stability. Different genes exhibit different
rate limiting steps for efficient expression. Multiple strategies are available to obtain high level expression in mammalian
cells. This article reviews vector design for expression of foreign genes in mammalian cells. 相似文献
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Shrikant Mali 《Indian journal of human genetics》2013,19(1):3-8
The structure of DNA was unraveled by Watson and Crick in 1953, and two decades later Arber, Nathans and Smith discovered DNA restriction enzymes, which led to the rapid growth in the field of recombinant DNA technology. From expressing cloned genes in bacteria to expressing foreign DNA in transgenic animals, DNA is now slated to be used as a therapeutic agent to replace defective genes in patients suffering from genetic disorders or to kill tumor cells in cancer patients. Gene therapy provides modern medicine with new perspectives that were unthinkable two decades ago. Progress in molecular biology and especially, molecular medicine is now changing the basics of clinical medicine. A variety of viral and non-viral possibilities are available for basic and clinical research. This review summarizes the delivery routes and methods for gene transfer used in gene therapy. 相似文献
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Delivery systems for gene therapy 总被引:11,自引:0,他引:11
Introduction of foreign genes into mammalian cellsin vitro has been accomplished previously by a variety of methods. The few techniques that have been developed for transfection of mammalian cellsin vivo, are technically difficult or lack cell specificity.We have developed a soluble, targetable DNA carrier system consisting of an asialoglycoprotein covalently coupled to a polycation. The strategy was based on: 1) the presence of unique receptors on hepatocytes which internalize galactose-terminal (asialo-)glycoproteins; 2) polycations can bind DNA in a non-covalent, non-damaging interaction. Using chloramphenicol acetyltransferase (CAT) as a marker gene, specific delivery and expression of CAT was demonstratedin vitro using asialoglycoprotein receptor ( +) and (-) cell lines.Intravenous injection of conjugate-DNA complexes in rats resulted in detection of CAT DNA sequences in liver 10 min later by dot blots with a CAT cDNA probe. CAT enzyme activity 24 hrs later was found specifically in liver but no other tissues or control livers. Targeted hepatic CAT expression was transient, maximal at 24 hrs but declined to barely detectable levels by 96 hrs. Persistent foreign gene expression was achieved by injection of DNA complex followed by 67% partial hepatectomy. High levels of hepatic CAT activity were detected through 11 weeks post-hepatectomy.The data indicate that a targetable gene delivery system can permitin vivo expression of an exogenous gene after simple intravenous injection. The foreign gene expression can be enhanced and made to persist by induction of hepatocyte replication. 相似文献
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At the gene therapy session of the ICCXV Chromosome Conference (2004), recent advances in the construction of engineered chromosomes and de novo human artificial chromosomes were presented. The long-term aims of these studies are to develop vectors as tools for studying genome and chromosome function and for delivering genes into cells for therapeutic applications. There are two primary advantages of chromosome-based vector systems over most conventional vectors for gene delivery. First, the transferred DNA can be stably maintained without the risks associated with insertion, and second, large DNA segments encompassing genes and their regulatory elements can be introduced, leading to more reliable transgene expression. There is clearly a need for safe and effective gene transfer vectors to correct genetic defects. Among the topics discussed at the gene therapy session and the main focus of this review are requirements for de novo human artificial chromosome formation, assembly of chromatin on de novo human artificial chromosomes, advances in vector construction, and chromosome transfer to cells and animals. 相似文献
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Locus control region of the human CD2 gene in a lentivirus vector confers position-independent transgene expression
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Vectors derived from murine leukemia virus (MLV) have been used in many human gene therapy clinical trials. However, insertion of the locus control regions (LCRs) derived from the beta-globin gene locus or the CD2 gene into MLV vectors frequently led to vector rearrangement. Since the human immunodeficiency virus (HIV) sequence diverges significantly from the MLV sequence, we tested whether the LCR sequence is more stable in the context of an HIV vector. Clones derived from human fibrosarcoma line HT1080 cells transduced with an HIV vector containing the T-cell-specific CD2 LCR exhibit the same wide range of transgene expression as clones lacking the LCR. In contrast, Jurkat and primary T-cell clones derived from the transduction of the LCR-containing vector show, on average, a three- to fourfold increase in transgene expression relative to that of the control vector. This is consistent with previous observations that the CD2 LCR contains a T-cell-specific enhancer. In addition, the clones derived from the LCR-containing vector have a much lower clonal variation in transgene expression than those derived from the control vector. We also demonstrate that the level of transgene expression is proportional to the vector copy number. These results suggest that the human CD2 LCR sequence is compatible with HIV vector sequences and confers enhanced integration site-independent and copy number-dependent expression of the transgene. Thus, HIV vectors may represent the ideal vehicle to deliver genes controlled by various cis-acting elements such as LCRs. 相似文献
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Gene therapy is a promising and rapidly developing field of modern medicine and is expected to improve or even cure the diseases that are incurable with classical therapies. The logics of the development of gene therapy in the nearest future will require the systems wherein a regulation is possible for expression of therapeutic genes. The review considers the currently available regulated gene therapeutic systems, which can be divided into two main classes. One includes the systems wherein external inducers are used to trigger therapeutic gene expression. Systems of the other class are autoregulated and function without an external inducer. The most important first-class expression systems are based on the regulation by tetracycline, rapamycin derivative-induced dimerization, steroid hormones, regulatory RNAs, and physical factors. The most important systems of the second class are regulated by oxygen or glucose levels. 相似文献
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Lentivirus vector gene expression during ES cell-derived hematopoietic development in vitro
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Hamaguchi I Woods NB Panagopoulos I Andersson E Mikkola H Fahlman C Zufferey R Carlsson L Trono D Karlsson S 《Journal of virology》2000,74(22):10778-10784
The murine embryonal stem (ES) cell virus (MESV) can express transgenes from the long terminal repeat (LTR) promoter/enhancer in undifferentiated ES cells, but expression is turned off upon differentiation to embryoid bodies (EBs) and hematopoietic cells in vitro. We examined whether a human immunodeficiency virus type 1-based lentivirus vector pseudotyped with the vesicular stomatitis virus G protein (VSV-G) could transduce ES cells efficiently and express the green fluorescent protein (GFP) transgene from an internal phosphoglycerate kinase (PGK) promoter throughout development to hematopoietic cells in vitro. An oncoretrovirus vector containing the MESV LTR and the GFP gene was used for comparison. Fluorescence-activated cell sorting analysis of transduced CCE ES cells showed 99.8 and 86.7% GPF-expressing ES cells in the VSV-G-pseudotyped lentivirus (multiplicity of infection [MOI] = 59)- and oncoretrovirus (MOI = 590)-transduced cells, respectively. Therefore, VSV-G pseudotyping of lentiviral and oncoretrovirus vectors leads to efficient transduction of ES cells. Lentivirus vector integration was verified in the ES cell colonies by Southern blot analysis. When the transduced ES cells were differentiated in vitro, expression from the oncoretrovirus LTR was severely reduced or extinct in day 6 EBs and ES cell-derived hematopoietic colonies. In contrast, many lentivirus-transduced colonies, expressing the GFP gene in the undifferentiated state, continued to express the transgene throughout in vitro development to EBs at day 6, and many continued to express in cells derived from hematopoietic colonies. This experimental system can be used to analyze lentivirus vector design for optimal expression in hematopoietic cells and for gain-of-function experiments during ES cell development in vitro. 相似文献
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Gene therapy aims at transferring a therapeutic gene into human somatic cells in order to treat a disease. Originally addressed to hereditary genetic disorders, gene therapy has found therapeutic applications in cancer, infectious diseases and degenerative disorders, particularly those of the nervous system. Although gene transfer into humans has been demonstrated in several clinical trials, with more than 300 currently underway worldwide, there is still no single outcome that undoubtedly showed a consistent benefit for the patient. Nevertheless, the expectations for gene therapy are still high, and the prospects of future clinical success are increasing together with the growing of the field. The development of better delivery systems specifically tailored to individual diseases, with sustained expression of the therapeutic gene in the appropriate cells, will in the end make possible true therapeutic applications of human gene transfer. 相似文献
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Chapdelaine P Moisset PA Campeau P Asselin I Vilquin JT Tremblay JP 《Protein engineering》2000,13(9):611-615
Transfection and transduction studies involving the use of the full-length dystrophin (11 kb) or the truncated mini-gene (6 kb) cDNAs are hampered by the large size of the resulting viral or non-viral expression vectors. This usually results in very low yields of transgene-expressing cells. Moreover, the detection of the few transgene-expressing cells is often tedious and costly. For these reasons, expression vectors containing the enhanced green fluorescent protein (EGFP) fused with the N-termini of mini- and full-length human dystrophin were constructed. These constructs were tested by transfection of Phoenix cells with Effectene, resulting after 48 h in a green fluorescent signal in 20% of cells. Analysis of the cell extracts by immunoblotting with the use of a monoclonal antibody specific to the dystrophin C-terminus confirmed the expression of EGFP-mini- (240 kDa) and EGFP-full-length human dystrophin (450 kDa) fusion proteins. Moreover, following the in vivo electroporation of the plasmids containing the EGFP-mini- and full-length dystrophin in mouse muscles, both fluorescent proteins were observed in cryostat sections in their normal location under the plasma membrane. This indicates that the fusion of EGFP to dystrophin or mini-dystrophin did not interfere with the normal localization of the protein. In conclusion, the fusion of EGFP provides a good tool for the search of the best methods to introduce mini- or full-length dystrophin cDNA in the cells (in vitro) or muscle fibers (in vivo) for the establishment of a treatment by gene therapy of Duchenne muscular dystrophy patients. 相似文献
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PURPOSE OF REVIEW: Atherosclerosis is a chronic inflammatory disease that is the primary cause of morbidity and mortality in the developed world. Many studies have shown that macrophages and T-cells play critical roles in multiple aspects of the pathogenesis of the disease. Given that these cells are ultimately derived from bone marrow precursors, the concept of performing gene therapy for atherosclerosis through the retroviral transduction of hematopoietic stem cells has received much attention. This review will highlight recent advances that will help bring this goal closer. RECENT FINDINGS: The clinical application of retroviral gene transfer into hematopoietic stem cells has been hampered, in part, by the absence of vectors that can direct long-lasting, cell-type specific gene expression. In this review we will detail recent developments in the design of novel retroviral and lentiviral vectors that appear to overcome these problems, offering approaches to express therapeutic genes in specific cell-types within atherosclerotic lesions. We will also highlight advances in our understanding of the pathogenesis of atherosclerosis that may offer new gene therapeutic targets. SUMMARY: The use of retroviral transduction of hematopoietic stem cells for treatment of patients with atherosclerosis still remains a long-term goal. However, the recent development of retroviral vectors capable of directing expression to specific cell types within the lesion will allow more targeted therapeutic strategies to be devised. In addition, these vectors will provide powerful experimental tools to further our understanding of the pathogenesis of the disease. 相似文献
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Marking and gene expression by a lentivirus vector in transplanted human and nonhuman primate CD34(+) cells
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An DS Wersto RP Agricola BA Metzger ME Lu S Amado RG Chen IS Donahue RE 《Journal of virology》2000,74(3):1286-1295
Recently, gene delivery vectors based on human immunodeficiency virus (HIV) have been developed as an alternative mode of gene delivery. These vectors have a number of advantages, particularly in regard to the ability to infect cells which are not actively dividing. However, the use of vectors based on human immunodeficiency virus raises a number of issues, not the least of which is safety; therefore, further characterization of marking and gene expression in different hematopoietic lineages in primate animal model systems is desirable. We use two animal model systems for gene therapy to test the efficiency of transduction and marking, as well as the safety of these vectors. The first utilizes the rhesus animal model for cytokine-mobilized autologous peripheral blood CD34(+) cell transplantation. The second uses the SCID-human (SCID-hu) thymus/liver chimeric graft animal model useful specifically for human T-lymphoid progenitor cell reconstitution. In the rhesus macaques, detectable levels of vector were observed in granulocytes, lymphocytes, monocytes, and, in one animal with the highest levels of marking, erythrocytes and platelets. In transplanted SCID-hu mice, we directly compared marking and gene expression of the lentivirus vector and a murine leukemia virus-derived vector in thymocytes. Marking was observed at comparable levels, but the lentivirus vector bearing an internal cytomegalovirus promoter expressed less efficiently than did the murine retroviral vector expressed from its own long terminal repeats. In assays for infectious HIV type 1 (HIV-1), no replication-competent HIV-1 was detected in either animal model system. Thus, these results indicate that while lentivirus vectors have no apparent deleterious effects and may have advantages over murine retroviral vectors, further study of the requirements for optimal use are warranted. 相似文献