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1.
Steven Froelich Leslie Ziegler Katie Stroup Pin Wang 《Biotechnology and bioengineering》2009,104(1):206-215
The development of a lentiviral system to deliver genes to specific cell types could improve the safety and the efficacy of gene delivery. Previously, we have developed an efficient method to target lentivectors to specific cells via an antibody–antigen interaction in vitro and in vivo. We report herein a targeted lentivector that harnesses the natural ligand–receptor recognition mechanism for targeted modification of c‐KIT receptor‐expressing cells. For targeting, we incorporate membrane‐bound human stem cell factor (hSCF), and for fusion, a Sindbis virus‐derived fusogenic molecule (FM) onto the lentiviral surface. These engineered vectors can recognize cells expressing surface CD117, resulting in efficient targeted transduction of cells in an SCF‐receptor dependent manner in vitro, and in vivo in xenografted mouse models. This study expands the ability of targeting lentivectors beyond antibody targets to include cell‐specific surface receptors. Development of a high titer lentivector to receptor‐specific cells is an attractive approach to restrict gene expression and could potentially ensure therapeutic effects in the desired cells while limiting side effects caused by gene expression in non‐target cells. Biotechnol. Bioeng. 2009; 104: 206–215 © 2009 Wiley Periodicals, Inc. 相似文献
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Background
A number of properties have relegated the use of Moloney murine leukemia virus (Mo‐MLV)‐based retrovirus vectors primarily to ex vivo protocols. Direct implantation of retrovirus producer cells can bypass some of the limitations, and in situ vector production may result in a large number of gene transfer events. However, the fibroblast nature of most retrovirus packaging cells does not provide for an effective distribution of vector producing foci in vivo, especially in the brain. Effective development of new retrovirus producer cells with enhanced biologic properties may require the testing of a large number of different cell types, and a quick and efficient method to generate them is needed.Methods
Moloney murine leukemia virus (Mo‐MLV) gag‐pol and env genes and retrovirus vector sequences carrying lacZ were cloned into different minimal HSV/AAV hybrid amplicons. Helper virus‐free amplicon vectors were used to co‐infect glioma cells in culture. Titers and stability of retrovirus vector production were assessed.Results
Simultaneous infection of two glioma lines, Gli‐36 (human) and J3T (dog), with both types of amplicon vectors, generated stable packaging populations that produced retrovirus titers of 0.5–1.2×105 and 3.1–7.1×103 tu/ml, respectively. Alternatively, when cells were first infected with retrovirus vectors followed by infection with HyRMOVAmpho amplicon vector, stable retrovirus packaging populations were obtained from Gli‐36 and J3T cells producing retrovirus titers comparable to those obtained with a traditional retrovirus packaging cell line, ΨCRIPlacZ.Conclusions
This amplicon vector system should facilitate generation of new types of retrovirus producer cells. Conversion of cells with migratory or tumor/tissue homing properties could result in expansion of the spatial distribution or targeting capacity, respectively, of gene delivery by retrovirus vectors in vivo. Copyright © 2002 John Wiley & Sons, Ltd.3.
病毒基因工程疫苗是以活病毒为载体将一段外源基因导入机体细胞内,并使外源基因维持较高水平的表达。通过使用复制型或复制缺陷型载体能使表达的抗原诱生机体产生相应的体液抗体,并能引起机体产生细胞介导的免疫反应及粘膜免疫反应。本文主要介绍有可能用于基因工程疫苗的DNA及RNA病毒载体构建及其应用。 相似文献
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Saller RM Indraccolo S Coppola V Esposito G Stange J Mitzner S Amadori A Salmons B Günzburg WH 《The journal of gene medicine》2002,4(2):150-160
Background
Because gene therapy of the future will primarily take an in vivo approach, a number of problems associated with its current implementation exist. Currently, repeated delivery of a vector in vivo is necessary to ensure adequate transfer of the therapeutic gene. This may lead to the development of an immune response against the vector, thus interfering with gene delivery. To circumvent this problem, retroviral vector packaging cells that permanently produce recombinant retroviral vector particles have been encapsulated.Methods
Vector (pBAG)‐producing amphotropic cells were encapsulated in beads composed of polymerized cellulose sulphate. These capsules were analysed in vitro for expression of the vector construct using X‐gal staining, as well as for the release of particles by performing RT‐PCR from culture supernatant. Infectivity studies were performed in vitro and in vivo. The latter was assayed using histological sections of the microcapsule and the surrounding area stained for β‐galactosidase activity and by RT‐PCR.Results
In culture, the virus‐producing cells inside the capsules remained viable and released virus into the culture medium for at least 6 weeks. To test whether these capsules, upon implantation into mice, also release vector virions that infect the surrounding cells, two different models were used. In the first, capsules were implanted in the fat pad of the mammary gland of Balb/c mice. The capsules were well tolerated for at least 6 weeks and a self‐limiting inflammatory reaction without any other gross immune response was observed during this period. Furthermore, the virus‐producing cells remained viable. In the second model, SCID mice were immunologically reconstituted by subcutaneous implantation of thymus lobes from MHC‐identical Balb/c newborn mice and gene transfer into lymphoid cells was achieved by retroviral vectors released by co‐implanted capsules.Conclusion
The implantation of such capsules containing cells that continually produce retroviral vector particles may be of use for in vivo gene therapy strategies. The data presented demonstrate the feasibility of the concept. Copyright © 2002 John Wiley & Sons, Ltd.6.
Dreier B Mikheeva G Belousova N Parizek P Boczek E Jelesarov I Forrer P Plückthun A Krasnykh V 《Journal of molecular biology》2011,405(2):410-426
Adenoviruses (Ads) hold great promise as gene vectors for diagnostic or therapeutic applications. The native tropism of Ads must be modified to achieve disease site-specific gene delivery by Ad vectors and this should be done in a programmable way and with technology that can realistically be scaled up. To this end, we applied the technologies of designed ankyrin repeat proteins (DARPins) and ribosome display to develop a DARPin that binds the knob domain of the Ad fiber protein with low nanomolar affinity (KD 1.35 nM) and fused this protein with a DARPin specific for Her2, an established cell-surface biomarker of human cancers. The stability of the complex formed by this bispecific targeting adapter and the Ad virion resulted in insufficient gene transfer and was subsequently improved by increasing the valency of adapter-virus binding. In particular, we designed adapters that chelated the knob in a bivalent or trivalent fashion and showed that the efficacy of gene transfer by the adapter-Ad complex increased with the functional affinity of these molecules. This enabled efficient transduction at low stoichiometric adapter-to-fiber ratios. We confirmed the Her2 specificity of this transduction and its dependence on the Her2-binding DARPin component of the adapters. Even the adapter molecules with four fused DARPins could be produced and purified from Escherichia coli at very high levels. In principle, DARPins can be generated against any target and this adapter approach provides a versatile strategy for developing a broad range of disease-specific gene vectors. 相似文献
7.
In order to facilitate the procedure of mammalian gene targeting, we have produced and functionally tested a series of generic vectors. Homologous recombination has been achieved with each vector. The vectors are designed for both replacement and insertional recombination, are suitable for hit and run strategies and contain all necessary genetic elements for both positive-negative and promoterless/gene fusion enrichment of homologous integrations. Multiple unique restriction sites are included to simplify the incorporation of genomic targeting sequences. 相似文献
8.
Martin J. Cline M.D. 《Molecular and cellular biochemistry》1984,59(1-2):3-10
Summary A number of techniques are available for insertion of new genetic information into mammalian cells. Some of these have been used successfully for genetic modification of germ line cells and somatic cells of living animals. Some of these techniques may be applicable to treatment of some of the genetic diseases of man, once problems related to the control of expression of introduced genes are solved. 相似文献
9.
The majority of clinical trials for gene therapy currently employ retroviral-mediated gene delivery. This is because the life
cycle of the retrovirus is well understood and can be effectively manipulated to generate vectors that can be efficiently
and safely packaged. Here, we review the molecular technology behind the generation of recombinant retroviral vectors. We
also highlight the problems associated with the use of these viruses as gene therapy vehicles and discuss future developments
that will be necessary to maintain retroviral vectors at the forefront of gene transfer technology. 相似文献
10.
Methods for construction of adenovirus vectors 总被引:40,自引:0,他引:40
Adenoviruses are attracting increasing attention as general purpose mammalian cell expression vectors, as recombinant vaccines,
and potentially as vectors for gene therapy. Not only is the adenovirus genome relatively easy to manipulate by recombinant
DNA techniques, but adenovirus vectors are relatively stable, grow to high titers, and can transduce a variety of cell types
in cell culture and in vivo. Vectors can be designed that are either replication competent or replication defective and, in
the latter case, are highly efficient at delivering and expressing genes in mammalian cells without resulting in cell killing.
Methods are described for growing, titrating, and purifying adenoviruses, for extracting viral DNA from purified virions and
from infected cells, for rescuing inserts of foreign DNA into the viral genome, and for assessing expression of inserted genes
in adenovirus vectors. 相似文献
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Gene therapy has developed to a technology which rapidly moved from the laboratory bench to the bedside in the clinic. This
implies safe, efficient and targeted gene transfer systems for suitable application to the patient. Beside the development
of such gene transfer vectors of viral or nonviral origin, improvement of cell type specific and inducible gene expression
is pivotal for successful gene therapy leading to targeted gene action. Numerous gene therapy approaches for treatment of
cancer and retroviral infections utilize cell type specific and/or regulatable promoter and enhancer sequences for the selective
expression of therapeutic genes in the desired cell populations and tissues. In this article the recent developments and the
potential of expression targeting are reviewed for gene therapy approaches of cancer and retroviral infections. 相似文献
16.
Novel tumour-specific promoters for transcriptional targeting of hepatocellular carcinoma by herpes simplex virus vectors 总被引:1,自引:0,他引:1
Foka P Pourchet A Hernandez-Alcoceba R Doumba PP Pissas G Kouvatsis V Dalagiorgou G Kazazi D Marconi P Foschini M Manservigi R Konstadoulakis MM Koskinas J Epstein AL Mavromara P 《The journal of gene medicine》2010,12(12):956-967
17.
基因治疗为治疗先天性遗传疾病和严重后天获得性疾病提供了一条新途径.目前,基因载体分为两类:病毒载体和非病毒载体.病毒载体转染效率高,但由于某些病毒载体存在免疫原性、致癌性、宿主DNA插入整合等缺点,从而限制了它们的应用.非病毒载体具有价格低、制备简单、安全有效、无免疫原性等优点,成为基因载体研究的热点.阳离子多聚物是非病毒载体的典型代表.文中综述近年来阳离子多聚物作为基因载体的研究现状和进展,重点介绍了阳离子多聚物基因载体的分类和与DNA的相互作用和传递机制. 相似文献
18.
The delivery of genetic material to mammalian cells is of great importance for modern fundamental biology, biomedicine, biotechnology, agriculture, and veterinary medicine. The development of new efficient techniques of gene transfer to human cells led to the advent of gene therapy, a novel approach to treating severe metabolic disorders, some viral infections (including HIV infection), autoimmune diseases, and genetic defects causing cancer. The review considers the main principles of constructing gene transfer and expression systems based on lentiviruses, a powerful tool for human gene therapy and transgenic research, with a special focus on the genome structure and life cycle of lentiviruses and the design and safety of lentiviral vector systems. 相似文献
19.
Gene transfer of constitutively active caspase-3 induces apoptosis in a human hepatoma cell line 总被引:3,自引:0,他引:3
Cam L Boucquey A Coulomb-L'hermine A Weber A Horellou P 《The journal of gene medicine》2005,7(1):30-38
BACKGROUND: The caspase-3 gene is expressed at significantly lower levels in human hepatocellular carcinomas than in normal hepatocytes. Gene transfer technologies offer the possibility to restore caspase-3 gene expression. We explored the interest for cancer gene therapy of a constitutively active recombinant caspase-3 (RevCasp3) obtained by rearranging its subunits. METHODS: An amphotropic retroviral vector was used to express the RevCasp3 gene. HuH7 cells were infected 1 and 2 days after plating. Caspase-3 activity was measured every 24 h for the following 6 days. The level of poly (ADP-ribose) polymerase cleavage induced by caspase-3 was measured by Western blot. The percentage of apoptotic cells was estimated after Hoechst-acridine orange and TUNEL stainings. RESULTS: Caspase-3 activity significantly increased from days 4 to 7 after infection. Caspase-3 activity peaked on day 7, and was 5.4-fold higher in RevCasp3-transduced HuH7 cells than in control cells. Poly (ADP-ribose) polymerase cleavage was first detected 6 days after the first infection. Hoechst-acridine orange and TUNEL stainings showed that most infected HuH7 cells were apoptotic. CONCLUSIONS: Apoptosis was selectively induced following infection of HuH7 cells with RevCasp3, demonstrating that retroviruses expressing RevCasp3 are of potential interest for the treatment of hepatocellular carcinomas and other tumour types. 相似文献
20.
The rare hereditary disease xeroderma pigmentosum (XP) is clinically characterized by extreme sun sensitivity and an increased predisposition for developing skin cancer. Cultured cells from XP patients exhibit hypersensitivity to ultraviolet (UV) radiation due to the defect in nucleotide excision repair (NER), and other cellular abnormalities. Seven genes identified in the classical XP forms, XPA to XPG, are involved in the NER pathway. In view of developing a strategy of gene therapy for XP, we devised recombinant retrovirus-carrying DNA repair genes for transfer and stable expression of these genes in cells from XP patients. Results showed that these retroviruses are efficient tools for transducing XP fibroblasts and correcting repair-defective cellular phenotypes by recovering normal UV survival, unscheduled DNA synthesis, and RNA synthesis after UV irradiation, and also other cellular abnormalities resulting from NER defects. These results imply that the first step of cellular gene therapy might be accomplished successfully. 相似文献