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1.
The utility of conditionally replicative adenoviruses (CRAds) in cancer gene therapy is based on their ability to destroy tumor cells by oncolysis. However, in order to achieve adequate therapeutic response, CRAds have to spread through the tumor tissue by replication. Thus, to study the potency of these viruses to replicate and penetrate in tumors, we created a herpes simplex virus type 1 thymidine kinase-green fluorescent protein fusion gene (TK-GFP) encompassing CRAd (Ad5Delta24TK-GFP), whose tumor selectivity is mediated by a retinoblastoma (Rb)-binding site mutation. In addition, we evaluated the oncolytic efficacy of Ad5Delta24TK-GFP in combination with the TK/ganciclovir (GCV) system. Based on our results, Ad5Delta24TK-GFP replicates in cancer cells resulting in oncolysis and can efficiently penetrate into tumors. Additionally, the combination of GCV with Ad5Delta24TK-GFP augmented cell death in vitro but this was not observed in vivo: tumor growth was significantly reduced by oncolysis when compared to non-replicative virus (p<0.001), but administration of GCV did not significantly enhance oncolysis. This suggests that in certain conditions, TK/GCV-mediated cell killing may be counterproductive to replication and oncolysis, which on the other hand might be useful feature for clinical trials in case of replication-associated toxicity.  相似文献   

2.
Gene therapy has opened new doors for treatment of neoplastic diseases. This new approach seems very attractive, especially for glioblastomas, since treatment of these brain tumors has failed using conventional therapy regimens. Many different modes of gene therapy for brain tumors have been tested in culture and in vivo. Many of these approaches are based on previously established anti-neoplastic principles, like prodrug activating enzymes, inhibition of tumor neovascularization, and enhancement of the normally weak anti-tumor immune response. Delivery of genes to tumor cells has been mediated by a number of viral and synthetic vectors. The most widely used paradigm is based on the activation of ganciclovir to a cytotoxic compound by a viral enzyme, thymidine kinase, which is expressed by tumor cells, after the gene has been introduced by a retroviral vector. This paradigm has proven to be a potent therapy with minimal side effects in several rodent brain tumor models, and has proceeded to phase 1 clinical trials. In this review, current gene therapy strategies and vector systems for treatment of brain tumors will be described and discussed in light of further developments needed to make this new treatment modality clinically efficacious.  相似文献   

3.
Adenovirus-mediated gene therapy for hematopoietic malignancies, especially those derived from B cells, is difficult due to systemic nature of these diseases. More importantly, most tumor cells derived from B cell lineage express a very low level of the adenovirus receptor hCAR; thus, warranting the design of adenoviral vectors with high affinity to abundant B cell surface molecules. To mimic this approach and to test the validity of adenoviral vectors in gene therapy of disseminated malignancies, we created an hCAR-expressing follicular lymphoma B cell line. The cell line was generated with the aid of a lentivirus vector carrying a novel fusion gene with EGFP replacing the cytoplasmic domain of hCAR. After verifying that this cell line was expressing the hybrid receptor in a correct manner and enrichment of the bright EGFP positive population, the cells were transduced with adenoviruses expressing the red fluorescent protein DsRed2. It was shown that regular transduction with a low viral dose (1 pfu/cell) increased the gene transfer rate by a factor of 5. Furthermore, experiments with adenovirus vector carrying the HSV-TK-GFP transgene demonstrated that the modified follicular lymphoma B cells became sensitive to ganciclovir while the parental cells remained virtually resistant to this form of gene therapy. In summary, we show here with this simple model system that adenoviral gene therapy of B cell malignancies is possible provided that correct receptors for adenovirus attachment are present on the surface of the target cells. Thus, our results warrant further modifications of adenovirus capsid to obtain vectors with specific affinity to B cell epitopes.  相似文献   

4.
Won YW  Kim KM  An SS  Lee M  Ha Y  Kim YH 《Biomaterials》2011,32(36):9766-9775
Suicide gene therapy based on a combination of herpes simplex virus-thymidine kinase (HSV-tk) and ganciclovir (GCV) has obstacles to achieving a success in clinical use for the treatment of cancer due to inadequate thymidine kinase (TK) expression. The primary concern for improving anticancer efficacy of the suicide gene therapy is to develop an appropriate carrier that highly expresses TK in vivo. Despite great advances in the development of non-viral vectors, none has been used in cancer suicide gene therapy, not even in experimental challenge. Reducible poly (oligo-D-arginine) (rPOA), one of the effective non-viral carriers working in vivo, was chosen to deliver HSV-tk to spinal cord tumors which are appropriate targets for suicide gene therapy. Since the system exerts toxicity only in dividing cells, cells in the central nervous system, which are non-proliferative, are not sensitive to the toxic metabolites. In the present study, we demonstrated that the locomotor function of the model rat was maintained through the tumor suppression resulting from the tumor-selective suicide activity by co-administration of rPOA/HSV-tk and GCV. Thus, rPOA plays a crucial role in suicide gene therapy for cancer, and an rPOA/HSV-tk and GCV system could help promote in vivo trials of suicide gene therapy.  相似文献   

5.
We explored the possibility of entrapping retroviral vector producing cells (VPC) within porous 3D matrix to induce a local and sustained release of viral particles to the malignant milieu. PA317/STK, which constantly shed retroviral vectors, was used to transduce cancer cells with the herpes simplex virus thymidine kinase (HSV-tk) gene. Once HSV-tk is expressed, it preferentially phosphorylates nucleoside analog prodrugs, such as ganciclovir (GCV) and N-methanocarbathymidine (N-MCT), to their active triphosphate metabolites, which when incorporated into cellular DNA cause cell death. PA317/STK cells were seeded within 3D alginate scaffold at two different cell densities via static seeding procedure. In vitro assays determined that PA317/STK seeded at high-cell density in scaffolds maintained constant cell number, low cell leakage, and spheroid morphology with viral vector transfection activity. Postcell-seeding viral vector activity was confirmed by transfection of murine colon cancer cells (MC38) with conditioned media originated from VPC-containing scaffolds and the subsequent ability to generate N-MCT triphosphate. Preliminary in vivo transplantation of VPC-containing scaffolds into the peritoneal cavity of mice bearing intraperitoneal MC38 tumors with 2 weeks subsequent GCV administration resulted in a significantly higher survival rate relative to control groups. Our results demonstrate the feasibility of employing alginate scaffolds to efficiently entrap and support PA317/STK cells for cancer gene therapy.  相似文献   

6.
Polyethylenimine (PEI) is the polymer most commonly used for transferring plasmids into eukaryotes, but its gene-transfer efficiency is lower compared to viral vectors. Receptors targeting PEI combined with ligands can enhance efficiency of gene transfer into the corresponding receptor-positive cells. Using the double-receptor-mediated pathway of viral infection, in this study we synthesized a novel non-viral vector based on PEI combined with two peptides recognizing FGF receptors (peptide YC25) and integrins (peptide CP9) on the cell surface. The dual targeting vector showed a physicochemical character similar to that of PEI, such as pDNA formation, particle size, zeta potential and lower toxicity. In vitro gene transfer showed that the dual-receptor targeted vector (YC25-PEI-CP9) exhibited a markedly higher transgene efficiency in cell lines with positive expression of FGF receptors and integrins, compared with single-peptide-modified PEI or unmodified PEI. In the cells with only integrin-positive expression, YC25-PEI-CP9 mediated a higher transgene expression than PEI but lower than CP9-PEI. The corresponding free peptides could inhibit the transgene efficiency of the peptide-coupled PEI. In vivo gene transfer in tumor-bearing nude mice also demonstrated that the dual-targeting vectors showed a significantly enhanced transfection efficiency in tumors with positive expression of FGF receptors and integrins. The synthesized polymer YC25-PEI-CP9 has the prospect to act as a novel kind of non-viral vector in gene therapy.  相似文献   

7.
Osteosarcoma and chondrosarcoma, the most prevalent primary malignant tumors of the bone, have been demonstrated to be potential target diseases for herpes simplex virus type 1 thymidine kinase (HSV-TK)/ganciclovir (GCV) suicide gene therapy. However, the utility of this gene therapy form for bone tumor cells has not been studied systematically. In this report we show, with the aid of three osteosarcoma cell lines (Saos-2, U-2-OS and MG-63) and one chondrosarcoma cell line (SW1353) that: i) these tumor cells were permissive for adenovirus- or lentivirus-mediated gene delivery; ii) the cell lines appeared to be good or excellent targets for HSV-TK/GCV gene therapy; and iii) the extent of HSV-TK/GCV cytotoxic effect correlated with the presence of the 'bystander effect' in these cells. Our results also suggest that lentiviruses are potential vectors for bone cancer gene therapy. They transduced all four cell lines with high efficiency and provided HSV-TK expression level that was sufficient for cytotoxicity and bystander effect comparable to that obtained with adenovirus vectors.  相似文献   

8.
Efficient virus and non-virus vector systems for gene transfer to tumor cells have been developed and tested in cell culture and in animal experiments. With some of the earliest and most comprehensively evaluated vectors, such as retroviruses, advanced clinical trials were performed in tumor patients. Malignant primary brain tumors (gliomas) have been chosen for the first clinical studies on novel gene therapy approaches because these tumors are non-metastatic and develop on the largely postmitotic background of normal glial and neuronal tissue. However, the human cancer gene therapy studies performed so far were not as successful as preclinical animal experiments. Furthermore, the clinical studies did not address major limiting factors for in vivo gene therapy, such as insufficient gene transfer rates to the tumor with the used local delivery modalities, and the resulting inability of a particular transgene-prodrug system to confer permanently eradicating cytotoxicity to the whole neoplasm. Critical evaluation of gene transfer and therapy studies has led to the conclusion that, even using identical vectors, the anatomical route of vector administration can dramatically affect both the efficiency of tumor transduction and its spatial distribution, as well as the extent of intratumoral and intracerebral transgene expression. This review concentrates on different physical methods for vector delivery to malignant primary brain tumors in experimental or clinical settings: stereotactic or direct intratumoral injection or convection-enhanced bulk-flow interstitial delivery; intrathecal and intraventricular injection; and intravascular infusion with or without modification of the blood-tumor-barrier. The advantages and drawbacks of the different modes and delivery routes of in vivo vector application, and the possibilities for tumor targeting by modifications of the native tropism of virus vectors or by using tissue-specific or inducible transgene expression are summarized.  相似文献   

9.
Polyethylenimine (PEI) is the polymer most commonly used for transferring plasmids into eukaryotes, but its gene-transfer efficiency is lower compared to viral vectors. Receptors targeting PEI combined with ligands can enhance efficiency of gene transfer into the corresponding receptor-positive cells. Using the double-receptor-mediated pathway of viral infection, in this study we synthesized a novel non-viral vector based on PEI combined with two peptides recognizing FGF receptors (peptide YC25) and integrins (peptide CP9) on the cell surface. The dual targeting vector showed a physicochemical character similar to that of PEI, such as pDNA formation, particle size, zeta potential and lower toxicity. In vitro gene transfer showed that the dual-receptor targeted vector (YC25-PEI-CP9) exhibited a markedly higher transgene efficiency in cell lines with positive expression of FGF receptors and integrins, compared with single-peptide-modified PEI or unmodified PEI. In the cells with only integrin-positive expression, YC25-PEI-CP9 mediated a higher transgene expression than PEI but lower than CP9-PEI. The corresponding free peptides could inhibit the transgene efficiency of the peptide-coupled PEI. In vivo gene transfer in tumor-bearing nude mice also demonstrated that the dual-targeting vectors showed a significantly enhanced transfection efficiency in tumors with positive expression of FGF receptors and integrins. The synthesized polymer YC25-PEI-CP9 has the prospect to act as a novel kind of non-viral vector in gene therapy.  相似文献   

10.
Abstract. The efficacy of single and combination suicide gene therapy was evaluated using a Herpes simplex virus thymidine kinase/ganciclovir system and Escherichia coli cytosine deaminase/5-fluorocytosine system on the rat prostate tumor cell line R3327 AT-1. The wild-type R3327 AT-1 cell line was transfected with a bifunctional fusion gene CDglyTK, which had the advantage that the resulting R3327 AT-1/CDglyTK cell line has the same amount of cytosine deaminase and thymidine kinase molecules. The percentage of viable R3327 AT-1/CDglyTK cells after 96 h incubation with 0.1 µg/ml ganciclovir or 10 µg/ml 5-fluorocytosine were 85% and 52% of controls, respectively. The cell viability when both suicide genes systems were activated was 43%. For in vivo analysis, Copenhagen rats were injected subcutaneously with R3327 AT-1 or R3327 AT-1/CDglyTK cells and treated with 30 mg/kg ganciclovir, 500 mg/kg 5-fluorocytosine, or both prodrugs together. A survival of 83% with the thymidine kinase/ganciclovir and 57% with the CD/5-FC could be observed. Only co-administration of thymidine kinase- and cytosine deaminase-specific prodrugs resulted in a 100% recurrence-free survival of the Copenhagen rats with a Dunning R3327 AT-1/CDglyTK prostate tumor and showed an additive cytotoxic effect. Calculation of the degree of activation and the potential of activation can be used to predict the success of a suicide gene therapy. In our case, the cytosine deaminase/5-fluorocytosine system had a low degree of activation (value 40), which is also found in the low response to 5- fluorocytosine in vivo (57% tumor free).  相似文献   

11.
Use of the herpes simplex virus thymidine kinase gene/ganciclovir (HSV-tk/GCV) system is one of the promising approaches in the rapidly growing area of gene therapy. The "bystander effect," a phenomenon in which HSV-tk+ cells exposed to GCV are toxic to adjacent HSV-tk- cells, was reported to play an important role in suicide gene therapy. However, the mechanism by which HSV-tk/GCV induces the bystander effect is poorly understood. We monitored the activation of caspase-3 in living cells induced by the HSV-tk/GCV system using a genetically encoded fluorescence resonance energy transfer (FRET) probe CD3, , a caspase-3 recognition site fused with a cyan fluorescent protien (CFP) and a red fluorescent protein (DsRed) which we reported and named in a previous paper. Fluorescence protein (FP)-based multicolor cellular labeling, combined with the multichannel fluorescence imaging and FRET imaging techniques, provides a novel and improved approach to directly determine whether the activation of caspase-3 involved in the HSV-tk/GCV system induces cell apoptosis in tk gene-expressing cells and their neighboring cells. FRET ratio images of CD3, and fluorescence images of the fusion protein of thymidine kinase linked with green fluorescent protein (TK-GFP), indicated that HSV-tk/GCV system-induced apoptosis in human adenoid cystic carcinoma (ACC-M) cells was via a caspase-3 pathway, and the activation of caspase-3 was not involved in the bystander effect of HSV-tk/GCV system.  相似文献   

12.
The efficacy of single and combination suicide gene therapy was evaluated using a Herpes simplex virus thymidine kinase/ganciclovir system and Escherichia coli cytosine deaminase/5-fluorocytosine system on the rat prostate tumor cell line R3327 AT-1. The wild-type R3327 AT-1 cell line was transfected with a bifunctional fusion gene CDglyTK, which had the advantage that the resulting R3327 AT-1/CDglyTK cell line has the same amount of cytosine deaminase and thymidine kinase molecules. The percentage of viable R3327 AT-1/CDglyTK cells after 96 h incubation with 0.1 micro g/ml ganciclovir or 10 micro g/ml 5-fluorocytosine were 85% and 52% of controls, respectively. The cell viability when both suicide genes systems were activated was 43%. For in vivo analysis, Copenhagen rats were injected subcutaneously with R3327 AT-1 or R3327 AT-1/CDglyTK cells and treated with 30 mg/kg ganciclovir, 500 mg/kg 5-fluorocytosine, or both prodrugs together. A survival of 83% with the thymidine kinase/ganciclovir and 57% with the CD/5-FC could be observed. Only co-administration of thymidine kinase- and cytosine deaminase-specific prodrugs resulted in a 100% recurrence-free survival of the Copenhagen rats with a Dunning R3327 AT-1/CDglyTK prostate tumor and showed an additive cytotoxic effect. Calculation of the degree of activation and the potential of activation can be used to predict the success of a suicide gene therapy. In our case, the cytosine deaminase/5-fluorocytosine system had a low degree of activation (value 40), which is also found in the low response to 5- fluorocytosine in vivo (57% tumor free).  相似文献   

13.
韩海勃  赵威  张志谦 《解剖学报》2011,42(6):756-760
Objective To establish Tetracycline-induced gene expression system for gene therapy based on third generation lentivirus system.Methods The mutant of the reverse Tet transactivator (rtTA and M2rtTA) was subcloned into a lentiviral vector with neo selection marker named as pELNS-rtTA-IRES-Neo and pELNS-M2rtTA-IRES-Neo. The Tet-responsive element (TETO and TREpitt) and green fluorescence proteint (GFP) were subcloned into a lentiviral vector with blasticidin selection marker named as plenti6-TETO-GFP and plenti6-TREpitt-GFP.293 cells were contransfect with pELNS-rtTA-IRES-Neo and plenti6-TETO-GFP, or with pELNS-M2rtTA-IRES-Neo and plenti6-TREpitt -GFP. Cells were treated by Dox to induce GFP expression. After 48hours, GFP expression in the co-transfected cells was observed under a fluorescent microscope.Results The first generation of Tetracycline-induced gene expression system named pELNS-rtTA-IRES-Neo and plenti6-TETO-GFP vectors were successfully set up. GFP expression in cotransfected cells induced with Dox was about 90% positive, while there was 30% positive GFP expression observed in no Dox inducing group. The second generation of Tetracycline-induced gene expression system named pELNS-M2rtTA-IRES-Neo and plenti6-TREpitt-GFP vectors were successfully set up. GFP expression in cotransfected cells induced with Dox was about 95% positive, while there was no positive GFP expression observed in no Dox inducing group.Conclusion Tetracycline-induced gene expression system based on lentivirus was successfully set up, which can induce gene expression effectively and tightly without obvious side-effects on cells by using the second Tetracycline-induced elements.  相似文献   

14.
Q Zhao  B Lu  SK George  JJ Yoo  A Atala 《Biomaterials》2012,33(29):7261-7271
Pluripotent stem cells provide an unlimited cell source for cell therapy. However, residual pluripotent stem cells after differentiation can form tumors. Modifying stem cells with suicide constructs through integrating plasmid DNA and viral vectors has been attempted to specifically eliminate residual pluripotent stem cells after differentiation. However, integration of foreign DNA has the potential of insertional mutagenesis, position effects and silencing. Scaffold/matrix attachment region (S/MAR)-based plasmid DNA can be maintained extra-chromosomally, offering a safer alternative to integrating vectors for this purpose. Here, we report the design of an S/MAR-based suicide construct capable of episomal maintenance and specifically killing pluripotent stem cells but not differentiated cells in the presence of ganciclovir. Treating cells differentiated from episomal suicide construct-modified stem cells with ganciclovir reduces the tumor formation risk after cell transplantation. Tumors formed by such modified pluripotent stem cells could be inhibited by ganciclovir administration. This episomal suicide construct enables negative selection of residual pluripotent stem cells in?vitro and control of tumors formed from residual pluripotent stem cells in?vivo.  相似文献   

15.
Suicide gene therapy is a promising concept in oncology. We have recently introduced a novel suicide gene, TK.007, which was shown to excel established herpes simplex virus thymidine kinase (HSVtk) variants when used for donor-lymphocyte modification in adoptive immunotherapy models. Here, the potential of TK.007 in killing cancer cells was studied. Initially, we transduced tumour cell lines derived from different neoplasias (glioblastoma, melanoma, lung cancer, colon cancer) with lentiviral LeGO vectors encoding TK.007 or the splice-corrected (sc)HSVtk together with an eGFP/Neo-marker. Based on direct in vitro comparison, we found that TK.007 facilitates more efficient tumour cell killing at significantly lower ganciclovir doses in all tumour cell lines tested. Also, using different readout systems, we found a significantly stronger bystander effect of TK.007 as compared to scHSVtk. Importantly, in vitro data were confirmed in vivo using a subcutaneous G62 glioblastoma model in NOD/SCID mice. In mice transplanted with scHSVtk-positive tumours, treatment with low (10 mg/kg) or standard (50 mg/kg) ganciclovir doses resulted only in short-term growth inhibition or transient tumour remission, respectively. In striking contrast, in the TK.007 group, all animals achieved continuous complete remission after both standard and low-dose ganciclovir. Finally, a substantial bystander effect for TK.007 was also confirmed with the G62 model in vivo, where significantly prolonged survival for mice bearing tumours containing only 10% or 50% TK.007-expressing cells was observed. In summary, our data indicate strongly improved anti-tumour activity of TK.007 as compared to conventional HSVtk. We therefore suppose that TK.007 is an excellent candidate for cancer suicide gene therapy.  相似文献   

16.
Progress in the use of adeno-associated viral vectors for gene therapy   总被引:9,自引:0,他引:9  
The development of safe and efficient gene transfer vectors is crucial for the success of gene therapy trials. A viral vector system promising to meet these requirements is based on the apathogenic adeno-associated virus (AAV-2), a member of the parvovirus family. The advantages of this vector system is the stability of the viral capsid, the low immunogenicity, the ability to transduce both dividing and non-dividing cells, the potential to integrate site specifically and to achieve long-term gene expression even in vivo, and its broad tropism allowing the efficient transduction of diverse organs including the skin. All this makes AAV-2 attractive and efficient for in vitro gene transfer and local injection in vivo. This review covers the progress made in AAV vector technology including the development of AAV vectors based on other serotypes, summarizes the results obtained by AAV targeting vectors and outlines potential applications in the field of cutaneous gene therapy.  相似文献   

17.
Over the past few years, technological procedures have been developed for utilizing stem cells in transplantation medicine. Human embryonic stem (ES) cells can produce an unlimited number of differentiated cells and are, therefore, considered a potential source of cellular material for use in transplantation medicine. However, serious clinical problems can arise when uncontrolled cell proliferation occurs following transplantation. To avoid these potential problems, we genetically engineered human ES cell lines to express the herpes simplex virus thymidine kinase (HSV-tk) gene. Expression of the HSV-tk protein renders the ES cells sensitive to the U.S. Food and Drug Administration-approved drug ganciclovir, inducing destruction of HSV-tk(+) cells at ganciclovir concentrations that are nonlethal to other cell types. The reversion rate of engineered cells was low even under prolonged selection with ganciclovir. The HSV-tk(+) clones retained a normal karyotype and the ability to differentiate to cells from all three germ layers. Most importantly, tumors that arose in mice following subcutaneous injection of HSV-tk(+) human ES cells could be ablated in vivo by administration of ganciclovir. By utilizing these cell lines, safety levels can be improved in transplantations involving tissues derived from human ES cells.  相似文献   

18.
Cancer arises as a direct result of genetic mutations. It therefore stands to reason that cancer should be well suited for the correction through gene therapy. Recent advances in the understanding of the molecular pathogenesis of cancer and the rapid development of recombinant DNA technology have made cancer gene therapy feasible in the clinical setting. The current efforts for cancer gene therapy mainly focus on immunogene therapy, chemogene therapy, restoration of tumor suppressor gene function, and oncolytic virus therapy. Central to all these therapies is the development of efficient vectors for gene delivery--this remains a work in progress. These vectors can be classified as viral and non-viral vectors. This paper will concentrate on viral vectors because of their practical advantages over non-viral vectors. Of the viral vectors, by far the most important are the human adenoviruses as is reflected by the enormous data and literature accumulated by studies relating to animal tumor models and clinical trials. In this review, we examine the recent progress in adenovirus-mediated cancer gene therapy with regard to cytokine gene, tumor suppressor gene, chemogene, and oncolytic adenovirus. We also discuss the current limitations of the adenoviral vector system and how they may be circumvented in future developments relating to targeted gene delivery.  相似文献   

19.
20.
Researchers have conducted numerous pre-clinical and clinical gene transfer studies using recombinant viral vectors derived from a wide range of pathogenic viruses such as adenovirus, adeno-associated virus, and lentivirus. As viral vectors are derived from pathogenic viruses, they have an inherent ability to induce a vector specific immune response when used in vivo. The role of the immune response against the viral vector has been implicated in the inconsistent and unpredictable translation of pre-clinical success into therapeutic efficacy in human clinical trials using gene therapy to treat neurological disorders. Herein we thoroughly examine the effects of the innate and adaptive immune responses on therapeutic gene expression mediated by adenoviral, AAV, and lentiviral vectors systems in both pre-clinical and clinical experiments. Furthermore, the immune responses against gene therapy vectors and the resulting loss of therapeutic gene expression are examined in the context of the architecture and neuroanatomy of the brain immune system. The chapter closes with a discussion of the relationship between the elimination of transgene expression and the in vivo immunological synapses between immune cells and target virally infected brain cells. Importantly, although systemic immune responses against viral vectors injected systemically has thought to be deleterious in a number of trials, results from brain gene therapy clinical trials do not support this general conclusion suggesting brain gene therapy may be safer from an immunological standpoint.  相似文献   

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