Production of high-titer helper-free retroviruses by transient transfection.

The generation of high-titer, helper-free retroviruses by transient transfection has been achieved by using the highly transfectable 293T cell line into which are stably introduced constructs that express retroviral packaging functions. The resulting ecotropic virus packaging cell line BOSC 23 produces infectious retrovirus at > 10(6) infectious units/ml of supernatant within 72 hr after CaPO4-mediated transfection. A stringent assay for replication-competent virus showed that no helper virus was present. The system can produce high titers of retroviral vectors expressing genes that are extremely difficult to propagate at high titer in stable producer lines. This method should facilitate and extend the use of helper-free retroviral gene transfer, as well as be useful for gene therapy.     

乙型肝炎病毒(adr亚型)重组逆转录病毒包装及滴度测定

目的包装乙型肝炎病毒（adr亚型）重组逆转录病毒并进行滴度测定，为下一步构建乙型肝炎病毒（adr亚型）转基因小鼠奠定基础。方法将乙型肝炎病毒（adr）全基因组插入到逆转录病毒载体pLNCL中获得了乙型肝炎（adr）重组逆转录病毒载体，鉴定正反向后，脂质体转染PA317包装细胞，G418抗性压力下进行筛选得到阳性细胞克隆，扩大培养后进行滴度测定，选取产毒率高细胞克隆株，然后扩大培养、浓缩，进行滴度测定。结果分别获得HBV（adr）全基因组正向和反向插入的重组逆转录病毒。正连重组逆转录病毒载体经PA317细胞包装，经筛选、浓缩后滴度为10^6CFU／ml，反连重组逆转录病毒载体经PA317细胞包装，经筛选、浓缩后滴度为10^5CFU／ml。结论PA317细胞包装了含乙型肝炎病毒（adr）全基因组的重组逆转录病毒。     

Efficient infection of a human T-cell line and of human primary peripheral blood leukocytes with a pseudotyped retrovirus vector.

Peripheral blood lymphocytes (PBLs) are an important target for gene transfer studies aimed at human gene therapy. However, no reproducibly efficient methods are currently available to transfer foreign, potentially therapeutic genes into these cells. While vectors derived from murine retroviruses have been the most widely used system, their low infection efficiency in lymphocytes has required prolonged in vitro culturing and selection after infection to obtain useful numbers of genetically modified cells. We previously reported that retroviral vectors pseudotyped with vesicular stomatitis G glycoprotein (VSV-G) envelope can infect a wide variety of cell types and can be concentrated to titers of greater than 10(9) infectious units/ml. In this present study, we examined the ability of amphotropic and pseudotyped vectors expressing a murine cell surface protein, B7-1, to infect the human T-cell line Jurkat or human blood lymphocytes. Limiting dilution analysis of transduced Jurkat cells demonstrated that the pseudotyped vector is significantly more efficient in infecting T cells than an amphotropic vector used at the same multiplicity of infection (moi). To identify the transduction efficiency on PBLs, we examined the levels of cell surface expression of the B7-1 surface marker 48 to 72 hr after infection. The transduction efficiency of PBLs with the pseudotyped vector increased linearly with increasing moi to a maximum of approximately 16-32% at an moi of 40. This relatively high efficiency of infection of a T-cell line and of blood lymphocytes with VSV-G pseudotyped virus demonstrates that such modified pseudotyped retrovirus vectors may be useful reagents for studies of gene therapy for a variety of genetic or neoplastic disorders.     

Generation of packaging cell lines for pseudotyped retroviral vectors of the G protein of vesicular stomatitis virus by using a modified tetracycline inducible system.

We have previously shown that the G protein of vesicular stomatitis virus (VSV-G) can be incorporated into the virions of retroviruses. Since expression of VSV-G is toxic to most mammalian cells, development of stable VSV-G packaging cell lines requires inducible VSV-G expression. We have modified the tetracycline-inducible system by fusing the ligand binding domain of the estrogen receptor to the carboxy terminus of a tetracycline-regulated transactivator. Using this system, we show that VSV-G expression is tetracycline-dependent and can be modulated by beta-estradiol. Stable packaging cell lines can readily be established and high-titer pseudotyped retroviral vectors can be generated upon induction of VSV-G expression.     

Safe and efficient generation of recombinant retroviruses with amphotropic and ecotropic host ranges.

We have constructed a set of packaging cell lines useful for the generation of helper-free recombinant retroviruses with amphotropic and ecotropic host ranges. To eliminate the problems of transfer of packaging functions and helper virus formation encountered with the previously available packaging systems, two mutant Moloney murine leukemia virus-derived proviral genomes carrying complementary mutations in the gag-pol or env regions were sequentially introduced into NIH 3T3 cells by cotransformation. Both genomes contained a deletion of the psi sequence necessary for the efficient encapsidation of retroviral genomes into virus particles and additional alterations at the 3' end of the provirus. We show that the resulting packaging cell lines psi CRIP and psi CRE can be used to isolate clones that stably produce high titers (10(6) colony-forming units/ml) of recombinant retroviruses with amphotropic and ecotropic host ranges, respectively. More importantly, we demonstrate that viral producers derived from the packaging cell lines do not transfer the packaging functions, or yield helper virus, even under conditions where existing packaging cell lines can be shown to yield transfer of packaging functions and/or helper virus. These properties of the psi CRIP and psi CRE packaging lines make them particularly valuable reagents for in vivo gene transfer studies aimed at cell lineage analysis and the development of human gene replacement therapies.     

Vesicular stomatitis virus G glycoprotein pseudotyped retroviral vectors: concentration to very high titer and efficient gene transfer into mammalian and nonmammalian cells.

The restricted host-cell range and low titer of retroviral vectors limit their use for stable gene transfer in eukaryotic cells. To overcome these limitations, we have produced murine leukemia virus-derived vectors in which the retroviral envelope glycoprotein has been completely replaced by the G glycoprotein of vesicular stomatitis virus. Such vectors can be concentrated by ultracentrifugation to titers > 10(9) colony-forming units/ml and can infect cells, such as hamster and fish cell lines, that are ordinarily resistant to infection with vectors containing the retroviral envelope protein. The ability to concentrate vesicular stomatitis virus G glycoprotein pseudotyped vectors will facilitate gene therapy model studies and other gene transfer experiments that require direct delivery of vectors in vivo. The availability of these pseudotyped vectors will also facilitate genetic studies in nonmammalian species, including the important zebrafish developmental system, through the efficient introduction and expression of foreign genes.     

A general method for the generation of high-titer, pantropic retroviral vectors: highly efficient infection of primary hepatocytes.

Retroviral vectors have been central components in many studies leading to human gene therapy. However, the generally low titers and inefficient infectivity of retroviral vectors in human cells have limited their use. We previously reported that the G protein of vesicular stomatitis virus can serve as the exclusive envelope protein component for one specific retroviral vector, LGRNL, that expresses vesicular stomatitis virus G. We now report a more useful general transient transfection scheme for producing very high-titer vesicular stomatitis virus G-enveloped pseudotypes from any Moloney murine leukemia-based retroviral vector without having to rely on the expression of the cytotoxic G protein from the retroviral vector itself. We also demonstrate very high efficiency of infection with a pseudotyped lacZ vector in primary mouse hepatocytes. We suggest that pseudotyped retroviral vectors carrying reporter genes will permit genetic studies in many previously inaccessible vertebrate and invertebrate systems. Furthermore, because these vectors represent retroviral vectors of sufficiently high titer to allow efficient direct retroviral-mediated in vivo gene transfer, we also suggest that pseudotyped vectors carrying potentially therapeutic genes will become useful to test the potential for in vivo gene therapy.     

端粒酶逆转录酶激活脐静脉内皮细胞端粒酶活性

目的　探讨外源性端粒酶逆转录酶 (hTERT)能否激活人脐静脉内皮细胞端粒酶活性 ,为建立体外人脐静脉内皮细胞系奠定基础。方法　用逆转录病毒转染法 ,将编码hTERT片段逆转录病毒载体与VSV G共转染 2 93 GP2 细胞 ,产生病毒上清液 ,感染原代分离的人脐静脉内皮细胞 (hUVEC) ,以嘌呤霉素筛选。观察细胞生长曲线、第Ⅷ因子、CD34、β 半乳糖苷酶染色、逆转录 聚合酶链反应 (RT PCR)等指标。 结果　细胞生长曲线显示培养至 30代的转染细胞生长速度基本与原代培养脐静脉内皮细胞一致但快于原代培养传至第 10代内皮细胞 ;RT PCR结果转染细胞有特异扩增 ;衰老指标 β 半乳糖苷酶染色显示转染后细胞胞浆内着染阳性细胞数明显少于第 10代内皮细胞。结论　外源性hTERT转入原代培养人脐静脉内皮细胞 ,可重现其端粒酶活性 ,从而阻止人脐静脉内皮细胞老化     

Construction of retroviral vectors to induce a strong expression of human class Ⅰ interferon gene in human hepatocellular carcinoma cells in vitro

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Development of packaging cell lines for generation of adeno-associated virus vectors by lentiviral gene transfer of trans-complementary components

Adeno-associated virus (AAV) vector system has several useful advantages with regard to in vitro and in vivo gene transfer. However, their usages have been limited by cumbersome and labor-intensive vector production in the traditional method. To overcome limitations in AAV production, in this report, we explored the possibility of generating AAV packaging cell line, 293T R/C.VA.E2A.E4. cells, by using lentivirus-mediated transduction of Rep/Cap gene of AAV-2, VA RNA, E2A, and E4 genes of Ad5 into 293T cells. In packaging cell lines, it is important that supply of the AAV vector can be stably performed for long time. We showed that the 293T R/C.VA.E2A.E4. cells have stably maintained the transduced components after more than 10 passages and yielded high-titer AAV vectors, and the titer of AAV vectors did not decline even if culture of the packaging cells was continued for long time. The Rep/Cap and E4 gene products caused no remarkable cytotoxicity. The 293T R/C.VA.E2A.E4. cells might be able to tolerate the Rep/Cap and E4 gene products, or have less copy numbers of the Rep/Cap and E4 genes than the traditional method. Moreover, we showed that the AAV vectors derived from 293T R/C.VA.E2A.E4. cells infected the primary human CD34+ haematopoietic progenitor cells with high efficiency (50-70%). In the 293T R/C.VA.E2A.E4. cells, the AAV vectors can be generated by the transfection of one AAV vector plasmid, and large-scale AAV production can be easily achieved. It is important that cumbersome, variable, and costly transfection is avoided.     

A retrovirus carrying an MDR1 cDNA confers multidrug resistance and polarized expression of P-glycoprotein in MDCK cells.

A full-length cDNA for the human multidrug resistance gene 1 (MDR1) has been inserted into a retroviral vector containing a murine Harvey sarcoma virus from which the viral oncogene was deleted. Ecotropic and amphotropic virus was produced after transfection of this vector into psi-2 and PA-12 packaging cell lines. This virus conferred the full phenotype of multidrug resistance on mouse and human cell lines. Viral titers of up to 2 X 10(5) drug-resistant colonies per ml were observed. Infected cells became resistant to colchicine, vinblastine, doxorubicin, VP16 (etoposide), and puromycin, but not cisplatin, indicating that the presence of the human MDR1 gene is sufficient to cause multidrug resistance. When the dog kidney cell line MDCK was infected with the MDR1 virus, P-glycoprotein was expressed in a polarized manner on the upper surface of the cells, showing that the cloned cDNA also encodes information for polarized expression of P-glycoprotein. The MDR1 virus should be useful for introducing this drug resistance gene into a variety of cell types for biological experiments in vitro and in vivo.     

Protection of hematopoietic cells from O6-alkylation damage by O6-methylguanine DNA methyltransferase gene transfer: studies with different O6-alkylating agents and retroviral backbones

Overexpression of O(6)-methylguanine DNA methyltransferase (MGMT) can protect hematopoietic cells from O(6)-alkylation damage. To identify possible clinical applications of this technology we compared the effect of MGMT gene transfer on the hematotoxicity induced by different O(6)-alkylating agents in clinical use: the chloroethylnitrosoureas ACNU, BCNU, CCNU and the tetrazine derivative temozolomide. In addition, various retroviral vectors expressing the MGMT-cDNA were investigated to identify optimal viral backbones for hematoprotection by MGMT expression. Protection from ACNU, BCNU, CCNU or temozolomide toxicity was evaluated utilizing a Moloney murine leukemia virus-based retroviral vector (N2/Zip-PGK-MGMT) to transduce primary murine bone marrow cells. Increased resistance in murine colony-forming units (CFU) was demonstrated for all four drugs. In comparison to mock-transduced controls, after transduction with N2/Zip-PGK-MGMT the IC50 for CFU increased on average 4.7-fold for ACNU, 2.5-fold for BCNU, 6.3-fold for CCNU and 1.5-fold for temozolomide. To study the effect of the retroviral backbone on hematoprotection various vectors expressing the human MGMT-cDNA from a murine embryonic sarcoma virus LTR (MSCV-MGMT) or a hybrid spleen focus-forming/murine embryonic sarcoma virus LTR (SF1-MGMT) were compared with the N2/Zip-PGK-MGMT vector. While all vectors increased resistance of transduced human CFU to ACNU, the SF1-MGMT construct was most efficient especially at high ACNU concentrations (8-12 microg/ml). Similar results were obtained for protection of murine high-proliferative-potential colony-forming cells. These data may help to optimize treatment design and retroviral constructs in future clinical studies aiming at hematoprotection by MGMT gene transfer.     

Retroviral transfer of genes into canine hemopoietic progenitor cells in culture: a model for human gene therapy.

Amphotropic retroviral vectors containing either a mutant dihydrofolate reductase gene (DHFR) or the bacterial neomycin phosphotransferase gene (neo) were used to infect canine hemopoietic cells. We report successful transfer and expression of the DHFR and neo genes in canine hemopoietic progenitor cells (colony-forming units, granulocyte/macrophage) as measured by the ability of the viruses to confer resistance to either methotrexate or the aminoglycoside G418, respectively. Transfer was achieved in the absence of helper virus by using retrovirus packaging cell lines. Successful transfer of these genes into canine hemopoietic progenitor cells in vitro indicates the feasibility of gene transfer into canine marrow for autologous reconstitution. Studies of transfer of new genetic information into a large, outbred animal such as the dog will provide a preclinical model for future gene therapy in humans.     

Generation of targeted retroviral vectors by using single-chain variable fragment: an approach to in vivo gene delivery.

We report the generation of a retroviral vector that infects human cells specifically through recognition of the low density lipoprotein receptor. The rationale for this targeted infection is to add onto the ecotropic envelope protein of Moloney murine leukemia virus, normally trophic for murine cells, a single-chain variable fragment derived from a monoclonal antibody recognizing the human low density lipoprotein receptor. This chimeric envelope protein was used to construct a packaging cell line producing a retroviral vector capable of high-efficiency transfer of the Escherichia coli beta-galactosidase gene to human cells expressing low density lipoprotein receptor. This approach offers a generalized plan to generate cell and tissue-specific retroviral vectors, an essential step toward in vivo gene therapy strategies.     

Selection of transduced CD34+ progenitors and enzymatic correction of cells from Gaucher patients, with bicistronic vectors.

The gene transfer efficiency of human hematopoietic stem cells is still inadequate for efficient gene therapy of most disorders. To overcome this problem, a selectable retroviral vector system for gene therapy has been developed for gene therapy of Gaucher disease. We constructed a bicistronic retroviral vector containing the human glucocerebrosidase (GC) cDNA and the human small cell surface antigen CD24 (243 bp). Expression of both cDNAs was controlled by the long terminal repeat enhancer/promoter of the Molony murine leukemia virus. The CD24 selectable marker was placed downstream of the GC cDNA and its translation was enhanced by inclusion of the long 5' untranslated region of encephalomyocarditis virus internal ribosomal entry site. Virus-producing GP+envAM12 cells were created by multiple supernatant transductions to create vector producer cells. The vector LGEC has a high titer and can drive expression of GC and the cell surface antigen CD24 simultaneously in transduced NIH 3T3 cells and Gaucher skin fibroblasts. These transduced cells have been successfully separated from untransduced cells by fluorescence-activated cell sorting, based on cell surface expression of CD24. Transduced and sorted NIH 3T3 cells showed higher GC enzyme activity than the unsorted population, demonstrating coordinated expression of both genes. Fibroblasts from Gaucher patients were transduced and sorted for CD24 expression, and GC enzyme activity was measured. The transduced sorted Gaucher fibroblasts had a marked increase in enzyme activity (149%) compared with virgin Gaucher fibroblasts (17% of normal GC enzyme activity). Efficient transduction of CD34+ hematopoietic progenitors (20-40%) was accomplished and fluorescence-activated cell sorted CD24(+)-expressing progenitors generated colonies, all of which (100%) were vector positive. The sorted, CD24-expressing progenitors generated erythroid burst-forming units, colony-forming units (CFU)-granulocyte, CFU-macrophage, CFU-granulocyte/macrophage, and CFU-mix hematopoietic colonies, demonstrating their ability to differentiate into these myeloid lineages in vitro. The transduced, sorted progenitors raised the GC enzyme levels in their progeny cells manyfold compared with untransduced CD34+ progenitors. Collectively, this demonstrates the development of high titer, selectable bicistronic vectors that allow isolation of transduced hematopoietic progenitors and cells that have been metabolically corrected.     

Expression of human class II major histocompatibility complex antigens using retrovirus vectors

Retrovirus vectors [direct orientation (DO) vectors] that permit the simultaneous expression of an inserted protein-coding sequence and a dominant-acting selectable marker have been constructed. In these vectors, an internal simian virus 40 or human metallothionein promoter sequence serves to drive the expression of the bacterial neomycin phosphotransferase or guanine-xanthine phosphoribosyltransferase genes, whereas the viral long terminal repeat sequences are utilized to promote expression of inserted sequences. In some of the vectors, the viral 5' splice site, normally used in the biogenesis of the subgenomic env-encoding mRNA, has been eliminated. These vectors yield high transient and stable titers of virus after transfection of viral packaging cell lines, show little or no depression of virus titer with a variety of inserts, and faithfully transmit recombinant proviral sequences to recipient cells. To characterize the expression potential of these vectors, a variety of inserts encoding the alpha and beta subunits of the human major histocompatibility complex class II antigen HLA-DR have been introduced into these vectors. NIH 3T3 cells infected by viruses containing HLA-DR alpha or beta cDNAs express these proteins as shown by immunoprecipitation of metabolically labeled extracts. In addition, through the sequential infection of cells with retrovirus constructions expressing two different selectable markers, both subunits of the class II antigen have been introduced into NIH 3T3 cells. Such infected cells express HLA-DR molecules at the cell surface.