Stable expression of the ecotropic retrovirus receptor in amphotropic packaging cells facilitates the transfer of recombinant vectors and enhances the yield of retroviral particles.

Retroviral vectors are used widely as gene transfer vehicles. Vector particles are generated by packaging cell lines, which supply the structural proteins gag, pol and env needed to package the retroviral vector RNA. The most efficient way to introduce the vector genome into the packaging cell line is cross-infection with a retroviral vector. Since the infection of a packaging cell line by the produced virus is blocked due to the down regulation of the retrovirus receptor by the envelope glycoprotein, the vector genome should be introduced by a virus with a host tropism different from the one of the packaging cell line. The murine ecotropic retrovirus receptor was expressed in the human amphotropic packaging cell line FLYA13 to generate a cell line which can be infected by murine ecotropic retroviruses. Vector transfer can now be facilitated by cross-infection with the appropriate ecotropic retroviral vectors and provides a simple and efficient method for the generation of amphotropic packaging lines.     

Characterization of recombinant helper retroviruses from moloney-based vectors in ecotropic and amphotropic packaging cell lines

We have characterized the recombinant replication-competent retrovirus (RCV) arising from p delta N2-derived vectors in the packaging cell lines psi 2 (ecotropic) and PA317 (amphotropic). Detailed restriction patterns and sequence of the envelope region of these RCVs has indicated that they arose from recombination events between the virus plasmids used to create the packaging cell line and the vectors. There was no evidence of recombination involving endogenous murine retroviral sequences in the packaging cell line or in transduced hematopoietic cells. In addition, we have confirmed that the mutation of the start codon of the pXM5(N2) derivatives gag+ sequence drastically decreased the occurrence of RCV production. These results offer encouragement that the risk of RCV production can be adequately decreased in gene therapy applications of defective retrovirus vectors.     

高滴度产病毒PA317细胞的制备及基因转移效率的研究

逆转录病毒载体的病毒滴度是其基因转移效率的最关键因素之一。用产病毒载体ψ－２细胞克隆的上清重复感染产病毒载体的ＰＡ３１７细胞克隆和用这两种产病毒细胞克隆进行乒乓上清感染，均能使ＰＡ３１７细胞克隆的病毒滴度提高１－２个数量级且无辅助病毒产生。面同样的造血生长因子应用下，以不同滴度的病毒载体重复感染人骨髓单核细胞，Ｓｏｕｔｈｅｒｎ迷杂交显示，，只有高滴度的病毒载体才能将目的基因较多地转移到人骨髓骨髓核     

Characterization of recombination events leading to the production of an ecotropic replication-competent retrovirus in a GP+envAM12-derived producer cell line

Replication-competent retrovirus (RCR) was identified in a GP+envAM12-derived producer cell, containing the MFG-S-Neo retroviral vector, using a marker rescue assay. Studies were undertaken to determine the origin and structure of this RCR. Receptor interference assays demonstrated that the virus was pseudotyped with an ecotropic envelope. Molecular analysis demonstrated the presence of a MoMLV ecotropic env recombinant where the neomycin resistance gene of the MFG-S-Neo vector was replaced by MoMLV ecotropic env. Additional recombinants linking the retroviral pol gene to neo and the neo gene to MoMLV env were also identified. A full-length MoMLV retroviral genome was detected by nested PCR in the contaminated amphotropic producer cells and in cells infected with its supernatant. Unexpectedly, this was also present in the GP+E86 packaging cells together with a previously undescribed envelope construct possessing a full 5' and 3' LTR, although these cells were consistently negative for the presence of RCR. These anomalies in the GP+E86 packaging cell line result in increased homology with the MFG-S-Neo vector, leading to an increased risk for the production of RCR. Our findings point to a need for increased vigilance when using these packaging lines to generate replication-defective retrovirus.     

Biological effects of a murine retrovirus carrying an activated N-ras gene of human origin

We have introduced a genomic DNA clone of a mutated human N-ras gene from a T-cell leukemia cell line into a retroviral vector equipped with a neo resistance gene and with SV40 and pBR322 origins of replication. The helper free N-ras virus, which was recovered after transfection of the construction in the psi 2 packaging cell line, contained a correctly spliced N-ras gene. Proviral DNA was amplified in cos cells and subsequently cloned in bacteria. Nucleic acid sequence analysis of the activated N-ras gene revealed a point mutation at codon 12 resulting in a glycine to aspartic acid substitution. The N-ras virus was able to transform mouse fibroblastic cell lines, but failed to fully transform mouse primary embryo fibroblasts. MoMuLV or amphotropic 4070A pseudotypes of the virus were injected intraperitoneally into newborn mice. The MoMuLV pseudotype produced only helper-virus-induced leukemias. The amphotropic pseudotype caused fibrosarcomas after a long latent period. The results of these and other in vivo experiments are discussed in relation to known pathogenic effects of other retroviruses carrying H-ras or K-ras genes.     

Amplified and tissue-directed expression of retroviral vectors using ping-pong techniques

Ping-pong amplification is an efficient process by which helper-free retrovirions replicate in cocultures of cell lines that package retroviruses into distinct hostrange envelopes [11]. Transfection of a retroviral vector DNA into these cocultures results in massive virus production, with potentially endless cross-infection between different types of packaging cells. Because the helperfree virus spreads efficiently throughout the coculture, it is unnecessary to use dominant selectable marker genes, and the retroviral vectors can be simplified and optimized for expressing a single gene of interest. The most efficient ping-pong vector, pSFF, derived from the Friend erythroleukemia virus, has been used for high-level expression of several genes that could not be expressed with commonly employed two-gene retroviral vectors. Contrary to previous claims, problems of vector recombination are not inherent to ping-pong methods. Indeed, the pSFF vector has not formed replication-competent recombinants as shown by stringent assays. Here we review these methods, characterize the ping-pong process using the human erythropoietin gene as a model, and describe a new vector (pSFY) designed for enhanced expression in T lymphocytes. Factors that limit tissue-specific expression are reviewed.Abbreviations CAT Chloramphenicol acetyl transferase - Epo Erythropoietin - LTR Long terminal repeat - SFFV Spleen focus-forming virus     

Characterization of R peptide of murine leukemia virus envelope glycoproteins in syncytium formation and entry

Summary The C-terminal R peptide of ecotropic murine leukemia virus (MLV) envelope protein (Env) negatively controls membrane fusion activity. The R peptide cleavage during virion maturation activates its fusogenicity and is required for viral entry. We analyzed fusogenicity and transduction efficiency of mutant Env proteins of ecotropic, amphotropic, polytropic, and xenotropic MLVs. As the result, we found that the hydrophobic amino acid residues around the R peptide cleavage site are important for membrane fusion inhibition by the R peptide. In addition, we found that Env complexes with R peptide-truncated and -containing Env proteins have lower fusogenicity and transduction efficiency than those with the R-peptide-truncated Env alone, suggesting that efficient R peptide cleavage is required for efficient MLV vector transduction. The role of R peptide cleavage in amphotropic, polytropic, and xenotropic MLV infection has not been investigated. We found in this study that the R peptide cleavage is required for amphotropic, xenotropic, and polytropic MLV vector transduction, like with ecotropic MLV. The R-peptide-truncated Env proteins of the xenotropic and polytropic MLVs, however, had much lower fusogenicity than those of the ecotropic and amphotropic MLVs. These results provide valuable information for construction of efficient MLV vectors and for understanding the retroviral entry mechanism.     

Can the use of HIV-1 derived gene transfer vectors for clinical application be justified?

Vectors derived from human immunodeficiency virus type 1 (HIV-1) are an attractive option for many gene therapy applications as they can transduce non-cycling cell populations, and can integrate their genome into the host cell chromosome. The rationale underlying the design of most retroviral vector systems is to segregate the viral cis sequences, which are required for transfer of the viral genome, from the trans sequences that encode viral proteins. This allows the efficient production of replication incompetent virus and has been successfully applied to the generation of HIV-1 vectors. Nonetheless, the possibility that recombination events in the vector production system can generate replication-competent virus, combined with the pathogenic nature of HIV-1, raises major bio-safety issues. Numerous HIV-1 vectors have now been reported, with each generation significantly improved in ways designed to reduce the risk of replication-competent virus being produced. However, progress in vector design needs to be complemented by the development of methods for the quantitation of the probability of replication competent virus being produced. Assaying individual events in the multi-step pathway that can lead to the production of replication-competent virus, rather than relying on the detection of replication-competent virus per se, will be important for quality control purposes. This review will specifically examine the approaches to HIV-1 vector design that have been postulated as increasing bio-safety, possible methods for evaluating bio-safety and whether these approaches are likely to be sufficient to overcome resistance to the use of HIV-1 for clinical application. In addition, we discuss the possible justifications for developing vectors from lentiviruses other than HIV-1.     

逆转录病毒介导DNA修复蛋白在人骨髓细胞中的表达和抗性研究

目的增强造血细胞对化疗药物的耐药表型,探讨逆转录病毒介导的基因转移效率及耐药基因的特性和在造血细胞保护性基因治疗中的作用和意义.方法应用RT-PCR从人肝组织中获得编码六氧甲基鸟嘌呤-DNA-甲基转移酶(MGMT)cDNA,将其克隆于pGEM-T质粒载体并构建了逆转录病毒载体G1Na-MGMT,应用脂质体LipofectAMINE基因转移法将后者导入GP+E86和PA317病毒包装细胞,以BCNU加压筛选后的阳性克隆上清经乒乓效应后继而感染K562细胞和人造血细胞.应用PCR,Southernblot,RT-PCR,Westernblot及MTT法检测人MGMT基因在细胞中的转移和表达.结果酶切鉴定及DNA测序证实其MGMTcDNA克隆的正确性,脂质体介导方法成功将其导入病毒包装细胞,BCNU加压筛选和乒乓感染法使病毒效价达8.6×106CFU/ml,逆转录病毒载体介导的MGMT基因在K562细胞及人造血细胞中获得有效转移和表达.结论MGMT耐药基因的成功克隆并导入骨髓造血细胞且获高效表达对开展肿瘤基因治疗的临床研究奠定了实验基础.     

Improved retroviral packaging lines derived from spleen necrosis virus

Using highly efficient gene expression vectors, we constructed new retroviral packaging lines derived from spleen necrosis virus. Core proteins are expressed from the murine leukemia virus promoter and enhancer followed by the tripartite leader sequence of an adenovirus. Using different plasmids for envelope expression, we found that the efficiency of vector transduction is dependent on the level of gag-pol expression. The level of envelope expression did not have a measurable impact on vector virus titers. The new helper cell lines do not contain any sequences homologous to vector genomes. They transduce standard retrovirus vectors with titers up to 10(6) colony forming units per milliliter of supernatant tissue culture medium. No replication-competent virus was observed.     

Isolation and characterization of new ecotropic murine leukemia viruses after passage of an amphotropic virus in NIH Swiss Mice

Amphotropic murine leukemia viruses (MuLV-A) cause mainly lymphoma in newborn inoculated NIH Swiss mice after a long latent period of 6-12 months. Rarely, however, about 1% of the inoculated mice develop hind limb paralysis and progressive central nervous system disease. The biological properties and RNase T1-resistant oligonucleotide fingerprints of the recovered viruses from tissues of both lymphomatous and paralyzed mice inoculated with MuLV-A were analyzed. These results indicate that serial in vivo passages of MuLV-A in NIH Swiss mice lead to generation of new MuLVs of both amphotropic and ecotropic host ranges. The recovered amphotropic viruses are highly lymphomagenic and are recombinants of MuLV-A-specific oligonucleotides and endogenous mouse sequences. The ecotropic viruses fall into two groups: (1) recombinants of MuLV-A genes and NIH Swiss mouse viral or cellular sequences and (2) new ecotropic viruses with oligonucleotide fingerprints not related to any of the known MuLVs. The naturally occurring ecotropic MuLVs of the wild mice produce both lymphoma and paralysis in NIH Swiss mice. The viruses recovered from in vivo passages are mainly of ecotropic host range although dual-tropic virus activity is occasionally seen in the spleens but not in the brains or spinal cords of the lymphomatous or paralyzed mice. Oligonucleotide fingerprinting of the recovered MuLV-Es from paralyzed mice are identical to the input MuLV-Es, indicating that the parental MuLV-E alone, without recombination, is responsible for the paralytic disease.     

Mouse mammary tumor virus mediated transfer and expression of neomycin resistance to infected cultured cells

The mammary gland specific, glucocorticoid controlled expression of MMTV makes it an ideal candidate for the basis of a nonpromiscuous regulated retroviral vector system. We have previously constructed an MMTV proviral variant that gives rise to virus particles upon introduction into cultured cells. This was used to construct a defective MMTV provirus in which the MMTV env gene was replaced by the neomycin resistance gene under the control of the herpes simplex thymidine kinase promotor. The defective provirus was packaged after transfection into two distinct MMTV producing cell lines. Conditioned medium from these cells contains virus particles which are able to infect cells of fibroblast and epithelial origin and to confer neomycin resistance upon them. This indicates that the defective MMTV provirus contains the sequences required for packaging of the genomic viral RNA. Transfection of the same MMTV-neo recombinant provirus into the MoMLV packaging cell line, psi 2, did not result in any infectious virus particles. Thus the packaging signals for MMTV and MoMLV appear to be distinct. Analysis of the MMTV infected cells reveals the presence of the MMTV-neo recombinant provirus. Expression of both MMTV and neomycin is detectable and augmented when the infected cells are grown in the presence of glucocorticoid hormone.     

逆转录病毒载体介导乙型肝炎病毒反义基因的转录表达

为了探索在真核细胞内转录表达乙型肝炎病毒（ＨＢＶ）反义核酸的方法，用基因重组技术将ＨＢＶ前Ｃ／Ｃ基因（ＰｒｅＣ／Ｃ）和前Ｓ／Ｓ基因（ＰｒｅＳ／Ｓ）片段反向插入逆转录病毒载体质粒，再将重组体分别转染ＰＡ３１７包装细胞，进而获得能够介导ＨＢＶ反义基因向小鼠ＮＩＨ３Ｔ３细胞转移表达的重组逆转录病毒。经分子杂交试验表明，含有ＨＢＶ反义基因的重组逆转录病毒序列已经整合到转染的ＰＡ３１７细胞染色体上；转导的ＮＩＨ３Ｔ３细胞内有ＨＢＶ反义ＲＮＡ转录表达。结论：逆转录病毒载体包装细胞系统能够介导ＨＢＶ反义基因在真核细胞中转录表达，因而有可能利用反义技术和基因转移方法进行抗－ＨＢＶ基因治疗     

反义HLA-A2和GDNF共表达逆转录病毒载体的构建和鉴定

为构建GDNF和反义HLA -A2共表达的逆转录病毒载体,将人GDNF酶切片段(XhoI/SalI)克隆于逆转录病毒载体pLNCX2 的XhoI位点,再将HLA A2cDNA片段反向克隆于上述重组载体的HindIII/SalI位点,对其作酶切鉴定和测序后转染PA317包装细胞系进行病毒的包装。收获重组病毒感染的NIH3T3并进行病毒滴度的测定,GDNF及HLA- A2表达的RT -PCR检测,进一步感染人胚肺成纤维细胞,观察GDNF分泌情况。结果表明:GDNF和反义HLA -A2两片段的序列和插入方向完全正确,包装后获得的重组病毒的平均滴度为5×105 CFU/ml。RT PCR显示:在小鼠源性的PA317细胞中有人GDNF和HLA A2的表达,ELISA法测得病毒感染人胚肺成纤维细胞上清液中GDNF含量为450pg/ml。通过本实验,我们获得能共表达GDNF和反义HLA -A2的重组逆转录病毒,其转染的人成纤维细胞具有合成GDNF的能力。