High-efficiency gene transfer into CD34+ cells with a human immunodeficiency virus type 1-based retroviral vector pseudotyped with vesicular stomatitis virus envelope glycoprotein G.

Currently, amphotropic retroviral vectors are widely used for gene transfer into CD34+ hematopoietic progenitor cells. The relatively low levels of transduction efficiency associated with these vectors in human cells is due to low viral titers and limitations in concentrating the virus because of the inherent fragility of retroviral envelopes. Here we show that a human immunodeficiency virus type 1 (HIV-1)-based retroviral vector containing the firefly luciferase reporter gene can be pseudotyped with a broad-host-range vesicular stomatitis virus envelope glycoprotein G (VSV-G). Higher-efficiency gene transfer into CD34+ cells was achieved with a VSV-G-pseudotyped HIV-1 vector than with a vector packaged in an amphotropic envelope. Concentration of virus without loss of viral infectivity permitted a higher multiplicity of infection, with a consequent higher efficiency of gene transfer, reaching 2.8 copies per cell. These vectors also showed remarkable stability during storage at 4 degrees C for a week. In addition, there was no significant loss of titer after freezing and thawing of the stock virus. The ability of VSV-G-pseudotyped retroviral vectors to achieve a severalfold increase in levels of transduction into CD34+ cells will allow high-efficiency gene transfer into hematopoietic progenitor cells for gene therapy purposes. Furthermore, since it has now become possible to infect CD34+ cells with pseudotyped HIV-1 with a high level of efficiency in vitro, many important questions regarding the effect of HIV-1 on lineage-specific differentiation of hematopoietic progenitors can now be addressed.     

Variable stability of a selectable provirus after retroviral vector gene transfer into human cells.

Human lymphoblasts deficient in the enzyme hypoxanthine-guanine phosphoribosyltransferase (HPRT) were infected with an amphotropic helper-free retroviral vector expressing human HPRT cDNA. The stability and expression of the HPRT provirus in five cell lines with different proviral integration sites were examined by determining HPRT mutation and reversion frequencies and by blot hybridization studies. Mutation to the HPRT-negative phenotype occurred at frequencies of approximately 4 X 10(-5) to 3 X 10(-6) per generation. Most mutations in each of the five cell lines were associated with partial or complete deletions or rearrangements of the provirus. Several mutants retained a grossly intact HPRT provirus, and in one such mutant HPRT shutdown resulted from a revertible epigenetic mechanism that was not associated with global changes in proviral methylation. Therefore, mutation and shutdown of the HPRT provirus in human lymphoblasts result from mechanisms similar to those reported for several other avian and mammalian replication-competent retroviruses.     

High efficiency gene transfer into murine T cell clones using a retroviral vector

To establish a gene transfer and expression system for murine T cell clones, we have introduced the neomycin phosphotransferase gene encoding resistance to the neomycin analogue, G418, into non-neoplastic inducer T cell clones by using a replication-defective retroviral vector. This method allowed highly efficient gene transfer (20 to 40%) into two inducer T cell clones. The level of viral RNA expression in G418r T cells was 0.1% of poly(A)+ RNA. The infected G418r cells retained physiologic responsiveness to specific antigen as judged by antigen-specific proliferation and production of IL 3.     

A new yeast artificial chromosome vector designed for gene transfer into mammalian cells

This report describes the construction of a new yeast artificial chromosome (YAC) vector designed for gene transfer into mammalian cells. For ease of use, the two arms of the vector were cloned separately. The vector harbours the Neo and Hyg genes for dominant selection in mammalian cells, a putative human origin of replication, a synthetic matrix attachment region and two loxP sites (one on each arm). The cloning ability of the vector was demonstrated by successful propagation of the cDNA of the cystic fibrosis gene, CFTR, as a YAC in Saccharomyces cerevisiae. A YAC containing the entire CFTR gene was also constructed by retrofitting the two arms of a pre-existing clone (37AB12) with the two arms of the novel vector. Both the cDNA and entire gene containing YACs were circularized in yeast by inducible expression of the Cre recombinase. Recombination occurred very specifically at the loxP sequences present on the two arms of the YAC. Applications of the vector to gene transfer are discussed.     

靶向WNT5A基因的shRNA逆转录病毒表达载体的构建与鉴定

目的:构建人WNT5A shRNA逆转录病毒表达载体.方法:根据人WNT5A基因mRNA序列设计并合成两条互补的DNA单链寡核苷酸,将退火后形成的双链连接于pSUPER Retro RNAi质粒,构建WNT5A shRNA逆转录病毒表达载体,经脂质体介导入GPG293细胞,包装成逆转录病毒.用该逆转录病毒感染人鼻咽癌细胞,Western blot法和RT-PCR检测细胞WNT5A的表达.结果:目的序列成功连接于载体并包装成逆转录病毒,免疫印迹检测和RT-PCR检测结果表明构建的WNT5A shRNA逆转录病毒表达载体能显著抑制鼻咽癌细胞WNT5A的表达.结论:成功构建人WNT5A shRNA逆转录病毒表达载体.     

Highly efficient gene transfer into antigen-specific primary mouse lymphocytes with replication-deficient retrovirus expressing the 10A1 envelope protein

Background

Introduction of recombinant genes in the genome of primary lymphocytes by virtue of a replication‐deficient retrovirus can be used in immunological studies and for cell‐based gene therapy.

Methods

Packaging cells GP+E86 producing replication‐deficient retrovirus incorporating the genes of enhanced green fluorescent protein (eGFP), C2γ or C2ξ, were generated by calcium phosphate‐mediated transfection. Clones with the highest titres of retrovirus vector were isolated from them and their supernatants were used for transduction of PT67 cells. Primary mouse lymphocytes and T‐cell hybridoma MD.45 were transduced by centrifugation with retroviral stock. The retroviral content of packaging cell supernatants was determined by dot blotting and hybridization with a DNA probe.

Results

PT67 cells produced ～50 times more retrovirus vector than the original GP+E86 clones. When retroviral stocks of PT67 and GP+E86 cells were used at 1/50 dilution and undiluted, respectively (to normalize them forretroviral RNA content), the transduction efficiency of mouse T‐cell hybridoma was 40% and 5%, respectively. Centrifugation of target cells with retroviral stock at 2000 g for 60 min increased the percentage of transduced cells two‐ to three‐fold. Within a population of cells isolated from the draining lymph nodes of an immunized mouse and reactivated with an antigen, up to 60% of CD4⁺ T cells and up to 80% of B cells could be transduced with a transgene in replication‐deficient retrovirus packaged by PT67 cells using the optimized gene transfer protocol.

Conclusions

This protocol allows for the generation of packaging cells producing high titres of retrovirus vector. The 10A1 envelope protein is superior to the ecotropic one for the transduction of mouse lymphocytes. Copyright © 2002 John Wiley & Sons, Ltd.

     

A customized retroviral vector confers marker gene expression in osteoclast lineage cells

Osteoclasts play a seminal role in many skeletal diseases and therefore are candidates for cell-based gene delivery systems to treat disorders of bone. As an initial step toward developing osteoclast-mediated gene delivery systems, we have made and analyzed a customized Molony-Murine leukemia virus (MMLV)-based retroviral vector containing elements of the osteoclast-specific tartrate-resistant acid phosphatase (TRAP) gene. RAW 264.7 cells were transduced with the customized vector (E3) and differentiated along macrophage or osteoclast lineages. E3 contained a truncated form of the human nerve growth factor receptor (NGFR) as a reporter gene. NGFR expression increased with RANK-ligand (RANK-L) treatment but not with macrophage (gamma-IFN/LPS treatment) differentiation. Enhanced NGFR expression peaked 48 h after RANK-L treatment. Electrophoretic mobility shift assays (EMSA) analysis of the TRAP gene regulatory elements in E3 identified a single 27 bp DNA probe, which specifically bound protein from RANK-L-treated cells. DNA sequence revealed AP-1 binding sites, and analysis with mutant probes implied that the sites were functional. EMSA supershift analysis identified Fos protein interacting with the 27 bp probe. In summary, insertion of sequence -962 to -868 from the TRAP gene into the U3 region of the MMLV LTR confers RANK-L induced retroviral gene expression via Fos family protein interaction at AP-1 sites.     

Gene transfer into hemopoietic stem cells using retroviral vectors

Gene transfer to hemopoietic cells offers a variety of new approaches to the experimental hematologist as well as potentially providing a means for correcting a number of genetic disorders of humans. From the experimental viewpoint, gene transfer utilizing retroviral vectors introduces new methods for analyzing hemopoietic cell lineages, and the effects of over-expression of genes affecting hemopoietic cell proliferation and differentiation. The unique properties of retroviral vectors and the optimized methods currently in use to infect hemopoietic cells are represented as a brief review of a rapidly expanding new field of experimental hematology.     

O-linked glycosylation of retroviral envelope gene products.

Treatment of [3H]glucosamine-labeled Friend mink cell focus-forming virus (FrMCF) gp70 with excess peptide:N-glycanase F (PNGase F) resulted in removal of the expected seven N-linked oligosaccharide chains; however, approximately 10% of the glucosamine label was retained in the resulting 49,000-Mr (49K) product. For [3H]mannose-labeled gp70, similar treatment led to removal of all the carbohydrate label from the protein. Prior digestion of the PNGase F-treated gp70 with neuraminidase resulted in an additional size shift, and treatment with O-glycanase led to the removal of almost all of the PNGase F-resistant sugars. These results indicate that gp70 possesses sialic acid-containing O-linked oligosaccharides. Analysis of intracellular env precursors demonstrated that O-linked sugars were present in gPr90env, the polyprotein intermediate which contains complex sugars, but not in the primary translation product, gPr80env, and proteolytic digestion studies allowed localization of the O-linked carbohydrates to a 10K region near the center of the gp70 molecule. Similar substituents were detected on the gp70s of ecotropic and xenotropic murine leukemia viruses and two subgroups of feline leukemia virus, indicating that O-linked glycosylation is a conserved feature of retroviral env proteins.     

Expression of the human beta-globin gene after retroviral transfer into murine erythroleukemia cells and human BFU-E cells.

Replication-defective amphotropic retrovirus vectors containing either the human beta-globin gene with introns or an intronless beta-globin minigene were constructed and used to study beta-globin expression following gene transfer into hematopoietic cells. The beta-globin genes were marked by introducing a 6-base-pair insertion into the region corresponding to the 5' untranslated region of the beta-globin mRNA to allow detection of RNA encoded by the new gene in human cells expressing normal human beta-globin RNA. Introduction of a virus containing the beta-globin gene with introns into murine erythroleukemia cells resulted in inducible expression of human beta-globin RNA and protein, while the viruses containing the minigene were inactive. The introduced human beta-globin gene was 6 to 110% as active as the endogenous mouse beta maj-globin genes in six randomly chosen cell clones. Introduction of the viruses into human BFU-E cells, followed by analysis of marked and unmarked globin RNAs in differentiated erythroid colonies, revealed that the introduced beta-globin gene was about 5% as active as the endogenous genes in these normal human erythroid cells and that again the minigene was inactive. These data are discussed in terms of the potential treatment of genetic disease by gene therapy.     

Enhancing glycoprotein sialylation by targeted gene silencing in mammalian cells

Recombinant glycoproteins produced by mammalian cells represent an important category of therapeutic pharmaceuticals used in human health care. Of the numerous sugars moieties found in glycoproteins, the terminal sialic acid is considered particularly important. Sialic acid has been found to influence the solubility, thermal stability, resistance to protease attack, antigenicity, and specific activity of various glycoproteins. In mammalian cells, it is often desirable to maximize the final sialic acid content of a glycoprotein to ensure its quality and consistency as an effective pharmaceutical. In this study, CHO cells overexpressing recombinant human interferon gamma (hIFNγ) were treated using short interfering RNA (siRNA) and short‐hairpin RNA (shRNA) to reduce expression of two newly identified sialidase genes, Neu1 and Neu3. By knocking down expression of Neu3 we achieved a 98% reduction in sialidase function in CHO cells. The recombinant hIFNγ was examined for sialic acid content that was found to be increased 33% and 26% respectively with samples from cell stationary phase and death phase as compared to control. Here, we demonstrate an effective targeted gene silencing strategy to enhance protein sialylation using RNA interference (RNAi) technology. Biotechnol. Bioeng. 2010;105: 1094–1105. © 2009 Wiley Periodicals, Inc.     

Encapsulated cells producing retroviral vectors for in vivo gene transfer

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.

     

Development of targeted gene transfer into human primary T lymphocytes and macrophages using high-titer recombinant HIV vectors

Primary human lymphocytes and macrophages are an important target cells for human immunodeficiency virus (HIV). For targeted gene transfer into CD4(+) lymphocytes and macrophages, we constructed HIV vectors with envelope glycoprotein (gp120) from the T-cell tropic BH10 strain and the macrophage tropic SF162, and developed an improved strategy for preparation of high-titer HIV vectors. Among several possible procedures, we found that ultrafiltration using CENTRIPREP columns was highly effective to concentrate HIV particles. The titer could be increased four orders of magnitudes. The total recovery was more than 80%. No replication-competent cytopathic HIV was detected in concentrated vector preparation. Using the high-titer HIV vector carrying the enhanced green fluorescent protein (EGFP) gene, we transduced human primary lymphocytes and macrophages. FACS analysis showed that the T-cell tropic vector could transduce 40-80% of CD4(+) T-cells stimulated with IL2 plus PHA and 20-50% of unstimulated cells. The macrophage tropic vector was shown to transduce approximately 20% of terminally differentiated macrophages. These results represent the initial report of targeted gene transfer into terminally differentiated macrophages. These results also indicate that these HIV vectors are useful for the manipulation of gene expression in HIV infectable cells and the development of gene therapy targeting lymphocytes and macrophages.     

逆转录病毒载体在血友病A基因治疗中的研究进展

基因治疗是彻底治愈血友病A的最理想方法,逆转录病毒是最为常用的载体之一,本文对逆转录病毒在血友病A基因治疗中的研究进展作一综述。     

Maturation of HIV envelope glycoprotein precursors by cellular endoproteases

The entry of enveloped viruses into its host cells is a crucial step for the propagation of viral infection. The envelope glycoprotein complex controls viral tropism and promotes the membrane fusion process. The surface glycoproteins of enveloped viruses are synthesized as inactive precursors and sorted through the constitutive secretory pathway of the infected cells. To be infectious, most of the viruses require viral envelope glycoprotein maturation by host cell endoproteases. In spite of the strong variability of primary sequences observed within different viral envelope glycoproteins, the endoproteolytical cleavage occurs mainly in a highly conserved domain at the carboxy terminus of the basic consensus sequence (Arg-X-Lys/Arg-Arg downward arrow). The same consensus sequence is recognized by the kexin/subtilisin-like serine proteinases (so called convertases) in many cellular substrates such as prohormones, proprotein of receptors, plasma proteins, growth factors and bacterial toxins. Therefore, several groups of investigators have evaluated the implication of convertases in viral envelope glycoprotein cleavage. Using the vaccinia virus overexpression system, furin was first shown to mediate the proteolytic maturation of both human immunodeficiency virus (HIV-1) and influenza virus envelope glycoproteins. In vitro studies demonstrated that purified convertases directly and specifically cleave viral envelope glycoproteins. Although these studies suggested the participation of several enzymes belonging to the convertases family, recent data suggest that other protease families may also participate in the HIV envelope glycoprotein processing. Their role in the physiological maturation process is still hypothetical and the molecular mechanism of the cleavage is not well documented. Crystallization of the hemagglutinin precursor (HA0) of influenza virus allowed further understanding of the molecular interaction between viral precursors and the cellular endoproteases. Furthermore, relationships between differential pathogenicity of influenza strains and their susceptibility to cleavage are molecularly funded. Here we review the most recent data and recent insights demonstrating the crucial role played by this activation step in virus infectivity. We discuss the cellular endoproteases that are implicated in HIV gp160 endoproteolytical maturation into gp120 and gp41.     

A retroviral vector for siRNA expression in mammalian cells

Utilization of RNA interference (RNAi) for knockdown of gene expression has become a standard tool for the study of gene function. Short hairpin RNAs (shRNAs) expressed from RNA polymerase III promoters are widely used to achieve stable knockdown of gene expression by RNAi. We have constructed a retroviral-based shRNA expression vector, pSiRPG, as a tool for shRNA-based functional genomic studies. This vector is based on a widely used shRNA expression system and was modified to harbor an enhanced green fluorescent protein (EGFP) and a puromycin selection marker. The functionality of the elements in the pSiRPG vector was validated. The H1(TetO₂) promoter in the vector facilitates doxycycline-inducible shRNA expression, which was demonstrated in cells expressing the Tet repressor (TetR). However, we also demonstrated limited efficiency of the inhibition of shRNA expression in an uninduced TetR-expressing cell line. This observation strongly indicates that the H1(TetO₂) promoter, which is used in a wide range of vectors, is not optimal for tightly regulated shRNA expression. Stable repression of the NDRG1 protein level was observed when introducing pSiRPG constructs expressing shRNAs targeting NDRG1 into two mammary epithelial cell lines by retroviral delivery. This vector should therefore facilitate functional studies in breast cell lines that are hard to transfect with conventional plasmid-based methods.     

Galactose receptors and presentation of HIV envelope glycoprotein to specific human T cells.

Recognition of viral Ag and of the envelope glycoprotein of HIV (gp120) in particular by human Th cells is critical in the immune response to the viral Ag which includes antibody production and generation of cytotoxic cells. Procedures to increase antigenicity of gp120 are highly desirable in a vaccine perspective. Therefore, to induce activation of gp120-specific T cells by a liminal dose of Ag we enhanced uptake of gp120 by exploiting the galactose receptors on APC. Terminal sialic acid residues were removed by neuraminidase treatment from the carbohydrate side chains of the heavily glycosylated gp120. Galactose residues were exposed and hence recognized by galactose receptors on APC. The experiments demonstrated that 1) human monocytes and dendritic cells, but not cells of the B lineage, bear galactose receptor; 2) galactose receptors are indeed involved because enhanced presentation is inhibited by galactose and acetylgalactosamine and competed for by other asialoglycoproteins; 3) galactose receptors mediate internalization of Ag in intracellular compartments that intersect the processing and presenting pathways, resulting in activation of specific T cells; 4) antigenicity of gp120 for specific T cells can be enhanced by the exposure of galactose residues.