A potential approach for gene therapy targeting hepatoma using a liver-specific promoter on a retroviral vector.

Recent technological advances made in molecular biology and in vitro culture of human and other mammalian cells have led to broad medical and scientific acceptance of the feasibility of gene therapy for genetic diseases. Cancer might practically be one of the attractive targets for such therapy. For the treatment of cancer, it is important to manipulate the gene of interest such that it is expressed solely in cancer cells. We have developed a tissue-specific gene expression system, based on a tissue-specific promoter on a retroviral vector. A murine ecotropic retroviral vector was constructed in which the Escherichia coli beta-galactosidase gene served as a reporter; it was expressed under control of the albumin enhancer element and promoter. The tissue specificity of this vector was first assessed in vitro, and beta-galactosidase activity was detected exclusively in hepatoma cell lines. This recombinant retrovirus was injected directly into a subcutaneous tumor composed of transplantable murine MH-134 hepatoma cells, and expression of the gene was observed in vivo. Then this recombinant retrovirus was injected via the spleen or directly into the liver, resulting in the gene expression in dividing hepatocytes in partially hepatectomized mice, but not in nondividing hepatocytes in normal mice. Gene transfer specific to dividing hepatocytes and expression by means of retroviral vectors should possess high potential for selective elimination of hepatoma cells surrounded by nondividing normal hepatocytes.     

Application of HSVtk suicide gene to X-SCID gene therapy: ganciclovir treatment offsets gene corrected X-SCID B cells

Recently, a serious adverse effect of uncontrolled clonal T cell proliferation due to insertional mutagenesis of retroviral vector was reported in X-SCID gene therapy clinical trial. To offset the side effect, we have incorporated a suicide gene into therapeutic retroviral vector for selective elimination of transduced cells. In this study, B-cell lines from two X-SCID patients were transduced with bicistronic retroviral vector carrying human gamma c chain cDNA and Herpes simplex virus thymidine kinase gene. After confirmation of functional reconstitution of the gamma c chain, the cells were treated with ganciclovir (GCV). The gamma c chain positive cells were eliminated under low concentration without cytotoxicity on untransduced cells and have not reappeared at least for 5 months. Furthermore, the gamma c chain transduced cells were still sensitive to GCV after five months. These results demonstrated the efficacy of the suicide gene therapy although further in vivo studies are required to assess feasibility of this approach in clinical trial.     

Recombinant adeno-associated virus-mediated high-efficiency, transient expression of the murine cationic amino acid transporter (ecotropic retroviral receptor) permits stable transduction of human HeLa cells by ecotropic retroviral vectors.

Adeno-associated virus has a broad host range, is nonpathogenic, and integrates into a preferred location on chromosome 19, features that have fostered development of recombinant adeno-associated viruses (rAAV) as gene transfer vectors for therapeutic applications. We have used an rAAV to transfer and express the murine cationic amino acid transporter which functions as the ecotropic retroviral receptor, thereby rendering human cells conditionally susceptible to infection by an ecotropic retroviral vector. The proportion of human HeLa cells expressing the receptor at 60 h varied as a function of the multiplicity of infection (MOI) with the rAAV. Cells expressing the ecotropic receptor were efficiently transduced with an ecotropic retroviral vector encoding a nucleus-localized form of beta-galactosidase. Cells coexpressing the ecotropic receptor and nucleus-localized beta-galactosidase were isolated by fluorescence-activated cell sorting, and cell lines were recovered by cloning at limiting dilution. After growth in culture, all clones contained the retroviral vector genome, but fewer than 10% (3 of 47) contained the rAAV genome and continued to express the ecotropic receptor. The ecotropic receptor coding sequences in the rAAV genome were under the control of a tetracycline-modulated promoter. In the presence of tetracycline, receptor expression was low and the proportion of cells transduced by the ecotropic retroviral vector was decreased. Modulation of receptor expression was achieved with both an episomal and an integrated form of the rAAV genome. These data establish that functional gene expression from an rAAV genome can occur transiently without genome integration.     

Long-term in vivo expression of genes introduced by retrovirus-mediated transfer into mammary epithelial cells.

Nonimmortalized mouse mammary epithelial cells expressing Escherichia coli beta-galactosidase from a murine amphotropic packaged retroviral vector were injected into the epithelium-divested mammary fat pads of syngeneic mice. Mammary glands formed from the injected mammary epithelial cells contained ductal and lobular cells, both of which expressed beta-galactosidase when examined in situ more than 12 months later. These results indicate that stable recombinant gene expression can be achieved in vivo in the mammary gland without altering the growth properties of normal mammary epithelium.     

重组逆转录病毒载体GTK的构建及HyTK基因转移的研究

用逆转录病毒载体将单纯疱疹病毒胸苷激酶基因（ＨＳＶｔｋ）导入恶性肿瘤细胞,随后可应用药物９－（１,３－二羟基－丙氧基－甲基）鸟嘌呤（ｇａｎｃｉｃｌｏｖｉｒ,ＧＣＶ）选择性地杀死肿瘤细胞．将ＨｙＴＫ基因替换逆转录病毒载体ＧｌＮａ中的ｎｅｏ基因,构建成重组逆转录病毒载体ＧＴＫ,转染混合包装细胞（双噬性ＰＡ３１７细胞和单噬性ＧＰ＋Ｅ－８６细胞）,通过“乒乓效应”获得高滴度重组病毒．用该重组病毒转染小鼠恶性黑色素瘤细胞系Ｂ１６细胞,用ｈｙｇｒｏｍｙｃｉｎＢ筛选出阳性细胞克隆（ＨｙＴＫ＋）,经ＰＣＲ方法检测证明ＨｙＴＫ基因已成功地导入肿瘤细胞中,且不含可复制的辅助病毒．分别用不同浓度的ＧＣＶ作用于ＨｙＴＫ－及ＨｙＴＫ＋的Ｂ１６细胞,光镜下观察２４ｈ和４８ｈ后细胞形态及进行活细胞计数．结果表明,ＧＣＶ浓度大于０．１μｍｏｌ／Ｌ时即对Ｂ１６／ＨｙＴＫ＋细胞有显著的杀伤作用     

Recombinant adeno-associated virus mediates a high level of gene transfer but less efficient integration in the K562 human hematopoietic cell line.

We tested the ability of a recombinant adeno-associated virus (rAAV) vector to express and integrate exogenous DNA into human hematopoietic cells in the absence of selection. We developed an rAAV vector, AAV-tNGFR, carrying a truncated rat nerve growth factor receptor (tNGFR) cDNA as a cell surface reporter under the control of the Moloney murine leukemia virus (MoMuLV) long terminal repeat. An analogous MoMuLV-based retroviral vector (L-tNGFR) was used in parallel, and gene transfer and expression in human hematopoietic cells were assessed by flow cytometry and DNA analyses. Following gene transfer into K562 cells with AAV-tNGFR at a multiplicity of infection (MOI) of 13 infectious units (IU), 26 to 38% of cells expressed tNGFR on the surface early after transduction, but the proportion of tNGFR expressing cells steadily declined to 3.0 to 3.5% over 1 month of culture. At an MOI of 130 IU, nearly all cells expressed tNGFR immediately posttransduction, but the proportion of cells expressing tNGFR declined to 62% over 2 months of culture. The decline in the proportion of AAV-tNGFR-expressing cells was associated with ongoing losses of vector genomes. In contrast, K562 cells transduced with the retroviral vector L-tNGFR expressed tNGFR in a constant fraction. Integration analyses on clones showed that integration occurred at different sites. Integration frequencies were estimated at about 49% at an MOI of 130 and 2% at an MOI of 1.3. Transduction of primary human CD34+ progenitor cells by AAV-tNGFR was less efficient than with K562 cells and showed a declining percentage of cells expressing tNGFR over 2 weeks of culture. Thus, purified rAAV caused very high gene transfer and expression in human hematopoietic cells early after transduction, which steadily declined during cell passage in the absence of selection. Although the efficiency of integration was low, overall integration was markedly improved at a high MOI. While prolonged episomal persistence may be adequate for gene therapy of nondividing cells, a very high MOI or improvements in basic aspects of AAV-based vectors may be necessary to improve integration frequency in the rapidly dividing hematopoietic cell population.     

Efficient gene transfer into nondividing cells by adeno-associated virus-based vectors.

Gene transfer vectors based on adeno-associated virus (AAV) are emerging as highly promising for use in human gene therapy by virtue of their characteristics of wide host range, high transduction efficiencies, and lack of cytopathogenicity. To better define the biology of AAV-mediated gene transfer, we tested the ability of an AAV vector to efficiently introduce transgenes into nonproliferating cell populations. Cells were induced into a nonproliferative state by treatment with the DNA synthesis inhibitors fluorodeoxyuridine and aphidicolin or by contact inhibition induced by confluence and serum starvation. Cells in logarithmic growth or DNA synthesis arrest were transduced with vCWR:beta gal, an AAV-based vector encoding beta-galactosidase under Rous sarcoma virus long terminal repeat promoter control. Under each condition tested, vCWR:beta Gal expression in nondividing cells was at least equivalent to that in actively proliferating cells, suggesting that mechanisms for virus attachment, nuclear transport, virion uncoating, and perhaps some limited second-strand synthesis of AAV vectors were present in nondividing cells. Southern hybridization analysis of vector sequences from cells transduced while in DNA synthetic arrest and expanded after release of the block confirmed ultimate integration of the vector genome into cellular chromosomal DNA. These findings may provide the basis for the use of AAV-based vectors for gene transfer into quiescent cell populations such as totipotent hematopoietic stem cells.     

Use of helper-free retroviral vector to direct a high expression of porcine growth hormone in mouse fibroblast cells.

A retroviral vector has been employed to express the cDNA coding for porcine growth hormone (pGH) in the mouse fibroblast cell NIH 3T3 in large quantity. In this study, a single gene vector which contained no selectable marker was used. We have coinfected NIH 3T3 cells with pGH retrovirus and Neo(r) retrovirus to obtain a stable, high-expression clone. Using a superinfection strategy, we further increased the copy number of proviral DNA in the host chromosome, thus increasing the pGH secretion from 22 to 55 micrograms/10(6) cells/24 h. The recombinant pGH produced from mouse fibroblast cells was heterogeneous at the N-terminus, which mimicked the situation with bovine growth hormone either from natural sources or from recombinant products derived from mouse fibroblasts. This technology is useful for many biologically important genes to be stably transduced by retroviral vector into mammalian cells and highly expressed.     

Targeted and stable gene delivery into muscle cells by a two-step transfer system

We developed a muscle-specific gene delivery system based on two-step gene transfer. The first step involved adenovirus-mediated transfer of the ecotropic retrovirus receptor (EcoRec) gene driven by the muscle-specific desmin promoter. Both human primary myoblasts and fibroblasts were efficiently transduced with this adenovirus vector. However, expression of EcoRec was detected only in myoblasts. In the second step, EcoRec-expressing myoblasts could be stably transduced with the ecotropic retroviral vector with the beta-galactosidase gene. Approximately 15% of myoblasts were transduced by this two-step strategy. When the transduced myoblasts were differentiated into myotubes, extensive cell-cell fusion occurred, and the apparent number of beta-galactosidase-positive cells increased to 28%. These results indicate that our two-step gene delivery system could be used for targeted and stable gene transfer into muscle cells.     

Recombinant gene expression in human umbilical vein endothelial cells transduced by retroviral vectors

Endothelial cells are attractive targets for gene transfer because of their immediate contact with the bloodstream, and, therefore, they might serve as vehicles for therapeutic drug delivery. Recently, we and others reported that endothelial cells of animal origin efficiently express both secretory and nonsecretory recombinant proteins. We now show that human endothelial cells are also capable of expressing a recombinant gene following transduction with retroviral vectors. Human umbilical vein endothelial cells were transduced with either the N2 or the SAX vector. Following selection with G418, cells transduced by both vectors were found to express neophosphotransferase activity, the product of the neomycin resistance gene. The fact that a recombinant gene can be readily inserted and efficiently expressed into human endothelial cells suggests that these cells may be able to serve a role in human gene therapy.     

Adeno-associated virus type 2-mediated transfer of ecotropic retrovirus receptor cDNA allows ecotropic retroviral transduction of established and primary human cells.

The cellular receptors that mediate binding and internalization of retroviruses have recently been identified. The concentration and accessibility of these receptors are critical determinants in accomplishing successful gene transfer with retrovirus-based vectors. Murine retroviruses containing ecotropic glycoproteins do not infect human cells since human cells do not express the receptor that binds the ecotropic glycoproteins. To enable human cells to become permissive for ecotropic retrovirus-mediated gene transfer, we have developed a recombinant adeno-associated virus type 2 (AAV) vector containing ecotropic retroviral receptor (ecoR) cDNA under the control of the Rous sarcoma virus (RSV) long terminal repeat (LTR) promoter (vRSVp-ecoR). Established human cell lines, such as HeLa and KB, known to be nonpermissive for murine ecotropic retroviruses, became permissive for infection by a retroviral vector containing a bacterial gene for resistance to neomycin (RV-Neo(r)), with a transduction efficiency of up to 47%, following transduction with vRSVp-ecoR, as determined by the development of colonies that were resistant to the drug G418, a neomycin analog. No G418-resistant colonies were present in cultures infected with either vRSVp-ecoR or RV-Neo(r) alone. Southern and Northern blot analyses revealed stable integration and long-term expression, respectively, of the transduced murine ecoR gene in clonal isolates of HeLa and KB cells. Similarly, ecotropic retrovirus-mediated Neo(r) transduction of primary human CD34+ hematopoietic progenitor cells from normal bone marrow was also documented, but only following infection with vRSVp-ecoR. The retroviral transduction efficiency was approximately 7% without prestimulation and approximately 14% with prestimulation of CD34+ cells with cytokines, as determined by hematopoietic clonogenic assays. No G418-resistant progenitor cell colonies were present in cultures infected with either vRSVp-ecoR or RV-Neo(r) alone. These results suggest that sequential transduction of primary human cells with two different viral vectors may overcome limitations encountered with a single vector. Thus, the combined use of AAV- and retrovirus-based vectors may have important clinical implications for ex vivo and in vivo human gene therapy.     

Molecular imaging of embryonic stem cell misbehavior and suicide gene ablation

Numerous studies have demonstrated the potential use of stem cells for the repair and regeneration of injured tissues. However, tracking transplanted stem cell fate and function in vivo remains problematic. To address these issues, murine embryonic stem (ES) cells were stably transduced with self-inactivating lentiviral vectors carrying either a triple fusion (TF) or double fusion (DF) reporter gene construct. The TF consisted of monomeric red fluorescence protein (mrfp), firefly luciferase (Fluc), and herpes simplex virus truncated thymidine kinase (HSV-ttk) reporter genes. The DF consisted of enhanced green fluorescence protein (egfp) and Fluc reporter genes but lacked HSV-ttk. Stably transduced ES-TF or ES-DF cells were selected by fluorescence activated cell sorting based on either mrfp (TF) or egfp (DF) expression. Afterwards, cells were injected subcutaneously into the right (ES-TF cells) and left (ES-DF cells) shoulders of adult female nude mice. Cell survival was tracked noninvasively by bioluminescence and positron emission tomography imaging of Fluc and HSV-ttk reporter genes, respectively. Imaging signals progressively increased from day 2 to day 14, consistent with ES cell survival and proliferation in vivo. However, teratoma formation occurred in all nude mice after 5 weeks. Administration of ganciclovir (GCV), targeting the HSV-ttk gene, resulted in selective ablation of teratomas arising from the ES-TF cells but not ES-DF cells. These data demonstrate the novel use of multimodality imaging techniques to (1) monitor transplanted ES cell survival and proliferation in vivo and (2) assess the efficacy of suicide gene therapy as a backup safety measure against teratoma formation.     

High sensitivity of neonatal rat hepatocytes to retroviral-mediated gene transfer and their transplantation into the spleen of adult rat.

To optimize conditions for retroviral-mediated transfer of a recombinant gene to hepatocytes, the pMNSM-Tk-lacZ vector, which we had constructed to express the bacterial beta-galactosidase gene, was transduced to rat hepatocytes under various conditions, and the expression of beta-galactosidase activity was examined by cytochemical staining. Compared to the hepatocytes of adult rats, those of newborns were about 50-100 times more sensitive to transduction with the beta-galactosidase gene in vitro. The sensitivity was high in the newborn hepatocytes when the virus was infected on days 1 and 2 after initiation of culture. However, the sensitivity to infection did not correlate with the DNA synthetic activity. The gene transfer was feasible not only to hepatocytes in monolayer culture but also to those in spheroid culture. The spheroidal aggregates containing hepatocytes transduced with the beta-galactosidase gene could be transplanted into the spleen of syngenic adult rat, although the expression was very low.     

Lentiviral and MLV based retroviral vectors for ex vivo and in vivo gene transfer

Retroviral vectors have become an important tool for gene transfer in vitro and in vivo. Classical Moloney murine leukemia virus (MLV) based retroviral vectors have been used for over 20 years to transfer genes into dividing cells. Cell lines for production of retroviral vectors have become commonly available and modifications in retroviral vector design and use of envelope proteins have made the production of high titer, helper-free, infectious virus stocks relatively easy. More recently, lentiviral vectors, another class of retroviruses, have been modified for in vitro and in vivo gene transfer. The ability of lentiviral vectors to transduce non-dividing cells has made them especially attractive for in vivo gene transfer into differentiated, non-dividing tissues. Several improvements in helper plasmids and vectors have made lentivirus a safe vector system for ex vivo and in vivo gene transfer. This review will briefly summarize the background of these vector systems and provide some common protocols available for the preparation of MLV based retroviral vectors and HIV-1 based lentiviral vectors.     

Spleen necrosis virus-derived C-type retroviral vectors for gene transfer to quiescent cells

Gene therapy applications of retroviral vectors derived from C-type retroviruses have been limited to introducing genes into dividing target cells. Here, we report genetically engineered C-type retroviral vectors derived from spleen necrosis virus (SNV), which are capable of infecting nondividing cells. This has been achieved by introducing a nuclear localization signal (NLS) sequence into the matrix protein (MA) of SNV by site-directed mutagenesis. This increased the efficiency of infecting nondividing cells and was sufficient to endow the virus with the capability to efficiently infect growth-arrested human T lymphocytes and quiescent primary monocyte-derived macrophages. We demonstrate that this vector actively penetrates the nucleus of a target cell, and has potential use as a gene therapy vector to transfer genes into nondividing cells.     

In Vivo Assessment of Gene Delivery to Keratinocytes by Lentiviral Vectors

For skin gene therapy, introduction of a desired gene into keratinocyte progenitor or stem cells could overcome the problem of achieving persistent gene expression in a significant percentage of keratinocytes. Although keratinocyte stem cells have not yet been completely characterized and purified for gene targeting purposes, lentiviral vectors may be superior to retroviral vectors at gene introduction into these stem cells, which are believed to divide and cycle slowly. Our initial in vitro studies demonstrate that lentiviral vectors are able to efficiently transduce nondividing keratinocytes, unlike retroviral vectors, and do not require the lentiviral accessory genes for keratinocyte transduction. When lentiviral vectors expressing green fluorescent protein (GFP) were directly injected into the dermis of human skin grafted onto immunocompromised mice, transduction of dividing basal and nondividing suprabasal keratinocytes could be demonstrated, which was not the case when control retroviral vectors were used. However, flow cytometry analysis demonstrated low transduction efficiency, and histological analysis at later time points provided no evidence for progenitor cell targeting. In an alternative in vivo method, human keratinocytes were transduced in tissue culture (ex vivo) with either lentiviral or retroviral vectors and grafted as skin equivalents onto immunocompromised mice. GFP expression was analyzed in these human skin grafts after several cycles of epidermal turnover, and both the lentiviral and retroviral vector-transduced grafts had similar percentages of GFP-expressing keratinocytes. This ex vivo grafting study provides a good in vivo assessment of gene introduction into progenitor cells and suggests that lentiviral vectors are not necessarily superior to retroviral vectors at introducing genes into keratinocyte progenitor cells during in vitro culture.     

High-throughput, library-based selection of a murine leukemia virus variant to infect nondividing cells

Gammaretroviruses, such as murine leukemia virus (MLV), are functionally distinguished from lentiviruses, such as human immunodeficiency virus, by their inability to infect nondividing cells. Attempts to engineer this property into MLV have been hindered by an incomplete understanding of early events in the viral life cycle. We utilized a transposon-based method to generate saturated peptide insertion libraries of MLV gag-pol variants with nuclear localization signals randomly incorporated throughout these overlapping genes. High-throughput selection of the libraries via iterative retroviral infection of nondividing cells led to the identification of a novel variant that successfully transduced growth-arrested cells. Vector packaging by cotransfection of the gag-pol.NLS variant with wild-type gag-pol produced high-titer virions capable of infecting neurons in vitro and in vivo. The capacity of mutant virions to transduce nondividing cells could help to elucidate incompletely understood mechanisms of the viral life cycle and greatly broaden the gene therapy applications of retroviral vectors. Furthermore, the ability to engineer key intracellular viral infection steps has potential implications for the understanding, design, and control of other post-entry events. Finally, this method of library generation and selection for a desired phenotype directly in a mammalian system can be readily expanded to address other challenges in protein engineering.     

High-efficiency identification of genes by functional analysis from a retroviral cDNA expression library.

Retroviral gene transfer efficiently delivers genes of interest stably into target cells, and expression cDNA cloning has been shown to be highly successful. Considering these two advantages, we now report a method by which one can identify genes stimulating cell growth through functional analysis. The first step requires the construction of a retroviral cDNA expression library and the optimization of transfection of vector DNA into virus packaging cells. The second step involves the cocultivation of target cells with libraries of retrovirus-producing cells, resulting in the amplification of target cells transduced with a gene(s) stimulating cell growth. Under standardized conditions of transfection, we detected an average of 4,000 independent clones per dish, among which expression of a retroviral beta-galactosidase gene at an abundance of 0.2% could be detected. Next, we demonstrated the augmentation of the sensitivity of the assay by retroviral infection and functional analysis. We did this by cocultivating factor-dependent (FD) cells with dishes of GP/E cells transfected with plasmids containing various molar ratios of pN2-IL3 DNA and retroviral library cDNA and by determining the highest dilution of pN2-IL3 which still resulted in the conversion of FD cells to factor independence. The retroviral interleukin-3 gene at an abundance as low as 0.001% could be detected. Indeed, we were able to detect from FD cells the development of factor-independent colonies with different phenotypes after retroviral transfer of cDNAs from an immortalized hemopoietic stem cell line. Thus, the combination of a standardized high-efficiency DNA transfection and retrovirus-mediated gene transfer should facilitate the identification of genes capable of conferring to target FD cells a detectable new function or phenotype. By scaling up the size of the experiment realistically during screening, the assay can detect cDNA at an abundance of lower than 0.0001%.