Antibody-directed targeting of retroviral vectors via cell surface antigens

Targeted stable transduction of specific cells is a highly desirable goal for gene therapy applications. We report an efficient and broadly applicable approach for targeting retroviral vectors to specific cells. We find that the envelope of the alphavirus Sindbis virus can pseudotype human immunodeficiency virus type 1- and murine leukemia virus-based retroviral vectors. When modified to contain the Fc-binding domain of protein A, this envelope gives a significant enhancement in specificity in combination with antibodies specific for HLA and CD4 relative to that without antibody. Unlike previous targeting strategies for retroviral transduction, the virus titers are relatively high and stable and can be further increased by ultracentrifugation. This study provides proof of principle for a targeting strategy that would be generally useful for many gene therapy applications.     

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.     

Advances in retroviral transduction of hematopoietic stem cells for the gene therapy of atherosclerosis

PURPOSE OF REVIEW: Atherosclerosis is a chronic inflammatory disease that is the primary cause of morbidity and mortality in the developed world. Many studies have shown that macrophages and T-cells play critical roles in multiple aspects of the pathogenesis of the disease. Given that these cells are ultimately derived from bone marrow precursors, the concept of performing gene therapy for atherosclerosis through the retroviral transduction of hematopoietic stem cells has received much attention. This review will highlight recent advances that will help bring this goal closer. RECENT FINDINGS: The clinical application of retroviral gene transfer into hematopoietic stem cells has been hampered, in part, by the absence of vectors that can direct long-lasting, cell-type specific gene expression. In this review we will detail recent developments in the design of novel retroviral and lentiviral vectors that appear to overcome these problems, offering approaches to express therapeutic genes in specific cell-types within atherosclerotic lesions. We will also highlight advances in our understanding of the pathogenesis of atherosclerosis that may offer new gene therapeutic targets. SUMMARY: The use of retroviral transduction of hematopoietic stem cells for treatment of patients with atherosclerosis still remains a long-term goal. However, the recent development of retroviral vectors capable of directing expression to specific cell types within the lesion will allow more targeted therapeutic strategies to be devised. In addition, these vectors will provide powerful experimental tools to further our understanding of the pathogenesis of the disease.     

Susceptibility of cell populations to transduction by retroviral vectors

Retroviral transduction efficiency is related to the multiplicity of infection and the physiological state of the target cells. It is generally not known what proportion of a cell population is susceptible to transduction. We used coinfection with two retroviral vectors containing the marker genes for green fluorescent protein and the truncated human nerve growth factor receptor. In the CD34+ cell line TF-1 or human primary CD34+ hematopoietic progenitor cells, it was found that cells transduced with one vector had a better than random chance of transduction by the other vector. A probability model was developed to estimate target cell susceptibility; susceptibility was calculated as the product of the proportions of transgene-positive cells divided by the proportion of double-positive cells. By using this relationship, it was found that susceptibility was related to the target cell type and culture conditions but not the retroviral titer or the retroviral packaging envelope protein used in this study. Cotransduction with two vectors is a relatively simple procedure that provides a means to assess the maximum transduction level possible in a given cell population.     

Production of High-Titer Human Immunodeficiency Virus Type 1 Pseudotyped with Vesicular Stomatitis Virus Glycoprotein

We describe a method for the production of high-titer stocks of human immunodeficiency virus type 1 (HIV-1) pseudotyped with vesicular stomatitis virus envelope glycoprotein (VSV G). VSV G pseudotypes provide several advantages over other retroviral envelope proteins. The VSV G envelope is mechanically stable, enabling ultracentrifugal concentration of virions to high titers, and VSV G has a broad host range, enabling infection of many mammalian and nonmammalian cell types. VSV G pseudotypes of HIV-1 are useful for the study of HIV infection and replication kinetics and for the study of the function of specific viral proteins. We describe applications for the study of HIV-1 using VSV G pseudotypes. Additionally, we describe a method for pseudotyping retroviral vectors with VSV G. The same advantages of VSV G pseudotypes of HIV-1 apply to retroviral vectors; VSV G pseudotyped retroviral vectors may be used to introduce genes of interest into a wide variety of cell lines.     

Development of Improved Adenosine Deaminase Retroviral Vectors

A series of adenosine deaminase (ADA) retroviral vectors were designed and constructed with the goal of improved performance over the PA317/LASN vector currently used in clinical trials. First, the bacterial selectable-marker neomycin phosphotransferase (neo) gene was removed to create a “simplified” vector. Second, the Moloney murine leukemia virus long terminal repeat (LTR) promoter used for ADA expression was replaced with either the myeloproliferative sarcoma virus (MPSV) or SL3-3 LTR. Supernatant from each ADA vector was used to transduce ADA-deficient (ADA⁻) B- and T-cell lines as well as primary peripheral blood mononuclear cells (PBMC) from an ADA⁻ severe combined immunodeficiency patient. Total ADA enzyme activity and ADA activity per integrant in the transduced cells demonstrated that the MPSV LTR splicing vector design provided the highest level of ADA expression per cell. This ADA(MPSV) vector was then tested in packaging cell lines containing either the gibbon ape leukemia virus envelope (PG13 cells), the murine amphotropic envelope (FLYA13 cells), or the feline endogenous virus RD114 envelope (FLYRD18 cells). The results indicate that FLYRD18/ADA(MPSV), a simplified ADA retroviral vector with the MPSV LTR, provides a 17-fold-higher level of ADA expression in human lymphohematopoietic cells than the PA317/LASN vector currently in use.Retroviral vectors have been the most common gene transfer vehicles in clinical gene therapy trials (15). These vectors can integrate into the host genome to provide permanent transgene expression in the targeted cells (20). The first generation of retroviral vectors have been useful in demonstrating the feasibility of gene therapy approaches, but vectors capable of higher levels of gene transfer and transgene expression would be beneficial. For example, gene transfer levels achieved by first-generation retroviral vectors in large mammals (28) and in human gene therapy trials (7, 13) have been disappointing. There are at least two avenues for improving retroviral vectors. First, molecular changes can be made in the retroviral vector sequence. Second, different packaging cell lines could be tested to modify the host range, increase transduction in a given cell type, and/or render the virions resistant to inactivation by human complement.A clinically useful model for improving retroviral vector design is the vector LASN packaged in the amphotropic line PA317. PA317/LASN was the first therapeutic vector used in a gene therapy clinical trial (1). This vector has yielded gene transfer levels of generally less than 10% in peripheral blood T cells of adenosine deaminase-deficient (ADA⁻) severe combined immunodeficiency (SCID) patients. Two possibilities to improve this vector include eliminating the dominant selectable marker gene and changing the long terminal repeat (LTR) promoter to optimize expression. LASN, like many of the retroviral vectors used in clinical trials to date, contains two genes: the therapeutic gene (the ADA gene) and a dominant selectable marker gene (the bacterial neomycin phosphotransferase II gene; neo). Dominant selectable marker genes have historically been included to facilitate the generation, isolation, and titration of retroviral producer cell clones and to permit the evaluation and selection of successfully targeted cells. neo is the most commonly used selectable marker gene, although other genes have been used, including a mutant dihydrofolate reductase gene (dhfr) (19), the multidrug resistance gene (mdr) (10), and genes for cell surface markers such as cd24 (24) and the human nerve growth factor receptor (2). Vectors carrying dominant selectable marker genes, particularly those of nonhuman origin, have two theoretical disadvantages. First, careful analysis of some patients has revealed an immune response directed against the dominant selectable marker protein expressed from the retroviral integrant (20a, 25). Second, the more complex retroviral genomes required to express two separate genes may result in lower titers or suboptimal expression of the therapeutic gene product due to promoter interference (8, 29). On the other hand, cloning and determining the titers of useful retroviral vectors without selectable markers have been laborious. Using a recently developed rapid-screening procedure, we have been able to identify a number of “simple” ADA retroviral vectors which lack dominant selectable markers (23).Different packaging cell lines may also improve gene transfer of retroviral vectors into specific target cells. Retroviral vectors are limited by the host range specified by the envelope protein on the surface of the retrovirus. Most gene therapy trials have used retroviruses with a murine amphotropic (4070A) host range. However, packaging cell lines with the gibbon ape leukemia virus (GALV) envelope (PG13 cells) (18) and the cat endogenous virus RD114 envelope (FLYRD18 cells) (5) have become available; these may improve transduction frequencies into various target cell populations. For example, there is evidence that GALV-pseudotyped retroviral vectors may facilitate gene transfer into human peripheral blood T cells with greater efficiency than vectors with an amphotropic envelope (3). Packaging cell lines derived from murine cells have the additional disadvantage that they produce retroviruses which are inactivated by complement in human sera. Packaging cell lines of human origin (FLYA13 and FLYRD18) (5) produce vectors which are complement resistant. Testing both new simple retroviral vector designs and new packaging cells may therefore improve retrovirus-mediated gene transfer.We report the construction and characterization of three simplified ADA vectors by using either the Moloney murine leukemia virus (MLV) LTR, the myeloproliferative sarcoma virus (MPSV) LTR, or the SL3-3 LTR. We tested these vectors to determine which LTR provided the highest level of ADA expression in our target cells of interest: human ADA⁻ lymphohematopoietic cells. The ADA retroviral vector with the highest level of transduction/expression was then evaluated in different packaging cell lines including PG13, FLYA13, and FLYRD18.     

Self-contained, tetracycline-regulated retroviral vector system for gene delivery to mammalian cells.

Retroviral vectors that contain the tetracycline-inducible (Tet) system were developed. The two components of the Tet system were organized within the vectors in a manner that stringently maintains tetracycline-dependent regulation. Regulated expression of an indicator gene inserted into the retroviral vectors was examined in several different cell types. In infected NIH 3T3 cells, levels of induction in the absence of tetracycline were observed to be as much as 336-fold higher than levels in the presence of tetracycline, which were extremely low. Tetracycline-dependent regulation was observed in all other transduced cell types and ranged from 24- to 127-fold. The generation of retroviral vectors containing regulatory elements that allow for the regulated expression of heterologous genes and that have the ability to infect virtually all dividing target cells should greatly facilitate the biochemical and genetic examination of a broad range of genes. Moreover, these inducible retroviral vectors should prove useful in gene therapy applications.     

Toolbox for retrovectorologists

Retroviral vectors have actively contributed to the advent of gene therapy as a realistic approach in human therapeutics. At the beginning, the use of retroviral vectors was thought to be as simple as the collection of a viral supernatant that was applied to the desired cell. Rapidly, target resistance to transduction appeared in various conditions, ex vivo as well as in vivo. At that time, retrovectorologists entered an active "back to the bench" era. This phase was thought to have reached its conclusion with the generation of theoretically safe lentiviral vectors and when, in 2000, a first clinical trial using retroviral vectors proved to be successful. Unfortunately, recent developments have shown that we still need to improve our knowledge of several steps in the retroviral life cycle before we can accurately adapt vectors to target specific cells. In this review we will first briefly detail key features of the life cycle of wild-type retroviruses. Thereafter, an overview of the minimal requirements needed to generate retroviral vectors will be followed by the relevant developments in this rapidly moving field. Of note, we have highlighted the crucial biosafety issues in a specific section.     

A transient three-plasmid expression system for the production of hepatocytes targeting retroviral vectors

Targeting of retroviral vectors to specific cells was attempted through modifying the surface protein of the murine leukemia viruses （MLVs）, but in many cases the protein function was affected, and it is difficult to achieve the targeted delivery. In this study, we have tried to engineer ecotropic Moloney murine leukemia viruses （MoMLV）-based retroviral vectors to transduce hepatocytes. A chimeric envelope （Env） expression plasmid was constructed containing the hepatitis B virus PreS2 peptide fused to aa ＋1 at the Nterminus of Env. Following simultaneous transfection of pgag-pol, pLEGFP and chimeric env plasmids into 293T cells, helper-free retrovirus stocks with the titer of approximately 104 infectious units/ml were achieved at 48 h post-transfection. These pseudotype vectors showed the normal host range of retrovirus, infecting host NIH 3T3 cells, although the efficiency was reduced compared with that of virions carrying wild-type ecotropic MoMLV envelope. In addition, the resultant pseudotype viruses could transduce human hepatoma cells mediated by polymerized human serum albumin with relatively high titers in comparison with those transductions without polymerized human serum albumin. This approach can be used to target hepatocytes selectively.     

Oncoretrovirus and Lentivirus Vectors Pseudotyped with Lymphocytic Choriomeningitis Virus Glycoprotein: Generation, Concentration, and Broad Host Range

Lymphocytic choriomeningitis virus (LCMV) is a noncytopathic arenavirus shown to infect a broad range of different cell types. Here, we combined the beneficial characteristics of the LCMV glycoprotein (LCMV-GP) and those of retroviral vectors to generate a new, safe, and efficient gene transfer system. These LCMV-GP pseudotypes were systematically compared with vectors containing the widely used amphotropic murine leukemia virus envelope (A-MLVenv) or the vesicular stomatitis virus G protein (VSV-G). Production of LCMV-GP-pseudotyped oncoretroviral and lentiviral vectors by transient transfection resulted in vector titers similar to those with A-MLVenv or VSV-G. In contrast to A-MLVenv particles, LCMV-GP pseudotypes could be efficiently concentrated by ultracentrifugation without loss of vector titer. Unlike the cell-toxic VSV-G, a stable retroviral packaging cell line constitutively expressing LCMV-GP could be established. Vectors pseudotyped with LCMV-GP efficiently transduced many cell lines from different species and tissues relevant for gene therapy. Transduction of human glioma cells was studied in detail. These cells are a major target for cancer gene therapy and were transduced more efficiently with LCMV-GP-pseudotyped vectors than with the generally used A-MLVenv particles. The high stability, low toxicity, and broad host range make LCMV-GP-pseudotyped vectors attractive for gene transfer applications. The recombinant LCMV-GP-pseudotyped vectors will also allow functional characterization of naturally occurring and recombinant LCMV-GP variants.     

Gene transfer in bovine blastocysts using replication-defective retroviral vectors packaged with gibbon ape leukemia virus envelopes

With this work we demonstrate that murine leukemia virus (MLV)-based replication-defective retroviral vectors encapsidated with Gibbon ape leukemia virus (GaLV) envelopes are significantly more infectious to bovine embryonic trachea (EBTr) cells than vectors encapsidated with murine xenotropic envelope proteins. In a test of internal promoter activity in an MLV retroviral vector, the rat β-actin promoter was shown to be better than the herpes simplex virus type 1 thymidine kinase (TK) and human cytomegalovirus (CMV) immediate early promoters for the expression of an E. coli β-galactosidase marker gene in bovine target cells. By co-culture of bovine blastocysts and virus-producing cells, or by culture of embryos in the medium harvested from virus-producing cells, we transferred the E. coli β-galactosidase gene into trophoblasts and also into inner cell mass (ICM) cells of a bovine embryo through the infection of the MLV-based replication-defective retroviruses encapsidated with GaLV envelope proteins. The infection was confirmed by the expression of the E. coli β-galactosidase gene under a β-actin internal promoter. In addition, co-culture of ICM cells with virus-producing cells resulted in differentiation of ICM cells into embryoid bodies expressing the marker genes. © 1993 Wiley-Liss, Inc.     

Excision of specific DNA-sequences from integrated retroviral vectors via site-specific recombination.

Vectors for gene transfer and gene therapy were developed which combine the advantages of the integrase and recombinase systems. This was achieved by inserting two loxP sites for specific DNA excision into an MESV based retroviral vector. We show that this 'retroviral lox system' allows the infection of cells and the expression of transferred genes. In addition, we constructed an efficient retrovirus-based expression system for a modified Cre recombinase. Functional tests for DNA excision from integrated retroviral lox vectors were performed by the use of a negative selectable marker gene (thymidine kinase). Cre expression in cells infected with retroviral lox vectors and subsequent BrdU selection for cells in which site-specific recombination has occurred results in large numbers of independent cell clones. These results were confirmed by detailed molecular analysis. In addition we developed retroviral suicide vectors in which the enhancer/promoter elements of both LTRs were replaced by lox sequences. We show that lox-sequences located in the LTRs of retroviral vectors are stable during retroviral replication. Potential applications of this system would be the establishment of revertants of retrovirus-infected cells by controlled excision of nearly the complete proviral DNA.     

Gene delivery to adult neural stem cells

Neural stem cells may present an ideal route for gene therapy as well as offer new possibilities for the replacement of neurons lost to injury or disease. However, it has proved difficult to express ectopic genes in stem cells. We report methods to introduce genes into adult neural stem cells using viral and nonviral vectors in vitro and in vivo. Adenoviral and VSV-G-pseudotyped retroviral vectors are more efficient than plasmid transfection or VSV-G lentiviral transduction in vitro. We further show that adult neural stem cells can be directed to a neuronal fate by ectopic expression of neurogenin 2 in vitro. Plasmids can be delivered in vivo when complexed with linear polyethyleneimine, and gene expression can be targeted specifically to neural stem or progenitor cells by the use of specific promoters. These techniques may be utilized both to study the function of various genes in the differentiation of neural stem cells to specific cell fates and, ultimately, for gene therapy or to generate specific differentiated progeny for cell transplantation.     

A retroviral vector capable of targeted gene transfer into cells expressing HIV envelope glycoprotein

An expression vector encoding a chimeric envelope protein composed of CD4 and ecotropic retroviral envelope glycoprotein was constructed with the aim of accomplishing targeted gene transfer into HIV-1-infected cells. The chimeric protein was efficiently expressed and transported to the surfaces of various cell types and supported HIV-1 entry into human cells. A packaging cell line producing retroviral vectors carrying chimeric envelope proteins was then established. The vector particles produced were shown to be capable of specific gene transfer into human cells expressing HIV envelope glycoprotein. Blocking experiments confirmed that the vector particles entered the cells via an interaction between the chimeric and HIV envelope proteins. This targeting vector may thus be a useful tool with which to develop effective gene therapies against HIV infection.     

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.     

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.     

Construction of Retroviral Vectors with Improved Safety, Gene Expression, and Versatility

Murine leukemia virus (MLV)-based retroviral vectors are the most frequently used gene delivery vehicles. However, the current vectors are still not fully optimized for gene expression and viral titer, and many genetic and biochemical features of MLV-based vectors are poorly understood. We have previously reported that the retroviral vector MFG, where the gene of interest is expressed as a spliced mRNA, is superior in the level of gene expression with respect to other vectors compared in the study. As one approach to developing improved retroviral vectors, we have systematically performed mutational analysis of the MFG retroviral vector. We demonstrated that the entire gag coding sequence, together with the immediate upstream region, could be deleted without significantly affecting viral packaging or gene expression. To our knowledge, this region is included in all currently available retroviral vectors. In addition, almost the entire U3 region could be replaced with the heterologous human cytomegalovirus immediately-early promoter without deleterious effects. We could also insert internal ribosome entry sites (IRES) and multicloning sites into MFG without adverse effects. Based on these observations, we have constructed a series of new, improved retroviral constructs. These vectors produced viral titers comparable to MFG, expressed high levels of gene expression, and stably transferred genes to the target cells. Our vectors are more convenient to use because of the presence of multicloning sites and IRESs, and they are also more versatile because they can be readily converted to various applications. Our results have general implications regarding the design and development of improved retroviral vectors for gene therapy.