Retroviral Vector Particles Research Articles

Strategies for Targeting Retroviral Integration for Safer Gene Therapy: Advances and Challenges.

Retroviruses are obligate intracellular parasites that must integrate a copy of the viral genome into the host DNA. The integration reaction is performed by the viral enzyme integrase in complex with the two ends of the viral cDNA genome and yields an integrated provirus. Retroviral vector particles are attractive gene therapy delivery tools due to their stable integration. However, some retroviral integration events may dysregulate host oncogenes leading to cancer in gene therapy patients. Multiple strategies to target retroviral integration, particularly to genetic safe harbors, have been tested with limited success. Attempts to target integration may be limited by the multimerization of integrase or the presence of host co-factors for integration. Several retroviral integration complexes have evolved a mechanism of tethering to chromatin via a host protein. Integration host co-factors bind chromatin, anchoring the complex and allowing integration. The tethering factor allows for both close proximity to the target DNA and specificity of targeting. Each retrovirus appears to have distinct preferences for DNA sequence and chromatin features at the integration site. Tethering factors determine the preference for chromatin features, but do not affect the subtle sequence preference at the integration site. The sequence preference is likely intrinsic to the integrase protein. New developments may uncouple the requirement for a tethering factor and increase the ability to redirect retroviral integration.

Efficient Gene Delivery to Quiescent Interleukin-2 (IL-2)–Dependent Cells by Murine Leukemia Virus-Derived Vectors Harboring IL-2 Chimeric Envelopes Glycoproteins

AbstractInterleukin-2 (IL-2) is a cytokine that induces the proliferation of certain IL-2 receptor expressing quiescent cells. Human IL-2 was fused to the amino-terminus of amphotropic murine leukemia virus (MLV) envelope glycoproteins. Retroviral vectors were pseudotyped with both the IL-2 chimeric envelope and the wild-type amphotropic MLV envelope. The chimeric IL-2 glycoproteins were incorporated on retroviral vectors and the IL-2–displaying vector particles could bind specifically to cell surface IL-2 receptors. In addition, the IL-2–displaying vectors could infect proliferating cells through amphotropic receptors irrespective of whether the cells expressed the IL-2 receptor. IL-2–displaying vector particles could also transiently stimulate the cell cycle entry and proliferation of several IL-2–dependent cell lines. Finally, retroviral vectors displaying IL-2 could efficiently transduce G0/G1-arrested cells expressing the IL-2 receptor at a 34-fold higher efficiency compared with vectors with unmodified envelopes. This new strategy, whereby C-type retroviral vector particles display a ligand that activates the cell cycle of the target cells at the time of virus entry, may represent an alternative to lentivirus-derived retroviral vectors for the infection of quiescent cells. In addition, upon infection of an heterogeneous population of nonproliferating cells, MLV-retroviral vectors that display cytokines/growth factors will allow the transgene of interest to be integrated specifically in quiescent cells expressing the corresponding cytokine/growth factor receptor.

Immune Protection of Retroviral Vectors Upon Molecular Painting with the Complement Regulatory Protein CD59.

Glycosylphosphatidylinositol anchoring is a type of post-translational modification that allows proteins to be presented on the exterior side of the cell membrane. Purified glycosylphosphatidylinositol-anchored protein can spontaneously re-insert into lipid bilayer membranes in a process termed Molecular Painting. Here, we demonstrate the possibility of inserting purified, recombinant CD59 into virus particles produced from a murine retroviral producer cell line. CD59 is a regulator of the complement system that helps protect healthy cells from the lytic activity of the complement cascade. In this study, we could show that Molecular Painting confers protection from complement activity upon murine retroviral vector particles. Indeed, increased infectivity of CD59-modified virus particles was observed upon challenge with human serum, indicating that Molecular Painting is suitable for modulating the immune system in gene therapy or vaccination applications.Electronic supplementary materialThe online version of this article (doi:10.1007/s12033-016-9944-z) contains supplementary material, which is available to authorized users.

Pseudotype-Independent Nonspecific Uptake of Gammaretroviral and Lentiviral Particles in Human Cells

The effective entry of retroviruses into target cells depends on the presence of viral envelope (Env) proteins and cognate cellular receptors, such as the murine cationic amino acid transporter-1 (mCAT-1) for the ecotropic murine leukemia virus (MLV-E). Here, we examined whether human cells internalize MLV-E or other retroviral pseudotypes irrespective of the presence of a specific receptor. Using fluorescently tagged Gag to monitor viral internalization, and treating cells with chloroquine or bafilomycin A1, we show that endocytosis is the main pathway for productive transduction with ecotropic particles, but endocytosis of retroviral particles itself does not depend on a suitable receptor or Env. Nonspecific endosomal uptake and lysosomal degradation occurred with all "illegitimate" envelope-receptor combinations tested: MLV particles pseudotyped with the ecotropic envelope or measles virus H and F proteins as well as "ecotropic" or "bald" HIV-1 particles. Kinetic studies in cell lines and primary human T lymphocytes showed the persistence of Gag-GFP signals for more than 10 days after exposure to retroviral vector particles, even in the absence of a suitable receptor. Further studies testing the Gag-mediated transfer of protein or retroviral mRNA revealed that nonspecific endocytosis prevented the release of functional particle-associated proteins and nucleic acids into the cytosol. We conclude that receptor-targeted retroviral particles are unlikely to escape nonspecific cellular uptake unless appropriate protective principles are discovered. Conversely, as lysosomal degradation was found to inactivate mRNA and proteins embedded into retroviral particles, receptor targeting is a useful strategy for both transient and permanent cell modification by retrovirus-like particles.

Retroviral Vectors Induce Epigenetic Chromatin Modifications and IL-10 Production in Transduced B Cells via Activation of Toll-like Receptor 2

The immune response toward viral vectors used for gene therapy and genetic vaccination appears to be critically important in determining the therapeutic outcome. However, the mechanisms that control the immune response following gene transfer are poorly understood. Unexpectedly, we found that integrating retroviral vector particles induce stable interleukin-10 (IL-10) production in murine (BALB/c H-2(d)) transduced B cells. This requires a novel mechanism whereby the interaction of retroviral vector particle with its cognate cellular receptor activates intracellular signaling pathways resulting in stable epigenetic modifications. Murine B cells exposed to retroviral vector particles triggered the colocalization of the retroviral cellular receptor [mouse cationic amino acid transporter 1 (mCAT1)] and Toll-like receptor 2 (TLR2) into lipid microrafts, which in turn activated TLR2 signaling pathways. TLR2 activation induced STAT3 phosphorylation and increased phosphorylated histone 3 (H3) at the STAT3-binding site of the IL-10 promoter. In addition, TLR2 activation during transduction activates nuclear factor of κ light polypeptide gene enhancer in B-cells inhibitor, α (NFKBIA), thereby preventing the translocation of the nuclear factor-κB (NF-κB) complex to the nucleus and the transcription of proinflammatory cytokines. These findings open new perspectives for controlling immune responses following gene therapy and genetic vaccination.

Entry kinetics and cell–cell transmission of surface‐bound retroviral vector particles

Transduction with recombinant HIV-1 derived lentivirus vectors is a multi-step process initiated by surface attachment and subsequent receptor-directed uptake into the target cell. We previously reported the retention of vesicular stomatitis virus G protein pseudotyped particles on murine progenitor cells and their delayed cell-cell transfer. To examine the underlying mechanism in more detail, we used a combination of approaches focused on investigating the role of receptor-independent factors in modulating attachment. The investigation of synchronized transduction reveals cell-type specific rates of vector particle clearance with substantial delays during particle entry into murine hematopoietic progenitor cells. The observed uptake kinetics from the surface of the 1 degrees cell correlate inversely with the magnitude of transfer to 2 degrees targets, corresponding with our initial observation of preferential cell-cell transfer in the context of brief vector exposures. We further demonstrate that vector particle entry into cells is associated with the cell-type specific abundance of extracellular matrix fibronectin. Residual particle-extracellular fibronectin matrix binding and 2 degrees transfer can be competitively disrupted by heparin exposure without affecting murine progenitor homing and repopulation. Although cellular attachment factors, including fibronectin, aid gene transfer by colocalizing particles to cells and disfavoring early dissociation from targets, they also appear to stabilize particles on the cell surface. The present study highlights the inadvertent consequences for cell entry and cell-cell transfer.

Retroviral vectors encoding ADA regulatory locus control region provide enhanced T-cell-specific transgene expression

BackgroundMurine retroviral vectors have been used in several hundred gene therapy clinical trials, but have fallen out of favor for a number of reasons. One issue is that gene expression from viral or internal promoters is highly variable and essentially unregulated. Moreover, with retroviral vectors, gene expression is usually silenced over time. Mammalian genes, in contrast, are characterized by highly regulated, precise levels of expression in both a temporal and a cell-specific manner. To ascertain if recapitulation of endogenous adenosine deaminase (ADA) expression can be achieved in a vector construct we created a new series of Moloney murine leukemia virus (MuLV) based retroviral vector that carry human regulatory elements including combinations of the ADA promoter, the ADA locus control region (LCR), ADA introns and human polyadenylation sequences in a self-inactivating vector backbone.MethodsA MuLV-based retroviral vector with a self-inactivating (SIN) backbone, the phosphoglycerate kinase promoter (PGK) and the enhanced green fluorescent protein (eGFP), as a reporter gene, was generated. Subsequent vectors were constructed from this basic vector by deletion or addition of certain elements. The added elements that were assessed are the human ADA promoter, human ADA locus control region (LCR), introns 7, 8, and 11 from the human ADA gene, and human growth hormone polyadenylation signal. Retroviral vector particles were produced by transient three-plasmid transfection of 293T cells. Retroviral vectors encoding eGFP were titered by transducing 293A cells, and then the proportion of GFP-positive cells was determined using fluorescence-activated cell sorting (FACS). Non T-cell and T-cell lines were transduced at a multiplicity of infection (MOI) of 0.1 and the yield of eGFP transgene expression was evaluated by FACS analysis using mean fluorescent intensity (MFI) detection.ResultsVectors that contained the ADA LCR were preferentially expressed in T-cell lines. Further improvements in T-cell specific gene expression were observed with the incorporation of additional cis-regulatory elements, such as a human polyadenylation signal and intron 7 from the human ADA gene.ConclusionThese studies suggest that the combination of an authentically regulated ADA gene in a murine retroviral vector, together with additional locus-specific regulatory refinements, will yield a vector with a safer profile and greater efficacy in terms of high-level, therapeutic, regulated gene expression for the treatment of ADA-deficient severe combined immunodeficiency.

Positive selection of DNA-protein interactions in mammalian cells through phenotypic coupling with retrovirus production

Through the shuffling of predefined modular zinc finger (ZF) domains with predictable target site recognition in vitro, we have generated a large repertoire of artificial transcription factors (ATFs) with five ZF domains (TFZFs). Here we report an effective strategy for the selection of ATF libraries through the coupling of the expression of transcriptional activators of the promoter of interest to the enhanced production of retroviral vector particles transferring the gene encoding the TFZF. Using this strategy, we successfully selected specific TFZFs that upregulate the expression of the γ-globin promoter. Selected transcription factors induced the expression of γ-globin when coupled to an activation domain and reduced expression when linked to a repression domain. This novel retroviral approach might be used to select other TFZFs but also might be generalized for the selection of other protein and small molecule interactions.

Identification of Host Proteins Associated with Retroviral Vector Particles by Proteomic Analysis of Highly Purified Vector Preparations

The Moloney murine leukemia virus (MMLV) belongs to the Retroviridae family of enveloped viruses, which is known to acquire minute amounts of host cellular proteins both on the surface and inside the virion. Despite the extensive use of retroviral vectors in experimental and clinical applications, the repertoire of host proteins incorporated into MMLV vector particles remains unexplored. We report here the identification of host proteins from highly purified retroviral vector preparations obtained by rate-zonal ultracentrifugation. Viral proteins were fractionated by one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis, in-gel tryptic digested, and subjected to liquid chromatography/tandem mass spectrometry analysis. Immunogold electron microscopy studies confirmed the presence of several host membrane proteins exposed at the vector surface. These studies led to the identification of 27 host proteins on MMLV vector particles derived from 293 HEK cells, including 5 proteins previously described as part of wild-type MMLV. Nineteen host proteins identified corresponded to intracellular proteins. A total of eight host membrane proteins were identified, including cell adhesion proteins integrin beta1 (fibronectin receptor subunit beta) and HMFG-E8, tetraspanins CD81 and CD9, and late endosomal markers CD63 and Lamp-2. Identification of membrane proteins on the retroviral surface is particularly attractive, since they can serve as anchoring sites for the insertion of tags for targeting or purification purposes. The implications of our findings for retrovirus-mediated gene therapy are discussed.

A heterologous system for assembly of retroviral gene vectors: Intracellular budding in yeast?

Mammalian retroviral gene vectors are of particular importance in gene therapy because of their efficient chromosomal integration and resulting stable maintenance of transgenes. These vectors are currently produced in mammalian cells, with low yield and at substantial expense. Therefore, a more efficient heterologous production system for retroviral vectors would be desirable. With their impressive track record in biotechnology, various yeast species appear like ideal organisms to generate retroviral vectors. Typically, retroviral vector particles emerge from mammalian cells after budding at the plasma membrane. However, in yeast, viral budding at the plasma membrane is blocked by the cell wall. At the same time, mass production of enveloped viral vectors in yeast protoplasts is technically challenging. Recent reports indicated the generation of infectious virions via intracellular budding for some combinations of retroviruses and mammalian cells. Relying on these data I hypothesise that a successful assembly of the retroviral transducing particles can be accomplished intracellularly inside yeast cells with a normal cell wall. Firstly, it is possible that some of the intracellular yeast compartments have all the necessary host factors that are required for successful RNA packaging, budding and maturation of infectious retroviral vector particles. Secondly, it might be possible to improve intracellular viral vector production by artificially targeting viral cores to bud at specific intracellular vesicular structures using appropriate targeting or retention signals. A suitable envelope protein, conferring infectivity and specific cellular tropism to the vector particles, can be expressed in yeast or, alternatively, 'bald' viral particles without envelope protein can be produced in yeast and later complexed with a desired envelope protein in vitro. Retroviral budding on yeast intracellular membranes can rely on the same host factors that are used by yeast retrotransposons driving intracellular formation of non-infectious virus-like particles. It is likely that optimal vector packaging system can be found as a result of the dedicated screening of the various retroviral vectors against an array of yeast species. The central implication of the hypothesis is that budding of retroviral vector particles inside yeast cells, with no need for obtaining and maintaining yeast spheroplasts or protoplasts, can be an efficient and economical method of mass production of these valuable gene therapy vectors.

Predictable and efficient retroviral gene transfer into murine bone marrow repopulating cells using a defined vector dose

Objective Current protocols of retroviral gene transfer into murine hematopoietic stem cells (HSC) result in variable gene transfer efficiency and involve various procedures that are not clinically applicable. We developed and evaluated a reliable transduction protocol that is more related to clinical methods. Materials and methods HSC were enriched from steady-state bone marrow by magnetic cell sorting (lineage depletion) and cultured in defined serum-free medium containing an improved growth factor cocktail (Flt3-ligand, stem cell factor, interleukin-3, interleukin-11). Cell-free ecotropic retroviral vector particles, generated by transient transfection of human 293T-based packaging cells, were preloaded at defined titers on CH296-coated tissue culture plates, thus largely avoiding serum contamination. These conditions were evaluated in 17 experiments involving 29 transduction cultures and 185 recipient mice. Results After two rounds of infection, the gene marking rates in cultured mononuclear cells and stem/progenitor cells (Lin −c-Kit +) were 15 to 85% (53.7%±21.7%, n = 23) and 30 to 95% (69.8%±20.4%, n = 17), respectively. Even after one round of infection, gene transfer was efficient (31.2%±15.1%, n = 12). Using identical conditions, gene transfer rates were highly reproducible. Average transgene expression in reconstituted animals correlated well with pretransplant data. Using a moderate multiplicity of infection, the majority of transduced cells carried less than three transgene copies. In addition, coinfection was possible to establish two different vectors in single cells. Conclusion The protocol described here achieves efficient retroviral transduction of murine bone marrow repopulating cells with a defined gene dosage, largely avoiding procedures that decrease stem cell output and repopulating capacity. This protocol may help to improve the predictive value of preclinical efficiency/toxicity studies for gene therapeutic interventions and basic research.

Poxviral/retroviral chimeric vectors allow cytoplasmic production of transducing defective retroviral particles.

AbstractRetroviral vectors have become important tools in gene therapy as a result of a number of highly desirable properties, including stable genomic integration, which are rarely found with other gene delivery vectors. However, after two decades of experience with this technology, some shortcomings still remain to be solved. Retroviral vectors cannot be grown to as high titers, as for example, adenoviruses; they tend to be unstable and are sensitive to lysis by complement when transfused into patients. The search for more robust retroviral delivery systems has led to the development of hybrid viral vectors to combine the broadly estimated features of retroviral vectors with advantageous properties of another viral vector system. Chimeric systems with adeno- or alphavirus vectors have already been reported (1–3). Furthermore, herpesvirus amplicons expressing gag, pol, and env genes were shown to rescue defective lacZ retro virus in a cell line carrying a corresponding pro virus (4). This chapter is dedicated to vaccinia virus, one of the most widely used vectors in molecular and cell biology (5,6) as a chimeric carrier for small RNA viruses, such as retroviruses. The methods provided in this chapter disclose the necessary steps for the generation of such hybrid vectors and for their use to produce retroviral vector particles in packaging cells. However, the system offers a number of interesting opportunities that will require individual cloning concepts. To fully exploit the possibilities of the hybrid vector system, it is therefore advisable to consider the life cycle and the specific properties of the two vectors that are fused in this chimera.KeywordsLong Terminal RepeatVaccinia VirusPackaging CellPackaging Cell LineHybrid VirusThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

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.

Encapsulated cells producing retroviral vectors for in vivo gene transfer

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. 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 beta-galactosidase activity and by RT-PCR. 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. 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.

Telomerase Can Inhibit the Recombination-based Pathway of Telomere Maintenance in Human Cells

Telomere length can be maintained by telomerase or by a recombination-based pathway. Because individual telomeres in cells using the recombination-based pathway of telomere maintenance appear to periodically become extremely short, cells using this pathway to maintain telomeres may be faced with a continuous state of crisis. We expressed telomerase in a human cell line that uses the recombination-based pathway of telomere maintenance to test whether telomerase would prevent telomeres from becoming critically short and examine the effects that this might have on the recombination-based pathway of telomere maintenance. In these cells, telomerase maintains the length of the shortest telomeres. In some cases, the long heterogeneous telomeres are completely lost, and the cells now permanently contain short telomeres after only 40 population doublings. This corresponds to a telomere reduction rate of 500 base pairs/population doubling, a rate that is much faster than expected for normal telomere shortening but is consistent with the rapid telomere deletion events observed in cells using the recombination-based pathway of telomere maintenance (Murnane, J. P., Sabatier, L., Marder, B. A., and Morgan, W. F. (1994) EMBO J. 13, 4953-4962). We also observed reductions in the fraction of cells containing alternative lengthening of telomere-associated promyelocytic leukemia bodies and extrachromosomal telomere repeats; however, no alterations in the rate of sister chromatid exchange were observed. Our results demonstrate that human cells using the recombination-based pathway of telomere maintenance retain factors required for telomerase to maintain telomeres and that once the telomerase-based pathway of telomere length regulation is engaged, recombination-based elongation of telomeres can be functionally inhibited.

Properties of wild-type, C-terminally truncated, and chimeric maedi-visna virus glycoprotein and putative pseudotyping of retroviral vector particles.

We have characterized the properties of the maedi-visna virus (MVV) glycoprotein, which has a long cytoplasmic C-terminal domain, and of a panel of C-terminally truncated and C-terminally chimeric MVV-Env constructs. Cells expressing wild-type MVV glycoprotein form syncytia with target cells from many different species and tissues, demonstrating that the MVV-Env cellular receptor is widely distributed. Similar to the situation with other lentiviral glycoproteins, truncation of the C-terminal domain of MVV-Env significantly increases its membrane fusion capacity. However, despite their presence in a fusogenic form at the cell surface, neither the wild-type nor any of the C-terminally modified MVV-Env constructs, these latter lacking sterically inhibitory C termini, were able to successfully pseudotype murine leukemia virus- or human immunodeficiency virus-derived vector particles.

Identification of Regions in the Moloney Murine Leukemia Virus SU Protein That Tolerate the Insertion of an Integrin-Binding Peptide

Targeting of retroviral vectors to specific cells has been attempted through engineering of the surface (SU) protein of the murine leukemia viruses (MuLVs), but in many cases this has adversely affected protein function and targeted delivery has been difficult to achieve. In this study, we have inserted a 15-mer peptide that binds specifically to the αvβ3 integrin into the Moloney MuLV SU protein, including regions that are surface exposed in the crystal structure of the ecotropic receptor-binding domain. We have concentrated in particular on the variable regions VRA, VRB, and VRC, which are responsible for the use of distinct cellular receptors by different MuLV subtypes and therefore may be more likely to accommodate a heterologous binding moiety. Despite these considerations, only 8 of 26 insertion sites were tolerated, including two separate regions in VRA, a cluster of sites in VRC, and previously identified sites at the N-terminus of the protein and in the proline-rich region immediately downstream of the receptor-binding domain. When expressed on retroviral vector particles, all of the viable proteins retained the ability to bind to and transduce murine cells, although the VRC mutants and an insertion in VRA gave reduced binding and titer. Finally, although all of the viable chimeras could bind to αvβ3 in a solid-phase binding assay, we were unable to demonstrate expanded tropism for αvβ3-expressing human cells. This study highlights the difficulty of engineering the Moloney MuLV SU protein, even when structural information is available, and provides guidelines for the insertion of peptide ligands into the SU protein.

In vivo cell type-specific gene delivery with retroviral vectors that display single chain antibodies.

Cell type-specific gene delivery will be essential for in vivo gene therapy. Our laboratory has previously developed retroviral vector particles, derived from spleen necrosis virus, SNV, which display the antigen-binding site of an antibody on the viral surface. Such particles infected only human cells in vitro, which expressed a receptor recognized by the antibody. To test cell type-specific gene delivery in vivo, a mouse model system has been developed. Antibiotic resistant human target and non-target cells were injected into the peritoneum of SCID mice. Subsequently, a vector solution containing 106 infectious particles, which display scAs against the human her2neu cell surface protein, was injected. Cells were recovered from the peritoneum, subjected to antibiotic selection, and tested for the expression of a lacZ gene transduced by the retroviral vector. We found that human target cells, which express her2neu, were infected in vivo. However, neither human cells that do not express her2neu, nor normal mouse cells were infected by such viral particles. These data give proof of principle that retroviral vector-mediated, cell type-specific gene delivery can be obtained in vivo.

A genetically engineered spleen necrosis virus-derived retroviral vector that displays the HIV type 1 glycoprotein 120 envelope peptide.

We reported that SNV-derived retroviral vectors, which display single-chain antibodies on the viral surface, enable cell type-specific gene delivery into various human cells. In particular, the SNV cell type-specific gene delivery vector system appears to be well suited to transduce genes into cells of the human hematopoietic system (Jiang et al., J. Virol. 72:10148-10156, 1998). Here, we report the construction of SNV vector particles that display the complete gp120 surface unit of the envelope protein of human immunodeficiency virus type 1 (HIV-1) on the viral surface. The complete gp120-coding region of a T cell-tropic HIV-1 strain (LAI/BRU) was fused to a short peptide spacer coding region [(Gly4Ser)3] linking it to the SNV TM-coding region. The corresponding protein was expressed as a single 145-kDa peptide as expected. This peptide was nontoxic and could be stably expressed in dog D17 SNV-derived packaging cells. Particles harvested from stable packaging lines infected CD4+ human hematopoietic cells with titers exceeding 10(5) CFU/ml supernatant tissue culture medium. Titers in other, CD4- cell lines expressing various coreceptors of HIV-1 were 100-fold lower than titers obtained in CD4+ cells. Specificity of infection was demonstrated by antibody inhibition assays or by preincubating cells with SDF-1alpha, the ligand, which binds to the CXCR4 coreceptor, to which this gp120 binds. Our data indicate that binding of the HIV-1 gp120 to either CD4 or CXCR4 is sufficient to enable infection of human cells with SNV vector particles. We constructed retroviral vector particles that display chimeric HIV-1-SU-SNV-TM proteins plus wild-type SNV envelope on the viral surface. Such particles allowed efficient infection of CD4-positive human T lymphocytes, and, at a lower efficiency, also cells expressing CXCR4 without CD4. These data coincide with our earlier hypothesis that the chimeric envelope is required only to bind the vector particle to a cell surface receptor of the target cell, while membrane fusion is mediated by wild-type Env, which alone is not sufficient to enable infection of human cells.

Human serum-resistant retroviral vector particles from galactosyl (α1–3) galactosyl containing nonprimate cell lines

Retroviral vector particles (RVP) which are resistant to inactivation by human serum will be needed for many in vivo gene therapy applications. Murine-based producer cell lines generate RVP which are inactivated by human serum, reportedly due to the presence of the galactosyl (alpha1-3) galactosyl carbohydrate moiety (alphaGal) on these and other nonprimate producer cells and RVP. Consequently, human cells (which lack the alphaGal moiety) have been developed as producer cell lines for generation of human serum-resistant RVP. In this study, we report that contrary to earlier reports, the presence of the alphaGal moiety on producer cells and RVP does not necessarily correlate with cell killing or RVP inactivation by human serum. We show that the alphaGal-positive ferret brain cell line, Mpf, is an excellent basal cell line for generation of RVP which have titers and serum resistance levels equal to or greater than RVP produced in human cell lines such as HT1080. Therefore, packaging cell lines need not be limited to those of human or primate origin for production of human serum-resistant RVP.