Altering the tropism of lentiviral vectors through pseudotyping.
TL;DR: This review samples a few of the more prominent examples from the ever-expanding list of published lentiviral pseudotypes, noting comparisons made with pseudotypes involving VSV-G in terms of titer, viral particle stability, toxicity, and host-cell specificity.
Abstract: The host range of retroviral vectors including lentiviral vectors can be expanded or altered by a process known as pseudotyping Pseudotyped lentiviral vectors consist of vector particles bearing glycoproteins (GPs) derived from other enveloped viruses Such particles possess the tropism of the virus from which the GP was derived For example, to exploit the natural neural tropism of rabies virus, vectors designed to target the central nervous system have been pseudotyped using rabies virus-derived GPs Among the first and still most widely used GPs for pseudotyping lentiviral vectors is the vesicular stomatitis virus GP (VSV-G), due to the very broad tropism and stability of the resulting pseudotypes Pseudotypes involving VSV-G have become effectively the standard for evaluating the efficiency of other pseudotypes This review samples a few of the more prominent examples from the ever-expanding list of published lentiviral pseudotypes, noting comparisons made with pseudotypes involving VSV-G in terms of titer, viral particle stability, toxicity, and host-cell specificity Particular attention is paid to publications of successfully targeting a specific organ or cell types
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TL;DR: This protocol describes how lentiviral vectors can be produced, purified and titrated for in vitro and in vivo gene delivery.
Abstract: Lentiviral vectors offer unique versatility and robustness as vehicles for gene delivery. They can transduce a wide range of cell types and integrate into the host genome in both dividing and post-mitotic cells, resulting in long-term expression of the transgene both in vitro and in vivo. This protocol describes how lentiviral vectors can be produced, purified and titrated. High titer suspensions can be routinely prepared with relative ease: a low-titer (106 viral particles/ml) unpurified preparation can be obtained 3 d after transfecting cells with lentiviral vector and packaging plasmids; a high-titer (109 viral particles/ml) purified preparation requires 2 more days.
939 citations
TL;DR: Recent advances of the three major genome editing technologies are reviewed and the applications of their derivative reagents as gene editing tools in various human diseases and potential future therapies are discussed, focusing on eukaryotic cells and animal models.
Abstract: Based on engineered or bacterial nucleases, the development of genome editing technologies has opened up the possibility of directly targeting and modifying genomic sequences in almost all eukaryotic cells. Genome editing has extended our ability to elucidate the contribution of genetics to disease by promoting the creation of more accurate cellular and animal models of pathological processes and has begun to show extraordinary potential in a variety of fields, ranging from basic research to applied biotechnology and biomedical research. Recent progress in developing programmable nucleases, such as zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeat (CRISPR)–Cas-associated nucleases, has greatly expedited the progress of gene editing from concept to clinical practice. Here, we review recent advances of the three major genome editing technologies (ZFNs, TALENs, and CRISPR/Cas9) and discuss the applications of their derivative reagents as gene editing tools in various human diseases and potential future therapies, focusing on eukaryotic cells and animal models. Finally, we provide an overview of the clinical trials applying genome editing platforms for disease treatment and some of the challenges in the implementation of this technology.
858 citations
Cites background from "Altering the tropism of lentiviral ..."
...For example, lentiviral vectors are typically pseudotyped with glycoprotein G from vesicular stomatitis virus (VSV-G), extending the vector tropism to a wide range of host cells.(334) By controlling the ratio of assembled wild-type viral capsid to protease-activatable subunits, the overall transduction level of protease-activatable viruses (PAVs) increased....
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TL;DR: Substantial progress in modifying viral vectors using diverse techniques now allows targeting to many cell types in vitro and, although important challenges remain for in vivo applications, the first clinical trials with targeted vectors have already begun to take place.
Abstract: To achieve therapeutic success, transfer vehicles for gene therapy must be capable of transducing target cells while avoiding impact on non-target cells. Despite the high transduction efficiency of viral vectors, their tropism frequently does not match the therapeutic need. In the past, this lack of appropriate targeting allowed only partial exploitation of the great potential of gene therapy. Substantial progress in modifying viral vectors using diverse techniques now allows targeting to many cell types in vitro. Although important challenges remain for in vivo applications, the first clinical trials with targeted vectors have already begun to take place.
661 citations
Additional excerpts
...Pseudotyping has been used most extensively to modulate the host-cell tropisms of retroviral (including lentiviral) vectors because they are highly permissive for incorporation of heterologous attachment glycoprotein...
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TL;DR: Improved protocols to generate highly concentrated lentiviral vector pseudotypes involving different envelope glycoproteins are described, which result in high-titer vector preparations that show reduced toxicity compared with lentIViral vectors produced using standard protocols involving ultracentrifugation-based methods.
Abstract: Over the past decade, lentiviral vectors have emerged as powerful tools for transgene delivery. The use of lentiviral vectors has become commonplace and applications in the fields of neuroscience, hematology, developmental biology, stem cell biology and transgenesis are rapidly emerging. Also, lentiviral vectors are at present being explored in the context of human clinical trials. Here we describe improved protocols to generate highly concentrated lentiviral vector pseudotypes involving different envelope glycoproteins. In this protocol, vector stocks are prepared by transient transfection using standard cell culture media or serum-free media. Such stocks are then concentrated by ultracentrifugation and/or ion exchange chromatography, or by precipitation using polyethylene glycol 6000, resulting in vector titers of up to 10(10) transducing units per milliliter and above. We also provide reliable real-time PCR protocols to titrate lentiviral vectors based on proviral DNA copies present in genomic DNA extracted from transduced cells or on vector RNA. These production/concentration methods result in high-titer vector preparations that show reduced toxicity compared with lentiviral vectors produced using standard protocols involving ultracentrifugation-based methods. The vector production and titration protocol described here can be completed within 8 d.
597 citations
Cites background from "Altering the tropism of lentiviral ..."
..., although a wide range of alternative glycoproteins have the ability to associate with the vector's membrane, a process that is referred to as pseudotypin...
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TL;DR: It is shown that the LDL receptor (LDLR) serves as the major entry port ofVSV and of VSV-G-pseudotyped lentiviral vectors in human and mouse cells, whereas other LDLR family members serve as alternative receptors.
Abstract: Vesicular stomatitis virus (VSV) exhibits a remarkably robust and pantropic infectivity, mediated by its coat protein, VSV-G. Using this property, recombinant forms of VSV and VSV-G-pseudotyped viral vectors are being developed for gene therapy, vaccination, and viral oncolysis and are extensively used for gene transduction in vivo and in vitro. The broad tropism of VSV suggests that it enters cells through a highly ubiquitous receptor, whose identity has so far remained elusive. Here we show that the LDL receptor (LDLR) serves as the major entry port of VSV and of VSV-G-pseudotyped lentiviral vectors in human and mouse cells, whereas other LDLR family members serve as alternative receptors. The widespread expression of LDLR family members accounts for the pantropism of VSV and for the broad applicability of VSV-G-pseudotyped viral vectors for gene transduction.
472 citations
Additional excerpts
...Cytokine Growth Factor Rev 18(5-6):525–533....
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References
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TL;DR: The ability of HIV-based viral vectors to deliver genes in vivo into nondividing cells could increase the applicability of retroviral vectors in human gene therapy.
Abstract: A retroviral vector system based on the human immunodeficiency virus (HIV) was developed that, in contrast to a murine leukemia virus-based counterpart, transduced heterologous sequences into HeLa cells and rat fibroblasts blocked in the cell cycle, as well as into human primary macrophages. Additionally, the HIV vector could mediate stable in vivo gene transfer into terminally differentiated neurons. The ability of HIV-based viral vectors to deliver genes in vivo into nondividing cells could increase the applicability of retroviral vectors in human gene therapy.
5,076 citations
TL;DR: The ability to concentrate vesicular stomatitis virus G glycoprotein pseudotyped vectors will facilitate gene therapy model studies and other gene transfer experiments that require direct delivery of vectors in vivo, and facilitate genetic studies in nonmammalian species, including the important zebrafish developmental system.
Abstract: The restricted host-cell range and low titer of retroviral vectors limit their use for stable gene transfer in eukaryotic cells. To overcome these limitations, we have produced murine leukemia virus-derived vectors in which the retroviral envelope glycoprotein has been completely replaced by the G glycoprotein of vesicular stomatitis virus. Such vectors can be concentrated by ultracentrifugation to titers > 10(9) colony-forming units/ml and can infect cells, such as hamster and fish cell lines, that are ordinarily resistant to infection with vectors containing the retroviral envelope protein. The ability to concentrate vesicular stomatitis virus G glycoprotein pseudotyped vectors will facilitate gene therapy model studies and other gene transfer experiments that require direct delivery of vectors in vivo. The availability of these pseudotyped vectors will also facilitate genetic studies in nonmammalian species, including the important zebrafish developmental system, through the efficient introduction and expression of foreign genes.
1,503 citations
TL;DR: A human 293-derived retroviral packaging cell line (293GPG) capable of producing high titers of recombinant Moloney murine leukemia virus particles that have incorporated the vesicular stomatitis virus G (VSV-G) protein is generated.
Abstract: We have generated a human 293-derived retroviral packaging cell line (293GPG) capable of producing high titers of recombinant Moloney murine leukemia virus particles that have incorporated the vesicular stomatitis virus G (VSV-G) protein. To achieve expression of the retroviral gag-pol polyprotein, the precise coding sequences for gag-pol were introduced into a vector which utilizes totally nonretroviral signals for gene expression. Because constitutive expression of the VSV-G protein is toxic in 293 cells, we used the tetR/VP 16 transactivator and teto minimal promoter system for inducible, tetracycline-regulatable expression of VSV-G. After stable transfection of the 293GPG packaging cell line with the MFG.SnlsLacZ retroviral vector construct, it was possible to readily isolate stable virus-producing cell lines with titers approaching 10(7) colony-forming units/ml. Transient transfection of 293GPG cells using a modified version of MFG.SnlsLacZ, in which the cytomegalovirus IE promoter was used to drive transcription of the proviral genome, led to titers of approximately 10(6) colony-forming units/ml. The retroviral/VSV-G pseudotypes generated using 293GPG cells were significantly more resistant to human complement than commonly used amphotropic vectors and could be highly concentrated (> 1000-fold). This new packaging cell line may prove to be particularly useful for assessing the potential use of retroviral vectors for direct in vivo gene transfer. The design of the cell line also provides at least theoretical advantages over existing cell lines with regard to the possible release of replication-competent virus.
1,034 citations
TL;DR: A peripheral membrane protein that is interactive with lymphocytic choriomeningitis virus (LCMV) was purified from cells permissive to infection and determined to be alpha-dystroglycan (alpha-DG).
Abstract: A peripheral membrane protein that is interactive with lymphocytic choriomeningitis virus (LCMV) was purified from cells permissive to infection. Tryptic peptides from this protein were determined to be alpha-dystroglycan (alpha-DG). Several strains of LCMV and other arenaviruses, including Lassa fever virus (LFV), Oliveros, and Mobala, bound to purified alpha-DG protein. Soluble alpha-DG blocked both LCMV and LFV infection. Cells bearing a null mutation of the gene encoding DG were resistant to LCMV infection, and reconstitution of DG expression in null mutant cells restored susceptibility to LCMV infection. Thus, alpha-DG is a cellular receptor for both LCMV and LFV.
647 citations
TL;DR: It is reported that a single injection of a VEGF-expressing lentiviral vector into various muscles delayed onset and slowed progression of ALS in mice engineered to overexpress the gene coding for the mutated G93A form of the superoxide dismutase-1 (SOD1G93A).
Abstract: Amyotrophic lateral sclerosis (ALS) causes adult-onset, progressive motor neuron degeneration in the brain and spinal cord, resulting in paralysis and death three to five years after onset in most patients. ALS is still incurable, in part because its complex aetiology remains insufficiently understood. Recent reports have indicated that reduced levels of vascular endothelial growth factor (VEGF), which is essential in angiogenesis and has also been implicated in neuroprotection, predispose mice and humans to ALS. However, the therapeutic potential of VEGF for the treatment of ALS has not previously been assessed. Here we report that a single injection of a VEGF-expressing lentiviral vector into various muscles delayed onset and slowed progression of ALS in mice engineered to overexpress the gene coding for the mutated G93A form of the superoxide dismutase-1 (SOD1(G93A)) (refs 7-10), even when treatment was only initiated at the onset of paralysis. VEGF treatment increased the life expectancy of ALS mice by 30 per cent without causing toxic side effects, thereby achieving one of the most effective therapies reported in the field so far.
638 citations