The use of self-inactivating (SIN) γ-retroviral vectors without enhancer–promoter sequences in the U3 region of the viral long terminal repeat (LTR) present several important advantages, the major ones being the increase of the internal promoter autonomy and a reduction of the risk of insertional upregulation of neighboring cellular genes.15, 16 However, several types of murine leukemia virus γ-retroviral SIN vectors were reported to yield lower viral vector titers, depending on the vector context.17, 18 This has been associated to an abundant transcription from the internal promoter driving transgene expression, which may occur at the expense of full-length viral RNA transcription originating from the LTR in packaging cells.18, 19 Various approaches were taken to overcome these limitations, such as the introduction of the post-transcriptional element from Woodchuck hepatitis virus into the 3′-untranslated region of SIN vectors,20, 17 or the incorporation of upstream polyadenylation enhancer elements derived from viral or cellular genes into the 3′U3 region of the LTR, allowing partial recovery of high viral vector titers.21 Recently, it has been observed that the use of a strong internal enhancer/promoter in SIN vectors may increase transgene expression but that this may also increase host cell transformation.22, 23 Thus, despite recent progress showing high titers for some SIN vectors,24 the production of high viral titers and the expression of the therapeutic gene at high levels without compromising safety may still remain a limitation for clinical use. Correspondence to Retroviral vector-mediated gene transfer has been central to the development of gene therapy. Retroviral vector-mediated gene transfer has been central to the development of gene therapy. However, the finding that the viral RNA levels were increased much more than the viral titer also implied that the mere viral RNA level is not the sole determinant of vector titer. γ-Retrovirus (gamma-retrovirus) is an RNA virus comprised of its genome and several structural and enzymatic proteins, including reverse transcriptase and … This also suggested that clones mediating high vector titers may be obtained more easily from these polyclonal populations.
An episomally replicating vector binds to the nuclear matrix protein SAF-A in vivo. mouse retrovirus vector caused leukemia in recipient animals because of two rare events: insertional mutagenesis and cooperation between the activated host gene and the introduced transgene. Lipps HJ, Jenke AC, Nehlsen K, Scinteie MF, Stehle IM, Bode J . Single sorted cells were cultured in conditioned medium containing hygromycin and puromycin as above. Gene therapy of X-linked severe combined immunodeficiency by use of a pseudotyped gammaretroviral vector.
Improving transcriptional termination of self-inactivating gamma-retroviral and lentiviral vectors.
To compensate for potential variations in the transfection efficiency, the GFP fluorescence was normalized to that of DsRed, as expressed from a cotransfected reference plasmid. The viral genes responsible for viral replication are separated from the rest of the genome, reducing the risk of infectious particle recombination, and thus retrovirus vector is a highly safe viral vector. This may be explained by the known competition of internal and upstream promoters in the clones showing very high GFP expression.48.
Viruses are natural vehicles for genetic information because they are obligate cellular parasites.
Gene Ther 2010; 17: 272–280. MAR elements regulate the probability of epigenetic switching between active and inactive gene expression.
Dr Mermod is a cofounder and owns stocks of the company. Harraghy N, Gaussin A, Mermod N . Retroviruses have several distinct advantages over other vectors, especially when permanent gene transfer is the preferred outcome.
Hum Gene Ther 1999; 10: 123–132. Specific primers were designed from the pRetro-GFP and pRetro-SEAP vectors to distinguish the full-length viral RNA from the shorter transgene mRNAs from pRetro-GFP transfected cells (forward primer, 5′-TTTAGTGAACCGTCAGATCCG-3′; reverse primer, 5′-CATTCACATCGCCATTCAGC-3′) and viral RNA from pRetro-SEAP transfected cells (forward primer, 5′-TTTAGTGAACCGTCAGATCCG-3′; reverse primer, 5′-GTTCCAGAAGTCCGGGTTC-3′). Packaging cell populations were transfected with pRetroQ-SEAP and derivative vectors and selection in complete medium containing 1.5 μg ml−1 puromycin (Invitrogen Life Technologies) for 2 weeks. You are using a browser version with limited support for CSS.
The medium was changed 10–12 h after transfection. Applied Bioengineering in Tissue Reconstruction, Replacement, and Regeneration.
Google Scholar. The objective of the present study was to assess whether epigenetic regulatory elements may be beneficial to generate SIN retroviral vectors.
1980;151:542–552. Click image to refresh the verification code. Ecotropic HEK-293 retroviral vector producer cells were transiently transfected with pRetro-GFP or with plasmid derivatives containing the human MAR elements 1–6 and 1–68 and X-29, as indicated. Fluorescence-activated cell sorting of high and low GFP-expressing cells was performed on cells treated with trypsin and washed with PBS containing 5% fetal bovine serum. Wilhelm C, Scherzad A, Bregenzer M, Meyer T, Gehrke T, Kleinsasser N, Hagen R, Hackenberg S. Oncol Lett.
Once inside the host cell's cytoplasm, the virus uses its own reverse transcriptase enzyme to produce DNA from its RNA genome, the reverse of the usual pattern, thus retro (backwards). Article
Equal potency of gammaretroviral and lentiviral SIN vectors for expression of O6-methylguanine-DNA methyltransferase in hematopoietic cells. Self-amplifying mRNA (SAM) Vaccine Platform, Fluorescent Virus-Like Particles Production, Outer Membrane Vesicles (OMVs) Platform for Vaccine Design, Epitope Predicting Algorithm for Vaccine Design, epiVacKit-Epitope-driven Vaccine Design Toolkit, SuperStable™ Thermostability Technology for Stable Storage of Vaccines, Design of Experiment (DOE)-based Formulation Platform, Suspension Culture for Pseudorabies Vaccine Production, Suspension Culture for PCV2 Vaccine Production, Suspension Culture for Avian Influenza Vaccine Production, Suspension Culture for Newcastle Disease Vaccine Production, Suspension Culture for Porcine Parvovirus Vaccine Production, Suspension Culture for Rabies Vaccine Production, Suspension Culture of Insect Cells for Subunit Vaccine Production, Cutaneous Squamous Cell Carcinoma Vaccines, Fully Synthetic Carbohydrate Based Cancer Vaccines, Cytomegalovirus-based Cancer Immunotherapy, Exosome-based Therapeutic Cancer Vaccines, Cytokine Development to Enhance Antigen Presentation, Microfluidic Cell-squeezing Technology for B Cell-based Vaccine, Antibody-antigen Fusion Technology for Cancer Vaccine, MHC Class II-associated li Protein Suppression Technology, Biomaterial-based Implantable Cancer Vaccine. This was assessed by measuring the viral RNA levels in packaging cells transfected with/without MAR using reverse transcription and quantitative PCR using primers targeting specifically the viral vector genomic RNA but not the transgene mRNAs (Figure 1). Levels of GFP expression were recorded from six individual clones that were selected to show different GFP expression levels, as represented in the original polyclonal population. This is a safety device commonly used for viral vectors.
Nat Rev Genet 2000; 1: 91–99. Internet Explorer). Retroviruses have several distinct advantages over other vectors, especially when permanent gene transfer is the preferred outcome. Recently, retroviral vector-mediated gene transfer, as well as the broader gene therapy field, has been re-invigorated with the development of a new class of retroviral vectors which are derived from lentiviruses.
Gene 2003; 304: 23–33. Although GFP expression levels were raised from 6- to 16-fold in MAR-containing clones, the viral RNA levels were increased by 32- or 38-fold, which translated to a 4.6- or 5-fold increase in vector titer when using 1–6M and 1–68M, respectively.
Ctr, control. page 3 Retroviral Vector Safety Acceptance of some low level risk may be justified when attempting to treat a life threatening disease. Ecopack and Amphopack packaging cell lines derived from human embryo kidney HEK293 cells were used for retroviral vector production as described by the provider (EcoPack2-293 and AmphoPack-293; Clontech Laboratories Inc.). Goetze S, Gluch A, Benham C, Bode J .
Knowledge about retroviral replication and virion assembly mechanisms provides the basis for the development of retroviral vectors. Karlen Y, McNair A, Perseguers S, Mazza C, Mermod N .
Google Scholar.
Europe Fig.2 Production of recombinant viral vectors. Adv Cancer Res 2003; 88: 53–99. Leukemia 2008; 22: 1519–1528.
Blood 2005; 105: 4235–4246. USA 1980;44:1265–1268. Packaging cells originally transfected with the 1–68M-containing plasmid yielded the highest retroviral vector titer, whereas a smaller but significant increase was also noted when using 1–6M (Figure 2d). Stimulation of Tat-independent transcriptional processivity from the HIV-1 LTR promoter by matrix attachment regions.
National Center for Biotechnology Information, Unable to load your collection due to an error, Unable to load your delegates due to an error.
This correlated with a prominent increase of the viral vector RNA levels in populations generated with the MARs (Figure 3d).
To determine whether similarly favorable effects may also be obtained when using other transgenes and/or packaging cell lines, we extended our analysis to amphotropic packaging cells, which express envelope proteins capable of transducing human cells. Oncogenicity of AKR endogenous leukemia viruses. Heng HH, Goetze S, Ye CJ, Liu G, Stevens JB, Bremer SW et al.
Genome-wide prediction of matrix attachment regions that increase gene expression in mammalian cells. Leukemias following retroviral transfer of multidrug resistance 1 (MDR1) are driven by combinatorial insertional mutagenesis.
The pRetroQ-transgene vector encodes puromycin-resistance gene and the GFP or the SEAP, as expressed from internal CMV and PGK promoters (dashed arrow), whereas the retroviral RNA is expressed from the hybrid (CMV/MSV) enhancer and promoter (P) fused to the γ-retroviral 5′LTR R and U5 sequences (long solid arrow).
Semin Liver Dis. Expression of another transgene was assessed, namely the human secreted alkaline phosphatase (SEAP) in amphotropic packaging cells.
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