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Gene Transfer into Human Hematopoietic Stem Cells
Published in Richard K. Burt, Alberto M. Marmont, Stem Cell Therapy for Autoimmune Disease, 2019
Serguei Kisselev, Tatiana Seregina, Richard K. Burt, Charles J. Link
Various laboratories have designed constructs and methods to address the aforementioned problems of clinical grade retroviral vectors. The vector opsonization problem during culturing in human serum-containing media may be avoided by using human instead of murine VPC. The introduction of picornavirus-derived internal ribosome entry site (IRES) followed by the Zeocin resistance gene sequence into the genome of helper virus simplifies the selection of transfected packaging cells and prevents the silencing of helper virus related to 5’methylated cap formation in that the envgene derived from amphotropic virus is inserted downstream from the IRES sequence.53 Transfection of the packaging cell lines with plasmid coding for the vector of interest for the clinical trial may also contain an IRES followed by a different selectable marker such as the Neomycin resistance gene sequence. Cloning of mixed populations of VPC in the presence of Zeocin and Neomycin allows for monoclonal human VPC with reasonably high titers ranging from 2.0 to 3.0 × 106 CFU/ml. Cloning of individual VPC is a desirable step because it permits selection of clones with favorable integration sites within the genome. This allows for stable long-term expression of both helper virus and vector.
VNPs as Tools for Nanomedicine
Published in Nicole F Steinmetz, Marianne Manchester, Viral Nanoparticles, 2019
Nicole F Steinmetz, Marianne Manchester
In another example, VLPs from the bacteriophage MS2 were engineered to package anti-sense RNA against the 5′-untranslated region and internal ribosome entry site of the Hepatitis C virus (HCV). Packaging was accomplished by fusion of the anti-sense RNA sequence to the stem loop triggering encapsulation (recall Section 5.1.1). Cell delivery and penetration was achieved making use of cell-penetrating peptides that were chemically attached to surface Lys side chains present on the VLP. Inhibitory effects on gene expression of HCV were shown in vitro using an established reporter system (Wei et al., 2009). The utility of non-human pathogens as viral vectors for gene or RNAi delivery opens a new sector in viral nanotechnology. Future studies evaluating transfection efficiency as well as non-desired side effects comparing non-human viral vectors, viral vectors, and non-viral delivery systems are expected to give further insights into the feasibility of utilizing non-human VLPs for gene delivery.
Virus-Based Nanobiotechnology
Published in Yubing Xie, The Nanobiotechnology Handbook, 2012
Magnus Bergkvist, Brian A. Cohen
Genetic modifications of MS2 have also been used for the purpose of generating drug delivery constructs. In 2009, Wei et al. developed an antisense RNA delivery system using MS2 capsids by a two plasmid coexpression system. One plasmid encoded for the MS2 maturase and coat protein, and the other encoded for the operator sequence alongside the antisense RNA for 5′-untranslated region (UTR) and internal ribosome entry site (IRES) of the hepatitis C virus (HCV) (Wei et al. 2009). These antisense-carrying MS2 “virus-like particles” (VLPs) were delivered to cells containing an HCV-luciferase reporting system by conjugating the human immunodeficiency virus (HIV-1) TAT peptide to lysine residues on the capsid exterior via a succinimide-based heterobifunctional cross-linker. Their results showed a decrease in luciferase activity in Huh-7 cells with increasing concentrations of TAT decorated MS2 VLPs loaded with HCV antisense RNAs.
Evaluation of different vector design strategies for the expression of recombinant monoclonal antibody in CHO cells
Published in Preparative Biochemistry & Biotechnology, 2018
Hadi Bayat, Saghar Hoseinzadeh, Eśhagh Pourmaleki, Roshanak Ahani, Azam Rahimpour
Accordingly, different vector design strategies have developed for expression of whole mAbs in CHO cells each presenting their strengths and weaknesses. While early studies were more focused on the use of two independent vectors each containing light chain (LC) or heavy chain (HC) coding sequences; however, in this system, the expression level of each polypeptide can be affected by their transfection efficiency, genomic integration site, and copy number.[7] The use of single vectors with two independent promoters for each polypeptide solves this problem although the adjacent promoters in a single vector can affect the transcription of one another; the phenomenon known as transcription interference.[8] Bicistronic-expression single vectors based on internal ribosome entry sites (IRES) have been extensively used for coexpression of different proteins in mammalian cells.[9] The bicistronic vector systems offer the advantage of using one single promoter for the expression of both polypeptides. When placed between two coding sequences, IRES elements facilitate cap-independent translation initiation of the downstream gene. However, it has been shown that the activity of IRESes can be affected by the cell type and variable expression can be observed for the downstream coding sequence.[10]