Here, we set out to design and validate a DTA-encoding lentiviral vector that expresses DTA under the control of a constituitive promoter to allow for expression of DTA in a variety of cell types.
However, for use in a variety of different cell types or experimental systems, the vectors must be re-engineered and the conditions for induction optimized. These strategies allow for expression of the transgene only in cells where the tissue-specific promoter is functional or that are treated with a drug for induction of the inducible promoter. To achieve targeted expression, many laboratories have used tissue-specific or inducible promoters 18, 21– 24. Delivery using a lentiviral vector is particularly attractive due to the ability to encode the gene within the viral genome, which is then integrated into the host genome. Two important considerations for each method of delivery are the quantity of delivered cargo and the ability to target the cargo to specific cells to avoid nonspecific cell death. Several methods for delivery of suicide genes have been tested, including conjugation to cell penetrating peptides (CPPs) 10, encapsidation of the protein into viral like particles (VLPs) 11, 12, delivery via liposomes and nanoparticles 13– 15, and use of viral vectors 16– 21. Interestingly, disruption of diphthamide biosynthesis does not affect protein translation or cell viability 7– 9.ĭespite the obvious experimental and therapeutic potential of suicide genes, their inability to cross biological membranes and enter cells remains a significant barrier to their use.
#YOUTUBE SNAPGENE INFUSION CLONING SERIES#
Biosynthesis of diphthamide consists of stepwise modifications to His715 by a series of proteins, Dph1-7 7. The A fragment inhibits eukaryotic translation elongation factor 2 (eEF2) through adenosine diphosphate (ADP)-ribosylation of a modified histidine residue, diphthamide, at His715, which blocks protein translation and leads to cell death 4– 7. Diphtheria toxin (DT) is a 62 kDa protein secreted by the gram positive bacillus, Corynebacterium diphtheria 4, 5.
One commonly used suicide gene is the catalytic diphtheria toxin fragment A gene (DTA). Suicide genes encode proteins that are toxic to host cells at very low levels of expression, and have been widely investigated for their utility as therapeutic agents and as tools for in vitro negative selection strategies 1– 3. Thus, we also detail development of DTA-resistant cell lines, engineered through CRISPR/Cas9-mediated knockout of the diphthamide 1 (DPH1) gene, which enable both robust virus production by transfection and evaluation of DTA-expressing virus infectivity.
DTA exerts its toxic activity through inhibition of eukaryotic translation elongation factor 2 (eEF2) via adenosine diphosphate (ADP)-ribosylation of a modified histidine residue, diphthamide, at His715, which blocks protein translation and leads to cell death. Here, we present the design and validation of a diphtheria toxin A (DTA)-encoding lentiviral vector expressing DTA under the control of a constituitive promoter to allow for expression of DTA in a variety of cell types, with specificity provided via selection of glycoproteins for pseudotyping of the lentiviral particles. Delivery using a lentiviral vector is particularly attractive due to the ability to encode the gene within the viral genome, as well as the ability to limit off-target effects by using cell type-specific glycoproteins. Two important considerations for delivery are the quantity of delivered cargo and the ability to target the cargo to specific cells.
Several methods for delivery of suicide genes have been explored. Suicide genes have been widely investigated for their utility as therapeutic agents and as tools for in vitro negative selection strategies.
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