Challenges in Delivering Gene Therapy
Yashwant Pathak in Gene Delivery, 2022
For gene therapy to work, DNA much be delivered to the target tissue and then transported to the nucleus for protein expression. Within this problem there is another underlying problem. The first problem is that a system must be established to deliver DNA to the target tissues and must be preventive to degradation. The second problem stems from the establishment of another system to build a DNA construct, and then allow the target cell to express that protein at efficient therapeutic levels [25, 26]. To establish a system which delivers DNA to the tissues and nucleus, a mechanism to circumvent extra and intracellular barriers must be taken into account. Some of these barriers include stability of the biological environment, cellular uptake, lysosomal escape, cytosolic transport, and gene unpacking [27–31]. These are some of the barriers that must be overcome, and such solutions must be provided with trials and experimentation.
Gene Targeting Models of Epilepsy: Technical and Analytical Considerations
Steven L. Peterson, Timothy E. Albertson in Neuropharmacology Methods in Epilepsy Research, 2019
The targeting construct is typically linearized and introduced into ES cells by electroporation. In this step, cells are subjected to an electrical current that facilitates the internalization and integration of the DNA construct. The cells are then plated onto feeder layers. One day following electroporation, double drug selection is applied with geneticin and FIAU or gancyclovir. Those cells that had failed to incorporate the targeting construct are killed by the addition of geneticin to the culture medium (positive selection). The majority of the surviving cells have incorporated the entire DNA construct (including the thymidine kinase gene) at random sites throughout the genome. By contrast, during homologous recombination, nonhomologous regions of the construct that are not flanked by homologous sequences are excluded from the integration event. Therefore, homologous recombinant clones will not contain the thymidine kinase gene. Thus, the addition of a second drug, gancyclovir, will selectively kill cells that have randomly incorporated the construct (negative selection), thereby enriching for targeted clones. In our experience, this strategy typically provides a 2- to 30-fold enrichment for targeted clones. These clones are expected to be heterozygous for the targeted allele (except when targeting X-linked genes in male ES cell lines).
Experimental perturbations to investigate cardiovascular physiology
Neil Herring, David J. Paterson in Levick's Introduction to Cardiovascular Physiology, 2018
By transfecting a DNA construct that contains two homologous regions of DNA identical to sequences each side of the gene of interest into mouse ESCs, homologous recombination between these regions can result in a sequence of DNA from the construct being introduced into the host genome as demonstrated in Figure 20.3. The introduced sequence may contain a modified version of the gene or even encode an additional fluorescent tag that is knocked in or may contain no functioning gene at all resulting in knockout. Gain of function can also be achieved by introducing extra copies of a gene of interest or manipulating its promoter to upregulate gene expression. Introducing a sequence containing an antibiotic-resistant gene will help select for cells that have undergone successful recombination during subsequent culture. ESCs incorporating the alteration are then injected into blastocysts and implanted into surrogate mothers. Offspring are genotyped and interbred to identify homo- and heterozygotes for the manipulation, and control wild-type mice.
A poly-neoantigen DNA vaccine synergizes with PD-1 blockade to induce T cell-mediated tumor control
Published in OncoImmunology, 2019
Elena Tondini, Tsolere Arakelian, Koen Oosterhuis, Marcel Camps, Suzanne van Duikeren, Wanda Han, Ramon Arens, Gerben Zondag, Jeroen van Bergen, Ferry Ossendorp
We first analyzed whether these five artificially connected sequences lead to the generation of the expected peptide epitopes and their presentation on MHC molecules. Upon transfection of the designed DNA construct, the translated protein product needs to be processed in such a way that the T cell epitopes are generated and presented by MHC molecules. MC38 cells, which do not express the ovalbumin gene, were transfected with the neoantigen construct and the presentation of the ovalbumin CTL epitope SIINFEKL was detected by staining the SIINFEKL/H2-Kb complex with the 25-D1.16 antibody (Figure 1(b), upper panel). Transfection with the poly-neoantigen construct, but not with a control GFP-encoding construct, displayed positive staining for SIINFEKL/H2-Kb complexes. Moreover, after transfection with the neoantigen construct, cells were recognized by the hybridoma T cell line B3Z, which express a TCR specific for SIINFEKL/H2-Kb (Figure 1(b), lower panel).
Dual-targeting triplebody 33-16-123 (SPM-2) mediates effective redirected lysis of primary blasts from patients with a broad range of AML subtypes in combination with natural killer cells
Published in OncoImmunology, 2018
Todd A. Braciak, Claudia C. Roskopf, Sarah Wildenhain, Nadja C. Fenn, Christian B. Schiller, Ingo A. Schubert, Uwe Jacob, Annemarie Honegger, Christina Krupka, Marion Subklewe, Karsten Spiekermann, Karl-Peter Hopfner, Georg H. Fey, Michael Aigner, Stefan Krause, Andreas Mackensen, Fuat S. Oduncu
The DNA construct coding for SPM-2 was synthesized by a commercial provider (Eurofins/MWG-Operon). To create this construct, the scFv subunits were disulfide-stabilized78-80 humanized81-83 and stability-engineered84 by standard procedures. The TransIT®-LT1 transfection reagent (Mirus Bio LLC, catalog # MIR 2300) was used for transfection according to the manufacturer´s protocol to generate a stable cell pool of FreestyleTM 293F cells (ThermoFisher Scientific, cat. # R79007) for protein expression. Cells were then cultured under continuous selection with hygromycin C. SPM-2 was captured from cell culture supernatants via its C-terminal hexahistidine tag by immobilized zinc ion affinity chromatography followed by anion exchange chromatography using a 1 ml (bed volume) HiTrap Q Sepharose HP column (GE Healthcare). The third purification step was a cation exchange (CEX) chromatography. Here, a 1 ml HiTrap SP Sepharose HP column (GE Healthcare) was used and connected to an Äkta liquid chromatography system (GE Healthcare). SPM-2 preparations were analyzed by size exclusion chromatography (Superdex S200 5/150 GL column, GE Healthcare) for quality control and by SDS polyacrylamide gel electrophoresis (SDS-PAGE) after the final purification step. The protein concentration was determined by UV absorption at 280 nm using a NanoDrop spectrometer (PeqLab). The theoretical extinction coefficient was calculated from the primary amino acid sequence with the computer program ProtParam (www.expasy.ch). A final yield after purification of approx. 2.5 to 5 mg SPM-2 per L of culture medium was achieved in several independent experiments.
Actin stabilizer TAGLN2 potentiates adoptive T cell therapy by boosting the inside-out costimulation via lymphocyte function-associated antigen-1
Published in OncoImmunology, 2018
Bu-Nam Jeon, Hye-Ran Kim, Yun Shin Chung, Bo-Ra Na, Hyunkyung Park, Chorong Hong, Yasmin Fatima, Hyeonju Oh, Chang-Hyun Kim, Chang-Duk Jun
Next, we generated a retroviral DNA construct containing wild-type TAGLN2 and eGFP genes or eGFP (empty vector [EV]) alone (Figure 2(a)). Retroviral particles containing TAGLN2 or EV were then produced from host plat E cells and infected into mouse primary CD8+ T cells for determination of the viral transduction efficiency by flow cytometry. The efficiency was generally over 80% (Figure 2(b)) and was also confirmed by western blotting (Figure 2(c)). Next, we questioned whether TAGLN2 expression may influence CD8+ T cell adhesion to cancer cells via the LFA-1/ICAM-1 interaction, as we observed for T-cell conjugation with APCs (Figure 1(c)). To this end, we examined the expression levels of ICAM-1 in two cancer cell lines. Interestingly, B16F10 melanoma expressed little ICAM-1, whereas E0771 breast cancer cells expressed relatively high amounts of ICAM-1 (Figure 2(d)). By performing conjugation assays, we observed that OTI TCR+ CD8+ T cells overexpressing TG2 (OTI TG2-CD8+ T cells) showed significant increases in the numbers of conjugates when the cells were incubated with OVA257-264 peptide-loaded E0771 (OVA-E0771) cells, but not with OVA-loaded B16F10 (OVA-B16F10) cells (Figure 2(e) and Figure S2), suggesting that the costimulatory LFA-1/ICAM-1 interaction was critical for cytotoxic T-cell adhesion to ICAM-1+ cancer target cells. This conclusion was further corroborated by utilizing anti-LFA-1 antibodies; anti-LFA-1, but not control IgG, antibodies significantly reduced the number of conjugates between TG2-CD8+ T cells and E0771 cells (Figure 2(e)).
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