1/19/2023 0 Comments Texmacs gmp medium![]() ![]() This thesis aims to explore these avenues by developing and applying advanced decisional tools that analyse the gene therapy supply chain systematically whilst capturing multiple stakeholder perspectives. Given the relative infancy of the sector, there is a strong drive towards adopting technologies that minimise costs and supply chain complexity. chimeric antigen receptor T-cell therapy – CAR T), hence the supply chain of these products is highly complex. Most of today’s gene therapy products are viral vector-based, typically relying on plasmid DNA supply for their production, and many are autologous ex vivo applications (e.g. Gene therapy products have tremendous therapeutic potential for indications such as cancer and even curative potential for some genetic diseases. We conclude that CCR5-Uco-hetTALEN transfected by mRNA electroporation facilitates specific, high-efficiency CCR5 gene-editing (30%–56%) and it is highly suited for clinical translation subject to further characterisation of off-target effects. Using single-cell HRMCA, we discovered high rates of TALEN-induced biallelic gene editing of CCR5, which translated in large numbers of CCR5-negative cells resistant to HIVenv-pseudotyped lentiviral vectors. We optimised in vitro mRNA production and showed that CCR5-on- and CCR2 off-target activities of CCR5-Uco-hetTALEN were limited to the first 72 and 24–48 h post-mRNA electroporation, respectively. ![]() Simultaneous cutting at CCR5 and CCR2 induced rearrangements, mainly 15-kb deletions between the cut sites, in up to 2% of T cells underlining the necessity to restrict TALEN expression. Notably, the CCR5Δ55–60 protein resulting from the 18-bp deletion was almost completely retained in the cytosol. ![]() Prevailing 18- and 10-bp deletions at the on-target site revealed microhomology-mediated end-joining as a major repair pathway. The novel variant, CCR5-Uco-hetTALEN features an obligatory heterodimeric Fok1-cleavage domain, which resulted in complete abrogation of off-target activity at previously found homodimeric as well as 7/8 in silico predicted, potential heterodimeric off-target sites, the only exception being highly homologous CCR2. Here we optimised our previously described CCR5-Uco-TALEN and its delivery by mRNA electroporation. Blue triangles indicate time points of sample collection on day 0 before and after activation, on day 3 prior to electroporation, on days 4–7 and after harvest on day 12.ĭisruption of the C-C-Chemokine-receptor-5 (CCR5) gene induces resistance towards CCR5-tropic HIV. During the whole process, the cell culture was regularly agitated to ensure sufficient gas support. Day 12: Harvest of cells in final formulation buffer. Medium and waste bags were exchanged during these days. Day 4–11: Expansion phase with feeding or media replacing steps with supplemented TexMACS GMP Medium. Day 3: Automated electroporation of cells in CliniMACS Electroporation Buffer with TALEN mRNA and incubation of cells at 32 ☌ for 24 h. CD4⁺ cells were separated after positive magnetic labelling with CliniMACS CD4 Reagent from fresh buffy coats and subsequently activated using MACS GMP T cell TransAct. Day 0: Installation of tubing sets (TS 520 + EP-2), integrity test and priming after attachment of supplemented CliniMACS PBS/EDTA (0.5 % human albumin) and TexMACS GMP Medium (MACS GMP Recombinant Human IL-7 and IL-15 and 3% human serum). Workflow of the automated T Cell Engineering (TCE) process and set up of the tubing sets at the CliniMACS ProdigyĬCR5-edited CD4⁺-T cells are manufactured during a 12-day process after single installation of a fused tubing set. ![]() In conclusion, transfection of primary T cells with CCR5-Uco-hetTALEN mRNA is readily scalable for GMP-compatible production and hence suitable for application in HIV gene therapy. Of note, about 40% of total large-scale produced cells showed a biallelic CCR5 editing, and between 25 and 42% of produced cells had a central memory T-cell phenotype. The automated process reliably produced high amounts of CCR5-edited CD4+-T cells (>1.5 × 109 cells with >60% CCR5 editing) within 12 days. Here we present a GMP-compatible mRNA electroporation protocol for the automated production of CCR5-edited CD4+-T cells in the closed CliniMACS Prodigy system. Clinical translation of the knockout approach requires up-scaling of the manufacturing process to clinically relevant cell numbers in accordance with good manufacturing practice (GMP). To this end, we have developed and optimised a CCR5-targeting TALE nuclease, CCR5-Uco-hetTALEN, mediating high-efficiency knockout of C-C motif chemokine receptor 5 (CCR5), the HIV co-receptor essential during initial infection. One of those strategies is directed at the protection of CD4+-T helper cells from HIV infection in HIV-positive individuals. Ex-vivo gene editing in T lymphocytes paves the way for novel concepts of immunotherapy. ![]()
0 Comments
Leave a Reply. |
AuthorWrite something about yourself. No need to be fancy, just an overview. ArchivesCategories |