![]() Moreover, although ART suppresses HIV replication below detectable levels, it is not able to fully prevent chronic immune activation, inflammation and slow lymphoid tissue damage. Importantly, ART does not eliminate the HI virus. Įven though anti-retroviral therapy (ART) has turned AIDS from a deadly into a chronic, largely manageable disease with nearly normal life expectancy, gene therapy is still an attractive treatment option. Second, reported cases of obviously complete virus elimination in individuals with an established HIV infection after haematopoietic stem cell transplantation from CCR5Δ32-homozygous donors. Two observations have made CCR5 a favourite target for HIV gene therapy: First, the above mentioned almost complete protection of homozygous individuals from HIV. Indeed, the essential role of CCR5 for successful HIV infection was established by the identification of a homozygous, naturally occurring 32 bp deletion in the open reading frame of CCR5 ( CCR5Δ32) in multiply-exposed, but non-infected individuals. ![]() CCR5 came into focus, since, besides its physiological function, it serves as an HIV co-receptor on the surface of CD4 +-T cells and macrophages. In fact, the very first human gene therapy application of genome editing was based on ZFN-mediated knockout of the C-C motif chemokine receptor 5. The potential of targeted genome modification using designer nucleases for human gene therapy has been already recognised for zinc-finger nucleases (ZFNs). In the last years, genome editing has rapidly turned from a niche technology to a broadly used approach in basic and applied science. 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 × 10 9 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.
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