Automated production of CCR5-negative CD4+-T cells in a GMP-compatible, clinical scale for treatment of HIV-positive patients

• Published in: Gene therapy Vol. 28; no. 9; pp. 572 - 587
• Main Authors: Schwarze, Lea Isabell, Sonntag, Tanja, Wild, Stefan, Schmitz, Sabrina, Uhde, Almut, Fehse, Boris
• Format: Journal Article
• Language: English
• Published: London Nature Publishing Group UK 01.09.2021; Nature Publishing Group
• Subjects: 13/109; 13/31; 13/56; 38/77; 631/250/251; 692/699/255; Automation; Biomedical and Life Sciences; Biomedicine; CCR5 protein; CD4 antigen; CD4-Positive T-Lymphocytes; Cell Biology; Chemokine receptors; Electroporation; Gene Editing; Gene Expression; Gene Therapy; Genome editing; Good Manufacturing Practice; Helper cells; HIV; HIV Infections - therapy; Human Genetics; Human immunodeficiency virus; Humans; Immunological memory; Immunotherapy; Lymphocytes; Lymphocytes T; Memory cells; mRNA; Nanotechnology; Nuclease; Phenotypes; Receptors, CCR5 - genetics; T-Lymphocytes; Transfection
• ISSN: 0969-7128; 1476-5462
• DOI: 10.1038/s41434-021-00259-5

Ex-vivo gene editing in T lymphocytes paves the way for novel concepts of immunotherapy. One of those strategies is directed at the protection of CD4 + -T helper cells from HIV infection in HIV-positive individuals. 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. 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). 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. 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. 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. 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.