Feasibility of Extracorporeal Delivery of Fusosomes to Generate CAR T Cells In Vivo

• Published in: Blood 2023-11, Vol.142 (Supplement 1), p.3631-3631
• Main Authors: Green, Jesse, Tucker, Andrew, Vakil, Aesha, Scott-Skandera, Matt, Vagin, Vasily, Dingar, Dharmesh, Elgendi, Tarik, Proano, Christina, Dolinski, Brian, Lunning, Matthew A, Koepsell, Scott A, Branson, Charles A, Enger, Kerrie, Quarles, Danielle, Kouros-Mehr, Hosein, Miller, Sarah, Shamashkin, Misha, Elpek, Kutlu G
• Format: Article
• Language: English
• ISSN: 0006-4971; 1528-0020
• DOI: 10.1182/blood-2023-186317

Fusosomes are viral vectors pseudotyped with modified paramyxovirus envelopes targeting specific cell types. A CD8-targeted fusosome delivering a CD19CAR transgene has the potential to provide an off-the-shelf therapeutic approach to generate in vivo CD19-directed chimeric antigen receptor (CAR) T cells in patients. For an in vivo gene delivery platform there are several approaches for administration that could be considered including direct intravenous (IV) administration or extracorporeal delivery (ECD). With ECD, patients would first undergo apheresis, then fusosomes would be retained with patient peripheral blood mononuclear cells (PBMCs) in the apheresis bag for a brief period of time before the entire contents of the bag is IV delivered into the same patient. Here, we demonstrate the feasibility of this approach to generate in vivo CAR T cells in a clinical setting with a mock infusion and discuss the translation of these findings to direct IV administration. A healthy donor was apheresed to collect PBMCs using standard apheresis equipment. No further cell enrichment, separation, or washing was performed. CD8-targeted fusosome formulated in an isotonic cryoprotective buffer was administered into the apheresis bag by gravity via an infusion set at a dose of ~0.5 IU/cell, gently mixed and retained for 30 min. The contents of the bag were mock infused into sample collection bags at a controlled flow rate using an infusion pump. Samples were collected before and after fusosome addition, after 30 min retention, and every 30 min during the mock infusion. Cell counts and viability were measured by complete blood count (CBC) and flow cytometry analyses. Fusosome binding to CD8 + T cells was measured by flow cytometry. Cytokines and complement components in supernatants were measured by ELISA or MSD. Residual fusosome in the supernatant was measured by ddPCR. Transduction of CD8 + T cells was measured by flow cytometry and vector copy number (VCN) analysis after PBMCs were washed, activated with CD3/CD28 beads, and cultured for 8 days. Early transcription events were measured by ddPCR using specific amplicons targeting different DNA species generated during reverse transcription. The CD8-targeted fusosome was successfully deliveredusing aseptic techniques to a freshly collected apheresis bag.PBMC viability stayed high (>%95) throughout the process. There were no major changes in white blood cell, platelet, or red blood cell levels as measured by CBC analysis.