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. 2023 May 16;31(7):2206–2219. doi: 10.1016/j.ymthe.2023.05.011

Figure 4.

Figure 4

TAF1-32i XDP signature derived from ENoMi-transduced neural progenitor cells implanted in mouse brains and detected in brain tissue and plasma extracellular vesicles

(A) Schematic of workflow for ENoMi-transduced hNPCs (control and XDP) implantation in nude mouse brain. Post-implantation, EVs were isolated from brain slices and plasma derived from cardiac blood draws (CBDs). exRNA was isolated from brain tissue and plasma of mice to analyze the levels of TAF1-32i and copGFP in EVs. copGFP was used as a unique control for transgene expression by hNPC. (B) ENoMi-EVs were found predominantly near the implantation site of ENoMi-transduced patient-derived hNPCs. Mouse brain slices derived from the hNPC injection site and a distal site were enzymatically digested. Brain digest solutions were separated based on SEC. Representative bars of NanoLuc levels in each SEC fraction of EVs from injected site (red) and EVs from non-injected site (blue) (n > 4 for each group). (C) XDP disease-related exRNA in ENoMi-EVs secreted by ENoMi-transduced patient-derived hNPCs in mouse brain. ENoMi-EVs were isolated from enzymatic mouse brain digests by serial affinity pulldown with magnetic anti-FLAG-tag beads followed by anti-CD81 beads. The non-bound EV condition represents the brain EVs not captured by either anti-FLAG-tag or -CD81. Mice were implanted with either XDP-patient-derived hNPCs (n = 4 mice for each group) or control hNPCs (n = 4). Bar graphs represent TAF1-32i normalized to copGFP RNA levels, both analyzed with ddPCR. N.D. indicates non-detectable levels of TAF1-32i. (D) Electron microscopic image of ENoMi-EVs derived from brain digests. ENoMi-EVs were isolated by pulldown with anti-FLAG-tag magnetic beads, followed by elution with FLAG tag peptide. Black arrows represent eluted EVs after negative staining. Scale bar, 100 nm. (E) SEC-isolated blood EVs secreted by ENoMi-transduced patient-derived hNPCs in mouse brain contain detectable TAF1-32i transcript. On day 21 after implantation, TAF1-32i transcript was detected in mouse plasma (CBD) EVs isolated by SEC injected with XDP hNPCs and not in control hNPCs (n = 4 mice for each cell type). Data were analyzed with an unpaired t test, p < 0.05. (F) TAF1-32i transcript carried by ENoMi-EVs accumulates over time in the blood. Time course on levels of TAF1-32i detected in mouse plasma from CBD on days 7, 14, and 21 after implantation of XDP hNPCs in the brain. EVs were isolated with anti-FLAG-tag beads in mice injected with XDP hNPCs (n = 4 mice for each time point). Two-way ANOVA statistics were performed on data, p < 0.05. N.D. indicates non-detectable levels of TAF1-32i. (G) Electron microscopic image of ENoMi-EVs derived from plasma after negative stain. Plasma EVs from CBD were isolated by pulldown with anti-FLAG-tag magnetic beads and elution with FLAG tag peptide. Black arrows represent eluted EVs. Scale bar, 100 nm. (H) Blood EVs from patient-derived hNPCs in a mouse brain can be isolated from retro-orbital blood draw (ROBD). exRNA was isolated from EVs derived from pooled plasma from ROBD on days 7, 14, and 21. Bar graphs represent TAF1-32i normalized to copGFP levels, both detected with ddPCR. TAF1-32i-containing EVs were isolated with exodisc and subsequently anti-human CD81 capture beads (n = 3 for Control and XDP groups). N.D. indicates non-detectable levels of TAF1-32i.