EFF-Nanotransfected Fibroblast Injection Could Treat Ischemic Stroke

April 5, 2021
Victoria Johnson
Victoria Johnson

Victoria Johnson, Assistant Editor for NeurologyLive, joined the MJH Life Sciences team in October 2020. Follow her on Twitter @VictoriaJNeuro or email her at vjohnson@neurologylive.com

Researchers found a 70% infarct resolution and 90% motor recovery in mice injected with the EFF-nanotransfected cells.

Data from a recent study suggest that vasculogenic cell therapies based on nanotransfection-driven (i.e., nonviral) cellular reprogramming have potential to treat ischemic stroke (IS).1

Researchers found that intracranial delivery of fibroblasts nanotransfected with Etv2, Foxc2, and Fli1 (EFF) led to a dose-dependent increase in perfusion 14 days after injection. Magnetic resonance imaging (MRI) and behavioral tests revealed around 70% infarct resolution and up to around 90% motor recovery in mice treated with the fibroblasts.

“We can rewrite the genetic code of skin cells so that they can become blood vessel cells,” said senior author Daniel Gallego-Perez, PhD, assistant professor, biomedical engineering and surgery, Ohio State University, in a statement.2 “When they’re deployed into the brain, they’re able to grow new, healthy vascular tissue to restore normal blood supply and aid in the repair of damaged brain tissue.”

Gallago-Perez and colleagues used transwell inserts to electrotransfect primary mouse embryonic fibroblasts (pMEFs) with a cocktail of plasmids encoding for EFF at a 1:1:1 ratio. pMEFs transfected with empty plasmids were used as controls. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) analyses at days 1 and 7 after nanotransfection showed a robust increase of EFF expression as compared to controls and immunofluorescence and tube formation assays at day 7 confirmed phenotypic changes consistent with fibroblast-to-induced endothelial cell (iEC) transitions and ability to assemble into blood vessel-like vascular networks in vitro.

READ MORE: IND Submitted for ImmCelz Regenerative Stroke Immunotherapy

Researchers found a marked increase in intracranial perfusion 7 days after intracranial injection of pMEFs into the subarachnoid space of 9-week-old C57BL/6 mice, 24 hours after EFF nanotransfection compared to controls as confirmed by laser speckle imaging (LSI).

They then intracranially injected EFF- or sham-nanotransfected pMEFs into the subarachnoid space of mice on day 7 after induced 30-minute IS. Mice were monitored for 21 days after stroke, and LSI was used to evaluate cerebral perfusion through the intact skull.

LSI analyses revealed a positive correlation between the number of EFF-nanotransfected cells and the perfusion levels for both the stroke-affected (ipsilateral) hemisphere, with an R2 value of 0.480 (adj. R2, 0.422; P = .018; n = 11) compared to 0.002 (adj. R2, 0.000; P = .894; n = 10) for number of sham-nanotransfected cells delivered, as well as contralateral hemisphere, with an R2 value of 0.696 (adj. R2, 0.662; P = .001; n = 11) compared to 0.002 (adj. R2 = 0.000; P =.906; n = 10) for sham-nanotransfected cells delivered.

“The implications of these observations are twofold, as it suggests that increased intracranial perfusion could potentially be modulated, in part, by a combination of (1) iEC-directed reprogramming of a proportion of the injected pMEFs, some of which appear to cross over to the contralateral hemisphere, and (2) exosome-driven autocrine and/or paracrine vasculogenesis or angiogenesis from preexisting vascular tissue,” Gallego-Perez and colleagues wrote in the paper. 

All told, mice treated with EFF-nanotransfected pMEFs demonstrated a non-significant trend (P = .1) of greater stroke volume reduction compared to mice injected with control pMEFs. Researchers also found data to support that at least a portion of the nanotransfected pMEFs is structurally involved in the intracranial de novo formation of induced vasculature (iVas).

Gallago-Perez identified weight loss as a confounder for stroke volume reduction, as mice with weight loss below 17% correlated with similar levels of spontaneous reduction in infarct volume regardless of treatment. When weight loss exceeded 17%, mice treated with the nanotransfected cells had significantly improved infarct resolution compared to mice treated with control. 

“Together, our results suggest that intracranial deployment of EFF-nanotransfected cells can lead to an overall improvement in gross motor skills in stroke-affected mice. However, additional studies are needed for a more in-depth evaluation of the impact of this potential therapeutic strategy on fine motor skills, which are also markedly affected by the stroke lesion in the motor cortex,” Gallago-Perez and colleagues concluded their paper.

Quantitative analyses show that compared to controls, mice injected with EFF-nanotransfected fibroblasts had a significantly higher count of neurons. Reduced glial fibrillary acidic protein (GFAP) immunoreactivity also suggests considerably less astroglial scarring in the stroke-affected hemisphere.

Significant motor improvements were seen 14 and 21 days after stroke in mice injected with the EEF-nanotransfected cells compared to those injected with sham-nanotransfected cells. These differences were seen in parameters such as average speed, traveled distance, time mobile, and counterclockwise rotations.

“This is a proof of concept in a biological system, in a relevant model of preclinical stroke, that demonstrates the potential that, you can have a stroke and then in a delayed fashion, you can receive this treatment and you can have an improvement in your outcome,” added study co-author Shahid M. Nimjee, MD, PhD, neurological surgeon, Wexner Medical Center, Ohio State University, to the statement.

REFERENCES
1.Lemmerman LR, Balch MHH, Moore JT, et al. Nanotransfection-based vasculogenic cell reprogramming drives functional recovery in a mouse model of ischemic stroke. Sci. Adv. 2021; 7(12) doi: 10.1126/sciadv.abd4735
2. New technology ‘retrains’ cells to repair damaged brain tissue in mice after stroke. News release. Ohio State University Wexner Medical Center. Published online March 19, 2021. http://osuwmc.multimedia-newsroom.com/index.php/2021/03/19/study-retraining-cells-may-reverse-brain-damage-after-stroke/