Engineered extracellular vesicles as a therapeutic strategy for neurofibromatosis type I.

Introduction:: Neurofibromatosis type 1 (NF1) is an autosomal dominant disorder caused by the loss of function of neurofibromin protein due to mutations in the NF1 gene. Neurofibromin is a tumor suppressor protein expressed in all the tissues. The loss of function of Neurofibromin results in the over-activation of RAS followed by an over-activation of the mitogen-activated protein kinases (MAPK) pathway, leading to uncontrolled cellular proliferation and tumor development. NF1 patients developed benign cutaneous and plexiform neurofibromas (cNF and pNF respectively). Patients with pNFs also have a higher incidence of developing malignant peripheral nerve sheath tumors (MPNST), which are neurogenic sarcomas that arise from nerve peripheral cells. Several strategies have been developed for the treatment of NF1. Among these, the most promising is the use of inhibitors of the RAS associated pathways which have shown promising results in-vitro but have failed on clinical and preclinical translational studies. NF1 gene therapies are based on the use of viral vectors to deliver NF1 but also this approach has shown some limitations in the delivery of full-length NF1 and low rates of transfection efficiency in cells. Engineered extracellular vesicles (eEVs) have emerged as promising non-viral carriers for gene therapies. EVs can package large molecular cargo and have the natural ability to pass through biological barriers compared to other viral carriers. For this reason, our research focuses on the restoration of neurofibromin protein function via delivery of NF1 (e.g., mRNA, protein, and NF1 encoded plasmid) via eEVs as a novel therapeutic strategy for Neurofibromatosis type 1.


Materials and Methods:: Materials and methods: Human dermal fibroblast cells (HDFs) were transfected using non-viral methods with plasmids encoding for human NF1 and the eEVs were isolated from the culture media. Effective transfection of the donor cells was evaluated via absolute RT-PCRs and IF imaging. Packing of the NF1 genetic material inside the eEVs was confirmed using aRT-PCR. eEV size distribution and concentration were analyzed via Nanosight. HDFs were exposed to NF1 eEVs and the expression levels of NF1 were evaluated via aRT-PCRs. HDFs transfected with an empty plasmid were used to generate control eEVs.

Results, Conclusions, and Discussions:: Results: Our preliminary results showed that HDFs cells can be nanotransfected with NF1-encoding plasmids, as evidenced by the positive expression of the DDK tag in the nanotransfected cells (Figure 1A). Absolute RT-PCR analysis confirmed a sustained upregulation of NF1 transcripts in the donor cells (Figure 1B). Characterization of the eEVs showed significant loading with NF1 compared to control eEVs (Figure 1C). Nanosight analysis revelaed eEV concentrations in the range of 1x10¹⁰ eEVs/mL for NF1 and control eEVs (Figure 1D). The particle size remained consistent for both NF1 and control eEVs (Figure 1E). Western blot characterization confirmed the presence of the EV markers CD63 and β-tubulin for both NF1 and control eEVs (Figure 1E). NF1 eEVs were effectively captured and incorporated by the recipient HDFs cells after 24 hours after treatment (Figure 1F). Absolute RT-PCRs confirmed that the recipient cells were successfully transfected with the NF1 cargo and showed a sustained upregulation of NF1 (Figure 1G).

Discussion: Our preliminary results showed that we can engineer NF1-loaded eEVs from nanotransfected HDF. In general, this data showed the ability of engineered EVs to transfer exogenous NF1 mRNA to target cells. Moreover, the delivered mRNA can be translated in the recipient cells to a functional neurofibromin protein. Currently studies include evaluating the potential of NF1 eEVs to rescue of neurofibromin-deficient cells derived from human patients, and their ability to reduce tumor progression (i.e., tumor size) by restoring the dysregulated pathways using an NF1 mouse model .

Conclusion: These findings demonstrate that EVs can be effectively engineered to pack NF1 molecular cargo, and that NF1 eEVs have the ability to deliver mRNA transcripts to recipient cells. Moreover, we observed that cells treated with NF1 eEVs showed a robust upregulation of NF1 compared to cells treated with control eEVs. Overall, our data shows the potential of NF1 eEVs as a novel gene therapy strategy for the rescue of neurofibromin protein function that can alleviate the outcome of patients with neurofibromatosis type 1.


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