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Proteins are essential in moderating functions and regulating the cell cycle to ensure appropriate cell production and death. Neurofibromatosis type 1 (NF1) is a genetic disorder resulting in truncated, non-functional, production of the neurofibromin protein. The protein is essential in cell cycle regulation by down regulating the RAS pathway, a part of the cell cycle's division pathway. The uncontrolled cell growth induces formation of benign and malignant tumors – cutaneous neurofibromas (cNFs) and plexiform neurofibromas (pNFs). Cutaneous neurofibromas are benign tumors associated with discomfort and emotional distress and pNFs grow along nerves, affecting the spinal cord, muscles, and eyes. PNFs can develop into malignant peripheral nerve sheath tumors – an aggressive tumor with high recurrence. Current treatments rely on the ability to obtain clean margins for resection while preserving the nerve. Unfortunately, it's difficult to get clear margins on pNFs and treatment frequently results in tumor regrowth. Here we explore the use of engineered extracellular vesicles (EVs) loaded with the healthy NF1 gene for treatment by upregulating neurofibromin. For this method of treatment of other diseases the current standard uses Adeno-Associated Viruses (AAV), but they are limited by size and cannot deliver treatment as large as NF1, 13KBp.

To engineer the EVs, human dermal fibroblast (HDFs) cells were nanotransfected with NF1 encoding plasmids, pCMV6 empty plasmid was used to transfect the cells as a control. The transfection efficiency for HDFs and PMEFs was evaluated via RT-PCRs and immunostaining. The engineered EVs (eEVs) were isolated from collected cell media using two different methods – exosome reagent and size exclusion chromatography. The NF1 mRNA cargo from the eEVs was characterized using RT-PCR and the presence of neurofibromin protein within the eEVs was then confirmed using Western Blot. To evaluate the functional cargo delivery of NF1 transcripts using NF1 loaded EVs, healthy HDFs were exposed to 3000 EVs per cell for 24 hours and the effective delivery of the cargo was evaluated using RT-PCRs. Preliminary results show that we can effectively engineer extracellular vesicles loaded with the full length of NF1 mRNA and encoded plasmid. Furthermore, Western Blot results confirmed that the eEVs are loaded with functional neurofibromin protein. The uptake experiments exhibited the potential of NF1 eEVs to transfer exogenous NF1 mRNA to healthy recipient cells. These findings demonstrate the feasibility of the NF1 loaded eEVs as a gene therapy strategy to treat Neurofibromatosis type 1. Current experiments are focused on evaluating the ability of NF1 loaded eEVs in the rescue of neurofibromin protein function in two human NF1 deficient cell lines sNF95.11b and sNF952.6. Preliminary data of this experiment shows that cells exposed to NF1 eEVs had a significant decrease in the proliferation in comparison to their untreated counterparts.