Gene therapy has gained significant attention due to the possibility of selectively correcting altered genes. The recent emergence of CRISPR/Cas9 technologies have strengthened the field even further. For this reason, this approach holds much promise for the treatment of several diseases, including Sandhoff disease, Phenylketonuria, and Mucopolysaccharidosis. A significant limitation is to assure that significant amounts of the genetic material reach the target cells without jeopardizing other cellular functions. This is mainly because of physiological barriers such as the spleen, kidney, liver, phagocyte system, the cell membrane, and the formed endosomes once they reach the intracellular space. Moreover, they may face quick degradation, interaction with serum proteins, nucleases, and cytoplasmic structures. An innovative solution to improve stability and transit during delivery is using nanostructured magnetic materials such as magnetite, which has been widely used as a delivery vehicle of biomolecules. Also, magnetite can be directed to target tissues by magnetic fields, showing high biocompatibility without significant accumulation. Therefore, this study aims to develop a platform for the delivery of nucleic acids with applications in gene therapy for the treatment of the previously mentioned diseases. The developed vehicle based on magnetite nanoparticles is functionalized with an organosilane, a surface spacer, and a molecule that contains a disulfide bond. The thiolated moiety is key conjugate a thiol-terminated DNA tag, which is complementary to a sequence within the CRISPR/Cas9 delivery vector. Additionally, this bond reduces as the vehicle reaches intracellular space, delivering the genetic material. In addition, a translocation protein with potent endosomal escape capabilities, called OmpA, was co-immobilized on the vehicle to increase transfection efficiency. We conducted a proof-of-concept delivery experiment <i>in vitro</i> by conjugating rhodamine B and Cysteine to the vehicle's thiolated terminal. The results confirm a functional disulfide bond and allowed a delivery of 86% of the loaded cysteine molecules, after two hours. Based on our results, we decided to deliver rhodamine B to Neuroblastoma and Vero cells, where the endosomal escape reached 89% as calculated by co-localization. Future experiments will focus on delivering the sequence encoding for mCherry to evaluate transfection efficiency.

Functionalization of cell-penetrating magnetite-OmpA vehicles: Efficient delivery of linearized CRISPR/Cas9 editing systems

ACS Fall 2021

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