An in vivo Cell-Based Delivery Platform for Zinc Finger Artificial Transcription Factors in Pre-clinical Animal Models.

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Public Summary:
Gene editing has emerged as a powerful method for making changes to DNA sequence and turning gene on or off. However, getting the proteins that do the gene editing into the cell of a living organism ("delivery") has been a major challenge for this field. The two major current methods are to use viral vectors or lipid nanoparticles to deliver the editor proteins into the cells of the body. Like small molecule drugs, vary large amounts of viral vectors or lipid nanoparticles must be created in order to get the editors into enough cells in the body, leading major challenges in production and safety of the high doses required. Here, we explore a very different idea. Unlike drugs, gene editors are made of protein and RNA that are things that the cells of the body can produce themselves. If we can get a few cells in the body to produce the editing proteins that can then get into the other cells of the body, that would avoid having to make and deliver high amounts of viral or lipid nanoparticles, resulting in simpler and less expensive production, lower doses for administration, and reduced toxicity. We demonstrate this approach using a type of gene editor called an engineered zinc finger (ZF) transcription factor that is expressed in a particular type of cell called a mesenchymal stem/stromal cell (MSC), which is know to be good at secreting large amounts of protein. The ZF-MSC where introduced into mice and rhesus macaques, and the MSC secrete the ZF into the space around the MSC. The ZF are also contain a cell-penetrating peptide that allows them to be be taken up by neighboring cells. Our data show that this works, and can improve motor deficits in a mouse model of Angelman Syndrome.
Scientific Abstract:
Zinc finger (ZF), transcription activator-like effectors (TALE), and CRISPR/Cas9 therapies to regulate gene expression are becoming viable strategies to treat genetic disorders, although effective in vivo delivery systems for these proteins remain a major translational hurdle. We describe the use of a mesenchymal stem/stromal cell (MSC)-based delivery system for the secretion of a ZF protein (ZF-MSC) in transgenic mouse models and young rhesus monkeys. Secreted ZF protein from mouse ZF-MSC was detectable within the hippocampus 1 week following intracranial or cisterna magna (CM) injection. Secreted ZF activated the imprinted paternal Ube3a in a transgenic reporter mouse and ameliorated motor deficits in a Ube3a deletion Angelman Syndrome (AS) mouse. Intrathecally administered autologous rhesus MSCs were well-tolerated for 3 weeks following administration and secreted ZF protein was detectable within the cerebrospinal fluid (CSF), midbrain, and spinal cord. This approach is less invasive when compared to direct intracranial injection which requires a surgical procedure.