Protein Delivery of an Artificial Transcription Factor Restores Widespread Ube3a Expression in an Angelman Syndrome Mouse Brain.

Angelman syndrome (AS) is a neurological genetic disorder caused by loss of expression of the maternal copy of UBE3A in the brain. Due to brain-specific genetic imprinting at this locus, the paternal UBE3A is silenced by a long antisense transcript. Inhibition of the antisense transcript could lead to unsilencing of paternal UBE3A, thus providing a therapeutic approach for AS. However, widespread delivery of gene regulators to the brain remains challenging. Here, we report an engineered zinc finger-based artificial transcription factor (ATF) that, when injected i.p. or s.c., crossed the blood-brain barrier and increased Ube3a expression in the brain of an adult mouse model of AS. The factor displayed widespread distribution throughout the brain. Immunohistochemistry of both the hippocampus and cerebellum revealed an increase in Ube3a upon treatment. An ATF containing an alternative DNA-binding domain did not activate Ube3a. We believe this to be the first report of an injectable engineered zinc finger protein that can cause widespread activation of an endogenous gene in the brain. These observations have important implications for the study and treatment of AS and other neurological disorders.

In Vivo Applications of Cell-Penetrating Zinc-Finger Transcription Factors.

Artificial transcription factors based on zinc finger, TALE, and CRISPR/Cas9 programmable DNA-binding platforms have been widely used to regulate the expression of specific genes in cultured cells, but their delivery into organs such as the brain represents a critical challenge to apply such tools in live animals. In previous work, we developed a zinc-finger-based artificial transcription factor harboring a cell-penetrating peptide (CPP) that could be injected systemically, cross the blood-brain barrier, and alter expression of a specific gene in the brain of an adult mouse. Importantly, our mode of delivery produced widespread distribution throughout the brain. Here we describe methods for the production and purification of the factor, testing CPP activity in cells, and testing CPP activity in mice.