UBASH3A deficiency accelerates type 1 diabetes development and enhances salivary gland inflammation in NOD mice.

Recent advances in genetic analyses have significantly refined human type 1 diabetes (T1D) associated loci. The goal of such effort is to identify the causal genes and have a complete understanding of the molecular pathways that independently or interactively influence cellular processes leading to the destruction of insulin producing pancreatic ß cells. UBASH3A has been suggested as the underlying gene for a human T1D associated region on chromosome 21. To further evaluate the role of UBASH3A in T1D, we targeted Ubash3a in NOD mice using zinc-finger nuclease mediated mutagenesis. In both 10-week-old females and males, significantly more advanced insulitis was observed in UBASH3A-deficient than in wild-type NOD mice. Consistently, UBASH3A-deficient NOD mice developed accelerated T1D in both sexes, which was associated with increased accumulation of ß-cell autoreactive T cells in the spleen and pancreatic lymph node. Adoptive transfer of splenic T cells into NOD.Rag1-/- mice demonstrated that UBASH3A deficiency in T cells was sufficient to promote T1D development. Our results provide strong evidence to further support a role of UBASH3A in T1D. In addition to T1D, UBASH3A deficiency also promoted salivary gland inflammation in females, demonstrating its broad impact on autoimmunity.

Non-viral Delivery of Zinc Finger Nuclease mRNA Enables Highly Efficient In Vivo Genome Editing of Multiple Therapeutic Gene Targets.

It has previously been shown that engineered zinc finger nucleases (ZFNs) can be packaged into adeno-associated viruses (AAVs) and delivered intravenously into mice, non-human primates, and most recently, humans to induce highly efficient therapeutic genome editing in the liver. Lipid nanoparticles (LNPs) are synthetic delivery vehicles that enable repeat administration and are not limited by the presence of preexisting neutralizing antibodies in patients. Here, we show that mRNA encoding ZFNs formulated into LNP can enable >90% knockout of gene expression in mice by targeting the TTR or PCSK9 gene, at mRNA doses 10-fold lower than has ever been reported. Additionally, co-delivering mRNA-LNP containing ZFNs targeted to intron 1 of the ALB locus with AAV packaged with a promoterless human IDS or FIX therapeutic transgene can result in high levels of targeted integration and subsequent therapeutically relevant levels of protein expression in mice. Finally, we show repeat administration of ZFN mRNA-LNP after a single AAV donor dose results in significantly increased levels of genome editing and transgene expression compared to a single dose. These results demonstrate LNP-mediated ZFN mRNA delivery can drive highly efficient levels of in vivo genome editing and can potentially offer a new treatment modality for a variety of diseases.