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

Abstract

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.

Lipid nanoparticle technology for therapeutic gene regulation in the liver

“…They showed sustained 12-month circulating TTR knockdown (97%) following a single administration of 3 mg/kg RNA body weight in a murine model with~70% editing in the liver (~70% liver cells are hepatocytes) [206]. Similarly, LNP-mediated delivery of mRNA encoding ZFN targeting ttr and pcsk9 resulted in > 90% knockout at mRNA doses 10-fold lower than reported previously [137]. In the same study, co-delivery of LNP-mRNA encoding ZFN targeting the albumin gene and a viral vector for templates of promotor-less human IDS or FIX resulted in integration of those templates at the albumin locus and generated therapeutically relevant levels of those proteins in murine models.…”

“…The lipid tails contribute to making the molecule sufficiently hydrophobic to promote incorporation into LNPs while endowing either stabilizing or destabilizing properties. The above lipids are classified into three broad categories: (i) ionizable cationic lipids such as DLinDMA[133], DLin-KC2-DMA[30], and DLin-MC3-DMA[31]; (ii) lipidoids like cKK-E12[134] and C12-200[29]; and (iii) next-generation lipids including the biodegradable molecules L319[130], TT3[135], and ssPalmE[136] as well as lipids from proprietary libraries belonging to Acuitas (A9)[137] and Moderna (L5)[138].…”

Role of Lipid-Based and Polymer-Based Non-Viral Vectors in Nucleic Acid Delivery for Next-Generation Gene Therapy

“…Conway et al used LNP-based formulation to deliver ZFN-mRNA targeting TTR (transthyretin) and PCSK9 gene in mice by systemic delivery. This led to 80% and 90% protein reduction levels respectively at 10-fold lower mRNA doses [ 141 ]. CRISPR/Cas9 genome editing, a novel delivery system developed by Sun et al, explored employing self-assembling nanoparticles comprising of DNA nanoclews (NCs), which are designed to contain sequences that are partially complimentary to sgRNA enabling the efficient loading of the sgRNA–Cas9 complex.…”

Nanomedicines to Deliver mRNA: State of the Art and Future Perspectives

“…This repair is useful to correct genomic mutations or to insert new sequences encoding therapeutic proteins. NHEJ eliminates the target region by binding DSBs; it can be used to silence or correct an anomalous gene [223,224].…”