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.

Lipid nanoparticle encapsulated nucleoside-modified mRNA vaccines elicit polyfunctional HIV-1 antibodies comparable to proteins in nonhuman primates.

The development of an effective AIDS vaccine remains a challenge. Nucleoside-modified mRNAs formulated in lipid nanoparticles (mRNA-LNP) have proved to be a potent mode of immunization against infectious diseases in preclinical studies, and are being tested for SARS-CoV-2 in humans. A critical question is how mRNA-LNP vaccine immunogenicity compares to that of traditional adjuvanted protein vaccines in primates. Here, we show that mRNA-LNP immunization compared to protein immunization elicits either the same or superior magnitude and breadth of HIV-1 Env-specific polyfunctional antibodies. Immunization with mRNA-LNP encoding Zika premembrane and envelope or HIV-1 Env gp160 induces durable neutralizing antibodies for at least 41 weeks. Doses of mRNA-LNP as low as 5 µg are immunogenic in macaques. Thus, mRNA-LNP can be used to rapidly generate single or multi-component vaccines, such as sequential vaccines needed to protect against HIV-1 infection. Such vaccines would be as or more immunogenic than adjuvanted recombinant protein vaccines in primates.

Lipid nanoparticle encapsulated nucleoside-modified mRNA vaccines elicit polyfunctional HIV-1 antibodies comparable to proteins in nonhuman primates.

Development of an effective AIDS vaccine remains a challenge. Nucleoside-modified mRNAs formulated in lipid nanoparticles (mRNA-LNP) have proved to be a potent mode of immunization against infectious diseases in preclinical studies, and are being tested for SARS-CoV-2 in humans. A critical question is how mRNA-LNP vaccine immunogenicity compares to that of traditional adjuvanted protein vaccines in primates. Here, we found that mRNA-LNP immunization compared to protein immunization elicited either the same or superior magnitude and breadth of HIV-1 Env-specific polyfunctional antibodies. Immunization with mRNA-LNP encoding Zika premembrane and envelope (prM-E) or HIV-1 Env gp160 induced durable neutralizing antibodies for at least 41 weeks. Doses of mRNA-LNP as low as 5 µg were immunogenic in macaques. Thus, mRNA-LNP can be used to rapidly generate single or multi-component vaccines, such as sequential vaccines needed to protect against HIV-1 infection. Such vaccines would be as or more immunogenic than adjuvanted recombinant protein vaccines in primates.