The first safe and effective DNA-loaded lipid nanoparticles (DNA-LNPs)

About This Grant

ABSTRACT / PROJECT SUMMARY Here we continue our development of the first safe & effective DNA-loaded lipid nanoparticles (DNA-LNPs) for the treatment of common chronic diseases such as atherosclerosis, emphysema, etc. DNA-LNPs are built on the billion-patient success of the mRNA-LNP vaccines for COVID, which showed LNPs’ unprecedented ability to drive protein expression. However, mRNA as a cargo has 2 major limitations that prevent it from being used for common chronic diseases: i) mRNA has a very short half-life of hours; ii) mRNA does not naturally encode cell- type-specific expression, which causes off-target expression. We solved both of these deficiencies in mRNA by loading LNPs with DNA, in a paper in revision at Nature Biotechnology. DNA has long been the goal of LNP delivery, but for 20 years DNA-LNPs proved “too toxic”, as we and others showed that IV injection of standard DNA-LNPs kill 100% of healthy mice within 2 days. We discovered this toxicity is caused by massive inflammation induced when LNPs’ delivery of DNA activates the cytosolic DNA sensor system cGAS-STING. We solved this by loading DNA-LNPs with our bodies’ natural inhibitor of STING, the nitrated lipid NOA, which is produced after viral infection to resolve STING-induced inflammation. NOA-DNA-LNPs completely abrogate STING activation and brought DNA-LNPs’ mortality from 100% to 0%. Further, NOA-DNA-LNPs produce high levels of protein expression for ~6 months in vivo and even accommodate large plasmids (>10kb). DNA-LNPs are poised to treat common chronic diseases in ways no other vector can: express any protein (not limited in size like AAV’s <4.4kb limit) or knockdown any protein (expressing shRNA); achieve cell-type-specificity via cell-type-specific promoter sequences (which do not fit in AAV) and LNPs’ other methods of cell-targeting; express for 6 months per dose; and non-immunogenic compared to AAVs. While our DNA-LNPs have great potential, we need to increase their expression by 2 orders of magnitude to reach levels achieved by mRNA-LNPs and AAV. Increasing expression will open up many additional diseases for treatment, and lower the required dose and thus side effects. Our central hypothesis is 3 hurdles inhibit DNA-LNPs’ expression: i) residual activation of cytosolic DNA-sensors (Aim 1); ii) degradation of DNA by intracellular DNases (Aim 2); & iii) inefficient nuclear transport of DNA (Aim 3). In each Aim, we will engineer “chemical approaches” (loading molecules into DNA-LNPs) & “genetic approaches” (deliver new sequences). The Deliverable by the end of this R01 is a new generation of DNA-LNPs that drives protein expression equivalent to mRNA-LNPs and AAV. This would enable DNA-LNPs to become the next pillar of genetic medicine, alongside mRNA-LNPs, AAV, siRNA, and CRISPR.