Summary:

Advances in molecular biology have led to great interest and potential in applying gene therapy to correct/ regulate expression of disease-related genes that are otherwise undruggable. Gene therapy is one of the great pillars of the newly emerging field of personalized medicine, allowing for customized patient care and treatments. Plasmid constructs are desirable gene therapy agents that can be developed to express genes of interest and thus compensate for protein loss-of-function or introduce new genes into the system.  They are advantageous over viral vectors since they are easier to generate, usually less immunogenic and pose a lower risk of integration into the host genome. Gene delivery and therapy usually require delivery vehicles (like Neuro9^TM Lipid Nanoparticles) to protect the integrity of cargo, minimize negative side effects and enhance uptake. However, due to their large size and their need for intranuclear delivery, they pose challenges to researchers interested in delivering plasmids to study and treat diseases. Previously, Lipid Nanoparticle (LNP) systems have been well established for delivery of small interfering RNA (siRNA). With siRNA being a very different nucleic acid species from plasmids, the LNP systems require optimization for effective encapsulation and delivery of plasmid DNA. In this article, the Cullis lab optimized LNP systems for transfection by modifying lipid composition and plasmid-to-lipid ratio. The authors used the NanoAssemblr^TM Benchtop to formulate a plasmid sequence into an LNP carrier and demonstrated highly efficient uptake and exogenous gene expression in vitro and in vivo.

One of the greatest challenges in gene therapy is the instability of nucleic acids in biological systems and immune system responses when administered systemically. Additionally, on their own, most nucleic acid molecules cannot transfect cells since they are poorly taken up by them. Plasmids need to cross the nucleolus membrane following cellular uptake to be effective. The siRNA-LNP systems have been optimized for endosomal release of siRNA. Plasmids are however larger than siRNA by orders of magnitude (thousands of base-pairs vs tens of base pairs), rendering the encapsulation and endosomal release processes more challenging. The NanoAssemblr Benchtop is a flexible tool to systematically explore multiple formulation parameters to optimize nanoparticle formulations. The plasmid-LNPs developed using the NanoAssemblr have great transfection potency and are significantly less toxic than other conventional in vitro transfection methods. Additionally, the plasmid-LNPs have shown great in vivo potential as demonstrated by the Cullis lab.

To optimize the LNP system for plasmid delivery, Cullis lab modified the well-established siRNA-LNP system by replacing the helper lipid – a saturated phosphatidylcholine (PC) - with unsaturated varieties that are thought to promote structures that enhance membrane fusion and intracellular delivery. They used a plasmid construct with a luciferase reporter to detect intracellular uptake and expression. The results demonstrated significantly higher cell transfection efficiency with unsaturated PCs. Additionally, they found out that while LNPs containing the lipid DLin-MC3-DMA were the most efficacious for siRNA delivery, plasmid transfection was highest when the LNPs were formulated with DLin-KC2-DMA. The said formulation was also optimized for the most efficacious plasmid/ lipid ratio and formulations were tested in multiple cell lines and primary cells. The results indicated that transfection was greatly improved and plasmid-LNP was highly superior to that of lipofectamine in terms of safety and efficacy. Ultimately, the authors assessed the efficacy of the optimized plasmid-LNP in vivo in a chick embryo model using a GFP plasmid construct for detection purposes. Successful plasmid transfection in vivo indicated that plasmid-LNP can provide a flexible and efficacious tool for delivering genome-editing tools in chick embryo and is an important step towards using exogenous genes in therapeutic applications. Overall, the plasmid-LNPs formulated with NanoAssemblr technology can be used for nucleic acid delivery in vitro and in vivo to further our understanding of genetic ailments and develop new genetic therapies.

Abstract: 

Lipid nanoparticles (LNPs) containing distearoylphosphatidlycholine (DSPC), and ionizable amino-lipids such as dilinoleylmethyl-4-dimethylaminobutyrate (DLin-MC3-DMA) are potent siRNA delivery vehicles in vivo. Here we explore the utility of similar LNP systems as transfection reagents for plasmid DNA (pDNA). It is shown that replacement of DSPC by unsaturated PCs and DLin-MC3-DMA by the related lipid DLin-KC2-DMA resulted in highly potent transfection reagents for HeLa cells in vitro. Further, these formulations exhibited excellent transfection properties in a variety of mammalian cell lines and transfection efficiencies approaching 90% in primary cell cultures. These transfection levels were equal or greater than achieved by Lipofectamine, with much reduced toxicity. Finally, microinjection of LNP-eGFP into the limb bud of a chick embryo resulted in robust reporter-gene expression. It is concluded that LNP systems containing ionizable amino lipids can be highly effective, non-toxic pDNA delivery systems for gene expression both in vitro and in vivo.