The biggest challenge facing the deployment of gene therapies for the treatment of aging and age-related disease is that delivery systems are lacking. There is no well established way to safely and robustly deliver a payload of sufficient size to most organs (or all organs) without multiple direct injections, an approach that bears an unacceptable risk when deployed across very large numbers of relatively healthy people. When delivering a gene therapy via intravenous injection, the bloodstream carries most of the payload to the liver and lungs, and this limits the amount of drug that can be introduced to any given other organ because delivery systems are toxic at higher doses. The amount that ends up in the liver dramatically limits what can be done.
A connected issue is that the options for selective expression of a transgene by tissue type are also limited. Selective expression requires introducing a transgene and an associated promoter structure either into the genome or as a plasmid into the cell nucleus; the promoter structure can usually be cleverly tailored to condition expression of the transgene to the desired cell type. The well-trodden options for vectors today are (a) viral vectors and (b) various forms of lipid nanoparticles (LNPs) delivering messenger RNA (mRNA). Viral vectors have the issue noted in the first paragraph above: while you can specify expression by cell type by tailoring the payload of the virus, if one wants sufficient amount of vector to be delivered to a specific tissue, one either risks toxicity in the liver and lungs or undertakes direct injection. LNP-mRNA vectors have two issues: firstly that selective delivery is only well solved for the liver, and secondly that mRNA cannot perform selective expression. It will express in every cell delivered to.
People are working on these problems. One of the most promising near future advances, generally agreed upon to be possible in principle, would be some form of viral vector or LNP delivering DNA plasmids rather than mRNA that more smoothly distributes to the whole body following intravenous injection. No vast buildup of vector inside liver cells or lung cells with minimal delivery to smaller organs, but a distribution that is closer to being even. In the case of LNP-DNA therapies, this hypothetical improvement would also require a novel technology to allow DNA plasmids to safely and effectively enter the cell nucleus. Clearly this also is possible in principle, as it is exactly what an adeno-associated virus (AAV) does. But engineering one of the existing widely used DNA plasmid structures to do this once dropped into the cell cytosol via uptake of an LNP remains an unsolved problem.
Today's open access paper from the Entos Pharmaceuticals team reports on their progress towards a better LNP, one that is very much less toxic than present standards, and delivers more smoothly throughout the body. To the degree that an LNP has very low toxicity, one can accept more of an excess delivery to the liver, provided that one is delivering DNA plasmids that will only express the transgene in the desired organ. Promisingly, the work reported here is relevant to both mRNA and DNA delivery. Specific optimizations of LNP composition and manufacture will differ between delivery of mRNA and DNA, but the general strategy should work in both cases.
Safe and effective in vivo delivery of DNA and RNA using proteolipid vehicles
Non-viral delivery vehicles such as lipid nanoparticles (LNPs) have been widely used for RNA-based therapeutic approaches and have cost, manufacturing, and immunogenicity advantages over viral vectors. The approval of patisiran (Onpattro) as a systemic therapy and the more recent success of LNP mRNA COVID-19 vaccines has set the stage for the development of numerous LNP-based nucleic acid therapies. LNPs are formulated with ionizable lipids, which facilitate endosomal escape. However, formulations containing ionizable lipids are also associated with tolerability challenges such as potentiation of apoptotic cell death and dose-limiting liver toxicity following systemic delivery.
Given the strengths and limitations of current viral and non-viral approaches, we developed a proteolipid vehicle (PLV) platform that incorporates an engineered viral fusion protein into a lipid-based formulation to achieve intracellular delivery of nucleic acid cargoes with low immunogenicity and high tolerability. The PLV platform utilizes fusion-associated small transmembrane (FAST) proteins derived from the non-enveloped fusogenic orthoreovirus. At 100-200 residues in length, FAST proteins are the smallest known viral fusogens. These fusion proteins are expressed inside virus-infected cells and are trafficked to the plasma membrane where they facilitate cell-cell membrane fusion, generating multinucleated syncytia to facilitate viral transmission. FAST proteins function at physiological pH and do not require specific cell receptors, allowing them to fuse almost all cell types.
We previously showed in proof-of-concept experiments that FAST protein-containing liposomes induce liposome-cell fusion and facilitate intracellular delivery of encapsulated membrane-impermeable cargo. Here, we evaluated a panel of chimeric FAST protein constructs for fusion activity to identify a high-activity FAST protein chimera that was formulated into a PLV comprised of well-tolerated lipids. We demonstrate that FAST-PLVs comprise a nucleic acid delivery platform that mediates effective delivery and expression of encapsulated mRNA and DNA in vitro and in vivo, while maintaining excellent tolerability, low immunogenicity, and favorable biodistribution in rodent and non-human primate (NHP) models.
Systemically administered FAST-PLVs showed broad biodistribution and effective mRNA and DNA delivery in mouse and non-human primate models. FAST-PLVs show low immunogenicity and maintain activity upon repeat dosing. Systemic administration of follistatin DNA gene therapy with FAST-PLVs raised circulating follistatin levels and significantly increased muscle mass and grip strength. These results demonstrate the promising potential of FAST-PLVs for redosable gene therapies and genetic medicines.
View the full article at FightAging
