Protein-based vehicles for biomimetic RNAi delivery

Abstract

Broad translational success of RNA interference (RNAi) technology depends on the development of effective delivery approaches. To that end, researchers have developed a variety of strategies, including chemical modification of RNA, viral and non-viral transfection approaches, and incorporation with delivery vehicles such as polymer- and lipid-based nanoparticles, engineered and native proteins, extracellular vesicles (EVs), and others. Among these, EVs and protein-based vehicles stand out as biomimetically-inspired approaches, as both proteins (e.g. Apolipoprotein A-1, Argonaute 2, and Arc) and EVs mediate intercellular RNA transfer physiologically. Proteins specifically offer significant therapeutic potential due to their biophysical and biochemical properties as well as their ability to facilitate and tolerate manipulation; these characteristics have made proteins highly successful translational therapeutic molecules in the last two decades. This review covers engineered protein vehicles for RNAi delivery along with what is currently known about naturally-occurring extracellular RNA carriers towards uncovering design rules that will inform future engineering of protein-based vehicles.

Keywords: Arc; Argonaute; Drug delivery; Lipoprotein; Protein engineering; RNAi.

Fig. 1

Barriers to RNA delivery. Left: RNA in circulation is vulnerable to RNase degradation and phagocytosis, and access to targeted tissue is blocked by physical barriers (e.g. endothelial and epithelial layers) and renal and hepatic clearance. Right: Cytoplasmic delivery is impaired by the plasma membrane, degradation within lysosomes, and nonspecific dsRNA immune activation. The latter can occur within the endosome by activating a Toll-like receptor (TLR) or in the cytoplasm by activating RIG1 or Protein kinase R (PKR). Images courtesy of Louisa Howard at Dartmouth University

Fig. 2

Overview of native extracellular RNA (exRNA) carriers. Unprotected RNAs are rapidly degraded in the extracellular space. Argonaute 2 (Ago2)-miRNA is found in circulation but its secretion mechanism is unknown, and it delivers via the receptor Neuropilin 1 (Nrp1). Apolipoprotein A-1 (ApoA1) is secreted by mainly the liver and intestine, and interacts with ABCA1, ABCG1, and SR-B1 in peripheral tissue to accumulate cholesterol and phospholipids. Discoidal nascent High Density Lipoprotein (HDL) is matured into spherical HDL through LCAT, PLTP, and CETP. Mature HDL is loaded with miRNA through an unknown mechanism. Nascent and mature HDL can interact with SR-B1 to deliver RNA and lipids, and lipid-free ApoA1 is released back into circulation. Spontaneous lipid transfers also play a large role in HDL function. EVs such as exosomes and microvesicles deliver RNA, as well as Ago2-miRNA and the retroviral Gag-like protein Arc. Arc has been found to mediate mRNA transport in the brain; non-exosomal Arc retains function but its prevalence is unknown. ABCA1, ATP-binding cassette subfamily A member 1; ABCG1, ATP-binding cassette subfamily G member 1; SR-B1, scavenger receptor class B type 1; LCAT, lecithin–cholesterol acyltransferase; CETP, cholesteryl ester transfer protein; PLTP, cholesteryl ester transfer protein; ARC, activity-regulated cytoskeleton-associated protein

Fig. 3

Trends in pharmacokinetic behavior of therapeutic proteins. a Glomerular sieving coefficient, which is representative of renal clearance, is inversely related to molecular weight, so smaller molecules are excreted faster. b Terminal slope of pharmacokinetic profile, which corresponds to rate of systemic clearance from the body, is inversely related to hydrodynamic radius, so smaller molecules are cleared faster (open dot represents IgG which utilizes FcRn pathway). c Systemic clearance is related to molecular charge, so more negative molecules are cleared faster (higher pI corresponds to more negative charge). d Half-life is related to binding affinity, so molecules with worse affinity are cleared faster. Reprinted with permission pending from [30]. Reproduced with permission from Springer Nature, Journal of Pharmacokinetics and Pharmacodynamics (Pharmacokinetic and pharmacodynamic considerations for the next generation protein therapeutics, Dhaval K. Shah, copyright (2015)