Functional Delivery of Lipid-Conjugated siRNA by Extracellular Vesicles.

Extracellular vesicles (EVs) are cell-derived, membranous nanoparticles that mediate intercellular communication by transferring biomolecules, including proteins and RNA, between cells. As a result of their suggested natural capability to functionally deliver RNA, EVs may be harnessed as therapeutic RNA carriers. One major limitation for their translation to therapeutic use is the lack of an efficient, robust, and scalable method to load EVs with RNA molecules of interest. Here, we evaluated and optimized methods to load EVs with cholesterol-conjugated small interfering RNAs (cc-siRNAs) by systematic evaluation of the influence of key parameters, including incubation time, volume, temperature, and EV:cc-siRNA ratio. EV loading under conditions that resulted in the highest siRNA retention percentage, incubating 15 molecules of cc-siRNA per EV at 37°C for 1 hr in 100 µL, facilitated concentration-dependent silencing of human antigen R (HuR), a therapeutic target in cancer, in EV-treated cells. These results may accelerate the development of EV-based therapeutics.

Transfer of genetic therapy across human populations: molecular targets for increasing patient coverage in repeat expansion diseases.

Allele-specific gene therapy aims to silence expression of mutant alleles through targeting of disease-linked single-nucleotide polymorphisms (SNPs). However, SNP linkage to disease varies between populations, making such molecular therapies applicable only to a subset of patients. Moreover, not all SNPs have the molecular features necessary for potent gene silencing. Here we provide knowledge to allow the maximisation of patient coverage by building a comprehensive understanding of SNPs ranked according to their predicted suitability toward allele-specific silencing in 14 repeat expansion diseases: amyotrophic lateral sclerosis and frontotemporal dementia, dentatorubral-pallidoluysian atrophy, myotonic dystrophy 1, myotonic dystrophy 2, Huntington's disease and several spinocerebellar ataxias. Our systematic analysis of DNA sequence variation shows that most annotated SNPs are not suitable for potent allele-specific silencing across populations because of suboptimal sequence features and low variability (>97% in HD). We suggest maximising patient coverage by selecting SNPs with high heterozygosity across populations, and preferentially targeting SNPs that lead to purine:purine mismatches in wild-type alleles to obtain potent allele-specific silencing. We therefore provide fundamental knowledge on strategies for optimising patient coverage of therapeutics for microsatellite expansion disorders by linking analysis of population genetic variation to the selection of molecular targets.

Exosomes and the emerging field of exosome-based gene therapy.

Exosomes are a subtype of membrane vesicle released from the endocytic compartment of live cells. They play an important role in endogenous cell-to-cell communication. Previously shown to be capable of traversing biological barriers and to naturally transport functional nucleic acids between cells, they potentially represent a novel and exciting drug delivery vehicle for the field of gene therapy. Existing delivery vehicles are limited by concerns regarding their safety, toxicity and efficacy. In contrast, exosomes, as a natural cell-derived nanocarrier, are immunologically inert if purified from a compatible cell source and possess an intrinsic ability to cross biological barriers. Already utilised in a number of clinical trials, exosomes appear to be well-tolerated, even following repeat administration. Recent studies have shown that exosomes may be used to encapsulate and protect exogenous oligonucleotides for delivery to target cells. They therefore may be valuable for the delivery of RNA interference and microRNA regulatory molecules in addition to other single-stranded oligonucleotides. Prior to clinical translation, this nanotechnology requires further development by refinement of isolation, purification, loading, delivery and targeting protocols. Thus, exosome-mediated nanodelivery is highly promising and may fill the void left by current delivery methods for systemic gene therapy.