Diverse lipid conjugates for functional extra-hepatic siRNA deliveryin vivo

Abstract

Small interfering RNA (siRNA)-based therapies are proving to be efficient for treating liver-associated disorders. However, extra-hepatic delivery remains challenging, limiting therapeutic siRNA utility. We synthesized a panel of fifteen lipid-conjugated siRNAs and systematically evaluated the impact of conjugate on siRNA tissue distribution and efficacy. Generally, conjugate hydrophobicity defines the degree of clearance and the liver-to-kidney distribution profile. In addition to primary clearance tissues, several conjugates achieve significant siRNA accumulation in muscle, lung, heart, adrenal glands and fat. Oligonucleotide distribution to extra-hepatic tissues with some conjugates was significantly higher than with cholesterol, a well studied conjugate, suggesting that altering conjugate structure can enhance extra-hepatic delivery. These conjugated siRNAs enable functional gene silencing in lung, muscle, fat, heart and adrenal gland. Required levels for productive silencing vary (5–200 μg/g) per tissue, suggesting that the chemical nature of conjugates impacts tissue-dependent cellular/intracellular trafficking mechanisms. The collection of conjugated siRNA described here enables functional gene modulation in vivo in several extra-hepatic tissues opening these tissues for gene expression modulation. A systemic evaluation of a panel of conjugated siRNA, as reported here, has not previously been investigated and shows that chemical engineering of lipid siRNAs is essential to advance the RNA therapeutic field.

Current Development of siRNA Bioconjugates: From Research to the Clinic

“…The biodistribution of various lipophilic siRNA conjugates has been extensively studied in a recent paper (Biscans et al, 2018). It was shown that cholesterol conjugates were more effectively retained in the body (62%) compared with other lipophilic conjugates (27–62%).…”

“…Following subcutaneous injection, the cholesterol conjugates accumulated in almost all organs: liver, kidney, adrenal glands, spleen, pancreas, heart, muscle, fat, thymus, lung, injection site, ovaries, and testes. At the same time, cholesterol conjugates accumulated most effectively in the liver, adrenal glands, spleen, and in the skin at the site of administration (Biscans et al, 2018). Cholesterol derivatives accumulated in other organs with the same or lesser efficiency than other lipophilic conjugates; e.g., it was shown that a conjugate of siRNA and saturated fatty acid (docosanoic, C21) accumulated more efficiently than a cholesterol conjugate and inhibited expression of the target gene ( Htt or Ppib ) in muscles (20 and 30% inhibition of Htt and Ppib , respectively) and fat (50% and 30% inhibition of Htt and Ppib , respectively) after subcutaneous injection (20 mg/kg) (Biscans et al, 2018).…”

“…In this study, it was shown that the main factor determining the nature of the biodistribution of conjugates is their lipophilicity. Conjugates of siRNA with lower lipophilicity; i.e., derivatives of retinoic acid, lithocholic acid, and docosahexanoic acid with greater efficiency than cholesterol conjugates accumulated in the kidneys, bladder, and lungs of the mouse after subcutaneous injection (Biscans et al, 2018). This fact is consistent with previous data that showed that more lipophilic conjugates bind more efficiently to serum components, and thus are not excreted by the kidneys (Wolfrum et al, 2007; Osborn et al, 2018).…”

“…Lipophilic derivatives after subcutaneous or intravenous injection do not penetrate the blood-brain barrier (BBB) (Biscans et al, 2018). Therefore, attempts were made to directly inject derivatives into the brain of the mouse to suppress gene expression in the brain (Alterman et al, 2015; Nikan et al, 2016, 2017).…”

DNA Nanotechnology Enters Cell Membranes

“…Efficient and stable insertion into live cell membranes allows amphiphilic DNA conjugates to cross the cell membrane . Importantly, these DNA amphiphiles can be modified with additional functional groups that enable specific targeting and biocompatibility in vivo, providing them with a tremendous potential for biomedicine …”