Edition 2 / December 2018
Lipid nanoparticles (LNP) are the leading delivery systems for enabling the therapeutic potential of small interfering RNA (siRNA), mRNA for systemic applications or CRISPR. These systems are relatively nontoxic, leading to therapeutic indices in mice approaching 1000. Cationic lipids have been extensively investigated for the delivery of nucleic acids, with a particular focus in the past several years on mRNA and siRNA delivery. They are amphiphilic molecules, comprising an amine, positively-charged head group, and a hydrocarbon chain or a cholesterol derivate, attached via a linker (e.g. glycerol). The positively charged head group of the lipid can electrostatically interact with negatively charged nucleic acids and allow their entrapment in a lipid-based nanoparticle. LNP siRNA systems containing optimized ionizable cationic lipids can exhibit remarkable in vivo potencies at doses as low as 0.02 mg siRNA/kg body weight for silencing liver (hepatocyte) target genes in rodents following intravenous injection.
The utilization of cationic lipids for in-vitro transfection of mRNA was first described more than 20 years ago, by complexing mRNA in a liposome containing the synthetic cationic lipid N-[1-(2,3-dioleyloxy)propyl-N,N,N-trimethylammonium chloride (DOTMA) and the helper lipid dioleoylphosphatidylethanolamine (DOPE), achieving high levels of the translated protein.
The most common siRNA formulations contain four components: an amine-containing lipid or lipid-like material, phospholipid, cholesterol, and lipid-anchored polyethylene glycol, the relative ratios of which can have profound effects on the formulation potency. ONPATTROTM for example, which is indicated for the treatment of the polyneuropathy of hereditary transthyretin-mediated amyloidosis in adults, is administered via intravenous (i.v.) infusion at a dosage of 0.3 mg/kg once every 3 weeks (patients weighing less than 100 kg). The LNP is composed of siRNA (ALN-18328), cholesterol USP, DLin-MC3-DMA, DSPC, PEG2000-C-DMG in a ratio (2:6.2:13:3.3:1.6), and some salts (potassium phosphate, monobasic anhydrous NF, sodium chloride USP, sodium phosphate dibasic heptahydrate USP) in Water For Injection (WFI). More recently, Patisiran is being developed to target hepatic production of wild type (wt) and mutant TTR using a very similar LNP formulation.
Ionizable Lipids & Lipid Nanoparticles: Initiatives to Allow Lower Dose
Significant effort has been applied to discover and develop vehicles which can guide small interfering siRNA through the many barriers guarding the interior of target cells. Improvements in delivery efficacy were required to fulfill the broadest potential of RNA interference therapeutics. Through the combinatorial synthesis and screening of a different class of materials, a formulation has been identified that enables siRNA-directed liver gene silencing in mice at doses below 0.01 mg∕kg. The potential of this formulation was further validated in non-human primates, where high levels of knockdown of the clinically relevant gene transthyretin were observed at doses as low as 0.03 mg∕kg. A screening of different tails and amine groups was evaluated, reporting interesting structure-activity results. With respect to tail length, most of the top performing structures possessed tails consisting of 14-carbons in length. Additionally, no compounds with tails less than 12-carbons in length mediated silencing greater than 30%. Regarding amine head groups, a tertiary amine was present in the top performing compounds. With the top 3 lipoids, an in vivo delivery of siRNA to hepatocytes in mice was conducted and a dose dependent gene silencing was obtained. One compound in particular, C12-200, demonstrated over two orders-of-magnitude higher potency when compared to LNP01.
CordenPharma Switzerland, located near Basel in Liestal, CH, have mastered the total, chemical synthesis of complex phospholipid derivatives from multi-gram to multi-kilogram scale (i.e. DSPG, DPPC, DSPC, SCS, MPEG derivatives) and are renowned for their contract lipid and phospholipid manufacturing expertise. Recently, we developed a manufacturing process for C12‑200, which is now ready for evaluation by potential customers looking for a breakthrough in LNP and an IP-free lipoid vehicle. C12‑200 is available with high purity and the process is robust enough to allow for a GMP campaign.
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