Conjugation of oligonucleotide therapeutics, including small interfering RNAs (siRNAs) or antisense oligonucleotides, to N -acetylga-lactosamine (GalNAc) ligands has become the primary strategy for hepatocyte-targeted delivery, and with the recent approvals of GIVLAARI (givosiran) for the treatment of acute hepatic porphyria, OXLUMO (lumasiran) for the treatment of primary hyperoxaluria, and Leqvio (inclisiran) for the treatment of hypercholesterol-emia, the technology has been well validated clinically. Although much knowledge has been gained over decades of development, there is a paucity of published literature on the drug metabolism and pharmacokinetic properties of GalNAc-siRNA. With this in mind, the goals of this minireview are to provide an aggregate analysis of these nonclinical absorption, distribution, metabolism, and excretion (ADME) data to build confidence on the translation of these properties to human. Upon subcutaneous administration, GalNAc-conjugated siRNAs are quickly distributed to the liver, resulting in plasma pharmacokinetic (PK) properties that reflect rapid elimination through asialoglycoprotein receptor – mediated uptake from circulation into hepatocytes. These studies confirm that liver PK, including half-life and, most importantly, siRNA levels in RNA-induced silencing complex in hepatocytes, are better predictors of pharmacodynamics (PD) than plasma PK. Several in vitro and in vivo nonclinical studies were conducted to characterize the ADME properties of GalNAc-conjugated siRNAs. These studies demonstrate that the PK/PD and ADME properties of GalNAc-conju-gated siRNAs are highly conserved across species, are largely predictable, and can be accurately scaled to human, allowing us to identify efficacious and safe clinical dosing regimens in the absence of human liver PK profiles. (anti-sense single-strand single

Therapeutics based on short interfering RNAs (siRNAs) delivered to hepatocytes have been approved, but new delivery solutions are needed to target additional organs. Here we show that conjugation of 2′- O -hexadecyl (C16) to siRNAs enables safe, potent and durable silencing in the central nervous system (CNS), eye and lung in rodents and non-human primates with broad cell type specificity. We show that intrathecally or intracerebroventricularly delivered C16-siRNAs were active across CNS regions and cell types, with sustained RNA interference (RNAi) activity for at least 3 months. Similarly, intravitreal administration to the eye or intranasal administration to the lung resulted in a potent and durable knockdown. The preclinical efficacy of an siRNA targeting the amyloid precursor protein was evaluated through intracerebroventricular dosing in a mouse model of Alzheimer’s disease, resulting in amelioration of physiological and behavioral deficits. Altogether, C16 conjugation of siRNAs has the potential for safe therapeutic silencing of target genes outside the liver with infrequent dosing.

RNA interference (RNAi) therapeutics are a new class of medicines that can address unmet medical needs by silencing disease-causing gene transcripts. While delivery of short interfering RNAs (siRNAs) to hepatocytes has yielded multiple drug approvals, novel delivery solutions are needed to expand the reach of RNAi therapeutics. Here we report that conjugation of 2'- O -hexadecyl (C16) to siRNAs enables efficient silencing in the central nervous system (CNS), eye, and lung of multiple nonclinical species with broad cell type specificity. Intrathecally delivered C16-siRNAs are active across CNS regions and cell types, with sustained silencing for at least three months, which is an especially important outcome considering the challenging dosing route. Similarly, intravitreal and intranasal administration of C16-siRNAs resulted in potent and sustained knockdown in the eye and lung, respectively. Efficient delivery facilitated through C16 conjugation to optimized siRNA designs has enabled candidate selection for investigational human clinical trials assessing therapeutic silencing beyond the liver with infrequent (e.g. bi-annual) dosing.

The Saccharomyces cerevisiae vacuole contains five ATP-binding cassette class C (ABCC) transporters, including Ycf1p, a family member that was originally characterized as a Cd(2+) transporter. Ycf1p has also been found to physically interact with a wide array of proteins, including factors that regulate vacuole homeostasis. In this study, we examined the role of Ycf1p and other ABCC transporters in the regulation of vacuole homotypic fusion. We found that deletion of YCF1 attenuated in vitro vacuole fusion by up to 40% relative to wild-type vacuoles. Plasmid-expressed wild-type Ycf1p rescued the deletion phenotype; however, Ycf1p containing a mutation of the conserved Lys-669 to Met in the Walker A box of the first nucleotide-binding domain (Ycf1p(K669M)) was unable to complement the fusion defect of ycf1Δ vacuoles. This indicates that the ATPase activity of Ycf1p is required for its function in regulating fusion. In addition, we found that deleting YCF1 caused a striking decrease in vacuolar levels of the soluble SNARE Vam7p, whereas total cellular levels were not altered. The attenuated fusion of ycf1Δ vacuoles was rescued by the addition of recombinant Vam7p to in vitro experiments. Thus, Ycf1p contributes in the recruitment of Vam7p to the vacuole for efficient membrane fusion.