Conjugation of triantennary N-acetyl galactosamine (GalNAc) to oligonucleotide therapeutics results in marked improvement in potency for reducing gene targets expressed in hepatocytes. In this report ...we describe a robust and efficient solution-phase conjugation strategy to attach triantennary GalNAc clusters (mol. wt. ∼2000) activated as PFP (pentafluorophenyl) esters onto 5′-hexylamino modified antisense oligonucleotides (5′-HA ASOs, mol. wt. ∼8000 Da). The conjugation reaction is efficient and was used to prepare GalNAc conjugated ASOs from milligram to multigram scale. The solution phase method avoids loading of GalNAc clusters onto solid-support for automated synthesis and will facilitate evaluation of GalNAc clusters for structure activity relationship (SAR) studies. Furthermore, we show that transfer of the GalNAc cluster from the 3′-end of an ASO to the 5′-end results in improved potency in cells and animals.
The extra hepatic delivery of antisense oligonucleotides (ASOs) remains a challenge and hampers the widespread application of this powerful class of therapeutic agents. In that regard, pancreatic ...beta cells are a particularly attractive but challenging cell type because of their pivotal role in diabetes and the fact that they are refractory to uptake of unconjugated ASOs. To circumvent this, we have expanded our understanding of the structure activity relationship of ASOs conjugated to Glucagon Like Peptide 1 Receptor (GLP1R) agonist peptide ligands. We demonstrate the key role of the linker chemistry and its optimization to design maleimide based conjugates with improved in vivo efficacy. In addition, truncation studies and scoping of a diverse set of GLP1R agonists proved fruitful to identify additional targeting ligands efficacious in vivo including native hGLP1(7–36)NH2. Variation of the carrier peptide also shed some light on the dramatic impact of subtle sequence differences on the corresponding ASO conjugate performance in vivo, an area which clearly warrant further investigations. We have confirmed the remarkable potential of GLP1R agonist conjugation for the delivery of ASOs to pancreatic beta cell by effectively knocking down islet amyloid polypeptide (IAPP) mRNA, a potential proapoptotic target, in mice.
Abstract
Enhancing the functional uptake of antisense oligonucleotide (ASO) in the muscle will be beneficial for developing ASO therapeutics targeting genes expressed in the muscle. We hypothesized ...that improving albumin binding will facilitate traversal of ASO from the blood compartment to the interstitium of the muscle tissues to enhance ASO functional uptake. We synthesized structurally diverse saturated and unsaturated fatty acid conjugated ASOs with a range of hydrophobicity. The binding affinity of ASO fatty acid conjugates to plasma proteins improved with fatty acid chain length and highest binding affinity was observed with ASO conjugates containing fatty acid chain length from 16 to 22 carbons. The degree of unsaturation or conformation of double bond appears to have no influence on protein binding or activity of ASO fatty acid conjugates. Activity of fatty acid ASO conjugates correlated with the affinity to albumin and the tightest albumin binder exhibited the highest activity improvement in muscle. Palmitic acid conjugation increases ASO plasma Cmax and improved delivery of ASO to interstitial space of mouse muscle. Conjugation of palmitic acid improved potency of DMPK, Cav3, CD36 and Malat-1 ASOs (3- to 7-fold) in mouse muscle. Our approach provides a foundation for developing more effective therapeutic ASOs for muscle disorders.
Abstract
We determined the effect of attaching palmitate, tocopherol or cholesterol to PS ASOs and their effects on plasma protein binding and on enhancing ASO potency in the muscle of rodents and ...monkeys. We found that cholesterol ASO conjugates showed 5-fold potency enhancement in the muscle of rodents relative to unconjugated ASOs. However, they were toxic in mice and as a result were not evaluated in the monkey. In contrast, palmitate and tocopherol-conjugated ASOs showed enhanced potency in the skeletal muscle of rodents and modest enhancements in potency in the monkey. Analysis of the plasma-protein binding profiles of the ASO-conjugates by size-exclusion chromatography revealed distinct and species-specific differences in their association with plasma proteins which likely rationalizes their behavior in animals. Overall, our data suggest that modulating binding to plasma proteins can influence ASO activity and distribution to extra-hepatic tissues in a species-dependent manner and sets the stage to identify other strategies to enhance ASO potency in muscle tissues.
Huntington's disease (HD) is a fatal neurodegenerative disease, caused by expansion of polyglutamine repeats in the Huntingtin gene, with longer expansions leading to earlier ages of onset. The HD ...iPSC Consortium has recently reported a new in vitro model of HD based on the generation of induced pluripotent stem cells (iPSCs) from HD patients and controls. The current study has furthered the disease in a dish model of HD by generating new non-integrating HD and control iPSC lines. Both HD and control iPSC lines can be efficiently differentiated into neurons/glia; however, the HD-derived cells maintained a significantly greater number of nestin-expressing neural progenitor cells compared with control cells. This cell population showed enhanced vulnerability to brain-derived neurotrophic factor (BDNF) withdrawal in the juvenile-onset HD (JHD) lines, which appeared to be CAG repeat-dependent and mediated by the loss of signaling from the TrkB receptor. It was postulated that this increased death following BDNF withdrawal may be due to glutamate toxicity, as the N-methyl-d-aspartate (NMDA) receptor subunit NR2B was up-regulated in the cultures. Indeed, blocking glutamate signaling, not just through the NMDA but also mGlu and AMPA/Kainate receptors, completely reversed the cell death phenotype. This study suggests that the pathogenesis of JHD may involve in part a population of 'persistent' neural progenitors that are selectively vulnerable to BDNF withdrawal. Similar results were seen in adult hippocampal-derived neural progenitors isolated from the BACHD model mouse. Together, these results provide important insight into HD mechanisms at early developmental time points, which may suggest novel approaches to HD therapeutics.
The efficient synthesis, antiviral activity, and duplex‐stabilizing properties of both isomers of the 2′‐fluoro analogue of Northern methanocarbathymidine (N‐MCT), 2 and 3, are reported. We show that ...2′‐F incorporation on the N‐MCT scaffold has a strong stabilizing effect on duplex thermal stability.
The power of conviction: The efficient synthesis, antiviral activity, and duplex‐stabilizing properties of both isomers, A and B, of the 2′‐fluoro analogue of N‐methanocarbathymidine (N‐MCT) are reported. Incorporation of the fluorine substituent at the 2′‐position of the N‐MCT scaffold was found to have a strong positive effect on duplex thermal stability.
Autosomal dominant diseases such as Huntington's disease (HD) are caused by a gain of function mutant protein and/or RNA. An ideal treatment for these diseases is to selectively suppress expression ...of the mutant allele while preserving expression of the wild-type variant. RNase H active antisense oligonucleotides (ASOs) or small interfering RNAs can achieve allele selective suppression of gene expression by targeting single nucleotide polymorphisms (SNPs) associated with the repeat expansion. ASOs have been previously shown to discriminate single nucleotide changes in targeted RNAs with ∼5-fold selectivity. Based on RNase H enzymology, we enhanced single nucleotide discrimination by positional incorporation of chemical modifications within the oligonucleotide to limit RNase H cleavage of the non-targeted transcript. The resulting oligonucleotides demonstrate >100-fold discrimination for a single nucleotide change at an SNP site in the disease causing huntingtin mRNA, in patient cells and in a completely humanized mouse model of HD. The modified ASOs were also well tolerated after injection into the central nervous system of wild-type animals, suggesting that their tolerability profile is suitable for advancement as potential allele-selective HD therapeutics. Our findings lay the foundation for efficient allele-selective downregulation of gene expression using ASOs-an outcome with broad application to HD and other dominant genetic disorders.
Antisense oligonucleotides (ASOs) have the potential to discriminate between subtle RNA mismatches such as SNPs. Certain mismatches, however, allow ASOs to bind at physiological conditions and result ...in RNA cleavage mediated by RNase H. We showed that replacing DNA nucleotides in the gap region of an ASO with other chemical modification can improve allele selectivity. Herein, we systematically substitute every position in the gap region of an ASO targeting huntingtin gene (HTT) with fluorinated nucleotides. Potency is determined in cell culture against mutant HTT (mtHTT) and wild-type HTT (wtHTT) mRNA and RNase H cleavage intensities, and patterns are investigated. This study profiled five different fluorinated nucleotides and showed them to have predictable, site-specific effects on RNase H cleavage, and the cleavage patterns were rationalized from a published X-ray structure of human RNase H1. The results herein can be used as a guide for future projects where ASO discrimination of SNPs is important.
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A convenient method for the synthesis of several triantennary GalNAc clusters based on a nitromethanetrispropionic acid core was developed. The synthetic approach involves ...pentafluorophenolic ester intermediates which can be used in a one-pot, seven reaction procedure to quickly prepare a variety of triantennary GalNAc conjugated ASOs. The GalNAc clusters were conjugated to the 5′-end of an antisense oligonucleotide and evaluated for activity in primary mouse hepatocytes where they showed ∼10-fold improvement in activity.
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