John J. Turner

Peptide-based delivery of nucleic acids: design, mechanism of uptake and applications to splice-correcting oligonucleotides.

CPPs (cell-penetrating peptides) have given rise to much interest for the delivery of biomolecules such as peptides, proteins or ONs (oligonucleotides). CPPs and their conjugates were initially thought to translocate through the cell membrane by a non-endocytotic mechanism which has recently been re-evaluated. Basic-amino-acid-rich CPPs first interact with cell-surface proteoglycans before being internalized by endocytosis. Sequestration and degradation in endocytotic vesicles severely limits the cytoplasmic and nuclear delivery of the conjugated biomolecules. Accordingly, splicing correction by CPP-conjugated steric-block ON analogues is inefficient in the absence of endosomolytic agents. New arginine-rich CPPs allowing efficient splicing correction by conjugated PNAs (peptide nucleic acids) or PMO (phosphorodiamidate morpholino oligomer) steric blockers in the absence of endosomolytic agents have recently been defined in our group and are currently being characterized. They offer promising leads for the development of efficient cellular delivery vectors for therapeutic steric-block ON analogues.

Increased RNAi is related to intracellular release of siRNA via a covalently attached signal peptide

In the last decade short interfering RNA (siRNA) became an important means for functional genomics and the development of gene-specific drugs. However, major technical hurdles in the application of siRNA include its cellular delivery followed by its intracellular trafficking and its release in order to enter the RNA interference (RNAi) machinery. The novel phosphorothioate-stimulated cellular uptake of siRNA contrasts other known delivery systems because it involves a caveosomal pathway in which large amounts of siRNA are delivered to the perinuclear environment, leading to measurable though moderate target suppression. Limited efficacy seems to be related to intracellular trapping of siRNA. To study the role of intracellular trafficking of siRNA for biological effectiveness we studied whether a signal peptide for trans-membrane transport of bacterial protein toxins, which is covalently attached to siRNA, can promote its release from the perinuclear space into the cytoplasm and thereby enhance its biological effectiveness. We show that attachment of the peptide TQIENLKEKG to lamin A/C-directed siRNA improves target inhibition after its PS-stimulated delivery. This is related to increased efflux of the siRNA-peptide conjugate from the ER-specific perinuclear sites. In summary, this study strongly suggests that intracellular release of siRNA leads to increased biological effectiveness. Thus covalent peptide-siRNA conjugates are proposed as new tools to study the relationship between intracellular transport and efficacy of siRNA.

ANTIVIRAL ACTIVITY OF STERIC-BLOCK OLIGONUCLEOTIDES TARGETING THE HIV-1 TRANS-ACTIVATION RESPONSE AND PACKAGING SIGNAL STEM-LOOP RNAS

Mixmer oligonucleotides consisting of residues of both 2′-O-methylnucleosides (OMe) and locked nucleic acids (LNA) were designed targeting two stem-loops in the 5′-UTR of HIV-1 RNA, the trans-activation response region (TAR), which is the site of binding of the Tat protein, and the SL3 loop, which is the primary packaging element that binds the Gag polyprotein. These oligonucleotides were found to inhibit syncitia formation dose- and sequence-dependently when delivered to HeLa T4 LTR β-Gal cells and subsequently infected with HIV-1.

Mitsunobu glycosylation of nitrobenzenesulfonamides: Novel route to Amadori rearrangement products

Abstract Amino-acid derived 2-nitrobenzenesulfonamides were successfully condensed under Mitsunobu conditions with 2,3,4,6-tetra-O-acetyl- d -glucose to afford the fully protected glucosylamines in excellent yield. Upon total deprotection, these compounds rearranged to provide the corresponding Amadori products in good overall yield.

Disulfide Conjugation of Peptides to Oligonucleotides and Their Analogs

Peptide conjugation of oligonucleotides and their analogs is being studied widely towards improving the delivery of oligonucleotides into cells. Amongst the many possible routes of conjugation, the disulfide linkage has proved to be the most popular. This reversible linkage may have advantages for cell delivery, since it is likely to be cleaved within cells, thus releasing the oligonucleotide cargo. It is straightforward to introduce thiol functionalities into both oligonucleotide and peptide components suitable for disulfide conjugation. However, severe difficulties have been encountered in carrying out conjugations between highly cationic peptides and negatively charged oligonucleotides because of aggregation and precipitation. Presented here are reliable protocols for disulfide conjugation that have been verified for both cationic and hydrophobic peptides as well as oligonucleotides containing deoxyribonucleosides, ribonucleosides, 2′‐O‐methylribonucleosides, locked nucleic acid (LNA) units, as well as phosphorothioate backbones. Also presented are reliable protocols for disulfide conjugation of peptide nucleic acids (PNAs) with peptides.

Anti‐HIV Activity of Steric Block Oligonucleotides

Abstract:  The unabated increase in spread of HIV infection worldwide has redoubled efforts to discover novel antiviral and virucidal agents that might be starting points for clinical development. Oligonucleotides and their analogs targeted to form complementary duplexes with highly conserved regions of the HIV RNA have shown significant antiviral activity, but to date clinical studies have been dominated by RNase H‐inducing oligonucleotide analog phosphorothioates (GEM 91 and 92) that have specificity and efficacy limitations. However, they have proven the principle that oligonucleotides can be safe anti‐HIV drugs. Newer oligonucleotide analogs are now available, which act as strong steric block agents of HIV RNA function. We describe our ongoing studies targeting the HIV‐1 trans‐activation responsive region (TAR) and the viral packaging signal (psi) with steric block oligonucleotides of varying chemistry and demonstrate their great potential for steric blocking of viral protein interactions in vitro and in cells and describe the first antiviral studies. Peptide nucleic acids (PNA) disulfide linked to cell‐penetrating peptides (CPP) have been found to have particular promise for the lipid‐free direct delivery into cultured cells and are excellent candidates for their development as antiviral and virucidal agents.

A convenient route to α-amino acids with β-alkyne substituents from a serine derived aziridine

Abstract 1-[(S)-1-(2-Nitrobenzenesulfonyl)-aziridin-2-yl]-4-methyl-2,6,7-trioxabicyclo[2.2.2]octane 5 was ring opened regioselectively with a variety of lithium acetylides to give α-amino acids bearing γ,δ-unsaturation in very good to excellent yields. The 2-nitrobenzenesulfonyl and OBO ester protecting groups were removed in excellent overall yield.