Eukaryotic chromosomes are capped with repetitive telomere sequences that protect the ends from damage and rearrangements. Telomere repeats are synthesized by telomerase, a ribonucleic acid (RNA)-protein complex. Here, the cloning of the RNA component of human telomerase, termed hTR, is described. The template region of hTR encompasses 11 nucleotides (5'-CUAACCCUAAC) complementary to the human telomere sequence (TTAGGG)n. Germline tissues and tumor cell lines expressed more hTR than normal somatic cells and tissues, which have no detectable telomerase activity. Human cell lines that expressed hTR mutated in the template region generated the predicted mutant telomerase activity. HeLa cells transfected with an antisense hTR lost telomeric DNA and began to die after 23 to 26 doublings. Thus, human telomerase is a critical enzyme for the long-term proliferation of immortal tumor cells.

We propose a novel model for the regulation of the p85/p110␣ phosphatidylinositol 3-kinase. In insect cells, the p110␣ catalytic subunit is active as a monomer but its activity is decreased by coexpression with the p85 regulatory subunit. Similarly, the lipid kinase activity of recombinant glutathione S-transferase (GST)-p110␣ is reduced by 65 to 85% upon in vitro reconstitution with p85. Incubation of p110␣/p85 dimers with phosphotyrosyl peptides restored activity, but only to the level of monomeric p110␣. These data show that the binding of phosphoproteins to the SH2 domains of p85 activates the p85/p110␣ dimers by inducing a transition from an inhibited to a disinhibited state. In contrast, monomeric p110 had little activity in HEK 293T cells, and its activity was increased 15-to 20-fold by coexpression with p85. However, this apparent requirement for p85 was eliminated by the addition of a bulky tag to the N terminus of p110␣ or by the growth of the HEK 293T cells at 30°C. These nonspecific interventions mimicked the effects of p85 on p110␣, suggesting that the regulatory subunit acts by stabilizing the overall conformation of the catalytic subunit rather than by inducing a specific activated conformation. This stabilization was directly demonstrated in metabolically labeled HEK 293T cells, in which p85 increased the half-life of p110. Furthermore, p85 protected p110 from thermal inactivation in vitro. Importantly, when we examined the effect of p85 on GST-p110␣ in mammalian cells at 30°C, culture conditions that stabilize the catalytic subunit and that are similar to the conditions used for insect cells, we found that p85 inhibited p110␣. Thus, we have experimentally distinguished two effects of p85 on p110␣: conformational stabilization of the catalytic subunit and inhibition of its lipid kinase activity. Our data reconcile the apparent conflict between previous studies of insect versus mammalian cells and show that p110␣ is both stabilized and inhibited by dimerization with p85.Phosphatidylinositol (PI) 3Ј-kinases constitute a family of enzymes that mediate intracellular signaling initiated by receptor tyrosine kinases and heterotrimeric G-protein-coupled receptors. Activation of PI 3Ј-kinase leads to increases in the intracellular levels of PI[3,4]P 2 and PI[3,4,5]P 3 , which are presumed second messengers (4). PI 3Ј-kinases have been implicated in the control of proliferation, cytoskeletal organization, apoptosis, and vesicular trafficking (6,16,20,31,46).A classification of PI 3Ј-kinases has been described by Zvelebil and coworkers (48). The class I enzymes are heterodimeric proteins that are composed of separate regulatory and catalytic subunits and that utilize PI, PI[4]P, and PI[4,5]P 2 as substrates. Class I enzymes include the p85/p110 PI 3Ј-kinase, which is activated by binding to phosphotyrosyl proteins, and the p101/ p120 PI 3-kinase-␥ isoform, which is activated by ␤␥ subunits from trimeric G proteins (11,13,38,39). Class II PI 3Ј-kinases contain C-terminal C2 domains and preferentially utiliz...

Triantennary N-acetyl galactosamine (GalNAc, GN3), a high-affinity ligand for the hepatocyte-specific asialoglycoprotein receptor (ASGPR), enhances the potency of second-generation gapmer antisense oligonucleotides (ASOs) 6–10-fold in mouse liver. When combined with next-generation ASO designs comprised of short S-cEt (S-2′-O-Et-2′,4′-bridged nucleic acid) gapmer ASOs, ∼60-fold enhancement in potency relative to the parent MOE (2′-O-methoxyethyl RNA) ASO was observed. GN3-conjugated ASOs showed high affinity for mouse ASGPR, which results in enhanced ASO delivery to hepatocytes versus non-parenchymal cells. After internalization into cells, the GN3-ASO conjugate is metabolized to liberate the parent ASO in the liver. No metabolism of the GN3-ASO conjugate was detected in plasma suggesting that GN3 acts as a hepatocyte targeting prodrug that is detached from the ASO by metabolism after internalization into the liver. GalNAc conjugation also enhanced potency and duration of the effect of two ASOs targeting human apolipoprotein C-III and human transthyretin (TTR) in transgenic mice. The unconjugated ASOs are currently in late stage clinical trials for the treatment of familial chylomicronemia and TTR-mediated polyneuropathy. The ability to translate these observations in humans offers the potential to improve therapeutic index, reduce cost of therapy and support a monthly dosing schedule for therapeutic suppression of gene expression in the liver using ASOs.

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