BACKGROUNDTransthyretin amyloidosis, also called ATTR amyloidosis, is a life-threatening disease characterized by progressive accumulation of misfolded transthyretin (TTR) protein in tissues, predominantly the nerves and heart. NTLA-2001 is an in vivo gene-editing therapeutic agent that is designed to treat ATTR amyloidosis by reducing the concentration of TTR in serum. It is based on the clustered regularly interspaced short palindromic repeats and associated Cas9 endonuclease (CRISPR-Cas9) system and comprises a lipid nanoparticle encapsulating messenger RNA for Cas9 protein and a single guide RNA targeting TTR. METHODSAfter conducting preclinical in vitro and in vivo studies, we evaluated the safety and pharmacodynamic effects of single escalating doses of NTLA-2001 in six patients with hereditary ATTR amyloidosis with polyneuropathy, three in each of the two initial dose groups (0.1 mg per kilogram and 0.3 mg per kilogram), within an ongoing phase 1 clinical study. RESULTSPreclinical studies showed durable knockout of TTR after a single dose. Serial assessments of safety during the first 28 days after infusion in patients revealed few adverse events, and those that did occur were mild in grade. Dose-dependent pharmacodynamic effects were observed. At day 28, the mean reduction from baseline in serum TTR protein concentration was 52% (range, 47 to 56) in the group that received a dose of 0.1 mg per kilogram and was 87% (range, 80 to 96) in the group that received a dose of 0.3 mg per kilogram. CONCLUSIONSIn a small group of patients with hereditary ATTR amyloidosis with polyneuropathy, administration of NTLA-2001 was associated with only mild adverse events and led to decreases in serum TTR protein concentrations through targeted knockout of TTR. (Funded by Intellia Therapeutics and Regeneron Pharmaceuticals; ClinicalTrials.gov number, NCT04601051.

The role of the abundant stress protein Hsp90 in protecting cells against stress-induced damage is not well understood. The recent discovery that a class of ansamycin antibiotics bind specifically to Hsp90 allowed us to address this problem from a new angle. We find that mammalian Hsp90, in cooperation with Hsp70, p60, and other factors, mediates the ATP-dependent refolding of heatdenatured proteins, such as firef ly luciferase. Failure to refold results in proteolysis. The ansamycins inhibit refolding, both in vivo and in a cell extract, by preventing normal dissociation of Hsp90 from luciferase, causing its enhanced degradation. This mechanism also explains the ansamycin-induced proteolysis of several protooncogenic protein kinases, such as Raf-1, which interact with Hsp90. We propose that Hsp90 is part of a quality control system that facilitates protein refolding or degradation during recovery from stress. This function is used by a limited set of signal transduction molecules for their folding and regulation under nonstress conditions. The ansamycins shift the mode of Hsp90 from refolding to degradation, and this effect is probably amplified for specific Hsp90 substrates.Exposure of prokaryotic and eukaryotic cells to heat and other stresses induces several classes of highly conserved stress proteins, including the members of the Hsp70, Hsp60, and Hsp90 families (1-3). These proteins are generally thought to act as molecular chaperones in preventing the aggregation of nonnative polypeptides and in aiding their correct folding. Although significant progress has been made in understanding the chaperone mechanisms in de novo protein folding, surprisingly little is known about the role of chaperones under stress conditions. This lack of knowledge is particularly apparent for the Hsp90s, the most abundant constitutively expressed stress proteins in the eukaryotic cytosol. Although Hsp90 can prevent protein aggregation in vitro (4-6) and is required for the survival of yeast at elevated temperature (7), its actual role in protein refolding and repair under stress has remained elusive (2, 8). Instead, current thinking views Hsp90 as part of a specific chaperone system for the conformational maturation and regulation of signal transduction molecules, such as several potentially oncogenic protein kinases and the nuclear receptors of steroid hormones (8-11). In the mammalian cytosol, these proteins are found in heterocomplexes containing Hsp90, Hsp70͞Hsc70, the Hsp70 regulator Hip (p48), p60, various immunophilins, and the small acidic protein p23.A recent study proposed that the benzoquinone ansamycins geldanamycin (GA) and herbimycin A (HA), originally classified as tyrosine kinase inhibitors (12), do not execute their biological effects directly by inhibiting kinase activities, but rather indirectly by acting on Hsp90 (13). Thus, these agents provide a powerful tool to explore the physiological role of Hsp90. We show that Hsp90, in cooperation with Hsc70, p60, and other factors, mediates the refolding of t...

The insulin receptor substrate-1 (IRS-1), a docking protein for both the type 1 insulin-like growth factor receptor (IGF-IR) and the insulin receptor, is known to send a mitogenic, anti-apoptotic, and anti-differentiation signal. Several micro RNAs (miRs) are suggested by the data base as possible candidates for targeting IRS-1. We show here that one of the miRs predicted by the data base, miR145, whether transfected as a synthetic oligonucleotide or expressed from a plasmid, causes down-regulation of IRS-1 in human colon cancer cells. IRS-1 mRNA is not decreased by miR145, while it is down-regulated by an siRNA targeting IRS-1. Targeting of the IRS-1 3-untranslated region (UTR) by miR145 was confirmed using a reporter gene (luciferase) expressing the miR145 binding sites of the IRS-1 3-UTR. In agreement with the role of IRS-1 in cell proliferation, we show that treatment of human colon cancer cells with miR145 causes growth arrest comparable to the use of an siRNA against IRS-1. Taken together, these results identify miR145 as a micro RNA that down-regulates the IRS-1 protein, and inhibits the growth of human cancer cells.The insulin receptor substrate-1 (IRS-1) 2 is one of the major substrates of both the type 1 insulin-like growth factor receptor (IGF-IR) and the insulin receptor (InR). IRS-1 plays an important role in cell growth and cell proliferation (1). IRS-1, especially when activated by the IGF-IR, sends an unambiguous mitogenic, anti-apoptotic, and anti-differentiation signal (2, 3). IRS-1 levels are often increased in human cancer (4), and they are low or even absent in differentiating cells (1,5,6). Overexpression of IRS-1 causes cell transformation, including the ability to form colonies in soft agar and tumors in mice (7,8). Transgenic expression of IRS-1 in the mammary gland of mice causes mammary hyperplasia, tumorigenicity, and metastases (9). Conversely, down-regulation of IRS-1 (by antisense or siRNA procedures) reverses the transformed phenotype (10 -12). The IRS proteins are conserved during evolution, and a gene described in Drosophila, called chico, is the equivalent of IRS-1 to IRS-4 in mammalian cells. IRS proteins play an important role in cell size. Deletion of chico reduces fly weight by 65% in females and 55% in males (13). Mice with a targeted disruption of the IRS-1 genes are also smaller than their wild-type littermates (14), and ectopic expression of IRS-1 increases rRNA synthesis and doubles cell size in cells in culture (7,15). Thus, IRS-1 seems to play important roles in cell growth (cell size), cell proliferation, and differentiation.Micro RNA (miRs) are RNAs of ϳ22-nucleotides long, that arise from one arm of longer endogenous hairpin transcripts. The characteristics of miRs have been summarized in several reviews (16 -19). Briefly, miRs are cleaved from one arm of a longer endogenous double-stranded precursor (70 -100 nt in length) by Drosha and Dicer enzymes (RNase III family). They are transcribed by RNA polymerase II (20) as long primary transcripts (pri-miRNAs), which ...

The inhibition of KSP causes mitotic arrest by activating the spindle assembly checkpoint. While transient inhibition of KSP leads to reversible mitotic arrest, prolonged exposure to a KSP inhibitor induces apoptosis. Induction of apoptosis by the KSP inhibitor couples with mitotic slippage. Slippage-refractory cells show resistance to KSP inhibitor-mediated lethality, whereas promotion of slippage after mitotic arrest enhances apoptosis. However, attenuation of the spindle checkpoint confers resistance to KSP inhibitor-induced apoptosis. Furthermore, sustained KSP inhibition activates the proapoptotic protein, Bax, and both activation of the spindle checkpoint and subsequent mitotic slippage are required for Bax activation. These studies indicate that in response to KSP inhibition, activation of the spindle checkpoint followed by mitotic slippage initiates apoptosis by activating Bax.