Elizabeth J. Ackermann

Pleiotropic cell-division defects and apoptosis induced by interference with survivin function.

Here we investigate the role of the control of apoptosis in normal cell division. We show that interference with the expression or function of the apoptosis inhibitor survivin causes caspase-dependent cell death in the G2/M phase of the cell cycle, and a cell-division defect characterized by centrosome dysregulation, multipolar mitotic spindles and multinucleated, polyploid cells. Use of a dominant-negative survivin mutant or antisense survivin complementary DNA disrupts a supramolecular assembly of survivin, caspase-3 and the cyclin-dependent-kinase inhibitor p21Waf1/Cip1 within centrosomes, and results in caspase-dependent cleavage of p21. Polyploidy induced by survivin antagonists is accentuated in p21-deficient cells, and corrected by exogenous expression of p21. These findings show that control of apoptosis and preservation of p21 integrity within centrosomes by survivin are required for normal mitotic progression.

Suppression of Vascular Endothelial Growth Factor-Mediated Endothelial Cell Protection by Survivin Targeting

The protective genes that mediate endothelial cell (EC) survival during angiogenesis have not been completely characterized. Here, we show that an antisense oligonucleotide to the apoptosis inhibitor survivin suppressed de novo expression of survivin in ECs by vascular endothelial cell growth factor (VEGF). In contrast, the survivin antisense oligonucleotide did not affect anti-apoptotic bcl-2 levels in endothelium. When assessed in cell death assays, antisense targeting of survivin abolished the anti-apoptotic function of VEGF against tumor necrosis factor-alpha- or ceramide-induced cell death, enhanced caspase-3 activity, promoted the generation of a approximately 17-kd active caspase-3 subunit, and increased cleavage of the caspase substrate, polyADP ribose polymerase. In contrast, the survivin antisense oligonucleotide had no effect on EC viability in the absence of VEGF. Antisense oligonucleotides to platelet-endothelial cell adhesion molecule-1 (PECAM-1, CD31), lymphocyte function-associated molecule-3 (LFA-3, CD58), or intercellular adhesion molecule-1 (ICAM-1, CD54) did not reduce the anti-apoptotic function of VEGF in endothelium. When tested on other angiogenic activities mediated by VEGF, survivin antisense treatment induced rapid regression of three-dimensional vascular capillary networks, but did not affect EC migration/chemotaxis. These data suggest that the anti-apoptotic properties of VEGF during angiogenesis are primarily mediated by the induced expression of survivin in ECS: Manipulation of this pathway may increase EC viability in compensatory angiogenesis or facilitate EC apoptosis and promote vascular regression during tumor angiogenesis.

Clinical development of an antisense therapy for the treatment of transthyretin-associated polyneuropathy.

Transthyretin (TTR)-associated amyloidosis is a late-onset autosomal-dominant genetic disease. Over 100 amyloidogenic mutations have been identified in TTR which destabilize the TTR tetramer thereby inducing the formation of amyloid fibrils in tissues such as the heart and peripheral nerves. This disease mainly affects peripheral nerves, causing familial amyloid polyneuropathy (FAP) or heart, causing familial amyloid cardiomyopathy (FAC). Circulating TTR is predominantly produced by liver, and the only widely available clinical treatment for FAP is orthotopic liver transplantation (OLT), whereas no treatment currently exists for FAC. Using second-generation antisense technology, we identified an antisense oligonucleotide (ASO) targeting TTR, ISIS-TTRRx, for the treatment of TTR-associated amyloidosis. When tested in a human TTR transgenic mouse model (hTTR Ile84Ser), ISIS-TTRRx showed a dose-dependent reduction of human TTR (up to >80%) at both the mRNA and protein levels. In cynomolgus monkeys, ISIS-TTRRx treatment produced a time-dependent reduction in plasma TTR levels. After 12 weeks of treatment in monkey, liver TTR mRNA and plasma TTR protein levels were reduced by ~80%. As expected, treatment with ISIS-TTRRx also produced a significant decrease in plasma RBP4 levels that correlated with reductions in TTR levels. ISIS-TTRRx treatment was well tolerated in both rodents and monkeys and produced a PK/PD profile consistent with prior experiences using this chemistry platform. ISIS-TTRRx is currently under evaluation in a Phase 1 clinical trial in normal healthy volunteers, and interim results of this trial will be presented.

Suppressing transthyretin production in mice, monkeys and humans using 2nd-Generation antisense oligonucleotides

Abstract Transthyretin amyloidosis (ATTR amyloidosis) is a rare disease that results from the deposition of misfolded transthyretin (TTR) protein from the plasma into tissues as amyloid fibrils, leading to polyneuropathy and cardiomyopathy. IONIS-TTRRx (ISIS 420915) is a 2nd-Generation 2′-O-(2-methoxyethyl) modified “2′-MOE” antisense oligonucleotide (ASO) that targets the TTR RNA transcript and reduces the levels of the TTR transcript through an RNaseH1 mechanism of action, leading to reductions in both mutant and wild-type TTR protein. The activity of IONIS-TTRRx to decrease TTR protein levels was studied in transgenic mice bearing the Ile84Ser human TTR mutant, in cynomolgus monkeys and in healthy human volunteers. Robust (>80%) reductions of plasma TTR protein were obtained in all three species treated with IONIS-TTRRx, which in mice and monkeys was associated with substantial reductions in hepatic TTR RNA levels. These effects were dose-dependent and lasted for weeks post-dosing. In a Phase 1 healthy volunteer study, treatment with IONIS-TTRRx for four weeks was well tolerated without any remarkable safety issues. TTR protein reductions up to 96% in plasma were observed. These nonclinical and clinical results support the ongoing Phase 3 development of IONIS-TTRRx in patients with ATTR amyloidosis.

The transthyretin amyloidoses: advances in therapy

There are two forms of transthyretin (TTR) amyloidosis: non-hereditary and hereditary. The non-hereditary form (ATTRwt) is caused by native or wild-type TTR and was previously referred to as senile systemic amyloidosis. The hereditary form (ATTRm) is caused by variant TTR which results from a genetic mutation of TTR. The predominant effect of ATTRwt amyloidosis is on the heart, with patients having a greater left ventricular wall thickness at presentation than the devastating form which is light chain (AL) amyloidosis. ATTRm amyloidosis is broadly split into two categories: a type that predominantly affects the nervous system (often called familial amyloid polyneuropathy (FAP)) and one with a predilection for the heart (often called familial amyloid cardiomyopathy (FAC)). Approximately half of all TTR mutations known to express a clinical phenotype cause a cardiomyopathy. Since the introduction of orthotopic liver transplantation for ATTRm amyloidosis in 1991, several additional therapies have been developed. These therapies aim to provide a reduction or elimination of TTR from the plasma (through genetic approaches), stabilisation of the TTR molecule (to prevent deposition) and dissolution of the amyloid matrix. We describe the latest developments in these approaches to management, many of which are also applicable to wild-type amyloidosis.

Assessing mNIS+7Ionis and International Neurologists' Proficiency in an FAP Trial.

Polyneuropathy signs (Neuropathy Impairment Score, NIS), neurophysiologic tests (m+7Ionis), disability, and health scores were assessed in baseline evaluations of 100 patients entered into an oligonucleotide familial amyloidotic polyneuropathy (FAP) trial.

Assessing mNIS+7Ionis and international neurologists' proficiency in a familial amyloidotic polyneuropathy trial

Polyneuropathy signs (Neuropathy Impairment Score, NIS), neurophysiologic tests (m+7Ionis), disability, and health scores were assessed in baseline evaluations of 100 patients entered into an oligonucleotide familial amyloidotic polyneuropathy (FAP) trial.

Treatment of transthyretin cardiomyopathy with a TTR-specific antisense oligonucleotide (IONIS-TTRRx)

Transthyretin amyloidosis (ATTR) is a systemic form of amyloidosis associated with at least 130 genetic mutations in TTR. Most forms of ATTR are associated with neuropathy, but cardiomyopathy may be a significant factor in longevity. In addition, amyloid cardiomyopathy associated with wildtype TTR is now appreciated to be an aging phenomenon with individuals present with this condition in their 8th to 9th decade. Liver transplantation is used to treat ATTR and has met with some success mainly in patients with the Val30Met mutation but with less success in patients with many of the other mutations, in particular, those that give predominantly cardiomyopathy [1]. Liver transplantation, of course, would not be considered to be of any therapeutic value in patients with ATTR wild-type. We have previously studied nine subjects with hereditary (familial) amyloid polyneuropathy (FAP) in which we were able to show significant progression of thickening of the left ventricular wall over a 12-month period if the left ventricular wall thickness had progressed to the point of 1.3 cm [2]. Individuals with left ventricular wall thickness less than 1.3 cm did not progress significantly over a one-year period and clinical experience suggests that 5, 10, or maybe 15 years may pass before an individual with a TTR amyloid gene mutation progresses from normal to the 1.3 cm level.

Evaluation of Therapeutic Oligonucleotides for Familial Amyloid Polyneuropathy in Patient-Derived Hepatocyte-Like Cells.

Familial amyloid polyneuropathy (FAP) is caused by mutations of the transthyretin (TTR) gene, predominantly expressed in the liver. Two compounds that knockdown TTR, comprising a small interfering RNA (siRNA; ALN-TTR-02) and an antisense oligonucleotide (ASO; IONIS-TTRRx), are currently being evaluated in clinical trials. Since primary hepatocytes from FAP patients are rarely available for molecular analysis and commercial tissue culture cells or animal models lack the patient-specific genetic background, this study uses primary cells derived from urine of FAP patients. Urine-derived cells were reprogrammed to induced pluripotent stem cells (iPSCs) with high efficiency. Hepatocyte-like cells (HLCs) showing typical hepatic marker expression were obtained from iPSCs of the FAP patients. TTR mRNA expression of FAP HLCs almost reached levels measured in human hepatocytes. To assess TTR knockdown, siTTR1 and TTR-ASO were introduced to HLCs. A significant downregulation (>80%) of TTR mRNA was induced in the HLCs by both oligonucleotides. TTR protein present in the cell culture supernatant of HLCs was similarly downregulated. Gene expression of other hepatic markers was not affected by the therapeutic oligonucleotides. Our data indicate that urine cells (UCs) after reprogramming and hepatic differentiation represent excellent primary human target cells to assess the efficacy and specificity of novel compounds.

The ISIS-TTRRx-CS2 phase 3 study in patients with familial amyloid polyneuropathy: Baseline results of the first 100 patients for the NIS, NIS+7 and mNIS+7 using different methods of scoring: identification of consistencies and key differences

Background Familial amyloid polyneuropathy (FAP) is a devastating disease with continuous progression over time leading to complete disability, bedridden status, and ultimately death within 10 to 15 years from symptom onset. In order to document disease progression, it is necessary to have valid instruments which allow assessment of the severity of the sensory, motor and autonomic neuropathy components of the disease. Over the last decades, multiple related instruments have been used and modifications developed to measure disease progression in FAP patients in clinical trial settings. These include the Neuropathy Impairment Score (NIS), originally called Neuropathy Disability Score, the Neuropathy Impairment Score for the Lower Limbs (NIS-LL), the Neuropathy Impairment Score +7 (NIS+7) and the modified NIS +7 (mNIS+7). The NIS scores were first used to quantitate neuropathic impairment in a series of therapeutic trials in chronic inflammatory demyelinating polyradiculoneuropathy and were also used as primary endpoints in clinical trials for FAP in the completed diflunisal (NIS+7) and tafamidis (NIS-LL) Phase 3 trials. Building on experience from these trials, modifications to the NIS+7 were developed (Suanprasert et al, 2014, J Neurol Sci) and are currently being applied in the ongoing ISIS-TTRRx-CS2 (mNIS+7 Isis) and patisiran (mNIS+7 ALN) Phase 3 trials. Even though the mNIS+7 Isis and mNIS+7 ALN scores are very similar, they are not identical. The details in the assessment and scoring for the various versions of the NIS based scores are described and their scoring methods applied to baseline data collected on the first 100 patients in the ISIS-TTRRx-CS2 trial to better understand and illustrate the key differences.