- Isis Pharma will make several presentations at the Society for Neuroscience (SFN) Annual Meeting November 12-16, 2011 - see complete abstracts and info below.
- Find detailed pipeline and company info on the ISIS research page.
- See complete calendar of Upcoming scientific and investor events, plus link to search for other abstracts at the SFN meeting.
1) Oral presentation: Sunday, Nov 13, 2011, 2:00 PM - 2:15 PM
Allele-specific antisense oligonucleotide silencing of mutant huntingtin expression
*A. L. SOUTHWELL1, N. SKOTTE1, J. CARROLL1, C. DOTY1, S. GREENLEE2, S. WARBY3, S. M. FREIER2, G. HUNG2, X. W. YANG4, C. F. BENNET2, M. R. HAYDEN1;
1CMMT, UBC-CMMT, Vancouver, BC, Canada; 2ISIS Pharmaceuticals, Carlsbad, CA; 3Stanford Univ., Palo Alto, CA; 4UCLA, Los Angeles, CA
In striking contrast to the simplicity of HD genetics, the convoluted disease mechanisms in this disorder have hindered development of disease-modifying therapies. The huntingtin protein (HTT) plays roles in numerous, seemingly unrelated cellular processes. In HD, mutant HTT (mHTT) fails to adequately perform these essential roles and also acquires toxic functions leading to a complex network of dysfunction. Preventing generation of this toxic protein by gene silencing should prevent all subsequent pathology and delay or prevent the onset of HD. Interventions that selectively silence mHTT while sparing wildtype HTT (wtHTT) expression are preferable, because long-term reduction of wtHTT may not be well-tolerated.
Facilitating this allele-specific approach is the finding that numerous single nucleotide polymorphisms (SNPs) are significantly enriched in human mHTT alleles compared to wtHTT alleles. Combinatorial analysis suggests that a panel of silencing reagents targeting as few as 3 of these HD-SNPs would provide a therapeutic option for up to 85% of the HD population. We have generated a large number of antisense oligonucleotides (ASOs) targeting HD-SNPs. ASOs silence gene expression post-transcriptionally and can discriminate between SNP alleles anywhere in the pre-mRNA, including introns. Moreover, naked ASOs can be effectively delivered throughout the adult primate CNS via ICV or intrathecal infusion [see abstract #3 below].
We next sought to validate candidate ASOs in primary murine neurons. In the case of our top 6 ranked ASOs, BACHD mice share target alleles with the majority of HD patients, while YAC18 mice do not. Screening of lead candidates in paired cortical neuron cultures from YAC18 and BACHD mice resulted in mHTT knockdown between 39% and 68% and negligible wtHTT knockdown. To generate a suitable mouse line for in vivo testing, we have cross bred BACHD, YAC18 and Hdh-/- mice. The resulting humanized line, Hu97/18, is the first murine model of HD that fully genetically recapitulates human HD. We are currently screening ASOs after acute delivery to the brains of Hu97/18 mice to assess in vivo activity and to select the ASOs with the greatest therapeutic potential. Preliminary data suggests that lead ASOs can potently and selectively silence mHTT expression in the Hu97/18 brain without induction of overt CNS toxicity. These results support the viability of a SNP-targeted, ASO-mediated, allele-specific mHTT gene silencing strategy.
Allele specific silencing of the mHtt gene is the most targeted, upstream therapeutic strategy for the treatment of HD. If successful in mice, this therapy could be rapidly translated into a similar approach for humans.
2) Oral Presentation: Monday, Nov 14, 2011, 2:15 PM - 2:30 PM
Targeting Transthyretin for the treatment of Transthyretin-associated polyneuropathy using antisense technology
*S. GUO1, S. BOOTEN1, L. ALVARADO1, A. SIWKOWSKI1, M. BENSON2, B. MONIA1;
1ISIS Pharmaceuticals, Carlsbad, CA; 2Dept. of Pathology and Lab. Med., Indiana Univ. Sch. of Med., Indianapolis, IN
Transthyretin (TTR)-associated amyloidosis is a late-onset autosomal-dominant genetic disease. It is the most prevalent form of hereditary systemic amyloidosis. Over 100 amyloidogenic mutations have been identified in TTR which destabilize the TTR tetramer thereby inducing the formation of amyloid fibrils in various tissues including peripheral nerves and cardiac muscle. This disease mainly affects peripheral nerves as in familial amyloidotic polyneuropathy (FAP) or heart as in familial amyloid cardiomyopathy (FAC). TTR is mainly produced by liver, and the only available clinical treatment for TTR-associated amyloidosis is orthotopic liver transplantation (OLT).
Using second generation antisense technology, we identified an antisense oligonucleotide (ASO) targeting TTR, ISIS-TTRRx, for the treatment of TTR-associated amyloidosis. This compound was first identified through in vitro screens in a human hepatocellular carcinoma cell line HepG2. When tested in a human TTR transgenic mouse model (hTTR Ile84Ser), ISIS-TTRRx showed a dose-dependent reduction of human TTR at both the mRNA and protein levels. In a 12-week study in cynomolgus monkeys, ISIS-TTRRx treatment produced a time-dependent reduction in plasma TTR levels. After 12 weeks of treatment, liver TTR mRNA and plasma TTR protein levels were reduced by ~80%. Since TTR is a plasma carrier for retinol-binding protein 4 (RBP4) and prevents RBP4 from renal filtration, we also monitored RBP4 plasma levels and observed a significant decrease in RBP4 levels that correlated with TTR reduction. 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 normal volunteers in a Phase I clinical trial.
3) Poster presentation: Tuesday, Nov 15, 2011, 9:00 AM -10:00 AM
Second generation Oligonucleotide CNS delivery via IT administration in mice, rats and Monkey
*G. HUNG1, C. MAZUR1, E. WANCEWICZ2, D. NORRIS2, F. BENNETT1;
1Isis Pharmaceuticals, CARLSBAD, CA; 2Isis Pharmaceuticals, carlsbad, CA
Introduction: We have previously demonstrated efficacy of antisense oligonucleotides (ASOs) targeting huntingtin (HTT) mRNA when administered ICV as a treatment for HD in different species. We aim to evaluate the CNS distribution and pharmacology of ASOs following intrathecal (IT) administration in mice, rats, and Rhesus monkeys.
Methods: In separate studies, mice, rats, and monkeys, received either a single bolus of ASO at different dosages or (rats and monkeys) received repeated doses directly into the subarachnoid space. Following dosing, select CNS tissues, plasma and cerebrospinal fluid samples were collected and analyzed for ASO concentrations by capillary gel electrophoresis (CGE). CNS distribution of ASO was determined by immunohistochemistry. HTT mRNA knockdown in different brain regions was measured by quantitative PCR.
Results: Dose dependent HTT mRNA knockdown in the various regions of the CNS was consistent with measured ASO concentrations in all tested species. Higher ASO concentrations, , were observed in the spinal cord after IT dosing compared to brain tissue. These higher concentrations were associated with greater HTT mRNA knockdown. The select CNS tissues displayed good PK/PD correlations. In different species, greater than 50% HTT mRNA knockdown in the distal regions of the CNS were achieved following single bolus IT administration. ASO cellular uptake was observed at CNS sites distant from the site of administration, and no cell type specific uptake was observed.
Conclusions: Similar PK/PD profiles were seen between species following IT administration. IT bolus administration can be an efficient method to deliver ASO for therapeutic intervention of CNS disease.
4) Poster presentation: Wednesday, Nov 16, 2011, 1:00 PM - 2:00 PM
Mis-localization of neuronal proteins due to loss of HIP14 palmitoylation
*S. S. SANDERS1, S. M. FREIER2, A. SALIM2, G. HUNG2, C. F. BENNETT2, M. R. HAYDEN1,3;
1Ctr. for Mol. Med. and Therapeut., The Univ. of British Columbia, Vancouver, BC, Canada; 2ISIS Pharmaceuticals, Inc., Carlsbad, CA; 3Child and Family Res. Inst., British Columbia Children's Hosp., Vancouver, BC, Canada
Palmitoylation is a post-translational modification of proteins with the lipid palmitate and is catalyzed by a class of enzymes called palmitoyl acyltransferases (PATs). It is an important mechanism for regulating protein trafficking and function, including that of huntingtin (htt) the protein mutated in Huntington disease (HD). HD is caused by a CAG expansion in exon 1 of the htt gene and is characterized by striatal degeneration and the associated motor, cognitive, and psychiatric symptoms. In the presence of the HD mutation, the interaction between htt and HIP14 (DHHC-17), a PAT for htt, is disturbed, resulting in reduced palmitoylation of htt, as well as other HIP14 substrates, and increased neuronal toxicity. HIP14 deficient mice recapitulate many features of HD, including striatal volme loss and motor deficits and exhibit decreased palmitoylation in the same group of neuronal proteins as seen in the YAC128 HD model. To determine the concequence of loss of HIP14 palmitoylation on the localization of these neuronal proteins; namely SNAP25, PSD95, GluR1, GluR2, NR2B, and CASP6, HIP14 antisense oligonucleotides (ASOs) were used to reduce HIP14 expression in cortical-striatal co-cultures. Up to 95% knockdown of HIP14 was achieved after 7 days of treatment and HIP14 substrates were in turn, mislocalized.