rnai therapeutics targeting pcsk9 for ldlc lowering in ... · rnai therapeutics targeting pcsk9 for...

RNAi Therapeutics Targeting PCSK9 for LDLc Lowering in Hypercholesterolemia M. D. Frank-Kamenetsky1, T. Racie1, A. Akinc1, N. Anderson2, L. Nechev1, S. Shulga-Morskaya1, K. Mills1, S. Millstein1, T. Lei1, R. Duncan1, A. Grefhorst2, G. Wang1, R. Kallanthottathil1, M. Maier1, S. Semple3, A. Lee3, L. Jeffs3, E. Yaworski3, P. Lutwyche3, I. MacLachlan3, C. Gamba-Vitalo1, M. Kretschmer1,M. Jayaraman1, R. Myers1, K. Charisse1, D. Anderson4, P. Cullis5, J. Horton2, M. Manoharan1, V. Kotelianski1, A. de Fougerolles1, K. Fitzgerald1

1Alnylam Pharmaceuticals Inc., Cambridge, Massachusetts, USA; 2UTSW Medical Center, Dallas, Texas, USA; 3Tekmira Pharmaceuticals Corp, Vancouver, Canada; 4MIT, Cambridge, Massachusetts, USA; 5UBC/AlCana Technologies, Vancouver, Canada

Conclusions�RNAi�therapeutic�targeting�PCSK9:� Highly active lead molecules – pM and specific with no measured cytokine induction or off-target effects LNP platform has improved dramatically – Efficacy improved from 5mg/kg to ~0.1mg/kg dose Efficacious in mice, rats, LDLR heterozygous and hCETP/hApoB 100 humanized transgenic mice Non-Human Primate Model: – Significant PCSK9 transcript and protein lowering – LDLc decreased by ~40-60% with no effects on HDL cholesterol at doses >0.1mg/kg Targeting multiple genes for the treatment of metabolic syndrome – Combinations in LNP (up to ten genes at once) – Dual targeting siRNA

AbstractsiRNA�Based�Treatments�For�Hyperlipidemia:Proprotein convertases subtilisin/kexin type 9 (PCSK9) is a protease involved in the regulation of low density lipoprotein receptor (LDLR) levels and function. Loss of PCSK9 protein activity (in murine models, as well as, in human individuals) increases LDLR levels while excess PCSK9 activity decreases LDLR levels. These changes in LDLR levels coincide with opposite changes in circulating levels of LDLc or total cholesterol over the lifetime of these individuals or animals. We have developed several cross-species siRNA inhibitors capable of targeting a combination of murine, rat, non-human primate (NHP), or human PCSK9. These inhibitors are highly potent in vitro and have been modified to inhibit their ability to stimulate the immune response, or produce off-target effects. We have demonstrated that liver specific silencing of PCSK9 in rodent and NHP species results in an acute lowering of LDLc. In NHP LDLc levels are lowered >50% without changes in HDLc at doses >0.1mg/kg. Additional dose sparing paradigms have also been investigated. PCSK9 silencing in LDLR heterozygous mice results in the expected phenotypic changes, specifically, in total serum cholesterol lowering, indicating that a single wt copy of LDLR is sufficient for the PCSK9 mechanism to be effective. These findings indicate that a PCSK9 RNAi therapy will likely be effective in LDLR heterozygous individuals with familial hypercholesterolemia. Finally, we have begun to investigate the liver specific silencing of gene combinations in order to address metabolic syndrome. We have shown that a single formulation containing siRNAs targeting up to 10 different genes or a single siRNA targeting two separate genes are effective at silencing each gene. Silencing the combination of PCSK9 and a second gene of interest believed to be involved in lipid metabolism with a single dual targeting siRNA lead to efficient lowering of total cholesterol in mice.

(A) A proprietary algorithm based on Alnylam’s large historical database of sequences is used to help choose highly active and specific siRNAs. Designed siRNAs have at least 3 mismatches with all other transcripts in the target species transcriptome. The top most likely off-target genes (those just below the threshold cut off on the list) are tested for silencing. There is no silencing of transcripts containing 3 mismatches to the selected siRNA sequence even at multiple log order higher siRNA concentrations than the IC50 for the target gene.(B) A number of internal chemical modifications (2’OMe, 2’ F among others) are utilized to avoid siRNA stimulation of immune responses. Chemically modified siRNAs do not induce cytokine responses in hPBMCs from 13 different donors across a panel of 7 cytokines: IFN-α, IP-10, IFN-γ, TNFα, IL-6, IL-1ra, G-CSF compared to positive scoring controls.

Fig 2. Off-Target Silencing and Cytokine Induction by PCS-B2 siRNA

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Fig 8. Silencing Multiple Genes by a Single Dosing (Mice)

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Patients with metabolic syndrome by definition have multiple disease parameters, and their disease may need to be controlled by modulating multiple pathways or targets. Newer generation formulations allow targeting multiple targets/pathways using several RNAi therapeutics formulated together: 5 genes (A) and 10 genes (B) are simultaneously silenced. Formulated Luciferase siRNA was used as a control.

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Fig 6. Silencing of PCSK9 in LDLR -/+ Transgenic Results in Total Cholesterol Lowering (Mice)

There remains a large unmet need for treatment of hypercholesterolemia in patients that are heterozygous for the LDLR gene. These individuals have one mutant and one wt copy of LDLR and as a result, have significantly elevated LDLc levels and higher incidence/risk of cardiovascular evens. Given that PCSK9 is proposed to work through up regulation of wt LDLR we investigated whether silencing of PCSK9 in LDLR heterozygous mice (which have high total cholesterol) would significantly decrease their total cholesterol. ED50 of PCSK9 transcript silencing in LDLR heterozygous mice appeared to be very similar to that in Wt mice. PCSK9 silencing in LDLR heterozygous mice results in phenotypic changes, specifically, a lowering of total serum cholesterol, indicating that a single wt copy of LDLR is sufficient for the PCSK9 mechanism to be effective.

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Fig 7. LNP Dosing Regimens Can Be Optimized (Rat)

RNAi therapeutics with a single bolus dose of 3mg/kg (of an earlier generation formulation) followed by weekly injections of 0.3mg/kg (10% of the original dose) are able to maintain lower total cholesterol levels over time.

Rats were bled one day prior to repeated dosingMaintenance: 1x wk Initial

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Fig 1. RNAi Therapy for Hypercholesterolemia

Hypercholesterolemia/PCSK9 Well validated target Gain and loss of function mutations • Rodent and Human (gf) and (lf) mutations • PCSK9 expressed in liver (delivery) • Early clinical markers of activity possible (PCSK9, LDLc, ApoB)

Alnylam�PCSK9�program Discovery of pM active PCSK9 siRNAs Formulations or conjugates for delivery Proof of concept – Multiple rodent models, NHP’s Collaboration with UT Southwestern – Horton, Hobbs, Brown and Goldstein

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(A) PCSK9 is an exceedingly well validated target for Low Density Lipoprotein cholesterol lowering. Genetic evidence in Mice, and Humans indicates that increased levels of PCSK9 lead to increased cholesterol levels and lower levels of PCSK9 lead to lower LDLc levels.(B) The transformation of synthetic siRNA into therapeutics is a step-wise process of sequential optimization and chemical modification. Our philosophy is to create safe and efficacious therapies by incorporating the minimal number of modifications needed.

Fig 3. Lipid Nanoparticles (LNP) for Systemic RNAi in Liver

Multi-component lipid formulation – Cationic lipid – Structural lipid – PEG lipid – Cholesterol Highly efficient for liver delivery – Hepatocyte-specific gene silencing achieved

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(A) Lipid containing formulations encapsulating PCSK9 siRNA and directing a large percentage of the injected dose to liver are used as delivery vehicles.(B) Multiple novel lipid nanoparticle compositions demonstrate dramatic (50-100 fold) potency improvements in gene silencing as compared to first generation LNPs (Factor VII gene silencing is utilized as a model system).(C) Highly active LNPs display ~ single digit ug/kg ED50.(D) The 2nd generation of lipid nanoparticle formulations of PCS-A2 siRNA demonstrates PCSK9 silencing and total cholesterol lowering in rats with ED50 ~0.1mg/kg.

Fig 5. PCSK9 Non-Human Primate Study with 2nd Generation LNPs

N=4/group30 minute infusion on dosing day 1Day 3 ultrasound guided liver biopsies for PCSK9 transcript Days -3, -1 pre-dose and 3, 4, 5, 7, 9,11,12 post-dose blood collectionsDirect measurements of blood PCSK9 protein, LDLc, HDLc

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(A) Treatment with LNPX-PCS-B2 results in dose dependent PCSK9 transcript silencing.(B) PCSK9 transcript silencing leads to rapid and durable PCSK9 protein reduction that translates into >50% lower LDLc levels in a dose dependent manner. These effects were found to be very potent, with ED50 dose levels between 30 and 100 micrograms per kilogram.(C) Silencing of PCSK9 in NHP with an RNAi therapeutic does not result in HDLc lowering.

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Fig 4. Silencing of PCSK9 LDL Particle Number Lowering in hCETP/hApoB100 Transgenic Mice

Rodents are poor models of human LDL and HDL cholesterol metabolism and we have therefore utilized transgenic mice with human–like lipoprotein profiles that overexpress human CETP and ApoB100. Silencing of PCSK9 in this model (A) results in dramatic decrease of LDL, but not HDL particle number (B).

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Simultaneous targeting of two genes with a single siRNA (or dual targeting construct). The dual targeting construct is as potent as the individual siRNAs targeting the genes in vitro (A) and efficiently silences both intended target genes in vivo (B). Gene X is a novel target thought to be involved in cholesterol metabolism and whose mechanism of affecting cholesterol should be additive or synergistic with PCSK9. The dual targeting construct significantly lowers cholesterol in mice (C).

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rnai therapeutics targeting pcsk9 for ldlc lowering in ... · rnai therapeutics targeting pcsk9 for...

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