Divalent siRNA for prion disease | JoVE Visualize

Area of Science:

Neuroscience
Oligonucleotide therapeutics
Prion disease research

Background:

Prion protein (PrP) lowering is a validated strategy for prion disease treatment in animal models and is under clinical investigation.
Existing PrP-lowering drug candidates require enhancement in potency and duration of action for improved therapeutic efficacy.
Divalent short interfering RNA (siRNA) represents a novel oligonucleotide modality for central nervous system (CNS) drug development.

Purpose of the Study:

To discover and develop novel, potent PrP-lowering drug candidates using a divalent siRNA approach.
To identify and characterize effective siRNA sequences targeting human and mouse PrP.
To evaluate the therapeutic potential of lead siRNA candidates in preclinical models of prion disease.

Main Methods:

Generation of transgenic mouse lines (Tg25109, Tg26372) expressing human PRNP for in vivo screening.
Design and synthesis of divalent siRNA molecules targeting mouse and human PrP, including optimization of chemical scaffolds (e.g., scaffold s4).
Assessment of PrP lowering efficacy in wild-type and prion-infected mice, including survival studies and pharmacokinetic analyses following cerebrospinal fluid (CSF) delivery.
Conducting Good Laboratory Practices (GLP) toxicology studies and preparing for clinical trials.

Main Results:

A mouse Prnp-targeting divalent siRNA, 1682-s4, reduced brain PrP to 49% residual expression and increased survival time by 2.7-fold in RML prion-infected mice with pre-symptomatic treatment.
A human PRNP-targeting siRNA, 2439-s4, demonstrated significant potency, reducing human PrP to 17% residual levels with a single dose in transgenic mice.
Scaffold optimization, specifically the fixed UU tail and extended nucleic acid linkages in scaffold s4, enhanced PrP knockdown by up to 15.9 percentage points.
CSF delivery of 2439-s4 showed efficient brain retention (1-2% of dose), with a median effective tissue concentration of 1.2 µg/g.
GLP toxicology studies revealed no significant liabilities, and an Investigational New Drug (IND) application for 2439-s4 was cleared by the U.S. FDA.

Conclusions:

Divalent siRNA molecules, particularly 2439-s4, represent a highly potent and promising new class of PrP-lowering therapeutics for prion diseases.
The developed transgenic mouse models are valuable tools for in vivo discovery of human PRNP-targeting oligonucleotides.
The favorable preclinical data and FDA IND clearance position 2439-s4 for clinical evaluation in human prion disease patients.