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Technology Overview

Intradigm’s RNAi Nanoplex (NPX) Delivery Technology

The challenges of systemic RNAi delivery are well documented and include numerous biological barriers that limit an RNAi therapeutic’s ability to reach its “target.”  These include:

• Rapid urinary excretion via the kidney due to the molecule’s small size
• Rapid enzymatic degradation in the serum and tissues
• Non-specific tissue distribution
• Challenges of intracellular uptake upon reaching the target cell
• Challenges of endosomal escape to achieve release of siRNA into the cytoplasm for incorporation into RNA-Induced Silencing Complex (RISC) and gene silencing

Intradigm is dedicated to solving the systemic delivery challenges related to RNAi therapeutics.  The company’s RNAi NPX delivery technology is a modular, multi-component delivery vector that carries active siRNA molecules in its core with the flexibility to attach a Polyethylene Glycol (PEG) layer and/or a targeting ligand to the polymer-siRNA core to improve its pharmacological properties.  The flexibility of Intradigm’s RNAi NPX delivery technology allows the company to build a broad range of proprietary RNAi therapeutic candidates, known as RNAi NPXs.

There are three components that can be used to build a proprietary RNAi NPX:

1.	The core component consists of the negatively charged active siRNA sequences that are designed to “silence” one or more disease causing gene product(s).  These siRNA sequences are complexed through electrostatic bonds with a cationic polymer to form a proprietary RNAi NPX.  Through its work with its RNAi NPX delivery technology, Intradigm has examined and evaluated multiple polycationic polymers.  The company’s current pre-clinical candidate incorporates a peptide-based, chemically homogeneous, and fully biodegradable polymer (PolyTran™) designed to facilitate cell internalization and endosomal release of its siRNA payload in the cytoplasm.
2.	A hydrophilic steric polymer can be added to the RNAi NPX usually in the form of a Polyethylene Glycol (PEG) layer. This component helps reduce non-specific tissue interaction, increase circulation time, and minimize immunogenic potential.  At the same time, this PEG layer can also enhance siRNA distribution to tumor tissue through the phenomenon of Enhanced Permeability and Retention (EPR) in the often leaky tumor vasculature.
3.	A binding ligand can be attached to guide the RNAi NPX to receptors expressed in the specifically targeted disease tissue.  In addition to enhancing selective tissue distribution, these ligands permit a receptor-mediated uptake by the cells of interest.  Targeting ligands are readily interchangeable depending on the disease and siRNA payload. The company is working on multiple ligands with its initial pre-clinical candidate incorporating an RGD (Arginine, Glycine, Aspartic Acid) peptide ligand that binds to activated integrins on tumor vasculature endothelial cells, particularly αvβ3 integrins.

Therapeutic Activity of the RNAi Nanoplex Delivery Technology

Intradigm believes that its RNAi NPX delivery technology is one of the most versatile siRNA delivery technologies currently in development.  The company anticipates that the RNAi NPX delivery technology will function as outlined below after systemic administration to a patient:

Step 1: Sequester and Protect Active siRNA Molecule
The active siRNA molecules contained in the RNAi NPX are protected from degradation in the blood stream and from renal elimination by the creation of a roughly spherical nanoparticle in which the nucleic acid is sequestered and which are large enough (~100nm) to prevent renal elimination. The addition of a hydrophilic polymer to the polycation/siRNA core may also improve the pharmacokinetic and biodistribution properties of the RNAi NPX.

Step 2: Binding of the RNAi NPX to the Targeted Tissue and cell internalization
Accumulation of the RNAi NPX in target tissue occurs through specific (ligand-mediated) interaction with the ligand receptors on the surface of the target cells. Cell internalization is facilitated by receptor-mediated endocytosis but also occurs through non-specific adhesion to the cell surface facilitated by the positive charge of the RNAi NPX.

Step 3: Release of siRNA Molecule from Polycation Nucleic Acid Carrier into Cytoplasm of Target Cell
The polycation nucleic acid carrier has been selected to facilitate the release of the active siRNA molecules into the cytoplasm of the target cells by exploiting the lower pH in the endosomal environment.  

Step 4: Silencing of Target Gene
The introduction of the siRNA molecule into the cytoplasm of the cell triggers the cell’s natural RNAi process through which an endogenous gene-silencing mechanism called RISC (RNA-Induced Silencing Complex) is activated.  RISC is a protein complex that, upon encountering the double-stranded siRNA, unwinds it leaving only the antisense strand.  The RISC complex then binds to the siRNA antisense strand and proceeds to automatically silence messenger RNA (mRNA) that it recognizes as having the complimentary sequence.  The process is auto-catalytic in that the RISC complex is capable of “silencing” multiple copies of the same mRNA. By silencing the undesirable mRNA, the cell’s natural RNAi process inhibits the activity of the targeted disease gene. The siRNA sequences used in an RNAi NPX are carefully selected to maximize their impact on the target gene mRNA thereby improving their efficacy and simultaneously limiting off-target toxicity.