The complete mapping of the human genome yielded numerous novel targets for gene therapy, and led to the emergence of powerful technologies for the modulation of gene expression, such as small interfering RNA (siRNA), antisense oligonucleotides, and tools for DNA editing (e.g., CRISPR–Cas9). However, the major hurdle currently preventing the clinical implementation of these breakthroughs, and their translation into novel therapeutic strategies for numerous medical disorders, is the lack of adequate tools for the delivery of these oligonucleotide constructs across biological membranes into cells in the clinical setting. For example, siRNA is a large macromolecule (molecular weight (MW) of 13 kDa) that comprises numerous negative charges. The delivery of such molecules across hydrophobic phospholipid membranes requires huge activation energies. To date, despite large investments by large pharma and biotech companies, this delivery barrier has not been broken.
An electric field available within any phospholipid membrane
Aposense is a publicly traded Israeli biopharmaceutical company (APOS in the Tel Aviv Stock Exchange (TASE)) pioneering energizing drug delivery by means of a recently discovered, constitutive, and extremely powerful electrostatic field (10 –10 V/m) that resides in the depth of any phospholipid membrane. It is related to the membrane electric dipole potential, generated by the unique architecture of the phospholipid bilayers. Aposense develops first-in-class Molecular NanoMotors (MNMs) (Fig. 1a), which are novel small chemical entities (MW: ~800 Da) capable of ‘energy mining’ from the intramembrane electric field. Attached to a genetic drug, the MNM converts this electrostatic energy into kinetic energy and uses it to move within the membrane core, thus delivering the drug across the cell membrane into the cytoplasm.
The complete mapping of the human genome yielded numerous novel targets for gene therapy, and led to the emergence of powerful technologies for the modulation of gene expression, such as small interfering RNA (siRNA), antisense oligonucleotides, and tools for DNA editing (e.g., CRISPR–Cas9). However, the major hurdle currently preventing the clinical implementation of these breakthroughs, and their translation into novel therapeutic strategies for numerous medical disorders, is the lack of adequate tools for the delivery of these oligonucleotide constructs across biological membranes into cells in the clinical setting. For example, siRNA is a large macromolecule (molecular weight (MW) of 13 kDa) that comprises numerous negative charges. The delivery of such molecules across hydrophobic phospholipid membranes requires huge activation energies. To date, despite large investments by large pharma and biotech companies, this delivery barrier has not been broken.
An electric field available within any phospholipid membrane
Aposense is a publicly traded Israeli biopharmaceutical company (APOS in the Tel Aviv Stock Exchange (TASE)) pioneering energizing drug delivery by means of a recently discovered, constitutive, and extremely powerful electrostatic field (10 –10 V/m) that resides in the depth of any phospholipid membrane. It is related to the membrane electric dipole potential, generated by the unique architecture of the phospholipid bilayers. Aposense develops first-in-class Molecular NanoMotors (MNMs) (Fig. 1a), which are novel small chemical entities (MW: ~800 Da) capable of ‘energy mining’ from the intramembrane electric field. Attached to a genetic drug, the MNM converts this electrostatic energy into kinetic energy and uses it to move within the membrane core, thus delivering the drug across the cell membrane into the cytoplasm.
Figure 1: The Apo-Si amphibian autonomous vehicle. (a) The structure of the Apo-Si Molecular NanoMotor (MNM). (b) The structure of the Apo-Si amphibian autonomous vehicle loaded with siRNA.
Apo-Si: amphibian autonomous vehicle
Apo-Si is a platform technology for the delivery of genetic drugs, such as siRNA, into cells. The Apo-Si construct (Fig. 1b) comprises MNMs conjugated to a siRNA cargo drug, and it has two major functional features: it is amphibian, and it is autonomous.
The Apo-Si construct’s amphibian properties include the ability to travel effectively through aqueous environments, such as the blood, extracellular fluid, and cytoplasm, as well as through hydrophobic milieus, such as phospholipid cell membranes. Whereas movement through aqueous media is accomplished by diffusion or convection and is not associated with an energetic cost, owing to the highly hydrophilic nature of RNA, passage of the construct through lipid membranes is associated with a very large unfavorable energetic gradient. The MNM is unique in its ability to recruit this energy from the membrane, thus enabling transmembrane delivery of the cargo drug, without the need for additional external resources. This feature therefore renders the Apo-Si platform entirely autonomous.
Upon reaching the cytoplasm, a redox-sensitive detachment unit installed within the construct disengages the MNMs. Consequently, the genetic drug is liberated to exert its gene-silencing effects at the respective cytoplasmatic protein complexes: the Dicer endonuclease, and the RNA-induced silencing complex.
Performance profile of the Apo-Si platform
To date, the Apo-Si platform has successfully completed the major parts of its core development:
• molecular design and lead optimization;
• development of the synthetic chemistry for the production of the MNMs, the redox-sensitive detachment unit, and a dedicated click chemistry for rapid and efficient assembly of the Apo-Si construct with any oligonucleotide sequence;
• proof-of-concept studies substantiating the mechanism of action and its relation to the internal membrane electric field; and
• biological assessment in cell cultures in vitro, as well as in vivo after systemic administration.
Two major functional aspects of the Apo-Si construct were characterized: delivery into cells, and gene silencing. In the delivery aspect, the Apo-Si construct manifests robust delivery of siRNA across cell membranes and into target organs.
• Delivery in cell culture in vitro
• Delivery is universal into practically all examined cell types, including cell lines, primary cell cultures, cells of neuronal origin, and hematopoietic cells.
• Delivery is very rapid and highly efficacious into all cells in the sample.
• Delivery occurs in a low-nanomolar concentration range, in a dose–response manner.
• Delivery is not associated with signs of toxicity.
• Delivery after intravenous administration in vivo
• Bio-distribution studies in rodents revealed wide distribution of the Apo-Si construct across various organs, with subsequent gradual and slow clearance over time, and stability in the plasma—all prerequisite features of a systemically administered genetic drug.
• Gene silencing
• The Apo-Si construct provides gene silencing both in vitro in cell cultures and in vivo after intravenous administration.
• Silencing is achieved at low nanomolar concentrations, in a dose-responsive manner.
• Effective and specific silencing of both reporter and disease-related genes is achieved.
The Apo-Si amphibian autonomous vehicle therefore utilizes a unique opportunity, enabled by co-localization in the cell membrane, to address both a large unmet need (the delivery of macromolecule drugs) and a novel solution (a strong intramembrane electric field capable of energizing the delivery process). By providing an interface between these phenomena, the Apo-Si platform achieves the desired transmembrane delivery of genetic drugs. These hallmarks, combined with the compact, versatile, and modular structure of the MNM, may conceivably qualify the Apo-Si platform to serve as a future unified industry standard for the transmembrane delivery of genetic drugs, thus translating genetic therapeutics into a ‘druggable’ reality.