Nanoparticle Delivered siRNA
Calando Pharmaceuticals is developing a nano-based delivery system for siRNA, something that James Hamilton, M.D., CEO, said could enable siRNA as an entirely new class of therapeutics.
The company is in Phase I trials recruiting for initial compound CALAA-01. Dr. Hamilton claimed it is “the only systemically delivered siRNA in the clinic using a nanoparticle drug delivery system.” CALAA-01 targets the M2 subunit of ribonucleotide reductase.
Calando uses the same delivery system for multiple siRNA therapeutics, making it easy for the company to rapidly develop new drug candidates. A second siRNA therapeutic, CALAA-02, is in the preclinical phase and is expected to enter clinical trials in 2009. CALAA-02 targets hypoxia inducible factor-2 alpha, which is overexpressed in many cancers and is instrumental in tumorigenesis. This intracellular target has been difficult to inhibit with monoclonal antibodies or traditional therapeutics, according to Dr. Hamilton.
Calando employs a three-part delivery system called Rondel™ (RNAi Oligonucleotide Nanoparticle Delivery). The system mixes an excipient vial consisting of a linear, polycationic cyclodextin polymer backbone, a stabilizing agent, and a targeting ligand with a second vial containing anionic siRNA.
When combined, spherical particles measuring about 70 nm self-assemble, and protect the siRNA from degradation in serum. “The use of a modular excipient vial provides flexibility with regards to type of targeting ligand or type of oligonoucleotide payload,” added Dr. Hamilton.
Early studies indicate that the particles do not trigger an immune response, appear stable, and are nontoxic in animal studies. “Calando uses transferrin to target nanoparticles against transferrin receptors, which are often upregulated on the surface of a variety of tumor cell types. The binding of transferring facilitates endocytosis of the siRNA payload,” explained Dr. Hamilton. However, once the nanoparticle delivery system releases the siRNA inside the cell, the payload is highly specific for the gene knockdown target.
Preclinical mouse work showed marked tumor inhibition. For example, when athymic nude mice containing subcutaneous HT144 (human melanoma) tumors were treated with CALAA-01, tumor size was well under 500 cubic millimeters for the 31 days of treatment. In contrast, the tumors in mice treated with D5W grew only slightly slower than those of the untreated mice. Those tumors reached the 500 cubic millimeter size in about seven days, and continued growing.
“A second-generation Rondel system could include a wide variety of cellular targeting ligands,” said Dr. Hamilton. “Theoretically, using other targeting systems such as aptamers or Fab fragments would be easy to incorporate.” The therapy also has the potential to expand beyond oncology to target autoimmune inflammatory conditions and intraocular diseases.
Throughout the field, “there is considerable interest in multivalent drugs that can bind to multiple closely spaced binding sites on a target protein or protein multimer,” related Don Bergstrom, Ph.D., professor of medicinal chemistry at Purdue University’s Birck Nanotechnology Center. “The high avidity associated with such binding should result in increased drug potency. We are looking for efficient ways to construct such peptide multimers.”
His approach uses synthesized peptide-conjugated peptide nucleic acids synthesized on automated peptide synthesizers, yielding combined molecules in which the peptide portion contains a sequence that will bind a target, and the peptide nucleic acid contains a sequence that facilitates self-assembly. This work is at the early stages.
“We have constructed and characterized the complexes and have data showing their apparent uptake into MCF-7 cells, but no drug molecules have been tested.”
Dr. Bergstrom is working with Rashid Bashir, Ph.D., director of the micro and nanotechnology laboratory at the University of Illinois–Urbana-Champaign, which has constructed silicon-based field-effect nano-plate sensors for label-free electrical detection of binding. The group has covalently attached PNA to the sensors and is exploring thermally controlled hybridization of solution oligonucleotides to the sensors. Each sensor in the array can be individually heated by applying AC current.
Dr. Bergstrom said the particularly high affinity of LNA to PNA hybridization results in robust capture sequences. “The same PNA sequence is attached at every address and allowed to hybridize with the complementary inert protecting sequence. We are now working to control the heating so that only a single-heated nanoplate will undergo an exchange hybridization with a sensing sequence.”
The sensing sequence contains separate regions for binding PNA and miRNA, and allows the sequential attachment of different sensing sequences to different addresses.