The Delivery Dilemma
The synthesis and purification of double-stranded RNAi compounds presents some unique challenges. Most evident is the need to manufacture two separate strands of RNA (and the time and cost required to do this) and allow them to anneal. RNAi duplexes represent a thermodynamically unstable system. In general, RNA is less stable than DNA and at greater risk for contamination on contact with glassware and instrumentation.
Dr. McCormac points to several other obstacles: a less developed raw material supply chain; more expensive building blocks; more complicated chemistry for the isolation processes; enhanced chemical hazard management on scale-up; and more challenging purification processes that require higher pressure chromatography.
The greater complexity of RNA synthesis compared to DNA rests largely on the need for a second deprotection step to reveal the extra hydroxyl group in the sugar.
Aptamers typically range from 25–50 nucleotides in length. They behave similar to mAbs and can exert a therapeutic effect without entering the cell. Aptamers are challenging molecules to synthesize because they are highly structured, with well-defined 2-D and 3-D structures. Additionally, their lengths push the resolution limits of traditional ion exchange chromatography, yielding multiple peaks on chromatographic purification. They also tend to require longer deprotection times.
With the exception of aptamers and immunostimulatory oligos, which do not need to get inside cells to exert a therapeutic effect, delivery of oligo-based drugs to their targets remains a significant problem.
“An unresolved, important question is how to get these molecules into cells,” says Dr. Ayguen of BioSpring. “This is a bottleneck for the whole technology.” Solving this problem would dramatically change the therapeutic oligos business, as it would provide “a platform technology to develop a broad spectrum of drugs and activities.”
“Oligonucleotide therapeutics have always been a good idea,” says Dr. Sproat, “people just didn’t take the delivery issue seriously enough.”
“Nobody has yet cracked systemic delivery,” says James Powell, general manager of the nucleic acid solutions division at Agilent. This has led to a growing focus on local delivery, including topical, intraocular, and inhaled delivery mechanisms, as well as injection of anticancer oligos directly into tumors. Delivery work with nanoparticles and lipid formations is ongoing.
Innovative clinical delivery strategies are contributing to more diverse chemistry, “which requires more flexible manufacturing assets to deal with different downstream chemistry and purification,” says Dr. McCormac, citing examples such as pegylation, RNA cleavage, duplexation, and high pressure/high temperature purification.
A potential side benefit of novel delivery mechanisms might be a reduced need for modified oligos, enabling the use of more natural-looking DNA and RNA in drug formulations.
DNA Therapeutics (www.dna-therapeutics.com) is focusing its efforts on developing oligonucleotide-based therapeutics called short-inhibiting DNA, or siDNA, which interfere with DNA damage-repair mechanisms in cells. The company’s lead compound, Dbait, mimics a DNA double-strand break to “divert and disorganize” the repair system responsible for recognizing and fixing DNA damage on chromosomes, explains Jian-Sheng Sun, Ph.D., chairman and CEO of DNA Therapeutics. The intent of treatment is to compromise the ability of cancer cells to repair the chromosomal damage inflicted by traditional anticancer therapies.
Following up on MRI and cytologic analysis showing that Dbait enhances irradiation-induced necrosis and apoptosis, the company describes molecular studies demonstrating that Dbait specifically activates DNA-PK, a protein kinase that phosphorylates H2AX, an isoform of the histone H2A after exposure of a cell to ionizing radiation. Phosphorylated H2AX (or gamma-H2AX) in a cell nucleus indicates a focus of DNA repair activity to mend double-stranded breakpoints. The distribution of gamma-H2AX in cells transfected with Dbait differs substantially from that observed in irradiated cells without Dbait, leading the company to conclude that Dbait acts by “baiting” and “hijacking” enzymatic repair complexes and causing disorganization of the repair system.
Dbait is a long, single-stranded hairpin oligo measuring 64 nucleotides and, notes Dr. Sun, the main “challenge in GMP manufacturing and scale-up is process development to ensure maximal yield.” Improvements in process development, such as the use of multiple-nucleotide building blocks instead of single nucleotides, “could help increase the overall yield and thus lower manufacturing costs,” says Dr. Sun. As for any oligo therapeutic that has to penetrate cells to exert its effects, including antisense and siRNA drugs, siDNA compounds must also overcome delivery issues, which can impact formulation strategies.
DNA Therapeutics is first focusing on local delivery to serve the interventional oncology field and will then pursue systemic and targeted delivery. The company’s clinical development strategy is to first demonstrate the efficacy of its Dbait technology in indications where standard of care treatment has failed to control cancer progression, and then to focus on other segments of the oncology market.