NEW YORK (GenomeWeb) – Researchers from Alnylam Pharmaceuticals this week published a study describing the company's development of GalNAc conjugates, which Alnylam is using with all of its newest drug candidates to deliver siRNAs into the liver.
The technology is based on N-acetylgalactosamine ligands, which have a high affinity for the asialoglycoprotein (ASGPR) receptors expressed on the surface of hepatocytes. While the concept of using these ligands for drug delivery in not new, doing so has been a challenge.
But in recent years, Alnylam has had success conjugating siRNAs to GalNAc ligands and delivering them selectively to hepatocytes, so much so that the company has made GalNAc the go-to delivery system for nearly all of its drug candidates. In the process, Alnylam is leaving behind the lipid nanoparticle-based delivery approach that has long been used by virtually every RNAi drug developer.
In a paper appearing in the Journal of the American Chemical Society, Alnylam scientists along with academic collaborators detailed the development of the firm's GalNAc-siRNA conjugates for subcutaneous delivery. They also highlighted their compatibility with solid-phase oligonucleotide synthesis, making their manufacture efficient and practical for therapeutic applications.
As noted in the JACS paper, a number of groups have reported the development of ASGPR ligand mimics, and Arrowhead Research scientists have demonstrated in vivo that cholesterol-bound siRNAs targeting hepatitis B virus could be delivered into hepatocytes and suppress their target when co-injected with GalNAc-melittin-like peptide conjugates. These molecules, which Arrowhead calls dynamic polyconjugates, are being used in its Phase II HBV drug ARC-520.
But two-component formulations such as dynamic polyconjugates, the Alnylam team wrote, are limited to intravenous administration. By conjugating its chemically modified siRNAs to a modified ASGPR ligand, however, Alnylam has been able to create RNAi molecules that are stable against nucleases, offer improved target knockdown compared with unconjugated siRNAs, and can trigger robust gene silencing after subcutaneous administration, according to the JACS report.
"Well-characterized bi- and triantennary GalNAc ligands were reengineered to facilitate covalent conjugation to siRNAs," they wrote. "Appropriately protected bi- and triantennary GalNAc monomers, which are compatible with solid-phase oligonucleotide synthesis and deprotection conditions, were synthesized." Meanwhile, a trans-4-hydroxyprolinol moiety was introduced in the design to enable site-specific conjugation of the ligand at any position of an oligo during solid-phase synthesis.
The researchers noted that the overall synthesis yield for their GalNAc conjugates was on par with those for standard oligonucleotides.
When tested in animal models, the Alnylam team found their GalNAc-siRNA conjugates capable of inhibiting their targets. However, refinement of the siRNA chemistry led to a five-fold improvement in efficacy in vivo, with a median effective dose of 1 mg/kg after a single dose.
This, they added, allowed for subcutaneous administration at therapeutically relevant doses at volumes at or less than 1 mL. Further supporting the clinical use of GalNAc conjugates was their apparent safety in rodents when administered weekly for over nine months.
On these and other data, Alnylam has pushed its GalNAc technology forward, and last month released Phase II data showing that its GalNAc-enabled TTR-mediated amyloidosis therapy revusiran could knock down its target protein by as much as 98.2 percent in patients. A Phase III trial remains on track to begin before year-end.
And in May, the company announced top-line Phase I data showing its hemophilia treatment ALN-AT3, which uses an enhanced version of the GalNAc technology, could silence its target after a single dose.
Alnylam and Arrowhead aren't the only firms employing GalNAc conjugates for delivery. Regulus Therapeutics, a spinoff of Alnylam and Isis Pharmaceuticals developing microRNA-targeting drugs, has a license to the technology and is using it with its hepatitis C therapy RG-101. In October, Regulus released better-than-expected Phase II data on the drug, showing that it could reduce patient viral loads on par with current treatments after a single dose.
And Dicerna Pharmaceuticals recently disclosed that it is working on its own GalNAc conjugate technology, which it expects to employ in upcoming liver disease programs.