Researchers from Alnylam Pharmaceuticals and the Massachusetts Institute of Technology this week published data showing how they used a novel class of lipid-based molecules to systemically deliver siRNAs and microRNA-targeting antisense oligos into a variety of tissue types in multiple animal models.
The data, which appears in the advance online version of Nature Biotechnology, show that the molecules, called lipidoids, can be used to trigger potent, specific, and long-lasting effects on gene expression in the liver, lung, and peritoneal macrophages, Alnylam said. The molecules were tested in mice, rats, and non-human primates.
“It is clear that more efficient delivery of RNAi therapeutics will require the discovery of new biomaterials and formulations,” Daniel Anderson, an MIT researcher and Alnylam collaborator, said in a statement. “We believe the lipidoids we have developed greatly expand the collection of available delivery materials and … hold significant promise as delivery agents for both siRNA and miRNA therapeutics.”
As first reported by RNAi News, an Alnylam official said last year that the company plans to use the so-called lipidoid delivery technology in both its hypercholesterolemia and liver cancer programs (see RNAi News, 6/28/2007). This week, however, a company spokeswoman told RNAi News that the firm has not “provided guidance on the specific formulations” that will be used with the two drug candidates.
In the Nature Biotechnology paper, the investigators noted that although efforts are ongoing to optimize lipid-based delivery systems for siRNAs, material synthesis remains problematic.
“Conventional lipid synthesis typically requires individually optimized, multiple-step synthesis, including time-intensive procedures such as chemical protection and deprotection, use and removal of catalysts, solvent exchanges, and purification,” they wrote. “The customization of each synthetic reaction and the multiple steps required limit throughput and, correspondingly, limit the ability to generate substantial library size and diversity.”
To address these issues, the investigators developed a method to rapidly synthesize the lipidoids based on the conjugate addition of alkyl-acrylates or alkyl-acrylamides to primary or secondary amines.
“This particular chemistry, unlike many traditional lipid synthesis chemistries, allows for reactions in the absence of solvent or catalysts, results only in lipidoid product, and thereby eliminates the need for protection and deprotection steps, purification, or concentration steps,” according to the paper.
“We believe the development of this library of lipid-like materials represents an important expansion of the diversity and collection of intracellular delivery materials. This one-step synthetic scheme enables the straightforward parallel generation of large libraries of delivery material.”
The research team used this method to create libraries of over 1,200 lipidoids, which were screened for their ability to deliver siRNAs to a HeLa cell line that expresses firefly and Renilla luciferase. Top-performing lipidoids were then examined in vitro in a human liver cancer cell line and primary bone marrow-derived mouse macrophages, and found to be “relatively non-cytotoxic” at effective concentrations.
To examine the therapeutic potential of the lipioids, the researchers conducted a screen to identify ones that facilitate high levels of siRNA-mediated gene silencing in hepatocytes. Seventeen of the most effective lipidoids were formulated to deliver siRNAs against the blood-clotting protein Factor VII.
When delivered to mice, seven of the lipidoid/siRNA formulations were found to cause “significant reduction of serum Factor VII protein levels,” with one cutting levels of the factor by more than 90 percent.
In rats, treatment with a single, intravenous injection of Factor VII-targeting siRNAs formulated with lipidoids led to dose-dependent reductions in liver Factor VII mRNA levels with 40 percent, 80 percent, and more than 90 percent silencing at 1.25 mg/kg, 2.5 mg/kg, and 5 mg/kg dose levels, respectively.
“As would be expected, significantly reduced serum Factor VII levels produced a phenotypic effect in the treated animals,” namely impaired clotting. This effect was determined to be specific and not attributable to the delivery vehicle since no clotting impairment was observed in control animals.
In an effort to explore the versatility of the lipidoids, the investigators tested the local administration of formulated siRNAs to the lung following intranasal administration in a mouse model of respiratory syncytial virus.
Alnylam is developing a siRNA-based treatment for RSV infection, which recently entered its second phase II trial (see RNAi News, 4/10/2008).
“In separate experiments, we confirmed the absence of gene silencing in the liver and kidney after local pulmonary administration of the formulation,” the paper’s authors wrote, adding that treatment with lipidoid-formulated siRNAs led to a greater than two log reduction in viral plaques compared with a roughly one log reduction with naked siRNAs.
The investigators also tested whether lipidoids could be used to deliver single-stranded 2’-O-methyl oligos targeting miRNAs. Lipidoid-formulated antagomir targeting miR-122, delivered intravenously into mice daily at 5 mg/kg on three consecutive days, led to greater silencing of the target miRNA than a cholesterol-conjugated version of the same oligo administered at 80 mg/kg for three days.
Control oligos did not have any effect on miR-122 levels.
Lastly, the Alnylam and MIT team evaluated the ability of lipidoids to deliver siRNAs against apolipoprotein B in non-human primates.
Cynomolgus monkeys were treated with a single IV injection of lipidoid-formulated siRNAs targeting apolipoprotein B at doses of 2.5 and 6.25 mg/kg, according to the Nature Biotechnology paper.
“Silencing of apoB was observed in a dose-dependent manner, with maximal serum apoB reduction of up to 75 percent relative to pre-dose levels,” the investigators wrote. “In contrast, no significant silencing was observed in … control groups.”
The greatest level of apoB silencing was observed three days after administration, and silencing of about 50 percent lasted two weeks.
In a follow-up cynomolgus monkey study using a lipidoid formulation optimized to maximize siRNA loading, treatment at doses of 2.5 mg/kg and 6.25 mg/kg silenced apoB up to 85 percent, corresponding to a maximal reduction in serum apoB protein of up to 74 percent relative to pre-dose levels.
No adverse effects were observed with this treatment.
“We believe the development of this library of lipid-like materials represents an important expansion of the diversity and collection of intracellular delivery materials,” the research team concluded. “This one-step synthetic scheme enables the straightforward parallel generation of large libraries of delivery material.”
Still, “further studies are warranted to investigate lipidoid-based delivery of RNA and other drugs to extend this technology for the broadest applications of RNAi therapy and drug delivery,” it added.