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UMMS Researchers Show How RNAi Delivery Technology Can Cut ApoB Levels In Vivo

Researchers from the University of Massachusetts Medical School have systemically delivered nanoparticle-siRNA complexes into mice to silence a gene encoding apolipoprotein B, a protein associated with low-density lipoprotein production and cholesterol metabolism, according to a recent paper.
Delivered at relatively low doses, the so-called interfering nanoparticles were also able to reduce levels of apoB and total cholesterol in plasma without triggering an interferon response.
“Our results show that these [complexes] can silence disease-related endogenous genes in clinically acceptable and therapeutically affordable doses,” the investigators wrote in the paper, which appears in this week’s online version of ACS Chemical Biology.
The data also mark a key milestone for RXi Pharmaceuticals, a subsidiary of CytRx, which exclusively licensed the interfering nanoparticle technology in January from UMMS for all therapeutic applications.
RXi, which calls the technology nanotransporters, has said it aims to advance RNAi therapeutic technology that its parent firm had in recent years put on the backburner in favor of more advanced small-molecule drugs and a DNA-based HIV vaccine (see RNAi News, 1/11/2007).
RXi President Tod Woolf told RNAi News in an e-mail this week that the firm expects to use the nanotransporters in its systemic RNAi programs, and said they will “fit in well” with the company’s metabolic disease program, which is focused on diabetes and obesity.
Currently, RXi has not publicly disclosed when this project, its lead program in amyotrophic lateral sclerosis, or a proposed cancer effort will move into phase I testing, Woolf wrote.
Woolf added that “given the long timeline of approval of diabetes and obesity drugs, we will likely be looking to partner this program with a large pharma company.”
Delivery Quandary
Researchers have taken major strides in recent years to overcome the delivery hurdle facing potential RNAi-based therapeutics.
For example, Protiva Biotherapeutics’ stable nucleic acid lipid particles, or SNALPs, have proven quite promising in preclinical studies as vehicles for siRNAs (see RNAi News, 7/29/2005 and 3/30/2006) — so much so that the technology is at the center of a heated legal battle over its ownership (see RNAi News, 3/22/2007).
In 2004, Alnylam Pharmaceuticals published data showing that cholesterol-conjugated siRNAs targeting apoB delivered intravenously into mice could cut apoB mRNA levels in the liver and jejunum, decrease plasma levels of apoB protein, and reduce total cholesterol (see RNAi News, 11/11/2004).
Nonetheless, “new chemistry and approaches are greatly needed to systemically silence disease-causing genes in a tissue-specific manner with high efficiencies and at clinically achievable doses,” the UMMS researchers wrote in ACS Chemical Biology, citing the large amounts of siRNA used in the Alnylam study and the sophistication required for SNALP assembly.
“We reasoned that an ideal delivery vehicle for siRNA should have at least three functions: to efficiently assemble siRNA; to be non-immunogenic; and to provide functional groups for covalent attachment of tissue-specific moieties and for modulation of pharmacological properties for future investigations,” they added.
To meet these requirements, the UMMS researchers constructed lysine-containing nanoparticles, conjecturing that the use of natural amino acids would make the nanoparticles less toxic and non-immunogenic. “In addition, amino acid-based synthesis of nanoparticles allows better control over specific synthetic steps to modulate physical and pharmacological properties of the nanomaterials,” they wrote.

“Our results show that these [complexes] can silence disease-related endogenous genes in clinically acceptable and therapeutically affordable doses.”

To enhance in vivo uptake, the investigators also modified the surface functional groups with lipid chains, while the siRNA component of the molecule was chemically modified using rules they had established in previous studies.
After designing the nanoparticle’s siRNA payload against apoB, the UMMS researchers administered the drug to mice via tail-vein injection. Doses of siRNAs ranged from 1 mg/kg to 5 mg/kg.
An analysis of liver tissue revealed that apoB levels were significantly reduced in animals treated with the siRNA-nanoparticle complex versus controls. “The maximum silencing effect of [the RNAi agent] was reached at 1 mg/kg,” the researchers wrote. For reasons unknown, increasing the siRNA dose did not boost in vivo RNAi efficiency.
Further study revealed that nanoparticle treatment cut plasma apoB levels more than 70 percent compared with controls, including at a level of 1.25 mg/kg, “a clinically feasible dose for RNAi therapeutic applications,” they added.
“To investigate the physiological effects of apoB mRNA silencing on cholesterol metabolism, we measured total plasma cholesterol levels in mice 24 [hours] after the final injection,” the researchers stated. While mice receiving control treatments or nanoparticles with mismatched siRNAs experienced no changes in cholesterol levels, treatment with the active siRNA-nanoparticle led to a significant reduction of total cholesterol.
Histological analyses revealed no differences between mice treated with the siRNA-nanoparticle complex and those receiving control treatments, demonstrating that the RNAi therapy “did not induce an immune response … and caused no apparent toxic effects,” they added.
“Together, these findings demonstrate that … targeting of apoB [with the siRNA-nanoparticle technology] could provide a clinically significant new approach to reducing cholesterol levels in patients with hypercholesterolemia,” the researchers concluded. Furthermore, the delivery technology could be used to administer therapeutic siRNAs for a variety of other indications in affordable and clinically acceptable doses.

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