Expanding RNA interference (RNAi) therapeutics from the lab to the clinic

It is now possible to deliver therapeutics based on short interfering RNAs to hepatocytes; however, new delivery solutions are necessary to target additional organs. In a new report now published in Nature Biotechnology, a team of researchers including Kirk M. Brown, Jayaprakash K. Nair, and Maja M. Manas, led by Vasant Jadhav at Alnylam Pharmaceuticals Cambridge MA, U.S., discussed the safe conjugation of 2'-O-hexadecyl (C16) to small interfering RNAs (siRNAs) for potent and durable silencing in target organs of rodents and non-human primates, with broad cell specificity. The experiments delivered sustained RNA interference activity for at least three months. The team observed intravitreal and intranasal administration, which relied on potent and durable knockdown. They investigated the preclinical efficacy of the siRNA targeting amyloid precursor protein via intracerebroventricular dosing in a mouse model of Alzheimer's disease, which ameliorated physiological and behavioral defects of the disease. The team showed the safety of C16 conjugation of siRNAs for therapeutic silencing of target genes outside the liver.

RNAi-based therapeutics

RNA interference (RNAi) therapeutics are based on an endogenous mechanism where short interfering RNAs (siRNAs) direct an RNA-induced silencing complex for genetic knockdown or genetic elimination. In this work, Brown et al built on nearly two decades of siRNA design and chemistry optimization to harness the RNA interference pathway in extrahepatic tissues, including the central nervous system, eye and lung. Patients with central nervous system diseases represent some of the highest unmet clinical needs with greatest therapeutic challenges, including gain-of-function mutations that make them suited for RNAi-based silencing. Researchers had recently conducted experiments with chemically modified siRNAs for potent silencing in preclinical models via an invasive intracerebroventricular administration approach, unsafe for repeated dosing in humans.

Biochemists are currently developing approaches to enable siRNA delivery across the blood brain barrier that are still in the early stages of discovery. Brown et al showed how the conjugation of 2'-O-hexadecyl (C16) can enhance the delivery and siRNA uptake onto the alveolar and bronchiolar epithelium. They combined a C16 lipophilic modification with chemically modified and metabolically stable siRNAs for efficient delivery to target organs for robust and durable gene silencing in rodents and non-human primates. The experiments provided a favorable safety profile to generate multiple candidates to investigate clinical safety and efficacy.

Brown et al carefully optimized the lipophilicity of chemically modified siRNAs to enhance the intracellular delivery of particles for broad and safe distribution. During the process, they harnessed the molecular position of the ribose sugar backbone to introduce the siRNA duplex and monitored RNA interference activity of siRNA conjugates in the central nervous system. The known siRNA design elements increased potency and specificity in the central nervous system to demonstrate the best activity after combining with vinyl phosphonate and C16, with up to 90 to 75 percent mRNA knockdown in the spinal cord and brain, respectively. The functional knockdown data of the conjugates showed superior brain biodistribution after a single injection in rats, compared to its unconjugated version.

Efficient siRNA delivery in the rodent central nervous system (CNS)

The bioengineers designed small interfering RNAs against cell-type-specific targets to understand the C16-siRNA uptake and RNA interference activity across major cell types in the central nervous system. Using immunohistochemistry staining, they noted robust knockdown in neurons, astrocytes and microglial cells to highlight how C16-siRNAs were effectively taken up by most cell types of therapeutic relevance in the central nervous system. Brown et al noted dose-dependent knockdown of a gene encoding the enzyme superoxide dismutase (Sod1) in the spinal cord and brain, and assessed the process for a duration of six months. They also examined dose-dependent silencing in the frontal cortex with an siRNA half-life for up to three to four months, with capacity to examine in a pharmacokinetic model.

The potential impact of small interfering RNAs (C16-siRNAs) in non-human primates

The team assessed the potency of the conjugated compound in non-human primates via an amyloid precursor protein, targeting small interfering RNA. The siRNA containing both vinyl phosphonate and C16 showed best activity across the central nervous system, without side-effects on the liver or kidney at 3-months of drug therapy. The team used cerebrospinal fluid samples to study the duration of silencing in non-human primates and showed how dosing variability led to the observation of a robust exposure response relationship across the analyzed brain regions. The team then tested the safety and tolerability of the drug dose in non-human primates via histopathological evaluations of regions in the central nervous system at different time points, post-drug administration.

Brown et al used transgenic mice with mutations specific to Alzheimer's disease to assess preclinical efficacy of the compound developed during research. The team showed how a single dose (60 µg) of the compound reduced the mutant protein levels in the ventral cortex and in cerebrospinal fluid, at a dose-variable timeframe. The scientists studied the dynamics of compound administration and used proton magnetic spectroscopy to determine decreased protein deposition and inflammation levels, to restore neuronal function. The team noted the reduction of specific metabolites in a CVN mouse strain, where the knockdown of specific genes normalized specific metabolites to bring it back to the same level of the wild type strain. The researchers further demonstrated the potent and durable activity of the compound in the eye and lung.

Outlook: Clinical treatment developments

In this way, Kirk M. Brown, Jayaprakash K. Nair, and Maja M. Manas and colleagues designed small interfering (siRNAs) for extrahepatic applications. The team combined lipophilic siRNA conjugates with additional design features for widespread distribution in the central nervous system of preclinical animal models via a clinically favorable safe dosing route. They conducted a proof-of-concept study in a preclinical mouse model to normalize behavioral deficits, with outcomes suited for clinical developments to treat early onset Alzheimer's disease and cerebral amyloid angiopathy. The conjugation strategy is also applicable in the eye and lung after local delivery. The team will conduct further studies to understand the precise mechanism behind the intracellular delivery of C16 for uptake into various cell types.