Complexes cross BBB, knock down CNS genes in rodents | 2021-08-18

A Japanese study has discovered a new means of regulating endogenous gene expression in the CNS, systemically administered antisense oligonucleotides (ASOs) in rodents, which facilitates the development of ASO-based therapies for patients with neurological disorders that require prolonged treatment.

The study showed that cholesterol-functionalized DNA / RNA heteroduplex oligonucleotides (Chol-HDOs) can cross the blood-brain barrier (BBB) ​​and destroy genes in the CNS of rats and mice, the authors reported in the issue of August 12, 2021 of natural biotechnology.

“This is the first study to show that Chol-HDOs can cross the BBB and turn off genes in the central nervous system of rodents,” said study leader Takanori Yokota, professor and chair of the Department of Neurology and Neurological Science at Tokyo Medical and Dental University.

“This has a major impact on the management of neurological conditions that require long-term ASO treatment, including Alzheimer’s disease (AD), Parkinson’s disease, amyotrophic lateral sclerosis (ALS), multiple sclerosis, stroke and epilepsy,” Yokota told BioWorld Science.

For example, in patients with such CNS disorders, a lumbar puncture / intrathecal injection of ASO could be given to rapidly achieve therapeutic levels and regulate endogenous CNS gene expression, followed by maintenance therapy with intravenous or subcutaneous Chol HDO injection.

Single-stranded ASOs

Recently, single-stranded ASOs have been extensively studied for the treatment of CNS disorders. For example, the ASO Spinraza (Nusinersen; Biogen Inc.), which is approved for the treatment of spinal muscular atrophy, has been shown to modulate the splicing of the SMN2 gene pre-mRNA.

However, single-stranded ASOs cannot normally enter the CNS after systemic administration and must be injected intrathecally directly into the CSF.

“Because they are highly polar, when administered systemically, ASOs generally do not enter brains with intact vascular barriers without treatments that open the BBB,” noted Yokota.

In clinical practice, Spinraza intrathecally was well tolerated by most patients, but may be difficult to use in people with severe lumbar scoliosis or in combination with anticoagulant therapy.

Therefore, an effective method of delivering ASOs from the blood to the brain would be clinically beneficial.

An oligonucleotide-based drug used to regulate gene expression in the CNS after systemic injection must be able to cross both the BBB and blood CSF barriers, which normally only small hydrophobic molecules can penetrate.

However, several recent strategies for the delivery of oligonucleotides into the CNS have not proven effective.

For example, studies have shown that delivery of DNA / RNA-HDO technology to the brain was negligible, although it has been shown to enable efficient RNA breakdown in the liver of mice and non-human primates when injected intravenously.

The authors of the new Nature Biotechnology study therefore tried to address this limitation by screening several lipid ligands that can conjugate with HDOs at a higher dose than in previous protocols.

“Screening about 60 different lipid ligands found that DNA / RNA HDOs conjugated to cholesterol or alpha-tocopherol could reach the CNS after subcutaneous or intravenous administration in rats and mice,” Yokota said, noting that “this would allow patients to do so to avoid the need. ” for invasive lumbar puncture. “

The HDOs were distributed in the brain, spinal cord and peripheral tissues and suppressed the expression of four target genes in the CNS by up to 90%.

“The HDO distribution in the spinal cord was slightly higher than that in the cerebellum, with the greatest suppression observed in the long non-coding RNA of metastatic lung adenocarcinoma transcript 1 (Malate-1),” said Yokota, noting that single-stranded ASOs conjugated to cholesterol showed only limited activity in this regard.

In addition, gene knockdown was observed in key CNS cell types and was greatest in neurons and microglial cells, “suggesting that this approach may be most effective in neuroinflammatory diseases such as AD and ALS,” said Yokota.

A potential safety problem in the treatment of diseases whose pathogenesis is primarily confined to the CNS is that the biodistribution of Chol-HDO can be greater in peripheral tissues than in the brain, potentially causing adverse side effects.

However, while the doses required to observe target knockdown in the CNS with Chol-HDO were high, the compounds were generally well tolerated. Side effects such as thrombocytopenia and focal cerebral necrosis could be reduced by subcutaneous administration or by dividing the intravenous injection doses.

Together, these results demonstrate that by effectively traversing the BBB, Chol HDOs can overcome the limited effectiveness of CNS-targeting ASOs without the need for invasive intrathecal administration.

Looking ahead, Yokota said, “we will work to increase potency, which includes ligand optimization of cholesterol and tocopherol derivatives and identification of the specific molecular pathways that underlie CNS delivery.”