Gene Silencing in the Right Place at the Right Time
Authors: Anna Gutkin, Dan Peer
Journal: Molecular Therapy (2018)

The CNS in general and the brain, in particular, remain difficult to target for drug or gene delivery. This is mainly due to the presence of a highly restrictive barrier that lines the blood vessels within the brain, termed the blood-brain barrier (BBB). The BBB protects the brain from invading organisms and neurotoxins, regulates the uptake of essential nutrients, and also prevents the entrance of therapeutic agents.1Banks W.A. From blood-brain barrier to blood-brain interface: new opportunities for CNS drug delivery.Nat. Rev. Drug Discov. 2016; 15: 275-292Crossref PubMed Scopus (586) Google Scholar In recent years, there has been a tremendous effort to develop new methods for delivering different types of therapeutic agents to the brain but, unfortunately, without much success.1Banks W.A. From blood-brain barrier to blood-brain interface: new opportunities for CNS drug delivery.Nat. Rev. Drug Discov. 2016; 15: 275-292Crossref PubMed Scopus (586) Google Scholar In this issue of Molecular Therapy, Godinho et al.2Godinho B.M.D.C. Henninger N. Bouley J. Alterman J.F. Haraszti R.A. Gilbert J.W. Sapp E. Coles A.H. Biscans A. Nikan M. et al.Transvascular Delivery of Hydrophobically Modified siRNAs: Gene Silencing in the Rat Brain upon Disruption of the Blood-Brain Barrier.Mol. Ther. 2018; 26 (this issue): 2580-2591Abstract Full Text Full Text PDF Scopus (25) Google Scholar report the delivery of small interfering RNA (siRNA)-lipid conjugates following BBB disruption as a means to deliver a therapeutic payload. Their work combines gene silencing, BBB disruption using mannitol, and specific brain targeting strategies. The strategy may pave the way for more efficient and selective delivery of therapeutics to the brain. RNAi technologies, mainly siRNAs, have been widely studied as a therapeutic strategy to silence key genes in different diseases to reduce the expression of important proteins, including those considered "undruggable." On August 10th, 2018, the US Food and Drug Administration (FDA) approved the first ever RNAi drug, called Onpattro (Patisiran), to treat polyneuropathy in patients with hereditary ATTR amyloidosis, a potentially fatal condition that affects an estimated 50,000 people worldwide.3Adams D. Gonzalez-Duarte A. O'Riordan W.D. Yang C.C. Ueda M. Kristen A.V. Tournev I. Schmidt H.H. Coelho T. Berk J.L. et al.Patisiran, an RNAi Therapeutic, for Hereditary Transthyretin Amyloidosis.N. Engl. J. Med. 2018; 379: 11-21Crossref PubMed Scopus (1387) Google Scholar Nevertheless, siRNA faces multiple challenges for safe and effective delivery. These include protecting the siRNA from degradation in the bloodstream, avoiding rapid renal clearance, minimizing off-target effects, and limiting liver, kidney, and immune toxicity issues that could result in death.3Adams D. Gonzalez-Duarte A. O'Riordan W.D. Yang C.C. Ueda M. Kristen A.V. Tournev I. Schmidt H.H. Coelho T. Berk J.L. et al.Patisiran, an RNAi Therapeutic, for Hereditary Transthyretin Amyloidosis.N. Engl. J. Med. 2018; 379: 11-21Crossref PubMed Scopus (1387) Google Scholar To overcome these challenges, Godinho and colleagues2Godinho B.M.D.C. Henninger N. Bouley J. Alterman J.F. Haraszti R.A. Gilbert J.W. Sapp E. Coles A.H. Biscans A. Nikan M. et al.Transvascular Delivery of Hydrophobically Modified siRNAs: Gene Silencing in the Rat Brain upon Disruption of the Blood-Brain Barrier.Mol. Ther. 2018; 26 (this issue): 2580-2591Abstract Full Text Full Text PDF Scopus (25) Google Scholar conjugated siRNA to phosphocholine (PC) docosahexaenoic acid (DHA), the most abundant fatty acid in the brain. This allows the siRNA to be protected from degradation in the bloodstream, thereby enhancing circulation time and reducing renal clearance. Furthermore, such siRNA-lipid conjugates facilitate cellular uptake in neuronal cultures, promote mRNA silencing, and accumulate and distribute throughout the brain following local injection, as was previously shown by the same group. Systemic administration of this conjugate achieved only limited distribution to the CNS, which was restricted to the choroid plexus and the conjugate failed to cross the BBB.4Nikan M. Osborn M.F. Coles A.H. Biscans A. Godinho B.M.D.C. Haraszti R.A. Sapp E. Echeverria D. DiFiglia M. Aronin N. Khvorova A. Synthesis and Evaluation of Parenchymal Retention and Efficacy of a Metabolically Stable O-Phosphocholine-N-docosahexaenoyl-l-serine siRNA Conjugate in Mouse Brain.Bioconjug. Chem. 2017; 28: 1758-1766Crossref PubMed Scopus (27) Google Scholar, 5Nikan M. Osborn M.F. Coles A.H. Godinho B.M. Hall L.M. Haraszti R.A. Hassler M.R. Echeverria D. Aronin N. Khvorova A. Docosahexaenoic Acid Conjugation Enhances Distribution and Safety of siRNA upon Local Administration in Mouse Brain.Mol. Ther. Nucleic Acids. 2016; 5: e344Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar Various strategies to breach the BBB have been devised in the last decade. These include invasive approaches, such as intracranial or intraparenchymal injections, or non-invasive approaches, such as intravenous (i.v.) injection or intranasal administration.6Gutkin A. Cohen Z.R. Peer D. Harnessing nanomedicine for therapeutic intervention in glioblastoma.Expert Opin. Drug Deliv. 2016; 13: 1573-1582Crossref Scopus (36) Google Scholar siRNA encapsulated in lipid nanoparticles (LNPs) induced therapeutic gene silencing following intracranial injection in a glioblastoma (GBM)-bearing mouse model.7Cohen Z.R. Ramishetti S. Peshes-Yaloz N. Goldsmith M. Wohl A. Zibly Z. Peer D. Localized RNAi therapeutics of chemoresistant grade IV glioma using hyaluronan-grafted lipid-based nanoparticles.ACS Nano. 2015; 9: 1581-1591Crossref PubMed Scopus (128) Google Scholar In the latter model, the researchers showed an increased survival of mice treated with siRNA against Polo-like kinase 1 (G2M cell cycle regulator) compared to all other controls. However, intracranial injections require surgery, and therefore are tedious, and could result in further complications. The intranasal administration route holds great promise for delivering therapeutics to the brain because it is simple, rapid, and reduces systemic exposure and adverse effects.8Henkin R.I. Intranasal delivery to the brain.Nat. Biotechnol. 2011; 29: 480Crossref Scopus (21) Google Scholar Systemic administration by i.v. injections or oral administration is considered to be the most trivial way of administering drugs to patients. Another way to facilitate the movement of therapeutics from the circulation to the brain parenchyma involves utilizing the receptors and transporters located on the apical membrane of the BBB, which offers substantial potential for drug development9Abbott N.J. Rönnbäck L. Hansson E. Astrocyte-endothelial interactions at the blood-brain barrier.Nat. Rev. Neurosci. 2006; 7: 41-53Crossref PubMed Scopus (3739) Google Scholar. Many studies focused on binding targeting moieties, such as antibodies, small peptides, or ligands to different types of nanoparticles to induce transcytosis.6Gutkin A. Cohen Z.R. Peer D. Harnessing nanomedicine for therapeutic intervention in glioblastoma.Expert Opin. Drug Deliv. 2016; 13: 1573-1582Crossref Scopus (36) Google Scholar Another strategy is to use viral vectors as drug carriers to the brain. In this way, the therapeutic payload can penetrate through the BBB by mechanisms of receptor/transporter-mediated transcytosis1Banks W.A. From blood-brain barrier to blood-brain interface: new opportunities for CNS drug delivery.Nat. Rev. Drug Discov. 2016; 15: 275-292Crossref PubMed Scopus (586) Google Scholar. Another field of study focuses on BBB disruption to enhance delivery, such as by using MRI-guided focused ultrasound (MRI-gFUS). In this method, microbubbles are injected systemically and under the guidance of MRI, a specific area in the brain is stimulated by the ultrasound waves, thus causing local disruption of the BBB to allow drug carriers to pass through.10Burgess A. Hynynen K. Noninvasive and targeted drug delivery to the brain using focused ultrasound.ACS Chem. Neurosci. 2013; 4: 519-526Crossref PubMed Scopus (84) Google Scholar, 11McDannold N. Arvanitis C.D. Vykhodtseva N. Livingstone M.S. Temporary disruption of the blood-brain barrier by use of ultrasound and microbubbles: safety and efficacy evaluation in rhesus macaques.Cancer Res. 2012; 72: 3652-3663Crossref PubMed Scopus (422) Google Scholar Other methods to temporarily disrupt the BBB include the use of the hyperosmotic agent, mannitol,12Kemper E.M. Boogerd W. Thuis I. Beijnen J.H. van Tellingen O. Modulation of the blood-brain barrier in oncology: therapeutic opportunities for the treatment of brain tumours?.Cancer Treat. Rev. 2004; 30: 415-423Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar or the vasoactive agent, bradykinin.13Liu L.B. Xue Y.X. Liu Y.H. Bradykinin increases the permeability of the blood-tumor barrier by the caveolae-mediated transcellular pathway.J. Neurooncol. 2010; 99: 187-194Crossref PubMed Scopus (37) Google Scholar Godinho et al.2Godinho B.M.D.C. Henninger N. Bouley J. Alterman J.F. Haraszti R.A. Gilbert J.W. Sapp E. Coles A.H. Biscans A. Nikan M. et al.Transvascular Delivery of Hydrophobically Modified siRNAs: Gene Silencing in the Rat Brain upon Disruption of the Blood-Brain Barrier.Mol. Ther. 2018; 26 (this issue): 2580-2591Abstract Full Text Full Text PDF Scopus (25) Google Scholar utilized mannitol to transiently disrupt the BBB and promote the delivery of siRNA conjugated to PC-DHA. The use of mannitol for BBB disruption has previously been employed for the delivery of drug payloads such as chemotherapy14Joshi S. Ergin A. Wang M. Reif R. Zhang J. Bruce J.N. Bigio I.J. Inconsistent blood brain barrier disruption by intraarterial mannitol in rabbits: implications for chemotherapy.J. Neurooncol. 2011; 104: 11-19Crossref PubMed Scopus (62) Google Scholar, nanoparticles15Rousseau V. Denizot B. Pouliquen D. Jallet P. Le Jeune J.J. Investigation of blood-brain barrier permeability to magnetite-dextran nanoparticles (MD3) after osmotic disruption in rats.MAGMA. 1997; 5: 213-222Crossref PubMed Scopus (30) Google Scholar, viral vectors,16Foley C.P. Rubin D.G. Santillan A. Sondhi D. Dyke J.P. Crystal R.G. Gobin Y.P. Ballon D.J. Intra-arterial delivery of AAV vectors to the mouse brain after mannitol mediated blood brain barrier disruption.J. Control. Release. 2014; 196: 71-78Crossref PubMed Scopus (58) Google Scholar and recombinant proteins17Brown R.C. Egleton R.D. Davis T.P. Mannitol opening of the blood-brain barrier: regional variation in the permeability of sucrose, but not 86Rb+ or albumin.Brain Res. 2004; 1014: 221-227Crossref PubMed Scopus (68) Google Scholar (Figure 1). Godinho et. al.2Godinho B.M.D.C. Henninger N. Bouley J. Alterman J.F. Haraszti R.A. Gilbert J.W. Sapp E. Coles A.H. Biscans A. Nikan M. et al.Transvascular Delivery of Hydrophobically Modified siRNAs: Gene Silencing in the Rat Brain upon Disruption of the Blood-Brain Barrier.Mol. Ther. 2018; 26 (this issue): 2580-2591Abstract Full Text Full Text PDF Scopus (25) Google Scholar delivered siRNA to the ipsilateral brain hemisphere upon intracarotid injection of mannitol followed by injection of the siRNA- PC-DHA conjugate. The delivery of siRNA achieved efficient and rapid gene silencing in multiple areas within the brain. In the absence of BBB disruption, the siRNA conjugates did not significantly pass through the BBB and reach the brain. Although the authors showed no local or systemic toxicity upon BBB disruption, the question of toxicity following repetitive injections of mannitol, which would be required for sustained mRNA knockdown, remains unclear and should be addressed. Lastly, a demonstration of a therapeutic effect using this method of delivery in a disease model also remains to be shown. Godinho et al.2Godinho B.M.D.C. Henninger N. Bouley J. Alterman J.F. Haraszti R.A. Gilbert J.W. Sapp E. Coles A.H. Biscans A. Nikan M. et al.Transvascular Delivery of Hydrophobically Modified siRNAs: Gene Silencing in the Rat Brain upon Disruption of the Blood-Brain Barrier.Mol. Ther. 2018; 26 (this issue): 2580-2591Abstract Full Text Full Text PDF Scopus (25) Google Scholar provide a proof-of-concept that temporary disruption of the BBB can serve as an effective strategy to deliver siRNAs to the brain. Such studies are essential to provide hope for many neuropathology patients that lack adequate treatment methods due to the absence of appropriate delivery strategies. RNAi therapeutics will now gain more attention since the FDA approval of the first RNAi drug, Patisiran, for a rare hereditary disease. The study by Godinho et. al.2Godinho B.M.D.C. Henninger N. Bouley J. Alterman J.F. Haraszti R.A. Gilbert J.W. Sapp E. Coles A.H. Biscans A. Nikan M. et al.Transvascular Delivery of Hydrophobically Modified siRNAs: Gene Silencing in the Rat Brain upon Disruption of the Blood-Brain Barrier.Mol. Ther. 2018; 26 (this issue): 2580-2591Abstract Full Text Full Text PDF Scopus (25) Google Scholar opens new avenues for exploring different delivery strategies for RNAi therapeutics in general and for brain delivery in particular. D.P. has financial interests in Quiet Therapeutics. A.G. has no conflicts of interest. This work was supported in part by grants from the Israel Cancer Research Fund. A.G. thanks the Dr. Albert and Doris Fields Trust for fellowship. Transvascular Delivery of Hydrophobically Modified siRNAs: Gene Silencing in the Rat Brain upon Disruption of the Blood-Brain BarrierGodinho et al.Molecular TherapyAugust 7, 2018In BriefGodinho et al. demonstrate the utility of transient blood-brain barrier (BBB) disruption using hyperosmolar mannitol as a delivery strategy for fully modified lipid-conjugated hydrophobic siRNAs (hsiRNAs). Phosphocholine-docosahexanoic acid (PC-DHA)-conjugated hsiRNAs broadly distribute in the brain after BBB disruption, enabling potent gene silencing without major neurotoxicity. Full-Text PDF Open ArchiveGene Silencing in the Right Place at the Right TimeGutkin et al.Molecular TherapyNovember 16, 2018In Brief(Molecular Therapy 26, 2539–2541; November 2018) Full-Text PDF Open Archive