Delivery is key: lessons learnt from developing splice‐switching antisense therapies
2017; Springer Nature; Volume: 9; Issue: 5 Linguagem: Inglês
10.15252/emmm.201607199
ISSN1757-4684
AutoresCaroline Godfrey, Lourdes R. Desviat, Bård Smedsrød, France Piétri‐Rouxel, Michela A. Denti, Petra Disterer, Stéphanie Lorain, Gisela Nogales‐Gadea, Valentina Sardone, Anwar Rayan, Samir EL Andaloussi, Taavi Lehto, Bernard Khoo, Camilla Brolin, Willeke M. C. van Roon‐Mom, Aurélie Goyenvalle, Annemieke Aartsma‐Rus, Virginia Arechavala‐Gomeza,
Tópico(s)Advanced biosensing and bioanalysis techniques
ResumoReview13 March 2017Open Access Delivery is key: lessons learnt from developing splice-switching antisense therapies Caroline Godfrey Caroline Godfrey Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford, UK Search for more papers by this author Lourdes R Desviat Lourdes R Desviat Centro de Biología Molecular Severo Ochoa UAM-CSIC, CIBERER, IdiPaz, Universidad Autónoma de Madrid, Madrid, Spain Search for more papers by this author Bård Smedsrød Bård Smedsrød Department of Medical Biology, University of Tromsø, Tromsø, Norway Search for more papers by this author France Piétri-Rouxel France Piétri-Rouxel UPMC, INSERM, UMRS 974, CNRS FRE 3617, Institut de Myologie, Paris, France Search for more papers by this author Michela A Denti Michela A Denti Centre for Integrative Biology, University of Trento, Trento, Italy Search for more papers by this author Petra Disterer Petra Disterer Centre for Amyloidosis and Acute Phase Proteins, Division of Medicine, University College London, London, UK Search for more papers by this author Stéphanie Lorain Stéphanie Lorain UPMC, INSERM, UMRS 974, CNRS FRE 3617, Institut de Myologie, Paris, France Search for more papers by this author Gisela Nogales-Gadea Gisela Nogales-Gadea Grup d'Investigació en Malalties Neuromusculars i Neuropediatriques, Institut d' Investigació en Ciències de la Salut Germans Trias i Pujol, Badalona, Barcelona, Spain Search for more papers by this author Valentina Sardone Valentina Sardone Dubowitz Neuromuscular Centre and Developmental Neuroscience Programme, Institute of Child Health, University College London, London, UK Search for more papers by this author Rayan Anwar Rayan Anwar Drug Discovery Informatics Lab, Qasemi-Research Center, Al-Qasemi Academic College, Baka El-Garbiah, Israel Drug Discovery and Development Laboratory, Institute of Applied Research, Galilee Society, Shefa-Amr, Israel Search for more papers by this author Samir EL Andaloussi Samir EL Andaloussi Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford, UK Department of Laboratory Medicine, Karolinska Institute, Stockholm, Sweden Search for more papers by this author Taavi Lehto Taavi Lehto Department of Laboratory Medicine, Karolinska Institute, Stockholm, Sweden Institute of Technology, University of Tartu, Tartu, Estonia Search for more papers by this author Bernard Khoo Bernard Khoo Centre for Neuroendocrinology, Division of Medicine, University College London, London, UK Search for more papers by this author Camilla Brolin Camilla Brolin Department of Cellular and Molecular Medicine, University of Copenhagen, Copenhagen, Denmark Search for more papers by this author Willeke MC van Roon-Mom Willeke MC van Roon-Mom Department of Human Genetics, Leiden University Medical Center, Leiden, The Netherlands Search for more papers by this author Aurélie Goyenvalle Aurélie Goyenvalle INSERM U1179, UFR des sciences de la santé, Université Versailles Saint Quentin, Montigny-le-Bretonneux, France Search for more papers by this author Annemieke Aartsma-Rus Annemieke Aartsma-Rus Department of Human Genetics, Leiden University Medical Center, Leiden, The Netherlands Search for more papers by this author Virginia Arechavala-Gomeza Corresponding Author Virginia Arechavala-Gomeza [email protected] orcid.org/0000-0001-7703-3255 Neuromuscular Disorders Group, BioCruces Health Research Institute, Barakaldo, Bizkaia, Spain Search for more papers by this author Caroline Godfrey Caroline Godfrey Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford, UK Search for more papers by this author Lourdes R Desviat Lourdes R Desviat Centro de Biología Molecular Severo Ochoa UAM-CSIC, CIBERER, IdiPaz, Universidad Autónoma de Madrid, Madrid, Spain Search for more papers by this author Bård Smedsrød Bård Smedsrød Department of Medical Biology, University of Tromsø, Tromsø, Norway Search for more papers by this author France Piétri-Rouxel France Piétri-Rouxel UPMC, INSERM, UMRS 974, CNRS FRE 3617, Institut de Myologie, Paris, France Search for more papers by this author Michela A Denti Michela A Denti Centre for Integrative Biology, University of Trento, Trento, Italy Search for more papers by this author Petra Disterer Petra Disterer Centre for Amyloidosis and Acute Phase Proteins, Division of Medicine, University College London, London, UK Search for more papers by this author Stéphanie Lorain Stéphanie Lorain UPMC, INSERM, UMRS 974, CNRS FRE 3617, Institut de Myologie, Paris, France Search for more papers by this author Gisela Nogales-Gadea Gisela Nogales-Gadea Grup d'Investigació en Malalties Neuromusculars i Neuropediatriques, Institut d' Investigació en Ciències de la Salut Germans Trias i Pujol, Badalona, Barcelona, Spain Search for more papers by this author Valentina Sardone Valentina Sardone Dubowitz Neuromuscular Centre and Developmental Neuroscience Programme, Institute of Child Health, University College London, London, UK Search for more papers by this author Rayan Anwar Rayan Anwar Drug Discovery Informatics Lab, Qasemi-Research Center, Al-Qasemi Academic College, Baka El-Garbiah, Israel Drug Discovery and Development Laboratory, Institute of Applied Research, Galilee Society, Shefa-Amr, Israel Search for more papers by this author Samir EL Andaloussi Samir EL Andaloussi Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford, UK Department of Laboratory Medicine, Karolinska Institute, Stockholm, Sweden Search for more papers by this author Taavi Lehto Taavi Lehto Department of Laboratory Medicine, Karolinska Institute, Stockholm, Sweden Institute of Technology, University of Tartu, Tartu, Estonia Search for more papers by this author Bernard Khoo Bernard Khoo Centre for Neuroendocrinology, Division of Medicine, University College London, London, UK Search for more papers by this author Camilla Brolin Camilla Brolin Department of Cellular and Molecular Medicine, University of Copenhagen, Copenhagen, Denmark Search for more papers by this author Willeke MC van Roon-Mom Willeke MC van Roon-Mom Department of Human Genetics, Leiden University Medical Center, Leiden, The Netherlands Search for more papers by this author Aurélie Goyenvalle Aurélie Goyenvalle INSERM U1179, UFR des sciences de la santé, Université Versailles Saint Quentin, Montigny-le-Bretonneux, France Search for more papers by this author Annemieke Aartsma-Rus Annemieke Aartsma-Rus Department of Human Genetics, Leiden University Medical Center, Leiden, The Netherlands Search for more papers by this author Virginia Arechavala-Gomeza Corresponding Author Virginia Arechavala-Gomeza [email protected] orcid.org/0000-0001-7703-3255 Neuromuscular Disorders Group, BioCruces Health Research Institute, Barakaldo, Bizkaia, Spain Search for more papers by this author Author Information Caroline Godfrey1, Lourdes R Desviat2, Bård Smedsrød3, France Piétri-Rouxel4, Michela A Denti5, Petra Disterer6, Stéphanie Lorain4, Gisela Nogales-Gadea7, Valentina Sardone8, Rayan Anwar9,10, Samir EL Andaloussi1,11, Taavi Lehto11,12, Bernard Khoo13, Camilla Brolin14, Willeke MC Roon-Mom15, Aurélie Goyenvalle16, Annemieke Aartsma-Rus15 and Virginia Arechavala-Gomeza *,17 1Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford, UK 2Centro de Biología Molecular Severo Ochoa UAM-CSIC, CIBERER, IdiPaz, Universidad Autónoma de Madrid, Madrid, Spain 3Department of Medical Biology, University of Tromsø, Tromsø, Norway 4UPMC, INSERM, UMRS 974, CNRS FRE 3617, Institut de Myologie, Paris, France 5Centre for Integrative Biology, University of Trento, Trento, Italy 6Centre for Amyloidosis and Acute Phase Proteins, Division of Medicine, University College London, London, UK 7Grup d'Investigació en Malalties Neuromusculars i Neuropediatriques, Institut d' Investigació en Ciències de la Salut Germans Trias i Pujol, Badalona, Barcelona, Spain 8Dubowitz Neuromuscular Centre and Developmental Neuroscience Programme, Institute of Child Health, University College London, London, UK 9Drug Discovery Informatics Lab, Qasemi-Research Center, Al-Qasemi Academic College, Baka El-Garbiah, Israel 10Drug Discovery and Development Laboratory, Institute of Applied Research, Galilee Society, Shefa-Amr, Israel 11Department of Laboratory Medicine, Karolinska Institute, Stockholm, Sweden 12Institute of Technology, University of Tartu, Tartu, Estonia 13Centre for Neuroendocrinology, Division of Medicine, University College London, London, UK 14Department of Cellular and Molecular Medicine, University of Copenhagen, Copenhagen, Denmark 15Department of Human Genetics, Leiden University Medical Center, Leiden, The Netherlands 16INSERM U1179, UFR des sciences de la santé, Université Versailles Saint Quentin, Montigny-le-Bretonneux, France 17Neuromuscular Disorders Group, BioCruces Health Research Institute, Barakaldo, Bizkaia, Spain *Corresponding author. Tel: +34 946007967; E-mail: [email protected] EMBO Mol Med (2017)9:545-557https://doi.org/10.15252/emmm.201607199 See the Glossary for abbreviations used in this article. PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract The use of splice-switching antisense therapy is highly promising, with a wealth of pre-clinical data and numerous clinical trials ongoing. Nevertheless, its potential to treat a variety of disorders has yet to be realized. The main obstacle impeding the clinical translation of this approach is the relatively poor delivery of antisense oligonucleotides to target tissues after systemic delivery. We are a group of researchers closely involved in the development of these therapies and would like to communicate our discussions concerning the validity of standard methodologies currently used in their pre-clinical development, the gaps in current knowledge and the pertinent challenges facing the field. We therefore make recommendations in order to focus future research efforts and facilitate a wider application of therapeutic antisense oligonucleotides. Glossary Antisense oligonucleotides Antisense oligonucleotides (AONs) are short strands of DNA or RNA that can bind to RNA through Watson–Crick base pairing and can modulate the function of the target RNA. Different types of AONs, defined by their chemical structure, are mentioned in this article: 2′ O-methyl phosphorothioate oligonucleotides (2OMe), locked nucleic acids (LNAs), phosphorodiamidate morpholino oligomers (PMO) and peptide nucleic acids (PNAs). These "naked" antisense oligonucleotides can be combined with several moieties to increase their delivery, such as cell-penetrating peptides (CPPs). These conjugated AONs include vivo-morpholinos (VMO) or peptide phosphorodiamidate morpholino oligomers (PPMO). When AONs are used to disrupt RNA splicing, they are referred to as splice-switching oligonucleotides (SSO), irrespective of their chemical structure. Drug delivery systems Drug delivery systems (DDS) are strategies to enhance delivery of drugs to target sites of pharmacological actions. Lipid nanoparticles (LNPs) or adeno-associated virus (AAV) may be considered DDS. Induced pluripotent stem cells Induced pluripotent stem cells (IPSCs) are cells generated directly from adult cells, which may give rise to every other cell type in the body, and can propagate indefinitely. Kupffer cells and liver sinusoidal endothelial cells Both Kupffer cells (KCs) and liver sinusoidal endothelial cells (LSECs) constitute the hepatic sinusoidal lining, KCs are resident liver macrophages and form the greater part of the mononuclear phagocyte system, while LSECs are specialized endothelial cells with unsurpassed clathrin-mediated endocytosis and endo-lysosomal processing, enabling efficient scavenging of blood-borne oligonucleotides, peptides, large macromolecules and nanoparticles. The space of Disse is the space between the liver sinusoidal lining and hepatocytes. Access to it is provided through fenestrae in LSECs or following transport through LSECs Introduction Antisense oligonucleotides (AONs) are therapeutically attractive compounds; their mechanism of action is usually through hybridization to target sequences in pre-mRNA or mRNA, and as such, AONs are highly specific. They can be manufactured at large scale in a standardized manner, and do not face many of the challenges of other genetic therapies such as gene addition and genome editing which need viral vector-mediated delivery. Thus, it is not surprising that AON therapy development is a dynamic and active field. To date, four AON compounds have received marketing authorization and more than 100 clinical trials with antisense compounds are listed on ClinicalTrials.gov (Aartsma-Rus, 2016; Fig 1). Figure 1. FDA-approved antisense drugs1 Download figure Download PowerPoint One type of AON aims to modulate splicing; these so-called splice-switching oligonucleotides (SSOs) have been shown to restore protein expression in multiple clinical trials. However, following systemic administration the clinical benefit has been marginal and thus gaining regulatory approval has proved difficult. The ability of SSOs to induce sufficient levels of splice modulation in target tissues is limited by their poor delivery. Once in circulation, unmodified charged-neutral AONs such as peptide nucleic acids (PNAs) and phosphorodiamidate morpholino oligomers (PMOs) are excreted rapidly via the kidney mainly as intact molecules typically with half-lives of less than a few hours (McMahon et al, 2002; Amantana et al, 2007). It is assumed that on average < 1% of AONs reach the correct cellular compartment. Furthermore, due to the body's tissue barriers, the circulation of AONs is restricted, for example, most AONs are not able to reach the central nervous system (CNS) after systemic delivery (Fig 2). Significantly enhancing cell-specific delivery of AONs is challenging due to our lack of knowledge of cellular uptake and subcellular mechanisms of transport and metabolism. Figure 2. Barriers in AON delivery Download figure Download PowerPoint This manuscript focuses on AON delivery using systemic and localized administration, uptake mechanisms and model systems (Box 1). The aim of this publication is not to review what is currently known in the field of AON delivery; for this, we refer the reader to another recently published review (Juliano, 2016), rather we will use selected examples from both literature and experience to illustrate current challenges, problems and gaps in knowledge (Box 2). Using this approach, we outline what lessons can be learnt from previous work and suggest areas on which to focus future research efforts (Box 3). Box 1: Background to this manuscript The delivery challenges facing AON therapy were recognized by the Cooperation of Science and Technology (COST) Action BM1207 (Networking towards clinical application of antisense-mediated exon skipping for rare diseases [www.exonskipping.eu]). In order to address these challenges, four workshops were organized; the participants of which were both pre-clinical and clinical researchers working on many aspects of AON therapy development. This manuscript is a result of discussions held at these meetings and focuses on AON delivery using systemic and localized administration, uptake mechanisms and model systems. Box 2: Key challenges of AON delivery Target/off target effects. Toxicity due to AONs in entrapping tissues. Toxicity due to chemical modifications. Liver and kidney as a barrier. Tissue-specific barriers (e.g. BBB for CNS). Box 3: Recommendations (or possible solutions?) Make the most of "encapsulated tissues". Develop efficient and safe drug delivery systems. Find specific receptor ligands. Delivery hurdles and how to make the most of them The problem of delivery As with the development of any treatment, the therapeutic agent need only be effective in a subset of cells in the body. Most AON clinical trials have used the systemic intravenous administration route which results in the majority of AONs distributing to the liver, kidney, bone marrow, lymph nodes and a small part accumulating in adipocytes (Martin-Armas et al, 2006; Geary et al, 2015). It is important to note however that as tissues consist of a mixture of cell types, not all cells within a tissue will take up equal amounts of AON. Increasing the administered dose in order to deliver sufficient amounts of AON to specific target cells is inherently limited by associated toxicities. Therefore, a detailed knowledge of what constitutes effective delivery within a specific disease context is essential in order to obtain sufficient potency with minimal toxicity. Increases in the efficiency of AON delivery have been achieved through chemical modification, conjugation to other moieties as well as the development of new chemical backbones (Fig 3). While these modifications provide some benefits, questions surrounding pre-clinical and clinical toxicity remain unresolved, and thus, it is important for scientists, toxicologists and pathologists as well as regulatory reviewers to be familiar with these issues. Figure 3. Strategies for improving delivery Download figure Download PowerPoint AON toxicity The main toxicological challenges facing AON development programmes include: proinflammatory effects (vasculitis/inflammatory infiltrates), nephrotoxicity, hepatotoxicity and thrombocytopenia (Frazier, 2015). These types of toxicity are often called AON chemistry-dependent toxicities, and represent effects that are not due to the Watson–Crick base pairing between an AON and an RNA sequence. These toxicities may still exhibit some sequence dependency despite the fact they do not involve base pairing. Such sequence-specific toxicity has been observed with locked nucleic acids (LNAs) which, depending on their sequence, can cause profound hepatotoxicity as measured by serum transaminases as well as mild to severe liver lesions (Swayze et al, 2007; Stanton et al, 2012; Burdick et al, 2014; Kakiuchi-Kiyota et al, 2014). This suggests that it may not be possible to define the toxicological profile of a new chemistry based on a limited number of sequences. Hybridization-independent toxicities fall into three general subcategories: AON accumulation effects, proinflammatory mechanisms (including immune complexes) and aptameric binding (as a consequence of AON interactions with extracellular, cell surface and/or intracellular proteins). The proinflammatory, aptameric binding effects are acute, while the accumulation effects are long term. As such, the relevance of the accumulation effect may depend on the type of treatment required by the pathology (high-dose, short-term treatment for cancer will not face the same cumulative effects as a lifelong therapy required for chronic diseases such as muscular dystrophies). The mechanisms underlying these toxicities are also related to the specific chemical class of AON involved, and each class of agent has specific toxicity profiles. Phosphorothioate AONs have well-characterized toxicities such as proinflammatory responses potentially related to their protein binding properties (Henry et al, 2002; Frazier, 2015), whereas neutral AONs such as PMOs do not interact to any significant extent with cellular proteins and tend to have fewer systemic toxicities. Both the chemical backbone and specific sequence should therefore be taken into account when evaluating toxicological profiles of novel AONs. A number of reviews have now been published highlighting guidelines and summarizing consensus opinion on the appropriate strategies to use when assessing potential adverse AON-mediated effects (Kornbrust et al, 2013; Engelhardt et al, 2015; Frazier, 2015). AON in the liver With a blood flow of about 2 l/min and a sinusoidal blood lining surface area the size of a tennis court, the liver is one of the most vascularized tissues in the body. It is responsible for the clearance of large molecules and nanoparticles from blood, a function which is often counterproductive to the successful delivery of therapeutic compounds to other tissues or even specific cells within the liver. Several studies show that oligonucleotides (unmodified or conjugated) will end up in the liver to a far higher extent than the intended target tissue, although the rates vary between studies. In one study intravenous administration of an AON resulted in 40% and 18% accumulation in the liver and kidneys respectively (Bijsterbosch et al, 1997), whilst intravenous administration of CpG oligonucleotides resulted in 50% and 40% accumulation in the liver and kidneys respectively (Martin-Armas et al, 2006). The main cellular site of liver uptake are the extremely active scavenger liver sinusoidal endothelial cells (LSECs) (Sorensen et al, 2015) followed by hepatocytes and Kupffer cells (KCs; (Bijsterbosch et al, 1997), however the degree of uptake in hepatocytes can vary from 40% in the first study to no apparent uptake in the CpG study. Similarly, a histological study revealed that phosphorothioate oligonucleotides accumulated mainly in KCs and LSECs (Butler et al, 1997). KCs specialize in phagocytic clearance of blood-borne particles larger than 200 nm while LSECs mediate the clearance of smaller particles such as oligonucleotides, peptides, large macromolecules and nanoparticles via rapid and powerful clathrin-mediated endocytosis (Sorensen et al, 2012). LSECs contain fenestrations with numerous open pores of 50–150 nm in diameter, enabling access into the underlying perisinusoidal space and therefore to hepatocytes. However, LSECs are also able to endocytose from the perisinusoidal space and do so at a much higher speed than hepatocytes (Magnusson & Berg, 1989). Stabilin is most likely the main receptor responsible for uptake of oligonucleotides in LSECs (Martin-Armas et al, 2006). Our experience suggests that delivery reagents are necessary for the successful use of AON therapeutics in the liver, particularly targeting hepatocytes (e.g. for the treatment of hyperlipidaemia, hepatitis C or inherited metabolic disorders with major hepatic expression) (Disterer et al, 2013; Yilmaz-Elis et al, 2013; Perez et al, 2014). AON-mediated liver toxicity, monitored as increased liver enzymes in the circulation, is generally considered a hepatocyte-specific event (Kakiuchi-Kiyota et al, 2014). However, it has been suggested that LSECs also play a significant role in the generation of liver toxicity caused by AONs. Firstly, as LSECs rapidly accumulate very high intracellular concentrations of AON due to the unsurpassed scavenger function of these cells, the adverse effects of oligonucleotides would be far more pronounced in these cells compared to other cell types. Secondly, it is known that initial damage to LSECs caused by certain drugs subsequently causes damage to the hepatocytes (DeLeve, 2007). It is therefore reasonable to assume that AON-mediated liver toxicity is, at least in part, caused by initial damage to LSECs with subsequent injury to hepatocytes. Clearly, future attempts to unravel the mechanism of AON-mediated hepatotoxicity must investigate LSECs in addition to hepatocytes and other types of liver cells. AON in the kidney Renal blood flow through the glomerular capillary system efficiently clears a large portion of AONs from the bloodstream in a short time (up to 40% with some AONs). AONs appear to enter by receptor-mediated endocytosis primarily at the brush border of the epithelium, although the specific receptor is as yet unknown. The fenestrated capillary endothelium provides a vast surface area for AON clearance, and in addition, AONs that are filtered through the glomerulus are reabsorbed by the proximal tubular epithelium via unidentified specific receptors, contributing to the high AON accumulation (Engelhardt, 2016). Following uptake, AONs are found in endosomes and lysosomes and high doses can result in the formation of cytoplasmic basophilic granules with or without vacuolation. The kidney accumulates one of the highest concentrations of AON following systemic administration in rodents, non-human primates and humans, and this could make it the primary organ for toxicity. However, for 2′ O-methyl phosphorothioate oligonucleotides, the histological changes seen in toxicity studies in animal models do not correlate with the data from multiple clinical trials that indicate no effect on renal function (Crooke et al, 2016; Engelhardt, 2016). Other chemistries may result in renal toxicity as has been described in clinical studies of LNA oligonucleotides (Engelhardt, 2016). Comparative pre-clinical and clinical studies on renal toxicity are thus necessary for each AON chemistry, as well as basic research into delivery agents that target the specific cell type while minimizing renal clearance. Encapsulated tissues: a barrier has two sides Recently renewed interest in the use of AONs to treat CNS diseases is based on the concept of the CNS as an encapsulated tissue; the same barriers that hamper delivery to the CNS after systemic delivery may trap therapeutic compounds once they reach the CNS. With standard systemic delivery, AONs have to cross the blood–brain barrier (BBB) or blood–cerebrospinal barrier, before they can distribute within the CNS. This barrier is comprised of a monolayer of endothelial cells, the basement membrane and either astrocytes or choroid cells which form tight junctions through interactions between these components (Palmer, 2010). Within the CNS, AONs benefit from a remarkably widespread distribution and exhibit efficient cellular uptake mechanisms (Whitesell et al, 1993; Rigo et al, 2014). The systemic route of delivery into the CNS includes diffusion (Banks et al, 2001) and receptor-mediated endocytosis (Lee et al, 2002; Kozlu et al, 2014). Direct delivery of AONs to the CNS is the most commonly used method of bypassing the BBB and can be achieved through intracerebroventricular or intrathecal (IT) injection. Due to the BBB preventing leakage of the AONs into peripheral circulation, relatively low doses can be administered less frequently (as half-lives are increased), thus minimizing the risks of toxicity. To date, two phase I clinical trials have been completed using IT injection of AONs, one in amyotrophic lateral sclerosis (ALS; Miller et al, 2013) and one in spinal muscular atrophy (SMA) patients (Chiriboga et al, 2016) with encouraging results. Recently reported interim results from two phase III trials with nusinersen, the SMA therapeutic, were so positive that both trials were stopped early and all participants rolled over onto treatment immediately (Ionis Pharma press releases, currently accessible at http://ir.ionispharma.com/phoenix.zhtml?c=222170&p=irol-newsArticle&ID=2191319 and http://ir.ionispharma.com/phoenix.zhtml?c=222170&p=irol-newsArticle&ID=2220037). Nusinersen treatment was able to significantly improve achievement of motor milestones in infantile-onset SMA (the most severe form of SMA) as well as in later-onset (type II) SMA. The investigators were able to assess the uptake of nusinersen into the tissues of three infants that died during the trial and found that there was significant uptake of the AON into the CNS (including the target motor neurons, but also non-neuronal cells) along with SMN2 exon inclusion and expression of SMN protein (Finkel et al, 2016). It should be noted, however, that repeated IT therapy is a relatively expensive method of administration, necessitating specialist expertise and hospital visits. A promising delivery approach is intranasal administration; molecules can be transported along the olfactory and trigeminal nerve pathways and the rostral migratory stream (Goyenvalle et al, 2015). Clinical trials utilizing this delivery route have resulted in improved cognition in Alzheimer's disease patients following application of intranasal insulin (Claxton et al, 2013). Among the CNS, the retina is becoming increasingly important as a target tissue for AON therapies (Bacchi et al, 2014). The eye is a small, enclosed, easy to access compartment and an immune-privileged organ (Stein-Streilein, 2008). Intravitreal, subretinal or suprachoroidal injections have been used (Thrimawithana et al, 2011). A well-established example is the intravitreal treatment of cytomegalovirus-associated retinitis in immunocompromised patients (Vitravene Study, 2002a,b,c), while topical and periocular routes are promising less invasive alternatives. Recently, a phase III study on a topical inhibitor of corneal angiogenesis (Cursiefen et al, 2009) significantly inhibited corneal neovascularization in patients with keratitis (Cursiefen et al, 2014). However, nucleic acids are retained by the superficial tissues and do not significantly penetrate intraocularly (Oliver, 1975; Bochot et al, 1998; Berdugo et al, 2003). Negatively charged AONs are potential candidates to be delivered into the eye by iontophoresis, which relies on applying a local electrical current (Andrieu-Soler et al, 2006; Pescina et al, 2013). It has been demonstrated that a number of modified AONs or those conjugated to different moieties (e.g. cell-penetrating peptide [CPP]-based delivery systems) (El-Andaloussi et al, 2005; Du et al, 2011; Kang et al, 2014) can induce splice modulation in the CNS following systemic administration albeit at very low levels. To date, the most successful of these are tricyclo-DNA (tcDNA) oligonucleotides (Goyenvalle et al, 2015). AAV vectors as an alternative delivery strategy for antisense sequences An alternative way of anti
Referência(s)