Delivery of oligonucleotide-based therapeutics : challenges and opportunities

Funding Information: This work was supported by funding from Cooperation of Science and Technology (COST) Action CA17103 (networking grant to V.A-G). V.A-G holds a Miguel Servet Fellowship from the ISCIII [grant reference CPII17/00004] that is part-funded by the European Regional Development Fund (ERDF/FEDER) and also acknowledges funding from Ikerbasque (Basque Foundation for Science). S.M.H is funded by the Medical Research Council and Muscular Dystrophy UK. A.A-R receives funding from amongst others the Duchenne Parent Project, Spieren voor Spieren, the Prinses Beatrix Spierfonds, Duchenne UK and through Horizon2020 project BIND. A.G and R.W.J.C are supported by several foundations including the Algemene Nederlandse Vereniging ter Voorkoming van Blindheid, Stichting Blinden-Penning, Landelijke Stichting voor Blinden en Slechtzienden, Stichting Oogfonds Nederland, Stichting Macula Degeneratie Fonds, and Stichting Retina Nederland Fonds (who contributed through UitZicht 2015-31 and 2018-21), together with the Rotterdamse Stichting Blindenbelangen, Stichting Blindenhulp, Stichting tot Verbetering van het Lot der Blinden, Stichting voor Ooglijders, and Stichting Dowilvo; as well as the Foundation Fighting Blindness USA, grant no. PPA-0517-0717-RAD. R.A.M.B is supported by Hersenstichting Nederland Grant DR-2018-00253. G.G. is supported by Ministry of Research and Innovation in Romania/National Program 31N/2016/PN 16.22.02.05. S.A is supported by Project PTDC/BBB-BMD/6301/2014 (Funda??o para a Ci?ncia e a Tecnologia?MCTES, Portugal). L.R.D. is supported by Fundaci?n Ram?n Areces Grant XVII CN and Spanish Ministry of Science and Innovation (MICINN, grant PID2019-105344RB-I00). T.L is supported by Estonian Research Council grant PSG226. S.K is supported by the Friedrich-Baur-Stiftung. C.F is funded by The Danish Council for Independent Research, Technology and Production Sciences (grant number DFF-4184-00422). W.vRM is supported by ZonMw Programme Translational Research 2 [Project number 446002002], Campaign Team Huntington and AFM Telethon [Project number 20577]. S.E.B is supported by the H2020 projects B-SMART, Grant number 721058, and REFINE, Grant number 761104. A.T.G is supported by the Institut National de la sant? et la recherche m?dicale (INSERM) and the Association Monegasque contre les myopathies (AMM). L.E. is founded by the Association Monegasque contre les myopathies (AMM). Publisher Copyright: © 2021 The Authors. Published under the terms of the CC BY 4.0 licenseNucleic acid-based therapeutics that regulate gene expression have been developed towards clinical use at a steady pace for several decades, but in recent years the field has been accelerating. To date, there are 11 marketed products based on antisense oligonucleotides, aptamers and small interfering RNAs, and many others are in the pipeline for both academia and industry. A major technology trigger for this development has been progress in oligonucleotide chemistry to improve the drug properties and reduce cost of goods, but the main hurdle for the application to a wider range of disorders is delivery to target tissues. The adoption of delivery technologies, such as conjugates or nanoparticles, has been a game changer for many therapeutic indications, but many others are still awaiting their eureka moment. Here, we cover the variety of methods developed to deliver nucleic acid-based therapeutics across biological barriers and the model systems used to test them. We discuss important safety considerations and regulatory requirements for synthetic oligonucleotide chemistries and the hurdles for translating laboratory breakthroughs to the clinic. Recent advances in the delivery of nucleic acid-based therapeutics and in the development of model systems, as well as safety considerations and regulatory requirements for synthetic oligonucleotide chemistries are discussed in this review on oligonucleotide-based therapeutics.publishersversionPeer reviewe

Positioning Europe for the EPITRANSCRIPTOMICS challenge

The genetic alphabet consists of the four letters: C, A, G, and T in DNA and C,A,G, and U in RNA. Triplets of these four letters jointly encode 20 different amino acids out of which proteins of all organisms are built. This system is universal and is found in all kingdoms of life. However, bases in DNA and RNA can be chemically modified. In DNA, around 10 different modifications are known, and those have been studied intensively over the past 20 years. Scientific studies on DNA modifications and proteins that recognize them gave rise to the large field of epigenetic and epigenomic research. The outcome of this intense research field is the discovery that development, ageing, and stem-cell dependent regeneration but also several diseases including cancer are largely controlled by the epigenetic state of cells. Consequently, this research has already led to the first FDA approved drugs that exploit the gained knowledge to combat disease. In recent years, the ~150 modifications found in RNA have come to the focus of intense research. Here we provide a perspective on necessary and expected developments in the fast expanding area of RNA modifications, termed epitranscriptomics.SCOPUS: no.jinfo:eu-repo/semantics/publishe

A microcell hybrid based elimination test to identify human chromosome 3 regions that antagonize tumor growth

Deletions at multiple sites on the short arm of chr3 have been detected in many different types of human tumors. Each of the deleted regions is expected to harbor a tumor antagonizing/suppressor gene(s) (TSG). The earlier studies showed that the tumorigenicity of malignant cells could be suppressed by introducing a single human chr3. However monochromosomal hybrids system in tumorigenicity tests suffers the frequent loss of the introduced chromosome or its parts. We have therefore chosen the identification of the regions that are regularly lost in the course of mouse tumor passage. In the first study we have shown that two monochromosomal A9 mouse fibrosarcoma microcell hybrids (MCHs) lost the introduced cytogenetically intact normal human chr3 during progressive growth in SCID mice. FISH showed only fragments translocated to mouse chromosomes. PCR analysis had revealed that markers spanning the 3p21-p24 region were regularly lost/absent in all of the 13 tumors derived from 5 MCHs. We suggested that our findings might be related to the postulated presence of TSG(s) in the 3p21-p24 as indicated by the frequent deletion of this region in solid tumors. We propose that the identification of tumor antagonizing genes may be supplemented by a microcell hybrids based approach, referred as the Elimination test (Et). In the following studies, by increasing the number of tumors (27 from 5 MCHs) and chr3 markers (53), we have identified a common eliminated region (CER) of about 7 cM at 3p21.3, between D3S1260 and D3S643/D3F15SF, and a frequently eliminated region (FER) at 3p14.2-p21, between D3S1235 and D3S1067, that was absent in 21 of the 27 tumors. Using a panel of 30 SCID tumors derived from 5 MCHs that carried chr3 from the same donor, we have defined within CER at it centromeric border, at 3p21.33 between D3S1029 and LRRC2 gene, a common eliminated region 1 (CER1) of about 2.4 Mb. A common eliminated region 2 (CER2) of about 1.1 Mb was mapped within CER at it telomeric border at 3p22 between RH94338 and SHGC-154057. Unlimited availability of tumor material and the utility of non-polymorphic DNA markers in high-resolution deletion mapping are important advantages of Et. Subsequently, we have created human-human MCHs by transferring a normal human chr3 by microcell fusion into KH39 RCC cells that contained uniparentally disomic chr3. In SCID tumors derived from four of these human-human MCHs, we could identify the same eliminated regions on 3p (hCER1 and hFER) as in the human-murine model, but the centromeric border of hCER1 was shifted to the CCR2 gene away from the LRRC2 gene in human-murine CER1. Later, from the one sub-clone of the previously studied and two new A9-based mono-chromosomal MCHs that carried cytogenetically normal human chr3 from three different donors, we generated 9 SCID mice tumors that remained positive for all of the 120 3p-specific PCR-markers tested ("chr3+" tumors). These tumors were analyzed by FISH chromosome painting and by RT-PCR for the expression of 14 human chr3p genes: 5 from CER1 (LIMD1, CCR1, CCR2, CCR3, CCR5), 5 from regions that are often homozygously deleted (HD) in carcinomas (ITGA4L, LUCA1, PTPRG, FHIT, DUTT1), and 4 other cancer-related genes (VHL, MLH1, TGM4, UBE1L). Alone among the 14 genes examined, FHIT showed a tumor growth-associated change. It was expressed in MCH lines in vitro, but all of the 9 chr3+ tumors analyzed lost the FHIT gene transcript. Further we have examined the human-murine "chr3+" and human-human SCID tumors by RT-PCR for the expression of 20 chr3p genes including 7 genes from CER1 at 3p21.33 and 9 candidate TSGs from the LUCA region at 3p21.31 defined by overlapping HDs in lung cancer cell lines. We have found that in addition to the FHIT gene, the LTF gene from CER1 has also lost its mRNA expression in chr3+ tumors. Moreover, SCL38A3 from the LUCA region and DRR1 at 3p21-14.3, have down-regulated, reduced or lost, their expression after SCID passage. In the human-human system the endogenous/exogenous FHIT transcript was maintained in the SCID tumors, but the level of the Fhit protein was reduced. In contrast to the loss of mRNA expression from human-murine MCHs during SCID passage, in the human-human system, three out of the 20 examined genes, LTF, LRRC2 (neighboring LTF), and SCL38A3 (from LUCA region), expressed in the donor mouse A9-based MCH line, were suppressed, with regard to mRNA expression, after the transfer of chr3 into recipient human tumor cell line KH39. DRR1 mRNA expression was reduced in all of the 6 analyzed tumors after SCID growth. At least four of the 20 examined chr3p genes, FHIT, LTF, SCL38A3, and DRR1, showed in common a tumor-associated impairment: in human-murine system the loss or down-regulation of mRNA expression after growth in SCID mice and in human-human system the down-regulation of the expression (FHIT and DRR1) in tumors or loss of the expression (LTF and SCL38A3) already after the transfer of chr3 into the human tumor cells