NEXT GENERATION ASO CHEMISTRIES AND CHEMICAL ASO CONJUGATES - SUZAN M HAMMOND, PHD DEPARTMENT OF PAEDIATRICS UNIVERSITY OF OXFORD 25-FEBRUARY-2019
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Next generation ASO chemistries
and chemical ASO conjugates
Suzan M Hammond, PhD
Department of Paediatrics
University of Oxford
25-February-2019
Functions of Antisense Oligonucleotides
Starting with the birth of ASOs >40 years ago to the discovery
of siRNAs and the recent surge of mRNAs, The Problem has
remained the same:
Delivery! Delivery! Delivery!
~Dowdy and Levy 2018 NAT
Lundin et al., (2015)
Hum Gene Ther. 26(8):475-85.
What’s the problem? • Less than 1% reach the correct cellular compartment • Rapid excretion via kidney • No delivery to brain and spinal cord • Cellular uptake into the endosome / lysosome
1. Survive serum degradation
2. Translocate across the blood endothelium
Juliano (2016) 3. Avoid uptake by the liver/kidney
Nucleic Acids Res. 44(14):6518-6548
Cytoplasmic Trafficking
1. Endocytosis
2. Intracellular trafficking
3. Endosomal escape –
endosomal entrapment is
the main rate-limiting step
in AON delivery
4. Nuclear entry
Juliano (2016)
Nucleic Acids Res. 44(14):6518-6548
Modifications to chemical structure of nucleic acid
BASE
INTERNUCLEOTIDE
LINKAGE
SUGAR
Chemical structures for charged ASOs
conformationally constrained oligonucleotides
Chemical structure of charged neutral ASOs
Next Generation ASOs - TriycloDNA
(Modified Sugar)
Improved In Vivo Efficacy over
2’OMe and PMO ASOs
Goyenvalle et al., 2015
Nature Medicine 21:270-275
Next Generation ASO – Mesyl
(Internucleotide Linkages)
Nuclease Resistant
Methanesulfonyl phosphoramidate group
Hybridization
RNase H Activity
to miR-21
Miroshnichenko et al., (2019)
PNAS 116:1229-1234Chemical Conjugates of ASOs
Cell Penetrating Peptides
Imre Mager, Characterization of cell-
penetrating peptides: assessment of
cellular internalization kinetics,
mechanisms, and bioactivity.
Carbohydrates: (GalNAc) Antibody and Antibody Fragments
Freskgard and Urich (2017)
Neuropharmacology 120:38-55
Rajeev et al., 2015
ChemBioChem 16:903-908GalNAc
N-acetyl galactosamine
GalNAc improves efficacy of MOE and cEt Oligos GalNAc directs MOE into the hepatocytes
Prakash TP et al., 2014 NAR 42:8796-8807GalNac receptor, asialoglycoprotein receptor, rapidly internalizes large quantities of GalNAc-modified oligos
so that even a low percentage of escaping cargo is sufficient
Dowdy SF 2017
Nature Biotechnology 35:222-229Cell Penetrating Peptides CPP Sequence Reference Transportan GWTLNSAGYLLGKINLKALAALAKKIL Chaubey B et al. Virology. (2005) Oligoarginine RRRRRRRRR Moulton et al., Bioconjug Chem. (2004) pTat Peptide CGRKKRRQRRR Penetratin RQIKIWFQNRRMKWKK Rogers et al., Mol. Ther. (2012) B Peptide RXRRBRRXRRBRXB Wu et al., PNAS (2008) B-MSP RXRRBRRXRRBRXB ASSLNIAX Yin et al., Hum Mol Gen (2009) Pip6a RXRRBRRXR YQFLI RXRBRXRB Betts et al., Mol Ther - Nucl Acids (2012) (RXR)4 RXRRXRRXRRXR Gomez et al., J Clin Invest (2014) F3 Henke et al., Nat Biotechnol (2008) RKKKRKV RKKKRKV Boffa et al., Cancer Gene Ther (2007) Lys4 KKKK Fabani et al., Nucleic Acids Res 2010 (RFF)3RXB RFFRFFRFFRXB Tilley et al., Antibicrob. Chemother (2007) (KFF)3K KFFKFFKFFK Tan et al., Agents Chemother (2005) R9F2 RRRRRRRRRFF Nelson et al., Bioconjug. Chem. (2005) T-cell-derived YARVRRRGPRGYARVRRRGPRRC Wesolowski et al., PNAS (2011) CPP
CPPs enhance in vivo efficacy of ASO therapy Hammond et al (2016) PNAS 113(39):10962-7
Looking Forward: improving safety
Brain Spinal Cord
(Cervical)
KIM-1
Skeletal Muscle Liver 200
Quadriceps
R e la tiv e K IM - 1 /C r e a t in in e
150
100
50
0
S a lin e D ay 2 D ay 7 D ay 2 D ay 7
D P E P 5 -6 D P E P 5 -1 7
Dr. Graham McCloreyAntibodies and Antibody Fragments. https://absoluteantibody.com/antibody-resources/antibody- engineering/antibody-fragments/
Antibody-siRNAs
Antibody: anti-CD71 Fab′ fragment
Payload: siRNA toward HPRT
i.v. administration at 10mg/kg.
Sugo et al., 2016
J Control Release 237:1–13Antibody-ASOs for treating PreB Leukemia engrafted leukemia cells in mice apoptotic leukemia cells murine cells (CD22 negative),
ANTIBODY–DRUG CONJUGATES
Collaborators
Michael Gait
SMA Group Prof. Matthew Wood
• Graham McClorey
• Larissa Goli
• Jessica Stoodley
• Anna Kordala
• Audrey Winkelsas
• Nina Ahlskog Frank Abendroth
PMO ELISA
Miguel Varela
David Seoane Miraz
Andrey Arzumanov% Uptake based
On ELISA data from
SMA and DM1
Live ScienceFunctions of Antisense Oligonucleotides
Lundin KE, Gissberg O, Smith CI. 2015
Hum Gene Ther. 26(8):475-85.“The main obstacle impeding the clinical translation of [splice-switching antisense therapy] is the relatively poor delivery of antisense oligonucleotides to target tissues after systemic delivery”
Next Generation ASOs - TriycloDNA
(Modified Sugar)
200 mg/kg/week 15 umol/kg/week
Goyenvalle et al., 2015
Reference Nature Medicine 21:270-275Next generation ASO chemistries and
1. Next Generation ASOs
various functionalized chemical ASO
• Various uses of ASOs
conjugates (like peptide-PMOs, Galnac-
• Steric blockage
ASOs etc). How do they improve the
• Knockdown gapmers
distribution and efficacy in various
• WAVE Stereoisomers
tissues/disease models and their
• Dimitri ASOs
impact on potentially improving on the
• IONIS next generation ASOs
previous generation naked ASOs. What
• Tricyclo DNA
are the advantages and limitations
2. Chemical ASO Conjugates
with such ASO conjugates. To which
• CPPs – cell penetrating peptides
extent the targeting can be achieved
• Antibodies
with present state-of-the-art methods.
• Gal-NAc – N-acetylgalactosamine (Akinc 2010; Rajeev 2015)
• Cyclic peptides – octaguanidine dendrimerExamples of CPPs directly conjugated to oligonucleotides and their applications. Peptide Sequence Application Reference Transportan-PNA GWTLNSAGYLLGKINLKALAALAKKIL Galanin receptor antagonist [123] B-PMO RXRRBRRXRRBRXB DMD [51] B-MSP-PMO RXRRBRRXRRBRXB ASSLNIAX DMD [52] Pip5e-PMO RXRRBRRXR ILFQY RXRBRXRB DMD [53] Pip6a-PMO RXRRBRRXR YQFLI RXRBRXRB DMD [54] T-cell-derived CPP-PMO YARVRRRGPRGYARVRRRGPRR Antibacterial [66] D. Wesolowski, H.S. Tae, N. Gandotra, P. Llopis, N. Shen, S. Altman, Basic peptidemorpholino oligomer conjugate that is very effective in killing bacteria by genespecific and nonspecific modes, Proc. Natl. Acad. Sci. U. S. A. 108 (2011) 16582–16587, http://dx.doi.org/10.1073/pnas.1112561108. (RXR)4-PMO RXRRXRRXRRXR Antibacterial [68] B.L. Geller, K. Marshall-Batty, F.J. Schnell, M.M. McKnight, P.L. Iversen, D.E. Greenberg, Gene-silencing antisense oligomers inhibit acinetobacter growth in vitro and in vivo, J. Infect. Dis. 208 (2013) 1553–1560, http://dx.doi.org/10. 1093/infdis/jit460. R. Abes, H.M. Moulton, P. Clair, S.T. Yang, S. Abes, K. Melikov, P. Prevot, D.S. Youngblood, P.L. Iversen, L.V. Chernomordik, B. Lebleu, Delivery of steric block morpholino oligomers by (R-X-R)4 peptides: structure–activity studies, Nucleic Acids Res. 36 (2008) 6343–6354, http://dx.doi.org/10.1093/nar/gkn541. (RXR)4XB-PMO RXRRXRRXRRXRXB Antiviral [60,61] S.-H. Lai, D.A. Stein, A. Guerrero-Plata, S.-L. Liao, T. Ivanciuc, C. Hong, P.L. Iversen, A. Casola, R.P. Garofalo, Inhibition of respiratory syncytial virus infections with morpholino oligomers in cell cultures and in mice, Mol. Ther. J. Am. Soc. Gene Ther. 16 (2008) 1120–1128, http://dx.doi.org/10.1038/mt.2008.81.
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