Oligonucleotides Flashcards

Question

Tell me about the role of CpG oligonucleotides with the innate immune response

Answer 1

CpG oligonucleotides and innate immune response (Toll-like receptors - TLR9) causing release of cytokines (^MeCpG) in eukaryotes.

Answer 2

How would you demonstrate that it is? Scrambled/mutated oligos. Mutate the target. Some may act as aptamers (folded structures with specific binding sites).

Answer 3

Deprotect to leave a reactive hydroxyl group Coupling with reactive phosphoramidite and tetrazole (essentially a catalyst) Capping (99% efficient but protects unreacted molecules attached to solid support – prevents incorrect coupling in following cycle) and oxidation of phosphite (to generate phosphate) Repeat A phosphoramidite nucleoside is a derivative of a natural or synthetic nucleotide, with a N ́N ́-diisiopropyl phosphoramidite group attached to the 3 ́-hydroxyl. It is more reactive than the naturally occurring nucleotides and commonly used during ON synthesis. To prevent undesired reactions, the phosphoramidite nucleosides have their functional groups protected by an acid-labile dimethoxyltrityl or base-labile 2 ́-cyanoethyl groups. The synthesis proceeds via cycles containing the following steps: deprotection, coupling, capping and stabilization. Each cycle results in the attachment of an additional nucleotide from the first nucleotide attached to the solid support made of controlled pore glass (CPG). First, during deprotection, the dimethoxytrityl group protecting the 5 ́-hydroxyl is removed with trichloroacetic acid in an inert solvent (toluene or dichloromethane), leaving a reactive hydroxyl group to attach the following nucleotide. In the coupling step, reaction between the hydroxyl group and a tetrazole-activated phosphoramidite creates a 5 ́-3 ́ linkage to attach the additional nucleotide. After reaction, excess of reagent and tetrazole are washed out. Although this step is usually 99% efficient, a small amount of hydroxyl group remains freely reactive. To avoid further reaction in the following coupling steps, a capping step is performed with acetic anhydride to block the free hydroxyls. The last step in the cycle is the stabilization that results in the oxidation of the phosphite into a phosphate using iodine and water

Answer 4

Depends on base pairing and stacking interactions (length/ sequence) UV/ fluorescence melting allow comparison of melting temperatures (Tm ) between duplexes

Answer 5

RNA helices form A-type structure DNA helices form B-type structures

Answer 6

Sugar pucker is a ribose ring which sugar attaches. It is unable to lie flat S-type DNA lies below the ring RNA N-type the 3’ carbon sticks up More likely to have N-type over S-type as we want the most stable complexes with RNA The RNA-DNA complex is less stable than the DNA-DNA helix

Answer 7

* Universal * Automated synthesis * Simple drug design * Easy to adjust affinity/ selectivity

Answer 8

* Poor targeting/ uptake * Degraded by nucleases * CpG can induce immune response

Answer 9

Can change the **base, sugar, backbone** (can replace the atoms to alter the charge distributions, in some you can get rid of backbone all together), and **conjugates** (e.g., cholesterol, and other which can increase the affinity as well as the uptake)

Answer 10

They increase affinity and duplex stability * **5-methyl-C** * **2-amino-A** * **G-clamp** improved interactions: * hydrogen bonding * hydrophobic/ stacking * charge * etc.

Answer 11

They improve nuclease resistance ways in which this can be done; forming... * **phosphorothioate:** changing the oxygens for sulphur atoms * **Methylphosphonate:** Me group added * **Phosphoramidate:** N added and also can be used in combination where an S is used in place of an O

Answer 12

1st generation In general, these modifications generate less stable duplexes due to steric hindrance (e.g., S atom or lack of stereospecific synthesis) Most common is changing the Oxygens for sulphurs (makes much more resistant to nuclear degradation). Presence of sulphur means it binds more to serum proteins, preventing the clearance of oligonucleotides and being less charged facilitates the uptake into cells Chiral centre (R and S forms) formed if only one sulphur is swapped in. All those made for commercial use could be an R or an S form If charged removed and Me added, these are useful clinically, far less soluble as charged has been removed, restrictions in concs made, no charge means it crosses membrane more easily N added, could be combined with other modifications i.e., swapping O for S All these modifications give great resistance against nucleases Very useful analogues Don’t activate RNaseH Methyl phosphonate and phosphoramidate do not activate RNaseH?

Answer 13

Improve nuclease resistance 2nd generation Add things in order to force the North/ up configuration Generates a more stable duplex The different ways: * **2'-deoxyribose** * **2'-methoxy (O-Me)** * **2'-O-(2-methoxy)ethyl (MOE)** * **Locked/bridge nucleic acid (LNA/ BNA)** Strongest way of doing this (not used clinically) is locked/bridged nucleic acids; links 2’ and 4’ carbon with methylene bridge across which forces 3’ carbon into north config., which increases stability. Would want to use in every other position otherwise structural constraint would be too great

Answer 14

Improve nuclease resistance (and affinity) 3rd generation **Morpholino:** sugar is a 6 ring, still P in backbone, expensive, clinically useful structure **PNA:** backbone is not a peptide, has extra C, side groups are the bases, based on pseudopeptide structure, no charge, forms strong complexes with RNA targets, downside is it’s not soluble, have to add charged groups to be soluble

Answer 15

**Chimeric 'gap-mer' oligonucleotides** (RNase H compatible) * Since sugar and backbone modifications reduce susceptibility to nuclear digestion they can also decrease/ prevent RNase H cleavage * Make modified antisense, the ends are modified and protected, central region is normal DNA (6-10 nts), RNaseH will still recognise and cleave target mRNA * Gap-mer is part modified and part standard

Answer 16

**Minor groove binders:** Netropsin **Intercalators:** Anthraquinone and Pyrene (flat molecules that stack tightly between base pairs, protect from degradation and increase affinity) **Edge binders:** Spermine and Spermidine (highly charged, common small molecules in all cells that bind to DNA, +ve charge to neutralise the DNA, protonated at physiological PH, increase stability)

Answer 17

**Cross-linking agents** **Cleavage agents**

Answer 18

Oligonucleotides are larger (10 kDa) than traditional small-molecule drugs (\<1 kDa) and are highly polyanionic (negatively charged)

Answer 19

Due to poor diffusion across the hydrophobic bilayer

Answer 20

Can gain access through non-specific binding to cell surface proteins internalisation by receptor-mediated endocytosis release from endosomes (Not always productive as can become trapped in endosomes)

Answer 21

Using backbone modifications that lack the negative charge

Answer 22

2ʹ-H, 2ʹ-deoxy; 2′-MOE, 2ʹ-O-methoxyethyl; ALT, alanine aminotransferase; apoB-100, apolipoprotein B-100; apoC-III, apolipoprotein C-III; ATTR, transthyretin amyloidosis; CMV, cytomegalovirus; CNS, central nervous system; DMD, Duchenne muscular dystrophy; FCS, familial chylomicronaemia syndrome; HoFH, homozygous familial hypercholesterolaemia; ISR, injection site reaction; IT, intrathecal; ITV, intravitreal; IV, intravenous; LDL-C, low-density lipoprotein cholesterol. MHLW, Japanese Ministry of Health, Labour and Welfare; PMO, phosphorodiamidate morpholino oligomer; SMA, spinal muscular atrophy; SC, subcutaneous. TTR, transthyretin; VLDL, very-low-density lipoprotein. All drugs are modified with phosphorothioate linkages, except for the PMOs. All 2ʹ-MOE chemistries include 2ʹ-deoxy sugar residues to support RNase H1 activity, unless specified as fully modified. Information in the table adapted with permission from ref.48, Elsevier.

Answer 23

* **Vitravene (fomivirsen, ISIS 2922)** * **Alicaforsen (ISIS-2302)** * **Mipomersen (kynamro; ISIS-301012)** * **Genasense (oblimersen)**

Answer 24

The first antisense oligonucleotide to be licensed for clinical use. **Active against CMV retinitis** (AID patients who were susceptible to this virus due to compromised immune system, leading to blindness with this virus) **21-mer phosphorothioate** (has sulphurs in each of the links) 5'-GCGTTTGCTCTTCTTCTTGCG-3' Targets cytomegalovirus (CMV) mRNA encoding for the CMV polymerase CMV causes retinitis in AIDS patients **Administered intravenously** (**intraocular-** direct injection into the eye) Approved by the FDA and available commercially since 1998 **Not used now** (still works well), but other therapies for AIDs are so successful that the incidence of CMV retinitis is almost zero so it’s not required Structure has a mixed chirality as it has R or S forms

Answer 25

**First generation 20-mer phosphorothioate** oligonucleotide 5’-GCCCAAGCTGGCATCCGTCA Targets the 3’-untranslated region of ICAM-1 mRNA ICAM-1 is a glycoprotein involved in cell trafficking in inflammatory bowel pathophysiology Crohn's disease (failed phase III) Ulcerative colitis (passed phase III- approved by FDA) **Administered intravenously or topologically** (enema formulation!) Available on named patient supply from 2007 (unlicensed) **Activates RNaseH** and causes a down regulation of mRNA

Answer 26

Second generation 20-mer phoshorothioate oligonucleotide containing ten 2’-MOE modifications (Approved by the FDA October 2013) **G\*^mC\*^mC\*U\*^mC\***AGT^mCTG^mCTT^m**CG\*^mC\*A\*C\*C\*** **(2’-MOE = Gapmer)** First ASO to exhibit success through systemic administration (by subcutaneous injection). Targets the coding region of human apolipoprotein B (apoB) mutations in apoB cause hypercholesterolemia Acts by steric block / RNaseH activation Treats patients with hypercholesterolemia with increased plasma concentrations of low-density lipoprotein (LDL) particles Administered intravenously or subcutaneously

Answer 27

* Targeted against bcl2 (antiapoptotic gene- inhibits apoptosis) * Aim is to sensitise cancer cells to the effects of other chemotherapeutic agents. * Failed in clinical trials on phase III as an anti-cancer drug for lung cancer

Answer 28

Splice-switcing oligonucleotides (SSOs)

Answer 29

Interactions between pre-mRNA, small nuclear ribonucleoproteins, and splicing factor proteins, and relies on multiple levels of regulation

Answer 30

Splicing occurs in the nucleus so it’s got to cross not only the plasma membrane but also the nuclear membrane

Answer 31

Hybridisation of ASOs to **splice sites**, **enhancer elements** within the pre-mRNA transcript can be used for **exon skipping** Whilst hybridisation to **silencer elements** can be used for **exon inclusion** (i.e., restoration of a splicing pattern)

Answer 32

**Exondys 51^(R) (Etepilrsin)**

Answer 33

5’-CTCCAACATCAAGGAAGATGGCATTTCTAG **(30-mer morpholino)** (3rd gen) Approved for marketing by the FDA in 2016 but costs $300k/patient/year! Treats patients with **Duchenne muscular dystrophy (DMD)**, an **X-linked** disease caused by mutations in gene encoding dystrophin (out-of-frame / premature termination) Dystrophin helps connect cytoskeleton of a muscle fibre to surrounding extracellular matrix Targets splice site of exon 51 causing it to be skipped during splicing, yielding a truncated in-frame protein that is 50% functional (misses an intron in the middle) **No RNase H activation** and must also enter nucleus

Answer 34

**Spinraza® (Nusinersin)**

Answer 35

5’-TmCAmCTTTmCATAATGmCTGG (18-mer fully modified with PS / 2′-O-MOE) (2nd gen) Spinal muscular atrophy (SMA) caused by mutations in the ‘survival of motor neuron’ gene (SMN1) --\> no SMN1 protein We possess a second copy of the gene (SMN2) but it contains a Unique Silencing Element (USE) which prevents the inclusion of exon 7 --\> protein degraded Blocks the USE resulting in the inclusion of exon 7 and synthesis of a fully functional protein (see over page) Note: **NO RNase H activation and must enter the nucleus** Delivered by a lumbar puncture directly into the cerebrospinal fluid (CNS compatible)

Answer 36

Antisense reviews (blackboard uploads) Antisense reviews (blackboard uploads): Molecular mechanisms of antisense oligonucleotides. Crooke (2017) Nucleic Acid Therapeutics. 27; 70. Chemistry, mechanism and clinical status of antisense oligonucleotides. Shen et al. (2018) Nucleic Acids Research. 46; 1584. Antisense oligonucleotides: modifications and clinical trials. Sharma et al. (2014) Medicinal Chemistry Communications. 5; 1545. New momentum for the field of oligo therapeutics. Aartsma-Rus (2016) Molecular Therapy. 24; 193. FDA-approved oligonucleotide therapies in 2017. Stein et al. (2017) Molecular Therapy. 25; 1069. Tegsedi. (2019) Pharmaceuticals. 12; 78 Waylivra (https://doi.org/10.1007/s40265-019-01168-z) Recent news commentary: https: //www.bbc.co.uk/news/health-42308341 (Huntington’s) https: //www.theguardian.com/science/2017/dec/11/excitement-as-huntingtons-drug-shown-to-slow-progress-of-devastating-disease (Huntington’s) Big Pharma: http: //www.ionispharma.com/ (website) https: //www.mixcloud.com/discover/nature+science+scientist/ (Crooke podcast)

Answer 37

endogenous RNA interference

Answer 38

A biological process in which dsRNA molecules (miRNAs) inhibit gene expression by degrading target mRNA molecules

Answer 39

Natural long RNA molecules which is degraded and cut at the ends dsRNA in RISC complex is separated into two strands; **guide strand** (useful), one released as **passenger strand** and is degraded RISC finds complementary sequence to mRNA and binds to it, recognising the target The RISC then leads to degradation of RNA target so its removed Guide strand is degraded, RISC with guide can find another mRNA with the same sequence leading to degradation of RNA target RNA is less stable than DNA. When RNA is in ds form it is relatively stable

Answer 40

Making synthetic silencing RNA: company called **Alnylam** has a clinical development pipeline with a number of compounds in trial. Some have made to commercial use e.g., Patisiran

Answer 41

5’G**U**AA**CC**AAGAG**U**A**UUC**CA**U**dTdT 3’dTdTCAUUGGUUCUCAUAAGGUA (21-mer duplex, **thio phosphoramidate**)-

Answer 42

First siRNA to be approved for therapy by RNAi (in **2018**) Treats patients with **hereditary transthyretin-mediated amyloidosi**s that exhibit nerve damage and heart problems due to the build-up of misfolded transthyretin Targets mRNA encoding for the **mutant transthyretin** preventing aggregation (acts by RISC-mediated cleavage)- Mutant (that forms amyloidosis) and WT forms Delivered via **intravenous injection** as a **lipid nanoparticle formulation** Selectively targeted to **liver** where transthyretin is synthesised **Note:** ASO Tegsidi® has also been approved that acts on the same mRNA

Answer 43

By triplex formation

Answer 44

**Rules:** * Simple rules that C binds to GC and T binds to AT * The C has to be protonated though when binding **Cons:** * The PK increases from 4 to about 5-6 but this is still physiological- unstable complexes * Cytosine derivatives have the same bonding pattern but higher PKs that work close to physiological * strand targeting only contains purines * Limited by PH and the sequences with what they can target

Answer 45

* be addressed using oligonucleotide chemistry * These are less stable than if just recognising a long string of purines * Works at physiological PH * Once they bind and form a 3-strand complex, is it silenced? TF cannot bind and cannot be read. Non-physiological complex formed in 3-strand (selectively switching off this gene)

Answer 46

* Only two copies of the duplex target per cell (number of targets is low) * Directed modifications to the duplex target can invoke permanent alterations to gene activity

Answer 47

* Target gene sequence must contain an oligopurine tract. * Cannot bind a physiological pH * Lower stability than duplex formation * Must also permeate the nuclear membrane (antisense oligonucleotides only have to pass plasma membrane to work in cytoplasm, triplex technology or splicing that works in nucleus have to pass two membranes)

Answer 48

* Vasquez et al. showed TFOs can induce mutations at specific genomic sites in adult mice * Mutations introduced due to recognition and mis-repair by DNA repair pathways (e.g., NER) * Mice were given daily intraperitoneal injections with 1 mg/day of oligonucleotide * 5-fold rate of mutation in the cells * Other target genes are mutated to a lower degree * Shows it gets to sequence it supposed to (whether it’s used clinically will be told in time)

Answer 49

An oligonucleotide version of an antibody Aptamers are short, single-stranded DNA or RNA (ssDNA or ssRNA) molecules that can selectively bind to a specific target, including proteins, peptides, carbohydrates, small molecules, toxins, and even live cells. Aptamers assume a variety of shapes due to their tendency to form helices and single-stranded loops.

Answer 50

Aptamers are **DNA or RNA oligonucleotides** (25-80 nts) that **fold into 3D structures** which are unique (lowest energy form, and best internal base pairing) and bind to target molecules with **high affinity and selectivity** (KD = 10^-9 M)

Answer 51

A natural aptamer in existance is **riboswitches** These regulate gene expression a riboswitch is a regulatory segment of a messenger RNA molecule that binds a small molecule, resulting in a change in production of the proteins encoded by the mRNA.

Answer 52

Lack the **immunogenicity** of proteins (e.g., antibodies)- they have the same toxicity as if treating people with a piece of DNA

Answer 53

Generate **random pool of oligonucleotides** **Each has a unique sequence** which can fold into its own structure

Answer 54

Created by in vitro selection from a large random sequence pool by **SELEX** (see over page but don’t need to learn in detail)

Answer 55

* Generate a **synthetic piece of RNA** which has **constant regions** at either end and a **random region** in the centre * Every strand that is made has a **unique sequence** at a low concentration. However, even though there is a low concentration of each unique sequence the overall pool has lots of combinations * Out of all of the combinations, **maybe one will bind to our targe**t and the rest will fold into structures which are irrelevant and not useful * Once you add the target, one should bind and those which are unbound should be **washed** away as it is now useless material * You take the sequence that bound to the target and **reverse transcribe the RNA into DNA** * go thorugh a thorugh sequences of **PCR** in order to generate more of the wanted sequence * Then **sequence it** (might be lucky if you get more than one sequence that binds to the target) * The one which binds to the target will bind with a **very high affinity**

Answer 56

* DNA sequence that binds to thrombin (involve in blood clotting) * Short sequence which binds selectively and tightly to thrombin * Folds into **G-quartet and quadruplexes** * Has a **metal ion in the middle** of the quartet * This sequence folds into unique structure which has a **pocket which binds to region of thrombin**

Answer 57

**Macugen (Pegaptinib)** Used for treating **macular degeneration** (degeneration in the visual system, only have a narrow point of focus, caused by vascularisation at back of eye)

Answer 58

Active against the **VEGF-165 isoform** ## Footnote **Kd ~ 50 pM (high affinity)**

Answer 59

administered by intravitreal injection (into eye) every six weeks.

Answer 60

5’-**PEG**-CGGAAUCAGUGAAUGCUUAUACAUCCG-**dT** 27-mer 2’-O-Me oligonucleotide containing **polyethylene glycol (PEG)** at its 5’-end and 3’-3’ terminal **dT** linkage to help with transport and nuclease resistance

Answer 61

Treats patients with age-related **macular degeneration of the retina**

Answer 62

Targets the **heparin binding domain of VEGF-165** preventing its interaction with VEGFR1 and VEGFR2, reducing angiogenesis and disease progression (KD of 50 pM)

Answer 63

VEGF is a signalling protein that promotes the growth of new blood vessels VEGF is critical in **angiogenesis** and can lead to ocular degeneration **Angiogenesis**: this is the formation of new blood vessels. This process involves the migreation, growth and differentiation of endothelial cells, which line the inside of wall of blood vessels This process is controlled by chemical signals in the body