CH20 - Nucleic Acid Therapeutics

rare, monogenic, undruggable

Nucleic acid therapeutics (or genetic drugs) are very suitable for what kinds of diseases?

• treats ___ or ___ disorders, as well as diseases caused by ____ protein targets

backbone, sugar, base

Main types of modifications that improve the properties of nucleic acid drugs (3)

Phosphorothioates

_____ (PSP) are analogs of natural DNA oligonucleotides in which one of the oxygen atoms of the phosphate group not involved in a phosphodiester bridge is replaced by a sulfur atom

Phosphorothioates, immune, lower

Backbone modification in nucleic acid therapeutics: ____ ( O → S )

• ____ stimulatory

• ____ target binding affinities than the unmodified counterparts

uncharged, RNase H

Backbone modification in nucleic acid therapeutics - PMOs

• ___ , which DEC serum protein binding and circulation lifetime

• Do not activate ____: relies exclusively on steric blockade-enabled regulatory mechanism to impact the target

improved, Rnase H

Sugar modifications of nucleic acid therapeutics - 2’ substituents

• influences ASO molecular conformation, resulting in ____ RNA target binding affinity and INC nuclease resistance.

• Do not recruit _____

most , stable

Sugar modifications of nucleic acid therapeutics - Locked nucleic acid (LNA)

• Drastically REDUCES the plasticity of the molecule and locks the ribose in the ( most / least ) favorable conformation to form long double-stranded molecules.

• Oligonucleotides containing one or more LNA-modified residues form RNA/LNA and DNA/LNA hybrids that are very ___.

Locked nucleic acid

_____ (LNA)

• Sugar modifications of nucleic acid therapeutics

• analogs in which the ribose ring is locked by a methylene bridge connecting the 2’-oxygen with the 4’-carbon

5mC, immunostimulatory

Base modification in nucleic acid therapeutics

• ___

• Reducing ____ effects w/o compromising Watson-Crick base-pairing

ribozymes, antisense, RNAi, mRNA, Aptamers, Gene

Types of nucleic acid therapeutics

• ____

• ____ (ASO)

• ____

• ____

• ____

• ____ Therapy

Ribozymes

catalytically active RNA molecules

Antisense

oligonucleotides designed to bind to the target RNA via base-pairing;

RNAi

the agents of RNA interference;

mRNA

in vitro transcribed (IVT) mRNA

Aptamers

DNA/RNAs that bind proteins and other molecular ligands

Gene therapy

the agents that modify genes via disruption, correction, or replacement

cleave, mRNA, Heptazyme, Angiozyme, HERzyme

Ribozymes

• Catalytically active RNA molecules that ____ target ____ in a site-specific manner

• _____: targets 5’-untranslated region of hepatitis C virus

• _____ : targets VEGF receptor VEGFR1 (Flt-1) mRNA in solid tumors (esp kidney cancer)

• _____ : targets human epidermal growth factor-2 in breast and ovarian cell carcinomas

blockade, splicing, RNase H, argonaute 2

Anti-sense oligonucleotides (ASO)

• About 13-30 nucleotides with backbone and/or sugar/base modifications

• Hybridize with RNA to regulate gene expression

• mRNA: steric blockade of translational machinery

• Pre-mRNA: modify its processing and splicing

• Target RNA cleavage

  • ____: DNA:RNA duplex

  • ____: RNA:RNA duplexes

Mipomersen

an antisense oligonucleotide (ASO) inhibitor of apo B

apo B, HoFH, single, RNase H, cleavage, dec

Mipomersen:

• an antisense oligonucleotide (ASO) inhibitor of _____

• Approved in 2013 as an orphan drug for homozygous familial hypercholesterolemia ( __ )

• short ___ -stranded DNA that complements mRNA of apo B-100;

  • the hybridized mipomersen and mRNA results in the activation of ____, which catalyzes RNA ____ and ___ apo B concentration.

• e

Nusinersen

_____ (Spinraza): an ASO for spinal muscular atrophy (SMA) by targeting SMN2

SMN2

Nusinersen (Spinraza):

• an ASO for spinal muscular atrophy (SMA) by targeting ___

SMN1, SMN2, Nusinersen, doesn’t

SMA is an autosomal recessive disorder caused by loss-of-function mutations in ____ gene, which results in motor neuron degeneration;

• ___ is a paralogue of SMN1 with a base substitution, resulting in SMN2 mRNA lacking exon 7 and producing truncated, non-functional SMN protein;

• ___ binds SMN2 pre-mRNA, modifies splicing to promote exon 7 inclusion, leading to the translation of functional SMN protein;

• Because the ASO does not target the causative gene for SMA, ASO effect ( does / doesn’t ) depend on SMN1 mutation type, making it a viable treatment option for all SMA patients.

double, RNase Dicer, siRNA, RISC, cleaved, inactive, RISC, translation

RNA interference (RNAi)

• RNAi is based on ___ stranded RNA (dsRNA), which triggers a cellular defense mechanism

• The dsRNA is degraded by an ______ into small fragment (~22 bp), including 2-nt long 3’-overhangs, which is called ___ (small interfering RNA)

• siRNA binds to ____ (RNA induced silencing complex) and is processed to 1 siRNA strand

  • If an mRNA with a sequence complementary to the siRNA is encountered by this complex, the mRNA is cleaved by an RNase and thereby rendered inactive

  • If the complementarity is not perfect, ____ may only bind to the mRNA which also blocks ____

GalNAc-siRNA , LNP

What are some technologies that allow efficient delivery of RNAi into cells?

GalNAc, ASGPR, liver, ApoE, LDLR

What are some technologies that allow efficient delivery of RNAi into cells?

• GalNAc-siRNA

  • Based on specific interaction between ____ and ___

  • Perfect for ___ hepatocytes

• LNP (lipid nano-particle)

  • Based on interaction between ____ and ____

Givosiran

a GalNAc-siRNA that inhibits ALAS1 expression in hepatocytes

ALAS1, AHP, subcutaneous

Givosiran

• GalNAc-siRNA that inhibits ____ expression in hepatocytes

• it’s indication is ___( acute hepatic porphyria )

• administration: ____

ALAS1

Increased ___ expression causes a build-up of ALA (aminolevulinic acid) and porphobilinogen, toxic intermediates that harm nervous tissue, contributing to life-threatening abdominal pain & neuropathy

Onpattro

a RNAi packaged into LNP to silence TTR gene in hepatocyte

LNP, TTR, intravenous

Onpattro

• RNAi packaged into ___ to silence __ gene in hepatocytes

• administration: ____

hATTR

TTR-mediated amyloidosis, is a genetic disease caused by mutations in the TTR gene

TTR

hATTR patients accumulate misfolded ____ protein

Aptamers

Oligonucleotides (RNA or ssDNA) that are selected for high-affinity binding to molecular targets

Macugen

Pegaptanib ( ____ ) is the first aptamer drug approved by FDA for treating diseases

Age Related Macular Degeneration

What disease/condition does Macugen treat?

• Abnormal collections of extracellular material build up in the macula, which is the center of the retina.

SELEX

Systematic Evolution of Ligands by Exponential enrichment

SELEX

laboratory method used to identify aptamers, which are short DNA or RNA sequences that bind specifically to a target molecule such as a protein, drug, or cell.

more, wider

ssDNA vs RNA aptamers:

• DNA is ___ stable than RNA, which makes the selection process easier;

• on the other hand, RNA may create a ___ set of three dimensional structures and can be synthesized inside the cells.

library, exposed, kept, PCR, repeated

In SELEX:

1. A large ____ of random nucleic acid sequences is created.

2. The sequences are ___ to the target molecule.

3. Sequences that bind the target are ___ , while nonbinding sequences are removed.

4. The bound sequences are amplified (usually by ___ ).

5. The process is ____ multiple times to enrich for the strongest binders.

The key step that makes SELEX so powerful is the iterative selection and amplification process. Repeatedly enriching high-affinity binders allows researchers to isolate aptamers with very high specificity and strong binding to the target.

spiegelmer

a “mirror-image aptamer.”

• It is made from L-nucleic acids (the mirror form of natural D-RNA/D-DNA), and it binds a natural biological target with high specificity.

mirror, L

spiegelmer is a “ ___ image aptamer.”

• It is made from ___ nucleic acids (the mirror form of natural D-RNA/D-DNA), and it binds a natural biological target with high specificity.

mirror, SELEX, spiegelmer, L

How you obtain a spiegelmer

• You first synthesize a ___ image (D-form) version of the target molecule (usually a protein or peptide).

• You perform standard in vitro selection using ___ to find a D-aptamer that binds the mirror target.

• Then you chemically synthesize the mirror-image of that aptamer (L-form) → this is the ____ .

• The ___ aptamer binds the natural (D-form) target in the real body

AAV

adeno-associated virus

long

one advantage of AAV-mediated gene

• Can induce ___ lasting effects

• That’s because Adeno-associated virus delivers genetic material into cells, where it can persist as an episome and continuously express the therapeutic gene

temporarily

ASO and RNAi drugs only work ( long / temporarily ) and usually need repeated dosing.

5, neutralizing

What are some limitations of AAV-mediated gene therapy?

• Have a packaging limit of < ___ kb (not good for large genes)

• ____ antibodies are widely prevalent

Zolgensma

Most Expensive Drug for SMA

SMN1, AAV, intravenous

Zolgensma

• gene target is ____ ( AAV9)

• ailment is ____

• administration: _____

Lenmeldy

World’s Most Expensive Drug is ____

MLD, ARSA, sulfatides

Lenmeldy

• Gene therapy for ____ : metachromatic leukodystrophy, an autosomal recessive genetic disorder.

  • caused by a deficiency of the enzyme Arylsulfatase-A (___ ).

  • w/o this enzyme, ____ build up, eventually destroying the myelin sheath of the nervous system.

ARSA

Lenmeldy: Patient’s own CD34+ cells (hematopoietic stem cells) transfected with a lentivirus carrying a functional ___ gene.

Ex vivo

Delivery of CRISPR therapy ( ____ )

• target cells extracted from the patient, cultured and expanded in vitro,

• delivery of the CRISPR components to yield the desired edits, selection, and expansion of edited cells, and

• finally reintroduction of the edited cells into the patient

In vivo

Delivery of CRISPR therapy ( ____ )

• vehicles can be delivered via IV infusions to the patient, where the CRISPR cargo travels through the bloodstream via arteries leading to the target tissue,

• or locally delivered with injections directly to target tissue.

• Once delivered, the edits are facilitated in vivo to provide therapeutic benefit.

CRISPR/Cas9

• allows direct editing of DNA in cells, most commonly by cutting the genome at a specific site so the cell repairs it.

• This can be used to knock out faulty genes or correct mutations in diseases like sickle cell disease.

Base editors

chemically change a single DNA base (e.g., C → T or A → G) without making a double-strand break