6.

Nucleic Acids as
Therapeutic Agents
q In vivo and ex vivo gene therapy

-(A) For in vivo gene therapy, a therapeutic gene is introduced by either a viral or
nonviral delivery system into a targeted tissue of the patient
-(B) Ex vivo gene therapy entails collecting and culturing cells from an affected
individual, introducing the therapeutic gene into these cultured cells, growing the cells
with the therapeutic gene and then either infusing or transplanting these cell back in to
the patient.
• Targeting Specific mRNA and
DNA sequences

• Viral delivery system

• Nonviral delivery system

• Gene therapy
 Targeting Specific mRNA and
DNA sequences
§ Antisense RNA
- is an RNA sequences that is complementary to all or part of a functional RNA
(usually mRNA).

-To be a useful therapeutic agent, an antisense RNA or an RNA oligonucleotide

must be bind to a specified mRNA and prevent translation of the encoded protein,
with the RNA duplex eventually being transcribed.
 Targeting Specific mRNA and
DNA sequences

-A chemically synthesized antisense oligonucleotide must be readily taken up into

cells, resist degradation by cellular nuclease, and hybridize to an accessible
nucleotide sequence on the target mRNA.
 Targeting Specific mRNA and
DNA sequences
Example:
Spinal Muscular Atrophy and Antisense RNA
Spinraza®, the first FDA-approved therapy for SMA, is a treatment that targets
the SMN2 gene.
Spinraza® is an antisense oligonucleotide (ASO) approved for all ages and types of SMA.
 Targeting Specific mRNA and
DNA sequences
§ Aptamers
- are short nucleic acid sequences, either RNA or DNA, that fold into a unique
tertiary structure and bind every tightly to proteins, amino acids, drugs, or other
molecules.
 Targeting Specific mRNA and
DNA sequences

Comparison of some of the properties of aptamers with

monoclonal antibodies
 Targeting Specific mRNA and
DNA sequences
- SELEX (systematic evolution of ligands by exponential
enrichment)
Example:
Pegaptanib for ocular vascular disease: The first
aptamer therapeutic was approved by the FDA in 2004

- Pegaptanib is a pegylated (to improve its nuclease resistance) 27-

nucleotide-long RNA aptamer that targets vascular endothelial growth factor
(VEGF) and binds to the protein with an extremely high affinity (Kd=0.05nM).
a variety of distinct compounds
that inhibit angiogenesis
 Targeting Specific mRNA and
DNA sequences
-Until recently, it was not possible to select RNA aptamers that were able to
distinguish a mutant protein from its wild-type counterpart that differed in a single
amino acid.
-Selectively enrich a mixture of aptamers until several aptamers that bound more
tightly to the mutant to the native form
 Targeting Specific mRNA and
DNA sequences
§ Ribozymes and DNAzymes
- Ribozymes are naturally occurring catalytic RNA molecules (RNA metalloenzymes)
that bind to and cleave RNA molecules.
-typically 40-50 nucleotides in length

Hammerhead ribozyme Hairpin ribozyme

 Targeting Specific mRNA and
DNA sequences

- By altering the nucleotide sequences within the substrate-binding domain, a

ribozyme can be engineered to specifically cleave any mRNA sequence

Two different ribozymes bound to two different sites on

the same mRNA with the subsequence cleavages of
the mRNA at both site
 Targeting Specific mRNA and
DNA sequences
- DNAzyme: in fact, the kinetics parameters of the DNA zyme on RNA targets were
superior to those their RNA counterparts, and some DNA zymes are as efficient as
protein enzymes in terms of catalytic efficiency
 Targeting Specific mRNA and
DNA sequences
§ Interfering RNA
- RNA interference is a biological process in which RNA molecules are involved in
sequence-specific suppression of gene expression by double-stranded RNA through
translational or transcriptional repression
- miRNA
 Targeting Specific mRNA and
DNA sequences

- siRNA(small interfering RNA), shRNA (small hairpin RNA)

 Targeting Specific mRNA and
DNA sequences
Example:
Partisiran, a first RNAi drug approved in 2018
-Hereditary transthyretin amyloidosis is an autosomal (상염색체) dominant disease
- This relatively rare disease is characterized by a buildup of abnormal amyloid
protein in peripheral nerves, causing pain, paresthesia, muscular weakness, and
autonomic nervous system dysfunction
 Targeting Specific mRNA and
DNA sequences

- The drug, which works by shuttingdown the production of transthyretin, was

administerted once every 3 weeks for 1.5 years
- treatment cost approximately $ 450,000 U.S. per year
 Targeting Specific mRNA and
DNA sequences
§ Zinc Finger Nuclease
-Instead of treating a genetic disease with a therapeutic agent or providing an
additional “corrective “gene, it might be advantageous to directly repair a cells’
defective genes.
-This has become possible using either of two recently developed techniques, zinc
finger nuclease or the CRISPR-Cas system.
-Zinc finger nuclease: are engineered proteins that contain two domains- a zinc
finger domain that binds to a target DNA sequences and a nuclease domains that
cleaves the DNA

FokI nuclease ( derived from

Flavobacterium okeanokoites)
 Targeting Specific mRNA and
DNA sequences
-Double Strand Break (DSB)
-Repair system (NHEJ, HR)

-This technology has been used to disrupt/correct genes in cultured CHO cells
and in fruit flies, zebra fish, rat, human cells.
- The possibility of using zinc fingers to correct a wide range of genetic
mutations has been limited by the lack of simple procedures for producing and
selecting specific zinc fingers.
 Targeting Specific mRNA and
DNA sequences
§ CRISPR-CAS system
-The simplest form of this technology consists of a single guide RNA molecules
(sgRNA), where part of the molecule matches the target DNA sequences, and an
endonuclease from the bacterium Streptococcocus pyogenes(CAS9).

Schematic of the RNA-guided Cas9 nuclease

 Targeting Specific mRNA and
DNA sequences

- DSB repair promotes gene editing

 Targeting Specific mRNA and
DNA sequences
Example:
CASGEVY: the first CRISPR/CAS9 genome
editing therapy approved in 2023
-Sickle cell disease (SCD): is a group of inherited red blood cell disorders that affect
hemoglobin, the protein that carries oxygen through the body.
- Normally, red blood cells are disc-shaped and flexible enough to move easily through
the blood vessels.
CRISPR/CAS9 genome editing
 Targeting Specific mRNA and
DNA sequences

§ Gene editing beyond CRISPR–CAS9Base

editors