5302 - Oligonucleotide Drugs & Gene Therapy

These degrade mRNAs or interfere w/ their processing

-Antisense oligonucleotides (ASOs)**

-Small interfering RNAs (siRNAs)**

-MicroRNA (miRNA)

-SINGLE stranded DNA or RNA molecules

-Short: 15-25 nucleotides in length

-Complementary sequence to the target mRNA; specific binding via the Watson-Crick base pairing

Antisense Oligonucleotides (ASOs)

-Degradation of Target mRNA

-Inhibition of Target mRNA Translation

-Alteration of Target mRNA Splicing

3 Mechanisms of ASOs to Modulate Gene Expression

-ASOs bind to the target RNA, forming a DNA/RNA hybrid

-RNase H1 recognizes this hybrid and degrades the RNA strand, reducing the target RNA levels

ASO Mech: Degradation of Target mRNA

An endogenous ribonuclease that hydrolyzes the RNA strand of an RNA/DNA duplex

RNaseH1

-ASO binding prevents the access of the ribosome to the target mRNA (tldr ASO BLOCKS ribosome binding to the target mRNA)

-mRNA translation is silenced, thus no proteins

ASO Mech: Inhibition of Target mRNA Translation

-ASOs bind to pre-mRNA and alter splicing

-The expression of proteins implicated in disease is thus altered

ASO Mech: Alteration of Target mRNA Splicing

A process that removes introns from pre-mRNA and connects exons to form a mature mRNA that can be translated into proteins

mRNA Splicing

Non-coding sequences

Introns

Coding sequences

Extrons

-DOUBLE stranded RNA molecules

-Short: 21-23 nucleotides in length

-Naturally-occurring

Small Interfering RNA (siRNA) Therapeutics

1. Dicer Processing

2. RISC Assembly

3. Target Recognition

4. mRNA Degradation

RNA Interference (RNAi) Steps

These work by specifically targeting and silencing certain genes at the mRNA level, thereby inhibiting the production of disease-containing proteins

siRNA Therapeutics

-Instability (rapidly hydrolyzed and degraded by nucleases in bloodstream)

-Requiring delivery systems (cannot easily cross cell membranes)

-Off-target effects (imperfect base-pairing may lead to unintended gene-silencing)

-Immunogenicity (can be recognized by TLRs, leading to inflammation, etc)

Limitations of Oligonucleotide Drugs

-Mipomersen

-Nusinersen

Oligonucleotide Drug examples

-Rituxan

-Humira

Monoclonal Antibodies examples

Induced by a mutation in hemoglobin (a protein delivering oxygen to the body's tissues)

-Causes a block in blood vessels and limits oxygen delivery

Sickle Cell Disease

-AAV (adeno-associated virus)

-RV (retrovirus)

-AdV (adenovirus)

-LNP (lipid nanoparticles)

Common Viral and Non-viral vectors used for delivering genes into cells

Which viral/non-viral vectors can be integrated into the genome?

-RV (retrovirus)

-LV (lentivirus)

Which viral/non-viral vectors can be long-term?

-RV (retrovirus)

-LV (lentivirus)

-AAV (adeno-associated virus)

Steps to Using Lentiviral Vector (LV) to Deliver Normal Hemoglobin Genes to Patient HSCs (Hematopoietic stem cells)

1. Autologous Stem Cell Collection

2. Gene Modification by Viral Vectors

-Reinfusion and Engraftment

1. Targeting

2. Cutting

3. Repairing

CRISPR/Cas9 Gene Editing Steps

-Adult

-Fetal

Forms of Hemoglobin

This drug uses CRISPR/Cas9 to disrupt BCL11A

Casgevy

Without BCL11A, what happens to the blood cell?

Goes. back to produce the fetal form of hemoglobin

In SCD patients, this form of hemoglobin is defective while the other is normal. Which one is defective?

Adult form

BCL11A

Switches fetal form of hemoglobin to adult form

This drug uses a Lentiviral (LV) vector for SCD?

Lyfgenia

Compared to oligonucleotide drugs (which only TEMPORARILY silence the faulty gene), this only requires ONE treatment for a LIFETIME

Gene Therapy

-High Cost (~2M per treatment)

-Potential Risk of Off-Target effect

-Complex ethical and regulatory issues (germline editing ***)

Limitations and Concerns of Gene Therapy

Can be passed down to future generations; prohibited but this is when they use CRISPR/Cas9 on the embryo stem cells

Germline Editing