Treatment of Genetic Disease

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In treating Genetic Disease, there are four levels we can work at:

Function

Protein

mRNA

DNA

Small molecules

Drugs that are approved to

either: treat other diseases (these can compensate for the functional deficits caused by genetic disorders)

or: treat the specific genetic disorder

EXAMPLE - Marfan Syndrome - a disorder of fibrillin, heart defects, skeletal defects, abnormally tall/thin stature

Treatment: Losartan

Angiotensin II Type I receptor antagonist

Widely used to treat hypertension

Clinical trials underway for Marfan Syndrome

Outcomes so far: reduction in enlargement of aortic root, reduction in serum TGF-beta levels (too high in Marfans because of lack of sequestering of TGF-beta by fibrillin)

Molecular Chaperons

Proteins that can: stabilise unfolded proteins, unfold them, translocate them across membranes, take them to be degraded, or assist with their proper folding or assembly.

They play a role in proteostasis:

"The concept that there are competing and integrated biological pathways within cells that control the biogenesis, folding, trafficking and degradation of proteins present within and outside the cell"

EXAMPLE - Cystic Fibrosis - caused by mutation in CFTR gene causing faulty CFTR ion channels that move sodium and chloride across epithelial membranes

F508Del - most common mutation

Affects folding of the CFTR protein and trafficking to the cell surface

HSC70 and HSP70 are molecular chaperons

HSC70 ubiquitinates mutant CFTR so that it is degraded

HSP70 helps fold CFTR to rescue some of the mutant phenotype

These molecular chaperons can be modulated to reduce some of the symptoms of CF:

Chemical Matrine downregulates the expression of HSC70, rescuing some of the mutant CFTR from degradation

Restores CFTR trafficking

Monoclonal Antibody (mAbs)

Target specific proteins

Inhibit or activate protein function

Nonsense Suppression

Normally a nonsense mutation will induce nonsense mediated decay

Aminoglycosides allow readthrough of the premature termination codon (PTC) through insertion of a novel aa

E.g. Alturen

This drug has been trialled on those with CF and DMD, still debate as to whether it works

Also being tried with siRNAs, see nonsense mediated decay section.

Exon Skipping

Method where the aim is to skip an exon during splicing

So, this exon is excised from the primary transcript

This can be done using an antisense oligonucleotide or a molecular patch

EXAMPLE

In DMD, a deletion has resulted in a frameshift, meaning that a severely truncated dystrophin protein is produced that is degraded by nonsense mediated decay

It is possible to create an in-frame transcript by inducing another exon deletion that cancels out the first (exon skipping)

This will result in a truncated but partially functional dystrophin protein being produced - BMD phenotype

RNA Interference (RNAi)

siRNA inhibits gene expression

this is beginning to be used to treat macular degeneration - shown to increase visual acuity and improve foveal thickness

Zinc-Finger Nuclease

Artificial restriction enzyme

Formed by a zinc finger (small protein motif) and a DNA cleavage domain (usually FOK1 - bacterial restriction endonuclease)

The zinc finger domain can be engineered to target specific sequences

ZFNs must dimerise to cleave so two different molecules must be used for non-palindromic sequences

Can be used to disrupt a gene, correct a gene, or insert a section of DNA

NHEJ - non homologous end joining - no homologous donor segment, break in dsDNA is repaired but ends do not have to be homologous

HDR - homology directed repair - double stranded break is repaired, incorporating the homologous donor segment

TALENs

Transcription Activator-Like Effector Nucleases

Artificial restriction endonuclease

Has FOKI cleavage domain - nonspecific, must dimerise to function

TAL Effector DNA binding domain - can be engineered to target specific DNA sequences

A = Zinc Finger

B = TALENs

Crispr

Developed from microbrial immune system

Intro

Function

mRNA > Protein

mRNA

DNA