Treatment of Genetic Disease

In treating Genetic Disease, there are four levels we can work at: Function Protein mRNA DNA

Small moleculesDrugs that are approved to either: treat other diseases (these can compensate for the functional deficits caused by genetic disorders)or: treat the specific genetic disorderEXAMPLE - Marfan Syndrome - a disorder of fibrillin, heart defects, skeletal defects, abnormally tall/thin statureTreatment: 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 ChaperonsProteins 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 SkippingMethod where the aim is to skip an exon during splicingSo, this exon is excised from the primary transcriptThis 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 expressionthis 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 

TALENsTranscription 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 FingerB = TALENs

CrisprDeveloped from microbrial immune system

Intro

Function

mRNA > Protein

mRNA

DNA