Treatment of Genetic Disease

What percentage of rare diseases have treatments?

Fewer than 6% have approved treatment option  

How are genetic vs non genetic treatments made?

• Strategies is different from the non-genetic version of the disease  

• For the non-genetic versions, the treatment focuses on manipulating the protein  

• Can be done for some genetic diseases  

• For most genetic the protein centred treatment might not work as well as the mutant proteins are continually produced  

What are the different treatment targets?

• DNA

• RNA

• Translation

• Protein function

How can DNA be targeted?

Could use gene therapy or gene editing  

How can RNA be targeted?

Exon skipping, RNAi and antisense oligonucleotides  

How can translation be targeted?

Nonsense suppression  

How can protein function be targeted?

Small molecules, chaperones, mAbs, replacement therapy  

What are the features of small molecule drugs?

• Repurpose the FDA approved small molecule  

• Drugs that are already used for the treatment for other diseases  

• Develop targeted drugs based on the molecular mechanism of the genetic disease  

• Gleevec for leukaemia and ivacaftor for cystic fibrosis  

What are the features of Marfan syndrome?

• A disorder that affects the connective tissue  

• Caused by mutations in the FBN1 gene encoding Fibrilin-1  

• Produces tall thin figure, long limbs and fingers, dislocated lens, dilation of aorta or aortic aneurism, sudden death 

What causes Marfan syndrome?

• Normally have an elevated level of serum TGF-B  

• TGF-B is secreted as in inactive form and binds to the laten TGF binding protein associated with fibirilin-1  

• Fibrilin-1 potentially regulates the release and activation of TGF-B  

• Mutant protein fails to sequester TGF-B and leads to elevated TGF-B serum levels  

How are small molecules used for Marfan syndrome?

• Key regulator of laten TGF-B regulator is thrombospondin-1  

• Produced from the activation of the Ang II signalling via the AT1 receptor  

• Block the TSP1 can downregulate the TGF-B activation  

• Losartan is a potent inhibitor of the AT1 receptor and is used for hypertension and became a candidate for Marfan's Syndrome  

What did Losartan clinical trials show?

• Initial data was very promising – showed decreased serum levels and reduced aortic root growth  

• More recent trials showed no beneficial effect compared to beta blocker treatment  

• Showed an additive benefit on top of the beta blocker for the paediatric trials  

• Losartan could be a useful and low-risk alternative to beta-blockers in the long term management due to reduced side effects  

What are the features of HGPS?

• Premature aging and accelerated and severe atherosclerosis  

• Caused by mutation in LMNA which encodes Lamin A 

• Lamin A is important for a proper nuclear membrane structure and function  

• Mutant has characteristic nuclear blebbing which are small protuberances of the nuclear membrane  

What are the features of progerin?

• Progerin is the mutant form of the protein  

• C to T at 1284 position impacts the normal splicing  

• 50 amino acids that have an enzyme binding site are missing in exon 11  

• ZMPSTE24 is critical for the C terminal processing and the maturation of Lamin A  

How is progerin edited?

• A farnesyl group is added to the CAAX motif by farnesyl transferase  

• Anchors the protein to the ER membrane  

• The enzyme cleaves the AAX and the protein is carboxymethylated  

• Lamin A then undergoes a second cleavage in the C-terminal globular domian and releases the mature protein 

How does progerin cause disease?

• Due to the abnormal splicing there is no cleavage site so it is 5' farnesyl and tethered to the membrane and accumulates at the membrane  

• Therapeutic blocking of the attachment to the nuclear membrane  

• Using a farnesyl transferase inhibitor  

How are small molecules used for HGPS?

• FTI treatment improves the nuclear shape and reduces the blebbing  

• Could prevent the onset of the cardiovascular phenotype by preventing VSCM loss in the aorta  

• Vascular stiffness, bone structure and audiological status were vastly improved  

How is chronic myeloid leukaemia caused?

• Reciprocal translocation of chromosome 9 and 22  

• Creates a fusion gene BCRABL  

• ABL is a tyrosine kinase, the fusion with BCR causes it to be in the locked active position due to ATP structure  

• Causes uncontrolled cell cycle progression causing Leukaemia  

How is a small molecule inhibitor used for AML?

• Gleevec can bind to the ATP binding site to the kinase and blocks the ATP binding  

• Inactivates the kinase activity  

• First line treatment for Philadelphia chromosome positive AML  

What are molecular chaperones?

• Chaperones are a large group of unrelated protein families whose role is to stabilise unfolded proteins, unfold them for translocation across membranes for segregation and assist their correct folding and assembly  

• Key regulator in protein homeostasis  

What do chaperones do?

• Around 180 chaperone components  

• Together with regulators are used for proteostasis  

• Involves protein folding, unfolding, refolding, remodelling, disaggregation and degradation  

What causes cystic fibrosis?

• Cystic fibrosis is caused by a mutation in the CFTR gene for the chloride channel  

• F508del occurs in 90% of CF cases  

• Stops the protein being able to fold properly and can't target the cell membrane  

How are chaperones used in CF?

• HSP70 and HSC70 are involved in processing of CFTR  

• HSC70 binds to the ubiquitinated mutant protein for proteosome degradation  

• HSP70 helps the folding of CFTR to rescue the mutant  

How is matrine used for CF?

• Matrine can downregulate HSC70 to stop the mutant degradation  

• Accumulated mutant is then targeted by HSP70 for proper folding and increases release from the ER  

• Helps the travelling to the cell membrane  

• Increased cellular CFTR and stopped the trapping of the mutant CFTR from the ER  

How do CFTR modulators work?

• Correctors are the chaperones and help the folding  

• Potentiators can help opening of the defective chloride channels  

• Amplifiers that enhance protein transcription  

• Stabilisers that increase the cell surface protein stability  

• To maximise the effect different modulators are used in combination  

What are mAbs used for?

• Target specific protein molecules  

• Regulate the protein function by inhibiting or activating  

How do mutation specific mAbs work?

• Would be good for genetic disease if the antibody was defined for the function of the mutant protein  

• Designed against Lamins with R453W and R482W that cause EDMD and FPLD  

• EDMD causes skeletal and or cardiac muscular dystrophies  

What causes haemophilia A?

• Produced by the lack of factor 8 for blood coagulation  

• Factor 8 is cleaved by thrombin to produce factor 8a  

• These subunits bind to factor 9a and factor 10 to bring the factors into proximity and house the catalytic activity of factor 9a 

How does emicizumab work?

Antibody that can bind factor 9a and factor 10 mimicking the function of factor 8a  

What are the features of LSD?

• LSDs are heterogenous group of genetic disorders characterised by the accumulation of undigested or partially digested macromolecules  

• Non degraded molecules accumulate in lysosomes and distort them damaging cell function  

How does enzyme replacement therapy work in LSD?

Alpha-glucosidase has been used for the treatment of Pompe disease 

How does nonsense suppression work in DMD?

• Aminoglycosides can supress the effect of the nonsense mutation 

• Incorporates a random amino acid at the PTC site for read through  

• Antibiotics have severe side effects 

• In mice used for DMD and restores dystrophin expression  

How was nonsense suppression used for CF?

• Nonaminoglycoside suppressor PTC124 was produced  

• Used for cystic fibrosis and increases the CFTR expression  

What are the limitations of nonsense supression?

• Doesn't correct NMD  

• Limited number of mRNA available for the correction  

• The random amino incorporation at the PTC might not restore the protein function  

• Especially if the conservation of the amino acid at that residue is important  

• Efficiency of the read through can change depending on the stop codon  

• Could read through a physiologically determined stop codon leading to unexpected outcomes  

What is used in exon skipping?

• Skipps an exon in mRNA splicing  

• Done using specifically designed antisense oligonucleotides  

How is DMD caused?

• Breakpoint of the deletion is normally in an intron and causes exons to be removed and shifts the reading frame  

• Further deletion of other exons can then cause the mutation to be in frame  

How does exon skipping work in DMD?

• DMD normally has exon 50 deleted causing the frame shift and NMD  

• Deleting 51 causes 49 and 52 to join there is no frameshift  

• PRO051 oligonucleotides binds to exon 51 and masks its exclusion  

What drugs are used for exon skipping?

• Clinical trials produce varying results  

• PRO051 had a low clinical benefit and toxicity  

• Eteplirsen was approved  

What are the limitations of exon skipping?

• Efficient delivery of antisense oligonucleotides  

• Short duration of the effect 

• Diversity of mutations  

• Efficacy and toxicity   

What are the features of RNAi?

• Reduces the mRNA of the mutant proteins with dominant negative effects 

• RNA induced gene silencing  

• miRNAs are endogenous non-coding RNAs of 22nt  

• siRNAs are mostly synthetic miRNAs of 20-25nt  

How does RNAi work in the cell?

• Primary microRNA is transcribed by RNA polymerase II and has a hairpin structure  

• Primary RNA is processed by DROSHA to produce precursor micro-RNA and exported to the cytoplasm 

• Cleaved by DICER to produce microRNA and unwinds to single strand  

• The guide strand is then incorporated into the RISC complex which guides the degradation of the target mRNA  

How are RNAi added to the cell?

Short hairpin RNAs can be delivered into the nucleus by viral vectors or siRNA directly to the cytoplasm for targeted gene silencing  

How is RNAi used in therapy?

• Patisiran for amyloidosis TTP protein  

• Is in lipid nanoparticles and intravenously administered  

• Taken in by the liver cells and siRNAs are loaded onto the RISC  

• Antisense binds to the complementary TTP mRNA for mutant and wild type mRNA leading to degradation  

What are RNAi limitations?

• Specificity as could have off target effects  

• Efficiency of delivery  

• Duration of the gene silencing  

How do antisense oligonucleotides work?

• ASOs are synthetic DNA oligonucleotides that are 15-25 bp  

• Designed in antisense that is complementary to the RNA of interest  

• Delivered into the cells and bind to RNA  

• Produce RNA-DNA heteroduplexes  

• This is a substrate for RNase H and the target RNA is disrupted  

• This means that no protein is translated  

How are ASOs used for ALS?

• 12-20% of familial ALS cases are caused by mutations in the SOD1 gene  

• Tofersen is an ASO that targets the SOD1  

• After a year SOD1 protein levels are reduced and reduced rate of decline in lung function and mobility  

How is genome editing used for genetic disease?

• When there is a double strand break the pathways are triggered to repair through NHEJ, HDR  

• NHEJ happens when there is no homologs DNA template and produces an insertion or deletion causing disruption  

• Can be used to disrupt a gene that is used in disease pathology 

• HDR can be used to correct the mutation if there is a WT, or a new gene can be inserted  

How are zinc finger nucleases used?

• Artificial restriction enzymes that are created by fusing zinc finger DNA binding domain and a non specific cleavage domain  

• Cleavage needs to dimerise to be able to snip the DNA  

• FokI is brough together by the two zinc finger domains and produce a double stranded break  

• Triggers DNA repair NHEJ or HDR  

How are zinc fingers used in HIV?

• Used to inactive CCR5 which is the HIV receptor  

• Treated CD4 T cells and showed reduced HIV RNA in the patient blood  

How is TALENs used?

• DNA recognition domain has a TAL effector DNA binding domain  

• Recognition is made by 34 amino acids  

• Has the right and left that each recognise 16-17nt so need to match before and after  

• Is a lot more precise  

• Has the same Fokl domain for the cleavage 

How is TALENs used in CAR-T?

• CAR-T cells for the CD19 disrupted the T cell receptor for the HLA mediated response and inactive CD52  

• Edited the T cells to the patient  

How does Cas9 work?

• Specific recognition uses guide RNAs  

• Has the guide RNA and the Cas9 endonuclease  

• Cas9 cleaves on both strand to produce the double strand break  

How is CRIPSR used for DMD?

• Two guide RNAs delivered by AAV two produce the double strand break  

• Remove the exon 23 and the joining of 22 and 24 brought back the reading frame  

How is CRISPR used in sickle cell?

• Can't target the genes as they are caused by loss of function mutations  

• Targets the transcription factor that represses the foetal haemoglobin BCL11A 

How is CRISPR used in Marfan?

• Introduction of a point mutation without a double strand break  

• Uses base editing  

• 6 embryos had WT genome so shows high accuracy  

What are IPSC used for?

Can be used as a drug discovery and testing/screening platform for developing the treatment of genetic diseases  

How are IPSC produced?

• Adult somatic cells are reprogrammed back to the embryonic stage  

• Transfect with pluripotent factors (OCT4, SOX2, KLF4, c-MYC) 

• Produce iPSCS that can differentiate into almost all cell types in the human body  

• Can also be used for drug screening 

How are IPSC used for genetic conditions?

• For genetic conditions can be patient specific for the mutation  

• Can be corrected and transplanted back to the patient for cell based therapy