Treatment Of Genetic Disease Flashcards
What makes a genetic disease treatable?
Diseases where a see relatable protein can be taken up by and complement other cells
Disease where defect is membrane protein, can to raffia to its site of action or into correct cells
What is a lysosomal storage disease?
Cell produces substrate for use within or outside cells
Internalised and broken down ( possibly in lysosomes)
Lack of enzyme or protein leads to accumulation of undegraded substrates in lysosomes
Can affect brain, bones, joints, muscles and other organs
Outline the principle of cross correction
Lysosomal enzymes are glycosylated in the ER
Have a secretory signal and are further modified in the Golgi with M-6-P
M-6-P receptors target them to lysosomes where acidic pH activates them
Some are secreted
Receptors on cell surface pick them up and traffic through endoscopes to lysosomes to be activated
What are the treatments for LSDs?
Enzyme replacement therapy (ERT)
Haematopoietic stem cell transplant (HSCT)
Substrate reduction therapy (SRT)
Genetic therapy (GT)
How does ERT work?
Enzyme delivered to blood steam can be take up by affected cells and correct disease
BBB limits delivery to brain and makes it ineffectual for neuronopathicdisease
Won’t work in LSDs where enzyme not secreted
Mammalian cells lines used for correct M-6-P tags
What are the limitations?
£144,000/patient/year in UK EARLIER TREATMENT BETTER BBB Joints and growth plate of bone poorly connected to blood stream M6P slow compared to MR Antibody response
What is future of ERT?
Bypass barrier by physical injection
Never production of enzyme or drug may improve delivery
Modify enzymes to improve uptake
Tolerisation regiment to limit antibody response
Outline HSCT
Delivery of enzyme from blood cells
Monocytes enter brain and release enzyme
HSCT donor cells repopulate blood system
What are the limitations of HSCT?
Early intervention essential
Few LSDs implicated as treatable this way
Some risk of morbidity/mortality
Insufficient brain enzyme produced in some diseases
Outline SRT
Reduction of primary storage material or restoring down alternate pathways
Drugs must reduce without being toxic
Drug must be able to reach all affected cells including brain
Not likely to raise antibody response
Discuss an example SRT for Gaucher
Miglustat
Immunosugar inhibiting glicosylceramide synthase
Blocks first step in glycosphingolipid production
Developed as treatment to Gaucher type 1 : reduces phosphosphingolipids
Also used for niemann pick C patients
Discuss an example SRT for muccopolysaccharidosis
Genistein Tyrosine kinase inhibitor Purified from soy beans Blocks GAG production 10% crosses BBB
What are the limitations of SRT?
Reduction of substrate can never cure disease
Primarily delays symptom onset
Low drug toxicity and BBB permeability essential
What are the four types of gene therapy?
Gene addition
Gene repair
Gene inhibition
Cell killing
Outline gene addition
Remove viral genes and package RNA/DNA therapeutic gene and promoter in their place
Can be episomal from a plasmid or more stable from viral vector or integration into host genome
What are barriers to gene addition?
Body resists entry vis skin, immune system, membrane, BBB etc.
What are the two routes of delivery?
Direct - intravenous, intracranial, intraventricular or intraocular. Targeting specificity achieved by deluxe site. AAV main choice due to high titres. Limites= immunogenicity, pre existing immunity and scale up
Ex Vivo- transduce cells in lab and them reintroduce as stem cells. Lenti viral vectors. Unlimited self renewal. Limitations= require space to re-engraft and hence damage target organs to achieve it
What are AAV?
Adeno-associated viral vectors Simple ssDNA More than 10 serotypes that can infect different tissues Replace viral genes with therapeutic transgene and promoter to stop replication Small packaging capacity Mostly remain outside nucleus Great for immune privileged sites AAV9 can cross BBB Immune response common
How have AAV gene therapies been used?
Haemophilia B = AAV8 against severe factor IX deficiency. Steady state of 1-6% of normal levels achieved.
Sanfillippo disease IIIA/B = direct Brian injection. Shows stabilisation of disease and improvement with more injections
Batten disease = brain injection. Stabilisation in some patients
Parkinson disease = brain ejection. 23% improvement in treated vs 12% in non-treated
Blindness= inject to eye. Visual improvement. Vector persists as eye is immune privileged site.
What are retrovirus/lentiviral vectors?
RNA viral genome
Reverse transcription and random integration
Can infect stem cells
Viral envelope provides specificity for cell types
Vector creation - replace viral genes with therapeutic ones. Removes LTRs and replace with SIN vectors. Replace promoter with CMV and remove ability to replicate.
Where have retro/lentiviral vectors been used?
HSC Gene therapy for immunodeficiency
Limited to patients with no other therapy options and no donor available
How could gene repair be achieved?
Zinc finger nucleases, transcription activator-like effector nucleases (TALEN), CRISPR/Cas9 guided endonuclease system
All introduce DS break at targeted location with the guide of homologous binding proteins or RNA
What are the future options?
Increase enzyme delivery target organs Novel substrate inhibitors STOP codon read through Antiinflamatories Gene theory clinical trials Combination therapies
What can be targeted in treatment of genetic disease?
Protein Function
Protein translation
mRNA
DNA
What is non-sense suppression?
Nonsense mutations cause nonsense mediated decay
Amino glycosides can suppress the effect by inducing incorporation of random amino acid to achieve read through
Used in cystic fibrosis
Limitations : not able to restore protein function if the aa is essential
Efficiency of read through varies depending on nature of STOP a codon
Read through of a physiological STOP codon could be detrimental
How can RNA be targeted?
Manipulation of splicing - exon skipping
RNA interference
How has exon skipping been used?
Treatment of DMD
Skip exon 3 to produce BMD protein - not as detrimental
What is RNAi?
Natural physiological sequence-specific gene silencing phenomenon mediated by double stranded RNA
miRNA and siRNA are essential
How has RNAi been implemented?
Retinitis pigmentosa
GCAP1 mutations cause RP
Target mutant product with siRNA in mice to rescue function of retina
What are challenges of siRNA?
Specificity
Delivery
Duration of silencing
What types of gene treatments exist?
Genome editing
Gene therapy
What three platforms exist for genome editing?
Zinc finger nucleases
Transcription activator like effector nucleases
Clustered regulatory inter spaced Short palindromic repeats/CRISPR associated protein 9 (CRISPR/Cas9)
What are ZFNs?
Artificial restriction enzymes
Fusion protein with Zinc finger domain that is targeted for specific DNA sequences
3amino acid recognise a single base and cleavage domain forms DSB for NHEJ or HR
What are TALENs?
Fusion protein with TAL effector DNA binding domain
Stretch of diresidues that can target specific sequence
Cleavage domain forms DSB
What is CRISPR/Cas9?
Originated in bacteria as defence against phage
Guide RNA is designed for recognising site and Cas9 is used to generate DSB
Cas9 requires crRNA and trancRNA to function. Chimeric crRNA and trancRNA form gRNA
What four factors are used for iPSC?
Oct4, sox2, KLF4 and c-Myc
