Gene therapy for Parkinsons Flashcards
What is gene therapy?
Use of bacterial virus to transport genetic material into cells/tissues
What is the aim of gene therapy?
Halt the process of neurodegeneration in inherited neurological disorders
What does gene therapy treat?
Gene therapy will affect motor symptoms & target other symptoms due to dysfunction of brain circuits eg speech
What is DNA-mediated gene rediting represented by?
CRISPR-cas9
What 3 things does RNA-mediated protein silencing involve?
siRNA
shRNA + dicer
antisense oligonucleotides
What is CRISPR?
Technique that allows precise alterations to be made in genomic DNA sequences by a process known as genome editing
What does CRISPR give bacteria?
ability to recognize precise genetic sequences
What are the enzymes associated with CRISPR called?
Cas
What is MoA of Cas9?
cut target sequence
What does the synthetic dsRNA contain?
Complementary sequence and targets place where there is a mutation
Why is non-homologous end joining bad?
Prone to errors
Could potentially disrupt gene function
Why is HDR better?
Template provided with specific change in DNA sequence
Change will be inserted in genome
Repair double stranded break
New experimental DNA with sequence we want
Describe the CRISPR + Cas9 genome diting technique?
RNAs direct the nuclease Cas9 to selected sequences of genomic DNA
What is Cas9?
Molecular scissors recruited to cut both strands at a precise location & repair or remove mutation
What is a benefit of CRISPR?
Repair any mutation at any location in genomic DNA
What is used as vector to reach nuceli?
Adeno-associated virus
What are 2 types of delivery of CRISPR Cas9?
Viral eg. adenovirus
Non-viral eg. nanoparticles
What 3 things does muscle-restricted Cas9 expression enable?
Editing of mutation
Multiexon deletion
Complete gene correction via homologous recombination
How is CRISPR-Cas9 used to treat PD?
Elicit down regulation of erroneous SNCA gene expression levels
What did CRISPR-Cas9 restore in PD?
Physiological levels of SNCA mRNA -> allowed dopaminergic neurons to maintain function
What are 3 challenges of CRISPR-Cas9?
Non-specific editing of DNA in other locations
Accidental changes that can be permanent
Unwanted immune response
What 2 things does Cas-9 trigger in the immune response?
Humoral response
Specific antigen T-cells
Why is RNA gene therapy better than DNA gene editing?
Only temporary regulations of gene expression
In RNA therapy what are 2 options if there are accidental adverse effects?
Discontinuation possible
Rapid degradation of RNA nucleotides
What is the MoA of dsRNA?
dsRNA -> transfect cells -> binds to complimentary mRNA -> protein depletion
What is the MoA of siRNA?
RNA molecules inhibit gene expression or translation by neutralizing mRNA molecules
What complex is siRNA incorporated in?
RISC
What can siRNA gene therapy be used to treat?
AD
What are 4 challenges of siRNA?
unwanted immune response
poor diffusion within brain tissue
requires injections of liposomes or polymers
delivery procedures & stable expression
Why are nanoparticles used?
carriers for siRNAs with controlled delivery efficiency & low toxicity profiles
How are shRNAs delivered?
AAV
How is shRNA different to siRNA?
Integrated into host genome - continually expressed for months
Uses endogenous processing that exists in the cell
What are 3 challenges of shRNA?
Risk of overdose due to excessively strong promoters
clog up miRNA
shRNA-mediated toxicity
What are antisense oligonucleotides?
single stranded nucleotides that complementary bind mRNA to suppress mutant protein expression
How long is mutant protein expression suppressed for after ASO therapy?
12 weeks
What is an advantage of ASO?
Good diffusion in brain
absorbed efficiently by cells
How is ASO administered?
Lumbar injection
What 3 things does ASO suppress in PD?
LRRK2
alpha syn synthesis
amelioration of motor symptoms
What is LRRK2 linked to?
alphasyn synthesis
What are 3 benefits of ASO?
No strong immunological response
low cost
negligible side effects
What 2 things does ASO gene therapy require?
evaluation of degree of disease modifying effect
and long term efficiency
