Gene therapy for Parkinsons

Question 1

What is gene therapy?

Answer 1

Use of bacterial virus to transport genetic material into cells/tissues

Question 2

What is the aim of gene therapy?

Answer 2

Halt the process of neurodegeneration in inherited neurological disorders

Question 3

What does gene therapy treat?

Answer 3

Gene therapy will affect motor symptoms & target other symptoms due to dysfunction of brain circuits eg speech

Question 4

What is DNA-mediated gene rediting represented by?

Answer 4

CRISPR-cas9

Question 5

What 3 things does RNA-mediated protein silencing involve?

Answer 5

siRNA
shRNA + dicer
antisense oligonucleotides

Question 6

What is CRISPR?

Answer 6

Technique that allows precise alterations to be made in genomic DNA sequences by a process known as genome editing

Question 7

What does CRISPR give bacteria?

Answer 7

ability to recognize precise genetic sequences

Question 8

What are the enzymes associated with CRISPR called?

Answer 8

Cas

Question 9

What is MoA of Cas9?

Answer 9

cut target sequence

Question 10

What does the synthetic dsRNA contain?

Answer 10

Complementary sequence and targets place where there is a mutation

Question 11

Why is non-homologous end joining bad?

Answer 11

Prone to errors
Could potentially disrupt gene function

Question 12

Why is HDR better?

Answer 12

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

Question 13

Describe the CRISPR + Cas9 genome diting technique?

Answer 13

RNAs direct the nuclease Cas9 to selected sequences of genomic DNA

Question 14

What is Cas9?

Answer 14

Molecular scissors recruited to cut both strands at a precise location & repair or remove mutation

Question 15

What is a benefit of CRISPR?

Answer 15

Repair any mutation at any location in genomic DNA

Question 16

What is used as vector to reach nuceli?

Answer 16

Adeno-associated virus

Question 17

What are 2 types of delivery of CRISPR Cas9?

Answer 17

Viral eg. adenovirus
Non-viral eg. nanoparticles

Question 18

What 3 things does muscle-restricted Cas9 expression enable?

Answer 18

Editing of mutation
Multiexon deletion
Complete gene correction via homologous recombination

Question 19

How is CRISPR-Cas9 used to treat PD?

Answer 19

Elicit down regulation of erroneous SNCA gene expression levels

Question 20

What did CRISPR-Cas9 restore in PD?

Answer 20

Physiological levels of SNCA mRNA -> allowed dopaminergic neurons to maintain function

Question 21

What are 3 challenges of CRISPR-Cas9?

Answer 21

Non-specific editing of DNA in other locations
Accidental changes that can be permanent
Unwanted immune response

Question 22

What 2 things does Cas-9 trigger in the immune response?

Answer 22

Humoral response
Specific antigen T-cells

Question 23

Why is RNA gene therapy better than DNA gene editing?

Answer 23

Only temporary regulations of gene expression

Question 24

In RNA therapy what are 2 options if there are accidental adverse effects?

Answer 24

Discontinuation possible
Rapid degradation of RNA nucleotides

Question 25

What is the MoA of dsRNA?

Answer 25

dsRNA -> transfect cells -> binds to complimentary mRNA -> protein depletion

Question 26

What is the MoA of siRNA?

Answer 26

RNA molecules inhibit gene expression or translation by neutralizing mRNA molecules

Question 27

What complex is siRNA incorporated in?

Answer 27

RISC

Question 28

What can siRNA gene therapy be used to treat?

Answer 28

AD

Question 29

What are 4 challenges of siRNA?

Answer 29

unwanted immune response poor diffusion within brain tissue requires injections of liposomes or polymers delivery procedures & stable expression

Question 30

Why are nanoparticles used?

Answer 30

carriers for siRNAs with controlled delivery efficiency & low toxicity profiles

Question 31

How are shRNAs delivered?

Answer 31

AAV

Question 32

How is shRNA different to siRNA?

Answer 32

Integrated into host genome - continually expressed for months Uses endogenous processing that exists in the cell

Question 33

What are 3 challenges of shRNA?

Answer 33

Risk of overdose due to excessively strong promoters clog up miRNA shRNA-mediated toxicity

Question 34

What are antisense oligonucleotides?

Answer 34

single stranded nucleotides that complementary bind mRNA to suppress mutant protein expression

Question 35

How long is mutant protein expression suppressed for after ASO therapy?

Answer 35

12 weeks

Question 36

What is an advantage of ASO?

Answer 36

Good diffusion in brain absorbed efficiently by cells

Question 37

How is ASO administered?

Answer 37

Lumbar injection

Question 38

What 3 things does ASO suppress in PD?

Answer 38

LRRK2 alpha syn synthesis amelioration of motor symptoms

Question 39

What is LRRK2 linked to?

Answer 39

alphasyn synthesis

Question 40

What are 3 benefits of ASO?

Answer 40

No strong immunological response low cost negligible side effects

Question 41

What 2 things does ASO gene therapy require?

Answer 41

evaluation of degree of disease modifying effect and long term efficiency