Muscle and Exercise Lecture 2.1 : Gene therapy for DMD: Non - viral strategies Flashcards
Name 6 muscle wasting conditions.
-Disuse atrophy, when not in use (limb casting/best rest).
-Denervation - spinal cord injury
-Sepsis - systemic infection (in blood)
-Sarcopenia - age related wasting
-Cancer cachexia - cancer related wasting
Muscular dystropy - genetic cause, genetic theraphy most helpful.
What is the best therapy for a disease with genetic causes?
Genetic therapy is best.
Pharmacological treatment prevents secondary consequences only.
What does genetic therapy do (name 3)?
Restores or replaces the defective gene. Can also introduce new gene to help fight diease.
What is used in cell therapy?
Myoblast transfer.
What is non-viral mediated therapy best for?
Altering a gene
What is viral mediated best for? What is needed to carry it out?
Replacing a defective gene. A vector is needed to carry it out.
What is a common vector?
Retrovirus.
What is a gene expression construct? What is it made up of (3)?
It is the intervention body.
It needs a promotor, the gene of interest and a stabilising element.
What is the promotor in a gene expression construct?
Promotor is used to induce high levels of expression, or make it cell specific by making it inducible in only certain cells.
In making a promotor for DMD, it is best to make it specific to which tissue?
Muscle tissue (including cardiac).
Aside from inducing high expression levels, what else can a promotor do?
Allows you to induce manually, such that one can induce when needed and turn off when uneeded.
How can the gene of interest in a gene expression construct be easily identified?
Adding a tag.
What is the purpose of a stabilising element in a gene expression construct?
Prolongs lifespan of the construct, allowing it to enter the cell.
How does cell based therapy differ to genetic therapy?
The cell is already modified to express the new gene, is injected into the affected area to replace defective cells.
In treating DMD, what must the treatment used have to do, and what percentage of muscles must be restored to begin improving function?
Must be able to enter both skeletal and cardiac cells, must be specific to them, and must be safe.
Only need 20% restoration to improve quality of life.
Whats special about the dystrophin gene? Why is this an issue? How can it be resolved?
Its the largest gene in humans. Hard to deliver the entire corrected gene. Can cut out large parts of the gene and still have a functional protein, is truncated.
What are the two main non-viral therapy techniques?
Unencapsulated plasmid delivery
Dystrophin restoration.
What is unencapsulated plasmid delivery, and give a pro/con.
Form a plasmid vector with the corrected gene, then inject directly to muscles.
Is safe and simple, but very low efficiency, only 1% of localised cells (injection site, not the whole muscle) epress the gene.
How can unencapsulated plasmid delivery be improved (5)?
Can combine with other therapies, add a carrier molecule (lipid) and add a DNA stabiliser.
Can use ultrasound to increase permeability.
Also electroporation, but damaging to cells.
How can hyaluronidase be used to improve unencapsulated plasmid delivery?
It breaks down the extracellular matrix, and improves efficiency when paired with electroporation, not localised to injection site, but the whole muscle.
Can unencapsulated plasmid delivery transfer the entire dystrophin gene? Is it permanent?
Yes, it can. It is not permanent, is only local to injection site, and repeat injections are needed.
Name 2 dystrophin restoration techniques.
Stop codon read through
Exon skipping
What is stop codon read through? What mutation does it work with?
Only works with nonsense mutation, 10-15% of DMD cases.
Is a premature stop codon, truncating the protein.
Stop codon read through uses a drug to allow ribosome to bypass the stop codon and keep reading.
What are two drugs in stop codon read through, and what do they interact with? Are they effective?
Gentimycin - ribosomal subunit 140s, drops CK levels too, moderately effective.
Ataluren - ribosomal subunit 60s
