Gene therapy successes Flashcards
What is duchenne muscular dystrophy?
X-linked recessive disorder.
Degeneration and regeneration of skeletal
and cardiac muscles.
Associated with severe progressive muscle
weakness.
Affected boys cannot walk by 10 years of
age.
Life expectancy is around 20 to 35 years,
death due to heart or respiratory failures.
Due to mutations in dystrophin gene.
What is the dystrophin gene?
The dystrophin gene is one of the longest human genes identified.
Codes for linear cytoplasmic protein that supports muscle fibres.
Major protein in the dystrophin-associated protein complex (DAPC).
The DAPC connects cytoskeleton of muscle fibres to extracellular matrix through the cell membrane.
Important for muscle fibre strength and flexibility.
What types of mutations lead to duchenne muscular dystrophy?
Deletion hotspots around exons 45-53 (cluster I) or exons 2-20 (cluster II).
Missense mutations distributed throughout, introducing premature stop codons.
Duplications.
Out-of-frame mutations or premature stop codons lead to no functional protein.
Becker’s muscular dystrophy:
Protein-binding N and C-terminal domains still present as no change in reading frame. The shorter protein has some functionality.
Why is duchenne muscular dystrophy suitable for gene therapy?
It is a monogenic recessive disorder.
Mapped to mutations in a single gene.
Currently no cure for DMD.
Physiotherapy, corticosteroids, creatine medication to improve muscle strength and slow progress of muscle weakening.
Translarna (Ataluren) available to some patients with nonsense mutation – allows ribosome to read-through premature stop codon in mRNA.
What is the process of exon skipping?
Exon skipping occasionally occurs during splicing of primary transcript, observed in revertant
fibres in DMD.
Can be induced with antisense oligonucleotides that mask splice sites.
Protein with partial deletions can still have functional benefits.
ON1 binding to exon 44 improved phenotype in mouse model.
What are the obstacles with oligonucleotide drugs?
Method of administration:
Lack of effective distribution away from the injection site.
> 300 muscles make up the total musculature in the human body.
Systemic delivery system required.
What are the obstacles with AAV vectors?
Potential problems:
High dose of virus needed to express required amounts of protein.
Immune responses against AAV may limit effectiveness of subsequent deliveries.
How can this be overcome?
Transient immunosuppression.
Recombinant AAV that are less immunogenic.
Why is cystic fibrosis suitable for gene therapy?
CF is a monogenic disorder.
Mapped to mutations in a single gene.
CFTR modulators target the underlying cause of the disease but only work on some mutations.
Gene replacement therapies are most promising hope for a cure.
How are adenoviral vectors used to deliver CFTR genes?
Ex vivo transfer of CFTR gene using adenovirus and retrovirus vectors caused cells to produce the CFTR protein.
Adenoviruses cause common colds.
Genetically engineered to non-harmful versions that carried the target gene.
Sequence does not integrate into host genome.
What are the disadvantages of adenovirus vectors?
No dramatic changes in the long-term.
Sequence does not integrate, and so treatment must be repeated.
Host immune system was producing a response against the adenovirus and destroying the vector.
How can delivery be improved?
Next-generation AAV vectors that have increased infectivity to reach more cells in the lung.
Shorter CFTR gene to allow additional stronger promoter sequences in the vector.
Lentiviruses that integrate the target gene into host genome.
Lipid nanoparticles instead of viral vectors.
What is the process of CRISPR/Cas 9 gene editing?
Patient’s CFTR gene can be edited to correct the
mutations.
Organoids taken from CF patients with no expression
of CFTR protein.
CRISPR/Cas9 gene editing of the CFTR gene.
Function of CFTR protein restored in the organoids.
What are the different parts of the HIV genome?
gag: viral capsid.
pol: reverse transcriptase.
vpr: accessory protein involved in immune evasion.
tat: transactivator essential for gene expression, binds
TAR sequence.
rev: essential for export of unspliced mRNA into the cytoplasm.
env: binds CD4 and CCR5 or CXCR4 for entry.
nef: accessory protein downregulates CD4 and MHC1 expression.
What are the obstacles with current therapies?
Requires adherence.
Development of resistance.
Viral genome is integrated into host genome and therefore current therapies are not a cure.
How can HIV be treated with gene/cell therapy strategies?
Targeting the virus.
Targeting host proteins.
Activating host immunity.
