FINAL exam (3 lectures) Flashcards
Three complications in treating monogenic disorders
1.) gene may not have been identified
2.) Fetal damage
3.) most severe clinical phenotypes are less ammenable to intervention
Levels of intervention for treatment strategy
-mutant gene modification
-mutant mRNA modification
-mutant protein modification
-disease-specific compensation
-clinical phenotype modification (medical/surgical)
-genetic counseling
Long-term complications of gene therapy
1.) deficiencies have a late onset
2.) successful in one tissue but harmful to another
3.) side effects have a late onset
Dominant negative
effect of genetic heterogeneity on therapy, new wildtype replacements my be “poisoned” by a still-present mutant gene
The most successful gene therapy
treatment of metabolic deficiencies (strategies include: dietary modification, avoidance, diversion, inhibition. and depletion)
Biotinidase deficiency
prenatal biotin administration
Cobalamin-responsive methylmalonic adicuria
prenatal maternal cobalamin administration
Congenital adrenal hyperplasia
Dexamethasone, a cortisol analogue
Phosphoglycerate dehydrogenase (PGDH) deficiency, a disorder of L-serine synthesis
Prenatal L-serine administration
Protein mutation genetic therapies
1.) add a cofactor
2.) replace the defective protein
Problems:
1.) proteins with a short half-life require frequent treatments and replacement –> insufficent supplies and harmful immune responses
Ivacaftor
For CF patients, restores lung function by permitting CTFR protein to be integrates into the cell membrane (augmentation)
Gaucher Disease
Lysosomal storage disease, mutation in glucocerebrosidase causing accumulation of glucocerebrosides in reticuloendothelial system
treated successfully with enzyme replacement (imiglucerase, miglustat) therapy (expensive and weekly infusions)
Beta-globin deficiencies– sickle cell
inducing the expression of gamma-globin (noramally only found in fetus) can partially rescue beta-globin deficiency phenotype
Hematopoietic stem cell (HSC) transplantation
bone marrow transplant, is risky due to infection
placental cord blood is more desirable over bone marrow, rich in HSCs, graft vs host risk is greatly reduced
gene therapy
modification of cells to produce a therapeutic effect, based on recombinant DNA technology that permits the introduction of new genes and possibly the removal of damaged genes
reasons to do gene therapy
- compensate for a mutant cellular gene with a loss of function mutation
- replace or inactivate a dominant mutant gene
- Pharmacological effect (like cancer)
Ex vivo gene transfer
transfer outside the body or a stem cell, followed by introduction into the body
ADVANTAGES: does not require an efficient means to enter a cell because it can be engineered for
DISADVANTAGES: difficult and time consumig
In vivo gene transfer:
direct injection into the body using a vector
ADVANTAGES: quick and easy
DISADVANTAGES: targeting proper cells, immune responses, safety
Retroviral vectors
-Can enter virtually all target cells
-made simple and replication-defective
-Easy to engineer
-Introduce DNA into host genome
-accomodate large transgenes
Problems:
-require dividing cells to introduce DNA into host genome (lentiviruses, HIV can get around this)
- Safety due to mutagenesis
adenoviral vectors
Advantages:
- generated at high titer
- infect wide range of cell types
- accomodate large genes
Disadvantages:
- does not integrate into the genome
- expression is transient
-strong immune responses
- result in cell toxicity sometimes
Adeno-associated vectors (AAVs)
Advantages:
- can infect both dividing and non-dividing cells
- integrate host genome
- EX: CF, Factor IX, muscular dystrophy, CNS diseases
Disadvantages:
- Small transgenes (5KB)
—- Preferred viral vector for clinical trials
non-viral vectors
Advantages:
- lack biological risks associated with viral vectors
Disadvantages:
- not successful, DNA degraded in lysosomes
EX: naked DNA, liposomes, protein-DNA conjugates, artificial chromosomes, nanoparticles
Herpesviruses
central nervous system tropism, have large packing capacity, however they have strong inflammation and neurotoxic responses
CRISPR/CAS-9 example
in vivo-gene editing of transthyretin amyloidosis (ATTR), KO the mutant gene with 87% response positive
siRNA gene therapy
designed to target a range of tissues
ex studies: SARS, pathology of lungs in CF, KO of Huntington mRNA
Hemophilia B gene therapy
- Factor IX replacement
- Liver-specific
-AAV2, AAV9
and Hemophilia A (factor VIII)
Lysosomal storage disease gene therapy
- enzyme replacement therapy, Gaucher disease)
- hematopoietic cell transplantation
- AAVs and other vectors
Parkinson’s disease gene therapy
- Viral vectors targeting the neuronal population that is affected in PD
Leber congenital amaurosis gene therapy
early onset photoreceptor degeneration
- RPE65 gene (RA metabolism)
-AAV2 vectors
DMD gene therapy
-mini-dystrophin genes delivered by AAVs
- mutation suppression by STOP codon read through
- Exon skipping
SCID gene therapy treatment
OTC deficiency gene therapy, Jesse Gelsinger, X-SCID –> fatality and Leukemia (LMO2 gene)
Terminal differentiation
As cells differentiate, they usually stop dividing permanently.
