Lecture 24- Genetic Therapies Flashcards
Translation of research
Human disease gene has been identified > Determine the gene’s NORMAL function and it’s disease state > This info then used > info used to design novel therapies
-Conventional drug-based approach
-molecular genetics
Gene therapy
- Transfer of DNA or RNA into the cells of an organism to treat disease or mark/follow it.
- Only somatic gene therapy currently undertaken (changes not transmitted via germ cell to future generations)
- Augmentation (overproduction of protein to get around damaged gene) easier then gene replacement
- Targeted approaches to gene therapy using engineered nucleases, ‘TALENs, CRISPR
Criteria for using gene therapy on a disease.
1) A life-threatening condition with no effective treatment
2) Cause of disorder is a single, identified gene
3) Regulation of the gene does not need to be precise to return to healthy function
4) Technical problems associated with gene delivery/expression must be resolvable
Two ways of Gene delivery
Ex vivo: cells removed from patient, maipulated in the lab and then reinserted into patient in a sterile manner.
Generally easier, but raises a few extra risks (contamination, extra mutations)
In Vivo: delivered GT in patient. Everything has to be assembled so that the therapy can just be delivered to the blood/lymph
Two major approaches to getting genes into cells
1) Physical: electroporation (holes drilled into cells), microinjection, lipofection
2) Viral: hijacking viruses, which are professionals at getting their DNA into cells. Retroviral, adenovirus
Target cells for gene therapy
- Haematopoietic stem cells
- lymphocytes
- Respiratory epithelium
- Hepatocytes
X-SCID
illustrates the RISK of gene therapies.
X-linked severe combined immunodeficiency
Common cytokine receptor affected.
Therapy (gene replacement) had initially good outcomes, but subsequently leukaemia developed in many children.
Transcription factor was turned on.
10+ children cured by GT replacing the IL2 common gamma chain
However at least 4 developed leukemia
Main issue with gene therapy
If we replace a gene with multiple effects and don’t know the full extent of its function and effect on other genes, we have no control of the consequences that could result
This is proven by the x-linked SCID
Other issues with gene therapy.
- GT not always permanent
- Immune response to the therapy
- issues with viral vectors used to get into cells
- True single gene disorders are extremely rare.
- Potentially turning on an oncogene or pathology
- Potential for misuse
Mutation correction In Vivo
May be required for gene therapy of ‘gain-of-function mutations’, where augmentation of normal gene function ISN’T effective (eg; marfans syndrome). You can either…
-Repair at DNA level homologous recombination (swapping DNA), promising but not effective in GT
-Repair at a RNA level
Therapeutic RNA editing with complementary RNA oligonucleotide.
RNAs are regularly changed and modified as part of normal function.
Targeted inhibition of gene expression
where you are having too much of the gene
Cancer (inhibit oncogenes)
Immune disorders (Immune response)
Infectious diseases
Gene disorder with GOF mutations
Strategies are targeted inhibition at the DNA, RNA and protein level
Recombinant Pharmaceuticals
Drugs used to treat patients that are produced by CLONING. Insert piece of DNA or RNA, and insert it into another piece of DNA/RNA that can be grown in a mammalian cel or bacteria (hijacking to produce large amounts of a protein grown from a template we designed ourselves)
Done in
- microorganisms: alterations will be different/may not occur
- Mammalian cell lines: Addition of lots of different things
- transgenic livestock
Large amounts of protein generated. This is valuable where extracting and purifying that protein from animals isn’t possible
Reduces risk of pathogen contamination
eg) in haemophilia theres AIDS/Hep C risk
Avoids side-effects/immunogenicity of closely related proteins
Examples of recombinant proteins used
Factors VIII and IX (haemophilia A and B)
Insulin
Growth Hormone
Erythropoietin (anaemia)
Genetically Engineered Antibodies
Artificially produced therapeutic ABs are designed to recognise specific disease associated antigens, Leads to killing of disease cells
Targets:
Cancer
Infectious diseases
Auto-immune disorders
Monoclonal(specific) antibodies produced in animals cause immune response in patients (short half life)
-mouse infected, mounts immune response, spleen taken and b-lymphocytes taken and grown> used therapeutically.
But when used in people there was cross-species barrier
Generation of human monoclonal ABs technically difficult
Cloning of immunoglobullin genes, engineered in a way that no IR occurred.
Chimeric rodent-human ABs
(humanized ABs)
eg) AB for TNF-a
Fully human ABs (made in vitro)
Modified ABs for cancer
eg) put cytokines on ABs the turn on IR when ABs bind to a tumor cell
