Genetic Therapies Flashcards
Translation of Research from “Bench to Benchside”
A human disease has been identified (group of people with disease sharing common mutation/familial) –>
The gene’s role in normal biology (in healthy tissue) (precise mutations) and the disease state is determined –>
This information then used to design novel therapies–>
a) conventional drug-based approaches (targeted therapy)
b) molecular genetic-based therapeutics (gene therapy)
Gene Therapy
The transfer of DNA or RNA into the cells of an organism to treat disease or to mark cell populations (follow/investigate to get further information)
In humans only somatic cell gene therapy undertaken at present (where the target is not a germ cell, so that changes cannot be transmitted to future generations)
Gene augmentation more often considered currently than gene replacement (disabled, easier to put in another gene to produce protein to overcome the lack of disabled gene)
-Marfan syndrome Fibrillin 1 gene causing dominant -ve effect. Not helpful in dominant inherited disorders. gene replacement required.
Targeted approaches to gene therapy using engineered nucleases (zinc finger nucleases, TALENs, CRISPR) are rapidly moving forward
Disorders where gene therapy approaches may be considered
-Gaucher’s disease + other storage disorders
• Immunodeficiencies
• Eye disorders (e.g. Leber’s congenital amaurosis)
• Cystic fibrosis
• Familial hypercholesterolaemia
• Storage disorders (Gaucher’s disease)
• Haemophilias
• Haemoglobinopathies (thalassaemia, sickle cell disease)
• Cancer (melanoma, brain tumours, lymphoma)
• Vascular disease
• Neurological disorders (Parkinson’s disease, Alzheimer’s disease)
• HIV (knockout of CCR5 in T cells)
Criteria proposed for identification of genetic disorders amenable to gene therapy
- A life threatening condition with no effective treatment
- Cause of the disorder is a single gene, that has been identified (cloned/known) (easily targeted/replaced)
- Regulation of the gene does not need to be precise to return healthy function (when replaced. just express large amounts. more difficult if need to be produced in certain fashions/ in response to certain stimuli/ in specific anatomical sites)
- Technical problems associated with gene delivery and expression must be resolvable (sometime the most difficult problem)
- this is an intensely regulated field because of the risks manipulating the human genome.
Gene Delivery Strategies
- Ex vivo- cells removed from patient, grown and manipulated in laboratory and then returned to patient
- In Vivo- requires targeting to correct cells (deliver gene therapy inside patient)
In vivo gene therapy
requires targeting to correct cells (deliver gene therapy inside patient)
assembled so can inject into patients’ blood stream/particular organ
Ex vivo gene therapy
- cells removed from patient, grown and manipulated in laboratory and then returned to patient
1. Take cells outside of patient
2. introducing new gene into cells (e.g. viruses)
3. growing the cells in lab
4. return to patient in sterile manner - easier
- risks: culturing cells in lab contamination. extra cell replication stress condition of labs cause other mutations to form/alter gene expression patterns
Getting genes into cells
Two major approaches:
- Physical (electroporation (drill holes into cells via small electric pulses), microinjection, lipofection (gene put into lipid bound vesicles, and fuse with membrane))
- Viral (retrovirus, adeno-associated virus, lentivirus, adenovirus, herpes simplex 1 virus)
- common to hijack viruses (already professional at doing so)
Viral gene insertion example
Using an adenovirus “vector” to insert a new gene into a cell”
- Mediated DNA injected into vector (viral genome)
- Vector binds to cell membrane (of a particular cell. virus often trophic for one lineage of body cells)
- Vector is packaged in membrane bound vesicle
- Vector injects new gene into nucleus (DNA encorporate into genome or be replicated outside)
- Cell makes protein using new gene
Target cells for gene transfer
- Haematopoietic stem cells
-take bone marrow biopsy. sorting stem cells based on antibody markers and growing in labs - Lymphocytes
- Respiratory epithelium
- Hepatocytes
5, Fibroblast skin cells - Skeletal muscle
What is the real struggle with gene therapy?
Easy to use with technology
Struggle has been regarding putting it all into practice
Example case X-SCID
X linked severe combined immunodeficiency (genetic workshop)
-caused by single gene
-no good treatment
-way of delivering potential gene therapy to patients
The gene encoding the gamma y common chain of several cytokine receptors is affected
-stem cells respond to signalling molecules/cytokines. Bind to their external receptors encouraging differentiation down a particular lineage (bone marrow development)
- mutation leads to stop receptor transmitting cytokine signals into cell. protein chain common to several different cytokine receptors.
-protein chain encoded by single gene, this gene can be replaced.
Initially good outcomes, but subsequent development of leukaemia in some children
-illustrates risks of gene therapy
- manipulation of cells, and encorporation of virus, turn on transcription factor LMO2
X linked SCID stats
Well over 10 children cured of X-linked SCI by gene therapy to replace the IL2 receptor common gamma chain
However at least 4 of the gene therapy recipients have gone on to develop T-cell ALL (Luekemia)
-“the bottom line here is if you replace a gene that has multiple effects, you have to know more about its regulation and its ability to affect other genes, and that requires extensive preclinical work and a much more careful analysis” - unexpected things may happen.
-is a promising and life changing area, but carries massive risks
-partly experimental. many barriers to be overcome
Researchers have overcome many problems for gene therapy: others remain a challenge
- Lack of permanence (stop reproducing encoded protein) (or cells preferentially killed off due to natural mechanisms)
- Immune response (to cells which produce the protein, or protein itself)
- Problems with viral vectors (induce immune response)
- Rarity of true single gene disorders (not amenable to simple gene therapy)
- Effects of insertional mutagenesis and therapeutic protein expression (inserted into area which can turn on an oncogene or cause chromosome break which can lead to cancer)
- or the protein being repordiced can have unexpected consequences leading to tumour or subsequent pathology - Potential for misuse (even though inducing change to somatic cells only but sometimes this change is passed on through germ line)
- Possibility that the Weismann barrier is sometimes permeable
Mutation Correction In Vivo
- correct changes in patient
- Fibrilin 1 (autosomally dominant inherited mutation leading to Marfan’s syndrome. building block protein, no end of additional blocks will help, as will have mischapened bricks which weaken the wall)
- May be required for gene therapy of gain-of-function/dominant -ve mutations where augmentation or normal gene function is not effective
