Gene Therapy Strategies (#12) Flashcards
Cloning a mammal
A. Scottish blackface ewe:
1. Isolate egg cells from ovaries
2. Remove haploid nucleus using micromanipulation
B. Finn Dorset ewe
1. Isolate diploid somatic cells from mammary gland
2. Induce quiescence (G0) by growth in low serum
A + B. Combine the two
1: Fuse cell membranes by electroporation
2. 6 days in culture
3. Uterine implantation
4. 150 days gestation
5. Dolly
Examples of proteins in milk of transgenic animals
- Factor Ix = blood clotting protein treatment of hemophilia B in sheep milk
- α-1-antitrypsin = protease inhibitor treatment of emphysema and cystic fibrosis in sheep milk
- Tissue plasminogen activator = Dissolves blood clots, used as an acute treatment of heart attacks in goat milk
- lactoferrin = iron transport protein, infant formula additive in cow milk
- protein C = anticoagulant, treatment of hemophilia and used for surgery in pig milk
What is gene therapy
The ability to genetically modify the cells of a patient for therapeutic purposes. The ultimate medical application of gene cloning
Two classes of gene therapy
- in vivo = transgene introduced directly into disease affected cells in the patient
- ex vivo = transgene introduced into disease affected cells after they are removed from the body. ‘fixed’ cells can then be re-introduced into the patient
Targeted cells for gene therapy
- Somatic cell therapy - diseased cells are targeted for therapy; however, the cells will not be passed on to offspring
- Germ line therapy - alters both somatic and germ cells (egg and sperm); this is a heritable change that will be passed on to offspring
Gene therapy in practice
- Hemophilia A and B = target cells (liver, muscle, bone marrow cells, fibroblasts). Transfected genes = Factor VIII and Factor IX
- Familial hypercholesterolemia (FH) = target cells (liver). Transfected genes = Low-density lipoprotein receptor
- Sever combined immunodeficiency (SCID) = target cells (bone marrow cells, T cells). Transfected genes = Adenosine deaminase (ADA)
- Cystic fibrosis (CF) = target cells (lung airway cells). Transfected genes = Cystic fibrosis gene (CFTR)
- Cancer = target cells (tumor cells). Transfected genes = p53, Rb, interleukins, growth-inhibitory genes, apoptosis genes
Some important considerations for gene delivery methods into cells/organisms
- Efficiency of gene delivery?
- Delivery to specific cell type?
- Does vehicle provoke and immune response? Is it immunogenic?
- Is mutant form of gene in cells dominant or recessive?
Types of gene therapy (Slides 10-13)
- Gene augmentation therapy. Insert Gene X into diseased cells = normal phenotype (increase in gene X product)
- Direct killing of disease cells = insert toxic gene into diseased cells (cells killed by expressed toxin) or insert Prodrug gene into diseases cells, add drug. (Cells killed by drug).
- Assisted killing of disease cells by immune system cells = insert foreign antigen gene or cytokine gene, disease cells express antigen (Killed bey enhanced immune response).
- Targeted inhibition of gene expression. Insert antisense gene and block expression of pathogenic gene
Why gene therapy has progressed slowly?
- Short lived nature of treatment
- Immune response
- Problems with viral vectors
- Difficulty in targeting only disease cells
- Prevalence of multi gene disorders (heart disease, Alzheimer’s disease, Arthritis)
Using DNA as a marker for human disease
Any changes in DNA sequence have the potential to be used as a marker for that region of DNA, or in some cases, a genetic marker for disease
common ways:
1. Restriction fragment length polymorphism (Southern blot). Can be used for diagnosis of diseases that have a mutation generating a difference in restriction fragment lengths (sickle cell)
2. PCR analysis. Similar to forensics, STRs can be used to diagnose disease (Huntington’s Disease). Allelic specific oligonucleotides (ASO) or allelic discrimination can also be used.
Using DNA as marker for Huntington’s
All humans have the gene Huntington (HTT).
The gene contains a number of trinucleotide (CAG) repeats, and it has been demonstrated that having too many repeats(36-39 = somewhat affected and >39 = fully affected) can lead to Huntington’s Disease
Why is studying gene expression important?
Gene expression = transcription of a gene.
- What regions of DNA is necessary for regulation of gene expression?
- In what cell types is a gene expressed?
- How is gene expression affected by changes in environmental/cellular conditions?
- How much gene expression is occuring
Reporter genes
produce and enzyme or fluorescent module that can be detected = indicates that the gene was expressed.
Typically used to study gene promoters and regulatory sequences.
Examples = lacZ, CAT, GUS, SEAP, Luciferase, GRP
A reporter gene must:
1. Be unique or have an activity that is unique to the cell
2. Not interfere with cellular processes
3. Provide for a sensitive assay
mRNA
Messenger RNA - transcript that will encode for a gene
tRNA
Transfer RNA - contains an anticodon and is attached to a specific amino acid
