Genetic Egeneering B Flashcards
Steps in Gene Therapy:
- Identification of the defective gene.
2.Cloning of normal healthy gene, (many copies). - Identification of target cell / tissue / organ.
4.Insertion of the normal functional gene into the host DNA. - Activate the gene so that transcription & translation take place.
Types of Gene Therapy:
1.Somatic cell gene therapy.
• Will no be inherited to the offsprings.
2.Germ cell gene therapy.
• Therapeutic genes transferred into the germ cells,
as sperm / ovum fertilized egg
Results in expression of modified features in
germ cells of the offspring. somatic as well as
• So, itis inherited merited and pass on to later generation.
• Considered unethical, it is not being attempted till now.
Ways for Transgene Delivery:
• Two ways to deliver genes:
1.Ex vivo approach: means outside the body.
To avoid immune system rejection,
It is only applicable to tissues that can be rémoved from the body, altered genetically & returned back where they engraft & survive For aa long period of time.
Therapeutic genes are delivered by using vectors.
2.in vivo approach: transgene delivered directly into host.
Cloned therapeutic gene is introduced directly into the affected tissue, without removing cells from the body.
Methods of Gene Delivery:
- PHYSICAL METHODS:
o Parenteral injection.
• Microinjection.
o Aerosol.
• Gene gun.
-CHEMICAL METHODS:
• Calcium phosphate.
o DEAE-Dextran Liposomes
- BIOLOGICAL METHODS; Viral Vectors like:
• Retrovirus.
• Adenovirus.
• Herpes simplex virus HSV. - Neo-organ implants.
-Tlissue transplantation: - Human artificial chromosomes.
- Others:
-Receptor mediated delivery. - Virally directed enzyme prodrug therapy.
Various Strategies for Gene Therapy:
•Gene augmentation therapy.
- Targeted mutation correction.
- Inhibition of gene expression.
- Gene therapy to achieve pharmacological effects.
What is the Strategies for Gene Therapy:
• If a disease is caused by a mutation causing loss of function, introduction of Functional copy of the GENE
into the cell will restore the normal function of the gene.
Gene Augmentation Therapy:
Examples:
• Adenosine deaminase (ADA).
• Hemophilia.
What is the Strategies for Gene Therapy:
Correction of mutation by
changing the mutated nucleotide sequence to normal
Practically, it’s difficult, but in principle can be done by
Targeted Mutation Correction:
homologous
recombination
What is the Strategies for Gene Therapy:
• In case of mutations that have a negative dominant effect, the
expression of the mutated gene can be blocked at the DNA RNA protein level.
• This strategy is useful in ……
Inhibition of gene expression.
Cancers
What is the Strategies for Gene Therapy:
Examples:
- Introduction of a gene that makes cancer cells susceptible to anticancer drugs.
- Introduction of a toxic gene whose expression kills cancer cells.
- Genes of cytokines can be introduced into cells of immune system to enhance their potential to kill diseased cells .
Gene Therapy To Achieve Pharmacological Effects:
Characters of the Ideal Vectors
-Integrate the gene in the cells.
- Activate the gene
-Should protect the gene from degradation by nucleases in extracelluiar matrix.
- Easy & reproducible.
-Target the right cells.
- Production in high concentration/mL
- Avoid harmful side effects.
- Long term gene expression through insertion into the genome or stable episomal persistence
-Controllable gene expression.
-Low immunogenicity.
Retro Viruses (RNA Viruses) :
Advantages:
Disadvantages:
Retroviruses used in gene therapy are made incapable of independent replication, to prevent side effects associated with infectivity.
• Retroviruses are used ONLY in EX VIVO THERAPY.
Advantages:
- Chromosomal integration& stable modification of target cells.
Disadvantages:
- Uncontrolled integration; may be oncogenic.
- Cannot infect non-dividing cells.
Adeno Viruses (DNA Viruses):
• Adenoviruses can be produced at high titers in cultures.
•Advantages:
- Can infect non-dividing cells, thus suitable for gene therapy of Cystic fibrosis, Duchenne muscular dystrophy (DMD).
- Does not integrate into host genome. Avoids the risks of uncontrolled integration
- Efficient gene transfer.
- Nononcogenic.
Disadvantages:
- Transient expression of gene due to episomal integration.
- Induces immune response.
Liposomes:
Advantages:
- Safer when compared to Viral vectors
- Can carry large DNA molecules.
Disadvantages:
- Inefficient transfer.
- Transient expression.
Leber’s congenital amaurosis:
- It’s caused by an abnormality in a gene called
RPE65.
- Without the protein produced by this gene, photoreceptors have severely impaired responses to light and ultimately
degenerate.
- The condition appears at birth or in the first few months of life and causes progressive worse and loss of vision.
Success in Parkinson Disease:
• Main-overactive brain region; the subthalamic nucleus should be introduced with gene that would produce aGABA (inhibitory neuro- transmitter), then they could potentially quiet that brain region and
alleviate tremors.
Performed with local anesthesia, used a harmless, inactive (AAV
2 GAD].
Deliver the gene of glutamic acid decarboxylase_(GAD) into the
patient’s subthalamic nucleus.
