Gene Therapy

Question 1

What is the aim of gene therapy?

Answer 1

To deliver gene in order to overcome a disease or infection.
Immunotherapy can be used to reprogram the patients immune system to target disease.

Question 2

What is in situ therapeutic protein production?

Answer 2

Trastuzamab – herceptin, present in serum. Tumour size reduced within one dose.
Gene for protein drug delivered into the patient and the patients cell produces the proteins themselves.

Question 3

What are suicide genes?

Answer 3

Gene directed enzyme prodrug therapy. Often used in combination of HSV thymidine kinase and ganciclovir.

Question 4

What are the strategies for nucleic acid based therapies?

Answer 4

Block transcription by binding to a transcription factor. Direct antisense to the cytoplasm.
siRNA can knock down gene expression of the sequence is known, readily designed and synthesised.
One strand binds to mRNA from the virus and this binding induces cleavage of the RNA. Analogues designed to do this and the strands unwind.

Question 5

What is direct delivery of a gene?

Answer 5

The therapeutic gene is packaged into a delivery vehicle eg a retrovirus and injected into the patient.

Question 6

What are the challenges to delivery of genetic material?

Answer 6

Complex formation, transport from delivery site, cellular uptake, endosome release, trafficking, nuclear entry, mRNA export, translation.

Question 7

Advantages of nonviral vectors?

Answer 7

Easy to prepare and modify, reduced immunogenicity, large capacity.

Question 8

Disadvantages of nonviral vectors?

Answer 8

Poor efficiency

Transient

Question 9

What are dendrimers?

Answer 9

They are non viral vectors which have a wide range of chemical approaches and functional groups. Surface groups can be functionalised for targeting. Can be designed to release under specific conditions. Amine rich dendrimers for complexation with negatively charged nucleic acids.

Question 10

What are cell penetrating peptides?

Answer 10

They are short sequence proteins which are able to cross the membrane. They are arginine rich and have various uptake mechanisms.

Question 11

What is the role of anti-angiogenic strategies?

Answer 11

They block angiogenesis which can halt and limit tumour progression which can be achieved by using genes or oligonucleotides. The targets include VEGF, TGF-Beta, Bfgf, PDGF.

Question 12

What are the approaches for anti-angiogenic strategies?

Answer 12

Antisense RNA or siRNA to block the translation of GF or its receptor.
Oligonucleotides to block transcription of GF or its receptor.
Gene therapy to induce production of soluble receptor or mAb.

Question 13

What is direct delivery of genes?

Answer 13

The treatment or missing gene is added to a vector eg AAV and delivered into the patient by injection.

Question 14

What is cell based delivery of genes?

Answer 14

Treatment or missing gene is added to a harmful retrovirus, at the same time the patients stem cells are removed from the body and cultured. They are introduced to the isolated stem cells which now contain the treatment gene and are returned to the patient.

Question 15

What is the purpose of hematopoietic stem cell transplantation?

Answer 15

To build up the patients immune system. The stem cells can be harvested from a suitably matched donor and the patient has the same hematopoietic system as the donor.

Question 16

What is T cell therapy?

Answer 16

Allogenic T cells can be transfected with HSV-TK which is a suicide gene used to boost the patients immune system and prevent host vs graft disease. It engrafts and helps reconstitute the immune system alongside HSCT from the same donor and improves overall survival and non relapse mortality.
Ganciclovir is administered if graft versus host disease develops. It activates the suicide genes and kills off the T cells. It gives the patient time to recover.

Question 17

What is cell based immunotherapy?

Answer 17

Cytotoxic T cells are activated by antigen presenting cells which prime them with specific antigens for target cells. Cytotoxic T cells recognise and kill cells which express these antigens via release of perforin and granzyme which induce apoptosis.

Question 18

What are antigen presenting cells?

Answer 18

Cells or microparticles with antigens in order to prime T cells. They stimulate T cells to recognise specific antigens.

Question 19

What are CAR T cells?

Answer 19

T cells genetically engineered to express a chimeric antigen receptor. They proliferate and kill tumour cells upon contact with the antigen they recognise. They are genetically engineered from the patient.

Question 20

What is mesenchymal stem cell therapy?

Answer 20

They have the ability to home to the site of injury with anti-inflammatory properties and are hypoimmunogenic.
The effect is done by vesicles and loading vesicles at the tumour site.
They are loaded with genes to secrete molecules that interfere with angiogenesis, they can be transfected with genes that deliver cytokine and can induce apoptosis in tumour cells. They can be loaded with miRNAs which are released through the vesicles and can up and down-regulate gene transcription.

Question 21

What is CRISPR/Cas 9 gene editing?

Answer 21

Gene editing system based on components of the adaptive immune systems of certain bacteria and other microbes. They deliver in vivo and in vitro.
Can be engineered to increase specificity or change its mode of action, eg dCas9 has a mutation which deactivates the nuclease activity and enables it to be used to control gene expression.
They can guide RNA so that target sites which differ by SNP can be detected and cleaved to inactive them.