Lecture 10: gene therapy

Question 1

What are the two approaches when it comes to inserting dna inside cells?

Answer 1

1. Germ Cells gene therapy: gene introduces into germ cell (highly experimental and not yet approved for human application!)
2. Somatic Cells gene therapy: gene introduces into somatic cells/tissues to treat or just to relieve symptoms

Question 2

What is in vivo gene therapy and what is it suitable for?

Answer 2

1. Administering the gene of interest locally or
   systemically
2. Suitable for easily accessible tissues

Question 3

What is ex vivo gene therapy and what is it commonly used for?

Answer 3

1. Introducing the gene of interest into cells taken from the body, then re-infusion/re-implantation of altered cells back into patient
2. Commonly use for diseases of the hematopoietic system

Question 4

Why is gene therapy more beneficial than conventional drug therapy?

Answer 4

1. The effect of conventional drug therapy is transient but gene therapy can result in sustained or even permanent change to the genetic constitution
2. Unlike conventional drugs, gene therapy has the potential to achieve outstanding therapeutic value by directly targeting the “cause” of the disease rather than just providing “symptomatic” relief

Question 5

What kind of genetic disorders are treated by gene replacement?

Answer 5

1. Genetic disorders caused by a defective or completely non-functional gene by providing cells/tissues with the normal gene that can express adequate functional protein
2. Usually single cell defects

(note: defective protein is still present in the cells/tissues).

Question 6

What kind of genetic disorders are treated by gene addition?

Answer 6

Cancer treatment:
1. Introduction of tumor suppressor genes to treat localized tumors
2. Introduce cytokine genes to cancer cells to induce inflammatory reaction to destroy cancer cells in primary tumor and distant metastases.
3. Introduction of genes to express proteins that can neutralize the toxic effects of chemotherapy
4. Personalized neoantigen vaccines that can target the cancer cells in the patient very specifically

Question 7

What is the end point once the DNA goes into the cell

Answer 7

1. Gets integrated into the host cell genome to produce therapeutic protein forever
2. Episomal system, where the gene remains outside the chromosome

Question 8

What are the two major categories of methods for delivery genes in gene therapy?

Answer 8

1. Non viral
2. Viral

Question 9

What is stable gene transfer?

Answer 9

1. The gene is integrated into the host cell chromosome and is stable
2. The therapeutic gene is transmitted to daughter cells after cell division

Question 10

What is transient gene transfer?

Answer 10

1. The therapeutic gene is inside the nucleus but doesn’t get integrated into the chromosome
2. The episomal DNA may be lost upon multiple round of cell division

Question 11

What is the process of non viral gene transfer?

Answer 11

1. Direct injection of plasmid DNA into tissues (usually skin or muscle).
2. Electroporation: strong electric pulses are applied to the cell culture which transiently opens up the cell membrane which allows the DN to enter the cell
3. Gene gun: a high pressure or electrical discharge device that can deliver microscopic gold/tungsten particles coated with DNA into tissues/cells. The particles penetrate the cell membrane and release dna
4. The DNA is stored inside liposomes, and enters by endocytosis and enclosed in an endosome

Question 12

What are the advantages of non viral gene transfer?

Answer 12

1. Easy to prepare and scale up
2. Safer for in vivo delivery
3. No strong immune response
4. Capable of delivering large DNA fragments (~ 50 kb)

Question 13

What are the disadvantages of non viral gene transfer?

Answer 13

1. Much lower efficiency than viral methods → but in theory can be improved by synthesis of novel cationic lipids and polymers in future.
2. Expression usually transient (hence, need to repeat frequently for maintaining the effect)

Question 14

What is done to insert the gene during viral gene transfer?

Answer 14

Gene of therapeutic value is incorporated into the viral genome for achieving gene transfer.

Question 15

What are the criteria for using viruses for gene transfer?

Answer 15

1. They have to be replication defective (i.e. inactivating the disease-causing property of the viruses).
2. They should not possess undesirable properties (i.e. minimal effect on the physiology of the infected host cell; genetic stability).
3. The viral genome must be able to accommodate the foreign gene

Question 16

What are the common major classes of viral vector for gene therapy applications?

Answer 16

1. Retrovirus
2. Adenovirus

Question 17

What are the key considerations in choosing viral vector for gene delivery?

Answer 17

1. Safety
2. Vector associated toxicity and immunogenicity
3. Integration into the host genome
4. Size limitations of the gene it can accommodate
5. Purity and titer of the vector
6. Transduction efficiency
7. Type of cell it infects
8. Long term expression of transgenes

Question 18

What form of nucleic acid is inside retroviral vectors, and how many copies?

Answer 18

RNA
2 copies

Question 19

What happens to the nucleic acid after the retroviral vector infects the cell?

Answer 19

Retroviral RNA introduced into the host will be
converted into double-stranded retroviral DNA by reverse transcriptase present in the viral core.

Question 20

What is the size limit of genetic material that retroviral viruses can accommodate?

Answer 20

Less than 9kb

Question 21

Describe the process by which a gene gets inserted into the nucleus by a retroviral vector.

Answer 21

1. Viral envelope glycoproteins interact with specific host-cell membrane protein receptor, after which the virus fuses directly with host cell’s plasma membrane
2. Viral core enters cell cytoplasm, and then the viral RTase copies ssRNA genome of virus into a dsDNA copy
3. The retroviral DNA is transported to nucleus and integrated by viral integrase enzyme into one of many possible sites in host cell chromosomal DNA . The integrated DNA is now called the provirus.
4. The DNA is transcribed and the mRNA is translated to form glycoproteins and viral core proteins. The viral glycoproteins are then exposed on the host cell membrane
5. Viral proteins packaged with the viral RNA generated by host to produce retrovirus particles. Retrovirus particles bud from host cell
   membrane (forms the retroviral envelope) → virions pinched off.

Question 22

What do the cis sequences of the normal retrovirus genome consist of?

Answer 22

1. A non coding sequence called LTR: Long Terminal Repeat which contains promoter sequences for making RNA from provirus.
2. Packaging sequence found downstream of left (5’) LTR which directs RNA genome packaging into virions.

Question 23

What do the trans sequences of the normal retrovirus genome consist of?

Answer 23

1. Gag gene: Codes for retroviral core proteins.
2. Pol gene: encodes RTase and integrase enzymes.
3. Env gene: encodes viral envelop proteins (for interaction of virions with specific cell surface receptors on target cell membrane) .

Question 24

What is the process of producing a retroviral vector?

Answer 24

1. The packaging cell is engineered to contain the LTR-gagpol-env-LTR sequence without the packaging sequence Ψ
2. A vector with the LTR-Ψ-foreign gene-LTR is inserted into the cell
3. The galpolenv gene allows for viral proteins, enzymes and membrane to be formed but the gene sequence cant be put into the capsid because of the lack of Ψ
4. Since the vector genes have Ψ that sequence is packaged into the virus and released from the cell and collected to be adminstered to the patient.

Question 25

What makes the retroviral particle non infectious?

Answer 25

It doesnt contain the gagpolenv genes so they cant replicate

Question 26

What kind of cells are used as the retroviral packaging cell?

Answer 26

Examples: mouse 3T3 fibroblasts, human embryonic kidney (HEK293) cells are commonly used.

Question 27

What is an application of retroviral gene therapy?

Answer 27

1. ADA deficiency: Ex vivo gene transfer carried out on lymphocytes, bone marrow or umbilical cord blood cells, then re-infused into the patient
2. Cancer gene therapy: tumour genes are removed form the patient and cultured –> genes that stimulate inflammation & systemic immunity are inserted into the cell ( IL-2, TNF-a or GM-CSF genes) which leads to systemic immunity and destruction of tumor cells in vivo.
3. CAR T cell therapy: T cells are removed from the blood –> gene therapy to insert gene coding for chimeric antigen receptor (CAR)–> replicated –> IV infusion into patient–> bind to antigens on cancer cells and kill them

Question 28

What are the reasons that ADA deficiency is potentially an ideal disease for the first gene therapy trial?

Answer 28

1. Disease results from the loss of function of a single gene.
2. ADA levels vary widely in the normal population so tight control of the introduced gene activity is not crucial.
3. ADA gene is small and easy to manipulate in the laboratory.
4. Target cells for therapy are lymphocytes, which are accessible, easy to culture and easy to put back into the body of the patient
5. Also Hemtapoietic stem and progenitor cells
   (HSPCs) have the ability to reconstitute a new
   hematopoietic system when transplanted into
   an immunodepleted patient

Question 29

What are lentiviruses and are they currently used in gene therapy?

Answer 29

They are a subclass of retrovirus
lentiviral based-vectors are the most popular viral gene delivery system used in research, but its applications for ex vivo gene therapy are gaining momentum

Question 30

Are retroviruses enveloped or naked viruses?

Answer 30

Enveloped

Question 31

Are adenoviruses enveloped or naked viruses?

Answer 31

Naked

Question 32

What is the genetic material in adenoviruses and what’s the size of it?

Answer 32

linear double-stranded DNA genome (~ 36 kb).

Question 33

Does the gene get incorporated into the chromosome when using adenoviruses and retroviruses?

Answer 33

Retroviruses: integrated into chromosome
Adenovirus: not integrated, remains as episomal DNA in the nucleus

Question 34

What is the process by which adenovirus infects a cell?

Answer 34

Adhesion and uptake by endocytosis–> endosomal disruption–> releases DNA into cytoplasm –> episomal DNA

Question 35

What are the advantages of using an adenoviral vector?

Answer 35

1. The biology of adenovirus is well characterized.
2. The adenovirus is not associated with severe human pathology (in humans, associated with mild respiratory disease).
3. The virus is efficient in introducing its DNA into wide range of host cell types (can be used in both ex vivo and in vivo applications).
4. Adenoviral DNA is not integrated into host cell genome → risk associated with insertional mutagenesis is avoided.
5. The adenovirus is able to infect non-dividing, terminally differentiated cells.
6. Ease of production of viruses in large quantities, facilitated by virus particle stability. → adenoviral vectors purified and concentrated and stored frozen until use

Question 36

What are the disadvantages of using an adenoviral vector?

Answer 36

1. Low gene transfer efficiency- adenovirus and adenoviral associated virus are common in the human population; presence of circulating neutralizing antibodies as a result of virus exposure. → reduce gene transfer efficiency.
2. Transient correction. Episomal viral genome not replicated when the host cell divides. Loss of viral DNA in descendent cells.
3. Host cell receptor used for infection by adenovirus is not expressed on some cells (e.g. smooth muscle cells).
4. Expression of adenovirus proteins could cause an immune reaction that leads to lysis of target cells. So you lose the expression of the gene and you get an undesirable inflammatory reaction

Question 37

What are the applications of Adenovirus Vector?

Answer 37

1. Cystic fibrosis: transfer the CFTR gene
2. Muscular dystrophy: transfer the dystrophin gene
3. Cancer gene therapy: transient expression
   of “therapeutic” genes like tumour suppressor genes to destroy tumor cells.

Question 38

How are