Viral Gene Therapy

Question 1

What is the most effective treatment for cancer currently as was demonstrated in the case study regarding 83% complete remission rate in B-cell acute lymphoblastic leukemia patients?

Answer 1

CAR-T Immunotherapy

Complete remission means that tests, physical exams, and scans show that all signs of your cancer are gone.

Question 2

What three gene therapy products did the U.S. Food and Drug Administration (FDA) approve for sale in the United States in 2017?

Answer 2

Luxturna (Adeno-associated virus) for mutation in RPE65 eye protein that cause blindness

CAR-T Immunotherapy includes 2:

-Tisagenlecleucel
Kymriah for B-cell precursor ALL
Pediatric
Relapsed/refractory diffuse large B-cell lymphoma (DLBCL)

-Axicabtagene Cioleucel-Yescarta (B-cell lymphoma in
adults)

Question 3

What is gene therapy?

Answer 3

Human gene therapy refers to products that introduce genetic material into a person’s cells to replace faulty or missing genetic material, thus treating a disease or abnormal medical condition.

Question 4

Introduction of genetic material into a person’s cells can do what in terms of gene therapy?

Answer 4

• Modification of the expression level of an individual gene
• Correction of abnormal genes
• Delivery of oncolytic or toxic genes

Question 5

What kinds of diseases are investigated as candidates for gene therapy?

Answer 5

Most are monogenic inherited diseases like cystic fibrosis, hemophilia, muscular dystrophy; delivery of normal gene or removal of mutated gene

Cancer: oncolytic vectors or toxic genes; effective killing of most cancer cells without damaging normal cells

Infectious diseases like AIDS

Question 6

Insertion of a viral vector into the target cell is called what for bacterial cells, eukaryotic cells, and insertion of a viral vector respectively?

Answer 6

Transformation for bacterial cells
Transfection for eukaryotic cells
Transduction for insertion of a viral vector.

Question 7

What is the most effective way to deliver genetic material into the patient’s cells?

Answer 7

through a viral vector

Question 8

Vector refers to__________ DNA that consists of the therapeutic gene, backbone, and is most often used as a vehicle to insert genetic material into the patient’s cell.

Answer 8

artificial

Question 9

What are the two most commonly used viral vectors in clinical trials especially for that of cancer treatment?

Answer 9

Adenoviral and retroviral vectors (23% and 20% respectively)

Most clinical trials have been cancer treatment (64%).

Question 10

What is the viral vector for gene therapy?

Answer 10

It is a recombinant vector meaning it is HUMAN-MADE product which is made by modification of a virus. It is not a natural phenomenon.

Question 11

What do viral vectors for gene therapy consist of?

Answer 11

• therapeutic gene AKA transgene
• signals for transcription and translation
• part of the viral genome with packaging signals AKA backbone
• normal or modified viral capsid

Question 12

What types of viruses are used for gene therapy?

Answer 12

• adeno-associated virus
• lentiviruses
• adenoviruses
• retroviruses
• alphaviruses
• herpes simplex virus

The first four are primarily used in clinical trials.

Question 13

An ideal gene therapy should have what qualities?

Answer 13

• allow efficient and selective transduction of the target cells
• the vector is maintained inside the cells for necessary time
• expresses gene of interest at levels necessary for achieving therapeutic effects
• safe

Question 14

Vectors made for hemophilia should be expressed at exact levels and so there is a narrow window to achieve for the level of expression. If the level is too low you get excessive bleeding. If the level is too high you get clotting. What aspect of an ideal gene therapy does this reflect?

Answer 14

-expresses gene of interest at levels necessary for achieving therapeutic effects

Question 15

What is the best design for gene therapy?

Answer 15

-First we have to choose a target gene. Not all genes are target genes (eg. cardiovascular disease genes).

-Second we think about administration:
Specificity of vector: specific vector for a specific purpose
Efficiency
Rout of delivery: is it local or systemic injection?
Time of expression
Therapeutic gene-base approach

Most important we need to know the safety and precautions.

Question 16

Leber congenital amaurosis is an eye disorder that primarily affects the retina, which is the specialized tissue at the back of the eye that detects light and color. People with this disorder typically have severe visual impairment beginning in infancy. What FDA-approved drug can be used to treat this condition?

Answer 16

Luxturna
consists of a subretinal injection of adeno-associated virus (AAV) containing a gene (AAV2-hRPE65v2) encoding a protein needed for the isomerohydrolase activity of the retinal pigment epithelium in the worst eye

Question 17

Adeno-associated virus (AAV) are part of the ________family, is a helper __________virus, and _________pathogenic. It consists of ________DNA that is either positive or negative-sensed around the size of 4.7Kb.

Answer 17

parvovirus
dependent
single stranded

Question 18

What is the purpose of inverted terminal repeats (ITRs) that flank both ends of the AAV DNA?

Answer 18

The Inverted Terminal Repeat (ITR) required for synthesis of the second DNA strand, viral multiplication and integration

Question 19

Which virus that in the presence of helper virus will produce infectious particles but in the absence of helper virus will induce a latent infection with integration in a unique noncoding site on chromosome 19?

Answer 19

Adeno-associated virus

Question 20

Which type of adeno-associated virus is used when producing a recombinant adeno-associated virus vector for gene therapy? What are the rep and cap proteins replaced with?

Answer 20

• AAV type 2

- rep and cap genes are replaced with the transgene or therapeutic gene

Question 21

AAV type 2 is mostly used for vector generation

Rep and Cap genes are replaced with_________. Only ITRs required in_____ for genome packaging

Structural (cap) and packaging (rep) genes are expressed in_______ in producing cell line

Ad genes is used for helper function to produce AAV infectious particles

Answer 21

transgene
cis
trans

Question 22

AAV type 2 is mostly used for vector generation

Rep and Cap genes are replaced with_________. Only ITRs required in_____ for genome packaging

Structural (cap) and packaging (rep) genes are expressed in_______ in producing cell line

Ad genes is used for helper function to produce AAV infectious particles.

Answer 22

transgene (therapeutic gene)
cis
trans

Question 23

AAV type 2 is mostly used for vector generation

Rep and Cap genes are replaced with_________. Only ITRs required in_____ for genome packaging

Structural (cap) and packaging (rep) genes are expressed in_______ in producing cell line

Ad genes is used for helper function to produce AAV infectious particles.

Answer 23

transgene (therapeutic gene)
cis
trans

Productive AAV infection requires co-infection with helper viruses that provide functions that aid in AAV replication, including larger DNA viruses such as Ad and HSV.

Question 24

What are the pros for using AVV for gene therapy? What are the cons?

Answer 24

Pros:

• non-cytotoxic and non-pathogenic without helper virus
• transduces non-dividing cells
• absence of all viral genes in the vector
• low immunogenicity
• long-term transduction as it can be integrated into the patient’s genome
• broad spectrum of infection

Cons:

• preexisting immunity to the virus
• small packaging capacity -4. kb; mRNA for most humans is significantly larger than 4Kb in size
• difficult to produce high yield of virus (depends on plasmid transfection); remember Insertion of a viral vector into eukaryotic cells is transfection
• difficult to purify

Question 25

This vector product only have exons, no introns.

Answer 25

AAV vector for gene therapy

Question 26

Lipoprotein lipase deficiency (LPLD), a rare inherited disorder which can cause severe pancreatitis can be treated with what viral gene therapy that is now expired because of high expenses?

Answer 26

Glybera: an AAV-based gene therapy

This disease is of low prevalence and costs $1.2 million for a single dose.

Question 27

Replication-Defective Adenovirus Vectors with Multiple Deletions Do Not Induce Measurable Vector-Specific T Cells in Human Trials.

Answer 27

Adenovirus vectors can be replication-defective; certain essential viral genes are deleted and replaced by a cassette that expresses a foreign therapeutic gene.

Question 28

What are the pros and cons of using adenoviral vectors for gene therapy?

Answer 28

Pros:

• high titer virus stock is possible
• high level of gene expression

Cons:

• pre-existing immunity to the virus
• strong immunogenicity
• transient expression, no genome integration

Question 29

You cannot predict what the immune response you will receive from a patient. A young men in OTC gene therapy trail died as a result of a severe systemic inflammatory reaction to the injected adenoviral vector. Why does this point to the limitations in using information from animal studies?

Answer 29

This case points to the limitations of animal studies in predicting human responses, the steep toxicity curve for replication defective adenovirus vectors, substantial subject-to-subject variation in host responses to systemically administered vectors, and the need for further study of the immune response to these vectors.

Investigation of this case led to the development of federal regulations for gene therapy.

Question 30

What is the mechanism of CAR T-cell immunotherapy for cancer?

CAR= chimeric antigen receptor

Answer 30

1. Remove blood from patient to get T cells
2. Make CAR T cells in the lab
3. Grow millions of CAR T cells
4. Infuse CAR T cells into patient
5. CAR T cells bind to cancer cells and kill them

Question 31

T-cell isolates from patients for CAR T-cell immunotherapy are transduced with what type of virus carrying the chimeric antigen receptor?

Answer 31

retrovirus and lentivirus carrying the chimeric antigen receptor

Question 32

T-cell immunotherapy is _________therapy, but cells are modified by viral delivery of artificial gene.

Answer 32

cell-based

Question 33

The artificial chimeric antigen receptor is produced by fusion containing what elements?

Answer 33

1. CD3 TCR domain- T cell activation
2. CD28 or 4-1BB co-stimulatory domain
3. Hinge and transmembrane domain
4. Antibody domain recognizes CD19 B-cell antigen (patient specific)

Question 34

Murine leukemia virus (MLV) is the most commonly used vector from what virus?

Answer 34

gamma retrovirus, simple retrovirus virus

Question 35

Production of retroviral vectors
consist of all viral genes being replaced by__________ and exogenous promoter.

The viral genes required for replication and packaging are provided in______ in packaging cell line.

Answer 35

transgene

trans

Question 36

What are the pros and cons of retroviral vectors?

Answer 36

Pros:

• Long-term expression possible, genome integration
• Ability to pseudotype with other viral envelope proteins to increase cell target range

Cons:

• Inability to infect non-dividing cells
• Sites of integration may lead to tumor formation

Question 37

Lentivirus is a complex___________ (HIV). Production of vectors is similar to retroviral vectors.
All viral genes replaced by_________.
The gag-pol and env genes are expressed in a ____________ or transfected.
Rev gene is required for packaging, RRE- rev responsible element is included into the vector

Answer 37

retrovirus
transgene
packaging line

Question 38

What are the pros and cons of lentiviral vectors?

Answer 38

Pros:

• Ability to infect both dividing and non-dividing cells
• Long -term expression possible, genome integration
• Highly specific for particular cell type (HIV-1 – CD4 cells)
• Ability to pseudotype with other viral envelope proteins to increase cell target range (Vesicular stomatitis virus VSV-G)

Cons:

• Safety concerns
• Sites of Integration, latency
• Difficult to develop packaging line due to viral gene toxicity

Question 39

What two products based on lentiviral vector were approved by FDA in 2017?

Answer 39

Kymriah (Tisagenlecleucel, Novartis): for treatment of
B-cell precursor ALL (Pediatric) and Relapsed/refractory diffuse large B-cell lymphoma (DLBCL)

Yescarta (Axicabtagene Cioleucel, Gilead Sciences Inc.) for treatment of adult B-cell lymphoma

Question 40

What is the advantages and cons for Kymriah and Yescarta ?

Answer 40

Pros:
- Patient-specific product.
Cons:
-High failure rate of CAR T-cell manufacturing
-Heterogeneity of antitumor response
-Severe toxicity (cytokine storm, neurotoxicity, B-cell aplasia)
-High price of vector production

Question 41

To optimize CAR-T expression after infusion, what is induced in patients by treatment with cyclophosphamide or fludarabine?

Answer 41

lymphodepletion

Question 42

Innate immunity and antigen-specific adaptive immune responses against vector-derived antigens reduce the efficacy and stability of _______gene transfer.

Answer 42

in vivo

A number of vectors are derived from viruses that humans encounter through natural infection (Ad, AAV), resulting in pre-existing antibodies and T and B- cells memory responses against vector antigens.