Review questions

Question 1

Which sentence better defines gene therapy?
(a) A method to cure genetic disorders
(b) A method to deliver a gene
(c) A method to correct a defective gene
(d) A method to silence the expression of a defective gene
(e) All the previous options

Answer 1

e)

Question 2

Which of the following points is not essential in the development of a gene therapy clinical trial?
(a) Delivery system
(b) Target cell/organ
(c) Immune response
(d) Civil status
(e) Therapy cost

Answer 2

d)

Question 3

To have a successful gene therapy, the healthy gene should be inserted in the target cell and:
(a) Destroy the defective gene
(b) Produce the correct amount of normal protein
(c) Bind to mRNA molecules in the cell
(d) Be inserted in the mitochondria
(e) None of the previous

Answer 3

b)

Question 4

From the following, which is a disadvantage of gene therapy?
(a) Eradicate diseases
(b) Prevent diseases
(c) High cost
(d) Enormous potential
(e) Correct genetic defects

Answer 4

c)

Question 5

The first clinical trial and the first death caused by gene therapy had as a disease target (choose the correct answers):
(a) Adenosine deaminase deficiency
(b) Severe combined immunodeficiency
linked to X chromosome
(c) Leber’s congenital amaurosis
(d) Ornithine transcarbamylase deficiency
(e) Lipoprotein lipase deficiency

Answer 5

a), d)

Question 6

Alix, flotillin, TSG101, and CD63 proteins are:
(a) Markers used to identity high-density lipoprotein-mimicking systems
(b) Markers used to identify microvesicles such as exosomes
(c) Markers used to identify high-density lipoprotein-mimicking systems and microvesicles such as exosomes
(d) Used to confer stealth properties to “smart liposomes”
(e) None of the above

Answer 6

b)

Question 7

Polyethylene glycol (PEG) is:
(a) A hydrophilic polymer used as a shielding reagent in polymer-based and lipid-based systems
(b) Added to the formulation with the purpose of promoting cellular targeting
(c) Added to the formulation with the purpose of decreasing the bloodstream circulation times
(d) A steric promoter of the interaction and binding of blood components (like the complement system elements) to the vector surface
(e) None of the above

Answer 7

a)

Question 8

Examples of “smart liposomes” are:
(a) Liposomes able to deliver their cargo to a specific cell population
(b) Liposomes able to deliver their cargo under the influence of a specific stimulus
(c) Liposomes incorporating ligands that specifically recognize and interact with certain cell surface components allowing the cargo to be selectively delivered
(d) Liposomes taking advantage of specific differentiating conditions such as endogenous pH levels and the redox environment or exogenous factors such as magnetic fields, ultrasound, and light to deliver their cargo
(e) All of the above
(f) None of the above

Answer 8

e)

Question 9

There are several physical methods used to
deliver nucleic acids, such as:
(a) Hydrodynamic delivery, in which hydrostatic pressure rise will enlarge the fenestrae enabling the delivery
(b) Microinjection, which is a very straightforward method applied to normal-sized cells
(c) Electroporation and nucleofection, that are based on the use of an electric pulse to
open transient pores in the cell membrane and nuclear membrane, respectively
(d) All of the above
(e) Answers (a) and (c) are correct
(f) None of the above

Answer 9

e)

Question 10

Many inorganic compounds have been used to make inorganic nanoparticles, including:
(a) Quantum dots, that commonly include noble metals such as gold and silver in their composition
(b) Single-walled carbon nanotubes, composed of a single graphene sheet, having 0.4–3 nm of diameter
(c) Silica-based systems, that are made up of silica (SiO3) and that can be used to deliver nucleic acids, drugs, and dyes
(d) All of the above
(e) None of the above

Answer 10

b)

Question 11

Which of the following viral vectors induces a potentially high inflammatory profile?
(a) Adenoviral vectors
(b) Herpes simplex virus vectors
(c) Recombinant AAV vectors
(d) Lentiviral and gamma retroviral vectors
(e) Gutless adenoviral vectors and herpes simplex virus vectors

Answer 11

b)

Question 12

Which of the following viral vectors has the
highest cloning capacity?
(a) Recombinant AAV vectors
(b) Gamma retroviral vectors
(c) Herpes simplex virus vectors
(d) Second-generation adenoviral vectors
(e) Lentiviral vectors

Answer 12

c)

Question 13

Which of the following sequences enumerates virus in the order of increasingly difficult genetic engineering?
(a) AAV, vaccinia, adenovirus
(b) Vaccinia, baculovirus, lentivirus
(c) AAV, lentivirus, adenovirus
(d) Gamma retrovirus, AAV, vaccinia
(e) Vaccinia, lentivirus, herpes simplex vector

Answer 13

c)

Question 14

Which of the following describes a vector with the ability to transduce post-mitotic cells, has a long-term expression in vivo, and has an
easy production process?
(a) Gutless adenoviral vectors
(b) Lentiviral vectors
(c) Herpes simplex virus vectors
(d) Baculovirus vectors
(e) Gamma retrovirus vectors

Answer 14

b)

Question 15

Which of the following sequences enumerates virus in the order of increasing oncolytic potential?
(a) Lentivirus, AAV, baculovirus
(b) AAV, herpes simplex virus, gamma retrovirus
(c) Gutless adenovirus, lentivirus, herpes simplex virus
(d) Lentivirus, adenovirus, AAV
(e) AAV, adenovirus, herpes simplex virus

Answer 15

e)

Question 16

The development of strategies to inactivate retroviral vectors constituted a major advance in the improvement of their safety profile. How was this accomplished?
(a) Deletion or modification of the U3 region of the LTRs
(b) Reverse transcriptase inactivation
(c) Integrase modification
(d) gal and pol deletion
(e) Modification of envelope proteins

Answer 16

a)

Question 17

Lentiviral vectors can lead to transgene integration. Is it possible to revert this viral feature? How?
(a) No, use AAV as an alternative
(b) Yes, by having integrase protein without packaging signal
(c) Yes, by mutating the integrase protein
(d) No, use non-viral vectors as an alternative
(e) Yes, by deleting the LTRs

Answer 17

c)

Question 18

Which of the following features better define an oncolytic vector?
(a) With replication, with transgene integration and with expression in every cell
(b) Without replication, without transgene integration and with expression in tumor cells
(c) With replication, hard to produce and with expression in every cell
(d) Without replication, easy to produce and with expression in tumor cells
(e) None of the above

Answer 18

c)

Question 19

Why is the immune response a particularly important extracellular barrier when using viral vectors to gene delivery?
(a) Because viral vectors enter the cell using membrane receptors
(b) Because viral proteins elicit a strong immune response
(c) Because viral vectors only activate the innate immune response
(d) Because viral proteins elicit immune suppression
(e) Because viral vectors cannot deliver genes into immune-privileged sites

Answer 19

b)

Question 20

Which of the following strategies is not used to enhance the ability of non-viral vectors to escape the endo-lysosome pathway?
(a) Binding with agents that create pores in the endocytic vesicles
(b) Use of agents prone to protonation
(c) Use of neutralizing antibodies
(d) Use of viral peptides
(e) Use of bacterial proteins

Answer 20

c)

Question 21

The delivery of a transgene using a non-viral vector with 50 nm to nondividing cells failed to reach the nucleus. Which of the following
features could explain the failure?
(a) The use of non-viral vectors is exclusive to dividing cells
(b) The nuclear pore complex does not allow particles of the size used
(c) The gene was not integrated in the cell genome
(d) The gene did not have a nuclear localization signal
(e) The gene size was more than 250 kb

Answer 21

b)

Question 22

Which of the following is not considered a technical barrier to gene delivery?
(a) Physical restrictions
(b) Unspecific interactions
(c) Cellular targeting
(d) Gene persistence
(e) Sustainable gene expression

Answer 22

b)

Question 23

Stem cells are non-differentiated and unspecialized cells characterized by their ability to self-renew, maintain the pool of stem cells and differentiate into mature specialized cells. Which of the following sentences is correct?
(a) There are different types of stem cells, namely the pluripotent neural stem cells and embryonic stem cells.
(b) Pluripotent stem cells can be isolated from the inner cell mass of the blastocyst.
(c) Pluripotent stem cells can be obtained by transdifferentiation.
(d) All of the above
(e) None of the above

Answer 23

b)

Question 24

Neural stem cells can be obtained from:
(a) Isolation from fetal and adult brains
(b) Direct chemical induction using valproic acid, CHIR99021 and RepSox
(c) Neural induction of pluripotent stem cells
(d) All of the above
(e) None of the above

Answer 24

d)

Question 25

Cell reprogramming enables obtaining new types of cells to be used as human cell models and as a source of cells for cell-based therapies. Choose the option that best applies. (a) Somatic cell nuclear transfer (SCNT) and cell fusion are strategies that allow cell reprogramming (b) To obtain induced pluripotent stem cells (iPSC) it is mandatory to use reprogramming factors, by delivering them with either viral or non-viral systems (c) Through direct reprogramming, it is possible to obtain differentiated cells such as neurons or multipotent stem cells like NSC (d) Answers (a) and (c) (e) Answers (b) and (c)

Answer 25

d)

Question 26

Fully differentiated somatic cells can be reprogrammed into a pluripotent state. The resulting cells are designated as induced pluripotent stem cells (iPSC), which are able to originate other cell types. Which of the following sentences is correct? (a) iPSC are very similar to the natural pluripotent ESC, namely in terms of morphology, cell potency, immortal growth and ability to originate embryoid bodies and to form teratomas (b) Several factors dictate the reprogramming efficiency, namely the type/source of cells to be reprogrammed (c) The four reprogramming factors described by Yamanaka (Sox-2, Klf4, Oct4 and c-Myc) play important roles in stem cell pluripotency and control cell proliferation and apoptosis (d) The reprogramming efficiency of somatic cells into iPSC is very low (e) All of the above (f) None of the above

Answer 26

e)

Question 27

The development of new disease models and platforms such as organoids and 3D bioprinting has been galvanized by the discovery of iPSC. Which of the following sentences is correct? (a) Organoids are three-dimensional (3D) cellular clusters that self-organize themselves into structures that resemble miniorgans, which are very promising to study, for example, pathways under the influence of the immune system, such as neurogenesis (b) Organoids can be obtained from different sources of cells (aNSC, ESC and iPSC) and have been used as disease models and also to study and manipulate tissue development and regeneration (c) Chitosan, alginate, hyaluronic acid, nanocellulose, poloxamer and polyethylene glycol (PEG)-conjugated synthetic polymers are examples of compounds used in 3D bioprinting (d) Answers (a) and (c) (e) Answers (b) and (c) (f) All of the above

Answer 27

e)

Question 28

Classify as true or false each one of the following sentences: (a) Gene augmentation therapy can only be applied in monogenic diseases (b) Gene augmentation therapy involves the delivery of a protein-coding gene (c) Gendicine® is a gene therapy product based on gene replacement therapy (d) Autophagy is the only cellular degradation pathway (e) The activation of autophagy is a gene therapy strategy useful for neurodegenerative diseases

Answer 28

(a) F (b) T (c) F (d) F (e) T

Question 29

In a gene therapy study targeting a monogenic recessive disorder, which of the following is crucial to ensure the success of the wild-type inserted gene? (a) Remove the mutant gene (b) Produce more cytokines (c) Ensure that the correct amount of protein is produced (d) Insert the gene in the mitochondria (e) Use specifically a gene addition strategy

Answer 29

c)

Question 30

Which one(s) of the following are aims of gene addition therapy? (a) Focus on reducing mutant protein levels (b) Target monogenic dominant disorders (c) Deliver protein-coding genes (d) Improve cellular homeostasis (e) Cure the genetic cause of the disease

Answer 30

c), d), e)

Question 31

Why autophagy activation by gene therapy approaches is a valuable target for neurodegenerative diseases? (a) Because there is no other option (b) Because autophagy degrades misfolded aggregate proteins (c) Because the ubiquitin-proteasome system does not function in neurons (d) Because there are no pharmacological drugs to activate autophagy (e) Because autophagy is not a dynamic process

Answer 31

b)

Question 32

Which of the following features is common between ASOs (antisense oligonucleotides) and siRNAs (small interfering RNAs)? (a) Composed of more than 100 nucleotides (b) Only of synthetic origin (c) Degradation involves the RISC complex (d) Degradation involves RNase H (e) High specificity

Answer 32

e)

Question 33

Which of the following features is not improved in third-generation ASOs compared with the second generation? (a) Nuclease resistance (b) Stability (c) Target affinity (d) Cellular uptake

Answer 33

d)

Question 34

Which of the following is not a functional action mechanism of ASOs? (a) RNAse H-mediated degradation (b) Translational arrest (c) Splicing modulation (d) Inhibition of transcription (e) Inhibition of RNA-binding proteins

Answer 34

e)

Question 35

Which of the following is not a feature of microRNAs? (a) Encoded by their own genes (b) Fully complementary to mRNA (c) Multiple mRNA targets (d) Different mechanisms of gene expression regulation (e) 19–25 nucleotide RNA duplex

Answer 35

b)

Question 36

Which of the following has/have one single mRNA target? (a) siRNA (b) siRNA and shRNA (c) shRNA and microRNA (d) microRNA (e) dsRNA and microRNA

Answer 36

b)

Question 37

Which of the following elements does not belong to the siRNA pathway of the RNA interference mechanism? (a) Argonaute (b) dsRNAs (c) RISC (d) Exportin (e) Dicer

Answer 37

d)

Question 38

In the context of gene therapy, gene editing systems can be used to: (a) Disrupt a mutated gene (b) Substitute a malfunctioning gene (c) Regulate the expression of a mutated gene (d) None of the above (e) All of the above

Answer 38

e)

Question 39

From the following, select the ones that apply to the CRISPR-Cas system of gene editing: (a) Direct binding to the target DNA through a protein domain (b) DNA recognition motif composed of several repeated units (c) Ligation of target DNA to RNA through base complementarity (d) Protein with only one site with nuclease activity (e) Derived from a prokaryote “immune” system (f) Requires a guide RNA sequence (g) Consists necessarily of a fusion protein (h) Has the ability to induce DNA doublestrand breaks

Answer 39

c), e), f), h)

Question 40

In the CRISPR-Cas system, what is the role of the gRNA? (a) It ensures that the cell recognizes that the DNA is damaged and tries to repair it (b) It determines the site at which the Cas9 enzyme cuts the genome (c) It uses the DNA repair machinery to introduce changes to one or more genes (d) It makes a cut across both strands of the DNA

Answer 40

b)

Question 41

From the following, select the ones that apply to the zinc-finger nucleases and TALENs systems of gene editing: (a) Direct binding to the target DNA through a protein domain (b) DNA recognition motif composed of several repeated units (c) Function in pairs (d) Use fusion proteins (e) Can lead to the introduction of indels (f) Require a guide RNA sequence

Answer 41

a), b), c), d), e)

Question 42

What is the main aim of a phase I clinical trial testing of a gene therapy strategy? (a) Access therapy safety (b) Access therapy efficacy (c) Compare the therapy with the gold standard therapy (d) Access the long-term effect of the therapy (e) None of the above

Answer 42

a)

Question 43

Which of the following is not a gene therapy strategy targeting cancer? (a) Suicide gene therapy (b) Oncolytic gene therapy (c) Tumor-suppressor gene therapy (d) Immunomodulatory gene therapy (e) Neurotrophic gene therapy

Answer 43

e)

Question 44

Which feature makes the eye a good target for gene therapy? (a) Difficult access (b) Immunological privilege (c) Is part of the nervous system (d) Non-viral vectors can be used to mediated delivery (e) Has only one type of cells

Answer 44

b)

Question 45

Which of the following viral vectors is more suitable for a direct administration of a gene therapy targeting the brain neurons? (a) Lentiviral vectors (b) Adenoviral vectors (c) Gamma retrovirus-based vectors (d) Adenovirus gutless vectors (e) Baculovirus-based vectors

Answer 45

a)