Lecture 9: Gene Therapy and DTC Genetic Services

Question 1

____: The use of recombinant DNA technology to correct a genetic defect in a mutant cell carrying defective gene with purpose of therapeutic outcome

Answer 1

Gene Therapy

Question 2

True or False: In order to approach a disease for gene therapy potential, it must be a trigenic disease

Answer 2

False - must be monogenic

Question 3

True or False: Missense mutations can lead to loss or gain of function due to changes in amino acid sequence while a non-sense mutation can lead to truncated mRNA or protein due to insertion of a stop codon

Answer 3

True

Question 4

Early stop codon, shifting of the reading frame, or indels of 1 or 2 nucleotides are a result of which mutation?
A. Missense
B. Nonsense
C. Frameshift

Answer 4

C. Frameshift

Question 5

Unstable mutant mRNA/proteins, or mRNA skipping exon, or mutation at junction of intron/exon is a result of which type of mutation?
A. Splicing site mutation
B. Frameshift mutation
C. Nonsense mutation
D. Missense mutation

Answer 5

A. Splicing site mutation

Question 6

What happens when aberrant methylation occurs?

Answer 6

• Gene is repressed (DNA methylation occurs at promoter)
• There is no mutation in gene body

Question 7

Which is the only mutation that does not result in decreased expression of mRNA?

Answer 7

Missense mutations

Question 8

_____: Re-introduction of normal (wild type) version of mutated gene to correct a specific condition. Typically used when lack of expression or LOF mutation of a gene causes pathology.

Answer 8

Gene Augmentation/Replacement Therapy

Note: Viral Vectors containing recombined human genes are commonly used!

Question 9

True or False: Once viral vectors are administered for gene augmentation/replacement therapy, they can replicate in other human tissues

Answer 9

False - they cannot replicate or infect other cells

Question 10

For Gene Augmentation, integration or non-integration of virus depends on type of ___

Answer 10

virus

Question 11

____ expression: When a virus is not integrated

Answer 11

Episomal

Question 12

True or False: All the viruses used in gene augmentation/replacement therapy are integrated

Answer 12

False - they are not ALL

Question 13

True or False: There are size restrictions to consider when attempting gene augmentation/repair

Answer 13

True

Question 14

What is a potential problem associated with gene augmentation/replacement?

Answer 14

Insertional mutagenesis

Question 15

True or False: Exogenous control of gene expression is lost in the transgene

Answer 15

False - endogenous control is lost

Question 16

Why is it possible that proliferating tissues may lose the transgene?

Answer 16

Non-proliferating tissues are hard to transduce

Question 17

Which condition is autosomal recessive and associated with adenosine deaminase deficiency?

Answer 17

SCID (severe combined immunodeficiency)

Question 18

ADA (adenosine deaminase) is required for which pathway?

Answer 18

Purine salvage pathway

Question 19

People with SCID’s have trouble making ___, which leads to lymphocyte development problems
A. pyramidines
B. purines
C. nucleotides

Answer 19

B. purines

Question 20

Although bone marrow transplants and extracted lymphocyte gene replacement were somewhat successful at treating SCIDS, which treatment provided long-lasting success?

Answer 20

Extracted bone marrow stem cells viral gene replacement therapy

Question 21

Why do ~20% of SCID’s patients develop leukemias?

Answer 21

Insertional mutagenesis

Question 22

Strimvelis is the EMA approved drug to treat which condition?

Answer 22

SCID

Question 23

True or False: Transgenes typically have a weak promoter/enhancer, both of which are typically euchromic

Answer 23

False - transgenes typically have STRONG promoter/enhancer, both of which are EUCHROMIC

Question 24

The introduction of exogenous DNA into cells comes with the possibility the exogenous DNA will be incorporated into genome via _____ _____

Answer 24

non-homologous end joining (NHEJ)

Question 25

Where can exogenous virus DNA land?

Answer 25

1. Promoter of oncogene (over-expression) or repressed gene (over-expression) 2. Within sequence of TS (thereby: inactivating it)

Question 26

_____ is the most common form of childhood blindness. It occurs when RPE65 (isomerohydrolase) is mutated.

Answer 26

Leber Congenital Amaurosis

Question 27

True or False: Patients with mutated RPE65 are unable to regenerate chromophores for vision

Answer 27

True

Question 28

Luxturna is a gene therapy for treatment of which condition? A. Leber Congenital Amaurosis B. SCID's

Answer 28

A. Leber Congenital Amaurosis

Question 29

Adeno-associated virus (AAV) vectors encodes a WT copy of RPE65 and is used to treat which condition?

Answer 29

Leber Congenital Amaurosis (LCA)

Question 30

Which type of therapy is employed when a disease state arises due to expression of a mutated gene (dominant negative or GOF) or the expression of a WT gene that should be repressed? A. Gene Augmentation/Replacement Therapy B. Gene Blocking Therapy

Answer 30

B. Gene Blocking Therapy

Question 31

Both Anti-sense and RNAi or siRNA are a type of ______ therapy

Answer 31

Gene Blocking Therapy

Question 32

Which type of therapy involves DNA oligonucleotide that is complement to target mRNA, which leads binding of oligo to target mRNA to block translation? A. RNAi B. siRNA C. Anti-sense Therapy

Answer 32

C. Anti-sense Therapy

Question 33

Which type of therapy involves RNA oligonucleotide complementary to target mRNA. Following this, RISC (RNA-induced silencing complex) binds to oligo, searches for target mRNA, and DEGRADES it? A. RNAi/siRNA B. Anti-sense Therapy

Answer 33

A. RNAi/siRNA

Question 34

True or False: Therapeutic efficacy will decrease with time when gene-blocking therapy is used

Answer 34

True

Question 35

What are the four main problems with Anti-sense and RNAi/siRNA?

Answer 35

1. Off target effects 2. RNA/DNA degradation 3. Organ targeting 4. Limited durability

Question 36

CRISPER/Cas9 system is made up of which two components?

Answer 36

sgRNA and Cas9 nuclease (generates ds break!)

Question 37

True or False: Cas9 endonuclease, used in CRISPER/Cas9, can be guided to a genomic locus by DNA

Answer 37

False - guided by RNA

Question 38

To use CRISPR, what is the only component needed to be designed for a specific locus? A. RNA fragment B. DNA fragment C. Protein fragment D. cDNA

Answer 38

A. RNA fragment

Question 39

True or False: CRISPR/Cas9 is efficient and accurate

Answer 39

True

Question 40

Which part of the CRISPR machinery generates a double stranded break? A. SgRNA B. Cas9

Answer 40

B. Cas9

Question 41

In CRISPR, how is double stranded break repaired?

Answer 41

NHEJ

Question 42

Once indels are generated using CRISPR, the indels generate a ____ mutation

Answer 42

frameshift mutation

Question 43

True or False CRISPR allows for the activation of target gene

Answer 43

False - allows for inactivation

Question 44

True or False: Gene inactivation is possible in the lab, but gene editing is more commonly used (as in CRISPR)

Answer 44

False - CRISPR uses gene inactivation but gene editing is only possible in the lab

Question 45

True or False: Gene editing has low efficiency, non-proliferating cells do not do HR, and a template must be reintroduced

Answer 45

Tru

Question 46

Problems with CRISPR?

Answer 46

1. Template must be introduced 2. Template / CRISPR/CAS9 pushes capacity of delivery system 3. Many cells that need to be corrected are differentiated and quiescent (not cycling) - meaning HR is greatly reduced

Question 47

Mutations in HBB can lead to ____ disease

Answer 47

Sickle Cell Disease

Question 48

Retroviral transduction with wild-type ___ and wild-type ___ can be used to treat SCD

Answer 48

HBF and HBB

Question 49

In sickle cell disease, CRISPR/Cas9 inactivation of BCL___, a repressor of HBF (fetal hemoglobin), can be used for treatment A. BCL10A B. BCL11A C. BCL12A

Answer 49

B. BCL11A

Question 50

True or False: Retroviral vectors integrate into host genome

Answer 50

True

Question 51

Which virus is a transduction vector that allows for episomal expression?

Answer 51

Adenoviral Vectors and AAV (adeno-associated virus vectors)

Question 52

True or False: Lentiviral vectors are RNA viruses that integrate into host's genome (e.g HIV)

Answer 52

True

Question 53

What is the main problem with viral delivery?

Answer 53

Insertional mutagenesis

Question 54

Are changes created using gene therapy permanent? Inheritable?

Answer 54

Permanent, but not inheritable

Question 55

Which technique does 23 and me use for genetic testing?

Answer 55

Genotyping by array - gene probes allow for detection of KNOWN variants

Question 56

Which genetic testing technique is expensive, but allows one to sequence all exons in a sample and identify gene variants, mutations, etc.? A. Genotyping by array B. Exome Sequencing C. Whole Genome Sequencing

Answer 56

B. Exome Sequencing

Question 57

True or False: Whole genome sequencing is the most expensive form of genetic testing. It allows for sequencing ALL DND in a sample and identify gene variants, mutations, etc.

Answer 57

True