Lecture 9: Gene Therapy and DTC Genetic Services Flashcards
____: The use of recombinant DNA technology to correct a genetic defect in a mutant cell carrying defective gene with purpose of therapeutic outcome
Gene Therapy
True or False: In order to approach a disease for gene therapy potential, it must be a trigenic disease
False - must be monogenic
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
True
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
C. Frameshift
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
A. Splicing site mutation
What happens when aberrant methylation occurs?
- Gene is repressed (DNA methylation occurs at promoter)
- There is no mutation in gene body
Which is the only mutation that does not result in decreased expression of mRNA?
Missense mutations
_____: 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.
Gene Augmentation/Replacement Therapy
Note: Viral Vectors containing recombined human genes are commonly used!
True or False: Once viral vectors are administered for gene augmentation/replacement therapy, they can replicate in other human tissues
False - they cannot replicate or infect other cells
For Gene Augmentation, integration or non-integration of virus depends on type of ___
virus
____ expression: When a virus is not integrated
Episomal
True or False: All the viruses used in gene augmentation/replacement therapy are integrated
False - they are not ALL
True or False: There are size restrictions to consider when attempting gene augmentation/repair
True
What is a potential problem associated with gene augmentation/replacement?
Insertional mutagenesis
True or False: Exogenous control of gene expression is lost in the transgene
False - endogenous control is lost
Why is it possible that proliferating tissues may lose the transgene?
Non-proliferating tissues are hard to transduce
Which condition is autosomal recessive and associated with adenosine deaminase deficiency?
SCID (severe combined immunodeficiency)
ADA (adenosine deaminase) is required for which pathway?
Purine salvage pathway
People with SCID’s have trouble making ___, which leads to lymphocyte development problems
A. pyramidines
B. purines
C. nucleotides
B. purines
Although bone marrow transplants and extracted lymphocyte gene replacement were somewhat successful at treating SCIDS, which treatment provided long-lasting success?
Extracted bone marrow stem cells viral gene replacement therapy
Why do ~20% of SCID’s patients develop leukemias?
Insertional mutagenesis
Strimvelis is the EMA approved drug to treat which condition?
SCID
True or False: Transgenes typically have a weak promoter/enhancer, both of which are typically euchromic
False - transgenes typically have STRONG promoter/enhancer, both of which are EUCHROMIC
The introduction of exogenous DNA into cells comes with the possibility the exogenous DNA will be incorporated into genome via _____ _____
non-homologous end joining (NHEJ)
Where can exogenous virus DNA land?
- Promoter of oncogene (over-expression) or repressed gene (over-expression)
- Within sequence of TS (thereby: inactivating it)
_____ is the most common form of childhood blindness. It occurs when RPE65 (isomerohydrolase) is mutated.
Leber Congenital Amaurosis
True or False: Patients with mutated RPE65 are unable to regenerate chromophores for vision
True
Luxturna is a gene therapy for treatment of which condition?
A. Leber Congenital Amaurosis
B. SCID’s
A. Leber Congenital Amaurosis
Adeno-associated virus (AAV) vectors encodes a WT copy of RPE65 and is used to treat which condition?
Leber Congenital Amaurosis (LCA)
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
B. Gene Blocking Therapy
Both Anti-sense and RNAi or siRNA are a type of ______ therapy
Gene Blocking Therapy
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
C. Anti-sense Therapy
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
A. RNAi/siRNA
True or False: Therapeutic efficacy will decrease with time when gene-blocking therapy is used
True
What are the four main problems with Anti-sense and RNAi/siRNA?
- Off target effects
- RNA/DNA degradation
- Organ targeting
- Limited durability
CRISPER/Cas9 system is made up of which two components?
sgRNA and Cas9 nuclease (generates ds break!)
True or False: Cas9 endonuclease, used in CRISPER/Cas9, can be guided to a genomic locus by DNA
False - guided by RNA
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
A. RNA fragment
True or False: CRISPR/Cas9 is efficient and accurate
True
Which part of the CRISPR machinery generates a double stranded break?
A. SgRNA
B. Cas9
B. Cas9
In CRISPR, how is double stranded break repaired?
NHEJ
Once indels are generated using CRISPR, the indels generate a ____ mutation
frameshift mutation
True or False CRISPR allows for the activation of target gene
False - allows for inactivation
True or False: Gene inactivation is possible in the lab, but gene editing is more commonly used (as in CRISPR)
False - CRISPR uses gene inactivation but gene editing is only possible in the lab
True or False: Gene editing has low efficiency, non-proliferating cells do not do HR, and a template must be reintroduced
Tru
Problems with CRISPR?
- Template must be introduced
- Template / CRISPR/CAS9 pushes capacity of delivery system
- Many cells that need to be corrected are differentiated and quiescent (not cycling) - meaning HR is greatly reduced
Mutations in HBB can lead to ____ disease
Sickle Cell Disease
Retroviral transduction with wild-type ___ and wild-type ___ can be used to treat SCD
HBF and HBB
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
B. BCL11A
True or False: Retroviral vectors integrate into host genome
True
Which virus is a transduction vector that allows for episomal expression?
Adenoviral Vectors and AAV (adeno-associated virus vectors)
True or False: Lentiviral vectors are RNA viruses that integrate into host’s genome (e.g HIV)
True
What is the main problem with viral delivery?
Insertional mutagenesis
Are changes created using gene therapy permanent?
Inheritable?
Permanent, but not inheritable
Which technique does 23 and me use for genetic testing?
Genotyping by array
- gene probes allow for detection of KNOWN variants
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
B. Exome Sequencing
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.
True
