gene therapy 2

Question 1

What is Tay-Sachs disease relate to ?

Answer 1

Lipids

Question 2

What are the steps to determine the genetic cause of IEMs?

Answer 2

Tissue sample taken from suffer
Chromosomal DNA extracted
DNA analysed for known/unknown mutations

Question 3

Name the 2 types of sampling in a fetus and at what week can these be done?

Answer 3

Amniocentesis- 12th week +

Chorionic Villus Sampling 8-12th week

Question 4

What cells are extracted from an amniocentisis?

Answer 4

Fetal skin cells in the amniotic fluid.

The centrifuge and culture

Question 5

What test is performed on newborns?

Answer 5

Guthrie test

tests for PKU, CF, sickle cell anaemia

Question 6

How do we obtain a tissue/ DNA sample in an adult?

Answer 6

Blood sample

Skin biopsy

Question 7

How is a tissue/DNA sample processed?

Answer 7

Cells are washed and nuclear DNA extracted and purified by centrifugation and ethanol precipitation

Question 8

What test is conducted to test for Tay-Sachs?

Answer 8

Blood test for low levels/absence of beta-hexosaminidase activity

Question 9

What genes are looked at for Familial hypercholesterolaemia?

Answer 9

LDLR
APOB
PCSK9

Question 10

Describe the screening process for unknown IEM

Answer 10

Suspected IEM
Lyse cells and extract DNA
DNA sequencing and compare to image for that mutation

Question 11

Describe the screening process for known IEM mutation

Answer 11

Suspect IEM
Lyse cells and extract DNA
PCR and run on gel 
ARMS
DNA sequencing to compare to known image of that mutation

Question 12

Increasing PCR cell cycle number above what has little positive effect?

Answer 12

35

Question 13

What are the 3 steps and temperatures for the PCR?

Answer 13

95C- denature template DNA to ssDNA w/ heat
55C- lower temp to allow primers to anneal
72C- DNA polymerase extends primer

Question 14

Describe how PCR detects known mutations

Answer 14

Cells are pelleted lysed and lysate used for PCR

2 primers are designed to anneal to either end of the gene

Question 15

How does PCR test for base substitutions?

Answer 15

Using ARMS- Amplification refractory mutation system

Question 16

Describe how ARMS works

Answer 16

2 PCR reaction
Primer A +c amplify wild type
Primer B + c amplifies mutant DNA

Question 17

Name 4 ways to scan genes for unknown mutations

Answer 17

a) Single-strand conformational polymorphisms (SSCP)
b) Denaturing gradient gel electrophoresis (DGGE)
c) Protein truncation test
d) DNA sequencing

Question 18

How does DNA sequencing work?

Answer 18

Run sequence of sample and known sample and compare

Question 19

How can IEMS be treated

Name 3 examples

Answer 19

Can’t be treated
Drugs
Supply of missing metabolite
Control of metabolite consumption

Question 20

How is a corrective nucleic acid prepared?

Answer 20

PCR up a normal of the gene
Need carrier for gene and promoter
Insert into plasmid vector

Question 21

What is the difference between Ex-vivo and In Vivo

Answer 21

Ex-vivo= GM outside the body
In-vivo= injection of vector encoding corrective gene

Question 22

What are the 3 bennifits to Ex vivo gene therapy?

Answer 22

No immune response
No off target effects
Integration of transgenic DNA into the genome

Question 23

What are 3 negatives to Ex-vivo therapy?

Answer 23

May require surgery
Some cells challenging to culture
Poor engraftment rate

Question 24

What are the 2 bennifits to In vivo gene therapy?

Answer 24

May only require an injection

Easy to treat

Question 25

What re the 2 negatives ot In vivo gene therapy?

Answer 25

Non specific targeting | Immune response to the vector

Question 26

Name the non-viral and viral gene transfer vectors

Answer 26

``` Non-viral= Liposomes Viral- adenovirus AAV Retrovirus Lentivirus ```

Question 27

Name the advantages and disadvantages to liposomes

Answer 27

Advantages of liposomes Non-toxic. Disadvantages Inefficient transfer of DNA to target cells. Non-specific uptake - mostly endothelial. Cannot target particular cell types. Poor expression.

Question 28

What does TNS stand for?

Answer 28

Therapeutic Nucleotide Sequence

Question 29

Describe the parts of the Adeno-associated virus TNS

Answer 29

``` DNA Enhancer Promoter Intron Transgene Poly-A signal ITR-inverted terminal repeats ```

Question 30

What is the role of the ITR-inverted terminal repeats in Adeno-associated virus TNS

Answer 30

packaging of the TNS into the virus

Question 31

Describe the parts of the Retrovirus TNS

Answer 31

``` RNA Enhancer Promoter Intron Transgene Poly-A signal Long terminal repeats Psi- packaging signal RRE ```

Question 32

Name the advantages to Viral vectors

Answer 32

Gets into cells Gets into nucleus Gets expressed

Question 33

Name the disadvantages tp Viral Vectors

Answer 33

``` Toxicity Immune response Limited duration Small inserts Insertional mutagenesis ```

Question 34

Describe the steps in CRISPR/Ca9

Answer 34

finds then replaces or disables defective genes. : system produces DNA cleavage at a desired genomic target. : insertion of a donor template can lead to gene correction via the homology directed repair (HDR) pathway. : can be used for both in vivo and ex vivo gene therapy.

Question 35

What is the disadvantage to CRISPR

Answer 35

Risk of introducing off target mutations in genome regions bearing similar sequence identity to the target site