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A BIOL1024 Part 2 > gene therapy 2 > Flashcards

gene therapy 2 Flashcards

1
Q

What is Tay-Sachs disease relate to ?

A

Lipids

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2
Q

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

A

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

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3
Q

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

A

Amniocentesis- 12th week +

Chorionic Villus Sampling 8-12th week

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4
Q

What cells are extracted from an amniocentisis?

A

Fetal skin cells in the amniotic fluid.

The centrifuge and culture

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5
Q

What test is performed on newborns?

A

Guthrie test

tests for PKU, CF, sickle cell anaemia

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6
Q

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

A

Blood sample

Skin biopsy

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7
Q

How is a tissue/DNA sample processed?

A

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

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8
Q

What test is conducted to test for Tay-Sachs?

A

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

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9
Q

What genes are looked at for Familial hypercholesterolaemia?

A

LDLR
APOB
PCSK9

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10
Q

Describe the screening process for unknown IEM

A

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

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11
Q

Describe the screening process for known IEM mutation

A 
Suspect IEM
Lyse cells and extract DNA
PCR and run on gel 
ARMS
DNA sequencing to compare to known image of that mutation
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12
Q

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

A

35

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13
Q

What are the 3 steps and temperatures for the PCR?

A

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

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14
Q

Describe how PCR detects known mutations

A

Cells are pelleted lysed and lysate used for PCR

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

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15
Q

How does PCR test for base substitutions?

A

Using ARMS- Amplification refractory mutation system

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16
Q

Describe how ARMS works

A

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

17
Q

Name 4 ways to scan genes for unknown mutations

A

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

18
Q

How does DNA sequencing work?

A

Run sequence of sample and known sample and compare

19
Q

How can IEMS be treated

Name 3 examples

A

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

20
Q

How is a corrective nucleic acid prepared?

A

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

21
Q

What is the difference between Ex-vivo and In Vivo

A 
Ex-vivo= GM outside the body
In-vivo= injection of vector encoding corrective gene
22
Q

What are the 3 bennifits to Ex vivo gene therapy?

A

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

23
Q

What are 3 negatives to Ex-vivo therapy?

A

May require surgery
Some cells challenging to culture
Poor engraftment rate

24
Q

What are the 2 bennifits to In vivo gene therapy?

A

May only require an injection

Easy to treat

25
Q

What re the 2 negatives ot In vivo gene therapy?

A

Non specific targeting

Immune response to the vector

26
Q

Name the non-viral and viral gene transfer vectors

A 
Non-viral= Liposomes
Viral- adenovirus
AAV
 Retrovirus
Lentivirus
27
Q

Name the advantages and disadvantages to liposomes

A

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.

28
Q

What does TNS stand for?

A

Therapeutic Nucleotide Sequence

29
Q

Describe the parts of the Adeno-associated virus TNS

A 
DNA
Enhancer
Promoter
Intron
Transgene
Poly-A signal
ITR-inverted terminal repeats
30
Q

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

A

packaging of the TNS into the virus

31
Q

Describe the parts of the Retrovirus TNS

A 
RNA
Enhancer
Promoter
Intron
Transgene
Poly-A signal
Long terminal repeats
Psi- packaging signal
RRE
32
Q

Name the advantages to Viral vectors

A

Gets into cells
Gets into nucleus
Gets expressed

33
Q

Name the disadvantages tp Viral Vectors

A 
Toxicity 
Immune response 
Limited duration
Small inserts
Insertional mutagenesis
34
Q

Describe the steps in CRISPR/Ca9

A

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.

35
Q

What is the disadvantage to CRISPR

A

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

A BIOL1024 Part 2 (18 decks)

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