Gene Therapy

Question 1

Define Gene Therapy

Answer 1

Delivery of genetic material into a patient’s cells as a drug

Question 2

Describe the 4 ways gene therapy can work

Answer 2

1) Mutation in coding sequence - mutant protein
2) Mutation in promotor - more or less mRNA
3) Mutation in splice site - incorrectly spiced mRNA
4) Mutation in regulatory element in UTR - more or less protein

Question 3

How do mutations in coding sequence work?

Example of a disease

Answer 3

Changes amino acid sequence affecting production or function of protein.
CF - F508 deletion, loss of phenylalanine causing miscoding and degradation
Premature termination - truncated protein
Frameshift - incorrect sequence downstream

Question 4

How do mutations in promoters work?

Answer 4

Change expression level - remove site for transcription factor

Question 5

How do mutations in splice sites work?

Answer 5

5’ or 3’ UTR leading to changes in regulation of expression by slicing or trans-acting factors e.g. - binding site prevents binding increasing production

Question 6

Four challenges for gene therapy

Answer 6

1) Delivery to target cell
2) Maintenance in cell
3) High cost
4) Only suitable for diseases caused by reduced production of protein as can’t achieve precise level of expression.

Question 7

Differences between in vivo and ex vivo

Answer 7

in - Directly into patient
ex - target cells out, introduce gene, return. Good for haematopoietic system. Precise targeting and increased efficiency

Question 8

Pro’s and Con’s of Viral delivery

Answer 8

Pro’s - Efficient uptake, persist in cells modified to remove pathogenicity and tumorigenesis
Con’s - Immune response, pathogenicity, tumourigenesis, size of gene

Question 9

Pro’s and Con’s of Non-Viral delivery

Answer 9

Pro’s - Low immunogenicity, Large scale production, larger DNA molecules
Con’s - Less efficient, difficult to target specific tissues, sustained expression

Question 10

Viral delivery must…..

Answer 10

Attack and enter cell
Transport material to nucleus/maintenance as DNA
Sustained expression
Lack of toxicity/immunogenicity

Question 11

Describe Retrovirus

Answer 11

Encodes reverse transcriptase (copies viral RNA to DNA) integrated into host genome.
Mutate to remove pathogenic element
Incorporation of gene into host genome and maintenance during devision
can’t control site of integration (can cause cancer)

Question 12

Describe Lentiviruses

Answer 12

BETTER
Highly modified viruses
can integrate into non-dividing cells
Lower immunogenicity

Question 13

Describe AAV

Answer 13

Non pathogenic/low immunogenicity
Dividing and non-dividing cells
Needs helper virus to replicate
DNA retained in cell nucleus - specific site, rep and cap genes deleted, retained as emisomal concatemer
serotypes = many cell types
small - limited size of gene

Question 14

What is CAR-T immunotherapy and what is it used for?

Answer 14

Adaptive T-Cell transfer
ALL - produce chimeric antigen receptors which recognise antigens on cancer cells
Retroviral
Cytokine release syndrome

Question 15

How can we use gene therapy for haemophilia

Answer 15

Lack of clotting factors, Hepatocyte targeted AAV introduce factor IV to liver
Expression of enough - 7X more effective
Immunity - modifications of capsid
Liver cell damage - short term corticosteroids

Question 16

Pro’s of mRNA therapy

Answer 16

Less stable expression, no danger of integration

Avoid immune response/degradation

Question 17

What is CRISPR

Answer 17

Prokaryotic immune system, site-specific genome modification, most flexible and easy to use.
Off-target effects
Bacterial genome contains CRISPR - short repeated sequences with short sequences from viruses that affect bacterium inserted between.
Repeat transcribed to crRNA

Question 18

Describe process of CRISPR

Answer 18

1) Double strand break at target site by cas9
2) Repaired with cellular machinery
3) NHEJ - error prone, small insertions/deletions disrupting target gene. Efficient but imprecise
4) Homology-directed repair - Template supplied matching cleavage site, desired mutation included. Precise but not efficient.

Question 19

Describe TALENs

Answer 19

Transcription activator like effector nucleases
Amino acid sequence of DNA binding domain shows strong association with cleavage at specific nucleotides.
FOK1 nuclease to mediate site-specific dsDNA cleavage
Large, repetitive genes - difficult to deliver AAV or lentivirus
More specific, more complex and harder to engineer

Question 20

How do antisense technologies work

Answer 20

1) Introduce oligonucleotide complementary to sequence on mRNA of interest
2) Prevents protein/RNA regulators by steric hindrance
3) Chemically modified bases/backbone
- Increased RNA binding
- Protect from nuclease degradation
- Improve cell entry

Question 21

TWO ways antisense can be used

Answer 21

1) Splicing- mask sequence recognised by splicing factor

2) Translational inhibition - Bind across start codon

Question 22

How can we use antisense for DMD

Answer 22

Exon51 frameshift
Exondys51 - causes exon51 skipping restoring reading frame
only partially functional - small deletion
Morpholine antisense oligonucleotide that targets splice enhancer preventing inclusion of exon

Question 23

How can we use antisense for SMA

Answer 23

SMN2 almost identical except for splice site mutation(10%)

spirRAZA - modulate splicing of SMN2 making more production

Question 24

How do siRNA’s work?

Answer 24

dsRNA processed by dicer, generate 21-23nt dsRNA duplexes with 2nt 3’ overhang.
One strand of duplex incorporated into RNAi-induced silencing complex. Binds to fully complementary RNA sequences and cleaves RNA

Question 25

What can siRNA’s be used for

Answer 25

Reduce mRNA levels of over expressed gene
Reduce incorrectly spliced mRNA
Target viral RNA

Question 26

Formulation of siRNA’s

Answer 26

antiviral mechanisms activated by dsDNA longer than 30 = siRNA’s too short.
Short hairpin RNA’s in vectors - processed by DICER allows viral delivery targeting specific organs

Question 27

Issues with siRNA’s

Answer 27

1) Specificity
2) Efficiency (don’t completely knock out and viruses can envelope)
3) Delivery - maintenance difficult, some organs easier

Question 28

Oligonucleotide delivery needs to achieve…

Answer 28

1) stability against serum nucleases

2) Entry into target cells

Question 29

Oligonucleotide chemical modification

Answer 29

1) Phosphorothioate modification of backbone increased stability
2) GalNAc conjugates enhance uptake by hepatocytes

Question 30

micro RNA’s background

Answer 30

Decrease in lin-14 protein necessary for normal development. Mediated by lin-4 = ss 22nt non-coding RNA
Binds to partially complementary sequence in lin-14 3’UTR repressing lin-14 expression

Question 31

Describe how micro RNA’s work

Answer 31

1) Encoded in genome
2) Transcribed as part of longer RNA (pri-miRNA’s)
3) Nuclear processing to pre-miRNA hairpin by Drosha and exportin 5
4) In cytoplasm dsRNA hairpin processed by dicer to 21-23nt dsRNA duplex
5) one strand retained as mature miRNA

Question 32

How are miRNA’s involved in cancer?

Answer 32

CLL = loss of heterozygosity at 13q14.

Tumour suppressor genes = miR-15a & 16-1 which target BCL2 which is anti-apoptotic

Question 33

How are miRNA’s involved in HepC?

Answer 33

Highly specific liver miR-22 interacts directly with 5’UTR required for replication