Gene Therapy

Question 1

What is gene therapy?

Answer 1

The delivery of genetic material into a patient’s cells to act as a drug and treat diseases associated with genetic mutation or changes in gene expression.

Question 2

Briefly describe the protein production process?

Answer 2

1. Promoter recruits a transcription factor to target gene.
2. RNA polymerase transcribes gene.
3. A cap is added to the 5’ end to stabilise the mRNA.
4. Splicing removes introns, leaving the mRNA with just exons, including the UTRs and start/stop codons.
5. Polyadenylation occurs to the end to add a tail which further stabilises the mRNA.
6. Coding sequence is translated from start codon to stop codon, to produce a protein.

Question 3

Following transcription, capping, splicing, and polyadenylation, what is the structure of mRNA?

Answer 3

m7G cap - 5’UTR - Start codon - coding sequence - Stop codon - 3’ UTR - polyadenyl tail

Question 4

What can a mutation in a coding sequence result in?

Answer 4

Changes to amino acid sequence resulting in production of mutant protein

Question 5

What can a mutation in a promoter result in?

Answer 5

A change in the level of production of mRNA, and therefore protein. E.g., could result in removal of transcription factor binding site.

Question 6

What can a mutation in a splice site result in?

Answer 6

Production of incorrectly spliced mRNA.

Question 7

What can a mutation in a regulatory element in a UTR result in?

Answer 7

Change in level of production of a protein. For example, a mutation in the miRNA binding site will prevent miRNA binding to a target mRNA, and prevent it from negatively regulating its expression, thus leading to an increase in protein production.

Question 8

What is an example of a disease caused by a mutation in the coding sequence of a gene?

Answer 8

Sickle cell disease (SCD), an autosomal recessive disease caused by a mutation to both copies of the beta-globin gene, where a change of GAG to GTG causes glutamic acid to be replaced with valine at position 6 in protein (E6V). This results in production of haemoglobin S instead of haemoglobin A which causes red blood cells to form a sickle shape which leads to severe pain, anaemia, and a reduced life expectancy.

Question 9

What is a frameshift mutation?

Answer 9

When a deletion or addition, unless in multiple of 3, alters the reading frame, meaning all the codons downstream are changed and read as different amino acids.

Question 10

What are nonsense mutations?

Answer 10

Where a mutation (subsitution, addition, or deletion) causes premature coding of a stop codon, leading to production of a truncated protein.

Question 11

Describe transcription.

Answer 11

1. RNA polymerase binds to promoter sequence
2. RNA polymerase separates the DNA strands, providing the single-stranded template needed for transcription
3. RNA polymerase “reads” this template one base at a time (3’ to 5’), and builds an RNA molecule out of complementary nucleotides, making a chain that grows from 5’ to 3’. The “read” DNA to close back up and form a double helix.
4. Terminator sequences signal that the RNA transcript is complete. The terminator DNA encodes a region of RNA forms a hairpin structure followed by a string of U nucleotides. The hairpin structure in the transcript causes the RNA polymerase to stall. The U nucleotides that come after the hairpin form weak bonds with the A nucleotides of the DNA template, allowing the transcript to separate from the template and ending transcription.

Question 12

Describe translation

Answer 12

1. mRNA interacts with small ribosomal subunit. Iniator tRNA attaches to m7G cap on 5’ end of mRNA.
2. This complex moves down mRNA until it reaches the start codon.
3. Iniator tRNA carrying methionone complementarily binds to start codon.
4. Large ribosomal subunit attaches to complex.
5. tRNA enters ribosome and complementarily binds to next codon.
6. Amino acid on this tRNA forms peptide bond with methionone on iniatior tRNA.
7. mRNA shifts by 1 codon.
8. Initiator tRNA leaves. New tRNA enters ribosome and complementarily binds to next codon.
9. Amino acid on this tRNA forms peptide bond with previous amino acid tRNA.
10. This continues until stop codon.
11. Release factor binds to stop codon instead of tRNA and mess with the enzyme that normally forms peptide bonds: they make it add a water molecule to the last amino acid of the chain.
12. This reaction separates the chain from the tRNA, and the newly made protein is released.

Question 13

Name 3 single-gene disorders.

Answer 13

Cystic fibrosis
Sickle cell disease
Huntington’s disease

Question 14

What are some issues with developing gene therapy?

Answer 14

o Delivery to correct target cell
o Maintenance of delivered gene in target cell
o High cost of drug development
o Only suitable for diseases that are caused by reduced production of a protein product.
o Can’t achieve a precise level of expression of delivered gene, so only practicable if expression level doesn’t matter too much.

Question 15

What are the 2 classes of gene delivery?

Answer 15

• Ex vivo – remove target cells from patient, introduce gene, and return them to the patient. This approach is good for cells in the haematopoietic system and allows precise targeting and increased efficiency.
• In vivo – introduce gene directly into patent via a method appropriate for the target tissue. For example, aerosol delivery to the lung.

Question 16

What is viral gene delivery?

Answer 16

Viral delivery exploits the natural delivery of viral genetic materials during infection by modifying the viral genome to include a gene of interest which will then be expressed in the host, thus avoid issues with pathogenicity and tumorigenesis.

Question 17

What are some pros and cons of viral gene delivery?

Answer 17

Pros:
* Efficient uptake
* Selectivity for specific cell types
* Can persist in cells.

Cons:
* Insertional mutagenesis risk (some viruses)
* Limit on size of gene incorporated.
* Immune response.

Question 18

What are some pros and cons of retroviral gene delivery?

Answer 18

Pros:
Can be mutated to remove all pathogenic elements, so that only genes necessary to reverse transcribe and integrate DNA remain.
Incorporation of the delivered gene into the host genome, and therefore maintenance during cell division.

Cons:
Since we cannot control the site of integration, this may lead to issues concerning insertional mutagenesis.

Question 19

What is insertional mutagenesis?

Answer 19

Where, following viral gene delivery, the gene integrates within an important gene (e.g., a tumour suppressor) and disrupts it, or a strong viral promoter is introduced next to a host oncogene, leading to dysregulation of expression. Essentially, this method of gene therapy can induce cancer.

Question 20

What are some pros and cons of lentivirus gene delivery?

Answer 20

Pros:
Can integrate into non-diving cells.
Lower immunogenicity
Modified to minimise the risk of insertional mutagenesis

Cons:
Insertional mutagenesis is still a potential problem.

Question 21

What are the pros and cons of AAV gene delivery?

Answer 21

Pros:
Can infect dividing and non-diving cells
Has low immunogenicity

Cona:
Cannot replicate without a helper virus. Do not integrate into host cell DNA. Instead, the viral DNA is retained inside the cell nucleus as episomal concatemer for the lifetime of a non-diving cell, or until it is lost through cell division (so not appropriate for rapidly dividing cells).
Small virus which can limit the size of the gene which can be delivered

Question 22

What is CAR-T immuno therapy?

Answer 22

A form of ex vivo immunotherapy involving adoptive T-cell transfer. T-cells are taken from the patient, and, usually using a lentiviral vector, the genome is engineered to produce chimeric antigen receptors (CARs) which recognise antigens on cancer cells. The modified T-cells are returned to the patient where they multiply, attack, and kill cancer cells.

Question 23

What can CAR-T therapy be used for?

Answer 23

Acute lymphoblastic leukaemia
Diffuse large B cell lymphoma
Potentially other cancers

Question 24

What are some issues with CAR-T therapy?

Answer 24

Risk of serious side effects such as cytokine release syndrome
Difficulty in targeting solid tumours
Precisely modifying CAR requires refinements including base editing.

Question 25

Name a gene therapy for use in the retina.

Answer 25

Luxturna (Spark, FDA approved 2017) to treat biallelic RPE65 mutation-associated retinal dystrophy. An AAV vector is injected directly into the eye and introduces a functioning RPE65 gene to the retinal cells.

Question 26

Describe how gene therapy can be used for haemophilia?

Answer 26

Haemophilia A - lack of clotting factor VIII.
Haemophilia B - lack of clotting factor IX.

Both can be treated using hepatocyte-targeted AAV vectors to introduce the respective factors to the liver.

Question 27

What were some issues which were solved during trials for haemophilia gene therapy?

Answer 27

Insufficient expression of clotting factor - Used a gain-of-function mutant of factor IX, which was 7-fold more active than the wildtype.
Pre-existing immunity to AAV in 30-50% of people - Included modifications to the AAV capsid to prevent secondary immune response. However, this will require more work to be suitable for all patients.
Transient liver cell damage in 25% of treated patients - Short term corticosteroid treatment.

Question 28

What are some advantages of mRNA therapeutics?

Answer 28

mRNA is delivered to cytoplasm and immediately translated to protein, then degraded over time.
Can avoid immune response and drapid degradation by modification of individual nucleosides such as 1-methyl-pseudouridine instead of uridine.
Can be rapidly developed using a single platform which can be easily adapted to any mRNA sequence, as opposed to protein drugs which require individualised development.

Question 29

What are some uses of mRNA therapeutics?

Answer 29

Vaccines:
COVID-19
Zika virus
Infleunza A
Personalised anti-tumour vaccines.

CVD and wound healing using vEGF mRNA.

Question 30

How does CRISPR Cas9 work?

Answer 30

CRISPR contains a guide sequence of gene-specific RNA (sgRNA) which interacts with the target DNA sequence, while the Cas9 endonuclease induces a double strand break (DSB) at the target gene. This break is subsequently repaired by either non-homologous end joining (NHEJ) or homology-directed repair (HDR).

Question 31

How does NHEJ work?

Answer 31

Resection of the strand ends recruitment of factors to the damage site, including DNA-PKcs which phosphorylates various substrates to facilitate the recruitment of ligation enzymes by trimming the overhanging nucleotides. Indels are produced by the removal or addition of DNA bases by DNA polymerases and nucleases. This process is efficient but error prone, which can be useful as it disrupts the gene, making it non-functional

Question 32

How does HDR work?

Answer 32

During the late S phase or G2 phase of the cell cycle, HDR uses the sister chromatid as a template for accurate repair of the gene. However, by introducing an exogenous DNA strand with “homology arms”, HDR can be exploited to insert a specific gene.

Question 33

In what ways can CRISPR be improved?

Answer 33

• Prime editing – allows precise single nucleotide changes
• Different Cas proteins originating from different bacteria can achieve different effects, eg targeting RNA instead of DNA
• Can be used to direct regulatory proteins (such as transcription factors) to specific site on DNA without cleavage

Question 34

WHat are the 4 main methods of genome modification?

Answer 34

• Meganucleases - endodeoxyribonucleases characterized by a large recognition site (double-stranded DNA sequences of 12 to 40 base pairs). Endonucleases cleave phosphodiester bonds in DNA. Deoxyribonucleases (Dnases) catalyses cleavage of phosphodiester linkages in DNA backbone.
• TALENs - nucleases bind to the DNA using TAL effector repeats that are ligated together to generate arrays, allowing recognition of target DNA sequences. TALENs bind with dimeric FokI (nuclease domain) that cleaves target DNA for generating double stranded breaks which is repaired by NHEJ to produce indels.
• Zinc-finger nucleases (ZFNs) - engineered DNA-binding proteins that facilitate targeted editing of the genome by creating double-strand breaks in DNA specified locations
• CRISPR/Cas9

Question 35

What are the 2 types of genome modification?

Answer 35

• Somatic - changes to genome in any cell other than a gamete, gametocyte, germ cell or undifferentiated stem cell. Permanent change to individual’s genome but cannot be passed on to offspring. Delivery and maintenance can be difficult.
• Germline - changes to genome in germ cells (egg or sperm) or early embryo that will be passed on to an individual’s descendants. Potential to eliminate an inherited disease. Ethically difficult to justify and very controversial.

Question 36

Why is genome editing not used for reproduction?

Answer 36

• High chance of off-target effects
• Inefficient
• Disease causing mutations can already be avoided by preimplantation genetic diagnosis during IVF.
• Ethics – eugenics?

Question 37

What is antisense technology?

Answer 37

The introduction of an oligonucleotide that is complementary to a sequence on the mRNA of interest. These antisense oligonucleotides (ASOs) are short, synthetic, single-stranded RNA strands which can influence RNA processing to reduce, restore, and modify protein expression, as well as cause steric hindrance (the stopping of a chemical reaction which might be caused by a molecule’s structure).

Question 38

How do antisense oligonucleotides work?

Answer 38

Either:
* Preventing protein or RNA regulators that should bind to this site from interacting, by steric block.
* Inducing RNA degradation by RNAse H recruitment

In order to:
* Improve RNA binding
* Protect from nuclease degradation
* Improve entry into cells
* Many different possible modifications

Question 39

How do oligonucleotides modulate splicing?

Answer 39

They can be designed to mask a sequence that is recognised by a splicing factor in order to promote exon inclusion or exon skipping of pre-mRNA. Many disease-associated genes contain multiple exons and introns. Antisense nucleotides can be designed to bind to splice sites or site enhances to alter the pattern of splicing.

Question 40

How can modulation of splicing using oligonucleotides be used to treat Duchenne muscular dystrophy?

Answer 40

The DMD gene has many exons, and drugs to induce exon skipping of the one with a premature stop codon can restore a partially functional protein with a small internal deletion.

Question 41

What is SMA?

Answer 41

Spinal muscular atrophy is a fatal condition in babies caused by an autosomal recessive mutation in the SMN1 gene which causes insufficient SMN protein production, leading to loss of spinal motor neurons and gradual paralysis.

Question 42

How does Spiranza work for SMA?

Answer 42

SMA is caused by mutation in SMN1. The SMN2 gene is almost identical to SMN1, except for a splice site mutation which skips exon 7, meaning only 10% of mRNAs produce the functional SMN protein.
Spiranza uses steric block ASOs to modulate splicing of SMN2, which causes inclusion of exon 7 and increases production of the functional SMN protein from this gene.

Question 43

How does Zolgensma work for SMA?

Answer 43

SMA is caused by a mutation in SMN1. Zolgensma delivers a functional SMN1 gene in an AAV9 vector.

Question 44

How do oligonucleotides work for RNA degradation?

Answer 44

Using ASOs known as gapmers where the DNA is in a gap between some strongly binding modified nucleotides (e.g., LNA) which confer stable binding to the sequence of interest to form an RNA-DNA hybrid. This recruits RNase H, an endonuclease that specifically cleaves RNA at RNA-DNA hybrids.

Question 45

Name all the RNA types and their functions.

Answer 45

• Messenger RNA (mRNA) – carries genetic information.
• Transfer RNA (tRNA) – decodes mRNA in the ribosome.
• Ribosomal RNA (rRNA) – central to ribosomal function.
• Small nucleolar RNAs (snoRNAs) – RNA processing
• Telomerase RNA – template function in telomerase
• Small nuclear RNAs (snRNAs) – important in splicing
• Viral RNA – RNA genomes of viruses
• Ribozymes – RNA enzymes

Question 46

What are siRNAs?

Answer 46

Short interfering RNA - exogenous 21-23 nucleotide RNA molecules used to regulate gene expression by binding to complementary targets.

Question 47

How do siRNA molecules regulate gene expression?

Answer 47

1. dsRNA processed by DICER which cleaves it into siRNA and miRNA.
2. The siRNA formed are 21-23 nucleotide duplexes with 2 nucleotide 3’ overhangs.
3. siRNA complementary to target mRNA delivered to cell.
4. One strand of the duplex is incorporated into the RNAi-induced silencing complex (RISC).
5. siRNA strand binds exactly complementary sequence in target mRNA.
6. Argonaute 2 endonuclease in RISC cuts target mRNA.
7. Target mRNA degraded by cellular machinery following cleavage, resulting in decrease of protein production.

Question 48

What is the basis of siRNA therapeutics?

Answer 48

Utilise siRNA binding to mRNA to decrease expression of a particular protein, particularly those translated from mutanyt mRNA.

Question 49

What is the main limitation of siRNA?

Answer 49

It’s not possible to target only mutant mRNA unless the exact mutation is known, therefore, the wildtype mRNA is also impacted, so both the mutant and normal protein is decreased.

Question 50

What are some issues with siRNA therapeutics?

Answer 50

Specificity - Off-target binding is a problem e.g., to wildetype mRNA

Efficiency
o siRNAs do not completely knock out a damaging gene (unlike genome modification), but only knock it down.
o Viruses can evolve to prevent siRNA cleavage (single nucleotide change sufficient). Combinations of multiple siRNAs targeting same virus can reduce this problem

Delivery
o Some organs are easier to target than others, e.g. liver is easier than brain
o For cancer treatment, we want to target the siRNA just to the cancer cells, not to the normal cells surrounding the tumour.
o Maintenance in cells also difficult.

Question 51

What are the 2 forms of siRNA delivery?

Answer 51

As siRNA
As dsRNA - as short hairpinRNAs in vectors which can be processed by DICER.

Question 52

How can delivery of oligonucleotides, (e.g., ASOs, siRNA, and anty-miRNA) be achieved with stability against serum nucleases and entry into target cells?

Answer 52

Encapsulation into lipid nanoparticles.

Chemical modification
o Phosphorothioate modification of backbone enhances stability.
o Ligand conjugation to enhance uptake into specific cell types. For example, GalNAc (N-acetylgalactosamine) binds to the asialoglycoprotein receptor on hepatocytes, allowing specific targeting of oligonucleotide drugs to liver

Question 53

What was the first approved siRNA drug?

Answer 53

Patisiran for treatment of h-ATTR.

Question 54

How does Patisiran work for treatment of hereditary transthyretin amyloidosis?

Answer 54

In h-ATTR, a mutant TTR protein is made in the liver from a faulty copy of the gene inherited from a parent (autosomal dominant). This accumulates in other organs and is eventually fatal. Patisiran (Onpattro) is an Alnylam siRNA therapy targeting TTR mRNA (both WT and mutant) to reduce expression of the TTR protein.

Question 55

What are GalNAc-conjugated siRNAs?

Answer 55

Hepatocyte-targeted siRNAs.
GalNAc can avidly bind to ASGPR, which is predominately expressed on hepatocytes. Upon reaching systemic circulation, GalNAc-conjugated siRNA molecules can be efficiently taken into the liver through ASGPR-mediated endocytosis, and thus accumulate in endosomes.

Question 56

What are miRNAs?

Answer 56

microRNAs are endogenous 21-23nt RNA molecules that bind to imperfectly complementary targets in 3’UTR of mRNAs and reduce production of encoded protein.

Question 57

How are miRNAs produced?

Answer 57

1. miRNAs are encoded in the genome and are transcribed as part of longer RNAs (pri-mRNAs).
2. Pri-mRNA undergoes a nuclear processing by Drosha, a specific endoribonuclease involved in initial step of miRNA biogenesis. This produces a pre-miRNA hairpin.
3. This pre-miRNA then undergoes nuclear export by Exportin 5.
4. In the cytoplasm, the dsRNA hairpin is processed by Dicer to form a 21-23nt dsRNA duplex.
5. One strand is retained as the mature miRNA and associates with RISC to bind target mRNAs.

Question 58

Why are the targets of miRNA therapeutics difficult to predict?

Answer 58

Imperfect complementarity
Each miRNA can target around 200 mRNAs.
60% of mRNAs have miRNA target sites.
Some mRNAs have multiple target sites for different miRNAs.

Question 59

How is miRNA mRNA repression different to that of siRNA?

Answer 59

miRNAs are chemically identical to siRNAs but have different outcomes due to differences in target binding:
* Complete base pairing to the site anywhere in RNA – RNA cleavage is similar to that of siRNAs but is followed by RNA degradation. This is the predominant mechanism in plants and is very rare in animals.
* Partial base pairing to 3’UTR sites – results in translational repression and RNA degradation in processing bodies (cytoplasmic ribonucleoprotein granules composed of translationally repressed mRNAs and proteins related to mRNA decay). This is the predominant mechanism in animals.

Question 60

How does miRNA repress protein synthesis?

Answer 60

1. Inhibition of translation initiation by binding to target mRNA.
2. mRNA decay by removal of polyadenine tail by Ago and GW182.

Question 61

What are some similarities and differences between miRNA and siRNA?

Answer 61

Similarities:
* Both 21-23nt RNAs, processed from larger dsRNA precursors by Dicer
* Both cleave an exactly complementary target RNA

Differences:
* miRNAs have many targets (around 200 mRNAs) while siRNAs have one target.
* miRNA reduces but does not abolish protein production (i.e., it modulates gene expression). siRNA destroys mRNA leading to a strong reduction in the production of the encoded protein.
* miRNAs are encoded in the genome (endogenous. siRNAs are derived from external dsRNA (viral infection, dsRNA transfection) (exogenous)
* Most animal miRNAs have imperfect complementarity to target sites in 3’UTR of mRNAs, and repress gene expression at the level of translation and/or RNA stability

Question 62

How can miRNA (+ siRNA) be targeted in therapeutics?

Answer 62

• Inhibit a damaging mRNA (e.g., oncogenes) using chemically modified complementary oligonucleotides (antagomins) which sequester miRNA and prevent it binding to the mRNA.
  Single stranded oligonucleotides complementary to miRNA have the potential to act as miRNA inhibitors via steric block. These oligonucleotides often have to undergo modification of their bases and backbone in order to improve affinity to miRNA, reduce off-target effects, and improve stability against serum nucleases. Some common modifications include 2’-O-methyl and locked nucleic acid (LNA).
• Overexpress a beneficial miRNA (e.g., tumour suppressor).
• Modulate levels of miRNAs associated with particular diseases. miRNAs are encoded in the genome, so we could also use gene therapy/genome modification approaches to increase or decrease expression.

Question 63

How can miRNAs be targeted in treating HepC?

Answer 63

Hepatitis C virus (HCV) replication requires miR-122, which is a liver specific miRNA that binds directly to the 5’UTR of HCV RNA and positively regulates viral replication. Inhibition of miRNA could slow disease progression.