pharmacogenetics & Gene Therapy

Question 1

Define Pharmacogenomics

Answer 1

The study of the relationship between genetic variations and inter individual response to medications. It involves assessing variation in a wide spectrum of pharmacogenes

Question 2

Define precision medicine

Answer 2

An emerging approach for disease diagnosis, treatment and prevention that takes into account individual variability in genes, environment and lifestyle

Question 3

What is therapeutic range

Answer 3

Everything between minimum effective concentration and minimum toxic concentration

Question 4

What are ADME genes

Answer 4

Group of genes play a crucial role in the absorption, distribution, metabolism and excretion and transport of drugs in the human body

Question 5

Advances in next generation technology have facilitated recent PGx genomic mining in Africa

Answer 5

1. Increasing availability of genomic datasets involving African participants.
2. Bioinformatics tools and pipelines for NGS data analysis
3. Developing more collaborative PGx research projects in Africa which helps with data generation and facilitates functional characterisation of African specific alleles

Question 6

Limitations of Key PGX

Answer 6

1. Most African ethnolinguistics groups are yet to be included in genomic studies
2. Affordable technologies only assess known/common variants
3. Comprehensive NGS technologies are relatively expensive.
4. Few PG studies across africa have performed functional assay to ascertain the impact of variants in key pharmacogenes
5. Difficult to assess the impact of rare variants
6. Factor other than variation within ADME genes could alter drug response phenotypes.
7. Lack of proper electronic health records and phenotype data across Africa
8. Limited access to essential; medication across most clinical setting in Africa.

Question 7

What are cart T cell

Answer 7

CAR T cells (Chimeric Antigen Receptor T cells) are a type of immunotherapy used primarily to treat certain types of cancers, particularly blood cancers like leukemia and lymphoma. They work by genetically modifying a patient’s T cells to better recognize and attack cancer cells.

Question 8

Steps of cart T cell therapy

Answer 8

Here’s a step-by-step overview of how CAR T cells work:

Collection of T cells: Blood is drawn from the patient, and T cells, a type of immune cell, are separated from the rest of the blood components.
Genetic modification: In the lab, the T cells are genetically engineered to express a chimeric antigen receptor (CAR) on their surface. This CAR is designed to recognize a specific protein (antigen) found on the surface of cancer cells.
Expansion: The modified T cells are then multiplied in the lab to create millions of CAR T cells.
Infusion: The CAR T cells are infused back into the patient’s bloodstream.
Targeting cancer cells: Once inside the patient’s body, the CAR T cells recognize and bind to the specific antigen on the surface of cancer cells.
Killing cancer cells: After binding, the CAR T cells are activated and release chemicals called cytokines, which signal the immune system to destroy the cancer cells.

Question 9

Advantages of using mRNA as an effector in gene therapy

Answer 9

1. Cell-free production which means it can be rapidly manufactured
2. Antigen expression in situ
   - not limited by protein solubility or location: This means the antigen doesn’t have to be extracted and purified outside of its natural location, so issues like protein solubility and stability, which can be problematic when working with purified or recombinant proteins, are less of a concern.
   - host-associated post translational modifications: When antigens are expressed in situ, the body’s own cells perform the necessary PTMs, which are often species- or tissue-specific.
   In contrast, if proteins are produced in simpler organisms like bacteria or yeast, they may not undergo the same PTMs or fold properly. This can lead to structural differences that may affect the immunogenicity of the antigen.
   - transient expression in cytoplasm :
3. Very good to used in vaccines: mRNA stays in the cytoplasm of the cell and does not need to enter the nucleus, where the cell’s DNA is located. This avoids the risk of genotoxicity (damage to the DNA) or permanent changes to the genome, which can occur with viral vectors or DNA-based gene therapies.

Question 10

Types of gene therapy effectors gene editors

Answer 10

1. Zinc Finger Protein ‘
2. Mega nuclease
3. TALE
4. CRISPR

Question 11

Explain how Zinc Finger protein are used for gene therapy

Answer 11

Zinc Finger proteins (ZFPs) are engineered DNA-binding proteins used in gene therapy to target specific genes. They consist of a series of zinc finger domains, each recognizing a specific DNA sequence. By fusing ZFPs with other functional domains, like nucleases (ZFN, Zinc Finger Nucleases), they can be used to precisely cut DNA at a targeted location. This allows for the correction of mutations, the disruption of harmful genes, or the insertion of new genes.

Question 12

Explain how Meganuclease are used for gene therapy

Answer 12

Meganucleases are a type of engineered endonuclease used in gene therapy to achieve precise genome editing. These enzymes recognize and cut long, specific DNA sequences, typically 12-40 base pairs, which makes them highly selective for targeting unique sites within the genome. After the meganuclease cuts the DNA at the desired location, the cell’s natural repair mechanisms, like non-homologous end joining (NHEJ) or homologous recombination (HR), are triggered. This can result in the repair of a gene mutation, the disruption of a harmful gene, or the insertion of new genetic material.

Question 13

Explain how TALE are used for gene therapy

Answer 13

TALEs (Transcription Activator-Like Effectors) are proteins engineered to bind specific DNA sequences and are used in gene therapy to edit or regulate genes. Each TALE contains a series of repeat domains, where each domain recognizes a single DNA base, allowing highly customizable targeting of specific gene regions. When fused to nucleases, such as TALENs (TALE Nucleases), they create double-strand breaks at precise locations in the genome. This enables the repair or modification of genes through the cell’s natural repair processes.

Question 14

Characterise Non-viral vectors

Answer 14

1. Synthetic
2. Low immunogenicity
3. Amenable to large scale production using in vitro chemical synthesis

Question 15

Characterise Viral VECTORS

Answer 15

1. Highly efficient
2. Immunogenicity
3. May be produced in large scale, but require intensive tissue culture, which may be expensive

Question 16

Advantages and disadvantage of non-viral vectors

Answer 16

-Reduced Immunogenicity: Lower risk of immune reactions compared to viral vectors.
-Safety: No risk of insertional mutagenesis or oncogenesis, as they do not integrate into the genome.
-Ease of Production: Generally simpler and cheaper to produce compared to viral vectors.
-Broad Range of Applications: Can be used for various types of cells and tissues.

Disadvantages of Non-Viral Vectors:
-Lower Efficiency: Often less efficient at delivering genetic material into cells compared to viral vectors.
-Transient Expression: May result in short-lived expression of the therapeutic gene.
-Delivery Challenges: May require specialized techniques to achieve effective delivery and uptake.
-Limited Payload Capacity: Typically have a smaller capacity for carrying genetic material compared to some viral vectors.

Question 17

Advantage and disadvantage of viral vectors

Answer 17

-High Efficiency: Capable of delivering genetic material into cells with high efficiency.
-Stable Expression: Often achieve long-term or permanent gene expression through integration into the host genome.
-Large Payload Capacity: Can carry larger amounts of genetic material compared to non-viral vectors.
-Targeted Delivery: Some viral vectors can be engineered to target specific cell types or tissues.

Disadvantages of Viral Vectors:
-Immunogenicity: Higher risk of triggering immune responses against the vector or transduced cells.
-Risk of Insertional Mutagenesis: Potential for disrupting host genes, which could lead to oncogenesis or other complications.
-Complex Production: More challenging and expensive to produce compared to non-viral vectors.
-Limited Reuse: Repeated use can lead to immunity against the vector, reducing efficacy in subsequent treatments.

Question 18

Advantages of adenovirus as viral vectors

Answer 18

-Efficient transduction of target cells
-sustained expression possible with use of helper-dependent vectors
-amenable to polymer modification
-HD Ads have very largely transgene capacity

Question 19

Advantages of AAV

Answer 19

-Serotype-dependent tropism
-low immunogenicity
-efficient transduction
-limited/no toxicity
-sustained expression of transgene

Question 20

Advantage lentiviruses

Answer 20

-Broad tropism
-low immunogenicity
-durable expression of transgenes

Question 21

Disadvantages of Ads

Answer 21

-Strongly immunostimulatory
-severe innate immunostimulation precludes systemic administration

Question 22

Disadvantages of AAVs

Answer 22

Limited capacity of transgene

Question 23

Disadvantage of lentiviruses

Answer 23

Potential for genotypic effects

Question 24

Characterise haemophilia A

Answer 24

Caused by a deficiency in Factor VIII, this is the most common form of haemophilia. The condition is typically the result of mutations in the F8 gene located on the X chromosome. Since it’s X-linked, it primarily affects males, while females can be carriers.

Question 25

Characterise haemophilia B

Answer 25

Also known as Christmas disease, it is caused by a deficiency in Factor IX. The condition results from mutations in the F9 gene, also located on the X chromosome, and follows a similar X-linked inheritance pattern as Haemophilia A.

Question 26

What’s the two most recent replacement therapies.

Answer 26

1st use of recombinant clotting factor
1st use of extended half-life clotting factor

Question 27

Pros of replacement therapy

Answer 27

1. Reduce anxiety
2. Increase factors levels
3. Reduce burden of disease

Question 28

Cons of replacement therapy

Answer 28

1. Treatment burden and adherence
2. Immunogenicity
3. Maintaining adequate factor levels
4. Poor bleed protection on prophylaxis
5. Deteriorating musculoskeletal outcomes

Question 29

7 things Patient need to be made aware of and educated about before gene therapy

Answer 29

1. Not all patients may benefit from treatment and patient response is unpredictable
2. Treatment will in most cases,require co-treatment with corticosteroids
3. Patients should be available for regular blood tests to check response to valoctocogene raxaparvovec and assess liver health
4. Lifestyle restrictions after gene therapy; abstain from alcohol for more than a year and double barrier contraception for 6 months
5. Potential risk of horizontal and germline transmission, development of factor VIII inhibitors and malignancy
6. Long-term treatment effect cannot be predicted, cannot be readministered at this time
7. Need long term surveillance

Question 30

What makes a patient not eligible for haem gene therapy. Explain

Answer 30

Men over 18- kids livers are still growing we do not know how their livers wil process the therapy
No detectable anti-AAV5 - anti AAV means that there is a built immunity thus it wont work
No history of factor VII/IX inhibitors- kind self explanatory
No known significant empathic fibrosis or cirrhosis- damaged liver/ liver with other priorities will focus on priorities and not transcribing inserted gene

Question 31

After gene therapy what outcomes have we seen for haemophilia?

Answer 31

-85% reduction in treated bleeds
-98% reduction in need for infusions
- when checked at 208 weeks (4 years) factor 8 was still being produced, patients seem to still be producing factor 8 at 11 years post infusion
-Annualized Bleeding Rate IS reduced by 65%<

Question 32

Plausible reasons for the variability of expression

Answer 32

1. Efficiency of expression
2. Efficiency of gene transfer
3. Immune response
4. Epigenetic regulation

Question 33

What aspects of efficiency of expression may be the reason for variability of expression

Answer 33

1. Codon Optimization:
   Explanation: Different organisms have preferences for certain codons that code for the same amino acid, known as codon bias. Codon optimization involves altering the codon usage in a gene to match the preferred codons of the host organism without changing the protein sequence.
2. Hyperactive Variants:
   Explanation: Hyperactive variants are engineered or naturally occurring versions of proteins that have enhanced activity or stability compared to the wild-type version. These variants often include mutations that improve the folding, stability, or catalytic efficiency of the protein.
3. B-Domain Deleted:
   Explanation: This specifically refers to the removal of the B-domain in certain proteins, like Factor VIII used in hemophilia treatment. The B-domain is a non-essential region that does not contribute to the protein’s functional activity but can hinder expression and secretion.

Question 34

What aspects of efficiency of gene transfer may be the reason for variability of expression

Answer 34

1. Type of capsid
2. Vector dose
3. Vector manufacturing

Question 35

What aspects of efficiency of immune response may be the reason for variability of expression

Answer 35

-Pre-existing vector exposure: Prior exposure to the viral vector can trigger an immune response, reducing the effectiveness of the therapy by attacking the vector before it delivers the gene.
-Host immune response: Individual differences in immune systems can lead to varying degrees of reaction to gene therapy, affecting how well the therapy works.
-Transamination immunosuppression: Immunosuppressive treatments can reduce immune activity, influencing the efficiency of the immune response and causing variability in how well the gene therapy expresses the target gene.

Question 36

What aspects of efficiency of epigenetic regulation may be the reason for variability of expression

Answer 36

1. DNA methylation
2. Histone modification
3. Non-coding RNA

Question 37

Challenges of using AVV for mediated gene therapy

Answer 37

1. Can it be administered to those with active HIV, HBV and HCV
2. Is it safe for children with growing livers
3. Potential for insertional mutagenesis
4. We don’t know reasons why some patients develop transamination whilst others do not.
5. Durability of factor expression
6. Variable factor expression is poorly understood
   7 can’t be re-administered

Question 38

Why is it important to study African populations

Answer 38

Studying African populations is vital due to their high genetic diversity, offering insights into human evolution, disease susceptibility, and drug response. Research in these populations can reduce health disparities, uncover novel genes, and improve precision medicine. It also promotes global health equity by ensuring that advances in genomics benefit all populations.

Question 39

Why is it important to identify genes that are under selection pressures

Answer 39

Signatures of selection differ across the continent and across populations, so we need to understand the differences . Example genes associated with malaria or lactose intolerance

Question 40

What was found in the steroid resistant nephrotic syndrome in black South African children with focal glomerulosclerosis

Answer 40

1. Looking at autosomal recess trait. NPHS2 gene encodes podocin. Podocin protein is a component of the filtration slits of podocytes (valine [V] to glutamic acid [E]).
2. Steroid resistant have all genotypes (V/V, V/E, E/E)
3. Steroid sensitive have only one genotype (V/V)
4. Utility: prognostic indicator, treatment guidance, genetic counselling and reproductive options.

Question 41

What do we have to take into consideration when looking at complex disorders (Performing GWAS) in African populations.

Answer 41

1. Most GWAS arrays are designed with SNPs common in European populations (H3Africa array more African appropriate)
2. Imputation reference panel
3. High false discovery rate if you do not correct for population structure (need to understand how African cluster on PCA plots)
4. Lead SNPs at the same locus can be different in different populations, SNPs can have different effect size in different populations
5. Africans have smaller haplotype; so a lead SNP associated with a disease gene in European. But in African populations haplotypes are smaller thus lead SNP can be on a different haplotype that disease gene.
6. Different allele frequencies
7. Different causative genes

Question 42

WHY is it important to do GWAS studies for African populations in Africa

Answer 42

PRS prediction in a target population is highest if discovery (source) data is from the same population or from a multi-ancestry (all)

Question 43

Give the cause of sickle cell anemia and the gene therapy

Answer 43

Cause**: A single mutation (A-T at codon 6 of the beta-globin gene) changes glutamic acid to valine, leading to abnormal hemoglobin folding.
- Gene Therapy: CRISPR gene therapy repairs the mutation in the hemoglobin gene, allowing the production of normal adult hemoglobin.

Question 44

Describe the cause of beta thalassemia and gene therapies

Answer 44

Cause**: Mutations in the beta-globin gene result in poor beta-globin production, leading to anemia and red cell production issues.
- Gene Therapy: Hematopoietic stem cell transplantation using lentiviral vectors to introduce a β-globin gene. Other approaches involve inducing fetal hemoglobin or targeting iron metabolism.

Question 45

Describe the cause of gaucher disease and gene therapies

Answer 45

Cause**: Deficiency of the enzyme acid β-glucosylceramidase leads to substrate accumulation in lysosomes, particularly in macrophages.
- Gene Therapy: Gene therapy approaches include antisense oligonucleotides that target mRNA of mutant enzyme genes.

Question 46

Describe the cause of Tuberous Sclerosis and gene therapies

Answer 46

Cause**: Mutations in TSC1 or TSC2 disrupt the regulation of the mTOR pathway, leading to uncontrolled cell growth.
- Gene Therapy: mTOR inhibitors like Rapamycin have been repurposed for treatment.

Question 47

Describe the cause of duchenne muscular dystrophy and gene therapies

Answer 47

Cause**: Mutations in the dystrophin gene result in absent or truncated dystrophin protein, affecting muscle structure.
- Gene Therapy: Therapies focus on exon skipping and microdystrophin gene therapy to restore dystrophin function.

Question 48

Describe the cause of spinal muscular atrophy and gene therapies

Answer 48

Cause**: Deletions in the SMN1 gene lead to motor neuron degeneration and muscle atrophy.
- Gene Therapy: ASO therapy, such as Spinraza (Nusinersen), increases SMN2 mRNA transcripts that include exon 7, improving motor neuron function.

Question 49

Describe the cause of huntington disease and gene therapies

Answer 49

Cause**: A triplet repeat expansion in the huntingtin gene causes neurodegeneration.
- Gene Therapy: Antisense oligonucleotides target the toxic protein production, though trials are ongoing.

Question 50

Describe the cause of achondroplasia disease and gene therapies

Answer 50

Cause**: Mutations in the FGFR3 gene impair cartilage replacement with bone, leading to short stature.
- Gene Therapy: Vosoritide stimulates bone growth by targeting natriuretic peptide receptor-B, counteracting the FGFR3 mutation.