W14LECT - Gene therapy Flashcards
What is Gene therapy?
- Any intervention aimed to manipulate specific genes: augmentation, replacements, eradication, repair, affecting expression
- Delivery of nucleic acid into a patient’s cells with therapeutic or profilactic purposes
What are the 2 types of gene therapy?
- Somatic gene therapy
- Germline gene therapy
What are the features of Somatic gene therapy?
- Replacement or correction of defective gene
- Genome editing
- Influencing gene expression
- Not inherited
What are the features of Germline gene therapy?
- All cells modified
- Inherited
- prohibited
Which disease can be treated with gene therapy?
- Genetic cause (gene, mutation) has to be known
- Phenotype has to be known, too (protein, tissue etc.)
- Is gene transfer possible into target tissue?
- Major disease classes
- Cancers
- Inheriteddisorders:monogenic and complex diseases
- Infectiousdiseases
How are Nucleis acids used in gene therapy?
How is recombinant DNA introduced into target cell?
- Vector is a construct to transfer a foreign genetic material into another cell
- ”Supporting structure”
(large DNA sequence) which consists of an inserted DNA
What is the Function of vectors?
- Uptake
- Protection of DNA/RNA insert
- Stabilization
- Amplification (cloning vector)
- Expression (expressing vector)
- Provide the selection of the
modified host
Why can Plasmid be a vector to transfer a gene to the host cells?
- Small circular double-stranded DNA molecule
- Occur naturally in e.g. bacterial cells
- Separated from chromosomal DNA
- Replicate independently
Expression vector
1. What are the characteristics of Expression vector?
- To produce stable gene product (RNA, protein)
- Optimal promoter, terminator and enhancer are needed
- Gene for expression does not contain intron
- Selection - Antibiotic resistance gene: allowing the survival of vector containing cells in the presence of antibiotics
Expression vector
2. What are the Applications of Expression vector?
- Production of medically useful proteins (biopharmaceuticals, microbial antigenes for vaccines)
- Genetherapy
- Determine gene function
Approaches for gene therapy
1. What are the 5 Approaches for gene therapy?
- Gene replacement
- Replacing the function of defective genes by introducing an intact gene - Gene silencing
- Suppression of overactive genes - Gene editing
- Changing DNA - Direct killing of disease cells
- Assisted killing of disease cells by immuncells
Approaches for gene therapy
2. Features of Direct killing of disease cells?
Approaches for gene therapy
3. Features of Assisted killing of disease cells by immuncells?
Approaches for gene therapy
4. What are the features of Gene replacement?
- Replacing the function of defective genes by introducing an intact gene
- Applied to disorders caused by loss of function mutation
− Recessive disorders
− Autosomal dominant
(haploinsufficiency) disorder
Approaches for gene therapy
5. What are the Basic structural elements of expression plasmid?
- Replication site (Ori)
- Antibiotic resistance gene
- Gene to be expressed behind a promoter
Approaches for gene therapy
6. What are the features of Gene delivery approaches - Transfection?
Viral and non-viral vectors for gene therapy
=> Give the examples of Vectors used in gene therapy clinical trials
Strategies for gene therapy
1. What are the 2 Strategies for gene therapy?
- In vivo gene therapy
- Ex vivo gene therapy
Strategies for gene therapy
2A. What is In vivo gene therapy?
A therapy in which The cells targeted by the gene therapy will be modified in the patient’s body.
Strategies for gene therapy
2B. What are the advantages of In vivo gene therapy?
- technically easier
- many cells and cell types can be
targeted
Strategies for gene therapy
2C. What are the disadvantages of In vivo gene therapy?
- requires very efficient vectors
- limited controllability
- often limited therapeutic impact
Strategies for gene therapy
3A. What is Ex vivo gene therapy?
The target cells are isolated from the patient, modified under laboratory conditions, selected and then reintroduced back into the patient’s body.
Strategies for gene therapy
3B. What are the advantages of Ex vivo gene therapy?
- Less effective vectors can be used
- highly controlled, directed to a specific cell
- stable effects
Strategies for gene therapy
3C. What are the disadvantages of Ex vivo gene therapy?
- more complicated implementation
- not all cell and tissue types can be used for this purpose
- tissue stem cells often need to be modified
What’s the role of Viral vectors?
- Making the virus vector harmless
* Remove harmful genes => “cripple” the virus (gutless viral vector)
* Vectors must be propagated in large numbers in cell culture with the aid of a helper virus - Following the gene transfer
- May integrate into the host genome -> permanent effect
- Remain as extrachromosomal genetic elements - episodes -> transient effect
What are the 4 Most commonly used viral vectors?
What are General features of viral vectors?
- Only transient effect, if it is not integrated into genom
- Integration into essential gene causing mutation (e.g. retrovirus)
- Entering not only in target cells
- May induce immun response (e.g. adenovirus)
Give the Comparison of viral vectors?
Viral vectors I.: Adenovirus vectors (Ad)
1. What are the features of Viral vectors I.: Adenovirus vectors (Ad)?
- Genom: double-stranded DNA
- After introduction it is located episomally (does not integrate into the genome)
- 36-38 kb DNA can be introduced (relatively high capacity)
- Adenoviruses are used in most gene therapy trials
- The first released Ad product: GENDICINE deliver p53 into cells, applied in head and neck cancer
Viral vectors I.: Adenovirus vectors (Ad)
2. What are the Consequences of using an adenovirus vector?
- For the treatment of ornithine transcarbamylase deficiency
- Urea cycle disorder
- X-linked dominant
- High concentration of ammonia - Adenovirus vector delivery–> cytokine strom-> he has died after 4 days in disseminated intravascular coagulation (DIC)
- Main disadvantage of Ad vectors: immunogenicity
Viral vectors II: Adeno-associated viruses (AAV)
1. What are the features of Adeno-associated viruses (AAV)?
- Small DNA virus that cannot replicate by itself, does not cause disease in humans
- Generally not integrated
- Dividing and non-dividing cells can be
targeted - The safest virus vectors known today! (less immunogenic)
- Disadvantage: small capacity
Viral vectors II: Adeno-associated viruses (AAV)
1. What are the features of Adeno-associated viruses (AAV)?
- Small DNA virus that cannot replicate by itself, does not cause disease in humans
- Generally not integrated
- Dividing and non-dividing cells can be
targeted - The safest virus vectors known today! (less immunogenic)
- Disadvantage: small capacity
Viral vectors II: Adeno-associated viruses (AAV)
2. The role of AAV in Hemophilia
- AAV as a coagulation factor 8 and 9 gene delivery vector
- Hemophilia is a good target for gene therapy
- It can be modified to deliver the gene to the liver
- Low immunogenicity
- Even existing antibody production has been eliminated!
- Modified F8, F9 genes: stronger gene expression
- Liver-specific promote
Viral vectors III.: Retrovirus vectors
1. What are the features of Retrovirus vectors?
- viral RNA is reverse transcribed into cDNA
- random integration
- only dividing cells can be targeted
- relatively immunogenic
- very efficient vectors
- capable of introducing relatively large amounts of DNA
Viral vectors III.: Retrovirus vectors
2. Describe X-SCID treatment with gamma-retrovirus vector
- X-linked severe combined immunedeficiency:
- γ-chain of IL-2 receptor is mutated
- Retroviral gene therapy was very effective
- T-cell, B-cell function were detectable even after 10 years, the patients did not need
immunoglobulin infusion, even they were vaccinable - But: five of them had leukemia
=> Possible causes of leukemia - integration occurs close to an oncogene
- transfected cells express abnormal amount of IL-2Rg -> unlimited cell proliferation occurs
(Retroviral gene therapy can be dangerous)
Describe a Successful gene therapy trial with retrovirus
- Mutation of adenosine deaminase: autosomal recessive form of SCID
- ADA deficiency → dATP accumulation → destruction of T cells → weakened immunsystem
- Standard treatment is based on enzyme replacement
- Ex vivo gene therapy is extremely successful, free from side effects (no oncogenic effect)
Viral vectors IV.: Lentiviral vectors
1. What are the features of Lentiviral vectors?
- HIV based virus - retrovirus
- Target not only dividing cells
- integration avoids the oncogenic sites
- larger transgenes can be incorporated
What are the features of Targeted influencing gene expression? (somatic gene therapy)
- Inhibition of overexpression of a gene at RNA or protein level
- Target disorders:
- Dominant disorders (dominant negative or gain of function mutation)
- Cancers
- infectious diseases
What are the features of RNA interference - RNAi?
- Process by which dsRNA silences gene expression
- Degradation of mRNA or translation inhibition
Gene silencing by small interfering RNA (siRNA) and micro RNA (miRNA)
1. What are the features of siRNA pathway?
- Usually exogeneous origin
- Tailor-made dsRNA
- The Dicer enzyme cuts it short (about 21 bp)
- Activate RISC complex and bind specifically to the target mRNA
- Degradation of target mRNA
Gene silencing by small interfering RNA (siRNA) and micro RNA (miRNA)
2. What are the features of miRNA pathway?
- Endogeneous non-coding RNA
- After processing, bind to the 3’UTR of target mRNA
- Inhibition of translation or degradation of mRNA
- Regulation of gene expression
Explain siRNA targeting PCSK9 in familial hypercholesterolaemia
- siRNA: inclisiran
- Against familial hypercholesterolaemia caused by PCSK9
Explain Application of antisense nucleotids to inhibit gene expression
- Sense RNA: mRNA is single-stranded, called “sense” because it results in a gene product (protein)
- Antisense Oligonucleotides (ASO) are single-stranded oligonucleotides (RNA or DNA) which are complementary to a target RNA molecule
– stimulate or inhibit protein synthesis at the mRNA level;
– aid the inclusion or exclusion of certain exons into mature mRNA
What is Antisense Oligonucleotides (ASO)?
Antisense Oligonucleotides (ASO) are single-stranded oligonucleotides (RNA or DNA) which are complementary to a target RNA molecule
– stimulate or inhibit protein synthesis at the mRNA level;
– aid the inclusion or exclusion of certain exons into mature mRNA
Explain Succesful antisense therapy in SMA
Spinal muscular atrophy (SMA)
* Most common genetic cause of death in childhood (8000-12000/infants)
* Autosomal recessive
* One in 50 people is a carrier
* Progressive death of motor neurons
* Mutation in SMN1 gene
* SMN2 gene: silent gene copy
* Due to a SNV in exon 7 undergo alternative splicing
* Nusinersen or Spinraza: ASO therapy
Explain Antisense oligonucleotid therapy
- nusinersen prevents the alternative splicing of the SMN2 gene
- Treatment is effective if the copies of SMN2 gene >2
Antisense oligonucleotid therapy
1. How do SMN1 and SMN2 gene work in healthy individuals?
- SMN1: produce functional SMN protein
- SMN2: due to alternative splicing, exon 7 is almost always deleted from the mRNA
Antisense oligonucleotid therapy
2. How do SMN1 and SMN2 gene work in SMA patient:?
- Mutation in SMN1: no functional mRNA and protein
- SMN2 targeting with an antisense oligonucleotide (ASO)
- ASO binds intron 7 and prevents the alternative splicing of exon7.
Genome editing
1. What are the features of Genome editing?
- DNA is inserted, deleted or replaced in the genome using nucleases by creating site-specific double or single strand breaks
- breaks are repaired through non-homologous end-joining or homologous recombination
Genome editing
2. What is the result of Genome editing?
targeted mutation correction (gene therapy)
Genome editing
3. What are the target diseases of Genome editing?
- Autosomal dominant diseases caused by gain of function or dominant negative mutation
- Cancers
- Infections
Genome editing
4. What is the example of Genome editing?
CRISPR/Cas9 system
Genome editing
5. What are the Main elements of CRISPR/Cas9 system?
- CRISPR: Clustered Regularly-Interspaced Short Palindromic Repeats
- Cas9: CRISPR associated system: endonucleaseend-joining or homologous recombination
Genome editing
6. What are the 2 Applications of CRISPR/Cas9 system for genome editing?
- Gene inactivation
- Introducing new gene
Genome editing
7. Application of CRISPR/Cas9 system for genome editing
-> What are the features of Gene inactivation by CRISPR/Cas9 system for genome editing?
– Sequences in spacer bind to target gene
– Cas9 cuts the target gene
– Breaks are repaired by non-homologous end joining (NHEJ) => leads to insertions or deletions
– Both in dividing and non-dividing cells
Genome editing
8. Application of CRISPR/Cas9 system for genome editing
-> What are the features of Introducing new gene
by CRISPR/Cas9 system for genome editing?
– Breaks are repaired by homology directed repair (HDR)
– From a template new DNA can be copied in the genome
– Only in dividing cells
Genome editing
8. Application of CRISPR/Cas9 system for genome editing
-> Give an example of Gene inactivation by CRISPR/Cas9 system for genome editing
- Inactivation of dominant negative mutations in AD diseases
- E.g. Achondroplasia (FGFR3 mutations)
- (in mice for now)
Genome editing
9. Describe base editors
Genome editing
10. What are Advantages of CRISPR/Cas9 system?
- Precise targeting and modifications
- Multiple spacers can be used at the same time, allowing multiple genes to be modified.
- No foreign, unwanted (e.g. antibiotic resistance gene) sequence is introduced into the genome.
- Easy to use
