I. Fixing broken genes by cutting-edge gene therapy and gene editing
a. Introduction
Complete human genome was sequenced in 2000
- 3 billion nucleotides: real challenge to sequence a whole genome
Took decades, now can be achieved in a week
o Was a competition between the public sector, the industry (C. Venter) and academia
(F. Collins)
▪ Bill Clinton: ‘With this profound new knowledge, humankind is on the
verge of gaining immense, new power to heal. It will revolutionize the
diagnosis, prevention and treatment of most, if not all, human diseases.’
Genetic diseases
- Just 1 point-mutation in 3 billion bp can cause a potentially life-threatening disorder like
hemophilia, Duchenne muscular dystrophy, cystic fibrosis, …
- Genetic diseases of high unmet medical need for which gene therapy might be an answer
Hemophilia A and B
Duchenne muscular dystrophy
Pompe disease
Myotubular myopathy
Huntington’s disease
Cystic fibrosis
- Examples of genetic editing curing diseases
Leukemia
o Got weaponized killer T cells that could recognize the cancer cells and kill them
o The girl was able to fight off the cancer and survive
Can these broken genes be fixed by gene therapy?
- Gene addition
Adding a functional normal copy of a gene (cDNA) that compensates for or complements
the mutated gene
Great for recessive genetic disorders
- Gene editing
Correcting the mutation directly in situ within the defective gene itself
o Can be done with high efficiency
Great for dominant and recessive genetic disorders
Gene transfer in somatic cells
- There is no gene transfer to the next generation
- If you were to transfer genes to germline cells you would generate a genetically modified human
Not ethical!
o Doesn’t it sound ethical to cure a disease for every generation to come? But, you
need to have given consent for treatment + we cannot foresee all the consequences
1
,Gene therapy/editing mantra
- ‘Delivery, delivery, delivery’
We need to deliver the genes efficiently and safely into cells
o Sometimes there are safe methods, but very inefficient and sometimes there are
efficient methods, but might kill the patient in the process
Optimizing gene therapy for clinical translation
- A molecular 3-pronged strategy
Vector
o Needed because both DNA and the cell membrane is negatively charged
(electrostatic repulsion) + lipid bilayer cannot be crossed by DNA
Gene
o Usually a cDNA optimized gene is used
Promoter
o Ideally if you want to treat a liver disease, it is better to use a promoter that is
expressed at high levels in hepatocytes
o Choose promoter depending on the application
▪ There a lot of choices and you have to make the right choice for the right
disease
How to perform gene therapy
- In vivo therapy
Directly administering the gene with a vector to the patient
- Ex vivo therapy
Taking the cells from the patient and genetically modify them in the lab and readminister
the genetically engineered cells
When is this preferred
o If the cells are rare in the body, you will first collect them and enrich them to use
as ex vivo therapy
o E.g. T cell therapy: in vivo is very hard to do (T cells will not be edited), but ex
vivo the cells can be enriched and purified
b. Non-viral gene transfer techniques
Advantages
- Production can be controlled
- Cell-independent production, synthetic production
- Relatively safe
No risk for replication of competent viruses
- Usually limited immunogenicity
- Prolonged expression in muscle (naked DNA)
- Useful for genetic vaccination
E.g. mRNA vaccines
Limitations
- Relatively inefficient
For the moment, it is not possible to cure for example Duchenne disease using non-viral
gene transfer methods, yet
- Usually transient expression
Expression will not last: gene is not stably integrated into the genome of the cell
Selective marker is needed to select for those cells in which the DNA has integrated into
the chromosome by chance
o E.g. use antibiotic resistance selectable markers
2
,Calcium phosphate transfection
- Old technique, but still used (robust)
- Mix DNA with CaCl2 and use a phosphate buffer (Hepes) at pH 7.05
Obtain calcium phosphate precipitates with DNA (forms long concatemers)
o Can be taken up by the cells
- More than 50% transfection efficiency
Electroporation
- Cuvets with electrodes gives electric shock to the cells
Membrane is permeabilized: allows DNA to be taken up
c. Viral gene transfer techniques
Viruses are able to inject their own genetic information into a cell very efficiently
- Interact with specific receptors on the surface of the target cells
Genetic information part of the viral particle will be released into the cell
Viral genes encode viral proteins
o E.g. proteins making up the envelope
Results in new viral particles
- Productive infection
Each of these new viruses are capable of infecting a new cell or can be transmitted from
one person to another
Can we use viruses to deliver genes of interest
- Last thing you want to do is to inject a real virus in a patient
Eliminate the viral genes and replace it with GOI (therapeutic gene)
- Viral vector = virus that do not contain viral genes, but a therapeutic gene
Viral vector still exploits the mechanism for entry like the original virus from which it is
derived
o Therapeutic gene is delivered into the cell and therapeutic protein is produced
o No viral particles will be made
This mechanism is called transduction
Generation of replication-deficient viral vectors by complementation
- Viral life cycle
Cell contains at least one viral gene that encodes a viral protein
o Material can be ss, ds, DNA, RNA, …
o Viral gene has to be expressed by a promoter
o Psi: packaging sequence (molecular signal)
▪ This sequence of the virus allows the genetic material to be recognized by
the cellular machinery that is destined for packaging
▪ Without this element the cell will not know that this is a viral gene
• There will be empty viral particles
Virus will burst out of the cell and the viral gene will be packaged into this viral particle
because of psi
- How to make a viral vector
Make the next construct: promoter + psi + therapeutic gene of interest
o Because of psi the gene will be packaged into the particles
o With only this construct there will be no viral particles
Add construct: promoter + viral gene
o Psi is eliminated from the construct
Result
o Particles that resemble wild life viral particles, but the therapeutic gene is inserted
instead of the viral genes
3
, Clinically relevant technologies
- Adeno associated viral vectors (ssDNA)
Stable expression of GOI
Genes are mostly not integrated in the genome
o You don’t necessarily need integration in order to have stable expression
▪ But there will be deletion of the gene after division
Pre-existing immunity
o A lot of people (> 50%) have natural exposure to the wild type virus, but not
believed to cause any pathologies: considered as a non-pathogenic human virus
▪ Dependo virus: depends on other viruses for its own replication (so small
that it needs help)
Limited capacity (< 5 kb)
o AAV are very small: disadvantage for gene therapy
Finding the right ‘molecular key’ to enter the target cells
o Corresponds to some of the protrusions on the outside of the AAV
o There is an enormous diversity of AAVs: different serotypes
▪ Depending on the serotype there is a different primary and secondary
receptor
▪ How is the different receptor interaction important for gene therapy
• You can transduce different cell types because expression of
receptors varies from cell to cell
o Different serotypes have different clinical applications
• E.g. AAV6 works well in the muscle, AAV9 is not specific
anymore therefore going everywhere in the body
o Based on mouse studies: some of these patterns do not
always correlate to the results in patient
▪ Looking at AAV9: found that it expresses its gene
in the heart
▪ Can we generate AAV vectors with defined tissue or organ-specificity?
• Impose a selective advantage that allows one species to survive:
leads to selective expansion
• Insert peptides in specific locations in the capsid structure of the
AAV (at the level of viruses, not viral vectors)
o Using genetic fusions: inside capsid gene encoding for the
capsid protein a library of random sequences (encoding
for peptides) can be cloned
• Positive selection for the virus that does enter the cell of interest
o After repeating the process a few times, the virus that
enters the cells the best and replicates the best will win
▪ AAV capsid shuffling
• Say you have a virus with the desired tropism, but isn’t efficient
and a virus that is efficient, but doesn’t have the desired tropism
o Generate a capsid library
o In vivo selection: you take a mouse and repopulate the
mouse with human hepatocytes
▪ Selection for the right virus
- Retroviral vectors/lentiviral vectors
Stable expression
Integrated into the chromosome
o Therapeutic gene will be passed on after division
No pre-existing immunity
o Incidence of HIV in the population is very low, but even then the envelope of the
vector is a non-HIV envelope so we will get around immunity issues
o Viral vector used is from a mouse leukemia virus which does not infect humans
4
a. Introduction
Complete human genome was sequenced in 2000
- 3 billion nucleotides: real challenge to sequence a whole genome
Took decades, now can be achieved in a week
o Was a competition between the public sector, the industry (C. Venter) and academia
(F. Collins)
▪ Bill Clinton: ‘With this profound new knowledge, humankind is on the
verge of gaining immense, new power to heal. It will revolutionize the
diagnosis, prevention and treatment of most, if not all, human diseases.’
Genetic diseases
- Just 1 point-mutation in 3 billion bp can cause a potentially life-threatening disorder like
hemophilia, Duchenne muscular dystrophy, cystic fibrosis, …
- Genetic diseases of high unmet medical need for which gene therapy might be an answer
Hemophilia A and B
Duchenne muscular dystrophy
Pompe disease
Myotubular myopathy
Huntington’s disease
Cystic fibrosis
- Examples of genetic editing curing diseases
Leukemia
o Got weaponized killer T cells that could recognize the cancer cells and kill them
o The girl was able to fight off the cancer and survive
Can these broken genes be fixed by gene therapy?
- Gene addition
Adding a functional normal copy of a gene (cDNA) that compensates for or complements
the mutated gene
Great for recessive genetic disorders
- Gene editing
Correcting the mutation directly in situ within the defective gene itself
o Can be done with high efficiency
Great for dominant and recessive genetic disorders
Gene transfer in somatic cells
- There is no gene transfer to the next generation
- If you were to transfer genes to germline cells you would generate a genetically modified human
Not ethical!
o Doesn’t it sound ethical to cure a disease for every generation to come? But, you
need to have given consent for treatment + we cannot foresee all the consequences
1
,Gene therapy/editing mantra
- ‘Delivery, delivery, delivery’
We need to deliver the genes efficiently and safely into cells
o Sometimes there are safe methods, but very inefficient and sometimes there are
efficient methods, but might kill the patient in the process
Optimizing gene therapy for clinical translation
- A molecular 3-pronged strategy
Vector
o Needed because both DNA and the cell membrane is negatively charged
(electrostatic repulsion) + lipid bilayer cannot be crossed by DNA
Gene
o Usually a cDNA optimized gene is used
Promoter
o Ideally if you want to treat a liver disease, it is better to use a promoter that is
expressed at high levels in hepatocytes
o Choose promoter depending on the application
▪ There a lot of choices and you have to make the right choice for the right
disease
How to perform gene therapy
- In vivo therapy
Directly administering the gene with a vector to the patient
- Ex vivo therapy
Taking the cells from the patient and genetically modify them in the lab and readminister
the genetically engineered cells
When is this preferred
o If the cells are rare in the body, you will first collect them and enrich them to use
as ex vivo therapy
o E.g. T cell therapy: in vivo is very hard to do (T cells will not be edited), but ex
vivo the cells can be enriched and purified
b. Non-viral gene transfer techniques
Advantages
- Production can be controlled
- Cell-independent production, synthetic production
- Relatively safe
No risk for replication of competent viruses
- Usually limited immunogenicity
- Prolonged expression in muscle (naked DNA)
- Useful for genetic vaccination
E.g. mRNA vaccines
Limitations
- Relatively inefficient
For the moment, it is not possible to cure for example Duchenne disease using non-viral
gene transfer methods, yet
- Usually transient expression
Expression will not last: gene is not stably integrated into the genome of the cell
Selective marker is needed to select for those cells in which the DNA has integrated into
the chromosome by chance
o E.g. use antibiotic resistance selectable markers
2
,Calcium phosphate transfection
- Old technique, but still used (robust)
- Mix DNA with CaCl2 and use a phosphate buffer (Hepes) at pH 7.05
Obtain calcium phosphate precipitates with DNA (forms long concatemers)
o Can be taken up by the cells
- More than 50% transfection efficiency
Electroporation
- Cuvets with electrodes gives electric shock to the cells
Membrane is permeabilized: allows DNA to be taken up
c. Viral gene transfer techniques
Viruses are able to inject their own genetic information into a cell very efficiently
- Interact with specific receptors on the surface of the target cells
Genetic information part of the viral particle will be released into the cell
Viral genes encode viral proteins
o E.g. proteins making up the envelope
Results in new viral particles
- Productive infection
Each of these new viruses are capable of infecting a new cell or can be transmitted from
one person to another
Can we use viruses to deliver genes of interest
- Last thing you want to do is to inject a real virus in a patient
Eliminate the viral genes and replace it with GOI (therapeutic gene)
- Viral vector = virus that do not contain viral genes, but a therapeutic gene
Viral vector still exploits the mechanism for entry like the original virus from which it is
derived
o Therapeutic gene is delivered into the cell and therapeutic protein is produced
o No viral particles will be made
This mechanism is called transduction
Generation of replication-deficient viral vectors by complementation
- Viral life cycle
Cell contains at least one viral gene that encodes a viral protein
o Material can be ss, ds, DNA, RNA, …
o Viral gene has to be expressed by a promoter
o Psi: packaging sequence (molecular signal)
▪ This sequence of the virus allows the genetic material to be recognized by
the cellular machinery that is destined for packaging
▪ Without this element the cell will not know that this is a viral gene
• There will be empty viral particles
Virus will burst out of the cell and the viral gene will be packaged into this viral particle
because of psi
- How to make a viral vector
Make the next construct: promoter + psi + therapeutic gene of interest
o Because of psi the gene will be packaged into the particles
o With only this construct there will be no viral particles
Add construct: promoter + viral gene
o Psi is eliminated from the construct
Result
o Particles that resemble wild life viral particles, but the therapeutic gene is inserted
instead of the viral genes
3
, Clinically relevant technologies
- Adeno associated viral vectors (ssDNA)
Stable expression of GOI
Genes are mostly not integrated in the genome
o You don’t necessarily need integration in order to have stable expression
▪ But there will be deletion of the gene after division
Pre-existing immunity
o A lot of people (> 50%) have natural exposure to the wild type virus, but not
believed to cause any pathologies: considered as a non-pathogenic human virus
▪ Dependo virus: depends on other viruses for its own replication (so small
that it needs help)
Limited capacity (< 5 kb)
o AAV are very small: disadvantage for gene therapy
Finding the right ‘molecular key’ to enter the target cells
o Corresponds to some of the protrusions on the outside of the AAV
o There is an enormous diversity of AAVs: different serotypes
▪ Depending on the serotype there is a different primary and secondary
receptor
▪ How is the different receptor interaction important for gene therapy
• You can transduce different cell types because expression of
receptors varies from cell to cell
o Different serotypes have different clinical applications
• E.g. AAV6 works well in the muscle, AAV9 is not specific
anymore therefore going everywhere in the body
o Based on mouse studies: some of these patterns do not
always correlate to the results in patient
▪ Looking at AAV9: found that it expresses its gene
in the heart
▪ Can we generate AAV vectors with defined tissue or organ-specificity?
• Impose a selective advantage that allows one species to survive:
leads to selective expansion
• Insert peptides in specific locations in the capsid structure of the
AAV (at the level of viruses, not viral vectors)
o Using genetic fusions: inside capsid gene encoding for the
capsid protein a library of random sequences (encoding
for peptides) can be cloned
• Positive selection for the virus that does enter the cell of interest
o After repeating the process a few times, the virus that
enters the cells the best and replicates the best will win
▪ AAV capsid shuffling
• Say you have a virus with the desired tropism, but isn’t efficient
and a virus that is efficient, but doesn’t have the desired tropism
o Generate a capsid library
o In vivo selection: you take a mouse and repopulate the
mouse with human hepatocytes
▪ Selection for the right virus
- Retroviral vectors/lentiviral vectors
Stable expression
Integrated into the chromosome
o Therapeutic gene will be passed on after division
No pre-existing immunity
o Incidence of HIV in the population is very low, but even then the envelope of the
vector is a non-HIV envelope so we will get around immunity issues
o Viral vector used is from a mouse leukemia virus which does not infect humans
4
