5. CRISPR therapies Flashcards
Define genomic editing
Genomic editing - type of genetic engineering in which DNA is inserted / deleted / replaced in the genome of a living organism using engineered nucleases
DNA clevage -> insert target sequence
What are the uses of genome engineering?
Use of genomic engineering:
- gene surgery: genome editing in patients’ cells - in vitro / in situ
- drug development
- animal model creation for research
- genetic variation
- materials
- food
- fuel
What are the types of gene editing that have been used historically?
Engineered DNA binding motifs in enzymes for gene specific editing:
- Zinc fingers: 1990s-2000s, hard to design, hard to synthesise
- TAL effectors: 2010-2014, easy to design, hard to synthesise
- CRISPR: 2013-now, easy to design, easy to synthesise - unlike other use RNA as a DNA recognition sequence - easier to synthesise
How exactly does CRISPR work?
What are the advantages of CRISPR compared with other DNA editing technologies?
- no protein engineering needed
- higher gene editing rates
- large scale experimnets with different nucleases possible
Besides gene editing what else can be achieved with synthetic DNA binding proteins?
Synthetic DNA binding proteins based on their fusion protein to Cas9 can perform:
- gene editing
- transcriptional activation / repression
- Fluorescence
- DNA cooping factors (??)
- Cytidine deamination (epigenetics)
- Reverse transcriptase
What type of DNA cut is performed in CRISPR gene editing?
DNA is cut via double strand break (DSB)
- DSBs in cells naturally recognised as DNA damage - can be highly toxic to cells
-> then use elaborate mechanisms to sense and repair DSBs
What are the DSB repair mechanisms used in gene editing CRISPR?
DNA DSBs repaired by inserting the target sequence - mutagenesis performed by DSB repair via:
- non-homologous end joining (NHEJ)
- homology directed repair (HDR)
Explain NHEJ in DSB repair
Non-homologous end joining (NHEJ) error prone:
1. DSB DNA ends processed by endonucleases -> change ORF - introduce mutations
2. Ends joined
3. Mutation introduced - deletion / insertion of variable length
=> can be used in research to create gene KOs - null -/- mutations - useful for gene KO but not for precise gene editing
Explain HDR in DSB repair
HDR DBS repair - precise sequence correction - rely on a template:
1. DSB DNA ends bound by enzymes directing search for homology between DSB and template sequence
2. Bind the template - synthesise complimentary strand using the template
3. Introduce sequences of the template into the strand
HDR good for precise gene editing / correction using the tenplate sequence
See MOG for good NHEJ, HDR explanations
Are DSB repair mechanisms exclusive?
No, work in equilibrium to repair DSBs - equilibrium between gene KO and gene correction but preferred in different cell cycle phases
Compare and contrast somatic vs germline gene therapy effects
What are the technical challenges of treating genetic disease with gene editing technologies?
Technical challenges of treating genetic disease with gene editing technologies:
- delivering necessary molecules to target cells
- avoiding off-target mutagenesis
- achieving desired genetic change at intended target site
What aspects need to be considered in adressing the challenge of delivering necessary molecules to the right cells in genome ediitng technologies to treat disease?
What are the different approaches ind delivering Cas9 and sgRNA in gene editing? What are their pros and cons
Can be delivered in different forms:
- DNA
- RNA
- Protein
What are the current clinical trials that use CRISPR?
Common theme - in all therapies disease cells are accessible for editing
What is a common approach in CRISPR therapies for treating cell related disease?
Gene editing in appropriate cells is done ex vivo:
- cell environment can be tightly controlled for better effect
- cells with editing outcomes can be selected
- cells with off target mutations can be excluded
Why are blood cells an ideal target cells for editing in CRISPR therapies?
A lot of clinical trials focus on editing blood cells because they are easily accessible - haematopoietic stem cells which can be isolated from blood can also give rise to all blood cell types
Ex. T cells
How are CRISPR therapies developed if ex vivo delivery is impossible?
Some cells are edited in vivo - ex:
- eye cells are externally accessible even in vivo
- molecules injected into blood get taken up efficiently by the liver - can use bloodstream as a delivery method
What makes a tissue hard to each with CRISPR?
Tissue is hard to get with CRISPR when:
- high cell turnover - ex. lung epithelium - treated cells are replaced by defected cells quickly after therapy
- inaccessible tissues - ex. heart, prostate, brain
How are CRISPR reagents delivered into cells?
CRISPR agents must transverse the cell membrane to get into cells:
- transfection - through lipid transport
- electroporation - using electrical current
Often are inefficient with varied levels of success in each experiment
Molecules need to find the right tissue first -> then transverse the membrane
What are the viral vectors for somatic gene therapy?
Viruses - good at evading immune system and delivering genetic cargo into human cells
Different viruses have different properties - choose vector depending on the therapy:
- adenovirus (AAV): ssDNA, 5kb, no chr integration
- retrovirus: RNA, 8kb, chr integration
- lentivirus: RNA, 8kb, chr integration
- Herpes simpex virus: dsDNA, 40kb, no chr integration
AAV used for CRISPR delivery - but rather small cargo allowed
What are the possible types of vectors for gene editing therapies?
Types of vectors for gene editing therapies:
- viral
- nanoparticles
Explain nanoparticles as vectors for somatic gene therapy?
Nanoparticles - alternative to viral vectors - inject into tissue - take up by cells in endocytosis - release cargo - integrate into genome by HDR
What are the current efforts for combating the challenge of gene therapy delivery?
Delivery challenge depends on the disease - current efforts trying to improve:
- more cargo
- different cell specificities
- lower toxicity
What are the current efforts for combating the challenge of off target mutagenesis?
Off target mutagenesis challenge increases the chance of cancer - by inactivation of tumour suppression genes, activation of oncogenes - what is done trying to reduce Cas9 induced off target mutations:
- careful nuclease design - screening for sgRNA sequence against genome - see if any other homologous sequences exist
- preclinical testing of candidate sgRNA molecules - check for toxicity
- re-engineering system to reduce the tolerance for mismatches - increase Cas9 specificity
Why does CRISPR Cas9 system tolerate sequence mismatch?
CRISPR evolved as bacterial immune system - recognises invading parasitic DNA - parasites evolve rapidly to evade immune defense - some flexbility in sequence recognition desirable to catch up with parasite evolution - but for therapeutic gene editing want to cut once in the genome at the very specific site 100% matching the sgRNA
What are the current efforts for combating the challenge of achieving the desired edit at the exact intended site?
Depends on the therapy desired outcome - if we want to correct:
- an existing gene + correct one, both, either of parental alleles (target maternal / paternal)
- knock it out to disable it
What is done:
- disease selection: focus on those that can be corrected by NHEJ / by transplantable cells (when cultured ex vivo, successful cells can be selected)
- use alternative editing mechanisms that don’t induce DSBs - less risk: base editing, prime editing
What must be considered when targeting the allele for editing / knockout?
Consider cell cycle stages for each of these:
- Correct by editing at precise sites - via HDR - active in S/G2/M phases of the cell cycle
- KO - via NHEJ - active through the whole cell cycle
For DSB repair - need to go into mitosis because G0 not dividing - cannot deal with DNA breaks
Explain how base editing works
Base editing - a gene-editing technique - enables precise, single-nucleotide changes in DNA w/o inducing DSBs - modifies individual nucleotides through chemical conversion
- Fusion of Cas9 and deaminase enzyme
- Targeting a specific base using sgRNA
- Chemical conversion of the base - cytosine base editing (CBE) or adenine base editing (ABE)
- DNA repair and base pairing - recognises the modified base and converts the mismatch into the desired nucleotide through mismatch repair / DNA replication
-> no DSBs induced in the editing
Explain how prime editing works
Prime editing - versatile and precise genome-editing technique - without inducing DSBs - relies on a modified Cas9 protein combined with a reverse transcriptase enzyme and a prime editing guide RNA (pegRNA)
- Modified Cas9 nickase (nCas9) cuts one strand of DNA
- Nickase is fused with reverse transcriptase - synthesises DNA directly onto the target site
- pegRNA is specific for prime editing: has a targeting sequence (directs nCas9) + template sequence (encodes desired genetic change along with a primer binding site for reverse transcription)
- Reverse transcription and DNA editing via synthesis directly onto the nick - seals the gap
Disadv:
- not very efficient
- big proteins needed
What are the advantages and disadvantages of base editing
What target gene therapies have been developed using gene editing nucleases?
> 80 CRISPR-mediated therapies in clinical trials:
- blood disorders
- solid tumours
- genetic blindness
- cardiovascular disease
- diabetes
- muscular dystrophy
Explain how CAR-T cell therapy works
T cells - circulating cells in bloodstream - detect and destroy things that should not be in our bodies
An antigen receptor on T cells allows them to distinguish self vs non-self (ex pathogens, tumours)
Ex. Anti-CD19 CAR-T therapy: T cells can be engineered to express CD19 linked to cytoplasmic T cell activation domain -> activates T cells against CD19+ B cell malignancies
Explain how CAR-T cells are produced and delivered to patients
Blood taken from the patient -> T cells isolated and engineered via retroviral delivery -> engineered cells cultivated and expanded -> injection back into the patient
CAR expression levels in therapeutic cells matters for efficiency - bad if too high, too low
Explain the use of gene editing in CAR-T therapy development
Gene editing is used for integration of chimeric antigen receptor (CAR) at naive TCR locus - enhances anti-tumour activity in vivo - editing made ex vivo T cell culture before transplant - can utilise NHEJ to integrate CAR at endogenous locus - targets long-lived as memory T cells persit for a long time
Active trials use TALENs/ CRISPR for editing
Explain how gene editing can be used in sickle cell anemia therapy
In sickle cell anemia misshaped erythrocytes (RBCs) due to mutation in beta-globin gene - harder for misshaped RBC to carry O2 -> blood vessel blockage, pain, anemia - in populations not selected againts because of heterozygote advantage for prevention against malaria parasite invasion
Gene editing can be used to correct the beta-globin gene: purify haematopoietic stem cells (HSCs) from blood - use AAV vector for beta-globin correction - select successfully edited cells -> transplant back into the patient => reduced RBC sickling
What are the diseases associated with beta globin gene mutations?
Problems with beta globin gene cause:
- sickle cell anemia - single mutation
- beta-thalassemia - many different mutations in Hbb gene
Both can be cured by bone marrow transplant
Explain human globin switch in development
In human development the fetus uses alpha + gamma subunits in HbF hemoglobin - more efficient than post-natal alpha+beta subunit HbA hemoglobin - switch gamma->beta occurs at birth
GWAS found BCL11A to repress HbF - idea to activate fetal gamma globin in adults to overcome defective beta globin - target BCL11A KO at erythroid specific enhancer - so only in target cells with editing would occur => functional haemoglobin in beta-thalassemia patients
What are the advantages of BCL11A targeting for beta-globin diseases
- Delivery - HSCs isolated from blood, cultured ex vivo and transplanted back into bloodstream - naturally go back to bone marrow, don’t have to specifcally place them
- Avoiding off target mutations - high fidelity Cas9 can be used, successfully edited cells selected before transplantation
- Achieving desired edit - targeting BCL11A instead of the target gene Hbb can use gene KO instead of editing - easier to achieve w/o DSBs + restricted to target cells
What are the results from the clinical trial activating detal hemoglobin as a treatment for defective beta-globin in SCD?
What is the current main problem in gene editing therapies?
Financial cost - therapy exists but inaccessible due to high financial costs
What are the current debates on germline gene editing?
- some diseases cannot be cured via somatic editing - germline would be the only option - ex. HIV
- risk of inducing germline mutations that will persist in future generations - high risk
- what diseases are worth curing and which not - tricky boundary - could turn ointo eugenics
