5. CRISPR therapies Flashcards

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1
Q

Define genomic editing

A

Genomic editing - type of genetic engineering in which DNA is inserted / deleted / replaced in the genome of a living organism using engineered nucleases

DNA cleavge -> insert target sequence

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2
Q

What are the uses of genome engineering?

A

Use of genomic engineering:
- gene surgery: genome editing in patients’ cells - in vitro / in situ
- drug development
- animal models
- genetic variation
- materials
- food
- fuel

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3
Q

What are the types of gene editing that have been used historically?

A

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

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4
Q

How exactly does CRISPR work?

A
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5
Q

What are the advantages of CRISPR compared with other DNA editing technologies?

A
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6
Q

Besides gene editing what else can be achieved with synthetic DNA binding proteins?

A

Syntehtic 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

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7
Q

What type of DNA cut is performed in CRISPR gene editing?

A

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

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8
Q

What are the DSB repair mechanisms used in gene editing CRISPR?

A

DNA DSBs repaired by inserting the target sequence - mutagenesis performed by DSB repair via:
- non-homologous end joining (NHEJ)
- homology directed repair (HDR)

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9
Q

Explain NHEJ in DSB repair

A

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

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10
Q

Explain HDR in DSB repair

A

HDR DBS repair - precise sequence correction - rely on template:
1. DSB DNA ends bound by enzymes directing search for homology between DSB and template sequence
2. Bind the templte - 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

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11
Q

Are DSB repair mechanisms exclusive?

A

No, work in equilibrium to repair DSBs - equilibbrium between gene KO and gene correction but preferred in different cell cycle phases

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12
Q

Compare and contrast somatic vs germline gene therapy effects

A
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13
Q

What are the technical challenges of treating genetic disease with gene editing technologies?

A

Technical challenges of treating genetic disease with gene editing technologies:
- delivering necessary molecules to right cells
- avoiding off-target mutagenesis
- achieving desired genetic change at intended target site

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14
Q

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?

A
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15
Q

What are the different approaches ind delivering Cas9 and sgRNA in gene editing? What are their pros and cons

A

Can be delivered in different forms:
- DNA
- RNA
- Protein

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16
Q

What are the current clinical trials that use CRISPR?

A

common theme - in all therapies disease cells are accessible for editing

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17
Q

What is a common approach in CRISPR therapies for treating cell related disease?

A

Gene editing in appropriate cells is done ex vivo:
- cell environment can be tightly controlled for better effect
- celss with editing outcomes can be selected
- cells with off taregt mutations can be excluded

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18
Q

Why are blood cells an ideal target cells for editing in CRISPR therapies?

A

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

19
Q

How are CRISPR therapies developed if ex vivo delivery is impossible?

A

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

20
Q

What makes a tissue hard to each with CRISPR?

A

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
21
Q

How are CRISPR reagents delivered into cells?

A

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

22
Q

What are the viral vectors for somatic gene therapy?

A

Viruses - good at evading immune system and delivering genetic cargo into human cells

Different viruses have different properties - choose 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

23
Q

What are the possible types of vectors for gene editing therapies?

A

Types of vectors for gene editing therapies:
- viral
- nanoparticles

24
Q

Explain nanoparticles as vectors for somatic gene therapy?

A

Nanoparticles - alternative to viral vectors - inject into tissue - take up by cells in endocytosis - release cargo - integrate into genome by HDR

25
Q

What are the current efforts for combating the challenge of gene therapy delivery?

A

Delivery challenge depends on the disease - current efforts trying to improve:
- more cargo
- different cell specificities
- lower toxicity

26
Q

What are the current efforts for combating the challenge of off target mutagenesis?

A

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
27
Q

Why does CRISPR Cas9 system tolerate sequence mismatch?

A

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

28
Q

What are the current efforts for combating the challenge of achieving the desired edit at the exact intended site?

A

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

29
Q

What must be considered when targeting the allele for editing / knockout?

A

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

30
Q

Explain how base editing works

A

Base editing- a gene-editing technique - enables precise, single-nucleotide changes in DNA without inducing DSBs - modifies individual nucleotides through chemical conversion

  1. Fusion of Cas9 and deaminase enzyme
  2. Targeting a specific base using sgRNA
  3. Chemical conversion of the base - cytosine base editing (CBE) or adenine base editing (ABE)
  4. 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
31
Q

Explain how prime editing works

A

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)

  1. Modified Cas9 nickase (nCas9) cuts one strand of DNA
  2. Nickase is fused with reverse transcriptase - synthesises DNA directly onto the target site
  3. 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)
  4. Reverse transcriptiona nd DNA editing via synthesis directly onto the nick - seals the gap

Disadv:
- not very efficient
- big proteins needed

32
Q

What are the advantages and disadvantages of base editing

A
33
Q

What target gene therapies have been developed using gene editing nucleases?

A

> 80 CRISPR-mediated therapies in clinical trials:
- blood disorders
- solid tumours
- genetic blindness
- cardiovascular disease
- diabetes
- muscular dystrophy

34
Q

Explain how CAR-T cell therapy works

A

T cels - 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 between 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

35
Q

Explain how CAR-T cells are produced and delivered to patients

A

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

36
Q

Explain the use of gene editing in CAR-T therapy development

A

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 as memory T cells persit for a long time

Active trials use TALENs/ CRISPR for editing

37
Q

Explain how gene editing can be used in sickle cell anemia therapy

A

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 advanatge 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 -> trasnplant back into the patient => reduced RBC sickling

38
Q

What are the diseases associated with beta globin gene mutations?

A

Problems with beta globin gene cause:
- sickle cell anemia - single mutation
- beta-thalassemia - many different mutations in Hbb gene

Bopth can be cured by bone marrow transplant

39
Q

Explain human globin switch in development

A

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 erythroiud specific enhancer - so only in target cells with ediitng would occur => functional haemoglobin in beta-thalassemia patients

40
Q

What are the advantages of BCL11A targeting for beta-globin diseases

A
  1. 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
  2. Avoiding off target mutations - high fidelity Cas9 can be used, successfully edited cells selected before transplantation
  3. 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
41
Q

What are the results from the clinical trial activating detal hemoglobin as a treatment for defective beta-globin in SCD?

A
42
Q

What is the current main problem in gene editing therapies?

A

Financial cost - therappy exists but inaccessible due to high financial costs

43
Q

What are the current debates on germline gene editing?

A
  • 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