5. CRISPR therapies

Question 1

Define genomic editing

Answer 1

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

Question 2

What are the uses of genome engineering?

Answer 2

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

Question 3

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

Answer 3

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

Question 4

How exactly does CRISPR work?

Answer 4

Question 5

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

Answer 5

• no protein engineering needed
• higher gene editing rates
• large scale experimnets with different nucleases possible

Question 6

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

Answer 6

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

Question 7

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

Answer 7

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

Question 8

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

Answer 8

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

Question 9

Explain NHEJ in DSB repair

Answer 9

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

Question 10

Explain HDR in DSB repair

Answer 10

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

Question 11

Are DSB repair mechanisms exclusive?

Answer 11

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

Question 12

Compare and contrast somatic vs germline gene therapy effects

Answer 12

Question 13

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

Answer 13

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

Question 14

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?

Answer 14

Question 15

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

Answer 15

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

Question 16

What are the current clinical trials that use CRISPR?

Answer 16

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

Question 17

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

Answer 17

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

Question 18

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

Answer 18

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

Question 19

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

Answer 19

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

Question 20

What makes a tissue hard to each with CRISPR?

Answer 20

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

Question 21

How are CRISPR reagents delivered into cells?

Answer 21

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

Question 22

What are the viral vectors for somatic gene therapy?

Answer 22

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

Question 23

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

Answer 23

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

Question 24

Explain nanoparticles as vectors for somatic gene therapy?

Answer 24

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

Question 25

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

Answer 25

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

Question 26

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

Answer 26

**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**

Question 27

Why does CRISPR Cas9 system tolerate sequence mismatch?

Answer 27

**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

Question 28

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

Answer 28

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**

Question 29

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

Answer 29

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

Question 30

Explain how base editing works

Answer 30

**Base editing** - a gene-editing technique - enables **precise**, **single-nucleotide** changes in DNA **w/o 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

Question 31

Explain how prime editing works

Answer 31

**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 transcription** and DNA editing via **synthesis directly onto the nick** - seals the gap Disadv: - **not very efficient** - **big proteins** needed

Question 32

What are the advantages and disadvantages of base editing

Answer 32

Question 33

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

Answer 33

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

Question 34

Explain how CAR-T cell therapy works

Answer 34

**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**

Question 35

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

Answer 35

**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

Question 36

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

Answer 36

**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

Question 37

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

Answer 37

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

Question 38

What are the diseases associated with beta globin gene mutations?

Answer 38

**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**

Question 39

Explain human globin switch in development

Answer 39

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

Question 40

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

Answer 40

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

Question 41

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

Answer 41

Question 42

What is the current main problem in gene editing therapies?

Answer 42

**Financial cost** - therapy exists but **inaccessible due to high financial costs**

Question 43

What are the current debates on germline gene editing?

Answer 43

- **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