2.) Genome Modification and Antisense

Question 1

Describe the strategies for gene therapy that consider genome modification and antisense therapy.

Answer 1

Genome modification (editing):
- Change sequence of genomic DNA to correct a mutation (v. novel)

Antisense:
- Use of oligonucleotides that binds to mRNA e.g. to modify splicing pattern of a disease-associated gene

Question 2

What technologies exist that allow site-specific genome targeting and editing?

Answer 2

• CRISPR/Cas9
• TALENs
• ZInc-finger nucleases
• Meganucleases

Question 3

What is the therapeutic goal of genome modification approach to genome therapy?

Answer 3

• Potential to correct disease-associated mutations
• Restoring expression of a faulty gene (e.g. cystic fibrosis)
  »> Find particular mutated nucleotide sequence and modify
  »> Precise modification of human genome

Question 4

Explain how individuals with mutations in coreceptor CCR5 have aided genome editing as a therapeutic?

Answer 4

Mutations in coreceptor CCR5 yield resistance to HIV infection:
- In 2009, the ‘Berlin patient’ (HIV patient w/leukaemia) was given a bone marrow transplant from donor with protective CCR5 mutation
- HIV was cured; protective CCR5 mutation (membrane protein receptor) yielded resistance to HIV
• Introducing protective CCR5 mutation ex vivo in patient’s T cells used in 2014 PI clinical trial (success)
• Genome editing via zinc-finger nuclease technology (ZFN - older than CRISPR)
• T-cells removed from patient, CCR5 gene disrupted, modified HIV-resistant T-cells then infused into HIV patient

Question 5

What type of mutation is the approach of disrupting a gene appropriate for? (as opposed to correcting a gene)

Answer 5

• Nonspecific deletions

- Gene disruption used in HIV+ patient’s T-cells; using ZFN technology ex vivo to disrupt HIV coreceptor CCR5

Question 6

Briefly describe what CRISPR and TALENs entail, and how they compare.

Answer 6

CRISPR:
- An adaptive prokaryotic immune system (vs. eukaryotic in human)
- Repurposed to allow site-specific genome modification in mammalian cells
- Most flexible/easy use of genome editing technologies
»> Problems with off-target effects

TALENs:
- Engineering bacterial DNA (also) binding proteins to allow site-specific targeting of dsDNA nuclease (cleaving enzyme)
- Hence fewer off-target effects than CRISPR due to higher specificity
»> Harder to engineer

Question 7

What are the limitations re. genome editing technology approaches (e.g. CRISPR/TALENs)?

Answer 7

• Off-target effects (even TALENs); do not want to be modifying genome sequence elsewhere (hence only ex vivo so far - NOT in vivo yet)
• It is less efficient engineering in a precise nucleotide sequence change than at introducing a non-specific deletion
  »> Ethics of genome modification

Question 8

What does CRISPR stand for? What does the process involve?

EXTRA

Answer 8

Clustered Regularly Interspaced Short Palindromic Repeats:

• Bacterial genome (e.g. Strep pyogenes) contain a CRISPR array; a series of short repeated sequences, with short sequences from viruses/bacteriophages that infect/have infected the bacterium inserted between the repeats (immune memory/secondary response effect)
• Repeat region is transcribed to crRNA (CRISPR RNA)
• crRNA guide Cas9 nuclease enzyme to specific exogenous genetic material (which must contain a protospacer adjacent motif (PAM - in type/group II systems); species-specific sequence (complementary vibes)
• CRISPR complex (crRNA:tracrRNA:Cas9) binds to the foreign DNA and cleaves it to destroy the invader, with the complex then unbinding after the ds-break (near the PAM)

https://www.addgene.org/crispr/history/

Question 9

How prevalent is the CRISPR system in prokaryotes?

Answer 9

• 40% of bacteria
• 90% of archaea
• DIfferent systems classified based on differences in cas genes

Question 10

Which CRISPR system/subtype does mammalian genome modification utilise most?

Answer 10

Group/Type II:

• Simplest system
• Requires PAM (protospacer adjacent motif) to allow complementary binding of CRISPR complex to exogenous genetic material

Question 11

How has CRISPR been adapted as a tool for genome modification

Answer 11

• 2012: minimal CRISPR system first used directing site-specific cleavage of genomic DNA in other organisms
• crRNA and tracrRNA combined to give single guide RNA which targets sequence of interest (which would be exogenous DNA in native prokaryotes)
• Guide RNA is then coexpressed in cells with Cas9 endonuclease (cleaves dsDNA)

Question 12

What are the two ways CRISPR is used to edit genes?

Answer 12

• Double strand breakages (DSBs) firstly induced at targeted site by Cas9 cleavage
• Cells DO NOT like DSBs; breakage is repaired by cellular machinery…

Non Homologous End Joining (NHEJ):

• dsDNA Ends are directly ligated without need for homologous template (fast, but error-prone method)
• Results in small insertions or deletions of nucleotides (INDELs), disrupting gene of interest

Homology-directed repair (HDR):
• Wild-type template supplied matching cleavage site, with the desired mutation included in template
• Mutation then incorporated as dsDNA break is repaired
• Allows PRECISE mutations to be made (changing out mutant to wild-type gene/allele)
• However, it is inefficient with a 10% cell yield

Question 13

What are INDELs? What process does it belong to?

Answer 13

Insertions or Deletions of bases in genome:

• End results of DSBs induced by CRISPR system, where error-prone NHEJ results in INDELs
• Thus disrupting the (mutant) gene of interest

Question 14

Name examples of indications where CRISPR genome editing has been used in clinical trials.

Answer 14

(July 2016) First human PI trial using CRISPR/Cas9 given ethics approval:
• Ex vivo trial in T cells targeting lung cancer (China)
• CRISPR used to inactivate (gene disruption via NHEJ) PD-1 which otherwise suppresses immune response and allows cancers to proliferate
• Inactivation (disruption) of PED-1 results in over/increased activation of immune system against cancer

Question 15

What is TALENs? What does it entail?

Answer 15

Modification of genome technology (akin to CRISPR):
• Transcription Activator Like Effector Nucleases
• Based on bacterial TAL effectors
• (AA sequence of DNA binding domain shows strong association with cleavage at specific nucleotides; allowing engineering of proteins with specific DNA recognition sequence)
• TAL effector (TALE) sequence-specific DNA binding domain (engineered to bind to any NT sequence of interest), when combined with a (non-specific) nuclease can cut DNA at specific locations
• TAL effector DNA-binding domain is fused to Fok 1 nuclease (DNA-cleavage domain) mediating site-specific dsDNA cleavage
»> As with CRISPR, DSBs are repaired by NHEJ or HDR

Question 16

What needs to be considered with TALENs delivery?

Answer 16

• They are large, repetitive genes

- Thus difficult to deliver via AAV or lentivirus (small space to pack genetic material)

Question 17

How is delivery of genome modification agents achieved? Advantages over traditional gene therapy?

Answer 17

• CRISPR requires a protein (Cas9) and an RNA (guide RNA); both can be encoded in one viral vector e.g. AAV
• TALENs is just a single protein; can also be encoded in a viral vector (though it is a large protein; lots of genetic material)

> > > Genome modification agents do not need to be in cells for extended length; just requires entry, alters the gene then it’s aighhhhht (change will then be expressed thereafter)

Question 18

Where do we currently stand with genome modification agents? Are they ready for clinic?

Answer 18

• For CRISPR to be useful in gene therapy, improvements in efficiency and specificity are needed for human therapeutics
• TALENs has better specificity, but are more complex to engineer and harder to deliver (BIG ass genes - one big protein) complex)
• Technologies being constantly refined, major improvements likely in 5-10 year window
  »> Major ethical implications to potential use in patients

Question 19

What are the ethical implications for genome modification? Compare somatic and germline modification.

Answer 19

Transient introduction of genetic material (gene therapy) vs. genome modification (somatic vs. germline):

Somatic:
• Changes to genome in any cell other than a gamete, gametocyte, germ cells or undifferentiated stem cell
• Results in permanent change to person’s genome, but it is not passed on to offspring (change is for as long as cell survives in patient)
• Thus delivery and maintenance can be difficult

GermlineL
• Changes to genome in germ cells/early embryo that will be passed on to descendants (permanent change to whole DNA)
• Potential to eliminate an inherited disease
- Ethically difficult to justify and controversial AF
- ‘Designer babies’ (rich vs. poor accessibility, medical vs. societal implications)

Question 20

What are the current hurdles for germline genome modification?

Answer 20

• High chance of off-target effects (specificity too poor atm)
• Inefficient
• Disease causing mutations can already be avoided by preimplantation genetic diagnosis during IVF (screened embryo)

Question 21

Is genome editing in embryos (germline) achievable currently?

Answer 21

• Practical hurdles (specificity, inefficient etc.)
• Strong global consensus against it for germline modification, though increasing prevalence in research (see Nature)
• Consequences for disabled community if disabilities can be eliminated
• Focus could shift from single gene mutation currently towards more complex characteristics influenced by many genes e.g. intelligence (how to restrict to diseases?)
  • New technologies will be developed/improve

Question 22

What is antisense technology?

Answer 22

• Introducing an oligonucleotide (short synthetic RNA/DNA strand) that is complementary to a sequence on mRNA of interest
• ‘Anti-sense’: prevents base-pairing e.g. tRNA, preventing proteins/RNA regulators from binding/interacting to this site via STERIC HINDRANCE

Question 23

What are the goals of antisense technology?

Answer 23

Chemically modified bases/backbone to:

• Improve RNA binding
• Protect from nuclease degradation
• Improve entry into cells
• Many different modifications e.g. morpholino antisense oligonucleotide

Question 24

What are the ways that antisense technology can be used to modulate gene expression?

Which are in the clinic?

Answer 24

Modulation of splicing:

• Usually highly regulated
• Oligonucleotide designed to mask a sequence that is recognised by a splicing factor: preventing interaction with splisosome, leaving extra intron e.g.
• Clinic

Translation inhibition

• Oligonucleotide designed to bind across start codon, preventing ribosome access and recognition of start codon
• Lab

Question 25

How do antisense oligonucleotides modulate splicing? How is this used therapeutically?

Answer 25

• Many disease-associated genes contain multiple exons and introns
• Antisense oligonucleotides can be designed to bind to splice sites or splice enhancers, altering pattern of splicing

Therapeutics:
Approach to treat disease is v gene-specific
- Duchenne muscular dystrophy (DMD) = induce exon skipping
- Spinal muscular atrophy (SMA) = exon inclusion

Question 26

What is duchenne muscular dystrophy (DMD)? Gene responsible?

Answer 26

• Muscular dystrophy caused by mutations in large dystrophin gene (79 exons)
• X-linked recessive (boys)
• Dystrophin protein normally protects membrane of muscle firbes from damage during contraction
• Progressive muscle wastage and early death (from 4-20)

• 13% of boys with DMD have mutation in exon 51 = frameshift mutation (changing AA sequence downstream of mutated site; severe mutation, codes for non-functional protein)

Question 27

How can DMD be treated with splice modulation?

Answer 27

• 13% of DMD = mutated exon 51
• Exondys51 (drug - morpholino antisense oligonucleotide) causes exon 51 skipping by targeting splice enhancer sequence within exon 51, restoring reading frame (correct codons)
• Allow production of functional dystrophin protein with small deletion
• Exon 51-skipped mRNA codes for wildtype dystrophin protein lacking short internal sequence
• Protein is only PARTLY functional (not generating a new wild-type), but symptoms now similar to Becker muscular dystrophy (much milder phenotype)
• Sept 2016: FDA approval

Question 28

What is spinal muscular atrophy (SMA)? Gene responsible?

Answer 28

• Most common death in babies (by 2 years; gradual paralysis)
• Caused by autosomal recessive mutation in SMN1 gene; insufficient SMN protein production leads to loss of spinal motor neurons hence gradual paralysis
• Different types of SMA relate to how many copies of SMN2 gene are present; SMA1 is worst, with 1-2 copies of SMN2
  »> With splice modulation, children walking/running at 2 y/o who would have normally died

Question 29

How is SMA treated via splice modulation?

Considerations?

Answer 29

• SMN2 gene exists which is almost identical to SMN1 except for splice site mutation - only 10% of mRNAs produce functional SMN protein
• Not enough SMN2 to substitute for mutated SMN1 normally

December 2016:
• Antisense oligonucleotide (Spinraza/nusinersen developed by Ionis/Biogen) approved by FDA
• Modulated splicing of SMN2, increasing production of fundamental SMN protein from SMN2 gene (more functional mRNA made = more SMN made)

• Delivered intrathecally every 4 months
• Available in UK to children meeting specific criteria through Expanded Access Programme
• List price $125,000 per injection

Alternative (November 2017):
> Gene therapy approach for SMA1 = v. high doses of SMN1 delivered in AAV9 vectir
> Excellent results in clinical trials
»> Single IV dose effective