Flashcards in 2.) Genome Modification and Antisense Deck (29):
Describe the strategies for gene therapy that consider genome modification and antisense therapy.
Genome modification (editing):
- Change sequence of genomic DNA to correct a mutation (v. novel)
- Use of oligonucleotides that binds to mRNA e.g. to modify splicing pattern of a disease-associated gene
What technologies exist that allow site-specific genome targeting and editing?
- ZInc-finger nucleases
What is the therapeutic goal of genome modification approach to genome therapy?
- 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
Explain how individuals with mutations in coreceptor CCR5 have aided genome editing as a therapeutic?
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
What type of mutation is the approach of disrupting a gene appropriate for? (as opposed to correcting a gene)
- Nonspecific deletions
- Gene disruption used in HIV+ patient's T-cells; using ZFN technology ex vivo to disrupt HIV coreceptor CCR5
Briefly describe what CRISPR and TALENs entail, and how they compare.
- 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
- 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
What are the limitations re. genome editing technology approaches (e.g. CRISPR/TALENs)?
- 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
What does CRISPR stand for? What does the process involve?
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)
How prevalent is the CRISPR system in prokaryotes?
- 40% of bacteria
- 90% of archaea
- DIfferent systems classified based on differences in cas genes
Which CRISPR system/subtype does mammalian genome modification utilise most?
- Simplest system
- Requires PAM (protospacer adjacent motif) to allow complementary binding of CRISPR complex to exogenous genetic material
How has CRISPR been adapted as a tool for genome modification
- 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)
What are the two ways CRISPR is used to edit genes?
• 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
What are INDELs? What process does it belong to?
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
Name examples of indications where CRISPR genome editing has been used in clinical trials.
(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
What is TALENs? What does it entail?
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
What needs to be considered with TALENs delivery?
- They are large, repetitive genes
- Thus difficult to deliver via AAV or lentivirus (small space to pack genetic material)
How is delivery of genome modification agents achieved? Advantages over traditional gene therapy?
- 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)
Where do we currently stand with genome modification agents? Are they ready for clinic?
- 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
What are the ethical implications for genome modification? Compare somatic and germline modification.
Transient introduction of genetic material (gene therapy) vs. genome modification (somatic vs. germline):
• 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
• 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)
What are the current hurdles for germline genome modification?
- High chance of off-target effects (specificity too poor atm)
- Disease causing mutations can already be avoided by preimplantation genetic diagnosis during IVF (screened embryo)
Is genome editing in embryos (germline) achievable currently?
- 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
What is antisense technology?
- 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
What are the goals of antisense technology?
Chemically modified bases/backbone to:
- Improve RNA binding
- Protect from nuclease degradation
- Improve entry into cells
- Many different modifications e.g. morpholino antisense oligonucleotide
What are the ways that antisense technology can be used to modulate gene expression?
Which are in the clinic?
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.
- Oligonucleotide designed to bind across start codon, preventing ribosome access and recognition of start codon
How do antisense oligonucleotides modulate splicing? How is this used therapeutically?
- 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
Approach to treat disease is v gene-specific
- Duchenne muscular dystrophy (DMD) = induce exon skipping
- Spinal muscular atrophy (SMA) = exon inclusion
What is duchenne muscular dystrophy (DMD)? Gene responsible?
- 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)
How can DMD be treated with splice modulation?
• 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
What is spinal muscular atrophy (SMA)? Gene responsible?
- 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