Gene Thearapy + Genome editting Flashcards
What causes many diseases
Over 10,000 diseases are caused by mutations in a single gene
Goal of Gene Therapy + Genome editing
To introduce DNA to correct the function of abnormal gene
Gene therapy
Addition of a correct copy of the gene into cells in the target organ or tissue
- Take gene and put in cells
- Introduce a gene into living cells to get correct copy of the gene into the cell (if the disease is caused by a broken one) – give a good copy of gene
Genome editting
Alternation of the genome at a specific location to alter the genetic sequence
- Change gene
- Integrated into genome so daughter cells get the good copy of the gene
***Requires DNA repair – cut genome –> put something in –> repair it
2 main challenges in Gene Thearapy + genome editting
- Gene delievery –> how to get the new peice of DNA into target cells
- Gene editting –> How do you midify the genome where you want it to be modified
Deleiver techniques
- Virus – Lentivirus + retroviral
- There are a lot of viral modes used to deliver the good copy of the gene
- Electroporation
- cell Squeezing
- Nanoparticles (lipids + polymer + gold)
- Blast nano particles into cell
Gene editting techniques
- Transposons
- Designer nucleases
- CRISPR/Cas9
What does Genome editting rely on
Genome editting relies on DNA repair systems
Relies on:
1. NHEJ
2. Homologous repair
Who discovered CRISPR/Cas9
Emmanuelle Charpentier + Jennifer Doudna
One of the biggest advancements since discovering DNA + PCR
CRIPR
First found DNA –> then how to replicate DNA –> NOW how to change it
Idea behind Crispr
Gene therapy
Introduced gene in gene therapy
NOT integrated into the genome – just have plasmid that has the normal copy
Two main challenges in gene therapy
- How do you get DNA into the cells you want to fix
- Technologies to try and deleiver peices of DNA – including a loy of viral modes used to develier the godo copy of gene
- Some technologies use nanopartciles – blast nanopartciles intp the cell
- How do you edite the genome once have DNA in cell – how do you edit genome itself (this is where CRIPSR comes in)
CRISPR (overall)
Cluster Regularly Interspaced Short Palindromic repeats/Cascade Protein 9
Overall – A tol to precisely edit a genome
History of genome editting
Start – David Vetter –> Boy in plastic bubble
- Severe combined Immunodeficiency (SCID) – X-linked recessive
DesilVa –> Idea for gene therapy when David went under gene therapy – took cell out of bone marrow used a viral delivery to present good copy of gene and then infused edited cells back into her
- She is still alive
- She because the 1st human to undergo a gene therapy trial to treat SCID
- her immune system was strengthened = success BUT T-cells didn’t establish a permanent populations – additional transfusions were needed
Set backs in gene therapy
- Viral delivery systems gives the good copy of the gene BUT not into specific location in the body – don’t know where the genes are inserted
- Viral delivery systems inserted the good copy of the gene into random part of the genome (our genomes have a lot of non-coding regions)
- Might inject the good copy of the gene into a region of genome that prevents us from getting cancer
***Did trial – 5 out of 9 kinds in a SCID gene in the trails developed luekemia –> the good copy of the SCID gene seemed to be targeting region of the genome that caused cancer
SCID Setbacks (gene therapy set back)
Jesse Gaklisnger – instead of taking cells out of the body they wanted to inject straight into the liver – he had a huge immune response and dies
- Jesse = mosaic of cells – some cells contained X-linked mutation causing liver disease and some did not
- He was injected with Adenoviral vector
- He suffered a massive immune response and dies within 4 days
- His parents = seemed unaware of the big risk with the technology
Everyone got upset –> work halted all clinical trials
AFTER – US and world halted all gene therapy clinical trails for 20 years
Research after Jesse’s death
Focused on identifying novel vectors for delivering gene therapeutic agents using model organisms
- Took 20 years to get better deleivery systems + figure out editting THEn Crispr was discovered
(Looked at DNA viriuses + RNA virsus + Lipsosomes + Cationic polymers + Dendrimers + Cell penetrating peptides)
Improving techniques for genome editting (Before CRISPR)
Zinc finger proteins and TALENs (Transcription Activator-like Effector nucleases) - bind to specific sequences of DNA and clease the dsDNA
To guide this cut the proteins must be engineered to bind to specific sequneces in the Genome
Aaron Klug
Invenyed gene editting using Zinc fingers
Worked with Rosalin Franklin on Viral Structures
Nobel Prize for invention of Tomography (CT scans)
Challenges with ZN fingers + Talens
- Time – Need to engineer and purify a specific bvarinet of the ZN/TALEN
- 2 - 4 weeks in a reallly good lab to syntehsize + need longer to test and preform quality control
- Efficiency – Not all ZN and TALEN engineered actually work
- About 1/2 of TALENS have good cleavage activity
- Efficacy – Both have a lot of non-specific binding
CRISPR is a…
Ribozyme – enzyme –> It is a ribozyme that can be engirneered to cleave a specific target of DNA
What is included with CRIPSR
Need Cas 9 protein + guide RNA –> Guide RNA goes to region of the genome and cites the DNA at a precice location
Pros of CRIPSR
NO need to purofy proteins = quick
High efficiency (>90% of cells provided with CRIPSR/Cas9 will be eidtted)
High Efficacy (BUT off target can still occur)
History of CRISPR
Lots of work indiscivering CRIPSR
What was Emmanuelle studying
Emanielle – she was NOT trying to make a tool to edit genomes she was trying to understand how do bacterial cells become immune
SHE knew you could infect bacteria with viral then clone the cells and expose the generation later to virus – bacteria remembered they had encountered the virus – they remembered the virus
Looking at how daughter cells remember the virus
She was working on CRIPSR system –> Contacts Jeneffer
How do bacterial cells remember a virus
Observation – Bacteria could be infected then cured of virus BUT the daughter cells were resistent to new exposures
Question – how does bacteria remeber that it has previously seen a virus
System: Have a virus that injects DNA/RNA into the cell – when injected the RNA/DNA is recongized by proteins (cas system) – it is inserted into the genome of the bacteria and is flanked by repetative sequneces
- Each time bectaeria is injected with virus it insertes the DNA/RNA into the genome (inserted into the CRISPR region of bacterial genome)
ONCE inserted into the genome – NOW have a locus in the bacterial genome that had information about the virus
- The information can be inherited by daughter cells
The part inserted into the genome = transcribed to create RNA – then cleave to make little RNAs
- The little RNAs bind to Cas proteins – Cas + RNA is what can get recognized – NOW if virus a virus that the bacteria had encountered before injects its genome again then the little RNAs + Cas can bind to it –> forms dsDNA
Cells = hate dsDNA – they are targeted for degradation = get rid of virus
Where is virus DNA inserted in bacteria genome
Using the Cas System it is inserted into the CRISPR region
Function of Little RNAs
The little RNAs bind to Cas proteins – Cas + RNA is what can get recognized – NOW if virus a virus that the bacteria had encountered before injects its genome again then the little RNAs + Cas can bind to it –> forms dsDNA
dsDNA in cells
Cells = hate dsDNA – they are targeted for degradation = get rid of virus
Different CRISPR systems
Cells have different CRISPR systems but they all have the same basic steps
Basic Steps of CRISPR pathways
- Aquistitions – A portion of the forgein DNA is insterted into the CRISPR array (region)
- Expression – The CRISPR region is transcribed and cleaned to form many small crRNAs
- Interferences – The crRNAs contain homologies to infecting agents
- IF the crRNAS/Cas complex recongznies an infection DNA or RNA = it will bind to it and target it for degredation
What was Jenefier working on
RNAi
RNAi
ONce have transcripts – cam lower the amount of protein from transcript if detsroy some of the transcript
- Cells have evoloved RNAi for gene regulation
Can destroy transcripys if can get thngs to be complementary to RNA to get dsRNA to be degraded in the cell
***Researchers have adaoted RNAi to control gene expression in the lab
Jenifer + Emmanuelle
Their research collided – together they disovered CRIPSR system
Their discivery – a Second RNA (tracerRNA) was also required – when Cas9/tracer/crRNA were together the complex would find the viral DNA sequences and cleave the dsDNA in a precise location
Realized that if made a synthetic RNA with tracer region + region that caise cas to cut DNA (put in any sequnece) – delover cas system to any place in genome and cut the genome right there at a specific target sequence
- Realized that by fusing the tracr and crRNA – create a Guide RNA + replacing viral sequence with any target sequence
- Realized they could cleave dsDNA precisley at any location on the genome
What was Emmanuele working with
Emmanual was working on Cas system with only 1 Cas protein (very simple) - she chose system with only 1 cas protein → She found cas protein + have RNA (have tracer RNA that enables the cas protein to find its target)
- She chose to work with the simplest CRIPSR system – only 1 Cas that binds to crRNAs
What is needed in CRIPSR seystem
IN bacterial cell = have cas 9 protein + crRNA (peice of RNA expressed from the CRISPR locus) + Tracer RNA
What does tracer RNA do
Forms dsRNA that is protected by cas9 complex – THIS is what you need to recognize RNA and chop it
What does tracr guide RNA need
Needs to find a sequence in the genome –> needs the PAM sequence
- Need to have the PAM
What is needed in genome for CRIPSR to work
Need Target + PAM sequence
What is in Guide RNA
Have tracr + crRNAs
CRISPR off target effects
CRISPR = highly effective at targeting the correct sequence BUT will sometimes cut other off target regions of the genome
PAM sequence
Any BP and 2 G (5’ NGG 3’)
Cas goes to genome and scans the genome first looking for PAM sequenece –> Genome has many NGG sequences in genome –> Any time sees GG it will look next to see if the DNA is the same DNA that it has – of not it moves and looks for another PAm sequnce + looks for complementary sequnece
What does Cas look for in genome
Cas goes to genome and scans the genome first looking for PAM sequenece –> Genome has many NGG sequences in genome –> Any time sees GG it will look next to see if the DNA is the same DNA that it has – of not it moves and looks for another PAM sequnce + looks for complementary sequnece
Cas looking for PAM
Cas 9 looking for PAM and looking to see if the guide matches the sequnece on the genome –> When finds PAM – Cas unwinds the template DNA to see if the DNA is complementary to the guide that it is carrying – IF the guiode is complementary to that region target binds to guide RNA –> get DNA/RNA hybrid = cas 9 has shift in protein and cas 9 cuts DNA –> leaves a peice of the genome cut a few BP down from PAM sequnce
Overall: CRIPSR finds region on genome and cuts it
How do cells fix cut DNA
- Homologous recombination
- NHEJ
Rely on these systems to fix the damage that CRISPR caused
- CRISPR cuts where we want it to and these fix it
NHEJ
Ligates two blunt ends together
- Sloppy porcess and sometimes chews the DNA to get blunt ends (because most ds breaks are ragged)
- NHEJ might not check if need to get DNA = will just cleave and hook back together
Issues in NHEJ
Often get small insertion + Deletion – don’t always get back same thing
NHEJ – will take out a few nucleoties = can change coding sequneces = get non-functional protein or might prevent the protein from being made
Homologous Directed repair + CRISPR
Instead of only add CRIPSR to cells might also want to get gene –> CRIPSR can make the cut AND CAN add a tenplate that promotes homologous recombination = can get healthy DNA into cell
- Allows cell to use homologous recombination to inster the gene into genome
CRIPSR cutting other PAM
GET off-target effects
There are lots of PAM sequneces – CRISPR might sometiems cut other PAM – if this is fixed and mutation occurs while bieng fixed = can get off target effects
If hits important gene this might matter = can’t ignore off targets
CRISPR signature
Does not leave a big signature when done
Simplicity of CRISPR
SO simple – of get plamsid –> Plasmid has cas system + Guide RNA + Can add in homologous sequnce to direct the repair
- All you need ius a plasmid that conatined the gene encoding cas 9 and a guide RNA (and maybe a second plasmid that has a donor DNA for Homologous repair)
- Just introduce the plasmid into a cell and let the cell do the rest of the work
CRISPR + Healthcare
CRISPR will revolutionize healthcare
Overall: Remove cells from body –> change them –> add them back in
Example:
Victoria Grey – gene therapy
- She had sickle cell anemia – they took out cells –> changed via CRISPR –> Infused cells back –> worked very well
- She was the first attenmpt to use gene editting technique CRIPSR to treat a genetic disorder in US
Used Somatic cells = not mieosis
Is a CURE NOT a fix – not something you have to repeat
***There are many clincal trials now – all single gene mutatins + all in cells you can take to
CRISPR in next genertaion
If want to affect next generation = need to affect germ line cells + do in mieosis
Somatic = not affecting kids
CRISPR in brain
Harder to do in brains because can’t take out cells from brain BUT maybe can do things that are blood born
What did Jenifer see when found CRIPSR
She saw potential negitive effects it could have –> Immediately upon discovery she led a world wide ethics conferance to discuss the ethics of CRISPR
- Made a worldwide agreement that they will not use CRISPR to edit germline cells
She said we shoudl pause
There was a world wide agreement that CRISPR should NOT be used to modify germ line cells (following Nuremberg code principals – no human testing without informed consent)
Technology + policy
Technology proceeds policy – need to think about what we need tp consider before using the technology
He Jianki
Announced that he had created twins that were CRISPR editted
- Claims of CRISPR being used to edit the genomes of twin girls
- he was very proud
- Reponse was bad
Issue in editting embryoes
How would an embryo give conset
Respnse to He jianki
BAD – response from scientific community was immediate and negitive
Chinease govermnment suspended his reserach + he was fired from SUSTech + He was sentances to 3 years in jail and fined 4.5 Million US$
What Allele from Jianki edit
Gene called CCR5 – known varaition of gene that leads to increased resistence to HIV
People with variation (deleted 32 Amino Acvids) – HIV binds to proteins –> needed for entery into cell
Varient - chops off binding domains = reduces ability of HIV to infect cells
He Jianki claimed to use CRISPR to aklter the CCR5 gene in the twins
Sequneces from He Jianki
He presented his work he cliamed to use CRISPR to alter the CCR5 gene in twins
Sequences:
Have PAM
delta 32 –> have PAM and then have a delation (have deletion that is frame of the protein
Lulu –> Missing PAM + other nucleotides – leads to protein different than d32 vareint
- Changes reading frame
Nana –> has 4 BP deletion after PAM – chnages sequences
- She is heterozygous – One chromosome with change in reading frame = get different protein
Twin girl sequneces are different AND the sequences are NOT the delta 32 varaient – not that same varainet that exists in nature
- The girls are missing diferent Base pairs
***Data has not been varified
Twin girl sequneces
Twin girl sequneces are different AND the sequences are NOT the delta 32 varaient – not that same varainet that exists in nature
- Girls are missing different base pairs
What type of repair was involoved in He Jianki’s work
NHEJ – because have random deletions + inserations – hallmark of NHEJ
- Don’t know what sequence you will get with NHEJ
Appears that he Jianki relied on NHEJ
Homologous Direct repair making new things
Shouldn’t create new things – should copy what sis there and make 2 copies – will mostly copy identical sequnece
Update on Twins
We do not know anything about twins now
What happened after Jianki’s paper was published
ALL retracted immediately
We can only go on what he presented – there is no peer reviewed paper
AFTER = had a new meeting about use of CRISPR and still concluded not to use un germline cells
