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
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
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
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
