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307BMS Gene Therapy and Editing short
Terms in this set (61)
Define gene therapy
Any procedure intended to treat or alleviate disease by genetically modifying the cells of a patient
The simplest form of gene therapy is _____________________________ in which the ________________ copy of a gene is introduced to _________________________.
The simplest form of gene therapy is gene replacement in which the wildtype copy of a gene is introduced to restore function.
Germ-line therapy involves modification of what cells?
gametes, zygote or early embryo (before cell becomes differentiated)
Germ-line therapy involves what kind of modification and to what cells?
permanent alteration to ALL body cells
Somatic gene therapy involves modification of what cells?
somatic (body) cells
The modification in somatic gene therapy __________ be passed on to the offspring and is ________________________________________
The modification in somatic gene therapy cannot be passed on to the offspring and is restricted to targeted cell type
Modification can be done (2)
Modification can be done ex vivo and in vivo
Ex vivo: gene is inserted into explanted or cultured cells, which are then transplanted into the patient
In vivo: gene is administered directly to the patient
Direct delivery in vivo involves the theraputic transgene being __________________________________________. there are _________________________ to ensure transgene __________________________________
Direct delivery in vivo involves the theraputic transgene being packaged into a delivery vechile. there are different vechicles to ensure transgene arrives at desired tissue
Episomal gene is when the transgene is ...
Episomal gene is when the transgene is in the cell but not part of the genome.
problem with episomal gene transfer rather than integrated gene transfer?
When cell divided the episomal gene may not be replicated, or may not segregate into both daughter cells. Therefore transgene expression may be more temporary
Integrated gene transfer is the ideal situation although ...
Integrated gene transfer is the ideal situation although there are some cases when episomal is desired
___________ can be used in gene therapy as ___________ because they _________________________________________________________________. Therefore they can be used to ______________________________________________________ and create desired trait
Viruses can be used in gene therapy as vectors because they replicate by inserting their DNA into host cell. Therefore they can be used to insert genes that encode for desired protein and create desired trait
Drawbacks of viral vectors (4)
Host Immune response
Tissue Specificity (virus must be specific to cell type you want to modify, cells must express receptors on cell surface to allow virus to infect desired cells)
Non-integrative (gene expression is short lived)
Integrative (might cause damage if inserted in middle of functional gene, in oncogene could cause tumourgenesis) insertion is random and not controlled - insertional mutagenesis
Adenoviral vectors ________ insert into ______________. Are ____________, lack __________________ and ______________________________________________________ so they are not ideal
Adenoviral vectors do not insert into genome. Are transient, lack specificity and hosts can mount a strong immune response so they are not ideal
Adeno-associated viral vectors are ...
Adeno-associated viral vectors are modified versions of adenoviral vectors.
Adeno-associated viral vectors transduce what kind of cells?
non-dividing cells, in muscle, liver, retina, brain and lungs
Adeno-associated viral vectors are mostly ___________, but _________________________________ _______________
Adeno-associated viral vectors are mostly episomal, but can integrate into host genome
Do adeno-associated viral vectors induce an immune response?
yes but it is milder than adenoviral vectors with a safer response
Problem with adeno-associated viral vectors
can only carry small amounts of DNA (up to 4.2kb)
Retroviruses can carry much bigger genes. the maximum insert size is ___________
Retroviruses can carry much bigger genes. the maximum insert size is 7-7.5kb
Retroviruses create ___________ copies from the viral RNA genome
maximum insert = ___________
Retroviruses create cDNA copies from the viral RNA genome
Integrate into the human genome
maximum insert = 7-7.5kb
Pros of retroviruses (2)
can carry much bigger genes
preexisting host immunity is unlikely
Cons of retroviruses (2)
can only transduce dividing cells (e.g. bone marrow)
cannot control site of integration (insertional mutagensis)
Non viral vectors such as ___________________ where the ____________________ helps ____________ with cell membrane
Non viral vectors such as liposomes where the lipid membrane helps fusion with cell membrane
Advantages of non-viral vectors
no immune response
can be made tissue specific
Disadvantages of non-viral vectors
cells will not actively take them up, need delivery methods
non-viral vector delivery method
Gene gun method. DNA coated on to ...
heavy metal particles
Gene gun problems:
relatively few cells take up DNA (only cells in immediate area exposed to particle)
High level of gene expression is short-lived
Gene editing tools needed:
A way to target the DNA sequence (signpost)
A way to cut the DNA at target sequence (nuclease)
DNA template to copy correct sequence (heterozygote can use normal copy to correct mutated version. Or exogenous DNA template)
DNA repair mechanisms
Genome editing with engineered nucleases: (3)
Zinc Finger nucleases (ZFNs)
TALE Nucleases (TALENs)
CRISPR RNA-guided Nucleases
Gene editing tools have 2 features:
1) Recognise specific DNA sequences
2) Cut DNA 'Nuclease' then a scar is left behind
Problem with TALE Nucleases
Very expensive - have to engineer proteins for each specific DNA sequence
How is the cut DNA repaired?
Endogenous repair mechanisms
Endogenous repair mechanisms (2)
Non-homologous end-joining (NHEJ)
Homology-directed repair (HDR)
Non-homologous end joining
Cleaved ends of DNA are directly re-ligated.
Indels introduced - results in a loss of information at the site of repair.
non-homoglous end joining is good when...
knocking out gene function, introduction of INDEL does not matter
the cellular DNA repair pathway that uses homologous DNA sequence to repair break by copying template
When is homology-directly repair used? (2)
Mutation correction or modification
Problems with endogenous repair mechanisms
Need to design new binding proteins for each sequence
Need better targeting mechanisms, which is where __________ comes in
Need better targeting mechanisms, which is where CRISPR comes in
CRISPR stands for...
Clustered Regularly Interspaced Short Palindromic Repeats
a collection of RNA-guided nucleases that tells Cas9 exactly where to cut
CRISPR-Cas system is a form of acquired immunity found in bacteria. Guide RNA directs the Cas9 protein to a target site. Creating guide RNA is very simple
CRISPR-Cas system is a form of ____________________ found in ______________. ____________________ directs the Cas9 protein to a target site. Creating _______ __________ is very simple
Pros of CRISPR/Cas9
Can be multiplexed (target several genes at once)
Cheap - only need to synthesise guide RNA
Gene deletion/knockout using _________ could be used to delete segments of a gene. For example:
___________________ in DMD gene to restore _________________
Deletion of r_________________________________________ in Huntingtons
Deletion of entire ____________ gene to protect ________________________from ______ infection (___________ enters lymphocytes by binding to __________ receptors)
Gene deletion/knockout using NHEJ could be used to delete segments of a gene. For example:
Exon deletion in DMD gene to restore reading frame
Deletion of repeat expansion of HTT gene in Huntingtons
Deletion of entire CCR5 gene to protect blood cell lineages from HIV infection (HIV enters lymphocytes by binding to CCR5 receptors
Gene addition using HDR. Pros over retroviruses (2)
More site specific
low risk of insertional mutatgensis/inactivation
Example of early-phase clinical trial for gene addition
Beta-thalassaemia by addition of functional copy of beta-globin gene
Challenges with gene addition (5)
Will enough cell have the added gene.
Will added gene make enough product to correct the disease phenotype
Is the clinical benefit short lived or durable (divided cells must also carry edit)
Does the gene addition change the function of any other genes in genome - unknown consequences
Cell within major organs are harder to edit as need to introduce CRISPR/Cas9
Gene editing of point mutations promises a permanent repair of disease causing mutation. Requires ___________________to copy correct sequence (not an issue in _____________________________________________________ __________________, otherwise ____________________________ must be provided)
Benefiical change is restricted to _________________ (_________________________)
Gene editing of point mutations promises a permanent repair of disease causing mutation. Requires template DNA to copy correct sequence (not an issue in heterozygote as wildtype copy on other chromosome, otherwise exogenous template DNA must be provided)
Benefiical change is restricted to gene of interest (highly specific)
Challenges of specific editing:
Point mutation reliant on ________but ___________ is _____________________ but __________________________
Cannot guarentee ____________________________________________________________
Editing may ________________________________________________________
Challenges of specific editing:
Point mutation reliant on HDR but NHCG is more efficient but introduces lots of IDENLs.
Cannot guarentee high frequency of edited cells with HDR
Editing may not be specific enough (off target editing)
Mismatches between DNA sequence and guideRNA. Cas9 cuts in wrong place. Could be deleterious/damaging if happen in vivo
To prevent off-target editing ____________________________________________________ is needed to check for _____________________
To prevent off-target editing extensive sequencing of edited DNA is needed to check for off-target edits
Delivery of gene editing nucleases is necessary to deliver ___________ and ______________________ (and __________________) into host cells so it is expressed in cells you want
Delivery of gene editing nucleases is necessary to deliver nuclease and DNA-binding system (and template DNA) into host cells so it is expressed in cells you want
Delivery of gene editing nucleases uses...
Viral vector examples (2)
pro of AAV
Safer (less immune response)
con of AAV
small, carries up to 4.7kb and cas9 cDNA is 4.2 so limited capacity and not enough space for DNA template
Pro of Retrovirus
con of retrovirus
can trigger host immune response
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