Precision Medicine Flashcards
Definition
A situation in which the total level of a gene product (a particular protein) produced by the cell is about half of the normal level and that is not sufficient to permit the cell to function normally
Haploinsufficiency
What are the three common site-direced nucleases that can be used to target specific genes?
Zinc finger nucleases (ZFNs)
Transcription activator-like effector nucleases (TALENs)
RNA-guided engineered nucleases (CRISPR-Cas9)
What levels of pathogenicity and immunogenicity do AAV vectors have?
Not pathogenic and very low immunogenicity
What types of cells can rAAV transduce?
rAAV can transduce both diving and non-diving cells, with stable transgene expression for years in postmitotic tissue
True or False:
AAV vectors have an integration promoting gene
False
AAV vectors lack the integration-promoting gene and therefore only rarely and randomly integrate into the human genome
What are the types of therapeutic gene editing?
Define
Precision medicine
medical care designed to optimize efficiency or therapeutic benefit for particular groups of patients, especially by using genetic or molecular profiling
What are the Ex vivo, In vivo and other general problems for somatic cell editing?
Ex vivo limitations
- Isolation and culture/maintenance of specific cells
- Potential for mutations in cell culture
In vivo limitations
- Targeting the right organ is difficult
- Life span of the cells targeted
Other general problems
- Homology directed repair doesn’t take place in non-dividing cells
Define
Homologous recombination (HR)
a type of genetic recombination in which nucleotide sequences are exchanged between two similar or identical molecules of double-stranded or single-stranded nucleic acids (usually DNA as in cellular organisms but may be also RNA in viruses).
Define
INDELs
Small insertions/deletions of typically 5-20 nucleotides
Define
Zinc-finger nucleases
artificial restriction enzymes generated by fusing a zinc finger DNA-binding domain to a DNA-cleavage domain
Definition
short, synthetic, single-stranded oligodeoxynucleotides that can alter RNA and reduce, restore, or modify protein expression through several distinct mechanisms
Antisense oligonuleotides (ASO)
Definition
restriction enzymes that can be engineered to cut specific sequences of DNA. They are made by fusing a TAL effector DNA-binding domain to a DNA cleavage domain (a nuclease which cuts DNA strands)
TALENs
What are Transcription activator-like effector nucleases (TALENs)?
Series of protein modules that bind individual nucleotides bringing Fok1 nuclease to site (dimer becomes active)
BCL11A (the gene involved in sickle cell anemia) plays an important role in neuron development and B-cell lymphopoiesis so its expression can’t be turned of system-wide.
How do we turn off its production only in RBCs?
Deletion of the BCL11A erythroid enhancer by genome editing results in specific loss of BCL11A expression in erythroid precurors (not in neurons or B-cells). This increases fetal hemoglobin production
Define
TALENs
restriction enzymes that can be engineered to cut specific sequences of DNA. They are made by fusing a TAL effector DNA-binding domain to a DNA cleavage domain (a nuclease which cuts DNA strands)
What are RNA-guided engineered nucleases (CRISPR/Cas9)?
RNA molecule (CRISPR) designed that binds nucleotides and targets Cas9 nuclease to site
Definition
a type of genetic recombination in which nucleotide sequences are exchanged between two similar or identical molecules of double-stranded or single-stranded nucleic acids (usually DNA as in cellular organisms but may be also RNA in viruses).
Homologous recombination (HR)
Define
gRNA
the RNAs that guide the insertion or deletion of uridine residues into mitochondrial mRNAs in kinetoplastid protists in a process known as RNA editing
Define
Inteins
a segment of a protein that is able to excise itself and join the remaining portions (the exteins) with a peptide bond in a process termed protein splicing
Definition
a pathway that repairs double-strand breaks in DNA. It is referred to as “non-homologous” because the break ends are directly ligated without the need for a homologous template
Non-homologous end joining (NHEJ)
Definition
a unique technology that enables geneticists and medical researchers to edit parts of the genome by removing, adding or altering sections of the DNA sequence. It is currently the simplest, most versatile and precise method of genetic manipulation and is therefore causing a buzz in the science world
CRISPR/Cas9
Define
Episome
a genetic element inside some bacterial cells, especially the DNA of some bacteriophages, that can replicate independently of the host and also in association with a chromosome with which it becomes integrated
Define
Antisense oligonuleotides (ASO)
short, synthetic, single-stranded oligodeoxynucleotides that can alter RNA and reduce, restore, or modify protein expression through several distinct mechanisms
What three ways do ASOs exert their effect?
ASOs exert their therapeutic effects in various ways depending on their chemical structure.
- RNA degradation
- Preventing protein translation
- Modifying RNA splicing
Define
Mitochondrial donation
involves removing the nuclear DNA from a patient’s egg containing faulty mitochondria and inserting it into a healthy donor egg, which has had its nuclear DNA removed. This can be done before the egg is fertilised (maternal spindle transfer) or post fertilisation (pronuclear transfer)
Definition
a segment of a protein that is able to excise itself and join the remaining portions (the exteins) with a peptide bond in a process termed protein splicing
Inteins
Definition
a protein which plays a vital role in the immunological defense of certain bacteria against DNA viruses and plasmids and which is heavily utilized in genetic engineering applications
Cas9 nuclease
How can ASOs be used to treat Duchenne muscular dystrophy?
ASO masks the splice site so the splicing machinery moves over this exon to the splice site in the next exon
What is an adeno-associated virus?
A single-stranded, DNA virus that has a “simple” genome packaged in an icosahedral capsid. The genome can be “gutted” for biotech, so that precious cargo space is opened for gene delivery and for safety
What are zinc finger nucleases (ZFNs)?
Series of protein modules that bind 3 nucleotides bringing Fok1 nuclease to site (dimer becomes active)
How can knockout cell/animal models be made using genome editing?
How does pronuclear transfer (PNT) work?
- involves fertilizing an oocyte from an ‘affected’ woman with her partner’s sperm to create a zygote (early embryo).
- The pronuclei (the nuclei of the sperm and egg during fertilization, prior to them fusing) are then removed and placed into another zygote that has been created using a donor oocyte and the partner’s sperm, but which has had the pronuclei removed.
- This embryo then begins to develop and is transferred into the woman as for MST above.
- In PNT the manipulation occurs after fertilization.
What is the genetic cause of Duchenne muscular dystrophy and why can it cause such severe symptoms?
How does altering the fitness of a cell modified through gene editing affect therapeutic effect?
Describe an experimental example of gene drives
How does maternal spindle transfer (MST) occur?
- the mitotic spindle from an oocyte of a woman with mutant mtDNA is transfered into a donor oocyte provided by a healthy donor, which has had its nucleus removed (an enucleated oocyte).
- This oocyte is then fertilized using sperm from the woman’s partner
- this technique leads to the creation of an oocyte with ‘healthy’ mitochondria containing a nucleus obtained from an oocyte that has ‘unhealthy’ mitochondria.
- the manipulation of mitochondria and gametes occurs prior to fertilization.
