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)
What is needed to target a sequence of nucleotides with CRISPR/Cas9?
A complementary gRNA to the sequence and a PAM sequence near the site of the double-stranded break
Definition
artificial restriction enzymes generated by fusing a zinc finger DNA-binding domain to a DNA-cleavage domain
Zinc-finger nucleases
Definition
the RNAs that guide the insertion or deletion of uridine residues into mitochondrial mRNAs in kinetoplastid protists in a process known as RNA editing
gRNA
Define
CRISPR/Cas9
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
Define
Pronuclear transfer (PNT)
the transfer of the pronuclei (the still separate maternal and parental nuclear DNA from the fertilised egg) from the affected zygote to the donor zygote
Spinal muscular atrophy is caused by loss-of-function mutations in a single gene: SMN1. An SMN2 gene is present but this only generates ~10-20% of the normal levels of function of SMN protein. SMN2 usually favours skipping of exon 7, resulting in an unstable protein.
How does the ASO SPINRAZA aim to treat SMA?
SPINRAZA binds to a specific sequence in the intron downstream of exon 7 of the SMN2 transcript. This prevents splicing of exon 7 resulting in a full-length, stable SMN protein
What is an antisense oligonucleotide (ASO)?
Short, single-stranded sequence of DNA or RNA designed to target specific RNAs to modify gene expression
Definition
Small insertions/deletions of typically 5-20 nucleotides
INDELs
What are the disadvantages of CRISPRs over ZFN/TALENs?
- Overall recognition sequence is small, making off-target effects a potential issue
What is a current idea on how to split Cas9 to fit into AAVs?
Inteins, “protein intron” that splice out autocatalytically from host polypeptides to generate functional proteins, would allow smaller sections of Cas9 to be packaged in seperate AAVs and rejoin automatically into the full, functional protein
Why are AAV genetic modifications not germ-line mutations?
When the virus enters the nucleus, it usually forms an episome, which replicates independently of the nucleus and is therefore not passed on
Define
Non-homologous end joining (NHEJ)
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
What are the two main mitochondrial donation approaches?
Materal spindle transfer (MST)
Pronuclear Transfer (PNT)
What is genome editing being used for?
Basic research: making cell and animal models (knocking out/modifying genes)
Screens to identify new therapeutic targets
Gene drives
Potential therapeutics to treat disease
How have AAVs been used to treat Spinal muscular atophy?
AAVs containing a transgene encoding the human SMN protein, under the control of a cytomegalovirus enhancer and a chicken-B-actin hybrid promoter, enables the producting of SMN protein
Definition
medical care designed to optimize efficiency or therapeutic benefit for particular groups of patients, especially by using genetic or molecular profiling
Precision medicine
What are the advantages of CRISPRs over ZFN/TALENs?
- DNA binding is by classic base pairing rules (not protein:DNA interactions)
- Making the custon gRNA used by CRISPRs is much easier, cheaper, quicker
How can knockout cell/animal models be made using genome editing?
Definition
small viruses that infect humans and some other primate species. They are not currently known to causes disease and several additional features make them an attractive candidate for creating viral vectors for gene therapy
Adeno-associated virus (AAV)
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.
Define
CCR5
A protein on the surface of certain immune system cells, including CD4 T lymphocytes (CD4 cells). It can act as a coreceptor (a second receptor binding site) for HIV when the virus enters a host cell
Definition
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
Episome
Definition
the transfer of the pronuclei (the still separate maternal and parental nuclear DNA from the fertilised egg) from the affected zygote to the donor zygote
Pronuclear transfer (PNT)
What is the genetic cause of Duchenne muscular dystrophy and why can it cause such severe symptoms?
Definition
a 2–6-base pair DNA sequence immediately following the DNA sequence targeted by the Cas9 nuclease in the CRISPR bacterial adaptive immune system
Protospacer adjacent motif (PAM)
What is the aim of precision medicine and how does it work?
Precision/personalised meicine aims to determine which medical treatments will work best for each patient. Precision medicine uses medical interventions to alter molecular mechanisms, often genetic, that cause disease or influence a patient’s response to certain treatments
Define
Adeno-associated virus (AAV)
small viruses that infect humans and some other primate species. They are not currently known to causes disease and several additional features make them an attractive candidate for creating viral vectors for gene therapy
Define
Protospacer adjacent motif (PAM)
a 2–6-base pair DNA sequence immediately following the DNA sequence targeted by the Cas9 nuclease in the CRISPR bacterial adaptive immune system
How are ZFN used to manage HIV?
ZFN create a double straded break in the CCR5 gene resulting in an indel and no CCR5 being produced. Since CCR5 is not produced, researchers theorised that the virus was unable to infect and destroy the altered cells enabling modified T-cells to proliferate
How does altering the fitness of a cell modified through gene editing affect therapeutic effect?
Define
Heteroplasmy
the presence of more than one type of organellar genome (mitochondrial DNA or plastid DNA) within a cell or individual
Define
Maternal spindle transfer (MST)
a technique in which the spindle shaped group of chromosomes containing the mother’s nuclear DNA, known as the ‘maternal spindle’, is extracted from one of the mother’s eggs (oocytes) and transferred to an unfertilised donor egg from which the maternal spindle has been removed and that contains healthy mtDNA
What is the limiting factor of an AAV?
Size of insertion: only small amounts of genetic material can fit in the virus
Describe an experimental example of gene drives
How can gene editing treat sickle cell anemia?
CRISPR induced disruption of the expression of BCL11A prevents the switching of adult hemoglobin from gamma to beta-globin. Gamma-globin is resistant to polymerisation caused by sickle cell anemia so symptoms are reduced
Why is in vivo use of CRISPR/Cas9 proving to be difficult?
Both the gRNA and the Cas9 protein must fit into the the viral vector in order to target a gene in a cell. These genes are too large to fit into a virus
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.
What are the two types of double-strand breaks?
Non-homologous end joining
Homologous recombination
Define
Haploinsufficiency
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
Definition
a technique in which the spindle shaped group of chromosomes containing the mother’s nuclear DNA, known as the ‘maternal spindle’, is extracted from one of the mother’s eggs (oocytes) and transferred to an unfertilised donor egg from which the maternal spindle has been removed and that contains healthy mtDNA
Maternal spindle transfer (MST)
Definition
A protein on the surface of certain immune system cells, including CD4 T lymphocytes (CD4 cells). It can act as a coreceptor (a second receptor binding site) for HIV when the virus enters a host cell
CCR5
Define
Cas9 nuclease
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
Definition
the presence of more than one type of organellar genome (mitochondrial DNA or plastid DNA) within a cell or individual
Heteroplasmy
Definition
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)
Mitochondrial donation
