GEN 10: New and Future Developments Flashcards
1
Q
Observe the learning outcomes of this session
A
2
Q
What are the four eras of the development of genetics and genomics?
Include some dates
A
3
Q
What is the current era of genetic and genomics doing?
A
- it features new methods for ‘reading’ and ‘writing’ the genome
- Increasingly fast and affordable DNA and RNA sequencing methods leading to huge expanding and readily accessible ‘omics’ databases.
- Increasingly efficient, precise and versatile methods for genetically modifying cells and organisms
4
Q
What is a new ‘third generation’ sequencing method that follows next generation sequencing (NGS)?
What is the significance of this?
A
- long read sequencing
- these methods focus on sequencing the genome from small amounts of DNA using much longer sequencing reads (10 - 100 kb or more)
- significance:
- expected to soon fill the remaining gaps in the otherwise complete human genome sequence
- permit other genomes of similar or greater complexity to be fully sequenced
- e.g. many agriculturally important plants have large, complex genomes that are being sequenced
- complete genome sequences will facilitate genome engineering projects designed to improve human health (crops with increased nutritional content, edible vaccines)
5
Q
What new method allows the genomes or transcriptomes of individual cells to be sequenced?
Why was this not able to be done before?
A
- before, any genome or transcriptome sequencing required a relatively large amount of DNA
- single-cell RNA-seq is revealing unprecedented insight into the cellular hierarchies existing within individual tissues
- previously unknown cell types existing within organs have been discovered
- see image for an example in the brain
6
Q
Summarise the uses of CRISPR/Cas9 for gene editing
A
7
Q
What are some limitations of CRISPR-based methods?
A
- specificity:
- Cas9 may occasionally cut DNA at off-target sites
- safety:
- even on-target cuts can damage the genome unpredictably
- e.g. by promoting chromosomal translocations
- efficiency:
- homology-directed repair-based edits are usually swamped by NHEJ-based edits
- versatility:
- editing is restricted to target loci with an adjacent PAM
8
Q
Discuss the limitation of specificity in CRISPR/Cas9 gene editing and how it can be addressed?
A
- if a gRNA binds to an off-target site in the genome, even with a small number of mismatches, Cas9 may generate double-strand breaks (DSBs) where they are not wanted
- to minimise this, online tools should be used to search for genomic sequences with similarity to each gRNA and select only those with the fewest significantly similar sequences in the genome
- factors concerning gRNA sequences that can influence off-target cleavage:
- the GC-content of the gRNA:DNA heteroduplex
- high GC content correlated with higher specificity in some systems, while in otehrs there is an optimum GC content
- the position of gRNA:DNA mismatches relative to the PAM
- effects are stronger the closer residues are to the PAM
- the chromatin status of potential genomic targets
- the sequence of the scaffold RNA
- extra dsRNA helices in the scaffold regions increases specificity
- Increased specificity is thought to reflect a reduced stability of the R-loop at off-target sites compared to the on-target site (R-loop is the unwound region of target DNA bound by the gRNA)
9
Q
Discuss the limitation of safety in CRISPR/Cas9 gene editing and how it can be addressed?
A
- in addition to small indels or knock-ins DSBs can occasionally induce large (several kb) insertion or deletions, and gross chromosomal rearrangements, such as translocations
- these likely depend on an alternative NHEJ-based pathway (alt-NHEJ)
- identifying and temporarily suppressing an alt-NHEJ pathway component may therefore be a way to minimise this
- the best safeguard to probably to avoid making DSBs altogether
- Base Editing is a way to fuse catalytically inactive or dead Cas9 (dCas9) to a base-editor, such as cytosine deaminase, which then edits a CG base-pair at the target site to AT
- in this way, DNA bases can be edited as needed in specific experiments, without DSBs being introduced
10
Q
Discuss the limitation of efficiency in CRISPR/Cas9 gene editing and how it can be addressed?
A
- on one approach to facilitating homology-directed repair (HDR), temporary inhibition of NHEJ is useful
- i.e. inhibiting NHEJ transiently, only while CRISPR is present
- HDR and NHEJ are thought to compete with each other, so inhibiting NHEJ should channel DSBs into the HDR pathway
11
Q
Discuss the limitation of versatility in CRISPR/Cas9 gene editing and how it can be addressed?
A
- most CRISPR applications to date have employed Cas9 from S. pyogenes
- but CRISPR-Cas systems from other bacteria and archaea have been characterised
- meaning that the choice of CRISPR targets will be less restricted
- see image for different systems
12
Q
Match the problem with the solution
A
13
Q
What is gene therapy?
A
- gene therapy involved introducing a therapeutic gene, or other genetic change, into the relevant patient tissue in a way that can be stably maintained, preferably for the lifetime of the patient
- it offers an enticing possibility that a one-off treatment could provide life-long benefits, or even cures, for a wide range of diseases
14
Q
What kind of diseases could gene therapy help to treat or c-
A
- cancer:
- genetic mutations that promote tumour growth could be targeted
- monogenic diseases:
- when a single gene is known to have a mutation that causes a disease, gene therapy offers an attractive way of correcting this
- viral infections
15
Q
How can therapeutic cells be delivered into cells?
A
- mostly viral vectors:
- efficient delivery of a therapeutic gene into a cell
- electroporation:
- not as efficient as viral delivery and it also cannot be used for in vivo delivery in patients
- it can be used to deliver DNA to patients’ cells ex vivo
16
Q
It is essential that gene therapy is delivered into the nucleus of the cell.
Is it necessary that the DNA integrates with the genome of the cell?
A
- sometimes
- for some cell types, it is important for the DNA to integrate into the genome of the cell, but not for others
17
Q
There are some factors to think about with integrating therapeutic genes into a host genome
Which is not a likely issue?
A
Not a concern:
- rupture of the cell membrane during treatment:
- when gene therapy is delivered via viral vectors, they are experts at entering the cells and are not likely to rupture the cell membrane in the process
Concerns:
- unwanted immune response:
- the body may raise an unwanted immune response to the viral vector used
- genome damage:
- could cause genome damage or off-target effects
18
Q
Give examples of cells that do not need integrating into the genome to be expressed
A
- non-dividing cells:
- e.g. nerves
- they must be delivered to the nucleus but no need to be integrated into the genome
19
Q
Give examples of cells that do need to be integrated into the genome to be expressed
A
- target tissues that turn over rapidly
- e.g. blood
- gene therapy must integrate inot the genome of stem cells, so that all new cells have the corrected genotype
20
Q
Give an example of a successful gene therapy related to haematopoietic stem cells (HSCs)
A
- gene therapy altered the haematopoietic stem cells (HSCs) of patients with X-linked severe combined immunodeficiency (XI-SCID)
21
Q
How is XI-SCID treated with gene therapy?
A
- HSCs are extracted from the patient and a therapeutic strategy known as cell-based delivery is used
- see image
- patients’ HSCs were infected with a retroviral vector stripped of its normal genes, but carrying the cDNA encoding IL2Rg, the gene lacking in XI-SCID
- this retroviral vector can enter the patients’ cells and integrate its genome, including the IL2Rg gene, allowing for long-term expression
- most XI-SCID patients treated with gene therapy developed full T-cell immunity and live relatively normal lives
- however, a small number developed T-cell leukaemia
22
Q
Describe two gene delivery strategies for gene therapy
A
- direct delivery
- cell-based delivery
23
Q
Which do you think is the most likely cause of leukaemia in Xl-SCID patients treated with gene therapy?
How could this be mitigated?
A
- integration of the retroviral genome resulted in oncogenesis
- Examination of the malignant cells showed that, in all cases, the retrovirus had integrated its genome close to a known proto-oncogene
- This gene (LMO2) had already been implicated in the generation of T-cell leukaemia as a result of chromosomal translocations that cause it to be upregulated
- Retroviral genomes contain long terminal repeats (LTRs) with strong enhancer elements that can upregulated host genes close to their integration sites.
- More recent trials using vectors that lack LTR enhancers are encouraging
- Nevertheless, uncontrolled genomic integration of vector DNA remains a concern for this and many other gene therapy studies.
24
Q
What is targeted gene therapy?
A
- the use of gene editing to correct or modify an existing gene in patients’ cells
- this distinguishes from the non-targeted introduction of an additional gene into the genome
25
What are some advantages of targeted gene therapy?
- safety:
- there is a reduced chance of random gene insertion that could potentially lead to oncogene activation
- efficacy:
- a corrected disease mutation is more likely to restore normal expression levels than a randomly integrated extra gene
- versatility:
- genes can be edited in specific ways
- e.g. in dominant genes, gene inactivation would be therapeutic
26
What is personalised medicine?
What are the benefits of it?
- personalised medicine aims to move away from treating all patients with a particular condition in the same way
- see figure for benefits
27
What was the 100,000 Genomes Project?
- a project by the NHS to sequence 100,000 human genomes of patients with cancer or rare diseases
28
What is pharmacogenomics?
- using the genetic information to ensure the right drugs are prescribed at the right time and at the right dosage
29
Why is pharmacogenomics so important?
- The NHS drugs budget is second only to its staff budget.
- 90% of drugs only work in 30–50% of the population.
- 6.5% of hospital admissions are due to adverse drug reactions.
30
How is personalised medicine and pharmacogenomics particularly relevant for cancer?
- every tumour is genetically unique
- e.g. in lung cancer, if the tumour tests positive for a mutated form of the *EGFR-TK* gene, it is more likely to respond to particular drugs (e.g. gefitinib)
- more work is needed to determine:
- Which mutations affect sensitivity to which drug.
- Which mutational combinations determine sensitivity to a drug
31
Give an example of why personalised treatment for cancer does not guarantee success
- in melanoma, where tumours with activating mutations in the *BRAF* gene responding well to treatment with B-raf inhibitors often become resistant
- More genomic research is needed to characterise how tumours evolve to become drug-resistant and provide more scope for developing successful personalised treatments
32
Define risk when it comes to developing a disease
- the expected probability that a patient will develop a disease within a given time interval
33
What is screening for diseases?
- the testing of apparently healthy individuals to identify those at greatest risk of developing a disease, or even to diagnose as early as possible
34
How can we better understand the intrinsic genetic component of disease risk?
- studying large numbers of genomes
- combinatorial effects need to be evaluated
35
What type of profiling is part of screening technology and why is it helpful?
- genetic, epigenomic and transcriptomic profiling of patient samples
- these essays may be able to detect the molecular changes underpinning disease development or progression
- some molecular aspects of disease development may be pharmacologically reversible, so patients can be offered preventative medicine
36
What are three areas of engineering non-human organisms for human health?
1. Genome engineering for better antibody drugs
2. Humanised pigs for xenotransplantation
3. CRISPR-Cas9 gene drive in mosquitos for malaria eradication
37
Describe genome engineering for better antibody drugs and what it is used for
- antibodies can be raised in animals against any antigens and be used therapeutically
- e.g. to target cancer cells
- however, since animal antibodies are recognised as foreign in humans, they must first be modified to look like human antibodies 'humanized'
38
Describe the humanisation of animal antibodies
- traditional
- new
- traditional approaches, seen in the image 1, start with an antibody that has been raised in normal mice
- they engineer key regions into a human anitbody
- but this places limits on the resulting antibody affinities
- achieving humanisation without impairing antigen binding affinity is very challenging
- new and very promising approaches use extensive genome engineering to make a strain whose complex array of antibody genes has been partly or wholly humanised
- the mouse's immune system can make high affinity antibodies, even when the resulting antibody is humanised
39
Describe how pigs are humanised for xenotransplantation
- barriers
- solutions
- xenotransplantation is the process of transplanting organs between two different species, such as between pigs and humans
- However, there are two main barriers to this:
- Pigs have genes whose products can promote transplant rejection in humans.
- The pig genome contains hundreds of integrated porcine endogenous retroviral (PERV) genomes, and these may be reactivated to infect human cells.
- Until recently, the sheer amount of gene editing needed to knockout enough of these genes, as well as the PERV genes, seemed overwhelming
- With the advent of CRISPR gene editing, however, this is no longer the case, and some are predicting that suitably engineered pig organs may be available for transplantation very soon!
40
Describe how CRISPR-CAs9 gene drive in mosquitos may eradicate malaria
- define gene drive
- Gene drive refers to processes whereby an allele is preferentially transmitted during meiosis so that it spreads throughout the population.
- Gene drives are being developed in insect populations to prevent diseases such as dengue fever, zika, and malaria.
- Recently, for example, CRISPR endonucleases have been used in mosquitoes to spread an infertility gene in laboratory mosquito populations
- If used in the wild, this should reduce the ability of mosquitos to transmit the malaria parasite in human populations.
- This gene drive works by editing a mosquito chromosomal locus so that one allele encodes an infertility gene as well as a CRISPR-Cas9 endonuclease gene
- The endonuclease is designed to cleave the wild-type allele during meiosis which is then repaired by homologous recombination, using the modified allele as a template
- In this way, the infertility allele and CRISPR-Cas9 genes are both copied onto the second allele, making a homozygous genotype.
- As the population grows, homozygous germ cells are rapidly generated, so the target gene spreads through the population
41
Give a hypothesis that suggests what underlies ageing
- One attractive hypothesis is that accumulating DNA damage in the genome of our somatic cells underlies much of the declining physiology of ageing.
- Consistent with this, it is established that mutations do indeed accumulate in our ageing somatic cells.
- Furthermore, patients with rare inherited syndromes that cause premature ageing have been shown to have mutations in genes encoding DNA repair proteins of the kind described in GEN8
42
Observe the diagram of how the accumulation of DNA damage can play a central role in the ageing process
43
What is problematic about replenishing telomeres by upregulating telomerase?
- while telomerae decreases risks of some diseases
- too much telomerase can increases risk of cancers
44
What are some gene therapies being developed to extend lifespan?
- since the diseases which shorten our lives (e.g. heart disease, cancer) are known to have genetic components, research is aimed at editing these genes
45
What are some ethical implications of editing the human genome?
- humans have been editing genomes of plants and domestic animals by artificial selection for a long time
- but we have never done this with humans
- designer babies
- will be passed onto the next generation
46
Which two of the following statements are incorrect?
