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Year 3 Sem 2 > 2. Molecular Diagnostic & Biological Therapies > Flashcards

2. Molecular Diagnostic & Biological Therapies Flashcards

1
Q

What is Genetic Screening regarding cancer?

A
  • Genetic analysis can identify the specific changes to the genome which have resulted in cancer
  • Can indicate whether a particular drug is suitable or not
  • May identify mutations which may one day result in cancer
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2
Q

What are biomarkers regarding cancer?

A
  • Substances, whose production is increased in cancer cells, or by healthy cell in response to cancer
  • May be secreted and found in blood or other clinical samples - blood, urine, stool sample, biopsy
  • Can be used to diagnose, monitor and manage treatment
    : concentration often related to prognosis; reduction may indicate efficacy of treatment
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3
Q

What is Hybridization regarding cancer?

A
  • Most genetic analysis techniques rely on the hybridization
    : the joining of two complementary strands of nucleic acids
  • Complementary
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4
Q

What is Hybridization?

A
  • Most genetic analysis techniques rely on the hybridization
    : joining of two complementary strands of nucleic acids
  • Complementary probe can bind to DNA/RNA to detect presence of a specific sequence
  • Complementary primer can bind to DNA/RNA to amplify a specific sequence
  • Occurs below the melting temperature of the nucleic acid
  • DNA and RNA sequences can hybridize together
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5
Q

What is FISH?

A
  • Fluoresence In Situ Hybridization
  • Modern method for looking at gross changes to genes and chromosomes
  • Probe (fluorescent or labeled) hybridized to a specific target
  • Must be sufficient size but not too large
  • Diagnostic tool
    : gene and chromosome duplications or loss
  • Numerous types of sample can be used
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6
Q

Describe how FISH technique is used in cancer?

A
  • Many cancers have significant alterations to the structure of genes or chromosomes, which FISH can detect
  • These changes often affect the activity of a tumour suppressor gene, or turn on a signalling pathway resulting in aberrant cell growth
    : type of genetic change can dictate treatment
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7
Q

FISH

: Describe an example of Gene Amplification

A

HER2
- HER2 is over-expressed in 25-30% of breast cancers

  • 90%-95% due to gene amplification
  • Results in a shorter disease-free survival and overall survival
  • Therapy influenced by HER2 status (use of anti-HER2 mAbs)
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8
Q

FISH

: Describe an example of Chromosome Translocation

A

Leukaemia
- In a number of leukaemias, chromosomal translocation resuelts in the fusion of the genes ABL and BCR

  • The result is the ‘Philadelphia chromosome’ which leads to the BCR-ABL fusion protein, an ‘always on’ tyrosine kinase
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9
Q

FISH

: Describe an example of Chromosome Inversion/Fusion

A

ALK
- a number of changes in the ALK (anaplastic lymphoma kinase) gene have been implicated in a variety of cancers

  • ALK is a receptor tyrosine kinasee
    : its activity can be altered by mutation of its gene, gene amplification or chromosomal rearrangement
  • a well known fusion/inversion of ALK is with the EML4 gene, implicated in 2-5% of NSCLC
  • altered ALK activity can be targeted with an ALK inhibitor
  • The EML4-ALK fusion can be screened for using a FISH ‘break apart’ assay
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10
Q

What is PCR and what is it used for?

A

Polymerase Chain Reaction
- developed in mid 1980s by Kary Mullis

  • method for detecting and amplifying DNA (and RNA)
  • PCR allows the DNA from a selected region of a genome to be amplified by more than a million-fold, provided that at least part of its nucleotide sequence is already known
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11
Q

What are the ingredients for PCR?

A
  1. Template
    : DNA of any origin (e.g genomic, DNA, plasmid) or cDNA (sequence complementary to the corresponding RNA obtained by ‘reverse transcription)
  2. Primers
    - Two synthetic oligonucleotides, usually 18-22 nucleotides, complementary to the 3 end region of each strand of template to be amplified
  3. DNA polymerase
    - replicates the template DNA from 3’ end of each primer
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12
Q

Describe steps involved in PCR (Polymerase Chain Reaction)

A

Step 1. Denaturation
- heat above melting point

Step 2. Annealing

  • cooling of the DNA to 30-65degrees in the presence of a large excess of complimentary primers
  • ~30 seconds
  • temperautre depends on Tm of primers

Step 3. DNA Synthesis
- synthesis of new DNA complimentary to the target sequence
: addition of dNTPs catalysed by DNA polymerase
- 67-75 degrees (Thermostable enyme)
- 2-5 minutes

Repeat…

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13
Q

What is Reverse Transcriptase PCR (RT-PCR)?

A
  • A technique for amplifying a specific sequence of RNA by initial conversion to its cDNA
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14
Q

What is cDNA?

A
  • DNA synthesized from a single-stranded RNA (e.g., messenger RNA (mRNA) or microRNA) template in a reaction catalyzed by the enzyme reverse transcriptase
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15
Q

Describe 2 ways of detecting PCR products

A
  1. Slab Gel Elctrophoresis
    - Traditionally, ethidium bromide is used to stain nucleic acid PCR products in agarose gels followed by visualisation under UV light
    - Alternatively, primers with a fluorescent label or conjugated enzyme can be used
    - Multiple products can be detected on a single gel if their sizes are sufficiently different i.e more than one set of primers can be used per tube (multiplex reaction)
  2. Capillary Gel Electrophoresis
    - PCR products seperated by size within a capillary
    - Can be combined with the use of primers tagged with different fluorophores
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16
Q

What is Real-Time PCR?

A
  • Regular PCR doesn’t give any information regarding the amount of DNA (or RNA) of interest present in the initial sample
  • Real-time PCR enables qualification of the product in real-time
  • Also referred to as quantitative PCR (q-PCR)
  • Several types of probe used
    : TaqMan, Molecular Beacons, Scorpion probes and SYBR Green
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17
Q

Describe how Real-time PCR is obtained

A
  • Forwarad and reverse primers, DNA pol & TaqMan probe are added to the DNA sample
  • Following denaturing and annealing of primers and probe, DNA pol synthesizes new DNA
  • Reverse strand synthesised normally
  • On forward strand, DNA pol encounters the TaqMan probe
  • 5’ nuclease activity degrades the probe, releasing the fluorescent reporter
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18
Q

Mutations in the genome can increase the probability of developing a certain cancer or may affect the choice of treatment for a patient with cancer
: certain drugs may be more or less effective depending on the specific mutations present

Describe how mutations are detected using PCR

A

PCR can be readily used to detect different types of mutations, e.g:

  • Point mutations / SNPs
  • Deletions
  • Insertions

PCR methods rely on reaction products being of different sizes in the absence or presence of a mutation, or the reaction not working at all when using a mutated template

Some mutations result in the creation or deletion of a restriction site (i.e the sequence recognised by a particular restriction enzyme)
: this generates restriction fragment length polymorphisms (RFLPs) when the PCR products are incubated with the enzyme

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19
Q

What is a Point mutation (PM)?

A
  • defined as an alteration in a single nucleotide pair in the DNA and usually leads to a change in only one biochemical function
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20
Q

What is Single Nucleotide Polymorphism (SNPs)?

A
  • defined as loci with alleles that differ at a single base, with the rarer allele having a frequency of at least 1% in a random set of individuals in a population
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21
Q

Describe how PCR is used to detect mutations in EGFR (Epidermal Growth Factor Receptor) especially regarding NSCLC (Non-small cell lung carcinoma)

A
  • NSCLC acocunts for~80% of all lung cancers
  • These mutations increase sensitivity to 1st 2nd and 3rd generation EGFR TKIs and can inform treatment options
  • Sequencing is costly and can be time consuming so PCR can be used to detect these changes by designing primers to give different sized PCR products in the absence and presence of the mutation
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22
Q

What is Allel-specific PCR (ASPCR)?

A
  • ASPCR can detect point mutations without relying on changes in restriction sites
  • Relies on the fact that the correct base at the 3’ end of a primer is crucial for DNA synthesis
  • Carefully designed primer sets are differentiated between wild type and mutant alleles
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23
Q

What is Micrroarray Technologies?

A
  • Microarrays are microchips or flow cells containing many microscopic spots of different DNA or RNA probes
  • Used to assess expression of large numbers genes, determine genotype or sequence nucleic acids
  • Probes are specific sequences of DNA or RNA used to capture complementary sequences from the test sample
  • The number of probes ranges from double digits to >1 million
  • Applications in areas such as
    : Basic research, including target identification
    : Human diagnostics
    : Personalised medicines
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24
Q

Why is DNA sequencing important?

A
  • Recent advances in sequencing technologies mean that sequencing genes, or even genomes, is becoming a routine practice
  • Sequencing results in a huge amount of information which can inform clinical decisions, even before the development of cancer
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25
Q

Describe BRCA mutations regarding DNA sequencing

A
  • BRCA1 and BRCA2 are just two of many genes which, when mutated, are implicated in the development of cancer
    : have tumour suppressor activity
  • Both BRCA1 and BRCA2 proteins are involved in the homologous recombination DNA repair (HRR) pathway
  • They are autosomal dominant
  • Carrying certain mutations in these genes can significantly increase the risk of developing, in particular, breast and ovarian cancers
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26
Q

Describe First Generation DNA sequencing - Sanger Sequencing

A
  • Dideoxy NTPs are chain terminators
  • Each labelled with a different fluorophore
  • Result is end-labelled sequences of DNA with different lengths
  • Readily detected by CGE, enabling sequence to be determined
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27
Q

Summary of Molecular diagnostics 2(JUST READ)

A
  • Cancer results from genetic changes, which can be readily detected using modern screening technologies
  • Some mutations affect drug efficacy; genetic testing can aid in clinical decision making
  • FISH can detect gross changes in genes and chromosomes
  • PCR can greatly amplify genetic material and be employed in detecting a range of mutations
  • Sequencing DNA sequences, genes, or even the whole genome is becoming more routine and gives a huge amount of information involved in detection and treatment of cancer
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28
Q

What are Biomarkers?

A
  • substances whose production is increased in cancer cells, or by healthy cells in response to cancer
  • May be secreted and found in blood or other clinical samples
    : blood, urine, stool sample, biopsy
  • Can be used to diagnose, monitor and manage treatment
    : concentration often related to prognosis, reduction may indicate efficacy of treatment
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29
Q

What is Immunodetection?

A
  • detection of a specific antigen using antibody binding
  • used in a number of different techniques
    : immunihistorchemistry / immunocytochemistry
    : western blot
    : ELISA
    : Flow cytometry
  • The antigen (protein) being detected can be in situ, in solution, on a membrane/well plate, on a microparticle or in a gel
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30
Q

What is ELISA?

A

Enzyme-Linked Immunosorbent Assay

  • used to detect a specific antigen or antibody in a sample (quantitative or qualitative)
  • useful in research and clinically to detect infection or disease markers
  • involved adsorption of antigen or antibody to multiwell plates (direct ELISA)
  • Secondary antibody has an enzyme conjugated to it
  • Substrate converted by enzyme to a chromogenic or fluorogenic product that can be detected using a plate reader
  • Alkaline phosphatase (AP) and horseradish peroxidase (HRP) commonly used
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31
Q

What is Sandwich ELISA?

A
  • Direct ELISAs only really suited to fairly ‘clean’ samples or for proteins at high concentrations
  • Low concentrations of protein can be detected using a ‘sandwich’ ELISA
  • Analogous approaches, i.e capturing the analyte, are now used for high-throughput screening of patient samples using microbeads instead of plates
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32
Q

Describe how ELISA is applied in numerous cases

A
  • numerous ELISA kits are available, many for disease markers
  • e.g Altered erythropoietin levels result from a number of conditions (anaemias, renal disorders, malignancies, infectious disease and inflammatory disorders)
    : readily assayed by ELISA
  • Antibodies to various pathogens can be detected by ELISA
    : e.g, HIV, hepatitis viruses etc
  • In many cancers, high levels of proteases can result in degradation of fibrin and tehse breakdown products can be detected by ELISA
  • For example, the commercially available DR-70 kit can identify 13 different tumour types including lung, breast, colon and uterus
  • Positive ELISA result = further tests to verify
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33
Q

Describe Immunosorbent Assay Technology

A
  • Self-testing
  • Prostate specific antigen (PSA) is an enzyme secreted by prostate epithelial cells
  • High (>4ng/mL) levels in the blood may be a sign of prostate cancer
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34
Q

What is immunohistorychemistry?

A
  • involves the process of selectively identifying antigens in cells of a tissue section by exploiting the principles of antibodies binding specifically to antigens in biological tissues.
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35
Q

What is CISH?

A

Chromogenic In Situ Hybridization
- essentially a combination of FISH and IHC
- can dual-stain a sample using probes with different enzymes
: horseradish peroxidase and alkaline phosphatase commonly used
- Enzyme reaction can be used to cause certain ions to precipitate
: e.g gold-faciliated in situ hybridization

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36
Q

What is Flow Cytometry?

A
  • method for detecting specific molecules on and within cells
  • can also be used for sorting/isolating specific sub-populations of cells
  • powerful analytical technique for cell biology research
  • important in medicine
    : immunophenotyping, diagnosis, cell sorting
  • Multiple antigens can be quantified simultaneously
  • Extra- and intracellular
  • mainly used for proteins, but DNA and RNA can also be detected
37
Q

What are the examples of Flow Cytometry application?

A
  • diagnosis
    : levels of specific proteins are altered in many disease states
  • immunophenotyping
  • cell enumeration
  • basic research
  • cell sorting
    : purification of cell populations for therapeutic applications
38
Q

Describe Electrochemiluminescence

A
  • rapid detection and quantification using low volumes
  • intensity of signal proportional to analyte concentration
  • High-throughput
39
Q

What is Luminex Technology?

A
  • multiplex immunoassay system
    : many targets can be identified and quantified per well of a multiwell plate
  • each type of bead is tagged with a different coloured fluorescent marker and captures a specific protein
  • secondary antibody has a PE (phycoerythrin) tag
  • beads are read in a flow-based system or if magnetic, in a layer at the bottom of a well
  • one laser classifies the type of bead, the second quantifies the PE signal
40
Q

JUST READ (Molecular diagnostic 3)

A
  • Early detection = better prognosis
  • Current detection/diagnosis based on imaging and cytology
  • The only widely used blood test for early detection of cancer is PSA
  • Routine blood test would be revolutionary
  • Must be sensitive and specific
  • There are a wealth of rapid, sensitive and accurate techniques which are capable of detecting and quantifying clinically-relevant proteins
  • Protein levels are used to diagnose and monitor disease states, including cancers
  • Huge ongoing research effort to identify new, specific biomarkers
    : proteins, nucleonic acids, fats, metabolites etc
  • Testing for combinations of biomarkers is likely to enable early identification of disease, with obvious socioeconomic benefits
41
Q

What do we know about human cancer?

What is cancer and its different types?

A
  • Cells have the ability to divide
  • The rate of that division is controlled by external cues
  • Cancer results when a mechanism in cells that regulate division lose control

No two cancers that are the same

  • sarcoma (mesenchymal)
  • carcinoma (epithelial)
  • adenocarcinoma (glands)
  • lymphoma (lymphoid)
  • leukemia (myeloid)
  • teratoma

Two aspects to cancers causes

  • nature = genetics
  • nurture = environment -> epigenetics

Why are humans so susceptible to cancer?
- hyper-mutable

42
Q

Describe staging of a general cancer

A

Stage 1. Early stage

  • a small, invasive mass or tumour has been found
  • no spread to lymph nodes or other tissues
  • sometimes called early-stage or ‘localised cancer

Stage 2. Localised

  • cancer has started to affect nearby tissue
  • mass may have grown in size
  • spread to lymph nodes near the mass

Stage 3. Regional spread

  • cancer affects more surrounding tissue
  • mass may have grown in size
  • spread to distant lymph nodes away from the mass

Stage 4. Distant spread

  • cancer has spread to other tissues or organs beyond the region where it originated
  • sometimes called advanced or ‘metastatic’ cancer
43
Q

Describe endogenous cancer suppressors

A
  • considered to be a tumour suppressor, p53 has cellular roles in genome stability, DNA repair, apoptosis/cell death, and cell metabolism
44
Q

What are the strategies to treat cancers?

A

Intracellular targets

  • suppress kinase function
  • suppress cell division

Extracellular targets

  • antibodies
  • Aptamers
  • delivery of effectors
45
Q

What are the examples of Tumorigenic phenotypes?

A
  • Survival
  • Motility
  • Evading antitumour immunity
  • Proliferation
  • DNA damage checkpoints
  • Angiogenesis
  • Metabolism
46
Q

Describe activation of genomic alterations of kinases

A

A) Activating point mutations in genes coing for kinases lead to the expression of a constitutively activated kinase

B) Chromosomal amplification leads to increased kinase transcription and downstream pathway become over-activated

C) Chromosomal alterations (deletions or translocations) can localise a kinase gene in proximity to another gene and lead to a constitutively activated chimeric or truncated kinase

47
Q

What are the 4 types of FDA approved small molecule kinase inhibitors?

A

Type I
- Active conformation

Type II
- Inactive conformation

Type III
- Allosteric adjacent

Type IV
- Allosteric remote

48
Q

Small molecules suppressing cell division

- Describe Chemotherapy drug categories

A

Alkylating agents
- bind DNA stop replication

Antimetabolites
- block use of nutrients

Anti-microtubule agents
- interfere with cell division

Antibiotics
- inhibit RNA synthesis to slow or block mitosis

49
Q

Extracellular antibody strategies

Due to their physicochemical nature, antibiotics do not access the cell cytoplasm but have many extracellular actions.

What are they?

A

A) Antibody-directed cell killing
B) Solid tumour growth requires angiogenesis
C) T-lymphocytes function to kill intruding cells that do not have protective surface markers
D) Radiation in mutagenic
E) Toxins can be directed
F) Effector cells can be brought into close proximity to cancer cells
G) CAR T technology
: outgrowth of how biopharmaceuticals can be used to target & kill cancer cells

50
Q

What are the pros and cons of antibody directed to cancer cells for tumour killing?

A

Pros

  • tremendous selectivity
  • flexible nature for coupling of toxic and other agents
  • can select different affinity
  • long systemic residence
  • relatively stable

Cons

  • Doses are very high due to massive volume of distribution
  • Hard to find a truly tumour-restricted antigen
  • Can have off-target outcomes
51
Q

What is Vascular and Stromal ablatio and its pros&cons?

A

Using radiofrequency or laser energy to cauterize and close varicose veins that are faulty

Pros
- Multiple new targets
Cons
- Unexpected outcomes

52
Q

How does someone die from influenza?

A
  • Time course of the human immune system getting fully engaged to induce a systemic inflammatory response syndrome without contaminating pathogens, endotoxin, or underlying disease.
53
Q

More strategies to treat cancers?

A

Vaccination
- Some success, lots of unknowns

Stem cells
- No consensus yet

Future Therapy possibilities - Pros and cons

  • Crispr/Cas9
  • RNA-based drugs

Induce re-differentiation
- a fix, a stalemate, or a mistake?

54
Q

What are the ways of attraction of effector cells using bi-specifics

A

Antibody-dependent cell-mediated cytotoxicity (ATCC)

Antibody-dependent cellular phagocytosis (ADPC)

Triggers involve CD16A engagement by TCR/CD3 or V9V2 engagement or blockade of immunocheckpoints

55
Q

Describe stem cell proliferation and cancers (Teratoma)

A
  • all cells in the body are generated by stem cells
  • some tissues turn over more rapidly than others, pushing the function of their stem cells to a greater extent
  • a correlation exists between the dividing behaviour of these stem cells and frequency of cancers observed in that cell type
56
Q

What is stem cell cancer hypothesis?

A
  • based on the observation that tumours, like adult tissues, arise from cells that exhibit the ability to self-renew as well as give rise to differentiated tissue cells
57
Q

What is CRISPR0-Cas9 and how does it work?

A
  • short for ‘clustered regularly interspaced short palindromic repeats and CRSPR-associated protein 9’
  • adapted from a naturally occurring genome editing system in bacteria
  • The bacteria capture snippets of DNA from invading viruses and use them to create DNA segments known as CRISPR arrays.
  • The CRISPR arrays allow bacteria to remember the viruses so if the virus attacks again, the bacteria produce RNA segments from the CRISPR arrays to target the viruses’ DNA.
  • Bacteria then use Cas9 or a similar enzyme to out the DNA apart, which disables the virus
58
Q

Describe the 2 types of Gene Therapy Vectors

A

Non-viral vectors

  • Naked DNA
  • Liposomes/DNA
  • Polymer/DNA complex (polypex)
  • Liposom/Polymer/DNA (lipopolyplex)
Viral vectors
- DNA viruses 
\: adenoviruses
\: Herpes Simplex Virus
- RNA viruses
\: retrovirus
59
Q

GalNAc-siRNA and Asialoglycoprotein Receptor (ASGPR) are leading commercial strategies.

Describe how they work

A

GaINAc-siRNA

  • GaINAc ligand conjugated to chemically modified siRNA to mediate targeted delivery
  • Trivalent GaINAc carbohydrate cluster has nM affinity for ASGPR

Asiaglycoprotein Receptor (ASGPR)

  • Clears serum glycoproteins via clathrin-mediated endocytosis
  • Well suited for receptor-mediated, targeted delivery
  • Conserved across species
60
Q

What controls solid tumour onset?

A
  • Epithelia express tight junctions and associate at their base with a complex of proteins known as the basement membrane
  • Epithelial cells lacking TJ structures and functional intracellular signaling mechanisms can undergo an epithelial
    : mesenchymal transformation (EMT)
  • Following EMT, cell division continues uncontrolled with secretion of enzymes that reduce basement membrane integrity
  • Continued cell replication and enzymatic basement membrane disruption provides conditiosn for tumour growth and metastatic disease
    : cancer aggressiveness correlates with a loss of functional TJs
61
Q

Describe Gene therapy strategies

A
  • Aims to deliver a gene in order to overcome a disease or infection
  • Disease cab be acquired or an underlying genetic condition
  • Immunotherapy can be used to reprogram the patient’s immune system to target disease
  • More recently, gene editing has become a very hot topic
  • In addition to these strategies, a patient’s cells can be transfected with a gene which encodes a protein drug such as an antibody
62
Q

Describe Suicide genes

A
  • known as gene-directed enzyme prodrug therapy (GDEPT)
  • Often uses a combination of HSV thymidine kinase and ganciclovir
  • HSV-TK has >1,000-fold greater efficiency in phosphorylating ganciclovir than mammalian kinases
63
Q

Describe Oligonucleotides in terms of

  • RNA interference
  • miRNA
  • miRNA and cancer
A

RNA interference

  • small interfering RNA (siRNA)
  • 21-25 bp long
  • can ‘knock down’ gene expression if sequence is known
  • readily designed
  • readily synthesised

miRNA

  • another type of RNAi
  • Micro RNA (miRNA) is ~22 nucelotide ssRNA
  • Derived from genes that specifically code miRNAs, not proteins
  • important in regulating gene expression (~1000 miRNAs regulate about one third of our genes)
  • Don’t usually cause mRNA destruction, just repress or destabilise target mRNA
  • Strong evidence of involvement in a number of disease states, including cancer

miRNA and cancer

  • DNA coding under-expressed or missing miRNAs could be delivered to cells to replace the missing miRNAs
  • Over-expressed miRNAs can be inhibited by anti-miRNAs, complementary nucleotides (~22 bases) that bind to the problem miRNA
64
Q

Describe Current Genetic Therapy Targets in..

  • Cancer
  • Monogenic disease
  • Infectious disease
  • Cardiovascular disease
A

Cancer

  • Antigen
  • Anti-angiogenesis
  • Antibodies
  • Tumour suppressor genes
  • Suicide genes
  • Immunotherapy

Monogenic disease
- Gene mutation -> disease e.g CF, sickle cell anaemia

Infectious diseases
- Target viral/bacterial genes or host receptor genes, especially HIV-1

Cardiovascular disease
- Anti-angiogenesis

65
Q

Describe direct delivery of Genes & Nucleic acids

A
  • Therapeutic gene is packaged into a delivery vehicle such as a retrovirus
  • injected into the patient and reaches target organ (e.g liver)
66
Q

Describe Cell-based delivery of Genes & Nucleic acids

A
  • Therapeutic gene is packaged into a delivery vehicle such as a retrovirus and introduced into the cells
  • Adult stem cells is introduced
  • The genetically modified cells are reinforced into the patient
67
Q

What are the challenges to delivery of genetic materials?

A
  1. Complex formation
  2. Transport from delivery site
  3. Cellular uptake
  4. Endosome release
  5. Trafficking
  6. Nuclear entry
  7. Transcription
  8. (m)RNA export
  9. Translation
68
Q

What is Retroviruse?

A
  • e.g HIV (genus = lentivirus)
  • ssRNA genome
  • Genome 8-11 kb (x2)
  • ~100nm diameter
  • some infect only dividing cells (not lentiviruses)
  • can be used to target specific cells
  • possible immune response
  • risk of tumour development
69
Q

Describe Adenoviruses

A
  • common cold
  • dsDNA genome
  • 60-90 nm diameter
  • Genome 30-38 kbp
  • Infects dividing and non-dividing cells
  • Doesn’t integrate
  • Can be used to target specific cell types
  • Possible immune response
70
Q

What is Oncorine?

A
  • first licensed oncolytic virus
    : virus that selectively infects and kills tumour cells
  • oncolytic viruses are engineered to take advantage of key regulatory factors of the cell cycle and the virus life cycle
  • Oncorine can’t replicate in normal cells due induction of p53, but can do so in p53-deficient tumour cells
71
Q

Describe Adeno-associated Viruses

A
  • no known disease
  • ssDNA genome
  • Genome ~5kbp
  • ~20nm diameter
  • Infects dividing and non-dividing cells
  • Specific integration
  • Can be used to target specific cell types
  • Little immune response

Glybera (alipogene tiparvovec) developed for treatment of lipoprotein lipase deficiency (LPLD) which is a rare disease causing dramatically increased fat levels in the blood

72
Q

Describe summary of viral vectors and its Advantages/Disadvantages

A

Retrovirus

  • ssRNA, 8-10kbp, Integration exists, Extended duration
  • Dividing cells, possibly oncogenic

Adenovirus

  • dsDNA, ~36kbp, No integration, Transient duration
  • High capacity, many cell types, immunogenic

AAV

  • ssDNA, ~5kbp, Integration exists, Extended duration
  • Small, low inflammatory response

Advantages

  • High transfer efficiency
  • Some inherent tissue tropisms

Disadvantages

  • Limited DNA/RNA capacity
  • Difficult to produce
  • Can be immunogenic
73
Q

What techniques of Non-viral Nucleic acid delivery exist?

A

Physical techniques

  • Direct injection
  • Gene gun
  • Ultrasound
  • Electroporation

Chemical techniques

  • Encapsulation
  • Complexation (nanoscale particulates)
74
Q

What are the advantages and disadvantages of Non-viral vectors?

A

Advantages

  • Easy to prepare and modify e.g targeted
  • Reduced immunogenicity
  • Large capacity

Disadvantage

  • Poor efficiency
  • Transient
75
Q

Describe Non-viral vector: Dendrimers

A
  • Wide range of chemical approaches and functional groups
  • Surface groups can be functionalised for targeting, stealth etc.
  • Can be designed to release under specific conditions
  • Amine-rich dendrimers for complexation with negatively charged nucleic acids
76
Q

Describe Non-viral Vector: Cell-Penetrating Peptides

A
  • Short sequence of these are able to cross membranes
  • Potential delivery of a variety of cargoes (including liposomes, nanoparticles, etc for biological entities and traditional drugs)
  • Low cytotoxicity, dose-dependent, can be conjugated to carriers
  • Tend to be arginine rich
  • Various uptake mechanisms, including direct translocation and the different types of endocytosis
  • Particularly promising for oligonucleotides
    : packaging and delivery in one
77
Q

What is an ideal non-viral vector?

A
  • Small
  • Stable in serum
  • Low toxicity
  • Specific cell targeting
  • Cytosolic release of cargo
  • Nuclear targeting
  • High transfection efficiency
78
Q

Summary of Gene Therapy (JUST READ)

A

Many disease, including cancer, can be treated by delivery of

  • genes that code for defective proteins
  • genes that code for therapeutic proteins (e.g antibodies, receptors, growth factors, enzymes, ribozymes)
  • suicide genes
  • genes that code for RNA interference molecules
  • RNA oligonucleotides

Delivery limits success of therapeutic nucleic acids

Delivery can be ‘in vivo’ or ‘in vitro’

Viral vectors
- good expression, potential toxicity, limited capacity
Non-viral vectors
- poor expression, lower toxicity, higher capacity

Combining the positives of both may lead to more effective treatments

79
Q

Describe anti-angiogenesis strategies briefly and its targets

A
  • Blocking angiogenesis can halt or limit tumour progression and this can be achieved using genes or oligonucleotides
  • Targets include VEGF, TGF-beta, bFGF, PDGF
80
Q

What are the possible approaches of Anti-angiogenico strategies?

A
  1. Anti-sense RNA or siRNA to block translation of GF or its receptor
  2. Oligonucleotides to block transcription of GF or its receptor
  3. Gene therapy to induce production of soluble receptor or mAb (against receptor of GF)
81
Q

Describe TK (Thydimine Kinase of the HSV, namely TK) Cell Therapy

A
  • Allogenic T cells transfected with HSV-TK
  • Engrafts and helps reconstitute the immune system alongside HSCT from same donor, improves overall survival and non-relapse mortality
  • Ganciclovir adminisered if graft versus host disease develops
82
Q

Describe Cell-based immunotherapy

A
  • Cytotoxic T cells (Tc cells) are activated by antigen presenting cells such as dendritic cells, which prime them with specific antigens for target cells (e.g infected cells, cancers)
  • Tc cells then recognise and kill cells which express these antigens via release of perforin and granzyme, which induce apoptosis
  • The efficacy of this process can be massively improved using cell engineering approaches
83
Q

What are CAR T cells?

A
  • T cells genetically engineered to express a chimeric antigen receptor (CAR)
  • CAR T cells proliferate and kill tumour cells upon contact with the antigen they recognise
84
Q

Describe how CAR T Cells are made and administered into patients

A
  1. White blood cells obtained from patient through leukapheresis
  2. Cells are sent to manufacturing facility where antibody-coated beads are used to activate the T cells
  3. Activated T cells are reprogrammed using retroviruses to express chimeric antigen receptors (CARs)
  4. The CAR T cells are expanded ex vivo
  5. The cells are sent back to the treatment center for treatment
  6. Patient receives lymphodepleting chemotherapy prior to T cell treatment
  7. CAR T cells are transfused back into the patient
85
Q

Describe Mesenchymal Stem Cell Therapy. What characteristics make them suitable for therapeutic cells?

A

MSCs have a number of characteristics which makes them suitable as therapeutic cells

  • Ability to home to site of injury
  • Anti-inflammatory properties
  • Hypoimmunogenic

Lots of options

  • Anti-angiogenesis
  • Cytokine delivery
  • miRNAs
  • Vehicle for anti-cancer drugs
86
Q

What is CRISPR/Cas9?

A
  • gene editing system based on components of the adaptive immune systems of certain bacteria and other microbes
  • Abbreviations from
    : Clustered Regularly Interspaced Short Palindromic Repeats
    : CRISPR-associated (Cas) genes
87
Q

Describe CRISPR/Cas9 variations

A

CRISPR/Cas9 can be engineered to increase specificity or change its mode of action
- e.g dCas9 has a mutation which deactivates nuclease activity and enables it to be used to control gene expression

88
Q

What are the advantages and disadvantages of Gene editing - CRISPSR/Cas9

A

Advantages

  • Small (potentially easy to deliver)
  • Inexpensive
  • Specific
  • Versatile

Disadvantages

  • Delivery (especially in vivo)
  • Off-target effects
  • Abuse
89
Q

Gene Therapy 2 Summary (JUST READ)

A
  • Genetic modification of a patient’s cells in vivo or in vitro is a powerful technique which can aid the treatment of cancer in combination with other treatment options
  • Immunotherapy boosts the immune system in order to fight cancer and can be personalised to a specific tumour
  • CAR T cells are an emerging therapeutic options
    : currently for ‘liquid tumours’, with solid tumour treatments under developement
  • Gene editing and regulation using CRISPR/Cas9 has the potential to revolutionise all areas of medicine, including oncology, enabling the precise modification of the genome in numerous scenarios to treat or even prevent disease

Year 3 Sem 2 (12 decks)

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