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PHARM 321 > PCR Technology and Gene Therapy > Flashcards

PCR Technology and Gene Therapy Flashcards

1
Q

What is PCR?

A
  • Method to produce enormous copies of any segment of DNA without need for cloning
  • Exploits and adapts characteristics of DNA replication (mimics helicase, primase, polymerase)
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2
Q

What are the two types of PCR?

A
  • Qualitative = Gene is there, yes or no

- Quantitative = Amount of gene there

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3
Q

What are the main features of PCR?

A
  • DNA polymerase, the replicating enzyme, can be directed to amplify specific DNA segments “in vitro”
  • Both strands of DNA serve as template simultaneously
  • Initiation of PCR reaction requires specific complementary “primers”
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4
Q

What are the requirements of PCR?

A
  • Template DNA (ssDNA/dsDNA)
  • Need to know portion of target on that DNA sequence in order to design “primers” (small oligonucleotides of homology that find and bind to target DNA region)
  • Synthetic “pair” of complementary oligonucleotide primers (need both “forward” and “reverse” primers to prime both strands of DNA)
  • dNTPs (deoxy-nucleotide triphosphates)
  • Heat stable polymerase
  • Contaminating DNA needs to be minimized
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5
Q

What are the reaction steps of PCR?

A
  • Extract DNA from sample
  • Heat denature to separate DNA strands
  • Primer “hybridization” (annealing) of single strand target DNA with specific primers in excess (to avoid self-annealing) followed by cooling
  • Primer extension with polymerase and dNTPs
  • Repeat cycle of heating (to denature), cooling to Tm for primers (to anneal), then allow cDNA to extend
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6
Q

Heat denaturing in PCR replaces the function of which enzyme?

A
  • Helicase
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7
Q

Annealing step of PCR replaces the function of which enzyme?

A
  • Primase
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8
Q

Describe the PCR cycles

A
  • Denaturation (break hydrogen bonds) (95C for 3 minutes, then 30 more seconds)
  • Annealing (primer binds at 3’ ends) (55C for 30-45 seconds)
  • Extension by DNA polymerase of both strands simultaneously (72C for 1 min per kb gene, then an extra 5 minutes)
  • Hold for 15 minutes at 4C
  • Repeat for about 30 cycles
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9
Q

In PCR, the number of copies is indicated by what mathematical equation?

A

2^n

Where n = number of cycles

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10
Q

What does reverse transcription-PCR use as a template and primer?

A
  • Template = mRNA

- Primer = oligo-dT

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11
Q

Why is RNA not used in reverse transcriptase?

A
  • Too heat labile
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12
Q

Describe the steps in RT-PCR

A
  • mRNA template (AAAAAA - 3’) (TTTTTT - 5’ - binded to mRNA 3’ end)
  • 1st strand cDNA synthesis (use viral enzyme reverse transcriptase)
  • Now have mRNA-DNA hybrid
  • Denature mRNA, use “random” hexamer primer, then conduct PCR
  • Now have dsDNA
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13
Q

RT-PCR - enzymes and end result?

A

Uses thermoactive reverse transcriptase –> Synthesis of complementary DNA (cDNA) from mRNA/RNA viruses –>l DNA polymerase based PCR –> Millions of cDNA copies

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14
Q

What are the advantages of PCR?

A
  • Specific (even single bp mutations can be found)
  • Fast (30 cycle amplification in 3h)
  • Ease of use (with thermocyclers)
  • Quantity (multiple sample analysis)
  • Purity of sample (tolerant to poor sample quality)
  • Versatility (can introduce site-directed mutations in certain applications)
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15
Q

What are the disadvantages of PCR?

A
  • Need to know/predict (degenerate primers) partial sequence
  • Size of amplified fragment is 0.1-3kb (ideal conditions generate 35kb)
  • Number of cycles may have limits due to NTPs
  • Contamination can lead to false positives
  • Need stringent positive and negative controls
  • Inhibitors of polymerase
  • Non-specific amplification
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16
Q

What are some sources of DNA polymerase for PCR?

A
  • Originally E. coli (but too heat labile and needed to be added after every cycle - cost and labour)
  • Thermus aquaticus (Taq) DNA Polymerase allows PCR at 72C decreases non-specific annealing as well, but no proof reading function to ensure fidelity
  • Pyrococcus aquaticus (Vent) polymerase (100C growth optimum) has proof reading functions
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17
Q

What are the advantages of using Taq and Vent polymerases?

A
  • Can be added once and remain active for all cycles

- Allows for automation of PCR by thermal cyclers which are programmed heat blocks

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18
Q

What is agarose gel electrophoresis?

A
  • Separates DNA fragments of different sizes
  • Gel matrix restricts random diffusion
  • At neutral pH, DNA has negative charge
  • Smaller fragments move more readily towards the positive terminal (cathode) than larger fragments
  • Visualize DNA bands with fluorescent dye
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19
Q

What are some applications for PCR?

A
  • Ultrasensitive diagnostics
  • Evolutionary studies - extant and extinct species
  • Diagnostic pathology for diseased cells
  • Research
  • DNA fingerprinting in criminology
  • Anthropology and archaeology
  • Viral load assay for therapeutics
  • Detection in archival paraffin tissue blocks/dried blood on filter paper
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20
Q

What is hot start PCR?

A
  • Used to ensure high stringency of primer annealing

- Most common method

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21
Q

What is booster PCR?

A
  • Low levels of primer to start to decrease mis-priming, with subsequent primer addition to boost exponential amplification
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22
Q

What is touchdown PCR?

A
  • Early gradual decrease of temperature (until Tm) for perfect hybridization
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23
Q

What is nested PCR?

A
  • Poor quality/low abundance DNA. Two PCRs with first external pair of primers and second internal (nested) pair
  • Used in rare and viral infections
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24
Q

What is gene therapy?

A
  • Introduction of “corrected genes” into patients
  • Essentially, DNA is being used as drug (“new age” pharmaceutical)
  • Genetic basis of inherited disorders has been mapped and causative genes have been identified and cloned (monogenetic disorders - manifest in one organ/tissue)
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25
Q

Why is gene therapeutics not currently accepted in society?

A
  • Ethics

- Scientific reasons

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26
Q

What are current gene therapy clinical trials based on?

A
  • Addition of intact gene and not replacement
27
Q

What is ex vivo gene therapy?

A
  • Cells from various organs and tissues or tumours are removed and cultured in vitro
  • During culture, therapeutic gene is added and modified cells are re-infused or implanted in patient (locally or systemically)
28
Q

What is in vivo gene therapy?

A
  • Brain, heart, lungs are less suited for ex vivo strategies and culture
  • The in vivo delivery of genes locally/systemically is only option
  • Use vector to locally or systemically deliver in body
29
Q

What does in vitro refer to?

A
  • Conducted in laboratory
30
Q

What are the advantages of ex vivo therapy?

A
  • Maximize gene delivery to selected cell/tissue cluster
  • Toxicity of gene delivery vectors (vehicles) to the whole body is avoided
  • Electroporation can be done without using chemicals
  • Selection of gene transformed cells can be made using specific assays (e.g., drug resistant markers)
31
Q

What disease are targeted for gene therapy?

A
  • Inheritable genetic disorders (intact gene introduced to correct inadequate gene that caused disease)
  • Highly localized disorders, where not all organs are involved in phenotypic manifestation of gene defect (e.g., lungs in CF, liver in hemophilia)
32
Q

What is transient vs. permanent gene expression? Which conditions require transient vs. permanent gene expression?

A
  • Permanent gene expression = used to rectify inherited disorders where permanent integration and expression of therapeutic gene is required
  • Transient gene expression = Used in cancer and other acute diseases where transient expression may be all that is needed
33
Q

Describe permanent gene transfer into the genome

A
  • Therapeutic gene is stably integrated into the host cell chromosome - important that cell is able to divide!
  • Therapeutic gene is then transmitted to daughter cells after cell division
34
Q

What viruses would mediate permanent gene transfer?

A
  • Retroviruses/adeno-associated virus mediated gene transfer

- Low efficiency after non-viral mediated DNA transfer

35
Q

Describe episomal/epigenetic stable gene transfer

A
  • Therapeutic gene is in episomal vector (can replicate autonously in cytoplasm or as part of chromosome) that contains ori
  • Therapeutic gene remains episomal when incorporated into cell
  • Therapeutic gene transmitted to daughter cells after cell division
  • Stable gene transfer and transmittance to progeny cells also occur when episomal vectors containing ori are used
36
Q

What is the problem with episomal stable gene transfer?

A
  • How do we get the gene into the right place in the chromosome?
37
Q

Describe episomal gene transfer without an ori

A
  • Therpeutic gene without ori
  • Therapeutic gene incorporated into cell but remains episomal
  • Therapeutic gene will be lost during cell division
38
Q

Describe what happens in transient gene transfer

A
  • Transient gene transfer occurs when therapeutic gene remains episomal and is not integrated into host chromosomal DNA
  • Episomal DNA is lost upon cell division
39
Q

What are some non-viral gene transfer methods?

A
  • Injection of naked DNA for skin and muscle disorders
  • Particle bombardment using a “gene gun” for deeper tissues (gold or tungsten micro-particles, ~1um in diameter, use helium propellant)
  • Liposome and nano-particles for systemic delivery
40
Q

Describe liposomal gene delivery

A
  • Liposomes are cationic (have a positive charge)
  • DNA is anionic (has a negative charge)
  • Liposomes and DNA will form a complex and fuse with the membrane of a cell
  • DNA is released into the cell and can eventually enter the nucleus
41
Q

Describe gene transfer using recombinant viruses (viral vectors)

A
  • Viruses have natural capacity to infect and enter nucleus and deliver viral genes to nucleus, often via cellular receptors
  • Rates of gene delivery are more efficient than non-viral methods
  • Viral vectors with therapeutic genes need to be modified to control replication and unwanted side effects (need to control life cycle)
42
Q

What are retroviral vectors?

A
  • Infect variety of cells stably as provirus during cell division
  • Use MuLV of rodents because rodent viruses are not associated with human pathology
  • Viral replication sequences can be separated as cis- and trans-acting elements
  • Virus particle has core proteins complexed with two single copies of RNA molecules
  • Surrounded by lipid envelope derived from cell membrane by budding but has viral envelope proteins
43
Q

What are cis-acting elements?

A
  • Act within (i.e., information is found on one)
44
Q

What are trans-acting elements?

A
  • Act across (i.e., information for gene is from a second introduced genome)
45
Q

What are the requirements for recombinant retroviral systems?

A

a) Recombine retroviral vector DNA to transfer desired gene to cells
b) Vector should be replication deficient (IMPORTANT)
c) No undesirable properties stemming from virus itself
d) Need to employ retroviral packaging cell to produce replication deficient vectors
e) Need to accommodate significant fragments of DNA for gene therapy

46
Q

Briefly describe the retrovirus life cycle

A
  • Following adhesion, virion enters a cell and its RNA is reverse transcribed to DNa, which is integrated into cellular chromosome to form provirus
  • Provirus is transcribed and translated into viral proteins with full length RNA molecules. Encapsidated viral RNA buds from cell to give progeny virus particles
47
Q

Describe retroviral mediated gene transfer

A
  • Packaging cell generates all viral proteins, but makes no viable virus particles
  • Retroviral shuttle vector carrying all in-cis requirements and gene(s) of interest is introduced into packaging cell (e.g., by physical transfection procedures)
  • Transcripts from shuttle vector can be packaged by virus proteins present and form infections virus
  • Recombinant retroviruses are replication defective such that they can only undergo one cycle of infection
48
Q

What are three important retrovirus aspects?

A
  • Integrates into genome
  • Initially an RNA genome
  • Amplify many copies, then shed them off
49
Q

Describe the production of recombinant retroviruses

A
  • Packaging cells have defective viruses that produce all proteins but lack ability to produce infections particles on their own
  • 3T3 cells are commonly used as platforms to develop packaging cells
  • Co-infection of recombinant retrovirus DNA vector into a packaging cell by transfection results in RNA copy of the vector generated and packaged as infectious particles
  • Virus particles are released in culture medium at 1 particle/hr/cell
  • Infection of target cells results in generation of DNA copy which integrates as a provirus to correct the gene defect
  • Since vector does not have viral genes, it cannot replicate
50
Q

Describe adenoviral vectors

A
  • Icosahedral DNA virus with linear dsDNA of 36kb
  • No DNA integration**
  • Subgroup C viruses cause mild respiratory disease
  • Early (E) and late (L) region genes are recognized based on their transcription before/after DNA replication
  • Genome has short inverted terminal repeat (ITR) segment necessary for viral replication
51
Q

Describe the adenovirus life cycle

A
  • Unlike retroviruses, replication causes lysis of the cell
  • Virus binds to cell surface receptor and is internalized
  • Particle enters endosome that fuses with lysosome
  • Low pH induces conformational change that disrupts endosomes
  • DNA is released, transported to nucleus
  • DNA remains as episome (extra chromosome) without integration
52
Q

What are the advantages of adenoviruses?

A
  • Biology of AV is well characterized
  • Not associated with severe human pathology
  • Extremely efficient at introducing DNA into host
  • Infect wide variety of cells both resting and dividing
  • Can be produced in large quantities at ease and stored frozen
  • Unlike retrovirus, AV can transduce terminally differentiated, non-dividing cells
53
Q

What are the limitations of adenovirus vectors?

A
  • DNA is not integrated into host cell genome (unlike retroviral vectors) therefore therapeutic gene can be lost after cell division
  • Lifelong treatment requires repeated injections
  • AV elicits humoral immune response that inhibits subsequent delivery of therapeutic gene
  • Some cells show low levels of AV proteins in absence of E1 and induce inflammatory response to eliminate the cells
54
Q

What are adeno-associated virus vectors?

A
  • Members of parvovirus family (icosahedral shaped virus, lack envelope)
  • Resistant to heat, chloroform, alcohol
  • 5kb strand of DNA
  • Not associated with human disease
  • “Dependo” virus (i.e., needs another virus to replicate like AV or herpes virus)
  • AAV establishes latent infection and integrates into DNA to region 19q13.3 of chromosome 19
55
Q

Describe AAV recombinant vectors

A
  • Entire protein coding domain can be replaced by the gene(s) of interest
  • TRs (on either side of gene of interest) are the only cis-acting elements required for all steps of life cycle, including replication, chromosomal integration, and packaging
  • Such vectors are packaged into virions by providing AAV proteins in trans
  • Vector and packaging plasmid is transfected into wild type AV infected cells
  • Mixture of AV and AAV can be resolved by heat inactivation to clear AV (AAV is heat stable, so will remain)
56
Q

Describe AAV vector integration

A
  • AAV vectors integrate into host chromosome with high efficiency
  • However, the fidelity of site specific integration of the wild type virus is not seen in the recombinant vector
  • Risk of transformation or oncogenesis is issue and needs to be addressed
  • Since entire AAV protein domain can be deleted, host immunity to proteins is avoided
57
Q

What are some applications of AAV vectors?

A
  • Gene transfer to lung cells seen for around 3 months
  • Hematopoietic stem cells constructed ex vivo for Beta-thalassemia and sickle cell anemia
  • Gene copies can correct Beta-globin defects seen in these diseases
58
Q 
For retroviruses, describe:
Genome transfer
Maximum size of recombinant gene
In vivo use
Integration
Non-dividing cells
A 
Genome transfer = RNA
Maximum size of recombinant gene = 8kb
In vivo use = No* (retrovirus producer cells are used in vivo)
Integration = Yes
Non-dividing cells = No
59
Q 
For adenoviruses, describe:
Genome transfer
Maximum size of recombinant gene
In vivo use
Integration
Non-dividing cells
A 
Genome transfer = DNA
Maximum size of recombinant gene = 7.5kb
In vivo use = Yes
Integration = No
Non-dividing cells = Yes
60
Q 
For AAV, describe:
Genome transfer
Maximum size of recombinant gene
In vivo use
Integration
Non-dividing cells
A 
Genome transfer = DNA
Maximum size of recombinant gene = 5kb
In vivo use = Yes
Integration = Yes
Non-dividing cells = Yes
61
Q 
For naked DNA, describe:
Genome transfer
Maximum size of recombinant gene
In vivo use
Integration
Non-dividing cells
A 
Genome transfer = DNA
Maximum size of recombinant gene = At least 50kb
In vivo use = Yes
Integration = Low
Non-dividing cells = Probably
62
Q 
For liposome-mediated vectors, describe:
Genome transfer
Maximum size of recombinant gene
In vivo use
Integration
Non-dividing cells
A 
Genome transfer = DNA or RNA
Maximum size of recombinant gene = At least 50kb
In vivo use = Yes
Integration = Low
Non-dividing cells = Probably
63
Q

Clinical studies target what types of diseases?

A
  • Adenosine deaminase deficiency
  • Cancer
  • Genetic disorders
  • Immune disorders
  • Cardiovascular disorders

PHARM 321 (9 decks)

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