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Genetics 2017 > 19. Gene therapy (p12) > Flashcards

19. Gene therapy (p12) Flashcards

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1
Q

Gene therapy

A

Any intervention aimed to manipulate specific genes replacements, eradication, repair, affecting expression

  • Real gene therapy
  • Modification of gene expression
  • May be: in vivo, ex vivo, in utero
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2
Q

Real gene therapy

A

Replacement of a lacking gene correction of a specific mutation

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3
Q

Key questions in gene therapy

A
  • Which disease can be treated?
  • How can it be treated? By which nucleic acid?
  • How is the nucleic acid introduced in the organisms?
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4
Q

Which diseases can be treated by gene therapy?

A

1) Monogenic diseases => lack of/nonfunctional protein (loss-of-function mutation, AR)
- Gene replacement

2) Monogenic diseases => malfunctional protein (gain-of-function mutation, AD)
- Gene replacement, expression inhibition

3) Complex diseases in which role of a specific gene/protein in the pathogenesis of the disease has been identified (e.g CML - BCR/ABL)

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5
Q

Complex diseases with specific gene/protein identified examples

A
  • CML: BCR/ABL
  • Colon cancer: p53
  • Obesity: leptin
  • RA: IL-1R
  • Wet macular degeneration: VEGF
  • Heart failure: sarcoplasmic reticulum Ca channel
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6
Q

Nucleic acids applied in gene therapy

A

1) DNA (plasmid, linearDNA-viral, artificial chromosome)
- Replacement therapy

2) RNA (antisense RNA, ribozyme, silencing RNA, miRNA, aptamer)
- Silencing function/expression of gene

3) Other modified nucleic acids (can be more stable)

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7
Q

DNA plasmid use and advantage

A

Usually occurs in bacteria

  • Can be used as tool itself in gene therapy
  • Often used to prod. viruses to be used in gene therapy
  • Advantage: easy amplification by help of bacteria
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8
Q

DNA plasmid components

A

1) Replication site: Ori
2) Antibiotic resistance gene - easy selection of bacteria
3) Promoter - promoter/enhancer/silencer
4) Gene to be expressed

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9
Q

Different DNA plasmids for different use

A

1) Expression plasmid (in lack-of-function mutations)
=> mRNA => synthesis of protein (to replace)

2) Silencing plasmid (in gain-of-function mutations)
=> shRNA (short hairpin) => inhibition of specific mRNA

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10
Q

Mechanisms of shRNA’s

A

1) DNA plasmid transcribed to shRNA
2) shRNA processed by Dicer enzyme => double-stranded silencing RNA (siRNA)
3) siRNA binds to RISC complex => single-stranded
4) Single-stranded siRNA binds to target mRNA => degradation

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11
Q

FANG vaccine

A

An anti-tumor plasmid with 2 components/functions:

1) GMCSF (gene for colony stimulating factor)
- Activate immune system (antigen presenting cells)

2) shRNA => silencing of furin protein
- Furin protein is needed for TGFβ (immune suppression

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12
Q

Medical application of shRNAs

A

1) FANG vaccine

2) FAP: familiar adenomatosus polyposis

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13
Q

shRNA in FAP

A

FAP: familiar adenomatosus polyposis

  • Use shRNAs against β-catenin mRNA (oncogene)
  • β-catenin functions: enter nucleus and incr. cyclins and cMYC
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14
Q

shRNA targeting with E.Coli

A
  • Plasmid encoding listeriolysin and shRNA introduced into E.Coli
  • E.coli endocytosed => E.Coli lysed => listeriolysin exposed => degrades endosome membrane and release shRNA to cytoplasm
  • Genes in plasmid (3 main): gene for cell surface receptor, listeriolysin (release gene) and shRNA gene
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15
Q

RNA molecules in gene therapy

A
  • Antisense RNA (arteficial RNA molecule)
  • Ribozyme: RNA w/enzymatic activity(cut target mRNA)
  • miRNA
  • siRNA
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16
Q

Antisense RNA

A

Arteficial RNA molecule which is complementer to specific mRNA

  • Inhibitory effect
  • Side effects: too long antisense RNA may induce antiviral responses (dsRNA formation)
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17
Q

miRNA pathway

A

Endogenous!

  • Pol II => pri-mRNA => Drosha => pre-miRNA
  • Exportin 5: export pre-miRNA from nucl=>cytoplasm
  • Pre-miRNA cut by Dicer => miRNA => RISC complex => single-stranded miRNA-RISC bind to target mRNA
  • *mRNA inhibited or degraded (regulation of gene exp.)
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18
Q

siRNA pathway

A

Exogenous! (virus or transposon)

  • Cut by Dicer => RISC complex => RISC complex => single-stranded siRNA-RISC bind to mRNA
  • *mRNA degraded (!) (antiviral defence)
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19
Q

Medical application of siRNAs (target mRNA)

A
  • TGF-β
  • VEGF
  • Cox-2
  • Viral RNA
  • EGFR
  • mTOR
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20
Q

Aptamers

A

RNA molecules that bind to a small molecule or a protein

  • Downregulation of gene expression (binding inhibit function of target molecule)
  • Similar function to Ab’s
  • Usually RNA molecules
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21
Q

Production of aptamers

A

SELEX cycle (similar to production of Ab’s)

1) Random nucleic acid pool (RNA molecules)
2) Target binding test (incubation)
3a) Removal of non-binding molecules
3b) Find the one with best affinity
4) Re-amplification

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22
Q

Aptamer example

A

Pegatinib (Macugen)

  • Inhibit VEGF
  • Used in wet macula degeneration
23
Q

How to introduce nucleic acids into cells?

A

1) Naked DNA (plasmid) - electroporation or sonoporatinol (US)
2) DNA binding positively charged nanoparticles - gene gun
3) Liposome (artificial double phospholipid layer)
4) Bacterium (E.coli)
5) Virus

24
Q

Most commolnly used viral vectors

A

1) Adenovirus (dsDNA)
2) Adeno-associated virus (ssDNA)
3) Retrovirus (ssRNA)
4) Lentivirus (ssRNA)

25
Q

Adenovirus (viral vector) - characteristics, entry, advantage/disadvantage, example

A

dsDNA

  • Does not integrate into genome
  • Mainly used in gene therapy trial
  • In vitro receptor: CAR => internalisation via integrins => endosome => microtubules => nucleus
  • Advantage: large capacity (Can contain large DNA)
  • Disadvantage: immunogenicity (cytokine storm)
  • Gendicine: deliver p53 into cells in head/neck cancer
26
Q

Adeno-associated virus (AAV) (viral vector) - characteristics, infection, advantage/disadvantage, example

A

ssDNA

  • Cannot replicate itself - no disease caused in humans
  • Does not integrate (if it does: into chr. 19 - non-oncogenic)
  • 2 forms of infection: Lytic (helper virus) or Latent (no helper virus)
  • Advantage: not immunogenic
  • Disadvantage: small capacity
  • Treatment of Leber congenital amaurosis with recombinant AAV vectors
27
Q

Leber congenital amaurosis

A

Usually AR

  • Thinned retina with visible choroid veins
  • Starts in childhood => progressive visual loss
  • Destruction of photoreceptors
  • Most common mut: RPE65 gene (retinol transformation)
  • Successful gene threapy: AAV
28
Q

Retroviral vectors - characteristics, infection, advantage/disadvantage, example

A

ssRNA - retrovirus

  • Can transcribe its RNA to DNA by reverse transcription
  • DNA integrates into genome! (provirus)
  • Infect only dividing cells
  • Disadvantage: dangerous (tumor initiation)
  • X-SCID treatment and ADA def. treatment
  • *Virulence factors removed - stops after DNA integr.
  • **Retroviral therapy only used ex-vivo!
29
Q

X-SCID

A

“Bubble boy syndrome” (gamma-retrovirus vector 1 - murine leukemia virus)

  • γ-chain of IL2-R mutated (T cell prolif)
  • Decreased immunity
  • Retroviral gene therapy successful, but some patients got leukemia
  • Best option: BM translplant or gene therapy
30
Q

Problems with retroviral vectors

A
  • Random where retrovirus integrate - can lead to tumor induction by activating oncogene or deactivate tumor suppressor gene
  • LTR: retroviral promoter - can be enhancer of far genes
  • Solution: SIN (self-inactivating virus) - decreased LTR function (gag, pol, env genes knocked out and LTRs partially deleted
  • Retroviral therapy only used ex-vivo!
31
Q

ADA deficiency

A

AR, Severe combined immunodeficiency

  • 20 % of cases: mut. of adenosine deaminase gene (ADA) => nuclear acid disorder => abnorm. prolif of lymphocytes
  • Treatment: enzyme replacement
  • Gene therapy: retroviral (very successful - no oncogenic effect)
32
Q

Lentiviral vectors - characteristics, infection, advantage/disadvantage, example

A
  • HIV-based virus
  • Transgene carrying virus-does not have wild-type HIV viral proteins
  • Surface modified - so not similar to HIV
  • Advantages:
    1. integration avoids oncogenic sites
    2. affects not only dividing cells
    3. SIN virus can be created - safe
    4. Can incorporate larger transgenes than other
    retroviruses
    *Promising vectors of future
33
Q

CRISPR/Cas9 system overview

A
  • From bacteria
  • Cas9: endonuclease - can cut both ss and ds
  • Guide RNA (crRNA) bind to Cas9 and target sequence
  • 2 types of genome engineering
    1. Genome engineering with Cas9 nuclease
    2. Genome engineering by double-nicking with
    paired Cas9 nickases
34
Q

CRISPR abbreviation

A

“Clustered Regularily-Interspaced Short Palindromic Repeats”

35
Q

CRISPR advantages

A
  • Precise targeting of modification
  • Application of more spacers allows modification of more genes at the same time
  • No foreign sequence is introduced into the genome
  • Easy to use
36
Q

Application of CRISPR/Cas9 system

A

Ex-vivo gene therapy - HIV

  • Hematopoietic stem cells isolated from HIV-patients
  • CCR5 gene inactivated (needed for HIV infection)
  • Cells administered back to patient
37
Q

Genome engineering with Cas9 nuclease

A

ds cleavage by Cas9 - can either result in:

1) Non-homologous end joining - no template (we can destroy gene)
2) Homology directed repair - template (we can introduce sequence and/or destroy)

38
Q

Genome engineering by double-nicking with paired Cas9 nickases

A

Cas9 nickases: modified to make ss cuts

  • ss cleavage by 2 diff Cas9/CRISPR
  • HDR: homology directed repair - we can introduce new sequence and/or destroy
39
Q

Therapeutic nucleic acids that remain and and act directly in the cytoplasm

A
  • siRNA
  • miRNA
  • Oligonucleotides
  • Ribozymes
40
Q

Therapeutic nucleic acids located in the nucleus, but not integrated

A
  • AAV
  • Adenovirus
  • Plasmids
41
Q

Therapeutic nuclear acids integrating into genome

A
  • Retrovirus
  • Lentivirus
  • Sleeping Beauty
  • ΦC31
42
Q

Therapeutic nucleic acids suitable to replace gene

A
  • Expression plasmids
  • DNA in viral vectors
  • (integrating systems)
43
Q

Therapeutic nucleic acids to reduce the expression of genes

A
  • siRNA
  • miRNA
  • Ribozymes
  • Antisense RNA
  • Plasmid-encoded shRNA
44
Q

Therapeutic nucleic acids with short-term effect

A
  • siRNA
  • miRNA
  • Ribozymes
  • Antisense RNA
  • E.g cancer therapy
45
Q

Therapeutic nucleic acids with medium-term effect

A
  • Expression plasmid
  • shRNA
  • E.g acute diseases, tumors
46
Q

Therapeutic nucleic acids with long-term effect

A
  • Episomal plasmids delivered by AAV vectors

E.g chronic diseases

47
Q

Therapeutic nucleic acids with final effect

A
  • Integrated systems (danger: irreversible effect)

E.g monogenic diseases

48
Q

AAV genome constituents

A

ssDNA

  • ITR (inverted terminal repeat) - regulates transcription, promoter, enhancer
  • Rep: genes encoding replication and transcription proteins
  • Cap: genes for capsid protein
49
Q

Preparing AAV vectors

A

3 plasmids involved:

  1. rAAV vector: carry transgene (betw. ITR regions)
  2. AAV packaging plasmid: with AAV structural protein genes
  3. Adenovirus helper plasmid - encodes proteins
    * AAV vectors may be introduced into different tissues - targeted cell type determined by serotype (AAV2/1 = muscle, RPE, lung)
50
Q

AAV vector composition

A
  • ITR: packing + transcription
  • CMV transcriptional enhancer element
  • Actin promoter, transcription start site
  • Exon, intron
  • hRPE65 transgene (for example)
  • SV40 virus-derived polydenylation signal
51
Q

Retroviral genome constituents

A
  • LTR: essential for integration+transcription. Promoter and enhancer at same time
  • Psi (Ψ): packing sequence
  • Gag: encode proteins
  • Pol: encode reverse transcriptase and integrase enz
  • Env: encode capsid proteins
  • When used as vector: gag, pol, env are knocked out and replaced by transgene
    • LTR may be partially deleted in vector (SIN)
52
Q

Adenovirus generations

A

3 generations:

1st: short insert
2nd: more deletions, short insert
3rd: deletetion of all viral genome, large insert
* 3rd generation used today - called “gutless” or “helper-dependent”
* *ITR and psi(Ψ) are kept

53
Q

How to prepare vector (adenovirus) for gene therapy

A

1) Amplification of transgene-carrying plasmid in bacteria
2) A cell infected with helper virus and the plasmid is also delivered
3) Enzyme in the cell inhibits package of helper virus genome
4) Thus, only the transgene can enter the otherwise normally produced viral proteins
5) The produced adenovirus contain transgene, but not viral genes (can only infect once) - “gutless vector”

Genetics 2017 (19 decks)

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