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Y2S1 302 > 15: Gene Therapy > Flashcards

15: Gene Therapy Flashcards

1
Q

T or F: germline gene therapy requires targeting

A

F

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2
Q

which type of gene therapy involvevs insertion of genes into diploid cells

A

somatic

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3
Q

somatic gene therapy can be done in 3 ways

A

ex vivo, in situ, in vivo

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4
Q

which of the following involves changing a person’s own immune cells to fight abnormal cells inside their body

  1. CART cell therapy
  2. introducing a new gene
  3. inserts of specific pieces of existing DNA
  4. replacing a mutated gene
A

1

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5
Q

what are some physicla methods of getting genes into cells

A 
ultrasound
electroporation
magnetofection
gene gun
naked DNA directly into cells
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6
Q

____ is a nonviral vector, while ____ are viral vectors for systemic deliveries

  1. cationic nanoparticles, micelles
  2. cationic polymers, liposomes
  3. adenovirus, lentivirus
  4. micelles, retrovirus
A

4

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7
Q

list some extracellular barriers present in inserting gene therapy

A
  • nuclease attack
  • tightly packed endothelial cells
  • nonspecific plasma/ vessel protein interaction
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8
Q

what are some intracellular barriers

A

must be recognized and endocytosed + be able to escape endosome

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9
Q

list the 7 desired properties of a vector

A 
protection of cargo
easy administration
serum stability
targetability to specific cell types
ease internalization
efficient unpackaging
non toxic, nonimmunogenic, nonpathogenic
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10
Q

what vectors are most commonly used in clinical trials

A

viral

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11
Q

how are RNA viruses adapted for transduction

A

all viral genes will be replaced with a transgene, so the retroviral vector genome can integrate into host genome

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12
Q

which infects proliferating cells only , and which integrates into both dividing and nondividing cells

A 
retrovirus = proliferating only
lentivirus = both
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13
Q

T or F: for retrovirus and lentivirus, there is potential for insertional mutagenesis, esp in immune compromised patients

A

T

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14
Q

how to target viral vectors

A

change the tropism by replacing the viral attachment protein (pseudotyping)
adding adapters that will be recognized by different cells

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15
Q

what is pseudotypping

A

adding different proteins from different viruses onto a virus so it has a higher capacity of being recognized and uptake = higher capacity of infection

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16
Q

list some disadvantages of liposomes

A 
low transfection and integration efficacy
small degree of toxicity
low solubility 
short half life
oxidation or hydrolysis rx
cost
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17
Q

how do cationic polymers work

A

complex with anionic pDNA via electrostatic interactions + provides protection against nucleases and enable cellular uptake

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18
Q

cationic lipids include (select all that apply)

  1. multilaminar vesicles
  2. multi vesicular vesicles
  3. cationic polymers
  4. unilaminar vesicles
A

1, 2, 4

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19
Q

list some disadvantages of cationic polymers

A

IV administration can result in particle instability
low transfection efficacy
toxicity
nonspecific interactions with blood
opsonization, aggregation of RBCs, platelet aggregation

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20
Q

organic nanoparticles include

  1. silica
  2. polymer micelle
  3. CRISPR gold
  4. DOTAP
A

1

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21
Q

disadvantage of organic nanoparticles

A

increase induction of cytokines and immune response

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22
Q

5 points of design criteria for a nonviral vector

A 
overcome extracellullar barriers
block access of nucleolytic enzymes
balance between binding strength and ability to release plasmid
DNA/ RNA unpackging
cell specific targeting
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23
Q

how do nonviral vectors block access of nucleolytic enzymes

A

condense DNA

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24
Q

T or F: strong binding and efficient DNA condensation directly correlate with gene transfection efficiency

A

F- do not correlate directly

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25
Q

stability of polyplexes depend on the _____ and on the DNA/ RNA polymer ________

A

polymer structure

DNA/RNA polymer charge ratio

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26
Q

which polyplexes are more likely to remain in solution

  1. negative
  2. positive
  3. neutral
A

positive

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27
Q

negatively charged polyplexes tend to adsorb to ____, causing further aggregation and can lead to _______________

A

adsorb albumin

can lead to rapid clearance of the polyplexes

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28
Q

how to reduce polymer/ RNA/ DNA binding strength

A

reduce number of positive charges
reduce conjugation of PEG chains
decreasing polymer molecular mass
increase gene expression

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29
Q

how to do cell specific targeting for nonviral vector

A

attach targeting moieties that allow increased cell uptake and specificity

30
Q

liposomes use _____ to get into cells and release genetic material, while polyplexes use _______

A 
liposomes = membrane destabilization 
polyplex = proton sponge effect with osmotic lysis
31
Q

a vector with high stability and gene transduction but limited packaging capacity describes ______

A

viral vectors

32
Q

describe NHEJ

A

nonhomologous end joining- break ends are directly ligated without need for homologous template

high error

33
Q

describe HDR

A

homology directed repair

repairs according to template = less errors

34
Q

4 CRISPR mechanisms

A

inhibition of RNA polymerases

activation
epigenetic modulators
induction

35
Q

advantages of CRISPR

A

target design simplicity
efficiency
multiplexed mutations

36
Q

disadvantages of CRISPR

A

off target effects need to be defined on a genome wide scale

37
Q

list 3 limitations of gene therapy and what we need to improve on

A 
enhance transfection efficacy
vector establishment
vector maintenance in host cell nucleus
decrease plasmid loss
cell toxicity
production costs
38
Q

when using viral vectors in clinical trials, there is the concern of _______ from integrating vectors

A

genotoxicity and immune responses

39
Q

cancer gene therapy may use vectors to transfer _________ or to inhibit _______

A

transfer tumor suppressor genes

use gene therapy to inhibit oncogenes/ other genes that promote tumor growth/ progression

40
Q

immunotherapies for cancer gene therapy

A

genetically modify T cells
genetic vaccine strategies- transfer genes encoding tumor associated antigens
induce susceptibility to other therapies

41
Q

describe genetic vaccine strategies

A

transferring genes encoding tumor associated antigens

42
Q

______ gene is mutated in 60-80% of all human cancers

A

p53

43
Q

p53 functions

A

induces cell cycle arrest, senescence, apoptosis, autophagy = prevents accumulation of genetic mutations within the cell

44
Q

T or F: gendicine shows better efficacy combined with chemo and radiotherapy compared to either monotherapy

A

T

45
Q

what is gendicine

A

recombinant adenoviral vector expressing p53

46
Q

what has gendicine been used for

A

head and neck cancer
breast cancer
liver cancer

47
Q

luxterna is a ______ based gene therapy that carries a functional copy of the _____ gene

A

AAC

RPE65

48
Q

what is the process that luxterna takes to help people see

A

RPG65 gene delivery
RPG65 protein production
restoring the visual cycle

49
Q

neovasculgen is a ________ encoding _______. it causes _______

A

plasmid encoding VEGF with a CMV promoter

it causes growth of collateral blood vessels

50
Q

collategene is a ______ encoding _________

A

plasmid encoding human HGF with CMV promoter

has potentially angiogenic activity for treatment of ischemic disease

51
Q

microRNAs mediate _______ of a particular mRNA and _______ for imperfectly complementary targets

A

translational repression

transcript degradation

52
Q

siRNA sequence specific cleavage of _______________ __

A

perfectly complementary mRNA

53
Q

what are the 2 types of off target effects needed to be avoided or minimized

A

silencing of genes sharing partial homology to the siRNA

immune stimulation due to recognition of certain siRNAs by the innate immune system

54
Q

activation of the endogenous gene silencing pathway can happen in 2 ways

A

viral vector expressing short hairpin RNA that resembles microRNA precursors
introducing siRNAs that mimic the Dicer cleavage product into the cytoplasmhow do siRNAs work

55
Q

how do siRNAs work

A

how do microRNAs workcomplements mRNA seuence, causing cleavage

56
Q

how do microRNAs work

A

associates with mRNA and causes translational repression, then degradation

57
Q

____ = mRNA cleavage, ______ = translational repression and mRNA degradation

A

siRNA

microRNA

58
Q

which is less likely to interfere with gene regulation, siRNA or microRNA

A

siRNA

59
Q

3 steps of turning siRNA into drugs

A
  1. select siRNA
  2. stabilize siRNA with 2’ base mod and P=S backbone mod
  3. select delivery method ex- nacked, liposomes, antibodies
60
Q

in stabilized siRNA, what does the P=S backbone linkage do

A

protects against exonuclease degradation

61
Q

which end is the P=S backbone linkage at in optimized siRNA

A

3’

62
Q

the 2’ sugar modification in siRNA provides ________

A

endonuclease resistance

63
Q

therapeutic mRNA are highly ___ and do not induce _________

A

translatable

serious inflammation

64
Q

what mRNA modifications have been made in vaccines to reduce toxicity and improve translation

A

incorporation of modified nucleosides (esp uridine)
optimized coding sequences
stringent purification

65
Q

most important innovations in mRNA vaccine technology include

A

engineering of mRNA sequences
development of methods that enable simple, rapid, and large scale cGMP production of mRNA
development of highly efficient and safe mRNA vaccine delivery materials

66
Q

describe the ex vivo prpocess of anti cancer vaccines

A

load dendritic cells with mRNAs that are either synthetic or have been isolated from whole tumor cells

67
Q

nonreplicating or conventional mRNA based vaccines encode the ________ and contain ________

A

encode antigen of interest

5’ and 3’ untranslated regions

68
Q

self amplifying RNAS encode ________ and ________

A

antigen and viral replicating machinery that enables intracellular RNA amplification

69
Q

describe the process of CAR-T or adoptive cell transfer based immunotherapy

A

taking T cells specific for a tumor associated antigen (health donor T cell + editing), amplifying it, and transferring it into a tumor bearing host

70
Q

T cells (in adoptive cell transfer based immunotherapy), can be genetically engineered to express _______ receptors or ________ antigen receptors. These receptors can recognize tumor cell surfaces

A

alphabeta receptors

chimeric antigen receptors

71
Q

what are some limitations of current gene therapy

A

knockout efficiencies are low, so higher delivery efficiency is needed to compensate
vectors that provide transient expression or highly efficient off switch are needed

Y2S1 302 (10 decks)

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