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Y2S1 302 > 15: Gene Therapy > Flashcards

15: Gene Therapy Flashcards

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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
stability of polyplexes depend on the _____ and on the DNA/ RNA polymer ________
polymer structure | DNA/RNA polymer charge ratio
26
which polyplexes are more likely to remain in solution 1. negative 2. positive 3. neutral
positive
27
negatively charged polyplexes tend to adsorb to ____, causing further aggregation and can lead to _______________
adsorb albumin | can lead to rapid clearance of the polyplexes
28
how to reduce polymer/ RNA/ DNA binding strength
reduce number of positive charges reduce conjugation of PEG chains decreasing polymer molecular mass increase gene expression
29
how to do cell specific targeting for nonviral vector
attach targeting moieties that allow increased cell uptake and specificity
30
liposomes use _____ to get into cells and release genetic material, while polyplexes use _______
``` liposomes = membrane destabilization polyplex = proton sponge effect with osmotic lysis ```
31
a vector with high stability and gene transduction but limited packaging capacity describes ______
viral vectors
32
describe NHEJ
nonhomologous end joining- break ends are directly ligated without need for homologous template high error
33
describe HDR
homology directed repair | repairs according to template = less errors
34
4 CRISPR mechanisms
inhibition of RNA polymerases activation epigenetic modulators induction
35
advantages of CRISPR
target design simplicity efficiency multiplexed mutations
36
disadvantages of CRISPR
off target effects need to be defined on a genome wide scale
37
list 3 limitations of gene therapy and what we need to improve on
``` enhance transfection efficacy vector establishment vector maintenance in host cell nucleus decrease plasmid loss cell toxicity production costs ```
38
when using viral vectors in clinical trials, there is the concern of _______ from integrating vectors
genotoxicity and immune responses
39
cancer gene therapy may use vectors to transfer _________ or to inhibit _______
transfer tumor suppressor genes | use gene therapy to inhibit oncogenes/ other genes that promote tumor growth/ progression
40
immunotherapies for cancer gene therapy
genetically modify T cells genetic vaccine strategies- transfer genes encoding tumor associated antigens induce susceptibility to other therapies
41
describe genetic vaccine strategies
transferring genes encoding tumor associated antigens
42
______ gene is mutated in 60-80% of all human cancers
p53
43
p53 functions
induces cell cycle arrest, senescence, apoptosis, autophagy = prevents accumulation of genetic mutations within the cell
44
T or F: gendicine shows better efficacy combined with chemo and radiotherapy compared to either monotherapy
T
45
what is gendicine
recombinant adenoviral vector expressing p53
46
what has gendicine been used for
head and neck cancer breast cancer liver cancer
47
luxterna is a ______ based gene therapy that carries a functional copy of the _____ gene
AAC | RPE65
48
what is the process that luxterna takes to help people see
RPG65 gene delivery RPG65 protein production restoring the visual cycle
49
neovasculgen is a ________ encoding _______. it causes _______
plasmid encoding VEGF with a CMV promoter | it causes growth of collateral blood vessels
50
collategene is a ______ encoding _________
plasmid encoding human HGF with CMV promoter | has potentially angiogenic activity for treatment of ischemic disease
51
microRNAs mediate _______ of a particular mRNA and _______ for imperfectly complementary targets
translational repression | transcript degradation
52
siRNA sequence specific cleavage of _______________ __
perfectly complementary mRNA
53
what are the 2 types of off target effects needed to be avoided or minimized
silencing of genes sharing partial homology to the siRNA | immune stimulation due to recognition of certain siRNAs by the innate immune system
54
activation of the endogenous gene silencing pathway can happen in 2 ways
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
how do siRNAs work
how do microRNAs workcomplements mRNA seuence, causing cleavage
56
how do microRNAs work
associates with mRNA and causes translational repression, then degradation
57
____ = mRNA cleavage, ______ = translational repression and mRNA degradation
siRNA | microRNA
58
which is less likely to interfere with gene regulation, siRNA or microRNA
siRNA
59
3 steps of turning siRNA into drugs
1. select siRNA 2. stabilize siRNA with 2' base mod and P=S backbone mod 3. select delivery method ex- nacked, liposomes, antibodies
60
in stabilized siRNA, what does the P=S backbone linkage do
protects against exonuclease degradation
61
which end is the P=S backbone linkage at in optimized siRNA
3'
62
the 2' sugar modification in siRNA provides ________
endonuclease resistance
63
therapeutic mRNA are highly ___ and do not induce _________
translatable | serious inflammation
64
what mRNA modifications have been made in vaccines to reduce toxicity and improve translation
incorporation of modified nucleosides (esp uridine) optimized coding sequences stringent purification
65
most important innovations in mRNA vaccine technology include
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
describe the ex vivo prpocess of anti cancer vaccines
load dendritic cells with mRNAs that are either synthetic or have been isolated from whole tumor cells
67
nonreplicating or conventional mRNA based vaccines encode the ________ and contain ________
encode antigen of interest | 5' and 3' untranslated regions
68
self amplifying RNAS encode ________ and ________
antigen and viral replicating machinery that enables intracellular RNA amplification
69
describe the process of CAR-T or adoptive cell transfer based immunotherapy
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
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
alphabeta receptors | chimeric antigen receptors
71
what are some limitations of current gene therapy
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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