15: Gene Therapy Flashcards
T or F: germline gene therapy requires targeting
F
which type of gene therapy involvevs insertion of genes into diploid cells
somatic
somatic gene therapy can be done in 3 ways
ex vivo, in situ, in vivo
which of the following involves changing a person’s own immune cells to fight abnormal cells inside their body
- CART cell therapy
- introducing a new gene
- inserts of specific pieces of existing DNA
- replacing a mutated gene
1
what are some physicla methods of getting genes into cells
ultrasound electroporation magnetofection gene gun naked DNA directly into cells
____ is a nonviral vector, while ____ are viral vectors for systemic deliveries
- cationic nanoparticles, micelles
- cationic polymers, liposomes
- adenovirus, lentivirus
- micelles, retrovirus
4
list some extracellular barriers present in inserting gene therapy
- nuclease attack
- tightly packed endothelial cells
- nonspecific plasma/ vessel protein interaction
what are some intracellular barriers
must be recognized and endocytosed + be able to escape endosome
list the 7 desired properties of a vector
protection of cargo easy administration serum stability targetability to specific cell types ease internalization efficient unpackaging non toxic, nonimmunogenic, nonpathogenic
what vectors are most commonly used in clinical trials
viral
how are RNA viruses adapted for transduction
all viral genes will be replaced with a transgene, so the retroviral vector genome can integrate into host genome
which infects proliferating cells only , and which integrates into both dividing and nondividing cells
retrovirus = proliferating only lentivirus = both
T or F: for retrovirus and lentivirus, there is potential for insertional mutagenesis, esp in immune compromised patients
T
how to target viral vectors
change the tropism by replacing the viral attachment protein (pseudotyping)
adding adapters that will be recognized by different cells
what is pseudotypping
adding different proteins from different viruses onto a virus so it has a higher capacity of being recognized and uptake = higher capacity of infection
list some disadvantages of liposomes
low transfection and integration efficacy small degree of toxicity low solubility short half life oxidation or hydrolysis rx cost
how do cationic polymers work
complex with anionic pDNA via electrostatic interactions + provides protection against nucleases and enable cellular uptake
cationic lipids include (select all that apply)
- multilaminar vesicles
- multi vesicular vesicles
- cationic polymers
- unilaminar vesicles
1, 2, 4
list some disadvantages of cationic polymers
IV administration can result in particle instability
low transfection efficacy
toxicity
nonspecific interactions with blood
opsonization, aggregation of RBCs, platelet aggregation
organic nanoparticles include
- silica
- polymer micelle
- CRISPR gold
- DOTAP
1
disadvantage of organic nanoparticles
increase induction of cytokines and immune response
5 points of design criteria for a nonviral vector
overcome extracellullar barriers block access of nucleolytic enzymes balance between binding strength and ability to release plasmid DNA/ RNA unpackging cell specific targeting
how do nonviral vectors block access of nucleolytic enzymes
condense DNA
T or F: strong binding and efficient DNA condensation directly correlate with gene transfection efficiency
F- do not correlate directly
stability of polyplexes depend on the _____ and on the DNA/ RNA polymer ________
polymer structure
DNA/RNA polymer charge ratio
which polyplexes are more likely to remain in solution
- negative
- positive
- neutral
positive
negatively charged polyplexes tend to adsorb to ____, causing further aggregation and can lead to _______________
adsorb albumin
can lead to rapid clearance of the polyplexes
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
how to do cell specific targeting for nonviral vector
attach targeting moieties that allow increased cell uptake and specificity
liposomes use _____ to get into cells and release genetic material, while polyplexes use _______
liposomes = membrane destabilization polyplex = proton sponge effect with osmotic lysis
a vector with high stability and gene transduction but limited packaging capacity describes ______
viral vectors
describe NHEJ
nonhomologous end joining- break ends are directly ligated without need for homologous template
high error
describe HDR
homology directed repair
repairs according to template = less errors
4 CRISPR mechanisms
inhibition of RNA polymerases
activation
epigenetic modulators
induction
advantages of CRISPR
target design simplicity
efficiency
multiplexed mutations
disadvantages of CRISPR
off target effects need to be defined on a genome wide scale
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
when using viral vectors in clinical trials, there is the concern of _______ from integrating vectors
genotoxicity and immune responses
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
immunotherapies for cancer gene therapy
genetically modify T cells
genetic vaccine strategies- transfer genes encoding tumor associated antigens
induce susceptibility to other therapies
describe genetic vaccine strategies
transferring genes encoding tumor associated antigens
______ gene is mutated in 60-80% of all human cancers
p53
p53 functions
induces cell cycle arrest, senescence, apoptosis, autophagy = prevents accumulation of genetic mutations within the cell
T or F: gendicine shows better efficacy combined with chemo and radiotherapy compared to either monotherapy
T
what is gendicine
recombinant adenoviral vector expressing p53
what has gendicine been used for
head and neck cancer
breast cancer
liver cancer
luxterna is a ______ based gene therapy that carries a functional copy of the _____ gene
AAC
RPE65
what is the process that luxterna takes to help people see
RPG65 gene delivery
RPG65 protein production
restoring the visual cycle
neovasculgen is a ________ encoding _______. it causes _______
plasmid encoding VEGF with a CMV promoter
it causes growth of collateral blood vessels
collategene is a ______ encoding _________
plasmid encoding human HGF with CMV promoter
has potentially angiogenic activity for treatment of ischemic disease
microRNAs mediate _______ of a particular mRNA and _______ for imperfectly complementary targets
translational repression
transcript degradation
siRNA sequence specific cleavage of _______________ __
perfectly complementary mRNA
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
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
how do siRNAs work
how do microRNAs workcomplements mRNA seuence, causing cleavage
how do microRNAs work
associates with mRNA and causes translational repression, then degradation
____ = mRNA cleavage, ______ = translational repression and mRNA degradation
siRNA
microRNA
which is less likely to interfere with gene regulation, siRNA or microRNA
siRNA
3 steps of turning siRNA into drugs
- select siRNA
- stabilize siRNA with 2’ base mod and P=S backbone mod
- select delivery method ex- nacked, liposomes, antibodies
in stabilized siRNA, what does the P=S backbone linkage do
protects against exonuclease degradation
which end is the P=S backbone linkage at in optimized siRNA
3’
the 2’ sugar modification in siRNA provides ________
endonuclease resistance
therapeutic mRNA are highly ___ and do not induce _________
translatable
serious inflammation
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
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
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
nonreplicating or conventional mRNA based vaccines encode the ________ and contain ________
encode antigen of interest
5’ and 3’ untranslated regions
self amplifying RNAS encode ________ and ________
antigen and viral replicating machinery that enables intracellular RNA amplification
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
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
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
