Gene Therapy Flashcards
gene therapy
a set of approaches to treatment of human disease based on the transfer of genetic material (DNA or RNA) into an individual
genetic disease and gene therapy
require life-long expression of a corrective gene
acquired disease and gene therapy
may only require expression of corrective gene for as long as the disease persists
uses of gene therapy
1) Alter or supplement the function of a mutated gene by providing a copy of the normal gene (gene augmentation-adding)
2) Directly alter and/or repair the mutated gene
3) provide a gene that adds missing functions or regulates the expression of another gene (make cells express toxin, become suceptible to a drug, or targets of the immune system)
4) can also be examples of silencing or “inhibiting” genes (siRNA expression)
two factors for the success of gene therapy
ability to get the gene into an appropriate cell and ability to express the transgene
in vivo gene therapy
direct administration of viruses or DNA encoding therapeutic gene to blood or tissues
dividing and nondividing cells
episoma, unstable, and treatment needs to be repeated
ex vivo gene therapy
indirectly through the introduction of cells manipulated in the laboratory to harbour foreign DNA
gene transfer requires cell division
integrates into host genome, stable
gene persists for life
What do we need to conduct a gene therapy protocol?
identification of the gene responsible for the disease - knowledge of protein function
a wild-type allele of the gene (normal functioning copy)
a convenient method to target the functional wild-type gene to appropriate sites
features of an ideal gene delivery vehicle
safety
reach target sites efficiently
express only in target sites
express at optimum levels
long-term therapeutic effect
relatively simple, commercially viable
integrating vectors
retrovirus - long-term, stable integration, permanent, only dividing cells
lentivirus - long-term, stable integration, dividing and non-dividing cells
non-integrating vectors
herpes virus - episoma, long-term
adenovirus - episomal, temporary
adeno-associated virus - stable, long-term, episomal
naked DNA - mostly, episomal, not efficient and temporary
liposomes as methods of gene delivery
episomal, many tissues, temporary, genes get trapped in endosomes
cell tropism
the way in which different virueses/pathogens have evolved to preferentially target specific host species, or specific cell types within those species
important because this defines the ability of a viral vector to transduce/enter a particular cell type
pseudo-typed
changing the envelope protein from what it would normally be used to something else
essential genes removed in virual vectors
gag, pol, and env
gets rid of important proteins for cell function, use packaching and producer cells to create the virus that won’t spread
advantages and disadvantages of retroviruses
advantages - 10kb cloning capacity, integrate into genome of target cell, does not transfer virus protein coding sequences
disadvantages - only integrates into actively dividing cells, integration could lead to gene activation or inactivation, low titer
characteristics of lentiviruses
linear single-stranded RNA genome
viral particles is diploid
four proteins are required for assembly of function HIV-1 based vectors
lentiviral vector
contains only minimal HIV sequences
defective virus is packaged into viral proteins using an unrelated virus caspid protein
advantage - transcduces non-dividing cells as well as dividing cells, many different cell types
disadvantages - it’s HIV
adenovirus
non-enveloped icosohedrial virion
dsDNAgenome of 36kb
over 47 different serotypes identified
early region 1 (E1) essential for replication, first region transcrived after infection
E1 can be removed and replaced with therapeutic gene
new vectors contain no viral coding sequences and have coding capacity of 36kb
adenovirus vector characteristics
advantages - replication defective, high transduction efficiency, both dividng an dnon-dividing cells, many different cell types and tissues, relatively large cloning capacity, can be grown to high titer
disadvantages - replication competent adenovirus. inflammatory and immune responses, transient transgene expression
adeno-associated viruses
non-enveloped virus, productive infetion requires helper virus
linear single-stranded DNA genes
inverted terminal repeats
AAV2 is the most commonly used serotype for rAAV vectors to date
AAV-2
wild-type preferentially integrates into human chromosome 19, though recombinant AAV vectors are now accepted as being episomal
non-pathogenic - wild type virus has not been implicated as etiological agent for any human disease
broad tissue tropism and host cell range - rAAV vectors have been shown to transduce many primary and cultured cells
AAV to rAAV
ITRs (inverted terminal repeats) flank the coding segments of the AAV genome
replication coding region and caspid coding region replaced with the therapeutic cDNA or gene
DNA gene therapy
direct injection of plasmid DNA can lead to expression of transgene
advantages - simple, relatively safe, ease of large-scale production, lack of specific immune response
disadvantages - relatively inefficient because not enough gets into the cell or cell nucleus, typically transient expression, “patchy”
good as DNA vaccines
bystander effect
when the thymadine kinase/gancyclovir(prodruge) system is used, very few cells expressing the TK gene are sufficient to eradicate the whole tumor even though the majority of cells do not express TK
