Gene therapy Flashcards
Types of gene therapy
- Gene substitution in nucleus: external gene donated by vector
- Gene correction in nucleus: Zn-finger nucleases/CRISPR
- Gene knock out: prevention of gene expression by small RNA
Ideal vector
- specifically targeting
- non-harmful to other cells
- permanently expressed in genetic diseases
- expressed only under treatment
- non-immunogenic
- adequate carrying capacity
- efficiently sufficient
Retroviruses
- enzymes: reverse transcriptase, protease, integrase
- structure: lipid envelope, glycoproteins, transmembrane glycoprot., capsid prot.
- genome: 8-11kB, 2sense mRNA + 2tRNA
- advantage: high mutation rate, recombination of proviruses, high efficiency
- disadvantage: infect only dividing cells, might lead to mutagenesis and cancer
Retroviruses can be replication defective or competent
defective:
- infect target cell and deliver payload
- fail to continue lytic pathway -> cell lysis
competent:
-continue to propagate themselves once infection occurs
Lentivirus
- subclass of retrovirus
- advantage: ability to integrate to genome of non-dividing cells
- disadvantage: site of integration is unpredictable, can disturb function of cellular genes, can cause cancer
Adenovirus
- dsDNA, pathogenic
- can not integrate into genome and is not replicated during cell division
- gene therapy and vaccination
- receptor-mediated endocytosis
- wild range of host cells
Adeno-associated virus
-ssDNA, nonpathogenic
-advantage:
infect both dividing and non-dividing cells
transduce different kind of cell types
requires helper virus
little immune response, no cytotoxity
-disadvantage:
small (5kb)
-receptors: heparan-S, FGFR, integrin, PDGFR
Non-viral vectors: liposomes and aposomes
entrance mechanism depends on cationic molecule
- both liposomal membrane and vector degraded in lysosome
- nuclear direction needs signal and helper molecule
- liposomes are not immunogenic
Transposons
- retrotransposon: RNA to DNA
- DNA transposon: cut and paste
Stem cells
Allogenic (from other person)
- totipotent (embryonic)
- umibilical cord, fetal tissue
Autologous (same person)
- multipotent
- limited differentiation lineage, low immunogenicity
- cardiac stem cell, adipose derived stem cell, bone-marrow
Induced pluripotent stem cell
programmed somatic cell to which certain genes are inserted
ZN-finger nucleases
- recognizes specific nucleotide bases in DNA
- FoKI domains cut both strands to cause double strand break
- Zn2+ coordinated by 2Cys and 2His
Non-coding small RNAs
RNAi:
- Rna interference, can recognize and bind to other RNA-> RNA degradation/translation inhibition
- siRNA, miRNA, aptamer
Ribozyms:
- recognize and cut other RNAs
- shRNA,snRNA,pRNA
Antisense RNA:
-complementer to a part of mRNA. binds to it, prevents translation
