BIOEN 502 Week 4
Terms
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- Why deliver nucleic acid?
- Gene therapy, downregulating proteins, label cells, create new cell lines
- What are the 4 kinds of nucleic cadids that can be delivered to cells?
- plasmid dna, artificial (yacs, bacs), antisense DNA or RNA, double stranded RNA (RNAi)
- What are the three barriers to NA delivery?
- Extracellular transport/degradation, cell membrane entry, endosome
- Why is it easier for RNA delivery than DNA delivery?
- RNA needs to only get to cytoplasm, DNA needs to get to the nucleus
- What is Ex-vivo gene therapy?
- gene transfer to cells in culture and replaced into person
- what is in vivo gene transfer?
- local or system delivery right into body
- What are the six considerations of gene delivery vector design?
- efficiency, toxicity, immunogenicity, duration of expression, size and type of nucleic acid delivered, ease and cost of production
- What are the four physical methods for non-viral delivery?
- electroporation, gene gun, microinjection, ultrasound
- What is electroporation and what is its drawback?
- Shock opens pores for DNA/RNA to get into cell, but results in a high cell death rate
- What is the gene gun?
- DNA on a gold particle and shot into cell
- What is microinejction
- Injection of recombinant DNA into zygotes, for generation of transgenic mice
- What is ultrasound-mediated gene delivery?
- High energy ultrasound waves tgransiently creates pores in cell membranes, used with microbubble blasting, enhances transfer of naked DNA in muscle
- What is calcium phosphate precipitation for gene delivery?
- DNA is mixed with calcium chloride and added to phosphate buffered solution, fine precipitate forms and is attached to the cells, glycerol shock used to increase efficiency. inexpensive, but low efficiency
- Why are cationic polymers used?
- condense DNA/RNA
- What are commonly used cationic polymers?
- Polyethlemimine (PEI), superfectin (dendrimer), DEAE-Dextran
- What are the limitations of cationic polymers
- limited success in vivo, high expression high toxicity
- What cationic lipids for?
- DNA condensation
- What are common cationic lipids?
- DOTAP, lipofectamine, FuGene
- What are limitations of cationic lipids
- toxic for in-vivo
- What are 5 advantages of non-viral gene delivery
- Not limited with type or size of nucleic acid, low to no immunogenicity, possible toxicity, low efficiency, no integration
- Improvements to non-viral gene delive yvectors
- Targeting cell receptors, enhancing endosomal escape (fusogenic peptides, ph sensitive), inclusion of nuclear localization signal peptides
- advantages of Viral vectors for gene delivery
- very efficient
- What is a retrovirus vectors
- RNA genome is reverse-transcribed and integrated into the genome, vectors produced by co-transfection of packaging cells, part of the genome replaced with expression cassette
- non-viral delivery and viral delivery names
- transfection and transductionq
- adenovirus vectors
- double stranded DNA, does not integrate to genome, easily produced , higher packing capacity of cassette
- What are the advantages and disadvantages of adenoviruses?
- infects all dividing and nondividing cells , high efficiency, but highly immunogenetic
- adeno-associated virus viruses
- small nonpathogenic, can only replicate with helper virus, only 4.7 kb
- herpes simplex virus vectors
- enveloped double stranded DNA virus, large genome, does not integrate but has a latent stage
- What would capsid modifications be done for?
- Get viral vectors to be non-immunogenic, and get targeting
- What are the five considerations of viral vectors?
- High efficiency, highly selective without modification, can mediate integration for long term expression, packaging capacity is limited, but can be highly immunogenic
- What are the 4 major gene therapy strategies?
- Gene addition, site-specific genome modification, gene silencing, splice-switching oligonucleotides
- What composes of gene addition vectors? (3)
- Promoter, cDNA, polydenylation signal
- Where are the two modes of functions for gene addition?
- Intracellular proteins (structural, enzymes), secreted proteins (clotting factors, insulin)
- What are fluorescent protein reporter genes?
- GFP, RFP, YFP
- What are enzyme reporter genes?
- luciferase, beta-galactosidase, alkaline phosphatase
- What are the four major concerns of gene addition?
- Efficacy, persistence, regulation of expression, safety
- What determines gene delivery efficacy?
- Number of cells gene is delivered to and how much is expressed in each transfected cell
- What are 5 ways of administrating gene therapy vectors?
- Intravenous injection, direct injection to tissue, intraperitoneal injection, transdermal, intranasal, injection in eye, retrograde transport in neurons, implantation of biomaterials with nucleic acids
- What vectors are used for ex vivo gene therapy?
- retro-Viral vectors, or non-viral vectors with transposases
- Where do Sleeping Beauty transposons integrate?
- AT repeats, not specific
- How can the Sleeping Beauty transposon be made specific?
- Fusion with zinc finger DNA binding domain
- where does ntegration with integrase occur?
- attB sitse
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what are four methods of transient gene addition?
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DNA vaccines, Cancer gene therapy, regenerative medicine, generation of induced pluripotent cells
- What is a DNA vaccine?
- Elicits cellular and humoral response
- What is cancer immunotherapy?
- Deliver adenovirus that has immunomodulatory genes
- What cells can be targeted by cancer immunotherapy
- can target cancer cells or immune cells
- What cancer suicide gene therapy?
- delivery pro-drug converting enzyme, then deliver pro-drug
- What is gene therapy for tissue regeneration for?
- skeletal muscle, cardiac muscle, bone, nerve
- What can done about skeletal muscle regeneration?
- IGF (insulin-like growth factor), inhibition of myostatin
- What can be done about cardiac muscle regeneration?
- VEGF, FGF, HGF, IGF, E2F2
- What can be done about bone regeneration?
- BMPs, RUNX2, VEGF
- What can be done about nerve regeneration?
- NGF, GNDF, IGF
- What is gene transfer of pluripotency?
- Expression of some combination of pluripotency genes in differentiated cells
- what are two ways of deliverying siRNA?
- delivery expression of short repeating sequences and turned into siRNA, or delivery siRNA itself