Lec 5- Nucleic acid based systems Flashcards

Question

Vectors used in nucleic acid delivery systems

Answer 1

* A) **Electroporation:** Uses short pulses of high voltage of electric current to carry DNA across the cell membrane * This shock causes the temporary formation of pores in the cell membrane, allowing DNA molecules to pass through * B) **Gene gun:** DNA is coated with gold particles and loaded into a device which generates a force to achieve penetration of DNA/gold into the cells

Answer 2

* C) **Sonoporation:** Uses ultrasonic frequencies to deliver DNA into cells * The process of acoustic cavitation is thought to disrupt the cell membrane and allow DNA to enter into cells * D) **Magnetofection:** DNA is complexed to magnetic particles, and a magnet is placed underneath the tissue culture dish to bring DNA complexes into contact with a cell monolayer * Pulling nanoparticles into the cells with the magnets

Answer 3

* A virus is a small infectious agent that can replicate only inside the living cells of organisms * Viruses have a natural ability to infect cells * Virus particles (virions) * Genetic material made from either DNA or RNA * Protein coat that protects these genes * Lipids that surrounds the protein coat

Answer 4

* Influenza virus becomes attached to a target epithelial cell * The cell engulfs the virus by endocytosis * Viral contents are released. Viral RNA enters the nucleus where it is replicated by the viral RNA polymerase * Viral mRNA is used to make viral proteins * New viral particles are made and released into the extracellular fluid. The cell, which is not killed in the process, continues to make new viruses

Answer 5

* Involves the use of attenuated or defective viruses * A) A retrovirus (An RNA virus) * X-linked severe combined immunodeficiency (X-SCID)- the most successful application of gene therapy to date * Problem: * Insert the genetic material into any arbitrary position in the genome of the host (Not targeted). Five children in the trial have developed leukaemia as a result of insertional mutagenesis by the retroviral vector

Answer 6

* B) Adenovirus (DNA virus) * Gendicine, adenoviral p53-based gene therapy was approved by the Chinese FDA in 2003 for head and neck cancer- very safe, decreased efficacy * Advexin, a similar gene therapy approach from Introgen, but turned down by USFDA in 2008 * Death of Jesse Gelsinger in 1999 while participating in a gene therapy trial

Answer 7

* Envelope protein pseudotyping of viral vectors * Herpes simplex virus (HSV)

Answer 8

* The EMA approved Glybera * For the treatment of lipoprotein lipase deficiency * LPLD: A very rare inherited condition that is associated with increased levels of fat in the blood * Glybera introduces a normal, healthy LPL gene into the body so that it can make functional LPL protein * It consists of the LPL gene packaged in a non-replicating adeno-associated virus (AAV) which has a particular affinity for muscle cells- which is its active site * It is administered via one-tome series of up to 60 small intramuscular injections in the legs

Answer 9

* Cationic- charges won't repel each other * Steric stabilisation- prevent degradation * targeting ligands- look at what ligands (including proteins) make up the membrane

Answer 10

* Condenses the size of the nucleic structure * Helps protect from nuclease degradation- smaller less likely to interact as well as different shape for nuclease enzymes * These enzymes bind to negatively charged nucleic acid systems * Help improve circulation time * Avoids recognition by the kupffer cells * Improve cellular uptake * This is the end goal/main focus- all of these process are improve to increase uptake

Answer 11

* Due to the anionic nature of the nucleic acids, cationic liposomes are used to electrostatically bind to the nucleic acids * Lots of NH2 groups * This can * Condense DNA (reduce the size) * Mask its anionic nature * Protect it from nuclease degradation * Very well packaged- no external interactions, less chance of breaking

Answer 12

1. A hydrophobic lipid anchor group which helps in forming liposomes and can interact with cell membranes 2. A linker group 3. A positively charged head group 1. DOTMA 2. DOTAP 3. DC-ChE 4. Often co-lipid DOPE is included in formulations * These charged head groups, give a similar structure to standard phospholipid layer * This means interactions between surface of liposomes and cells that it approaches

Answer 13

* Co-lipid commonly used in liposomes used for nucleic acid delivery * Though to facilitate the release pDNA (plasmid DNA) from endosome (Efficient release of the pDNA from the endosomal compartment is a limiting step in gene expression) * When DOPE-containing liposomal-DNA complexes are taken up by the endosome, all of the cationic lipid headgroups are neutralised by the anionic lipids in the endosomal membrane * Promote membrane breakdown in acidic conditions (e.g. endosomes)

Answer 14

* Step A: Cationic liposome/nucleic acid complex is endocytosed * Step B: In the early endosome, membrane destabilisation results in anionic phospholipid flip-flop * Step C: The anionic lipids diffuse into the complex and form charge-neutral ion-pairs with the cationic lipids * Step D: The nucleic acid dissociates from the complex and is released into the cytoplasm

Answer 15

* Red= part of original cell membrane * membranes fuse together

Answer 16

* Correctly target required site * Use targeting groups on the surface of liposomes, e.g. Ab's * Protect against degradation * Neutralise charge and protect with a lipid coat * Enhanced cellular uptake * Improve stability and improve the amount of reaching cells * Cell uptake thought to be via endocytosis (Improve using receptor-mediated endocytosis)- DOPE, flip flop * Improved exit from sub-cellular compartments * Incorporate DOPE in the formulation which improves release via flip-flop mechanism * Improve entry into the nucleus if required for action * Free DNA released from endosome may enter the nucleus through pores in the nuclear membrane

Answer 17

* Protonated amine group (NH₃⁺) interact with phosphate groups of the NA's * This can condensing DNA, reduce the size, mask its anionic nature and protect it from nuclease degradation * Vary in terms of their molecular mass, their shape * Their backbone can also be modified by the introduction of side chains or target-specific moieties

Answer 18

* The cationic polymer (such as PEI) soaks up the H+ ions and becomes more cationic at low pH, leading to inflow H+ and Cl- and water into the endosome * This causes osmotic swelling, burst open the endosome and releasing the DNA

Answer 19

* Composed of: Primary amine; Secondary amines; **Tertiary amines** * **Lots of hydrogen** * This increase in protonation as the pH drops can have a buffering effect inside the endosome

Answer 20

* Disulphide- linked polymers are not stable intracellular due to high glutathione concentrations, but stable extracellular due to low glutathione concentrations * Positive charge on the surface= better uptake into the cell

Answer 21

* Very low transfection efficiency * Rapidly cleared by the reticuloendothelial system (RES) * After IV injection high expression in the lungs has been noted * Clearance may be reduced by pegylation * Local injection of complexes have shown to be ineffective