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PH4701- clinical pharmaceutics > Lec 5- Nucleic acid based systems > Flashcards

Lec 5- Nucleic acid based systems Flashcards

1
Q

The central dogma

A
  • Target early in this pathway and you can take advantage of the amplification mechanism
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2
Q

Introduction to gene therapy

A
  • Gene therapy is the insertion, alteration or removal of genes within individual cells and biological tissues to treat diseases caused by genetic disorders
  • A number of human diseases are known to be genetic in origin e.g. CF, Huntington’s and cancer
  • The therapeutic genes: DNA, oligonucleotides, siRNA and mRNA
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3
Q

Indications addressed by gene therapy clinical trials

A
  • Most diseases treat via this pathway is cancer- genetic factor, lots of funding
  • Monogenic disease- single change in a gene leading to a disease- far easier to target than other diseases
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4
Q

History of gene therapy

A
  • 1998, Fomivirsen was the first antisense oligonucleotide approved by the FDA. It was used in the treatment of CMV in immunocompromised patients
  • In 2003, the Chinese FDA approved the controversial and first adenovirus-based gene therapy for head and neck squamous cell carcinoma
  • 2004, Pegaptanib anti-VEGF aptamer, was marketed in the USA for age-related macular degeneration
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5
Q

From Lab to the clinic: progress so far

A
  • NB- Large failure rate
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6
Q

Mechanism of gene therapy

A
  • Potency potential of gene therapy
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7
Q

Cell transfection

A
  • Genetic material enters into the cell- (Similar to virus)
  • We will then interupt the centeral dogma- either put correct gene in or knock out deffective gene
  • In the endosome the DNA is not technically in the cell yet so we need to be able to release it
  • Then cross the nuclear membrane
  • Transcrption of functional protein
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8
Q

Types of gene therapy

A
  • Bind to genomic DNA in the nucleus and thus block transcription
  • The guide strand of siRNA activates the RNA-induced silencing complex (RISC) and then degrade the mRNA (RNA silence)
  • A complimentary (antisense) oligonucleotide bind their target (sense) mRNA and block the translation
  • Plasmid= insert a functional gene/ missing gene (Autoimmune disease)- must be in the nucleus
  • Antisense oligonucleotides/siRNA- block gene
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9
Q

Example 1: Pegaptanib

A
  • The first aptamer to be successfully developed as a therapeutic agent in humans- a milestone in drug development
  • In 2004, FDA approved pegaptanib an Anti-Vascular Endothelial Growth Factor (anti-VEGF), RNA aptamer
  • The treatment of all types of age-related macular degeneration
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10
Q

Biological barriers to gene delivery

A
  • Once in the endosome, we need to ensure it stays stable
  • And we need to ensure that we can get it out of the endosome to enter to the cytoplasm or transfer into the nucleus
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11
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A
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12
Q

Potential disadvantages and problems

A
  • Short-lived nature of gene therapy: susceptible to degradation
  • Trigger immune response- put DNA into a cell and recognises the new DNA (it shouldn’t be there, the immune cells will destroy the cells- do more damage)
  • Multigene disorders: high blood pressure, Alzheimer’s disease, arthritis and diabetes
    • More than one genetic factor can complicate things
  • Polyanions
  • Surfaces of cells and DNA are both negative therefore is a difficult membrane to cross
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13
Q

Summary of oligo action

A
  • Site of action
  • Genomic DNA- active site= nucleus
  • mRNA- active site= cytoplasm
  • Protein- active site= cytoplasm
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14
Q

Chemical modification of nucleic acids

A
  • Unmodified plasmid DNA, siRNA and phosphodiester (PO) backbone oligos are rapidly degraded by enzymes in biological fluids
  • To overcome the instability of oligos, chemically modified oligos have been developed
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15
Q

Phosphodiester backbone

A
  • Phosphorothioate oligos
  • Non-Bridging oxygen is replaced with sulphur
  • Swap out unbound oxygen with sulphur (Disulphide bond more stable)- less sensitive to degradation so is stable invivo and hydrophobic= partition more into the membrane
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16
Q

Example 2: Fomivirsen

A
  • The first antisense oligomer approved by the FDA in Aug 1998
  • A synthetic 21 member oligonucleotide with phosphorothioate linkages (which are resistant to degradation by nucleases
  • Sequence: 5’-GCGTTTGCTCTTCTTCTTGCG-3’
  • Treatment CMV in immunocompromised patients, including those with AIDS
  • Mechanism: Bind to the complementary sequence of the mRNA to block translation of viral mRNA
  • Administrated by intraocular injection
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17
Q

Example 3: Mipomersen

A
  • It is a cholesterol-reducing drug candidate
  • It targets the messenger RNA for apolipoprotein B
  • A second-generation antisense oligonucleotide
    • The nucleotides are linked with phosphorothioate linkages
    • The sugar parts are deoxyribose in the middle part of the molecule and 2’-O-methoxyethyl-modified ribose at the two ends
      • combination of RNA and DNA- the steric arrangement is different therefore the change of shape = won’t fit inactive site- change function
  • These modifications make the drug-resistant to nucleases
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18
Q

Modification of sugar

A
  • Losing the hydrophobicity may actually be benefitial
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19
Q

Locked Nucleic acids (LNA)

A
  • The ribose ring is locked by a methylene bridge connecting the 2’-O atom and the 4’-C atom- strengthening the overall molecule
  • Discovery in 1997 by Danish scientists in Danish
  • The locked ribose conformation enhances base stacking and backbone pre-organisation
  • More stable
  • Increase the sensitivity and specificity
20
Q

Peptide Nucleic Acid (PNA)

A
  • PNA: Consisting of repeating N-(2-aminoethyl) glycine units linked by peptide bond, the bases are attached to the backbone through methylene carbonyl linkages
  • PNA is being produced to match DNA
21
Q

PNA features

A
  1. The stronger binding between PNA/DNA strands then between DNA/DNA strands
    • H-TGTACGTCACAACTA-NH2 , It’s Tm is 69.5 ‘C - more stable
    • The corresponding DNA-DNA duplex Tm is 53.3 ‘C
    • 1’C higher per base pair on average
  2. A mismatch is a PNA/DNA duplex usually causes more destabilisation than a mismatch in DNA/DNA duplex
    • E.g. for a 15 per PNA, it’s average deltaTm is 15’C, whereas average deltaTm for the corresponding DNA/DNA duplex is 11’C
  3. Significantly higher rate of hybridisation in assays where either the target or the probe is immobilised
  4. Stable to nucleases and proteases as it is neither a DNA nor a peptide
  5. Recent research in our lab found out that an 18-mer antisense PNA inhibited BCL-2 protein with liposome as the delivery vehicle
22
Q

Melting point of DNA duplex

A

Don’t worry about this slide

23
Q

Modification of heterocycles

A
  • Very similar structure
  • Changes can change stability
24
Q

Nucleic acid delivery systems

Vectors for gene delivery

A
  • Physical methods for gene delivery microinjection, gene gun
    • => Disadvantage- practicability problem in the human body
  • Viral vectors, Adenoviruses, retroviruses, HSV
    • => Inherent drawbacks- Strong immune response, Risk of oncogenesis
  • Non-viral vectors lipid, PLL, PEI, PAMAM
    • Limitations- Low transfection efficiency, High toxicity
25
Q

Vectors used in nucleic acid delivery systems

A
26
Q

Physical methods in gene delivery

A
  • 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
27
Q

Physical methods in gene delivery

A
  • 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
28
Q

Viral-vectors in gene delivery

A
  • 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
29
Q

Pathway of viral infection

A
  • 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
30
Q

Viral-vectors in gene delivery

A
  • 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
31
Q

Viral-vectors in gene delivery

B)

A
  • 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
32
Q

Viral-vectors in Gene Delivery

C+D)

A
  • Envelope protein pseudotyping of viral vectors
  • Herpes simplex virus (HSV)
33
Q

Glybera

A
  • 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
34
Q

Non-viral vectors in gene delivery

A
  • Cationic- charges won’t repel each other
  • Steric stabilisation- prevent degradation
  • targeting ligands- look at what ligands (including proteins) make up the membrane
35
Q

Masking of anionic charges

Advantages of masking anionic charges of nucleic acids by cationic delivery systems

A
  • 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
36
Q

Cationic Liposomes

(Masking of anionic charges)

A
  • 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
37
Q

Basic components of a cationic lipid

A
  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
38
Q

Dioleolyl phospatidylethanolamine (DOPE)

A
  • 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)
39
Q

Membrane fusion theory- Endosome escape

A
  • 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
40
Q

Flip flop

A
  • Red= part of original cell membrane
  • membranes fuse together
41
Q

Cationic liposomes: Delivery objectives

A
  • 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
42
Q

Polymer-based gene delivery system

A
  • 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
43
Q

Proton sponge hypothesis- endosome escape

A
  • 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
44
Q

Cationic polymers: PEI

A
  • 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
45
Q

Bio-reducible disulphide-linked polymer

A
  • 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
46
Q

Disadvantages of non-viral systems

A
  • 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

PH4701- clinical pharmaceutics (20 decks)

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