NUCLEIC ACID AND CELL BASED PHARMACEUTICALS Flashcards
What are the three translation-inhibiting oligonucleotides?
siRNA, miRNA and ribozymes.
Describe triple helix forming oligonucleotides (TFO)
Bind genomic DNA specifically in major groove.
Act on transcription of RNA by preventing initiation or elongation by binding to promotor, gene or regulatory DNA regions.
Used to mediate site-specific genome modification.
How do TFOs mediate site-specific genome modification?
Bind as third strand with sequence specificity, resulting in a triple helix which provokes repair or recombination (direct mutagenesis, recombination and possibly gene correction).
Describe transcription factor decoys:
Oligonucleotides that match attachment site for a transcription factor.
These decoys bind to the transcription factor and prevent gene activation.
Limitations for use as pharmaceuticals arise because many genes can be controlled by a single transcription factor.
Describe atopic dermatitis. Which transcription factor has a role in this disease?
Chronic skin condition which is traditionally treated with topical corticosteroids which have systemic and local side effects.
NF-kB transcription factor family has a central role in atopic dermatitis by –> transcription of inflammatory cytokines and adhesion molecules.
Describe how the topical NF-kB decoy oligonucleotide is used to combat atopic dermatitis?
The decoy binds to NF-kB preventing the transcription of key cytokines and adhesion molecules, this having anti-inflammatory action.
Describe translation-inhibiting oligonucleotides.
Single stranded DNA or RNA molecules (13-25 nucleotides).
May be mRNA blocking (physically prevent splicing or translation by binding complementary mRNA sequences), or mRNA cleaving (induce mRNA degradation by binding complementary mRNA and recruiting cytoplasmic RNase H. siRNA combines with proteins to form RISC complex –> cleavage.
Describe antisense oligonucleotides.
introduce a short oligonucleotide to complementary piece of mRNA.
Oligonucleotide binds to mRNA and reduce expression of the protein.
Eg. vitravene is an antisense oligonucleotide used to treat CMV retinitis in AIDS patients.
Describe CMV retinitis and it’s treatment with an antisense oligonucleotide.
Cytomegalovirus. Usually a latent infection, however may undergo reactivation if host immunity is compromised (e.g. in AIDS).
Fomivirsen (vitravene) is a nuclease resistant antisense oligonucleotide. It inhibits human CMV replication by acting on major immediate-early region 2 (IE2) transcriptional unit.
describe si RNA and it’s control of mRNA stability
Small, ds RNA molecules (20-25 nucleotides). Can regulate gene expression.
Involved in cell defence, and activated by the presence of dsRNA in cells (e.g. after cell infected by virus).
dsRNA binds to DICER which cleaves dsRNA into smaller fragments. One of the RNA strands is loaded into a RISC complex and links the complex to the mRNA strand by base pairing. mRNA is cleaved and destroyed. No protein can be synthesised.
Describe the use of siRNA to treat disease.
cells can be transfected with siena by electroporation or lipofection (difficult in human cells).
Viral-mediated delivery of siRNA can silence mRNAs in liver of mice.
siRNA has been shown to inhibit Fas-mediated cell death (reduces Fas levels) in several liver diseases and be life saving. Reduces development of liver fibrosis in mice (in chronic hepatitis)
Describe aptamers and riboswitches.
Involved in direct binding to non-nucleic acids.
Single stranded DNA or RNA ~60 nucleotides long. Bind to target molecule with high affinity.
Riboswitches occur naturally, while aptamers are synthetic.
Describe the identification of aptamers.
Pool of randomised oligonucleotides is subjected to selection steps whereby the oligonucleotides with higher activity become enriched in the population.
By increasing stringency of the election steps and competition between oligonucleotides, molecular evolution towards oligonucleotides with higher binding affinity takes place.
What is pegaptanib?
AKA Macugen. An anti-angiogenic treatment for macular degeneration.
Pegylated anti-VEGF aptamer. binds to VEGF-165 protein which is involved in blood vessel formation and increased BV permeability.
Briefly describe the steps in gene therapy.
DIRECT DELIVERY: therapeutic transgene is packaged into a delivery vehicle such as a virus and injected into patient where is reaches the target organ via the bloodstream.
CELL BASED DELIVERY: therapeutic transgene is packaged into a delivery vehicle and then introduced into a delivery cell, such as a stem cell, that is derived from the patient.
The genetically-modified cells are cultured and then read ministered to the patient.
