E3L6P1 - Nucleic Acids as Therapeutic Agents

In vivo gene replacement

Designed to modify genes in somatic cells in whole organism with virus.

Ex vivo gene replacement

Modification of stem cells extracted from whole organism. Modification occurs in vitro with therapeutic nucleic acid. The modified cells can then be reintroduced into the organism.

Aptamers

small synthetic nucleic acids, bind enzymes, prevent function. They allow the target molecule to be bound to and inhibited/have the therapeutic agent delivered.

Inhibition of Translation with Anti-sense RNA

Gene of interest cloned with PCR primers that have endonuclease sites, to easily insert into an expression vector. The coding and template strands are inverted in the expression vector, and an Anti-sense RNA is now produced. This strand of antisense RNA hybridizes with processed mRNA, and translation is inhibited.

Short anti-sense oligonucleotides can be used as well. They can target the 5’ end of mRNA, preventing initiation of protein synthesis. Targeting introns, prevents RNA slicing and mRNA processing. Targeting 3’ sequence prevents initiation of protein synthesis.

Treatment of Congenital Hypercholesterolemia

Individuals afflicted with homozygous familial hypercholesterolemia express high levels of apolipoprotein B (apoB). Increasing apoB will increase cholesterol levels. Anti-sense oligonucleotide decreases apoB, and consequently, cholesterol levels drop.

Inhibition of Retroviral Genome Replication

Genome replication of retroviruses reverse flow of genetic information. Viral RNA primed with tRNA derived from host, complementary to viral genome U3 sequence, RT uses 3’-OH to synthesize cDNA Resulting product viral RNA/cDNA heteroduplex, RNAse H degrades regions following poly-purine tract (PPT). These fragments can be used as primers for RT to make complementary DNA seq. The dsDNA is then inserted into host genome. Viral genomes then expressed with host RNApol. Antisense RNA complementary to PPT and U3 seqs. Heteroduplex may prematurely activate RNAase H and destroys viral genome before RT is activated.

Systematic Evolution of Ligands by Exponential Enrichment (SELEX)

Many random nucelotide sequences cloned into expression vector. All clones expressed and RNA produced is incubated w/ target protein. Target immobilized on sepharose bead and the highest affinity binding RNAs are enriched for each cycle. Results in 1 or a few high affinity and high specificity nuceic acid sequences.

Increasing Stability and Delivery of Aptamers

RNAse is incredibly durable and degrades extracted RNA. Modifications are needed to prevent degradation. 2’-OH on purines can be replaced with a 2’-O-methyl residue. The 3’ ends can be capped with a deoxythymidine or deoxyribonucleotide. Phosphates can be modified to have a sulfur added. These modifications increased RNA stability without affecting their function.

Treatment of Age-Related Macular Degeneration

Retinal cells senesce due to lack of nutrient transport in elderly individuals. Vascular endothelial growth factor (VEGF) must be expressed and activated to prevent this. VEGF165 is associated with macular degeneration. Aptamer Pegaptanib binds the heparin binding domain and inhibits VEGF165. Degeneration stops, but the condition is not reversed to a less degenerated state.

Inhibition of Mutant Proteins in Hetrozygote

p53 – tumor suppressor, activated in response to extreme DNA damage, induces apoptosis. P53 must be a tetramer to be active. Variant p53/R175H HetDom is incapable of binding to DNA, oncogenesis ensues. The method below is used to produce an inhibitor that binds specifically to P53/R175H.

Modified SELEX procedure employed. p53/R175H bound to Sepharose Iron bead, WT p53 bound to Sepharose bead. Aptamers added. Centrifuge to remove WT bound aptamers. p53/R175H binding aptamers retained by using a magnet to bind to the iron beads. Repeated for several cycles to produce an aptamer that binds specifically to P53/R175H and prevents multimerization in mice. Therefore no tumor formation.