Gene and cell therapy - Add? Flashcards
(14 cards)
What are nucleic acids composed of?
Made up of a 5-carbon sugar (ribose or deoxyribose), phosphate group and nitrogenous base (purine or pyrimidine)
DNA is a nucleotide polymer composed of 2 anti-parallel strands of DNA, held together by hydrogen bonds between complementary base pairs. Strings of nucleotides are coupled by phosphodiester bonds
mRNA is composed of -
5’ cap – capping efficiency, cap structure, innate sensing, protein synthesis. Increases stability
5’ untranslated region – regulatory elements modulate the translation efficiency with their length and structure
Open reading frame – codon optimization, sequence modifications, increases protein expression
3’ untranslated region
Poly(A) tail – tail elongation (length) impacts the stability and translational efficiency
what are Antisense oligonucleotides (ASOs)?
targets RNA and alters its expression and can modulate protein translation.
Short stretches (13-25 nucleotides) of single stranded modified nucleic acids by hybridizing with pre-mRNA or mRNA using classic Watson-Crick base pairing.
Post-transcriptionally regulates gene expression by promoting degradation of bound target RNA, sterically blocking translation to inhibit protein synthesis, or modifying pre-mRNA processing and splicing
ASOs can recruit RNase H to the hybrid and induce cleavage and degradation of the mRNA, thereby reducing protein expression/translation:
ASO binds to RNA to generate a DNA-RNA hybrid.
DNA-RNA hybrid recruite RNase H which is present in the cytoplasm.
Gapmer ASOs – made up of a central 8-10 base DNA gap which forms the hybrid which is recognised by the RNase H, and two highly modified RNA arms which resist nucleases and increase affinity for its target
ASO nucleic acid modifications -
Phosphoro/thio/ate - increases nuclease resistance, decreases hydrophilicity (increases serum protein binding), recruits RNase H, immunostimulatory, lower binding affinity.
Phosphoro/di/amidate morpholino oligos – superior target affinity, increased stability, uncharged so decreases serum protein binding, does not activate RNase H.
2’ ribose modifications – improve binding affinity, increases nuclease resistance, no RNase H recruitment.
Nucleobase modifications – reduces immunostimulatory effects.
What are short interfering RNAs and short hairpin RNAs?
target mRNA
RNA interference – a biological mechanism to regulate gene expression
It is the process by which the expression of a target gene is effectively silenced or knocked down by the selective inactivation (degradation) of its corresponding mRNA by double-stranded RNA (dsRNA)
RNAi can be activated by dsRNA species delivered to cells. These dsRNAs are either siRNAs or shRNAs which have a sequence complementary to their target mRNA and 3’ overhangs
ShRNA and siRNA processing -
DNA encoding shRNAs are introduced into the nuclei of target cells using either bacterial or viral vectors that can stably integrate into the genome in some cases
Pre-shRNAs are then transcribed by either RNA polymerase II or III, depending on the promoter driving their expression
Pre-shRNAs are processed by Drosha (a class 2 ribonuclease III enzyme) resulting in the shRNAs, which are then exported to the cytoplasm
The shRNA is the cleaved by Dicer removing the hairpin and creating a 20-25 nucleotide double-stranded siRNA with 2 nucleotide 3’ overhangs at each end
This active siRNA is then loaded onto the RNA-induced silencing complex (RISC)
The RISC promotes mRNA recognition and degradation
SiRNA nucleic acid modifications -
Phosphodiester modification – increases nuclease resistance, decreases hydrophilicity, immunostimulatory, lower binding affinity
2’ ribose modifications – improved binding affinity increases nuclease resistance
SiRNA conjugates – to enhance efficacy/targeting
Inclusiran – an approved siRNA therapy
Treats atherosclerotic cardiovascular disease and risk equivalents. Caused by mutations in the PCSK9 gene
These mutations increase its protease activity, reducing low density lipoprotein receptor levels and preventing uptake of cholesterol into cells
Inclisiran targets the PCSK9 mRNA and reduces its expression
What are RNA aptamers?
target proteins
Short single-stranded oligonucleotides usually 20-80 nucleotides long which fold into a 3D structure. Aptamers are highly specific and bind tightly to their target protein (like antibodies) and can modulate their activity/function
Aptamers can be linked with drugs, radioisotopes, RNA oligonucleotides or even nanostructures to specifically delivery drugs for targeted therapy
Pegaptanib -
Used to treat age-related macular degeneration in which blood vessels penetrate the retina and cause vision to deteriorate
Pegaptanib binds to and blocks the function of a mutant isoform of vascular endothelial growth factor165, an isoform most associated with pathologic ocular neovascularization. Blocking VEGF165 leads to a reduction in the growth and permeability of blood vessels in the eye
How are mRNAs used as a therapeutic?
Enzyme replacement therapy - substituting or replenishing specific enzyme deficiencies, the enzyme may previously be absent or non-functional due to mutations
Immunotherapy or vaccine - mRNA can be transfected and produce an immune response by direct injection, and has no risk of accidental infection or mutagenesis unlike viral vaccines
How are RNA therapies delivered via lipid nanoparticles?
Liposomes are used to deliver drugs into cells, they are vesicles composed of a lipid bilayer that forms in the shape of a hollow sphere encompassing an aqueous phase, shielding the drug from the body’s immune system
Lipid nanoparticles are liposome-like structures especially geared towards encapsulating nucleic acids
LNPs that are used to deliver genes are primarily synthesized using cationic (+) lipids that associate with anionic (-) nucleic acids
LNPs are often PEGylated – surrounded by polyethylene glycol (PEG) polymer covalently attached to the head of a phospholipid. PEGylated phospholipids reduce immune recognition and increase stability of the particle
What are the two types of target cells in gene therapy?
Germline cells – stem cells (sperm/egg) are altered, leading to long term change with ‘functional’ genes integrated into genome. ‘corrected’ genes are then passed onto future offspring
Somatic cells – all cells present in the body except reproductive cells. Effect is only restricted to patient, ‘faulty’ gene can still be passed onto future offspring
What is gene therapy?
‘Capacity for gene improvement by means of correction of altered/mutated genes or site-specific modifications that lead to therapeutic treatment’
Healthy version of gene of interest is inserted into a vector & then added to the person/their cells to replace defective gene
What is genome editing?
Uses enzymatic scissors to cute DNA at specific gene loci, induce a double strand break in the DNA and then using the cells own endogenous DNA repair mechanism to repair break with the correct sequence
What is ZFN?
A genome editing technique (zinc-finger nucleases).
ZFNs consist of n-terminal DNA binding domain (array of multiple zinc-finger domains which recognize 3 base pairs each) and c-terminal endonuclease (non-specific cleavage domain from bacterial Fok1 endonuclease)
Some 3 base pair sequences are not recognized by zinc-finger domains, requires protein engineering to design arrays that flank target sequence
Used to treat Hunter’s syndrome which has X-linked recessive inheritance
What are TALENs?
A genome editing technique (transcription activator-like effector nuclease).
TALENs consist of n-terminal DNA binding domain (highly conserved with variations at specific positions conferring sequence specificity) and c-terminal Fok1 endonuclease domain
Multiple TALE domains can flank the gene of interest with endogenous DNA repair machinery employed close to the DNA break
Easier to engineer and good cytotoxic profile compared to ZFN, doesn’t work when thymidine is not part of a sequence or for highly methylated regions
What is CRIPSR-Cas9?
A genome editing technique.
Adapted from prokaryotes adaptive resistance to invading plasmids or viruses. Bacteria capture snippets of DNA from invading viruses and creates DNA segments known as CRISPR arrays
CRISPR arrays allow bacteria to “remember” the viruses and if infected again, the bacteria produce CRIPSR RNA arrays to target the viruses’ DNA
Bacteria then use Cas9 together with CRISPR to cut the DNA, disabling virus
Used to treat Hepatitis B/C liver disease – can be used to create new Hep B or Hep C resistant cells via the following methods;
Support iPS differentiate into hepatic cells
CRIPSR-Cas9 editing against mechanism used by Hep-B/C virus to replicate
Produce iPSs/hepatic stem cells immune to hepatitis
Hep B/C resistance iPSs/stem cells could repopulate the liver as the old infected cells die
What is the Lentivirus system for distributing gene editing tools?
Modified form of HIV is the most commonly used, as it can infect quiescent (dividing) cells
can be used to co-deliver larger molecules such as CRISPR-Cas proteins
low immunogenicity depending on genes delivered
more frequently used than adenovirus system
What is the adenovirus system for distributing gene editing tools?
Small dsDNA virus that is non-pathogenic, only weakly immunogenic
Does not integrate into genome or at neutral sites
