Gene Therapy and Oligonucleotide Thrapy (EXAM 3) Flashcards
What is Gene therapy used for?
-Replacing a missing or defective gene (CFTR - cystic fibrosis)
-silencing overactive genes (STAT 3)
-repair genes - genome editing
-prosthetic gene therapy
Two types of Gene therapy
Targets somatic cells
Targets germ cells (eggs, sperm) - not in development
How are genes delivered for gene therapy?
Means of delivery: Vectors or non-viral approaches
Mode of delivery:
Direct (in vivo): IV into the tissue or giving a drug
Indirect (ex vivo): cell is isolated from the body (in vitro) -> editing -> put back into the patient (in vivo)
What happens with the virus once it delivers the gene into the host cell?
-integration into the host genome
-episomal: replication of viral genes without integrating
How can viruses be used as a vector for gene therapy?
-Retrovirus: integrating virus, stable modification of target cells bc integrated, cant infect non-dividing cells (no dividing - no integration), mild immunogenicity
-Adenovirus: episomal - non-intergrating (uses machinery for replication), brief activity, can infect a non-dividing cell, high immunogenicity
-Adeno-associated Virus: integrating and episomal, can infect non-dividing cells, duration of gene expression for a long period, mild immunogenicity
How can viruses be used as a vector for gene therapy?
-Retrovirus: integrating virus, stable modification of target cells bc integrated, cant infect non-dividing cells (no dividing - no integration), mild immunogenicity
-Adenovirus: episomal - non-integrating (uses machinery for replication), brief activity, can infect a non-dividing cell, high immunogenicity
-Adeno-associated Virus: integrating and episomal, can infect non-dividing cells, duration of gene expression for a long period, mild immunogenicity
How is the replication of delivered viruses (vectors) ensured?
-remove genes that is essential for viral replication and replace it with the gene of interest -> place the altered virus + vector with essential genes separately into HEK cells -> the virus can duplicate bc of the essential genes on the plasmid -> harvest the viruses and use it to deliver genes into human -> the virus is safe because it doesn’t have the genes to replicate independently
Risks of virus-based gene delivery
-Immune reaction
-virus may insert the gene of interest in the wrong place
-affect other genes, inactivate genes
What is a tool used for ex vivo editing?
CRISPR-CAS -> correction of mutant beta-globin gene to correct sickle cell anemia
What are non-viral approaches to delivering genes?
-Synthetic liposomes mimicking the cell membrane
-DNA is negatively charged, the quaternary lipid amines are positively charged -> liposomes protect DNA/RNA from nucleases
-Compared to viral delivery the transfection is not as efficient -> with liposomes the DNA/RNA enters the nucleus randomly (when a cell divides pores open up)
Advantages, and Disadvantages of non-viral delivery
Advantage:
-non-immunogenic
-multiple administration
-less chance of mutagenesis
-can be manipulated easily
Disadvantages:
-low transduction efficacy, non-specific uptake of the vector
-poor delivery
-gene expression is not sustained
Chimeric antigen therapy (CAR-T)
EX-VIVO Gene transfer
T-cells harvested from patient -> ex vivo gene transfer: equip T-cells with CD19 receptors -> now able to recognize cancerous B-cells (lymphoma) -> plant it back to the patient
-Human T-cell: KYMRIAH, YESCARTA
How does Luxturna work?
Lux (latin): light
-cells processing the light are degenerating
-adenovirus carries the gene -> cells (non-dividing) take up the virus and replace the “broken” gene
Gene Therapy for Spinal muscular Atrophy
SMA = protein required for motor proteins -> degenerates when not sufficient
What are ways to use Oligonucleotides in therapy?
-SiRNA
-miRNA
-Aptamers
-CpG
-Antisense (ASO)
MOA of Antisense Oligos
-ASO binds and promotes endonuclease digestion
-ASO binds and produces an altered protein
Antisense treatment: Fomiversen
-15-40% of HIV patients (low immune system) suffer from CMV-retinitis (blindness)
-ASO binds to viral mRNA and mediates endonuclease
Duchenne Muscular Distrophy
alteration of dystrophin causes progressive muscle degeneration and weakness
-engineered ASO binds to frameshift mutation site -> leading to Exon Skipping: the exon causing the mutation is skipped during transcription due to ASO binding there
-the altered product is party functional
-helps 13% of DMD patients
small interference RNA interference: siRNA
-siRNA ad ds and can bind specifically to mRNA
-bind to protein complex containing the mRNA = RISC
-bind to their target and prevent ribosome from translation + mark the mRNA for destruction
Patisiran: siRNA
-gain of function mutation -> liver produces more TTR protein than it should -> TTR binds to Amyloid fibrils -> plaques deposited in organs (heart, lungs)
-destroys mRNA and prevents amyloid accumulation
What are Aptamers?
-RNA and DNA that takes 3D structure (protein-like)
Able to bind to proteins specifically with high affinity like antibodies
-easier to make
-not immunogenic
Aptamer: Pegaptanib (MACUGEN)
-used for wet Macular degeneration
-VEGF causes blood vessel generation -> becomes leaky and fluid builds up and damages the retina causing blindness
-Pegaptanib is PEGylated and has a longer-half-life
How does PEGylation prolong the half-life of a drug?
-Digesting enzymes cant bin readily
-drugs cant be filtered well in the glomerulus due to enlargement due to PEGylation
What are CpG Oligonucleotides?
-(bacterial CpG can trigger immune system)
-unmethylated cytosine-phosphodiester-guanine (CpG) acting as immunostimulants to cells with TLR9 -> B-cells, macrophages, dendritic cells, monocytes
-this triggering factor can be used as adjuvant
Barriers of Antisense Oligonucleotides (AON)
-susceptible to nucleases
-accumulate in kidneys, liver, bone marrow, muscles
-poor cellular uptake
-short half-life
