Replication strategies & gene therapy Flashcards
Telomeres
Protect the ends of linear chromosomes b/c during replication, the end regions can’t be replicated by DNA polymerase & RNA primers degraded
Somatic cells: As cell proliferates, telomeres shorten
- limited to 20-60 divisions
- advantage- prevents accumulation of mutations
- cancer cells have active telomerase
Germ cells: Active telomerase
- Telomerase is a specialized reverse transcriptase
- Enzyme contains RNA template
- RNA-primed DNA synthesis
DNA viruses
(3)
ds DNA
- Herpes simplex virus
- Large: encode own replication apparatus
- origin-binding protein, helicase, primase, helix-destabilizing protein, DNA pol
- encodes enzymes in nucleotide metabolism
- Papilloma virus
- Small: use host replicaiton system
- HPV: E6 & E7 bind to tumor supressors p53 & Rb → S phase
- Pox virus
RNA viruses
(4)
- Require an RNA-dependent RNA polymerase (replicase)
- Negative strand must have replicase packaged in virion
- Positive strand can be translated to synthesize replicase
- No mechanism for repairing errors; evolves into new strains to evade host immune system
A. Reovirus: ds RNA
B. Influenza: multiple ss RNA copies (negative strand)
C. Togavirus: ss RNA (positive strand)
D. HIV retrovirus: RNA (positive strand)
- two identical RNA copies of retroviral genome
- tRNA from host
- reverse transcriptase
Retroviruses
replication cycle
- Positive RNA strands contain unique end seq U3/U5 and R sequences that flank provirus
- tRNA binds to 5’ end of 1st RNA strand as primer.
- RT synthesizes
Herpes simplex virus (HPV)
treatments
Acyclovir: acycloguanosine
- HSV → thymidine kinase phosphorylates acyclovir → acycloguanosin-MP → acycloguanosineTP
- When this analog is incorporated into viral DNA by DNA polymerase → chain termination
*Cellular mechanism (host) cannot use acyclovir as a substrate, so drug is specific for HSV infected cells
HIV infections
treatments
3’-azido-2’,3’-dideoxythymidine (AZT) & 2’,3’-dideoxyinosine (DDI)
- Cellular kinases phosphorylate → AZT triphosphate
- For reverse transcriptase, AZT-TP has a higher affinity than dTTP → integration causes chain terminaiton
- For DNA pol, dTTP has a higher affinity than AZT-TP
*Unfortunately, it affects bone marrow cell → anemia
Gene therapy
Viruses as vectors to introduce fxnal genes
- SCID: Introduction of ADA gene to lymphocytes
- Lesch-Nyhan syndrome: introduction of gene encoding hypozanthine-guanine phosphoribosyltransferase
- Acquired diseases/cancer: introduction of genes that help kill cells = tumor necrosis factor (TNF) to tumor-infiltrating lymphocytes → stimulate immune system
Advantages of Viral Vectors
(3)
- Efficient introduction of genetic information to cells
- Retroviral vectors allow gene to be stably integrated into cellular genome
- Defective for replication so do not disseminate
Disadvantages of Viral Vectors
- Target cells must be replicating cells. Cannot be used to transfer genes to differentiated somatic cells
* Cannot be introduced or manipulated in fertilized cells for ethical regions → transgenic animals - There is always the possibilty of an outbreak of replication-competent retrovirus & mutations introduced *only requires a rare trigger
- Mutagenic: integrated at random sites; always a possibility of activation of oncogene
SCID gene therapy design
- Harvest T lymphocytes from child with ADA deficiency
- Infect with retroviral vector containing functional ADA gene and neomycin resistance genes
- Select for antibiotic resistance encoded by neo gene in vitro to collect T cells that have taken up vector
- Expand positive clones and infuse back into patient → expand lymphocytes and restore ADA
- DNA replication can take place readily → restores immune fxn
