J.Ingram - 200743868 - Emerging Topics - Dr Chen - Paper Seminar
1. Emerging Topics – Dr Chen
J. Ingram 200743868
Paper Seminar
Combinatorial Modification of Degradable
Polymers Enables Transfection of Human Cells
Comparable to Adenovirus
Jordan J. Green, Gregory T. Zugates, Nathan C. Tedford, Yu-hung Huang, Linda
G. Griffith, Douglas A. Lauffenburger, Janet A. Sawicki, Robert Langer, And
Daniel G. Anderson
2. Introduction: Viral vs. Non-Viral
Gene Delivery Methods
V i r a l Tr a n s f e c t i o n Ve c t o r s Previous Non-Viral
(VTVs) Tr a n s f e c t i o n Ve c t o r s ( N V T V s )
• High efficacy • Low efficacy
• Safety risks – infection and cell • Simple to produce
death, immunogenicity and • Easier to deliver
targeting issues.
• Tuneable
• Production is difficult;
• Larger nucleic acid cargo capacity
• Limited nucleic acid cargo
capacity
http://www.virology.net/big_virology/BVDNAadeno.html http://products.invitrogen.com/ivgn/product/11668019
7. Testing: Human Umbilical Vein Endothelial
Cells (HUVECs) Transfection 2
PEI Adenovirus – MOI = 500 C32-103
24 hours after transfection, cells exposed to C32-103 show far higher
expression of GFP than equivalent exposure to PEI, or even high
concentrations of Adenovirus.
8. Testing: Ovarian Cancer Transfection
OvT C32-OH • C32-OH vs. C32-117 as vectors to deliver
pCAGluc gene to ovarian cancers in MISIIR/TAg
transgenic mice.
• C32-117 results in luciferase expression 2 orders of
magnitude higher than C32-OH
1.6
Luciferase Expression 6h Post-
1.4 Exposure
1.2 10000000
RLU/pixel (x106)
OvT C32-117 1 Photons/sec
0.8 1000000
0.6
100000
0.4
0.2
10000
0 C32-OH n=6 C32-117 n=9
9. Further Flaws in the Paper
• Unexplained sections:
• GFP Histogram: heterogenous
populations.
• Ambiguities of Polymer:DNA Ratios
• Some mentioned, some in the
experimental section, some have to be
calculated, some just missing.
• What happened to D60?
Histogram of GFP Expression in
HUVECs 48h Post Exposure
10. Conclusions
• Managed to make a non-viral
vector which is able to transfect by
a similar degree to adenovirus,
C32-117 even at high MOI.
• Non-viral vectors are generally
safer than viral ones – further
testing required to test this.
• Some lack of clarity in reasoning
and usage.
• Some sections feel incomplete, e.g.
ovarian model not tested against
other vectors.
http://fineartamerica.com/featured/adenovirus-36-russell-kightley.html