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Crossing the blood brain barrier:
AAV-2 based therapeutics using
rational mutagenesis approach
Dave Ousterout
Retreat presentation
Jin Lab
08/15/08
Background
Adeno-associated virus 2 (AAV2) is a small, nonpathogenic virus
 Sensitive to even small genomic insertions
 Native tropism through heparan-sulfate
proteoglycan
 Tropism effectively altered in previous research
(Girod, etc.) to specifically target cells, lower
efficiency
 Crystal structure solved in 2002 by Xie, et. al.
 AA 587 most commonly researched and used
insertion
 Use L14 (Integrin) and RVG (N. Ach-R) peptides

Aims for my research
Evaluate and select efficient strategies for production
and purification of wild type and mutant AAV-2
vectors
 Evaluate the infectious properties of mutant virus
using the aa 453 genomic insertion site using the
L14 and RVG peptide ligands
 Analyze different genomic insertion sites for efficient
presentation of a peptide ligand on the capsid
surface
 Modify the AAV2 virus to efficiently cross the blood
brain barrier using RVG

Primary hypothesis
Using the RVG peptide presented on its
surface, the AAV-2 virus should be able to
uniquely and specifically infect cells that will
allow it to cross the blood brain barrier.
Progress
• Produced partially purified wild type, RVG453
and L14453 mutants
• Using purification protocol for AAV-2
developed by Dr. Colin Parrish
• New method of transfection
• Isolated HSPG-deificient mutant plasmids,
currently producing for small scale
transfection
PCR for presence of virus
•First, need to show that PCR techniques can
detect virus through GFP gene amplification
•Tested wild type virus using GFP primers
•Tested dilutions against GFP plasmid to
determine saturation point and ‘ballpark’
relative virus amount
PCR for presence of virus
GFP plasmid
Virus
GFP plasmid
Neg.
1 10 102 103 104 105 1 10 102 103 104 105
1
10 102 103
Treated virus solution with benzonase (50 U/1 mL)
(negative control shown)
Important to note: no virus shows up in first well.
-Too much junk from crude lysate interfering
PCR reaction?
with
'Endocytosis' assay
• The purpose of this assay is to determine if
virus is successfully endocytosed by target
cell line
• Not concerned with other aspects of
infectious pathway (i.e. make sure the virus
“gets in” first)
'Endocytosis' assay
Cells at confluency
Aspirate media and add virus
Incubate for 1hr at 37C
Wash cells to remove unbound virus
Lyse cells (Buffer P2 + 2X F/T)
PCR lysis products for GFP
Preliminary endocytosis assay
results
• Buffer P2 used for cell lysis – found to
interfere with PCR up to certain dilution, then
the reaction works
• It was found that some PBS stocks were
contaminated (positive for GFP expression) –
hence why no virus control shows positive
result – contamination was isolated and
eliminated
• Need to run fresh assay, but preliminary
results are encouraging
Preliminary endocytosis assay
results
RVG
wt
HeLa
N.V.
RVG
wt
Neuro-2a
N.V. RVG
HT1080
wt
N.V.
Preliminary endocytosis assay
results
RVG wt N.V. RVG wt N.V RVG wt N.V
HeLa
Neuro-2a HT1080
Heparin-binding deficient mutants
• In order to eventually prove that RVG is solely
responsible for virus transduction,
heparin/HSPG binding deficient mutants must
be produced
• R585A, R588A mutations shown to remove
HSPG binding (Opie, et. al. 2006)
• Currently producing this deficiency in all
mutants for future comparison
Future work
Continued use of PCR detection methods for
evaluating virus
wt
wt hb
RVG453
RVG453
hb
HeLa
+
-
+
-
M17
0
-
+
+
Neuro-2a
0
-
+
+
HT1080
+
-
+
-
With HSPG binding deficient mutants, we would like to show that
RVG-AAV2 can efficiently bind and transduce target cells relative
to wt and infect cell lines wt cannot.
Evaluation of adjacent sites
Move peptides upstream and downstream, from amino
acids 448  456
The idea is that a small change maybe cause a different
folding and presentation of the loop with inserted peptide
Evaluation of adjacent sites
Generating mutants:
1.Q.C. PCR – two rounds to shift flanking
amino acid, thereby shifting ‘insert’
2.Cross-over PCR
The mutants can be compared by the following:
1.PCR dilution (saturation level)
2.RT-PCR genomic titering
3.Fluorescence assay?
The long term picture
• Test AAV-2-RVG virus capability to package
and deliver different genes for therapeutic
applications
• Modify virus to evade host antibody
neutralization
Other peptides or insertions?
If an optimized insertion site is found, then it
could be used for presenting a signal
sequence for exocytosis of the virus –
complementing vesicle transport of the virus
through the blood brain barrier to neuronal
cells
In and out!
(Transcytosis)
RVG
BBB
N. Ach-R
Virus is
encapsulated in
liposome coated
with RVG peptide
RVG binds to
nicotinic
acetylcholine
receptor on blood
brain barrier
In and out!
(Transcytosis)
Signal
sequence
Liposome endocytosed into cell,
releases virus; signal sequence
presented on virus capsid ‘rescues’
virus from normal infectious
pathway to an exocytosis pathway
Signal
sequence
Virus can now
invade other target
cells, having
successfully
crossed the blood
brain barrier
Significance of work
Lays ground work for:
Novel method of crossing blood brain barrier
using AAV-2, with or without vesicle transport
Identification of a new, more efficient insertion
site for AAV-2 retargeting