Download Dave Ousterout – Jin Lab – Project Proposal 08/14/08 Adeno

Dave Ousterout – Jin Lab – Project Proposal 08/14/08
Adeno-associated virus serotype 2 (AAV-2) has shown many promising applications in the field
of drug delivery. It is a relatively small (~4.7kb) and easily manipulated non-pathogenic virus, making
it an ideal therapeutic vector. Its small size does limit its therapeutic packaging capability and ability to
tolerate genetic insertions. However, it has many encouraging properties that make it an interesting
vector to research. Previous research has shown that a novel tropism can be introduced by genetically
modifying the capsid structure of the virus by inserting a peptide presented on the virus capsid surface
that interacts with a host receptor specific to target cells or tissues (Girod, et. al. 1999). The wild type
virus utilizes heparin sulfate proteoglycan (HSPG) as its primary receptor. The L14 ligand, an RGDcontaining peptide targeting integrins (Ruoslahti, et. al. 1996), has been shown to effectively retarget
AAV-2 when inserted into amino acid position 587 within the cap open reading frame of the AAV-2
genome (Girod, et. al. 1999).
My research aims:
 to evaluate, select, and develop efficient strategies for production and purification of wild type
and mutant AAV-2 vectors
 in the short term, to evaluate the infectious properties of mutant virus using the aa 453 genomic
insertion site using the L14 and RVG peptide ligands
 to analyze different genomic insertion sites for efficient presentation of a peptide ligand on the
capsid surface
 to modify the AAV2 virus to efficiently cross the blood brain barrier using RVG
 in the long term, to use a genetically modified vector as a therapeutic delivery system to specific
tissue
The aa 453 site has shown promise in previous research, but has not been fully evaluated to date
(Lochrie, et. al. 2005 and Wu, et. al. 2000). I have looked at the site using the solved crystal structure
for AAV-2 (Xie, et. al. 2002) and it appears that the putative loop containing aa 453 may better present
inserted peptides as compared to the previously used aa 587 site (Figure 1). There is a loop from an
adjacent subunit that lies between the two putative loops shown, which may appear to hinder the 587
loop. I want to show that the putative loop containing the 453 insertion site is a viable and perhaps
superior alternative to the commonly used 587 site.
My research subgroup currently uses the L14 peptide, described above, as well as the RVG
peptide. The rabies virus glycoprotein (RVG) peptide has been shown to bind efficiently to
acetylcholine receptors expressed in neuronal cells (Gatska, et. al. 2006). My research is centered on
engineering AAV2 to use the RVG peptide presented on its surface to uniquely and specifically infect
cells that will allow it to cross the blood brain barrier. Additionally, I use techniques such as PCRbased cell binding and endocytosis assays, and fluorescence microscopy to characterize the infectious
properties of generated mutants. My research subgroup is also analyzing the viability of using the RVG
peptide for neuronal cell transduction by fusing the protein to free GFP protein (Waldo GFP) and
characterizing cell transduction through fluorescence microscopy.
We will be pursuing further avenues to test the AAV-2 mutants in the near future.
I want to quantify our virus, possibly using RT-PCR techniques to determine genomic titers. I am
currently producing AAV-2 wild type virus, as well as the L14- and RVG-453 mutants, with a heparinbinding deficiency. I hope to use this to show that the mutants are efficiently and exclusively
transducing target cells through their genetically introduced peptide ligand. I am currently conducting
assays with the goal osf showing that the AAV-2 RVG mutants can efficiently transduce the cell lines
M17 (human neuronal cell) and Neuro-2a (murine neuronal cell), which the wild type virus should
minimally transduce, if at all. I am exploring this using an 'endocytosis' assay. This assay first allows
the virus to be taken up by the cells, which are then lysed and assayed for GFP expression indicating
the presence of the virus within the cell. I also want to explore the adjacent insertion sites to aa 453
and generate a library of mutants over a tight range upstream and downstream to that site. I want to
produce these mutants on a small scale and quantify their infectious properties using techniques similar
to those mentioned above. The purpose of this is to identify the optimal insertion site in the adjacent
putative loop to present a peptide ligand using rational design techniques. This may be used to present a
multitude of useful short peptides, which could further fine-tune the tropism of the adeno-associated
virus. One particularly useful application of this would be to present a signal sequence to allow
exocytosis of the virus from the cell after crossing the blood brain barrier using an intermediary, such
as an RVG-coated vesicle.
Selected references:
Ruoslahti, Erkki, et. al. “RGD and other recognition sequences for integrins.” Annual review of cell
and developmental biology, vol 12, pp. 697-715. 1996.
Girod, Anne, et. al. “Genetic capsid modifications allow efficient re-targeting of adeno-associated virus
type 2.” Nature Medicine, vol. 5 no. 9, pp. 1052-1056. 1999.
Lochrie, Michael, et. al. “Mutations on the external surfaces of adeno-associated virus type 2 capsids
that affect transduction and neutralization.” Journal of Virology, vol. 80 no. 2, pp. 821-834. 2005.
Wu, Pei, et. al. “Mutational analysis of the adeno-associated virus type 2 (AAV2) capsid gene and
construction of AAV2 vectors with altered tropism.” Journal of Virology, vol. 74 no. 18, 2000.
Xie, Qing, et. al. “The atomic structure of adeno-associated virus (AAV-2), a vector for human gene
therapy.” PNAS, 2002 vol. 99 no. 16, pp. 10405-10410. 2002.
Gatska, Maria, et. al. “Rabies virus binding to the nicotinic acetylcholine receptor {alpha} subunit
demonstrated by virus overlay protein binding assay. ' J. Gen. Virology, vol. 77, pp, 2437 - 2440.
1996.