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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