Gene therapy vectors based on adeno-associated virus (AAV) are currently in clinical trials for numerous disease targets, such as muscular dystrophy, hemophilia, Parkinson's disease, Leber's congenital amaurosis and macular degeneration. Despite its considerable promise and emerging clinical success, several challenges impede the broader implementation of AAV gene therapy, including the prevalence of neutralizing antibodies in the human population, low transduction of a number of therapeutically relevant cell and tissue types, an inability to overcome physical and cellular barriers in vivo and a relatively limited carrying capacity. These challenges arise as the demands we place on AAV vectors are often different from or even at odds with the properties nature bestowed on their parent viruses. Viral-directed evolution—the iterative generation of large, diverse libraries of viral mutants and selection for variants with specific properties of interest—offers an approach to address these problems. Here we outline progress in creating novel classes of AAV variant libraries and highlight the successful isolation of variants with novel and advantageous in vitro and in vivo gene delivery properties.
This is a preview of subscription content, access via your institution
Open Access articles citing this article.
Intraocular neutralizing antibodies against aflibercept in patients with age related macular degeneration
BMC Ophthalmology Open Access 10 January 2023
Journal of Neurodevelopmental Disorders Open Access 18 May 2018
BioDrugs Open Access 01 July 2017
Subscribe to Journal
Get full journal access for 1 year
only $9.92 per issue
All prices are NET prices.
VAT will be added later in the checkout.
Tax calculation will be finalised during checkout.
Get time limited or full article access on ReadCube.
All prices are NET prices.
Knipe DM, Howley PM, Griffin DE, Lamb RA, Martin MA, Roizman B et al. Field's Virology. 5th edn. Lippincott Williams & Wilkins: Philadelphia, PA, USA, 2006.
Sonntag F, Schmidt K, Kleinschmidt JA . A viral assembly factor promotes AAV2 capsid formation in the nucleolus. Proc Natl Acad Sci 2010; 107: 10220–10225.
Sonntag F, Köther K, Schmidt K, Weghofer M, Raupp C, Nieto K et al. The assembly-activating protein promotes capsid assembly of different adeno-associated virus serotypes. J Virol 2011; 85: 12686–12697.
Lux K, Goerlitz N, Schlemminger S, Perabo L, Goldnau D, Endell J et al. Green fluorescent protein-tagged adeno-associated virus particles allow the study of cytosolic and nuclear trafficking. J Virol 2005; 79: 11776–11787.
Sonntag F, Bleker S, Leuchs B, Fischer R, Kleinschmidt JA . Adeno-associated virus Type 2 capsids with externalized VP1/VP2 trafficking domains are generated prior to passage through the cytoplasm and are maintained until uncoating occurs in the nucleus. J Virol 2006; 80: 11040–11054.
Nonnenmacher M, Weber T . Adeno-associated virus 2 infection requires endocytosis through the CLIC/GEEC pathway. Cell Host Microbe 2011; 10: 563–576.
Ferrari FK, Samulski T, Shenk T, Samulski RJ . Second-strand synthesis is a rate-limiting step for efficient transduction by recombinant adeno-associated virus vectors. J Virol 1996; 70: 3227–3234.
Kotin RM, Siniscalco M, Samulski RJ, Zhu XD, Hunter L, Laughlin CA et al. Site-specific integration by adeno-associated virus. Proc Natl Acad Sci 1990; 87: 2211–2215.
Duan D, Sharma P, Yang J, Yue Y, Dudus L, Zhang Y et al. Circular intermediates of recombinant adeno-associated virus have defined structural characteristics responsible for long-term episomal persistence in muscle tissue. J Virol 1998; 72: 8568–8577.
Nakai H, Storm TA, Kay MA . Recruitment of single-stranded recombinant adeno-associated virus vector genomes and intermolecular recombination are responsible for stable transduction of liver in vivo. J Virol 2000; 74: 9451–9463.
Flotte TR . Gene therapy progress and prospects: recombinant adeno-associated virus (rAAV) vectors. Gene Therapy 2004; 11: 805–810.
Schaffer DV, Koerber JT, Lim K-i . Molecular engineering of viral gene delivery vehicles. Annu Rev Biomed Eng 2008; 10: 169–194.
Wu Z, Asokan A, Samulski RJ . Adeno-associated virus serotypes: vector toolkit for human gene therapy. Mol Therapy 2006; 14: 316–327.
Maguire AM, High KA, Auricchio A, Wright JF, Pierce EA, Testa F et al. Age-dependent effects of RPE65 gene therapy for Leber's congenital amaurosis: a phase 1 dose-escalation trial. Lancet 2009; 374: 1597–1605.
Maguire AM, Simonelli F, Pierce EA, Pugh Jr EN, Mingozzi F, Bennicelli J et al. Safety and efficacy of gene transfer for Leber's congenital amaurosis. N Engl J Med 2008; 358: 2240–2248.
Bainbridge JWB, Smith AJ, Barker SS, Robbie S, Henderson R, Balaggan K et al. Effect of gene therapy on visual function in Leber's congenital amaurosis. N Engl J Med 2008; 358: 2231–2239.
Nathwani AC, Tuddenham EGD, Rangarajan S, Rosales C, McIntosh J, Linch DC et al. Adenovirus-associated virus vector–mediated gene transfer in hemophilia B. N Engl J Med 2011; 365: 2357–2365.
Edelstein M . Gene Therapy Clinical Trials Worldwide. John Wiley and Sons: New York, 2011.
Girod A, Ried M, Wobus C, Lahm H, Leike K, Kleinschmidt J et al. Genetic capsid modifications allow efficient re-targeting of adeno-associated virus type 2. Nat Med 1999; 5: 1052–1056.
Zhong L, Li B, Mah CS, Govindasamy L, Agbandje-McKenna M, Cooper M et al. Next generation of adeno-associated virus 2 vectors: point mutations in tyrosines lead to high-efficiency transduction at lower doses. Proc Natl Acad Sci 2008; 105: 7827–7832.
Romero PA, Arnold FH . Exploring protein fitness landscapes by directed evolution. Nat Rev Mol Cell Biol 2009; 10: 866–876.
Perabo L, Endell J, King S, Lux K, Goldnau D, Hallek M et al. Combinatorial engineering of a gene therapy vector: directed evolution of adeno-associated virus. J Gene Med 2006; 8: 155–162.
Maheshri N, Koerber JT, Kaspar BK, Schaffer DV . Directed evolution of adeno-associated virus yields enhanced gene delivery vectors. Nat Biotechnol 2006; 24: 198–204.
Asuri P, Bartel MA, Vazin T, Jang J-H, Wong TB, Schaffer DV . Directed evolution of adeno-associated virus for enhanced gene delivery and gene targeting in human pluripotent stem cells. Mol Therapy 2011; 20: 329–338.
Bowles DE, Rabinowitz JE, Samulski RJ . Marker rescue of adeno-associated virus (AAV) capsid mutants: a novel approach for chimeric AAV production. J Virol 2003; 77: 423–432.
Grimm D, Lee JS, Wang L, Desai T, Akache B, Storm TA et al. In vitro and in vivo gene therapy vector evolution via multispecies interbreeding and retargeting of adeno-associated viruses. J Virol 2008; 82: 5887–5911.
Koerber JT, Jang J-H, Schaffer DV . DNA shuffling of adeno-associated virus yields functionally diverse viral progeny. Mol Therapy 2008; 16: 1703–1709.
Li W, Asokan A, Wu Z, Dyke TV, DiPrimio N, Johnson JS et al. Engineering and selection of shuffled AAV genomes: a new strategy for producing targeted biological nanoparticles. Mol Therapy 2008; 16: 1252–1260.
Yang L, Jiang J, Drouin LM, Agbandje-Mckenna M, Chen C, Qiao C et al. A myocardium tropic adeno-associated virus (AAV) evolved by DNA shuffling and in vivo selection. Proc Natl Acad Sci 2009; 106: 3946–3951.
Müller OJ, Kaul F, Weitzman MD, Pasqualini R, Arap W, Kleinschmidt JA et al. Random peptide libraries displayed on adeno-associated virus to select for targeted gene therapy vectors. Nat Biotechnol 2003; 21: 1040–1046.
Varadi K, Michelfelder S, Korff T, Hecker M, Trepel M, Katus HA et al. Novel random peptide libraries displayed on AAV Serotype 9 for selection of endothelial CELL-directed gene transfer vectors. Gene Therapy 2011; e-pub ahead of print 29 September 2011;
Koerber JT, Schaffer DV . Transposon-based mutagenesis generates diverse adeno-associated viral libraries with novel gene delivery properties. Methods Mol Biol 2008; 434: (Design and Characterization of Gene Transfer Vectors) 161–170.
Koerber JT, Jang J-H, Yu JH, Kane RS, Schaffer DV . Engineering adeno-associated virus for one-step purification via immobilized metal affinity chromatography. Hum Gene Therapy 2007; 18: 367–378.
Koerber JT, Klimczak R, Jang J-H, Dalkara D, Flannery JG, Schaffer DV . Molecular evolution of adeno-associated virus for enhanced glial gene delivery. Mol Therapy 2009; 17: 2088–2095.
Perabo L, Buning H, Kofler DM, Ried MU, Girod A, Wendtner CM et al. In vitro selection of viral vectors with modified tropism: the adeno-associated virus display. Mol Therapy 2003; 8: 151–157.
Waterkamp DA, Muller OJ, Ying Y, Trepel M, Kleinschmidt JA . Isolation of targeted AAV2 vectors from novel virus display libraries. J Gene Med 2006; 8: 1307–1319.
Michelfelder S, Lee M-K, deLima-Hahn E, Wilmes T, Kaul F, Muller O et al. Vectors selected from adeno-associated viral display peptide libraries for leukemia cell–targeted cytotoxic gene therapy. Exp Hematol 2007; 35: 1766–1776.
Sellner L, Stiefelhagen M, Kleinschmidt JA, Laufs S, Wenz F, Fruehauf S et al. Generation of efficient human blood progenitor–targeted recombinant adeno-associated viral vectors (AAV) by applying an AAV random peptide library on primary human hematopoietic progenitor cells. Exp Hematol 2008; 36: 957–964.
Russell DW, Hirata RK . Human gene targeting by viral vectors. Nat Genet 1998; 18: 325–330.
Jang J-H, Koerber JT, Kim J-S, Asuri P, Vazin T, Bartel M et al. An evolved adeno-associated viral variant enhances gene delivery and gene targeting in neural stem cells. Mol Therapy 2011; 19: 667–675.
Khan IF, Hirata RK, Wang P-R, Li Y, Kho J, Nelson A et al. Engineering of human pluripotent stem cells by AAV-mediated gene targeting. Mol Therapy 2010; 18: 1–8.
McPhee SWJ, Janson CG, Li C, Samulski RJ, Camp AS, Francis J et al. Immune responses to AAV in a phase I study for Canavan disease. J Gene Med 2006; 8: 577–588.
Halbert CL, Standaert TA, Wilson CB, Miller AD . Successful readministration of adeno-associated virus vectors to the mouse lung requires transient immunosuppression during the Initial exposure. J Virol 1998; 72: 9795–9805.
Jiang H, Couto LB, Patarroyo-White S, Liu T, Nagy D, Vargas JA et al. Effects of transient immunosuppression on adeno-associated, virus-mediated, liver-directed gene transfer in rhesus macaques and implications for human gene therapy. Blood 2006; 108: 3321–3328.
Manning WC, Zhou S, Bland MP, Escobedo JA, Dwarki V . transient immunosuppression allows transgene expression following readministration of adeno-associated viral vectors. Hum Gene Therapy 1998; 9: 477–485.
Manno CS, Pierce GF, Arruda VR, Glader B, Ragni M, Rasko JJE et al. Successful transduction of liver in hemophilia by AAV-factor IX and limitations imposed by the host immune response. Nat Med 2006; 12: 342–347.
Stroes ES, Nierman MC, Meulenberg JJ, Franssen R, Twisk J, Henny CP et al. Intramuscular administration of AAV1-lipoprotein LipaseS447X lowers triglycerides in lipoprotein lipase-deficient patients. Arterioscler Thromb Vasc Biol 2008; 28: 2303.
Boutin S, Monteilhet V, Veron P, Leborgne C, Benveniste O, Montus MF et al. Prevalence of serum IgG and neutralizing factors against adeno-associated virus (AAV) Types 1, 2, 5, 6, 8, and 9 in the healthy population: implications for gene therapy using AAV vectors. Hum Gene Therapy 2010; 21: 704–712.
Calcedo R, Vandenberghe LH, Gao G, Lin J, Wilson JM . Worldwide epidemiology of neutralizing antibodies to adeno-associated viruses. J Infect Dis 2009; 199: 381–390.
Maersch S, Huber A, Büning H, Hallek M, Perabo L . Optimization of stealth adeno-associated virus vectors by randomization of immunogenic epitopes. Virology 2009; 397: 1–9.
Excoffon KJDA, Koerber JT, Dickeya DD, Murtha M, Keshavjeed S, Kaspar BK et al. Directed evolution of adeno-associated virus to an infectious respiratory virus. Proc Natl Acad Sci 2009; 106: 3865–3870.
Li W, Zhang L, Johnson JS, Zhijian W, Grieger JC, Ping-Jie X et al. Generation of Novel AAV variants by directed evolution for improved CFTR delivery to human ciliated airway epithelium. Mol Therapy 2009; 17: 2067–2077.
Nagele RG, Wegiel J, Venkataraman V, Imaki H, Wang KC . Contribution of Glial cells to the development of amyloid plaques in Alzheimer's disease. Neurobiol Aging 2004; 25: 663–674.
Nagai M, Re DB, Nagata T, Chalazonitis A, Jessell TM, Wichterle H et al. Astrocytes expressing ALS-linked mutated SOD1 release factors selectively toxic to motor. Neurons Nat Neurosci 2007; 10 (5): 615–622.
Maguire CA, Gianni D, Meijer DH, Shaket LA, Wakimoto H, Rabkin SD et al. Directed evolution of adeno-associated virus for glioma cell transduction. J Neurooncol 2010; 96: 337–347.
Michelfelder S, Kohlschutter J, Skorupa A, Pfennings S, Muller O, Kleinschmidt JA et al. Successful expansion but not complete restriction of tropism of adeno-associated virus by in vivo biopanning of random virus display peptide libraries. PLoS One 2009; 4: e5122 (1–13).
Perrault SD, Walkey C, Jennings T, Fischer HC, Chan WC . Mediating tumor targeting efficiency of nanoparticles through design. Nanoletters 2009; 9: 1909–1915.
Klimczak RR, Koerber JT, Dalkara D, Flannery JG, Schaffer DV . A novel adeno-associated viral variant for efficient and selective intravitreal transduction of rat muller cells. PLoS One 2009; 4: e7467.
Dalkara D, Kolstad KD, Guerin KI, Hoffmann NV, Visel M, Klimczak RR et al. AAV mediated GDNF secretion from retinal glia slows down retinal degeneration in a rat model of retinitis pigmentosa. Mol Therapy 2011; 19: 1602–1608.
Dalkara D, Klimczak R, Visel M, Schaffer D, Flannery J . Developing Photoreceptor Targeted AAV Variant by Directed Evolution, in American Society of Gene and Cell Therapy's 14th Annual Meeting. Seattle: WA, 2011.
Cabrera-Salazar MA, Roskelley EM, Bu J, Hodges BL, Yew N, Dodge JC et al. Timing of therapeutic intervention determines functional and survival outcomes in a mouse model of late infantile batten disease. Mol Therapy 2007; 15: 1782–1788.
Muramatsu S, Fujimoto K, Kato S, Mizukami H, Asari S, Ikeguchi K et al. A Phase I study of aromatic L-amino acid decarboxylase gene therapy for Parkinson's disease. Mol Therapy 2010; 18: 1731–1735.
Gray SJ, Blake BL, Criswell HE, Nicolson SC, Samulski RJ, McCown TJ . Directed evolution of a novel adeno-associated virus (AAV) vector that crosses the seizure-compromised blood-brain barrier (BBB). Mol Therapy 2010; 18: 570–578.
Mueller C, Flotte TR . Clinical gene therapy using recombinant adeno-associated virus vectors. Gene Therapy 2008; 15: 858–863.
Bowles DE, McPhee SWJ, Li C, Gray SJ, Samulski JJ, Camp AS et al. Phase 1 gene therapy for duchenne muscular dystrophy using a translational optimized AAV vector. Mol Therapy 2011; 20 (2): 443–455.
The authors declare no conflict of interest.
About this article
Cite this article
Bartel, M., Weinstein, J. & Schaffer, D. Directed evolution of novel adeno-associated viruses for therapeutic gene delivery. Gene Ther 19, 694–700 (2012). https://doi.org/10.1038/gt.2012.20
- directed evolution
- adeno-associated virus
- viral vector
- gene delivery
- gene targeting
This article is cited by
Intraocular neutralizing antibodies against aflibercept in patients with age related macular degeneration
BMC Ophthalmology (2023)
Journal of Neurodevelopmental Disorders (2018)
Virus Genes (2017)
Molecular Therapy (2016)