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Review
. 2013 Apr;24(2):59-67.
doi: 10.1089/hgtb.2012.243. Epub 2013 Apr 3.

Pre-existing anti-adeno-associated virus antibodies as a challenge in AAV gene therapy

Vedell Louis Jeune  1 , Jakob A Joergensen, Roger J Hajjar, Thomas Weber
Affiliations
Review

Pre-existing anti-adeno-associated virus antibodies as a challenge in AAV gene therapy

Vedell Louis Jeune et al. Hum Gene Ther Methods. 2013 Apr.

Abstract

Adeno-associated virus (AAV)-based vectors are promising tools for gene therapeutic applications, in part because AAVs are nonpathogenic viruses, and vectors derived from them can drive long-term transgene expression without integration of the vector DNA into the host genome. AAVs are not strongly immunogenic, but they can, nonetheless, give rise to both a cellular and humoral immune response. As a result, a significant fraction of potential patients for AAV-based gene therapy harbors pre-existing antibodies against AAV. Because even very low levels of antibodies can prevent successful transduction, antecedent anti-AAV antibodies pose a serious obstacle to the universal application of AAV gene therapy. In this review, we discuss the current knowledge of the role of anti-AAV antibodies in AAV-based gene therapy with a particular emphasis on approaches to overcome the hurdle that they pose.

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Figures

FIG. 1.
FIG. 1.
Creation of libraries of AAV variants and directed evolution for the isolation of AAV variants with increased resistance to neutralizing antibodies. (A) Error-prone PCR: Creation of libraries of AAV variants through the introduction of mutations into the AAV capsid by error-prone PCR. The fidelity of the PCR reaction is either reduced by the use of a DNA polymerase with decreased accuracy or by modification of the PCR conditions. Examples include altered nucleotide composition, the addition of high amounts of magnesium or the substitution of magnesium by manganese. (B) DNA shuffling: Libraries of AAV variants with chimeric capid proteins composed of stretches of multiple AAV serotypes are created by limited DNAse digestion of the capsid DNA of several AAV serotypes followed by PCR amplification. (C) Staggered extension PCR: AAV variants with chimeric capsid proteins are created by repetitive PCR reaction with very short extension times. For simplicity, only one strand of two serotypes are depicted. However, staggered extension PCR can, at least in principle, be used to assemble AAV capsids with capsid proteins composed of stretches of multiple AAV serotypes. (D) Directed evolution to isolate AAV variants with increase resistance to neutralizing antibodies: A library of AAV variants is incubated with high amounts of neutralizing antibodies, for example, human IVIG, and used to infect permissive cells. Adenovirus superinfection triggers the amplification of AAV genomes that successfully reached the nucleus. The capsid genes from these successful AAV variants can then be isolated by PCR and subcloned to generate a secondary AAV library. Optionally, the diversity of this secondary library can be increased by additional mutagenesis. After several rounds of selection, individual variants are isolated by PCR amplification and subcloning.
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