Structure-Function of AAV - a Viral Gene Therapy Vector

描述（由申请人提供）：腺相关病毒（AAV）是体内和体外基因治疗的主要候选载体，用于细胞递送DNA以纠正遗传错误，缺陷或疾病易感性。我们的长期目标是了解介导AAV进入特定细胞和组织的效率的病毒与宿主的相互作用。该基金会将支持广泛寻求的目标，即靶向天然非允许细胞，并减少转基因表达在非靶组织中的不利影响。Aim 1的重点是细胞进入的第一步，与主要受体的附着，主要受体是几种人aav的硫酸肝素（HS）蛋白多糖。它建立在我们的AAV-2， -3和-6的结构上，以及这些同源病毒的HS结合位点的意想不到的多样性。晶体学和电子显微镜（EM）将用于表征在AAV中进化的HS相互作用的多样性，将结构与测量的肝素亲和力、细胞附着和细胞进入（通过体外转基因表达测量）相关联。我们的目标是阐明调节进入效率和特定细胞类型的差异敏感性的附着的结构亲和力决定因素。我们的理解将受到工程学的检验

DESCRIPTION (provided by applicant): Adeno-associated virus (AAV) is a leading candidate vector for in vivo and ex vivo gene therapy, for cellular delivery of DNA to correct genetic errors deficiencies or predispositions to disease. Our long term goal is to understand the virus-host interactions that mediate the efficiency with which AAV enters specific cells and tissues. This foundation will support the widely sought goals of targeting naturally non-permissive cells, and reducing the adverse effects of transgene expression in off-target tissues. Aim 1 focuses on the first step in cell entry, attachment to the primary receptor, which is heparan sulfate (HS) proteoglycan for several human AAVs. It builds on our structures of AAV-2, -3 & -6, and unexpected diversity in the HS binding site of these otherwise homologous viruses. Crystallography and electron microscopy (EM) will be used to characterize the diversity of HS interactions that has evolved in AAV, correlating structure with measured heparin affinity, cell attachment and cell entry, measured by in vitro transgene expression. Our goal is to elucidate the structure-affinity determinants of attachment that modulate the efficiency of entry and the differential susceptibility of specific cell types. Our understanding will be tested by engineering vectors with altered HS interactions, and characterizing their attachment and entry in a panel of representative cell lines. Aim 2 will elucidate the major antigenic determinants in a human polyclonal neutralizing antibody response. Our structure of AAV-DJ, a chimeric recombinant selected to escape pooled neutralizing serum, shows change localized around a likely dominant AAV2 monoclonal epitope visualized by EM. It opens a direct path to human polyclonal interactions: selection for neutralizing serum escape from a library of random single-site mutants. Dominant antigenic sites will be mapped through clustering of mutations on the AAV structure. Aim 2 will have impact in the design of neutralization-resistant vectors for in vivo human gene therapies, and will guide structural studies of MAb complexes to those that best model interactions that are important in human patients. Aim 3 focuses on co-receptors involved in the second step of cell entry. Our first goal is to rationalize a growing list of integral membrane proteins that have been implicated by various lines of evidence. Genomic approaches, including gene trapping, cDNA expression cloning and RNA interference, will be used to rank the functional importance of interactions to viral entry. They will also help distinguish direct AAV interactions from modulation of cellular pathways indirectly affecting rAAV transgene expression. Following confirmation by complementation and other methods, ecto-domain constructs will be expressed for measurement of binding affinities and for structural characterization by cryo-EM or footprinting by hydrogen / deuterium exchange mass spectrometry. Thus, Aim 3 will advance the basic understanding of a poorly understood aspect of AAV biology that will likely also prove important in cellular targeting of vectors.