Utility of AAV serotypes for gene delivery in treatment of chronic joint disease

描述(由申请人提供)：关节炎是一种慢性的、严重的疾病，没有治愈的方法。我们和其他人已经证明，在动物模型中，直接关节内注射某些重组病毒载体可以提供足以阻止关节炎的治疗性转基因表达。此外，我们最近已经证明，使用免疫相容的载体和cDNA，外源转基因可以在实验动物的关节中无限表达。慢性关节疾病基因疗法临床转化的主要障碍是缺乏合适的载体系统。在这方面，重组腺相关病毒(AAV)具有许多优点：它是非致病性的，免疫原性低，可以在许多应用中实现持续的转基因表达。AAV2载体的交叉包装或伪型能力显著扩展了该系统的通用性，为增强转基因表达提供了机会，也为逃避先前存在的免疫提供了可能性。目前，正在开发9种AAV衣壳血清型，作为基因治疗应用的载体。最近发展的双链、自互补(Sc)AAV载体克服了以前关节组织中第二链DNA合成效率低下的限制，使得AAV载体可以现实地用于人类应用。除了AAV载体将外源基因运送到关节组织并实现短期功能表达的能力外，人们对该系统在关节环境中的生物学特性知之甚少。因此，本项目的目标是在关节内了解AAV载体1-9型的药代动力学，以促进该技术以安全、合理和有效的方式在临床上应用。在具体目标1中，使用裸鼠模型来避免免疫干扰问题，我们将在关节内给药后建立局部和系统的AAV血清型1-9的转基因传递和表达谱。在目标2中，我们将在关节炎模型中进行类似的实验，以确定与炎症性疾病相关的形态变化如何影响AAV介导的基因传递和表达。对于目标3，我们将使用具有免疫能力的动物，通过重复载体注射和使用替代载体血清型来确定增强或挽救AAV介导的转基因表达的能力。在目的4中，我们将确定先前自然感染野生型AAV2对相同和交替血清型的重组AAV载体的转基因传递和表达的影响。

DESCRIPTION (provided by applicant): Arthritic diseases are chronic, crippling conditions for which there are no cures. We, and others, have shown that direct intra-articular injection of certain recombinant viral vectors can provide expression of therapeutic transgenes at levels sufficient to halt arthritis in animal models. Further, we have recently demonstrated that with the use of immunologically compatible vectors and cDNAs, exogenous transgenes can be expressed indefinitely in the joints of experimental animals. A primary barrier to clinical translation of gene therapies for chronic joint disease has been the lack of suitable vector systems. In this regard, recombinant adeno- associated virus (AAV) offers many advantages: it is nonpathogenic, of low immunogenicity, and enables persistent transgene expression in many applications. The capacity to cross-package, or pseudotype, the AAV2 vector has significantly expanded the versatility of this system, providing the opportunity to enhance transgenic expression as well as the potential to evade pre-existing immunity. Currently 9 AAV capsid serotypes are being developed as vectors for gene therapy applications. The recent development of double- stranded, self-complementary (sc) AAV vectors overcomes previous limitations associated with inefficient second strand DNA synthesis in articular tissues such that AAV vectors can be realistically considered for human application. Beyond the capacity of AAV vectors to deliver exogenous genes to joint tissue and achieve short-term functional expression, very little is known specifically of the biology of this system in the context of the articular environment. Thus, the goal of this project is to develop an understanding of the pharmacokinetics of AAV vector serotypes 1-9 intra-articularly to facilitate the clinical application of this technology in a safe, rational and effective manner. In Specific Aim 1, using a nude rat model to avoid issues of immune interference, we will establish local and systemic transgene delivery and expression profiles for AAV serotypes 1-9 following intra-articular administration. In Aim 2 we will perform similar experiments in an arthritic model to determine how morphologic changes associated with inflammatory disease affect AAV-mediated gene delivery and expression. For Aim 3, using immunologically competent animals we will determine the capacity to enhance or rescue AAV-mediated transgene expression by repeat vector administration and the use of alternate vector serotypes. In Aim 4 we will determine the impact of prior natural infection with wild type AAV2 on transgene delivery and expression of recombinant AAV vectors of the same and alternate serotypes.