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

描述（由申请人提供）：关节炎是一种无法治愈的慢性致残疾病。我们和其他人已经证明，在动物模型中，直接在关节内注射某些重组病毒载体可以提供治疗性转基因的表达，其水平足以阻止关节炎。此外，我们最近已经证明，通过使用免疫相容载体和cdna，外源转基因可以在实验动物的关节中无限表达。慢性关节疾病基因治疗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，并且能够在许多应用中进行持续的转基因表达。AAV2载体的交叉包装或伪型的能力大大扩展了该系统的多功能性，提供了增强转基因表达的机会，并有可能逃避预先存在的免疫。目前，有9种AAV衣壳血清型正在被开发作为基因治疗应用的载体。最近发展的双链自互补（sc） AAV载体克服了先前在关节组织中低效率的第二链DNA合成的局限性，使得AAV载体可以实际考虑用于人类应用。除了AAV载体将外源基因传递到关节组织并实现短期功能表达的能力之外，我们对该系统在关节环境中的生物学特性知之甚少。因此，本项目的目标是了解AAV载体血清型1-9在关节内的药代动力学，以促进该技术的安全、合理和有效的临床应用。在Specific Aim 1中，为了避免免疫干扰问题，我们将使用裸鼠模型，在关节内给药后，建立AAV血清型1-9的局部和全身转基因传递和表达谱。在目的2中，我们将在关节炎模型中进行类似的实验，以确定与炎症疾病相关的形态学变化如何影响aav介导的基因传递和表达。在Aim 3中，我们将使用免疫能力强的动物，通过重复载体给药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aims 4中，我们将确定先前自然感染野生型AAV2对相同和替代血清型重组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.