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

描述（由申请人提供）：关节炎疾病是一种慢性、致残性疾病，无法治愈。我们和其他人已经证明，直接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，其水平足以阻止动物模型中的关节炎。此外，我们最近证明，通过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期地表达。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，并且能够在许多应用中持续表达转基因。 AAV2 载体的交叉包装或假型能力显着扩展了该系统的多功能性，提供了增强转基因表达的机会以及逃避预先存在的免疫的潜力。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。最近开发的双链、自互补（sc）AAV载体克服了先前与关节组织中第二链DNA合成效率低下相关的限制，使得AAV载体可以实际考虑用于人类。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，人们对该系统在关节环境中的生物学知之甚少。因此，本项目的目标是加深对AAV载体血清型1-9体内药代动力学的了解，以促进该技术安全、合理、有效的临床应用。在具体目标 1 中，使用裸鼠模型以避免免疫干扰问题，我们将在关节内给药后建立 AAV 血清型 1-9 的局部和全身转基因递送和表达谱。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，通过重复载体施用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。在目标 4 中，我们将确定先前野生型 AAV2 自然感染对相同和替代血清型的重组 AAV 载体的转基因递送和表达的影响。

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.