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

描述（由申请人提供）：关节炎疾病是慢性的，致残的条件，有没有治愈。我们和其他人已经证明，在动物模型中，某些重组病毒载体的直接关节内注射可以提供足以停止关节炎的治疗性转基因表达水平。此外，我们最近已经证明，使用免疫相容性载体和cDNA，外源性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）提供了许多优点：它是非致病性的，具有低免疫原性，并且在许多应用中能够实现持久的转基因表达。交叉包装或假型化AAV 2载体的能力显著扩展了该系统的通用性，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。双链、自身互补（sc）AAV载体的最新发展克服了与关节组织中低效的第二链DNA合成相关的先前限制，使得AAV载体可以实际地考虑用于人类应用。除了腺相关病毒载体将外源基因递送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，本项目的目标是了解AAV载体血清型1-9关节内的药代动力学，以促进该技术以安全、合理和有效的方式临床应用。在具体目标1中，使用裸大鼠模型以避免免疫干扰问题，我们将在关节内施用后建立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，以确定与炎性疾病相关的形态学变化如何影响AAV介导的基因递送和表达。对于目标3，使用免疫活性动物，我们将确定通过重复载体施用和使用替代载体血清型来增强或拯救AAV介导的转基因表达的能力。在目的4中，我们将确定野生型AAV 2的先前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.