A structure-based approach to accelerated recombinase engineering

项目总结/摘要 HIV和HTLV都是逆转录病毒，在数百万人中引起危及生命的不治之症。 世界各地的个人。这些逆转录病毒将它们的DNA插入细胞的基因组中 他们感染。一部分被感染的细胞不产生病毒。很少有，除了 前病毒DNA，将这些潜伏感染的细胞与未感染的细胞区分开来。在 与目前靶向病毒蛋白的治疗剂（即HIV蛋白酶抑制剂或逆转录酶抑制剂）相反， 转录酶抑制剂），我们正在开发一类新的逆转录病毒疗法， 原病毒DNA本身我们的方法使用工程重组酶， 位于整合前病毒侧翼的末端重复序列（LTR）。我们已经证明， 重组酶可以有效地从患者来源的细胞中去除HIV前病毒， 慢病毒载体我们还表明，当工程化的、抗- HIV重组酶在转基因小鼠中组成型表达。与CRISPR/Cas9相比， 靶向前病毒DNA的其他基于核酸酶的方法，重组酶与单核苷酸 精确性，从而使它们本质上比核酸酶更适合于此目的。(The不可预测 与抗HIV CRISPR/Cas9相关的插入缺失似乎提高了病毒逃逸的速率。 该提议针对工程重组酶方法的主要限制，即 改变重组酶的特异性是非常耗时的。迄今为止，新工程 重组酶依赖于随机诱变作为遗传多样性的来源。这是高度 因为筛选的绝大多数突变发生在蛋白质的区域中， 与DNA识别无关。此外，考虑到大量的潜在突变， 在整个蛋白质中，随机筛选必然会对可用的序列空间进行采样， 稀疏地。在这个应用中，我们提出使用一种新的，结构引导的方法来识别 重组酶的特异性改变。我们将再次筛选数百万个克隆体以寻找所需的活性， 但我们筛选的遗传多样性将仅限于蛋白质的关键区域， 在这些关键位置处的氨基酸的子集将是文库的一部分。(To明确指出， 我们将确定抗HIV和抗HTLV的晶体结构 重组酶与其LTR靶复合，并探测特定突变位点的功能作用 在重组酶中）。我们预计，最相关区域的密集采样， 序列空间将极大地简化重组酶设计过程。这将使我们能够 快速靶向我们目前最好的重组酶无法切除的HIV和HTLV毒株。

Project Summary/Abstract HIV and HTLV are both retroviruses that cause life-threatening, incurable disease in millions of individuals throughout the world. These retroviruses insert their DNA into the genomes of the cells they infect. A fraction of the the infected cells do not produce virus. There is little, other than the proviral DNA, that distinguishes these latently-infected cells from those that have not been infected. In contrast to current therapeutics, which target viral proteins (i.e. HIV protease inhibitors or reverse transcriptase inhibitors), we are developing a new class of retroviral therapeutics that target the proviral DNA itself. Our approach uses engineered recombinases that specifically act on the Long Terminal Repeats (LTRs) that flank the integrated provirus. We have already demonstrated that such recombinases can efficiently remove HIV provirus from patient-derived cells when delivered using a lentiviral vector. We have also shown that there are no deleterious effects when the engineered, anti- HIV recombinase is constitutively expressed in transgenic mice. In contrast to CRISPR/Cas9 and other nuclease-based approaches to targeting proviral DNA, recombinases act with single-nucleotide precision thereby making them intrinsically better for this purpose than nucleases. (The unpredictable indels associated with anti-HIV CRISPR/Cas9 appear to enhance the rate of viral escape.) This proposal targets the primary limitation of the engineered recombinase approach, namely that altering recombinase specificity is extremely time consuming. To date, engineering of new recombinases has relied on random mutagenesis as a source of genetic diversity. This is highly inefficient since the overwhelming majority of mutations screened occur in regions of the protein that are not associated with DNA recognition. Moreover, given the vast number of potential mutations across the entire protein, random screening, necessarily, samples the available sequence-space very sparsely. In this application, we propose to use a new, structure-guided approach to identifying recombinases with altered specificity. We will again screen millions of clones for the desired activity, but the genetic diversity in our screens will be limited to key regions of the protein, and only a sensible subset of the amino acids at these key positions will be part of the library. (To clarify precisely which amino acids should be varied, we will determine crystal structures of anti-HIV and anti-HTLV recombinases in complex with their LTR targets and probe the functional role of specific mutation sites within the recombinases.) We anticipate that dense-sampling of the most relevant regions of sequence-space will dramatically streamline the recombinase design process. This will allow us to quickly target HIV and HTLV strains that our current-best recombinases cannot excise.