Modification of Conserved Influenza Genes Enabling their Elevated Expression

描述(申请人提供)：哺乳动物基因可以根据GC含量分为五个等轴线，L-等轴线(低GC)基因的表达水平一致低于H-等轴线基因(高GC)。我们的初步数据强烈表明，流感基因，类似于其他病毒的基因，都一致偏向于L等位基因，因此，在体内很可能表达不足，无论是在自然感染期间，还是在重组载体的背景下都是如此。我们建议通过将标准DNA载体中流感病毒基因的密码子使用从L1-Isochore改变为H3-Isochore(H3-Isochore是最高表达的)来提高它们的表达水平。我们建议根据这个算法修改流感病毒的四个保守基因，它们是已知具有很强诱导交叉保护免疫潜力的基因(NP、M1、NS1或M2)。我们之前在小鼠和鸡身上展示了有限的、但在统计上显著的对异型流感病毒攻击的保护作用，这些疫苗是用编码NP、M1和NS1基因的质粒组合接种的。所有这些基因都天生偏向于L1-Isochore。我们将用与高表达的H3-Isochore的密码子偏好一致的同义密码子取代具有L1偏向(占总编码序列的16%-20%)的野生型密码子。然后，我们将在体外比较修饰的保守流感基因和它们的野生型对应基因的表达水平。如果表达水平提高，我们将建议在此拨款的第二阶段利用修饰的基因来提高DNA疫苗的免疫原性和保护效力。我们已经开发了一种用于同义密码子改变的电子技术，它允许产生符合特定等长基因家族密码子使用的基因修饰。众所周知，许多病毒蛋白在各种载体系统中的表达是次优的。这种现象严重阻碍了针对包括流感在内的不同病毒的重组疫苗的开发。公认的病毒序列密码子修饰(即人源化)方法并不一定会导致表达增加，可能是因为从未考虑过等长序列适应。因此，如果被证实，我们的方法将导致一种新的重组基因修饰的范例，以增强体内表达，无论是疫苗应用还是非疫苗应用。

DESCRIPTION (provided by applicant): Mammalian genes can be grouped into five isochores based on their GC content, with genes of L-isochores (low GC) uniformly expressed at a lower level than the genes of H- isochores (high GC). Our preliminary data strongly suggest that influenza genes, similarly to genes of other viruses, are uniformly biased towards L-isochores and thus, are likely to be under-expressed in vivo, both during natural infection and especially if introduced in the context of a recombinant vector. We propose to increase the level of influenza viral genes' expression in a standard DNA plasmid vector by altering their codon usage from L1- to H3-isochore (the latter being most highly expressed). We propose to modify according to this algorithm, four conserved genes of influenza virus that are known to possess strong potential for induction of cross-protective immunity (NP, M1, NS1 or M2). We previously demonstrated limited, yet statistically significant protection against heterotypic influenza viral challenge in mice and chicken vaccinated with a combination of plasmids encoding NP, M1 and NS1 genes. All of these genes are naturally biased towards L1-isochore. We will substitute wild-type codons that possess such an L1-bias (16-20% of total coding sequence) with synonymous ones that will adhere to the codon preference of the highly expressed H3-isochore. We will then compare the expression levels of modified conserved influenza genes to their wild-type counterparts in vitro. If the expression level is enhanced, we will propose to utilize the modified genes to increase the immunogenicity and protective efficacy of the DNA vaccine during Phase II of this grant. We have already developed an in silico technology for synonymous codon changes, that allows the generation of gene modification that adheres to the codon usage of a specific isochore family. It is known that the expression of many viral proteins in various vector systems is suboptimal. This phenomenon profoundly hinders the development of recombinant vaccines against different viruses including influenza. Accepted means of codon modification (i.e., humanization ) of viral sequences do not necessarily lead to increased expression, probably because isochore adaptation has never been taken into account. Therefore, if proven, our approach will lead to a novel paradigm for recombinant gene modifications for enhanced expression in vivo, both for vaccine and non-vaccine applications.