Modification of Conserved Influenza Genes Enabling their Elevated Expression

描述（由申请人提供）：哺乳动物基因可根据其GC含量分为5个等粒体，其中L-等粒体（低GC）的基因以低于H-等粒体（高GC）的基因的水平均匀表达。我们的初步数据有力地表明，流感基因，类似于其他病毒的基因，是均匀地偏向于L-等位基因，因此，很可能是在体内表达不足，无论是在自然感染，特别是如果在重组载体的情况下引入。我们建议通过将流感病毒基因的密码子使用从L1-等容区改变为H3-等容区（后者表达最高）来增加流感病毒基因在标准DNA质粒载体中的表达水平。我们建议根据该算法修改已知具有诱导交叉保护性免疫的强大潜力的流感病毒的四个保守基因（NP、M1、NS 1或M2）。我们以前证明了有限的，但在统计学上显着的保护，对异型流感病毒的攻击，在小鼠和鸡与编码NP，M1和NS 1基因的质粒的组合接种。所有这些基因都自然偏向L1-等容线。我们将用与高度表达的H3等排区的密码子偏好一致的同义密码子替换具有这种L1偏好（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与其野生型对应基因的表达水平。如果表达水平得到提高，我们会建议在第二阶段拨款期间，利用经修饰的基因，以增加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计算机技术，该技术允许产生遵循特定等容线家族的密码子使用的基因修饰。已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。公认的密码子修饰方式（即， 人源化）不一定导致表达增加，可能是因为从未考虑过等容线适应。因此，如果得到证实，我们的方法将导致一个新的范式重组基因修饰增强体内表达，无论是疫苗和非疫苗应用。

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.