化学与生命科学新闻简报 - 2022 年 6 月 by HLK

化学与生命科学新闻简报 2022 年 6 月

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一切始于大爆炸！ – 新 WIPO 序列表标准（ST.26）

And it all Started with a Big Bang! – The New WIPO Standard for Sequence Listings (ST.26)

专利申请中公开的生物序列（例如 DNA、RNA、肽）要求使用符合适用 WIPO 标准的序列表提供。对该类信息实施国际标准化，旨在方便 IPO 开展搜索工作、简化申请人的申请流程并允许与公众共享序列。

There is a requirement that biological sequences (e.g., DNA, RNA, peptides) disclosed in patent applications be provided in a sequence listing, which conforms with the applicable WIPO standard. The international standardisation of this information is intended to facilitate searching by IPOs, simplify filing for applicants and allow sequences to be shared with the public.

现行标准 ST.25 自 1998 年通过后一直沿用至今。但是，该项标准不符合国际核酸序列数据库联盟 (INSDC) 的要求，因此在公共数据库中输入数据时会丢失数据。此外，当前的常见序列类型（例如核苷酸类似物、D-氨基酸、支链序列）未被 ST.25 所涵盖，因此不在可检索的数据库中。新标准 ST.26 将解决这些问题。大爆炸日期自 2022 年 7 月 1 日（“大爆炸”日期）起，ST.25 将被 ST.26 代替，点击查看 ST.26。两项标准之间没有过渡期，因此在 2022 年 7 月 1 日或之后提交的包含序列信息的新申请都需要符合 ST.26 格式。

The current standard, ST.25, was adopted in 1998 and has been in force ever since. However, this standard is not compliant with the International Nucleotide Sequence Database Collaboration (INSDC) requirements meaning that data may be lost when entered into public databases. Further, sequence types that are common today (such as nucleotide analogs, D-amino acids, branched sequences) are also not covered by ST.25 and therefore are not present in searchable databases. The new standard, ST.26, will remedy these issues. The Big Bang Date From 1 July 2022, denoted the “Big Bang” date, ST.25 will be replaced with new standard ST.26, which can be found here. There is no transition period between the standards, so new applications comprising sequence information filed on or after 1 July 2022 will need to comply with the ST.26 format. Priority-claiming applications will also require ST.26 compliant sequence listings filed after 1 July 2022, even if the priority application has a filing date prior to 1 July 2022. For divisional applications and regional/ national phase entry, implementation of the new standard may vary between patent offices. For example: The EPO has indicated that they WILL require ST.26 compliant sequence listings

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对于主张优先权的申请，还需在 2022 年 7 月 1 日之后提交符合 ST.26 的序列表，即使优先权申请的提交日期早于 2022 年 7 月 1 日。对于分案申请和进入地区/国家阶段的申请，视各专利局对新标准的实施情况而定。例如： EPO 要求在 2022 年 7 月 1 日之后提交的分案申请包含符合 ST.26 的序列表，即使母申请的提交日期早于 2022 年 7 月 1 日。

但 UKIPO 称，如果母申请的提交日期在 2022 年 7 月 1 日之前，他们不会要求分案申请包含符合 ST.26 的序列表。 EPO 和 UKIPO 均表示，如果 PCT 申请的提交日期在 2022 年 7 月 1 日之前，

他们不会在进入地区/国家阶段时要求提供符合 ST.26 的序列表。辞旧迎新

•

The UKIPO however, has indicated that they WILL NOT require ST.26 compliant sequence listings for divisional applications if the parent application has a filing date before 1 July 2022. Both the EPO and UKIPO have indicated that they WILL NOT require a ST.26 compliant sequence listing at regional/ national phase entry, if the PCT application has a filing date before 1 July 2022. Out with the old and in with the new In addition to compatibility with INSDC requirements and inclusion of additional sequence types, many other updates have been made. Some key changes include: •

The requirement of an XML format as opposed to the TXT format required for ST.25

•

Sequences with < 10 nucleotides or < 4 amino acids can no longer be included

•

Sequence identifiers can now be more precisely defined with the mandatory mol_type qualifier (e.g., RNA can now be annotated as mRNA, tRNA etc.)

•

对必填“生物体”限定符的名称作了一些变更（“人工序列”更名为“合成构建体”，“未知”更名为“未识别”）

Some changes made to the nomenclature for the mandatory “organism” qualifier (“artificial sequence” renamed “synthetic construct”, “unknown” renamed “unidentified”)

•

“T”代表 RNA 序列中的尿嘧啶和 DNA 序列中的胸腺嘧啶

“T” to be used to represent both uracil in RNA and thymine in DNA

•

Amino acid sequences will now be represented by one letter abbreviations

•

Mixed mode sequences are no longer allowed (i.e. separated sequences must be submitted for an amino acid sequence and the corresponding nucleotide sequence)

•

Further options/qualifiers for

除兼容 INSDC 要求并纳入新的序列类型外，新标准还作出了多项更新。部分重大变更包括： •

for divisional applications filed after 1 July 2022, even if the parent application has a filing date before 1 July 2022.

要求使用 XML 格式，而 ST.25 要求使用 TXT 格式不再包括核苷酸少于 10 个或氨基酸少于 4 个的序列现在可以使用必填的 mol_type 限定符更加精确地定义序列标识符（例如，现在可以将 RNA 标注为 mRNA、tRNA 等）

现在，氨基酸序列用一个字母的缩写来表示不再允许混合模式序列（即必须为氨基酸序列和相应的核苷酸序列提交单独的序列）提供更多用于序列注解的选项/限定符

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•

不再需要定义 “x”或“n” ，“x”或“n” 具有默认值（例如，n 是 T、A、G、C；x 是任何天然氨基酸）

•

序列表现在只能包括一个申请人的详细信息和最早主张的优先权的详细信息。

“x” or “n” no longer need to be defined and have a default value (e.g., n is T, A, G, C and x is any natural amino acid)

•

The sequence listing can now only include details of one applicant and details of the earliest claimed priority.

ST.26 还纳入了一些附件，用于为新标

准提供指导。

例如，附件四提供了一个广泛的序列示例表，详细解释了它们在符合 ST.26 的序列表中如何表示。附件七提供了关于将序列表从 ST.25 转换为 ST.26 的有用信息。 WIPO 序列在实施 ST.26 的同时，WIPO 还发布了一款新的软件 WIPO Sequence，这是唯一一款可以编译序列表和生成符合 ST.26 标准的 XML 文件的软件。该软件还提供转换功能，可以将 ST.25 TXT 文件转换为 ST.26 XML 文件（但是由于两种格式之间存在部分不兼容性，所以强烈建议进行人工检查）。新软件可以从 WIPO 网站上下载。该网站还提供了详细的用户手册和测试序列（点击查看）。除了关于新标准的网络研讨会，WIPO 还发布了关于新软件的多语言在线网络研讨会。如需了解更多信息，请点击该网络研讨会链接。在下一篇探讨大麻相关发明专利的文章中，我们将重点介绍该领域的工业生物技术的发展：对植物或宿主生物体进行基因改造，以产生大麻素通路中的关键中间体，或终止大麻素的产生。

annotation of sequences are available

ST.26 also has a number of Annexes that provide guidance on the new standard. For example, Annex IV provides an extensive list of sequence examples with detailed comments on their representation in an ST.26 compliant sequence listing. Annex VII provides useful information on the conversion of ST.25 sequence listings to ST.26 compliant sequence listings. WIPO Sequence In conjunction with the implementation of ST.26, WIPO has released a new software, WIPO Sequence, which is the only software that can be used to compile sequence listings and generate XML files that are ST.26 compliant. The software also provides a conversion feature, which converts an ST.25 TXT file into a ST.26 XML file (however, manual checking is strongly recommended, as there are some incompatibilities between the two formats). The new software can be downloaded from the WIPO website. A detailed user manual and test sequences are also provided (see here). An online webinar, available in multiple languages, on the new software has also been released by WIPO, in addition to further webinars on the new standard. A link to this webinar can be found here, if you require further information.

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从 EPO 角度解读生物序列权利要求 - 大麻素案例研究在下一篇探讨大麻相关发明专利的文章中，我们将重点介绍该领域的工业生物技术的发展：对植物或宿主生物体进行基因改造，以产生大麻素通路中的关键中间体，或终止大麻素的产生。近来，商业界和医学界对大麻素的兴趣日渐增加，并研发了从大麻植物提取物中提取相关大麻素的技术，并在此基础上提出了专利申请。这些技术本身会受到植物天然产生的活性化合物或其前体的数量的限制：由于植物必须在安全的环境中种植，并且种植植物和分离大麻素产品需要大量的能源和水，所以必须提供充足的植物材料，以满足需求。因此，在当今这个气候变化和能源需求备受关注的世界中，人们的兴趣自然而然转向了工业过程，特别是利用基因工程来提高大麻素通路内的酶转化率，无论是在大麻植物中（用以提高提取后的产率），还是在宿主系统（例如大肠杆菌或酵母）中。利用大麻素通路大麻素的天然生物合成过程如图 1 所示，并且包含橄榄醇酸环化酶和异戊烯基转移酶等酶，用于制造关键中间体大麻萜酚酸（CBGA）。CBGA 随后被大麻素合成酶转化为 Δ9-THCA 或 CBDA，中间体分别被非酶转化为 Δ9-四氢大麻酚（大麻的精神活性成分）和大麻二酚。

Interpreting Biological Sequence Claims at the EPO – a Cannabinoid Case Study In this next article looking at patenting cannabis-related inventions, our focus is on the growth in industrial biotechnology in this area: genetically modifying plants or host organisms to produce key intermediates in the cannabinoid pathway, or end cannabinoids themselves. Historically, as commercial and medical interest in cannabinoids increased, technologies, and resulting patent filings, focussed on methodologies for extracting relevant cannabinoids from cannabis plant extracts. Such techniques are inherently limited by the quantities of active compounds, or their precursors, naturally produced by the plants: supply of sufficient levels of plant materials has to keep up with the demand, while being constrained by the requirement to only grow plants in a secure environment, and the large quantities of energy and water required to grow the plants and isolate the cannabinoid products. In a world concerned with climate change and energy demand, interest has therefore naturally turned to more industrial processes, in particular, the use of genetic engineering to increase enzymatic turnover within the cannabinoid pathway, either in cannabis plants for increased yield after extraction, or in host systems such as E.coli or yeast. Exploiting the cannabinoid pathway The biosynthesis of cannabinoids in nature is shown in Figure 1, and includes the enzymes olivetolic acid cyclase and prenyltransferase, to make the key intermediate cannabigerolic acid (CBGA). CBGA is then converted by cannabinoid synthases to either Δ9THCA or CBDA, intermediates which are non-enzymatically converted to Δ9tetrahydrocannabinol (the psychoactive component of cannabis) and cannabidiol respectively.

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橄榄醇酸己酰辅酶 A

橄榄醇酸环化酶（OAC）

3 x 丙二酰辅酶A

牻牛儿基二磷酸盐

芳香异戊烯基转移酶

THC 合成酶

CBDA 合成酶非酶转化 (-CO2)

Figure 1: Cannabinoid Pathway 图 1：大麻素通路

Genomatica 和 Ginkgo Bioworks 等公司

正在围绕这些通路中涉及的变体酶和表达这些酶的工程细胞开发广泛的专利组合。近年来，Genomatica 先后公布了分别针对橄榄醇合成酶变体、橄榄醇酸环化酶变体、大麻素合成酶变体和异戊烯基转移酶变体的专利 WO2020/214951 、WO2020/247741、WO2021/211611 和 WO2021/046367。 Ginkgo Bioworks 近期公布的专利有 WO2021/195520（针对变体末端合成酶）、WO2021/257915（针对橄榄醇酸环化酶变体）、WO2022/011175（针对变体末端合成酶）和 WO2022/081615（针对

异戊烯基转移酶变体和相关的嵌合异戊烯基转移酶和融合多肽）。保护变体由于该领域的专利活动众多，尤其是美国公司，那么专利申请人在编制专利申请时需要考虑哪些因素，才能最大限度地提高他们在欧洲获得广泛且有效的商业保护机会呢？

寻求地域覆盖范围广泛的保护时，向欧洲专利局（EPO）提交欧洲专利申请的性价比最高。但是，在提交美国临时申请和/或 PCT 申请之前，稍作思考和规划也能增加在欧洲获得保护范围广泛的权利要求的机会。虽然 EPO 设定了一项基本要求，限定只 6

Companies such as Genomatica and Ginkgo Bioworks are developing extensive patent portfolios around variant enzymes along these pathways, and engineered cells which express these enzymes. In recent years, Genomatica’s WO2020/214951, WO2020/247741, WO2021/211611, and WO2021/046367 have published, directed to olivetol synthase variants, olivetolic acid cyclase variants, cannabinoid synthase variants, and prenyltransferase variants respectively. Ginkgo Bioworks’ recent publications include WO2021/195520 (directed to variant terminal synthases), (WO2021/257915 (directed to olivetolic acid cyclase variants), WO2022/011175 (directed to variant terminal synthases, and WO2022/081615 (directed to prenyltransferase variants and related chimeric prenyltransferases and fusion polypeptides). Protecting variants With so much patenting activity in this area, notably from US companies, what do patent applicants need to think about when preparing patent applications, to maximise their chances of obtaining broad, commercially useful protection in Europe? The most cost-effective way of obtaining broad geographical coverage is of course via a European patent application at the www.hlk-ip.cn

European Patent Office (EPO). However, a little forethought and planning before filing a US provisional application, and/ or a PCT application, can increase the chances of obtaining allowance of claims with a usefully broad scope in Europe. While there is a basic requirement at the EPO that a gene sequence is only patentable if it is capable of industrial application, this is easily met for most cases – particularly in the cannabinoid sector – in which there is a recognised therapeutic application requiring industrial production of active compounds: a simple statement in the specification setting out the industrial applicability usually suffices.

有能够实现工业应用的基因序列才可以申请专利，但在大多数情况下，这个要求很容易满足——尤其是在大麻素领域——该领域的公认治疗应用需要对活性化合物进行工业生产：通常只需在说明书中简单指明工业适用性即可。转基因生物领域的专利权利要求通常涉及变体酶的多肽序列，以及编码变体酶的基因的对应核苷酸序列，通过引用在作为专利申请一部分或与专利申请一起提交的序列表中公开的一个或多个特定序列来定义。例如：一种与 SEQ ID NO:1 具有至少 80% 相同序列的核酸，该核酸编码具有橄榄醇酸环化酶活性的多肽。包含一个与 SEQ ID NO:2 一致性/相似性至少达 80% 的序列的多肽。与 SEQ ID NO:2 具有至少 80% 同源性的多肽。序列一致性——具体何意？ “序列一致性”是指在给定比对中，两个不同序列在定义长度内完全匹配的核苷酸/氨基酸的数量或百分比，而“序列相似性”是指两个序列在比较时的相似度。基于上述示例性权利要求的公开语言（“具有”、“包含”、“包括”）， 7

Patent claims in the field of genetically modified organisms are typically directed to polypeptide sequences of variant enzymes, and corresponding nucleotide sequences of the genes that encode the variant enzymes, with the claims defined by reference to one or more specific sequences disclosed in a sequence listing filed as part of or with the patent application. Examples of such claims are: A nucleic acid having a sequence that is at least 80% identical to SEQ ID NO:1 and wherein the nucleic acid encodes a polypeptide having olivetolic acid cyclase activity. A polypeptide comprising a sequence that is at least 80% identical / similar to SEQ ID NO:2. A polypeptide including at least 80% homology to SEQ ID NO:2. Sequence identity – what does it even mean? “Sequence identity” refers to the number or percentage of nucleotides/amino acids that match exactly between two different sequences over a defined length in a given alignment, while “sequence similarity” refers to a resemblance between two sequences when compared. Based on the open language of the above example claims (“having”, “comprising”, “including”), the sequence identity can be determined in different ways, depending,

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序列一致性可以使用不同的方式进行确定，具体取决于标的序列与查询序列的长度是否相同以及序列的对齐方式等。评估此类权利要求的新颖性时，权利要求和说明书对此的定义方式不同，结果可能会大相径庭。此外，EPO 可能还会认为这类权利要求不明确，从而驳回申请。图 2 显示了为什么建议（在非必要情况下）指明所用的算法，或者至少指明一致性的定义方式：

for example, on whether the subject and query sequences have the same length or different lengths, or how the sequences are aligned. How this is defined in the claims and specification can lead to very different results when it comes to assessing novelty of such a claim. In addition, the EPO may also consider such claims unclear and thus not allowable. Figure 2 shows why it is advisable, if not essential, to specify the algorithm used, or at the very least how the identity is to be defined: 比对 - 200 nt，4 nt 不匹配

查询： 300 nt

标的：400 nt不匹配

Figure 2: A sequence alignment 图 2：序列比对

查看图 2 中的假设示例，如果仅按 200 nt 序列的比对结果来定义一致性，则一致性比例为 98%（计算方式 (2004)/200 = 98 %) 。但是，如果按 300 nt 查询序列（例如，输入到算法中的序列）来定义一致性，就有 104 nt 不匹配，查询序列的一致性比例降至 65%。而如果按照 400 nt 标的序列（即算法序列数据库内的 hit 序列）定义一致性，就有 204 nt 不匹配，标的序列的一致性比例进一步降至 49% 。 FastA 和 BLAST 等算法基于最佳局部比对计算一致性，而 GLSearch 基于查询序列计算一致性，GSSearch 基于标的序列计算一致性，GGsearch 基于全局比对

计算一致性。

EPO 在其审查指南 F-IV, 4.24 中解释了“

序列一致性”和“序列相似性”的解读方法，并明确指出，如果未在申请中提供或定义算法、计算方法或相似性评分矩阵，将使用截至相关申请日期已知的任何合理算法、计算方法或矩阵对这些条款作出最宽解释。此外，如果相似性或同源性是区分权利要求主题与现有技术的唯一特征，EPO 8

Looking at the hypothetical example in Figure 2, if the identity is defined with reference just to the alignment over the 200 nt sequence, then the percentage identity is calculated as being 98% (with the calculation being (200-4)/200 = 98%). However, if the identity is defined with reference to a 300 nt query sequence (e.g., the sequence inputted into the algorithm), then there are 104 mismatches, and the % identity over the query drops to 65%. Further still, if the identity is defined with reference to a 400 nt subject sequence (i.e., hits within the algorithm sequence database), then there are 204 mismatches and the % identity over the subject sequence drops further to 49%. Algorithms such as FastA and BLAST provide identity over the best local alignment, whereas GLSearch provides identity over the query sequence, GSSearch provides identity over the subject sequence and GGsearch identity over a global alignment. The EPO’s Guidelines for Examination at F-IV, 4.24 explain how “sequence identity” and “sequence similarity” are interpreted, and unequivocally state that if no algorithm, calculation method or similarity-scoring matrix is provided or defined in the www.hlk-ip.cn

将提出明确性异议，除非在提交的申请中明确定义了同源性比例的确定或计算方式。以上述示例为例，如果专利申请人打算使用的序列（例如 BLAST 局部比对）与 EPO 可能搜索的序列（更大的标的序列比对）不一致，并且没有定义算法，那么 EPO 可能会认定变体序列缺乏新颖性和/或不明确。因此，如果未在起草阶段预留适当的备选位置（例如，算法或序列长度），可能就无法修改权利要求，重获新颖性。产率更高、转化更快或对映选择性更高的工程酶或突变酶在任何工业环境中都很受欢迎，并且属于需要保护的商品或资产。如果想要保障自己在欧洲的竞争优势，专利申请人一定不能忽视欧洲专利局在由核苷酸或氨基酸序列定义的生物技术发明方面的审查实践（或者 ST26，实际上 ST26 将于 2022 年 7 月 1 日生效，它与序列表的制备有关——点击查看）。我们的生命科学和医疗保健团队可随时为您提供指导，帮您避免该领域专利申请易犯的错误。如有任何问题，请随时与我们联系。

application, the broadest interpretation of these terms will be applied, using any reasonable algorithm, calculation method or matrix known at the relevant filing date. In addition, if the similarity or homology is the only feature to distinguish the subjectmatter of a claim from the prior art, the EPO will raise a clarity objection unless the determination or calculation of the percentage of homology is clearly defined in the application as filed. Using the above example, loss of identity between what a patent applicant intends (for example BLAST local alignment) and what the EPO might search (alignment over a larger subject sequence) could result in a variant sequence being considered to lack novelty and/or be unclear if no algorithm was defined. As a result, without suitable fallback positions (e.g., to the algorithm, or the length of sequence) included at the drafting stage it may not be possible to amend the claims to restore novelty. Engineered or mutant enzymes that have higher yields, faster turnover, or higher enantioselectivity are sought after in any industrial setting and are commodities or assets that need to be protected. It is important that patent applicants do not lose sight of the European Patent Office’s examination practices for biotechnological inventions defined by nucleotide or amino acid sequence (or, indeed, the entry into force on 1 July 2022 of ST26 relating to the preparation of sequence listings – see here) if a competitive edge in Europe is to be secured. Our Life Sciences and Healthcare team are always on hand to guide you through the pitfalls of patenting in this area. Feel free to reach out to us with any questions you might have.

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