Hepatitis B is an infectious disease caused by hepatitis B virus (HBV) infection in human, and is mainly manifested as liver lesions. Nucleoside analogs and interferon drugs are generally used to treat patients with chronic hepatitis B virus infection. However, these drugs often require long-term or even lifelong administration, and might face tolerance problems.

RNA interference (RNAi) is an emerging therapy, which acts by destroying the messenger RNA (mRNA) of target genes with RNA molecules to inhibit the expression of disease-related genes. RNAi therapy has been widely used in exploring gene function and in the field of gene therapy for infectious diseases and malignant tumors. RNAi therapy is of great significance to the research of new drugs for hepatitis B and is expected to become a new therapy that can effectively cure hepatitis B.

Hepatitis B virus has four open reading frames (ORFs) encoding the pre-core/core protein, polymerase, surface protein and X protein, respectively. The four ORFs have overlapping sequences and the same polyadenylation signal at the 3' end of the core protein coding region. This allows a single siRNA to interfere with all the four HBV transcripts. A great deal of research has been conducted on RNAi therapy for hepatitis B virus, resulting in patented technologies, which are promising candidate RNAi therapies for developing new hepatitis B drugs. The patent number ZL201580072874.0 titled "Hepatitis B Virus (HBV) iRNA Composition and Methods of Use Thereof" in the name of Alnylam Pharmaceuticals Inc. is one of these patented technologies.

The petitioner Zhang Yuan submitted a request for invalidation against the patent ZL201580072874.0 to the China National Intellectual Property Administration (CNIPA) on December 31, 2021 on the grounds that claims 1-41 are unclear, claims 1-10 and 12-41 are not supported by the description, the description fails to make sufficient disclosure, and claims 1-41 lack inventiveness.

In response to the petitioner's request for invalidation, the patentee amended the claims by adding the technical features of claim 2 to claim 1, and deleting the terms "a deoxy-nucleotide", "a locked nucleotide", "an abasic nucleotide", "a 2'-hydroxyl-modified nucleotide", "a 2'-O-alkyl-modified nucleic acid" and "a non-natural base comprising nucleotide " in previous claim 3.

The CNIPA conducted examination on this patent and made an Invalidation Decision on October 10, 2022, which states that the patent is partially upheld based on the amended claims submitted by the patentee.

The amended claims 1 and 2 are as follows:

"1. A double stranded RNAi agent for inhibiting expression of hepatitis B virus (HBV) in a cell, wherein said double stranded RNAi agent comprises a sense strand and an antisense strand forming a double stranded region,

“wherein said sense strand comprises 5'-GUGUGCACUUCGCUUCACA-3' (SEQ ID NO: 39) and said sense strand has no more than 21 nucleotides in length, and said antisense strand comprises 5'-UGUGAAGCGAAGUGCACACUU-3' (SEQ ID NO: 40) and said antisense strand has no more than 23 nucleotides in length,

“wherein all of the nucleotides of said sense strand and all of the nucleotides of said antisense strand are modified nucleotides,

“wherein said sense strand is conjugated to a ligand attached at the 3'-terminus, and

“wherein the ligand is one or more GalNAc derivatives attached through a divalent or trivalent branched linker.

“2. The double stranded RNAi agent of claim 1, wherein at least one of said modified nucleic acids is selected from the group consisting of a 3'-terminal deoxy-thymine (dT) nucleotide, a 2'-O-methyl modified nucleotide, a 2'-fluoro modified nucleotide, a 2'-deoxy-modified nucleotide, a locked nucleotide, an unlocked nucleotide, a conformationally restricted nucleotide, a constrained ethyl nucleotide, an abasic nucleotide, a 2'-amino-modified nucleotide, a 2'-O-allyl-modified nucleotide, a 2'-C-alkyl-modified nucleotide, a 2’-hydroxly-modified nucleotide, a 2'-methoxyethyl modified nucleotide, a morpholino nucleotide, a phosphoramidate, a tetrahydropyran modified nucleotide, a 1,5-anhydrohexitol modified nucleotide, a cyclohexenyl modified nucleotide, a nucleotide comprising a phosphorothioate group, a nucleotide comprising a methylphosphonate group, a nucleotide comprising a 5'-phosphate, and a nucleotide comprising a 5'-phosphate mimic.”

Based on the amended claims, the patent claims focus on a double stranded RNAi sequence defined in an open-ended manner. The patent claims define the core sequence of the double stranded RNAi sense strand and antisense strand and the sequence length. They also define that all of the nucleotides of said sense strand and all of the nucleotides of said antisense strand comprise a modification, and the sense strand is conjugated to a ligand at the 3'-terminus. The type of ligand and the conjugation manner are defined.

This patent description provides experimental data on two specific double stranded RNAi. The two specific double stranded RNAi have a dsRNA primary sequence as defined in claim 1 and have a sense strand conjugated to one or more GalNAc ligands at the 3'-terminus, and all the nucleotides comprise a modification which is a 2'-O-methylation and a 2'-fluoro phosphorothioate group.

This case focuses on the following issues: whether the protection scope of the patent related to double stranded RNAi drugs has support in the description, whether the description sufficiently discloses the invention, and whether the invention possesses inventiveness.

Specifically, the petitioner proposed the following opinions:

1) The claims are not supported by the description. More specifically, the claims of this patent define that all of the nucleotides of said sense strand and all of the nucleotides of said antisense strand comprise a modification, but do not define the specific type, position, and number of the modification. Therefore, the protection scope defined by the claims is unclear. The double stranded RNAi sequence is defined by an open-ended term “comprise” in the patent claims, and thus those skilled in the art cannot determine which sequence(s) other than the verified ones can maintain RNA interference activity similar to that of the modified sequences as verified. The claims define that the sense strand is conjugated to one or more GalNAc derivatives at the 3'-terminus. However, those skilled in the art cannot beforehand determine which ligands attached through other branched linkers than the L967 ligand recited in the description can cause the modified double stranded RNAi agent to achieve a similar technical effect.

2) The description fails to sufficiently disclose the invention. More specifically, the description does not disclose the technical solution that substantial all of the nucleotides in the sense strand or antisense strand of the double stranded RNAi agent comprise a modification as defined in claim 1. The description does not disclose a parallel technical solution having modification types other than 2'-O-methylation, 2'-fluoro, and phosphorothioate linkage groups. There are contradictions between different experimental results, and thus it is reasonable to doubt the authenticity of the above experimental data, and in turn the technical effect of the present invention.

3) The claims possess no inventiveness. More specifically, Evidence 13 discloses candidate dsDNAs for inhibiting hepatitis B virus expression, wherein there is a dsDNA which differs from the double stranded RNAi defined in the patent claims by only one base pair of the core sequence in the primary sequence, and two bases at the terminus of the antisense strand. Evidence 13 and other reference documents give a motivation of combining these distinguishing features. Moreover, the reference documents also give a motivation of combining other distinguishing features (i.e., the modification manner, and the types and conjugation manner of the ligands).

On these issues, the opinions in the Invalidation Decision are as follows.

1) Regarding the protection scope of the patents related to double stranded RNAi sequence.

Regarding the issue whether a claim is clear, if those skilled in the art can understand the meaning of the technical features defined in the claim based on the conventional technical knowledge in the field and the disclosure in the patent application documents, the technical features will not cause the protection scope of the claim to be unclear. Regarding the issue whether a claim is supported by the description, the evaluation should be made from the perspective of those skilled in the field, based on the technical knowledge he or she possesses, with full reference to relevant prior art information and consideration of the entire content of the description, but not only the content of the Detailed Description of the Preferred Embodiments.

In this case, regarding the issue of clarity, the amended claims define that all of the nucleotides of said sense strand and all of the nucleotides of said antisense strand comprise a modification. The patent description discloses the purposes, roles and main types of nucleic acid modifications on the sense strand and antisense strand of iRNA (such as dsRNA) in the patent technical solutions, and explicitly discloses the technical concept of making chemical modification to enhance stability or other beneficial properties. The patent description also exemplifies the alternative modification types in detail and provides corresponding documents in prior art for reference. Based on the conventional technical knowledge in the field and the disclosure in patent application documents, those skilled in the art can understand the exact meaning of the expression "all of the nucleotides comprises a modification" recited in the claims.

Regarding the support issue, the claims define the core sequences of the sense and antisense strands of the double stranded RNAi sequence and their lengths. In particular, the claims define 19 nucleotides of the core sequence of the sense strand of the double stranded RNAi sequence and the sense strand has no more than 21 nucleotides in length. Also, they define 21 nucleotides of the core sequence of the antisense strand of the double stranded RNAi sequence and the antisense strand has no more than 23 nucleotides in length. The claims also define that the sense strand is conjugated to a ligand attached at the 3'-terminus, and further define that the conjugated ligand is one or more GalNAc derivatives attached through a divalent or trivalent branched linker.

Firstly, it is well known in the art that the in vitro silencing effect of an siRNA mainly depends on its primary sequence. Where the structure of the primary sequence of an siRNA is determined, in order to enhance the stability of the siRNA, those skilled in the art can select a particular modification group according to the technical guidance in the prior art. Secondly, the treatment by double stranded RNAi is based on the sequence complementarity between the corresponding dsRNA core sequence and the target RNA. Based on the systematic disclosure of this patent description and in combination with conventional technical means for selection and verification, those skilled in the art have the ability to add one or two nucleotides at either terminus of the dSRNA core sequence to form a structure, such as overhang, while maintaining the corresponding basic function. Finally, this patent description discloses the types, properties, conjugation manner and other information of alternative ligands. The research on GalNAc derivatives as siRNA ligands in the art can also be taken as reference. Thus, those skilled in the art have ability to obtain suitable GalNAc ligands for siRNA and the conjugation manner thereof, while maintaining the corresponding basic function of siRNA.

2) Regarding sufficient disclosure of an invention by the description.

Sufficient disclosure of an invention means that those skilled in the technical field can implement the technical solution(s), solve the technical problem(s), and produce the expected technical effect(s) based on the technical content disclosed in the application.

As far as this case is concerned, as the claims have defined the primary sequence of dsRNA, those skilled in the art can expect that modified dsRNA with the sequence defined in the claims can effectively inhibit the expression of hepatitis B virus (HBV) in cells; and the description have sufficiently disclosed the expected technical effect(s) of the modified dsRNA with the sequence defined in the claims. Biomedicine is a typical experimental science, and thus the authenticity and probative force of the experimental data in the description should be comprehensively evaluated with all factors affecting the experimental data being fully taken into account, usually including the common technical knowledge in the field, the overall prior-art status, and the experimental conditions and measurement standards disclosed in the description, etc. The fact that there is a certain deviation between the experimental data recited in the description and the theoretical expectations does not necessarily render the experimental data completely unreliable.

3) Regarding Inventiveness level of the Invention.

When evaluating the technical motivation related to the inventiveness of an invention, one needs to focus on whether those skilled in the field, without knowing the technical solution of the invention, would be motivated to improve the prior art to obtain the technical solution of the invention based only on the teachings of the prior art, but not focus on considering the possibility or feasibility of making such improvements from the prior art after knowing the technical solution of the invention. Also, when evaluating whether there is motivation to combine two or more prior-art documents to obtain the technical solution of an invention, one should fully analyze whether there is reasonable expectation of success in introducing the distinguishing technical feature(s) to solve the technical problem to be actually solved by the invention. If the results after the two or more prior-art documents are combined cannot be expected, it is usually difficult to produce motivation to purposefully combine these prior-art documents.

As far as this case is concerned, there are four distinguishing technical features between the claims of this patent and the closest reference document. The four distinguishing technical features are: one different base pair in the core sequence, two different bases at the terminus of the antisense strand, different modification manners, and presence of a conjugated ligand or not. Although these distinguishing technical features are described in different reference documents, including the closest reference document, they should not be considered separately. These distinguishing features involve several different aspects of sequence structural characteristics, and only a comprehensive consideration thereof can objectively analyze the structure-activity relationship of an siRNA molecule. If starting from the dsRNA modification sequence targeting the hepatitis B virus gene disclosed in the closest reference document, it is difficult for those skilled in the art to make structural improvements based on such sequence, so as to obtain a double stranded RNAi agent effectively inhibiting the expression of hepatitis B virus (HBV) in cells. As far as the primary sequence design of siRNA is concerned, the reference document only generally discloses alternative siRNA design rules, but it neither teaches their use in the recited specific dsRNA modification sequences, nor implicitly discloses the priority order and way of application of these rules. The reference document does disclose the rules for making selection among many possible siRNAs based on a target sequence, but they are not a determining principle for designing an siRNA sequence, and thus further selection and confirmation are still needed in the practice of applying these rules in siRNA design. Moreover, the dsRNA modification sequences in the closest prior art for this patent do not satisfy many of the selection rules listed in the above evidence. Therefore, those skilled in the art would have no motivation to make structural improvements and modifications based on the closest prior art, with reference to the selection rules disclosed in the above reference documents. The double stranded RNAi agents as claimed in the claims of this patent can effectively inhibit the expression of hepatitis B virus (HBV) in cells, which is resulted from a large number of experimental screenings conducted in a targeted and directional manner in this patent, and cannot be predicted from the prior art. Furthermore, this patent achieves balance between activity and in vivo stability through the combined application of core technical means, such as primary sequence reconfiguration, nucleotide modification, and ligand selection, effectively extending the duration of in vivo activity.

Through this case, we can understand the principles of determining the reasonable boundary of the protection scope of a claim involving gene sequence. For an invention involving gene sequence, the difficulties in patent drafting lie in inventiveness of the invention defined by gene sequence, support of the invention in the description, and sufficient disclosure of the invention by the description. During the substantive examination stage, the examiner often requires that inventions involving gene sequence are defined by the sequences whose effects are verified in the examples. Inventions defined in such a manner usually have protection scope that is too narrow to match the inventor's technical contribution to the prior art, and thus the rights obtained by the applicant/patentee would also be greatly compromised. In this case, the Invalidation Decision states that analysis should be made starting from technical principles, the protection scope should be interpreted through the technical principles, and factors such as "the function to be achieved by sequence", "whether it is usual to add a further sequence to either end(s)" and "the impact of the sequence to be added on its original function" should be considered when evaluating the protection scope of such claims. This case ultimately supports sequence extension to the core sequence in the claims, and appropriate extension of the protection scope of the claims based on the inventor's technical contribution. This principle of determining the boundary of the protection scope of a claim is also applicable to antibody-related invention patents. In antibody-related invention patents, it is a common way to define a claimed antibody by using six CDR sequences.

This case provides the manner of evaluating whether the difference(s) in gene sequence as compared with the prior art could bring inventiveness to the technical solution. From the aspect of the structure of the short basic sequence alone, this patent differs from the closest prior art by only one nucleotide. When evaluating the technical motivation related to gene sequence difference(s), one needs to focus on whether those skilled in the art, without knowing the technical solution of the invention, would be motivated to improve the prior art to obtain the technical solution of the invention based only on the teachings of the prior art. Also, when evaluating whether there is motivation to combine two or more prior-art documents to obtain the technical solution of an invention, one should fully analyze whether there is a reasonable expectation of success in introducing the sequence difference(s) to solve the technical problem to be actually solved by the invention. At the same time, for the inventiveness of the technical solution(s) involving gene sequence, in addition to the difference(s) in primary sequence as compared with the prior art, one should also comprehensively consider the difference(s) of the gene sequence(s) in nucleotide modification and conjugation ligand as compared with the prior art. One should consider whether the technical effects brought about by all distinguishing technical features could be expected and whether they would exceed the reasonable expectation of those skilled in the art. Therefore, for inventions related to gene sequence, one should consider not only the structural characteristics of the primary sequence, but also other structural characteristics, as well as the technical effects brought about by the combination of these structural characteristics.