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RésuméAnne Marie ClarkAnne Marie is a Senior Searcher on theCAS IP Services team. Her particularsubject expertise is small molecules,sequences, and formulations, and she isskilled in patent searching, analysis andlandscaping, and competitivebenchmarking.

Peter BlasiPeter leads business development forthe CAS IP Services team, introducingclients to the value of CAS IP Servicesand bringing them together withseasoned search professionals to meettheir information needs.

32 THE PATENT LAWYER CTC Legal Media

With the approval of two smallinterfering RNA (siRNA) biopharma-ceuticals and 52 clinical trials spanning

multiple therapeutic areas in progress, the valueand complexity of the patent landscape aroundRNA interference (RNAi) is increasing. Challengeswith searching siRNA include non-standardizedterminology, drug delivery that is claimedseparately from the molecules, and the limitationsof familiar biosequence search methodologies.Due to these challenges, RNAi searches requirespecialized expertise in search techniques andinformation sources where those techniquesshould be implemented.

This article examines how RNAi inventions are claimed and described in the specification,which information sources ensure the mostcomprehensive retrieval, and the best search

techniques to use. These examples and techniquesaim to demystify the process for finding relevantpatent and non-patent literature related toefficiently retrieving these sequences.

RNA interference discovery and mechanismIn 1998, Andrew Fire and Craig Mello discoveredRNA interference (RNAi), sometimes called Post-Transcriptional Gene Silencing (PTGS), for whichthey received the Nobel Prize in 2006. Smallinterfering RNAs (siRNAs) selectively silence theexpression of a target gene by degrading itsmRNA. The silenced gene is specified by thesmall RNA component, which recognizes thetarget by base pairing.

RNAi allows a brand-new class of drugs.Sequence-specific gene silencing can eliminatethe root cause of a disease, and can halt orreverse the disease progression, rather thanslowing it or only treating the symptoms. Thereare two common types of RNAi molecules:micro RNA (miRNA) and small interfering RNA(siRNA). Both are non-coding RNAs, as they donot code for a protein. miRNA are small RNAmolecules which silence genes by binding totarget messenger RNAS (mRNAs). Mechanismsinclude:

1) Cleavage of the mRNA strand into two pieces,

2) Destabilization of the mRNA throughshortening of its poly(A) tail, and

3) Less efficient translation of the mRNAinto proteins by ribosomes.

Since their discovery in 1993, miRNA have beenfound in all eukaryotic cells conserved acrossthe species. miRNAs are small non-coding RNAmolecules (containing about 22 nucleotides) andoften have hairpin structures as seen in Figure 1.

Demystifying RNAifor stronger patentsearches

Peter Blasi and Anne Marie Clark of CAS IP Services discuss avoiding pitfallsin patentability and freedom to operate searches.

Peter Blasi

Anne Marie Clark

RNAi

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siRNA, sometimes known as short interferingRNA or silencing RNA, is a class of double-strandedRNA, typically 20-25 base pairs in length, similarto miRNA. Typically, siRNA duplexes have 21-ntsense and 21-nt antisense strands, paired with a 2-nt 3' overhang as seen in Figure 2. Intherapeutic siRNAs, 2'-deoxynucleotides in the 3'overhangs are used instead of ribonucleotidessince they are cheaper to synthesize and probablymore nuclease resistant.

RNAi Drug DiscoveryThere are 52 clinical trials on RNAi-based drugsspanning multiple therapeutic areas in progress.Successful drugs must be safe and effectivewith relatively convenient dosing. For therapeutics,siRNA development challenges include off-target transcript silencing (causing safetyconcerns), delivery to target cells, and degradationby nucleases causing the drug to be degradedbefore achieving a therapeutic dose. Chemicalmodification and bioconjugation can helpovercome these problems.

The US Food and Drug Administration recentlyapproved two siRNA drugs: Onpattro (Patisiran)and Givlaari (Givosiran). Both drugs were developedby Alnylam Pharmaceutical and are shownbelow in Figures 5 and 6. Patisiran is a chemicallymodified, double-stranded small interferingribonucleic acid (siRNA), formulated as a lipidcomplex for delivery to hepatocytes and is usedfor the treatment of transthyretin-mediatedamyloidosis. Patisiran specifically binds to agenetically conserved sequence in the 3’untranslated region (3’UTR) of mutant and wild-type transthyretin (TTR) messenger RNA(mRNA).

Source: US Patent Application No. 20050144669

Figure 1. miRNA hairpin structure binding a target sequence.

Figure 2 Structure of siRNA

Figure 4: Top 10 companies filing RNAi-related patent publications since 1991

Source: US Patent No. 9260470

siRNAs are 21-23 nucleotide double stranded RNA

(dsRNA) duplexes with symmetric 2-3 nucleotide 3’

overhangs and 5’ phosphate and 3’ hydroxyl groups

Growth in RNAi Patent PublicationsSince the discovery of RNAi, there has been steadygrowth in the volume of worldwide patentpublications that disclose its different forms. Thefollowing two figures show the number of worldwidepatent publications and the companies with themost RNAi-related patent publications since 1991.

Figure 3: Number of worldwide RNAi-related patent publications by year

(Source: CAS content collection)

Note: Due to the elapsed time between patent application and publication, the last

two years of data are incomplete.

(Source: CAS content collection).

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How RNAi is Represented in PatentsRNAi is claimed in a variety of ways within patents. siRNA orRNAi terminology is not always used, and chemical modificationscan be described a variety of ways which means that there is nosingle strategy that can be used to efficiently retrieve thisinformation from different databases. For example, US PatentNo. 8,334,373 does not use the term siRNA or claim specificsequences. It uses double-stranded ribonucleic acid terminologyand claims chemical modifications as sequence motifs makingboth retrieval and analysis challenging. Claim 1 is shown below.

In US Patent No. 9,101,643, lipid-formulated compositionsand methods for inhibiting expression of transthyretin (ttr) areclaimed, and analysis of the full patent document is requiredto determine the scope of the claims, as shown below.

However, as seen in Figure 8 and Table 2, neither the claimnor the sequence listing describes the chemical modifications.The chemical modifications are described in Tables 3 and 4,making analysis challenging.

Figure 5: Chemical Structure of Patisiran

(Source: US Food and Drug Administration)

Givosiran is an aminolevulinate synthase 1-directed small interfering

RNA (siRNA), covalently linked to a ligand containing three

N-acetylgalactosamine (GalNAc) residues to enable delivery of the siRNA

to hepatocytes. Givosiran is used to treat acute hepatic porphyria and it is

both chemically modified and bioconjugated.

(Source: US Food and Drug Administration)

Abbreviations: Af = adenine 2'-F ribonucleoside;

Cf = cytosine 2'-F ribonucleoside; Uf = uracil 2'-F ribonucleoside; Am =

adenine 2'-OMe ribonucleoside; Cm = cytosine 2'-OMe ribonucleoside;

Gf=guanine 2'F ribonucleoside; Gm = guanine 2'-OMe ribonucleoside;

Um = uracil 2'-OMe ribonucleoside;

L96 = triantennary GalNAc (N-acetylgalactosamine)

Figure 6: Chemical Structure of Givosiran

Figure 7 Claim in Patent No. 8,334,373

Table 1: Summary of the Invention in the '373 Patent

Figure 8: Claim 1 from '643 Patent

Table 2: SEQ ID No. 1009

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Search Techniques to RetrieveRNAi ReferencesA comprehensive search for RNAi prior art shouldinclude both a sequence search and a text search,because of the inconsistencies of how RNAi-related inventions are claimed. There are a varietyof different human-curated and algorithmically-curated databases with information for bothsequences and text. A typical workflow for apatent search is: start with sequences withhuman-curated sequence databases such as CAS BIOSEQUENCESTM first, then searchalgorithmically-curated sequence databases.After the sequence search, search text terms inhuman-curated databases, such as CAplusSM,and then finally search full-text patent databases.An siRNA search should start by searching theunmodified base sequence in a biosequencedatabase.

Some common methodologies for sequencesearches include:

• Exact sequence - retrieves sequenceswith exact match, and same length.

• Subsequence - retrieves sequenceswhere the query sequence may beembedded.

• Motif search - retrieves sequences with.repeats, alternatives, spacers, etc.

• BLAST (Basic Local Alignment SearchTool) - searches sequence similarity(generally expressed as % of sequenceidentity, or %ID).

siRNA and miRNA sequences are usually firstsearched using a motif allowing specific variablesor exact search where 100% matches are found.

The following questions should be consideredby the searcher and attorney:

• Should overhanging nucleotides betrimmed from the strategy? Theoverhanging nucleotides can bereplaced with bioconjugates or modifiednucleotides.

• Will the strategy retrieve chemicalmodifications or bioconjugates?

• Will the bioconjugate molecule besearched separately?

Conducting an RNAi sequence search usinghuman-curated databases can yield preciseresults. When searching text, the strategy shouldinclude such human-curated databases likeCAplus which offer the increased power andprecision of controlled vocabulary as well asfull-text patent databases. When developing asearch strategy, consider multiple ways the siRNAcan be described such as modified dsRNA,oligonucleotides, oligomers, RNA interference,RNAi. For chemical modifications, the modificationpattern, not just a specific sequence, can beclaimed and should be searched. Claims canalso be target-focused rather than substance-focused; so, that aspect may also be searched.To ensure the most reliable results, RNAi searchesshould cover different features, uses, andcharacteristics of the siRNA/miRNA.

ConclusionA high-quality, professional search performedusing multiple information sources and searchtechniques specific to RNAi is extremely valuableto support drafting the strongest possible RNAiclaims. A search performed by a reliableprofessional with database and strategy knowledgeis the key first-step to assist attorneys who areadvising their clients on patentability andfreedom-to-operate.

Theseexamplesandtechniquesaim todemystifythe process.

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ContactCAS IP ServicesTel: 866-360-0814www.scienceip.orgwww.cas.org

Table 3: Abbreviations of Nucleotide Monomers Used in Nucleic Acid Sequence

Representation

Table 4: Sequences With Chemical Modifications

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