EP Patent status and claim 1 of Broad’s CRISPR-Cas9 Patents.

‘執筆用メモ

欧州におけるBroad Instituteの特許の状況。～2018年10月初旬までに成立済のものまで。

EP2764103：口頭審理待ち（2019/03/04、補正クレーム提出済）

1.  An engineered, non-naturally occurring Clustered Regularly Interspersed Short Palindromic Repeats (CRISPR)-CRISPR associated (Cas) (CRISPR-Cas) vector system comprising one or more vectors comprising:

     a) a first regulatory element operably linked to one or more nucleotide sequences encoding one or more CRISPR-Cas system guide RNAs that hybridize with target sequences in polynucleotide loci in a eukaryotic cell, the guide RNA comprising a guide sequence, a tracr sequence, and a tracr mate sequence,

     b) a second regulatory element operably linked to a nucleotide sequence encoding a Type II Cas9 protein, said protein comprising a nuclear localization signal (NLS);

     wherein components (a) and (b) are located on same or different vectors of the system, wherein the tracr sequence is 30 or more nucleotides in length, and

     whereby the one or more guide RNAs target the polynucleotide loci in a eukaryotic cell and the Cas9 protein cleaves the polynucleotide loci,

     whereby sequence of the polynucleotide loci is modified; and, wherein the Cas9 protein and the one or more guide RNAs do not naturally occur together.

EP2771468：特許取消→審判請求

1.  A non-naturally occurring or engineered composition comprising:   a Clustered Regularly Interspersed Short Palindromic Repeats (CRISPR)-CRISPR associated (Cas) (CRISPR-Cas) system chimeric RNA (chiRNA) polynucleotide sequence, wherein the polynucleotide sequence comprises

    (a) a guide sequence of between 10 - 30 nucleotides in length, capable of hybridizing to a target sequence in a eukaryotic cell,

    (b) a tracr mate sequence, and

    (c) a tracrRNA sequence

  wherein (a), (b) and (c) are arranged in a 5' to 3' orientation,

  wherein when transcribed, the tracr mate sequence hybridizes to the tracrRNA sequence and the guide sequence directs sequence-specific binding of a CRISPR complex to the target sequence,

  wherein the CRISPR complex comprises a Type II Cas9 protein complexed with (1) the guide sequence that is hybridized to the target sequence, and (2) the tracr mate sequence that is hybridized to the tracrRNA sequence,

  wherein the tracrRNA sequence is 50 or more nucleotides in length.

・優先権無効→D3およびD4に基づく新規性欠如

D3:MALI P. ET AL, "RNA-GUIDED HUMAN GENOME ENGINEERING VIA CAS9", SCIENCE, (20130215), vol. 339, no. 6121, doi:10.1126/SCIENCE.1232033, pages 823 - 826, XP055111247, DOI:   http://dx.doi.org/10.1126/science.1232033

D4：WOONG Y HWANG ET AL, "EFFICIENT GENOME EDITING IN ZEBRAFISH USING A CRISPR-CAS SYSTEM", NATURE BIOTECHNOLOGY, (201303), vol. 31, no. 3, doi:10.1038/NBT.2501, pages 227 - 229, XP055086625, DOI:   http://dx.doi.org/10.1038/nbt.2501

EP2784162：特許取消（2019/02/13）→審判請求（2019/02/15）

1.  An engineered, non-naturally occurring Clustered Regularly Interspersed Short Palindromic Repeats (CRISPR)-CRISPR associated (Cas) (CRISPR-Cas) vector system comprising one or more vectors comprising:

  a) a first regulatory element operably linked to one or more nucleotide sequences encoding one or more CRISPR-Cas system polynucleotide sequences comprising a guide sequence, a tracr RNA, and a tracr mate sequence, wherein the guide sequence hybridizes with one or more target sequences in polynucleotide loci in a eukaryotic cell ,

  b) a second regulatory element operably linked to a nucleotide sequence encoding a Type II Cas9 protein, wherein components (a) and (b) are located on same or different vectors of the system,

  wherein the CRISPR-Cas system comprises two or more nuclear localization signals (NLSs) expressed with the nucleotide sequence encoding the Cas9 protein,

   whereby the one or more guide sequences target the one or more polynucleotide loci in a eukaryotic cell and the Cas9 protein cleaves the one or more polynucleotide loci,

   whereby the sequence of the one or more polynucleotide loci is modified.

・こちらも優先権が争われていたので、優先権無効→新規性or進歩性欠如か？

EP2825654：口頭審理未定（補正クレーム提出済（2018/09/03））

1.  A non-naturally occurring or engineered composition comprising a vector system comprising one or more vectors comprising

  I. a first regulatory element operably linked to a CRISPR-Cas system chimeric RNA (chiRNA) polynucleotide sequence, wherein the polynucleotide sequence comprises

    (a) a guide sequence capable of hybridizing to a target sequence in a eukaryotic cell,     (b) a tracr mate sequence, and

    (c) a tracr sequence, and

  II. a second regulatory element operably linked to an enzyme-coding sequence encoding a CRISPR enzyme comprising at least one or more nuclear localization sequences (NLSs) in the proximity of a terminus of the CRISPR enzyme, wherein (a), (b) and (c) are arranged in a 5' to 3' orientation,

  wherein components I and II are located on the same or different vectors of the system,

  wherein when transcribed, the tracr mate sequence hybridizes to the tracr sequence and the guide sequence directs sequence-specific binding of a CRISPR complex to the target sequence,

   wherein the CRISPR complex comprises the CRISPR enzyme complexed with (1) the guide sequence that is hybridized to the target sequence, and (2) the tracr mate sequence that is hybridized to the tracr sequence, and

   wherein the chimeric RNA polynucleotide sequence comprises two or more hairpins.

EP2840140：口頭審理未定（補正クレーム提出済（2018/07/23））

1.  A method of selecting one or more prokaryotic cell(s) by introducing one or more mutations in one or more prokaryotic cell(s), the method comprising:

   introducing one or more vectors into the prokaryotic cell(s);

   wherein the one or more vectors drive expression of one or more of: a CRISPR enzyme, a guide sequence linked to a tracr mate sequence, a tracr sequence, and an editing template for recombination into a targeted chromosomal locus comprising a target polynucleotide;   and all of a CRISPR enzyme, a guide sequence linked to a tracr mate sequence, and a tracr sequence, and an editing template are produced in the prokaryotic cell(s);

   introducing the editing template into a target polynucleotide through recombination,

   wherein the editing template comprises the one or more mutations of the target polynucleotide that alter either the protospacer-adjacent motif (PAM) sequence or the protospacer sequence, and that abolish CRISPR enzyme cleavage of the target polynucleotide;

   allowing a CRISPR complex to bind to the target polynucleotide to effect cleavage of the target polynucleotide;

   wherein the CRISPR complex comprises the CRISPR enzyme complexed with (1) the guide sequence that is hybridized to a target sequence within the target polynucleotide, and (2) the tracr mate sequence that is hybridized to a tracr sequence;

   wherein cleavage of the target polynucleotide by the CRISPR complex induces cell death;

   thereby allowing one or more prokaryotic cell(s) in which one or more mutations have been introduced to be selected.

・対象が原核生物（重要性低）

EP2931892：特許許可（2018/08/06）

1.  A computer implemented method for selecting a CRISPR complex for targeting and/or cleavage of a candidate target nucleic acid sequence within a cell, comprising the steps of:

  (a) determining amount, location and nature of mismatch(es) of guide sequence of potential CRISPR complex(es) and the candidate target nucleic acid sequence,

  (b) determining contribution of each of the amount, location and nature of mismatch(es) to hybridization free energy of binding between the target nucleic acid sequence and the guide sequence of potential CRISPR complex(es) from a training data set,

  (c) based on the contribution analysis of step (b), predicting cleavage at the location(s) of the mismatch(es) of the target nucleic acid sequence by the potential CRISPR complex(es), and

  (d) selecting the CRISPR complex from potential CRISPR complex(es) based on whether the prediction of step (c) indicates that it is more likely than not that cleavage will occur at location(s) of mismatch(es) by the CRISPR complex wherein step (b) is performed by defining a thermodynamic model having a set of weights linking effective free energy of hybridization Z to local free energies G;

   defining a training set of guide RNA/target DNA sequence pairs; inputting known values of local free energies G for each guide RNA/target DNA sequence pair in the training set; calculating a value of effective free energy of hybridization Z for each guide RNA/target DNA sequence pair in the training set;

   determining the weights using a machine learning algorithm and outputting the weights whereby the weights can be used to estimate the free energy of hybridization for any sequence.

・機械学習を用いた設計用プログラム

EP2931897：口頭審理未定（補正クレーム提出済（2019/01/08））

1.  A composition comprising a CRISPR complex comprising

  a) a tracr mate RNA polynucleotide,

  b) a tracr RNA polynucleotide,

  c) a guide RNA polynucleotide capable of hybridizing to a target sequence in a eukaryotic cell or a cell in a multi-cellular eukaryotic organism; and

  d) a Cas9 comprising one or more nuclear localization sequences, for use in therapy.

・CRISPR-Cas9複合体を含む第一医薬用途組成物。なぜこのクレームで通る…。

EP2898075：口頭審理待ち（2019/07/04、補正クレーム提出済（2018/05/09））

1.  A composition comprising:

  CRISPR complex components comprising:

       I. CRISPR-Cas system polynucleotide sequence(s) which comprise(s):

           (a) an engineered guide sequence comprised of RNA and capable of hybridizing to a target sequence in a polynucleotide locus,

           (b) a tracr mate sequence comprised of RNA, and

           (c) a tracrRNA sequence comprised of RNA, and wherein (a), (b) and (c) are arranged in a 5' to 3' orientation,

       II. a Type II Cas9 protein, wherein the tracr mate sequence hybridizes to the tracrRNA sequence and the guide sequence directs sequence-specific binding of a CRISPR complex to a target sequence,

  wherein the CRISPR complex comprises the Type II Cas9 protein complexed with (1) the guide sequence that is hybridized to the target sequence, and (2) the tracr mate sequence that is hybridized to the tracrRNA sequence, and

  wherein the Type II Cas9 protein is or comprises a Staphylococcus aureus Cas9 (SaCas9).

・SaCas9タイプ（小さいCas9）のII型CRISPR-Cas9複合体、JPに対応する特許があったはず。

EP2931898：口頭審理待ち（2019/05/15、補正クレーム提出済（2018/03/27））

1.  A non-naturally occurring or engineered composition comprising:

  a delivery system operably configured to deliver CRISPR-Cas complex components or polynucleotide sequences comprising or encoding said components into a eukaryotic cell, wherein said CRISPR-Cas complex is operable in the eukaryotic cell;

       I. one or more CRISPR-Cas complex polynucleotide sequences comprising or encoding for expression in the eukaryotic cell:

           (a) a guide sequence capable of hybridizing to a target sequence in a eukaryotic cell,

           (b) a tracr mate sequence, and

           (c) a tracr sequence, and

       II. a CRISPR enzyme comprising one or more heterologous functional domains or a polynucleotide encoding a CRISPR enzyme comprising one or more heterologous functional domains for expression in the eukaryotic cell; wherein:

    the tracr mate sequence hybridizes to the tracr sequence,

    the guide sequence directs sequence-specific binding of a CRISPR complex to the target sequence,

    the CRISPR complex comprises the CRISPR enzyme complexed with (1) the guide sequence that is hybridized to the target sequence, and (2) the tracr mate sequence that is hybridized to the tracr sequence,

    the CRISPR enzyme comprises one or more mutations, such that the enzyme has altered nuclease activity compared with the wild type enzyme,

    wherein the one or more heterologous functional domains comprises at least one or more Nuclear Localization Signals NLS(s).

・Cas9以外のCas（CRISPR enzyme）を包含する特許がついに…。サポート要件との兼ね合いがどうなるか。UC Berkeleyの出願とは異なり、パイオニア発明では無いので、もっと厳しく判断されてもいいものであるが。