ToolGen Incorporated v Fisher (No 2)
[2023] FCA 794
•14 July 2023
FEDERAL COURT OF AUSTRALIA
ToolGen Incorporated v Fisher (No 2) [2023] FCA 794
File number: NSD 1909 of 2018 Judgment of: NICHOLAS J Date of judgment: 14 July 2023 Catchwords: PATENTS – appeal against decision of delegate of Commissioner of Patents upholding opposition to patent application (“PA”) for compositions and methods using the CRISPR/Cas9 system for genome editing in eukaryotic cells – identity of person skilled in the art (“PSA”) – whether PSA comprises a team including a microbiologist with expertise in CRISPR/Cas system in prokaryotes – meaning of phrase “nucleic acid encoding a guide RNA” in the claims – interrelationship between independent claim and dependant claim – whether dependent claim lacks clarity due to inconsistency between it and independent claim – whether priority document (“P1”) provides an enabling disclosure of the invention as required by s 43(2A) of the Patents Act 1990 (Cth) (“the Act”) – whether invention disclosed by P1 when read in light of the common general knowledge at date of filing of P1 – whether certain publications were common general knowledge at date of filing of P1 – whether disclosure clear enough and complete enough for invention to be performed by a person skilled in the art – whether work required of PSA would be an undue burden – consideration of priority date – whether claims lack novelty or do not involve an inventive step at the deferred priority date – whether PA provides an enabling disclosure of invention of the claims as required by s 40(2)(a) of the Act – whether work required of PSA would be an undue burden – whether claims supported by matter disclosed in specification as required by s40(3) of the Act – consideration of who should determine the appellant’s foreshadowed application to amend the specification
Held: dependent claim lacks clarity – invention of claims not disclosed by P1 – no enabling disclosure by P1 of invention of claims – work required of PSA would be an undue burden – claims not entitled to priority based on P1 claims – claims lack novelty and/or do not involve an inventive step – no enabling disclosure by PA of invention of claims – claims not supported by matter disclosed in the specification – any application to amend the specification should be heard and determined by the Court
Legislation: Intellectual Property Laws Amendment (Raising the Bar) Act 2012 (Cth)
Patents Act 1990 (Cth) ss 7(1), 7(2), 7(3), 18(1)(b)(i), 40, 40(2)(a), 40(3), 43(1), 43(2), 43(2A), 43(2A)(b), 43(3), 49, 60(3A), 60(4), 105(1A), 112A
Patents Regulations 1992 (Cth) reg 3.12(4), 3.13A
Patents Act 1977 (UK) ss 14(3), 14(5), 14(5)(c), 72(1)(c)
Explanatory Memorandum to the Intellectual Property Laws Amendment (Raising the Bar) Act 2012 (Cth)
Cases cited: Aktiebolaget Hässle v Alphapharm Pty Ltd (2002) 212 CLR 411
Allsop Inc v Bintang Ltd (1989) 15 IPR 686
Apotex Pty Ltd v Warner-Lambert Company LLC (No 2) (2016) 122 IPR 17
British Acoustic Films Ltd v Nettlefold Productions (1936) 53 RPC 221
Catnic Components Ltd v Hill & Smith Ltd [1982] RPC 183
Commissioner of Patents v Sherman (2008) 172 FCR 394
Eli Lilly & Co Ltd v Apotex Pty Ltd (2013) 100 IPR 451
Eli Lilly & Co v Pfizer Overseas Pharmaceuticals (2005) 64 IPR 506
Eli Lilly & Co v Human Genome Sciences Inc [2008] RPC 29
Encompass Corporation Pty Ltd v InfoTrack Pty Ltd (2018) 130 IPR 387
EXXON/Fuel Oils (T-409/91) [1994] OJ EPO 653
Fisher v ToolGen Inc (2018) 144 IPR 315
Freeman v TJ and FL Pohlner Pty Ltd (1994) 30 IPR 377
General Tire & Rubber Company v Firestone Tyre & Rubber Company Limited [1972] RPC 457
Genentech I/Polypeptide expression (T292/85) 27 January 1988
Gilead Sciences Pty Ltd v Idenix Pharmaceuticals LLC (2016) 117 IPR 252
GlaxoSmithKline Consumer Healthcare Investments (Ireland) (No 2) Limited v Generic Partners Pty Limited (2018) 264 FCR 474
Halliburton Energy Services Inc v Smith International (North Sea) Ltd [2006] EWCA Civ 1715
HTC Corp v Gemalto SA [2014] EWCA Civ 1335
Icescape Ltd v Ice-World International BV [2019] FSR 5
Idenix Pharmaceuticals LLC v Gilead Sciences Pty Ltd (2017) 134 IPR 1
Jupiters Ltd v Neurizon Pty Ltd (2005) 222 ALR 155
Kimberly-Clark Australia Pty Limited v Multigate Medical Products Pty Limited (2011) 92 IPR 21
Kimberly-Clark Australia Pty Ltd v Arico Trading International Pty Ltd (2001) 207 CLR 1
Kirin-Amgen Inc v Hoechst Marion Roussel Ltd (2004) 64 IPR 444
Lockwood Security Products Pty Ltd v Doric Products Pty Ltd (2004) 217 CLR 274
Lockwood Security Products Pty Ltd v Doric Products Pty Ltd(No 2) (2007) 235 CLR 173
Meat and Livestock Australia Limited v Branhaven LLC (2020) 281 FCR 640
MedImmune Ltd v Novartis Pharmaceuticals UK Ltd [2013] RPC 27
Mentor Corp v Hollister Inc [1993] RPC 7
Merck & Co Inc v Arrow Pharmaceuticals Ltd (2006) 154 FCR 31
Merck Sharp & Dohme Corporation v Wyeth LLC (No 3) (2020) 155 IPR 1
Minnesota Mining and Manufacturing Company v Beiersdorf (Australia) Limited (1980) 144 CLR 253
Nicaro Holdings Pty Ltd v Martin Engineering Co (1990) 16 IPR 545
Novartis AG v Johnson & Johnson Medical Ltd [2009] EWHC 1671
Novozymes A/S v Danisco A/S (2013) 99 IPR 417
Patent Gesellschaft AG v Saudi Livestock Transport and Trading Company (1997) 37 IPR 523
Pfizer Overseas Pharmaceuticals v Eli Lilly & Co (2005) 225 ALR 416
Ranbaxy Laboratories Ltd v AstraZeneca AB (2013) 101 IPR 11
RD Werner & Co Inc v Bailey Aluminium Products Pty Ltd (1989) 25 FCR 565
Regeneron Pharmaceuticals Inc v Kymab Ltd [2020] UKSC 27
Schering Biotech Corp’s Application [1993] RPC 249
Schlumberger Holdings Ltd v Electromagnetic Geoservices AS [2010] RPC 33
Valensi v British Radio Corporation [1973] RPC 337
Wake Forest University Health Sciences v Smith & Nephew Pty Ltd (No 2) (2011) 92 IPR 496
Warner-Lambert Co LLC v Apotex Pty Ltd (2018) 129 IPR 205
Warner-Lambert LLC v Generics (UK) Ltd t/a Mylan [2018] UKSC 56
Welch Perrin & Co Pty Ltd v Worrell (1961) 106 CLR 588
Winner v Ammar Holdings Pty Ltd (1993) 41 FCR 205
Division: General Division Registry: New South Wales National Practice Area: Intellectual Property Sub-area: Patents and associated Statutes Number of paragraphs: 436 Date of hearing: 21-25, 28-30 September 2020 Counsel for the Appellant/Cross-Respondent: Mr T Cordiner QC with Mr P Flynn SC Solicitor for the Appellant/Cross-Respondent: Jones Day Counsel for the Respondents/ Cross-Appellants: Mr C Dimitriadis SC with Ms C Cunliffe Solicitor for the Respondents/ Cross-Appellants: Ashurst Australia
Table of Corrections 19 July 2023 [95] and [96] delete the word “Type II” where appearing in those paragraphs [196] replace reference to (f)-(g) with (a)-(b) [260] and [261] corrections to formatting [334] replace reference to (d)-(j) with (a)-(g) [408] insert the words “in relation to P1” in penultimate sentence and the words “they say” in the last sentence ORDERS
NSD 1909 of 2018 BETWEEN: TOOLGEN INCORPORATED
Appellant
AND: GRANT FISHER
First Respondent
ACN 004 552 363 PTY LTD
Second Respondent
AND BETWEEN: GRANT FISHER
First Cross-Appellant
ACN 004 552 363 PTY LTD
Second Cross-AppellantAND: TOOLGEN INCORPORATED
Cross-Respondent
ORDER MADE BY:
NICHOLAS J
DATE OF ORDER:
14 JULY 2023
THE COURT ORDERS THAT:
1.The appellant file and serve any interlocutory application seeking an order directing that the complete specification be amended pursuant to s 105(1A) of the Patents Act 1990 (Cth) together with any affidavit in support of such application by 4.00pm, 11 August 2023.
2.The proceeding be stood over to 9.30am on 17 August 2023 for the making of:
(a)final orders in the event no application is filed pursuant to order 1;
(b)for the making of further orders in relation to any application filed pursuant to order 1; and
(c)other orders (including in relation to costs) as may be considered appropriate.
Note: Entry of orders is dealt with in Rule 39.32 of the Federal Court Rules 2011.
REASONS FOR JUDGMENT
INTRODUCTION
[1]
PRINCIPAL ISSUES
[10]
ONUS OF PROOF
[14]
BACKGROUND TO TECHNOLOGY
[20]
Eukaryotic and prokaryotic cells
[20]
DNA and RNA
[21]
Protein expression
[25]
Cellular expression of proteins
[30]
Nuclear localisation sequence
[33]
RNA interference, Zinc finger nucleases and TALEN nucleases
[35]
CRISPR/Cas system
[36]
WITNESSES
[46]
ToolGen’s Witnesses
[47]
Respondents’ Witnesses
[49]
Joint Expert Reports
[51]
THE EARLIEST PRIORITY DOCUMENT (P1)
[52]
THE PATENT APPLICATION
[56]
Body of the Specification
[56]
The Claims
[73]
THE NOTIONAL SKILLED ADDRESSEE
[76]
Background
[76]
P1
[82]
THE PATENT APPLICATION
[91]
COMMON GENERAL KNOWLEDGE
[97]
PRINCIPLES OF CONSTRUCTION
[101]
CONSTRUCTION ISSUES
[113]
“a nucleic acid encoding”
[113]
“paired Cas9 nickases”
[146]
RELEVANT LEGISLATIVE PROVISIONS
[160]
THE DISCLOSURE REQUIREMENT
[167]
P1 – DISCLOSURE AND ENABLEMENT
[194]
A nucleic acid encoding a guide RNA
[199]
A Type II CRSIPR/Cas system from a bacterial species other than S. pyogenes
[213]
Chimeric guide RNA other than sgRNA (+48)
[366]
Nuclear localisation sequences (NLSs) and their location
[371]
Single guide RNA fusion other than with a GAAA linker
[377]
Use of “paired Cas9 nickases” and Cas9 endonucleases that create staggered-ended double-stranded DNA breaks
[379]
PATENT APPLICATION – DISCLOSURE AND ENABLEMENT
[383]
PATENT APPLICATION – SUPPORT
[391]
THE PRIORITY DATE
[412]
NOVELTY
[415]
INVENTIVE STEP
[423]
AMENDMENT
[432]
DISPOSITION
[434]
NICHOLAS J
INTRODUCTION
This proceeding concerns an opposed patent application for what is now a well-known gene editing system known as the CRISPR/Cas9 system. The CRISPR/Cas9 system described in the application can be used to edit target DNA sequences in eukaryotic cells so as to disable or modify gene expression through (inter alia) the deletion or insertion of such sequences using an RNA-guided endonuclease.
The appellant (“ToolGen”) is the applicant in Australian Patent Application 2013335451 (“the patent application”). The patent application relates to compositions and methods involving a system for introducing a site-specific double-stranded break (or cleavage) at a target nucleic acid sequence in a eukaryotic cell comprising a nucleic acid encoding a Cas9 polypeptide and a nucleic acid encoding a guide RNA specific for the target DNA. The system disclosed in the patent application is referred to as a “Type II Clustered Regularly Interspaced Short Palindromic Repeats/Cas system” or “CRISPR/Cas system”. The patent application has 21 claims including independent claim 1 for a composition and independent claim 10 for a method.
The patent application was filed on 23 October 2013 and relies on an earliest priority date of 23 October 2012 based on US Provisional Patent Application 61/717,324 (“P1”). There are two other priority documents referred to in the patent application being US Provisional Patent Application 61/803/599 (“P2”) with a filing date of 20 March 2013 and US Provisional Patent Application 61/837,481 (“P3”) with a filing date of 20 June 2013. No submissions were made by either party in relation to P2 or P3 and none of the experts were questioned about them. I will say a little more about them later in these reasons. It is sufficient to say at this point that P2 is incapable of conferring priority on any claim in the patent application and P3 was filed after the publication date of various journal articles that deprive the claims of novelty or any inventive step.
The first respondent opposed the patent application before the Commissioner of Patents. That opposition was successful in relation to claims 1-8 and 10-18 of the patent application, which the Delegate of the Commissioner of Patents (“Delegate”) found were not novel and did not involve an inventive step in circumstances where none of those claims was entitled to priority from P1. Claim 19 was found to lack clarity. Claim 21 was also found to not involve an inventive step. The Delegate indicated that she would allow ToolGen two months to propose appropriate amendments. (See Fisher v ToolGen Inc (2018) 144 IPR 315, [2018] APO 65).
ToolGen appealed the Delegate’s decision pursuant to s 60(4) of the Patents Act 1990 (Cth) (“the Act”). The second respondent was named as an additional respondent. The respondents filed a cross-appeal in relation to claims 9 and 20. They have also raised additional grounds of invalidity which were rejected by the Delegate.
Even though this proceeding is referred to as an appeal, it is well-established that it is not an appeal in the strict sense but is conducted as a hearing de novo in the original jurisdiction of the Court: Commissioner of Patents v Sherman (2008) 172 FCR 394 at [18].
It is common ground that if claims 1-8 and 10-18 are not entitled to priority from P1, they are not novel and lack an inventive step. As to claims 9 and 19-21, the respondents contend that they also lack novelty and do not involve an inventive step if they are not entitled to priority from P1.
In these reasons I refer to various publications in the scientific literature. Sometimes I refer to these publications by their full citation but more often than not it is sufficient to identify them by the lead authors name and the year of publication (eg. Wang (2013)). Full details of these publications are set out in the Bibliography in Annexure A to these reasons.
The Primer (Exhibit A) which was agreed between the parties was of considerable assistance to me. It covers a range of topics including the basics of cell biology, the genetic code, molecular biology, and gene editing. The matters described in paras 14-101 of the Primer (which I need not reproduce) are elementary and were very well known to molecular biologists before the priority date.
PRINCIPAL ISSUES
The parties agreed on a lengthy and detailed statement of issues which I found helpful and have had regard to, even though I have chosen not to frame my reasons for judgment around it.
Broadly stated, the principal issues addressed in these reasons are as follows:
(a)who is the skilled addressee of P1 and the patent application and what was the common general knowledge of the skilled addressee (or skilled team) as at 23 October 2012?
(b)what construction should be given to claims 1 and 10 (and their dependent claims) of the patent application, including to the phrase “nucleic acid encoding a guide RNA”?
(c)do claims 1 and 10 (and their dependent claims) extend to “paired Cas nickases”?
(d)does claim 19 of the patent application lack clarity?
(e)does P1 provide an enabling disclosure of the invention claimed in each of the claims of the patent application?
(f)if the priority date is deferred, does the invention claimed in each of claims 9, 19 and 20 of the patent application lack novelty in light of Wang (2013)?
(g)if the priority date is deferred, does the invention claimed in each of claims 9, 19, 20 and 21 of the patent application not involve an inventive step in light of the common general knowledge at the relevant date considered together with each of Cong (2013), Mali (2013) and Wang (2013) (taken separately)?
(h)does the complete specification of the patent application comply with s 40(2)(a) of the Act in respect of the invention claimed in each claim?
(i)is each claim of the patent application supported in accordance with s 40(3) of the Act by matter disclosed in the complete specification?
ToolGen accepts that if the priority date is deferred, then claims 1-8 and 10-18 will lack novelty and an inventive step. ToolGen makes no such concession in relation to claims 9, 19, 20 or 21.
With regard to issues (e), (h) and (i), the respondents contend that these questions should be answered in the negative because neither P1 nor the patent application discloses the invention as claimed or, alternatively, does not enable its use across the full scope of each claim. These issues raise questions as to the proper construction and application of s 40(2)(a), s 40(3) and s 43(2A) in the form they have taken since the Act was amended by the RTB Act.
For the reasons that follow I have concluded:
(a)None of the claims are entitled to priority based on P1 (s 43(2A)).
(b)All of the claims lack novelty or do not involve an inventive step (s 18(1)(b)).
(c)The complete specification does not provide an enabling disclosure of the invention (s 40(2)(a)).
(d)The claims are not supported by matter disclosed in the specification (s 40(3)).
(e)Claim 19 lacks clarity (s 40(3)).
ONUS OF PROOF
Each party made submissions concerning the onus of proof. ToolGen emphasised that the legal burden on all issues is on the opponent. The respondents did not dispute that they carry the legal burden in this proceeding. However, they drew attention to the following observations in the Explanatory Memorandum to the Intellectual Property Laws Amendment (Raising the Bar) Act 2012 (Cth) (“RTB Act”) concerning the amendment to s 40(2)(a) of the Act and the requirement that there be an enabling disclosure:
A specification that provides a single example of the invention may satisfy the requirements, but only where the skilled person can extend the teaching of the specification to produce the invention across the full width of the claims, without undue burden, or the need for further invention.
However, it is expected to be more likely that, where the claims are broad, the specification will need to give a number of examples or describe alternative embodiments or variations extending over the full scope of the claims. This ensures that the monopoly extends only to that which could reasonably be said to be disclosed and no further.
If, on its face, the specification would appear to the skilled person to lack sufficient disclosure, the onus of establishing that the invention is described in enough detail lies with the applicant (see item 14).
The statement concerning onus in the Explanatory Memorandum appears to be directed to the onus at the examination stage. I note that the reference to item 14 is to a proposed amendment to s 49 of the Act. That section is concerned with acceptance of a patent request rather than the hearing and determination of any opposition to the grant of a patent following acceptance. With regard to the opposition, s 60(3A) of the Act provides:
(3A)If the Commissioner is satisfied, on the balance of probabilities, that a ground of opposition to the grant of the standard patent exists, the Commissioner may refuse the application.
In their submissions the respondents referred to the shifting of the evidentiary onus to ToolGen in circumstances where, in their submission, P1 does not on its face, purport to provide an enabling disclosure extending to, for example, use of a Type II CRISPR/Cas9 system derived from bacterial species other than S. pyogenes. They submitted, in effect, that in these circumstances the evidentiary onus shifted to ToolGen to establish that there was an enabling disclosure.
I do not consider it helpful to speak of a shifting evidential onus in this case. Ultimately, it is for the respondents to persuade the Court that P1 does not provide an enabling disclosure. To the extent it is necessary to resolve a disputed issue of fact in determining whether that objection is established, the issue is to be determined on the balance of probabilities.
In deciding whether there is an enabling disclosure, the Court will necessarily have regard to the content of P1 when read in light of the common general knowledge, the cogency of the evidence relied on by each side as to adequacy of the information made available, the difficulties that would be faced by the skilled addressee in seeking to perform the invention, and whether the work involved amounts to an undue burden. The determination of that question involves an evaluative judgment based on a consideration of both the nature of the technology and the work required of the skilled addressee to perform the invention across the scope of the claims.
Even though the burden of proof is on the respondents, circumstances may still arise in which ToolGen’s failure to adduce any evidence or any sufficient evidence on some particular matter (eg. a fact which it asserts was common general knowledge) may ultimately lead the Court to conclude, on the totality of the relevant evidence, that the invention cannot be performed across the full scope of the claims without undue burden. This may be particularly true in relation to matters in respect of which P1 is wholly silent.
BACKGROUND TO TECHNOLOGY
Eukaryotic and prokaryotic cells
Eukaryotes are organisms comprised of one or more eukaryotic cells. A eukaryotic cell has a defined nucleus which is an organelle that contains DNA enclosed within a nuclear envelope (double membrane). Mammals (including humans) are classified as eukaryotes as they are comprised of eukaryotic cells. Prokaryotes are unicellular organisms comprised of a prokaryotic cell. A prokaryotic cell has no nucleus or membrane bound organelles, and DNA in prokaryotic cells is found in the form of supercoiled circular DNA that is not enclosed by a nuclear membrane. Bacteria are an example of a prokaryote.
DNA and RNA
DNA (deoxyribonucleic acid) and RNA (ribonucleic acid) are both nucleic acids consisting of a series (or string) of nucleotides. The nucleotides in DNA and RNA each contain a sugar, a nitrogenous base and a phosphate group. In DNA, the sugar is deoxyribose whereas in RNA the sugar is ribose. The nitrogenous base in each nucleotide in a DNA molecule is either a purine (being adenine (A) or guanine (G)) or a pyrimidine (being cytosine (C) or thymine (T)). The nitrogenous bases in RNA are the same as those in DNA except that uracil (U) is substituted for thymine (T). Nucleotides are joined together to form a long chain of DNA or RNA. This long chain is termed a polynucleotide.
In DNA, the sequence in which the four bases (A, C, G, T) are arranged in the polynucleotide chain comprises the DNA code. The bases in one polynucleotide chain of DNA pair with complementary bases in the other polynucleotide chain of DNA (so called “base pairing”) to form a double-stranded helical structure. In the double-stranded DNA structure, the nitrogenous base guanine (G) base pairs with cytosine (C), while the nitrogenous base adenosine (A) base pairs with thymine (T).
In biological cells, molecules of RNA predominantly consist of a single polynucleotide chain or strand. However, the sugar-phosphate backbone of the chain is flexible and can fold so that self-complementary sequences within the RNA pair with each other (to form a duplex, or double-stranded structure).
The nucleotide sequence of both DNA and RNA can be identified by a technique known as sequencing.
Protein expression
Proteins are produced (or “expressed”) in a cell by the processes of transcription (DNA to RNA) and translation (RNA to protein). In a eukaryotic cell, transcription occurs in the nucleus (where DNA is located) and translation occurs in the cytoplasm (where ribosomes are located). In a prokaryotic cell, transcription and translation occurs in the cytoplasm (where DNA and ribosomes are located). The cytoplasm is the gelatinous liquid that fills the inside of a cell that is comprised of water, salts and other organic molecules, and a ribosome is an organelle within the cytoplasm that is the site of protein synthesis.
During transcription, the DNA double-stranded helix unwinds and one of the two strands (the template or non-coding strand) acts as a template for the synthesis of a single-stranded RNA molecule. Transcription generates a synthesised RNA (pre-mRNA) molecule with bases complementary to the template DNA strand and with bases identical (with the exception that “U” is substituted for “T”) to the coding DNA strand. Pre-mRNA is then processed to form mature messenger RNA (mRNA).
During translation, mRNA acts as a template for the synthesis of a polypeptide chain (a sequence of amino acids joined together by peptide bonds) which make up a protein. The mRNA sequence is read consecutively in groups of three nucleotides, known as codons. Each codon specifies either one amino acid or comprises a start or stop codon that starts and ends the translation process.
There are only 20 amino acids that are commonly found in proteins, however, there are 64 possible combinations of nucleotide triplets to make up a codon (given that there are four different nucleotides which could be in each position in the triplet). This is because the same amino acid can be coded for by more than one codon. This is referred to as the degeneracy or redundancy of the genetic code.
The codons in a molecule of mRNA are recognised by small RNA molecules known as transfer RNA (tRNA) that are located in ribosomes. One region of the tRNA (the anticodon) binds complementarily to an mRNA codon while another region of the tRNA binds to the amino acid that matches the mRNA codon attached to the tRNA. As the mRNA sequence is read from start codon to stop codon, the amino acids coded for by the intervening codons are brought together to form a polypeptide chain (protein).
Cellular expression of proteins
Proteins can be expressed in eukaryotic cells (including mammalian cells) via recombinant DNA technology using vectors. Vectors can be plasmids (small circular pieces of DNA from bacteria) or phages (viruses) that transfer foreign DNA into a cell. The insertion of a foreign DNA sequence into the vector enables the DNA sequence to be propagated (cloning vectors) or used to express a protein or RNA (expression vectors).
The foreign DNA inserted into a vector can comprise a DNA fragment of a particular size, including DNA synthesised outside the cell (in vitro), a section of DNA from another clone to be subcloned (that is, taking a smaller part of the larger fragment), a section of DNA produced using restriction enzymes (enzymes that cut DNA) or a PCR fragment.
In a plasmid vector, the plasmid and foreign DNA insert are both cut with restriction enzymes which generate compatible 5’ and 3’ ends. The plasmid and insert are then combined and ligated (stitched together). The newly formed plasmid (with DNA insert) is transformed (delivered) into bacteria and selected for using antibiotic-containing growth medium.
Nuclear localisation sequence
As set out above, proteins are produced by the process of translation which occurs in the ribosome in the cytoplasm of cells. In eukaryotic cells, the protein must pass into the nucleus through the nuclear membrane in order for a protein to access and interact with chromosomal DNA. Nuclear proteins (ie. proteins that function in the nucleus) commonly enter the nucleus by passing through a nuclear pore channel. This can be contrasted to prokaryotic cells, where chromosomal DNA is found in the cytoplasm.
As of October 2012, scientists were using a number of different nuclear localisation sequences (“NLS”) as modular tags to deliver proteins or protein fragments from the cytoplasm to the nucleus. A NLS is a short peptide derived from proteins which enter the nucleus of a cell (nuclear proteins). For example, the sequence PKKKRKV is an NLS which was widely used and studied prior to October 2012.
RNA interference, Zinc finger nucleases and TALEN nucleases
The patent application is set against the backdrop of tools and methodologies used prior to October 2012 for introducing mutations into DNA sequences. The patent application describes how the CRISPR/Cas system is used to recognise and silence exogenous genetic elements in a manner analogous to the process of RNA interference (RNAi) in eukaryotic organisms. RNAi is a biological system in which RNA molecules inhibit gene expression by neutralising targeted mRNA molecules. RNAi was known to those in the field of genetic engineering well before October 2012. The patent application and P1 also refer in particular to the use of other gene-editing tools known as Zinc finger nucleases (ZFNs) and TALEN nucleases (TALENs) that are derived from eukaryotic transcription factors.
CRISPR/Cas system
CRISPR is an acronym for “Clustered Regularly Interspaced Short Palindromic Repeats”. Cas9 is the CRISPR associated protein 9, which is a prokaryotic dual RNA-guided DNA endonuclease (an enzyme that cuts DNA within the internal part of the DNA sequence). These components are associated with the CRISPR/Cas adaptive immune system found in bacteria, an example of which is the Type II CRISPR/Cas system which is characterised by (inter alia) its use of Cas9 protein (or polypeptide). In essence, these systems are a defence mechanism which protects the bacteria from invading viruses by cleaving the DNA of the virus and thereby disabling it.
Type II CRISPR/Cas systems were known to exist in certain prokaryotic cells and function in the genomes of bacteria as part of their acquired bacterial immune system. Specifically, Type II CRISPR/Cas systems were understood to confer bacterial resistance to exogenous (external) genetic elements such as plasmids (small circular DNA found in bacteria that replicate in bacterial cells) and phages (viruses that infect and replicate in cells).
This bacterial resistance is achieved by way of short segments of plasmid/phage DNA, called spacers, which are incorporated into the bacterial genome between (and separate) CRISPR repeats (short palindromic sequences). Together, these spacers and repeats make up what is known as the CRISPR array. The CRISPR spacers serve as a memory of past exposure to plasmids and phages and are used to recognise and silence foreign DNA from invading bacteria and phages.
Figure 2(A) in Horvath (2010) (reproduced below) shows the process of spacer incorporation which is taking DNA from an invading virus or plasmid and incorporating this into the CRISPR array as spacer units. Figure 2(B) illustrates the function of CRISPR-Cas in targeting and cleaving invading DNA that is cognate to a spacer. This is done by transcribing the CRISPR repeat-spacer array into “pre-crRNA” which is then processed through RNA cleavage into individual “crRNA” (CRISPR RNA) units incorporating the spacer and sequence derived from the repeat. The spacer sequence in the crRNA unit guides the crRNA-Cas complex to the invading nucleic acid. The crRNA unit complexed with Cas protein(s) is the active form of the CRISPR defence system which performs the cleavage of the invading DNA.
The patent application describes how the Cas9 component of the Type II CRISPR/Cas system forms an active endonuclease when complexed with two RNA molecules designated CRISPR RNA (crRNA) and trans-activating crRNA (tracrRNA) that are fused together to form a chimeric (single) guide RNA that guide the CRISPR/Cas 9 complex to its target DNA sequence. The RNA guided endonuclease is then able to break foreign genetic elements in invading phages or plasmids and protect the host cell (bacteria) from infection.
The composition and role of the crRNA and tracrRNA that make up the single guide RNA (“sgRNA”) are of some importance to understanding the background to the CRISPR/Cas9 system. The crRNA is transcribed from the spacer sequence of the CRISPR array into pre-crRNA which is further processed into mature crRNA that is complementary to the target DNA sequence. This complementarity is used to guide the Cas9 to the target DNA sequence of interest where it will cleave. The tracrRNA is transcribed from a gene outside of the CRISPR array but it is complementary to the repeat sequences of the CRISPR array. This complementarity is used to process pre-crRNA into mature crRNA so that it can perform its guiding function.
The invention described in the specification involves the use of individual components of the CRISPR/Cas9 system as a programmable system for introducing a site-specific double-stranded break in a target nucleic acid of a eukaryotic cell (i.e. outside of a prokaryotic system). The system can be used in vitro (outside the cell) or in vivo (in the cell) to introduce mutations into DNA sequences including in so-called “gene-editing” experiments or research.
The reference to a site-specific double-stranded break is a reference to the ability of the system to target and cleave a precise site which is within a DNA target sequence that is complementary to the variable part of the guide RNA, and which contains a PAM recognised by Cas9. PAM refers to “protospacer adjacent motif” which is a nucleotide sequence adjacent to the target DNA sequence (known as the protospacer) to be cleaved by Cas9. The PAM sequence is the means by which a Cas9 recognises where to cleave DNA. Each Cas9 derived from a bacterial species recognises a specific PAM sequence. For example, the patent application discloses that Cas9 derived from Streptococcus pyogenes recognises a “NGG” or “NAG” PAM sequence where “N” stands for any nucleotide and “GG” stands for two guanine nucleotides. S. pyogenes Cas9 will therefore cleave DNA adjacent to the nucleotide sequence “NGG” where the Cas9 is complexed with a guide RNA that has a variable region of the crRNA that is complementary to the target DNA sequence.
The sgRNA depicted in Figure 1a of the patent application and Figure 1A of P1 are identical and reproduced below. The target DNA sequence is shown in green. The PAM sequence “CGG” recognised by Cas9 is shown in orange and the triangles indicate cleavage sites. Cas9 is shown in yellow and the sequences of the guide RNA derived from crRNA and tracrRNA are shown in red and blue, respectively. Vertical bars between the target DNA and crRNA sequence and between the crRNA and tracrRNA denote complementarity. The coloured boxes do not appear in Figure 1a of the patent application or Figure 1A of P1 and have been added to assist explanation of the figure.
Figure 1A/ Figure 1a
In summary, the particular CRISPR/Cas9 system the subject of the patent application (and P1) is said to be comprised of a Cas9 polypeptide, that when complexed with a chimeric (single) guide RNA, has endonuclease activity in eukaryotic cells.
WITNESSES
There were four principal witnesses each of whom provided written and oral evidence.
ToolGen’s Witnesses
Associate Professor Ron Firestein
Associate Professor Firestein made two affidavits dated 13 September 2019 (“Firestein 1”) and 25 March 2020 (“Firestein 2”). He also annexed to Firestein 1 his Declaration in the Patent Office opposition proceedings. He is the Head of the Centre for Cancer Research at the Hudson Institute of Medical Research, and a consulting pathologist in molecular genetic pathology at Monash Health. By his expertise and training he can be described as a molecular biologist. At October 2012, Associate Professor Firestein was generally aware of the existence of bacterial innate immunity but did not have knowledge of the mechanistic details of the system, nor was this work of interest to him because the potential impact of the CRISPR/Cas9 system for molecular biology and gene editing had not been identified. He has used the CRISPR/Cas9 system since mid-2013.
Professor Philip Giffard
Professor Giffard made two affidavits dated 13 September 2019 (“Giffard 1”) and 16 March 2020 (“Giffard 2”). He is the Head of Laboratory Science at the Menzies School of Health Research and Professor and Associate Dean for Research and Innovation in the College of Health and Human Sciences at Charles Darwin University. He has specialised knowledge in the field of bacterial genetics and physiology, molecular bacteriology, bioinformatics and molecular microbiology. By his expertise and training he can be described as a microbiologist. Before October 2012, he co-authored two papers reporting research into the CRISPR loci of the bacterial species C. jejuni and the Staphylococcus bacterial strain MSHR1132.
Respondents’ Witnesses
Professor Paul Thomas
Professor Thomas made two affidavits dated 8 April 2019 (“Thomas 1”) and 20 August 2020 (“Thomas 2”). He is a Professor of Biochemistry at the University of Adelaide and is the Head of the Genome Editing Laboratory at the South Australian Health and Medical Research Institute. Since 1995, he has engaged in genetic research in eukaryotes, including gene targeting to inactivate genes of interest using homologous recombination, the development of mouse models with genetic changes, and using molecular biology technology to screen for genetic mutations. By his expertise and training he can be described as a molecular biologist. At October 2012, Professor Thomas was generally aware of the CRISPR/Cas9 system in bacteria, but not to a high level of detail. He did not actively follow literature developments relating to bacterial systems. He became interested in using CRISPR/Cas9 systems in mid-2013 after reading Mali (2013) and Wang (2013). Since then, he has produced more than 60 novel mouse models using CRISPR/Cas9 systems, as well as cell lines with modified genomes.
Associate Professor Marco Herold
Associate Professor Herold made two affidavits dated 4 April 2019 (“Herold 1”) and 20 December 2019 (“Herold 2”). He is a molecular biologist and the Laboratory Head at the Walter and Eliza Hall Institute of Medical Research. From 2001, he focused on the molecular regulation of cell death, including by introducing foreign genomic material into the genome of host cells using retroviruses. From 2005, he worked on genetic manipulation using RNA interference (RNAi) technology to silence or “knock down” particular genes in eukaryotes, using both mouse models and in vitro systems. At October 2012, Associate Professor Herold was generally aware of CRISPR/Cas9 systems in bacteria after reading Jinek, but was not aware of the specific details of the system. He started working with CRISPR/Cas9 systems in eukaryotic cells and organisms in May 2013, after reading Cong (2013), Mali (2013) and Wang (2013). Since then, he has made around 220 mouse models using CRISPR/Cas9 systems.
Joint Expert Reports
There were two expert conclaves held prior to the hearing and two concurrent sessions of expert evidence at the hearing. The first expert conclave included the molecular biologists Associate Professor Firestein, Associate Professor Herold and Professor Thomas who prepared a Joint Expert Report dated 1 September 2020 (“JER 1”). These experts also gave evidence in a concurrent session. The second expert conclave included the microbiologist Professor Giffard, Professor Thomas and Associate Professor Herold. They prepared a Joint Expert Report dated 2 September 2020 (“JER 2”) and also gave evidence in another concurrent session.
THE EARLIEST PRIORITY DOCUMENT (P1)
It is common ground that P1 was filed on 23 October 2012. P1 is a relatively short document which resembles an unpublished journal article to which has been added an additional paragraph headed “Summary of the Invention”. Nothing turns on the purpose for which P1 was prepared. P1 states at pages 1-6:
[Page 1]
Abstract:
We present a novel genome editing technology based on RNA-guided Cas9 endonucleases (RGENs). Cas9 is a sequence-specific endonuclease in type II CRISPR/Cas systems, which confer prokaryotes with adaptive immunity against invading phages and plasmids. Cas9 recognizes and cleaves target DNA sequences complementary to small synthetic guide RNAs embedded in this protein, generating site-specific DNA double-strand breaks in vitro and in human cells, whose spontaneous repair induces targeted genome modifications at high frequencies. Unlike ZFNs and TALENs, which are used widely in research and biotechnology, RGENs are customized without any cloning step, making them a broadly useful, scalable and expeditious platform for genome engineering in cells and organisms.
Summary of the Invention
In some embodiments, the present invention provides compositions and methods for research, clinical and screening applications for genome editing. In some embodiments, the present invention provides nucleic acids encoding RNA-guided Cas9 endonucleases, vectors comprising Cas-9 endonucleases, Cas-9 polypeptides, and uses of such compositions.
Additional embodiments are described herein.
[Page 2]
Main Text:
We exploited the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated protein (Cas) system (1), an adaptive immune response in bacteria and archaea, to develop a novel genome editing technology based on RNA-guided endonucleases (RGENs). Cas9, an essential protein component in the Type II CRISPR/Cas system, forms an active endonuclease when complexed with two RNAs termed CRISPR RNA (crRNA) and transactivating crRNA (tracrRNA), thereby slicing foreign genetic elements in invading phages or plasmids to protect the host cells. crRNA is transcribed from the CRISPR element in the host genome, which was previously captured from such foreign invaders. Recently, Jinek et al. (2) elegantly demonstrated that a single-chain chimeric RNA produced by fusing an essential portion of crRNA and tracrRNA could replace the two RNAs in the Cas9/RNA complex to form a functional endonuclease, raising the possibility of using this system for genome editing in cells and organisms. Here, we present the first evidence that RGENs can indeed induce site-specific genome modifications in mammalian cells at high frequencies.
We first tested the DNA cleavage activity of Cas9 derived from Streptococcus pyogenes in the presence or absence of a chimeric guide RNA in vitro. To this end, we used recombinant Cas9 protein that was expressed in and purified from E. coli to cleave a predigested or circular plasmid DNA that contained the 23-base pair (bp) human CCR5 target sequence. A Cas9 target sequence consists of a 20-bp DNA sequence complementary to crRNA or a chimeric guide RNA and the trinucleotide (5'-NGG-3') protospacer adjacent motif (PAM) recognized by Cas9 itself (Fig. 1A); Cas9 cleaved the plasmid DNA efficiently at the expected position only in the presence of the synthetic RNA and did not cleave a control plasmid that lacked the target sequence (Fig. 1B).
Next, we used a RFP-GFP reporter to investigate whether the Cas9/guide RNA complex can cleave the target sequence incorporated between the RFP and GFP sequences in mammalian [Page 3] cells. In this reporter, the GFP sequence is fused to the RFP sequence out-of-frame (3). The active GFP is expressed only when the target sequence is cleaved by site-specific nucleases, which causes frameshifting small insertions or deletions (indels) around the target sequence via error-prone non-homologous end-joining (NHEJ) repair of the double-strand break (DSB). We co-transfected the Cas9-encoding plasmid, the guide RNA, and the RFP-GFP reporter plasmid into human embryonic kidney (HEK) 293T cells, and found that GFP-expressing cells were obtained only when the cells were co-transfected with the Cas9 plasmid and the guide RNA (Fig. 2), demonstrating that RGENs could recognize and cleave the target DNA sequence in cultured human cells.
To test whether RGENs could be used for targeted disruption of endogenous genes in mammalian cells, we analyzed genomic DNA isolated from transfected cells using T7 endonuclease I (T7E1), a mismatch-sensitive endonuclease that specifically recognizes and cleaves heteroduplexes formed by the hybridization of wild-type and mutant DNA sequences (4). We found that mutations were induced only when the cells were co-transfected with both Cas9 and guide RNA (Fig. 3). Mutation frequencies (Indels (%) in Fig. 3A) estimated from the relative DNA band intensities were RNA-dosage dependent, ranging from 1.3% to 5.1%. DNA sequencing analysis of the PCR amplicons corroborated the induction of RGEN-mediated mutations at the endogenous sites. Indels and microhomologies, characteristic of error-prone NHEJ, were observed at the target site. The mutation frequency measured by direct sequencing was 7.3% (= 7 mutant clones/96 clones), on par with those obtained with zinc finger nucleases (ZFNs) or transcription-activator-like effector nucleases (TALENs).
Both ZFNs and TALENs have been successfully developed to disrupt the human CCR5 gene (4-7), which encodes a G-protein-coupled chemokine receptor, an essential co-receptor of HIV infection. A CCR5-specific ZFN is now under clinical investigation in the US for the treatment of AIDS (8). These ZFNs and TALENs, however, have off-target effects, inducing both local [Page 4] mutations at sites whose sequences are homologous to the on-target sequence (7, 9-11) and genome rearrangements that arise from the repair of two concurrent DSBs induced at on-target and off-target sites (12-13). The most striking off-target sites associated with these CCR5-specific engineered nucleases reside in the CCR2 locus, a close homolog of CCR5, located 15-kbp upstream of CCR5. To avoid off-target mutations in the CCR2 gene and unwanted deletions, inversions, and duplications of the 15-kbp chromosomal segment between the CCR5 on-target and CCR2 off-target sites, we intentionally chose the target site of our CCR5-specific RGEN to recognize a region within the CCR5 sequence that has no apparent homology with the CCR2 sequence.
We investigated whether the CCR5-specific RGEN had off-target effects. To this end, we searched for potential off-target sites in the human genome by identifying sites that are most homologous to the intended 23-bp target sequence. As expected, no such sites were found in the CCR2 gene. Instead, we found four sites, each of which carries 3-base mismatches with the on-target site (Fig. 4A). The T7E1 assays showed that mutations were not detected at these sites (assay sensitivity, ⁓0.5%), demonstrating exquisite specificities of RGENs (Fig. 4B). Furthermore, we used PCR to detect the induction of chromosomal deletions in cells separately transfected with plasmids encoding the ZFN and RGEN specific to CCR5. Whereas the ZFN induced deletions, the RGEN did not (Fig. 4C). Although we did not detect any off-target effects with RGENs in this study, deep sequencing of candidate sites and whole genome or exome sequencing may reveal off-target mutations induced by RGENs.
Next, we reprogrammed RGENs by replacing the CCR5-specific guide RNA with a newlysynthesized RNA designed to target the human C4BPB gene, which encodes the beta chain of C4b-binding protein, a transcription factor. This RGEN induced mutations at the chromosomal target site in K562 cells at high frequencies (Fig. 38): Mutation frequencies measured by the T7E1 assay and by direct sequencing were 14% and 8.3% (= 4 mutant clones/48 clones), [Page 5] respectively. Out of four mutant sequences, two clones contained a single-base or two-base insertion precisely at the cleavage site, a pattern that was also observed at the CCR5 target site. These results indicate that RGENs cleave chromosomal target DNA at expected positions in cells.
ZFNs and TALENs enable targeted mutagenesis in mammalian cells (14-16), model organisms (17-20), plants (21-23), and livestock (24-25), but the mutation frequencies obtained with individual nucleases are widely different from each other. Furthermore, some ZFNs and TALENs fail to show any genome editing activities (26-29). DNA methylation may limit the binding of these engineered nucleases to target sites (30). In addition, it is technically challenging and time-consuming to make custom nucleases. In this regard, RGENs based on Cas9 could provide useful options for genome editing. Compared to ZFNs and TALENs, RGENs can be more readily customized because only the synthetic RNA component is replaced to make a new genome-editing nuclease: No sub-cloning steps are involved to make customized RGENs. Furthermore, the relatively small size of the Cas9 gene (4.2 kbp) as compared to a pair of TALEN genes (⁓6 kbp) provides an advantage for this system in some applications such as virus-mediated gene delivery. These features will make RGENs scalable, versatile, and convenient tools for genome engineering in cells and organisms.
The specificity of DNA recognition by RGENs is somewhat limited by the requirement for a 5'-GG-3' dinucleotide in the PAM sequence. This motif is recognized by the Cas9 protein but not by the guide RNA. Thus, RGENs can be designed to cleave DNA once per 8 bp (= 4x4/2) on average. This limitation might be relieved by engineering Cas9 or employing Cas9 derived from other species.
Unlike Fokl-based ZFNs and TALENs, which produce 4- to 6-base 5' overhangs at cleavage sites, RGENs yield blunt ends rather than cohesive ends (2). Our results show that DSBs with blunt ends can also be readily repaired in mammalian cells. It would be interesting to investigate [Page 6] how and whether blunt DSB ends would be differentially repaired by endogenous end-joining processes.
Taken together, these findings indicate that RGENs are a new member in the family of genome editing tools that have revolutionized basic and biomedical research but with their own unique features that make them an ideal platform in many applications. We propose that RGENs should find broad utility in research, biotechnology, and medicine in the post-genomic era.
These three sections of P1 are followed by 30 references to various journal articles, the second of which is the article first published in Science online on 28 June 2012 and published in print on 17 August 2012 by Jinek et al that is of some importance to the issue of enablement. The article is by Martin Jinek, Krzysztof Chylinski, Ines Fonfara, Michael Hauer, Jennifer A. Doudna, and Emmanuelle Charpentier, and is entitled “A Programmable Dual-RNA-Guided DNA Endonuclease in Adaptive Bacterial Immunity” Science 337, 816 (2012) (“Jinek”). The question whether the invention involves an inventive step in light of Jinek does not arise in this proceeding.
The 30 references are then followed by a description of various figures reproduced in P1 including Figure 1A which includes a schematic representation of a guide RNA and a Cas9 protein used to cleave plasma DNA in vitro. The PAM sequence recognised by the Cas9 protein is identified in Figure 1A as CGG which is a PAM sequence recognised by Cas9 derived from S. pyogenes (ie. a 5’-NGG-3’ PAM where N stands for any nucleotide A, T, C or G). Triangles in Figure 1A depict the site of the intended break which is in a complimentary position on each DNA strand. Figure 3 provides mutation frequencies for RGEN-driven mutations at sites on the CCR5 gene and the C4BPB gene. The authors state that they reprogrammed the guide RNA used with the Cas9 protein to target the CCR5 gene with a different guide RNA to target the C4BPB gene. Accordingly, there is a specific disclosure of the use of two different guide RNAs targeting these two different genes in mammalian cells.
The description of the figures is followed by a section of P1 headed “Materials and Methods” which describes (inter alia) the construction of Cas9 encoding plasmids derived from S. pyogenes strain M1 GAS and the preparation of RNA in vitro using a MEGAshortscript T7 kit (Ambion). This is followed by a description of the genome-editing assay whereby mammalian cells (K562 cells) were transfected with 20µg of Cas9-encoding plasmid followed (after 24 hours) by the introduction of 10-40µg of in vitro transcribed chimeric RNA.
THE PATENT APPLICATION
Body of the Specification
The complete specification (“the Specification”) is entitled “COMPOSITION FOR CLEAVING A TARGET DNA COMPRISING A GUIDE RNA SPECIFIC FOR THE TARGET DNA AND CAS PROTEIN-ENCODING NUCLEIC ACID OR CAS PROTEIN, AND USE THEREOF”. The Specification is divided into a number of different sections. The first section contains a brief description of the “Technical Field” of the invention. The Specification states at [1]:
The present invention relates to targeted genome editing in eukaryotic cells or organisms. More particularly, the present invention relates to a composition for cleaving a target DNA in eukaryotic cells or organisms comprising a guide RNA specific for the target DNA and Cas protein-encoding nucleic acid or Cas protein, and use thereof.
The second section is headed “Background Art” and includes a brief discussion at [3]-[9] of the relevant technology including some prior art. The prior art referred to includes Jinek.
The Specification states at [3]-[4]:
[3]CRISPRs (Clustered Regularly Interspaced Short Palindromic Repeats) are loci containing multiple short direct repeats that are found in the genomes of approximately 40% of sequenced bacteria and 90% of sequenced archaea. CRISPR functions as a prokaryotic immune system, in that it confers resistance to exogenous genetic elements such as plasmids and phages. The CRISPR system provides a form of acquired immunity. Short segments of foreign DNA, called spacers, are incorporated into the genome between CRISPR repeats, and serve as a memory of past exposures. CRISPR spacers are then used to recognize and silence exogenous genetic elements in a manner analogous to RNAi in eukaryotic organisms.
[4]Cas9, an essential protein component in the Type II CRISPR/Cas system, forms an active endonuclease when complexed with two RNAs termed CRISPR RNA (crRNA) and trans-activating crRNA (tracrRNA), thereby slicing foreign genetic elements in invading phages or plasmids to protect the host cells. crRNA is transcribed from the CRISPR element in the host genome, which was previously captured from such foreign invaders. Recently, Jinek et al. (1) demonstrated that a single-chain chimeric RNA produced by fusing an essential portion of crRNA and tracrRNA could replace the two RNAs in the Cas9/RNA complex to form a functional endonuclease.
The Cas9 protein is an essential part of the genome editing technology described. It is an enzyme which, when complexed with a suitable guide RNA, provides a functional endonuclease that cuts each strand of DNA within the internal part of its sequence (rather than at its end) so as to generate a double-stranded break. A nuclease is an enzyme that cleaves DNA. An endonuclease is an enzyme that cuts DNA within the internal part of its sequence (in comparison to an exonuclease which trims the DNA at its ends). Endonucleases can generate two types of “ends”. One type of endonuclease cuts the DNA at the same position on the sense and antisense strands of the DNA to generate what is known as a “blunt” end. Another type of endonuclease cuts DNA at different positions on the sense and antisense strands to generate a “staggered” (or “sticky”) end. This second type of double-stranded break, sometimes referred to as “composite” double-stranded break, will have single stranded overhangs on either the 5’ or 3’ side of the DNA formation.
Two well-known gene editing tools discussed in the Specification are Zinc finger nucleases (ZFNs) and TALEN nucleases (TALENs). Zinc finger technology uses artificial restriction enzymes which can be used to cleave (cut) DNA strands generated by fusing a zinc finger DNA-binding domain to a DNA-cleavage domain. Zinc finger nucleases can be engineered to target specific desired DNA sequences and this enables them to target unique sequences within complex genomes. TALEN is an acronym for “Transcription Activator-like Effector Nuclease”, which are DNA-binding proteins that can be engineered to cut specific sequences of DNA. “RFLP” is an acronym for “Restriction Fragment Length Polymorphism”. This refers to the presence of a difference in the nucleotide sequence between two homologous DNA sequences that can be detected by use of a restriction enzyme that will preferentially recognise (and cut) only one variant of those two sequences. RFLP analysis allows polymorphisms to be identified based on the differences in the cutting activity of the restriction enzyme.
The Specification states at [5]-[9]:
[5]CRISPR/Cas systems offer an advantage to zinc finger and transcription activator-like effector DNA-binding proteins, as the site specificity in nucleotide binding CRISPR-Cas proteins is governed by a RNA molecule instead of the DNA-binding protein, which can be more challenging to design and synthesize.
[6]However, until now, a genome editing method using the RNA-guided endonuclease (RGEN) based on CRISPR/Cas system has not been developed.
[7][BLANK]
[8]Meanwhile, Restriction fragment length polymorphism (RFLP) is one of the oldest, most convenient, and least expensive methods of genotyping that is still used widely in molecular biology and genetics but is often limited by the lack of appropriate sites recognized by restriction endonucleases.
[9]Engineered nuclease-induced mutations are detected by various methods, which include mismatch-sensitive T7 endonuclease I (T7El) or Surveyor nuclease assays, RFLP, capillary electrophoresis of fluorescent PCR products, Dideoxy sequencing, and deep sequencing. The T7El and Surveyor assays are widely used but are cumbersome. Furthermore, theses enzymes tend to underestimate mutation frequencies because mutant sequences can form homoduplexes with each other and cannot distinguish homozygous bi-allelic mutant clones from wildtype cells. RFLP is free of these limitations and therefore is a method of choice. Indeed, RFLP was one of the first methods to detect engineered nuclease-mediated mutations in cells and animals. Unfortunately, however, RFLP is limited by the availability of appropriate restriction sites. It is possible that no restriction sites are available at the target site of interest.
The “Technical Problem” to which the invention is directed is described as follows at [11]-[13]:
Disclosure of Invention
Technical Problem
[11]Until now, a genome editing and genotyping method using the RNA-guided endonuclease (RGEN) based on CRISPR/Cas system has not been developed.
[12]Under these circumstances, the present inventors have made many efforts to develop a genome editing method based on CRISPR/Cas system and finally established a programmable RNA-guided endonuclease that cleave DNA in a targeted manner in eukaryotic cells and organisms.
[13]In addition, the present inventors have made many efforts to develop a novel method of using RNA-guided endonucleases (RGENs) in RFLP analysis. They have used RGENs to genotype recurrent mutations found in cancer and those induced in cells and organisms by engineered nucleases including RGENs themselves, thereby completing the present invention.
The section of the Specification headed “Solution to the Problem” includes at [15] a lengthy statement of what are said to be objects of the invention. At least some of these are in a form that reflects the language of the claims including claims 1 and 10. For convenience, the ten paragraphs that make up [15] have been numbered as [15.1]-[15.10]. All of these paragraphs but for the first appear to have been introduced by amendments made in 2016. The first ten objects are said to be:
[15.1]It is an object of the present invention to provide a composition for cleaving target DNA in eukaryotic cells or organisms comprising a guide RNA specific for target DNA or DNA that encodes the guide RNA, and Cas protein-encoding nucleic acid or Cas protein.
[15.2]It is another object of the present invention to provide a composition for inducing targeted mutagenesis in eukaryotic cells or organisms, comprising a guide RNA specific for target DNA or DNA that encodes the guide RNA, and Cas protein-encoding nucleic acid or Cas protein.
[15.3]It [sic] an object of the present invention to provide a composition comprising a Type II Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas system for use in introducing a site-specific, double stranded break at a target nucleic acid sequence in a eukaryotic cell, said CRISPR/Cas system comprising (i) a nucleic acid encoding a Cas9 polypeptide comprising a nuclear localization sequence, and (ii) a nucleic acid encoding a guide RNA that hybridizes to a target nucleic acid, wherein the guide RNA is a chimeric guide RNA comprising a CRISPR RNA (crRNA) portion fused to a trans activating crRNA (tracrRNA) portion.
[15.4]It is still another object of the present invention to provide a kit for cleaving a target DNA in eukaryotic cells or organisms comprising a guide RNA specific for target DNA or DNA that encodes the guide RNA, and Cas protein-encoding nucleic acid or Cas protein.
[15.5]It is still another object of the present invention to provide a kit for inducing targeted mutagenesis in eukaryotic cells or organisms comprising a guide RNA specific for target DNA or DNA that encodes the guide RNA, and Cas protein-encoding nucleic acid or Cas protein.
[15.6]It is still another object of the present invention to provide a method for preparing a eukaryotic cell or organism comprising Cas protein and a guide RNA comprising a step of co-transfecting or serial-transfecting the eukaryotic cell or organism with a Cas protein-encoding nucleic acid or Cas protein, and a guide RNA or DNA that encodes the guide RNA.
[15.7]It is an objection [sic] of the present invention to provide a method of introducing a site-specific, double-stranded break at a target nucleic acid sequence in a eukaryotic cell, the method comprising introducing into the eukaryotic cell a Type II Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas system, wherein the CRISPR/Cas system comprises:
a)a nucleic acid encoding a Cas9 polypeptide comprising a nuclear localization signal, wherein the nucleic acid is codon-optimized for expression in eukaryotic cells, and
b)a nucleic acid encoding a guide RNA that hybridizes to the target nucleic acid, wherein the guide RNA is a chimeric guide RNA comprising a CRISPR RNA (crRNA) portion fused to a trans activating crRNA (tracrRNA) portion, wherein the target nucleic acid sequence comprises a first strand that binds to the crRNA portion and a second strand having a trinucleotide protospacer adjacent motif (PAM),
and wherein the Cas9 polypeptide and the guide RNA form a Cas9/RNA complex in the eukaryotic cell, whereby a site-specific, double stranded break at the target nucleic acid sequence is introduced.
[15.8]It is still another object of the present invention to provide a eukaryotic cell or organism comprising a guide RNA specific for target DNA or DNA that encodes the guide RNA, and Cas protein-encoding nucleic acid or Cas protein.
[15.9]It is still another object of the present invention to provide a method for cleaving a target DNA in eukaryotic cells or organisms comprising a step of transfecting the eukaryotic cells or organisms comprising a target DNA with a composition comprising a guide RNA specific for target DNA or DNA that encodes the guide RNA, and Cas protein-encoding nucleic acid or Cas protein.
[15.10]It is still another object of the present invention to provide a method for inducing targeted mutagenesis in a eukaryotic cell or organism comprising a step of treating a eukaryotic cell or organism with a composition comprising a guide RNA specific for target DNA or DNA that encodes the guide RNA, and Cas protein-encoding nucleic acid or Cas protein.
(sub-paragraph numbering added)
Mutagenesis refers to changes in DNA (either naturally occurring or artificially engineered) that result in gene mutation.
It is apparent that sub-paragraph [15.1] distinguishes between a composition comprising (inter alia) “a guide RNA” that targets a particular DNA sequence and “DNA that encodes the guide RNA”. Sub-paragraph [15.3], which mirrors the language of claim 1, also refers to nucleic acid “encoding” a Cas9 polypeptide and nucleic acid “encoding” a guide RNA. The Specification makes similar use of the word “encoding” at [156] where there is reference to “a component in the form of a protein or in the form of a nucleic acid encoding Cas protein.”
Further objects are set out at [31], [33], [35], [37]-[38], [40], [42], [44], [46], [48], [50], [52], [54], [56], [58], [60], [62], [64], [66] and [68]. The advantageous effects of the invention are described as follows at [69]:
The present composition for cleaving a target DNA or inducing a targeted mutagenesis in eukaryotic cells or organisms, comprising a guide RNA specific for the target DNA and Cas protein-encoding nucleic acid or Cas protein, the kit comprising the composition, and the method for inducing targeted mutagenesis provide a new convenient genome editing tools. In addition, because custom RGENs can be designed to target any DNA sequence, almost any single nucleotide polymorphism or small insertion/deletion (indel) can be analyzed via RGEN-mediated RFLP, therefore, the compostion [sic] and method of the present invention may be used in detection and cleaving naturally-occurring variations and mutations.
In the section entitled “Best Mode for Carrying out the Invention” the Specification states at [137] – [145]:
[137]In accordance with one aspect of the invention, the present invention provides a composition for cleaving target DNA in eukaryotic cells or organisms comprising a guide RNA specific for target DNA or DNA that encodes the guide RNA, and Cas protein-encoding nucleic acid or Cas protein. In addition, the present invention provides a use of the composition for cleaving target DNA in eukaryotic cells or organisms comprising a guide RNA specific for target DNA or DNA that encodes the guide RNA, and Cas protein-encoding nucleic acid or Cas protein.
[138][BLANK]
[139]In the present invention, the composition is also referred to as a RNA-guided endonuclease (RGEN) composition.
[140][BLANK]
[141]ZFNs and TALENs enable targeted mutagenesis in mammalian cells, model organisms, plants, and livestock, but the mutation frequencies obtained with individual nucleases are widely different from each other. Furthermore, some ZFNs and TALENs fail to show any genome editing activities. DNA methylation may limit the binding of these engineered nucleases to target sites. In addition, it is technically challenging and time-consuming to make customized nucleases.
[142][BLANK]
[143]The present inventors have developed a new RNA-guided endonuclease composition based on Cas protein to overcome the disadvantages of ZFN s and TALENs.
[144][BLANK]
[145]Prior to the present invention, an endonuclease activity of Cas proteins has been known. However, it has not been known whether the endonuclease activity of Cas protein would function in an eukaryotic cell because of the complexity of the eukaryotic genome. Further, until now, a composition comprising Cas protein or Cas protein-encoding nucleic acid and a guide RNA specific for the target DNA to cleave a target DNA in eukaryotic cells or organisms has not been developed.
The Specification refers at [153] to the three types of CRISPR-Cas system found in bacteria including the Type II system involving the Cas9 protein. However, as will been seen, the claims all involve compositions or methods that make use of the Type II system and the Cas9 protein which is integral to that system.
According to the Specification at [158]-[159], in the present invention, the Cas protein may be any Cas protein provided that it has an endonuclease or nickase activity when complexed with a guide RNA, but preferably, it is Cas9 protein or variants thereof. The Specification states at [161]-[162]:
[161]Further, Cas protein may be the one isolated from an organism such as Streptococcus sp., preferably Streptococcus pyogens or a recombinant protein, but it is not limited thereto.
[162]The Cas protein derived from Streptococcus pyogens may recognizes NGG trinucleotide. The Cas protein may comprise an amino acid sequence of SEQ ID NO: 109, but it is not limited thereto.
(Errors in original).
Streptococcus pyogenes (“S. pyogenes”) is a particular species of bacteria which recognises the PAM sequence “NGG” (5’-NGG-3’ PAM) where N designates any nucleotide and “GG” represents two guanine nucleotides running in the 5’ to 3’ direction.
The Specification also states at [176]-[179]:
[176]The guide RNA may be transferred into a cell or an organism in the form of RNA or DNA that encodes the guide RNA. The guide RNA may be in the form of an isolated RNA, RNA incorporated into a viral vector, or is encoded in a vector. Preferably, the vector may be a viral vector, plasmid vector, or agrobacterium vector, but it is not limited thereto.
[177]A DNA that encodes the guide RNA may be a vector comprising a sequence coding for the guide RNA. For example, the guide RNA may be transferred into a cell or organism by transfecting the cell or organism with the isolated guide RNA or plasmid DNA comprising a sequence coding for the guide RNA and a promoter.
[178]Alternatively, the guide RNA may be transferred into a cell or organism using virus-mediated gene delivery.
[179]When the guide RNA is transfected in the form of an isolated RNA into a cell or organism, the guide RNA may be prepared by in vitro transcription using any in vitro transcription system known in the art. The guide RNA is preferably transferred to a cell in the form of isolated RNA rather than in the form of plasmid comprising encoding sequence for a guide RNA. As used herein, the term “isolated RNA” may be interchangeable to “naked RNA”. This is cost- and time-saving because it does not require a step of cloning. However, the use of plasmid DNA or virus-mediated gene delivery for transfection of the guide RNA is not excluded.
Transfection refers to the introduction of foreign DNA into a cell. The first sentence of [176] indicates that the guide RNA may take the form of isolated (or naked) RNA introduced into the cell or, alternatively, guide RNA encoded by DNA in the cell. The DNA may be transfected (introduced) into the cell using a vector (i.e. a plasmid). Once inside the cell, the transcription process will be initiated by a promoter included in the plasmid and the DNA will then transcribe the guide RNA. Thus, as [179] indicates, the guide RNA can be prepared in vitro before it is introduced into the cell in the form of “isolated” or “naked” RNA, or the guide RNA can be prepared in vivo after a plasmid containing the RNA-encoding DNA is transfected into the cell. The use of isolated RNA prepared in vitro is said to be preferable to the use of RNA-encoding plasmid DNA prepared in vivo because the former is the cheaper and less time consuming alternative and it does not involve a cloning step of inserting a target DNA fragment into a plasmid.
A nickase is an enzyme that cuts only one of the two strands of DNA to create a single strand break in the DNA. A Cas9 nickase is a mutant version of the wild-type Cas9 protein. A “paired Cas nickase” as defined in the Specification “may refer to the guide RNA and the Cas protein functioning as a pair” which may be used to make two breaks at the same or different locations on each of the complementary DNA strands. The Specification suggests that there may be advantages in using paired Cas9 nickases. The discussion in the Specification concerning Example 7 suggests that paired Cas9 nickases may produce composite double-stranded breaks which trigger DNA repair leading to efficient mutagenesis (ie. the generation of mutations) and a doubling in the specificity of Cas9-based genome editing. Various other possible advantages are also discussed.
The Claims
Claims 1 to 21 are as follows:
1A composition comprising a Type II Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas system for use in introducing a site-specific, double stranded break at a target nucleic acid sequence in a eukaryotic cell, said CRISPR/Cas system comprising (i) a nucleic acid encoding a Cas9 polypeptide comprising a nuclear localization sequence, and (ii) a nucleic acid encoding a guide RNA that hybridizes to a target nucleic acid, wherein the guide RNA is a chimeric guide RNA comprising a CRISPR RNA (crRNA) portion fused to a trans activating crRNA (tracrRNA) portion.
2.The composition of claim 1, wherein said Cas9 polypeptide is a Streptococcus Cas9 polypeptide.
3.The composition of claim 2, wherein said Cas9 polypeptide is a Streptococcus pyogenes Cas9 polypeptide.
4.The composition of any one of claims 1-3, wherein said nucleic acid encoding a Cas9 polypeptide is codon-optimized for expression in eukaryotic cells.
5.The composition of claim 4, wherein said nucleic acid encoding a Cas9 polypeptide is codon-optimized for expression in mammalian cells.
6.The composition of any one of claims 1-5, wherein said nuclear localization sequence is located at the C terminus of the Cas9 polypeptide.
7.The composition of any one of claims 1-5, wherein the target nucleic acid is an endogenous target nucleic acid.
8.The composition of any one of claims 1-5, wherein the guide RNA is in the form of a vector.
9.The composition of any one of claims 1-5, wherein said guide RNA comprises 2 additional guanine nucleotides at the 5' end.
10.A method of introducing a site-specific, double-stranded break at a target nucleic acid sequence in a eukaryotic cell, the method comprising introducing into the eukaryotic cell a Type II Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas system, wherein the CRISPR/Cas system comprises:
(a)a nucleic acid encoding a Cas9 polypeptide comprising a nuclear localization signal, wherein the nucleic acid is codon-optimized for expression in eukaryotic cells, and
(b)a nucleic acid encoding a guide RNA that hybridizes to the target nucleic acid, wherein the guide RNA is a chimeric guide RNA comprising a CRISPR RNA (crRNA) portion fused to a trans activating crRNA (tracrRNA) portion, wherein the target nucleic acid sequence comprises a first strand that binds to the crRNA portion and a second strand having a trinucleotide protospacer adjacent motif (PAM),
and wherein the Cas9 polypeptide and the guide RNA form a Cas9/RNA complex in the eukaryotic cell, whereby a site-specific, double stranded break at the target nucleic acid sequence is introduced.
11.The method of claim 10, wherein the Cas9 polypeptide is a Streptococcus Cas9 polypeptide.
12.The method of claim 11, wherein the Cas9 polypeptide is a Streptococcus pyogenes Cas9 polypeptide.
13.The method of any one of claims 10-12, wherein the nucleic acid encoding the Cas 9 polypeptide is codon-optimized for expression in mammalian cells.
14.The method of any one of claims 10-13, wherein the nuclear localization signal is located at the C terminus of the Cas9 polypeptide.
15.The method of any one of claims 10-14, wherein the eukaryotic cell is a mammalian cell.
16.The method of claim 15, wherein the mammalian cell is a human cell.
17.The method of any one of claims 10-16, wherein the target nucleic acid sequence is a genomic sequence located at its endogenous site in the genome of the eukaryotic cell.
18.The method of any one of claims 10-16, wherein the nucleic acid encoding the guide RNA is a vector.
19.The method of any one of claims 10-16, wherein the nucleic acid encoding the guide RNA is in vitro transcribed RNA.
20.The method of any one of claims 10-16, wherein said guide RNA comprises 2 additional guanine nucleotides at the 5' end.
21.The method of any one of claims 10-16, wherein the nucleic acid encoding the Cas9 polypeptide is introduced into the eukaryotic cell before introducing the nucleic acid encoding the guide RNA into the eukaryotic cell.
The claims refer to a “Cas9 polypeptide” rather than a “Cas9 protein” but, as used in both the description of the invention and the claims, these terms have the same meaning and are used interchangeably.
The following points should also be noted:
(1)Each of the independent claims 1 and 10 also refer to “nucleic acid encoding” a chimeric guide RNA. There is a question as to whether these words encompass a guide RNA that is prepared in vitro (outside the cell) and introduced into the cell in naked (or isolated) form or whether the claim limits what is described to guide RNA encoded by DNA in vivo (in the cell).
(2)Each of the independent claims 1 and 10 also refer to a composition or method for introducing “a site-specific, double-stranded break”. There is a question as to whether these words are apt to describe not only a blunt end break made by a single active endonuclease, but also a break having staggered ends of the kind made using “paired Cas nickase” in which there will be two Cas9 polypeptides each with its own guide RNA and each producing its own single strand break.
(3)Neither of the independent claims is limited to a Cas9 polypeptide derived from S. pyogenes (although some dependent claims are) that recognises the 5’-NGG-3’ PAM.
(4)Claim 8 refers to the relevant composition wherein the guide RNA “is in the form of a vector”. It is common ground, and I accept, that this should be understood as guide RNA encoded by DNA in a vector.
THE NOTIONAL SKILLED ADDRESSEE
Background
The question of who is the notional skilled addressee (or person skilled in the art) arises both in relation to P1 and the patent application.
There was a dispute between the parties as to the identity of the notional skilled addressee both in relation to the patent application and P1. ToolGen contends that the notional skilled addressee comprises a team that includes a molecular biologist such as Associate Professors Firestein and Herold and Professor Thomas and a microbiologist such as Professor Giffard. The respondents says that the skilled team does not include a microbiologist. On that basis they contend that Professor Giffard’s evidence is not relevant.
The notional skilled addressee is a legal construct and a tool of analysis framed by reference to the available evidence. This will include the patent specification and, typically, evidence of persons with knowledge and experience in the field of the invention.
The notional skilled addressee is a person who is likely to have a practical interest in the subject matter of the invention: Catnic Components Ltd v Hill & Smith Ltd [1982] RPC 183 at 242 per Lord Diplock. A person may have a practical interest in an invention at a number of levels. He or she may have an interest in using the products or methods of the invention, making the products of the invention, or making products used to carry out the methods of the invention either alone or in collaboration with others having such an interest: Apotex Pty Ltd v Warner-Lambert Company LLC (No 2) (2016) 122 IPR 17 (“Warner-Lambert”) at [27]. Broadly speaking, the skilled addressee will be a person who also has knowledge and experience in the field of the invention and who will bring to the reading of the relevant document the background knowledge and experience available to those working in that field.
In General Tire & Rubber Company v Firestone Tyre & Rubber Company Limited [1972] RPC 457 (“General Tire”) the English Court of Appeal referring to both the construction of the patent in suit and relevant prior art said at 485:
… If the art is one having a highly developed technology, the notional skilled reader to whom the document is addressed may not be a single person but a team, whose combined skills would normally be employed in that art in interpreting and carrying into effect instructions such as those which are contained in the document to be construed. We have already described the composite entity deemed to constitute the notional skilled addressee.
In some cases involving complex technology in which the notional skilled addressee is a team, the composition of the team may vary depending on the issue under consideration. As observed by Jacob LJ in Schlumberger Holdings Ltd v Electromagnetic Geoservices AS [2010] RPC 33 at [44] “… the notional team for considering obviousness may have wider skills than the team required for sufficiency” (original emphasis). Referring to Genentech Inc’s Patent [1989] RPC 147, his Lordship observed at [45]:
On the facts the patent was held obvious. The important point to note for present purposes is that the team for obviousness included a protein chemist whereas the team for implementation (sufficiency) did not need him. Different teams for different purposes.
P1
The invention described in P1 is said to be a novel genome editing technology. The system described is said to be based on RNA-guided endonucleases (RGENs). The RGENs described in P1 use a Type II CRISPR/Cas system in which the Cas9 protein, when complexed with a guide RNA (crRNA) and trans-activating RNA (tracrRNA), forms an active endonuclease. It is apparent from the opening paragraphs of P1 that it follows on from Jinek which P1 describes as raising the possibility of using the system disclosed in that publication for genome editing in cells and organisms. It is clear from P1 that the focus of the inventors was on the use of their invention in genome editing in eukaryotic cells and in human cells in particular.
In support of its submission that the notional skilled addressee would comprise a team including a microbiologist, ToolGen relied on the reference to Jinek in P1 and evidence given by Associate Professor Firestein that the reference to Jinek was directing him to an important paper regarding the development and repurposing of CRISPR/Cas9 technology.
I do not consider that P1 directs the notional skilled addressee to Jinek at least not as a source of anything more than general background that gives context to the invention described in P1.
It was submitted by ToolGen that the reference to Jinek in P1 must be regarded as part of the disclosure of P1, because the “draftsman had adopted the cross-referencing system solely as a shorthand means of incorporating a writing disclosing the invention”. The authority relied upon by ToolGen in support of that proposition is a passage in the judgment of Lockhart J in Nicaro Holdings Pty Ltd v Martin Engineering Co (1990) 16 IPR 545. In the context of considering whether a prior publication disclosed all the features of the invention of the patent in suit, his Honour said at 549:
The invention must appear in a single disclosure, so it is not permissible to make a pattern or mosaic of or to read together various pieces of prior art in different patents. It is however, permissible, to refer not only to the patent relied on as the source of disclosure but to another patent or other patents incorporated by reference provided that it is plain that the incorporation by reference unequivocally and plainly demonstrates that the draftsman has adopted the cross-referencing system solely as a shorthand means of incorporating a writing disclosing the invention: George C Warner Laboratories Pty Ltd v Chemspray Pty Ltd (1967) 37 AOJP 2513 at 2516; Blanco White, 5th ed, at para 4.107 and Gratwick, “Having Regard to What was Known and Used” (1972) 88 LQR 341 at 343.
In my view, the reference to Jinek in P1 falls well short of meeting that test.
The evidence of the molecular biologists made clear that the invention disclosed in P1 could be performed without recourse to Jinek. However, ToolGen contended that the notional skilled team (which on its case includes a microbiologist) would refer to Jinek for the purpose of producing a RGEN derived from a bacterial species other than S. pyogenes. The difficulty with this argument is that the reference in P1 to “Cas9 derived from other species” is a mere conjecture that does not form part of the invention described in P1. Importantly, the inventors do not state that other species can be used to perform the invention. The suggestion is much more tentative and does no more than refer to the possibility that other species might prove useful in overcoming the need for a 5’-GG-3’ dinucleotide in the PAM sequence. The fact that a microbiologist with expertise in bacterial CRISPR/Cas systems might be engaged by a molecular biologist who was interested in exploring that possibility is in my opinion not of itself sufficient to justify a finding that such a person would be part of the skilled team. In any event, all of the molecular biologists said that they would not have sought to establish if Cas9 from bacteria other than S. pyogenes recognises a non-NGG PAM on reading P1.
In my opinion, the notional skilled addressee to whom P1 is addressed is a molecular biologist with expertise in the field of gene editing in eukaryotic cells. The reference to Jinek in P1 does not justify a finding that a microbiologist would be part of the skilled team.
The molecular biologist (working alone or with other molecular biologists and laboratory assistants) will be a highly qualified scientist with a PhD in the field of molecular biology with expertise in gene screening, targeting and manipulation in eukaryotes including in mouse models and in vitro systems. They will be engaged in high level research in a well-resourced laboratory in a medical research institute.
If, contrary to my finding, there was a microbiologist on the team, they would have a PhD in the field of microbiology or bacteriology with expertise in the CRISPR/Cas systems found in bacteria and archaea. This member of the team would most likely be engaged in academic research within the biology faculty or department of a University.
The Patent Application
In the present case, ToolGen submitted that the patent application is directed to the repurposing of bacterial Type II CRISPR/Cas systems for cleaving target DNA in eukaryotic cells or organisms and that the skilled addressee will comprise a team that includes a person with knowledge of these systems. Hence, ToolGen says that the notional team will have expertise in the field of gene editing in eukaryotic cells, and the expertise of a microbiologist with expertise in bacterial CRISPR/Cas systems. It is on this basis that ToolGen contends that the team will include persons with the knowledge and experience of Associate Professors Herold and Firestein and Professor Thomas (as experts in gene editing in eukaryotic cells) and also a person like Professor Giffard (an expert in bacterial CRISPR/Cas systems). In support of its submissions, ToolGen refers to the description in the patent application of Type II CRISPR/Cas systems involving Cas9 and crRNA and tracrRNA and, in particular, the statements to the effect that the invention is not limited to such systems from S. pyogenes.
The respondents submitted that the person who has a practical interest in the subject matter of the invention is a molecular biologist with an interest in genome editing in eukaryotic cells. They submitted that this reflects the “field of the invention” specified in the patent application as well as the express purpose of the invention as described and claimed. On that basis they submitted that a microbiologist such as Professor Giffard, with expertise in relation to bacteria and prokaryotes (not eukaryotic cells) would not form part of the team. They submitted that a microbiologist would have no interest in the subject matter of the invention as described and claimed and that such a person would not be interested in making or using the compositions or methods of the claims for genome editing in eukaryotic cells.
The invention disclosed in the patent application is not limited to a system which uses a Cas9 protein derived from S. pyogenes. The Specification makes clear at [161]-[162] that the Cas9 protein which is integral to the Type II CRISPR/Cas system may be derived from S. pyogenes but is not limited to that bacteria and that it may be derived from a different organism. The claims also make clear that claims 1 and 10 are not limited to Cas9 derived from S. pyogenes and that they may be derived from other species. I think it must follow that the invention, as described and claimed, is directed to a notional team including a person with expertise in identifying Cas9 derived from other bacterial species.
The respondents submitted that the notional skilled addressee should not be defined in order to fill a gap in the disclosure of the patent application. It submitted that it was not legitimate to attempt to make up for an absence of disclosure by artificially adding to the skilled team a person whose skills might be thought to assist with the development of products or methods that are not disclosed.
As I have explained, the patent application makes clear that the invention can be performed using different species of bacteria. Unlike P1, the statements to that effect are clear and unequivocal and indicate that the invention extends to systems that use Cas9 derived from other bacterial species. Molecular biologists reading the patent application would necessarily understand that the invention is not confined to a system that uses Cas9 derived from one species only. If they wished to perform the invention using Cas9 derived from a different bacterial species they would turn to a microbiologist with expertise in CRISPR/Cas systems who could assist in identifying a suitable substitute for S. pyogenes. For that reason I think such a person should be taken to form part of the notional skilled team.
In my opinion, the notional skilled addressee to whom the patent application is addressed is a team comprising a molecular biologist with expertise in the field of gene editing in eukaryotic cells and a microbiologist with expertise in CRISPR/Cas systems.
COMMON GENERAL KNOWLEDGE
Second, the respondents accepted that the patent application, unlike P1, discloses an invention that comprises “a nucleic acid encoding a guide RNA”. They do not contend that the invention of the claims is, in this particular respect, not sufficiently enabled.
Third, the patent application differs from P1 in that it discloses the existence of a nucleic acid encoding a Cas9 polypeptide derived from S. pyogenes which in Example 9 is shown to recognise and bind to a 5’-NAG-3’ PAM sequence. It is important to note, however, that Example 9 used Cas9 derived from S. pyogenes. There is no Example in which CRISPR/Cas9 components from other bacterial species are used. The respondents submitted that the patent application does not disclose the invention in each claim in a manner that is clear enough and complete enough for it to be performed by a person skilled in the relevant art because the patent application does not enable an invention comprising a system (or components of a system) derived from a bacterial species other than S. pyogenes without undue burden. I accept that submission essentially for the reasons given in relation to P1. I also accept that the patent application does not enable the use of engineered S. pyogenes derived Cas9 in the compositions or methods of the claims, essentially for the same reasons given in relation to P1.
Fourth, ToolGen relied on similar arguments to those which I have previously considered in support of its contention that the length of the sgRNAs disclosed in the patent application are not confined by the single example shown in Figure 1a of the patent application, being a sgRNA (+48). Figure 1a of the patent application reproduces Figure 1A of P1. There is no material difference between the disclosures of P1 and the patent application regarding the design of the sgRNA including its tracrRNA component. In my opinion, the patent application does not provide an enabling disclosure of a sgRNA having a length different from that shown in Figure 1a of the patent application.
Fifth, ToolGen relied on similar arguments to those which I have previously considered in support of its submission that the NLSs disclosed in the patent application are not limited to the PKKKRKV NLS and that all other suitable NLSs are implicitly disclosed. I accept that submission. In my opinion, the patent application implicitly discloses an invention that uses any suitable NLS apart from the PKKKRKV NLS both in terms of its sequence and location. I consider that their use would be routine and straightforward, and not such as to create undue burden for the skilled addressee.
Sixth, the respondents did not press their ground of opposition based on the disclosure of the GAAA linker in the context of the patent application which makes the same disclosure in Figure 1a of the patent application as is made by Figure 1A in P1.
PATENT APPLICATION – SUPPORT
The support requirement is found in s 40(3) of the Act which relevantly provides that the “claims must be supported by the matter disclosed in the specification”. The support requirement in s 40(3) was discussed in the Explanatory Memorandum to the RTB Act. The Explanatory Memorandum states:
Overseas law generally requires there to be a relationship between the claims and the description, and between the claims and any document from which priority is being claimed. This is expressed by the requirement that a claim be ‘supported by’ or ‘fully supported by’ the description. Broadly speaking, the terms ‘support’ and ‘full support’ pick up two concepts:
•there must be a basis in the description for each claim; and
•the scope of the claims must not be broader than is justified by the extent of the description, drawings and contribution to the art.
Despite the underlying concept and policy between fair basis and support being similar, the different terminology has produced different substantive law in different countries.
The difference in substantive law in different countries causes unnecessary complexity and uncertainty for applicants seeking protection in Australia and other jurisdictions. As discussed above (see item 7), having different standards in different countries imposes costs on global innovators, who must familiarise themselves with the varying requirements.
This item is intended to align the Australian requirement with overseas jurisdictions’ requirements (such as the UK). Overseas case law and administrative decisions in respect of the ‘support’ requirement will be available to Australian courts and administrative decision-makers to assist in interpreting the new provision.
In Merck Sharp & Dohme Corporation v Wyeth LLC (No 3) (2020) 155 IPR 1 (“Merck”) Burley J, after referring to the relevant extrinsic material (including the Explanatory Memorandum referred to above), observed at [514]:
Having regard to the content of the secondary materials, there can be little doubt that Parliament considers that it is appropriate for the Court to have regard to the law in the European Union and the United Kingdom in considering the scope of the requirement for “support”: Acts Interpretation Act 1901 (Cth) s 15AB.
His Honour went on to consider the law in Europe and the United Kingdom concerning Art 84 of the EPC and s 14(5) of the UK Act. Article 84 of the EPC, which is enshrined in s 14(5), provides:
The claims shall define the matter for which protection is sought. They shall be clear and concise and be supported by the description.
Subparagraph (c) of s 14(5) of the UK Act requires that the claims “be supported by the description”.
The effect of Burley J’s analysis of the European and UK law is that the “support” requirement or what he called the “claim support obligation” requires that the technical contribution to the art disclosed by the specification justify the breadth of the claim. His Honour referred at [546] to the judgment of Aldous J in Schering Biotech Corp’s Application [1993] RPC 249 (“Schering Biotech”) where his Lordship said at 252-3:
In my view the correct approach under the 1977 Act is to consider the description and claims in the specification through the eyes of the skilled man in the art. Under section 125(1) the invention is that specified in the claims. Thus to decide whether the claims are supported by the description it is necessary to ascertain what is the invention which is specified in the claims and then compare that with the invention which has been described in the specification. Thereafter the court's task is to decide whether the invention in the claims is supported by the description. I do not believe that the mere mention in the specification of features appearing in the claim will necessarily be a sufficient support. The word "support" means more than that and requires the description to be the base which can fairly entitle the patentee to a monopoly of the width claimed. This approach is I believe consistent with the decision of the European Patent Office's Technical Board of Appeals in the Riogen case T301l87 of 16 February 1989 [Biogen NV v Hoffmann-La Roche & Co. AG (Decision T30l/87), [19901 Official Journal E.P.O. 335.]. They said: “The scope of protection in the claims must be fair having regard to the way in which the invention is described and having regard to the information which the skilled person has been given in the description as to how the invention can be carried out”.
Burley J said at [547]:
That approach encapsulates broadly the claim support obligation under s 40(3). To it may be added the requirement that the technical contribution to the art must be ascertained. Where it is a product, it is that which must be supported in the sense that the technical contribution to the art disclosed by the specification must justify the breath [sic] of the monopoly claimed.
I agree with Burley J that the approach by Aldous J in Schering Biotech at 252-3 broadly encapsulates the support obligation under s 40(3) of the Act.
The question whether a disclosure in a patent application fairly entitles the patentee to a monopoly of the width claimed calls for an assessment of the patentee’s contribution to the art, which must be weighed against the scope of the patentee’s monopoly as defined by the claims. The monopoly, according to UK and European authorities, must be justified by the technical contribution to the art that arises from the disclosure of the specification.
At least two difficulties can arise in applying the test. First, as the UK authorities show, determining the patentee’s technical contribution to the art is often not easy. While it has been suggested that the technical contribution may correspond with the inventive step, there will be situations in which this is not so including where, for example, the invention of the claim involves a very significant inventive step and yet, by reason of some deficiency in the disclosure of the specification, the public is deprived of its side of the patent bargain. Leaving aside what the UK authorities sometimes refer to as “classical insufficiency” (broadly corresponding to the requirements of s 40(2)(a) of the Act), this may arise when the specification discloses how to perform the invention across the scope of the claims (eg. by using a known pharmaceutical compound in a new method of treatment) but not the basis upon which the invention of the claim might reasonably be expected to work (ie. by delivering the relevant therapeutic effect). The practice of claiming inventions that are not shown to have a sufficiently plausible or credible justification or support is sometimes referred to as speculative claiming.
An important decision in the UK that was directly concerned with the support requirement enshrined in Art 84 of the EPC and s 14(5)(c) of the UK Act is the UK Supreme Court’s decision in Warner-Lambert LLC v Generics (UK) Ltd t/a Mylan [2018] UKSC 56 (“Warner-Lambert UK”) . The leading judgment was given by Lord Sumption (with whom Lord Reed agreed). Much of the discussion in Warner-Lambert UK focused on “second use” claims to new methods of treatment using known pharmaceutical compounds and related Swiss-style claims. But it also includes more general discussion regarding the support requirement albeit in the context of the somewhat different statutory scheme which Burley J elucidated in Merck at [527].
Lord Sumption referred at [17] to the “patent bargain” which he described as the foundation of modern patent law both in the UK and the EPO. In this regard, his Lordship referred to the following statement from the decision in EXXON/Fuel Oils (T-409/91) [1994] OJ EPO 653 at paras 3.3 and 3.4 in which the EPO Technical Board observed that it was:
… the general legal principle that the extent of the patent monopoly, as defined by the claims should correspond to the technical contribution to the article in order for it to be supported, or justified. … This means that the definitions in the claims should essentially correspond to the scope of the invention as disclosed in the description. … Although the requirements of articles 83 and 84 are directed to different parts of the patent application, since article 83 relates to the disclosure of the invention, whilst article 84 deals with the definition of the invention by the claims, the underlying purpose of the requirement of support by the description, insofar as its substantive aspect is concerned, and of the requirement of sufficient disclosure is the same, namely to ensure that the patent monopoly should be justified by the actual technical contribution to the art.
His Lordship went on to discuss the problem of speculative claiming and the patentee who attempts to claim a monopoly more extensive than could be justified by his or her contribution to the art. This may arise in different contexts including in cases involving claims to wide classes of chemical compounds or cases involving second use patents where known compounds are the subject of claims for methods of treatment or Swiss-style claims directed to new indications. Warner-Lambert UK, which concerned the UK patent for a method of treating pain using pregabalin, was such a case. Lord Sumption, having referred to the discussion in the Court of Appeal’s judgment regarding speculative claiming, said at [22]-[23]:
[22]The Court of Appeal’s reference to “armchair inventors” suggests that what they meant by speculative claiming was claiming by persons who had done nothing new or inventive at all but had simply sought to patent abstract possibilities. That may well be a particular risk in the case of patents for new uses of known compounds, especially when they are commercially successful in their existing use. In reality, however, speculative claiming of this kind is simply one of a number of ways in which a patentee may attempt to claim a monopoly more extensive than anything which is justified by his contribution to the art. Other ways in which this can happen include claiming a monopoly wider than the disclosure in the patent can support. An over-broad claim will not necessarily be speculative. The inventor may really have invented something corresponding to the full breadth of the claim. Research may subsequently demonstrate this. But the claim will still exceed his contribution to the art if that contribution is not sufficiently disclosed in the patent.
[23]The concept of plausibility originates in the case law of the EPO as a response to over-broad claims, in particular claims to whole classes of chemical compounds supported by a description which fails to show which compounds can be expected to work. The Technical Board of Appeal treats the condition of sufficiency under EPC article 83 as satisfied if it is possible to work the invention across the scope of the claim from the information in the specification, interpreted in the light of common general knowledge at the priority date. It addresses the broader question whether the disclosed contribution to the art is commensurate with the monopoly claimed under EPC article 56, in the context of inventive step. In that context, its case law requires the formulation of a problem which the claims of the patent could be said to solve: see T 939/92 AGREVO/Triazole sulphonamides [1996] EPOR 171. It imports a requirement that the patent should disclose not just what the invention is and how to replicate it, but some reason for expecting that it will work. Plausibility was the standard to which the patentee was expected to demonstrate this.
It can be seen that the concept of plausibility has been developed in the UK authorities as a check on speculative claiming and to ensure that the patentee’s monopoly is no more extensive than the contribution to the art made by the relevant disclosure.
Lord Sumption referred in some detail to the distinction drawn in the UK cases between so-called “classical insufficiency” (where the skilled person is unable to perform the invention from the information disclosed in the specification) and so-called Biogen insufficiency (where the claim is said to be too broad, because it exceeds the disclosed contribution to the art). The expression Biogen insufficiency is derived from the decision of the House of Lords in Biogen Inc v Medeva Plc [1997] RPC 1. His Lordship said of Biogen insufficiency at [25]:
… The House of Lords imported into section 14(3) of the Act a concept similar to the former requirement of fair basis in section 32(1)(i) of the Patents Act 1949 (“that any claim of the complete specification is not fairly based on the matter disclosed in the specification”). It held that if the claim extended beyond the technical contribution to the art disclosed in the patent, it failed for insufficiency independently of any objection based on want of an inventive step and notwithstanding that the skilled person could perform the invention across the whole scope of the claim. Lord Hoffmann, delivering the leading speech, said at p 50:
But the fact that the skilled man following the teaching of Biogen 1 would have been able to make HBcAg and HBsAg in bacterial cells, or indeed in any cells, does not conclude the matter. I think that in concentrating upon the question of whether Professor Murray’s invention could, so to speak, deliver the goods across the full width of the patent or priority document, the courts and the EPO allowed their attention to be diverted from what seems to me in this particular case the critical issue. It is not whether the claimed invention could deliver the goods, but whether the claims cover other ways in which they might be delivered: ways which owe nothing to the teaching of the patent or any principle which it disclosed.
He went on to make the same point in the context of the objection of insufficiency. Adopting the statement of principle cited above from EXXON/Fuel oils, he pointed out, at p 54, that the purpose of requiring sufficiency of disclosure could not be limited to enabling the public to work the invention after the patent had expired:
Section 72(1)(c) of the 1977 is not only intended to ensure that the public can work the invention after expiration of the monopoly. It is also intended to give the court in revocation proceedings a jurisdiction which mirrors that of the Patent Office under section 14(3) or the EPO under article 83 of the EPC, namely, to hold a patent invalid on the substantive ground that, as the EPO said in Exxon/Fuel Oils (T 409/91) [1994] OJ EPO 653, para 3.3, the extent of the monopoly claimed exceeds the technical contribution to the art made by the invention as described in the specification.
Lord Hoffmann was not, in these observations, addressing the question of second use patents. But such patents raise a similar problem. If it is enough to disclose how to make a known compound and for what conditions, the patentee has acquired a monopoly without adding anything to the sum of knowledge. He will have satisfied the condition of sufficiency but without satisfying its purpose.
Lord Sumption set out at [37] a number of propositions relevant to the concept of plausibility. Some of these were expressed in terms most relevant to claims for methods of treatment, Swiss-style claims and suggested therapeutic effects, but they are also relevant to the concept of support more generally. His Lordship observed that the proposition that a product is efficacious for the treatment of a particular condition is not made plausible by a bare assertion to that effect. He went on to consider what information may render an assertion that a product is efficacious plausible. His Lordship observed:
… the claimed therapeutic effect may well be rendered plausible by a specification showing that something was worth trying for a reason, ie not just because there was an abstract possibility that it would work but because reasonable scientific grounds were disclosed for expecting that it might well work. The disclosure of those grounds marks the difference between a speculation and a contribution to the art. This is in substance what the Technical Board of Appeal has held in the context of article 56, when addressing the sufficiency of disclosure made in support of claims extending beyond the teaching of the patent …
ToolGen submitted that while claim 1 is ostensibly to a product, namely a Type II CRISPR/Cas system, it is limited by the requirement that it be suitable to make a double-stranded break in a target DNA sequence in a eukaryotic cell. I have already addressed this argument. I do not accept it. The claim is not limited to those compositions capable of making a double-stranded break. Whether or not the composition would make such a break in use would depend on a variety of factors including whether the sgRNA and Cas9 complex was successful in locating and interacting with the target DNA.
ToolGen also submitted that the inventors’ technical contribution to the art is, in substance, use of a bacterial Type II CRISPR/Cas system to achieve a double-stranded cut in DNA in a eukaryotic cell using a Cas9 polypeptide from that Type II system with the other components referred to in the claim. It submitted that no one had previously described the use of a CRISPR/Cas system in eukaryotic cells with that ability, and the disclosure of that system represented a substantial contribution to the art. It further submitted that the technical contribution was one of general application and that the width of claims 1 and 10 could be supported on that basis.
The respondents submitted that the technical contribution to the art made by the patent application is at best a disclosure of a particular Type II CRISPR/Cas systems derived from S. pyogenes having the following features and characteristics:
(i)a requirement for a 5’-NGG-3’ or 5’-NAG-3’ PAM sequence;
(ii)a chimeric guide RNA having the crRNA and tracrRNA portions identified in Figure 1a a of the patent application and the examples;
(iii)a GAAA linker fusing the crRNA and tracrRNA components in the guide RNA; and
(iv)a particular NLS (PKKKRKV) located at the C-terminus of the Cas9 polypeptide.
They submitted that the scope of the monopoly claimed far exceeds the extent of the contribution to the art given that no Type II CRISPR/Cas9 system is illustrated in the patent application except for the particular system based on S. pyogenes. The last two of these features and characteristics identified by the respondents (i.e. (iii) and (iv)) can be disregarded in light of my previous findings.
It is useful to refer back to the patent application and the sections of the specification entitled “Technical Problem” and “Solution of the Problem”. The technical problem disclosed was that a genome editing method for using RNA guided endonuclease based on the CRISPR/Cas system had not been developed. It is then stated that the inventors made many efforts to develop such a system “… and finally established a programmable RNA-guided endonuclease that cleave DNA in a targeted manner in eukaryotic cells and organisms”.
The patent application includes statements in [161] that the Cas protein may be isolated from a Streptococcus species, preferably, S. pyogenes, or a recombinant protein, but is not limited to Cas protein so derived. There are also statements in [158] that any Cas protein may be used provided that it has endonuclease or nickase activity when complexed with a guide RNA. The statements that any such Cas protein can be used can be put aside because claim 1 is limited to a composition that uses a Cas9 polypeptide. As I have mentioned, there are no examples given in the patent application of the invention using components derived from any species other than S. pyogenes.
In closing submissions ToolGen relied on the decision in Warner-LambertUK including, in particular, Lord Sumption’s observation at [36] that the test of plausibility is “relatively undemanding” while at the same time expressing a preference for the dissenting judgment of Lady Black. However, ToolGen also stated that it understood the respondents did not rely on any lack of plausibility on the face of the patent application. Having reviewed the respondents’ submissions on this point closely, I think ToolGen’s understanding is correct. This is because the respondents say (as I have already found in relation to P1) that the patent application does not disclose any principle of general application. Accordingly, they say the issue of plausibility does not arise.
The respondents submitted that the support requirement in s 40(3) in combination with the requirement of disclosure in s 40(2)(a), operates to ensure that there is an enabling disclosure, and if the disclosure does not enable the invention to be performed to the full extent of the claim, the claim will lack the support required by s 40(3).
The facts in Warner-Lambert UK show how a claim might meet the requirement of s 40(2)(a) by providing an enabling disclosure, but not meet the support requirement of s 40(3). However, it is difficult to see how a claim to an invention for which there was no enabling disclosure could meet the support requirement. In such circumstances, the scope of the monopoly defined by the claim could not be justified by the technical contribution to the art. The two requirements are closely interrelated and not wholly distinct in their fields of operation.
In the present case, the broadest claims (ie. claims 1, 2, 10 and 11) fail to meet the requirements of both s 40(2)(a) and s 40(3) of the Act due to the fact that they are not confined to the use of Cas9 derived from S. pyogenes. Further, all of the claims, insofar as they encompass other sgRNAs of different lengths to that shown in Figure 1a of the patent application (i.e. sgRNA (+48)), also fail to meet the requirements of both s 40(2)(a) and s 40(3) of the Act.
THE PRIORITY DATE
The hearing of the appeal was conducted on the premise that if the claims were not entitled to priority based on P1, then a deferred date of 20 June 2013 would apply (“the deferred priority date”). Although the evidence included two US provisional patent applications filed on 20 March 2013 (P2) and 20 June 2013 (P3), none of the evidence or submissions of the parties made reference to them. P2 is a provisional application entitled “Genotyping with CRISPR/Cas-derived RNA-guided endonucleases” which appears to be solely concerned with a method of using RNA-guided endonucleases in restriction fragment length polymorphism analysis. It does not disclose the invention of any of the claims in the patent application. P3 is a provisional application entitled “Composition for cleaving a target DNA comprising a guide RNA specific for the target DNA and Cas protein-encoding nucleic acid or Cas protein”. The parties did not make any submissions in relation to P3 presumably because it is common ground that even if there are claims entitled to priority based on P3, it was filed too late in time to save the claims of the patent application in light of various journal articles published between the filing of P1 (23 October 2012) and the filing of P3 (20 June 2013).
The following publications are relevant to the issues of novelty and inventive step:
·Cong et al, “Multiplex Genome Engineering Using CRISPR/Cas Systems” (2013) Science 339, 819-823 and Supplementary Materials;
·Mali et al, “RNA-Guided Human Genome Engineering via Cas9” (2013) Science 339, 823-826 and Supplementary Materials; and
·Wang et al, “One-Step Generation of Mice Carrying Mutations in Multiple Genes by CRISPR/Cas-Mediated Genome Engineering” (2013) Cell 153, 910-918 and Supplementary Information.
ToolGen has admitted that Cong (2013) was published on 15 February 2013 and that the Supplementary Materials for Cong (2013) were published on 13 June 2014. However, there is other evidence which shows, and I find, that the Supplementary Materials for Cong (2013) were published on 3 January 2013. ToolGen has admitted that Mali (2013) and its Supplementary Materials were published on 15 February 2013. ToolGen has also admitted that Wang (2013) was published on 9 May 2013 and that its Supplementary Information was published on 2 May 2013.
NOVELTY
Section 18(1)(b)(i) of the Act provides that an invention is a patentable invention, for the purposes of a standard patent, if the invention, so far as claimed in any claim, when compared with the prior art base as it existed before the priority date, is novel. Section 7(1) of the Act provides that an invention is taken to be novel when compared with the prior art base unless it is not novel in light of relevant prior art information (as defined in the Act).
The test for whether a patent claim lacks novelty by reason of a prior publication (or is “anticipated” by a prior publication) has been described as follows in General Tire at 486:
To anticipate the patentee’s claim the prior publication must contain clear and unmistakable directions to do what the patentee claims to have invented … A signpost, however clear, upon the road to the patentee's invention will not suffice. The prior inventor must be clearly shown to have planted his flag at the precise destination before the patentee.
(Citations omitted.)
As previously mentioned, ToolGen accepts that if claims 1-8 and 10-18 are not entitled to priority from P1 then the Delegate’s findings that those claims lacked novelty will stand.
With regard to claims 9, 19 and 20, the respondents contend that if, contrary to my findings, those claims include the use of in vitro transcribed sgRNA, then they are anticipated by Wang 2013 which discloses systems and methods within those claims.
With regard to claims 9 and 20, Associate Professor Herold and Professor Thomas gave unchallenged evidence that T7 promoters were well-known and widely used to drive RNA transcription in vitro and that these produced an RNA molecule with two guanine (G) nucleotides at the 5’ end.
There was unchallenged evidence given by Associate Professor Herold in relation to Wang (2013) and its disclosure including the use of a guide RNA with two additional guanine nucleotides at the 5’ end. He said in Herold 1:
The experiments reported in Table 2 that targeted Tet3 were conducted using sgRNA with two extra guanine molecules at the 5' end. I understand this from the bottom of Table S3, which includes the oligonucleotides used to add the T7 promoter to the sgRNA template for in vitro transcription of the sgRNA (see Experimental Section: “Production of Cas9 mRNA and sgRNA” on page 916 and Table S3).
ToolGen did not adduce any evidence in answer or make any submission in relation to this evidence. If, as I found, the patent application is not entitled to priority based on P1, and if, contrary to my findings, claims 9 and 20 include the use of in vitro transcribed guide RNA, then those claims, so construed, would lack novelty based on the publication of Wang 2013.
Further, if claim 19 (when read with claim 10) does not lack clarity and includes the use of in vitro transcribed guide RNA then that claim would also lack novelty based on the publication of Wang 2013.
INVENTIVE STEP
An invention is a patentable invention if the invention, when compared with the prior art base, involves an inventive step: see s 18(1)(b)(ii) of the Act. Sections 7(2) and (3) of the Act identify the nature of the enquiry. They provide:
(2)For the purposes of this Act, an invention is to be taken to involve an inventive step when compared with the prior art base unless the invention would have been obvious to a person skilled in the relevant art in the light of the common general knowledge as it existed (whether in or out of the patent area) before the priority date of the relevant claim, whether that knowledge is considered separately or together with the information mentioned in subsection (3).
(3) The information for the purposes of subsection (2) is:
(a)any single piece of prior art information; or
(b)a combination of any 2 or more pieces of prior art information that the skilled person mentioned in subsection (2) could, before the priority date of the relevant claim, be reasonably expected to have combined.
A “scintilla of invention” can sustain a valid patent, but there must be “some difficulty overcome, some barrier crossed” or something “beyond the skill of the calling”: Lockwood Security Products Pty Ltd v Doric Products Pty Ltd(No 2) (2007) 235 CLR 173 at [52]; RD Werner & Co Inc v Bailey Aluminium Products Pty Ltd (1989) 25 FCR 565 at 574; Allsop Inc v Bintang Ltd (1989) 15 IPR 686 at 701.
ToolGen accepts that if claims 1-8 and 10-18 are not entitled to priority from P1 then the Delegate’s finding that those claims lack an inventive step will stand.
With regard to claims 9 and 20, I accept it would have been a simple and routine matter for the skilled addressee to use T7 promoters and, by so doing, produce two additional guanine nucleotides at the 5’ end of the guide RNA. Use of T7 promoters would not require any inventive capacity or imagination.
Having regard to the unchallenged evidence on this topic, I accept that if, contrary to my findings, the Court was to construe claims 9, 19 and 20 as including the use of in vitro transcribed guide RNA then the evidence shows that these claims would have been obvious in light of each of Wang (2013), Cong (2013) and Mali (2013) taken together with the common general knowledge as at the deferred priority date.
On the construction of claims 9 and 20 which limits them to the use of a nucleic acid (such as plasmid DNA) encoding a guide RNA, then the evidence also establishes that each of those claims is obvious in light of each of Cong (2013) and Mali (2013) taken together with the common general knowledge as at the deferred priority date. Cong (2013) and Mali (2013) each describe experiments in which the guide RNA is encoded by a plasmid DNA.
Claim 21 introduces the additional step to the method described in any of claims 10-16 wherein the nucleic acid encoding the Cas9 polypeptide is introduced into the eukaryotic cell before introducing the nucleic acid encoding the guide RNA. ToolGen made some very brief oral submissions in relation to claim 21 in which it drew attention to what was said by Associate Professor Herold in Herold 1 concerning claim 21. ToolGen submitted the evidence filed by the respondents does not reach the level required to demonstrate a lack of inventive step for claim 21.
The respondents submitted that to introduce the components of the system described in claim 10 in a stepwise fashion would be a simple and routine matter. In their oral evidence each of Professor Thomas, Associate Professor Firestein and Associate Professor Herold agreed that it would be possible to employ the method of the claims using a stepwise approach with a DNA plasmid that encoded for the guide RNA being introduced as a second step. None of the witnesses was asked whether it would be desirable or routine to adopt that approach nor was any of them asked whether it would have been an obvious thing to do.
The patent application does not provide any indication that there is anything added by claim 21 to what is claimed in claims 10-20 which could renders claim 21 inventive if (as is the case) none of those claims involves an inventive step. I find that it would be obvious to the skilled addressee that the method of claim 10 (which was itself obvious in light of each of Wang (2013), Cong (2013) and Mali (2013)) could be performed in a stepwise fashion in the manner described in claim 21. Adoption of that method would not require any inventive capacity or imagination or the exercise of skill beyond that of the calling. I therefore find that claim 21 does not involve an inventive step.
AMENDMENT
For the reasons explained each of the claims would, if granted, be invalid. In its closing submissions, ToolGen indicated that it may wish to amend the patent application in the event that I was to find that any one or more of the claims was invalid. Presumably, this would involve amendments aimed at narrowing the scope of the claims and aligning them with the disclosure of P1.
The Court has power to hear and determine an application to amend in this case: see s 105(1A) and s 112A of the Act and Meat and Livestock Australia Limited v Branhaven LLC (2020) 281 FCR 640 at [17]-[20], [91]-[93]. ToolGen submitted that it may be appropriate to remit the matter to the Patents Office so that the Delegate could consider the amendment application. I do not think that would be desirable. Given the complexity of this matter, I think ToolGen should make any application to amend the patent application to this Court so that that application may be determined before any final order is made or any application for leave to appeal any such final order is filed. It is plainly desirable that any application to amend the claims be heard and determined in advance of any appeal so that the Full Court may give consideration not only to the issues addressed in these reasons, but any further issues arising out of the foreshadowed amendment application. In this regard, I note that the respondents have already foreshadowed that they will oppose any application that may be made by ToolGen to amend the claims.
DISPOSITION
The only orders I propose to make at this time are procedural orders relating to any application to amend the patent application. In the event that no application to amend is filed within 28 days, I propose to make orders dismissing the appeal and allowing the cross-appeal together with an order directing the Commissioner to refuse the patent application.
There does not appear to be any reason why the appellant should not pay the respondents’ costs of the appeal and cross-appeal. I will hear from the parties in relation to costs when the proceeding is next before the Court.
Orders accordingly.
I certify that the preceding four hundred and thirty-six (436) numbered paragraphs are a true copy of the Reasons for Judgment of the Honourable Justice Nicholas. Associate:
Dated: 14 July 2023
Annexure A
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