EnviroLogix Inc v Ionian Technologies, Inc
[2021] APO 44
•9 November 2021
IP AUSTRALIA
AUSTRALIAN PATENT OFFICE
EnviroLogix Inc v Ionian Technologies, Inc. [2021] APO 44
Patent Application: 2015202439
Title:Nicking and extension amplification reaction for the exponential amplification of nucleic acids
Patent Applicant: Ionian Technologies, Inc.
Opponent:EnviroLogix Inc
Delegate:Felix White
Decision Date: 9 November 2021
Hearing Date: 18 August 2021, by videoconference
Catchwords: PATENTS – Final Determination – claims as amended correspond to principle of general application as recognised in opposition decision – application to proceed to grant.
Representation: Counsel for the applicant: Mr Craig Smith
Patent attorney for the applicant: Dr Adam Denley, FPA Patent Attorneys
Counsel for the opponent: Mr Greg Arthur
Patent attorney for the opponent: Dr Caryn DeHoratius, Southern Cross Intellectual Property
IP AUSTRALIA
AUSTRALIAN PATENT OFFICE
Patent Application: 2015202439
Title:Nicking and extension amplification reaction for the exponential amplification of nucleic acids
Patent Applicant: Ionian Technologies, Inc.
Date of Decision: 9 November 2021
DECISION
The grounds raised in the opposition decision have been overcome, and no new issues have been raised by amendment. I direct that the application proceed to grant.
REASONS FOR DECISION
Australian patent application 2015202439 (“the application”) in the name of Ionian Technologies, Inc. (“the Applicant”) was opposed under s59 of the Patents Act 1990 (“the Act”) by EnviroLogix Inc. (“the Opponent”). A decision was issued on 17 April 2020[1] which found the opposition to be successful on the grounds of lack of sufficiency and lack of support, but which gave the Applicant an opportunity to file amendments to address these grounds.[2]
[1] EnviroLogix Inc v Ionian Technologies, Inc [2020] APO 18, “the Decision”
[2] Decision at [171]
The Applicant filed voluntary amendments under s104 on 17 June 2020. These amendments were allowed and were advertised in the Official Journal on 24 December 2020. These amendments were not opposed. The amended specification published as AU2015202439 C1 is the subject of this decision. The amended claims (showing markups for convenience) are attached as an annex to this decision.
It is well established that a decision of a delegate in opposition proceedings is final and determines all the issues arising from the notice of opposition so far as they are capable of determination at the time.[3] The Deputy Commissioner in Novozymes A/S v DSM IP Assets B.V.[4] considered the law on final determinations, and concluded that: “... I cannot revisit any findings that were part of the original decision, but issues that were not considered in the original decision can be decided (for the first time) in a final determination. However, the decision in the final determination should be consistent with any relevant findings in the original decision.”
[3] R v Smith; Ex parte Mole Engineering Pty Ltd [1981] HCA 25; (1981) 147 CLR 340
[4] [2018] APO 2
The questions for final determination are does the amended specification overcome the grounds identified in the Decision, and does the amended specification introduce any new issues.
In this regard, the Opponent requested to be heard on 23 December 2020 and the Applicant also requested to be heard on 4 January 2021. An oral hearing took place by videoconference on 18 August 2021.
It is convenient at this point to provide a brief summary of the issues addressed in the Decision.
The application is directed to an improved “nicking strand displacement amplification” method. Nicking SDA[5] was a known technology but the application demonstrated that this could be performed without an initial thermal denaturation step, which was thought to be required to enable the SDA templates to bind to the target and allow the reaction to commence. The application at p. 37 lines 22-25 stated that this could occur by the templates interacting with their target during the normal breathing of double-stranded DNA, or that the target may also be generated by single or double nick sites in the target genome.
[5] Summarised in the Decision at 23-24
In terms of the disclosure of the specification, in the Decision I found that
The description prima facie appears to provide a principle of general application that short templates can initiate nicking SDA on a nondenatured target, with an appropriate choice of template length, target size and reaction conditions providing some “wriggle room” around those exemplified in the thirty examples, i.e. with a single nickase and single polymerase.[6]
[6] Decision at 151
I further found that the specification provided significant guidance with regard to the optimisation of fifteen empirical factors for amplification in the presence of one nickase and polymerase.[7]
[7] Decision at 149
10.However I found that the specification did not provide any guidance with regards to the optimisation required to perform the claimed methods when additional reagents such as bumper primers, additional nickases or additives such as Tm modifying reagents or strand-opening enzymes are utilised,[8] and that performing the invention over the full scope of the claims when such additional reagents are present would require a “research project”[9] in contravention of the s40 requirements as set out in Evolva.[10]
[8] Decision at 154-155
[9] Decision at 160
[10] Evolva SA [2017] APO 57 (14 November 2017)
11.The claims as accepted were directed to methods of nicking SDA characterised by lacking an initial denaturation step, with differences between various independent claims relating to the nature of the target and the way the reaction products were detected.[11] In the Decision I found that the independent claims were
[11] Decision at 36
open with respect to presence of additional reagents;
not particularly limited by template lengths or reaction temperature; and
“capable of” performing an amplification, i.e. must be capable of working.[12]
[12] Decision at 159
12.I then found that
… there is a principle disclosed that can be applied to the situation where the templates access the target through natural breathing, but given that the claims are broader than this, I do not accept that it does in fact have “general application” over the entire scope of the claims, which permit the presence of additional reagents affecting primer access or DNA duplex stability.[13]
[13] Decision at 159
13.To sum up, in the Decision I found that there was an area of claim scope that was enabled and which satisfied the sufficiency and support requirements, but the opposed claims exceeded it. The question then is, do the claims now correspond to (or fall within) the enabled scope.
14.It is fair to say from the Decision that the enabled scope included optimisation of parameters to allow initiation of amplification without denaturation, when initiation occurs by breathing.
15.The independent claims have been relevantly amended as follows (the amended claims can be found in an annex to this decision, and the relevant extract of claim 1 is shown; the other independent claims have been amended in a corresponding fashion or deleted).
subjecting the reaction mixture formed by the step of combining to essentially isothermal conditions to allow the template recognition regions of the templates to interact with the corresponding target nucleotide sequences during breathing of the double stranded target nucleic acid and to amplify the target nucleotide sequence
16.All claims now specify that the templates are allowed access to the target by breathing. As the second issue relating to stabilising regions has also been resolved (see paragraph 32 below), prima facie the claims correspond to the enabled area.
17.The Opponent’s written submissions disputed that “natural breathing” was a principle of general application within the meaning of Biogen.[14] The Opponent’s position that the mere mention of a principle in a claim does not render the claim automatically sufficient[15] is sound. However as set out above the principle of general application recognised in the Decision and demonstrated by the specification was that short templates can initiate nicking SDA on a nondenatured target via access through natural breathing.
[14] Biogen Inc v Medeva plc [1997] RPC 1
[15] OS at 45
18.At the hearing the Opponent further disputed that the scope of the claims as amended corresponded to that principle by using a three layered argument as follows:
1) The method as amended does not require breathing to occur at all;
2) Even if breathing is required, other reagents and mechanisms are not excluded;
3) And finally even if breathing is the only mechanism, this raises new issues in terms of how to carry out the method using breathing given that nothing in the specification directly demonstrates breathing.
I will deal with each of these in turn.
The requirement for breathing to occur:
19.The Opponent submitted that the term “allows” in the amended claim language merely requires that if breathing is taking place the templates can interact with the target, and there is no requirement for breathing to occur at all.[16]
[16] OS at 16, elaborated on during the oral submissions
20.At this point I should say a little about the understanding in the art (and the disclosure in the specification) regarding breathing. Dr Todd understood that breathing is the transient separation of short regions of double stranded nucleic acids below the melting temperature.[17] The specification only explicitly mentions breathing at two points, but does refer to transient interactions between the template and the target in its discussion of the reaction mechanism at p. 37 line 14.[18] Dr Edwards disputed that these “transient interactions” are due to breathing[19] but did not provide any further interpretation as to what that passage referred to. Dr Edwards’ other evidence regarding breathing was that he was not aware that primers could access DNA during natural breathing[20] (which comes across as evidence of the inventiveness of the present invention); that the claimed method “may partially take advantage” of breathing but breathing is not required.[21] Dr Edwards went on to state that
[17] Todd at 78
[18] Referred to by Dr Todd at 81
[19] Edwards #2 at 47
[20] Edwards #2 at 45
[21] Edwards #2 at 52
“If the authors of the Opposed Application wanted to show the importance of the mechanism of normal breathing of DNA, I would expect them to have performed the method without a nicking enzyme, or using some other means of making single stranded DNA available.”
However later in his declaration Dr Edwards did acknowledge that it is only at the initial step that breathing is required,[22] which is consistent with my understanding of the invention.
[22] Edwards #2 at 76
21.Given that DNA breathing is a transient and random process, it does not make sense to say that breathing “does” or “does not” occur. I can appreciate that the Opponent may wish to challenge the construction of the term “allows” in this context. However I believe the only sensible construction of “allows” is that when breathing occurs at the target site (which it will do from time to time, at a rate depending on factors such as the temperature and the GC content) the templates must be able to access it in order for the reaction to commence.[23]
[23] Cf the Decision at 42 “if there is target nucleotide sequence present, the reagents and reaction conditions must be such as to be able to amplify it”.
The potential for other mechanisms and reagents:
22.The Opponent further submitted that there is no requirement for breathing to be the only mechanism employed.[24] Again this is a reasonable challenge of construction for the Opponent to make: does the claim exclude access of the templates to the target via techniques that owe nothing to the invention disclosed? Certainly if additional reagents still fell within the scope of the claims then the problem identified at para 159 of the Decision would not be resolved.
[24] OS at 16, elaborated on during the oral submission
23.Firstly, in view of the disclosure of the specification at p. 37 lines 22-25 mentioned above, it seems reasonable to conclude that the drafter’s intention was to specify that the templates access the target during normal breathing and not as a result of nicking. However this does not necessarily mean that the claims need be absolutely closed with respect to additional reagents. The Applicant’s submissions in response to this point[25] appear reasonable: if the claim was required to exclude any and all additional reagents then the claim scope could be avoided by the addition of even small amounts of additional reagents. On the other hand if strand opening additives were present in effective amounts such that the templates did not access the target via natural breathing the claims would not be infringed, which is appropriate since that situation would no longer owe anything to the invention disclosed.
[25] AS at 25-27
24.Although there is no further evidence as to this point I am satisfied that the skilled worker would be able to distinguish between these two situations: the former falls within the scope of the claims (and would not seem to affect the amount of experimentation required to optimise the invention) whereas the latter does not.
The requirement for breathing to be demonstrated:
25.The Opponent submitted that the specification does not provide any examples of carrying out the invention using breathing without nicking, and as such breathing cannot be a principle of general application,[26] and furthermore the Opponent asserts that the feature of breathing is not supported for this reason.
[26] OS at 50-51, 53 and 56-58, with reference to Edwards #2 at 45, 52 and 88
26.Although it is true that the specification does not have any examples of carrying out the invention without the presence of nicking enzymes, this is only because the nicking SDA reaction requires nicking of the amplified target (at the introduced nicking site) for the reaction to proceed. The access via breathing is only required for the reaction to commence. There is no suggestion that the claimed method could amplify a target through breathing alone.
27.On the other hand, the fact that the reaction was demonstrated in the Examples to proceed successfully on various targets while using the same nicking enzyme satisfies me on balance that the templates were able to access the target through breathing rather than accidental nicking. This is consistent with my finding in the Decision that initiation of the reaction via natural breathing was a principle of general application.
Other new issues related to breathing:
28.The Opponent made some further submissions that breathing was not disclosed in isothermal conditions or in relation to dsRNA.[27] Neither of these are strictly “new issues” in that they were already present (at least implicitly with respect to dsRNA) in the claims as accepted and were not criticised in the Decision, so it is not open to me to find any new deficiencies. However for completeness I will address them as follows.
[27] OS at 54-55, 59-60
29.The presence of the term “isothermal” appears to me to merely confirm the absence of a thermal denaturation step. Nicking SDA conventionally takes place in isothermal conditions. The isothermal conditions merely need to be suitable for breathing to occur i.e. not too cold.
30.While dsRNA breathing is not discussed in the specification, breathing is a property of all double stranded nucleic acids. No evidence has been presented that dsRNA would not breathe or that the skilled worker could not adjust the conditions appropriately to enable the invention to work with a dsRNA target if required.
Stabilising Region
31.In the Decision I found that there was no disclosure of how to perform the claimed methods without the presence of a stabilising region on both templates, and that claims lacking the feature of a stabilising region were insufficiently disclosed and not supported.[28] The amended claims now recite that the templates have a stabilising region.
[28] Decision at 164 (and 168)
32.I note that the Opponent challenged that the structural features of the stabilising regions were still unspecified.[29] However in the Decision I found that the skilled worker would understand the characteristics of a stabilising region to make the invention work,[30] so no further structural definition of the stabilising regions is necessary. I consider this ground has been overcome.
[29] OS at 36-39
[30] Decision at 64
Conclusion
33.None of the points raised by the Opponent have convinced me, on the balance of probabilities, that the claims as amended do not correspond to the scope found to be enabled. Therefore I am of the opinion that the claims as amended comply with s40(2)(a) and s40(3).
Residual Issues: Novelty and Inventive Step in view of D6 and D10
34.The Decision also noted that two documents not pressed in the Opposition were potentially relevant to novelty and inventive step of the claims as accepted[31] and noted that should subject matter relating to amplification of single stranded targets or non-thermal denaturation remain after amendment then the Commissioner may consider whether to initiate re-examination.[32]
[31] Decision at 135
[32] Decision at 141 and 171
35.The claims have been amended to specify that the target must be double stranded. Furthermore, the requirement in the claims that the templates are allowed to interact with the target during DNA breathing also seems to exclude non-thermal denaturation, because if DNA is denatured then breathing cannot occur.
36.Therefore I will not be referring the application for consideration of whether to initiate re-examination on these grounds prior to grant.
Costs
37.It is normal that costs follow the event. I therefore award costs according to Schedule 8 against the Opponent.
Felix White
Delegate of the Commissioner of Patents
Annex: Changes made to the claims as amended.
Due to deletion of some dependent claims, numbering of the remaining dependent claims has been modified accordingly (not shown)
1. A method for nucleotide sequence amplification, which comprises:
obtaining, from an animal, plant, or food, a sample comprising a double-stranded target nucleic acid, the target nucleic acid comprising a target nucleotide sequence,
without first subjecting the target nucleic acid molecule to a denaturation step associated with amplification of the target nucleotide sequence, combining, in a single step, the obtained sample directly with an amplification reagent mixture to form a reaction mixture or diluting the obtained sample and combining, in a single step, the diluted sample with an amplification reagent mixture to form a reaction mixture, in either case, the amplification reagent mixture comprising:
(i) a polymerase,
(ii) a first template nucleic acid that hybridizes to a first strand of the target nucleotide sequence, wherein the first template comprises a nucleic acid sequence comprising a first template recognition region at the 3' end that is complementary to the 3' end of the first strand of the target nucleotide sequence, a nicking enzyme binding site and a nicking site upstream of said recognition region, and a stabilizing region upstream of said nicking site; and
(iii) a second template nucleic acid that hybridizes to the complement of the first strand of the target nucleotide sequence, wherein the second template comprises a nucleotide sequence comprising a second template recognition region at the 3' end that is complementary to the 3' end of the complement of the first strand of the target nucleotide sequence, a nicking enzyme binding site and a nicking site upstream of said recognition region, and a stabilizing region upstream of said nicking site, and
(iv) one or more nicking enzymes;
subjecting the reaction mixture formed by the step of combining to essentially isothermal conditions to allow the template recognition regions of the templates to interact with the corresponding target nucleotide sequences during breathing of the double stranded target nucleic acid and to amplify the target nucleotide sequence; and
detecting amplified target nucleotide sequence in real time within 10 minutes of subjecting the reaction mixture formed by the step of combining to essentially isothermal conditions.
(Claims 5, 7, 10-11, 33-34 deleted)
86. The method of any one of claims 1 to 45, wherein the target nucleic acid is
double stranded RNA.
3529. A method for amplifying a double-stranded nucleic acid target sequence without a heat denaturation step, comprising
a) directly adding an optionally diluted sample from an animal, plant, or food, said sample comprising a target DNA molecule comprising a double-stranded target sequence, having a sense strand and an antisense strand, with an amplification reagent mixture comprising a forward template and a reverse template, a first nicking enzyme, a second nicking enzyme and a polymerase to form a reaction mixture, wherein:
i) said forward template comprises a nucleic acid sequence comprising a recognition region at the 3' end that is complementary to the 3' end of the target sequence antisense strand; a nicking enzyme binding site and a nicking site upstream of said recognition region, and a stabilizing region upstream of said nicking site;
ii) said reverse template comprises a nucleotide sequence comprising recognition region at the 3' end that is complementary to the 3 ' end of the target sequence sense strand, a nicking enzyme binding site and a nicking site upstream of said recognition region, and a stabilizing region upstream of said nicking site; and
wherein said first nicking enzyme nicks upstream, downstream, or at the nicking site of said forward template, and does not nick within said target sequence;
and said second nicking enzyme nicks upstream, downstream, or at the nicking site of said reverse template and does not nick within said target sequence; and
b) subjecting the reaction mixture to essentially isothermal conditions to allow the recognition regions of the templates to interact with the corresponding target sequences during breathing of the target DNA molecule and to amplify the double-stranded nucleic acid target sequence;
wherein amplification is performed by multiple cycles of said polymerase extending said forward and reverse templates along said target sequence producing a double-stranded nicking site, and said nicking enzymes nicking at said nicking sites, or amplified copies of said sites, producing an amplification product.
(Claims 36, 39 deleted)
4335. A method for amplifying a double-stranded nucleic acid target sequence without a heat denaturation step, comprising
a) directly contacting an optionally diluted sample from an animal, plant, or food, said sample comprising a target DNA molecule comprising a double-stranded target sequence, having a sense strand and an antisense strand, with an amplification reagent mixture comprising a forward template and a reverse template, a first nicking enzyme, a second nicking enzyme and a polymerase to form a reaction mixture, wherein:
i) said forward template comprises a nucleic acid sequence comprising a recognition region at the 3' end that is complementary to the 3' end of the target sequence antisense strand; a nicking enzyme binding site and a nicking site upstream of said recognition region; and a stabilizing region upstream of said nicking site;
ii) said reverse template comprises a nucleotide sequence comprising a recognition region at the 3' end that is complementary to the 3' end of the target sequence sense strand; a nicking enzyme binding site and a nicking site upstream of said recognition region; and a stabilizing region upstream of said nicking site; and
wherein said first nicking enzyme nicks at the nicking site of said forward template, and does not nick within said target sequence; and
said second nicking enzyme nicks at the nicking site of said reverse template and does not nick within said target sequence; and
b) subjecting the reaction mixture to essentially isothermal conditions to allow the recognition regions of the templates to interact with the corresponding target sequences during breathing of the target DNA molecule and to amplify the double-stranded nucleic acid target sequence, wherein amplification is performed by multiple cycles of said polymerase extending said forward and reverse templates along said target sequence producing a double-stranded nicking site, and said nicking enzymes nicking at said nicking sites, or amplified copies of said sites, producing an amplification product,
wherein at least a 1 X 107 fold amplification of a 22-35 nucleotide long target sequence is obtained when the amplification reaction is run for twelve minutes.
4840. The method of claim 4739, wherein the animal pathogen is a single-stranded
DNA virus,double-stranded DNA virus, or single-stranded RNA virus.
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