LanzaTech New Zealand Limited

Patent Trials and Appeals Board

Dec 9, 2021

2021001216 (P.T.A.B. Dec. 9, 2021)

UNITED STATES PATENT AND TRADEMARK OFFICE
UNITED STATES DEPARTMENT OF COMMERCE
United States Patent and Trademark Office
Address: COMMISSIONER FOR PATENTS
P.O. Box 1450
Alexandria, Virginia 22313-1450
www.uspto.gov
APPLICATION NO. FILING DATE FIRST NAMED INVENTOR ATTORNEY DOCKET NO. CONFIRMATION NO.
15/442,695 02/26/2017 Shilpa Nagaraju LT122US1 3968
19135 7590 12/09/2021
LanzaTech NZ, Inc.
8045 Lamon Ave, Suite 400
Skokie, IL 60077
EXAMINER
BROWN, MINDY G
ART UNIT PAPER NUMBER
1636
NOTIFICATION DATE DELIVERY MODE
12/09/2021 ELECTRONIC
Please find below and/or attached an Office communication concerning this application or proceeding.
The time period for reply, if any, is set in the attached communication.
Notice of the Office communication was sent electronically on above-indicated "Notification Date" to the
following e-mail address(es):
ip@lanzatech.com
PTOL-90A (Rev. 04/07)
UNITED STATES PATENT AND TRADEMARK OFFICE
__________

BEFORE THE PATENT TRIAL AND APPEAL BOARD
__________

Ex parte SHILPA NAGARAJU and MICHAEL KOEPKE
__________

Appeal 2021-001216
Application 15/442,695
Technology Center 1600
__________

Before ERIC B. GRIMES, JEFFREY N. FREDMAN, and
RACHEL H. TOWNSEND, Administrative Patent Judges.

FREDMAN, Administrative Patent Judge.

DECISION ON APPEAL
This is an appeal1 under 35 U.S.C. § 134 involving claims to a method
of genetically engineering a C1-fixing bacterium using the CRISPR/Cas
system. The Examiner rejected the claims as obvious. We have jurisdiction
under 35 U.S.C. § 6(b). We affirm.

1 We use the word “Appellant” to refer to “applicant” as defined in 37
C.F.R. § 1.42. Appellant identifies the Real Party in Interest as Lanza Tech
New Zealand Limited (see Appeal Br. 2). We have considered the
Specification of Feb. 26, 2017 (“Spec.”); Final Office Action of Oct. 3, 2019
(“Final Action”); Appeal Brief of June 3, 2020 (“Appeal Br.”); and
Examiner’s Answer of Sept. 21, 2020 (“Ans.”).
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Statement of the Case
Background
“The CRISPR/Cas system has a wide variety of applications, e.g.,
deleting, inserting, translocating, inactivating, or activating DNA” (Spec.
¶ 6). “[T]he CRISPR/Cas system may be used to express an exogenous
gene” (id. ¶ 8). “A ‘C1-fixing microorganism’ is a microorganism that has
the ability to produce one or more products from a C1-carbon source.
Typically, the microorganism of the invention is a C1-fixing bacterium” (id.
¶ 48). “[A]ttempts to transform the C1-fixing bacterium C.
autoethanogenum with a plasmid carrying cas9 under the control of a native
constitutively-expressed phosphotransacetylase-acetate kinase (Ppta-ack)
promoter were not successful” (id. ¶ 16).
The Specification teaches the “CRISPR/Cas system of the invention
utilizes an inducible promoter, instead of a constitutive promoter, which
renders it suitable for use in C1-fixing bacteria” (Spec. ¶ 16).
The Claims
Claims 1–18 are on appeal. Claim 1 is an independent claim, is
representative and reads as follows:
1. A method of genetically engineering a C1-fixing
bacterium comprising introducing into a C1-fixing bacterium
containing a DNA molecule comprising a target sequence an
engineered, non-naturally occurring Clustered Regularly
Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-
associated (Cas) (CRISPR/Cas) system comprising one or more
vectors comprising:
(a) a nucleotide sequence encoding a guide RNA that
hybridizes with the target sequence and
(b) a nucleotide sequence encoding a type-II Cas9 protein
under the control of an inducible promoter;
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wherein the Cas9 interacts with the DNA molecule;
wherein expression of a gene product or a nucleotide
sequence of the DNA molecule is altered.

The Rejection
The Examiner rejected claims 1–18 under 35 U.S.C. § 103 as obvious
over Xu,2 Green Biologics,3 Basu,4 Doudna,5 Frisch,6 and Sander7 (Final
Act. 2–4).
The Examiner finds it obvious
to have used the well known and well studied, Cas9 CRISPR
system, with Cas9 enzyme, as disclosed by Xu et al., in the
related C. autothanogenum [sic] or C. ljungdahlii, since one
would have expected the Cas9 system, which has been used in a
wide variety of organisms, would function in the useful bacteria
C. autoethanogenum and C. ljundahlii, as taught by Basu.
(Final Act. 3–4). The Examiner further finds incorporation of an inducible
promoter obvious because “inducible promoters allow the control of the
expression of a gene of interest, including Cas9” (id. at 4). The Examiner
finds “a reasonable expectation of success to result in the claimed invention”
(id.).

2 Xu et al., Efficient Genome Editing in Clostridium cellulolyticum via
CRISPR-Cas9 Nickase, 81 Applied Environmental Microbiology 4423–31
(2015).
3 Green Biologics, International Application PCT/GB2015/051153,
published as WO 2015/159087 A1 on Oct. 22, 2015. We will rely on the
published version.
4 Basu et al., WO 2015/054507 A1, published Apr. 16, 2015.
5 Doudna et al., US 2016/0289659 A1, published Oct. 6, 2016.
6 Frisch et al., US 2017/0369866 A1, published Dec. 28, 2017.
7 Sander et al., CRISPR-Cas systems for genome editing, regulation and
Targeting, 32 Nature Biotechnology 347–55 (2014).
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The issue with respect to this rejection is: Does a preponderance of the
evidence of record support the Examiner’s conclusion that the prior suggests
the method of claim 1?
Findings of Fact
1. The Specification teaches in “a preferred embodiment, the C1-
fixing bacterium is Clostridium autoethanogenum, Clostridium ljungdahlii,
or Clostridium ragsdalei” (Spec. ¶ 11). The Specification further teaches
“one or more elements of a CRISPR system is derived from a particular
organism comprising an endogenous CRISPR system, such as Streptococcus
pyogenes” (id. ¶ 36).
2. Xu teaches “the use of the Streptococcus pyogenes CRISPR-
Cas9 system in editing the genome of Clostridium cellulolyticum, a model
microorganism for bioenergy research” (Xu 4423, abstract).
3. Xu teaches “[t]o demonstrate genome editing by gRNA-guided
Cas9, a pyrF gene encoding orotidine-5’-phosphate decarboxylase
(Ccel_0614) in C. cellulolyticum was chosen as our first target gene since
inactivation of this gene would generate uracil auxotrophic and 5-
fluoroorotic acid (5-FOA)-resistant phenotypes, which are easily monitored”
(Xu 4426, col. 1).
4. Xu teaches “a highly efficient strategy for genome editing in C.
cellulolyticum using Cas9n-mediated single-nick generation and HR. This
SNHR strategy is capable of circumventing the DSB[8] lethality to allow
versatile editing in hosts with inefficient DSB repair systems” (Xu 4429, col.
1).

8 DSB stands for double-strand break.
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5. Xu teaches “the SNHR strategy has a very wide targeting space
with a median interval distance of 6 to 14 bp in the multiple bacterial
genomes analyzed in this study. It also allows editing of over 95% of genes
in multiple genomes, demonstrating the great versatility of this editing
system” (Xu 4430, col. 1).
6. The Examiner acknowledges that Xu does not teach “a C-1
fixing bacteria, such as C. autothanogenum [sic] or Clostridium ljungdahlii”
and a Cas9 under control of “an inducible promoter” (Final Act. 3).
7. Green Biologics teaches “a process for producing a mutation in
an Intended Mutagenesis Region (IMR) within a bacterial genome, wherein
the bacteria comprise a CRISPR/Cas system” (Green Biologics 6:22–24).
8. Green Biologics teaches in “preferred embodiments, the
bacteria are of the genus Clostridium. Preferred Clostridium species include
. . . C. ljungdahlii, C. autoethanogenum” (Green Biologics 9:22–27).
9. Basu teaches
“CRISPR” or the “CRISPR system” refers collectively to
nucleic acids, proteins and other elements involved in the
expression of or directing the activity of Clustered Regularly
Interspaced Short Palindromic Repeats (“CRISPR”) and
CRISPR-associated (“Cas”) genes, including sequences
encoding a Cas gene, a tracr (trans-activating CRISPR)
sequence (e.g. tracrRNA or an active partial tracrRNA), a tracr-
mate sequence (encompassing a “direct repeat” and a
tracrRNA-processed partial direct repeat in the context of an
endogenous CRISPR system), a guide sequence (also referred
to as a “spacer” in the context of an endogenous CRISPR
system), or other sequences and transcripts from a CRISPR
locus.
(Basu ¶ 158).
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10. Basu teaches type II CRISPR where “the CRISPR/Cas-like
protein of the fusion protein is derived from a Cas9 protein” where “suitable
organisms include . . . Clostridium ljungdahlii” (Basu ¶¶ 160, 165).
11. Doudna teaches in “Type II CRISPR-Cas systems, Cas9
functions as an RNA-guided endonuclease that uses a dual-guide RNA
consisting of crRNA and trans-activating crRNA (tracrRNA) for target
recognition and cleavage” (Doudna ¶ 3).
12. Doudna teaches “[i]n some embodiments, a nucleotide
sequence encoding a guide nucleic acid and/or a Cas9 polypeptide is
operably linked to an inducible promoter. In some embodiments, a
nucleotide sequence encoding a guide nucleic acid and/or a Cas9
polypeptide is operably linked to a constitutive promoter” (Doudna ¶ 340).
13. Doudna teaches “[e]xamples of inducible promoters include,
but are not limited toT7 RNA polymerase promoter, T3 RNA polymerase
promoter, Isopropyl-beta-D-thiogalactopyranoside (IPTG)-regulated
promoter, lactose induced promoter, heat shock promoter, Tetracycline-
regulated promoter, Steroid-regulated promoter, Metal-regulated promoter,
estrogen receptor-regulated promoter, etc.” (Doudna ¶ 343).
14. Doudna teaches:
In some embodiments, a subject guide nucleic acid and a
subject Cas9 polypeptide are used as an inducible system for
shutting off gene expression in cells. For example, in some
cases, nucleic acids encoding an appropriate guide nucleic acid
and/or an appropriate Cas9 polypeptide and/or a PAMmer can
be incorporated into the chromosome of a target cell and are
under control of an inducible promoter. When the guide
nucleic acid and/or the PAMmer and/or the site-directed
polypeptide are induced, the target nucleic acid is cleaved (or
otherwise modified) at the location of interest, when the
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PAMmer, the guide nucleic acid and the Cas9 polypeptide are
present and bind the single stranded target nucleic acid.
(Doudna ¶ 457).
15. Frisch teaches “a DNA sequence encoding a guide RNA and a
circular polynucleotide modification template to an E.coli cell comprising a
Cas9 endonuclease DNA sequence operably linked to an inducible
promoter” (Frisch ¶ 9).
16. Sander teaches CRISPR technology is a “new approach for
generating RNA-guided nucleases, such as Cas9, with customizable
specificities. Genome editing mediated by these nucleases has been used to
rapidly, easily and efficiently modify endogenous genes in a wide variety of
biomedically important cell types and in organisms that have traditionally
been challenging to manipulate genetically” (Sanders 347, abstract).
17. Sander teaches the
type II CRISPR system from S. pyogenes has been adapted for
inducing sequence-specific DSBs and targeted genome editing.
In the simplest and most widely used form of this system, two
components must be introduced into and/or expressed in cells
or an organism to perform genome editing: the Cas9 nuclease
and a guide RNA.
(Sander 348, col. 2 (footnote omitted)).
18. Sander teaches “a flurry of papers published in 2013 showed
that this platform also functions efficiently in a variety of cells and
organisms. Initial proof-of-principle studies showed that Cas9 could be
targeted to endogenous genes in bacteria, cultured transformed human
cancer cell lines and human pluripotent stem cells in culture” (Sander 348,
col. 2 (footnote omitted)).
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Table I recites a variety of different organisms which have been successfully
modified by Cas9.
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Principles of Law
“The combination of familiar elements according to known methods
is likely to be obvious when it does no more than yield predictable results.”
KSR Int’l Co. v. Teleflex Inc., 550 U.S. 398, 416 (2007).
“Obviousness does not require absolute predictability of success . . .
all that is required is a reasonable expectation of success.” In re Kubin, 561
F.3d 1351, 1360 (Fed. Cir. 2009) (citation and emphasis omitted).
Analysis
We adopt the Examiner’s findings of fact and conclusion of law (see
Final Act. 2–4; FF 1–19) and agree that the combination of prior art renders
the claims obvious. We address Appellant’s arguments below.
Appellant contends
the teachings of Xu with respect to C. cellulolyticum cannot be
reasonably extended to C1-fixing bacteria such as C.
autoethanogenum and C. ljungdahlii since CRISPR/Cas9
systems function very differently across these species. In fact,
the entire purpose of Xu is to describe a strategy for
overcoming unexpected effects of using standard CRISPR/Cas9
systems in a particular clostridial species.
(Appeal Br. 6).
We find this argument unpersuasive because the Examiner does not
solely rely upon Xu alone. Doudna, and Frisch, who teach genetic
engineering of clostridium species using a target specific guide RNA
sequence and type II-cas9 that alters DNA (FF 2–5) where the Cas9 is
controlled by an inducible promoter (FF 11–15). Sander demonstrates that
the CRISPR/Cas9 system functions in dozens of different species and that
“proof-of-principle studies showed that Cas9 could be targeted to
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endogenous genes in bacteria, cultured transformed human cancer cell lines
and human pluripotent stem cells in culture” (FF 18–19).
Moreover, the Examiner cites Green Biologics and Basu to evidence
that the CRISPR/Cas9 system was expected to specifically function on
bacteria including C. autoethanogenum and C. ljungdahlii (FF 8, 10) that are
identified as C1-fixing bacteria by the Specification (FF 1). Thus, we
balance evidence presented by the Examiner that CRISPR/Cas9 has both a
general expectation of working in almost any cell type (FF 18–19), specific
evidence that suggests CRISPR/CAS functions in C1-fixing bacteria such as
C. autoethanogenum and C. ljungdahlii (FF 8, 10), with attorney argument.
“[A]ttorney argument [is] not the kind of factual evidence that is required to
rebut a prima facie case of obviousness.” In re Geisler, 116 F.3d 1465, 1470
(Fed. Cir. 1997).
Appellant contends “the use of a constitutive promoter was found to
be toxic to C1-fixing bacteria (paragraph 0016 of the present specification).
Accordingly, the CRISPR/Cas9 system of Xu would be toxic to the C1-
fixing bacteria of the present invention” (Appeal Br. 6).
We find this argument unpersuasive because both Doudna and Frisch
teach that either constitutive or inducible promoters may be selected for
expression (FF 12, 15) and inducible promoters may be selected to control
gene expression in cells (FF 14). While inducible promoters were well
known for use in situations expressing lethal proteins, Doudna’s motivation
to control gene expression also represents a different reason to use inducible
promoters. And “the motivation in the prior art to combine the references
does not have to be identical to that of the applicant to establish
obviousness.” In re Kemps, 97 F.3d 1427, 1430 (Fed. Cir. 1996).
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Moreover, as shown by the parallel reference to inducible and
constitutive promoters in Doudna in a single paragraph (FF 12), the claims
“recite[] a combination of elements that were all known in the prior art, and
all that was required to obtain that combination was to substitute one well-
known . . . agent for another.” Wm. Wrigley Jr. Co. v. Cadbury Adams USA
LLC, 683 F.3d 1356, 1364 (Fed. Cir. 2012). Thus, the prior art provides
persuasive reasons to select known inducible promoters to allow for control
of gene expression.
Appellant contends
even if the CRISPR/Cas9 system of Xu would somehow
function in C1-fixing bacteria despite the toxicity of
constitutive Cas9 expression, Xu fails to provide any suggestion
whatsoever to use the CRISPR/Cas9 system described therein
in any other species, much less a C1-fixing bacterium such as
C. autoethanogenum or C. ljungdahlii.
(Appeal Br. 7). Appellant contends that “Green Biologics and Basu merely
disclose C. autoethanogenum and C. ljungdahlii in laundry lists of
microorganisms and Sander simply highlights the successful use of
CRISPR/Cas9 systems in particular eukaryotic and prokaryotic species”
(id.). Appellant contends “many of the cited references themselves
acknowledge the challenges of adapting CRISPR/Cas9 systems to different
species” (id.).
We find these arguments unpersuasive for a number of reasons. First,
“[n]on-obviousness cannot be established by attacking references
individually where the rejection is based upon the teachings of a
combination of references. . . . [The reference] must be read, not in
isolation, but for what it fairly teaches in combination with the prior art as a
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whole.” In re Merck & Co., 800 F.2d 1091, 1097 (Fed. Cir. 1986). Here,
the argument fails to read the references together, all of which focus on the
CRISPR/Cas9 system, but rather attempts to read them individually without
recognizing that the combination reasonably suggests successfully applying
the CRISPR/Cas9 system to a variety of cells, including mammalian cells,
insect cells, plant cells, bacteria, yeast, and fungi. (FF 2–19).
Second, we find the laundry list argument unpersuasive because
“specific disclosure, even in a list, makes this case different from cases
involving disclosure of a broad genus without reference to the potentially
anticipating species.” Perricone v. Medicis Pharm. Corp., 432 F.3d 1368,
1377 (Fed. Cir. 2005). Simply because the prior art “discloses a multitude
of effective combinations does not render any particular formulation less
obvious.” Merck & Co. v. Biocraft Labs., Inc., 874 F.2d 804, 807 (Fed. Cir.
1989). In Corkill, an obviousness rejection was affirmed in light of prior art
teachings that “hydrated zeolites will work” in detergent formulations, even
though “the inventors selected the zeolites of the claims from among
‘thousands’ of compounds.” In re Corkill, 771 F.2d 1496, 1500 (Fed. Cir.
1985).
As applied here, the specific disclosure in both Basu and Green
Biologic of C. autoethanogenum and C. ljungdahlii (FF 8, 10) as useful in
CRISPR/Cas9 (FF 7, 9) reasonably suggests that these organisms would
function. Appellant provides no persuasive evidence, as opposed to attorney
argument, to rebut factual reference teachings relied on by the Examiner.
See In re Pearson, 494 F.2d 1399, 1405 (CCPA 1974) (“Attorney’s
argument in a brief cannot take the place of evidence.”).
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And to the extent that the Examiner’s position is in part that it would
be obvious to try to modify the CRISPR/Cas9 system to C1 fixing bacteria
like C. autoethanogenum and C. ljungdahlii specifically identified in the
prior art as usable in the system (FF 7–10), the evidence in the prior art that
the system is highly efficient in other clostridium species (FF 4), has great
versatility in multiple genomes (FF 5), and has been shown to function
“efficiently in a variety of cells and organisms” (FF 18) including many
different species (FF 19) supports the Examiner’s reasonable expectation of
success position. And “[o]bviousness does not require absolute
predictability of success . . . all that is required is a reasonable expectation of
success.” In re Kubin, 561 F.3d 1351, 1360 (Fed. Cir. 2009) (citation and
emphasis omitted). As noted, Appellant provides no persuasive evidence, as
opposed to attorney argument, to address the Examiner’s fact findings.
Conclusion of Law
A preponderance of the evidence of record supports the Examiner’s
conclusion that the prior suggests the composition of claim 1.
DECISION SUMMARY
In summary:
Claim(s)
Rejected
35
U.S.C. §
Reference(s)/Basis Affirmed Reversed
1–18 103 Xu, Green Biologics,
Basu, Doudna, Frisch,
Sander
1–18
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No time period for taking any subsequent action in connection with
this appeal may be extended under 37 C.F.R. § 1.136(a).

AFFIRMED