SGI-DNA, INC.

Patent Trials and Appeals Board

Oct 30, 2020

2020001253 (P.T.A.B. Oct. 30, 2020)

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.
15/051,463 02/23/2016 Vladimir N. Noskov CODEX1270-3 5766
95432 7590 10/30/2020
Synthetic Genomics, Inc.
c/o DLA Piper LLP (US)
4365 Executive Drive
Suite 1100
San Diego, CA 92121-2133
EXAMINER
HILL, KEVIN KAI
ART UNIT PAPER NUMBER
1633
NOTIFICATION DATE DELIVERY MODE
10/30/2020 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):
gtdocket@dlapiper.com
PTOL-90A (Rev. 04/07)

UNITED STATES PATENT AND TRADEMARK OFFICE
____________

BEFORE THE PATENT TRIAL AND APPEAL BOARD
____________

Ex parte VLADIMIR N. NOSKOV and RAY-YUAN CHUANG
____________

Appeal 2020-001253
Application 15/051,463
Technology Center 1600
____________

Before DONALD E. ADAMS, ERIC B. GRIMES, and
ULRIKE W. JENKS, Administrative Patent Judges.

ADAMS, Administrative Patent Judge.

DECISION ON APPEAL
Pursuant to 35 U.S.C. § 134(a), Appellant1 appeals from Examiner’s
decision to reject claims 1–6 and 10–21 (Appeal Br. 4). We have
jurisdiction under 35 U.S.C. § 6(b).
We REVERSE.

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 “Synthetic
Genomics, Inc.” (Appellant’s July 25, 2019 Appeal Brief (Appeal Br.) 4).
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STATEMENT OF THE CASE
Appellant’s disclosure relates to “methods, nucleic acids, and systems
for transfer (cloning) of donor nucleic acids into host cells, for manipulation
(e.g., modification) of donor nucleic acids, e.g., within host cells, and for
transplantation of modified donor nucleic acids into recipient cells” (Spec.
¶ 9). Appellant’s claim 1 is representative and reproduced below:
1. A method for seamlessly introducing a modification in a
target nucleic acid molecule present in a host cell, comprising:
a. introducing a mutagenesis construct and a host vector
into the host cell whereby the host vector recombines with the
mutagenesis construct in the host cell,
wherein the mutagenesis construct contains
a first portion of homology that is homologous to a
sequence of the target nucleic acid molecule upstream or
downstream of a target gene to be modified in the target
region on the target nucleic acid molecule, wherein upon
recombination the first portion of homology generates a
tandem repeat with the homologous sequence of the
target nucleic acid molecule;
a second portion of homology that is homologous
to a 5’ portion of the target region on the target nucleic
acid molecule upstream of the modification;
a gene encoding an endonuclease having an
inducible promoter;
an endonuclease recognition site for the encoded
endonuclease; and
a selectable marker;
a third portion of homology that is homologous to
a 3’ portion of the target region on the target nucleic acid
molecule downstream of the modification;
wherein the second and/or third portions of
homology comprise one or more nucleotide
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modifications compared to the homologous portion in the
target nucleic acid;
and
b. incubating the cells under conditions whereby the
mutagenesis construct is integrated into the target nucleic acid
molecule and tandem repeats are generated that flank the target
region on the target nucleic acid molecule between the first
portion of homology and a sequence upstream or downstream
of the target region; and
inducing the inducible promoter, whereby the
endonuclease is expressed and promotes one or more double-
strand break cleavages in the target nucleic acid molecule;
i. whereby recombination occurs between the first
portion of homology and the upstream or downstream
portion of the target region on the target nucleic acid
molecule that is homologous to the first portion of
homology, thereby seamlessly removing a portion of the
construct;
whereby a modification is seamlessly introduced
into the target nucleic acid molecule.
(Appeal Br. 15–16.)

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Claims 1–6 and 10–21 stand rejected under 35 U.S.C. § 103(a) as
unpatentable over the combination of Storici,2 Pόsfai,3 Seiko,4 Gibson,5
Ripoll,6 Golic,7 Akada,8 Hirashima,9 and Scherer.10

ISSUE
Does the preponderance of evidence relied upon by Examiner support
a conclusion of obviousness?

2 Storici et al., US 7,314,712 B2, issued Jan. 1, 2008.
3 Gyӧrgy Pόsfai et al., Markerless gene replacement in Escherichia coli
stimulated by a double-strand break in the chromosome, 27 Nucleic Acids
Research 4409–4415 (1999).
4 Ryu Seiko et al., JP 2007-111015, published May 10, 2007, machine
translated.
5 Daniel G. Gibson et al., Complete Chemical Synthesis, Assembly, and
Cloning of a Mycoplasma genitalium Genome, 319 Science 1215–1220
(2008).
6 Pierre-Jean Ripoll et al., A new yeast artificial chromosome vector
designed for gene transfer into mammalian cells, 210 Gene 163–172 (1998).
7 Golic et al., US 2003/0175968 A1, published Sept. 18, 2003.
8 Rinji Akada et al., PCR-mediated seamless gene deletion and marker
recycling in Saccharomyces cerevisiae, 23 Yeast 399–405 (2006).
9 Kyotaro Hirashima et al., A simple and effective chromosome modification
method for large-scale deletion of genome sequences and identification of
essential genes in fission yeast, 34 Nucleic Acids Research 1–7 (2006),
available at doi:10.1093/nar/gnj011.
10 Stewart Scherer & Ronald W. Davis, Replacement of chromosome
segments with altered DNA sequences constructed in vitro, 76 Proc. Natl.
Acad. Sci. 4951–4655 (1979).
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FACTUAL FINDINGS (FF)
FF 1. Examiner finds that individuals of ordinary skill in this art
will be highly educated individuals such as medical doctors,
scientists, or engineers possessing advanced degrees, including
M.D.’s and Ph.D.’s . . . [who] most likely will be
knowledgeable and well-read in the relevant literature and have
the practical experience in molecular biology, genetics,
methods of gene editing and chromosome manipulation.
Therefore, the level of ordinary skill in this art is high.
(Ans. 9).
FF 2. Storici “relates to methods of in vivo site-directed mutagenesis,
particularly mutagenesis mediated by oligonucleotides” (Storici 1:15–17
(see generally Ans. 3)).
FF 3. Storici discloses a “modification[] of the delitto perfetto
[mutagenesis] system that provides . . . improved recombination efficiency,
in some embodiments over 1000-fold increase in efficiency. These
embodiments, using methods termed generally delitto perfetto-DSB, exploit
the use of induced site-specific DSBs in the genome to increase
oligonucleotide targeted mutagenesis” (Storici 2:59–65; see id. at 5:4–5
(Storici defines the term “DSB” as “a double-strand break”)).

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FF 4. Storici’s Figure 7 is reproduced below:

[Storici’s] FIG. 7 illustrates an embodiment of the delitto
perfetto-DSB system, which utilizes integrative recombinant
oligo-nucleotides (IROs) to generate mutations.
The DSB-CORE cassette[, which] contains the
COunterselectable and REporter genes plus the I-SceI
endonuclease under the control of an inducible promoter, such
as the illustrated GAL 1/10 promoter. Expression of I-SceI
leads to a double-strand break (DSB) at the unique target site,
also contained in the cassette.
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Step 1: A DSB-CORE cassette is inserted by standard
DNA targeting procedures at a desired site. The insertion site is
anywhere in the sequence which has been chosen to be deleted
or is close to a site where a specific mutation, or multiple
mutations, is to be created. The DSB-CORE cassette contains
the following:
i) Gal 1/10 promoter fused to I-SceI open reading frame
(GAL 1/10::I-SceI). The I-SceI creates a DSB at a cut site
included on the cassette. Any inducible (ie., on/off) promoter
can be used and any DSB site-specific cutting enzyme can be
included in the DSB-CORE cassette, provided that the only
DNA site that is cut in the cell is located in the cassette.
ii) I-SceI cut site (or other unique cut site that is the
target of a DSB site-specific cutting enzyme) located at one end
of the cassette or internally.
iii) COunterselectable REporter genes (i.e., the
counterselectable gene K1-URA3+ the kanMX4 reporter gene).
Step 2: Immediately prior to transformation with IRO(s),
cells are grown in the presence of galactose, which induces I-
SceI endonuclease expression. The enzyme targets its single
site in the cassette and generates a DSB.
Step 3: Transformation of cells with IROs leads to loss
of the cassette, creation of the desired mutation(s), or deletion
of the desired region. Oligonucleotide targeting in the vicinity
of the DSB (delitto perfetto-DSB method) is increased up to
1000-fold, compared to oligo-mediated changes without the
DSB (delitto perfetto method).
(Storici 4:65–5:34; see Ans. 3–4.)

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FF 5. Examiner finds that Storici discloses that a “mutagenesis construct
may be used to modify a host vector in the host cell whereby the host vector
recombines with the mutagenesis construct in the host cells, e.g.
modification of yeast artificial chromosomes (YACs)” (Ans. 8 (citing Storici
27:45–29:34)).
FF 6. Examiner finds that Storici fails to disclose a “mutagenesis construct
[that] . . . comprise[s] a portion of homology that is homologous to a
sequence of the target nucleic acid molecule upstream or downstream of a
target region on the target nucleic acid molecule” (Ans. 4).
FF 7. Pόsfai discloses:
A simple and efficient gene replacement method, based on the
recombination and repair activities of the cell, . . . [that] permits
the targeted construction of markerless deletions, insertions and
point mutations in the Escherichia coli chromosome. A suicide
plasmid, carrying the mutant allele and the recognition site of
meganuclease I-SceI, is inserted into the genome by
homologous recombination between the mutant and the wild-
type (wt) alleles. Resolution of this cointegrate by
intramolecular recombination of the allele pair results in either
a mutant or a wt chromosome which can be distinguished by
allele-specific PCR screening. The resolution process is
stimulated by introducing a unique double-strand break (DSB)
into the chromosome at the I-SceI site. Cleavage by the
nuclease not only enhances the frequency of resolution by two
to three orders of magnitude, but also selects for the resolved
products.
(Pόsfai, Abstract.)

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FF 8. Pόsfai’s Figure 1 is reproduced below:

[Pόsfai’s] Figure 1 [illustrates a] [g]eneral scheme of the DSB-
stimulated gene replacement procedure. Cointegrates of the
chromosome and the suicide plasmid can form via homologous
recombination between the mutant and the wt alleles of the
target gene. The plasmid carries a temperature-sensitive (ts)
replicon, an antibiotic (Ab) resistance gene and the recognition
site for I-SceI. Cointegrates are selected by their Ab resistance
at the non-permissive temperature for plasmid replication.
Next, cleavage of the chromosome by I-SceI is induced. The
DSB stimulates recombination between the duplications
resulting either in a reversion to the wt chromosome or in a
markerless gene replacement event.
(Pόsfai 4411; see generally Ans. 4; see also Ans. 7 (Examiner finds that “the
integration of [Pόsfai’s] mutagenesis cassette[, i.e. suicide plasmid,] creates
a tandem duplicated region of the homologous sequence from which the
modification is seamlessly produced”).)
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FF 9. Seiko discloses “a method for deleting a selective marker gene used
in selecting a transformant simultaneously with deleting a DNA fragment of
a host DNA” (Seiko, Abstract; see generally Ans. 5).
FF 10. Seiko’s Figure 1 is reproduced below:

Seiko’s Figure 1 illustrates a process involving two homologous
recombination events. The first homologous recombination results in the
insertion of donor DNA into a host DNA sequence, wherein the donor DNA
comprises from 5’ to 3’: (a) a DNA sequence that is homologous to the host
DNA region that is 3’ of the first homologous recombination insertion site,
(b) a cat gene, and (c) an araR gene (see Seiko ¶¶ 54–57; see generally Ans.
5). The second homologous recombination involves the recombination of
DNA sequence (a) with its homologous 3’ host DNA sequence (see Seiko
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¶ 57; see generally Ans. 5). The second recombination results in the
deletion of the cat gene, araR gene, and a portion of host DNA, which is
illustrated in Figure 1 with hatching (see Seiko ¶ 57; see generally Ans. 5–
6).
FF 11. Examiner finds that Seiko’s “mutagenesis construct . . . comprise[s]
a portion of homology that is homologous to a sequence of [a] target nucleic
acid molecule upstream or downstream of a target region . . . or target gene”
(Ans. 5 (citing Seiko, Figures 1 and 4)).
FF 12. Examiner relies on Gibson to disclose
a method of introducing a modification in a target nucleic acid
molecule present in a host cell, the method comprising the step
of introducing a donor construct, e.g. a second YAC clone, and
a host vector, e.g. a first YAC clone, into the host cell, e.g. a
yeast spheroplast, whereby the host vector recombines with the
mutagenesis construct in the host cell via homologous
recombination.
(Ans. 8 (citing Gibson 1218, Figure 6; Gibson 1218: cols. 1–2).)
FF 13. Examiner finds that Ripoll discloses
a method of introducing a modification in a target nucleic acid
molecule present in a host cell, the method comprising the step
of introducing a donor construct . . . into the host cell . . .
whereby the host vector recombines with the mutagenesis
construct in the host cell via homologous recombination.
(Ans. 8 (citing Ripoll, Abstract, §§ 2.4, 3.3, 3.4, and Figure 4).)
FF 14. Examiner finds that Golic discloses
a method of introducing a modification in a target nucleic acid
molecule present in a host cell, the method comprising a
mutagenesis construct comprising:
a first portion of homology to a 5' portion of the target
nucleic acid molecule upstream of the modification;
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an endonuclease recognition site, e.g. an I-Sce1 or Cre
endonuclease recognition site;
a selectable marker; and
another portion of homology that is homologous to a 3'
portion of the target region on the target nucleic acid molecule
downstream of the modification, the mutagenesis construct
further comprising tandem repeat regions, e.g. LoxP or FRT
sites, that flank the target region or portion thereof on the target
nucleic acid molecule, including the selectable marker.
(Ans. 8–9 (citing Golic ¶ 165 and Figures 1, 3, 21, and 22); see also Ans. 9
(citing Golic Figure 9) (Examiner finds that Golic discloses a “mutagenesis
construct ha[ving] a first portion of homology that is homologous to a
sequence upstream or downstream of a target region to be modified, wherein
upon recombination the first portion of homology generates a tandem repeat
with the homologous sequence of the target nucleic acid molecule.”).)
FF 15. Akada discloses a method of “[s]eamless gene deletion” (Akada
402).

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FF 16. Akada’s Figure 1 is reproduced below:

Akada’s Figure 1 illustrates:
A short sequence (black arrows) copied from the adjacent
region of the targeted locus is inserted into a disruption
fragment containing a counter-selectable URA3 marker and
homologous targeting sequences. PCR 1, chromosomal
sequence amplification. PCR 2, URA3 marker amplification
with sequences for targeting (dotted bars), a repeat sequence
copied from the chromosome (black arrows) and an annealing
sequence for fusion PCR (grey bars with an arrow inside). PCR
3, fusion PCR producing a chromosomal sequence upstream of
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HIS3 attached to the URA3 fragment. Integration of the
construct simultaneously generates gene disruption and direct
repeats on both sides of URA3 in the chromosome, making it
possible to delete URA3 without any extraneous sequences
being left behind in the chromosome.
(Akada 402, Figure 1 legend; see generally Ans. 6.)
FF 17. Hirashima discloses a “chromosome modification method for large-
scale deletion of genome sequences and identification of essential genes in
fission yeast” (Hirashima, Abstract).
FF 18. Hirashima’s Figure 1B is reproduced below:

Hirashima’s Figure 1B is a “[s]chematic representation of the deletion
(recycling) of [a] selectable marker using direct repeats. The gray squares
show the direct repeats. The portions enclosed with a dotted line show the
introduced modification fragments. . . . [T]he ura4 and target genes are
placed between direct repeats on the chromosome,” which facilitate the
recombination event and resulting deletion of nucleic acid between the direct
repeats in the deletant (Hirashima, Figure 1, legend; see Ans. 6 (Examiner
finds that “[h]omologous recombination [according to Hirashima’s method]
occurs between the tandem repeats to remove the undesired construct
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elements while achieving the desired modification in the target gene/target
region”)).
FF 19. Scherer discloses a method “for the stable introduction of foreign
sequences or deletions, constructed in vitro, into the chromosomes of
Saccharomyces cerevisiae” (Scherer, Abstract).
FF 20. Scherer’s Figure 1 is reproduced below:

Scherer’s Figure 1 illustrates the “[r]eplacement of a chromosome segment
with an altered DNA sequence,” wherein
[t]he transforming DNA . . . recombines with the homologous
DNA sequence in the chromosomes and results in a direct,
nontandem duplication separated by the vector and marker
DNAs. A second recombinational event, on the other side of
the sequence alteration relative to the first, generates a
chromosome with the altered sequence replacing those in the
untransformed cell. The vector and marker DNAs are lost.
Homologous sequences on both sides of the alteration must be
present in the transforming DNA but the molecule need not be
capable of autonomous replication in the organism where the
replacement will occur. The classical prokaryotic examples of
this type of manipulation would be transduction of mutant
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galactose gene with phage λ as the vector and immunity as the
selection.
(Scherer, Figure 1, legend.)
FF 21. Examiner finds that Scherer’s
mutagenesis construct has a first portion of homology (a' and/or
c') that is homologous to a sequence (a and/or c) upstream or
downstream of a target gene to be modified, wherein upon
recombination the first portion of homology generates a tandem
repeat with the homologous sequence of the target nucleic acid
molecule. Homologous recombination occurs between the
tandem repeats (c' and c are illustrated) to remove the undesired
elements (loss of marker, a', b regions) while achieving the
desired modification in the target gene/target region (black box,
b'). . . . Thus, the black box (b') illustrated in Figure 1 may
reasonably be interpreted as a “target region” or a “target gene”.
Segments a' and c' are tandem repeats upstream and
downstream, respectively, of the “target region” or “target
gene”.
(Ans. 6–7 (citing Scherer 4951 and Figure 1).)
ANALYSIS
Based on the combination of Storici, Pόsfai, Seiko, Gibson, Ripoll,
Golic, Akada, Hirashima, and Scherer, Examiner concludes that, at the time
Appellant’s invention was made, it would have been prima facie obvious to
modify Storici’s in vivo site-directed mutagenesis to include a tandem repeat
region upstream or downstream of a target gene to be modified (see Ans. 12;
see generally id. at 9–13 and 14–38). We are not persuaded.
On this record, Examiner identified a number of documents, which
disclose various mutagenesis methods (see FF 1–21). Examiner failed,
however, to adequately articulate a reason why a person of ordinary skill in
this art would have, absent hindsight, combined the various mutagenesis
methods in the manner necessary to arrive at Appellant’s claimed invention.
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“[E]xaminer bears the initial burden, on review of the prior art or on any
other ground, of presenting a prima facie case of unpatentability.” In re
Oetiker, 977 F.2d 1443, 1445 (Fed. Cir. 1992).
Obviousness requires more than a mere showing that the
prior art includes separate references covering each separate
limitation in a claim under examination. KSR Int’l Co. v.
Teleflex Inc., 550 U.S. 398, 418, 127 S.Ct. 1727, 167 L.Ed.2d
705 (2007). Rather, obviousness requires the additional
showing that a person of ordinary skill at the time of the
invention would have selected and combined those prior art
elements in the normal course of research and development to
yield the claimed invention. Id. at 421, 127 S.Ct. 1727.
Unigene Laboratories, Inc. v. Apotex, Inc., 655 F.3d 1352, 1360 (Fed. Cir.
2011). In presenting a prima facie case of unpatentability, “[c]are must be
taken to avoid hindsight reconstruction by using ‘the patent in suit as a guide
through the maze of prior art references, combining the right references in
the right way so as to achieve the result of the claims in suit.’” In re NTP,
Inc., 654 F.3d 1279, 1299 (Fed. Cir. 2011) (Fed. Cir. 2011) (citations
omitted). Further, “rejections on obviousness grounds cannot be sustained
by mere conclusory statements; instead, there must be some articulated
reasoning with some rational underpinning to support the legal conclusion of
obviousness.” In re Kahn, 441 F.3d 977, 988 (Fed. Cir. 2006).
As Appellant explains, “Seiko does not appear to teach or suggest
any method utilizing a mutagenesis construct having a gene encoding an
endonuclease and an endonuclease recognition site, nor the use of such
endonuclease to promote a double-stranded break, thereby provoking a
recombination event, as claimed” (Appeal Br. 9). In addition, Appellant
explains that “Storici does not teach or suggest generating a tandem repeat
upon introduction of the construct between a sequence on the target nucleic
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acid and a homologous sequence on the mutagenesis construct” (Appeal Br.
7). In Storici’s method “the purpose of the CORE cassette is to carry the
endonuclease . . . so that it can be induced to perform the double-stranded
break. This increases the successful targeting of the IRO containing the
desired modification by 1000-fold” (id. at 10 (citing Storici 5:29–34)). In
addition, “the CORE cassette is [subsequently] eliminated [in Storici’s
method] by homologous recombination between the IRO cassette and a
sequence on the target nucleic acid” (id. (citing Storici 21:3–16 and Figure
7)). Thus, as Appellant explains, if Examiner’s proposed modification
“were on the CORE cassette . . . it would be lost with the CORE cassette”
and, in the alternative, if the basis of Examiner rejection is to “eliminate
[Seiko’s] IRO or incorporate it on the CORE cassette, then how would the
CORE cassette be removed” (id.).
We acknowledge Examiner’s assertion that Pόsfai
taught the use of the first portion of homology to introduce the
construct comprising the cassette encoding endonuclease target
site . . ., the artisan’s modification of interest, a first portion of
homology upstream of the target region, a second portion of
homology 5’ to the target region, and a third portion of
homology 3’ to the target region[, wherein] [u]pon introduction
of the endonuclease . . . homologous recombination is
stimulated to yield the seamless modification in the target
nucleic acid molecule.
(Ans. 32 (citing Pόsfai, Figure 1); cf. Appeal Br. 7 (Appellant contends that
Pόsfai “does not teach or suggest” “a first portion of homology that is
homologous to a sequence upstream or downstream of the target gene to be
modified,” but instead “replaces the gene with an amended copy and
removes the suicide plasmid through homology with the gene copy itself . . .
, not with a sequence ‘upstream or downstream’ of the target gene”).) We
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find, however, that even if Examiner correctly interpreted Pόsfai as
including a region of homology upstream or downstream of a target gene;
Examiner failed to adequately explain why a person of ordinary skill in this
art would have modified Storici’s method to include features disclosed by
Pόsfai, Seiko, Gibson, Ripoll, Golic, Akada, Hirashima, and Scherer (see
Appeal Br. 10–11 (Appellant contends that “the skilled person finds no
motive to include the sequence modification on the CORE cassette as the
Office proposes. The proposed modification of Storici does not appear to
result in any benefit and would require substantial, inventive changes to the
methods disclosed by both Storici and Pόsfai” and none of Seiko, Gibson,
Ripoll, Golic, Akada, Hirashima, and Scherer make up for this deficiency);
see generally id. at 11).
CONCLUSION
The preponderance of evidence relied upon by Examiner fails to
support a conclusion of obviousness. The rejection of claims 1–6 and 10–21
under 35 U.S.C. § 103(a) as unpatentable over the combination of Storici,
Pόsfai, Seiko, Gibson, Ripoll, Golic, Akada, Hirashima, and Scherer is
reversed.
DECISION SUMMARY
In summary:
Claims
Rejected
35 U.S.C. § Reference(s)/Basis Affirmed Reversed
1–6,
10–21
103 Storici, Pόsfai,
Seiko, Gibson,
Ripoll, Golic,
Akada, Hirashima,
Scherer
1–6,
10–21

REVERSED