Valton, Julien et al.

Patent Trials and Appeals Board

Mar 27, 2020

14235759 - (D) (P.T.A.B. Mar. 27, 2020)

UNITED STATES PATENT AND TRADEMARK OFFICE
UNITED STATES DEPARTMENT OF COMMERCE
United States Patent and Trademark Office
Address: COMMISSIONER FOR PATENTS
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www.uspto.gov
APPLICATION NO. FILING DATE FIRST NAMED INVENTOR ATTORNEY DOCKET NO. CONFIRMATION NO.
14/235,759 05/07/2014 Julien Valton DI2011-04US1 5698
76392 7590 03/27/2020
ARRIGO, LEE, GUTTMAN & MOUTA-BELLUM LLP
2200 Pennsylvania Ave NW
Suite 400E
WASHINGTON, DC 20037
EXAMINER
WEILER, KAREN S
ART UNIT PAPER NUMBER
IPLA
NOTIFICATION DATE DELIVERY MODE
03/27/2020 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
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PTOL-90A (Rev. 04/07)
UNITED STATES PATENT AND TRADEMARK OFFICE
____________

BEFORE THE PATENT TRIAL AND APPEAL BOARD
____________

Ex parte JULIEN VALTON, ALEXANDRE JUILLERAT, and
PHILIPPE DUCHATEAU
____________

Appeal 2019-004044
Application 14/235,759
Technology Center IP00
____________

Before ERIC B. GRIMES, DEBORAH KATZ, and
ULRIKE W. JENKS, Administrative Patent Judges.

JENKS, Administrative Patent Judge.

DECISION ON APPEAL
Pursuant to 35 U.S.C. § 134(a), Appellant1,2 appeals from Examiner’s
decision to reject claims 40–42, 45, 49, 53, 55, and 57. 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(a). Appellant identifies the real party in interest as Cellectis.
Appeal Br. 3.
2 An oral hearing was held on March 2, 2020.
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STATEMENT OF THE CASE
Claims 40–42, 45, 49, 53, 55, and 57 are on appeal, and can be found
in the Claims Appendix of the Appeal Brief. Claim 40, reproduced below,
is illustrative of the claimed subject matter:
40. A method of generating and assembling polynucleotides
comprising arrays of at least two transcription activator-like
effector (TALE) DNA binding repeat polynucleotide modules
which each encodes for 30 to 42 amino acids, comprising the
steps of:
a) Generating “n” polynucleotide building blocks, each
comprising at least:
one TALE DNA binding repeat polynucleotide module;
a single cleavage site for a first type IIS restriction
enzyme A, placed on one side of the polynucleotide
module;
a single cleavage site for a second type IIS restriction
enzyme B, placed on the other side of the polynucleotide
module;
wherein A and B can produce compatible cohesive ends;
wherein cleavage of each of said polynucleotide
building blocks with restriction enzyme A results in a
polynucleotide comprising a polynucleotide module
flanked on one side by a cohesive end that can be re-
ligated with a polynucleotide building block cleaved by
restriction enzyme B without restoring a sequence
cleavable by restriction enzyme A and/or B;
wherein cleavage of each of said polynucleotide
building blocks with restriction enzyme B results in a
polynucleotide comprising a polynucleotide module
flanked on one side by a cohesive end that can be re-
ligated with a polynucleotide building block cleaved by
restriction enzyme A without restoring a sequence
cleavable by restriction enzyme A and/or B;
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b) Generating “n” polynucleotides linked to a solid
phase by: b1) linking each of the polynucleotide building
blocks generated according to step a) to a solid phase so that
the cleavage site for the first restriction enzyme A is placed on
the side of the polynucleotide module that is linked to the solid
phase and b2) cleaving said polynucleotide building block with
restriction enzyme B;
c) Generating one C-terminal polynucleotide building
block comprising at least:
one TALE DNA binding repeat polynucleotide module;
a single cleavage site for a first restriction enzyme A,
placed on one side of the polynucleotide module;
a single cleavage site for a second restriction enzyme B,
placed on the other side of the polynucleotide module;
wherein cleavage of said C-terminal polynucleotide
building block with restriction enzyme A results in a
polynucleotide comprising a polynucleotide module
flanked on one side by a cohesive end that can be re-
ligated with a polynucleotide building block of step a)
cleaved by restriction enzyme B without restoring a
sequence cleavable by restriction enzyme A; and
wherein cleavage of said C-terminal polynucleotide
building block with restriction enzyme B results in a
polynucleotide comprising a polynucleotide module
flanked on one side by a cohesive end that cannot be re-
ligated with a polynucleotide building block of step a)
cleaved by restriction enzyme A and/or B;
d) Cutting said one C-terminal polynucleotide building
block of c) with restriction enzyme A;
e) Ligating the resulting C-terminal polynucleotide
module with the free end of one polynucleotide of b)
immobilized on a solid phase, thereby producing a new
immobilized polynucleotide comprising one additional
polynucleotide module;
f) Cutting the resulting new immobilized polynucleotide
with restriction enzyme A, thus producing a new
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polynucleotide having a free end compatible with the cohesive
ends resulting from cleavage of a polynucleotide building
block of step a) with restriction enzyme B;
g) Ligating the new polynucleotide with the free end of
one polynucleotide of b) immobilized on a solid phase thus
producing a new immobilized polynucleotide comprising one
additional polynucleotide module.
Appeal Br. 24–27 (Claims Appendix).
REFERENCE(S)
The prior art relied upon by Examiner is:
Name Reference Date
Cease et al. (“Cease”) US 5,827,704 Oct. 27, 1998
Joung et al. (“Joung”)3 US 2014/0274812 Al Sept. 18, 2014
Schatz et al. (“Schatz”) US 2006/0194202 Al Aug. 31, 2006
REJECTION(S)
Appellant requests review of the following grounds of rejection made
by Examiner:
I. Claims 40–42, 45, 49, 55 and 57 under 35 U.S.C. § 103(a) as
unpatentable over Joung in view of Schatz.
II. Claims 40–42, 45, 49, 53, 55 and 57 under 35 U.S.C. § 103(a) as
unpatentable over Joung in view of Schatz and further in view of
Cease.
OBVIOUSNESS OVER JOUNG AND SCHATZ
Since both of these rejections rely upon Joung and Schatz for
teaching “a polynucleotide building block with a TALE DNA binding

3 Examiner’s rejection relies on the earliest filing date of Joung’s
provisional application No. 61/508,366 filed on July 15, 2011, herein
referred to as “J-Provisional”.
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repeat polynucleotide module and two different, but compatible restriction
sites on each side of the module,” the same issue is dispositive for both
rejections, so we will consider the rejections together.
Findings of Fact
FF1. Joung teaches the assembly of an elongated polynucleotide sequence
containing TALE repeat sequences. Figure 1 of Joung’s provisional
application, reproduced below, shows “[a]n exemplary method of
assembling a TALE repeat domain array” by the addition of
preassembled sequences to the C-terminal end TALE sequence. J-
Provisional 8:13.

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Figure 1 of Joung’s provisional application, reproduced above, shows
the following steps:
(1) attachment of a single biotinylated PCR product
encoding one single N-terminal TALE repeat domain[]
to a solid support (in the example shown here, a
magnetic streptavidin coated bead is used but other solid
supports might also be utilized as well as other ways of
tethering the initial DNA fragment to the solid support),
(2) creation of an overhang at the 3' end of the anchored
DNA (e.g., using a Type IIS restriction enzyme),
(3) ligation of a second fragment containing four TALE
repeat domain[s],
(4) additional cycles of steps (2) and (3) to create a long
array,
(5) in the final cycle performing ligation of a piece of
DNA encoding one, two, three, or four TALE repeat
domains depending upon the length of the desired final
array, and
(6) release of the extended DNA encoding the TALE
repeats from the solid support (e.g., by using a Type IIS
restriction enzyme whose site is built in at the 5' end of
the initial biotinylated DNA product). The final
fragment can then be prepared for ligation to an
appropriate expression plasmid.
See J-Provisional 8:13–26 (formatting added); Joung ¶ 59; see
Ans. 8.
FF2. Joung teaches that “ligatable ends can be produced by cutting with a
restriction endonuclease (e.g., a type II or type IIS restriction
endonuclease) or by ‘chewing back’ the end using an enzyme (or
enzymes) with exonuclease and polymerase activities in the presence
of one or more nucleotides.” J-Provisional 10:12–15; Joung ¶ 65; see
Ans. 8.
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FF3. Joung’s example 1 teaches assembly of TALE repeat arrays using
streptavidin coated magnetic beads. The assembly method contains
the following steps:
(1) amplification of α-type TALE repeat to insert biotin and digestion
of the amplified fragment with BsaI HF4 (a type IIS restriction
endonuclease) and purification. J-Provisional 48:1–12; Joung
¶ 193.
(2) cutting a plasmid containing a four TALE repeat domain subarray
unit (βγδϵ) with BbsI and purifying the fragment.
J-Provisional 48:13–22; Joung ¶ 194.
(3) ligating the biotin containing α-subunit polynucleotide and the
βγδϵ-subunit containing polynucleotide to create a nucleic acid
containing αβγδϵ TALE subunits. J-Provisional 48:23–49:5;
Joung ¶ 195.
(4) attaching the ligated αβγδϵ polynucleotide to streptavidin
containing beads, washing, and digesting the bead bound
polynucleotide with BsaI HF. J-Provisional 48:23–49:5; Joung
¶ 195.
(5) extending the bead bound nucleic acid by adding TALE repeat
domain subarray unit (βγδϵ) and ligating to create a
polynucleotide containing TALE subunits αβγδϵβγδϵ.
J-Provisional 49:6–25; Joung ¶ 196. This cycle is repeated one
more time to create a bead bound polynucleotide containing
TALE subunits αβγδϵβγδϵβγδϵ. Finally, a TALE subarray

4 Appellant does not dispute that BsaI is a type IIS endonuclease. See
Appeal Br. 22.
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containing βγδ is added to the bead bound polynucleotide. J-
Provisional 49:6–25; Joung ¶ 195.
(6) digesting the bead bound polynucleotide with BsaI HF to create a
3' end for cloning into an expression vector and digesting the bead
bound polynucleotide with BbsI to cleave the 5' end to release the
product from the bead. J-Provisional 49:26–50:3; Joung ¶ 197.
FF4. Schatz teaches methods of producing nucleic acid sequences by
stringing together elongation blocks of nucleic acids. Schatz’s
method contains the following steps:
(a) providing a first at least partially double-stranded
oligonucleotide which has a modification allowing the
oligonucleotide to be coupled to a surface, whereby the
oligonucleotide comprises a recognition site for a first type IIS
restriction enzyme which cuts outside its recognition site, and
which oligonucleotide comprises a single-stranded overhang,
b) immobilising the first oligonucleotide to the surface via the
modification,
c) providing a second at least partially double-stranded
oligonucleotide whereby the oligonucleotide comprises a
recognition site or a part thereof for a second type IIS
restriction enzyme which cuts outside its recognition site, and
which second oligonucleotide comprises a single-stranded
overhang,
d) ligating the first and the second oligonucleotide via their
overhangs generating a first ligation product,
e) cutting the immobilised ligation product with the first type IIS
restriction enzyme thus releasing an elongated oligonucleotide
having an overhang,
f) providing a further at least partially double-stranded
oligonucleotide which has a modification allowing the
oligonucleotide to be specifically coupled to a surface,
whereby the oligonucleotide contains a recognition site for a
further type IIS restriction enzyme and a single-stranded
overhang,
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g) immobilising the further at least partially double-stranded
oligonucleotide on a surface via its modification,
h) combining the elongated oligonucleotide with the immobilised
further oligonucleotide, and ligating them via their overhangs
forming a further ligation product,
i) cutting the resulting further ligation product with the further
type IIS restriction enzyme releasing an elongated
oligonucleotide having an overhang, and
j) optionally, repeating steps f) to i).
Schatz ¶¶ 21–30, see also ¶¶ 93–101, see Figure 1.

Principle of Law
“If the claim extends to what is obvious, it is invalid under § 103.”
KSR Int’l Co. v. Teleflex Inc., 550 U.S. 398, 419 (2007).

Analysis
Examiner finds that Joung teaches generating a polynucleotide using
building blocks comprising TALE repeat domains. Final Act. 3; FF1–FF3.
Examiner finds that Joung teaches a “PCR product containing a single N-
terminal TALE repeat domain having type IIS restriction enzyme sites at
the 5' and 3' ends.” Final Act. 3 (citing ¶¶ 59, 65). Examiner acknowledges
that “the assembly process of Joung [] involves successive ligation of
TALE repeat modules to the growing ‘C-terminal’ end of the repeat array
(distal to the support), rather than insertion of TALE repeat modules at the
‘N-terminal’ end of the repeat array (proximal to the support), as per instant
claim 40.” Final Act. 4; FF1–FF3. Examiner relies on Schatz for
teach[ing] ligating an immobilized ‘N-terminal’ repeat module
to an elongated ‘C-terminal’ repeat module array (e.g.
releasing the first ligation product (elongated oligonucleotide)
by cleavage with a type IIS enzyme and ligating it to an
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immobilized further oligonucleotide, which in turn can be
released and ligated to another immobilized further
oligonucleotide.
Final Act. 4 (citing Schatz ¶¶ 25–30); FF4.
Examiner concludes that one of ordinary skill in the art would have
been motivated to apply Schatz’s reverse solid phase synthesis (RSPS)
approach using Joung’s TALE nucleic acid building blocks because the
RSPS method results in an almost complete removal of side products. Final
Act. 4–5 (citing Schatz ¶ 116).
Appellant contends that example 5 of Joung is not prior art to
Appellant’s claims (Appeal Br. 13, Reply Br. 2–3) and that example 1 of
Joung’s provisional application does not teach many aspects of the claimed
invention (Appeal Br. 13). Specifically, Appellant contends that Joung does
not teach the limitation
‘wherein cleavage of each of said polynucleotide building
blocks with restriction enzyme A results in a polynucleotide
comprising a polynucleotide module flanked on one side by a
cohesive end that can be re-ligated with a polynucleotide
building block cleaved by restriction enzyme B without
restoring a sequence cleavable by restriction enzyme A and/or
B’ is not discussed anywhere in the Office Action.
Appeal Br. 16, 17. Appellant contends that there is no specific mention of a
second enzyme site that is compatible with the first. Id. at 20. Appellant
contends that the disclosure in Joung is not sufficient to establish that the
TALE polynucleotide modules contain different but comparable restriction
enzyme sites on each side of the module. Id. at 22.
Although we agree with Appellant that example 5 is not disclosed in
Joung’s provisional application, we, however, do not agree with
Appellant’s contention that example 1 of Joung is missing the disputed
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aspects of the claimed invention. In the Answer, Examiner directs attention
to passages in Joung that are supported in Joung’s provisional application
that disclose adding multiple TALE repeat domain subarray unit (βγδϵ) into
a growing polynucleotide chain. See FF1–FF3. Specifically, Examiner
directs our attention to paragraph 59 and 65 of Joung, which are fully
supported in Joung’s provisional application, for teaching the cyclic
assembly process. See Ans. 8 (“These paragraphs describe the cyclic
assembly (i.e. cutting and ligation) process to create a long array, and that
the ligatable ends can be produced using type IIS restriction enzymes.”).
Examiner finds that the “growth of the polynucleotide [in Joung] takes
place in the direction corresponding to N terminus → C terminus of the
encoded polypeptide.” Ans. 6. Examiner identifies “[e]nzyme ‘A’ of claim
40 corresponds to the type IIS restriction endonuclease BbsI of the working
examples [including example 1], and enzyme ‘B’ of claim 40 corresponds
to the type IIS restriction endonuclease BsaI of the working examples
[including example 1].” Ans. 9; FF3.
Joung teaches cutting a plasmid containing TALE repeat domain
subarray unit (βγδϵ) with BbsI and purifying the fragment. FF3 (step 2). In
the first cycle the purified TALE repeat domain subarray unit (βγδϵ) is
added to the α-subunit containing biotin and the two units are ligated. FF3
(step 3). The ligated product containing TALE subunits αβγδϵ
polynucleotide is mixed with streptavidin containing beads, washed, and the
bound polynucleotide is digested with BsaI HF (FF3 (step 4)), thereby,
creating a ligatable C-terminal end to receive another TALE repeat domain
subarray unit (βγδϵ). FF3 (step 2). Because the same TALE subarray unit
(βγδϵ) can be added one or more times to the attached array, we agree with
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Examiner that this reasonably indicates that 3′ and 5′ ends cut with different
enzymes produce cohesive ends. Ans. 9.
We are not persuaded by Appellant’s argument that example 1 does
not teach a polynucleotide building block with a TALE DNA binding repeat
polynucleotide having two different but compatible restriction sites. See
Appeal Br. 21. The plasmid carrying the subarray unit (βγδϵ) meets the
building block limitation. See FF3 (steps 2, 4, 5). The building block
plasmid contains the BbsI and BsaI HF type IIS restriction endonuclease
sites. Id. Because the subarray unit (βγδϵ) is added multiple times the ends
created by the restriction endonuclease BbsI and BsaI HF are cohesive.
Here, the digestion with BbsI creates an N-terminus in the subarray unit
(βγδϵ) that is ligated to the C-terminus created by the BsaI HF digestion.
We are not persuaded that more information is required in order to
demonstrate that the BbsI and BsaI HF cleavage products create cohesive
ends. Here, Joung’s growing polynucleotide chain indicates that it has an
end that is acceptable for ligating the next subarray unit (βγδϵ) to form the
chain.
Examiner further explains that “the extended polynucleotide can not
retain an ‘internal’ site that is recognized by the first [and second] type IIS
restriction endonuclease because such a recognition site would result in
cleavage 3' of the α repeat (releasing the ligated fragment) at each cycle,
precluding polynucleotide growth.” Ans. 9. The second type IIS restriction
endonuclease (FF3 (step 4)) is also lost during the growth of the
polynucleotide chain. Examiner finds that Joung’s teaching of creating an
extended polynucleotide chain reasonably suggests “that the recognition
sequence of the first type IIS enzyme (i.e. “B”) is removed by digestion and
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not reformed during ligation, as additional cycles of cleavage and ligation
could not otherwise create a long array.” Ans. 9. We find no error with
Examiner’s explanation that Joung’s teaching of adding a multiple TALE
subarray unit (βγδϵ) reasonably suggests that a type IIS enzyme (i.e. “B” =
BsaI) site is not part of the elongated array because this enzyme is used to
prepare a cohesive end to which another TALE subarray unit (βγδϵ) is
ligated in order to grow the chain. See Ans. 9, 11 (“the use of BsaI (i.e. “B”)
to cleave the new 3' end of the extended polynucleotide evidences the
presence of BsaI at the 3' end of each TALE repeat sub-array unit, and its
absence at the site of ligation of the a [sic] unit with the initial TALE repeat
sub-array unit”); FF3. Examiner explains that
the library of TALE repeat subarrays functions as the ‘building
blocks’. Cleavage of each TALE repeat sub-array plasmid with
BbsI results in a 5' end that is compatible for ligation with the 3'
end of the growing polynucleotide, which is produced by
cleavage with BsaI. Notably, the growing polynucleotide
comprises multiple TALE repeat sub-arrays [(βγδϵ)]; the new 3'
end formed at each cycle corresponds to the 3' end of the most
recently added TALE repeat sub-array [(βγδϵ)].
Ans. 12.
We are not persuaded by Appellant’s contention that there is
insufficient disclosure in Joung’s provisional application to suggest
building blocks having “compatible restriction sites on each side of the
module.” Appeal Br. 22. Examiner relies on Joung’s teaching of “digestion
of the TALE repeat sub-array plasmids with BbsI for 2 hrs, thereby
indicating [] the presence of the BbsI recognition sequence in the cloned
repeat unit.” Ans. 12–13. Examiner notes that “a BsbI [sic] recognition site
is not present in the pUC cloning vector,” thus, the BbsI recognition site is
part of the TALE polynucleotide sequence inserted into the plasmid.
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Ans. 12. Examiner notes that the presence of a BsaI recognition sequence in
the cloned repeat unit of the library plasmids is not explicitly described, but
is readily apparent in view of the TALE assembly method exemplified in
example 1. Ans. 13; FF3. In particular, after adding a TALE repeat sub-
array to the 3' end of the growing polynucleotide, the ‘new’ 3' end is
cleaved with BsaI. Ans. 13. We agree with Examiner’s position that, even
though, the disclosure in Joung’s provisional application does not recite the
vector sequences, Joung’s teaching that the subarray unit (βγδϵ) is ligated to
form the growing polynucleotide chain sufficiently supports the position
that the digested polynucleotide ends are compatible. See FF3.
Thus, notwithstanding Appellant’s contentions to the contrary,
knowledge of the specific sequences of the plasmids containing TALE are
not necessary to establish that the resulting enzyme cleavage products have
ligatable ends. Here, the evidence further supports that the cleavage site for
enzymes A and B are not present in the ligated product forming the
elongated polynucleotide chain, because if such enzyme recognition and
cleavage sites were present the polynucleotide, the chain would not grow.
One skilled in the art must be presumed to know something about the art
apart from what the references disclose. In re Jacoby, 309 F.2d 513, 516
(CCPA 1962); In re Sovish, 769 F.2d 738, 743 (Fed. Cir. 1985); see also
KSR, 550 U.S. at 418 (An analysis under 35 U.S.C. § 103 “need not seek
out precise teachings directed to the specific subject matter of the
challenged claim, for a court can take account of the inferences and creative
steps that a person of ordinary skill in the art would employ.”). At the time
of the invention, one of ordinary skill in the art would have been aware of
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the mechanics of the Golden Gate cloning protocol. The Golden Gate
protocol provides that
a DNA fragment of interest can be subcloned with very high
efficiency in one step and one tube from one plasmid to
another []. The principle of the cloning strategy is based on the
ability of type IIs restriction enzymes to cut outside of their
recognition site. Two DNA ends can be designed to be flanked
by a type IIs restriction site such that digestion of the
fragments removes the enzyme recognition sites and generates
ends with complementary 4 nt overhangs; such ends can be
ligated seamlessly, creating a junction that lacks the original
site. . . . This property allows cloning to be performed using a
one-step restriction-ligation. This strategy was shown to result
in the conversion of more than half of all input plasmids
present into the desired recombinant product in just a 30
minutes restriction ligation. Subcloning was also found to be
very efficient when two and three inserts were subcloned, but
the total amount of recombined plasmid was lower.
Engler5 2 (emphasis added), see Spec. 3. Here, example 1 of Joung teaches
that the same βγδϵ subarray unit is added in multiple rounds to the
polynucleotide attached to the bead, and with each round the polynucleotide
chain grows. FF3 (steps 2 and 5), see also FF1. Even if Joung does not
disclose the sequence of the plasmids containing the polynucleotide
building blocks, one of ordinary skill in the art would ensure that digestion
with the type IIs restriction results in ends that are compatible for ligation
purposes in order to grow the polynucleotide chain. Accordingly, we are not
persuaded by Appellant’s contention that Joung’s example 1 provides
insufficient information to establish that Joung teaches a polynucleotide

5 Engler et al., Golden Gate Shuffling: A. One-Pot DNA Shuffling Method
Based on Type Us Restriction Enzymes, 4 PloS ONE eSSS3, 1–9 (2009)
(“Engler”) submitted Jan. 28, 2014 with Information Disclosure Statement.
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building block having two different restriction sites on each side of the
module that contain compatible ends.
Considering the totality of the cited evidence and arguments, we
conclude that the preponderance of the evidence supports Examiner’s
conclusion of obviousness with respect to claim 40, and Appellant has not
provided sufficient rebuttal evidence that outweighs the evidence
supporting Examiner’s conclusion. As Appellant does not argue the claims
separately, claims 41, 42, 45, 49, 53, 55, and 57 fall with claim 40. 37
C.F.R. § 41.37 (c)(1)(iv).
DECISION SUMMARY
In summary:
Claims
Rejected
35 U.S.C.
§
Reference(s)/Basis Affirmed Reversed
40–42, 45,
49, 55, 57
103 Joung, Schatz 40–42, 45,
49, 55, 57

40–42, 45,
49, 53, 55,
57
103 Joung, Schatz,
Cease
40–42, 45,
49, 53, 55,
57

Overall
Outcome
40–42, 45,
49, 53, 55,
57

TIME PERIOD FOR RESPONSE
No time period for taking any subsequent action in connection with
this appeal may be extended under 37 C.F.R. § 1.136(a). See 37 C.F.R.
§ 1.136(a)(1)(iv).

AFFIRMED