Ex Parte Murphy et al

Board of Patent Appeals and Interferences

Nov 3, 2011

11583223 (B.P.A.I. Nov. 3, 2011)

UNITED STATES PATENT AND TRADEMARK OFFICE
__________

BEFORE THE BOARD OF PATENT APPEALS
AND INTERFERENCES
__________

Ex parte WILLIAM L. MURPHY and SIYOUNG CHOI
__________

Appeal 2011-008457
Application 11/583,223
Technology Center 1600
__________

Before ERIC GRIMES, MELANIE L. McCOLLUM, and
JEFFREY N. FREDMAN, Administrative Patent Judges.

FREDMAN, Administrative Patent Judge.

DECISION ON APPEAL
This is an appeal under 35 U.S.C. § 134 involving claims to a cell
culture system. The Examiner rejected the claims as obvious. We have
jurisdiction under 35 U.S.C. § 6(b). We reverse.

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Application 11/583,223

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Statement of the Case
Background
The Specification teaches that “spatial and temporal control [of DNA
delivery] offers particular utility when applied in the context of cell
differentiation, especially in bioengineering three-dimensional matrices such
as tissues or organs containing multiple differentiated cell types that derive
from a single stem cell precursor” (Spec. 1 ¶ 0004).
The Claims
Claims 1-23 are on appeal. Claim 1 is representative and reads as
follows:
1. A cell culture system comprising:
a substrate;
a nucleic acid molecule non-covalently attached to
an oligonucleotide linker tethered to the substrate;
a target cell capable of taking up the nucleic acid
molecule by endocytosis, the cell being adhered to the
substrate via a cell adhesion linker; and
a culture medium compatible with survival of the
target cell.

The issues

A. The Examiner rejected claims 1-3, 5-10, 12-16, 19, 20, and 22 under
35 U.S.C. § 103(a) as obvious over Yamauchi,
1
Bamdad,
2
Segura,
3
and
Hickman
4
(Ans. 5-7).

1
Yamauchi et al., Micropatterned, self-assembled monolayers for
fabrication of transfected cell micro arrays, 1672 BIOCHIMICA ET
BIOPHYSICA ACTA 138-147 (2004).
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B. The Examiner rejected claims 1-16, 19, 20, and 22 under 35 U.S.C. §
103(a) as obvious over Yamauchi, Bamdad, Segura, Hickman, Iyer,
5
and
Cherng
6
(Ans. 8-9).
C. The Examiner rejected claims 1-3, 5-10, 12-17, 19, 20, and 22 under
35 U.S.C. § 103(a) as obvious over Yamauchi, Bamdad, Segura, Hickman,
and Harrison
7
(Ans. 9-10).
D. The Examiner rejected claims 1-3, 5-10, 12-16, 18-20, and 22 under
35 U.S.C. § 103(a) as obvious over Yamauchi, Bamdad, Segura, Hickman,
and Hosseinkhani
8
(Ans. 10-11).
E. The Examiner rejected claims 1-3, 5-10, 12-16, and 19-23 under 35
U.S.C. § 103(a) as obvious over Yamauchi, Bamdad, Segura, Hickman, and
Tanabe
9
(Ans. 11).

2
Bamdad, C., A DNA Self-Assembled Monolayer for the Specific
Attachment of Unmodified Double- or Single-Stranded DNA, 75
BIOPHYSICAL J. 1997-2003 (1998).
3
Segura et al., US 6,890,556 B1, issued May 10, 2005.
4
Hickman, J., US 7,266,457 B1, issued Sep. 4, 2007.
5
Iyer et al., Accelerated Hybridization of Oligonucleotides to
Duplex DNA, 270 J. BIOL. CHEMISTRY, 14712-14717 (1995).
6
Cherng et al., Effect of DNA topology on the transfection
efficiency of poly((2-dimethylamino)ethyl methacrylate)-plasmid
complexes, 60 J. CONTROLLED RELEASE 343-353 (1999).
7
Harrison et al., Screening for oligonucleotide binding affinity by a
convenient fluorescence competition assay, 27 NUCLEIC ACIDS
RESEARCH e14 i-v (1999).
8
Hosseinkhani et al., Ultrasound Enhances the Transfection of
Plasmid DNA by Non-viral Vectors, 4 CURRENT PHARMACEUTICAL
BIOTECHNOLOGY 109-122 (2003).
9
Tanabe et al., WO 92/00995 A1, published Jan. 23, 1992.
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A. 35 U.S.C. § 103(a) over Yamauchi, Bamdad, Segura, and Hickman
The Examiner finds that Yamauchi teaches “a method for the delivery
of nucleic acids to target cells by using a cell culture system comprising a
self-assembled monolayer (SAM) substrate, plasmid DNA attached to SAM
by electrostatic interactions (i.e., non-covalently), target cells adhered to the
substrate, and a culture medium containing factors necessary for the survival
of the target cells” (Ans. 5). The Examiner finds that “Bamdad teaches non-
covalent nucleic acids attachment to SAM via oligonucleotide linkers
tethered to SAM” (id.). The Examiner finds that “Segura et al. teach that
transfection occurs by nucleic acids uptake via endocytosis” (id. at 6). The
Examiner finds that Hickman teaches “cell culture systems comprising
SAMs and cells attached to SAMs via RGD peptides” (id. at 7).
The Examiner concludes that it would have been obvious “to modify
the method of Yamauchi et al. by non-covalently attaching their plasmid
DNA to SAM via an oligonucleotide tethered to SAM as taught by Bamdad,
with a reasonable expectation of success” (id. at 6). The Examiner finds that
one “of skill in the art would have been motivated to do so in order to use
the system for controlled gene delivery at desired times” (id.).
Appellants contend that Yamauchi fails “to disclose or suggest a
nucleic acid molecule non-covalently attached to an oligonucleotide linker
tethered to a substrate, or a target cell capable of taking up the nucleic acid
molecule by endocytosis that is adhered to the substrate via a cell adhesion
linker” (App. Br. 6). Appellants contend that “Bamdad fails to disclose or
suggest transfection, a target cell adhered via a cell adhesion linker to the
same substrate as the DNA, or a culture medium compatible with survival of
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the target cell” (id. at 7). Appellants also contend that Segura and Hickman
fail “to disclose or suggest a cell culture system comprising a nucleic acid
molecule non-covalently attached to an oligonucleotide linker tethered to a
substrate” (id. at 8).
Appellants contend that “nothing in Bamdad discloses or suggests that
one skilled in the art would reasonably expect that the anti-sense strand of
the duplex DNA could be used for cell transfection because Bamdad teaches
that it is dissociated away to leave single-stranded DNA presented on the
surface for hybridization studies” (App. Br. 11). Appellants contend that
“[m]odifying Bamdad in the manner suggested by the Office would render
Bamdad inoperable for this purpose as everything Bamdad discloses is
directed to covalently attaching single-stranded or double-stranded DNA
onto the SAM surface” (id. at 11-12).
Appellants contend that “Yamauchi et al. provide definitive
experimental data suggesting that substituting Bamdad’s DNA-SAM may
render Yamauchi’s et al. method inoperable” (id. at 13). Appellants contend
that “based on Yamauchi’s et al. explicit statement and Yamauchi’s et al.
own results, Applicants submit that one skilled in the art would be led away
from modifying and or combining Yamauchi et al. with references such as
Bamdad” (id. at 14).
Appellants contend that the skilled artisan “reading the Segura et al.
reference would thus actually be lead away from using the DNA-SAMs in
the method of Yamauchi et al., as the DNA in Bamdad is not packaged into
a complex, as suggested by Segura et al” (id. at 16).
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The issue with respect to this rejection is: Does the evidence of record
support the Examiner’s conclusion that Yamauchi, Bamdad, Segura, and
Hickman would have rendered obvious the cell culture system of claim 1?
Findings of Fact
1. Figure 1 of Yamauchi is reproduced below:

Fig. 1. Scheme for the preparation of a plasmid DNA
microarray by the alternate adsorption process. (A) An
alkanethiol monolayer presenting different terminal groups
was formed within circular spots of 1-mm diameter. The
regions between the spots were the methyl-terminated SAM.
(B) A solution of lipid DNA (LD) complex was pipetted to
the spots. (C) Plasmid DNA was pipetted to the LD-
adsorbed spots. The procedures (B) and (C) were repeated to
obtain a plasmid DNA microarray (D). The alternate
adsorption was always ended with adsorption of LD
complex.

(Yamauchi 140, figure 1 legend.)
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2. Yamauchi teaches that a “cell suspension (2 ml) was plated to
the microarray placed on the bottom of a 35-mm polystyrene cell culture
dish. The cells were incubated for 72 h at 37°C under 5% CO2, and the
culture medium was replaced every 3 days” (Yamauchi 140, col. 2).
3. Yamauchi teaches that the “efficiency of transfection was 80-
85% when determined from the microphotographs . . . These photographs
also show no significant cytotoxic effects” (Yamauchi 144, col. 1).
4. Yamauchi teaches that in
the case of an amine-terminated SAM, naked DNA, rather
than the LD complex, could be loaded to the spot probably
due to the same reason (electrostatic interactions). The
results also suggest that in the second and the later cycles,
DNA or LD complex adsorbed through electrostatic
interactions with the previously loaded components with an
opposite sign of charges.

(Yamauchi 145, col. 2.)
5. Yamauchi teaches that “[a]lthough the immobilization of DNA
onto material surfaces has been investigated extensively in the field of DNA
microarray, most studies aimed at covalent binding or firm adsorption of
DNA which is not expected to detach from the surface” (Yamauchi 145, col.
2).
6. Bamdad teaches that “[s]ince the anti-sense strand is not
covalently attached to the SAM, it can be dissociated by heat or chemical
treatment, leaving ssDNA presented on the surface for hybridization studies”
(Bamdad 2002, col. 1).

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7. Bamdad teaches

Bamdad teaches that “our strategy was to form a SAM that presented
relatively short strands of DNA (10-15 bases); then, if necessary, to
enzymatically attach longer strands of DNA to the preassembled surface
(Scheme 1)” (Bamdad 1999-2000).
8. Bamdad teaches that “DNA surfaces described herein are
compatible with diverse sensing technologies. They can be used with SPR
devices to detect hybridization, protein-protein, or protein-DNA
interactions” (Bamdad 2003, col. 2).
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9. Figure 1 of Segura is reproduced below:

“FIG. 1 is a schematic depiction of DNA condensation (ionically localized)
with polymeric delivery to tether DNA complexes to the surface of a
hydrogel” (Segura, col. 5, ll. 38-40).
10. Segura teaches “controlled delivery of a nucleic acid to a target
cell or cells that combines nucleic acid condensation with polymeric
delivery by tethering or immobilizing nucleic acid complexes to a surface
that supports cell adhesion” (Segura, col. 2, ll. 20-24).
11. Segura teaches that the “gene delivery system of the invention
allows both temporal and spatial control of gene delivery due to efficient
internalization of the immobilized complex” (Segura, col. 2, ll. 25-28).
12. Segura teaches a “condensed nucleic acid in the form of a
nucleic acid-polylinker complex immobilized on the surface of a support
substrate; the complex is released from the surface of the support substrate
into the cell microenvironment, and is capable of being internalized by the
cell” (Segura, col. 6, l. 67 to col. 7, l. 4).

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13. Hickman teaches the
use of a surface modifying agent, preferably comprising a
self-assembling monolayer. Examples of suitable surface
modifying agents include, but are not limited to, silanes,
thiols, isocyanides, polyelectrolytes and the like, or
combinations thereof. More preferably, the system
incorporates an intervening layer that further comprises cell
anchorage molecules. Suitable cell anchorage molecules
include, but are not limited to, antibodies, antigens, receptor
ligands, receptors, lectins, carbohydrates, enzymes, enzyme
inhibitors, biotin, avidin, streptavidin, cadherins, RGD-type
peptides.

(Hickman, col. 7, ll. 4-14.)
Principles of Law
“In rejecting claims under 35 U.S.C. § 103, the examiner bears the
initial burden of presenting a prima facie case of obviousness. Only if that
burden is met, does the burden of coming forward with evidence or
argument shift to the applicant.” In re Rijckaert, 9 F.3d 1531, 1532 (Fed.
Cir. 1993).
An invention
composed of several elements is not proved obvious merely
by demonstrating that each of its elements was,
independently, known in the prior art…. [I]t can be
important to identify a reason that would have prompted a
person of ordinary skill in the relevant field to combine the
elements in the way the claimed new invention does.

KSR Int’l Co. v. Teleflex Inc., 550 U.S. 398, 418 (2007).
Analysis
We agree with Appellants “that one skilled in the art simply would
have no reason to even look to the Bamdad reference at all when reading the
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Yamauchi et al. reference as a whole, much less have a reason to modify the
Yamauchi et al” (Reply Br. 1).
In our opinion, even if the ordinary artisan were provided the
Yamauchi, Bamdad, Segura, and Hickman references for use in designing an
invention, the artisan would have found no reason in the references to
substitute Bamdad’s DNA linker for either the lipid-DNA spots of
Yamauchi or the covalent tether of Segura. The Examiner acknowledges
that Yamauchi does “not teach non-covalent attachment of the plasmid via
an oligonucleotide linker tethered to SAM” (Ans. 5). Segura never suggests
or provides a reason that DNA itself can function as a tethering component,
whether as a functional group, a bifunctional polylinker, a tether or
polylinker (see Segura, col. 7, ll. 8-64). Bamdad provides no reason to use
the DNA surface for release of a captured DNA strand, much less release
into a cell for transfection (see, e.g., FF 8). Hickman provides no teaching
of a DNA tether.
The Examiner’s reasoning is essentially that “at the time of filing non-
covalent attachment of nucleic acids to SAM via oligonucleotide was known
in the prior art” (Ans. 5). However, this does not represent a reason to use
the non-covalent attachment of DNA in methods of transfection of cells.
The mere existence of a technology is not sufficient, by itself, to represent a
reason to combine. Rather, at least a specific rationale based on the
teachings of the prior art or the knowledge of those skilled in the art is
required (see, e.g., MPEP § 2141). The rejection lacks such a rationale and
no reason to combine these references is evident from the references
themselves.
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The Examiner notes that Appellants also contend that “there is no
reason to modify Yamauchi et al. by using a cell adhesion linker” (Ans. 22).
The Examiner finds that “cell culture systems comprising SAMs and
cells attached to SAMs via RGD peptides was taught by the prior art; the
prior art also teaches that such culture systems are suitable for introducing
nucleic acids into the attached cells” (id. at 7). The Examiner relies upon
“Hickman et al., column 6, lines 1-19; column 7, lines 1-15; column 18,
lines 42-67; column 9, lines 1-13; column 17, lines 26-32 and 59-67; column
35, lines 25-57” (id.).
We agree with Appellants that the Examiner has not provided a reason
or rationale to combine the use of cell adhesion linkers with the transfection
method of Yamauchi. In particular, none of the cited portions of Hickman
teach or suggest the introduction of nucleic acids into attached cells.
Hickman teaches at column 6, lines 1-19, cell culture with microelectrodes.
Hickman teaches at column 7, lines 1-15, cell anchorage molecules, but does
not directly teach attachment of the cells, and does not teach transfection of
nucleic acids. Hickman, column 18, lines 42-67 teaches an example of
cardiac myocytes on SAMs, but no attachment or nucleic acid transfection is
disclosed. Hickman, column 9, lines 1-13 and column 17, lines 26-32 and
59-67 discusses the study of cells already transfected by DNA, but do not
provide any suggestion to use attached cells or to use a surface to transfect
DNA. Hickman, column 35, lines 25-27 discusses measurement of cell
surface receptors and has no teaching regarding attachment or transfection.
Thus, Hickman provides no reason and supports no rationale to attach
cells for transfection in the method of Yamauchi or Segura.
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Conclusion of Law
The evidence of record does not support the Examiner’s conclusion
that Yamauchi, Bamdad, Segura, and Hickman would have rendered
obvious the cell culture system of claim 1.
B.-E. 35 U.S.C. § 103(a)
These rejections each rely upon the underlying obviousness rejection
over Yamauchi, Bamdad, Segura, and Hickman which we reversed above.
Having reversed the obviousness rejection over Yamauchi, Bamdad, Segura,
and Hickman for a failure to present a prima facie case of obviousness for
Claim 1, we necessarily reverse each of these obviousness rejections as the
Iyer, Cherng, Harrison, Hosseinkhani, and Tanabe references do not provide
the obviousness rationale missing from the rejection over Yamauchi,
Bamdad, Segura, and Hickman.
SUMMARY
In summary, we reverse the rejection of claims 1-3, 5-10, 12-16, 19,
20, and 22 under 35 U.S.C. § 103(a) as obvious over Yamauchi, Bamdad,
Segura, and Hickman.
We reverse the rejection of claims 1-16, 19, 20, and 22 under 35
U.S.C. § 103(a) as obvious over Yamauchi, Bamdad, Segura, Hickman, Iyer,
and Cherng.
We reverse the rejection of claims 1-3, 5-10, 12-17, 19, 20, and 22
under 35 U.S.C. § 103(a) as obvious over Yamauchi, Bamdad, Segura,
Hickman, and Harrison.
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We reverse the rejection of claims 1-3, 5-10, 12-16, 18-20, and 22
under 35 U.S.C. § 103(a) as obvious over Yamauchi, Bamdad, Segura,
Hickman, and Hosseinkhani.
We reverse the rejection of claims 1-3, 5-10, 12-16, and 19-23 under
35 U.S.C. § 103(a) as obvious over Yamauchi, Bamdad, Segura, Hickman,
and Tanabe.

REVERSED

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