Ex Parte Hamm et al

Board of Patent Appeals and Interferences

Jul 19, 2011

11750401 (B.P.A.I. Jul. 19, 2011)

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.
11/750,401 05/18/2007 Sarah E. Hamm LCO 4941.4 1028
49328 7590 07/19/2011
BRYAN CAVE LLP
211 NORTH BROADWAY
SUITE 3600
ST. LOUIS, MO 63102-2750
EXAMINER
LEAVITT, MARIA GOMEZ
ART UNIT PAPER NUMBER
1633
MAIL DATE DELIVERY MODE
07/19/2011 PAPER
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.
PTOL-90A (Rev. 04/07)

UNITED STATES PATENT AND TRADEMARK OFFICE
__________

BEFORE THE BOARD OF PATENT APPEALS
AND INTERFERENCES
__________

Ex parte SARAH E. HAMM and WILLIAM P. LEINERT
__________

Appeal 2011-003267
Application 11/750,401
Technology Center 1600
__________

Before LORA M. GREEN, 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
composition for transfecting Chinese hamster ovary cells. The Examiner
rejected the claims as obvious. We have jurisdiction under 35 U.S.C. § 6(b).
We reverse.

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Application 11/750,401

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Statement of the Case
Background
“[T]he present invention relates to processes for the transfection of
CHO [Chinese hamster ovary] cells suspended in an aqueous medium using
a transfection composition containing nucleic acid and a polycationic
composition” (Spec. 1 ¶ 0002).
The Claims
Claims 21-23, 25, and 33 are on appeal. Claims 21-23 and 33 are
representative and read as follows:
21. A composition for transfecting Chinese hamster
ovary (CHO) cells, the transfection composition
containing a population of particles comprising linear
polyethyleneimine and nucleic acid, the population
having an average particle size and a polydispersity with
the polydispersity being between 0 and 0.4, and the ratio
of polyethyleneimine nitrogen moieties (N) to nucleic
acid phosphate moieties (P) in the particles being 15:1 to
30:1 (N/P).

22. The composition as set forth in claim 21 wherein
the population has an average particle size of from about
300 to about 900 nm.

23. The composition as set forth in claim 21 wherein
the polydispersity of the population is less than 0.3.

33. The composition as set forth in claim 21 wherein
the ratio of polyethyleneimine nitrogen moieties (N) to
phosphate moieties (P) in the particles is 23:1 (N/P).
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The issue
1

The Examiner rejected claims 21-23, 25, and 33 under 35 U.S.C.
§ 103(a) as obvious over Derouazi
2
and Dunlap
3
(Ans. 4-9).
The Examiner finds that “Derouazi et al., discloses optimization of the
DNA-PEI complexes including ratios of 2:1, 1:1, 1:2 and 1:3 (w: w)” (Ans.
4). The Examiner finds that “Derouazi et al., states that complete DNA
condensation at DNA:PEI ratio (1:0.3) corresponds to a N/P ratio (PEI
nitrogen to DNA phosphate) of 2 . . . Derouazi discloses other optimized
N/P ratios including optimal ratios from 9 to 13” (id. at 4-5). The Examiner
finds that “the 25kD-PEI polymers and nucleic acid complexes of Derouazi
are formed by mixings at room temperature aqueous solutions at pH of 7.0
of PEI and nucleic acid to form an aqueous transfection reagent to transfect
CHO cells” (id. at 5). The Examiner finds that this “is the same process
used in the instant invention to form DNA: PEI particles in a population
having an average particle size and various the N/P ratio (Specification
paragraph [0131], for example), then the polydispersity being between 0 and
0.4 is implicitly anticipated by the population of DNA:PEI particles of
Derouazi” (id.).

1
The Examiner withdrew the rejection under 35 U.S.C. § 112,
second paragraph (Ans. 3).
2
Derouazi et al., Serum-Free Large-Scale Transient Transfection
of CHO Cells, 87 BIOTECHNOLOGY BIOENGINEERING 537-545
(2004).
3
Dunlap et al., Nanoscopic structure of DNA condensed for gene
Delivery, 25 NUCLEIC ACIDS RES. 3095-3101 (1997).
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The Examiner finds that “Derouazi et al., does not particularly teach
compositions having an effective diameter of from about 300 to about
900nm or the N/P ratio of 15:1 to 30:1” (Ans. 5).
The Examiner finds that “Dunlap teaches that the majority of the
25kD-PEI and nucleic acid condensates were 20-40 nm in diameter and that
a small number of larger condensates of 25kD-PEI were also found” (id. at
6). The Examiner finds that “Dunlap states that positively charged
condensates produce more homogeneous, condensed plasmids, the size of
which are probably affected by the ionic strength of the solution” (id.).
The Examiner finds that the ordinary artisan
would have a reasonable expectation of success in
optimizing with a N/P ratio from 15:1 to 30:1 the N/P ratio
from 9:1 to 13:1 of Derouazi in an attempt to improve the
efficacy of the positive surface charges reflected in the
behavior of the transfection active particle. Moreover, one of
ordinary skill in the art, at the time the invention was made,
would have a reasonable expectation of success in
substituting a population having an average size from about
300 to 900 nm for the population having 20-40 nm in
diameter and larger of Dunlap in an attempt to provide an
improved formulation of the population of particles for
transfection of CHO cells . . .

(id. at 6-7).
Appellants contend that “the Examiner has failed to
articulate any reason why a population of particles satisfying the
compositional requirements of claim 21 and the polydispersity requirements
of claim 21 would have been obvious to a person o[f] ordinary skill” (App.
Br. 6). Appellants contend that
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Derouazi et al. point out that the findings of Dunlap et al.
indicate that particle size is influenced by the DNA:PEI
ratio, with size decreasing as the ratio of PEI to DNA is
increased, and that smaller particles are actually less efficient
for transfection. Thus, Derouazi et al. and Dunlap et al.
actually teach away from compositions having N/P ratios
greater than 13:1

(App. Br. 7). Appellants contend that “a person of ordinary skill could not
have predicted the benefit of having an N:P ratio as required by claim 21”
(id.).
The issue with respect to this rejection is: Does the evidence of record
support the Examiner’s conclusion that Derouazi and Dunlap render obvious
the method of claim 21?
Findings of Fact
1. Derouazi teaches that “we demonstrate the efficient expression
of r-proteins in suspension-adapted CHO cells using PEI-mediated gene
delivery in serum-free conditions” (Derouazi 538, col. 1).
2. Derouazi teaches that the “highest level of GFP expression was
observed in cultures transfected with linear 25 kDa PEI” (Derouazi 539, col.
1).
3. Derouazi teaches that “[s]everal parameters that are critical to
the formation of DNA-PEI complexes, including the amount of DNA, the
DNA:PEI ratio, the solution conditions, and the length of the incubation
period were evaluated” (Derouazi 539, col. 2).
4. Derouazi teaches that:
Nearly complete DNA condensation has also been found
with a branched 25 kDa PEI at an N/P ratio of 3 . . . . In our
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hands, however, successful gene transfer was only observed
with N/P ratios of 6 or more, and the optimal N/P ratio for
the transfection of CHO cells was found to be 13. Likewise,
an optimal N/P ratio from 9 to 13 has previously been shown
for the transfection of various cell lines with PEI.

(Derouazi 543, col. 2.)
5. Derouazi teaches that
we consistently observed higher r-protein yields following
transfections with a DNA:PEI ratio of 1:2 than with a ratio
of 1:3 or higher (cf. Figs. 1, 3, and 4). These results may be
explained by an increase in cell death following transfection
with the higher amounts of PEI. Each of these properties of
PEI and DNA-PEI complexes may therefore pose
constraints on the critical parameters for PEI-mediated
transfection of cells in serum-free medium.

(Derouazi 544, col. 1.)
6. The Examiner finds that “Derouazi et al., does not particularly
teach compositions having an effective diameter of from about 300 to about
900nm or the N/P ratio of 15:1 to 30:1” (Ans. 5).
7. Dunlop teaches that “PEI saturated condensates were prepared
with 1.6 equivalents of 22 kDa PEI : 1 equivalent DNA which produced
slightly positively charged condensates” (Dunlop 3099, col. 1).
8. Dunlop teaches that the “vast majority of condensates were 20-
40 nm in diameter for both the linear 22 kDa and branched 25 kDa PEI
polymers, although a small number of much larger condensates resulted
from condensation with branched 25 kDa PEI” (Dunlop 3099, col. 1-2).

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9. Figure 4 of Dunlop is reproduced below:

“Figure 4. A histogram of condensate diameters. . . . PEI condensates were
preapred with 1.6 equivalents : 1 equivalent DNA in 150 mM NaCl”
(Dunlop 3099, col. 2).
10. Dunlop teaches that when “condensates are used in transfection,
excess amounts of cationic lipids or polycations are usually necessary to
yield positively charged condensates. The present data indicate that such
ratios produce more homogeneous, condensed plasmids the sizes of which
are probably affected by the ionic strength of the solution” (Dunlop 3101,
col. 1).
11. Dunlop teaches that “some PEI-condensed plasmids seemed to
be coated by polymer rather than bundled in folded loops. Perhaps these
forms constitute more effective transfection agents” (Dunlop 3101, col. 1).
12. The Specification teaches that the “optimal N/P ratio for CHO-
S cells was observed to be 23:1. This N/P ratio gave the highest transfection
efficiency (95%) and maintained cell viabilities greater than 90%” (Spec. 55
¶ 0151).
13. The Specification teaches that with “N/P ratios from 18:1 to
28:1, about 90% of the cells were transfected. . . . At lower N/P ratios of
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15:1 or less, the percent of GFP positive cells decreased to 70% and less”
(Spec. 55 ¶ 0151).
Principles of Law
“[R]ejections 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.” KSR Int’l Co. v. Teleflex lnc., 550 U.S. 398, 418 (2007).
“[A] prima facie case of obviousness exists when the claimed range
and the prior art range do not overlap but are close enough such that one
skilled in the art would have expected them to have the same properties.” In
re Peterson, 315 F.3d 1325, 1329 (Fed. Cir. 2003).
Analysis
There is no dispute that Derouazi teaches a composition for
transfecting Chinese hamster ovary cells comprising linear
polyethyleneimine and nucleic acid with an optimal ratio of
polyethleyenimine nitrogen moieties to nucleic acid phosphate moieties of
13 and a range of 9-13 (FF 1-5).
The Examiner acknowledges that “Derouazi et al., does not
particularly teach compositions having an effective diameter of from about
300 to about 900nm or the N/P ratio of 15:1 to 30:1” (Ans. 5; FF 6).
We agree with Appellants that there is no teaching or suggestion in
Derouazi or Dunlop for a population with an average particle size of about
300 to about 900 nm (App. Br. 7-8).
We also agree that the Examiner provides no reason to use an N/P
ratio of 15:1 to 30:1 (see Ans. 6). At best, the Examiner cites Dunlop’s
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teaching that “excess amounts of cationic lipids or polycations are usually
necessary to yield positively charged condensates” (Dunlop 3101, col. 1; FF
10). However, this does not provide any specific teaching to use an N/P
ratio greater than the “optimal” ratio of 13 determined by Derouazi (FF 4).
The Examiner has not argued or demonstrated any expectation that the
N/P value of 15:1 would be expected to have the same or similar properties
as the disclosed optimal value of 13:1 of Derouazi (FF 4).
That conclusion is supported by the instant Specification, which
suggests that there are differences expected based on these values.
According to the Specification, the “optimal N/P ratio for CHO-S cells was
observed to be 23:1. This N/P ratio gave the highest transfection efficiency
(95%) and maintained cell viabilities greater than 90%” (Spec. 55 ¶ 0151;
FF 12). The Specification teaches that with “N/P ratios from 18:1 to 28:1,
about 90% of the cells were transfected. . . . At lower N/P ratios of 15:1 or
less, the percent of GFP positive cells decreased to 70% and less” (Spec. 55
¶ 0151; FF 13).
We thus conclude that the Examiner has not shown that the ordinary
artisan would have expected the same properties in a N/P ratio of 13:1 as is
found in the range of 15:1 to 30:1 of claim 21. We are therefore constrained
to reverse this rejection.
Conclusion of Law
The evidence of record does not support the Examiner’s conclusion
that Derouazi and Dunlap render obvious the method of claim 21.
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SUMMARY
In summary, we reverse the rejection of claims 21-23, 25, and 33
under 35 U.S.C. § 103(a) as obvious over Derouazi and Dunlap.

REVERSED

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