Ex Parte Fahrenkrug et al

Patent Trial and Appeal Board

Jan 30, 2019

13025373 (P.T.A.B. Jan. 30, 2019)

UNITED STA TES p A TENT AND TRADEMARK OFFICE
APPLICATION NO. FILING DATE FIRST NAMED INVENTOR
13/025,373 02/11/2011 Scott C. Fahrenkrug
153885 7590 02/01/2019
RECOMBINETICS - WILSON, SONSINI, GOODRICH & ROSATI
650 PAGE MILL ROAD
PALO ALTO, CA 94304-1050
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
ATTORNEY DOCKET NO. CONFIRMATION NO.
5054.05US02 2541
EXAMINER
WEHBE, ANNE MARIE SABRINA
ART UNIT PAPER NUMBER
1633
NOTIFICATION DATE DELIVERY MODE
02/01/2019 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):
patentdocket@wsgr.com
PTOL-90A (Rev. 04/07)
UNITED STATES PATENT AND TRADEMARK OFFICE
BEFORE THE PATENT TRIAL AND APPEAL BOARD
Ex parte SCOTT C. FAHRENKRUG and DANIEL F. CARLSON1
Appeal2017-002215
Application 13/025,373
Technology Center 1600
Before TONI R. SCHEINER, JEFFREY N. FREDMAN, and
DAVID COTTA, Administrative Patent Judges.
SCHEINER, Administrative Patent Judge.
DECISION ON APPEAL
This is an appeal under 35 U.S.C. § 134(a) from the final rejection of
claims directed to a transgenic swine and a transfected swine cell line. The
Examiner rejected the claims as obvious. We have jurisdiction under 35
U.S.C. § 6(b ).
We affirm.
1 Appellants identify Recombinetics, Inc. as the real party in interest.
Appeal Br. 3.
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Application 13/025,373
BACKGROUND
"Swine are an important agricultural commodity and biomedical
model" and "[g]enetic engineering can also expand the utility of pigs for
modeling human disease, developing clinical treatment methodologies, or
donating tissues for xenotransplantation." Spec. 1.
The Specification describes a stably transfected swine cell "with a
knockout for the low density lipoprotein receptor [gene] (LDLR) ... in male
and female domestic and miniature swine cells." Id. at 2. The cells may be
fetal or adult fibroblasts, and "[a] transgenic swine may be prepared by
nuclear transfer of such a cell." Id. at 3. "An embodiment of the invention
is a transgenic swine comprising a genomic disruption of an endogenous
gene," e.g., "a Low-Density Lipoprotein Receptor gene (LDLR) ... for
preventing expression of a functional protein and/or preventing expression
[ of] any protein." Id. at 2. "The swine may be homozygous or heterozygous
for said disrupted gene" (id.), and "may exhibit a phenotype chosen from the
group consisting ofhypercholesterolemia, atherosclerosis, and
atherosclerotic lesions" (id).
The Specification teaches that transgenic swine may be produced by
exposing swine cells in vitro to "a transfection agent that comprises an
exogenous nucleic acid [construct] during a first culture time period" (id. at
3), and "subsequently adding a second group of swine cells to the first group
for a second culture time period, wherein the second group of cells have not
been exposed to the transfection agent" (id.). The Specification also teaches
that "[ o ]ther elements that can be included on a nucleic acid construct"
include "a sequence encoding a selectable marker ... flanked by loxP
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recognition sequences for a recombinase such as, e.g., Cre or Flp ... such
that the selectable marker can be excised from the construct." Id. at 30.
The Specification discloses that:
[T]ransgenic swine cells were made with a defect in LDLR
expression .... In brief, the production of a functional LDLR
gene product was disrupted by introduction of a stop cassette
within LDLR exon 4 by Adeno-associated virus (rAAV)
homologous recombination (HR). An rAA V HR cassette
(rAAVLDLR-E4-stop) was generated with a PGK-Neo
selection cassette inserted within LDLR exon 4 at the XhoI
restriction site (Figure I). Replacement of LDLR exon 4 with
rAA V-LDLR-E4-stop resulted in a truncated, non-functional
LDLR protein product. Several AA V constructs were designed
and created that either targeted other exons or that avoided
certain repetitive elements around exon 4. Colonies with
LDLR nonexpression were identified from three separate
transductions.
Id. at 7-8.
In addition to disruption of LDLR exon 4, the Specification teaches
that "frame-shift mutation, truncation, or introduction of single amino acid
changes throughout the LDLR gene are expected to disrupt LDLR function."
The Specification teaches that "[ t ]argeting such changes would simply
require the acquisition of the pig LDLR sequences available in Genbank,
ENSEMBLE, or as described in SEQ ID NO: l "2 (id. at 11 ), and "the
application of methods for homologous recombination, allele conversion, or
the introduction of an indel using zinc finger nucleases, meganucleases, or
2 The Specification indicates that SEQ ID NO: 1 is "LDLR Partial CDS
[ coding sequence] from MARC library est sequenced by Applicants." Spec.
6. "SEQ ID NO: 1 was used to generate a probe for ... screening and
recovery of a genomic clone containing the pig LDLR gene." Id. at 12.
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T AL effector nucleases or any other targeted method for DNA
breakage/modification" (id.).
According to the Specification, "tests verified that cells were made
with LDLR knockouts." Id. at 10. The Specification does not exemplify a
transgenic pig comprising a genomic knockout of the endogenous low-
density lipoprotein receptor gene, but teaches that the knockout "cells may
be cloned into male and female pigs" (id.), and "[ t ]hese founders may then
be bred to create pigs homozygous for knockout of the LDLR gene" (id.).
Further according to the Specification, "[ v ]arious techniques known in the
art can be used to introduce nucleic acid constructs into non-human animals
to produce founder lines, in which the nucleic acid construct is integrated
into the genome" (id. at 24), including "somatic cell nuclear transfer" (id. at
24--25).
STATEMENT OF THE CASE
Claims 1, 3, 6, 7, 19--22, 26, 27, 35, and 39 are on appeal; claims 2, 4,
5, 8-18, 23-25, 28-34, and 36-38 have been canceled. Claims 1 and 21 are
representative and read as follows:
1. A transgenic swine comprising a genomic
knockout of an endogenous Low-Density Lipoprotein Receptor
gene (LDLR), wherein the animal is free of selectable marker
genes.
21. An isolated transfected somatic swine cell
comprising a gene knockout of an endogenous Low-Density
Lipoprotein Receptor gene (LDLR), wherein the transfected cell
is free of selectable marker genes.
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The Examiner relies on the following evidence:
Bentzon et al. WO 2008/106982
("Bentzon et al.")
Bentzon et al. US 8,546,643
("Bentzon '643")
Sept. 12, 2008
Oct. 1, 2013
S. Ishibashi et al., Hypercholesterolemia in low density
lipoprotein receptor knockout mice and its reversal by
adenovirus-mediated gene delivery, 92 J. CLIN. INVEST. 883-
893 (1993) ("Ishibashi").
Katalin Banki et al., Cloning and Expression of the Human
Gene for Transaldolase, 269 J. BIOL. CHEM. 2847-2851 (1994)
("Banki").
Xiaoling Xu et al., Direct Removal in the Mouse of a Floxed
Neo Gene From a Three-loxP Conditional Allele by Two Novel
Approaches, 30 GENESIS 1---6 (2001) ("Xu").
Christopher S. Rogers et al., Production of CFTR-null and
CFTR-!'1F508 heterozygous pigs by adeno-associated virus-
mediated gene targeting and somatic cell nuclear transfer, 118
J. CLIN. INVEST. 1571-1577 (2008) ("Rogers I").
Li Li et al., Production of a reporter transgenic pig for
monitoring Cre recombinase activity, 3 82 BIOCHEM. BIO PHYS.
RES. COMM. 232-235 (2009) ("Li").
Appellants rely, in relevant part, on the following additional evidence:
Declaration of Dr. Eric A. Hendrickson, dated June 25, 2015,
originally entered into the record with the Amendment of
September 17, 2015 ("Hendrickson Deel.").
Declaration of Dr. Daniel F. Carlson, dated August 3, 2015,
originally entered into the record with the Amendment of
September 17, 2015 ("Carlson Deel.").
Liangxue Lai et al., Production of a-1,3-Galactosyltransferase
Knockout Pigs by Nuclear Transfer Cloning, 295 SCIENCE
1089-1092 (2002) ("Lai").
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Application 13/025,373
Christopher S. Rogers et al., Disruption of the CFTR Gene
produces a Model of Cystic Fibrosis in Newborn Pigs, 321
SCIENCE 1837-1841 (2008) ("Rogers II").
J. Ramsoondar et al., Targeted disruption of the porcine
immunoglobulin kappa light chain locus, Transgenic Res.
Published online: 26 Sept. 2010 ("Ramsoondar").
The claims stand rejected under 35 U.S.C. § 103(a) as follows: 3
Claims 1, 3, 6, 19-22, 26, 27, 35, and 39 as unpatentable over
Bentzon et al. or Bentzon '643, 4 in view of Xu and Li; and
Claim 7 as unpatentable over Bentzon et al. or Bentzon '643, in view
of Xu, Li, and Ishibashi.
THE PRIOR ART
Bentzon '643
Bentzon '643 "relates to a genetically modified pig as a model for
studying atherosclerosis, wherein the pig model expresses at least one
phenotype associated with said disease and/or a modified pig comprising at
3 According to the Examiner, rejections of previously pending claims 1-3,
5, 6, 8, 19-23, 26, and 27 as anticipated by Bentzon et al. under 35 U.S.C.
§ 102(b), and of the same claims as anticipated by Bentzon '643 under
35 U.S.C. § 102(e), "were withdrawn due to the amendment of the claims to
recite that the transfected swine cell or transgenic pig is free of a selectable
marker gene" "in the Non-Final Office Action mailed ... on 8/1/14." Ans.
2-3.
4 Throughout the Answer, the Examiner relies on both Bentzon et al. and
Bentzon '643 for the same disclosures. Ans., passim. Appellants
characterize these references, as well US 2010/0138939 (the initially
published version of Bentzon '643), as "counterparts of each other," and
indicate that their "[r ]eferences to 'Bentzon' herein refer to the group of
them." Appeal Br. 9. For the sake of simplicity, our citations in this decision
refer to Bentzon '643, unless otherwise indicated.
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least one mutation in the ... endogenous ApoE gene and/or ... endogenous
LDL receptor gene." Bentzon '643, 3:41--48. Bentzon '643 further "relates
to a genetically modified porcine donor cell." Id. at 4:49--50.
Bentzon '643 teaches that the donor cells may be somatic cells
"originat[ing] from fetal or adult tissue ... and [ e ]specially preferred
somatic cells are those of foetal origin." Id. at 19:25-31. "Preferably, the
somatic cells are fibroblast cells as the[y] can be obtained both from
developing fetuses and adult animals in large quantities ... [ and] may
furthermore be easily propagated in vitro." Id. at 19:53-56.
Bentzon '643 teaches that "[t]he genetic modifications are introduced
in the somatic cell prior to cell nuclear transfer." Id. at 9:39--40. Bentzon
'643 further teaches that the modifications "may be targeted to a specific
region in the porcine genome by homologous recombination of a targeting
construct or by gene editing procedures," and "could be inactivation ( e.g.
knock-out) of specific genes that will cause a disease or phenotype" (id. at
9:65-10:3). In addition, although Bentzon '643 does not explicitly disclose
transfected cells or transgenic pigs that are free of selectable marker genes,
the reference does teach that such genes are removable. For instance,
Bentzon '643 discloses that "the recombinant vector construct may also
comprise at least one site for Cre recombinase" (id. at 21: 11-12), and "[t]he
presence of two Cre recombination sites allows Cre recombinase-mediated
cassette exchange ... thereby facilitating, if needed, removal of plasmid
sequences and selection genes" (id. at 49:9--13).
According to Bentzon '643,
[I]mproved procedures [ are provided] for cloning pigs by
nuclear transfer which refers to the introduction of a full
complement of nuclear DNA from one cell to an enucleated
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cell. The genetically modified pig of the present invention may
be produced using any technique in which modified genetic
material, transcriptional product and/ or translational product or
part thereof is transferred from [a] donor cell to a host cell, such
as an enucleated oocyte. A number of techniques exist such as
introducing genetic material from a genetically modified
somatic cell into an enucleated oocyte by for example
microinjection or by nuclear transfer.
In cloning, the transfer of the nucleus of a somatic (body)
cell or somatic cell into an egg cell ( oocyte) which has had its
own nucleus removed ( denucleated or enucleated) is called
somatic cell nuclear transfer. The new individual will develop
from this reconstructed embryo and be genetically identical to
the donor of the somatic cell.
Id. at 16: 17-32.
Example 2 of Bentzon '643 demonstrates production of ApoE
knockout porcine fibroblasts by adeno-associated virus-mediated
homologous recombination, initial screening of locus-specific targeting
events by PCR, and verification of locus-specific events by Southern blot.
Id. at 38:25--41:29. According to Bentzon '643, "[t]he resulting transgenic
ApoE knockout porcine fibroblasts were subsequently used for somatic cell
nuclear transfer (SCNT) by handmade cloning" (id. at 41 :30-32), but no
results are reported regarding production of ApoE knockout pigs.
Example 4 ofBentzon '643 describes a method of making LDLR
knockout porcine fibroblasts pig by homologous recombination, for use in
producing LDLR knockout pigs:
Part of the pig LDL receptor coding sequence has been
sequenced (GenBank accession no. AF065990). A targeting
vector containing part of the gene sequence is created with a
promoter-less neomycin resistance gene cassette inserted into
one of the exons to disrupt gene function. The targeting vector
is linearized and transfected into Yucatan fetal fibroblasts.
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Fibroblasts are cultured in the presence of G418. Resistant
clones are screened for homologous recombination by PCR.
Yucatan minipigs with knockout of one or both apoE alleles are
created from recombinant cells by "hand-made" cloning.
Id. at 43:20-43.
Xu
Xu teaches that "[ c ]onditional knockouts in mice have been used
successfully to overcome embryonic lethality." Xu, 1.
Id.
To produce conditional knockouts, loxP sites are placed by
homologous recombination using a cotransfer type-targeting
construct ... into two introns that surround the coding sequence
or a critical protein domain. One of the loxP sites is associated
with a positive selection marker, most commonly a neo gene,
which is flanked by two loxP sites (floxed neo) to allow its
removal. Such a three lox construct can be directly introduced
into the desired locus by homologous recombination in ES cells
and then into the germline by standard techniques. However,
the presence of the neo gene in the intron often affects
endogenous gene expression and results in the reduction or
complete inactivation of the floxed genes .... Thus, it is
important to be able to remove the floxed-neo gene from
targeted loci whenever it is necessary.
Xu discloses two different approaches for direct removal of the ploxP-
neo-loxP cassette from introns in mice. Id., Abstract.
Li
Li discloses a reporter pig strain containing the EGFP gene driven by
the CMV promoter, where the EGFP gene is expressed only after Cre-
mediated excision of loxP-flanked stop sequences. Li, 232.
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DISCUSSION
Rejection I
Claims 1, 3, 6, 19--22, 26, 27, 35, and 39 stand rejected under
35 U.S.C. § 103(a) as unpatentable over Bentzon et al. or Bentzon '643, Xu,
and Li. Ans. 2-5.
As Appellants do not argue the claims separately, we focus our
analysis on independent claims 1 and 21, and claims 3, 6, 19, 20, 22, 26, 27,
35, and 39 will stand or fall with those claims. 37 C.F.R. § 41.37 (c)(l)(iv);
see also Appeal Br. 22-24 (indicating that the dependent claims are
patentable "for the same reasons as set forth with respect to claim 1 ").
Issue
The dispositive issue raised by this rejection is whether the Examiner
has established by a preponderance of the evidence that the teachings of
Bentzon et al. or Bentzon '643, together with the teachings of Xu and Li,
render obvious the production of the claimed transfected swine cells and
transgenic swine.
Discussion
The Examiner finds, in relevant part, that Bentzon '643 describes a
method of making a transfected swine cell comprising a knockout of an
endogenous LDLR, where the transfected cell is a fetal or adult pig
fibroblast, as well as a method of using the transfected cell to produce a
transgenic miniature pig comprising a knockout of an endogenous Low-
Density Lipoprotein Receptor Gene (LDLR). Ans. 3 (citing Bentzon '643,
cols. 3, 4, 19, 43 (Example 4)). The Examiner finds that Bentzon '643
teaches that the transgenic pig can be produced by somatic cell nuclear
transfer of the transgenic donor cell. Id. (citing Bentzon '643, cols. 19, 43).
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The Examiner also finds that Bentzon '643 "teaches to delete/disrupt
an exon of the endogenous LDL receptor gene in a pig, and ... the use of a
ere-lox system to genetically modify pigs and in particular a system which
allows for removal of a selection marker" (id. at 4 ( citing Bentzon '643, 21,
49) ), but acknowledges that the reference "does not specifically teach to use
the ere/lox system to delete/disrupt an exon in the LDL receptor gene in the
pig wherein the resulting pig is free of selectable marker genes." Ans. 4.
Nevertheless, the Examiner finds:
[ A ]t the time of filing, the prior art reported a number of
strategies for disrupting/deleting coding exons in genes of
interest in a transgenic animal, including pigs. Xu et al., for
example, teaches several inducible cre-loxP based knockout
approaches of a gene allele, Brea 1, in mice which further result
in the removal of the selection marker (Xu et al., pages 1---6).
Although the strategies taught by Xu et al. were exemplified in
mice, Li et al. demonstrates that Cre-loxP strategies can be used
successfully in the generation of transgenic pigs (Li et al., page
232).
Ans. 4.
The Examiner concludes that "it would have been prima facie obvious
... to delete/disrupt an exon in the LDL receptor gene in a pig using an
inducible cre-loxP system, and to further remove any inserted selection
marker gene with a reasonable expectation of success" (id.), "in view of the
teachings of Bentzon ['643] to delete/disrupt an exon of the endogenous
LDL receptor gene in a pig, the further teachings of Bentzon ['643] for the
use of a ere-lox system for targeted genetic modification of pigs and for the
removal of selectable markers" (id. at 7), together with "the teachings of Xu
et al. for cre-loxP based knockout approaches of a gene allele which result in
removal of selectable marker genes, and the demonstration by Li et al. that
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Cre-loxP strategies can be used successfully in the generation of transgenic
pigs" (id.).
Appellants do not dispute the Examiner's representation of Bentzon
'643 's disclosure. Rather, Appellants contend "the evidence in the Record
plainly demonstrates that the prior art was not enabling for the claimed
inventions." Appeal Br. 9. Based on an analysis of the Wands factors 5
(Appeal Br. 10-15), and relying on the Carlson and Hendrickson
Declarations, Appellants contend "there [was] no reasonable expectation of
success when using the references or state of the art at the time of filing for
making the claimed invention." Id. at 10. In addition, Appellants contend
"the claimed invention is not merely a predictable use of the prior art" (id.),
and the claimed invention would have required undue experimentation (id. at
11).
With respect to the Wands factors, Appellants contend "[t]he state of
knowledge of the art and its predictability pointed to a general expectation of
non-success for gene knockouts in pigs." Id. at 11. Appellants contend
"although cloning was well known (q.v. Dolly the Sheep), it effectively
required a primary somatic cell" and "[ w ]orking with primary cells is
difficult because genetic changes had to be made and verified ... before the
5 In re Wands, 858 F.2d 731, 737 (Fed. Cir. 1988). Factors to be weighed in
determining whether a disclosure is in compliance with the enablement
requirement include: (1) the quantity of experimentation necessary to
practice the invention; (2) the amount of direction or guidance presented; (3)
the presence or absence of working examples; (4) the nature of the
invention; ( 5) the state of the prior art; ( 6) the relative skill of those in the
art; (7) the predictability or unpredictability of the art; and (8) the breadth of
the claims.
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cells senesced." Id. (citing Carlson Deel. ,r 7). Appellants contend, in
practice, "some gene knockouts are fatal; primary cells have to be edited and
screened quickly but prior art processes were severely limiting; processes
commonly used in mice are not useful in the pig; and customary selection
processes were severely limiting." Id. at 12 (citing Carlson Deel. ,r 8).
In addition, Appellants contend that "chromosomal DNA is the
starting point for making a knockout" (id. at 13), but, according to Dr.
Hendrickson, "[ a ]t the time of Bentzon et al. there was no more than a
partial cDNA sequence available for the LDLR gene" (Hendrickson Deel.
3: 86-87). Moreover, Appellants contend that "conventional processes used
homology arms that have homology to target DNA ... typically 3000-8000
bases long" (id. at 14 ( citing Hendrickson Deel. 2: 60-71, 3: 110-111) ), but
these conventional processes failed-probably because repetitive elements
located within the homology arm promoted promiscuous hybridization
(Hendrickson Deel. 3:101-105). "[I]nstead, a short arm of298 bases" was
unexpectedly effective and successful. Appeal Br. 14 ( citing Hendrickson
Deel. 3: 116-117).
Furthermore, Appellants contend it was well known that the efficiency
of targeting DNA sequences varies from locus to locus (id. at 12 (citing
Carlson Deel. ,r 8) ), and "whether or not a knockout at a particular locus
would be successful or not was unknown" (id. at 13 (quoting Hendrickson
Deel. 4: 127-134)).
Finally, Appellants contend "[ t ]here are no working examples of the
experimental procedure in the cited references" with respect to the pig
LDLR gene (id. at 14}-that is, Bentzon '643 merely "aspires to knockout
LDLR but does not demonstrate making an animal with an LDLR knockout
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or a cell that could be used to make such an animal" (id.). Appellants
acknowledge that Bentzon '643 reports production of somatic cells with
"knockouts of a different gene, ApoE," but Appellants contend "by the time
the experiments were done, the somatic cells were high-passage cells, which
are not expected to be useful for cloning." Reply Br. 4. Indeed, Appellants
contend "knockouts in pigs were almost unknown----despite very many
attempts" (Appeal Br., 12), and "[o]nly a very few genes had been knocked
out in swine; roughly 4 of 20,000, which is about 0.02%" (id. at 13). In this
regard, Dr. Carlson states he is "aware of only four genes for which there
had been a report of a successful creation of a knockout pig by methods such
as those taught in the [Bentzon '643] reference" (Carlson Deel., 10), e.g., a-
1,3-galactosyltransferase, cystic fibrosis transmembrane receptor, and IgG
kappa light chain, disclosed by Lai, Rogers II, and Ramsoondar, 6
respectively (id.).
According to Appellants, "[t]his lack of efficiency very quickly
translates into undue experimentation and a discouragement to artisans to
make such attempts." Id. at 11. Furthermore, "[t]he success of targeting a
particular locus by the [Bentzon '643] reference methods is not expected
because it cannot be predicted." Id. at 12-13.
During patent prosecution, an examiner is entitled to reject claims
over a prior art publication or patent without conducting an inquiry into
whether or not that prior art reference is enabling, and, "[a]s long as an
examiner makes a proper prima facie case ... by giving adequate notice
under§ 132, the burden shifts to the applicant to submit rebuttal evidence of
6 Lai, Rogers II, and Ramsoondar accompanied Dr. Carlson's Declaration.
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nonenablement." In re Antor Media Corp., 689 F.3d 1282, 1289 (Fed. Cir.
2012).
Here, Bentzon '643 explicitly, albeit prophetically, teaches knocking
out the LDLR gene in miniature pig fetal fibroblasts by homologous
recombination, and using those transfected cells to produce a knockout pig
to serve as a model for atherosclerosis. Accordingly, we find that the
Examiner has established a proper prima facie case with respect to
enablement of Bentzon '643, shifting the burden to Appellants to establish
that Bentzon '643 is not enabling for producing a transfected somatic swine
cell comprising a gene knockout of an endogenous LDLR gene, or a
transgenic swine comprising a genomic knockout of an endogenous LDLR
gene. We determine that Appellants have not carried that burden.
An enabling specification "must teach those skilled in the art how to
make and use the full scope of the claimed invention without 'undue
experimentation."' In re Wright, 999 F.2d 1557, 1561 (Fed. Cir. 1993).
"That some experimentation may be required is not fatal; the issue is
whether the amount of experimentation required is 'undue."' In re Vaeck,
947 F.2d 488,495 (Fed. Cir. 1991). Some experimentation, even a
considerable amount, is not "undue" if, for example, the specification
provides a reasonable amount of guidance as to the direction in which the
experimentation should proceed. Wands, 858 F.2d at 737.
Again, Bentzon '643 explicitly teaches knocking out the LDLR gene
in miniature pig fetal fibroblasts by homologous recombination in order to
produce a knockout pig to serve as a model for atherosclerosis. Moreover,
we note that Bentzon '643 exemplifies the successful production of fetal pig
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fibroblasts with a knockout of the ApoE gene (see Example 2, discussed
above). 7
Although Appellants argue persuasively that transfecting somatic pig
cells is difficult, time consuming, and costly, that many gene knockouts are
fatal (and therefore unsuccessful), and that "the artisan must contemplate a
need to 'bum' through many many pigs with a feedback loop of
success/failure/modify/retry of at least four months per round of cloning"
(Appeal. Br. 11 ), these considerations relate principally to the amount and
cost of experimentation likely required, rather than the direction in which the
experimentation should proceed. As discussed above, even a considerable
amount of experimentation is not "undue" if the specification provides a
reasonable amount of guidance as to the direction in which the
experimentation should proceed. Wands, 858 F.2d at 737.
With respect to Appellants' contention that Bentzon '643 discloses
cDNA for the pig LDLR, rather than genomic DNA, and that Appellants'
initial attempts to obtain the genomic sequence through PCR failed because
of repetitive elements in the pig LDLR sequence, we find that the Examiner
has provided persuasive evidence that "there were many methods considered
routine in the art for obtaining genomic sequence using partial cDNA
sequence that do not use PCR." Ans. 11. Specifically, the Examiner
provides evidence that "Southern blot cloning of cDNA and genomic DNA
7 We note Appellants' argument that "the distinctions between a Gain-of-
Function versus a Knockout have to be appreciated" (Appeal Br. 11 ), but we
are not persuaded the distinctions are relevant here, as Bentzon '643
discloses gene knockouts as well as gain-of-function modifications (see
Example 2 of Bentzon '643).
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libraries was considered routine at the time of filing and did not require
foreknowledge of a complete cDNA sequence," and "has been further
demonstrated to work successfully in isolating genomic clones for genes
with highly repetitive elements." Id. (citing Banki8). Appellants have not
addressed this evidence.
With respect to Appellants' assertion that only about four of 20,000
swine genes had been knocked out at the time of filing, we agree with
Appellants that the calculation has little or no meaning absent some evidence
of the number of pig genes subjected to knockout attempts (see Reply Br. 3).
Appellants, through Dr. Carlson, submitted several references (e.g., Lai,
Rogers II, and Ramsoondar (Carlson Deel. 10-11)) as evidence of the rarity
of knockouts in pigs. Nevertheless, as we find the Examiner has established
a prima facie case that Bentzon '643 is enabling, it is Appellants' burden to
establish that number of successful knockouts was or was not reasonably
proportional to the number of attempts (see Antor Media, 689 F.3d at 1289).
Appellants have not done so.
Moreover, we agree with the Examiner that the art cited by Appellants
shows that "unrelated and substantially different genes were successfully
targeted in pig donor cells which were then used to make transgenic
knockout pigs" using somatic cell nuclear transfer (Ans. 15), and that neither
the Carlson Declaration nor the Hendrickson Declaration "provides any
specific evidence that the LDLR gene differs sign[i]ficantly from any of
these other gene loci such that the skilled artisan would not have had a
8 The Examiner provided Banki as rebuttal evidence with the non-final
Office Action of October 6, 2015.
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reasonable expectation of success in making an LDLR knockout pig at the
time of filing" (id. at 14--15). By way of example, we note that Rogers II
"used homologous recombination in fibroblasts of outbred domestic pigs to
disrupt the CFTR gene and somatic cell nuclear transfer to generate
CFTR+I- pigs." Rogers II, 2. Similarly, Lai discloses production of a-1,3-
galactosyltransferase knockout pigs by nuclear transfer cloning using clonal
fetal fibroblasts (Lai, 1089), and also teaches that "[t]he ability to use
cryopreserved donor cells without further culture ... [is] advantageous, as it
extends the number of potential donor lines available for use in nuclear
transfer" (id. at 1091).
Appellants have not established persuasively that producing a
transfected somatic swine cell comprising a gene knockout of an
endogenous LDLR gene, or a transgenic swine comprising a genomic
knockout of an endogenous LDLR gene, would have required undue
experimentation, given Bentzon '643 's teachings and the state of the art at
the time of filing. Therefore, Appellants have not carried their burden of
establishing that Bentzon '643 does not enable production of transfected
swine cells and transgenic swine.
Turning to the claims' requirement that the transfected cells and
transgenic animals are "free of selectable marker genes," Appellants argue
that "the Cre/loxP or FR T /FLP systems ... are not directed to specific target
sites." Appeal Br. 16. Rather, "the loxP or FRT site is inserted without
specificity and then the CRE or FLP reacts specifically with the loxP or FR T
sites." Id. Appellants argue that the Examiner's "rationale errs in fact by
incorrectly assuming that ere-lox can be used to target a specific gene such
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as LDLR and knock it out," but "the lox site is necessarily inserted
randomly." Reply Br. 6. Appellants contend:
Id.
For instance, in Li et al., a gene cassette flanked by lox is
randomly inserted. Then ere can be used to remove the gene
cassette. Cre acts with specificity towards lox but for nothing
else. Xu does not cure these defects because it uses the Cre-lox
systems in mice, which is useless for the claimed pig, in part
because embryonic stem (ES) cells in mice can be easily
manipulated but there are no comparable techniques in swine.
Appellants' arguments are not persuasive. The Examiner's rationale
is not, as Appellants' argue, that ere-lox can be used to target a specific gene
... and knock it out." Reply Br. 6. Rather the Examiner contends Bentzon
'643, in addition to "teach[ing] knocking out ... gene alleles using
homologous recombination in the donor cell followed by nuclear transfer"
(Ans. 3), "also teaches that site-specific modification can be utilize[d] [for]
the insertion of recombination sites for ... enzymes such as FLP or CRE,
and can be further combined to allow removal of selection genes" (id. at 3--4
(citing Bentzon '634, 21, 49)). Indeed, the Examiner confirms that "Li was
not cited for using cre-loxP to knock out a gene in a pig," but "for teaching
that cre-loxP systems can be successfully introduced into the genome of pigs
and that expressed ere is functional in mediating loxP excision in transgenic
pigs" (Ans. 17). The Examiner's findings are consistent with the teaching in
Bentzon '643 that the Cre-Lox system may be used for removal of selection
genes. See Bentzon '643, 49:9--13.
Accordingly, the rejection of claims 1, 3, 6, 19--22, 26, 27, 35, and 39
as unpatentable over Bentzon et al. or Bentzon '643, in view of Xu and Li is
affirmed.
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Re} ection II
Claim 7 stands rejected under 35 U.S.C. § 103(a) as unpatentable over
Bentzon '643, Xu, Li, and Ishibashi (Ans. 5-7).
Claim 7 depends from claim 1 and specifies that the LDLR gene is
disrupted at exon 4.
Appellants contend that "Claim 7 is patentable for the same reasons as
set forth with respect to claim 1," and "Ishibashi does not make up for the
defects of the other references." Appeal Br. 24.
As we are not persuaded that claim 1 is patentable over the cited art
for the reasons discussed above, the rejection of claim 7 under 35 U.S.C.
§ 103(a) as unpatentable over Bentzon '643, Xu, Li, and Ishibashi is
affirmed for the same reasons.
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SUMMARY
The rejection of claims 1, 3, 6, 19-22, 26, 27, 35, and 39 as
unpatentable over Bentzon et al. or Bentzon '643, in view of Xu and Li is
affirmed; and
The rejection of claim 7 as unpatentable over Bentzon et al. or
Bentzon '643, in view of Xu, Li, and Ishibashi is affirmed.
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
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