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Institut Pasteur & Universite Pierre Et Marie Curie v. Focarino

United States Court of Appeals, Federal Circuit

December 30, 2013

Margaret A. FOCARINO, Commissioner for Patents, Appellee, and Precision Biosciences, Inc., Appellee.

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Thomas H. Jenkins, Finnegan, Henderson, Farabow, Garrett & Dunner, LLP, of Washington, DC, argued for appellant. With him on the brief was Kenneth J. Meyers, of Washington, DC and Amelia F. Baur, of Boston, Massachusetts.

Mary L. Kelly, Associate Solicitor, United States Patent and Trademark Office, of Alexandria, Virginia, argued for appellee Margaret A. Focarino, Commissioner for Patents. With her on the brief were Nathan K. Kelley, Acting Solicitor, and Kristi L.R. Sawert, Associate Solicitor.

Michael J. Twomey, Wilmer Cutler Pickering Hale and Dorr, LLP, of Boston, Massachusetts, argued for appellee Precision BioSciences, Inc. Of counsel was Andrej Barbic.

Before NEWMAN, CLEVENGER, and TARANTO, Circuit Judges.

TARANTO, Circuit Judge.

The Institut Pasteur owns U.S. Patent Nos. 7,309,605, 6,610,545, and 6,833,252, which claim methods and tools for the site-directed insertion of genes into eukaryotic chromosomes. Precision BioSciences requested inter partes reexamination of each of the patents, and the Patent and Trademark Office examiner rejected a number of Pasteur's claims for obviousness, under 35 U.S.C. ยง 103. On appeal, the Board of Patent Appeals and Interferences (now the Patent Trial and Appeal Board) affirmed the rejections, concluding that the claimed inventions were obvious extensions of two prior-art references disclosing similar methods of targeting non-chromosomal DNA in prokaryotic cells. Pasteur appeals the Board's conclusions to this court. For the '605 patent, we dismiss Pasteur's appeal as moot, because Pasteur presented only substantively amended claims to the Board and to this court, and amended claims cannot be entered now that the

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patent has expired. For the '545 patent, we reverse the Board's conclusion as based on factual findings unsupported by substantial evidence and an erroneous obviousness analysis, including an improper discounting of Pasteur's objective indicia of non-obviousness. For the '252 patent, we vacate the Board's decision and remand for consideration of what motivation, if any, a skilled artisan at the relevant time would have had to pursue the claimed invention.


In the early 1990s, scientists at the Institut Pasteur made a series of inventions that allowed for the insertion, deletion, or modification of genes at targeted locations in the chromosomes of living cells. Specifically, Pasteur discovered a class of enzymes— group I intron-encoded (GIIE) endonucleases— that cleave both backbones of the DNA double helix at the location of a specific nucleotide sequence, called a recognition site. Pasteur established, first, that GIIE endonucleases can cleave DNA in the chromosomes of eukaryotic (nucleus-possessing) cells and, second, that eukaryotic cells can successfully repair such cleavages by initiating a process known as homologous recombination. This process uses a DNA template whose sequence is, in certain places, highly similar ( i.e., homologous) to that of the cleaved chromosomal DNA. It requires that the DNA template be homologous only at two portions, which must, respectively, match the regions of the pieces of the broken DNA on either side of the break. When such matching occurs, the whole template sequence, including the nucleotides lying between the matching portions, gets copied to become part of a rejoined DNA molecule in a chromosome. The cell faithfully reproduces the DNA template, including the interior nucleotides, which need not match the broken DNA at all. In this way, it is possible to design a DNA template that adds a DNA sequence to the chromosome or makes specific alterations to its sequence.

GIIE endonucleases are particularly well suited for genetic engineering. Their recognition sites, i.e., the nucleotide sequences at which they cleave DNA, are much longer than most other endonucleases. Whereas other classes of endonucleases recognize and bind to DNA sequences as short as four to eight nucleotides long, the recognition sites of GIIE endonucleases extend over eighteen nucleotides. This makes GIIE endonucleases much more discriminating in where they cleave a DNA molecule. By sheer probability, a given sequence of eight nucleotides occurs much more frequently than one of eighteen nucleotides. So whereas other endonucleases tend to cleave an organism's DNA at many different sites, GIIE endonucleases provide far superior specificity.

Pasteur first encountered GIIE endonucleases in yeast mitochondria, which are membrane-enclosed structures found within most eukaryotic cells. Although most of an organism's DNA is contained in chromosomes located in the cell nucleus— a different membrane-enclosed cellular structure— mitochondria have a small amount of their own DNA. Pasteur identified a specific DNA sequence that is repeatedly copied from one location in the mitochondrial DNA to another location in that DNA, i.e., a mobile genetic sequence. The mobile DNA sequence that Pasteur identified resided in the introns of mitochondrial genes, i.e., those nucleotides in a gene that generally do not code for the protein that the gene expresses. After analyzing this mobile intronic sequence, Pasteur determined that it coded for an endonuclease, an enzyme that cleaves DNA backbones. Pasteur named the newly discovered endonuclease " I-SceI," and

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the new class became known as group I intron-encoded endonucleases.

Pasteur recognized that GIIE endonucleases could be useful laboratory tools and set out to determine whether they could cleave DNA other than the yeast mitochondrial DNA that they naturally cleave. First, Pasteur inserted an artificial GIIE endonuclease recognition site into a yeast chromosome and demonstrated that the GIIE endonuclease cleaved yeast chromosomes after they had been extracted from yeast cells and purified. See, e.g., '545 patent, col. 15, line 63, to col. 16, line 8. Next, Pasteur created a plasmid— a small DNA molecule that is separate from and can replicate independently of chromosomal DNA— containing an artificial GIIE endonuclease recognition site and injected the plasmid into the yeast nucleus. The GIIE endonuclease successfully cleaved the plasmid. See, e.g., id., col. 18, lines 35-39. Finally, and of particular significance, Pasteur found that GIIE endonucleases could cleave chromosomes in living cells and that, if DNA homologous to the cleavage site was present or added, the cell could repair the break using homologous recombination. See, e.g., id., col. 18, lines 42-53.

Pasteur filed a series of patent applications relating generally to GIIE endonucleases and methods of using them to insert DNA at a targeted location in an organism's DNA. All of the patents at issue here— the '605, '545, and ' 252 patents— claim priority to the same patent application, which was filed on May 5, 1992, and is now abandoned. All three patents expired on May 6, 2012. The '605 and '252 patents have a common specification, which differs in only minor ways from the specification of the '545 patent. Each specification describes first introducing a GIIE recognition site into a cell's chromosomal DNA. See, e.g., '545 patent, col. 18, line 43, to col. 19, line 64. By introducing into the cell both (a) a GIIE endonuclease and (b) a plasmid that is homologous to the cleavage site and contains the DNA sequence to be inserted, the Pasteur inventors taught, it is possible to achieve the " site specific insertion of a DNA fragment from a plasmid into a chromosome." Id., col. 19, lines 42-44.

The '605 patent claims methods for using a GIIE endonuclease to cleave DNA at a specific location and (for some claims) the subsequent insertion of DNA. During reexamination, Pasteur proposed amendments to claim 1 and the dependent claim now at issue, claim 14. Specifically, the proposed amendments would limit the claims to targeting chromosomal DNA in viable cells. Claim 1 reads as follows, with the amendments underlined:

1. A method for inducing at least one site directed double-stranded break in the chromosomal DNA of an organism comprising:
(a) providing an isolated, viable cell of said organism containing at least one Group I intron encoded endonuclease recognition site at a location in the chromosomal DNA of the cell,
(b) providing said Group I intron encoded endonuclease to said cell by genetically modifying the cell with a nucleic acid comprising said Group I intron encoded endonuclease or by introducing said Group I intron encoded endonuclease protein into the cell such that the Group I intron encoded endonuclease cleaves said Group I intron encoded endonuclease site at the location in the DNA of the cell.

'605 patent, col. 69, lines 22-35; see also J.A. 79 (as amended). Although claim 1 does not claim the insertion of new DNA sequences into the cleaved DNA, dependent claim 14, the only claim now at issue, adds that element. It reads (also with a proposed amendment underlined):

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14. The method of claim 1, wherein said method further comprises providing to said cell

a plasmid comprising a DNA sequence homologous to the sequence of the chromosome, which allows homologous recombination, and

a modified sequence,
wherein said Group I intron encoded endonuclease cleaves the Group I intron encoded endonuclease recognition site,
whereby said cleavage promotes the insertion of said modified sequence into said chromosomal DNA of said cell at a specific site by homologous recombination.

'605 patent, col. 70, lines 26-39; see also J.A. 4029 (as amended).

The '545 patent similarly claims methods for the sitespecific insertion of DNA sequences into the chromosomes of living cells. As amended during reexamination, Claim 7 reads:

7. A method for in vivo site directed genetic recombination in an organism comprising:
(a) providing a transgenic eukaryotic cell having at least one Group I intron encoded endonuclease recognition site inserted at a unique location in a chromosome;
(b) providing an expression vector that expresses said endonuclease in said transgenic cell;
(c) providing a plasmid comprising a gene of interest and a DNA sequence homologous to the sequence of the chromosome, ...

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