DETAILED ACTION
Notice of Pre-AIA or AIA Status
1. The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .2. This action is in response to the amendment filed on 03 February 2026. Applicant's arguments and amendments to the claims have been fully considered but do not place the application in condition for allowance. All rejections not reiterated herein are hereby withdrawn.
Claim Status
3. Note that in the reply filed on 12 September 2025, Applicant elected without traverse the F8, F9 and VWF genes and the combination of primers of SEQ ID NO: 1 and SEQ ID NO: 173 for the Factor VIII (F8) gene; SEQ ID NO: 88 and SEQ ID NO: 260 for Factor IX (F9) gene; and SEQ ID NO: 105 and SEQ ID NO: 277 for VWF gene.
Claims 1-4, 6-11 and 13-20 are pending and have been examined herein to the extent that the claims encompass that the detecting step requires each of the primers of SEQ ID NO: 1 and 173 for the F8 gene; SEQ ID NO: 88 and 260 for the F9 gene and SEQ ID NO: 105 and 277 for the VWF gene. The claims encompass the non-elected species of the additionally recited combinations of primers. Prior to the allowance of claims, any non-elected subject matter which has not been rejoined with the elected subject matter will be required to be removed from the claims.
Non-Compliant Amendment / Response
4. In the reply of 03 February 2026, Applicant included the “Remarks” section immediately following claim 20. However, as set forth in MPEP 714, each section of a response, including the section with claim amendments and the section with remarks, should appear on a separate page. See MPEP 714:
“(h) Amendment sections. Each section of an amendment document (e.g., amendment to the claims, amendment to the specification, replacement drawings, and remarks) must begin on a separate sheet.”
New / Modified Claim Rejections - 35 USC § 112(b) - Indefiniteness
5. The following is a quotation of 35 U.S.C. 112(b):
(b) CONCLUSION.—The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the inventor or a joint inventor regards as the invention.
The following is a quotation of 35 U.S.C. 112 (pre-AIA ), second paragraph:
The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the applicant regards as his invention.
Claims 1-4, 6-11 and 13-20 are rejected under 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph, as being indefinite for failing to particularly point out and distinctly claim the subject matter which the inventor or a joint inventor (or for applications subject to pre-AIA 35 U.S.C. 112, the applicant), regards as the invention.
Claims 1-4, 6-11 and 13-20 are indefinite and vague because it is not clear as to whether “the subject” recited in steps (b), (c) and (d) is limited to a human subject (as recited in (a)) or encompasses any subject as recited in the preamble of the claims.
Claims 1-4, 6-11 and 13-20 are indefinite over the recitation in (b) of “a sample obtained from a subject” because it is not clear as to whether the sample obtained from a subject in (b) is the same as or distinct from the “sample derived from a human subject” in (a).
Claims 6, 7, 13, 14, 19 and 20 are further indefinite over the recitation that steps (b)-(d) are performed within 48 hours or 5 days of receipt of the sample derived from the subject because while the claims as amended recite a step of receiving a sample derived from the subject, steps (b)-(d) are performed using “a sample obtained from the subject.” Since the claims do not recite a nexus between the received sample derived from a human subject and a sample obtained from the subject, it is unclear as to when the sample in (b) is obtained and by whom it is received and thereby it is unclear as to when the time period of 48 hours to 5 days begins.
Response to Remarks:
The response states “Claims 1, 8 and 15 have been amended to recite the step of receiving a sample derived from a subject. claims 6, 7, 13, 14, 19 and 20 have been amended to clarify that the steps are performed within 48 hours or 5 days from receipt of the sample.”
However, this amendment does not obviate the prior rejection. As set forth above, the claims as amended recite a step (a) of receiving a sample derived from a human subject and a step (b) of “a sample obtained from the subject.” The claims do not recite a nexus between the sample of (a) and the sample of (b). If the sample of (b) is intended to be different from the sample in (a), then it remains unclear as to who receives the sample obtained from the subject (i.e., a subject per se and not necessarily a human subject) and it is unclear as to whether the sample obtained from the subject may be stored for any period of time and then later received at a laboratory and the time range recited in the claims is that which occurs between receipt of the sample by the laboratory and when steps (b)-(d) are performed. Thus, it is unclear as to what constitutes the time period of 48 hours to 5 days.
Maintained / Modified Claim Rejections - 35 USC § 112(a) - Written Description
6. The following is a quotation of the first paragraph of 35 U.S.C. 112(a):
(a) IN GENERAL.—The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same, and shall set forth the best mode contemplated by the inventor or joint inventor of carrying out the invention.
The following is a quotation of the first paragraph of pre-AIA 35 U.S.C. 112:
The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same, and shall set forth the best mode contemplated by the inventor of carrying out his invention.
Claims 1-4, 6-11 and 13-20 are rejected under 35 U.S.C. 112(a) or 35 U.S.C. 112 (pre-AIA ), first paragraph, as failing to comply with the written description requirement. The claim(s) contains subject matter which was not described in the specification in such a way as to reasonably convey to one skilled in the relevant art that the inventor or a joint inventor, or for applications subject to pre-AIA 35 U.S.C. 112, the inventor(s), at the time the application was filed, had possession of the claimed invention. This is a written description rejection.
In analyzing the claims for compliance with the written description requirements of 35 U.S.C. 112, first paragraph, a determination is made as to whether the specification contains a written description sufficient to show they had possession of the full scope of their claimed invention at the time the application was filed.
To provide adequate written description and evidence of possession of a claimed genus, the specification must provide sufficient distinguishing identifying characteristics of the genus. The factors to be considered include disclosure of a complete or partial structure, physical and/or chemical properties, functional characteristics, structure/function correlation, and any combination thereof.
In the present situation, with respect to the elected species, the claims are drawn to methods for determining a subject’s risk for developing hemophilia A, hemophilia B, or von Willebrand disease (VWD) comprising amplifying nucleic acids from the Factor 8 (F8) gene, the Factor IX (F() gene, and the von Willebrand factor (VWF) gene using combination of SEQ ID NO: 1 and SEQ ID NO: 173 for the F8 gene; SEQ ID NO: 88 and SEQ ID NO: 260 for F9 gene; and SEQ ID NO: 105 and SEQ ID NO: 277 for VWF gene, comparing the amplified F8, F9, and VWF genes with amplified F8, F9 or VWF gene sequences from a normal control reference sequence, and determining a subject’s risk of developing hemophilia A, hemophilia B or VWD based on detecting a “pathogenic or likely pathogenic” germline alterations in the F8, F9 or VWF genes in the sample that are not present in a normal reference sequence.
The claims encompass detecting a potentially significantly large genus of germline alterations in the F8, F9 or VWF genes, and particularly pathogenic or likely pathogenic germline mutations that are diagnostic of hemophilia A, hemophilia B, or VWF, which are not defined in terms of any specific structural properties or any other relevant identifying characteristics.
The size of the genus is further expanded by the fact that the claims encompass detecting the gene alterations in any subject, including any non-human subjects. It is well accepted that genes and gene alterations that cause or are associated with diseases may vary between organisms. Thereby, the germline alterations in the F8, F9 and VWF genes that are correlated with hemophilia A, hemophilia B, and/or VWD, in human subjects may be distinct from those present in other mammals, such as dogs, cats, goats and horses. Note that the claims were amended to recite a step (a) of receiving a sampled derived from a human subject, steps (b)-(d) of the claims recite more generally “the subject.” Since the preamble of the claim recites “a subject” - i.e., a human or non-human subject and the sample in (b)-(d) is not necessarily the same sample as in (a), the claims still encompass methods wherein the subject is a non-human subject.
The specification does not identify any particular germline alterations that are present in the amplification products generated by the nucleic acids of SEQ ID NO: 1, 173, 88, 260, 105 and 277. While Table 1 of the specification discloses a limited number of alterations that may be detected using some of the primer pairs set forth therein, this Table does not list any germline alterations that are present in the F8 gene sequences amplified by the combination of primers of SEQ ID NO: 1 and 173, in F9 gene sequences amplified the combination of primers of SEQ ID NO 88 and 260, or in the VWF gene amplified by the combination of the primers of SEQ ID NO: 105 and 277. Nor does the specification clearly indicate that the alterations listed in Table 1 are necessarily germline alterations, as opposed to somatic alterations, and that each of these alterations are diagnostic of hemophilia A, hemophilia B, and/or VWD.
The specification discloses the detection of a c.2440C>T, p.Arg814Ter variant in the F8 gene, generating a premature stop codon, which variant is known in the prior art to be associated with hemophilia A (para [0037]).
The present application is a CIP of U.S. Application 16/676,041 and discloses additional mutations in the F9 gene. In Example 2, the specification teaches using each of the primers of SEQ ID NO: 1-344, in pools of 57 or 58 sets of primers to amplify the F8, F9 and VWF genes.
One of the alterations detected in the above method is a c.580A>G, p.Thr194Ala alteration in the F9 gene. The specification (para [0040] states that “(t)his polymorphism is present in about 15% of the general population. The polymorphism was detected in 48% of the total reads, indicating this donor is heterozygous for this variant. This polymorphism does not correlate with hemophilia.”
The method detected in patient 3 a nonsense variant (c.100C>T, p.Arg34Ter) in exon 3 of the VWF gene, which had been previously reported (para [0043]). A nonsense variant (c.880C>T, p.Arg294Stop) in exon 8 of F9 was detected in patient 4, which had also been previously reported as being a pathogenic mutation, and is known as Arg248Ter. A missense variant (c.1537G>A, p.Gly513Ser) was detected in exon 10 of the F8 gene in patient 5 and was classified as “likely pathogenic” Patient 6 had a heterozygous, missense variant (c.1481T>G, p.11e494Ser) in exon 10 of the F8 gene and the variant was classified as “likely pathogenic” using the ACMG-AMP criteria. Lastly, patient 7 was found to harbor a hemizygous, missense variant (c.2167G>A, p.A1a723Thr) in exon 14 of F8, which has been previously identified and is also known as p.A1a704Thr. It is stated that “(t)his variant is classified as pathogenic using the ACMG-AMP criteria and the ClinVar database” (para [0047]).
The prior art also teaches mutations in the F8, F9 and VWF genes that are correlated with the occurrence or risk of hemophilia A, hemophilia B and VWD, respectively.
However, the specification does not teach that the 6 disclosed pathogenic or “likely pathogenic” mutations, or any mutations known in the prior art, are in the regions of the F8, F9 and VWF genes amplified by the primers of SEQ ID NO: 1 and 173, 88 and 206, or 105 and 277. While the specification lists the exon location at which 5 of the mutations occur, there is no disclosure in the specification as to the particular nucleotide position in the F8, F9 and VWF genes at which the elected primer sequences hybridize and it does not appear that the primers hybridize to nucleotide sequences that generate an amplification product that includes the 6 disclosed mutations, or a representative number of additional mutations that may be disclosed in the prior art. Note again, that the mutations disclosed in the specification were obtained using the combination of all of the primers of SEQ ID NO: 1-344 to amplify the sequences of the F8, F9 and VWF genes.
Additionally, it is noted that not all alterations detecting by comparing a subject’s nucleic acid sequences for the F8, F9 or VWF genes with a control sample will constitute germline mutations diagnostic of hemophilia A, hemophilia B, and/or VWD. For example, see para [0040] of the specification which states:
“A common polymorphism, c.580A>G, p.Thr194Ala, was detected in the F9 gene from Patient 2. This polymorphism is present in about 15% of the general population. The polymorphism was detected in 48% of the total reads, indicating this donor is heterozygous for this variant. This polymorphism does not correlate with hemophilia.”
It is acknowledged that the specification teaches the general methodology for screening for alterations in nucleic acid sequences. However, possession may not be shown by merely describing how to obtain possession of members of the claimed genus or how to identify their common structural features.
Herein, no common structure has been disclosed to identify F8, F9 and VWF gene sequences which meet the criteria of being a pathogenic or likely pathogenic germline alteration that is diagnostic of hemophilia A, hemophilia B, and/or VWD within the region amplified by the elected primer pairs.
With respect to the present invention, there is no record or description which would demonstrate conception of a representative number of germline alterations within the broadly claimed genus. Therefore, the claims fail to meet the written description requirement because the claims encompass a significantly large genus of germline mutations within the region amplified by the elected primer pairs which are not described in the specification. Response to Remarks:
In the response, Applicant points to the specification as teaching 1 pathogenic mutation in the F9 gene; 1 pathogenic mutation in the VWF gene, and 4 pathogenic mutations in the F8 gene. It is argued that the specification (para [0033]) states that sequence variations can be classified as pathogenic or predicted pathogenic as recommended by the American College of Medical Genetics. It is stated “This provides written description for the amended limitation requiring determination based on pathogenic or likely pathogenic germline alterations.”
However, while the specification provides literal basis for the amendment to the claims to recite pathogenic or likely pathogenic germline alterations, the cited teachings in the specification do not establish that Applicant was in possession of a representative number of the pathogenic or likely pathogenic germline alterations in the F8, F9 and VWF genes encompassed by the claims which are amplified by the primers of SEQ ID NO: 1 and 173, 88 and 206, or 105 and 277. The response does not indicate that the 6 mutations disclosed in the specification and addressed in the response occur in the regions amplified by the elected primer pairs of SEQ ID NO: 1 and 173, SEQ ID NO: 88 and 206, or SEQ ID NO: 105 and 277. As discussed above, the 6 mutations disclosed in the specification were obtained using the combination of all of the primers of SEQ ID NO: 1-344 to amplify the sequences of the F8, F9 and VWF genes. Further, the response does not establish that the 6 mutations disclosed in the specification are representative of the broadly claimed genus of any pathogenic or likely pathogenic germline alteration that is present at any location in the coding or non-coding sequences of the F8, F9 and VWF genes and which are diagnostic of hemophilia A, hemophilia B or von Willebrand disease (VWD).
Maintained / Modified Claim Rejections - 35 USC § 103
7. In the event the determination of the status of the application as subject to AIA 35 U.S.C. 102 and 103 (or as subject to pre-AIA 35 U.S.C. 102 and 103) is incorrect, any correction of the statutory basis for the rejection will not be considered a new ground of rejection if the prior art relied upon, and the rationale supporting the rejection, would be the same under either status.
The following is a quotation of 35 U.S.C. 103 which forms the basis for all obviousness rejections set forth in this Office action:
A patent for a claimed invention may not be obtained, notwithstanding that the claimed invention is not identically disclosed as set forth in section 102, if the differences between the claimed invention and the prior art are such that the claimed invention as a whole would have been obvious before the effective filing date of the claimed invention to a person having ordinary skill in the art to which the claimed invention pertains. Patentability shall not be negated by the manner in which the invention was made.
This application currently names joint inventors. In considering patentability of the claims the examiner presumes that the subject matter of the various claims was commonly owned as of the effective filing date of the claimed invention(s) absent any evidence to the contrary. Applicant is advised of the obligation under 37 CFR 1.56 to point out the inventor and effective filing dates of each claim that was not commonly owned as of the effective filing date of the later invention in order for the examiner to consider the applicability of 35 U.S.C. 102(b)(2)(C) for any potential 35 U.S.C. 102(a)(2) prior art against the later invention.
Claims 1-4, 6-11 and 13-20 are is/are rejected under 35 U.S.C. 103 as being unpatentable over Bastida et al (October 2016. Thromb Haemost. 117: 66-74) in view of Ero et al (U.S. 20150337377), GenBank Accession No AY769950 (NCBI Database, National Library of Medicine, NIH (Bethesda, MD, USA),available via URL: < ncbi.nlm.nih.gov/nucleotide/AY769950.1?report=genbank&log$=nucltop&blast_rank=2&RID=DNGTAF6U014>, 11 Dec 2004), GenBank Accession No AF536327 (NCBI Database, National Library of Medicine, NIH (Bethesda, MD, USA), available via URL: < ncbi.nlm.nih.gov/nucleotide/AF536327.1?report=genbank&log$=nucltop&blast_rank=2&RID=DNJRFSJA015>, 21 August 2002), GenBank Accession No AH005287 (NCBI Database, National Library of Medicine, NIH (Bethesda, MD, USA), available via URL: < ncbi.nlm.nih.gov/nucleotide/AH005287.2?report=genbank&log$=nucltop&blast_rank=2&RID=DNKPZ023014>, 10 June 2016) and Sigma-Aldrich (qPCR Technical Guide. 2008 Available via url: <gene-quantification.com/SIAL-qPCR-Technical-Guide.pdf>).
Bastida et al teaches a method for determining a subject’s risk of having or developing hemophilia A, hemophilia B, or VWD comprising obtaining a sample of genetic material from the subject, amplifying the genetic material using primers specific for the F8, F9 and VWF genes, determining the sequence of the amplified genetic material and detecting genetic alterations in the amplified genetic material as indicative of the subject having or at an increased likelihood of having, or as being a carrier of a risk allele for hemophilia A, hemophilia B, or VWD (see “Sanger sequencing (SS)” p. 68, col. 2). The PCR / Sanger Sequencing method of Bastida does not include a step of comparing the sequence determined for the F8, F9 and VWF genes of the subject to that of a reference control sequence. Further, Bastida teaches that this method is more expensive and has a longer turnaround time as compared to next generation sequencing (NGS) methods (p. 72, col 2 final para).
Bastida et al teaches a second method for determining a subject’s risk of having or developing hemophilia A, hemophilia B, or VWD comprising obtaining a sample of genetic material from the subject, performing targeted capture with probes specific for the F8, F9 and VWF genes, determining the sequence of the captured genetic material, comparing the determined sequence to that of healthy control subjects and to normal control sequences provided in public databases, and detecting genetic alterations in the F8, F9 and VWF captured genetic material as indicative of the subject having or at an increased likelihood of having, or as being a carrier of a risk allele for hemophilia A, hemophilia B, or VWD (see, e.g., p. 68-69 “DNA isolation”, “Library preparation and sequencing” and “NGS data analysis” and p. 72, col. 2). Bastida teaches that the total turnaround time for the NGS approach is “at least one week” after the samples have been collected (p. 72, col. 1). Bastida discusses including the target capture / NGS assay with the classic SS approach to improve molecular diagnosis (p. 72, col. 1).
The target capture / NGS method of Bastida does not include a step of amplifying the target F8, F9 and VWF genes using primers specific for the F8, F9 and VWF genes and is not a method in which a diagnosis or determination can be made within 5 days or 48 hours after the sample has been obtained.
However, Ero et al teaches methods for diagnosing a disease by simultaneously detecting mutations and polymorphisms in multiple target genes. The method comprises the steps of: obtaining a sample of genetic material from a patient; amplifying the genetic material in the sample using primers specific for the disease associated gene; determining the genetic sequence of the amplified genetic material of step; comparing the sequence of the amplified genetic material with a normal reference sequence; and identifying variations between the sample amplified genetic material and the normal reference sequence, wherein a variation between the sample amplified genetic material and the normal reference sequence is indicative of a risk for the patient for developing the disease (para [0009]). The method is characterized as one in which a diagnosis can be obtained within 48 hours or 5 days from receipt of the sample from the subject (para [0009]).
Ero teaches the advantages of the PCR assay stating:
“[0026] During the PCR process, the DNA generated is used as a template for replication. This sets in motion a chain reaction in which the DNA template is exponentially amplified. PCR can amplify a single or few copies of a piece of DNA by several orders of magnitude, generating millions or more copies of the DNA piece. PCR can be extensively modified to perform a wide array of genetic manipulations, as known by one of skill in the art.”
Ero further teaches that the advantage of the PCR / NGS method described therein is that it allows for the rapid diagnosis of disease so that patient management can be initiated more quickly (para [0007 - 0009]).
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have modified the method of Bastida so as to have performed an amplification step of the target genes using primer pairs specific for the F8, F9 and VWF genes, in place of the target capture step, and then to have performed next-generation sequencing on the amplified target DNA sequences, as is taught by Ero. One would have been motivated to have done so in order to have achieved the advantage set forth by Ero that the PCR amplification / NGS assay can be performed very rapidly, with results obtained within 48 hours or 5 days of obtaining a sample from a patient. Thereby, such a modification would have allowed practioners to have more quickly initiated treatment of those patients diagnosed as having or at risk of developing hemophilia A, hemophilia B, or VWD, or would have permitted practioners to rapidly alert those patients needing to know if they are genetic carriers for hemophilia A, hemophilia B, or VWD.
Regarding the amendment to the claims to recite that the germline alteration is pathogenic or likely pathogenic, Bastida teaches that the methods disclosed therein aloe pathogenic variants in HA, HB and VWD patients to be detected (e.g., Figure 1 and legend for Figure 1). Bastida (p. 68, col. 2) states:
Pathogenic variants were identified by searching the HGMD professional database and locus-specific mutation databases (LSDBs): HA (http: //www.factorviii-db.org), HB (http: //www.factorix.org) and/or http: //www.cdc.gov/ncbddd/haemophilia/champs.html) and VWD (https: //grenada.lumc.nl/LOVD2/VWF/home.php?select_db=VWF). The structural pathogenic impact of novel missense variants was defined according to the standards and guidelines of the consensus of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology.”
Secondly, Bastida teaches that previously designed / disclosed primers were used for PCR amplification of the F8, F9 and VWF gene sequences (p. 68, final para). Bastida does not teach that the primers for amplifying the F8 gene consist of the sequence of present SEQ ID NO: 1 and 173; the primers for amplifying the F9 gene consist of the sequence of present SEQ ID NO: 88 and 260 and the primers for amplifying the VWF gene consist of the sequence of present SEQ ID NO: 105 and 277.
However, the nucleotide sequences of the F8, F9 and VWF genes were known in the prior art and these nucleotide sequences include the sequences of the primers of present SEQ ID NO: 1, 173, 88, 260, 105 and 277.
For example, GenBank Accession No AY769950.1 (available via URL: < ncbi.nlm.nih.gov/nucleotide/AY769950.1?report=genbank&log$=nucltop&blast_rank=2&RID=DNGTAF6U014>, 11 Dec 2004) discloses the complete sequence of the human F8 gene which includes the sequence of the inverse complement of present SEQ ID NOs: 1 (“Query”) and the complete sequence of SEQ ID NO: 173 (“Query”), as shown in the alignments below:
SEQ ID NO: 1:
PNG
media_image1.png
200
850
media_image1.png
Greyscale
SEQ ID NO: 173:
PNG
media_image2.png
188
860
media_image2.png
Greyscale
GenBank Accession No. AF536327 discloses the sequence of the F9 gene, which includes the sequence of present SEQ ID NO: 88 (“Query”) and the inverse complement sequence of present SEQ ID NO: 260 (“Query”), as shown in the alignments below:
SEQ ID NO: 88:
PNG
media_image3.png
194
1104
media_image3.png
Greyscale
SEQ ID NO: 260:
PNG
media_image4.png
204
1070
media_image4.png
Greyscale
GenBank Accession No. AH005287 discloses the sequence of the VWF gene, which includes the inverse complement of the sequence of present SEQ ID NO: 105 (“Query”) and the sequence of present SEQ ID NO: 277 (“Query”), as shown in the alignments below:
SEQ ID NO: 105:
PNG
media_image5.png
202
892
media_image5.png
Greyscale
SEQ ID NO: 277:
PNG
media_image6.png
222
858
media_image6.png
Greyscale
Moreover, the prior art provides extensive guidance, direction and motivation to select primers to amplify a target nucleic acids.
In particular, Sigma-Aldrich provides a review of the parameters which effect PCR sensitivity and specificity and discloses the parameters for the design of optimal PCR primer sequences to be used in quantitative PCR (e.g., p. 10). It is disclosed that primers may be designed to be at least 17 nucleotides, with primers of a higher complexity ensuring a reduction in random priming (p. 10). It is disclosed that software programs are available to assist with primer and probe selection (e.g., p. 11 and 18). Sigma-Aldrich also teaches routine assays that may be performed to ensure that the optimal primers have been selected (e.g., p. 17 and 25-26). Sigma-Aldrich also teaches providing kits comprising the reagents necessary to perform PCR amplification and probe detection assays (e.g., p. 28).
Thus, the teachings of Sigma-Aldrich establish that it was well known in the prior art and well within the skill of the ordinary artisan to design PCR primers of optimum length and specificity for amplifying target nucleic acids.
Designing primers which are equivalents to those taught in the art requires only routine experimentation. The parameters and objectives involved in the selection of primers were well known in the art at the time the invention was made. Software programs were readily available which aid in the identification of conserved and variable sequences and in the selection of optimum primer pairs. The prior art is replete with guidance and information necessary to permit the ordinary artisan to design additional primers for the amplification of the F8, F9 and VWF nucleic acid sequences.
Accordingly, it would have been obvious to one of ordinary skill in the art at the time the invention was made to have further modified the method of Bastida so as to have amplified the F8, F9 and VWF genes using alternative primers consisting of the known sequences of the F8, F9 and VWF genes, including primers for amplifying the F8 gene consisting of the sequence of present SEQ ID NO: 1 and 173, primers for amplifying the F9 gene consisting of the sequence of present SEQ ID NO: 88 and 260, and primers for amplifying the VWF gene consisting of the sequence of present SEQ ID NO: 277. One would have been motivated to have done so in order to have facilitated the amplification of the F8, F9 and VWF genes and the detection of germline alterations in these genes. One would have had more than a reasonable expectation of success of generating such primers since the sequences of the F8, F9 and VWF genes were disclosed in the prior art and the prior art provides extensive guidance as to how to select primers to known sequences to amplify target nucleic acids.
Note that the present disclosure has not established any unexpected results obtained by performing the recited methods using the primers of SEQ ID NO: 1, 173, 88, 260, 105 and 277 and the claims do not require detecting any particular mutations with these primers.
Regarding claims 2-4, 9-11, and 16-18, Bastida teaches assaying for DNA sequence alterations that are mutations, polymorphisms and structural variants in the F8, F9 and VWF genes (see, e.g., Tables 1-3 and p. 71-72). Further, Ero teaches that the PCR amplification / NGS assay can detect mutations, polymorphisms and deletions in target gene sequences (e.g., para [0009-0011]). Accordingly, modification of the method of Bastida as set forth above would have resulted in a method that detects DNA sequence alterations in the F8, F9 and VWF genes that are mutations, polymorphisms or structural variants.8. Claims 1-4, 6-11 and 13-20 are is/are rejected under 35 U.S.C. 103 as being unpatentable over Tan et al (CN 109207573, published 15 January 2019; translation provided) in view of Ero et al (U.S. 20150337377), GenBank Accession No AY769950 (NCBI Database, National Library of Medicine, NIH (Bethesda, MD, USA),available via URL: < ncbi.nlm.nih.gov/nucleotide/AY769950.1?report=genbank&log$=nucltop&blast_rank=2&RID=DNGTAF6U014>, 11 Dec 2004), GenBank Accession No AF536327 (NCBI Database, National Library of Medicine, NIH (Bethesda, MD, USA), available via URL: < ncbi.nlm.nih.gov/nucleotide/AF536327.1?report=genbank&log$=nucltop&blast_rank=2&RID=DNJRFSJA015>, 21 August 2002), GenBank Accession No AH005287 (NCBI Database, National Library of Medicine, NIH (Bethesda, MD, USA), available via URL: < ncbi.nlm.nih.gov/nucleotide/AH005287.2?report=genbank&log$=nucltop&blast_rank=2&RID=DNKPZ023014>, 10 June 2016) and Sigma-Aldrich (qPCR Technical Guide. 2008 Available via url: <gene-quantification.com/SIAL-qPCR-Technical-Guide.pdf>).
Tan et al teaches a method for determining a subject’s risk of having or developing hemophilia A, hemophilia B, or VWD comprising obtaining a sample of genetic material from the subject, amplifying the genetic material using primers specific for the F8, F9 and VWF genes, determining the sequence of the amplified genetic material and detecting DNA sequence alterations in the amplified genetic material as indicative of the subject having or at an increased likelihood of having, or as being a carrier of a risk allele for hemophilia A, hemophilia B, or VWD (see e.g., abstract and para [0013], [0015], [0056] and [0058] of the translation). Tan teaches comparing the DNA sequence alterations detected in the subject’s sample to DNA sequence alterations known to be associated with hemophilia A, hemophilia B, or VWD, and particularly those set forth in databases of mutations correlated with hemophilia A, hemophilia B, or VWD (e.g., para [0007-0008] of the translation).
Tan also teaches identifying those mutations in the F8, F9 and VWF genes which are pathogenic (e.g., para [0005-0007], [0009] and [0061] of the translation).
Tan does not teach that the method comprises a step of comparing the sequence determined for the F8, F9 and VWF genes of the subject to that of a control, normal reference sequence.
However, Ero et al teaches methods for diagnosing a disease by simultaneously detecting mutations and polymorphisms in multiple target genes. The method comprises the steps of: obtaining a sample of genetic material from a patient; amplification of the sample of genetic material using primers specific for the disease associated gene; determining the genetic sequence of the amplified genetic material of step; comparing the sequence of the amplified genetic material with a normal reference sequence; and identifying variations between the sample amplified genetic material and the normal reference sequence, wherein a variation between the sample amplified genetic material and the normal reference sequence is indicative of a risk for the patient for developing the disease (para [0009]).
Further, Tan does state that new mutation sites are continually being identified in disease-causing genes. It is stated that mutation sites are highly polymorphic in the population and that disease-associated mutations are often population dependent, such that the different mutations are identified in different regions (e.g. para [0010] of the translation).
Accordingly, it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have modified the method of Tan so as to have also included a step of comparing the DNA sequence determined for the subject to that of a normal control reference sequence, as well as to the DNA sequences in databases, in order to identify new pathogenic DNA sequence alterations that are correlated with the occurrence or risk of hemophilia A, hemophilia B, or VWD.
Secondly, Tan does not teach that the primers for amplifying the F8 gene consist of the sequence of present SEQ ID NO: 1 and 173; the primers for amplifying the F9 gene consist of the sequence of present SEQ ID NO: 88 and 260 and the primers for amplifying the VWF gene consist of the sequence of present SEQ ID NO: 105 and 277.
However, Tan does exemplify primers for amplifying each of the F8, F9 and VWF gene sequences, including sequences of each of the exons, splicing regions and part of the promoter and intron regions (para [0030] of the translation and the Sequence Listing therein). Tan exemplifies a primer (SEQ ID NO: 298 therein; “Db” in alignment below) which is 25 nucleotides in length and comprises the inverse complement sequence of nucleotides 1-20 of present SEQ ID NO: 88 (“Qy” below)
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Further, the nucleotide sequences of the F8, F9 and VWF genes were well-known in the prior art.
For example, GenBank Accession No AY769950.1 (available via URL: < ncbi.nlm.nih.gov/nucleotide/AY769950.1?report=genbank&log$=nucltop&blast_rank=2&RID=DNGTAF6U014>, 11 Dec 2004) discloses the complete sequence of the human F8 gene which includes the sequence of the inverse complement of present SEQ ID NOs: 1 (“Query”) and the complete sequence of SEQ ID NO: 173 (“Query”), as shown in the alignments below:
SEQ ID NO: 1:
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SEQ ID NO: 173:
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860
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GenBank Accession No. AF536327 discloses the sequence of the F9 gene, which includes the sequence of present SEQ ID NO: 88 (“Query”) and the inverse complement sequence of present SEQ ID NO: 260 (“Query”), as shown in the alignments below:
SEQ ID NO: 88:
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SEQ ID NO: 260:
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GenBank Accession No. AH005287 discloses the sequence of the VWF gene, which includes the inverse complement of the sequence of present SEQ ID NO: 105 (“Query”) and the sequence of present SEQ ID NO: 277 (“Query”), as shown in the alignments below:
SEQ ID NO: 105:
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SEQ ID NO: 277:
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Moreover, the prior art provides extensive guidance, direction and motivation to select primers to amplify a target nucleic acids.
In particular, Sigma-Aldrich provides a review of the parameters which effect PCR sensitivity and specificity and discloses the parameters for the design of optimal PCR primer sequences to be used in quantitative PCR (e.g., p. 10). It is disclosed that primers may be designed to be at least 17 nucleotides, with primers of a higher complexity ensuring a reduction in random priming (p. 10). It is disclosed that software programs are available to assist with primer and probe selection (e.g., p. 11 and 18). Sigma-Aldrich also teaches routine assays that may be performed to ensure that the optimal primers have been selected (e.g., p. 17 and 25-26). Sigma-Aldrich also teaches providing kits comprising the reagents necessary to perform PCR amplification and probe detection assays (e.g., p. 28).
Thus, the teachings of Sigma-Aldrich establish that it was well known in the prior art and well within the skill of the ordinary artisan to design PCR primers of optimum length and specificity for amplifying target nucleic acids.
Designing primers which are equivalents to those taught in the art requires only routine experimentation. The parameters and objectives involved in the selection of primers were well known in the art at the time the invention was made. Software programs were readily available which aid in the identification of conserved and variable sequences and in the selection of optimum primer pairs. The prior art is replete with guidance and information necessary to permit the ordinary artisan to design additional primers for the amplification of the F8, F9 and VWF nucleic acid sequences.
Accordingly, it would have been obvious to one of ordinary skill in the art at the time the invention was made to have further modified the method of Tan so as to have amplified the F8, F9 and VWF genes using alternative or additional primers consisting of fragments of the known sequences of the F8, F9 and VWF genes, including primers for amplifying the F8 gene consisting of the sequence of present SEQ ID NO: 1 and 173, primers for amplifying the F9 gene consisting of the sequence of present SEQ ID NO: 88 and 260, and primers for amplifying the VWF gene consisting of the sequence of present SEQ ID NO: 277. One would have been motivated to have done so in order to have facilitated the amplification of the F8, F9 and VWF genes and the detection of germline alterations in these genes. One would have had more than a reasonable expectation of success of generating such primers since the sequences of the F8, F9 and VWF genes were disclosed in the prior art and the prior art provides extensive guidance as to how to select primers to known sequences to amplify target nucleic acids.
Note that the present disclosure has not established any unexpected results obtained by performing the recited methods using the primers of SEQ ID NO: 1, 173, 88, 260, 105 and 277 and the claims do not require detecting any particular mutations with these primers.
Regarding claims 2-4, 9-11, and 16-18, the method of Tan assays for DNA sequence alterations that are mutations, polymorphisms and structural variants in the F8, F9 and VWF genes (see, e.g., the tables at para [0031], [0044] and [0046] of the original document).
Regarding claims 6, 7, 13, 14, 19 and 20, Tan states that the method is one that provides rapid diagnosis of hemophilia A, hemophilia B, or VWD (e.g., para [0005], but Tan does not specifically state that the method is one in which a diagnosis or determination that a subject is at risk of having or developing, or being a genetic carrier for, hemophilia A, hemophilia B, or VWD can be made within 48 hours or 5 days after providing the sample from a subject.
However, since the method steps disclosed by Tan are the same as those recited in the present claims with respect to the amplification and sequencing steps, in the absence of evidence to the contrary, the method of Tan is also expected to have the property that the subject’s risk for having or developing, or being a genetic carrier of, hemophilia A, hemophilia B, or VWD. It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have performed the method of Tan such that a diagnosis or determination of a subject having or developing or being a genetic carrier for hemophilia A, hemophilia B, or VWD is made within a short time period, including 48 hours to 5 days, in order to provide the benefit that the treatment can be quickly initiated in those subjects diagnosed as having or at risk of developing hemophilia A, hemophilia B, or VWD, and to provide this essential information in those subjects in need of knowing if they are genetic carriers for hemophilia A, hemophilia B, or VWD.
Alternatively, the method of PCR amplification and sequencing disclosed by Ero is characterized as one in which a diagnosis or determination can be obtained within 48 hours or 5 days from receipt of the sample from the subject (para [0009]).
Ero teaches that the advantage of the PCR / NGS method described therein is that it allows for the rapid diagnosis of disease so that patient management can be initiated more quickly (para [0007 - 0009]).
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have modified the method of Tan so as to have performed the amplification and sequencing steps using the assay of Ero. Such a modification would have necessarily resulted in a method in which the diagnosis or determination of risk of a subject having or developing or being a carrier of risk allele for hemophilia A, hemophilia B, or VWD could be obtained within 48 hours or 5 days of obtaining a sample from a patient. One would have been motivated to have made this modification so as to have ensured that practioners could make the fastest possible diagnosis and then quickly initiated treatment of those patients diagnosed as having hemophilia A, hemophilia B, or VWD or quickly provided this information to those patients needing to know if they are a genetic carrier for hemophilia A, hemophilia B, or VWD.Response to remarks regarding the rejections under 35 U.S.C. 103:
The response traverses the rejections by arguing the references separately. The response states “Nothing in Bastida teaches or suggests replacing its capture-based methodology with a primer-specific PCR approach using the recited primer sets. The Office proposes modifying Bastida to employ Tan's PCR amplification methods; however, Tan likewise does not disclose or suggest the particular multi-gene amplification architecture required by the present claims, nor does Tan teach the use of the claimed primer sequences for the coordinated amplification of all three genes in a single diagnostic workflow.” The response states that the rejection relies on Ero for teaching comparing a test sequence to a reference sequence but asserts that Ero’s teachings are broad and “without the claimed clinical decision constraint requiring pathogenicity classification.”
These arguments and the amendments to the claims have been fully considered but are not persuasive.
In response to applicant's arguments against the references individually, one cannot show nonobviousness by attacking references individually where the rejections are based on combinations of references. See In re Keller, 642 F.2d 413, 208 USPQ 871 (CCPA 1981); In re Merck & Co., 800 F.2d 1091, 231 USPQ 375 (Fed. Cir. 1986). Herein, it is maintained that the cited references when considered as a whole would have suggested the claimed invention and would have provided both the motivation and guidance to arrive at the claimed invention.
Regarding the particularly recited primers, the rejection addresses the obviousness of these primers in view of the cited prior art. As noted in the rejection, the present disclosure has not established any unexpected results obtained by performing the recited methods using the primers of SEQ ID NO: 1, 173, 88, 260, 105 and 277 and the claims do not require detecting any particular mutations with these primers.
Regarding the amendment to the claims to recite that the determined sequences are compared to a control reference sequence, as acknowledged by Applicant, Ero does teach comparing the determined test sequence to a control, reference sequence. The teachings of Ero, while broad and not limited to a pathogenic mutation in the F8, F9 or VWF genes, is analogous art and applicable herein since Ero teaches methods for identifying pathogenic mutations diagnostic of a disease. Further, contrary to Applicant’s assertion, Ero does teach that the comparison of the test sequence to a reference sequence is part of the process for identifying a pathogenic mutation. For instance, Ero (para [0031]) states:
“Based on the sequence alignments and mapping results, sequence variants are identified based upon available information including for example, information from human mutational databases and other reference databases. Furthermore, any sequence variations, including aHUS-associated mutations, disease-associated polymorphisms, benign polymorphisms and other known variants of undetermined significance can be determined to be homozygous or heterozygous. Any variations in the gene analyzed as compared to the normal control gene can be classified as pathogenic, predicted pathogenic, uncertain, predicted benign or benign, as recommended by the American College of Medical Genetics (ACMG).”
Additionally, Bastida does teach identifying mutations in the F8, F9 and VWF genes that are pathogenic. For example, Bastida (p. 68, col. 2) states:
Pathogenic variants were identified by searching the HGMD professional database and locus-specific mutation databases (LSDBs): HA (http: //www.factorviii-db.org), HB (http: //www.factorix.org) and/or http: //www.cdc.gov/ncbddd/haemophilia/champs.html) and VWD (https: //grenada.lumc.nl/LOVD2/VWF/home.php?select_db=VWF). The structural pathogenic impact of novel missense variants was defined according to the standards and guidelines of the consensus of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology.”
Similarly, Tan teaches identifying those mutations in the F8, F9 and VWF genes which are pathogenic (e.g., para [0005-0007], [0009] and [0061] of the translation).
The response argues that “the present claims require that a risk determination or diagnosis be made only when the detected alteration is classified as "pathogenic or likely pathogenic" and is absent from a normal human control reference sequence. This two- part gating requirement-combining pathogenicity classification with absence from a normal control-constitutes a particular analytical framework that is not disclosed in either Bastida or Tan.” It is also argued that the claims require “that a determination be made only when a pathogenic or likely pathogenic alteration is detected that is absent from the normal control.”
However, the claims do not recite this “two-part gating requirement.” Rather, claim 1, for instance, recites “comparing the sequences obtained in step (a) with corresponding sequences of the amplified F8, F9, and VWF genes obtained from a normal control reference sequence subject; and (d) determining that the subject has a risk of developing hemophilia A, hemophilia B, or VWD when one or more pathogenic or likely pathogenic germline alterations are present.” Thus, the claims require the step of comparing the determined sequences with a normal control reference sequence and determining that the subject is at risk of developing (claim 1) or diagnosing (claim 8) or determining that the subject is a genetic carrier of (claim 15) hemophilia A, hemophilia B, or VWD when a germline alteration is detected in the subject which alteration has the property of being pathogenic or likely pathogenic. The claims do not require the asserted limitations of classifying an alteration as pathogenic or likely pathogenic and do not require that the final determining step occurs “only when a pathogenic or likely pathogenic alteration is detected that is absent from the normal control.” Even if the claims did require such limitations, the combined teachings would have suggested both comparing the determined sequences to a control reference sequence, identifying differences in the determined sequence which are absent from the control reference sequence and which have the property of being pathogenic or likely pathogen and then determining that the subject is at risk of hemophilia A, hemophilia B or VWF based on the identified pathogenic or likely pathogenic alterations in the F8, F9 or VWF genes which are absent from the normal, reference control since Ero teaches that the amplifying / sequencing methods disclosed therein for diagnosing a disease based on the presence of a pathogenic genetic alteration require performing each of these steps. And Bastida and Tan each teach detecting pathogenic germline alterations in the F8, F9 and VWF genes as diagnostic of hemophilia A, hemophilia B and VWF.
Regarding the limitation in claims 6, 7, 13, 14, 19 and 20 regarding the “48-hour or 5-day limitations,” the response states:
”The Office asserts that such timeframes would have been expected, but no cited reference teaches or suggests performing the claimed multi-gene analysis within these specific time constraints.”
Applicant’s statement does not accurately characterize the rejection. The rejection clearly relies on Ero as teaching the limitation that the amplifying, sequencing, comparing and determining steps are performed within 48 hours to 5 days of receiving the sample from the subject. Ero teaches that the advantage of the PCR / next-generation sequencing method described therein is that it allows for the rapid diagnosis of disease so that patient management can be initiated more quickly (para [0007- 0009] and [0027]). Ero specifically states that the results can be obtained within 48 hours to 5 days from receiving the sample. See, for example, para [0009-0010] and [0012], set forth below:
“The method also includes comparing the amplified DNA sequences with DNA sequences from a normal control subject. Any variations in the patient's DNA sequence as compared to the normal control subject indicates that the subject has a risk of complement-mediated thrombic microangiopathy. The variation in the subject's DNA sample can be a mutation, a polymorphism, or a deletion. The determination can be made within 48 hours, or 5 days, from receipt of the sample of genetic material from the patient.”
The rejection is maintained for the reasons set forth above.
Conclusion
Applicant's amendment necessitated the new ground(s) of rejection presented in this Office action. Accordingly, THIS ACTION IS MADE FINAL. See MPEP § 706.07(a). Applicant is reminded of the extension of time policy as set forth in 37 CFR 1.136(a).
A shortened statutory period for reply to this final action is set to expire THREE MONTHS from the mailing date of this action. In the event a first reply is filed within TWO MONTHS of the mailing date of this final action and the advisory action is not mailed until after the end of the THREE-MONTH shortened statutory period, then the shortened statutory period will expire on the date the advisory action is mailed, and any nonprovisional extension fee (37 CFR 1.17(a)) pursuant to 37 CFR 1.136(a) will be calculated from the mailing date of the advisory action. In no event, however, will the statutory period for reply expire later than SIX MONTHS from the mailing date of this final action.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to CARLA J MYERS whose telephone number is (571)272-0747. The examiner can normally be reached M-Th 6:30-5:00 EST.
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/CARLA J MYERS/Primary Examiner, Art Unit 1682
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