DETAILED ACTION
Notice of Pre-AIA or AIA Status
The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
Continued Examination Under 37 CFR 1.114
A request for continued examination under 37 CFR 1.114, including the fee set forth in 37 CFR 1.17(e), was filed in this application after final rejection. Since this application is eligible for continued examination under 37 CFR 1.114, and the fee set forth in 37 CFR 1.17(e) has been timely paid, the finality of the previous Office action has been withdrawn pursuant to 37 CFR 1.114. Applicant's submission filed on February 2, 2026 has been entered.
Effective Filing Date
The present application was filed on August 12, 2022 and does not claim the benefit of, or priority to, any previously filed applications.
Claim Status and Action summary
Claims 1-5, 7-11, 14-18 and 21-25 are pending in the present application. Claims 6, 12-13 and 19-20 were canceled by applicant. Claims 21-23 were newly added in the amendment filed on August 26, 2025. Claims 24 and 25 were newly added in the amendment filed on February 2, 2026.
Claims 1-5, 7-11, 14-18 and 21-25 are under examination.
This action is in response to the papers filed February 2, 2026.
Any objections and rejections not reiterated below are hereby withdrawn.
Claim Rejections - 35 USC § 112(a) –Enablement
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.
Claims 17-18 are/remain rejected under 35 U.S.C. 112(a) as failing to comply with the enablement requirement because the specification, while being enabling for specific, treatable X-linked genetic disease, hemophilia, does not reasonably provide enablement for the genus “muscular dystrophy”, comprising Duchenne Muscular Dystrophy (DMD). The specification does not enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the invention commensurate in scope with these claims.
Factors to be considered in determining whether a disclosure meets the enablement requirements of 35 USC 112, first paragraph, have been described by the court in In re Wands, 8 USPQ2d 1400 (CA FC 1988). Wands states at page 1404, “Factors to be considered in determining whether a disclosure would require undue experimentation have been summarized by the board in Ex parte Forman. They include (1) the quantity of experimentation necessary, (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.”
The nature of the invention and the breadth of the claims:
Claims 17-18 are broadly drawn to a method for simultaneous detection of Y chromosome-specific target DNA of the SRY, DAZ2, and TSPY1 genes and control DNA of the ACTB gene comprising steps of simultaneously amplifying the recited genes by PCR, detecting the presence or absence of corresponding PCR amplicons, selecting a sample based on the presence or absence of the PCR amplicons, and “treating a [male or female] fetus for hemophilia or muscular dystrophy.”
The state of the art:
The invention is in a class of invention which the CAFC has characterized as “the unpredictable arts such as chemistry and biology.” Mycogen Plant Sci., v. Monsanto Co., 243 F.3d 1316, 1330 (Fed. Cir. 2001).
The post-filing date art teaches that “muscular dystrophy” is a very large genus of genetic disorders comprising diverse phenotypic presentations involving at least 50 genes (Bozzi et al., “Diagnosis, Pathogenesis and Treatment of Muscular Dystrophy” Biomedicines 2025, 13, 1820). The art further teaches that at least 110 distinct “muscular dystrophies” are recognized, not all of which have single genes associated with the phenotype within the chromosome band segregating with the disease phenotype (Benarroch et al., “The 2025 version of the gene table of neuromuscular disorders (nuclear genome)” Neuromuscular Disorders 46 (2025), 105261). Furthermore, Benarroch et al. teach that of the 110 distinct “muscular dystrophies”, only 3 are known to be X-linked (“Duchenne muscular dystrophy; Becker muscular dystrophy”, “Emery-Dreifuss muscular dystrophy, X-linked, type 1”, and “Emery-Dreifuss muscular dystrophy 6, X-linked”), while the other members of the genus exhibit autosomal patterns of inheritance and are attributed to genes/loci on autosomes (Benarroch et al., Gene table of monogenic neuromuscular disorders (nuclear genome only), see Group 1 and 2 in the table).
The post-filing date art (Bozzi (2025)) teaches that there is no definitive cure for any of the known muscular dystrophies. Bozzi teaches that there are experimental/supportive therapies (Glucocorticoid therapy, and several drugs in clinical trials). However, Bozzi teaches glucocorticoid therapy, while helpful for delaying disease progression, has significant side effects including adrenal suppression, growth delay, weakened bones, and metabolic syndrome. Bozzi additionally teaches that the experimental drugs in clinical trials demonstrated broad safety profiles, but with limited demonstrated effectiveness (Bozzi, page 1, paragraphs 3-4). Furthermore, none of the experimental treatments (or glucocorticoid treatment) reviewed by Bozzi appear to have been investigated for safety or effectiveness for fetal subjects.
Other approaches in the post-filing date art such as in utero gene editing using lipid nanoparticles (LNPs) as delivery vehicles for Cas9 mRNAs and sgRNAs have been attempted in mouse models (Gao et al., “In utero delivery of mRNA to the heart, diaphragm, and muscle with lipid nanoparticles”, Bioactive Materials (2023) 25, pp. 387-398) and (Palanki et al., “In utero delivery of targeted ionizable lipid nanoparticles facilitates in vivo gene editing of hematopoietic stem cells” PNAS (2024) 121 (32) e2400783121). However, these studies caution that further study is needed to “achieve clinically meaningful mRNA delivery rates” (Gao et al., page 388, column 2, paragraph 2), and “further work is necessary to fully characterize… safety of… LNP[s]” (Palanki et al., page 9, column 1, paragraph 2).
Furthermore, several examples in the art teach examples of Muscular Dystrophies for which there are no effective clinical treatments, much less treatments suitable for administration to a fetus.
Zhong et al., 2021 teaches Duchenne Muscular Dystrophy (DMD) is a fatal X-linked muscular disease for which there is no effective clinical treatment (Zhong, background). Furthermore, Kihara et al., “In utero transplantation of myoblasts and adipose-derived mesenchymal stem cells to murine models of Duchenne muscular dystrophy does not lead to engraftment and frequently results in fetal death” Regenerative Therapy (2022) Oct20;21:486-493 teach experimental in utero cell therapies in mouse models comprising fetal intraperitoneal transplantation and transplacental transplantation with allogenic myoblasts or adipose-derived stem cells (ASCs). Kihara et al. found that in-utero transplanted cells were not viable, and that the interventions showed high fetal mortality rates (Kihara et al., table 1 and page 490-491 bridging paragraph).
Most recently, Farmer et al., “Feasibility and safety of cellular therapy for in-utero repair of myelomeningocele (CuRe Trial): a first-in-human, phase 1, single-arm study” The Lancet (2026) 407, pp.867-875 demonstrated improved immediate safety findings of administration of allogenic placenta-derived stem cells (PMSCs) onto exposed fetal spinal cords during experimental fetal surgical repair of myelomeningocele (spina bifida). However, it is not clear whether transplanted PMSCs will remain safe in longer term follow up studies of the very small trial cohort (6 patients), as Farmer et al. only assessed birth outcomes and acknowledged the possibility of malignant or proliferative transformation of the transplanted PMSCs (Farmer et al., page 873, column 1-2 bridging paragraph). Furthermore, Farmer et al. teach that other genetic disorders requiring long-term engraftment of therapeutic stem cells following fetal administration have not been successful and continue (as of 2026) to be optimized (Farmer et al., page 873, column 2, paragraph 2).
Guidance in the specification and Working Examples
The specification broadly teaches that prenatal treatments may comprise: maternal dietary supplementation with amino acids or sugars, fetal blood transfusion, administration of thyroxin or other enzymes or hormones, or in utero stem cell transplantation (Specification, page 38, line 10-16).
The specification does not teach any working examples of treating a fetus for any condition.
Quantity of Experimentation
Claims 17-18 are broadly drawn to a method comprising treating a fetus for “hemophilia or muscular dystrophy”. The specification teaches “Over a hundred X-linked inherited diseases have been discovered in humans” (specification, page 1, line 19-20). However, the specification does not teach any exemplary treatments that may be administered to a fetus. The prior art teaches that “muscular dystrophy” is a very large genus of known heritable phenotypes comprising at least 110 distinct “muscular dystrophies”, not all of which have single genes associated with the phenotype within the chromosome band segregating with the disease phenotype (Benarroch et al.). The prior art further recognizes that there are no effective cures or effective treatments for any of the known muscular dystrophies (Bozzi, page 1, paragraphs 3-4) at any developmental stage, much less during fetal development. Furthermore, even in early-stage mouse models, an attempted prenatal treatment for Duchenne muscular dystrophy failed to correct the disease, as donor myoblast or adipose-derived stem cells failed to engraft into fetal muscle tissue and high fetal mortality was observed for the attempted surgical intervention. Muscular dystrophies are not treated with any routine therapies. Therefore, it is unpredictable whether particular effective treatments for the broadly claimed “hemophilia or muscular dystrophy” can be administered to a fetus.
The art also teaches several studies where the skilled artisan was unable to treat Duchenne Muscular Dystrophy (see above, Bozzi et al. and Kihara et al). The specification teaches that prenatal therapies encompass maternal dietary supplementation with amino acids or sugars, fetal blood transfusion, administration of thyroxin or other enzymes or hormones, or in utero stem cell transplantation (Specification, page 38, line 10-16). The art further suggests that gene therapy or gene editing based therapies may eventually be able to treat Duchenne Muscular Dystrophy (Zhong, 2021 page 8, column 1, paragraph 5). Furthermore, the post-filing date art (Bozzi et al., Gao et al., and Palanki et al.) demonstrate that while in utero delivery of lipid nanoparticles comprising CRISPR-Cas9 components for activating reporter gene expression is possible in mice (Gao et al., 2023), this line of research remains active, there is no demonstration of therapeutic editing during fetal development for any muscular dystrophy, and the therapies have not been shown to be safe or effective in human fetuses.
Therefore, the skilled artisan would be required to perform additional undue and unpredictable experimentation to determine an effective prenatal treatment for the broad genus of “muscular dystrophy or hemophilia”. The diseases within this broad genus differ in genetic causes, knowledge of a molecular basis, and proposed prenatal therapeutic strategies upon which a skilled artisan may begin to form hypotheses of potential treatments. The skilled artisan would have to perform additional unpredictable and undue experimentation for each known muscular dystrophy with known (or unknown) genetic bases to arrive at a candidate treatment that may be administered to a developing fetus, after which, they would have to perform extensive clinical experimentation on developing male or female fetuses affected by each muscular dystrophy to resolve unpredictable factors such as dosing, effective route of administration, safety, and long-term efficacy, with no expectation of success at the clinical stage, as demonstrated by the failed in utero cell therapy for Duchenne muscular dystrophy in mouse models taught by Kihara et al. Furthermore, the art teaches Duchenne Muscular Dystrophy (DMD) (i.e. “muscular dystrophy” is a fatal X-linked muscular disease for which there is no effective clinical treatment (Zhong, background). Even more, despite the improved safety demonstrated by the 2026 spina bifida trial (Farmer et al.), the in-utero LNP gene editing platform taught by Gao et al., 2023, does not appear to have been followed up on by the study’s authors in the intervening 2+ years between the publication of Gao et al. and the writing of this office action (March 2026), much less as a demonstrated therapeutic for in-utero treatment of any muscular dystrophy or hemophilia. In fact, Farmer et al. caution that, regarding other recent studies, “in-utero intravenous transplantation of maternal bone marrow-derived hematopoietic stem cells for thalassaemia and of allogeneic fetal liver-derived stem cell for osteogenesis imperfecta appeared safe, but long-term engraftment for genetic conditions is challenging and continues to be optimised. In contrast, long-term engraftment is not warranted for myelomeningocele, which is a structural birth defect with a defined anatomical area requiring transient regeneration and repair.” (Farmer et al., page 873, column 2, paragraph 2).
The quantity of experimentation in this area is very large because there are many unknown, and unpredictable variables that would likely be unique to each muscular dystrophy or hemophilia. Each disease would require extensive genetic and molecular experimentation to discover, test, and administer candidate therapies without an expectation of success at each intervening step. Furthermore, there is no guidance in the specification or art that supports the claim scope: “treating a fetus for hemophilia or muscular dystrophy” as broadly claimed.
Level of Skill in the art
The level of skill in the art is deemed to be high.
Conclusion
In the instant case, given the breadth of the claim to treating a fetus for any muscular dystrophy or hemophilia, the lack of guidance provided in the specification and prior art as to how the skilled artisan might treat a fetus with many of the diseases within the claimed genus, the very large quantity of experimentation in the art, lack of demonstrated working examples, the presence of negative teachings in the art within the broadly claimed genus, and the unpredictability of the art, balanced only against the high level of skill in the art, it is the position of the examiner that it would require undue experimentation for one of skill in the art to perform the method of claims 17-18 as broadly written.
Response to arguments
The response traverses the scope of enablement rejection under 112(a) on the grounds that the amendments to claim 1 are asserted to narrow the claim scope to “specific X-linked diseases such as hemophilia or muscular dystrophy”. These arguments have been thoroughly reviewed but are not persuasive. First, as is emphasized in the rejection updated above for the amended claim scope, and as was noted in the rejection of record, Duchenne Muscular Dystrophy (DMD) (i.e. “a muscular dystrophy”) is a fatal X-linked muscular disease for which there is no effective clinical treatment (Zhong, background). Furthermore, it is noted that “muscular dystrophy” is a broad genus of genetic disorders recognized by the art comprising more than 100 distinct conditions with distinct genotypic and phenotypic characteristics, only a minority of which exhibit X-linked patterns of inheritance.
Claim Rejections - 35 USC § 112(b) – Indefiniteness
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.
Claims 25 is rejected under 35 U.S.C. 112(b) as being indefinite for failing to particularly point out and distinctly claim the subject matter which the inventor or a joint inventor, regards as the invention.
The term “intensity” in claim 25 is a relative term which renders the claim indefinite.
The term “intensity” is not defined by the claim, the specification does not provide a standard for ascertaining the requisite degree, and one of ordinary skill in the art would not be reasonably apprised of the scope of the invention. The metes and bounds of the claim are unclear, because it is unclear whether the claim is intended to cover any method of measuring relative abundance of PCR products amplified by the method recited in claim 1. It is similarly unclear how the “intensity” is to be measured and what standard units the detection threshold set forth in claim 1 (“≥1,000,000 intensity” or “lower than 1,000,000 intensity”) is to be compared to.
Response to arguments
The response asserts that the rejection is moot because of the removal of “intensity” from claim 1. The rejection has been updated, as the previously rejected indefinite claim language has been relocated to a new dependent claim of claim 1 (claim 25) in the amended claims.
Claims 1-5, 7-11, 14-18 and 21-25 are rejected under 35 U.S.C. 112(b) as being indefinite for failing to particularly point out and distinctly claim the subject matter which the inventor or a joint inventor regards as the invention.
Claim 1 recites “A multiplex polymerase chain reaction (PCR) method for simultaneous detection of Y chromosome-specific target DNA of the SRY, DAZ2 and TSPYI genes and control DNA of the ACTB gene in a sample containing DNA, comprising: ---”.
Claim 17 recites “The method of claim 1, wherein the sample is a fetal sample, and wherein said method further comprises treating a male fetus for hemophilia or muscular dystrophy”.
Claim 18 recites “The method of claim 1, wherein the sample is a fetal sample, and wherein said method further comprises treating a female fetus for hemophilia or muscular dystrophy”.
The treating step recited in claims 17 and 18 does not relate back to the preamble of claim 1 directed to “A multiplex polymerase chain reaction (PCR) method for simultaneous detection of …” recited in claim 1. Accordingly, the metes and bounds of claim 1 and its dependent claims 2-5, 7-11, 14-18 and 21-25 cannot be determined.
Claim Rejections - 35 USC § 103
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, 9, 11, 14, 16, 21, and newly added claims 24 and 25 are/remain rejected under 35 U.S.C. 103 as being unpatentable over Kolialexi et al., “Early non-invasive detection of fetal Y chromosome sequences in maternal plasma using multiplex PCR” European Journal of Obstetrics and Gynecology and Reproductive Biology. 161 34-37, 2012 in view of Fernandez-Martinez et al., “Noninvasive fetal sex determination in maternal plasma: a prospective feasibility study” Genetics in Medicine. 14, 1, 101-106 (2012), Xie et al., “Designing highly multiplex PCR primer sets with Simulated Annealing Design using Dimer Likelihood Estimation (SADDLE)” Nature Communications, published April 11, 2022, Rychlik (Nucleic Acids Research, Vol. 17, No. 21, Pg 8543-8551, 1989), Buck (Biotechniques, Vol. 27, Pg. 528-536, 1999), and Genbank. Bethesda (MD): National Library of Medicine (US), National Center for Biotechnology Information; 2004 – [cited March 18, 2025].
Regarding claim 1, Kolialexi teaches multiplex PCR detection of SRY, DYS14 (i.e. TSPY1), and ACTB (Kolialexi, page 35, column 2, paragraph 3) for early non-invasive detection of fetal Y chromosome DNA in maternal plasma (i.e. detecting the presence of amplicons of different lengths (Kolialexi, Table 1), and selecting a sample containing Y chromosome DNA or a sample not containing Y chromosome DNA (Kolialexi, Fig 1).
Kolialexi does not teach amplifying and detecting DAZ2 with the multiplexed markers of SRY, TSPY1, and ACTB.
However, Fernandez-Martinez teaches amplifying DAZ in addition to the two Y-specific markers of Kolialexi. Fernandez-Martinez teaches: “we chose to use a combination of 3 Y chromosome sequences to maximize the accuracy of the test… [and] demonstrate that the detection of 3 markers is highly accurate for clinical use” (Fernandez-Martinez, page 105, column 2, paragraph 1).
Therefore, it would have been prima facie obvious prior to the effective filing date of the claimed invention for one of ordinary skill in the art to modify the multiplex PCR sex-determination method taught by Kolialexi by amplifying and detecting DAZ2 in addition to SRY and TSPY1, as taught by Fernandez-Martinez. The ordinary artisan would have been motivated to modify the method of Kolialexi with additional primers to detect DAZ2 in addition to the other Y specific markers SRY and TSPY1 and the control sequence ACTB because Fernandez-Martinez teaches that detection of only two Y chromosome markers (SRY and TSPY1) is prone to error (Fernandez-Martinez, page 105, column 1, paragraph 2) and that inclusion of a third marker (DAZ2) improves the accuracy of sex determination (Fernandez-Martinez, page 105, column 2, paragraph 1). The ordinary artisan would have been reasonably confident that addition of an additional set of primers specific to DAZ2, as taught by Fernandez-Martinez, to the multiplex sex-determination method taught by Kolialexi would have improved the accuracy of sex determination.
Kolialexi does not teach primers comprising the nucleotide sequences recited by claim 1 as amended. However, Genbank teaches the complete nucleotide sequences of the human genes SRY (GenBank: L10102.1, published 1993), ACTB (GenBank: M10277.1, published 1985), TSPY1 (GenBank: X74029.1, published 1993), and DAZ2 (AC000021.1, published 1996). Rychlik teaches it is routine and predictable to make primers for DNA amplification wherein primers are designed to a known oligonucleotide sequence. Rychlik teaches criteria to design/choose primers for DNA amplification (see whole document and Abstract). Buck provides evidence of the equivalence of primers. Specifically, Buck invited primer submissions from a number of labs (Pg. 532, column 3), with 69 different primers being submitted (Pg 530, column 1). Buck also tested 95 primers spaced at 3 nucleotide intervals along the entire sequence at issue, thereby testing more than 1/3 of all possible 18-mer primers on the 300 base pair sequence (Pg 530, column 1). When Buck tested each of the primers selected by the methods of the different labs, Buck found that every single primer worked (Pg. 533, column 1). Further, every single control primer functioned as well (Pg. 533, column 1). Buck expressly states, “The results of the empirical sequencing analysis were surprising in that nearly all of the primers yielded data of extremely high quality” (Pg. 535, column 2). Therefore, Buck provides direct evidence that all primers would be expected to function, and in particular, all primers selected according to the ordinary criteria. This clearly shows that every primer would have a reasonable expectation of success. Finally, Xie teaches a publicly available computer program for selecting highly multiplexed combinations of primer pairs for qPCR assays. (Xie, page 2, column 2) Xie demonstrates a successful example of a set of 60 primers designed by their program to detect 56 gene fusions that frequently occur in non-small cell lung cancer. (Xie, page 6, column 2) Xie teaches that highly multiplex qPCR assays, designed by the program disclosed by their paper, are useful for highly multiplex molecular diagnostics where the cost of next generation sequencing assays cannot be economically justified (Xie, page 8, column 2).
Therefore, it would have been prima facie obvious prior to the effective filing date of the claimed invention for one of ordinary skill in the art to design primers capable of amplifying segments of the known sequences of the Y specific DNA target genes, taught by Kolialexi in view of Fernandez-Martinez by the ordinary methods for multiplex primer design taught by Rychlik, Buck, and Xie. The ordinary artisan would have been motivated to design multiplex compatible primers by the methods of Rychlik, Buck, and Xie because of the teaching of Xie that that highly multiplex qPCR assays, are useful for molecular diagnostics where the cost of next generation sequencing assays cannot be economically justified (Xie, page 8, column 2). The ordinary artisan would have been reasonably confident that a multiplex primer set, designed by the method of Xie, for the four target genes taught by Kolialexi in view of Fernandez-Martinez would have successfully detected the presence or absence of the target genes because Xie teaches a successful 60 primer multiplex qPCR panel targeting 56 gene fusions.
Regarding claim 2, Kolialexi teaches DNA is stored at -80°C (i.e. is frozen) (Kolialexi, page 35, column 2, paragraph 2).
Regarding claim 3, Kolialexi teaches the sample comprises fetal DNA (Kolialexi, abstract, paragraph 1).
Regarding claim 4, Kolialexi teaches the sample comprises maternal cell-free plasma (Kolialexi, page 35, column 2, paragraph 2).
Regarding claim 9, Kolialexi teaches isolating target DNA from other components (Kolialexi, page 35, column 2, paragraph 2).
Regarding claim 11, Fernandez-Martinez teaches contacting a sample with TaqMan minor-groove binding probes (i.e. sequence-specific DNA probes to SRY, DAZ2, and TSPY1) (Fernandez-Martinez, page 104, column 2, paragraph 1-2).
Regarding claim 14, Kolialexi teaches the fetal DNA is extracted form maternal blood where one or both parents have or are carriers of an X-linked genetic disease, namely hemophilia, Duchenne muscular dystrophy, or chronic granulomatous disease (Kolialexi, page 35, column 1, paragraph 4).
Regarding claim 16, Kolialexi teaches further testing the fetus for a causative mutation in male fetuses at risk for hemophilia (Kolialexi, page 37, column 1, paragraph 3)
Regarding claim 21, Kolialexi teaches sequencing the amplicons (Kolialexi, page 35, column 2, paragraph 3).
Regarding newly added claim 24, Fernandez-Martinez teaches that detection of only two Y chromosome markers (SRY and TSPY1) is prone to error (Fernandez-Martinez, page 105, column 1, paragraph 2) and that inclusion of a third marker (DAZ2) improves the accuracy of sex determination (Fernandez-Martinez, page 105, column 2, paragraph 1) (i.e. male gender is determined when amplicons for SRY, TSPY1, and DAZ2 are detected).
Regarding newly added claim 25, each of Fernandez-Martinez teaches comparing amplification of sample DNA to positive and negative controls, with Ct cutoffs at 32, 39, and 40 (i.e. AN=Ao*2N; where AN=cycle copy number, Ao=initial copy number, and N=number of cycles). Therefore, where N=32, AN/Ao =2N =232=4.29*109, an approximately 4.3 billion fold increase in the number of molecules relative to the starting amount. If Ao =1 copies (i.e. for a single copy locus unique to the Y chromosome), then A32=4.29*109 is the defined minimum number of molecules required for a positive result.
While it is unclear how “an intensity of at least 1,000,000” is meant to be interpreted (see the 112(b) rejection above and of record), it is clear that Ct=32 results in an approximately 4.3 billion-fold increase in DNA copy number relative to input (or greater if relative to a negative control).
Claims 5, 7, 10, and 15 are/remain rejected under 35 U.S.C. 103 as being unpatentable over Kolialexi et al., in view of Fernandez-Martinez et al., “Noninvasive fetal sex determination in maternal plasma: a prospective feasibility study” Genetics in Medicine. 14, 1, 101-106 (2012), Xie et al., “Designing highly multiplex PCR primer sets with Simulated Annealing Design using Dimer Likelihood Estimation (SADDLE)” Nature Communications, published April 11, 2022, Rychlik (Nucleic Acids Research, Vol. 17, No. 21, Pg 8543-8551, 1989), Buck (Biotechniques, Vol. 27, Pg. 528-536, 1999), and Genbank. Bethesda (MD): National Library of Medicine (US), National Center for Biotechnology Information; 2004 – [cited March 18, 2025] as applied to claims 1-4, 9, 11, 14, 16, and 21 above, and further in view of Zhang et al., “Frequency-enhanced transferrin receptor antibody-labelled microfluidic chip (FETAL-Chip) enables efficient enrichment of circulating nucleated red blood cells for non-invasive prenatal diagnosis” Lab Chip, 2018, 18, 2749-2756 (2018).
Regarding claim 5, 7, and 10, the method of non-invasive prenatal sex determination by multiplex PCR of Y chromosome-specific genes from fetal DNA isolated from peripheral maternal blood, taught by Kolialexi in view of Fernandez-Martinez, Xie et al., Rychlik, Buck, and Genbank does not teach additional steps of isolating fetal cells expressing CD71 from maternal blood. However, Zhang teaches a method for enriching fetal nucleated red blood cells from maternal circulation for non-invasive prenatal genetic testing, including prenatal sex determination by qPCR of the SRY gene (Zhang, page 2750, column 1, paragraph 3-column 2, paragraph 1 and figure 7) (i.e. Zhang teaches the isolating fetal cells expressing CD71 from peripheral maternal blood). Zhang further teaches that “highly fragmented cffDNA and a huge background of maternal DNA affect the accuracy of [non-invasive prenatal testing] and limit the application in other genetic disorders such as microdeletions and duplications” (Zhang, page 2749, column 2, paragraph 1) and circulating mononuclear fetal red blood cells hold complete genetic information as well as proteins which can be used for fetal analysis (Zhang, page 2750, column 1, paragraph 3).
Therefore, it would have been prima facie obvious prior to the effective filing date of the claimed invention for one of ordinary skill in the art to combine the method of multiplex PCR detection of SRY, DAZ2, TSPY1, and ACTB in peripheral maternal blood for non-invasive prenatal sex determination, taught by Kolialexi in view of Fernandez-Martinez, Xie et al., Rychlik, Buck, and Genbank, with the method of isolating CD71 positive fetal cells from peripheral maternal blood for non-invasive prenatal diagnosis, comprising an SRY qPCR assay for sex determination, taught by Zhang. The ordinary artisan would have been motivated to combine the method of Kolialexi in view of Fernandez-Martinez, Xie et al., Rychlik, Buck, and Genbank with the method of Zhang because of the teaching of Zhang that assays based on cell-free fetal DNA detection are limited in their application to detecting genetic disorders because of the presence of maternal DNA and the highly fragmented nature of cffDNA (Zhang, page 2749, column 2, paragraph 1). The ordinary artisan would have been reasonably confident that purifying CD71-positive fetal cells from peripheral maternal blood by the method of Zhang would have improved the assay taught Kolialexi in view of Fernandez-Martinez, Xie et al., Rychlik, Buck, and Genbank by decreasing background due to maternal DNA.
Regarding claim 15, Zhang teaches imaging DAPI-stained captured fetal cells (Zhang, fig 5) (i.e. determining the karyotype of the fetus).
Claim 8 is rejected under 35 U.S.C. 103 as being unpatentable over Kolialexi et al., in view of Fernandez-Martinez et al., “Noninvasive fetal sex determination in maternal plasma: a prospective feasibility study” Genetics in Medicine. 14, 1, 101-106 (2012), Xie et al., “Designing highly multiplex PCR primer sets with Simulated Annealing Design using Dimer Likelihood Estimation (SADDLE)” Nature Communications, published April 11, 2022, Rychlik (Nucleic Acids Research, Vol. 17, No. 21, Pg 8543-8551, 1989), Buck (Biotechniques, Vol. 27, Pg. 528-536, 1999), and Genbank. Bethesda (MD): National Library of Medicine (US), National Center for Biotechnology Information; 2004 – [cited March 18, 2025] as applied to claims 1-4, 9, 11, 14, 16, and 21 above, and further in view of McMichael et al., “DNA from Buccal Swabs Suitable for High-Throughput SNP Multiplex Analysis” Journal of Biomolecular Techniques 20:232-235 (2009).
Regarding claim 8, Kolialexi in view of Fernandez-Martinez, Xie et al., Rychlik, Buck, and Genbank do not teach isolating maternal and paternal DNA from buccal cells.
However, McMichael et al. teach buccal swabs (i.e. collecting buccal cells) are a “convenient and reliable method for collection of genetic material that is inexpensive and noninvasive and suitable for self-collection” providing “DNA of sufficient quantity and quality for high-throughput SNP multiplex analysis” (McMichael et al., Abstract). McMichael et al. demonstrate successful multiplex amplification and analysis using three different multiplex PCR assays comprising 25, 11, or 3 SNPs (McMichael et al., page 233, column 2, paragraph 2 and page 235, column 1, paragraph 4).
Therefore, it would have been prima facie obvious prior to the effective filing date of the claimed invention for one of ordinary skill in the art to modify the method of multiplex PCR detection of SRY, DAZ2, TSPY1, and ACTB in peripheral maternal blood for non-invasive prenatal sex determination, taught by Kolialexi in view of Fernandez-Martinez, Xie et al., Rychlik, Buck, and Genbank, with the method of multiplex amplification, detection, and analysis of SNPs comprising cheap, convenient, and rapid collection of high-quality DNA from buccal samples of adults, taught by McMichael et al. The ordinary artisan would have been motivated to modify the method taught by Kolialexi in view of Fernandez-Martinez, Xie et al., Rychlik, Buck, and Genbank to detect the claimed Y chromosome-specific markers in maternal and paternal buccal cell samples, rather than in peripheral maternal blood, because of the relative ease with which buccal cell samples may be collected compared to peripheral blood. The ordinary artisan would have been relatively confident that parental buccal swab samples comprising buccal cells would have been a suitable sample for multiplex PCR detection of Y chromosome specific markers because of the teaching of McMichael et al that multiplex detection of 3, 11, or 25 SNPs was readily attainable from buccal samples.
Claims 1 and 17-18 are rejected under 35 U.S.C. 103 as being unpatentable over Kolialexi et al., in view of Fernandez-Martinez et al., “Noninvasive fetal sex determination in maternal plasma: a prospective feasibility study” Genetics in Medicine. 14, 1, 101-106 (2012), Xie et al., “Designing highly multiplex PCR primer sets with Simulated Annealing Design using Dimer Likelihood Estimation (SADDLE)” Nature Communications, published April 11, 2022, Rychlik (Nucleic Acids Research, Vol. 17, No. 21, Pg 8543-8551, 1989), Buck (Biotechniques, Vol. 27, Pg. 528-536, 1999), and Genbank. Bethesda (MD): National Library of Medicine (US), National Center for Biotechnology Information; 2004 – [cited March 18, 2025] as applied to claims 1-4, 9, 11, 14, 16, and 21 above, and further in view of Iizuka et al., “Neonatal Gene Therapy for Hemophilia B by a Novel Adenovirus Vector Showing Reduced Leaky Expression of Viral Genes” (Molecular Therapy-Methods Clinical Development. 2017 Jul 8; 6:183-193).
This obviousness rejection is over the scope of claims 17 and 18 identified as enabled in the 112(a) rejection above.
Regarding claim 1, as described above, Kolialexi et al. in view of Fernandez-Martinez et al., Xie et al., Rychlik, Buck, and Genbank teach methods comprising multiplex detection of the recited Y-chromosome specific and control DNA markers for detecting the presence of Y-chromosome DNA in a (male or female) fetal sample.
Regarding claims 17-18, Kolialexi et al. in view of Fernandez-Martinez et al., Xie et al., Rychlik, Buck, and Genbank do not teach further treating a fetus for hemophilia.
However, Iizuka et al. teach approaches for treating hemophilia in neonatal animal models comprising gene transfer for hemophilia B (Iizuka et al., abstract).
Therefore, it would have been prima facie obvious prior to the effective filing date of the claimed invention for one of ordinary skill in the art to have combined the methods taught by Kolialexi et al. in view of Fernandez-Martinez et al., Xie et al., Rychlik, Buck, and Genbank comprising detecting Y-chromosome specific DNA sequences in fetal samples with the methods taught by Iizuka et al. comprising treating neonatal animal models for hemophilia. The ordinary artisan would have been motivated to treat a fetus diagnosed with hemophilia and determined as having, or lacking, Y-chromosome DNA by the teachings of Kolialexi et al. that fetal sex and hemophilia status can be determined from cell free fetal DNA in the first trimester of pregnancy and by the teachings of Iizuka et al. that a single administration of a gene therapy vector for Hemophilia B rescued the bleeding phenotypes of neonatal hemophilia B mice for more than 100 days.
Claim 22 is rejected under 35 U.S.C. 103 as being unpatentable over Kolialexi et al., in view of Fernandez-Martinez et al., “Noninvasive fetal sex determination in maternal plasma: a prospective feasibility study” Genetics in Medicine. 14, 1, 101-106 (2012), Xie et al., “Designing highly multiplex PCR primer sets with Simulated Annealing Design using Dimer Likelihood Estimation (SADDLE)” Nature Communications, published April 11, 2022, Rychlik (Nucleic Acids Research, Vol. 17, No. 21, Pg 8543-8551, 1989), Buck (Biotechniques, Vol. 27, Pg. 528-536, 1999), and Genbank. Bethesda (MD): National Library of Medicine (US), National Center for Biotechnology Information; 2004 – [cited March 18, 2025] as applied to claims 1-4, 9, 11, 14, 16, and 21 above, and further in view of EP 1201768 A2 (Laird, published 2002).
Kolialexi in view of Fernandez-Martinez, Xie et al., Rychlik, Buck, and Genbank do not teach that primers for multiplex PCR comprise one or more modified nucleotides.
However, Laird teaches PCR amplification using primers modified with 2’-O-methyl-, 2’-fluoro-, 2’-amino- or arabinose nucleotides (Laird, paragraph 0032-0033) reduce the formation of non-specific amplification, such as primer dimers (Laird, paragraph 0037).
Xie teaches that one of the major factors in designing multiplex compatible primer sets is reducing the frequency of primer dimer formation (Xie, Abstract).
Therefore, it would have been prima facie obvious prior to the effective filing date of the claimed invention for one of ordinary skill in the art to modify the method of multiplex PCR amplification of SRY, DAZ2, TSPY1, and ACTB, taught by Kolialexi in view of Fernandez-Martinez, Xie, Rychlik, Buck, and Genbank by including 2’-O-methyl-, 2’-fluoro-, 2’-amino- or arabinose nucleotides in the primers, as taught by Laird. The ordinary artisan would have been motivated to include modified nucleotides in the primers as taught by Laird by the suggestion of Xie that reduction of primer dimer formation is a major factor in designing multiplex-optimized primer sets (Xie, Abstract), and by the teaching of Laird that inclusion of modified nucleotides reduces the formation of primer dimers and off-target primer binding (Laird, paragraph 0037). The ordinary artisan would have been reasonably confident that inclusion of 2’-O-methyl-, 2’-fluoro-, 2’-amino- or arabinose nucleotides in the multiplex optimized primers designed by the method of Xie would have successfully further reduced the formation of primer dimers and off target amplification, thus reducing background signal in the multiplex Y chromosome detection method taught by Kolialexi in view of Fernandez-Martinez, Xie, Rychlik, Buck, and Genbank.
Claim 23 is rejected under 35 U.S.C. 103 as being unpatentable over Kolialexi et al., in view of Fernandez-Martinez et al., “Noninvasive fetal sex determination in maternal plasma: a prospective feasibility study” Genetics in Medicine. 14, 1, 101-106 (2012), Xie et al., “Designing highly multiplex PCR primer sets with Simulated Annealing Design using Dimer Likelihood Estimation (SADDLE)” Nature Communications, published April 11, 2022, Rychlik (Nucleic Acids Research, Vol. 17, No. 21, Pg 8543-8551, 1989), Buck (Biotechniques, Vol. 27, Pg. 528-536, 1999), and Genbank. Bethesda (MD): National Library of Medicine (US), National Center for Biotechnology Information; 2004 – [cited March 18, 2025] as applied to claims 1-4, 9, 11, 14, 16, and 21 above, and further in view of Tounta et al., “A Multiplex PCR for Non-invasive Fetal RHD Genotyping Using Cell-free Fetal DNA” In vivo May 2011, 25(3) 411-417 and Piovesan et al., “On the length, weight, and GC content of the human genome” BMC Research Notes 2019 Feb 27; 12:106.
Kolialexi in view of Fernandez-Martinez, Xie et al., Rychlik, Buck, and Genbank do not teach the multiplex PCR method detects 0.2 ng of Y chromosome.
However, Tounta et al. teach multiplex PCR of 5 loci in circulating cell free fetal DNA comprising SRY and ACTB (Tounta et al., abstract). Tounta et al. further teach that multiplex amplification of the target loci were observed from single cells isolated from blood (Tounta et al., page 414, column 1, paragraph 1) after optimization of parameters comprising annealing temperatures and primer concentrations (Tounta et al., page 412, column 2, paragraph 2). Piovesan et al. teach the (human) male nuclear diploid genome weighs approximately 6.41 pg (Piovesan et al., abstract) and the Y chromosome weighs approximately 0.06 pg (Piovesan et al., table 2). Therefore, the teaching of Tounta et al. of optimized multiplex PCR detection of multiple targets comprising Y chromosome specific SRY sequences from a single circulating cell teaches multiplex PCR detection of 0.06 pg of Y chromosome DNA (i.e. far less than the recited 0.2 ng of Y chromosome DNA).
Therefore, it would have been prima facie obvious prior to the effective filing date of the claimed invention for one of ordinary skill in the art to have optimized the method comprising multiplex detection of the Y chromosome specific- and internal control- markers taught by Kolialexi in view of Fernandez-Martinez, Xie et al., Rychlik, Buck, and Genbank using the teachings of Tounta et al. and Piovesan et al. that multiplex PCR for circulating fetal cell free DNA for detection of target loci comprising Y-chromosome specific markers can be optimized to detect an amount of DNA equivalent to as little as a single diploid genome equivalent. The ordinary artisan would have been motivated to have optimized the multiplex PCR assay taught by Kolialexi in view of Fernandez-Martinez, Xie et al., Rychlik, Buck, and Genbank using the optimizable parameters taught by Tounta et al. to minimize the amount of genetic material required as input to perform the assay.
Response to arguments
The response asserts that the specific sequences of the primers recited by claim 1 are distinct from the longer sequences of the genes from which they are derived, which are known in the art on the grounds that the ordinary artisan would not have been motivated to select the specific primers claimed and that the particular primers claimed provide a specific, non-obvious solution for amplifying portions of the recited genes and asserts that the primers claimed result with superior detection properties compared to the cited prior art. The response further asserts that: a) consumption of polymerase and nucleotides, b) the possibility of introducing primer-primer interactions, and c) the repetitive nature of the Y chromosome constitutes unpredictable factors. These arguments have been reviewed but are not persuasive.
As described above, Buck, and Rychlik teach first principles and demonstrate interchangeability of primers for a given known target sequence. Xie et al. teach publicly available software for searching for sets of compatible multiplex primers and demonstrate examples wherein as many as 60 primer pairs are successfully used in multiplex PCR. Furthermore, the claimed primer sequences are simply fragments of the extremely well-known Y-chromosome specific sequences taught by the prior art (SRY, DAZ2, TSPY1) and the well-known and widely used internal control sequence ACTB, the complete sequences of which are taught by Genbank. Therefore, to summarize the detailed rejections above, the prior art, taken together, renders obvious the claimed invention as follows. It would have been obvious to the ordinary artisan to make a simple combination of markers used by Kolialexi et al. and Fernandez-Martinez et al. in multiplex PCR assays for prenatal diagnosis for which a number of suitable primers could have been selected using the known sequences in Genbank using the known methods taught by Xie, Buck, and Rychlik. Absent some unexpected result present from the specific claimed primers, it is the position of the examiner that selection of any combination of primers specific to the well-known sequences of the genetic markers widely used for fetal sex determination would have been obvious to the ordinary artisan. Even in the instance that some “manual” screening or “adjustment” (i.e. BLAST search to confirm no significant homology to off-target sequences or inputting specific desired melting temperatures into the program taught by Xie et al.) was performed to optimize the specific primers, optimization of these parameters are well known and routine to those of ordinary skill in the art.
To further demonstrate that the claimed primers are merely obvious variants of those known in the art, the examiner has prepared comparative alignments of each of the amplicons that may reasonably be expected to be produced by the claimed primers and those expected to be produced by the primers taught in the art (see below).
Comparing the ACTB amplicons expected from the claimed primers to those from the primers taught by Kolialexi: it is clear that the naturally occurring sequences of claimed primers are simply an alternative to those known in the art readily selected by the ordinary artisan by the methods discussed above, absent evidence of unexpected results.
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Comparing the SRY amplicons expected from the claimed primers to those from the primers taught by Kolialexi: it is clear that the naturally occurring sequences of claimed primers are simply an alternative to those known in the art readily selected by the ordinary artisan by the methods discussed above, absent evidence of unexpected results.
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Comparing the DAZ amplicons (repeats amplified in DAZ 1-4 by both primer sets) expected from the claimed primers to those from the primers taught by Fernandez-Martinez et al., it is clear that the naturally occurring sequences of claimed primers are simply an alternative to those known in the art readily selected by the ordinary artisan by the methods discussed above, absent evidence of unexpected results.
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Comparing the TSPY amplicons (repeats amplified in TSPY1-4, 6P, 7P, 8-10, and 11P by both primer sets) expected from the claimed primers to those from the primers taught by Kolialexi et al., it is clear that the naturally occurring sequences of claimed primers are simply an alternative to those known in the art readily selected by the ordinary artisan by the methods discussed above, absent evidence of unexpected results.
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Therefore, for the reasons detailed in the rejections and summarized here, the claims are/remain rejected under U.S.C. 103 over the cited combinations of prior art references.
Conclusion
No claim is allowed.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to ZACHARY MARK TURPIN whose telephone number is (703)756-5917. The examiner can normally be reached Monday-Friday 8:00 am - 5:00 pm.
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/Z.M.T./Examiner, Art Unit 1682
/WU CHENG W SHEN/Supervisory Patent Examiner, Art Unit 1682