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and the fee set forth in 37 CFR 1.17(e) has been timely paid, the finality of the previous Office action has been withdrawn pursuant to 37 CFR 1.114.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 8/19/2025 has been entered.
Claim Status and Formal maters
The instant application is in response to 8/19/2025.
Claims 8-15 have been added by amendment.
Applicant’s election without traverse of detecting type II diabetes mellitus risk factors of elevated blood glucose levels for normal weight individuals] in the reply filed on 10/1/2024 is acknowledged.
All previous ground of rejection have been withdrawn as all previous claims have been canceled.
Priority
The instant application was filed 12/19/2022 and is a continuation of 15570573 , filed 10/30/2017, which is a National Stage entry of PCT/EP2016/059556 , International Filing Date: 04/28/2016 and claims foreign priority to DE10 2015 208 083.8, filed 04/30/2015 the foreign priority document is not in English.
Applicant’s claim for the benefit of a prior-filed application under 35 U.S.C. 119(e) or under 35 U.S.C. 120, 121, 365(c), or 386(c) is acknowledged. Applicant has not complied with one or more conditions for receiving the benefit of an earlier filing date under 35 U.S.C. 119(e). 120, 121, 365(c), or 386(c) as follows:
The later-filed application must be an application for a patent for an invention which is also disclosed in the prior application (the parent or original nonprovisional application or provisional application). The disclosure of the invention in the parent application and in the later-filed application must be sufficient to comply with the requirements of 35 U.S.C. 112(a) or the first paragraph of pre-AIA 35 U.S.C. 112, except for the best mode requirement. See Transco Products, Inc. v. Performance Contracting, Inc., 38 F.3d 551, 32 USPQ2d 1077 (Fed. Cir. 1994).
The disclosure of the prior-filed application, Application No. 15570573, PCT/EP2016/059556 , International Filing Date: 04/28/2016, and DE10 2015 208 083. fails to provide adequate support or enablement in the manner provided by 35 U.S.C. 112(a) or pre-AIA 35 U.S.C. 112, first paragraph for one or more claims of this application. The claims from which the application claims benefit do not support, “diagnosing the subject with type II diabetes mellitus when:1) the gene expression level of HMGA2 is elevated, the gene expression level of PPAR-gamma is non-elevated; or 2) the gene expression level of HMGA2 is non-elevated, the gene expression level of PPAR-gamma is non-elevated;wherein the gene expression level of HMGA2 or PPAR-gamma is elevated when the gene expression level of HMGA2 or PPAR-gamma exceeds the gene expression level of the housekeeping gene by at least 5%; wherein the gene expression level of HMGA2 or PPAR-gamma is non-elevated when the gene expression level of HMGA2 or PPAR-gamma is reduced with respect to the gene expression level of the housekeeping gene by at least 5%.;.” While the specification teaches, “"Elevated relative gene expression level" in this context means that the gene expression level elevated with respect to the mean of the gene expression levels of a group of patients (preferably n greater than 100). Within the framework of the relative quantification, the expression of the target genes is ascertained in relation to a so-called endogenous control. Ubiquitously expressed housekeeping genes serve as an endogenous control, preferably selected from the group consisting of HPRT, 18S, GAPDH, GUSB, PBGD, B2M, ABL, RPLPO, wherein most particularly HPRT is preferred. Such a gene expression level value preferably counts as elevated if it deviates from the respective reference value at the top by at least 5%, further preferably at least 10%, particularly preferably at least 20%. A non-elevated gene expression level within the scope of the present application is in contrast one such that moves in the order of magnitude of one or more of the afore- mentioned (selected) reference parameters (that is, +/- 2% from the corresponding value) or - which is preferred - one such which is reduced with respect to one or more of the reference values, i.e., is reduced by at least 5%, preferably at least 10% and further preferably at least 20%.” However this is with respect to a reference group, not housekeeping genes. Further review and searching did not reveal support for diagnosing type II diabetes when ) the gene expression level of HMGA2 is elevated, the gene expression level of PPAR-gamma is non-elevated; or 2) the gene expression level of HMGA2 is non-elevated, the gene expression level of PPAR-gamma is non-elevated Thus the amendment is not supported.
Response to Arguments
This is a new ground of objection necessitated by amendment..
Claim Objections
Claims 8-15 are objected to because of the following informalities:
Claim 8 is objected to as it recites “HMGA2 and PPAR-gamma” but does not recite the full terminology for the acronym (or abbreviation). Claims are more concise when the first time an acronym (or abbreviation) is presented the full terminology is also presented. Finally an acronym (or abbreviation) may have alternative meanings to an artisan.
Claim 11 is objected to as it recites “HPRT, 18S, GAPDH, GUSB, PBGD, B2M, ABL, and RPLPO” but does not recite the full terminology for the acronym (or abbreviation). Claims are more concise when the first time an acronym (or abbreviation) is presented the full terminology is also presented. Finally an acronym (or abbreviation) may have alternative meanings to an artisan.
Appropriate correction is required.
Response to Arguments
This is a new ground of objection necessitated by amendment.
.Claim Rejections - 35 USC § 112
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 8-15 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.
MPEP 2163 IB New or amended claims section II
With respect to newly added or amended claims, applicant should show support in the original disclosure for the new or amended claims. See, e.g., Hyatt v. Dudas, 492 F.3d 1365, 1370, n.4 (Fed. Cir. 2007) (citing MPEP § 2163.04 which provides that a "simple statement such as ‘applicant has not pointed out where the new (or amended) claim is supported, nor does there appear to be a written description of the claim limitation ‘___’ in the application as filed’ may be sufficient where the claim is a new or amended claim, the support for the limitation is not apparent, and applicant has not pointed out where the limitation is supported."); see also MPEP §§ 714.02 and 2163.06 ("Applicant should ... specifically point out the support for any amendments made to the disclosure."); and MPEP § 2163.04
Claim 1 has been amended to recite, “diagnosing the subject with type II diabetes mellitus when:1) the gene expression level of HMGA2 is elevated, the gene expression level of PPAR-gamma is non-elevated; or 2) the gene expression level of HMGA2 is non-elevated, the gene expression level of PPAR-gamma is non-elevated;wherein the gene expression level of HMGA2 or PPAR-gamma is elevated when the gene expression level of HMGA2 or PPAR-gamma exceeds the gene expression level of the housekeeping gene by at least 5%; wherein the gene expression level of HMGA2 or PPAR-gamma is non-elevated when the gene expression level of HMGA2 or PPAR-gamma is reduced with respect to the gene expression level of the housekeeping gene by at least 5%.;.” While the specification teaches, “"Elevated relative gene expression level" in this context means that the gene expression level elevated with respect to the mean of the gene expression levels of a group of patients (preferably n greater than 100). Within the framework of the relative quantification, the expression of the target genes is ascertained in relation to a so-called endogenous control. Ubiquitously expressed housekeeping genes serve as an endogenous control, preferably selected from the group consisting of HPRT, 18S, GAPDH, GUSB, PBGD, B2M, ABL, RPLPO, wherein most particularly HPRT is preferred. Such a gene expression level value preferably counts as elevated if it deviates from the respective reference value at the top by at least 5%, further preferably at least 10%, particularly preferably at least 20%. A non-elevated gene expression level within the scope of the present application is in contrast one such that moves in the order of magnitude of one or more of the afore- mentioned (selected) reference parameters (that is, +/- 2% from the corresponding value) or - which is preferred - one such which is reduced with respect to one or more of the reference values, i.e., is reduced by at least 5%, preferably at least 10% and further preferably at least 20%.” However this is with respect to a reference group, not housekeeping genes. Further review and searching did not reveal support for diagnosing type II diabetes when the gene expression level of HMGA2 is elevated, the gene expression level of PPAR-gamma is non-elevated; or 2) the gene expression level of HMGA2 is non-elevated, the gene expression level of PPAR-gamma is non-elevated Thus the amendment is not supported
Further claim 14 has been amended to recite, “wherein the test subject has a BMI between 23.4 kg/m2 and 36 kg/m2.” The response does not identify where the amendment is supported. However, review and searching of the specification revealed, “The BMI status of the diabetics ranged from normal-weight (diabetic 1: BMI 23.4 kg/m2) via overweight (diabetic 2: BMI 26.5 kg/m2) to class I obesity (diabetic 3: BMI 31.5 kg/m2) and class II obesity (diabetic 4: BMI 36.0 kg/m2). “ Thus the amendment does not specifically envision the claimed range. Further the teachings of the specification with the two end points of the range are limited to diabetic subjects. Thus the amendment has introduced new matter.
Response to Arguments
This is a new grounds of rejection necessitated by amendment.
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 8-15 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.
Claim 8is indefinite because it lacks a positive active step relating back to the preamble. The preamble recites is drawn preparing a sample for having analyzed gene expression as to diagnosis of type II diabetes mellitus, however the last positive active step is drawn to diagnosing the subject with type II diabetes mellitus. Therefore it is unclear as to whether the method is drawn preparing a sample for having analyzed gene expression as to diagnosis of type II diabetes mellitus preparing a sample for having analyzed gene expression as to diagnosis of type II diabetes mellitus.
Further claim 1 recites, “wherein the gene expression level of HMGA2 or PPAR-gamma is elevated when the gene expression level of HMGA2 or PPAR-gamma exceeds the gene expression level of the housekeeping gene by at least 5%; wherein the gene expression level of HMGA2 or PPAR-gamma is non-elevated when the gene expression level of HMGA2 or PPAR-gamma is reduced with respect to the gene expression level of the housekeeping gene by at least 5%.” The claim is confusing and unclear as the specification teaches, “Elevated relative gene expression level" in this context means that the gene expression level elevated with respect to the mean of the gene expression levels of a group of patients (preferably n greater than 100). Within the framework of the relative quantification, the expression of the target genes is ascertained in relation to a so-called endogenous control. Ubiquitously expressed housekeeping genes serve as an endogenous control, preferably selected from the group consisting of HPRT, 18S, GAPDH, GUSB, PBGD, B2M, ABL, RPLPO, wherein most particularly HPRT is preferred. Such a gene expression level value preferably counts as elevated if it deviates from the respective reference value at the top by at least 5%, further preferably at least 10%, particularly preferably at least 20%. A non-elevated gene expression level within the scope of the present application is in contrast one such that moves in the order of magnitude of one or more of the afore- mentioned (selected) reference parameters (that is, +/- 2% from the corresponding value) or - which is preferred - one such which is reduced with respect to one or more of the reference values, i.e., is reduced by at least 5%, preferably at least 10% and further preferably at least 20%.” “ Thus the specification appears to teach normalization using housing keeping genes to identify 5% increase of normalized expression relative to a reference population is elevated and 5% decrease in normalized expression relative to a reference population is non-elevated.
Claim 14 recites, “ the test subject.” Neither claim 14 nor claim 8 from which it depends previously recite test subject. Thus the metes and bounds are unclear what test subject the claim is referencing. This rejection can be overcome by amending claim 8 to recite,” test subject.”
Claim 15 recites, “ the test subject.” Neither claim 15 nor claim 8 from which it depends previously recite test subject. Thus the metes and bounds are unclear what test subject the claim is referencing. This rejection can be overcome by amending claim 8 to recite,” test subject.”
Response to Arguments
This is a new grounds of rejection necessitated by amendment.
Claim Rejections - 35 USC § 101
35 U.S.C. 101 reads as follows:
Whoever invents or discovers any new and useful process, machine, manufacture, or composition of matter, or any new and useful improvement thereof, may obtain a patent therefor, subject to the conditions and requirements of this title.
Claims 8-15 are rejected under 35 U.S.C. 101 because the claimed invention is directed to a natural correlation and mental step without significantly more. The claim(s) recite(s) the abstract idea or mental step in the wherein clause of determining elevated or non-elevated. Further the claim recites, “diagnosis of type II diabetes mellitus comprises:i) measuring gene expression level of HMGA2 and PPAR-gamma via polymerase chain reaction (PCR);ii) measuring gene expression level of a housekeeping gene via PCR; andiii) diagnosing the subject with type II diabetes mellitus when:1) the gene expression level of HMGA2 is elevated, the gene expression level of PPAR-gamma is non-elevated; or 2) the gene expression level of HMGA2 is non-elevated, the gene expression level of PPAR-gamma is non-elevated;wherein the gene expression level of HMGA2 or PPAR-gamma is elevated when the gene expression level of HMGA2 or PPAR-gamma exceeds the gene expression level of the housekeeping gene by at least 5%; wherein the gene expression level of HMGA2 or PPAR-gamma is non-elevated when the gene expression level of HMGA2 or PPAR-gamma is reduced with respect to the gene expression level of the housekeeping gene by at least 5%., “ which is a natural law or correlation. This judicial exception is not integrated into a practical application because steps depend from or otherwise integrate the judicial exception. The claim(s) does/do not include additional elements that are sufficient to amount to significantly more than the judicial exception because the steps of obtaining a sample and assaying RNA expression is routine and conventional and/or data gathering steps required to practice the invention.
Claim analysis
The instant claim 8 is directed towards a method of preparing a sample for having analyzed gene expression as to diagnosis of type II diabetes mellitus comprising: a) puncturing subcutaneous abdominal adipose tissue of a subject; wherein during step a) negative pressure is generated using a syringe and a cannula attached to the syringe is moved back and forth thereby aspirating cells of the adipose tissue; b) extracting nucleic acid from the adipose tissue sample, wherein the sample mass is < 50 mg and wherein the nucleic acid is RNA; c) denaturing the RNA; d) reverse transcribing the denatured RNA into cDNA; ande) pre-amplifying the cDNA; wherein diagnosis of type II diabetes mellitus comprises: i) measuring gene expression level of HMGA2 and PPAR-gamma via polymerase chain reaction (PCR);ii) measuring gene expression level of a housekeeping gene via PCR; and iii) diagnosing the subject with type II diabetes mellitus when:1) the gene expression level of HMGA2 is elevated, the gene expression level of PPAR-gamma is non-elevated; or 2) the gene expression level of HMGA2 is non-elevated, the gene expression level of PPAR-gamma is non-elevated; wherein the gene expression level of HMGA2 or PPAR-gamma is elevated when the gene expression level of HMGA2 or PPAR-gamma exceeds the gene expression level of the housekeeping gene by at least 5%; wherein the gene expression level of HMGA2 or PPAR-gamma is non-elevated when the gene expression level of HMGA2 or PPAR-gamma is reduced with respect to the gene expression level of the housekeeping gene by at least 5%.
The diagnosing step is a mental step and/or a natural phenomenon or natural correlation and requires a comparison in view of the wherein clause.
Dependent claims set forth further limitations to about the individual being a human. Sample mass, normalizing expression (another judicial exception). The BMI of individual.
According to the 2019 Patent Eligibility Guidance an initial two step analysis is required for determining statutory eligibility.
Step 1. Is the claim directed to a process, machine, manufacture, or composition of matter? In the instant case the Step 1 requirement is satisfied as the claims are directed towards a process.
Step 2A Prong one. Does the claim recite a law of nature, a natural phenomenon or an abstract idea? Yes, abstract idea and law of nature or natural phenomena.
With regards to claim 8, the claim recites, “ diagnosis of type II diabetes mellitus comprises :i) measuring gene expression level of HMGA2 and PPAR-gamma via polymerase chain reaction (PCR);ii) measuring gene expression level of a housekeeping gene via PCR; an diii) diagnosing the subject with type II diabetes mellitus when:1) the gene expression level of HMGA2 is elevated, the gene expression level of PPAR-gamma is non-elevated; or 2) the gene expression level of HMGA2 is non-elevated, the gene expression level of PPAR-gamma is non-elevated ;wherein the gene expression level of HMGA2 or PPAR-gamma is elevated when the gene expression level of HMGA2 or PPAR-gamma exceeds the gene expression level of the housekeeping gene by at least 5%; wherein the gene expression level of HMGA2 or PPAR-gamma is non-elevated when the gene expression level of HMGA2 or PPAR-gamma is reduced with respect to the gene expression level of the housekeeping gene by at least 5%.” These are an abstract idea or mental step and/or natural correlation.
Step 2A prong two. Does the claim recite additional elements that integrate the judicial exception into a practical application? The answer is no as the claim requires no additional steps.
Step 2B. Does the claim recite additional elements that are significantly more than the judicial exceptions? No
With regards to claims 8 the claim requires puncturing, extracting, denaturing, reverse transcribing, pre-amplifying and measuring are considered to be an active steps. The specification in paragraphs 0075-0080 teaches detection of HMGA2 and PPAR gamma were by known methods, using commercially available reagents. The claims requiring determining expression of the recited genes in adipose, which is routine and conventional in view of the teachings of Bullderdiek (WO2014/006163A1, published 1/9/2014). Dependent claims limit the sample, methods of detecting expression which are routine and conventional over the art of Bullderdiek (WO2014/006163A1, published 1/9/2014), Campbell (Cancer Prev Research (2009) volume 2, pages 37-42), Lafuente-Lafuente (Br J Clin Pharmacol / (2009)67:5 / 511–519 / 51), Santosa (Int J Obes (Lond). 2015 May ; 39(5): 874–876. doi:10.1038/ijo.2014.185.) Ritthaler (In J Sports Med (1980) volume 1, pages 50-51), and Stahlberg (Methods (2010) volume 50, pages 282-288)..
Thus the claims do not provide additional steps which are significantly more.
Response to arguments
The response traverses the rejection in view of the amendment.
The response continues traversing the prior rejection in view of Illumina, Inc. V. Ariosa Diagnostics. (Fed Circ 2020). This argument has been thoroughly reviewed but is not considered persuasive as the fact pattern between the instant case and Illumina, Inc. V. Ariosa Diagnostics. (Fed Circ 2020) are different as the Federal Circuit decision states, “ This is not a diagnostic case. And it is not a method of treatment case.” However the instant claims provide for a diagnosis and thus are materially different.
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.
Claim 8-9, 11-15 is/are rejected under 35 U.S.C. 103 as being unpatentable over Bullderdiek (WO2014/006163A1, published 1/9/2014), Campbell (Cancer Prev Research (2009) volume 2, pages 37-42), Lafuente-Lafuente (Br J Clin Pharmacol / (2009)67:5 / 511–519 / 51), Santosa (Int J Obes (Lond). 2015 May ; 39(5): 874–876. doi:10.1038/ijo.2014.185.) Ritthaler (In J Sports Med (1980) volume 1, pages 50-51), and Stahlberg (Methods (2010) volume 50, pages 282-288)..
With regards to claim 1, Bullderdiek teaches determination of gene expression via qRT-PCR for genes including HMGA2 and PPARgama (page 17, last paragraph). Bullderdiek teaches the samples were taken from subcutaneous adipose (page 18, top).
Bullderdiek while teaching determination of expression of HMGA2 and PPARgama based on BMI and HMGA2 and PPARgama expression.
Bullderdiek teaches that in the investigation BMI greater than 25 is defined as overweight (page 17).
Bullderdiek teaches reduced levels of PPAR gamma as well as elevated levels of HMGA2 can be assumed to reflect immature fat cells (page 17).
Bullderdiek teaches in the figures there are error bars, thus demonstrating populations of patients were examined.
Bullderdiek teaches in figure 1 there is high expression of HMGA2 and low expression of HMGA2
Bullderdiek teaches in figure 1b separating subjects based on diabetes, no diabetes, over weight and normal weight with HMGA2 expression.
Bullderdiek teaches examining correlation of BMI with HMGA2 expression (figure 2).
Bullderdiek teaches PPargamma and HMGA2 in that are inversely correlated (figures 4 and 5).
Bullderdiek teaches examining correlation of BMI with PPAR gamma expression (figure 6).
Bullderdiek teaches, “The term "prognosis" as used herein refers to a prediction of the probable course and outcome of a clinical condition or disease. A prognosis of a patient is usually made by evaluating factors, markers, and/or symptoms of a disease that are indicative of a favorable or unfavorable course or outcome of the disease. Preferably herein prognosis involves determining the probability that an early diabetes will develop into a late stage diabetes. In the early stage of type 2, the predominant abnormality is reduced insulin sensitivity. At this stage, hyperglycemia can be reversed by a variety of measures and medications that improve insulin sensitivity or reduce glucose production by the liver. Hence, prognosis herein means assessing the probability of progression into a late stage diabetes, and/or the development of an early stage diabetes.” (page 2, 3rd paragraph).
Bullderdiek teaches elevated expression of HMGA2 is diagnostic or prognostic of diabetes (page 6, lines 7-14). Bullderdiek teaches greater than 5% increase figure 1B.
Bullderdiek teaches, “Thus, reduced levels of PPARgamma expression as well as elevated levels of HMGA2 expression can be assumed to reflect an increased level of immature fat cells” (page 17, bottom).
Bullderdeik teaches, “HPRT served as endogenous control as described before (Markowski et al., 2010).” (page 18, last paragraph)
While Bullderdiek teaches obtaining samples from surgery and fine needle aspirates from subcutaneous tissue which encompass both encompass cutting (or puncturing) the subcutaneous adipose tissue.(examples)
Bullderdiek does not explicitly teach puncturing of subcutaneous abdominal tissue of the individual or test subjects to obtain a sample of adipose tissue and detecting both PPAR-gamma and HMGA2 RNA.
However, Campbell teaches, “Subcutaneous abdominal adipose samples were obtained by the same trained physician (K.F.S.) from superficial abdominal adipose tissue. The biopsy methods and sample processing steps are outlined in Fig. 1. A sample was collected from an area in the lower quadrant (10-12 cm from the umbilicus) by one method and repeated on the contralateral side in succession, using the second method. The method done first (i.e., A or B) and the location (i.e., right or left) were alternated and recorded. Method A used a 14-gauge needle and required a <0.5-cm scalpel incision, whereas method B used a 16-gauge needle without incision. In both cases, the Yale needle was attached to a 20-mL syringe filled with sterile saline and was passed through the subcutaneous fat several times while applying negative pressure. The collected adipose tissue was processed immediately following sampling. From method A, approximately half of the sample was flash frozen on dry ice, whereas the remainder was processed for tissue culture. From method B, the entire sample was flash frozen on dry ice. Subjects provided informed consent and the study was approved by the Fred Hutchinson Cancer Research Center Institutional Review Board. Potential side effects were reviewed by participants and included some discomfort, small risk of bleeding, mild bruising, and a small risk of infection. Participants reported method preference, extent of side effects, and any unanticipated symptoms in a follow-up phone call (7-10 d after procedure) by one of the investigators (K.C.). This scripted interview asked about the presence and extent of potential side effects and also biopsy method preference (i.e., the sample done on the right or left).” (page 38, 2nd column).
Campbell teaches, “RNA yield per milligram of tissue was equivalent for methods A and B (14.2 ± 7.8 versus 16.2 ± 8.3ng of RNA/mg of tissue, respectively; P = 0.95) and RNA quality was good for both methods.” (gene expression, page 40).
Therefore it would have been prima facie obvious to one of ordinary skill in the art prior to the effective filing date of the claims to obtain a sample of subcutaneous abdominal adipose by aspiration for test subjects and the individual, detecting expression of the recited PPAR-gamma and HMGA2 and examine expression of both mRNAs to examine with diabetes and identify subjects with increased HMGA2 and unchanged or decreased expression of PPAR-gamma as diagnosed with increased risk of type II diabetes. The artisan would be motivated to use the method of Campbell as it demonstrates multiple genes can be analyzed. The artisan would have a reasonable expectation of success as the artisan is merely using known methods to get enough RNA to be examine multiple transcripts.
The teachings of Bullderdiek, Campbell are set forth above.
While Bullderdiek teach the use of needle biopsies for human subcutaneous fat sampling (example 1)., Bullderdiek, Campbell do not teach the mass of the sample obtained.
However, Ritthaler teaches there are two biopsy methods for human subcutaneous adipose which includes needle biopsies. Ritthaler teaches needle biopsies delivered a mean 20mg to 50 mg (page 50, bottom, 2nd column)..
Therefore it would been prima facie obvious to one of ordinary skill in the art prior to the effective filing date that needle biopsy of Bullderdiek is 20 mg to 50 mg. The artisan would be motivated as the Ritthaler teaches needles biopsies are typically 20 mg to 50 mg. The artisan would have a reasonable expectation of success as the artisan is merely using known methods to obtain samples of normal size.
The teachings of Bullderdiek, Campbell and Ritthaler teach obtaining samples. While Campbell teaches the use of negative pressure, it does not specifically teach moving back and forth.
However, Santosa teaches , “2-4 g of subcutaneous abdominal adipose tissue was collected by surgical excision and needle aspiration from adjacent sites. Surgically excised tissue was removed by cauterization. For needle aspiration, 0.9% saline was first injected subcutaneously to mimic the research approach we employ and adipose tissue was aspirated using a 12 gauge, monoeye cannula (Mayo Clinic Department of Engineering, Rochester, MN) with negative pressure generated by a 10 mL syringe” (page 2, general protocol)
Lafuente-Lafuente teaches, “Subcutaneous adipose tissue was obtained by needle aspiration following the technique of Beynen and Katan [18]: skinfold is gripped with one hand and a 18-G needleinserted at 45°, then connected to vacuum using a 7-mlevacuated tube (BD Vacutainer Systems, Plymouth, UK) andgently pushed back and forth a few times in the adiposelayer to obtain a few fragments of tissue. To test if concen-trations varied depending on the body region, sampleswere obtained at two different points: the abdominal walland low lumbar region.) (page 512-513)
Therefore it would have been prima facie obvious to one of ordinary skill in the art prior to the effective filing date of the claims to use the back and forth movement of the syringe of Santosa and Lafuente-Lafuente to collect adipose tissue. The artisan would be motivated as Massry teaches other methods may damage tissue. The artisan would have a reasonable expectation of success of using known methods to harvest know tissue samples.
Bullderdiek, Campbell, Ritthaler, Santosa and Lafuente-Lafuente do not specifically teach preamplifcation as part of detection in RT-PCR.
However, Stahlberg teaches, “ Pre-amplification of single-cell cDNA allows virtually limitless number of measured genes, and has been used on single cells in concert with microarrays [29]. In a pioneering work by Brady and Iscove [30], it was shown that polyA-cDNA can be globally amplified, followed by gene-specific PCR on the resulting pool. Alternatively, a multiplex PCR on the single-cell cDNA, using low concentrations of all gene-specific primers, results in a population of PCR products that are quantified by qPCR. This way, up to 40 [31] and 100s (TaqMan PreAmp Master Mix, p/n 4391128, Applied Biosystems) of transcripts have been analyzed in a single cell.”(page 286, 2nd column, 1st full paragraph).
Therefore it would have been prima facie obvious to one of ordinary skill in the art prior to the effective filing date of the claims to pre-amplify cDNA reverse transcribed from the tissue. The artisan would be motivated as Stahlberg teaches pre-amplification allows for the detection of a limitless number of genes from as little as a single cell. The artisan would have a reasonable expectation of success as the artisan is merely using a known method of preamplification as part of real time PCR.
With regards to claim 9, Bullderdiek teaches the use of human subcutaneous abdominal adipose white tissue (page 5, 1st full paragraph). Bullderdiek teaches aspirates by use of a syringe (which is a puncture).
With regards to claim 11, Bullderdiek teaches, “In order to determine the express10n level of a gene it is necessary to compare the expression level to a control. Preferably, the control is selected from the group of housekeeping genes as e.g. HPRT, 18S, GAPDH, GUSB, PBGD, B2M, ABL, RPLPO” (page 12).
While, Campbell teaches, “ RNA yield per milligram of tissue was equivalent for methods A and B (14.2 ± 7.8 versus 16.2 ± 8.3ng of RNA/mg of tissue, respectively; P = 0.95) and RNA quality was good for both methods.” (gene expression, page 40). Campbell does not specifically teach an RNA concentration of less than or equal 25 ng/microliter.
However, it would have been prima facie obvious to one of ordinary skill in the art prior to the effective filing date of the claims to provide RNA at concentrations of f less than or equal 25 ng/microliter. The artisan would be motivated to provide RNA at f less than or equal 25 ng/microliter to allow consistency of volume for addition to preamplification, amplification and detection methods. The artisan would have a reasonable expectation of success as the artisan is merely providing known reagents at known concentrations.
With regards to claims 14-15 Bullderdiek teaches the following investigations overweight was defined as a BMI >25 (page 18).
Response to Arguments
The response traverses the rejection in view of the teachings of Massry and Livaoglu being directed to fat grafting. These arguments have been thoroughly reviewed but is not considered persuasive as Massry and Livaoglu are no longer relied upon.
Claim 10 is/are rejected under 35 U.S.C. 103 as being unpatentable over Bullderdiek (WO2014/006163A1, published 1/9/2014), Campbell (Cancer Prev Research (2009) volume 2, pages 37-42), Lafuente-Lafuente (Br J Clin Pharmacol / (2009)67:5 / 511–519 / 51), Santosa (Int J Obes (Lond). 2015 May ; 39(5): 874–876. doi:10.1038/ijo.2014.185.) Ritthaler (In J Sports Med (1980) volume 1, pages 50-51), and Stahlberg (Methods (2010) volume 50, pages 282-288) as applied to claims 18-9, 11-15 in further view of Healy (Methods in molecular biology (2002) volume 193, pages 349-361) and Ventura (European Journal of Obstetrics & Gynecology and Reproductive Biology, Volume 169, Issue 1, July 2013, Pages 28-32.)
The teachings of Bullderdiek, Campbell, Ritthaler, , Lafuente-Lafuente, Santosa, and Stahlberg are set forth above.
While Bullderdiek, Campbell, Ritthaler, , Lafuente-Lafuente, Santosa, and Stahlberg teach the use of needle biopsies for human subcutaneous fat sampling (example 1), and needle biopsies with masses of 30-50 mg. Further Stahlberg detection of RNA from a single cell.
Bullderdiek, Campbell, Ritthaler, , Lafuente-Lafuente, Santosa, and Stahlberg do not teach 5 mg samples.
However, Healy teaches, “If the amount of biological material is limited and resultant poly A+ RNA yields are low (<500 ng), the conventional approach is not an option. This problem will arise, for example, if one is attempting to construct cDNA libraries from a small number of cells, or indeed a single cell, from a tissue with a large number of cellular phenotypes such as the brain.” (abstract).
Cambpell teaches, “Campbell teaches, “ RNA yield per milligram of tissue was equivalent for methods A and B (14.2 ± 7.8 versus 16.2 ± 8.3ng of RNA/mg of tissue, respectively; P = 0.95) and RNA quality was good for both methods.” (gene expression, page 40).”
Stahlberg detection of RNA from a single cell.
Ventura teaches isolation and reverse transcription of RNA extracted from 5 mg of tissue. (page 29, 2.3-2.4)
Therefore it would been prima facie obvious to one of ordinary skill in the art prior to the effective filing date of the claims a biopsy 5mg or less could be used to examine gene expression. The artisan would be motivated as the Healy and Stahlberg suggest the use of a single cell, while Ventura demonstrates the use of 5 mg of tissue for RNA isolation and RT-PCR were known.. The artisan would have a reasonable expectation of success as the artisan is merely using known methods to obtain samples of normal size.
Response to Arguments
The response traverses the rejection in view of the amendment to the independent claim. This argument is not persuasive for the reasons of record.
Summary
No claims are allowed.
Conclusion
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/Steven Pohnert/Primary Examiner, Art Unit 1683
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