DETAILED ACTION
Election/Restrictions
Applicant’s election without traverse of Group I, claims 2, 6, 7, 12, 14, 18, 20, 25-26, 28, 30, 31, 34, 35, 39-41, 53, and 54, drawn to a method of detecting an abnormal level of mature and/or active mitochondrial transcription factor A (TFAM) protein in a sample and species steps of claim 14 in the reply filed on 02/26/2026 is acknowledged.
Claims 12 and 23 withdrawn from further consideration pursuant to 37 CFR 1.142(b) as being drawn to a nonelected Group II and species, there being no allowable generic or linking claim. Election was made without traverse in the reply filed on 02/26/2026.
Claims Status
Claims 2,6-7,12,14,18,20,23,26,28,30-31,34-35,39-41 and 53-54 are pending.
Claims 12 and 23 are withdrawn.
Claims 2,6-7,14,18,20,25-26,28,30-31,34-35,39-41 and 53-54 are currently under examination.
Priority
This application is the National Phase Application of International Application No. PCT/EP2021/082385, filed November 19, 2021, which claims priority to U.S. Provisional Application No. 63/126,703, filed December 17, 2020, and European Patent Application No. 20208932.2, filed November 20, 2020. Accordingly, the priority date of claim set filed on November 27,2023, is determined to be November 20, 2020.
Drawings
Specification
The abstract of the disclosure is objected to because the abstract is not on a separate sheet in accordance with 37 CFR 1.52(b)(4) and 1.72(b). A corrected abstract of the disclosure is required and must be presented on a separate sheet, apart from any other text. See MPEP § 608.01(b).
The following guidelines illustrate the preferred layout for the specification of a utility application. These guidelines are suggested for the applicant’s use.
Arrangement of the Specification
As provided in 37 CFR 1.77(b), the specification of a utility application should include the following sections in order. Each of the lettered items should appear in upper case, without underlining or bold type, as a section heading. If no text follows the section heading, the phrase “Not Applicable” should follow the section heading:
(a) TITLE OF THE INVENTION.
(b) CROSS-REFERENCE TO RELATED APPLICATIONS.
(c) STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT.
(d) THE NAMES OF THE PARTIES TO A JOINT RESEARCH AGREEMENT.
(e) INCORPORATION-BY-REFERENCE OF MATERIAL SUBMITTED ON A READ-ONLY OPTICAL DISC, AS A TEXT FILE OR AN XML FILE VIA THE PATENT ELECTRONIC SYSTEM.
(f) STATEMENT REGARDING PRIOR DISCLOSURES BY THE INVENTOR OR A JOINT INVENTOR.
(g) BACKGROUND OF THE INVENTION.
(1) Field of the Invention.
(2) Description of Related Art including information disclosed under 37 CFR 1.97 and 1.98.
(h) BRIEF SUMMARY OF THE INVENTION.
(i) BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWING(S).
(j) DETAILED DESCRIPTION OF THE INVENTION.
(k) CLAIM OR CLAIMS (commencing on a separate sheet).
(l) ABSTRACT OF THE DISCLOSURE (commencing on a separate sheet).
(m) SEQUENCE LISTING. (See MPEP § 2422.03 and 37 CFR 1.821 - 1.825). A “Sequence Listing” is required on paper if the application discloses a nucleotide or amino acid sequence as defined in 37 CFR 1.821(a) and if the required “Sequence Listing” is not submitted as an electronic document either on read-only optical disc or as a text file via the patent electronic system.
The disclosure is objected to because of the following informalities: section headers should appear in upper case, without underlining or bold type, as a section heading.
Appropriate correction is required.
Claim Objections
Claims 2 and 6 are objected to because of the following informalities:
Regarding claim 2, step “(c)” (ln 8) should be modified to “(d)”. Claim 53, in which claim 2 depends on already comprises a step (c).
Regarding claim 6, step “(c)” (ln 8) should be modified to “(d)”. Claim 53, in which claim 6 depends on already comprises a step (c).
Appropriate correction is required.
Claim Rejections - 35 USC § 112(b)
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 7 is 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 7, is indefinite over the limitations “intensive care unit (ICU) freedom within one week” (ln 2) and “no ICU freedom for at least one week” (ln 3). It is unclear as to the metes and bounds meant by the phrases as having abnormal levels of TFAM, and organ dysfunction is not the only scenario that would have someone in the ICU.
Claims 6-7 are rejected under 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph, as being incomplete for omitting essential steps, such omission amounting to a gap between the steps. See MPEP § 2172.01. The omitted steps are: a step that would cause for the outcome to become positive, if the abnormal level is less than 40% lower than the reference. Claim 7 depend on claim 6.
Claim Rejections - 35 USC § 112(d)
The following is a quotation of 35 U.S.C. 112(d):
(d) REFERENCE IN DEPENDENT FORMS.—Subject to subsection (e), a claim in dependent form shall contain a reference to a claim previously set forth and then specify a further limitation of the subject matter claimed. A claim in dependent form shall be construed to incorporate by reference all the limitations of the claim to which it refers.
The following is a quotation of pre-AIA 35 U.S.C. 112, fourth paragraph:
Subject to the following paragraph [i.e., the fifth paragraph of pre-AIA 35 U.S.C. 112], a claim in dependent form shall contain a reference to a claim previously set forth and then specify a further limitation of the subject matter claimed. A claim in dependent form shall be construed to incorporate by reference all the limitations of the claim to which it refers.
Claim 7 is rejected under 35 U.S.C. 112(d) or pre-AIA 35 U.S.C. 112, 4th paragraph, as being of improper dependent form for failing to further limit the subject matter of the claim upon which it depends, or for failing to include all the limitations of the claim upon which it depends. Claims 14, 53 or 6 do not indicate a patient is in the ICU. Applicant may cancel the claim(s), amend the claim(s) to place the claim(s) in proper dependent form, rewrite the claim(s) in independent form, or present a sufficient showing that the dependent claim(s) complies with the statutory requirements.
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 Interpretations: Regarding claims 2,6,14,18,20,26,28,30-31,34-35,39-40 and 53-54, “and/or” reads on any the limitation clause as an individual limitation or in combination with the any other limitation clause listed in the claim.
Claims 2, 6, 14, 18, 20, 25-26, 30, 40-41 and 53 are rejected under 35 U.S.C. 103 as being unpatentable over Gunst et al. (“Gunst”; (2013). Insufficient autophagy contributes to mitochondrial dysfunction, organ failure, and adverse outcome in an animal model of critical illness. Critical care medicine, 41(1), 182-194.).
Regarding claim 14, Gunst teaches a method comprising “Antibodies were purchased from Abcam, Cambridge, UK…. mitochondrial transcription factor A… DakoCytomation, Glostrup, Denmark (horseradish peroxidase–conjugated goat–antirabbit or goat–antimouse antibodies).” (Pg. 185, Immunoblotting, Para. 1). Gunst teaches a method comprising “Mitochondrial repair pathways in liver and kidney of surviving vs. nonsurviving hyperglycemic critically ill rabbits. Relative expression levels of key proteins… TFAM = mitochondrial transcription factor A” (Pg. 187, Figure 4 legend). Gunst teaches a method comprising “Expression of mitochondrial transcription factor A, a key transcription factor in mitochondrial biogenesis, was lowered in the liver of nonsurviving critically ill rabbits, whereas in survivors it remained low-normal (Fig. 4)” (Pg. 188, Mitochondrial Biogenesis, Para. 1; Fig. 4 (see below)). Furthermore, as depicted in Figure 4, the levels of TFAM protein expression were decreased about 20%-60% in Non-survivor patient samples compared to the Healthy patient sample. “mitochondrial transcription factor A” reads on mature and/or active TFAM.
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Thus, Gunst suggests a method of detecting an abnormal level of mature and/or active mitochondrial transcription factor A (TFAM) protein in a sample from a patient, wherein said method comprises (a) measuring the level of mature and/or active TFAM protein in the sample; and(b) determining whether the level in the sample is abnormal, wherein the level in the sample is determined to be abnormal if the level of mature and/or active TFAM protein is at least about 20%, at least about 50%, or at least about 70% lower than the amount determined for a reference sample.
The teachings of Gunst are documented above in the rejection of claim 14 under 35 U.S.C. 103. Claims 18, 30, 40, 41 and 53 depend on claim 14. Claims 2 and 6 depends on claim 53, which depend on claim 14. Claims 20, and 25-26 depend on claim 18 which depend on claim 14.
Regarding claims 18 and 53, Gunst teaches a method wherein “our data implicate insufficient autophagy in the pathogenesis of organ failure and risk of death during critical illness” (Pg. 191, Col. 1, Para. 1). Gunst teaches a method wherein “increasing evidence implicates mitochondrial dysfunction as a culprit in multiple organ failure” (Pg. 182-183, Introduction, Para. 1). Gunst teaches a method wherein “nonsurvivors revealed multiple organ damage” (Pg. 186, Mitochondrial Repair in Relation to Survival Status, Para. 1). Thus, Gunst suggests a method wherein an abnormal level of mature and/or active TFAM protein in the sample is indicative of the presence of an organ dysfunction; and a method further comprising (c) determining that an organ dysfunction is present in the patient when the level of mature and/or active TFAM protein is abnormal, and/or determining that no organ dysfunction is present in the patient when the level of mature and/or active TFAM protein is not abnormal.
Regarding claims 2, 6 and 20, Gunst teaches a method comprising “Expression of mitochondrial transcription factor A, a key transcription factor in mitochondrial biogenesis, was lowered in the liver of nonsurviving critically ill rabbits, whereas in survivors it remained low-normal (Fig. 4)” (Pg. 188, Mitochondrial Biogenesis, Para. 1; Fig. 4 (see above)). Furthermore, as depicted in Figure 4, the levels of TFAM protein expression were decreased about 20%-60% in Non-survivor patient samples compared to the Healthy patient sample. “nonsurvivor” reads on negative outcome. Thus, Gunst suggests a method wherein a level of mature and/or active TFAM protein that is at least about 40% or at least about 80% lower in the sample from said patient than the amount determined for said reference sample is indicative of the presence of very severe organ dysfunction; a method further comprising (c) determining a degree of severity of the organ dysfunction in the patient, wherein (i) the degree of severity is low when the level of mature and/or active TFAM protein is abnormal but less than 40% lower in the sample from said patient than the amount determined for said reference sample, and/or (ii) the degree of severity is high when the level of mature and/or active TFAM protein is at least about 40% or at least about 80% lower in the sample from said patient than the amount determined for said reference sample; and method further comprising (c) prognosing the outcome of said organ dysfunction in the patient, wherein (i) a positive outcome is prognosed when the level of mature and/or active TFAM protein is abnormal but less than 40% lower in the sample from said patient than the amount determined for said reference sample, and/or (ii) a negative outcome is prognosed when the level of mature and/or active TFAM protein is at least about 40% or at least about 80% lower in the sample from said patient than the amount determined for said reference sample.
Regarding claim 25, Gunst teaches a method wherein “A study on muscle biopsies found an early biogenesis response in surviving but not in nonsurviving patients with severe sepsis” (Pg. 192, Col. 2. Para.2). Thus, Gunst suggests a method wherein the organ dysfunction is sepsis.
Regarding claim 26, Gunst teaches a method wherein “organ failure is a leading cause of morbidity and mortality in prolonged critically ill patients (1). The pathogenesis is multifactorial involving a complex interplay among inflammatory, hemodynamic, metabolic, endocrine, and immunological disturbances (2).” (Pg. 182, Para. 1). Thus, Gunst suggests a method wherein (a) the organ dysfunction is associated with and/or caused by an infection, and/or wherein the patient is suffering from an infection; and/or (b) wherein the organ dysfunction is associated with and/or caused by an inflammation, and/or wherein the patient is suffering from an inflammation.
Regarding claim 30, Gunst teaches a method comprising “Antibodies were purchased from Abcam, Cambridge, UK…. mitochondrial transcription factor A… DakoCytomation, Glostrup, Denmark (horseradish peroxidase–conjugated goat–antirabbit or goat–antimouse antibodies).” (Pg. 185, Immunoblotting, Para. 1). Gunst teaches a method comprising “Mitochondrial repair pathways in liver and kidney of surviving vs. nonsurviving hyperglycemic critically ill rabbits. Relative expression levels of key proteins… TFAM = mitochondrial transcription factor A” (Pg. 187, Figure 4 legend). “mitochondrial transcription factor A” reads on mature TFAM protein. Thus, Gunst suggests a method wherein the level of mature and/or active TFAM protein corresponds to (i) the amount of mature TFAM protein,(ii) the number of interactions of TFAM protein with TFB2M protein, and/or (iii) the ratio of the amount of mature TFAM protein over the amount of immature TFAM protein.
Regarding claim 40, Gunst teaches a method wherein “Relative expression levels of
key proteins and messenger RNA involved in mitochondrial repair” (Pg. 187, Figure 4 legend.). Thus, Gunst suggests a method wherein the level of mature and/or active TFAM protein corresponds to the amount of mature TFAM protein in the mitochondria.
Regarding claim 41, Gunst teaches a method wherein “blood sampling” (Pg.184, Col. 1, Para. 1) and “A 4-mL blood sample” (Pg.184, Col. 2, Para. 3). Thus, Gunst suggests a method wherein said sample is a blood sample.
Claim 54 is rejected under 35 U.S.C. 103 as being unpatentable over Gunst et al. (“Gunst”; (2013). Insufficient autophagy contributes to mitochondrial dysfunction, organ failure, and adverse outcome in an animal model of critical illness. Critical care medicine, 41(1), 182-194.) as applied to claim 14 above, and further in view of Hood et al. (“Hood”; Patent Pub. US 8586006 B2, Nov. 19, 2013).
The teachings of Gunst are documented above in the rejection of claims 14 under 35 U.S.C. 103. Claim 54 depends on claim 14. Gunst does not explicitly teach the limitations of claim 54.
Hood discloses methods for identifying and using organ-specific proteins and transcripts. The present invention further provides compositions comprising organ-specific proteins and transcripts encoding the same, detection reagents for detecting such proteins and transcripts, and diagnostic panels, kits and arrays for measuring organ-specific proteins/transcripts in blood, biological tissue or other biological fluid.
Regarding claim 54, Hood teaches SEQ ID No: 4625 which has 100% sequence identity to SEQ ID 4 as shown below in the search result.
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“and/or” reads on the limitation “ and/or wherein the mature and/or active TFAM protein does not contain at the N-terminus the sequence set forth in SEQ ID NO:40 or a sequence that has at least 90% sequence identity to the sequence set forth in SEQ ID NO:40 as an optional limitation. Thus, Gunst and Hood suggest a method wherein the mature and/or active TFAM protein is a human mature TFAM protein with the sequence set forth in SEQ ID NO:4 or SEQ ID NO:8, or a protein that has at least 90% sequence identity to the sequence set forth in SEQ ID NO:4 or SEQ ID NO:8; and/or wherein the mature and/or active TFAM protein does not contain at the N-terminus the sequence set forth in SEQ ID NO:40 or a sequence that has at least 90% sequence identity to the sequence set forth in SEQ ID NO:40.
Gunst and Hood are both considered to be analogous to the claimed invention because they are in the same field of detecting proteins in a biological sample. Therefore, it would have been obvious to someone of ordinary skill in the art before the effective filing date of the claimed invention to have modified the method of detecting an abnormal level of mature and/or active mitochondrial transcription factor A (TFAM) protein in a sample from a patient as taught by Gunst to incorporate the method wherein TFAM sequence comprises SEQ ID NO:4 as taught by Hood and provide a method of detecting an abnormal level human mature TFAM protein with the sequence set forth in SEQ ID NO:4. These claim elements were known in the art and one of skill in the art could have combined these elements by known methods with no change in their respective functions, and the combination would have yielded the predictable outcome according to the limitations of claim 54. Doing so would allow for detection of active TFAM protein level.
Claim 28 is rejected under 35 U.S.C. 103 as being unpatentable Gunst et al. (“Gunst”; (2013). Insufficient autophagy contributes to mitochondrial dysfunction, organ failure, and adverse outcome in an animal model of critical illness. Critical care medicine, 41(1), 182-194.) as applied to claim 14 above, and further in view of Shenoy, S. (“Shenoy”; (2020). Coronavirus (Covid-19) sepsis: revisiting mitochondrial dysfunction in pathogenesis, aging, inflammation, and mortality. Inflammation research, 69(11), 1077-1085.).
The teachings of Gunst are documented above in the rejection of claims 14 and 18 under 35 U.S.C. 103. Claim 28 depends on claim 18, which depends on claim 14. Gunst does not explicitly teach the limitations of claim 28.
Shenoy discloses “Evidence indicates that mitochondria in senescent cells may be dysfunctional and unable to keep up with hypermetabolic demands associated with Covid-19 sepsis”. (Abstract-Results, first sent.)
Regarding claim 28, Shenoy teaches “The molecular mechanisms for this remain unclear. With cell recovery, mitochondrial biogenesis is initiated and there are upregulation of markers of mitochondrial biogenesis such …Transcription factor A for mitochondria (TFAM)” (Pg. 1080, Col. 2, Para. 2). Shenoy teaches “the relevance of mitochondrial dysfunction in pathogenesis of multi-organ failure associated with Covid-19 sepsis continues to be under active investigation” (Pg. 1083, Col. 2, Last Para.). Shenoy teaches “Excessive Covid-19 virus-related sepsis and mortality may be due to decline in mitochondrial efficiency and respiratory chain functions with aging. Furthermore, recent evidence also indicates that the virus may manipulate the mitochondria to maintain its life cycle.” (Pg. 1078, Col. 1, Para. 2) and “mitochondrial disruption hinders an effective immune response, increases inflammation, and severity in the Covid-19-related sepsis” (Pg. 1078, Col. 2, Para. 1). Shenoy also teaches “Sepsis is defined as a life-threatening organ dysfunction caused by dysregulated host response to an infection” (Pg. 1077, Col. 1, Para. 1) Thus, Gunst and Shenoy suggest a method wherein the organ dysfunction is associated with and/or caused by COVID19 and/or an infection with SARS-CoV-2.
Gunst and Shenoy are both considered to be analogous to the claimed invention because they are in the same field of mitochondrial dysfunction. Therefore, it would have been obvious to someone of ordinary skill in the art before the effective filing date of the claimed invention to have modified the method of detecting an abnormal level of mature and/or active mitochondrial transcription factor A (TFAM) protein in a sample from a patient as taught by Gunst to incorporate suggestion that organ dysfunction is associated with COVID19 taught by Shenoy and provide a method of detecting an abnormal level TFAM protein in a sample from a patient, wherein an abnormal level of mature and/or active TFAM protein in the sample is indicative of the presence of an organ dysfunction and wherein the organ dysfunction is associated with and/or caused by COVID19 and/or an infection with SARS-CoV-2. These claim elements were known in the art and one of skill in the art could have combined these elements by known methods with no change in their respective functions, and the combination would have yielded the predictable outcome according to the limitations of claim 28. Doing so would allow for the molecular mechanism of mitochondrial dysfunction in pathogenesis of multi-organ failure associated with Covid-19 sepsis to be elucidated.
Claim 31 is rejected under 35 U.S.C. 103 as being unpatentable over Gunst et al. (“Gunst”; (2013). Insufficient autophagy contributes to mitochondrial dysfunction, organ failure, and adverse outcome in an animal model of critical illness. Critical care medicine, 41(1), 182-194.) as applied to claim 14 above, and further in view of Sarkar et al. (“Sarkar”; Patent App. Pub. US 20110033480 A1, Feb. 10, 2011).
The teachings of Gunst are documented above in the rejection of claims 14 under 35 U.S.C. 103. Claim 31 depends on claim 14. Gunst does not explicitly teach the limitations of claim 31.
Sarkar discloses “mitochondrial transcription stimulatory factors Tfam, TBF1M and TFB2M facilitate mtRNA-polymerase activity to initiate transcription of the mtDNA-encoded genes such as several subunits of the OxPhos subunits, tRNA and rRNA genes, essential for mitochondrial integrity, function and mitochondrial DNA replication.”(Para. 305).
Regarding claim 31, Sarkar teaches a method wherein “Tfam, and TFB2M were evaluated… by Western blotting” (Para. 306). “Tfam” reads on mature and/or active TFAM protein. “Western blotting” reads on measuring the levels of protein. “Tfam … and TFB2M facilitate mtRNA-polymerase activity” reads on TFAM interaction with TFB2M. Thus, Gunst and Sarkar suggests a method wherein the level of mature and/or active TFAM protein corresponds to the number of interactions of TFAM protein with TFB2M protein, and/or wherein measuring the level of mature and/or active TFAM protein comprises quantifying the interaction of TFAM protein with TFB2M protein.
Gunst and Sarkar are both considered to be analogous to the claimed invention because they are in the same field of detecting proteins in a biological sample. Therefore, it would have been obvious to someone of ordinary skill in the art before the effective filing date of the claimed invention to have modified the method of detecting an abnormal level of mature and/or active mitochondrial transcription factor A (TFAM) protein in a sample from a patient as taught by Gunst to incorporate the method of measuring the level TFAM protein with TFB2M protein as taught by Sarkar and provide a method for detecting an abnormal level TFAM protein in a sample from a patient, wherein measuring the level of mature and/or active TFAM protein comprises quantifying the interaction of TFAM protein with TFB2M protein. These claim elements were known in the art and one of skill in the art could have combined these elements by known methods with no change in their respective functions, and the combination would have yielded the predictable outcome according to the limitations of claim 31. Doing so would provide insight to the levels of TFAM protein with TFB2M that is able to facilitate mitochondrial function.
Claim 34-35 and 39 are rejected under 35 U.S.C. 103 as being unpatentable over Gunst et al. (“Gunst”; (2013). Insufficient autophagy contributes to mitochondrial dysfunction, organ failure, and adverse outcome in an animal model of critical illness. Critical care medicine, 41(1), 182-194.) in view of Sarkar et al. (“Sarkar”; Patent App. Pub. US 20110033480 A1, Feb. 10, 2011).as applied to claim 31 above, and further in view of Alam, Muhammad (“Alam”;. "Proximity ligation assay (PLA)." Current protocols in immunology 123.1 (2018): e58.).
The teachings of Gunst and Sarkar are documented above in the rejection of claims 14 and 31 under 35 U.S.C. 103. Claim 39 depends on claim 35, which depends on claim 34, which depends on claim 31. Gunst does not explicitly teach the limitations of claim 34-35 and 39.
Alam discloses “Proximity ligation assay (PLA), also referred to as Duolink® PLA technology, permits detection of protein-protein interactions in situ (at distances <40 nm) at endogenous protein levels. It exploits specific antibodies identifying (either directly or indirectly) the two proteins of interest and utilizes specific DNA primers covalently linked to the antibodies. A hybridization step followed by DNA amplification with fluorescent probes permit visualization of spots of proximity by fluorescence microscopy. Since the development of PLA in 2002, it has been increasingly used to detect the interaction between two proteins with high sensitivity and specificity. It is a simple and sensitive technique to study protein-protein interaction in cells.” (Abstract)
Regarding claim 34-35 and 39, Alam teaches a method wherein “Duolink R PLA technology, allows one to detect protein-protein interactions at endogenous protein levels with high sensitivity and specificity (Fredriksson et al., 2002; Gullberg et al., 2004; Soderberg et al., 2006). Cells must be stained with two immunohistochemistry or immunofluorescence compatible primary antibodies to the target proteins. The two primary antibodies must be generated from different species (i.e., mouse/rabbit, rabbit/goat, or mouse/goat). Cells are then stained with secondary antibodies (2°-Ab) known as PLA probes (one PLUS and one MINUS). The PLA probes that bind to the constant regions of the primary antibodies contain a unique DNA strand. If the proteins of interest interact with each other, the DNA probes hybridize to make circular DNA. This DNA can be amplified and visualized by fluorescently labeled complementary oligonucleotide probes. The schematic diagram of Duolink® PLA is shown in Figure 1. Interactions are visualized as dots, and the number and intensity of the dots can be quantified by fluorescence microscopy.” (Pg. 1, Para. 2). “primary antibodies to the target proteins” reads on a pair of binding molecules wherein one specifically bind TFAM protein and the other specifically binds TFB2M protein. Thus, Gunst, Sarkar and Alam suggests a method wherein quantifying the interaction of TFAM protein with TFB2M protein comprises the steps of (a) contacting said sample with a pair of binding molecules, wherein one of said binding molecules specifically binds TFAM protein, and wherein the other of said binding molecules specifically binds TFB2M protein, and(b) generating a detectable signal when said two binding molecules are in close proximity to each other; wherein one of the binding molecules is an antibody specifically binding TFAM protein and the other binding molecule is an antibody specifically binding TFB2M protein, wherein at least one of said binding molecules is an antibody conjugated to an oligonucleotide, and/or wherein at least one of said binding molecules is specifically bound by an antibody conjugated to an oligonucleotide; and/or wherein said step (b) of generating a detectable signal comprises the steps of (i) generating an oligonucleotide template when said two binding molecules are in close proximity to each other, and(ii) amplifying and/or extending said oligonucleotide template; and wherein said interaction is quantified by a proximity ligation assay (PLA) and/or a proximity- dependent initiation of hybridization chain reaction (proxHCR).
Gunst and Alam are both considered to be analogous to the claimed invention because they are in the same field of detecting proteins in a biological sample. Therefore, it would have been obvious to someone of ordinary skill in the art before the effective filing date of the claimed invention to have modified the method of detecting an abnormal level of mature and/or active mitochondrial transcription factor A (TFAM) protein in a sample from a patient as taught by Gunst to incorporate the method of quantifying the interaction of TFAM protein with TFB2M protein as taught by Alam and provide a method for detecting an abnormal level TFAM protein in a sample from a patient, wherein measuring the level of mature and/or active TFAM protein comprises quantifying the interaction of TFAM protein with TFB2M protein and wherein quantifying the interaction of TFAM protein with TFB2M protein comprises the steps of (a) contacting said sample with a pair of binding molecules, wherein one of said binding molecules specifically binds TFAM protein, and wherein the other of said binding molecules specifically binds TFB2M protein, and (b) generating a detectable signal when said two binding molecules are in close proximity to each other. These claim elements were known in the art and one of skill in the art could have combined these elements by known methods with no change in their respective functions, and the combination would have yielded the predictable outcome according to the limitations of claim 34-35 and 39. Doing so would provide insight to the protein-protein interactions of TFAM protein with TFB2M that is able to facilitate mitochondrial function in situ.
Conclusion
No claims are in condition for allowance.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to KENDRA R VANN-OJUEKAIYE whose telephone number is (571)270-7529. The examiner can normally be reached M-F 9:00 AM- 5:00 PM.
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/KENDRA R VANN-OJUEKAIYE/Examiner, Art Unit 1682
/WU CHENG W SHEN/Supervisory Patent Examiner, Art Unit 1682