DETAILED ACTION
Notice of Pre-AIA or AIA Status
1. The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
2. Applicant’s election of Group 1 in the reply filed on May 3, 2026 is acknowledged. Because applicant did not distinctly and specifically point out the supposed errors in the restriction requirement, the election has been treated as an election without traverse (MPEP § 818.01(a)).
Claims 1-10 and 13-22 are currently pending.
Claim 14 is withdrawn from further consideration pursuant to 37 CFR 1.142(b) as being drawn to a nonelected invention, there being no allowable generic or linking claim. Election was made without traverse in the reply filed on May 3, 2026.
Claim Rejections - 35 USC § 103
3. 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.
4. Claims 1-5, 9-10, and 13 are rejected under 35 U.S.C. 103 as being unpatentable over Baird (US 2017/0306404 Pub 10/26/2017) in view of Mazzucco (Nature Communications 2020 11:5297) and Cawthon (Nucleic Acids Research 2002, Vol 30 No 10 e47).
Regarding Claim 1 Baird teaches a high throughput method for determining telomere length of mammalian chromosomal DNA. The method is called HT-STELA and comprises: i) annealing a primer sequence to a region adjacent a telomere repeat array of chromosomal DNA wherein said region is between 3843 bp-30 bp from said telomere repeat array; ii) PCR amplifying 20 ng-35 ng of said chromosomal DNA using 21-23 cycles to generate an amplification product; and iii) detecting the length of the amplification product. Baird teaches that reference to a region 3843 bp-30 bp from said telomere repeat array is reference to a region upstream from the start of the array (characterized predominantly by TTAGGG repeats) that caps the chromosome end (paras 0008-0013). Baird teaches that it has been found that pre-digesting the chromosomal DNA prior to performing HT-STELA reduces the complexity of sample DNA providing a simpler target for amplification. Therefore, in a further embodiment chromosomal DNA is digested with a restriction endonuclease that cleaves non-telomeric DNA (paras 0025-0027). Thus Baird teaches a method comprising digesting DNA from the subject or sample therefrom with at least one restriction enzyme, wherein the at least one restriction enzyme cuts proximal to, but does not cut within, a telomeric sequence; and amplifying a telomeric- specific sequence.
Regarding Claim 13 Baird teaches a method wherein the sample is a blood sample (para 0022).
Baird does not teach a method further comprising separating the digested DNA by size and isolating from the separated DNA, polynucleotide fragments shorter in length than an average telomere length for the sample (clm 1). Baird does not teach a method wherein the DNA is digested with multiple restriction enzymes (clm 2). Baird does not teach a method wherein the digested DNA is separated by gel electrophoresis (clm 3). Baird does not teach a method wherein the polynucleotide fragments are isolated from the gel (clm 4). Baird does not teach a method wherein the polynucleotide fragments are isolated from the gel by removing a gel section containing the fragments, and eluting the DNA from the gel section (clm 5).
However Mazzucco teaches that they have developed a two-step procedure for the large-scale purification of telomeric repeats from mammalian cells. Mazzucco teaches that genomic DNA (∼2.5 mg) from SV40LT-immortalized MEFs was digested with HinfI and MspI. The digested DNA was separated by centrifugation on a sucrose gradient. Seven fractions were collected and an aliquot (∼1/500) of each fraction was loaded on an agarose gel. The gel was blotted onto a membrane and hybridized with a TTAGGG repeats probe to verify that telomeric repeats remained in the high molecular weight (HMW) fractions. The HMW DNA, contained in the last four fractions of the sucrose gradient was recovered and digested with RsaI, AluI, MboI, HinfI, MspI, HphI, and MnlI. The digested DNA was separated on a preparative agarose gel and the DNA migrating in the area above 5 kb was extracted from the gel. Mazzucco teaches that the high molecular weight DNA recovered from the agarose gel shows a ∼1000-fold increase in telomeric repeats compared to the starting material, while more abundant mouse long interspersed repeats (L1 repeats) are undetectable (Fig. 1c). Telomere enrichment was confirmed in single-molecule IF-FISH analysis, where over 80% of the DNA molecules from enriched samples are recognized by a telomeric probe, while <1 in 1000 telomeric fibers were present in non-enriched samples (Fig. 1d). (see page 2, col 1-2, Fig 1 inset).
Accordingly, it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have modified the method of Baird by further separating the digested DNA by size and isolating from the separated DNA, polynucleotide fragments short in length than an average telomere length as suggested by Mazzucco. As discussed above, Baird teaches that it has been found that pre-digesting the chromosomal DNA prior to performing HT-STELA reduces the complexity of sample DNA providing a simpler target for amplification. One of skill in the art would have been motivated to separate the digested DNA by size and isolate polynucleotide fragments short in length and the average telomere length for the benefit of being able to further reduce the complexity of sample and enrich for telomeres having a desired length in the sample.
The combined references do not teach amplifying from the isolated fragments (a) a telomeric specific sequence and (b) a non-telomeric single copy sequence; quantitating the products of (a) and (b); and determining an amount of critically short telomeres by dividing the quantitated amount of (a) by the quantitated amount of (b). The combined references do not teach a method wherein amplification and quantitation of (a) and (b) is by quantitative PCR (clm 9). The combined references do not teach a method wherein the quantitative PCR is real time PCR or digital PCR (clm 10).
However Cawthon teaches a method of telomere measurement by real time quantitative PCR. Cawthon teaches that their strategy for determining relative telomere lengths by quantitative PCR was to measure, for each DNA sample, the factor by which the sample differed from a reference DNA sample in its ratio of telomere repeat copy number to single copy gene copy number. This ratio should be proportional to the average telomere length (page 1). Cawthon teaches that Figure 5 shows the strong correlation between T/S ratio and TRF length. Cawthon teaches that the correlation between relative T/S ratios measured by quantitative PCR and relative TRF lengths measured by the traditional Southern blot strongly supports the conclusion that the new PCR method does indeed measure relative telomere lengths (page 4).
Accordingly, it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have modified the method of Baird and Mazzucco by amplifying from the isolated fragments (a) a telomeric specific sequence and (b) a non-telomeric single copy sequence; quantitating the products of (a) and (b); and determining an amount of critically short telomeres by dividing the quantitated amount of (a) by the quantitated amount of (b) as suggested by Cawthon. In the instant case Cawthon teaches that the correlation between relative T/S ratios measured by quantitative PCR and relative TRF lengths measured by the traditional Southern blot strongly supports the conclusion that the new PCR method does indeed measure relative telomere lengths. One of skill in the art would have been motivated to perform the steps taught by Cawthon for the benefit of being able to measure relative telomere lengths.
5. Claims 6-7 are rejected under 35 U.S.C. 103 as being unpatentable over Baird (US 2017/0306404 Pub 10/26/2017) in view of Mazzucco (Nature Communications 2020 11:5297), Cawthon (Nucleic Acids Research 2002, Vol 30 No 10 e47) as applied to claim 1 above and in further view of Harley (US 10,316,366 Issued 6/11/2019).
The teachings of Baird, Mazzucco, and Cawthon are presented above. The combined references do not teach a method wherein the isolated polynucleotide fragments are no larger than about 1.5 kilobases or about 3.0 kilobases in length (clm 6). The combined references do not teach a method wherein the isolated polynucleotide fragments are between about 0.5 kilobases and about 1.5 kilobases in length, between about 1.0 kilobases and about 1.5 kilobases, or between about 1.5kilobases and about 3.0 kilobases in length.
However Harley teaches a method of measuring short telomere abundance. Harley teaches that short telomeres are telomeres having length no more than about than about 0.5 kb, than about 1 kb, than about 2 kb, than about 3 kb, than about 4 kb or than about 5 kb. The methods involve producing a population of copies of chromosomal fragments only from chromosomes having telomeres within a pre-defined length range (e.g., all telomeres no longer than a certain length, e.g., shorter than about 5 kbp) (col 15, lines 60-68).
Accordingly, it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have modified the method of Baird, Mazzucco, and Cawthon by isolating polynucleotides fragments having the claimed sizes recited in claims 6 and 7 as suggested by Harley. One of skill in the art interested in detecting the abundance of short telomeres would have been motivated to isolate polynucleotide fragments having the claimed fragment sizes since Harley teaches that short telomeres are telomeres having length no more than about than about 0.5 kb, than about 1 kb, than about 2 kb, than about 3 kb, than about 4 kb or than about 5 kb.
6. Claim 8 is rejected under 35 U.S.C. 103 as being unpatentable over Baird (US 2017/0306404 Pub 10/26/2017) in view of Mazzucco (Nature Communications 2020 11:5297) and Cawthon (Nucleic Acids Research 2002, Vol 30 No 10 e47) as applied to claim 1 and in further view of Cawthon (Nucleic Acids Research 2009, Vol 37 No 3 e21).
The teachings of Baird, Mazzucco, and Cawthon (2002) are presented above.
The combined references do not teach a method wherein (a) and (b) are amplified in the same PCR reaction.
However Cawthon (2009) teaches telomere length measurement by a novel monochrome multiplex quantitative PCR method. Cawthon teaches that for multiplex QPCR, the telomere primer pair telg and telc (final concentrations 900 nM each), were combined either with the albumin primer pair albu and albd (final concentrations 900 nM each), or with the beta-globin primer pair hbgu and hbgd, (final concentrations 500 nM each) in the master mix (page 2).
Accordingly, it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have modified the method of Baird, Mazzucco, and Cawthon (2002) by using multiplex PCR to quantitate (a) and (b) as suggested by Cawthon (2009). One of skill in the art would have been motivated to perform multiplex PCR since Cawthon (2009) teaches that multiplexing this assay is desirable, because variation in the amount of DNA pipetted would no longer contribute to variation in T/S, since T and S would be collected within each reaction, from the same input DNA. Multiplexing also increases throughput and lowers costs, since half as many reactions are needed (abstract).
7. Claims 15-22 are rejected under 35 U.S.C. 103 as being unpatentable over Baird (US 2017/0306404 Pub 10/26/2017) in view of Mazzucco (Nature Communications 2020 11:5297), Cawthon (Nucleic Acids Research 2002, Vol 30 No 10 e47), and Harley (US 10,316,366 Issued 6/11/2019).
Regarding Claim 15 Baird teaches a high throughput method for determining telomere length of mammalian chromosomal DNA. The method is called HT-STELA and comprises: i) annealing a primer sequence to a region adjacent a telomere repeat array of chromosomal DNA wherein said region is between 3843 bp-30 bp from said telomere repeat array; ii) PCR amplifying 20 ng-35 ng of said chromosomal DNA using 21-23 cycles to generate an amplification product; and iii) detecting the length of the amplification product. Baird teaches that reference to a region 3843 bp-30 bp from said telomere repeat array is reference to a region upstream from the start of the array (characterized predominantly by TTAGGG repeats) that caps the chromosome end (paras 0008-0013). Baird teaches that it has been found that pre-digesting the chromosomal DNA prior to performing HT-STELA reduces the complexity of sample DNA providing a simpler target for amplification. Therefore, in a further embodiment chromosomal DNA is digested with a restriction endonuclease that cleaves non-telomeric DNA (paras 0025-0027). Thus Baird teaches a method comprising digesting DNA from the subject or sample therefrom with at least one restriction enzyme, wherein the at least one restriction enzyme cuts proximal to, but does not cut within, a telomeric sequence; and amplifying a telomeric- specific sequence.
Baird does not teach a method further comprising separating the digested DNA by size and isolating from the separated DNA, polynucleotide fragments shorter in length than an average telomere length for the sample (clm 15).
However Mazzucco teaches that they have developed a two-step procedure for the large-scale purification of telomeric repeats from mammalian cells. Mazzucco teaches that genomic DNA (∼2.5 mg) from SV40LT-immortalized MEFs was digested with HinfI and MspI. The digested DNA was separated by centrifugation on a sucrose gradient. Seven fractions were collected and an aliquot (∼1/500) of each fraction was loaded on an agarose gel. The gel was blotted onto a membrane and hybridized with a TTAGGG repeats probe to verify that telomeric repeats remained in the high molecular weight (HMW) fractions. The HMW DNA, contained in the last four fractions of the sucrose gradient was recovered and digested with RsaI, AluI, MboI, HinfI, MspI, HphI, and MnlI. The digested DNA was separated on a preparative agarose gel and the DNA migrating in the area above 5 kb was extracted from the gel. Mazzucco teaches that the high molecular weight DNA recovered from the agarose gel shows a ∼1000-fold increase in telomeric repeats compared to the starting material, while more abundant mouse long interspersed repeats (L1 repeats) are undetectable (Fig. 1c). Telomere enrichment was confirmed in single-molecule IF-FISH analysis, where over 80% of the DNA molecules from enriched samples are recognized by a telomeric probe, while <1 in 1000 telomeric fibers were present in non-enriched samples (Fig. 1d). (see page 2, col 1-2, Fig 1 inset).
Accordingly, it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have modified the method of Baird by further separating the digested DNA by size and isolating from the separated DNA, polynucleotide fragments short in length than an average telomere length as suggested by Mazzucco. As discussed above, Baird teaches that it has been found that pre-digesting the chromosomal DNA prior to performing HT-STELA reduces the complexity of sample DNA providing a simpler target for amplification. One of skill in the art would have been motivated to separate the digested DNA by size and isolate polynucleotide fragments short in length and the average telomere length for the benefit of being able to further reduce the complexity of sample and enrich for telomeres in the sample.
The combined references do not teach amplifying from the isolated fragments (i) a telomeric specific sequence and (ii) a non-telomeric single copy sequence; quantitating the products of (i) and (ii); and determining an amount of critically short telomeres by dividing the quantitated amount of (i) by the quantitated amount of (ii).
However Cawthon teaches a method of telomere measurement by real time quantitative PCR. Cawthon teaches that their strategy for determining relative telomere lengths by quantitative PCR was to measure, for each DNA sample, the factor by which the sample differed from a reference DNA sample in its ratio of telomere repeat copy number to single copy gene copy number. This ratio should be proportional to the average telomere length (page 1). Cawthon teaches that Figure 5 shows the strong correlation between T/S ratio and TRF length. Cawthon teaches that the correlation between relative T/S ratios measured by quantitative PCR and relative TRF lengths measured by the traditional Southern blot strongly supports the conclusion that the new PCR method does indeed measure relative telomere lengths (page 4).
Accordingly, it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have modified the method of Baird and Mazzucco by amplifying from the isolated fragments (i) a telomeric specific sequence and (ii) a non-telomeric single copy sequence; quantitating the products of (i) and (ii); and determining an amount of critically short telomeres by dividing the quantitated amount of (i) by the quantitated amount of (ii) as suggested by Cawthon. In the instant case Cawthon teaches that the correlation between relative T/S ratios measured by quantitative PCR and relative TRF lengths measured by the traditional Southern blot strongly supports the conclusion that the new PCR method does indeed measure relative telomere lengths. One of skill in the art would have been motivated to perform the steps taught by Cawthon for the benefit of being able to measure relative telomere lengths.
The combined references do not teach a method further comprising f) repeating steps a) to e), wherein the DNA is obtained from the subject at a later time point in comparison to the DNA used in steps a) to e); and g) comparing the amount of the critically short telomeres determined in step f) with the amount of the critically short telomeres determined in step e) (clm 15). The combined references do not teach a method wherein the later time point is hours days, months, or years (clm 16). The combined references do not teach a method wherein step f) is repeated at one or more additional later time points (clm 17).
However Harley teaches a method comprising: determining a rate of change in a measure of short telomere abundance in cells from a plurality of subject samples, each sample taken at different times; and correlating the rate of change with: (1) a measure of health; (2) a risk of a pathological condition; (3) a telomeric disease or (4) drug responsiveness (col 7, lines 3-13).
Accordingly, it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have modified the method of Baird, Mazzucco, Cawthon by repeating steps a) to e), using a sample obtained from the subject at a later time point and comparing the amounts of the critically short telomeres as suggested by Harley. One of skill in the art would have been motivated to determine how the amounts are changing over time for the benefit of being able to correlate the rate of change with (1) a measure of health; (2) a risk of a pathological condition; (3) a telomeric disease or (4) drug responsiveness.
The combined references do not teach a method wherein the subject is undergoing a treatment for a disease or condition associated with increased critically short telomeres, and wherein a decrease in the amount of the critically short telomeres over time indicates efficacy of treatments for the disease or condition (clm 18). The combined references do not teach a method wherein the subject is undergoing a treatment for a disease or condition associated with decreased critically short telomeres, and wherein an increase in the amount of critically short telomeres over time indicates efficacy of treatments for the disease or condition (clm 19). The combined references do not teach a method wherein the disease is a cancer, and wherein the therapy is chemotherapy (clm 20).
However Harley teaches that the present disclosure is useful in monitoring effectiveness of therapeutics or in screening for drug candidates affecting telomere length or telomerase activity. The ability to monitor telomere characteristics can provide a window for examining the effectiveness of particular therapies and pharmacological agents. The drug responsiveness of a disease state to a particular therapy in an individual may be determined by the method of the present disclosure. For example, the present disclosure finds use in monitoring the effectiveness of cancer therapy since the proliferative potential of cells is related to the maintenance of telomere integrity (clm 31, lines 45-65). Further Harley teaches methods for determining measures of short telomere abundance in a population of chromosomes and of using these measures for determining measures of health and effects of interventions that increase or decrease telomere length and, hence, increase or decrease health, or conversely decrease or increase risk of future disease or death, respectively (col 15, lines 55-67). Harley discloses diseases associated with increased critically short telomeres and those associated with decreased critically short telomeres (col 27 line 44 to col 28 line 67). Harley discloses chemotherapy for cancer treatment (col 32, lines 1-5).
Accordingly, it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have modified the method of Baird, Mazzucco, and Cawthon by further using the amount of critically short telomeres to monitor the effects of a treatment (such as chemotherapy) for a disease that is associated with telomere length (such as cancer) as suggested by Harley. One of skill in the art would have been motivated to do this since Harley teaches that that it was known in the art at the time of the invention that determining measures of short telomere abundance in a population of chromosomes was useful to determine measures of health and effects of interventions that increase or decrease telomere length and, hence, increase or decrease health, or conversely decrease or increase risk of future disease or death, respectively.
The combined references do not teach a method wherein the isolated polynucleotide fragments are no larger than about 1.5kilobases or about 3.0 kilobases in length (clm 21). The combined references do not teach a method wherein the isolated polynucleotide fragments are between about 0.5 kilobase and about 1.5 kilobases in length, between about 1.0 kilobases and about 1.5kilobase, or between about 1.5kilobases and about 3.0 kilobases in length (clm 22).
However Harley teaches a method of measuring short telomere abundance. Harley teaches that short telomeres are telomeres having length no more than about than about 0.5 kb, than about 1 kb, than about 2 kb, than about 3 kb, than about 4 kb or than about 5 kb. The methods involve producing a population of copies of chromosomal fragments only from chromosomes having telomeres within a pre-defined length range (e.g., all telomeres no longer than a certain length, e.g., shorter than about 5 kbp) (col 15, lines 60-68).
Accordingly, it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have modified the method of Baird, Mazzucco, and Cawthon by isolating polynucleotides fragments having the claimed sizes recited in claims 6 and 7 as suggested by Harley. One of skill in the art interested in detecting the abundance of short telomeres would have been motivated to isolate polynucleotide fragments having the claimed fragment sizes since Harley teaches that short telomeres are telomeres having length no more than about than about 0.5 kb, than about 1 kb, than about 2 kb, than about 3 kb, than about 4 kb or than about 5 kb.
8. Any inquiry concerning this communication or earlier communications from the examiner should be directed to AMANDA HANEY whose telephone number is (571)272-8668. The examiner can normally be reached Monday-Friday, 8:15am-4:45pm EST.
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If attempts to reach the examiner by telephone are unsuccessful, the examiner’s supervisor, Wu-Cheng Shen can be reached at 571-272-3157. The fax phone number for the organization where this application or proceeding is assigned is 571-273-8300.
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/AMANDA HANEY/Primary Examiner, Art Unit 1682
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