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 .
DETAILED ACTION
1. The Office acknowledges the receipt of Applicant’s restriction election filed April 22, 2026. Applicant elects Group I for examination. As no traverse is presented, this restriction election is considered to be made without traverse. Claims 1-6, 8, 9, 11, 12 and 14-23 are pending. Claim 23 is withdrawn from examination. Claims 1-6, 8, 9, 11, 12 and 14-22 are examined in the instant application. The restriction is made FINAL.
Specification
2. The disclosure is objected to because of the following:
The disclosure is objected to because it contains an embedded hyperlink and/or other form of browser-executable code. Applicant is required to delete the embedded hyperlink and/or other form of browser-executable code; references to websites should be limited to the top-level domain name without any prefix such as http:// or other browser-executable code. See MPEP § 608.01. See p. 97 ln. 14, and p. 100 ln. 3, for example. On page 111 of the specification, Applicant states that URL addresses are provided as non-browser-executable codes, with periods of the respective web address in parentheses, and the actual web addresses do not contain the parentheses. However, the periods of the respective web addresses are not in parentheses. It is suggested the “https://” be deleted.
On page 91, “Solution CS” should be spelled out.
Appropriate correction is required.
Claim Objections
3. Claims 9, 15-17 and 19 are objected to because of the following:
In claims 9 and 15, a comma should be inserted after “wherein prior to (b)”.
In claims 16, 17 and 19, a comma should be inserted after “wherein in ____”. Appropriate correction is required.
Claim Rejections - 35 USC § 112(b)
4. 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.
5. Claims 9, 12 and 17 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 pre-AIA the applicant regards as the invention.
In claim 9, it is unclear how “fixed” is defined. It is unclear whether “fixed” means the cells are immobilized/attached to a surface, or the cells have been subjected to a particular treatment.
In claim 12, the metes and bounds of “about 11.8” are unclear. The specification does not define the range encompassed by the recitation of “about 11.8”. It is suggested “about” be deleted.
In claim 17, it is unclear whether “plasmid safe DNase” refers generically to DNase that is safe for use with plasmids, or Applicant is referring to ATP-dependent Plasmid Safe DNase (P.S. DNase). The specification appears to use these terms interchangeably.
Clarification and/or correction is required.
Claim Rejections - 35 USC § 112(a)
6. The following is a quotation of the first paragraph of 35 U.S.C. 112(a):
(a) IN GENERAL.—The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same, and shall set forth the best mode contemplated by the inventor or joint inventor of carrying out the invention.
The following is a quotation of the first paragraph of pre-AIA 35 U.S.C. 112:
The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same, and shall set forth the best mode contemplated by the inventor of carrying out his invention.
7. Claims 1-6, 8, 9, 11, 12 and 14-22 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.
Applicant’s disclosure is as follows.
Example 1 discloses a three-step extrachromosomal circular DNA (eccDNA) enrichment method. First, crude eccDNAs are extracted by alkaline lysis at pH 11.8 to prevent irreversible denaturation or breakage of circular DNA. Secondly, a rare-cutter PacI restriction enzyme is used to linearize mitochondrial DNA (mtDNA) before ATP-dependent plasmid safe DNase (P.S DNase, an exonuclease) is used to digest linear DNA. Thirdly, a solution (Solution A) is used to selectively recover circular, but not linear, DNA on silica beads to further exclude any remaining linear DNA that escapes exonuclease digestion.
Example 2 discloses that eccDNAs are mapped to widespread locations across the entire genome and their abundance are correlated with genomic copy numbers.
Example 3 discloses that eccDNAs are generated by random ligation of nucleosome-size genomic DNA fragments. Example 3 further discloses that apoptotic DNA fragmentation (ADF) is necessary for eccDNA generation, and that DNase γ nuclease, but not Endonuclease G (EndoG), is responsible for ADF, as evidenced by the abrogation of ADF in DNase γ knockout cells.
Example 4 discloses that the Lig3 nuclear, not mitochondria, isoform (NucLig3-I-) is the main DNA ligase for eccDNA generation, as determined by knockout of NucLig3-I-. Knockout of Lig1 or Lig4 alone or in combination did not significantly affect eccDNA generation. Knockout of Lig1 and Lig3 is lethal.
Example 5 discloses that eccDNAs are more potent immunostimulants of bone marrow derived dendritic cells (BMDCs) and bone marrow derived macrophages (BMDMs) than linear DNA..
Example 6 discloses that the circular nature of eccDNAs is critical for their strong immunostimulant activity. Circular DNA, not linear DNA of the same sequences, greatly induced cytokine gene transcription. The increased immunostimulatory potency of circular DNA is not due to its increased stability and transfection efficiency, because linear DNA having protected ends have the same transfection efficiency and stability.
Example 7 discloses that eccDNAs present in apoptotic medium can be sensed by BMDCs. Specifically, eccDNA are generated in apoptotic cells and released into the culture medium. When BMDCs were co-incubated with the cell-free apoptotic supernatant of WT mESCs (mouse embryonic stem cells) cells, the supernatant of apoptotic medium of WT cells (not DNase γ-/- mESCs cells, which are deficient in eccDNA generation) stimulates IFN-α and IFN-β expression. These data indicates that eccDNAs, rather than linear DNAs, mitochondrial DNAs, or RNAs in the supernatant of apoptotic medium, are responsible for the induced immune response, and that eccDNAs can be actively sensed by BMDCs without transfection. These results indicate that eccDNAs are potent damage-associated molecular patterns (DAMPs) of the innate immune system.
Example 8 discloses that STING (stimulator of interferon genes) is required for eccDNA-triggered immune response. Specifically, BMDCs transfected with purified eccDNAs, but not linear genomic DNA fragments, significantly increased the expression of cytokines, chemokines and type I interferons, indicating that eccDNA is a potent innate immunostimulant that can increase innate immune response. Similar results are shown for eccDNAs in BMDMs. Mouse BMDCs deficient for STING completely abrogates the capacity of BMDC to respond to eccDNAs, while BMDCs deficient for Myd88 (Toll-like receptor (TLR)) does not affect the capacity of BMDC to respond to eccDNAs. Thus, the sensing pathway is STING and not TLR. Table 1 shows the genes that are up-regulated in BMDCs treated with eccDNA. Table 2 shows the genes that are up-regulated in BMDMs treated with eccDNA. Table 3 shows differentially expressed genes in BMDCs in response to STING or Myd88 (TLR) knockout with eccDNA treatment.
Example 9 discloses an optimized method for eccDNA purification. The size heterogeneity and low abundance of eccDNA relative to linear chromosomal DNA make obtaining high purity eccDNA difficult. Previous purification methods are laborious, in which crude DNA circles are extracted from samples by Hirt or alkaline lysis during which large size chromosome DNAs are co-precipitated and removed with proteins. Then linear contaminant DNAs within the crude circular DNA are digested with an exonuclease such as ATP-dependent Plasmid Safe DNase (P.S. DNase), exonuclease V (RecBCD) or exonuclease III. However, the low amount of isolated eccDNA may be lost due to trace amounts of contaminating endonucleases or endonuclease activity in the exonuclease, or the exonuclease activity can be inhibited or blocked by damaged nucleosides, cross linkages, special structures, or their digestion products. The crude circular DNA from Hirt, alkaline lysis or exonuclease treatment could be further purified by cesium chloride-ethidium bromide (CsCl-EB) gradient ultracentrifuge. Applicant’s method utilizes a 3-step eccDNA purification (3SEP) technique as described in Example 1. While previous techniques require one week, the 3SEP procedure can be completed in one day. Given its high reproducibility, the method can also be used for evaluating the eccDNA yield by mass quantification, visualization in agarose gel or other DNA quantification techniques, with or without further amplification. The 3SEP DNA recovery uses magnetic separation. Reagents, equipment and buffers for use in the 3SEP protocol are listed. The 3SEP protocol has three steps: crude circular DNA isolation, digest linear DNA, and selectively recover eccDNA. Table 4 discloses guide RNA targeting sequences and PCR primers for DNase γ and Endonuclease G in mESC/E14 cells and CH12F3 cells. Table 5 discloses synthetic small DNA and its PCR amplification primer sequences.
The claimed invention lacks adequate written description for the following reasons.
In claim 1(b), “an enzyme that linearize mitochondrial DNA (mtDNA), under conditions under which mtDNA is linearized, thereby producing a mixture of linearized DNA and eccDNA” lacks adequate written description. The specification discloses only one enzyme that linearizes mtDNA--a rare-cutter PacI restriction enzyme. No other enzyme is disclosed. One skilled in the art cannot predict the structures of other enzymes for linearizing mtDNA without destroying eccDNA from the disclosure of the PacI enzyme. No function and structure relationship is disclosed or recognized for enzymes that linearize mtDNA without also linearizing eccDNA. Applicant is pre-empting and capturing innovations beyond what is described. The claim encompasses embodiments that Applicant did not contemplate as well as yet-to-be discovered inventions. Accordingly, Applicant has not provided adequate written description for the broadly claimed “an enzyme that linearizes mitochondrial DNA (mtDNA), under conditions under which mtDNA is linearized, thereby producing a mixture of linearized DNA and eccDNA.” See also claim 22(b).
In claim 1(c), “an enzyme that digests linear DNA, thereby producing a mixture of digested linear DNA and eccDNA” lacks adequate written description. The working examples disclose exonuclease ATP-dependent Plasmid Safe DNase (P.S. DNase). As described in Example 9, the specificity and efficacy of the exonuclease used must be carefully determined because the low amount of isolated eccDNA may be lost due to trace amounts of contaminating endonucleases or endonuclease activity in the exonuclease. However, the claim does not exclude enzymes that digest linear DNA without endonuclease contamination. From the disclosure of P.S. DNase, one skilled in the art cannot predict the structures of other enzymes that would digest linear DNA without destroying eccDNA. There is no disclosed or recognized structure and function relationship for these enzymes. Accordingly, “an enzyme that digests linear DNA, thereby producing a mixture of digested linear DNA and eccDNA” lacks adequate written description. See also claim 22(c).
8. Claims 1-6, 8, 9, 11, 12 and 14-22 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 enablement requirement. The claim(s) contains subject matter which was not described in the specification in such a way as to enable one skilled in the art to which it pertains, or with which it is most nearly connected, to make and/or use the invention.
Enablement factors to consider include (1) the quantity of experimentation necessary, (2) the amount of direction or guidance presented, (3) the presence or absence of working examples, (4) the nature of the invention, (5) the state of the prior art, (6) the relative skill of those in the art, (7) the predictability or unpredictability of the art, and (8) the breadth of the claims. In re Wands, 858 F.2d 731, 737 (Fed. Cir. 1988).
Applicant’s disclosure is as set forth above. The claimed invention is not enabled for the following reasons.
In claim 1, “a sample comprising unenriched eccDNA” does not exclude cells comprising unenriched eccDNA. However, there is no lysis step of the cells to release the eccDNA. It is unclear how step (b) can be performed absent the lysis step. Moreover the lysis step must be performed at pH 11.8 to prevent irreversible denaturation or breakage of circular DNA as described in Example 1. However, the claimed method does not recite the lysis step nor the alkaline pH conditions required. Applicant has no working example of performing the claimed method with a cell sample or a different pH. Thus, absent the lysis step at pH 11.8, one skilled in the art cannot make and use the claimed invention without undue experimentation. See also claim 22.
In claim 1, separating eccDNA from the digested linear DNA in a mixture of digested linear DNA and eccDNA to produce enriched eccDNA is not fully enabled. Applicant’s working example for separating the eccDNA from the digested linear DNA is by using a solution (Solution A) on magnetic silica beads. It is unpredictable how digested DNA can be separated from eccDNA other than by the disclosed method. There is no disclosure as to what conditions are necessary to separate them. It is not known what is present in Solution A. As the eccDNAs are of different sizes and have different sequence structures, separating the eccDNAs from linear DNA by size or by chemical means would not separate the eccDNA from digested linear DNA, especially given their low abundance compared to linear DNA. The specification lists different ways of separating eccDNA from linear DNA, however, none of them addresses the lack of specificity in size and sequence of the eccDNA population within the sample. See also claim 22.
In claim 21, “rolling circle amplification” is not enabled because no primer sequences are disclosed to enrich the eccDNA. The specification discloses eccDNAs are not sequence specific. The claims are not limited to a particular sequence structure. Absent a disclosure of a target eccDNA structure and primers for use in the rolling circle amplification to enrich said target eccDNA, one skilled in the art cannot enrich eccDNA by rolling circle amplification without undue experimentation.
Given these difficulties, notwithstanding a relatively high level of ordinary skill of those in the art, the amount of experimentation would likely be extensive and undue. Weighing all the Wands factors based on the totality of the record as discussed above, the Office determines that it would require undue experimentation for a person of ordinary skill in the art to make and use the invention as claimed.
Claim Rejections - 35 USC § 102
9. In the event the determination of the status of the application as subject to AIA 35 U.S.C. 102 and 103 (or as subject to pre-AIA 35 U.S.C. 102 and 103) is incorrect, any correction of the statutory basis (i.e., changing from AIA to pre-AIA ) for the rejection will not be considered a new ground of rejection if the prior art relied upon, and the rationale supporting the rejection, would be the same under either status.
10. The following is a quotation of the appropriate paragraphs of 35 U.S.C. 102 that form the basis for the rejections under this section made in this Office action:
A person shall be entitled to a patent unless –
(a)(1) the claimed invention was patented, described in a printed publication, or in public use, on sale, or otherwise available to the public before the effective filing date of the claimed invention.
11. Claims 1, 2, 4-6, 8, 9, 16, 17, 21 and 22 are rejected under 35 U.S.C. 102(a)(1) as being anticipated by Iwasaki et al. (FEBS Letters 363, 1995, pp. 239-245 (Applicant’s IDS)).
With regard to claims 1, 2, 4-6, 8, 16, 17 and 22, Iwasaki teaches a method for enriching eccDNA from a human cell (HeLa) sample comprising (a) obtaining a sample comprising unenriched eccDNA; (b) treating the sample with PacI, an enzyme that linearizes mitochondrial DNA, to produce a mixture of linearized DNA and eccDNA; (c) treating the mixture with ATP-dependent DNase (same as Applicant’s plasmid safe DNase), an enzyme that digests linear DNA to produce a mixture of digested linear DNA and eccDNA; and (d) separating eccDNA from the digested linear DNA in the mixture, thereby producing enriched eccDNA (p. 239, “2.2 Purification of EC DNA from human cells”.
With regard to claim 9, prior to step (b), the cells are lysed with proteinase K (p. 239, “2.2 Purification of EC DNA from human cells”).
With regard to claim 21, the enriched eccDNA is amplified by rolling circle amplification (p. 240, “2.5 Inverse PCR”).
Accordingly, the claimed invention is anticipated by the prior art.
12. Claims 1-6, 8, 9, 15-17 and 20-22 are rejected under 35 U.S.C. 102(a)(1) as being anticipated by Moller et al. (Methods in Molecular Biology, 2020, Vol. 2119, pp. 165-181 (U)).
With regard to claims 1, 2, 4-6, 9, 16, 17, 20 and 22, Moller teaches a method for enriching eccDNA from a human muscle cell sample comprising (a) obtaining a sample comprising unenriched eccDNA from lysed cells (p. 166, “2.1 Cell Lysis”); (b) treating the sample with PacI, an enzyme that linearizes mitochondrial DNA, to produce a mixture of linearized DNA and eccDNA (p. 176, first paragraph); (c) treating the mixture with Plasmid-Safe ATP-dependent DNase (p. 167, “2.3 Linear DNA Removal”), an enzyme that digests linear DNA to produce a mixture of digested linear DNA and eccDNA; and (d) separating eccDNA from the digested linear DNA in the mixture using magnetic silica beads (p. 168, “2.5 Concentrate DNA”; p. 172, “Clean Up and Concentrate DNA”; Note: Agencourt AMPure XP beads are magnetic silica-coated beads), thereby producing enriched eccDNA.
With regard to claim 3, Moller also teaches a blood biological sample (p. 176, Note 4) and plasma biological sample (p. 177, Note 6).
With regard to claim 8, Moller teaches cultured mammalian cells (p. 166, second full paragraph).
With regard to claim 15, Moller teaches prior to step (b), unenriched eccDNA is bound to magnetic silica beads to separate eccDNA from lysed cells in the sample (p. 177, Note 10).
With regard to claim 21, Moller teaches amplifying the enriched eccDNA by rolling circle amplification (Abstract, p. 168 “2.6 Rolling-Circle Amplification & Sequencing”).
Accordingly, the claimed invention is anticipated by the prior art.
Claim Rejections – 35 USC § 103
13. 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.
14. Claim 3 is rejected under 35 U.S.C. 103 as being unpatentable over Iwasaki et al. (FEBS Letters 363, 1995, pp. 239-245 (Applicant’s IDS)) in view of Lo et al. (US Pub. No. 20200407799 (Applicant’s IDS)).
The teachings of Iwasaki have been discussed above.
Iwasaki teaches the biological sample is HeLa cells. Iwasaki does not teach that the biological sample is from blood or plasma.
Lo teaches a method of enriching eccDNA from plasma [0091].
It would have been prima facie obvious to one skilled in the art at the time of filing to substitute the HeLa cells of Iwasaki with the plasma of Lo, depending on the desired target eccDNA population to be enriched. The choice of biological sample to enrich eccDNA from is a matter of design choice well within the means of one skilled in the art with a reasonable expectation of success.
15. Claims 11 and 12 are rejected under 35 U.S.C. 103 as being unpatentable over Iwasaki et al. (FEBS Letters 363, 1995, pp. 239-245 (Applicant’s IDS)) in view of Thatcher et al. (CA 2244115 (L), paragraph bridging pp. 26-27).
The teachings of Iwasaki have been discussed above.
Iwasaki does not teach lysing cells in a buffered alkaline solution at a pH of about 11.8.
Thatcher teaches cell lysis in a 0.075M NaOH and 1% SDS solution to raise the pH to approximately 11.9 and raising the pH to a value of between 12.1 and 12.9, depending upon the irreversible denaturation characteristics of the plasmid being isolated. Plasmid DNA is extrachromosomal circular DNA.
It would have been prima facie obvious to one skilled in the art at the time of filing to lyse the cells in a biological sample in a buffered alkaline solution such as 11.9 or slightly higher as taught by Thatcher to prevent the irreversible denaturation of the plasmid DNA, which is an extrachromosomal circular DNA. The prior art teaches different methods of lysing cells to obtain eccDNA. Iwasaki teaches one method. Thatcher teaches another method with the advantage that the buffered alkaline solution of Thatcher does not cause irreversible denaturation of plasmid DNA. The choice of methods to lyse cells is a matter of design choice well within the means of one skilled in the art with a reasonable expectation of success.
16. Claim 18 is rejected under 35 U.S.C. 103 as being unpatentable over Iwasaki et al. (FEBS Letters 363, 1995, pp. 239-245 (Applicant’s IDS)) in view of Eccleston et al. (US Pub. No. 20180087089 (A)).
The teachings of Iwasaki have been discussed above.
Iwasaki does not teach conducting steps (b) and (c) concurrently.
However, using multiple enzymes that act on different targets is well-known in the prior art. Eccleston teaches multiple restriction enzymes may be used concurrently to introduce targeted cuts into DNA sequences [0083].
It would have been prima facie obvious to one skilled in the art at the time of filing to conduct steps (b) and (c) concurrently because they are different enzymes that target different DNA sequences. The enzyme of step (b) linearizes mtDNA, and the enzyme of step (c) digests linear DNA. There is no evidence that the presence of one would adversely affects the activity of the other. Additionally, conducting steps (b) and (c) together is less time-consuming than conducting steps (b) and (c) separately, and one skilled in the art would expect some loss of eccDNA with each purification / enrichment step. Accordingly, one skilled in the art would have been motivated to do so with a reasonable expectation of success.
17. Claims 15, 19 and 20 are rejected under 35 U.S.C. 103 as being unpatentable over Iwasaki et al. (FEBS Letters 363, 1995, pp. 239-245 (Applicant’s IDS)) in view of Wisniewski et al. ((US Pub. No. 20120178918 (B)).
Iwasaki teaches separation of eccDNA from digested linear DNA by a two-step process: extraction with phenol then purification with ethanol (p. 239, “2.2 Purification of EC DNA from human cells”). However, Iwasaki does not teach prior to step (b), the unenriched eccDNA is bound to magnetic silica beads to separate eccDNA from lysed cells in the sample. Iwasaki also does not teach using phenol/chloroform/isoamyl alcohol or magnetic silica beads to separate eccDNA from digested linear mtDNA.
Wisniewski teaches magnetic silica beads bind to DNA ([0211], [0214], [0221], [0232]). Wisniewski further teaches that phenol/chloroform/isoamyl alcohol and magnetic silica beads are better suited for extracting larger strands of nucleic acids ([0004], [0063]).
It would have been prima facie obvious to one skilled in the art at the time of filing to utilize the magnetic silica beads of Wisniewski to separate the unenriched eccDNA from lysed cell material to remove non-DNA contaminants from the sample, as Wisniewski teaches that magnetic silica beads bind to DNA. It would also have been prima facie obvious to one skilled in the art at the time of filing to substitute the two-step method of separating the eccDNA from the digested linear mtDNA of Iwasaki for the one-step method of Wisniewski (phenol/chloroform/isoamyl alcohol or magnetic silica beads) for increased efficiency and efficacy (each additional purification / enrichment step causes loss of eccDNA). One skilled in the art would have been motivated to do so with a reasonable expectation of success.
18. Claim 14 is rejected under 35 U.S.C. 103 as being unpatentable over Iwasaki et al. (FEBS Letters 363, 1995, pp. 239-245 (Applicant’s IDS)) in view of van Agthoven et al. (US Pub. No. 20080026475 (C)).
The teachings of Iwasaki have been discussed above.
Iwasaki does not teach the buffered alkaline lysis is conducted in the presence of pyrrolidine, which inherently has an acid dissociation pH (pKa) between 11.0 and 12.3.
van Agthoven teaches lysis of erythrocytes (red blood cells) using pyrrolidine because unlike ammonium chloride lysis reagent, pyrrolidine does not have a toxic effect on blood cells [0008].
It would have been prima facie obvious to one skilled in the art at the time of filing to utilize the pyrrolidine of van Agthoven as a cell lysis reagent because it does not have a toxic effect on the cells. Iwasaki teaches using Proteinase K enzyme. Van Agthoven teaches pyrrolidine chemical lysis. The choice of methods to lyse cells is a matter of design choice well within the means of one skilled in the art with a reasonable expectation of success.
Conclusion
19. No claim is allowed.
20. Any inquiry concerning this communication or earlier communications from the examiner should be directed to PHUONG T BUI whose telephone number is (571)272-0793. The examiner can normally be reached on M-F 8am-5pm.
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/PHUONG T BUI/Primary Examiner, Art Unit 1663
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