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 .
Election/Restrictions
Applicant’s election without traverse of Group II in the reply filed on 9/4/2025 is acknowledged.
Claims 1-6, 8, 11-30 and 34 are withdrawn from further consideration pursuant to 37 CFR 1.142(b) as being drawn to nonelected inventions, there being no allowable generic or linking claim.
The election is made FINAL.
Claim Status
Claims 1-34 are pending.
Claims 1-6, 8, 11-30 and 34 are withdrawn from examination being drawn to nonelected inventions.
Claims 7, 9-10 and 31-33 are being examined.
Claim Objections
Claim 10 is objected to because of the following informalities:Claim 10 lacks a period “.” at the end of the claim/sentence.
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.
Claim 10 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 10 recites, “… wherein said VirD2 variant is encoded by SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 42 and SEQ ID NO: 43”. It is not clear if the Applicant intended to include all the said sequences in a VirD2 variant. It is apparent that any specific VirD2 variant in a specific XYA105Pt Agrobacterium strain can have only one of the sequences recited.
It is suggested to replace the “and” (underlined above) with “or”.
Claim Rejections - 35 USC § 103
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.
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.
The factual inquiries for establishing a background for determining obviousness under 35 U.S.C. 103 are summarized as follows:
1. Determining the scope and contents of the prior art.
2. Ascertaining the differences between the prior art and the claims at issue.
3. Resolving the level of ordinary skill in the pertinent art.
4. Considering objective evidence present in the application indicating obviousness or nonobviousness.
Claims 7, 9-10 and 31-33 are rejected under 35 U.S.C. 103 as being unpatentable over Sorokin et al. (US 2019/0203214 A1; published in 2019) in view of van Kregten et al. (Agrobacterium-Mediated T-DNA Transfer and Integration by Minimal VirD2 Consisting of the Relaxase Domain and a Type IV Secretion System Translocation Signal, 2009, Molecular plant-microbe interactions, 22:1356-1365).
Claim 7 is drawn to a variant of an isolated Agrobacterium strain XYA105Pt comprising a reconstituted virD operon encoding a VirD2 variant selected from SEQ ID NO: 45, SEQ ID NO: 46 and SEQ ID NO: 47.
The isolated Agrobacterium strain (XYA105Pt) is derived from a known agrobacterium strain EHA101 (Spec, page 17, line 17-18), which is widely used in plant transformation since long. The endogenous VirD2 gene comprising nuclear localizing signals (NLSs) in EHA101 strain is deleted, resulting in the Agrobacterium strain XYA105ΔvirD2 (Spec, page 17, line 22-24). Different PtVirD2 variants, as part of a separate binary vector, are created by fusing specific plastid transit peptides, including tobacco Rubisco small subunit (SSU) transit peptide, with a VirD2 polypeptide devoid of any NLS and a T4SS signal (polypeptide sequence involved in Type IV secretion system). A specific PtVirD2 sequence is then introduced into the XYA105ΔvirD2 strain to obtain a specific XYA105Pt strain, wherein PtVirD2 can be any polynucleotide or gene listed in Table 1 (page 7, line 18-26; Fig. 13A-13D).
SEQ ID NO:45 comprises a specific Pt-virD2 variant sequence has the tobacco SSU Transit peptide and the VirD2-T4SS signal, and the whole sequence is flanked by 500 bp of virD1 and 500 bp of virD3 (page 26, line 36-38; and description in sequence listing). The Pt-VirD2 polypeptide, encoded by SEQ ID NO: 27 (as recited in claim 10), comprises the VirD2 protein fused with tobacco Rubisco SSU completed with the 5 amino acid residues (as part of matured Rubisco SSU) at its N-terminal end and the VirD2 T4SS signal at the C-terminal end (page 25, line 26-28).
The VirD2 protein encoded by the native agrobacterium strain EHA101 is well-known (GenBank Accession No. KY000035.1; published in 2017). The relevant N-terminal 204 amino acid long Relaxase domain, the NLS sequences (van Kregten et al., page 1358, Fig. 2A), including the N-terminal nuclear localization signal (RKGR)1, the signal (i.e., sequence) for Type IV secretion system (T4SS) (van Kregten et al., page 1, right column, para 2, line 6-23; page 5, left column, para 2, line 2-5) in the VirD2 protein are well characterized and known before the instant invention. Commonly used chloroplast transit peptide from Tobacco Rubisco small subunit (rbc-SSU TP) is also well characterized (Uniprot Accession No: P69249) and is having 100% sequence identity to the tobacco Rubisco small subunit transit peptide in SEQ ID NO: 27 (as recited in claim 10), which is turn has 100% sequence identity to instant SEQ ID NO: 45 (sequence alignment is not shown here).
Sorokin et al. describes a method of transforming a plant cell with a DNA of interest via agrobacterium mediated transformation by introducing into a binary vector a virD2 gene fused to a plant organelle transit peptide outside of the T-DNA region (claim 58; page 6, para 0050, line 5-8), wherein the organellar transit peptide is a plastid transit peptide (claim 47). The proteins encoded by the genes in the Vir operon including VirD2 act in trans and does not need to be integrated into the chloroplast genome. Sorokin et al. describes an Agrobacterium strain comprising a knockout mutant of native virD2, an Agrobacterium binary vector comprising a modified ViD2 DNA sequence, a DNA sequence encoding an organellar transit peptide fused to the 5' end of a VirD2 DNA sequence (page 1, para 0005, line 3-11). The small subunit transit peptide from tobacco rubisco is widely used in the art for targeting heterologous proteins to chloroplast, as described by Sorokin et al. (page 1, para 0007, line 8-10).
Sorokin et al. also describes infiltration of tobacco leaves with specific agrobacterium strain (page 41, para 0134, line 14-17) transforming leaf explants from three different plant species including tobacco and maize/corn, as recited in claim 31 (page 42, para 0146, line 1-2). It also describes using a selection media comprising spectinomycin as a selection agent to isolate transplastomatic cells leading to recovering or regenerating plants (as recited in claim 32) containing the transgene in the chloroplasts in all three plant species (page 42, para 0148-0149), as recited in claim 9.
The Agrobacterium mediated chloroplast transformation method described by Sorokin et al. overcomes the challenges associated with chloroplast transformation via particle bombardment followed by many rounds of selection to achieve homoplastic state of transformation with the goal to produce pharmaceutical and other heterologous proteins in plants (page 1, para 0002, line 8-9). It is prudent to mention here that Sorokin et al. does not describe using and/or modifying the endogenous T4SS signal sequence in the VirD2 protein, yet successfully transforms and introduces transgenes into the chloroplast of several plant species (page 8, para 0076; page 8, para 0080; page 8, para 0085, line 1-2) by agrobacterium mediated transformation.
However, Sorokin et al. does not describe any T4SS signal sequence or a sequence identical to instant SEQ ID NO: 45.
As explained above, SEQ ID NO: 45 comprises the polynucleotide sequence encoding VirD2 polypeptide fused with the tobacco (Rubisco Small Subunit) SSU plastid transit peptide (Pt) at the N-terminal and wild type VirD2 T4SS signal peptide (T4SS) at the C-terminal, and the whole fused sequence (Pt-VirD2-T4SS polypeptide) is flanked with 500 bp VirD1 and 500 bp of VirD3 sequence (Spec, page 26, line 36-38).
van Kregten et al. describes that the VirD2 protein of Agrobacterium tumefaciens, comprising a relaxase domain at the N terminus and a bipartite nuclear localization signal (NLS), being essential for processing and transport of the T-DNA (abstract). It also describes the type IV secretion system (T4SS) and the T4SS sequence in VirD2 in relation to nuclear targeting of the VirD2-T-strand complex (T-complex). It teaches that after its formation, the T-complex is transported to cytoplasm and the transport/translocation events are mediated by the type IV secretion system (T4SS) of the Agrobacterium (page 1, right column, para 2, line 1-7). VirD1 and VirD2 are both required for recognition of the border repeat sequences flanking the T-strand but it is the relaxase domain of the VirD2 protein which actually cleaves the LB and RB sequence on one of the DNA strands, enabling the release of the T-strand (page 1, left column, para 1, last 4 lines, and page 1, right column, para 1, line 1). The recognition and transfer of T-DNA by the T4SS rather than the relaxase (in VirD2) is the predominant reason for transport of relaxase domain (into host cytoplasm) (page 1, right column, para 2, line 20-23).
Before the effective filing date of the invention, it would have been obvious to an ordinarily skilled artisan to develop a more efficient method to transform plastids/chloroplasts using agrobacterium mediated transformation, as described by Sorokin et al., while using relevant information regarding various VirD2 sequences including the T4SS, as discussed by van Kregten et al., as explained below. The tobacco SSU fused with the VriD2 at its N-terminal end and T4SS is fused at the C-terminal end would give rise to SEQ ID NO: 45 or a functional equivalent of SEQ ID NO: 45, depending on the different variants of VirD2, SSU, and T4SS sequences.
It is long known in the art that VirA detects plant signaling molecules and phosphorylates VirG, which activates transcription of other vir genes on the Ti plasmid. VirD2, an endonuclease, in concert with VirD1, nicks the left and right border sequences of the T-DNA, and VirD2 covalently attaches to the 5′ end of single-stranded T-DNA (T-strand). The VirD2/T-strand complex (T-complex) along with other effector proteins including VirE2, VirE3, VirF and VirD5 are translocated to the plant cell through a Type IV secretion system (T4SS) composed of VirB 1-11 and VirD4 (Spec, page 2, line 6-12). In the plant cytoplasm, VirE2 covers the T-strand of the VirD2/T-strand complex, forming the T-complex to protect it from degradation. The T-complex is directed to the nucleus by VirD2, which has nuclear localization signals (Spec, page 2, line 13-15). Sorokin et al. makes it clear that once the T-complex enters the host cytoplasm, its organelle transport (to nucleus, mitochondria, or chloroplast) depends exclusively on the transit/signal peptide in the VirD2 protein (Fig. 19). Considering the information above, it would have been obvious to an ordinarily skilled artisan to knockout all the NLSs in VirD2 and introduce a chloroplast targeting signal (transit peptide) to enable any heterologous protein (as described by Sorokin et al.) and/or polynucleotide that covalently attaches itself with the VirD2 protein (as occur in nature during agrobacterium infection) to enter the chloroplast using the chloroplast transit peptide. Use of a suitable T4SS sequence including the one present in native VirD2 or VirF, as described by van Kregten et al. (page 5, left column, para 2, line 2-9), allows translocation of the VirD2 complex into the cell (more precisely into the cytoplasm). Once the VirD2-TDNA complex (T-complex) is inside the cytoplasm, the chloroplast transit peptide then enables the T-complex to enter into the chloroplast, as shown by Sorokin et al.
It is an experimental design choice of an ordinarily skilled artisan to use any functional equivalent agrobacterium strain including EHA101 or GV3101 (as described by Sorokin et al.; page 41, para 0134, line 1-2) and different sequences encoding VirD2, chloroplast transit peptide (including rbc-SSU TP), and T4SS that enables successful targeting of the T-complex into a chloroplast in a plant cell. Use of floral dip method to transform various plants including Arabidopsis and Brassica sp. (as recited in claim 31) and harvesting transformed seeds (as recited in claim 33) are standard practice in the art2. It would have been obvious to an ordinarily skilled artisan to try floral dip method by exposing the flowers in Arabidopsis and/or Brassica plants to the agrobacterium strain XYA105Pt and/or its variants including XYA105Pt45 with a predictable and realistic expectation of success to obtain transplastomic plants and with the goal to produce pharmaceutical and other heterologous proteins in the plants, as described by Sorokin et al.
Before the effective filing date, an ordinarily skilled artisan would have been motivated to modify an agrobacterium strain (e.g., XYA105Pt45), as discussed above, to transform chloroplasts using the modified agrobacterium strain with a realistic goal to simplify the process of chloroplast transformation with the goal to produce pharmaceutical and other heterologous proteins in the plants with transformed chloroplast.
Conclusion
No claim is allowed.
Contact Information
Any inquiry concerning this communication or earlier communications from the examiner should be directed to JAY CHATTERJEE whose telephone number is (703)756-1329. The examiner can normally be reached (Mon - Fri) 8.30 am to 5.30 pm..
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Jay Chatterjee
Patent Examiner
Art Unit 1662
/Jay Chatterjee/ Examiner, Art Unit 1662
/BRATISLAV STANKOVIC/ Supervisory Patent Examiner, Art Units 1661 & 1662
1 Chen et al. (Targeting Specificity Modification of Agrobacterium tumefaciens VirD2 Protein, 2012, Journal of Agricultural Biotechnology, 20:331-336) describes the NLS sequences including the N-terminal RKGR sequence (page 7, Fig. 2).
2 Hu et al. (Agrobacterium-mediated vacuum infiltration and floral dip transformation of rapid-cycling Brassica rapa, 2019, BMC Plant Biology, 19:246) provides the evidence that floral dip method to transform various plants including Arabidopsis and Brassica sp. is a well-known standard process (abstract).