DETAILED ACTION
The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
Claims 1-13 are currently pending in the application and under examination. Claims 1, 6, 7, 8, 9 and 13 are independent claims.
An action on the merits follows. The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
Priority
The present application is a 35 U.S.C. 371 national stage filing of International Application No. PCT/CN2021/143731, filed 31 December 2021.
Thus, the earliest possible priority for the instant application is 31 December 2021.
Information Disclosure Statement
The information disclosure statements filed 11 January 2024 have been considered by the Examiner.
The information disclosure statement filed 27 May 2025 fails to comply with the provisions of 37 CFR 1.98(a)(4) because it lacks the appropriate size fee assertion. It has been placed in the application file, but the information referred to therein has not been considered as to the merits.
Claim Objections
Claims 1-5, 7-10, and 12-13 are objected to because of the following informalities:
Claim 1 recites abbreviations “gag1”, “gag2”, “pol1”, “pol2”, “LTR1”, “LTR2”, “HIV-1”, and “MS2” without first writing out the terms for which they are abbreviations. Claims 2-5, 7-10, and 12-13 likewise recite some or all of the abbreviations recited in claim 1 without first reciting the terms for which they are abbreviations. Appropriate correction is required.
Additionally, claims 1, 7-8, and 13 each also recite “regions on genes from HIV-1”, which appears to be a typographical error for “regions of genes from HIV-1”. 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 1-13 are rejected under 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph, as being indefinite for failing to particularly point out and distinctly claim the subject matter which the inventor or a joint inventor (or for applications subject to pre-AIA 35 U.S.C. 112, the applicant), regards as the invention.
Claim 1 recites, “conserved sequence fragments of gag1, gag2, pol1, pol2, LTR1, LTR2 regions”, which is indefinite because the list does not have an indication of whether the individual regions are linked by “and”, “or”, or “and/or”, and so it is unclear whether all regions are required to be comprised in the vector.
Moreover, the phrase “sequence of gene from phage MS2” in claim 1 and 4 is grammatically incorrect and appears to be missing a definite or indefinite article preceding the term “gene”. Appropriate correction is requested.
Claim 8 also recites, “conserved sequence fragments of gag1, gag2, pol1, pol2, LTR1, LTR2 regions”, which is also indefinite because the list does not have an indication of whether the individual regions are linked by “and”, “or”, or “and/or”, and so it is unclear whether all regions are required to be comprised in the vector.
As such, the metes and bounds of the claims cannot be determined.
Claims 2 and 12 each recite that the MS2 and HIV-1 sequences “respectively are located between any restriction sites behind the 3’ ends of two T7 promoters”, which has multiple issues of indefiniteness. Firstly, “behind” is a relative term which is not an art recognized term for a relationship between sequences in a vector. As such, it is unclear what is meant to be encompassed by a location “behind” the 3’ ends of the promoters. Secondly, “the 3’ ends of two T7 promoters” is indefinite because it is unclear whether the same sequence is meant to be repeated twice in the vector to be “behind” each of two T7 promoters, whether two T7 promoters are adjacent to each other with the inserted sequence “behind” the 3’ end of the combined tandem T7 promoters, or whether some of the recited sequences are “behind” one of the T7 promoters and other of the recited sequences are “behind” the other T7 promoter. Thirdly, use of the term “respectively” is indefinite in that the claim does not recite two or more values which would be related “respectively” to the recited sequences. As such, the metes and bounds of the claims cannot be determined. Additionally, claim 2 recites “any restriction sites” in line 4 which lacks proper antecedent bases. As such, the metes and bounds of the claims cannot be determined.
Claim 3 recites “wherein the gag1 is the sequence set forth in SEQ ID NO: 1, the gag2 is the sequence set forth in SEQ ID NO: 2”, which is indefinite because claim 1 recites “gag1, gag2… regions on genes from HIV-1”, which would be understood by an ordinarily skilled artisan to indicate the HIV-1 gag gene sequence. However, as taught by GenBank, the sequence of SEQ ID NO: 1 only partially comprises gag gene sequences and is fully encompassed by the pol gene in that SEQ ID NO: 1 starts within the overlapping sequence of the gag and pol open reading frames and extends into the pol-only sequence [GenBank 1996, Human immunodeficiency virus type 1, strain CM240, complete proviral genome, Accession: U54771.1, retrieved on 15 January 2026 from: <Human immunodeficiency virus type 1, strain CM240, complete proviral g - Nucleotide - NCBI>, published 13 September 1996]. Additionally, GenBank further teaches that SEQ ID NO: 2 is fully comprised within the pol coding sequence outside of the overlapping gag-pol sequence. As such, it is unclear how the gag1 and gag2 regions are the sequences of SEQ ID NO: 1 and SEQ ID NO: 2. As such, the metes and bounds of the claim cannot be determined.
Claim 4 recites, “wherein the sequence of gene from phage MS2 is SEQ ID NO: 7”, which is indefinite because although a sequence of gene can be a sequence according to SEQ ID NO: 7, it is unclear how a sequence of gene can be a sequence identifier itself. As such, the metes and bounds of the claim cannot be determined.
Claims 5 and 12 each recite that the sequences “are sequentially connected in direct”, which is indefinite because it is unclear what sequence of sequences is being claimed and what is meant by the phrase “connected in direct”. The adjective “direct” is missing a noun to modify. The specification defines this phrase as follows:
“As used herein, the phrase "sequentially connected in direct" refers to the direct synthesis of fragments in the foregoing order of the fragments using techniques known in the art.” [page 6 ¶ 2].
However, this teaching of the specification does not provide a sufficiently limiting or sufficiently definite definition for the phrase. For example, it is still unclear whether the MS2 gene sequence and the HIV-1 sequences are meant to be directly adjacent to each other in the vector, or whether they can be separated by other elements, such as restriction sites, promoters, terminators, or other regulatory sequences. Additionally, the plain meaning of “sequentially connected in direct” would not be understood to require the “foregoing order” of the fragments. Also, it is unclear whether the MS2 gene sequence is meant to be encompassed as a “fragment” according to the definition provided in the specification.
As such, the metes and bounds of the claim cannot be determined.
Claim 6 recites, “An expression vector, comprising the sequences of SEQ ID NOs: 1-7 of the recombinant expression vector according to claim 3, which is indefinite because it is unclear in what way the sequences of SEQ ID NOs: 1-7 are “of the recombinant expression vector according to claim 3”, particularly given that claim 3 only recites wherein the gag1, gag2, pol1, pol2, LTR1, and LTR2 regions are the sequences of SEQ ID NOs: 1-6 and does not require inclusion of the full sequences of SEQ ID NOs: 1-6. Additionally, neither claim 3 nor claim 1, upon which claim 3 depends, recite the sequence of SEQ ID NO: 7. As such, the metes and bounds of the claim cannot be determined.
Claim 7 recites, “by expressing the recombinant expression vector according to claim 1”, which is indefinite because although gene sequences can be expressed, a vector is not expressed. Additionally, it is unclear in what way the vector is to be expressed in that there is no recitation of any cell or expression system for expressing the genes/sequences comprised in the vector. As such, the metes and bounds of the claim cannot be determined.
Claim 8 recites, “a mixture, comprising a recombinant expression vector the pseudovirus particles”, which is indefinite because it is unclear whether the mixture is meant to comprise a recombinant expression vector [“and”, “or”, or “and/or”] the pseudovirus particles. As such, the metes and bounds of the claim cannot be determined.
Independent claim 9 has multiple issues of indefiniteness.
Firstly, claim 9 recites, “directly synthesizing the target fragments and the sequence of gene from phage MS2 respectively” in lines 4-5, which is indefinite because it is unclear what is meant to be applied “respectively” to the recited components. For example, it is unclear whether “respectively” is meant to indicate that the fragments are directly synthesized separately from each other and/or separately from the MS2 gene sequence, or whether “respectively” is meant to apply a list of attributes to be applied to the recited components. If the latter, it is unclear which attributes are meant to be applied “respectively”.
Secondly, it is unclear how the method of claim 9 is meant to prepare the pseudovirus particles of claim 7 in that the method does not recite that the recombinant vector comprises any HIV-1 fragments. Additionally, the method does not comprise any steps which would produce a pseudovirus particle in that the method steps only produce a recombinant vector without any transfection or transduction steps which would allow expression and assembly of the pseudovirus components into a pseudovirus particle.
Thirdly, claim 9 contains the trademark/trade name “pETDuet”. Where a trademark or trade name is used in a claim as a limitation to identify or describe a particular material or product, the claim does not comply with the requirements of 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph. See Ex parte Simpson, 218 USPQ 1020 (Bd. App. 1982). The claim scope is uncertain since the trademark or trade name cannot be used properly to identify any particular material or product. A trademark or trade name is used to identify a source of goods, and not the goods themselves. Thus, a trademark or trade name does not identify or describe the goods associated with the trademark or trade name. In the present case, the trademark/trade name is used to identify/describe a starting vector into which particular sequences are inserted and, accordingly, the identification/description is indefinite.
Claims 10-12 additionally recite “pETDuet” and are likewise indefinite for reciting a trademark or trade name as addressed for claim 9.
Claim 10 additionally recites, “endonucleases BamH I/Hind III”, which is indefinite because it is unclear whether “BamH I/Hind III” is meant to indicate a combination of individual BamHI and HindIII enzymes, whether “BamH I/Hind III” is meant to indicate alternative enzymes, or whether BamH I/Hind III” is meant to claim a BamHI-HindIII fusion or some other combined endonuclease entity.
Claim 11 recites, “endonucleases EcoR V/Kpn I” ”, which is indefinite because it is unclear whether “EcoR V/Kpn I” is meant to indicate a combination of EcoRV and KpnI, whether “EcoR V/Kpn I” is meant to indicate alternative enzymes, or whether it is meant to claim a EcoRV-KpnI fusion or some other combined endonuclease entity.
Claims 10 and 11 also contain the trademark/trade name “EasyGeno Assembly Cloning kit”. Where a trademark or trade name is used in a claim as a limitation to identify or describe a particular material or product, the claim does not comply with the requirements of 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph. See Ex parte Simpson, 218 USPQ 1020 (Bd. App. 1982). The claim scope is uncertain since the trademark or trade name cannot be used properly to identify any particular material or product. A trademark or trade name is used to identify a source of goods, and not the goods themselves. Thus, a trademark or trade name does not identify or describe the goods associated with the trademark or trade name. In the present case, the trademark/trade name is used to identify/describe a kit for restriction endonuclease cloning and, accordingly, the identification/description is indefinite.
As such, the metes and bounds of the claims cannot be determined.
Claim 13 has been included in this rejection due to its dependence on and/or encompassing of claim 1, 3, and 6.
Claim Interpretation
The terms “gag1”, “gag2”, “pol1”, “pol2”, “LTR1”, and “LTR2”, as recited in claims 1-3, 5, 7, 8, and 13 have been interpreted such that the designations of “1” or “2” indicate a first sequence region and a second sequence region, respectively, for each of the indicated gag, pol, and LTR gene region sequences.
Note that claim 1 recites a vector “comprising conserved sequence fragments of gag1, gag2, pol1, pol2, LTR1, LTR2 regions on genes from HIV-1”. Claim 3 recites the vector of claim 1, “wherein the gag1 is the sequence set forth in SEQ ID NO: 1, the gag2 is the sequence set forth in SEQ ID NO: 2, the pol1 is the sequence set forth in SEQ ID NO: 3, the pol2 is the sequence set forth in SEQ ID NO: 4, the LTR1 is the sequence set forth in SEQ ID NO: 5, and the LTR2 is the sequence set forth in SEQ ID NO: 6.” Therefore, claim 3 is limiting the gag1, gag2, pol1, pol2, LTR1, and LTR2 sequence regions themselves, not the specific fragments of gag1, gag2, pol1, pol2, LTR1, and LTR2. As such, any sequence which comprises any fragment having 2 or more nucleotides of the sequences set forth in the recited SEQ ID NOs: 1-6 satisfies the limitations of claim 3.
Claim 1 also recites, “a sequence of gene from phage MS2”. Claim 4 further limits the sequence of gene from phage MS2 such that “the sequence of gene from phage MS2 is SEQ ID NO: 7.” Therefore, claim 1 has been interpreted to comprise any sequence of any gene from the phage MS2. Claim 4 has been interpreted such that the sequence of gene from phage MS2 must be a sequence having 100% identity to the full-length sequence according to SEQ ID NO: 7.
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.
Claim(s) 1-3, 5, and 7-12 are rejected under 35 U.S.C. 103 as being unpatentable over Barouch [US20120076812A1, published 29 March 2012]; in view of Sun et al. [2011, Biochemical and Biophysical Research Communications, 407, 124-128]; Agilent [2015, pESC Yeast Epitope Tagging Vectors: Instruction Manual, Agilent Technologies, Revision C.0, © 2015, 1-21]; Biocompare [2010, Biocompare: The Buyer’s Guide for Life Scientists, retrieved on 14 January 2026 from: <https://www.biocompare.com/Product-Reviews/40993-Co-expression-with-pETDuet-1-Duet-Expression-System-From-Novagen/>, published 18 January 2010]; GenBank [1996, Human immunodeficiency virus type 1, strain CM240, complete proviral genome, Accession: U54771.1, retrieved on 15 January 2026 from: <Human immunodeficiency virus type 1, strain CM240, complete proviral g - Nucleotide - NCBI>, published 13 September 1996]; Roebuck & Saifuddin [1999, Gene Expression, 8(2), 67-84]; Addgene [2020, Vector Database: pETDuet-1, retrieved on 14 January 2026 from: <https://web.archive.org/web/20200918130300/https://www.addgene.org/vector-database/2659/>, archived 18 September 2020]; and Feng et al. [2018, Plant Biotechnology Journal, 16, 1848-1857].
Regarding claim 1, Barouch teaches a recombinant expression vector comprising conserved sequence fragments encoding HIV-1 Gag and Pol, along with HIV-1 LTR sequences [0003, 0007, 0042, 0057-0058, 0064]. Barouch further teaches that the mosaic Gag and Pol genes comprised in the vector are mosaic blends of fragments of an arbitrary set of naturally-occurring viral gene product sequences provided as inputs [0056], and as such represent at least a fragment 1 and a fragment 2 for each of the Gag and Pol genes (e.g., a gag1, gag2, pol1, and pol2 fragment) [Table 5, SEQ ID NOs: 3, 4, 5, 6]. Additionally, the limitation “conserved sequence fragments” does not limit the size of the fragment for each sequence fragment. As such, a teaching to include the full length HIV-1 LTR sequence necessarily is teaching inclusion of at least a first and second fragment of the sequence which would combine to produce the full sequence, thereby teaching to include conserved sequence fragments of LTR1 and LTR2.
Barouch does not teach wherein the recombinant expression vector comprises a sequence of a gene from phage MS2.
Sun teaches a vaccine platform based on MS2 virus-like particles (VLPs)/pseudovirus particles, wherein MS2 VLPs packaging HIV-1 gag mRNAs were produced, used to immunize BALB/c mice, and shown to elicit strong antigen-specific antibody responses [abstract, column 7 ¶ 1]. Sun also teaches a recombinant expression vector comprising sequences encoding the MS2 capsid protein and the HIV-1 Gag protein for production of the VLPs [column 2 ¶ 2, column 3 ¶ 2]. Sun further teaches that MS2 VLPs have remarkably high stability in that the HIV-1 gag mRNA was protected by the capsid protein of MS2 from degradation by DNase I and RNase A [abstract, column 6 ¶ 3]. Sun teaches that the MS2 VLPs can be used in the design and construction of novel and safe phage-based mRNA vaccines [abstract, column 7 ¶ 1]. Therefore, an ordinarily skilled artisan at the time of filing the instant application would have been motivated to use an MS2 VLP particle to deliver antigenic HIV-1 sequences, wherein the HIV-1 gene sequences and the MS2 capsid gene sequence are comprised in the same expression vector for generation of the VLPs/pseudoviruses, particularly for the generation of safe phage-based HIV-1 mRNA vaccines.
Regarding claim 2, the combination of Barouch and Sun teach the limitations of claim 1. Barouch further teaches that the vector comprises a promoter [0019, 0064, 0069, 0072, 0074]. Barouch also teaches that the vector has multiple cloning sites with restriction enzyme cleavage loci for cloning the viral polypeptides into the vector [0064, 0066]. Sun also teaches wherein the MS2 capsid cDNA sequence and HIV-1 Gag cDNA sequence are each inserted into a pESC-Ura vector from Stratagene to generate first a pMS vector and then pMS-GAG vector [column 2 ¶ 2]. Agilent teaches that the pESC-Ura vector has GAL10 and GAL1 promoters in opposing orientation with each having a multiple cloning site (MCS) downstream of the promoter to facilitate target gene sequence insertion for expression of one or two cloned genes in yeast [page 3 ¶ 1, page 5 plasmid map and sequence].
Barouch, Sun, and Agilent do not teach that the conserved sequence fragments from HIV-1 and the sequence of gene from phage MS2 are located between any restriction sites behind the 3’ ends of two T7 promoters in the recombinant expression vector.
However, Biocompare teaches a pETDuetTM-1 DUETTM Expression System for co-expression of target genes in E. coli, wherein the system satisfies all the requirements for multiple target gene expression [page 1 ¶ 1]. Biocompare further teaches that the pETDuetTM-1 vector has two expression units each controlled by a T7 promoter for high-level protein expression of two target genes [page 1 ¶ 2]. Biocompare also teaches that the vector comprises two multiple cloning sites (MCSs), wherein each MCS follows a T7 promoter [page 1 ¶ 3]. Biocompare teaches that use of the pETDuetTM-1 vector allowed for overcoming issues of low solubility of a target protein by co-expressing a binding partner and that it is very easy to generate protein complexes by using this system, such that the system is a very useful and efficient tool for purification and structure studies [page 1 ¶ 5-6]. Therefore, an ordinarily skilled artisan at the time of filing the instant application would have been motivated to use the pETDuetTM-1 vector for the cloning and expression of multiple coding sequences, wherein the sequences are inserted into the pETDuetTM-1 vector within the MCSs which are located downstream of each of the two T7 promoters of the vector.
Regarding claim 3, the combination of Barouch and Sun teach the limitations of claim 1. Barouch also teaches the amino acid sequences of Gag and Pol included in the expression vector [e.g., SEQ ID NOs: 1-39], and to include a mosaic sequence to generate positive immune responses [Figure 1, 15], and that mosaics have potential advantages over the monovalent vaccines because mosaics have a better chance of stimulating a response that reacts with more common variants and also stimulate multiple responses to the different forms that are present in the cocktail, thereby giving them the potential to block common escape routes [0027].
Barouch further teaches amino acid sequences encoded by the gag and pol sequences included in the recombinant expression vector. Barouch teaches the mosaic pol sequence of SEQ ID NO: 6, which has 96.3% identity to the sequence encoded by the sequence of instant SEQ ID NO: 1:
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Additionally, the mosaic pol sequence of Barouch SEQ ID NO: 5 has 97.9% identity to the sequence encoded by the sequence of instant SEQ ID NO: 2:
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The mosaic pol sequence of Barouch SEQ ID NO: 5 also has 95.0% identity to the sequence encoded by the sequence of instant SEQ ID NO: 3:
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The mosaic pol sequence of Barouch SEQ ID NO: 5 also has 97.9% identity to the sequence encoded by the sequence of instant SEQ ID NO: 4:
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Therefore, Barouch teaches to include nucleotide sequences which encode sequences with 100% sequence identity to sequences encoded by fragments of instant SEQ ID NOs: 1-4.
Barouch and Sun do not teach any specific nucleotide sequences for the conserved sequence fragments of gag1, gag2, pol1, pol2, LTR1, or LTR2 regions on genes from HIV-1.
Note that, as described above in the Claim Interpretation section, claim 3 is limiting the gag1, gag2, pol1, pol2, LTR1, and LTR2 sequences themselves, not the fragments of gag1, gag2, pol1, pol2, LTR1, and LTR2. As such, any sequence which comprises a fragment of each of the sequences set forth in SEQ ID NOs: 1-6 satisfies the limitations of claim 3.
GenBank teaches a complete proviral genome for HIV-1 which comprises sequences which are 100% identical to the full length sequences according to instant SEQ ID Nos: 1 and 2:
SEQ ID NO: 1:
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SEQ ID NO: 2:
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GenBank also teaches sequences which align with instant SEQ ID NOs: 3 and 4, having 96.0% and 99.4% sequence identity, respectively:
SEQ ID NO: 3:
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SEQ ID NO: 4:
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Although GenBank does not teach the full length sequences of instant SEQ ID NOs: 3 and 4 at 100% identity, note that GenBank teaches a sequence comprising sequences which are 100% identical to fragments of instant SEQ ID NOs: 3 and 4.
GenBank also teaches sequences which align with instant SEQ ID NOs: 5 and 6, having 55.2% and 54.7% sequence identity, respectively:
SEQ ID NO: 5:
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SEQ ID NO: 6:
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Although GenBank does not teach the full length sequences of instant SEQ ID NOs: 5 and 6 at 100% identity, note that GenBank teaches a sequence comprising sequences which are 100% identical to fragments of instant SEQ ID NOs: 5 and 6.
GenBank also teaches that nucleotides 355-1866 correspond to the gag coding sequence, nucleotides 1644-4670 correspond to the pol coding sequence, and that nucleotides 9014-9084 comprise conserved regulatory sequences (e.g., NFkappaB binding site, SP1 binding sites, and a TATA box) [page 2 lines 5-8, 21-24, page 3 lines 53-66]. Therefore, GenBank teaches that the sequences of instant SEQ ID NOs: 1-6 correspond to sequences of the gag and pol genes as well as the untranslated LTR terminal region.
GenBank further teaches that the sequence of HIVU54771.1 is the full-length sequence for the complete proviral genome of a human HIV-1 isolate from Thailand which is an example of a mosaic of a clade A virus and a clade E virus [page 1 lines 23-24, 40-50].
Given the teachings of Barouch to construct a recombinant expression vector comprising sequence fragments of HIV-1 gag, pol, and LTR; the further teaches of Barouch that mosaics have a better chance of stimulating a response that reacts with more common variants, stimulate multiple responses to the different forms that are present in the cocktail, and have the potential to block common escape routes; the teachings of Barouch of mosaic pol sequences which comprise sequences encoded by fragments of instant SEQ ID NOs: 1-4; and the teachings of GenBank of a complete proviral mosaic genome sequence comprising gag, pol, and LTR sequences; an ordinarily skilled artisan would have been motivated to select sequence from the mosaic proviral genome taught by GenBank as corresponding to HIV-1 gag, pol, and LTR, which comprise sequences having 100% identity to fragments of the sequences according to instant SEQ ID NOs: 1-6.
Regarding claim 5, the combination of Barouch and Sun teach the limitations of claim 1. Barouch further teaches rAD26 vectors expressing Gag, Pol, and Env [0100], as well as vectors expressing fusion proteins of GagPol GagPolNef [0099, SEQ ID NOs: 19-29]. As such, Barouch teaches that at least the gag1, gag2, pol1, and pol2 sequences are sequentially positioned and directly connected within the expression vector. Additionally, given the teaching of Barouch to also include the HIV-1 LTR in the expression vector, it would have been obvious to an ordinarily skilled artisan to include the LTR sequence sequentially with the GagPol gene in that Barouch does not teach to interrupt the Gag-Pol sequences with the LTR sequences, and so including the LTR before and/or after the Gag-Pol sequence would represent selecting from a very limited number of options.
Additionally, Roebuck teaches that HIV comprises two LTR sequences, a 5’LTR and a 3’LTR, which each comprise unique as well as repeat regions [Figure 1]. Roebuck further teaches that the LTR comprises the HIV-1 promoter which is essential for virus transcription, including trans-activation by Tat, and which comprises binding sites for numerous transcription factors [column 3 ¶ 2- column 4 ¶ 1, Figure 2, 3]. Therefore, an ordinarily skilled artisan would have been motivated to include the two LTR sequences (e.g., the 5’ LTR and the 3’LTR) in an expression vector for expressing HIV-1 proteins to facilitate expression of the HIV-1 proteins, wherein the two LTR sequences flank the additional HIV-1 sequences (e.g., gag and pol).
Regarding claims 7 and 8, the combination of Barouch and Sun teach the limitations of claim 1. Additionally, Sun teaches to make VLPs/pseudovirus particles by expressing the genes included in the recombinant expression vector as described above [column 3 ¶ 2]. Additionally, in making the pseudovirus particles, Sun teaches a mixture comprising the recombinant expression vector described above and the pseudovirus particles generated by expressing the genes included in the recombinant expression vector in that the production of the MS2 VLPs/pseudovirus particles comprises transferring the recombinant expression vector into a cell for producing the particles, wherein once the cell produces particles, the cell necessarily comprises both the expression vector and the particles.
Regarding claim 9, the combination of Barouch and Sun teach the limitations of claims 1 and 7. Additionally, Barouch teaches a method for preparing virus particles, the method comprising designing and selecting target fragments [0003, 0029, 0056]; directly synthesizing the target fragments [0059]; and inserting the target fragments into a vector to obtain a vector comprising the target fragments (e.g., the nucleotide sequence of two or more HIV-1 viral polypeptides) [0059-0066].
Further, Sun teaches a method for preparing VLPs/pseudovirus particles, the method comprising designing and selecting target fragments and the sequence of capsid gene from phage MS2 [column 1 ¶ 2- column 2 ¶ 1]; synthesizing the target fragments and the sequence of gene from phage MS2 (e.g., amplifying the cDNA encoding the MS2 capsid protein and the cDNA encoding HIV-1 Gag) [column 2 ¶ 2]; inserting the sequence of gene from phage MS2 into a pESC-Ura vector to obtain a pMS vector [column 2 ¶ 2]; and inserting the target fragments into the pMS vector to obtain a pMS-GAG recombinant expression vector [column 2 ¶ 2].
Barouch and Sun do not teach wherein the starting vector is a pETDUet-1 vector.
However, as described above, Biocompare teaches a pETDuetTM-1 DUETTM Expression System for co-expression of target genes in E. coli, wherein the system satisfies all the requirements for multiple target gene expression [page 1 ¶ 1]. Biocompare further teaches that the pETDuetTM-1 vector has two expression units each controlled by a T7 promoter for high-level protein expression of two target genes [page 1 ¶ 2]. Biocompare also teaches that the vector comprises two multiple cloning sites (MCSs), wherein each MCS follows a T7 promoter [page 1 ¶ 3]. Biocompare teaches that use of the pETDuetTM-1vector allowed for overcoming issues of low solubility of a target protein by co-expressing a binding partner and that it is very easy to generate protein complexes by using this system, such that the system is a very useful and efficient tool for purification [page 1 ¶ 5-6]. Therefore, an ordinarily skilled artisan at the time of filing the instant application would have been motivated to use the pETDuet-1 vector for the cloning and expression of multiple coding sequences, such as the MS2 capsid protein coding sequence and the HIV-1 gag/pol/LTR sequences as described above.
Regarding claims 10-11, the combination of Barouch, Sun, and Biocompare teach the limitations of claim 9. Biocompare teaches a plasmid map for the pETDuetTM-1vector, but the image resolution is too low to teach the specific restriction enzyme sites comprised within each multiple cloning site. Addgene teaches a plasmid map for the pETDuetTM-1 vector, wherein the MCS immediately downstream of the upstream T7 promoter comprises both a BamHI restriction enzyme cleavage site and an HindIII restriction enzyme cleavage site, and wherein the MCS immediately downstream of the downstream T7 promoter comprises both an EcoRV restriction enzyme cleavage site and a KpnI restriction enzyme cleavage site [page 1 plasmid map]. Therefore, it would have been obvious to an ordinarily skilled artisan to select any two restriction sites from each of the MCSs for the insertion of the transgene sequences to be expressed under the control of the T7 promoter, wherein the first step of the recombinant cloning process is to linearize the plasmid by double digestion with the corresponding restriction enzymes and to subsequently ligate a transgene with compatible ends to the linearized plasmid.
Additionally, in selecting which cloning site to use for each of the two transgenes (e.g., the MS2 capsid protein and the HIV-1 conserved sequence fragments), it would have been obvious to an ordinarily skilled artisan to use either site for either transgene, in that the specific site-transgene combinations represent selection from a limited number of options (e.g., only 2 options).
Further, Feng teaches the use of the Easygeno Assembly Cloning Kit from TianGen to attach a linearized heterologous nucleic acid sequence to a purified linear vector fragment to generate a circular recombinant plasmid, e.g., for insertion of a promoter into the plasmid p35S-Cas9-SK at a restriction enzyme XhoI site to generate pDmc1-Cas9-SK and for insertion of a Dmc1-Cas9-SK transgene into the binary vector pTF10.1.1 digested with restriction enzymes XmaI and EcoRI to generate pDmc1-Cas9 [column 15 ¶ 5- column 16 ¶ 1]. Therefore, given the teachings of Feng of the successful use of the Easygeno Assembly Cloning Kit in a recombinant cloning procedure for the ligation of linearized vector with linearized insertion sequences to generate a recombinant circular plasmid, an ordinarily skilled artisan at the time of filing the instant application would have been motivated to use the Easygeno Assembly Cloning Kit in a method performed using recombinant cloning techniques to combine a linearized plasmid with a heterologous insertion sequence to generate a recombinant expression vector.
Regarding claim 12, the combination of Barouch, Sun, and Biocompare teach the limitations of claim 9. As discussed above for claim 2, Biocompare teaches the motivation to use the pETDuetTM-1 vector for the cloning and expression of multiple coding sequences, wherein the sequences are inserted into the pETDuetTM-1 vector within the MCSs which are located downstream of each of the two T7 promoters of the vector.
Additionally, as discussed above for claim 5, Barouch teaches that at least the gag1, gag2, pol1, and pol2 sequences are sequentially positioned and directly connected within the expression vector. Additionally, given the teaching of Barouch to also include the HIV-1 LTR in the expression vector, it would have been obvious to an ordinarily skilled artisan to include the LTR sequence sequentially with the GagPol gene in that Barouch does not teach to interrupt the Gag-Pol sequences with the LTR sequences, and so including the LTR before and/or after the Gag-Pol sequence would represent selecting from a very limited number of options.
Additionally, Roebuck teaches the motivation to include the two LTR sequences (e.g., the 5’ LTR and the 3’LTR) in an expression vector for expressing HIV-1 proteins to facilitate expression of the HIV-1 proteins, wherein the two LTR sequences flank the additional HIV-1 sequences (e.g., gag and pol).
Given the motivation taught by Sun to use an MS2 VLP particle to deliver antigenic HIV-1 sequences, wherein the HIV-1 gene sequences and the MS2 capsid gene sequence are comprised in the same expression vector for generation of the VLPs/pseudoviruses; the motivation taught by Biocompare to use the pETDuetTM-1 vector for the cloning and expression of multiple coding sequences, wherein the sequences are inserted into the pETDuetTM-1 vector within the MCSs which are located downstream of each of the two T7 promoters of the vector; the motivation taught by Barouch and GenBank to select sequence from the mosaic proviral genome taught by GenBank as corresponding to HIV-1 gag, pol, and LTR, which comprise sequences having 100% identity to fragments of the sequences according to instant SEQ ID NOs: 1-6; the motivation taught by Roebuck to include the two LTR sequences (e.g., the 5’ LTR and the 3’LTR) in an expression vector for expressing HIV-1 proteins to facilitate expression of the HIV-1 proteins, wherein the two LTR sequences flank the additional HIV-1 sequences (e.g., gag and pol); the motivation taught by Biocompare to use the pETDuet-1 vector for the cloning and expression of multiple coding sequences, such as the MS2 capsid protein coding sequence and the HIV-1 gag/pol/LTR sequences as described above; the motivation taught by Feng to use the Easygeno Assembly Cloning Kit in a method performed using recombinant cloning techniques to combine a linearized plasmid with a heterologous insertion sequence to generate a recombinant expression vector; it would have been prima facie obvious to an ordinarily skilled artisan to modify the expression vector and method of Barouch such that the vector comprises sequences which comprise fragments of instant SEQ ID NOs: 1-6, that the vector further comprises an MS2 capsid protein coding sequence, and that the method comprises packaging the expression vector sequences into a pseudovirus particle comprising an MS2 capsid protein with a reasonable expectation of success.
Conclusion
No claim is allowed.
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DR. KATIE L. PENNINGTON
Examiner
Art Unit 1634
/KATIE L PENNINGTON/Examiner, Art Unit 1634
/MARIA G LEAVITT/Supervisory Patent Examiner, Art Unit 1634