DETAILED ACTION
Applicant’s response to the restriction/election requirement received on 7/6/26 has been entered. Claims 1-2, 7-8, 11, 13-15, 17-18, 20-21, 32-36, 38-39, and 42-43 are pending in this application.
Applicant’s election of the species “CMV” as the species of constitutive promoter and “nucleic acid encoding a viral protein” as the species of nucleic acid present in the third expression cassette without traverse is acknowledged.
The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
Information Disclosure Statement
The information disclosure statements (IDS) submitted on 12/13/23, 1/15/24, and 10/1/24 are in compliance with the provisions of 37 CFR 1.97 and 1.98. Accordingly, the information disclosure statements have been considered by the examiner and initialed and signed copies of the 1449s are attached to this action.
Claim Rejections - 35 USC § 112
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 8 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 8 recites an expression system where the coumermycin-inducible promoter comprises the nucleotide sequence set forth in SEQ ID NO:11 or a functional variant thereof. The recitation of “functional variants” of the promoter sequence set forth in SEQ ID NO:11 is indefinite as it is unclear what sequences may qualify as “functional variants” of SEQ ID NO:11. Claim 8 depends on claim 7 which recites that the coumermycin-inducible promoter comprises an adenoviral tripartite leader (TPL) and/or an adenoviral major late promoter (MPL) enhancer. The specification discloses that the coumermycin inducible promoter set forth in SEQ ID NO:11 has 13 copies of the lambda operator (13xlambdaOP), a CMV minimal promoter, as adenovirus tripartite leader (TPL) and the enhancer of the adenovirus major late promoter (MLP Enhancer) within a small intron. However, the specification does not provide a specific definition of what is encompassed by a “functional variant” of coumermycin inducible promoter such as SEQ ID NO:11. The specification states in paragraph 99 that, “In one embodiment, the present disclosure includes functional variants to the nucleic acid sequences disclosed herein. The functional variants include nucleotide sequences that hybridize to the nucleic acid sequences set out above, under at least moderately stringent hybridization conditions”. This disclosure, however, simply provides an example of a “functional variant” without actually providing a definition of what other types of sequences may be considered “functional variants” of the coumermycin inducible promoter set forth in SEQ ID NO:11. Further, the specification does not describe which functional feature of SEQ ID NO:11 may be varied and still qualify as a “functional variant”. For example, do any sequences which exhibit the same or similar coumermycin inducible promoter activity as SEQ ID NO:11 qualify as functional variants of SEQ ID NO:11? Or alternatively, is the ability of a sequence to hybridize to SEQ ID NO:11 all that is required to qualify as a “functional variant”, thus including sequences that may hybridize to SEQ ID NO:11 but which do not exhibit coumermycin inducible promoter activity. It is further noted that paragraph 100 of the specification discloses that by “at least moderately stringent hybridization conditions" it is meant that conditions are selected which promote selective hybridization between two complementary nucleic acid molecules in solution where hybridization may occur to all or a portion of a nucleic acid sequence molecule., where the hybridizing portion is typically at least 15 (e.g. 20, 25, 30, 40 or 50) nucleotides in length. As such, does any sequence which can hybridize to at least 15 or more nucleotides qualify as a “functional variant”, or do only sequences with coumermycin inducible promoter activity which hybridize to at last 15 or more nucleotides qualify as a “functional variant” of SEQ ID NO:11. Note that SEQ ID NO:11 is 904 nucleotides in length, Therefore, it is unclear which sequences may or may not qualify as a “functional variant” of SEQ ID NO:11 such that the metes and bounds of claim 8 cannot be determined.
In the interests of compact prosecution, claim 8 has been given its broadest reasonable interpretation in regards to the phrase “functional variant” of SEQ ID NO:11 as encompassing any sequence that can act as a coumermycin inducible promoter.
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.
Claim 8 is 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.
Claim 8 recites an expression system where the coumermycin-inducible promoter comprises the nucleotide sequence set forth in SEQ ID NO:11 or a functional variant thereof. The specification fails to provide adequate written description for the genus of “functional variants” of the promoter sequence set forth in SEQ ID NO:11. Claim 8 depends on claim 7 which recites that the coumermycin-inducible promoter comprises a tripartite leader (TPL) and/or a major late promoter (MPL) enhancer. The specification discloses that the coumermycin inducible promoter set forth in SEQ ID NO:11 has 13 copies of the lambda operator (13xlambdaOP), a CMV minimal promoter, a adenovirus tripartite leader (TPL) and the enhancer of the adenovirus major late promoter (MLP Enhancer) within a small intron. However, the specification does not provide a specific definition of what is encompassed by a “functional variant” of coumermycin inducible promoter such as SEQ ID NO:11. The specification states in paragraph 99 that, “In one embodiment, the present disclosure includes functional variants to the nucleic acid sequences disclosed herein. The functional variants include nucleotide sequences that hybridize to the nucleic acid sequences set out above, under at least moderately stringent hybridization conditions”. This disclosure, however, simply provides an example of a “functional variant” without actually providing a definition of what other types of sequences may be considered “functional variants” of the coumermycin inducible promoter set forth in SEQ ID NO:11. Further, the specification does not describe which functional feature of SEQ ID NO:11 may be varied and still qualify as a “functional variant”. For example, do any sequences which exhibit the same or similar coumermycin inducible promoter activity as SEQ ID NO:11 qualify as functional variants of SEQ ID NO:11? Or alternatively, is the ability of a sequence to hybridize to SEQ ID NO:11 all that is required to qualify as a “functional variant”, thus including sequences that may hybridize to SEQ ID NO:11 but which do not exhibit coumermycin inducible promoter activity. It is further noted that paragraph 100 of the specification discloses that by “at least moderately stringent hybridization conditions" it is meant that conditions are selected which promote selective hybridization between two complementary nucleic acid molecules in solution where hybridization may occur to all or a portion of a nucleic acid sequence molecule., where the hybridizing portion is typically at least 15 (e.g. 20, 25, 30, 40 or 50) nucleotides in length. This disclosure suggests that any sequence which can hybridize to at least 15 or mor nucleotides may qualify as a “functional variant”. SEQ ID NO:11 is 904 nucleotides in length. Thus, the genus of “functional variants” of SEQ ID NO:11 comprises potentially thousands of fragments of this sequence which are at least 15 or more nucleotides and which can hybridize under moderately stringent conditions in addition to an unknown number of additional sequences which differ from the sequence of SEQ ID NO:11 or a fragment of SEQ ID NO:11 and which can hybridize to SEQ ID NO:11, and further potentially including any other nucleotide which may not be capable of hybridizing to SEQ ID NO:11 but which exhibits coumermycin inducible promoter activity. Of this potentially enormous genus of “functional variants” of SEQ ID NO:11, the specification only provides a description of SEQ ID NO:11, and possibly the related sequences SEQ ID NOS 9, 10, and 12 which are taught to have coumermycin inducible promoter activity and which comprise a 12Xlambda sequence but do not comprise MPL or TPL sequences (SEQ ID NO:9), an 11Xlambda sequence that includes MPL and TPL (SEQ ID NO:12), or a 13xlambda that lack MPL and TPL sequence (SEQ ID NO:10). However, even if these three disclosed sequences qualify as “functional variants” of SEQ ID NO:11, the disclosure of three species of the potentially enormous genus of sequences which qualify as “functional variants” of SEQ ID NOS:11 is not sufficient to adequately describe and demonstrate possession of either genus of variants as claimed.
The following guidance provided in MPEP 2163 is informative. To satisfy the written description requirement, a patent specification must describe the claimed invention in sufficient detail that one skilled in the art can reasonably conclude that the inventor had possession of the claimed invention. See, e.g., Moba, B.V. v. Diamond Automation, Inc., 325 F.3d 1306, 1319, 66 USPQ2d 1429, 1438 (Fed. Cir. 2003); Vas-Cath, Inc. v. Mahurkar, 935 F.2d at 1563, 19 USPQ2d at 1116. The written description requirement for a claimed genus may be satisfied through sufficient description of a representative number of species by actual reduction to practice (see i)(A) above), reduction to drawings (see i)(B) above), or by disclosure of relevant, identifying characteristics, i.e., structure or other physical and/or chemical properties, by functional characteristics coupled with a known or disclosed correlation between function and structure, or by a combination of such identifying characteristics, sufficient to show the applicant was in possession of the claimed genus (see i)(C) above). See Eli Lilly, 119 F.3d at 1568, 43 USPQ2d at 1406. An invention described solely in terms of a method of making and/or its function may lack written descriptive support where there is no described or art-recognized correlation between the disclosed function and the structure(s) responsible for the function. In other words, describing a composition by its function alone typically will not suffice to sufficiently describe the composition. See for example Eli Lilly, 119 F.3 at 1568, 43 USPQ2d at 1406 (Holding that description of a gene’s function will not enable claims to the gene "because it is only an indication of what the gene does, rather than what it is."); see also Fiers, 984 F.2d at 1169-71, 25 USPQ2d at 1605-06 (discussing Amgen Inc. v. Chugai Pharm. Co., 927 F.2d 1200, 18 USPQ2d 1016 (Fed. Cir. 1991)). An adequate written description of a chemical invention also requires a precise definition, such as by structure, formula, chemical name, or physical properties, and not merely a wish or plan for obtaining the chemical invention claimed. See, e.g., Univ. of Rochester v. G.D. Searle & Co., 358 F.3d 916, 927, 69 USPQ2d 1886, 1894-95 (Fed. Cir. 2004) (The patent at issue claimed a method of selectively inhibiting PGHS-2 activity by administering a non-steroidal compound that selectively inhibits activity of the PGHS-2 gene product, however the patent did not disclose any compounds that can be used in the claimed methods. While there was a description of assays for screening compounds to identify those that inhibit the expression or activity of the PGHS-2 gene product, there was no disclosure of which peptides, polynucleotides, and small organic molecules selectively inhibit PGHS-2. The court held that "[w]ithout such disclosure, the claimed methods cannot be said to have been described."). Furthermore, written description issues may also arise if the knowledge and level of skill in the art would not have permitted the ordinary artisan to immediately envisage the claimed product arising from the disclosed process. See, e.g., Fujikawa v. Wattanasin, 93 F.3d 1559, 1571, 39 USPQ2d 1895, 1905 (Fed. Cir. 1996). While it has been held that what is conventional or well known to one of ordinary skill in the art need not be disclosed in detail, for inventions in emerging and unpredictable technologies, or for inventions characterized by factors not reasonably predictable which are known to one of ordinary skill in the art, more evidence is required to show possession.
As discussed above, the specification fails to provide a specific definition of what types of sequences qualify as “functional variants” of SEQ ID NO:11 such that the skilled artisan would not be apprised as to how to identify any such sequence from sequences known in the prior art, or how to screen for such sequences based on any specific functional or structural features. Further, as noted above, the potential genus of “functional variants” of SEQ ID NO:11 is extremely large and the specification only provides a description of three sequences, SEQ ID NOS 9, 10, and 12, which may be “functional variants” of SEQ ID NO:11. As such, the specification fails to provide a description of a representative number of species by actual reduction to practice (see i)(A) above), reduction to drawings (see i)(B) above), or by disclosure of relevant, identifying characteristics, i.e., structure or other physical and/or chemical properties, by functional characteristics coupled with a known or disclosed correlation between function and structure, or by a combination of such identifying characteristics, sufficient to show the applicant was in possession of the claimed genus of “functional variants” of SEQ ID NO:11 at the time of filing.
Claim Rejections - 35 USC § 103
The following is a quotation of 35 U.S.C. 103 which forms the basis for all obviousness rejections set forth in this Office action:
A patent for a claimed invention may not be obtained, notwithstanding that the claimed invention is not identically disclosed as set forth in section 102, if the differences between the claimed invention and the prior art are such that the claimed invention as a whole would have been obvious before the effective filing date of the claimed invention to a person having ordinary skill in the art to which the claimed invention pertains. Patentability shall not be negated by the manner in which the invention was made.
This application currently names joint inventors. In considering patentability of the claims the examiner presumes that the subject matter of the various claims was commonly owned as of the effective filing date of the claimed invention(s) absent any evidence to the contrary. Applicant is advised of the obligation under 37 CFR 1.56 to point out the inventor and effective filing dates of each claim that was not commonly owned as of the effective filing date of the later invention in order for the examiner to consider the applicability of 35 U.S.C. 102(b)(2)(C) for any potential 35 U.S.C. 102(a)(2) prior art against the later invention.
Claims 1-2, 7-8, 11, 13-15, 17-18, 20, 32-36, 38-39, and 42-43are rejected under 35 U.S.C. 103 as being unpatentable over Sudomoina et al. (2004) BMC Biotech., Vol. 4:9; biomedcentral.com/1472-6750/4/9, page 1-9, in view of Ma et al. (2016) Sci. Reports., Vol. 6:26617; doi:10.1038/srep26617, pages 1-13, Mullick et al. (2006) BMC Biotech., Vol. 6:43; doi:10.1186/1472-6750-6-43, pages 1-18, and Broussau et al. (2008) Mol. Ther., Vol. 16(3), 500-507.
Sudomoina et al. teaches a dual drug multi-layer expression system for mammalian cells. Sudomoina et al. teaches a multi- plasmid system which uses a combination of a dimerizer-regulated two-hybrid system and a tetracycline regulatory element system in an ordered cascade to place the expression of endpoint reporters under the control of two distinct drugs (Sudomoina et al., page 1). Sudomoina et al. teaches it has been previously shown efficient regulation of gene expression can be achieved through the use of artificial promoters dependent on the action of small molecule modulators, including tetracycline and derivatives; streptogramin and macrolide antibiotics; combined coumermycin and novobiocin systems; ecdysone; and dimerizer molecules such as FK1012 and other rapamycin-related derivatives (Sudomoina et al., page 2). Sudomoina et al. teaches that while each of these systems offers specific advantages, in order to generate flexible tools for general usage in achieving fine control of gene expression, it would be useful to be able to combine elements of these different systems (Sudomoina et al., page 2). Sudomoina et al. teaches that sequential cascades combining different groups of transcription factors, with localization and activity regulated by multiple inputs from modifying factors represents a standard means of gene regulation in naturally occurring living organisms where the complexity arising from the multiple inputs leads to more fine-tuning of output, and enables a more complex biological response (Sudomoina et al., page 2). Sudomoina et al. teaches the successful combination of a dimerizer-regulated two-hybrid system and a tetracycline regulatory element system to regulate the expression of two proteins of interest. More specifically, Sudomoina et al. teaches a first plasmid which expresses fusion proteins of DBD and AD, which interact only in the presence of a small molecule dimerizer; a second plasmid which comprises an expression cassette transcriptionally responsive to the first DBD which directs expression of the Tet-OFF protein; and a third plasmid which encodes a reporter gene transcriptionally responsive to binding by Tet-OFF (Sudomoina et al., page 1). More specifically, Sudomoina et al. teaches the vectors pZBS-Tet-OFF, a pRetro-OFFvector which has been modified to place the tTA (tetracycline repressor fused to viral VP16 protein) under transcriptional control of 12 ZFHD1-binding sites in a minimal promoter context derived from IL2, the vector pBI-EGFP-SEAP where the tetracycline responsive promoter was used to bidirectionally express pEGFP and the secreted alkaline phosphatase (SEAP) gene, and the plasmids pAR-DBD and pAR-AD which were constructed as derivatives of pC4N2-RHS/ZF3 by eliminating one of the two coexpressed components initially expressed, such that ZFHD-FKBP12 (pAR-DBD) or p65-FRAP (pAR-AD) is separately expressed under the control of the CMV promoter (Sudomoina et al., page 8). Sudomoina et al. successfully demonstrated combining the two discrete drug-regulated expression systems in an ordered cascade.
Sudomoina et al. differs from the instant claimed dual regulated system by using a combination of a dimerizer-regulated two-hybrid system and a tetracycline regulatory element system instead of the cumate inducible system and coumermycin inducible system recited in the instant claims. However, as noted above, Sudomoina et al. teaches a number of alternative inducible systems, including the coumermycin inducible expression system. Ma et al. supplements Sudomoina et al. by teaching the combined use of a tet-on inducible system and a regulated mammalian coumermycin inducible system to express both Sox9 and bcl2 in a mammalian cell (Ma et al., pages 1-2). Ma et al. teaches mammalian cells comprising four expression vectors, including a first vector encoding the rtTA transactivator under operative control of a constitutive CMV promoter, second vector encoding Sox9 under operative control of a tetracycline responsive promoter, a third vector which is a pREG neo vector encoding a coumermycin chimeric transactivator fusion protein comprising the VP16 transcriptional activator domain that can bind to the λ operator sequence under operative control of a promoter, and fourth vector which is a pF12K RM Flexi vector which contains tandem λ operator sequences and a minimal CMV promoter that responds to the transactivator encoded in the pREG neo vector operably linked to the anti-apoptotic gene Bcl-2 (Ma et al., pages 5-6 and Figure 2). Ma et al. teaches two different coumermycin responsive promoters which were both commercially available at the time of filing from Promega, the 6Xlambda minimal CMV promoter and the 12Xlambda minimal CMV promoter (Ma et al., page 6, Figure 2, and Supplemental Table S1). Ma et al. shows that both Sox9 expression and Bcl-2 expression were effectively induced by doxycycline or coumermycin at a concentration of 2.5 nM respectively both in cells in vitro and in vivo (Ma et al., pages 5, and 7-8).
Mullick et al. further teaches another inducible expression system for use in regulating transgene expression in mammalian cells, the cumate operon system based on the bacterial operons cmt and cym which operates similarly to the Tet operon system (Mullick et al., abstract). Mullick et al. teaches that in the repressor configuration, regulation is mediated by the binding of the repressor (CymR) to the operator site (CuO), placed downstream of a CMV5 constitutive promoter, where the addition of cumate relieves the repression thus allowing gene expression (Mullick t al., page 1). Mullick et al. further teaches that in the transactivator configuration, chimaeric transactivator (cTA) protein, formed by the fusion of CymR with the activation domain of VP16, is able to activate transcription when bound to multiple copies of CuO placed upstream of the CMV minimal promoter where addition of cumate, at a concentration of 200 mg/ml, abrogates DNA binding and transactivation by cTA (Mullick et al., page 1 and Figures 1 and 2). In one embodiment, Mullick et al. further teaches a CuO operator sequence which is 6X-CuO upstream of a minimal promoter element and the Ad TPL sequence (Mullick et al., page 13). Mullick et al. teaches that this cumate system is similar to the well known Tet operon based system which utilizes a chimeric transactivator tTA composed of a repressor TetR fused to the VP16 activation domain and linked to seven repeats of the Tet operator sequence to the minimal CMV promoter, or reverse chimeric transactivator rtTA o either repress or activate gene expression in the presence of tetracycline (Mullick et al., page 2). Mullick et al. teaches that it is not only desirable but necessary to have a wide variety of regulatory systems since an increasing number of applications benefit from the possibility of controlling simultaneously the expression of several genes (Mullick et al., page 2). Mullick et al. further teaches that combinations of these gene switches now allows for sophisticated multilevel gene regulation in mammalian cells (Mullick et al., page 2).
Therefore, in view of the motivation provided by Sudomoina et al. for generating and using a dual drug multi-layer expression system for expressing a desired protein in mammalian cells in order to recapitulate the type of a complex sequential cascades present cells in order to fine tune gene expression, the further teachings of Sudomoina et al. that a number of inducible expression systems were known in the art, the teachings of Ma et al. for a regulated mammalian coumermycin inducible system which can be used in conjunction with a second inducible system such as the Tet system to regulate the expression of two genes, and the teachings of Mullick et al. for a coumermycin inducible expression system which operates similarly to the Tet operon system, it would have been prima facie obvious to the skilled artisan at the time of filing to modify the dual drug layer expression system of Sudomoina et al. to utilize the cumate expression system of Ma et al. to regulate the expression of coumermycin expression system of Mullick et al. with a reasonable expectation of success. More specifically, based on the specific elements and mechanism of the cumate system taught by Mullick et al. which involves the use of a first plasmid which encodes a vector encoding a cumate repressor protein CymR under operative control of a CMV5 promoter, wherein the expressed CymR binds to and represses the CuO operator sequence present in a second vector which contains the CuO operator sequences and a minimal CMV promoter in the absence of cumate, wherein addition of cumate releases CymR to induce expression of an operably linked to a gene of interest, and the specific elements and mechanism of the coumermycin system taught by Ma et al. which involve the use of a vector encoding the chimeric transactivator for coumermycin which only binds and activates the lambda operator sequence in a second vector in the presence of coumermycin to induce expression of a gene of interest, it would have been prima facie obvious to the skilled artisan at the time of filing to prepare a first vector comprising a strong promoter such as the CMV promoter taught by Sudomoina et al. operatively liked to a cumate repressor protein CymR, a second vector comprising a cumate inducible promoter comprising CuO and the minimal CMV promoter operatively linked to sequence encoding the coumermycin chimeric transactivator, and a third plasmid comprising a coumermycin inducible promoter such as the 12Xlambda minimal CMV promoter, which may further comprise the TPL sequence as taught by Mullick et al., operatively linked to a gene of interest with a reasonable expectation of success.
Sudomoina et al., Ma et al., and Mullick et al. further differ from the instant claims by not specifying the presence of a polyA sequence in each vector, and further by not teaching that the gene of interest encodes a viral protein. Further, Sudomoina et al., Ma et al., and Mullick et al. do not teach to use the dual system to express the three viral proteins required for lentiviral packaging, Rev, envelope, and gag/pol, in a packaging cell for production of lentivirus.
Broussau et al. supplements Sudomoina et al., Ma et al., and Mullick et al. by teaching that because of the reported cytotoxicity of the lentiviral protease, and also of the glycoprotein of vesicular stomatitis virus (VSV-G) that is often utilized to pseudotype LV, it has been difficult to make stable lentiviral producer cell lines (Broussau et al., pages 500 ). Broussau et al. teaches that this problem of cytotoxicity can be solved by regulating the expression of VSV-G and of some additional lentiviral components using an inducible transcription system that is activated at the time of production (Broussau et al., page 500). Broussau et al. teaches that the tetracycline (Tet)–dependent regulatory system and the inducible ecdysone system have be successfully employed for producing LV packaging cell lines (Broussau et al., page 500). However, Broussau et al. teaches that in some cases poor stability of the packaging cells has been reported caused by leaky expression of VSV-G, such as was documented when using a Tet-Off system (Broussau et al., page 502). Broussau et al. teaches the development of a packaging cell line 293SFPacLV, a cell line derived from the human HEK 293 cells, that can produce lentiviral vectors (LVs) in serum free suspension cultures where expression of VSV-G and Rev are tightly regulated at the transcription level using the optimized Tet-On switch in conjunction with an inducible cumate switch derived from the p-cym operon, where induction is promoted by the addition of two inducers, doxycycline (dox) and cumate to the growth medium (Broussau et al., page 502). More specifically, Broussau et al. teaches that the stable packaging cell line derived from 293SF cells comprises five vector expression constructs, wherein the first vector encodes the repressor (CymR) of the cumate switch operatively linked to the constitutive CMV promoter and polyadenylation (pA) sequence, the second vector encodes rtTA2-M2 operatively linked to the CMV promoter and a pA sequence, the third vector encodes gag-pol operatively linked to the CMV promoter and a pA sequence, the fourth vector encodes Rev operatively linked to both the TR5 (7XtetOATA) tetracycline responsive promoter and the CuO cumate switch and a pA sequence, and the fifth vector encodes VSV-G also operatively linked to both the TR5 promoter and a CuO cumate switch and a pA sequence where expression of Rev and VSV-G is tightly regulated by the cumate and Tet switches by dox which promotes binding of the reverse tetracycline transactivator (rtTa2s-m2) to the TR5 promoter, and cumate which promotes the release of the cumate repressor (CymR) from the CuO operator (Broussau et al., pages 2-3, Table I, and Figure 1). Finally, in order to generate LV particles, the stable packaging cell line was transfected with the lentiviral transfer vector which encodes a gene of interest encoding GFP under operative control of either the constitutive RSV or CMV promoters (Broussau et al., page 4).
Therefore, in view of the motivation provided by Broussau et al. to utilize a system involving the use of two inducible promoters, where one of the inducible systems includes cumate, to express the protein elements of a lentivirus in a packaging cell for production of a lentivirus in suspension culture, and the further teachings of Broussau et al. that vectors encoding each element of the system utilize a pA sequence 3’ of the protein coding sequence, it would have been prima facie obvious to the skilled artisan at the time of filing to prepare a vector system according to Sudomoina et al., Ma et al., and Mullick et al. where the gene encoded by the third vector is a protein involved in lentiviral packaging, more specifically Rev or VSV-G in order to achieve multi-layered fine tuned expression of these toxic in mammalian cells and particularly a packaging cell with a reasonable expectation of success. Further, based on the motivation provided by Broussau et al. to express all three of Rev, gag/pol, and VSV-G, where at least Rev and VSV-G are under inducible expression due to their cytotoxicity, it would have been further prima facie obvious to the skilled artisan at the time of filing to include a fourth and fifth vector encoding Rev or VSV-G and gag/pol respectively where at least the third and fourth vector are under the inducible control of the coumermycin inducible promoter, such as the 12Xlambda minimal CMV promoter, with a reasonable expectation of expressing all three viral proteins in a packaging cell in the presence of both cumate and coumermycin, and of producing a lentiviral particle from the packaging cell where the cell further comprises a lentiviral transfer vector.
Claim 21 is rejected under 35 U.S.C. 103 as being unpatentable over Sudomoina et al. (2004) BMC Biotech., Vol. 4:9; biomedcentral.com/1472-6750/4/9, page 1-9, in view of Ma et al. (2016) Sci. Reports., Vol. 6:26617; doi:10.1038/srep26617, pages 1-13, Mullick et al. (2006) BMC Biotech., Vol. 6:43; doi:10.1186/1472-6750-6-43, pages 1-18, and Broussau et al. (2008) Mol. Ther., Vol. 16(3), 500-507, as applied to claims 1-2, 7-8, 11, 13-15, 17-18, 20, 32-36, 38-39, and 42-43 above, and further in view of Farson et al. (2004) J. Gene Med., Vol. 6, 1369-1381, and Holsher et al. (1995) J. Virol., Vol. 69(11), 6800-6885.
Sudomoina et al., Ma et al., Mullick et al., and Broussau et al. provides the teachings and motivation to prepare a dual drug multi-layer expression system for expressing viral proteins in mammalian packaging cells, such as lentiviral proteins and in particular Rev and VSV-G, in order to fine tune gene expression of potentially cytotoxic viral proteins, where the system comprises a first vector comprising a strong promoter such as the CMV promoter taught by Sudomoina et al. operatively linked to a cumate repressor protein CymR, a second vector comprising a cumate inducible promoter comprising CuO and the minimal CMV promoter operatively linked to the coumermycin chimeric transactivator, and a third plasmid comprising a coumermycin inducible promoter such as the 12Xlambda minimal CMV promoter operatively linked to a gene encoding a viral protein such as Rev, and further a fourth vector also comprising a coumermycin inducible promoter such as the 12Xlambda minimal CMV promoter operatively linked to a gene encoding a viral protein such as VSV-G, or vice versa for the third and fourth vectors, and a fifth vector encoding gag/pol operatively linked to a CMV promoter as set forth in detail above with a reasonable expectation of success.
While Broussau et al. provides the teaching and motivation to express lentiviral proteins in a packing cell line, including Rev, VSV-G, and gag/pol, Broussau et al. does not teach to express adeno-associated viral proteins such as Rep40 or Rep52 in the third vector, or Rep68 or Rep 78 in the fourth vector. Farson et al. supplements Sudomoina et al., Ma et al., Mullick et al., and Broussau et al. by teaching that the toxicity of the AAV rep genes is an obstacle to the generation of stable AAV packaging cell lines, and that regulation of expression of the rep genes can diminish this toxicity (Farson et al., page 1370). Holsher et al. further supplements Sudomoina et al., Ma et al., Mullick et al., and Broussau et al. by investigating the individual contributions of the Rep proteins of AAV in AAV packaging in Hela cells (Holsher et al., pages 6885-6886). Holsher generated four separate plasmid encoding AAV Rep78, Rep68, Rep52, or Rep40 operatively linked to a constitutive CMV promoter (Holsher et al., page 6886). Holsher et al. demonstrates that the combination of a plasmid encoding Rep78, a plasmid encoding Rep52, a rep-negative AAV-2 genome construct pTAV2-3, and Ad2 helper virus expressed in HeLa cells resulted in efficient and high titer production of the rep-negative virus (Holsher et al., page 6886). Holsher et al. further teaches the expression of Rep78 using a glucocorticoid inducible promoter responsive to dexamethasone, where contact of a HeLa derived cell lines which stably express Rep40, and which comprise a glucocorticoid receptor expression construct, an AAV genome vector, Ad2 helper virus, and the inducible Rep78 vector with dexamethasone resulted in production of infections AAV particles (Holsher et al., pages 6881-6882).
Therefore, in view of the teaching of Farson et al. that the toxicity of the AAV rep genes, similar to the toxicity of the Rev and VSV-G genes taught by Broussau et al., is an obstacle to the generation of stable AAV packaging cell lines, and the teachings of Holsher et al. for generating packaging cell lines for AAV which comprise a plasmid encoding Rep78 operatively linked to an inducible promoter and a plasmid encoding Rep40 which are capable of producing infectious AAV particles in a Hela cell which further comprises an AAV genome vector and Ad2 helper virus in the presence of the inducer, it would have been prima facie obvious to the skilled artisan at the time of filing to express the AAV viral proteins Rev40 and Rev78 in the third and fourth vectors respectively of the dual drug multi-layer expression system for expressing viral proteins taught by Sudomoina et al., Ma et al., Mullick et al., and Broussau et al. with a reasonable expectation of success.
No claims are allowed.
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Dr. A.M.S. Wehbé
/ANNE MARIE S WEHBE/Primary Examiner, Art Unit 1634
