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 Invention Group I in the reply filed on 08/25/2025 is acknowledged. Accordingly, claims 1-8,14 and 15 are under examination and Group II, claims 12,13, 16-17 are withdrawn from further consideration pursuant to 37 CFR 1.142(b) as being drawn to a nonelected Invention (Group II), there being no allowable generic or linking claim.
Priority
This application claims priority to foreign parent to Application 20170133.1 EP filed 4/17/2020. Receipt is acknowledged of certified copies of papers required by 37 CFR 1.55.
Information Disclosure Statement
The information disclosure statements (IDS’s) submitted on 01/17/2023 is in compliance with the provisions of 37 CFR 1.97. Accordingly, the information disclosure statements is being considered by the examiner.
Drawings
The drawings, filed 10/14/2022, are accepted.
Claim Objections
Claim 1 is objected to because of the following informalities:
As presented, the claim syntax is challenging to understand. The claim is interpreted to reference an isolated nucleic acid comprising at least two copies of a sequence that are operably linked to a transgene. However, as written, it is challenging to understand what “operably linked” modifies, given its position in the claim, since the relevant phrase is not adjacent to the modifier. a Appropriate correction is required to improve the clarity of the claim.
Claim Interpretation
In evaluating the patentability of the claims presented in this application, the claims will be given their broadest reasonable interpretation, in view of the specification, and as set forth at MPEP§ 2111. For clarity of the record, Claim 1 references two copies selected “independently” from one another. This independence is interpreted to mean that if one copy is selected there is no relationship between that and the second copy type that is selected.
Claim 1 references a heterologous transgene linked to SEQID(s) in an isolated nucleic acid. Since SEQIDs are mouse sequence, this will be interpreted to mean the heterologous transgene is not from mouse and is linked to SEQID from mouse.
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 1-8, 14 and 15 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 is indefinite in recitation of, “An isolated nucleic acid comprising, operably linked to a heterologous transgene, at least two copies of a sequence selected from the group consisting of SEQID NO:1, SEQID NO: 2 and SEQID NO:3 ” because it is unclear exactly what two copies references. This could be interpreted to mean the copies of sequence may / must come from: a) two different SEQID NO: 1, for example, and since SEQID NO: 1 is degenerate there are many possible variants, OR the phrase could refer to b) two copies of the same SEQID NO:1 variant, OR c) two copies could refer to one from SEQID 1 and one from SEQID 2, for example (or any other combination of SEQ ID Nos), OR d) something other than the options recited could be the true intent. This ambiguity makes it challenging to establish the metes and bounds of the claim. This claim also recites and isolated nucleic acid, then two copies of sequence linked to a heterologous transgene, but no species are recited. Claims 2-8, 14 and 15 depend from claim 1 and are rejected for the same reason.
Claim 1, 2, and 6 are indefinite in the recitation of “operably linked” since the functional relationship is not stated (could be on/off, or a quantity, or something different for claims 1 and 2), and so it is difficult to known how to clearly interpret what “operably linked” means, when it happens, or how it is measured, which is particularly true for all of these claims when the molecule comprises a fragment or variant, thus making the metes and bounds of the claim unclear. Claims 2-8, 14 and 15 depend from claim 1, and Claim 7 and 8 depend from claim 6, and are rejected for the same reason.
Claim 2 and 6 are indefinite in the recitation of “active fragment” since it is not clear what particular activity is being referenced for a fragment, so interpretation of when/how it is considered ‘active’, to establish metes and bounds, cannot be made, making the scope of the claim unclear. Similarly unclear is the particular definition of ‘variant’ – it is not clear how to determine when something is, or is no longer, considered to be a ‘variant’. Specifically, what (e.g. a gene, a protein, something different) this is a ‘variant’ of, is also unclear. Claim 7 and 8 depend from claim 6, and are rejected for the same reason.
Claim Rejections - 35 USC § 112(a)
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.
Claims 1-8, 14 and 15 are rejected under 35 U.S.C. 112(a) or 35 U.S.C. 112 (pre-AIA ), first paragraph, because the specification, while being enabling for a composition of one to three copies of SEQID NO:3 in mouse stem cells, with a heterologous luciferase reporter gene, and further comprising the BANP gene, does not reasonably provide enablement for an operable association of a motif of SEQ ID Nos: 1 and/or 2 to a heterologous transgene and/or with a BANP gene, and/or in cells, other than mouse stem cells. Nor is there enablement for claim 3, a heterologous transgene that is a(ny) chimeric antigen receptor. The specification does not enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the invention commensurate in scope with these claims. This is a scope of enablement rejection.
The MPEP §2164.01(a) recites, “There are many factors to be considered when determining whether there is sufficient evidence to support a determination that a disclosure does not satisfy the enablement requirement and whether any necessary experimentation is undue.” In re Wands, 858 F.2d 731, 737, 8 USPQ2d 1400, 1404 (Fed. Cir. 1988) points to these factors, which include but are not limited to
A. Nature of the Invention
B. Breadth of the Claims
C. Amount of direction provided by Inventor
D. Existence of Working Examples
E. State of the Art
F. Level of Predictability in the Art
G. Quantity of experimentation needed to make or use the invention based upon the disclosure
Per MPEP 2164.01(a) compliance with enablement asks, is there undue experimentation necessary for one of skill in the art to make and use the claimed invention based upon what is known in the art and what is disclosed in the specification?
Nature of the Invention: The claims are directed to a composition that comprises an orphan motif, where more than one copy of the motif may be present. The motif has an operable link (to regulate gene expression) with a transgene, and the motif is bound by the BANP protein, which serves as a transcriptional activator. The motif may consist of one of three motifs, two of which are degenerate. The claims include introducing a construct into a vector and into an isolated cell. Accordingly, enablement requires that one of ordinary skill in the art be able to construct and use the isolated nucleic acid, where there is a operative functional working relationship between the transgene and the motif(s), introduce the composition into cells stably, and introduce it into a vector.
B. Breadth of Claims: The claims are broadly directed to an isolated nucleic acid, copies of a motif and a transgene. Claim 1 discloses at least two copies of the motif: SEQ ID NO 3 (TCTGCGAGA) or the degenerate sequences (SEQ ID 1: BMYCGCRBV or SEQ ID NO 2: YMYCGCRKV). The Genus of permutations that exists for each of the degenerate motifs is large. Further, there are no limitations recited that disclose which genes comprise the very large Genus of any and all possible “transgenes”. Limitations could include reference to taxonomic species and could recite the gene(s). Claims 2, and 6 (to a kit), narrow the invention only by adding the BANP gene to the aforementioned “isolated nucleic acid”; claim 6 adds that BANP is linked to a promotor. Claim 3 narrows the invention, such that the heterologous gene is a chimeric antigen receptor. However, CAR’s are themselves a large Genus of thousands of receptors. Claim 4, and 7 (the kit) broadly discloses any vector comprising the “isolated nucleic acid” of claim 1; Claim 5 narrows this, to a suite of vectors. Claim 8 employs use of two vectors. Claim 14 is a (any) isolated cell of any taxonomic species, comprising claim 1. Claim 15 narrows claim 14, by indicating stable integration, in any cell.
C. and D. Amount of direction from Inventor and Existence of working Examples
There are no working examples that clearly depict SEQID NO: 1 and 2 as the most enriched peaks (motifs), after the (RNA-seq) and ChIP seq analysis. The Specification (Pg 25) particularly discloses at the end of the working examples, that potential target genes of BANP were genes with promotors that contained a perfect match to the motif SEQID NO 3: TCTCGCGAGA. This motif is also depicted in FIG 3 as the predominant, enriched motif identified after ChIP-Seq analysis, per detection with HOMER (Fig 3). No equivalent data set is presented for SEQID NO1: bmycgcgrbv, or SEQID NO: 2: ymycgcgrkv. The alternate nucleotides of SEQID NO: 1 and 2 are found only in much lower frequency, in the image of the HOMER analysis.
FIG 3 actually depicts enrichment for individual nucleotides at individual positions and does not show the presence of any singular sequence.
Working examples depict:
Oligo-IP and mass spectrometry were used to detect proteins binding to the orphan motif after molecular footprinting in mouse embryonic stem cells. To validate BANP locates the orphan motif, ChIP-seq was conducted, then top enriched motifs identified with HOMER.
Oligo-bound beads (motif or control) were constructed. Nuclear lysate (protein extract of mouse cells) was generated and protein quantified. Oligo immunoprecipitation was conducted to capture binding proteins from mouse cell lysate.
BANP and scrambled controls were cloned upstream of a luciferase gene in plasmid that was transfected into mouse stem cells. Luciferase activity was measured (and normalized), and increase in luciferase activity for BANP-motif constructs calculated. ChIP-seq analysis was then preformed to identify transcription factor binding sites, as well as RNA-seq (aligning to mouse) for identification of gene expression results.
Figures: (1) relates to BANP protein binding an orphan activator and detection by mass spectrometry, (2) BANP protein, as a transgene activator, upstream of a reporter luciferase, relative to a scrambled motif, in mouse stem cells, depicts 1, 2 and 3 motifs, correlated with increased luciferase activity. (3) BANP locates orphan motif; HOMER motif from common peaks is SEQID NO 3.
E. and F. State of the Prior Art and Predictability:
The art does not disclose the degenerate motifs, SEQ ID 1: BMYCGCRBV or SEQ ID NO 2: YMYCGCRKV, or an operable link of these motifs to any transgene.
Evidence shows that mutations have significant, varied impacts on motif functionality such that such that motif degeneracy would contribute to unpredictability in functionality. For example, Donello (Donello, J. et al., Woodchuck Hepatitis Virus Contains a Tripartite Posttranscriptional Regulatory Element, ,1998, Jour Virol, 5085–5092) identified that mutation of a single post-transcriptional regulatory element (WPRE) in woodchuck hepatitis virus decreases WPRE activity by over 40% (Pg 5091, right col, 3rd para) and mutation of two residues can cause even greater activity reduction (Pg 5091, right col, 1st para). The sub element quantity also leads to alterations in functionality, and mutations in these result in unpredictable activity changes.
Regarding mutations to instant SEQIDNO:3, as are depicted in instant SEQIDNOs: 1 and 2, the art (Mikula (Mikula, M. et al., Comprehensive Analysis of the Palindromic Motif TCTCGCGAGA: A Regulatory Element of the HNRNPK Promoter, 2010, DNA RESEARCH 17, 245–260) demonstrated unpredictability and a change in functionality of the motif of SEQID NO;3 with mutation
Mikula designed constructs with 1-3 mutations ( Pg 250, right col, para 2) which resulted in marked luciferase activity decreases in cells transfected with constructs containing the mutants (Pg 250, right col, para 2). A single point mutation in the motif resulted in >80% loss of promotor activity, and triple mutations reduced activity to near basal levels (Pg 250, right col, para 2), for HNRNPK promotor activity (Pg 250, right col, para 2).
In considering the Genus of any and all potential transgenes, even narrowed just to the claim 3 limitation of the gene being a CAR, Zhang (Zhang, C., et al., Engineering CAR-T cells 2017, Biomarker Research (2017) 5:22) disclosed functional differences in CARs, meaning one simply cannot be replaced with another with the expectation of identical results. First generation constructs, did not produce enough IL2 (to kill tumor cells) (Pg 2 right col, first full para)
Second generation CARs added signaling domains improving proliferation and prolonging life of CAR-T cells (Pg 3, left col, para two). For these however, CD28 mediated co-stimulation is important for regulation of proliferation and survival (Pg 3 left col para 2).
Fourth generation CARs added IL-12 to the base of second generation constructs and have universal cytokine-mediated killing (Pg 3 right col, para 2). The effect of these in shaping tumor environment by inducible release of transgenic immune modifiers arises as a concept in this work.
Regarding vector claims (e.g. claim 4 and 5), Zhang also addressed tools for delivering foreign genes into human cells and differences in vector traits that impact their interchangeability and predictability. Zhang noted that viruses have high transfer efficiency, and for T cells, allow cell numbers accrue quickly. Different viruses have different expression characteristics. Retroviruses for gene delivery have safety risks, where insertion mutations create problematic issues including tumorigenesis, toxicity, and limited carrier capacity exists (Pg 3 right col, penultimate para). Non-viral vectors have higher efficiency, target specificity and are not infectious (Pg 3 right col final para). Minicircle DNA vectors are non-viral and can persistently express transgene with high levels in vivo (Pg 4 left col, para 1).
Re AAV and lentivirus, Powell (Powell S. et al., Viral expression cassette elements to enhance transgene target specificity and expression in gene therapy, 2015, Discov Med 2015 Jan;19(102):49-57) pointed out that these expression cassettes need to be designed to meet specific needs, and key differences relate to target specificity in promotors, and these vectors are space limited (Abstract). In these vectors, transgene expression can be modulated by adding a CMV enhancer (or other elements) (Pg 2 para 1). Even these two virus vectors have different packaging size constraints, with AAV having about half the size availability of lentiviruses (Pg 2, para 1). \ Thus multiple factors matter for results including the virus used, the cell type used, the promotor used with a given cell type vs another cell type, all relevant to the breadth of the claims in the instant application.
G. Experimentation needed: The necessary experimentation would be high, given the variability and unpredictability identified in the art, including for motif mutations and their impact, a lack of examples with predictable results for multiple motif use, prior lack of success in deorphanizing motifs, the breadth of cell and vector use presented in the claims, relative to the impact varying these has on functionality, the limited detailed disclosure in the specification, with data only presented for SEQID 3 in mouse cells, heterologous genes not clearly disclosed, no use of SEQ ID NO: 1 and 2. Thus after weighing the above factors, there is insufficient support for complete enablement of the claims, no articulated stable integration into the genome of a cell, inclusive of at least two copies of any/all of the SEQID Nos recited, no support for ‘any vector, or use of multiple vectors, no obvious reveal of a transgene that is a particular chimeric antigen receptor. The support provided for a scope of enablement, is inclusive of SEQID3 in mouse cells, with demonstrated luciferase expression differences revealed for use of 1-3 motifs of this sequence, and data for BANP locating this motif in mouse stem cells.
Claims 1-8, 14 and 15 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 (possession) 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.
MPEP 2163.II.A.3(a) i) states, “Whether the specification shows that applicant was in possession of the claimed invention is not a single, simple determination, but rather is a factual determination reached by considering a number of factors... including level of skill and knowledge in the art, partial structure, physical and/or chemical properties, functional characteristics alone or coupled with a known or disclosed correlation between structure and function, and the method of making the claimed invention”.
For claims drawn to a genus, MPEP § 2163 states 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, reduction to drawings, 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 Eli Lilly, 119 F.3d at 1568, 43 USPQ2d at 1406.
Nature of the Invention: Claim 1 is directed to an isolated nucleic acid comprising at least two copies of a sequence selected from the group consisting of SEQ ID NOs 1, 2, and 3 operably linked to a heterologous transgene. SEQ ID NOs 1 is defined as the nucleotide sequence BMYCGCRBV and SEQ ID NO 2 is defined as the nucleotide sequence YMYCGCGRKV. The nucleotide symbol B refers to A or G or T/U, the symbol M refers to A or C, the symbol Y refers to T/U or C, the symbol R refers to G or A, the symbol V refers to A or G or C, and the symbol K refers to G or T/U. Therefore, SEQ ID NO 1 refers to a genus of 216 nucleotide sequences and SEQ ID NO 2 refers to a genus of 96 nucleotide sequences. The recitation in claim 1 that these sequences are “operably linked” to a heterologous transgene is a functional limitation of those sequences that requires that, for the purposes of this rejection, that they regulate the transcription of the heterologous transgene. However, the specification has not adequately described the genus of such sequences that have the corresponding function of regulating the transcription of the heterologous transgene as discussed below.
Guidance provided by applicant and specific examples: The specification describes a ChIP-Seq experiment (see page 24) in which an antibody was used to pull down BANP, which is a transcription factor, from mouse embryonic stem cells. The specification teaches that the de novo motif finder HOMER was used to identify the top 500 peaks (see page 24). Figure 3 illustrates these results, which shows the relative nucleotide enrichment at each individual position. It clearly shows that there is a strong preference for SEQ ID NO 3: TCTCGCGAGA. However, it also suggests that BANP motif also tolerates alternative nucleotides as positions 1-3 and 6-10, which form the basis for the genus of nucleotides encompassed by SEQ ID NOs 1 and 2. However, this figure does not adequately describe the genus of sequences covered by SEQ ID NOs 1 and 2 or further that each sequence would be capable of regulating the transcription of a heterologous gene as discussed below.
First, since Figure 3 shows the relative nucleotide abundance at positions 1-10, where the relative abundance of the nucleotides at each position is independent of the relative abundance of nucleotides at each other position, this is not evidence that all possible nucleotide sequences encompassed by SEQ ID NOs 1 and 2 even exist in the tested sample. For example, there is no evidence that the sequence CACCGCGGTG, which comprises non-preferred nucleotides illustrated in Figure 3 and encompassed by SEQ ID NO 2, even exists in the sample tested. As a result, the illustration in Figure 3 is not a description of all possible sequences encompassed by SEQ ID NOs 1 and 2.
Second, the specification does not describe testing such variants to determine whether they would be capable of binding to BANP sufficient to regulate transcription of a heterologous gene. Given the strong preference for the motif represented by SEQ ID NO 3: TCTCGCGAGA, it would be unpredictable whether a nucleotide sequence comprised of a sequence comprising non-preferred nucleotides would be capable of binding to BANP sufficient to regulate transcription of a heterologous gene as required by the claims.
State of the art: As articulated for the enablement rejection, the state of the art is unpredictable in general, for a number of features in the claims, as discussed above, and particularly unpredictable for results arising from degenerate SEQID NO 1 and 2, for which no data is presented, nor is it known in the art in any reliable form.
Conclusion regarding possession: Taking into consideration the factors outlined above, including the nature of the invention, the state of the art, the guidance provided by the applicant and the specific examples, it is the conclusion that Applicant does not possess the genus of nucleotide sequences of SEQ ID NOs 1 and 2 operably linked to a heterologous gene as recited in the claims. There is not sufficient specific written example in the Specification that would lead one with ordinary skill in the art to a different conclusion. The null expectation from the art is for unpredictability, and change, in functionality, when modifying these species of motif.
Conclusion
All claims rejected.
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/LISA HORTH/ Examiner, Art Unit 1681
/NEIL P HAMMELL/ Supervisory Patent Examiner, Art Unit 1636