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 .
Applicant’s submission filed on July 24, 026 has been entered and considered. Rejections and/or objections not reiterated from the previous action mailed April 24, 2026 are hereby withdrawn. The following rejections and/or objections are either newly applied or are reiterated and are the only rejections and/or objections presently applied to the instant application. The text of those sections of Title 35, U.S. Code not included in this action can be found in a prior Office action.
Applicant has amended claim 1 to include the limitations as recited in previously dependent claim 3 pertaining to the Ca2+ binding domain “…wherein the Ca2+-sensing GvpC protein comprises a Ca2+-binding domain, wherein the Ca2+-binding domain is capable of binding 1, 2, 3, 4, 5, or 6 Ca2+ ions, wherein the Ca2+-sensing GvpC protein is capable of undergoing a first allosteric conformational change upon the Ca2+-binding domain binding Ca2+ that causes the Ca2+ sensing GV to change from a GV stiff state to a GV soft state, wherein the Ca2+-binding domain binding Ca2+ comprises the Ca2+_ binding domain binding 1, 2, 3, 4, 5, or 6 Ca2+ ions, wherein the Ca2+-sensing GvpC protein is capable of undergoing a second allosteric conformational change upon the Ca2+-binding domain releasing bound Ca2+ that causes the Ca2+-sensing GV to change from a GV soft state to a GV stiff state, wherein the Ca2+-binding domain releasing bound Ca2+ comprises the Ca2+-binding domain releasing 1, 2, 3, 4, 5, or 6 bound Ca2+ ions, wherein one or more of the mechanical and acoustic properties of the Ca2+ sensing GV differ between the GV soft state and the GV stiff state” and limitations as recited in previously dependent claim 6 pertaining to the interaction domain “…and wherein the Ca2+-sensing GvpC protein comprises an interaction domain configured to: bind the Ca2+-binding domain upon the Ca2+-binding domain binding Ca2+, wherein the first allosteric conformational change comprises the interaction domain binding the Ca2+-binding domain; and detach from the Ca2+-binding domain upon the Ca2+-binding domain releasing bound Ca2+, wherein the second allosteric conformational change comprises the interaction domain detaching from the Ca2+-binding domain.” Previously dependent claims 3 and 6 have been canceled.
Claims 1-2, 4-5, and 7-17 are examined on the merits.
Election/Restrictions
Applicant’s election without traverse of Group 1, claims 1-17, in the reply filed on February 13, 2026 is acknowledged.
Claims 18-20 were previously withdrawn from further consideration pursuant to 37 CFR 1.142(b) as being drawn to a nonelected groups, there being no allowable generic or linking claim.
Claim Objections
Claims 12, 16, and 17 are objected to because of the following informalities:
Claim 12 recites the abbreviation CaMKI in line 2 without first using the full spelling of the term. Upon first usage, terms should be recited by their full name followed by the abbreviation in parentheses. Appropriate correction is required.
Claim 16, which depends from claim 1, recites “The nucleic acid composition of claim 1, where in one or more GV polynucleotides comprises…” which appears to be missing an article before one or more GV polynucleotides. Appropriate correction is required. It is recommended that Applicant amend claim 16 to recite “the one or more GV polynucleotides”.
Claim 17 recites the abbreviations AAV in line 6, MVA in line 8, and VEE in line 10 without first using the full spelling of the term. Upon first usage, terms should be recited by their full name followed by the abbreviation in parentheses. Appropriate correction is required.
New Claim Rejections - 35 USC § 112(a) – Written Description
This is a new rejection necessitated by Applicant’s amendment. However, this rejection is based on a similar interpretation as set forth in the office action dated April 24, 2026 and provides additional interpretation of the claims as amended. Any aspect of Applicant’s traversal that pertains to the rejection as newly set forth will be provided following the new statement of rejection.
Claims 1-2, 4-5, and 7-17 are rejected under 35 U.S.C. 112(a) or 35 U.S.C. 112 (pre-AIA ), first paragraph, as failing to comply with the written description requirement. The claim(s) contains subject matter which was not described in the specification in such a way as to reasonably convey to one skilled in the relevant art that the inventor or a joint inventor, or for applications subject to pre-AIA 35 U.S.C. 112, the inventor(s), at the time the application was filed, had possession of the claimed invention.
Independent claim 1 encompasses a genus of nucleic acids comprising one or more promoters operably connected to one or more GV polynucleotides comprising one or more GVA genes encoding one or more GVA proteins; one or more GVS genes encoding one or more GVS proteins selected from GvpA and GvpB; and a Ca2+ sensing GvpC gene encoding a Ca2+ sensing GvpC protein wherein the one or more GVA and GVS proteins and the Ca2+-sensing GvpC protein are capable of forming Ca2+ sensing GVs in a cell or cell-like environment,
wherein the Ca2+-sensing GvpC protein comprises a Ca2+-binding domain capable of binding 1-6 Ca2+ ions, and wherein the Ca2+-sensing GvpC protein is capable of undergoing a first allosteric conformational change causing the GV to change from a GV “stiff state” to a GV “soft state” in response to 1-6 Ca2+ ion binding; and where in Ca2+-sensing GvpC protein is capable of undergoing a second allosteric conformational change in response to the Ca2+ binding domain releasing 1-6 bound Ca2+ ions causing the GV to change from a GV “soft state” to a GV “stiff state” and wherein one or more of the mechanical and acoustic properties differs between the GV soft state and GV stiff state,
and wherein the Ca2+ sensing GvpC protein comprises an interaction domain configured to bind the Ca2+ binding domain in response to the Ca2+ binding domain binding Ca2+ wherein the first allosteric conformational change comprises the interaction domain binding the Ca2+ binding domain, and detach from the Ca2+ binding domain upon release of bound Ca2+ ions from the Ca2+ binding domain wherein the second allosteric conformational change comprises the interaction domain detaching from the Ca2+ binding domain.
Dependent claim 2 encompasses a genus of nucleic acid compositions of claim 1 wherein the Ca2+ sensing GV comprises gas enclosed by a protein shell comprising the Ca2+ sensing GvpC protein and a GVS protein selected from GvpA and GvpB.
Dependent claim 4, encompasses a genus of nucleic acid compositions of claim 1 wherein the acoustic contrast of the Ca2+ sensing GV is capable of reversibly changing in response to Ca2+ concentrations.
Dependent claim 5, encompasses a genus of nucleic acid compositions of claim 1 wherein the Ca2+ sensing GV is capable of binding and releasing 1-6 Ca2+ ions recurrently in response to Ca2+ dynamics.
Dependent claim 7 encompasses a genus of nucleic acid compositions of claim 1 wherein the Ca2+ sensing GvpC protein is derived from Anabaena flos-aquae, Halobacterium salinarum, Halobacterium mediterranei, Microchaete diplosiphon, Nostoc sp., or a combination thereof, and wherein the Ca2+ sensing GvpC protein comprises one or more truncations, insertions, and mutations as compared to the parental GvpC protein from which it is derived.
Dependent claim 8 encompasses a genus of nucleic acid compositions of claim 1 wherein the Ca2+ sensing GvpC proteins comprising an amino acid sequence which is at least 90% identical to any one of SEQ ID NOs: 38 and 43-45.
Dependent claims 9 and 12 encompass genus of nucleic acid compositions of claim 1 comprising a genus of interaction domains comprising amino acid sequences which are at least 90% identical to any one of SEQ ID NOs: 46-51 and wherein the interaction domain comprises or is derived from CaMKI.
Dependent claims 10 and 11 encompass a genus of nucleic acid compositions of claim 1 comprising a genus of Ca2+ binding domains comprising an amino acid sequence which is at least 90% identical to any one of SEQ ID NOs: 52-58 and wherein the Ca2+ binding domain comprises calmodulin (CaM) or a derivative thereof.
Dependent claims 13-15 encompass physical properties and related indicators of alterations in acoustic contrast based on Ca2+ dependent allosteric conformational changes and changes between GV stiff and GV soft states of the Ca2+ sensing GVs produced by the genus of nucleic acid of claim 1.
Dependent claim 16 encompasses a genus of nucleic acid compositions of claim 1 wherein one or more GV polynucleotides comprises:
two or more GVS genes derived from different prokaryotic species;
GVA genes and/or GVS genes from Bacillus Megaterium, Anabaenaflos-aquae, Serratia sp., Bukholderia thailandensis, B. megaterium, Frankia sp, Haloferax mediaterranei, Halobacterium sp, Microchaete diplosiphon, Nostoc sp, Halorubrum vacuolatum, Microcystis aeruginosa, Methanosarcina barkeri, Streptomyces coelicolor, and/or Psychromonas ingrahamii;
gvpB, gvpN gvpF, gvpG, gvpL gvpS, gvpK, gvpJ, and/or gvpU from B. megaterium;
gvpA, gvpN, gvpJ, gvpK, gvpF, gvpG, gvpV, and/or gvpW from Anabaena flos-aquae;
gvpR, gvpN, gvpF, gvpG, gvpL, gvpS, gvpK, gvpJ, gvpT and/or gvpU from B. megaterium and gvpA from Anabaenaflos-aquae;
gvpA from Anabaena flos-aquae, and gvpN, gvpF, gvpG, gvpL, gvpS, gvpK, gvpJ, and/or gvpU from B. megaterium; and/or
gvpA and/or gvpN from Anabaena flos-aquae, and gvpF, gvpG, gvpL, gvpS, gvpK, gvpJ, and/or gvpU from B. megaterium.
Dependent claim 17 encompasses a genus of nucleic acid compositions of claim 1 wherein the nucleic acid composition is, comprises, or further comprises, one or more vectors, wherein at least one of the one or more vectors is a viral vector, a plasmid, a transposable element, a naked DNA vector, a lipid nanoparticle (LNP), or any combination thereof,
wherein the viral vector is an AAV vector, a lentivirus vector, a retrovirus vector, an adenovirus vector, a herpesvirus vector, a herpes simplex virus vector, a cytomegalovirus vector, a vaccinia virus vector, a MVA vector, a baculovirus vector, a vesicular stomatitis virus vector, a human papillomavirus vector, an avipox virus vector, a Sindbis virus vector, a VEE vector, a Measles virus vector, an influenza virus vector, a hepatitis B virus vector, an integration-deficient lentivirus (IDLV) vector, or any combination thereof, and
wherein the transposable element is piggybac transposon or sleeping beauty transposon.
The Court of Appeals for the Federal Circuit has held that a “written description of an invention involving a chemical genus, like a description of a chemical species, ‘requires a precise definition, such as by structure, formula [or] chemical name,’ of the claimed subject matter sufficient to distinguish it from other materials.” University of California v. Eli Lilly and Co., 1997 U.S. App. LEXIS 18221, at *23, quoting Fiers v. Revel, 25 USPQ2d 1601, 1606 (Fed. Cir. 1993). To fully describe a genus of genetic material, which is a chemical compound, applicants must (1) fully describe at least one species of the claimed genus sufficient to represent said genus whereby a skilled artisan, in view of the prior art, could predict the structure of other species encompassed by the claimed genus and (2) identify the common characteristics of the claimed molecules, e.g., structure, physical and/or chemical characteristics, functional characteristics when coupled with a known or disclosed correlation between function and structure, or a combination of these (paraphrased from Enzo Biochemical).
“[T]he purpose of the written description requirement is to ‘ensure that the scope of the right to exclude, as set forth in the claims, does not overreach the scope of the inventor’s contribution to the field of art as described in the patent specification.’” Ariad Pharm., Inc. v. Eli Lilly & Co., 598 F.3d 1336, 1353-54 (Fed. Cir. 2010) (en banc) (quoting Univ. of Rochester v. G.D. Searle & Co., 358 F.3d 916, 920 (Fed. Cir. 2004)). To satisfy the written description requirement, the 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. Vas-Cath, Inc. v. Mahurkar, 935 F.2d 1555, 1562-63, 19 USPQ2d 1111 (Fed. Cir. 1991). See also MPEP 2163.04.
ACTUAL REDUCTION TO PRACTICE
In regard to claim 1 encompasses a genus of nucleic acids comprising one or more promoters operably connected to one or more GV polynucleotides comprising one or more GVA genes encoding one or more GVA proteins; one or more GVS genes encoding one or more GVS proteins selected from GvpA and GvpB; and a Ca2+ sensing GvpC gene encoding a Ca2+ sensing GvpC protein wherein the one or more GVA and GVS proteins and the Ca2+-sensing GvpC protein are capable of forming Ca2+ sensing GVs in a cell or cell-like environment,
wherein the Ca2+-sensing GvpC protein comprises a Ca2+-binding domain capable of binding 1-6 Ca2+ ions, and wherein the Ca2+-sensing GvpC protein is capable of undergoing a first allosteric conformational change causing the GV to change from a GV “stiff state” to a GV “soft state” in response to 1-6 Ca2+ ion binding; and where in Ca2+-sensing GvpC protein is capable of undergoing a second allosteric conformational change in response to the Ca2+ binding domain releasing 1-6 bound Ca2+ ions causing the GV to change from a GV “soft state” to a GV “stiff state” and wherein one or more of the mechanical and acoustic properties differs between the GV soft state and GV stiff state,
and wherein the Ca2+ sensing GvpC protein comprises an interaction domain configured to bind the Ca2+ binding domain in response to the Ca2+ binding domain binding Ca2+ wherein the first allosteric conformational change comprises the interaction domain binding the Ca2+ binding domain, and detach from the Ca2+ binding domain upon release of bound Ca2+ ions from the Ca2+ binding domain wherein the second allosteric conformational change comprises the interaction domain detaching from the Ca2+ binding domain, the specification details four embodiments of nucleic acids comprising Ca2+ sensing GvpC proteins, i.e. UGECIs where the modified GvpC genes correspond to SEQ ID NOs: 38 and 43-45 (i.e., UGECIs 281, 141, 241, and 381, respectively, see Table 1).
Regarding the Ca2+ binding domain capable of binding and releasing 1-6 Ca2+ ions and undergoing reversible allosteric conformational changes due to binding to and detaching from an interaction domain which changes GVs from soft to stiff states, the specification discloses exemplary SEQ ID NOs: of 52-58 (Table 1) corresponding to the Ca2+ binding domain. However,
Applicant has not clearly disclosed which of the SEQ ID NOs of Ca2+ binding domains were reduced to practice related to functional ability, beyond reference that the “proof-of-concept biosensor” has “a mutant human CaM” attached at the C terminus (Para. [0188]). Likewise, since the Ca2+ binding domains of the instant invention are not sufficiently described, the number of Ca2+ ions capable of being bound is also not sufficiently described. Applicant has demonstrated reversibility based on Ca2+ concentration for only one embodiment (See Fig. 2E), which based on Applicant’s disclosure that Fig. 2E represents the “first generation of UGECI” (Para. [0188]), is understood to be related to expression of the original UGECI, UGECI-281 (Para. [0192]) corresponding to the GvpC represented by SEQ ID NO: 38 which comprises the Ca2+ binding domain of SEQ ID NO: 55, mutant calmodulin CaM6f-EF1 (see Table 1). UGECIs 241 (SEQ ID NO: 44) and 381 (SEQ ID NO: 45) also appear to comprise the Ca2+ binding domain of SEQ ID NO: 55 (CaM6f-EF1) while UGECI 141 appears to comprise the Ca2+ binding domain of SEQ ID NO: 57, also a mutant calmodulin (CaM6f-EF3). Applicant has not disclosed or reduced to practice any other Ca2+ binding domains.
Regarding the interaction domain which is able to reversibly bind and detach the Ca2+ binding domain in response to the Ca2+ binding domain binding 1-6 Ca2+ ions, Applicant has disclosed exemplary interaction domains having SEQ ID NOs: 46-51, with the interaction domain of SEQ ID NO: 50 found in UGECI-281 (SEQ ID NO: 38) and UGECI-241 (SEQ ID NO: 44), (“CBP2”, which based on the specification is “part of CaMKI”), the interaction domain of SEQ ID NO: 49 found in UGECI-141 (SEQ ID NO: 43) (“CPB1”, which is modified from M13-skMLCK), and the interaction domain of SEQ ID NO: 5I found in UGECI-381 (SEQ ID NO: 45) (“CBP3”, modified from M13-skMLCK) (See Table 1).
In regard to dependent claim 2 encompassing a genus of nucleic acid compositions of claim 1 wherein the Ca2+ sensing GV comprises gas enclosed by a protein shell comprising the Ca2+ sensing GvpC protein and a GVS protein selected from GvpA and GvpB, the instant specification discloses that the exemplary Ca2+ sensing GVs comprise a GvpA protein, specifically from Anabaena flos-aquae (Para. [0190]).
In regard to dependent claim 4, encompassing a genus of nucleic acid compositions of claim 1 wherein the acoustic contrast of the Ca2+ sensing GV is capable of reversibly changing in response to Ca2+ concentrations, as detailed supra, Applicant has only demonstrated reversibility for a single embodiment of the Ca2+ sensing GV, which is understood to correspond to UGECI-281 having SEQ ID NO: 38 (See Fig. 2E).
In regard to dependent claim 5, encompassing a genus of nucleic acid compositions of claim 1 wherein the Ca2+ sensing GV is capable of binding and releasing 1-6 Ca2+ ions recurrently in response to Ca2+ dynamics, as detailed supra, Applicant has provided exemplary embodiments of SEQ ID NOs corresponding to Ca2+ sensing GVs (SEQ ID NO: 38, 43-45). These exemplary Ca2+ sensing GVs comprise Ca2+ binding domains as represented by SEQ ID NO: 55 or SEQ ID NO: 57, both of which appear to be a modified calmodulin (See Table 1) which would be understood by a skilled artisan to be capable of binding of up to four Ca2+ ions. The specification is silent as to the number of Ca2+ ions the modified calmodulin Ca2+ binding domains are capable of binding as well as whether binding of less than four Ca2+ ions is sufficient in order to result in the functional ability to interact with an interaction domain in order to affect conformational change.
In regard to dependent claim 7 encompassing a genus of nucleic acid compositions of claim 1 wherein the Ca2+ sensing GvpC protein is derived from Anabaena flos-aquae, Halobacterium salinarum, Halobacterium mediterranei, Microchaete diplosiphon, Nostoc sp., or a combination thereof, and wherein the Ca2+ sensing GvpC protein comprises one or more truncations, insertions, and mutations as compared to the parental GvpC protein from which it is derived, the instant specification discloses use of a truncated three-repeat GvpC protein derived from the native five-repeat GvpC protein of Anabaena flos-aquae and, specifically, the interaction domain is incorporated into the second repeat. (Para. [0188]).
In regard to dependent claim 8, encompassing a genus of Ca2+ sensing GvpC proteins comprising an amino acid sequence which is at least 90% identical to any one of SEQ ID NOs: 38 and 43-45 or a portion thereof. As detailed above, Applicant discloses a fixed number of Ca2+ sensing GvpC proteins corresponding to SEQ ID NOs: 38 and 43-45.
In regard to dependent claims 9 and 12, encompassing a genus of interaction domains comprising amino acid sequences which are at least 90% identical to any one of SEQ ID NOs: 46-51 (claim 9) or which comprises or is derived from CaMKI (claim 12), as stated supra, Applicant has disclosed a fixed number of interaction domains, SEQ ID NOs: SEQ ID NOs: 46-51, with the interaction domain of SEQ ID NO: 50 found in UGECI-281 (SEQ ID NO: 38) and UGECI-241 (SEQ ID NO: 44), (“CBP2”, which based on the specification is “part of CaMKI”), the interaction domain of SEQ ID NO: 49 found in UGECI-141 (SEQ ID NO: 43) (“CPB1”, which is modified from M13-skMLCK), and the interaction domain of SEQ ID NO: 5I found in UGECI-381 (SEQ ID NO: 45) (“CBP3”, modified from M13-skMLCK) (See Table 1).
In regard to dependent claims 10 and 11, encompassing a genus of Ca2+ binding domains comprising an amino acid sequence which is at least 90% identical to any one of SEQ ID NOs: 52-58 (claim 10) or which comprises calmodulin (CaM) (claim 11) or a derivative thereof, as stated supra, Applicant has not clearly disclosed which of the SEQ ID NOs of Ca2+ binding domains were reduced to practice related to functional ability, beyond reference that the “proof-of-concept biosensor” has “a mutant human CaM” attached at the C terminus (Para. [0188]). Likewise, since the Ca2+ binding domains of the instant invention are unclear, the number of Ca2+ ions capable of being bound is also unclear. Applicant has demonstrated reversibility based on Ca2+ concentration for only one embodiment (See Fig. 2E), which based on Applicant’s disclosure that Fig. 2E represents the “first generation of UGECI” (Para. [0188]), is understood to be related to expression of the original UGECI, UGECI-281 (Para. [0192]) corresponding to the GvpC represented by SEQ ID NO: 38 which comprises the Ca2+ binding domain of SEQ ID NO: 55, mutant calmodulin CaM6f-EF1 (see Table 1). UGECIs 241 (SEQ ID NO: 44) and 381 (SEQ ID NO: 45) also appear to comprise the Ca2+ binding domain of SEQ ID NO: 55 (CaM6f-EF1) while UGECI 141 appears to comprise the Ca2+ binding domain of SEQ ID NO: 57, also a mutant calmodulin (CaM6f-EF3).
In regard to dependent claims 13-15, encompassing physical properties and indicators of alterations in acoustic contrast related to Ca2+ dependent allosteric conformational changes and changes between GV stiff and GV soft states of the Ca2+ sensing GVs, Applicant has disclosed stiffness measurements for all exemplary UGECIs (See Fig. 4A) and alterations in nonlinear contrast and contrast to noise ratio for SEQ ID NO: 38 (see Figs. 2E-F 3C-D).
In regard to dependent claim 16 encompassing a genus of nucleic acid compositions of claim 1 wherein one or more GV polynucleotides comprises:
two or more GVS genes derived from different prokaryotic species;
GVA genes and/or GVS genes from Bacillus Megaterium, Anabaenaflos-aquae, Serratia sp., Bukholderia thailandensis, B. megaterium, Frankia sp, Haloferax mediaterranei, Halobacterium sp, Microchaete diplosiphon, Nostoc sp, Halorubrum vacuolatum, Microcystis aeruginosa, Methanosarcina barkeri, Streptomyces coelicolor, and/or Psychromonas ingrahamii;
gvpB, gvpN gvpF, gvpG, gvpL gvpS, gvpK, gvpJ, and/or gvpU from B. megaterium;
gvpA, gvpN, gvpJ, gvpK, gvpF, gvpG, gvpV, and/or gvpW from Anabaena flos-aquae;
gvpR, gvpN, gvpF, gvpG, gvpL, gvpS, gvpK, gvpJ, gvpT and/or gvpU from B. megaterium and gvpA from Anabaenaflos-aquae;
gvpA from Anabaena flos-aquae, and gvpN, gvpF, gvpG, gvpL, gvpS, gvpK, gvpJ, and/or gvpU from B. megaterium; and/or
gvpA and/or gvpN from Anabaena flos-aquae, and gvpF, gvpG, gvpL, gvpS, gvpK, gvpJ, and/or gvpU from B. megaterium,
Applicant has disclosed UGECIs which comprise a gene encoding a modified GvpC protein derived from Anabaena flos-aquae (Para. [0188]), a GvpA gene from Anabaena flos-aquae, as well as other GV encoding genes from Anabaena flos-aquae: GvpN, GvpJ, GvpK, GvpF, GvpG, GvpW and GvpV (Para. [0190]).
In regard to dependent claim 17 encompassing a genus of nucleic acid compositions of claim 1 wherein the nucleic acid composition is, comprises, or further comprises, one or more vectors, wherein at least one of the one or more vectors is a viral vector, a plasmid, a transposable element, a naked DNA vector, a lipid nanoparticle (LNP), or any combination thereof,
wherein the viral vector is an AAV vector, a lentivirus vector, a retrovirus vector, an adenovirus vector, a herpesvirus vector, a herpes simplex virus vector, a cytomegalovirus vector, a vaccinia virus vector, a MVA vector, a baculovirus vector, a vesicular stomatitis virus vector, a human papillomavirus vector, an avipox virus vector, a Sindbis virus vector, a VEE vector, a Measles virus vector, an influenza virus vector, a hepatitis B virus vector, an integration-deficient lentivirus (IDLV) vector, or any combination thereof, and
wherein the transposable element is piggybac transposon or sleeping beauty transposon,
Applicant discloses use of a 3-plasmid system (Para. [0190]) for transient transfection (Para. [0193]) in order to test UGECI in mammalian cells.
However, as stated supra, Applicant was only in possession of a limited number of nucleic acid compositions comprising Ca2+ sensing GvpC proteins, Ca2+ binding domains, and interaction domains (see Table 1 of the instant specification).
DISCLOSURE OF STRUCTURE
The Applicant has provided sequence listings of SEQ ID NOs for four embodiments of the nucleic acid compositions comprising Ca2+ sensing GvpC proteins (SEQ ID NOs: 38 and 43-45), which comprise several embodiments of interaction domains (SEQ ID NOs: 49-51) and several embodiments of Ca2+ binding domains (SEQ ID NO: 55 and 57) (See Table 1). Certainly, with the help of a computer, a skilled artisan could identify the sequences of the GvpCs, Ca2+ binding domains, and interaction domains yet the prior art is silent as to being able to determine which combinations of GvpCs, Ca2+ binding domains, and interaction domains and alterations thereof also result in alterations of mechanical and acoustic properties related to the GvpCs making up the GV “shell”.
Furthermore, neither the specification nor the art indicate a relationship between the structure of the claimed genus of nucleic acid compositions comprising Ca2+ sensing GvpCs, Ca2+ binding domains, and interaction domains; and the ability to produce Ca2+ sensing GVs which are capable of reversibly changing from a stiff to soft state based on 1-6 Ca2+ ions binding to a Ca2+ binding domain causing interaction with an interaction domain which results in a reversible allosteric conformational change of a Ca2+ sensing GvpC protein and the resultant mechanical and acoustic properties based on the change from GV stiff to GV soft states.
SUFFICIENT RELEVANT IDENTIFYING CHARACTERISTICS
As mentioned in above, the complete amino acid sequence corresponding to the SEQ ID NOs of several variant GvpC proteins which each comprise specific SEQ IN NOs corresponding to variant interaction domains and Ca2+ binding domains which are able to produce a Ca2+ sensing GV when a Ca2+ binding domain is present which result in changes to GV stiffness in the presence of increased Ca2+ are provided.
Although instant specification indicates 100 variants were tested (Para. [0189]) and use of a library of UGECI GvpCs comprising different Ca2+ binding motifs in different positions, Applicant has reduced to practice only four variant UGECIs: 281 (SEQ ID NO: 38), 141 (SEQ ID NO: 43), 241 (SEQ ID NO: 44), and 381 (SEQ ID NO: 45 ), each of which encompass specific Ca2+ binding domains and interaction domains, which appear to have alterations in mechanical stiffness in response to Ca2+ (See Fig. 4A). However, Applicant has shown reversibility for only the single embodiment comprising the GvpC of SEQ ID NO: 38 (See Fig. 2E).
Regarding the disclosed SEQ ID NOs of the Ca2+ sensing GvpC protein, Applicant’s instant claims encompass embodiments having 90% sequence identity to SEQ ID NOs: 38 and 43-45, which encompasses 30 or more amino alterations in the Ca2+ sensing GvpC while still maintaining the instantly claimed functional ability. Likewise, related to the Ca2+ binding domain, Applicant’s instant claims encompass embodiments having 90% sequence identity to SEQ ID NOs: 52-58, which encompasses alterations of 14 amino acids in modified calmodulins (CaM) while maintaining the functional ability to bind to 1-6 Ca2+ ions. Related to the interaction domains, Applicant’s instant claims encompass embodiments having 90% sequence identity to SEQ ID NOs: 46-51, which, for SEQ ID NOs 46-47 and 49-51 encompasses alterations of 1 or 2 amino acids for these modified calcium binding proteins, and for SEQ ID NO 48 encompasses alterations of 37 amino acids in CaMKI, while maintaining the functional ability to interact with the Ca2+ binding domain and cause allosteric conformational change of the GV. Applicant has not defined what 10% of these amino acid sequences can be altered and while still maintaining functional effect.
Further, all reduced to practice Ca2+ binding domains appear to be derived from calmodulin which a skilled artisan would expect to allow for binding of up to four Ca2+ ions. Applicant is silent as to the specific number of Ca2+ ions which the exemplary Ca2+ binding domains are able to bind and what number of bound or released Ca2+ ions would trigger the functional binding and detachment interaction between the Ca2+ binding domain and interaction domain which is required for alterations of mechanical and acoustic properties.
It is also noted that although Applicant’s claims indicate that GvpC genes and various combinations of GV genes from various species can be utilized (see claims 7 and 16), Applicant’s reduced to practice Ca2+ sensing GVs are derived from GV genes from a single species, Anabaena flos-aquae and utilizes a truncated three-repeat GvpC protein.
Thus, a skilled artisan would not be able, from the instant specification, to easily determine which combinations of GvpC protein, Ca2+ binding domain, and interaction domains could be used nor which amino acid alterations could be made while still maintaining the claimed functionality of GV formation, binding of Ca2+, interacting with an interaction domain, and undergoing an allosteric conformational change then releasing Ca2+ and returning to the original conformation in order to generate Ca2+ sensing GVs which can transition from stiff to soft states (based on binding/release of as little as 1 Ca2+ ion) thereby altering mechanical and acoustic properties of the GVs which can be detected via imaging methods (i.e., ultrasound).
Accordingly, if the skilled artisan sought to generate the claimed genus of nucleic acids compositions which produce the Ca2+ sensing GV having functional properties as instantly claimed, they would first need to know which sequences in the GvpC protein, Ca2+ binding domain, and interaction domain could be chosen and still be able to predictably produce a functional Ca2+ sensing GV which is able to undergo alterations in stiffness based on binding and release of 1-6 Ca2+ ions to the Ca2+ binding domain and binding/detachment from the interaction domain. Hence, based on the written description guidelines, the Examiner should conclude that the applicant was not in possession of the claimed genus of nucleic acids beyond the sequences described in the specification.
The breadth of the claims encompass a genus of nucleic acid compositions operably connected to any promoter; any GVA gene; any GvpA or GvpB gene; comprising any variant GvpC having any Ca2+ binding domain able to bind 1-6 Ca2+ ions and any interaction domain; which is capable of a reversible allosteric conformational change based on binding of as few as a single Ca2+ ion and in the presence of any concentration of Ca2+ which results in a transition from “stiff” GVs to “soft” GVs which alters of any mechanical and acoustic property of the GV which then is able to be detected by ultrasound. The present specification provides no guidance nor description to any rational in choosing the identified sequences, therefore the skilled artisan would not know what rational approach to take to make modifications with any predictable outcome on Ca2+ sensing properties based on reversible allosteric conformational change resulting in transition from stiff to soft state and resultant change in mechanical and acoustic properties. Therefore, it is incumbent on the applicant to provide this nexus between structure and function, in order to be given credit for possession of a larger genus of nucleic acid compositions related to those comprising these individual species. Otherwise, the Written Description guidelines suggest that the applicant is entitled to only the species specifically recited as having this activity. Moreover, even when several species are disclosed, these are not necessarily representative of the entire genus. AbbVie Deutschland GMBH v. Janssen Biotech, 111 USPQ2d 1780, 1790 (Fed. Cir. 2014) (“The ’128 and ’485 patents, however, only describe species of structurally similar antibodies that were derived from Joe-9. Although the number of the described species appears high quantitatively, the described species are all of the similar type and do not qualitatively represent other types of antibodies encompassed by the genus.”). Thus, when there is substantial variation within the genus, one must describe a sufficient variety of species to reflect the variation within the genus to provide a "representative number” of species.
An applicant may show that an invention is complete by disclosure of sufficiently detailed, relevant identifying characteristics which provide evidence that applicant was in possession of the claimed invention, i.e., complete or partial structure, other physical and/or chemical properties, functional characteristics when coupled with a known or disclosed correlation between function and structure, or some combination of such characteristics. Enzo Biochem, 323 F.3d at 964, 63 USPQ2d at 1613.
STATE OF THE ART & QUANTITY OF EXPERIMENTATION
The method of making the claimed invention is not well established. Although the generation of nucleic acid compositions is routine and conventional, one of skill in the art would neither expect nor predict the appropriate functioning of Ca2+ sensing GVs which are capable of reversibly transitioning between stiff and soft states due to allosteric conformational change which occurs based on 1-6 Ca2+ ions binding to a Ca2+ binding domain and subsequent interaction with an interaction domain which results in detectible changes in GV properties according to the claimed genus of nucleic acid compositions as broadly as currently claimed.
It is well known that even minor changes in the amino acid sequence can drastically alter protein function. With regard to Ca2+ binding, Haiech et al. (1991, Restoration of the calcium binding activity of mutant calmodulins toward normal by the presence of a calmodulin binding structure. J. of Bio. Chem., 266(6), 3427-3431) teaches that point mutations in CaM, including a single amino acid substitution, can completely abolish Ca2+ binding (Abstract, Pg. 3427, left col. 2nd para.). With regard to gas vesicle proteins, Strunk et al. (2011. Structural model of the gas vesicle protein GvpA and analysis of GvpA mutants in vivo. Molec. Microbio., 81(1), 56-68.) teaches that point mutations of critical amino acids results in lack of formation of gas vesicles (Pg. 61, right col., last col.). Additionally, Lakshmanan 2020 discloses that alterations of the GvpC amino acid sequence can alter GV mechanical features and that shortening or removing GvpC makes GV less rigid which allows for easier buckling under acoustic pressure (Pg. 988, bridging para. left and right col.). Since the reversible allosteric conformation which results in transition between GV stiff and GV soft states is based on GV buckling (See Fig. 2D), a skilled artisan would need to recognize that the ability to buckle reversibly would require a GvpC which is not too rigid or too soft, and which modifications could be predictably made in order to achieve the unexpected results of a Ca2+ sensing GvpC protein.
Applicant has claimed a genus of nucleic acid compositions which function as Ca2+ sensing GVs, yet the specification has only managed to identify very specific sequences that can be used in the formation of the aforementioned GVs. Independent of how these specific sequences were arrived upon by Applicant, a GvpC protein, Ca2+ binding domain, and interaction domain cannot have any position modified and predictably produce proteins having the instantly claimed functional ability. Because Applicant has no manner a priori to predict which GvpC protein, Ca2+ binding domain, and interaction domain can be modified and used to make a functioning Ca2+ sensing GV with the claimed properties, the genus of nucleic acid compositions claimed by Applicant cannot be predictably made or used by the ordinary artisan.
Not knowing, absent further experimentation and screening, which modifications affect function when, as set forth above, even a single change of an encoded amino acid can unpredictably affect structure and function, leads to one having no predictability or expectation of success for the function of any given modification to the Ca2+ sensing GvpC, Ca2+ binding domain, and interaction domain. Such random experimentation to identify at a later time what structure or variant or modification is or is not functional and is embraced by Applicant’s claims is undue experimentation. Furthermore, functionally defined genus claims (i.e., a Ca2+ sensing GV) can be inherently vulnerable to invalidity challenge for lack of written description support, especially in technology fields that are highly unpredictable, where it is difficult to establish a correlation between structure and function for the whole genus or to predict what would be covered by the functionally claimed genus. See ABBVIE DEUTSCHLAND GMBH & 2 CO. v. JANSSEN BIOTECH, INC., Appeals from the United States District Court for the District of Massachusetts in Nos. 09-CV-11340-FDS, 10-CV-40003-FDS, and 10-CV-40004-FDS, Judge F. Dennis Saylor, IV. See also Ariad, 598 F.3d at 1351 (“[T]he level of detail required to satisfy the written description requirement varies depending on the nature and scope of the claims and on the complexity and predictability of the relevant technology.”); see also Centocor Ortho Biotech, Inc. v. Abbott Labs., 636 F.3d 1341, 1352 (Fed. Cir. 2011) (noting the technical challenges in developing fully human antibodies of a known human protein).
CONCLUSION
Therefore, the examiner concludes that there is insufficient written description of the instantly claimed genus. Specifically, there is limited description of the structure-function relationship between the claimed genus of nucleic acid compositions comprising GVA genes/proteins; GVS genes/proteins selected from GvpA or GvpB; and Ca2+ sensing GvpC gene/proteins comprising Ca2+ binding domains and interaction domains; and their ability to produce functional Ca2+ sensing GVs which are able to reversibly change between “soft” and “stiff” states thereby altering mechanical and acoustic properties of the Ca2+ sensing GV and resulting in changes detectible by ultrasound, and the Examiner further concludes a skilled artisan would find the specification inadequately describes the nucleic acid compositions encompassed by the very large claimed genus of Ca2+ sensing GVs.
Response to Arguments
Applicant's arguments filed July 24, 2026 have been fully considered but they are not persuasive.
Claims 1, 3, 6, 9-12, and 14-15 were rejected under 35 U.S.C. 112(a) for failing to comply with written description.
Applicant has amended claim 1 to incorporate limitations of previously dependent claims 3 and 6 and, as such, the instantly claimed nucleic acid comprises one or more GVA genes, one or more GVS genes encoding GvpA or GvpB proteins, and a Ca2+ sensing GvpC gene encoding a Ca2+ sensing GvpC protein which are capable of forming a Ca2+ sensing GV upon expression in a cell or cell-like environment wherein the GvpC protein comprises a Ca2+ binding domain capable of binding 1-6 Ca2+ ions and an interaction domain which is configured to bind the Ca2+ binding domain upon Ca2+ binding and detach from the Ca2+ binding domain upon Ca2+ release and wherein this interaction causes an allosteric conformational change which is results in changes between GV soft and GV stiff states and related mechanical and acoustic properties. Applicant asserts that this amendment removes prior claim embodiments of Ca2+ sensing GvpC proteins which did not require both the interaction domain and a Ca2+ binding domain. Applicant traverses that the specification describes the claimed coupling mechanism of the interaction domain which is configured to bind the Ca2+ binding domain upon Ca2+ binding thereby causing the first allosteric conformational change, and to detach upon Ca2+ release thereby causing the second allosteric conformational change and that Example 1 supports the Ca2+ responsive mechanism and related acoustic alterations. Applicant further asserts that the disclosed working examples, architecture, and design guidance demonstrate possession of the Ca2+ responsive switching mechanism (Pg. 12).
Applicants’ traversal has been fully considered but it not persuasive.
As stated supra, although Applicant has amended to further clarify the instantly claimed Ca2+ sensing gas vesicle, there is still a high degree of unpredictability regarding the structure of the nucleic acids encoding the GV proteins, including the GvpC protein, the Ca2+ binding domain, and the interaction domain and the relationship between the nucleic acids and the claimed functional limitations as broadly as instantly claimed. Although Applicant discloses testing of over 100 variants (Para. [0189]) and screening of a library of UGECI (Para. [0191]), Applicant has disclosed only four embodiments of UGECI which each have different characteristics (Para. [0192]). Thus, it appears from the instant specification as well as the prior art that alterations in the amino acid sequences and framework of UGECIs can cause alterations in functional ability and it is not clear which particular nucleic acids encoding various portions of GVs could be pieced together within Applicant’s framework (See Fig. 2H) and still produce Ca2+ gas vesicles which are able to undergo reversible allosteric conformational changes which alters the GV mechanical and acoustic properties.
Conclusion
Applicant's amendment necessitated the new ground(s) of rejection presented in this Office action. Accordingly, THIS ACTION IS MADE FINAL. See MPEP § 706.07(a). Applicant is reminded of the extension of time policy as set forth in 37 CFR 1.136(a).
A shortened statutory period for reply to this final action is set to expire THREE MONTHS from the mailing date of this action. In the event a first reply is filed within TWO MONTHS of the mailing date of this final action and the advisory action is not mailed until after the end of the THREE-MONTH shortened statutory period, then the shortened statutory period will expire on the date the advisory action is mailed, and any nonprovisional extension fee (37 CFR 1.17(a)) pursuant to 37 CFR 1.136(a) will be calculated from the mailing date of the advisory action. In no event, however, will the statutory period for reply expire later than SIX MONTHS from the mailing date of this final action.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to ERIN V PAULUS whose telephone number is (571)272-6301. The examiner can normally be reached Mon-Fri 8 AM-5 PM.
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/ERIN V PAULUS/Examiner, Art Unit 1631
/ARTHUR S LEONARD/Examiner, Art Unit 1631
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