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 .
Change in Examiner
The examiner of your application in the PTO has changed. To aid in correlating any papers for this application, all further correspondence regarding this application should be directed to Stephanie Sullivan, Art Unit 1635.
Election/Restrictions
Applicant’s election without traverse of Group II (claims 54-56,60,63,67-69,71,83,84 and 92-94) in the reply filed on 03/06/2026 is acknowledged.
Claims 1,2,4,8,11,15-17,19,32,33,42-44,103,108,111,114-117,120,121,124 and 127 are withdrawn from further consideration pursuant to 37 CFR 1.142(b) as being drawn to a nonelected invention, there being no allowable generic or linking claim. Election was made without traverse in the reply filed on 03/06/2026.
Claims 54-56,60,63,67-69,71,83,84 and 92-94 are under examination.
Priority
This application is a 371 of PCT/US21/65682, filed 12/30/2021 which claims benefit of 63/132,316, filed 12/30/2020 and claims benefit of 63/179,083, filed 04/23/2021, and claims benefit of 63/208,556, filed 06/09/2021, and claims benefit of 63/270,388, filed 10/21/2021 as reflected by the most recent filing receipt.
Claim Objections
Claims 54,60,68,69,83,92 and 93 are objected to because of the following informalities: The above claims recite various acronyms, including KCNH2, IRES, P2A, LQTS, SQTS, LQT2, and APD which are not defined in the claims. When an acronym is used in a claim set, it should be defined the first time it appears in the claims.
The examiner is interpreting KCNH2 to be Potassium Voltage-Gated Channel Subfamily H Member 2, LQTS to be congenital long QT syndrome (page 1), SQTS to be short QT syndrome (page 2), , LQT2 to be long QT syndrome Type 2, APD to be action potential duration (page 6).
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 60 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 60 recites the limitation "said cDNA" in line 4. Claim 60 depends on claim 54 which does not recite cDNA. There is insufficient antecedent basis for this limitation in the claim.
Claim Rejections-Scope of Enablement
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 68,69,71,83,84 and 92-94 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 the in vitro treatment of cardiac cells having a mutation in KCNH2 (hERG) comprising administering the recited nucleic acid construct to the cardiac cells in vitro and resulting in suppression of the mutation in KCNH2 and replacement of the normal protein product of KCNH2, does not reasonably provide enablement for a method of treating a mammal having any congenital cardiac disease, reducing APD in cardiac cells within a mammal or reducing symptoms of LQTS in a mammal, the method comprising administering by any route of administration to the mammal the recited nucleic acid construct. The specification does not enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to use the invention commensurate in scope with these claims.
As stated in MPEP §2164.01(a), “there are many factors to consider when determining whether there is sufficient evidence to support a determination that a disclosure does not satisfy the enablement requirement and whether any experimentation is ‘undue’.” These factors include, but are not limited to:
1. The breadth of the claims;
2. The nature of the invention;
3. The state of the prior art;
4. The level of skill in the art;
5. The level of predictability in the art;
6. The amount of direction provided by the inventor;
7. The presence or absence of working examples;
8. The quantity of experimentation necessary needed to make or use the invention based on the disclosure.
See In re Wands USPQ 2d 1400 (CAFC 1988).
The Breadth of the Claims and The Nature of the Invention
Claim 68 encompasses treating any mammal, including humans, having any congenital heart disease by administering by any route of administration to the mammal a nucleic acid construct comprising any first nucleic acid sequence encoding any RNAi molecule capable of hybridizing to a target sequence encoding an endogenous KCNH2 polypeptide within any cell and suppressing expression of the endogenous KCNH2 polypeptide in the cell, and any second nucleic acid sequence encoding a KCNH2 polypeptide that comprises a target sequence identical to the target sequence of the first nucleotide sequence with the exception of 1-13 wobble position variants compared to the target sequence of the first nucleotide sequence, and wherein said RNAi molecule does not suppress expression of the KCNH2 polypeptide from the second nucleotide sequence within the cell. Claim 69 limits the congenital heart disease to LQTS, SQTS or LQT2. However, the instant specification discloses regarding a congenital heart disorder include, LQTS (e.g., LQT1, LQT2, LQT3, LQT4, LQT5, LQT6, LQT7, LQT8, LQT9, LQT10, LQT11, LQT12, LQT13, LQT14, LQT15, LQT16, or LQT17), Brugada syndrome (BrS), catecholaminergic polymorphic ventricular tachycardia (CPVT), arrhythmogenic cardiomyopathy (ACM), hypertrophic cardiomyopathy (HCM), dilated cardiomyopathy (DCM), SQTS, Timothy syndrome, left ventricular non-compaction cardiomyopathy (LVNC), skeletal myopathy, Andersen-Tawil syndrome (ATS), familial hypercholesterolemia (FH), cardiomyopathies, atrial fibrillation, and Triadin knockout syndrome (TKOS) (pages 29-30). Therefore, claims 68 and 71 encompass treating a large genus of congenital heart diseases in any mammal.
Claim 83 encompasses reducing APD in cardiac cells within a mammal by any route of administration to the mammal a nucleic acid construct comprising any first nucleic acid sequence encoding any RNAi molecule capable of hybridizing to a target sequence encoding an endogenous KCNH2 polypeptide within cardiac cells of the mammal and suppressing expression of the endogenous KCNH2 polypeptide in the cardiac cells, and any second nucleic acid sequence encoding a KCNH2 polypeptide that comprises a target sequence identical to the target sequence of the first nucleotide sequence with the exception of 1-13 wobble position variants compared to the target sequence of the first nucleotide sequence, and wherein said RNAi molecule does not suppress expression of the KCNH2 polypeptide from the second nucleotide sequence within the cell.
Claim 92 encompasses reducing one or more symptoms of LQTS in any mammal including humans, by any route of administration to the mammal a nucleic acid construct comprising any first nucleic acid sequence encoding any RNAi molecule capable of hybridizing to a target sequence encoding an endogenous KCNH2 polypeptide within any cell and suppressing expression of the endogenous KCNH2 polypeptide in the cell, and any second nucleic acid sequence encoding a KCNH2 polypeptide that comprises a target sequence identical to the target sequence of the first nucleotide sequence with the exception of 1-13 wobble position variants compared to the target sequence of the first nucleotide sequence, and wherein said RNAi molecule does not suppress expression of the KCNH2 polypeptide from the second nucleotide sequence within the cell and claim 93 limits the LQTS to LQT2.
The State of the Prior Art
Bezzerides et al. (Cardiovasc Res. 2020 April 22, 116 (9): 1635-1650) cited on an IDS, teach for cardiovascular gene therapy, the most commonly used viral vector is based on AAV that is not known to cause human disease and important strengths of rAAV are that they are safe, non-integrating, maintain expression for many years, and efficiently transduce many cell types, including cardiomyocytes (page 1638). Bezzerides et al. teach regarding oligonucleotides that can be used to perturb gene expression in target cells, modifications to selectively transduce hepatocytes are available, but efficient or selective targeting of most other parenchymal cell types, including cardiomyocytes is not yet possible (page 1639, left column).
Bezzerides et al. teach that regarding gene therapies for inherited arrhythmias, moving from proof-of-concept to clinical translation requires addressing several important challenges, including robust expression in the heart and minimal extra-cardiac expression are key factors for selection of promoters to direct cardiac gene therapy, but that the CMV promoter has widespread activity and cardiac targeting by AAV isotype selection is insufficient to restriction expression to the heart (page 1646, right column).
Bezzerides et al. teach that therapies that modulate cardiac rhythm carry the risk of pro-arrhythmia in the case of AAV gene therapy which will create inhomogeneities within the myocardium as a result of cardiomyocytes that have and have not been transduced by AAV, and activation of the innate immune response has been reported in large animals that have received large AAV doses (page 1646-1647). Challenges for cardiac gene therapy using viral vectors include demonstration of efficacy at transduction efficiencies achievable in humans, unforeseen off target effects, development of pro-arrhythmia, durability of therapy, and lack of reversibility after treatment remain important concerns for translation (Conclusion, page 1647).
Li et al. (Experimental and Therapeutic Medicine 10: 395-400, 2015, “ RNA interference-based therapeutics for inherited long QT syndrome (review)”), cited on an IDS, teach current treatment options for LQTS include the administration of β-adrenoceptor antagonists, the implantation of pacemakers or implantable cardioverter defibrillators (ICDs), and left cardiac sympathetic denervation (page 395, right column).
Li et al. teach a number of groups have reported the successful generation of iPSCs from inherited LQTS cells, and iPSCs derived from patients with LQTS may be differentiated into patient-specific iPSC-derived cardiomyocytes (iPS-CMs), offering a potentially unlimited source of materials for biomedical study. iPS-CMs may be used to recapitulate complex physiological phenotypes, probe toxicological testing and drug screening, clarify novel mechanistic insights and provide alternative strategies to rectify genetic defects at the cellular and molecular level. Human iPS-CMs have been used to investigate the disease-inducing biophysical mechanisms of LQT2-associated mutation in a hERG in vitro model, to evaluate gene-based therapeutics for the treatment of inherited LQTS (page 397, right column).
Li et al. teach there are numerous problems and barriers to be solved prior to the use of RNAi therapy in clinical practice, including specificity for the target gene, delivery to the correct cell or tissues, the duration of RNAi activity and the stability of the target mRNA and encoded protein. Efficient delivery to target cells and tissues is the primary challenge for RNAi-based therapeutics, and cardiac targeting has emerged as an extremely difficult task that has not been achievable in a simple and efficient manner using earlier gene transfer systems. A second key issue is the stability of the therapeutic vector. Small regulatory RNAs are inherently unstable, and viral vectors are currently the only available tools for non-topical in vivo therapy using these molecules. In addition, even assuming that the delivery problems can be solved, toxicity of small hairpin (sh)RNA may result from competition with the normal cellular miRNA processing system. shRNAs may disturb cellular miRNA pathways and thereby result in adverse effects. The off-target effects of regulatory RNAs are another possible source of toxicity and are difficult to assess and therefore the side-effects at the cellular level and in vivo require further investigation to assess the possible toxicity of the RNAi-based therapeutics (page 399, left column).
Wallace et al. (Pediatric Cardiology (22 Aug 2019) 40: 1419-1430), cited on an IDS, taught long QT syndrome (LQTS) is an inherited primary arrhythmia syndrome that may represent with malignant arrhythmia and rarely, risk of sudden death (Abstract). Wallace et al. taught LQT2 is the second most subtype affecting 25-30% of LQTS individuals, and hERG or KCNH2 codes for the voltage-gated pore forming alpha- subunit of the inwardly rectifying potassium channel subunit, and there are mutations that exert a dominant-negative effect and loss-of-function mutations (page 1420, right column). Wallace et al. teach standard of care therapy for LQT1,2 and 3 includes non-cardioselective beta-blockers (page 1422, right column). Wallace et al. teach the therapeutic potential of RNAi based therapeutics for LQTS, however taught it remains to be established if this technology works in vivo (page 1426, right column). Wallace et al. teach the ability to generate cardiomyocytes from skin biopsies using iPSC technology has enhanced the capacity to model LQTS phenotypes in vitro, and patient-specific iPSC-derived CMs offer the potential to understand individual disease processes and to potentially target in vitro models of genotype rescue using CRISPR/Cas9 editing technology, and recent advances in iPSC, and RNAi offer hope for development of gene-based therapies for treatment of LQTS but substantial challenges remain to be overcome (Conclusion, page 1427).
Therefore, the state of the art shows promise for RNAi and gene therapy in vivo however there are challenges and problems that must be addressed, including for cardiac gene therapy using viral vectors include demonstration of efficacy at transduction efficiencies achievable in humans, unforeseen off target effects, development of pro-arrhythmia, durability of therapy, and lack of reversibility after treatment remain important concerns for translation, and for RNAi, efficient delivery to target cells and tissues is the primary challenge for RNAi-based therapeutics, and cardiac targeting has emerged as an extremely difficult task that has not been achievable in a simple and efficient manner.
The Level of Predictability in the Art
The instant claimed invention is highly unpredictable due to the claims encompassing that the recited method occurs in any mammal, including humans, and that the nucleic acid construct is administered by any route of administration, and claims 68,69,71 and 92-94 encompass any cell of said mammal. In addition, it is noted that none of the method claims recite using a particular viral vector or a specific promoter that would help target the nucleic acid construct to the right cells (cardiac cells).
It would be unpredictable that oral or topical administration would result in the nucleic acid construct as being delivered to the appropriate target cells, as KCNH2 is only expressed in the heart, and therefore only certain routes of administration and/or using particular vectors and serotypes known to target heart tissue would be predictable as being able to target cells in the heart to suppress and replace the KCNH2 gene. In addition, the state of the art cited above, teach that challenges regarding treatment of LQTS with RNAi and gene-based therapies remain to be overcome, including for cardiac gene therapy using viral vectors include demonstration of efficacy at transduction efficiencies achievable in humans and efficient delivery to target cells and tissues is the primary challenge for RNAi-based therapeutics, and cardiac targeting has emerged as an extremely difficult task. Therefore, the level of predictability of treating a mammal having a congenital heart disease, reducing APD in cardiac cells within a mammal and reducing symptoms of LQTS in a mammal with the recited method remains highly unpredictable, particularly in humans.
The Amount of Direction Provided by the Inventor and
The Presence or Absence of Working Examples
Regarding claims 68,69,71,83,84 and 92-94, the specification does not enable any person skilled in the art to which it pertains to make and/or use the invention commensurate in scope with the claims. The instant specification discloses the RNAi nucleic acid can be shRNA, siRNA or miRNA and can have any appropriate length, and can target a region of disease associated allele that does not contain the pathogenic mutation or other genetic polymorphisms, or can target the region of disease associated allele that contains the pathogenic mutations (page 30). The instant specification discloses the suppressive component/corrective component combinations and the suppressive component can be designed to target different regions including the 5’ or 3’ UTR since the corrective component does not need to contain the UTRs but endogenous transcription of mRNA does contain the UTRs, and the corrective component does not need to contain silent variants since the suppressive component is targeted to a UTR. The suppressive component can also target a sequence near the 5’ or 3’ end of the coding sequence and the corrective component can include a truncated cDNA that does not contain the sequence targeted by the suppressive component (page 31).
Table 1B on pages 35-36 shows examples of shRNA and shIMM sequences targeted to KCHN2 as well as the KCHN2 sequence of NCBI NM_000238. SEQ ID NOs: 27 and 29 are included in the table among other sequences.
Examples 12-18 pertain to materials and methods for LQT2 SupRep, and Example 12 on page 168 shows that for the final KCNH2-SupRep gene therapy vector, KCNH2 sh#4 was selected as the lead candidate and is referred to as shKCNH2 and then a DNA fragment containing 10 synonymous variants within the KCNH2 sh#4 target sequence of the KCNH2-WT cDNA was synthesized. Example 13 discloses 17 shRNAs targeting KCNH2 were tested and one shRNA candidate (SEQ ID NO: 27) was identified that suppressed KCNH2 alleles with 80% knockdown efficiency (FIG. 22), and then a SupRep construct containing the shRNA and an shRNA-immune (SEQ ID NO: 29) was generated as a version of the KCNH2 cDNA and the shIMM sequence had alterations at the wobble base of each codon within the shRNA target sequence.
Example 14 discloses in vitro treatment and testing of iPSC-CMs having different mutations with the KCNH2-SupRep regarding cardiac APD. Results for some of the variants resulted in overcorrection in APD shortening (pages 177-178).
Example 16 discloses testing of the KCNH2-SupRep gene therapy on if it knocked down and replaced KCNH2 in a variant independent manner in TSA201 cells (human kidney cell line), with results showing that KCNH2-SupRep knocked down LQT2 disease-causing KCNH2 missense variants and replaced them with KCNH2-shIMM (page 179-180).
Example 18 discloses showing that KCNH2-SupRep can rescue both LQT2 and short QT (SQT1) disease phenotypes in iPSC-CMs, and treatment of SQT1 iPSC-CMs with KCNH2-SupRep resulted in APD90 prolongation and for KCNH2-N588K, KCNH2 Sup-Rep corrected the shortened APD90 and APD50 as compared to isogenic controls treated with shCT (page 181).
Therefore, there are no examples pertaining to the recited methods occurring in vivo in any mammal, including humans. As the examples only pertain to in vitro testing, and with the unpredictability shown in the state of the art with regards to the recited methods being carried out in vivo in any mammal including humans, the specification does not enable the methods as recited.
The Quantity of Experimentation Necessary
Regarding claims 68,69,71,83,84 and 92-94, in light of the unpredictability surrounding the breadth of the claimed method, one wishing to practice the presently claimed invention would be unable to do so without engaging in undue experimentation. In vivo experiments would need to be performed in mammalian animal models, and therefore disease appropriate animal models would need to be generated and testing of different suppression-replacement constructs in different variants in KCNH2 would be needed. Given the unpredictability in the state of the art regarding the ability to treat humans with gene suppression and replacement therapy in general, and given the lack of guidance present in the specification for the recited method, further experimentation would be required and would be considered undue.
Conclusion of 35 U.S.C. 112(a) (Enablement) Analysis
After applying the Wands factors and analysis to claims 68,69,71,83,84 and 92-94, in view of the applicant’s entire disclosure, it is concluded that the specification is not enabled for the full scope as discussed above. Therefore, claims 68,69,71,83,84 and 92-94 are rejected under 35 U.S.C. §112(a) for failing to disclose sufficient information to enable a person of skill in the art to use the invention commensurate in scope with these claims.
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.
The factual inquiries for establishing a background for determining obviousness under 35 U.S.C. 103 are summarized as follows:
1. Determining the scope and contents of the prior art.
2. Ascertaining the differences between the prior art and the claims at issue.
3. Resolving the level of ordinary skill in the pertinent art.
4. Considering objective evidence present in the application indicating obviousness or nonobviousness.
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.
Claim Interpretation
“KCNH2” and “hERG” are the same genes and used synonymously. In the interest of compact prosecution, as claims 68,69,71,83,84 and 92-94 as instantly recited are not enabled as explained above in the Scope of Enablement rejection, the examiner is interpreting claims 68,69,71,83,84 and 92-94 for the purpose of art rejections as an in vitro method of suppressing and replacing KCNH2 in cardiac cells comprising administering the recited nucleic acid construct to cardiac cells in vitro.
Claims 54,56,60,63,68,69,83,92 and 93 are rejected under 35 U.S.C. 103 as being unpatentable over Farrar et al. (US 20040234999, Published 25 Nov. 2004) in view of Shim et al. (US 20160033481, Published 4 Feb 2016) and Keating et al. (US 20010034024, Published 25 Oct 2001).
Regarding claim 54, Farrar et al. teach compositions and methods for gene suppression and replacement that exploit the degeneracy of the genetic code, thereby circumventing the difficulties and expenses associated with the need to specifically target disease mutations. In particular, the invention relates to suppression of the expression of mutated genes that give rise to a dominant or deleterious effect or contributes towards a disease. Farrar et al. teach a suppression effector targets either the disease allele or normal allele or targets both the disease allele and normal allele. In a particular embodiment of the invention, a replacement nucleic acid is provided that is altered at one or more degenerate or wobble bases from the endogenous wild type gene but will code for the identical amino acids as the wild type gene. In another embodiment, the replacement nucleic acid encodes a beneficial replacement nucleic acid (e.g., which encodes a more active or stable product than that encoded by the wild-type gene). The replacement nucleic acid provides expression of the normal protein product when required to ameliorate pathology associated with reduced levels of wild type protein. The same replacement nucleic acid can be used in conjunction with the suppression of many different disease mutations within a given gene (paragraph 0010).
Farrar et al. teach the strategy circumvents the need for a specific therapy for every disease-causing mutation within a given gene, and the invention has the advantage that the same suppression effector can be used to suppress many mutations in a gene, which is particularly relevant when any one of a large number of mutations within a single gene can cause disease pathology. The compositions and methods of the invention allow greater flexibility in choice of target sequence for suppression of expression of a disease allele (paragraph 0012).
Farrar et al. teach the replacement nucleic acid encodes a wild-type or non-disease causing allele that comprises at least one degenerate/wobble nucleotide that is altered so that the replacement nucleic acid is not suppressed, or is only partially suppressed, by the siRNA (paragraph 0019). Farrar et al. teach RNAi can be used to suppress expression of the target nucleic acid, and a replacement nucleic acid is provided that is altered around the RNAi target site at degenerate (wobble) positions such that it escapes suppression by the RNAi at least in part but the amino acid sequence it encodes is normal. Replacement nucleic acids thereby escape, at least in part, suppression by the RNAi. The sequence specificity of RNAi suppression may be dependent on the individual structures of siRNA molecules and their targets (paragraph 0123). Farrar et al. teach the suppression effector and replacement nucleic acid are operatively linked to an expression vector, such as a bacterial or viral expression vector (paragraph 0021).
Farrar et al. do not teach the suppression effector (first nucleotide sequence) which can be siRNA targets an endogenous KCNH2 polypeptide within a cell, and do not teach the replacement nucleic acid (second nucleotide sequence) encodes a KCNH2 polypeptide.
Before the effective filing date, Shim et al. taught genetic disorders caused by mutations in ion channel subunits, including hereditary long QT syndrome (LQTS) caused by mutations in one or more ion channels expressed in the heart, and that LQTS is characterized by delayed or prolonged cardiac repolarization in electrocardiogram with increased risks of developing polymorphic ventricular tachycardia (torsade de pointes, TdP), syncope and sudden cardiac death. To date, LQTS has been associated with over 500 different mutations in at least 13 genes encoding cardiac ion channel proteins (paragraph 0002). Shim et al. taught LQTS2 implicates hERG (human ether-a-go-go related gene), a gene (KCNH2) that codes for a protein known as Kv11.1, which constitutes the pore-forming α subunit of the rapidly-activating delayed rectifier potassium current (IKr). Heterozygote KCNH2 mutations exert a dominant-negative effect on wild-type hERG channel associated IKr currents by impaired trafficking pathways or altered channel kinetics of the resulting co-assembled hERG heterotetramers (paragraph 0003). Shim et al. taught the terms “hERG”, “KCNH2” and “Kv11.1” may be used interchangeably in the context of the invention (paragraph 0044). Shim et al. taught an hERG gene modulator is hERG gene-specific siRNA (paragraph 0079).
Keating et al. taught long QT syndrome (LQT), has been associated with specific genes including HERG, SCN5A, KVLQT1 and KCNE1, may be hereditary and due to specific mutations in the above genes or it may be acquired. It had previously been shown that the HERG gene encodes a K+ channel which is involved in the acquired form of LQT (Paragraph 0003). Keating et al. also taught gene therapy by supplying wild-type HERG function to a cell which carries mutant HERG alleles, which should allow normal functioning of the recipient cells. The wild-type gene or a part of the gene may be introduced into the cell in a vector such that the gene remains extrachromosomal (paragraph 0193). Keating et al. taught the HERG gene or fragment may be employed in gene therapy methods in order to increase the amount of the expression products of such genes in cells. It may also be useful to increase the level of expression of a given LQT gene even in those heart cells in which the mutant gene is expressed at a “normal” level, but the gene product is not fully functional (paragraph 0193).
Regarding claim 56, Farrar et al. teach cDNA encoding human rhodopsin expressed from a T7 promoter (paragraph 0026-0028) and vectors/plasmids containing an siRNA targeting a target gene and driven by a promoter (H1 promoter) (paragraphs 0076, 0241, 0243).
Regarding claim 60, Farrar et al. teach a replacement gene cloned in a vector from which fusion transcripts carrying both the target sequence and a reporter gene (EGFP) sequence are transcribed and suppression was evaluated using the enhanced green fluorescent protein as a marker (paragraph 0073)..
Regarding claim 63, Farrar et al. teach vectors containing one or more suppression effectors in the form of nucleic acids that target coding sequence(s) or combinations of coding and non-coding sequences of a target nucleic acid and a vector containing a replacement nucleic acid sequence to which nucleic acids for suppression are unable to bind, and vectors can be DNA or RNA vectors derived from viruses, and exemplary viral vectors that may be used in the practice of the invention include those derived from adenovirus, and adenoassociated virus (AAV) (paragraph 0089).
Regarding claims 68,69,83,92 and 93, Farrar et al. teach co-transfection of targeting and RNAi in COS-7 cells including co-transfection of the COL1A1 target and siRNA targeting COL1A1, and that although siRNA has been used in the demonstration of the invention, other suppression effectors could be used to achieve gene silencing. Again replacement nucleic acids with altered sequences (at wobble positions) could be generated so that transcripts from these constructs are protected partially or completely from gene silencing and provide the wild type (or beneficial) gene product. (paragraph 0232), and therefore teaches administering a suppression and replacement construct to cells in vitro.
Farrar et al. do not teach administering the suppression-replacement construct to cardiac cells, or wherein the suppression effector (first nucleotide sequence) which can be siRNA targets an endogenous KCNH2 polypeptide within a cell, and wherein the replacement nucleic acid (second nucleotide sequence) encodes a KCNH2 polypeptide.
Before the effective filing date, Shim et al. taught genetic disorders caused by mutations in ion channel subunits, including hereditary long QT syndrome (LQTS) caused by mutations in one or more ion channels expressed in the heart, and that LQTS is characterized by delayed or prolonged cardiac repolarization in electrocardiogram with increased risks of developing polymorphic ventricular tachycardia (torsade de pointes, TdP), syncope and sudden cardiac death. To date, LQTS has been associated with over 500 different mutations in at least 13 genes encoding cardiac ion channel proteins (paragraph 0002). Shim et al. taught LQTS2 implicates hERG (human ether-a-go-go related gene), a gene (KCNH2) that codes for a protein known as Kv11.1, which constitutes the pore-forming α subunit of the rapidly-activating delayed rectifier potassium current (IKr). Heterozygote KCNH2 mutations exert a dominant-negative effect on wild-type hERG channel associated IKr currents by impaired trafficking pathways or altered channel kinetics of the resulting co-assembled hERG heterotetramers (paragraph 0003). Shim et al. taught the terms “hERG”, “KCNH2” and “Kv11.1” may be used interchangeably in the context of the invention (paragraph 0044). Shim et al. taught an hERG gene modulator is hERG gene-specific siRNA (paragraph 0079).
Shim et al. taught a preferred embodiment of the method, in step (i) the hERG gene modulator is hERG gene-specific siRNA and any method known in the art suitable for introducing siRNA into the cardiomyocytes or EBs to inhibit activity of a hERG gene allele is intended to fall within the scope of the invention. For example, siRNAs can be introduced by transfection (paragraph 0079).
Additionally, Keating et al. taught long QT syndrome (LQT), has been associated with specific genes including HERG, SCN5A, KVLQT1 and KCNE1, may be hereditary and due to specific mutations in the above genes or it may be acquired. It had previously been shown that the HERG gene encodes a K+ channel which is involved in the acquired form of LQT (Paragraph 0003). Keating et al. also taught gene therapy by supplying wild-type HERG function to a cell which carries mutant HERG alleles, which should allow normal functioning of the recipient cells. The wild-type gene or a part of the gene may be introduced into the cell in a vector such that the gene remains extrachromosomal (paragraph 0193). Keating et al. taught the HERG gene or fragment may be employed in gene therapy methods in order to increase the amount of the expression products of such genes in cells. It may also be useful to increase the level of expression of a given LQT gene even in those heart cells in which the mutant gene is expressed at a “normal” level, but the gene product is not fully functional (paragraph 0193).
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have used the hERG gene-specific siRNA of Shim et al. as the gene suppression component and the gene therapy HERG gene or fragment thereof of Keating et al. as the gene replacement component in the gene suppression and replacement vector of Farrar et al. with a reasonable expectation of success as this would have amounted to combining prior art elements according to known methods to yield predictable results. One of ordinary skill in the art would have been motivated to do so because Farrar et al. teach that gene suppression and replacement circumvents the difficulties and expenses associated with the need to specifically target disease mutations and has the advantage that the same suppression effector can be used to suppress many mutations in a gene, which is particularly relevant when any one of a large number of mutations within a single gene can cause disease pathology. Farrar et al. teach RNAi can be used to suppress expression of the target nucleic acid, and a replacement nucleic acid is provided that is altered around the RNAi target site at degenerate (wobble) positions such that it escapes suppression by the RNAi at least in part but the amino acid sequence it encodes is normal. One of ordinary skill in the art would have been motivated to use an RNAi molecule that targets HERG of Shim et al. as the gene suppression component of Farrar et al. because Shim et al. taught genetic disorders caused by mutations in ion channel subunits, including hereditary long QT syndrome (LQTS) caused by mutations in one or more ion channels expressed in the heart and that LQTS has been associated with over 500 different mutations in at least 13 genes encoding cardiac ion channel proteins and that LQTS2 implicates hERG (human ether-a-go-go related gene), a gene (KCNH2) that codes for a protein known as Kv11.1 and taught an hERG gene modulator is hERG gene-specific siRNA. Therefore, since Shim et al. teach that LQTS has been associated with over 500 different mutations, including mutations in HERG there would be motivation to use the HERG siRNA of Shim et al. in the gene suppression and replacement vector of Farrar et al. because Farrar et al. teach the same suppression effector can be used to suppress many mutations in a gene, which is particularly relevant when any one of a large number of mutations within a single gene can cause disease pathology. One of ordinary skill in the art would have been motivated to use the gene therapy HERG gene or fragment in the gene suppression and replacement vector of Farrar et al. because Keating et al. taught long QT syndrome (LQT), has been associated with specific genes including HERG which may be hereditary and due to specific mutations or it may be acquired and taught gene therapy by supplying wild-type HERG function to a cell which carries mutant HERG alleles, which allows normal functioning of the recipient cells, and may also be useful to increase the level of expression of a given LQT gene even in those heart cells in which the mutant gene is expressed at a “normal” level, but the gene product is not fully functional.
Accordingly, the limitations of claims 54,56,60 and 63 would have been prima facie obvious to one of ordinary skill in the art before the effective filing date.
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to modify the in vitro suppression-replacement method of Farrar et al. based on the teachings of Shim et al. and Keating et al. regarding the method being carried out in vitro in cardiac cells, using the hERG gene-specific siRNA of Shim et al. and the gene therapy HERG gene or fragment thereof of Keating et al. with a reasonable expectation of success as this would have amounted to combining prior art elements according to known methods to yield predictable results.
One of ordinary skill in the art would have been motivated to do so because Farrar et al. teach that gene suppression and replacement circumvents the difficulties and expenses associated with the need to specifically target disease mutations and has the advantage that the same suppression effector can be used to suppress many mutations in a gene, which is particularly relevant when any one of a large number of mutations within a single gene can cause disease pathology. Farrar et al. teach RNAi can be used to suppress expression of the target nucleic acid, and a replacement nucleic acid is provided that is altered around the RNAi target site at degenerate (wobble) positions such that it escapes suppression by the RNAi at least in part but the amino acid sequence it encodes is normal. One of ordinary skill in the art would have been motivated to use the gene suppression-replacement method of Farrar et al. regarding in vitro COS-7 cells including co-transfection of the COL1A1 target and siRNA targeting COL1A1, and apply the method to other genes and appropriate cells based on the teachings of Shim et al. and Keating et al. One of ordinary skill in the art would have been motivated to use an RNAi molecule that targets HERG as the gene suppression component of Farrar et al. because Shim et al. taught genetic disorders caused by mutations in ion channel subunits, including hereditary long QT syndrome (LQTS) caused by mutations in one or more ion channels expressed in the heart and that LQTS has been associated with over 500 different mutations in at least 13 genes encoding cardiac ion channel proteins and that LQTS2 implicates hERG (human ether-a-go-go related gene), a gene (KCNH2) that codes for a protein known as Kv11.1 and taught an hERG gene modulator is hERG gene-specific siRNA. One of ordinary skill in the art would be motivated to administer such a suppression-replacement construct to cardiac cells in vitro, because Shim et al. taught a method for introducing hERG gene-specific siRNA into the cardiomyocytes to inhibit activity of a hERG gene allele and siRNAs can be introduced by transfection. Since Shim et al. teach that LQTS has been associated with over 500 different mutations, including mutations in HERG there would be motivation to use the HERG siRNA of Shim et al. in the gene suppression and replacement vector of Farrar et al. because Farrar et al. teach the same suppression effector can be used to suppress many mutations in a gene, which is particularly relevant when any one of a large number of mutations within a single gene can cause disease pathology.
Additionally, Keating et al. taught gene therapy by supplying wild-type HERG function to a cell which carries mutant HERG alleles, which should allow normal functioning of the recipient cells, and that the wild-type gene or a part of the gene may be introduced into the cell in a vector such that the gene remains extrachromosomal (paragraph 0193), and that it may also be useful to increase the level of expression of a given LQT gene even in those heart cells in which the mutant gene is expressed at a “normal” level, but the gene product is not fully functional (paragraph 0193).
Accordingly, the limitations of claims 68,69,83,92 and 93 would have been prima facie obvious to one of ordinary skill in the art before the effective filing date.
Claims 55,71,84 and 94 are rejected under 35 U.S.C. 103 as being unpatentable over Farrar et al. in view of Shim et al. and Keating et al. as applied to claims 54,56,60,63,68,69,83,92 and 93 above and further in view of GenBank Accession No. NM_000238.4 (16 Dec 2019), cited on an IDS.
The teachings of Farrar et al. Shim et al. and Keating et al. as applicable to claims 54,56,60,63,68,69,83,92 and 93 have been described above.
Farrar et al, Shim et al. and Keating et al. do not teach the first nucleotide sequence comprises the sequence set forth in SEQ ID NO: 27 and the second nucleotide sequence comprises the sequence set forth in SEQ ID NO: 29.
Before the effective filing date, the mRNA sequence of homo sapiens potassium voltage-gated channel subfamily H member 2 (KCNH2) of NM_000238.4 was publicly available.
An NCBI Blast of instant SEQ ID NO: 27 shows that nucleotides 1-29 of instant SEQ ID NO: 27 align with nucleotides 3102-3130 of NM_00238.4.
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Alignment of instant SEQ ID NO: 29 (Qy) to the mRNA sequence of NM_00238.4 (Db) shows alignment of nucleotides 2-29 of instant SEQ ID NO: 29 to nucleotides 3103-3130 of NM_00238.4, including 9 mismatches which would indicate placement of the wobble position variants.
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It would have been obvious to one of ordinary skill in the art before the effective filing date, to have arrived at instant SEQ ID NO: 27 for the nucleotide sequence of the first sequence encoding the hERG gene-specific siRNA of Shim et al. as the gene suppression component and to arrive at instant SEQ ID NO: 29 for the second nucleotide sequence encoding a KCNH2 polypeptide comprising a target sequence identical to the target sequence of the first nucleotide and comprising 1-13 wobble position variants compared to the target sequence of the first nucleotide sequence as the gene therapy for the HERG gene or fragment thereof of Keating et al. in the gene suppression and replacement vector of Farrar et al. with a reasonable expectation of success. There would be a reasonable expectation of success because the mRNA sequence of homo sapiens potassium voltage-gated channel subfamily H member 2 (KCNH2) of NM_000238.4 was publicly available, and therefore an ordinary artisan could have designed an RNAi molecule that hybridizes to a target sequence of KCNH2 using the publicly available mRNA sequence of KCNH2 of NM_000238.4, and arrived at the sequence of instant SEQ ID NO: 27. One of ordinary skill in the art would have been motivated to provide an RNAi molecule targeting KCNH2 because Shim et al. taught the association of genetic disorders caused by mutations in ion channel subunits, including hereditary long QT syndrome (LQTS) caused by mutations in one or more ion channels expressed in the heart and that LQTS has been associated with over 500 different mutations in at least 13 genes encoding cardiac ion channel proteins and that LQTS2 implicates hERG (human ether-a-go-go related gene), a gene (KCNH2) that codes for a protein known as Kv11.1 and taught an hERG gene modulator is hERG gene-specific siRNA. Therefore, there would be motivation to use the HERG siRNA of Shim et al. in the gene suppression and replacement vector of Farrar et al. because Farrar et al. teach the same suppression effector can be used to suppress many mutations in a gene, which is particularly relevant when any one of a large number of mutations within a single gene can cause disease pathology.
Likewise, an ordinary artisan knowing the mRNA sequence of NM_000238.4 would have then been able to design and provide a second gene therapy nucleotide sequence encoding KCNH2 that comprises a target sequence identical to the target sequence of the RNAi molecule of the first nucleotide sequence, and would have been motivated to optimize the number of wobble bases and positions thereof and arrive at instant SEQ ID NO: 29 based on the teachings of Farrar et al. which taught a replacement nucleic acid is provided that is altered around the RNAi target site at degenerate (wobble) positions such that it escapes suppression by the RNAi at least in part but the amino acid sequence it encodes is normal and the teachings of Keating et al. regarding gene therapy by supplying wild-type HERG function to a cell which carries mutant HERG alleles, which allows normal functioning of the recipient cells. Absent demonstration of the criticality of the number and position of the wobble variants in the second nucleotide sequence encoding KCNH2, an ordinary artisan would have engaged in routine experimentation to determine optimal or workable ranges that produce expected results. Where the general conditions of a claim are disclosed in the prior art, it is not inventive to discover the optimum or workable ranges by routine experimentation. In re Aller, 220 F. 2d 454, 105 USPQ 233 (CCPA 1955). NOTE: MPEP 2144.05.
Accordingly, the limitations of claims 55,71,84 and 94 would have been prima facie obvious to one of ordinary skill in the art before the effective filing date.
Claim 67 is rejected under 35 U.S.C. 103 as being unpatentable over Farrar et al. in view of Shim et al. and Keating et al. as applied to claims 54,56,60,63, 68,69,83,92 and 93 above and further in view of Yan et al. (Appl Microbiol Biotechnol (2015) 99:10415-10432).
The teachings of Farrar et al. Shim et al. and Keating et al. as applicable to claims 54,56,60,63,68,69,83,92 and 93 have been described above.
Farrar et al, Shim et al. and Keating et al. do not teach a virus particle comprising the nucleic acid construct.
Before the effective filing date, Yan et al. taught virus like particles (VLPs) are easy to be produced in large-scale quantities using the existing expression systems either as enveloped or nonenveloped VLPs, VLPs are capable of targeting the corresponding cell transport with its surface ligands by modification on the gene level (gene insertion) or the protein level (chemical coupling), VLPs have good carrying capacity because of its large surface area and numerous amino acid residues on the surface, and VLPs are self-assembled by viral structure proteins under proper conditions which looks more like a protein cage with a large cavity space that can encapsulate numerous biological molecules, and as a result, expanded these molecules’ applications (page 10422, left column). Yan et al. taught VLPs have thermodynamic stability and have emerged as multifunctional platform systems for the development of bioderived nanomaterials and have good potential for application in drug delivery, genetic therapy, and other fields (page 10422, right column).
Therefore, it would have been obvious to one of ordinary skill in the art before the effective filing date, to have modified the suppression effector and replacement vector of Farrar et al. in view of Shim et al. and Keating et al. to have been in the form of a virus particle, including a VLP, with a reasonable expectation of success. There would be a reasonable expectation of success because Farrar et al. taught exemplary viral vectors that may be used in the practice of the invention include those derived from adenovirus, and adenoassociated virus (AAV) (paragraph 0089), and Yan et al. also pertains to VLPs as a delivery vehicle for applications in genetic therapy. One of ordinary skill in the art would have been motivated to modify the suppression effector and replacement vector of Farrar et al. in view of Shim et al. and Keating et al. to have been in the form of a virus particle, including a VLP, based on the teachings of Yan et al. who taught benefits of VLPs including being easy to be produced in large-scale quantities, being capable of targeting the corresponding cell transport with its surface ligands by modification on the gene level (gene insertion) or the protein level, having good carrying capacity because of its large surface area and a large cavity space that can encapsulate numerous biological molecules, good thermodynamic stability, and is a multifunctional platform system with good potential for application in drug delivery and genetic therapy.
Accordingly, the limitations of claim 67 would have been prima facie obvious to one of ordinary skill in the art before the effective filing date.
Conclusion
Claims 54-56,60,63,67-69,71,83,84 and 92-94 are rejected.
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/STEPHANIE L SULLIVAN/Examiner, Art Unit 1635
/ABIGAIL VANHORN/Primary Examiner, Art Unit 1636
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