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 .
This action is in response to the amendment filed 02/04/2026, in which claims 29 were amended, claims 29-41 were previously presented and claims 42-46 were newly added. Claims 29-46 are currently pending.
Applicant’s arguments have been thoroughly reviewed, but are not persuasive for the
reasons that follow. Any rejection and objections not reiterated in this action have been
withdrawn. This action is FINAL.
Priority
Acknowledgment is made of applicant’s claim for priority based on a provisional application filed as 62/939,795 on 11/25/2019.
Acknowledgment is made of applicant’s claim for PCT/US20/61605 filed on 11/20/2020.
All claims are given the priority date of 11/25/2019.
Drawings
The previous objection to the drawings has been withdrawn in view of Applicants submission of substitute drawings filed on 02/04/2026.
Claim Rejections - 35 USC § 112
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.
This is a previous rejection mailed on 08/11/2025 and rewritten to address the amendments and arguments filed by Applicant on 02/04/2026.
Claims 29-46 are rejected under 35 U.S.C. 112(a) or 35 U.S.C. 112 (pre-AIA ), first paragraph, because the specification, while being enabling for a method comprising,
(a) delivering to the liver of an individual an effective amounts of a first polynucleotide and a second polynucleotide, wherein
the first polynucleotide comprises an expression cassette, said expression cassette comprising a therapeutic polynucleotide linked to a sequence encoding an essential gene product polynucleotide, wherein the essential gene product is selected from the group consisting of fumarylacetoacetate hydrolase (FAH), dehydrodolichyl diphosphate synthase subunit (DHDDS), 3-hydroxy-3-methylglutaryl Co-enzyme A reductase (HMGCR),UDP glucuronosyltransferase family 1 member Al (UGTJAJ), and methylmalonyl coA mutase (MMUT), wherein said cassette comprises one or more sequences capable of integrating at least part of the cassette at an endogenous locus selected from the group consisting of ApoAl (APOAJ), albumin (ALB), haptoglobin (HP), serum amyloid al (SAAJ), orosomucoid 1 (ORM]), ferritin light chain (FTL), Apolipoprotein C3 (APOC3), fibrinogen beta chain (FGB), fibrinogen gamma chain (FGG), serpin family A member 1 (SERPINAJ) and fumarylacetoacetate hydrolase (FAH), and
the second polynucleotide comprises one of (i) and (ii):
(i) a second polynucleotide comprising a targeting region capable of knocking down expression of RNA expressed from the endogenous locus,
(ii) a second polynucleotide comprising a targeting region that targets integration at the endogenous locus to disrupt expression of the locus; and
(b) after delivering, maintaining on one or more nutritional or pharmacological agents selected from the group consisting of nitisinone, cholesterol , 2-(2-nitro-4-trifluoromethylbenzoyl)-1,3-cyclohexanedione (NTBC) and mevalonic acid, resulting in selective expansion of liver cells harboring the therapeutic polynucleotide, does not reasonably provide enablement for a method of effecting gene therapy in an individual comprising the step of delivering to the liver of the individual an effective amounts of the first claimed polynucleotide and second claimed polynucleotide; expressing any fusion of a therapeutic polypeptide and any essential gene product; and wherein following delivery of the first and second polynucleotides to the liver of an individual, expression of the essential gene product is disrupted at any endogenous liver locus, and wherein the disruption is therapeutically treatable by delivering to the liver of an individual an effective amount of one or more nutritional or pharmacological agents to substitute for absence of the any essential gene product. 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.
Enablement is considered in view of the Wands factors (MPEP 2164.01(A)). These include: the breadth of the claims, the nature of the invention, the state of the prior art, the level of one of ordinary skill, the level of predictability in the art, the amount of direction provided by the inventor, the existence of working examples, and the quantity of experimentation needed to make or use the invention. All of the Wands factors have been considered with regard to the instant claims, with the most relevant factors discussed below.
Nature of the invention: The claims are drawn to a method of effecting gene therapy of an individual comprising delivering to an individual an effective amount of a first and second polynucleotide. The claims continue to state that the first polynucleotide comprises an expression cassette wherein the expression cassette comprises a therapeutic polynucleotide linked to any essential gene product polynucleotide wherein the cassette comprises one or more sequences capable of integrating at least part of the cassette at any first endogenous liver locus. The claims continues that the second polynucleotide has to either comprise a target region capable of inhibiting, knocking down or disrupting expression of any second endogenous liver locus and/or activity of a gene product or a targeting region that targets integration at any second endogenous liver locus to disrupt expression of the second endogenous locus and/or the activity of a gene product. The nature of the invention is complex in that the claims require a therapeutic outcome, yet at the same time expression of an essential gene product is inhibited or lost, which could potentially cause the occurrence of disease being that the gene product is defined as essential. Even if the essential gene product is fumarylacetoacetate hydrolase (Fah), the nature of the invention is complex in that this gene product can be linked to any therapeutic product for the treatment of any disease based on expression of the therapeutic gene product being required in a tissue other than liver for the purpose of treatment.
Breadth of the claims: The claims broadly encompass a method of administering to the liver of an individual a first and second polynucleotide in order to disrupt a gene in an endogenous liver locus in order to knock out any essential gene product. The first polynucleotide can include any therapeutic polynucleotide for treatment of any disease, where the therapeutic polynucleotide is required to be present in any cell, tissue or organ of the body to exert its therapeutic effect. The first polynucleotide can encode any essential gene product, which is essential in any one or more cells of the liver. The second polynucleotide must knock down, inhibit or disrupt expression of the endogenous liver locus that encodes the any essential gene product in an endogenous form. The complex nature of the subject matter of this invention is greatly exacerbated by the breadth of the claims.
Guidance of the specification and existence of working examples: The specification describes a method of administering to the liver of an individual a first and second polynucleotide in order to disrupt a gene in an endogenous locus in order to knock out the essential gene product. The specification does not teach the predictability or safe administration of a method of administering to the liver of an individual the first and second polynucleotide in order to disrupt a gene in any endogenous liver locus to knock out any essential gene product. (Emphasis added). The specification envisions methods of treating an individual for a medical condition by subjecting the individual to the system of the disclosure wherein the individual has a medical condition related to the therapeutic polynucleotide, such that correction of the corresponding endogenous gene of the therapeutic polynucleotide treats at least one symptom of the medical condition, wherein the individual has a liver medical condition and the essential gene product is fumarylacetoacetate hydrolase (Fah) [0058]. When the loss of Fah in cells transfected with the first and second polynucleotides is not needed in the individual with the liver medical condition, the individual is provided an effective amount of 2- (2-nitro-4-trifluoromethylbenzoyl) -l ,3-cyclohexanedione (NTBC) and on the contrary, when the loss of Fah in cells transfected with the first and second polynucleotides is needed in the individual with the liver medical condition, the individual is provided an effective amount of a high protein diet [0058].
The specification teaches the essential gene product is fumarylacetoacetate hydrolase (Fah), fumarylacetoacetate hydrolase (FAH), dehydrodolichyl diphosphate synthase subunit (DHDDS), or 3-hydroxy-3-methylglutaryl Co-enzyme A reductase (HMGCR), UDP glucuronosyltransferase family 1 member Al (UGTJAJ), ormethylmalonyl coA mutase (MMUT) [0016]. The specification teaches the first endogenous locus is ApoAl (APOAJ), albumin (ALB), haptoglobin (HP), serum amyloid al (SAAJ), orosomucoid 1 (ORM]), ferritin light chain (FTL), Apolipoprotein C3 (APOC3), fibrinogen beta chain (FGB), fibrinogen gamma chain (FGG), serpin family A member 1 (SERPINAJ) or fumarylacetoacetate hydrolase (FAH) [0012].
Example 1 (Pages 19-28) discloses APOA1 targeting and promotion of durable expression of therapeutic transgenes in mice. The specification teaches the limitations of liver-directed AAV gene therapy as well as the current inventions claim to targeting the ApoA1 locus with AAV and CRISPR/Cas9 [0066 and 0071]. Example 1 teaches the process of targeting the ApoA1 locus with CRISPR/Cas9, gRNA to the 3’ untranslated region of ApoA1, downstream of the stop codon designed within an AAV8 vector also comprising a liver-specific promoter along with a second promoter less AAV8 vector was constructed to enable insertion of a far-red fluorescent protein reporter (mKate2) into the Apoa1 locus, using a P2A skipping peptide [0071]. The results described in example 1 outlined that the mice receiving both polynucleotide sequences showed more fluorescence when compared to the mice that only received the “rescue cassette” (containing only SaCas9/gRNA alone) showing that the single polynucleotide “rescue cassette” sequence was not capable of targeting the intended locus and thus no recombination with the intended target gene [0076-0077].
Example 2 (Pages 28-37) teaches the system of the disclosure is utilized with respect to FAH and Hereditary Tyrosinemia Type I (HT-I) [0089]. The specification continues patients with HT-I present with severe liver failure in the neonatal period, requiring liver transplantation where toxic metabolites accumulate in the absence of FAH activity (i.e. succinylacetone) which cause hepatocyte apoptosis, necrosis, and repeated cycles of liver regeneration and if untreated, liver injury will progress to cirrhosis and hepatocellular carcinoma and death at an early age [0089]. The specification teaches to examine the feasibility of using FAH for positive selection in the liver, AAV vectors were generated expressing SaCas9 and a gRNA targeting the Ldlr gene in mice [0091]. Example 2 continues to show the gRNA targets Exon 14 of the Ldlr gene, and was designed to promote targeted integration of the remainder of the Ldlr coding sequence (CDS) [0091]. The specification teaches the AAV repair template includes homology arms, the remainder of the Ldlr CDS, fused to a 2A skipping peptide, human FAH cDNA, followed by another 2A, an mKate2 reporter gene, and poly A signal (FIG. 8A) and the correct integration of this repair cassette through HDR is expected to restore Ldlr expression, and also allow for expansion of these cells that also express FAH in mice [0091]. The specification continues to demonstrate that half of the mice were administered and maintained on 100% of NTBC where the other half were cycled on and off NTBC to apply selective pressure and it was resulted that the uncycled group (100% NTBC, no selective pressure) showed rare individual hepatocytes with FAH expression (FIG. 8B) whereas in contrast, the cycled group (NTBC cycling, strong selective pressure) had impressive outgrowth of colonies of FAH+ hepatocytes [0091].
The specification describes optimizing FAH disruption as the “poison pill” for selection where the essential gene (i.e. Fah) is removed efficiently in the rest of the liver to allow for selective expansion and next it was tested whether it is possible to remove Fah from the liver in mice using an AAV-CRISPR vector [0092]. The specification continues that the wild type C57BL6/J mice were injected with AAV vectors encoding SaCas9 and a gRNA targeting Fah and the animals were maintained on 100% NTBC to prevent any injury or selection, and then sacrificed one month later [0092]. The working example describes a method for treating a mouse model with a defective Ldlr gene by disrupting the gene encoding the essential gene product, FAH, in order to apply an expression cassette for targeting both the Ldlr gene and the human FAH cDNA along with a pharmacological agent, such as NTBC [0091 and 0101].
No working example teaches the method of disruption within a human of a gene in an endogenous locus for the purposeful loss of FAH activity in order to administer another gene editing system to correct a separate inherited or endogenous defective gene along with the correction of the engineered knock out of the gene responsible for FAH activity. No working example teaches the inhibition or disruption of any other essential gene in an individual.
Predictability and state of the art: For some relevant background, Cheng et al (Mol Cell Biol. 2016 May 16;36(11):1628-38) teaches the splicing factor SRSF2 is critically required for liver homeostasis and that its loss leads to early death in mice with acute liver failure (Page 1635, Column 2 bridging Page 1636, Column 1). Cheng teaches that SRSF2 not only is involved in splicing regulation of a large number of transcripts but also activates transcription of metabolism-related genes and transcription factors (Page 1636, Column 2 bridging Page 1637, Column 1). Therefore, not all essential liver genes would be able to be removed and still retain viability of the organism as well as unpredictability of the downstream effects of other genes effected by the removal of the essential gene.
Hora et al (Cells. 2023 Aug 22;12(17):2129) teaches how specific proteins contribute to the process of liver regeneration such as Itgb1 which was shown to be essential for liver regeneration, they also found that Itgb1 cooperates with growth factor signaling components—HGF, c-Met, and EGFR—which are known to play crucial roles in liver regeneration (Page 7, Paragraph 2). Hora teaches when c-Met or EGFR was deleted in the liver, it led to diminished hepatocyte proliferation and impaired liver regeneration (Page 7, Paragraph 2). Therefore, essential genes such as Itgb1, HGF, c-Met, and EGFR would not be able to be deleted and/or removed without significant impact to the organism’s liver.
One of skill in the art would have recognized the unpredictability of decreasing expression of or disrupting any essential liver gene in a subject.
Amount of experimentation necessary: In order to practice the full scope of the claimed invention, an immense amount of experimentation would be required. First one would be required to knock out any essential liver gene and retain viability of the liver as well as the organism as a whole. Next, one would need to successfully find a nutritional or pharmacological agent capable of maintaining the organism without the production of the essential gene product. Finally, one would need to be able to successfully correct the genetic deficiency without the organism succumbing to the loss of any essential liver gene product. Therefore, it would require immense amount of unpredictable experimentation to practice the claimed invention with such variants in the possible result.
In view of the breadth of the claims and the lack of guidance provided by the specification as well as the unpredictability of the art, the skilled artisan would have required an undue amount of experimentation to make and/or use the claimed invention. Therefore, claims 29-41 are not considered to be fully enabled by the instant disclosure.
Response to Arguments - Claim Rejections - 35 USC § 112
The previous rejection of claims 29-41 under 35 U.S.C. 112(a) has been maintained in view of Applicant’s arguments filed on 02/04/2026.
Applicant’s arguments have been fully considered but are not found to be persuasive. Applicant argues that the office incorrectly applies organism-level lethality studies. Applicant continues to argue that the claims are drawn to the loss of an essential gene product at a cellular level and not an organismal level. Applicant argues the claimed invention provides methods of somatic genetic engineering of an essential gene that could be expected to result in mosaicism and not complete organismal knockout. Applicant continues to argue that the office does not provide evidence that mosaic targeting of somatic cells would lead to the same lethal effects as germline knockouts and it is not appropriate for the office to rely on “common knowledge” without evidentiary support on the record.
However, it is unclear by the instant claims as well as the instant specification if the essential gene loss is by total knockout or mosaic knockout by the current recitation of the claims. The current recitation of the instant claims does not provide clearly that the intended method is somatic genetic engineering of an essential gene that could be expected to result in mosaicism and not complete organismal knockout. Evidence was provided of the enablement issues that were found in the broadly claimed invention.
Applicant argues that the specification provides direct evidence of safety, specifically the specification describes that even a small degree of Fah activity (1-5%) can maintain liver function and therefore, a method that results in mosaic knockout of an essential gene thus could be reasonably expected by a POSITA to be safe enough for gene therapy.
This argument is acknowledged by the Office and the essential gene FAH was included in the original and re-written scope of enablement. The Office has recognized that the specification describes that even a small degree of Fah activity (1-5%) can maintain liver function and therefore, mosaic knockout of FAH would not result in the death of the organism. As previously stated above, the current recitation of the instant claims does not provide clearly that the intended method is somatic genetic engineering of an essential gene that could be expected to result in mosaicism and not complete organismal knockout.
Applicant continues to argue that the specification reasonably demonstrates treatment of disease, specifically that hemophilia B, hemophilia A, Crigler-Najjar syndrome, Huner's Syndrome, Hurler's Syndrome, mucopolysaccharidosis I, mucopolysaccharidosis II, hyperlipidemia, and hereditary tyrosinemia type I have been treated by restoring deficient gene expression or activity. Applicant argues that it is also taught that only partial restoration of gene expression or activity is required. Thus, Applicant argues that a POSITA could reasonably expect that even little restoration of expression of a therapeutic polynucleotide in an individual with a disease in need of said therapeutic polynucleotide would provide a therapeutic effect. FIGs. 12A-12I show that a gene of interest (e.g., mKate2, which can be swapped with a therapeutic gene) can be expressed in the liver via the claimed methods. Applicant continues to argue that the Declaration under 37 C.F.R. § 1.132 by Dr. William Lagor (the "Declaration"), submitted herewith, the claimed method has been confirmed to result in production of a therapeutic gene in models of hemophilia B, Fabry disease, and mucopolysaccharidosis type II, resulting in levels higher than is recognized as therapeutic in the art and thus, the specification discloses multiple therapeutic applications spanning different diseases, all unified by the general principle of selective expansion of cells expressing both therapeutic and essential genes such that this principle has been described across at least several metabolic disorders, FAH, DHDDS, Factor IX, ApoE, and LDLR, establishing a reasonable correlation to the claimed scope and confirmed by Applicant with post-filing data.
The declaration submitted has been considered and acknowledged. However, the current rejection is in regards to the claim recitation does not provide clearly that the intended method is somatic genetic engineering of an essential gene that could be expected to result in mosaicism and not complete organismal knockout as well as the fact that any essential liver gene knockdown would be capable of mosaic somatic genetic engineering without resulting in death of the organ and/or organism.
Applicant argues that multiple essential genes are disclosed and demonstrated wherein the working examples provide evidence for DHDDS and FAH as the essential genes and therefore shows enablement. Applicant continues to argue that because the specification discloses several essential genes and the working examples provide successful experimentation with two of the essential genes therefore, any experimentation that could be required to practice the claimed invention is routine and well within the skill of the art. Applicant continues to argue that detailed guidance of methods for practicing the claimed method are provided and furthermore, an experimental design workflow is disclosed such that the specification also discloses a list of essential genes and safe harbor loci with mechanistic explanations, discussion of treatable disorders, and examples of delivery to an individual.
However, due to the working examples only providing successful experimentation with two of the essential genes does not enable the invention for any essential gene. This is because it is unlikely that the intended method of somatic genetic engineering of an essential gene to result in mosaicism such that any essential liver gene knockdown would be capable of mosaic somatic genetic engineering without resulting in death of the organ and/or organism.
Therefore, the current rejection is maintained.
Claim Rejections - 35 USC § 103
In the event the determination of the status of the application as subject to AIA 35 U.S.C. 102 and 103 (or as subject to pre-AIA 35 U.S.C. 102 and 103) is incorrect, any correction of the statutory basis (i.e., changing from AIA to pre-AIA ) for the rejection will not be considered a new ground of rejection if the prior art relied upon, and the rationale supporting the rejection, would be the same under either status.
The following is a quotation of 35 U.S.C. 103 which forms the basis for all obviousness rejections set forth in this Office action:
A patent for a claimed invention may not be obtained, notwithstanding that the claimed invention is not identically disclosed as set forth in section 102, if the differences between the claimed invention and the prior art are such that the claimed invention as a whole would have been obvious before the effective filing date of the claimed invention to a person having ordinary skill in the art to which the claimed invention pertains. Patentability shall not be negated by the manner in which the invention was made.
The factual inquiries for establishing a background for determining obviousness under 35 U.S.C. 103 are summarized as follows:
1. Determining the scope and contents of the prior art.
2. Ascertaining the differences between the prior art and the claims at issue.
3. Resolving the level of ordinary skill in the pertinent art.
4. Considering objective evidence present in the application indicating obviousness or nonobviousness.
Claims 29-33, 35-39, 41, 42 and 46 are rejected under 35 U.S.C. 103 as being unpatentable over Grompe et al (Genes Dev. 7(12A):2298-307; 1993) in view of Wangensteen et al (Hepatology, Vol 68, No 2, Pgs. 663-676; 2018). This rejection was made in the Office action mailed 08/11/2025 and re-written to address the amendments to the claims filed on 02/04/2026.
Regarding claims 29-31, 35-39, 42 and 46, Grompe teaches producing mice models that are FAH deficient showing that the loss-of-function of an essential gene product resulted in death of the mice (Page 2298, Abstract). Grompe teaches a polynucleotide, targeting construct pD10, comprising a targeting region capable of knocking down therefore disrupting expression of the FAH locus and the loss of function activity of FAH (Page 2300, Figure 2).
Grompe does not teach the first polynucleotide comprising an expression cassette, said expression cassette comprising a therapeutic polynucleotide linked to an essential gene product polynucleotide, wherein said cassette comprises one or more sequences capable of integrating at least part of the cassette at a first endogenous locus.
Wangensteen teaches the method of producing a mouse model for the purpose of screening for treatment methods of HT1. Wangensteen teaches the mouse models are produced by injection of polynucleotide sequence comprising a gRNA complementary to the locus, the dCas9 protein, and a TA protein that binds to the SunTag domain of dCas9 where the mice were then injected with an AAV-Cre comprising a Cas9 protein to remove the floxed stop cassette from the dCas9 allele (Page 667, Column 2). Wangensteen teaches once the dCas9 protein was specifically expressed within the mouse hepatocytes upon injection of the AAV-Cre, the mice were crossed with FAH-/- to produce the FAH-/-; dCas9+ mice (Page 667, Column 2). Wangensteen teaches to prevent liver injury during treatment, the mice were provided with continuous nitisinone (NTBC) therapy until FAH-encoding plasmids were injected (Page 667, Column 2). Wangensteen teaches the Fah–/– and Fah–/–; Cas9 mice were maintained on nitisinone (NTBC) until the time of injection with multiple “sleeping beauty” transposon-containing plasmids comprising gRNA, TA and an FAH expression cassette and post-injection the mice were withdrawn from the nitisinone (Page 667, Column 2 bridging Page 669, Column 1 and Page 668, Figure 1a). Wangensteen teaches nodules of FAH-positive hepatocytes were found in the livers, consistent with the clonal expansion of stably transduced hepatocytes seen with Sleeping Beauty– mediated gene therapy in Fah–/– mice (Page 671, Column 1, Paragraph 1). Wangensteen teaches that their CRISPRa system successfully activates target gene expression in repopulating hepatocytes in vivo (Page 671, Column 1, Paragraph 1). Wangensteen teaches MYC expression corresponded with FAH-positive nodules, indicating a robust and specific activation of expression (Page 671, Column 1). Wangensteen teaches Myc gRNAs were enriched up to 10-fold compared to a Fah–/– control mouse or the input plasmid pool and thus demonstrated hepatocyte clonal expansion (Page 671, Column 1 bridging Column 2). Wangensteen teaches their system allows for genetic epistasis experiments, showing that up-regulation of an endogenous oncogene locus promotes cell proliferation and initiation of carcinogenesis and conversely that targeting of tumor suppressors inhibits tumorigenesis leading to the treatment of genetic liver cancer (Page 665, Column 1).
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 teachings of Grompe to include the first polynucleotide comprises an expression cassette, said expression cassette comprising a therapeutic polynucleotide linked to an essential gene product polynucleotide, wherein said cassette comprises one or more sequences capable of integrating at least part of the cassette at a first endogenous locus because Grompe teaches it is within the ordinary skill in the art to use expression cassettes administered to mice in order to disrupt the FAH gene in order to knockout the FAH activity and Wangensteen teaches using mice previously constructed without FAH activity where the mice were provided with NTBC in order to protect the liver from deterioration and further liver injury and administering to the mice an expression construct to target MYC expression for repopulation of liver cells.
One would have been motivated to make such a modification in order to receive the expected benefit of regain of the loss of function of the essential gene product, FAH, through targeted expression of MYC for liver cell repopulation as taught by Wangensteen.
Regarding claim 32, Grompe teaches FAH deficient mice can be kept alive by pharmacologically by inhibiting p-OH phenylpyruvate dioxygenase using NTBC to disrupt the tyrosine catabolic pathway upstream to FAH (Page 2304, Column 1).
Regarding claims 33 and 41, Grompe does not teach wherein the one or more nutritional or pharmacological agents are delivered to the individual to effect negative selective pressure on cells lacking the first polynucleotides and the delivering step comprises nanoparticle delivery, transfection, electroporation or hydrodynamic delivery.
Wangensteen teaches a schema of the experimental design to first inject AAV-Cre to activate dCas9 specifically within hepatocytes, followed by hydrodynamic injection of plasmid pools and removal of nitisinone to induce liver injury and repopulation (Page 668, Figure 1a description).
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 teachings of Grompe to include the NTBC are delivered to the individual to effect negative selective pressure on cells lacking the first polynucleotides wherein the first polynucleotide is delivered via hydrodynamic injection because Grompe teaches it is within the ordinary skill in the art to use expression cassettes administered to mice in order to disrupt the FAH gene in order to knockout the FAH activity and Wangensteen teaches using mice previously constructed without FAH activity where the mice were provided with NTBC in order to protect the liver from deterioration and further liver injury and administering to the mice an expression construct to target MYC expression for repopulation of liver cells.
One would have been motivated to make such a modification in order to receive the expected benefit of regain of the loss of function of the essential gene product, FAH, through targeted expression of MYC for liver cell repopulation and successful delivery via hydrodynamic injection as taught by Wangensteen.
Claim 34 is rejected under 35 U.S.C. 103 as being unpatentable over Grompe et al (Genes Dev. 7(12A):2298-307; 1993) in view of Wangensteen et al (Hepatology, Vol 68, No 2, Pgs. 663-676; 2018) as applied to claims 29-33, 35-39 and 41 above, and further in view of Hickey et al (Cell Transplantation, Vol 28 No 1, pgs. 79-88; 2019). This rejection was made in the Office action mailed 08/11/2025.
The teachings of Grompe and Wangensteen as described and applied above.
Regarding claim 34, Wangensteen teaches to prevent liver injury during treatment, the mice were provided with continuous nitisinone (NTBC) therapy until FAH-encoding plasmids were injected (Page 667, Column 2).
Grompe and Wangensteen do not teach wherein the one or more nutritional or pharmacological agents are delivered to the individual to effect positive selective pressure on cells harboring the polynucleotides.
Hickey teaches the demonstration of curative ex-vivo gene and cell therapy using a lentiviral vector to express FAH in autologous hepatocytes within pig models (Page 79, Abstract). Hickey teaches hepatocytes were isolated and transduced with a lentiviral vector expressing the porcine FAH cDNA under the control of a hepatocyte-specific promoter (Page 81, Column 2). Hickey teaches the LV-transduced autologous hepatocytes were transplanted back to the same pig and the pig was cycled on/off of the protective drug NTBC for four cycles and became NTBC-independent 95 days after transplantation (Page 81, Column 2). Hickey teaches liver biopsies and biochemical analyses revealed complete amelioration of symptoms characteristic of HT1 (Page 81, Column 2).
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 teachings of Grompe and Wangensteen to include the nutritional or pharmacological agent to effect positive selective pressure on cells harboring the polynucleotides as taught by Hickey because Grompe teaches it is within the ordinary skill in the art to use expression cassettes administered to mice in order to disrupt the FAH gene in order to knockout the FAH activity, Wangensteen teaches using mice previously constructed without FAH activity where the mice were provided with NTBC in order to protect the liver from deterioration and further liver injury and administering to the mice an expression construct to target MYC expression for repopulation of liver cells and Hickey teaches the LV-transduced autologous hepatocytes were transplanted back to the same pig and the pig was cycled on/off of the protective drug NTBC for four cycles and became NTBC-independent 95 days after transplantation.
One would have been motivated to make such a modification in order to receive the expected benefit of hepatocyte protective qualities during the treatment phase for the hepatocytes to repopulate the liver as taught by Hickey.
Claim 40 is rejected under 35 U.S.C. 103 as being unpatentable over Grompe et al (Genes Dev. 7(12A):2298-307; 1993) in view of Wangensteen et al (Hepatology, Vol 68, No 2, Pgs. 663-676; 2018) as applied to claims 29-33, 35-39, 41, 42 and 46 above, and further in view of Byson et al (Yale Journal of Biology and Medicine 90, pgs. 553-566; 2017). This rejection was made in the Office action mailed 08/11/2025.
The teachings of Grompe and Wangensteen as described and applied above.
Regarding claim 40, Grompe and Wangensteen do not teach wherein when the loss of Fah in cells transfected with the first and second polynucleotides is needed in the individual, the individual is provided an effective amount of a high protein diet.
Bryson teaches that the standard treatment for HT1 consists of protein-restricted diet with the drug 2-(2-nitro-4-trifluoromethylbenzoyl)-1,3 cyclohexane dione (NTBC) where NTBC blocks the accumulation of toxic metabolites by inhibiting the 4-OH phenylpyruvate dioxygenase (HPD) enzyme upstream of FAH and liver transplantation is the only curative therapy available for HT1 (Page 556, Column 1, Paragraph 1). Bryson teaches the hydrodynamic injection of plasmids encoding for the Cas9 system and corrective short ssDNA donor template to mouse models resulting in robust enrichment of gene corrected hepatocytes where the initial percentage was low but in conjunction of withdrawal from NTBC the percentage increases (Page 561, Column 1). Bryson teaches the delivery of AAV8 vectors containing the Cas9 system and donor template in neonatal OTC mice resulted in 10 percent gene correction in hepatocytes along with a 40 percent reduction in blood ammonia levels as well as enhanced survival on a high protein diet (Page 561, Column 1, Page 3).
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 teachings of Grompe and Wangensteen to include that when the loss of Fah in cells transfected, the individual is provided an effective amount of a high protein diet as taught by Bryson because Grompe teaches it is within the ordinary skill in the art to use expression cassettes administered to mice in order to disrupt the FAH gene in order to knockout the FAH activity, Wangensteen teaches using mice previously constructed without FAH activity where the mice were provided with NTBC in order to protect the liver from deterioration and further liver injury and administering to the mice an expression construct to target MYC expression for repopulation of liver cells and Bryson teaches mice administered with AAV8 vectors containing the Cas9 system and donor template to produce mice with disruption of the FAH gene that were also administered a high protein diet.
One would have been motivated to make such a modification in order to receive the expected benefit of enhanced survival for mice receiving a high protein diet as taught by Bryson.
Claim 43 is rejected under 35 U.S.C. 103 as being unpatentable over Grompe et al (Genes Dev. 7(12A):2298-307; 1993) in view of Wangensteen et al (Hepatology, Vol 68, No 2, Pgs. 663-676; 2018) as applied to claims 29-33, 35-39, 41, 42 and 46 above, and further in view of Ohmori et al (Sci Rep. 2017 Jun 23;7(1):4159). This is a NEW rejection.
The teachings of Grompe and Wangensteen as described and applied above.
Regarding claim 43, Grompe and Wangensteen do not specifically teach the subject has hemophilia B.
Ohmori teaches effectively delivered genome editing components including Cas9 and sgRNA into hepatocytes using a single AAV8 vector, and were able to restore hemostasis in a mouse model of hemophilia Busing three different strategies (Page 2, Paragraph 2).
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 teachings of Grompe and Wangensteen to include the treatment of a subject with hemophilia B as taught by Ohmori because Grompe teaches it is within the ordinary skill in the art to use expression cassettes administered to mice in order to disrupt the FAH gene in order to knockout the FAH activity, Wangensteen teaches using mice previously constructed without FAH activity where the mice were provided with NTBC in order to protect the liver from deterioration and further liver injury and administering to the mice an expression construct to target MYC expression for repopulation of liver cells and Ohmori teaches successful treatment of hemophilia B using an AAV vector comprising at least a Cas9.
One would have been motivated to make such a modification in order to receive the expected benefit of successful treatment of hemophilia B as taught by Ohmori.
Claim 44 is rejected under 35 U.S.C. 103 as being unpatentable over Grompe et al (Genes Dev. 7(12A):2298-307; 1993) in view of Wangensteen et al (Hepatology, Vol 68, No 2, Pgs. 663-676; 2018) as applied to claims 29-33, 35-39, 41, 42 and 46 above, and further in view of Tian et al (Nat Commun 10, 1785 (2019); Pgs. 1-13). This is a NEW rejection.
The teachings of Grompe and Wangensteen as described and applied above.
Regarding claim 44, Grompe and Wangensteen do not specifically teach the subject has Fabry disease.
Tian teaches using CRISPR/Cas9 to knock out specific glycosyltransferases and hydrolases in CHO cells to optimize the N-glycosylation and mannose-6-phosphate (M6P) processing of human GLA (Page 2, Column 2 bridging Page 3, Columns 1 and 2). Tian teaches engineer Chinese Hamster Ovary (CHO) cells to produce improved therapeutic enzymes for Fabry disease over enzyme replacement therapy (Page 9, Columns 1 and 2).
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 teachings of Grompe and Wangensteen to include the treatment of a subject with Fabry disease as taught by Tian because Grompe teaches it is within the ordinary skill in the art to use expression cassettes administered to mice in order to disrupt the FAH gene in order to knockout the FAH activity, Wangensteen teaches using mice previously constructed without FAH activity where the mice were provided with NTBC in order to protect the liver from deterioration and further liver injury and administering to the mice an expression construct to target MYC expression for repopulation of liver cells and Tian teaches successful treatment of Fabry disease using CRISPR/Cas9 technology in CHO cell lines.
One would have been motivated to make such a modification in order to receive the expected benefit of successful treatment of Fabry disease as taught by Tian.
Claim 45 is rejected under 35 U.S.C. 103 as being unpatentable over Grompe et al (Genes Dev. 7(12A):2298-307; 1993) in view of Wangensteen et al (Hepatology, Vol 68, No 2, Pgs. 663-676; 2018) as applied to claims 29-33, 35-39, 41, 42 and 46 above, and further in view of Sharma et al (Blood. 2015; 126(15):1777-1784)). This is a NEW rejection.
The teachings of Grompe and Wangensteen as described and applied above.
Regarding claim 45, Grompe and Wangensteen do not specifically teach the subject has mucopolysaccharidosis II.
Sharma teaches using zinc-finger nucleases (ZFNs) to insert the iduronate 2-sulfatase (IDS) gene into the albumin locus in liver cells acts as a "safe harbor," providing a permanent treatment for Hunter Syndrome (MPS II) by restoring enzyme production and reducing stored GAGs (Page 1777, Column 2 and Page 1782, Column 1).
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 teachings of Grompe and Wangensteen to include the treatment of a subject with MPS II, also known as Hunters syndrome, as taught by Sharma because Grompe teaches it is within the ordinary skill in the art to use expression cassettes administered to mice in order to disrupt the FAH gene in order to knockout the FAH activity, Wangensteen teaches using mice previously constructed without FAH activity where the mice were provided with NTBC in order to protect the liver from deterioration and further liver injury and administering to the mice an expression construct to target MYC expression for repopulation of liver cells and Sharma teaches using zinc-finger nucleases (ZFNs) to insert the iduronate 2-sulfatase (IDS) gene into the albumin locus in liver cells acts as a "safe harbor," providing a permanent treatment for Hunter Syndrome (MPS II) by restoring enzyme production and reducing stored GAGs.
One would have been motivated to make such a modification in order to receive the expected benefit of successful treatment of Fabry disease as taught by Sharma.
Response to Arguments - Claim Rejections - 35 USC § 103
The previous rejection of Claims 29-33, 35-39 and 41 under 35 U.S.C. 103 as being unpatentable has been maintained in view of Applicant’s arguments filed on 02/04/2026.
The previous rejection of Claim 34 under 35 U.S.C. 103 as being unpatentable has been maintained in view of Applicant’s arguments filed on 02/04/2026.
The previous rejection of Claim 40 under 35 U.S.C. 103 as being unpatentable has been maintained in view of Applicant’s arguments filed on 02/04/2026.
All of Applicant’s arguments have been fully considered but have not been found persuasive.
As it related to the rejection of claims 29-33, 35-39 and 41, Applicant argues the cited art fails to disclose every element of the claimed invention, specifically Grompe relates to genetic engineering of embryonic stem cells to generate germline knockouts; it does not knockout a gene in an individual. Applicant continues that Wangensteen relates to the use of activating dead Cas9, a catalytically inactive form of Cas9; it does not knockout a gene in an individual.
However, in response to applicant's arguments against the references individually, one cannot show nonobviousness by attacking references individually where the rejections are based on combinations of references. See In re Keller, 642 F.2d 413, 208 USPQ 871 (CCPA 1981); In re Merck & Co., 800 F.2d 1091, 231 USPQ 375 (Fed. Cir. 1986).
Specifically, Grompe teaches producing mice models that are FAH deficient showing that the loss-of-function of an essential gene product resulted in death of the mice (Page 2298, Abstract). Grompe teaches a polynucleotide, targeting construct pD10, comprising a targeting region capable of knocking down therefore disrupting expression of the FAH locus and the loss of function activity of FAH (Page 2300, Figure 2).
Grompe does not teach the first polynucleotide comprising an expression cassette, said expression cassette comprising a therapeutic polynucleotide linked to an essential gene product polynucleotide, wherein said cassette comprises one or more sequences capable of integrating at least part of the cassette at a first endogenous locus.
Wangensteen teaches the method of producing a mouse model for the purpose of screening for treatment methods of HT1. Wangensteen teaches the mouse models are produced by injection of polynucleotide sequence comprising a gRNA complementary to the locus, the dCas9 protein, and a TA protein that binds to the SunTag domain of dCas9 where the mice were then injected with an AAV-Cre comprising a Cas9 protein to remove the floxed stop cassette from the dCas9 allele (Page 667, Column 2). Wangensteen teaches once the dCas9 protein was specifically expressed within the mouse hepatocytes upon injection of the AAV-Cre, the mice were crossed with FAH-/- to produce the FAH-/-; dCas9+ mice (Page 667, Column 2). Wangensteen teaches to prevent liver injury during treatment, the mice were provided with continuous nitisinone (NTBC) therapy until FAH-encoding plasmids were injected (Page 667, Column 2). Wangensteen teaches the Fah–/– and Fah–/–; Cas9 mice were maintained on nitisinone (NTBC) until the time of injection with multiple “sleeping beauty” transposon-containing plasmids comprising gRNA, TA and an FAH expression cassette and post-injection the mice were withdrawn from the nitisinone (Page 667, Column 2 bridging Page 669, Column 1 and Page 668, Figure 1a). Wangensteen teaches nodules of FAH-positive hepatocytes were found in the livers, consistent with the clonal expansion of stably transduced hepatocytes seen with Sleeping Beauty– mediated gene therapy in Fah–/– mice (Page 671, Column 1, Paragraph 1). Wangensteen teaches that their CRISPRa system successfully activates target gene expression in repopulating hepatocytes in vivo (Page 671, Column 1, Paragraph 1). Wangensteen teaches MYC expression corresponded with FAH-positive nodules, indicating a robust and specific activation of expression (Page 671, Column 1). Wangensteen teaches Myc gRNAs were enriched up to 10-fold compared to a Fah–/– control mouse or the input plasmid pool and thus demonstrated hepatocyte clonal expansion (Page 671, Column 1 bridging Column 2).
Therefore in combination, 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 teachings of Grompe to include the first polynucleotide comprises an expression cassette, said expression cassette comprising a therapeutic polynucleotide linked to an essential gene product polynucleotide, wherein said cassette comprises one or more sequences capable of integrating at least part of the cassette at a first endogenous locus because Grompe teaches it is within the ordinary skill in the art to use expression cassettes administered to mice in order to disrupt the FAH gene in order to knockout the FAH activity and Wangensteen teaches using mice previously constructed without FAH activity where the mice were provided with NTBC in order to protect the liver from deterioration and further liver injury and administering to the mice an expression construct to target MYC expression for repopulation of liver cells. One would have been motivated to make such a modification in order to receive the expected benefit of regain of the loss of function of the essential gene product, FAH, through targeted expression of MYC for liver cell repopulation as taught by Wangensteen.
As it relates to the rejection of claim 34, Applicant argues that the rejection of independent claim 29 is not proper and is allowable therefore, the dependent rejections should also be withdrawn due to being proper as well.
However, as stated above, the rejection of independent claim 29 is proper and therefore the rejection is maintained.
As it relates to the rejection of claim 40, Applicant argues that the rejection of independent claim 29 is not proper and is allowable therefore, the dependent rejections should also be withdrawn due to being proper as well.
However, as stated above, the rejection of independent claim 29 is proper and therefore the rejection is maintained.
Conclusion
No claims are allowed.
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.
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/ALEXANDRA ROSE LIPPOLIS/Examiner, Art Unit 1637
/CELINE X QIAN/Primary Examiner, Art Unit 1637
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