DETAILED ACTION
The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
Applicant’s response to restriction requirement and amendments to the claims filed on August 20, 2025 have been received and entered. Claims 1-25 have been canceled. Claims 26-35 are newly added. Claims 26-35 are pending in the instant application.
Election/Restrictions
Applicant’s election of claims 26 (group III) in the reply filed on August 20, 2025 is acknowledged. It is noted that claims 1-7, 11-13, 16-17, 21-25 link to invention of group III. Applicant further elects the species of (i) iPSC, (ii) recessive mutation and (iii) wherein the recombinant nucleic acid is not transmitted to progeny and (iv) genetic mutation is Tex11. Because applicant did not distinctly and specifically point out the supposed errors in the restriction requirement, the election has been treated as an election without traverse (MPEP § 818.01(a)). Claims 26-35 read on elected invention.
Priority
This application is a continuation of 16/630589 filed on 01/13/2020 that is a 371 of PCT/US2018/043948 filed on 07/26/2018, which claims priority from US provisional application no 62/537,370 filed on 07/26/2017.
Information Disclosure Statement
The information disclosure statements (IDS) submitted on 08/28/2023 is in compliance with the provisions of 37 CFR 1.97. Accordingly, the information disclosure statement has been considered by the examiner.
Claims 26-35 are under consideration.
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.
Claims 26-35 are rejected under 35 U.S.C. 112(a) or 35 U.S.C. 112 (pre-AIA ), first paragraph, as failing to comply with the enablement requirement. The claim(s) contains subject matter which was not described in the specification in such a way as to enable one skilled in the art to which it pertains, or with which it is most nearly connected, to make and/or use the invention.
In determining whether Applicant’s claims are enabled, it must be found that one of skill in the art at the time of invention by applicant would not have had to perform “undue experimentation” to make and/or use the invention claimed. Such a determination is not a simple factual consideration, but is a conclusion reached by weighing at least eight factors as set forth in In re Wands, 858 F.2d at 737, 8 USPQ 1400, 2d at 1404. Such factors are: (1) The breadth of the claims; (2) The nature of the invention; (3) The state of the art; (4) The level of one of ordinary skill in the art; (5) The level of predictability in the art; (6) The amount of direction and guidance provided by Applicant; (7) The existence of working examples; and (8) The quantity of experimentation needed to make and/or use the invention.
The office has analyzed the specification in direct accordance to the factors outlines in In re Wands. MPEP 2164.04 states: “[W]hile the analysis and conclusion of a lack of enablement are based on factors discussed in MPEP 2164.01(a) and the evidence as whole, it is not necessary to discuss each factor in written enablement rejection.” These factors will be analyzed, in turn, to demonstrate that one of ordinary skill in the art would have had to perform “undue experimentation” to make and/or use the invention and therefore, applicant’s claims are not enabled.
Nature of the Invention:
The claims are directed to a method of treating non-obstructive azoospermia (NOA) in a male subject caused by a genetic mutation, comprising: introducing a recombinant nucleic acid molecule into induced pluripotent stem cells (iPSCs) of the male subject, wherein the nucleic acid molecule corrects the genetic mutation causing the NOA, thereby generating transformed iPSCs; isolating transformed iPSCs that are heterozygous or hemizygous for the genetic mutation, thereby generating isolated transformed iPSCs; differentiating the isolated transformed iPSCs into primordial germ cell-like cells (PGCLCs); and transplanting the PGCLCs into the testes of the male subject or differentiating the PGCLCs into sperm in vitro, thereby treating NOA in the subject. Dependent claim limits the recombinant nucleic acid molecule is not transmitted to progeny of the male subject and wherein the recombinant nucleic acid molecule is operably linked to a promoter. Dependent claim method further comprises introducing a Cas9 protein into the iPSCs in culture and wherein the recombinant nucleic acid molecule targets an endogenous native locus associated with NOA. Claims 34 and 35 limits the method wherein the genetic defect causing the NOA comprises a homozygous recessive mutation or wherein the genetic defect causing the NOA comprises a TEX11 mutation or an androgen receptor (AR) mutation. Thus, the nature of Applicant's invention is the application of transplanting the genetically modified PGCLCs into the testes of the male subject or differentiating the PGCLCs into sperm in vitro with or without genomic editing to treat non-obstructive azoospermia (NOA) in any male subject including human subject with or without transmitting the therapeutic gene to offspring of the infertile subject.
Breadth of the claims:
The claims are broadly directed to a method of treating non-obstructive azoospermia (NOA) in any male subject that includes a human, a monkey or other non-human primate, mouse, rat, rabbit, pig, goat, sheep, dog, cat, boar, bull, horse, or cow (see page 22 of the specification) caused by any genetic mutation that encompass homozygous or heterozygous dominant or recessive mutation, comprising: introducing any recombinant nucleic acid molecule into induced pluripotent stem cells (iPSC) of the male subject, wherein the nucleic acid molecule corrects the genetic mutation causing the NOA, thereby generating transformed iPSC; isolating transformed iPSC that are heterozygous or hemizygous for the genetic mutation, thereby generating isolated transformed iPSCs; and differentiating the isolated transformed iPSCs into primordial germ cell-like cells (PGCLCs); and transplanting the PGCLCs into the testes of the male subject or differentiating the PGCLCs into sperm in vitro, thereby treating NOA in the subject. The specification contemplated that the subjects that can be treated include human and veterinary mammals, such as primates, mice, rats, rabbits, bulls, horses, cows, pigs, and sheep (see page 19, lines 13-21 of the specification).
The dependent claims limit the genetic defect causing the NOA comprises any homozygous recessive mutation. The dependent claims further limit the method, wherein the method further includes: introducing by any mean a Cas9 protein or Cas9 encoding nucleic acid molecule into the iPSC and wherein the recombinant nucleic acid molecule targets any endogenous native locus associated with NOA, or targets or any known or yet to identified native locus associated with NOA. The dependent claims further limit to the genetic mutation causing the NOA comprises any mutation in TEX11, or androgen receptor (AR).
Instant rejection is based on: 1) the absence of an enabling disclosure to address the issues of unpredictability with introducing nucleic acid molecule into iPSC derived from any species o the male subject that corrects any known or yet to be identified genetic mutation causing NOA, 2) isolating iPSC derived from plurality of different subject that have heterozygous or hemizygous genetic mutation that results in NOA , 3) culturing and maintaining genetically modified iPSC derived from any subject with NOA or genetically modified iPSC that contains nucleic acid molecule that corrects plurality of mutation as embraced by the breadth of the claim; 4) a predicable model for NOA comprises any recessive or any dominant mutation subsequently limiting to the genetic mutation causing the NOA comprises a mutation in TEX11, or androgen receptor (AR), 5) the recombinant nucleic acid molecule targets any known or yet to be identified endogenous native locus associated with NOA and 6) germline editing using Crispr/Cas system in iPSC derived from any subject intended to treat NOA and 7) transplanting the PGCLCs into the testes or differentiating the PGCLCs into sperm in vitro to treat NOA in a male subject caused by a mutation. The disclosure provided by the applicant, in view of prior art, must encompass a wide area of knowledge to a reasonably comprehensive extent. In other word each of these, aspect must be shown to a reasonable extent so that one of the ordinary skills in the art would be able to practice the invention without any undue burden being on such Artisan.
Guidance of the Specification and The Existence of Working Examples:
The specification prophetically provides a general description of a method for in vivo somatic cell gene therapy (see fig. 1A-1C). Figure 2A-D discloses a SCARKO mice model of NOA. Figures 3-5 teaches somatic gene therapy by delivering Adeno-EF1a-eGFP-AR gene therapy vector to express eGFP reporter gene in Sertoli cells thereby restoring sperm production in SCARKO mice. The specification explicitly teaches Adeno-EF1a-eGFP-hAR transduces Sertoli cells, but not germ cells. Therefore, claim 1 as presented is not enabling for in vivo or in utero introduction of introduction of a recombinant nucleic acid molecule into iPSCs. Figure 8-9 and 11 prophetically disclose the schematic for an ex vivo germline gene therapy for autosomal recessive or dominant disorder by inserting therapeutic gene into an endogenous locus. Figure 10 discloses constructs that can be used for ex vivo gene editing of spermatogonial stem cells (SSCs) from the testis or ex vivo gene editing of male or female patient-derived induced pluripotent stem cells (iPSCs). Figure 12 and 13 teaches efficient transfection of the mouse SSC and validation of sgRNA targeting human SOHLH1 and TEX 11 gene that target the c.346-1G>A mutation of SOHLH1 intron 3, whereas the sgRNA targeting TEX11 was designed to target the a c.792+1G->A. Applicant example only prophetically provides a schematic of treatment of male infertility, including non-obstructive azoospermia (NOA) and comorbid diseases, with or without transmitting the therapeutic gene to offspring of the infertile subject. The specification provides no teaching that sperm were ever produced in any of the working examples following transplantation of a transformed iPSCs derived PGCLCs that are heterozygous or hemizygous for the genetic mutation and that infertility was treated in any male mammal.
State of the Art and Predictability of the Art and the Amount of Experimentation Necessary:
The state of the art before the effective filing date of instant invention teaches genetic and epigenetic variations in iPSCs that raised concern as these variations may compromise the utility of iPSCs (see Liang et al Cell Stem Cell. 2013 August 1; 13(2): 149–159 abstract). The art teaches an iPSC genome may harbor a wide range of variations, including aneuploidy, subchromosomal copy number variation (CNV), and single nucleotide variations (SNVs). These variations can be introduced into the iPSCs from different sources during iPSC generation and maintenance (Figure 1) (see page 149, col. 1, para. 2) . The art further teaches that reprogramming process may be mutagenic, which potentially introduces de novo variations (Figure 1B). Third, like ESCs, prolonged culturing of iPSCs may introduce or select for genetic alterations that facilitate cell propagation (Figure 1C). In addition to these causes, certain variations may arise from innate genetic instability of the in vitro pluripotent state (see page 149, col. 2, para. 1). Liang concludes that these variations may alter the differentiation potential of iPSCs, cause phenotypic changes in iPSC-derived somatic cells, or increase the tumorigenicity and immunogenicity of iPSCs and their derivatives. These adverse changes directly affect the utility of iPSCs. In the instant case, claims as presented are not enabling for introducing a recombinant nucleic acid molecule introducing a recombinant nucleic acid molecule into induced pluripotent stem cells (iPSCs) of the male subject having NOA caused by any genetic mutation. The guidance provided in the specification is limited to transfecting EF1a-eGFP in an isolated mouse SSC using PEI (see example and figure 10). The claims encompass introducing a recombinant nucleic acid molecule into iPSCs derived from any species of male subject having NOA than isolating transformed iPSCs that are heterozygous or hemizygous for the genetic mutation that is then differentiated into transformed PGCLCs for introduction of said isolated transformed PGCLCs into the testes of a male subject. The specification teaches efficient transfection of the mouse SSC and validation of sgRNA targeting human SOHLH1 and TEX 11 gene that target the c.346-1G>A mutation of SOHLH1 intron 3. The specification prophetically contemplates genetically modifying SSC using any of a variety of transfection/transduction reagents. The guidance provided in the specification is limited to use of polyethyleneimine (PEI) to genetically modify SSC that could be transplanted to a mouse testis to produce colonies of spermatogenesis (see example 2, fig. 12). The specification is silent on treating NOA caused by any known or yet to identified mutation by transplanting any transformed PGCLCs that are heterozygous or hemizygous for the genetic mutation into the male subject as specification has not disclosed any sperm ever produced under any condition or that spermatogenesis had occurred in the seminiferous tubule in any male subject having non-obstructive azoospermia (NOA) that is caused by a genetic mutation in any predictable animal model. Fang (Frontiers in Cell and Developmental Biology, 2020, 8:432, 1-12) in a post filing publication teaches “NOA patient-specific iPSCs exhibit poor response to PGCLC induction in vitro, and the compromised differentiation potential for germ cell fate might be correlated with apoptosis mechanism. Moreover, PGCLC specification in vitro cannot completely reconstitute the development of gonadal PGCs in vivo” (see page 10, col. 1, para. 1). Fang teaches epigenetic modifications is involved in the etiology of male idiopathic infertility, such as DNA methylation, modification of histones, and non-coding RNAs (see page 9, col. 1, para. 2). These studies suggest iPSC reprogramming can leave behind epigenetic memory of the original somatic cell type, which can be a barrier to proper germline specification. In view of foregoing, it is apparent that any resulting PGCLCs generated from iPSC derived from a male subject having NOA caused by a genetic mutation in vitro may have abnormal epigenetic profiles. The specification fails to teach how to generate any transformed iPSCs from a male subject having NOA caused by any genetic mutation. The specification is further silent separating mature PGCLCs using any specific surface marker from any remaining undifferentiated iPSCs that is critical to prevent any contamination of the subsequent transplant of a mixed population of PGCLCs with pluripotent cells derived from a make subject having NOA caused by a genetic mutation. Additionally, specification provide no guidance on transplanting PGCLCs of any species derived from iPSC obtained from a male subject having NOA caused by a genetic mutation that is capable of producing sperm for reproduction or restoring fertility. Furthermore, it would be unpredictable to establish complete spermatogenesis and fertility following transplantation of PGCLCs into the testes of the male subject or differentiating the PGCLCs into a functional sperm in vitro to treat NOA in a male subject caused by a genetic mutation, particularly since art teaches NOA patient-specific iPSCs exhibit poor response to PGCLC induction in vitro Sosa (Nature Communication, 2018, 1-13) in post filing publication reported immature rPGCLCs once transplanted into an adult gonadal niche commit to differentiate towards late rPGCs that initiate epigenetic reprogramming but do not complete the conversion into ENO2-positive spermatogonia (see abstract). It is emphasized that specification is silent in producing any sperm following transplanting the PGCLCs into the testes of the male subject or differentiating the PGCLCs under in vivo or in vitro in any of the working examples and no evidence of spermatogenesis has occurred following the transplantation of transformed PGCLCs that are heterozygous or hemizygous for a genetic mutation that cause NOA in a predictable animal model of NOA. The method is not enabling for differentiating the PGCLCs into sperm in vitro, as there are no teachings in the art or the specification that without first producing sperm in the NOA treated subject or without undergoing spermatogenesis to generate sperm in the NOA treated subject. An artisan would have to perform undue experimentation to make and use the experimentation, without reasonable expectation of success.
The lack of guidance in the specification would force the skilled practitioner to first produce iPSC from a male subject (encompasses human and other mammalian species) having NOA caused by genetic mutation, maintain and transform iPSCs to produce transformed IPSC that differentiate into a mature PGCLC. One of ordinary skill in the art would have to perform undue experimentation to first characterize the resulting PGCLs then select PGCLC s that are heterozygous or hemizygous for the genetic mutation before transplanting the PGCLCs into the testes of the male subject or differentiating the PGCLCs into sperm in vitro for treating NOA, without reasonable expectation of success. Applicant should note that “case law requires that the disclosure of an application shall inform those skilled in the art how to use applicants’ alleged discovery, not to find out how to use it for themselves.” In re Gardner 166 USPQ 138 (CCPA) 1970.
Regarding male infertility, the art teaches that male infertility can have many different causes. Specifically, Raheem et al. (Trends in Urology & Men's Health, September/October, 2011, 8-11) teaches that male infertility can be cause by a variety of mutations in non-obstructive causes. For instance, a deletion in the AZF region of the Y chromosome, the claimed method non-obstructive azoospermia to be treated by the transplantation of genetically modified PGCLCs that corrects the mutation causing NOA into the seminiferous tubules. Raheem continue to teach that there are numerous other genes not yet identified that regulate sperm production, hormone production and hormone receptors. Any defect in such genes will impair fertility (see page 9, col. 2, last para.). The art taches “NOA is not a single genetic condition, but rather a clinical endpoint of a spectrum of alternative pathological processes and sub-phenotypes. Given the large number of genes implicated in spermatogenesis and testicular function, a high heterogeneity in monogenic defects that may cause NOA is expected. Alternative forms of genetic inheritance of the condition are to be considered. NOA may manifest itself through autosomal recessive (AR) mutations inherited from fertile parents and combined to a pathogenic genotype in the homozygous, heterozygous or hemizygous form. Alternatively, NOA can be caused by maternally inherited or de novo mutations in X-chromosomal or dosage sensitive autosomal dominant (AD) genes” (see Kasak et al Human Genetics (2021) 140:135–154, page 137, col. 1, para. 2). However, the claimed method would not treat NOA caused by a mutation taught Kasak and Raheem since the specification has not taught that produces functional sperm were produced by the claimed method following transplantation of transformed PGCLSs that are heterozygous or hemizygous for the genetic mutation causing NOA into the male subject. An artisan would have to perform undue experimentation to make and use the invention, without reasonable expectation of success.
The claims encompass method wherein the recombinant nucleic acid molecule targets any endogenous native locus associated with NOA. The prophetic guidance provided in the specification is limited to a mouse endogenous (non-functional) SOHLH1 locus or safe harbor ROSA26 locus (see figure 12-13). However, prior art teaches stably integrating any nucleic acid at any locus would not necessarily yield predictable gene expression. For instance, Bronson applied homologous recombination to introduce a single transgene copy into the HPRT locus. However, several reports implied that “targeted transgenesis” at the HPRT locus is not suitable to avoid unpredictable position effects in transgenic animals: Insertion of a lacZ gene under the control of the polyoma enhancer/HSV thymidine kinase promoter into Hprt resulted in variable beta-galactosidase expression that was both orientation and cell-type dependent (Shaw- White et al., Transgenic Res. 1: 1-13 (1993)). Transgenes under the control of the human and the chicken B-actin gene promoter showed widespread expression when inserted into the Hprt locus. Unexpectedly, however, the level of transcripts varied strongly in different tissues and expression of these transgenes, in contrast to the endogenous HPRT gene, appeared to be low or undetectable in kidney and liver (Bronson et al., Proc. Natl. Acad. Sci, USA, 93(17): 9067-72 (1996)). Hatada et al. demonstrated that the HPRT locus suppresses the |activity of both, the haptoglobin gene promoter as well as the herpes simplex thymidine kinase promoter in several tissues of mice (Hatada et al., J. Biol., Chem., 274(2):948-55, 1999). Likewise, a human eNOS promoter-LacZ reporter gene placed in the Hprt locus was found to be inactive in hepatic vessels that otherwise express the endogenous eNOS gene (Guillot et al., Physiol. Genomics, Mar. 13, (2):77-83, 2000). In a recent study, Ordova´s et al (Stem Cell Reports, 2015, 5, 918-931) teach AAVS1 locus cannot be considered a universally safe harbor locus for reliable transgene expression in vitro (see abstract) (emphasis added). Ordova´s states that “our studies demonstrate that the AAVS1 locus is not as ‘‘safe’’ as suggested, as transgene expression was variable in both undifferentiated and hepatocyte committed ESC progeny in vitro. The AAVS1 has been described as an open chromatin locus by DNase hypersensitive assays and ChIP, but these studies did not cover the region targeted by the ZFNs in PSCs” (see page 927, col. 1, para. 2). Ordova´s et al continue to “demonstrate that the region targeted by the ZFNs used in this study, but also in other studies and by TALENs and CRISPR/Cas9), can exert silencing in vitro via induction of de novo DNA methylation in undifferentiated hESC (see page 927, col. 1, para. 2). The claim as such do not specify any promoter to drive gene expression. Klatt (Human Gene Therapy, 2020, 31, 3, 199-209) teaches correct promoter choice is essential to achieve sufficient gene expression, for example, in a therapeutic setting. Moreover, the inclusion of insulators and ubiquitous chromatin opening elements could help to overcome transgene silencing for promoters that are prone to epigenetic silencing through DNA methylation or addition of repressive histone marks (see page 209, col. , 1, para. 1). This is critical because silenced or overactive promoter could disrupt spermatogenesis. The art teaches while although genome editing tools (GETs) are efficient at inducing specific double-strand breaks (DSBs), the outcome of DNA repair remains unpredictable. Teboul (see Molecular Therapy, 2020, 20, 1422-1431) teaches GETs need to be assessed in the cellular context(s) for which they are intended in therapy, as the mechanism by which targeted cells repair specific DSBs may differ among cell types, cell-cycle phase, metabolic status, and level of differentiation (see Box 1). The specification fails to teach either by southern blot or whole genome sequencing to screen for clones with random insertion of the transgene or for other off target insertion to repair the mutation. There is no detection of gene-corrected clones either at nucleic acid or protein level as compared to the unmodified clone confirming the expression of transgene under control of any promoter at any locus of an IPSC derived from a male subject having NOA. The specification provides limited guidance on validating the ssRNAs in 293AD cells that are transfected with plasmid DNA containing the sgRNA and Cas9 sequences using PEI. There specification is silent on assessing the specific targeting in the IPSC or resulting differentiated cell as art teaches repair specific DSBs may differ among cell types, cell-cycle phase, metabolic status, and level of differentiation. In view of foregoing, it is apparent that several factors confounded the method of targeted transgenesis at known or yet to be identified safe harbor locus or endogenous nonfunctional locus. The specification fails to provide any guidance to overcome art recognized unpredictability for the breadth of the claims. The specification is further silent on inserting any recombinant nucleic acid molecule that targets any endogenous native locus associated with NOA, or targets or a safe harbor locus in the treatment of NOA caused by any genetic mutation. An artisan would have to perform undue experimentation without reasonable expectation of success in order to make and use the invention.
The claims encompass introducing recombinant nucleic acid molecule, such as a cDNA encoding a therapeutic gene, can express a protein that is missing or downregulated in the resulting transformed cell. Claims are also directed to introducing a recombinant nucleic acid molecule that includes a recombinant DNA template to direct homology directed modification of the treated subject's genome. The art teaches that variation in the efficiency with which a specific gRNA directed Cas9-mediated ablation suggesting that on-target efficiency of site directed mutation is highly gRNA dependent (Hsu et al Nat Biotechnology. 2013 Sep;31(9):827-32, IDS). This is further supported by Cowan et al (USPGPUB 20180141992) who teaches that variation in the efficiency with which a specific gRNA directed Cas9-mediated ablation is observed, even between gRNAs targeting the same exon or nearly overlapping sites (FIG. 5A-E) indicating that on-target efficiency of site directed mutation is highly gRNA dependent as previously noted by Hsu et al (Nat Biotechnology. 2013 Sep;31(9):827-32). In a post filing art, Devkota (BMB Rep. 2018; 51(9): 437-443) raise several issues including (i) immune reaction that is already activated in humans against the most widely used forms of Cas9, (ii) role of p53 after the generation of DSB as p53 is activated in response to DNA damage whether it is induced under natural circumstances or experimentally by Cas9 and (iii) off-target activity of Cas9 and the possibility of abnormal DNA rearrangement after DSB (see page 441, col. 2, para 1). Dow et al (Trends in Molecular Medicine, 2015, 21, 609-621) reported “Despite significant effort to design better sgRNAs, the potential for off-target DNA cleavage or, in the case of dCas9 variants, undesired changes to gene expression or chromatin patterns is an ongoing concern. It is further disclosed that studies have revealed significant variability in the degree of off-target cleavage between individual sgRNAs, implying that it may be difficult to accurately predict off-target potential a priori.(see page 618, last para.).In a recent publication, Kosicki et al (Nature Biotechnology, 2018, 765-771) report significant on-target mutagenesis, such as large deletions and more complex genomic rearrangements at the targeted sites in mouse embryonic stem cells, mouse hematopoietic progenitors and a human differentiated cell line. Using long-read sequencing and long-range PCR genotyping, we show that DNA breaks introduced by single-guide RNA/Cas9 frequently resolved into deletions extending over many kilobases. Furthermore, lesions distal to the cut site and crossover events were identified. The observed genomic damage in mitotically active cells caused by CRISPR—Cas9 editing may have pathogenic consequences (see abstract). In the instant case, specification as well as claims fails to provide any information as to the structural elements required for correcting any genetic mutation that causing infertility has a dominant or recessive mode of inheritance, which can be corrected using the disclosed methods by genetic modification of the germline with germline transmission to progeny. The specification prophetically provides schematic as how to insert the corrective transgene into the endogenous locus that will allow regulation of expression from the endogenous promoters, as the CRISPR/Cas9 technology enables precise genomic integration (see fig. 9-11 of the specification). However, specification fails to provide any evidence that induced pluripotent stem cells (iPSCs) of the male subject having NOA caused by a genetic mutation could be maintained, transformed, and then differentiating the isolated transformed iPSC to genetically modify via any mean to correct any recessive or dominant known or yet to be identified heterozygous or homozygous mutation that causes NOA. It should be noted that while iPSCs cells are promising and likely to enable genomic correction in near future, however, art teaches human iPSCs may change (epi)genetically when exposed to an in vitro environment. DNA methylation of maternal and paternal imprinted genes (see Liang above). Cox et al (Nature Medicine, 2015, 21, 121-131) with respect to genome editing describe “success of a given strategy will depend on the ease with which a therapeutic modification 'threshold' is achieved, a criterion that is governed by the fitness of edited cells; the DSB repair pathway used to edit the genome; and the efficiency of delivery of genome editing molecules to target cell types. If edited cells have an increased fitness relative to unedited cells, this will result in a selective advantage for edited cells, reducing the number of cells that initially needs to be edited to reverse disease symptoms (Fig. 2, see page 124, col. l to col. 2). Benjamin et al continue to teach that “Inefficient modification of target loci will be compounded by any inefficiencies in delivery, making tissues lacking robust delivery platforms particularly difficult to treat with this mode of therapy” (see page 127, col. 2, para. 3). Benjamin Cox et al also raise issue of “Genetic modifications are permanent, and deleterious off-target mutations could create cells with oncogenic potential, reduced fitness or functional impairment. Furthermore, oncogenic mutations resulting from off-target editing may lead to expansion of edited cells, and thus even low levels of off-target mutagenesis may have devastating consequences” (see page 128, col. 1, para. 6). Applicants fail to correlate the donor sequences with homology arms described in the specification and art at the time of filing to the “any recombinant nucleic acid molecule” as broadly encompassed by claims. Applicants fail to indicate expression of homology directed modification of mutant protein in the IPSCs of any subject was of a threshold level (specifically of a level capable of treating any genetic condition (eg NOA) as discusses supra in Benjamin Cox et al. Applicants fail to teach the amount of exogenous nucleic acid that corrects any mutation causing NOA in the iPSCs of any species that is required to treat the specific NOA condition as embraced by the breadth of the claims. If the expression in iPSCs that are differentiated to PGCLCs are used to treat a patient with NOA caused by a genetic mutation, it is unclear that any donor sequences with homology arms will target the specific sites in therapeutic effective level that result correction of NOA. The art teaches NOA are believed to be caused by an array of genes, which renders genetic correction substantially more difficult as it would require simultaneous targeting of various loci (see 569, col. 1, para. 3). One of skill in the art would have to (i) identify the mutations causing NOA and then generate a predictable animal model carrying NOA patient-originated mutations, (ii) analyze testicular phenotypes of males, (iii) isolate and in vitro culture of iPSCs, (iv) correction of the genetic defect by CRISPR-Cas9-mediated HDR by designing sgRNA, (v) derivation of repaired iPSCs lines through single-cell expansion, (vii) off-target effect analysis by whole-genome sequencing, (viii) differentiation of iPSCs into the testes of the male subject or differentiating the PGCLCs into sperm in vitro to restore spermatogenesis, The specification fails to treat any subject having NOA caused by any mutation. An artisan would have to perform undue experimentation to make and use the invention without reasonable expectation of success. Given the unpredictability of germline gene therapy taken with the lack of guidance in the specification and the art before the effective filing of instant application, it would have required those of skill undue experimentation to try correcting the mutation causing NOA in iPSCs at random or an endogenous locus such that nucleic acid that corrects the genetic mutation is integrated into the cleaved gene and the transgene is expressed at a level sufficient to produce differentiated PGCKCs to treat NOA of different etiology and pathology as required by the claims. An artisan would have to perform undue experimentation to make and use the invention without reasonable expectation of success.
In conclusion, in view of breadth of the claims and absence of a strong showing by Applicant, in the way of specific guidance and direction, and/or working examples demonstrating the same, such invention as claimed by Applicant is not enabled for the claimed inventions. An artisan of skill would have required undue experimentation to practice the invention because the art of germline gene therapy was unpredictable at the time of filing of this application as supported by the observations in the art record.
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.
Claims 26-35 are rejected under 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph, as being indefinite for failing to particularly point out and distinctly claim the subject matter which the inventor or a joint inventor (or for applications subject to pre-AIA 35 U.S.C. 112, the applicant), regards as the invention.
Claim 26 in part is vague and indefinite to the extent there is no nexus between recitation of alternative embodiment of differentiating the PGCLCs into sperm in vitro to treating NOA in a male subject caused by a genetic mutation. Claims 27-35 are included in the rejection because they directly or indirectly depend from the rejected base claim. Appropriate correction and/or clarification is required.
Conclusion
No claims allowed.
The prior art made of record and not relied upon is considered pertinent to applicant's disclosure. Ramathal et al (US US20160137975, dated 7/10/2014). teaches a method of treating non-obstructive azoospermia (NOA) in a male subject caused by a genetic mutation (para (0009), for example a deletion, in the (AZF) region", para (0042) "a male with azoospermia, which may be non-obstructive azoospermia (NOA) comprising: introducing a recombinant nucleic acid molecule into pluripotent stem cells from the male subject, wherein the nucleic acid molecule corrects the genetic mutation causing the NOA, thereby generating transformed stem cells (para (0081) "genes are optionally introduced into the pluripotent cells prior to performing the method , isolating transformed stem cells that are heterozygous or hemizygous for the genetic mutation, thereby generating isolated transformed stem cells (para (0081) and he pluripotent cells are injected directly into the seminiferous tubules of the donor, where the pluripotent cells are brought into contact with a Sertoli cell environment and induced to differentiate into male germ cells, including sperm cells (see para. 9).
Irie et al (Cell, 2015, 160, 253-268) teaches isolation of hPGCLCs from competent hiPSCs (see fig. 7).
Wnag et al (Scientific Reports (2016), 6, 1-13,) teaches defined culture system for the induction of primordial germ cell-like cells (PGCLCs) from porcine induced PSCs (piPSCs)
Hamra et al (USPGPUB 20190134227, dated 5/9/2019, filed on 4/5/2017, EFD 4/18/2016) teach a method for generating a germline modification in the genome of a mammal comprising contacting the spermatogonial stem cells (SSCs) in said mammal with Cas9 and at least one guide RNA, wherein gerrmline modification comprises insertion of the single-stranded oligonucleotide by non-homologous end joining (NHEJ)-mediated insertion repair or homology-directed repair (HDR).
Orwig et al “Gene therapy in and around the germline: International Summit on Human Gene Editing: A Global Discussion,” Washington, DC, December 1-3, 2015 (18 pages), IDS)..
Wu et al (Cell Research (2015) 25:67-79, IDS) teach correction of a genetic disease by CRISPR-Cas9 mediated gene editing in mouse SSC.
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/ANOOP K SINGH/ Primary Examiner, Art Unit 1632