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 .
DETAILED ACTION
The text of those sections of Title 35, U.S. Code not included in this action can be found in a prior Office action.
This Action is in response to the papers filed on June 2, 2026. Pursuant to the amendment filed on June 2, 2026, claims 2-5, 9-10 and 15 are currently pending. Claim 15 has been amended in Applicant’s Amendments filed on June 2, 2026. No claims have been added, and no claims have been cancelled in the Amendment.
Therefore, claims 2-5, 9-10, and 15 are currently under examination to which the following grounds of rejection are applicable.
Response to Arguments
Maintained Objections/Rejections in response to Applicants’ arguments or amendments:
New Grounds of Rejection:
Claim Rejections - 35 USC § 103
Claims 2, 4, 5, 9, and 15 remain rejected under 35 U.S.C. 103 as being unpatentable over Biffi et al. (Science 341, 1233158 (2013); of record IDS filed on February 21, 2023; hereinafter ‘Biffi’) in view of Krawetz et al. (Tissue Engineering Part C: Methods 16.4 (2010): 573-582; hereinafter ‘Krawetz’; of record) and Zanta-Boussif et al. (Gene Therapy 16.5 (2009): 605-619; of record IDS filed on February 21, 2023; hereinafter ‘Zanta-Boussif’) as evidenced by STIC Sequence Search for SEQ ID NO:3; of record). The STIC Sequence Search has been supplied in the Office Action dated May 15, 2025.
Claim 15 is directed to a method of maintaining the multipotency for lentiviral vector (LV) infection of a hematopoietic stem cell transduced with a recombinant LV comprising: an arylsulfatase A (ARSA) gene encoding an arylsulfatase A polypeptide, wherein said LV is a self-inactivating (SIN) lentiviral vector, wherein the recombinant LV comprises a modified Woodchuck Post-transcriptional Regulatory Element having a nucleic acid sequence as set forth in SEQ ID NO: 3, the method comprising incubating hematopoietic stem cells that comprise the recombinant LV in a media comprising thrombopoietin (TPO), stem cell factor (SCF), FMS-like tyrosine kinase-3 (FLT3), and Interleukin 3 (IL-3) factors, and wherein the media further comprises kinase inhibitors that inhibit mammalian target of rapamycin (mTOR) and Rho-associated protein kinase (ROCK) activity.
Regarding claim 15, Biffi teaches CD34+ cells/ hematopoietic stem cells (HSCs) that are isolated from a patient which were then placed in culture media containing interleukin-3 (IL-3), thrombopoietin (TPO), stem cell factor (SCF), and Flt3 ligand, and the cells were then transduced with a self-inactivating lentiviral vector comprising the ARSA gene ((p 9, col 1), “The vector used in this study (pCCLsin.cPPT.hPGK.hARSA.WPREmut6 - PGK.ARSA.LV) is a self-inactivating lentiviral vector produced with a third-generation split packaging system”; p 8, col 2-3); HSC gene marking in vivo revealed evidence of self-renewal and multilineage potential of the transduced engrafted HSCs suggesting maintenance of multipotent potential (p 5, col 2- p 6, col 1).
Biffi does not teach any kinase inhibitors with the transduced HSCs, more specifically kinase inhibitors that inhibit mTOR and ROCK activity, and furthermore the vector employing the same WPRE comprised in SEQ ID NO: 3.
Krawetz teaches that ROCK inhibitors (ROCKi) increase the survival of dissociated, single human embryonic stem cells, and moreover, facilitates the formation of aggregates in static cultures (p 573, col 2, par 1-2). This is vital because hES cells do not survive when transitioned directly from static culture to a suspension culture, and therefore the addition of ROCKi would increase aggregate formation, and subsequently cell survival (p 576, col 1, par 1). Furthermore, the use of Rapamycin, a mTOR inhibitor, revealed at high concentrations the hES cell cultures no longer expanded, but at lower concentrations spontaneous differentiation no longer occurred (p 580, col 1, par 2).
Biffi and Krawetz do not teach the LV comprises a modified Woodchuck Posttranscriptional Regulatory Element having a nucleic acid sequence as set forth in SEO ID NO: 3.
Zanta-Boussif teaches a modified WPRE, specifically mut1, consisting of a single nucleotide mutation in the WHX protein translation start site (Fig. 1). The WPRE sequence overlaps with that of the woodchuck hepatitis virus X protein (WHX), which is a transcriptional activator of about 150 AA implicated in the development of liver tumors. The WPRE inserted in most vectors contains the WHX gene promoter and an open-reading frame coding for the first 61 AA of WHX in its 3’ region (p 606, col 1). Zanta-Boussif found that the mut1 version was as efficient in expressing high levels of the transgene in comparison to the wild-type WPRE sequence, and therefore functioned equivalently despite losing the ability to translate the WHX polypeptide(abstract, p 606, col 2). The mut1 version is displayed below in Fig. 1a:
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The STIC Sequence Search has provided no alignments that are 100% identical, in particular the closest identical sequence is around 99.7% identical as seen below for alignments compared across other Published Applications, specifically the differences are a single nucleotide difference in the same location described by Zanta-Boussif. Provided below is a snapshot of the most similar alignments, in addition to Result #3 (filing date of 03-03-2010) sequence alignment that depicts the same mismatch as Mut1.
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The specification describes SEQ ID NO: 3 as WPRE mut 1 (same name used by Zanta-Boussif) wherein a thymine is mutated to guanine as seen in bold “("t" to "G" mutation site in BOLD)” (p 6, lines 22-24). The region highlighted for SEQ ID NO: 3 is 575 nt in length (sites 1698-2273) wherein the mutation occurs at site 2107 which is the 409th nt: tccaGggct as depicted in instant Figure 11. It appears based on the location and specific mutation made, that Zanta-Boussif teaches this mutation wherein the WHX translation is abrogated, and expression levels of the transgene are maintained.
It would have been prima facie obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have been motivated to include kinase inhibitors to inhibit ROCK and mTOR based on the teachings of Krawetz that describe the combination being necessary for moving stem cells from a static culture to a suspension culture. In particular, the use of a ROCK inhibitor at low concentrations promoted aggregation and the mTOR inhibitor blocked spontaneous differentiation by maintaining pluripotency. Therefore, there would a reasonable expectation that using such inhibitors would have similar outcomes with the HSCs described in the claimed invention wherein multipotency is maintained.
Secondly, it would have been prima facie obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have modified the WPRE of the vector to be have a mutation in the WHX translation start site because Zanta-Boussif teaches the same mutation wherein translation of the WHX is prevented which is implicated in liver tumors, and expression of the transgene is maintained relative to the wild-type version, and altogether functioned equivalently. Therefore, there is a reasonable expectation of success that incorporation of this mut1 sequence by Zanta-Boussif would lead to similar outcomes for the claimed invention in maintaining high expression of the ARSA gene in the self-inactivating (SIN) lentiviral vector.
Claim 2, dependent on claim 15, recites wherein the vector comprises a recombinant ARSA gene under the control of an ARSA gene 5' promoter and an ARSA 3' enhancer.
Biffi teaches lentiviral vectors “(LVs) encoding the human ARSA cDNA under the control of the human phosphoglycerate kinase promoter (PGK)” (p 1233158-1, col 3, par 3). Moreover, Biffi teaches Metachromatic leukodystrophy (MLD) is an inherited lysosomal storage disease caused by mutations in the Arylsulfatase A gene, the reference treated patients diagnosed with MLD wherein gene replacement was accomplished by using lentiviral vectors within hematopoietic stem cells (HSCs).
Biffi does not teach employing the 5’ promoter and 3’ enhancer of ARSA, nor a recombinant ARSA.
It would have been prima facie obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have employed the endogenous ARSA promoter in addition to the enhancer regions found within the genomic sequence based on these sequences being well-known at the time of filing of the claimed invention. In particular, the goal of MLD treatment is to restore ARSA expression with a functional copy, and therefore, it would be obvious to employ all the same endogenous regulatory elements, i.e. promoters, enhancers, absent any evidence of unexpected results. Secondly, in respect to the lentivirus vector encoding a recombinant ARSA, it would have been obvious to modify the coding sequence in view of the system/ organism being used in such as animal studies.
Regarding claim 4, dependent on claim 15, Zanta-Boussif teaches a self-inactivating lentiviral vector that comprises a Rev Responsive Element (RRE) (Fig. 1b).
Regarding claim 5, dependent on claim 15, Biffi teaches the vector contains a central polypurine tract (“cPPT”), in addition to containing a post-translational regulatory element, specifically a modified Woodchuck Post-transcriptional Regulatory Element (WPRE) (“WPREmut6”) (p 5, col 2). Moreover, Zanta-Boussif teaches a self-inactivating vector that comprises a central polypurine tract (Fig. 1).
Regarding claim 9, dependent on claim 15, Biffi teaches the vector encodes the ARSA gene, that is the human ARSA gene (“CD34+ cells were stimulated ex vivo with early acting cytokines in serum-free medium and transduced with purified third-generation LVs encoding the human ARSA cDNA under the control of the human phosphoglycerate kinase promoter (PGK) (fig. S2).”; p 1, col 3, par 3).
Claim 3 remains rejected under 35 U.S.C. 103 as being unpatentable over Biffi et al. (Science 341, 1233158 (2013); of record IDS filed on February 21, 2023; hereinafter ‘Biffi’) in view of Krawetz et al. (Tissue Engineering Part C: Methods 16.4 (2010): 573-582; hereinafter ‘Krawetz’; of record) and Zanta-Boussif et al. (Gene Therapy 16.5 (2009): 605-619; of record IDS filed on February 21, 2023; hereinafter ‘Zanta-Boussif’) as applied to claim 15 above, and further in view of Malik (US-2019/0276844-A1; of record).
The disclosure of Biffi in view of Krawetz and Zanta-Boussif is applied as in the 103 rejections above, the content of which is incorporated herein, in its entirety.
Regarding claim 3, dependent on claim 15, Biffi does not teach the vector used includes an insulator.
Malik teaches a modified self-inactivating (SIN) lentiviral vector for transduction into hematopoietic stem cells (HSCs) that comprises a cHS4 chromatin insulator that permits higher titer expression of the vector and an env fragment containing a rev response element (RRE) that is essential for efficient assembly/packaging of lentivirus particles as opposed to mRNA transport (0020, 0027, 0104, claim 27).
It would have been prima facie obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have modified the lentiviral vector taught by Biffi is to include both an insulator and RRE sequences in order to improve outcomes related to gene expression and viral particle assembly as Malik has described with such sequences. Furthermore, there is an expectation that the combination of these sequences as taught in a lentiviral vector would lead to the predictable outcome of the claimed lentiviral vector having improved gene expression and number of viral particles due to these sequences’ taught characteristics.
Claim 10 remains rejected under 35 U.S.C. 103 as being unpatentable over Biffi et al. (Science 341, 1233158 (2013); of record IDS filed on February 21, 2023; hereinafter ‘Biffi’; of record) in view of Krawetz et al. (Tissue Engineering Part C: Methods 16.4 (2010): 573-582; hereinafter ‘Krawetz’; of record) and Zanta-Boussif et al. (Gene Therapy 16.5 (2009): 605-619; of record IDS filed on February 21, 2023; hereinafter ‘Zanta-Boussif’) as applied to claims 9 and 15 above, and further in view of Oshikawa et al. (Molecular vision 15 (2009): 482.; STIC Sequence Search Result; hereinafter ‘Oshikawa’; of record). The STIC Sequence Search has been supplied in Office Action dated May 15, 2025.
The disclosure of Biffi in view of Krawetz and Zanta-Boussif’ is applied as in the 103 rejections above, the content of which is incorporated herein, in its entirety.
Claim 10, dependent on claim 9, is directed to wherein the ARSA gene has a nucleic acid sequence as set forth in SEQ ID NO: 2. Biffi teaches the human ARSA gene within the vector, “the vector used in this study (pCCLsin.cPPT.hPGK.hARSA.WPREmut6 - PGK.ARSA.LV) is a self-inactivating lentiviral vector produced with a third-generation split packaging system” (p 8, col 2); yet Biffi does not teach the particular ARSA sequence.
The STIC Sequence Search determined the instant SEQ ID No: 2 is 100% identical to Result #6, ID# AB448736 listed as “Homo sapiens ARSA mRNA for arylsulfatase A, complete cds.” (The full alignment has been previously provided). The sequence was published by Oshikawa et al wherein the reference describes the purpose as “The aim of this study was to characterize the arylsulfatase I (ARSI) gene that has been shown to be preferentially expressed in the human retinal pigment epithelium cell line ARPE-19 and to propose it as a candidate gene responsible for inherited eye diseases such as retinitis pigmentosa (RP).” (Abstract).
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It would have been prima facie obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have employed SEQ ID No: 2 for the ARSA coding sequence of the disclosed vector based on this sequence being well-known at the time of the filing of the instant application as the human ARSA coding sequence.
Response to Applicants' Arguments as they apply to rejection of claims 2-5, 9-10, and 15 rejected under 35 USC § 103
Starting on page 4 of the remarks filed on June 2, 2026, Applicants essentially argue the following:
In relation to claim 15, Applicants' describe starting on page 5, that (1) Biffi does not disclose the use of media comprising kinase inhibitors to incubate hematopoietic stem cells that comprise a recombinant LV, (2) Krawetz does not refer to use of hematopoietic stem cells as host cells, let alone hematopoietic stem cells transduced with a recombinant L V comprising an ARSA gene and WPRE having a nucleic acid sequence as set forth in SEQ ID NO: 3.
“A person of ordinary skill in the art (POSITA) would not have been motivated to combine Biffi and Krawetz in view of their disparate aims, in which Biffi's aim is to develop a therapeutic for metachromatic leukodystrophy and Krawetz's aim is to develop a protocol to expand human embryonic stem cells.”
“Moreover, there would not have been a reasonable expectation of success in combining the teachings of Biffi and Krawetz because a POSITA would not have assumed that ROCKi and rapamycin would have the same or similar effects on hematopoietic stem cells transduced with a recombinant LV as they do on human embryonic stem cells that were not transduced with a recombinant LV.”
“Therefore, a skilled worker with Zanta-Boussif in-hand would have used mut6 WPRE and would have had no motivation to use a mutated mut1 WPRE in an LV.”
The arguments have been fully considered but are not persuasive due to the following reasons:
Regarding the first presented argument, the argument is directed to how each reference, Biffi and Krawetz does not teach the entirety of the limitations presented in claim 15. 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). The rejection of claim 15 makes it clear as to which limitations each reference teaches, for example the kinase inhibitors are taught by Krawetz and Biffi teaches hematopoietic stem cells as host cells transduced with a recombinant LV comprising an ARSA gene with a WPRE, particularly mut6. Altogether, it is the combination of the references that teach the limitations presented in claim 15. Regarding the second presented argument, despite the focuses of the references being different it remains clear to use these reference teachings to arrive at the claimed invention because of the findings therein. In particular, Krawetz demonstrates the use of a ROCK inhibitor at low concentrations promoted aggregation and the mTOR inhibitor blocked spontaneous differentiation by maintaining pluripotency, and therefore the combination of kinase inhibitors are necessary for moving stem cells from a static culture to a suspension culture. In view of the claimed invention, it remains obvious to use these inhibitors when culturing the hematopoietic stem cell transduced with the taught recombinant LV in order to maintain multipotency.
Regarding the third presented argument, wherein Applicant states there is not a reasonable expectation that ROCKi and rapamycin would have the same or similar effects on hematopoietic stem cells transduced with a recombinant LV as they do on human embryonic stem cells that were not transduced with a recombinant LV, Applicants present no evidence where different outcomes are to be expected than observed with the claimed inhibitors used on non-transduced ESC.
Evidence of unexpected properties may be in the form of a direct or indirect comparison of the claimed invention with the closest prior art which is commensurate in scope with the claims. See In re Boesch, 617 F.2d 272, 205 USPQ 215 (CCPA 1980) and MPEP § 716.02(d) - § 716.02(e). An affidavit or declaration under 37 CFR 1.132 must compare the claimed subject matter with the closest prior art to be effective to rebut a prima facie case of obviousness. In re Burckel, 592 F.2d 1175, 201 USPQ 67 (CCPA 1979). “A comparison of the claimed invention with the disclosure of each cited reference to determine the number of claim limitations in common with each reference, bearing in mind the relative importance of particular limitations, will usually yield the closest single prior art reference.” In re Merchant, 575 F.2d 865, 868, 197 USPQ 785, 787 (CCPA 1978) (emphasis in original). Where the comparison is not identical with the reference disclosure, deviations therefrom should be explained, In re Finley, 174 F.2d 130, 81 USPQ 383 (CCPA 1949), and if not explained should be noted and evaluated, and if significant, explanation should be required. In re Armstrong, 280 F.2d 132, 126 USPQ 281 (CCPA 1960) (deviations from example were inconsequential). See also MPEP 716.02e.
Regarding the fourth presented argument, Applicant cites from Zanta-Boussif how the WPRE mutant, i.e. mut6, is recommended, and therefore one would not be motivated to use mut1 for which the claims are directed, i.e. SEQ ID NO: 3. The finding of mut6 were similar to mut1 in relation to transgene expression and lentiviral production (Table 2, Fig. 4a, b). The paper then further explored in vivo work with only one of the mutants, for which they employed mut6. These findings do not take away from the previous findings for mut1, because there is no evidence that mut1 performed worse than mut6, but rather was not included in all of the experiments tested as seen in Fig 4c, d; Fig 5-6; and Tables 3-5. The reason for only using mut6 in both the in vivo and ex vivo studies is not expanded upon, yet based on the findings presented in Fig. 2, 3 and Tables 1 and 2, there is a likely expectation that they would have performed similarly since both mutants have similar outcomes for transgene expression and vector production. Altogether, the findings presented by Zanta-Boussif for mut1 are sufficient for the incorporation of this sequence into the claimed LV.
Conclusion
Claims 2-5, 9-10, and 15 are rejected No claims are allowed.
THIS ACTION IS MADE FINAL. Applicant is reminded of the extension of time policy as set forth in 37 CFR 1.136(a).
A shortened statutory period for reply to this final action is set to expire THREE MONTHS from the mailing date of this action. In the event a first reply is filed within TWO MONTHS of the mailing date of this final action and the advisory action is not mailed until after the end of the THREE-MONTH shortened statutory period, then the shortened statutory period will expire on the date the advisory action is mailed, and any nonprovisional extension fee (37 CFR 1.17(a)) pursuant to 37 CFR 1.136(a) will be calculated from the mailing date of the advisory action. In no event, however, will the statutory period for reply expire later than SIX MONTHS from the mailing date of this final action.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to MICHAEL A RIGA whose telephone number is (571)270-0984. The examiner can normally be reached Monday-Friday (8AM-6PM).
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/MICHAEL ANGELO RIGA/ Examiner, Art Unit 1634
/TERESA E KNIGHT/ Primary Examiner, Art Unit 1634
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