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 .
Applicant’s amendment filed on 07/14/2026 has been entered.
Amended claims 99-118 are pending in the present application.
Applicant elected previously without traverse of Group I. Applicant failed previously to elect any of the required species in the Restriction Requirement dated 01/22/2026 (top of page 8). However, in the Amendment dated 07/14/2026, Applicant elected the following species: (i) miRNA; and (ii) miR-124 (related to the elected Group I). However, the examiner has examined both miR-124 and siRNA against the fusion breakpoint of a FGFR3-TACC3 gene fusion product in the Non-Final Office action dated 04/14/2026.
Claims 111-118 were withdrawn previously from further consideration because they are directed to a non-elected invention.
Accordingly, amended claims 99-110 are examined on the merits herein.
Priority
The present application is a 371 of PCT/US2022/029684, filed on 05/17/2022; which claims benefit of the provisional application 63/191202, filed on 05/20/2021.
Upon review of the specifications of the above PCT/US2022/029684 and the provisional application, it is determined that claims under examination have the effective filing date of 05/20/2021.
Response to Amendment
1. The rejection under 35 U.S.C. 102(a)(1) as being anticipated by Shpall et al (WO 2019/099927; IDS) was withdrawn in light of currently amended independent claim 99, particularly with the new limitation “wherein steps (a) and (b) utilize media comprising L-alanyl-L-glutamine dipeptide”.
2. The rejection under 35 U.S.C. 102(a)(1) as being anticipated by Lang et al (Neuro-Oncology 20:380-390, 2018; IDS) and evidenced by the Lonza Product Overview for MSCs (2018) was also withdrawn in light of currently amended independent claim 99, particularly with the new limitation “wherein steps (a) and (b) utilize media comprising L-alanyl-L-glutamine dipeptide”.
3. All 103 rejections that were set forth in the Non-Final Office action dated 04/14/2026 were also withdrawn in light of currently amended independent claim 99, particularly with the new limitation “wherein steps (a) and (b) utilize media comprising L-alanyl-L-glutamine dipeptide”.
Claim Rejections - 35 USC § 103
The following is a quotation of 35 U.S.C. 103 which forms the basis for all obviousness rejections set forth in this Office action:
A patent for a claimed invention may not be obtained, notwithstanding that the claimed invention is not identically disclosed as set forth in section 102, if the differences between the claimed invention and the prior art are such that the claimed invention as a whole would have been obvious before the effective filing date of the claimed invention to a person having ordinary skill in the art to which the claimed invention pertains. Patentability shall not be negated by the manner in which the invention was made.
Amended claims 99-103 and 105-107 are rejected under 35 U.S.C. 103 as being unpatentable over Shpall et al (WO 2019/099927; IDS) in view of Kent Lia (Cell Culture Basics: Stem Cell Media-The “What and “Why”, 8 pages, 2016). This is a new ground of rejection necessitated by Applicant’s amendment.
The instant claims encompass a method of producing therapeutic exosomes comprising the steps of: (a) culturing mesenchymal stem cells (MSCs); and (b) collecting the exosomes from the culture; wherein the collected exosomes are electroporated to load one or more therapeutic agents into the exosomes; and wherein steps (a) and (b) utilize media comprising L-alanyl-L-glutamine dipeptide.
Shpall et al already disclosed at least a method of manufacturing clinical grade exosomes derived from mesenchymal stem cells (MSCs) ( e.g., bone marrow-derived MSCs, adipose-derived MSCs), the method comprises: (i) culturing the MSCs in a functionally closed bioreactor (e.g., a hollow fiber bioreactor, a Terumo cell expansion system) to confluency (e.g., 75-95% or 80-90% confluency) in media comprising human platelet lysate (PLT); (ii) further culturing the cells in media essentially free of PLT (e.g., free of PLT); (iii) collecting conditioned media fractions from the bioreactor; (iv) isolating exosomes from the conditioned media fractions; and further (v) loading the exosomes with a therapeutic agent (e.g., one or more cytokines, chemotherapeutic drugs, nucleic acids, small molecules, RNA such as siRNA, miRNA, shRNA, or preferably KRAS siRNA), wherein loading comprises electroporating the exosomes in FDA-approved, sterile, and non-pyrogenic Plasmalyte-A buffer (see at least Abstract; Summary; particularly paragraphs [0005], [0007], [0009], [0017]-[0019], [0023]-[0025], [0045]). Shpall et al also taught that isolating comprises filtration and ultracentrifugation of the pooled fractions to obtain an exosome-containing pellet and resuspending the exosome-containing pellet in a buffer such as Plasmalyte-A (paragraphs [0017]-[0018]). Shpall et al also disclosed that the cells may be seeded in the bioreactor in any suitable cell culture media such as alpha MEM media supplemented with L-glutamine, and the media may be supplemented with one of more of growth factors, cytokines, hormones, or B27, antibiotics, vitamins and/or small molecule drugs; and particularly the media is serum-free (paragraphs [0081]-[0082] and [00180]). Shpall et al further taught that the cells may be incubated at room temperature, and the incubator/bioreactor may be humidified and have an atmosphere that is about 5% CO2 and about 1% O2, in some embodiments, the CO2 may range from about 1-20%, 2-10%, or 3-5% and O2 concentration may range from about 1-20% (paragraph [0083]). Please note that the rest of the atmosphere in the incubator/bioreactor is predominantly nitrogen. Shpall et al described that hollow fiver bioreactors may have the cells embedded within the lumen of the fibers, with the medium perfusing the extra-lumenal space or, alternatively, may provide gas and medium perfusion through the hollow fibers, with the cells growing within the extraluminal space; and hollow fiber bioreactors may include and are not limited to the Caridian (Terumo)BCT Quantum Cell Expansion System (an automated system) (paragraph [0090]). Shpall et al further taught that the bioreactor may be primed prior to seeding of the cells, and the priming may also comprise coating the bioreactor with an extracellular matrix protein, such as fibronectin (paragraph [0092]). In an exemplification, Shpall et al disclosed electroporation of exosomes in the range of 2.5x1010 to 2.5x1012 exosomes with specific siRNA using the 4D Nucleofactor system with each of the three different Nucleofector Solutions SE, SF and SG in combination with 16 different Nucleofector Programs; and the efficiency of each condition to efficiently incorporate the siRNA into MSC-derived exosomes was evaluated by apoptosis of recipient cells, induced by MSC-exosomes carrying siRNA in Fig. 7 (% apoptotic cells ranges from 2% to 50%; paragraph [0037]; and Example 2); as well as demonstrating that Plasmalyte-A produced the optimal electroporation result in comparison with Nucleofector Solutions SE, SF and SG with about 75% of apoptotic cells (paragraphs [0038], [00190]; Example 3; and Fig. 8A).
Shpall et al did not teach explicitly that the method utilizes media comprising L-alanyl-L-glutamine dipeptide in step (a) of culturing mesenchymal stem cells and in step (b) of collecting the exosomes from the culture.
Before the effective filing date of the present application (05/20/2021), Kent already disclosed that all stem cell media essentially contain the same basic components: a basal medium, buffer system, glutamine, serum (or serum alternative), specific growth factors, and additional supplements; and looked at a few of the main media components to better understand their importance and influence on cell cultures (Introduction paragraph at page 1). Kent specifically taught that L-alanyl-L-glutamine is a dipeptide that is stable in cell culture medium over longer periods of time, even at 37 0C, due to strong bonds within L-alanyl-L-glutamine that stabilize the compound from degradation while the L-glutamine itself is still readily available to the cells; and it is used to combat the rapid breakdown and increase glutamine stability within cell culture media (see section titled “L-Alanyl-L-Glutamine” at page 3). Kent further taught that supplementing stem cell media with L-alanyl-L-glutamine can extend the shelf-life of the media at 4 0C and greatly reduce the problems associated with the breakdown of glutamine into ammonia waste (last sentence of last paragraph at page 3).
Accordingly, it would have been obvious for an ordinary skill in the art to modify the teachings of Shpall et al by also utilizing the L-alanyl-L-glutamine dipeptide in the culture media used for steps (a) and (b), in light of the teachings of Kent as set forth above.
An ordinary skilled artisan would have been motivated to carry out the above modification because Kent taught various advantages for the inclusion of the L-alanyl-L-glutamine dipeptide in stem cell culture media instead of glutamine, such as enhanced the shelf-life of the media at 4 0C, greatly reduced the problems associated with the breakdown of glutamine into ammonia waste (ammonia waste adversely affects cell growth), and a stable source of glutamine for stem cell culture.
An ordinary skilled artisan would have a reasonable expectation of success in light of the teachings of Shpall et al and Kent; coupled with a high level of skill for an ordinary skilled artisan in the relevant art.
The modified method resulting from the combined teachings of Shpall et al and Kent as set forth above is indistinguishable and encompassed by the presently claimed method.
Therefore, the claimed invention as a whole was prima facie obvious in the absence of evidence to the contrary.
Claim 104 is rejected under 35 U.S.C. 103 as being unpatentable over Shpall et al (WO 2019/099927; IDS) in view of Kent Lia (Cell Culture Basics: Stem Cell Media-The “What and “Why”, 8 pages, 2016) as applied to claims 99-103 and 105-107 above, and further in view of Zeringer et al (World J Methodol 3:11-18, 2013).
The combined teachings of Shpall et al and Kent were presented above. However, none of the cited references teach explicitly that the modified method further comprising the step of extracting a sample from the system and testing the sample for one or more characteristics of the exosomes.
Before the effective filing date of the present application (05/20/2021), Zeringer et al already disclosed at least a fast and efficient extraction of exosomes from HeLa cell culture media, and samples were analyzed by Western blots with an antibody specific to CD63- a well characterized exosomal marker– to confirm that clean exosome population was recovered, along with sizing and quantitation of exosomes were performed with the NanoSight LM10 instrument to track the size and number of the nanoparticles (Abstract; and particularly Section titled “Extraction of exosomes from cell media and serum” at page 14).
Accordingly, it would have been obvious for an ordinary skill in the art to further modify the combined teachings of Shpall et al and Kent by also extracting a sample from the system at one or more of the 6 collection times, including at days 9 and/or 19, to test for one or more characteristics of the exosomes to track at least the size, quantitation and proper markers of generated exosomes, in light of the teachings of Zeringer et al as set forth above.
An ordinary skilled artisan would have been motivated to further carry out the above modification because Zeringer et al already disclosed at least a fast and efficient extraction of exosomes from HeLa cell culture media, and samples were analyzed by Western blots with an antibody specific to CD63- a well characterized exosomal marker– to confirm that clean exosome population was recovered, along with sizing and quantitation of exosomes were performed with the NanoSight LM10 instrument to track the size and number of the nanoparticles. Moreover, it is noted that the primary Shpall et al already taught extracting 2 mL conditioned media to test for sterility, endotoxin and mycoplasma at collection days 9 and 19.
An ordinary skilled artisan would have a reasonable expectation of success in light of the teachings of Shpall et al, Kent and Zeringer et al; coupled with a high level of skill for an ordinary skilled artisan in the relevant art.
The modified method resulting from the combined teachings of Shpall et al, Kent and Zeringer et al as set forth above is indistinguishable and encompassed by the presently claimed method.
Therefore, the claimed invention as a whole was prima facie obvious in the absence of evidence to the contrary.
Claims 108-110 are rejected under 35 U.S.C. 103 as being unpatentable over Shpall et al (WO 2019/099927; IDS) in view of Kent Lia (Cell Culture Basics: Stem Cell Media-The “What and “Why”, 8 pages, 2016) as applied to claims 99-103 and 105-107 above, and further in view of Lasorella et al (WO 2016/105517), Lang et al (Neuro-Oncology 20:380-390, 2018; IDS) and Pomatto et al (Molecular Therapy 13:133-144, 2019).
The combined teachings of Shpall et al and Kent were presented above. However, none of the cited references teach explicitly that one or more therapeutic agents is miR-124 or siRNA against the fusion breakpoint of a FGFR3-TACC3 gene fusion product, wherein the concentration of the miRNA or the siRNA for loading is between 1 ug/mL to 200 ug/mL; or the loading efficiency of one or more therapeutic agents is at least 30%.
Before the effective filing date of the present application (05/20/2021), Lasorella et al already identified a highly expressed class of gene fusions in glioblastoma (GBM) which join the tyrosine kinase domain of FGFR genes to the TACC domain of TACC1 or TACC3, and disclosed at least a siRNA that specifically targets a FGFR-TACC fusion gene for treating patients with GBM (Summary of the Invention, particularly last full paragraph at page 1; lines 20-36 at page 5; Section titled “FGFR fusion molecule inhibitors” at pages 42-43; particularly lines 18-23 at page 43). Lasorella et al stated “In another embodiment, an siRNA directed to a human nucleic acid sequence comprising a breakpoint of an FGFR fusion molecule can be generated…..In one embodiment, the hairpin sequences targeting the FGFR3 gene comprise SEQ ID NOS: 182, 183, or 184” (lines 20-23 at page 43).
Additionally, Lang et al also identified miR-124a as the most effective anti-glioma agent against glioma stem cell (GSC) lines, and taught that exosomes containing miR-124a (Exo-miR124) that were isolated from medium of MSCs transduced with lentivirus vectors containing miR-124a resulted in a significant reduction in the viability and clonogenicity of GSCs compared with controls in vitro, as well as in vivo treatment of mice harboring intracranial GSC267 with systemically delivered Exo-miR124 resulted in 50% of animals living long term (Abstract).
Moreover, Pomatto et al already disclosed improved loading of plasma-derived extracellular vesicles (EVs) to encapsulate antitumor miRNAs by electroporation, and they found that the optimized electroporation protocol (750 V and 10 pulses) allowed loading efficiency of 31.63± 5.94% molecules/EV for a synthetic miRNA (cel-39) derived from Caenorhabditis elegans (Abstract; Section titled “Optimization of electroporation protocol to load miRNA in EVs” at page 134; particularly third paragraph on right column at page 134). Pomatto et al also taught that EVs and miRNA were mixed, and the final volume was adjusted to 10 uL using the electroporation buffer; and ratios of 3 x 109 EVs and different miRNA doses were used: 5, 10, or 20 pmol (Section titled “EV loading protocols” on right column at page 141). Under the above disclosed EV loading conditions and assuming the average MW for a standard miRNA is about 7,000 g/mol and the average MW for a standard siRNA is about 13,300 g/mol, the concentration of loaded miRNA and siRNA at a dose of 20 pmol/10 uL for electroporation is about 15 ug/mL and 27 ug/mL, respectively.
Accordingly, it would have been obvious for an ordinary skill in the art to further modify the combined teachings of Shpall et al and Kent by also selecting miR124 and/or siRNA against the fusion breakpoint of a FGFR3-TACC3 gene fusion product as a therapeutic agent to be loaded into MSC-derived exosomes, including at the concentration of 15 ug/mL and 27 ug/mL, respectively, and with a loading efficiency of about 32%, in light of the teachings of Lasorella et al, Lang et al and Pomatto et al as set forth above.
An ordinary skilled artisan would have been motivated to further carry out the above modifications because: (i) Lasorealla et al already taught at least a siRNA that specifically targets a FGFR-TACC fusion gene for treating patients with GBM, including an siRNA directed to a human nucleic acid comprising a breakpoint of an FGFR fusion molecule; (ii) Lang et al also identified miR-124a as the most effective anti-glioma agent in vitro and in vivo; and (iii) Pomatto et al already disclosed the optimized electroporation protocol (750 V and 10 pulses) that allowed loading efficiency of 31.63± 5.94% molecules/EV for a synthetic miRNA (cel-39) derived from Caenorhabditis elegans, and the exemplary EV loading conditions that resulted in the concentration of loaded miRNA and siRNA at a dose of 20 pmol/10 uL for electroporation to be about 15 ug/mL and 27 ug/mL, respectively (assuming the average MW for a standard miRNA is about 7,000 g/mol and the average MW for a standard siRNA is about 13,300 g/mol).
An ordinary skilled artisan would have a reasonable expectation of success in light of the teachings of Shpall et al, Kent, Lasorella et al, Lang et al and Pomatto et al; coupled with a high level of skill for an ordinary skilled artisan in the relevant art.
The modified method resulting from the combined teachings of Shpall et al, Kent, Lasorella et al, Lang et al and Pomatto et al as set forth above is indistinguishable and encompassed by the presently claimed method.
Therefore, the claimed invention as a whole was prima facie obvious in the absence of evidence to the contrary.
Double Patenting
The nonstatutory double patenting rejection is based on a judicially created doctrine grounded in public policy (a policy reflected in the statute) so as to prevent the unjustified or improper timewise extension of the “right to exclude” granted by a patent and to prevent possible harassment by multiple assignees. A nonstatutory double patenting rejection is appropriate where the conflicting claims are not identical, but at least one examined application claim is not patentably distinct from the reference claim(s) because the examined application claim is either anticipated by, or would have been obvious over, the reference claim(s). See, e.g., In re Berg, 140 F.3d 1428, 46 USPQ2d 1226 (Fed. Cir. 1998); In re Goodman, 11 F.3d 1046, 29 USPQ2d 2010 (Fed. Cir. 1993); In re Longi, 759 F.2d 887, 225 USPQ 645 (Fed. Cir. 1985); In re Van Ornum, 686 F.2d 937, 214 USPQ 761 (CCPA 1982); In re Vogel, 422 F.2d 438, 164 USPQ 619 (CCPA 1970); In re Thorington, 418 F.2d 528, 163 USPQ 644 (CCPA 1969).
A timely filed terminal disclaimer in compliance with 37 CFR 1.321(c) or 1.321(d) may be used to overcome an actual or provisional rejection based on nonstatutory double patenting provided the reference application or patent either is shown to be commonly owned with the examined application, or claims an invention made as a result of activities undertaken within the scope of a joint research agreement. See MPEP § 717.02 for applications subject to examination under the first inventor to file provisions of the AIA as explained in MPEP § 2159. See MPEP § 2146 et seq. for applications not subject to examination under the first inventor to file provisions of the AIA . A terminal disclaimer must be signed in compliance with 37 CFR 1.321(b).
The filing of a terminal disclaimer by itself is not a complete reply to a nonstatutory double patenting (NSDP) rejection. A complete reply requires that the terminal disclaimer be accompanied by a reply requesting reconsideration of the prior Office action. Even where the NSDP rejection is provisional the reply must be complete. See MPEP § 804, subsection I.B.1. For a reply to a non-final Office action, see 37 CFR 1.111(a). For a reply to final Office action, see 37 CFR 1.113(c). A request for reconsideration while not provided for in 37 CFR 1.113(c) may be filed after final for consideration. See MPEP §§ 706.07(e) and 714.13.
The USPTO Internet website contains terminal disclaimer forms which may be used. Please visit www.uspto.gov/patent/patents-forms. The actual filing date of the application in which the form is filed determines what form (e.g., PTO/SB/25, PTO/SB/26, PTO/AIA /25, or PTO/AIA /26) should be used. A web-based eTerminal Disclaimer may be filled out completely online using web-screens. An eTerminal Disclaimer that meets all requirements is auto-processed and approved immediately upon submission. For more information about eTerminal Disclaimers, refer to www.uspto.gov/patents/apply/applying-online/eterminal-disclaimer.
Amended claims 99-110 are rejected on the ground of nonstatutory double patenting as being unpatentable over claims 1-37 of U.S. Patent No. 11,766,402 in view of Shpall et al (WO 2019/099927; IDS), Kent Lia (Cell Culture Basics: Stem Cell Media-The “What and “Why”, 8 pages, 2016), Lasorella et al (WO 2016/105517), Lang et al (Neuro-Oncology 20:380-390, 2018; IDS), Pomatto et al (Molecular Therapy 13:133-144, 2019) and Zeringer et al (World J Methodol 3:11-18, 2013). This is a new ground of rejection necessitated by Applicant’s amendment.
Claims 1-37 of U.S. Patent No. 11,766,402 are drawn to a method of manufacturing exosomes from mesenchymal stem cells (MSCs) comprising: (a) culturing the MSCs (e.g., bone marrow-derived MSCs or adipose-derived MSCs; dependent claims 4-5) in a functionally closed bioreactor (e.g., a hollow fiber bioreactor; dependent claim 8) to 75-95% confluency in media comprising human platelet lysate (PLT); (b) further culturing the cells in media essentially free of PLT; (c) collecting one or more conditioned media fractions from the bioreactor wherein conditioned media fractions are collected every 24-48 hours; and (d) isolating exosomes from the conditioned media fractions; the same method further comprising loading the exosomes with a therapeutic agent (e.g., siRNA, miRNA, or shRNA) via electroporating the exosomes (dependent claims 31-35).
The instant claims differ from claims 1-37 of U.S. Patent No. 11,766,402 in reciting specifically “wherein steps (a) and (b) utilize media comprising L-alanyl-L-glutamine dipeptide” (independent claim 99), “wherein the culturing step (a) occurs in the presence of 5% CO2, 20% O2, and conditions balanced with nitrogen” (claim 100); “wherein the method occurs in an automated system configured to comprise continuous perfusion of medium through at least part of the system or a bioreactor having one or more inside surfaces modified to allow adherence of cells” (claim 102); “wherein the one or more surfaces inside the bioreactor are modified to comprise one or more extracellular matrix proteins” (claim 103); “further comprising the step of extracting a sample from the system and testing the sample for one or more characteristics of the exosomes” (claim 104); “wherein step (a) utilizes media that lacks platelet lysate; and wherein step (a) utilizes alpha MEM media, heparin, human platelet lysate, or a combination thereof” (claim 105); “wherein the collected exosomes are electroporated to load one or more therapeutic agents into exosomes, and wherein the collected exosomes are suspended in a sterile, isotonic, non-pyrogenic buffer prior to electroporation” (claim 106); “wherein the one or more therapeutic agents is miR-124, miR-148a, miR-let7i, miR-135a-2, miR-668, miR-942, or miR-657, and wherein the concentration of miRNA for loading is between 1 ug/mL and 200 ug/mL” (claim 108); “wherein the one or more therapeutic agents is siRNA against the fusion breakpoint of a FGFR3-TACC3 gene fusion product, and wherein the concentration of siRNA for loading is between 1 ug/mL and 200 ug/mL” (claim 109); and “wherein the loading efficiency of the one or more therapeutic agents is at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90%” (claim 110).
Before the effective filing date of the present application (05/20/2021), Shpall et al already disclosed at least a method of manufacturing clinical grade exosomes derived from mesenchymal stem cells (MSCs) ( e.g., bone marrow-derived MSCs, adipose-derived MSCs), the method comprises: (i) culturing the MSCs in a functionally closed bioreactor (e.g., a hollow fiber bioreactor, a Terumo cell expansion system) to confluency (e.g., 75-95% or 80-90% confluency) in media comprising human platelet lysate (PLT); (ii) further culturing the cells in media essentially free of PLT (e.g., free of PLT); (iii) collecting conditioned media fractions from the bioreactor; (iv) isolating exosomes from the conditioned media fractions; and further (v) loading the exosomes with a therapeutic agent (e.g., one or more cytokines, chemotherapeutic drugs, nucleic acids, small molecules, RNA such as siRNA, miRNA, shRNA, or preferably KRAS siRNA), wherein loading comprises electroporating the exosomes in FDA-approved, sterile, and non-pyrogenic Plasmalyte-A buffer (see at least Abstract; Summary; particularly paragraphs [0005], [0007], [0009], [0017]-[0019], [0023]-[0025], [0045]). Shpall et al also taught that isolating comprises filtration and ultracentrifugation of the pooled fractions to obtain an exosome-containing pellet and resuspending the exosome-containing pellet in a buffer such as Plasmalyte-A (paragraphs [0017]-[0018]). Shpall et al also disclosed that the cells may be seeded in the bioreactor in any suitable cell culture media such as alpha MEM media supplemented with L-glutamine, and the media may be supplemented with one of more of growth factors, cytokines, hormones, or B27, antibiotics, vitamins and/or small molecule drugs; and particularly the media is serum-free (paragraphs [0081]-[0082] and [00180]). Shpall et al further taught that the cells may be incubated at room temperature, and the incubator/bioreactor may be humidified and have an atmosphere that is about 5% CO2 and about 1% O2, in some embodiments, the CO2 may range from about 1-20%, 2-10%, or 3-5% and O2 concentration may range from about 1-20% (paragraph [0083]). Please note that the rest of the atmosphere in the incubator/bioreactor is predominantly nitrogen. Shpall et al described that hollow fiver bioreactors may have the cells embedded within the lumen of the fibers, with the medium perfusing the extra-lumenal space or, alternatively, may provide gas and medium perfusion through the hollow fibers, with the cells growing within the extraluminal space; and hollow fiber bioreactors may include and are not limited to the Caridian (Terumo)BCT Quantum Cell Expansion System (an automated system) (paragraph [0090]). Shpall et al further taught that the bioreactor may be primed prior to seeding of the cells, and the priming may also comprise coating the bioreactor with an extracellular matrix protein, such as fibronectin (paragraph [0092]). Fig. 2 is a schematic representation of the strategy for the production of conditioned media containing EVs from MSCs cultured on a bioreactor, in which 6 conditioned media fractions (e.g., 250 mL fraction each) are sequentially collected over days 9-19 in sealed bags and cryopreserved until isolation of exosomes, with 2 mL conditioned media are tested for sterility, endotoxin and mycoplasma at days 9 and 19 (paragraphs [0093]-[0095]; and Fig. 2).
Additionally, Kent already disclosed that all stem cell media essentially contain the same basic components: a basal medium, buffer system, glutamine, serum (or serum alternative), specific growth factors, and additional supplements; and looked at a few of the main media components to better understand their importance and influence on cell cultures (Introduction paragraph at page 1). Kent specifically taught that L-alanyl-L-glutamine is a dipeptide that is stable in cell culture medium over longer periods of time, even at 37 0C, due to strong bonds within L-alanyl-L-glutamine that stabilize the compound from degradation while the L-glutamine itself is still readily available to the cells; and it is used to combat the rapid breakdown and increase glutamine stability within cell culture media (see section titled “L-Alanyl-L-Glutamine” at page 3). Kent further taught that supplementing stem cell media with L-alanyl-L-glutamine can extend the shelf-life of the media at 4 0C and greatly reduce the problems associated with the breakdown of glutamine into ammonia waste (last sentence of last paragraph at page 3).
Lasorella et al also already identified a highly expressed class of gene fusions in glioblastoma (GBM) which join the tyrosine kinase domain of FGFR genes to the TACC domain of TACC1 or TACC3, and disclosed at least a siRNA that specifically targets a FGFR-TACC fusion gene for treating patients with GBM (Summary of the Invention, particularly last full paragraph at page 1; lines 20-36 at page 5; Section titled “FGFR fusion molecule inhibitors” at pages 42-43; particularly lines 18-23 at page 43). Lasorella et al stated “In another embodiment, an siRNA directed to a human nucleic acid sequence comprising a breakpoint of an FGFR fusion molecule can be generated…..In one embodiment, the hairpin sequences targeting the FGFR3 gene comprise SEQ ID NOS: 182, 183, or 184” (lines 20-23 at page 43).
Lang et al also identified miR-124a as the most effective anti-glioma agent against glioma stem cell (GSC) lines, and taught that exosomes containing miR-124a (Exo-miR124) that were isolated from medium of MSCs transduced with lentivirus vectors containing miR-124a resulted in a significant reduction in the viability and clonogenicity of GSCs compared with controls in vitro, as well as in vivo treatment of mice harboring intracranial GSC267 with systemically delivered Exo-miR124 resulted in 50% of animals living long term (Abstract).
Moreover, Pomatto et al already disclosed improved loading of plasma-derived extracellular vesicles (EVs) to encapsulate antitumor miRNAs by electroporation, and they found that the optimized electroporation protocol (750 V and 10 pulses) allowed loading efficiency of 31.63± 5.94% molecules/EV for a synthetic miRNA (cel-39) derived from Caenorhabditis elegans (Abstract; Section titled “Optimization of electroporation protocol to load miRNA in EVs” at page 134; particularly third paragraph on right column at page 134). Pomatto et al also taught that EVs and miRNA were mixed, and the final volume was adjusted to 10 uL using the electroporation buffer; and ratios of 3 x 109 EVs and different miRNA doses were used: 5, 10, or 20 pmol (Section titled “EV loading protocols” on right column at page 141). Under the above disclosed EV loading conditions and assuming the average MW for a standard miRNA is about 7,000 g/mol and the average MW for a standard siRNA is about 13,300 g/mol, the concentration of loaded miRNA and siRNA at a dose of 20 pmol/10 uL for electroporation is about 15 ug/mL and 27 ug/mL, respectively.
Furthermore, Zeringer et al already disclosed at least a fast and efficient extraction of exosomes from HeLa cell culture media, and samples were analyzed by Western blots with an antibody specific to CD63- a well characterized exosomal marker– to confirm that clean exosome population was recovered, along with sizing and quantitation of exosomes were performed with the NanoSight LM10 instrument to track the size and number of the nanoparticles (Abstract; and particularly Section titled “Extraction of exosomes from cell media and serum” at page 14).
Accordingly, it would have been obvious for an ordinary skilled artisan before the effective filing date of the present application to modify the method of manufacturing exosomes from MSCs in claims 1-37 of U.S. Patent No. 11,766,402 by also having the features recited in the instant claims; in light of the teachings of Shpall et al, Kent, Lasorella et al, Lang et al, Pomatto et al and Zeringer et al as set forth above with a reasonable expectation of success.
An ordinary skilled artisan would have been motivated to carry out the above modifications because: (i) Shpall et al already taught at least a method of manufacturing clinical grade exosomes derived from mesenchymal stem cells (MSCs) using the Caridian (Terumo)BCT Quantum Cell Expansion System (an automated hollow fiber bioreactor system), wherein the bioreactor may be primed prior to seeding of the cells, and the priming may also comprise coating the bioreactor with an extracellular matrix protein; loading comprises electroporating the exosomes in FDA-approved, sterile, and non-pyrogenic Plasmalyte-A buffer; and that the cells may be incubated in an incubator/bioreactor having an atmosphere that is about 5% CO2 and about 1% O2, and in some embodiments, the CO2 may range from about 1-20%, 2-10%, or 3-5% and O2 concentration may range from about 1-20%. Please note that the rest of the atmosphere in the incubator/bioreactor is predominantly nitrogen; (ii) Kent taught various advantages for the inclusion of the L-alanyl-L-glutamine dipeptide in stem cell culture media instead of glutamine, such as enhanced the shelf-life of the media at 4 0C, greatly reduced the problems associated with the breakdown of glutamine into ammonia waste (ammonia waste adversely affects cell growth), and a stable source of glutamine for stem cell culture; (iii) Lasorealla et al already taught at least a siRNA that specifically targets a FGFR-TACC fusion gene for treating patients with GBM, including an siRNA directed to a human nucleic acid comprising a breakpoint of an FGFR fusion molecule; (iv) Lang et al also identified miR-124a as the most effective anti-glioma agent in vitro and in vivo; (v) Pomatto et al already disclosed the optimized electroporation protocol (750 V and 10 pulses) that allowed loading efficiency of 31.63± 5.94% molecules/EV for a synthetic miRNA (cel-39) derived from Caenorhabditis elegans, and the exemplary EV loading conditions that resulted in the concentration of loaded miRNA and siRNA at a dose of 20 pmol/10 uL for electroporation to be about 15 ug/mL and 27 ug/mL, respectively (assuming the average MW for a standard miRNA is about 7,000 g/mol and the average MW for a standard siRNA is about 13,300 g/mol); and (vi) Zeringer et al already disclosed at least a fast and efficient extraction of exosomes from HeLa cell culture media, and samples were analyzed by Western blots with an antibody specific to CD63- a well characterized exosomal marker– to confirm that clean exosome population was recovered, along with sizing and quantitation of exosomes were performed with the NanoSight LM10 instrument to track the size and number of the nanoparticles. Moreover, it is noted that Shpall et al already taught extracting 2 mL conditioned media to test for sterility, endotoxin and mycoplasma at collection days 9 and 19.
The modified method resulting from claims 1-37 of U.S. Patent No. 11,766,402 along with teachings of Shpall et al, Kent, Lasorella et al, Lang et al, Pomatto et al and Zeringer et al is indistinguishable and encompassed by the presently claimed invention.
Therefore, the claimed invention as a whole was prima facie obvious in the absence of evidence to the contrary.
Amended claims 99-110 are provisionally rejected on the ground of nonstatutory double patenting as being unpatentable over claims 1, 3, 5-7, 11, 16-21, 23, 27, 29, 33 and 43 of copending Application No. 18/000,414 (reference application) in view of Shpall et al (WO 2019/099927; IDS), Lasorella et al (WO 2016/105517), Lang et al (Neuro-Oncology 20:380-390, 2018; IDS), Pomatto et al (Molecular Therapy 13:133-144, 2019) and Zeringer et al (World J Methodol 3:11-18, 2013). This is a modified rejection necessitated by Applicant’s amendment.
Claims 1, 3, 5-7, 11, 16-21, 23, 27, 29, 33 and 43 of copending Application No. 18/000,414 (reference application) are drawn to a method of producing exosomes from mesenchymal stromal cells (MSCs) comprising the steps of: (a) culturing MSCs in the presence of an effective amount of interferon (IFN)y, tumor necrosis factor (TNF)α, interleukin (IL)-1 β, and IL-17; and (b) collecting the exosomes from the culture; the same method wherein the exosomes are loaded to comprise one or more therapeutic agents (dependent claim 43); wherein step (b) utilizes media that lacks platelet lysate and/or wherein step (b) utilizes media that comprises L-alanyl-L-glutamine dipeptide (dependent claim 27); wherein the culture in step (a) further comprises media that comprise L-alanyl-L-glutamine dipeptide and/or wherein the culture in step (a) further comprises alpha MEMTM media, heparin, human platelet lysate and L-alanyl-L-glutamine dipeptide (dependent claim 29).
The instant claims differ from claims 1, 3, 5-7, 11, 16-21, 23, 27, 29, 33 and 43 of copending Application No. 18/000,414 in reciting specifically at least “wherein the culturing step (a) occurs in the presence of 5% CO2, 20% O2, and conditions balanced with nitrogen” (claim 100); “further comprising the step of extracting a sample from the system and testing the sample for one or more characteristics of the exosomes” (claim 104); “wherein the collected exosomes are electroporated to load one or more therapeutic agents into exosomes, and wherein the collected exosomes are suspended in a sterile, isotonic, non-pyrogenic buffer prior to electroporation” (claim 106); “wherein the one or more therapeutic agents is miR-124, miR-148a, miR-let7i, miR-135a-2, miR-668, miR-942, or miR-657, and wherein the concentration of miRNA is between 1 ug/mL and 200 ug/mL” (claim 108); “wherein the one or more therapeutic agents is siRNA against the fusion breakpoint of a FGFR3-TACC3 gene fusion product, and wherein the concentration of siRNA is between 1 ug/mL and 200 ug/mL” (claim 109); and “wherein the loading efficiency of the one or more therapeutic agents is at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90%” (claim 110).
Before the effective filing date of the present application (05/20/2021), Shpall et al already disclosed at least a method of manufacturing clinical grade exosomes derived from mesenchymal stem cells (MSCs) ( e.g., bone marrow-derived MSCs, adipose-derived MSCs), the method comprises: (i) culturing the MSCs in a functionally closed bioreactor (e.g., a hollow fiber bioreactor, a Terumo cell expansion system) to confluency (e.g., 75-95% or 80-90% confluency) in media comprising human platelet lysate (PLT); (ii) further culturing the cells in media essentially free of PLT (e.g., free of PLT); (iii) collecting conditioned media fractions from the bioreactor; (iv) isolating exosomes from the conditioned media fractions; and further (v) loading the exosomes with a therapeutic agent (e.g., one or more cytokines, chemotherapeutic drugs, nucleic acids, small molecules, RNA such as siRNA, miRNA, shRNA, or preferably KRAS siRNA), wherein loading comprises electroporating the exosomes in FDA-approved, sterile, and non-pyrogenic Plasmalyte-A buffer (see at least Abstract; Summary; particularly paragraphs [0005], [0007], [0009], [0017]-[0019], [0023]-[0025], [0045]). Shpall et al also taught that isolating comprises filtration and ultracentrifugation of the pooled fractions to obtain an exosome-containing pellet and resuspending the exosome-containing pellet in a buffer such as Plasmalyte-A (paragraphs [0017]-[0018]). Shpall et al further taught that the cells may be incubated at room temperature, and the incubator/bioreactor may be humidified and have an atmosphere that is about 5% CO2 and about 1% O2, in some embodiments, the CO2 may range from about 1-20%, 2-10%, or 3-5% and O2 concentration may range from about 1-20% (paragraph [0083]). Please note that the rest of the atmosphere in the incubator/bioreactor is predominantly nitrogen. Fig. 2 is a schematic representation of the strategy for the production of conditioned media containing EVs from MSCs cultured on a bioreactor, in which 6 conditioned media fractions (e.g., 250 mL fraction each) are sequentially collected over days 9-19 in sealed bags and cryopreserved until isolation of exosomes, with 2 mL conditioned media are tested for sterility, endotoxin and mycoplasma at days 9 and 19 (paragraphs [0093]-[0095]; and Fig. 2).
Additionally, Lasorella et al already identified a highly expressed class of gene fusions in glioblastoma (GBM) which join the tyrosine kinase domain of FGFR genes to the TACC domain of TACC1 or TACC3, and disclosed at least a siRNA that specifically targets a FGFR-TACC fusion gene for treating patients with GBM (Summary of the Invention, particularly last full paragraph at page 1; lines 20-36 at page 5; Section titled “FGFR fusion molecule inhibitors” at pages 42-43; particularly lines 18-23 at page 43). Lasorella et al stated “In another embodiment, an siRNA directed to a human nucleic acid sequence comprising a breakpoint of an FGFR fusion molecule can be generated…..In one embodiment, the hairpin sequences targeting the FGFR3 gene comprise SEQ ID NOS: 182, 183, or 184” (lines 20-23 at page 43).
Lang et al also identified miR-124a as the most effective anti-glioma agent against glioma stem cell (GSC) lines, and taught that exosomes containing miR-124a (Exo-miR124) that were isolated from medium of MSCs transduced with lentivirus vectors containing miR-124a resulted in a significant reduction in the viability and clonogenicity of GSCs compared with controls in vitro, as well as in vivo treatment of mice harboring intracranial GSC267 with systemically delivered Exo-miR124 resulted in 50% of animals living long term (Abstract).
Moreover, Pomatto et al already disclosed improved loading of plasma-derived extracellular vesicles (EVs) to encapsulate antitumor miRNAs by electroporation, and they found that the optimized electroporation protocol (750 V and 10 pulses) allowed loading efficiency of 31.63± 5.94% molecules/EV for a synthetic miRNA (cel-39) derived from Caenorhabditis elegans (Abstract; Section titled “Optimization of electroporation protocol to load miRNA in EVs” at page 134; particularly third paragraph on right column at page 134). Pomatto et al also taught that EVs and miRNA were mixed, and the final volume was adjusted to 10 uL using the electroporation buffer; and ratios of 3 x 109 EVs and different miRNA doses were used: 5, 10, or 20 pmol (Section titled “EV loading protocols” on right column at page 141). Under the above disclosed EV loading conditions and assuming the average MW for a standard miRNA is about 7,000 g/mol and the average MW for a standard siRNA is about 13,300 g/mol, the concentration of loaded miRNA and siRNA at a dose of 20 pmol/10 uL for electroporation is about 15 ug/mL and 27 ug/mL, respectively.
Furthermore, Zeringer et al already disclosed at least a fast and efficient extraction of exosomes from HeLa cell culture media, and samples were analyzed by Western blots with an antibody specific to CD63- a well characterized exosomal marker– to confirm that clean exosome population was recovered, along with sizing and quantitation of exosomes were performed with the NanoSight LM10 instrument to track the size and number of the nanoparticles (Abstract; and particularly Section titled “Extraction of exosomes from cell media and serum” at page 14).
Accordingly, it would have been obvious for an ordinary skilled artisan before the effective filing date of the present application to modify the method of producing exosomes from MSCs in claims 1, 3, 5-7, 11, 16-21, 23, 27, 29, 33 and 43 of copending Application No. 18/000,414 by also having the recited features of the present claims; in light of the teachings of Shpall et al, Lasorella et al, Lang et al, Pomatto et al and Zeringer et al as set forth above with a reasonable expectation of success.
An ordinary skilled artisan would have been motivated to carry out the above modifications because: (i) Shpall et al already taught at least a method of manufacturing clinical grade exosomes derived from mesenchymal stem cells (MSCs) in which loading the isolated exosomes with a therapeutic agent (e.g., RNA such as siRNA, miRNA, or shRNA) by electroporating the exosomes in FDA-approved, sterile, and non-pyrogenic Plasmalyte-A buffer; and that the cells may be incubated in an incubator/bioreactor having an atmosphere that is about 5% CO2 and about 1% O2, and in some embodiments, the CO2 may range from about 1-20%, 2-10%, or 3-5% and O2 concentration may range from about 1-20%. Please note that the rest of the atmosphere in the incubator/bioreactor is predominantly nitrogen; (ii) Lasorealla et al already taught at least a siRNA that specifically targets a FGFR-TACC fusion gene for treating patients with GBM, including an siRNA directed to a human nucleic acid comprising a breakpoint of an FGFR fusion molecule; (iii) Lang et al also identified miR-124a as the most effective anti-glioma agent in vitro and in vivo; (iv) Pomatto et al already disclosed the optimized electroporation protocol (750 V and 10 pulses) that allowed loading efficiency of 31.63± 5.94% molecules/EV for a synthetic miRNA (cel-39) derived from Caenorhabditis elegans, and the exemplary EV loading conditions that resulted in the concentration of loaded miRNA and siRNA at a dose of 20 pmol/10 uL for electroporation to be about 15 ug/mL and 27 ug/mL, respectively (assuming the average MW for a standard miRNA is about 7,000 g/mol and the average MW for a standard siRNA is about 13,300 g/mol); and (v) Zeringer et al already disclosed at least a fast and efficient extraction of exosomes from HeLa cell culture media, and samples were analyzed by Western blots with an antibody specific to CD63- a well characterized exosomal marker– to confirm that clean exosome population was recovered, along with sizing and quantitation of exosomes were performed with the NanoSight LM10 instrument to track the size and number of the nanoparticles. Moreover, it is noted that Shpall et al already taught extracting 2 mL conditioned media to test for sterility, endotoxin and mycoplasma at collection days 9 and 19.
The modified method resulting from claims 1, 3, 5-7, 11, 16-21, 23, 27, 29, 33 and 43 of copending Application No. 18/000,414 along with teachings of Shpall et al, Lasorella et al, Lang et al, Pomatto et al and Zeringer et al is indistinguishable and encompassed by the presently claimed invention.
Therefore, the claimed invention as a whole was prima facie obvious in the absence of evidence to the contrary.
This is a provisional nonstatutory double patenting rejection.
The prior art made of record and not relied upon is considered pertinent to applicant's disclosure.
Imamoto et al (Cytotechnology 65:135-143, 2013) disclosed advantages of AlaGln as an additive to cell culture medium: use with anti-CD20 chimeric antibody-producing POTELLIGENTTM CHO cell lines (see Abstract).
Conclusions
No claim is 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.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to Quang Nguyen, Ph.D., at (571) 272-0776.
If attempts to reach the examiner by telephone are unsuccessful, the examiner’s acting SPE, James Douglas (Doug) Schultz, Ph.D., may be reached at (571) 272-0763.
To aid in correlating any papers for this application, all further correspondence regarding this application should be directed to Group Art Unit 1631; Central Fax No. (571) 273-8300.
Any inquiry of a general nature or relating to the status of this application or proceeding should be directed to (571) 272-0547.
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/QUANG NGUYEN/Primary Examiner, Art Unit 1631
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