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 .
Claims 1-23 are pending.
Claim Interpretation
Claims 19-23 recites a modified cell made by the method of claim 1. The limitation “made by the method of claim 1” is interpreted as a product-by-process claim. Therefore, claims 19-23 does not require the modified cell to be distinguished solely by the recited process steps. Rather this product-by-process will be examined according to the structure of the modified cell that is produced by recited method. See. MPEP 2113.
Claim Rejections - 35 USC § 103
The following is a quotation of 35 U.S.C. 103 which forms the basis for all obviousness rejections set forth in this Office action:
A patent for a claimed invention may not be obtained, notwithstanding that the claimed invention is not identically disclosed as set forth in section 102, if the differences between the claimed invention and the prior art are such that the claimed invention as a whole would have been obvious before the effective filing date of the claimed invention to a person having ordinary skill in the art to which the claimed invention pertains. Patentability shall not be negated by the manner in which the invention was made.
The factual inquiries for establishing a background for determining obviousness under 35 U.S.C. 103 are summarized as follows:
1. Determining the scope and contents of the prior art.
2. Ascertaining the differences between the prior art and the claims at issue.
3. Resolving the level of ordinary skill in the pertinent art.
4. Considering objective evidence present in the application indicating obviousness or nonobviousness.
This application currently names joint inventors. In considering patentability of the claims the examiner presumes that the subject matter of the various claims was commonly owned as of the effective filing date of the claimed invention(s) absent any evidence to the contrary. Applicant is advised of the obligation under 37 CFR 1.56 to point out the inventor and effective filing dates of each claim that was not commonly owned as of the effective filing date of the later invention in order for the examiner to consider the applicability of 35 U.S.C. 102(b)(2)(C) for any potential 35 U.S.C. 102(a)(2) prior art against the later invention.
Claims 1, 4-5, 10, and 12-23 are rejected under 35 U.S.C. 103 as being unpatentable over Charlesworth (Charlesworth et al., Molecular Therapy Nucleic Acids, (2018), 12:89-104, cited in IDS of 03/22/2024) in view of Tandon (WO2019183239A1, Published 26 September 2019, cited in IDS of 03/22/2024).
Charlesworth teaches a method of modifying cells comprising selecting human hematopoietic stem and progenitor cells (HSPCs) cells for CRISPR/cas9 and rAAV6-mediated gene targeting (see abstract).
Regarding claim 1, Charlesworth teaches that the cells were obtained from blood samples via the Binns Program for Cord Blood Research at Sandford University. The cells were isolated, cultured, and prepared for electroporation (see “cell culture” section under material and method). Charlesworth teaches concentrating the cells prior to modification by resuspending the collected HSPCs at defined cell concentration before electroporation (see “electroporation and targeting of cells” section of materials and method). Charlesworth also teach about rAAV-6 virus for use in transduction (abstract).
Charlesworth further teaches electroporation of cells and subsequent viral transduction, wherein rAAV6 is delivered immediately following electroporation. Transducing HSPCs with rAAV6 post-electroporation results in greater than a two-fold increase in electroporation aided transduction, thereby demonstrating enhanced viral transduction (abstract).
Regarding claims 4-5, Charlesworth teaches that electroporation enhances AAV6-mediated transduction of CD34+ HSPCs using a GFP-encoding self-complementary AAV6 (scAAV6-SFFV-GFP) vector (see “EAT of rAAV6 in CD34+ HSPCs” section of Result). Figure 2A of Charlesworth clearly shows that more than 20% of the electroporated cells express the desired protein (scAAV6-SFFV-GFP) as compared to un-electroporated cells. Regarding claim 5, figure 2A of Charlesworth teaches that approximately 35% of the electroporated cells were GFP-positive after 2-hours of exposure to scAAV6.
Regarding claim 10, Charlesworth et al. teaches the concentration of cells to be modified (e.g., HSPCs cell) to be 1x105 cells/mL (see abstract). Furthermore regarding, cell plating density, Charlesworth teaches that cells were plated at 5x105 – 1x106 cells/mL (see “electroporation and targeting of cells” section under materials and method), which falls within the claimed range of cell population.
Regarding claim 12, Charlesworth teaches HBB-edited CD34+ HSPCs were transplanted into mice after ex vivo gene editing (See “Transplantation of CD34+ HSPCs into NSG Mice” section under Materials and Method). Charlesworth teaches that tail vein and intrafemoral injections were used to administer the edited cells.
Regarding claim 13, Tandon teaches about existing approaches for co-localization of viral vectors and cells and mentioned that centrifugation or spinoculation of cells and virus as a method for co-localization (paragraph 008). Accordingly, it was well known in the art that centrifugation was a commonly used technique for concentration of cells prior to downstream manipulation.
Regarding claims 14 and 15, Charlesworth teaches that the cells-to-be-modified comprise human CD34+ Hematopoietic Stem and Progenitor Cells (HSPCs) obtained from cord blood and peripheral blood for ex vivo gene editing (see “Cell Culture” section under Material and Method).
Regarding claim 16, Charlesworth teaches that the cells-to-be-modified comprise human CD34+ HSPCs obtained from cord blood and peripheral blood (see “Cell Culture” section under Material and Method). CD34+ HSPCs are obtained by isolating cells from the cord blood mononuclear cells (CBMCs) fraction. Therefore, Charlesworth teaches cells derived from CBMCs.
Regarding claim 17, Charlesworth teaches Hematopoietic Stem and Progenitor Cells (HSPCs) (Abstract). HSPCs include hematopoietic progenitor cells, which is one of the recited cell types in claim 17.
Regarding claim 18, Charlesworth teaches that the cells were resuspended in either P3 buffer (Lonza) or buffer IM before performing electroporation (see “Electroporation and Targeting of Cells” section under Material and Method).
Regarding claim 19, Charlesworth teaches a modified CD34+ hematopoietic stem and progenitor cell (HSPC) produced by ex vivo CRISPR)-Cas9 and rAAV6-mediated genome editing (see abstract).
Regarding claims 20 and 21, Charlesworth teaches that the cells-to-be-modified comprise human CD34+ Hematopoietic Stem and Progenitor Cells (HSPCs) obtained from cord blood and peripheral blood for ex vivo gene editing (see “Cell Culture” section under Material and Method).
Regarding claim 22, Charlesworth teaches that the cells-to-be-modified comprise human CD34+ HSPCs obtained from cord blood and peripheral blood (see “Cell Culture” section under Material and Method). CD34+ HSPCs are obtained by isolating cells from the cord blood mononuclear cells (CBMCs) fraction. Therefore, Charlesworth teaches cells derived from CBMCs.
Regarding claim 23, Charlesworth teaches Hematopoietic Stem and Progenitor Cells (HSPCs) (Abstract). HSPCs include hematopoietic progenitor cells, which is one of the recited cell types in claim 23.
Charlesworth does not teach combining the cells with virus to form a mixture and simultaneously performing electroporation and viral transduction as recited in claim 1.
However, Tandon teaches a viral transduction and electroporation device comprising a membrane, electroporation electrodes, and an electrical voltage source wherein cells, viruses for transduction, and cargo for electroporation are co-localized at the membrane (Figure 1A-1B, abstract, claim 1).
Figures 1A and 1B of Tandon teaches cross-sectional views showing how the dual-purposes viral transduction and electroporation device can function by co-localizing target cells and vector on a membrane. Tandon teaches that both modes, electroporation and transduction, can be used sequentially or simultaneously (paragraph 0021).
Tandon teaches trapping both cells and vectors against a membrane using electrophoresis by applying an electrical field across the membrane (paragraph 0055). Tandon further teaches that electrically charged virus are directed towards the membrane for enhanced contact with the cells, after which permeabilization of the cells is conducted by electroporation (paragraph 0055, Figure 1C-E). Thus, Tandon teaches mixing cells and viral vectors in a membrane and performing electroporation simultaneously to form one or more co-electroporated cells.
Therefore, it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have modified method of Charlesworth by combining viral vectors and cells-to-be-modified and performing electroporation and viral transduction simultaneously as taught by Tandon because Tandon teaches that electroporation and transduction can be performed simultaneously and co-localization of cells and virus enhance their contact, thereby increasing viral transduction. A person of ordinary skill in the art would have been motivated to do in order to reduce the number of separate handling steps, thereby saving time required to perform experiment and simplifying the workflow.
In view of the foregoing, claims 1, 4-5, 10, and 12-23 taken as a whole would have been prima facie obvious before the effective filing date.
Claims 2 and 3 are rejected under 35 U.S.C. 103 as being unpatentable over Charlesworth (Charlesworth et al., Molecular Therapy Nucleic Acids, (2018), 12:89-104, cited in IDS of 03/22/2024) and Tandon (WO2019183239A1, Published 26 September 2019, cited in IDS of 03/22/2024) as applied to claims 1, 4-5, 10, and 12-23 above, and further in view of Henley (US20200354743A1, published 12 November 2020).
Neither Charlesworth nor Tandon teach virus or viral vectors is co-electroporated with gene editing agents as recited in claims 2 and 3.
However, Henley teaches viral and non-viral vectors for delivery of gene-editing agents. Specifically, paragraph 0323 of Henley teaches that vector systems include plasmid vectors, retroviral vectors, lentiviral vectors, adenovirus vectors, adeno-associated virus vectors, etc. Henley teaches that these vectors may comprise CRISPR, TALEN, transposon-based, ZFN, meganuclease, or Mega-TAL molecules and/or transgenes (paragraph 0323). Henley teaches that a vector can be pre-complexed with Cas protein prior to electroporation (paragraph 0327). Henley further teaches that Cas protein can be pre-mixed with guide RNA and plasmid prior to electroporation (paragraph 0327).
Therefore, it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have modified the method of Charlesworth by co-electroporating viral vectors and gene editing agents (e.g., CRISPR-cas9 protein) as taught by Henley because Henley teaches that viral vectors and gene editing agents (e.g., Cas protein) can be pre-mixed prior to electroporation. An artisan would have been motivated to do so to get further advantage of better contact between viral vectors and gene editing agents, thereby increasing the electroporation efficiency.
In view of the foregoing, claims 2-3 taken as a whole would have been prima facie obvious before the effective filing date.
Claims 6-9 are rejected under 35 U.S.C. 103 as being unpatentable over Charlesworth (Charlesworth et al., Molecular Therapy Nucleic Acids, (2018), 12:89-104, cited in IDS of 03/22/2024) and Tandon (WO2019183239A1, Published 26 September 2019, cited in IDS of 03/22/2024) as applied to claims 1, 4-5, 10, and 12-23 above, and further in view of Li (US 20190247436A1, published 15 August 2019).
Neither Charlesworth nor Tandon teach the claimed ranges of reduction in cell viability recited in claims 6-9.
However, Li teaches electroporation-based genome editing methods in which cell viability is maintained following electroporation. Li teaches that primary cells for use in genome editing comprise peripheral blood mononuclear cells (PBMC), peripheral blood lymphocytes (PBLs) and other blood cell (see paragraph 0166). Li further teaches that viability of electroporated cells can be at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% (or any range derivable therein), as compared to the viability of starting or unelectroporated population (see paragraph 0187), thereby teaching all limitations of claim 6-9.
Therefore, it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have modified the method of Charlesworth by Li because optimization of electroporation conditions to achieve desired balance between transduction and cell viability was a routine aspect of electroporation protocol. Furthermore, Li teaches that the density of cells during electroporation is a controlled variable and may be varied according to, but not limited to, cell type, desired electroporation efficiency or desired viability of resultant electroporated cells (paragraph 0200).
In view of the foregoing, claims 6-9 taken as a whole would have been prima facie obvious before the effective filing date.
Claim 11 is rejected under 35 U.S.C. 103 as being unpatentable over Charlesworth (Charlesworth et al., Molecular Therapy Nucleic Acids, (2018), 12:89-104, cited in IDS of 03/22/2024) and Tandon (WO2019183239A1, Published 26 September 2019, cited in IDS of 03/22/2024) as applied to claims 1, 4-5, 10, and 12-23 above, and further in view of Benitez (Benitez et al., Frontiers in Genome Editing, (2020), 2: 601541).
Regarding claim 11, Neither Charlesworth nor Tandon teach the recited volume of cells for use in electroporation.
However, Benitez teaches pelleting the cells and resuspending in 100 μl of BTXpress electroporation buffer prior to electroporation, which constitutes concentration the cells-to-be-modified to a volume within the claimed range of 10 μl to 1 L (see “CD34+ HSPC Electroporation with Cas9 Variants” section under Materials and Method).
Therefore, it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have modified electroporation method of Charlesworth by concentrating the volume of cells-to-be-modified as taught by Benitez because Benitez teaches pelleting the cells and resuspending them in 100 μl of electroporation buffer before electroporation. An artisan would have been motivated to do so because selecting a resuspension volume within the disclosed range is a routine optimization of an electroporation protocol and would have been expected to yield predictable results.
In view of the foregoing, claim 11 taken as a whole would have been prima facie obvious before the effective filing date.
Conclusion
No claims are allowed.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to SURAJ SAPKOTA whose telephone number is (571)270-0842. The examiner can normally be reached Monday-Thursday 7am-5pm.
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If attempts to reach the examiner by telephone are unsuccessful, the examiner’s supervisor, Ram R Shukla can be reached at (571) 272-0735. The fax phone number for the organization where this application or proceeding is assigned is 571-273-8300.
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Suraj Sapkota
Patent Examiner
AU 1635
/RAM R SHUKLA/Supervisory Patent Examiner, Art Unit 1635
Prosecution Insights