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 Status
Claims 1,9, and 22 are amended.
Claims 12-21, and 25 are cancelled.
Claims 22-24 are withdrawn from consideration.
Claims 26-33 are newly added.
Claims 1-11, and 26-33 are currently under examination.
Withdrawn Rejections
Rejections under 35 USC § 102
The rejection of claims 1-9 under 35 U.S.C. 102(a)(1) as being anticipated by Bjorklund et al ( WO 2019/158619 A1) is withdrawn in light of Applicants amendment. It is noted that claim 1 recites in step (c) “ performing single-cell RNA sequencing on the plurality of transduced cells” to determine the sequence of the unique identifier sequence in each transduced cell. Bjorklund does not disclose, teach, or suggest performing single-cell RNA sequencing.
Rejections under 35 USC § 103
The rejection of claims 1-11 under 35 U.S.C. 103 as being unpatentable over Bjorklund et al ( WO 2019/158619 A1), in view of Boye et al (US 2016/0369299 A1) is withdrawn in light of Applicants amendment.
Response to Amendments
Applicant’s arguments have been carefully considered and found persuasive as noted
above. The edited ground of rejection below addresses the deficiencies raised by Applicant with
respect to the amended claims
Edited Rejections Necessitated by Claims Amendments
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.
Claims 1-11, and 27-32 are rejected under 35 U.S.C. 103 as being unpatentable over Bjorklund et al ( WO 2019/158619 A1), in view of Greenberg et al (WO 2020/118012 A1, with a filing date of 12/05/2019), and Boye et al (US 2016/0369299 A1)
Regarding claim 1-3, Bjorklund et al disclose a method for designing and manufacturing a library of modified viral vectors comprising a modified capsid protein, wherein the modified capsid is generated by inserting a polynucleotide fragment in the capsid gene. Bjorklund et al teach that the said insertion generates a viral capsid with increased tropism and/or infectivity for a given cell type. (See page 12 lines 27-35). Bjorklund et al also disclose a method for identifying an engineered capsid protein exhibiting a preferential tropism to a desired cell type. Also provided is a method of improving tropism of a viral vector or particle toward a target cell. ( See page14, lines 25-27; and page 6, lines 7-8). According to Bjorklund et al, the modified viral particles may encapsulate a transgene and a barcode polynucleotide (i.e. identifier sequence) to be delivered to a target cell, wherein the transgene and the barcode polynucleotide may be inserted in the viral genome, i.e. between the terminal repeat sequences, this reads on step (a) of instant claim. (See page 35, line 8, and page 20, lines 20-21). The method of Bjorklund also involves a step of identifying a capsid protein that exhibit a preferential tropism to a desired cell type, which is achieved by contacting a cell population with the modified viral vectors either in vivo or in vitro, this reads on step (b) of instant claim. ( See page 29 lines 27-32, and page 30, lines 1-3). Bjorklund et al also teach a step of determining the sequence of the barcode to identify the capsid protein present in each cell type, and thereby identifying the candidate polynucleotides(i.e. the polynucleotide fragment that was inserted in the capsid gene) responsible for the desired tropism and the corresponding candidate polypeptides, this reads on step (c) and (e) of instant claim. ( See page 19, lines 14-32). The method of Bjorklund also involves the step of determining the cell type of each transduced cell from (b) by monitoring marker expression and selecting the cells wherein marker expression follows a desired pattern, this reads on step (d) of instant claim.(See page 30, lines 1-11, and lines 28-31).
It is noted that the Bjorklund does not include the use of single-cell RNA sequencing to determine the sequence of the unique identifier sequence in each transduced cell. Rather, Bjorklund infers capsid tropism by correlating bulk barcode readouts with marker expression patterns in target cell populations.
Greenberg et al supplement Bjorklund et al by teaching a screening platform for the development of cell-type-specific viruses, including for example AAVs specific for the brain. The method involves constructing individual viral vectors comprising putative cell-type-restricted enhancer elements with a unique barcode sequences (i.e. identifier sequence) incorporated into viral transcripts. Greenberg et al further teach administering a pooled viral library to tissue, performing single-cell RNA sequencing, identifying the cellular identity of individual cells based on endogenous transcriptomic profiles (this reads on claim 3), recovering barcode-containing viral transcripts from the sequenced cells, and correlating barcode identity with the identified cell type. Greenberg et al further teach determining cell-type specificity of individual viral constructs based upon the recovered barcode information and transcriptionally assigned cell identities. (See paragraphs [0011], and [0046]). In one of the embodiment, Greenberg et al state that “ the method of screening is for capsid sequences. In some embodiments of any of the aspects, one or more, including a library, of capsid DNA is encoded in viral genome and its expression detected in scRNA-seq to ID the cell-type-specificity and magnitude of expression of each virus carrying a unique capsid. In some embodiments of any of the aspects, capsids are barcoded to generate a library of capsids detected as one or more, including a library of barcodes. In some embodiments of any of the aspects, capsids include a variable region modified to generate the library of capsids detected as one or more, including a library of barcodes. In some embodiments of any of the aspects, the one or more barcodes is associated with a capsid structure, function, or both.” ( See paragraphs [0051] ).Greenberg et al also state that “ In some embodiments of any of the aspects, the method of screening for capsid sequences comprises substantially the same steps as screening for a cell-type specific GRE, comprising replacing the GRE sequence with a capsid sequence”. ( See paragraph [00113] on page 29). As such, Greenberg et al expressly suggest that the single cell RNA sequencing can be adapted to identify the cell-type-specificity and magnitude of expression of each virus carrying a unique capsid. Thus, it would have been prima facie obvious to one with ordinary skill in the art at the time the invention was filed to modify the method of Bjorklund to employ the single-cell RNA sequencing and barcode-correlation techniques taught by Greenberg as Greenberg et al expressly teach that such technique permits simultaneous evaluation of large numbers of viral constructs and enable determination of cell types in which individual barcoded viral constructs are active. (See paragraph [0046] line 5-10). As well as, clearly suggest that the screening method can be employed to screen for engineered capsid sequences to identify the cell type and viral tropism. In other words, claim 1 would have been obvious to one of ordinary skill in the art, as there was some teaching, suggestion, or motivation in the prior art that would have led one of ordinary skill to modify the prior art reference or to combine prior art reference teachings to arrive at the claimed invention. There is a reasonable expectation of success when building the barcoded viral constructs of Bjorklund to apply the known single-cell transcriptomic analysis methods of Greenberg to the viral screening platform of Bjorklund to produce high-resolution information regarding cell-type specificity and viral tropism.
Regarding claim 4, following the discussion above, the combine teachings of Bjorklund et al in view of Greenberg et al render obvious claim 1-3. Bjorklund et al disclose that the viral vector contain a transgene encoding a detectable marker (e.g. reporter). ( See page 2 ,lines 28-29).
Regarding claim 5, following the discussion above, Bjorklund et al also disclose utilizing a transgene encoding for a reporter protein to identify cells transduced with an r AAV. The identification method of Bjorklund, for example, involves contacting a cell population with viral vectors comprising of a modified viral capsid encapsulating a transgene encoding for a reporter, and then a method of monitoring the reporter expression and selecting the cells wherein marker expression follows a desired pattern. ( See page 3, lines 5-12).
Regarding claim 6, following the discussion above, Bjorklund et state that “ The cells to be targeted for transgene delivery are preferably cells in which expression of the transgene is desired. The target cells may for example be neurons”. (See page 35 lines 12-13).
Regarding claims 7,27, and 32, following the discussion above, Bjorklund et al also disclose generating a viral particle comprising modified viral capsid ( termed as AAV-MNM001-024) which display an improved tropism to primary glial cells, including astrocytes and microglia. (See page 54, lines 21-25).
Regarding claim 8, following the discussion above, Bjorklund et al further disclose that the engineered capsid protein is generated by inserting a polynucleotide fragment within the capsid gene so that it can be transcribed and translated into a polypeptide fragment displayed on the capsid. ( See page 15 lines 25-36, and page 16 lines 1-3).
Regarding claim 9, following the discussion above, Bjorklund et al state that the “ The insertion site may be
at the N-terminus of VP2. It may also be at the vertices of the assembled capsid, e.g. centered around amino acid residue 587 of the Cap gene of AAV2 or around amino acid residue 588 of the AAV9 cap gene.”
Regarding claim 10-11, following the discussion above, Bjorklund in view of Greenberg et al render obvious a method of engineering a modified capsid protein displaying a preferential tropism to a desired cell type. However, neither Bjorklund et al nor Greenberg teach generating a modified capsid protein comprising of an AAV2 capsid protein containing the Y444F, Y500F, Y730F, T491V, R585S, R588T, R487G amino acid substitutions or a combination thereof.
Boye et al teach an a rAAV viral vector comprising an engineered capsid protein, wherein the engineered capsid protein is an AAV2 capsid protein containing the amino acid substitutions of Y444F, Y500F ,T491V, R585S, R588T, R487G, and Y730F. According to Boye et al, a rAAV vector harboring the engineered capsid protein with such amino acid substitutions has an enhanced ability to selectively and exclusively target photoreceptors or retinal pigmented epithelial cells (RPE). (See paragraphs [0009],[0014], and [0018-0019]. Therefore, claims 10-11 would have been obvious to one of ordinary skill in the art, as there was some teaching, suggestion, or motivation in the prior art that would have led one of ordinary skill to modify the prior art reference or to combine prior art reference teachings to arrive at the claimed invention. Because Bjorklund et al teach a method for engineering a modified capsid protein with a preferential tropism to a desired cell type using the method a peptide insertion. Boye et al teach an engineered AAV2 capsid protein with enhanced tropism to photoreceptors and RPE cells. Thus, it would have been obvious to one with ordinary skill in the art at the time the invention was filed to combine the teachings of Bjorklund, Greenberg, and Boye et al and engineer an AAV2 capsid comprising the amino acid substitutions of Boye et al. There would be a reasonable expectation of success in doing so will generate a rAAV viral vector for targeting photoreceptor and RPE cells.
Regarding claim 28, following the discussion above, Bjorklund in view of Greenberg et al render obvious claim 1-3. Greenberg et al state that “In some embodiments of any of the aspects, the method of screening for capsid sequences comprises substantially the same steps as screening for a cell-type specific GRE, comprising replacing the GRE sequence with a capsid sequence.” ( See paragraph [00113]). In other words, Greenberg et al’s method involves performing single-cell RNA sequencing to identify the cellular identity of individual cells based on endogenous transcriptomic profiles (this reads on claim 3), recovering barcode-containing viral transcripts from the sequenced cells, and correlating barcode identity with the identified cell type. Greenberg et al further teach determining cell-type specificity of individual viral constructs based upon the recovered barcode information and transcriptionally assigned cell identities. Meaning that, capsid tropism in the screening method of Greenberg is also based on the infectivity of the cell-type. (See paragraphs [0011], and [0046]).
Regarding claim 29,31, as discussed above, Bjorklund et al teach evaluating modified AAV capsids based on transduction efficiency as an indicator of altered cellular tropism and targeting. ( See page 2 lines 1-7). Although Bjorklund et al do not expressly disclose selecting the variants exhibiting at least 4-fold increase in transduction efficiency relative to any other cell type, Bjorklund however teaches screening engineered capsids and identifying variants demonstrating improved transduction. Thus, an ordinary skill in the art would have recognized that the magnitude of transduction enhancements represent a result-effective variable that may be optimized through routine experimentation. Furthermore, none of the prior art expressly teach the exclusion criteria set in claim 31. However, the claimed limitations are also result effective variable that can be optimized through routine experimentation.
As per the MPEP. "[W]here the general conditions of a claim are disclosed in the prior
art, it is not inventive to discover the optimum of workable ranges by routine experimentation.
The "discovery of an optimum value of a result effective variable in a known process is ordinarily
within the skill of the art." Application of Boesch, 617 F.2d 272, 276, 205 USPQ 215, 218-219
(C.C.P.A. 1980). See MPEP 2144.05
Regarding claim 30, following the discussion of claim 1 above. Greenberg et al teach that the transcriptional profile is obtained by either single-cell RNA sequencing (sc-RNA seq) or single-nucleus RNA sequencing (sn-RNA seq) and involves analyzing the expression levels of at least 866 unique genes per cell. ( See paragraph [00327]).
Claim 26 is rejected under 35 U.S.C. 103 as being unpatentable over Bjorklund et al ,Greenberg et al, and Boye et al as applied to claims 1-11, and 27-32 above, and further in view of Dattwyler et al ( US 2004/0033236 A1), and Garcia et al ( Infection and Immunity, 2005).
Regarding claim 26, the teachings of Bjorklund, Greenberg, and Boye are set forth above. Bjorklund et al in view of Greenberg and Boye render obvious a method for identifying an engineered viral capsid exhibiting a preferential tropism to a desired cell type. However, none of the cited prior arts teach generating an engineered viral capsid comprising a peptide insertion with an amino acid that is at least 90% identity to SEQ ID NO:2.
It is noted that the claimed sequence correspond to the amino acid sequence comprising the outer surface lipoprotein OspB of Borreliella burgdorferi. Dattwyler et al teach an isolated and engineered recombinant protein comprising the outer surface lipoproteins of Borreliella burgdorferi, including OspA variants and related outer-surface lipoprotein such as OspB, and provides the amino acid sequences of these proteins to use in vaccines development. Specifically, Dattwyler et al teach a recombinant protein of OspB represented by “ SEQ ID NO 22 ” that is 99.4% identical to SEQ ID NO 2 of instant claim. (See abstract, and paragraphs [0013], and [0210-0211]), (See alignment below). Thus, establishing that the OspB is a well-characterized, surface-exposed bacterial antigen that can be used in molecular engineering and peptide derivation.
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Furthermore, Garcia et al teach that that B. Burgdorferi outer surface proteins OspA and OspB directly bind complement receptor 3 (CR3) found on mammalian target cells. ( See abstract). Garcia et al demonstrate that OspB is a functional host-adhesion ligand capable of engaging mammalian cell surface receptors involved in cellular attachment and pathogen-host interaction. ( See Fig.3). Thus an ordinary skill in the art would be motivated to combine the teachings of Bjorklund, Greenberg, Dattwyler, and Garcia and use OspB derived sequences, disclosed by Dattwyler in view of Garcia et al to generate an engineered AAV capsid protein comprising of a peptide insertion corresponding to the amino acids of OspB that is capable of binding CR3-expressing cells. Because Bjorklund et al in view of Greenberg and Boye render obvious a method for designing and manufacturing a library of modified viral vectors comprising a modified capsid protein, wherein the modified capsid is generated by inserting a peptide fragment in the capsid, wherein said insertion generates a viral capsid with increased tropism and/or infectivity for a given cell type, but fail to teach a modified capsid protein comprising OspB peptide. Dattwyler et al in view of Garcia et al supplement the cited prior arts by establishing that the OspB is a well-characterized, surface-exposed bacterial adhesin/antigen that can be used in molecular engineering and peptide derivation. In other words, claim 26 is combining prior art elements according to known methods to yield predictable results, namely the predictable result being the use of OspB derived sequence, disclosed by Dattwyler , to generate a modified AAV capsid comprising of OspB that is capable of binding CR3-expressing cells. Because the prior art collectively teach that OspB is a surface adhesin that binds CR3-expressing cell, thereby providing a predictable mechanism for generating a viral construct with an increased tropism to CR3-expressing cells.
Claim 31 is rejected under 35 U.S.C. 103 as being unpatentable over Bjorklund et al ,Greenberg et al, and Boye et al as applied to claims 1-11, and 27-32 above, and further in view of Fritz et al ( US 2005/0063978 A1), and Lee et al (Biochemical Society, 2006).
Regarding claim 26, the teachings of Bjorklund, Greenberg, and Boye are set forth above. Bjorklund et al in view of Greenberg and Boye render obvious a method for identifying an engineered viral capsid exhibiting a preferential tropism to a desired cell type. However, none of the cited prior arts teach generating an engineered viral capsid comprising a peptide insertion with an amino acid that is at least 90% identity to SEQ ID NO:154.
Fritz et al supplement the cited prior art by teaching a recombinant construct comprising the helicobacter pylori-derived peptide known as HP(2-20) peptide. (See paragraphs [0100] the table on page 9). Specifically, Fritz et al teach a recombinant protein of HP(2-20) peptide represented by “ SEQ ID NO 6 ” that is 100% identical to SEQ ID NO 154 of instant claim. (See alignment below). Thus, establishing identifying HP(2-20) as a known biologically active peptide available for incorporation into recombinant systems.
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It is noted that Fritz et al do not teach or suggest the use of this peptide to generate a modified AAV capsid with an enhanced tropism toward a specific cell type. Lee et al teach that HP (2-20) adopt amphipathic a-helical structure in membrane-mimetic environments and interacts strongly with phospholipids membranes. ( See Fig.9). Specifically, Lee et al show membrane association of the peptide through CD, fluorescence, and NMR studies, indicating that HP(2-20) is a membrane active peptide suitable for applications where enhanced interaction with cellular membrane is desired. ( See abstract, and the results section). In other words, the documented membrane-binding characteristic of the HP(2-20) would have suggested that the peptide as a candidate for incorporation into delivery vehicles where the enhanced interaction with target cell membrane is desired. Thus, it would have prima facie obvious to one with ordinary skill in the art at the time the invention was filed to substitute the HP (2-20) peptide taught by Fritz et al for one of the heterologous peptides utilized in the engineered AAV capsids of Bjorklund. One would be motivated to make such substitution because Lee et al teach that HP (2-20) possess membrane-binding and membrane-interacting properties that would have been expected to promote interactions between the viral capsid and cellular membranes. An ordinary skill in the art would have reasonably expected that the incorporation of a known membrane-active peptide into a surface-exposed region of an AAV capsid would alter or enhance vector-cell interactions and thereby affect cellular uptake, or tissue tropism consistent with the teachings expressly taught by Bjorklund in view of Greenberg and Boye.
Response to Arguments
Applicant's arguments filed 04/11/2026 have been fully considered but they are not
persuasive.
Applicants argue that neither Bjorklund nor Boye et al disclose, teach, or suggest
performing single-cell RNA sequencing.
Examiner’s Response to Traversal: Applicant’s arguments have been carefully
considered but are not found persuasive. This is because Applicants amended claim 1 to include the feature of using “ single-cell RNA sequencing on the plurality of transduced cells”, while Bjorklund in view of Boye do not teach the limitation, however the edited ground of rejection over Bjorklund in view of Greenberg et al ,and Boye et al covers all of the added claim limitations (as discussed above).
Conclusion
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 FATIMAH KHALAF MATALKAH whose telephone number is (703)756-5652. The examiner can normally be reached Monday-Friday,7:30 am-4:30 pm EST.
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/FATIMAH KHALAF MATALKAH/Examiner, Art Unit 1638
/Tracy Vivlemore/Supervisory Primary Examiner, Art Unit 1638
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