DETAILED ACTION
The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
Claims 1-21 are pending. It is noted that claim 21 is numbered twice and under 37 CFR 1.121, all of the claims presented in a claim listing shall be presented in ascending numerical order. Hence, the cancelled claims are renumbered herein as 22-135.
The instant application claims the benefit of U.S. provisional 63/210,786 filed 6/15/2021.
Information Disclosure Statement
Information disclosure statements filed 11/1/2023, 9/20/2024, 3/12/2025 and 7/17/2025. have been identified and the documents considered. The corresponding signed and initialed PTO Form 1449s for the IDS filed 11/1/2023 and 9/20/2024 has been mailed with this action. Initials indicate that the document has been considered even if the reference is lined through. In the case that only an English abstract was identified, this is indicated.
The information disclosure statement 3/12/2025 and 7/17/2025 fails to comply with the provisions of 37 CFR 1.98(a)(4) because it lacks the appropriate size fee assertion. It has been placed in the application file, but the information referred to therein has not been considered as to the merits.
Specification
The disclosure is objected to because of the following informalities: on page 6, ¶4, “editstrand” requires a space between the two words. Page 135, ¶1, “andprogrammed” requires a space. Appropriate correction is required.
Claim Objections
Claims 2, 6, 11-13 and 21 are objected to because of the following informalities: Once an abbreviation has been established i.e. claim 1, it is not needed to be spelled out thereafter i.e. claim 2. This also occurs with NLS in claim 20 versus claim 21. Alternatively, once an abbreviation is established, it provides consistency to use it thereafter Claims 2, line 11, claim 11and 12 refer to “primer binding site” but this was abbreviated in claim 2, line 7 to PBS.
Claim 6 appears to inadvertently have the term “to” at the end.
Claim 13, is grammatically incorrect. It is missing the phrase “of a” prior to substitutions. As well because the claim states “one or more” it is not necessary to recite “and/or”.
Appropriate correction is required.
Claim 3 is objected to under 37 CFR 1.75 as being a substantial duplicate of claim 2. When two claims in an application are duplicates or else are so close in content that they both cover the same thing, despite a slight difference in wording, it is proper after allowing one claim to object to the other as being a substantial duplicate of the allowed claim. See MPEP § 608.01(m). Claim 3 recites that the nicking results in a 5’ and a 3’ end. This is an inherent consequence of nicking.
Claim 5 is objected to under 37 CFR 1.75 as being a substantial duplicate of claim 4. When two claims in an application are duplicates or else are so close in content that they both cover the same thing, despite a slight difference in wording, it is proper after allowing one claim to object to the other as being a substantial duplicate of the allowed claim. See MPEP § 608.01(m). Since claim 4 establishes that the 3’ end is free, the DPO would inherently extend the 3’ end. This given the intended edits means the target polynucleotide is inherently altered.
Claim Rejections - 35 USC § 112, second paragraph
The following is a quotation of 35 U.S.C. 112(b):
(b) CONCLUSION.—The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the inventor or a joint inventor regards as the invention.
The following is a quotation of 35 U.S.C. 112 (pre-AIA ), second paragraph:
The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the applicant regards as his invention.
Claim 1 recites the limitation "an editing target sequence in the edit strand" in claim 1, line 17 and claim 2, line 21. There is insufficient antecedent basis for this limitation in the claim. It is unclear what the editing target strand is. As to strands, the DNA comprises a target strand and an edit strand. As to potential editing target strands, the Peg comprises a DNA segment with edits to be incorporated into the target strand but there is no mention of a double-strand or strands. Hence, it is not clear to what the claims refer.
Claim 1, line 17, claim 2, line 22 and claim 6 recite the limitation “the double stranded polynucleotide” in claim 5. There is insufficient antecedent basis for this limitation in the claim. There is a provided double stranded polynucleotide and a nicked double stranded polynucleotide as well as a hybridized double stranded polynucleotide. As the method uses the terminology comprising which means as recited there is no explicit order and as the method requires sequential steps, it is important to distinguish which component is used and hence generic reference to the double stranded polynucleotide is unclear.
Claim 5 recites the limitation "the nicked edit strand" in 4. There is insufficient antecedent basis for this limitation in the claim. There are two nicked edit strand references. There is the nicked strand by the nickase but then in claim 4, the PBS hybridizes to the nicked edit strand. As this is a method, it is critical to know which of the two the DPA extends. Because the claim does not distinguish between the two, the metes and bounds of the claim are unclear.
Claim Rejections - 35 USC § 112 ¶4 rejection
The following is a quotation of the fourth paragraph of 35 U.S.C. 112:
Subject to the [fifth paragraph of 35 U.S.C. 112 prohibiting improper multiple dependent claims], a claim in dependent form shall contain a reference to a claim previously set forth and then specify a further limitation of the subject matter claimed. A claim in dependent form shall be construed to incorporate by reference all the limitations of the claim to which it refers.
Claim 10-12 are rejected under 35 U.S.C. 112, 4th paragraph, as being of improper dependent form for failing to further limit the subject matter of the claim upon which it depends, or for failing to include all the limitations of the claim upon which it depends. Claim 10 refers to an orientation of the DNA sequence and the RNA sequence that is contradictory to the orientation established in claim 9 from which claim 10 depends. Claims 11 which depends from 10 reflects that in claim 9 while claim 12 which depends from claim 11 reflects that in claim 10. However, their dependency upon one another renders them improper by referring to claim limitations that read outside of the original scopes. Hence by claiming variants thereof, applicants have recited limitations outside of the scope of claim 2. .Applicant may cancel the claim(s), amend the claim(s) to place the claim(s) in proper dependent form, rewrite the claim(s) in independent form, or present a sufficient showing that the dependent claim(s) complies with the statutory requirements.
Claim Rejections - 35 USC § 112, first paragraph
The following is a quotation of the first paragraph of 35 U.S.C. 112(a):
(a) IN GENERAL.—The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same, and shall set forth the best mode contemplated by the inventor or joint inventor of carrying out the invention.
Claims 1-21 are rejected under 35 U.S.C. 112(a) or 35 U.S.C. 112 (pre-AIA ), first paragraph, as failing to comply with the written description requirement. The claim(s) contains subject matter which was not described in the specification in such a way as to reasonably convey to one skilled in the relevant art that the inventor or a joint inventor, or for pre-AIA the inventor(s), at the time the application was filed, had possession of the claimed invention.
The claims are drawn to a method of editing a double stranded target polynucleotide. The method uses a number of molecules that are claimed in terms that of the function that is to be performed by each. The structures, to the contrary, are recited in the claims broadly and incompletely as descriptive elements.
Claim 1 recites a method of editing that requires use of an editing molecule called an napDNAbp which comprises a nickase protein to which the RNA of the Chimeric Peg guide is “capable” of binding. As an initial issue, the napDNAbp does not “result” in a nick but “nicks” the edit strand. Critical to the napDNAbp function is a chimeric PEg polynucleotide guide (also called chipGNA in the disclosure). This molecule comprises a chimeric DNA/RNA segments. The sequences are “at least partially complementary” to a “portion” of the edit and target strands respectively and the RNA segment binds to the napDNAbp. Hence, the overall picture is need for the RNA segment to act as a guide RNA to direct the napDNAbp to the desired target. Once there the RNA segment hybridizes to the nicked edit strand. However, the RNA is said to be complementary to the target strand. Hence, these actions are in contrast to one another and to claim 2 and appear to be a mistake. Claim 1 concludes that the DNA polymerase synthesizes a single stranded DNA that replaces “an editing target sequence in the edit strand.
Claim 2 follows the same format using a ds polynucleotide with an edit and target strand and an napDNAbp that is capable of nicking the edit strand. The distinction is the nature of the chimeric Peg. The RNA segment which is described as having an invariable region that is “capable” of binding the napDNAbp and a variable region that at least partially complementary to a portion of the target strand. The DNA segment comprises an editing template and a primer binding site (PBS) which is at least partially complementary to the edit strand. Hence, in this case, the target/edit complementarity is opposite that of claim 1.
These claims have several description issues. First, the impact of only being partially complementary to the target and edit sequences such that one can know how much complementarity is had. However, their binding to the correct target is critical. This renders sequences recited broadly wherein the function is quite narrow. This according to the disclosure requires rather the guide RNA (RNA segment) bind to a specific sequence within the ds target polynucleotide. As well, the guide RNA must be bound to the napDNAbp and not just be capable of binding. Next, the method requires that this complementarity lead to hybridization of the RNA segment to the nicked ds polynucleotide strand. As an initial issue, it is unclear how nicking is ensured to occur prior to hybridization such that the ds polynucleotide has a nicked edit strand. But, considering that the nicked strand is available to the method, the RNA hybridizes and it appears this is designed to bring the DNA template within range of the nicked end such that the DPO can synthesize the complement of the DNA strand which comprises the edits to be inserted into the target ds polynucleotide. Hence, the location of hybridization would appear to be critical. And finally, once the DPO synthesized single strand is synthesized, it is not clear how it is inserted into the nicked, RNA bound ds polynucleotide.
The disclosure teaches only use of a nCas9 (Cas9 nickase). nCas9 is fused to the DPO and as such called a prime editor fusion protein. The ability of the prime editor fusion was tested with a chipGNA to target a test sequence. The primer editor binds the RNA segment to form a riboprotein. The specification states (emphasis added)-
“The chipGNA directs the nCas9 to a specific location of a double stranded DNA target polynucleotide (e.g. genomic DNA) to be edited through annealing of the spacer sequence in the chipGNA to the search target sequence on a target strand. The nCas9 then nicks the edit strand (the opposite strand of the target strand) of the double-stranded DNA target polynucleotide and generates a free 3' end on the edit strand. Subsequently, the primer binding site sequence (PBS) in the extension arm of the chipGNA (e.g., in a DNA segment) binds to a complementary portion of the nicked strand, as shown in FIG. 1(ii). Next, the DNA polymerase of the prime editor synthesizes a new strand of DNA, primed by the sequence in the edit strand annealed with the PBS, using the editing template sequence of the DNA segment of the chipGNA as a synthesis template, as shown in FIG. 1(iii).
This makes clear that 1) the RNA must have complementarity to the region TO BE NICKED as there is no way to ensure nicking prior to hybridization. 2) The complementarity must be more than partial. It must be specific to the nicking region and to this end the disclosure provides generic, broad guidance. However, the art teaches 20 nucleotides of complete complementarity are necessary for this binding (see Fu et al, page 1, ¶1). 3) napDNAbp that function with a guide sequence are limited to Cas molecules. These are known to bind to sequences and be directed to the sequence targeted by the guide. 4) Claim 1 appears to in error recite that the guide portion (RNA segment) hybridizes to the nicked edit strand and the DNA edit strand of the chipGNA to target strand. It is the reverse. 5) The DNA sequence (PBS in claim 2) with partial complement to the edit strand is actually complementary to the target strand. It binds to the target sequence (complementary portion of the nicked strand). This DNA has edits to the sequence and serves as a primer for the DPO. Hence, the RNA and DNA must actually be complementary to the sequence to be edited in the ds polynucleotide wherein the RNA is complementary to the target and the DNA to the edit strand. The napDNAbp is a Cas molecule that is bound by the RNA of the chipGNA.
Claims 3-7 refer to outcomes wherein each lack adequate description. Claim 3 recites that the nicking results in a 5’ and a 3’ end. This is an inherent consequence of nicking and hence the only description of this claim is found in claim 2. Claim 4 recites an outcome without structure which demonstrates how the PBS hybridizes to the 3’ end of the nicked edit strand. It is essentially a desire without a description. Claim 5 recites that the PBS binds to the 3’ end which would mean the 3’ end is available for extension by the DPO. This is, therefore, an inherent consequence as recited of the method. By providing a nicked 3’ end as recited and a DPO, extension is a natural consequence and hence there are no steps performed. Rather, the description provided in claim 4 is all that applies to claim 5. Claim 6 adds a step of “further comprises repairing the target” with a DNA repair protein and claim 8 “nicking” the target strand with a nickase. These steps are provided as actions to be performed, actions of repairing and nicking. This requires structurally that a DNA repair protein be included in the method of repair and that a pair of Cas9 nickases be used in the method of nicking both strands.
This is a limited descriptive element wherein the claims broadly and incompletely claim the inventive elements. The claims lack adequate description to link structure to these required functions. The Court indicated that while applicants are not required to disclose every species encompassed by a genus, the description of a genus is achieved by the recitation of a precise definition of a representative number of members of the genus, such as by reciting the structure. Structural features that could distinguish the compounds of the claimed genus from others not encompassed by the genus are missing from the disclosure. In this case, there are specific elements referenced but the claims reference these structures with broad generic functional terms that represent a large and diverse genus of elements.
Claim Rejections - 35 USC § 112, first paragraph
The following is a quotation of the first paragraph of 35 U.S.C. 112(a):
(a) IN GENERAL.—The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same, and shall set forth the best mode contemplated by the inventor or joint inventor of carrying out the invention.
Claims 1-21 are rejected under 35 U.S.C. 112, first paragraph, because the specification, while being enabling for a method for editing a double stranded polynucleotide in a cell in vitro wherein the method comprises contacting the double-stranded target polynucleotide with a nucleic acid sequence comprising an expression regulatory sequence operably linked to each of a chimeric prime editing guide polynucleotide (chimeric PEg polynucleotide) coding sequence, a nucleic acid programmable DNA binding protein (napDNAbp) which is Cas9 having nickase activity (nCas9) coding sequence, and a DNA polymerase coding sequence; wherein the double-stranded target polynucleotide comprises a target strand and an edit strand; wherein the chimeric PEg polynucleotide comprises i) a deoxyribonucleic acid (DNA) segment comprising one or more intended nucleotide edits to be incorporated into the double- stranded target polynucleotide which comprises at least 20 nucleotides that are 100% complementary to the site to be edited on the target strand of the double-stranded target polynucleotide, and ii) a ribonucleic acid (RNA) segment bound to the nCas9 and with at least 20 nucleotides with 100% complementary to the site to be edited on the edit strand of the double-stranded target polynucleotide; wherein after the PEg RNA guides the nCas9 to the site to be edited the edit strand of the double-stranded target polynucleotide is nicked; the RNA segment hybridizes to the nicked edit strand of the double-stranded target polynucleotide and the DNA segment binds to complementary portion of the nicked strand to act as a DNA as a synthesis template and thereafter incorporated into the target polynucleotide by DNA repair, thereby editing the double-stranded target polynucleotide, does not reasonably provide enablement for any other embodiment. The specification does not enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the invention commensurate in scope with these claims.
The test of enablement is whether one skilled in the art could make and use the claimed invention from the disclosures in the patent coupled with information known in the art without undue experimentation (United States v. Telectronics, Inc., 8 USPQ2d 1217 (Fed. Cir. 1988)). Whether undue experimentation is required is not based on a single factor but is rather a conclusion reached by weighing many factors (See Ex parte Forman, 230 USPQ 546 (Bd. Pat. App. & Inter, 1986) and In re Wands, 8USPQ2d 1400 (Fed. Cir. 1988); these factors include the following:
1) Nature of invention. The instant claims are drawn to a general method of editing target sequences to incorporate “intended nucleotide edits”. The method is performed using gene editing components.
2) Scope of the invention. The scope of the invention is extremely broad in that the method as performed does not designate the whether the method is performed in vivo or in vitro. The method uses generally any napDNAbp, any DNA or RNA so long as it is partially complementary, and any target.
3) Number of working examples and guidance. The disclosure teaches only use of a nCas9 (Cas9 nickase). nCas9 is fused to the DPO and as such called a prime editor fusion protein. The ability of the prime editor fusion was tested with a chipGNA to target a test sequence. The primer editor binds the RNA segment to form a riboprotein. The specification states (emphasis added)-
“The chipGNA directs the nCas9 to a specific location of a double stranded DNA target polynucleotide (e.g. genomic DNA) to be edited through annealing of the spacer sequence in the chipGNA to the search target sequence on a target strand. The nCas9 then nicks the edit strand (the opposite strand of the target strand) of the double-stranded DNA target polynucleotide and generates a free 3' end on the edit strand. Subsequently, the primer binding site sequence (PBS) in the extension arm of the chipGNA (e.g., in a DNA segment) binds to a complementary portion of the nicked strand, as shown in FIG. 1(ii). Next, the DNA polymerase of the prime editor synthesizes a new strand of DNA, primed by the sequence in the edit strand annealed with the PBS, using the editing template sequence of the DNA segment of the chipGNA as a synthesis template, as shown in FIG. 1(iii).
4) State of the art. The art as claimed embraces use of a napDNAbp to edit mutant sequences. The method, as recited, requires a nucleic acid, a protein and an enzyme be delivered to a target polynucleotide. It would appear the method is performed in a cell despite no mention of location. Thus, the method requires a mix of protein therapy and gene therapy using a direct reading of the claims. Most often these therapies use nucleic acids encoding each which are delivered to the cell. For delivery to humans, this is often an issue. Shim et al., 2017 (Current Gene Therapy, Vol. 17, No. 5, p. 1-18) teaches that in all gene therapy applications, delivery modes are essential for adequate levels of gene delivery wherein even simple methods are complicated as nucleic acids are highly polar macromolecules and cannot diffuse through cell membranes. For the delivery of nucleic acids into target cells, viral and nonviral delivery agents exist but each have advantages and disadvantages. Despite success, viral vectors still suffer from various challenges, including cytotoxicity, immune response, tumorigenicity, cargo capacity and production problems (e.g. p. 1, right column, ¶2). “Although nonviral methods have many advantages, including safety, the reasons these methods are falling behind viral methods with regard to outcomes might still be a matter of “delivery”, including passing in vivo physiological barriers, cellular/nuclear uptake, and endosomal release... Behavior in the physiological environment is the most important hurdle for vectors” (e.g. p. 13, left column, ¶4). Thus, viral vector delivery of nucleic acid still suffer from various challenges, including cytotoxicity, immune response, tumorigenicity, cargo capacity and production problems. Nonviral delivery of nucleic acid still face the hurdle of passing in vivo physiological barriers, cellular/nuclear uptake, and endosomal release.
5) Unpredictability of the art. The claims embrace a therapeutic based in gene editing with genome editing components has a number of obstacles which are frustrating clinical use. To this end, the claims due to several broadly recited elements exacerbate the unpredictability of the method as claimed. As a first issue, there is no specificity of the chimeric PEg sequence as the claims refer generically to sequences that are partially complementary to the target ds polynucleotide. However, the art has determined that the specificity and selectivity is critical for the method of gene editing (Hunt, Human Genetics, 2023, page 706, col 1).
The primary concern when considering the clinical application of any gene therapy is the potential for unintended genome alterations that may create genomic instability or interfere with regular gene function. Accordingly, it is important to be aware that the CRISPR-Cas system may generate undesired, genotoxic side effects. For example, CRISPR-Cas gRNAs may tolerate small DNA mismatches and cause DNA cleavage and thus INDELs at off-target sites (Han et al. 2020). Furthermore, the improper repair of any DSBs, either at on- or off-target sites has the potential to induce larger genomic aberrations (>50 bp) known as structural variants (SVs).
There is a great need for specificity of gRNA to avoid this and the claims by being partially complementary to a portion of the target strand do not provide any specificity.
Secondly, the method does not indicate where the target would be found, in vitro, in vivo or ex vivo. When considering in vivo use, delivery is the beginning and the end of all obstacles. Development of delivery vehicles has been a field of intense interest. Lenzi et al., 2014 (NCBI Bookshelf, pages 1-16) discuss scientific hurdles of gene transfer in vivo. Some scientific hurdles, such as the absence of efficient delivery systems, difficulty with sustained expression, insertional mutagenesis and host immune reactions, remain formidable challenges to the field of gene transfer. Many of the hurdles have to do with providing efficient gene delivery. This is complicated by the lack of disclosure as to how the components will be delivered. Currently , the art teaches a variety of viral and non-viral vectors. Lenzi teaches, “Although nonviral methods have many advantages, including safety, the reasons these methods are falling behind viral methods with regard to outcomes might still be a matter of “delivery”, including passing in vivo physiological barriers, cellular/nuclear uptake, and endosomal release... Behavior in the physiological environment is the most important hurdle for vectors” (Lenzi, p. 13, left column, 4 full paragraph). Thus, viral vector delivery of nucleic acid still suffer from various challenges, including cytotoxicity, immune response, tumorigenicity, cargo capacity and production problems. Nonviral delivery of nucleic acid still face the hurdle of passing in vivo physiological barriers, cellular/nuclear uptake, and endosomal release”. Hence, the art of therapeutic delivery in vivo is faced with a number of obstacles.
Applicants do not provide any in vivo results. There is simply provided prophetic exemplification. It is noted that a number of related mouse studies have been performed. But, the inability to translate the results other than as proof of principle is well established in the field. Gutman et al, J of Clincial Investigation, 2006, pages 847- teaches, page 847-852, col 1, ¶1.
While straightforward in principle, executing preclinical studies in mice that allow for meaningful and immediate application to the treatment of human cancer is difficult. Moreover, the potential use of GEM cancer models to accelerate the process of bringing effective new treatments to patients is largely theoretical, as few examples exist in which mouse preclinical data has been successfully translated to clinical practice.
Sharma (Molecular Therapy, 2021), page 578, col 2, states
Although both preclinical work and clinical trials focusing on curative therapies are proceeding globally, the clinical translation of CRISPRCas9-mediated gene correction is associated with unpredictable outcomes.186 Factors affecting the success rate of CRISPR-Cas9- mediated gene editing in humans includes off-target effects and cargo delivery methods. It has been observed that off-target effects are principally guided by sgRNAs, and thus rational designs of sgRNAs are necessary to ensure the efficiency of CRISPR-Cas9 gene-editing technology. It was observed that off-target effects were common in human cell culture with persistent Cas9 expression.187,188 while these effects were less common in in vivo models.189 It might be plausible that the occurrence of off-target effects in cell cultures are due to the influence of various factors, such as cell type, expression level, transfection method, cell culture maintenance, consecutive nuclease expression, guide sequence, and repair events.18
What does stop the direct translation of the preclinical models and what is sought in the clinical trials is overcoming the barriers to achieve efficient and adequate gene delivery. These trials seek to determine vector/delivery mode that is necessary to understand for each of the disorders. The trials are set up to determine this. Sharma, page 580, col 1,
As the treatment of human diseases needs to be tissue-specific, it is essential to efficiently deliver the CRISPR-Cas9 cargo into target tissue. Therefore, additional consideration should be given to the suitable delivery system that is based on the charge, size, and content of the CRISPR-Cas9 cargo. CRISPR-Cas9 cargo may be of three types, i.e., plasmid DNA encoding sgRNA and Cas9, a combination of sgRNA and Cas9 mRNA, and a combination of sgRNA and Cas9 protein. Various physical, viral, and non-viral systems have been used as vectors for the delivery of CRISPR-Cas9
6) Undue experimentation. The claims have been evaluated in light of the art at the time of filing and found not to be commensurate in scope with the specification. The MPEP teaches, "However, claims reading on significant numbers of inoperative embodiments would render claims non- enabled when the specification does not clearly identify the operative embodiments and undue experimentation is involved in determining those that are operative. Atlas Powder Co. v. E.I. duPont de Nemours & Co., 750 F.2d 1569, 1577, 224 USPQ409, 414 (Fed. Cir. 1984); In re Cook, 439 F.2d 730, 735, 169 USPQ 298,302 (CCPA 1971). (see MPEP 2164.08(b). In this case, the following issues add up to a significant number of inoperative embodiments such that undue experimentation would have been required. Because the art is nascent as to delivery of genome editing structures to humans for targeted therapy and because the claims rely as a critical factor on an underdeveloped aspect of the art, the claims lack enablement.
Claim Rejections - 35 USC § 102
In the event the determination of the status of the application as subject to AIA 35 U.S.C. 102 and 103 (or as subject to pre-AIA 35 U.S.C. 102 and 103) is incorrect, any correction of the statutory basis (i.e., changing from AIA to pre-AIA ) for the rejection will not be considered a new ground of rejection if the prior art relied upon, and the rationale supporting the rejection, would be the same under either status.
The following is a quotation of the appropriate paragraphs of 35 U.S.C. 102 that form the basis for the rejections under this section made in this Office action:
A person shall be entitled to a patent unless –
(a)(1) the claimed invention was patented, described in a printed publication, or in public use, on sale, or otherwise available to the public before the effective filing date of the claimed invention.
Claims 1-21 are rejected under 35 U.S.C. 102(a)(1) as being anticipated by Liu et al (WO 2020191249, 24 September 2020).
Liu et al teach a method of editing a double stranded DNA using a napDNAbp and a chimeric PEg polynucleotide. This polynucleotide can be a chimeric or hybrid PEgRNA which comprise an RNA portion (guide RNA and hence binds to the nickase Cas9) and a DNA extension arm.
For example, chemical synthesis can be used to synthesize a hybrid RNA/DNA PEgRNA molecule for use in prime editing, wherein the extension arm of the hybrid PEgRNA is DNA instead of RNA. In such an embodiment, a DNA-dependent DNA polymerase can be used in place of a reverse transcriptase to synthesize the 3ʹ DNA flap comprising the desired genetic change that is formed by prime editing.
The full method is shown in Figure 1B and described in ¶0008 and 0343.
To transfer information from the PEgRNA to the target DNA, the mechanism of prime editing involves nicking the target site in one strand of the DNA to expose a 3¢-hydroxyl group. The exposed 3’-hydroxyl group can then be used to prime the DNA polymerization of the edit-encoding extension on PEgRNA directly into the target site. In various embodiments, the extension—which provides the template for polymerization of the replacement strand containing the edit—can be formed from RNA or DNA. In the case of an RNA extension, the polymerase of the prime editor can be an RNA-dependent DNA polymerase (such as, a reverse transcriptase). In the case of a DNA extension, the polymerase of the prime editor may be a DNA-dependent DNA polymerase.
The steps are binding of the gRNA to the target sit wherein the nCas9 nicks the strand(¶0069) thereafter the polymerase uses the DNA as set forth above for priming and extension.
Claim 2 uses terms for the RNA and DNA sequences but they are functional terms that are found in Liu et al. The extension portion of the guide RNA (¶0034, as set forth above can be RNA or DNA even though the following passage uses RNA as the model). The RNA portion binds the nCas and the target site (see e.g. ¶0033 and 0069) thus meeting the limitations of the variable and invariable domains.
As recited in claim 3, the nicking leads to a free 3’ end (see e.g. ¶0017).
As recited in claim 4 and 5, the PBS hybridizes to the 3’ end and the extends with the polymerase (see Figure 1L and text ¶0080).
(¶0080) The extension arm comprises in the 3’ to 5’ direction a primer binding site and a DNA synthesis template (comprising both an edit of interest and regions of homology (i.e., homology arms) that are homologous with the 5’ ended single stranded DNA immediately following the nick site on the PAM strand. As shown, once the nick is introduced thereby producing a free 3’ hydroxyl group immediately upstream of the nick site, the region immediately upstream of the nick site on the PAM strand anneals to a complementary sequence at the 3’ end of the extension arm referred to as the “primer binding site,” creating a short double-stranded region with an available 3’ hydroxyl end, which forms a substrate for the polymerase of the prime editor complex.
The replacement strand anneals and is repaired by e.g. FEN1 (see e.g. ¶0011) as recited in claim 6 and 8.
Both strands can be nicked as recited in calim 8 (see e.g. ¶0050). While claims 9-12 recite components in orders that render them rejected un 35 USC 112, 4th, the meaning appears to be that claim 11 should depend from 9, Claim 10 from claim 1 and claim 12 from claim 10. With this in mind, it is taught by Liu that alternative arrangements are possible which in ¶0159 and figure 72 includes the arrangements claimed.
Depicts alternative designs for PEgRNAs that can be achieved through known methods for chemical synthesis of nucleic acid molecules. For example, chemical synthesis can be used to synthesize a hybrid RNA/DNA PEgRNA molecule for use in prime editing, wherein the extension arm of the hybrid PEgRNA is DNA instead of RNA…. For example, and as shown for a PEgRNA in the 5ʹ-to- 3ʹ orientation and with an extension attached to the 3ʹ end of the sgRNA scaffold, the DNA synthesis template is orientated in the opposite direction, i.e., the 3ʹ-to-5ʹ direction. This embodiment may be advantageous for PEgRNA embodiments with extension arms positioned at the 3’ end of a gRNA. By reverse the orientation of the extension arm, the DNA synthesis by the polymerase (e.g., reverse transcriptase) will terminate once it reaches the newly orientated 5’ of the extension arm and will thus, not risk using the gRNA core as a template.
Claims 13-19 detail the edits to the sequence wherein these edits are found in Liu. Substitutions, insertions and deletions are envisioned as recited in claim 13 (see e.g. ¶0010). When describing deletions, 1-100 nucleotides inserted or deleted are envisioned thus meeting the limitations of claims 14, 15, 18 and 19. The changes can be random i.e. non-consecutive or consecutive (see ¶0010) as recited in claims 16 and 17.
As recited in claims 20 and 21, the napDNAbp and the DPO can be linked to NLS (see e.g. ¶0546). 2021
Conclusion
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/MARIA MARVICH/Primary Examiner, Art Unit 1634