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 .
Withdrawn Rejections/Objections
The objection to the nucleic acid/amino acid sequence, contained in this application does not comply with the requirements for such a disclosure as set forth in 37 C.F.R. 1.821 - 1.825, is withdrawn. Applicant filed a new Sequence Listing with SEQ ID NOS:54-59. No new matter was added because they were present in the original specification.
The rejection of claim 49, under 35 U.S.C. 112(d) or pre-AIA 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the claim upon which it depends, or for failing to include all the limitations of the claim upon which it depends, is withdrawn. Claim 49 has been canceled by amendment.
The rejection of claim(s) 1-3, 5, 9, 10, and 24, under 35 U.S.C. 102(a)(1) as being anticipated by Verhoeyen (Verhoeyen et al. Blood 106(10): 3386-3395, 2005), is withdrawn. Vehoeven does not disclose “a least one mutation that diminishes its native viral tropism, wherein the viral envelope protein is a VSV-G envelope protein.
The following new rejection is necessitated by the amendments to the claims:
Claim Rejections - 35 USC § 112
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.
The following is a quotation of the first paragraph of pre-AIA 35 U.S.C. 112:
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 of carrying out his invention.
Claims 1, 2, 4-13, 20-21, and 24-26 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 applications subject to pre-AIA 35 U.S.C. 112, the inventor(s), at the time the application was filed, had possession of the claimed invention.
When determining if a recited genus has adequate written description for a genus:
(1) the broadest reasonable interpretation of the genus is determined;
(2) the disclosure is examined to determine if the specification has provided a representative number of species to describe the complete structure of the genus;
(3) the disclosure is examined to determine whether a representative number of species have been sufficiently described by other relevant characteristics, specified features and functional attributes that would distinguish different members of the claimed genus; and
(4) the state of the art is examined to the determine if it supports/supplement the genus description in the specification in a manner that would demonstrate the application was in possession of the claimed genus at the time of effectively filing.
Claim 1 as amended recites, “a viral envelope protein comprising at least one mutation that diminishes its native tropism, wherein the viral envelope protein is a VSV-G envelope protein”. Thus the amended claim comprises the genus, a mutant VSV-G envelope protein with diminished tropism (Genus 1)
Claim 26 recites, “a viral envelope protein comprising at least one mutation that diminishes its native tropism, wherein the viral envelope protein is a VSV-G envelope protein, a measles virus envelop protein, a nipah virus envelop protein, or cocal virus envelop protein”. Thus the amended claim comprise the genus, a mutant viral envelope protein with diminished tropism (Genus 2).
Broadest Reasonable Interpretation of the Genus:
Genus 1: a mutant VSV-G envelope protein with diminished tropism:
VSV-G viruses have three envelop proteins (VP1, VP2, and VP3). As such, the breadth of the genus encompasses mutations in any of the three envelop proteins. The genus as recites comprises at least one mutation. As such, the breadth encompasses at least one of the amino acids is deleted, substituted, or added up to any all amino acid resides are substituted, added, or deleted in any one, two, or three of the VSV-G envelope proteins. The genus also recites that the at least one mutation “diminishes its native viral tropism”. “Diminish” means that to make less. However, diminish in and of itself does not impart any particular degree of making less. As such, the claimed at least one mutation encompasses diminishing viral tropism to any degree, which is a broad spectrum of functionality, from slightly less viral tropism, significant decrease in viral tropism, reducing almost all viral tropism, to completely ablating viral tropism of any one, two, or three of the VSV-G viral envelops. As such, the breadth of the claimed genus is vastly broad comprises an enormous number of structurally different VSV-G envelop proteins that have a spectrum of different viral tropism functionality.
Genus 2: a mutant viral envelope protein with diminished tropism:
Genus 2 has the same vast breadth as Genus 1 discussed above. However, Genus 2 is even broader because it is not limited to VSV-G viral envelope proteins but further includes all the viral envelope proteins for measle virus, nipah virus, and cocal virus G. As such, Genus 2 has an enormous number of possible species.
Specification Description: Citations from the pre-grant publication
[0011] In some embodiments, the retrovirus enters or infects the cell during (ii). In some embodiments, the retrovirus is a lentivirus. In some embodiments, the viral envelope protein is a VSV-G envelope protein or a cocal virus G protein. In some embodiments, at least one mutation of a VSV-G envelope protein is a mutation selected from the group consisting of H8, 141, K47, Y209, and R354. In some embodiments, the at least one mutation of the measles virus envelope protein is a mutation selected from the group consisting of Y481, R533, 5548, and F549. In some embodiments, the at least one mutation of the nipah virus envelope protein is a mutation selected from the group consisting of E501, W504, Q530, and E533. In some embodiments, the at least one mutation of the cocal virus G protein is a mutation selected from the group consisting of K64 and R371.
[0050] In some embodiments, a mutated VSV-G envelope protein comprises a mutation at H8, 141, K47, Y209, and/or R354. The position for an amino acid substitution in the mutated VSV-G envelope protein is identified in reference to the wildtype VSV-G envelope protein without the leader sequence, for example as provided in SEQ ID NO: 13. In some embodiments, a mutated VSV-G envelope protein comprises a HBA, I41L, K47A, K47Q, Y209A, R354A, and/or R354Q mutation. In some embodiments, a mutated VSV-env protein comprises an I41L, K47Q, and R354A mutation, such as a mutated VSV-env protein set forth in SEQ ID NO: 16. In some embodiments, a mutated VSV-env protein comprises a K47Q and R354A mutation, such as a mutated VSV-env protein set forth in SEQ ID NO: 17. In some embodiments, a mutated VSV-G envelope protein is as described in Nikolic et al., “Structural basis for the recognition of LDL-receptor family members by VSV glycoprotein.” Nature Comm., 2018, 9:1029, the relevant disclosures of which are incorporated by reference herein for this particular purpose.
[0051] In some embodiments, a mutated measles virus envelope protein comprises a mutation at Y481, R533, 5548, and/or F549. In some embodiments, a mutated measles virus envelope protein comprises a Y481A, R533A, S548L, and/or F549S mutation. In some embodiments, a mutated measles virus envelope protein comprises the mutated measles virus envelope protein set forth in SEQ ID NO: 21.
[0052] In some embodiments, a mutated Nipah virus envelope protein comprises a mutation at E501, W504, Q530, and/or E533. In some embodiments, a mutated measles virus envelope protein comprises a E501A, W504A, Q530A, and/or E533A mutation. In some embodiments, a mutated Nipah virus envelope protein comprises the mutated Nipah virus envelope protein set forth in SEQ ID NO: 23.
[0053] In some embodiments, a mutated cocal virus G protein comprises a mutation at K64 and/or R371. In some embodiments, a mutated cocal virus G protein comprises a mutation at K64Q and/or R371A. The position for an amino acid substitution in the mutated cocal virus G protein is identified in reference to the wildtype cocal virus G protein, for example as provided in SEQ ID NO: 24. In some embodiments, a mutated cocal virus G protein comprises a K64Q and R371A mutation, such as the mutated cocal virus G protein set forth in SEQ ID NO: 26.
[0055] In some embodiments, a viral envelope protein comprising at least one mutation comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more mutations. In some embodiments, a viral envelope protein comprising at least one mutation comprises a nucleotide sequence and/or amino acid sequence that is at least 50%, 60%, 70%, 80%, 90%, 95%, or 97% identical to a wild-type viral envelope protein. In some embodiments, a viral envelope protein comprising at least one mutation that diminishes its native function retains less than 95%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, or 10% of the function of a wild-type viral envelope protein. In some embodiments, a viral envelope protein comprising at least one mutation lacks all of its native function. In some embodiments, a retrovirus comprising a viral envelope protein comprising at least one mutation that diminishes its native function comprises less than 95%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, or 10% of the cellular infectivity of a retrovirus comprising a wild-type viral envelope protein.
As such, while Genus 1 generically contemplates a great number of possible mutations, it only describes 4 species point mutations in the VSV-G. While Genus 2 generically contemplates a great number of possible mutation, the specification only provides 4 species of point mutations for each of env proteins from measles, nipah virus, and cocal virus G proteins. Given the great breadth of Genus 1 and Genus 2, describing 4 point mutations for each fails to provide a representative number of species examples to each the complete structure of Genus 1 and Genus 2. Further, the specification does not provide any further description to other relevant types of mutations that will diminish the tropism or to what degree the tropism should be diminished. As such the specification fails to provide descriptions of relevant trails or characteristics of the species members to describe the full breadth of Genus 1 and Genus 2.
State of the Art: Regarding Genus 2, VSV-G protein is the still the elected species. As such, the discussion of the search art is limited to the elected species. However, the issue of mutations that “diminish” native tropism discussed belong do have applicability to the non-elected species.
As previously stated in the enablement rejection of record, Around the time of effective filing of the instant application Gutierrez-Guerrero et al. (Viruses 2020 12(1016) pp. 1/20 to 20/20) report, “it has been shown that VSV-G needs to traffic through the endosomal network of the cell and requires a low pH to fuse and eject its LV content into the cytoplasm before the viral RNA can be retrotranscribed and migrate into the nucleus and integrate…. It is the variation in the levels of the LDL-R expression that explains the low efficiency of the LVs pseudotyped with VSV-G in certain cell types. For example, the levels of LDL-R in unstimulated human T, B and hematopoietic stem and progenitor cells (CD34+ cells) are very low. “ See p. 4, 2nd to last paragraph. Regarding gene editing, they report, “In the context of gene therapy, HSCs are the targets of choice for gene editing-based therapies. The variability of efficiency of gene editing in cells is related to their repair pathway. It has been reported that adult primary cells use the error prone NHEJ instead of the DHR pathway due to their non-dividing stage… For the previously mentioned studies, different methods for delivery the editing tools have been utilized such as electroporation, adenoviruses and LVs conferring different degrees of efficiency, toxicity and off-target effects.” See page 13, section 4.
As such, Gutierrez-Guerrero et al. also teach that the use of VSV-G envelops in lentiviral vectors for delivery to HSC were hindered and unpredictable to due low efficiently.
Regarding specific mutated species of VSV-G envelop protein as claimed in claims 12 and 13, Albertini (US 12,091,434 B2 effectively published in Pre-grant application 7/9/2020) reports, “The invention is based on the unexpected observation made by the inventors that a substation of at least one amino acid at positions 8, 47, 209, or 354, or a combination of two or three or four amino acids, affects the ability of VSV G protein to interact with its receptor (LDL membrane receptor) but retain its property to induce membrane fusion in particular at low pH.”. Col 7, lines 36-42. Further Albertini discloses a mutant VSV-G envelop protein sequence, SEQ ID NO:175 that has 100% identity with SEQ ID NO:17 of claim 13 in the instant application. These disclosures by Albertini provide implications for mutating the VSV-G protein for use in the claimed retrovirus. However, Albertini does not go has far as demonstrate that it can effectively transduce the HSC or transduce to a level that that overcomes the unpredictabilites in the specification of the instant application and the art at the time of the application’s effective filing.
As such, the art teaches that VSV-G env has variable consequence on trophism of a virus and that mutations have been provided in the art that change binding its natural ligand, thus providing unknown degree and variable impact on tropism of such immune cells and hematopoietic stem cells. As such, the state of the regarding the structural/functional relationship between the breadth of the structural limitations of “at least one mutation” in the viral VSV-G envelope protein and the breadth of the functional limitations “that diminishes native viral tropism” as newly claimed in unpredictable.
In conclusion, the great breadth of any “at least one mutation” in a VSV-G protein “that diminishes nature viral tropism” lacks adequate written description. The breadth of the number of possible mutations encompassed by the breadth of “at least one mutation” with the requisite function of “diminishing” native viral tropism and then ultimately “delivering one or more nucleic acids to a hematopoietic stem cell” is not supported by the description of a few species and whose level of “diminishing native viral tropism” has not be described. The prior art does not describe some substitution mutations that alter the ability to bind to its cognate receptor and retain some degree of function but does not provide any description of means of any level of diminishing native tropism and still being involved in delivering one or more nucleic acids to a HSC. Further, the art teaches that altering the structure of the VSV-G does alter ligand binding in variable ways and the efficiency of VSV-G protein tropism already does not predictably transfect HSC due to demonstrated low levels of transduction efficiency. As such, the prior art does not supplement the shortage of descriptions by the specification. Further, an artisan could not envision the time of mutations and the degree of diminishing VSV-G native tropism from could predictably be used in the claimed method from the combined descriptions of the specification and existing art. As such, it appears the application was not in possession of the great breadth of ‘at least one mutation that diminishes its native viral tropism at the time of effective filing.
The following rejections of record are modified to take into consideration the amendments to the claims:
Claim Rejections - 35 USC § 112
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.
Improper Markush Grouping
(1) Claim 2 is rejected on the basis that it contains an improper Markush grouping of alternatives. See In re Harnisch, 631 F.2d 716, 721-22 (CCPA 1980) and Ex parte Hozumi, 3 USPQ2d 1059, 1060 (Bd. Pat. App. & Int. 1984). A Markush grouping is proper if the alternatives defined by the Markush group (i.e., alternatives from which a selection is to be made in the context of a combination or process, or alternative chemical compounds as a whole) share a “single structural similarity” and a common use. A Markush grouping meets these requirements in two situations. First, a Markush grouping is proper if the alternatives are all members of the same recognized physical or chemical class or the same art-recognized class, and are disclosed in the specification or known in the art to be functionally equivalent and have a common use. Second, where a Markush grouping describes alternative chemical compounds, whether by words or chemical formulas, and the alternatives do not belong to a recognized class as set forth above, the members of the Markush grouping may be considered to share a “single structural similarity” and common use where the alternatives share both a substantial structural feature and a common use that flows from the substantial structural feature. See MPEP § 2117.
The Markush grouping of extracellular targeting domain of SCF, FLT3L, TPO is improper because the alternatives defined by the Markush grouping do not share both a single structural similarity and a common use for the following reasons: each of these extracellular targeting domains have different primary amino acid sequences that bind distinctly different ligands and result in distinctly different signaling intracellularly. As such they do not have a common structural similarity that lead to a common use that flows from that structural similarity because the extracellular domain will bind to a structurally different ligand and impart functionally different signaling pathway.
To overcome this rejection, Applicant may set forth each alternative (or grouping of patentably indistinct alternatives) within an improper Markush grouping in a series of independent or dependent claims and/or present convincing arguments that the group members recited in the alternative within a single claim in fact share a single structural similarity as well as a common use.
(2) Claim 1 is rejected on the basis that it contains an improper Markush grouping of alternatives. See In re Harnisch, 631 F.2d 716, 721-22 (CCPA 1980) and Ex parte Hozumi, 3 USPQ2d 1059, 1060 (Bd. Pat. App. & Int. 1984). A Markush grouping is proper if the alternatives defined by the Markush group (i.e., alternatives from which a selection is to be made in the context of a combination or process, or alternative chemical compounds as a whole) share a “single structural similarity” and a common use. A Markush grouping meets these requirements in two situations. First, a Markush grouping is proper if the alternatives are all members of the same recognized physical or chemical class or the same art-recognized class, and are disclosed in the specification or known in the art to be functionally equivalent and have a common use. Second, where a Markush grouping describes alternative chemical compounds, whether by words or chemical formulas, and the alternatives do not belong to a recognized class as set forth above, the members of the Markush grouping may be considered to share a “single structural similarity” and common use where the alternatives share both a substantial structural feature and a common use that flows from the substantial structural feature. See MPEP § 2117.
The Markush grouping of a protein on the surface of HSC comprising CD34, CD90, CD133, CD201, c-Kit, FLT3, or TPO receptor is improper because the alternatives defined by the Markush grouping do not share both a single structural similarity and a common use for the following reasons: Each of these HSC surface proteins has a distinctly different amino acid structure that allows it to bind uniquely to a structurally different cognate. The end result that comes from each of these different surface proteins is a structurally distinct binding capacity and infectivity of the retrovirus having the surface protein capability. As such, the surface proteins do not have a common structural similarity form which a common use flows.
To overcome this rejection, Applicant may set forth each alternative (or grouping of patentably indistinct alternatives) within an improper Markush grouping in a series of independent or dependent claims and/or present convincing arguments that the group members recited in the alternative within a single claim in fact share a single structural similarity as well as a common use.
(3) Claim 4 is rejected on the basis that it contains an improper Markush grouping of alternatives. See In re Harnisch, 631 F.2d 716, 721-22 (CCPA 1980) and Ex parte Hozumi, 3 USPQ2d 1059, 1060 (Bd. Pat. App. & Int. 1984). A Markush grouping is proper if the alternatives defined by the Markush group (i.e., alternatives from which a selection is to be made in the context of a combination or process, or alternative chemical compounds as a whole) share a “single structural similarity” and a common use. A Markush grouping meets these requirements in two situations. First, a Markush grouping is proper if the alternatives are all members of the same recognized physical or chemical class or the same art-recognized class, and are disclosed in the specification or known in the art to be functionally equivalent and have a common use. Second, where a Markush grouping describes alternative chemical compounds, whether by words or chemical formulas, and the alternatives do not belong to a recognized class as set forth above, the members of the Markush grouping may be considered to share a “single structural similarity” and common use where the alternatives share both a substantial structural feature and a common use that flows from the substantial structural feature. See MPEP § 2117.
The Markush grouping is improper because the alternatives defined by the Markush grouping do not share both a single structural similarity and a common use for the following reasons: each SEQ ID NO:54-59 comprises a structurally different primary, secondary, and tertiary structure that allows it to bind to a structurally different ligand that imparts distinctly different signally pathways.
To overcome this rejection, Applicant may set forth each alternative (or grouping of patentably indistinct alternatives) within an improper Markush grouping in a series of independent or dependent claims and/or present convincing arguments that the group members recited in the alternative within a single claim in fact share a single structural similarity as well as a common use.
Response to Arguments
Applicant's arguments filed 10/9/2025 have been fully considered but they are not persuasive.
Applicant disagrees that the claims recite improper Markush groupings. Applicant submits claims 2 and 4 both recite groups of compounds having common structural properties and, within the context of the claim, each member could be substituted one for the other, with the expectation that the same intended result would be achieved. Claim 2 recites three alternative proteins (SCG, FLT3L and TPO), members of the class of cytokines involved in regulating hematopoiesis by binding to proteins on the surface of hematopoietic cells. Cytokines are a well-known class of proteins. Each of the three cytokines listed in claim 2 is known to be interact with proteins on the surface of hematopoietic cells (intended result). Thus, each of SCG, FLT3L and TPO meet the requirements of a proper Markush claim. Claim 4 recites a group of 6 proteins that are extracellular targeting domains which bind to the proteins present on a cell surface. In the context of claim 4, each of the proteins shares the common features of the class of well-known extracellular targeting domains that bind to a target cell surface and thus meet the requirements of a proper Markush claim.
In response, the extracellular targeting domain that binds to CD90 has a unique structure of the extracellular targeting domain that binds each of CD133, CD49f, CD201, c-Kit, Flts or thrombopoietin receptor that only allows for each of these species to bind the one specific ligand. For example that extracellular targeting domain that binds c-Kit can only bind the ligand SCF and the extracellular targeting domain that binds thrombopoietic receptor is thrombopoietin. As such, the extracellular targeting domain that binds c-Kit on HSC surface is not interchangeable to an extracellular targeting domain that binds thrombopoietin receptor on HSC because they are binding two structurally different ligands on HSC. As such, each of the claimed extracellular targeting domains cannot be substituted one for the other because the cells it targets are required to express structurally different proteins cognate. As the extracellular targeting domain species so not have a common structure from with a common function flows.
Applicant submits that it is alleged in the office action that the proteins of claims 2 and 4 are not proper members of a Markush claim because they don't share the same primary and secondary structure and bind to different ligands (Office Action pages 6-7). The Patent Trial and Appeal Board has dismissed similar arguments in Ex parte Narva (Appeal 2018-006168, Application No. 14/577,811), where an improper Markush rejection was reversed. In the decision, the Board indicated that a grouping of nucleic acids was a proper Markush group regardless of the fact that the nucleic acids had distinct primary sequences. The PTAB noted that the grouping of nucleic acids was not improper, as the claimed nucleic acid sequences "belong to the same recognized chemical class of polyribonucleotides" and encode ROP proteins. Similarly, the instant claimed compounds are all proteins, made up of amino acids and function by binding to cell surface proteins, as required by the claims.
In response, Ex parte Narva does not have the same fact pattern as the instant claims. Ex parte Narva states, “We agree with Appellants that the claimed Markush group is not improper, as the claimed species share a single structural similarity and a common use. (See Guidelines). The nucleic acid sequences recited in the rejected claims belong to the same recognized chemical class of polyribonucleotides that are hybridized in dsRNA molecules and encode ROP proteins. While the individual sequences differ because they are drawn to ROP sequences of different insects (e.g., SEQ ID NO:1 and 115) or different portions of the ROP sequence (e.g., SEQ ID NO:120, 131, and 133), all of the sequences share the common use of silencing ROP proteins.” (page 4 of Board decision). However, Ex parte Narva teaches that the polynucleotides with different sequences still have structural commonality to hybridize (i.e. bind) to the same gene encoding the same ROP proteins. In contrast, the claimed extracellular targeting domains do not bind same protein but structural different protein. As discussed above each extracellular targeting domain targets a different cognate molecule. As such, one cannot be substitute for the other as is the fact patter in Ex parte Narva. As such, contrary to the decision in Ex Parte Narva, the Markush grouping is proper because the extracellular target domain species unique and specifically bind structurally different cognates.
Claim Rejections - 35 USC § 112
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.
The following is a quotation of the first paragraph of pre-AIA 35 U.S.C. 112:
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 of carrying out his invention.
Claims 1-2, 4-13, 20-21, 24-26, as amended or previously presented, are rejected under 35 U.S.C. 112(a) or 35 U.S.C. 112 (pre-AIA ), first paragraph, because the specification, while being enabling for a method of delivering one or more nucleic acids to a human HSC comprising transducing a human HSC with a recombinant lentivirus comprising one or more nucleic acids of interest, an mutated VSV-G envelop protein, and a membrane bound SCF polypeptide comprising an extracellular targeting domain that binds to the surface of HSC, wherein the recombinant lentivirus transduces the HSC, wherein the mutated VSV-G envelop protein comprises the amino acid sequence of SEQ ID NO:16 or 17, does not reasonably provide enablement for a method of gene editing a HSC using a retrovirus comprising any VSV-G envelope protein mutant that diminishes its native function. 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.
While determining whether a specification is enabling, one considers whether the claimed invention provides sufficient guidance to make and use the claimed invention, if not, whether an artisan would require undue experimentation to make and use the claimed invention and whether working examples have been provided. When determining whether a specification meets the enablement requirements, some of the factors that need to be analyzed are: the breadth of the claims, the nature of the invention, the state of the prior art, the level of one of ordinary skill, the level of predictability in the art, the amount of direction provided by the inventor, the existence of working examples, and whether the quantity of any necessary experimentation to make and use the invention based on the content of the disclosure is “undue”.
Nature of the Invention: The invention is directed to a gene delivery method and a gene editing method that utilizes recombinant retroviruses pseudotyped with mutated forms of an envelope proteins having tropism for HSC and non-viral ligand extracellular targeting domains specific for surface proteins on HSC.
Breadth of the Claims: The elected invention is mutated VSV-G envelop protein with a species election to SCF as the extracellular targeting domain. As such, the scope of enablement pertains to the elected invention, more than the broader limitations recited in claim 1 and some of its dependents that encompass subject matter of non-elected inventions.
The breadth of the claims encompasses using any retrovirus comprising at least one mutation to any VSV-G envelope protein that diminishes any function of the envelope protein. The types of mutations encompass any one or more point mutations anywhere in the VSV-G envelop proteins and any two or more contiguous or non-contiguous mutation of any amino acid sequence anywhere in VSV-G envelop proteins sequences. The types of mutations also comprise amino acid(s) deletions, insertions, or substitutions. The mutated VSV-G envelop proteins in combination with the rest of the retrovirus also have the capacity to specifically deliver the intended nucleic acid payloads to HSC or more specifically gene edit the HSC.
Regarding claim 12, it recites, “a VSV-G envelope protein is a mutation selected from H8, I41, K47, Y209, and R354”. The claim does not provide a reference sequence for the VSV-G protein being mutated at these point mutations. As such, the breadth of the reference species encompasses any VSV-G protein of any length. It can be a wild-type sequence of any length having any part of the VSV-G protein present. It can also be a variant sequence of any.
Specification Guidance: (citations from Pre-grant publication)
[0046] Retrovirus and lentivirus constructs are well known in the art and any suitable retrovirus can be used to construct the retrovirus (or a plurality or library of retroviruses) as described herein. Non-limiting examples of retrovirus constructs include lentiviral vectors, human immunodeficiency viral (HIV) vector, avian leucosis viral (ALV) vector, murine leukemia viral (MLV) vector, murine mammary tumor viral (MMTV) vector, murine stem cell virus, and human T cell leukemia viral (HTLV) vector. These retrovirus constructs comprise proviral sequences from the corresponding retrovirus.
[0048] Viral envelope protein The retroviruses described herein comprise a viral envelope protein comprising at least one mutation that diminishes its native function (e.g., wild-type function of a non-mutated viral envelope protein). In some embodiments, a viral envelope protein is any viral envelope protein of any retrovirus (e.g., lentivirus). A viral envelope protein may be a VSV-G envelope protein, a measles virus envelope protein, a nipah virus envelope protein, or a cocal virus G protein. In some embodiments, a wild-type or non-mutated VSV-G envelope protein has the amino acid sequence of SEQ ID NO: 12 (with leader sequence) or SEQ ID NO: 13 (without leader sequence). In some embodiments, a wild-type or non-mutated measles virus envelope protein has the amino acid sequence of SEQ ID NO: 19 (with leader sequence). In some embodiments, a wild-type or non-mutated cocal virus G protein has the amino acid sequence of SEQ ID NO: 24. In some embodiments, the native function that is diminished by a mutation of a viral envelope protein is viral tropism (e.g., ability to infect cells, bind to cells, etc.).
[0050] In some embodiments, a mutated VSV-G envelope protein comprises a mutation at H8, 141, K47, Y209, and/or R354. The position for an amino acid substitution in the mutated VSV-G envelope protein is identified in reference to the wildtype VSV-G envelope protein without the leader sequence, for example as provided in SEQ ID NO: 13. In some embodiments, a mutated VSV-G envelope protein comprises a HBA, I41L, K47A, K47Q, Y209A, R354A, and/or R354Q mutation. In some embodiments, a mutated VSV-env protein comprises an I41L, K47Q, and R354A mutation, such as a mutated VSV-env protein set forth in SEQ ID NO: 16. In some embodiments, a mutated VSV-env protein comprises a K47Q and R354A mutation, such as a mutated VSV-env protein set forth in SEQ ID NO: 17. In some embodiments, a mutated VSV-G envelope protein is as described in Nikolic et al., “Structural basis for the recognition of LDL-receptor family members by VSV glycoprotein.” Nature Comm., 2018, 9:1029, the relevant disclosures of which are incorporated by reference herein for this particular purpose.
[0056] Non-viral membrane-bound protein The retroviruses described herein comprise a non-viral membrane-bound protein. A non-viral membrane-bound protein may comprise a membrane-bound domain and an extracellular targeting domain that binds to a protein on the surface of a hematopoietic stem cell (HSC). In some embodiments, a non-viral membrane-bound protein is a chimeric protein comprising sequences from at least two different proteins. In some embodiments, a non-viral membrane-bound protein is a full-length or truncated protein comprising sequence from a single protein.
[0060] In some embodiments, an extracellular targeting domain is any protein or peptide that has an amino acid sequence and is a binding partner for a target molecule or ligand (e.g., a cognate protein) on a surface of a hematopoietic stem cell (HSC). When present in the extracellular environment beyond the interior of the retrovirus, an extracellular targeting domain is capable of binding to an HSC. In some embodiments, an extracellular targeting domain binds or targets to a cognate protein or ligand (e.g., a protein receptor present on a target HSC) that is present on the cellular surface of an HSC or a subset of a population of HSCs. In some embodiments, an extracellular targeting domain binds to a cognate protein or ligand that is present on the cell surface of a single HSC or a subset of a population of HSCs. In some embodiments, a binding interaction between an extracellular targeting domain of a retrovirus and a cognate protein or ligand of a cell enables the retrovirus to enter the HSC.
[0063] In some embodiments, an extracellular targeting domain is stem cell factor (SCF), FMS-like tyrosine kinase 3 ligand (FLT3L), or thrombopoietin (TPO). In some embodiments, an extracellular targeting domain comprises the amino acid sequence set forth in any one of SEQ ID NOs. 54-59. [SEQ ID NO:58 is a truncated SCF amino acid sequence]
[0064] In some embodiments, an extracellular targeting domain binds to a protein on the surface of the HSC selected from the group consisting of: CD34, CD90, CD133, CD49f, CD201, c-Kit, FMS-like tyrosine kinase 3 (FLT3), and thrombopoietin receptor.
[0083] Described herein are methods of gene editing in a target cell (e.g., a hematopoietic stem cell (HSC)) comprising (i) providing a retrovirus comprising one or more nucleic acids encoding a gene editing composition, a viral envelope protein comprising at least one mutation that diminishes its native function, and a non-viral membrane-bound protein comprising an extracellular targeting domain that binds to a protein on the surface of the target cell; and (ii) contacting the retrovirus with the target cell such that the one or more nucleic acids encoding a gene editing composition are delivered to the target cell, wherein the gene editing composition specifically targets a section of the chromosomal DNA of the target cell to cause a genetic modification.
[0084] In some embodiments, the gene editing composition comprises one or more nucleic acids, wherein the one or more nucleic acids encode a gene editing protein and/or a guide RNA. In some embodiments, the gene editing composition comprises one or more nucleic acids, wherein the one or more nucleic acids encode a gene editing protein. In some embodiments, the gene editing composition comprises one or more nucleic acids, wherein the one or more nucleic acids encode a gene editing protein and a guide RNA.
[0085] In some embodiments the gene may be used to correct or ameliorate gene deficiencies, which may include deficiencies in which normal genes are expressed at less than normal levels or deficiencies in which the functional gene product is not expressed. Alternatively, the gene may provide a product to a cell which is not natively expressed in the cell type or in the host. A type of gene sequence encodes a therapeutic protein or polypeptide which is expressed in a host cell. The invention further includes using multiple genes. In certain situations, a different gene may be used to encode each subunit of a protein, or to encode different peptides or proteins. This is desirable when the size of the DNA encoding the protein subunit is large.
Working Examples:
Example 1. Expression of SCFa and S4-3a PStalk and IgG4Hinge constructs
[0124] Targeted lentiviruses were generated by polyethylenimine (PEI) transfection of HEK293T cells using plasmids encoding a mutated VSV-G (VSVd) or wild-type VSV-G (VSVwt). Constructs containing wild-type murine stem cell factor (mSCFa),with endogenous affinity for the cKIT receptor, and S4-3a, an affinity matured version of SCF which has been shown to exhibit more efficient viral entry (Ho C C et al., Cell 2017) were generated using the procedure described in International Patent Publication WO 2020/236263. Monomeric and pre-dimeric versions of the constructs were created. In the monomeric versions, mSCF was tethered to the PDGFR stalk and transmembrane protein (mSCFa-Pstalk (SEQ ID NO: 32), mS4-3a-Pstalk (SEQ ID NO: 28)). In the pre-dimeric versions, mSCF was tethered to an IgG4 hinge linker protein (mSCFa-IgG4hinge (SEQ ID NO: 42), mS4-3a-IgG4hinge (SEQ ID NO: 36)). The constructs were exposed to VSVd or VSVwt along with a fluorescently labeled antibody (HA-tag: AF647) and tested for expression on the surface of a HEK viral packaging line. As shown in FIG. 1 (mSCFa) and FIG. 2 (mS4-3a), all the constructs were found to express on HEK cells.
Example 4. Primary HSC transduction with engineered SCF and FLT3 specific virus
[0127] Engineered SCF and FLT3 virus constructs were tested to determine whether they were specific and efficient at delivering GFP protein to murine hematopoietic stem cells in the presence or absence of exogenous cytokines (SCF and FLT3) (FIG. 10).
[0128] Whole bone marrow cells (WBM) were isolated from B6 mice at 7 weeks. An aliquot of the isolated cells for was removed for further specificity testing in WBM. cKIT enrichment was performed and another aliquot was removed for further specificity testing in the cKIT enriched population. The cells were then sorted into three HSC populations according to the following criteria: 1-Lineage negative, cKIT positive; 2-Lineage negative, Sca-1 positive, cKIT positive (LSK); 3-Lineage negative, Sca-1 positive, cKIT positive, FLT3 positive. The cells were then cultured in media, with or without cytokines, for respective groups. The normal media for all HSC primary cells included FLT3L (50 ng/mL), TPO (50 ng/mL), and SCF (50 ng/mL). 24 hours after sorting, the cells (1M/mL) were incubated with concentrated virus at a ratio of 1:2. After 24 hours, the virus was removed, and cells were plated in cytokine complete media. 48 hours later, cells were stained, and flow panel was run to determine GFP expression within certain populations.
[0131] SCF-mutant was tested against SCF-WT in LSK (Lin-, Sca-1+, cKIT+), cKIT enriched, lineage depleted, and WBM (FIGS. 12A-12B). Results showed that GFP+ cells predominantly fell in the lineage—negative “immature” cell fraction. SCF-WT virus had slightly higher transduction efficiency than SCF-mutant. However, even in the purified population (LSK) efficiency was low.
[0132] SCF specificity was then examined in cKIT enriched cells in media with and without SCF (FIGS. 13A-13B). Results show that viability and expansion was not greatly changed by short term culture depletion of SCF. Additionally, withholding SCF did not seem to significantly change % GFP-positive fraction
[0133] SCF virus specificity was determined in the LSK (Lin-, Sca-1+, cKIT+), lineage depleted, and WBM cell populations (FIG. 14). Results show GFP+ cells predominantly fell into the cKIT-positive quadrant. Additionally, it was shown that cKIT expression can be lost in culture, as all cells in LSK culture were at one-point cKIT+ and therefore the small fraction of GFP+cKIT-cells could have been due to a specific infection. Finally, relative specificity (#GFP+ cells that are cKIT+compared to cKIT-) scaled regardless of starting cells in culture.
[0137] Taken together these results show that the engineered SCF and FLT3 lentiviruses demonstrate low efficiency transduction but fairly specific targeted integration. Although efficiency is low, there seems to be good specificity where cells that were successfully transduced (with both SCF and FLT3 viruses) are cKIT positive. Generally, the removal of a single cytokine from initial culture conditions did not seem to impede expansion and viability of cells. Overall, WBM did not perform well in culture conditions, suggesting that it may require a different set up for transduction-transplantation.
Thus, while the specification generically contemplates the use of retroviruses comprising a broad genus of mutated VSV-G envelop proteins and a SCF extracellular targeting domain to deliver or gene edit HSC, the specification, particularly the working examples, solely provide specific guidance to one species of lentiviral vector comprising one VSB-G envelope protein sequence and one truncated SCF extracellular targeting domain sequence that transduces human HSC. Further, the working examples does teach that the lentivirus specific did transduce HSC with specificity. However, transduction efficiency was with low efficiency, which demonstrates unpredictability. Further, while the specification does contemplate gene editing HSC with the claimed delivery method, the specification and working examples do not provide any specific guidance to a method going a step further with the delivery method and successfully gene editing any HSC. As such, while the specification provide sufficient specific guidance to a method of using the species of lentivirus in a method of delivery, the low efficiency of such a delivery method suggest unpredictability of extrapolating to a gene editing method, because it requires a predictable degree of transduction efficiency to provide enough of the gene editing payload for payload expression in an amount capable of predictably exacting gene editing. As such, the specification fails to provide predictable enabling guidance to the breadth of the claims encompassing the genus of the claimed retrovirus for gene editing an HSC.
State of the Art: Around the time of effective filing of the instant application Gutierrez-Guerrero et al. (Viruses 2020 12(1016) pp. 1/20 to 20/20) report, “it has been shown that VSV-G needs to traffic through the endosomal network of the cell and requires a low pH to fuse and eject its LV content into the cytoplasm before the viral
RNA can be retrotranscribed and migrate into the nucleus and integrate [34,35]. It is the variation in the levels of the LDL-R expression that explains the low efficiency of the LVs pseudotyped with VSV-G in certain cell types. For example, the levels of LDL-R in unstimulated human T, B and hematopoietic stem and progenitor cells (CD34+ cells) are very low. “ See p. 4, 2nd to last paragraph. Regarding gene editing, they report, “In the context of gene therapy, HSCs are the targets of choice for gene editing-based therapies. The variability of efficiency of gene editing in cells is related to their repair pathway. It has been reported that adult primary cells use the error prone NHEJ instead of the DHR pathway due to their non-dividing stage… For the previously mentioned studies, different methods for delivery the editing tools have been utilized such as electroporation, adenoviruses and LVs conferring different degrees of efficiency, toxicity and off-target effects.” See page 13, section 4.
As such, Gutierrez-Guerrero et al. also teach that the use of VSV-G envelops in lentiviral vectors for delivery to HSC were hindered and unpredictable to due low efficiently. Further, they teach that effectively delivering gene editing tools add another layer of unpredictability intrinsic to the gene editing tools that render low efficiency of gene editing of cells even if transduced. As such, art at the time of the invention teaches that bread of the claimed method is highly unpredictable.
Regarding specific mutated species of VSV-G envelop protein as claimed in claims 12 and 13, Albertini (US 12,091,434 B2 effectively published in Pre-grant application 7/9/2020) reports, “The invention is based on the unexpected observation made by the inventors that a substation of at least one amino acid at positions 8, 47, 209, or 354, or a combination of two or three or four amino acids, affects the ability of VSV G protein to interact with its receptor (LDL membrane receptor) but retain its property to induce membrane fusion in particular at low pH.”. Col 7, lines 36-42. Further Albertini discloses a mutant VSV-G envelop protein sequence, SEQ ID NO:175 that has 100% identity with SEQ ID NO:17 of claim 13 in the instant application. These disclosures by Albertini provide implications for mutating the VSV-G protein for use in the claimed retrovirus. However, Albertini does not go has far as demonstrate that it can effectively transduce the HSC or transduce to a level that that overcomes the unpredictabilites in the specification of the instant application and the art at the time of the application’s effective filing.
Thus the art at the time of effective filing does not overcome the unpredictabilites described in the specification and even mirrors its unpredictabilites by described additional hinderances to the breadth of the claimed invention.
Amount of Experimentation: As discussion above, both the instant application and the state of the art demonstrate a high degree of unpredictability before and at the time of effective filing of the instant application. Additionally the breadth of the claimed retroviral vector with a vastly large number of possible mutated VSV-G proteins and SCF protein variants would require an extensive amount of post-filing experimentation to discover what aspects of the VSV-G proteins are hindering their use as a means to effectively pseudostyle retrovirus for predictable transduction of to a degree that would allow for predictable gene editing. This type and degree of experimentation goes beyond routine experimentation and would be considered undue.
As such, the breadth of the claims to a method using any retrovirus comprising any mutated form of VSV-G protein and any SCF extracellular targeting domain to deliver a nucleic acid to and gene edit a HSC lacks enablement, the specification solely provides specific guidance to one species example of a lentivirus comprising one specific VSV-G protein mutant and one specific truncated form of SCF that transduces HSC with low efficiency, implying unpredictability. The specification does not provide any guidance to a predictable gene editing method as well. Further, the art also teaches unpredictabilites and fails to provide predictable supplementation to the disclosure of the instant application that overcome its unpredictable shortcomings. Even further, a significant amount of addition post-filing experimentation would be required to make discoveries to overcome the shortcomings in predictability and guidance by the specification and art. This level of experimentation is considered undue. As such, the claims lack enablement.
Response to Arguments
Applicant's arguments filed 10/9/2025 have been fully considered but they are not persuasive.
Applicant submits that retrovirus is a well-known tool for non-specifically delivering nucleic acids to target cells. All retroviruses are envelope viruses, with viral envelopes being of such recombinant viruses being derived from same or similar packaging cell lines. Thus, one of ordinary skill in the art would view the envelope of a recombinant lentivirus as representative of the genus of recombinant retrovirus envelops and expect the claimed mutated VSV-G protein and no-viral membrane tropism molecules to be present and function similarly in any recombinantly produced enveloped virus, and certainly any recombinant retrovirus. VSV-G protein having mutations resulting in diminished native tropism are also know to the skilled artisan. Further, the specification provides reductions to practice.
In response, Applicant is not considering the great breadth of “at least one mutation that diminishes it native viral tropism”. The breadth of this term is discussed above in the new written description rejection. The rejection acknowledges the reduction to practice are enabled because the scope designated as enables is to the reductions to practice. While the structure/function of retroviruses and VSV-G proteins are known and the specification and art do provide a small subset of mutations that can be made to the VSV-G proteins that disrupt their function, the breadth of diminish is vastly larger. The art teaches that altering the structure of a VSV-G protein alters its ability to bind its cognate in unpredictable ways and the few examples provided by the reduction to practice and the art does not providing enabling guidance to the vast number of other species encompasses by the claims. The art also does not supplement the guidance of the specification with predictable means of arrive at the great breadth of the claimed recombinant retrovirus. Thus the claims still lack enablement for the breadth of the amended claims.
Applicant address the art cited in the enablement provided to demonstrate unpredictability. Applicant refers to the Gutierrez-Guerrero cited by the rejection and submits that this reference teaches low efficiency of transfection not unpredictability. Applicant refers to Albertini and submits it teaches VSV-G mutations that ablate its ability to bind LDL-R, but preserve it fusion activity binding activity. Albertini fuses a mutated VSV-G with targeting moiety but does not provide a non-viral membrane-bound proteins comprising an extracellular targeting domain as the claims require. The fact that Albertini does not transduce HSC in not relevant since they are not using the same retrovirus.
In response, Applicant is not considering the art teachings in the context of the full rejection. The rejection refers to teaching from the specification that teach that VSV-G protein trophism has to do with its ability to bind its receptor. Low efficiency of VSV-G protein results in low efficiency most likely because of the low level of its ligand’s presence on the HSC. The art provided also teaches such obstacles to efficiency and that VSV-G with “diminished” function of varying degrees will not predictably bind to HSC because HSC so not predictably produce cell surface ligand. Albertini was provide to teach mutation to VSV-G also later the binding capacity it is cognate in ways that cannot be predicted. As such, mutations with a wide range of degrees of “diminishing” tropism in conjunction with the added extracellular targeting domain will have unpredictable impacts trophism. Given this unpredictability exist and neither the prior art and the specification provide sufficient enablement guidance to overcome this unpredictability for the great breadth of VSV-G protein, the claims are only enabled for the reduction to practice taught in the specification.
No claims are 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 MARCIA STEPHENS NOBLE whose telephone number is (571)272-5545. The examiner can normally be reached M-F 9-5:30.
Examiner interviews are available via telephone, in-person, and video conferencing using a USPTO supplied web-based collaboration tool. To schedule an interview, applicant is encouraged to use the USPTO Automated Interview Request (AIR) at http://www.uspto.gov/interviewpractice.
If attempts to reach the examiner by telephone are unsuccessful, the examiner’s supervisor, Peter Paras can be reached at 571-272-4517. The fax phone number for the organization where this application or proceeding is assigned is 571-273-8300.
Information regarding the status of published or unpublished applications may be obtained from Patent Center. Unpublished application information in Patent Center is available to registered users. To file and manage patent submissions in Patent Center, visit: https://patentcenter.uspto.gov. Visit https://www.uspto.gov/patents/apply/patent-center for more information about Patent Center and https://www.uspto.gov/patents/docx for information about filing in DOCX format. For additional questions, contact the Electronic Business Center (EBC) at 866-217-9197 (toll-free). If you would like assistance from a USPTO Customer Service Representative, call 800-786-9199 (IN USA OR CANADA) or 571-272-1000.
MARCIA S. NOBLE
Primary Examiner
Art Unit 1632
/MARCIA S NOBLE/Primary Examiner, Art Unit 1632
Prosecution Insights