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 .
A request for continued examination under 37 CFR 1.114, including the fee set forth in 37 CFR 1.17(e), was filed in this application after final rejection. Since this application is eligible for continued examination under 37 CFR 1.114, and the fee set forth in 37 CFR 1.17(e) has been timely paid, the finality of the previous Office action has been withdrawn pursuant to 37 CFR 1.114. Applicant's submission filed on 05/28/2025 has been entered.
Amended claims 137, 139-149, 151, 156, 158, 160-164 and new claims 165-166 are pending in the present application.
Applicant elected previously with traverse the Invention of Group I, which is drawn to an LNP composition comprising the components recited in independent claim 137.
Applicant also elected previously with traverse the following species: (i) Lipid A as a species of an ionizable lipid; (ii) cholesterol as a species of a helper lipid; (iii) DSPC as a species of a neutral lipid; and (iv) PEG2k-DMG as a species of a stealth lipid.
Claims 160-163 were withdrawn previously from further considerations because they are directed to a non-elected invention.
Accordingly, amended claims 137, 139-149, 151, 156, 158 and 164-166 are examined on the merits herein with the above elected species.
Claim Rejections - 35 USC § 112
The following is a quotation of pre-AIA 35 U.S.C. 112, fourth paragraph:
Subject to the following paragraph [i.e., the fifth paragraph of pre-AIA 35 U.S.C. 112], 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 158 is rejected 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. This is because dependent claim 158 recites the limitation “wherein the guide RNA nucleic acid is an sgRNA”, but amended independent claim 137 already recites “wherein the guide RNA nucleic acid is a single guide RNA (sgRNA)”. 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. This is a new ground of rejection.
Claim Rejections - 35 USC § 103
The following is a quotation of 35 U.S.C. 103 which forms the basis for all obviousness rejections set forth in this Office action:
A patent for a claimed invention may not be obtained, notwithstanding that the claimed invention is not identically disclosed as set forth in section 102, if the differences between the claimed invention and the prior art are such that the claimed invention as a whole would have been obvious before the effective filing date of the claimed invention to a person having ordinary skill in the art to which the claimed invention pertains. Patentability shall not be negated by the manner in which the invention was made.
Amended claims 137, 139-149, 151, 156, 158 and 164-166 are rejected under 35 U.S.C. 103 as being unpatentable over Zhang et al (US 2014/0179770; IDS) in view of Brito et al (WO 2015/095340; IDS) and Yin et al (WO 2015/191693; IDS). This is a modified rejection.
The instant claims encompass a lipid nanoparticle (LNP) composition comprising: (a) an mRNA encoding a Cas nuclease, wherein the Cas nuclease is Cas9 nuclease or Cpf1 nuclease; (b) a guide RNA nucleic acid, wherein the guide RNA nucleic acid is a single guide RNA (sgRNA); and (c) a plurality of component lipids comprising: Lipid A which is (9Z,12Z)-3-((4,4-bis(octyloxy)butanoyl0oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl-octadeca-9,12-dienoate, (ii) a helper lipid (with cholesterol as the elected species), (iii) a neutral lipid (with DSPC as the elected species), and (iv) a stealth lipid (with PEG2k-DMG as the elected species), wherein the LNP composition includes a ratio of mRNA encoding the Cas nuclease to guide RNA nucleic acid of about 10:1 to about 1:10 (w/w), and wherein the mRNA encoding the Cas nuclease and the guide RNA nucleic acid are co-encapsulated in the LNP composition.
With respect to the elected species, Zhang et al already taught at least a composition comprising components of a CRISPR-Cas system (e.g., type II CRISPR-Cas system such as the CRISPR-Cas9 system) for manipulation of sequences and/or activities of target sequences (e.g., editing or modifying a target site in a genomic locus of interest) in a eukaryotic cell (see at least Abstract; Summary of the Invention; particularly paragraphs [0008]-[0010], [0020], [0026], [0037]-[0039], [0226]-[0232], [0337]; and Fig. 1). Zhang et al specifically taught PCSK9 (proprotein convertase subtilisin kexin 9) gene which is primarily expressed by the liver as a target for CRISPR, and PCS9K-targeted CRISPR may be formulated in a lipid particle and for example administered intravenously in humans with gain of function mutations in PCSK9 (paragraph [0337]). Zhang et al stated explicitly “Any or all of the polynucleotide sequence encoding a CRISPR enzyme, guide sequence, tracr mate sequence or tracr sequence, may be RNA. The polynucleotides encoding the sequence encoding a CRISPR enzyme, the guide sequence, tracr mate sequence or tracr sequence may be RNA and may be delivered via liposomes, nanoparticles, exosomes, microvesicles, or a gene-gun” (paragraph [0026]); ”To enhance expression and reduce toxicity, the CRISPR enzyme and/or guide RNA can be modified using pseudo-U or 5-Methyl-C. mRNA delivery methods are especially promising for liver delivery currently…CRISPR enzyme mRNA and guide RNA may be delivered simultaneously using nanoparticles or lipid envelopes” (paragraphs [0225]-[[0228]); “In another embodiment, lipid nanoparticles (LNPs) are contemplated. In particular, an antitranstheretin small interfering RNA encapsulated in lipid nanoparticles (see, e.g., Coelho et al., N Engl J Med 2013; 369:819-29) may be applied to the CRISPR Cas system of the present invention….LNPs have been shown to be highly effective in delivering siRNAs to the liver (see, e.g., Tabemero et al., Cancer Discovery, April 2013, Vol. 3, No. 4, pages 363-470) and are therefore contemplated for delivering CRISPR Cas to the liver” (paragraphs [0236]-[0237]); and “Preparation of LNPs and CRISPR Cas encapsulation may be used/and or adapted from Rosin et al, Molecular Therapy, Vol. 19, no.12, pages 1286-2200, December 2011)….The specific CRISPR Cas RNA may be encapsulated in LNPs containing DLinDAP, DLinDMA, DLinK-DMA, and DLinKC2-DMA (cationic lipid:DSPC:CHOL:PEGS-DMG or PEG-C-DOMG at 40:10:40:10 molar ratios)…..The particle size for all three LNP systems may be about 70 nm in diameter” (paragraph [0239]). Please note that DLinDAP, DLinDMA, DLinK-DMA and DLinKC2-DMA are ionizable cationic lipids (paragraphs [0238]) while DSPC is a neutral lipid, CHOL is a helper lipid, and PEGS-DMG ((3-o-[2’-(methoxypolyethyleneglycol 2000) succinoyl]-1,2-dimyristoyl-sn-glycol) or PEG-C-DOMG (R-3-[(w-methoxy-poly(ethylene glycol)2000) carbamoyl]—1,2-dimyristyloxlpropyl-3-amine) is a stealth lipid. Zhang et al also taught that the CRISPR-Cas system may be administered in liposomes such as stable nucleic acid-lipid particles (SNALP) which have been proven to be effective delivery molecules to highly vascularized HepG2-derived liver tumors; and that the SNALP liposomes are about 80-100 nm in size and may be composed of Cholesterol/D-Lin-DMA/DSPC/PEG-C-DMA in the 48:40:10:2 molar ratio (paragraphs [0269]-[0270]). Zhang et al also stated “FIG. 1 shows a schematic model of the CRISPR system. The Cas9 nuclease from Streptococcus pyogenes (yellow) is targeted to genomic DNA by a synthetic guide RNA (sgRNA) consisting of a 20-nt guide sequence (blue) and a scaffold (red). The guide sequence base-pairs with the DNA target (blue), directly upstream of a requisite 5’-NGG protospacer adjacent motif (PAM; magenta), and Cas9 mediates a double-stranded break (DSB)-3 bp upstream of the PAM (red triangle)” (paragraph [0086]); and “CRISPR enzyme mRNA and guide RNA might also be delivered separately….Alternatively, CRISPR enzyme mRNA and guide RNA can be administered together” (paragraphs [0327]-[0328]).
Zhang et al did not teach explicitly at least a lipid nanoparticle (LNP) composition comprising the ionizable Lipid A (9Z,12Z)-3-((4,4-bis(octyloxy)butanoyl0oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl-octadeca-9,12-dienoate, a helper lipid, a neutral lipid, and a stealth lipid; wherein Cas9 nuclease mRNA and a sgRNA are co-encapsulated in the LNP composition and wherein the LNP composition includes a ratio of mRNA encoding Cas9 nuclease to sgRNA of about 10:1 to about 1:10 (w/w).
Before the effective filing date of the present application (03/30/2016), Brito et al already taught a lipid nanoparticle with a diameter in a range of about 50-150 nm and comprised of a cationic lipid (e.g., Lipid A which is (9Z,12Z)-3-((4,4-bis(octyloxy)butanoyl0oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl-octadeca-9,12-dienoate at the 55-40 lipid molar ratio), a helper lipid (e.g., cholesterol or cholesterol hemisuccinate at the 55-40 lipid molar ratio), a neutral lipid (e.g., DSPC at the 15-5 lipid molar ratio), and a stealth lipid (e.g., S010, S024 or PEG-DMG at the 10-1 lipid molar ratio) to deliver biological active agents (e.g., DNA or RNA agents such as mRNA, siRNA) to cells and tissues, including to the liver (Abstract; Summary of the Invention; particularly page 15, lines 23-24; page 19, lines 23-24; page 30, line 1 continues to line 8 on page 37; page 38, lines 13-16; page 39, line 1 continues to line 17 on page 40; Example 13 on page 86; pages 162-157, especially Table 2 on page 164 and Table 3 on page 167; pages 169-171, especially Table 4 on pages 170-171). Please noting that Lipid A contains a tertiary amine head group, and therefore it is an ionizable cationic lipid. Brito et al stated clearly “The lipid nanoparticles have a size of about 1 to about 2,500 nm, about 10 to about 1,500 nm, about 20 to about 1,000 nm, in a sub-embodiment about 5 to about 600 nm, in a sub-embodiment about 50 to about 400 nm, in a sub-embodiment about 50 to about 250 nm, and in a sub-embodiment bout 50 to about 150 nm. Unless indicated otherwise, all sizes referred to herein are the average sizes (diameters) of the fully formed nanoparticle, as measured by dynamic light scattering on a Malvern Zetasizer” (page 38, lines 13-18); and “In certain embodiments the PEG (e.g., conjugated to a lipid, such as a stealth lipid) is a “PEG-2K”, also termed “PEG-2000”, which has an average molecular weight of about 2000 daltons” (page 37, lines 1-3). Brito et al also stated clearly “The present invention provides a cationic lipid scaffold that demonstrates enhanced efficacy along with lower toxicity (improved therapeutic index) as a result of lower sustained lipid levels in the relevant tissues, and for local delivery applications (eye, ear, skin, lung); delivery to muscle (i.m.), fat, or subcutaneous cells (s.c. dosing)” (page 3, lines 7-10). In an exemplification, the ionizable cationic lipid in Example 13 (Lipid A) shown in Table 2 on page 164 exhibits an 90.7% encapsulation efficiency for Leptin mRNA and 88.1% encapsulation efficiency for siRNA FVII. Table 3 on pages 253-254 showed the cationic lipid in Example 13 (Lipid A) exhibits an encapsulation efficiency of 95.4%, 96.4%, 98.5%, 98.2% and 88.1% for Leptin mRNA, hFIX mRNA, mEPO mRNA, pDNA and FVII siRNA, respectively; and a lipid nanoparticle size ranging from 88.1 nm to 127.6 nm. Additionally, the cationic lipid in Example 13 (Lipid A) also exhibits 97% FVII knock-down following intravenous injection of encapsulated siRNA in mice (Table 4 on page 171).
Additionally, Yin et al also disclosed a CRISPR-Cas9 system and various delivery systems (e.g., viral and/or non-viral delivery systems) for the components of the CRISPR-Cas9 system to achieve an efficient in vivo gene editing/modification of a target nucleotide sequence (see at least Abstract; Summary of the Invention; particularly paragraphs [0006], [0008]-[0009], [0011, [0043]-[0051], [0054]-[064], [0070]-[0072], [0088], [00117]-[00122], [0138]-[0142]; examples 1, 4; and Figures 1-2 and 9-10). Yin et al stated clearly “[t]he present disclosure provides delivery systems comprising (i) one or more gRNA covalently or noncovalently bound to a repair template and (ii) a nucleic acid editing system, wherein (i) and (ii) are present on the same or different delivery vehicles” (paragraph [0006]); “The delivery vehicles provided herein may be viral vectors or non-viral vectors, or RNA conjugates. In some embodiments, the gRNA and the nucleic acid editing system are provided in the same type of delivery vehicle, wherein the delivery vehicle is a viral vector or a non-viral vector” (paragraph [0051]); and “In another embodiment, each of the components of the delivery systems provided herein (e.g., the nucleic acid editing system, gRNA and, optionally, repair template) are each contained in the same or in different nanoparticles” (paragraph [0088]). Yin et al specifically taught that the ratio of Cas9:sgRNA is from about 1:100 to about 100:1, or about 1:50 to about 50:1, or about 1:25 to about 25:1, or about 1:10 to about 10:1, or about 1:5 to about 5:1, or about 1:2 to about 2:1, or about 1:1 for consistent delivery to the target sequence (last sentence in paragraph [00112]). Yin et al also disclosed that the delivery vehicle can be a non-viral vector in the form of a lipid-based or polymeric vector such as liposomes, lipid encapsulation systems, nanoparticles and small nucleic acid-lipid particle (SNALP), particularly the lipid encapsulation comprising one or more of a phospholipid, cholesterol, polyethyelene glycol (PEG)-lipid, and a lipophilic compound (e.g., C12-200 and cKK-E12), including the lipid encapsulation composed of cKK-E12, DOPE, cholesterol and C14-PEG2000 for efficient in vivo editing in liver tissue (paragraphs [0011] and [0063]-[0064]). Yin et al also taught that the lipid nanoparticle is between about 1 and about 100 nanometers in size (paragraph [0054], and nanoparticles are formulated with Cas9 mRNA chemically modified to reduce TLR responses (paragraph [0060]). Yin et al further taught that the gRNA is also chemically modified at the 5’ end, middle portion of the RNA or the 3’ end (paragraphs [0070]-[0072]). In an exemplification, Yin et al demonstrated an efficient CRISPR-Cas9-mediated correction of a Fumarylacetoacetate hydrolase (Fah) mutation in a mouse model of the human disease hereditary tyrosinemia gene defect using Cas9 mRNA encapsulated in lipid nanoparticles composed of cKK-E12, DOPE, Cholesterol and C14-PEG2000 and rAAV2/8 virus expressing gRNA against a mutant FAH gene and a correct FAH repair template; and Yin et al concluded that systemic delivery of Cas9 mRNA by lipid nanoparticle can effectively mediate genome editing in vivo (paragraphs [00117]-[00118], [00138]-[00142] and Figs. 9-10).
Accordingly, it would have been obvious for an ordinary skilled artisan before the effective filing date of the present application to modify the teachings of Zhang et al by also utilizing the disclosed lipid nanoparticles containing the Lipid A of Brito et al, including at least LNPs composed of a cationic Lipid A, DSPC, cholesterol and PEG2k-DMG at their respective molar ratios in the range of 55-40%:15-5%:55-40%:10-1%, that have an average particle size of about 75 nm to about 150 nm to co-encapsulate sgRNA and CRISPR-Cas9 mRNA, including at the ratio of CRISPR-Cas9 mRNA to sgRNA at a ratio of about 10:1 to about 1:10 (w/w), for editing or modifying a target sequence in a eukaryotic cell such as a liver cell, in light of the teachings of Brito et al and Yin et al as presented above.
An ordinary skilled artisan would have been motivated to carry out the above modifications because Brito et al provided LNPs with a diameter in a range of about 50-150 nm and comprised of: (i) the ionizable cationic Lipid A ((9Z,12Z)-3-((4,4-bis(octyloxy)butanoyl0oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl-octadeca-9,12-dienoate) at the 55-40 lipid molar ratio; (ii) the helper lipid cholesterol at the 55-40 lipid molar ratio; (iii) the neutral lipid DSPC at the 15-5 lipid molar ratio, and (iv) the stealth lipid PEG2k-DMG at the 10-1 lipid molar ratio for delivering biological active agents to cells and tissues, that have enhanced efficacy along with lower toxicity (improved therapeutic index). Additionally, Yin et al already demonstrated successfully that systemic delivery of Cas9 mRNA by lipid nanoparticle can effectively mediate genome editing in vivo; and taught explicitly that both gRNA and mRNA encoding Cas9 can be present on the same delivery vehicle/lipid nanoparticle as well as the ratio of Cas9:sgRNA is from about 1:10 to about 10:1, or about 1:5 to about 5:1, or about 1:2 to about 2:1, or about 1:1 for consistent delivery to the target sequence. Please note that the primary Zhang reference also taught that CRISPR enzyme mRNA and guide RNA can be administered together.
An ordinary skilled artisan would have a reasonable expectation of success in light of the teachings of Zhang et al, Brito et al and Yin et al, coupled with a high level of skill for an ordinary skilled artisan in the relevant art.
The modified LNP composition resulting from the combined teachings of Zhang et al, Brito et al and Yin et al is indistinguishable from and encompassed by the presently claimed invention.
Therefore, the claimed invention as a whole was prima facie obvious in the absence of evidence to the contrary.
Response to Arguments
Applicant’s arguments related to the above modified 103 rejection in the Amendment filed on 05/28/2025 (pages 6-11) have been fully considered, but they are respectfully not found persuasive for the reasons discussed below.
A. Applicant argued basically that the claimed LNP composition is not taught or suggested by Zhang, Britto or Yin, alone or in combination. With respect to Zhang, Applicant argued that the reference provides several examples of delivery system known to be useful for formulating RNA cargo, e.g., siRNA; but it does not indicate which of these systems provide for successful coformulation of mRNA and a gRNA. Applicant also argued that Zhang does not provide any specific guidance with respect to co-formulating an mRNA and a gRNA in LNPs, and only speculative paragraphs that would not lead one of ordinary skill to conclude that mRNA and a guide RNA could be successfully formulated together. Applicant also argued that neither Zhang nor Yin teaches a lipid nanoparticle composition comprising the ionizable cationic Lipid A, and the Examiner attempts to cure this deficiency with Brito. Applicant argued that Brito discloses 77 ionizable lipids among which Lipid A is one example, and that the data presented by Brito does not provide motivation for the skilled artisan to select Lipid A specifically. This is because Applicant notes that Example 13 (Lipid A) does not provide the best in vitro encapsulation data for either mRNA or siRNA in Table 2; while in Table 3 Lipid A did not provide consistent performance for encapsulation efficiency data and polydispersity since it provides the worst encapsulation efficiency for hLeptin mRNA while having the best polydispersity and yet the best encapsulation efficiency for pDNA while having the worst polydispersity. Thus, a person of ordinary skill in the art would have lacked the motivation to select Lipid A specifically from the plethora of lipids disclosed therein. Applicant also argued that there is no reasonable expectation of success for modifying the lipids described by Zhang with Lipid A described in Brito because the structure of Lipid A is substantially different than those cationic lipids (DLinKC2-DMA, DLinKDMA, DLinDMA, and DLinDAP) taught by Zhang, particularly the closest in structure to Lipid A is DLinDAP that is the least potent of the cationic lipids discussed by Zhang. Applicant also argued that Yin fails to cure the deficiency in the combination of Zhang and Brito as Yin does not teach or suggest Lipid A, or its selection for use in a formulation for co-encapsulation of an mRNA and a gRNA. Applicant also argued that the data in Yin would not motivate one of ordinary to co-formulate Cas9 mRNA and gRNA into a single LNP composition, as Yin only demonstrates successful gene editing when the two components are delivered as separate formulations in Examples 1 and 4. In contrast, Applicant has demonstrated successfully delivery of a LNP composition comprising both Cas9 mRNA and a sgRNA. Applicant further argued that one of ordinary skill in the art would not be able to predict that the specifically claimed LNP composition could effectively encapsulate Cas9 mRNA and sgRNA, or such a composition could achieve effective delivery and gene editing in vivo, as demonstrated by Applicant.
First, please note that since the above rejection was made under 35 U.S.C. 103 none of the cited references have to teach every limitation of the instant claims. For example, neither Zhang nor Yin has to teach an LNP comprising Lipid A for delivery the components of a CRISPR-Cas9 system; nor does Brito have to teach using an LNP comprising Lipid A to co-encapsulate mRNA encoding a Cas9 nuclease and a gRNA nucleic acid (e.g., a gRNA mRNA or a DNA encoding a gRNA). It appears that Applicant considered each of the cited references in total isolation one from the others, and did not consider the specific combination of Zhang, Brito and Yin as set forth in the above modified 103 rejection.
Second, with respect to the primary Zhang reference there is no legal requirement whatsoever that the reference has to provide experimental data (a working example) on any particular disclosed lipid nanoparticle system for successful coformulation of a mRNA and a gRNA. Zhang clearly teaches that the specific CRISPR Cas RNA may be encapsulated in LNPs containing an ionizable cationic lipid DLinDAP, DLinDMA, DLinK-DMA, and DLinKC2-DMA, with the cationic lipid:DSPC:CHOL:PEGS-DMG or PEG-C-DOMG are at 40:10:40:10 molar ratios, and wherein DSPC is a neutral lipid, CHOL is a helper lipid, and PEGS-DMG ((3-o-[2’-(methoxypolyethyleneglycol 2000) succinoyl]-1,2-dimyristoyl-sn-glycol) or PEG-C-DOMG (R-3-[(w-methoxy-poly(ethylene glycol)2000) carbamoyl]—1,2-dimyristyloxlpropyl-3-amine) is a stealth lipid. Zhang also teaches specifically that the CRISPR-Cas system may be administered in liposomes such as stable nucleic acid-lipid particles (SNALP) which have been proven to be effective delivery molecules to highly vascularized HepG2-derived liver tumors; and that the SNALP liposomes are about 80-100 nm in size and may be composed of Cholesterol/D-Lin-DMA/DSPC/PEG-C-DMA in the 48:40:10:2 molar ratio. Moreover, Zhang teaches CRISPR enzyme mRNA and guide RNA may be delivered simultaneously using nanoparticles or lipid envelopes. An ordinary skill in the art would readily recognize that a lipid nanoparticle that is suitable for delivery of siRNA is also suitable for delivery of at least a gRNA and/or a mRNA encoding a Cas9 nuclease with a reasonable expectation of success; particularly before the effective filing date of the present application Yin already demonstrated successfully that systemic delivery of Cas9 mRNA by an exemplary lipid nanoparticle composed of cKK-E12 (an ionizable cationic lipid), DOPE, cholesterol and C14-PEG2000 can effectively mediate genome editing in vivo. Additionally, Yin also disclosed the use of Nano.Cas9 that is formulated with C12-200, cholesterol, C14-PEG 2000, DSPC and Cas9 mRNA in a weight ratio of 50:20:10:10 for successfully delivering Cas9 mRNA to mediate genome editing in vivo, and the nano.Cas9 is less toxic in comparison with Lipofectamine 2000 (Example 4; paragraphs [00136]-[00137]; Fig. 12d-e and Fig. 13). Moreover, the prior art in the form of Hoge et al (WO 2015/006747; IDS) also demonstrated that the lipid nanoparticle formulation of composed of Dlin-MC3-DMA (another ionizable cationic lipid), DSPC, cholesterol and PEG-DOMG at the respectively molar ratio of 50:10:38.5:1.5 successfully delivered Cas9 mRNA in mice via intravenous injection for Cas9 expression in vivo (see paragraphs [00658]-[00661]; Table 3 and Figure 5); as well as Lee et al (US 10,626,393) successfully delivered CRISPR therapeutics with lipid nanoparticles (Abstract; Examples 1-2; and issued claims 1-15). Lee et al also stated clearly “In certain embodiments, the lipid particles comprise both gRNA and an mRNA encoding a Cas9” (col. 1, lines 61-62). Furthermore, Brito already demonstrated the cationic lipid in Example 13 (Lipid A) exhibits an encapsulation efficiency of 95.4%, 96.4%, 98.5%, 98.2% and 88.1% for Leptin mRNA, hFIX mRNA, mEPO mRNA, pDNA and FVII siRNA, respectively; and the cationic lipid in Example 13 (Lipid A) also exhibits 97% FVII knock-down following intravenous injection of encapsulated siRNA in mice.
Third, Brito et al stated clearly “The present invention provides a cationic lipid scaffold that demonstrates enhanced efficacy along with lower toxicity (improved therapeutic index) as a result of lower sustained lipid levels in the relevant tissues, and for local delivery applications (eye, ear, skin, lung); delivery to muscle (i.m.), fat, or subcutaneous cells (s.c. dosing)” (page 3, lines 7-10); and this is a provided motivation for an ordinary skill in the art. Please also note that Lipid A is a preferable lipid that Brito selected for further characterization in exemplifications. An ordinary skill in the art would hardly consider an encapsulation efficiency in a range of 95.45%-98.5% for hLeptin mRNA, hFIX mRNA, FLuc mRNA, Gluc mRNA and mEPO mRNA by Lipid A is “inferior” under any standard, particularly Lipid A also encapsulates FVII siRNA with an efficiency of 88.1% (Table 3) that mediates 97% FVII knock-down following intravenous injection of encapsulated siRNA in mice (Table 4). Accordingly, based on the functional characterization of Lipid A presented in Tables 2-4 of the Brito reference, an ordinary skill in the art would also similarly select Lipid A as a preferred lipid.
Fourth, please note that Yin’s teachings are not necessarily limited only to Examples 1 and 4. Yin stated clearly “In another embodiment, each of the components of the delivery systems provided herein (e.g., the nucleic acid editing system, gRNA and, optionally, repair template) are each contained in the same or in different nanoparticles” (paragraph [0088]). Moreover, in addition to the exemplary lipid nanoparticle composed of cKK-E12 (an ionizable cationic lipid), DOPE, cholesterol and C14-PEG2000 in Example 1, Yin also disclosed the use of Nano.Cas9 that is formulated with C12-200, cholesterol, C14-PEG 2000, DSPC and Cas9 mRNA in a weight ratio of 50:20:10:10 for successfully delivering Cas9 mRNA to mediate genome editing in vivo, and the nano.Cas9 is less toxic in comparison with Lipofectamine 2000 (Example 4; paragraphs [00136]-[00137]; Fig. 12d-e and Fig. 13). As already noted above the prior art in the form of Hoge et al (WO 2015/006747; IDS) demonstrated successfully that the lipid nanoparticle formulation composed of Dlin-MC3-DMA (another ionizable cationic lipid), DSPC, cholesterol and PEG-DOMG at the respectively molar ratio of 50:10:38.5:1.5 successfully delivered Cas9 mRNA in mice via intravenous injection for Cas9 expression in vivo (see paragraphs [00658]-[00661]; Table 3 and Figure 5); as well as Lee et al (US 10,626,393) successfully delivered CRISPR therapeutics (Cas9 mRNA and/or a gRNA) in lipid nanoparticles (Abstract; Examples 1-2; and issued claims 1-15). There is no factual evidence of record indicating or suggesting that any lipid nanoparticle formulation is incapable of co-encapsulating a Cas9 mRNA and a sgRNA for any reason, let alone for the 4-component lipid nanoparticle containing Lipid A as a cationic lipid that is taught by Brito.
B. Previously Applicant also argued that in contrast to the cited references, Applicant has experimentally demonstrated that the claimed compositions comprising Lipid A are capable of delivering Cas9 mRNA and sgRNA encapsulated together; and the data in Figure 7 demonstrate robust in vitro editing efficiency of Neuro2A cells. Additionally, the data in Figs. 8-11 show that these formulations produce robust in vivo editing efficiency, resulting in phenotypic changes (e.g., large decrease of up to approximately 75% in serum TTP levels observed in animals treated with LNPs targeting a TTR sequence as shown in Fig. 10). Applicant submitted that these results would not have been expected in light of the cited references. Applicant further argued that the data in the specification show superior properties of LNPs comprising Lipid A for delivery of Cas9 mRNA and sgRNA relative to the commercial transfection agent Lipofectamine as shown in Fig. 19.
First, with respect to the robust in vitro editing efficiency in Figure 7, the robust in vivo editing efficiency in Figs. 8-11, and the “superior” data in Fig. 19 of the as-filed specification, the examiner notes that these results were obtained using at least a lipid nanoparticle with the specific formulation of Lipid A (45 mol-%), cholesterol (44 mole-%), DSPC (9 mol-%) and PEG2k-DMG or PEG2k-C11 (2 mol-%); together with the ratio of mRNA:sgRNA was approximately 1:1 by weight of the RNA component, wherein the sgRNA has 2’-O-methyl modifications and phosphorothioate linkages at and between the three terminal nucleotides at both the 5’ and 3’ ends of the sgRNA that specifically targets a particular target sequence in a target gene (e.g., a TTR gene) (see at least Examples 1-7). Please note that any “surprising/unexpected” result must be commensurate with the scope of the claims. The instant specification already demonstrated that altered ratios of mRNA:gRNA resulted in drastic changes in editing efficiencies in vivo with an editing efficiency of nearly 60% for the mRNA:gRNA ratio of 1:1, while editing efficiencies of about 32% and 17% for mRNA:gRNA ratio of 1:10 and 10:1, respectively (Example 8). Fig. 7 of the instant specification also shows variations in in vitro editing efficiency that depend on different concentrations used and/or different targeting sequences (FVII or TTR sequences). The instant specification also stated explicitly “Large increase in editing were measured for both targets when using chemically modified sgRNA-LNPs co-transfected with Cas9 mRNA-LNPs, when compared to the dgRNA LNP formulations that were tested” (paragraph [212]; and Fig. 5). With respect to the superior properties of LNPs comprising Lipid A for delivery of Cas9 mRNA and sgRNA relative to the commercial transfection agent Lipofectamine as shown in Fig. 19, please note that Yin already disclosed the use of Nano.Cas9 that is formulated with C12-200, cholesterol, C14-PEG 2000, DSPC and Cas9 mRNA in a weight ratio of 50:20:10:10 for successfully delivering Cas9 mRNA to mediate genome editing in vivo, and that nano.Cas9 is less toxic in comparison with Lipofectamine 2000 (Example 4; paragraphs [00136]-[00137]; Fig. 12d-e and Fig. 13).
Second, with respect to pending claims under rejection please note that the standard under 35 U.S.C. 103 is a “reasonable” expectation of success.
Amended claims 137, 139-149, 151, 156, 158 and 164-166 are rejected under 35 U.S.C. 103 as being unpatentable over Lee et al (US 10,626,393) in view of Brito et al (WO 2015/095340; IDS) and Yin et al (WO 2015/191693; IDS). This is a new ground of rejection.
The instant claims encompass a lipid nanoparticle (LNP) composition comprising: (a) an mRNA encoding a Cas nuclease, wherein the Cas nuclease is Cas9 nuclease or Cpf1 nuclease; (b) a guide RNA nucleic acid, wherein the guide RNA nucleic acid is a single guide RNA (sgRNA); and (c) a plurality of component lipids comprising: Lipid A which is (9Z,12Z)-3-((4,4-bis(octyloxy)butanoyl0oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl-octadeca-9,12-dienoate, (ii) a helper lipid (with cholesterol as the elected species), (iii) a neutral lipid (with DSPC as the elected species), and (iv) a stealth lipid (with PEG2k-DMG as the elected species), wherein the LNP composition includes a ratio of mRNA encoding the Cas nuclease to guide RNA nucleic acid of about 10:1 to about 1:10 (w/w), and wherein the mRNA encoding the Cas nuclease and the guide RNA nucleic acid are co-encapsulated in the LNP composition.
With respect to the elected species, Lee et al already disclosed at least a lipid particle comprises both gRNA and an mRNA encoding a Cas9 that are capable of inhibiting or silencing target gene expression in vitro and in vivo, wherein the lipid particle has a median diameter of from about 30 nm to about 150 nm, and wherein the lipid particle comprises: (i) a cationic lipid (e.g., DLinDMA, DLenDMA, DLin-MP-DMA) from about 48 mol% to about 62 mol% of the total lipid present in the particle, (ii) a phospholipid (e.g., DPPC, DSPC) from about 7 mol% to about 17 mol% of the total lipid present in the particle, (iii) a cholesterol or a cholesterol derivative from about 25 mol% to about 40 mol% of the total lipid present in the particle, and (iv) a conjugated lipid (e.g., PEG-DAG, PEG-DAA, PEG-Cer) that inhibits aggregation of particles from about 0.5 mol% to about 3 mol% of the total lipid present in the particle in the various Formulations A-Z (see at least Abstract; Brief Summary; particularly col. 1, lines 61-62; col. 3, line 6 continues to line 16 on col. 4; and col. 16, lines 42-54). Lee et al stated clearly “In certain embodiments, the lipid particles comprise both gRNA and an mRNA encoding a Cas9” (col. 1, lines 61-62), and “In certain embodiments, the nucleic acid-lipid particle further comprises a mRNA sequence encoding a CRISPR associated protein 9 (Cas9)” (col. 3, lines 13-15). In Example 1, Lee et al described successfully CRISPR/Cas9-induced gene editing of an endogenous gene following intravenous delivery of a LNP formulation of mRNA for the Cas9 protein (2 mg/kg body weight) and a separate LNP formulation of a gRNA (0.42 mg/kg) to a mouse liver in vivo, with an apparent mass ratio of Cas9 mRNA to gRNA of 4.8:1.0.
Lee et al did not teach explicitly at least a lipid nanoparticle (LNP) composition comprising the ionizable Lipid A (9Z,12Z)-3-((4,4-bis(octyloxy)butanoyl0oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl-octadeca-9,12-dienoate, a helper lipid, a neutral lipid, and a stealth lipid; wherein Cas9 nuclease mRNA and a sgRNA are co-encapsulated in the LNP composition and wherein the LNP composition includes a ratio of mRNA encoding Cas9 nuclease to sgRNA of about 10:1 to about 1:10 (w/w).
Before the effective filing date of the present application (03/30/2016), Brito et al already taught a lipid nanoparticle with a diameter in a range of about 50-150 nm and comprised of a cationic lipid (e.g., Lipid A which is (9Z,12Z)-3-((4,4-bis(octyloxy)butanoyl0oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl-octadeca-9,12-dienoate at the 55-40 lipid molar ratio), a helper lipid (e.g., cholesterol or cholesterol hemisuccinate at the 55-40 lipid molar ratio), a neutral lipid (e.g., DSPC at the 15-5 lipid molar ratio), and a stealth lipid (e.g., S010, S024 or PEG-DMG at the 10-1 lipid molar ratio) to deliver biological active agents (e.g., DNA or RNA agents such as mRNA, siRNA) to cells and tissues, including to the liver (Abstract; Summary of the Invention; particularly page 15, lines 23-24; page 19, lines 23-24; page 30, line 1 continues to line 8 on page 37; page 38, lines 13-16; page 39, line 1 continues to line 17 on page 40; Example 13 on page 86; pages 162-157, especially Table 2 on page 164 and Table 3 on page 167; pages 169-171, especially Table 4 on pages 170-171). Please noting that Lipid A contains a tertiary amine head group, and therefore it is an ionizable cationic lipid. Brito et al stated clearly “The lipid nanoparticles have a size of about 1 to about 2,500 nm, about 10 to about 1,500 nm, about 20 to about 1,000 nm, in a sub-embodiment about 5 to about 600 nm, in a sub-embodiment about 50 to about 400 nm, in a sub-embodiment about 50 to about 250 nm, and in a sub-embodiment bout 50 to about 150 nm. Unless indicated otherwise, all sizes referred to herein are the average sizes (diameters) of the fully formed nanoparticle, as measured by dynamic light scattering on a Malvern Zetasizer” (page 38, lines 13-18); and “In certain embodiments the PEG (e.g., conjugated to a lipid, such as a stealth lipid) is a “PEG-2K”, also termed “PEG-2000”, which has an average molecular weight of about 2000 daltons” (page 37, lines 1-3). Brito et al also stated clearly “The present invention provides a cationic lipid scaffold that demonstrates enhanced efficacy along with lower toxicity (improved therapeutic index) as a result of lower sustained lipid levels in the relevant tissues, and for local delivery applications (eye, ear, skin, lung); delivery to muscle (i.m.), fat, or subcutaneous cells (s.c. dosing)” (page 3, lines 7-10). In an exemplification, the ionizable cationic lipid in Example 13 (Lipid A) shown in Table 2 on page 164 exhibits an 90.7% encapsulation efficiency for Leptin mRNA and 88.1% encapsulation efficiency for siRNA FVII. Table 3 on pages 253-254 showed the cationic lipid in Example 13 (Lipid A) exhibits an encapsulation efficiency of 95.4%, 96.4%, 98.5%, 98.2% and 88.1% for Leptin mRNA, hFIX mRNA, mEPO mRNA, pDNA and FVII siRNA, respectively; and a lipid nanoparticle size ranging from 88.1 nm to 127.6 nm. Additionally, the cationic lipid in Example 13 (Lipid A) also exhibits 97% FVII knock-down following intravenous injection of encapsulated siRNA in mice (Table 4 on page 171).
Additionally, Yin et al also disclosed a CRISPR-Cas9 system and various delivery systems (e.g., viral and/or non-viral delivery systems) for the components of the CRISPR-Cas9 system to achieve an efficient in vivo gene editing/modification of a target nucleotide sequence (see at least Abstract; Summary of the Invention; particularly paragraphs [0006], [0008]-[0009], [0011, [0043]-[0051], [0054]-[064], [0070]-[0072], [0088], [00117]-[00122], [0138]-[0142]; examples 1, 4; and Figures 1-2 and 9-10). Yin et al stated clearly “[t]he present disclosure provides delivery systems comprising (i) one or more gRNA covalently or noncovalently bound to a repair template and (ii) a nucleic acid editing system, wherein (i) and (ii) are present on the same or different delivery vehicles” (paragraph [0006]); “The delivery vehicles provided herein may be viral vectors or non-viral vectors, or RNA conjugates. In some embodiments, the gRNA and the nucleic acid editing system are provided in the same type of delivery vehicle, wherein the delivery vehicle is a viral vector or a non-viral vector” (paragraph [0051]); and “In another embodiment, each of the components of the delivery systems provided herein (e.g., the nucleic acid editing system, gRNA and, optionally, repair template) are each contained in the same or in different nanoparticles” (paragraph [0088]). Yin et al specifically taught that the ratio of Cas9:sgRNA is from about 1:100 to about 100:1, or about 1:50 to about 50:1, or about 1:25 to about 25:1, or about 1:10 to about 10:1, or about 1:5 to about 5:1, or about 1:2 to about 2:1, or about 1:1 for consistent delivery to the target sequence (last sentence in paragraph [00112]). Yin et al also disclosed that the delivery vehicle can be a non-viral vector in the form of a lipid-based or polymeric vector such as liposomes, lipid encapsulation systems, nanoparticles and small nucleic acid-lipid particle (SNALP), particularly the lipid encapsulation comprising one or more of a phospholipid, cholesterol, polyethyelene glycol (PEG)-lipid, and a lipophilic compound (e.g., C12-200 and cKK-E12), including the lipid encapsulation composed of cKK-E12, DOPE, cholesterol and C14-PEG2000 for efficient in vivo editing in liver tissue (paragraphs [0011] and [0063]-[0064]). Yin et al also taught that the lipid nanoparticle is between about 1 and about 100 nanometers in size (paragraph [0054], and nanoparticles are formulated with Cas9 mRNA chemically modified to reduce TLR responses (paragraph [0060]). Yin et al further taught that the gRNA is also chemically modified at the 5’ end, middle portion of the RNA or the 3’ end (paragraphs [0070]-[0072]). In an exemplification, Yin et al demonstrated an efficient CRISPR-Cas9-mediated correction of a Fumarylacetoacetate hydrolase (Fah) mutation in a mouse model of the human disease hereditary tyrosinemia gene defect using Cas9 mRNA encapsulated in lipid nanoparticles composed of cKK-E12, DOPE, Cholesterol and C14-PEG2000 and rAAV2/8 virus expressing gRNA against a mutant FAH gene and a correct FAH repair template; and Yin et al concluded that systemic delivery of Cas9 mRNA by lipid nanoparticle can effectively mediate genome editing in vivo (paragraphs [00117]-[00118], [00138]-[00142] and Figs. 9-10).
Accordingly, it would have been obvious for an ordinary skilled artisan before the effective filing date of the present application to modify the teachings of Lee et al by also utilizing the disclosed lipid nanoparticles containing the Lipid A of Brito et al, including at least LNPs composed of a cationic Lipid A, DSPC, cholesterol and PEG2k-DMG at their respective molar ratios in the range of 55-40%:15-5%:55-40%:10-1%, that have an average particle size of about 75 nm to about 150 nm to co-encapsulate sgRNA and CRISPR-Cas9 mRNA, including at the ratio of CRISPR-Cas9 mRNA to sgRNA at a ratio of about 10:1 to about 1:10 (w/w), for editing or modifying a target sequence in a eukaryotic cell such as a liver cell, in light of the teachings of Brito et al and Yin et al as presented above.
An ordinary skilled artisan would have been motivated to carry out the above modifications because Brito et al provided LNPs with a diameter in a range of about 50-150 nm and comprised of: (i) the ionizable cationic Lipid A ((9Z,12Z)-3-((4,4-bis(octyloxy)butanoyl0oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl-octadeca-9,12-dienoate) at the 55-40 lipid molar ratio; (ii) the helper lipid cholesterol at the 55-40 lipid molar ratio; (iii) the neutral lipid DSPC at the 15-5 lipid molar ratio, and (iv) the stealth lipid PEG2k-DMG at the 10-1 lipid molar ratio for delivering biological active agents to cells and tissues, that have enhanced efficacy along with lower toxicity (improved therapeutic index). Additionally, Yin et al also demonstrated successfully that systemic delivery of Cas9 mRNA by lipid nanoparticle can effectively mediate genome editing in vivo; and taught explicitly that both gRNA and mRNA encoding Cas9 can be present on the same delivery vehicle/lipid nanoparticle as well as the ratio of Cas9:sgRNA is from about 1:10 to about 10:1, or about 1:5 to about 5:1, or about 1:2 to about 2:1, or about 1:1 for consistent delivery to the target sequence. Please note that the primary Lee reference also taught a lipid nanoparticle comprising both gRNA and an mRNA encoding a Cas9 that are capable of inhibiting or silencing target gene expression in vitro and in vivo; and using a mass ratio of Cas9 mRNA to gRNA of 4.8:1.0 in an exemplification.
An ordinary skilled artisan would have a reasonable expectation of success in light of the teachings of Lee et al, Brito et al and Yin et al, coupled with a high level of skill for an ordinary skilled artisan in the relevant art.
The modified LNP composition resulting from the combined teachings of Lee et al, Brito et al and Yin et al is indistinguishable from and encompassed by the presently claimed invention.
Therefore, the claimed invention as a whole was prima facie obvious in the absence of evidence to the contrary.
Double Patenting
The nonstatutory double patenting rejection is based on a judicially created doctrine grounded in public policy (a policy reflected in the statute) so as to prevent the unjustified or improper timewise extension of the “right to exclude” granted by a patent and to prevent possible harassment by multiple assignees. A nonstatutory double patenting rejection is appropriate where the conflicting claims are not identical, but at least one examined application claim is not patentably distinct from the reference claim(s) because the examined application claim is either anticipated by, or would have been obvious over, the reference claim(s). See, e.g., In re Berg, 140 F.3d 1428, 46 USPQ2d 1226 (Fed. Cir. 1998); In re Goodman, 11 F.3d 1046, 29 USPQ2d 2010 (Fed. Cir. 1993); In re Longi, 759 F.2d 887, 225 USPQ 645 (Fed. Cir. 1985); In re Van Ornum, 686 F.2d 937, 214 USPQ 761 (CCPA 1982); In re Vogel, 422 F.2d 438, 164 USPQ 619 (CCPA 1970); In re Thorington, 418 F.2d 528, 163 USPQ 644 (CCPA 1969).
A timely filed terminal disclaimer in compliance with 37 CFR 1.321(c) or 1.321(d) may be used to overcome an actual or provisional rejection based on nonstatutory double patenting provided the reference application or patent either is shown to be commonly owned with the examined application, or claims an invention made as a result of activities undertaken within the scope of a joint research agreement. See MPEP § 717.02 for applications subject to examination under the first inventor to file provisions of the AIA as explained in MPEP § 2159. See MPEP § 2146 et seq. for applications not subject to examination under the first inventor to file provisions of the AIA . A terminal disclaimer must be signed in compliance with 37 CFR 1.321(b).
The filing of a terminal disclaimer by itself is not a complete reply to a nonstatutory double patenting (NSDP) rejection. A complete reply requires that the terminal disclaimer be accompanied by a reply requesting reconsideration of the prior Office action. Even where the NSDP rejection is provisional the reply must be complete. See MPEP § 804, subsection I.B.1. For a reply to a non-final Office action, see 37 CFR 1.111(a). For a reply to final Office action, see 37 CFR 1.113(c). A request for reconsideration while not provided for in 37 CFR 1.113(c) may be filed after final for consideration. See MPEP §§ 706.07(e) and 714.13.
The USPTO Internet website contains terminal disclaimer forms which may be used. Please visit www.uspto.gov/patent/patents-forms. The actual filing date of the application in which the form is filed determines what form (e.g., PTO/SB/25, PTO/SB/26, PTO/AIA /25, or PTO/AIA /26) should be used. A web-based eTerminal Disclaimer may be filled out completely online using web-screens. An eTerminal Disclaimer that meets all requirements is auto-processed and approved immediately upon submission. For more information about eTerminal Disclaimers, refer to www.uspto.gov/patents/apply/applying-online/eterminal-disclaimer.
Amended claims 137, 139-149, 151, 156, 158 and 164-166 are provisionally rejected on the ground of nonstatutory double patenting as being unpatentable over claims 3-10, 12-16, 19-21, 23-26, 28, 30, 32-49, 53-59, 61-63 and 76-84 of copending Application No. 16/651,911 (reference application). This is a modified rejection.
Although the claims at issue are not identical, they are not patentably distinct from each other because a lipid nanoparticle (LNP) composition comprising: an RNA component (e.g., a Cas9 nuclease mRNA and a gRNA/sgRNA; dependent claims 14, 21, 23, 32-37), including the LNP composition in which the gRNA and Class 2 Cas nuclease mRNA are present in a ratio ranging from about 10:1 to about 1:10 by weight (dependent claims 32-37); and a lipid component, wherein the lipid component comprises: about 40-60 mol-% amine lipid having the structural formula shown in independent claim 7 (e.g., Lipid A; dependent claim 49); about 0-10 mol-% neutral lipid (e.g., DSPC; dependent claim 57); about 1.5-10 mol-% PEG lipid (e.g., PEG-DMG or PEG2k-DMG; dependent claims 61-62), wherein the remainder of the lipid component is helper lipid (e.g., cholesterol; dependent claim 56) and wherein the N/P ratio of the LNP composition is about 3-10 in claims 3-10, 12-16, 19-21, 23-26, 28, 30, 32-49, 53-59, 61-63 and 76-84 of copending Application No. 16/651,911 encompasses a LNP composition in the application being examined and, therefore, a patent to the genus would, necessarily, extend the rights of the species or sub- should the genus issue as a patent after the species of sub-genus. It is noted that since the LNP composition of the co-pending Application No. 16/651,911 contains the same components in the same respective molar ranges, such lipid composition would also possess an average particle size from 75 nm to 150 nm, as well as an encapsulation efficiency ranging from about 70% to about 100% as those of the LNP composition of the application being examined.
This is a provisional nonstatutory double patenting rejection because the patentably indistinct claims have not in fact been patented.
Amended claims 137, 139-149, 151, 156, 158 and 164-166 are provisionally rejected on the ground of nonstatutory double patenting as being unpatentable over claims 1, 3-4, 6, 8, 10, 12-13, 16-18, 21-24, 26-27, 30, 32, 37, 49, 51, 60, 63, 65 and 67 of copending Application