DETAILED ACTION
Status of the Claims
Claims 60-77 are pending in the instant application and are being examined on the merits in the instant application.
Advisory Notice
The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
All rejections and/or objections not explicitly maintained in the instant office action have been withdrawn per Applicants’ claim amendments and/or persuasive arguments.
Priority
The U.S. effective filing date has been determined to be 05/15/2020, the filing date of the U.S. Provisional Application No. 63/025,355.
Claim Rejections - 35 USC § 103
The text of those sections of Title 35, U.S. Code not included in this action can be found in a prior Office action.
Claims 60-64, 67-72 and 74-77 are rejected under 35 U.S.C. 103 as being unpatentable over DEROSA (US 2016/0038432; published February, 2016) in view of LEAVITT (WO 2020/077007 A1; published 16-APR-2020; with priority to 62/743,116 filed 09-OCT-2018); ASKEW (US 2019/0192688 A1; June, 2019); Schmitz et al. (“Purification of nucleic acids by selective precipitation with polyethylene glycol 6000,” 2006; ELSEVIER; Analytical Biochemistry, Vol. 354, pp. 311-313); SMITH (WO 2017/218704 A1; published December, 2017); BOLOTIN (US 2008/0015263; published January, 2008); PARELLA (WO 2020/041793 A1; published 27-FEB-2020) and Chen et al. (“Polyethylene glycol and solutions of polyethylene glycol as green reaction media,” 2005, RSC; Green Chemistry, Vol. 7. pp. 64-82).
Applicants Claims
Applicant claims a process for encapsulating messenger RNA (mRNA) in lipid nanoparticles (LNPs) comprising a step of mixing (a) an mRNA solution comprising one or more mRNAs with (b) a lipid solution comprising one or more cationic lipids, one or more non-cationic lipids, and one or more PEG-modified lipids, to form mRNA encapsulated within LNP’s (mRNA-LNPs) in a LNP formulation solution, wherein the lipid solution is prepared in an amphiphilic polymer solution consisting essentially of poloxamers, polyvinyl pyrrolidone, polyvinyl alcohol, polyethylene glycol (PEG), or combinations thereof and does not comprise volatile organic compounds, and wherein the lipid solution does not comprise pre-formed LNPs or wherein the LNPs are not pre-formed prior to mixing, wherein the mRNA solution and the lipid solution are mixed at a ratio (v/v) of 1:1 to 2:1 (instant claim 60).
Applicants have elected the following species in the reply filed 10/27/2022: (a)(i) one or more cationic lipids is ML-2 (cKK-E12), (a)(ii) one or more non-cationic lipids is distearoylphosphaditylcholine (DSPC), (a)(iii) one or more PEG-modified lipids is DMG-PEG2K, and (a)(iv) a species of an amphiphilic polymer is PEG (instant claim 61) and more specifically mTEG (instant claim 61).
Determination of the scope
and content of the prior art (MPEP 2141.01)
DEROSA teaches encapsulation of mRNA (title, see whole document), and particularly that “The present invention provides an improved process for lipid nanoparticle formulation and mRNA encapsulation. In some embodiments, the present invention provides a process of encapsulating messenger RNA (mRNA) in lipid nanoparticles comprising a step of mixing a mRNA solution and a lipid solution, wherein the mRNA solution and/or the lipid solution are at a pre-determined temperature greater than ambient temperature.” [emphasis added](abstract).
DEROSA teaches their process includes “A suitable mRNA solution may be any aqueous solution containing mRNA to be encapsulated at various concentrations.” ([0056] through [0063]), a Lipid solution including ethanol ([0064] through [0066]), cationic lipids ([0067] through [0081]), non-cationic/helper lipids ([0082]-[0083]), cholesterol-based lipids ([0084]), and Pegylated lipids ([0085]-[0086]). DEROSA teaches that: “The present invention is based on the discovery of unexpected effect of temperature on the mRNA encapsulation efficiency and recovery rate. Thus, in some embodiments, the present invention provides a process of encapsulating messenger RNA (mRNA) in lipid nanoparticles by mixing a mRNA solution and a lipid solution, described herein, wherein the mRNA solution and/or the lipid solution are heated to a pre-determined temperature greater than ambient temperature. As used herein, the term "ambient temperature" refers to the temperature in a room, or the temperature which surrounds an object of interest (e.g., a mRNA solution or lipid solution) without heating or cooling. In some embodiments, the ambient temperature refers to temperature ranging from about 20-25° C.” ([0085]).
DEROSA teaches that the one or more non-cationic lipids are selected from disteroylphosphatidylcholine (DSPC), among others ([0019])(instant claims 64).
DEROSA teaches that: “Suitable flow rates for mixing may be determined based on the scales. In some embodiments, a mRNA solution is mixed at a flow rate ranging from about 40-400 ml/minute […].” And further that: “In some embodiments, a lipid solution is mixed at a flow rate ranging from about 25-75 ml/minute, […].” ([0094]-[0095]). The mixing flow rates clearly encompass a 1:1 mixing ratio (v/v)(e.g. 50 ml/min for each of the mRNA solution and the lipid solution), and is thus considered prima facie obvious. “In the case where the claimed ranges "overlap or lie inside ranges disclosed by the prior art" a prima facie case of obviousness exists.” (MPEP 2144.05-I)(instant claims 60 and 76).
DEROSA teaches that: “A suitable mRNA solution may be any aqueous solution containing mRNA to be encapsulated at various concentrations. For example, a suitable mRNA solution may contain a mRNA at a concentration of or greater than about 0.01 mg/ml, 0.05 mg/ml, 0.06 mg/ml, 0.07 mg/ml, 0.08 mg/ml, 0.09 mg/ml, 0.1 mg/ml, 0.15 mg/ml, 0.2 mg/ml, 0.3 mg/ml, 0.4 mg/ml, 0.5 mg/ml, 0.6 mg/ml, 0.7 mg/ml, 0.8 mg/ml, 0.9 mg/ml, or 1.0 mg/ml.” ([0056])(instant claim 66, greater that about 1g of mRNA per 12 L of the mRNA solution > 1000mg/12,000ml = 0.083 mg/mL). “In the case where the claimed ranges "overlap or lie inside ranges disclosed by the prior art" a prima facie case of obviousness exists.” (MPEP 2144.05-I).
DEROSA teaches that: “In some embodiments, a suitable mRNA solution may have a pH ranging from about 3.5-6.5, 3.5-6.0, 3.5-5.5, 3.5-5.0, 3.5-4.5, 4.0-5.5, 4.0-5.0, 4.0-4.9, 4.0-4.8, 4.0-4.7, 4.0-4.6, or 4.0-4.5.” ([0059])(instant claim 67). “In the case where the claimed ranges "overlap or lie inside ranges disclosed by the prior art" a prima facie case of obviousness exists.” (MPEP 2144.05-I).
DEROSA does not expressly teach a step of incubating the mRNA-LNPs however, the claimed limitation is generic to incubating for any amount of time. One of ordinary skill would have clearly recognized that the produced mRNA-LNPs would have been incubated for at least a small amount of time prior to diafiltration with TFF (see, Figures 1-3 & 5; [0096]-[100]). Therefore it is reasonably implied based on the disclosure of DEROSA that the mRNA-LNPs would have been incubated prior to diafiltration (MPEP §2144.01)(instant claim 69).
DEROSA teaches the Lipid Solution includes Cholesterol-Based Lipids (([0064] and [0084])(instant claim 70).
DEROSA further teaches methods for purification of messenger RNA (mRNA) including tangential flow filtration (TFF)([0100] through [0104])(instant claim 71).
DEROSA teaches that: “A process according to the present invention results in more homogeneous and smaller particle sizes ( e.g., less than 100 nm), as well as significantly improved encapsulation efficiency and/or mRNA recovery rate as compared to a prior art process.” ([0106]). And that: “the purified nanoparticles, have a size less than about 100 nm (e.g., less than about 95 nm, about 90nm, about 85 nm, about 80 nm, about 75 nm, about 70 nm, about 65 nm, about 60 nm, about 55 nm, or about 50 nm).” ([0107])(instant claim 72).
DEROSA teaches that: “A mRNA solution and a lipid solution may be mixed at various flow rates. Typically, the mRNA solution may be mixed at a rate greater than that of the lipid solution[…].” ([0093]). And that: “Suitable flow rates for mixing may be determined based on the scales. In some embodiments, a mRNA solution is mixed at a flow rate ranging from about 40-400 ml/minute, 60-500 ml/minute, 70-600 ml/minute, 80-700 ml/minute, 90-800 ml/minute, 100-900 ml/minute, 110-1000 ml/minute, 120-1100 ml/minute, 130-1200 ml/minute, 140-1300 ml/minute, 150-1400 ml/minute, 160-1500 ml/minute, 170- 1600 ml/minute, 180-1700 ml/minute, 150-250 ml/minute, 250-500 ml/minute, 500-1000 ml/minute, 1000-2000 ml/minute, 2000-3000 ml/minute, 3000-4000 ml/minute, or 4000-5000 ml/minute. In some embodiments, the mRNA solution is mixed at a flow rate of about 200 ml/minute, about 500 ml/minute, about 1000 ml/minute, about 2000 ml/minute, about 3000 ml/minute, about 4000 ml/minute, or about 5000 ml/minute.” ([0094])(instant claim 74).
DEROSA teaches “The effect of temperature on the nanoparticle formulation process was evaluated for size, size dispersity, encapsulation efficiency and yield (or recovery). Exemplary data shown in Table 1.” ([0122]), including encapsulation efficiencies of “greater than 70%” at ambient temperature (25 °C)(Table 1, col. 6 - “Encapsulation”)(instant claims 62-63, 77).
DEROSA teaches the mRNA solution can include a buffer such as citrate in a broad range: “suitable concentration of the buffering agent may range from about 0.1 mM to 100 mM, 0.5 mM to 90 mM, 1.0 mM to 80 mM, 2 mM to 70 mM, 3 mM to 60 mM, 4 mM to 50 mM, 5 mM to 40 mM, 6mMto 30mM, 7mMto 20mM, 8mM to 15mM, or 9 to 12 mM.” ([0057])(instant claim 77, “wherein the mRNA solution comprises less than 5 mM”).
Ascertainment of the difference between
the prior art and the claims (MPEP 2141.02)
The difference between the rejected claims and the teachings of DEROSA is that DEROSA does not expressly teach: (1) substituting the organic solvent (e.g. ethanol) with a polymer solution such as PEG or specifically mTEG; or (2) the cationic species ML-2.
LEAVITT teaches compositions and systems comprising transfection-component vesicles (TCVs) free of organic solvents and detergents and related methods (see whole document, particularly the title). LEAVITT teaches “Lipid-based vesicles, typically herein called transfection competent vesicles (TCVs), configured to safely and efficiently deliver DNA, RNA, other nucleic acid and protein cargoes into target cells. The safety and efficiency are each, and both, achieved in part by eliminating organic solvents such as ethanol and detergents such as sodium dodecyl sulfate from the TCV loading processes (i.e., inserting a cargo into the TCV), TCV storage processes, and/or TCV delivery processes.” (abstract).
LEAVITT teaches that: “One of the important areas for scientific research and medical treatments is the desire to selectively and efficiently deliver RNA, DNA, other nucleic acids and/or protein cargo to target sites such as specific target cells. This can be helpful for a variety of reasons including improved patient treatments such as gene therapy and for treatment of cancer and other conditions.” ([0002]). And further that: “Turning to a more scientific discussion of the delivery of DNA and other nucleic acids into target sites such as diseased cells in the brain, existing methods for such delivery include lipid particles, in some cases called lipid nanoparticles ("LNPs") or liposomes. The term lipid nanoparticles or "LNPs" is used to describe lipid-based particles at about neutral pH that typically contain nucleic acid and have an electron dense core. Liposomes, also known as vesicles, are lipid-based structures with a single bilayer and an aqueous core. Typical established processes of LNP formation load the vesicle with specific cargo at time of initial vesicle formation. These processes further use specialized instrumentation, organic solvents and/or detergents, require large amounts of material, and constitute processing times on the order of days, all of which severely hamper utility, accessibility and therapeutic usability.” ([0003]).
LEAVITT teaches that: “LNP formulations can be generated through rapid-mixing of the lipid components dissolved in ethanol with an acidic aqueous phase consisting of the nucleic acid cargo ([…]). An established rapid mixing process for LNP manufacture includes microfluidic mixing through a staggered herringbone micromixer (SHM) ([…]), or T-junction mixing with specialized pumps ([…]) or a more dated approach of ethanol-/detergent-destabilised loading of pre-formed vesicles ([…]). In all three methods, an ethanolic solution (or detergent) is required to provide sufficient membrane fluidity for lipid reorganization and entrapment to occur, and in the case of the SHM and T-junction techniques, particle formation also occurs upon dilution of the ethanolic solution into the aqueous phase ([…]). However, the resulting suspension is not "ready-to-use" due to the organic solvent and acidic pH and thus the resulting suspension requires substantial downstream processing. In terms of material costs and time, these approaches have significant impediments to achieving a transfection competent formulation at lab-scales for in vitro applications or for or therapeutic levels for direct administration.” ([0005]). And further that: “There remains a need for transfection reagents that effectively deliver nucleic acid and protein cargo into mammalian cells in a non-toxic manner, including for cultured mammalian primary cells ([…]). While the importance of using primary cells and their advantages over the use of cell lines is well-understood, the difficulty encountered in transfecting such cells has precluded their use almost entirely from any type of discovery or validation studies requiring selective gene knockdown. Furthermore, a move towards personalized medicine is pushing for functional genomic screening and validation to be done in primary patient cells, increasing the need for robust and non-toxic transfection methods for these hard-to-transfect cell types.” ([0006]).
ASKEW teaches treatment of Ornithine transcarbamylase (OTC) deficiency (an X-linked genetic disorder characterized by mutations in the gene for OTC) by mRNA delivery compositions, the mRNA encoding an omithine transcarbamylase protein formulated in a lipid nanoparticle (see whole document, particularly the title, abstract, [0002], [0005]-[0006]). ASKEW teaches that “In some embodiments, the mRNA is formulated in a lipid nanoparticle. In some embodiments, the mRNA is encapsulated in the lipid nanoparticle. In some embodiments, the lipid nanoparticle comprises one or more cationic lipids. In some embodiments, the one or more cationic lipids are selected from the group consisting of […] cKK-E12 (ML2) […].” [emphasis added] ([0019], [0172]-[0173], [0247], [0248]-[0250])(instant claim 1, cationic lipid, elected species ML2). The examiner notes that “Polynucleotide molecules (e.g., DNA or RNA) are large, hydrophilic macromolecules with a negative charge.” (Bondi et al.1; “Solid lipid nanoparticles for applications in gene therapy: a review of the state of the art,” 2010, Infroma; Expert Opinion on Drug Delivery, Vol. 7, No. 1, pp. 7-18; see §Introduction, 2nd paragraph, lines 1-2), therefore the obvious reason to incorporate cationic species such as cKK-E12 (ML2) in an mRNA nanoparticles is because negative and positive charges attract, and in order to achieving a charge balance between the positively charged (cationic) species and the negatively charged mRNA would have enhanced the stability (relative to the absence of a cationic lipid) of the encapsulated mRNA, as one of ordinary skill in the art would have clearly recognized.
ASKEW teaches that “mRNA can be purified from natural sources, produced using recombinant expression systems and optionally purified” ([0059]).
Schmitz et al. teaches purification of nucleic acids by selective precipitation with polyethylene glycol 6000 (see whole document), and particularly that “Precipitation with PEG6000 can also be very useful for the fractionation of naturally occurring nucleic acid mixtures (Fig. 2B).” (p. 313, col. 2, lines 16-18). Schmitz et al. further teaches that: “While PEG’s potential for the fractionation of nucleic acids has been described more than 30 years ago, this reagent is commonly used only in the large-scale preparation of plasmid DNA. The results outlined in the present study will hopefully encourage others to employ it for the routine purification of nucleic acids, just as the authors have successfully done for years.” (p. 313, col. 2, last full paragraph). Schmitz et al. does not teach the PEG is triethylene glycol monomethyl ether (mTEG).
With regards to the use of an amphiphilic polymer in the process for production of LNPs, SMITH teaches stabilized formulations of lipid nanoparticles (see whole document), and particularly includes an amphiphilic polymer ([0005]), such as poloxamers (Pluronic®), poloxamines (Tetronic®), polyoxyethylene glycol sorbitan alkyl esters (polysorbates) and polyvinyl pyrrolidones (PVPs)([0020]), as a lyoprotectant ([0019] & [0071]). SMITH teaches the inclusion of an amphiphilic polymer lowers the immunogenicity ([0047] & [0061]) and increases the therapeutic index ([0048]) of the resulting formulation. SMITH teaches including the amphiphilic polymer in process of making mRNA-containing lipid nanoparticles ([0062]-[0065]). SMITH teaches that: “On a macroscopic level, LNP dispersions are physically stabilized by the combination of charge interactions (i.e., Coulombic repulsion of like-charges) and by steric stabilization imparted by surface-localized hydrophilic moieties. At elevated concentrations of nanoparticles, stability of the dispersion can be derived from inter-particle interactions or nanoparticle associations with other hydrophobic interfaces in their environment. Those interactions can drive lipid reorganization, fouling, and aggregation. Steric stabilization of the LNP may be improved by increasing the concentration of surface-exposed hydrophilic polymers that can bind to the surface. Amphiphilic polymers selectively partition to hydrophobic interfaces, whereas hydrophilic polymeric regions of the amphiphilic polymers remain oriented towards the bulk aqueous solution. Without wishing to be bound by the theory, through this interaction, the amphiphilic polymer serves as a steric stabilizer that may reduce inter-molecular interactions between nanoparticles and hydrophobic interfaces, which may lead to improved stability of lipid nanoparticles for use in therapy involving nucleic acids and oligonucleotides (including mRNA, siRNA, miRNA, lncRNA, etc.).” [emphasis added]([00103]). SMITH further teaches that “It is surprisingly discovered that lipid nanoparticles containing nucleic acid are rendered more stable throughout its life cycle with the addition of an effective amount of amphiphilic polymers.” ([00111]). SMITH does not expressly teach PEG and more specifically mTEG as the amphiphilic polymer, however, BOLOTIN teaches examples of suitable examples of organic solvents that are miscible in water include alcohols such as ethanol and polyethylene glycol (100 to 4000 g/mol) including monohydroxalkyl ethers such as triethylene glycol monomethyl ether and N-vinylpyrrolidones (PVPs)([0092]), in the context of forming drug carriers (i.e. microemulsions, liposomes, and micellar and reverse micellar structures)([0039], [0085]-[0091])(instant claims 1-3, amphiphilic polymer, polyethylene glycol (PEG), and triethylene glycol monomethyl ether (mTEG), respectively).
Consistent with the disclosure of BOLOTIN, PARELLA teaches that: “In some embodiments, the step of precipitating mRNA comprises use of an organic solvent to precipitate the mRNA. In some embodiments, the step of precipitating the mRNA comprises use of ethanol to precipitate the mRNA.” (p. 4, [0020]). And that: “In some embodiments, the step of precipitating mRNA does not comprise an organic solvent. In some embodiments, the step of precipitating mRNA does not comprise an organic solvent and the mRNA is precipitated using polyethylene glycol (PEG). In some embodiments, the step of precipitating mRNA comprises using PEG to precipitate the mRNA.” (p. 4, [0021]). And that: “ In some embodiments, the step of precipitating mRNA does not comprise an organic solvent and comprises triethylene glycol (TEG) to precipitate the mRNA. In some embodiments, the step of precipitating mRNA comprises use of TEG to precipitate the mRNA.” ([0022]).
And that: “In some embodiments, the step of precipitating mRNA does not comprise an organic solvent and comprises triethylene glycol monomethyl ether (MTEG) to precipitate the mRNA. In some embodiments, the step of precipitating mRNA comprises use of MTEG to precipitate the mRNA.” (p. 4, [0023])(instant claim 1, “wherein the lipid solution is prepared in an amphiphilic polymer solution).
Furthermore, Chen et al. teaches the aqueous solution properties of PEG including that: “Polyethylene glycol, PEG: HO–(CH2CH2O)n–H, is available in a variety of molecular weights from 200 to tens of thousands. At room temperature, the water soluble and hygroscopic polymer is a colorless viscous liquid at molecular weight <600 and a waxy, white solid at molecular weights >800. The numerical designation of PEGs generally indicates the number average molecular weight (e.g., PEG-2000), although typically they are not monodisperse polymers. Liquid PEG is miscible with water in all proportions, and solid PEG is highly soluble in water, for example, PEG-2000 has a solubility of about 60% in water at 20 uC. Lower molecular weight liquid PEGs can be used as solvents in their own right with or without addition of water. These we define loosely here as low molecular weight PEG.
PEG has a number of benign characteristics that underlie, for example, its application in bioseparations. PEG is on the FDA’s GRAS list, (compounds Generally Recognized as Safe) and has been approved by the FDA for internal consumption. PEG is weakly, if at all, immunogenic, a factor which has enabled the development of PEG–protein conjugates as drugs. Aqueous solutions of PEG are biocompatible and are utilized in tissue culture media and for organ preservation.
Unlike VOCs, low molecular weight liquid PEGs are nonvolatile. The vapor density for low molecular weight PEG is greater than 1 relative to air according to available MSDS data, and this is consistent with the industry standard for selection of alternative solvents to VOCs. PEG also has low flammability, and is biodegradable.” (p. 65, col. 1, §2).
Regarding the limitation “wherein the step of mixing the mRNA solution and the lipid solution yields the amphiphilic polymer solution at a concentration of greater than 25% (v/v)” the combination of cited prior art clearly suggest PEG and particularly mTEG as an alternative solvent to ethanol. Therefore, substitution of the ethanol solvent with mTEG (“an amphiphilic polymer solution consisting essentially of […] PEG […].”) would have more likely than not resulted in “the amphiphilic polymer solution at a concentration of greater than 25% (v/v)” given that the primary reference, DEROSA, teaches a process of encapsulating messenger RNA (mRNA) in lipid nanoparticles comprising a step of mixing a mRNA solution and a lipid solution.
Finding of prima facie obviousness
Rationale and Motivation (MPEP 2142-2143)
It would have been prima facie obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to produce a lipid nanoparticle composition including mRNA and a process of loading the mRNA into the lipid nanoparticles, as taught by DEROSA, and that excludes organic solvents alcohols such as ethanol, as suggested by LEAVITT, by substitution of the ethanol therein by PEG/TEG/mTEG amphiphilic polymer in the organic (lipid) phase as these species are described as “organic solvents” (BOLOTIN: [0092]) or as a substitute for ethanol (PARELLA: p. 4, [0019] to [0023]), and further to utilize a known cationic lipid species such as cKK-E12 (ML2) and to include purified mRNA, as suggested by, ASKEW, the mRNA purified by a process that uses PEG as described by Schmitz et al., and SMITH teaches specific advantages of including an amphiphilic polymer in mRNA-LNPs (steric stabilization, lyoprotectant, etc.), and further to utilize mTEG as an obvious variant of PEG/PVP, as suggested by BOLOTIN, in order to produce the most efficient method for introduction of mRNA into lipid nanoparticles, mTEG being available as a homogeneous material in contrast to PEG which is a mixture2, and it would have been prima facie obvious to include the PEG and/or mTEG as an green reaction media with favorable properties (FDA approved and GRAS, high aqueous solubility, non-volatile), as suggested by Chen et al., for a green reaction media.
From the teachings of the references, it is apparent that one of ordinary skill in the art would have had a reasonable expectation of success in producing the claimed invention because it would have required no more than an ordinary level of skill in the art to produce an mRNA lipid nanoparticle production process using known ingredients and method steps, as evidence by the cited prior art. Therefore, the invention as a whole would have been prima facie obvious to one of ordinary skill in the art before the effective filing date of the claimed invention, as evidenced by the references, especially in the absence of evidence to the contrary.
In light of the forgoing discussion, the Examiner concludes that the subject matter defined by the instant claims would have been obvious within the meaning of 35 USC 103.
Claims 65, 66 and 73 are rejected under 35 U.S.C. 103 as being unpatentable over DEROSA in view of LEAVITT;ASKEW; Schmitz et al.; SMITH; BOLOTIN; PARELLA and Chen et al., as applied to claims 60-64, 67-72 and 74-77 above, and further in view of MANOHARAN (US 2013/0338210; published December, 2013).
Applicants Claims
Applicant claims a process for encapsulating messenger RNA (mRNA) in lipid nanoparticles (LNPs), as discussed above.
Applicants have elected the following species in the reply filed 10/27/2022: (a)(i) one or more cationic lipids is ML-2, (a)(ii) one or more non-cationic lipids is distearoylphosphaditylcholine (DSPC), (a)(iii) one or more PEG-modified lipids is DMG-PEG2K, and (a)(iv) a species of an amphiphilic polymer is PEG (instant claim 2) and more specifically mTEG (instant claim 61).
Claim interpretation: the “N/P” recited in instant claim 73 is being interpreted as follows: “N” is a nitrogen atom and “P” is a phosphorus atom, thus the ratio defines a nitrogen/phosphorus ratio.
Determination of the scope
and content of the prior art (MPEP 2141.01)
DEROSA teaches encapsulation of mRNA, as discussed above and incorporated herein by reference.
LEAVITT teaches compositions and systems comprising transfection-component vesicles (TCVs) free of organic solvents and detergents and related methods, as discussed above and incorporated by reference.
ASKEW teaches lipid nanoparticles, wherein the lipid nanoparticle comprises one or more cationic lipids such as cKK-E12 (ML2), as discussed above and incorporated herein by reference.
Schmitz et al. teaches purification of nucleic acids by selective precipitation with polyethylene glycol 6000, as discussed above and incorporated herein by reference.
SMITH teaches stabilized formulations of lipid nanoparticles including amphiphilic lipids for steric stabilization, among other advantages, as discussed above and incorporated herein by reference.
BOLOTIN teaches examples of suitable examples of organic solvents including PEGs, PVPs, and mTEG, as discussed above and incorporated herein by reference.
PARELLA teaches triethylene glycol monomethyl ether (MTEG) to precipitate the mRNA, as discussed above and incorporated herein by reference.
Chen et al. teaches the properties of polyethylene glycols, as discussed above and incorporated herein by reference.
Ascertainment of the difference between
the prior art and the claims (MPEP 2141.02)
The difference between the rejected claims and the teachings of LEAVITT is that LEAVITT does not expressly teach: (1) including trehalose in the mRNA solution (instant claim 65); (2) a N/P ratio of between 1 to 10 (instant claim 73).
MANOHARAN teaches compositions for delivery of nucleic acids including charge lipids (see whole document). MANOHARAN teaches that “In another aspect, a storage-stable composition can include a cryoprotectant selected from sucrose, trehalose, […].” ([0057] & [0358]-[0359])(instant claims 13 & 65).
MANOHARAN teaches that “In accordance with the invention, the lipid mixture is combined with a buffered aqueous solution that may contain the nucleic acids. […] The amount of nucleic acid in buffer can vary, but will typically be from about 0.01 mg/mL to about 200 mg/mL, more preferably from about 0.5 mg/mL to about 50 mg/mL.” ([0597])(instant claims 15 & 66: 1g per 12 L being ~0.083 mg/mL, of which 0.5 mg/mL is “greater than about”).
MANOHARAN teaches that “The N/P ratio is the ratio of number of molar equivalent of cationic nitrogen (N) atoms present in the lipid particle to the number of molar equivalent of anionic phosphate (P) of the nucleic acid backbone. For example, the N/P ratio can be in the range of about 1 to about 50. In one example, the range is about 1 to about 20, about 1 to about 10, about 1 to about 5. […] The particles can be formulated with a nucleic acid therapeutic agent so as to attain a desired N/P ratio. The N/P ratio is the ratio of number of moles cationic nitrogen (N) atoms (i.e., charged lipids) to the number of molar equivalents of anionic phosphate (P) backbone groups of the nucleic acid. For example, the N to P ratio can be in the range of about 5: 1 to about 1:1.” ([0431]-[0432])(instant claims 39 & 73).
Finding of prima facie obviousness
Rationale and Motivation (MPEP 2142-2143)
It would have been prima facie obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to produce a lipid nanoparticle composition including mRNA and a process of loading the mRNA into the lipid nanoparticles, and that excludes organic solvents alcohols such as ethanol, as discussed above, and further to include a cryoprotectant such as trehalose, a suitable concentration of mRNA (e.g. 0.5 mg/mL), and to ensure a suitable N/P ratio in the produced lipid nanoparticles such as about 1 to about 10, as suggested by MANOHARAN, in order to produce a storage-stable product including a suitable amount of a cationic lipid.
From the teachings of the references, it is apparent that one of ordinary skill in the art would have had a reasonable expectation of success in producing the claimed invention because it would have required no more than an ordinary level of skill in the art to produce an mRNA lipid nanoparticle production process using known ingredients and method steps, as evidence by the cited prior art. Therefore, the invention as a whole would have been prima facie obvious to one of ordinary skill in the art before the effective filing date of the claimed invention, as evidenced by the references, especially in the absence of evidence to the contrary.
In light of the forgoing discussion, the Examiner concludes that the subject matter defined by the instant claims would have been obvious within the meaning of 35 USC 103.
Response to Arguments:
Applicant's arguments filed 10/15/2025 have been fully considered but they are not persuasive.
Applicant argues that: “none of the references teach or disclose the unexpected results discussed in the present application, which demonstrates that the process of encapsulating mRNA in LNPs without the use of a volatile organic compound (e.g. ethanol) and in low volumes, such as a ratio of 1: 1 to 2: 1 (v/v) of mRNA solution to lipid solution, improves mRNA stability and encapsulation efficiency relative to ethanol-prepared mRNA-LNP formulations in low volume.
DEROSA teaches that: “Suitable flow rates for mixing may be determined based on the scales. In some embodiments, a mRNA solution is mixed at a flow rate ranging from about 40-400 ml/minute […].” And further that: “In some embodiments, a lipid solution is mixed at a flow rate ranging from about 25-75 ml/minute, […].” ([0094]-[0095]). The mixing flow rates clearly encompass a 1:1 mixing ratio (v/v)(e.g. 50 ml/min for each of the mRNA solution and the lipid solution), and is thus considered prima facie obvious. “In the case where the claimed ranges "overlap or lie inside ranges disclosed by the prior art" a prima facie case of obviousness exists.” (MPEP 2144.05-I)(instant claims 60 and 76).
The process of encapsulating mRNA in LNPs in low volumes not only reduces downstream processing in manufacturing but is also beneficial for dosing patients by enabling low-volume administration routes such as subcutaneous and intramuscular administration.” (p. 7, 2nd paragraph). And that: “The instant application discloses that mixing a 100% ethanol solution comprising lipids with an aqueous solution comprising mRNA at a low (v/v) ratio, such as a 1:1 ratio, can result in unstable mRNA solubility and in some cases mRNA precipitation due to the high concentrations (50% vol/vol) of ethanol in the resulting mixture. See as-filed specification, Example 3, paragraph [0290]. Thus, prior to the present invention, approaches to address this problem included diluting the ethanol component in the ethanol-lipid solution (which may impact lipid solubility), increasing the volume of aqueous-mRNA solution, and/or adding a third stream of aqueous solution to yield a lower ethanol concentration in the resulting mixture to avoid mRNA instability and possible mRNA precipitation. Id. Unfortunately, these approaches all require mixing larger volumes than would be necessary, typically with substantially diluted amounts of lipid and mRNA in the respective solutions, which at large-scale processing levels can significantly increase time and cost, and require subsequent concentration steps, which further increase time and cost, in addition to ethanol removal steps.” (paragraph bridging pp. 7-8). And further that: “The instant application teaches that when an amphiphilic polymer solution (such as 100% triethylene glycol monomethyl ether (mTEG)) comprising lipids is mixed with an aqueous solution comprising mRNA at low volumes (such as a 1: 1 (v/v) ratio) to generate mRNA-LNPs, the high concentration of mTEG (50% v/v) in the resulting mixture does not appear to result in mRNA instability or precipitation. Id. at paragraph [0291]. These mRNA-LNP formulations prepared at a large-scale using optimized low volumes can be advantageous in reducing processing volumes and thereby increasing ease of manufacturing processes.” (p. 8, 2nd paragraph).
Applicant points “Example 3 of the present application demonstrates the significant advantage of using an amphiphilic polymer solution (such as mTEG) versus an ethanol solvent for lipids in the preparation of mRNA-LNPs, particularly with respect to lowering the volumes in preparing mRNA-LNP formulations (e.g., for dosing). See id., Example 3, paragraph [0290]. Table 4 of Example 3 indicates that low-volume mRNA-LNP formulations prepared using lipids including ML-2 as the cationic lipid and dissolved in mTEG achieved a 69% encapsulation efficiency." Id. at paragraph [0293], Table 4. Further, low-volume mRNA-LNP formulations prepared using lipids including MC-3 as the cationic lipid and dissolved in mTEG achieved a 99% encapsulation efficiency. Id. at paragraph [0294], Table 4. In contrast, low-volume mRNA-LNP formulations prepared using lipids including ML-2 as the cationic lipid and "dissolved in ethanol could not be stably produced in a low volume formulation and showed precipitation following mixing." Id. at paragraph [0293], Table 4 (emphasis added). The results indicate that amphiphilic polymer solvents are suitable for use in low-volume, ethanol-free mRNA-LNP formulations and provide improved mRNA stability after mixing and improved encapsulation efficiency relative to ethanol-prepared LNP formulations. Id. at paragraph [0295]. None of the references cited by the Office teach or disclose these unexpected results.” (p. 8, 3rd paragraph).
In response to Applicants allegation of unexpected results, instant claim 60, the sole independent claim, is not limited to mTEG or ML-2 used for producing, therefore claim 60 is clearly not commensurate with Example 3. Additionally, the claims are not limited by any volume – a ratio of volumes “wherein the mRNA solution and the lipid solution are mixed at a ratio of (v/v) 1:1 to 2:1.” (claim 60, last two lines) does not the absolute volume(s). Additionally, the claims are broadly drafted relying on exclusionary language rather than limit the claimed invention. For example the limitation that the lipid solution does not contain volatile organic solvents (instant claim 60, lines 3-8), DEROSA teaches that: “In another embodiment, a suitable lipid solution is dimethylsulfoxide-based.” Where dimethylsulfoxide is not a volatile organic solvent (claim 60), and is not ethanol (instant claim 68). Therefore Applicants position that: “The results indicate that amphiphilic polymer solvents are suitable for use in low-volume, ethanol-free mRNA-LNP formulations and provide improved mRNA stability after mixing and improved encapsulation efficiency relative to ethanol-prepared LNP formulations.” is not a convincing argument with respect to DEROSA which does not require ethanol.
Conclusion
Claims 60-77 are pending and have been examined on the merits. Claims 60-77 are rejected under 35 U.S.C. 103. No claims allowed at this time.
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.
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/IVAN A GREENE/Examiner, Art Unit 1619
/TIGABU KASSA/Primary Examiner, Art Unit 1619
1 Copy of record as cited by the Examiner on 03/14/2024.
2 King et al.; “Monoclonal Antibody Conjugates of Doxorubicin Prepared with Branched Peptide Linkers: Inhibition of Aggregation by Methoxytriethylene glycol chains,” 2002, ACS: Journal of Medicinal Chemistry, Vol. 45, pp. 4336-4343; see p. 438, col. 1, lines 1-5.