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 .
Applicant’s election of Group I, claims 6, 8, 9, and 45-61 in the reply filed on 11/14/2025 is acknowledged. Because applicant did not distinctly and specifically point out the supposed errors in the restriction requirement, the election has been treated as an election without traverse (MPEP § 818.01(a)).
Information Disclosure Statement
The information disclosure statement (IDS) submitted on 09/03/2024 was the submission is in compliance with the provisions of 37 CFR 1.97. Accordingly, the information disclosure statement is being considered by the examiner.
Drawings
The drawings were received on 01/19/2024. These drawings are acknowledged.
Claim Rejections - 35 USC § 103
The following is a quotation of 35 U.S.C. 103 which forms the basis for all obviousness rejections set forth in this Office action:
A patent for a claimed invention may not be obtained, notwithstanding that the claimed invention is not identically disclosed as set forth in section 102, if the differences between the claimed invention and the prior art are such that the claimed invention as a whole would have been obvious before the effective filing date of the claimed invention to a person having ordinary skill in the art to which the claimed invention pertains. Patentability shall not be negated by the manner in which the invention was made.
The factual inquiries for establishing a background for determining obviousness under 35 U.S.C. 103 are summarized as follows:
1. Determining the scope and contents of the prior art.
2. Ascertaining the differences between the prior art and the claims at issue.
3. Resolving the level of ordinary skill in the pertinent art.
4. Considering objective evidence present in the application indicating obviousness or nonobviousness.
Claim(s) 6, 8, 9, and 45-58 and 60 are rejected under 35 U.S.C. 103 as being unpatentable over Mya S. Thu et al., (Nature Medicine, 18, 463-467, 2012) in view of Fred Reynolds et al. (Bioconjugate Chemistry, 16, 1240-1245) and Masayuki Ishihara et al. (Int. J Med, 5, 147-155, 2010).
Mya discloses a straightforward magnetic cell labeling by administering a combination of ferumoxytol, heparin and protamine (HPF) to form self-assembling nanocomplexes that effectively label cells for in vivo magnetic resonance imaging. Mya observed that the HPF nanocomplexes were stable in serum free cell culture media and exhibited a three-fold increase in T2 relaxivity compared to F. Electron
Microscopy revealed internalized HPF within endosomes, confirmed by Prussian blue staining of labeled cells (abstract). Combining heparin, protamine and ferumoxytol results in the formation of a self-assembling nanocomplexes (HPF) and used to able stem cells or immune cells for MRI (page 463). The HPF nanocomplexes show ferumoxytol as electron-dense iron nanoparticles of approximately 150-200 nm in diameter (Fig. 16, c) and average iron content per cell 2.12+ 0.11 pg (page 463). Heparin, protamine and ferumoxytol are routinely used for specific clinical indications and the HPF nanocomplexes have similar biochemical properties to SPIONs, which have been shown to label cells and biodegrade through the iron metabolic pathway. Optimization of the HPF protocol by modifying the amount of heparin or protamine or by increasing the amount of ferumoxytol may result in higher intracellular iron concentrations, providing basis for the rapid translation into clinical trials (page 466). The HPF complexes were prepared by sequentially adding FDA approved drugs-heparin at 2 IU ml−1, protamine at 60 µg ml−1 and ferumoxytol at 50 µg ml−1 in either sterile water for physiochemical characterization or in serum-free RPMI-1640 medium for cell-culture studies would obviously result a composition comprising no unbound heparin, protamine, or iron-based imaging agent (supplemental material). Additional disclosure includes that one of the main advantages of complexing ferumoxytol with heparin and protamine to label cells is that these drugs are used clinically; therefore, extensive safety testing of the drugs should not be necessary, and the time required for evaluating HPF for an investigative new drug application should be shortened.
Mya fails to disclose imaging contrast agent conjugated to heparin or protamine and composition is lyophilized.
Fred Reynolds discloses synthesis of fluorescent protamines (contrast agent) such as protamine labeled with tetramethyl rhodamine (Pro(Rh), fluorescein-labeled protamine (Pro(Fl) by reacting the N-hydroxysuccinimidyl esters of 5-carboxytetramethylrhodamine and 5-carboxyfluorescein with protamine (abstract and Table 1). In one embodiment discloses the time course of internalization of the (Pro(Rh) and (Pro(Fl) and compared it with tat peptide (TatFl) as a function of time and concentration and found that internalization of Pro(Rh) was complete within 1 hour and did not show saturation behavior with respect to concentration (Fig 3A and 3B). Reynolds discloses that, when Pro(Ph) was covalently attached to the amino-CLIO nanoparticle, the cellular uptake of the nanoparticle was greatly enhanced and the internalization of protamine was strongly dependent on the attached fluorochrome, with the uptake of Pro(Rh) being far greater than Pro(Fl) (abstract and Page 1242). Additional disclosure includes that polymer-coated nanoparticles such as CLIO (Cy5.5) are not significantly internalized by non-phagocytic cells such as HeLa cells and the attachment of protamine yielded a nanoparticle, Pro(Rh)-CLIO-(Cy5.5), that was internalized at 4-5 times higher levels than that of the nanoparticle with the tat peptide attached, Tat(Fl)-CLIO.
Ishihara discloses production of low-molecular-weight heparin/protamine nanoparticles (LMV-H/P NPs) as carrier for heparin-binding growth factors, such as fibroblast growth factor-2 (FGF-2) (abstract). The solution of LMV-H/P MPs was then subjected to washing twice with phosphate-buffered saline to remove unreacted materials and further in order to remove unreacted materials and salts, and the dialysate was lyophilized by adding 2 mg/mL dextran and remained soluble after lyophilization of dialyzed LMV-H/P NPs (page 148). Additional disclosure includes that LMW-H/P NPs may serve as an effective nanocarrier for various heparin-binding cytokines, including FGF-2. Particularly in the local application of heparin-binding cytokines.
It would have been obvious to one of ordinary skill in the art at the invention to incorporate fluorescent protamines such as protamine labeled with tetramethyl rhodamine (Pro(Rh), fluorescein-labeled protamine (Pro(Fl) into Mya’s composition. The person of ordinary skill in the art would have been motivated to make those modifications because Reynold teaches that nanoparticle, Pro(Rh)-CLIO-(Cy5.5), was internalized at 4-5 times higher levels than that of the nanoparticle with the tat peptide attached, Tat(Fl)-CLIO, and protamine like tat peptide can be conjugated to nanoparticles, and the resulting peptide-nanoparticle conjugated is readily internalized by cells (page 1243) and reasonably would have expected success because Reynold teaches that protamine, a protein in regular clinical use, might be used for the design of novel membrane translocating/nuclear localizing pharmaceuticals whose development was initiated with other membrane-translocating peptides and in addition, the fluorescent protamines developed might be used to further our understanding of this important pharmaceutical.
It would have been obvious to one of ordinary skill in the art at the invention to incorporate the method of lyophilization into Mya’s composition. The person of ordinary skill in the art would have been motivated to make those modifications because Ishihara teaches that lyophilization of LMV-H/P NPs was carried out to remove unreacted materials and salts, and the dialysate and addition of dextran stabilizes the LMV-H/P NPs and LMV-H/P MPs in solution, and allows preparations of stable and resoluble freeze- dried LMV-H/P MPs and LMV-H/P NPs (page 150) and reasonably would have expected success because the LMV-H/P NPs may serve as an effective nanocarrier for various heparin-binding cytokines, including FGF-2, particularly in the local application of heparin- binding cytokines.
Claim(s) 59 and 61 is/are rejected under 35 U.S.C. 103 as being unpatentable over Mya S. Thu et al., (Nature Medicine, 18, 463-467, 2012) in view of Fred Reynolds et al. (Bioconjugate Chemistry, 16, 1240-1245) and Masayuki Ishihara et al. (Int. J Med, 5, 147-155, 2010) as applied to claims 6, 8, 9, and 45-58 and 60 above, and further in view of Hugo Groult et al. (Biomarcomolecules, 18, 3156-3167, 2017) and Ming Liu et al. Nanoscale Research Letters, 10:34, 2015).
The teachings of Mya S. Thu, Fred Reynolds and Masayuki Ishihara are delineated above.
None of these teach nanoparticles encapsulating imaging contrast agent and nucleic acid instantly claimed.
Hugo discloses iron-oxide nanoparticles (HEP-IONP) coated with different heparins of distinct anticoagulant/anti-heparanase activity ratios and suitable for positive contrast in magnetic resonance imaging (abstract). In one embodiment, discloses a one-step microwave-assisted rapid synthesis of IONP to produce heparin functionalized HEP-IONP that could be used for the T1-positive contrast agents in MRI (page 3157). Hugo discloses that, when functionalized on the IONP surface, the bioactivity of these modified heparins was preserved with slight modifications that can be taken advantage of for more specificity, for instance, as a promising candidate for thromboembolism clinical MRI diagnosis gather with immediate anticoagulant treatment (page 3165). Additional disclosure includes that, taking the advantage of its excellent T1 enhanced blood pool MRI contrast, HEP-IONP may represent a good antiangiogenic agent with possibility of magneto-guidance and magnetic resonance angiography (MRA) based angiogenesis characterization.
Ming Liu discloses preparation of protamine nanoparticles encapsulating small hairpin RNA (shRNA) by desolvation method (abstract). The shRNA -expressing plasmid DNA targeting the Bcl-2 gene (shBcl-2) as a model gene was encapsulated in nanoparticles to treat cancer more efficiently (page 1). After preparation of the loaded nanoparticles, the difference between the amount of the initially added shRNA and ShRNA in the supernatant was measured at detecting absorbance to determine the encapsulation efficiency of shRNA in nanoparticles (page 2). Liu discloses that shRNA was efficiently protected from the enzyme’s degradation by encapsulation in NPs. Additional disclosure includes that shBcl-2 loaded protamine nanoparticles increased their cytotoxicity of A549 and induced cell apoptosis by silencing Bcl-2 and taken together, protamine nanoparticle could be a promising nonviral nanodevice for improving the targeting delivery of gene in cancer therapy.
It would have been obvious to one of ordinary skill in the art at the invention to incorporate the bioactive heparin-coated iron oxide nanoparticles into Mya’s composition. The person of ordinary skill in the art would have been motivated to make those modifications because Hugo teaches that, after injection of LOV-IONP or usHEP-IONP, the main vascular architecture was brightened on the magnetic resonance angiography (MRA) acquisitions, demonstrating that the nanoparticles can enhance T1 relaxation in the circulating system that can provide high-resolution images from sequences of long acquisition times (page 3162) and reasonably would have expected success because Hugo showed that IONP coated with a commercial LMWH suggested as a theranostic translational probe for MRA diagnostic and treatment of thrombosis, and an antitumor IONP coated with a specific depolymerized heparin to be used in targeted therapy and diagnostic modalities.
It would have been obvious to one of ordinary skill in the art at the invention to incorporate the preparation of protamine nanoparticles encapsulating shRNA into Mya’s composition as taught by Liu. The person of ordinary skill in the art would have been motivated to make those modifications because Liu teaches that shRNA loaded in nanoparticles was protected effectively from the degradation of DNase 1 and serum and significantly improved the efficiency of transfection of shRNA in vitro in A549 cells and increased its cytotoxicity and induced more cell apoptosis by silencing Bcl-2 (abstract) and reasonably would have expected success because protamine nanoparticles can efficiently encapsulate DNA, RNA, peptide nucleic acid (PNA), and double-stranded RNA (dsRNA0 and prevent them from degradation by enzymes.
Conclusion
No claims are allowed at this time.
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/J.R.S/Examiner, Art Unit 1618
/Michael G. Hartley/Supervisory Patent Examiner, Art Unit 1618
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