DETAILED ACTION
The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
This Office action is in response to the communications filed 1-30-26.
Claims 1, 2, 5, 6, 9, 11-13, 18, 20, 21, 23, 24, 26, 29, 33, 38, 39 and 45 are pending in the instant application.
Election/Restrictions
Claims 1, 2, 5, 6, 9, 11-13, 18, 20, 21, 23, 24, and 45 are directed to an allowable product. Pursuant to the procedures set forth in MPEP § 821.04(B), claims 26, 29, 33, 38 and 39, directed to the process of making or using an allowable product, previously withdrawn from consideration as a result of a restriction requirement, are hereby rejoined and fully examined for patentability under 37 CFR 1.104.
Because all claims previously withdrawn from consideration under 37 CFR 1.142 have been rejoined, the restriction requirement as set forth in the Office action mailed on 8-1-25 is hereby withdrawn. In view of the withdrawal of the restriction requirement as to the rejoined inventions, applicant(s) are advised that if any claim presented in a divisional application is anticipated by, or includes all the limitations of, a claim that is allowable in the present application, such claim may be subject to provisional statutory and/or nonstatutory double patenting rejections over the claims of the instant application. Once the restriction requirement is withdrawn, the provisions of 35 U.S.C. 121 are no longer applicable. See In re Ziegler, 443 F.2d 1211, 1215, 170 USPQ 129, 131-32 (CCPA 1971). See also MPEP § 804.01.
Withdrawn Rejections
Any rejections not repeated in this Office action are hereby withdrawn.
New Objections/Rejections Necessitated by Amendments
Claim Objections
Claim 39 is objected to because of the following informalities: Claim 39 recites “producing of an EMN” in line 1, which appears grammatically incorrect (e.g. perhaps deleting “of” would be remedial).
Appropriate correction is required.
Claim Rejections - 35 USC § 112
The following is a quotation of the first paragraph of 35 U.S.C. 112(a):
(a) IN GENERAL.—The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same, and shall set forth the best mode contemplated by the inventor or joint inventor of carrying out the invention.
The following is a quotation of the first paragraph of pre-AIA 35 U.S.C. 112:
The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same, and shall set forth the best mode contemplated by the inventor of carrying out his invention.
Claims 26, 29, 33, 38 are rejected under 35 U.S.C. 112(a) or 35 U.S.C. 112 (pre-AIA ), first paragraph, because the specification, while being enabling for the synthesis and characterization of the PLGA EPC-EM nanovesicles described in the specification, does not reasonably provide enablement for providing treatment, or for diagnostic and therapeutic kits. The specification does not enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and/or use the compositions claimed,
The following factors have been considered in determining that the specification does not enable the skilled artisan to make and/or use the invention over the broad scope claimed.
The breadth of the claims:
The claims are broadly drawn to therapeutic or diagnostic kits, methods of preventing or treating any vascular, neuronal or hyperinflammatory disease or condition in a subject, and methods for rescuing any neuron, endothelial or lung cell in a subject or in vitro, comprising the administration of compositions comprising exosome mimicking nanovesicles (EMN).
The teachings in the specification:
The instant specification teaches the following:
Optimal nanovesicle loading was obtained when lipid raft vesicles were loaded with 0.5mg/mL FITC-BSA and FITC-Biotin. Further, the EMNs that were loaded with 0.5 mg/mL concentrated conditioned medium and imaged using transmission electron microscopy displayed structure and shape similar to that of exosomes. The production of EMNs could be scaled up to produce 3.78 x 10° vesicles from 10 million hPMSCs. Addition of the conditioned medium loaded EMNs to neurons undergoing apoptosis in vitro indicated that the EMNs could also rescue apoptotic neurons.
hPMSC Culture [0207] 1x10° hPMSCs were cultured in T150 tissue culture treated flask with DS media containing Dulbecco’s modified eagle’s medium (DMEM) with high glucose, 5% fetal bovine serum (FBS), 20ng/mL fibroblast growth factor (FGF) and 20ng/mL epithelial growth factor (EGF) at 37 °C, 5% CO2 for 7 days until they reached 90% confluence and are between 6-7 10° cells. The cells were washed with 10 mL phosphate-buffered saline (PBS) and lifted off using 6 mL of TrypLe, neutralized with 18mL DMEM and centrifuged at 470 x g until the cells pelleted at the bottom. The pellet was re-suspended in 5 mL D5 media, 10 pL of the suspension was mixed with 10 uL Trypan Blue and counted using trypan blue exclusion method. 4000 cells/cm” were seeded on six 150 mm dishes with 15 mL of media and cultured at 5% CO2 and 37 °C for 7 days until the plates were 95% confluent.
Isolation of Lipid Rafts [0208] As the composition of lipid rafts is mainly lipid, they can be effectively separated on a hydrophilic sucrose gradient. The increased presence of lipids and proteins within the rafts makes them float to the low-density regions of the sucrose gradient, thus they are commonly found as a band between the 5 and 30%.
[0209] In one aspect, seven 150-mm dishes with 90-95% confluent hPMSCs were washed with 7 mL of ice cold PBS (4° C). Then 7mL of ice cold PBS was added to the dishes and the cells were gently scraped using a cell scraper and the supernatant was collected to a 50 mL conical centrifuge tube. The plates were washed with 3 mL of fresh ice cold PBS and was added to the 50 mL conical centrifuge tube. The cell suspension was centrifuged at 470 x g in the Sorvall RT 6000D centrifuge. The pellet was re-suspended in 5 mL ice cold PBS and the cells were pooled into one 50 mL conical centrifuge tube. The cells were counted as described earlier and 20-25 x 10° cells were pelleted down and resuspended in 2 mL of lysis buffer (pH of 6.5; Table 2) containing 50 mM MES, 150 mM NaCl, 0.5% Triton-X-100 and protease inhibitor cocktail and incubated on ice for 30 minutes. Following the lysis, 378 uL of the cell lysate was mixed with a 522 uL of 60% OptiPrep™ to obtain a final concentration of 35% OptiPrep™ gradient and added to the bottom of a 5 mL Beckman Coulter ultracentrifuge tube. Then 900 wL of Optiprep™ gradients were sequentially added in the following order: 30%, 25%, 20% and 0% (Table 3). Care was taken not to mix the gradients while adding. The samples were centrifuged at 200,000 x g at 4°C for 4 hours using a SW 55 rotor and Beckman L7 ultracentrifuge. After centrifugation, the gradients from three ultracentrifuge tubes were collected in 500 uL fractions starting from the top to bottom and transferred to nine 1.5 mL Eppendorf tubes—these tubes were subjected to dot-blot analysis. In the remaining three ultra-centrifuge tubes, the lipid rafts viewed as a ring between 20-30% gradient were collected and transferred to a new ultracentrifuge tube. 4 mL of PBS was added to the tubes with the lipid rafts and centrifuged at 200,000 x g for 40 minutes. The supernatant was aspirated and 1 mL of fresh PBS was added. The addition of PBS caused the lipid rafts to float up like a thin film and the Eppendorf tube containing the floating lipid raft was stored at -80 °C.
[0213] In another aspect, hPMSCs are cultured, at 37°C and 5% CO2, until they are 80% confluent. Cells are pelleted, lysed and subjected to sucrose gradient centrifugation. The sucrose gradients are 80%, 30% and 5% and centrifuged at 270000 x g for 16 h to obtain lipid rafts situated between the 5% and 30% gradient. Lipid rafts are characterized by assessing the presence of raft-specific markers such as flotillin 1, caveolin 1, cell membrane-specific markers such as integrins and Annexins and exosome markers such CD 9/63/81, Alix and TSG101 by Western blotting. Flotillin-1 and caveolin-1 to ensure successful raft isolation…
[0218] In a further aspect, a lipid raft pellet was re-suspended in 1 mL fluorescein isothiocyanate bovine serum albumin (FITC-BSA) and FITC-Biotin solutions at concentrations 0.25, 0.5 and I mg/mL, respectively, and extruded through a mini-extruder according to the manufacturer’s instructions (FIG. 3). The lipid raft-FITC-BSA/ FITC-Biotin samples were extruded successively 30 times through each membrane of pore sizes 400 nm, 200 nm and 100 nm. After extrusion the sample was collected and stored in black centrifuge tubes to prevent the loss of fluorescence. 50 uwL of the FITC-BSA loaded vesicles were filtered through a Pierce BSA depletion column according to the manufacturer’s instruction. The FITC-Biotin loaded vesicles were spun down at 16,000 = g for 10 minutes. The supernatant was collected and the vesicles at the bottom were washed with 500 uL of PBS and re-centrifuged at 16,000 = g for a total of 5 washes. The FITC-BSA and FITC-Biotin vesicles were read in Nanodrop!™ 2000 after blanking with unloaded vesicles extruded with water. The absorbance of FITC-BSA and Biotin was measured before loading and the absorbance of the loaded vesicle was subtracted from the initial value to obtain the loading efficiency of the sample. The loading efficiency was highest at a concentration of 0.5 mg/mL and this was fixed as a loading concentration for conditioned medium-loaded nanovesicles.
Conditioned Medium Collection and Concentration [0220] In one aspect, PMSCs were seeded on to 150 mm tissue culture treated dishes at 100,000 cells/cm? in 20 mL D5 media and cultured at 5% CO2 and 37°C for 48 hours. After 48 hours, the conditioned medium was collected and spun down at 470 x g to remove cell debris. The supernatant was transferred to a clean ultracentrifuge tube and centrifuged at 112,600 x g in SW 28 rotor for 90min to deplete native exosomes. The supernatant was then concentrated by centrifuging through an Amicon Ultra-15 centrifugal 3 kDa filter unit for 90 minutes until the conditioned medium was concentrated to 20 times. The BSA present in the concentrated conditioned medium was removed by using the HiTrap™ Blue HP albumin depletion kit, according to the manufacturer’s instructions. The D5 media, concentrated conditioned medium before BSA depletion, BSA depleted medium, the albumin entrapped in the column and albumin standard were loaded onto a 4-12% Bis-Tris NuPAGE gel and stained using Imperial’™ protein stain to determine the effect of BSA depletion.
[0221] In another aspect, PMSCs are seeded at 20,000 cells/cm? for T1s0 flask with exosome-depleted FBS containing DS media for 48h at 5% COn at 37°C. Condition medium is then collected by centrifuging at 1500 x g for 20 min. Media is concentrated using Amicon Ultra-15 centrifugal filter units with a 3 kDa molecular weight cutoff and stored at -80 °C until use.
Synthesis of EMNs and Nanoparticle Tracking Analysis [0222] In one aspect, the exosome-depleted conditioned media obtained from hPMSCs is concentrated and subjected to ELISA to detect the presence of BDNF, HGF and VEGF. The lipid rafts are mixed with the varying concentrations of conditioned media and extruded through a Mini Extruder to form EMNs containing the conditioned media. Following synthesis, the morphology of EMNs is measured using TEM and the size distribution and concentration of EMNs is analyzed by NTA. Since neuronal damage, via apoptosis, 1s a common occurrence during the progression of neurological diseases, the neuroprotective ability of EMNs is assessed by using established methods. Subsequently, the neurites are assessed for branching points, circuitry length and segments by using WimNeuron Analysis (Wimasis).
[0223] In a further aspect, the lipid raft pellet was resuspended in the concentrated conditioned medium and extruded using the Mini Extruder with polycarbonate filters of reducing pore size (400-100 nm). The formed EMNs were concentrated by centrifuging at 16000 x g and the EMNs pelleted in the bottom 50 uL fraction were collected. The EMNs were subjected to nanoparticle tracking analysis to obtain the concentration and size distribution. 50 uL of the EMNs sample was added to 950 uL of 0.22 um triple-filtered water and loaded on to the stage of the Nano Sight LM10 with a 404-nm laser and imaged using the sCMOS camera provided with the instrument. Using the NTA software v 3.0 software, three 90-second videos captured at a screen gain of 10, detection threshold of 3 and camera level of 12 were analyzed to determine the size and concentration of the EMNs.
Exosome collection (Control) [0225] PMSCs are seeded at 20,000 cells/cm in for T1s0 flask with exosome-depleted FBS containing D5 media for 48h at 5% CO2 at 37°C. The media is then centrifuged at 300 x g for 10 min, 2000 x g for 20 min and passed through 0.2um filter. The media is also concentrated using Amicon Ultra-15 centrifugal filter units with a 100kDa filter. After being transferred to a thick wall polypropylene tube and centrifuging at 8836 x g, the following steps are performed once or repeated: the supernatant is then further centrifuged at 112,700 x g for 90 min and the pellet is resuspended in PBS (this supernatant is conditioned media free of exosomes).
Transmission Electron Microscopy [0226] In one aspect, the surface morphology of the EMNs was studied using an established negative protocol for characterizing exosomes (Thery et al., 2006). 50 uL of the conditioned medium loaded-EMNs was mixed with equal volume of 4% paraformaldehyde and 5 uL of this mixture was added on to three Formvar-carbon coated electron microscopy (EM) grids each. The grids were washed with a Parafilm strip containing 100 uL PBS by gently touching the grid of the drop edge with the help of a pair of forceps. The grid was touched to 50-uL drop of 1% glutaraldehyde and incubated for 5 minutes following which the grids were washed for 8 times with 100 uL of distilled water by allowing the grid to stay immersed in the water for 2 minutes. The grids were then transferred to a 50-uL drop of uranyl-oxalate (pH, 7.0) for 5 minutes. The grids were transferred to a 50-uL drop of methyl cellulose UA solution and incubated for 10 minutes on ice. Finally, the sides of the grids were gently tapped against a filter paper and were imaged at 80 V using a CM120 transmission electron microscope.
Isolation and characterization of lipid rafts from human placental mesenchymal stem cells hPMSCs [0230] Briefly, the lipid rafts from hPMSCs are isolated using sucrose gradient centrifugation. Following isolation, the lipid rafts are characterized for lipid raft-specific, cell-specific and exosome-specific markers. Without wishing to be bound by the theory, the lipid ring located at a certain (for example, between the 5% and 30 % or about 20% to about 30% sucrose gradient consists of lipid rafts, which having a composition similar to that of hPMSC cell membrane.
[0231] To obtain the lipid rafts the hPMSC cell lysate was subjected to density gradient centrifugation using an OptiPrep™ lysed (FIG. 4A). During ultracentrifugation, the various cell components of the cell lysate fractionate based on their density (FIG. 4A & FIG. 4B). The gradients between 20% and 30% contained a white ring-like structure that contained the lipid rafts. The gradient fractions between 0%-35% gradients (collected as 500 uL aliquots) when assessed by dot blot indicated a positive signal for Caveoilin-1, thus confirming the location of lipid rafts between the 20% and 30% gradient (FIG. 4C). Since the raft-specific markers were detected between 20% and 30% gradients the lipid raft ring at this location was precipitated and probed for exosome-specific markers ALIX, TSG101, CD9 and CD63 and failed to express endoplasmic reticulum marker Calnexin, suggesting that the lipid raft isolation was complete and that the vesicles share some of the markers present on native hPMSC exosomes (FIG. 4D). The presence of Integrin a4 and 81 indicate that the lipid raft vesicles contain cell surface receptors that can assist in targeted delivery similar to that of native exosomes (FIG. 4D).
Determination of loading efficiency [0232] The isolated lipid rafts along with fluorescein isothiocyanate-labelled bovine serum albumin (FITC-BSA) were extruded through the Mini Extruder. The morphological feature was analyzed using transmission-electron microscopy (TEM) and the size distribution and concentration of the EMNs were measured using nanoparticle tracking analysis (NTA) Subsequently, the concentration of the FITC-BSA within the EMNs was measured using a microplate reader to confirm the loading efficiency of these EMNs…
[0233] The results from the loading show that the EMNs were able to encapsulate the FITC-BSA and FITC-Biotin and that the optimum loading concentration was at 0.5mg/mL (FIG. 5A). 50 uL of the loaded EMNs were diluted in 950 pL of Triple-filtered water and analyzed using the Nano Sight LM10. The NTA analysis indicated that EMNs had an average size range of 187.62 +5.1 nm and concentration of 4.896 10° +1.43 x108 vesicles/mL (FIG. 5B). TEM imaging showed that the 0.5mg/mL FITC-BSA loaded EMN had a circular morphology with a smooth edge (FIG. 5C) unlike the cup-shaped structure of native exosomes (Thery et al., 2006).
Concentrating the conditioned medium [0234] Previous studies from Applicant’s lab have shown that the conditioned medium obtained at 24-hour time point is known to contain significant levels of BDNF, HGF and VEGF (Kumar et al., 2019). Since BSA is found in FBS used in the culture medium, the hPMSC secretome was concentrated up to 20 times and subjected to BSA depletion using the HiTrap™ column. Subsequent gel electrophoreses showed that the BSA band 66-kDa band corresponding to BSA was reduced to 1/3 the amount compared to medium control (lane 5 and 6 compared to lane 2; FIG. 6A). The albumin rich fraction of the BSA that was entrapped within the column formed a larger band at 66 kDa compared to the depleted fraction (lane 3 and 4 compared to lane 5 and 6; FIG. 6A). Recent studies in Applicant’s lab have shown that the hPMSC secretome contains BDNF, HGF and VEGF that play an important role in neuroprotection (Kumar et al., ((2019). In order to confirm the presence of these growth factors, Applicant analyzed the secretome using enzyme-linked immunosorbent assay (ELISA). The levels of BDNFsecreted by hPMSC was 1420.48 pg/mL (FIG. 6B), HGF was 6229.54 pg/mL and VEGF was 1169.65 pg/mL (FIG. 6C & FIG. 6D). The level of BDNF was increased 2 times indicating that the presence of BSA hindered the detection of BDNF. However, the levels of VEGF decreased by 100 folds and HGF decrease by 1.3 folds likely because these growth factors are being bound to the depletion column in a non-specific manner. Since storage affects the stability of proteins, Applicant tested the effects of storage on the levels of BDNF at 24 hours to ensure that the BDNF levels can be normalized to the initial cell seeding density… The levels of BDNF was 2 times higher in 48-hour conditioned medium as opposed to the conditioned medium collected 30 days prior (stored at -80°C) or conditioned medium obtained at 24 hours. (FIG. 6E).
Synthesis of EMNs and neuroprotection assay [0235] Using the optimal conditions standardized above, EMNs were loaded with 0.5mg/mL concentrated conditioned medium had a size range of ~135.7 + 4.8nm and a concentration of ~3.78 x 10°+/- 1.05 x10? particles/ml (FIG. 7A). TEM images of the conditioned medium loaded EMNs displayed a circular morphology different from the characteristic cup-shaped morphology of native exosomes (FIG. 7B). .. The apoptotic SH-SYSY cells were treated 1000, 2000, 4000 and 8000 EMNs/cell. The cells treated with 1000, 2000 and 4000 EMNs/cell showed an increase in the number of cells similar in morphology to normal SH-SYSY cells when compared to the PBS-only treated cells that had more rounded morphology typical of dying apoptotic cells and a low number of surviving cells (FIG. 7C). Cells treated with 8000 EMN+CM/cell had more rounded cells suggesting a dose dependency in the neuroprotective function of EMNs loaded with hPMSC conditioned medium and the subsequent pellet was washed 2 times with ice-cold 1X PBS. Cells were then be resuspended in 4 mL of hypotonic lysis buffer (20 mM Tris-HCl, pH=7.5, 10 mM KCl, 2 mM MgCl2) with 5 uL of protease inhibitor cocktail to preserve protein function and incubated on ice for 1 hour. The cell lysate were homogenized on ice using a Dounce homogenizer for 30 passes and then incubated on ice for 5 minutes. The homogenized lysate was ultracentrifuged at 10,000 x g at 4°C for 20 minutes to pellet the cell nuclei and other organelles. The pellet was discarded and the supernatant was ultracentrifuged at 100,000 x g at 4°C for 35 minutes. The resulting pellet was the plasma membrane fraction and was resuspended in 1X PBS at a concentration of 1 mg/mL, and stored at -80°C.
EPC-EM synthesis [0243] PM and PLGA cores were mixed together at different PM:PLGA ratios (0:1, 0.25:1, 0.5:1, 1:1, 1:0.5, 1:0.25, and 1:0) in deionized water for a total volume of 1 mL. The combined solution was coextruded through a 200 nm polycarbonate membrane using the Avanti MiniExtruder for 15 passes. The resulting EPC-EMs were centrifuged at 9,500 rpm for 20 minutes to remove excess PM fragments and passed through a 0.2 um filter to remove any contaminants.
SILY functionalization [0244] SILY-azide was conjugated to the PM coating of the EPC-EM using a combination of sulfo-NHS ester chemistry and copper-free Click chemistry. This conjugation was mediated by the biochemical linker dibenzocyclooctyne-sulfo-N-hydroxysuccinimidy] ester (DBCO-sulfo-NHS). DBCO-sulfo-NHS was prepared at a 1 mg/mL solution in PBS and mixed with EPC-EMs for a 40X molar excess of DBCO. The DBCO-sulfo-NHS/EM solution was incubated on a shaker at room temperature for 1 hour. The excess DBCO-sulfo-NHS was neutralized by reacting with Tris-HCl, pH 8 and removed using ultrafiltration. The azide-SILY was then added to the DBCO-EM conjugate at a 2:1 (for example weight ratio) azide: DBCO molar ratio and incubated overnight at 4°C. Excess azide-SILY was removed using dialysis tubing with a 14kDa cutoff for 24 hours at 4°C.
[0249] Plasma membrane (PM) was successfully isolated from cord-blood derived EPCs using a combination of hypotonic lysis, mechanical homogenization, and serial ultracentrifugation. Western blot analysis (FIG. 8A) revealed presence of the plasma membrane marker caveolin-1 and the diminished presence of the endoplasmic reticulum marker calnexin (negative control). EPC surface marker CD31 was detected, indicating a preservation of parent cell identity. Finally, common EV markers of CD9, CD63, CD81, and ALIX were retained on the plasma membrane surface, indicating physical similarity to EV membrane structure. Next, proteomic analysis of isolated plasma membrane was conducted using tandem mass spectrophotometry. A total of ~3472 proteins in 2781 clusters were identified using cluster analysis via Scaffold software (FIG. 8B).
[0250] PLGA nanoparticles loaded with miR126 were synthesized using a modified nanoprecipitation method … These particles were found to be highly homogenous, with an average size of 77.11 + 12.1 nm (comparable to empty PLGA nanoparticles which were measured to be 71.5 + 0.325 nm) and a loading efficiency of 44.4% + 3.5. Preliminary release kinetics studies revealed a burst release of miR126 from PLGA nanoparticles followed by a sustained release profile (FIG. 9). A 44% miRNA release was observed on Day 1 followed by slower sustained release over the next nine days. A cumulative release of about 60% was released over a period of 10 days.
[0251] Following successful PLGA nanoparticle synthesis and PM isolation, EPC-EMs were synthesized by coating PM around the PLGA particles. Florescence microscopy used to confirm the coating (FIG. 10). For visualization, PLGA particles were loaded with 1,1'- dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine (Dil) (excitation: 549 nm, emission: 565 nm), a red dye, while the PM was labeled green using PKH67 (excitation: 490 nm, emission 502 nm). Composite images show their colocalization of the two as yellow particles, confirming the coating.
[0252] Various PM:PLGA ratios were tested and the ratio was seen to impact size and stability of the EPC-EM (data not shown). Higher PM:PLGA ratio improves EPC-EM stability, with a 2:1 weight ratio being optimal. At this ratio, EPC-EM size was 112.3 + 1.6 nm, which was comparable to native EPC EV size which was measured to be 113.5 + 9.1 nm. The membrane thickness at this ratio is estimated to be about 21 nm. The stability of EPC- EMs was assessed by monitoring the hydrodynamic size of the particles in water at 4°C over a period of 28 days (FIG. 11). Dynamic light scattering revealed size and polydispersity index changes between PLGA nanoparticles, EMs, and PM vesicles over time. Addition of a PLGA core decreases particle aggregation and indicates improved stability as indicated by the limited increase and size and PDI for the EPC-EMs. Meanwhile, the PM vesicles, which contained no PLGA core, steadily increased in size and PDI, suggesting vesicle aggregation over time.
[0259] The loading efficacy/encapsulation efficiency and/or yield was measure. One representative result show that encapsulation efficiency= (44x10-12) mol/mg PLGA x 6.02 x 1023 copies/mol= 8.4 x 1012 copies/mg PLGA. While 1 mg PLGA= 2.53 x 109 particles (n=1), 8.4.x 1012 copies/mg PLGA) x 1 mg/2.53 x 109 particles x 106 = 3.3 x 109 copies per 106 particle. In one embodiment, the yield is 2.53 x 109 particles/mL for 1 mg of PLGA.
The specification, on pages 92-9,5 proposes animal models for treating various injuries, e.g., spinal cord injury, traumatic brain injury, stroke etc., but these are prophetic teachings.
[Emphases added][Citations omitted].
The teachings in the instant specification fail to provide proper guidance for providing treatment in any subject, and further whereby therapeutics and diagnostics also have been provided. Since the specification fails to provide the particular guidance for providing treatment effects in any subject, and since determination of the factors for providing treatment in a subject is highly unpredictable, it would require undue experimentation to practice the invention over the full scope claimed.
Allowable Subject Matter
Claims 1, 2, 5, 6, 9, 11-13, 18, 20, 21, 23, 24 and 45 are allowed.
Claim 39 is objected to as indicated above. Claim 39 appears free of the prior art searched and of record. As allowable subject matter has been indicated, applicant's reply must either comply with all formal requirements or specifically traverse each requirement not complied with. See 37 CFR 1.111(b) and MPEP § 707.07(a).
Conclusion
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.
Certain papers related to this application may be submitted to Art Unit 1637 by facsimile transmission. The faxing of such papers must conform with the notices published in the Official Gazette, 1156 OG 61 (November 16, 1993) and 1157 OG 94 (December 28, 1993) (see 37 C.F.R. ' 1.6(d)). The official fax telephone number for the Group is 571-273-8300. NOTE: If Applicant does submit a paper by fax, the original signed copy should be retained by applicant or applicant's representative. NO DUPLICATE COPIES SHOULD BE SUBMITTED so as to avoid the processing of duplicate papers in the Office.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to Jane Zara whose telephone number is (571) 272-0765. The examiner’s office hours are generally Monday-Friday, 10:30am - 7pm. If attempts to reach the examiner by telephone are unsuccessful, the examiner's supervisor, Jennifer Dunston, can be reached on (571)-272-2916. Any inquiry of a general nature or relating to the status of this application should be directed to the Group receptionist whose telephone number is (703) 308-0196.
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Jane Zara
2-20-26
/JANE J ZARA/Primary Examiner, Art Unit 1637