GDF11 biomarkers, methods, and compositions for treating stroke

Abstract

The disclosure relates to methods of treating stroke, dosing regimens of GDF11, and post-stroke treatment with GDF11.

Description

P390200. W0.01GDF11 BIOMARKERS, METHODS, AND COMPOSITIONS FOR TREATING STROKE CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U. S. Provisional Application No. 63 / 753,849, filed on February 4, 2025, which is hereby incorporated by reference in its entirety.TECHNICAL FIELD

[0002] The disclosure relates to biomarkers of GDF11 administration, modes of administration, and compositions.BACKGROUND

[0003] Growth differentiation factor (GDF11) has been demonstrated to broadly and consistently stimulate regenerative capacity in multiple different tissue systems (see, e.g., International Patent Publication Nos. WO 2013 / 142114; WO 2014 / 168973; WO 2014 / 201143; WO 2015 / 070076; and WO 2021 / 236824).

[0004] In 2013, approximately 6.9 million people had an ischemic stroke, and 3.4 million people had a hemorrhagic stroke (Global Burden of Disease Study 2013 Collaborators (August 2015) The Lancet 386(9995): 743-800). Stroke has been cited as the second most frequent cause of death after coronary artery disease, accounting for 11% of the total (GBD 2015 Mortality and Causes of Death Collaborators (October 2016) The Lancet 388(10053): 1459-1544). The majority of stokes are ischemic since it is difficult to ascertain if a hemorrhagic stroke was initiated by an ischemic event.

[0005] Current medical intervention and treatment of stroke is time-sensitive. In acute ischemic stroke, the gold standard of thrombolysis, such as with recombinant tissue plasminogen activator (rtPA), when administered within three hours of symptom onset provides an overall benefit of about 10% with respect to living without disability but does not improve chances of survival (see, e.g., Wardlaw et al. (July 2014) The Cochrane Database of Systemic Reviews 7(7): CD000213 and Emberson et al. (2014) The Lancet 384(9958): 1929-1935). The desirability of administration of thrombolytics between three and four and a half to five hours of symptom onset is subject to debate as regards to whether it provides therapeutic benefit or causes potential further damage. Intra-arterial fibrinolysis, where a catheter is passed up an artery into the brain and medication is injected at the site of thrombosis has shown benefit in improving outcomes in acute ischemic stroke (see, e.g., Lee et al. (2010) Stroke 41(5): 932-937). Mechanical removal of a blood clot causing ischemic stroke (mechanical thrombectomy) represents another potential treatment for occlusion of a large artery, e.g., the middle cerebral artery), and published reviews have reported the safety and efficacy of such procedures in reducing disability if performed withinP390200. W0.01up to 24 hours of the onset of symptoms, but again without improving chances of survival (see, e.g., Sardar et al. (2015) European Heart Journal 36(35): 2373-2380; Saver et al. (2016) JAMA 316(12): 1279-1288; Goyal et al. (2016) The Lancet 387(10029): 1723-1731; Mistry et al. (2017) Stroke 48(9): 2450-2456; and Powers et al. (2018) Stroke 49(3): e46-e110).

[0006] To date, there are no approved pharmaceuticals for repair or rejuvenation of neurological tissue and / or systems damaged by a stroke event. There is currently an unmet need for therapeutics that can promote recovery of neurological function and provide regenerative benefits beyond neuroprotection for post-ischemic stroke. This is particularly important more than 24 hours after the stroke, when patients are in the hospital and stabilized with baseline functions assessed, but outside the treatment time windows for conventional Tissue Plasminogen Activator (tPA) and endovascular therapies.1SUMMARY

[0007] In one aspect, the disclosure is directed to a method of treating stroke or reducing a symptom thereof in a human subject in need thereof. GDF11 is administered to a human subject having experienced a stroke event a dosing regimen of GDF11 on intermittent days. In some variations, the intermittent dosing begins 24 hours + / - 8 hours after the stroke event. In further variations, the intermittent dosing is administered to the subject separated by a 24 + / - 8, + / - 4, or + / - 2 hour period without a dose. In still further variations, the intermittent dosing is administered to the subject separated by a 48 + / - 8, + / - 4, or + / - 2 hour period without a dose.

[0008] In a second aspect, the disclosure is directed to methods of treating stroke or reducing a symptom thereof in a human subject in need thereof. In a human subject to which has been administered a first dosing regimen of GDF11, the second dosing regimen of GDF11 is administered to a subject, who following the first dosing regimen has an increased abundance of one or more biomarkers having a SomaScan Aptamer sequence selected from 19197-95, 9843-5, 9844-138, 15312-14, 15323-112, 15627-83, 2867-52, 23302-19, 5360-9, 14685-17, 15523-9, 18185-118, 2811-27, 2602-2, 13660-76, 13621-31, 12583-77, 14748-31, 13573-5, 5870-23, 24237-115, 11130-158, 3351-1, 9840-2, 2819-23, 7959-34, 4203-50, 14029-42, 15321-8, 4976-57, 15527-90, 20913-27, 25105-87, 19259-176, 24723-58, 18829-4, 22984-10, 18824-7, 25287-7, 4230-1, 6036-78, 8039-41, 4393-3, 4130-71, 4487-1, 4394-71, 4989-7, 13384-110, 11171-25, 11245-43, 13122-19, 7713-102, 7713-50, 13740-51, 14587-16, 2765-4, 18233-10, 23903-3, 5464-52, 3236-12, 24050-26, 18900-37, 3317-33, 9816-37, 11816-84, 20073-22, 3453-87, 3381-24, 2864-2, 22041-26, 3115-64, 5007-1, 2855-49, 15604-18, 9760-13, 21768-9, 21813-171, 23693-4, 14036-116, 19373-3, 19122-47, 20105-7, 21817-5, 13636-20, 19188-21, 4292-5, 20584-4, 9869-28, 8778-3, 5846-24, 5542-22, 3214-3, 6590-54, 15387-44, 8295-16, 19377-14, 13719-19, 25249-33,P390200. W0.0125947-116, 21430-4, 11354-21, 24909-40, 25949-3, 10070-22, 15545-13, 3466-8, 21483-155, 12479-50, 25463-3, 2644-11, 5475-10, 3379-29, 9504-19, 17516-7, 23329-52, 17205-21, 19222-124, 14287-6, 18950-13, 16769-20, 14332-3, 22547-17, 18373-13, 21713-11, 17764-108, 12540-25, 21752-10, 22578-17, 5116-62, 3469-74, 3868-8, 13242-134, 16043-30, 5272-55, 19630-2, 13707-27, 4467-49, 15433-4, 5488-74, 12351-25, 10346-5, 10354-57, 19553-14, 18308-30, 17355-56, 21539-139, 3339-33, 14111-15, 14082-56, 5440-26, 24320-3, 9997-12, 10756-34, 5275-28, 9830-109, 7952-2, 3050-7, 25451-39, and 4560-34, and / or a decreased abundance of one or more biomarkers having a SomaScan aptamer sequence selected from 18922-27, 4337-49, 25306-51, 10025-1, 10801-11, 14124-6, 22580-29, 2615-60, 5658-64, 2443-10, 19570-12, 17166-4, 14757-144, 5581-28, 2841-13, 5755-29, 10426-21, 13547-5, 13738-8, 19622-7, 2748-3, 5193-51, 5242-37, 7655-11, 3723-1, 9380-2, 21380-77, 20546-71, 9099-19, and 9970-7. In some variations, the biomarkers can be Uniprot IDs or proteins identified by Uniprot ID of protein name or identifier, respectively, and as described herein.

[0009] In a third aspect, the disclosure is directed to methods of treating stroke or reducing a symptom thereof in a human subject in need thereof. In a human subject to which has been administered a first dosing regimen of GDF 11, the second dosing regimen of GDF 11 is administered to a subject, who following the first dosing regimen has an increased abundance of one or more biomarkers having a SomaScan Aptamer sequence selected from 19197-95, 9843-5, 9844-138, 15312-14, 15323-112, 15627-83, 2867-52, 23302-19, 5360-9, 14685-17, 15523-9, 18185-118, 2811-27, 2602-2, 13660-76, 13621-31, 12583-77, 14748-31, 13573-5, 5870-23, 24237-115, 11130-158, 3351-1, 9840-2, 2819-23, 7959-34, 4203-50, 14029-42, 15321-8, 4976-57, 3516-60, 15527-90, 20913-27, 25105-87, 19259-176, 24723-58, 18829-4, 22984-10, 18824-7, 25287-7, 4230-1, 6036-78, 8039-41, 4393-3, 4130-71, 4487-1, 4394-71, 4989-7, 13384-110, 11171-25, 11245-43, 13122-19, 7713-102, 7713-50, 13740-51, 14587-16, 2765-4, 18233-10, 23903-3, 5464-52, 3236-12, 24050-26, 18900-37, 3317-33, 9816-37, 11816-84, 20073-22, 3453-87, 3381-24, 2864-2, 22041-26, 3115-64, 5007-1, 2855-49, 15604-18, 9760-13, 21768-9, 21813-171, 23693-4, 14036-116, 19373-3, 19122-47, 20105-7, 21817-5, 13636-20, 19188-21, 4292-5, 20584-4, 9869-28, 8778-3, 5846-24, 5542-22, 3214-3, 6590-54, 15387-44, 8295-16, 19377-14, 13719-19, 25249-33, 25947-116, 21430-4, 11354-21, 24909-40, 25949-3, 10070-22, 15545-13, 3466-8, 21483-155, 12479-50, 25463-3, 2644-11, 5475-10, 3379-29, 9504-19, 17516-7, 23329-52, 17205-21, 19222-124, 14287-6, 18950-13, 16769-20, 14332-3, 22547-17, 18373-13, 21713-11, 17764-108, 12540-25, 21752-10, 22578-17, 5116-62, 3469-74, 3868-8, 13242-134, 16043-30, 5272-55, 19630-2, 13707-27, 4467-49, 15433-4, 5488-74, 12351-25, 10346-5, 10354-57, 19553-14, 18308-30, 17355-56, 21539-139, 3339-33, 14111-15, 14082-56, 5440-26, 24320-3, 9997-12, 10756-34, 5275-28, 9830-109, 7952-2, 3050-7, 25451-39, andP390200. W0.014560-34, and / or a decreased abundance of one or more biomarkers having a SomaScan aptamer sequence selected from 18922-27, 4337-49, 25306-51, 10025-1, 10801-11, 14124-6, 22580-29, 2615-60, 5658-64, 2443-10, 19570-12, 17166-4, 14757-144, 5581-28, 2841-13, 5755-29, 10426-21, 13547-5, 13738-8, 19622-7, 2748-3, 5193-51, 5242-37, 7655-11, 3723-1, 9380-2, 21380-77, 20546-71, 9099-19, and 9970-7. In some variations, the biomarkers can be Uniprot IDs or proteins identified by Uniprot ID of protein name or identifier, respectively, and as described herein.

[0010] In some variations, the second dosing regimen begins at least 3 weeks following the final dose of the first dosing regimen. In other variations, for example, the second dosing regimen begins at least 2 months following the final dose of the first dosing regimen.

[0011] In a third aspect, the disclosure is directed to a method of treating stroke or reducing a symptom thereof in a human subject to whom has been administered a first dosing regimen of GDF11.

[0012] In a fourth aspect, the disclosure is directed to a method of treating stroke or reducing a symptom thereof in a human subject in need thereof by administering a first dosing regimen of GDF11 to the human subject, and administering a second dosing regimen of GDF11 depending on whether the biomarker abundance is increased and / or decreased.

[0013] In a fifth aspect, the disclosure is directed to a method of treating stroke or reducing a symptom thereof in a human subject in need thereof by identifying the human subject as increased and / or decreased abundance of the one or more biomarkers.

[0014] In a sixth aspect, the disclosure is directed to a method of improving therapeutic efficacy for treatment of stroke or a symptom thereof with GDF11 by determining an increased and / or decreased abundance of the one or more biomarkers in a human subject to whom GDF11 has been administered. The increase abundance and / or decrease in the abundance of the one or more biomarkers corresponds to improved therapeutic efficacy of stroke treatment or symptom thereof with GDF11.

[0015] In a seventh aspect, the disclosure is directed to a method of determining effectiveness of treatment of stroke or a symptom thereof with GDF11 by determining an increase and / or decrease in abundance of the one or more biomarkers in a human subject to whom GDF11 has been administered. The increase and / or decrease in abundance corresponds to improved therapeutic efficacy of stroke treatment or symptom thereof with GDF11.

[0016] In an eighth aspect, the disclosure is directed to a method of selecting one or more human subjects with increased responsiveness to treatment of stroke or symptom thereof. An increased and / or decreased abundance of one or more biomarkers is determined. The increase and / or decrease in the abundance of the one or more biomarkers in a subject in theP390200. W0.01group of human subjects corresponds to improved therapeutic efficacy of stroke treatment or symptom thereof with GDF11 in that subject.

[0017] In some variations, the determining or identifying step comprises a proteomic test. In some variations, the determining or identifying step comprises a genetic test. In further variations, the determining or identifying step comprises testing a blood sample. In still other variations, the determining or identifying step comprises testing a blood serum sample.BRIEF DESCRIPTION OF THE FIGURES

[0018] The foregoing summary, as well as the following detailed description, will be better understood when read in conjunction with the appended figures. For the purpose of illustrating the invention, the figures demonstrate embodiments. It should be understood, however, that the invention is not limited to the precise arrangements, examples, and instrumentalities shown.

[0019] Figure 1. Recombinant GDF11 (rGDF11) is Active In Vitro and In Vivo. A.Representative Coomassie blue stained protein gel of CHO expressed rGDF11 isolated as a stable dimer of the mature domain. B. Normalized fold activation of internally HEK Expi293 generated (GDF11, blue) and commercial Peprotech (GDF11-Pep, black) rGDF11 employing a SMAD2 / 3 responsive luciferase reporter activity assay. EC50s were determined using a non-linear four parameter curve fit (dotted vertical line): Internally produced rGDF11 - 0.1792nM and commercial Peprotech rGDF11 - 0.2273nM. B-F. Internally produced rGDF11 was transiently expressed and purified from HEK Expi293 cells (ThermoFisher Scientific). C-F. Graphs depicting phosphorylated Smad2 / 3 (pSmad2 / 3) levels in mouse spleen (C), liver (D), pancreas (E) and heart (F) 0.25, 1, 3, 6, 12, 24 and 48h after 1mg / kg rGDF11 treatment. Individual animals are shown with mean + / -SEM. Stats: For each graph, 2 way ANOVA was run and showed interaction significance for all graphs: (C) F(6,27) = 41.96 & p< 0.0001, (D) F(6,27)=34 & p<0.0001, (E) F(6, 28) = 2.997 & p=0.0217, (F) F(6,28) = 4.111 & p=0.0044. Unpaired t-test with Welsch’ correction between rGDF11 and vehicle: rGDF11 0.25h and 1h were compared to Vehicle Oh; rGDF11 3h and 6h were compared to Vehicle 3h; rGDF11 12h and 24h were compared to Vehicle 12h; rGDF11 48h was compared to Vehicle 48h. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, #p<0.1. NR (nonreduced); R (reduced); GDF11 (internally produced GDF11); Pep (Peprotech).

[0020] Figure 2. rGDF11 Decreases Age-Related Cardiac Hypertrophy. A. Heart weight normalized to tibial length for young (n=8) and aged (n=10) mouse groups treated once daily with vehicle (black) and 1mg / kg rGDF11 (blue) for 15 days. Treatment started on day 0 (red arrow). B, D. Graphs depicting body weight measurements in vehicle (black) and rGDF11-treated (blue) (B) aged mice (open symbols, n=10) or (D) young mice (closed symbols, n=8). C, E. Percent change in body weight of (C) aged and (E) young mice after 15 days of dailyP390200. W0.01administration of vehicle or 1mg / kg rGDF11. A-E. rGDF11 was transiently expressed and purified from HEK Expi293 cells (ThermoFisher Scientific). Stats: Data plotted as Mean + / -SEM. A. Multiple unpaired t-test with Welsch correction. B-E. 2-way ANOVA repeated measure for day 1 until day 16 interval with multiple comparison (for each day compare Vehicle to GDF11) and Sidak post hoc correction. B. F(15, 240) = 0.8126 p = 0.6632. C. F(15, 240) = 0.9837 p = 0.4730. D. F(15, 224) = 0.5531 p = 0.9076. E. F(15, 210) = 5.059 p < 0.0001. For all graphs, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

[0021] Figure 3. rGDF11 Improves Glucose Tolerance in Young and Aged Mice. A. Blood glucose concentration in the glucose tolerance test (GTT) for control (black circle, n=10) or rGDF11 (blue triangle) groups of aged (open symbols) mice, at pre-treatment baseline (dashed line) or after treatment with vehicle (black solid line) or 1 mg / kg rGDF11 (blue solid line, n=9). B. Blood glucose concentration in the insulin sensitivity test (ITT) for control (black circle, n=10) or rGDF11 (blue triangle, n=8) groups of aged mice, at pretreatment baseline (dashed line) or after treatment with vehicle (black solid line) or 1 mg / kg rGDF11 (blue solid line). C. Blood glucose concentration in the glucose tolerance test (GTT) for control (black circle, n=10) or rGDF11 (blue triangle) groups of young (closed symbols) mice, at pre-treatment baseline (dashed line) or after treatment with vehicle (black solid line) or 1 mg / kg rGDF11 (blue solid line, n=9). D. Blood glucose concentration in the insulin sensitivity test (ITT) for control (black circle, n=8) or rGDF11 (blue triangle, n=8) groups of young (closed symbols) mice, at pre-treatment baseline (dashed line) or after treatment with vehicle (black solid line) or 1mg / kg rGDF11 (blue solid line). A-E. rGDF11 was transiently expressed and purified from HEK Expi293 cells (ThermoFisher Scientific). Stats: A-D. 2-way ANOVA repeated measure with multiple comparison (for each timestamp compare baseline with treatment: *p<0.05, **p<0.01, ****p<0.0001, or gdf 11 with control: #p<0.05, ### p<0.001) and Sidak post hoc correction. E. Timeline showing days of dosing, days performed GTT and ITT, and tissue harvest day.

[0022] Figure 4. Intravenous Dose Regimen Optimization and Dose Dependency for Daily Dosing vs Single Dosing. A-C. Motor function analysis for Study 6 (green number), daily dosing on day 1 to 5 (grey squares represent dosing days) after a stroke event (red arrow). Dose groups were 0 (Vehicle), 0.3 and 1 mg / kg rGDF11. Graphs show animal body swing (A), forelimb placement (B) or hindlimb placement (C) with baseline measurement performed on day -1, stroke procedure performed on day 0 and post-stroke measurements on day 1, 3, 5, 7 and 14. D-F. Motor function analysis for Study 6, single dose on day 1 (grey squares represent dosing day) after a stroke event (red arrow). Dose groups were 0 (Vehicle), 0.3 and 1 mg / kg rGDF11. Graphs show rat body swing (D), forelimb placement (E) or hindlimb placement (F) with baseline measurement performed on day -1, stroke procedure performed on day 0 and post-stroke measurements on day 3, 5, 7 and 14. Stats:P390200. W0.01Data plotted as Mean + / - SEM with n=10 per group except for the single 0.3 mg / kg dose group (n=9). 2-way ANOVA repeated measure with multiple comparison (for each day compare doses to vehicle) and Dunnett post hoc correction. For all graphs, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

[0023] Figure 5. Body Weight Changes Associated with Different Intravenous Dosing Regimens. A. Body weight measurements for Study 6 (green number) encompassing all dosing regimens at 1 mg / kg rGDF11 treatment: (i) single dose on day 1 (orange), (ii) intermittent dosing on day 1, 3 and 5 (red) and (iii) daily dosing on day 1-5 (blue) after a stroke event (red arrow) for 1 mg / kg rGDF11 (continuous lines) or vehicle (dotted lines). The small squares show the dosing days for each group with their respective color. B-D. Body weight measurements for Study 6 (green number) for rats exposed to 0 (Vehicle), 0.3 and 1 mg / kg rGDF11 administered as (B) single dose on day 1, (C) intermittent dosing on day 1, 3 and 5 and (D) daily dosing on day 1-5 after a stroke event (red arrow). The small squares show the dosing days for each graph. All graphs show body weights with baseline measurement performed on day -1, stroke procedure performed on day 0 and post-stroke measurements on day 1, 3, 5, 7 and 14. Stats: Data plotted as Mean + / - SEM with n=10 per group except for the single 0.3 mg / kg dose group (n=9). A. 2-way ANOVA repeated measure with multiple comparison (for each dosing regimen, compare vehicle vs treatment) and Sidak post-hoc correction B-D. 2-way ANOVA repeated measure with multiple comparison (for each day compare doses to vehicle) and Dunnett post hoc correction. For all graphs, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

[0024] Figure 6. Vascular Morphometric Analysis Provides Evidence of Enhanced Vascularization in rGDF11 Treated Rats (Study 4). Quantitative analysis of CD31-positive vessels using AngioTool20is presented in the following graphs: (A) total vessel length, (B) average vessel length, (C) junction density, and (D) percent vessel area. One way ANOVA: (A) F (3,20) =1.842 & p=0.1720, (B) F (3,20) =1.510 & p=0.2426, (C) F (3,20) =1.587 & p=0.2238, and (D) F (3,20) =1.491 & p=0.2475. (A-D) Parametric unpaired t-tests were conducted for each measurement with *p<0.05, #p<0.1. Data plotted as individual value with Mean + / - SEM.

[0025] Figure 7. Representative images showing CD31 -positive vessels (Study 4). Representative images corresponding to Figure 6. (A) Regions of interest placed within 3 mm of the glial scar of the infarct (white boxes) in DAPI-positive coronal section. Scale bar = 1 mm. (B) CD31 -positive vessels on the left and overlay of CD31 and DAPI on the right. Scale bar = 100 pm.

[0026] Figure 8. Vascular Morphometric Analysis of Functional Vessels Reveals Evidence of Enhanced Vascularization in rGDF11 Treated Rats (Study 5). Quantitative analysis of CD31-positive vessels using AngioTool20is presented in the following graphs: (A)P390200. W0.01total vessel length, (B) average vessel length, and (C) junction density. One way ANOVA: (A) F (3,11) =1.915 & p=0.1857, (B) F (3,11) =0.1571 & p=0.9229, and (C) F (3,11) =3.944 & p=0.0391. (A-C) Parametric unpaired t-tests were conducted for each measurement with *p<0.05, #p<0.1. Data plotted as individual value with Mean + / - SEM.

[0027] Figure 9. Representative images showing CD31 -positive and Lectin-positive vessels (Study 5). Representative images corresponding to Figure 8. (A) regions of interest placed within 1 mm of the glial scar of the infarct (white boxes) in DAPI-positive coronal section. Scale bar = 1 mm. (B) Lectin-positive vessels on the left, CD31 -positive vessels in the middle and overlay of Lectin, CD31, and Dapi on the right. Scale bar = 50pm.

[0028] Figure 10. Canonical Pathway Analysis of Study 6 SomaScan Data. Heat maps showing the most activated canonical pathways and relative levels of circulating proteins in the top 5 canonical pathways identified using Ingenuity Pathway Analysis software. A.Paired comparison of day 2 (D2), day 3 (D3), or day 5 (D5)) post-stroke and pre-stroke (DO) for either vehicle (V) or rGDF11 (G) was used, as well as unpaired comparison of rGDF11 and vehicle for each day. Initial comparison threshold was p<0.01 and FC>1.5 and then canonical analysis threshold filter was p<0.0001 and Z>4. Dots indicate 1.96> Z>-1.96. B. Relative levels of circulating proteins for the top 5 canonical pathways are also shown, with a red gradient indicting increasing levels and green gradient indicating decreasing levels.

[0029] Figure 11. Path Explorer Analysis of Study 6 SomaScan Data. Following analysis of rGDF11 vs. Vehicle for days 2, 3, and 5, proteins with Bonferroni corrected significance less than 0.05 from day 2 and 5 analysis and less than 0.1 from day 3 analysis were used as seed proteins (inside circle). Path and predicted relationship were evaluated for neurogenesis, angiogenesis, axonogenesis, developmental process of synapse, and inflammation of nervous system using Ingenuity Pathway Analysis software.

[0030] Figure 12. Candidate Stroke Recovery-Related Biomarkers Decreased in Circulation in Response to rGDF11 (Study 6). Graphs representing the SomaScan aptamer measured circulating levels of (A) RTN4RL1, (B) CD68, (C) INHBA, (D) FGL1, and (E) NPPB after 1 mg / kg rGDF 11 treatment for intermittent dosing day 1, 3, 5 (red), sham intermittent dosing (green), daily dosing day 1-5 (blue) after a stroke event (red arrow). The small squares show the dosing days for each group with their respective color. 2-way ANOVA repeated measure with multiple comparison (for each day, compare treatment vs their respective vehicle for sham and daily dose) and Sidak post-hoc correction. Intermittent 1 mg / kg (red) was compared to vehicle daily dosing (solid black) of the respective day (day 5 or 7) since intermittent vehicle samples were not analyzed by SomaScan. Secreted proteins are indicated by a ‘0’ sign following protein name. For all graphs data plotted as Mean + / -SEM, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, #p<0.1.P390200. W0.01

[0031] Figure 13. Candidate Stroke Recovery-Related Biomarkers Increased in Circulation in Response to rGDF11 (Study 6). Graphs representing the SomaScan aptamer measured circulating levels of (A) FAM177A1, (B) UCN3, (C) NRP1, (D) NOG, (E) SPARCL1, (F) THBS2 after 1 mg / kg rGDF11 treatment for intermittent dosing day 1, 3, 5 (red), sham intermittent dosing (green), daily dosing day 1-5 (blue) after a stroke event (red arrow). The small squares show the dosing days for each group with their respective color.2-way ANOVA repeated measure with multiple comparison (for each day, compare treatment vs their respective vehicle for sham and daily dose) and Sidak post hoc correction. Intermittent 1 mg / kg (red) was compared to vehicle daily dosing (solid black) of the respective day (day 5 or 7) since intermittent vehicle samples were not analyzed by SomaScan.Secreted proteins are indicated by a ‘0’ sign following protein name. For all graphs data plotted as Mean + / -SEM, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, #p<0.1.

[0032] Figure 14. Elevated Levels of Vascular Modulators in Circulation in Response to rGDF11 (Study 6). Graphs representing the SomaScan aptamer measured expression levels of (A) ANGPT 1, and (B) ANGPT2 after 1 mg / kg rGDF11 treatment for intermittent dosing day 1, 3, 5 (red), sham intermittent dosing (green), daily dosing day 1-5 (blue) after a stroke event (red arrow). The small squares show the dosing days for each group with their respective color. 2-way ANOVA repeated measure with multiple comparison (for each day, compare treatment vs their respective vehicle for sham and daily dose) and Sidak post-hoc correction. Intermittent 1 mg / kg (red) was compared to vehicle daily dosing (solid black) of the respective day (day 5 or 7) since intermittent vehicle samples were not analyzed by SomaScan. For all graphs data plotted as Mean + / - SEM, *p<0.05, #p<0.1.

[0033] Figure 15a. Study Designs for Evaluating the Efficacy of rGDF11 in the pMCAO Rat Model. A-C. Experimental designs for Studies 4, 5, and 6, detailing the timing of permanent middle cerebral artery occlusion (pMCAO) or sham surgeries (indicated by red arrows), dosing initiation, dosing frequency, dosing duration, behavioral testing (BT), and study termination. A. Study 4: IP Dose Ranging. Once daily intraperitoneal (IP) dosing for 5 days. Dosing days are shown as gray squares. B. Study 5: IV Dose Ranging. Once daily Intravenous (IV) dosing for 7 days. Dosing days are shown as gray squares. C. Study 6: IV Dose and Regimen Optimization. A single IV dose on Day 1 is marked by an orange square; intermittent dosing on Days 1, 3, and 5 is marked by red squares; and daily dosing from Days 1-5 is marked by blue squares. For all studies, animals received only one dose per day on dosing days. On days when multiple activities were performed, behavioral tests were conducted first, followed by drug administration, and then blood draws

[0034] Figure 15b. IP rGDF11 Improves Motor Function and Increases Radial Glial Cell Numbers. A-C. Motor function analysis for Study 4 (green number): One dose daily for 5 days, on days 1-5 (grey squares represent dosing days) after a stroke event (red arrow) withP390200. W0.01n=10 rats per group. Dose groups were 0 (Vehicle), 0.1, 0.5, 1, 2 and 4mg / kg rGDF11. Graphs show rat body swing (A), forelimb placement (B), or hindlimb placement (C) with baseline measurement performed on day-1, stroke procedure performed on day 0 and poststroke measurements on day 1, 3, 5, 7, 14, 21 and 28. D. Graph showing the number of neuronal progenitor cells in rat ipsilateral and contralateral brain slides after 0 (vehicle), 1, 2 or 4 mg / kg rGDF11 treatment 28 days following pMCAO. Stats: A-C. Data plotted as Mean + / - SEM. 2-way ANOVA repeated measure with multiple comparison (for each day compare doses to vehicle) and Dunnett post hoc correction. (A) For 2mg / kg, we also show a body swing score at day 21 and day 28 comparable to pre-stroke measurements (day -1). For the latter, we ran a 2-way ANOVA repeated measure with multiple comparison (for each dose to compare days to d-1) and Dunnett post hoc correction. (D) One way ANOVA non-significant: F (3,6) =2.682 & p=0.0818 and parametric unpaired t-test with *p<0.05. Data plotted as individual value with Mean + / - SEM. For all graphs, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, #p<0.1.

[0035] Figure 16. IV rGDF11 Improves Motor Function and Increases Vascularization. A-C. Motor function analysis for Study 5 (green number): One dose daily for 7 days, on day 1-7 (grey squares represent dosing days) after a stroke event (red arrow) with n=10 rats per group. Dose groups were 0 (Vehicle), 0.1, 0.3 and 1 mg / kg rGDF11. Graphs show rat body swing (A), forelimb placement (B) or hindlimb placement (C) with baseline measurement performed on day -1, stroke procedure performed on day 0 and post-stroke measurements on day 1, 3, 5, 7, 14 and 21. D. Graph showing the percent area in rat ipsilateral brains with CD31+ Lectin+ endothelial progenitor cells after 0 (vehicle), 0.1, 0.3 or 1 mg / kg rGDF11 treatment 21 days following pMCAO. E. CRP concentration measured in rat serum collected 15 minutes after 0 (vehicle), 0.1, 0.3 or 1 mg / kg rGDF11 treatment on day 7. The shaded green area represents the normal CRP concentration range in healthy rats.30F. GDF11 concentration measured via ELISA assay on rat serum on day 7 after a stroke event, 15min after 0 (vehicle), 0.1, 0.3 or 1 mg / kg rGDF11 treatment. Stats: A-C. Data plotted as Mean + / -SEM. 2-way ANOVA repeated measure with multiple comparison (for each day compare doses to vehicle) and Dunnett post hoc correction. (A) For 1 mg / kg, we also show a body swing score at day 14 and 21 comparable to pre-stroke measurements (day-1). For the latter, we ran a 2-way ANOVA repeated measure with multiple comparison (for each dose compare days to d-1) and Dunnett post hoc correction. D-E. Data plotted as individual value with Mean + / - SEM. (D) One way ANOVA non-significant: F (3, 11) =2.429 & p=0.1203 and parametric unpaired t-test with *p<0.05. (E-F). 2-way ANOVA with multiple comparison (compare each dose with all other doses) with Tukey post hoc correction, n = 12 per group for 0.1, 0.3, and 1 mg / kg doses and n = 4 for the Vehicle group. For all graphs, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, #p<0.1.P390200. W0.01

[0036] Figure 17. IV rGDF11 Dose Regimen Optimization and Dose Dependency. A-C.Motor function analysis for Study 6 (green number) encompassing all intravenous dosing regimens at 1mg / kg rGDF11 treatment: (i) single dose on day 1 (orange), (ii) intermittent dosing on day 1, 3 and 5 (red) and (iii) daily dosing on day 1-5 (blue) after a stroke event (red arrow) for 1mg / kg rGDF11 (continuous lines) or vehicle (dotted lines). The small squares show the dosing days for each group with their respective color. D-E. Motor function analysis for Study 6, intermittent dosing only on day 1, 3 and 5 (grey squares represent dosing days) after a stroke event (red arrow). Dose groups were 0 (Vehicle), 0.3 and 1mg / kg rGDF11. Graphs show rat body swing (D), forelimb placement (E) or hindlimb placement (F) with baseline measurement performed on day -1, stroke procedure performed on day 0 and post-stroke measurements on day 1, 3, 5, 7 and 14. Stats: Data plotted as Mean + / - SEM with n=10 per group. A-C. 2-way ANOVA repeated measure with multiple comparison (for each dosing regimen, compare vehicle vs treatment) and Sidak post hoc correction. D-F. 2-way ANOVA repeated measure with multiple comparison (for each day compare doses to vehicle) and Dunnett post hoc correction. For all graphs, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, #p<0.1.

[0037] Figure 18. IV rGDF11 Treatment Decreases Serum CRP Concentrations Post Injury. CRP levels normalized to baseline for Study 6 (green number) encompassing all dosing regimens at 1mg / kg rGDF11 treatment: (A) single dose on day 1, (B) intermittent dosing on day 1, 3 and 5 and (C) daily dosing on day 1-5 after a stroke event (red arrow) and (D) intermittent dosing on day 1, 3, and 5 after sham (green arrow). The grey squares show the dosing regimen for each graph. Stats: All graphs were analyzed for 2-way ANOVA repeated measure with multiple comparison (each treatment compared to vehicle) and Sidak post hoc correction. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

[0038] Figure 19. Biomarker analysis. A. PCA analysis of biomarkersdiscovered using SomaScan analysis. Circles represent individual rats exposed to daily dosing on day 1 -5 with either Omg / kg (vehicle) or 1 mg / kg rGDF 11. SomaScan analysis was run on rat serum on day 2, 3, and 5 after a stroke event. The different colors correspond to rats treated with either rGDF 11 or vehicle and from which serum was harvested on day 2, 3, and 5 respectively as indicated in the associated legend. B-D. Volcano plot showing the distribution of SomaScan biomarkers for day 2 (B), day 3 (C) or day 5 (D) after a stroke event with the specific names of proteins related to stroke. Biomarkers with a fold change >1.5 and p<0.01 when compared to vehicle control, are highlighted in “green” (decreased in circulation) or red (increased in circulation). Biomarkers marked with a “*” are specific to each day (B: Day 2; C: Day 3; and D: Day 5) except for FAM177A1 which is increased on both Day 3 and Day 5. All other highlighted biomarkers green or red are day 2, 3 and 5.P390200. W0.01

[0039] Figure 20. rGDF11 Promotes Recovery Post Injury in a Mouse ICH Model.Behavioral outcomes including (A) Neuroseverity Score and (B) Rotarod were evaluated before and after ICH. (A) and (B) Data Plotted as Mean + / - SEM and analyzed using Kolmogorov-Smirnov test. CatWalk analysis including (C) forelimb base of support and (D) average speed were conducted 7 days post ICH. (C) and (D) Data Plotted as Mean + / - SEM and analyzed using unpaired t-test with Holm-Sidak correction. *** p<0.001; ** p<0.01; * p<0.05.

[0040] Figure 21. rGDF11 Reduces Microglial Density and Increases Vascular Area Post Injury in a Mouse ICH Model. Unbiased quantitative stereology of brain sections 28 days post ICH using (A) F4 / 80 staining for microglia density and (B) Cd31 staining for vascular area. Data Plotted as Mean + / - SEM and analyzed using unpaired t-test with Holm-Sidak correction. ** p<0.01; * p<0.05; ns - not significant; Ipsi - Ipsilateral; Contra -Contralateral.

[0041] Figure 22. rGDF11 Promotes Recovery Post Injury in a Mouse TBI Model.Behavioral outcomes including (A) Neuroseverity Score and (B) Rotarod were evaluated before and after ICH. (A) and (B) Data plotted as Mean + / - SEM. Analyzed using a 2-way ANOVA repeated measures with multiple comparison (for each day compare rGDF11 to vehicle) with Sidak post hoc correction. *** p<0.001; ** p<0.01; * p<0.05; ns - not significant.

[0042] Figure 23. In Vivo Stroke Study Designs. Table describing the route of administration (RoA) being either IP (intra-peritoneal) or IV (intravenous), the dosing frequency, duration and experimental design for studies 1-6. pMCAO (permanent middle cerebral artery occlusion); FLP (Forelimb placing); HLP (Hindlimb placing); BS (Body swing); sig days (significant days).

[0043] Figure 24. Biomarker Study 6 Design. Table describing the different study groups (pMCAO - colors or sham - grey), their rGDF11 treatment frequency (single dose day 1 -yellow; intermittent dose day 1, 3, 5 - orange; daily dose day 1-5 - blue), the treatment groups (V - vehicle; L - low dose 0.3mg / kg; H - high dose 1 mg / kg), and the monitoring schedule. Baseline motor function measurement was performed on day -1, stroke / sham procedure performed on day 0 and post-stroke measurements on day 1, 3, 5, 7 and 14. Rat blood was sampled on day -1 as baseline and post stroke on day 3, 5, 7 and 14. Serum samples from 14 time points (red box borders) were analyzed for their biomarker composition with SomaScan. pMCAO (permanent middle cerebral artery occlusion); tx (treatment).

[0044] Figure 25. Summary of Results from Rat Toxicology Studies Performed with rGDF11. Safety and toxicology studies in male and female rats suggest at least a 10-Fold therapeutic and safety window above the optimized efficacious dose (1 mg / kg) in the rat pMCAO Model: No Adverse Events Observed at Any Dose Tested. HED (Human EquivalentP390200. W0.01Dose); RoA (Route of Administration); IV (Intravenous); NOAEL (No Observed Adverse Effects Level); * (Includes Toxicokinetic Study Animals).

[0045] Figure 26. Characterization of Serum Biomarkers Using SomaScan Analysis (Study 6: IV Dose and Regimen Optimization). Table describing the overall statistical outcomes of the SomaScan biomarker analysis. SomaScan analysis was run across different study groups (pMCAO or sham), dose regimens (Daily dosing day 1-5 vs intermittent dosing day 1, 3, 5) and treatment groups (Vehicle vs rGDF11). Statistical tests were run for specific treatment groups between two measurement days (top half of table) or for specific days between two treatment groups (bottom half of table), depicted by the <->’ sign. For comparisons between post and pre-treatments for each treatment group we performed a paired t-test. For comparisons of rGDF11 to Vehicle for each day we performed an unpaired t-test. Corrections were made using the False Discovery Rate (FDR adjusted p-value) and Bonferroni correction (BF). For each of the conditions being tested (different rows), we showed the number of biomarkers increased (red) or decreased (green) in circulation and fitting within the threshold of nominal significance of p<0.05 (nom); number of biomarkers with FDR significance p<0.05 (FDR); number of biomarkers with Bonferroni significance p<0.05 (BF). Biomarkers that were selected for further pathway analysis (Pathway analysis) had a normalized significance of p<0.01 and a fold change (FC) > 1.5 (=log(0.5849)). The color scale for both increased (red) and decreased (green) circulating proteins spans from 0 to 500 with 50th percentile midpoint. pMCAO (permanent middle cerebral artery occlusion) nom sig (nominal significant); D (day).DETAILED DESCRIPTION

[0046] Growth differentiation factor 11 (GDF11), a circulating blood factor and member of the transforming growth factor-beta (TGF-β) superfamily, can stimulate regeneration in multiple tissues and organs and has been demonstrated to have beneficial effects in some aging disease models.23The specific impacts of GDF 11 on tissue repair and aging appears to be both concentration and context dependent, emphasizing the importance of comprehensive assessment of its effects across a broad range of doses and in biologically relevant aging-related disease models.

[0047] Studies in aged mice demonstrated that systemic administration of exogenous recombinant GDF11 (rGDF11) increases neovascularization broadly in the brain and enhances neurogenesis in the both the subventricular zone and hippocampus.45In addition, in the APP / PS1 mouse model of Alzheimer’s Disease, rGDF11 improves cognitive function and increases cerebrovascular blood flow.6rGDF11 administered once daily for seven days promoted neurogenesis, increased neovascularization, and improved sensorimotor motor function in young mice post middle cerebral artery occlusion (MCAO), a model of ischemicP390200. W0.01stroke.7Similarly, Hudobenko and colleagues demonstrated that rGDF11 supplementation increased neovascularization, improved sensorimotor function and white matter integrity, and reduced brain atrophy, gliosis, inflammation, and mortality post-MCAO in aged mice.8

[0048] As described herein, GDF11 is used to treat stroke and / or improve one or more symptoms, including functional sensorimotor recovery, following a stroke event. Dosing regimens, including timing of dosing initiation post-injury, duration of dosing, and / or dose range are provided. Outcomes include limb placement, which primarily evaluates sensorimotor cortical function, and body swing, which reflects subcortical function.910

[0049] This disclosure provides biomarkers of GDF11 treatment efficacy, methods of treating ischemic stroke with GDF11. Specifically, biomarkers that promote recovery post¬ stroke, and circulating biomarkers whose levels were modified in response to systemic rGDF11 administration, providing a panel of candidate biomarkers for improved administration.Definitions

[0050] Described herein are several definitions. Such definitions are meant to encompass grammatical equivalents. Unless otherwise required by context, singular terms as used herein and in the claims shall include pluralities and plural terms shall include the singular.

[0051] The use of “or” means “and / or” unless stated otherwise. Furthermore, the use of the terms “comprising,” “having,” “including,” as well as other forms, such as “includes” and “included,” are intended to be inclusive and mean that there may be additional elements other than the listed elements. Also, terms such as “element” or “component” encompass both elements and components comprising one unit and elements and components that comprise more than one subunit unless specifically stated otherwise.

[0052] As various changes could be made in the above-described compositions, methods, and kits without departing from the scope of the disclosure, it is intended that all matter contained in the above description and in the examples given below, shall be interpreted as illustrative and not in a limiting sense.

[0053] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the disclosure. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are also encompassed within the disclosure, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure.P390200. W0.01

[0054] As used throughout this specification and the appended claims, the singular forms “a,” “an” and “the” include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to “a pharmaceutically acceptable carrier, excipient or vehicle” includes a mixture of two or more such entities, and the like.

[0055] Generally, nomenclatures used in connection with, and techniques of, cell and tissue culture, molecular biology, immunology, microbiology, genetics, protein, and nucleic acid chemistry and hybridization described herein are those well-known and commonly used in the art. The methods and techniques of the disclosure are generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification unless otherwise indicated. See, e.g., Sambrook et al. Molecular Cloning: A Laboratory Manual, 2d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N. Y. (1989); Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates (1992, and Supplements to 2002); Harlow and Lan, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N. Y. (1990); Principles of Neural Science, 4th ed., Eric R. Kandel, James H. Schwart, Thomas M. Jessell eds. McGraw-Hill / Appleton & Lange: New York, N. Y. (2000); The Merck Manual of Diagnosis and Therapy, 19th Edition, published by Merck Research Laboratories (2006) (ISBN 0-911910-19-0), Robert S. Porter et al. eds., The Encyclopedia of Molecular Biology, published by Blackwell Science Ltd. (1994) (ISBN 0-632-02182-9); and Current Protocols in Protein Sciences (2009) Wiley Intersciences, Coligan et al., eds.

[0056] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the disclosure, representative illustrative methods, and materials are now described.

[0057] As used herein, the terms “administer”, “administering”, and “administered” refer to providing a therapeutically active amount of GDF11 (in some variation as “dose”) to the subject being treated. Administration of a dose of the GDF11 molecule can be carried out on any suitable basis. In some variations, the dose can be provided in a single time of administration, such as once daily (QD) basis, or divided between multiple administrations, such as twice daily (BID), three times daily (TID), four times daily (QID), and each day of treatment can be the same or different over the course of treatment. In variations, the GDF11 molecule is administered in the form of a liquid solution or suspension that is introduced to the subject via normal intraperitoneal, subcutaneous, or intravenous delivery techniques, but can include microinjection, stereotactic injection, and / or direct application to a particular site in the subject.P390200. W0.01

[0058] The term “controlled release” refers to a pharmaceutical dosage form or composition that provides for the delayed, slowed over a period of time, continuous, discontinuous, or sustained release of a therapeutically active molecule.

[0059] The terms “increase”, “increased”, “enhance”, and “enhanced” are interchangeably used herein generally to mean an increase by a statistically significant amount relative to a control amount or reference amount (p < 0.05).

[0060] The terms “decrease”, “reduce”, “reduced”, “reduction”, “decrease”, and “inhibit” are all used interchangeably herein generally to mean a decrease by a statistically significant amount relative to a control amount or reference amount (p < 0.05).

[0061] The phrase “dose of GDF11” or “dose of GDF11 molecule”, as used interchangeably herein, denotes the quantity of GDF11 administered to a subject over within a single 24 hour period. Such quantity can be measured as relative to the body weight of the subject being treated.

[0062] As used herein, a “moderate dose of GDF11” means moderate application of a broad range of GDF11 molecule to a subject over a 24 hour period, typically in the range of from 0.001 mg / kg up to and including 0.5 mg / kg body weight daily in a subject alternatively at 0.1 mg / kg body weight daily in a rodent subject and as normally extrapolated to other larger mammalian species up to and including human subjects. In a human subject, the predicted Human Equivalent Dose (HED) can be calculated as described in Nair and Jacob, J Basic Clin Pharm, 7(2):27-31 (Mar. 2016), incorporated herein by reference in its entirety. For example, the human dose can be calculated from rats based on allometric scaling by dividing the rat dose (mg / kg) by 6.2. A 1 mg / kg dose in rat corresponds to a 0.16 mg / kg predicted HED. The conversion for mice to predicted HED is achieved by dividing the mouse dose (mg / kg) by 12.3. As noted above, published reports of in vivo administration of moderate doses of GDF11 have avoided the high end (0.5 mg / kg), and instead concentrated on a specific moderate dose of 0.1 mg / kg. A moderate dose of GDF11 includes the vast majority of published in vivo GDF11 dosing studies targeting from about a 1 to 4 fold increase in circulating GDF11 in the treated subject, where mouse or rat subjects are treated with GDF 11 administered daily at a typical amount of about 0.1 mg / kg body weight, on a daily basis. See, e.g., Zhang et al (2016) Scientific Reports 6(1): 34624; Mei et al. (2016) Molecular Therapy: the Journal of the American Society of Gene Therapy 24(11): 1926-1938; Du et al. (2017) Basic Research in Cardiology 112(1): 7; Li et al. (2017) Diabetes 66(7): dbl70086-1927; Onodera et al. (2017) Thorax (April); Bajikar et al. (2017) Developmental Cell 43(4): 418-435; Harper et al. (2018) Circulation Research 7 Sep 2018; Zhang et al. (2018) Diabetes; Ma et al. (2018) Brain Research Bulletin 139(February): 38-47; Zhang et al. (2018) Journal of Alzheimer ’s Disease 62(2); Lu et al. (2018) Front Cell Neurosci 12: 205; Wang et al. (2018) Am J Physiology Gastrointest Liver Physiology andP390200. W0.01Wang et al. (2018) Inflammation, all of which are included by reference in their entirety. Such moderate dose administration of GDF11 has been characterized as extending from 0.001 mg / kg to up to 0.5 mg / kg body weight (see, e.g, U. S. Patent No. 9,434,779; and U. S. Patent Publication Nos. US2016 / 074477 and US2016 / 220640, all of which are incorporated by reference in their entirety).

[0063] The term “high dose of GDF11” as used herein defines a dose of a GDF11 molecule administered to a subject that falls in between a moderate dose of GDF11 and an excess dose of GDF11 in the relevant subject and that has, surprisingly, demonstrated an unexpected beneficial effect in the treatment of stroke, ICH, and TBI as set forth in this disclosure. A high dose of GDF11 molecule is generally defined herein as encompassing a bracketed range of doses starting from greater than the top end of reported moderate doses of GDF11, to less than the bottom reported excess dose of GDF11, both such moderate and excess doses of GDF11 as reported on a daily basis and in a relevant subject. More particularly, a standard high dose of GDF11 is defined herein as equivalent to about 1 mg / kg body weight of a rodent subject (mouse or rat) on a daily basis, accordingly about 1 order of magnitude greater than the normal moderate dose of GDF11 in such species (i.e., about 0.1 mg / kg). Accordingly, a “minimal high dose of GDF11” is at least about 0.8 mg / kg (body weight) in a rodent species, and the same such dose in a larger mammalian species, normalized to the molecular weight of rhGDF11 as defined by SEQ ID NO: 1. In like manner, a “maximal high dose of GDF11 ” is about 4 mg / kg (body weight) in a rodent species and the same such dose in a larger mammalian species, normalized to the molecular weight of rhGDF11 as defined by SEQ ID NO: 1. In a human subject, for example, the predicted Human Equivalent Dose (HED) can be calculated as described in Nair and Jacob, J Basic Clin Pharm, 7(2):27-31 (Mar. 2016) incorporated herein by reference in its entirety and as described elsewhere herein. In this regard, the maximal high dose of GDF 11 is that which avoids adverse side effects in the treated subject. It is to be noted that specific dosage values can vary with the severity of the stroke, ICH, and TBI to be addressed. For any particular subject, specific dosing regimens can be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the administration of the present GDF11 compositions and the concentration ranges set forth herein are exemplary only and are not intended to limit the scope or practice of the claimed methods. Exact dosing can be predicted empirically by testing in in vitro and in vivo systems well known to those of skill in the art and then extrapolated therefrom for use in subjects including human subjects. Human doses are then typically fine-tuned in clinical trials and titrated to response. For the purposes of this disclosure, a high dose of GDF11, as defined in a rodent subject, is at least about 0.8 mg / kg to about 4 mg / kg, or at least about 0.9 mg / kg to about 4 mg / kg, in some cases at least about 1 mg / kg to about 3 mg / kg, and in certainP390200. W0.01aspects of the disclosure at least about 1 to 2 mg / kg, and encompasses the corresponding dose in any larger mammalian species.

[0064] An “excess dose of GDF11 ” is a dose of GDF11 that, although providing some or no therapeutic benefit, may result in generating adverse or otherwise unacceptable side effect or side effect profile in the subject being treated with GDF11. As used herein, a “side effect” is any pharmacological or physiological effect of GDF11 administration that is secondary to the one intended, and an “adverse side effect” is any such secondary effect that is undesired and / or harmful, and that manifests in an outcome such as morbidity, mortality, loss of function or any other pathological change in a treated subject where it may be reversible or irreversible. A number of published in vivo GDF11 dosing studies targeting excess doses of GDF11, where mouse or rat subjects were treated with GDF11 administered daily at a typical amount of about 5 mg / kg to 10 mg / kg body weight or greater, on a daily basis, have reported that such supraphysiological doses of GDF11 may give rise to significant adverse effects including cachexia, muscle atrophy, anorexia, fibrosis or even death (see, e.g., Hammers et al. (2017) EMBO Molecular Medicine 9(4): 531-544; Pons et al. (2018) Surgery (May); and Jones et al. (2018) Cell Reports 22(6): 1522-1530).

[0065] As used herein, the term “GDF11” refers to “Growth and Differentiation Factor 11” (NCBI Gene ID No: 10220), which is a member of the Transforming Growth Factor-beta superfamily of growth factors. As used herein, “GDF11” can include the mature disulfide linked 109 amino acid homodimer in humans (SEQ ID NO: 1), as described in WO 2021 / 236824, which is incorporated herein by reference in its entirety. The 109 amino acid sequences of GDF11 is SEQ ID NO: 1 of WO 2021 / 236824. For human GDF11, the propeptide plus signal sequence (e.g. the precursor polypeptide) is 407 amino acids long. Cleavage of the 24 amino acid signal peptide generates a pro-peptide of 383 amino acids and cleavage of the pro-peptide results in a mature GDF11 polypeptide of 109 amino acids that corresponds to the C-terminal 109 amino acids of the pro-peptide. The mature polypeptide forms the mature disulfide-linked homodimer. Accordingly, “GDF11” can include the human precursor GDF11 polypeptide (SEQ ID NO: 2) (Depicted in FIG. 5 of U. S. Patent No. 10,980,857, incorporated herein by reference in its entirety; NCBI Ref Seq: NP _005802); the human GDF11 pro-peptide (SEQ ID NO: 3) (Depicted in FIG. 6 of U. S. Patent No. 10,980,857, incorporated herein by reference in its entirety); the human N-terminal polypeptide (SEQ ID NO: 4) (Depicted in FIG. 8 of U. S. Patent No. 10,980,857, incorporated herein by reference in its entirety).

[0066] As used herein, the term “hydrogel” is used in its usual manner within the art, for example to refer to a polymer that swells in the presence of water or other aqueous system, shrinks in the absence or reduction of the amount of water, is able to retain a significant fraction of water within its structure, and typically does not dissolve in water. One ordinarilyP390200. W0.01skilled in the art will appreciate that there are a number of standard tests that one can employ in order to determine if a polymer or polymer system will act as a hydrogel, e.g., form a hydrogel, when immersed in an aqueous system such as when it is implanted or otherwise delivered in vivo into a mammalian subject.

[0067] An “implant” or “implantable composition or device”, as used herein, refers to any implantable system for use in the delivery of a therapeutically active substance to a subject. Common implantable devices allow local (site specific) and / or systemic administration of the agent of interest and examples include solid structures such as stents or wafers that can be left behind on or in tissue at a surgical site, rods or microparticles that can be administered via subcutaneous or intramuscular injection with a needle and syringe or trocar, and implantable drug pump devices. Solid implantable compositions or devices are commonly formed using bioerodible polymers that can provide for controlled release of an agent of interest. Injectable implantable compositions or devices can be provided in the form of viscous liquid carriers, hydrogel compositions, nanoparticle compositions, microspheres or microparticles, or plasticized polymer carriers.

[0068] The term, “kit” as used herein, means any manufacture (e.g., a package or container) including at least one therapeutically active agent (a GDF11 molecule). In certain kits the manufacture may be promoted, distributed, or sold as a unit for performing the methods of the disclosure.

[0069] As used herein, the term “liposome” refers to a spherical vesicle having at least one lipid bilayer formed by certain lipids (phospholipids such as phosphatidylcholine) that are filled with an aqueous core that may contain other components including a therapeutically active agent of the disclosure. A liposome can be up to about 10 microns in size, however, the liposomes employed in the practice of the compositions and methods of the disclosure are sub-micron in size, i.e., in the form of a nanoparticle.

[0070] The term “local” or “locally” as used herein means, with respect to delivery or administration of a therapeutically active agent to a subject, that such agent is delivered to a localized site in the subject but may not be detectable at a biologically significant level in the blood plasma of the subject.

[0071] A “nanoparticle” refers to a particulate material or a population of such particles with sizes generally ranging between 1 and 100 nm and can include nanospheres, for example lipid systems such as liposomes and micelles, nanocrystals and nanoparticles.

[0072] Nanospheres can contain pharmacological agents (molecules or compounds) as well as other materials such as inorganic nanoparticles like gold or magnetic particles, or nanoparticles may contain or be formed from polymers such as biodegradable polymers. In the practice of the compositions and methods of the disclosure, synthetic polymers such as polyvinyl alcohol, poly-L-lactic acid, polyethylene glycol and poly(lactic-co-glycolic acid andP390200. W0.01natural polymers such as alginate and chitosan can be used in the nanofabrication of nanoparticles to provide nanospheres or nanocapsules. Nanoparticles generally remain in the blood circulatory system for a prolonged period, thus enabling the extended release of agents and extended pharmacological agent life cycle. Due to their nanosize, nanoparticle structures readily penetrate tissue systems and facilitate easy uptake by cells to achieve efficient delivery at targeted locations.

[0073] The term “pharmaceutically acceptable” refers to a material that has been approved or is approvable for pharmaceutical use by a regulatory agency of a relevant federal or state government and / or is listed in the U. S. Pharmacopeia or another generally recognized pharmacopeia for use in animal subjects, and more particularly in humans. A “pharmaceutically acceptable carrier, excipient or vehicle” refers to any vehicle, diluent, adjuvant, excipient or carrier with which a therapeutically active compound is administered.

[0074] A “pharmaceutically acceptable salt" refers to a salt of a therapeutically active molecule or compound that is pharmaceutically acceptable and that possesses the desired pharmacological activity of the parent molecule or compound. Pharmaceutically acceptable salts of the therapeutically active agents described herein include those salts derived from pharmaceutically acceptable inorganic and organic acids and bases. Examples of suitable acid salts include acetate, adipate, alginate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, citrate, camphorate, camphorsulfonate, cyclopentanepropionate, digluconate, dodecyl sulfate, ethanesulfonate, formate, fumarate, glucoheptanoate, glycerophosphate, glycolate, hemisulfate, heptanoate, hexanoate, hydrochloride, hydrobromide, hydroiodide, 2-hydroxyethanesulfonate, lactate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oxalate, palmoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, salicylate, succinate, sulfate, tartrate, thiocyanate, tosylate and undecanoate salts. Other acids, such as oxalic, while not in themselves pharmaceutically acceptable, may be employed in the preparation of salts useful as intermediates in obtaining pharmaceutically acceptable acid addition salts. Salts derived from appropriate bases include alkali metal (e.g., sodium and potassium), alkaline earth metal (e.g., magnesium), ammonium and salts.

[0075] As used herein, “polymer” intends any polymer, copolymer and blends unless otherwise expressly defined. The polymers for use in connection with the compositions and methods of the disclosure can be produced using standard polymerization and copolymerization techniques, such as graft copolymerization, polycondensation and polyaddition, optionally with an appropriate catalyst. These techniques can be carried out in conventional manner well known in the polymer art as regards to time, temperature and other parameters. Alternatively, the polymers used herein can be produced using standardP390200. W0.01blending techniques of polymers or blending of copolymers, again carried out in conventional manners well known in the polymer art.

[0076] As used herein, the terms “protein” and “polypeptide” are used interchangeably to designate a series of amino acid residues connected to the other by peptide bonds between the alpha-amino and carboxy groups of adjacent residues. The terms thus refer to a polymer of protein amino acids, including modified amino acids (e.g., phosphorylated, glycated, glycosylated, etc.) and amino acid analogs, regardless of size or function. Protein and polypeptide are often used in reference to relatively large polypeptides, whereas the term “peptide” is often used in reference to small polypeptides, but usage of these terms in the art overlaps. The terms “protein” and “polypeptide” are used interchangeably herein when referring to a gene expression product and fragments thereof. Thus, exemplary polypeptides or proteins include gene expression products, naturally occurring proteins, homologs, orthologs, paralogs, fragments and other equivalents, variants, derivatives, fragments, and analogs of the foregoing.

[0077] As used herein, a “stroke event” in a subject refers to a medical condition where inadequate blood flow to the brain results in neurological cell death. Stroke events are classified as two main types: ischemic (resulting from an interruption of blood supply to the brain); and hemorrhagic (resulting from rupture of a blood vessel or an abnormal vascular structure. Both types of stroke event result in parts of the brain not functioning properly. An ischemic stroke event is typically caused by blockage of a blood vessel, where blood supply to part of the brain is decreased, leading to dysfunction of the brain tissue in that area. There are four causes for such ischemia; thrombosis; embolism; systemic hypoperfusion; and cerebral venous sinus thrombosis. A hemorrhagic stroke event is caused by either bleeding directly into the brain or into the space between the brain’s membranes, wherein such bleeding may occur due to a ruptured brain aneurysm. There are two main types of hemorrhagic stroke, intracerebral hemorrhage (bleeding within the brain itself); and subarachnoid hemorrhage (bleeding outside the brain tissue but within the brain cavity).

[0078] Signs and symptoms of a stoke event occurring in a subject are well known in the medical arts and can include sudden-onset face weakness, arm drift, abnormal speech, an inability to move or feel on one side of the body, difficulty in understanding, dizziness or vertigo, loss of vision to one side, and severe headache. An ischemic stroke event can be classified as total anterior circulation infarct (TACI); partial anterior circulation infarct (PACI); lacunar infarct (LACI); or posterior circulation infarct (POCI) that predict the extent of the stroke event, the area of brain affected, the underlying cause and the prognosis. Clinical diagnoses of a stroke event are also well known in the medical arts and are typically based on physical and neurological examination (such as the NIHSS) supported by medicalP390200. W0.01imaging techniques such as CT scan, MRI scan, Doppler ultrasound and arteriography and often supported by ancillary tests such as electrocardiogram (ECG) and blood tests.

[0079] The terms “subject”, “patient”, and “individual” are used interchangeably herein, and mean any mammalian animal (e.g., including, but not limited to humans, primates, dogs, cattle, cows, horses, kangaroos, pigs, sheep, goats, cats, rabbits, rodents), transgenic nonhuman animals, which are to be the recipient of a particular method of treatment, dose, or pharmaceutical composition.

[0080] The terms “systemic” or “systemically” as used herein mean, with respect to delivery or administration of a therapeutically active agent to a subject, that such agent is detectable at a biologically-significant level in the blood plasma of the subject. Systemic administration includes intravenous (i.v.), intraperitoneal (i.p.), and subcutaneous (s.c.). The term includes oral or parenteral administration of a therapeutically active agent to a subject.

[0081] As used herein, the term “therapeutically active" may refer to an activity of a GDF11 molecule or compound whose effect is consistent with a desirable therapeutic outcome in an intended subject. The terms “therapeutically active agent”, “therapeutically active GDF11 molecule” or a “therapeutically active derivative, variant or modified GDF11” are used interchangeably herein and refer to a molecule having a therapeutic activity whose effect is consistent with a desirable outcome in a subject and, in the case of a variant, derivative and / or modified molecule, is consistent with the pharmacological activity of the parent molecule. Therapeutic activity may be measured using in vitro or in vivo methodology well known to those of skill in the relevant art, for example a desirable therapeutic effect can be assayed in cell culture.

[0082] A “therapeutically effective amount” refers to the amount of a therapeutically active GDF11 that, when administered to a subject, is sufficient to affect a desired treatment for the disease, condition, complication or disorder present in the subject. The “therapeutically effective amount” of a therapeutically active agent for use in any particular method herein will vary depending on the molecule or compound, the disease, condition, complication or disorder, and its severity and the age and weight of the subject. The full therapeutic effect may not necessarily occur by administration of one single dose of the therapeutically active agent (molecule or compound) and may occur only after administration of a series of doses thereof. A therapeutically effective amount may also vary depending on the identity of the active agent(s), the disease, condition, disorder or complication being addressed (and the severity thereof), as well as the age, weight, adsorption, distribution, metabolism and excretion of the relevant active agent in the subject. Thus, a therapeutically effective amount may need to be administered in one or more administrations to the subject. An appropriate therapeutically effective amount of a therapeutically active molecule or compound can be determined according to any one of several well-established protocols known to those ofP390200. W0.01ordinary skill in the relevant art. For example, animal studies, such as studies using mice, rats or larger mammals, can be used to determine an appropriate dose of a pharmaceutical compound. The results from such animal studies can then be extrapolated to determine doses for use in other species, such as for example, humans.

[0083] The terms “treating” or “treatment” refer to any amelioration, rehabilitation, rejuvenation, improvement, decrease or mitigation of any one or more affect, complication, decrease in normal or preexisting function or capacity, disability or disorder arising from a stroke event, ICH, and / or TBI in a subject and / or progression or exacerbation of such affect, complication, decrease in normal or preexisting function or capacity, disability or disorder, or of at least one clinical symptom thereof (e.g., stabilization of a discernible symptom), physiologically (e.g., stabilization of a physical parameter), or both, and / or inhibiting at least one physical parameter which may not be discernible to the subject. “Treating” or “treatment” as used herein also refers to the potential to prohibit a future or further stroke event, ICH, and / or TMI in the subject.

[0084] The effectiveness of treating a stroke event, ICH, and / or TBI or a symptom thereof by administering GDF11 can be measured by a Modified Rankin Scale (mRS). The mRS is 7 point global end point scale that measures range of disability (see Figure 2 Saver et al., Stroke. 2021;52:3054-3062, which is incorporated herein by reference in its entirety. The mRS is further described in Pozarowszczyk et al., Frontiers in Neurology, 10.3389 / fneur.2023.1064642, incorporated herein by reference in its entirety.

[0085] In some variations, the effectiveness of treating a stroke event, ICH, and / or TBI or a symptom thereof by administering GDF11 can be measured by a NIH Stroke Scale (NIHSS). The NIHSS measures a variety of outcomes, including motor function (arm and leg), as described in www_ninds.nih.gov / sites / default / files / 2024-05 / KnowStroke_NIHStrokeScale_May2024_508c_pdf, which is incorporated herein by reference in its entirety.

[0086] mRS and NIHSS scores are further described in Lin et al., Stroke 2018 December; 49(12): 3107-3114, Lin et al. Neurology 96(21) May 25, 2021, which is incorporated herein by reference in its entirety.

[0087] In some variations, the effectiveness of treating stroke ora symptom thereof can be measured using the Fugl-Meyer Assessment as described in www_gu_se / en / neuroscience-physiology / fugl-meyer-assessment, and www_strokengine_ca / en / assessments / fugl-meyer-assessment-of-sensorimotor-recovery-after-stroke-fma / , both of which are incorporated herein by reference in their entirety. An example of use of the Fugl-Meyer Assessment may be found at Wolf et al, Frontiers in Neurology, June 2021 Vo. 12 Article 675255, incorporated herein by reference in its entirety.P390200. W0.01

[0088] In some variations, the effectiveness of treating stroke or a symptom thereof can be measured using the Action Research Arm Test (ARAT). The ARAT may be used as described at www_sralab_org / rehabilitation-measures / action-research-arm-test or www_strokengine_ca / en / assessments / action-research-arm-test-arat / , both of which are incorporated herein in their entirety. An example of use of the ARAT may be found at Wolf et al, Frontiers in Neurology, June 2021 Vo. 12 Article 675255. In some variations, cognition can be measured as described in Cramer et al., Stroke 2023 Jan;54(1):5-9, incorporated herein by reference in its entirety. Intracranial Hemorrhage (ICH) refers to bleeding inside the skull, which can occur in different locations (e.g., subdural, epidural, subarachnoid, or intracerebral). Symptoms depend on the severity and location of the bleeding but may include: sudden severe headache (thunderclap headache) loss of consciousness or altered mental status, nausea and vomiting, seizures, weakness, numbness, paralysis (one-sided or bilateral), speech difficulties (aphasia), visual disturbances (blurry vision, double vision), unequal pupil size or non-reactive pupils, and hypertension and bradycardia (including Cushing’s reflex).

[0089] Traumatic Brain Injury (TBI) can range from mild (concussion) to severe. Symptoms may vary depending on the severity. Mild TBI (Concussion) symptoms can include headache, dizziness or loss of balance, nausea or vomiting, confusion and / or loss of memory, sensitivity to light and / or noise, and mood changes including irritability and depression). Moderate to Severe TBI symptoms can include persistent and worsening headache, repeated vomiting, slurred speech, seizures, loss of consciousness (minutes to hours), limbs weakness and / or numbness, agitation and / or profound confusion, and coma.

[0090] The effectiveness of GDF11 therapy in treating ICH and TBI can be measured by one or more measures. For example, the Glasgow Coma Scale (GCS) evaluates consciousness level (including eye, verbal, and motor response with a score ranging from 3 to 15). The NIH Stroke Scale (NIHSS) can be used to assess neurological deficits. The modified Rankin Scale (mRS) as described herein can be used to measure disability and functional independence. The Glasgow Outcome Scale (GOS) can evaluate TBI recovery outcomes (including, but not limited to, death, vegetative state, and severe disability).Functional Independence Measure (FIM) can assess daily living abilities. Post-Concussion Symptom Scale (PCSS) can track concussion symptoms overtime. Imaging & Objective Measures can include CT and / or MRI scans used to monitor hematoma size, resolution, or secondary injury (e.g., edema, herniation). Intracranial Pressure (ICP) monitoring can be used in severe cases to manage swelling. Cerebral Blood Flow (CBF) measurements can be used to assess brain perfusion recovery. Cognitive and functional evaluations can also be performed. Neuropsychological testing can be used to assess memory, attention, executive function, and processing speed. Return-to-Work and / or school assessments can be used toP390200. W0.01evaluate readiness for daily activities. Rehabilitation milestones can include improvement in speech, movement, and / or independent living skills.

[0091] For global outcome tracking of ICH or TBI, GCS, GOS-E, mRS, or NIHSS can be used. For motor function WMFT, Box & Block Test, or 9-Hole Peg Test can be used. For cognitive function, MoCA, TMT, or RBANS can be used. For daily function & rehab planning the Barthel Index and FIM can be used purely motor-focused tools. For spasticity & tonethe modified Ashworth Scale or Tardieu Scale can be used.

[0092] In the practice of the methods of the disclosure, treating a stroke event, ICH, and / or TBI entails administering a therapeutically effective amount of a GDF11 molecule (both as defined herein) to a subject using a dosing regimen. The dose can be any dose described herein, including, for example, a moderate dose, a minimal high dose, or a high dose. As discussed herein above, such techniques are typically based on physical and neurological (behavioral) examination (such as the NIHSS) to assess improved body motor function and / or cognitive function in the subject, and can be supported by medical imaging techniques such as CT scan, MRI scan (e.g., spin-echo MRI or cine magnetic resonance), Doppler ultrasound and arteriography and often supported by ancillary tests such as electrocardiogram (ECG) and blood tests. Using such techniques, the ordinarily skilled person can assess successful stroke, ICH, and / or TBI treatment in a subject by way of visualizing neovascularization, neurogenesis, improved cerebrovascular structure, and / or function or blood flow at or near the site of stroke, ICH, and / or TBI in a subject. Here again, for clarity, successful stroke, ICH, and / or TBI therapy using the methods of the disclosure can be established by assessing any one or more (and any combination thereof) of the above-noted criteria and / or by employing any one or more of the above-noted diagnostic and imaging techniques.

[0093] “Percent (%) amino acid sequence identity” with respect to a protein sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the specific (parental) sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared. One particular program is the ALIGN-2 program outlined at paragraphs

[0279] to

[0280] of U. S. Pub. No. 20160244525, hereby incorporated by reference.P390200. W0.01

[0094] Sequence identity between two similar sequences can be measured by algorithms such as that of Smith, T. F. & Waterman, M. S. (1981) “Comparison Of Biosequences,” Adv. Appl. Math. 2:482 [local homology algorithm]; Needleman, S. B. & Wunsch, C D. (1970) “A General Method Applicable To The Search For Similarities In The Amino Acid Sequence Of Two Proteins,” J. Mol. Biol. 48:443 [homology alignment algorithm], Pearson, W. R. & Lipman, D. J. (1988) “Improved Tools For Biological Sequence Comparison,” Proc. Natl. Acad. Sci. (U. S. A.) 85:2444 [search for similarity method]; or Altschul, S. F. et al, (1990) “Basic Local Alignment Search Tool,” J. Mol. Biol. 215:403-10, the “BLAST” algorithm, see blast. ncbi.nlm.nih_gov / Blast.cgi. When using any of the aforementioned algorithms, the default parameters (for Window length, gap penalty, etc.) are used. In one embodiment, sequence identity is done using the BLAST algorithm, using default parameter.

[0095] The degree of identity between an amino acid sequence of the disclosure (“disclosure sequence”) and the parental amino acid sequence is calculated as the number of exact matches in an alignment of the two sequences, divided by the length of the “disclosure sequence," or the length of the parental sequence, whichever is the shortest. The result is expressed in percent identity. In some embodiments, two or more amino acid sequences are at least 50%, 60%, 70%, 80%, or 90% identical. In some embodiments, two or more amino acid sequences are at least 95%, 97%, 98%, 99%, or even 100% identical.

[0096] “Therapeutically effective amount” corresponds to a dose that produces the effects for which it is administered. The does may cure, alleviate, or partially arrest the clinical manifestations of a given disease or disorder and its complications. The exact dose will depend on the purpose of the treatment as well as the weight and general state of the human subject, and will be ascertainable by one skilled in the art using known techniques. It will be understood that determination of an appropriate dosage may be achieved, using routine experimentation, by constructing a matrix of values and testing different points in the matrix, all of which is within the ordinary skills of a trained physician or clinical scientist.

[0097] It should be understood that this disclosure is not limited to the particular methodology, protocols, and reagents, etc., described herein and as such can vary. The terminology used herein is for the purpose of describing particular aspects only and is not intended to limit the scope of the disclosure, which is defined solely by the claims.

[0098] Administration of the GDF11 molecule can be systemic and / or local and is carried out via any suitable parenteral administration technique.GDF11 moleculesP390200. W0.01

[0099] The GDF11 molecule can be any therapeutically active form of a GDF11 molecule that can be the same or different in the compositions employed over the course of the dosing regimen(s) disclosed herein.

[0100] In some variations, GDF11 is the human form of a GDF11 polypeptide, whether natively purified GDF11 or recombinant GDF11.

[0101] In certain variations, the subject can be administered a modified GDF11 molecule comprising a second polypeptide moiety selected from Collectin kidney 1 (e.g. NCBI Gene ID No: 78989), Cathespin D (e.g. NCBI Gene ID No: 1509), Dickkopf-r elated protein 4 (e.g. NCBI Gene ID No: 27121), Erythrocyte membrane protein 4.1 (e.g. NCBI Gene ID No: 2035), esterase D (e.g. NCBI Gene ID No: 2098), hemoglobin (e.g. NCBI Gene ID No: 3043 or 3047), interleukin-1 receptor accessory protein (e.g. NCBI Gene ID No: 3556), natural killer group 2 member D (e.g. NCBI Gene ID No: 22914), Ras-related C3 botulinum toxin substrate 1 (e.g. NCBI Gene ID No: 5879), GTP -binding nuclear protein Ran (e.g. NCBI Gene ID No: 5901), tissue inhibitor of metalloproteases 3 (e.g. NCBI Gene ID No: 7078), or thymidylate synthase (e.g. NCBI Gene ID No: 7298).

[0102] In some variations, the GDF11 molecule can be a polypeptide obtained by mutations of native nucleotide sequences. DNA sequences encoding polypeptide molecules encompass sequences that comprise one or more additions, deletions, or substitutions of nucleotides when compared to a native or reference DNA sequence but encode a variant or derivative protein or polypeptide (or fragment thereof) that retains the relevant biological activity relative to the reference protein.

[0103] The variant or derivative amino acid sequence can be at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more, identical to a native or reference sequence. Any one of the percent identities described herein can be elected. The degree of homology (percent identity) between a native and a mutant sequence can be determined, for example, by comparing the two sequences using freely available computer programs commonly employed for this purpose on the world wide web. The variant amino acid can be at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more, similar to the sequence from which it is derived (referred to herein as an “original” sequence). The degree of similarity (percent similarity) between an original and a mutant sequence can be determined, for example, by using a similarity matrix. Similarity matrices are well known in the art and a number of tools for comparing two sequences using similarity matrices are freely available online, e.g. BLAST (available on the world wide web atP390200. W0.01blast. ncbi.nlm.nih_gov), with default parameters set. It is noted that the mature GDF11 polypeptide includes likely intrachain disulfide bonds between, e.g., amino acid 313 and 372; 341 and 404; and 345 and 406 (numbered relative to the full length polypeptide, including the signal sequence) and that amino acid 371 likely participates in interchain disulfide bonding.

[0104] A given amino acid can be replaced by a residue having similar physiochemical characteristics, e.g., substituting one aliphatic residue for another (such as lie, Vai, Leu, or Ala for one another), or substitution of one polar residue for another (such as between Lys and Arg; Glu and Asp; or Gin and Asn). Other such conservative substitutions, e.g., substitutions of entire regions having similar hydrophobicity characteristics, are well known. Polypeptides comprising conservative amino acid substitutions can be tested in any one of the assays described herein to confirm that a desired apoptotic activity of a native or reference polypeptide is retained. Conservative substitution tables providing functionally similar amino acids are well known in the art. Such conservatively modified variants are in addition to and do not exclude polymorphic variants, interspecies homologs, and alleles consistent with the disclosure. Typical conservative substitutions for one another include: 1) Alanine (A), Glycine (G); 2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine (N), Glutamine (Q); 4) Arginine (R), Lysine (K); and 5) Isoleucine (I), Leucine (L), Methionine (M).

[0105] Any cysteine residue not involved in maintaining the proper conformation of the polypeptide also can be substituted, generally with serine, to improve the oxidative stability of the molecule and prevent aberrant crosslinking. Conversely, cysteine bond(s) can be added to the polypeptide to improve its stability or facilitate oligomerization.

[0106] The GDF11 polypeptide molecule administered to the subject can also comprise one or more amino acid substitutions, modifications or additions. For example, substitutions and / or modifications or additions can be used to prevent or reduce proteolytic degradation and / or prolong half-life of the GDF11 molecule in the subject.

[0107] Suitable GDF11 polypeptide molecules for use herein can be synthesized by using well known methods including recombinant methods and chemical synthesis.

[0108] Recombinant methods of producing a peptide through the introduction of a vector including nucleic acid encoding the peptide into a suitable host cell are well known in the art, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual, 2d Ed, Vols 1 to 8, Cold Spring Harbor, N. Y. (1989); M. W. Pennington and B. M. Dunn, Methods in Molecular Biology Peptide Synthesis Protocols, Vol 35, Humana Press, Totawa, N. J. (1994).P390200. W0.01Suitable polypeptides can also be chemically synthesized using methods well known in the art (see, e.g., Merrifield et al. (1964) J Am. Chem. Soc. 85: 2149; Bodanszky, M. (1984) Principles of Peptide Synthesis, Springer-Verlag, New York, N. Y.; Kimmerlin et al. (2005) Pept. Res. 65: 229-260; Nilsson et al. (2005) Annu. Rev. Biophys. Biomol. Struct. 34: 91-118; W. C. Chan and P. D. White (Eds.) Fmoc Solid Phase Peptide Synthesis: A Practical Approach, Oxford University Press, Cary, N. C. (2000); N. L. Benoiton, Chemistry of Peptide Synthesis, CRC Press, Boca Raton, Fla. (2005); J. Jones, Amino Acid and Peptide Synthesis, 2. sup. nd Ed, Oxford University Press, Cary, N. C. (2002); and P. Lloyd-Williams, F. AIbericio, and E. Giralt, Chemical Approaches to the Synthesis of Peptides and Proteins, CRC Press, Boca Raton, Fla. (1997). Peptide derivatives can also be prepared as described in U. S. Pat. Nos. 4,612,302; 4,853,371 and 4,684,620; and in U. S. Pat. App. Pub. No.2009 / 0263843. In various aspects, GDF11 can be prepared as described in, for example, in Pepinsky et al,Biochemistry 56(33) 2017, or Walker et al., BMC Biology 15(19) 2017, both of which are incorporated by reference herein in their entirety.

[0109] Alterations of the original amino acid sequence of a GDF11 molecule can be accomplished by any of a number of techniques known to one of skill in the art. Mutations can be introduced, for example, at particular loci by synthesizing oligonucleotides containing a mutant sequence, flanked by restriction sites permitting ligation to fragments of the native sequence. Following ligation, the resulting reconstructed sequence encodes an analog having the desired amino acid insertion, substitution, or deletion. Alternatively, oligonucleotide-directed site-specific mutagenesis procedures can be employed to provide an altered nucleotide sequence having particular codons altered according to the substitution, deletion, or insertion required. Techniques for making such alterations include those disclosed in US Patent No. 9,434,779, incorporated herein by reference in its entirety.Pharmaceutical Compositions

[0110] In some embodiments, GDF11 is administered in a pharmaceutical composition. As used herein, the term "pharmaceutical composition” refers to the active agent in combination with a pharmaceutically acceptable carrier commonly used in the pharmaceutical industry. The phrase “pharmaceutically acceptable” is employed herein to refer to those compounds, materials, compositions, and / or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio.P390200. W0.01

[0111] As discussed herein above, routes of administration of pharmaceutical compositions are parenteral, e.g., via intravenous, intramuscular, intraperitoneal, intradermal or subcutaneous injection. Solutions or suspensions used for such parenteral application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. The pH of a composition can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. Parenteral preparations can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.

[0112] Pharmaceutical compositions suitable for injection include sterile aqueous solutions (where water soluble) or dispersions, emulsions or suspensions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable carriers comprise physiological saline, bacteriostatic water, Cremophor EL. TM. (BASF, Parsippany, N. J.) or phosphate buffered saline (PBS). In all cases, the composition must be sterile and should be fluid to the extent that easy syringability exists. It should be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier or vehicle can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyetheylene glycol, and the like), and suitable mixtures thereof. The proper fluidity of a composition can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the selected particle size in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanoi, phenol, ascorbic acid, thimerosal, and the like. In some cases, isotonic agents are included in the composition, for example, sugars, polyalcohols such as manitol, sorbitol, or sodium chloride. Prolonged absorption of an injectable composition can be achieved by including in the composition an excipient that delays absorption, for example, aluminum monostearate or gelatin.

[0113] Sterile injectable solutions can be prepared by incorporating the GDF11 molecule in a specified amount in an appropriate solvent with one or a combination of ingredients enumerated above, as needed, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the GDF 11 molecule into a sterile vehicle that contains a basic dispersion medium and other ingredients selected from those enumerated above or othersP390200. W0.01known in the art. In the case of sterile powders for the preparation of sterile injectable solutions, the methods of preparation include vacuum drying and freeze-drying which yields a powder of the GDF11 molecule plus any additional desired ingredient from a previously sterile-filtered solution thereof.

[0114] In various aspects, for intravenous administration, the pharmaceutical composition can be between pH 5.0 and 5.5. For i.p. administration, the pharmaceutical composition can be between pH 3.0 and 5.5. The one variation, the pharmaceutical composition comprises sodium acetate and sorbitol, mannitol, or a combination of both. The sodium acetate can be at least 5 mM. The sodium acetate can be less than 50 mM. In some variations, the sodium acetate can be less than 10 mM. In some variations, the 10 mM Sodium acetate and 5 wt% Sorbitol pH5. In one variation, the sorbitol and / or mannitol is at least 1wt%. In a variation, the sorbitol and / or mannitol is less than or equal to 10 wt%. In a more particular variation, the pharmaceutical composition is 10mM sodium acetate, 5 wt% sorbitol, and pH5. In a more particular variation, the pharmaceutical composition is 25mM sodium acetate, 6% mannitol, and pH 5. In various aspects, the pharmaceutical composition can range from 8 - 10 wt% trehalose.

[0115] In other aspects of the disclosure, a single dose can be administered at one time or divided into multiple times on any suitable basis, such as on a once daily (QD) basis, twice daily (BID), three times daily (TID), four times daily (QID), hourly (“q_h” where “h” denotes the number of hours between doses), or the like, and each day of treatment can be the same or different over the course of treatment. In other aspects of the disclosure, treatment can be carried out once, or can entail any number of treatment regimens suitable for the specific treatment being contemplated.

[0116] In certain aspects, the dosing regimen entails classical titration of the GDF11 molecule in either ascending or descending doses, for example wherein the first administration is carried out at an initial dose of at least the minimal high dose of GDF11 on day 1 of the treatment period and finishes at a second, higher dose, with any number of different intervening doses carried out between such first and second doses. Alternatively, titration of the GDF11 molecule can entail an initial (day one) high dose of the GDF11 molecule and ending with a final dose of at least the minimal high dose of GDF 11, again with any number of different intervening doses carried out between such initial and final doses. In any titration strategy, it may be preferred to administer the GDF11 molecule at a first high dose approaching the median toxic dose (MTD) for that molecule, or at least approaching the maximum dose of the therapeutic window for the administered GDF11 molecule, followed by a subsequent dose (or doses) at lower level.P390200. W0.01

[0117] In some variations, the initial dose of GDF11 can be administered at or 24 hours after a stroke event, ICH, and / or TBI. The delay allows subjects baseline functions to be stabilized and monitored.Intermittent Dosing

[0118] In some variations, the GDF11 can be administered on intermittent days. In one variation, GDF11 is administered every second day (i.e., once every 48 hours + / - 8 hours). In another variation, GDF11 is administered every third day (i.e., once every 72 hours + / - 8 hours). In another variation, GDF11 is administered every fourth day (i.e., once every 96 hours + / - 8 hours).

[0119] In some variations, the intermittent dosing can be once every two days, once every three days, or once every four days, in any combination. For example, a dose can be administered, followed by a day without dosing, followed by a day of dosing, followed by a day without dosing, followed by day of dosing (using the shorthand dose - one day rest -dose - one day rest - dose). In some variations, the intermittent dosing is dose - one day rest - dose -two days rest - dose. In some variations, the intermittent dosing is dose - two days rest - dose - one day rest - dose. In some variations, the intermittent dosing is dose -two days rest - dose - two days rest - dose. In some variations, the intermittent dosing is dose - one day rest - dose - three days rest - dose. In some variations, the intermittent dosing is dose - three days rest - dose - one day rest - dose. In some variations, the intermittent dosing is dose - two days rest - dose - three days rest - dose. In some variations, the intermittent dosing is dose - three days rest - dose - three days rest - dose.

[0120] The days of rest can be + / - 8 hours, alternatively + / - 4 hours, alternatively + / - 2 hours measured from the 24 hours elapsed in a day. By way of example and not limitation, in a 2 -2-2 day dosing regimen, a first intermittent dose is administered 24 hours (beginning on the second day) + / - 8 hours after the stroke event, ICH, and / or TBI. A second dose is administered 72 hours (3 days, or +2 days) + / - 8 hours after the stroke event, ICH, and / or TBI. A third intermittent dose is administered 120 hours (3 days, or an additional +2 days) + / - 8 hours after a stroke event, ICH, and / or TBI.

[0121] In some variations, administration can be ceased at 5 days. In some variations, administration can be ceased at 6 days. In some variations, administration can be ceased at 7 days. In some variations, administration can be ceased at 8 days. In some variations, administration can be ceased at 5 days. In some variations, administration can be ceased at 9 days. In some variations, administration can be ceased at 10 days. In some variations, administration can be ceased at 11 days. In some variations, administration can be ceased at 12 days.Second GDF11 Dosing RegimenP390200. W0.01

[0122] In additional variations, a second GDF11 dosing regimen can be administered. The dosing regimen can be any regimen disclosed herein. In some variations, the second dosing regimen can be an intermittent dosing regimen, as described herein.

[0123] The second dosing regimen differs from a so-called “drug holiday” in that it is not a structured treatment interruption. Rather, the second dosing regimen provides a sufficiently long break between cessation of the first treatment regimen and second treatment regimen that it can be considered a new treatment.

[0124] In some variations, the second dosing regimen begins at least four weeks following the stroke event, ICH, and / or TBI. In some variations, the second dosing regimen begins at least six weeks following the stroke event, ICH, and / or TBI. In some variations, the second dosing regimen begins at least eight weeks following the stroke event, ICH, and / or TBI. In some variations, the second dosing regimen begins at least ten weeks following the stroke event, ICH, and / or TBI. In some variations, the second dosing regimen begins at least twelve weeks following the stroke event, ICH, and / or TBI.

[0125] In some variations, the second dosing regimen begins at least 3 months following the stroke event, ICH, and / or TBI. In some variations, the second dosing regimen begins at least 4 months following the stroke event, ICH, and / or TBI. In some variations, the second dosing regimen begins at least 5 months following the stroke event, ICH, and / or TBI. In some variations, the second dosing regimen begins at least 6 months following the stroke event, ICH, and / or TBI. In some variations, the second dosing regimen begins at least 7 months following the stroke event, ICH, and / or TBI. In some variations, the second dosing regimen begins at least 8 months following the stroke event, ICH, and / or TBI. In some variations, the second dosing regimen begins at least 9 months following the stroke event, ICH, and / or TBI. In some variations, the second dosing regimen begins at least 10 months following the stroke event, ICH, and / or TBI. In some variations, the second dosing regimen begins at least 11 months following the stroke event, ICH, and / or TBI. In some variations, the second dosing regimen begins at least 12 months following the stroke event, ICH, and / or TBI.

[0126] Alternatively, in some variations, the second dosing regimen begins at least three weeks following the final dose of the first dosing regimen. In some variations, the second dosing regimen begins at least four weeks following the final dose of the first dosing regimen. In some variations, the second dosing regimen begins at least six weeks following the final dose of the first dosing regimen. In some variations, the second dosing regimen begins at least eight weeks following the final dose of the first dosing regimen. In some variations, the second dosing regimen begins at least ten weeks following the final dose of the first dosing regimen. In some variations, the second dosing regimen begins at least twelve weeks following the final dose of the first dosing regimen.P390200. W0.01

[0127] In some variations, the second dosing regimen begins at least 3 months following the final dose of the first dosing regimen. In some variations, the second dosing regimen begins at least 4 months following the final dose of the first dosing regimen. In some variations, the second dosing regimen begins at least 5 months following the final dose of the first dosing regimen. In some variations, the second dosing regimen begins at least 6 months following the final dose of the first dosing regimen. In some variations, the second dosing regimen begins at least 7 months following the final dose of the first dosing regimen. In some variations, the second dosing regimen begins at least 8 months following the final dose of the first dosing regimen. In some variations, the second dosing regimen begins at least 9 months following the final dose of the first dosing regimen. In some variations, the second dosing regimen begins at least 10 months following the final dose of the first dosing regimen. In some variations, the second dosing regimen begins at least 11 months following the final dose of the first dosing regimen. In some variations, the second dosing regimen begins at least 12 months following the final dose of the first dosing regimen.Biomarkers and Identification Thereof

[0128] The disclosure is directed to a method of improving therapeutic efficacy for treatment of stroke event, ICH, and / or TBI. A different abundance of one or more biomarkers as described herein GDF11 compared to a control or reference value is predictive of effective treatment with GDF 11.

[0129] Biomarkers having reduced or increased abundance can be described by SomaScan Aptamer Sequences ID, protein name, UniProt ID, or Gene Symbol as described in Table 1. Any one or more biomarker can be used, in any combination. SomaScan can be found at www_menu.somalogic_com / ; Uniprot can be found at Uniprot.org; Entrez can be found at NCBI or gene GeneCards.org, all of which are incorporated by reference in their entirety.

[0130] In some variations, the biomarker is a SomaScan Aptamer sequence having decreased abundance selected from 18922-27, 4337-49, 25306-51, 10025-1, 10801-11, 14124-6, 22580-29, 2615-60, 5658-64, 2443-10, 19570-12, 17166-4, 14757-144, 5581-28, 2841-13, 5755-29, 10426-21, 13547-5, 13738-8, 19622-7, 2748-3, 5193-51, 5242-37, 7655-11, 3723-1, 9380-2, 21380-77, 20546-71, 9099-19, and 9970-7.

[0131] In some variations, the biomarker is a SomaScan Aptamer sequence having increased abundance selected from 19197-95, 9843-5, 9844-138, 15312-14, 15323-112, 15627-83, 2867-52, 23302-19, 5360-9, 14685-17, 15523-9, 18185-118, 2811-27, 2602-2, 13660-76, 13621-31, 12583-77, 14748-31, 13573-5, 5870-23, 24237-115, 11130-158, 3351-1, 9840-2, 2819-23, 7959-34, 4203-50, 14029-42, 15321-8, 4976-57, 15527-90, 20913-27, 25105-87, 19259-176, 24723-58, 18829-4, 22984-10, 18824-7, 25287-7, 4230-1, 6036-78, 8039-41, 4393-3, 4130-71, 4487-1, 4394-71, 4989-7, 13384-110, 11171-25, 11245-43,P390200. W0.0113122-19, 7713-102, 7713-50, 13740-51, 14587-16, 2765-4, 18233-10, 23903-3, 5464-52, 3236-12, 24050-26, 18900-37, 3317-33, 9816-37, 11816-84, 20073-22, 3453-87, 3381-24, 2864-2, 22041-26, 3115-64, 5007-1, 2855-49, 15604-18, 9760-13, 21768-9, 21813-171, 23693-4, 14036-116, 19373-3, 19122-47, 20105-7, 21817-5, 13636-20, 19188-21, 4292-5, 20584-4, 9869-28, 8778-3, 5846-24, 5542-22, 3214-3, 6590-54, 15387-44, 8295-16, 19377-14, 13719-19, 25249-33, 25947-116, 21430-4, 11354-21, 24909-40, 25949-3, 10070-22, 15545-13, 3466-8, 21483-155, 12479-50, 25463-3, 2644-11, 5475-10, 3379-29, 9504-19, 17516-7, 23329-52, 17205-21, 19222-124, 14287-6, 18950-13, 16769-20, 14332-3, 22547-17, 18373-13, 21713-11, 17764-108, 12540-25, 21752-10, 22578-17, 5116-62, 3469-74, 3868-8, 13242-134, 16043-30, 5272-55, 19630-2, 13707-27, 4467-49, 15433-4, 5488-74, 12351-25, 10346-5, 10354-57, 19553-14, 18308-30, 17355-56, 21539-139, 3339-33, 14111-15, 14082-56, 5440-26, 24320-3, 9997-12, 10756-34, 5275-28, 9830-109, 7952-2, 3050-7, 25451-39, and 4560-34P390200. W0.01

[0132] In some variations, the biomarker is a SomaScan Aptamer sequence having increased abundance selected from 19197-95, 9843-5, 9844-138, 15312-14, 15323-112, 15627-83, 2867-52, 23302-19, 5360-9, 14685-17, 15523-9, 18185-118, 2811-27, 2602-2, 13660-76, 13621-31, 12583-77, 14748-31, 13573-5, 5870-23, 24237-115, 11130-158, 3351-1, 9840-2, 2819-23, 7959-34, 4203-50, 14029-42, 15321-8, 4976-57, 3516-60, 15527-90, 20913-27, 25105-87, 19259-176, 24723-58, 18829-4, 22984-10, 18824-7, 25287-7, 4230-1, 6036-78, 8039-41, 4393-3, 4130-71, 4487-1, 4394-71, 4989-7, 13384-110, 11171-25, 11245-43, 13122-19, 7713-102, 7713-50, 13740-51, 14587-16, 2765-4, 18233-10, 23903-3, 5464-52, 3236-12, 24050-26, 18900-37, 3317-33, 9816-37, 11816-84, 20073-22, 3453-87, 3381-24, 2864-2, 22041-26, 3115-64, 5007-1, 2855-49, 15604-18, 9760-13, 21768-9, 21813-171, 23693-4, 14036-116, 19373-3, 19122-47, 20105-7, 21817-5, 13636-20, 19188-21, 4292-5, 20584-4, 9869-28, 8778-3, 5846-24, 5542-22, 3214-3, 6590-54, 15387-44, 8295-16, 19377-14, 13719-19, 25249-33, 25947-116, 21430-4, 11354-21, 24909-40, 25949- 3, 10070-22, 15545-13, 3466-8, 21483-155, 12479-50, 25463-3, 2644-11, 5475-10, 3379-29, 9504-19, 17516-7, 23329-52, 17205-21, 19222-124, 14287-6, 18950-13, 16769-20, 14332-3, 22547-17, 18373-13, 21713-11, 17764-108, 12540-25, 21752-10, 22578-17, 5116-62, 3469-74, 3868-8, 13242-134, 16043-30, 5272-55, 19630-2, 13707-27, 4467-49, 15433- 4, 5488-74, 12351-25, 10346-5, 10354-57, 19553-14, 18308-30, 17355-56, 21539-139, 3339-33, 14111-15, 14082-56, 5440-26, 24320-3, 9997-12, 10756-34, 5275-28, 9830-109, 7952-2, 3050-7, 25451-39, and 4560-34

[0133] In some variations, the biomarker is a protein having decreased abundance of a protein selected from Macrosialin, C-reactive protein, DAXX, Dihydrolipoyl dehydrogenase (mitochondrial), Ephrin-A2, Ephrin-A2, Ephrin-A2, Ephrin-A5, Coagulation factor XIII B chain, Fibroblast growth factor 8 isoform B, Fibroblast growth factor 8, Fibroblast growth factor 8 isoform F, Fibroblast growth factor 8 isoform B, Fibrinogen-like protein 1, Secreted frizzled-related protein 3, Gastric inhibitory polypeptide, Golgi SNAP receptor complex member 2, 5-hydroxytryptamine receptor 7, Inhibin beta A chain, Activin A, Activin A, GTPase KRas, Dual specificity mitogen-activated protein kinase kinase 4, N-terminal pro-BNP, Brain natriuretic peptide 32, Group XI IB secretory phospholipase A2-like protein, Queuine tRNA-ribosyltransferase, Reticulon-4 receptor-like 1, Synaptotagmin-5, and UBX domaincontaining protein 4: Cytoplasmic domain 2.

[0134] In some variations, the biomarker is a protein having increased abundance of a protein selected from Acetyl-CoA acetyltransferase (mitochondrial), Alpha-actinin-1, Alpha-actinin-2, Protein argonaute-1, Protein argonaute-3, RAC-alpha serine / threonine-protein kinase, RAC-alpha serine / threonine-protein kinase, Proline-rich AKT1 substrate 1, RAC-beta serine / threonine-protein kinase, RAC-beta serine / threonine-protein kinase, Delta-aminolevulinic acid dehydratase, Fructose-bisphosphate aldolase B, Angiopoietin-1,P390200. W0.01Angiopoietin-2, Angiopoietin-2, AP-2 complex subunit alpha-2, Serine / threonine-protein kinase A-Raf, Rho GTPase-activating protein 5, Actin-related protein 2 / 3 complex subunit 3, Bcl2-associated agonist of cell death, Voltage-dependent L-type calcium channel subunit beta-3, Voltage-dependent L-type calcium channel subunit beta-4, Calcium / calmodulin-dependent protein kinase type II subunit beta, Cell division control protein 42 homolog, Cadherin-5, Cadherin-7, Cofilin-1, COP9 signalosome complex subunit 2, Complexin-2, Adapter molecule crk, Dihydrolipoyl dehydrogenase (mitochondrial), Eukaryotic translation initiation factor 1, Eukaryotic translation initiation factor 2A, Eukaryotic translation initiation factor 2 subunit 1, Eukaryotic translation initiation factor 2 subunit 2, Eukaryotic initiation factor 4A-I, Eukaryotic initiation factor 4A-II, Eukaryotic initiation factor 4A-II, Eukaryotic translation initiation factor 4 gamma 1, Eukaryotic translation initiation factor 4 gamma 2, Ephrin type-A receptor 10, Protein FAM177A1, Fibroblast growth factor 16, Fibroblast growth factor 6, Fibroblast growth factor 7, Fibroblast growth factor 8 isoform A, Fibrinogen gamma chain, Fumarate hydratase (mitochondrial), Filamin-A: Calponin Homology 2, Filamin-A: Calponin Homology 1, Leucine-rich repeat transmembrane protein FLRT2, Fragile X mental retardation protein 1, Fragile X mental retardation protein 1, Secreted frizzled-related protein 3, Growth / differentiation factor 11, Growth / differentiation factor 11 / 8, Aspartate aminotransferase (mitochondrial), Aspartate aminotransferase (mitochondrial), Growth factor receptor-bound protein 2, Glycogen synthase kinase-3 beta, Glycogen synthase kinase-3 beta, GTPase HRas, Serine protease HTRA2 (mitochondrial), Isochorismatase domaincontaining protein 1, Tyrosine-protein kinase JAK2, GTPase KRas, Tyrosine-protein kinase Lyn, Tyrosine-protein kinase Lyn (isoform B), Dual specificity mitogen-activated protein kinase kinase 1, Dual specificity mitogen-activated protein kinase kinase 5, Mitogen-activated protein kinase 1, Mitogen-activated protein kinase 14, Mitogen-activated protein kinase 3, Mitogen-activated protein kinase 9, Mitogen-activated protein kinase 9, Alpha-1, 6-mannosylglycoprotein 6-beta-N-acetylglucosaminyltransferase A, Alpha-1,6-mannosylglycoprotein 6-beta-N-acetylglucosaminyltransferase A, MAP kinase-interacting serine / threonine-protein kinase 1, Multiple PDZ domain protein, Myosin regulatory light chain 12A, Myosin regulatory light chain 12B, Myosin light polypeptide 6, Myosin (light chain 9-regulatory), Nucleosome assembly protein 1 -like 1, nucleosome assembly protein 1-like 4, Alpha-soluble NSF attachment protein, Nectin-1 (isoform gamma: Extracellular domain), Nuclear factor NF-kappa-B p105 subunit, Noggin, Noggin, Neuropilin-1, Neuropilin-1, Neuropilin-2, Neuropilin-2, Noelin-2, Noelin-2, Serine / threonine-protein kinase PAK 4, Pyruvate dehydrogenase E1 component subunit alpha (testis-specific form (mitochondrial)), Phosphatidylinositol 3-kinase catalytic subunit type 3, Cytosolic phospholipase A2 alpha, 1-phosphatidylinositol 4,5-bisphosphate phosphodiesterase beta-1, 1 -phosphatidylinositol-4,5-bisphosphate phosphodiesterase delta-1, 1 -phosphatidylinositol 4,5-bisphosphateP390200. W0.01phosphodiesterase delta-3, 1 -phosphatidylinositol 4,5-bisphosphate phosphodiesterase gamma-2, Calcineurin subunit B type 1, cAMP-dependent protein kinase catalytic subunit alpha, cAMP-dependent protein kinase type l-alpha regulatory subunit, cAMP-dependent protein kinase type l-beta regulatory subunit, cAMP-dependent protein kinase type ll-beta regulatory subunit, Protein kinase C alpha type, Protein kinase C beta type (splice variant beta-ll), Protein kinase C iota type, Ras-related protein Rab-27A, Ras-related protein Rab-3A, Ras-related protein Rab-4B, Ras-related protein Rab-5A, Ras-related protein Rab-5B, Ras-related protein Rab-5C, Ras-related C3 botulinum toxin substrate 2, Ras-related C3 botulinum toxin substrate 3, Ras-related protein Ral-A, RNA-binding motif (single-stranded-interacting protein 1), Radixin, Reelin, Rho-related GTP-binding protein RhoC, Rho-related GTP-binding protein RhoG, Mitochondrial Rho GTPase 1, Roundabout homolog 2, Roundabout homolog 2, Ribosomal protein S6 kinase alpha-3, Small glutamine-rich tetratricopeptide repeat-containing protein alpha, SH3 and multiple ankyrin repeat domains protein 3, SHC-transforming protein 1 Phosphotyrosine Interaction Domain, SHC-transforming protein 1: Src Homology domain, Gamma-synuclein, SPARC-like protein 1, SPARC-like protein 1, Proto-oncogene tyrosine-protein kinase Src, Proto-oncogene tyrosineprotein kinase Src isoform 2, Signal transducer and activator of transcription 1-alpha / beta, Signal transducer and activator of transcription 3, Signal transducer and activator of transcription 3, Syntaxin- 1 A, Syntaxin-binding protein 6, Synaptotagmin-4, Synaptotagmin-6, Thrombospondin-2, Thrombospondin-2, Talin-2, Troponin I (fast skeletal muscle), Tripartite motif-containing protein 72, UBX domain-containing protein 4: Cytoplasmic domain 1, Urocortin-3, Proto-oncogene vav, Guanine nucleotide exchange factor VAV3, Vesicle transport through interaction with t-SNAREs homolog 1A, von Willebrand factor, Transcriptional coactivator YAP 1, and Tyrosine-protein kinase Lck.

[0135] In some variations, the biomarker has decreased abundance of a Uniprot ID selected from P34810, P02741, Q9UER7, P09622, 043921, 043921, 043921, P52803, P05160, P55075, P55075, P55075, P55075, Q08830, Q92765, P09681, O14653, P34969, P08476, P08476, P08476, P01116, P45985, P16860, P16860, Q9BX93, Q9BXR0, Q86UN2, O00445, Q92575

[0136] In some variations, the biomarker has increased abundance of a Uniprot ID selected from P24752, P12814, P35609, Q9UL18, Q9H9G7, P31749, P31749, Q96B36, P31751, P31751, P13716, P05062, Q15389, 015123, 015123, O94973, P10398, Q13017, O15145, Q92934, P54284, O00305, Q13554, P60953, P33151, Q9ULB5, P23528, P61201, Q6PUV4, P46108, P09622, P41567, Q9BY44, P05198, P20042, P60842, Q14240, Q14240, Q04637, P78344, Q5JZY3, Q8N128, 043320, P10767, P21781, P55075, P02679, P07954, P21333, P21333, 043155, Q06787, Q06787, Q92765, 095390, O95390|O14793, P00505, P00505, P62993, P49841, P49841, P01112, 043464, Q96CN7, 060674, P01116, P07948, P07948,P390200. W0.01Q02750, Q13163, P28482, Q16539, P27361, P45984, P45984, Q09328, Q09328, Q9BUB5, 075970, P19105, 014950, P60660, P24844, P55209, Q99733, P54920, Q15223, P19838, Q13253, Q13253, 014786, 014786, 060462, 060462, 095897, 095897, 096013, P29803, Q8NEB9, P47712, Q9NQ66, P51178, Q8N3E9, P16885, P63098, P17612, P10644, P31321, P31323, P17252, P05771, P41743, P51159, P20336, P61018, P20339, P61020, P51148, P15153, P60763, P11233, P29558, P35241, P78509, P08134, P84095, Q8IXI2, Q9HCK4, Q9HCK4, P51812, 043765, Q9BYB0, P29353, P29353, 076070, Q14515, Q14515, P12931, P12931, P42224, P40763, P40763, Q16623, Q8NFX7, Q9H2B2, Q5T7P8, P35442, P35442, Q9Y4G6, P48788, Q6ZMU5, Q92575, Q969E3, P15498, Q9UKW4, Q96AJ9, P04275, P46937, P06239

[0137] In some variations, the biomarker has decreased abundance of a protein selected from CD68, CRP, DAXX, DLD, EFNA2, EFNA2, EFNA2, EFNA5, F13B, FGF8, FGF8, FGF8, FGF8, FGL1, FRZB, GIP, GOSR2, HTR7, INHBA, INHBA, INHBA, KRAS, MAP2K4, NPPB, NPPB, PLA2G12B, QTRT1, RTN4RL1, SYT5, and UBXN4.

[0138] In some variations, the biomarker has decreased abundance of a protein selected from HBS2, NRP1, UCN3, SPARCL1, FAM177a1, ANGPT1, and NOG. Alternatively, the corresponding SomaLogic aptamer or Uniprot ID may be used.

[0139] In some variations, the biomarker has increased abundance, and a gene or protein symbol selected from ACAT1, ACTN1, ACTN2, AGO1, AG03, AKT1, AKT1, AKT1S1, AKT2, AKT2, ALAD, ALDOB, ANGPT1, ANGPT2, ANGPT2, AP2A2, ARAF, ARHGAP5, ARPC3, BAD, CACNB3, CACNB4, CAMK2B, CDC42, CDH5, CDH7, CFL1, COPS2, CPLX2, CRK, DLD, EIF1, EIF2A, EIF2S1, EIF2S2, EIF4A1, EIF4A2, EIF4A2, EIF4G1, EIF4G2, EPHA10, FAM177A1, FGF16, FGF6, FGF7, FGF8, FGG, FH, FLNA, FLNA, FLRT2, FMR1, FMR1, FRZB, GDF11, GDF11|MSTN, GOT2, GOT2, GRB2, GSK3B, GSK3B, HRAS, HTRA2, ISOC1, JAK2, KRAS, LYN, LYN, MAP2K1, MAP2K5, MAPK1, MAPK14, MAPK3, MAPK9, MAPK9, MGAT5, MGAT5, MKNK1, MPDZ, MYL12A, MYL12B, MYL6, MYL9, NAP1L1, NAP1L4, NAPA, NECTIN1, NFKB1, NOG, NOG, NRP1, NRP1, NRP2, NRP2, 0LFM2, 0LFM2, PAK4, PDHA2, PIK3C3, PLA2G4A, PLCB1, PLCD1, PLCD3, PLCG2, PPP3R1, PRKACA, PRKAR1A, PRKAR1B, PRKAR2B, PRKCA, PRKCB, PRKCI, RAB27A, RAB3A, RAB4B, RAB5A, RAB5B, RAB5C, RAC2, RAC3, RALA, RBMS1, RDX, RELN, RHOC, RHOG, RHOT1, ROBO2, ROBO2, RPS6KA3, SGTA, SHANK3, SHC1, SHC1, SNCG, SPARCL1, SPARCL1, SRC, SRC, STAT1, STAT3, STAT3, STX1A, STXBP6, SYT4, SYT6, THBS2, THBS2, TLN2, TNNI2, TRIM72, UBXN4, UCN3, VAV1, VAV3, VTI1A, VWF, YAP1, and LCK.

[0140] In some variations, the biomarker has increased abundance, and a gene or protein symbol selected from HBS2, NRP1, UCN3, SPARCL1, FAM177a1, ANGPT1, and NOG. Alternatively, the corresponding SomaLogic aptamer or Uniprot ID may be used.P390200. W0.01

[0141] The biomarker can have a SomaScan Aptamer sequence having decreased abundance. In some variations, the biomarker is the SomaScan Aptamer sequence 18922-27. In some variations, the biomarker is the SomaScan Aptamer sequence 4337-49. In some variations, the biomarker is the SomaScan Aptamer sequence 25306-51. In some variations, the biomarker is the SomaScan Aptamer sequence 10025-1. In some variations, the biomarker is the SomaScan Aptamer sequence 10801-11. In some variations, the biomarker is the SomaScan Aptamer sequence 14124-6. In some variations, the biomarker is the SomaScan Aptamer sequence 22580-29. In some variations, the biomarker is the SomaScan Aptamer sequence 2615-60. In some variations, the biomarker is the SomaScan Aptamer sequence 5658-64. In some variations, the biomarker is the SomaScan Aptamer sequence 2443-10. In some variations, the biomarker is the SomaScan Aptamer sequence 19570-12. In some variations, the biomarker is the SomaScan Aptamer sequence 17166-4. In some variations, the biomarker is the SomaScan Aptamer sequence 14757-144. In some variations, the biomarker is the SomaScan Aptamer sequence 5581-28. In some variations, the biomarker is the SomaScan Aptamer sequence 2841-13. In some variations, the biomarker is the SomaScan Aptamer sequence 5755-29. In some variations, the biomarker is the SomaScan Aptamer sequence 10426-21. In some variations, the biomarker is the SomaScan Aptamer sequence 13547-5. In some variations, the biomarker is the SomaScan Aptamer sequence 13738-8. In some variations, the biomarker is the SomaScan Aptamer sequence 19622-7. In some variations, the biomarker is the SomaScan Aptamer sequence 2748-3. In some variations, the biomarker is the SomaScan Aptamer sequence 5193-51. In some variations, the biomarker is the SomaScan Aptamer sequence 5242-37. In some variations, the biomarker is the SomaScan Aptamer sequence 7655-11. In some variations, the biomarker is the SomaScan Aptamer sequence 3723-1. In some variations, the biomarker is the SomaScan Aptamer sequence 9380-2. In some variations, the biomarker is the SomaScan Aptamer sequence 21380-77. In some variations, the biomarker is the SomaScan Aptamer sequence 20546-71. In some variations, the biomarker is the SomaScan Aptamer sequence 9099-19. In some variations, the biomarker is the SomaScan Aptamer sequence 9970-7.

[0142] the biomarker is a SomaScan Aptamer sequence having increased abundance. In some variations, the biomarker is the SomaScan Aptamer sequence 19197-95. In some variations, the biomarker is the SomaScan Aptamer sequence 9843-5. In some variations, the biomarker is the SomaScan Aptamer sequence 9844-138. In some variations, the biomarker is the SomaScan Aptamer sequence 15312-14. In some variations, the biomarker is the SomaScan Aptamer sequence 15323-112. In some variations, the biomarker is the SomaScan Aptamer sequence 15627-83. In some variations, the biomarker is the SomaScan Aptamer sequence 2867-52. In some variations, the biomarker is the SomaScanP390200. W0.01Aptamer sequence 23302-19. In some variations, the biomarker is the SomaScan Aptamer sequence 5360-9. In some variations, the biomarker is the SomaScan Aptamer sequence 14685-17. In some variations, the biomarker is the SomaScan Aptamer sequence 15523-9. In some variations, the biomarker is the SomaScan Aptamer sequence 18185-118. In some variations, the biomarker is the SomaScan Aptamer sequence 2811-27. In some variations, the biomarker is the SomaScan Aptamer sequence 2602-2. In some variations, the biomarker is the SomaScan Aptamer sequence 13660-76. In some variations, the biomarker is the SomaScan Aptamer sequence 13621-31. In some variations, the biomarker is the SomaScan Aptamer sequence 12583-77. In some variations, the biomarker is the SomaScan Aptamer sequence 14748-31. In some variations, the biomarker is the SomaScan Aptamer sequence 13573-5. In some variations, the biomarker is the SomaScan Aptamer sequence 5870-23. In some variations, the biomarker is the SomaScan Aptamer sequence 24237-115. In some variations, the biomarker is the SomaScan Aptamer sequence 11130-158. In some variations, the biomarker is the SomaScan Aptamer sequence 3351-1. In some variations, the biomarker is the SomaScan Aptamer sequence 9840-2. In some variations, the biomarker is the SomaScan Aptamer sequence 2819-23. In some variations, the biomarker is the SomaScan Aptamer sequence 7959-34. In some variations, the biomarker is the SomaScan Aptamer sequence 4203-50. In some variations, the biomarker is the SomaScan Aptamer sequence 14029-42. In some variations, the biomarker is the SomaScan Aptamer sequence 15321-8. In some variations, the biomarker is the SomaScan Aptamer sequence 4976-57. In some variations, the biomarker is the SomaScan Aptamer sequence 3516-60. In some variations, the biomarker is the SomaScan Aptamer sequence 15527-90. In some variations, the biomarker is the SomaScan Aptamer sequence 20913-27. In some variations, the biomarker is the SomaScan Aptamer sequence 25105-87. In some variations, the biomarker is the SomaScan Aptamer sequence 19259-176. In some variations, the biomarker is the SomaScan Aptamer sequence 24723-58. In some variations, the biomarker is the SomaScan Aptamer sequence 18829-4. In some variations, the biomarker is the SomaScan Aptamer sequence 22984-10. In some variations, the biomarker is the SomaScan Aptamer sequence 18824-7. In some variations, the biomarker is the SomaScan Aptamer sequence 25287-7. In some variations, the biomarker is the SomaScan Aptamer sequence 4230-1. In some variations, the biomarker is the SomaScan Aptamer sequence 6036-78. In some variations, the biomarker is the SomaScan Aptamer sequence 8039-41. In some variations, the biomarker is the SomaScan Aptamer sequence 4393-3. In some variations, the biomarker is the SomaScan Aptamer sequence 4130-71. In some variations, the biomarker is the SomaScan Aptamer sequence 4487-1. In some variations, the biomarker is the SomaScan Aptamer sequence 4394-71. In some variations, the biomarker is the SomaScan Aptamer sequence 4989-7. In some variations,P390200. W0.01the biomarker is the SomaScan Aptamer sequence 13384-110. In some variations, the biomarker is the SomaScan Aptamer sequence 11171-25. In some variations, the biomarker is the SomaScan Aptamer sequence 11245-43. In some variations, the biomarker is the SomaScan Aptamer sequence 13122-19. In some variations, the biomarker is the SomaScan Aptamer sequence 7713-102. In some variations, the biomarker is the SomaScan Aptamer sequence 7713-50. In some variations, the biomarker is the SomaScan Aptamer sequence 13740-51. In some variations, the biomarker is the SomaScan Aptamer sequence 14587-16. In some variations, the biomarker is the SomaScan Aptamer sequence 2765-4. In some variations, the biomarker is the SomaScan Aptamer sequence 18233-10. In some variations, the biomarker is the SomaScan Aptamer sequence 23903-3. In some variations, the biomarker is the SomaScan Aptamer sequence 5464-52. In some variations, the biomarker is the SomaScan Aptamer sequence 3236-12. In some variations, the biomarker is the SomaScan Aptamer sequence 24050-26. In some variations, the biomarker is the SomaScan Aptamer sequence 18900-37. In some variations, the biomarker is the SomaScan Aptamer sequence 3317-33. In some variations, the biomarker is the SomaScan Aptamer sequence 9816-37. In some variations, the biomarker is the SomaScan Aptamer sequence 11816-84. In some variations, the biomarker is the SomaScan Aptamer sequence 20073-22. In some variations, the biomarker is the SomaScan Aptamer sequence 3453-87. In some variations, the biomarker is the SomaScan Aptamer sequence 3381-24. In some variations, the biomarker is the SomaScan Aptamer sequence 2864-2. In some variations, the biomarker is the SomaScan Aptamer sequence 22041-26. In some variations, the biomarker is the SomaScan Aptamer sequence 3115-64. In some variations, the biomarker is the SomaScan Aptamer sequence 5007-1. In some variations, the biomarker is the SomaScan Aptamer sequence 2855-49. In some variations, the biomarker is the SomaScan Aptamer sequence 15604-18. In some variations, the biomarker is the SomaScan Aptamer sequence 9760-13. In some variations, the biomarker is the SomaScan Aptamer sequence 21768-9. In some variations, the biomarker is the SomaScan Aptamer sequence 21813-171. In some variations, the biomarker is the SomaScan Aptamer sequence 23693-4. In some variations, the biomarker is the SomaScan Aptamer sequence 14036-116. In some variations, the biomarker is the SomaScan Aptamer sequence 19373-3. In some variations, the biomarker is the SomaScan Aptamer sequence 19122-47. In some variations, the biomarker is the SomaScan Aptamer sequence 20105-7. In some variations, the biomarker is the SomaScan Aptamer sequence 21817-5. In some variations, the biomarker is the SomaScan Aptamer sequence 13636-20. In some variations, the biomarker is the SomaScan Aptamer sequence 19188-21. In some variations, the biomarker is the SomaScan Aptamer sequence 4292-5. In some variations, the biomarker is the SomaScan Aptamer sequence 20584-4. In some variations, the biomarker is the SomaScan AptamerP390200. W0.01sequence 9869-28. In some variations, the biomarker is the SomaScan Aptamer sequence 8778-3. In some variations, the biomarker is the SomaScan Aptamer sequence 5846-24. In some variations, the biomarker is the SomaScan Aptamer sequence 5542-22. In some variations, the biomarker is the SomaScan Aptamer sequence 3214-3. In some variations, the biomarker is the SomaScan Aptamer sequence 6590-54. In some variations, the biomarker is the SomaScan Aptamer sequence 15387-44. In some variations, the biomarker is the SomaScan Aptamer sequence 8295-16. In some variations, the biomarker is the SomaScan Aptamer sequence 19377-14. In some variations, the biomarker is the SomaScan Aptamer sequence 13719-19. In some variations, the biomarker is the SomaScan Aptamer sequence 25249-33. In some variations, the biomarker is the SomaScan Aptamer sequence 25947-116. In some variations, the biomarker is the SomaScan Aptamer sequence 21430-4. In some variations, the biomarker is the SomaScan Aptamer sequence 11354-21. In some variations, the biomarker is the SomaScan Aptamer sequence 24909-40. In some variations, the biomarker is the SomaScan Aptamer sequence 25949-3. In some variations, the biomarker is the SomaScan Aptamer sequence 10070-22. In some variations, the biomarker is the SomaScan Aptamer sequence 15545-13. In some variations, the biomarker is the SomaScan Aptamer sequence 3466-8. In some variations, the biomarker is the SomaScan Aptamer sequence 21483-155. In some variations, the biomarker is the SomaScan Aptamer sequence 12479-50. In some variations, the biomarker is the SomaScan Aptamer sequence 25463-3. In some variations, the biomarker is the SomaScan Aptamer sequence 2644-11. In some variations, the biomarker is the SomaScan Aptamer sequence 5475-10. In some variations, the biomarker is the SomaScan Aptamer sequence 3379-29. In some variations, the biomarker is the SomaScan Aptamer sequence 9504-19. In some variations, the biomarker is the SomaScan Aptamer sequence 17516-7. In some variations, the biomarker is the SomaScan Aptamer sequence 23329-52. In some variations, the biomarker is the SomaScan Aptamer sequence 17205-21. In some variations, the biomarker is the SomaScan Aptamer sequence 19222-124. In some variations, the biomarker is the SomaScan Aptamer sequence 14287-6. In some variations, the biomarker is the SomaScan Aptamer sequence 18950-13. In some variations, the biomarker is the SomaScan Aptamer sequence 16769-20. In some variations, the biomarker is the SomaScan Aptamer sequence 14332-3. In some variations, the biomarker is the SomaScan Aptamer sequence 22547-17. In some variations, the biomarker is the SomaScan Aptamer sequence 18373-13. In some variations, the biomarker is the SomaScan Aptamer sequence 21713-11. In some variations, the biomarker is the SomaScan Aptamer sequence 17764-108. In some variations, the biomarker is the SomaScan Aptamer sequence 12540-25. In some variations, the biomarker is the SomaScan Aptamer sequence 21752-10. In some variations, the biomarker is the SomaScan Aptamer sequence 22578-17. In some variations,P390200. W0.01the biomarker is the SomaScan Aptamer sequence 5116-62. In some variations, the biomarker is the SomaScan Aptamer sequence 3469-74. In some variations, the biomarker is the SomaScan Aptamer sequence 3868-8. In some variations, the biomarker is the SomaScan Aptamer sequence 13242-134. In some variations, the biomarker is the SomaScan Aptamer sequence 16043-30. In some variations, the biomarker is the SomaScan Aptamer sequence 5272-55. In some variations, the biomarker is the SomaScan Aptamer sequence 19630-2. In some variations, the biomarker is the SomaScan Aptamer sequence 13707-27. In some variations, the biomarker is the SomaScan Aptamer sequence 4467-49. In some variations, the biomarker is the SomaScan Aptamer sequence 15433-4. In some variations, the biomarker is the SomaScan Aptamer sequence 5488-74. In some variations, the biomarker is the SomaScan Aptamer sequence 12351-25. In some variations, the biomarker is the SomaScan Aptamer sequence 10346-5. In some variations, the biomarker is the SomaScan Aptamer sequence 10354-57. In some variations, the biomarker is the SomaScan Aptamer sequence 19553-14. In some variations, the biomarker is the SomaScan Aptamer sequence 18308-30. In some variations, the biomarker is the SomaScan Aptamer sequence 17355-56. In some variations, the biomarker is the SomaScan Aptamer sequence 21539-139. In some variations, the biomarker is the SomaScan Aptamer sequence 3339-33. In some variations, the biomarker is the SomaScan Aptamer sequence 14111-15. In some variations, the biomarker is the SomaScan Aptamer sequence 14082-56. In some variations, the biomarker is the SomaScan Aptamer sequence 5440-26. In some variations, the biomarker is the SomaScan Aptamer sequence 24320-3. In some variations, the biomarker is the SomaScan Aptamer sequence 9997-12. In some variations, the biomarker is the SomaScan Aptamer sequence 10756-34. In some variations, the biomarker is the SomaScan Aptamer sequence 5275-28. In some variations, the biomarker is the SomaScan Aptamer sequence 9830-109. In some variations, the biomarker is the SomaScan Aptamer sequence 7952-2. In some variations, the biomarker is the SomaScan Aptamer sequence 3050-7. In some variations, the biomarker is the SomaScan Aptamer sequence 25451-39. In some variations, the biomarker is the SomaScan Aptamer sequence 4560-34.

[0143] In some variations, the biomarker is a protein having decreased abundance. In some variations, the biomarker is the protein Macrosialin. In some variations, the biomarker is the protein C-reactive protein. In some variations, the biomarker is the protein DAXX. In some variations, the biomarker is the protein Dihydrolipoyl dehydrogenase (mitochondrial). In some variations, the biomarker is the protein Ephrin-A2. In some variations, the biomarker is the protein Ephrin-A2. In some variations, the biomarker is the protein Ephrin-A2. In some variations, the biomarker is the protein Ephrin-A5. In some variations, the biomarker is the protein Coagulation factor XIII B chain. In some variations, the biomarker is the proteinP390200. W0.01Fibroblast growth factor 8 isoform B. In some variations, the biomarker is the protein Fibroblast growth factor 8. In some variations, the biomarker is the protein Fibroblast growth factor 8 isoform F. In some variations, the biomarker is the protein Fibroblast growth factor 8 isoform B. In some variations, the biomarker is the protein Fibrinogen-like protein 1. In some variations, the biomarker is the protein Secreted frizzled-related protein 3. In some variations, the biomarker is the protein Gastric inhibitory polypeptide. In some variations, the biomarker is the protein Golgi SNAP receptor complex member 2. In some variations, the biomarker is the protein 5-hydroxytryptamine receptor 7. In some variations, the biomarker is the protein Inhibin beta A chain. In some variations, the biomarker is the protein Activin A. In some variations, the biomarker is the protein Activin A. In some variations, the biomarker is the protein GTPase KRas. In some variations, the biomarker is the protein Dual specificity mitogen-activated protein kinase kinase 4. In some variations, the biomarker is the protein N-terminal pro-BNP. In some variations, the biomarker is the protein Brain natriuretic peptide 32. In some variations, the biomarker is the protein Group XI IB secretory phospholipase A2-like protein. In some variations, the biomarker is the protein Queuine tRNA-ribosyltransferase. In some variations, the biomarker is the protein Reticulon-4 receptor-like 1. In some variations, the biomarker is the protein Synaptotagmin-5. In some variations, the biomarker is the protein UBX domain-containing protein 4: Cytoplasmic domain 2.

[0144] In some variations, the biomarker is a protein having increased abundance. In some variations, the biomarker is the protein Acetyl-CoA acetyltransferase (mitochondrial). In some variations, the biomarker is the protein Alpha-actinin-1. In some variations, the biomarker is the protein Alpha-actinin-2. In some variations, the biomarker is the protein Protein argonaute-1. In some variations, the biomarker is the protein Protein argonaute-3. In some variations, the biomarker is the protein RAC-alpha serine / threonine-protein kinase. In some variations, the biomarker is the protein RAC-alpha serine / threonine-protein kinase. In some variations, the biomarker is the protein Proline-rich AKT1 substrate 1. In some variations, the biomarker is the protein RAC-beta serine / threonine-protein kinase. In some variations, the biomarker is the protein RAC-beta serine / threonine-protein kinase. In some variations, the biomarker is the protein Delta-aminolevulinic acid dehydratase. In some variations, the biomarker is the protein Fructose-bisphosphate aldolase B. In some variations, the biomarker is the protein Angiopoietin-1. In some variations, the biomarker is the protein Angiopoietin-2. In some variations, the biomarker is the protein Angiopoietin-2. In some variations, the biomarker is the protein AP-2 complex subunit alpha-2. In some variations, the biomarker is the protein Serine / threonine-protein kinase A-Raf. In some variations, the biomarker is the protein Rho GTPase-activating protein 5. In some variations, the biomarker is the protein Actin-related protein 2 / 3 complex subunit 3. In some variations, the biomarker is the protein Bcl2-associated agonist of cell death. In some variations, theP390200. W0.01biomarker is the protein Voltage-dependent L-type calcium channel subunit beta-3. In some variations, the biomarker is the protein Voltage-dependent L-type calcium channel subunit beta-4. In some variations, the biomarker is the protein Calcium / calmodulin-dependent protein kinase type II subunit beta. In some variations, the biomarker is the protein Cell division control protein 42 homolog. In some variations, the biomarker is the protein Cadherin-5. In some variations, the biomarker is the protein Cadherin-7. In some variations, the biomarker is the protein Cofilin- 1. In some variations, the biomarker is the protein COP9 signalosome complex subunit 2. In some variations, the biomarker is the protein Complexin-2. In some variations, the biomarker is the protein Adapter molecule crk. In some variations, the biomarker is the protein Dihydrolipoyl dehydrogenase (mitochondrial). In some variations, the biomarker is the protein Eukaryotic translation initiation factor 1. In some variations, the biomarker is the protein Eukaryotic translation initiation factor 2A. In some variations, the biomarker is the protein Eukaryotic translation initiation factor 2 subunit 1. In some variations, the biomarker is the protein Eukaryotic translation initiation factor 2 subunit 2. In some variations, the biomarker is the protein Eukaryotic initiation factor 4A-I. In some variations, the biomarker is the protein Eukaryotic initiation factor 4A-II. In some variations, the biomarker is the protein Eukaryotic initiation factor 4A-II. In some variations, the biomarker is the protein Eukaryotic translation initiation factor 4 gamma 1. In some variations, the biomarker is the protein Eukaryotic translation initiation factor 4 gamma 2. In some variations, the biomarker is the protein Ephrin type-A receptor 10. In some variations, the biomarker is the protein Protein FAM177A1. In some variations, the biomarker is the protein Fibroblast growth factor 16. In some variations, the biomarker is the protein Fibroblast growth factor 6. In some variations, the biomarker is the protein Fibroblast growth factor 7. In some variations, the biomarker is the protein Fibroblast growth factor 8 isoform A. In some variations, the biomarker is the protein Fibrinogen gamma chain. In some variations, the biomarker is the protein Fumarate hydratase (mitochondrial). In some variations, the biomarker is the protein Filamin-A: Calponin Homology 2. In some variations, the biomarker is the protein Filamin-A: Calponin Homology 1. In some variations, the biomarker is the protein Leucine-rich repeat transmembrane protein FLRT2. In some variations, the biomarker is the protein Fragile X mental retardation protein 1. In some variations, the biomarker is the protein Fragile X mental retardation protein 1. In some variations, the biomarker is the protein Secreted frizzled-related protein 3. In some variations, the biomarker is the protein Growth / differentiation factor 11. In some variations, the biomarker is the protein Growth / differentiation factor 11 / 8. In some variations, the biomarker is the protein Aspartate aminotransferase (mitochondrial). In some variations, the biomarker is the protein Aspartate aminotransferase (mitochondrial). In some variations, the biomarker is the protein Growth factor receptor-bound protein 2. In some variations, the biomarker is the proteinP390200. W0.01Glycogen synthase kinase-3 beta. In some variations, the biomarker is the protein Glycogen synthase kinase-3 beta. In some variations, the biomarker is the protein GTPase HRas. In some variations, the biomarker is the protein Serine protease HTRA2 (mitochondrial). In some variations, the biomarker is the protein Isochorismatase domain-containing protein 1. In some variations, the biomarker is the protein Tyrosine-protein kinase JAK2. In some variations, the biomarker is the protein GTPase KRas. In some variations, the biomarker is the protein Tyrosine-protein kinase Lyn. In some variations, the biomarker is the protein Tyrosine-protein kinase Lyn (isoform B). In some variations, the biomarker is the protein Dual specificity mitogen-activated protein kinase kinase 1. In some variations, the biomarker is the protein Dual specificity mitogen-activated protein kinase kinase 5. In some variations, the biomarker is the protein Mitogen-activated protein kinase 1. In some variations, the biomarker is the protein Mitogen-activated protein kinase 14. In some variations, the biomarker is the protein Mitogen-activated protein kinase 3. In some variations, the biomarker is the protein Mitogen-activated protein kinase 9. In some variations, the biomarker is the protein Mitogen-activated protein kinase 9. In some variations, the biomarker is the protein Alpha-1. In some variations, the biomarker is the protein6-mannosylglycoprotein 6-beta-N-acetylglucosaminyltransferase A. In some variations, the biomarker is the protein Alpha-1. In some variations, the biomarker is the protein6-mannosylglycoprotein 6-beta-N-acetylglucosaminyltransferase A. In some variations, the biomarker is the protein MAP kinase-interacting serine / threonine-protein kinase 1. In some variations, the biomarker is the protein Multiple PDZ domain protein. In some variations, the biomarker is the protein Myosin regulatory light chain 12A. In some variations, the biomarker is the protein Myosin regulatory light chain 12B. In some variations, the biomarker is the protein Myosin light polypeptide 6. In some variations, the biomarker is the protein Myosin (light chain 9- regulatory). In some variations, the biomarker is the protein Nucleosome assembly protein 1-like 1. In some variations, the biomarker is the protein nucleosome assembly protein 1-like 4. In some variations, the biomarker is the protein Alpha-soluble NSF attachment protein. In some variations, the biomarker is the protein Nectin-1 (isoform gamma: Extracellular domain). In some variations, the biomarker is the protein Nuclear factor NF-kappa-B p105 subunit. In some variations, the biomarker is the protein Noggin. In some variations, the biomarker is the protein Noggin. In some variations, the biomarker is the protein Neuropilin-1. In some variations, the biomarker is the protein Neuropilin-1. In some variations, the biomarker is the protein Neuropilin-2. In some variations, the biomarker is the protein Neuropilin-2. In some variations, the biomarker is the protein Noelin-2. In some variations, the biomarker is the protein Noelin-2. In some variations, the biomarker is the protein Serine / threonine-protein kinase PAK 4. In some variations, the biomarker is the protein Pyruvate dehydrogenase E1 component subunit alpha (testis-specific formP390200. W0.01(mitochondrial)). In some variations, the biomarker is the protein Phosphatidylinositol 3-kinase catalytic subunit type 3. In some variations, the biomarker is the protein Cytosolic phospholipase A2 alpha. In some variations, the biomarker is the protein 1-phosphatidylinositol 4. In some variations, the biomarker is the protein5-bisphosphate phosphodiesterase beta-1. In some variations, the biomarker is the protein 1-phosphatidylinositol-4. In some variations, the biomarker is the protein5-bisphosphate phosphodiesterase delta-1. In some variations, the biomarker is the protein 1-phosphatidylinositol 4. In some variations, the biomarker is the protein5-bisphosphate phosphodiesterase delta-3. In some variations, the biomarker is the protein 1-phosphatidylinositol 4. In some variations, the biomarker is the protein5-bisphosphate phosphodiesterase gamma-2. In some variations, the biomarker is the protein Calcineurin subunit B type 1. In some variations, the biomarker is the protein cAMP-dependent protein kinase catalytic subunit alpha. In some variations, the biomarker is the protein cAMP-dependent protein kinase type l-alpha regulatory subunit. In some variations, the biomarker is the protein cAMP-dependent protein kinase type l-beta regulatory subunit. In some variations, the biomarker is the protein cAMP-dependent protein kinase type I l-beta regulatory subunit. In some variations, the biomarker is the protein Protein kinase C alpha type. In some variations, the biomarker is the protein Protein kinase C beta type (splice variant beta-ll). In some variations, the biomarker is the protein Protein kinase C iota type. In some variations, the biomarker is the protein Ras-related protein Rab-27A. In some variations, the biomarker is the protein Ras-related protein Rab-3A. In some variations, the biomarker is the protein Ras-related protein Rab-4B. In some variations, the biomarker is the protein Ras-related protein Rab-5A. In some variations, the biomarker is the protein Ras-related protein Rab-5B. In some variations, the biomarker is the protein Ras-related protein Rab-5C. In some variations, the biomarker is the protein Ras-related C3 botulinum toxin substrate 2. In some variations, the biomarker is the protein Ras-related C3 botulinum toxin substrate 3. In some variations, the biomarker is the protein Ras-related protein Ral-A. In some variations, the biomarker is the protein RNA-binding motif (single-stranded-interacting protein 1). In some variations, the biomarker is the protein Radixin. In some variations, the biomarker is the protein Reelin. In some variations, the biomarker is the protein Rho-related GTP-binding protein RhoC. In some variations, the biomarker is the protein Rho-related GTP-binding protein RhoG. In some variations, the biomarker is the protein Mitochondrial Rho GTPase 1. In some variations, the biomarker is the protein Roundabout homolog 2. In some variations, the biomarker is the protein Roundabout homolog 2. In some variations, the biomarker is the protein Ribosomal protein S6 kinase alpha-3. In some variations, the biomarker is the protein Small glutamine-rich tetratricopeptide repeat-containing protein alpha. In some variations, the biomarker is the protein SH3 and multiple ankyrin repeatP390200. W0.01domains protein 3. In some variations, the biomarker is the protein SHC-transforming protein 1 Phosphotyrosine Interaction Domain. In some variations, the biomarker is the protein SHC-transforming protein 1: Src Homology domain. In some variations, the biomarker is the protein Gamma-synuclein. In some variations, the biomarker is the protein SPARC-like protein 1. In some variations, the biomarker is the protein SPARC-like protein 1. In some variations, the biomarker is the protein Proto-oncogene tyrosine-protein kinase Src. In some variations, the biomarker is the protein Proto-oncogene tyrosine-protein kinase Src isoform 2. In some variations, the biomarker is the protein Signal transducer and activator of transcription 1-alpha / beta. In some variations, the biomarker is the protein Signal transducer and activator of transcription 3. In some variations, the biomarker is the protein Signal transducer and activator of transcription 3. In some variations, the biomarker is the protein Syntaxin-1 A. In some variations, the biomarker is the protein Syntaxin-binding protein 6. In some variations, the biomarker is the protein Synaptotagmin-4. In some variations, the biomarker is the protein Synaptotagmin-6. In some variations, the biomarker is the protein Thrombospondin-2. In some variations, the biomarker is the protein Thrombospondin-2. In some variations, the biomarker is the protein Talin-2. In some variations, the biomarker is the protein Troponin I (fast skeletal muscle). In some variations, the biomarker is the protein Tripartite motif-containing protein 72. In some variations, the biomarker is the protein UBX domain-containing protein 4: Cytoplasmic domain 1. In some variations, the biomarker is the protein Urocortin-3. In some variations, the biomarker is the protein Proto-oncogene vav. In some variations, the biomarker is the protein Guanine nucleotide exchange factor VAV3. In some variations, the biomarker is the protein Vesicle transport through interaction with t-SNAREs homolog 1 A. In some variations, the biomarker is the protein von Willebrand factor. In some variations, the biomarker is the protein Transcriptional coactivator YAP1. In some variations, the biomarker is the protein Tyrosine-protein kinase Lck.

[0145] In some variations, the biomarker has decreased abundance and can be identified by Uniprot ID. In some variations, the biomarker has the Uniprot ID P34810. In some variations, the biomarker has the Uniprot ID P02741. In some variations, the biomarker has the Uniprot ID Q9UER7. In some variations, the biomarker has the Uniprot ID P09622. In some variations, the biomarker has the Uniprot ID O43921. In some variations, the biomarker has the Uniprot ID O43921. In some variations, the biomarker has the Uniprot ID O43921. In some variations, the biomarker has the Uniprot ID P52803. In some variations, the biomarker has the Uniprot ID P05160. In some variations, the biomarker has the Uniprot ID P55075. In some variations, the biomarker has the Uniprot ID P55075. In some variations, the biomarker has the Uniprot ID P55075. In some variations, the biomarker has the Uniprot ID P55075. In some variations, the biomarker has the Uniprot ID Q08830. In some variations, the biomarker has the Uniprot ID Q92765. In some variations, the biomarker hasP390200. W0.01the Uniprot ID P09681. In some variations, the biomarker has the Uniprot ID O14653. In some variations, the biomarker has the Uniprot ID P34969. In some variations, the biomarker has the Uniprot ID P08476. In some variations, the biomarker has the Uniprot ID P08476. In some variations, the biomarker has the Uniprot ID P08476. In some variations, the biomarker has the Uniprot ID P01116. In some variations, the biomarker has the Uniprot ID P45985. In some variations, the biomarker has the Uniprot ID P16860. In some variations, the biomarker has the Uniprot ID P16860. In some variations, the biomarker has the Uniprot ID Q9BX93. In some variations, the biomarker has the Uniprot ID Q9BXR0. In some variations, the biomarker has the Uniprot ID Q86UN2. In some variations, the biomarker has the Uniprot ID O00445. In some variations, the biomarker has the Uniprot ID Q92575.

[0146] In some variations, the biomarker has increased abundance and can be identified by Uniprot ID. In some variations, the biomarker has the Uniprot ID P24752. In some variations, the biomarker has the Uniprot ID P12814. In some variations, the biomarker has the Uniprot ID P35609. In some variations, the biomarker has the Uniprot ID Q9UL18. In some variations, the biomarker has the Uniprot ID Q9H9G7. In some variations, the biomarker has the Uniprot ID P31749. In some variations, the biomarker has the Uniprot ID P31749. In some variations, the biomarker has the Uniprot ID Q96B36. In some variations, the biomarker has the Uniprot ID P31751. In some variations, the biomarker has the Uniprot ID P31751. In some variations, the biomarker has the Uniprot ID P13716. In some variations, the biomarker has the Uniprot ID P05062. In some variations, the biomarker has the Uniprot ID Q15389. In some variations, the biomarker has the Uniprot ID O15123. In some variations, the biomarker has the Uniprot ID O15123. In some variations, the biomarker has the Uniprot ID O94973. In some variations, the biomarker has the Uniprot ID P10398. In some variations, the biomarker has the Uniprot ID Q13017. In some variations, the biomarker has the Uniprot ID O15145. In some variations, the biomarker has the Uniprot ID Q92934. In some variations, the biomarker has the Uniprot ID P54284. In some variations, the biomarker has the Uniprot ID O00305. In some variations, the biomarker has the Uniprot ID Q13554. In some variations, the biomarker has the Uniprot ID P60953. In some variations, the biomarker has the Uniprot ID P33151. In some variations, the biomarker has the Uniprot ID Q9ULB5. In some variations, the biomarker has the Uniprot ID P23528. In some variations, the biomarker has the Uniprot ID P61201. In some variations, the biomarker has the Uniprot ID Q6PUV4. In some variations, the biomarker has the Uniprot ID P46108. In some variations, the biomarker has the Uniprot ID P09622. In some variations, the biomarker has the Uniprot ID P41567. In some variations, the biomarker has the Uniprot ID Q9BY44. In some variations, the biomarker has the Uniprot ID P05198. In some variations, the biomarker has the Uniprot ID P20042. In some variations, the biomarker hasP390200. W0.01the Uniprot ID P60842. In some variations, the biomarker has the Uniprot ID Q14240. In some variations, the biomarker has the Uniprot ID Q14240. In some variations, the biomarker has the Uniprot ID Q04637. In some variations, the biomarker has the Uniprot ID P78344. In some variations, the biomarker has the Uniprot ID Q5JZY3. In some variations, the biomarker has the Uniprot ID Q8N128. In some variations, the biomarker has the Uniprot ID 043320. In some variations, the biomarker has the Uniprot ID P10767. In some variations, the biomarker has the Uniprot ID P21781. In some variations, the biomarker has the Uniprot ID P55075. In some variations, the biomarker has the Uniprot ID P02679. In some variations, the biomarker has the Uniprot ID P07954. In some variations, the biomarker has the Uniprot ID P21333. In some variations, the biomarker has the Uniprot ID P21333. In some variations, the biomarker has the Uniprot ID 043155. In some variations, the biomarker has the Uniprot ID Q06787. In some variations, the biomarker has the Uniprot ID Q06787. In some variations, the biomarker has the Uniprot ID Q92765. In some variations, the biomarker has the Uniprot ID 095390. In some variations, the biomarker has the Uniprot ID O95390|O14793. In some variations, the biomarker has the Uniprot ID P00505. In some variations, the biomarker has the Uniprot ID P00505. In some variations, the biomarker has the Uniprot ID P62993. In some variations, the biomarker has the Uniprot ID P49841. In some variations, the biomarker has the Uniprot ID P49841. In some variations, the biomarker has the Uniprot ID P01112. In some variations, the biomarker has the Uniprot ID 043464. In some variations, the biomarker has the Uniprot ID Q96CN7. In some variations, the biomarker has the Uniprot ID 060674. In some variations, the biomarker has the Uniprot ID P01116. In some variations, the biomarker has the Uniprot ID P07948. In some variations, the biomarker has the Uniprot ID P07948. In some variations, the biomarker has the Uniprot ID Q02750. In some variations, the biomarker has the Uniprot ID Q13163. In some variations, the biomarker has the Uniprot ID P28482. In some variations, the biomarker has the Uniprot ID Q16539. In some variations, the biomarker has the Uniprot ID P27361. In some variations, the biomarker has the Uniprot ID P45984. In some variations, the biomarker has the Uniprot ID P45984. In some variations, the biomarker has the Uniprot ID Q09328. In some variations, the biomarker has the Uniprot ID Q09328. In some variations, the biomarker has the Uniprot ID Q9BUB5. In some variations, the biomarker has the Uniprot ID 075970. In some variations, the biomarker has the Uniprot ID P19105. In some variations, the biomarker has the Uniprot ID 014950. In some variations, the biomarker has the Uniprot ID P60660. In some variations, the biomarker has the Uniprot ID P24844. In some variations, the biomarker has the Uniprot ID P55209. In some variations, the biomarker has the Uniprot ID Q99733. In some variations, the biomarker has the Uniprot ID P54920. In some variations, the biomarker has the Uniprot ID Q15223. In some variations, the biomarker has the Uniprot ID P19838. In some variations,P390200. W0.01the biomarker has the Uniprot ID Q13253. In some variations, the biomarker has the Uniprot ID Q13253. In some variations, the biomarker has the Uniprot ID 014786. In some variations, the biomarker has the Uniprot ID 014786. In some variations, the biomarker has the Uniprot ID 060462. In some variations, the biomarker has the Uniprot ID 060462. In some variations, the biomarker has the Uniprot ID 095897. In some variations, the biomarker has the Uniprot ID 095897. In some variations, the biomarker has the Uniprot ID 096013. In some variations, the biomarker has the Uniprot ID P29803. In some variations, the biomarker has the Uniprot ID Q8NEB9. In some variations, the biomarker has the Uniprot ID P47712. In some variations, the biomarker has the Uniprot ID Q9NQ66. In some variations, the biomarker has the Uniprot ID P51178. In some variations, the biomarker has the Uniprot ID Q8N3E9. In some variations, the biomarker has the Uniprot ID P16885. In some variations, the biomarker has the Uniprot ID P63098. In some variations, the biomarker has the Uniprot ID P17612. In some variations, the biomarker has the Uniprot ID P10644. In some variations, the biomarker has the Uniprot ID P31321. In some variations, the biomarker has the Uniprot ID P31323. In some variations, the biomarker has the Uniprot ID P17252. In some variations, the biomarker has the Uniprot ID P05771. In some variations, the biomarker has the Uniprot ID P41743. In some variations, the biomarker has the Uniprot ID P51159. In some variations, the biomarker has the Uniprot ID P20336. In some variations, the biomarker has the Uniprot ID P61018. In some variations, the biomarker has the Uniprot ID P20339. In some variations, the biomarker has the Uniprot ID P61020. In some variations, the biomarker has the Uniprot ID P51148. In some variations, the biomarker has the Uniprot ID P15153. In some variations, the biomarker has the Uniprot ID P60763. In some variations, the biomarker has the Uniprot ID P11233. In some variations, the biomarker has the Uniprot ID P29558. In some variations, the biomarker has the Uniprot ID P35241. In some variations, the biomarker has the Uniprot ID P78509. In some variations, the biomarker has the Uniprot ID P08134. In some variations, the biomarker has the Uniprot ID P84095. In some variations, the biomarker has the Uniprot ID Q8IXI2. In some variations, the biomarker has the Uniprot ID Q9HCK4. In some variations, the biomarker has the Uniprot ID Q9HCK4. In some variations, the biomarker has the Uniprot ID P51812. In some variations, the biomarker has the Uniprot ID 043765. In some variations, the biomarker has the Uniprot ID Q9BYB0. In some variations, the biomarker has the Uniprot ID P29353. In some variations, the biomarker has the Uniprot ID P29353. In some variations, the biomarker has the Uniprot ID 076070. In some variations, the biomarker has the Uniprot ID Q14515. In some variations, the biomarker has the Uniprot ID Q14515. In some variations, the biomarker has the Uniprot ID P12931. In some variations, the biomarker has the Uniprot ID P12931. In some variations, the biomarker has the Uniprot ID P42224. In some variations, the biomarker has the Uniprot ID P40763. In someP390200. W0.01variations, the biomarker has the Uniprot ID P40763. In some variations, the biomarker has the Uniprot ID Q16623. In some variations, the biomarker has the Uniprot ID Q8NFX7. In some variations, the biomarker has the Uniprot ID Q9H2B2. In some variations, the biomarker has the Uniprot ID Q5T7P8. In some variations, the biomarker has the Uniprot ID P35442. In some variations, the biomarker has the Uniprot ID P35442. In some variations, the biomarker has the Uniprot ID Q9Y4G6. In some variations, the biomarker has the Uniprot ID P48788. In some variations, the biomarker has the Uniprot ID Q6ZMU5. In some variations, the biomarker has the Uniprot ID Q92575. In some variations, the biomarker has the Uniprot ID Q969E3. In some variations, the biomarker has the Uniprot ID P15498. In some variations, the biomarker has the Uniprot ID Q9UKW4. In some variations, the biomarker has the Uniprot ID Q96AJ9. In some variations, the biomarker has the Uniprot ID P04275. In some variations, the biomarker has the Uniprot ID P46937. In some variations, the biomarker has the Uniprot ID P06239.

[0147] In some variations, the biomarker has decreased abundance, and can be identified by gene or protein symbol. In some variations, the biomarker corresponds to CD68. In some variations, the biomarker corresponds to CRP. In some variations, the biomarker corresponds to DAXX. In some variations, the biomarker corresponds to DLD. In some variations, the biomarker corresponds to EFNA2. In some variations, the biomarker corresponds to EFNA2. In some variations, the biomarker corresponds to EFNA2. In some variations, the biomarker corresponds to EFNA5. In some variations, the biomarker corresponds to F13B. In some variations, the biomarker corresponds to FGF8. In some variations, the biomarker corresponds to FGF8. In some variations, the biomarker corresponds to FGF8. In some variations, the biomarker corresponds to FGF8. In some variations, the biomarker corresponds to FGL1. In some variations, the biomarker corresponds to FRZB. In some variations, the biomarker corresponds to GIP. In some variations, the biomarker corresponds to GOSR2. In some variations, the biomarker corresponds to HTR7. In some variations, the biomarker corresponds to INHBA. In some variations, the biomarker corresponds to INHBA. In some variations, the biomarker corresponds to INHBA. In some variations, the biomarker corresponds to KRAS. In some variations, the biomarker corresponds to MAP2K4. In some variations, the biomarker corresponds to NPPB. In some variations, the biomarker corresponds to NPPB. In some variations, the biomarker corresponds to PLA2G12B. In some variations, the biomarker corresponds to QTRT1. In some variations, the biomarker corresponds to RTN4RL1. In some variations, the biomarker corresponds to SYT5. In some variations, the biomarker corresponds to UBXN4.

[0148] In some variations, the biomarker has increased abundance, and can be identified by gene or protein symbol. In some variations, the biomarker corresponds to ACAT1. In someP390200. W0.01variations, the biomarker corresponds to ACTN1. In some variations, the biomarker corresponds to ACTN2. In some variations, the biomarker corresponds to AGO1. In some variations, the biomarker corresponds to AGO3. In some variations, the biomarker corresponds to AKT1. In some variations, the biomarker corresponds to AKT1. In some variations, the biomarker corresponds to AKT1S1. In some variations, the biomarker corresponds to AKT2. In some variations, the biomarker corresponds to AKT2. In some variations, the biomarker corresponds to ALAD. In some variations, the biomarker corresponds to ALDOB. In some variations, the biomarker corresponds to ANGPT1. In some variations, the biomarker corresponds to ANGPT2. In some variations, the biomarker corresponds to ANGPT2. In some variations, the biomarker corresponds to AP2A2. In some variations, the biomarker corresponds to ARAF. In some variations, the biomarker corresponds to ARHGAP5. In some variations, the biomarker corresponds to ARPC3. In some variations, the biomarker corresponds to BAD. In some variations, the biomarker corresponds to CACNB3. In some variations, the biomarker corresponds to CACNB4. In some variations, the biomarker corresponds to CAMK2B. In some variations, the biomarker corresponds to CDC42. In some variations, the biomarker corresponds to CDH5. In some variations, the biomarker corresponds to CDH7. In some variations, the biomarker corresponds to CFL1. In some variations, the biomarker corresponds to COPS2. In some variations, the biomarker corresponds to CPLX2. In some variations, the biomarker corresponds to CRK. In some variations, the biomarker corresponds to DLD. In some variations, the biomarker corresponds to EIF1. In some variations, the biomarker corresponds to EIF2A. In some variations, the biomarker corresponds to EIF2S1. In some variations, the biomarker corresponds to EIF2S2. In some variations, the biomarker corresponds to EIF4A1. In some variations, the biomarker corresponds to EIF4A2. In some variations, the biomarker corresponds to EIF4A2. In some variations, the biomarker corresponds to EIF4G1. In some variations, the biomarker corresponds to EIF4G2. In some variations, the biomarker corresponds to EPHA10. In some variations, the biomarker corresponds to FAM177A1. In some variations, the biomarker corresponds to FGF16. In some variations, the biomarker corresponds to FGF6. In some variations, the biomarker corresponds to FGF7. In some variations, the biomarker corresponds to FGF8. In some variations, the biomarker corresponds to FGG. In some variations, the biomarker corresponds to FH. In some variations, the biomarker corresponds to FLNA. In some variations, the biomarker corresponds to FLNA. In some variations, the biomarker corresponds to FLRT2. In some variations, the biomarker corresponds to FMR1. In some variations, the biomarker corresponds to FMR1. In some variations, the biomarker corresponds to FRZB. In some variations, the biomarker corresponds to GDF11. In some variations, the biomarker corresponds to GDF11 |MSTN. In some variations, the biomarkerP390200. W0.01corresponds to GOT2. In some variations, the biomarker corresponds to GOT2. In some variations, the biomarker corresponds to GRB2. In some variations, the biomarker corresponds to GSK3B. In some variations, the biomarker corresponds to GSK3B. In some variations, the biomarker corresponds to HRAS. In some variations, the biomarker corresponds to HTRA2. In some variations, the biomarker corresponds to ISOC1. In some variations, the biomarker corresponds to JAK2. In some variations, the biomarker corresponds to KRAS. In some variations, the biomarker corresponds to LYN. In some variations, the biomarker corresponds to LYN. In some variations, the biomarker corresponds to MAP2K1. In some variations, the biomarker corresponds to MAP2K5. In some variations, the biomarker corresponds to MAPK1. In some variations, the biomarker corresponds to MAPK14. In some variations, the biomarker corresponds to MAPK3. In some variations, the biomarker corresponds to MAPK9. In some variations, the biomarker corresponds to MAPK9. In some variations, the biomarker corresponds to MGAT5. In some variations, the biomarker corresponds to MGAT5. In some variations, the biomarker corresponds to MKNK1. In some variations, the biomarker corresponds to MPDZ. In some variations, the biomarker corresponds to MYL12A. In some variations, the biomarker corresponds to MYL12B. In some variations, the biomarker corresponds to MYL6. In some variations, the biomarker corresponds to MYL9. In some variations, the biomarker corresponds to NAP1L1. In some variations, the biomarker corresponds to NAP1L4. In some variations, the biomarker corresponds to NAPA. In some variations, the biomarker corresponds to NECTIN1. In some variations, the biomarker corresponds to NFKB1. In some variations, the biomarker corresponds to NOG. In some variations, the biomarker corresponds to NOG. In some variations, the biomarker corresponds to NRP1. In some variations, the biomarker corresponds to NRP1. In some variations, the biomarker corresponds to NRP2. In some variations, the biomarker corresponds to NRP2. In some variations, the biomarker corresponds to OLFM2. In some variations, the biomarker corresponds to OLFM2. In some variations, the biomarker corresponds to PAK4. In some variations, the biomarker corresponds to PDHA2. In some variations, the biomarker corresponds to PIK3C3. In some variations, the biomarker corresponds to PLA2G4A. In some variations, the biomarker corresponds to PLCB1. In some variations, the biomarker corresponds to PLCD1. In some variations, the biomarker corresponds to PLCD3. In some variations, the biomarker corresponds to PLCG2. In some variations, the biomarker corresponds to PPP3R1. In some variations, the biomarker corresponds to PRKACA. In some variations, the biomarker corresponds to PRKAR1A. In some variations, the biomarker corresponds to PRKAR1B. In some variations, the biomarker corresponds to PRKAR2B. In some variations, the biomarker corresponds to PRKCA. In some variations, the biomarker corresponds to PRKCB. In some variations, the biomarker corresponds to PRKCI. In someP390200. W0.01variations, the biomarker corresponds to RAB27A. In some variations, the biomarker corresponds to RAB3A. In some variations, the biomarker corresponds to RAB4B. In some variations, the biomarker corresponds to RAB5A. In some variations, the biomarker corresponds to RAB5B. In some variations, the biomarker corresponds to RAB5C. In some variations, the biomarker corresponds to RAC2. In some variations, the biomarker corresponds to RAC3. In some variations, the biomarker corresponds to RALA. In some variations, the biomarker corresponds to RBMS1. In some variations, the biomarker corresponds to RDX. In some variations, the biomarker corresponds to RELN. In some variations, the biomarker corresponds to RHOC. In some variations, the biomarker corresponds to RHOG. In some variations, the biomarker corresponds to RHOT1. In some variations, the biomarker corresponds to ROBO2. In some variations, the biomarker corresponds to ROBO2. In some variations, the biomarker corresponds to RPS6KA3. In some variations, the biomarker corresponds to SGTA. In some variations, the biomarker corresponds to SHANK3. In some variations, the biomarker corresponds to SHC1. In some variations, the biomarker corresponds to SHC1. In some variations, the biomarker corresponds to SNCG. In some variations, the biomarker corresponds to SPARCL1. In some variations, the biomarker corresponds to SPARCL1. In some variations, the biomarker corresponds to SRC. In some variations, the biomarker corresponds to SRC. In some variations, the biomarker corresponds to STAT1. In some variations, the biomarker corresponds to STAT3. In some variations, the biomarker corresponds to STAT3. In some variations, the biomarker corresponds to STX1A. In some variations, the biomarker corresponds to STXBP6. In some variations, the biomarker corresponds to SYT4. In some variations, the biomarker corresponds to SYT6. In some variations, the biomarker corresponds to THBS2. In some variations, the biomarker corresponds to THBS2. In some variations, the biomarker corresponds to TLN2. In some variations, the biomarker corresponds to TNNI2. In some variations, the biomarker corresponds to TRIM72. In some variations, the biomarker corresponds to UBXN4. In some variations, the biomarker corresponds to UCN3. In some variations, the biomarker corresponds to VAV1. In some variations, the biomarker corresponds to VAV3. In some variations, the biomarker corresponds to VTI1A. In some variations, the biomarker corresponds to VWF. In some variations, the biomarker corresponds to YAP1. In some variations, the biomarker corresponds to LCK.

[0149] It will be recognized that the abundance can also be detected by measuring the expression instead of protein abundance.Treating Stroke, ICH, and TBIP390200. W0.01

[0150] The disclosure is directed to one or more biomarkers that can be used to assess whether a stroke, ICH, or TBI subject respond to GDF11. Further, the disclosure is directed to one or more biomarkers that can be used to assess whether stroke, ICH, or TBI subjects are not responsive to GDF11.

[0151] In instances where a higher or lower abundance of a biomarker is measured, the biomarker alone or in combination with one or more other biomarkers can be detected or measured as part of any method described herein.

[0152] In some variations, after administration of the GDF11 to a subject, a response to GDF11 corresponds to increased abundance of biomarkers having increased abundance, as described above. In some variations, a response to GDF 11 corresponds to increased abundance of two or more, alternatively three or more, alternatively four or more, alternatively five or more, alternatively six or more, alternatively seven or more, alternatively eight or more, alternatively nine or more, alternatively ten or more.

[0153] In some variations, after administration of the GDF11 to a subject, a response to GDF11 corresponds to a decreased abundance of one or more biomarker having a decreased abundance, as described above. In some variations, a response to GDF11 corresponds to decreased abundance of two or more, alternatively three or more, alternatively four or more, alternatively five or more, alternatively six or more, alternatively seven or more, alternatively eight or more, alternatively nine or more, alternatively ten or more, or each biomarker.

[0154] The biomarker abundance can be determined from whole blood, blood serum, blood cells in cerebrospinal fluid (CSF), blood-derived pluripotent stem cells (iPSCs), and others. Alternatively, the abundance can be measured from different cell types that are separated, isolated, and / or increased in concentration in a sample. The sample may be subjected to processing following sample collection, such as cell lysis. In some variations, the expression of biomarkers can be determined from whole blood.

[0155] Another variation of the disclosure is a method of treating stroke, ICH, or TBI in a subject in need thereof by administering a therapeutically effective amount of GDF11. In certain embodiments, the one or more of the biomarkers are increased or decreased in the subject, as described herein. Yet another variation of the disclosure is use of a therapeutically effective amount of GDF11 for treating stroke, ICH, or TBI in a subject having increased abundance or decreased abundance of any of the biomarkers disclosed herein. In yet another variation of the disclosure is use of a therapeutically effective amount of GDF11 in the manufacture of a medicament for treating stroke, ICH, or TBI in a subject having increased or decreased abundance, of one or more biomarkers, as described herein.

[0156] As described further in the examples below, it has been surprisingly found that determining an increased abundance of one or more biomarkers disclosed herein providesP390200. W0.01increased efficacy of GDF11 to treat stroke, ICH, or TBI subjects. In some variations, those subjects have been previously treated with a dosing regiment of GDF11. In some variations, those subjects with increased or decreased abundance of the one or more biomarkers had experienced an improvement in one or more stroke, ICH, or TBI symptoms, as described herein.

[0157] There are many suitable methods that may be used to determine a biomarker abundance. Measurement of Somascan Aptamers described herein, for example, can be used to determine biomarker abundance. In various aspects, the methods may use a linear regression model. In another alternative, the level of biomarker expression can be compared with a threshold level for each biomarker in question. A suitable threshold level for a biomarker can be determined, for example, using SomaScan Assay data or qPCR expression data, and / or machine learning methods (such as logistic regression, support vector machines, or decision tree-based methods) to establish an abundance threshold that allows maximal separation of subjects.

[0158] Alternatively, the median abundance level of one or more biomarkers may be used as a control value, wherein the group consisted of subjects, in some variations at least 100, at least 50, or at least 10 subjects. For biomarkers having an increased abundance in Table 1, GDF11 is administered if these biomarkers are statistically above the median abundance level. For biomarkers having a decreased abundance in Table 1, GDF11 is administered if these biomarkers are statistically below the median abundance level.

[0159] For biomarkers having an increased abundance in Table 1, if the one or more biomarkers are measured to be below the abundance level, GDF11 may not be administered. Likewise, for biomarkers having an decreased abundance in Table 1, if the one or more biomarkers are measured to be above the abundance level, GDF11 may not be administered.

[0160] The level of abundance of biomarkers may be determined by any methods known in the art. As described herein, a SomaScan® Assay (SomaLogic Inc., Boulder, CO) can be performed. SOMAscan and related methods and reagents are described, for example, at U. S. Patent Nos. 5,843,653; 5,853,984; 5,989,823; 6,261,783; 6,329, 145; 6,531,286; 6,670, 132; 6,673,553; 6,706,482; 7,709, 192; 7,855,054; 7,964,356; 8,975,026 and 8,945,830, which are incorporated by reference in their entirety. Alternative, an expression level can be measured. For example, suitable techniques include: real time quantitative PCR (RT-qPCR), digital PCR, microarray analysis, whole transcriptome shotgun sequencing (RNA-SEQ), RNA-Seq by Expectation-Maximization (RSEM), and direct multiplexed gene expression analysis. A method of the disclosure may therefore comprise bringing a whole blood or blood serum sample obtained from an subject into contact with a reagent suitable for determining biomarker expression levels e.g. a reagent or reagents suitable forP390200. W0.01determining the expression level of two or more of said genes using RT-qPCR, digital PCR, microarray analysis, whole transcriptome shotgun sequencing, direct multiplexed gene expression analysis, ELISA, protein chips, flow cytometry, mass spectrometry, or Western blotting. For example, the reagent may be a pair or pairs of nucleic acid primers, suitable for determining the expression level of one or more of said genes using RT-qPCR, digital PCR, or whole transcriptome shotgun sequencing. Alternatively, the reagent may be an antibody suitable for determining the expression level of said one or more genes using ELISA or Western blotting. The level of expression of said genes is determined using RT-qPCR, digital PCR, microarray analysis, whole transcriptome shotgun sequencing, or direct multiplexed gene expression analysis. Alternatively level of expression of said genes is determined using RT-qPCR.

[0161] RT-qPCR allows amplification and simultaneous quantification of a target DNA molecule. To analyze gene expression levels using RT-qPCR, the total mRNA of a whole blood sample may first be isolated and reverse transcribed into cDNA using reverse transcriptase. For example, mRNA levels can be determined using e.g. Taqman Gene Expression Assays (Applied Biosystems) on an ABI PRISM 7900HT instrument according to the manufacturer’s instructions. Transcript abundance can then be calculated by comparison to a standard curve.

[0162] Digital PCR can also be used to detect biomarkers. Digital PCR works by partitioning a sample of DNA or cDNA into many subject, parallel PCR reactions; some of these reactions contain the target molecule (positive) while others do not (negative). A single molecule can be amplified a million-fold or more. During amplification, TaqMan® chemistry with dye-labeled probes is used to detect sequence-specific targets. When no target sequence is present, no signal accumulates. Following PCR analysis, the fraction of negative reactions is used to generate an absolute count of the number of target molecules in the sample, without the need for standards or endogenous controls. The use of a nanofluidic chip provides a convenient and straightforward mechanism to run thousands of PCR reactions in parallel. Each well is loaded with a mixture of sample, master mix, and TaqMan® Assay reagents, and analyzed to detect the presence (positive) or absence (negative) of an endpoint signal. To account for wells that may have received more than one molecule of the target sequence, a correction factor is applied using the Poisson model.

[0163] RNA-SEQ uses next-generation sequencing (NGS) for the detection and quantification of RNA in a biological sample at a given moment in time. An RNA library is prepared, transcribed, fragmented, sequenced, reassembled and the sequence or sequences of interest quantified.

[0164] NanoString technology uses unique color-coded molecular barcodes that can hybridize directly to many different types of target nucleic acid molecules, and offers a cost-P390200. W0.01effective way to analyze the expression levels of up to 800 genes simultaneously, with sensitivity comparable to qPCR.

[0165] Flow-FISH for RNA employs flow cytometry to determine the abundance of a target mRNA within a sample using fluorescently-tagged RNA oligos. This technique is described, for example, in Porichis et al., Nat Comm (2014) 5:5641. The advantage of this technique is that it can be used without the need to separate the cells present in a sample.

[0166] Microarrays allow gene expression in two samples to be compared. Total RNA is first isolated from, e.g. PBMCs or whole blood using, for example, Trizol or an RNeasy mini kit (Qiagen). The isolated total RNA is then reverse transcribed into double-stranded cDNA using reverse transcriptase and polyT primers and labelled using e.g. Cy3- or Cy5-dCTP. Appropriate Cy3- and Cy5-labelled samples are then pooled and hybridized to custom spotted oligonucleotide microarrays comprised of probes representing suitable genes and control features, such as the microarray described in (Willcocks et al., J Exp Med 205, 1573-82, 2008). Samples may be hybridized in duplicate, using a dye-swap strategy, against a common reference RNA derived from pooled PBMC or whole blood samples. Following hybridization, arrays are washed and scanned on e.g. an Agilent G2565B scanner. Suitable alternatives to the steps described above are well known in the art and would be apparent to the skilled person. The raw microarray data obtained can then be analyzed using suitable methods to determine the relative expression of any biomarkers disclosed herein.

[0167] Alternatively, enzyme-linked immunosorbent assays (ELISAs) allow the relative amounts of proteins present in a sample to be detected. The sample is first immobilized on a solid support, such as a polystyrene microtiter plate, either directly or via an antibody specific for the protein of interest. After immobilization, the antigen is detected using an antibody specific for the target protein. Either the primary antibody used to detect the target protein may be labelled to allow detection, or the primary antibody can be detected using a suitably labelled secondary antibody. For example, the antibody may be labelled by conjugating the antibody to a reporter enzyme. In this case, the plate developed by adding a suitable enzymatic substrate to produce a visible signal. The intensity of the signal is dependent on the amount of target protein present in the sample.

[0168] Protein chips, also referred to as protein arrays or protein microarrays, allow the relative amounts of proteins present in a sample to be detected. Different capture molecules may be affixed to the chip. Examples include antibodies, antigens, enzymatic substrates, nucleotides, and other proteins. Protein chips can also contain molecules that bind to a range of proteins. Protein chips are well known in the art and many different protein chips are commercially available.

[0169] Western blotting also allows the relative amounts of proteins present in a sample to be determined. The proteins present in a sample are first separated using gelP390200. W0.01electrophoresis. The proteins are then transferred to a membrane, e.g. a nitrocellulose or PVDF membrane, and detected using monoclonal or polyclonal antibodies specific to the target protein. Many different antibodies are commercially available and methods for making antibodies to a given target protein are also well established in the art. To allow detection, the antibodies specific for the protein(s) of interest, or suitable secondary antibodies, may, for example, be linked to a reporter enzyme, which drives a colorimetric reaction and produces a color when exposed to an appropriate substrate. Other reporter enzymes include horseradish peroxidase, which produces chemiluminescence when provided with an appropriate substrate. Antibodies may also be labelled with suitable radioactive or fluorescent labels. Depending on the label used, protein levels may be determined using densitometry, spectrophotometry, photographic film, X-ray film, or a photosensor.

[0170] Flow cytometry allows the relative amounts of proteins present in e.g. a PBMC or whole blood sample obtained from a subject to be determined. Flow cytometry can also be used to detect or measure the level of expression of a protein of interest on the surface of cells. Detection of proteins and cells using flow cytometry normally involves first attaching a fluorescent label to the protein or cell of interest. The fluorescent label may for example be a fluorescently-labelled antibody specific for the protein or cell of interest. Many different antibodies are commercially available and methods for making antibodies specific for a protein of interest are also well established in the art.

[0171] Mass spectrometry, e.g. matrix-assisted laser desorption / ionization (MALDI) mass spectrometry, allows the identification of proteins present in a sample obtained from a subject using e.g. peptide mass finger printing. Prior to mass spectrometry the proteins present in the sample may be isolated using gel electrophoresis, e.g. SDS-PAGE, size exclusion chromatography, or two-dimensional gel electrophoresis.Kits

[0172] Also disclosed is a kit for use in determining one or more biomarkers. The kit may include reagents for establishing the biomarker(s) expression levels. The kit may include reagents for establishing the abundance of three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, or ten or more biomarkers. For example, the reagents may be associated with the SomaScan methods. Alternatively, the reagents may be reagents suitable for establishing the expression of the genes in question using any technique described herein, such as RT-qPCR, digital PCR, microarray analysis, whole transcriptome shotgun sequencing, or direct multiplexed gene expression analysis. For example, the kit may comprise primers suitable for establishing the level of expression of the genes in question using e.g. RT-qPCR, digital PCR, whole transcriptome shotgun sequencing, or direct multiplexed gene expression analysis. The design of suitable primers is routine and well within the capabilities of the skilled person. A kit for direct multiplexedP390200. W0.01gene expression analysis may in addition, or alternatively, include fluorescent probes for establishing the level of expression of the genes in question. In addition to detection reagents, the kit may also include RNA extraction reagents and / or reagents for reverse transcription of RNA into cDNA.

[0173] A kit may also include one or more articles and / or reagents for performance of the method, such as buffer solutions, and / or means for obtaining the test sample itself, e.g. means for obtaining and / or isolating a sample and sample handling containers (such components generally being sterile). The kit may include instructions for use of the kit in a method for assessing whether to administer an GDF11 described herein to a subject.TABLE 1: BiomarkersSomaScan Aptamer Protein Name Up / DownSequence ID UniProtID Gene Symbol W / rGDF11 Day p-value 18922-27 Macrosialin P34810 CD68 Down Day 5 0.00037 4337-49 C-reactive protein P02741 CRP Down Day 5 0.0081 25306-51 DAXX Q9UER7 DAXX Down Day 5 0.000013Dihydrolipoyldehydrogenase,10025-1 mitochondrial P09622 DLD Down Day 5 0.033 10801-11 Ephrin-A2 043921 EFNA2 Down Day 5 0.0026 14124-6 Ephrin-A2 043921 EFNA2 Down Day 5 0.0061 22580-29 Ephrin-A2 043921 EFNA2 Down Day 5 0.038 2615-60 Ephrin-A5 P52803 EFNA5 Down Day 5 0.043Coagulation factor XIII5658-64 B chain P05160 F13B Down Day 5 0.0086Fibroblast growth2443-10 factor 8 isoform B P55075 FGF8 Down Day 5 0.000012Fibroblast growth19570-12 factor 8 P55075 FGF8 Down Day 5 0.0065P390200. W0.01SomaScan Aptamer Protein Name Up / DownSequence ID UniProtID Gene Symbol W / rGDF11 Day p-value Fibroblast growth17166-4 factor 8 isoform F P55075 FGF8 Down Day 5 0.033Fibroblast growth14757-144 factor 8 isoform B P55075 FGF8 Down Day 3 0.017Fibrinogen-like protein5581-28 1 Q08830 FGL1 Down Day 5 0.0046Secreted frizzled- 2841-13 related protein 3 Q92765 FRZB Down Day 5 0.012Gastric inhibitory5755-29 polypeptide P09681 GIP Down Day 2 0.0074Golgi SNAP receptor10426-21 complex member 2 O14653 GOSR2 Down Day 3 0.015-hydroxytryptamine13547-5 receptor 7 P34969 HTR7 Down Day 5 0.00045 13738-8 Inhibin beta A chain P08476 INHBA Down Day 5 0.00032 19622-7 ActivinA P08476 INHBA Down Day 5 0.00077 2748-3 ActivinA P08476 INHBA Down Day 5 0.001 5193-51 GTPase KRas P01116 KRAS Down Day 5 0.0039P390200. W0.01SomaScan Aptamer Protein Name Up / DownSequence ID UniProtID Gene Symbol W / rGDF11 Day p-value Dual specificitymitogen-activated5242-37 protein kinase kinase 4 P45985 MAP2K4 Down Day 5 0.00039 7655-11 N-terminal pro-BNP P16860 NPPB Down Day 5 0.00011Brain natriuretic3723-1 peptide 32 P16860 NPPB Down Day 5 0.03Group XI IB secretoryphospholipase A2-like9380-2 protein Q9BX93 PLA2G12B Down Day 5 0.013Queuine tRNA- 21380-77 ribosyltransferase Q9BXR0 QTRT1 Down Day 5 0.0000057Reticulon-4 receptor- 20546-71 like 1 Q86UN2 RTN4RL1 Down Day 5 0.0062 9099-19 Synaptotagmin-5 O00445 SYT5 Down Day 5 0.0005UBX domaincontaining protein4: Cytoplasmic domain9970-7 2 Q92575 UBXN4 Down Day 5 0.000016P390200. W0.01SomaScan Aptamer Protein Name Up / DownSequence ID UniProt ID Gene Symbol W / rGDF11 Day p-value Acetyl-CoAacetyltransferase,19197-95 mitochondrial P24752 ACAT1 Up Day 5 0.0096 9843-5 Alpha-actinin-1 P12814 ACTN1 Up Day 5 0.016 9844-138 Alpha-actinin-2 P35609 ACTN2 Up Day 5 0.0079 15312-14 Protein argonaute-1 Q9UL18 AGO1 Up Day 5 0.0011 15323-112 Protein argonaute-3 Q9H9G7 AGO3 Up Day 5 0.00074RAC-alphaserine / threonine- 15627-83 protein kinase P31749 AKT1 Up Day 5 0.000062RAC-alphaserine / threonine- 2867-52 protein kinase P31749 AKT1 Up Day 5 0.0024Proline-rich AKT123302-19 substrate 1 Q96B36 AKT1S1 Up Day 5 0.01RAC-betaserine / threonine- 5360-9 protein kinase P31751 AKT2 Up Day 5 0.0041P390200. W0.01SomaScan Aptamer Protein Name Up / DownSequence ID UniProtID Gene Symbol W / rGDF11 Day p-value RAC-betaserine / threonine- 14685-17 protein kinase P31751 AKT2 Up Day 5 0.016Delta-aminolevulinic15523-9 acid dehydratase P13716 ALAD Up Day 5 0.000027Fructose-bisphosphate18185-118 aldolase B P05062 ALDOB Up Day 5 0.00059 2811-27 Angiopoietin-1 Q15389 ANGPT1 Up Day 5 0.013 2602-2 Angiopoietin-2 015123 ANGPT2 Up Day 5 0.0054 13660-76 Angiopoietin-2 015123 ANGPT2 Up Day 5 0.0055AP-2 complex subunit13621-31 alpha-2 O94973 AP2A2 Up Day 5 0.0038Serine / threonine- 12583-77 protein kinase A-Raf P10398 ARAF Up Day 5 0.0012Rho GTPase-activating14748-31 protein 5 Q13017 ARHGAP5 Up Day 5 0.0000052Actin-related protein13573-5 2 / 3 complex subunit 3 O15145 ARPC3 Up Day 5 0.018P390200. W0.01SomaScan Aptamer Protein Name Up / DownSequence ID UniProtID Gene Symbol W / rGDF11 Day p-value Bcl2-associated5870-23 agonist of cell death Q92934 BAD Up Day 5 0.0084Voltage-dependent L- type calcium channel24237-115 subunit beta-3 P54284 CACNB3 Up Day 5 0.0069Voltage-dependent L- type calcium channel11130-158 subunit beta-4 O00305 CACNB4 Up Day 5 0.006Calcium / calmodulin- dependent proteinkinase type II subunit3351-1 beta Q13554 CAMK2B Up Day 5 0.000044Cell division control9840-2 protein 42 homolog P60953 CDC42 Up0.0024 2819-23 Cadherin-5 P33151 CDH5 Up Day 5 0.045 7959-34 Cadherin-7 Q9ULB5 CDH7 Up Day 5 0.0025 4203-50 Cofilin-1 P23528 CFL1 Up Day 5 0.0035COP9 signalosome14029-42 complex subunit 2 P61201 COPS2 Up Day 5 0.0036 15321-8 Complexin-2 Q6PUV4 CPLX2 Up Day 5 0.0037P390200. W0.01SomaScan Aptamer Protein Name Up / DownSequence ID UniProt ID Gene Symbol W / rGDF11 Day p-value 4976-57 Adapter molecule crk P46108 CRK Up Day 5 0.0019Stromal cell-derived3516-60 factor 1 P48061 CXCL12 Up Day 5 0.0044Dihydrolipoyldehydrogenase,15527-90 mitochondrial P09622 DLD Up Day 5 0.00046Eukaryotic translation20913-27 initiation factor 1 P41567 EIF1 Up Day 5 0.006Eukaryotic translation25105-87 initiation factor 2A Q9BY44 EIF2A Up Day 5 0.0099Eukaryotic translationinitiation factor 219259-176 subunit 1 P05198 EIF2S1 Up Day 5 0.0063Eukaryotic translationinitiation factor 224723-58 subunit 2 P20042 EIF2S2 Up Day 5 0.0066Eukaryotic initiation18829-4 factor 4A-I P60842 EIF4A1 Up Day 5 0.0085Eukaryotic initiation22984-10 factor 4A-II Q14240 EIF4A2 Up Day 5 0.00039P390200. W0.01SomaScan Aptamer Protein Name Up / DownSequence ID UniProtID Gene Symbol W / rGDF11 Day p-value Eukaryotic initiation18824-7 factor 4A-II Q14240 EIF4A2 Up Day 5 0.026Eukaryotic translationinitiation factor 425287-7 gamma 1 Q04637 EIF4G1 Up Day 5 0.0066Eukaryotic translationinitiation factor 44230-1 gamma 2 P78344 EIF4G2 Up Day 5 0.0071Ephrin type-A receptor6036-78 10 Q5JZY3 EPHA10 Up Day 5 0.011 8039-41 Protein FAM177A1 Q8N128 FAM177A1 Up Day 5 0.0095Fibroblast growth4393-3 factor 16 043320 FGF16 Up Day 5 0.0083Fibroblast growth4130-71 factor 6 P10767 FGF6 Up Day 5 0.017Fibroblast growth4487-1 factor 7 P21781 FGF7 Up Day 5 0.0087Fibroblast growth4394-71 factor 8 isoform A P55075 FGF8 Up Day 5 0.029P390200. W0.01SomaScan Aptamer Protein Name Up / DownSequence ID UniProtID Gene Symbol W / rGDFll Day p-value Fibrinogen gamma4989-7 chain P02679 FGG Up Day 7 0.044Fumarate hydratase,13384-110 mitochondrial P07954 FH Up Day 5 0.0011Filamin-A: Calponin11171-25 Homology 2 P21333 FLNA Up Day 5 0.023Filamin-A: Calponin11245-43 Homology 1 P21333 FLNA Up Day 5 0.044Leucine-rich repeattransmembrane13122-19 protein FLRT2 043155 FLRT2 Up Day 5 0.0000024Fragile X mental7713-102 retardation protein 1 Q06787 FMR1 Up Day 5 0.013Fragile X mental7713-50 retardation protein 1 Q06787 FMR1 Up Day 7 0.015Secreted frizzled- 13740-51 related protein 3 Q92765 FRZB Up Day 5 0.000002Growth / differentiation14587-16 factor 11 095390 GDF11 Up Day 5 0.0019P390200. W0.01SomaScan Aptamer Protein Name Up / DownSequence ID UniProt ID Gene Symbol W / rGDFll Day p-value Growth / differentiation 095390 | O 0.0000004 2765-4 factor 11 / 8 14793 GDF11| MSTN Up Day 5 7Aspartateaminotransferase,18233-10 mitochondrial P00505 GOT2 Up Day 5 0.0018Aspartateaminotransferase,23903-3 mitochondrial P00505 GOT2 Up Day 5 0.0054Growth factor receptor- 5464-52 bound protein 2 P62993 GRB2 Up Day 5 0.0095Glycogen synthase3236-12 kinase-3 beta P49841 GSK3B Up Day 5 0.0018Glycogen synthase24050-26 kinase-3 beta P49841 GSK3B Up Day 5 0.0041 18900-37 GTPase HRas P01112 HRAS Up Day 5 0.023Serine protease3317-33 HTRA2, mitochondrial 043464 HTRA2 Up Day 5 0.00067Isochorismatasedomain-containing9816-37 protein 1 Q96CN7 ISOC1 Up Day 5 0.00022P390200. W0.01SomaScan Aptamer Protein Name Up / DownSequence ID UniProt ID Gene Symbol W / rGDFll Day p-value Tyrosine-protein kinase11816-84 JAK2 060674 JAK2 Up Day 3 0.07 20073-22 GTPase KRas P01116 KRAS Up Day 5 0.017Tyrosine-protein kinase3453-87 Lyn P07948 LYN Up Day 5 0.023Tyrosine-protein kinase3381-24 Lyn, isoform B P07948 LYN Up Day 5 0.043Dual specificitymitogen-activated2864-2 protein kinase kinase 1 Q02750 MAP2K1 Up Day 5 0.0022Dual specificitymitogen-activated22041-26 protein kinase kinase 5 Q13163 MAP2K5 Up Day 5 0.0065Mitogen-activated3115-64 protein kinase 1 P28482 MAPK1 Up Day 5 0.0091Mitogen-activated5007-1 protein kinase 14 Q16539 MAPK14 Up Day 5 0.0084Mitogen-activated2855-49 protein kinase 3 P27361 MAPK3 Up Day 5 0.0013P390200. W0.01SomaScan Aptamer Protein Name Up / DownSequence ID UniProt ID Gene Symbol W / rGDFll Day p-value Mitogen-activated15604-18 protein kinase 9 P45984 MAPK9 Up Day 5 0.0000076Mitogen-activated9760-13 protein kinase 9 P45984 MAPK9 Up Day 5 0.00012Alpha-1, 6- mannosylglycoprotein6-beta-N- acetylglucosaminyltran21768-9 sferase A Q09328 MGAT5 Up Day 5 0.0000054Alpha-1, 6- mannosylglycoprotein6-beta-N- acetylglucosaminyltran21813-171 sferase A Q09328 MGAT5 Up Day 5 0.000039MAP kinase-interactingserine / threonine- 23693-4 protein kinase 1 Q9BUB5 MKNK1 Up Day 5 0.007Multiple PDZ domain14036-116 protein 075970 MPDZ Up Day 5 0.012Myosin regulatory light19373-3 chain 12A P19105 MYL12A Up Day 5 0.0046P390200. W0.01SomaScan Aptamer Protein Name Up / DownSequence ID UniProt ID Gene Symbol W / rGDFll Day p-value Myosin regulatory light19122-47 chain 12B 014950 MYL12B Up Day 5 0.0088Myosin light20105-7 polypeptide 6 P60660 MYL6 Up Day 5 0.004Myosin, light chain 9,21817-5 regulatory P24844 MYL9 Up Day 5 0.0079Nucleosome assembly13636-20 protein 1-like 1 P55209 NAP1L1 Up Day 5 0.0042 nucleosome assembly19188-21 protein 1-like 4 Q99733 NAP1L4 Up Day 5 0.0045Alpha-soluble NSF4292-5 attachment protein P54920 NAPA Up Day 5 0.0098Nectin-1, isoformgamma: Extracellular20584-4 domain Q15223 NECTIN1 Up Day 5 0.000049Nuclear factor NF- 9869-28 kappa-B pl05 subunit P19838 NFKB1 Up Day 5 0.00061 8778-3 Noggin Q13253 NOG Up Day 5 0.011 5846-24 Noggin Q13253 NOG Up Day 5 0.014P390200. W0.01SomaScan Aptamer Protein Name Up / DownSequence ID UniProtID Gene Symbol W / rGDFll Day p-value 5542-22 Neuropilin-1 014786 NRP1 Up Day 5 0.0055 3214-3 Neuropilin-1 014786 NRP1 Up Day 5 0.024 6590-54 Neuropilin-2 060462 NRP2 Up Day 5 0.000012 15387-44 Neuropilin-2 060462 NRP2 Up Day 5 0.0089 8295-16 Noelin-2 095897 OLFM2 Up Day 5 0.000014 19377-14 Noelin-2 095897 OLFM2 Up Day 5 0.00004Serine / threonine- 13719-19 protein kinase PAK 4 096013 PAK4 Up Day 5 0.0004Pyruvatedehydrogenase Elcomponent subunitalpha, testis-specific25249-33 form, mitochondrial P29803 PDHA2 Up Day 5 0.00737832-181 Protein disulfide- isomerase A3 P30101 PDIA3 Up Day 3 0.06 Phosphatidylinositol 3- kinase catalytic25947-116 subunit type 3 Q8NEB9 PIK3C3 Up Day 5 0.012P390200. W0.01SomaScan Aptamer Protein Name Up / DownSequence ID UniProtID Gene Symbol W / rGDFll Day p-value Cytosolicphospholipase A221430-4 alpha P47712 PLA2G4A Up Day 5 0.00541-phosphatidylinositol4,5-bisphosphatephosphodiesterase11354-21 beta-1 Q9NQ66 PLCB1 Up Day 5 0.000851-phosphatidylinositol- 4,5-bisphosphatephosphodiesterase24909-40 delta-1 P51178 PLCD1 Up Day 5 0.0281-phosphatidylinositol4,5-bisphosphatephosphodiesterase25949-3 delta-3 Q8N3E9 PLCD3 Up Day 5 0.00871-phosphatidylinositol4,5-bisphosphatephosphodiesterase10070-22 gamma-2 P16885 PLCG2 Up Day 5 0.024Calcineurin subunit B15545-13 type 1 P63098 PPP3R1 Up Day 5 0.00024P390200. W0.01SomaScan Aptamer Protein Name Up / DownSequence ID UniProt ID Gene Symbol W / rGDFll Day p-value cAMP-dependentprotein kinase catalytic3466-8 subunit alpha P17612 PRKACA Up Day 5 0.011 cAMP-dependentprotein kinase type I- alpha regulatory21483-155 subunit P10644 PRKAR1A Up Day 5 0.0053 cAMP-dependentprotein kinase type I- 12479-50 beta regulatory subunit P31321 PRKAR1B Up Day 5 0.02 cAMP-dependentprotein kinase type II- 25463-3 beta regulatory subunit P31323 PRKAR2B Up Day3 0.022Protein kinase C alpha2644-11 type P17252 PRKCA Up Day 5 0.0012Protein kinase C betatype (splice variant5475-10 beta-ll) P05771 PRKCB Up Day 5 0.00038Protein kinase C iota3379-29 type P41743 PRKCI Up Day 5 0.0034P390200. W0.01SomaScan Aptamer Protein Name Up / DownSequence ID UniProt ID Gene Symbol W / rGDFll Day p-value Ras-related protein9504-19 Rab-27A P51159 RAB27A Up Day 5 0.043Ras-related protein17516-7 Rab-3A P20336 RAB3A Up Day 5 0.025Ras-related protein23329-52 Rab-4B P61018 RAB4B Up Day 5 0.0099Ras-related protein17205-21 Rab-5A P20339 RAB5A Up Day 5 0.0037Ras-related protein19222-124 Rab-5B P61020 RAB5B Up Day 5 0.0042Ras-related protein14287-6 Rab-5C P51148 RAB5C Up Day 5 0.0033Ras-related C3botulinum toxin18950-13 substrate 2 P15153 RAC2 Up Day 5 0.0048Ras-related C3botulinum toxin16769-20 substrate 3 P60763 RAC3 Up Day 5 0.019Ras-related protein14332-3 Ral-A P11233 RALA Up Day 5 0.017P390200. W0.01SomaScan Aptamer Protein Name Up / DownSequence ID UniProt ID Gene Symbol W / rGDFll Day p-value RNA-binding motif,single-stranded- 0.0000008 22547-17 interacting protein 1 P29558 RBMS1 Up Day 5 6 18373-13 Radixin P35241 RDX Up Day 5 0.0056 21713-11 Reelin P78509 RELN Up Day 5 0.011Rho-related GTP- 17764-108 binding protein RhoC P08134 RHOC Up Day 5 0.000036Rho-related GTP- 12540-25 binding protein RhoG P84095 RHOG Up Day 5 0.025Mitochondrial Rho21752-10 GTPase 1 Q8IXI2 RHOT1 Up Day 5 0.0058 22578-17 Roundabout homolog 2 Q9HCK4 ROBO2 Up Day 5 0.0000034 5116-62 Roundabout homolog 2 Q9HCK4 ROBO2 Up Day 5 0.000012Ribosomal protein S63469-74 kinase alpha-3 P51812 RPS6KA3 Up Day 5 0.0015Small glutamine-richtetratricopeptiderepeat-containing3868-8 protein alpha 043765 SGTA Up Day 5 0.0028P390200. W0.01SomaScan Aptamer Protein Name Up / DownSequence ID UniProt ID Gene Symbol W / rGDFll Day p-value SH3 and multipleankyrin repeat domains13242-134 protein 3 Q9BYB0 SHANK3 Up Day 5 0.018SHC-transformingproteinl: Phosphotyrosine16043-30 Interaction Domain P29353 SHC1 Up Day 5 0.00073SHC-transformingprotein 1:Src Homology5272-55 domain P29353 SHC1 Up Day 5 0.0023 19630-2 Gamma-synuclein 076070 SNCG Up Day 5 0.0015 13707-27 SPARC-like protein 1 Q14515 SPARCL1 Up Day 5 0.00022 4467-49 SPARC-like protein 1 Q14515 SPARCL1 Up Day 5 0.0017Proto-oncogenetyrosine-protein kinase15433-4 Src P12931 SRC Up Day 5 0.00086Proto-oncogenetyrosine-protein kinase5488-74 Src isoform 2 P12931 SRC Up Day 5 0.014 12351-25 Signal transducer and P42224 STAT1 Up Day 5 0.0015 activator ofP390200. W0.01SomaScan Aptamer Protein Name Up / DownSequence ID UniProt ID Gene Symbol W / rGDFll Day p-value transcription 1- alpha / betaSignal transducer andactivator of10346-5 transcription 3 P40763 STAT3 Up Day 5 0.00032Signal transducer andactivator of10354-57 transcription 3 P40763 STAT3 Up Day 5 0.0018 19553-14 Syntaxin-1A Q16623 STX1A Up Day 7 0.027Syntaxin-binding18308-30 protein 6 Q8NFX7 STXBP6 Up Day 5 0.000021 17355-56 Synaptotagmin-4 Q9H2B2 SYT4 Up Day 5 0.011 21539-139 Synaptotagmin-6 Q5T7P8 SYT6 Up Day 5 0.0028 3339-33 Thrombospondin-2 P35442 THBS2 Up Day 5 0.000036 14111-15 Thrombospondin-2 P35442 THBS2 Up Day 5 0.0024 14082-56 Talin-2 Q9Y4G6 TLN2 Up Day 5 0.0022Troponin I, fast skeletal5440-26 muscle P48788 TNNI2 Up Day 5 0.003P390200. W0.01SomaScan Aptamer Protein Name Up / DownSequence ID UniProt ID Gene Symbol W / rGDFll Day p-value Tripartite motif-containing protein 72 Q6ZMU5 TRIM72 Up Day 5 0.0076UBX domaincontaining protein4: Cytoplasmic domain9997-12 1 Q92575 UBXN4 Up Day 5 0.0015 10756-34 Urocortin-3 Q969E3 UCN3 Up Day 5 0.0018 5275-28 Proto-oncogene vav P15498 VAV1 Up Day 5 0.018Guanine nucleotide9830-109 exchange factor VAV3 Q9UKW4 VAV3 Up Day 5 0.02Vesicle transportthrough interactionwith t-SNAREs7952-2 homolog 1A Q96AJ9 VTI1A Up Day 5 0.0071 3050-7 von Willebrand factor P04275 VWF Up Day 5 0.00069Transcriptional25451-39 coactivator YAP1 P46937 YAP1 Up Day 5 0.0067Tyrosine-protein kinase4560-34 Lek P06239 LCK Up Day 3 0.0098TABLE 2: SequencesSEQID NO. Sequence1 NLGLDCDEHSSESRCCRYPLTVDFEAFGWDWIIAPKRYKANYCSGQCEYMFMQKYPHTHL VQQANPRGSAG PCCTPTKMSPI N M LYFN DKQQIIYG KI PG MVVDRCGCS2 MVLAAPLLLGFLLLALELRPRGEAAEGPAAAAAAAAAAAAAGVGGERSSRPAPSVAPEPD GCPVCVWRQHSRELRLESIKSQILSKLRLKEAPNISREVVKQLLPKAPPLQQILDLHDFQ GDALQPEDFLEEDEYHATTETVISMAQETDPAVQTDGSPLCCHFHFSPKVMFTKVLKAQL WVYLRPVPRPATVYLQILRLKPLTGEGTAGGGGGGRRHIRIRSLKIELHSRSGHWQSIDF KQVLHSWFRQPQSNWGIEINAFDPSGTDLAVTSLGPGAEGLHPFMELRVLENTKRSRRNL GLDCDEHSSESRCCRYPLTVDFEAFGWDWIIAPKRYKANYCSGQCEYMFMQKYPHTHLVQ QANPRGSAGPCCTPTKMSPINMLYFNDKQQIIYGKIPG MVVDRCGCS3 AEGPAAAAAAAAAAAAAGVGGERSSRPAPSVAPEPDGCPVCVWRQHSRELRLESIKSQIL SKLRLKEAPNISREVVKQLLPKAPPLQQILDLHDFQGDALQPEDFLEEDEYHATTETVIS MAQETDPAVQTDGSPLCCHFHFSPKVMFTKVLKAQLWVYLRPVPRPATVYLQILRLKPLT GEGTAGGGGGGRRHIRIRSLKIELHSRSGHWQSIDFKQVLHSWFRQPQSNWGIEINAFDP SGTDLAVTSLGPGAEGLHPFMELRVLENTKRSRRNLGLDCDEHSSESRCCRYPLTVDFEA FGWDWIIAPKRYKANYCSGQCEYMFMQKYPHTHLVQQANPRGSAGPCCTPTKMSPINMLY F N DKQQI IYG KI PG MVVD RCG CS4 AEGPAAAAAAAAAAAAAGVGGERSSRPAPSVAPEPDGCPVCVWRQHSRELRLESIKSQIL SKLRLKEAPNISREVVKQLLPKAPPLQQILDLHDFQGDALQPEDFLEEDEYHATTETVIS MAQETDPAVQTDGSPLCCHFHFSPKVMFTKVLKAQLWVYLRPVPRPATVYLQILRLKPLT GEGTAGGGGGGRRHIRIRSLKIELHSRSGHWQSIDFKQVLHSWFRQPQSNWGIEINAFDPSGTDLAVTSLGPGAEGLHPFMELRVLENTKRSRRP390200. W0.01EXAMPLES

[0174] The following examples are included to demonstrate embodiments of the disclosure. It should be appreciated by those of skill in the art that the techniques disclosed in the examples that follow represent techniques discovered by the inventors to function well in the practice of the disclosure, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the disclosure Example 1

[0175] Protein Production rGDF11 (recombinant human mature domain dimer) was isolated and purified from mammalian cell culture supernatants either from transient expression in HEK Expi293 cells (ThermoFisher Scientific) or in a CHO cell line (CHOZN® GS- / - ZFN-modified, Millipore Sigma) engineered to stably express rGDF11 generated as described in the supplemental methods. Commercially available E. coli expressed rGDF11 was purchased from Peprotech (Cranbury, NJ) for comparison with internally produced material.Luciferase Assays

[0176] \HEK 293(CAGA)₁₂ cells containing a SMAD 2 / 3 responsive luciferase reporter gene were seeded in 96-well plates, treated with serial dilutions of proteins, and incubated as described in the supplemental methods. EC50 values were generated by analysis of luminescence data.11GDF11 / n Vivo Activity r

[0177] An in vivo activity study assessed exposure of internally produced HEK Expi293 cell expressed rGDF11 protein in C57BI / 6 mice. Intraperitoneal injections were administered, and tissue and serum samples were collected for analysis. Proteins were extracted from tissue samples and analyzed using phospho-Smad2 / 3 (pSmad2 / 3) ELISA and GDF11 serum ELISA assays. Animal work for this study was conducted at an independent CRO (Neosome Life Sciences, Billerica, MA) with animals randomized and investigators blinded to treatment assignment. Tissue and serum samples remained blinded for in-house processing and ELISA analysis.Cardiac hypertrophy, glucose and insulin tolerance

[0178] Male C57BL / 6 mice at 14 weeks old (young) or 84 weeks old (aged) received intraperitoneal (IP) injections of 1 mg / kg rGDF11 (HEK Expi293 cell produced) or vehicle once daily for 15 days. Baseline measurements for the Glucose Tolerance Test (GTT) and Insulin Tolerance Test (ITT) were performed on days -7 and -3, respectively, to be compared with readings on treatment days 10 and 14. Cardiac hypertrophy was assessed byP390200. W0.01calculating the ratio of heart weight to tibial length. All harvested tissues were weighed, measured, and flash-frozen on day 16. This study was conducted at an independent CRO (Neosome Life Sciences, Billerica, MA) with animals randomized and investigators blinded to treatment assignment throughout the study, including end point assessments.Stroke Efficacy Studies

[0179] Ischemic stroke recovery studies evaluating rGDF11 treatment were conducted to assess sensorimotor behavioral changes in 8-10 week old young adult male Sprague-Dawley rats following focal cerebral infarcts induced by permanently occluding the middle cerebral artery (pMCAO) by microbipolar coagulation. Limb placement and body swing tests were performed, with treatment groups blinded to the operator and rats randomized into different pMCAO or sham groups. All ischemic stroke recovery efficacy studies were conducted by an independent CRO (NeuroVasc Preclinical Services, Billerica, MA) employing CHO cell produced rGDF11. Serum samples from these studies were either analyzed in-house (study 5) using CRP and GDF11 ELISAs (supplemental methods, R& D Systems, Minneapolis, MN) or used for Somascan analysis (study 6). For all analyses samples were randomized and investigators blinded to treatment assignment throughout the study.Immunohistochemistry

[0180] Immunofluorescence staining was performed on brain tissue sections using antibodies against specific markers to assess cellular changes following pMCAO. To ensure robustness and validity of results, all samples were randomized and blinded in these analyses, including end point assessments.Proteomic Platform

[0181] Serum proteome profiling was carried out using the SomaScan® Assay (SomaLogic Inc., Boulder, CO), capable of measuring over 7,000 proteins. Serum samples from different time points in the dose optimization study (Study 6) were selected for analysis, and standard procedures were followed for sample preparation and data analysis.12This study was conducted at an independent CRO (SomaLogic Inc., Boulder, CO) and investigators were blinded to treatment assignment throughout the study, including end point assessments.rGDF11 activity in vitro and in vivo.

[0182] A representative Coomassie Blue stained protein gel of rGDF11 expressed and purified from CHO cells is presented in Figure 1A. To compare the potency of in house produced protein generated from transient expression in HEK Expi293 cells (denoted as GDF11 in Figure 1B) against commercial E. coli expressed rGDF11 purchased from Peprotech (Cranbury, NJ; denoted as GDF11-Pep in Figure 1B), cell-based assays employing a SMAD2 / 3 responsive luciferase reporter gene were conducted.11As shown in Figure 1B, internally produced (GDF11) and commercial (GDF11-Pep) rGDF11 hadP390200. W0.01comparable activities as demonstrated by their EC50 values of 0.18 and 0.23nM, respectively. All lots of rGDF11 (HEK Expi293 and CHO expressed) used in the studies presented here were characterized and qualified prior to use and had comparable purity and EC50s (data not shown).

[0183] To assess the in vivo function and biological activity of rGDF11, levels of phosphorylated Smad2 / 3 (pSmad2 / 3) were analyzed by ELISA in four different organs (spleen, liver, heart, and pancreas) of mice (Figure 1C) after a single intraperitoneal (IP) injection of 1 mg / kg purified protein. rGDF11 induced elevation of Smad2 / 3 phosphorylation in all organs tested. Phosphorylation levels were comparable in spleen and liver and began to rise at or before 0.25 hours post-dosing, peaking at approximately 1 hour and returning to baseline at the 12 hour-time point. rGDF11 mediated Smad2 / 3 phosphorylation in the heart was less pronounced than in the liver and spleen. In heart tissue, elevated pSmad2 / 3 levels returned to baseline by 6 hours post-dosing. Smad2 / 3 signaling in pancreatic tissue peaked at approximately 1 hour, was sustained for at least 6 hours and dropped back to baseline by 12 hours. Overall, the levels of pSmad2 / 3 were as follows: Spleen> Liver> Pancreas> Heart. Thus, the SMAD2 / 3 responsive luciferase reporter activity data coupled with in vivo pSmad2 / 3 ELISA data indicate that Epi293 cell produced rGDF11 exhibits high purity and potent activity both in vitro and in vivo, with EC50 comparable to commercially available E. coli-produced rGDF11 protein.rGDF11 decreased cardiac hypertrophy and improved glucose tolerance in aged mice

[0184] In some variations, the dosing regimen, including timing of dosing initiation postinjury, duration of dosing, and dose range improved efficacy in the permanent middle cerebral artery occlusion (pMCAO) model. In a particular variation, a dosing regimen of three intravenous doses of GDF11 are administered once every 48 hours over five days at a concentration of 1 mg / kg in rats after initiation of 24 hours following the stroke event, as described in Figure 3 A-F.

[0185] To validate the activity and efficacy of internally produced rGDF11 in vivo, its effects on cardiac hypertrophy and glucose metabolism in aged mice were evaluated. These effects are well-established experimental models in which rGDF11 effects have been studied previously.1314Young (14 weeks) and old (84 weeks) mice were treated with either vehicle or 1 mg / kg rGDF11 once daily for 15 days via intraperitoneal injection. Prior to treatment, baseline measurements of body weight (Figure 2 B, D), glucose tolerance (Figure 3 A, C) and insulin tolerance (Figure 3 B, D) were recorded. In response to rGDF11 treatment, aged mice exhibited a decrease in body weight, which was reduced by 15.5% at day 10 compared to the first day of treatment (day 1) (Figure 2 C). In contrast, young mice showed a maximum mean weight reduction of 10.1% on day 11. Percent body weight change wasP390200. W0.01significantly decreased for rGDF11 -treated versus vehicle-treated young mice starting on day 4 (Figure 2 E). Some changes in body weight were due to fasting, which commenced 16 hours before the GTT on days -7 and 10 (Figure 4E).

[0186] The heart weight to tibial length (HW / TL) ratios between rGDF11- vs vehicle-treated mice, as well as between aged and young mice (Figure 2A), were compared after 15 days of treatment.314HW / TL ratio was significantly higher in aged vehicle-treated mice compared to young vehicle-treated mice. Aged mice treated with rGDF11 had significantly lower HW / TL ratios compared to aged vehicle-treated mice, consistent with previously published data indicating that rGDF11 decreases age-related cardiac hypertrophy.314There were no differences in HW / TL ratios between rGDF11 -treated aged mice and either vehicle-treated or rGDF11 -treated young mice.

[0187] The in vivo effects of rGDF11 on glucose metabolism using glucose tolerance tests (GTT) and insulin tolerance tests (ITT) were evaluated in the young and aged mice.Consistent with previous findings,13rGDF11 treatment improved glucose tolerance in both young and aged mice (Figure 3 A, C). In aged mice, significant reductions in blood glucose were observed at 15 and 30 minutes after glucose injection in the rGDF11 -treated group compared to the vehicle-treated group. In young mice, significant blood glucose reductions were observed at 30- and 60- minutes post-glucose injection in the rGDF11 -treated group compared to the vehicle-treated group (Figure 3 C). ITT results showed no significant effect of rGDF11 treatment on insulin sensitivity in either age group, while a significant difference was noted at 90 minutes between baseline and day 10 and 14 for rGDF11 -treated aged group (Figure 3 B). These data rigorously validate our rGDF11 in established in vitro and in vivo models. The effects of this protein were studied in a validated model of ischemic stroke.

[0188] Example 2

[0189] Stroke Efficacy Studies

[0190] Ischemic stroke recovery studies evaluating rGDF11 treatment were conducted to assess sensorimotor behavioral changes in 8-10 week old young adult male Sprague-Dawley rats (Charles River Laboratories, Wilmington, MA) following focal cerebral infarcts induced by permanently occluding the middle cerebral artery (pMCAO) by microbipolar coagulation.13 Designs for all six independent stroke studies are described in Figure 23 and the Supplemental Methods. Dosingfor the IP Dose Ranging (Study 4), IV Dose Ranging (Study 5) and IV Dose and Regimen Optimization (Study 6), studies was initiated 24 post injury (pMCAO or Sham) employing daily, intermittent (every 48 hours), or single day dosing regimens for various dosing durations (1, 5, or 7P390200. W0.01days) with behavioral testing performed out to 14, 21, or 28 days and study termination as indicated in Figure 15a.

[0191] Limb placement and body swing tests were performed, with treatment groups blinded to the operator and rats randomized into different pMCAO or sham groups (Supplemental Methods). All ischemic stroke recovery efficacy studies were conducted by an independent CRO (NeuroVasc) employing CHO cell produced rGDF11. Serum samples from these studies were either analyzed in-house (Study 5: IV Dose Ranging) using CRP and GDF11 ELISAs on day 7 (Supplemental Methods, R& D Systems, Minneapolis, MN) or used for SomaScan® analysis on days -1, 2, 3, 5, and 7 (Study 6: IV Dose and Regimen Optimization, Figure 23, Figure 24, and Supplemental Methods). In all studies, animals were either exposed to pMCAO or sham surgery on day 0. For all analyses, samples were randomized and investigators blinded to treatment assignment throughout all the studies.A rGDF11 dosing regimen with optimized efficacy in promoting recovery post ischemic stroke

[0192] T o evaluate the benefits of rGDF 11 and develop an dosing regimen for promoting recovery post-stroke, six independent preclinical ischemic stroke rat experiments were performed, all with blinding and randomization (Figure 23).15

[0193] Figure 23 describes the route of administration (RoA) being either IP (intraperitoneal) or IV (intravenous), the dosing frequency, duration and experimental designs for studies 1-6. pMCAO (permanent middle cerebral artery occlusion); FLP (Forelimb placement); HLP (Hindlimb placement); BS (Body swing); sig days (significant days).

[0194] Young adult male rats were selected for these studies because of their relatively stable hormonal and physiological profiles which minimize variability in key outcome measures such as infarct volume, behavioral recovery, and drug pharmacokinetics.1617Additionally, age-related factors and hormonal cycles in females could introduce confounding variables,18complicating the interpretation of dose-response relationships and therapeutic effects during these studies designed to develop an optimized dosing regimen, characterize mechanism of action, identify pharmacodynamic and mechanistic biomarkers, and define a safety and therapeutic window for rGDF11 in the pMCAO model. Results from all six experiments showed improved sensorimotor function recovery in pMCAO rats treated with rGDF11 compared to vehicle treated controls. Functional behavioral results for three out of six studies are presented in Figure 15b A-C, 16 A-C, 17 A-F, and Figure 4 A-F. All six studies showed efficacy. One dosing regimen outperformed others with respect to improvingP390200. W0.01sensorimotor function (Figure 17 A-F) and mitigating rGDF11-induced reductions in body weight (Figure 5 A-D). Specifically, the dosing regimen administering three intravenous doses of rGDF11 administered once every 48 hours over five days at a concentration of 1 mg / kg and initiated 24 hours following pMCAO performed best (Figure 17 A-F) across the six studies.rGDF11 improved sensorimotor function, increased neural stem cell numbers, and enhanced neovascularization

[0195] Daily doses of vehicle or rGDF11 (0.1, 0.5, 1, 2, and 4 mg / kg) were administered intraperitoneally once daily for five days to pMCAO rats with dosing initiated 24 hours postinjury. Superior and sustained motor function recovery was observed for the 1, 2, and 4 mg / kg-treated rats compared to vehicle-treated animals in all behavioral assays, which included body swing, forelimb placement, and hindlimb placement over the 28 days post-pMCAO study period (Figure 15b A-C). Superior sustained body swing recovery was also observed for the 0.5 mg / kg rGDF11 dose (Figure 15b A). The effect of rGDF11 on neural stem cells post stroke was quantified using immunofluorescence analysis of the ventricular zone at study termination (28 days post-injury). In the ipsilateral ventricular zone, numbers of Sox2 (a marker for neural stem cells)19positive cells were increased for the 1, 2, and 4 mg / kg doses, whereas in the contralateral ventricular zone significant increases in progenitor cell number were only observed for the 2 mg / kg dose (Figure 15b D). This study also evaluated the impact of rGDF11 on vascularization in the peri-infarct region using CD31 staining. Vascular morphometric analysis demonstrated a significant increase in the percentage of vessel area and total vessel length in rats treated with 1 mg / kg of rGDF11 compared to vehicle-treated controls. Additionally, a marginally significant increase in junction density and average vessel length was observed in the 1 mg / kg treated group compared to the vehicle group (Figures 6 A-D).20The peri-infarct region used for vascularization analysis is shown in Figure 7A, along with representative images in Figure 7B.rGDF11 improved sensorimotor function, increased neovascularization, and decreased C-Reactive Protein (CRP) levels

[0196] A study of once daily IV administration of either vehicle alone, or 0.1, 0.3, or 1 mg / kg rGDF11 for 7 days was performed to determine an efficacious dose range in the rat pMCAO model. All three rGDF11 doses showed significant and sustained improvement of motor function in body swing (Figure 16 A) and hindlimb placement tests (Figure 16 C) tests compared to vehicle control, with the 1 mg / kg dose showing the greatest efficacy. In these two tests, motor function was significantly improved as early as day 3 after rGDF11 administration and exhibited persistent improvement through day 21. The 1 mg / kg dose showed superior improvement over the 0.1 and 0.3 mg / kg doses. The 1 mg / kg and 0.3P390200. W0.01mg / kg doses showed significant improvement in the forelimb placement test from day 3 to day 7 (Figure 16 B). The effect of rGDF11 on neovascularization 21 days post-pMCAO was assessed via transcardial perfusion of biotinylated tomato lectin prior to fixation.Immunofluorescent analysis of lectin and CD31 double positive cells indicated a rGDF11 mediated increase in functional vessels for the 0.3 mg / kg and 1 mg / kg doses in the ipsilateral cortex (Figure 16 D). Vascular morphometric analysis was also conducted to evaluate total vessel length, average vessel length, and junction density.20The analyses revealed a marginally significant increase in total vessel length in the 1 mg / kg dose group compared to the vehicle, with no significant changes observed in average vessel length (Figure 8 A, B). A significant increase in junction density was identified in the 0.3 mg / kg dose group compared to the vehicle. Additionally, both the 1 mg / kg and 0.3 mg / kg dose groups showed a marginally significant increase in junction density compared to the 0.1 mg / kg dose group (Figure 8 C). The peri-infarct region used for vascularization analysis is shown in Figure 9 A, along with representative images in Figure 9 B.

[0197] Serum levels of C-reactive protein (CRP) were also measured in order to evaluate the potential impact of rGDF11 on inflammation and to evaluate CRP as a candidate mechanistic biomarker. Serum samples collected 15 minutes after the final rGDF11 administration on day 7 were analyzed by CRP and rGDF11 ELISAs. Results showed a significant dose dependent decrease in CRP levels (Figure 16 E) with increasing rGDF11 treatment doses. rGDF11 ELISA of serum samples confirmed increased rGDF11 exposure in the serum for each dose (Figure 16 F). Pearson Correlation showed a negative correlation trend (r=-0.9190 p-value = 0.0810) of rGDF11 levels with CRP levels in the serum (data not shown). Once daily IV dosing with 1 mg / kg rGDF11 had the largest effect on sensorimotor recovery, neovascularization, and CRP post-pMCAO injury.Improved motor function recovery with acute intermittent dosing of rGDF11

[0198] We further optimized frequency of rGDF11 administration by treating pMCAO injured rats with either a single dose (day 1), three intermittent doses (day 1, 3 and 5), or five repeated daily doses (days 1-5) of 1mg / kg rGDF11. Body swing (Figure 17 A), forelimb placement (Figure 17 B), and hindlimb placement (Figure 17 C) behavioral tests were performed to assess motor function recovery post-stroke (Figure 24).

[0199] Intermittent dosing post-pMCAO showed comparable improvements in functional sensorimotor recovery to those observed for five daily doses as compared to vehicle controls in all functional outcome measures (Figure 17 A-C). Furthermore, intermittent and daily dosing of 0.3 mg / kg and 1 mg / kg showed dose-dependent behavioral improvements when compared to their respective vehicle-treated animals (Figure 17 D-F and Figure 4 A-C). Additionally, dose dependent sensorimotor function recovery was also observed after aP390200. W0.01single IV administration of rGDF11 (0.3 mg / kg and 1 mg / kg) (Figure 4 D-F) although to a lesser extent than intermittent and repeated daily dosing.

[0200] Body weight was systematically studied when considering an appropriate dose regimen. On days 3 through 7 post-stroke, 5 daily doses of rGDF11 showed a greater effect in reducing body weight than intermittent doses on days 1, 3 and 5, when compared to their respective vehicle controls (Figure 5). All dose regimens showed a recovery in body weight by day 14 post-stroke. Taken together, intermittent dosing showed a comparable efficacy and smaller body weight loss to daily dosing, which supported the design of safety and toxicology studies (Figure 25) in both male and female rats using the intermittent dosing approach. No adverse effects were observed in rat (Sprague Dawley) safety and toxicology studies for either sex employing an intermittent dosing regimen with 3 doses over a 5-day period at a concentration up to 10 mg / kg, suggesting a safety window of at least 10-fold above the optimized efficacious dose in rats (Figure 25).

[0201] To extend our findings demonstrating the effect of rGDF11 treatment on circulating stroke-associated inflammatory biomarkers, we performed CRP analysis on longitudinally collected serum from animals treated at 1 mg / kg for each dosing regimen. For the single dose, intermittent dose, and daily dose groups, ELISA analyses were performed on serum samples from pre-stroke (day -1), day 2, day 5, day 7, and day 14. All dosing regimens showed a decrease in circulating CRP levels in response to 1 mg / kg GDF11 treatment compared to vehicle controls, beginning on day 2 (1 day following first treatment) through day 7 (Figure 18 A-D).Serum analysis revealed a panel of stroke-specific biomarkers

[0202] Exogenous rGDF11 triggers signaling pathways that promote spatiotemporal gene expression, protein translation, and secretion.21A Somascan aptamer-based analysis was performed on longitudinally collected serum samples from the daily 1 mg / kg rGDF11 IV and vehicle treatment animal groups. We also included samples from 1 mg / kg intermittent dosing group to identify any overlapping candidates. Additionally, serum samples from sham animals exposed to 1 mg / kg rGDF11 and vehicle were analyzed to distinguish the stroke-associated candidate biomarkers, (see Figure 24 for full study design and samples selected). The number of proteins that surpassed our significance threshold for the pathway analysis (p<0.01 and fold change> |1,5|) was the lowest at day 2 and the highest at day 5 when comparing rGDF11 treatment to vehicle control. Furthermore, data from the intermittent dosing group and sham groups showed a limited number of significant changes compared to the day 5 daily dose group (Figure 26), supporting a decision to limit pathway analysis to the daily dose animals.P390200. W0.01

[0203] Principal component analysis (PCA) was performed from samples collected on days 2, 3 and 5 from both vehicle and 1 mg / kg treated animals from the daily dosing regimen. All samples from vehicle treated animals clustered together with no apparent separation, regardless of number of days of treatment. On the other hand, samples from the 1 mg / kg GDF11 treated animals showed clustering per day of treatment with increasing separation for increasing days of treatment (Figure 19 A). The increased variance of the day 5 daily dosing group that was seen in the PCA was further explored by comparing the volcano plots of GDF11 to Vehicle from day 2, day 3, and day 5 (Figure 19 B-D). The effect of rGDF11 treatment on the circulating proteome increased together with the number of treatment days, with more proteins showing significantly altered abundance at day 5 versus days 2 or 3 of dosing. In addition to the expected dosing-related increase in serum GDF11, several proteins showed consistent changes on all three days: seven proteins were increased (GDF11, Alad, Aldob, Fh, Olfm2, Htra2, and Isocl) and two decreased (Inhba and Rtn4rl1) in the serum on days 2, 3, and 5 in response to rGDF11 treatment (Figure 19 B-D).

[0204] Pathways analysis was performed to compare vehicle- to rGDF11 -treated animals for each day as well as pre-pMCAO to post-pMCAO animals. To maximize analysis power, data from serum collected on days 2, 3, and 5 from the daily dosing group was collected. IPA analysis identified differentially activated signaling pathways, many of which are associated with the hypothesized mechanism of stroke repair, including synaptogenesis, NGF signaling, ERK / MAPK signaling, and IGF-1 signaling (Figure 10). Under the tested comparison analyses thresholds (p-value<0.0001 and z-score>4), day 2 showed no activated pathways for the paired comparison of vehicle vs rGDF11 nor treatment vs pre-pMCAO. Activated pathways for day 3 were seen when rGDF11 was compared to pre-pMCAO animals and to vehicle control. Comparison of vehicle control to pre-pMCAO animals at day 3 did not show a proteomic signature that was associated with any activated pathways. Vehicle-treated animals at day 2 and day 3, when compared to pre-pMCAO animals, showed a trend towards inhibition of the synaptogenesis signaling pathway.

[0205] To further explore the mechanisms by which rGDF 11 -responsive proteins may facilitate stroke recovery, we analyzed proteins that showed a Bonferroni corrected significance for the 1 mg / kg rGDF11 daily dosing group compared to control. Path explorer analysis of these proteins from day 2, day 3, and day 5 predicted activation of functions in angiogenesis, neurogenesis, axon-genesis, and developmental process of synapses, together with inhibition of nervous system inflammation. While some of these proteins showed a direct path to the predicted functions, others may have an indirect role in pathway activation or inhibition (Figure 11).

[0206] Individual proteins that showed statistical significance were evaluated, a fold-change > |1.5| between GDF11 treatment and control, and known biological activity that is relevantP390200. W0.01to GDF11 function and / or stroke repair. Data from the intermittent dosing group were also incorporated to identify proteins that show similar trends; and included the data from the sham groups to determine if changes are dependent on pMCAO. Candidate biomarkers that showed a consistent decrease in response to rGDF11 treatment compared to the vehicle treated group were Rtn4rl1, Cd68, Inhba, Fgl1, and Nppb (Figure 12). Candidate biomarkers that were upregulated in response to rGDF11 treatment included Thbs2, Nrp1, Ucn3, Sparcl1, and Fam177a1 (Figure 13).

[0207] To further substantiate earlier findings of enhanced neovascularization in response to rGDF11, circulating vascular modulators that may contribute to vascular repair mechanisms were examined. On day 5, a significant increase in angiopoietin- 1 (Angptl) levels in the rGDF11 daily dosing group was observed compared to the vehicle control (Figure 14 A). Additionally, circulating levels of angiopoietin-2 (Angpt2) showed an upward trend on day 5; however, this increase reached only marginal significance after adjustment for multiple testing (Figure 14 B).

[0208] In vivo supplementation with GDF11 promotes recovery and regenerative processes in the brain post-stroke. We initially confirmed that rGDF11 reversed cardiac hypertrophy and improved glucose metabolism in aged mice and demonstrated that both these beneficial effects were observed using the same dosing regimen. We then completed an extensive investigation of the therapeutic potential of rGDF11 to promote recovery and regeneration post-stroke. Time of dosing initiation post-injury, administration frequency, duration, and range, and route of rGDF11 administration demonstrated that a short term-acute intermittent dosing paradigm initiated 24 to 72 hours post-injury improves sensorimotor function, promotes neurogenesis and neovascularization in the brain, and reduces inflammation postinjury.

[0209] C-reactive protein (CRP), a sensitive marker for inflammation,22was found to decrease dose-dependently in response to rGDF11 (Figure 16E). Elevated CRP increases secondary brain damage in animal models of focal cerebral ischemia23and elevated CRP levels are found in up to 75% of patients with ischemic stroke.24

[0210] An unexpected and striking finding the rapid effect of GDF11, with differences apparent within the first week after ischemic stroke. It is possible that multiple mechanisms are responsible for the observed effects on inflammation, neurogenesis, vascularization, and functional recovery.

[0211] Proteomics analysis identified several canonical pathways activated by rGDF11 treatment, particularly on days 3 and 5 post-stroke. Analysis of protein levels revealed consistent upregulation (Alad, Aldob, Fh, Olfm2, Htra2, and Isocl) and downregulation (Inhba and Rtn4rl1) of specific proteins. These persistent changes in protein levels provide a wide temporal window for biomarker measurements. The serum proteomics analysisP390200. W0.01focused on daily dosing study arms, with fewer significant changes observed in the intermittent dosing group, possibly due to the longer intervals between dosing and measurements. These findings highlight the need for further studies to investigate the role of these biomarkers in stroke recovery and validate them using orthogonal assays. We also believe that additional biomarker and molecular studies will be needed to understand the reproducible and surprising early benefit of rGDF11 during stroke recovery.

[0212] Young adult male rats were utilized to establish a controlled and reproducible model for investigating the effects of rGDF11 post pMCAO. Young adult males were chosen to minimize biological variability, as age- and sex-related differences in stroke pathology and recovery have been well-documented.1825Aged male and female rats exhibit distinct cerebrovascular physiology, hormonal influences, and immune responses that could introduce additional variables, complicating the interpretation of experimental outcomes.2628By focusing on a homogenous group, we aimed to ensure consistency and reliability in our findings, providing a robust foundation for future translational and clinical studies. We recognize the importance of developing stroke recovery therapeutics to address the diverse population of affected individuals particularly older patients and those with comorbidities such as hypertension, diabetes, and hypercholesterolemia.

[0213] These data demonstrate that systemically delivered rGDF11 enhances neovascularization, reduces inflammation, promotes neurogenesis, and improves sensorimotor function post-injury in a rat model of ischemic stroke.

[0214] The surprisingly rapid onset of sensorimotor benefits and the dose-dependent reduction in C-reactive protein underscore rGDFU’s potential to address key pathological processes in stroke recovery.30Coupled with the identification of an optimized and clinically-feasible dosing regimen, a panel of candidate pharmacodynamic and mechanistic biomarkers, and a broad therapeutic and safety window, the data provide an important foundational basis to support the clinical translation of rGDF11 as a stroke recovery therapeutic.Example 3Protein Production

[0215] In-house upstream process development work to produce recombinant human GDF11 mature dimer started with transient expression in HEK Expi293 cells using a rGDF11 expression construct. The rGDF11 expression construct was subsequently used for stable cell line development in the CHOZN® GS- / - ZFN-modified CHO cell line (CHOZN) (Millipore Sigma, Sandrasagra et. al, in prep). Briefly, the rGDF11 expression construct was incorporated into the CHOZN cell line via electroporation followed by the separation of the electroporated cells into minipools. The minipools were sub-cultured and screened by ELISAP390200. W0.01to identify pools with the highest expressing cells. Selected minipools were further characterized by both ELISA and western blot methods to confirm complete processing of the precursor protein into hGDF11 mature dimer. Single cell cloning of the selected minipool was then performed by limiting dilution. The best performing clone was then selected based on expression and doubling time. Following this, a research cell bank (RCB) and master cell bank (MCB) were generated and characterized at a Contract Drug Manufacturing Organization (Northway Biotech, Vilnius, LT). In-house downstream purification employed a multistep chromatography process to capture and isolate high quality active rGDF11 mature domain dimer (Sandrasagra et al, in prep.) used in the studies presented here.Luciferase Reporter Assays

[0216] Luciferase reporter activity assays were performed to compare the activities of commercial E. coli-produced GDF11 (Peprotech, Cranbury NJ) vs internally produced rGDF11 from HEK Expi 293 cells. The assays were performed using a HEK 293T cell line containing a luciferase reporter gene driven by the SMAD2 / 3-responsive CAGA promoter.1'1Briefly, HEK 293T CAGA cells were grown in complete media (DMEM containing 4.5 g / L glucose with L-gln, 200 ug / mL G418, 1% strep / pen, 10% FBS). On day 1 of the assay, cells were trypsinized, counted and seeded in 96-well white, flat bottom plates (Cat#3909, Corning®) at 30,000 cells / well followed by a 24-hour incubation at 37°C with 5% CO2. On day 2, serial dilutions of the proteins were performed and added along with vehicle controls in triplicate to the respective wells, in order to generate the desired dose concentration range. Cells were then cultured for another 24-hours. On the third and final day of the assay, culture media was removed from the wells. Cells were lysed using 1x Passive Lysis Buffer (Cat# E194A, Promega), transferred to a Luminescence plate (Cat# 6005030, Perkin Elmer) followed by addition of the luciferase assay reagent (Cat# E1501, Promega). Luminescence readings were done on a Molecular devices SpectraMax i3x plate reader. Data points were plotted in GraphPad Prism to determine EC50 values.GDF11 In Vivo Activity Study

[0217] To investigate rGDF11 activity in vivo, animal studies were conducted at Neosome Life Sciences (Lexington, MA), an independent Contract Research Organization (CRO). Tissue processing and pSmad2 / 3 ELISAs were performed in-house. Thirty-three (33) male C57BI / 6 mice (Jackson Labs), approximately 13 weeks of age at study initiation were used for this study. Single intraperitoneal (IP) injections of vehicle (n=12 mice) or rGDF11 protein (n=21 mice) internally produced from HEK Expi 293 cells. Sample size was determined based on historical data from similar studies performed by the CRO. The test material was supplied at 1 mg / mL and kept at 4°C. On the day of dosing, the protein was diluted to a working concentration of 0.1 mg / ml in diluent (4mM HCl). All doses were administered at 1mg / kg within 1-hour of preparation. Tissue samples were collected and flash frozenP390200. W0.01immediately from heart, pancreas, liver, and spleen at 0, 3, 12, 48-hours for the vehicle-treated group (n = 3 mice per group per time point) and at 0.25, 1, 3, 6, 12, 24, 48-hours for the GDF11-treated mice (n = 3 mice per group per time point). Study animals were randomized, and investigators were blinded to treatment assignment. The protocol was reviewed and approved by the NeoSome’s IACUC, with all animal welfare concerns addressed and documented. Treatment blinding was performed by a person who was not involved in the animal studies, analytical analysis, or data analyses and only unblinded the treatment after all data analyses were completed.Proteins were extracted from lysed tissue samples. Tissue lysis was done using a TissueLyser (Cat # 85300, Qiagen). Prior to beginning the lysis, the centrifuge and TissueLyser blocks were cooled to 4°C. Frozen tissue samples were cut on a glass plate on dry ice then placed into a clean, round bottom Eppendorf tube. Subsequently, frozen tissue samples were weighed, and the obtained weights recorded. Depending on the protein of interest, 20-50 mg of frozen tissue was used. Extra care was taken to ensure everything was kept on ice. To each tube containing tissue sample, 20 uL / mg of RIPA buffer containing protease and phosphatase inhibitor solution (Cat# 78441, ThermoFisher) was added. A single 5mm stainless steel bead (Cat# 69989, Qiagen) was added to each tube followed by homogenization using the TissueLyser as per manufacturer’s recommendations. Upon completion, the stainless-steel bead was removed, and the homogenized tissue was transferred to clean tubes on ice followed by centrifugation at 14,000 RPM for 10 min. The supernatant was carefully removed and placed into clean tubes. Lysates were analyzed for total protein using a BCA Assay kit (Cat# A53225, ThermoFisher) then stored at -80°C as aliquots to prevent multiple freeze / thaw cycles. Investigators were blinded to the groups for which animal tissue was assigned.

[0218] Frozen protein lysates were thawed on ice then analyzed using PathScan pSMAD2 / 3 kit (Cat# 12001, Cell Signaling Technology) as per manufacturer’s instructions. Briefly, the supplied microwell plates, pre-coated with SMAD2 / 3 mouse antibody, were equilibrated to room temperature before adding the cell lysates and incubating overnight at 4°C. The wells were washed thoroughly in between the steps. Detection antibody (Phospho-SMAD2 / 3) was then added and followed by HRP-Linked secondary antibody. Lastly, TMB substrate was added to the wells and incubated for 10-20 min at 25°C. Stop solution (2 M sulfuric acid) was added upon observing initial color of positive reaction (blue). Absorbance readings were taken at 450 nm and 540 nm within 30 mins of adding the stop solution. To ensure robustness and validity of our results, we randomized and blinded all samples in this experiment.GDF11 Serum ELISA AssaysP390200. W0.01

[0219] To quantify the amount of rGDF 11 in serum samples we ran an in-house developed ELISA. Microtiter plates (Cat#15041; Fisher Sci / Pierce) were coated with 6 µg / mL capture antibody (Cat# MAB19581; R& D Systems) diluted in coating buffer (50mM carbonate buffer pH 9.6), sealed, and incubated overnight at 4°C on a platform shaker. After the overnight incubation, the plates were washed 4 times with wash buffer (1X PBS + 0.1% Tween-20) then blocked with PBS-Casein Block 1X (Cat# PBSC-1000-1, SurModics) for an hour at room temperature. Following another wash step, serum was added to each well then incubated for 2 hours at room temperature. Recombinant GDF11 proteins were used to generate the standard curves in the concentration range from 200-0.09 ng / mL. Following incubation, plates were washed before adding detection antibody (Custom Ordered Biotinylated-mAb19581, R& D Systems) diluted to 1.5 ug / mL in blocking buffer (PBS-Casein 1x). After an hour incubation in detection antibody at 37°C, plates were washed followed by a 30-minute incubation with Streptavidin-HRP beads (Cat# 21134; Pierce) diluted 1:200 in blocking buffer. Subsequently, a wash step was completed before adding 1-step ultra TMB substrate solution (Cat#34028, ThermoFisher) to each well. Plates were kept at room temperature until desired color change was attained (approximately 10-20 mins). ELISA stop solution (2 M sulfuric acid) was promptly added and absorbance measurements were taken at 450 nm and 540 nm immediately. The 540nm reading were subtracted from the 450 nm reading for each sample and value was compared to standard curve. To ensure robustness and validity of our results, we randomized and blinded all samples in this experiment.CRP Serum ELISA Assays

[0220] Quantification of C-reactive protein (CRP) levels in serum samples was preformed using the Rat CRP DuoSet ELISA kit (R& D DY1744) according to manufacturer’s protocol. Briefly, serum was diluted 400,000 times with 1% BSA in 1x PBS to obtain a concentration in the linear range for all samples. Microtiter plates (Cat#15041; Fisher Sci / Pierce) were coated overnight with the CRP capture antibody diluted in 1x PBS and blocked with 1% BSA in 1x PBS. Plates were the washed, blocked, incubated with samples or standards and then incubated with detection antibody according to the manufacturer’s protocol. 1-step ultra TMB substrate solution (Cat#34028, ThermoFisher) was used for detection followed by stop solution (2 M sulfuric acid). Absorbance measurements were measured at 450 nm and 540 nm. A540 values were subtracted from A450 values for each sample and used to calculate the CRP concentration from the standard curve. For Figure 16 E CRP concentrations (ng / mL) were reported and in Figure 18, CRP concentrations on Days 2 through 14 were normalized to the baseline Day -1 (pre-pMCAO or sham) values for each animal. To ensure robustness and validity of our results, we randomized and blinded all samples in this experiment.Analysis of Cardiac Hypertrophy and Glucose and Insulin ToleranceP390200. W0.01

[0221] Male C57BL / 6 mice at either 14 weeks old (young) or 84 weeks old (aged) were divided into two groups to receive either intraperitoneal (IP) injections of 1 mg / kg rGDF11 (internally produced from HEK Expi 293 cells) or a vehicle once daily for 15 days (from day 1 to day 15; see Figure 3E for timeline). We included 8 mice per group for the young cohort and 10 mice per group for the aged cohort, totaling 4 groups and 38 mice. Sample size was determined based on previous studies conducted by Walker et al,13and Poggioli et al.14Baseline measurements for the Glucose Tolerance Test (GTT) and Insulin Tolerance Test (ITT) were taken on days -7 and -3, respectively, to be compared with readings on treatment days 10 and 14. GTT and ITT measurements were performed as previously published13with slight modifications. Briefly, GTT was performed after a 16-hour fasting period, while the ITT was conducted following a 4-hour fast. For GTT measurements, blood from nicked tails was directly placed onto glucose test strips, and glucose was then measured using a glucometer at 0, 15, 30, 60, and 120 minutes following a single intraperitoneal injection of 2 g / kg glucose. ITT was performed on the same cohort as described for GTT at 0, 15, 30, 45, 60, and 90 minutes following a single intraperitoneal injection of 0.75 units / kg insulin. Body weights were recorded daily. All harvested tissues were weighed, measured, and flash-frozen on day 16. Cardiac hypertrophy was assessed by calculating the ratio of heart weight to tibial length for each mouse. One mouse from the aged cohort, treated with rGDF11, had body weight deviation that were greater than 25% and thus, was excluded from the GTT analysis on day 10. Two mice from the aged cohort, treated with rGDF11, were found dead on day 11 and thus were not used for the ITT analysis on day 14, the body weight analysis, and tissue weights and measurements. This study was conducted at an independent CRO (Neosome Life Sciences, Lexington, MA). The protocol was reviewed and approved by the NeoSome’s IACUC, with all animal welfare concerns addressed and documented. Animals were randomized and investigators were blinded to treatment assignment throughout the study, including end point assessments. Treatment blinding was performed by a person who was not involved in the animal studies or data analyses and only unblinded the treatment after all data analyses were completed.Stroke Efficacy Studies

[0222] All six ischemic stroke recovery studies were performed by NeuroVasc Preclinical Services, Inc. (NeuroVasc, Lexington, MA) an independent CRO, in vivarium and laboratory space leased from and operated by NeoSome Life Sciences, LLC (Lexington, MA). The protocol was reviewed and approved by the NeoSome IACUC, and all animal welfare concerns were addressed and documented. For all studies, animals were randomized and investigators were blinded to treatment assignment. Treatment blinding for all studies were performed by a person who was not involved in the animal studies or data analyses and only unblinded the treatment after all data analyses were completed.P390200. W0.01

[0223] Male Sprague-Dawley rats, housing and diet were ordered 6-10 days prior to surgery from Charles River Laboratories (Wilmington, MA). Animals were housed in rooms provided with filtered air at a temperature of 21 ± 5° C and 50%± 30% relative humidity. The room was on an automatic timer for a light / dark cycle of 12 hours on and 12 hours off with no twilight. 1 / 4" Bed-o’Cobs® / Pure-o’Cel ©was used for bedding and Crink-l’NestTM, Nestlets by Ancare and 1or2 pieces of Nylon Bones (BioServ K3581) was put in each cage. Animals were fed with Lab Diet® 5001 chow. Water and food was provided ad libitum. The rats were housed 2 per cage and handled for behavioral assessment for five days prior to surgery for acclimation purposes. At the end of handling period, rats were randomized and assigned to different pMCAO groups. The animals were observed the day prior to surgery, and those appearing to be in poor health were excluded from the study. Rats were given unique identification numbers by tail marking.

[0224] Study design for all six studies including number of experimental groups, route of administration, dosing frequency, dosing duration, days of behavioral testing, and number of rats per dose is provided in Figure 23. Sample size was determined based on historical data from similar pMCAO model studies performed by the CRO. For Study 6, one animal from the single 0.3 mg / kg dose died 8 days after the surgery and was excluded from the study.

[0225] Focal cerebral infarcts were made by permanent occlusion of the proximal right middle cerebral artery (MCA) using a modification of the method of Tamura and colleagues.15Male Sprague-Dawley rats (300-400 g at the time of surgery) were anesthetized with 3% isoflurane in the mixture of N2O: O2 (2:1), and were maintained with 2-2.5% isoflurane in the mixture of N2O: O2 (2:1). The temporalis muscle was bisected and reflected through an incision made midway between the eye and the eardrum canal. The proximal MCA was exposed through a subtemporal craniectomy without removing the zygomatic arch and without transecting the facial nerve. The artery was then permanently occluded by microbipolar coagulation from just proximal to the olfactory tract to the inferior cerebral vein. Using a rectal temperature probe and a connected heating pad, targeted body temperature was set at 37.0 ± 1°C throughout the procedure. Cefazolin (40 mg / kg; West Ward Pharma) was given intraperitoneally, before surgery to prevent infections.Buprenorphine (~0.1 mg / kg, Simbadol) was also given subcutaneously before the pMCAO surgery as analgesia. For the sham operation, animals underwent the same procedure described above without the middle cerebral artery being coagulated. Following surgery, animals remained on a heating pad until they awakened from anesthesia. They were then returned to clean home cages and observed frequently on the day of pMCAO surgery and at least once daily thereafter.P390200. W0.01

[0226] Dosing solutions of vehicle and rGDF11 (internally produced from CHO cells) were delivered blinded with coded labels. The solutions were stored in 4°C and were kept on ice during dosing. For IP administration rGDF11 stocks in 4 mM hydrochloric acid were diluted ten times with phosphate buffered saline (1X PBS) on the day of dosing. For IV administration all solutions were diluted ten times with IV Solution (25 mM sodium-acetate with 10% T rehalose, adjusted to a pH of 5.0 with acetic acid) on the day of dosing, and given as a slow bolus at 5 ml / kg. The final composition of all IV dosing solutions including the vehicle was 22.5 mM sodium-acetate with 9% Trehalose, 0.4 mM hydrochloric acid, and 11.7 mM acetic acid.

[0227] For study 5 (IV Dose Ranging Study) blood was collected through the saphenous vein (>0.3 ml whole blood) at day 7 after pMCAO. For Study 6 (IV Dose Optimization Study) blood was collected through the saphenous vein (>0.3 ml whole blood) prior to the pMCAO surgery (Day -1), day 2, day 3, day 5 and day 7 after pMCAO. On day 3, day 5 and day 7, blood was collected after all behavior tests were done, and before i.v. dosing (if scheduled to be dosed). Blood was collected through the saphenous vein (>0.3 ml whole blood) prior to the sham surgery (Day -1), day 2, day 3 and day 5 after pMCAO. Blood was collected through heart puncture at the time of sacrifice for both pMCAO and sham groups after rats were anesthetized deeply with ketamine / xylazine (91 mg / kg ketamine, 9 mg / kg xylazine, respectably). After collection, blood was processed for serum collection by allowing whole blood (approximately 0.5 ml / sample) to clot at room temperature (15-30°C) for 30-60 min. If required, collection tubes were held at 2-8°C after clotting took place, before centrifugation was initiated. To isolate blood serum, clotted blood was spun down in a centrifuge for approximately 10 minutes at 2-8°C and the supernatant was transferred into newtubes. Serum was snap-frozen and stored at -71 to -78°C until further analyses were performed.

[0228] Functional activities were evaluated using limb placement and body swing behavioral tests for pMCAO animals only. Behavioral tests were performed one day before surgery (day -1 or Day-pre), and throughout the study duration before drug administration and blood collections on days when both were done. Please see Figure 23 for days of functional behavioral tests for each study.

[0229] Limb placement tests were divided into both forelimb and hindlimb tests. For the forelimb-placement test, the examiner held the rat close to a tabletop and scored the rat's ability to place the forelimb on the tabletop in response to whisker, visual, tactile, or proprioceptive stimulation. Similarly, for the hindlimb placement test, the examiner assessed the rat's ability to place the hindlimb on the tabletop in response to tactile and proprioceptive stimulation. Separate sub-scores were obtained for each mode of sensory input (half-pointP390200. W0.01designations possible), and added to give total scores (for the forelimb placement test: 0 = normal, 12 = maximally impaired; for the hindlimb placement test: 0 = normal; 6 = maximally impaired).

[0230] For the body swing test, the rat was held approximately one inch from the base of its tail. It was then elevated to an inch above a surface of a table. The rat was held in the vertical axis, defined as no more than 10° to either the left or the right side. A swing was recorded whenever the rat moved its head out of the vertical axis to either side. The rat must have returned to the vertical position for the next swing to be counted. Thirty (30) total swings were counted. A normal rat typically has an equal number of swings to either side. Following focal ischemia, the rat tends to swing to the contralateral (left) side. The test was performed at the same time of the Limb Placement test.ImmunohistochemistrypMCAO Study 4 analysis

[0231] Twenty-nine days (Day 29) after pMCAO, rats were anesthetized deeply with ketamine / xylazine (91 mg / kg ketamine, 9 mg / kg xylazine, respectively). After anesthetization, the rats were perfused transcardially with normal saline (with heparin 2 unit / ml) followed by 4% paraformaldehyde. Brains were removed and stored in 1X PBS with 4% paraformaldehyde overnight and then changed to 1xPBS and stored in 0-4°C. Animal work was conducted at an independent CRO (NeuroVasc, Billerica, MA) and investigators blinded to treatment assignment. The brains were then incubated in 1X PBS with 30% sucrose and kept overnight, followed by transfer to 10% sucrose in PBS for cryoprotection. The brains were later cut coronally into 3 mm slices using a rat brain matrix to make 4 slices. These slices were then put in optimal cutting temperature compound (Thermo Fisher Scientific, Waltham, MA) in cryomolds (4728 TissueTek, Torrance CA) and placed in -20°C as tissue blocks. The tissue blocks were sectioned coronally into 40 pm thick slices on a cryostat (CM1860UV Leica Biosystems, NJ) and mounted on glass slides (22-035813 Thermo Fisher Scientific, Waltham, MA).

[0232] Tissue sections were prepared as follows: Slides were allowed to air-dry overnight at room temperature (RT). Subsequently, fixation was carried out with 4% Paraformaldehyde (15714 Electron Microscopy Sciences, Hatfield PA) for 10 minutes at RT. For antigen retrieval, the slides were treated with 1X Citrate Buffer (Ab93678 Abeam, Cambridge, MA) at pH 6, heated to 90°C for 10 minutes. Following retrieval, the slides underwent a thorough wash with 1X PBS (MRGF6235 Growcells, Irvine CA), with three washes of 5 minutes each.

[0233] After primary antibody incubation, sections were washed three times with PBS containing 0.1% Triton X-100 (PBS-T), with each wash lasting five minutes. For secondary staining, sections were incubated for 1-2 hours at room temperature with donkey anti-goatP390200. W0.01Alexa Fluor 594 (ThermoFisher Scientific) diluted 1:1000 in PBS-T. Following this, sections were washed again with PBS-T and counterstained with DAPI (ThermoFisher Scientific) at a dilution of 1: 10,000. A final series of washes with PBS-T was performed to remove excess stains. The stained sections were mounted on glass slides using ProLong Gold Antifade Reagent (ThermoFisher Scientific) and cover-slipped with coverslips secured using nail lacquer for stability during imaging.

[0234] Images were acquired using an Olympus VS120 slide scanner. For quantitative analysis, three regions of interest (ROIs) were selected within the ipsilateral hemisphere of each of six brains per dose group. ROIs measuring 0.3 square millimeters were chosen within 3 mm of the glial scar surrounding the infarct (Figure 7 A). Vascular parameters, including percent vessel area, total vessel length, average vessel length, and junction density, were quantified using AngioTool software (NIH).20To ensure robustness and validity of our results, we randomized and blinded all samples in this analysis.

[0235] ForSox2 staining, blocking was achieved using 10% normal donkey serum (NDS, SD30-0500 VWR, Radnor PA) in 0.3% Triton (SLB2521 Sigma, Burlington MA) in 1X PBS-T. To demarcate the sections, a lipophilic pen (H-4000 Vector Laboratories, Newark, CA) was employed. The sections were then incubated overnight at 4°C with the primary Sox-2 antibody at 1 pg / mL (ab97959 Abeam, Cambridge, MA) with approximately 300 pl per section, on a shaking platform. Subsequently, slides were washed three times for 5 minutes each with 1X PBS + 0.3% Triton (1X PBS-T). Following this, incubation with the secondary antibody Goat anti-Rabbit at 1:1000 AF 488 (A32731 Thermo Fisher Scientific, Waltham, MA) was performed for 1 hour at RT. A wash with 300 pL DAPI (62248 Thermo Fisher Scientific, Waltham, MA) diluted to 1:10,000 + 1X PBS-T for 5 minutes was carried out, followed by three additional washes of 5 minutes each with 1X PBS-T. The slides were then left to completely dry in the dark. Cover slipping was accomplished using a mounting medium (P36930 Thermo Fisher Scientific, Waltham, MA), and the slides were sealed with Nail Lacquer (Amazon, WA). Imaging was conducted using a slide scanning microscope VS120 (Olympus, Tokyo, Japan) with a 20x objective. Cell counting analysis was performed using Fiji software. To ensure robustness and validity of our results, we randomized and blinded all samples in this analysis. ForCD31 staining, following antigen retrieval, tissue sections were blocked to minimize nonspecific binding using a blocking solution composed of 10% normal goat serum (NGS, Equitech Bio) and 0.3% Triton X-100 (Sigma-Aldrich) in phosphate-buffered saline (PBS). The primary antibody, anti-CD31 (Abeam), was diluted to a concentration of 7.5 pg / mL in PBS containing 1 % NGS. Sections were incubated with the primary antibody overnight at 4°C on a shaking platform.pMCAO Study 5 analysisP390200. W0.01

[0236] Twenty-one days (Day 21) after pMCAO, rats were anesthetized deeply with ketamine / xylazine (91 mg / kg ketamine, 9 mg / kg xylazine, respectably). After anesthetization, the rats were perfused transcardially first with biotinylated lycopersicon esculentum (tomato) lectin, then with ice cold PBS followed by 4% paraformaldehyde. Brains were removed and stored in 1X PBS with 4% paraformaldehyde overnight and then changed to 1xPBS and stored in 0-4°C. Animal work was conducted at an independent CRO (NeuroVasc, Billerica, MA) and investigators blinded to treatment assignment. Brains were cryoprotected by incubating them in 1X PBS with 30% sucrose at 4°C. Brains were cut into seven 2 mm thick coronal sections using a rat brain matrix (+4.7, +2.7, +0.7, -1.3, -3.3, -5.3 and -7.3, compared to bregma respectively). The 2 mm coronal sections were subsequently cut into 40 pm thick coronal sections using a cryostat and placed in 24-well plates containing PBS.

[0237] The tissue sections were processed as follows: first, the plate was rinsed in PBS to remove OCT and next, antigen retrieval was carried out by treating the sections with 1X Citrate Buffer (Ab93678 Abeam, Cambridge, MA) at 90°C for 10 minutes. Following this, blocking was performed using 10% NDS (SD30-0500 VWR, Radnor PA) in 0.3% Triton (SLB2521 Sigma, Burlington MA) in 1X PBS-T. The sections were then incubated with the CD31 primary antibody at 7.5 pg / mL (AF3628, R& D Systems, Minneapolis, MN) in 1% BSA in PBS on a shaking platform overnight at 4°C. Subsequently, a thorough wash was conducted with 1X PBS-T three times for 5 minutes each. The sections were further incubated with the secondary antibodies: Donkey anti-Goat FL647- AF at 1:2500 (A32849 Thermo Fisher Scientific, Waltham, MA) and Streptavidin Dylight 488 at 1:200 (21832, Thermo Fisher Scientific, Waltham, MA) in 1X PBS-T. Following another wash with DAPI (62248 Thermo Fisher Scientific, Waltham, MA) diluted to 1:10,000, a final wash with PBS-T was performed three times for 5 minutes each. The sections were mounted on glass slides (22-035813 Thermo Fisher Scientific, Waltham, MA) with a small amount of water, coverslipped using mounting medium (P36930 Thermo Fisher Scientific, Waltham, MA), and sealed with Nail Lacquer (Amazon, WA). Sections proximal to the bregma within the periinfarct region were selected for their proximity to the stroke site and the availability of viable tissue. Each experimental group comprised four animals (n=4), with one matched section examined per brain. The vehicle control group consisted of three animals (n=3) due to challenges in achieving consistent section matching, attributed to compromised tissue integrity in one of the control slides. ROIs were chosen within 1 mm from the glial scar of the infarct (Figure 9 A). Imaging was carried out utilizing a confocal microscope, TissueFAXS (TissueGnostics GmbH, Vienna Austria), employing a 40x objective, with each region of interest (ROI) measuring 0.4 square millimeters. Analysis of percent vessel area was executed using StrataQuest software (TissueGnostics GmbH, Vienna Austria). Other vascular parameters, including total vessel length, average vessel length and junctionP390200. W0.01density, were quantified using AngioTool software (NIH).20To ensure robustness and validity of our results, we randomized and blinded all samples in these analyses.Proteomic Platform

[0238] Profiling of the serum proteome was carried out using the SomaScan® Assay by an independent CRO (SomaLogic Inc., Boulder, CO). The CRO received blinded and randomized serum samples, and all data remained blinded through endpoint assessment. SomaScan is a highly multiplexed aptamer-based proteomic technology capable of making over 7,000 protein measurements. These chemically modified aptamers, called SOMAmer® Reagents, form complex three-dimensional shapes which bind to epitopes on their target protein with high affinity and specificity. This technology is based on proprietary Slow Off-rate Modified Aptamers (SOMAmer reagents) selected to bind to structural epitopes on proteins. During synthesis, a Cyanine-3 fluorophore, spacer, and photocleavable biotin are incorporated at the 5’ end of each SOMAmer reagent. These SOMAmer reagents are preimmobilized onto streptavidin beads and used to capture target proteins from biological samples during an incubation step. Unbound proteins are washed away, and captured proteins are biotinylated using NHS-biotin. UV light is used to cleave the photocleavable linker, releasing complexes back into solution in the presence of a high concentration of universal polyanionic competitor. This step takes advantage of solution kinetics to selectively enrich for specificity. Fast off-rate, non-specific complexes dissociate, and rebinding is blocked by the competitor. Slow off-rate, specific complexes do not dissociate on this timescale. Complexes and some free proteins that dissociated are captured onto new streptavidin beads. These dissociated proteins do not contribute to signal downstream since they are no longer bound to a SOMAmer reagent. After washing, SOMAmer reagents are eluted from the beads by denaturing the proteins with a chaotropic salt, sodium perchlorate. The eluate is placed onto a custom Agilent microarray with probes complimentary to each SOMAmer reagent for overnight hybridization. Slides are washed and read in an Agilent microarray scanner. The resulting RFU values correlate to the amount of target epitope in the initial samples.

[0239] SomaScan assay data are first normalized using hybridization controls to mitigate variation within the run that comes from the readout steps: transfer to Agilent slides, hybridization, wash, and scan. This is followed by median signal normalization across pooled calibrator replicates within the run to mitigate within-run technical variation in the calibrator signal prior to use in scaling calculations. The set of ratios of the calibrator reference value to the median of calibrator replicates for each SOMAmer reagent is calculated for plate scaling. Plate scaling adjusts for overall signal intensity differences between runs, mitigating platebased batch effects. Median signal normalization is finally performed using median normalization to a study-specific reference, adjusting for confounding differences betweenP390200. W0.01samples. For comparisons between post and pre-treatments for each treatment group we performed paired t-test. For comparisons of rGDF11 to Vehicle for each day we performed unpaired t-test. Corrections were made by the False Discovery Rate (FDR adjusted p-value) and Bonferroni correction (Figure 25).

[0240] For the Somascan analysis we selected samples from different time points in the Dose Optimization Study. Figure 24 shows all the dose regimen groups that were evaluated in this study with the 14 samples selected for Somascan analysis highlighted by the red box borders. We hypothesized that the Daily Dose regimen will provide the most significant differences in GDF11 -responsive protein levels when comparing the 1 mg / kg and vehicle groups, and thus mostly focused on these arms. From the Daily Dose 1 mg / kg and vehicle groups, serum from 5 animals per group per timepoint was used from Day -1, Day 2, Day 3, Day 5, and Day 7. From the Intermittent Dose 1 mg / kg group, serum from 5 animals per group per timepoint was used from Day 5, and Day 7. From the sham Intermittent dose 1 mg / kg group and vehicle groups, serum from 5 animals per group per timepoint was used from Day 2, and Day 5.STATISTICAL ANALYSES

[0241] Statistical analyses were performed using Prism GraphPad (San Diego, CA), with the exception of the SomaScan proteomics data, which was analyzed using the SomaLogic DataDelve Statistics tool (SomaLogic Inc., Boulder CO). Specific statistical tests and correction methods used for each analysis are detailed in the respective figure legends. For analyses involving multiple comparisons, adjusted p-values are repoted. Statistical significance was set at p<0.05 unless otherwise noted.

[0242] Methods: The therapeutic benefits of recombinant GDF11 (rGDF11) were evaluated using a rat ischemic stroke model, in which focal cerebral infarcts were induced in 8 -10 week-old young adult male Sprague-Dawley rats by permanently occluding the proximal right middle cerebral artery. Rats received single or multiple doses of rGDF11 (0.1-4 mg / kg) or vehicle 24-72 hours post-injury. Sensorimotor functions were evaluated, and brain and serum samples were examined to determine mechanism of action and identify biomarkers, using immunofluorescence, target-specific ELISAs, and an aptamer-based proteomics platform.

[0243] Results: We first confirmed rGDF11 activity in vitro and in established in vivo mouse models of cardiac hypertrophy and glucose metabolism. We then assessed the efficacy of rGDF11 treatment in six preclinical stroke experiments, using independent Contract Research Organizations with all study animals and treatment groups blinded. All six studies revealed that rGDF11 improved sensorimotor outcomes. rGDF11 -treated rats showed increased cortical vascularization and radial glia in the ventricular zone. Serum analysisP390200. W0.01revealed GDF11 dose-dependent decreases in C-reactive protein and identified novel pharmacodynamic biomarkers and pathways associated with potential mechanisms of action of rGDF11.

[0244] Conclusion: These results demonstrate that systemically delivered rGDF11 enhances neovascularization, reduces inflammation, promotes neurogenesis, and improves sensorimotor function post-injury in a rat model of ischemic stroke. More importantly, these data define an optimized and clinically-feasible rGDF11 dosing regimen for therapeutic development in ischemic stroke and identify a panel of candidate pharmacodynamic and mechanistic biomarkers to support clinical translation.REFERENCES:1. Wechsler LR, Adeoye O, Alemseged F, Bahr-Hosseini M, Deljkich E, Favilla C, Fisher M, Grotta J, Hill MD, Kamel H, et al. Most Promising Approaches to Improve Stroke Outcomes: The Stroke Treatment Academic Industry Roundtable XII Workshop. Stroke. 2023;54:3202-3213. doi: 10.1161 / STROKEAHA.123.0442792. Driss LB, Lian J, Walker RG, Howard JA, Thompson TB, Rubin LL, Wagers AJ, Lee RT. GDF11 and aging biology - controversies resolved and pending. J Cardiovasc Aging. 2023;3. doi: 10.20517 / jca.2023.233. Loffredo FS, Steinhauser ML, Jay SM, Gannon J, Pancoast JR, Yalamanchi P, Sinha M, DaH'Osso C, Khong D, Shadrach JL, et al. Growth differentiation factor 11 is a circulating factor that reverses age-related cardiac hypertrophy. Cell. 2013;153:828- 839. doi: 10.1016Zj.cell.2013.04.0154. Katsimpardi L, Litterman NK, Schein PA, Miller CM, Loffredo FS, Wojtkiewicz GR, Chen JW, Lee RT, Wagers AJ, Rubin LL. Vascular and neurogenic rejuvenation of the aging mouse brain by young systemic factors. Science. 2014;344:630-634. doi: 10.1126 / science.12511415. Ozek C, Krolewski RC, Buchanan SM, Rubin LL. Growth Differentiation Factor 11 treatment leads to neuronal and vascular improvements in the hippocampus of aged mice. Sci Rep. 2018;8:17293. doi: 10.1038 / s41598-018-35716-66. Zhang W, Guo Y, Li B, Zhang Q, Liu JH, Gu GJ, Wang JH, Bao RK, Chen YJ, Xu JR.GDF11 Rejuvenates Cerebrovascular Structure and Function in an Animal Model of Alzheimer's Disease. J Alzheimers Dis. 2018;62:807-819. doi: 10.3233 / JAD-170474 7. Lu L, Bai X, Cao Y, Luo H, Yang X, Kang L, Shi MJ, Fan W, Zhao BQ. Growth Differentiation Factor 11 Promotes Neurovascular Recovery After Stroke in Mice. Front Cell Neurosci. 2018; 12:205. doi: 10.3389Zfncel.2018.002058. Hudobenko J, Ganesh BP, Jiang J, Mohan EC, Lee S, Sheth S, Morales D, Zhu L, Kofler JK, Pautler RG, et al. Growth differentiation factor-11 supplementation improves survival and promotes recovery after ischemic stroke in aged mice. Aging (Albany NY).2020;12:8049-8066. doi: 10.18632Zaging.1031229. laci JF, Parry TJ, Huang Z, Finklestein SP, Ren J, Barrile DK, Davenport MD, Wu R, Blight AR, Caggiano AO. Dalfampridine improves sensorimotor function in rats with chronic deficits after middle cerebral artery occlusion. Stroke. 2013;44:1942-1950. doi: 10.1161 ZSTROKEAH A.111.00014710. Kawamata T, Dietrich WD, Schallert T, Gotts JE, Cocke RR, Benowitz LI, Finklestein SP. Intracisternal basic fibroblast growth factor enhances functional recovery and up- regulates the expression of a molecular marker of neuronal sprouting following focal cerebral infarction. Proc Natl Acad Sci U S A. 1997;94:8179-8184. doi: 10.1073Zpnas.94.15.8179P390200. W0.01Walker RG, Czepnik M, Goebel EJ, McCoy JC, Vujic A, Cho M, Oh J, Aykul S, Walton KL, Schang G, et al. Structural basis for potency differences between GDF8 and G DF 11. BMC Biol. 2017;15:19. doi: 10.1186 / s 12915-017-0350-1Simats A, Ramiro L, Montaner J, Garcia-Berrocoso T. Application of an Aptamer-Based Proteomics Assay (SOMAscan) in Rat Cerebrospinal Fluid. Methods Mol Biol.2019;2044:221-231. doi: 10.1007 / 978-1 -4939-9706-0_13Walker RG, Barrandon O, Poggioli T, Dagdeviren S, Carroll SH, Mills MJ, Mendello KR, Gomez Y, Loffredo FS, Pancoast JR, et al. Exogenous GDF11, but not GDF8, reduces body weight and improves glucose homeostasis in mice. Sci Rep.2020;10:4561. doi: 10.1038 / s41598-020-61443-yPoggioli T, Vujic A, Yang P, Macias-Trevino C, Uygur A, Loffredo FS, Pancoast JR, Cho M, Goldstein J, Tandias RM, et al. Circulating Growth Differentiation Factor 11 / 8 Levels Decline With Age. CircRes. 2016;118:29-37. doi: 10.1161 / CIRCRESAHA.115.307521 Tamura A, Graham DI, McCulloch J, Teasdale GM. Focal cerebral ischaemia in the rat: 1. Description of technique and early neuropathological consequences following middle cerebral artery occlusion. J Cereb Blood Flow Metab. 1981;1:53-60. doi: 10.1038 / jcbfm.1981.6Casals JB, Pieri NC, Feitosa ML, Ercolin AC, Roballo KC, Barreto RS, Bressan FF, Martins DS, Miglino MA, Ambrosio CE. The use of animal models for stroke research: a review. Comp Med. 2011;61:305-313.Macrae IM. Preclinical stroke research-advantages and disadvantages of the most common rodent models of focal ischaemia. Br J Pharmacol. 2011;164:1062-1078. doi: 10.1111 / j.1476-5381.2011.01398.xPark MJ, Pilla R, Panta A, Pandey S, Sarawichitr B, Suchodolski J, Sohrabji F. Reproductive Senescence and Ischemic Stroke Remodel the Gut Microbiome and Modulate the Effects of Estrogen Treatment in Female Rats. Transl Stroke Res.2020;11:812-830. doi: 10.1007 / sl 2975-019-00760-5Ellis P, Fagan BM, Magness ST, Hutton S, Taranova O, Hayashi S, McMahon A, Rao M, Pevny L. SOX2, a persistent marker for multipotential neural stem cells derived from embryonic stem cells, the embryo or the adult. Dev Neurosci. 2004;26:148-165. doi: 10.1159 / 000082134Zudaire E, Gambardella L, Kurcz C, Vermeren S. A computational tool for quantitative analysis of vascular networks. PloS one. 2011;6:e27385. doi: 10.1371 / journal, pone.0027385Katsimpardi L, KuperwasserN, Camus C, Moigneu C, ChicheA, Tolle V, Li H, Kokovay E, Lledo PM. Systemic GDF11 stimulates the secretion of adiponectin and induces a calorie restriction-like phenotype in aged mice. Aging Cell. 2020; 19:e13038. doi: 10.1111 / aceL13038Pepys MB, Hirschfield GM. C-reactive protein: a critical update. J Clin Invest.2003;111:1805-1812. doi: 10.1172 / JCI18921Gill R, Kemp JA, Sabin C, Pepys MB. Human C-reactive protein increases cerebral infarct size after middle cerebral artery occlusion in adult rats. J Cereb Blood Flow Metab. 2004;24:1214-1218. doi: 10.1097 / 01. WCB.0000136517.61642.99Smith CJ, Emsley HC, Vail A, Georgiou RF, Rothwell NJ, Tyrrell PJ, Hopkins SJ. Variability of the systemic acute phase response after ischemic stroke. J Neurol Sci.2006;251:77-81. doi: 10.1016 / j.jns.2006.09.011Wang RY, Wang PS, Yang YR. Effect of age in rats following middle cerebral artery occlusion. Gerontology. 2003;49:27-32. doi: 10.1159 / 000066505Liu F, Yuan R, Benashski SE, McCullough LD. Changes in experimental stroke outcome across the life span. J Cereb Blood Flow Metab. 2009;29:792-802. doi: 10.1038 / jcbfm.2009.5Brown CM, Suzuki S, Jelks KA, Wise PM. Estradiol is a potent protective, restorative, and trophic factor after brain injury. Semin Reprod Med. 2009;27:240-249. doi: 10.1055 / S-0029-1216277P390200. W0.0128. Buga AM, Di Napoli M, Popa-Wagner A. Preclinical models of stroke in aged animals with or without comorbidities: role of neuroinflammation. Biogerontology. 2013;14:651- 662. doi: 10.1007 / s10522-013-9465-029. Fisher M, Feuerstein G, Howells DW, Hurn PD, Kent TA, Savitz SI, Lo EH, Group S.Update of the stroke therapy academic industry roundtable preclinical recommendations. Stroke. 2009;40:2244-2250. doi: 10.1161 / STROKEAHA.108.54112830. de Beer FC, Baltz ML, Munn EA, Feinstein A, Taylor J, Bruton C, Clamp JR, Pepys MB. Isolation and characterization of C-reactive protein and serum amyloid P component in the rat. Immunology. 1982;45:55-70.Example 4

[0245] Intracerebral Hemorrhage (ICH) and Traumatic Brain Injury (TBI) and are significant contributors to long-term disability and mortality in the U. S. and worldwide, with both conditions representing major public health challenges. ICH involves the rupture of a blood vessel within the brain, leading to bleeding and subsequent neurological damage, while TBI occurs when external mechanical forces injure or damage the brain. Despite differences in etiology, both conditions share common pathological mechanisms, including inflammation, disruption of vascular integrity, neuronal death, and impaired recovery. These overlapping pathways highlight the need for therapies that target shared processes to promote neurological recovery.

[0246] Preclinical research to date provides evidence that systemically administered rGDF11 exhibits broad therapeutic effects by promoting vascular regeneration1, activating progenitor / stem cells1, and antagonizing inflammation1. GDF11 has consistently demonstrated enhanced neurological recovery in rodent models of ischemic stroke, and aging.1 6This research extends these findings to ICH and TBI models.

[0247] The efficacy of rGDF11 in improving recovery outcomes post - ICH and TBI was evaluated. Potential mechanisms by which rGDF11 exerts effects in stroke recovery were elucidated.

[0248] For ICH, intrastriatal collagenase injection was performed on mice as previously described by Lei et al., 2014.7ForTBI, the murine closed head injury model with a pneumatic impactor was employed as described by Laskowitz et al., 2017.8rGDF11 or vehicle was administered to male mice by intraperitoneal injection (1 mg / kg) starting 30 minutes after ICH or TBI and then every 24 hours for 7 days. For ICH, Neuroseverity Score (NSS)9and Rotarod Latency (RR)9behavioral assessments were conducted pre-ICH and on days 1, 2, 3, 4, 5, 6, 7, 14, 21, and 28 post-ICH. CatWalk (CW) assessment was performed on day 7 post-injury.10Brain section analyses of Microglia and vascular area were performed with F4 / 80 and Cd31 antibodies by immunofluorescence. For TBI, RR behavioral assessment was conducted pre-TBI and on days 1, 2, 3, 4, 5, 6, 7, 14, 21, and 28 post-TBI.P390200. W0.01NSS was conducted pre-TBI and on days 1 and 28 post-ICH. Total improvement was determined by subtracting day 1 score with day 28 score

[0249] rGDF11 improves functional recovery of neurobehavioral deficits post injury in preclinical rodent models of ICH and TBI. rGDF11 functions through multiple mechanisms of action for recovery post ICH including reducing inflammation and increasing vascularization. rGDF11 shows strong potential as a neurorestorative therapy for ICH and TBI. Its ability to improve motor and behavioral outcomes highlights its promise in promoting recovery through mechanisms such as neovascularization and anti-inflammatory effects, which are essential for brain repair and functional recovery. Further dose regimen optimization studies, including evaluating route of administration, dosing initiation, dosing frequency, and dose levels are planned.

[0250] Cohen et al., (2025). ‘Recombinant GDF11 Promotes Recovery in a Rat Permanent Ischmia Model of Subacute Stroke’, Stroke (in press).REFERENCES1. Cohen et al., (2025). ‘Recombinant GDF11 Promotes Recovery in a Rat Permanent Ischmia Model of Subacute Stroke’, Stroke (in press).2. Lu et al., (2018). 'Growth Differentiation Factor 11 Promotes Neurovascular Recovery After Stroke in Mice', Front Cell Neurosci.3. Ma et al., (2018). 'Growth differentiation factor 11 improves neurobehavioral recovery and stimulates angiogenesis in rats subjected to cerebral ischemia / reperfusion', Brain Res Bull.4. Hudobenko et al., (2020). 'Growth differentiation factor-11 supplementation improves survival and promotes recovery after ischemic stroke in aged mice', Aging.5. Zhao et al., (2020). 'The neuroprotective and neurorestorative effects of growth differentiation factor 11 in cerebral ischemic injury', Brain Res.6. Su et al., (2021). 'In situ slow-release recombinant growth differentiation factor 11 exhibits therapeutic efficacy in ischemic stroke’, Biomedicine & Pharmatherapeutic.7. Lei et al., (2014). ‘Intrastriatal Injection of Autologous Blood or Clostridial Collagenase as Murine Models of Intracerebral Hemorrhage’, J Vis Exp.8. Laskowitz et al., (2017). ‘Neuroprotective pentapeptide CN-105 is associated with reduced sterile inflammation and improved functional outcomes in a traumatic brain injury murine model’, Scientific Reports.9. Wang et al., (2021). ‘Neuroprotective Pentapeptide, CN-105, Improves Outcomes in Translational Models of Intracerebral Hemorrhage’, Neurocrit Care.10. Walter et al., (2020). ‘The CatWalk XT is a valid tool for objective assessment of motor function in the acute phase after controlled cortical impact in mice’, Behavioural Brain Research.EMBODIMENTSThe following list of embodiments is intended to complement, rather than displace or supersede, the previous descriptions.P390200. W0.01Embodiment 1. A method of treating stroke or reducing a symptom thereof in a human subject in need thereof, comprising:administering to the human subject having experienced a stroke event a dosing regimen of GDF11 comprising a dose of GDF11 on intermittent days.Embodiment 2. The method of embodiment 1, wherein the intermittent dosing begins 24 hours + / - 8 hours after the stroke event.Embodiment 3. The method of any one preceding embodiment, comprising three doses, each separated by a 24 + / - 8 hour period without a dose.Embodiment 4. The method of any one preceding embodiment, comprising three doses, each separated by 24 + / - 4 hour period without a dose.Embodiment 5. The method of any one preceding embodiment, comprising three doses, each separated by 24 + / - 2 hour period without a dose.Embodiment 6. The method of any one preceding embodiment, comprising three doses, each separated by 48 + / - 8 hour period without a dose.Embodiment 7. The method of any one preceding embodiment, comprising three doses, each separated by 48 + / - 4 hour period without a dose.Embodiment 8. The method of any one preceding embodiment, comprising three doses, each separated by 48 + / - 2 hour period without a dose.Embodiment 9. A method of treating stroke or reducing a symptom thereof in a human subject in need thereof, comprising:administering a second dosing regimen of GDF11 to a subject, who following the first dosing regimen has an increased abundance of one or more biomarkers having a SomaScan Aptamer sequence selected from 19197-95, 9843-5, 9844-138, 15312-14, 15323-112, 15627-83, 2867-52, 23302-19, 5360-9, 14685-17, 15523-9, 18185-118, 2811-27, 2602-2, 13660-76, 13621-31, 12583-77, 14748-31, 13573-5, 5870-23, 24237-115, 11130-158, 3351-1, 9840-2, 2819-23, 7959-34, 4203-50, 14029-42, 15321-8, 4976-57, 15527-90, 20913-27, 25105-87, 19259-176, 24723-58, 18829-4, 22984-10, 18824-7, 25287-7, 4230-1, 6036-78, 8039-41, 4393-3, 4130-71, 4487-1, 4394-71, 4989-7, 13384-110,P390200. W0.0111171-25, 11245-43, 13122-19, 7713-102, 7713-50, 13740-51, 14587-16, 2765-4, 18233-10, 23903-3, 5464-52, 3236-12, 24050-26, 18900-37, 3317-33, 9816-37, 11816-84, 20073-22, 3453-87, 3381-24, 2864-2, 22041-26, 3115-64, 5007-1, 2855-49, 15604-18, 9760-13, 21768-9, 21813-171, 23693-4, 14036-116, 19373-3, 19122-47, 20105-7, 21817-5, 13636-20, 19188-21, 4292-5, 20584-4, 9869-28, 8778-3, 5846-24, 5542-22, 3214-3, 6590-54, 15387-44, 8295-16, 19377-14, 13719-19, 25249-33, 25947-116, 21430-4, 11354-21, 24909-40, 25949-3, 10070-22, 15545-13, 3466-8, 21483-155, 12479-50, 25463-3, 2644-11, 5475-10, 3379-29, 9504-19, 17516-7, 23329-52, 17205-21, 19222-124, 14287-6, 18950-13, 16769-20, 14332-3, 22547-17, 18373-13, 21713-11, 17764-108, 12540-25, 21752-10, 22578-17, 5116-62, 3469-74, 3868-8, 13242-134, 16043-30, 5272-55, 19630-2, 13707-27, 4467-49, 15433-4, 5488-74, 12351-25, 10346-5, 10354-57, 19553-14, 18308-30, 17355-56, 21539-139, 3339-33, 14111-15, 14082-56, 5440-26, 24320-3, 9997-12, 10756-34, 5275-28, 9830-109, 7952-2, 3050-7, 25451-39, and 4560-34, and / ora decreased abundance of one or more biomarkers having a SomaScan aptamer sequence selected from 18922-27, 4337-49, 25306-51, 10025-1, 10801-11, 14124-6, 22580-29, 2615-60, 5658-64, 2443-10, 19570-12, 17166-4, 14757-144, 5581-28, 2841-13, 5755-29, 10426-21, 13547-5, 13738-8, 19622-7, 2748-3, 5193-51, 5242-37, 7655-11, 3723-1, 9380-2, 21380-77, 20546-71, 9099-19, and 9970-7,in a human subject to which has been administered a first dosing regimen of GDF 11.Embodiment 10. A method of treating stroke or reducing a symptom thereof in a human subject in need thereof, comprising:administering a second dosing regimen of GDF11 to a subject, who following the first dosing regimen has an increased abundance of one or more biomarkers having a SomaScan Aptamer sequence selected from 19197-95, 9843-5, 9844-138, 15312-14, 15323-112, 15627-83, 2867-52, 23302-19, 5360-9, 14685-17, 15523-9, 18185-118, 2811-27, 2602-2, 13660-76, 13621-31, 12583-77, 14748-31, 13573-5, 5870-23, 24237-115, 11130-158, 3351-1, 9840-2, 2819-23, 7959-34, 4203-50, 14029-42, 15321-8, 4976-57, 3516-60, 15527-90, 20913-27, 25105-87, 19259-176, 24723-58, 18829-4, 22984-10, 18824-7, 25287-7, 4230-1, 6036-78, 8039-41, 4393-3, 4130-71, 4487-1, 4394-71, 4989-7, 13384-110, 11171-25, 11245-43, 13122-19, 7713-102, 7713-50, 13740-51, 14587-16, 2765-4, 18233-10, 23903-3, 5464-52, 3236-12, 24050-26, 18900-37, 3317-33, 9816-37, 11816-84, 20073-22, 3453-87, 3381-24, 2864-2, 22041-26, 3115-64, 5007-1, 2855-49, 15604-18, 9760-13, 21768-9, 21813-171, 23693-4, 14036-116, 19373-3, 19122-47, 20105-7, 21817-5, 13636-20, 19188-21, 4292-5, 20584-4, 9869-28, 8778-3, 5846-24, 5542-22, 3214-3, 6590-54, 15387-44, 8295-16, 19377-14, 13719-19, 25249-33, 25947-116, 21430-4, 11354-21,P390200. W0.0124909-40, 25949-3, 10070-22, 15545-13, 3466-8, 21483-155, 12479-50, 25463-3, 2644-11, 5475-10, 3379-29, 9504-19, 17516-7, 23329-52, 17205-21, 19222-124, 14287-6, 18950-13, 16769-20, 14332-3, 22547-17, 18373-13, 21713-11, 17764-108, 12540-25, 21752-10, 22578-17, 5116-62, 3469-74, 3868-8, 13242-134, 16043-30, 5272-55, 19630-2, 13707-27, 4467-49, 15433-4, 5488-74, 12351-25, 10346-5, 10354-57, 19553-14, 18308-30, 17355-56, 21539-139, 3339-33, 14111-15, 14082-56, 5440-26, 24320-3, 9997-12, 10756-34, 5275-28, 9830-109, 7952-2, 3050-7, 25451-39, and 4560-34, and / ora decreased abundance of one or more biomarkers having a SomaScan aptamer sequence selected from 18922-27, 4337-49, 25306-51, 10025-1, 10801-11, 14124-6, 22580-29, 2615-60, 5658-64, 2443-10, 19570-12, 17166-4, 14757-144, 5581-28, 2841-13, 5755-29, 10426-21, 13547-5, 13738-8, 19622-7, 2748-3, 5193-51, 5242-37, 7655-11, 3723-1, 9380-2, 21380-77, 20546-71, 9099-19, and 9970-7,in a human subject to which has been administered a first dosing regimen of GDF 11.Embodiment 11. The method of embodiment 9 or 10, wherein the second dosing regimen begins at least 3 weeks following the final dose of the first dosing regimen.Embodiment 12. The method of embodiment 9 or 10, wherein the second dosing regimen begins at least 2 months following the final dose of the first dosing regimen.Embodiment 13. A method of treating stroke or reducing a symptom thereof in a human subject in need thereof, comprising:administering a second dosing regimen of GDF11 to a subject having an increased abundance of one or more biomarkers having a SomaScan Aptamer sequence selected from 19197-95, 9843-5, 9844-138, 15312-14, 15323-112, 15627-83, 2867-52, 23302-19, 5360-9, 14685-17, 15523-9, 18185-118, 2811-27, 2602-2, 13660-76, 13621-31, 12583-77, 14748-31, 13573-5, 5870-23, 24237-115, 11130-158, 3351-1, 9840-2, 2819-23, 7959-34, 4203-50, 14029-42, 15321-8, 4976-57, 15527-90, 20913-27, 25105-87, 19259-176, 24723-58, 18829-4, 22984-10, 18824-7, 25287-7, 4230-1, 6036-78, 8039-41, 4393-3, 4130-71, 4487-1, 4394-71, 4989-7, 13384-110, 11171-25, 11245-43, 13122-19, 7713-102, 7713-50, 13740-51, 14587-16, 2765-4, 18233-10, 23903-3, 5464-52, 3236-12, 24050-26, 18900-37, 3317-33, 9816-37, 11816-84, 20073-22, 3453-87, 3381-24, 2864-2, 22041-26, 3115-64, 5007-1, 2855-49, 15604-18, 9760-13, 21768-9, 21813-171, 23693-4, 14036-116, 19373-3, 19122-47, 20105-7, 21817-5, 13636-20, 19188-21, 4292-5, 20584-4, 9869-28, 8778-3, 5846-24, 5542-22, 3214-3, 6590-54, 15387-44, 8295-16, 19377-14, 13719-19, 25249-33, 25947-116, 21430-4, 11354-21, 24909-40, 25949-3, 10070-22, 15545-13, 3466-8, 21483-155, 12479-50, 25463-3, 2644-11, 5475-10, 3379-29, 9504-19, 17516-7, 23329-52, 17205-P390200. W0.0121, 19222-124, 14287-6, 18950-13, 16769-20, 14332-3, 22547-17, 18373-13, 21713-11, 17764-108, 12540-25, 21752-10, 22578-17, 5116-62, 3469-74, 3868-8, 13242-134, 16043-30, 5272-55, 19630-2, 13707-27, 4467-49, 15433-4, 5488-74, 12351-25, 10346-5, 10354-57, 19553-14, 18308-30, 17355-56, 21539-139, 3339-33, 14111-15, 14082-56, 5440-26, 24320-3, 9997-12, 10756-34, 5275-28, 9830-109, 7952-2, 3050-7, 25451-39, and 4560-34, and / ora decreased abundance of one or more biomarkers having a SomaScan aptamer sequence selected from 18922-27, 4337-49, 25306-51, 10025-1, 10801-11, 14124-6, 22580-29, 2615-60, 5658-64, 2443-10, 19570-12, 17166-4, 14757-144, 5581-28, 2841-13, 5755-29, 10426-21, 13547-5, 13738-8, 19622-7, 2748-3, 5193-51, 5242-37, 7655-11, 3723-1, 9380-2, 21380-77, 20546-71, 9099-19, and 9970-7,in a human subject to whom has been administered a first dosing regimen of GDF11.Embodiment 14. A method of treating stroke or reducing a symptom thereof in a human subject in need thereof, comprising:administering a second dosing regimen of GDF11 to a subject having an increased abundance of one or more biomarkers having a SomaScan Aptamer sequence selected from 19197-95, 9843-5, 9844-138, 15312-14, 15323-112, 15627-83, 2867-52, 23302-19, 5360-9, 14685-17, 15523-9, 18185-118, 2811-27, 2602-2, 13660-76, 13621-31, 12583-77, 14748-31, 13573-5, 5870-23, 24237-115, 11130-158, 3351-1, 9840-2, 2819-23, 7959-34, 4203-50, 14029-42, 15321-8, 4976-57, 3516-60, 15527-90, 20913-27, 25105-87, 19259-176, 24723-58, 18829-4, 22984-10, 18824-7, 25287-7, 4230-1, 6036-78, 8039-41, 4393-3, 4130-71, 4487-1, 4394-71, 4989-7, 13384-110, 11171-25, 11245-43, 13122-19, 7713-102, 7713-50, 13740-51, 14587-16, 2765-4, 18233-10, 23903-3, 5464-52, 3236-12, 24050-26, 18900-37, 3317-33, 9816-37, 11816-84, 20073-22, 3453-87, 3381-24, 2864-2, 22041-26, 3115-64, 5007-1, 2855-49, 15604-18, 9760-13, 21768-9, 21813-171, 23693-4, 14036-116, 19373-3, 19122-47, 20105-7, 21817-5, 13636-20, 19188-21, 4292-5, 20584-4, 9869-28, 8778-3, 5846-24, 5542-22, 3214-3, 6590-54, 15387-44, 8295-16, 19377-14, 13719-19, 25249-33, 25947-116, 21430-4, 11354-21, 24909-40, 25949-3, 10070-22, 15545-13, 3466-8, 21483-155, 12479-50, 25463-3, 2644-11, 5475-10, 3379-29, 9504-19, 17516-7, 23329-52, 17205-21, 19222-124, 14287-6, 18950-13, 16769-20, 14332-3, 22547-17, 18373-13, 21713-11, 17764-108, 12540-25, 21752-10, 22578-17, 5116-62, 3469-74, 3868-8, 13242-134, 16043-30, 5272-55, 19630-2, 13707-27, 4467-49, 15433-4, 5488-74, 12351-25, 10346-5, 10354-57, 19553-14, 18308-30, 17355-56, 21539-139, 3339-33, 14111-15, 14082-56, 5440-26, 24320-3, 9997-12, 10756-34, 5275-28, 9830-109, 7952-2, 3050-7, 25451-39, and 4560-34, and / orP390200. W0.01a decreased abundance of one or more biomarkers having a SomaScan aptamer sequence selected from 18922-27, 4337-49, 25306-51, 10025-1, 10801-11, 14124-6, 22580-29, 2615-60, 5658-64, 2443-10, 19570-12, 17166-4, 14757-144, 5581-28, 2841-13, 5755-29, 10426-21, 13547-5, 13738-8, 19622-7, 2748-3, 5193-51, 5242-37, 7655-11, 3723-1, 9380-2, 21380-77, 20546-71, 9099-19, and 9970-7,in a human subject to whom has been administered a first dosing regimen of GDF11.Embodiment 15. The method of embodiment 13 or 14, wherein the second dosing regimen begins at least 3 weeks following the first dosing regimen.Embodiment 16. The method of embodiment 13 or 14, wherein the second dosing regimen begins at least 2 months following the first dosing regimen.Embodiment 17. A method of treating stroke or reducing a symptom thereof in a human subject in need thereof, comprising:administering a first dosing regimen of GDF11 to the human subject;if the human subject has an increased abundance of one or more biomarkers having a SomaScan Aptamer sequence selected from 19197-95, 9843-5, 9844-138, 15312-14, 15323-112, 15627-83, 2867-52, 23302-19, 5360-9, 14685-17, 15523-9, 18185-118, 2811-27, 2602-2, 13660-76, 13621-31, 12583-77, 14748-31, 13573-5, 5870-23, 24237-115, 11130-158, 3351-1, 9840-2, 2819-23, 7959-34, 4203-50, 14029-42, 15321-8, 4976-57, 15527-90, 20913-27, 25105-87, 19259-176, 24723-58, 18829-4, 22984-10, 18824-7, 25287-7, 4230-1, 6036-78, 8039-41, 4393-3, 4130-71, 4487-1, 4394-71, 4989-7, 13384-110, 11171-25, 11245-43, 13122-19, 7713-102, 7713-50, 13740-51, 14587-16, 2765-4, 18233-10, 23903-3, 5464-52, 3236-12, 24050-26, 18900-37, 3317-33, 9816-37, 11816-84, 20073-22, 3453-87, 3381-24, 2864-2, 22041-26, 3115-64, 5007-1, 2855-49, 15604-18, 9760-13, 21768-9, 21813-171, 23693-4, 14036-116, 19373-3, 19122-47, 20105-7, 21817-5, 13636-20, 19188-21, 4292-5, 20584-4, 9869-28, 8778-3, 5846-24, 5542-22, 3214-3, 6590-54, 15387-44, 8295-16, 19377-14, 13719-19, 25249-33, 25947-116, 21430-4, 11354-21, 24909-40, 25949-3, 10070-22, 15545-13, 3466-8, 21483-155, 12479-50, 25463-3, 2644-11, 5475-10, 3379-29, 9504-19, 17516-7, 23329-52, 17205-21, 19222-124, 14287-6, 18950-13, 16769-20, 14332-3, 22547-17, 18373-13, 21713-11, 17764-108, 12540-25, 21752-10, 22578-17, 5116-62, 3469-74, 3868-8, 13242-134, 16043-30, 5272-55, 19630-2, 13707-27, 4467-49, 15433-4, 5488-74, 12351-25, 10346-5, 10354-57, 19553-14, 18308-30, 17355-56, 21539-139, 3339-33, 14111-15, 14082-56, 5440-26, 24320-3, 9997-12, 10756-34, 5275-28, 9830-109, 7952-2, 3050-7, 25451-39, and 4560-34, and / orP390200. W0.01a decreased abundance of one or more biomarkers having a SomaScan aptamer sequence selected from 18922-27, 4337-49, 25306-51, 10025-1, 10801-11, 14124-6, 22580-29, 2615-60, 5658-64, 2443-10, 19570-12, 17166-4, 14757-144, 5581-28, 2841-13, 5755-29, 10426-21, 13547-5, 13738-8, 19622-7, 2748-3, 5193-51, 5242-37, 7655-11, 3723-1, 9380-2, 21380-77, 20546-71, 9099-19, and 9970-7,administering a second dosing regimen to the subject.Embodiment 18. A method of treating stroke or reducing a symptom thereof in a human subject in need thereof, comprising:administering a first dosing regimen of GDF11 to the human subject;if the human subject has an increased abundance of one or more biomarkers having a SomaScan Aptamer sequence selected from 19197-95, 9843-5, 9844-138, 15312-14, 15323-112, 15627-83, 2867-52, 23302-19, 5360-9, 14685-17, 15523-9, 18185-118, 2811-27, 2602-2, 13660-76, 13621-31, 12583-77, 14748-31, 13573-5, 5870-23, 24237-115, 11130-158, 3351-1, 9840-2, 2819-23, 7959-34, 4203-50, 14029-42, 15321-8, 4976-57, 3516-60, 15527-90, 20913-27, 25105-87, 19259-176, 24723-58, 18829-4, 22984-10, 18824-7, 25287-7, 4230-1, 6036-78, 8039-41, 4393-3, 4130-71, 4487-1, 4394-71, 4989-7, 13384-110, 11171-25, 11245-43, 13122-19, 7713-102, 7713-50, 13740-51, 14587-16, 2765-4, 18233-10, 23903-3, 5464-52, 3236-12, 24050-26, 18900-37, 3317-33, 9816-37, 11816-84, 20073-22, 3453-87, 3381-24, 2864-2, 22041-26, 3115-64, 5007-1, 2855-49, 15604-18, 9760-13, 21768-9, 21813-171, 23693-4, 14036-116, 19373-3, 19122-47, 20105-7, 21817-5, 13636-20, 19188-21, 4292-5, 20584-4, 9869-28, 8778-3, 5846-24, 5542-22, 3214-3, 6590-54, 15387-44, 8295-16, 19377-14, 13719-19, 25249-33, 25947-116, 21430-4, 11354-21, 24909-40, 25949-3, 10070-22, 15545-13, 3466-8, 21483-155, 12479-50, 25463-3, 2644-11, 5475-10, 3379-29, 9504-19, 17516-7, 23329-52, 17205-21, 19222-124, 14287-6, 18950-13, 16769-20, 14332-3, 22547-17, 18373-13, 21713-11, 17764-108, 12540-25, 21752-10, 22578-17, 5116-62, 3469-74, 3868-8, 13242-134, 16043-30, 5272-55, 19630-2, 13707-27, 4467-49, 15433-4, 5488-74, 12351-25, 10346-5, 10354-57, 19553-14, 18308-30, 17355-56, 21539-139, 3339-33, 14111-15, 14082-56, 5440-26, 24320-3, 9997-12, 10756-34, 5275-28, 9830-109, 7952-2, 3050-7, 25451-39, and 4560-34, and / ora decreased abundance of one or more biomarkers having a SomaScan aptamer sequence selected from 18922-27, 4337-49, 25306-51, 10025-1, 10801-11, 14124-6, 22580-29, 2615-60, 5658-64, 2443-10, 19570-12, 17166-4, 14757-144, 5581-28, 2841-13, 5755-29, 10426-21, 13547-5, 13738-8, 19622-7, 2748-3, 5193-51, 5242-37, 7655-11, 3723-1, 9380-2, 21380-77, 20546-71, 9099-19, and 9970-7,administering a second dosing regimen to the subject.P390200. W0.01Embodiment 19. The method of embodiment 17 or 18 further comprising determining if the human subject has an increased abundance of one or more biomarkers having a SomaScan Aptamer sequence selected from 19197-95, 9843-5, 9844-138, 15312-14, 15323-112, 15627-83, 2867-52, 23302-19, 5360-9, 14685-17, 15523-9, 18185-118, 2811-27, 2602-2, 13660-76, 13621-31, 12583-77, 14748-31, 13573-5, 5870-23, 24237-115, 11130-158, 3351-1, 9840-2, 2819-23, 7959-34, 4203-50, 14029-42, 15321-8, 4976-57, 15527-90, 20913-27, 25105-87, 19259-176, 24723-58, 18829-4, 22984-10, 18824-7, 25287-7, 4230-1, 6036-78, 8039-41, 4393-3, 4130-71, 4487-1, 4394-71, 4989-7, 13384-110, 11171-25, 11245-43, 13122-19, 7713-102, 7713-50, 13740-51, 14587-16, 2765-4, 18233-10, 23903-3, 5464-52, 3236-12, 24050-26, 18900-37, 3317-33, 9816-37, 11816-84, 20073-22, 3453-87, 3381-24, 2864-2, 22041-26, 3115-64, 5007-1, 2855-49, 15604-18, 9760-13, 21768-9, 21813-171, 23693-4, 14036-116, 19373-3, 19122-47, 20105-7, 21817-5, 13636-20, 19188-21, 4292-5, 20584-4, 9869-28, 8778-3, 5846-24, 5542-22, 3214-3, 6590-54, 15387-44, 8295-16, 19377-14, 13719-19, 25249-33, 25947-116, 21430-4, 11354-21, 24909-40, 25949-3, 10070-22, 15545-13, 3466-8, 21483-155, 12479-50, 25463-3, 2644-11, 5475-10, 3379-29, 9504-19, 17516-7, 23329-52, 17205-21, 19222-124, 14287-6, 18950-13, 16769-20, 14332-3, 22547-17, 18373-13, 21713-11, 17764-108, 12540-25, 21752-10, 22578-17, 5116-62, 3469-74, 3868-8, 13242-134, 16043-30, 5272-55, 19630-2, 13707-27, 4467-49, 15433-4, 5488-74, 12351-25, 10346-5, 10354-57, 19553-14, 18308-30, 17355-56, 21539-139, 3339-33, 14111-15, 14082-56, 5440-26, 24320-3, 9997-12, 10756-34, 5275-28, 9830-109, 7952-2, 3050-7, 25451-39, and 4560-34, and / ora decreased abundance of one or more biomarkers having a SomaScan aptamer sequence selected from 18922-27, 4337-49, 25306-51, 10025-1, 10801-11, 14124-6, 22580-29, 2615-60, 5658-64, 2443-10, 19570-12, 17166-4, 14757-144, 5581-28, 2841-13, 5755-29, 10426-21, 13547-5, 13738-8, 19622-7, 2748-3, 5193-51, 5242-37, 7655-11, 3723-1, 9380-2, 21380-77, 20546-71, 9099-19, and 9970-7.Embodiment 20. The method of embodiment 17 or 19 further comprising determining if the human subject has an increased abundance of one or more biomarkers having a SomaScan Aptamer sequence selected from 19197-95, 9843-5, 9844-138, 15312-14, 15323-112, 15627-83, 2867-52, 23302-19, 5360-9, 14685-17, 15523-9, 18185-118, 2811-27, 2602-2, 13660-76, 13621-31, 12583-77, 14748-31, 13573-5, 5870-23, 24237-115, 11130-158, 3351-1, 9840-2, 2819-23, 7959-34, 4203-50, 14029-42, 15321-8, 4976-57, 3516-60, 15527-90, 20913-27, 25105-87, 19259-176, 24723-58, 18829-4, 22984-10, 18824-7, 25287-7, 4230-1, 6036-78, 8039-41, 4393-3, 4130-71, 4487-1, 4394-71, 4989-7, 13384-110, 11171-25, 11245-43, 13122-19, 7713-102, 7713-50, 13740-51, 14587-16, 2765-4, 18233-10, 23903-3, 5464-52, 3236-12, 24050-26, 18900-37, 3317-33, 9816-37, 11816-84,P390200. W0.0120073-22, 3453-87, 3381-24, 2864-2, 22041-26, 3115-64, 5007-1, 2855-49, 15604-18, 9760-13, 21768-9, 21813-171, 23693-4, 14036-116, 19373-3, 19122-47, 20105-7, 21817-5, 13636-20, 19188-21, 4292-5, 20584-4, 9869-28, 8778-3, 5846-24, 5542-22, 3214-3, 6590-54, 15387-44, 8295-16, 19377-14, 13719-19, 25249-33, 25947-116, 21430-4, 11354-21, 24909-40, 25949-3, 10070-22, 15545-13, 3466-8, 21483-155, 12479-50, 25463-3, 2644-11, 5475-10, 3379-29, 9504-19, 17516-7, 23329-52, 17205-21, 19222-124, 14287-6, 18950-13, 16769-20, 14332...

Claims

P390200. W0.01CLAIMSWhat is claimed is:

1. A method of treating stroke or reducing a symptom thereof in a human subject in need thereof, comprising:administering to the human subject having experienced a stroke event a dosing regimen of GDF11 comprising a dose of GDF11 on intermittent days.

2. The method of claim 1, wherein the intermittent dosing begins 24 hours + / - 8 hours after the stroke event.

3. The method of any one preceding claim, comprising three doses, each separated by a 24 + / - 8 hour period without a dose.

4. The method of any one preceding claim, comprising three doses, each separated by 24 + / - 4 hour period without a dose.

5. The method of any one preceding claim, comprising three doses, each separated by 24 + / - 2 hour period without a dose.

6. The method of any one preceding claim, comprising three doses, each separated by 48 + / - 8 hour period without a dose.

7. The method of any one preceding claim, comprising three doses, each separated by 48 + / - 4 hour period without a dose.

8. The method of any one preceding claim, comprising three doses, each separated by 48 + / - 2 hour period without a dose.

9. A method of treating stroke or reducing a symptom thereof in a human subject in need thereof, comprising:administering a second dosing regimen of GDF11 to a subject, who following the first dosing regimen has an increased abundance of one or more biomarkers having a SomaScan Aptamer sequence selected from 19197-95, 9843-5, 9844-138, 15312-14, 15323-112, 15627-83, 2867-52, 23302-19, 5360-9, 14685-17, 15523-9, 18185-118, 2811-27, 2602-2, 13660-76, 13621-31, 12583-77, 14748-31, 13573-5, 5870-23, 24237-115, 11130-158, 3351-1, 9840-2, 2819-23, 7959-34, 4203-50, 14029-42, 15321-8, 4976-57,P390200. W0.0115527-90, 20913-27, 25105-87, 19259-176, 24723-58, 18829-4, 22984-10, 18824-7, 25287-7, 4230-1, 6036-78, 8039-41, 4393-3, 4130-71, 4487-1, 4394-71, 4989-7, 13384-110, 11171-25, 11245-43, 13122-19, 7713-102, 7713-50, 13740-51, 14587-16, 2765-4, 18233-10, 23903-3, 5464-52, 3236-12, 24050-26, 18900-37, 3317-33, 9816-37, 11816-84, 20073-22, 3453-87, 3381-24, 2864-2, 22041-26, 3115-64, 5007-1, 2855-49, 15604-18, 9760-13, 21768-9, 21813-171, 23693-4, 14036-116, 19373-3, 19122-47, 20105-7, 21817-5, 13636-20, 19188-21, 4292-5, 20584-4, 9869-28, 8778-3, 5846-24, 5542-22, 3214-3, 6590-54, 15387-44, 8295-16, 19377-14, 13719-19, 25249-33, 25947-116, 21430-4, 11354-21, 24909-40, 25949-3, 10070-22, 15545-13, 3466-8, 21483-155, 12479-50, 25463-3, 2644-11, 5475-10, 3379-29, 9504-19, 17516-7, 23329-52, 17205-21, 19222-124, 14287-6, 18950-13, 16769-20, 14332-3, 22547-17, 18373-13, 21713-11, 17764-108, 12540-25, 21752-10, 22578-17, 5116-62, 3469-74, 3868-8, 13242-134, 16043-30, 5272-55, 19630-2, 13707-27, 4467-49, 15433-4, 5488-74, 12351-25, 10346-5, 10354-57, 19553-14, 18308-30, 17355-56, 21539-139, 3339-33, 14111-15, 14082-56, 5440-26, 24320-3, 9997-12, 10756-34, 5275-28, 9830-109, 7952-2, 3050-7, 25451-39, and 4560-34, and / ora decreased abundance of one or more biomarkers having a SomaScan aptamer sequence selected from 18922-27, 4337-49, 25306-51, 10025-1, 10801-11, 14124-6, 22580-29, 2615-60, 5658-64, 2443-10, 19570-12, 17166-4, 14757-144, 5581-28, 2841-13, 5755-29, 10426-21, 13547-5, 13738-8, 19622-7, 2748-3, 5193-51, 5242-37, 7655-11, 3723-1, 9380-2, 21380-77, 20546-71, 9099-19, and 9970-7,in a human subject to which has been administered a first dosing regimen of GDF 11.

10. A method of treating stroke or reducing a symptom thereof in a human subject in need thereof, comprising:administering a second dosing regimen of GDF11 to a subject, who following the first dosing regimen has an increased abundance of one or more biomarkers having a SomaScan Aptamer sequence selected from 19197-95, 9843-5, 9844-138, 15312-14, 15323-112, 15627-83, 2867-52, 23302-19, 5360-9, 14685-17, 15523-9, 18185-118, 2811-27, 2602-2, 13660-76, 13621-31, 12583-77, 14748-31, 13573-5, 5870-23, 24237-115, 11130-158, 3351-1, 9840-2, 2819-23, 7959-34, 4203-50, 14029-42, 15321-8, 4976-57, 3516-60, 15527-90, 20913-27, 25105-87, 19259-176, 24723-58, 18829-4, 22984-10, 18824-7, 25287-7, 4230-1, 6036-78, 8039-41, 4393-3, 4130-71, 4487-1, 4394-71, 4989-7, 13384-110, 11171-25, 11245-43, 13122-19, 7713-102, 7713-50, 13740-51, 14587-16, 2765-4, 18233-10, 23903-3, 5464-52, 3236-12, 24050-26, 18900-37, 3317-33, 9816-37, 11816-84, 20073-22, 3453-87, 3381-24, 2864-2, 22041-26, 3115-64, 5007-1, 2855-49, 15604-18, 9760-13, 21768-9, 21813-171, 23693-4, 14036-116, 19373-3, 19122-47, 20105-7, 21817-5,P390200. W0.0113636-20, 19188-21, 4292-5, 20584-4, 9869-28, 8778-3, 5846-24, 5542-22, 3214-3, 6590-54, 15387-44, 8295-16, 19377-14, 13719-19, 25249-33, 25947-116, 21430-4, 11354-21, 24909-40, 25949-3, 10070-22, 15545-13, 3466-8, 21483-155, 12479-50, 25463-3, 2644-11, 5475-10, 3379-29, 9504-19, 17516-7, 23329-52, 17205-21, 19222-124, 14287-6, 18950-13, 16769-20, 14332-3, 22547-17, 18373-13, 21713-11, 17764-108, 12540-25, 21752-10, 22578-17, 5116-62, 3469-74, 3868-8, 13242-134, 16043-30, 5272-55, 19630-2, 13707-27, 4467-49, 15433-4, 5488-74, 12351-25, 10346-5, 10354-57, 19553-14, 18308-30, 17355-56, 21539-139, 3339-33, 14111-15, 14082-56, 5440-26, 24320-3, 9997-12, 10756-34, 5275-28, 9830-109, 7952-2, 3050-7, 25451-39, and 4560-34, and / ora decreased abundance of one or more biomarkers having a SomaScan aptamer sequence selected from 18922-27, 4337-49, 25306-51, 10025-1, 10801-11, 14124-6, 22580-29, 2615-60, 5658-64, 2443-10, 19570-12, 17166-4, 14757-144, 5581-28, 2841-13, 5755-29, 10426-21, 13547-5, 13738-8, 19622-7, 2748-3, 5193-51, 5242-37, 7655-11, 3723-1, 9380-2, 21380-77, 20546-71, 9099-19, and 9970-7,in a human subject to which has been administered a first dosing regimen of GDF 11.

11. The method of claim 9 or 10, wherein the second dosing regimen begins at least 3 weeks following the final dose of the first dosing regimen.

12. The method of claim 9 or 10, wherein the second dosing regimen begins at least 2 months following the final dose of the first dosing regimen.

13. A method of treating stroke or reducing a symptom thereof in a human subject in need thereof, comprising:administering a second dosing regimen of GDF11 to a subject having an increased abundance of one or more biomarkers having a SomaScan Aptamer sequence selected from 19197-95, 9843-5, 9844-138, 15312-14, 15323-112, 15627-83, 2867-52, 23302-19, 5360-9, 14685-17, 15523-9, 18185-118, 2811-27, 2602-2, 13660-76, 13621-31, 12583-77, 14748-31, 13573-5, 5870-23, 24237-115, 11130-158, 3351-1, 9840-2, 2819-23, 7959-34, 4203-50, 14029-42, 15321-8, 4976-57, 15527-90, 20913-27, 25105-87, 19259-176, 24723-58, 18829-4, 22984-10, 18824-7, 25287-7, 4230-1, 6036-78, 8039-41, 4393-3, 4130-71, 4487-1, 4394-71, 4989-7, 13384-110, 11171-25, 11245-43, 13122-19, 7713-102, 7713-50, 13740-51, 14587-16, 2765-4, 18233-10, 23903-3, 5464-52, 3236-12, 24050-26, 18900-37, 3317-33, 9816-37, 11816-84, 20073-22, 3453-87, 3381-24, 2864-2, 22041-26, 3115-64, 5007-1, 2855-49, 15604-18, 9760-13, 21768-9, 21813-171, 23693-4, 14036-116, 19373-3, 19122-47, 20105-7, 21817-5, 13636-20, 19188-21, 4292-5, 20584-4, 9869-28, 8778-3, 5846-24, 5542-22, 3214-3, 6590-54, 15387-44, 8295-16, 19377-14, 13719-19, 25249-33,P390200. W0.0125947-116, 21430-4, 11354-21, 24909-40, 25949-3, 10070-22, 15545-13, 3466-8, 21483-155, 12479-50, 25463-3, 2644-11, 5475-10, 3379-29, 9504-19, 17516-7, 23329-52, 17205-21, 19222-124, 14287-6, 18950-13, 16769-20, 14332-3, 22547-17, 18373-13, 21713-11, 17764-108, 12540-25, 21752-10, 22578-17, 5116-62, 3469-74, 3868-8, 13242-134, 16043-30, 5272-55, 19630-2, 13707-27, 4467-49, 15433-4, 5488-74, 12351-25, 10346-5, 10354-57, 19553-14, 18308-30, 17355-56, 21539-139, 3339-33, 14111-15, 14082-56, 5440-26, 24320-3, 9997-12, 10756-34, 5275-28, 9830-109, 7952-2, 3050-7, 25451-39, and 4560-34, and / ora decreased abundance of one or more biomarkers having a SomaScan aptamer sequence selected from 18922-27, 4337-49, 25306-51, 10025-1, 10801-11, 14124-6, 22580-29, 2615-60, 5658-64, 2443-10, 19570-12, 17166-4, 14757-144, 5581-28, 2841-13, 5755-29, 10426-21, 13547-5, 13738-8, 19622-7, 2748-3, 5193-51, 5242-37, 7655-11, 3723-1, 9380-2, 21380-77, 20546-71, 9099-19, and 9970-7,in a human subject to whom has been administered a first dosing regimen of GDF11.

14. A method of treating stroke or reducing a symptom thereof in a human subject in need thereof, comprising:administering a second dosing regimen of GDF11 to a subject having an increased abundance of one or more biomarkers having a SomaScan Aptamer sequence selected from 19197-95, 9843-5, 9844-138, 15312-14, 15323-112, 15627-83, 2867-52, 23302-19, 5360-9, 14685-17, 15523-9, 18185-118, 2811-27, 2602-2, 13660-76, 13621-31, 12583-77, 14748-31, 13573-5, 5870-23, 24237-115, 11130-158, 3351-1, 9840-2, 2819-23, 7959-34, 4203-50, 14029-42, 15321-8, 4976-57, 3516-60, 15527-90, 20913-27, 25105-87, 19259-176, 24723-58, 18829-4, 22984-10, 18824-7, 25287-7, 4230-1, 6036-78, 8039-41, 4393-3, 4130-71, 4487-1, 4394-71, 4989-7, 13384-110, 11171-25, 11245-43, 13122-19, 7713-102, 7713-50, 13740-51, 14587-16, 2765-4, 18233-10, 23903-3, 5464-52, 3236-12, 24050-26, 18900-37, 3317-33, 9816-37, 11816-84, 20073-22, 3453-87, 3381-24, 2864-2, 22041-26, 3115-64, 5007-1, 2855-49, 15604-18, 9760-13, 21768-9, 21813-171, 23693-4, 14036-116, 19373-3, 19122-47, 20105-7, 21817-5, 13636-20, 19188-21, 4292-5, 20584-4, 9869-28, 8778-3, 5846-24, 5542-22, 3214-3, 6590-54, 15387-44, 8295-16, 19377-14, 13719-19, 25249-33, 25947-116, 21430-4, 11354-21, 24909-40, 25949-3, 10070-22, 15545-13, 3466-8, 21483-155, 12479-50, 25463-3, 2644-11, 5475-10, 3379-29, 9504-19, 17516-7, 23329-52, 17205-21, 19222-124, 14287-6, 18950-13, 16769-20, 14332-3, 22547-17, 18373-13, 21713-11, 17764-108, 12540-25, 21752-10, 22578-17, 5116-62, 3469-74, 3868-8, 13242-134, 16043-30, 5272-55, 19630-2, 13707-27, 4467-49, 15433-4, 5488-74, 12351-25, 10346-5, 10354-57, 19553-14, 18308-30, 17355-56, 21539-139, 3339-33, 14111-15, 14082-56,P390200. W0.015440-26, 24320-3, 9997-12, 10756-34, 5275-28, 9830-109, 7952-2, 3050-7, 25451-39, and 4560-34, and / ora decreased abundance of one or more biomarkers having a SomaScan aptamer sequence selected from 18922-27, 4337-49, 25306-51, 10025-1, 10801-11, 14124-6, 22580-29, 2615-60, 5658-64, 2443-10, 19570-12, 17166-4, 14757-144, 5581-28, 2841-13, 5755-29, 10426-21, 13547-5, 13738-8, 19622-7, 2748-3, 5193-51, 5242-37, 7655-11, 3723-1, 9380-2, 21380-77, 20546-71, 9099-19, and 9970-7,in a human subject to whom has been administered a first dosing regimen of GDF11.

15. The method of claim 13 or 14, wherein the second dosing regimen begins at least 3 weeks following the first dosing regimen.

16. The method of claim 13 or 14, wherein the second dosing regimen begins at least 2 months following the first dosing regimen.

17. A method of treating stroke or reducing a symptom thereof in a human subject in need thereof, comprising:administering a first dosing regimen of GDF11 to the human subject;if the human subject has an increased abundance of one or more biomarkers having a SomaScan Aptamer sequence selected from 19197-95, 9843-5, 9844-138, 15312-14, 15323-112, 15627-83, 2867-52, 23302-19, 5360-9, 14685-17, 15523-9, 18185-118, 2811-27, 2602-2, 13660-76, 13621-31, 12583-77, 14748-31, 13573-5, 5870-23, 24237-115, 11130-158, 3351-1, 9840-2, 2819-23, 7959-34, 4203-50, 14029-42, 15321-8, 4976-57, 15527-90, 20913-27, 25105-87, 19259-176, 24723-58, 18829-4, 22984-10, 18824-7, 25287-7, 4230-1, 6036-78, 8039-41, 4393-3, 4130-71, 4487-1, 4394-71, 4989-7, 13384-110, 11171-25, 11245-43, 13122-19, 7713-102, 7713-50, 13740-51, 14587-16, 2765-4, 18233-10, 23903-3, 5464-52, 3236-12, 24050-26, 18900-37, 3317-33, 9816-37, 11816-84, 20073-22, 3453-87, 3381-24, 2864-2, 22041-26, 3115-64, 5007-1, 2855-49, 15604-18, 9760-13, 21768-9, 21813-171, 23693-4, 14036-116, 19373-3, 19122-47, 20105-7, 21817-5, 13636-20, 19188-21, 4292-5, 20584-4, 9869-28, 8778-3, 5846-24, 5542-22, 3214-3, 6590-54, 15387-44, 8295-16, 19377-14, 13719-19, 25249-33, 25947-116, 21430-4, 11354-21, 24909-40, 25949-3, 10070-22, 15545-13, 3466-8, 21483-155, 12479-50, 25463-3, 2644-11, 5475-10, 3379-29, 9504-19, 17516-7, 23329-52, 17205-21, 19222-124, 14287-6, 18950-13, 16769-20, 14332-3, 22547-17, 18373-13, 21713-11, 17764-108, 12540-25, 21752-10, 22578-17, 5116-62, 3469-74, 3868-8, 13242-134, 16043-30, 5272-55, 19630-2, 13707-27, 4467-49, 15433-4, 5488-74, 12351-25, 10346-5, 10354-57, 19553-14, 18308-30, 17355-56,P390200. W0.0121539-139, 3339-33, 14111-15, 14082-56, 5440-26, 24320-3, 9997-12, 10756-34, 5275-28, 9830-109, 7952-2, 3050-7, 25451-39, and 4560-34, and / ora decreased abundance of one or more biomarkers having a SomaScan aptamer sequence selected from 18922-27, 4337-49, 25306-51, 10025-1, 10801-11, 14124-6, 22580-29, 2615-60, 5658-64, 2443-10, 19570-12, 17166-4, 14757-144, 5581-28, 2841-13, 5755-29, 10426-21, 13547-5, 13738-8, 19622-7, 2748-3, 5193-51, 5242-37, 7655-11, 3723-1, 9380-2, 21380-77, 20546-71, 9099-19, and 9970-7,administering a second dosing regimen to the subject.

18. A method of treating stroke or reducing a symptom thereof in a human subject in need thereof, comprising:administering a first dosing regimen of GDF11 to the human subject;if the human subject has an increased abundance of one or more biomarkers having a SomaScan Aptamer sequence selected from 19197-95, 9843-5, 9844-138, 15312-14, 15323-112, 15627-83, 2867-52, 23302-19, 5360-9, 14685-17, 15523-9, 18185-118, 2811-27, 2602-2, 13660-76, 13621-31, 12583-77, 14748-31, 13573-5, 5870-23, 24237-115, 11130-158, 3351-1, 9840-2, 2819-23, 7959-34, 4203-50, 14029-42, 15321-8, 4976-57, 3516-60, 15527-90, 20913-27, 25105-87, 19259-176, 24723-58, 18829-4, 22984-10, 18824-7, 25287-7, 4230-1, 6036-78, 8039-41, 4393-3, 4130-71, 4487-1, 4394-71, 4989-7, 13384-110, 11171-25, 11245-43, 13122-19, 7713-102, 7713-50, 13740-51, 14587-16, 2765-4, 18233-10, 23903-3, 5464-52, 3236-12, 24050-26, 18900-37, 3317-33, 9816-37, 11816-84, 20073-22, 3453-87, 3381-24, 2864-2, 22041-26, 3115-64, 5007-1, 2855-49, 15604-18, 9760-13, 21768-9, 21813-171, 23693-4, 14036-116, 19373-3, 19122-47, 20105-7, 21817-5, 13636-20, 19188-21, 4292-5, 20584-4, 9869-28, 8778-3, 5846-24, 5542-22, 3214-3, 6590-54, 15387-44, 8295-16, 19377-14, 13719-19, 25249-33, 25947-116, 21430-4, 11354-21, 24909-40, 25949-3, 10070-22, 15545-13, 3466-8, 21483-155, 12479-50, 25463-3, 2644-11, 5475-10, 3379-29, 9504-19, 17516-7, 23329-52, 17205-21, 19222-124, 14287-6, 18950-13, 16769-20, 14332-3, 22547-17, 18373-13, 21713-11, 17764-108, 12540-25, 21752-10, 22578-17, 5116-62, 3469-74, 3868-8, 13242-134, 16043-30, 5272-55, 19630-2, 13707-27, 4467-49, 15433-4, 5488-74, 12351-25, 10346-5, 10354-57, 19553-14, 18308-30, 17355-56, 21539-139, 3339-33, 14111-15, 14082-56, 5440-26, 24320-3, 9997-12, 10756-34, 5275-28, 9830-109, 7952-2, 3050-7, 25451-39, and 4560-34, and / ora decreased abundance of one or more biomarkers having a SomaScan aptamer sequence selected from 18922-27, 4337-49, 25306-51, 10025-1, 10801-11, 14124-6, 22580-29, 2615-60, 5658-64, 2443-10, 19570-12, 17166-4, 14757-144, 5581-28, 2841-13, 5755-29, 10426-21, 13547-5, 13738-8, 19622-7, 2748-3, 5193-51, 5242-37, 7655-11, 3723-1, 9380-2, 21380-77, 20546-71, 9099-19, and 9970-7,P390200. W0.01administering a second dosing regimen to the subject.

19. The method of claim 17 or 18 further comprising determining if the human subject has an increased abundance of one or more biomarkers having a SomaScan Aptamer sequence selected from 19197-95, 9843-5, 9844-138, 15312-14, 15323-112, 15627-83, 2867-52, 23302-19, 5360-9, 14685-17, 15523-9, 18185-118, 2811-27, 2602-2, 13660-76, 13621-31, 12583-77, 14748-31, 13573-5, 5870-23, 24237-115, 11130-158, 3351-1, 9840-2, 2819-23, 7959-34, 4203-50, 14029-42, 15321-8, 4976-57, 15527-90, 20913-27, 25105-87, 19259-176, 24723-58, 18829-4, 22984-10, 18824-7, 25287-7, 4230-1, 6036-78, 8039-41, 4393-3, 4130-71, 4487-1, 4394-71, 4989-7, 13384-110, 11171-25, 11245-43, 13122-19, 7713-102, 7713-50, 13740-51, 14587-16, 2765-4, 18233-10, 23903-3, 5464-52, 3236-12, 24050-26, 18900-37, 3317-33, 9816-37, 11816-84, 20073-22, 3453-87, 3381-24, 2864-2, 22041-26, 3115-64, 5007-1, 2855-49, 15604-18, 9760-13, 21768-9, 21813-171, 23693-4, 14036-116, 19373-3, 19122-47, 20105-7, 21817-5, 13636-20, 19188-21, 4292-5, 20584-4, 9869-28, 8778-3, 5846-24, 5542-22, 3214-3, 6590-54, 15387-44, 8295-16, 19377-14, 13719-19, 25249-33, 25947-116, 21430-4, 11354-21, 24909-40, 25949-3, 10070-22, 15545-13, 3466-8, 21483-155, 12479-50, 25463-3, 2644-11, 5475-10, 3379-29, 9504-19, 17516-7, 23329-52, 17205-21, 19222-124, 14287-6, 18950-13, 16769-20, 14332-3, 22547-17, 18373-13, 21713-11, 17764-108, 12540-25, 21752-10, 22578-17, 5116-62, 3469-74, 3868-8, 13242-134, 16043-30, 5272-55, 19630-2, 13707-27, 4467-49, 15433-4, 5488-74, 12351-25, 10346-5, 10354-57, 19553-14, 18308-30, 17355-56, 21539-139, 3339-33, 14111-15, 14082-56, 5440-26, 24320-3, 9997-12, 10756-34, 5275-28, 9830-109, 7952-2, 3050-7, 25451-39, and 4560-34, and / ora decreased abundance of one or more biomarkers having a SomaScan aptamer sequence selected from 18922-27, 4337-49, 25306-51, 10025-1, 10801-11, 14124-6, 22580-29, 2615-60, 5658-64, 2443-10, 19570-12, 17166-4, 14757-144, 5581-28, 2841-13, 5755-29, 10426-21, 13547-5, 13738-8, 19622-7, 2748-3, 5193-51, 5242-37, 7655-11, 3723-1, 9380-2, 21380-77, 20546-71, 9099-19, and 9970-7.

20. The method of claim 17 or 19 further comprising determining if the human subject has an increased abundance of one or more biomarkers having a SomaScan Aptamer sequence selected from 19197-95, 9843-5, 9844-138, 15312-14, 15323-112, 15627-83, 2867-52, 23302-19, 5360-9, 14685-17, 15523-9, 18185-118, 2811-27, 2602-2, 13660-76, 13621-31, 12583-77, 14748-31, 13573-5, 5870-23, 24237-115, 11130-158, 3351-1, 9840-2, 2819-23, 7959-34, 4203-50, 14029-42, 15321-8, 4976-57, 3516-60, 15527-90, 20913-27, 25105-87, 19259-176, 24723-58, 18829-4, 22984-10, 18824-7, 25287-7, 4230-1, 6036-78, 8039-41, 4393-3, 4130-71, 4487-1, 4394-71, 4989-7, 13384-110, 11171-25, 11245-43,P390200. W0.0113122-19, 7713-102, 7713-50, 13740-51, 14587-16, 2765-4, 18233-10, 23903-3, 5464-52, 3236-12, 24050-26, 18900-37, 3317-33, 9816-37, 11816-84, 20073-22, 3453-87, 3381-24, 2864-2, 22041-26, 3115-64, 5007-1, 2855-49, 15604-18, 9760-13, 21768-9, 21813-171, 23693-4, 14036-116, 19373-3, 19122-47, 20105-7, 21817-5, 13636-20, 19188-21, 4292-5, 20584-4, 9869-28, 8778-3, 5846-24, 5542-22, 3214-3, 6590-54, 15387-44, 8295-16, 19377-14, 13719-19, 25249-33, 25947-116, 21430-4, 11354-21, 24909-40, 25949-3, 10070-22, 15545-13, 3466-8, 21483-155, 12479-50, 25463-3, 2644-11, 5475-10, 3379-29, 9504-19, 17516-7, 23329-52, 17205-21, 19222-124, 14287-6, 18950-13, 16769-20, 14332-3, 22547-17, 18373-13, 21713-11, 17764-108, 12540-25, 21752-10, 22578-17, 5116-62, 3469-74, 3868-8, 13242-134, 16043-30, 5272-55, 19630-2, 13707-27, 4467-49, 15433-4, 5488-74, 12351-25, 10346-5, 10354-57, 19553-14, 18308-30, 17355-56, 21539-139, 3339-33, 14111-15, 14082-56, 5440-26, 24320-3, 9997-12, 10756-34, 5275-28, 9830-109, 7952-2, 3050-7, 25451-39, and 4560-34, and / ora decreased abundance of one or more biomarkers having a SomaScan aptamer sequence selected from 18922-27, 4337-49, 25306-51, 10025-1, 10801-11, 14124-6, 22580-29, 2615-60, 5658-64, 2443-10, 19570-12, 17166-4, 14757-144, 5581-28, 2841-13, 5755-29, 10426-21, 13547-5, 13738-8, 19622-7, 2748-3, 5193-51, 5242-37, 7655-11, 3723-1, 9380-2, 21380-77, 20546-71, 9099-19, and 9970-7.

21. A method of treating stroke or reducing a symptom thereof in a human subject in need thereof, comprising:identifying the human subject as an increased abundance of one or more biomarkers having a SomaScan Aptamer sequence selected from 19197-95, 9843-5, 9844-138, 15312-14, 15323-112, 15627-83, 2867-52, 23302-19, 5360-9, 14685-17, 15523-9, 18185-118, 2811-27, 2602-2, 13660-76, 13621-31, 12583-77, 14748-31, 13573-5, 5870-23, 24237-115, 11130-158, 3351-1, 9840-2, 2819-23, 7959-34, 4203-50, 14029-42, 15321-8, 4976-57, 15527-90, 20913-27, 25105-87, 19259-176, 24723-58, 18829-4, 22984-10, 18824-7, 25287-7, 4230-1, 6036-78, 8039-41, 4393-3, 4130-71, 4487-1, 4394-71, 4989-7, 13384-110, 11171-25, 11245-43, 13122-19, 7713-102, 7713-50, 13740-51, 14587-16, 2765-4, 18233-10, 23903-3, 5464-52, 3236-12, 24050-26, 18900-37, 3317-33, 9816-37, 11816-84, 20073-22, 3453-87, 3381-24, 2864-2, 22041-26, 3115-64, 5007-1, 2855-49, 15604-18, 9760-13, 21768-9, 21813-171, 23693-4, 14036-116, 19373-3, 19122-47, 20105-7, 21817-5, 13636-20, 19188-21, 4292-5, 20584-4, 9869-28, 8778-3, 5846-24, 5542-22, 3214-3, 6590-54, 15387-44, 8295-16, 19377-14, 13719-19, 25249-33, 25947-116, 21430-4, 11354-21, 24909-40, 25949-3, 10070-22, 15545-13, 3466-8, 21483-155, 12479-50, 25463-3, 2644-11, 5475-10, 3379-29, 9504-19, 17516-7, 23329-52, 17205-21, 19222-124, 14287-6, 18950-13, 16769-20, 14332-3, 22547-17, 18373-13, 21713-11, 17764-108, 12540-25, 21752-10,P390200. W0.0122578-17, 5116-62, 3469-74, 3868-8, 13242-134, 16043-30, 5272-55, 19630-2, 13707-27, 4467-49, 15433-4, 5488-74, 12351-25, 10346-5, 10354-57, 19553-14, 18308-30, 17355-56, 21539-139, 3339-33, 14111-15, 14082-56, 5440-26, 24320-3, 9997-12, 10756-34, 5275-28, 9830-109, 7952-2, 3050-7, 25451-39, and 4560-34, and / ora decreased abundance of one or more biomarkers having a SomaScan aptamer sequence selected from 18922-27, 4337-49, 25306-51, 10025-1, 10801-11, 14124-6, 22580-29, 2615-60, 5658-64, 2443-10, 19570-12, 17166-4, 14757-144, 5581-28, 2841-13, 5755-29, 10426-21, 13547-5, 13738-8, 19622-7, 2748-3, 5193-51, 5242-37, 7655-11, 3723-1, 9380-2, 21380-77, 20546-71, 9099-19, and 9970-7; andadministering a GDF11 dosing regimen to the subject.

22. A method of treating stroke or reducing a symptom thereof in a human subject in need thereof, comprising:identifying the human subject as an increased abundance of one or more biomarkers having a SomaScan Aptamer sequence selected from 19197-95, 9843-5, 9844-138, 15312-14, 15323-112, 15627-83, 2867-52, 23302-19, 5360-9, 14685-17, 15523-9, 18185-118, 2811-27, 2602-2, 13660-76, 13621-31, 12583-77, 14748-31, 13573-5, 5870-23, 24237-115, 11130-158, 3351-1, 9840-2, 2819-23, 7959-34, 4203-50, 14029-42, 15321-8, 4976-57, 3516-60, 15527-90, 20913-27, 25105-87, 19259-176, 24723-58, 18829-4, 22984-10, 18824-7, 25287-7, 4230-1, 6036-78, 8039-41, 4393-3, 4130-71, 4487-1, 4394-71, 4989-7, 13384-110, 11171-25, 11245-43, 13122-19, 7713-102, 7713-50, 13740-51, 14587-16, 2765-4, 18233-10, 23903-3, 5464-52, 3236-12, 24050-26, 18900-37, 3317-33, 9816-37, 11816-84, 20073-22, 3453-87, 3381-24, 2864-2, 22041-26, 3115-64, 5007-1, 2855-49, 15604-18, 9760-13, 21768-9, 21813-171, 23693-4, 14036-116, 19373-3, 19122-47, 20105-7, 21817-5, 13636-20, 19188-21, 4292-5, 20584-4, 9869-28, 8778-3, 5846-24, 5542-22, 3214-3, 6590-54, 15387-44, 8295-16, 19377-14, 13719-19, 25249-33, 25947-116, 21430-4, 11354-21, 24909-40, 25949-3, 10070-22, 15545-13, 3466-8, 21483-155, 12479-50, 25463-3, 2644-11, 5475-10, 3379-29, 9504-19, 17516-7, 23329-52, 17205-21, 19222-124, 14287-6, 18950-13, 16769-20, 14332-3, 22547-17, 18373-13, 21713-11, 17764-108, 12540-25, 21752-10, 22578-17, 5116-62, 3469-74, 3868-8, 13242-134, 16043-30, 5272-55, 19630-2, 13707-27, 4467-49, 15433-4, 5488-74, 12351-25, 10346-5, 10354-57, 19553-14, 18308-30, 17355-56, 21539-139, 3339-33, 14111-15, 14082-56, 5440-26, 24320-3, 9997-12, 10756-34, 5275-28, 9830-109, 7952-2, 3050-7, 25451-39, and 4560-34, and / ora decreased abundance of one or more biomarkers having a SomaScan aptamer sequence selected from 18922-27, 4337-49, 25306-51, 10025-1, 10801-11, 14124-6, 22580-29, 2615-60, 5658-64, 2443-10, 19570-12, 17166-4, 14757-144, 5581-28, 2841-13, 5755-P390200. W0.0129, 10426-21, 13547-5, 13738-8, 19622-7, 2748-3, 5193-51, 5242-37, 7655-11, 3723-1, 9380-2, 21380-77, 20546-71, 9099-19, and 9970-7; andadministering a GDF11 dosing regimen to the subject.

23. A method of improving therapeutic efficacy for treatment of stroke or a symptom thereof with GDF11, comprising:in a human subject to whom GDF11 has been administered, determining an increased abundance of one or more biomarkers selected from an increased abundance of one or more biomarkers having a SomaScan Aptamer sequence selected from 19197-95, 9843-5, 9844-138, 15312-14, 15323-112, 15627-83, 2867-52, 23302-19, 5360-9, 14685-17, 15523-9, 18185-118, 2811-27, 2602-2, 13660-76, 13621-31, 12583-77, 14748-31, 13573-5, 5870-23, 24237-115, 11130-158, 3351-1, 9840-2, 2819-23, 7959-34, 4203-50, 14029-42, 15321-8, 4976-57, 15527-90, 20913-27, 25105-87, 19259-176, 24723-58, 18829-4, 22984-10, 18824-7, 25287-7, 4230-1, 6036-78, 8039-41, 4393-3, 4130-71, 4487-1, 4394-71, 4989-7, 13384-110, 11171-25, 11245-43, 13122-19, 7713-102, 7713-50, 13740-51, 14587-16, 2765-4, 18233-10, 23903-3, 5464-52, 3236-12, 24050-26, 18900-37, 3317-33, 9816-37, 11816-84, 20073-22, 3453-87, 3381-24, 2864-2, 22041-26, 3115-64, 5007-1, 2855-49, 15604-18, 9760-13, 21768-9, 21813-171, 23693-4, 14036-116, 19373-3, 19122-47, 20105-7, 21817-5, 13636-20, 19188-21, 4292-5, 20584-4, 9869-28, 8778-3, 5846-24, 5542-22, 3214-3, 6590-54, 15387-44, 8295-16, 19377-14, 13719-19, 25249-33, 25947-116, 21430-4, 11354-21, 24909-40, 25949-3, 10070-22, 15545-13, 3466-8, 21483-155, 12479-50, 25463-3, 2644-11, 5475-10, 3379-29, 9504-19, 17516-7, 23329-52, 17205-21, 19222-124, 14287-6, 18950-13, 16769-20, 14332-3, 22547-17, 18373-13, 21713-11, 17764-108, 12540-25, 21752-10, 22578-17, 5116-62, 3469-74, 3868-8, 13242-134, 16043-30, 5272-55, 19630-2, 13707-27, 4467-49, 15433-4, 5488-74, 12351-25, 10346-5, 10354-57, 19553-14, 18308-30, 17355-56, 21539-139, 3339-33, 14111-15, 14082-56, 5440-26, 24320-3, 9997-12, 10756-34, 5275-28, 9830-109, 7952-2, 3050-7, 25451-39, and 4560-34, and / ora decreased abundance of one or more biomarkers having a SomaScan aptamer sequence selected from 18922-27, 4337-49, 25306-51, 10025-1, 10801-11, 14124-6, 22580-29, 2615-60, 5658-64, 2443-10, 19570-12, 17166-4, 14757-144, 5581-28, 2841-13, 5755-29, 10426-21, 13547-5, 13738-8, 19622-7, 2748-3, 5193-51, 5242-37, 7655-11, 3723-1, 9380-2, 21380-77, 20546-71, 9099-19, and 9970-7;wherein the increase abundance and / or decrease in the abundance of the one or more biomarkers corresponds to improved therapeutic efficacy of stroke treatment or symptom thereof with GDF 11.P390200. W0.0124. A method of improving therapeutic efficacy for treatment of stroke or a symptom thereof with GDF11, comprising:in a human subject to whom GDF11 has been administered, determining an increased abundance of one or more biomarkers selected from an increased abundance of one or more biomarkers having a SomaScan Aptamer sequence selected from 19197-95, 9843-5, 9844-138, 15312-14, 15323-112, 15627-83, 2867-52, 23302-19, 5360-9, 14685-17, 15523-9, 18185-118, 2811-27, 2602-2, 13660-76, 13621-31, 12583-77, 14748-31, 13573-5, 5870-23, 24237-115, 11130-158, 3351-1, 9840-2, 2819-23, 7959-34, 4203-50, 14029-42, 15321-8, 4976-57, 3516-60, 15527-90, 20913-27, 25105-87, 19259-176, 24723-58, 18829-4, 22984-10, 18824-7, 25287-7, 4230-1, 6036-78, 8039-41, 4393-3, 4130-71, 4487-1, 4394-71, 4989-7, 13384-110, 11171-25, 11245-43, 13122-19, 7713-102, 7713-50, 13740-51, 14587-16, 2765-4, 18233-10, 23903-3, 5464-52, 3236-12, 24050-26, 18900-37, 3317-33, 9816-37, 11816-84, 20073-22, 3453-87, 3381-24, 2864-2, 22041-26, 3115-64, 5007-1, 2855-49, 15604-18, 9760-13, 21768-9, 21813-171, 23693-4, 14036-116, 19373-3, 19122-47, 20105-7, 21817-5, 13636-20, 19188-21, 4292-5, 20584-4, 9869-28, 8778-3, 5846-24, 5542-22, 3214-3, 6590-54, 15387-44, 8295-16, 19377-14, 13719-19, 25249-33, 25947-116, 21430-4, 11354-21, 24909-40, 25949-3, 10070-22, 15545-13, 3466-8, 21483-155, 12479-50, 25463-3, 2644-11, 5475-10, 3379-29, 9504-19, 17516-7, 23329-52, 17205-21, 19222-124, 14287-6, 18950-13, 16769-20, 14332-3, 22547-17, 18373-13, 21713-11, 17764-108, 12540-25, 21752-10, 22578-17, 5116-62, 3469-74, 3868-8, 13242-134, 16043-30, 5272-55, 19630-2, 13707-27, 4467-49, 15433-4, 5488-74, 12351-25, 10346-5, 10354-57, 19553-14, 18308-30, 17355-56, 21539-139, 3339-33, 14111-15, 14082-56, 5440-26, 24320-3, 9997-12, 10756-34, 5275-28, 9830-109, 7952-2, 3050-7, 25451-39, and 4560-34, and / ora decreased abundance of one or more biomarkers having a SomaScan aptamer sequence selected from 18922-27, 4337-49, 25306-51, 10025-1, 10801-11, 14124-6, 22580-29, 2615-60, 5658-64, 2443-10, 19570-12, 17166-4, 14757-144, 5581-28, 2841-13, 5755-29, 10426-21, 13547-5, 13738-8, 19622-7, 2748-3, 5193-51, 5242-37, 7655-11, 3723-1, 9380-2, 21380-77, 20546-71, 9099-19, and 9970-7;wherein the increase abundance and / or decrease in the abundance of the one or more biomarkers corresponds to improved therapeutic efficacy of stroke treatment or symptom thereof with GDF 11.

25. A method of determining effectiveness of treatment of stroke or a symptom thereof with GDF11, comprising:in a human subject to whom GDF11 has been administered, determining an increase in abundance of one or more biomarkers having a SomaScan Aptamer sequence selected from 19197-95, 9843-5, 9844-138, 15312-14, 15323-112, 15627-83, 2867-52, 23302-19,P390200. W0.015360-9, 14685-17, 15523-9, 18185-118, 2811-27, 2602-2, 13660-76, 13621-31, 12583-77, 14748-31, 13573-5, 5870-23, 24237-115, 11130-158, 3351-1, 9840-2, 2819-23, 7959-34, 4203-50, 14029-42, 15321-8, 4976-57, 15527-90, 20913-27, 25105-87, 19259-176, 24723-58, 18829-4, 22984-10, 18824-7, 25287-7, 4230-1, 6036-78, 8039-41, 4393-3, 4130-71, 4487-1, 4394-71, 4989-7, 13384-110, 11171-25, 11245-43, 13122-19, 7713-102, 7713-50, 13740-51, 14587-16, 2765-4, 18233-10, 23903-3, 5464-52, 3236-12, 24050-26, 18900-37, 3317-33, 9816-37, 11816-84, 20073-22, 3453-87, 3381-24, 2864-2, 22041-26, 3115-64, 5007-1, 2855-49, 15604-18, 9760-13, 21768-9, 21813-171, 23693-4, 14036-116, 19373-3, 19122-47, 20105-7, 21817-5, 13636-20, 19188-21, 4292-5, 20584-4, 9869-28, 8778-3, 5846-24, 5542-22, 3214-3, 6590-54, 15387-44, 8295-16, 19377-14, 13719-19, 25249-33, 25947-116, 21430-4, 11354-21, 24909-40, 25949-3, 10070-22, 15545-13, 3466-8, 21483-155, 12479-50, 25463-3, 2644-11, 5475-10, 3379-29, 9504-19, 17516-7, 23329-52, 17205-21, 19222-124, 14287-6, 18950-13, 16769-20, 14332-3, 22547-17, 18373-13, 21713-11, 17764-108, 12540-25, 21752-10, 22578-17, 5116-62, 3469-74, 3868-8, 13242-134, 16043-30, 5272-55, 19630-2, 13707-27, 4467-49, 15433-4, 5488-74, 12351-25, 10346-5, 10354-57, 19553-14, 18308-30, 17355-56, 21539-139, 3339-33, 14111-15, 14082-56, 5440-26, 24320-3, 9997-12, 10756-34, 5275-28, 9830-109, 7952-2, 3050-7, 25451-39, and 4560-34, and / ora decrease in abundance of one or more biomarkers having a SomaScan aptamer sequence selected from 18922-27, 4337-49, 25306-51, 10025-1, 10801-11, 14124-6, 22580-29, 2615-60, 5658-64, 2443-10, 19570-12, 17166-4, 14757-144, 5581-28, 2841-13, 5755-29, 10426-21, 13547-5, 13738-8, 19622-7, 2748-3, 5193-51, 5242-37, 7655-11, 3723-1, 9380-2, 21380-77, 20546-71, 9099-19, and 9970-7;wherein the increase and / or decrease in abundance corresponds to improved therapeutic efficacy of stroke treatment or symptom thereof with GDF11.

26. A method of determining effectiveness of treatment of stroke or a symptom thereof with GDF11, comprising:in a human subject to whom GDF11 has been administered, determining an increase in abundance of one or more biomarkers having a SomaScan Aptamer sequence selected from 19197-95, 9843-5, 9844-138, 15312-14, 15323-112, 15627-83, 2867-52, 23302-19, 5360-9, 14685-17, 15523-9, 18185-118, 2811-27, 2602-2, 13660-76, 13621-31, 12583-77, 14748-31, 13573-5, 5870-23, 24237-115, 11130-158, 3351-1, 9840-2, 2819-23, 7959-34, 4203-50, 14029-42, 15321-8, 4976-57, 3516-60, 15527-90, 20913-27, 25105-87, 19259-176, 24723-58, 18829-4, 22984-10, 18824-7, 25287-7, 4230-1, 6036-78, 8039-41, 4393-3, 4130-71, 4487-1, 4394-71, 4989-7, 13384-110, 11171-25, 11245-43, 13122-19, 7713-102, 7713-50, 13740-51, 14587-16, 2765-4, 18233-10, 23903-3, 5464-52, 3236-12, 24050-26,P390200. W0.0118900-37, 3317-33, 9816-37, 11816-84, 20073-22, 3453-87, 3381-24, 2864-2, 22041-26, 3115-64, 5007-1, 2855-49, 15604-18, 9760-13, 21768-9, 21813-171, 23693-4, 14036-116, 19373-3, 19122-47, 20105-7, 21817-5, 13636-20, 19188-21, 4292-5, 20584-4, 9869-28, 8778-3, 5846-24, 5542-22, 3214-3, 6590-54, 15387-44, 8295-16, 19377-14, 13719-19, 25249-33, 25947-116, 21430-4, 11354-21, 24909-40, 25949-3, 10070-22, 15545-13, 3466-8, 21483-155, 12479-50, 25463-3, 2644-11, 5475-10, 3379-29, 9504-19, 17516-7, 23329-52, 17205-21, 19222-124, 14287-6, 18950-13, 16769-20, 14332-3, 22547-17, 18373-13, 21713-11, 17764-108, 12540-25, 21752-10, 22578-17, 5116-62, 3469-74, 3868-8, 13242-134, 16043-30, 5272-55, 19630-2, 13707-27, 4467-49, 15433-4, 5488-74, 12351-25, 10346-5, 10354-57, 19553-14, 18308-30, 17355-56, 21539-139, 3339-33, 14111-15, 14082-56, 5440-26, 24320-3, 9997-12, 10756-34, 5275-28, 9830-109, 7952-2, 3050-7, 25451-39, and 4560-34, and / ora decrease in abundance of one or more biomarkers having a SomaScan aptamer sequence selected from 18922-27, 4337-49, 25306-51, 10025-1, 10801-11, 14124-6, 22580-29, 2615-60, 5658-64, 2443-10, 19570-12, 17166-4, 14757-144, 5581-28, 2841-13, 5755-29, 10426-21, 13547-5, 13738-8, 19622-7, 2748-3, 5193-51, 5242-37, 7655-11, 3723-1, 9380-2, 21380-77, 20546-71, 9099-19, and 9970-7;wherein the increase and / or decrease in abundance corresponds to improved therapeutic efficacy of stroke treatment or symptom thereof with GDF11.

27. A method of selecting one or more human subjects with increased responsiveness to treatment of stroke or symptom thereof, comprising:in a group of human subjects to whom GDF11 has been administered, determining an increased abundance of one or more biomarkers having a SomaScan Aptamer sequence selected from 19197-95, 9843-5, 9844-138, 15312-14, 15323-112, 15627-83, 2867-52, 23302-19, 5360-9, 14685-17, 15523-9, 18185-118, 2811-27, 2602-2, 13660-76, 13621-31, 12583-77, 14748-31, 13573-5, 5870-23, 24237-115, 11130-158, 3351-1, 9840-2, 2819-23, 7959-34, 4203-50, 14029-42, 15321-8, 4976-57, 15527-90, 20913-27, 25105-87, 19259-176, 24723-58, 18829-4, 22984-10, 18824-7, 25287-7, 4230-1, 6036-78, 8039-41, 4393-3, 4130-71, 4487-1, 4394-71, 4989-7, 13384-110, 11171-25, 11245-43, 13122-19, 7713-102, 7713-50, 13740-51, 14587-16, 2765-4, 18233-10, 23903-3, 5464-52, 3236-12, 24050-26, 18900-37, 3317-33, 9816-37, 11816-84, 20073-22, 3453-87, 3381-24, 2864-2, 22041-26, 3115-64, 5007-1, 2855-49, 15604-18, 9760-13, 21768-9, 21813-171, 23693-4, 14036-116, 19373-3, 19122-47, 20105-7, 21817-5, 13636-20, 19188-21, 4292-5, 20584-4, 9869-28, 8778-3, 5846-24, 5542-22, 3214-3, 6590-54, 15387-44, 8295-16, 19377-14, 13719-19, 25249-33, 25947-116, 21430-4, 11354-21, 24909-40, 25949-3, 10070-22, 15545-13, 3466-8, 21483-155, 12479-50, 25463-3, 2644-11, 5475-10, 3379-29, 9504-19, 17516-7, 23329-P390200. W0.0152, 17205-21, 19222-124, 14287-6, 18950-13, 16769-20, 14332-3, 22547-17, 18373-13, 21713-11, 17764-108, 12540-25, 21752-10, 22578-17, 5116-62, 3469-74, 3868-8, 13242-134, 16043-30, 5272-55, 19630-2, 13707-27, 4467-49, 15433-4, 5488-74, 12351-25, 10346-5, 10354-57, 19553-14, 18308-30, 17355-56, 21539-139, 3339-33, 14111-15, 14082-56, 5440-26, 24320-3, 9997-12, 10756-34, 5275-28, 9830-109, 7952-2, 3050-7, 25451-39, and 4560-34, and / ora decreased abundance of one or more biomarkers having a SomaScan aptamer sequence selected from 18922-27, 4337-49, 25306-51, 10025-1, 10801-11, 14124-6, 22580-29, 2615-60, 5658-64, 2443-10, 19570-12, 17166-4, 14757-144, 5581-28, 2841-13, 5755-29, 10426-21, 13547-5, 13738-8, 19622-7, 2748-3, 5193-51, 5242-37, 7655-11, 3723-1, 9380-2, 21380-77, 20546-71, 9099-19, and 9970-7;wherein the increase and / or decrease in the abundance of the one or more biomarkers in a subject in the group of human subjects corresponds to improved therapeutic efficacy of stroke treatment or symptom thereof with GDF11 in that subject.

28. A method of selecting one or more human subjects with increased responsiveness to treatment of stroke or symptom thereof, comprising:in a group of human subjects to whom GDF11 has been administered, determining an increased abundance of one or more biomarkers having a SomaScan Aptamer sequence selected from 19197-95, 9843-5, 9844-138, 15312-14, 15323-112, 15627-83, 2867-52, 23302-19, 5360-9, 14685-17, 15523-9, 18185-118, 2811-27, 2602-2, 13660-76, 13621-31, 12583-77, 14748-31, 13573-5, 5870-23, 24237-115, 11130-158, 3351-1, 9840-2, 2819-23, 7959-34, 4203-50, 14029-42, 15321-8, 4976-57, 3516-60, 15527-90, 20913-27, 25105-87, 19259-176, 24723-58, 18829-4, 22984-10, 18824-7, 25287-7, 4230-1, 6036-78, 8039-41, 4393-3, 4130-71, 4487-1, 4394-71, 4989-7, 13384-110, 11171-25, 11245-43, 13122-19, 7713-102, 7713-50, 13740-51, 14587-16, 2765-4, 18233-10, 23903-3, 5464-52, 3236-12, 24050-26, 18900-37, 3317-33, 9816-37, 11816-84, 20073-22, 3453-87, 3381-24, 2864-2, 22041-26, 3115-64, 5007-1, 2855-49, 15604-18, 9760-13, 21768-9, 21813-171, 23693-4, 14036-116, 19373-3, 19122-47, 20105-7, 21817-5, 13636-20, 19188-21, 4292-5, 20584-4, 9869-28, 8778-3, 5846-24, 5542-22, 3214-3, 6590-54, 15387-44, 8295-16, 19377-14, 13719-19, 25249-33, 25947-116, 21430-4, 11354-21, 24909-40, 25949-3, 10070-22, 15545-13, 3466-8, 21483-155, 12479-50, 25463-3, 2644-11, 5475-10, 3379-29, 9504-19, 17516-7, 23329-52, 17205-21, 19222-124, 14287-6, 18950-13, 16769-20, 14332-3, 22547-17, 18373-13, 21713-11, 17764-108, 12540-25, 21752-10, 22578-17, 5116-62, 3469-74, 3868-8, 13242-134, 16043-30, 5272-55, 19630-2, 13707-27, 4467-49, 15433-4, 5488-74, 12351-25, 10346-5, 10354-57, 19553-14, 18308-30, 17355-56, 21539-139, 3339-33, 14111-15, 14082-P390200. W0.0156, 5440-26, 24320-3, 9997-12, 10756-34, 5275-28, 9830-109, 7952-2, 3050-7, 25451-39, and 4560-34, and / ora decreased abundance of one or more biomarkers having a SomaScan aptamer sequence selected from 18922-27, 4337-49, 25306-51, 10025-1, 10801-11, 14124-6, 22580-29, 2615-60, 5658-64, 2443-10, 19570-12, 17166-4, 14757-144, 5581-28, 2841-13, 5755-29, 10426-21, 13547-5, 13738-8, 19622-7, 2748-3, 5193-51, 5242-37, 7655-11, 3723-1, 9380-2, 21380-77, 20546-71, 9099-19, and 9970-7;wherein the increase and / or decrease in the abundance of the one or more biomarkers in a subject in the group of human subjects corresponds to improved therapeutic efficacy of stroke treatment or symptom thereof with GDF11 in that subject.

29. Use of a therapeutically effective amount of GDF11 in the manufacture of a medicament for treating stroke in a human subject having in a group of human subjects to whom GDF11 has been administered, determining an increased abundance of one or more biomarkers having a SomaScan Aptamer sequence selected from 19197-95, 9843-5, 9844-138, 15312-14, 15323-112, 15627-83, 2867-52, 23302-19, 5360-9, 14685-17, 15523-9, 18185-118, 2811-27, 2602-2, 13660-76, 13621-31, 12583-77, 14748-31, 13573-5, 5870-23, 24237-115, 11130-158, 3351-1, 9840-2, 2819-23, 7959-34, 4203-50, 14029-42, 15321-8, 4976-57, 15527-90, 20913-27, 25105-87, 19259-176, 24723-58, 18829-4, 22984-10, 18824-7, 25287-7, 4230-1, 6036-78, 8039-41, 4393-3, 4130-71, 4487-1, 4394-71, 4989-7, 13384-110, 11171-25, 11245-43, 13122-19, 7713-102, 7713-50, 13740-51, 14587-16, 2765-4, 18233-10, 23903-3, 5464-52, 3236-12, 24050-26, 18900-37, 3317-33, 9816-37, 11816-84, 20073-22, 3453-87, 3381-24, 2864-2, 22041-26, 3115-64, 5007-1, 2855-49, 15604-18, 9760-13, 21768-9, 21813-171, 23693-4, 14036-116, 19373-3, 19122-47, 20105-7, 21817-5, 13636-20, 19188-21, 4292-5, 20584-4, 9869-28, 8778-3, 5846-24, 5542-22, 3214-3, 6590-54, 15387-44, 8295-16, 19377-14, 13719-19, 25249-33, 25947-116, 21430-4, 11354-21, 24909-40, 25949-3, 10070-22, 15545-13, 3466-8, 21483-155, 12479-50, 25463-3, 2644-11, 5475-10, 3379-29, 9504-19, 17516-7, 23329-52, 17205-21, 19222-124, 14287-6, 18950-13, 16769-20, 14332-3, 22547-17, 18373-13, 21713-11, 17764-108, 12540-25, 21752-10, 22578-17, 5116-62, 3469-74, 3868-8, 13242-134, 16043-30, 5272-55, 19630-2, 13707-27, 4467-49, 15433-4, 5488-74, 12351-25, 10346-5, 10354-57, 19553-14, 18308-30, 17355-56, 21539-139, 3339-33, 14111-15, 14082-56, 5440-26, 24320-3, 9997-12, 10756-34, 5275-28, 9830-109, 7952-2, 3050-7, 25451-39, and 4560-34, and / ora decreased abundance of one or more biomarkers having a SomaScan aptamer sequence selected from 18922-27, 4337-49, 25306-51, 10025-1, 10801-11, 14124-6, 22580-29, 2615-60, 5658-64, 2443-10, 19570-12, 17166-4, 14757-144, 5581-28, 2841-13, 5755-29, 10426-P390200. W0.0121, 13547-5, 13738-8, 19622-7, 2748-3, 5193-51, 5242-37, 7655-11, 3723-1, 9380-2, 21380-77, 20546-71, 9099-19, and 9970-7.

30. Use of a therapeutically effective amount of GDF11 in the manufacture of a medicament for treating stroke in a human subject having in a group of human subjects to whom GDF11 has been administered, determining an increased abundance of one or more biomarkers having a SomaScan Aptamer sequence selected from 19197-95, 9843-5, 9844-138, 15312-14, 15323-112, 15627-83, 2867-52, 23302-19, 5360-9, 14685-17, 15523-9, 18185-118, 2811-27, 2602-2, 13660-76, 13621-31, 12583-77, 14748-31, 13573-5, 5870-23, 24237-115, 11130-158, 3351-1, 9840-2, 2819-23, 7959-34, 4203-50, 14029-42, 15321-8, 4976-57, 3516-60, 15527-90, 20913-27, 25105-87, 19259-176, 24723-58, 18829-4, 22984-10, 18824-7, 25287-7, 4230-1, 6036-78, 8039-41, 4393-3, 4130-71, 4487-1, 4394-71, 4989-7, 13384-110, 11171-25, 11245-43, 13122-19, 7713-102, 7713-50, 13740-51, 14587-16, 2765-4, 18233-10, 23903-3, 5464-52, 3236-12, 24050-26, 18900-37, 3317-33, 9816-37, 11816-84, 20073-22, 3453-87, 3381-24, 2864-2, 22041-26, 3115-64, 5007-1, 2855-49, 15604-18, 9760-13, 21768-9, 21813-171, 23693-4, 14036-116, 19373-3, 19122-47, 20105-7, 21817-5, 13636-20, 19188-21, 4292-5, 20584-4, 9869-28, 8778-3, 5846-24, 5542-22, 3214-3, 6590-54, 15387-44, 8295-16, 19377-14, 13719-19, 25249-33, 25947-116, 21430-4, 11354-21, 24909-40, 25949-3, 10070-22, 15545-13, 3466-8, 21483-155, 12479-50, 25463-3, 2644-11, 5475-10, 3379-29, 9504-19, 17516-7, 23329-52, 17205-21, 19222-124, 14287-6, 18950-13, 16769-20, 14332-3, 22547-17, 18373-13, 21713-11, 17764-108, 12540-25, 21752-10, 22578-17, 5116-62, 3469-74, 3868-8, 13242-134, 16043-30, 5272-55, 19630-2, 13707-27, 4467-49, 15433-4, 5488-74, 12351-25, 10346-5, 10354-57, 19553-14, 18308-30, 17355-56, 21539-139, 3339-33, 14111-15, 14082-56, 5440-26, 24320-3, 9997-12, 10756-34, 5275-28, 9830-109, 7952-2, 3050-7, 25451-39, and 4560-34, and / ora decreased abundance of one or more biomarkers having a SomaScan aptamer sequence selected from 18922-27, 4337-49, 25306-51, 10025-1, 10801-11, 14124-6, 22580-29, 2615-60, 5658-64, 2443-10, 19570-12, 17166-4, 14757-144, 5581-28, 2841-13, 5755-29, 10426-21, 13547-5, 13738-8, 19622-7, 2748-3, 5193-51, 5242-37, 7655-11, 3723-1, 9380-2, 21380-77, 20546-71, 9099-19, and 9970-7.

31. The method of any one of claims 13-30, wherein the determining or identifying step comprises a proteomic test.

32. The method of any one of claims 13-30, wherein the determining or identifying step comprises a genetic test.P390200. W0.0133. The method of any one of claims 13-30, wherein determining or identifying step comprises testing a blood sample.

34. The method of claim 33, wherein determining or identifying step comprises testing a blood serum sample.

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