RELATED APPLICATIONS

This application claims priority to U.S. provisional application 63/406,506, filed September 14, 2022, hereby incorporated by reference in its entirety.

GOVERNMENT SUPPORT

This invention was made with government support under CA247073, CA224088, AI056299, and AI108545, awarded by National Institutes of Health (NIH). The government has certain rights in this invention.

BACKGROUND

The coinhibitory receptor Programmed Cell Death-1 (PD-1, CD279) plays a critical role in T cell exhaustion during chronic infection and cancer. PD-1 pathway inhibitors are FDA-approved for treating over 20 cancer types and have shown tremendous success in a subset of patients. However, most cancer patients do not exhibit durable responses and patients with specific cancer types such as glioblastoma experience little to no benefit from PD-1 pathway inhibitors. Consequently, efforts are currently focused on identifying potential combination treatments that can synergize with PD-1 blockade to increase the breadth and durability of response.

Despite its widespread use in the clinic, there is a limited mechanistic understanding of PD-1 signaling. Ligation of PD-1 on CD8 ⁺ T cells by its ligands Programmed Death Ligand (PD-L)l and PD-L2 results in the attenuation of multiple cellular processes driven by engagement of the T cell receptor (TCR) and costimulatory receptor CD28. PD-1 plays a pivotal role in regulating the dephosphorylation of TCR-related proteins and in modulating T cell functions such as cytokine production, Ca ²⁺ flux, cytolysis, cytoskeletal rearrangements, migration and metabolism. PD-1 exerts its inhibitory functions, at least in part, through the recruitment of SH2-containing tyrosine phosphatases SHP2 (PTPN11) and to a lesser extent, SHP1 (PTPN6), which bind phosphotyrosine residues within the immunoreceptor tyrosine-based switch motif (ITSM) and inhibition motif (ITIM) on the PD-1 cytoplasmic tail. However, the precise mechanism by which PD-1 and SHP2/SHP1 counter T cell activation remain unclear. Recent studies have shown that T cell-specific SHP2 knockout (KO) mice respond to PD-1 blockade and T cells from these mice can become exhausted, showing that PD-1 inhibition can occur in the absence of SHP2. These data suggest that the consequences of phosphatase association with PD-1 are more complex than previously thought and that unidentified proteins and signaling pathways may play a role in mediating PD-1 function. Thus, it was hypothesized that additional unidentified proteins contribute to PD-1 -mediated CD8 ⁺ T cell inhibition and among these proteins potential targets for therapeutics may be identified that can augment PD-1 -based immunotherapy.

SUMMARY

In some aspects, provided herein are methods of treating cancer in a subject, the method comprising administering to the subject an agent that increases or stabilizes the activity or expression of PIEZO 1. The agent may be a small molecule agonist of PIEZO 1, such as Yodal, Jedi 1 , Jedi2, or a modulator of PIEZO1, such as Docosahexaenoic acid. The method may further comprise administering an immune checkpoint inhibitor to the subject.

In some aspects, provided herein are methods of treating cancer in a subject, the method comprising administering to the subject an agent that increases or stabilizes the activity or expression of PIEZO 1 and an immune checkpoint inhibitor. In some embodiments, the immune checkpoint inhibitor is an inhibitor of an immune checkpoint protein selected from CTLA-4, PD-1, VISTA, B7-H2, B7-H3, PD-L1, B7-H4, B7-H6, ICOS, HVEM, PD-L2, CD160, gp49B, PIR-B, KIR family receptors, TIM-1, TIM-3, TIM- 4, LAG-3, BTLA, SIRPalpha (CD47), CD48, 2B4 (CD244), B7.1, B7.2, ILT-2, ILT-4, TIGIT, HHLA2, butyrophilins, A2aR, and combinations thereof.

In some embodiments, the agent that increases or stabilizes the activity or expression of PIEZO 1 and the immune checkpoint inhibitor are administered conjointly. In some embodiments, the that agent increases or stabilizes the activity or expression of PIEZO 1 and the immune checkpoint inhibitor act synergistically when administered.

In some embodiments, the agent is a gRNA fused to a transcription activator, such as a gRNA that comprises a region that is complementary to a portion of a gene that encodes a PIEZO 1 protein. In some embodiments, the agent is a vector encoding a PIEZO 1 protein, such as a viral vector encoding a PIEZO 1 protein.

The agent may be an agent is administered systemically, intravenously, subcutaneously, or intramuscularly. The agent may be administered to the subject in a pharmaceutically acceptable formulation. The method may further comprise administering to the subject an additional agent, such as a chemotherapeutic agent or a cancer vaccine. The method may further comprise administering to the subject a cancer therapy, such as radiation. The subject may be refractory for immune checkpoint inhibitory therapy.

In some aspects, provided herein are methods of treating cancer in a subject unresponsive to immune checkpoint inhibitor therapy, the method comprising administering to the subject an agent that increases or stabilizes the activity or expression of PIEZO 1 (e.g., any agent that increases or stabilizes the activity or expression of PIEZO 1 disclosed herein) and an immune checkpoint inhibitor. The agent may be a small molecule agonist of PIEZO1, such as Yodal, Jedi 1 , Jedi2, or a modulator of PIEZO1, such as Docosahexaenoic acid. The method may further comprise administering an immune checkpoint inhibitor to the subject, such an inhibitor of PD-1 or PD-L1, or another immune checkpoint inhibitor disclosed herein.

In some aspects, provided herein are methods of treating cancer in a subject, the method comprising administering to the subject T-cells that have been treated ex vivo with an agent that increases or stabilizes the activity or expression of PIEZO1 (e.g., any agent that increases or stabilizes the activity or expression of PIEZO1 disclosed herein). The T- cells may be tumor infiltrating lymphocytes. In some embodiments, T-cells are autologous. In some embodiments, T-cells are allogeneic.

In some aspects, the subject is a human.

BRIEF DESCRIPTION OF THE FIGURES

Figs. 1A-1J show that quantitative proximity proteomics identifies PD-1 and PIEZO1 association following PD-L1 ligation. Figure 1 A shows a diagram of PD-1- APEX2 proximity labeling dynamics in Jurkat cells treated with TCR-PD-L1 and TCR- control beads. Figure IB shows an experimental schematic of PD-1-APEX2 proximity labeling time course with bead treatment. TMT ratios (TMT RA) over time (min) of (Fig. 1C) SHP2, (Fig. ID) PIEZO 1, (Fig. IE) SHP1, (Fig IF) CD3£ and (Fig. 1G) CD28 following stimulation of PD-l-APEX2-expressing Jurkat cells with TCR-PD-L1 (PD-L1) or TCR-control (mlgGl) beads. Data are presented as means from three independent experiments ± SD. Statistical significance was assessed using two-way ANOVA analysis comparing TCR-PD-L1 and TCR-control bead-treated groups. Only significant differences are indicated. Figure 1H shows hierarchical one-way clustering of the averaged TMT ratios from the top 25 proteins calculated from three independent experiments. Figure II shows that log-adjusted p-values of significantly enriched GO Terms in TCR-PD-L1 or TCR-control conditions identified from three independent experiments and calculated from fold changes comparing TCR-PD- L1 and TCR-control mean slope of each gene over time. The top 50 genes were selected for analysis using gProfiler g:Ost. Figure 1 J shows rank list of the top ten genes identified in TCR-PD-L1 or TCR-control conditions calculated from the mean slope of each gene in three independent experiments. Schematics created using BioRender.

Fig. 2A-Fig 2K shows that PD-1 inhibits TCR-induced PIEZO1 activity around F- actin rings. Figure 2A shows a schematic of the working principle of GenEPi reporter in which GCaMP fluorescence is increased by PIEZO 1 -specific Ca ²⁺ influx. Representative TIRF microscopy images of GenEPi Jurkat (AF488) cells stained with PD-1 (AF647) and F-actin dye (SPY555) and passed through flow cell chambers coated with anti-CD3/CD28 crosslinking antibodies and either Fig. 2b shows control mlgGi ligand or Fig. 2C shows PD-L1 to assess PIEZO 1 activity over 300 seconds of stimulation. Yellow arrows indicate F-actin rings and merged images contain GenEPi and PD-1 signal only. Normalized fluorescence intensity for Fig. 2D shows that PD-1 expression and Fig. 2E shows PIEZO1 activity calculated from the sum of pixel values within each region of interest (ROI) at 200 seconds of stimulation. ROIs comprise single cells. Figure 2F shows normalized PIEZO 1 activity over 200 seconds measured at intervals of 20 seconds. Data are presented as means of n=37-50 ROIs ± SEM. Statistical significance for a) and b) was measured using Student’s unpaired t-test and statistical significance for Figure 2C was measure using two- way ANOVA. * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001. Representative kymographs for Fig. 2G shows TCR-control and Fig. 2H shows TCR-PD-L1 conditions. Quantification of kymographs for local Fig. 21 shows PD-1 expression and Fig. 2J shows PIEZO 1 activity during stimulation. Fig. 2K shows normalized fluorescence intensity for local PIEZO 1 activity over 200 seconds calculated from the sum of pixel values within each ROI for TCR-control and TCR-PD-L1 conditions. ROIs comprise PD-1 clustering sites. Data are presented as means of n=5-l 1 ROIs ± SEM. Statistical significance for Fig. 21 and Fig. 2J was measured using Student’s unpaired t-test and statistical significance for k) was measured using two-way ANOVA Schematic created using BioRender.

Fig. 3A-3N shows that PIEZO 1 activity in CD8 ⁺ T cells regulates antitumor immunity. Fig. 3 A shows a schematic of E8i-Cre-ER ^T2 Piezo l^ ^x/:flx tumor growth experiment and TIL analysis. Tumor volumes (mm ³ ) were measured over time (days) for Fig. 3B shows MC38 and Fig. 3C shows B16-0VA tumors in Cre+ and Cre- E8i-Cre-ER ^T2 Piezol^* mice. Data are presented as means of n=10-l l for MC38 and n=12-18 for B16-0VA ± SD from two independent experiments for each tumor type including male and female mice. Frequencies of Fig. 3D and Fig. 3E shows CD8 ⁺ TILs and Fig. 3F and Fig. 3G shows frequencies of CD62L-expressing CD8 ⁺ TILs isolated from MC38 and B16-0VA tumors on day 15 post-implantation. Ratio of Fig. 3H and Fig. 31 shows frequencies of TIM-3 and Slamf6-expressing CD8 ⁺ TILs. Data are presented as means of n=18-26 from three independent experiments ± SD. Statistical significance was measured using Student’s unpaired t-test. * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001. Non-parametric t tests were used for graphs that did not exhibit Gaussian distribution. Fig. 3 J shows a schematic of tumor growth experiment with PD-1 blockade. Tumor volumes (mm ³ ) were measured over time (days) for Fig. 3K shows MC38 and Fig. 3L shows B16-0VA tumors in Cre+ and Cre- E8i-Cre-ER ^T2 Piezo l ^flx/flx mice with corresponding Fig. 3M shows MC38 and Fig. 3N shows B16-0VA survival analysis. Data are presented as means of n=5-8 ± SD from one experiment for each tumor type. Statistical significance for tumor growth curves was calculated using two-way ANOVA. * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001. Statistical significance of survival curves was measured using Log-rank Mantel Cox test. * p<0.0332, ** p<0.0021, *** p<0.002.

Fig. 4A-4K shows combined PD-1 blockade and PIEZO1 agonism promote tumor control. Fig. 4A shows a schematic of Yodal treatment in MC38 tumor bearing WT mice. Frequencies of b) CD8 ⁺ T cells, (Fig. 4C) CD62L-expressing, (Fig. 4D) granzyme B- expressing, (Fig. 4E) PD-1 -expressing and (Fig. 4F) perforin-expressing CD8 ⁺ T cells and (Fig. 4G) ratio of TIM-3/Slamf6 expressing CD8+ T cells in tumors assessed by flow cytometry. Data are presented as means of n=6-12 ± SD from one or two independent experiments. Statistical significance was measured using Student’s unpaired t-test. * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001. Fig. 4H shows a schematic of combined PD-1 blockade and PIEZO1 agonist Yodal administration to B16.F10 tumor-bearing mice. Fig. 41 shows tumor volumes (mm ³ ) were measured over time (days) and Figure 4J shows individual tumor growth curves from data depicted in Fig. 41. Fig. 4K shows corresponding survival analysis. Data are combined from two independent experiments and are presented as means of n=13-15. Statistical significance of tumor growth was measured using 3-way ANOVA. * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001. Statistical significance of survival curves was measured using Log-rank Mantel Cox test. * p<0.0332, ** p<0.0021, *** p<0.002.

Fig. 5A-5C shows PD-1-APEX2 construct design and cell line optimization. Fig. 5 A. Design of human and murine PD-1-APEX2 constructs. hPGK denotes the human phosphoglycerate kinase promoter. Fig. 5B. PD-1-APEX2 expression in Jurkat cells was measured by flow cytometry and compared to unstimulated, non-lentivirally transduced Jurkat cells. Fig. 5C. Labelling efficiency of PD-1-APEX2 probe shown via Western blot stained with Ponceau to assess protein loading and probed with streptactin-HRP.

Fig. 6A-6P shows Dynabead optimization. Tosyl-activated Dynabeads coated with various ratios of crosslinking-CD3/CD28 antibodies and recombinant murine PD-L1 or human IgGiK (hlgGiK) control ligand (x-axis) were incubated with murine CD8 ⁺ T cells for 48 h to assess percentages of cells expressing Fig. 6A) CD8[3, Fig. 6B) CD44, Fig. 6C) PD- 1, Fig. 6D) granzyme B, and percentage of Fig. 6E) live cells via flow cytometry. Cell culture supernatants were collected to assess concentrations of excreted cytokines Fig. 6F) IL-2, Fig. 6G) IFNy and Fig. 6H) TNFoc using CBA assay. The x-axis denotes specified protein ratios where “T” represents the combined percentage of TCR components CD3 and CD28 while the latter number represents percentage of murine PD-L1 or hlgGiK on the beads. Increasing concentrations of beads coated with 60% PD-L1 and 40% TCR components selected from the previous assay were incubated with primary murine CD8 ⁺ T cells for 48 h to assess percentages of cells expressing Fig. 61) CD8[3, Fig. 6J) CD44, Fig. 6K) PD-1, 1) granzyme B and percentage of Fig. 6M) live cells via flow cytometry. Cell culture supernatants were collected to assess levels of excreted cytokines Fig. 6N) IL-2, Fig. 60) IFNy, Fig. 6P) TNFoc using CBA assay. Data are presented as means of n=5 biological replicates ± SD. Statistical significance was assessed between control and PD-L1 groups using two-way ANOVA analysis, ns, * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001.

Fig. 7A-7I shows APEX2 proximity labelling identifies PIEZO 1 as target of murine PD-1. TMT ratios over time (min) of Fig. 7A) SHP2, Fig. 7B) PIEZO1, Fig. 7C) SHP1, Fig. 7D) CD3£, Fig. 7E) CD28, Fig. 7F) ZAP70, Fig. 7G) CD35, Fig. 7H) CD3E and Fig. 71) CD3y following stimulation of murine PD-1-APEX2 with murine TCR-PD-L1 (mPD- Ll) or TCR-control hlgGiK beads at indicated timepoints. Data are represented as means ± SD from two independent experiments using two-way ANOVA analysis comparing PD-L1 and control hlgGiK bead-treated groups. Only significant differences are indicated. * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001.

Fig. 8A-8E shows additional PD-1-APEX2 findings. TMT ratios over time (min) of Fig. 8A) CD38, Fig. 8B) CD3E, Fig. 8C) CD3y and Fig. 8D) ZAP70 following stimulation of PD-1-APEX2 with TCR-PD-L1 or TCR-control mlgGi beads. Data are presented as means from three independent experiments ± SD. Statistical significance was assessed using two-way ANOVA analysis comparing TCR-PD-L1 and TCR-control mlgGi bead- treated groups. Only significant differences are indicated. * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001. Fig. 8E) Hierarchical one-way clustering of the averaged TMT ratios of all identified proteins calculated from three independent experiments.

Fig. 9A-9G shows PD-L1 inhibits PIEZO1 activity in PD-1 -expressing GenEPi Jurkat cells in vitro Fig. 9A) Electroporated and blasticidin-selected GenEPi Jurkat cells were incubated with a dose titration of doxycycline (25-200ng/mL) for 24 h and treated with 1O|1M Yodal immediately prior to flow cytometric analysis. Numbers on plots indicate frequencies of FITC+ cells. Fig. 9B) Schematic of TIRF time lapse imaging experimental design. Raw integrated density reported as intensity for Fig. 9C) PD-1 expression and Fig. 9D) PIEZO 1 activity calculated from the sum of pixel values within each region of interest (ROI) at 200 seconds of stimulation presented as normalized values in Fig. 2. ROIs comprise single cells. Data are presented as means of n=37-50 ROIs ± SEM. Statistical significance was measured using Student’s unpaired t-test. ns, * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001. Flow cytometric analysis of PIEZO1 reporter activity in PD-1 -expressing GenEPi Jurkat cells treated with RPMI media, Yodal (5|1M), TCR- control or TCR-PDL1 beads for Fig. 9E) 10 s or Fig. 9F) 30 s acquisition. Fig. 9G) MFI of XLGenEPi reporter in all conditions following 30 s acquisition. Cells were treated with beads 20 min prior to acquisition and with Yodal (5|1M) immediately prior to acquisition. Data are presented as means of n=7-12 technical replicates ± SD. Statistical significance was assessed using one-way ANOVA analysis, ns, * p<0.05, ** p<0.01, *** p<0.001, **** pO.OOOl.

Fig. 10A-10G shows E8i-Cre-ER ^T2 PIEZO 1 KO is specific to CD8 ⁺ T cells. Fig. 10A) Schematic of E8i-Cre-ER ^T2 Piezo P' ^{x f} ' ^x mouse model. Fig. 10B) Relative fold change of PIEZO 1 expression in Cre+ and Cre- CD8 ⁺ T cells following 8 doses of lOmg/mL tamoxifen daily using qPCR and TaqMan probes recognizing specific Piezo 1 floxed regions. Data are presented as means of n=6 ± SD. Flow cytometric analysis comparing Cre expression using an eGFP reporter in Cre+ or Cre- Fig. IOC) murine CD8 ⁺ , CD4 ⁺ , CD1 lb ⁺ and CD1 lc ⁺ splenocytes following in vivo tamoxifen treatment and Fig. 10D) in murine CD8 ⁺ , CD4 ⁺ and B220 ⁺ splenocytes following in vitro 4-hydroxy (4-OH) tamoxifen treatment. Numbers on plots indicate frequencies of eGFP-expressing cells. Fig. 10E) Cre expression measured by eGFP and Fig. 10F) Cre activity measured by TD-tomato frequencies in CD8 ⁺ T cells isolated from the spleens of MC38 and B16-0VA tumor bearing mice at day 15 after tumor implantation. Data are presented as means of n=22-34 ± SD from four independent experiments. Fig. 10G) Ratio of bound/unbound Ca ²⁺ and fold change of Ca ²⁺ influx in non-treated versus Yodal-treated CD8 ⁺ T cells from Cre+ and Cre- mice stained with Indo-1 following tamoxifen dosing. Data are presented as means of n=3 ± SD. Statistical significance was measured using Student’s unpaired t-test. * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001.

Fig. 11A-11K shows E8i-Cre-ER ^T2 Piezo P ^{lx flx} CD8 ⁺ T cells show minimal phenotypic alterations at baseline. CD8 ⁺ T cells isolated from spleens of Cre+ and Cre- E8i- Cre-ER ^T2 Piezo l ^flx/flx mice were stimulated for 24 h with increasing doses of anti- CD3/CD28 crosslinking antibodies (0-10 ug/mL, x-axis) and assessed for frequencies of CD8 ⁺ Fig. 11 A) live cells, Fig. 1 IB) PD-1, Fig. 11C) CTLA-4, Fig. 1 ID) CD44, Fig. 1 IE) CD62L, Fig. 1 IF) granzyme B, Fig. 11G) Ki-67 and Fig. 11H) IFNy/TNFoc. Cytometric bead array assays were performed on the supernatants of stimulated Cre+ and Cre- CD8 ⁺ T cells to assess concentrations (pg/mL) of Fig. 1 II) IFNy, Fig. 11 J) TNFoc and Fig. 1 IK) IL- 2. Data are presented as means of n=3-4 ± SD. Statistical significance was assessed using Student’s unpaired t-test. Only significant differences are indicated. * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001.

Fig. 12A-12S shows flow cytometric analysis of TILs from tumor bearing E8i-Cre- ER ^T2 Pzezo/^’^ ^x mice. Fig. 12A) Gating strategy for CD8 TILs isolated from MC38 or B16- OVA tumors. Frequencies of Fig. 12B and E) CD3E ⁺ , Fig. 12C and F) total CD4 ⁺ T cells and Fig. 12D and G) CD8 ⁺ /CD4 ⁺ ratio (counts). Frequencies of CD8 ⁺ cells expressing Fig. 12H and K) PD-1, Fig. 121 and J) CTLA4, Fig. 12J and M) CD69, Fig. 12N and O) granzyme B, Fig. 12P and Q) Slamf6 and Fig. 12R and S) TIM-3 isolated from Cre+ and Cre- mice bearing MC38 or Bl 6-0 VA tumors on day 15 after tumor implantation. Data are presented as means of n=18-26 ± SD from three independent experiments. Statistical significance was measured using Student’s unpaired t-test. * p<0.05, ** p<0.01, *** p<0.001, **** pO.OOOl.

Fig. 13A-13B shows individual tumor growth curves of E8i-Cre-ER ^T2 mice. Individual tumor growth curves from data depicted in Figure 2.3k-l of Cre+ and Cre- E8i- Cre-ER ^T2 PIEZO l ^flx/flx mice treated with anti-PD-1 or isotype control antibody and bearing Fig. 13A) MC38 or Fig. 13B) Bl 6-0 VA tumors.

Fig. 14A-14D shows PIEZO agonism influences TIL phenotypes in WT mice. Tumor volume (mm ³ ) measured over time (days) for Fig. 14A) experiment 1 and Fig. 14B) experiment 2 of WT mice bearing MC38 tumors and treated with Yodal (7.5 mg/kg). Data are presented as means of n=4-8 ± SD. Statistical significance was assessed using 2-way ANOVA analysis. Frequencies of CD8 ⁺ TILs that are Fig. 14C) Slamf6+ TIM-3- and Fig. 14D) Slamf6- TIM-3+ assessed by flow cytometry. Data are presented as means of n=12 ± SD from one or two independent experiments. Statistical significance was assessed using Student’ s unpaired t test, ns, * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001.

DETAILED DESCRIPTION

The PD-1 pathway plays a critical role in mediating T cell exhaustion, and blockade of this pathway can promote antitumor immunity. While PD-1 inhibitors are revolutionizing cancer therapy, only a subset of patients respond and show durable remission, highlighting the need to better understand how the PD-1 pathway suppresses T cell functions. Here, PD- 1/PD-L1 ligation-induced dynamic changes of the local proteome proximal to PD-1 were quantified using unbiased multiplexed proximity proteomics. The mechanosensitive cation channel PIEZO 1 was identified as a primary target of PD-1 -mediated inhibition. Stimulation of CD8 ⁺ T cells through TCR and CD28 engagement triggered the activation of PIEZO1, while simultaneous PD-1 ligation countered this activation. Mice lacking PIEZO1 selectively on CD8 ⁺ T cells exhibited increased tumor growth marked by impaired CD8 ⁺ T cell function, which could not be rescued by PD-1 blockade. Conversely, mice treated with PIEZO 1 agonist showed increased numbers and function of CD8 ⁺ tumor-infiltrating lymphocytes compared to controls. Combined administration of PIEZO 1 agonist and anti- PD-1 significantly reduced tumor burden and improved survival in a tumor model unresponsive to PD-1 blockade. These findings identify PIEZO 1 inhibition as an important mechanism by which PD-1 signaling regulates CD8 ⁺ T cell functions and suggest PIEZO1 agonism and/or modulation as a novel approach for augmenting cancer immunotherapy. Provided herein is unbiased proximity-labeling as applied to the characterization of the dynamic behavior of proteins recruited to the cytoplasmic tail of PD-1 following ligation and identification of the mechanosensitive ion channel PIEZO 1 (FAM38a) as a primary target of PD-1 -mediated inhibition. PIEZO1 is expressed on a variety of cancers, which include epithelial and immune cells, where its activity can be regulated by external forces such as shear stress, cyclical hydrostatic pressure, and membrane deformation (force- from-lipid), as well as actomyosin contractility and extracellular matrix tethering (force- from-filament). Both external and internal forces contribute to PIEZO 1 gating, a process by which an ion channel transitions between its open and closed conformations as a means of regulating the passage of electrical current through the ion-conducting pore; this, in turn, induces downstream signaling. The stimulation of CD8 ⁺ T cells through TCR and CD28 engagement triggers the activation of PIEZO 1. Notably, it is also demonstrated that simultaneous ligation of PD-1 and engagement of TCR and CD28 significantly reduces PIEZO 1 -mediated Ca ²⁺ influx. Moreover, knockout mice (KO) deficient in PIEZO 1 only in CD8 ⁺ T cells exhibited markedly increased tumor growth. CD8 ⁺ tumor infiltrating lymphocytes (TILs) isolated from these mice were less abundant and less activated. CD8 ⁺ T cell-specific PIEZO 1 KO decreased the efficacy of PD-1 blockade in tumor-bearing mice. In complementary studies, stimulating PIEZO 1 activity in wild-type (WT) tumor-bearing mice by systemic treatment with the PIEZO1 agonist Yodal resulted in increased activation of effector CD8 ⁺ T cells in the tumor microenvironment (TME). Importantly, combined administration of anti-PD-1 and Yodal decreased tumor burden and improved survival in WT mice bearing tumors unresponsive to PD-1 blockade. These summarized results identify reduction of PIEZO1 activity as a novel mechanism by which PD-1 mediates its inhibitory functions and pharmacological agonism of PIEZO 1 as a potentially effective combination therapy to improve PD-1 inhibitors in cancer patients.

Definitions

For convenience, certain terms employed in the specification, examples, and appended claims are collected here.

The articles “a” and “an” are used herein to refer to one or to more than one (i.e. to at least one) of the grammatical object of the article. By way of example, “an element” means one element or more than one element. The term “agent” is used herein to denote a chemical compound, a small molecule, a mixture of chemical compounds and/or a biological macromolecule (such as a nucleic acid, an antibody, an antibody fragment, a protein or a peptide). The activity of such agents may render them suitable as a “therapeutic agent” which is a biologically, physiologically, or pharmacologically active substance (or substances) that acts locally or systemically in a subject.

Unless otherwise specified here within, the terms “antibody” and “antibodies” broadly encompass naturally-occurring forms of antibodies (e.g. IgG, IgA, IgM, IgE) and recombinant antibodies such as single-chain antibodies, chimeric and humanized antibodies and multi-specific antibodies, as well as fragments and derivatives of all of the foregoing, which fragments and derivatives have at least an antigenic binding site. Antibody derivatives may comprise a protein or chemical moiety conjugated to an antibody.

The term “antibody” as used herein also includes an “antigen-binding portion” of an antibody (or simply “antibody portion”). The term “antigen-binding portion”, as used herein, refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen (e.g., a biomarker polypeptide or fragment thereof). It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full- length antibody. Examples of binding fragments encompassed within the term “antigenbinding portion” of an antibody include (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CHI domains; (ii) a F(ab')2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the VH and CHI domains; (iv) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (v) a dAb fragment (Ward et al., (1989) Nature 341 :544-546), which consists of a VH domain; and (vi) an isolated complementarity determining region (CDR). Furthermore, although the two domains of the Fv fragment, VL and VH, are coded for by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent polypeptides (known as single chain Fv (scFv); see e.g., Bird et al. (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883; and Osbourn et al. 1998, Nature Biotechnology 16: 778). Such single chain antibodies are also intended to be encompassed within the term “antigen-binding portion” of an antibody. Any VH and VL sequences of specific scFv can be linked to human immunoglobulin constant region cDNA or genomic sequences, in order to generate expression vectors encoding complete IgG polypeptides or other isotypes. VH and VL can also be used in the generation of Fab, Fv or other fragments of immunoglobulins using either protein chemistry or recombinant DNA technology. Other forms of single chain antibodies, such as diabodies are also encompassed. Diabodies are bivalent, bispecific antibodies in which VH and VL domains are expressed on a single polypeptide chain, but using a linker that is too short to allow for pairing between the two domains on the same chain, thereby forcing the domains to pair with complementary domains of another chain and creating two antigen binding sites (see e.g., Holliger et al. (1993) Proc. Natl. Acad. Sci. U.S.A. 90:6444-6448; Poljak et al. (1994) Structure 2:1121- 1123).

An antibody for use in the instant invention may be a bispecific antibody. A bispecific antibody has binding sites for two different antigens within a single antibody polypeptide. Antigen binding may be simultaneous or sequential. Triomas and hybrid hybridomas are two examples of cell lines that can secrete bispecific antibodies. Examples of bispecific antibodies produced by a hybrid hybridoma or a trioma are disclosed in U.S. Patent 4,474,893. Bispecific antibodies have been constructed by chemical means (Staerz et al. (1985) Nature 314:628, and Perez et al. (1985) Nature 316:354) and hybridoma technology (Staerz and Bevan (1986) Proc. Natl. Acad. Sci. USA, 83: 1453, and Staerz and Bevan (1986) Immunol. Today 7:241). Bispecific antibodies are also described in U.S. Patent 5,959,084. Fragments of bispecific antibodies are described in U.S. Patent 5,798,229.

Bispecific agents can also be generated by making heterohybridomas by fusing hybridomas or other cells making different antibodies, followed by identification of clones producing and co-assembling both antibodies. They can also be generated by chemical or genetic conjugation of complete immunoglobulin chains or portions thereof such as Fab and Fv sequences.

Still further, an antibody or antigen-binding portion thereof may be part of larger immunoadhesion polypeptides, formed by covalent or noncovalent association of the antibody or antibody portion with one or more other proteins or peptides. Examples of such immunoadhesion polypeptides include use of the streptavidin core region to make a tetrameric scFv polypeptide (Kipriyanov et al. (1995) Human Antibodies and Hybridomas 6:93-101) and use of a cysteine residue, biomarker peptide and a C-terminal polyhistidine tag to make bivalent and biotinylated scFv polypeptides (Kipriyanov et al. (\99$)Mol. Immunol. 31 : 1047-1058). Antibody portions, such as Fab and F(ab')2 fragments, can be prepared from whole antibodies using conventional techniques, such as papain or pepsin digestion, respectively, of whole antibodies. Moreover, antibodies, antibody portions and immunoadhesion polypeptides can be obtained using standard recombinant DNA techniques, as described herein.

Antibodies may also be "humanized” which is intended to include antibodies made by a non-human cell having variable and constant regions which have been altered to more closely resemble antibodies that would be made by a human cell. For example, by altering the non-human antibody amino acid sequence to incorporate amino acids found in human germline immunoglobulin sequences. The humanized antibodies of the disclosure may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo), for example in the CDRs. The term “humanized antibody”, as used herein, also includes antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences.

The terms “cancer” or “tumor” refer to the presence of cells possessing characteristics typical of cancer-causing cells, such as uncontrolled proliferation, immortality, metastatic potential, rapid growth and proliferation rate, and certain characteristic morphological features.

Cancer cells are often in the form of a tumor, but such cells may exist alone within an animal, or may be a non-tumorigenic cancer cell, such as a leukemia cell. As used herein, the term “cancer” includes premalignant as well as malignant cancers. Cancers include, but are not limited to, B cell cancer, e.g., myelomas like multiple myeloma, Waldenstrom's macroglobulinemia, the heavy chain diseases, such as, for example, alpha chain disease, gamma chain disease, and mu chain disease, benign monoclonal gammopathy, and immunocytic amyloidosis, melanomas, breast cancer, lung cancer, bronchus cancer, colorectal cancer, prostate cancer, pancreatic cancer, stomach cancer, ovarian cancer, urinary bladder cancer, brain or central nervous system cancer, peripheral nervous system cancer, esophageal cancer, cervical cancer, uterine or endometrial cancer, cancer of the oral cavity or pharynx, liver cancer, kidney cancer, testicular cancer, biliary tract cancer, small bowel or appendix cancer, salivary gland cancer, thyroid gland cancer, adrenal gland cancer, osteosarcoma, chondrosarcoma, cancer of hematologic tissues, and the like. Other non-limiting examples of types of cancers applicable to the methods encompassed by the present disclosure include human sarcomas and carcinomas, e.g., fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, colorectal cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, liver cancer, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, bone cancer, brain tumor, testicular cancer, lung carcinoma, small cell lung carcinoma, bladder carcinoma, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma, retinoblastoma; leukemias, e.g., acute lymphocytic leukemia and acute myelocytic leukemia (myeloblastic, promyelocytic, myelomonocytic, monocytic and erythroleukemia); chronic leukemia (chronic myelocytic (granulocytic) leukemia and chronic lymphocytic leukemia); and polycythemia vera, lymphoma (Hodgkin's disease and non-Hodgkin's disease), myeloma, multiple myeloma, Waldenstrom's macroglobulinemia, and heavy chain disease. In some embodiments, cancers are epithlelial in nature and include but are not limited to, bladder cancer, breast cancer, cervical cancer, colon cancer, gynecologic cancers, renal cancer, laryngeal cancer, lung cancer, oral cancer, head and neck cancer, ovarian cancer, pancreatic cancer, prostate cancer, or skin cancer. In other embodiments, the cancer is breast cancer, prostate cancer, lung cancer, or colon cancer. In still other embodiments, the epithelial cancer is non-small-cell lung cancer, nonpapillary renal cell carcinoma, cervical carcinoma, ovarian carcinoma, or breast carcinoma. The epithelial cancers may be characterized in various other ways including, but not limited to, serous, endometrioid, mucinous, clear cell, Brenner, or undifferentiated. In some embodiments, the cancer comprises a solid tumor. In some embodiments, the tumor is an adenocarcinoma, an adrenal tumor, an anal tumor, a bile duct tumor, a bladder tumor, a bone tumor, a blood born tumor, a brain/CNS tumor, a breast tumor, a cervical tumor, a colorectal tumor, an endometrial tumor, an esophageal tumor, an Ewing tumor, an eye tumor, a gallbladder tumor, a gastrointestinal, a kidney tumor, a laryngeal or hypopharyngreal tumor, a liver tumor, a lung tumor, a mesothelioma tumor, a multiple myeloma tumor, a muscle tumor, a nasopharyngeal tumor, a neuroblastoma, an oral tumor, an osteosarcoma, an ovarian tumor, a pancreatic tumor, a penile tumor, a pituitary tumor, a primary tumor, a prostate tumor, a retinoblastoma, a Rhabdomyosarcoma, a salivary gland tumor, a soft tissue sarcoma, a melanoma, a metastatic tumor, a basal cell carcinoma, a Merkel cell tumor, a testicular tumor, a thymus tumor, a thyroid tumor, a uterine tumor, a vaginal tumor, a vulvar tumor, or a Wilms tumor.

As used herein, the phrase “ conjoint administration" refers to any form of administration of two or more different therapeutic agents such that the second agent is administered while the previously administered therapeutic agent is still effective in the body (e.g., the two agents are simultaneously effective in the subject, which may include synergistic effects of the two agents). For example, the different therapeutic agents can be administered either in the same formulation or in separate formulations, either concomitantly or sequentially. In certain embodiments, the different therapeutic agents can be administered within about one hour, about 12 hours, about 24 hours, about 36 hours, about 48 hours, about 72 hours, or about a week of one another. Thus, a subject who receives such treatment can benefit from a combined effect of different therapeutic agents. As used herein, any two agents and/or additional agents may be conjointly administered according to the methods provided herein.

PIEZO 1 is a mechanosensitive ion channel protein that in humans is encoded by the gene PIEZ01. It is a mechanosensitive non-specific cation channel. It plays a key role in epithelial cell adhesion by maintaining integrin activation through R-Ras recruitment to the ER, likely in its activated state, and subsequent stimulation of calpain signaling. Exemplary nucleotide and amino acid sequences of human PIEZO 1, which correspond to GenBank Accession numbers, are listed below in Table 1. In some embodiments, an agent described herein targets an amino acid sequence disclosed in Table 1 or at least partially encodes an sequence listed in Table 1. In some embodiments, an agent described herein targets a nucleic acid sequence described in Table 1 or at least partially encodes an sequence listed in Table 1. As used herein, PIEZO1. may comprise a sequence listed below in Table 1, or may comprise at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% homology to a sequence listed in Table 1. Table 1

SEO ID NO: 1 : Human PIEZO1 Amino Acid Sequence; NP 001136336,2

MEPHVLGAVLYWLLLPCALLAACLLRFSGLSLVYLLFLLLLPWFPGPTRCGLQGHTG RLLRALLGLSLLF LVAHLALQICLHIVPRLDQLLGPSCSRWETLSRHIGVTRLDLKDIPNAIRLVAPDLGILW SSVCLGICG RLARNTRQSPHPRELDDDERDVDASPTAGLQEAATLAPTRRSRLAARFRVTAHWLLVAAG RVLAVTLLAL AGIAHPSALSSVYLLLFLALCTWWACHFPISTRGFSRLCVAVGCFGAGHLICLYCYQMPL AQALLPPAGI WARVLGLKDFVGPTNCSSPHALVLNTGLDWPVYASPGVLLLLCYATASLRKLRAYRPSGQ RKEAAKGYEA RELELAELDQWPQERESDQHWPTAPDTEADNCIVHELTGQSSVLRRPVRPKRAEPREASP LHSLGHLIM DQSYVCALIAMMVWSITYHSWLTFVLLLWACLIWTVRSRHQLAMLCSPCILLYGMTLCCL RYVWAMDLRP ELPTTLGPVSLRQLGLEHTRYPCLDLGAMLLYTLTFWLLLRQFVKEKLLKWAESPAALTE VTVADTEPTR TQTLLQSLGELVKGVYAKYWIYVCAGMFIWSFAGRLWYKIVYMFLFLLCLTLFQVYYSLW RKLLKAFW WLWAYTMLVLIAVYTFQFQDFPAYWRNLTGFTDEQLGDLGLEQFSVSELFSSILVPGFFL LACILQLHY FHRPFMQLTDMEHVSLPGTRLPRWAHRQDAVSGTPLLREEQQEHQQQQQEEEEEEEDSRD EGLGVATPHQ ATQVPEGAAKWGLVAERLLELAAGFSDVLSRVQVFLRRLLELHVFKLVALYTVWVALKEV SVMNLLLWL WAFALPYPRFRPMASCLSTVWTCVI IVCKMLYQLKWNPQEYSSNCTEPFPNSTNLLPTEISQSLLYRGP VDPANWFGVRKGFPNLGYIQNHLQVLLLLVFEAIVYRRQEHYRRQHQLAPLPAQAVFASG TRQQLDQDLL GCLKYFINFFFYKFGLEICFLMAVNVIGQRMNFLVTLHGCWLVAILTRRHRQAIARLWPN YCLFLALFLL YQYLLCLGMPPALCIDYPWRWSRAVPMNSALIKWLYLPDFFRAPNSTNLISDFLLLLCAS QQWQVFSAER TEEWQRMAGVNTDRLEPLRGEPNPVPNFIHCRSYLDMLKVAVFRYLFWLVLVWFVTGATR ISIFGLGYL LACFYLLLFGTALLQRDTRARLVLWDCLILYNVTVI ISKNMLSLLACVFVEQMQTGFCWVIQLFSLVCTV KGYYDPKEMMDRDQDCLLPVEEAGI IWDSVCFFFLLLQRRVFLSHYYLHVRADLQATALLASRGFALYNA ANLKSIDFHRRIEEKSLAQLKRQMERIRAKQEKHRQGRVDRSRPQDTLGPKDPGLEPGPD SPGGSSPPRR QWWRPWLDHATVIHSGDYFLFESDSEEEEEAVPEDPRPSAQSAFQLAYQAWVTNAQAVLR RRQQEQEQAR QEQAGQLPTGGGPSQEVEPAEGPEEAAAGRSHWQRVLSTAQFLWMLGQALVDELTRWLQE FTRHHGTMS DVLRAERYLLTQELLQGGEVHRGVLDQLYTSQAEATLPGPTEAPNAPSTVSSGLGAEEPL SSMTDDMGSP LSTGYHTRSGSEEAVTDPGEREAGASLYQGLMRTASELLLDRRLRIPELEEAELFAEGQG RALRLLRAVY QCVAAHSELLCYFI I ILNHMVTASAGSLVLPVLVFLWAMLSIPRPSKRFWMTAIVFTEIAVWKYLFQFG FFPWNSHWLRRYENKPYFPPRILGLEKTDGYIKYDLVQLMALFFHRSQLLCYGLWDHEED SPSKEHDKS GEEEQGAEEGPGVPAATTEDHIQVEARVGPTDGTPEPQVELRPRDTRRISLRFRRRKKEG PARKGAAAIE AEDREEEEGEEEKEAPTGREKRPSRSGGRVRAAGRRLQGFCLSLAQGTYRPLRRFFHDIL HTKYRAATDV YALMFLADWDFI 11 IFGFWAFGKHSAATDITSSLSDDQVPEAFLVMLLIQFSTMWDRALYLRKTVLGK LAFQVALVLAIHLWMFFILPAVTERMFNQNWAQLWYFVKCIYFALSAYQIRCGYPTRILG NFLTKKYNH LNLFLFQGFRLVPFLVELRAVMDWVWTDTTLSLSSWMCVEDIYANIFI IKCSRETEKKYPQPKGQKKKKI VKYGMGGLI ILFLIAI IWFPLLFMSLVRSWGWNQPIDVTVTLKLGGYEPLFTMSAQQPSI IPFTAQAY EELSRQFDPQPLAMQFISQYSPEDIVTAQIEGSSGALWRISPPSRAQMKRELYNGTADIT LRFTWNFQRD LAKGGTVEYANEKHMLALAPNSTARRQLASLLEGTSDQSWIPNLFPKYIRAPNGPEANPV KQLQPNEEA DYLGVRIQLRREQGAGATGFLEWWVIELQECRTDCNLLPMVIFSDKVSPPSLGFLAGYGI MGLYVSIVLV IGKFVRGFFSEISHSIMFEELPCVDRILKLCQDIFLVRETRELELEEELYAKLIFLYRSP ETMIKWTREK E

SEO ID NO: 2: Human NM 001142864,4 Homo sapiens piezo type mechanosensitive ion channel component 1 (PIEZO 1\ mRNA

GGGAGCCGCCGTCCGGCCCAGCTCGGCCCCAGTGAGCCGAGCGCTGCGCTCCGCCGA GGGGCAGGGCGGT CGCCTGAGCGAGCGCGGGCCCGGGACGTCGGCACCGGCGGGGCGGCCGAAGGAGAAGGAG GAAGAGGAGA

AGGCGGCGCGCGGGTCCCCGCGGGTCAGCCATGGCGCGCCGGCCCCGGGGCCCCCGC ACCGCCCCATAGC

GCCGCGGCGTCCGCTCGGTCTGGGCCGGGCCCTGGGCCCTCCAGCCATGGAGCCGCA CGTGCTCGGCGCG

GTCCTGTACTGGCTGCTGCTGCCCTGCGCGCTGCTGGCTGCCTGCCTGCTCCGCTTC AGCGGACTCTCGC

TGGTCTACCTGCTCTTCCTGCTGCTGCTGCCCTGGTTCCCCGGCCCCACCCGATGCG GCCTCCAAGGTCA

CACAGGCCGCCTCCTGCGGGCATTGCTGGGCCTCAGCCTGCTCTTCCTGGTGGCCCA TCTCGCCCTCCAG

ATCTGCCTGCATATTGTGCCCCGCCTGGACCAGCTCCTGGGACCCAGCTGCAGCCGC TGGGAGACCCTCT

CGCGACACATAGGGGTCACAAGGCTGGACCTGAAGGACATCCCCAACGCCATCCGGC TGGTGGCCCCTGA

CCTGGGCATCTTGGTGGTCTCCTCTGTCTGCCTCGGCATCTGCGGGCGCCTTGCAAG GAACACCCGGCAG

AGCCCACATCCACGGGAGCTGGATGATGATGAGAGGGATGTGGATGCCAGCCCGACG GCAGGGCTGCAGG

AAGCAGCAACGCTGGCCCCTACACGGAGGTCACGGCTGGCCGCTCGTTTCCGAGTCA CGGCCCACTGGCT GCTGGTGGCGGCTGGGCGGGTCCTGGCCGTAACACTGCTTGCACTGGCAGGCATCGCCCA CCCCTCGGCC CTCTCCAGTGTCTACCTGCTGCTCTTCCTGGCCCTCTGCACCTGGTGGGCCTGCCACTTT CCCATCAGCA

CTCGGGGCTTCAGCAGACTCTGCGTCGCGGTGGGGTGCTTCGGCGCCGGCCATCTCA TCTGCCTCTACTG

CTACCAGATGCCCTTGGCACAGGCTCTGCTCCCGCCTGCCGGCATCTGGGCTAGGGT GCTGGGTCTCAAG

GACTTCGTGGGTCCCACCAACTGCTCCAGCCCCCACGCGCTGGTCCTCAACACCGGC CTGGACTGGCCTG

TGTATGCCAGCCCCGGCGTCCTCCTGCTGCTGTGCTACGCCACGGCCTCTCTGCGCA AGCTCCGCGCGTA

CCGCCCCTCCGGCCAGAGGAAGGAGGCGGCAAAGGGGTATGAGGCTCGGGAGCTGGA GCTAGCAGAGCTG

GACCAGTGGCCCCAGGAACGGGAGTCTGACCAGCACGTGGTGCCCACAGCACCCGAC ACCGAGGCTGATA

ACTGCATCGTGCACGAGCTGACCGGCCAGAGCTCCGTCCTGCGGCGGCCTGTGCGGC CCAAGCGGGCTGA

GCCCAGGGAGGCGTCTCCGCTCCACAGCCTGGGCCACCTCATCATGGACCAGAGCTA TGTGTGCGCGCTC

ATTGCCATGATGGTATGGAGCATCACCTACCACAGCTGGCTGACCTTCGTACTGCTG CTCTGGGCCTGCC

TCATCTGGACGGTGCGCAGCCGCCACCAACTGGCCATGCTGTGCTCGCCCTGCATCC TGCTGTATGGGAT

GACGCTGTGCTGCCTACGCTACGTGTGGGCCATGGACCTGCGCCCTGAGCTGCCCAC CACCCTGGGCCCC

GTCAGCCTGCGCCAGCTGGGGCTGGAGCACACCCGCTACCCCTGTCTGGACCTTGGT GCCATGTTGCTCT

ACACCCTGACCTTCTGGCTCCTGCTGCGCCAGTTTGTGAAAGAGAAGCTGCTGAAGT GGGCAGAGTCTCC

AGCTGCGCTGACGGAGGTCACCGTGGCAGACACAGAGCCCACGCGGACGCAGACGCT GTTGCAGAGCCTG

GGGGAGCTGGTGAAGGGCGTGTACGCCAAGTACTGGATCTATGTGTGTGCTGGCATG TTCATCGTGGTCA

GCTTCGCCGGCCGCCTCGTGGTCTACAAGATTGTCTACATGTTCCTCTTCCTGCTCT GCCTCACCCTCTT

CCAGGTCTACTACAGCCTGTGGCGGAAGCTGCTCAAGGCCTTCTGGTGGCTCGTGGT GGCCTACACCATG

CTGGTCCTCATCGCCGTCTACACCTTCCAGTTCCAGGACTTCCCTGCCTACTGGCGC AACCTCACTGGCT

TCACCGACGAGCAGCTGGGGGACCTGGGCCTGGAGCAGTTCAGCGTGTCCGAGCTCT TCTCCAGCATCCT

GGTGCCCGGCTTCTTCCTCCTGGCCTGCATCCTGCAGCTGCACTACTTCCACAGGCC CTTCATGCAGCTC

ACCGACATGGAGCACGTGTCCCTGCCTGGCACGCGCCTCCCGCGCTGGGCTCACAGG CAGGATGCAGTGA

GTGGGACCCCACTGCTGCGGGAGGAGCAGCAGGAGCATCAGCAGCAGCAGCAGGAGG AGGAGGAGGAGGA

GGAGGACTCCAGGGACGAGGGGCTGGGCGTGGCCACTCCCCACCAGGCCACGCAGGT GCCTGAAGGGGCA

GCCAAGTGGGGCCTGGTGGCTGAGCGGCTGCTGGAGCTGGCAGCCGGCTTCTCGGAC GTCCTCTCACGCG

TGCAGGTGTTCCTGCGGCGGCTGCTGGAGCTTCACGTTTTCAAGCTGGTGGCCCTGT ACACCGTCTGGGT

GGCCCTGAAGGAGGTGTCGGTGATGAACCTGCTGCTGGTGGTGCTGTGGGCCTTCGC CCTGCCCTACCCA

CGCTTCCGGCCCATGGCCTCCTGCCTGTCCACCGTGTGGACCTGCGTCATCATCGTG TGTAAGATGCTGT

ACCAGCTCAAGGTTGTCAACCCCCAGGAGTATTCCAGCAACTGCACCGAGCCCTTCC CCAACAGCACCAA

CTTGCTGCCCACGGAGATCAGCCAGTCCCTGCTGTACCGGGGGCCCGTGGACCCTGC CAACTGGTTTGGG

GTGCGGAAAGGGTTCCCCAACCTGGGCTACATCCAGAACCACCTGCAAGTGCTGCTG CTGCTGGTATTCG

AGGCCATCGTGTACCGGCGCCAGGAGCACTACCGCCGGCAGCACCAGCTGGCCCCGC TGCCTGCCCAGGC

CGTGTTTGCCAGCGGCACCCGCCAGCAGCTGGACCAGGATCTGCTCGGCTGCCTCAA GTACTTCATCAAC

TTCTTCTTCTACAAATTCGGGCTGGAGATCTGCTTCCTGATGGCCGTGAACGTGATC GGGCAGCGCATGA

ACTTTCTGGTGACCCTGCACGGTTGCTGGCTGGTGGCCATCCTCACCCGCAGGCACC GCCAGGCCATTGC

CCGCCTCTGGCCCAACTACTGCCTCTTCCTGGCGCTGTTCCTGCTGTACCAGTACCT GCTGTGCCTGGGG

ATGCCCCCGGCCCTGTGCATTGATTATCCCTGGCGCTGGAGCCGGGCCGTCCCCATG AACTCCGCACTCA

TCAAGTGGCTGTACCTGCCTGATTTCTTCCGGGCCCCCAACTCCACCAACCTCATCA GCGACTTTCTCCT

GCTGCTGTGCGCCTCCCAGCAGTGGCAGGTGTTCTCAGCTGAGCGCACAGAGGAGTG GCAGCGCATGGCT

GGCGTCAACACCGACCGCCTGGAGCCGCTGCGGGGGGAGCCCAACCCCGTGCCCAAC TTTATCCACTGCA

GGTCCTACCTTGACATGCTGAAGGTGGCCGTCTTCCGATACCTGTTCTGGCTGGTGC TGGTGGTGGTGTT

TGTCACGGGGGCCACCCGCATCAGCATCTTCGGGCTGGGCTACCTGCTGGCCTGCTT CTACCTGCTGCTC

TTCGGCACGGCCCTGCTGCAGAGGGACACACGGGCCCGCCTCGTGCTGTGGGACTGC CTCATTCTGTACA

ACGTCACCGTCATCATCTCCAAGAACATGCTGTCGCTCCTGGCCTGCGTCTTCGTGG AGCAGATGCAGAC

CGGCTTCTGCTGGGTCATCCAGCTCTTCAGCCTTGTATGCACCGTCAAGGGCTACTA TGACCCCAAGGAG

ATGATGGACAGAGACCAGGACTGCCTGCTGCCTGTGGAGGAGGCTGGCATCATCTGG GACAGCGTCTGCT

TCTTCTTCCTGCTGCTGCAGCGCCGCGTCTTCCTTAGCCATTACTACCTGCACGTCA GGGCCGACCTCCA

GGCCACCGCCCTGCTAGCCTCCAGGGGCTTCGCCCTCTACAACGCTGCCAACCTCAA GAGCATTGACTTT

CACCGCAGGATAGAGGAGAAGTCCCTGGCCCAGCTGAAAAGACAGATGGAGCGTATC CGTGCCAAGCAGG

AGAAGCACAGGCAGGGCCGGGTGGACCGCAGTCGCCCCCAGGACACCCTGGGCCCCA AGGACCCCGGCCT

GGAGCCAGGGCCCGACAGTCCAGGGGGCTCCTCCCCGCCACGGAGGCAGTGGTGGCG GCCCTGGCTGGAC

CACGCCACAGTCATCCACTCCGGGGACTACTTCCTGTTTGAGTCCGACAGTGAGGAA GAGGAGGAGGCTG

TTCCTGAAGACCCGAGGCCGTCGGCACAGAGTGCCTTCCAGCTGGCGTACCAGGCAT GGGTGACCAACGC

CCAGGCGGTGCTGAGGCGGCGGCAGCAGGAGCAGGAGCAGGCAAGGCAGGAACAGGC AGGACAGCTACCC

ACAGGAGGTGGTCCCAGCCAGGAGGTGGAGCCAGCAGAGGGCCCCGAGGAGGCAGCG GCAGGCCGGAGCC

ATGTGGTGCAGAGGGTGCTGAGCACGGCGCAGTTCCTGTGGATGCTGGGGCAGGCGC TAGTGGATGAGCT

GACACGCTGGCTGCAGGAGTTCACCCGGCACCACGGCACCATGAGCGACGTGCTGCG GGCAGAGCGCTAC

CTCCTCACACAGGAGCTCCTGCAGGGCGGCGAAGTGCACAGGGGCGTGCTGGATCAG CTGTACACAAGCC

AGGCCGAGGCCACGCTGCCAGGCCCCACCGAGGCCCCCAATGCCCCAAGCACCGTGT CCAGTGGGCTGGG CGCGGAGGAGCCACTCAGCAGCATGACAGACGACATGGGCAGCCCCCTGAGCACCGGCTA CCACACGCGC AGTGGCAGTGAGGAGGCAGTCACCGACCCCGGGGAGCGTGAGGCTGGTGCCTCTCTGTAC CAGGGACTGA TGCGGACGGCCAGCGAGCTGCTCCTGGACAGGCGCCTGCGCATCCCAGAGCTGGAGGAGG CAGAGCTGTT TGCGGAGGGGCAGGGCCGGGCGCTGCGGCTGCTGCGGGCCGTGTACCAGTGTGTGGCCGC CCACTCGGAG CTGCTCTGCTACTTCATCATCATCCTCAACCACATGGTCACGGCCTCCGCCGGCTCGCTG GTGCTGCCCG TGCTCGTCTTCCTGTGGGCCATGCTGTCGATCCCGAGGCCCAGCAAGCGCTTCTGGATGA CGGCCATCGT CTTCACCGAGATCGCGGTGGTCGTCAAGTACCTGTTCCAGTTTGGGTTCTTCCCCTGGAA CAGCCACGTG GTGCTGCGGCGCTACGAGAACAAGCCCTACTTCCCGCCCCGCATCCTGGGCCTGGAGAAG ACTGACGGCT ACATCAAGTACGACCTGGTGCAGCTCATGGCCCTTTTCTTCCACCGCTCCCAGCTGCTGT GCTATGGCCT CTGGGACCATGAGGAGGACTCACCATCCAAGGAGCATGACAAGAGCGGCGAGGAGGAGCA GGGAGCCGAG GAGGGGCCAGGGGTGCCTGCGGCCACCACCGAAGACCACATTCAGGTGGAAGCCAGGGTC GGACCCACGG ACGGGACCCCAGAACCCCAAGTGGAGCTCAGGCCCCGTGATACGAGGCGCATCAGTCTAC GTTTTAGAAG AAGGAAGAAGGAGGGCCCAGCACGGAAAGGAGCGGCAGCCATCGAAGCTGAGGACAGGGA GGAAGAAGAG GGGGAGGAAGAGAAAGAGGCCCCCACGGGGAGAGAGAAGAGGCCAAGCCGCTCTGGAGGA AGAGTAAGGG CGGCCGGGCGGCGGCTGCAGGGCTTCTGCCTGTCCCTGGCCCAGGGCACATATCGGCCGC TACGGCGCTT CTTCCACGACATCCTGCACACCAAGTACCGCGCAGCCACCGACGTCTATGCCCTCATGTT CCTGGCTGAT GTTGTCGACTTCATCATCATCATTTTTGGCTTCTGGGCCTTTGGGAAGCACTCGGCGGCC ACAGACATCA CGTCCTCCCTATCAGACGACCAGGTACCCGAGGCTTTCCTGGTCATGCTGCTGATCCAGT TCAGTACCAT GGTGGTTGACCGCGCCCTCTACCTGCGCAAGACCGTGCTGGGCAAGCTGGCCTTCCAGGT GGCGCTGGTG CTGGCCATCCACCTATGGATGTTCTTCATCCTGCCCGCCGTCACTGAGAGGATGTTCAAC CAGAATGTGG TGGCCCAGCTCTGGTACTTCGTGAAGTGCATCTACTTCGCCCTGTCCGCCTACCAGATCC GCTGCGGCTA CCCCACCCGCATCCTCGGCAACTTCCTCACCAAGAAGTACAATCATCTCAACCTCTTCCT CTTCCAGGGG TTCCGGCTGGTGCCGTTCCTGGTGGAGCTGCGGGCAGTGATGGACTGGGTGTGGACGGAC ACCACGCTGT CCCTGTCCAGCTGGATGTGTGTGGAGGACATCTATGCCAACATCTTCATCATCAAATGCA GCCGAGAGAC AGAGAAGAAATACCCGCAGCCCAAAGGGCAGAAGAAGAAGAAGATCGTCAAGTACGGCAT GGGTGGCCTC ATCATCCTCTTCCTCATCGCCATCATCTGGTTCCCACTGCTCTTCATGTCGCTGGTGCGC TCCGTGGTTG GGGTTGTCAACCAGCCCATCGATGTCACCGTCACCCTGAAGCTGGGCGGCTATGAGCCGC TGTTCACCAT GAGCGCCCAGCAGCCGTCCATCATCCCCTTCACGGCCCAGGCCTATGAGGAGCTGTCCCG GCAGTTTGAC CCCCAGCCGCTGGCCATGCAGTTCATCAGCCAGTACAGCCCTGAGGACATCGTCACGGCG CAGATTGAGG GCAGCTCCGGGGCGCTGTGGCGCATCAGTCCCCCCAGCCGTGCCCAGATGAAGCGGGAGC TCTACAACGG CACGGCCGACATCACCCTGCGCTTCACCTGGAACTTCCAGAGGGACCTGGCGAAGGGAGG CACTGTGGAG TATGCCAACGAGAAGCACATGCTGGCCCTGGCCCCCAACAGCACTGCACGGCGGCAGCTG GCCAGCCTGC TCGAGGGCACCTCGGACCAGTCTGTGGTCATCCCTAATCTCTTCCCCAAGTACATCCGTG CCCCCAACGG GCCCGAAGCCAACCCTGTGAAGCAGCTGCAGCCCAATGAGGAGGCCGACTACCTCGGCGT GCGTATCCAG CTGCGGAGGGAGCAGGGTGCGGGGGCCACCGGCTTCCTCGAATGGTGGGTCATCGAGCTG CAGGAGTGCC GGACCGACTGCAACCTGCTGCCCATGGTCATTTTCAGTGACAAGGTCAGCCCACCGAGCC TCGGCTTCCT GGCTGGCTACGGCATCATGGGGCTGTACGTGTCCATCGTGCTGGTCATCGGCAAGTTCGT GCGCGGATTC TTCAGCGAGATCTCGCACTCCATTATGTTCGAGGAGCTGCCGTGCGTGGACCGCATCCTC AAGCTCTGCC AGGACATCTTCCTGGTGCGGGAGACTCGGGAGCTGGAGCTGGAGGAGGAGTTGTACGCCA AGCTCATCTT CCTCTACCGCTCACCGGAGACCATGATCAAGTGGACTCGTGAGAAGGAGTAGGAGCTGCT GCTGGCGCCC GAGAGGGAAGGAGCCGGCCTGCTGGGCAGCGTGGCCACAAGGGGCGGCACTCCTCAGGCC GGGGGAGCCA CTGCCCCGTCCAAGGCCGCCAGCTGTGATGCATCCTCCCGGCCTGCCTGAGCCCTGATGC TGCTGTCAGA GAAGGACACTGCGTCCCCACGGCCTGCGTGGCGCTGCCGTCCCCCACGTGTACTGTAGAG TTTTTTTTTT AATTAAAAAATGTTTTATTTATACAAATGGACAATCAGA

SEO ID NO: 3 NG 042229, 1 Homo sapiens piezo type mechanosensitive ion channel component 1 (PIEZO1E RefSeqGene (LRG 1137) on chromosome 16

TAGTTTTATTTGAGATGAGGTTTTGCCATGTTGGCCAGGCTGGTCTCGAACTCGCCA CCTCAGGTGATCC TCCCACCTCGGCCTCCCAAAGTGCTGGGATTACAGGCACGAGCCACTGCACCCAGCCTGA AAATTGTTTT AACCACGTACATATTTCTTAAAATTGTCTATCATGGTCAAAGTACATAATATAAATGTAC CTTTTAAATC ATTTTTAAGTGTCCAATTTGGAGGCATTAAGCACATTTACAATGTTGTGTCAACACTACT ATTTCCAGAA CCTTTTTATCACCCCAAAGAGAAACTCTGCCACTGTTAAACAACAGCTGCCCACTTCCCC CTCTGGCCTG GTAACCACCCTCTGCTGTCTGTCTCCATAGACATTTCACGTACACAGAGCCACATGGTGT CTTCCTTCTG TGCCTGGCTCCCTTCCCTCCACGTCCCCCAGCCCCGTGCTGGCGCGGTGGCTCACACCTG TGATCCCAGC ACCTTGGGAGGCCAAGGCAGGAGGATCGCTTGAGCCTGGGAGTTTGAGACCAGCGTGGGC AATATAGCAA GACCCCATCTCTATGGGAAAAAAAATTGGCTGGGTGTGGCAGTGCATGCCTGTAGTTCCA GTTACTTGGG AGGCTGAGGTGGGAGGCTTCCTTGAGCCTGAGGTCCAGGTTACAGGGAGCCATGATTGCA CCACTGCAAT CTAGCCTGGTAGCCTGGGTGACAGAGCGGGACCCTGTCTCACACACACACACACAAGTTC ATCCCTGTTG TAGCGTACATCAGAGTTTCATTTCTTTTTATGGACAAATAATATCAGATTGTATGAACAG ACCACATTTT GTTTCTACACTCCATTAACAGACTCTGGGGTTGCTGCCTCTTGAAATTATTATTTATTTT ATTACTTTTT TTGAGACAGGGTCTTGCTCTTTCACCCAGGCAGGAGTGCAGTGGCATGATCTCAGCTCAC TGCAATCTCC ACCTCCTGGGATCAAGCGATTCTCCCGCCTCAGCCTCCCGAGTAGCTGGAACCACAGGCA TGCGCTACCA TGCCCGGCTAATTTTTGTATTTTTAGTAGAGACAGGGTTTCACCATGTTGGCCAGGCTGG TCTTGAACCC CTGACCTCAAGTGATCCACCCACCTCAGCCTCCCAAAGTGCTGGTGTTGCAGGTGTGAGC CACAGTGCCC GGCCAAAATTATTATTATTTTTGAGACAGGGTCTTGGCCTGTTGTCCAGGCCGGAGTGCA GTGGCGCCAT CACTACTCACTGCAGCCTCCACCTCCTGGGCTCTAGAGATCCTCCCACCTCAGCCTCCTA AGTAGCCGGG ACCACAGGCAGGCACCACCACATCTGGCTAATTTTTTTTTTTTTTGAGGCGGAGTCTCGC TCTGTCACCC AGGCTGGAGTGTAATGGCATGGTCTTGGCTCACTGCAACCTCTGCCTCCCGGGTTCAAGC GATTCTGCCT CAGCTTCCCGAGTAGCTGGGATTACAGGTGCCTGCTGCCACGCCTGGCTAATTTTTGTAT TTTAGTAGAG ACAGGGTTTCACCATGTTGGCCAGGCTGGTCTCGAACTCCTGACCCTGTGATCCACCCGC CTCAGCCTCC CAAAGTGCTGGGATTACAGGCATGAGGCACCATGCCTGCCTGGCATGCCTGGCTAATTTT TATTTTTTGT ATAGATGGGGTCTTGCTGTGTTGTCCAGGCTGGTCTCCAACTCCTGGCCACAAGCCATTC TCCTGCCTTA GGC C C C C AAAGTGTTGGGATTAC AGACGTGAGC C AC C AC AC C C AGC CTGAAATTAATTTTTAAGTATAGA CAAGTAATGGTGACTATGTTATCATTGGGGAAAAAGTTACAACCTGCATGATAAAGCTTA AAGTCCCTGT GACCCCTCTTGCCCTGGCTTTTCCTCTCTCCCAAGGGGACCTGACCATTGTTGCTCTCCC AAGACTAAGT CACTGGCCGGGCATGGTGGCTCACACCTGTAATCCCAGCAGTTTGGGAGGTCAAGGCAGG CGGATCATTT GAGGTCAGGAGTTCAAGACCTGGCCAACATTTTGAAATCCCATCTCTACTAAAAATACAA AAGTTGGCCA GGTGTGGTGGCACACGCCTGTGATCCCAGCTACTTGGGAGGTTGAGGCAGGAGAATCACT AGAACTCAGG AGGCGGAGGGTGCAGTGAGCCGAGATTGCTCCACTGCACTCCAGCCTGGACAACAGTGGC AGACTTTGTC TCAAGAAAATACAATAAAATAAAAAATAAATCACTGTTTTAAACAGTGACAAAAATAAAA AGCAACAGCA AGTTGAGAAAGGGCTCCCAGCTGTGGGTCAGTGGGACAGGAGTGACTTTGGACTTGGAAT CTGGAAAGCT GGGTGCAAAGGCCGGTCTATTCCTTTGGCAAATGGTTATCCCGCTCTGGCCCTTAGCGTC CTCTTCTGCA ACAGGGGTTGAGTTAGAACACGGTCCAAATTCCTGAACGGGCAGTGAACTGGGCACAGGT GGGCTCCCCT GCCCTTGCCCGCCCCACCTCTCCCTGGCTGGCAAGCAGAGATCTTTTTCAGAGTATTTGT CTCCATTTTG CTAATTTTATTTTGTGAATAAGTGAATTATGTATGAAGCAAAATATTAAAACAGCACCAA AGGGTATAGG TGAAGACCAGGTCTCCCTCCCACCCCAAGTGGGTGAGCGTGGCCCTGAGTGTAGCTGCTT GCTTTTCCCA ATTGTTAGCACTGTAACTTAGCAATTGTTCCTCCTTGATAACAGAGCAATTGCGGGATTC TCTCTAACCG CTGCTACAGAGTGCCATTGTATCTTAGCTTCATGTTGATACAAAACACCTAGCCGGATTC TGTTTTTTAC CTGCTGTGTAGCACTCCACTGTGAGACACGCTATCATTTATTTGACAAGCTTCTGTGGGC AGAGTTTTTC TGCTTTTTGCTGCTTGAACAATTCTAGGACACATACATCCTCTACCTTTGCAAGTCCATC TGCAGGGCAA GCTCCCAGACTTCCCCACCTCTGCAGCAACTTTTCAAAGCCTGACCAGAGGGTGAGACTT GCAGAGAAAG TAAATCAGCCTTTTTTTTTTTTCTTCTTTGAGATGGAGTCTCATTCTGTTGCCCAGGCTG GAGTGCAGTG GGGTGATCTCGGCTCACTGCAAGCTCCGCCTCCCGGGTTCACGCCATTCTCCTGCCTCAG CCTTCCGAGT AGCTGGGACTACAGGCGCCCGCCACCGCGCCCGGCTAATTTTTTGTATTTTTAGTAGAGA TGGGGTTTCA CCGTGTTAGCCAGGATGGTCTCCATCTGCTGACCTCGTGATCCACCCGCCTCGGCCTCCA AAAGTGCTGG GATTACAGGCGTGAGCCACCGCACCCGGCCTATATCAGCCTTTAAACACTAGACTTCCCA TGAAAATCTC CATTTCTGGGTGGTTTTTTTTTTTTTAATGGAGTTTCGTTCTTGTCACTCAGGCTGGAGT GCAATGGCAT GATCTCGGCTCACGGCAACCTCTGCCTCCCAGGTTCAAGCGATTTTCCTGCCTCGGCCTC CTGAGTAGTT GGGAATACAGGCGCCTGCCATCACGCCCGGCTAATTTTTGTATATTTATTTATTTATTTA TTTATTTATT TATTTATTTATTGAGACGGAGTCTGGCTCTGTCGCCCAGGCTGGAGTGCAGTGGCGCTAT CTCGGCTCAC TGCAAGCTCCGCCTCCTGGGTTGATGCCATTCTCCTGCCTCAGCCTCCCGAGTAGTTGGG ACTACAGGAG CCCGCCACCACGCCCGGCTAATTTTTTATGTTTTTAGTAGAGATGGGTTTTCACCGTGTT AGCCAGGATG GTCTCGATCTCCTGACCTGGTGATCCACCCGCCTCGGCCTCCCAAAGTGCTGGGATTACA GGCGTGAGCC ACCGCGCCCGGCCTAATTTTTGTATTTTTAGTAGAGACGGGGTTTCACCATGTTGGCCAG GCTGGTCTCG AACTCCTGACGTCAAGTGATCCCCCGACCCCCTACCCCCTTGCCTCAGCCCCGCAAAGTG CTGGGATTAC AGGCCTGAGCCACTGCCCCGCCATCTTTTTTTTTTTTTTTTTTTTTTGAGCTGGAGGGTG TGGGCTTGTG CTGGGCTCTCATGAGTTGCCAGTGCCTTGAGGCTCAGTGGCCACAGTCCCCCCAGCCCCA CTCCCTGCCT TACGCAGCTGAGTTTGAGAGCCATGAACTGAGCCCAATTAAACGGTGTAGAGCCCTTTGC CCTCTTTCTG TGCCCATCTCAAGGGCAGACGAAGGCCAAACTGACGTGGCCTGGGTGAGGGTCCCGAACA CCCTGGACTC AGACCCTACCTCTCCGTGGCAGGGCGCCGCGGCAGCAGGTGCACAGGCCGTGGCTCTCAC TCTCACCGTC TCACGCCAGAGCGACCCGGGCAAAGCCGCGCGCGGTGGCCGGCGTGGCTGGGAGCAGCCC CCGAGCCCCC GTCAGACCAACGCGGATCGCGGCACTGCTGCGACCCGAGCATCCCGCGTGCTCCCTCCGG GACCACTAGG CCCGGCCGCCGCGGGAACTCCGAGCCTCCCCACGGTCCCCAGGGGGCGCTGCGTGCTCGC GCCTCGGGGA GCAAAGCGAAAGTCGCTCGGGCACGCGGGGTGCAGTTGCGCGTGGGGCGCTGCCTGGGGG AGCCAGAGGC CCCGGGACCCCTTATGTAGCGCCCGGGCCCCAGCCGCAGCCCACCCAGCGAGAAGCGCCC CTGCCCACCG CCGCGCACGCTCGGAGCCGCGGGGCCAAGACTGCGGGAGGGGAGGGGTCCGCTCCGGGCC AGCGCCAGTC GGGGGTCTCCAGGCCCCAACGCACCAGGGCGGGCCGGAGGCGGGACGGGCCGCGCGGGAC AGCGGAGGGG GTGCCTGGGCGCGCCGGGCCGTCGGGGAAGCGCGGAGGCGGGCACGCCGGGGAGGGCGGG AGGGGGCCGA GCTTATAAAGGCCCGCGGGCGGAGGAGGGCGGGAGCCGCCGTCCGGCCCAGCTCGGCCCC AGTGAGCCGA

GCGCTGCGCTCCGCCGAGGGGCAGGGCGGTCGCCTGAGCGAGCGCGGGCCCGGGACG TCGGCACCGGCGG

GGCGGCCGAAGGAGAAGGAGGAAGAGGAGAAGGCGGCGCGCGGGTCCCCGCGGGTCA GCCATGGCGCGCC

GGCCCCGGGGCCCCCGCACCGCCCCATAGCGCCGCGGCGTCCGCTCGGTCTGGGCCG GGCCCTGGGCCCT

CCAGCCATGGAGCCGCACGTGCTCGGCGCGGTCCTGTACTGGCTGCTGCTGCCCTGC GCGCTGCTGGCTG

GTGAGTGGGGGGCGGGCGCCTGGGGGCGACGGGAGGGGGCTGCGTCTCGGCTCCCCA CGGCCTGGACACC

GGACGACGCCGGCCGGGGCGAGGGCTGCGGGCGAGCGGGCGCGGAAATTCCCAGGGA CGCGCGACCCGGG

CGCCCGCATTCCTGAAGCATGAGCGCGCCAGGCGGCGGCGGGGCTCCTGTCCCAGGG CCGGGCTGGAAGG

GCGGCGGCGGCTGGGGGAGACGGCACCGCGTGCCCACGGGGGCGGTCGAGCGAGCGC CGGGCATAGCGCG

GCTGGCGTCTCCGCCGGGGCGCTGCGGAGAGGAGGCCGCCGGGCGAGGCGGTGTTTG CCCCGGTGGGAAG

GGCCGCGGCGGTGGTGGGGGGAGCACGAATCTCTTTTTCTCTTTCGGGTTTAAAAAA AAAAGCGCAAAGT

TGCATCAGGACTTCCTGACAATCTGGGAGAAGGCGGGCTTCCTGCCTGGAGCTGTTT AATTTGGAGCTTC

CCGAGCCCAACGAACGTCCGTGCCCAGGGCCCAGCCCCGCTCACCGCTGCACCCCCC TCTGCCGGACTGA

GGCGGTCCCACACTTTGGAAAAAAATAGTGTGGGTTCCTCCCTGGTCCTCCCTTGCC CTACTGGGCTCAG

TTTCGCAGGGGCGGGGGCCGGCCTCTGCCCTGGTCTGGGGGAGGGGACACCCCCGGA GGCTGTGGCCTGG

TGTCAGGGCGGGGCAGGGGTCCCCAGTCCTGGCATCTGTGTTCCCTGCTTGCCGGGC AGTGGTGCCCCTT

TCGCGAAGCACACCCGGGTGGCTTGGTGCTGCACGGCCTGGCACCCCTACCCTTCCC CGACCCTGGCCTA

GCCGGGACCCAGGGTCCGCGCCCTCCGCCCGGGGGCTCCCCACGTGTGATTGATCTG GGAAGCAGTCGGA

TGGAATTAACCCACGGACAAGTGGGACGGTTTGCATTGGGAGTCCGCCATGGACACG GCAGGTGGGGCCT

TTTGATTGTAAAAGCCCTTTCGGAGCCCTTGCCTCGCTCCAGGTGGGAGCTCGCCCA GCGCTAGCTTTGG

GGATCTAGAGCCGCCTGCCTGAGGCTCCCAGACAGACTGCGTTTTGATCGGTCGCAC AGAAAGGTGGTGA

AACTTGGGGAAGATTTTCTAGACAGGAATCAATGAAAACCATTGAGGCTGGAGAGGA GAGGTTTTGAGCA

ACTCTCTTCAGTGCGGTCAGCCCTGTGTGGACTGGGCAGCCTGGGACCTGCTCCCAG TGCAGGGTCAGAT

GGGCCGTAAACAGGGCCCGGCTGTGTTCCTTCCTGTGCCTTGAAAACAAGCAGGACA GCCTGGCACAGAG

GCAGAGTCTAGAGCTGACAGGCCTTAGAGAGGGAAACAGGAAAGCTTCTGAAACGTC CCGTTCACACTGA

TCGTTCCATTTCCTCTTGTGTCTGAGTGGGAGCGGGTGTCCTCCCTGCAGGGAATGC CCCCCCTCTCAGA

TGGCAGCTGCTCCTTGGGCAGAGTTGGCAATGTTTTTCTTTAAATGACCAGATGGTA AATATTTTCATGT

CACAAAATCTTCTTCTTCTGGTATTTTTCCAGCCATTAAATGTAAAAGCCCTCCTGA GTTCATGGGCTGT

ACCTAAACAGGTGTTGAGCCCGATTCTGTGGCACATGTGGTTTGCTGCCCCCTGGGC ATTGGTCAGGGGG

CCTGGGTTCTGCCTTCTCGATTGCTATCCGCGTGGGGGATCTGGGGGAGGGATCACT GTTCTTCTTGCTT

TTGGCCTCCTTGGGGAGGATGGGGAGGTAGCCCAGGGGTGCTCACCCAGGCCCCGTG TCAGTCTTCTATG

AAACTTTTAAAGAATAGTGATGACTGACTGTCTGTCTGTATGGTACTTTCCTTAAAC CTAAAACTGGTCC

CAAATAAAGTCTCTTAATTTGAAAGATGCTGAAGCCCGGGCCATACCCCACACTGAT TCTGTGTCTGGGG

ATGGGGCGTGGGGCCTGGGCCTCACTCAGTGTTTTCTCTCAGTCACCTGGGGGAGAT GGAAGTGGAGCCG

GCCAAGAACCCTGCCTGCCTGCCTGCTGGCCGGGACTCCTGAGTCAGGCTCTCTGGC CCTGGGGTGTGGG

CAGCTCCAGATGGACCCGCGATGTGCAGGTTCAGCTGGCCTGGCCGGAGGTGGGACA CTGGCTTTGCTGT

CTTTGGAGTGCCCCCTCCCTCTCTGGCGAGCTTTGGCTGGAAGCAGTTCTACCGTGT TTTGGAAATGAAT

GAGGCCTTCAGAAGGCATTAGTCAGTGTGTGCCTGCGCTGGCTCAGACAGTGCCTGG TGAGGGTTTGAGT

CATCCTGGGGTGCCCCTGGCCCCCACGCCCTCCCTCTCCAGTGCAGGATCATTACCC AAAAATCTGGCAG

GGAGCTGCCCCACCCACAGGGAGCAGGGGCCTCCTTCAGCAGTCTCACCTAATGTTG CTGGAGCCTTGGG

GGATCAGGGCCCATCTCTTCTAGAGAGATGTCAGGGCAGGGCTGGGCGCGATGGCTC ACACCTGTAATCC

CAGAGCTTCGGGAGGCCAAGGTGGGAGGATTGCTTGAGCGTAGCCATTCGAGAGCAG CCTGGGCAACGTA

GAGATCCCCATCTCTATGAAAAATATTTAAAAATTAGCTGGGCATGGTGGTAGTGCA CCTGTAGTCCCAG

CTACTCAGGAGGCCAAGGTGGGATGATTGTTTGAGTCCAGGAGTTGGAGGCTACAGT GAGCCATGATTGT

ATGACTGCACTCCAGCCTGGGTGACAGACCCTGTCTCAAAAAAGAAAAAGGAAAAAA AAGGGCAGGACAT

TTGTGATTCTGATAATATGGGACCGCACCTCCAGTTCTATCAGTGGGAAATCTAGAC AGGGCTGCGGAAA

GCCAGCTGGTGCGAGAGGAGCCACCGTGTCACTGACTGTGGGACACCCACGTGGGCT GACAATATGGCTT

CTGCTTTTCAGGGTGCCTCGTGGCACAGCCTAGGGGCACACCCACGGCCGGCAAGCT GGGCGTCACCTCC

TTCAGGCTGATTGTCACTGAGAAGTGTCACCATTTAGCATGAGGGATGCTGCCTCCT TTTTCAGAACATT

GTCACCATCAGGGTCTCACCACTCCTGGGAGGCGGCCGAGAAGCTGGGGAACAGCAG GCACTCGGCTCAC

AGTTGCTCAGCAGTGCAGACCCTCTGAGCTGAGCATGGCAGAGTCACCCTTCGGAGG CCTGTGCCCGGGT

CTCAGGACCTGCACAGAACCCTGGCCTGTCCCATCCGAGGGTGCTGGGAAGAGCATG GCCGTGGCAGAGT

AGGGTGGGAGCTGCTTTCCTCTGTGGCTTGGGGGCCCCTTCTGAGCATCAGCTCCCT GGTGTGACAGAGG

GGCGCACTCTGTCCCCATGCTGGGCCTGGAGGCTGGATGAGTCAGCAGGAGAGCCTG GGGCCTGCCTCAC

AGCACCAAGGGCTGCAGGTGTGAGTGTGCACATGTGTGCGTGTTTGGGGAAGGGGCC AGGGACTGCCCAG

GAGCTGAGGATGGGTCACAGCGGGTGCTCGTCCCGCAGCGGGTCACTGGTGCCCAGG ACACAGGGAGCTC

CAGCCCCAGCTGCCAGGGTCCCACAGAGAGGAAGTTTCCTCTGGGGGTGGGTGGGGG CGCACAGTCTCTG

ATCCTGGCCCCAAGGCAGCTTCCTGGGCGGTGTCTCTCCTGTGCTGACTCGGCAGTG CATTTGCTTTCGG

TGCTCAAAGATGAAGGGGAACCACCGTGGGCCTTGACGGCCTCATCTGCCCGCTGCA GCCCACTCCTGAG ATGGGACCACCGCAGTCGTCAGGGTCCAGTGAGAGCCGCATCTTGCAGGAAGCCATTCCT GGCCTCTCTG

GCCTCAGAAATCCCCTTTTCAATTCAACAAAATGTTAGTCTTCTGTTTAGCTATTTT AGAAATAGACAGT

TAGGCTTTTTTCTCTTTTTTCTTAAAGACAGAGGCTCACTCTGTCGCCCAGGCTGGA GTGTAGCGGCGTG

ATTTTGGCTCCCTACAACTTCAGCCTCCAGGGCTCAAGCCATCCTCCTGCCTCAGCC TCCTGAGTAGCTG

AGATTACAGGTGTGTAGCACCATACCTAATTTTTGTATTTTTTGTAGAGACGGGGTT TCACCACTTTGGC

CAGGCTGGCCTTGAACTCCTGACCTTAAGCGATCTGCCCGCCTCAGCCTCCAAAAGT GCTGGGGTTACAG

GCATGAGCCACCGCGCCCGGCTGACAGTTAGACTTTTGCTTCTTTGTTTATATAAGC TTTTTCTTCTGGT

TCCAAAAGCAAGTTTGCCCTTCCTTGTGGTAGAGAATCCTAGCTCACAGAGCAGTTT AGAAGCCAGCACA

GTATCCCACACACACATCTGGCATGGACAGACCCTTCCTTGCTGGGTGTGGGTCCTG TGTTCTTCTGAAA

GGCAAGCTGTTCCCAGCCAACCCCTGCCCCTCTCTGCCTTAGCCTGCCTGGAGGCCT GAGTCTCCTGGGT

GACTGTGAGGTGGGACCCCCCCTTCCCCTACCCCCACCCACATCCTCTGTATCTGCC TTCTGTCCTGCTC

TTGACCTTTGAGCTCCTCTGCTGGCTTCAGGGTGCGGCTGTTGAGCCTATTTTTTGG ATTAGGACTCTGG

GGTGAGGGAAGTTAATTCACACACCCAAGATCACACTGGTGGGAAGGGACAGGCCCG GGGTGAAGGCTTC

TCCTCTCCTTGGCGGTTGAGTCCCACACCTGCTGGCCGAGGCACCTGAAGGGGACTT GGGGTCCAGGGTC

ACTGGGAGGACGGGGGCAGGCAGAGGGGTGGCCGACCTGGTGGCGGCTCGTGGGCAG CAGCCGACCCTAT

CTTGCTCTGAACGTGTGGGGCCCTCACCCCCTTCTCTGGGTCTGGGTTTCCTCCCCT GTAAGTGACACCG

TAAAAGCTTCACAGCCGCTTCCAAGTCTCAGGTCTCTCGGGCTTTGGATCTCACACC CAGGCTGGGTGGG

GGTAGGGGCGGGACAGCCGTCCCCCCCGGAAGGCTCAGAATTCCTCGCACAATCGTG GGGCCAGGAGACC

CGCAACACAGGCTTTCCCAGCTGCGCTGAGTGCCGCGGTGGCCGGGGGTCCGTCGGG CCTCCATGGAGCT

GAGGGGAAGGGGCCACTCACCGCCTGGTCCCGGAGCACACAGGGCAGCTCCCAGGAG CACAGAGGCTCCT

TGGGACCTGTGGGGCTGTCGGCCTCCCTCATGCTGCACACACAGCGCGTCCCCAGGG GTGTCTGCAGCCC

AGCCCATCCCACTGCAGATTCCCGACCACTCAGATTCATTCATGCATCCTCTCACGC CGGTCCTGTGGGA

CAGAGCTCTGGGCAGCAGCCAGAAGTCCAAGTTCTGGTTCAGGGCCAGTGGAGGTGG GTGTTGGGGTGGG

GCTGGAGCTCCCTGCTCTCCCTCCCAAGCTAGCCAGGAAAGGAGGTTGGGGGCCCCG CACGGTCATTGCT

GTTTATTCACAAAGCGCGATGCTGAGCACAGGCGGGGAAAGAAAAGTGCGATCAGTG CCAGGAAAATGGG

GCTCCCCCGACGCCGTCCAAAATGGGATCCCTTGCCGGGCGCGGTGGCTCACACCTG TAATCCCAGCACT

TTGGGAAGCCGAGGCAGGTGGATCACCTGAGGTCAGGAGTTCGAGACCAGCCTGACC AACATGGCAAAAC

TCCATCTCAGCTAAAAATACAAAAATTAGCTGGGCATGGTGGCGGGCACCTGTATTC CCAGCTGGGAAGG

CTGAGACAGGAGAATCGCCGGAACCCAGGAGGTAGAGGTTGCAGTGAGCCACAGTCG TGCCACTGCACTC

CAGCTTGGGGGCTTGGGGGACAGCAAGACTCCCTCTCAAAAAAAAAAAAAAAAAGTG AAGTCGTAAATCA

GATTAAATTCCCTTTTTAACCCTTTGAACCTCTGTCCTCCCCTGTTCCCAGCGGGAA GCCTCTGGTGAAC

GCGCCATGCACCCCACCTGCCCCCGCTCTCTGGGTCTCTCTCCCAGCTGGAACGGCC GCTTCCCCAGGTG

CCTTCCCTGGGCCACAGCCTTGTGCCTCGGCGGCTGCTGGATGCCTGGGTGTGTGGG TGGCTCCCAGTGT

GTGGGATGGCACACGAGCCTCTCGCCCTTCTGTGTGGGGTCGCACACCCACCGCAGG CCGTATTTTTGCT

CACGTTCATGTTTCTCCACGGTGGACGCTGGGTTGCAGGGACCCTTCCTGTGTGCGG GTGAGGATCTGGG

CAGCTGCTGGTGCCGGGCCCATGGGGACGCTGACCGTCCCGGGTGCCGGCTCTGAGG TGTGCAGTGGACG

GCTGTCCTGCCGGGCGCTGCCAGGGCCCCTTAGGCCGACGTGCGTGGCCACCCGATT CCCCGCCGTTGTC

TCAGGAACCTTTGCCGAGTGGGGTGGATCAATTTTTCGGGTGTGTTTTAATAGCATA ATTACAGGGAGTA

TTTCAGGCTCCCTCTGACGGGCCGGCAGGGTTTGGCTGCCGGCTGTTTACCAGGCTC CAATCTGCACACT

ATTTTTCTGTGGGTATATATAGCTGGGGCTGCTTCTCCTTCCTCAGGTTCAGGCTAA AGAGGGACAGCAG

CCGCCTCAGCCACCCCCTGTGGTTTCCTTTGCCTGTGGATGGGCGGCTAAAATGGGC CCAGGAAGAGTCA

AGAACAAGGCCGGCTCTCGGTGCCACAGCTCTACCCCCAAAAGCAGGAAGGGGGCTC GGGCCATGCCCAT

CTGTGAGCTACACCGGTCCGGGAGCGGCATCAGGCAGGGGAGTCCTGGACCCCCGCA GTGCTGGGGTGTG

TTTGTCCGCCCTCCCTCCCGTGTGTCTAGAAGCCTCCAGCCTCGGGGAAAACAATGA AACTCAACTGTGA

CTTAAACAGATTCCCAGGCCCGCAGGAGCTCCCGGAGGCTTGTGGCTGTGGCGAGAC CTGGAGGGCCATG

CGGGAGGGACAGACGCAGGTTTGCGGAGGCCGCCTGCCCAGGAGGGGCGTCAAAGGA GGGGACAGATGTG

GGTTTAGGGAGGCCACCTGCCCGGGAGGAGCCTCGAAGGAAGGCACAGGCGTGGGTT TGGGGCTGCCTGC

CCTGGAGGGGCCTCAAGGACCCCAGGTCTGGTCTTGGTCTCACTCACCTCCTGGACC CCCCAAGGCCTGC

AGTTTGCAATCTGTCGCCTGGGACCCCCACTGTCTGCCTTTACGCAGCTCAGCCACC ACGCGGCCCTCGC

TCCCTCATTTACTTGATTTCTGTTTATGGTTAAAGTACCGTTTAAAACGACACATCA TTAAAGCAACATG

AAAGGGAGTTTTGAAAAGGGAAGCCATCGTCCATCCCACTGCCCCTGCCTCAGCGGG GATTTACTTTTCC

TTTCCTGTCTGCGGGCCAGTGACAATGAGGACCCCGCAATGTGTCTGCGGGCCAGTG ACAATGAGGACCC

CGCAATGTGTCTGCGGGCCAGTGACAGTGAGGACCCCGCAATGTGTCTGCGGGCCAG TGACAGTGAGGAC

CCTGCAATGTGTCTGCGGGCCAGTGACAATGAGGACCCTGCAATGGGCGGCCTGTAA GGCTCTGCCCTGG

CCTCCGCTGCCTTCGCTTTCTCCCTCCTTGGTGGGTGCACGCCCTTGTGCTTTTCCT AAAAGAGCAGGTC

CTCCGGGCATGGTGGCTCACGCCGGTAATCCCAGCACTTTGGGAGGCCGAGACGGGT GGATCCCAAGGCC

AGGAGTTCAAGACCAGCCTGGCCAACATGGCAAAACCCTGTCCCTACTGAAAATACA AAAATTAGCTGGG

TATGGTGACAGGCATCTGTAATCCTAGCTACTCGGGAGGCTGAGGCAGGAGAATCAC TTGAATCCGGGAG

ACGGAGTTTGCAGTGAGCCGAGATCACACCATTGCACTCCAGCCTGGGCAACAAGAG CGAAACTCCATCT CAAAAAAGAAGAAGAAGAAAATCCTGACACTTCAGCCTGGTGCAGGCCCTTCCCTCCTTC TAGTCCCTGC

CAAGAAGTGAGCCGGGCCCAGATCTCCTGCCGGGCGGGGAATGAGCACACACATTCC CCTCTTGGGACAG

ACAGCAGCAGCAGCCCTGTTGCACACATGAGGACGTACAGGCTCAGGGGCCGTGGGT GGCAGAGAGGCTA

TCAGCGCCGGACTGGCCCGCCCCGAGCCAGGGTCCAGCCCCACAGTCCTGTCCCCAA GCCCTGGCCCTTC

TGCGGTCACTCCCGTCTGCTGAATCCCCTACTCTGCCCCTGGTGTGTGGCCCCCCAG TTCCCTCCTGTGT

TCATTCCCTGCTAACCTCCCGTGGCTTCGCCTCCCCAGATGCCCTGAGCACACAGCC TCTCCCCTTCCTC

CCCTCCTAGATGTGCACATAGGAGCCGCCCAAAGGCTGGGGCAGCTAGTGGGGCCCC TCCAAGGGAAGCT

GGGCCCCGGGCAATGCCCTGAGCCACCAGGTCCTGGCCCTGCGTCTCATCCCTTCTT TTTTTTTTTTGAG

ACAGAGTCTCGCTCTGTCGCCCTGGCTGGAGTGCAGTGGCCCAGTCTGGGCTCACTG CAAGCTCTGCCTC

CCAGGTTCACGCCATTCTCCTTCCTCAGCCTCCCGAGTAGCTGGGACTACAGGCGCC CACCACCACACTC

CGCTAATTTTTTGTATTTTTAGTAGAGACGGGGTTTCACCGTGTTAACCAGGATGGT CTCAATCTCCTGA

CCTCATGATCCACCCGCCTCGGCCTCCCAGAATGCTGGGATGACAGGCGTGAGCCAC CGCGCCCGGCCTC

ATTTGGTTCCTTCTGTGCACCACAGTGTGGGATCTCGGGTCCTGGGTGGCACGTGTT TAACCTGAAAGGA

AACTGCCCCTGCGCTCCCCAGCTTGTAGCCCGTGGGCCTGGCTCTGAGGCCCCGACT GCCCCACGGCCTG

TGCTGCGCATGCTGGAGCCAGGCTCCGGCTCTGGTTGGCGCCTCCCGTGGCTTTAAT CTGCAGTGACCTG

GGTGCTTAACGAGGGCCTTTGCCTCTGCGTGTGCACTGCCTTCTTCTGAGGAGTCTC CTCAAGTCTCCCA

GCTGTTTAAACAATGGGGTCTTTTGTCTTTTGACCTGTTGGTGCCATCAGCCAGCCT CCTGCACATACTC

TCCCTACCCTTGGCTGCCACTCCTGTCCTGCCCTTCGCCATGTCTTTTTTCTTTTCT TTTTTTTTTTTTT

TTTTGACAGCGTCTTATTCTGCCACCCAGGCTGTAGTGGAGTGGCACGATCTTGGCT CACTGCACCCTCC

GCCTCCCTGGTTCAACCGATTCTCCTGCCTTAGCCTCCCAAGTAGCTAGGACTACAG GTGCCCACCACCA

CACCCGGCTAATTTTTGTATTGTTAGTGGAGATGGAGTTTCACCATTTTGGCCAGGC TGGTCTCAAACTC

CTGACCTTAGGTGATCCGCCTGCCTTGGCCTCCCAAAGTGCTGGGATTACAGGCGTG AGCCACTGCGCCG

GGCCCCCGCCATGTGTTTTTAAGAGCAGGCATTTCATTCAGATGAAGCCCGGACACT CTTGGGGGTTCTG

CTCAGGGGCTCTACCTGCCACTGAACGTCCTCTCCCTGTGTGGGTGGCCATGGCATT CAGCCTGTGTTGG

GCCTTGTTCTGCCTGGCTACTGGCCGCTAGGTCTGCCCCGGCATCCTCTGTGTCCTG TGTGGGCGCCTCT

GCCTCCTGCTCCAGCCTGTGCCAGGCAATCCTGCTCACCTTCCAGGAGCCAGGCCTC TCCCCAGGGCCTG

CGTCCTGTCAGGGTCAGGGACGGCCCCTCTGCCATGCTCCGGAGTCCCTGGTCCCCT CACTCCGTTACGT

CCTGGGTGTCACGGGTACGGCCGGGAACTCTGTCGTCTTCACTGTCTGGGCCGGGGC CTCCCTGGGTGTC

TGCTGGATGGAGTGGGTGCCTTTGGGTCCCTGCAAAGTGAGCCTGCCTCCCAGCACC GCCCTGTCGTTAC

ATAGCCACTATCTTTGCGCCTGTTTTTCCTTCCTTTGACTGGTTCCTCTGGGGTTAA TTCCCAGGCCTGG

TATTACCATCTCTGAATGCCTGGGTGGTTTCAGCGCCCAGGAGGCTGTGCTGAGGTA TCTTAGACCATGT

GGGCACCGTTCGCTCCTGCGCAGCCGGCTCCGGGGTGCCCTGTTTTTGCGGAATCCT GCAGGGAACCCCA

TGTACCCTAAGAGTGCTCTCCCCAGCCACTGTGGCATAAGACAAGCGGTCTCTTTGC CCTTGGGCCCCAT

CCTTGTCTGGTCGGCCCTTCTTCATGGGTCCAGCGCGGGATTGCCGGCTTCCTTTTC AGGCTTCCTGGGA

CCCCCACTCAGACCTGCAGCTGGGCCAGCGATGCCCACCCGTTTCTCCTCCACGTGG TATACAGAGGTGC

CCAGGCTGCTGCTGGGGACTCTGGAGCCCAGGAGTGAGTCTCCTTGACCCTGAGCTG TCCTGGCTATCAC

AGCTGGGTCCTGTTTTCCCCTTCAGCACCCACGGGTGTCTTTCTCCAGTTTATATTG TTATTTTTATTTA

TTTACTTATCGAGACAGAGTCTCGCTCTGTCGCCCAAGCTGGTGTGCAGTGGCACAA TCTCGACTCATCG

CAAACTCCGCCTCCCGGGTTCAAGCGATTCTCATGCCTCAGCCTCCTGAGTAGCTGG GATTACAGGCATG

TATCACCAGGCTGGACCTCTAGTTTATATTATTACAGCCTGGTCAGGGAGTCACTGG GCGTCGCCATCCT

GTGGGTGGGAAGGGGGCCCAGGCAGAGGCCAGGAGGAGGTGACAGTCATGCGCTTAG AAGCTCAGGCCAC

GCCCACCCAGCCCTGCCTGGTGACCGGTTCCTGCTGTGTTGGGAGCTGCATCCCAGA CCTTATCGCCGGG

CACAGAACTTCCAAGCCAGGGGAGAGGGAGGCCTGGAGGGGCCCCAATCTCTGAAGG TCAGCAGTGGCGG

GGAGGAGGCTTGCGGTGCTGAAGGGACTCGGGGGACCTGCAGGGAGGTGTTGGTGGT CAGGGATGGTGGC

ACCTGAGGGGACTTTTGGCAGGGCCAGAAGTGCCCAGAGCAAGCTCCGGTGGGGCCC TGCACTGGAGGGC

TGGGGTCGAGTGACCCTCTTCCTAAGACCACCCAGGAGGACACTGGGTGCAGGGTGG CCGGAGCCCTCAC

CCCCAGTAGGCAGCTGCTGTCCACTCCGCCGACCTGCCTGTCACCCAGTAGGCAGCT GCTGTCCACTCCC

CCGACCTGCCTGTCACCCAGTAGGCAGCTGCTGTCCACTCCCCCGACCTGCCTGTCA CCCAATAGGCAGC

TGCTGTCCAGTCCCCCGACCTGCCTGTCAGCCTTTTCCCTGTCAGGCCCTGTTCCTA GGAGCCTGGAGAC

CTCAGGGGTGGCCTTGAGCCCCCAGGGTTTTTTTGAGGGGAAGCGCCAGCTGCTGTC TTCACCCTTCCCC

TAGTGAGGCCAGGCTGTGCAGGGCCACGTGGAGGCAGTCTGTGCTGCGCCCATCGGT CGCCTGGCTTCCT

GCCGACCCTCGGCCCCCAGCCACCTCTGGTCTCGGGCAAGGCCCCTCCCCCTGCCCA CCTCCTCCCTGGC

CCCCACGCCAGGTGGGCAGACCTCCTCTGCGGTTTTTATTCAGGGGGTCCCTCGTTG GCTGCCCACTCTT

GGAGGGCTGTCCTGACTCAGGCCTCCCCTCTACTCAGATCCCCTGAGCGAGGGCCTG GGCGTGACGCCGG

GAGTTACTGGGGGGCCAGAGGGGGAGGCTCAGCCTGAATCAATGAGACCCAGGAGGA AGGAGGCACGTGG

ACCTGAGGGCTGGCTGGAGCCGCTGGTGACAGATGGAGGAGTAATTGCTGCTTCCAG AGCACAGCGAGCT

CGAGCTCCCTGGAGTGCCAGAAGCTTCTGGGTGGACAGACAGGCCGCCTCACATTCC AGAGGCTGACACA

GTCTTCCAGGCACCCCTGGGGCCAGCTGGAAGCCATGTGCCTCCACTCACGTGTCCC GTGGGTGTTTGGA

GGGGAGGCTGGCCCTGCCATGGCCCACCCCAGCCTGCGGCCTCAGGAGCTTGCACAT TTGCAAGGGGGTG ACATGAAGACTGAGCCAGGGCTGCGGCGGGCATCCCCCTCAGAGAACAGGGAGGAGGGGG CACAGGCCTT _{TT TTTTTTTTTTTTTGACAGAGTCTCACTCTTT} GGCCAGGCTGGAATGCAGTGGCACGATCTCAGCTCAC TGCCACCTTCGCCTCCCAGGTTCAAGCGATTCTCCTGCCTCAGCCTCCCCAGTAGCTGAG ATTACAGGCA CCCACCATCACGCCCGGCTCATTTTTGTATTTTTAGTAGAGACGGGGTTTTACCATGTTG TCCAGGATGG TCTTGATCTGTTGACCTCGTGATCTGCTTGCCTCGGCCTCCCAAAGTGCTGGGATTACAG GCGTGAGCCA CTGTGCCCTGCCAGGCACGGGCCTTTTTATGAGGGAGGAAGCCGTGCCCTCTGCCACCTG CCTGTGGGCT GGGGCCTGCAGGCTCCCAGGGCCCTCAGCTCCAGCGTGGCTGTTGGTTATTGGCACCTGG GCCTCATGTC CACATGGAAAGTATATTCCGAGCTGGTGGCTAAGCCACAAGAGCCTGCTTACCTGTCCCA ACCCCCGGCT CACTTGCTGCCATGATACGTCTGGGATTCTGGGCCCTCCTTCTGAGCTTGGACACAGACT TGGGACTTCA GCGCTGGACACAGGTGCTGGATCTGGGGTCTCAATGGCACCAGGGATGGTCATTCTGACA GCCATGCCCA GAGTCTCAGGAGGGCCTGGGGCCGCAGGTGGACAGGCATCCTGACCAGAGTCTCAGGAGG GCCTGGGGCC GCGGGTGGACAGGCATAGAGTCTCAGGTGGGCCTGGGGCCGCGGGTGGACGGGCATAGAG TCTCAGGAGG GCCTGGGGCCGCGGGTGGATGGGCATAGAGTCTCAGGAGGGCCTGGGGCCGCGGGTGGAC GGGCATAGAG TCTCAGGTGGGCCTGGGGCCGCGGGTGGACGGGCATAGAGTCTCAGGAGGGCCTGGGGCC GCGGGTGGAC GGGCATAGAGTCTCAGGAGGGCCTGGGGCCGCGGGTGGACGGGCATAGAGTCTCAGGAGG GCCTGGGGCC GCGGGTGGACGGGCATAGAGTCTCAGGAGGGCCTGGGGCCGCGGGTGGACGGGCATAGAG TCTCAGGAGG GCCTGGGGCCGCGGGTGGACGGGCATCTGGACGTCGGCTCCACTAGTGTCACCTCTGCAA CCTTGTCCCA CCCAGCACCTCCCTATGGCCCTGCTCTGACTGTGGGCTGAGGGGGACCCCCATGGCTAGC TTGGGACCTC CGCTGGACACTGTCATAGGGCACTTGTCGAGTGGCCCTGCTGAGGTTCAGGCTTGGGCGT GTGTGGCCCC TGAGCCCAGCAGCTGCGTGAGCCGTGGGGAGCCGAGCCCCCAGTCAAGAAAGCTGTGTGC TCCTTACCCT GCCTGCTGGGCTTGGGGCCTGCAGGGCAAGGGAGGCGGTGGGATGGGAGCCCCGCCTGGC CTGGTTTCTT GTTGTCGCCGCACTCCTAGCTGTATGTAGTCAGATGAGGCTCCCAGTCCTGGCTCTGCTG CTGAGCACCA TTGGGCCGGTGGTTGGGTGAATCTCTCAGCCTCTCTGAGCCTCCAGCACTTCGTCCTTAA GAGTGGAACT GGTCTGGACCTCCCAAGGGGGGGTCACTGGGTGCAGAGTTCCTGCAAGGCTGGGCCCGGT GCCAGTAGCG GTCGCTCTGTGGGGCCTTTCGCACGGTTGATTTTTATCTCCCTGTGAGATTAGCGCCTGC CCTGCTCTGC CTCACGGCTCTGGCTCTCCGGGACTGAGACCACCTGCTAAGTGACAGGGGCGGAAGGACC TTGCAACACA GAGCAAGACGTGATAGGGTGGAGCTGCTCCGCCCAGGCCCCGCCTGCAGATGAGAGGGCC CAGAGCCTCT TAATCCCCTGCGCTCAGGGCATGTGACGGCAGGGAGTCTCTCCAGTGCCGCCCACCGGAT TCCTATAGTT AGAGCTCCTCGCCCAAGCCCCCAGGGCACCTCACTGGCCTCCCAGGTGAGGACATGAGGC CCAGCGACTC TGGTGGTGGCTGGTGTCACCTATGGCAGGGCCGCGCGGGGCCCAGTGGAGGACGCCTGGC CCGGACCTGA CTGCTCCGTGGGAACCGTGGGGCGTGCTCAGGGTGACTTGGTGCGGCTGGGCTGCCAGCC GGCCTGGCTG AGTCACTGGCAGTGAAGGCACCAGGTGGTGGACGTGCTCGGCAGCTGGCGGGAGAGTACC CCCACCCAGG ACCGTGTTCAGGCTGAGGGGGAAGGGAGCGGCCCAGGCAGGCACCGCCCATGGCGGGGCC CCCGCACTAA CGGCCACCTCTCCCGATGGACTGCAGTTCTGGAGCCGCCCCTGAGGAGAACGCCCTGCTC AGTGTCCTGG CAGACTCTGGACTCTTCACCGTGCCATCCTCGCTACAGCCTGGCCAGGCAGCCGCTCCCG TCCCCCCACC CCACAGACAGGAACTGCAGACTCCTAAAGGTCGAGCCAGCTCCCAAGTCCATAGCCAGAA AGTGATGCAG GCGGGGCCCGGCTGGGGGGCAGGTGTGACCGGGTCATGGGGGAGCGGGCTGTGCACAGGC ATCAGGCTGA GAGGTCTCGGGGATACGTCCTGGGAGCCGCCTGCCAGGCGTCCTGTGGGGCTGCCTGCAG GCCTCCTCTG GCTTTGGCAGGTTCTGTCCCGACAGACCGAGGCTTGGGAAGACCGGATTGTGCACTCTCT GCCTGGAGAG AGGGTGGGGAGGGGCTGGGGCGTGTGGGCAGGGAGAACACCCTGGGAGGCATCAGTCCTC TTCCCTGGGG ACGGGCCATGCGTCAACAGTCCCTCACTTCCTCTCTCAGGGGCAGAGGCGTGGGGGTCCC GAGCCTCCAG GTTGGAGTAGGTTAGGAGGGCCTCAGAGCCCCCTGCTGCCCCGCCATGTGGCATTGGAAG GGGTGTGACC CCCTAGGGGATCTGTTAACCACACTCCGCCCCATGCCGGCCTCTCTGTTCCCTCTAACCT GCGCAGGAGC AGGGCCAGCTCTGAGCTAGCAGAGGTGACATGAGGCTCGTCCTGGCCCCGCCCACCCTGG CCCCTCCCTG TTTCCCACCACGCTCCGCCCACCAGAGCTACATGTTGTGTTTGAGCCTCAGCCCCAGACC TGGCTCAGGT CCTCAGGAGGAAGCAGTGACAGCCAGGCCCGGGGTCCTCTCCCTGCTCTGCTCCCTGCGT GCTGACTGGA GGCTGCAGGTCCCCAGGCTTGGCCCTGACCCTCAGGAATGGAGCCGGCCGATGTGAGGTG GGGGCTCCGG TCATGTGCAGTGGTAGGAGAGGAGGCGGGACCGGTCCCACAGCTCCATTGCTGCCGAGGC GTTCCGCAGG TCTGTCTTCATATTGGTAAGGAAAATGCGAGGATGGTGTAGCCTGGGCATCCCAGTCCCC AAGTCCGAAA TCTAAATCCAAGCGGAAAATTCCAGGCCTGACTTCATACGCCAGGTCACAGTCAGAAGCC AGAATTATTG AAAGTATGCTCTGGGCGTGGTGGCCTCACACCTGTAATCCCAGCACTTTGGGAGGCCATG GTGGGAGGAT CACTTGAGGCCAGGAGTTTGAGAGCAGCCTGGGTAACATAGTGAGACCCCCGTCATTATT TACACAAAAT TATGTGACTGCCTTTAGGGTATGTGTACGAGGTATATATTGAAATGTGAGTAGAATTTTG TGTTGAGTCC CATCCCCAAGATACCTCATTATGCGTGTGCAAATATTCCAAAATCCAGAAAATCCTAAAC TGGAAATACT TCTGGCCCCTTTGGATGAGGGGCCCCCACTCTGCATAGCAAGTGTGGGCGCGGGTGCTGC TGTGTCCTAG CGTACTTCAGTGTGTGGCCTTTGCAGTGAATAGGGCTGGTGTCCTCACTGTACAGATGAG GAAACTGAGG CCCAGCTTGCTTTGCCAAGGTGCATCCGGCCCCGGCCATGGCTATTCTGGCTCCAGATCC CATGGTCTGC AGCCACAATACTGCTGTGCCCCGGACTGGGCCCTGCAGCTCGCGGTGTCGCTGGCCTGCC CATTGTGGGC ACCGCCCCCACCCCAGACTGGCCGAGGCCTAGAAGGGAGCAGGGCCTGGCTGAGGCTGCA GGGGTGGGGA CGGTCAGCCAGCCCCTCACTGCCAGGAAGGGCGCATCCATCCTGGCCTCTCCCCAGGGAG AAGGGAGGAG

CGGCTGAGAGGGAAGCGCTCTTGCCCTGTGGACGAGCTCCTGCCCCACGGACTAGGG AGCCCCCGCCCAC

AACCTGCTTGTCAGGGCCACCCGGGACCCCCGGGAGTTCGGCTGCTCGCTCTGCTGT TAGGAATTGGATT

AGTTTTCCATAAAAACAGGATGTGGTGGGTGAGAGGGCAGTGTGTCCGTCTTTCTCA CTCCCCTTTTTCC

AGGAACTGAGCACGCGCATAGGTTTTAGCCAGGGCCGTCCAGTCCCCTCCCCACCCC CCACAGGGAACAA

TCCACTCTCTGCTCTTAAGTGGCCACTTAATCAGCTTCTCCTCCTGGCCCGGGGAGC TTCTTGGAGCCGG

CCTGCCGTGGTGGGAACAGCTATGGGGACACCCTGCCATAAGGTCCAGCAGCTAAGC TGGGATGTGGGGG

GAGGGGCTGCGAGGCCCAGGCAGTGTGCCAGGCCGCACAAGAGGAGCCCAGCTCTTG CCCCACCAGCTGG

CAGCCCTGGACCGAGGTTGGGCCCGTGAGGTTGGCTGGGCCCTGGGCCCTGGGCCCC CCTCCCCAGGACA

CGACTGTGGTGGCACATGGCTTTGGGGGCTCGTGGGTCCCACTTTGCAGACCTCTGC TTTAAGGGGTCTG

GTCCACGGGGTCCCCTCTGGAGGGCCTGGGGGAAATCTCAGGGACCTGGGGTCTGGA CCCGGGGGAGGGA

GCGGGAGAAGCATGTGCGTGAGTCTCGTGCTGTCAGGGAGCCCGGGAAGTCTGCGAG GGCTTGGGGGTTG

TGTCAGGGAGTGTGGGTTTTGCCCTTCAGTGGTGGAAGCTGGCTTGAGTCCCCTCGA TCCCTCAAGGCTG

TAGTCCTGACTCGGGGCTGTAGGGCAGGGCAGGGTGGGGTCACCCTGAGGCAGAAGG CTCAGCGGAGATG

TTCTGGCTTGGCTCTGCCCCTCCCTGGCTGGGTGGCCTCACCCTGTGACCTTTGGGG ACCCTGGTTCCTC

TGAGCCAGGGGACAGCAGTGACTCCGCCTTCCTAGGTGGCTGAGGATGACATGGGCT CCCCCTGCAAATG

TGGGTTCTGGCTCAGTGGCCAAGTGTATGATGGTATGTGGCTCTTGGGGTCCTGAGA GAGATGGGAGAGG

AGCAGGGGTTTGTAGGGAAGCTGGCGGCTTCCACCCCAGCCAGTCACCTGCAGTGGG GGAGTTCCAAAGC

TGACTGAAGCTTCGACCTTGTGGCTGGTCCCCTTCCTTCCTGCCTCAGTCATTCTGG TCTCTGGGGGATC

AGGGCTGGGGGGCTCTGGGCTGTGGGGGCCTGTTTTTGTGACTTAAAGCTCTCCCAG CACAGCCCCCTGA

CCTCCTTCCTCATGGGCAGGACCTGGCCCAGGGGTCTCAGCACAGCCACAGGCCAGG GATGCCCTTGCAG

ATGGCCCTGGATGGAATTCCAGAACTCAGAAATGTCTCCTTCCCGTAAGGATGTCCC GAGACTCATGAGA

CCGTTTCCTTCTGGGAAGGGGGAGGAATGGGGAGATGAGTGAAGAGCGCACTGCAGC TCAATCCGGGAAG

AAGCTAATCAATCAATCAGGGAAGCCAATCGATCGGAGAAGCTGATGGGAAAGCTGT GCTTGGTAGAATA

GGCTCGTGCGGGCAGAGCAGTCGCGGCACTCACAGGCTGACTGTGAGGACCTTGGGT GTTACTTTGTGCT

TCTCTGCATTTTAAAAACTTTTGAAAGTGGAGAAGAGGAAAGTGAATGTTCTCTGAG ATTTCATGGAAAG

GGGAAACTGAGGCCCACTCTAGCCAGTTTGGGCCCAGGGTTCCAACCTGGGGTGGCC CCGGCCCTCGTGG

CCTGAGGTGATCGTCCCTGTGGCTCTGAGAGCAGCTGGGGCCGGGTCCCCGTTCTGG GGCTGGTGATCCT

GGGGAAGAGCCAGGCAGTGCCCTGCCCACCTAGTGGTTATGAGCCCAGAATGTTGAT TTTTTTCCCTTGG

TTGCTTCATGACTTTGTTGAATTTCCAGAGTATGTGTGGGGGCCCCCGGCGTCCCAC TCGCCCCAGCCCT

GTGGCAGCAGAGCTGGCTGTCAAGCTCAGTCAGCTGGGCCCAGGGCCCCGGAGGTAG GTGGGTGTGTGCC

TGAGCTTCCCCTTTGGGCCCTGCCAGGTGCTGGGAGGGACCACACAGGCGGCAGGAA CTCGGGGTCCCCA

GGCCTCGGCCACACCAGCCTGGTGCTTGTTATATATTGATATGTCTCTCTACCTGTG AAATGGGTATTTA

TTTTAAGGAGCTGACGCACGCGATTGTGGGGCTGTCACGTCTGAAGTTCGCAGGGCG CGCTGGCTGTCAA

GAGCCGCTGTTGCAGTCTCGAGTCCAGGCTGTGCGCTCAGGCCGGGCTTCTCTGTGG CGGTCCTGGGAAT

TCCTCCTCCGGGCCCTTAGGCGACACTCCCCTGCCTTCAGCTGATTGGATGAGGCCC TCATGCCGTCTGC

TGATTTAAATCAATCTCATTTTAAAAACACCTGCACACCAAATTGCGATTGGCGTTT GACTAAACTGGGC

AGCGTGGCCCAGCAGCGCTGACACATGAATCAACCCTCACACCCCACCCGCAGGCGT GACCACGGCACCC

ACGCTGCCGACTGAGAACACAGCCGTGCGCTGACTCGCATGTGATGTCTCTGGTCGC ACCTCCTCCATCC

TCAGCACCCTGTTCAGAATGGAGATAGCGGCCGGGTGCAGTGCTCACACCTGTATCC CCAGCACCTTGGG

AGGCCTGGGTGGGCGGGTCACCTGAGGTCAGGAGTTCGAGACCAGCCTGGCCAACAT GGTGAAACCCCAT

CTCTACTGAAAATAGAAAAATTAGCCGGGTGTGGTGGCAGGTGTCTGTAGTCCCAGC TACTCGGGAGGCT

GAGGCAGGAGAATCGCTTGAACCCAGGAGGTAGAGGTTGTAGTGCGCGAGATTGTGC CACTGCACTCCAG

CCTGGGCGGGCGACAGAGTGAGACTCTGTCTCAAAAAAAAAAAAAAAAAAAATTAGA GATAACGACTTGG

GCTCTCTAGAGACCAGGGAGCCAGGCTCCAGGCGCTGCTGTCCAGCCCCTTGCCAGC TATACGAGGTGCT

CACCTGGCCACGTGTCCGGGCAGTGCTTCTGGGGTGGCATCCACTGGGAGGGCAAGA TGGTTCTGGTACA

GGTGCCCAATTCTGTCCCCATTTCACTTACTGGGAACTCAAGGCACAGAGAGGGGAG GGCTGAGCTAGGA

CCAGACCCCAGTCTCCTGCAGAAGTACACAGACATGATGTACGACCATCCTGGACAC CTGCCCTGAGATT

CCCCTCCCTCTCCTGCCCTGTCCCAGTGGCCTGGGGGGAAGGGGAAGCAAGGTTCTG AAGGGAGTGTGAC

CAGACACCTGCCCGTGACACCCCCTCTCCAGGCTGCCTCCGAGTGGCTGGTGACTCC CCTCCTGCCTGCG

AGGGAGGTGGCCAGGTTGCATTCCTCTCTGAGTGCCGGGGAAGTCCCTAGAGAGCAG GCCAGCCTGTGAC

TGGGCCCTGGGGCAGTCTAGACAGGCCAGACTGGACAGGCCAGGGGGCTGGGTGCCG CTGGGTAAATCAC

AGGGTGAGGGCTCTGAGTCAGCACCCCATCTTCTGTCCTGGGTCCAGCACCGCTGAG GACACAGTGGGCA

GCCGGGTCTGCCAGGGCCAGGTAGCTGTGTTGAGAAGGCAGTGCTCCTGAGAGGCGG CTACCGGGAGGTT

TTCAATGGCCAGGCTTCTTAGGAAGCCCTTGTTGCCTCTCTGGGGTGAGTTGCTGGG GCCATGGTTGGAG

TGGTCGCCAGTGTCTGCCCCTGGTGCCGAGGGCGGAGTCCTCGTTTTGGGAGGTCAC GGCATGATGCTGG

GAGTCAAAGGCAGGCCGTGGCAGGGGCATTCCTTTTTTCTTTCTTTTTTTTTTTTTT TTGAGACGGAGTT

TCACTCTTGTCGCCCAGGCTGGAGTGCAGTGGTGCGATCCCAGCTCACTGGGATCCC AGGCTCCCAGGTT

CAAGCGATTCTCCTGCCTCAGCCTCCCGAGTAGCTGGGATTACAGGTACGTGCCACC ATGCCCGGCTAAT TTTTGTATTTTTAGTAGCGACGGGGTTTCGTCATGTTGGCCAGGCTGGTCTCGAACTCCT GACCTCATGA TCCACCCGCCTTAGCTTCCCAAAGTGTTGGCATTACAGGTGTGAGCCACCGCGCCCGGCC TGGCAGGGGA ATGTCGACGCGTGATCTCTGCCTGGAGAGCACGTTCATGTTTCCCAGAGGACACTTTAGA ACATGGCGCC TGGGTTTGGATGAACCTCAGCCTAAGAATCTACCTGCTCAGGATCCAGCGACGCTGGTGG TGTGGACTTC AGCTCTGGAGAATGGGTTATATGGAACCTGGGCGCCGGGAGGGCATTGCCACGTGCTTGC TGCTGGGGCT TCAAGAGGACCCCATCTCCTGTGGCCGAGACCCCGTGTCTCAAGGCACATCCCCTTTCGT ACCCCGCCCC ACCCTCCGCAGCTTCATGACCTCTGGGTTTCCCCCAGGACCTTCGCATCTGATGTTCCCA GATCCTTCCT GCCACTGGGTCCTGCTCTGGTGCCCCCCGGGGAAGCCTTCCCTGAGGACCCAGCTCAGCA TCTGGTGCTA ACTGTGGCTCATCGTGCACTTTGGCCCCCAGGAGGCTGTGGGCTCCTGAAACCCCTCTGA ACCACGGGCT TCCAACCACACCTGACCCCCTGCGTGCCGACCCTGTGTGCAGGTGTACAGGTGTGCCTGG GGCAGGATGG CTGTGACGGCCTCACAGAGCCCGGAGAGCTGCCTCCTAGCTTCCAAAGCCTTCATTTCAG GAATCTTACC CTCTCAATTAGTCTGGAATGCTGGGGGCGGGGCCAGCTCCAGGTCACAGAGCGACCTTGT TTACCCAGAC CTTAACATCGGCCCTTCCATGCTAATCAATGTAATCATTGTGGTCCATGCGGCCTCTGGA ATGTGCTGTC CACTCCCTGGGTCAGCCAGAGAGTGTCAGGGAGCACCTACCGGCTGTTACCCAGTGCTGC CCTGACCTGG TTCTTCACTCTGCACATTTGTATCACGCCAGACCCTGGCTGGCAGCTCCAGGTGACGAGG CATGTCAGTG CCTTCCTGTTCTGTTCTGTTTTGTTTTGTTTTTTAAATCGGGATGAGGCCTTCCTGTGTT GCCCAGGTTG GTCTTGAACTCTCAGGGTCAAGCGAACCTTCTGCCTTGGCCTCATAAACTGCTGGATTAC AGGCAGGAGT CACCATACCTGGCCCACTGCTACTTTCTAGATGAAGAGACAGAATCCCAGAGAAGAAGCA GGGGTTTGGC TGCTGGTCTGGAGCCGGTTCTGCTCACCTCCAGCTTCTGCCTTGGGCCGCCCTGTTCACA CAGGAGCTGC TCACAGGCTGAGACCTCGAGCAGGGCCCTCCTAGAGGAACTGGGCCCCCGTAAGTGCCCT GAGCCGCCAG GAGCCGGCCCTGCGTCTCATCCCTATCTCCGGAGGACATTGGCTGCTAGCTCACCAGCTG GCCCCTGGGC AGGCTTGAATCATGACCTGGAACGCCAGGTGTCTCTGGCTCTACCCCTGGACCTGCACCC TGTCCAAGTG CCCCAGGGCCAGACTGTTTTGGTTGCACCCTCTGGACGGGCTACCCCCATGATGGCTGCT CATGGAAAGC TGTGGTTCTTAGGGAGCTGCCAATTCCTAGTCCTGCAGCCTGGAGCTCCTGGGTATAAGG TGGGGGCTCG GGCGTATTGGGGACTGGGGGTCTCAGGAAAGAGCCTGTGGGACCTGTGAACTCATAGCTG CTGGCCGAGG GACCACCTTGTGGCTGTCCTTCTCAGCTAGGCCTGGTCAGGGCTTGTGTGCAGGGCGGCT GAAGCTGTGG GAGGCCACACTGTCCACACAGTGCCCTGTAAGCCCACCGTGCCTCAGTTTCCCCGTCTGA CAAGTGGCAC AACAGAAGCCACTTCCTGGCACACAGCTCAGGGTCAGGGCCGACACAGCACTTGTGGGCT GCCGGGAGAC CCGAGAGGCTGCCCCTTCCTTGCCTTGGCTGCCACGGGTGACCTGGCAAACCCCTCTGGC CGTGGCACCA CTGGGGGTCTACCCTTGGCAGTCAGGGTTGGCCGCTTGGCTGGGAGCCCCTTCTCCTCCC CAGACACATC CTCTCCTTGGGGCTGGAGGGGGTCCTGCCGTCCCCGGGATTGTCGAGCAGCAGGAATCCA GGAGGGCAGT GCCTGCAGCTCAGATGGGGGCCAGTGGGCAGGGCCGATCCAAGGGTGGCAGGAAAGTGCC CATCACTGAC CTCAGGTGGGGGAGGCCATGGTGTGTGAAGGAAGGAGAGCTGAGTGGGAGGTCTTACTTT GTCACCGCCC CTCTGAGTGCCTGCTGTGTGCAAAGGCCAGCGGGGGCCCTTTCTTCAGCTGGGCTCTGCC CAGACCCCTG AGCTCTGGGTGGGCCGGGAGGGAGACCTTGCTGCTCACAGAGGTGGCTCTGCCTGACCCA GCTCCCTTCC CAGGGCACAGTGGGGGCATAGGGTCGGCTCCGTCAGACATTCCGGGACCTGCGTCCTTCC TGGGGCCACA CCCTCTACCCACTGTCCCCCACCTATTTACCTGTATCTGCGCCAGAGATGGCTGCCCAGA TAAGCCCTGG TTTCCTCCCTTTCTGGAGAGGCTGCGGGGGCTGGCGAGGAACCCACCTGCGCAGAGAGTC AGGGGATTGC TCTGTGTGGAACGCAGGCCTCACCCATGCCCTGGAATCTGTCCCCTTCTCTGTTGCTGAG GGATGAGTCC CAGATCCCTGACCCTGCAGGGGAGCCCAGCACAGAAAAACTCTGAGGCCTCCACACCCTG GCAGCGCTGC TGGTCGTCTGTGGGGAAGGACAGGCCCTGGGAGGGAGGGGGAGGTGCGGAGGGCAGTGGG GAGGGTCAGG AGGAAGTGGGGGAAGGGCCACCCAGGCCCCGATGTGGGGGATGTCTCACGCGTGGGGTGG GGCATTCTCA TCTCTGCTTGGTCTCCTGCCATGCTGGGGGTCGTTCACTGCGGACCCCAAGTACCATGAA GATGGGGATG GGATGCTGAGCAGCATCGGGGAGAACGCAAAGGCACCTCCCAGCTCACCCGCCCCCACCC CGCAAGCACA ACCATTGCCATGGTGTGGGGACCGGAAGGCGGCGGCTTTGGGACCAGACTGCTTCTGCCT CGGGCCGTGC CGCTGGGCCTTTGGTCAGCACCATCGTGCCCTGCAAGTCACTCTTAGCCTGGTGCCCTCC TGGGCAGGGC AGTGCCACAAGAGCTCAGGCCCAGATGTGCAGGTGCCACTGTTCCACCCACCAGCAGGTC ACCTGGGGAA CCCTCCCCTCCTGCAGCCTCTGTGTGCTCATCTGTGAAATGGGCGTGGTGGAGTCACCCC TGGCTTGTGG GAGAATCCCAGGGGCTGATGCCTGCAGGACCCTGTGGGCTTTGCCCCGCTCCCTGGGCAG AGACAGTTCC CCCAGTCCCACCCACGTGGCTGTGTGCAGCAGGTGCCTGCTGACCCTCTGTTCCTGCACA GTCACCTTCT TCACAGACGGACCCCCACCCTGCCCTGCAAACCCCTCCAGGGGCTGCGGGCTGAGTGTGT CCGGGAGGGT GTCCTGACTCTCCACGCCAGCAGGTCTGAGAGCAGATGGCTGTGGCAGGTGCGGTGGGTG CCCAGCCCAC AGCAGCCACCAGGCCTGCAGGACCCTGCCCCGTGTAGGTCAGATGAGCCATAAAACTGAG TTTCCTGGAC ACTGAGCTAATTAAACCTGGACACCGAGCTAATTAAATGGTCCAGAAGCTCCTCAGTGCC CCAGGCTGCT GGCCGGGCTCCAAGTAGGTGAGGAACATTCCGTTTACCTCCTGCTGCGTCGAAGGCGGGT GGCTCCCCTC GGGCCCCTGCCTGTCCCGGGCCCCCTGGGTGCTGCTGCGTCAAAGGCGGGTGTCTCCGCT TGGGACCCTG CCTGTCCTGGGCTTCCTGGGTGCTGCTGCCTCGAAGACGGGTGGCTCCCCTCGGGCCCCT GCCTGTCCTG GGCTCCCTGGGTCCTGCTGCCTTAAAGGCGGGTCGTTCCCCTCGGATCCCTGCCTGTCTT GAGCTCCCTG GGTGCTGCCTGCCCTGTGGCCACGTCCCACTGTTGCCTTTGGACAAAGCTCTCCTAGGGC CTGGGGTTCT CCCATGAGATGACAGGGGTTGGTTCAAAGGTCTCTGAGGTTTTTAGAGCTTTGTGAAGTG TTCTAGAATC

CATTTTCTCTCTCATTGCTGGACAGAGAGTATAGACTGGGCTTTTTCTTGAGTTTCT CTCAACTCTTCTT

TGTCCATGAGGCAGGAAGAGGGGCTTCCTCAGTCCTCAGCTTGGGGTGGATTGGGAT GGACAGAGAACCG

TGCAGGATCCCAGCCTGAGACGGTCCAGCGCCCCGGGGGAGCGGAGCCCGTGGCTCA GCCGTCTGTAGCC

ACGGCCGGGGTCACTGTCACTGAGGGCACGGAGGCCCCGCCGGCGAGGTCCGAGGTG GGCGTGTGGGGTG

GTGGGCGCTGGAGGCAGGACCTCTGCTTGTGGAGGGTGGGCCAGGCGTGGACCAGGT GTCACGTCCGCTG

GGGCCTTTGAGGGGCAGGTGGGGATTGGCGCGGAGAAGGAGTGAAGCAGCTGGGGTT TTGGGAGCCTGTG

CGGAGCAGGCCCGGATGCCAGGGTGGCTGGTGAAGGTGGGCCTGGCCGGGCTCGGCC TCCATTGGGGGAG

CCCCCCAGGTCCCCTCCCCACACAGCTTCCCCCTCTGTCTGCTGTCCAAGGCTCCTG TGCTGGCACTCTG

GGGTGGTAGGCCATGCAGCCCGTGTGAACCTCCATAGACCTTGCTGTGAACGCTGCA CGGGGCGTCTGGG

GGCGGGCTGGCTTCCCCCCGTCCCCTGGGCCAACCTTGCCAGCCTTCTTCTTCCATA AAAGTGGGATCCG

TCTGAGCCCTCATGGCCCCTCTGGGTCTGGGTTTGCTTTTTGTGCTCTGCCTGGGGT CATGGCAGGGAAG

GCCCAGCAGGCTCCCTTGCAGAGCAGGGCAGAGCAGCGGCCGCCAGGAGCTGCCTGT ACCACGTCTGCTC

TTCTTCCTCTCTTCCTCTGCCCGGCCCCGCCCACCGTCGAAAGCACTAGCACGGGAG TGTTTATCGGTTT

CTCTTCCAAGCCAAATAAGGCAGAGAGCGCTCTTGCAGGAGTTGGAGCAGGCCAGGG GGAGGGCAGCTGG

GGCTTGGACCAGGACCCCGGCTCCCTGTAGCTGCCCTGGCCTGGCAGCTGTCATCCG CGAACTCTGATCC

TCGCCTGCCCTCACCCGGCCCCTTTCAGAGCTTCCCATGCTCAGCTGGTAGCAAAAG AAGTCAAGACCTA

GCAAGGTGCCTGCCCCGGCCAGGAGGCCCAGGTTCTAGCCCTGCAGCTGCCGCTGTG AGCTCTGGGGACT

CAGACGACTGATGTCCTTCCCAGCTTCTGTTTCCTTTGATGAGAGGAAGGTTGGACC AAGCGACCTGCCC

TGCTTCTGTCTGTTTCCTCCAGTCCTTTTCCTTTTTGACCAGTTCACTTGTTGATAA CTCCAGCAACGCA

TGAAAACATTCCCCTTGTAAAAAAGGGAAATGCCGTGGGTAGGGGGGACCAAGGACC TCTTCTGCAGGAG

CAGACGCAACAGCTTGGTGTGGATCTTCCTCAGCCTTTTTGTCCATCCTAGACATGT GGAATTTCAGTTT

TACACAAAAGGAATGGTAGGCGTCTGACTCTCTGTGGCTTGTATCACTTGGCAGTGC CACAGGGATTGGT

GCATGTTTGCTCTCTCATGCTATGTGTCAGGCACTGTTGTCTCTGCGACAATCCTGT GAGGAGGGAGTTA

TGGTTCTTGATTTATAGACGAACAAACTAAGACAGAGGAGATTGGGCTCATCTGTGG TCATCTCAGCAGT

TGTGAGAGGTCAGGGCCAGAGCCCAGTGGTGTCATTCTGCAGGGCACATACAGACAC GACCCACAGGGTT

CTGCCGCACCCCCTGCCGCTTGGCCTGCTCTGCAGGTGGGTGCCGAGGCTGTTCCTG CATCTGCTGTGTC

TTGTGAGTCCGTGGGTGCCTGTGGAGTGGGGGCAGTGGGTTTGCATTTCCAAGCACT GCCTCTGTCATAG

GCTCAGGGAGAAGCAGTTACGCATGAGCTGCTATGAGCCACGGGTATGACTGGCATC ACAGCAGGAGGAG

CCCTGGGGATGGCTGAGTGACGATGGGTGGGGACAGAGCTCACAGAAGCCAGGGTTG CCCCCAGGCCAGG

CCACTGCTGGGGCCAAGGCTGGGATGCAGCACGATGTTGGTCTGTCAGGGAGTGGCA GGAAGCCCACAGG

GTCAGCTGACACTTAACCCAGAGTGGTGGGAGCCTTGAATGCCAGACTGAGGGCCAC ACAGTTGGTCTCG

GTGCACAAGGACTTGGGCTGAGAGGGCTGTTGCTCCTCAATGGCCATCTCCTTGTGG GAGGAGAGTCCGG

GCAGCCGCTTCCAGGAACCCACCCATGCTTGAAATAGTGGGACCACAGGGCTGAGGA GGCTCCTGGGGAT

TCCTCCCAGGATTCAGCTGCTCTGTCCCCAACCCCGGAGGGCCTATTCGTCTAGCCC TATATCCTCTCTG

CCAGTTAGGTCAGGGCAGAGAAGTAGAGGCACAGAGGGTGGCGGAGGGGTGTCCAGC CAGGCCTTTCTGT

GGCACCTTCTGAGGCTGGCTGGAGTCTGTGAAATCTGTGATAACTGGCCCAGACCCT TCCTGCCTCCTGG

GGCATCTGCTGGAGCTGAGGCTGCTGGGGGAGTCTGCCTGTGACTTGAGTCTCTTGC TGGGCCATGCTGG

GCTCAGTTCGCCTGTCTGTGGGGTGGGATGGTGCCACCCTTAGGTTGTTGGGAGGAC CCAAGGAGAGTGA

TGCCTCCAGCCATGGCAGCTGGCCCAGCTCCGGCCCGCAGCCTGGCTCCTTCAGGGC CAGGAACCCCCAG

GTCATGGACTCCAACCCTCGGGCTCTCCTGCTTCCCAGGTGACGAAGCTTCAACCCA TGGGCTCTCCTTC

CTGGGTGTGGAAGCCCTGGAGAAGGTGGAGATGGGATCTCAGTCCAGCCCATTGGCT CCACTTCCTTTGA

GAAGCTCTTTCCCCCGTCTTGGCCCCCAGCCCTTGTTCAAGCACCTGTCCCTCTCCT GTCTCCTAGGCCC

CGTCAGTCCTTTTGGAGACCTGTTCCCACCCGCCCCTGCTCTCACAGGCTGCCAGTG TCCACACACTCTC

AGGCACTGTCATTGGAGCCTTTTAACCACACGGGAAAGCAGGCAGGTTAGGTAACTA CCCCCCACCCCCC

CGCCAGCAACGCCCCCTGCATACCCAGGGCCTGCACCAGGTGCTGCTGCGCTGCCAG CTCTCTCGAGGTC

CCTCCTCCTAACCTCAATGCATCGCGTCTTCCAGCCCCCGGCTCCGAGGGCTCAGCC TCCAGGTGGTCTA

ACCTGGAGTCTAAACCTGGATTGCCATCTGCCTGCCTCAGCCCTTCCCTTCCCCAGG GACCGAGGGCCAG

TGCGGCTCCCCAGCCACCCATTTTTTGTTTGTTTTGAGACGGAGTCTCGCTCTGTCG CCCAGGCTGGAGT

GCAGTGTCCTGATCTCGGCTCACTGCAAGCTCCATCTCCTGGGTTCACGCCATTCTC CTGCCTCAGCCCC

CCAAGTAGCTGGGACTACAGGTGCCCGCCACCACACCCGGCTACTTTTTTGTATTTT TAGTAGAGACAGG

GTTTCACCATGTTGGCCAGGATGGTCTCGATCTCCTGACCTTCTGATCTGCCCGCCT CGGCCTCCCAAAG

TGCTGGGATTACAGGCATGAGCCACTGCACCTGGCTTGTTTGTTTTTATTTTTATTG TTTTTTGAGACGG

AATCTTGCTCTATTGCCCAGGCTGGAGTGCAGTGACATAATCTCAGCTCACTGCAAC CTCCACCTCCCAG

GTTCAAGCGATTCTCTTGCCTCAGCCTCCCAAGTAGCTGGGACTACAGGCACCCACC ACCACACCCAGAT

ACATTTTGTATTTTTAGTAGAGATGGGGTTTCACCATGTTGGCCAGGCTGGTTTCGA ACTCCTGACCATG

AGATGGCGGGCTTGAGCCATCTTCCCGCCTCGGCCTCCCAAAGTGCTGGGATTACAG GCGTGAGCCACGG

TGCCCGGCCCCAGCCACCAGTTTTGTACAGCACTCAGGTGGGTGTGGTTCCTGAGGA CATGTCACACGAG

ATCCTTCCGGTCCATTAGCCCACCCCGCCCCATCAGTGGCCAGTGCGGCCATTGTGG GGTCCAGAGTCCC GAGGCAGAACGACTGACTCCAGGTTGTGGAGGGGCCTCTTAGGATCCTTCTAGGCACCCA GGTTGGGCAG GTGCCTCCATGCCTAAGACTGAGCTCCTGGTGGACATCGGGCTGGGTGTGGGCGTCCTGC ACACCCTCCC TGTGGCCTGGTGCCCGTGAGGAGTGGACAGTCACCCCGAGAGCATAGACCACCCTCGAGG CTCCCAGATG GCGGCCGCAGAGCCCCACTTCCCTGCCTGGTGTGTGATACCTTCCATGGCTGAGGACACC TCTGACTGCT CACTCCTGTGACCCTGGACCCAAGTGCTGGGCCACACCTGGACTCCAGGCCCCAGTCCTT CCTTCTTCTG CTGTCCTGGCTCCTGGGTGGCCCCCGCCCTCCCCGCTCCGAGGCTCTGCCCTGTGCTGGG AACCCAGGAA TTGCCACCCCCTGGCCCCGGTTTCTCATGTCAGGATCTGCTGGCCAGGTTGACTCCTGAG CGCCACTGTC TCCTCAGACTTGCTGGTCCCACCCGGTCCCCACCTGTCCACCGCCCGAGGCTCTCCTGTG AGGCCGCTCT GCAGCTCTTGGCCTGGGGGCCCCCGGCCTCTCATGGTGCCTTCTGCACTGTCCCCTTTGG AGATGGGAGC TTTGAGGGTAGGCGCCACCGTTCGGTGAAAAGGCGGTATAAGCAAAGCAACCCCCAGATA CTGAAGGAGC CGGGAAATAAAAGGAGGCAGACAGAGCAAGTGTGTTGGTACCCGCCTATTTACTGGCAGG AACTTACAGA CGGAAACATGGCCTCAGGCAGCCTCCAGGCAGGTAGAGCTCTGAACCCAAACGCCCGATC TGGGGCTCGT ATCGTGCCCTGGGAGGAATGTGCAGGTGGCTGGGAACGTGGCGGGTGGGGTGGCCAGATT CCTGCTGCAG CACCGGGTTTGTTTAGGAAGAAACGTACGAGGACTAGACACTCTTTTTTTTCTTTCCTGC ACGTGAATGC CAGGGAGTAGGTGCTCCTGCAGGAAGATGATGCATCAACCAGCCAGTCTGGAGGCATTCC GAGACCCGGG GTTAATCAGCAGATTAGCATTTAAATAAAGTTACTCTGTCCCCACAGCCAGCTAGGCCCG AGCCACATGG CGGGGGCGGCGGGGGCGGCTGTTCGCCGCATCCCAGGCTTGCTCCATCTCCTCGTCTGCA ATGTGGAGGT GACAGTCGTGGTCCTCGGCTAGTGCCACAGGACTGTACGGGACAGATCACCCAGCCCCGT GCCTGCACTG GGGGCGGCGGTGACGTGATGGCCGCGGCGGTGACCTGATGGCCGCAGGTGTGTCCACCCT CTGGTTAGGC CTCTCCTCTCCCTGGGGCGCAACAGGTGTCCTCGGGTGGGAGGTTCAGCTGCTGGGCCTG GGGCCCCAGC TTTCAGCTCCTGTGCAGGCGTGGAGCGGGACGGCACGCCGGGGCGTTCGTTCACACGATG CCCCCAGCTC GTCTCTGCCTGCTCTGTGATGCAGCCTCAGCTCCTGTGGGTCCCGGGTCACCCACCCGTG TGGGCCGCAG TCTTTCTGCACCACGTCCTGCTCTCGGCCAGCGCTGGCCTGCGGGCCCCGGCAGTGTGGG GTCACACACA GGCCGCACCGGCTGTTTCACGGAGCCCCCCTGCTCAAAATCCTGGCCTTGGGTCTCTGTG TGGCTCCTGG AGGGAGCATGTATGAGGTGTCCAGGTTGTGAAAGCCCCATCCGGCAGCCCTCCCTGTGAC AGTCCTTCAG GCCTCGTCAGGCTTGGTTCAGCCCGTGTTAGAGGAGGCCAGACAGCTCCTCCTCGATCAC CACAGGGAAC GAGGGTTGGGCAGTTTCTCCTCTCCCACCCGGGCGTGGGTCAGCTGCAAGGGTCTCATCC CAGGAACTGG ATCCTGGCAGGGGGGCTCAGGCATCTGACATCCCCTGGCAGGTCTTCCTATGTGCCAGTG GGAGTGGGTG GTGTCCAGCCCTCTTTGTGGAATGTTCCGGTTGAGTGGTGTGTGGTCCTGGTTACCACCA TGTGGCATTT GTGGCTTTCCTGGCCAGCACCCACTGAAAGCAGAGGCCAGCCCAGCCCTTGGACACCCAG GGGCCCAGCA GCTACCGTGGGGGCAGCCTCTCCCCTCCTGACTGTGCTCCTCTCAGCTGTAGGATGAAGG TCTGTGGATG GGTGATGGCTGGGCTCTCTGCTTCCCAAGGAGCTGGATGTGGGCCATAGTCAGGCGTGTG CTGGTGAGGC GAGGGGGTTGGGGGTTGGGAACCACAGCTCTCTCTGACCCCATCCCATTGCAGCTGACTC AGGAAAAGGC ATTACCCCCACGGGGTCACAGGCCCAGCCTGGGCAGCTCCTGATGGGAACATGGGAGCCA CAGGGCAGGG CAGGCTCCTGTCCCCGCTCCTTGGATTGTCTCAGGCCCCGCCTCCCCTTTGGCTGAATTT CTTCCTTCTC TGCCTGAGCCTGATCTGCGAAGCTTCCGACCTGCCCTGAGTTGGAGCCAGTCTTCAGGGC CAAAAGCAAT GCCGTCTCCTCCAGGCAGCCTGCTCTGAAGTCTCCCCCTCTGGCTTTAGCCCACAAAGTC ACAGCTGAGG CCACATCAACACACTTAGACCAAGAGCACTGCCGGCTGCATAGACAGCACAGGTAGATCC AAGGAGTGTT TCGAAAAGCCGTGGCCCTGCGGCCGGGATGCCCGGACCTGGGTCCTGACCACTCTTGGGA GCTCCTCCCC TCTCAGAGCCCATCCCATCTGAAAATGGGGGCATTGGAGCATTGGAGGAGTGCTCCTCAC TCCTGCCCTG CTAGCCGGCTACTCCGAGGACCCCAAGACCAGATGATGGGAGAAGTGGTGGGCTGGGCTG TGACCCTGAG CCCCCTTCTCATCCCACCCGTGAGGCTGCCCCTGCTTGGTGTCTGTGGCAAGGCTGACAG AAGATGGCGC CAGGGACCAGCCAGGGCCCTCCTGTCTCAGCCCGTCCTTGGGTGAGTGGGTGTGCTCTGG GGGTGGCAGG TGTGCTCCGGGGGCGGGAGGTGTGCTGCGGGGGGCCGGGTACGCTCTGGGGGGGCGGGTG AGCTCTGGGG GGCTGGGTGCGCTCTGGGGGGGCTGGTGTGCTCTGGGGATGGACAGGTGTGCTCTGGGGG GCGGGGTGTG CTCTGGGGATGGATAGGTGTGCTCTGGGGGGGCGGTTGTGCTCTGGGGGGGCAGGGGTGC TCCGGGGGTG AGTGTGCTCTGGGGGGCGGTGGGTGTGCTCTGGGGGCGGTGGGTGGGCTTTGGGCTAAGT AGCCCTCTTC CTCTCTGAGCCTTTTCCTTGTCTGTGTTGTTGGTGAGTGGCCGCCACCTGGGACAAGCCT TAGCTGTCTG TCCAACTGTCCACCATGTCCCCGGACCCTAGACCAAGCCCGCTGCCTGGTGGGGCCTGCT TGGTATTGAA TGAATGAATGAATGGATGAATGGATGAATGGATGAATGAATGAGTGAACAAACTCTGGGG GGCGTGTTGC TGATCACCCCAGGGCAGGCCCGTGGTGGGTCATGGTCTCAGGCAGGGGACTTGCCCTGCC GGGTGACGTT GGCCTGCCGGGGAACCCCTGCCAAGGTGCCTCCCATGCCCCCATCGTCCTCTGCCTTTGA GTCCACTCTG CCCCTCAAGGTTCCCCTCTGGCTCGGCCAGGGGCAGGGCGGTCAGGGAGGCCTCCAGCTC CAGCCCCTCC CTCATCTCCATCCTCAGCCCCTGCACCCGCCTCATCTCCAGCCCCAGCCCCTTGCAGCCT CAGGCTCGGG CTTCTGAGAATCCTTTGCAGATTTCAGGGCTGGTTATTTTTTTATTCTGGCAAAATATAC ATCCATAAAG CTTTGTTTCCTGAGATGATCTCACCCTGTCGCCCAGGCTGGAGTGCGGCAGCGTGATCAT GGCTCACTGC GGCCTCAGCCTCCTGGGCTCAAGTGATCCTCCTGCTCCAGCCTCCTGAGTAGCTGGGACC ACAGGCATGC ACCATCATCCCCGGCTAATTTTTGAATTTTTTTGTAGAGATGGGGTCTCTGTGTTGCCTG GTCTGGTCTC GAACTCCTGGGCTCAAGTGATCCGCCTGCCTTGGCCTCCCAAAGTGCTGGGATTACAGGC CTCAGCCACC ACACCCGGCCTAAAGTTTGTCACGTTAGCCGCATTGAAGGGCACAGGTCAGCAGCGGTAA GCATGTTCGC

ATTGTTGTGCAGCCATCACTGACACCCATCTCTAGAACTTCTTCGTTTTCCCAAAGG AAACTGCACGCAC

GGCACCACCTCCCTGGCCTCCCCACAGCCTCAGGGTCTGGTGACCTTCATCCCCAGG ACCTACGCTTCTG

ACTCCAGGAGCCCTGCCTGGTGTCCACACCACACTAAGGTGCCCTGCCACGGGCTTC GAACCCAACCCTG

GTGCACTTGTTGAGCATTGGACGTTCCTGGCAGGTCAGACCGTGAGCTCCTCTGCTC TGACGGATGCATG

GACGGCTGCCACCTGGCTGGACAGGTGCAGAGGGGAGGTGCCACCTTCTCTGGGGAC AGGACTGGGGCAG

AACTCACCCGGCTGAAAAGTGCAGCTGTGGAGAACTTCAGAACTTCAGAAGCCTTTG CTGTGAATGCATT

TTCCTCCTGCCTCTTGGGCCAGATCTTAGGGATTTTTTTTTTTTTTAATGAAAATGT ATAATCGTCAAAA

AACTTTAGATTTAAAAGCATGGAGCCGATGGCTATGCTTGGTGCGTTAATGGGAGGA AGAGGGGATCTTA

ACTTCAGGGAGGGCTCCTGGGGGAGCTGGGGGAGGCTGCTGTGTCCAGCAGGGCGGG CGGTGCCCCCCGG

AGCCCGGCACTCCGCGATGTGTGCGCTAAGCCGAGGCCGCCTTGAGCCCGTAGCAGC GCCGAGCGCGATT

CTTTCGTGTCTGCTTCCGGGAGGGTGGAAGGGTGAAGCTGTTGAGAGTGGGAAGGGA GGGGCTGTGCTTC

TTGAGTTTGCCGTGTGCCCCTGACCCTTTCAGGTGCAAGCGTCAGCTCCACGGTGCC AATGGGGAGATGG

GTCTGGAGGCCCCACCAGGCTGTGAAAGGCCCGGCCTCCTGTGCTGTCCGTGGAGGT CGGGGTCTCCCTC

TGCCCTGCGCCTCCTCTAACTTGCCCTGCGTTTTCCGTGCCCCTGGTCTAGGGTGAG GCTCGGGTCTTGG

GGACTGAGGGGCAGGTGGGTGTCAAGGTGAGAGCTGGGCTGGGCTGGGCCTCCCTCC AGAAGACTTCCCG

GGGCTCCTTTGGCACCAAGGATGGGGCAGATGGTGGCACGCATGGCCTCCCCTGGGC ACAGAGCCAGACT

ATGGAGGGATCTCGTTGGCTACATCCTGGCTGGGGCTTTGGGGTCATGGTGGGAGCA GCAGCCAGGGGTC

TCGCTTGGACACGGGTCTGAGCTCACTGTGGCCTCCTTCCTCAGCCTCCTGACCGGG GTCCTGAGGCTGG

AGGGTGGGTGGAGGGTGGAGGGTGGGTGGATAGTGGGCTGGAGGATGGGCTGCTGGC GCCCCTTAGGCAG

CATGAGGCGCCTTTCCAGTCCCAGTGTCCTCCCTGGTAGCCCCGGAAAGGCTGAGTG GTCCCTCAGGTCC

CCTGCGAGGAGACGACATCAGGCCCCACCCCTCATCTCCTGACAAACCTCACATTCC TAGGAGGAAGCAG

GCCTGGCTAGCACGTGGCAGGCACAGCCTCCCTGACTCCAGACCTTATCAGATCAGT GCAAGTGCCAGGG

AGACCCCGGGTGGGAGCTGTGTGGTTGGGACTGATGCAGGCACTGGCCGGACTCTCA GGATGAGGGTGGG

AGGCCAGGCCCCGGGACCTTCTGGGACAGGCAGGAGGGCAGACACGGGACATGGTGT GGGGGCTGTGGCG

AGTGCGGCCACGCAGAGCATTTGGGCTGAAGGCGCGCTGTGTCCCACTGCCCCATAC GTGTGCGGGGGCT

CCCAGACACGTGCTGGGGGCAGGAATCATAGCATTTTTTTGTTTGATTGTTTTTTGA GATGGAGTCTCGC

TCTGTCGCCCAGGCTGGAGTGCAGTGGCTCAATCTCGGCTCACTGCAACCTCCACCT CCCGGGTTCAAGC

GATTCTCCTGCCTCAGCCTCCTGAGTAGCTGGAATTACAGGTGCCCGCCACCACACC CAGCTAATTTTTG

TATTTTTTTGTCAAGACAGGGATTCACCATATTGGCCAGGCTGGTCTTGAACTCCTG ACCTCAGGTGATC

CTCCTGCTGCAGCCTCCCAAACTGCTGGGATTACAGGCGTGAGCCACCACACCCAGC CAAGAATAGCATT

TTCTTTCTTTCTCTTTTTGAGGCGGAGTCTTGCTCTGTTGCCCAGGCTGGAGTGCAG TGGTGCCATCTCA

GTGTAACCTCCGCCTCCCGGTTTCAAGCAATTCTTCTGCCTCAGCCTCCCGAGTAGC TGGGATTACAGGT

GCCCGCCACCATGCCCGGCTAATTTTTGTATTTTCGGTGGAGACAGGGTTTCACCAC GTTGGCCAGGCTG

GTCTTGAACTCCTGACCTCAAGTGATCCGCCCGCCTTGGCCTCCCAAAGTGCTGGGA TGACAGGTGTGAA

CCACTGCACCCAGCCCAAGAATAGCATTTTTAAAGGAAAAAAAGCAAGGTAGAAAGA TGGGTCAAAACCC

CAGCGCCCCGCTGGGAGGCTGGTGCCCGTGTCGTGGCGTGTGTGAGATATGTACGGA AGCGCGAGGCGGC

TTCCACGGCTCTGGCTCAGGGCAGCACTCACAGGGCACACGGCCTCGGCGGTGTGAC GAGCTGGGCCGTG

GGTGCGCTGTCCTCTGGGGTGGGGCTGGCCATCCTCTGTTGGCGACCTAGTTAACGC CCATCTCCCCGCA

GCCTGCCTGCTCCGCTTCAGCGGACTCTCGCTGGTCTACCTGCTCTTCCTGCTGCTG CTGCCCTGGTTCC

CCGGCCCCACCCGATGCGGCCTCCAAGGTAAGGCCAGGGGACCCTGCCCACGCTCTC AGGGTGGGAGGGG

GTGGGCATTCGTTTGGGGCCAAAATTCGTACAGCTTTCCTCACCTTTAACAGCCCGG GGTGGAAGATGAG

GGGTCCCGAAATAAAATTTTTTTTTTTTTTTGAGACGGAGTCTCGCTCAGTCGCCCA GGCTGGAGTGCAG

TGGCGCGATCTCTGCTCACTGCAGGCTTCGCCTCCCAGGTTCACGCCATTCTCCTGC CTCAGCCTCCCGG

GTAGCTGGGACTACAGGCGCCCGCCACCACGCCTGGCTTATTTTTTTTTGTATTTTT AGTAGAGACGGGG

TTTCACCGTGATAGCCAGGATGGTCTCGATCTCCTGACCTTGTGATCCGCCCGCCTC GGCCTCCCAAAGT

GCTGGGATTACAGACGTGAGCCACCGCGCCCGGCTTTTTTTTTTTTTTTTTTTTTTT TGAGACCGAGTCT

CGCTCTATCTCCCAGGCTGGAGTGCAGTGGCACAATCTTGGCTCACTGCAACCTCCG CATCCCAGGTTCA

AGTAATCCTCGCATCCCAGCCTCCCAAGTAGCTGCAATTACAGGTGTGAGCCACCAC GCCCAGCCATCAA

AGTTTCTTATTTTCATTGTAGACCCCAAGCTAAGCTCAGAATCTTTTCTGATCCGTG TTGCAGGGGCTGT

GAGGGGCGAGGAGGGTCATGGGGGTGGAGTCTCTGTGTCCCTTGGGAGGCCACAGGG AGTGGATGGGCCC

CAACTGCCCACTCTCTGCAGGCTCCAGGCACGTTTCCTGCCTGCTGAGGCTCTGGCA GCCTGGGAGTTCC

AGCCGCCTGGACCGCCCTCCCGCTGCCCGCTGTGGGGTTGGACCTCAGTTGACTTTG ATCCACTTGTGCG

GGGGGGGGGGGGAGCTGAGACCCCCCTTGGGAACCACCTTGGGGTCTGAGCTGGGCC AGGACTGTGTTGC

TCTCCCGTATCCTCCAAACAGAGGGCGGTGAGGCCACGTGCCAAGCCTGCAGCCTGG GTGACGAGGGTAG

CAGCAAACCCTCCATCCTGTGACATGTCACCTACCTGCCCTGTGGGGGCCATGTCCA TTTTGTGCCCACG

TTTTAGGAGAGCGCGTTTTGGAGCAAACACTCAGAGACCTGTCCCCAAAGGCCATGG GCCAAACGTGGGT

GGGGCCGGGCCCTCCGCTGGTCTTTGGGTTTGGAGGGTGAAGAGCCACTGAAAGGAA GGAAGGTCCCCAG

GGAGGGCAAGTGTAGGCAGCAAGAGACCTTCCCCAGCAGAGGGCAGAGAGGAGGCCA AGCAGGACCAGGG GGCGTGACTGTGGAGACAGGCTCTTGGGTGCCGCCCTTCTGGGAGGTGCTGGACGCATCA GGCTGGCAGC

GTCGGGGCCTGTCCATGGCTACGAGGTGTCCACCGGGCTGCGTTTTTCCAGGGGACT CTGGGGAGGATCT

GCTTCCTCTTCCAGCTGCTCCAGGTGCCTACCTTCCTGGGCTTGGGGCCCCTCCCTC CATCTCCAGAGCC

GGTGGGGTGGGGTCAGGATCACACCCTGTCTCTCCTGCTCCCTCTCCCCTGCGGTAA CCCTGTGGCTTCG

TCCCATCTGCCTCCCCGGCTAATCCAGGCCGACTTCCCTGGTTTAAGGTCAGCTGTT GGGCAGCCCCCAT

TTCCTCTGCTGCCTGGACTCGCCTCTGCATGTGAGCTTCTGCGTTCACAGATTCTGG GGACTGGGGTGCA

GGCATCATTGGGGTCCATCGTTCTGCCAGCTGCAGGTGGAAGGTCGCTCTCCGGCCT CGCGACATCTGGC

TGGAAGCATCCCAGAGGCTCCTCCTAGCTCGTGGTGCTGTGGGGTGGGCAATGGTCT TTTTTATTTATTT

TTTTTGAGATGGAGTCTCACTCTGTCACCCAGGCTGGAGTGCAATGGCACGATCTGG GCTCACTGCAACC

TCCGCCTCGCGGGTTCAAGCGATTCTCCCGCCTCGGCCTCCCGAGTAGCTGGGATTA CAGGCGCCCACCA

TCATGCCTGGCTAATTTTTGTGTTTTTGTAGAGACAGGGGTTTTACCATGTTGGCCA GGCTGGTCTTGAA

CTCCTGACCTCAGGTGATCTGCCCACCTCGGCCGAGACTACAGGCATGAGCTACCGT GCTTGGCTTTTTT

TTTTTTTTTCGAGTGGGGGTCTTGCTTGGTTGCCCAGGCTGGAGAGCAGTGGCATGA TCATGGCTCACTG

CAGCCTCCACCTCCTGGGCTCAAGCGATCCTCCTGCCTTAGCCTACCAAAGTGCTGA GATTACAGGTGTG

AGTCACTGTGCCCAGCGCAGGCACTGATCTTGGATCATGGGTAGAATTCAGGGGTGC AGAGAGGCATTTT

GGGAGGGGCCCAGTGCGGCCGAGTGCTGGGAGTCGCTGGGGCCCGAGGCCCAAACCC CTCCAGGATGCAT

GGCTGGGGTGGGTGGGCACCCCCACTCCCGGTCCCTATCCTGGGCCTCCCCTTTGCT CTGTGGAGCCGGT

TCTGTCTGTTCCCAGGCCCCTGGCGTTTCGCCGTTTGTTCCGTAAATATTTCATGCC CAGGGCGCAATTG

GAAACACTTTCCTTTAATCAGCAGTGGGGGAAGGCAGGCGCCCAGCCAGGCCAGGGG AGGAGCTGGGGTG

GGAAGATTTGGAGAGGACCCGGGAGGACTTCCCTGCCTGAGCCTCGTCAGAGGCCCT TCAGAGACGGAGA

TGCTGCCCAGTTTTCCGGGAGGGAGAGAGGAAAGTGTGAGGCTTGCCCGAGCCGAGT GCCCGGGGCTTTA

TGATTTGTGCAGCTGCTGGGCTTGGCGTGGCCCTGGTGGAAGCTCTGACGCCATCTG AGCCTTGGTCCCC

TTGTAGGCGTCAGCCTGATCCTTGGGGGGCTGGAGGTTGAGCTCAGACTGAACAGGG AGGAGAGGGGTAG

GGGCTGGGGCTGGGGCAGAGGGAACAGACCCTGGTGCTCCAGGCAGGGCTGCAGGCA GAGCCACAGGGGG

TGGCTCCCAGAGACCTGCTTGTCATTTAGGGACTCAGAAGCCCCATCCTGCCTTAGG ATAAAACCCACCT

CCAAGACTGACCCTGCTGTCTGGAGAGAGATCACCCCCCACCTCTTACCATGCCCCG TGCAGGGAGGCCC

TGCCACCCTCTCCAGCTTCACTGGTCCCCCGGCTCTGCCAGCCTTGTGTAGTCTCCA TCCTCTGTCTCAA

AAGGCCCCTCTCGTGGCCCTACCCCGCAGCTCAGCCCTAGCTGTCGCTGGGTGGGCC TCATGGCTCAGCC

TCAAGGCACCCTGGGTCCCTTGGCTGGTCCCATGCACACCCCATGGATCAGGTTCCA GGGGGCTCCCATG

CACACCCCACGGATCAGACTCAGGGCAACTCCACACTCCCCACAGAACAATCAGGCC CAGCCCTTGTCCG

GCAGGTGTGGGGATGGTGGCTATGGACCACGCATCAGGATGCTGCCGTGGTGGGGCC CCTCAGCCTTTGC

AGGCCGTGCCGGGGTCCAAGGGCTCCGGCTCTGGGGGAGGTCCATGCTCTGTCAACC ATCCGGTCTGCAA

TACCCTGGGGCCTCCCCGCCCCTTCCTGTTTCCTCCTGAGAGCCCTGCAAGCTGGAG GGGGAGCCCCGGC

CCCTTTTTTTTTTTTTTTTTTTGAGACGGAGTCTTGTTCTGTTGCCCAGGCTGGAGT GCAGTGGCGCGAT

CTCAACTCACTGCAACCTCCGCCTCCTGGGTTTAAGTGAAAGTGATTCTCCTGCCTC AGCCTCCTGAGTA

GCTGAGATTACAGGTGCCCATCACCACGCCTGGCTAATTTTTGTATTTTCAGTAGAA ACAGGGTTTCACC

ATGTTGGCCAGGCTGGTCTCAAACTCCTGACCTGAGGTGATCCACCCACCTCGGCCT CCCAAAGGTGTGA

ACCACCACGCCAGGCCCCCTGCACCTTCTTCTGGGTGGGCAAGAGCCATGCTGAGCC CCTCCCCTGCCAT

GGATAGGGGTGGCAAGAATAGTCCATTCCCCATGGGCTCCCTCCGGCAGGCAGCGGA TGGGTTGGGGCCC

ACACAGTGGTTCAGGAAAGGCTGAAGGGCGGTTGCCGGCCAGAGCTGGGCGGGTCCC TGGCCTGAGCCTC

CTCTCACTCCCTTTCCCAGGATGGAAAGGCCACTGTGAGCTGGGTGTGTCCTGCCAG GAGGGAGTTGGAG

TCGGGGGCAGGAGACCCACTGGGTCTCCTGGGCTCCTGGAAGAGGCAGCAGTGGGTC CCTGCGGAGGTGG

CCTGATCCCCGGCTCAGCCTGGTTTTCCCAGCTTTGCATTTGGGGGTGGGACCTGCA GGGGGAGAGTGAC

GGGGTGGGCGGGGACAGGGGCTGCAGGTGGAGGCACGAGAAAGCCACCCGACCCCTT GAGGTCATGCTGT

TTGCGTCGGAGAAGAGGGCCGGTGTCAGGGTGCCCCCTCGGCCTCTGTGTGCACCTC CCCGCTCCCCGCC

CCGTGCAGGTCAGCAGAGCCTCGATCCCTCTGCACGTTCCGGCCCCCTCCCATCTCC AGCAGCGTCTCTT

CCCGGGGTGCCCCTCGGACTCTCAGCTCCCTAGTCTGATCCAGCCCAGGCAGGGAGC GGGCGTGCCACGT

GGCCAGGAGTGGCCTCCAGGCCCAGCTGCGGCCCCTTCGACCCTGCGGAGGGATCAT GGCCCAGCTGTTC

TAACATGGCCAGACCAGGACACTCTGGCCGGCCCGAGAACTGAACCGGGAAGGAGGC AGGGAAAGGGGAG

GGAGGAAGACAATGGGGAAGCAGTGACATCCGAGATGTAGCCAGAGACGGACATCCT GGACTGTCGTGCA

GGGCAAGGCGGGTGGGCGGGAGCCAGGTGGCCTGAGAGCCCCTCCCTGCCCAGGGGT CTCTGGTCAGGCA

GTTCCTTCCCGGGCTGCTGGATCGTGTGTGCAGGGAACCCGTCAGCCTGGCTGCCCA AGGGCCCAGATGT

CCTCTCAGTGCCCGGGGGTCCTTGACAGCCCCAGCAGGAGCCCCACGTGCCTGTGGG GCAGGCCCCGCAG

GTCCCTCTCTGTAGGACTCAGAATACCTTCTCCAATGCCACGTGCTCTCCCTGAGTG CCCAGTGCCACAG

AGGGCCCCGCTGGCGAGGTTACTTCAACGTCAGGCTAGAGGGTGCAGCGAGCAGGAC TCACAGCCCAGGT

TCCCAGGCAGGTGTGGCAGGAGCCCATTCCCTGGGTCGGTGTTCTCATGTCACCCCA CGGTGACCCTGCT

GTACAGAGGCGGGGGCGCGGGGCCCCTGGCTGGTGCCCTCCTGCTGCCAGACTTGTG CTCTGCTGAAGAG

GGGCTGGCGCGGCAGGTATGAGCCCGGCACGGAGGTCGTGAGCAGTGAGAAGCCTGG CACCTATGGGTTC

GGGGCAGGGAGGCCCTGGAAGGTCCTGTCCTCCGGGAGCCCTGCACAGCACCCCCTG GTGGTGGCTCCTG TTCGTGTCGGGGCTGCAGCCTCCCTTCCTGGACGGTTTTCCATTCCTCCTTCCCACTCTC CCTGCTGCTC

ACCCCTCCCATCCTCCACAGGTCAGAGGTCATAGCTGCAGGGTCAGCAGGTCAGGGC CTGGGGCAACCCT

AGGAAGGTGTGAGTGTGAGAAGCTGGTCCAAACCTGCCTCGGCTGCCTGGTGGTGCG GACAGGGAGTCCT

GGGCATCCGTGAGGGCTGCTTCACGGTCAGGCTTAGGGATGTGCAGGGTGACTTGGA CGTGGGTCATGAG

TCTTTGCTCCAGAAAGAGGGGGCTGAGTGTCCAAGGCCAATCCCGAGTCTGTCACCT AACACCATTTGTG

CTCAAAAAACTGAACAGAGTGGACACAGGCCCTGAGTTTGCGCCCCGAGGCCGAGAG AGGGCAGATGTGG

TGGTGACATTCACCACCTTGGACCCAGACCCAGATGCTACCTGTCCCTGAGCTCCAG GAAGTTGTGAGAA

GGGCCTGAGCTGTTCTGCACTTTCTCGTGGCCGGGCGTGGCTGGTGGTGCAGGAGTT GCTGCCCCAGGGT

GAGGGCCCGGAAGCTCCTGCCAGCACGTGCCGGGGTGGAAAGGGAAGCTGTCCACAG CCCTGTCAGGACT

CAGAACCCGGTGGGTCAAGGACTTTGGTCCGGACCCCTGCTGAAGGGTGAGCTGTCC ACATGTGCGCCGA

GAGCAGAGGTGAAGCCAGGGCTCCTGAGTGCCCCCAGCCACAGGGTGCGCCCGCCCA GCCCCTGCCCTGC

AGCCGAAGGCCTCCCTGCTGGGGGGCTGAGTCCAGTGGGGCCACAGGCAGCTGGGAG CAGGACAAGGCTG

CCAGGCAACCAGATGGTGCTGCCGCTTCCTGCCAGGTGTGGGTGCACAGAGAGAGAG AGGATGCCGGTCT

GGGGCCTGACCTGGTGCACAGCAGGTGCCTGAAATGCCAGGGTGGCCATGGGGACTG GGTACCATGCATA

GGCCATGCATCGGGATGCAACTTCTCCTTGCAGCCCCTCAGCCCCAGGGAGGCAGCT GCCTGCCCCACTT

TTCTCCAGAGCCATCATGGCCCTGCTCCCACCCCAGCCACGGCTGCTCAGGGGCGCT CCGCATGCTCTGG

TCTCCATCCACCTGCAGCCCCCACTGGGGTGAGGTGGAGCTTCTTGCCTCTCCTTGT GTCTATTTCCTCT

GCTTCCACAACTGAACGGTGACAGGTATTTGCTGGATGAGGGAGCACACCCCAGGTG GTTTCCTCTGAGC

CTGGGAGGCCTTTTCCTGCCTGTGGGCCCCAGGCCCATCCTGCTGCCACCCCCAGGA GGATGCCCGGCTC

CTTGTGACAAGAGTGACCCTCGGGAGGCGTGGGGAGTGGGGCTGGCCGGCCTGCCTG ATGGGGTCCTGAG

TCCATGGCGGGTTTGCATCTCAGGCCTCTGGGCTCTGGCCGGGCTGGGGGCTATTGT CCGGCTGAGCGGC

CTGGGCTGCGGCCCCTCCCCGTCCCCGGGACCAGCCTCACCCACTCGCTCTGCCGCA GGTCACACAGGCC

GCCTCCTGCGGGCATTGCTGGGCCTCAGCCTGCTCTTCCTGGTGGCCCATCTCGCCC TCCAGATCTGCCT

GCATATTGTGCCCCGCCTGGACCAGCTCCTGGGACCCAGCTGTGAGTCGCTGAGGGG GCGGGGTAGGGAT

AGCCATCCTGGGGGTCAGGGAGAGGGCCCTGCAGTGACCCCGAGTCTCCTGGGGGGG TTGACTCAGCCTG

ATTTATGTCTGGCCTGGATGGTCCAGGTGAAACGCTCCAGGGATGACCAGGCCACGG TGCTGGCTGGGCA

GAGCCTGACCTGGGTTCCCCCGTCTTTCTCTGCAGGCAGCCGCTGGGAGACCCTCTC GCGACACATAGGG

GTCACAAGGTAAGACCATTCCTCCCACCCCCAACCAGCAAGCCTCCCTTGGGGATTT CAGGCCCCAGGAA

GTGGGGGGACCCAGGAGGGACAGAGGGGGACCTGGAGACTCATCCACACTCCCACCC ACACCTGGAGACC

CATCCACGCTCCCACCCACACCTGGAGACCCATCCACACTCTCACCCGCACCTGGAG ACCCATCCACACT

CCCACCCGCACCTGGAGACCCATCCACACTCCCACCCGCACCTGCAGACCCATCCAC ACTCTCACCCGCA

CCTGGAGACCCATCCACACTCCCACCCGCACCTGGAGACCCATCCACACTCCCACCC GCACCTGGAGACC

CATCCACACTCCCACCCGCACCTGGAGACCCATCCACACTCACACCCACACCTGTCC TCTTGGTCTGACC

GCGGCTGCTCCCTGCTCTCCGCAGGCTGGACCTGAAGGACATCCCCAACGCCATCCG GCTGGTGGCCCCT

GACCTGGGCATCTTGGTGGTCTCCTCTGTCTGCCTCGGCATCTGCGGGCGCCTTGCA AGGAACACCCGGC

AGAGCCCACATCCACGGGAGCTGGTGAGGGCAGCTGCGTCACCCGTGTGTCAGGGAG GTCATTCGAGAGC

TGTGGTCTCAGCCATTTTGAGGGTTATTTTAATCTTTTTAAAACAGATGTAGACGTT TTGGTTGTAAGTT

GGTGTTAAAGAGAGGAGGAAGTTCCAAATCCCACCCCGGGGCCCAGCCTGCAGTTCC ATCCGTTCAGACC

TGTTTCTACTCGGGCTCTGCCTCTAGTCAGAAACCTCCACGCCCCGACATGGCATCT GTGCCCTTAGGAA

CTCTTCACAGGGGAATTATTTGGGGCCACGCGGTGGTGGAAACCTGCAGTGCTGGGC AGTGGGTCTGGCT

GGAGAGCCACTGCAGAAGGGCTGAGAAGGGGCGGCCCCAGCAGGCCCCATGCACTTA CAGGCAAACAGGC

TGTCGGCCCAGAGCCCCAGCAGGGGCCTGGACCCCAGGAGCGACGGGCCTGAAGCAG GGCCTCTGTCCTC

GGAGTGGGAGGCAGAGTGAACTTTAGCTGCTACAAGACTTGGAGGTCCGGCCCCGGG AATCTCTGAGCTA

TGGCCCCCTCACACGGACAGGGCATGAGCTGGGGCCTGCGACACCCAACGTTGACTG CTCAGACCCTCTC

GTCCCTGCCTGGGCCCACACTTGCCATCCCCAGGCTCAGCCAGGATTTATGGCCACC TGGGTGTCCTGGC

CCACGAACTCCCTGCCCCGGGCACCCAGCCTCCTCCCACCTCACCCTCCCAGTGCCC ACTCGTCAGATAG

CTGATGCTCGCCACGCACATGGGCCTCAACTTCTAGAGGAGTCCCGAGAGGGAAAGG GGTTCCCCAAGCC

ACACGGTAGGTCGAGTACACCTGCTCAGCCTGCAGAGGCGGCCTGCCCTCCTGCCTG TCCTGTCGCCACC

CCATGGGGCAAGGCTGGTGCCCGCATTTCAGAAGTGGGAACGGGGACCTGGAGGTCA AGTGGCTTTTCCG

GGCATTTGAGGGGGTCCAGCGCTCATGAGGACTGTGCTGCTGCCTGTCCTCCAAGAG ACCCGCCCCTCCC

TCGTCAGCCCCTCCATGCTGATGGGGGCATGGGGCATGAGCTTCCTAGAGTTTGGCT GCTGGGAAGGGGC

TGGGTGGCTGGACTCTGGTCATCTTCATCTTGGTGTGATGGGAGCCTGAGTGTGCAG CTGCGCCCTGGGC

GGCCAGCGTGGACAGACTTCAGAGTGAGCTGGGGGCACCAGGCACCTGCAGGATACC GTTCCGAAAGATC

TAGAAGCCCCATGACCCGGCCTGGCAGGGCCCCCGGGCGACCCTCACTCCCACATAT GGGCAACACACAC

ACGTGTTTGCAGGCATGCGACGGAGTGTGCCAGCCAGCCCGAGGGGTGCTGGCCGCT AGAGGAGGGTGAG

GGGCGTCTGCAGCATGGCTGCGGCACGTGGGGCCCAAGTCAGGGCCGGGCCTGCCCC TCGGTGGCCTCAC

ACCCCAAGTCAGGGCCGGGCCTGCCCCTCGGTGGCCTCATACCCCAAGTCAGGGCCG GGCCTGCCCCTCG

GTGGCCTCACACCTGTTCTCTCACAGCAGCTGCCTCTGTTCTTTCCACTCCCAGAAA TGCTCATTTTTCA

CGGCTCTGCTCAGAAGATTTCCCAGGGCCAGGGCCAGGGCCAGGCTGTGGGTGGGGT GAGCGATGGCGCC CTGCGTTCTGATCCAGCCTCGCCCCCAGCTGCTGTGTGGTCTCTGAGAGGGGTGGGCTCA GGGCCTGGGC CTCTCCTGCCCTGATCTCCCTGTTACCCTGGCCTCGGAGCCTCCCAGCAGCCCCTGCAGC CCTGGAGGCC TGTGAGGACAGGACTGGTCTGGTTCATCTGTCCCGTCAAGCCCCCATCCCATTTGACAGA AGGGGAATAG GCTCCGAGAGGCTGTGACTTGCCTCGGCCTGCAGAGCACGGAGACTGTCAGAGCTGGGGT GGGGCCTATC GTCCAGCCCCACAGCCTGCGGTCTCAGCCCGGGCACCTGGCGCCTCAGCCACCAGGAGAG CCCCCGAGTC ACAGATGGGGACACTGGGCCGGTGGCAGCCAGGCCAGAGCCAGGGCATCTGACGACAGCC GTGTTCTTTC CACCACACCTGGCTTTCCCTGGTGTTTGGGAAATGGCTGTGTTCTGGGAAATCTTTAGTC AGGTCCAGGG AAAACAGGCCCGGCAGGTCTCTCTCCCCAGCCCCAGGCCGGCCTGTCACTTCTTCTAGGC AGCTGGCCAC CTCTGTCCCCCCAGAGGCTTGGGGAGGGCAGGAGTACCACCCCTATCTTCCAGGCAGAGC CACCCAGAAG GCTGGCAGGGGTACACAGGAGACCCTGGAGCCTGGCCATGTCCCTCAGGCCCCCTTGGTC TCTAGAGACC CCCCGGGGTATAGACAGGGCCCCGCTGCTCCCTGGGTGCGTGGTTCGGGGAGATGAGGTG GTATAGGACA GTCTGTGGTCTGTCTGTACCTGGCAAGGTCATCACGTGCCTGGGCTTGGCAGGACAGACC CTGGGTCTTC GGCCAGGGTGGGAGCTGCTACCAGGAAGGCCTGCAGGAACTGTGAGCTTGAGTGAGGAAG TAGGAAGGTG TCAGGCAGACCTCAGGGACGGCTGGGGCCTGTGCCCGGGGAGGCTGTCCTGTGGCCCTCA GAGGAGCAGC TGTGGATGTGGCCGCCTCCCACGCTCCTGGCTGGGCAGGTGTGGGCTGGAGAGGTGGCGT CAGTGCGATA CACCTGACCTTGCCCCTGTCCGTGACCTTGGCAGGATGATGATGAGAGGGATGTGGATGC CAGCCCGACG GCAGGGCTGCAGGAAGCAGCAACGCTGGCCCCTACACGGAGGTCACGGCTGGCCGCTCGT TTCCGAGTCA CGGCCCACTGGCTGCTGGTGGCGGCTGGGCGGGTCCTGGCCGTAACACTGCTTGCACTGG CAGGTACGCA CCGAGGCAGGGGGCACTGGCAGTCACACTGGGAGGGGTCTTGGGAGTTCCCTGATGACTG TGGAGACAGC GGGACACATGGCACTGGCCAGGTACCACCCTGTGTGCCCCTGCCCCGCAGGCATCGCCCA CCCCTCGGCC CTCTCCAGTGTCTACCTGCTGCTCTTCCTGGCCCTCTGCACCTGGTGGGCCTGCCACTTT CCCATCAGCA CTCGGGGCTTCAGCAGACTCTGCGTCGCGGTGGGGTGCTTCGGCGCCGGCCATCTCATCT GCCTCTACTG CTACCAGATGCCCTTGGCACAGGCTCTGCTCCCGCCTGCCGGCATCTGGGCTAGGTAACG GCTTGCCACA CAGCCCCTTTTTCCTGCCACCCTGGTCCCGCCCACCTGGCTCGTCTAGCCCCTGTGGCCC CACTGCCTCT GGGGTGGTAGGCTGTGACGGGTCTTCTCTGGACAGGGTGCTGGGTCTCAAGGACTTCGTG GGTCCCACCA ACTGCTCCAGCCCCCACGCGCTGGTCCTCAACACCGGCCTGGACTGGCCTGTGTATGCCA GCCCCGGCGT CCTCCTGCTGCTGTGCTACGCCACGGCCTCTCTGCGCAAGCTCCGCGCGTACCGCCCCTC CGGCCAGGTG AGCACCTGCCACCCATGGTGGGTGGGCTGAGGCCAGGCCATGGGGCTGGTCTCAGGACCT CCTGCCTCTG GGTGGGGTGTGGAGCTGGTTTGGGCTCAAGACGCTGGTCTCTGCAGAGGAAGGAGGCGGC AAAGGGGTAT GAGGCTCGGGAGCTGGAGCTAGCAGAGCTGGACCAGTGGCCCCAGGAACGGGAGTCTGAC CAGGTGAGCA GCCAGGCAGGTGGAGACGCCAGCGTGGGGGGCGCCCGGCCAGCCCGTGCATGGCTCAGCG CTGCTTGCCC ACAGCACGTGGTGCCCACAGCACCCGACACCGAGGCTGATAACTGCATCGTGCACGAGCT GACCGGCCAG AGCTCCGTCCTGCGGCGGCCTGGTGAGTACCGCACACTGCAAGGTATGGCTGGGTGCGGG GGGCGGGGCG GAGGCCGGTGCTGCCCCCTGGTGGCCGCCTGGCGCTCTCGCATGCTCGCGCCGCACCTCT GCCTGCCGCC CCCTCGGGGGCCCAGGACATCCACGGGTCGGTGTCAGTGACCCCCGAGACCCCCAGGGCA GCCGAGTGGC CATGTCACTGACCAACCCCCAAGACCCCCAGGGCAGCTGAGTGGCCGCGTCGTTGATCCC CAAGACCCCA TGGGGGGCCTCCAGGTCCCCCAACCCCTCCCCAGAGAATGTGGCTATGCTGTCTTGTGCT GTTAGCTCTG GGAGCTGCTCCAGGTGGCCCAGTGGCCCCAGGAGGCCGCTCGTCCAGGGCAGGGGCTGGC CTGGGAACTC TGTGTTGGCCACGTCGCCTTGGGAGGGCCTGGGGGCTCTTTCTGGCTACTTTCTTTCTTT ACCCTAACCC TTGATTTTCCATTTTGCAATGTGTTTCTGAATGAAGCAAATGAAGCCACGGCCCTGGGGT GGGGGTCCTG AGAGTCTTCAGGTGCGCAGAGCTGGAAAGGGGGTCAGGGCCACCTTTCCCACCCTTTCAA GGAAAGTGAG GCCCAGAGAACGGCAGGTGCTGGCAGGGCCATCCCTGACGCTCAGGGACGGTGTCAGCCC AATTGCCGGA GCCCTCGTGTTCTGCCCATAGCCCACCGGGGGCCTGTCTCTCCTGCTGTGTGCTTGCCCA GGGCCCAGAT TTTAGGGCATAGTCAGGGTGGGGAGGCCTGCAGATCAACCTGCCGAAGCTGACCGCTGTC CCCACCTGCA GTGCGGCCCAAGCGGGCTGAGCCCAGGGAGGCGTCTCCGCTCCACAGCCTGGGCCACCTC ATCATGGACC AGAGCTATGTGTGCGCGCTCATTGCCATGATGGTAGGCGGCTGTGGGGGTTGGGGTGGGC GGCCCCCTCT GCCGCGCAGGTGTGGGGCATCGCCTGGGTGGGGTGCGCTGGGCAGCTGTGCAGCCCCCTC TGCCGCGCAG GTGTGGGGCATCGCCTGGGTGGGGTGCGCTGGGCAGCTGTGCAGCCCCCTCTGCCGCGCA GGTGTGGGGC ATCGCCTGGGTGGGGTGCGCTGGGCAGCTGTGCAGCCCTCTCTGCCGCGCAGGTATGGAG CATCACCTAC CACAGCTGGCTGACCTTCGTACTGCTGCTCTGGGCCTGCCTCATCTGGACGGTGCGCAGC CGCCACCAAC TGGCCATGCTGTGCTCGCCCTGCATCCTGCTGTATGGGATGACGCTGTGCTGCCTACGCT ACGTGTGGGC CATGGACCTGCGCCCTGAGCTGCCCACCACCCTGGGCCCCGTCAGCCTGCGCCAGCTGGG GCTGGAGCAC ACCCGCTACCCCTGTCTGGACCTTGGTGCCATGGTGAGTGTGCACCACCACATCCGGGGG TGCCTGGGTG CGCAGACCCATCAGGGTTGTCGTCCTGTTCAATGTCCACTTGCCCGGGGGAGTGGCAGCG CCAAGAAGGC AGATGTGTCTGTCTGTCCCCTTCTGCCCACCCAGAGCCAGCCCAGAGTAGCTTCTCAGTG AGCGTTTGTT GACTGAATAAACAGACAACCTTGTGTTGGCACGGGCACCACCCCTGTGCCCTGACACTGT GTGAGCGTGG GCTCTGTTGGCACGGGCACCACCCCTGTGCCCCGACACTGTGTGTGAGCATGGGCTATGC CCATTGGCAC AGGCACCACCCCTGCGCCCCTGACAGTGTGTGTGAGCGTGGGCTCTGCCCATTGGCACCA TGCACAGCCC TGGGTCTCAGTGACAAGCTGTGCAGGCCATGTGTTCACAGGGTGCCTGCGTGTCCATGTG AAACGGGTGC CAGCATCGTGTCTGGACACCTGTTTGCAGGCCAGTGGGTCTCATCTTGTGAAACTTGTGA GCCTGTGTGC

CAACATATGCACCTGTGAGCTTGTGTGCATATGTGAGCACGCATGTGGGCAAACATG CACTCCAGCATGT

GGACATGTGTGCAGGTGCGTGCATGGATCTGTGCCCACGTGAACTAGTGAACCCGTG TGTGACTCTGCGT

GTGAGCGCAAGTGAACCCATGCACTCATCCATGGATGTGAGAGTGTGTGCTCGTGTG CCTCTGAGTGGGT

GTGAGCGAGAGGGTGTTCGGTGCCTGTGGGGAGGCTGCGGTGGATGGGCTGGTGCCA GCCGCCTGAGAGC

TCTTGCCCCCTGCTATAGGAGGGTGCTGGGTCCCCCGGCTGTGGGAGGGGTGCTGGG CCCCCCGGCTGAC

TGTGACACCCTGCGCTTGTCACAGTTGCTCTACACCCTGACCTTCTGGCTCCTGCTG CGCCAGTTTGTGA

AAGAGAAGCTGCTGAAGTGGGCAGAGTCTCCAGCTGCGCTGACGGAGGTCACCGTGG CAGACACAGGTGA

GTGGTGGGCCAGAGGCGGGGGTTGCCCTCCTGCCTGCCCGCCCTGATGCCATCGCCT GCCCCTGGCTTGG

CCCACAGAGCCCACGCGGACGCAGACGCTGTTGCAGAGCCTGGGGGAGCTGGTGAAG GGCGTGTACGCCA

AGTACTGGATCTATGTGTGTGCTGGCATGTTCATCGTGGTCAGCTTCGCCGGCCGCC TCGTGGTCTACAA

GATTGTCTACATGTTCCTCTTCCTGCTCTGCCTCACCCTCTTCCAGGTGGCTGGGGG GCCGGGATGGGGG

CTGGGGCACGGACCCTCCCCGCGGTCCTCACCACCCCCACCTCACCCGGCAGGTCTA CTACAGCCTGTGG

CGGAAGCTGCTCAAGGCCTTCTGGTGGCTCGTGGTGGCCTACACCATGCTGGTCCTC ATCGCCGTCTACA

CCTTCCAGTTCCAGGACTTCCCTGCCTACTGGCGCAACCTCACTGGCTTCACCGACG AGCAGTGAGTCCA

GGCTGGGGCGGTGGGGCAGGGGCGCCGAAACCCCGTGCACTTCCCCGGGGCTGCAGC GGCTCTGCCGGGG

GCCGGGCCGGTGCTGATGCTGCCCCTCCACAGGCTGGGGGACCTGGGCCTGGAGCAG TTCAGCGTGTCCG

AGCTCTTCTCCAGCATCCTGGTGCCCGGCTTCTTCCTCCTGGCCTGCATCCTGCAGC TGCACTACTTCCA

CAGGCCCTTCATGCAGCTCACCGACATGGAGCACGTGTCCCTGCCTGGCACGCGCCT CCCGCGCTGGGCT

CACAGGTGCGGCCCCGCCCTCCCTGTCCGGCCCTGGAGAGGTGTAGCCTCCTGGGCC AGGGAGGGAGCCA

GGTGGGAGTTGGACAGGAGCCACATCTTCCACCTTCAGATCCCAAGGGGCATTTGCT CATACCAAGGGGA

TGGCAGTAGCGTGGAGGTCACAGGGACAGTGGGCATGAGTTGCGACACAGCTGTGCA CCTGAACTGGCAG

CTGCAGCAGAAGCGGTGCCGACAGGGCTTCTTCCAGCCCCAGGAAATGAGGGGCAGG AACCCAGTTGGGA

GATGACATTTTCGGACCCTCTCCCAGGCAGGATGCAGTGAGTGGGACCCCACTGCTG CGGGAGGAGCAGC

AGGAGCATCAGCAGCAGCAGCAGGAGGAGGAGGAGGAGGAGGAGGACTCCAGGGACG AGGGGCTGGGCGT

GGCCACTCCCCACCAGGCCACGCAGGTGCCTGAAGGTGGGTTGGGCGGGCAGAGCAC AGCTGCCACCCAG

TCTGCTGTGCCATGTCCCAGCTCGGGGGGCGTTGGCAGAGTCCCCTCTGGGCTCCAG AGCCTCTTCCTCA

CAGGGGACCCGGGAATCCCCGTTTGTGCCCCGCACTGACCCTCACACCATCACAGGG GCAGCCAAGTGGG

GCCTGGTGGCTGAGCGGCTGCTGGAGCTGGCAGCCGGCTTCTCGGACGTCCTCTCAC GCGTGCAGGTGTT

CCTGCGGCGGCTGCTGGAGCTTCACGTTTTCAAGCTGGTGGCCCTGTACACCGTCTG GGTGGCCCTGAAG

GAGGTGAGTGTGGCAGGCAACTCAGCTTCCCATCTGGGGTGGGGTCGCTCTGGCCTG CCCAGCTGGCCTC

CCCAAGCCCAGCCCCACGTGCCCACTGCCCTCCCCAAGCCCAGCCCCACGTGCCCAC TGCCCTCAGGTGT

CGGTGATGAACCTGCTGCTGGTGGTGCTGTGGGCCTTCGCCCTGCCCTACCCACGCT TCCGGCCCATGGC

CTCCTGCCTGTCCACCGTGTGGACCTGCGTCATCATCGTGTGTAAGATGCTGTACCA GCTCAAGGTTGTC

AACCCCCAGGAGTATTCCAGCAACTGCACCGAGGTACCGGCCCCCGAGGGCTGGGAC GGGAGGAAGCTCC

AGGCAACTCTGTATTCGCAGCCCGACCCTCCTGGGGCAGCTGCCTCAGTGCAGTGGG GCCAGCAATGGAG

ATGGAGGACTCTCCCCTGGGGGCGCCAAGGGGGCTTCCTGGAGGCAGCATCCTTCGA CCTCAACTGTGGA

CCAGGGGCGCACTCCCTGCACACAAGGGTGTCCAGTAGGGGCGGAGTCCCAGGGTCT CCGGCAGTGAGGA

CGGGAGGGCCCCACCCCTGGACAGGGAGAGACAGTCAGGCATCTCTGCCTGGGACCT TCTCGCACATCCC

TCCTTCTCCCTGGACCTCTCTTCACTCCCCCAGCCCCTGCCCGTGGTCTCCCTGTTT CTCAAACACCTGG

TCCCCTTCCCCGTGAAGGTGGCTCCAAGGCTGGCAGCCCCCGTGTCCCTGGCTGGGG AGCAGTGGACCTG

CCCCAGAGCTGTGGCTGTGGTGGGCTCCGGGCAGGGCCAGGGGGCACTGTGGCCTGG GAGGGGGCACTGA

TGCCTGGCCTCTTGCCAGCCCTTCCCCAACAGCACCAACTTGCTGCCCACGGAGATC AGCCAGTCCCTGC

TGTACCGGGGGCCCGTGGACCCTGCCAACTGGTTTGGGGTGCGGAAAGGGTTCCCCA ACCTGGGCTACAT

CCAGGTGAGTTGAAGGGCTGGTGGGCGGCTGGGCGGGCGAGTACCCGGCTGCCCCCT GACCCTTGCCCTC

CGCAGAACCACCTGCAAGTGCTGCTGCTGCTGGTATTCGAGGCCATCGTGTACCGGC GCCAGGAGCACTA

CCGCCGGCAGCACCAGCTGGCCCCGCTGCCTGCCCAGGCCGTGTTTGCCAGCGGCAC CCGCCAGCAGCTG

GACCAGGATCTGCTCGGCTGCCTCAAGTACTTCATCAACTTCTTCTTCTACAAATTC GGGCTGGAGGTGA

GGCAAGGACATTGCCTCCCCCTGGGGCAGGGCTTGGCCTTCGGGAGGGAGGGACGGC TGCACCGTGCAGG

CACCGCAAGCCTGGCCCCACCTGGGTTTGCCTGGGCCACAGAGGGTGGGGGACTCAG GGCCAGGCACGGC

TTCCCTGGACTCCTGTGGTGTGTCGGTGCTGACAACAGGCAGGGGGCCAAGTTAGAT CTGCTCTACTGTA

CAGCCCACCTCCTGGAGCCTCAGTTTCCCCTGCACGATGGCAACTGCCAGCCACTCC TGCCCTCTTGACA

GCGCCGCTGGCCCTGTCCTTGCTTGATGCCCGCAGCCTCCAGGCAGGGCTGCTGCAA GCCTGAGGCCTGC

TGGGTGGGACAAGAAAGTCCCTCCCCCCAGACTCAGTGCATCCCCACACCCCGCCCT CTCCCCTCCCCAG

ATCTGCTTCCTGATGGCCGTGAACGTGATCGGGCAGCGCATGAACTTTCTGGTGACC CTGCACGGTTGCT

GGCTGGTGGCCATCCTCACCCGCAGGCACCGCCAGGCCATTGCCCGCCTCTGGCCCA ACTACTGCCTCTT

CCTGGCGCTGTTCCTGCTGTACCAGTACCTGCTGTGCCTGGGGATGCCCCCGGCCCT GTGCATTGGTGAG

GGGCACGTGGCTTGGGTGGGAGTGGGCTTTGTGGCTTTGTGGATGCCCGTGGGGGTG TTTCCCGCCTGCC

CCAGACTCCTGTCCACCCTCCTAGACTTAGCCTTGGCCTCCTCCAGTCCCTCCTCTC TGCTCCACATCCT ACCCAGGCGCCATCACCTGCATCCTGTCTCTCGGGGGCGGCCTGGCCCCCTCCAGGCTCT GCTGTTCTCT

TTTCCTTTTTTGCCCAGTATTCTATCTTGAAATATTTCAAACCTTCAGGAAAGTTGT GAGTCACACAAGG

AGCACTGCGTCCTCTCCGGGCCCCGTGCGGGTCGGCCGTGATGCCTCACACCCAAGT GCTGCCTCGAGCA

TGCGTCTCCTGGGTGAGGGTGTCTGAGCCCACAGCCCCACACCCGTGGTCCCGTCTC CTGGCAGTGCCAT

CGTCAAACGTGTCCTCTGCATTCAAACAGCCCCGGCCTCAGCACCCTTCTTTGTGGC CATTTGGTTTTCA

GACGGGATCTGGTCTGATGTTTGCTCTAGTCTCTTGTCTTGGGTCTAAGCCGCCCCC GCCTCTCCTGTCT

CTTGGGAAGTTCCGGAGGGAGGCCGGTAGCGTTGCTGACGCCGTGAGACTGGATTTG CGTGGCTGTCCTG

GTGCTGCAGGTCTGCTCAAGGCACACAGCACCCTGCGGTCTGAGATGGGGAGTCACA TTTGTGCACGTGG

CCGGCTCAGGGGCGTCCCACCTGCCCCACAGTGGCACCCAGCCTGTTGGCACTGGTG GGCTGTGTGGGGT

CAGCCTTGTGGTTTTACGAAACAGACTTTCTCTCTGCTGTCTCTGTGTGTCTGTCAG CTGGGATTCCCAT

CAAGGACAGCTGCCATTTGTTACTGGCTGCTTTCCAAGAATTCTGTCATCCGCAGAC CCTGGGCCTCCCC

TCTGCTGAGTGGGTCCTGGCCCCTCCGGCCACACACTGTTACATCATCTCCCCGTAT TTGGCTGGGCATG

GTGGTTCACGCCTGTGATGCCAGCACTTTGGGAGGCTGAGGCAGGAGCATCCCTTGA GGCCAGGAGTTTG

AAACCACTCTGGTCAACATAGCAAGAACCCTTTTTTTTTTTTTTTTTTTTTTTGAGA TGGAGTCTTGCTC

TGTCACCCAGGCTGGAGTACACTGGCACAATCTCGGCTCACTGCAGCCTCCGCCTCC CGGGTTCAAGCAA

TTCTCCTGTCTCAGCCTCCCTCGTGGCTAGGATTACAGGCAAGCACCACCAGGCCCG GCTAATTTTTGTA

TTTTTAGTAGAGACGGGGTTTCACCATCTTGGCCAGGCTGGTCTTGAACTCCTGACC TCAGGTGATCCAC

CCGCCTCCCAAATTACAGGCCTCCCTCCTGGGATTACAGGCGTGAGCTGCCACGCCC GGCCCCGTCTTGT

TTTCTGCTCCCAGGCGCTGCTGCCTCATCTTCTGCTACCCAGGCCCAGCCTTGTGCT CACAGCCATTGCT

CCAGGGAGCCCAATCGAGTTCTAGGAGCGTGAGGTTTAGAGCCCGGGGTCTGGGCGC TGGGTGTGCCTGT

TGCTACAGGGCTGCCTCAGCCTCTGGGCCCTCCAGCTCTTCCTTGTTGAAACATCTG CTTTCGAGCATCA

CCGAGGCCAGCTCCCCGTCTCCTGTCCACCTCTTCCTTGTTGAAATACCTGCTATCA AGCGTCACCTAGG

CCAGCTCCCCTTCTTCTGCCTCCTTCCACGCGGCTGCGCCATGCAGTCGCCATCCTG TGAGATCAGCATG

TCCTGGGTTCCCCAACATCGAGGGTAACTTTGTTTTTGTATCGTGAGGTTCCCTCTG TGGCAGATGGGGC

TGTGGGTTCAGCATGTCCTGGGTTCCCCAGCATCGAGGGTCACTTTGTTTTTGGGTC GCGAGGTTCCCTC

TGTGGCAGATGGGGCTGTGGGTTCAGCATGTCCTGGGTTCCCCAGCATCGAGGGTCA CTTTGTTTTTGTG

TCGCGAGGTTCCCTCTGTGGCAGATGGGGCTGTGGGTTCAGCATGTCCTGGGTTCCC CAGCATCGAGGGT

CACTTTGTTTTTGGGTCGCGAGGTTCCCTCTGTGGCAGATGGGGGCTGTGGGTTCAG AATGTCCTGGGTT

CCCCAGCATCGAGGGTCACTTTGTTTTTGGGTCACGAGGTTCCCTCTGTGGCAGATG GGGCTGTGGGTTC

AGCATGTCCTGGGTTCCCCAGCATCGAGGGTCACTTTGTTTTTGTGTCGCGAGGTTC CCTCTGTGGCAGA

TGGGGCTGTGAGATCAGCATGTCCTGGGTTCCCCAACATCGAGGGTCACTTTGTTTT TGGGTCGCGAGGT

TCCCTCTGTGGCAGATGGGGCTGTGGGTTCAGCATGTCCTGGGTTCCCCAGCATCGA GGGTCACTTTGTT

TTTGGGTCGCGAGGTTCCCTCTGTGGCAGATGGGGCTGTGGGTTCAGCATGTCCTGG GTTCCCCAGCATC

GAGGGTCACTTTGTTTTTGGGTCGCGAGGTTCCCTCTGTGGCAGATGGGGCTGTGGG TTCAGAATGTCCT

GGGTTCCCCAGCATCGAGGGTCACTTTGTTTTTGGGTCGCGAGGTTCCCTCTGTGGC AGATGGGGCTGTG

GGTTCAGCATGTCCTGGGTTCCCCAGCATCGAGGGTCACTTTGTTTTTGGGTCACGA GGTTCCCTCTGTG

GCAGATGGGGCTGTGGGTTCAGCATGTCCTGGGTTCCCCAACATCGAGGGTCACTTT GTTTTTGGGTCGC

GAGGTTCCCTCTGTGGCAGATGGGGCTGTGAGTTCAGCATGTCCTGGGTTCCCCAGC ATGGAGGGTCACT

TTGTTTTTGTGTCGCAAGGTTCCCTCTGTGGCAGATGGGGCTGTGAGTTCAGCATGT CCTGGGTTCCCCA

GCATGGAGGGTCACTTTGTTTTTGTGTCGCGAGGTTCCCTCTGTGGCAGATGGGGCT GTGAGTTCAGCAT

GTCCTGGGTTCCCCAGCATGGAGGGTCACTTTGTTTTTGTGTCGCGAGGTTCCCTCT GTGGCAGATGGGG

CTGTGAGTTCAGAATGTCCTGGGTTCCCCAGCATCGAGGGTCACTTTGTTTTTGTGT CGCGAGGTTCCCT

CTGTGGCAGATGGGGCTGTGAGATCAGCATGTCCTGGGTTCCCCAACATCGAGGGTC ACTTTGTTTTTGG

GTCGCGAGGTTCCCTCTGTGGCAGATGGGGCTGTGGGTTCAGCATGTCCTGGGTTCC CCAGCATCGAGGG

TCACTTTGCTTTTGGGTCGCGAGGTTCCCTCTGTGGCAGATGGGGCTGTGAGTTCAG CATGTCCTGGGTT

CCCCAGCATGGAGGGTCACTTTGTTTTTGTGTCGCGAGGTTCCCTCTGTGGCAGATG GGGCTGTGGGTTT

CGCAGATGCGTGGAGTCACATCCATGCCCTCAGTCCTTAGGGACCGACCCTCCCTGC CTCACACGCCTCC

CAGGAAGTGTGGCCGGGGGCCGGCAGTGCCACGGCTCCCTCCCCAGCAGGCCCCGGC CGCTCCCATCCCC

AGCACGTGGTCCTATCAGAACGCCACGTCAGCAGGACTCCCAGCAGGTGGCCTTTAG GTCTGGCTTCTTT

CACTTGGCAGAGCACACTGAGGTCTGTCTAGGCTGTCGCATGGATCCCGGTCCCACG TGCTGAGCAGCGC

GTTCCCAGCTGTGGTTGCTGCAGGTTGAACTTTTCCTGGCTGCAGGCGTCCGTGCAG CTTCTGGCCGTTG

TTTTCAGAGCTGTCCTATCACACGCACTGTCCTATCATGGAATATGACGCCGTGTGG GCCACAACTCAGG

CCCAGCAGCCCCCACCCCCGTGCTCTCTGGCCTCCTGCTCAGTTCCTTTGCCCCCAG GGGCTTGGTGCAG

AGTTGAAGGAATCTGTGTGTGTGAACACACAGGACACTAGAGCTGTCAGTTCTCGAG ACACCAGGTGTGC

GCGAGGTGATTCCCATGGACCCTGAGGGCTGGTGATAGACTCGGGTCAACGGGTGGG GACCGGGTGTCTC

AGGCCCCAGGCAGGCCCGGCCCTTCCTGACATGACACCCCTTCCCCCAGATTATCCC TGGCGCTGGAGCC

GGGCCGTCCCCATGAACTCCGCACTCATCAAGTGGCTGTACCTGCCTGATTTCTTCC GGGCCCCCAACTC

CACCAACCTCATCAGTGAGTGCCCCCCACCACCCCCGCCTCTGCAGAGGACCCTCAG AGTACATTCACGC

CCCCAAATCTGCTCACAAGTGTGCACACAGGCGTGCACGGGCGGAGGTGTGGTCAGG CACATGGCGGCCT GCAGGCCCTGACCTCGCACGCACGCACGCAGACCTCAGCCTGTGTGCACGGCAGCCCTTG TGCAGATGCC

CTCACACCGGGGCTCCCCCAGGGACACCCGGCCACTCACCCAGGCAGACGTGTGTCC GCTCCCAGCGGCT

GCACGCCGACAGGCCTGGGGTGGGAGGTGGGATTTATGCGCCGTGCCCACCTCGTGT GGGTCCCCGTGTG

GCACAGCGGCGGCTCCTGTGTCCTGCAGGCGACTTTCTCCTGCTGCTGTGCGCCTCC CAGCAGTGGCAGG

TGTTCTCAGCTGAGCGCACAGAGGAGTGGCAGCGCATGGCTGGCGTCAACACCGACC GCCTGGAGCCGCT

GCGGGGGGAGCCCAACCCCGTGCCCAACTTTATCCACTGCAGGTGGGTTCCACGTCA CCCTCCACGGGGA

ACCTTCTGGGAGGGGTGGCCGGGGCGCCCGCCCTGACGCTCCGGCCTGGCAGGTCCT ACCTTGACATGCT

GAAGGTGGCCGTCTTCCGATACCTGTTCTGGCTGGTGCTGGTGGTGGTGTTTGTCAC GGGGGCCACCCGC

ATCAGCATCTTCGGGCTGGGCTACCTGCTGGCCTGCTTCTACCTGCTGCTCTTCGGC ACGGCCCTGCTGC

AGAGGGACACACGGGCCCGCCTCGTGCTGTGGGACTGCCTCATTCTGTACAACGTCA CCGTCATCATCTC

CAAGAACATGCTGTCGGTGAGCCTCCGGCCCCCCCGCACCCACCGCCCTGGGGCCCC GCTGGCCCCGCTG

ACCCTGCTCTCCCCCAGCTCCTGGCCTGCGTCTTCGTGGAGCAGATGCAGACCGGCT TCTGCTGGGTCAT

CCAGCTCTTCAGCCTTGTATGCACCGTCAAGGGCTACTATGACCGTGAGTGGCCAGG ACGGTGGCGGGGG

AGGGCGTGGGGAAGCCCCCTGCTCCTGGGCCCTGGGCCTGACCCTTGCCGGTGCCTG CCTTGCAGCCAAG

GAGATGATGGACAGAGACCAGGACTGCCTGCTGCCTGTGGAGGAGGCTGGCATCATC TGGGACAGCGTCT

GCTTCTTCTTCCTGCTGCTGCAGCGCCGCGTCTTCCTTAGCCATTACTACCTGCACG TCAGGGCCGACCT

CCAGGCCACCGCCCTGCTAGCCTCCAGGCAAGCTTGGGCCCAGACACAGCCCAGAGC TCCCGTCTTGGGG

CTGGGAGGGGGCAATGGGAGGTTCCTCACTGTCTCAGGCCCCGGCCCGTGGAGGGCA GGCTCTGCCACTC

TGTGACATGGGCGTGTCATCTAGAGGGAGAATGAAGGCCGGCAGATCCCCGGCACCA TCACACTCTGCCC

CAGTGCTGGGTCTGTCAGAGACCACAGGCTGCAGTGCTGACGGTGGCTGGTGTCTCA CCCCCAGCCAACT

TTCCCACTAAGGGCTAAGTTTCTCCACCAGCGGGAGGGCCACTGTGTGGTGTCACGA CTGCCCCAGGGAG

GGGTTCTGGCTTGGGGCCAGCTTTGCCTTCTTCCCTGCAGCTGTGGTGGGGTGGGTG CCACCAGACGCCC

CTGCATCTGTACGGCAGAAGGGCCTGTCCTCGCCGCAGACAGCACGGAGGGTGGGGG CAGCAGATGCCTC

CCCCGTGGGTGCCTCTTGTCCAGCGTGGGCAGAGAGGAGCAGGCTGAGCTGTCCCGG GCTGAGCGGGGAG

CGGCGGCTGCCCATGTTGCTGGGGTCGAGTGCCTGGTGCTCACACCCCATCCCCGCC TCCCTACAGGGGC

TTCGCCCTCTACAACGCTGCCAACCTCAAGAGCATTGACTTTCACCGCAGGATAGAG GAGAAGTCCCTGG

CCCAGCTGAAAAGACAGTAGGTGCCTCTGGGGCGGGGACTCCCCGGCTCCTCCCCCC AATGCTCAGCATA

CCCCACCTTTCCCCACCACAGGATGGAGCGTATCCGTGCCAAGCAGGAGAAGCACAG GCAGGGCCGGGTG

GACCGCAGTCGCCCCCAGGACACCCTGGGCCCCAAGGACCCCGGCCTGGAGCCAGGT GAGTGCAGCTGGA

GTCGGGCACCCAGGGCCCCGTGTCCAGCATGTCTGTGCCTGCTGGCGTGTGCTGCGT CTGTGCCCATGTG

ACGTCCCACAGGGCTCCCAGCCCGCCTGTCCTGTCCGCATGATCACCCTCTGTCTGG CAGGCCCCATGGC

CGCCCTGTGACTGTCCGTCCACGCACATGGGCTCTGAGCCCCATGGCCCCACACGGC CCCCGTCACTGTG

GGTGTCCGTGTCTGTCTCCACCTATCCTGTCTCCAAGACGGGAGCACTCACAGCCCC GACCCCTCCTGGT

GGCTTGACTGCTGCCTCATGCTCACCCTGCCCCTCCACAGGGCCCGACAGTCCAGGG GGCTCCTCCCCGC

CACGGAGGCAGTGGTGGCGGCCCTGGCTGGACCACGCCACAGGTACCCCCAATTAGG CCGCCTGTGGCCA

CCCTCTCAGGCCCTCTGTGCCCCCATCTGTCCTCTGCCTGGCCTGCTATCTTCCCCT CCCTTCCCCCGAC

TCCCAGGCCCTGAGCGTCAGGACGTGCTCAGGCCTCCTGGGTCGGGGGGTGCCTCAC TGGCTGCAGACCC

CTGGGCTGACTATGTCCTCTCCTGGCTATGCCCCAGCCCTTCCAACAGTGGGAGTCT CGGAGCTTGCCCC

GATGACACATGGTGGTCGAGCAGCGATCTCACCTGGGACCCAGCAGCACTGCGTTAT TCTGTTTTTGTTT

CTTTTTGAGATGGAGTCTCGCTCTGTCACTGCAGGCTGGAGTGCAGTGGCATGATCT CAGCTCACTGCAA

TCTCTACCTCCCGGGTTCAAGTGATCCTCCTGCCTCAGCCTCCCAAGTAACTGAGAC TACAAGCATGTGC

CCCACTCCAGGCCTTTTTTTTTTTTTTTTGGAGACGGTGTCTTGCCCTGTCGCCCAG GCTACAGTGCAAT

GGCGTGATTGCGGCTCACTACAACCCCCACCTCCCAGGTTCAAAGGATTCTCCTGCC TCAGCCTCCCAAG

TAGCTGGGATTACAGGTGCCCGCCACCACGCCCAGCTAATTTTCGTATTTTTAGTAG AGATGGGGTTTCA

CCATATTGGCCAGGCTGGTCTCGAACCTCTGACTTCAGGTGATCCGCCCGCCTCAGC CTCCCACAGTGCT

GGGATTACAGGCGTCAGCCACCGTGCCCGGCCTGTTCTGTTTTTCTAACTCTCACAC AGCCTCCTGGGTT

TTCCCCGGTCCTCTGCAGTCGGCCCACTCTGCACCCCAGCCCGCGCTGGCTCTGCTC CTCAGCTGCCCTG

CCCACCTCTGTCTTGTCCCACCGCGCTGGCCTGTGTCTTGTGCCTGCACTGCTCCCG GCTACTCCGCATG

GGAAGGGTGGCTCTCGGGCCTTGGCCCATGCAGGCGGAGGGGGTCTGGCTGGGAGTC TCCCTGCATGGAA

GGCTGGCTCTCAGTGCTGCCTGCCCACAGTCATCCACTCCGGGGACTACTTCCTGTT TGAGTCCGACAGT

GAGGAAGAGGAGGAGGCTGTTCCTGAAGACCCGAGGCCGTCGGCACAGAGTGCCTTC CAGGTGAGGTGGG

AGAGCCCCGTCGGCCCCACTCCAACCACAGAGCTTGTGGTCCTGGACCAGGGCAGCA TAGAGGGTGTCAG

ATGCCCCCAGGGCCTGGGAGCCGAGCTCCTCCACCTCCAGTTAGCCCACCCCGCCCC ATCCAGGCCTCCC

AAGTCCCATGGGAAACCAGGCTACAGGGACATGGGTCATGTGTAGCCTGCTGCCCCA CGGTCTTGGCTCT

GACCACCCAGGTTCTGGTGGCTGCCCGTGGCCTGACCTGTGAGACCGGCCCAACACC TTTGTGCTGGCCG

CCTGGCTGTCCTGGGTCCATCTTTGGGCCCCTGGCTCTTGGTGTTAGACCAGCCCAC CCAACTCCTGAAT

GGGTGGGAGTCTTCCCCCACAGCCCCTCAGGGTCCCCATCCGGGAGGGGCTCAGGGA CACGGAGGTCCCT

GGGAGACACAGAGCAGGGATCTGGATCTGGCGCCCGGCTTGCCCAACCCCAGCTTCC CGCCTGGGTCTGA

TGGCTCGGGAGGCCGGGTCCTAACCCGGGGGCTGGCCGACAGCTGGCGTACCAGGCA TGGGTGACCAACG CCCAGGCGGTGCTGAGGCGGCGGCAGCAGGAGCAGGAGCAGGCAAGGCAGGAACAGGCAG GACAGCTACC

CACAGGTGAGCTGGGGGGCGTGGGGACTCTGAGGGGAAGCCGCGGGACTGCCAGTCA CTCACCAGCATCC

TGTGCCCAGGAGGTGGTCCCAGCCAGGAGGTGGAGCCAGCAGAGGGCCCCGAGGAGG CAGCGGCAGGTAC

GTGGGCCCGGGGCTGGGGAGTGGGAGGTCTCTCTTGGCCCCACAGGCTGCCCCTCCA GCGCCCCCTCCCG

CCCTCCCGCAGGCCGGAGCCATGTGGTGCAGAGGGTGCTGAGCACGGCGCAGTTCCT GTGGATGCTGGGG

CAGGCGCTAGTGGATGAGCTGACACGCTGGCTGCAGGAGTTCACCCGGCACCACGGC ACCATGAGCGACG

TGCTGCGGGCAGAGCGCTACCTCCTCACACAGGAGCTCCTGCAGGTGAGCCTGCCCG TGCACCACGCTCG

TCCCTGCTCTGCCTGACTACGCCCCTGCCTGCTTAACAGCCTAGTCCCGCGCCCACT GCACGAAACCCCG

TGTGGGGACAAGAGCTGGACGCAGCCCTGAGCCCCCTGCTGTGCCCTGCAGGGCGGC GAAGTGCACAGGG

GCGTGCTGGATCAGCTGTACACAAGCCAGGCCGAGGCCACGCTGCCAGGCCCCACCG AGGCCCCCAATGC

CCCAAGCACCGTGTCCAGGTAGGTGCGGGGGTGACCCGAGCCCCAGCTGCTGCCCCT GGTGTGTGGGCAT

CGCCTAGCCATCCCCGACCCTCGCCATTCCCTTGTACCCCAAAGGACCGTGGGCACT TTCCACCCTGACC

CTCCCTGTAGCCTGGGGTCAGGCCATAGAGCAGGATTCTCTGTGACTCGGCTTCCCT CCCCAGTGGGCTG

GGCGCGGAGGAGCCACTCAGCAGCATGACAGACGACATGGGCAGCCCCCTGAGCACC GGCTACCACACGC

GCAGTGGCAGTGAGGAGGCAGTCACCGACCCCGGGGAGCGTGAGGCTGGTGCCTCTC TGTACCAGGGACT

GATGCGGACGGCCAGCGAGCTGCTCCTGGACAGGTGGGGGCGGGACGCGCACAACAC CAGCCTCACCATG

GCCCTCGGGGAGCAGCCGAACAGGGGCAGGAGACTGACTGTGACCGGCAACAGATCG GGCCGTCATGCCT

TCGGGCAGTCCCAGACTCCCCCAAACACGCGGGTCTCCCTGTAGGCGCCTGCGCATC CCAGAGCTGGAGG

AGGCAGAGCTGTTTGCGGAGGGGCAGGGCCGGGCGCTGCGGCTGCTGCGGGCCGTGT ACCAGTGTGTGGC

CGCCCACTCGGAGCTGCTCTGCTACTTCATCATCATCCTCAACCACATGGTCACGGC CTCCGCCGGCTCG

CTGGTGCTGCCCGTGCTCGTCTTCCTGTGGGCCATGCTGTCGATCCCGAGGCCCAGC AAGCGCTTCTGGA

TGACGGCCATCGTCTTCACCGAGGTGGGCCGAGGCCGCGGGGGAGGGGGCGCCCGGC CCACCGCGCCGTG

ACCCTCCCCGCGTGCTGAGCCCCCTCCCCCACAGATCGCGGTGGTCGTCAAGTACCT GTTCCAGTTTGGG

TTCTTCCCCTGGAACAGCCACGTGGTGCTGCGGCGCTACGAGAACAAGCCCTACTTC CCGCCCCGCATCC

TGGGCCTGGAGAAGACTGACGGCTACATCAAGTACGACCTGGTGCAGCTCATGGCCC TTTTCTTCCACCG

CTCCCAGCTGCTGGTGAGTGTGAGCCTTGGCTGGCAATGCGGGGCTGGGCAGGCCCT CTGGGCACCTGTG

CTCTCCACCAGGGAGGCAAGGCCCCCTCACCACACCCTCCCGCCCCTCAGTGCTATG GCCTCTGGGACCA

TGAGGAGGACTCACCATCCAAGGAGCATGACAAGAGCGGCGAGGAGGAGCAGGGAGC CGAGGAGGGGCCA

GGGGTGCCTGCGGCCACCACCGAAGACCACATTCAGGTGGAAGCCAGGGTCGGACCC ACGGACGGGACCC

CAGAACCCCAAGTGGAGCTCAGGCCCCGTGATACGAGGCGCATCAGTCTACGTTTTA GAAGAAGGAAGAA

GGAGGGCCCAGCACGGAAAGGAGCGGCAGCCATCGGTATAAGCGCCCTGCCTCACAA CCTCCTGCCTACC

CAGTTTTCTGAGTGGGGCTACTGCAGGGAGGGTCTTTCTCAGATGAGACGGCCAAGC CCAGTGCGAGGCC

CACCTGGATCCCAGGAAGGTGCCACTTCTGAGCCACAGCTCCCGGCTCTGCCTACAG AGCCGTCCCTGAC

TGCTGCCCCCGGGGATGCTCCCCACGTGTAGGGTGACTGTTGGCCTGGGCTGGCCCC TCACAGTTGCCCC

AGACAGAGGACACAGCCCCAGCTGTCTCCTTGCCAGTGACACTGGGAGCTTTCCTGT GCTCCGTCTGCTT

GTCTGTCAAACAGGGAGAATGCCAGCCTCTTAGGGTGGTCAGGAGCCATGAGCCAGG CCCAGTCCCCAGG

GGGCCCAGGCAGAAGTCAGCTTTTCCCTACAGAAGCTGAGGACAGGGAGGAAGAAGA GGGGGAGGAAGAG

AAAGAGGCCCCCACGGGGAGAGAGAAGAGGCCAAGCCGCTCTGGAGGAAGAGTAAGG GCGGCCGGGCGGC

GGCTGCAGGGCTTCTGCCTGTCCCTGTGAGTGATGGCGGCCGGGGGCAGCTGGGGAG TGGGGGTGGGGAG

GCGGGTACTGGGCCCAGGCTGAGCGCCCCCTTCCGCAGGGCCCAGGGCACATATCGG CCGCTACGGCGCT

TCTTCCACGACATCCTGCACACCAAGTACCGCGCAGCCACCGACGTCTATGCCCTCA TGTTCCTGGCTGA

TGTTGTCGACTTCATCATCATCATTTTTGGCTTCTGGGCCTTTGGGGTGAGCCAGGC CCGGGACCCAAAC

CCAGTGTACGCAGAGCTCAGCAGCCACCCACATCCCCTGGGCTTGGCTCCCCCTGAC CTGTGCTCTCCTG

GCCACAGAAGCACTCGGCGGCCACAGACATCACGTCCTCCCTATCAGACGACCAGGT ACCCGAGGCTTTC

CTGGTCATGCTGCTGATCCAGTTCAGTACCATGGTGGTTGACCGCGCCCTCTACCTG CGCAAGACCGTGC

TGGGCAAGCTGGCCTTCCAGGTGGCGCTGGTGCTGGCCATCCACCTATGGATGTTCT TCATCCTGCCCGC

CGTCACTGAGAGGTGGGCCCACGCGTGGGGGCGCTCGGTCTCCAGGGGCGGGGCAGT GCAGGCTGGGGGC

CCTGCGGGGCTGTTTCTGATGGGGTCCTTGACCTGGCCATCCCGCCCCAGGATGTTC AACCAGAATGTGG

TGGCCCAGCTCTGGTACTTCGTGAAGTGCATCTACTTCGCCCTGTCCGCCTACCAGA TCCGCTGCGGCTA

CCCCACCCGCATCCTCGGCAACTTCCTCACCAAGAAGTACAATCATCTCAACCTCTT CCTCTTCCAGGGG

TGAGTGCAGGTCCGCCGGGGTGGGGGTCACGGCCCGGGCATGAGGGAGCCCACCTGA CGGGAACCCTGGC

TGTGGGCAGGTTCCGGCTGGTGCCGTTCCTGGTGGAGCTGCGGGCAGTGATGGACTG GGTGTGGACGGAC

ACCACGCTGTCCCTGTCCAGCTGGATGTGTGTGGAGGACATCTATGCCAACATCTTC ATCATCAAATGCA

GCCGAGAGACAGAGAAGGTGCCTGGGCCCAGGGCGGGGGCCGGGACAAGGGCCAGGG ATATGCCCTCTCC

CTAAGACAGAGGCACTGCTGCCACGAGAACCCGTGGTGCTGGAGGCCCTCCCAGGGC TCGGAGCCCATGG

GGACATGAGGCGAGCCCACCCACTAGCTGATCACGAGGCCAGTGATCTTGGCAGCTG CGAGTGAGTGCTG

GGCGCAGAAGTGGGCAGCGGAGTTGGTCCTGTTCCAGGCAGGCTGGCAGCAGAGCAG GGCCTGGTGCAGG

GAGGACCGGACAGCCACTGTTTGCTGCATTCTTGTTTAATGGCCTTTCTCAGAGAGA ATTCGTGCGTCAG

ACCACTCCCCCACGTAAAAAGTACAACTCAGGGGTTTCTAGTGGATTCACAGTTGAG CATCTGCCTTCTC ACCACTTCAAAAAGAAACCCCGGGCCGGGCACAGCGGCTCACCCCTATCATCCCAGCACT GTAAGAGACC CAGGAGGGAGGTACTGCTCAAGGCCAGGAGTTCAAGATCTGCACGGCCACAAGCGAGACC CTGCCTCAGC AAATGTAAAAATAAAACAATTAGCTGGCAATGGCAGCTCATGCCTATGGTCCCAGCACTT TGGGAGGCCA AAGGAGGAGGATCAGTTGAGGCCAGGAGTTTTAAGACCAGCCTGGGCAACATAGTGAGTG AGACTCTCTC TACAAAAAAATAAACGTTAGCTGGGTGTGATGGTGCACACTTGTGCTCCCAGCTACTCTG GAGGCTGAGG TGGGAGGATGGCTTGAGGCCAGGAGTACAGGGCTGCATTAAGCCTGATCACACCACTGCA CTCCAGCTTG GGCAACAGAGTGAGACCCTGTCTCTAAAAAAGTAAAAAGAAGAAACCCAGTGCCCATTGG CTGTCACTCG GTTTCCCCTCCCCTGGCCCCTAGTAACCCCTTGTCTAGGTCCTGGTTCTATGAGATTTGC CTACAGTGGA TATTTCATGAAAACAGGCTCAGACAGCGTTTGTCCTTTTGTAATCAGCTTTCCTTGCTCA GCATGGTGTC TCTGAGGTCCACCCACGTGGCAGTATGGCCTTCCTGTTTATGGGCAAATGATATTCTGTG GCATGGATGG ACCACAACATGCTCATCTGTTGATGGGCTGTTGCCCGTGATGCTGCCAGGCACGCCGGTG TACACGTCTG TGTGCCCGTGCCCCTGGTCCTGTGGCTGCACGCCAGGGCCGGAAGTGCTGGTGGTGGTTT CCATCATGAG GAGCTGCTGGTTTTCCATAGCAGCTCCACCATCTAAGGTTCCCACCACCAACACGAGGTT GCGGTTTCTC CACATCCTCAACAACCTGTTACTATGTCTTTTTTGTTCTGGCCATGCTGCTGAACGGGTG GCTTGCTGTG GGCTCTTCTCAGTTCCCTGTTGACCGACGCTGAGCGTCTTTTCATGTGCTTGGCCATTTG TGTATCTTCT CTGGGGAAATGTCTATTCAAATCCTTTCTCCATAGTTTAGTTGGGCTTTTGAGATAGGAT CTCAGGCTGA AGTGCAGTGGCATGACCTTCACTCACTGTAGCCTCTGCCTCCCAGGTTCAAGCGATTCTC CCACCTCAGC CTCCCGAGTAGCTGGGACTACAGGTGTGCACCACCAGGCCTGGCTAGTCTTTTGTATTTT TGGCCAGGTT CGTCTCAAACTCCTGACCTCAAAGTGCTGGGATGACAAGTGTGAGCCGCCACACCCAGCA GTTGGGTTGT TTTTTATTAGAGTTTTTTCTGTTTTTCTGCAGATACATTGGCTAGAAATGACTGAATTGG AAGAATTCTC TGTATTCTCTGGTTGCTAGAACCTTATCAATTAAAATTTGCAGAAAATTTCTCCAATTCT ATGGACTGTC TCTTAAATTTTCTTGGTGTCTTTGGAAGCACAAAGTATTTATTTTGGTAATATCTGGTTT ACTTTGTTTC CTTCGCCAAATCCAGGTTCATGAAGATTTGCCCGTTTTCTTCTAAAAGTTCTATAGTTTT AGCTCTGAAG TTTCGCTCTTTGATCCACTTTGAGGTAAATTTTGGCACATGGTATGAGGCAGGAGTTGCG TTTCATTCTC CTGCCTGGGGCGGTGCCTGCACCGTGTTGAAAAAGGTTGTCCGTTCCCACTGAACGGTCA CAGCTCCCTT GTCTAAGATCAACGACCCCTGAACATGAGGGTTCCAACTGGACTCTTAGTTCTACTCCAC TGGCCTGTGT CTGCCCACCATTACTACCGCTGTGCCATACTGAGGTCAGGCTGGGGCTTTTCTGGGCTGC TGGGTGAGCT GGAGAATGCGGTTGTGTGACCCGCAGGAAGGGCAGAGCTGAGCGTATGACCTGTGTCGTT CCCCTCCAGA AATACCCGCAGCCCAAAGGGCAGAAGAAGAAGAAGATCGTCAAGTACGGCATGGGTGGCC TCATCATCCT CTTCCTCATCGCCATCATCTGGTTCCCACTGCTCTTCATGTCGCTGGTGCGCTCCGTGGT TGGGGTTGTC AACCAGCCCATCGATGTCACCGTCACCCTGAAGCTGGGCGGCTATGAGGTGAGCATGTGT GGGTCCGCCT GTCCATTCCCATCCCCTGGGGGTTCTGGCCAAGGTGGTGCACCACCCCCAGCCGCTCCTC CACGCTCATC TTCGTGGCCCCGTGTCCCCGTGCCTGCCCCAGCCGCTGTTCACCATGAGCGCCCAGCAGC CGTCCATCAT CCCCTTCACGGCCCAGGCCTATGAGGAGCTGTCCCGGCAGTTTGACCCCCAGCCGGTAAG TGGCCTCTGC CCTGTGAAAGCTGGTGTGGGGAGGCGGCTGCAGTCACTGAGGGTGTCACTTGTACCCAGC TGGCCATGCA GTTCATCAGCCAGTACAGCCCTGAGGACATCGTCACGGCGCAGATTGAGGGCAGCTCCGG GGCGCTGTGG CGCATCAGTCCCCCCAGCCGTGCCCAGATGAAGCGGGAGCTCTACAACGGCACGGCCGAC ATCACCCTGC GCTTCACCTGGAACTTCCAGAGGTTCGTCCTGGACTTGGGGCAGTGCCTGGGTGGGTGGA CCCACTACAG TGGGTCACGCTGTGTTCCCACCCCCAGGGACCTGGCGAAGGGAGGCACTGTGGAGTATGC CAACGAGAAG CACATGCTGGCCCTGGCCCCCAACAGCACTGCACGGCGGCAGCTGGCCAGCCTGCTCGAG GGCACCTCGG ACCAGTCTGTGTGAGTGAAGGGCCCGGGTGGTGGGCAGGAGGGCTGTGCCAGGTTGGCTG GGCCAGGCCT GACCTGCCAGCACCTCCCTGCAGGGTCATCCCTAATCTCTTCCCCAAGTACATCCGTGCC CCCAACGGGC CCGAAGCCAACCCTGTGAAGCAGCTGCAGCCCAGTGAGTATGGGCGTGGGGGTTGGGGGA GGCTAGAGAG GGGTGACCTGCGGCCTCAACGATCTTCTCCCTCCATCCCAGATGAGGAGGCCGACTACCT CGGCGTGCGT ATCCAGCTGCGGAGGGAGCAGGGTGCGGGGGCCACCGGCTTCCTCGAATGGTGGGTCATC GAGCTGCAGG AGTGCCGGACCGACTGCAACCTGCTGCCCATGGTCATTTTCAGTGACAAGGTCAGCCCAC CGAGCCTCGG CTTCCTGGCTGGCTACGGGTGAGTGAGTGGCTGGGGGGGCACCCCGCAGCTCGGGGGGCT CCGGGCGGCC CCAGGACTCACCAGCTTCCCCCGCAGCATCATGGGGCTGTACGTGTCCATCGTGCTGGTC ATCGGCAAGT TCGTGCGCGGATTCTTCAGCGAGATCTCGCACTCCATTATGTTCGAGGAGCTGCCGTGCG TGGACCGCAT CCTCAAGCTCTGCCAGGACATCTTCCTGGTGCGGGAGACTCGGGAGCTGGAGCTGGAGGA GGAGTTGTAC GCCAAGCTCATCTTCCTCTACCGCTCACCGGAGACCATGATCAAGTGGACTCGTGAGAAG GAGTAGGAGC TGCTGCTGGCGCCCGAGAGGGAAGGAGCCGGCCTGCTGGGCAGCGTGGCCACAAGGGGCG GCACTCCTCA GGCCGGGGGAGCCACTGCCCCGTCCAAGGCCGCCAGCTGTGATGCATCCTCCCGGCCTGC CTGAGCCCTG ATGCTGCTGTCAGAGAAGGACACTGCGTCCCCACGGCCTGCGTGGCGCTGCCGTCCCCCA CGTGTACTGT AGAGTTTTTTTTTTAATTAAAAAATGTTTTATTTATACAAATGGACAATCAGAGGCCAGT CCCCCGTCCT TGCCTTCCGGCTCCAGTGTGGTGTACCAGGTGGCCACTGCCTGCTGTCCCGGCAAGCACG TCCTCACGCT CAGCTCTGGCCCGCCTCGTCGTCACTGTCCTCAATCAGACAGTCCCGGATCTCCTGCAAG CCCCAGGCCC TAGAGAGAAAGCTGCAGTGGGCACCAGCCCCGAGGCCCCTTACCCACGCGCCACGGCAGT GTGGGCCCCA GTACCTCTGTGTGCGGAGCTGGGCCTCAGCCAGGATGTACGGCGGCAGGGGGTCCAGTGA GGGCTGCAAC AGAGGCCGGTGTCGAGGGGCCAAGAAACCTGCCAGCACCCCCTCAAGCCACCCACGAGGT GACGGCCTTC CCGCGTCCCCAAGCCCGGCCCAGGACAGGTGGGCACGTACTCACCAAGTCCTTCATGTTC ACGCGGCAGC TGTAGCACAGCTGCTCCTCAATGCAGGCTTGGGGGTCCTCCCTGCGGGAGCAGAGCTGGC TGTGTGCCCA CCTTGGCACCTCAATGCCGTGCAGTGTGGGGTAAGGACAGGTCCCCCTCCCGCCCCATGC AGCTGGCCAT GGGTGGCAGGGACAGGGACTCACCTCCTGCAGGCCCCCTGGCCACAGCGCTGGGCCCAGC CCACCCCTGG AGAACAGCAGGGCCCCGGGGGTGTCCGGGTCTCAGTCAGGGGGATGGGTGACTGCATCTG GGAGAGACGC GAGGAGGTCTGAGCCCCAAAAGCCGTGGCACTGTCTGCAAGCAGAGAGGGGGTGACGGAG CCTGGGGCTG CTGAGCCCCCACACCCACTCCCTGGCTCCCTCCCCGCATCACCCCAAGAGCATGAAACAC AGACCAGCGG CGTCGACGTCCAGGGCACACATGCAGAGGAGGCAGCGGGGGCCGGAGTCGCCAGCAGCAG GGCCATCCCG GGGGCCCTTCACCAGCTTCTCACTTGTCCTGTGGCCGGATTGTGGGAGGAGCAGGTGAAT CACACGGGCT GGGGCTCCACCCTAAGCCCTGGCCTGAGCACGAGTGGTGAGCTCACCACCCGCACACACC CGCGCACAGC ACACACCCGCGCACCCCCCGCACACACCCACACACACCCACACCTGTACACAGTGCTGAC AGTGGAGGGG AACTGGGTCTGCAGCCTGAGGATGAAGGCCTCCATCAGCCGGTGGATGCTGGCCTTTTCA GGGGCCTAGG GACAATCAGATTGGAGGCTATGGGAAAGCCCAGAGGCCAGCCCCAGGCTGGGGCAGAGTC CCCAGGACAG GTGCCGTGGGTTCCACGCTGCTGGCCCACATGGGTCTGGCTTTGCTGCGACTGCTCAAGG CAGCCAGAGA ACCCGCTTCAGCAGTGCACCCACTGTGGGGTCCTCCTGTCACCCCCACACCCTCACGGCA CAGGGTGGGC TCAGAGGATGCAGCACTGCTGGGGGCCACAGCAGGAGTCACTGAAGGTGTGCTGGTGACC TGAGCCTGTG AGAGGCTGCCCAGCCCACATGGCCCACCCCGGTGTCAATGGTGAGAGAGGTTGCCCAGCC CACAAGGCCC ACCTTGGTGTCGACGGCTGGTGTGAAGACAGAAGGAACGGAGAACAGGCGGTTGTAGAAA GCGACCTCCT TCAGGGTGTGGTCCCGCATGGGCCGCACCACCACCACGTCCCCGTGCCGCTCATCCGAGA AGCCCTGCGG GAGGCAGGGGCTGTGTGGTCCAGGTCAAGGCCCGAATGTGCTTGCCCCTCCTCCCCTGCC AGGTAGGGGA GGTCACAGACCCCACCACCATCCGAGGCCGCAGGACTGATACGCGAGAGGCTACTCTCCA GTAAAGGTGG CCGAAGGGGTGAAGGTGAGGATGGGCCTCCTGAACACCCAGGCCCCTGCCTACCGTATCC CAGGCCAGGA AGGCCCCTCGACCCAGCGCCAGGTTGGTCATGAGCTTGATAGCCAAGCGTGTGCAGCTGT CCCCAGTCAT GACCTTGGAGTAGCCGTGGGCTC

It will be appreciated that specific sequence identifiers (SEQ ID NOs) have been referenced throughout the specification for purposes of illustration and should therefore not be construed to be limiting. Any marker encompassed by the present invention, including, but not limited to, the markers described in the specification and markers described herein, are well-known in the art and may be used in the embodiments encompassed by the present invention.

The phrase “pharmaceutically-acceptable carrier" as used herein means a pharmaceutically-acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, or solvent encapsulating material, involved in carrying or transporting the subject compound from one organ, or portion of the body, to another organ, or portion of the body.

The terms “polynucleotide”, and “nucleic acid” are used interchangeably. They refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof. Polynucleotides may have any three-dimensional structure, and may perform any function, known or unknown. The following are nonlimiting examples of polynucleotides: coding or non-coding regions of a gene or gene fragment, loci (locus) defined from linkage analysis, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers. A polynucleotide may comprise modified nucleotides, such as methylated nucleotides and nucleotide analogs. If present, modifications to the nucleotide structure may be imparted before or after assembly of the polymer. The sequence of nucleotides may be interrupted by non-nucleotide components. A polynucleotide may be further modified, such as by conjugation with a labeling component. The term “recombinant” polynucleotide means a polynucleotide of genomic, cDNA, semi synthetic, or synthetic origin which either does not occur in nature or is linked to another polynucleotide in a non-natural arrangement.

The terms “prevent, ” “preventing, ” “prevention, ” and the like refer to reducing the probability of developing a disease, disorder, or condition in a subject, who does not have, but is at risk of or susceptible to developing a disease, disorder, or condition.

The term “small molecule" is a term of the art and includes molecules that are less than about 1000 molecular weight or less than about 500 molecular weight. In one embodiment, small molecules do not exclusively comprise peptide bonds. In another embodiment, small molecules are not oligomeric. Exemplary small molecule compounds which can be screened for activity include, but are not limited to, peptides, peptidomimetics, nucleic acids, carbohydrates, small organic molecules (e.g., polyketides) (Cane et al. (1998) Science 282:63), and natural product extract libraries. In another embodiment, the compounds are small, organic non-peptidic compounds. In a further embodiment, a small molecule is not biosynthetic.

As used herein, the term “subject” means a human or non-human animal selected for treatment or therapy.

The “tumor microenvironment” is an art-recognized term and refers to the cellular environment in which the tumor exists, and includes, for example, interstitial fluids surrounding the tumor, surrounding blood vessels, immune cells, other cells, fibroblasts, signaling molecules, and the extracellular matrix.

The phrases "therapeutically-effective amount" and “effective amount” as used herein means the amount of an agent which is effective for producing the desired therapeutic effect in at least a sub-population of cells in a subject at a reasonable benefit/risk ratio applicable to any medical treatment. “Treating" a disease in a subject or “treating" a subject having a disease refers to subjecting the subject to a pharmaceutical treatment, e.g, the administration of a drug, such that at least one symptom of the disease is decreased or prevented from worsening.

In some aspects, provided herein are methods of treating cancer in a subject, the method comprising administering to the subject an agent that increases or stabilizes the activity or expression of PIEZO 1. The agent may be a small molecule agonist of PIEZO 1, such as Yodal, Jedi 1, Jedi2, or a modulator of PIEZO1, such as Docosahexaenoic acid. The method may further comprise administering an immune checkpoint inhibitor to the subject.

In some aspects, provided herein are methods of treating cancer in a subject, the method comprising administering to the subject an agent that increases or stabilizes the activity or expression of PIEZO 1 and an immune checkpoint inhibitor. In some embodiments, the immune checkpoint inhibitor is an inhibitor of an immune checkpoint protein selected from CTLA-4, PD-1, VISTA, B7-H2, B7-H3, PD-L1, B7-H4, B7-H6, ICOS, HVEM, PD-L2, CD160, gp49B, PIR-B, KIR family receptors, TIM-1, TIM-3, TIM- 4, LAG-3, BTLA, SIRPalpha (CD47), CD48, 2B4 (CD244), B7.1, B7.2, ILT-2, ILT-4, TIGIT, HHLA2, butyrophilins, A2aR, and combinations thereof. In some preferred embodiments, the immune checkpoint inhibitor is an inhibitor of PD-1 or PD-L1.

In some embodiments, the agent that increases or stabilizes the activity or expression of PIEZO1 and the immune checkpoint inhibitor are administered conjointly In some embodiments, the agent increases or stabilizes the activity or expression of PIEZO 1 and the immune checkpoint inhibitor act synergistically when administered. In some embodiments, the agent is a gRNA fused to a transcription activator, such as a gRNA that comprises a region that is complementary to a portion of a gene that encodes a PIEZO 1 protein. In some embodiments, the agent is a vector encoding a PIEZO 1 protein, such as a viral vector encoding a PIEZO1 protein. For example, without being bound by theory or methodology, the agent that increases or stabilizes the activity or expression of PIEZO 1 may be a small molecule agonist of PIEZO 1, such as Yodal, Jedi 1 , Jedi2, a modulator of PIEZO1, such as Docosahexaenoic acid, a gRNA disclosed herein, or any combination thereof.

The agent may be administered systemically, intravenously, subcutaneously, or intramuscularly. The agent may be administered to the subject in a pharmaceutically acceptable formulation. The method may further comprise administering to the subject an additional agent, such as a chemotherapeutic agent or a cancer vaccine. The method may further comprise administering to the subject a cancer therapy, such as radiation. The subject may be refractory for immune checkpoint inhibitory therapy.

In some aspects, provided herein are methods of treating cancer in a subject unresponsive to immune checkpoint inhibitor therapy, the method comprising administering to the subject an agent that increases or stabilizes the activity or expression of PIEZO 1 (e.g., any agent that increases or stabilizes the activity or expression of PIEZO 1 disclosed herein) and an immune checkpoint inhibitor. The agent may be a small molecule agonist of PIEZO1, such as Yodal, Jedi 1 , Jedi2, a modulator of PIEZO1, such as Docosahexaenoic acid, or any combination thereof. The method may further comprise administering an immune checkpoint inhibitor to the subject, such an inhibitor of PD-1 or PD-L1, or another immune checkpoint inhibitor disclosed herein.

In some aspects, provided herein are methods of treating cancer in a subject, the method comprising administering to the subject T-cells that have been treated ex vivo with an agent that increases or stabilizes the activity or expression of PIEZO1 (e.g., any agent that increases or stabilizes the activity or expression of PIEZO1 disclosed herein). The T- cells may be tumor infiltrating lymphocytes. In some embodiments, T-cells are autologous (z.e., derived from the subject). In some embodiments, T-cells are allogeneic (ie., derived from a doner).

In some aspects, the subject is a human.

Modulators of PIEZO 1

Provided herein are methods and compositions for preventing or treating cancer in a subject, the method comprising administering to the subject an agent that increases or stabilizes the activity or expression of PIEZO 1. The agent may increase the activity of PIEZO 1 by at least 5%, at least 10%, at least

15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100%. The agent may increase the expression of PIEZO 1 by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100%.

An agent disclosed herein may increase PIEZO 1 mRNA by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100%. Measurement of PIEZO 1 can be done in a biological sample or multiple biological samples taken from the subject over a period of time.

Polypeptide Agents

In some embodiments, the agent provided herein is a polypeptide agent (e.g., a polypeptide that binds to a PIEZO 1 protein). A polypeptide agent disclosed herein may increase or stabilize the expression or activity of PIEZO 1 by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100%. In some embodiments, the agent may be a chimeric or fusion polypeptide. In some embodiments, the agent may be a ligand or binding partner of PIEZO 1. A fusion or chimeric polypeptide can be produced by standard recombinant DNA techniques. For example, DNA fragments coding for the different polypeptide sequences are ligated together in-frame in accordance with conventional techniques, for example by employing blunt-ended or stagger-ended termini for ligation, restriction enzyme digestion to provide for appropriate termini, filling-in of cohesive ends as appropriate, alkaline phosphatase treatment to avoid undesirable joining, and enzymatic ligation. In another embodiment, the fusion gene can be synthesized by conventional techniques including automated DNA synthesizers. Alternatively, PCR amplification of gene fragments can be carried out using anchor primers which give rise to complementary overhangs between two consecutive gene fragments which can subsequently be annealed and re-amplified to generate a chimeric gene sequence (see, for example, Current Protocols in Molecular Biology, Ausubel et al., eds., John Wiley & Sons: 1992). Moreover, many expression vectors are commercially available that already encode a fusion moiety.

The polypeptides described herein can be produced in prokaryotic or eukaryotic host cells by expression of polynucleotides encoding a polypeptide(s). Alternatively, such peptides can be synthesized by chemical methods. Methods for expression of heterologous polypeptides in recombinant hosts, chemical synthesis of polypeptides, and in vitro translation are well known in the art and are described further in Maniatis et al., Molecular Cloning: A Laboratory Manual (1989), 2nd Ed., Cold Spring Harbor, N. Y.; Berger and Kimmel, Methods in Enzymology, Volume 152, Guide to Molecular Cloning Techniques (1987), Academic Press, Inc., San Diego, Calif.; Merrifield, J. (1969) J. Am. Chem. Soc. 91 :501; Chaiken I. M. (1981) CRC Crit. Rev. Biochem. 11 :255; Kaiser et al. (1989) Science 243: 187; Merrifield, B. (1986) Science 232:342; Kent, S. B. H. (1988) Annu. Rev. Biochem. 57:957; and Offord, R. E. (1980) Semisynthetic Proteins, Wiley Publishing, which are incorporated herein by reference.

Nucleic Acids

In certain embodiments, provided herein are agents that are vectors that contain the isolated nucleic acid molecules described herein, such as those that encode a PIEZO 1 peptide. As used herein, the term “vector,” refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of vector is a “plasmid”, which refers to a circular double stranded DNA loop into which additional DNA segments may be ligated. Another type of vector is a viral vector, wherein additional DNA segments may be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non- episomal mammalian vectors) can be integrated into the genome of a host cell upon introduction into the host cell, and thereby be replicated along with the host genome. Moreover, certain vectors are capable of directing the expression of genes. Such vectors are referred to herein as “recombinant expression vectors” (or simply, “expression vectors”). In certain embodiments, provided herein are cells that contain a nucleic acid described herein (e.g., a nucleic acid encoding an antibody, antigen binding fragment thereof, antibody-like molecule, or polypeptide described herein). The cell can be, for example, prokaryotic, eukaryotic, mammalian, avian, murine and/or human.

The interfering nucleic acids described herein may be contacted with a cell or administered to an organism (e.g., a human). Alternatively, constructs and/or vectors encoding the interfering RNA molecules may be contacted with or introduced into a cell or organism. In certain embodiments, a viral, retroviral or lentiviral vector is used.

In some embodiments, agents for increasing the expression or activity of PIEZO 1, are delivered to subjects by use of viral vectors. Viral vector systems which can be utilized with the methods and compositions described herein include, but are not limited to, (a) adenovirus vectors; (b) retrovirus vectors, including but not limited to lentiviral vectors, moloney murine leukemia virus, etc. \ (c) adeno-associated virus vectors; (d) herpes simplex virus vectors; (e) SV 40 vectors; (f) polyoma virus vectors; (g) papilloma virus vectors; (h) picornavirus vectors; (i) pox virus vectors such as an orthopox, e.g., vaccinia virus vectors or avipox, e.g. canary pox or fowl pox; and (j) a helper-dependent or gutless adenovirus.

In some embodiments, adenoviruses can be used to deliver nucleic acid agents for increasing the expression or activity of PIEZO 1. Adenoviruses have the advantage of being capable of infecting non-dividing cells. Kozarsky and Wilson, Current Opinion in Genetics and Development 3:499-503 (1993) present a review of adenovirus-based gene therapy.

In some embodiments, nucleic acid agents for increasing the expression or activity of PIEZO 1 can be delivered by adeno-associated virus (AAV) vectors. In some embodiments, a AAV vector that expresses a nucleic acid agent for increasing the expression or activity of PIEZO 1 is a recombinant AAV vector having, for example, either an U6 or Hl RNA promoter, or a cytomegalovirus (CMV) promoter. Suitable AAV vectors for use in agents, compositions, and methods described include, but are not limited to AAVs described in Passini et al., Methods Mol. Biol. 246: 225-36 (2004).

In another embodiments, the agent comprises a CRISPR activation agent and/or a sgRNA. In some embodiments, the agent is an sgRNA. An sgRNA combines tracrRNA and crRNA, which are separate molecules in the native CRISPR/Cas9 system, into a single RNA construct, simplifying the components needed to use CRISPR activation system. In some embodiments, the crRNA of the sgRNA has complementarity to PIEZO 1 DNA.

Antibody Agents

Antibodies may be polyclonal or monoclonal; xenogeneic, allogeneic, or syngeneic; or modified forms thereof (e.g. humanized, chimeric, etc.). Antibodies may also be fully human. The terms “monoclonal antibodies” and “monoclonal antibody composition”, as used herein, refer to a population of antibody polypeptides that contain only one species of an antigen binding site capable of immunoreacting with a particular epitope of an antigen, whereas the term “polyclonal antibodies” and “polyclonal antibody composition” refer to a population of antibody polypeptides that contain multiple species of antigen binding sites capable of interacting with a particular antigen. A monoclonal antibody composition typically displays a single binding affinity for a particular antigen with which it immunoreacts.

In certain embodiments, the methods and compositions provided herein relate to antibodies and antigen binding fragments thereof that bind specifically to a PIEZO 1 protein. Such antibodies can be polyclonal or monoclonal and can be, for example, murine, chimeric, humanized or fully human. In some embodiments, the agent may be a recombinant antibodies specific for a PIEZO 1 protein, such as chimeric or humanized monoclonal antibodies, can be made using standard recombinant DNA techniques. Such chimeric and humanized monoclonal antibodies can be produced by recombinant DNA techniques known in the art, for example using methods described in US Pat No. 4,816,567; US Pat. No. 5,565,332; Better et al. (1988) Science 240: 1041-1043; Liu et al. (\9KT) Proc. Natl. Acad. Sci. USA 84:3439-3443; Liu et al. (1987) J. Immunol. 139:3521-3526; Sun et al. (1987) Proc. Natl. Acad. Sci. 84:214-218; Nishimura et al. (1987) Cancer Res. 47:999- 1005; Wood et al. (1985) Nature 314:446-449; and Shaw et al. (1988) J. Natl. Cancer Inst. 80: 1553-1559); Morrison, S. L. (1985) Science 229: 1202-1207; Oi et al. (1986) Biotechniques 4:214; Winter U.S. Patent 5,225,539; Jones et al. (1986) Nature 321 :552- 525; Verhoeyan et al. (1988) Science 239: 1534; and Beidler et al. (1988) J. Immunol. 141 :4053-4060.

Human monoclonal antibodies specific for a PIEZO 1 protein can be generated using transgenic or transchromosomal mice carrying parts of the human immune system rather than the mouse system. For example, “HuMAb mice” which contain a human immunoglobulin gene miniloci that encodes unrearranged human heavy (p and y) and K light chain immunoglobulin sequences, together with targeted mutations that inactivate the endogenous p and K chain loci (Lonberg, N. et al. (1994) Nature 368(6474): 856 859). Accordingly, the mice exhibit reduced expression of mouse IgM or K, and in response to immunization, the introduced human heavy and light chain transgenes undergo class switching and somatic mutation to generate high affinity human IgGK monoclonal antibodies (Lonberg, N. et al. (1994), supra; reviewed in Lonberg, N. (1994) Handbook of Experimental Pharmacology 113:49 101; Lonberg, N. and Huszar, D. (1995) Intern. Rev. Immunol. Vol. 13: 65 93, and Harding, F. and Lonberg, N. (1995) Ann. N. Y Acad. Sci 764:536 546). The preparation of HuMAb mice is described in Taylor, L. et al. (1992) Nucleic Acids Research 20:6287 6295; Chen, J. et al. (1993) International Immunology 5: 647 656; Tuaillon et al. (1993) Proc. Natl. Acad. Sci USA 90:3720 3724; Choi et al. (1993) Nature Genetics 4: 117 123; Chen, J. et al. (1993) EMBO J. 12: 821 830; Tuaillon et al. (1994) J. Immunol. 152:2912 2920; Lonberg et al, (1994) Nature 368(6474): 856 859; Lonberg, N. (1994) Handbook of Experimental Pharmacology 113:49 101; Taylor, L. et al. (1994) International Immunology 6: 579 591; Lonberg, N. and Huszar, D. (1995) Intern. Rev. Immunol. Vol. 13: 65 93; Harding, F. and Lonberg, N. (1995) Ann. N.Y. Acad. Sci 764:536 546; Fishwild, D. et al. (1996) Nature Biotechnology 14: 845 851. See further, U.S. Pat. Nos. 5,545,806; 5,569,825; 5,625,126; 5,633,425; 5,789,650; 5,877,397; 5,661,016; 5,814,318; 5,874,299; 5,770,429; and 5,545,807.

Small Molecule Agents

Certain embodiments of the methods and compositions disclosed herein relate to the use of small molecule agents e.g., small molecule agents that increase or stabilize the expression or activity of a PIEZO 1 protein. Such agents include those known in the art and those identified using the screening assays described herein. A small molecule provided herein may have at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% specificity for a PIEZO 1 protein.

In some embodiments, assays used to identify agents (e.g., anti-cancer agents) in the methods described herein include obtaining a population of cells and a small molecule agent, wherein the cells are incubated with a small molecule agent and the level of a PIEZO 1 protein is measured. Agents identified via such assays, may be useful, for example, for increasing the expression or the activity of a PIEZO 1 protein in a subject.

Agents useful in the methods disclosed herein may be obtained from any available source, including systematic libraries of natural and/or synthetic compounds. Agents may also be obtained by any of the numerous approaches in combinatorial library methods known in the art, including: biological libraries; peptoid libraries (libraries of molecules having the functionalities of peptides, but with a novel, non-peptide backbone which are resistant to enzymatic degradation but which nevertheless remain bioactive; see, e.g., Zuckermann et al, 1994, J. Med. Chem. 37:2678-85); spatially addressable parallel solid phase or solution phase libraries; synthetic library methods requiring deconvolution; the 'one-bead one-compound' library method; and synthetic library methods using affinity chromatography selection. The biological library and peptoid library approaches are limited to peptide libraries, while the other four approaches are applicable to peptide, non-peptide oligomer or small molecule libraries of compounds (Lam, 1997, Anticancer Drug Des. 12: 145). Examples of methods for the synthesis of molecular libraries can be found in the art, for example in: DeWitt et al. (1993) Proc. Natl. Acad. Sci. U.S.A. 90:6909; Erb et al. (1994) Proc. Natl. Acad. Sci. USA 91 : 11422; Zuckermann et al. (1994). J. Med. Chem. 37:2678; Cho et al. (1993) Science 261 : 1303; Carrell et al. (1994) Angew. Chem. Int. Ed. Engl. 33:2059; Carell et al. (1994) Angew. Chem. Int. Ed. Engl. 33:2061; and in Gallop et al. (1994) J. Med. Chem. 37: 1233.

Libraries of agents may be presented in solution (e.g., Houghten, 1992, Biotechniques 13:412-421), or on beads (Lam, 1991, Nature 354:82-84), chips (Fodor, 1993, Nature 364:555-556), bacteria and/or spores, (Ladner, USP 5,223,409), plasmids (Cull et al, 1992, Proc Natl Acad Sci USA 89: 1865-1869) or on phage (Scott and Smith, 1990, Science 249:386-390; Devlin, 1990, Science 249:404-406; Cwirla et al, 1990, Proc. Natl. Acad. Sci. 87:6378-6382; Felici, 1991, J. Mol. Biol. 222:301-310; Ladner, supra.).

Pharmaceutical Compositions

In certain embodiments, provided herein is a composition, e.g., a pharmaceutical composition, containing at least one agent described herein together with a pharmaceutically acceptable carrier. In one embodiment, the composition includes a combination of multiple (e.g., two or more) agents described herein.

In some embodiments, the pharmaceutical composition is delivered locally or systemically. In some embodiments, the pharmaceutical composition may be administered to a tumor present in the subject. In some embodiments, the agent or pharmaceutical composition is administered with an additional cancer therapeutic agent. In some embodiments, the additional cancer therapeutic agent is a chemotherapeutic agent. In some embodiments, the pharmaceutical composition further comprises an additional agent for treatment of cancer. In some embodiments, the additional agent is a tumor vaccine. In certain embodiments, the additional therapeutic agent is a chemotherapeutic agents include alkylating agents such as thiotepa and cyclophosphamide (Cytoxan™); alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; emylerumines and memylamelamines including alfretamine, triemylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, and trimemylolomelamine; acetogenins (especially bullatacin and bullatacinone); a camptothecin (including synthetic analogue topotecan); bryostatin; cally statin; CC-1065 (including its adozelesin, carzelesin and bizelesin synthetic analogues); cryptophycins (articularly cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including the synthetic analogues, KW-2189 and CBI-TMI); eleutherobin; pancrati statin; a sarcodictyin; spongistatin; nitrogen mustards such as chlorambucil, chlornaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosoureas such as carmustine, chlorozotocin, foremustine, lomustine, nimustine, ranimustine; antibiotics such as the enediyne antibiotics (e.g., calicheamicin, especially calicheamicin gammall and calicheamicin phili); dynemicin, including dynemicin A; bisphosphonates, such as clodronate; an esperamicin; as well as neocarzinostatin chromophore and related chromoprotein enediyne antibiotic chromomophores), aclacinomysins, actinomycin, authramycin, azaserine, bleomycins, cactinomycin, carabicin, carrninomycin, carzinophilin, chromomycins, dactinomycin, daunorubicin, detorubicin, 6- diazo-5-oxo-L-norleucine, doxorubicin (Adramycin™) (including morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins such as mitomycin C, mycophenolic acid, nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; antimetabolites such as methotrexate and 5 -fluorouracil (5-FU); folic acid analogues such as demopterin, methotrexate, pteropterin, trimetrexate; purine analogs such as fludarabine, 6- mercaptopurine, thiamiprine, thioguanine; pyrimidine analogues such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine; androgens such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone; anti-adrenals such as aminoglutethimide, mitotane, trilostane; folic acid replinisher such as frolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine; hestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elformthine; elliptinium acetate; an epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidamine; maytansinoids such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidamol; nitracrine; pentostatin; phenamet; pirarubicin; losoxantrone; podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK™; razoxane; rhizoxin; sizofiran; spirogermanium; tenuazonic acid; triaziquone; 2, 2', 2"- tricUorotriemylamine; trichothecenes (especially T-2 toxin, verracurin A, roridin A and anguidine); urethane; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C"); cyclophosphamide; thiopeta; taxoids, e.g., paclitaxel (Taxol™, Bristol Meyers Squibb Oncology, Princeton, N.J.) and docetaxel (Taxoteret™, Rhone-Poulenc Rorer, Antony, France); chlorambucil; gemcitabine (Gemzar™); 6-thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisplatin and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitroxantrone; vancristine; vinorelbine (Navelbine™); novantrone; teniposide; edatrexate; daunomycin; aminopterin; xeoloda; ibandronate; CPT-11; topoisomerase inhibitor RFS 2000; difluoromethylornithine (DMFO); retinoids such as retinoic acid; capecitabine; and pharmaceutically acceptable salts, acids or derivatives of any of the above. Also included in the definition of “chemotherapeutic agent” are anti -hormonal agents that act to regulate or inhibit hormone action on tumors such as anti-estrogens and selective estrogen receptor modulators (SERMs), including, for example, tamoxifen (including Nolvadex™), raloxifene, droloxifene, 4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone, and toremifene (Fareston™); inhibitors of the enzyme aromatase, which regulates estrogen production in the adrenal glands, such as, for example, 4(5)-imidazoles, aminoglutethimide, megestrol acetate (Megace™), exemestane, formestane, fadrozole, vorozole (Rivisor™), letrozole (Femara™), and anastrozole (Arimidex™); and antiandrogens such as flutamide, nilutamide, bicalutamide, leuprohde, and goserelin; and pharmaceutically acceptable salts, acids or derivatives of any of the above. In some embodiments, the additional cancer therapeutic agent is an immune checkpoint inhibitor. Immune Checkpoint inhibition broadly refers to inhibiting the checkpoints that cancer cells can produce to prevent or downregulate an immune response. Examples of immune checkpoint proteins are CTLA-4, PD-1, VISTA, B7-H2, B7-H3, PD-L1, B7-H4, B7-H6, ICOS, HVEM, PD-L2, CD160, gp49B, PIR-B, KIR family receptors, TIM-1, TIM-3, TIM- 4, LAG-3, BTLA, SIRPalpha (CD47), CD48, 2B4 (CD244), B7.1, B7.2, ILT-2, ILT-4, TIGIT, HHLA2, butyrophilins, A2aR, and combinations thereof.

The pharmaceutical compositions and/or agents disclosed herein may be specially formulated for administration in solid or liquid form, including those adapted for the following: (1) oral administration, for example, drenches (aqueous or non-aqueous solutions or suspensions), tablets, e.g., those targeted for buccal, sublingual, and systemic absorption, boluses, powders, granules, pastes for application to the tongue; or (2) parenteral administration, for example, by subcutaneous, intramuscular, intravenous, intrathecal, intracerebral or epidural injection as, for example, a sterile solution or suspension, or sustained-release formulation. Methods of preparing pharmaceutical formulations or compositions include the step of bringing into association an agent described herein with the carrier and, optionally, one or more accessory ingredients. In general, the formulations are prepared by uniformly and intimately bringing into association an agent described herein with liquid carriers, or finely divided solid carriers, or both, and then, if necessary, shaping the product.

Pharmaceutical compositions suitable for parenteral administration comprise one or more agents described herein in combination with one or more pharmaceutically-acceptable sterile isotonic aqueous or nonaqueous solutions, dispersions, suspensions or emulsions, or sterile powders which may be reconstituted into sterile injectable solutions or dispersions just prior to use, which may contain sugars, alcohols, antioxidants, buffers, bacteriostats, solutes which render the formulation isotonic with the blood of the intended recipient or suspending or thickening agents.

Examples of suitable aqueous and nonaqueous carriers which may be employed in the pharmaceutical compositions include water, ethanol, dimethyl sulfoxide (DMSO), polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of coating materials, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.

Therapeutic Methods

In some aspects, provided herein are methods of treating a cancer by administering to a subject (e.g., to a tumor present in a subject) an agent and/or a pharmaceutical composition described herein. In some aspects, the subject is a human.

In some embodiments, the methods described herein may be used to treat any cancerous or pre-cancerous tumor. In some embodiments, the cancer includes a solid tumor. Cancers that may be treated by methods and compositions provided herein include, but are not limited to, cancer cells from the bladder, blood, bone, bone marrow, brain, breast, colon, esophagus, gastrointestine, gum, head, kidney, liver, lung, nasopharynx, neck, ovary, prostate, skin, stomach, testis, tongue, or uterus. In addition, the cancer may specifically be of the following histological type, though it is not limited to these: neoplasm, malignant; carcinoma; carcinoma, undifferentiated; giant and spindle cell carcinoma; small cell carcinoma; papillary carcinoma; squamous cell carcinoma; lymphoepithelial carcinoma; basal cell carcinoma; pilomatrix carcinoma; transitional cell carcinoma; papillary transitional cell carcinoma; adenocarcinoma; gastrinoma, malignant; cholangiocarcinoma; hepatocellular carcinoma; combined hepatocellular carcinoma and cholangiocarcinoma; trabecular adenocarcinoma; adenoid cystic carcinoma; adenocarcinoma in adenomatous polyp; adenocarcinoma, familial polyposis coli; solid carcinoma; carcinoid tumor, malignant; branchiolo-alveolar adenocarcinoma; papillary adenocarcinoma; chromophobe carcinoma; acidophil carcinoma; oxyphilic adenocarcinoma; basophil carcinoma; clear cell adenocarcinoma; granular cell carcinoma; follicular adenocarcinoma; papillary and follicular adenocarcinoma; nonencapsulating sclerosing carcinoma; adrenal cortical carcinoma; endometrioid carcinoma; skin appendage carcinoma; apocrine adenocarcinoma; sebaceous adenocarcinoma; ceruminous adenocarcinoma; mucoepidermoid carcinoma; cystadenocarcinoma; papillary cystadenocarcinoma; papillary serous cystadenocarcinoma; mucinous cystadenocarcinoma; mucinous adenocarcinoma; signet ring cell carcinoma; infiltrating duct carcinoma; medullary carcinoma; lobular carcinoma; inflammatory carcinoma; mammary paget's disease; acinar cell carcinoma; adenosquamous carcinoma; adenocarcinoma w/squamous metaplasia; malignant thymoma; malignant ovarian stromal tumor; malignant thecoma; malignant granulosa cell tumor; and malignant roblastoma; sertoli cell carcinoma; malignant leydig cell tumor; malignant lipid cell tumor; malignant paraganglioma; malignant extra-mammary paraganglioma; pheochromocytoma; glomangiosarcoma; malignant melanoma; amelanotic melanoma; superficial spreading melanoma; malignant melanoma in giant pigmented nevus; epithelioid cell melanoma; malignant blue nevus; sarcoma; fibrosarcoma; malignant fibrous histiocytoma; myxosarcoma; liposarcoma; leiomyosarcoma; rhabdomyosarcoma; embryonal rhabdomyosarcoma; alveolar rhabdomyosarcoma; stromal sarcoma; malignant mixed tumor; mullerian mixed tumor; nephroblastoma; hepatoblastoma; carcinosarcoma; malignant mesenchymoma; malignant brenner tumor; malignant phyllodes tumor; synovial sarcoma; malignant mesothelioma; dysgerminoma; embryonal carcinoma; malignant teratoma; malignant struma ovarii; choriocarcinoma; malignant mesonephroma; hemangiosarcoma; malignant hemangioendothelioma; kaposi's sarcoma; malignant hemangiopericytoma; lymphangiosarcoma; osteosarcoma; juxtacortical osteosarcoma; chondrosarcoma; malignant chondroblastoma; mesenchymal chondrosarcoma; giant cell tumor of bone; ewing's sarcoma; malignant odontogenic tumor; ameloblastic odontosarcoma; malignant ameloblastoma; ameloblastic fibrosarcoma; malignant pinealoma; chordoma; malignant glioma; ependymoma; astrocytoma; protoplasmic astrocytoma; fibrillary astrocytoma; astroblastoma; glioblastoma; oligodendroglioma; oligodendroblastoma; primitive neuroectodermal; cerebellar sarcoma; ganglioneuroblastoma; neuroblastoma; retinoblastoma; olfactory neurogenic tumor; malignant meningioma; neurofibrosarcoma; malignant neurilemmoma; malignant granular cell tumor; malignant lymphoma; Hodgkin's disease; Hodgkin's lymphoma; paragranuloma; small lymphocytic malignant lymphoma; diffuse large cell malignant lymphoma; follicular malignant lymphoma; mycosis fungoides; other specified non-Hodgkin's lymphomas; malignant histiocytosis; multiple myeloma; mast cell sarcoma; immunoproliferative small intestinal disease; leukemia; lymphoid leukemia; plasma cell leukemia; erythroleukemia; lymphosarcoma cell leukemia; myeloid leukemia; basophilic leukemia; eosinophilic leukemia; monocytic leukemia; mast cell leukemia; megakaryoblastic leukemia; myeloid sarcoma; and hairy cell leukemia.

In some embodiments, the subject has cancer. In some embodiments, the cancer comprises a solid tumor. In some embodiments, the tumor is an adenocarcinoma, an adrenal tumor, an anal tumor, a bile duct tumor, a bladder tumor, a bone tumor, a blood born tumor, a brain/CNS tumor, a breast tumor, a cervical tumor, a colorectal tumor, an endometrial tumor, an esophageal tumor, an Ewing tumor, an eye tumor, a gallbladder tumor, a gastrointestinal, a kidney tumor, a laryngeal or hypopharyngreal tumor, a liver tumor, a lung tumor, a mesothelioma tumor, a multiple myeloma tumor, a muscle tumor, a nasopharyngeal tumor, a neuroblastoma, an oral tumor, an osteosarcoma, an ovarian tumor, a pancreatic tumor, a penile tumor, a pituitary tumor, a primary tumor, a prostate tumor, a retinoblastoma, a Rhabdomyosarcoma, a salivary gland tumor, a soft tissue sarcoma, a melanoma (such as uveal melanoma (UVM), a metastatic tumor, a basal cell carcinoma, a Merkel cell tumor, a testicular tumor, a thymus tumor, a thyroid tumor, a uterine tumor, a vaginal tumor, a vulvar tumor, or a Wilms tumor. Actual dosage levels of the active ingredients in the pharmaceutical compositions or agents to be administered may be varied so as to obtain an amount of the active ingredient (e.g., an agent described herein) which is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient.

The compositions disclosed herein may be administered over any period of time effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient. The period of time may be at least 1 day, at least 10 days, at least 20 days, at least 30, days, at least 60 days, at least three months, at least six months, at least a year, at least three years, at least five years, or at least ten years. The dose may be administered when needed, sporadically, or at regular intervals. For example, the dose may be administered monthly, weekly, biweekly, triweekly, once a day, or twice a day. In certain embodiments, a dose of the composition is administered at regular intervals over a period of time. In some embodiments, a dose of the composition is administered at least once a week. In some embodiments, a dose of the composition is administered at least twice a week. In certain embodiments, a dose of the composition is administered at least three times a week. In some embodiments, a dose of the composition is administered at least once a day. In some embodiments, a dose of the composition is administered at least twice a day. In some embodiments, doses of the composition are administered for at least 1 week, for at least 2 weeks, for at least 3 weeks, for at least 4 weeks, for at least 1 month, for at least 2 months, for at least 3 months, for at least 4 months, for at least 5 months, for at least 6 months, for at least 1 year, for at least two years, at least three years, or at least five years.

The selected dosage level will depend upon a variety of factors including the activity of the particular agent employed, the route of administration, the time of administration, the rate of excretion or metabolism of the particular compound being employed, the duration of the treatment, other drugs, compounds and/or materials used in combination with the particular compound employed, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well known in the medical arts.

A physician having ordinary skill in the art can readily determine and prescribe the effective amount of the pharmaceutical composition required. For example, the physician could prescribe and/or administer doses of the compounds employed in the pharmaceutical composition at levels lower than that required in order to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved.

Exemplification

Example 1: Proximity proteomics identifies PD-1 and PIEZO1 association

Previous proteomic studies involving PD-1 pulldown have identified direct binding partners of PD-1 but have not been successful in tracking the time-resolved behavior of proteins recruited to PD-1 following PD-L1 ligation. To overcome these issues and to test if important factors in PD-1 -mediated CD8 ⁺ T cell inhibition remain unidentified, proximitylabeling combined with multiplexed quantitative mass spectrometry was utilized. This made possible time-resolved detection of proteins lying within ~10nm radius of the PD-1 cytoplasmic tail in the presence or absence of PD-L1 ligation (Fig. 1A). Jurkat cell lines stably expressing human or murine PD-1 fused to an ascorbate peroxidase 2 (APEX2) labeling probe via the C-terminus of the PD-1 cytoplasmic tail was utilized (Fig. 5A). PD- l-APEX2-expressing Jurkat cells were sorted and assessed for labeling efficiency using anti-streptavidin Western blotting (Fig. 5B,C). Tosyl-activated Dynabeads were covalently coupled to CD3 and CD28 crosslinking antibodies using either control murine IgGi ligand (mlgGi, TCR-control) or recombinant PD-Ll-Fc fusion protein (TCR-PD-L1) to provide TCR and co-stimulatory signals for T cell activation while also driving strong inhibitory signals through PD-1, respectively (Fig. 6). Following incubation in labeling media containing biotinyl tyramide, PD-l-APEX2-expressing Jurkat cells were co-cultured with TCR-PD-L1 or TCR-control beads for 2, 5, 10 and 20 min while the 0 min condition contained no beads (Fig. IB). Biotinylation of proteins proximal to the PD-1 cytoplasmic tail was induced by the addition of H2O2 to the celkbead mixture during the last minute of each timepoint (Fig. 1 A, Fig. 5C). Cell pellets were lysed and biotinylated proteins were enriched using streptavidin beads under denaturing conditions. Trypsin digest-derived peptides were labelled with tandem mass tags (TMT) and quantified by triple-stage mass spectrometry (MS ³ ) ³¹ .

To quantify proteins co-localized with PD-1 over time following PD-L1 ligation relative to TCR/CD28 stimulation alone, scaled TMT ratios (TMT RA) of identified peptides for each condition were calculated. A strong 2-3 -fold, time-dependent enrichment of SHP2 following TCR-PD-L1 ligation was observed as compared to TCR-control stimulation. Enrichment was observed using either human or murine PD-1 peroxidase fusion proteins, confirming that SHP2 is recruited to PD-1 following ligation (Fig. 1C, Fig. 7 A). A robust 2-3-fold enrichment of the mechanosensitive ion channel PIEZO1 following PD-L1 ligation compared to TCR-control stimulation using both human and murine PD-1 peroxidase fusion proteins was observed (Fig. ID, Fig. 7B): among 1247 quantified proteins, the peptides corresponding to PIEZO 1 fell within a small group that associated with PD-1 in a time-dependent manner following PD-L1 ligation. One-way hierarchical data clustering confirmed near-identical recruitment kinetics for SHP2 and PIEZO 1 to PD-1 (Fig. lH). Thus, following PD-L1 binding to PD-1, PIEZO1 associates with PD-1.

The recruitment kinetics of previously reported mediators and targets of PD-1 signaling, including SHP1, CD3 subunits, CD28 and Zap70 were also identified. While significant enrichment of SHP1 following 10 and 20 min of PD-1 ligation (as compared to TCR stimulation alone) was detected, the degree of enrichment was markedly lower as compared to SHP2 or PIEZO1 (Fig. IE, Fig. 7C). Although CD3 subunits, CD28 and ZAP70 were identified as binding to PD-1, significant increases in association of these proteins with PD-1 following PD-L1 ligation were not observed. These data suggest that CD3 subunits, CD28 and ZAP70 remain proximal to PD-1 at the immune synapse in the presence or absence of PD-L1 ligation (Fig. 1F-1G, Fig. 7D-I and Fig. 8A-D). One-way hierarchical clustering confirmed different recruitment kinetics for SHP1, CD3 subunits, CD28 or Zap70 (Fig. 1H, Fig. 8E). The limited changes in PD-1 binding by known mediators as compared to the robust, ligand-dependent association of PIEZO 1 and PD-1 implicates an uncharacterized mechanism of PD-1 that involves PIEZO1 function.

To further assess the landscape of protein localization changes proximal to PD-1, gProfiler g:OSt gene ontology (GO) term functional enrichment analysis was used to compare pathways that were enriched in TCR-PD-L1 or TCR-control stimulated conditions. It was found that clathrin-mediated endocytosis and membrane trafficking were among the most significantly enriched pathways in TCR-PD-L1 conditions as compared to TCR-controls, suggesting that PD-1 ligation may alter receptor clustering and turnover in the plasma membrane to prevent stable contact with antigen presenting cells at the immune synapse (Fig. II). As anticipated, signaling pathways involved in TCR and immune response activation, as well as actin cytoskeleton organization, were enriched in TCR- control conditions compared to those treated with TCR-PD-L1 beads (Fig. II). When the mean slope for each gene across all time points was calculated in both TCR-PD-L1 and TCR-control conditions as a means to rank the most highly enriched proteins, SHP2 and PIEZO 1 ranked highest along with sorting nexins involved in intracellular trafficking (SNX12), actin polymerizing subunits (ARPC2, TFG) and membrane transporters (SLC34A1, SLC44A1) (Fig. 1J). In addition, the observed enrichment for PD-1 following PD-L1 ligation signifies an increase in local PD-1 concentration, indicating clustering of PD-1 molecules that undergo enhanced biotinylation (Fig. 1 J). As expected, various TCR- and CD28-related genes enriched in the TCR-control -treated samples, including CD28, CD3^, ZAP70, VAV3 and PI3K-related proteins were identified (Fig. 1 J). Collectively, these data highlight the changing membrane dynamics that occur following TCR stimulation and PD-L1 ligation in T cells, which not only confirm previous findings, but also identify the association between PD-1 and PIEZO1.

Example 2: PD-1 ligation inhibits PIEZOl-mediated Ca ²⁺ influx

Since PIEZO 1 activation has been shown to promote optimal TCR signaling and PD-1 ligation attenuates T cell function, it was hypothesized that PD-1 association with PIEZO 1 would result in PIEZO 1 inhibition and thereby impair T cell activation. Upon TCR stimulation, there is a tightly regulated response to Ca ²⁺ influx, with Ca ²⁺ release-activated channels (CRAC) and voltage gated Ca ²⁺ channels being necessary for the TCR signaling cascade. Due to the involvement of multiple Ca ²⁺ channels, uncoupling the individual contribution of each ion channel during T cell activation is challenging. The role of PIEZO 1 in T cell activation and its regulation by PD-1 using a Jurkat cell line stably expressing an inducible, fluorescent PIEZO 1 -specific activity reporter (GenEPi) was exploreed. GenEPi contains a low affinity and high dynamic range GFP, calmodulin and Ml 3 peptide fusion protein (GCaMP) engineered to ensure the specificity of sensing PIEZO- 1 -mediated Ca ²⁺ influx. The GenEPi reporter was expressed using a Tet-On system and is comprised of GCaMP fused to the cytoplasmic tail of PIEZO 1 using a flexible linker (Fig. 2A). GenEPi Jurkat cells exhibited increasing levels of reporter expression and PIEZO 1 activity with increasing doses of doxycycline in the presence of the PIEZO 1 agonist Yodal, verifying the inducibility and functional specificity of GenEPi (Fig. 9A).

To visualize and compare changes in PIEZO1 activity in the presence or absence of PD-1 ligation, PD-1 -expressing GenEPi Jurkat cells were passed through a flow-cell chamber coated with crosslinking CD3 and CD28 antibodies and either PD-L1 or control mlgGi ligand (Fig. 9B). Using Total Internal Reflection Fluorescence (TIRF) time lapse imaging, the intensity of PIEZO 1 activity and PD-1 expression were assessed at the ligandcell interface at various times after TCR-control and TCR-PD-L1 stimulation (Fig. 2B and 2C and Fig. 9B). It was found that PIEZO 1 activation in GenEPi Jurkat cells ligated with PD-L1 was delayed and had lower intensity overall as compared to TCR-stimulated cells, while PD-1 intensity remained the same in both conditions, suggesting that PD-1 inhibits PIEZO 1 activation (Fig. 2B and 2E, Fig. 9C). Specifically, GenEPi Jurkat cells exhibited strong PIEZO 1 activity following 100 s of TCR stimulation that peaked at 200 s and persisted for 5 min (300 s), further confirming that TCR stimulation induces PIEZO1 activity (Fig. 2B, and 2E-2F, Fig. 9D, and captured via extended videos, incorporated herein by reference). This activity coincided with F-actin ring formation, where PIEZO1 activity appeared to localize around F-actin ring structures (Fig. 2B). A significant decrease in PIEZO1 activity over time in the presence of PD-L1 was observed; PIEZO1 activity was visible starting at 140 s, but remained less intense than in TCR-stimulated conditions throughout the time course (Fig. 2C, and 2E-2F, and captured via extended videos, incorporated herein by reference). Interestingly, local PIEZO 1 activity was significantly decreased upon PD-1 ligation in the presence of PD-L1 at PD-1 clustering sites during the process of immune synapse and F-actin ring formation implying that PD-1 ligation results in the attenuation of PIEZO 1 activity through physical association (Fig. 2C,2G-2K).

In complementary studies flow cytometry was used to assess changes in PIEZO 1 activity following PD-1 ligation by quantifying mean fluorescence intensity (MFI) of GCaMP fluorescence and frequency of GFP -positive, PD-1 -expressing GenEPi Jurkat cells treated with TCR-control or TCR-PD-L1 beads. PD-1 -expressing GenEPi Jurkat cells exhibited a significant decrease in PIEZO1 activity following exposure to TCR-PD-L1 beads compared to GenEPi Jurkat cells treated with TCR-control beads or PIEZO 1 agonist Yodal (Fig. 9E-G). Together, these data imply that contractile F-actin rings formed at the immune synapse provide sufficient force to gate PIEZO1 in an open conformation, thereby sustaining Ca ²⁺ influx for optimal TCR signaling (Fig. 2C). Importantly, it was demonstrated that this activity is inhibited by PD-1 ligation (Fig. 2C).

Example 3: CD8 ⁺ -specific PIEZO1 KO impairs antitumor immunity

To investigate PD-1 and PIEZO1 function specifically in CD8 ⁺ T cells, a tamoxifen- inducible CD8 ⁺ -specific PIEZO 1 KO mouse was developed by crossing Piezo l^ ^x mice with E8i-Cre-ER ^T2 Rosa26 tdTomato reporter mice (Extended Data Fig. 6a). Following tamoxifen treatment, the efficacy of PIEZO 1 deletion was -70% in Cre+ CD8 ⁺ T cells as compared to Cre- controls, as assessed by RT-qPCR using probes specific for the loxP regions flanking exons 20-23 of PIEZO 1 (Fig. 10B). The frequency of CD8 ⁺ T cells expressing Cre protein was consistent with decreased PIEZO 1 transcript levels assessed by RT-qPCR (Fig. 10B-D). As an additional control, the level of Cre reporter in CD8 ⁺ splenocytes was assessed on day 15 following tumor implantation and observed sustained Cre activity in Cre+ CD8 ⁺ T cells, confirming the efficacy of our inducible Cre system (Fig. 10E,F). Functionally, a significant decrease in PIEZO- 1 -specific Ca ²⁺ influx induced by Yodal treatment (visualized using the cell permeable Ca ²⁺ dye Indo-1) was observed in Cre+ CD8 ⁺ T cells compared to Cre- CD8 ⁺ T cells; Fig. 10G). In vitro stimulation of tamoxifen-treated Cre+ E8i-Cre-ER ^T2 Piezo l^ ^x/ ^ ^x CD?> ⁺ T cells with a titration of crosslinking CD3 and CD28 antibodies revealed slight decreases in viability, CD44, granzyme B and IFNy/TNFoc co-expression, as well as increased expression of CD62L at higher CD3/CD28 stimulation concentrations compared to controls (Extended Data Fig. 7a,d-f,h), suggesting that PIEZO 1 KO impairs CD8 ⁺ T cell activation and effector function. Significant differences were not observed in PD-1, CTLA4 and Ki-67 expression or production of IL-2, IFNy or TNFoc between the two groups (Fig. 11B-C, G, LK).

Given the increase in PIEZO 1 activity observed following TCR stimulation (Fig. 2D), it was hypothesized that loss of PIEZO 1 in CD8 ⁺ T cells would lead to more rapid tumor growth as a consequence of impaired T cell activation. Indeed, when MC38 or Bl 6- OVA tumor cells were implanted in tamoxifen-treated E8i-Cre-ER ^T2 Piezo mice and tumor growth was monitored, loss of PIEZO 1 in CD8 ⁺ T cells alone was sufficient to drive significantly increased tumor burden in Cre+ mice as compared to Cre- controls in both tumor models (Fig. 3A-3C). There also was a significant decrease in CD8 ⁺ T cell frequencies (Fig. 3D-3E and Fig. 12A), an increase in total CD4 ⁺ T cell frequencies and lower CD8 ⁺ /CD4 ⁺ T cell ratios in the tumor with no significant changes in CD3e frequencies (Fig. 12A-G). In addition, Cre+ CD8 ⁺ TILs from MC38 tumors expressed significantly more CD62L and significantly less PD-1, CTLA4 and CD69 compared to Cre- controls (Fig. 3F and Fig. 12H-J). Cre+ CD8 ⁺ TILs from B16-0VA tumors also expressed significantly more CD62L and significantly less CTLA4 as compared to Cre- controls but did not show significant differences in the frequencies of PD-1- or CD69-expressing cells although both trended toward decreased frequency (Fig. 3G and Fig. 12K-M). Moreover, Cre+ CD8 ⁺ TILs from B16-0VA tumors expressed significantly less granzyme B. CD8 ⁺ TILs from MC38 tumors also expressed less granzyme B, but this trend was not significant (Fig. 12N,O). Interestingly, PIEZO1 KO CD8 ⁺ TILs from MC38 and B16-0VA models consistently retained high expression of Slamf6 and had minimal expression of TIM-3 (Fig. 3H and 31, and Fig. 12P-S), suggesting that PIEZO1 is involved in regulating the transition of Slamf6 progenitor cells to TIM-3 effector-like CD8 ⁺ TILs, thereby controlling exhausted CD8 ⁺ T cell subpopulations. Together, these findings demonstrate that loss of PIEZO 1 activity in CD8 ⁺ T cells decreases their activation and acquisition of effector functions to impair antitumor immunity and promote tumor growth, similar to PD-1 -mediated inhibition of T cell activation.

To further study PD-1 regulation of PIEZO 1, E8i-CRE-ER ^T2 Pzezo7- ^/Zx ^ ^x Cre+ and Cre- tumor-bearing mice were treated with anti-PD-1 or isotype control antibody (Fig. 3J). Strikingly, administration of PD-1 blocking antibody on days 14 and 17 reduced clearance of MC38 tumors, which are highly sensitive to PD-1 blockade, in E8i-CRE-ER ^T2 Piezo P ^{lx f} ' ^x Cre+ mice compared to anti-PD-1 treated Cre- mice (Fig. 3K and Fig. 13A). Administration of PD-1 blockade on days 10 and 13 following tumor implantation did not significantly reduce growth of Bl 6-0 VA tumors in E8i-CRE-ER ^T2 Piezo l^ ^x/:flx CXQ+ mice in contrast to marked tumor control in anti-PD-1 treated Cre- mice (Fig. 3J,L, and Fig. 13B). PIEZO1 KO in CD8 ⁺ T cells significantly impaired overall survival in both tumor models regardless of PD-1 blockade (Fig. 3M and 3N). Thus, PD-l-mediated inhibition of PIEZO1 in CD8 ⁺ T cells contributes to immune evasion by tumors, underscoring the importance of PIEZO 1 function in shaping T cell responses.

Example 4: PIEZO1 agonism improves CD8 ⁺ T cell antitumor immunity

The impaired antitumor immunity observed in CD8 ⁺ -specific PIEZO 1 KO mice led us to ask whether PIEZO1 agonism would enhance antitumor immunity. To answer this question, MC38 tumors were implanted subcutaneously in WT mice and administered Yodal (7.5mg/kg) intraperitoneally (i.p.) (Fig. 4A). While Yodal treatment had variable effects on MC38 tumor growth in WT mice (Fig.14A-B), phenotypic analysis of the TILs at day 15 following MC38 tumor implantation revealed increased percentages of CD8 ⁺ TILs in Yodal-treated mice compared to controls (Fig. 4B). Additionally, Yodal-treated CD8 ⁺ TILs expressed more activation and effector proteins, including PD-1, granzyme B and perforin and lower expression of CD62L (Fig. 4C-F). PIEZO 1 -agonized CD8 ⁺ TILs expressed high levels of terminal exhaustion marker TIM-3 and low levels of progenitor stem-like marker Slamf6 compared to controls, further suggesting a role for PIEZO 1 in driving an effector-exhausted phenotype (Fig. 4G, Fig. l4C-D).

Next considered was if the increased CD8 ⁺ T cell effector function observed in PIEZO 1 -agonized CD8 ⁺ TILs would improve the efficacy of PD-1 blockade. To assess the effects of combined anti-PD-1 and PIEZO1 agonist Yodal treatment, the B16.F10 tumor model was chosen because it is unresponsive to PD-1 blockade (Fig. 4H). Mice treated with either PD-1 blockade or Yodal alone did not show decreased tumor growth (Fig. 41 and 4J). In marked contrast, tumor growth was significantly attenuated when PD-1 blockade and Yodal were administered together (Fig. 41 and 4J). Moreover, combined anti-PD-1 and Yodal treatment significantly improved survival in B16.F10 tumor-bearing mice as compared to single and negative controls, such that 40% of mice treated with combination therapy - but not mice treated with monotherapy - survived beyond 30 days (Fig. 4K). These findings point to PIEZO 1 agonism as a potential avenue for therapeutic intervention in cancer.

Example 5: Discussion

While PD-1 signaling counters T cell activation, the understanding of the signaling mechanisms by which PD-1 exerts its inhibitory functions is incomplete. Herein a novel function was identified for PD-1 in restraining T cell signals driven by a mechanosensitive ion channel. Specifically, PD-1 ligation induces co-localization of PD-1 with PIEZO 1 and this association impairs PIEZO 1 activation and subsequent Ca ²⁺ influx that drives T cell activation, acquisition of effector functions and key cell fate decisions. Knockout of PIEZO 1 in murine CD8 ⁺ T cells alone is sufficient to impair CD8 ⁺ TIL-mediated tumor control and this cannot be overcome by PD-1 blockade. Conversely, PIEZO 1 -agonism improves CD8 ⁺ T cell activation and function in the TME and has significant therapeutic benefit in mouse models when combined with PD-1 blockade to control tumor growth. These findings are the first to document coinhibitory receptor-mediated negative regulation of a mechanosensor and highlight PIEZO1 agonism in combination with PD-1 blockade as a possible therapeutic approach for targeting immune checkpoint blockade-resistant cancers.

Recent studies have demonstrated that PIEZO 1 -driven Ca ²⁺ influx in T cells leads to calpain activation and reorganization of cortical F-actin scaffolding, linking PIEZO 1 to optimal TCR signaling. In contrast, PD-1 ligation has been shown to impair F-actin clearance from the cellular interface, reduce Ca ²⁺ signaling and abrogate tumor cell killing through decreased tumor-T cell couplings. These findings confirm these opposing functions of PD-1 and PIEZO 1. Based on the data, PD-1 targets PIEZO 1 to prevent optimal T cell activation through inhibition of Ca ²⁺ influx and destabilization of the immune synapse (Fig. 2). This model is supported by the in vitro studies, which demonstrate that PD-1 ligation inhibits PIEZO 1 at the immune synapse, and the in vivo studies provided herein, which highlight the necessity of PIEZO 1 for promoting optimal CD8 ⁺ TIL function in tumor models and the inability of PD-1 blockade to reverse accelerated tumor growth resulting from PIEZO 1 KO in CD8 ⁺ T cells (Fig. 2, 3). These findings imply that expression of PIEZO1 is required for PD-1 to fully execute T cell inhibition (Fig. 3K-3N). Although PIEZO 1 agonism alone is not sufficient to consistently decrease tumor burden in WT mice (Fig. 14A,B), combining PD-1 blockade with PIEZO1 agonism improved tumor control in PD-1 blockade-unresponsive tumors (Fig. 4H-4K). The effectiveness of this combination may result from 1) prevention of PIEZO 1 inactivation as well as inhibitory phosphatase recruitment by PD-1 blockade and 2) PIEZO1 agonism further increasing the number and function of cytotoxic CD8 ⁺ T cells in the tumor, leading to enhanced tumor control.

PD-l-mediated inhibition of PIEZO1 is demonstrated herein (Fig. 2). PIEZO1 activity may be regulated by TCR-induced cytoskeletal rearrangements, specifically retrograde actin flow, which is supported by the data capturing PIEZO 1 activity concentrated around F-actin ring structures. The mechanism by which Ca ²⁺ influx is sustained to induce specific gene transcription following CRAC channel activation in T cells remains unclear. Previous studies have proposed a positive feedback loop between F- actin polymerization and Ca ²⁺ influx, which persists over hours at the immune synapse to maintain TCR signaling ⁷ . It is possible that actomyosin contractile forces modulate PIEZO 1 to sustain Ca ²⁺ influx, resulting in downstream gene transcription. Moreover, the proximity proteomics experiments demonstrate PD-1 co-localization with various proteins involved in actin polymerization, membrane trafficking and endocytosis (Fig. 1H-J). Thus, PD-1 sequestration of actin cytoskeletal regulatory proteins and proteins that mediate membrane turnover may play a role in impairing actin cytoskeletal dynamics and stability at the immune synapse, thereby attenuating actomyosin contractile forces that activate PIEZO 1.

Although previous proteomic methods have been used to characterize PD-l- mediated T cell inhibition, PD-1 co-localization with PIEZO1 and subsequent inhibition of PIEZO1 have never been documented. However, rather than studying PD-1 in isolation, insight into the spatial and temporal behavior of proteins involved in PD-1 signaling at the membrane was gained by using a multiplexed proximity proteomic approach that assesses changes in membrane dynamics, receptor clustering and protein recruitment immediately proximal to PD-1. Given the dynamic nature of immune cell receptor signaling, high- resolution proximity proteomic methods may be instrumental in studying additional macromolecular immunoreceptor complexes for which, despite clinical relevance, signaling remains largely unclear.

In summary, a new mechanism by which PD-1 exerts its inhibitory signals through negative regulation of the mechanosensitive ion channel PIEZO 1 is described herein. These studies are the first to demonstrate PD-1 co-localization with PIEZO 1 following PD-L1 ligation. This association results in the inhibition of PIEZO 1 -mediated Ca ²⁺ influx, while TCR/CD28 stimulation in the absence of PD-L1 ligation promotes PIEZO1 activity around F-actin rings. The selective inhibition of PIEZO 1 by PD-1 illuminates the significance of PIEZO 1 in regulating CD8 ⁺ T cell function and introduces a novel receptor target class. Moreover, these findings in mice show that modulating PIEZO 1 activity influences the anti-tumor activity of CD8 ⁺ T cells, subsequently affecting tumor growth. Strikingly, combined therapy using PIEZO1 agonist and PD-1 blockade improved tumor control in mice harboring tumors unresponsive to PD-1 blockade. Together, these studies expand our understanding of the diverse mechanisms by which PD-1 inhibits T cell function, identify a novel role for PIEZO 1 in controlling CD8 ⁺ T cell function and reveal a new combination therapeutic strategy using PD-1 blockade and PIEZO1 agonism. Further understanding of the mechanisms controlling PD-1 and PIEZO 1 association, the specific downstream pathways involved and the effects of external mechanical force on this biology should provide additional fundamental and therapeutic insights.

Example 6: Methods

Cell lines

Jurkat E6.1 and human PD-1 -expressing Jurkat E6.1 cells (35.8 line transduced with an ORF-T2A/IRES-GFP reporter) (a gift from N. Haining/K. Yates, Dana Farber Cancer Institute) were cultured in RPMI media supplemented with 10% FBS, 1% penicillin/streptomycin, 1% HEPES and 0.1% BME (denoted RIO). MC38-OVA (a gift from N. Haining/N. Collins, Dana-Farber Cancer Institute), B16-OVA (generated in collaboration with the N. Haining lab), MC38-WT (a gift from D. Vignali, University of Pittsburgh School of Medicine), B16.F10 (a gift from G. Dranoff, Novartis Institutes for Biomedical Research, Cambridge, MA) and 293x (a gift from C. Kadoch, Dana-Farber Cancer Institute) cells were cultured in DMEM supplemented with 10% FBS, 1% penicillin/streptomycin and 20 pg/mL gentamicin. MC38-OVA and B16-OVA cells were produced by transduction of parental MC38 and B16.F10 cells with the lentiviral vector TRC-pLX305 (Broad Institute) containing OVA protein.

PD-1-APEX2 plasmid design, cloning and stable expression

PD-1 sequences were obtained from NCBI and were modified using the codon optimization tool from Integrated DNA Technologies (IDT). APEX2 sequences have been previously described ¹⁷ . PD-1-APEX2 fusion sequences contain human or murine PD-1 linked to APEX2 on the C-terminal tail by an 8-residue serine-glycine linker (GGSSGGSS) and a FLAG-TAG added to the C-terminal tail of APEX2. All human and murine PD-1- APEX2 double-stranded gene fragments (gBlocks) were generated by IDT. Cloning of gBlock gene fragments into the pXPR_053 plasmid (Addgene #113591) was completed by GenScript.

To generate stable PD-l-APEX2-expressing Jurkat cell lines, 293x cells were transfected with PD-1-APEX2 fusion plasmids along with PsPax2 and MD2G packaging plasmids suspended in Optimem (Life Technologies #31985-062) and polyethylamine “Max” (PEI, Polysciences #24765-2), and supplemented with fresh media after 24 h. Lentivirus was harvested 48 h following media supplementation by ultracentrifugation for 2 h at 71,934.8 RCF at 4°C. Viral titer was calculated using serial dilutions of the viral stock on 293x cells in DMEM complete media containing PEI “Max”. A multiplicity of infection of 30 was used to spin transduce Jurkat cells for 2 h at 652 RCF at 37°C. Jurkat cells were stained with human PD-1 surface antibody (BioLegend, clone EH12.2H7, #329936) and evaluated for PD-1 expression using flow cytometry 24 h and 48 h following transduction. Highly expressing PD-1-APEX2 fusion clones were sorted on the BD Aria II. Stocks were frozen in RIO media containing 10% dimethyl sulfoxide (DMSO) for future experiments.

APEX2 proximity labeling and sample processing

PD-l-APEX2-expressing Jurkat cells were cultured at a concentration of 1 million cells/mL in labeling media (RPMI supplemented with 10% FBS, 1% penicillin/streptomycin, 1% HEPES, 0.1% BME and 500|iM biotinyl tyramide purchased from Toronto Research Chemicals) for 2 h. TCR-PD-L1 or TCR-control Dynabeads (see supplemental methods for PD-L1 bead generation) were added to PD-l-APEX2-expressing Jurkat cells at a ratio of 1 :4 cells/beads for the specified timepoints. No beads were added to the 0 min control to assess protein localization at baseline. Following each timepoint, hydrogen peroxide (H2O2, Sigma Aldrich) was added at a final concentration of ImM for 1 min to each condition not including the no H 2 O2 -treated control used to quantify background labelling. The labeling reaction was quenched using 2X quenching buffer (DPBS supplemented with 20mM sodium ascorbate, lOmM trolox and 20mM sodium azide) and cells were spun down immediately at 726 RCF for 2 min. Cells were immediately washed three times with ice-cold IX quenching buffer and spun down at 726 RCF for 2 min after each wash. Following the third IX quenching wash, cells were washed with 10 mL ice-cold PBS and 1 million cells were taken for Western blotting analysis. Cells were then spun down a final time at 726 RCF for 2 min. All PBS was removed from cell pellets, which were flash-frozen on dry ice and stored at -80°C until processing and streptavidin pulldown.

PD-l-APEX2-expressing Jurkat cell pellets were processed using previously described methods with slight modification ³ . Cell pellets were lysed in filtered 8M urea with 1% sodium dodecyl sulfate (SDS) at room temperature for 15 min. 55% ice-cold trichloroacetic acid (TCA) was added to lysates at a 1 : 1 ratio and left on ice for 15 min to precipitate the protein. Lysates were spun down at 20,817 RCF for 15 min at room temperature to prevent urea from precipitating out of solution. The supernatant was discarded, and protein pellets were washed 4X with ice-cold acetone and spun down at 20,817 RCF for 10 min following each wash. Protein pellets were then air-dried and resuspended in 8M urea lysis buffer supplemented with 1% SDS and lOmM tris(2- carboxyethyl)phosphine (TCEP, Sigma) and 100 mM NH4CO3. Pellets were water-bath sonicated for 3 cycles of 30 s and vortexed mixed at 37°C for 2 h until the pellets were completely resuspended. Redissolved pellets were checked for an alkaline pH and then spun down at 20,817 RCF for 15 min at room temperature. Clear supernatants were transferred to new microcentrifuge tubes and freshly prepared 50mM ammonium bicarbonate (NH4CO3) containing 400mM iodoacetamide was added to each sample at a final concentration of 20mM iodoacetamide. Samples were immediately vortexed and incubated in the dark at room temperature for 25 min. During the incubation period, streptavidin beads (Pierce) were washed twice with 4M urea and 0.5% SDS. Alkylation of cysteine resides was quenched using a solution of lOOmM dithiothreitol (DTT) containing streptavidin beads such that each sample received l OOpL beads and reached a final concentration of 50mM DTT, 4M urea and 0.5% SDS (1 : 1). Samples containing streptavidin beads were rotated overnight at 4°C and the following day were applied to the Dyna-Mag-2 to remove the supernatant. Beads were washed with 4M urea and 0.5% SDS and transferred to new Eppendorf tubes to decrease background signal. Beads were washed 3X more with 4M urea and 0.5% SDS followed by 3 washes with 4M urea to remove all detergent.

Mass spectrometry sample preparation

Streptavidin beads were digested with lysyl endopeptidase (LysC, 2mg/mL, Wako) in 50pL 200mM 3 -[4-(2-hydroxyethyl)piperazin-l-yl]propane-l -sulfonic acid (EPPS) pH 8.5 and 2% acetonitrile at 37°C. Trypsin (50pL stock, Promega #V5111, in EPPS buffer and at a final dilution of 1 : 100) was added for additional digest overnight at 37°C. Beads were removed with a magnetic rack and clear supernatants were transferred to new tubes. Peptide digest reactions were directly labelled with TMT1 Iplex (Thermo Fisher Scientific #A34808) reagents in 200mM EPPS pH 8.5, 30% acetonitrile for 1 h at room temperature. Labeling efficiency was measured by MS of mixed small aliquots of the labeling reactions and was >95%. Frozen and thawed TMT labeling reactions were quenched with 0.3% hydroxylamine for 15 min at room temperature. Reactions were then mixed, dried in a speed vac centrifuge to near completion and subjected to alkaline reversed phase fractionation (Thermo Fisher Scientific #84868) with 12 elution subsequent fractions of 10%, 12.5%, 15%, 17.5%, 20%, 25%, 30%, 35%, 40%, 50%, 65% and 80% acetonitrile. Fraction pairs 1+7, 2+8, 3+9, 4+10, 5+11, 6+12 were mixed, dried down to completion, desalted with Stage tips and run on Orbitrap Lumos mass spectrometers (Thermo Fisher Scientific).

Mass spectrometry analysis

Data collection followed a synchronous precursor selection (SPS) MS ³ TMT method ⁴⁹ . Peptides were separated prior to the electrospray ionization with a Proxeon EASY-nLC1200 system over a ~35cm capillary column of 100pm inner diameter packed with Accucore C18 beads (2.6 pm, 150A, Thermo Fisher Scientific). Service MS ¹ scans were performed in the Orbitrap at a resolution of 120,000, mass range 400-1400 thomson (Th). After collision induced dissociation (CID, CE=35) and using an isolation window of 0.4 m/z, MS ² was performed in the ion-trap with maximum injection times of 150-400 ms. For MS ³ quantification, precursors were selected following a Top 10 method followed by high-energy collision-induced dissociation (HCD, CE=65). Orbitrap MS ³ analysis was done at a resolution of 60,000 at 200 Th with varying injection times of up to 650 ms and chargestate dependent variable isolation windows from 0.7 to 1.2 Da as described previously ⁵⁰ . Peptide-spectrum matches (PSM) were obtained by a SEQUEST (v.28, rev.12) based software, searching a database with respective UniProt mouse and human reference proteomes with added common contaminants and reverse peptide sequences as decoy. After mzXML conversion of spectra, searches used a mass tolerance of 20 p.p.m. for precursors and a fragment-ion tolerance of 0.9 Da. Searches allowed for up to two missed trypsin cleavage sites with dynamic modification of oxidized methionines (+15.9949 Da) and static peptide N-terminal and lysine modifications with TMT11 (+229.1629 Da). PSM were filtered by linear discriminant analysis with a false discovery rate (FDR) of 1% and a following FDR of 1% for collapsed proteins. MS ¹ data were calibrated post search and searched again. TMT signal to noise quantification for peptides was filtered for an MS ² isolation specificity of 70% or greater and a summed signal to noise of 200 or greater for all TMT channels for each peptide. Details of TMT intensity quantification applied were described previously ⁵⁰ . Scaled quantification data were analyzed by one-way clustering (Ward’s method) using the JMP Pro statistical software package. Proteomics raw data and search results were deposited in the PRIDE archive ⁵¹ and can be accessed under ProteomeXchange ⁵² accession numbers PXD036136, PXD036207 and PXD036147 for human PD-1-APEX2 experiments and PXD036218 and PXD036216 for murine PD-1- APEX2 experiments.

Summary statistics and GO Term analysis

Raw data from three independent proximity labeling time course experiments were normalized to ACACA. Proteins that were not identified in all three experiments were excluded from the analysis. Each protein was then normalized to its respective 0 min control condition and the mean slope over time for TCR-control and TCR-PD-L1 conditions was calculated for each protein, where larger slope values indicate a stronger association with PD-1. The top 10 mean slope values for TCR-control and TCR-PD-L1 conditions were reported in Fig. Ij. The mean slope ratios between TCR-PD-L1 /TCR- control and TCR-control/TCR-PD-Ll conditions were then calculated for all proteins to assess protein enrichment per condition. The top 50 proteins were selected for GO Term functional enrichment analysis using g-Profiler g:GOSt functional profiling ³³ . Adjusted p- values of significantly enriched functions for TCR-control and TCR-PD-L1 conditions were reported in Fig. li.

Jurkat GenEPi nucleofection and stable clone selection

The GenEPi reporter plasmid (XLGenEPi) was generated by the Pantazis lab. GenEPi reporter expression was driven by a doxycycline-inducible Tet-On system. Jurkat cells were co-nucleofected with the XLGenEPi plasmid and pCMV_pBase at a molecular ratio of 1 : 1 using Lonza’s SE Cell line kit (cat. V4XC-2024) and corresponding CL-120 program on Lonza’s Amaxa-4D Nucleofector. The pCMV_pBase plasmid is a non-viral vector that expresses the piggyBac transposase (pBASe) which, when co-transfected, allows for the integration of the GenEpi transgene into the genome using two terminal repeat domains flanking the GenEPi reporter ⁵³ . This integration into the genome allows for the generation of stable GenEPi-expressing clones that can be selected for using blasticidin (BSD). 2pg of XLGenEpi and equimolecular amounts of pCMV_pBase plasmids were used to nucleofect 1 million Jurkat cells in cuvettes. Cells were rested in complete RPMI media (RPMI supplemented with 10% FBS, 1% penicillin/streptomycin, 1% HEPES, 0.1% BME, ImM sodium pyruvate and 4.5g/L of glucose) for 24 h following nucleofection. To generate stable clones, GenEPi nucleofected cells were selected with 8pg/mL BSD for 7 days. Cells were then rested in complete RPMI media for 24 h and treated with 200 ng/mL doxycycline for 24 h to induce GenEPi reporter expression. Selected Jurkat cells were then treated with 5pM Yodal and GFP -positive clones were immediately sorted on the BD FACS Aria and cultured to generate stable cell lines.

Flow cell chamber preparation and TIRE imaging

6-channel |i-Slide VI Glass Bottom slides (Ibidi #80607) were coated with Poly-D- Lysine for 1 h at room temperature. Excess Poly-D-Lysine was removed from the channels and the channels were washed 4X with PBS. Chambers were then coated with 2pg/mL CD3 and CD28 crosslinking antibodies and 4pg/mL PD-L1 or mlgGi overnight at 4°C. Slides were then washed 4X with PBS and 80pL of PBS was added to the flow cell chamber to prevent drying out prior to use. Stable GenEPi Jurkat cells were treated with ImmunoCult Human CD3/CD28 T cell activator (STEMCELL Technologies #10971) for 48 h prior to imaging to induce PD-1 expression. GenEPi Jurkat cells were treated with doxycycline (200ng/mL) for 24 h prior to imaging to temporally control and induce GenEPi expression. GenEPi Jurkat cells were then stained with F-actin SPY550-FastAct (1 :500, Cytoskeleton #CY-SC205) and antihuman PD-1 AF647 (1 :50, Biolegend #329910 clone EH12.2H7) for 1 h at 37°C in PBS supplemented with 10% FBS (PBS-F). Cells were washed twice with PBS-F and resuspended at a concentration of 1 million/mL in complete RPMI media.

TIRF imaging was performed using a fully motorized Nikon Ti inverted microscope equipped with a Nikon Ti-TIRF-EM Motorized Illuminator and a Nikon LUN-F Laser Launch with single fiber output (488nm, 90mW;561 nm, 70mW; 640nm, 65mW). To immobilize the sample, 80 pL of stained XLGenEPi Jurkat cells were flowed into one of the 6 coated |i-Slide channels (Ibidi #80607). Flow was established by capillarity using a Kimwipe. Imaging was performed under static conditions (e.g., not flow). Images were collected using an Apo TIRF 100x/1.49 DIC oil immersion objective lens with Nikon NF immersion oil, adjusting the correction collar to minimize spherical aberration. Images were captured with an Andor Zyla 4.2 Plus sCMOS monochrome camera using the 16-bit dual gain digitizer mode, 540 MHz readout rate and 2x2 pixel binning (resulting pixel size 0.1 um/px) and Nikon Elements Acquisition Software AR 5.02. The TIR angle for each channel was adjusted using fluorescence beads in the same flow chambers as used in the experiments and verified with a control sample. Signals from the different channels were acquired sequentially using a Chroma ZT 405/488/561/640 multi-band pass dichroic mirror mounted on a Nikon TIRF filter cube located in the filter cube turret, and band pass emission filters for GenEPi AF488 (Chroma ET525/50m), SPY5550-FastAct (Chroma ET 595/50m) and PD-1 AF647 (Chroma ET 6551p) channels, respectively, located on a Sutter emission filter wheel within the infinity space of the stand. Time-lapse imaging was performed with a time interval of 20 s and total acquisition time of 5 min. An ND 16 filter was introduced in the light-path to reduce irradiation. Imaging conditions were optimized to reduced photobleaching and phototoxicity. Fiji analysis for TIRF imaging

TIRF image analysis was completed using a custom workflow built in Fiji/ImageJ ⁵⁴ . The TIRF images were opened in Fiji and split into the different fluorescent channels. A 500 pixel rolling ball background subtraction was applied to reduce background intensities close to 0. The individual PD-1 and PIEZO1 channels were processed by performing a Tophat/difference of Gaussian filter to emphasize the small puncta. The processed images were segmented by intensity -based thresholding. A close filter followed by a size filter was applied to the thresholded masks to generate the final masks for PD-1 and PIEZO1. Regions of interest (ROI) were then drawn to demarcate cell boundaries to calculate the parameters on a per cell basis. The mask areas, the intensities of the signal and all ROIs were measured. Normalized fluorescence intensity for PIEZO 1 activity was calculated from the sum of pixel values within each ROI for TCR-control and TCR-PD-L1 conditions. Only cells expressing both PD-1 and the PIEZO1 GenEPi reporter were analyzed. Representative images were processed using Nikon Imaging Software (NIS) and Fiji/ImageJ. Al-denoise was applied to F-actin channels using NIS. A 25 pixel rolling ball background subtraction was then applied to F-actin channels in Fiji to reduce background. A 50 pixel rolling ball background subtraction was applied to GenEPi reporter and PD-1 channels in Fiji to reduce background. A median filter of 1 pixel was applied to GenEPi reporter and PD-1 channels in Fiji. Gamma adjustments of 0.8 for PD-1 and GenEPi channels and 1.0 for the F-actin channel were also applied to supplemental videos. Coloring for supplemental videos was adjusted such that PD-1 is displayed in magenta, GenEPi in yellow and F-actin in cyan to clearly convey changes in each channel when merged.

Mice

Seven- to ten-week-old age-matched female or male mice were used for all in vivo experiments and seven- to fourteen-week-old mice were used for CD8 ⁺ T cell isolation and in vitro experimentation. Wild type C57BL/6J mice were purchased from Jackson Laboratories. E8I-Cre-ER ^T2 mice were a generous gift from the Vignali lab. Homozygous E8I-Cre-ER ^T2 mice were crossed with homozygous Piezol ^flx/flx mice purchased from Jackson Laboratories (Stock #029213). Mice were crossed until they were fixed for the Pi ezol ^flx/flx mutant allele as assessed by the Transnetyx genotyping service. The Piezol-2 WT probe was used to detect the WT Piezol allele (forward primer:

CTGTCCCCTTCCCCATCAAG; reverse primer: GGGTCCAGGGTAGACAACAG). The L1L2-Bact-P MD probe (forward primer: GCTGGCGCCGGAAC; reverse primer: GCGACTATAGAGATATCAACCACTTTGT) was used to detect the LlL2_Bact_P cassette, composed of an FRT site followed by a lacZ sequence and a loxP site, confirming the integration of the floxed Piezol mutant allele. eGFP E8I-Cre-ER ^T2 and Rosa26-LSL TD tomato knock-in activity reporters were also quantified by Transnetyx using the eGFP (forward primer: CGTCGTCCTTGAAGAAGATGGT; reverse primer: CACATGAAGCAGCACGACTT) and tdRFP (forward primer: AGATCCACCAGGCCCTGAA; reverse primer: GTCTTGAACTCCACCAGGTAGTG) probes, respectively. Heterozygous Cre mice were bred to generate both Cre+ and Cre- littermates. To induce Piezol deletion, Cre+ and Cre- mice were intraperitoneally injected with 8 doses of lOmg/mL tamoxifen daily. Deletion efficiency was assessed using RT- qPCR with probes Mm01241547_gl spanning exons 21-22 (ThermoFisher #4351372) and Mm01241549_ml spanning exons 23-24 (ThermoFisher #4331182) to specifically assess the region containing loxP sites in exons 20 and 23. Results indicated that Cre+ mice required two copies of the Cre+ allele for -70% PIEZO 1 deletion. Cre+ and Cre- mice Piezol ^flx/flx mice were then bred separately to ensure the homozygous expression of Cre alleles for efficient gene deletion. All mice were housed in specific pathogen-free conditions and all animal experimentation was performed in accordance with regulations and animal care guidelines from the Harvard Medical School Standing Committee on Animals (IACUC) and the National Institute of Health.

Tumor implantation

Mice were anesthetized with 2.5% 2,2,2-tribromoethanol (Avertin, Sigma-Aldrich catalog no. T48402-25G) and injected in the flank subcutaneously with 2.5 * 10 ⁵ MC38, B16.F10 or B16-OVA tumor cells. Once palpable tumors were observed, tumors were measured every other day to calculate tumor volume over time. Tumor volume was calculated using the equation (L*W ² )/2 where L denotes tumor length and W denotes tumor width. Mice were monitored for body condition and weight loss. Mice with tumors that exceeded 2000mm ³ , that were severely ulcerated or that infiltrated the i.p. cavity were sacrificed. Tumor infiltrating lymphocyte isolation

Tumors were harvested at day 15 following implantation and processed for analysis. Extracted tumors were mechanically chopped and treated with collagenase type 1 (Worthington Biochemical, #LS004194) and mixed for 25 min at 37°C. Lymphocytes were enriched using a 40/70% Percoll gradient that was centrifuged at 805 RCF for 20 min (no brake). Immune cells were harvested from the interface between 40% and 70% Percoll and resuspended in MACS buffer (PBS with 1 % FBS and 2 mM EDTA) for staining for flow cytometry analyses.

Flow cytometry staining

Cells were surface stained with antibodies listed below at a 1 : 100 dilution (unless otherwise noted) in 96-well V-bottom plates for 45 min in the dark on ice. Samples were washed twice with MACS buffer and fixed for 20 min in the dark at room temperature and permeabilized with FoxP3/Transcription Factor Staining Buffer Set Kit according to the manufacturer’s protocol (eBioscience #00-5523-00). Cells were stained with intracellular antibodies listed below at a 1 : 100 dilution (unless otherwise noted) for 1 h in the dark on ice and washed twice with eBioscience Permeabilization Buffer. All washes used 200p.L of the specified buffers and spins were performed at 726 RCF for 2 min at 4°C. Fixed and stained samples were acquired the following day on the BD FACSymphony and were analyzed using FlowJo software.

Antibodies for flow cytometry and sorting

Flow cytometry analyses were performed on a BD FACSymphony and cell sorting was performed on a BD Aria II. The following fluorescent antibodies were purchased from Biolegend for flow cytometry and cell sorting: human PD-1 (clone EH12.2H7, #329936) and murine CD45.2 (APC-Cy7, clone 104, #109824), CD8b (Alexa Fluor700, clone 53-6.7, #00730), CD44 (APC, clone IM7, #103012), PD-1 (PE-Cy7, RMP1-30, #109110), CD69 (BV421, H1.2F3, #104528), CD62L (BV605, PerCPCy5.5, clone MEL-14), CD3e (BUV395, PerCpCy55, FITC, clone 145-2C11), perforin (PE, clone S16009A), granzyme B (BV421, clone GB11, #515408), CTLA4 (BV605, clone UC10-4B9, #106323), TIM-3 (BV711, BV421, clone RMT3-23, #119727), Slamf6 (PE, APC, clone 330-AJ), IFNy (APC, clone XMG1.2, #505810) and TNFoc (PerCP/Cy5.5, clone MP6-XT22, #506322). The following fluorescent antibodies were purchased from BD Biosciences: CD4 (BUV496, BUV737, clone GK1.5), LFA1 (BV786, clone M17/4) and Ki67 (PerCpCy55, clone B56). LIVE/DEAD Fixable Aqua dead cell stain (1 :600, L34957) and LIVE/DEAD Fixable Near-IR dead cell stain (1 :600, L34976) were purchased from Thermo Fisher Scientific.

In vivo PD-1 blockade and Piezo 1 agonist treatment

Two lOOpg doses of anti-PD-1 (Freeman lab or BioXcell, clone 29F.1A12, #BE0273) or rat IgG2a isotype control (BioXcell, clone 2A3, #BP0089) were administered interperitoneally to mice on days 10 and 13 (B16-OVA model) or 14 and 17 (MC38 model) following tumor implantation. Yodal (Tocris, #5586) was reconstituted in DMSO to reach a final stock concentration of 17 mg/mL. Reconstituted Yodal was freshly formulated with PBS and sonicated prior to each experiment to obtain a dose of 7.5mg/kg per mouse (250pg/200pL) administered interperitoneally 3-5 times between days 5 and 13.

Western blotting

Jurkat cells or tumor cells were lysed in Pierce RIPA Buffer supplemented with Halt Protease and Phosphatase Inhibitor Cocktail (100X) for 15 min on ice. Whole cell lysates were spun down at 20,817 RCF at 4°C for 15 min. Supernatants were collected and transferred to new Eppendorf tubes and I OpL of each lysate was taken for protein estimation using the Pierce BCA Protein Assay Kit to normalize for protein loading. Lysates were denatured with 4X Laemmli Sample Buffer (BioRad) or 4X NuPAGE LDS (Invitrogen) containing beta mercaptoethanol (BME) and boiled for 5 min at 95°C. 15-40pg of protein per lysate was loaded and run on a NuPAGE 4-12% Bis-Tris protein gel and then transferred onto a nitrocellulose membrane. Ponceau staining was performed to check transfer efficiency and protein loading. Membranes were then blocked for 1 h in TBS supplemented with 1% Tween (TBS-T) and 5% milk supplemented with 0.2% Tween at room temperature. Membranes were then incubated with primary antibody Streptactin-HRP (Bio-Rad, 1 :50,000, #1610381) and rocked overnight in blocking buffer at 4°C. Membranes were washed 3X for 5 min in TBS-T buffer prior to imaging. Membranes were then treated with SuperSignal West Pico PLUS Chemiluminescent Substrate (ThermoFisher #34580) and imaged using the Amersham Imager 600. PD-L1 bead generation

M-450 Tosyl-activated magnetic Dynabeads (Invitrogen #14013) were washed using the Dyna-Mag-2 in 0.1M sodium phosphate buffer, pH 7.4-8.0. Magnetic beads were then treated with a mixture of human or mouse CD3 crosslinking antibody (BioXcell, human clone OKT3 #BE0001-2, murine clone 145-2C11 #BE0001-l), CD28 (BioXcell, human clone 9.3 #BE0248, murine clone 37.51 #BE0015-l) crosslinking antibody and either recombinant human (for use in Jurkat cells, R&D Systems #156-B7-100) or murine (for use in primary murine CD8 ⁺ T cells, R&D Systems #1019-B7-100) PD-L1 fusion protein or recombinant human IgGiK (for use in primary murine CD8 ⁺ T cells, SouthernBiotech #015 lk-01) or mouse IgGi (for use in Jurkat cells, SouthernBiotech #0102-01) control protein depending on the PD-1 construct and origin of cells used. Multiple titrations of protein concentrations were tested to determine which ratio generated the most significant inhibition of T cells: 10/90%, 20/80%, 40/60%, 60/40% and 80/20% of TCR/CD28 antibodies and recombinant PD-L1 or control respectively. Antibodies and recombinant protein mixtures were crosslinked to beads using a total of 200pg of protein per ImL of beads. Beads were incubated and rotated for 16-24 h at room temperature. Beads were then applied to the Dyna-Mag-2 to remove supernatant and washed in PBS supplemented with 0.1% BSA and 2mM EDTA pH 7.4. To deactivate the remaining free tosyl groups on the beads, beads were rotated overnight at room temperature in 0.2M Tris- HCL supplemented with 0.1% BSA, pH 8.5. Beads were washed 3X more with PBS buffer supplemented with 0.1% BSA and 2mM EDTA pH 7.4 and reconstituted in this buffer at 100 million beads per ImL and stored at 4°C prior to use.

On-bead protein ratios and cell: bead ratios

Naive murine CD8 ⁺ T cells were isolated from spleens of wild type mice using negative selection magnetic-activated cell sorting (MACS) isolation (Miltenyi #130-096- 543). Naive CD8 ⁺ T cells were stimulated for 24 h on 96-well U-bottom plates coated with anti-CD3 and anti-CD28 crosslinking antibodies. After 24 h, cells were moved to a new U- bottom plate. Murine TCR-PD-L1 or TCR-control hlgGiK beads containing various protein ratios outlined above were added to CD8 ⁺ T cells at various celkbead ratios (1 :2, 1 :4, 1 :6) for 48 h. Supernatant was collected 48 h following the addition of beads for cytometric bead array (CBA) analysis (BD Biosciences). After 48 h, CD8 ⁺ T cells were stained and acquired the following day on the BD FACSymphony for flow cytometric analysis as described in Flow Cytometry Staining in Main Methods.

Indo-1 Ca ²⁺ flux assay

Naive CD8 ⁺ T cells were isolated from the spleens of WT mice or E8i-Cre-ER ^T2 Piezol ^flx/flx Cre+ or Cre- mice treated with lOmg/mL tamoxifen for 8 days using naive CD8 ⁺ T cell MACS. Purified CD8 ⁺ T cells were stimulated on 96-well U-bottom dishes coated with 4pg/mL of crosslinking murine CD3 and CD28 antibodies for 48 h. Stimulated CD8 ⁺ T cells were then rested for 4-12 h in RIO media and moved to a fresh, uncoated 96- well U-bottom plate. WT or E8i-Cre-ER ^T2 Piezol ^flx/flx CD8 ⁺ T cells were then stained in PBS containing 1% FBS for 1 h at room temperature with near IR fixable LIVE/DEAD stain and 1|1M of Indo-1 (Thermo Fisher Scientific #11226). Cells were then washed twice with 1% FBS PBS and resuspended in RIO media in 5mL round-bottom polystyrene tubes. TCR-PD-L1 or TCR-control beads were added at a ratio of 1 :5 celkbead and Yodal was added to samples at a final concentration of 5|1M. Following addition of PIEZO1 agonist or beads for specified timepoints, cells were immediately acquired for 1 min using tube mode on the BD FACSymphony or BD LSRII and analyzed using FlowJo software. Ca ²⁺ influx was quantified by calculating the ratio of bound (BUV395) to unbound (BUV496) Ca ²⁺ using Indo-1.

RT-qPCR

Murine CD8 ⁺ T cells were isolated from the spleens and inguinal lymph nodes of Cre+ and Cre- naive mice treated with lOmg/mL tamoxifen for 8 days using CD8 ⁺ T cell positive selection MACS (Miltenyi #130-117-044) or cell sorting. Isolated CD8 ⁺ T cells were washed with PBS and spun down at 453 RCF for 5 min. PBS was removed from the cell pellets and cell pellets were frozen at -80°C or immediately processed for RNA. RNA was isolated from purified CD8 ⁺ T cell pellets using Qiagen’s RNAeasy Mini Kit (#74104). RNA was quantified using the Qubit RNA HS Assay Kit (#Q32852) and RNA concentration was normalized across samples. Reverse transcription was perform using the Superscript VILO cDNA Synthesis Kit (Thermo Fisher Scientific #11-754-050) to generate cDNA. cDNA was diluted and mixed with Pzezof specific fluorescein amidites (FAM) Taqman probes (Thermo Fisher Scientific, Assay ID # Mm01241547_gl and #MmO1241549_ml) and control eukaryotic 18s rRNA FAM probe (Thermo Fisher Scientific, Assay ID #Hs99999901_sl) according to the Fast Advanced Mastermix real time PCR protocol. PCR mixtures were plated in Roche LightCycler 480 96-well plates (#04729692001) and sealed. Plates were run on the Roche LightCycler 480 with a 20 s polymerase activation step at 95°C followed by 40 cycles of 1 s of denaturing at 95°C and 20 s of annealing and extending at 60°C.

PIEZO 1 KO CD8 ⁺ T cell stimulation and flow cytometry

E8I-Cre-ER ^T2 Cre+ and Cre- mice were treated with 8 doses of lOOmg/mL tamoxifen. Naive CD8 ⁺ T cells were isolated from spleens of tamoxifen-treated mice using negative selection MACS isolation (Miltenyi). Naive CD8 ⁺ T cells were stimulated for 48 h on 96-well U-bottom plates coated with a titration of anti-CD3 and anti-CD28 crosslinking antibodies (0, 0.1, 1.0, 5.0 and 10|ig/mL). Supernatant from each sample was collected 48 h following the addition of beads for CBA analysis (BD Biosciences). After 48 h, CD8 ⁺ T cells were stained as described in Flow Cytometry Staining in Main Methods and analyzed on the BD FACSymphony or CytoFLEX (Beckman Coulter).

Incorporation by Reference

All publications, patents, and patent applications mentioned herein are hereby incorporated by reference in their entirety as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated by reference. In case of conflict, the present application, including any definitions herein, will control.

Equivalents

Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.