WO2015143113A1

WO2015143113A1 - Preparations of derived extracellular vesicles, assays, and methods to modify therapeutic outcomes using such preparations

Info

Publication number: WO2015143113A1
Application number: PCT/US2015/021394
Authority: WO
Inventors: Barb Ariel Cohen
Original assignee: Barb Ariel Cohen
Priority date: 2014-03-20
Filing date: 2015-03-19
Publication date: 2015-09-24

Abstract

A method for altering the properties of extracellular vesicles and exosomes for use in diagnosis and therapeutics is disclosed, through their production by primary cell culture under Applicant's specific controlled conditions. A mammalian ejaculate is provided and sperm therefrom are incubated, during which time aliquots are evaluated for production of vesicles engineered to contain the cargo of choice and targeted to the desired application through adjustment of the surface receptor complement. For example, vesicle-borne Fc receptors act as decoys to protect sperm from antibodies in the female tract and vesicles additionally deliver regulatory molecules to modulate tract function, enabling the ability to control release and to engineer sperm-derived vesicle/ exosome structures to be used in diagnosis of infertility and in production of sperm doses of superior performance.

Description

PREPARATIONS OF DERIVED EXTRACELLULAR VESICLES, ASSAYS, AND METHODS TO MODIFY THERAPEUTIC OUTCOMES USING SUCH

PREPARATIONS

CROSS-REFERENCE TO RELATED APPLICATIONS

[001] This application claims priority to U.S. Provisional Patent Application No. 61/955,938 filed March 20, 2014, which is incorporated by reference herein in its entirety.

FIELD OF THE DISCLOSURE

[002] The present invention relates to production of engineered extracellular vesicles including exosomes that are abundant and derived from a single cell type—sperm— under controlled conditions of incubation. These vesicles and exosomes have proven diagnostic predictive value and therapeutically they can be used to adjust cell states to produce improved medical outcomes. Additionally, they can receive cargo while cells are in culture and, because Fc receptors are present, these sperm-derived extracellular vesicles (SEVs) can be used in both directly- applied Fc-recep tor-based anti-inflammatory therapy and in therapies requiring antibody or ligand-based targeting to distant sites, achieved by attachment of specific antibodies to the Fc receptors in a way that allows the antibody specificity to target recipient cells.

BACKGROUND

[003] Extracellular vesicles (EVs) including exosomes are now recognized as important agents in the transfer of information between cells, both local and distant. Kriemer and colleagues (2013) provide the following definition for them: "circulating extracellular micro vesicles shed by cells comprise a heterogenous population of membrane enclosed vesicles varying in size (20 to 1000 nm) and content." In the literature, larger vesicles (> approx. 120nm) generally are referred to as extracellular vesicles, while smaller ones are referred to more specifically as exosomes.

[004] Extracellular vesicles communicate with cells to alter their states. At one extreme, EVs may induce receptivity of pre-metastatic niches to invasion by tumor cells (Shen et al., 2013; Luga et al., 2013; Katsuda et al, 2013). At another extreme, they may repair cellular damage (Meisner et al., 2013; Aliotta et al., 2013a; Lindoso et al., 2013; Corteling et al., 2013). Normal cells behave like tumors when mixed with tumor-derived exosomes (Atay et al., 2013; Beheshti et al., 2013; Harmati et al., 2013). Exosomes may play a role in cancer chemotherapeutic drug resistance (Aung et al., 2013). Diabetic-like insulin resistance may be induced in normal cells upon exposure to exosomes derived from intestinal microbes of animals fed a high-fat diet (Kim et al., 2013). Exosomes derived from rats with pulmonary hypertension may transfer this disease to healthy animals (Aliotta et al, 2013b).

[005] Through immune modulation— both up (Subramanian et al., 2013; Kahraman et al., 2013) and down (Dubin et al., 1991), exosomes may affect susceptibility to infection.

Parasites (Torrecilhas et al., 2013; Cabrera-Cabrera et al., 2013), bacteria (Pierson et al., 2013), viruses (Favoreel et al., 2003) and yeast (Nimrichter et al., 2013) may use them to evade the host immune response.

[006] One mechanism of evading immune destruction is through expression of Fc receptors (Ackerman and Nimmerjahn, 2014), a class of membrane-bound receptors that interact with the constant region of immunoglobulin molecules, the Fc region. These receptors play key roles in modulation of the immune response. To destroy a target, antibodies bind to it via their FAb domain. The antibody Fc domain then binds white blood cells to activate immune mechanisms of target destruction. This mechanism can be perverted if the target carries an Fc receptor and binds antibody in a neutralizing fashion. The target thereby becomes an antibody sink, silencing immune activating signals and promoting infectivity: Herpesvirus contain Fc receptors that protect virus from immune destruction (Dubin et al., 1991).

Mammalian sperm have been known to possess Fc receptors accessible on the sperm surface, where they were shown to participate in cell-surface immune reactions in vitro, however, the in vivo function remained unknown (Witkin et al., 1980). Witkin's subsequent publication stated that antibody binding to sperm was not mediated by Fc receptors, but by disulphide exchange (Richards and Witkin, 1984).

[007] Vesicles occur in semen. Vesicles derived from male accessory glands, the epididymis (Belleannee et al., 2013) and prostate (Poliakov et al., 2008) have been described. Vojtech and colleagues showed that exosomes purified from seminal plasma (but not further characterized as to source) were capable of entering antigen-presenting cells (2013).

Vesicles 150-300nm in diameter, which exceed the size range of exosomes, are produced by the hermaphrodite worm C. elegans in its developed and developing sperm (Kosinski et al., 2005). The sperm acrosome is shed as a single large vesicle (for example, 4,000 nm diameter in cattle) released from mature sperm during the acrosome reaction (Fraser, 2010). Kim and colleagues documented release of what they identified as soluble material from the acrosome (Kim et al., 2001) but did not report vesicles. Although Primakoff and colleagues (1980) characterized hybrid vesicles released from epididymal (not ejaculated) sperm that were provoked to undergo the acrosome reaction, they incorrectly stated that they were observing a process identical to the physiological one. In fact, the ionophore they used causes both the acrosome reaction and the size of vesicles associated with the sperm head to be abnormal as compared to the spontaneous process (Watson et al., 1992). In addition, the spontaneous process required long incubation—4 hours— before it could occur. Watson and colleagues did not report release of vesicles from the sperm head, nor release by freshly ejaculated sperm too immature to undergo the acrosome reaction.

[008] Vesicle release by sperm has been characterized further as resulting from the acrosome reaction (Zanetti and Mayorga, 2009). The acrosome reaction is reported to occur naturally in proximity to the egg (Yanagimachi, 2011), but only after after capacitation (Itach et al., 2011), a maturational process signaled by acquisition of hyperactivated motility that is associated with release of sperm from the oviductal storage reservoirs (Chang and Suarez, 2010) and with penetration of the egg. It is thus reported as a process that occurs late in sperm maturation.

[009] Vesicles could hold great promise as therapeutic agents—even in cases where they are not internalized (Zhang, et al., 2013), and they can also be effective cross-species (Buck et al., 2013).

[0010] Unfortunately, several key impediments diminish the utility of EVs (Wang et al., 2013). These include, for example, (a) inhomogeneity of complex admixtures obtained from blood and other sources such as cell culture in complex medium, (b) in many cases, lack of abundance, and (c) questions of stability and safety. Synthetic vesicles to date have not been a solution; they lack the complete biological structure of native EVs, have inferior therapeutic response, have poor uptake by target cells, have reduced half-life and cause allergic reactions (Marcus and Leonard, 2013).

[0011] Methods of engineering naturally- obtained EVs are needed to overcome these impediments. Such methods are being developed for delivery specifically of cargo, but are hampered by problems that adversely affect clinical translation (Marcus and Leonard, 2013). Specifically, although exosomes may be isolated from cell culture in conditioned medium (after 3-6 days of culturing) or from body fluids, methods are time-consuming, labor intensive and yield is very poor or contains contaminants. Cargo loading is hampered because, as noted in a November 2013(b) abstract by Marcus and Leonard, "the mechanisms by which a subset of cellular proteins and RNAs are sorted into exosomes are poorly understood" and

"biophysical constraints dramatically modulate RNA loading." Methods for targeting exosomes to recipient cells also are hampered by lack of universal targeting ligands on the exosomes to direct them to recipient cells.

[0012] Therefore, there is a need to accomplish the goal of producing abundant, safe, universally targetable exosomes and extracellular vesicles capable of being supplied with a variety of cargos in a natural process.

SUMMARY

[0013] Presently described are methods of producing, engineering and evaluating vesicles and exosomes derived from sperm is required. Applicant's work has revealed sperm to be an abundant and safe source of easily-engineered sperm-derived extracellular vesicles/ exosomes (SEVs).

[0014] Thus, in one aspect, the present disclosure provides a method for producing sperm- derived extracellular vesicles, the method comprising: collecting semen from a mammal; incubating the semen to provide a desired quantity of homogeneous sperm-derived extracellular vesicles; taking a sample at a pre-selected interval and assaying the sample for the presence of extracellular vesicles in the sample; repeating step d at pre-selected intervals until the desired quantity of extracellular vesicles is present in the sample; and processing the semen. [0015] In an additional embodiment, the present disclosure provides a method for producing sperm-derived extracellular vesicles, the method comprising: collecting semen from a mammal; isolating sperm from the semen; incubating the isolated sperm to provide a desired quantity of extracellular vesicles homogeneous sperm-derived extracellular vesicles;

processing the sperm to separate the sperm-derived extracellular vesicles.

[0016] T In an additional embodiment, the present disclosure provides a method for producing sperm-derived extracellular vesicles, the method comprising: collecting semen from a mammal; isolating sperm from the semen; incubating the isolated sperm to provide homogeneous sperm-derived extracellular vesicles; taking a sample at a pre-selected interval and assaying the sample for the presence of extracellular vesicles in the sample; repeating sampling and assaying at pre-selected intervals until a desired quantity of extracellular vesicles is present in the sample; and processing the sperm to separate the sperm-derived extracellular vesicles. In some preferred embodiments, the method further includes adding an agent to the incubation step to provide cargo to be incorporated with the extracellular vesicles. In certain preferred embodiments, the cargo is an antibody. In some preferred embodiments, the method further includes adding an agent to the incubation step to provide a means of targeting the extracellular vesicles to the desired recipient cells.

[0017] In an additional aspect, the present disclosure provides a method for treating a patient in need of immune modulation, the method comprising the steps of administering a therapeutically effective amount of extracellular vesicles, at least a portion of which have an active Fc receptor.

[0018] In another aspect, the present disclosure also includes a method for improving reproductive results in assisted reproductive therapy (ART), the method comprising:

collecting semen from a mammal; incubating the semen to provide extracellular vesicles in the semen; taking a sample at a pre-selected interval and assaying the sample for the presence of extracellular vesicles in the sample; repeating the sampling and assaying at pre-selected intervals until a desired quantity of extracellular vesicles is present in the sample; and using the semen containing the desired quantity of extracellular vesicles in the ART procedure.

[0019] In another aspect, the disclosure provides sperm-derived extracellular vesicles made according to any of the methods described herein. In certain embodiments, the sperm-derived extracellular vesicles can include cargo. In some preferred embodiments, the cargo can be a ligand attached to one or more of the extracellular vesicles by a Fc receptor on the extracellular vesicle. In certain preferred embodiments, the ligand can be an antibody.

BRIEF DESCRIPTION OF THE FIGURES

[0020] FIG. 1. Schematic of extracellular vesicle- and exosome-mediated cargo transfer between cells, (a) Many cell types produce extracellular vesicles with cargo that includes small molecules, proteins, and nucleic acids among other agents. These can be transferred from the cell that produces them to a recipient cell, triggering profound changes. (Figure from Raposo and Stoorvogel, 2013). (b) Mechanism of targeting microRNA transfer from exosomes to target cells (Figure from Stoorvogel, 2012).

[0021] FIG. 2. The male and female reproductive tracts as sources of extracellular vesicles. Eggs from the female are known to produce exosomes. Male accessory glands, among them the prostate gland, are known to produce exosomes. Sperm are known to produce large hybrid vesicles from the acrosome, as part of the acrosome reaction. Applicant now reports production of extracellular vesicles and exosomes from sperm in a new manner: production occurs after incubation of ejaculate but very early in sperm maturation (beginning within 30 minutes of ejaculation, with a peak of production often in l-2h and with greater accumulation of larger vesicles thereafter), (male and female [by T. Winslow] diagrams, respectively: www.web-books.com/eLibrary/Medicine/Physiology/Reproductive/Male.htm;

www.cancer.gov/cancertopics/pdq/treatment/unusual-cancers-childhood/Patient/page6).

[0022] FIG. 3. SEV production by sperm. As SEVs include vesicles in the size range associated with both extracellular vesicles and exosomes, both confocal

microscopy/cytometery and electron microscopy were used to ensure detection. Confocal microscopy of bull sperm beginning to bud Fc-receptor containing vesicles, which stain positive by the Applicant's Cohen Biomarker Assay (COBO Assay, see Module 4). (b) Confocal microscopy of bull sperm ejecting vesicles, with vesicles already present in the surrounding medium, (c) Electron microscopy of human sperm head and large vesicles of the size seen with confocal microscopy, (d) Electron microscopy of exosomes derived from washed human sperm incubated in defined medium. These were not detected without incubation, nor in control incubated samples from vasectomized men who have no sperm in the ejaculate (data not shown), indicating either a difference in their presence or their concentration, (e) Exosomes from incubated bull semen, surrounded by amorphous matrix of material that may be part of the acrosomal shroud. Small exosomes such as these provide opportunity for endocytosis by target cells as an uptake mechanism, (f) Large vesicles on head of human sperm. Note vesicle in upper left, where two smaller objects with circular outlines are present. Note detached object with outline similar to the attached vesicles at bottom of image, (g) Higher magnification of human sperm head showing larger vesicle and two smaller exosome-sized objects. Notches in larger vesicle (note especially to left of exosome-like objects) suggest sites of previous deployment of smaller objects, (h) Bull sperm with vesicles on head.

[0023] FIG. 4. Kinetics of vesicle production by human sperm, with size distribution. A fresh ejaculate was cooled and sperm were harvested by centrifugation. They were resuspended in a defined synthetic medium and incubated, with elapsed time from ejaculate production to the start of incubation in defined medium of about 30 minutes. Aliquots were taken into the COBO Assay at the start of incubation in defined medium and at 30 minute intervals thereafter, and analyzed by confocal imaging for vesicle content, (a) Magnified images from beginning (0 hours) and end (3.5 hours) of incubation, for comparison of vesicle density in defined medium from washed, and then incubated sperm, (b) Detailed time course (0.5 hour increments). See Figure 7 legend for microscopy conditions.

[0024] FIG. 5. SEV production requires the presence of sperm in an ejaculate, production does not occur in an ejaculate post-vasectomy that contains no sperm. It also requires time to occur and, unlike with prostatomes and epididymosomes, SEVs do not peak in abundance until hours after ejaculation—see also Figure 7.

[0025] FIG. 6. The timing of SEV appearance correlates to sperm maturation status and, like sperm maturation status, can be used to time ejaculate processing to create gender bias. An ejaculated stabilized 2h after peak sperm positivity produces female gender bias in dairy calf births. An ejaculated stabilized lh after SEVs including larger ones become very abundant produces female gender bias in dairy calf births. This makes sense, because sperm positivity for budding vesicles precedes vesicle shedding by about lh. Applicant showed in a prior application that fertility occurs at specific times relative to gender bias, therefore SEV production as measured by the Cohen Biomarker Assay can also be used to adjust fertility.

[0026] FIG. 7. Kinetics of SEV production, as measured by confocal microscopy. Sperm from a human ejaculate were cooled, purified by centrifugation through Sperm Prep medium, resuspended in a defined medium (HTF medium) and incubated. Aliquots were sampled over time to determine SEV size ranges and abundance. Images were collected on a Lica SP 8 x confocal system. Excitation was at 496nm, emission range was 510-600nm. The timegating function on the HyD detector was activated to reduce laser reflection from coverslips, to improve the signal to noise ratio. Z- stack images were acquired with a plan apo 63x/1.40 oil objective at the Nyquist-Shannon sampling rate in the axial plane, for a total of 19 slices in a depth of 5.29um (interval set to 294nm). Volume of each stack was 0.027 nanoliters. Based on volume sampled and the observed maximum number of small vesicles —about 1000, an activating cohort of sperm produces about 1 x 10(15) vesicles in the size range detectable by confocal microscopy (1000 vesicles in 0.027 nanoliters, taken from a ΙΟΟμΙ sample containing ΙΟμΙ input semen). Using the present invention it was found that excessive vesicle abundance at inappropriate times— especially too early— correlates with poor ejaculate quality in cattle.

[0027] FIG. 8. (a) SEV production, cargo loading and vesicle targeting modifications, from applicant's invention, (b) SEV production flow chart showing manufacturing modules.

[0028] FIG. 9. Examples of uses of engineered SEVs. Targeting to the epidermal growth factor receptor (EGFR) is shown, antibody specificity can be varied to target other ligands.

[0029] FIG. 10. (a) Illustration of Fc -receptor (artist' s rendering) binding to an antibody that is used to target the SEV to desired site through antibody specificity, (b) Monoclonal antibodies approved for cancer treatment and (c) Flow chart of decisions to be made during SEV production and engineering for different applications.

[0030] FIG. 11. COBO Assay results as shown in cytometer plots, (a) Screen shot of cytometer dashboard from bull sperm assay. Plot 2 shows negative and positive sperm pools. Plot 7 shows weak signals in the SEV signature region, which, depending on sample, extends from an FL1-A lower reading of about 6.2 - 6.7 to the highest reading on that axis, which is 7.2. (b) Screen shot of cytometer dashboard from human sperm assay of an intact man's ejaculate, from which sperm were purified by centrifugation of semen with Sperm Prep medium, after which the pellet was resuspended in HTF medium and incubated before running COBO Assay. The cell gate is indicated on the FSC x SSC plot as gate P3. The COBO assay result for positive and negative sperm detects events in that cell gate, (c) Screen shot of cytometer dashboard from human sperm assay of a vasectomized man's ejaculate, treated as was the intact ejaculate shown in (b). The SEV signature region for human sperm is seen to be similar to that in the plot shown for bull sperm.

[0031]

DETAILED DESCRIPTION

[0032] High fertility is a desired outcome for both veterinary and clinical practice. Livestock fertility is a primary economic driver of dairy farm productivity, without which dairy farms fail as businesses. Pork producer's profits increase with litter size. Infertility clinics are rated in their industry by fertility results, as nothing is more heartbreaking to couples trying to conceive than repeated failures.

[0033] Nonetheless, serious risk remains associated with use of ART. Use of ART in the human clinic results in decreased rates of normal live births and increased rates of low birth weight, preterm labor, admission to the neonatal ICU, need for surgery, hormonal abnormalities, chromosomal defects, epigenetic defects, and cardiac, urogenital and musculoskeletal birth defects (Alukal and Lipshultz, 2008).

[0034] Part of the risk of ART is that it bypasses normal selective methods that ensure fertilization by the best sperm. In natural mating, the female reproductive tract exerts intense selective pressure on sperm to ensure fertilization between healthy gametes. Mammalian sperm must demonstrate successive correct changes at the appropriate locations in the female tract to avoid destruction and to be permitted to reach the oviduct, where sperm storage reservoirs exist and where fertilization occurs. One of these changes is the elaboration of sperm-derived extracellular vesicles (SEVs).

[0035] Sperm cannot protect themselves from female immune surveillance, release agents that facilitate sperm movement to the oviduct, or ultimately fertilize an egg until they undergo a series of preparative steps that only occur after an ejaculate is produced (Fraser, 2010), with timing unique to each ejaculate (Cohen-Dayag et al., 1995) and unique to the conditions to which the ejaculate is exposed. One of these functions is production of SEVs through steps involving increased sperm permeability followed by exposure of SEV epitopes on the sperm surface, budding and ultimately release of SEVs. Because half of infertility cases are due to male-side or male-contributory issues, it is important to have good methods of diagnosing male reproductive competency and minimizing male- side iatrogenic risks from ART. These methods must include the ability to evaluate and modulate SEV production, composition and status, and to use SEVs therapeutically.

[0036] Male reproductive competency is generally evaluated by analysis of an ejaculate. Numerous sperm assays have been developed with the goal of predicting sperm fertility and reproductive outcome through evaluation of sperm and semen. These assays include measurement of cell number, motility, morphology, and cell staining properties. However, existing sperm assays are considered inadequate, with experts such as Barrett (2011) concluding: "Although the diagnostic and predictive value of traditional semen parameters has been debated for over 80 years, the inescapable conclusion remains that its clinical value is limited."

[0037] This and similar conclusions are based on the use of single point assays of sperm. Those methods cannot report the changes that sperm undergo from being infertile at ejaculation to acquiring fertility and other required competencies. Furthermore, the literature has not reported or even appeared to recognize that an early important event post-ejaculation is initiation of steps to produce SEVs—including Fc receptor-positive ones, which has been recognized herein. Assays to measure this important function have not been reported. Fc receptor presence and, in some cases deployment, is known to occur in parasites (Torrecilhas et al., 2013), bacteria (Pierson et al., 2013), viruses (Favoreel et al., 2003) and yeast

(Nimrichter et al., 2013) as foreign cells downregulating the host immune response. Like parasites, sperm also evoke a strong female immune response (Oren-Benaroya et al., 2008) and antibody-based sperm destruction in the female tract is a known cause of infertility (Bahraminejad et al., 1991).

[0038] Furthermore, because active, fertile cohorts of sperm live only 2-3h (Cohen-Dayag et al., 1995), and it is now recognized that only sperm at specific biological states can bud and shed SEVs , it is important to know the status (maturation) of both sperm and SEVs in an ejaculate, neither of which is reported by existing methods.

[0039] In use of ART, the ejaculated spermatozoa are vulnerable to the in vitro conditions, where they are exposed to factors that may cause iatrogenic sperm dysfunction (Mortimer, 1991). One example of creation of an iatrogenic issue is removal of shed SEVs from sperm by washing prior to intrauterine insemination, which compromises the ability of the washed sperm to survive the female immune response and induce their uterine contraction-derived transport to the oviducts once inseminated.

[0040] That sperm should be in different states of maturation depending on the type of ART used has been recognized, although with mixed clinical results (Huzzar et al., 2007; Gianaroli et al., 2010; Tarozzi et al., 2009; Morrell and Rodriguez-Martinez, 2011). By repeatedly monitoring sperm as they change, and adjusting them to the desired sperm state, one can achieve statistically significant fertility increases.

[0041] Except for the present application, there does not appear to have been any attempts to engineer and use SEVs for purposes of monitoring/ adjusting an ejaculate's status for use in specific types of ART, or to improve reproductive or other therapeutic outcomes.

Furthermore, in contrast to previous reports, the process described herein is spontaneous, produces vesicles in a different size range, and occurs much earlier in sperm maturation, often as early as 30 minutes post-ejaculation, at a time where sperm are not mature enough to undergo the spontaneous acrosome reaction (Jaiswal et al., 1998; Watson et al., 1992).

Although extracellular vesicles have been reported, they are not the ones now observed and utilized herein (Table 1). There is a stark contrast between the SEVs of this invention and previously described vesicles in how they arise kinetically. Vesicles in the scientific literature are reported to arise high in the reproductive tract, after capacitation/ hyperactivated motility is acquired, when sperm are able to acrosome react, SEVs arise shortly after ejaculation, when sperm are low in the female tract, before linear motility occurs, which precedes the hyperactivated motility of capacitation which precedes ability to acrosome react.

[0042] Vesicles reported in the scientific literature:

Ejaculation-^ Capacitation & hyperactivated motility-^ Ability to acrosome-react

(vesicles) [0043] SEVs describe here:

Ejaculation- SEV production/linear motility-^ capacitation & hyperactivated

motility-^ Ability to acrosome react (vesicles)

[0044] Table 1. Contrast between SEVs of instant invention and vesicles previously reported in semen.

[0045] Therefore, there remains a need in the art to provide a procedure, on which one can reliably depend, to provide a semen sample or useful component(s) of a semen sample such as engineered SEVs having a desirable trait such as, for example, vesicle status that is appropriate for the type of ART used. In preferred embodiments of the present invention, SEV engineering and evaluation can be performed without the need for a specialized laboratory and highly trained professional and would have broad commercial applicability (see Table 2).

[0046] Presently described are methods of producing, engineering and evaluating vesicles and exosomes derived from sperm is required. Applicant's work has revealed sperm to be an abundant and safe source of easily-engineered sperm-derived extracellular vesicles/ exosomes (SEVs).

[0047] Thus, in one aspect, the present disclosure provides a method for producing sperm- derived extracellular vesicles, the method comprising: collecting semen from a mammal; incubating the semen to provide a desired quantity of homogeneous sperm-derived extracellular vesicles; taking a sample at a pre-selected interval and assaying the sample for the presence of extracellular vesicles in the sample; repeating step d at pre-selected intervals until the desired quantity of extracellular vesicles is present in the sample; and processing the semen.

[0048] In an additional embodiment, the present disclosure provides a method for producing sperm-derived extracellular vesicles, the method comprising: collecting semen from a mammal; isolating sperm from the semen; incubating the isolated sperm to provide a desired quantity of extracellular vesicles homogeneous sperm-derived extracellular vesicles;

processing the sperm to separate the sperm-derived extracellular vesicles.

[0049] T In an additional embodiment, the present disclosure provides a method for producing sperm-derived extracellular vesicles, the method comprising: collecting semen from a mammal; isolating sperm from the semen; incubating the isolated sperm to provide homogeneous sperm-derived extracellular vesicles; taking a sample at a pre-selected interval and assaying the sample for the presence of extracellular vesicles in the sample; repeating sampling and assaying at pre-selected intervals until a desired quantity of extracellular vesicles is present in the sample; and processing the sperm to separate the sperm-derived extracellular vesicles. In some preferred embodiments, the method further includes adding an agent to the incubation step to provide cargo to be incorporated with the extracellular vesicles. In certain preferred embodiments, the cargo is an antibody. In some preferred embodiments, the method further includes adding an agent to the incubation step to provide a means of targeting the extracellular vesicles to the desired recipient cells.

[0050] In an additional aspect, the present disclosure provides a method for treating a patient in need of immune modulation, the method comprising the steps of administering a therapeutically effective amount of extracellular vesicles, at least a portion of which have an active Fc receptor.

[0051] In another aspect, the present disclosure also includes a method for improving reproductive results in assisted reproductive therapy (ART), the method comprising:

[0052] In another aspect, the disclosure provides sperm-derived extracellular vesicles made according to any of the methods described herein. In certain embodiments, the sperm-derived extracellular vesicles can include cargo. In some preferred embodiments, the cargo can be a ligand attached to one or more of the extracellular vesicles by a Fc receptor on the

extracellular vesicle. In certain preferred embodiments, the ligand can be an antibody.

[0053] In this application, we will refer to a certain type of vesicle as a "sperm-derived extracellular vesicle" (SEV), which includes all extracellular and budding vesicles produced by sperm upon either incubation under controlled conditions or upon provocation of maturation to simulate the effects of the incubation process. One example of a useful subset of SEVs of the instant invention is those that possess Fc receptors.

[0054] SEVs can be derived from a pure cell type in hours, not days. They already possess some established safety profiles for mucosal administration, typically being well tolerated by female mammals upon normal insemination. The presence of virus and other adventitious agents is easily minimized by semen analysis. This is because sperm—in contrast to other cell vesicle sources— are not genetically active and cannot support viral replication. For males, sperm also provide an abundant source of syngeneic vesicles, thereby minimizing hurdles to clinical use by having the donor provide his own biological therapeutic. Due to the way sperm synthesize SEVs, they possess surface structures and total composition that can be tailored to a desired use (according to Applicant's novel methods described herein) providing a customized or engineered SEV and enabling improved performance and application to previously unapproachable therapeutic needs. For example, engineered SEVs can be used for adjustment of semen dose properties to produce more desirable outcomes in terms of fertility and female gender bias in dairy cattle births. These adjustments also include reduced inflammatory responses and improved transport of materials and cells, due to the composition of the engineered SEVs.

[0055] Producing and evaluating customized SEVs, customized or modified to provide for the desired use, for example, enables production of sperm doses for assisted reproductive technologies according to the desired performance of the sample, where sample structure must be adjusted according to the type of Assisted Reproductive Technologies (ART) used.

Furthermore, a source of customized extracellular vesicles/ exosomes has great commercial utility. This is because of the diverse roles these structures play in biological processes and the ability to engineer desirable traits into the vesicles. These traits then positively affect biological processes such as, in preferred embodiments, cargo transport and vesicle targeting to the desired site(s), because SEVs have a surface receptor that allows universal attachment of antibodies via their Fc regions, so that the specificity-determining region of the antibody will direct SEVs to the desired target cites or target cells.

[0056] Sperm-derived extracellular vesicle and exosome (SEV) engineering has great utility for reasons unique to the ways the sperm system can be engineered. In the SEV production setting, SEV can be initially visualized when budding on the sperm cell surface (Figure 3a) and then are released (Figure 3b-h). Kinetics of SEV production can be measured and SEV status can be used to adjust the performance of sperm so that, once sperm are stabilized into doses, these doses will produce better outcomes: e.g., higher fertility or gender bias in dairy cattle births (Figure 6). [0057] The sperm system has other benefits as well. First, sperm can be isolated in abundance as a pure cell preparation. Second, they produce an extraordinary quantity of SEVs—a single maturing cohort of sperm can produce roughly 1 x 10¹⁵ larger SEVs detectable by confocal imaging, let alone SEVs so small they are visible only by electron microscopy (where they outnumbered the larger vesicles on the electron microscopy grids. Third, SEVs can be supplied with cargo during their production by sperm, by controlling and altering the sperm environment. Fourth, the Fc receptor on these SEVs makes them antiinflammatory. Alternatively, the Fc receptor can be bound to antibodies to target SEVs to the desired cell or organ (or the antibody can be used to recognize and bind ligands that do the targeting). Fifth, SEV purification is straightforward due to the presence on these SEVs of identifiable targets, such as Fc receptors. These receptors are able to serve as handles for highly efficient and commercially scalable isolation and purification steps using

immunoaffinity chromatography or magnetic bead capture. Sixth, SEVs can serve as vehicles for transfer of nucleic acids, without the viral system disadvantages of payload size limits or virally-induced persistent inflammation and tissue destruction. For these reasons, sperm- derived extracellular vesicles have great diagnostic, screening and therapeutic potential, but even more importantly, they have a safe biological profile, as shown by their typical routine and benign presence in females after normal mammalian insemination.

[0058] Because they are motile, sperm themselves can serve as vectors for transport. This may be preferred in some settings. Other approaches include use of SEVs or of soluble components derived from SEVs in suitable carriers or made synthetically. While it is also possible to use synthetic SEVs based on this discovery and disclosure, it is preferable to employ a subset of SEV components in such a setting, as the pharmacology of exosomes has been shown superior to agents from synthetic sources, with exosome tolerance even demonstrated across species (Marcus and Leonard, 2013a).

[0059] Preferred manufacturing methods in accord with the present invention include ways to isolate sperm prior to SEV production, to control SEV production, to detect SEV production, to supply cargo (if desired), to target SEVs to the appropriate site (if desired) and to evaluate SEVs for integrity. Finally, when required for specific areas of utility (e.g., diagnosis, therapy and screening), SEVs must be stabilized to be accessible. [0060] A preferred application of SEVs is for improving sperm stability, producing better performance including fertility enhancement and improved shelf life. Sperm fertility is profoundly affected by severe selective mechanisms in the female tract that are provided naturally to try to ensure the one male gamete that enters the egg is of high quality. Selective events in the female reproductive tract include a florid anti- sperm immune response that destroys sperm that are past their prime (Oren-Benaroya et al., 2007). It also is likely to destroy abnormal sperm that fail to deploy—at the right time and place— the normal sperm responses needed for immune evasion. Applicants have discovered that such normal sperm responses include expression of Fc receptors, budding of Fc-positive sperm-bound vesicles, and shedding of Fc receptors that function as antibody decoys on sperm-derived SEVs.

[0061] The coordination of these processes is elegant in nature— maturational changes in sperm and in SEV production happen in synch with the complementary selective mechanisms found at different sites in the female tract. This resembles computer games in which one must first acquire the right tool before advancing further, thereby gaining new attributes or items that are matched to each successive stage, in order to reach the highest level. Exosome production by the female tract, and interaction of these vesicles with sperm, is the

complementary fruitful area of great potential (e.g., with exosomes or vesicles obtained from uterine, cervical, vaginal or tubal flushing to harvest exosomes for uses such as described herein).

[0062] This elegant natural synchrony is perturbed in ART and in male- side infertility, making it difficult for healthy sperm to make their health known. In natural mating of mammals such as cattle and humans, sperm mature and acquire fertility (Fraser, 2010)—and, as discovered by Applicants, also the ability to express Fc receptors and shed them in SEVs— through processes that begin immediately after ejaculation. In contrast, washing steps in ART strip sperm of SEVs, and then introduce the sperm into the female tract (or even into Petri dishes!) at sites or times no longer synchronized with the normal stages of sperm or SEV development/deployment or of SEV concentrations naturally found at those sites and times after vaginal deposition of semen. The female tract (or eggs in Petri dishes!) may incorrectly interpret this asynchrony as abnormal sperm, with disastrous consequences. Despite this, in vitro use of rapid, repeat monitoring that reports the evolving state of sperm-bound and released SEVs in an ejaculate has not been available.

[0063] Multipoint rapid assays have been developed and employed by Applicant exclusively with sperm in a processing procedure that, although providing useful enhanced outcomes, now has been found to be an incomplete solution to reproductive issues. It has now been discovered that awareness of SEV production under controlled conditions and SEV use is also required to enable further improved outcome in ART. Use of SEVs can prevent economic losses and medical burdens by providing sperm and SEVs that are at the correct states of maturation and that have the correct structure and concentration for the type of ART used, enabling female- side selective processes to occur more favorably, preferably without the forced loss of discrimination between normal and abnormal sperm and the resulting issues of reduced or defective births currently observed in ART by Alukal and Lipshultz, 2008.

Preferred methods for SEV engineering, provided herein, enable production of SEVs in the appropriate state to produce improved outcomes.

[0064] Thus, the present invention provides methods for producing, evaluating and engineering SEVs, and purifying SEVs (if desired), for use in assisted reproductive technologies (ART) and in other applications of therapeutic, diagnostic and screening relevance. In accord with this invention, a method for engineering SEVs comprises some or all of the following steps: providing a mammalian ejaculate; incubating the ejaculate or isolated sperm derived therefrom under controlled conditions; assaying an aliquot of the sperm during incubation period to determine state of maturation by observing the populations of vesicle-budding sperm and the quantity of shed vesicles in the aliquot; optionally repeating the assaying step with successive aliquots at intervals during incubation to observe real time changes in the bound and free vesicle states; and processing the remaining ejaculate or components therefrom for the desired ART upon reaching a fixed time of incubation, or upon detection of the presence of the most highly positive sperm-bound vesicles for antibody binding in the latest aliquot being assayed (fertility), or when the percentage of positive events in the SEV signature region by cytometry in the latest aliquot being assayed increases by a factor of 1.5-fold relative to the prior timepoint (to harvest vesicles as an additive for ART), or when the largest proportion of antibody-positive shed vesicles appear in the latest aliquot being assayed by microscopy (to harvest vesicles or create gender bias). One can allow the SEV signature region ratio to greatly exceed 1.5, if desired, to increase yield. Note, as vesicle characteristics change with time, for some applications it can be desirable to harvest them immediately upon their first detection by cytometer, and for some applications it can be desired to harvest vesicles from older ejaculates, where the vesicles are generally larger in size and more abundant.

[0065] Another method for providing SEVs is to insert a large portion of the ejaculate into the ingredients for a COBO Assay (see, for example, Modules 4 and 5, set forth below), if it is desired to prepare SEVs directly from that type of incubation environment. In other words, by keeping proportions of ejaculate and reagents identical to the amounts used in the assay, SEVs will be shed and can be separated and purified.

[0066] Sperm can be incubated in seminal plasma without wash, in which case they can take up or be coated with seminal plasma components that may be useful. Or, in a preferred embodiment, the ejaculate is washed with SpermCare™ medium upper layer, which is a colloidal silica suspension in HTF (synthetic Human Tubal Fluid) that specifically purifies sperm from everything else. The sperm can also be washed in SpermCare™ lower layer. A wash step is preferred prior to incubation, because it provides a way to specifically isolate SEVs. Prostasomes and epididymosomes are present in seminal plasma at the time of collection, while SEVs only accumulate with time. A low speed spin, say 2,000 x g for 1-5 min (with large ejaculate volumes, time should be increased up to about 15 min), will pellet sperm, but can't pellet vesicles from the male tract. Hence, a purer preparation of vesicles is obtained—specifically, SEVs— than that which occurs in nature.

[0067] In certain preferred embodiments, the methods comprise the step of, for example, providing and/or collecting the ejaculate from a mammal using a collection device pre- equilibrated to a temperature at or below the body temperature of the mammal.

[0068] In certain embodiments, the method includes a step of incubating the ejaculate from a mammal. In certain embodiments, the incubating step includes controlling the temperature of the ejaculate at a temperature in the range of about 4° C to body temperature of the mammal. In additional embodiments, the incubating step includes controlling the temperature of the ejaculate at a temperature in the range of about 4° C to about 17° C. In certain additional embodiments, the incubating step includes harvesting sperm by centrifugation at about 2000 x g, washing them, and resuspending them in an incubation medium. In additional

embodiments, the centrifugation step includes harvesting sperm by pelleting them at about 2000 x g and aspirating the supernatant. In certain embodiments, the incubating step includes centrifugation and filtration steps to remove sperm and to partially purify the SEVs produced. In still additional embodiments, the incubating step includes harvesting sperm at about 2000 x g and then filtering the supernatant through a syringe filter of about 0.2μιη-0.8μιη, followed by centrifugation at about 100,000 x g for 90 minutes, with pellet resuspension in storage, targeting or deployment medium. In certain embodiments, the incubating step includes resuspension of the sperm in deployment medium comprised of lx phosphate buffered saline pH 7.0, human tubal fluid, embryo wash medium, semen, extender for diluting semen, 0.9% (w/v) physiological saline. In certain additional embodiments, the incubating step includes resuspension in storage medium comprised of lx phosphate buffered saline pH 7.0, human tubal fluid, embryo wash medium, semen, extender for diluting semen, 0.9% (w/v) physiological saline.

[0069] In certain embodiments, the method includes a step of washing the sperm. In certain embodiments, the washing step includes washing with a defined medium such as

commercially available lx phosphate buffered saline pH 7.0, human tubal fluid medium, embryo wash medium, 0.9% (w/v) physiological saline, or Sperm Prep medium.

[0070] In certain embodiments, the method includes a step of resuspending the sperm. In certain embodiments, the resuspending step includes resuspending the sperm in a medium. In certain additional embodiments, the medium is at least one of lx phosphate buffered saline pH 7.0, semen or components thereof, human tubal fluid medium, embryo wash medium, 0.9% (w/v) physiological saline, with optional addition of agent(s) to become cargo for the SEVs or to influence SEV production or composition or a combination thereof.

[0071] In any of the aspects or embodiments as described herein, the cargo is at least one member selected from the group consisting of antioxidants, aptamers, carbohydrates, DNA, enzymes, nucleic acids, proenzymes, proteins, reactive oxygen species or their precursors, RNA, small molecule drugs, toxins, and combinations thereof.

[0072] In any of the aspects or embodiments described herein, the mammal can be a human. [0073] In any of the aspects or embodiments described herein, the collection device is pre- equilibrated to a temperature in the range of about 4° C to about 40° C.

[0074] In certain embodiments, the method includes a step of assaying the ejaculate, sperm (e.g., resuspended sperm as described above) or both. In certain embodiments, the assaying step includes: mixing an ejaculate or sperm (e.g., resuspended sperm as described above) aliquot with at least one reagent capable of reacting with a marker indicative of SEV vesicle status, wherein the reaction produces fluorescence in connection with a positive reaction with a sperm-bound or free vesicle.

[0075] In certain embodiments, the reagent, including, e.g., an antibody or an antibody Fc region or carbohydrates similar enough to the Fc region's carbohydrates to be reactive, that interact with the target marker and the antibody or carbohydrate is labeled with a fluorescent label. In certain embodiments, the reagent includes a primary antibody, a secondary antibody or combination thereof that is labeled with a fluorescent label. In still additional

embodiments, the reagent includes a stabilizer for the sperm cells and/or an additive that may include preparations of SEVs.

[0076] In certain additional embodiments, the method includes a step of determining a percentage of vesicle-positive sperm or the quantity of free vesicles. In certain embodiments, the step of determining the percent positive cells or of SEVs is performed by a method selected from the group consisting of antibody-based, dye-based, motility-based, and optically based procedures.

[0077] In certain additional embodiments, the method includes a step of processing the ejaculate or sperm. In certain embodiments, the processing step includes adding agents to the ejaculate or sperm and thereby stabilizing the ejaculate or sperm for further processing, including for further processing for the desired ART.

[0078] In certain embodiments, the method includes a further processing step including making doses having a predetermined amount of sperm cells and/or SEVs of the desired state and stabilizing them for further diagnostic workup or as doses for ART.

[0079] As used herein, lx phosphate buffered saline pH 7.0, the PBS working solution has the following description: MP Biomedicals LLC, PBS Tablets cat # 092810305 (MP biomedicals cat #) without calcium without magnesium, Fisher Catalog 12821680 prepared according to the manufacturer's specifications. This preparation produces a PBS solution having the following attributes for lOx formulation— Inorganic Salts: Potassium Chloride [KC1]: 200.00 mg/L; Potassium Phosphate Monobasic [KH₂P0₄]: 200.00 mg/L; Sodium Chloride [NaCl]: 8000.00 mg/L; Sodium Phosphate Dibasic [Na₂HP0₄]: 1150.00 mg/L, pH 7.3-7.5 for 1 tablet in 100ml water (therefore pH is of working solution, not lOx formulation).

[0080] In accord with the present invention, semen in the ejaculate is collected and maintained in a tightly controlled environment.

[0081] Environmental control refers to both timing and temperature control. Timing must be prompt in step execution. Because biological events generally occur more rapidly at higher temperature, it is useful to have the collection temperature as low as practicable initially and, with rapid timing of cooling, insure a prompt temperature reduction. Slower rate of change enables superior control. A fast monitoring assay generally is run repeatedly post-ejaculation but before insemination, to monitor sperm and SEV maturation state and permit adjustment of an ejaculate's sperm and SEV condition to the different states needed for different types of diagnosis and/or treatment modalities, such as insemination, e.g., vaginal insemination, insemination into the uterus (IUI), in vitro fertilization (IVF) and intracytoplasmic sperm injection of the egg (ICSI). Sperm and/or SEVs then can be stabilized in the different required states and will thereby produce improved therapeutic outcomes, such as increased fertility or improved freeze/thaw survivability or extended shelf life of cooled semen.

[0082] Stabilization of the sperm and/or vesicles at different required states, as desired, in accord with the present invention can produce, for example, higher numbers of sperm in oviductal reservoirs, extend shelf life, increase resistance to freeze/thaw damage, improve fertility and/or skew the gender ratio when used in ART, and the data generated provides very useful diagnostic information. Thus, the profitability of agricultural operations can be increased. Further, in the clinic, simpler interventions can be made possible and the suffering associated with human infertility can be reduced.

[0083] Moreover, the immunomodulatory or targetable characteristics of the vesicles can be applied in non-reproductive therapies, and the ability to supply the vesicles with

therapeutically-relevant cargo can broaden vesicle utility. In addition, SEV production monitoring is a powerful screening modality for drug development and toxicity, as SEV production requires numerous pathways, which are highly sensitive indicators of toxicity but have been historically difficult to screen without the presently disclosed sperm and SEV engineering system.

[0084] The semen samples useful in the practice of the present invention are mammalian, preferably including, but not limited to human, bovine, swine, ovine, caprine, equine, canine, feline, camelid, exotic or endangered mammals and murine. Marker(s), useful in the practice of certain preferred embodiments of the present invention, that are being assayed before therapeutic use of an individual semen sample or components thereof such as vesicles or other additives, which is being adjusted for desired state of maturation according to the methods described herein, can be an Fc receptor.

[0085] An Fc receptor, as used herein, encompasses a ligand that binds to a region other than the variable domain of an antibody. Accordingly, an Fc receptor encompasses a ligand that binds to the constant region of an antibody, for example to a constant domain of an antibody (including, for example, IgA, IgM, IgG, IgE and their subtypes as well as fragments and engineered forms of ligands functioning like antibodies (such as aptamers, synthetic peptides, carbohydrates, etc.) and including carbohydrates of the region and including covalent attachment through sulfhydryl exchange and through binding to the Fc region of an antibody or antibody derivative.

[0086] In another embodiment, the assay comprises more than one marker. Markers or biomarkers useful herein provide expression correlating to sperm and vesicle maturation, reflected as a measurement over time of an expression pattern of one or more biomarkers against which maturation can be correlated.

[0087] The methods described herein can be applied to any of the class of SEVs containing Fc receptors is identified as those that become positive in the biomarker assay as previously described in patent filings and in this application. Positivity upon execution of the assay can be detected by cytometry (see, for example, Figure 5(a) in this application). Positivity can also be detected by microscopy (see, for example, Figure 4(a) and 4(b) in this application). For SEVs that are smaller in size than those detectable by cytometry or fluorescence microscopy, detection can occur by electron microscopy. In such a case, the biomarker assay is run with the substitution of a secondary antibody tagged with lOnm colloidal gold particles, as opposed to the assay for cytometry and fluorescence microscopy that uses a secondary antibody tagged with a fluorophore.

[0088] Electron microscopy labeling will occur, for example, as follows (to see exosome-size objects): Spin lOOul semen in a 1.5ml microcentrifuge tube containing 1.5ml SpermCare™ upper layer, at 2,000 x g for 1 minute. Aspirate supernatant, resuspend pellet in 1ml HTF medium, centrifuge again. Aspirate supernatant and resuspend sperm pellet in lOOul. To lOOul of Green 1, add 20ul Red 2, 5ul washed sperm and incubate for lOmin. Spin for lmin at 2,000 x g. Resuspend in 25ul Green 1 or HTF medium, add 0.5ul Protein A lOnm colloidal gold stock solution (Cell Microscopy Center, Department of Cell Biology, University Medical Center Utrecht, The Netherlands). (Protein A gold is also available from Sigma: G-7402, Affinity Isolated Antibody to Rabbit IgG (whole molecule) lOnm gold colloid labeled (monodisperse)—host is goat. This can be used if Red 2 antibody is a rabbit polyclonal, which is our preferred Red 2 embodiment.) Incubate PrA gold and sperm for up to lh, on ice pack, for transport to electron microscopy facility. As we see larger amounts of Fc-positive material accumulating over time, it is unlikely that prostasomes or epididymosomes possess Fc receptors—if they did have Fc receptors, we would see vesicles immediately in large amounts (or if they do, then the vesicles are not abundant). So the kinetics suggest that SEV either greatly outnumber other vesicle sources in semen, or that other vesicle sources lack Fc receptors. Green 1, Red 2 and Blue 3 reagents are described in US Publication No.

20120252000 (US Serial No. 13/436,701), the disclosure of which is hereby incorporated by reference. Further, solutions also are described below.

[0089] An alternative method for visualizing Fc receptor on exosomes is to rosette them: take an antibody labeled with colloidal gold (but with Fc region free) and mix with an exosome preparation. This can be done by pipetting 20 μΐ of an exosome preparation into 100 μΐ Green 1, adding the appropriate amount of colloidal gold-labeled antibody as described above (ideally in about 5ul of solution), and incubating at ambient temperature for 60 minutes before preparing grids for electron microscopy. Fc receptor-positive exosomes will bind these antibodies, producing a gold cluster of one or more particles surrounding the exosome. A variant of this rosetting assay may be carried out by coating red blood cells with antibody in such a way as to leave the Fc regions exposed, as described by Witkin et al., 1980. Upon addition of a suspension of exosomes at the correct concentration, RBCs should crosslink to each other through Fc-receptor/ exosome interactions.

[0090] Sperm cells are susceptible to damage from both excessive heat and from too-rapid cooling which produces coiled tails and is termed "cold shock" (Benson et al., 1967).

Accordingly, when cooling sperm cells such as promptly after collecting semen, a relatively slow cooling process is used that minimizes temperature stress. Preferably, a cooling ramp rate of about l-2°C/min. is used.

[0091] Terminology used in this application

[0092] In accord with this detailed description, the following abbreviations and definitions apply unless defined elsewhere herein.

[0093] It should be noted that as used herein, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "an antibody" includes a plurality of such antibodies, and so forth. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art (e.g., in cell culture, molecular genetics, nucleic acid chemistry, hybridization techniques, flow cytometry and biochemistry). Unless otherwise stated, all ranges described herein are inclusive of the specific endpoints and generally accepted measurement and manufacturing tolerances. The following definitions are provided below.

[0094] As used herein, the term "semen sample" includes any semen sample collected from an ejaculate or from a biopsy (e.g., testicular, epididymal, etc.) of any mammal, including, but preferably not limited to, human, cattle, goats, sheep, buffalo, swine, horses, cats, dogs, rat, mouse, rabbits, hamsters and endangered species of mammals. A semen sample can be obtained from both first and second ejaculates, sperm or sperm cell precursors extracted from the epididymis or testes by needle aspiration or other forms of retrieval, and electro ejaculated collections, for example from bull farms.

[0095] As used herein, the term "maturation" is the process of developmental changes that sperm undergo after ejaculation, whether in vitro or in vivo. Maturational changes begin before sperm are capacitated and include the capacitation process as part of the later stages of maturation. [0096] Capacitation is an imprecise term because definitions vary in the scientific literature. Some have broadly defined capacitation as the functional modifications that render sperm competent to fertilize an egg. Historically, more limited definitions restrict capacitation to the changes that occur in sperm within the female reproductive tract and/or to changes that occur at the later stages of sperm maturation. As used herein, the more restrictive definition of "capacitation" is used, i.e., changes that occur in vivo or in vitro in late maturity, in which sperm immediately become able to fertilize an egg.

[0097] As used herein, the term "fertility" with respect to sperm in a semen sample, refers to the ability of the sperm to fertilize an egg and produce a positive result in a sperm/egg penetration assay, and/or produce a viable embryo, fetus and live-born animal. This ability changes as the sperm age and it changes differentially with respect to whether the sperm is carrying an X chromosome or a Y chromosome.

[0098] As used herein, the term "room temperature" is meant to refer to an environment in which the assays of the invention are performed, typically in the range of about 17 - 25 °C.

[0099] The term "marker" and "biomarker" may be used interchangeably and includes, but is not limited to, a ligand, a lectin, an enzyme and a receptor, which is expressed on the surface of the sperm, or internally, or both, and/or in the seminal fluid. In some embodiments, the marker is a morphological change in an acrosome which can be viewed, for instance, using bright field or phase contrast microscopy. With respect to acrosome morphology, over time the surface of the acrosome' s membrane appears increasingly ruffled, with SEVs being released. In some embodiments a marker can be cryptic at some stages of metabolism, and not detected.

[00100] As used herein, the term "antibody," includes, but is not limited to a polypeptide substantially encoded by an immunoglobulin gene or immunoglobulin genes, an IgG antibody, an IgM antibody, or a portion thereof, which specifically binds and recognizes an analyte, antigen or antibody. An antibody or fragment thereof can be isolated from a natural source, for example, an animal, mammal, mouse or human. Alternatively, an antibody or antibody fragment can be produced using synthetic processes, including but not limited to recombinant methods and chemical synthesis. [00101] "Antibody" also includes, but is not limited to, a polypeptide substantially encoded by an immunoglobulin gene or immunoglobulin genes, which specifically binds and recognizes the antigen- specific binding region (idiotype) of antibodies produced by a host in response to exposure to the analyte or immunogen.

[00102] As used herein, the term "antibody," encompasses polyclonal and monoclonal antibody preparations, as well as preparations including monoclonal antibodies, polyclonal antibodies, hybrid antibodies, altered antibodies, F(ab')₂ fragments, F(ab) fragments, F(c) fragments, Fv fragments, single domain antibodies, chimeric antibodies, humanized antibodies, dual specific antibodies, bifunctional antibodies, trifunctional antibodies, single chain antibodies, and the like, and functional fragments and multimers thereof, which retain specificity for an analyte or antigen. For example, an antibody can include variable regions, or fragments of variable regions, and multimers thereof, which retain specificity for an analyte or antigen. See, e.g., Paul, Fundamental Immunology, 3rd Ed., 1993, Raven Press, New York, for antibody structure and terminology. Alternatively, the term "antibody" comprises a fragment thereof containing the constant region, in particular the Fc region. The antibody or portion thereof, may be derived from any mammalian species, e.g., from a mouse, goat, sheep, rat, human, rabbit, or cow antibody, or from a chicken antibody (e.g., IgY). An antibody or fragments thereof, may be produced synthetically by methods known in the art, including modification of whole antibodies or synthesis using recombinant DNA

methodologies, including using phage display libraries. Also included are structures that are not antibodies but can, like antibodies, function in ligand binding reactions, including but not limited to aptamers, oligosaccharides, oligopeptides, and nucleic acids.

[00103] As used herein, the term "label" includes a detectable indicator, including but not limited to labels which are soluble or particulate, metallic, organic, or inorganic, and includes radiolabels (such as, e.g., 14 C, 3 H, 32 P, and the like), latex or other beads, enzymatic labels (e.g., horseradish peroxidase, galactosidase, and other enzyme conjugates), spectral labels such as green fluorescent protein, quantum dots, polarimetric spin labels, fluorescent dyes (e.g., fluorescein and its derivatives, e.g., fluorescein isothiocyanate (FITC), Calcein AM (AnaSpec Cat. No. 89201) which is not fluorescent until the molecule is subject to enzymatic cleavage, Alexa Fluor® 488 Dye, which is a green-fluorescent dyes conjugate with nearly identical spectral properties and quantum yield as fluorescein isothiocyanate, rhodamine, Yo- Pro, a carbocyanine nucleic acid stain sold by Invitrogen, catalog Product V 13243, the green- fluorescent YO-PRO®-l), chemiluminescent compounds (e.g., luciferin and luminol), spectral colorimetric labels such as colloidal gold, or carbon particles, or colored glass or plastic (e.g. polystyrene, polypropylene, latex, etc.) beads, as well as dyes, including the cell- permeant pH indicator, carboxy SNARF®-1, an acetoxymethyl ester, acetate which has a pKa of -7.5 after deesterification and is sold by Invitrogen, as catalog # PPLM63- C1270, and the like. Where necessary or desirable, particle labels can be colored, e.g., by applying dye to particles. Thus, the label can be detected using colorimetric platforms with enzyme-produced color like in ELISA type tests. Luminometers also can be used. Fluorescence polarization can also be used. FRET (fluorescence resonance energy transfer) also can be used.

[00104] As used herein, the term "colored particle label" includes, but is not limited to colored or transparent (uncolored) latex (polystyrene) particles, metallic (e.g. gold) sols, non-metallic elemental (e.g. Selenium, carbon) sols and dye sols. In one preferred embodiment, a colored particle label is a colored particle that further comprises a member of a conjugate pair.

Examples of colored particles that may be used include, but are not limited to, organic polymer latex particles, such as polystyrene latex beads, colloidal gold particles, colloidal sulphur particles, colloidal selenium particles, colloidal barium sulfate particles, colloidal iron sulfate particles, metal iodate particles, silver halide particles, silica particles, colloidal metal (hydrous) oxide particles, colloidal metal sulfide particles, carbon black particles, colloidal lead selenide particles, colloidal cadmium selenide particles, colloidal metal phosphate particles, colloidal metal ferrite particles, any of the above-mentioned colloidal particles coated with organic or inorganic layers, protein or peptide molecules, or liposomes. For example, Quantum dots, sold by Life Technologies, are a type of label encompassed herein.

[00105] It should be noted that the extracellular vesicle/ exosome field is so new that the scientists tasked with nomenclature failed to reach agreement, instead publishing the following statement (Gould and Raposo, 2013) in a recent issue of the Journal of Extracellular Vesicles:

"In the face of these conflicting definitions, we feel that investigators should not be forced to concede their scientific independence, violate precedent or ignore compelling empirical data when it comes to their choice of nomenclature. As such, we offer 4 suggestions for authors, reviewers and editors. First, authors should state their use of terms explicitly, choose their terms based on precedent and logical argument, and apply them consistently throughout a piece of work. Second, authors should clearly state their method(s) of vesicle collection, how the method(s) relate to their use of terms and even the method(s) for obtaining and storing biological fluids prior to isolating vesicles. Third, reviewers and editors should respect authors' scientific freedom in their choice of vesicle nomenclature, so long as it follows precedent, logic and the authors' data. Fourth, authors should be encouraged to use the term 'extracellular vesicle' (EV) as a generic term for all secreted vesicles, and as a keyword in all publications."

[00106] Therefore, herein, the broad definitions cited by these authors are used, with the added awareness that the term exosome is generally used to refer to smaller vesicles (about 50- 120nm in size, although these boundaries differ according to different publications).

[00107] As used herein, the term "exosome" includes, but is not limited to, secreted vesicles of about 20-120nm in diameter that may serve a physiologic function.

[00108] As used herein, the term "extracellular vesicle" includes, but is not limited to, all vesicles released from cells by any mechanism, therefore including secreted and exocytosed vesicles, thereby encompassing exosomes, but also including vesicles released by ectosytosis, reverse budding, fission of membrane(s) (as, for example, multivesicular endosomes, ectosomes, micro vesicles and microparticles, see Barteneva et al. 2013), and release of apoptotic bodies and hybrid vesicles containing acrosomal and sperm plasma membrane components.

[00109] As used herein, the term "sperm-derived extracellular vesicle" includes all

extracellular and budding vesicles produced by sperm upon either incubation under controlled conditions or upon provocation of maturation to simulate the effects of the incubation process. One example of a useful subset of SEVs of the instant invention is those SEVs that possess Fc receptors.

[00110] As used herein, the term "cargo" includes, but is not limited to, any agent(s) that can be carried in the aqueous compartment of an EV, or inserted into the membrane of an EV, or tethered to the EV by any form of attachment (covalent or non-covalent). Examples of cargo include nucleic acids such as RNA (mRNA, regulatory RNA such as, for example, siRNA, miRNA, antisense RNA), DNA, enzymes, proteins such as antibodies, cytokines, soluble Fc receptors or fragments thereof including Fc receptors or fragments thereof incorporated into lipid bilayers, enzymes such as those of the Krebs citric acid cycle or the glycolytic pathway, phospholipases, reverse transcriptases or drug- activating/ inactivating enzymes such as cytochrome P450, carbohydrates such as heparin or other sulfated glycans, dyes such as Brilliant Blue, ions such as Ca⁺⁺, small molecules such as chelators, antibiotics, sugars, glycosides, cholesterol-loaded cyclodextrins, cholesterol depleted cyclodextrins, fluorophores, nanoparticles, drugs (e.g., the antiretroviral Maraviroc, NSAIDs, antioxidants,

chemotherapeutic agents and other similar species, including generics, as use of SEVs as a delivery system constitutes a novel use in conjunction with these agents). Examples of particles that may be used include, but are not limited to, organic polymer latex particles, such as polystyrene latex beads, colloidal gold particles, colloidal sulphur particles, colloidal selenium particles, colloidal barium sulfate particles, colloidal iron sulfate particles, metal iodate particles, silver halide particles, silica particles, colloidal metal (hydrous) oxide particles, colloidal metal sulfide particles, carbon black particles, colloidal lead selenide particles, colloidal cadmium selenide particles, colloidal metal phosphate particles, colloidal metal ferrite particles, any of the above-mentioned colloidal particles coated with organic or inorganic layers, protein or 30 peptide molecules, or liposomes. For example, Quantum dots sold by Life Technologies, is a type of particle encompassed herein.

[00111] As used herein, the term "targeting structure" includes, but is not limited to, antibodies (see preceding definition) including anti-transferrin antibody for targeted delivery into tumors and therapeutic monoclonal antibodies (that can be tethered to SEV-borne payloads to confer synergistic therapeutic response) such as those shown in Figure 10c, and any ligand that can be recognized by the target tissue, surface, or substrate toward which the SEV is targeted. Examples include proteins such as antibodies or fragments (synthetic or natural), cluster of differentiation (CD) proteins (for example, as targeted by Alemtuzumab for treatment of leukemia, lymphoma or in conditioning regimens for transplantation), adhesion molecules such as ankyrin, antibody mimetic proteins such as DARPins, where the precise one can be chosen for desired function from libraries having randomized potential target interaction residues— as is done for antibodies and their derivatives— through methods known to one of ordinary skill in the art, enzymes (such as glycosidases, sialidases, lipases, proteases) carbohydrates such as heparin or other sulfated glycans, cell adhesion molecules such as EpCAM (epithelial cell adhesion molecule), synthetic or natural oligomers, polymers or large ligands that can bind to the antibody on the SEV surface while still having ligand-binding sites exposed for recognition on the targeting structure (such as viral envelope glycoproteins, receptors or fragments thereof, cell adhesion molecules, proteins, carbohydrates, liposomes), cells including bacteria, viruses and cells of higher organisms.

[00112] Targeting structures can be compound in nature, that is, for example, an antibody can be reacted with the ligand it specifically recognizes, and that ligand (as opposed to the antibody) can react with the targeted structure. An example is coating the SEVs with an antibody directed against an O-glycan containing Sialyl Lewis antigen (for example found on solubilized ZP3 or ZP4 from the zona pellucida of the egg), and then reacting the antibody with a molecule containing Sialyl Lewis X antigen. The Sialyl Lewis X antigen can then be used to target the SEVs to cells containing receptors for the Sialyl Lewis sequence (sperm, for example). Antibodies bound to the SEVs can also be used to tether other antibodies in turn, where these other antibodies carry the recognition domain for the targeted substrate. One also can use an antibody to tether an enzyme or biologically active molecule to the SEV surface, to add functionality to the SEV in terms of its catalytic ability or biological activity.

[00113] One can clone, isolate and use only a Fc receptor or fragment(s) thereof in certain applications. However, a Fc fragment lacks much of the SEV functionality and can be less desirable therapeutically than the SEV. The isolated Fc fragment, by itself, cannot carry cargo, as do vesicles. The isolated Fc fragment will have different pharmacological absorbtion, distribution, metabolism, excretion and toxicity properties than a SEV, and potentially a shorter half-life in vivo or in vitro. These differences will affect efficacy because, unlike vesicles, the receptor or fragment(s) is not derived from a natural biological process of dose administration and metabolism that is already established as non-toxic. In addition, use of the Fc receptor to bind antibodies that can target SEVs to specific sites avoids use of antibodies that are free in solution, an advantage because free antibodies, although they can be therapeutically useful, ultimately are foreign proteins known to cause allergic reactions and immune complex glomerulonephritis in some patients.

[00114] Because EVs and exosomes including SEVs have existing mechanisms for cellular uptake and component recycling or degradation, antibodies bound to them do not have the same fate as the ultimately dangerous free antibodies or other proteins such as a Fc receptor and fragments thereof.

[00115] Treating and evaluating a semen sample according to the methods described herein enables adjustment of the timing for processing an ejaculate or its SEVs, and for modifying the structure of SEVs, for use in assisted reproductive technologies (and other applications), according to the desired performance of either the sample or an ejaculate to which vesicles are added, such performance being, for example, increased fertility or increased ability to resist antibody attack and create successful normal live births in ART, i.e., with babies healthy enough to bring home from the hospital (what the industry terms a high take -home baby rate).

[00116] Kinetic measurement, monitoring and engineering of SEV formation as a diagnostic or preparative method for therapy or screening also is broadly applicable to other sample types, as well as to cell types other than sperm, to cultured cell systems and to synthetic vesicle production. For example, depending on vesicle structure from other cells, one can merely substitute a different antibody in the assay, for example, to measure kinetics of vesicle production from non-sperm sources. One can change the timing or type of cargo added. One can attach surface ligands by making minor modifications within the skill in the art. All fundamental principles remain the same.

[00117] These observations question the accuracy of long held presumptions in the art that a certain number of sperm are needed for successful fertilization. Although not relying on theory, it now appears that a certain number of SEVs are needed to ensure proper function of the sperm that are present.

[00118] The present findings suggest significant commercial applicability (see Table 2).

[00119] Table 2. Products and functions derived from the instant invention when it is applied in manufacturing

sperm per dose for insemination, dose of semen to result in pregnancy. The more improving profitability, product is an doses per ejaculate, the greater the profit, since additive of SEVS or synthetic semen is sold on a per dose basis, and the lower agent(s) derived from them or related the labor costs.

to them or their functions

Fertility improvement, product is an Use at time of artificial insemination to down additive of SEVs or synthetic regulate female immune response

agent(s) derived from them or related

to them or their functions Use in IVF/ ICSI during in vitro incubation of germ cells to improve fertilization rate and embryo quality

Use with sperm having acrosomal

abnormalities, to enable them to fertilize

Gender bias in livestock, product is a Diagnostic to indicate proper time to test kit for sperm to detect SEVs stabilize/freeze sperm doses to produce desired reproductive outcome

Sperm stabilization and/or increased Use test kit and/or additive to prepare sperm survival for storage, cooled or frozen doses with increased shelf life, fertility, doses for use in assisted reproductive resistance to degradation/damage on storage technologies and/or or freeze-thaw, or other desirable

properties

Two products, additive and test kit

(test kit to optimize sperm state, prior

to mixing them with SEV

preparation as an additive)

Immunomodulation via Fc receptor Insemination, embryo transfer/implantation immune response silencing, product Transplantation

is an additive Dental and surgical implants

Inflammation reduction in brain, joints, periodontal structures, organs, etc.

Reduction of auto-immune responses

SEV targeting via binding antibodies Use SEV to carry cargo to desired site (note: to Fc receptors, to target both SEVs cargo can be a mixture of targeting and and their cargo, product is an therapeutic agents affixed to SEV surface, it engineered SEV additive or need not only be present in SEV membrane or component(s) or synthetic(s) derived internal locations), as with other vesicles may therefrom cross blood-brain or blood-retinal barriers Diagnostic evaluation of sperm for Enables faster rejection of abnormal doses, to normalcy of SEV biology, product is speed dose manufacture on farm or reduce cycle a test kit time between inseminations in clinic

Reproductive status monitoring after Doctors can more accurately advise patients chemotherapy, radiation, illness or about attempting conception after illness.

other insults, product is a test kit

Toxicity screening, drug As an early screening method, enables faster development, product is a test kit and identification of possible issues as potential the screening substrate (sperm, SEVs drugs move down the developmental pipeline or derivatives thereof)

Drug delivery to increase specificity Improved therapeutic performance and

of targeting, reduce dosage and/or formulations.

toxicity, preserve efficacy, increase

half-life, product is an engineered

SEV

SEV profiling, product is a test kit Evaluate SEVs for biomarkers predictive of interfaced with mass spec or other fertility issues (e.g., oxidative damage such as omics profiling platform or high- lipid oxidation) so that appropriate therapeutic content or high throughput platform interventions to improve fertility can be

implemented

[00120] SEVs are, thus, especially attractive as both diagnostic targets and—when adjusted to the appropriate maturation state and engineered in accord with the present invention— as an additive to facilitate good outcomes in ART and other therapeutic applications.

Diagnostically, SEVs can be prepared from a semen collection at different kinetic points in the assay curve observed by following the incubation, to determine if abnormalities are universal or localized to a single stage of the maturation process. SEVs isolated as described herein can be profiled in numerous ways, such as through nucleic acid analysis (for example, through use of Affymetrix genechip 2.0), by LC-mass spectrometry for protein analysis, by surface plasmon resonance for the presence of surface antigens such as Fc receptors, cluster differentiator antigens (for example, antigens reactive to CD63, this requires use modified antibodies lacking the Fc region, to insure target detection is through the FAb region), antigenic carbohydrates, and lipids. Profiling of oxidative status, to which fertilization is sensitive, can be carried out on SEV preparations by analysis of their lipid profiles in general and for oxidized lipids in particular, as well as by RNA and enzyme profiling to search for signatures associated with oxidative stress. [00121] Because attachment of antibodies to SEVs can alter antibody pharmacological properties such as toxicity and half-life, preparations of SEVs coated with therapeutic monoclonal antibodies or other therapeutic agents can offer improved outcomes not seen with the isolated antibody or therapeutic agent. The capacity to add cargo, in contrast to the inability seen with isolated antibodies or therapeutic agents, provides an additional opportunity for synergy in therapeutics.

[00122] In addition, because the Fc receptor binding event orients antibody so that its epitope recognition regions are exposed (Figure 10a), it is possible to apply a mixture of antibodies to the SEV surface and obtain highly specific targeting and binding of a second ligand. This can include, for example, one antibody to target the SEV to the recipient cell, and another to carry a pharmacological payload, for those situations where it is desired to have such an agent on the surface of the SEV.

[00123] Enzymatic profiling of SEV is of additional importance, as sperm have a very high metabolic rate and external support for their functions is important. Exosomes have been characterized that contain enough enzymes from the glycolytic pathway that they can generate ATP—these are from the prostate and are present in ejaculates (Ronquist et al., 2013). Their purification and use as an additive, as with SEVs, can be useful to enhance fertility and/or sperm performance (stability, freezing damage resistance, shelf life of sperm stored cooled but not frozen). Feeder exosomes, whether from sperm or the male or female tract, that are subsequently absorbed by sperm, support necessary metabolic functions as well as cell-cell communication (hence their use as additives has utility). This also makes their presence an important factor for normal fertilizing ability in sperm, and their normal or abnormal function can be reflected in the SEV signature of the sperm that have absorbed them. This is one reason why washing of sperm at the wrong time prior to use in ART can be

counterproductive. With vesicles and active molecules washed away, sperm cannot take up the proper metabolic machinery from prostasomes to support sperm viability, or take up active agents such as cytokines from semen that sperm can then extrude in vesicles or in soluble form to induce receptivity of the female tract.

[00124] As used herein, a stability agent is an agent that affects COBO kinetics by: (1) reducing the amplitude of positive signal, (2) retarding appearance of the positive signal. It also is an agent that slows or stops sperm metabolism, such as very cold temperature (as when, for example, sperm are frozen in liquid nitrogen) and/or an agent that preserves sperm viability for a longer time, such viability being defined by preservation of motility, preservation of the ability to swim up in the swim up test, ability to fertilize eggs in vitro or in females carrying them.

[00125] Stability can be achieved, for example, by the following mechanisms: one, by retarding capacitation and so keeping more sperm alive and potentially able to fertilize and two, by enhancing resistance to freeze/thaw damage, so that more live sperm come out of a dose post-thaw.

[00126] Examples of "stability" agents include: cholesterol, cholesterol-loaded dextrins, cool temperature, egg yolk, calcium chelators such as EGTA, kinase inhibitors such as Sutent (sunitinib maleate, SU11248), bicarbonate removal agents such as carbonic anhydrase, oviductal explants and components therefrom, pH (low), glycerol, trehalose, saturated fatty acids and their derivatives (by preventing gain of membrane fluidity associated with capacitation)—and also unsaturated fatty acids or polyunsaturated fatty acids (PUFA) because by increasing membrane fluidity pre-freezing, they may enhance survivability of sperm through freeze/thaw, bovine serum albumin or other albumins, especially cholesterol-loaded, lipoproteins, n-ethylmaleimide, botulin toxin and SNARE inhibitors, glutathione, 2- mercaptoethanol, spin labels with saturated fatty acid chains, .decapacitaiton factor(s) from seminal plasma and/or epididymal fluid, prostasomes, Rp-Adenosine-3',5'-Cyclic

Monophosphorothioate (cAMP antagonist), desmosterol sulfate, anesthetics such as lidocaine, heparin sulfate, sphingomyelin, capsaicin, glycerol, trehalose, Annexin V, proton pump inhibitors (including omeprazole, esomeprazole, lansoprazole, pantoprazole and rabeprazole), partial pressure of oxygen (oxygen tension), and the like.

[00127] As sperm can be subjected to conditions that mimic normal or aberrant female tract conditions, the ability of sperm from a healthy donor to fertilize in the face of female- side issues can also be evaluated by SEV diagnostics. One simply changes the conditions under which sperm are incubated and determines if they are resilient to the conditions—that is, they continue to produce normal signatures, or if they are susceptible to the conditions- developing abnormal signatures specifically in the face of the tested conditions, not the control conditions (see FIG. 5 examples, where "intact early" is a normal early signature and "intact late" is a normal late signature as measured by cytometry after running the COBO assay). Sperm that are resilient, preserving the normal signature, are more likely to be fertile despite the modifications of ART that are known to iatrogenically induce dysfunction

(Mortimer 1991). Sperm that change from the normal signature upon minor provocation are most likely to achieve fertility only after more complex ART interventions—such as ICSI— that better compensate for sperm frailty.

[00128] Profiling of inflammatory mediators is of particular import, as these are one of the selective mechanisms in the female repertoire to ensure fertilization by the healthiest sperm. Therefore, not all female-side anti-sperm inflammatory response is necessarily bad, as it is currently (mis)understood to be by the medical establishment. On the contrary, it helps to ensure a high quality conceptus will form. Sperm with abnormal biology, ones that are not capable of producing SEVs, become (rightfully so) targets for the female immune response. The proper level of inflammation is therefore useful and selective. Inflammation of the vasculature can support uptake of platelet-derived exosomes, and this induction of exosome uptake can apply to the tissues of the female tract when inflamed by the presence of sperm. This enhancement of communication potential can be an important component of successful fertilization, with the concomitant concern that use of anti-inflammatory approaches should be of very targeted types to prevent fertilization by substandard sperm that would otherwise be eliminated by the female immune response. Use of NSAIDS is detrimental to the normal process. However, use of SEVs, with their Fc receptors, as an antibody sink, can preserve other inflammatory processes intact and thereby improve embryo quality better than the blunt instrument of NSAIDS or related pharmaceuticals. SEVs prepared according to the present invention have great utility in such an application, as they offer a source of highly targeted immunomodulation. One of ordinary skill in the art will recognize that this concept can be extended to other systems where the immunomodulatory capacity of SEVs confers therapeutic utility and the ability to fine-tune the degree of immune response.

[00129] SEV diagnostics can also be run with other clinical tests on ill donors, to determine the correlation between normalization of other clinical tests and normalization of SEV diagnostic results as the donor recovers. One key application can be after recovery from a febrile illness, as these are known to damage sperm. However, cancer survivors, transplant survivors and others may wish to know their status as well, in order to initiate conception attempts when sperm have regained normal function, which includes normal SEV kinetics, composition and concentration. Such an approach serves men who wish to initiate a pregnancy, but only after their sperm biology has recovered fully. Doctors can test SEV profiles, after the needed time has elapsed for recovery from the type of insult sustained, and counsel men and their partners with respect to the best paternal status for attempting conception.

[00130] Sperm environmental sampling, which includes uptake of surrounding medium and of exosomes from the female or male tract or from other sperm, has demonstrated now that is possible to isolate sperm and analyze them to see the signature left by non-sperm derived exosomes or environmental fluid, to determine if this signature is normal. Sperm can be harvested by low speed centrifugation, washed, lysed and analyzed by mass spectrometry and compared to analysis of a sample of control sperm not exposed to exosomes, or sperm from healthy and infertile men can be profiled to stratify results into diagnostic categories.

[00131] Those of ordinary skill in the art can recognize that assays or agents, whose use and/or production is described herein, can be applied to high throughput screening platforms or similar methodology for measurement of diagnostic parameters of import in drug discovery, such as toxicity of drug candidates or hits. The COBO assay can be used as an end point measurement reporting enhanced or normal (or, in the presence of toxicity or drug activity, unsuccessful) budding and shedding of SEVs.

[00132] Because SEV production requires sperm to have correct functioning of metabolic pathways that are pharmaceutically-relevant, and sperm contain a large number of such pathways, a wide range of cellular metabolic pathways can be interrogated for impact of a drug or other chemical agent. This is especially true since reproductive functions are designed to fail rather than to produce a defective offspring, making measurement of reproductive function the canary in the mine for toxicity testing.

[00133] Additionally, when SEVs are prepared and stabilized as made possible by the present invention, they have significant utility in broader applications (Table 3). Sperm can be washed in a defined medium before they produce SEVs, thereby producing SEVs—within hours— to make a high purity product derived from a single cell type. Because sperm associate material from the environment with these vesicles, as shown by acquisition of COBO-assay-derived dye positivity in the cytometer vesicle signature region, their SEVs can carry various cargos that give these SEVs even greater therapeutic utility. They can be used as a biotherapeutic or the sperm producing them can be even be used as a live therapeutic.

[00134] Table 3. Partial list of SEV attributes

[00135] For example, SEVs can be used in immune suppression, for examples ranging from topical to transplantation, due to their ability to adsorb and inactivate large amounts of antibody in a localized fashion, without concomitant systemic immune suppression and its associated dangers, especially on newly-operated patients at risk for hospital-acquired drug- resistant infections. Alternatively, SEVs can be encapsulated in gel to alter dosage formulation, or provided with a cargo during or after their formation, coated with antibodies to the desired target (which, because SEVs contain Fc receptors, will retain the antibody' s target specificity without steric hindrance, because the Fab regions are exposed), and administered for homing to the desired organ or niche. IgM has been used successfully in this regard. SEV recognizes antibodies through carbohydrates and numerous antibody isotypes are glycosylated. Exosomes have even been shown to cross the blood-brain barrier (Lee et al., 2013), making their use in administration of CNS therapeutics of great interest. In one embodiment of the invention, the transferrin receptor can be targeted as the uptake mechanism by anchoring the desired transferrin structure to the SEV via antibody or direct attachment. Therapeutic use of exosomes has suffered greatly due to their lack of abundance in tissue cultured cells and to their mixed nature when obtained from biological sources such as blood. That is not an issue for sperm-derived SEVs. Abundant, single-cell-source SEVs are readily obtainable from isolated, washed sperm cells in vitro, provided there is a method to measure their appearance and to engineer their content and surface structure as provided by the present invention.

[00136] Additional means of cargo introduction to SEVs preferably include the use of colloid- stabilizing medium being preferred (Hood et al., 2014; Alvarez-Erviti et al., 2013). As aggregates are undesirable at the protein level also, at sufficient scale, hollow fiber filtration can be used in addition to ultracentrifugation to achieve superior purification (Suntres, 2013). Also, for example, cargo loading by exosome electroporation has been described in Nat Biotechnol. 2011 Apr: 29(4):341-5. doi: 10.1038/nbt.l807. Epub 2011 Mar 20.

[00137] SEVs have properties that make them particularly useful for both targeting to recipient cells and for loading of cargo. One method already described in the SOP examples below is to permit sperm to produce SEVs in defined medium. Under such conditions, SEVs become associated with agents from the medium. In a preferred embodiment, the association is illustrated with two agents through different mechanisms. One agent is antibodies—which can be both internal and external, through Fc receptors, which can function both when externally disposed on the SEV surface, or at the SEV interior. A second agent is dye, which can be integrated into the membrane or be incorporated into the SEV interior. The same mechanism can also function to trap antibodies internal to the SEV, in addition to the SEV capacity to bind antibody at its surface.

[00138] Another method of associating cargo with SEVs is the process of electroporation, which allows introduction of large, highly charged molecules into cells and other lipid- bilayer-containing structures. Two advantages of electroporation are that is preserves viability, and electroporation devices are commercially available. Sperm electroporation was used to introduce DNA into sperm well over a decade ago (Tsai, 2000). Thus, it is possible to use electroporation at the level of sperm or with isolated SEV preparations. Means of electroporating in the presence of agents that prevent precipitation have become available more recently, and can be used with isolated SEV preparations involving small vesicles (Hood et al., 2014; Alvarez-Erviti et al., 2013). [00139] As previously mentioned, SEVs can be separated from cargo by centrifugation as shown in examples. At large scale, hollow fiber filtration is an effective way to separate antibody- sized molecules from cells in monoclonal antibody manufacturing, and would also be straightforward in its application to separation of small cargo agents from SEVs. Dialysis can also be employed.

[00140] Examples: MODULES SHOWING STANDARD OPERATING PROCEDURES (SOPs). Figure 8(b) maps these modules onto a manufacturing flow chart.

[00141] Module 1 Semen collection

[00142] Module 2 Cargo loading into extracellular SEVs

[00143] Module 3 Targeting of SEVs to desire location through attachment of ligand- binding agent(s)

[00144] Module 4: COBO Biomarker Assay— production of SEVs (also see Figure 11)

[00145] Module 5: COBO Biomarker STAT Assay for fresh semen collection

[00146] Module 6: Use of SEV preparations in assisted reproductive technology (ART)

[00147] MATERIALS AND EQUIPMENT

[00148] Preparative ultracentrfuge, Beckman Coulter Optima L-90k;

[00149] Ultracentrifuge rotor, Beckman Coulter SW41 Ti rotor;

[00150] Ultracentrifuge tubes, Beckman Coulter 14mm x 89mm open-top thin wall polyallomar, cat. # 331327;

[00151] Syringe filter, 0.2μηι to 0.8μπι;

[00152] Ice bucket;

[00153] Chiller bath capable of maintaining bath temperature of 12°C;

[00154] Extender (semen diluent);

[00155] Cattle extender examples include: Bioxcell, CRYOBOS, Andromed CSS (Minitube), Triladyl (Minitube), OptiXcell (IMV), BlOXcell CSS I & II with and without antibiotics (IMV), BlOXcell, animal protein free extender (IMV), BULLXcell (IMV);

[00156] Human extender examples include: Spermprep TYB, Sperm Maintenance Medium (Irvine Scientific), Freezing Medium— TYB with Glycerol & Gentamicin (Irvine Scientific), CryoProtec II (Nidacon), Refrigeration Medium— TYB with Gentamicin; [00157] Boar extender examples include: Beltsville Thawing Solution (BTS) (IMV),

PRIMXcell (IMV), NUTRIXcell (IMV), TRIXcell+ (IMV), SAFECELL+ (IMV), BF-5, ZORPVA, KIEV, Vital®, Madena, MR-A, X-Cell®, Illinois Variable Temperature (IVT), MULBERRY III®, Reading, Zorlesco;

[00158] Cargo loading medium (can also serve as wash medium in some cases):

Antibody Diluent (Life Technologies, Part# 00-3118, 250 mL supplied as 5 x 50ml; Part# 00-3218, 500 mL);

[00159] PBS Tablets without calcium without magnesium (MP Biologicals LLC; Catalog 2810305);

[00160] HTF Medium (human tubal fluid medium) (Irvine Scientific; Catalog 90125);

[00161] mHTF Medium (Catalog 90126);

[00162] MultiBlast Medium (Catalog 90139);

[00163] PBS Buffer: 8 g NaCl; 0.2 g KCl; 1.44 g Na2HP04 · 7H20; 0.24 g KH2P04; H20 to

1 liter. pH 7.2;

[00164] VWR pH 7.2 (Catalog 95062-798);

[00165] PBS Tablets without calcium without magnesium (MP Biologicals LLC, Catalog 2810305);

[00166] Wash Medium (for washing sperm free of seminal plasma prior to resuspending in cargo loading medium);

[00167] PBS Buffer: 8 g NaCl; 0.2 g KCl; 1.44 g Na2HP04 · 7H20; 0.24 g KH2P04; H20 to 1 liter. pH 7.2;

[00168] PBS Tablets without calcium without magnesium (MP Biologicals LLC; Catalog 2810305);

[00169] VWR pH 7.2 (Catalog 95062-798);

[00170] 0.9% w/v physiological saline

[00171] Saline, Blood bank; Fisherbrand; Azide-free, preservative-free; 0.85% w/v isotonic NaCl solution; Buffered; pH 7.2 + 0.1; For in vitro diagnostic, laboratory use; Supplied in Cubitainer; 2.64 gal. (10L) (Fisher; Catalog # 23-312-651)

[00172] Antibody Diluent (Life Technologies; Part# 00-3118, 250 mL supplied as 5 x 50ml; Part# 00-3218, 500 mL); [00173] InVitroCare® Sperm Prep Media (Fertility Technology Resources, Inc.; Sperm wash medium, Catalog ivc2003); SpermCare™ Upper layer (45%), Catalog ivc2221; SpermCare™ Lower layer (90%) Catalog ivc2222);

[00174] HTF Medium (human tubal fluid medium) (Irvine Scientific; Catalog 90125);

[00175] mHTF Medium (Irvine Scientific; Catalog 90126);

[00176] MultiBlast Medium (Irvine Scientific; Catalog 90139);

[00177] Targeting Medium: Green 1, HTF, 1 x PBS or 0.9% w/v physiological saline (0.9%), containing antibody or ligand at a concentration of lmg protein/ml

[00178] Reagent Green 1 (When reagents are termed "premixed" this means a 1.5ml microcentrifuge tube contains ΙΟΟμΙ of Reagent Green 1 and 20μ1 of Reagent Red 2):

[00179] Green 1 is the following:

[00180] Red 2 (when reagents are termed "premixed" this means a 1.5ml microcentrifuge tube contains 100 μΐ of Reagent Green 1 and 20 μΐ of Reagent Red 2) is one of the following: Difco Salmonella H Antiserum Poly a-z, EN, G, L, Z, and 1 complexes and a-k, r-z, z6, zlO, z29 agglutinins or other Difco antibodies, where many lyophillized antibodies are available, examples from Voigt Global Distributors include Catalog numbers: Difco- 241049, 264010, 221001, 229701, 223021, 223001, 228151, 222641, 224061. Rabbit antibody-derived and purified Fc fragment was substituted for intact antibody and functioned well (Cohen et al., unpublished results), however, this is not a preferred embodiment due to its higher cost. Lyophillized whole antibody is reconstituted to the volume specified by the manufacturer, but using 1 x PBS.

[00181] Voigt Global Distributors (See also URL: http://voigtglobal.com/15-difco-antigens- and-antiserum).

[00182] Reagent Blue 3 (also called "Activator"):

Alexa Fluor Goat anti-rabbit IgG (H + L) Secondary Ab (Life Technologies

(ThermoFisher); Cat. No. A-11008).

[00183] Targeting ligands [00184] Targeting ligands can include antibodies or fragments thereof, antibodies to which the actual targeting ligand that will interact with the recipient cell type(s) is attached, and related structures. Examples include monoclonal and polyclonal antibodies, for example:

[00185] Those shown in Figure 10(b) used in cancer therapy and sourced from various pharmaceutical companies.

[00186] EpCAM oligoclonal antibody (clone 22 HCLC) ABfinity™ Recombinant antibody

(Novex®), Life Technologies catalog # 710524.

[00187] Transferrin Receptor Monoclonal Antibody, Mouse (clone 236-15375), Life

Technologies Catalog # A-l 1130.

[00188] Cluster of Differentiation (CD)-directed antibodies, a number of which are available for purchase from Life Technologies (note: unconjugated antibodies are preferred for use so that they will bind to the SEV Fc receptor without steric hindrance from a conjugated fluorophore) for example at the following URL and web pages related to it:

http://wwwMfetechnologiesxom/us/en/home/life-science/cell-analysis/flow- cytometry/antibodies-for-flow-cytometry/mouse-antibodies-for-flow-cytom

[00189] Buffer:

PBS Buffer: 8 g NaCl; 0.2 g KC1; 1.44 g Na2HP04 · 7H20; 0.24 g KH2P04; H20 to 1 liter; pH 7.2

[00190] PBS Tablets without calcium without magnesium (MP Biologicals LLC; Catalog 2810305);

[00191] VWR pH 7.2 (Catalog 95062-798);

[00192] 15 xl, 1 L, pH 7.4 (Fisher; Catalog* R58190001A);

[00193] 20 500 Tablets, each makes 100 mL; pH 7.4 (Fisher; Catalog* IC-N2810307);

[00194] 0.9% w/v physiological saline;

[00195] Saline, Blood bank; Fisherbrand; Azide-free, preservative-free; 0.85% w/v isotonic NaCl solution; Buffered; pH 7.2 + 0.1; For in vitro diagnostic, laboratory use; Supplied in Cubitainer; 2.64 gal. (10L), (Fisher; Catalog # 23-312-651).

[00196] Module 1. Semen collection

[00197] Example 01 - SOP: Collecting the Ejaculate, Cattle

[00198] 1. INSPECT DEVICE a. Visually inspect device (as disclosed, for example, in PCT/US2009/038134 published as WO/2009/123889 on October 8, 2009, the disclosure of which is hereby incorporated in its entirety by reference) for cracks or damage before using. Use only devices that are intact.

[00199] 2. BRING DEVICE TO OPERATING TEMPERATURE

a. Place device in 32°C water bath for at least 60 minutes. Make sure device is submerged in water up to the cap of the large tube, so the device warms uniformly. Devices may be left in bath overnight for use the next day.

[00200] 3. PERFORM COLLECTION AND BEGIN INCUBATION BEGIN INCUBATION FOR PRODUCTION OF SEVs (SPERM-DERIVED EXTRACELLULAR VESICLES/ EXOSOMES)

a. Use standard methods of attachment to the artificial vagina (AV) and of collection. Do not raise the AV temperature above 48°C—it is preferable to use 42°C. If device is out of water bath for more than 5 minutes between placement onto AV and collection, remove it from AV and replace with another device from the 32°C water bath, so that the collection device temperature will remain near 32°C. When assembling the AV, the collection cone should be at ambient temperature (at or below 25°C).

b. Within 1 minute of collection, retrieve device, cap and invert once, then place immediately into 12°C water bath. Failure to carry out these steps quickly may result in process failure.

c. Proceed to Module 2, Cargo loading into/onto SEVs

[00201] Example 02 - SOP: Collecting the Ejaculate, Human

[00202] 1. INSPECT COLLECTION CUP

a. Visually inspect cup for cracks or damage. Give donors only cups that are intact and have an unbroken safety seal.

[00203] 2. ASSEMBLE COLLECTION CUP AND INSULATOR (See US20120252000, which is incorporated herein by reference in its entirety)

a. Place the cup into the collection cup holder b. Place the cup and collection cup holder in an ice bucket filled with ambient temperature water (23°C) to a depth of 4cm.

[00204] 3. PERFORM COLLECTION AND BEGIN INCUBATION BEGIN INCUBATION FOR PRODUCTION OF SEVs (SPERM-DERIVED EXTRACELLULAR VESICLES/ EXOSOMES)

a. Ensure that the Laboratory has the donor's signed consent form and that it is properly filled in. If it is not, do not proceed until the form is correctly completed.

b. Instruct the donor to break the seal on the collection cup himself, or to have a member of the laboratory staff break the seal in the donor's presence. Inform donor that after sample production and delivery he may report to the

Laboratory when ready.

c. Give donor the directions to the Producing Room and instruct donor to produce a sample by masturbation, without use of lubricants, into the cup provided. Instruct donor that immediately upon sample production he is to cap cup, place in cup holder in bucket and place sample outside Producing Room door for retrieval by laboratory staff.

d. As soon as sample is placed outside the Producing Room door, retrieve bucket, and transfer cup holder containing cup into 12°C water bath. Failure to carry out these steps quickly may result in process failure. Note time of sample delivery in notebook, log or LIMS system.

e. When donor arrives at the Laboratory, inquire whether any portion of the

ejaculate was lost and record donor's answer in notebook, log or LIMS system. Inform donor that the donation process is complete and that he may leave. f. Proceed to Module 2, Cargo loading into/onto SEVs

[00205] Example 03 - SOP: Collecting the Ejaculate, Swine

[00206] 1. INSPECT DEVICES

a. Visually inspect devices for cracks or damage before using. Use only devices that are intact. In this SOP, the same device is used for cattle and for swine.

[00207] 2. BRING DEVICE TO OPERATING TEMPERATURE a. Place devices in 32°C water bath for at least 60 minutes. Make sure device is submerged in water up to the cap of the large tube, so the device warms uniformly. Devices may be left in bath overnight for use the next day.

b. Place a collection funnel on the top of each device.

[00208] 3. PERFORM COLLECTION AND BEGIN INCUBATION BEGIN INCUBATION FOR PRODUCTION OF SEVs (SPERM-DERIVED EXTRACELLULAR VESICLES/ EXOSOMES)

a. Using the gloved hand technique, begin semen collection.

(i) For collection of sperm-rich fraction: Direct the boar's penis into the collection funnels when this fraction is being produced, and direct the penis to a waste cup during production of the other fractions.

(ii) For collection of total ejaculate: Direct the boar's penis into the collection funnels as the ejaculate is being produced.

b. As each collection device is filled, move to the next funnel and device. As soon as device is filled, place immediately into 12°C water bath. (Note: use of a 17°C waterbath is permissible but not preferred.) Failure to carry out these steps quickly may result in process failure.

c. If it is desired to pool the cooled ejaculates, wait 15 minutes after the last

device that was filled is transferred to the 12°C bath, then quickly pour the contents of each device into a pre-cooled container (for example, a 500ml Erlenmeyer glass flask) that has been pre-cooled in a 12°C bath.

d. Proceed to Module 2, Cargo loading into/onto SEVs

[00209] Note: This collection process is also acceptable on strains of genetically modified pigs developed for xenotransplantation.

[00210] Module 2: Cargo loading into/ onto SEVs (Sperm-derived extracellular vesicles/ exosomes

[00211] Example 04 - SOP: Cargo loading, cattle, swine, human sperm

[00212] 1. DETERMINE CARGO SOURCE FOR CARGO LOADING OF ACTIVE

CARGO, INERT CARGO, OR COMBINATION THEREOF a. If the desired cargo is from seminal plasma, proceed to Step 2a.

b. If the desired cargo is not from seminal plasma, proceed to Step 2b.

[00213] 2. PREPARE SPERM FOR CARGO LOADING INTO/ONTO SEVs AND

INITIATE CARGO LOADING

a. Continue incubation of sperm in seminal plasma for 0.5h to overnight, with usual incubation time choice being 0.5-6h (more preferably 3-6h for larger vesicles or 0.5h-3h for smaller vesicles).

(i) It is recommended to immediately run a COBO Assay to

determine if collection is normal and may be used for SEV production (see Module 5, COBO Biomarker STAT Assay for fresh semen collection evaluation).

(ii) It is recommended to run the COBO Assay throughout incubation to ensure a good yield of SEVs (see Module 4, COBO Biomarker Assay for production of SEVs). To test for acquisition of a SEV signature, the preferred method is cytometry (Module 4).

Epiflouresence microscopy to examine fields for an increase in vesicle size, presence and other attributes may be used for cattle and pigs only, but is not preferred (Module 4). When the desired incubation period is complete per instructions in Module 4, proceed to Step 2c of this Module.

b. As previously instructed, the operator, immediately upon collection of the

ejaculate, placed semen in collection device into a 12°C water bath. Before taking next steps in this procedure, insure that collection has cooled in the 12°C bath for 15 minutes. Then, as shown below for next steps that are detailed according to sperm source, place semen into pre-cooled (12°C) centrifuge tubes of a size suitable for the amount of ejaculate being processed and centrifuge the semen so sperm can be purified and resuspended in medium. Carry out all steps at 12°C and with pre-cooled reagents also at 12°C.

Examples of tubes are given for the example amounts of ejaculate indicated below, and it is possible to scale the process by increasing volumes while maintaining the ratios that are shown below.

(i) Cattle sperm

1. Place 1ml semen in each 1.5ml microfuge tube.

2. Centrifuge in a microfuge at 2,000 x g for 1 minute

3. Aspirate supernatant, leaving pelleted sperm in tube

4. Resuspend sperm in in a volume of wash medium equal to the amount of ejaculate used.

5. Centrifuge in a microfuge at 2,000 x g for 1 minute

6. Aspirate supernatant

7. Resuspend sperm pellet in a volume of cargo loading medium equal to the initial volume of ejaculate used. Incubate for desired time (usually 0.5-6h— see (2a) above for the preferred method, which is application of the COBO Assay, to detect SEV signature during incubation).

(ii) Human sperm (For wash media other than SpermPrep Media)

1. Place 1ml semen in each 1.5ml microfuge tube.

2. Centrifuge in a microfuge at 2,000 x g for 1 minute

3. Aspirate supernatant, leaving pelleted sperm in tube

5. Centrifuge in a microfuge at 2,000 x g for 1 minute

6. Resuspend sperm pellet in cargo loading medium equal to the initial volume of ejaculate used.

Incubate for desired time (usually 0.5-6h— see (2a) above for the preferred method, which is application of the COBO Assay, to detect SEV signature during incubation).

of Sperm Prep Medium as wash medium Place 50ul semen in each 1.5ml microfuge tube. Add 1ml Sperm Prep Medium

Centrifuge in a microfuge at 2,000 x g for 1 minute Aspirate supernatant, leaving pelleted sperm in tube Resuspend sperm pellet in 200μ1 cargo loading medium (for example, HTF).

— sperm rich fraction

Place 10ml sperm-rich fraction in each 15ml conical tube.

Centrifuge in a swinging bucket centrifuge in 12°C cold room at 2,000 x g for 5 minutes

Aspirate supernatant, leaving pelleted sperm in tube

Resuspend sperm in in a volume of wash medium equal to the amount of ejaculate used.

Centrifuge in a microfuge at 2,000 x g for 5 minutes

Aspirate wash medium

Resuspend sperm pellet in cargo loading medium in a volume equal to the volume of the original sperm- rich ejaculate volume used. Incubate for desired time (usually 0.5-6h— see (2a) above for the preferred method, which is application of the COBO Assay, to detect SEV signature during incubation). — total ejaculate

Place 10ml semen in each 15ml conical tube.

Centrifuge in a swinging bucket centrifuge in 12°C cold room at 2,000 x g for 5 minutes 3. Aspirate supernatant, leaving pelleted sperm in tube

4. Resuspend sperm pellet in a volume of wash buffer equal to the amount of ejaculate used.

5. Centrifuge in a microfuge at 2,000 x g for 5 minutes

6. Aspirate wash medium

7. Resuspend sperm pellet in cargo loading medium in a volume equal to 20% of the volume of the ejaculate volume used (e.g., for 50ml of ejaculate resuspend in 10ml). Incubate for desired time (usually 0.5-6h— see (2a) above for the preferred method, which is application of the COBO Assay, to detect SEV signature during incubation).

c. Upon completion of incubation, centrifuge the incubated samples at 2,000 x g for 2 minutes. For larger volumes of semen (>lml), increase centrifugation time to 5-10 minutes.

d. Aspirate the supernatant containing SEVs and transfer to a clean tube(s) in an ice bucket containing ice. Depending on the type of cargo loaded and the importance of its concentration, it may be useful to measure cargo

concentration after SEV purification away from cargo loading medium.

Module 3 describes how to terminate cargo loading and prepare SEVs, including targeted SEVs containing surface ligand to direct their interaction with recipient cells, in medium that does not contain unloaded cargo, with Step 2b detailing the recommended ultracentrifugation protocols for SEV purification.

e. Proceed to Module 3, "Targeting of SEVs to desired location through

attachment of ligand-binding agent(s) and preparation of SEVs with unoccupied Fc receptors"

[00215] Module 3: Targeting of SEVs to desired location through attachment of ligand- binding agent(s) and preparation of SEVs with unoccupied Fc receptors

[00216] Example 05 [00217] 1. DETERMINE DESIRED TARGETING

[00218] Unoccupied Fc receptors will bind to generic antibodies in the environment to which they are introduced and can down-regulate the immune response by serving as an antibody sink. SEVs in this state are suitable for use in assisted reproductive technologies (ART) and applications where SEV anti-inflammatory properties are desirable. For specifically-targeted SEVs, coating with antibody that is specific to a given target will direct SEVs to bind to the antibody' s target, because the antibody' s Fc portion will bind to the SEV and the Fab regions that carry specificity for the targeted antigen will be externally oriented.

[00219] 2. TERMINATE CARGO LOADING AND PREPARE SEVs FOR TARGETING

a. If the desired SEV state for preparing targeted SEVs begins with them in the cargo-loading medium, proceed to Step 3a.

b. If the desired SEV state for preparing targeted SEVs begins with them purified from the loading medium, follow the instructions below before proceeding to Step 3a. All solutions and the SEV suspension should be on ice.

(i) Filter the SEV suspension through a 0.8μιη syringe filter (you may substitute a smaller filtration size, 0.4μιη or 0.2um, if desired, provided SEV yield and purity remains satisfactory for your application.) If the loading capacity of the filter is exceeded, you may use multiple filters.

(ii) Place the supernatant from Step 2b(i) into ultracentrifuge tubes, for example, Ultracentrifuge tubes, Beckman Coulter 14mm x 89mm open-top thin wall polyallomar, cat. # 331327 tubes/

Beckman Coulter SW41 Ti rotor. To ensure tubes are filled to prevent deformation during ultracentrifugation, fill remaining tube volume with lx PBS or 0.9% physiological saline to within 2-3mm of tube top, place tubes in swinging buckets and balance each pair of buckets that are opposite from each other on the rotor. Screw caps onto swinging buckets.

(iii) Attach swinging buckets to rotor. Centrifuge for 90 minutes at 100,000 x g at 4°C in an evacuated chamber, for example in a Beckman Coulter Optima L-90k preparative ultracentrifuge. If sucrose gradient purification is desired, proceed to Step 2b(iv) and follow instruction in italics. If no sucrose gradient purification is desired, proceed to Step 2b(v).

(iv) (optional) If additional purification is desired, one may use

sucrose gradient purification as in Perez-Gonsalez et ah, J. Biol. Chem. (2012) 287(51): 43108-43115, as shown.

1. Resuspend SEV pellet in 2 ml of 0.95 m sucrose solution.

2. Insert this inside a sucrose step gradient column (six 2 -ml steps starting from 2.0 m sucrose up to 0.25 m sucrose in 0.35 m increments, with the 0.95 m sucrose step containing the SEV).

3. Centrifuge the sucrose step gradient at 200,000 x g for 16 h at 4 °C.

4. Collect one -ml fractions from the top of the gradient, and pool fractions flanking the interphase separating two neighboring sucrose layers together, for a total of seven fractions (a, top 1 -ml fraction; b, 2 -ml; c, 2 -ml; d, 2 -ml; e, 2 -ml; f 2 -ml; and g, bottom 1 -ml fraction).

5. Dilute these fractions in cold PBS and centrifuge at 100,000 x g at 4 °Cfor 70 min.

(v) After run, aspirate supernatant and immediately proceed to Step 3b of this Module. Do not permit SEV pellets to dry in the ultracentrifuge tubes.

[00220] 3. INCUBATE SEVs WITH TARGETING MEDIUM AND PREPARE TARGETED SEVs FOR STORAGE OR DEPLOYMENT

[00221] SEVs can be targeted in two formats. SEVs can be mixed with the targeting ligand while still in cargo-loading medium, in which case a dilution step is used, but not a centrifugation step (go to Step 3a of this Module). Or SEVs can be harvested by ultracentrifugation and resuspended in Targeting Medium containing the targeting ligand (go to Step 3b of this Module). Examples of both methods below employ antibodies as the targeting ligand, although antibodies are not the only ligand choice available.

a. To prepare targeted SEVs within the cargo-loading medium (example uses 5μ1 of bull semen but can be scaled and can employ semen from other species).

(i) You should only use semen collected and cooled as instructed in the preceding examples. Semen should be at 12°C at the time of its use in this procedure.

(ii) To ΙΟΟμΙ of Green 1 in a 1.5ml microfuge tube, add 20μ1 of Targeting antibody, for example, a Difco antibody reconstituted as described: the lyophyllized Difco antibody should be reconstituted to the volume indicated by Difco, but instead of their recommended resuspension solution, use 1 x PBS.

(iii) Add 5μ1 of bull sperm collected and cooled as indicated in this application.

(iv) Incubate for 20 minutes at 12-26°C.

(v) Centrifuge at 2000 x g for 1 minute

(vi) Aspirate supernatant containing targeted SEVs (keep in mind that this will include free targeting agent as well). The supernatant can be further purified by centrifugation (see Step 2b of this Module for protocols, and depending on scaling, you may need to add more 1 x PBS so SEVs are in a large enough volume to be compatible with centrifugation), or can be prepared for deployment or storage (Step 4 of this Module).

(vii) Prepare targeted SEVs for deployment or storage (Step 4 of this Module).

b. To prepare targeted SEVs in a medium other than the cargo loading medium, resuspend the pellet from Step 2(b)v of this Module in the desired amount of Targeting Reagent. It is recommended to resuspend in a volume of targeting medium 2 times greater than the volume of ejaculate that was processed to produce the volume of SEVs being used. For example, if 1ml of ejaculate was processed, the SEVs should be resuspended in 2ml Targeting Medium.

c. Incubate for 20 minutes at 12-26°C. Rock gently to mix at least three times during incubation. It is preferred to use a tube rotator on low speed (about 2 revolutions/minute) .

d. Prepare targeted SEVs for deployment or storage (Step 4 of this Module).

[00222] 4. PREPARE TARGETED SEVs for DEPLOYMENT OR STORAGE

a. To prepare SEVs for storage: if desired, wash off Targeting Medium (or

medium in which SEVs with unoccupied Fc receptors are present) by following Steps in 2(b) of Module 3 for ultracentrifugation. Carry out final resuspension, at a volume equal to 10% of the volume of ejaculate or fraction thereof processed, in a Storage Medium for storage at 4°C. If freezing is desired, add cryoprotectant (BioXcell or other extender used for freezing sperm), treating the final resuspension volume of SEVs as equivalent to the volume of ejaculate in the manufacturer's recommendation for extender use, except keep SEVs at 4°C during processing. Freeze according to manufacturer's recommendation in 0.25cc straws (preferred) or 0.5ml straws (an example of a vendor is EVIV Technologies).

b. To deploy SEVs as an additive for use with extended frozen sperm doses, go to Module 6.

[00223] Module 4: COBO Biomarker Assay for production of SEVs

[00224] Example 06 - SOP: COBO Biomarker Assay for production of SEVs

[00225] Before running this procedure, it is preferred that semen is collected and incubated exactly as instructed. Obtain a baseline value at 30 minutes post-collection, and a test sample 3h-24h after collection at the time when cargo loading is terminated. You may repeat assay at 30min intervals to determine the time at which a given ejaculate or fraction thereof contains the highest proportion of events in the "SEV Signature Region." This is recommended because SEV production demonstrates sinusoidal kinetics and highest yields therefore vary with time. The exception is for aged ejaculates where larger vesicles build up in concentration and sperm are shedding not only SEVs but large fragments of acrosome. It is time-consuming and expensive to enhance SEV visualization by linking SEVs via their Fc receptors to an agent of size and optical properties that is easier to visualize by cytometry. Therefore, such an approach is not preferred but it is certainly feasible, should this be desired for a specific application.

[00226] 1. MIX SAMPLE WITH ASSAY REAGENTS AND INCUBATE

a. Add the following to an assay tube that is filled with premixed reagents:

(i) ΙΟμΙ sample

(ii) 5μ1 Activator

b. Incubate at ambient temperature for 15 minutes

c. Add 1ml Buffer and proceed in this Module to Step 2 for washing method (preferred) or to Step 3 for omission of the wash steps.

[00227] 2. WASH (Washing is preferred, but in some cases -especially with animal semen— washing is optional when scoring by cytometry. Washing is required for scoring by microscopy. Human sperm are very difficult to score by microscopy even with high-end microscopes, not usually found in clinics, so cytometry is required.)

a. Microfuge 30 seconds at 2000 x g

b. Carefully remove supernatant

c. Add 500μ1 PBS BUFFER to cell pellet and mix to resuspend cells if scoring by cytometry. Add 200μ1 PBS BUFFER to cell pellet and mix to resuspend cells if scoring by microscopy.

[00228] 3. SCORE

a. For cytometer: Place microfuge tube onto cytometer SIP tube and analyze on a calibrated cytometer using the "SEV Assay" template (see Figure 11 for images of cytometric plots and results) to determine the number of events in the "SEV Signature Region." When the number of events in the "SEV

Signature Region" has decreased and then increases to at least 1.5 times the intensity of the preceding timepoint, process sample as shown in the SOPs. The template is set to certain parameters to ensure successful scoring. For example, the cytometer must acquire at least 10,000 events in the cell gate (P3), before the operator scores fluorescence in the SEV signature region of the FL-1 channel. This is in part because the assay wash step causes a partial depletion of SEVs. Scoring a higher number of events in the cell gate partially compensates for the loss,

b. For microscopy (cattle and pig sperm only): Place 7μ1 aliquot of resuspended cells onto microscope slide, place cover slip and examine for appearance of a "milky way" signature. Magnification of 400x is recommended, however, microscopists may use what they prefer, so long as the described appearance is detectable. FIG. 3(b) is an early stage example. The fully developed milky way signature differs from FIG. 3(b) in several ways. It contains more fluorescent vesicles, including those of larger size. It contains large fluorescent fragments derived from sperm heads. It contains a higher percentage of sperm that are biomarker-positive but are missing fragments of positive material from their heads, resulting in a patchy appearance of the fluorescence on their anterior heads.

[00229] NOTE: Further clarification on running assay without a wash step: some applications may be suitable for use with an assay that does not include the centrifugation step, which lowers background but causes SEV loss. Assays without a wash step provide faster throughput in busy labs and are easier to run. To run the COBO Assay without a wash step, follow these instructions:

[00230] 1. MIX SAMPLE WITH ASSAY REAGENTS AND INCUBATE

a. Add the following to an assay tube that is filled with premixed reagents (these being ΙΟΟμΙ Green 1 and 20μ1 Red 2):

(i) ΙΟμΙ sample

(ii) 5μ1 Activator

b. Incubate at ambient temperature for 15 minutes

c. Add 1.3 ml Buffer. Rather than counting a fixed number of total events

(usually 5,000 or 10,000), you can use buffer to which Trucount beads (BD cat. No. 340334) are added, and count by acquiring a fixed number of

Trucount beads per sample. One of ordinary skill in cytometry can dissolve the Trucount bead pellet in its test tube and remove a suitable number of beads to mix with Buffer, such that acquisition of 10,000 beads corresponds roughly to 10,000 events in the cell gate of a collection of semen containing a normal concentration of sperm, by adjusting the cytometer parameters to acquire based on Trucount bead numbers. The advantage of Trucount beads is that changes in counts of events coming from the sample do not affect the data. Events are acquired based on number of Trucount beads present, which normalizes data. The disadvantage to Trucount beads in a manufacturing or clinical setting is the high cost per assay.

[00231] 2. SCORE

a. Place microfuge tube onto cytometer SIP tube and analyze on a calibrated cytometer using the "SEV Assay" template (see Figure 11 for images of cytometric plots and results) to determine the number of events in the "SEV Signature Region." When the number of events in the "SEV Signature Region" has decreased and then increases to at least 1.5 times the intensity of the preceding timepoint, process sample as shown in the SOPs of Module 3 for targeting, storage and deployment.

[00232] Module 5: COBO Biomarker STAT Assay for fresh semen collection evaluation

[00233] Example 07- SOP: COBO Biomarker STAT Assay for fresh semen collection

[00234] Before running this procedure, be sure that semen is collected and incubated exactly as instructed as described herein.

[00235] 1. MIX SAMPLE WITH ASSAY REAGENTS AND INCUBATE

a. Add the following to an assay tube that is filled with premixed reagents:

(i) ΙΟμΙ sample

(ii) 5μ1 Activator

b. Incubate at ambient temperature for 15 minutes

[00236] 2. WASH

a. Add lml PBS Buffer

b. Microfuge 30 seconds at 2000 x g

c. Carefully remove supernatant [00237] 3. SCORE

a. For cytometer: Add ~500μ1 PBS BUFFER to cell pellet and mix to resuspend cells. Place aliquot of resuspended cells onto cytometer SIP tube and analyze on a calibrated cytometer as shown in Step 4 of this Module. Collections that fail the STAT assay should not be used.

[00238] 4. START EQUIPMENT

a. Turn on computer

b. Turn on cytometer (Accuri, Lansing, MI)

c. If needed, empty waste bottle and fill sheath bottle with DI water

[00239] 5. OPEN TEMPLATE AND NAME FILE

a. Click COBO Assay Template for ejaculate's source, animal or human (For cattle and swine, all events from the FSC x SSC plot are scored on the SSC x FL-1 plot. Human ejaculates are lower in cell number and have more irrelevant events present on the FSC x SSC plot, therefore the FCS x SSC plot is gated so that only events in the cell gate of that plot are scored for fluorescence on the SSC x FL-1 plot.)

b. Select File>Save CFlow file

c. Name file by date by typing date code under File Name

d. Click Save

[00240] 6. COLLECT DATA

a. Under the red Collect tab, click on desired cytometer grid (1A is for first sample, IB is for second sample, etc.

b. Next to that sample's cytometer grid (e.g., A01) type sample information c. Check that stoplight shows green color. Load sample onto SIP tube on

cytometer and pull out plastic tube support underneath tube

d. Click Run

e. Remove sample from SIP tube

f. For the next sample, repeat process above starting with step 6a

g. After the samples for that hour are finished, click Backflush, wait for stoplight to show green, then click Unclog [00241] 7 ANALYZE DATA

a. Determine the percentage of positive sperm in FL-1 for the Cohen Biomarker.

If this is less than 20%, the ejaculate may be used. If this is equal to or greater than 20%, the ejaculate fails and should not be used.

[00242] 8 CLEAN AND SHUT DOWN EQUIPMENT

a. Between every sample, place towel under SIP, select Backflush or Unclog b. At the end of the day, place a tube of cleaning fluid on the cytometer, select well HI 1 and click Run. Run for 2 minutes. Then place a tube of water on the cytometer, select well HI 1 and click Run. Run for 2 minutes. Allow water to clean system for at least 2 minutes, or longer if desired.

c. Turn off cytometer, then turn off computer

d. Maintenance— follow instructions in Accuri manual for instrument cleaning.

This should include weekly extended cleaning of flow cell (found under the Instrument tab on the cytometer menu).

[00243] Module 6: Use of SEVs in assisted reproductive technologies (ART)

[00244] Example 08

[00245] Harvested SEV preparations can be used in a variety of ways. Deployment in ART, just before intrauterine insemination, is shown below. It is preferred where possible to mix SEVs with sperm prior to freezing, to simplify the downstream AI process. For additional uses, please contact manufacturer so that a deployment protocol can be customized to your specific application and infrastructure.

[00246] 1. PREPARE SEVs FOR DEPLOYMENT IN ART

If frozen, thaw a dose of SEVs at 37°C for 30 seconds. Deploy immediately. If cool, remove from cold storage. Deploy immediately.

[00247] 2. INSEMINATE SEMEN DOSE AND SEVs

a. For cattle, proceed to Step 2b. For humans, proceed to Step 2c. For swine, proceed to Step 2d. Note that use of the COBO Kinetic Assay (for example, as described in US2012/0252000A1, the disclosure of which is hereby incorporated in its entirety by reference) to adjust fertilizing potential of the ejaculate, when employed in concert with these methods, will further improve the reproductive outcome.

For cattle semen doses where SEV preparation was not introduced to sperm during extension

(i) If frozen, thaw sperm dose at 37°C for 30 seconds. A cooled dose may be used directly.

(ii) Mix thawed or cooled SEV preparation with sperm dose, re-load straws

(iii) Perform standard insemination into uterine body at juncture

between cervix and uterus. Administer hormones (if in use), detect heat and time insemination according to methods in standard use at farm or, preferably, with an ejaculate whose fertility is already improved through use of the COBO Kinetic Assay as described in US2012/0252000A1.

For human semen doses where SEV preparation was not introduced to sperm during extension

(i) Thaw semen dose or obtained prepared unfrozen semen dose for intrauterine insemination (IUI).

(ii) Mix thawed or cooled SEV preparation with sperm dose, load syringe and catheter

(iii) Immediately perform IUI (intrauterine insemination) according to standard practice for clinic. IUI takes place following administration of standard drug regimen to female patient, with sperm preparation from ejaculate and timing of insemination according to methods currently in use at the clinic or, preferably, with an ejaculate whose fertility is already improved through use of the COBO Kinetic Assay as described, for example, by applicant in US2012/0252000A1.

For pig semen doses where SEV preparation was not introduced to sperm during extension (i) Thaw sperm dose according to standard procedure at site, or remove dose from cooler.

(ii) Mix thawed or cooled SEV preparation with sperm suspension in insemination bottle and attach catheter.

(iii) Perform standard insemination into cervix or into uterus. Prior to insemination, administer hormones (if used) to sow or gilt, detect heat and time insemination according to methods in standard use at farm or, preferably, with preferably, with an ejaculate whose fertility is already improved through use of the COBO Kinetic Assay, for example, as described in US2012/0252000A1.

[00248] Module 7: Preparation of semen for overnight shipping prior to SEV manufacture

[00249] SEVs can be produced from semen shipped overnight on cold packs, although the preferred production method uses fresh collections. The method for use with bull semen is shown below.

[00250] 1. FOR SHIPPER: COLLECT AND SHIP SEMEN

a. Collect bull semen into a 15ml conical tube attached to the artificial vagina. b. Within 1 minute of collection, insert conical tube between two cold packs pre- chilled to 4°C and fastened together by rubber bands so that the conical tube can be wedged between the two cold packs. A suitable cold pack for this process is Uline model S-7361 8oz, 6x4x3/4" FDA compliant cold pack.

c. Place collection and cold packs into Styrofoam shipper and ship overnight for next day morning receipt.

[00251] 2. FOR RECEIVER: UPON RECEIPT, PREPARE SEMEN IMMEDIATELY FOR DOWNSTREAM PROCESSING

a. Upon receipt, open package and remove conical tube containing semen from between cold packs. Immediately place tube into 12°C water bath so that the top of the ejaculate is at or below the waterline. Treat as a fresh collection that has just been placed at 12°C. Also place a thermometer between the cold packs, where they tube had been, as quickly as possible. Record the temperature once the reading on the thermometer is stable. If the temperature is greater than 6°C, do not process ejaculate. If cold packs have arrived frozen, do not process ejaculate. If temperature is acceptable, the usual starting point for working with a shipped ejaculate is Module 2 Step 1.

[00252] 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. It is understood that the detailed examples and embodiments described herein are given by way of example for illustrative purposes only, and are in no way considered to be limiting to the invention. Various modifications or changes in light thereof will be suggested to persons skilled in the art and are included within the spirit and purview of this application and are considered within the scope of the appended claims. For example, the relative quantities of the ingredients may be varied to optimize the desired effects, additional ingredients may be added, and/or similar ingredients may be substituted for one or more of the ingredients described. Additional advantageous features and functionalities associated with the systems, methods, and processes of the present invention will be apparent from the appended claims. Moreover, 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.

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73

Claims

In the Claims:

1. A method for producing sperm-derived extracellular vesicles, the method comprising:

a. collecting semen from a mammal;

b. incubating the semen to provide a desired quantity of homogeneous sperm- derived extracellular vesicles;

c. taking a sample at a pre-selected interval and assaying the sample for the presence of extracellular vesicles in the sample;

d. repeating step (c) at pre-selected intervals until the desired quantity of extracellular vesicles is present in the sample; and

e. processing the semen.

2. A method for producing sperm-derived extracellular vesicles, the method comprising:

a. collecting semen from a mammal;

b. isolating sperm from the semen;

c. incubating the isolated sperm to provide a desired quantity of extracellular

vesicles homogeneous sperm-derived extracellular vesicles;

d. processing the sperm to separate the sperm-derived extracellular vesicles.

3. A method for producing sperm-derived extracellular vesicles, the method comprising:

a. collecting semen from a mammal;

b. isolating sperm from the semen;

c. incubating the isolated sperm to provide homogeneous sperm-derived extracellular vesicles;

d. taking a sample at a pre-selected interval and assaying the sample for the presence of extracellular vesicles in the sample;

e. repeating step d at pre-selected intervals until a desired quantity of extracellular vesicles is present in the sample; and

f. processing the sperm to separate the sperm-derived extracellular vesicles.

4. A plurality of sperm-derived extracellular vesicles provided by the method of claim 3.

5. Sperm-derived extracellular vesicles comprising cargo.

6. Sperm-derived extracellular vesicles according to claim 5, comprising a ligand attached to one or more of the extracellular vesicles by a Fc receptor on the extracellular vesicle.

7. The method of claim 3, further comprising: adding a ligand to the incubation step to provide cargo to be incorporated with the extracellular vesicles.

8. The sperm-derived extracellular vesicles according to claim 6 wherein the ligand is an antibody.

9. The method of claim 7 wherein the cargo is an antibody.

10. The method of claim 7 wherein the cargo is selected from the group consisting of antioxidants, aptamers, carbohydrates, DNA, enzymes, nucleic acids, proenzymes, proteins, reactive oxygen species or their precursors, RNA, small molecule drugs and toxins.

11. A method for treating a patient in need of immune modulation, the method comprising administering a treatment effective amount of extracellular vesicles, at least a portion of which have an active Fc receptor.

12. A method for improving reproductive results in assisted reproductive therapy (ART), the method comprising:

a. collecting semen from a mammal;

b. incubating the semen to provide extracellular vesicles in the semen; c. taking a sample at a pre-selected interval and assaying the sample for the

presence of extracellular vesicles in the sample;

d. repeating step (c) at pre-selected intervals until a desired quantity of extracellular vesicles is present in the sample; and e. using the semen containing the desired quantity of extracellular vesicles in the ART procedure.

13. A method for producing sperm-derived extracellular vesicles containing cargo, the method comprising:

a. collecting semen from a mammal;

b. isolating sperm from the semen;

c. incubating the isolated sperm in a medium containing cargo to provide a desired quantity of extracellular vesicles homogeneous sperm-derived extracellular vesicles;

d. processing the sperm to separate the sperm-derived extracellular vesicles.

14. A method for producing sperm-derived extracellular vesicles containing cargo, the method comprising:

a. collecting semen from a mammal;

b. isolating sperm from the semen;

c. incubating the isolated sperm in a medium containing cargo to provide

homogeneous sperm-derived extracellular vesicles;

f. processing the sperm to separate the sperm-derived extracellular vesicles.

15. A method for producing sperm-derived extracellular vesicles containing cargo, the method comprising:

a. collecting semen from a mammal;

b. isolating sperm from the semen;

c. incubating the isolated sperm to provide homogeneous sperm-derived

extracellular vesicles; d. taking a sample at a pre-selected interval and assaying the sample for the presence of extracellular vesicles in the sample;

e. repeating step d at pre-selected intervals until a desired quantity of

extracellular vesicles is present in the sample;

f. processing the sperm to separate the sperm-derived extracellular vesicles; and g. introducing cargo by electroporation.