U.S. patent application number 17/686352 was filed with the patent office on 2022-09-29 for process of delivering small rnas to sperm.
The applicant listed for this patent is UNIVERSITY OF MASSACHUSETTS. Invention is credited to Colin CONINE, Oliver RANDO, Upasna SHARMA.
Application Number | 20220307025 17/686352 |
Document ID | / |
Family ID | 1000006388009 |
Filed Date | 2022-09-29 |
United States Patent
Application |
20220307025 |
Kind Code |
A1 |
RANDO; Oliver ; et
al. |
September 29, 2022 |
PROCESS OF DELIVERING SMALL RNAS TO SPERM
Abstract
Methods and compositions directed to altering a population of
sRNAs in a sperm using vesicles isolated from an epididymosome are
provided. Methods and compositions directed to altering a
population of sRNAs in an oocyte using vesicles isolated from an
epididymosome are also provided. Methods for altering an sRNA
population in a sperm or an oocyte can be used to prevent, or
reduce the severity of, a disease, disorder, or condition that
would otherwise be inherited by progeny. For example, certain
epigenetic inherited conditions due to paternal effects, such as
certain metabolic and stress disorders and conditions, can be
ameliorated in progeny using sperm or oocytes having an altered
sRNA population.
Inventors: |
RANDO; Oliver; (Natick,
MA) ; SHARMA; Upasna; (Shrewsbury, MA) ;
CONINE; Colin; (Brighton, MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
UNIVERSITY OF MASSACHUSETTS |
Boston |
MA |
US |
|
|
Family ID: |
1000006388009 |
Appl. No.: |
17/686352 |
Filed: |
March 3, 2022 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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16315004 |
Jan 3, 2019 |
11299733 |
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PCT/US2017/041647 |
Jul 12, 2017 |
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17686352 |
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62363174 |
Jul 15, 2016 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12N 2320/30 20130101;
A61D 19/04 20130101; A61K 9/06 20130101; A61K 9/0034 20130101; A61B
17/435 20130101; C12N 15/111 20130101; A61B 17/43 20130101; C12N
5/061 20130101; A61D 19/02 20130101; C12N 2501/65 20130101; C12N
2510/00 20130101; C12N 2310/14 20130101; C12N 2310/141 20130101;
C12N 5/0604 20130101; C12N 15/113 20130101 |
International
Class: |
C12N 15/113 20060101
C12N015/113; A61B 17/43 20060101 A61B017/43; A61B 17/435 20060101
A61B017/435; A61D 19/02 20060101 A61D019/02; A61D 19/04 20060101
A61D019/04; A61K 9/00 20060101 A61K009/00; A61K 9/06 20060101
A61K009/06; C12N 5/073 20060101 C12N005/073; C12N 5/076 20060101
C12N005/076; C12N 15/11 20060101 C12N015/11 |
Goverment Interests
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH
[0002] This invention was made with government support under Grant
Nos. ES025458 and HD080224 awarded by the National Institutes of
Health. The Government has certain rights in the invention.
Claims
1-81. (canceled)
82. A pharmaceutical composition comprising an sRNA-containing
vesicle isolated from an epididymosome.
83. The pharmaceutical composition of claim 82, wherein the sRNA is
selected from the group consisting of a siRNA, a miRNA, a piRNA, a
snoRNA, a srRNA, a U-RNA, and a tRNA fragment.
84. The pharmaceutical composition of claim 83, wherein the tRNA
fragment is selected from the group consisting of a tRNA-Gly-CCC
fragment, a tRNA-Gly-TCC fragment, a tRNA-Gly-GCC fragment, a
tRNA-Val-CAC fragment, a tRNA-Glu-CTC fragment, a tRNA-Lys-CTT
fragment, and a tRNA-His-GTG fragment.
85. The pharmaceutical composition of claim 82, wherein the
pharmaceutical composition is a vaginal foam or a gel.
86. The pharmaceutical composition of claim 82, wherein the
epididymosome is selected from the group consisting of caput
epididymosome, corpus epididymosome, and cauda epididymosome.
87. The pharmaceutical composition of claim 82, wherein the vesicle
comprises a heterologous RNA.
88. The pharmaceutical composition of claim 87, wherein the
heterologous RNA comprises a small RNA (sRNA).
89. The pharmaceutical composition of claim 88, wherein the sRNA is
selected from the group consisting of a siRNA, miRNA, a piRNA, a
snoRNA, a srRNA, a U-RNA, and a tRNA fragment.
90. The pharmaceutical composition of claim 89, wherein the sRNA is
a tRNA fragment selected from the group consisting of a
tRNA-Gly-CCC fragment, a tRNA-Gly-TCC fragment, a tRNA-Gly-GCC
fragment, a tRNA-Val-CAC fragment, a tRNA-Glu-CTC fragment, a
tRNA-Lys-CTT fragment, and a tRNA-His-GTG fragment.
91. The pharmaceutical composition of claim 82, wherein the vesicle
comprises autologous RNA.
92. The pharmaceutical composition of claim 91, wherein the sRNA is
selected from the group consisting of a siRNA, miRNA, a piRNA, a
snoRNA, a srRNA, a U-RNA, and a tRNA fragment.
93. The pharmaceutical composition of claim 92, wherein the sRNA is
a tRNA fragment selected from the group consisting of a
tRNA-Gly-CCC fragment, a tRNA-Gly-TCC fragment, a tRNA-Gly-GCC
fragment, a tRNA-Val-CAC fragment, a tRNA-Glu-CTC fragment, a
tRNA-Lys-CTT fragment, and a tRNA-His-GTG fragment.
94. The pharmaceutical composition of claim 82, wherein the vesicle
comprises an artificial or a synthetic RNA.
95. The pharmaceutical composition of claim 94, wherein the sRNA is
selected from the group consisting of a siRNA, miRNA, a piRNA, a
snoRNA, a srRNA, a U-RNA, and a tRNA fragment.
96. The pharmaceutical composition of claim 95, wherein the sRNA is
a tRNA fragment selected from the group consisting of a
tRNA-Gly-CCC fragment, a tRNA-Gly-TCC fragment, a tRNA-Gly-GCC
fragment, a tRNA-Val-CAC fragment, a tRNA-Glu-CTC fragment, a
tRNA-Lys-CTT fragment, and a tRNA-His-GTG fragment.
97. The pharmaceutical composition of claim 82, wherein the vesicle
comprises a transgene.
98-141. (canceled)
Description
RELATED APPLICATIONS
[0001] This application is a division of U.S. patent application
Ser. No. 16/315,004, filed Jan. 3, 2019, which is a 35 U.S.C.
.sctn. 371 filing of International Patent Application No.
PCT/US2017/041647, filed Jul. 12, 2017, which claims priority to
U.S. Provisional Patent Application Ser. No. 62/363,174, filed Jul.
15, 2016, the contents of which are incorporated herein by
reference in their entirety.
TECHNICAL FIELD
[0003] The disclosed methods and compositions are directed to the
field of reproductive biology. Specifically, the disclosed methods
and compositions are directed to the delivery of small RNAs to
sperm to effectuate changes in sperm RNA content.
BACKGROUND
[0004] Mendelian laws of genetics govern most inheritance, and most
epigenetic modifications an organism may acquire are reset between
generations. However, there has recently been growing evidence to
support transgenerational epigenetic inheritance, where some
epigenetic phenotypes are maintained through generations (Lim and
Brunet, Trends Genet. 2013 29(3):176-186).
[0005] For example, animal studies and human cohort studies have
suggested that metabolic changes in parents can be inherited
epigenetically by offspring. In rats, a high fat diet in male
parents affects glucose metabolism in female offspring (Ng et al.,
Nature 2010; 467: 963-966). Overfeeding male mice also results in
the observation of metabolic changes in the two subsequent
generations of male offspring (Pentinat et al., Endocrinology 2010;
151: 5617-5623). When female rats are fed a high fat diet,
metabolism was also found to be altered in the next two
generations, with only the females of the third generation showing
this metabolic alteration, this latter phenotype being passed only
paternally (Dunn and Bale, Endocrinology 2009; 150: 4999-5009; Dunn
and Bale, Endocrinology 2011; 152: 2228-2236). If male rat parents
are fed a low protein diet, then the offspring show metabolic
alterations, such as lowered liver cholesterol (Carone et al., Cell
2010; 143: 1084-1096).
[0006] Human cohort studies also have proven to be the source of
striking observations regarding apparent epigenetic effects on
metabolism. For example, when mothers are exposed to famine during
pregnancy, metabolism in male offspring is affected (Lumey et al.,
Am. J. Clin. Nutr. 2009; 89: 1737-1743). Second generation
offspring also demonstrate alterations in metabolism, including a
predisposition to suffer metabolic disease (Painter et al., Bjog.
2008; 115: 1243-1249). In another study, low food intake during
adolescence correlated with an increase in survival of
grandchildren (Pembrey et al., Eur. J. Hum. Genet. 2006; 14:
159-166).
[0007] Another example of intergenerational transmission of
environmental information is the effect of stress experienced by
parents, which appears to affect stress-related behaviors, and
glucose metabolism, in offspring. For example, when parental male
mice were exposed to maternal separation and unpredictable maternal
stress (MSUS), depressive-like behaviors were observed in two
subsequent generations (Franklin et al., Biol. Psychiatry 2010; 68:
408-415; see also Gapp, K et al., Nature Neuroscience 2014; 17(5):
667-669).
[0008] The mechanisms responsible for epigenetic inheritance
patterns are just beginning to be understood. Mechanisms that have
been implicated in these inheritance patterns thus far include
histone modifications, DNA methylation, and non-coding RNAs,
including RNA interference (RNAi) machinery, small interfering RNAs
(siRNAs), Piwi-interacting RNAs (piRNAs) and microRNAs (miRNAs)
(Lim and Brunet, Trends Genet. 2013 29(3):176-186). For example,
there is evidence that paternal dietary conditions that affect
offspring metabolism also affect the sperm small RNA payload
(Sharma et al., Science 2016; 351(6271): 391-396). If purified
sperm RNAs are injected into naive one-cell embryos, alterations in
metabolism are observed in the resultant offspring (Grandjean et
al., Sci Rep 2015; 5:18193; see also Chen, Q et al., Science 2016;
351: 397-400). Likewise, when total sperm RNA from traumatized
males was injected into fertilized wild-type oocytes, the resultant
offspring displayed metabolic changes (Gapp et al., Nature
Neuroscience 2014; 17(5): 667-669). Finally, injecting nine
sperm-specific miRNAs into zygotes that were identified in a
paternal stress mouse model recapitulated the stress dysregulation
phenotype in offspring (Rodgers et al., PNAS 2015; 112(44):
13699-13704).
[0009] There is a need in the art to efficiently modify sperm RNA
payload to, for example, decrease the transmission of disease or
disorders.
SUMMARY
[0010] In a first aspect, disclosed herein is a method of altering
a population of sRNAs in a sperm of a subject, comprising
contacting the sperm with an sRNA-containing vesicle isolated from
an epididymosome to produce a sperm having an altered sRNA
population. In embodiments, the sRNA is selected from the group
consisting of a siRNA, a miRNA, a piRNA, a snoRNA, a srRNA, a
U-RNA, and a tRNA fragment. In further embodiments, the tRNA
fragment is selected from the group consisting of a tRNA-Gly-CCC
fragment, a tRNA-Gly-TCC fragment, a tRNA-Gly-GCC fragment, a
tRNA-Val-CAC fragment, a tRNA-Glu-CTC fragment, a tRNA-Lys-CTT
fragment, and a tRNA-His-GTG fragment. In an embodiment, the
epididymosome is selected from the group consisting of caput
epididymosome, corpus epididymosome, and cauda epididymosome.
[0011] In some embodiments, prior to contacting the sperm, the
sperm is immature and altering an sRNA population increases sperm
maturity. In other embodiments, prior to contacting the sperm, the
sperm is defective and altering an sRNA population diminishes at
least one defect. In such embodiments, the defective sperm can
comprise a defect selected from the group consisting of a reduced
level of sRNA, at least one aberrant sRNA, or absence of at least
one sRNA that is present in healthy mature sperm. In an embodiment,
the defective sperm comprises a defect in siRNA, miRNA, piRNA,
snoRNA, srRNA, U-RNA, and tRNA fragment content. In further
embodiments, the tRNA fragment content comprises a defect selected
from the group consisting of tRNA-Gly-CCC fragment content,
tRNA-Gly-TCC fragment content, tRNA-Gly-GCC fragment content,
tRNA-Val-CAC fragment content, tRNA-Glu-CTC fragment content,
tRNA-Lys-CTT fragment content, and tRNA-His-GTG fragment content.
In other embodiments, the defective sperm comprises a decrease in
at least one let-7 species of RNA when compared to a healthy
sperm.
[0012] In certain embodiments, after altering the RNA content, the
sperm is used to fertilize an oocyte.
[0013] In an embodiment, the subject is a mammal, such as a
primate, such as a human.
[0014] In certain embodiments, the sperm that is altered is
obtained from the subject's caput epididymis, corpus epididymis,
cauda epididymis, vas deferens, testis, or ejaculate. In further
embodiments, the sperm is obtained from the subject's caput
epididymis, corpus epididymis, or cauda epididymis using
microscopic or microsurgical epididymal sperm aspiration (MESA) or
percutaneous epididymal sperm aspiration (PESA). In yet other
further embodiments, the sperm is obtained from the subject's
testis using a technique selected from the group consisting of
needle aspiration (TESA), percutaneous or open surgical biopsy
(TESE), multibiopsy TESE, microdissection TESE, site-directed TESE
after fine needle aspiration mapping, and MicroTESE. Such
techniques are routinely used in assisted reproduction.
[0015] In certain embodiments, the subject whose sperm is altered
is experiencing a condition selected from the group consisting of a
stress-related disease or disorder, a dietary restriction, and
obesity. In an embodiment, the dietary restriction is protein
deficiency. In another embodiment, the stress-related disease or
disorder is selected from the group consisting of major depressive
disorder, dysthymia, bipolar disorder, generalized anxiety
disorder, a phobia, social anxiety disorder, separation anxiety
disorder, agoraphobia, and panic disorder.
[0016] In embodiments, the vesicle that is contacted to the sperm
is heterologous to the subject. In other embodiments, the vesicle
is autologous to the subject. In yet other embodiments, the vesicle
comprises a heterologous RNA; the heterologous RNA can comprise a
small RNA (sRNA). Such sRNA can be one selected from the group
consisting of a siRNA, miRNA, a piRNA, a snoRNA, a srRNA, a U-RNA,
and a tRNA fragment. In embodiments where the sRNA is a tRNA
fragment, the tRNA fragment can be selected from the group
consisting of a tRNA-Gly-CCC fragment, a tRNA-Gly-TCC fragment, a
tRNA-Gly-GCC fragment, a tRNA-Val-CAC fragment, a tRNA-Glu-CTC
fragment, a tRNA-Lys-CTT fragment, and a tRNA-His-GTG fragment. In
an embodiment where the sRNA is an miRNA, it can be selected from
the group consisting of miR-10a/b, miR-141, miR-143, miR-148 and
miR-200a. In other embodiments, the vesicle comprises autologous
RNA. Such a vesicle can comprise sRNA that can be selected from the
group consisting of a siRNA, miRNA, a piRNA, a snoRNA, a srRNA, a
U-RNA, and a tRNA fragment. In the case where the sRNA is a tRNA
fragment, the tRNA fragment can be selected from the group
consisting of a tRNA-Gly-CCC fragment, a tRNA-Gly-TCC fragment, a
tRNA-Gly-GCC fragment, a tRNA-Val-CAC fragment, a tRNA-Glu-CTC
fragment, a tRNA-Lys-CTT fragment, and a tRNA-His-GTG fragment. In
an embodiment where the sRNA is an miRNA, it can be selected from
the group consisting of miR-10a/b, miR-141, miR-143, miR-148 and
miR-200a. In other embodiments, the vesicle comprises an artificial
(synthetic) RNA. In such vesicles, the sRNA can be selected from
the group consisting of a siRNA, miRNA, a piRNA, a snoRNA, a srRNA,
a U-RNA, and a tRNA fragment. In the case where the sRNA is a tRNA
fragment, then the tRNA fragment can be selected from the group
consisting of a tRNA-Gly-CCC fragment, a tRNA-Gly-TCC fragment, a
tRNA-Gly-GCC fragment, a tRNA-Val-CAC fragment, a tRNA-Glu-CTC
fragment, a tRNA-Lys-CTT fragment, and a tRNA-His-GTG fragment. In
an embodiment where the sRNA is an miRNA, it can be selected from
the group consisting of miR-10a/b, miR-141, miR-143, miR-148 and
miR-200a. In other embodiments, the vesicle comprises a
transgene.
[0017] In certain embodiments, the altered sperm fertilizes an
oocyte in vitro. In other embodiments, the sperm is used in
intracytoplasmic sperm injection (ICSI). These embodiments can
further comprise implanting the fertilized oocyte to a second
subject (e.g., a non-human subject) to produce a progeny.
[0018] In other embodiments, the altered sperm fertilizes an oocyte
in vivo.
[0019] In embodiments, prior to contacting the sperm with a
vesicle, the sperm are frozen.
[0020] In a second aspect, disclosed herein is a method of treating
an epigenetically inheritable trait at risk of being transmitted to
a progeny of a subject, comprising altering a population of sRNAs
in a sperm from the subject by contacting the sperm with an
sRNA-containing vesicle isolated from an epididymosome and
fertilizing an oocyte with the sperm to produce the progeny. In
embodiments, the sRNA is selected from the group consisting of a
siRNA, a miRNA, a piRNA, a snoRNA, a srRNA, a U-RNA, and a tRNA
fragment. In embodiments where the sRNA is a tRNA fragment, the
tRNA fragment can be selected from the group consisting of a
tRNA-Gly-CCC fragment, a tRNA-Gly-TCC fragment, a tRNA-Gly-GCC
fragment, a tRNA-Val-CAC fragment, a tRNA-Glu-CTC fragment, a
tRNA-Lys-CTT fragment, and a tRNA-His-GTG fragment. In embodiments
where the sRNA is an miRNA, it can be selected from the group
consisting of miR-10a/b, miR-141, miR-143, miR-148 and miR-200a. In
other embodiments, the vesicle comprises a transgene.
[0021] In embodiments, the epididymosome is selected from the group
consisting of caput epididymosome, corpus epididymosome, and cauda
epididymosome.
[0022] In embodiments, the epigenetically inheritable trait is a
disease or disorder that is a metabolic or stress-related disease
or disorder. In some embodiments, the metabolic disease or disorder
comprises a glucose or hepatic metabolic disease or disorder. In
further embodiments, the hepatic metabolic disease or disorder
comprises reduced sterol biosynthesis. In yet further embodiments,
the reduced sterol biosynthesis comprises reduced cholesterol
biosynthesis. In even further embodiments, hepatic Sqle gene
expression is upregulated. In other embodiments, the stress-related
disease or disorder is selected from the group consisting of major
depressive disorder, dysthymia, bipolar disorder, generalized
anxiety disorder, a phobia, social anxiety disorder, separation
anxiety disorder, agoraphobia, and panic disorder.
[0023] In embodiments, the progeny lacks symptoms of the
epigenetically inheritable trait. In other embodiments, the progeny
has ameliorated symptoms of the epigenetically inheritable
trait.
[0024] In embodiments of this second aspect, the sperm comprises a
defect selected from the group consisting of a reduced level of
sRNA, at least one aberrant sRNA, or absence of at least one sRNA
that is present in healthy mature sperm. In some embodiments, the
sperm comprises a defect in siRNA, miRNA, piRNA, snoRNA, srRNA,
U-RNA, and tRNA fragment content. In those embodiments wherein the
defect is in tRNA content, the defect can be selected from the
group consisting of tRNA-Gly-CCC fragment content, tRNA-Gly-TCC
fragment content, tRNA-Gly-GCC fragment content, tRNA-Val-CAC
fragment content, tRNA-Glu-CTC fragment content, tRNA-Lys-CTT
fragment content, and tRNA-His-GTG fragment content. In other
embodiments, prior to contacting the sperm, the sperm comprises a
decrease in at least one let-7 species of RNA when compared to a
healthy sperm.
[0025] In embodiments, the subject is a mammal, such as a primate,
such as a human.
[0026] In embodiments, the sperm is obtained from the subject's
caput epididymis, corpus epididymis, cauda epididymis, vas
deferens, testis, or ejaculate. In such embodiments where the sperm
obtained from the subject's caput epididymis, corpus epididymis, or
cauda epididymis, microscopic or microsurgical epididymal sperm
aspiration (MESA) or percutaneous epididymal sperm aspiration
(PESA) is used. In other embodiments wherein the sperm is obtained
from the subject's testis, a technique selected from the group
consisting of needle aspiration (TESA), percutaneous or open
surgical biopsy (TESE), multibiopsy TESE, microdissection TESE,
site-directed TESE after fine needle aspiration mapping, and
MicroTESE can be used. Such techniques are routinely used in
assisted reproduction.
[0027] In embodiments of this second aspect, the subject is
experiencing a condition selected from the group consisting of a
stress-related disease or disorder, dietary restriction, and
obesity. In further embodiments, the dietary restriction is protein
deficiency. In other further embodiments, the stress-related
disease or disorder is selected from the group consisting of major
depressive disorder, dysthymia, bipolar disorder, generalized
anxiety disorder, a phobia, social anxiety disorder, separation
anxiety disorder, agoraphobia, and panic disorder.
[0028] In some embodiments, the vesicle is heterologous to the
subject. In other embodiments, the vesicle is autologous to the
subject. In other embodiments, the vesicle comprises a heterologous
RNA. In further embodiments, the heterologous RNA comprises a small
RNA (sRNA). In such embodiments, the sRNA can be selected from the
group consisting of a siRNA, miRNA, a piRNA, a snoRNA, a srRNA, a
U-RNA, and a tRNA fragment. In embodiments where the sRNA is a tRNA
fragment, the tRNA fragment can be selected from the group
consisting of a tRNA-Gly-CCC fragment, a tRNA-Gly-TCC fragment, a
tRNA-Gly-GCC fragment, a tRNA-Val-CAC fragment, a tRNA-Glu-CTC
fragment, a tRNA-Lys-CTT fragment, and a tRNA-His-GTG fragment. In
other embodiments, the vesicle comprises autologous RNA. In such
vesicles, the sRNA can be selected from the group consisting of a
siRNA, miRNA, a piRNA, a snoRNA, a srRNA, a U-RNA, and a tRNA
fragment. In those embodiments where the sRNA is a tRNA fragment,
the tRNA fragment can be selected from the group consisting of a
tRNA-Gly-CCC fragment, a tRNA-Gly-TCC fragment, a tRNA-Gly-GCC
fragment, a tRNA-Val-CAC fragment, a tRNA-Glu-CTC fragment, a
tRNA-Lys-CTT fragment, and a tRNA-His-GTG fragment. In other
embodiments, the vesicle comprises an artificial (synthetic) RNA.
In such vesicles, the sRNA can be selected from the group
consisting of a siRNA, miRNA, a piRNA, a snoRNA, a srRNA, a U-RNA,
and a tRNA fragment. In those embodiments where the sRNA is a tRNA
fragment, the tRNA fragment can be selected from the group
consisting of a tRNA-Gly-CCC fragment, a tRNA-Gly-TCC fragment, a
tRNA-Gly-GCC fragment, a tRNA-Val-CAC fragment, a tRNA-Glu-CTC
fragment, a tRNA-Lys-CTT fragment, and a tRNA-His-GTG fragment. In
other embodiments, the vesicle comprises a transgene.
[0029] In yet more embodiments of this second aspect, the sperm
fertilizes an oocyte in vitro. In other embodiments, the sperm is
used in intracytoplasmic sperm injection (ICSI).
[0030] Some embodiments comprise implanting the fertilized oocyte
to a second subject to produce a progeny. In other embodiments, the
sperm fertilizes an oocyte in vivo.
[0031] In embodiments, prior to contacting the sperm with an
vesicle, the sperm are frozen.
[0032] In a third aspect, disclosed herein are pharmaceutical
compositions comprising an vesicle comprising a small RNA molecule
(sRNA). In embodiments, the sRNA is selected from the group
consisting of a siRNA, a miRNA, a piRNA, a snoRNA, a srRNA, a
U-RNA, and a tRNA fragment. In those embodiments where the sRNA is
a tRNA fragment, the tRNA fragment can be selected from the group
consisting of a tRNA-Gly-CCC fragment, a tRNA-Gly-TCC fragment, a
tRNA-Gly-GCC fragment, a tRNA-Val-CAC fragment, a tRNA-Glu-CTC
fragment, a tRNA-Lys-CTT fragment, and a tRNA-His-GTG fragment.
[0033] In some embodiments, the pharmaceutical composition is a
vaginal foam or gel.
[0034] In some embodiments, the vesicle is an exosome; in yet
further embodiments, the exosome is an epididymosome. In further
embodiments, the epididymosome is selected from the group
consisting of caput epididymosome, corpus epididymosome, and cauda
epididymosome. In other embodiments, the vesicle is a seminosome or
a prostasome. In other embodiments, the vesicle is a
microvesicle.
[0035] In embodiments of this third aspect, the vesicle comprises a
heterologous RNA. In further embodiments, the heterologous RNA
comprises a small RNA (sRNA). In yet further embodiments, the sRNA
is selected from the group consisting of a siRNA, miRNA, a piRNA, a
snoRNA, a srRNA, a U-RNA, and a tRNA fragment. In those embodiments
where the sRNA is a tRNA fragment, the tRNA fragment can be
selected from the group consisting of a tRNA-Gly-CCC fragment, a
tRNA-Gly-TCC fragment, a tRNA-Gly-GCC fragment, a tRNA-Val-CAC
fragment, a tRNA-Glu-CTC fragment, a tRNA-Lys-CTT fragment, and a
tRNA-His-GTG fragment. In other embodiments, the vesicle comprises
autologous RNA. In such embodiments, the vesicle comprises an sRNA
that can be selected from the group consisting of a siRNA, miRNA, a
piRNA, a snoRNA, a srRNA, a U-RNA, and a tRNA fragment. In those
embodiments where the sRNA is a tRNA fragment, the tRNA fragment
can be selected from the group consisting of a tRNA-Gly-CCC
fragment, a tRNA-Gly-TCC fragment, a tRNA-Gly-GCC fragment, a
tRNA-Val-CAC fragment, a tRNA-Glu-CTC fragment, a tRNA-Lys-CTT
fragment, and a tRNA-His-GTG fragment. In other embodiments, the
vesicle comprises an artificial (synthetic) RNA. In such
embodiments, the vesicle comprises an sRNA that can be selected
from the group consisting of a siRNA, miRNA, a piRNA, a snoRNA, a
srRNA, a U-RNA, and a tRNA fragment. In those embodiments where the
sRNA is a tRNA fragment, the tRNA fragment can be selected from the
group consisting of a tRNA-Gly-CCC fragment, a tRNA-Gly-TCC
fragment, a tRNA-Gly-GCC fragment, a tRNA-Val-CAC fragment, a
tRNA-Glu-CTC fragment, a tRNA-Lys-CTT fragment, and a tRNA-His-GTG
fragment. In an embodiment, the vesicle comprises a transgene.
[0036] In certain embodiments of any of the aspects above in which
the sRNA is an miRNA, it may be selected from the group consisting
of miR-10a/b, miR-141, miR-143, miR-148 and miR-200a.
[0037] In a fourth aspect, disclosed herein is a method of altering
the sRNA population in an oocyte, comprising altering a population
of sRNA in a sperm by contacting a sperm with a vesicle isolated
from an epididymosome (e.g., a caput epididymosome, a corpus
epididymosome and/or a cauda epididymosome) to produce a sperm
having an altered sRNA population, and fertilizing the oocyte with
the sperm having an altered sRNA population. In certain
embodiments, the sRNA is selected from the group consisting of a
siRNA, a miRNA, a piRNA, a snoRNA, a srRNA, a U-RNA, and a tRNA
fragment. In certain embodiments, the tRNA fragment is selected
from the group consisting of a tRNA-Gly-CCC fragment, a
tRNA-Gly-TCC fragment, a tRNA-Gly-GCC fragment, a tRNA-Val-CAC
fragment, a tRNA-Glu-CTC fragment, a tRNA-Lys-CTT fragment, and a
tRNA-His-GTG fragment.
[0038] In certain exemplary embodiments, the sperm comprises a
defect selected from the group consisting of a reduced level of
sRNA, at least one aberrant sRNA, or absence of at least one sRNA
that is present in healthy mature sperm. In certain exemplary
embodiments, the sperm comprises a defect in siRNA, miRNA, piRNA,
snoRNA, srRNA, U-RNA, and tRNA fragment content.
[0039] In certain embodiments, the tRNA fragment content comprises
a defect selected from the group consisting of tRNA-Gly-CCC
fragment content, tRNA-Gly-TCC fragment content, tRNA-Gly-GCC
fragment content, tRNA-Val-CAC fragment content, tRNA-Glu-CTC
fragment content, tRNA-Lys-CTT fragment content, and tRNA-His-GTG
fragment content.
[0040] In certain embodiments, the miRNAs is selected from the
group consisting of miR-10a/b, miR-141, miR-143, miR-148 and
miR-200a. In certain embodiments, the vesicle comprises a synthetic
RNA and/or a transgene.
[0041] In certain exemplary embodiments, the sperm fertilizes an
oocyte in vitro or in vivo. In other embodiments, the sperm is used
in intracytoplasmic sperm injection (ICSI). In certain embodiments,
the method further includes the step of implanting the fertilized
oocyte into a second, non-human subject to produce a progeny. In
certain embodiments, the sperm are frozen prior to contacting the
vesicle.
[0042] In a fifth aspect, disclosed herein is a method of altering
a population of sRNAs in an isolated sperm, comprising contacting
the isolated sperm with an sRNA-containing vesicle isolated from a
caput epididymosome to produce a sperm having an altered sRNA
population.
[0043] In certain embodiments, the sperm having an altered sRNA
population exhibits an increase in the levels of miRNAs and/or tRNA
fragments compared to the levels of miRNAs and/or tRNA fragments in
the isolated sperm prior to contacting the sRNA-containing vesicle.
In other embodiments, a tRNA fragment is selected from the group
consisting of a tRNA-Gly-CCC fragment, a tRNA-Gly-TCC fragment, a
tRNA-Gly-GCC fragment, a tRNA-Val-CAC fragment, a tRNA-Glu-CTC
fragment, a tRNA-Lys-CTT fragment and a tRNA-His-GTG fragment. In
still other embodiments, a miRNA is selected from the group
consisting of miR-10a/b, miR-141, miR-143, miR-148 and
miR-200a.
[0044] In certain embodiments, the levels of tRNA fragments present
are increased by at least about 5%, at least about 6%, at least
about 7%, at least about 8%, at least about 9% or at least about
10% compared to levels of tRNA fragments in the isolated sperm
prior to contacting with the sRNA-containing vesicle. In other
embodiments, the levels of tRNA fragments present are increased by
at least about two-fold compared to levels of tRNA fragments in the
isolated sperm prior to contacting with the sRNA-containing
vesicle.
[0045] In certain embodiments, the caput epididymosome is between
about 100 nm and about 400 nm in diameter, between about 250 nm and
about 350 nm in diameter, between about 120 nm and about 170 nm in
diameter, or about 150 nm in diameter. In certain embodiments, the
caput epididymosome is isolated from an epididymal sample and/or is
isolated from the epididymal sample by ultracentrifugation.
[0046] In a fifth aspect, disclosed herein is a method of
correcting a developmental defect in a zygote comprising
microinjecting the zygote with a tRNA-Gly-GCC fragment to correct
the developmental defect. In certain embodiments, the expression
level of one or more genes associated with zygote development is
altered. In other embodiments, the expression level of one or more
genes associated with zygote development is downregulated.
BRIEF DESCRIPTION OF THE DRAWINGS
[0047] The foregoing and other features and advantages of the
present invention will be more fully understood from the following
detailed description of illustrative embodiments taken in
conjunction with the accompanying drawings.
[0048] FIGS. 1A-1D shows the characterization of small RNAs in
sperm. FIG. 1A shows small RNA sequencing data from mature cauda
sperm samples. Sizes of deep sequencing reads are shown for the
average of 12 small (<40 nt) cauda sperm RNA datasets. FIGS.
1B-1D show examples of abundant tRNA fragments (tRFs) in cauda
sperm. tRNA fragments from the 5' end of tRNA-Gly-GCC (FIG. 1B),
tRNA-Val-CAC (FIG. 1C), and tRNA-Glu-CTC (FIG. 1D) are shown
schematically, with arrows indicating dominant 3' ends.
[0049] FIGS. 2A-2C show that tRNA fragments are abundant in the
epididymis. FIG. 2A shows a Northern blot analysis of total RNA
isolated from testis, cauda epididymis, and caput epididymis, as
indicated. FIG. 2B shows the quantitation of Northern blot data.
Bars show levels of tRFs in testis, caput epididymis, and cauda
epididymis, normalized to testis levels. Error bars show s.e.m.
FIG. 2C shows pie charts showing the percentage of small RNAs
mapping to the indicated features, for each tissue. rRNA-mapping
reads are excluded.
[0050] FIGS. 3A-3B show that tRNA cleavage predominantly occurs
downstream of the testis. FIG. 3A shows that sperm RNA payload
diverges dramatically from the RNA population in testes. Small
(<40 nt) RNA data from cauda sperm and from testes were
normalized to parts per million (ppm) total reads (excluding
rRNA-mapping reads), and data are shown for all RNAs present at
greater than 5 ppm in the sperm or the testis averaged datasets.
Scatterplot shows abundance of small RNAs in testis (x axis, log 10
scale) vs. sperm (y axis, log 10 scale), with RNAs mapping to tRNA
genes, to microRNAs, to repeat elements/unique piRNAs, and to all
other transcripts (fragments of mRNAs, snoRNAs, etc.) all indicated
separately. FIG. 3B shows a schematic of murine epididymis. Sperm
exiting the testis first enter the proximal (caput) epididymis,
then proceed distally to the corpus and cauda epididymis, and exit
via the vas deferens.
[0051] FIG. 4 shows a cartoon showing testicular spermatogenesis
and post-testicular maturation in the epididymis. For each purified
gamete population, pie charts show the relative abundance of tRNA
fragments, microRNAs, piRNAs (defined as reads mapping to either
repeatmasker consensus sequences or to unique piRNA clusters), and
to Refseq (mRNA fragments), as indicated. Data are shown for three
separate fractions of purified testicular germ cells, and for sperm
isolated from caput and cauda epididymis, as indicated. Consistent
with the low levels of tRNA fragments found in intact testes,
spermatocytes and two populations of post-meiotic spermatids carry
extremely low levels of tRNA fragments, indicating that the absence
of tRNA fragments in intact testis is not a result of contamination
by testicular somatic cells.
[0052] FIGS. 5A-5B show changes in sperm tRF payload during
epididymal transit. FIG. 5A shows that tRNA fragments that are more
abundant in cauda, relative to caput, epididymis are also gained in
cauda sperm. The scatterplot shows the relative changes between
caput and cauda epididymis (x axis--positive values show
cauda-enriched RNAs) compared to relative changes between caput and
cauda sperm (y axis). Note that given the normalization to total
small RNA abundance, a "loss" of a given tRF present in caput sperm
could result from degradation of this tRF, or from constant
abundance of this tRF in the face of overall tRF gain. FIG. 5B
shows proximal-distal biases for tRFs in the epididymis and in
sperm samples, averaged for each anticodon. Only tRFs with an
average abundance of >100 ppm small RNAs are shown, and tRFs are
ordered by cauda/caput ratio for epididymis samples.
[0053] FIGS. 6A-6C show the characterization of cauda
epididymosomes. FIG. 6A show a transmission electron micrograph of
purified cauda epididymosomes, showing abundant vesicles of about
120-150 nm. FIG. 6B shows epididymosome size distribution.
Nanosight sizing data for two independent cauda epididymosome
preps. Data for 0-200 nm are shown in main panel, while inset shows
0-500 nm zoom-in. FIG. 6C shows that epididymosomal preparations
are not contaminated with free RNA, or with fragments of sperm.
tRNA fragments are protected from RNaseA treatment, indicating
their presence in vesicles. In addition, epididymosomes purified
from tdrdl-/- mice, which lack mature sperm, carry high levels of
tRNA fragments, indicating that our epididymosome preparations are
not simply fragments generated from maturing sperm such as the
residual body.
[0054] FIG. 7 shows a comparison of small RNA payloads of cauda
sperm vs. cauda epididymosomes.
[0055] FIGS. 8A-8E show the characterization of caput epididymosome
preparations. FIG. 8A shows a schematic of murine epididymis.
Circles represent epididymosomes. FIG. 8B shows a transmission
electron micrograph of purified cauda epididymosomes (top panel),
showing abundant vesicles of approximately 120-150 nm. Lower panel
shows vesicle size distributions for epididymosomes isolated from
cauda or from caput epididymis, obtained using nanosight sizing. A
subtle increase in approximately 250-300 nm vesicles is apparent in
cauda samples. FIG. 8C shows distributions for epididymosomal small
(<40 nt) RNA-Seq libraries, showing highly abundant 28-32 nt
(approximately 87% of reads) 5' tRNA fragments for cauda
epididymosomes. In contrast, caput epididymosomes primarily carry
microRNAs, with approximately 28% of reads mapping to 5' tRFs. FIG.
8D shows that small RNA populations in epididymosomes are highly
correlated with those in mature sperm. The scatterplot shows
abundance of various classes of small RNAs for sperm (x axis, log
scale) and epididymosomes (y axis, log scale). FIG. 8E shows the
consistent proximal-distal biases for specific RNAs in epididymis
and epididymosome samples. For all RNAs with a maximum abundance of
greater than 10 ppm in either caput or cauda samples, the log
2(cauda/caput) ratio is plotted for epididymis (x axis) vs.
epididymosomes (y axis).
[0056] FIGS. 9A and 9B show our ability to deliver small RNAs to
immature sperm via fusion with epididymosomes. FIG. 9A shows Taqman
analysis of the indicated tRFs in purified caput sperm and in
"reconstituted" caput sperm which have been incubated with cauda
epididymosomes, showing gain of tRFs relative to normalization
control let-7. FIG. 9B shows the results of deep sequencing of
sperm reconstitutions. In each case, tRFs are aggregated by codon,
and data are normalized to levels of tRF-Glu-CTC. For bull, data
shown mean and s.e.m. for four replicate reconstitutions
experiments. For mouse, deep sequencing libraries were under
sequenced approximately 100-200 thousand reads), and only two
replicates were sequenced, but the same trends seen between natural
caput and cauda sperm were recapitulated, with cauda-enriched tRFs
such as tRF-Val-CAC being delivered to caput sperm via fusion with
cauda epididymosomes.
[0057] FIGS. 10A-10F show that tRF-Gly-GCC regulates MERVL-driven
transcripts in the early embryo. FIG. 10A shows Affymetrix
microarray data for mRNA abundance in embryonic stem cells
transfected with an LNA antisense oligo targeting tRF-Gly-GCC. The
X axis shows abundance of mRNAs in anti-GFP knockdown cells, and
the y axis shows mRNA abundance for cells transfected with an LNA
antisense targeting the 5' end of tRF-Gly-GCC. Data represent
average of seven replicates. FIG. 10B shows the effect of
tRF-Gly-GCC knockdown on MERVL is isoacceptor-specific. Affymetrix
data for knockdown studies with the indicated LNA antisense oligos.
All comparisons are to GFP siRNA transfections done in the same
batch. Identical results are obtained when comparing to
mock-transfected ES cells. All genes showing abundance changes of
2-fold or greater in 2 or more samples are shown. FIG. 10C shows
RNA-Seq data for four pooled replicate samples of ES cells
transfected with shRNA against GFP, or with the anti-tRF-Gly-GCC
LNA oligo, as indicated.
[0058] FIG. 10D shows a schematic showing genomic context for four
tRF-Gly-GCC target genes, showing MERVL LTRs associated with all
target genes. Some additional target genes, such as the Tdpoz
cluster, are not as closely associated with MERVL LTRs, but instead
are located in large MERVL-rich genomic clusters, and have also
been shown to be part of the MERVL-regulated gene expression
program (Macfarlan, T S, et al. 2012. Nature 487: 57-63). FIG. 10E
shows that inhibition of tRF-Gly-GCC affects MERVL target
expression in 4-cell embryos. Control zygotes were generated via
IVF, and then either mock-injected or injected with an antisense
oligonucleotide targeting tRF-Gly-GCC. Embryos were then allowed to
develop to the 4-cell stage, and subject to single-embryo RNA-Seq.
Averaged single embryo RNA-Seq data for control (n=28) or
tRF-inhibited (n=27) embryos. Among genes upregulated at least
2-fold on average, those previously described as MERVL targets are
indicated separately. FIG. 10F shows examples of single embryo data
for two MERVL targets. Here, each bar represents mRNA abundance
from a single embryo, with embryos ordered from highest to lowest
expression for each condition.
[0059] FIGS. 11A-F show paternal dietary effects on preimplantation
development. FIG. 11A shows embryos generated by IVF that were
cultured for varying times, then subject to single embryo RNA-Seq.
FIG. 11B shows single-embryo data for preimplantation embryos
represented via PCA: first two principal components explain 74% of
dataset variance. FIG. 11C shows mRNA abundance in 2-cell embryos
generated via IVF using Control vs. Low Protein sperm (n=41 C and
39 LP). Cumulative distribution plots for tRF-Gly-GCC targets
(p=4.5.times.10-7, KS test), other MERVL targets (17)
(p=2.5.times.10-13), and all remaining genes, showing percentage of
genes with the average Log 2(LP/C) indicated on the x axis. Low
Protein embryos exhibit a significant shift to lower expression of
MERVL targets. Bottom panels show individual embryo data for two
targets. FIG. 11D shows small RNAs isolated from Control or Low
Protein cauda sperm were microinjected into control zygotes.
RNA-Seq (n=42 C and 46 LP embryos) reveals downregulation of
tRF-Gly-GCC targets (p=4.8.times.10-14) driven by Low Protein RNA.
FIG. 11E shows the effects of synthetic tRF-Gly-GCC on 2-cell gene
regulation, showing significant (p=0.0001) downregulation of target
genes in embryos injected with tRF-Gly-GCC (n=26) vs. GFP controls
(n=11). Inset shows effects of tRF-Glu-CTC (n=6). FIG. 11F shows
effects of epididymal passage on embryonic gene regulation. Intact
sperm isolated from rete testis (n=12), or cauda epididymis (n=9),
were injected into control oocytes, and mRNA abundance was analyzed
as above.
[0060] FIGS. 12A-12H show paternal dietary effects on
preimplantation development. FIG. 12A shows subjected cumulative
distribution plot for all genes encoding ribosomal protein genes
during the indicated stages. X axis shows the relative expression
of these genes in Low Protein IVF embryos, compared to Control.
Grey line shows distribution of dietary effects on all non-RPG
genes, for all four stages. Left shift at the 2-cell stage shows
downregulation of RPGs in Low Protein 2-cell embryos. FIGS. 12B-E
show GSEA plots for various sets of genes involved in ribosome
biogenesis at the indicated developmental stages. FIG. 12F shows an
example image of a blastocyst stained with DAPI and anti-Cdx2 to
image total cell number and trophectoderm cells. FIG. 12G shows
that Low Protein diet reproducibly alters developmental tempo. FIG.
12H shows aggregated data for three replicate experiments, showing
the number of blastocysts with the indicated number of cells, for
embryos generated via IVF using Control or Low Protein sperm, as
indicated.
[0061] FIGS. 13A-13H show dietary effects on tRNAs in testes. FIG.
13A shows a schematic illustrating assay for tRNA charging
analysis. FIG. 13B shows validation of tRNA charging protocol.
Changes in tRNA abundance for charged and uncharged tRNAs are shown
on the y axis, sorted by the change in charged tRNA abundance. FIG.
13C shows testicular tRNA abundance correlation with codon bias in
the mouse. The X axis shows intact tRNA abundance in testis in log
scale, and the y axis shows the corresponding codon abundance (in
codon frequency/1000) in all murine mRNAs, or in the 47 most-highly
expressed mRNAs in testis. FIG. 13D shows validation of tRNA
charging analysis. Scatterplot shows abundance of approximately
60-80 nt RNAs in the total RNA protocol (x axis, log scale)
compared to abundance of RNAs in the charged tRNA protocol (y axis,
log scale). FIGS. 13E-13G show Low Protein vs. Control effects on
tRNA levels for total (FIG. 13E), uncharged (FIG. 13F), and charged
(FIG. 13G) tRNA levels in testis. FIG. 13H shows that dietary
effects on sperm tRFs are not explained by effects on intact tRNA
abundance in testes.
[0062] FIGS. 14A and 14B show that there are consistent dietary
effects throughout the reproductive tract. FIG. 14A shows the
dietary effects on small RNA abundance in testes and caput and
cauda epididymis samples. Each heatmap shows log 2 of Low
Protein/Control RNA abundance for a pair of samples, showing RNAs
(rows) that exhibit consistent dietary effects across >75% of
samples. FIG. 14B shows the coherent dietary effects on tRF-Gly and
let-7 family members throughout the male reproductive tract. For
each RNA, bars show average and standard error of the mean for Low
Protein effects on the abundance of the RNA species in the
indicated tissue. Changes with a nominal p value of <0.05
(paired t test, not corrected for multiple testing) are indicated
with asterisks.
[0063] FIGS. 15A-15F show RNA populations in caput sperm. FIG. 15A
shows that unwashed caput sperm are contaminated with RNAs abundant
in caput epididymosomes. FIG. 15B shows a comparison of small RNA
payloads of cauda vs. caput sperm for all RNA species with an
abundance of at least 1 ppm in both sperm populations. FIG. 15C
shows the proximal-distal biases observed for epididymis (x axis)
are recapitulated in cauda vs. caput sperm samples (y axis). FIG.
15D shows that there is a gain in all four tRFs from caput to
cauda. Data from FIG. 15D are shown with tRF-Val-CAC normalized to
tRF-Glu-CTC rather than to microRNAs. FIG. 15E shows that
tRF-Val-CAC is strongly cauda-enriched in all three
preparations--epididymal epithelium, epididymosomes, and
sperm--examined. FIG. 15F depicts Northern blots showing that caput
sperm carry intact tRNAs
[0064] FIGS. 16A-16C show that dietary information is carried in
sperm. FIG. 16A shows the sperm from males consuming Control or Low
Protein diet which were used to fertilize oocytes gathered from
Control females. Sqle levels (normalized to Actb) are shown for all
offspring as individual points, with horizontal lines showing mean
expression. FIG. 16B shows the cumulative distribution of Sqle
expression for all offspring generated using Control or Low Protein
sperm, as indicated. FIG. 16C shows consistent litter effects. Sqle
levels were averaged for all offspring of a given litter.
[0065] FIGS. 17A and 17B show the mechanistic basis for tRF-Gly-GCC
regulation of MERVL. FIG. 17A shows RNA-Seq and ribosome
footprinting data for Sp110. FIG. 17B shows that RNA abundance and
ribosome footprinting data are highly correlated, indicating that
tRF-Gly-GCC does not affect MERVL elements as a secondary effect of
its effects on protein translation.
[0066] FIGS. 18A-18D show the reconstitution of small RNA delivery
to testicular sperm. FIG. 18A depicts an experimental schematic
showing purified testicular sperm, which carry extremely low levels
of tRFs, were incubated with caput epididymosomes for 2 hours, and
then extensively washed with detergent. Small RNAs were purified
from either mock-treated testicular sperm, or reconstituted sperm,
and deep sequenced. Pie charts show average levels of various small
RNA classes, revealing increased levels of microRNAs and tRFs
delivered to testicular sperm by epididymosomes. FIG. 18B shows the
delivery of two prominent tRFs to testicular sperm. Taqman q-RT-PCR
for the indicated tRFs, with individual replicates plotted (on aa
log 2 y axis) relative to the average level present in Mock-treated
testicular sperm. FIG. 18C shows the distribution of small RNA
changes upon reconstitution. The X axis shows the log 2-fold
difference between reconstituted and mock-treated sperm; positive
values indicate delivery by epididymosomes. The peak for piRNAs is
approximately -0.6, reflecting the assumption in genome-wide
normalization of equal numbers of molecules in both samples. Under
conditions of RNA delivery, pre-existing RNAs (shown), the piRNAs
that predominate in testicular sperm will appear to "decrease" in
abundance due to normalization. As such, all values over -0.6
indicate gain of RNA during the fusion protocol.
[0067] FIG. 18D shows a scatterplot of small RNA abundances from
deep sequencing data. The strong diagonal for piRNAs indicates RNAs
present in testicular sperm that are not affected by the delivery
process. Essentially all RNAs shown either lie along this diagonal,
indicating that they are either absent or present in low abundance
in caput epididymosomes, or above the diagonal, indicating
widespread delivery of many RNA species to testicular sperm during
reconstitution.
DETAILED DESCRIPTION
[0068] The present disclosure is directed to methods and
compositions that can alter sperm molecular content, such that a
disease, disorder, or condition that would otherwise be inherited
by offspring, are not, or such disease, disorder, or condition
severity is reduced.
[0069] The inventors discovered that as sperm mature in the male
reproductive tract, their molecular cargo changes. Specifically, as
sperm move through the epididymis, the small RNA molecule (sRNA)
content changes dramatically. The sperm sRNA content mirrors that
found in vesicles found in the different regions of the epididymis
("epididymosomes"). For example, a sperm located in the cauda
epididymis has a similar sRNA content as a caudal
epididymosome.
[0070] The inventors have discovered methods of altering the RNA
content of sperm, such as to increase the maturity of immature
sperm, to "rescue" defective mature sperm that have at least one
sRNA defect (e.g., a reduction or absence of at least one sRNA, or
at least one aberrant sRNA), and to decrease transmission of
epigenetically-transmitted diseases or disorders to progeny. These
methods involve contacting the target sperm with vesicles, such as
epididymosomes, to alter the sperm RNA content. Such treated sperm
can then be used to fertilize oocytes in vitro or in vivo.
I. Definitions
[0071] As used in the specification and in the claims, the term
"comprising" may include the embodiments "consisting of" and
"consisting essentially of" The terms "comprise(s)," "include(s),"
"having," "has," "may," "contain(s)," and variants thereof, as used
herein, are intended to be open-ended transitional phrases, terms,
or words that require the presence of the named ingredients/steps
and permit the presence of other ingredients/steps. However, such
description should be construed as also describing compositions or
processes as "consisting of" and "consisting essentially of" the
enumerated compounds, which allows the presence of only the named
compounds, along with any pharmaceutically acceptable carriers, and
excludes other compounds.
[0072] As used herein, approximating language may be applied to
modify any quantitative representation that may vary without
resulting in a change in the basic function to which it is related.
Accordingly, a value modified by a term or terms, such as "about"
and "substantially," may not be limited to the precise value
specified, in some cases. In at least some instances, the
approximating language may correspond to the precision of an
instrument for measuring the value. The modifier "about" should
also be considered as disclosing the range defined by the absolute
values of the two endpoints. For example, the expression "from
about 600 to about 2000" also discloses the range "from 600 to
2000." The term "about" may refer to plus or minus 10% of the
indicated number. For example, "about 10%" may indicate a range of
9% to 110%, and "about 1" may mean from 0.9 to 1.1. Other meanings
of "about" may be apparent from the context, such as rounding off,
so, for example "about 1" may also mean from 0.5 to 1.4.
[0073] As used herein, "treatment" or "treating," is defined as the
application or administration of a therapeutic agent to a subject,
or application or administration of a therapeutic agent to an
isolated tissue, cell line, or cell from a subject. "Therapeutic
agents" include vesicles, including epididymosomes.
[0074] As used herein, "patient," "individual" or "subject" refers
to a human or a non-human mammal. Non-human mammals include, for
example, livestock and pets, such as ovine, bovine, porcine,
canine, feline and murine mammals. Non-human mammals also include
primates. Preferably, the patient, subject, or individual is
human.
[0075] As used herein, "untreated sperm" means sperm that have not
been subjected to the application or administration of a
therapeutic agent as described in the disclosed methods. "Treated
sperm" means sperm that have been subjected to the application or
administration of a therapeutic agent as described in the disclosed
methods. In embodiments, an untreated sperm may have been exposed
to vesicles in the male reproductive tract, but become treated
sperm when exposed to vesicles that are autologous or heterologous
in vitro. In some embodiments, the untreated sperm are treated in
vivo when exposed to autologous or heterologous vesicles, which can
be comprised in a composition, such as a pharmaceutical
composition.
[0076] As used herein, "vesicle" means extracellular vesicles (EVs)
that cells shed from their plasma membrane, or from multivesicular
bodies. These vesicles are generally referred to as microvesicles,
ectosomes, shedding vesicles, or microparticles, as well as exosome
vesicles (or exosomes). Exosomes are extracellular vesicles that
originate from multivesicular endosomes (MVEs) that fuse with the
plasma membrane. However, circulating extracellular vesicles such
as epididymosomes also include microvesicles (MVs). Thus, unless
otherwise noted, the term "vesicles" includes MVs and exosomes. A
vesicle comprises at least one RNA molecule, such as a small RNA
(sRNA). Vesicles that originate from specific tissues or cells can
be designated by specific terms, such as epididymosomes, which
originate from the epididymis; seminosomes which originate from
seminal fluid, and prostasomes, which originate from the prostate.
The ExoCarta database (found on the world-wide web at exocarta.org)
contains the proteins, lipids, and RNA that have been found in EVs
from various sources.
[0077] As used herein, "altering the RNA content" means, such as
when applied to cells, such as sperm, to add or remove an RNA
molecule by treating the cells or sperm. For example, vesicles can
be used to deliver RNA cargo to sperm, thus altering the RNA
content of the sperm.
[0078] As used herein, "increasing sperm maturity" means that after
a treatment, the sperm takes on or improves in at least one
characteristic that indicates that the sperm has further matured.
An increase in sperm maturity is reflective of a healthy sperm that
has progressed to the same location or further in the male
reproductive tract relative to the untreated sperm.
[0079] As used herein, "a defective sperm" means a sperm that lacks
at least one characteristic in relation to its maturity by virtue
of its location in the male reproductive tract or ejaculate when
compared to a healthy ejaculated sperm. The altered characteristic
can be a difference in at least one molecule, such as an RNA
molecule or a polypeptide. The difference can be the absence of a
molecule, the presence of a molecule that is usually absent in
healthy sperm, or a changed molecule, such as a mutated or
mis-processed molecule. In some embodiments, the at least one
molecule is a sRNA. "Rescuing a defective sperm" means to add or
subtract the molecule that is different than healthy sperm, or
supplying a wild-type molecule of a changed molecule, to the sperm
by treating the sperm, so that the sperm resemble healthy sperm in
relation to its source of isolation from the male reproductive
tract or ejaculate. "Defective mature sperm" means a sperm that
appears to have matured by virtue of it completing its journey
through the male reproductive tract, but lacks at least one
characteristic of mature healthy sperm. A defective mature sperm is
not necessarily incapable of fertilizing an oocyte, but may instead
transmit a trait, condition, disease, or disorder to a resulting
progeny.
[0080] As used herein, "epigenetically-transmitted" means a trait,
condition, disease, or disorder transmitted by a parent to
offspring wherein the acquired trait, condition, disease, or
disorder is not the result of a mutation in DNA; that is, the trait
is transmitted in violation of Mendelian genetics. In such
intergenerational epigenetic inheritance, epigenetic phenotypes are
transmitted to at least one generation and may be
gender-specific.
[0081] As used herein, "healthy sperm" means a sperm that has the
characteristics of sperm found in healthy, fertile subjects in
relation to its maturity by virtue of its location in the male
reproductive tract or ejaculate.
[0082] As used herein, "sRNA" means "small RNA" and includes all
classes of small RNAs, including: small interfering RNAs (siRNAs),
Piwi-interacting RNAs (piRNAs), microRNAs (miRNAs), tRNA fragments
(tRF), small nucleolar RNA (snoRNA), small rDNA-derived RNA
(srRNA), and small nuclear RNA (U-RNA). Generally, sRNAs are about
200 nucleotides or less in length, such as 40 nucleotides in
length, or less. siRNAs are generally double-stranded pairs of RNAs
about 20-25 base pairs long, and can participate in the RNA
interference (RNAi) pathway (Hannon, G J and J J Rossi. 2004.
Nature, 431:371-378). piRNAs are non-coding RNA molecules of about
26-31 nucleotides long and form RNA-polypeptide complexes with piwi
proteins. These RNA molecules have been linked to epigenetic gene
silencing of "molecular parasites," such as transposons found in
germ line cells (Czech, B and G J Hannon. 2016. Trends Biochem Sci,
41: 324-337; Siomi M C, et al. 2011. Nat Rev Mol Cell Biol,
12:246-258). miRNAs are about 19-24 nucleotide long, non-coding RNA
molecules. They regulate protein-coding gene expression
translationally and post-transcriptionally (Virant-Klun, I., et al.
2016. Stem Cells Int. 2016:3984937). tRFs are fragments of tRNA
molecules that are about 28 to 34 nucleotides long, have a wide
variety of molecular effects on cells and are found enriched in,
for example, sperm (Peng, H., et al. 2012. Cell Res, 22:
1609-1612). snoRNA guide chemical modifications of other RNAs, such
as rRNAs, tRNAs, and snRNAs; these small non-coding RNAs fall into
two classes, one of about 60-90 nucleotides long ("box C/D"), and
another of about 120-140 nucleotides long ("box H/ACA")
(Dupuis-Sandoval, F, et al. 2015. Wiley Interdiscip Rev RNA, 6:
381-397). srRNAs map by sequence to rRNA coding regions in the
sense direction; coimmunoprecipitate with Argonaute proteins, and
are involved in various signaling pathways, and are thought to be
about 18-30 nucleotides long (Wei, H, et al. 2013. PLoS One, 8:
e56842). U-RNA molecules are about 150 nucleotides long and
function to process pre-mRNA in the nucleus (Zhang, L, et al. 2013.
Protein Sci, 22: 677-692).
[0083] An "aberrant" sRNA is an sRNA molecule that differs from a
wild-type sRNA. For example, the sRNA has a changed sequence, such
as one or more point mutations, deletions, insertions,
translocations; or is chemically modified, etc.
[0084] An "artificial RNA" or "synthetic RNA" is an RNA molecule
that is synthesized in vitro by any art-accepted method.
[0085] As used herein, "stress" means a state of physical, mental
or emotional strain or tension in an organism, such as a subject,
that results from adverse or demanding circumstances and causes
physiological alterations in the organism. The stress is often
applied repeatedly or continually. In some embodiments, the stress
may last for a period of time. In some cases, the physiological
alterations are present after the stress has been applied.
[0086] As used herein, "dietary restriction" means a diet that is
deficient in one or more components of a healthy diet, such as a
vitamin, a nutrient, a micronutrient, a fat, a simple carbohydrate,
a complex carbohydrate, and protein, or a calorie deficit (that is,
insufficient calories to support the health of an organism) such
that the physiology of an organism is altered. In some embodiments,
the restriction is repetitive or continual. In some embodiments,
the restriction may last for a period of time. In some cases, the
physiological change persists after the dietary restriction
stops.
[0087] As used herein, "overeating" means the condition of an
organism consuming more calories than is necessary to maintain the
normal health of the organism.
[0088] As used herein, "stress-related disease or disorder" means a
disease or disorder which symptoms in an organism can be triggered
or amplified by the application of stress to an organism. In some
embodiments, a stress-related disease or disorder is related to
mental health. In some embodiments, the mental health disease or
disorder is a form of depression, such as major depressive disorder
(also known as major depression or clinical depression), dysthymia,
and bipolar disorder (having a depressive phase). Other examples of
mental health disease or disorders include those that are
anxiety-based conditions, including generalized anxiety disorder, a
specific phobia, social anxiety disorder, separation anxiety
disorder, agoraphobia, and panic disorder.
[0089] As used herein, "metabolic disorder" means a disorder
wherein a component of metabolism is absent, up-regulated, or
down-regulated when compared to the metabolism of a healthy
organism, such as a subject. A metabolic disorder can manifest in
many forms. For example, the metabolic disorder can be a hepatic
metabolic disorder, which originates in the liver, or affects the
expression of a marker of liver-based metabolism, such as Sqle gene
expression. A manifestation of a hepatic metabolic disorder
includes a reduction in sterol biosynthesis, such as reduced
cholesterol biosynthesis. Pancreatic metabolic disorders include
type II diabetes.
[0090] As used herein, "reduced" means that the substance or
activity being measured is present in a lesser amount and/or lesser
activity than when compared to that of a healthy organism.
[0091] "Healthy" means that the organism, tissue, or cell has the
composition and activity of an organism, tissue or cell that falls
within the boundaries of wild-type expression, indicative of a
non-disease state.
II. Methods
[0092] In an aspect, disclosed herein is a method of altering the
RNA content of a sperm of a subject, comprising contacting the
sperm with a vesicle comprising a sRNA to produce a sperm having an
altered RNA content.
[0093] In a second aspect, disclosed herein is a method of treating
an epigenetically inheritable trait at risk of being transmitted to
a progeny of a subject, comprising altering the RNA content of a
sperm from the subject by contacting the sperm with a vesicle
comprising a sRNA and fertilizing an oocyte with the sperm to
produce the progeny.
[0094] In embodiments, the sRNA is selected from the group
consisting of a siRNA, a miRNA, a piRNA, a snoRNA, a srRNA, a
U-RNA, and a tRNA fragment. In further embodiments, the tRNA
fragment is selected from the group consisting of a tRNA-Gly-CCC
fragment, a tRNA-Gly-TCC fragment, a tRNA-Gly-GCC fragment, a
tRNA-Val-CAC fragment, a tRNA-Glu-CTC fragment, a tRNA-Lys-CTT
fragment, and a tRNA-His-GTG fragment. In an embodiment, the
vesicle is an exosome; in yet further embodiments, the exosome is
an epididymosome; the epididymosome can be selected from the group
consisting of caput epididymosome, corpus epididymosome, and cauda
epididymosome. In other embodiments, the vesicle is a seminosome or
a prostasome. In other embodiments, the vesicle is a
microvesicle.
[0095] In some embodiments, prior to contacting the sperm, the
sperm is immature and altering the RNA content increases sperm
maturity. In other embodiments, prior to contacting the sperm, the
sperm is defective and altering the RNA content diminishes at least
one defect. In such embodiments, the defective sperm can comprise a
defect selected from the group consisting of a reduced level of
sRNA, at least one aberrant sRNA, or absence of at least one sRNA
that is present in healthy mature sperm. In an embodiment, the
defective sperm comprises a defect in siRNA, miRNA, piRNA, snoRNA,
srRNA, U-RNA, and tRNA fragment content. In further embodiments,
the tRNA fragment content comprises a defect selected from the
group consisting of tRNA-Gly-CCC fragment content, tRNA-Gly-TCC
fragment content, tRNA-Gly-GCC fragment content, tRNA-Val-CAC
fragment content, tRNA-Glu-CTC fragment content, tRNA-Lys-CTT
fragment content, and tRNA-His-GTG fragment content. In other
embodiments, the defective sperm comprises a decrease in at least
one let-7 species of RNA when compared to a healthy sperm.
[0096] In embodiments, after altering the RNA content, the sperm
fertilizes an oocyte.
[0097] In an embodiment, the subject is a mammal, such as a
primate, such as a human.
[0098] In embodiments, the sperm that is altered is obtained from
the subject's caput epididymis, corpus epididymis, cauda
epididymis, vas deferens, testis, or ejaculate. In further
embodiments, the sperm is obtained from the subject's caput
epididymis, corpus epididymis, or cauda epididymis using
microscopic or microsurgical epididymal sperm aspiration (MESA) or
percutaneous epididymal sperm aspiration (PESA). In yet other
further embodiments, the sperm is obtained from the subject's
testis using a technique selected from the group consisting of
needle aspiration (TESA), percutaneous or open surgical biopsy
(TESE), multibiopsy TESE, microdissection TESE, site-directed TESE
after fine needle aspiration mapping, and MicroTESE. Such
techniques are routinely used in assisted reproduction.
[0099] In embodiments, the subject which sperm is altered is
experiencing a condition selected from the group consisting of a
stress-related disease or disorder, dietary restriction, and
obesity. In an embodiment, the dietary restriction is protein
deficiency. In another embodiment, the stress-related disease or
disorder is selected from the group consisting of major depressive
disorder, dysthymia, bipolar disorder, generalized anxiety
disorder, a phobia, social anxiety disorder, separation anxiety
disorder, agoraphobia, and panic disorder.
[0100] In embodiments, the vesicle that is contacted to the sperm
is heterologous to the subject. In other embodiments, the vesicle
is autologous to the subject. In yet other embodiments, the vesicle
comprises a heterologous RNA; the heterologous RNA can comprise a
small RNA (sRNA). Such sRNA can be one selected from the group
consisting of a siRNA, miRNA, a piRNA, a snoRNA, a srRNA, a U-RNA,
and a tRNA fragment. In embodiments where the sRNA is a tRNA
fragment, the tRNA fragment can be selected from the group
consisting of a tRNA-Gly-CCC fragment, a tRNA-Gly-TCC fragment, a
tRNA-Gly-GCC fragment, a tRNA-Val-CAC fragment, a tRNA-Glu-CTC
fragment, a tRNA-Lys-CTT fragment, and a tRNA-His-GTG fragment. In
other embodiments, the vesicle comprises autologous RNA. Such an
vesicle can comprise sRNA that can be selected from the group
consisting of a siRNA, miRNA, a piRNA, a snoRNA, a srRNA, a U-RNA,
and a tRNA fragment. In the case where the sRNA is a tRNA fragment,
the tRNA fragment can be selected from the group consisting of a
tRNA-Gly-CCC fragment, a tRNA-Gly-TCC fragment, a tRNA-Gly-GCC
fragment, a tRNA-Val-CAC fragment, a tRNA-Glu-CTC fragment, a
tRNA-Lys-CTT fragment, and a tRNA-His-GTG fragment. In other
embodiments, the vesicle comprises an artificial (synthetic) RNA.
In such vesicles, the sRNA can be selected from the group
consisting of a siRNA, miRNA, a piRNA, a snoRNA, a srRNA, a U-RNA,
and a tRNA fragment. In the case where the sRNA is a tRNA fragment,
then the tRNA fragment can be selected from the group consisting of
a tRNA-Gly-CCC fragment, a tRNA-Gly-TCC fragment, a tRNA-Gly-GCC
fragment, a tRNA-Val-CAC fragment, a tRNA-Glu-CTC fragment, a
tRNA-Lys-CTT fragment, and a tRNA-His-GTG fragment. In other
embodiments, the vesicle comprises a transgene.
[0101] In embodiments, the altered sperm fertilizes an oocyte in
vitro. In other embodiments, the sperm is used in intracytoplasmic
sperm injection (ICSI). These embodiments can further comprise
implanting the fertilized oocyte to a second subject to produce a
progeny.
[0102] In other embodiments, the altered sperm fertilizes an oocyte
in vivo.
[0103] In embodiments, prior to contacting the sperm with a
vesicle, the sperm are frozen.
[0104] Specifically in the second aspect, the epigenetically
inheritable trait is a disease or disorder that is a metabolic or
stress-related disease or disorder. In some embodiments, the
metabolic disease or disorder comprises a glucose or hepatic
metabolic disease or disorder. In further embodiments, the hepatic
metabolic disease or disorder comprises reduced sterol
biosynthesis. In yet further embodiments, the reduced sterol
biosynthesis comprises reduced cholesterol biosynthesis. In even
further embodiments, hepatic Sqle gene expression is upregulated.
In other embodiments, the stress-related disease or disorder is
selected from the group consisting of major depressive disorder,
dysthymia, bipolar disorder, generalized anxiety disorder, a
phobia, social anxiety disorder, separation anxiety disorder,
agoraphobia, and panic disorder.
[0105] In embodiments of this second aspect, the progeny lacks
symptoms of the epigenetically inheritable trait. In other
embodiments, the progeny has ameliorated symptoms of the
epigenetically inheritable trait.
[0106] In yet other embodiments of this second aspect, the subject
is experiencing a condition selected from the group consisting of a
stress-related disease or disorder, dietary restriction, and
obesity. In further embodiments, the dietary restriction is protein
deficiency. In other further embodiments, the stress-related
disease or disorder is selected from the group consisting of major
depressive disorder, dysthymia, bipolar disorder, generalized
anxiety disorder, a phobia, social anxiety disorder, separation
anxiety disorder, agoraphobia, and panic disorder.
[0107] Vesicles
[0108] Vesicles can be used to treat a subject, organ, tissue, or
cell (such as sperm). Vesicles can be used as found in their normal
milieu, such as a tissue fluid. In the case of epididymosomes,
fluid found in the epididymis contains vesicles and can be directly
applied to the target subject, organ, tissue, or cell (such as
sperm).
[0109] However, in preferable embodiments, vesicles are used at
least partially purified. "Purified" means to be substantially free
from other components normally associated with the purification
target in a native environment. Vesicle purification can be
accomplished by many procedures. For example, in the case of
cultured cells, differential ultracentrifugation can be used
(Raposo, G and W Stoorvogel. 2013. J Cell Biol., 200: 373-383). If
cultured cells are used as a source of vesicles, media components,
such as serum (e.g., fetal bovine serum), are depleted of EVs
before applying to the cells so as to not contaminate the cell
vesicle preparation with vesicles of other origins. To separate
vesicles from non-membranous particles (such as protein
aggregates), the relatively low buoyant density and differences in
floatation velocity can be used (Raposo, G, et al. 1996. J Exp
Med., 183: 1161-1172; Escola, J M, et al. 1998. J Biol Chem, 273:
20121-20127; Van Niel, G, et al. 2003. Gut, 52:1690-1697; Wubbolts,
R, et al. 2003. J Biol Chem, 278: 10963-10972; Aalberts, M, et al.
2012. Biol Reprod, 86:82. In some embodiments, vesicles can be
further purified through immunopurification by using a protein of
interest found on the surface of the target vesicle. A method of
purifying vesicles is also set out in the Examples.
[0110] Vesicles can also be isolated using a number of commercially
available kits, such as Total Exosome Isolation (Invitrogen
(ThermoFisher Scientific); Waltham, Mass.), ExoQuick-TC.TM. (System
Biosciences; Palo Alto, Calif.), ME.TM. (New England Peptide;
Gardner, Mass.), miRCURY.TM. (Exiqon; Woburn, Mass.); and
Exo-spin.TM. (Cell Guidance Systems; St. Louis, Mo.),
[0111] In some embodiments, physical methods can be used to produce
nanovesicles that have some of the features of vesicles (Gyorgy, B.
et al. 2015. Annu Rev Pharmacol Toxicol. 55: 439-464). For example,
cells comprising the target molecules to be transferred to the
target subject, organ, tissue, or cell can be extruded through
filters, which fragment the cells and generate vesicles (Jang, S C,
et al. 2013. ACS Nano, 7: 7698-7710). Alternatively, such cells can
be extruded through a microfluidic chamber (Jo, W, et al. 2014. Lab
Chip, 14: 1261-1269). Such formed vesicles are similar to
endogenous EVs in size, shape, and composition and can deliver RNA
molecules (Gyorgy, B. et al. 2015. Annu Rev Pharmacol Toxicol. 55:
439-464). In principle, vesicles can also be formed by sonicating,
lysing, electroporating and freeze-thawing cells (Gyorgy, B. et al.
2015. Annu Rev Pharmacol Toxicol. 55: 439-464).
[0112] In certain embodiments, vesicles (e.g., epididymosomes) of
the invention are between about 50 nm and about 400 nm in diameter,
between about 60 nm and about 350 nm in diameter, between about 70
nm and about 300 nm in diameter, between about 80 nm and about 250
nm in diameter, between about 90 nm and about 150 nm in diameter,
between about 110 nm and about 180 nm in diameter, between about
240 nm and 360 nm in diameter, or any ranges of diameters or
individual diameters between these ranges. In exemplary
embodiments, vesicles (e.g., epididymosomes) of the invention are
approximately 150 nm in diameter.
[0113] In embodiments, vesicles can be loaded with molecular cargo
(such as a sRNA), such as by using electroporation or co-incubation
(Alverez-Erviti, L, et al. 2011. Nat. Biotechnol. 29: 341-345;
El-Andaloussi, S, et al. 2012. Nat Protoc, 7: 2112-2126; Sun, D, et
al. 2010. Mol. Ther. 18:1606-1614; Tian, Y H, et al. 2014.
Biomaterials, 35:2383-2390). In some embodiments, the molecular
cargo includes a transgene. In other embodiments, the molecular
cargo includes an artificial (synthetic RNA). In some embodiments,
the molecular cargo includes an autologous molecule, such as an RNA
molecule (such as a sRNA); in other embodiments, the molecular
cargo includes a heterologous molecule.
[0114] In some embodiments, vesicles can be modified to incorporate
a molecule that targets a tissue or cell-specific molecule; e.g., a
molecule that binds to a sperm-specific membrane molecule, such as
a protein or a lipid.
[0115] In some embodiments, further characterization of the
vesicles may be desired to ensure that the targeted vesicles have
indeed been purified. Methods such as sizing (although both "true"
vesicles as well as MVs can be isolated in a single preparation;
size does not necessarily distinguish true vesicles from MVs),
immunoblotting, mass spectrometry, and imaging techniques can be
used to further characterize isolated vesicles (Raposo, G and W
Stoorvogel. 2013. J Cell Biol., 200: 373-383). Imaging techniques
include conventional transmission electron microscopy, whole mount
transmission electron microscopy, and cryo-electron transmission
electron microscopy. In addition, nanoparticle tracing analysis to
determine size distribution of the isolated vesicles can be
accomplished based on the Brownian motion of vesicles in suspension
(Soo, C Y, et al. 2012. Immunology, 136: 192-197). Furthermore,
individual vesicles can be analyzed using high resolution flow
cytometry methods when the vesicles are immunolabeled (Nolte-'t
Hoen, E N. 2012. Nanomedicine, 8:712-720; van der Vlist, E J, et
al. 2012. Nat Protoc, 7: 1311-1126).
[0116] Sperm Acquisition
[0117] In embodiments, untreated sperm, or untreated immature
sperm, or untreated defective mature sperm ("sperm") are obtained
from a subject, such as from a mammal, such as a primate or human.
A mammalian sperm, which may also be referred to as a "spermatid,"
"spermatozoon" or "spermatozoan," are produced through
spermatogenesis inside the testicle through meiotic division. Sperm
formed in the testis then enter the caput epididymis, progress
through the corpus epididymis region, and finally enter the cauda
epididymis. After exiting the testis, sperm mature by structurally
and functionally reorganizing the sperm membrane, which maturation
results in the acquisition of motility and fertilization
capabilities. However, sperm also lose their ability to synthesize
proteins (Barkalina, N, et al. 2015. Human Reprod Update, 21(5):
627-639). Epididymosomes fuse with sperm to deliver proteins,
including P34H (necessary for fertilization), ADAM-7 (a disintegrin
and metalloproteinase), glioma pathogenesis-related I-like protein;
epididymal sperm binding protein I (ELSPBPI), and plasma membrane
Ca.sup.2+-ATPase (Barkalina, N, et al. 2015. Human Reprod Update,
21(5): 627-639). Fusion not only changes the protein composition of
the sperm, but also its lipid composition (Barkalina, N, et al.
2015. Human Reprod Update, 21(5): 627-639).
[0118] During ejaculation, sperm flow from the cauda epididymis
through the vas deferens prior to entering the ejaculatory duct.
The sperm then pass through the prostate gland, enter the urethra,
and exit the body through the urethral opening in the seminal fluid
(also referred to as the ejaculate). Sperm for use in the disclosed
methods can be retrieved from any point along the reproductive
tract from the testis to the ejaculate, including the subject's
testis, epididymis (including the caput, corpus, or cauda
epididymis), vas deferens, or ejaculate. In the case of in vivo
fertilization, the sperm remain in the ejaculate for fertilization
of the oocyte.
[0119] In one embodiment, sperm can be obtained from a subject's
epididymis (including from the caput, corpus, and cauda epididymis)
using microscopic or microsurgical epididymal sperm aspiration
(MESA) or percutaneous epididymal sperm aspiration (PESA). In
another embodiment, sperm can be obtained from a subject's testis
using a technique selected from the group consisting of needle
aspiration (TESA), percutaneous or open surgical biopsy (TESE),
multibiopsy TESE, microdissection TESE, site-directed TESE after
fine needle aspiration mapping, and MicroTESE. Such techniques are
routinely used in assisted reproduction.
[0120] In one embodiment, sperm can be from the same subject or a
different subject than the source of vesicles for use in the
disclosed methods. In another embodiment, the sperm can be a donor
sperm, such as those available from a sperm bank.
[0121] The isolated, untreated sperm may comprise a condition such
as reduced levels of sRNA, at least one aberrant sRNA, or the
absence of at least one sRNA that is present in healthy sperm. In
other embodiments, there may be increased levels of an sRNA
compared to healthy sperm, or the presence of sRNAs that are not
usually present in healthy sperm. For example, untreated sperm can
have an absence or decrease in at least one sRNA selected from the
group consisting of tRNA-Gly-CCC fragments, tRNA-Gly-TCC fragments,
tRNA-Gly-GCC fragments, tRNA-Lys-CTT fragments, and tRNA-His-GTG
fragments; or aberrant forms of these molecules. A decrease in a
sRNA is one wherein such a decrease has an effect on an offspring,
such as in the case of an epigenetically transmitted condition. In
some embodiments, the sRNA that is absent, decreased, or is
aberrant is a let-7 species of miRNA.
[0122] In some embodiments, the sperm that is obtained is from a
subject that has experienced some form of stress, including mental
stress, dietary restriction, or overeating. For example, the
subject may suffer from a protein deficiency. In other embodiments,
the subject has a disease or disorder that is a metabolic or
stress-related disease or disorder. Such diseases and disorders can
be a hepatic metabolic disease or disorder, which can include
reduced sterol biosynthesis (such as reduced cholesterol
synthesis), or an upregulation or downregulation in hepatic Sqle
gene expression. In other embodiments, the stress-related disease
or disorder is a mental health disease or disorder, such as
depression.
[0123] In some embodiments, the sperm is frozen using
well-established techniques, such as those used by sperm banks, for
later use in the disclosed methods.
[0124] Oocyte Fertilization
[0125] A mammalian "oocyte," which may also be referred to as an
"ovocyte," "immature ovum" or "egg cell" for use in the disclosed
methods is produced through oogenesis by meiotic division.
[0126] An oocyte for use in in vitro fertilization can be retrieved
from a subject by any known method, including aspiration directly
from the ovarian follicles. An oocyte for use in in vivo
fertilization is not retrieved, but fuses with the sperm within the
subject prior to implantation in the uterus.
[0127] An oocyte may be fertilized by any known method, including
in vivo methods and in vitro methods. In certain embodiments, the
method of oocyte fertilization may be in vitro fertilization (IVF).
In one embodiment, the oocyte may be fertilized through
intracytoplasmic sperm injection (ICSI).
[0128] Vesicle Application
[0129] For in vitro application, partially or fully purified
vesicles can be resuspended in a buffered solution, such as
phosphate-buffered saline (PBS) or cell culture media (and if serum
is present, it is EV-depleted). Alternatively, the vesicles can be
formulated into a pharmaceutical composition comprising, for
example, a pharmaceutical excipient or carrier. To contact the
target tissue or cells, for example, the vesicles are applied to
the tissue or cells, and the components incubated for a sufficient
time to permit vesicle fusion and cargo delivery. For example,
solutions comprising vesicles can be incubated with the target
tissue or cells for minutes to hours to days, such as, in minutes,
1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55,
60, 120, 180, and 240; such as in hours, 5, 6, 7, 8, 9, 10, 11, 12,
13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 30, 36, 42, and 48;
such as in days, 3 and 4. One of skill in the art can ascertain the
time to incubate an vesicle-containing composition with the target
tissue or cells, adjusting variables of not only time, but also
variables concerning the applied concentration of vesicles (often
determined by quantifying total protein), temperature (typically
physiological temperatures, but above about 4.degree. C.), volume,
components of buffered solution used for resuspension, and number
of target cells. Vesicles can be applied to the target tissue or
cells multiple times.
[0130] Alternatively, vesicles can be injected into a subject in a
pharmaceutical composition, such as glucose (e.g., 5% glucose)
(Cooper, J M, et al. 2014. Mov Disord., 29(12): 1476-1485).
[0131] Pharmaceutical compositions comprising vesicles, dosage
forms, and dosing, are described in more detail below.
[0132] As defined above, vesicles comprise at least one RNA
molecule. Such RNA molecule can be a sRNA, such as siRNA, miRNA,
piRNA, snoRNA, srRNA, U-RNA, or tRNA fragment (tRF). Such RNA can
be heterologous or autologous. For example, in some embodiments,
the sperm to be treated are immature, whether by virtue of a
biological cause or isolation location, but the vesicles are
derived from cauda epididymis of the same subject (e.g., wherein
the RNA are therefore autologous in the case of isolating immature
sperm based on location), or derived from the cauda epididymis of
another subject (thus comprising heterologous RNA), which may be
desirable to treat immature sperm from healthy subjects as well as
for treating immature sperm from subjects having a biological cause
that results in immature sperm. In other embodiments, heterologous
RNA is introduced into vesicles isolated from the same subject as
the sperm donor. For example, such RNA can be one that is derived
from the epididymis, such as the cauda epididymis.
[0133] Pharmaceutical Compositions
[0134] Pharmaceutical compositions comprising vesicles are
expounded on in part, for example, in US 20160060652.
[0135] Pharmaceutical compositions that contain vesicles useful in
the disclosed methods can comprise a liquid medium. Examples of
liquid media include water, physiologically acceptable buffer
solutions (phosphate-buffered saline, etc.) and biocompatible
aqueous mediums such as propylene glycol and polyoxyethylene
sorbitan fatty acid ester. The media is desirably sterile and
adjusted to be isotonic to blood or other tissue fluid (e.g.,
epididymal) if necessary.
[0136] Pharmaceutical compositions can comprise a pharmaceutically
acceptable carrier. Examples of pharmaceutically acceptable
carriers include suspending agents, tonicity agents, buffers and
preservatives. Carriers can be used to facilitate formulation and
maintaining the dosage form and drug effects.
[0137] For example, glyceryl monostearate, aluminum monostearate,
methylcellulose, carboxymethylcellulose, hydroxymethylcellulose and
sodium lauryl sulfate can be used as suspending agents. Examples of
tonicity agents include sodium chloride, glycerin and D-mannitol.
Examples of buffers include phosphate, acetate, carbonate and
citrate. Examples of preservatives include benzalkonium chloride,
parahydroxybenzoic acid and chlorobutanol.
[0138] If necessary or desired, pharmaceutical compositions can
also comprise a corrigent, a thickener, a solubilizing agent, a pH
adjuster, a diluent, a surfactant, an expander, a stabilizer, an
absorption promoter, a wetting agent, a humectant, an adsorbent, a
coating agent, a colorant, an antioxidant, a flavoring agent, a
sweetener, an excipient, a binder, a disintegrant, a disintegration
inhibitor, a filler, an emulsifier, a flow control additive, or a
lubricant.
[0139] Pharmaceutical compositions useful in the disclosed methods
can also contain an additional drug without losing pharmacological
effects possessed by the vesicles. For example, the pharmaceutical
composition may contain an antibiotic.
[0140] Information directed to suitable formulations and additional
carriers can be found in, for example, Remington "The Science and
Practice of Pharmacy" (20th Ed., Lippincott Williams & Wilkins,
Baltimore Md.), which is incorporated by reference in its entirety
herein.
[0141] In some embodiments, a dosage form may be desired. A dosage
form of the pharmaceutical composition is not limited and can be
any form that does not inactivate the vesicle or its contents. The
dosage form of pharmaceutical compositions can be, for example, a
liquid, solid or semisolid form. Specific examples of dosage form
include parenteral dosage forms such as injections, suspensions,
emulsions, creams, ointments, gels and foams. In some embodiments,
the dosage form is a vaginal gel or foam.
[0142] A "pharmaceutically effective amount" refers to a dose
required for the vesicles contained in pharmaceutical compositions
to prevent, diminish, or treat the target disease or condition, or
alleviate symptoms, in a subject and/or in the subject's offspring
(or in some cases, the offspring's offspring). A specific dose
differs depending on the disease to be prevented, diminished,
and/or treated; the mechanism of action underlying the occurrence
of the disease, the dosage form used, information about a subject
and an administration route, etc. The range of the pharmaceutically
effective amount and a preferred administration route of the
pharmaceutical composition that is administered to a human subject
are generally set on the basis of data obtained from cell culture
assay and animal experiments. The final dose can be determined and
adjusted by the judgment of, for example, a physician. Information
about the subject to be taken into consideration can include the
degree of progression or severity of the disease, general health
conditions, age, body weight, sex, diet, drug sensitivity and
resistance to treatment, etc.
[0143] The pharmaceutical compositions can be administered twice or
more at predetermined intervals of time, for example, every hour, 3
hours, 6 hours or 12 hours; every day, every 2 days, 3 days or 7
days; or every month, 2 months, 3 months, 6 months or 12
months.
[0144] The administration of the pharmaceutical composition can be
systemic administration or local administration, and can be
appropriately selected according to the target organ, tissue, or
cell location. Local administration is preferred for in vivo
treatments because the vesicles can be administered in a sufficient
amount to the site (organ, tissue, or cells) to be effective in
treatment, but have no influence on other tissues. However, if the
vesicles are targeted to a specific organ, tissue, or cell-type
(e.g., by virtue of incorporating in the vesicle a protein or lipid
that binds a specific molecule on the target organ, tissue, or
cell), then systemic administration through, for example,
intravenous injection or the like can be used. Blood flow will
systemically transport the vesicles, which will then contact the
target organ, tissue, or cells.
[0145] In the case of administration by injection, the injection
site may be a site where the vesicle can exert its functions and
attain the purpose of the pharmaceutical composition. Examples of
injection sites include intravenous, intraarterial, intrahepatic,
intramuscular, intraarticular, intramedullary, intraspinal,
intraventricular, percutaneous, subcutaneous, intracutaneous,
intraperitoneal, intranasal, intestinal and sublingual sites. In
one embodiment, direct administration to the epididymis is
preferred.
III. Compositions and Kits
[0146] In yet another aspect, disclosed herein are pharmaceutical
compositions comprising an vesicle comprising a small RNA molecule
(sRNA). In embodiments, the sRNA is selected from the group
consisting of a siRNA, a miRNA, a piRNA, a snoRNA, a srRNA, a
U-RNA, and a tRNA fragment. In those embodiments where the sRNA is
a tRNA fragment, the tRNA fragment can be selected from the group
consisting of a tRNA-Gly-CCC fragment, a tRNA-Gly-TCC fragment, a
tRNA-Gly-GCC fragment, a tRNA-Val-CAC fragment, a tRNA-Glu-CTC
fragment, a tRNA-Lys-CTT fragment, and a tRNA-His-GTG fragment.
[0147] In some embodiments, the pharmaceutical composition is a
vaginal foam or gel.
[0148] In some embodiments, the vesicle is an exosome; in yet other
embodiments, the exosome is an epididymosome. In further
embodiments, the epididymosome is selected from the group
consisting of caput epididymosome, corpus epididymosome, and cauda
epididymosome. In other embodiments, the vesicle is a seminosome or
a prostasome. In other embodiments, the vesicle is a
microvesicle.
[0149] In embodiments of this third aspect, the vesicle comprises a
heterologous RNA. In further embodiments, the heterologous RNA
comprises a small RNA (sRNA). In yet further embodiments, the sRNA
is selected from the group consisting of a siRNA, miRNA, a piRNA, a
snoRNA, a srRNA, a U-RNA, and a tRNA fragment. In those embodiments
where the sRNA is a tRNA fragment, the tRNA fragment can be
selected from the group consisting of a tRNA-Gly-CCC fragment, a
tRNA-Gly-TCC fragment, a tRNA-Gly-GCC fragment, a tRNA-Val-CAC
fragment, a tRNA-Glu-CTC fragment, a tRNA-Lys-CTT fragment, and a
tRNA-His-GTG fragment. In other embodiments, the vesicle comprises
autologous RNA. In such embodiments, the vesicle comprises an sRNA
that can be selected from the group consisting of a siRNA, miRNA, a
piRNA, a snoRNA, a srRNA, a U-RNA, and a tRNA fragment. In those
embodiments where the sRNA is a tRNA fragment, the tRNA fragment
can be selected from the group consisting of a tRNA-Gly-CCC
fragment, a tRNA-Gly-TCC fragment, a tRNA-Gly-GCC fragment, a
tRNA-Val-CAC fragment, a tRNA-Glu-CTC fragment, a tRNA-Lys-CTT
fragment, and a tRNA-His-GTG fragment. In other embodiments, the
vesicle comprises an artificial (synthetic) RNA. In such
embodiments, the vesicle comprises an sRNA that can be selected
from the group consisting of a siRNA, miRNA, a piRNA, a snoRNA, a
srRNA, a U-RNA, and a tRNA fragment. In those embodiments where the
sRNA is a tRNA fragment, the tRNA fragment can be selected from the
group consisting of a tRNA-Gly-CCC fragment, a tRNA-Gly-TCC
fragment, a tRNA-Gly-GCC fragment, a tRNA-Val-CAC fragment, a
tRNA-Glu-CTC fragment, a tRNA-Lys-CTT fragment, and a tRNA-His-GTG
fragment. In an embodiment, the vesicle comprises a transgene.
[0150] Kits
[0151] In some embodiments, components necessary to perform the
methods disclosed herein are included in kits. For example,
vesicles can be formulated into pharmaceutical compositions and
supplied in a vessel for use as a vaginal foam or gel for in vivo
use. In other embodiments, vesicles can be supplied in a vessel
suspended in a buffer or media for in vitro or in vivo use, such as
would be suitable for contacting isolated sperm with the vesicles.
In some embodiments, the vesicles incorporate heterologous
molecular cargo. In some embodiments, this molecular cargo is an
RNA molecule (such as a sRNA) or a transgene.
[0152] Reagents included in kits can be supplied in containers of
any sort such that the life of the different components are
preserved and are not adsorbed or altered by the materials of the
container. For example, sealed glass ampules may contain
lyophilized components (such as vesicles), or buffers that have
been packaged under a neutral, non-reacting gas, such as nitrogen.
Suitable buffers include those that maintain the integrity of the
vesicles over time. Ampules may consist of any suitable material,
such as glass, organic polymers (i.e., polycarbonate, polystyrene,
etc.), ceramic, metal or any other material typically employed to
hold reagents. Other examples of suitable containers include simple
bottles that may be fabricated from similar substances as ampules,
and envelopes that may have foil-lined interiors, such as aluminum
or alloy. Other containers include test tubes, vials, flasks,
bottles, syringes, or the like. Containers may have a sterile
access port, such as a bottle having a stopper that can be pierced
by a hypodermic injection needle. Other containers may have two
compartments that are separated by a readily removable membrane
that upon removal permits the components to mix. Removable
membranes may be glass, plastic, rubber, etc.
[0153] Kits can also be supplied with instructional materials.
Instructions may be printed on paper or other substrate and/or may
be supplied as an electronic-readable medium, such as a floppy
disc, CD-ROM, DVD-ROM, DVD, SD card, videotape, audio tape, etc.
Detailed instructions may not be physically associated with the
kit; instead, a user may be directed to an internet web site
specified by the manufacturer or distributor of the kit, or
supplied as electronic mail.
EXAMPLES
Example 1--Mouse Husbandry
[0154] Mice used in this study were primarily FVB/NJ strains,
obtained from Jackson Laboratories (Bar Harbor, Me.). All animal
care and use procedures were in accordance with guidelines of the
Institutional Animal Care and Use Committee (University of
Massachusetts). Animals were raised on one of two diets--defined
control diet (AIN-93G; Bioserv; Flemington, N.J.) or a Low Protein
diet based on AIN-93g (10% of protein rather than 19%, remaining
mass made up with sucrose)--as previously described (Carone, B R,
et al. 2010. Cell 143: 1084-1096). Because it has been observed
that in natural matings that paternal dietary effects are
substantially less penetrant when using females from our long term
mouse colony, the experiments described herein have been restricted
to the use of female mice whose parents or grandparents had been
obtained from the animal vendor.
Example 2--Epididymosome Isolation
[0155] An adult male mouse (8-12 weeks old) was sacrificed using
double kill method (Isofluorane treatment followed by spinal
dislocation). Next, cauda epididymis was dissected out and placed
in a dish containing 1 ml Whitten's media (100 mM NaCl, 4.7 mM KCl,
1.2 mM KH.sub.2PO.sub.4, 1.2 mM MgSO.sub.4, 5.5 mM Glucose, 1 mM
Pyruvic acid, 4.8 mM Lactic acid (hemicalcium), and HEPES 20 mM)
pre-warmed at 37.degree. C. The epididymides were then gently
squeezed using forceps to isolate the epididymal luminal content.
The dish was then placed in an incubator set at 37.degree. C. with
5% CO.sub.2 for 15 minutes to allow any remaining epididymal
content to release from the tissue. Next, the media containing
epididymal luminal content was transferred to a 1.5 ml tube and
allowed to incubate for an additional 15 minutes. At the end of the
15 minutes, any tissue pieces or non-motile sperm settled down at
the bottom of the tube and all the contents of the tube except for
the bottom approximately 50 .mu.l were transferred to a fresh tube.
Next, the tube was spun in a tabletop centrifuge at 2000.times.g
for 5 minutes to pellet down sperm. The supernatant, which
contained epididymosomes, was then transferred to a fresh tube and
centrifuged at 10000.times.g for 30 minutes at 4.degree. C. to get
rid of any non-sperm cells and cellular debris. Supernatant from
this spin was then transferred to a polycarbonate thick wall tube
(13.times.56 mm, Beckman Coulter (Brea, Calif.), Catalog number
362305) and centrifuged at 120000.times.g for 2 hours at 4.degree.
C. in a table top ultracentrifuge (Beckman Optima TL) using a
TLA100.4 rotor. The pellet from this spin was then washed with 500
.mu.l 1.times.PBS and centrifuged for another 2 hours at
120000.times.g at 4.degree. C. Finally, the pellet containing
epididymosomes was resuspended in 50 .mu.l ice-cold 1.times.PBS,
transferred to a 1.5 ml tube, and flash frozen in liquid
nitrogen.
Example 3--Small RNA Cloning
[0156] Total RNA was combined with an equal volume of Gel Loading
Buffer II (Ambion; ThermoFisher Scientific; Carlsbad, Calif.),
loaded onto a 15% Polyacrylamide with 7M Urea and 1.times.TBE gel,
and run at 15 W in 1.times.TBE until the dye front was at the very
bottom of the gel (approximately 25 minutes for Criterion.TM.
minigels (Bio-Rad; Hercules, Calif.)). After staining with SYBR.TM.
Gold (Life Technologies; Carlsbad, Calif.) for 7 minutes, and
destaining in 1.times.TBE for 7 minutes, gel slices corresponding
to 18-40 nucleotides were then cut from the gel. Gel slices were
then ground using a pipette tip or plastic pestle and mixed with
750 .mu.l of 0.3 M NaCl-TE, pH 7.5 prior to incubation with shaking
on a thermomixer overnight at room temperature. The samples were
then filtered using a 0.4 .mu.m cellulose acetate filter
(Costar.RTM.; Corning; Corning, N.Y.) to remove gel debris. The
eluate was transferred to a new low binding microcentrifuge tube
and 20 .mu.g of glycoblue and 1 volume of isopropanol
(approximately 700 .mu.l) were added. Samples were precipitated for
30 or more minutes at -20.degree. C.
[0157] Size selection of the small RNAs was then followed by the
ligation of a 3' adaptor and then a barcoded 5' adaptor as
described by Gu et al (2009, Mol. Cell 36: 231-244). The libraries
were then converted to DNA using SuperScript III.RTM. reverse
transcriptase (Invitrogen; ThermoFisher Scientific) and amplified
by sequential rounds of PCR, to first add short primer tails and
then longer primer tails, providing the products with the correct
adaptor sequences for deep sequencing. Libraries were then
sequenced by Illumina HiSeq 2000 (Illumina; San Diego; CA) at the
University of Massachusetts Deep Sequencing Core (Worcester,
Mass.).
Example 4--Normalization and Data Analysis
[0158] For each small RNA library, rRNA-mapping reads (which were
highly abundant in testis and epididymis samples, but rare in
epididymosome and sperm samples) were removed. Remaining reads were
mapped to murine tRNAs, to the unique sequences present in the 467
defined pachytene piRNA clusters (Li, X Z, et al. 2013. Mol. Cell
50:67-81), to Repeatmasker (Institute for Systems Biology; Seattle,
Wash.) (tRNA entries from Repeatmasker were deleted to avoid
duplicating tRNA-mapping reads), to miRbase (Kozomara, A and S
Griffiths-Jones, 2011. NAR 39 (Database Issue): D152-D157) and to
Refseq (Pruitt, K C D et al., 2014. NAR 42(1): D756-D763) (using
RSEM (web link: deweylab.github.io/RSEM/) to separate distinct mRNA
isoforms). Non rRNA-mapping reads were normalized to parts per
million mapped reads.
Example 5--ES Cell Culture and Transfection
[0159] E14 Embryonic Stem Cell (ESC) lines were grown in DMEM
(Gibcom.TM.; ThermoFisher Scientific), and transfections were
carried out in in Opti-MEM.TM. (Gibco.TM.; ThermoFisher Scientific)
in 6 well plates (Fazzio, T G, et al. 2008. Cell 134: 162-174),
with 9.5 cm.sup.2 wells of ES cells seeded at a density of
2.3.times.10.sup.5 cells/mL. One ng of antisense LNA containing
oligonucleotides (synthesized by Exiqon; Woburn, Mass.) were
transfected using Lipofectamine.TM. 2000 (Invitrogen, ThermoFisher
Scientific) for 16 hours, then ESCs were allowed to recover for 32
hours. Controls included Lipofectamine.TM. (Fisher Scientific) only
(Mock) and anti-GFP shRNA transfections. RNA extraction was
performed at the end of 48 hours using the standard TRIzol.RTM.
(Ambion, Life Technologies; Carlsbad, Calif.) protocol. RNA
extracted from mouse ES cells was prepared for hybridization on
Mouse GeneChip.RTM. 2.0 ST arrays (Affymetrix; Santa Clara, Calif.)
using the GeneChip.RTM. WT PLUS kit from Affymetrix.
Example 6--In Vitro Fertilization, Embryo Culture, RNA
Microinjection, and Single Embryo RNA-Seq
[0160] In vitro fertilization (IVF) was performed according to Nagy
(Manipulating the Mouse Embryo: A Laboratory Manual (Cold Spring
Harbor Laboratory Press, Cold Spring Harbor, N.Y., ed. 3, 2003)).
FVB/NJ mice were used as egg donors and sperm was isolated from
males fed dietary regimes as previously described. Fertilization
took place in 250 .mu.l of HTF media covered in mineral oil,
pre-gassed in 5% CO.sub.2 at 37.degree. C.
[0161] IVF-derived control zygotes were placed in KSOM medium in 5%
CO.sub.2 5% O.sub.2 incubator for 2 hours after IVF. Embryos were
then washed twice in FHM medium containing 0.1% PVA, and subject to
micromanipulation. Embryos were microinjected with either H3.3-GFP
mRNA (control group) or H3.3-GFP mRNA+tRF-Gly-GCC antisense RNA
(experimental group). RNA concentrations used for microinjections
were: 100 ng/.mu.l for H3.3-GFP and 200 ng/.mu.l for tRF-Gly-GCC
antisense RNA. The sequence of the tRF-Gly-GCC antisense RNA is
5'GCG AGA AUU CUA CCA CUG AAC CAC CAA UGC 3' (SEQ ID NO:1). After
the microinjections, embryos were placed back into culture, and GFP
fluorescence was verified at the 2-cell stage. GFP-positive
injected embryos were cultured until the 4-cell stage, when they
were collected and processed for single-embryo RNA sequencing.
[0162] Single embryo RNA-Seq was carried out using the SMART-Seq
protocol (Ramskold, S, et al. 2012. Nat. Biotechnol. 30: 777-782;
Shalek, A K, et al. 2013. Nature 498: 236-240).
Example 7--Epididymal Delivery of Small RNAs to Immature Sperm
[0163] To interrogate the molecular mechanisms underlying
transmission of paternal dietary information to offspring, the
small (<40 nt) RNA repertoire of mouse sperm were characterized.
Sperm were isolated from the cauda epididymis and subjected to
several wash steps including a detergent wash with epithelial lysis
buffer, yielding preparations that were routinely >99.5% pure as
assessed by microscopy. Sperm RNA was isolated, subjected to size
selection (<40 nt), and small RNAs were characterized by cloning
(with or without "healing" of 3' ends by PNK treatment) and deep
sequencing as previously described (Gu, W. et al. 2013. Cell, 151:
1488-1500). The resulting sequencing libraries show a remarkable
abundance of approximately 28-32 nt tRNA fragments (tRFs) in mature
sperm (approximately 80% of all small RNAs with cloneable 3' ends),
as well as less abundant peaks of 19 nt and 22 nt RNAs (FIG. 1A).
The tRFs in the dataset were derived from the 5' ends of tRNAs (no
evidence was found for persistence of 3' fragments in mature
sperm), and untreated RNAs typically exhibit 2-4 predominant 3'
ends with a series of lower-abundance products potentially deriving
from degradation or alternative cleavage/processing sites (FIGS.
1B-1D). As previously reported (Peng, H et al. 2012. Cell Res.
22:1609-1612), 5' fragments of tRNA-Glu-CTC and tRNA-Gly-GCC were
particularly abundant, although the presently described dataset
demonstrated that levels of 5' tRFs derived from other tRNA-glycine
isoacceptors and from tRNA-valine isoacceptors were comparable to
those of tRF-Gly-GCC. 5' tRFs were also highly abundant in cauda
sperm obtained from B. taurus, revealing that extensive tRNA
cleavage in gametes is conserved among mammals.
Example 8--tRNA Fragments are Abundant in the Epididymis
[0164] This example shows that tRNA fragments are abundant in the
epididymis; the data are presented in FIG. 2. FIG. 2A shows a
Northern blot analysis of total RNA isolated from testis, cauda
epididymis, and caput epididymis, as indicated. Each panel (except
the right-most panel, missing testis samples) shows paired samples
in which each of the three tissues was obtained from the same
animal. For each pair of samples, the left sample was isolated from
a Control animal and right sample was isolated from a littermate
consuming a Low Protein diet. The approximately 55-60 nt band
observed in epididymis samples for tRNA-Gly-GCC and tRNA-Val-CAC
varied somewhat in abundance between samples. This band almost
certainly represents a T loop tRNA cleavage product rather than an
intact tRNA differing in size from the approximately 75 nt tRNA by
virtue of amino acid charging, as (1) it migrates at approximately
55-60 nt, which is too short to be an intact tRNA, (2) this band
was observed both when using RNAs isolated under acidic conditions
and using RNAs isolated under more basic deacylating conditions
(not shown), and (3) it is absent in testis samples. FIG. 2B shows
the quantitation of Northern blot data. For the indicated tRNAs,
levels of the approximately 30 nt tRNA fragment were quantitated
and normalized to 5S RNA abundance. Bars show levels of tRFs in
testis, caput epididymis, and cauda epididymis, normalized to
testis levels. Error bars show s.e.m. FIG. 2C shows pie charts
showing the percentage of small RNAs mapping to the indicated
features, for each tissue. rRNA-mapping reads are excluded. Here,
piRNAs refer to all small RNAs mapping either to Repeatmasker or to
unique piRNA clusters (Li, X Z et al. 2013. Molecular Cell, 50:
67-81).
Example 9--tRNA Processing in the Epididymis
[0165] To explore the biogenesis of tRNA fragments found in sperm,
small RNA sequencing data were generated for 18 testis samples from
10-12 week old males, and published data generated from testes of
animals at varying ages after birth were reanalyzed (Li, X. Z., et
al. 2013. Molecular Cell, 50:67-81). As previously reported (Peng,
H et al. 2012. Cell Res. 22:1609-1612), very few small RNAs from
testis mapped to tRNAs, with <8% of all <40 nt RNAs
(excluding rRNA-mapping reads) mapping to tRNAs (FIG. 3A). Northern
blots against 5' ends of tRNAs confirmed barely detectable levels
of tRNA cleavage products in testes. The spectrum of specific tRFs
also differed between the testis, proximal caput epididymis, and
distal cauda epididymis. Similar results were obtained with small
RNA profiles of various testicular sperm fractions, including
primary spermatocytes, early and late round spermatids, and
testicular spermatozoa (FIG. 4). In all four populations very low
levels of tRNA fragments were observed, raising the question of
where the tRNA fragments present in mature sperm might
originate.
[0166] In a Northern blot analysis of tRNA cleavage, samples of the
epididymis were also included. The epididymis is the convoluted
tubular structure in which sperm undergo post-testicular maturation
over the course of 1-2 weeks, moving from caput to corpus to cauda
epididymis. Curiously, abundant 5' tRFs were identified throughout
the epididymis, but not in testes--for both tRNAs analyzed the
approximately 30 nt 5' tRNA fragment that was previously sequenced
in sperm was observed (Peng, H et al. 2012. Cell Res. 22:1609-1612)
and FIG. 1). Levels of tRF-Gly-GCC were similar in the caput and
cauda epididymis samples, while tRF-Val-CAC was reproducibly more
abundant in the cauda epididymis than in the caput.
[0167] Deep sequencing of small RNAs from caput and cauda
epididymal samples confirmed high levels of 5' tRFs in the
epididymis. The overall tRF abundance increased from approximately
8% of all small RNA reads (excluding rRNA fragments) in testis to
approximately 39% in the caput epididymis to approximately 64% in
the cauda epididymis. Results for tRF-Glu-CTC, tRF-Gly-GCC, and
tRF-Val-CAC were further validated in additional samples by Taqman.
Not only do overall tRF levels increase dramatically more distally
in the male reproductive system, but the spectrum of specific tRFs
differs between testis, caput epididymis, and cauda epididymis
(FIGS. 5A and 5B). For instance, valine tRFs are generally more
abundant distally (cauda>caput), whereas levels of various
glycine isoacceptor tRFs either peak in the caput epididymis or are
high throughout the epididymis (consistent with the described
Northern blotting results).
Example 10--Epididymosomes Carry Abundant tRFs that Match the Cauda
Sperm RNA Repertoire
[0168] The finding of robust tRNA cleavage in the epididymis, but
not in testis, raises the surprising possibility that the abundant
tRFs in cauda sperm might originate from the epididymal epithelium
rather than during testicular spermatogenesis. How might such
trafficking from the epididymis to maturing sperm occur? During
transit through the epididymis sperm gain scores of proteins
(Dacheux, J L and F Dacheux. 2013. Reproduction, 147: R27-R42;
Sullivan, R, et al. 2013. Reproduction, 146: R21-R35) via fusion
with small extracellular vesicles known as epididymosomes
(Sullivan, R. et al. 2007. Asian J. Androl. 9: 483-491; Sullivan,
R, et al. 2013. Reproduction, 146: R21-R35). Because extracellular
vesicles carry functional RNAs in multiple systems (Valadi, H, et
al. 2007. Nat. Cell Biol. 9: 654-659; Regev-Rudzki, N, et al. 2013.
Cell, 153: 1120-1133; Gibbings, D and O Voinnet. 2010. Trends Cell
Biol. 20: 491-501), epididymosomes might be responsible for the
dramatic alterations in the sperm RNA payload that occur during
epididymal transit.
[0169] Epididymosomes were purified from the cauda epididymis of
6-12 week old male mice by differential centrifugation. Purified
epididymosomes were somewhat heterogeneous in size, with a major
size class centered around approximately 150 nm (that occasionally
revealed subpeaks of approximately 120 and approximately 170 nm),
as well as a far less abundant group of approximately 250-350 nm
vesicles (FIGS. 6A and 6B).
[0170] Deep sequencing of small RNA libraries prepared from cauda
epididymosome samples (n=15) revealed several size classes of small
RNAs, including highly abundant (approximately 87% of total reads)
28-34 nt tRNA fragments as well as lower levels of microRNAs and
piRNAs (FIG. 7). RNaseA treatment of epididymosomes prior to RNA
extraction had little effect on tRF abundance (FIG. 6C), consistent
with these RNAs being present in vesicles or otherwise protected,
rather than free in the epididymal lumen. In addition, to ensure
that the vesicles purified from the epididymis were not generated
from maturing sperm, epididymosomes were isolated from male
tdrd1-/- mice (in which spermatogenesis is impaired), confirming
high levels of tRFs in these vesicles (FIG. 6C).
[0171] Small RNAs found in epididymosomes closely mirrored those
found in cauda sperm. For example, the most abundant RNA species in
epididymosomes were 5' fragments of tRNA-Glu-CTC, followed by the
5' ends of tRNA-Val-CAC/AAC and tRNA-Gly-GCC/CCC. Overall, the
entire RNA payload of mature sperm was remarkably well-correlated
(r=0.98) with the RNAs present in purified epididymosomes (FIG. 7).
The small RNAs that were enriched in sperm relative to
epididymosomes included those mapping to repeat elements or unique
piRNA clusters, as well as fragments of mRNAs involved in
spermatogenesis (Prm1, for example). These RNAs thus almost
certainly reflect RNAs gained during spermatogenesis, but the
remaining RNAs, which represented the vast majority of RNAs in
cauda sperm, were found at similar levels in sperm and in
epididymosomes. Epididymosomes carried a highly similar RNA payload
to that found in sperm, suggesting that these vesicles may be
responsible for delivering tRNA fragments and perhaps other small
RNAs to maturing sperm.
Example 11--Vesicles Carrying tRNA Fragments Originate in the
Epididymis
[0172] As fluid flow in the epididymis proceeds from testis through
the epididymis and onwards to the vas deferens, luminal contents of
the cauda epididymis could reflect a mixture of species secreted
from a variety of upstream locations. In order to further
investigate the origin of tRF-containing vesicles in the
reproductive tract, epididymosomes were purified from the caput
epididymis (FIGS. 8A-8E). Purified caput epididymosomes had a
similar size distribution to that of cauda epididymosomes, although
the modal size of cauda epididymosomes was slightly larger than
that of caput epididymosomes, and cauda epididymosomes also
included larger (250-500 nm) particles. Small RNA populations from
caput epididymosomes (n=7) were isolated and subject to deep
sequencing as above. Intriguingly, cauda and caput epididymosomes
differed markedly in the relative abundance of microRNAs versus
tRNA fragments. The relatively low abundance of tRFs in caput
epididymosomes strongly argues for an epididymal origin for the
abundant tRFs in cauda epididymosomes, as any vesicles originating
in the testis should if anything be over-represented in the caput
epididymosome samples relative to the cauda.
[0173] Beyond these bulk changes in the abundance of general
classes of small RNAs, marked differences in the specific RNAs in
each epididymosome population were also observed. While not
particularly abundant overall, mRNA fragments as a class were
comparatively more abundant in caput epididymosomes, with the
greatest enrichment for fragments of mRNAs that are highly
expressed in the caput epididymis (Johnston, D S. 2005. Biol.
Reprod. 73: 404-413), such as Lcn5, Defb12, and Adam28. MicroRNAs
were overall more abundant in caput epididymosomes, with individual
microRNAs varying in their relative enrichment in the two samples.
tRNA fragments varied considerably in relative abundance as well,
with cauda epididymosomes gaining abundant tRF-Val-CAC,
tRF-Val-AAC, and tRF-Gly-CCC while exhibiting relative "loss" of
isoleucine and leucine tRFs. Overall, differences between caput and
cauda epididymosomes in small RNA abundance were moderately
well-correlated to the analogous differences between epididymal
epithelium samples, supporting the hypothesis that epididymosomes
from a given luminal region likely originate in the underlying
epithelium. Together, these observations strongly support a model
in which extracellular vesicles are secreted throughout the male
reproductive tract, with different sections of the tract releasing
different RNA cargos.
Example 12--Epididymosomes Deliver Small RNAs to Sperm
[0174] The strong correlation between the small RNA cargo of
epididymosomes and that of cauda sperm, along with published
evidence that epididymosomes can fuse with sperm and deliver other
macromolecular cargo (Sullivan, R. et al. 2007. Asian J. Androl. 9:
483-491; Sullivan, R, et al. 2013. Reproduction, 146: R21-R35),
suggests that epididymosomes are responsible for shaping the RNA
payload of maturing sperm. In order to isolate mature sperm that
had not yet completed epididymal transit, sperm from the caput
epididymis was purified and subjected them to small RNA-Seq. Caput
sperm (n=10) carried high levels of tRNA fragments indicating that
the dramatic increase in tRNA fragment abundance in sperm relative
to testis occurs either very late during testicular
spermatogenesis, or during the first approximately 3-5 days of
epididymal transit. That said, variation between caput and cauda
sperm samples revealed extensive differences in the abundance of
specific small RNAs. Examining tRNA fragment dynamics in detail,
proximal-distal biases for specific tRFs along the epididymis, and
in epididymosomes, were also reflected in tRF dynamics in maturing
sperm. Ratios of tRFs (as well as other small RNA classes) between
caput and cauda sperm were well-correlated with the caput/cauda
ratios observed in epididymosomes and epididymis (FIG. 5). This
does not result from artefactual contamination of caput sperm
samples with epididymosomes, as sequencing of small RNAs from caput
sperm samples isolated with or without a step of washing with
detergent revealed that this washing protocol easily removed
epididymosome-enriched RNAs.
[0175] In all three sample types analyzed--epididymis,
epididymosomes, and sperm--key tRFs exhibited consistent biases in
their enrichment along the proximal-distal axis of the epididymis.
A small subset of tRFs was generally enriched in the proximal
epididymis, with most leucine and isoleucine isoacceptors generally
being enriched in caput samples of epididymis, epididymosomes, and
sperm. In contrast, a dramatic apparent gain of tRF-Val-AAC/CAC
between caput and cauda samples was observed, which was validated
by Taqman in multiple independent samples. These data support the
hypothesis that fusion of caput sperm with cauda epididymosomes
results in gain of tRF-Val-CAC and other RNAs, but could also be
explained if small RNAs are globally degraded in sperm during
epididymal transit, with tRF-Val-CAC and related species being
resistant to this degradation.
[0176] To determine whether epididymosomes can deliver their RNAs
to caput sperm, caput sperm were stringently purified over Percoll
gradients, incubated them with cauda epididymosomes at 37.degree.
C. for 1 or 2 hours, then pelleted and washed the "reconstituted"
sperm (FIG. 9). Epididymosomal fusion with caput sperm was observed
to be sufficient to deliver both tRF-Val-CAC and multiple other
cauda-enriched tRFs to caput sperm (FIG. 9A), confirming that
tRF-bearing epididymosomes either are capable of fusing with sperm
to deliver their small RNA cargo (Caballero, J N et al. 2013. PLOS
ONES 8: e65364) or adhere to caput sperm strongly enough to resist
removal by several consecutive washing steps. These results were
repeated using the more abundant caput sperm samples obtainable
from the bull B. taurus, with cauda epididymosome fusion with caput
sperm (n=4) resulting in delivery of tRF-Val-CAC and other tRFs to
relatively immature caput sperm (FIG. 9B).
[0177] Taken together, these experiments are most consistent with a
mechanism of RNA biogenesis in mammalian sperm in which tRFs
generated in the epididymis are trafficked to sperm in
epididymosomes.
Example 13--Functions of Sperm tRNA Fragments in Stem Cells and in
Embryos
[0178] Next, potential downstream targets of the small RNAs in
sperm were considered, initially using embryonic stem (ES) cells as
an experimental system amenable to mechanistic analysis. Here, the
function of specific tRFs was interfered with using antisense
LNA-containing oligonucleotides in ES cell culture, and genome-wide
analysis of RNA abundance (using Affymetrix microarrays and
RNA-Seq) was carried out to assay the consequences of tRNA fragment
inhibition. The majority of antisense oligos had no effect on mRNA
abundance, suggesting that the targeted tRFs are not functional in
ES cells, or that they exerted regulatory effects that were not
assayed by mRNA abundance.
[0179] In contrast, interfering with tRF-Gly-GCC function using an
LNA-containing antisense oligonucleotide resulted in dramatic
upregulation of approximately 50 genes, with several genes being
upregulated over 10-fold (FIGS. 10A-10C). Upregulation of these
genes was consistently observed by microarray in seven separate
transfections, and further confirmed in four additional replicates
by RNA-Seq. These genes were unaffected by antisense LNA oligos
directed against the 5' end of tRNA-Ser-GCT, the 5' ends of other
tRNA-Gly isoacceptors, or against the middle or the 3' end of
tRNA-Gly-GCC (FIG. 10B). This last finding strongly suggests that
changes in gene expression caused by interfering with the 5'
fragment of tRNA-Gly-GCC are unlikely to be an artifact of
interfering with the function of the intact tRNA. Surprisingly, all
the genes upregulated in tRF-Gly-GCC knockdowns are highly
expressed in 2-cell and 4-cell embryos, and have been shown to be
regulated by the long terminal repeat (LTR) of an endogenous
retroelement known as MERVL (Macfarlan, T S, et al. 2011. Genes
Dev. 25:594-607; Macfarlan, T S, et al. 2012. Nature 487: 57-63)
(FIG. 10D). Transfection studies using ES cell lines carrying
fluorescent reporters driven by the LTR of MERVL revealed a modest
increase (approximately 25-40%) in the fraction of "MERVL positive"
cells upon tRF-Gly-GCC inhibition, independently confirming the
link between tRF-Gly-GCC and the MERVL LTR.
[0180] To determine whether the effects of tRF-Gly-GCC inhibition
observed in tissue culture also hold in a more physiological
context, zygotes (n=27) were microinjected with an antisense oligo
directed against tRF-Gly-GCC. These embryos were then allowed to
develop to the 4-cell stage and subjected to single embryo RNA-Seq
(Ramskold, D., et al. 2012. Nat. Biotechnol. 30: 777-782; Shalek, A
K et al. 2013. Nature, 498: 236-240). Strikingly, significant
upregulation of 72 transcripts in embryos subject to tRF-Gly-GCC
inhibition was observed compared to control embryos (n=28), with
the majority of upregulated genes having previously been identified
as MERVL targets (Macfarlan, T S, et al. 2012. Nature 487: 57-63)
(FIGS. 10E and 10F).
Example 14--Paternal Dietary Effects on Preimplantation
Development
[0181] This example shows that paternal diet effects
preimplantation development. The data are shown in FIGS. 11 and 12.
Given the robust connection between a diet-regulated small RNA and
a highly specific set of target genes, could tRF-Gly-GCC targets be
affected in preimplantation embryos generated using sperm from
animals consuming Control or Low Protein diet? Single-embryo
RNA-Seq (Ramskold, D., et al. 2012. Nat. Biotechnol., 30: 777-782;
Shalek, A K, et al. 2013. Nature, 498: 236-240) of individual
embryos cultured to various stages of development robustly
clustered embryos by developmental stage (FIGS. 11A-B), with the
first two principal components of the dataset representing
oocyte-derived transcripts (PC1), and embryonic genome activation
(PC2).
[0182] As single embryo RNA-Seq data are not suitable for
identification of modest changes in individual mRNAs, consistent
changes in larger genesets were searched: the subset of MERVL
targets that respond to tRF-Gly-GCC inhibition (FIG. 10) and
remaining MERVL targets (Macfarlan, T S, et al. 2012. Nature, 487:
57-63). At the 2-cell stage both tRF-Gly-GCC targets and remaining
MERVL targets were downregulated in Low Protein embryos relative to
Control (FIG. 11C), consistent with the hypothesis that tRF-Gly-GCC
in sperm affects expression of MERVL targets in early embryos.
Several independent tests of this hypothesis were carried out.
First, control zygotes were injected with <40 nt RNA populations
purified from Control and Low Protein sperm, finding that Low
Protein RNAs could inhibit tRF-Gly-GCC targets in 2-cell embryos
(FIG. 11D) indicating that paternal diet can affect preimplantation
gene regulation via RNAs in sperm. Second, further defining the
relevant RNA from Low Protein sperm, microinjection of a synthetic
tRF-Gly-GCC oligo resulted in repression of MERVL target genes in
2-cell embryos (FIG. 11E). Finally, as tRFs in sperm are gained
during epididymal transit, embryos were generated via
intracytoplasmic sperm injection (ICSI) using testicular
spermatozoa or cauda sperm. Consistent with the higher levels of
tRF-Gly-GCC in cauda sperm, embryos generated using cauda sperm
expressed MERVL targets at lower levels than embryos generated
using testicular sperm (FIG. 11F). Together, these findings all
support the hypothesis that tRF-Gly-GCC in sperm is capable of
delaying or repressing MERVL target expression in 2-cell
embryos.
[0183] Lastly, tRF-Gly-GCC is observed to be one of several
abundant RNAs regulated by Low Protein diet, and MERVL-driven genes
are not the only diet-responsive genes in preimplantation embryos.
Most notably, ribosomal protein genes (RPGs) were downregulated in
Low Protein embryos, and, correspondingly, Low Protein embryos
develop slower than Controls (FIG. 12; discussed below) (Mitchel,
M, et al. 2011. Fertil. Steril., 95: 1349-1353).
[0184] FIGS. 12A-12H show paternal dietary effects on
preimplantation development. FIG. 12A shows subjected cumulative
distribution plot for all genes encoding ribosomal protein genes. X
axis shows the relative expression of these genes in Low Protein
IVF embryos, compared to Control. Grey line shows distribution of
dietary effects on all non-RPG genes, for all four stages. Left
shift at the 2-cell stage shows downregulation of RPGs in Low
Protein 2-cell embryos. FIGS. 12B-E show GSEA plots for various
sets of genes involved in ribosome biogenesis at the indicated
developmental stages. FIG. 12F shows an example image of a
blastocyst stained with DAPI and anti-Cdx2 to image total cell
number and trophectoderm cells. FIG. 12G shows that Low Protein
diet reproducibly alters developmental tempo. FIG. 12H shows
aggregated data for three replicate experiments, showing the number
of blastocysts with the indicated number of cells, for embryos
generated via IVF using Control or Low Protein sperm, as
indicated.
Example 15--Dietary Effects on tRNAs in Testes
[0185] This example shows that when the levels of intact tRNAs are
assayed in the testis, there is no correlation between dietary
effects on testicular tRNA levels and tRF changes in cauda sperm.
The data are shown in FIG. 13. FIG. 13A shows a schematic
illustrating assay for tRNA charging analysis. RNA purified from a
given tissue was isolated under acidic conditions to preserve
charged tRNAs, and subjected to the three treatments shown to
enable deep sequencing characterization of charged, uncharged, and
total tRNA levels. FIG. 13B shows validation of tRNA charging
protocol. Budding yeast grown in the presence (+HIS) or absence
(-HIS) of histidine were subjected to the tRNA analysis shown in
FIG. 13A. Changes in tRNA abundance for charged and uncharged tRNAs
are shown on the y axis, sorted by the change in charged tRNA
abundance. As expected, charged tRNA-His levels dropped
dramatically after two hours of histidine starvation, while levels
of uncharged tRNA-His increased. FIG. 13C shows testicular tRNA
abundance correlation with codon bias in the mouse. The x axis
shows intact tRNA abundance in testis (total tRNA is shown here but
similar results hold for uncharged or charged tRNA datasets) in log
scale, and the y axis shows the corresponding codon abundance (in
codon frequency/1000) in all murine mRNAs, or in the 47 most-highly
expressed mRNAs in testis. Data for testis mRNA abundance is from
Carone et al. (Carone, B R, et al. 2010. Cell 143: 1084-1096). FIG.
13D shows validation of tRNA charging analysis. Scatterplot shows
abundance of approximately 60-80 nt RNAs in the total RNA protocol
(x axis, log scale) compared to abundance of RNAs in the charged
tRNA protocol (y axis, log scale). While tRNA levels are broadly
consistent between the two protocols (charged/uncharged ratios vary
up to approximately 10-fold between individual tRNAs), other RNAs
captured in the total RNA protocol, mostly snoRNAs (some of which
are of similar size to tRNAs), are approximately 20-100 fold less
abundant in the charged tRNA library. FIGS. 13E-G show Low Protein
vs. Control effects on tRNA levels for total (FIG. 13E), uncharged
(FIG. 13F), and charged (FIG. 13G) tRNA levels in testis. FIG. 13H
shows that dietary effects on sperm tRFs are not explained by
effects on intact tRNA abundance in testes. Log ratio between
Control and Low Protein males is shown for total tRNA levels in
testis (x axis) compared to tRNA fragment levels in cauda sperm (y
axis).
Example 16--Consistent Dietary Effects are Observed Throughout the
Reproductive Tract
[0186] This example shows that there are consistent dietary effects
throughout the reproductive tract. The data are presented in FIG.
14. FIG. 14A shows the dietary effects on small RNA abundance in
testes and caput and cauda epididymis samples. Each heatmap shows
log 2 of Low Protein/Control RNA abundance for a pair of samples,
showing RNAs (rows) that exhibit consistent dietary effects across
>75% of samples. FIG. 14B shows the coherent dietary effects on
tRF-Gly and let-7 family members throughout the male reproductive
tract. For each RNA, bars show average and standard error of the
mean for Low Protein effects on the abundance of the RNA species in
the indicated tissue. Changes with a nominal p value of <0.05
(paired t test, not corrected for multiple testing) are indicated
with asterisks.
Example 17--RNA Populations in Caput Sperm
[0187] In this example, the RNA populations in caput sperm are
detailed, showing that the RNA payload of caput sperm differs
substantially from that of cauda sperm. The data are shown in FIG.
15. FIG. 15A shows that unwashed caput sperm are contaminated with
RNAs abundant in caput epididymosomes. Caput sperm were isolated
with and without washing with an epithelial cell lysis buffer. RNA
isolated from unwashed caput sperm included numerous microRNAs that
were most abundant in caput epididymosomes. FIG. 15B shows a
comparison of small RNA payloads of cauda vs. caput sperm for all
RNA species with an abundance of at least 1 ppm in both sperm
populations. These changes in RNA abundance could result from
extant RNAs from caput sperm being degraded during further transit
through the epididymis, or from small RNAs being gained via
processing or trafficking during post-testicular maturation. FIG.
15C shows the proximal-distal biases observed for epididymis (x
axis) were recapitulated in cauda vs. caput sperm samples (y axis).
For clarity only RNAs are shown with at least 50 ppm abundance in
at least one of the four sample types (cauda or caput, sperm or
epididymis) are shown. The correlation coefficient for each RNA
class is shown adjacent to its label. FIG. 15D shows that there is
a gain in all four tRFs from caput to cauda. Data for the four tRFs
indicated was normalized to let-7b, and here the average
cauda/caput difference for each tissue is shown plus/minus the
standard deviation. Similar results were obtained using miR-21 as a
normalized control. In addition, nearly-identical results were
obtained using Taqman assays for the 23, 27, or 29 nt variants of
tRF-Gly-GCC (only data for 27 nt is shown). These data are
consistent either with a general gain of tRFs from caput to cauda
samples of all three tissue types--epididymis, epididymosomes, and
sperm--or loss of let-7 or miR-21. FIG. 15E shows that tRF-Val-CAC
is strongly cauda-enriched. Data from FIG. 15D are shown with
tRF-Val-CAC normalized to tRF-Glu-CTC rather than to microRNAs.
Northern blots performed against the 5'end of tRNA-Gly-GCC for
samples of bull caput sperm and bull cauda sperm, show that caput
sperm carry intact tRNAs (FIG. 15F).
Example 18--Dietary Information is Carried in Sperm
[0188] This example shows that metabolic gene expression is altered
in offspring generated via in vitro fertilization (IVF) using sperm
obtained from animals consuming a control or low-protein diet.
Despite the potential for IVF and embryo culture to obscure
paternal effects on offspring metabolism, it was found that,
compared with control IVF offspring, IVF-derived offspring of males
consuming a low-protein diet exhibited significant hepatic
upregulation of the gene encoding the cholesterol biosynthesis
enzyme squalene epoxidase (Sqle). These results are shown in FIG.
16.
[0189] FIGS. 16A-16C show that dietary information is carried in
sperm. FIG. 16A shows the sperm from males consuming Control or Low
Protein diet which were used to fertilize oocytes gathered from
Control females. Two-cell stage embryos were then implanted into
pseudopregnant females and allowed to develop to birth. At 3 weeks
of age, offspring were sacrificed (n=92 for Control, n=86 for Low
Protein), and livers were harvested for analysis of Sqle, a gene
previously shown to be upregulated in offspring of Low Protein
males relative to Control males Carone et al., Cell 2010; 143:
1084-1096). Sqle levels (normalized to Actb) are shown for all
offspring as individual points, with horizontal lines showing mean
expression. FIG. 12B shows the cumulative distribution of Sqle
expression for all offspring generated using Control or Low Protein
sperm, as indicated.
[0190] FIG. 16C shows consistent litter effects. Here, Sqle levels
were averaged for all offspring of a given litter. As sperm samples
were always obtained from male siblings split to different diets,
litter pairs resulting from paired fathers were compared, with each
dot representing the ratio of Sqle expression between appropriately
paired litters.
Example 19--Mechanistic Basis for tRF-Gly-GCC Regulation of MERVL
Targets
[0191] This example provides data supporting the mechanistic basis
for tRF-Gly-GCC regulation of MERVL targets. The data are shown in
FIG. 17. FIGS. 17A-B show the mechanistic basis for tRF-Gly-GCC
regulation of MERVL. FIG. 17A shows RNA-Seq and ribosome
footprinting data for Sp110. Genome browser view shows aggregated
data for four independent replicate ES cell transfections with
shRNAs targeting GFP, and an antisense oligo targeting tRF-Gly-GCC.
FIG. 17B shows that RNA abundance and ribosome footprinting data
are highly correlated. Scatterplot shows the effect of tRF-Gly-GCC
inhibition, expressed as the log 2 of the median of the four LNA
transfections divided by the median of the eight control replicates
(four mock, four GFP KD). Genes exhibiting a 2-fold difference
between GFP KD and mock, and genes with maximum abundance of <2
FPKM in any individual replicate, were excluded from this
scatterplot.
Example 20--Observations from Examples 1-19
[0192] The results from the previous examples show (1) that effects
of paternal diet on offspring are mediated via information found in
sperm (FIG. 15), (2) that diet alters the level of small RNAs,
including specific tRNA fragments, throughout the male reproductive
tract and in mature sperm (FIGS. 1 and 7), and (3) that tRNA
fragments can regulate expression of transcripts driven by
endogenous retroelements (FIGS. 8-9). The data also uncover the
temporal dynamics of small RNA biogenesis during post-testicular
maturation (FIGS. 2-4), and strongly suggest a role for
epididymosomes in transmitting small RNAs from somatic cells of the
epididymis to maturing gametes.
[0193] A role for epididymosomes in small RNA trafficking to sperm
Perhaps the most surprising hypothesis raised from the results of
these Examples is that epididymosomes deliver a payload of small
RNAs to maturing sperm. The idea that epididymal cells are partly
responsible for the RNA payload of sperm is compelling given the
increasing number of organisms in which gametogenesis involves a
key role for small RNA communication between germ cells and somatic
support cells (Bourc'his, D and O Voinnet. 2010. Science, 330:
617-622). Four observations support the hypothesis. First,
extremely low levels of tRNA fragments were found in the murine
testis, instead observing increasingly abundant tRFs throughout the
epididymis. Moreover, during epididymal transit, levels of a number
of tRFs increase in sperm between the proximal and distal segments.
Second, the small RNA payload of purified epididymosomes is a
remarkable match for the small RNAs found in cauda sperm. In the
very unlikely case that sperm tRNA fragments do not originate in
the epididymis, this observation would then either be an
astonishing coincidence if epididymosomal RNAs serve no regulatory
function, or more likely would hint at potential regulatory roles
of epididymosomal RNAs in lumicrine signaling or signaling to the
female reproductive tract (Bromfield, J J, et al. 2014. Proc. Natl.
Acad. Sci. USA, 111: 2200-2205; Vojitech, L, et al. 2014. Nucleic
Acids Res., 42: 7290-7304). Third, fusion of purified
epididymosomes with caput sperm in vitro delivers tRNA fragments to
the resulting "reconstituted" sperm, demonstrating that the
epididymosomes bearing tRFs either can fuse with caput sperm or
very stably adhere to sperm. Finally, although the major small RNAs
(glycine tRFs and let-7) that respond to diet in mature sperm are
also diet-regulated in the testis (as well as the epididymis),
other diet-responsive small RNAs in sperm only exhibit dietary
responses in epididymis but not in testis.
[0194] Dietary Effects on Small RNAs in Mammalian Sperm
[0195] The key changes in small RNA observed in sperm of animals
raised on Low Protein diet are observed throughout the male
reproductive tract. Generally, at least five levels at which diet
could exert effects on the levels of a given tRF in sperm can be
identified, by influencing: (1) intact tRNA abundance, either via
transcription or stability, (2) tRNA cleavage, regulated
potentially by tRNA charging status or by dietary signaling to
tRNA-modifying enzymes such as Dnmt2 or Nsun2, (3) tRF stability,
(4) tRF sorting into epididymosomes, or (5) sperm fusion with
epididymosomes--this category includes dietary regulation of
fusion-related cell surface proteins, but also mechanisms involving
changes in sperm maturation time or epididymis luminal flow rate
that could affect how long sperm spend in different parts of the
epididymis. At present, dietary effects on tRF processing,
stability, or trafficking appear to be the most likely scenario for
at least a subset of diet-regulated tRFs.
[0196] tRF Regulation of an Endogenous Retroelement
[0197] How might diet-regulated small RNAs in sperm have the
ability to impact the phenotype of offspring? tRF-Gly-GCC was the
focus of these studies thanks to its readily apparent role in
altering mRNA abundance in ES cells--other abundant tRFs such as
tRF-Gly-TCC may play roles in regulation of genes not expressed in
ES cells, or may exert regulatory effects that are not apparent in
mRNA abundance measures (e.g., on translation), and it will be
interesting to determine whether these other abundant tRFs in sperm
have effects on preimplantation development. tRF-Gly-GCC is
extremely unlikely to be uniquely responsible for the effects of
paternal Low Protein diet on offspring cholesterol metabolism, as
let-7 and many other small RNAs change abundance in Low Protein
sperm, and many more genes (such as RPGs) change in preimplantation
embryos fertilized using these sperm than just MERVL target genes
(FIG. 8E). Thus, we feel the likeliest scenario is that paternal
dietary effects are analogous to complex disease genetics, with
multiple separate factors each contributing a fraction of the
quantitative phenotype.
[0198] Inhibition of tRF-Gly-GCC, but not related tRFs, results in
dramatic derepression in both ES cells and in early embryos of a
subset (approximately 50 of approximately 500) of transcripts that
are regulated by dispersed LTRs of the endogenous retroelement
MERVL (Macfarlan, T S, et al. 2011. Genes Dev., 25: 594-607).
Moreover, embryos generated using sperm from Low Protein males
reveal significant changes in MERVL target mRNA abundance (FIG.
10), consistent with the idea that tRFs delivered by sperm could
affect gene regulation in the early embryo. The mechanistic basis
for the observed effects of tRF-Gly-GCC on repression of MERVL LTRs
is of interest--although tRNAs nearly universally act as primers
for retroelement replication, homology-driven tRF regulation of
MERVL is unlikely here as (1) MERVL utilizes tRNA-Leu, not
tRNA-Gly, to prime reverse transcriptase, (2) many of the genes in
our dataset are associated with isolated LTRs and appear to lack
the adjacent tRNA "primer binding sequence", (3) primer binding
sequences for ERVs typically have homology to the 3' end of tRNAs,
not the 5' end, and (4) transfection of the antisense LNA to
tRF-Gly-GCC does not affect levels of tRNA-Leu fragments in ES
cells. Shown in these examples is that regulation of MERVL targets
is unlikely to be a secondary effect of altered translation of
MERVL regulators by tRF-Gly-GCC, while reporter assays show that
removing the MERVL LTR from its genomic context--many of the
tRF-Gly-GCC targets are found in large chromosomal regions with
many MERVL LTRs nearby--does not completely eliminate the ability
of the LTR to respond to tRF-Gly-GCC inhibition.
[0199] The MERVL regulon provides an intriguing connection to
offspring metabolism. MERVL-driven genes are highly expressed in
totipotent early embryos (Kigami, D, et al. 2003. Biol. Reprod.,
68: 651-654), but a small fraction of otherwise pluripotent
embryonic stem cells also express the MERVL program, and MERVL
positive cells are functionally totipotent (Macfarlan, T S, et al.
2012. Nature, 487: 57-63). It is well known that alterations in
placental function (as induced by uterine artery ligation or
caloric restriction) lead to altered cholesterol and glucose
metabolism in offspring (Rando, O J and Simmons, R A. 2015. Cell,
161: 93-105). It is hypothesized that tRF-Gly-GCC regulation of the
MERVL program could alter the tempo of early development, or alter
cell fate allocation in the early embryo. While there was no
significant difference in the percentage of Cdx2-positive cells
between Control and Low Protein embryos (73+/-5% vs. 71+/-7%), Low
Protein embryos consistently exhibited delayed growth relative to
Control embryos. Interestingly, altered growth kinetics in early
embryogenesis have been shown to occur in response to paternal
obesity, which also has been linked to offspring metabolism
(McPherson, N O, et al. 2013. PLoS One, 8(8)e71459).
[0200] Future studies will shed further light on the role of the
epididymis in sensing environmental conditions, on the mechanistic
basis for regulation of RNA levels in sperm, and on effects of tRNA
fragments on preimplantation development and placentation.
Example 21--Caput Epididymosomes Deliver Small RNAs to Testicular
Spermatozoa
[0201] The Examples described herein demonstrate that testicular
spermatozoa have scarce levels of tRFs, and caput sperm are highly
abundant in these small RNAs. Epididymosomes secreted by the
epithelium of cauda epididymis have been found to have similar RNA
payload as that of the mature sperm and can deliver small RNAs to
the relatively "immature" caput sperm (see, e.g., Sharma et al.,
Science 2016; 351(6271): 391-396).
[0202] To test whether tRFs and other small RNAs are delivered to
testicular sperm upon entry into epididymis via fusion with
epididymosomes present in the caput epididymis, testicular
spermatozoa were reconstituted by fusing them with caput
epididymosomes (FIG. 18A). Testicular spermatozoa were incubated
with caput epididymosomes for two hours and then purified by
multiple washes to isolate "reconstituted" spermatozoa. Next, the
levels of specific small RNAs in reconstituted spermatozoa were
examined using TaqMan qRT-PCR assays.
[0203] It was determined that tRFs, such as tRF-Glu-CTC and
tRF-Val-CAC, which are highly abundant in caput epididymosomes,
were up-regulated more than 2-fold in reconstituted spermatozoa
compared to the mock fusions (FIG. 18B). Deep sequencing of small
RNAs from reconstituted spermatozoa revealed consistent results.
Higher levels of tRFs and miRNAs were observed in reconstituted
spermatozoa compared to mock controls (FIG. 18A, 18C-18D).
[0204] Several lines of evidence prove that the small RNA content
of reconstituted sperm is altered due to epididymosome
fusion/delivery of small RNAs: 1) as piRNAs are not expressed in
epididymis, there are scant levels of piRNAs in epididymosomes
(Sharma et al., Science 2016; 351(6271): 391-396), and no change in
the levels of piRNAs was detected in reconstituted spermatozoa
(piRNAs are all on the diagonal axis of the scatter plot in FIG.
18D); 2) an increase in the levels of small RNA was the primary
population of RNA detected, supporting that RNA is delivered to
sperm; and 3) reconstituted sperm showed an increase in miRNAs and
tRFs that were specifically highly abundant in epididymosomes, such
as, e.g., miR-10a/b, miR-148, miR-143, tRF-ValCAC, tRF-GluCTC,
tRF-GlyGCC and tRF-HisGTG.
[0205] The reconstitution of testicular sperm recapitulated
testicular sperm to caput sperm maturation step in vitro. For
instance, it was determined that the reconstituted sperm showed 10%
higher levels of tRFs compared to testicular spermatozoa (FIG.
18A). In addition, specific small RNA changes were also very well
recapitulated. Reconstituted sperm were found to have a higher
abundance of caput sperm-enriched microRNAs such as miR-10a/b,
miR-143, miR-141, and miR-200a. As such, caput epididymosomes were
capable of fusing with mature testicular spermatozoa to deliver
their small-RNA cargo. Without intending to be bound by scientific
theory, taken together these experiments are most consistent with a
mechanism of RNA biogenesis in mammalian sperm in which small RNAs
generated in the epididymis are trafficked to sperm in
epididymosomes.
Example 22--Analysis of Gene Regulation Effects in Embryos Made Via
ICSI Using Caput Sperm, and Cauda Sperm
[0206] To determine the effects of epididymal maturation on
phenotype in the following generation, experiments are carried out
in which zygotes are generated via intracytoplasmic sperm injection
(ICSI) using sperm obtained from the caput epididymis or from the
cauda epididymis. Such zygotes are then allowed to develop into
2-cell embryos, to blastocysts, or are implanted into females and
carried to term. Gene regulation is studied in the preimplantation
embryos, and metabolic traits are measured in grown offspring, to
identify the consequences of using immature sperm to fertilize
oocytes.
Example 23--Microinjection of Other Small RNAs into Zygotes; Early
Gene Regulation and Metabolic Sequelae Assays
[0207] To determine the effects of specific small RNAs on phenotype
in the following generation, experiments are carried out in which
control zygotes are injected with specific small RNAs, such as
tRF-Val-CAC, and allowed to develop into 2-cell embryos, to
blastocysts, or are implanted into females and carried to term.
Gene regulation is studied in the preimplantation embryos, and
metabolic traits are measured in grown offspring, to identify the
functions of specific small RNAs in early development and future
health.
[0208] The invention is not to be limited in scope by the specific
embodiments and examples described herein. Indeed, various
modifications of the invention in addition to those described will
become apparent to those skilled in the art from the foregoing
description and accompanying figures. Such modifications are
intended to fall within the scope of the appended claims.
[0209] All references (e.g., publications or patents or patent
applications) cited herein are incorporated herein by reference in
their entireties and for all purposes to the same extent as if each
individual reference (e.g., publication or patent or patent
application) was specifically and individually indicated to be
incorporated by reference in its entirety for all purposes. Other
embodiments are within the following claims.
Sequence CWU 1
1
1130RNAMus musculus 1gcgagaauuc uaccacugaa ccaccaaugc 30
* * * * *