U.S. patent application number 17/032233 was filed with the patent office on 2021-12-23 for trans-activated functional rna by strand displacement and uses thereof.
This patent application is currently assigned to Massachusetts Institute of Technology. The applicant listed for this patent is Massachusetts Institute of Technology. Invention is credited to Giulio Alighieri, Ron Weiss.
Application Number | 20210395732 17/032233 |
Document ID | / |
Family ID | 1000006010506 |
Filed Date | 2021-12-23 |
United States Patent
Application |
20210395732 |
Kind Code |
A9 |
Weiss; Ron ; et al. |
December 23, 2021 |
TRANS-ACTIVATED FUNCTIONAL RNA BY STRAND DISPLACEMENT AND USES
THEREOF
Abstract
The present disclosure, at least in part, relates to an
engineered RNA (e.g., microRNA and sgRNA), in the absence of an
input signal, that is engineered to have a large enough energy gap
between the formations of a first secondary structure, which is
unrecognizable by an actuator, and a second secondary structure,
which is recognizable by an actuator (e.g., Drosha and Cas
protein).
Inventors: |
Weiss; Ron; (Newton, MA)
; Alighieri; Giulio; (Cambridge, MA) |
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Applicant: |
Name |
City |
State |
Country |
Type |
Massachusetts Institute of Technology |
Cambridge |
MA |
US |
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Assignee: |
Massachusetts Institute of
Technology
Cambridge
MA
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Prior
Publication: |
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Document Identifier |
Publication Date |
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US 20210095286 A1 |
April 1, 2021 |
|
|
Family ID: |
1000006010506 |
Appl. No.: |
17/032233 |
Filed: |
September 25, 2020 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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62906248 |
Sep 26, 2019 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12N 9/22 20130101; C12N
2310/20 20170501; C12N 2310/122 20130101; A61K 31/7105 20130101;
C12N 15/113 20130101; C12N 2310/141 20130101 |
International
Class: |
C12N 15/113 20060101
C12N015/113; C12N 9/22 20060101 C12N009/22; A61K 31/7105 20060101
A61K031/7105 |
Goverment Interests
FEDERALLY SPONSORED RESEARCH
[0002] This invention was made with Government support under Grant
No. R01 CA207029 awarded by the National Institutes of Health. The
Government has certain rights in the invention.
Claims
1. An engineered RNA comprising, (i) an effector portion; and (ii)
a responder sequence, wherein the effector portion comprises a
coding sequence for a functional RNA, wherein, in the absence of an
input signal, the engineered RNA forms a first secondary structure
in which the engineered RNA is not capable of being recognized by
an actuator; and wherein, in the presence of the input signal, the
responder sequence is capable of responding to the input signal
such that the engineered RNA forms a second secondary structure,
not at its lowest energy state, in which the engineered RNA is
capable of being recognized by the actuator.
2. The engineered RNA of claim 1, wherein the effector portion
comprises the coding sequence for a pre-microRNA (pre-miRNA).
3. The engineered RNA of claim 2, comprising parts T-d-f-e-b-S-a-c,
wherein the coding sequence for a pre-miRNA comprises parts b-S-a;
wherein the responder sequence comprises parts T-d-f-e; wherein the
actuator is Drosha; wherein, in the absence of an input signal
which comprises an input RNA that is completely or partially
complementary to parts T-d-f, the engineered RNA forms a first
secondary structure in which part d completely or partially
hybridizes to part b, part e completely or partially hybridizes to
part f, part a partially hybridizes to part c, and parts a and b
are incapable of hybridizing with each other such that the
engineered RNA is not capable of being recognized by Drosha;
wherein, in the presence of the input RNA that is completely or
partially complementary to parts T-d-f, the engineered RNA forms a
secondary structure in which parts T-d-f form a double strand with
the input RNA, thereby releasing part e from part f and part b from
part d, and in which part a partially hybridizes to part b to form
a Drosha recognizable cleavage site not at its lowest energy
state.
4. The engineered RNA of claim 2, comprising parts T-f-d-c-a-S-b-e,
wherein the coding sequence for a pre-miRNA comprises parts b-S-a;
wherein the responder sequence comprises parts T-f-d and e; wherein
the actuator is Drosha; wherein, in the absence of an input signal
which comprises an input RNA that is completely or partially
complementary to parts T-f-d, the engineered RNA forms a first
secondary structure in which part d completely or partially
hybridizes to part b, part e completely or partially hybridizes to
part f, part a partially hybridizes to part c, and parts a and b
are incapable of hybridizing with each other such that the
engineered RNA is not capable of being recognized by Drosha;
wherein, in the presence of the input RNA that is completely or
partially complementary to parts T-f-d, the engineered RNA forms a
secondary structure in which parts T-f-d form a double strand with
the input RNA, thereby releasing part e from part f and part b from
part d, and in which part a partially hybridizes to part b to form
a Drosha recognizable cleavage site not at its lowest energy
state.
5. The engineered RNA of claim 2, comprising parts 5'
hairpin-toehold-antisense-ribozyme-stem-seed-sense-3' hairpin,
wherein the coding sequence for a pre-miRNA comprises parts
stem-seed-sense; wherein the responder sequence comprises parts
toehold-antisense-ribozyme; wherein the actuator is Drosha;
wherein, in the absence of an input signal which comprises an input
RNA that is completely or partially complementary to parts
toehold-antisense-ribozyme, the engineered RNA forms a first
secondary structure in which part 5' hairpin completely or
partially hybridizes to itself, part antisense completely or
partially hybridizes to part sense, part ribozyme completely or
partially hybridizes to itself, part stem completely or partially
hybridizes to itself, part seed completely or partially hybridizes
to itself, part 3' hairpin completely or partially hybridizes to
itself, and part stem is incapable of hybridizing to part sense,
such that the engineered RNA is not capable of being recognized by
Drosha; wherein, in the presence of the input RNA that is
completely or partially complementary to parts
toehold-sense-ribozyme, the engineered RNA forms a secondary
structure in which parts toehold-antisense-ribozyme hybridize with
the input RNA, resulting in ribozyme-mediated cleavage that
releases an RNA waste product comprising the input RNA hybridized
to parts 5' hairpin-toehold-sense and a portion of part ribozyme of
the engineered RNA, wherein, following the release of the RNA waste
product, the remaining portion of the engineered RNA forms a
secondary structure in which part stem partially or completely
hybridizes to part sense to form a Drosha recognizable cleavage
site not at its lowest energy state.
6. The engineered RNA of claim 2, wherein miRNA is therapeutic
miRNAs selected from the group consisting of miR-16, miR-29,
miR-34, miR-143, miR-145, and miR-200 family.
7. The engineered RNA of claim 1, wherein the effector portion
comprises the coding sequence for a single guide RNA (sgRNA).
8. The engineered RNA of claim 7, wherein the engineered RNA is an
engineered sgRNA comprising: parts S-g-a-c-T-d-f-e-b-h wherein the
coding sequence for sgRNA comprises part S-g-a and b-h, wherein the
responder sequence comprises parts c-T-d-f-e, wherein the actuator
is a Cas protein, wherein, in the absence of an input signal which
comprises an input RNA that is completely or partially
complementary to parts T-d-f, the engineered RNA forms a first
secondary structure in which part d partially hybridizes to part b,
part e completely or partially hybridizes to part f, part a
completely or partially hybridizes to part c, part g hybridizes to
part h, and parts a and b are incapable of hybridizing with each
other; wherein, in the presence of the input RNA that is completely
or partially complementary to parts T-d-f, the engineered RNA forms
a second secondary structure in which parts T-d-f form a double
strand with the input RNA, thereby releasing part e from part f and
part b from part d, and in which part a partially hybridizes to
part b to form a Cas protein binding site not in its lowest energy
state.
9. The engineered RNA of claim 7, wherein the engineered RNA is an
engineered sgRNA comprising: parts S-g-b-e-f-d-T-c-a-h; wherein the
coding sequence for sgRNA comprises part S-g-b and a-h; wherein the
responder sequence comprises parts e-f-d-T-c, wherein the actuator
is a Cas protein, wherein, in the absence of an input signal which
comprises an input RNA that is completely or partially
complementary to parts T-d-f, the engineered RNA forms a first
secondary structure in which part d completely or partially
hybridizes to part b, part e completely or partially hybridizes to
part f, part a completely or partially hybridizes to part c, part g
hybridizes to part h, and parts a and b are incapable of
hybridizing with each other; wherein, in the presence of the input
RNA that is completely or partially complementary to parts T-d-f,
the engineered RNA forms a second secondary structure in which
parts T-d-f form a double strand with the input RNA, thereby
releasing part e from part f and part b from part d, and in which
part a partially hybridizes to part b to form a Cas protein binding
site not at its lowest energy state.
10. The engineered RNA of claim 7, further comprising a nexus and
hairpins.
11. The engineered RNA of claim 7, wherein the Cas protein is: a)
selected from a group consisting of Cas9, saCas9, CjCas9, xCas9,
Cas13a/C2c2, Cas13b, Cpf1 and variants thereof; or b) a Cas9 fusion
protein selected from a group consisting of dCas9-transcription
factor, dCas9-VP64, dCas9-VPR, dCas9-Suntag, dCas9-P300,
dCas9-VP160, dCas9VP192, dCas9-KRAB and its derivatives,
dCas9-MXI1, dCas9-SID4X, dCas9-LSD1, dCas9-CIB1, dCas9-GFP, and
dCas9-RFP.
12. (canceled)
13. An engineered nucleic acid, comprising a promoter operably
linked to a nucleotide sequence encoding the engineered RNA of
claim 1.
14. A recombinant virus, optionally a recombinant AAV (rAAV),
lentivirus, adenovirus, or bacteriophage, comprising: a viral
capsid containing a promoter operably linked to a nucleotide
sequence encoding the engineered RNA of claim 1.
15-16. (canceled)
17. A host cell, comprising the engineered RNA of claim 1,
optionally wherein the host cell is: a) a prokaryotic cell,
optionally a bacterial cell; b) a eukaryotic cell, optionally a
fungal cell, plant cell, insect cell, or mammalian cell, optionally
a human cell; c) a diseased cell; and/or d) from a specific
tissue.
18-25. (canceled)
25. The host cell of claim 17, wherein the host cell comprises the
input signal, optionally wherein the host cell is capable of
processing the engineered RNA to produce the functional RNA.
26. (canceled)
27. A pharmaceutical composition, comprising the engineered RNA of
claim 1, optionally further comprising a pharmaceutically
acceptable carrier.
28. (canceled)
29. A method comprising delivering the engineered RNA of claim 1 to
a subject, optionally a human or non-human mammal, in need
thereof.
30. A method for delivering a functional RNA to a cell in a
subject, optionally a human or non-human mammal, in need thereof,
comprising administering to the subject an effective amount of the
engineered RNA of claim 1.
31. A method for treating a disease in a subject, optionally a
human or non-human mammal, in need thereof, comprising
administering to the subject an effective amount of the engineered
RNA of claim 1.
32. (canceled)
33. The method of claim 31, wherein a) the subject has or is at
risk of having Sickle Cell Disease, X-linked severe combined
immunodeficiency (SCID-X1), Hurler Syndrome, Gaucher Disease,
Wiskot-Aldrich syndrome, human immunodeficiency virus (HIV),
Hepatitis B, human papillomavirus (HPV), Herpesviruses, Cystic
Fibrosis, B-thalassemia, Retinitis Pigmentosa, amyotrophic lateral
sclerosis (ALS), BEST disease, Parkinson's Disease, Schizophrenia,
or severe combined immunodeficiency (SCID); b) the engineered RNA
is an engineered sgRNA, further comprising delivering Cas protein
prior to or concurrently with the engineered RNA; c) the Cas
protein is saCas9, and the saCas9 is delivered by a rAAV comprising
an rAAV capsid enclosing a promoter operably linked to a saCas9
coding sequence, the promoter and the saCas9 coding sequence being
flanked by AAV ITRs; and/or d) the Cas protein is not saCas9, and
the Cas protein is delivered by a first rAAV comprising an rAAV
capsid enclosing a promoter operably linked to a first portion of
saCas9 coding sequence, the promoter and the first portion of the
saCas9 coding sequence being flanked by AAV ITRs; and a second rAAV
comprising an rAAV capsid enclosing a promoter operably linked to a
second portion of saCas9 coding sequence, the promoter and the
second portion of the saCas9 coding sequence being flanked by AAV
ITRs.
34-36. (canceled)
Description
RELATED APPLICATION
[0001] This application claims the benefit under 35 U.S.C. .sctn.
119(e) of U.S. provisional application No. 62/906,248 filed Sep.
26, 2019, which is incorporated by reference herein in its
entirety.
BACKGROUND
[0003] The current engineered RNA based technologies designed to
respond to an input signal may be activated in the absence of the
input signal. This side effect poses an issue to RNA based gene
therapy where the activation of the engineered RNA needs to be
tightly controlled.
SUMMARY
[0004] The present disclosure, at least in part, relates to an
engineered RNA (e.g., microRNA and sgRNA), in the absence of an
input signal, that is engineered to have a large enough energy gap
between the formations of a first secondary structure at its lowest
energy state, which is unrecognizable by an actuator, and a second
secondary structure, which is recognizable by an actuator (e.g.,
Drosha and Cas protein). Such design provides the benefit of
decreasing unwanted activation of the engineered RNA when the input
signal is absent. When the input signal is present, it induces a
conformational change of the engineered RNA molecule, such that the
engineered RNA forms the second secondary structure not at its
lowest energy state, which is recognizable by an actuator.
[0005] The present disclosure relates to engineered RNAs that are
designed so that they can interact with an actuator only in the
presence of an input signal, and are thus more specific in exerting
their activity. An engineered RNA that is similar to those
described in the present disclosure but is less specific, and more
likely to refold into a conformation that could interact with an
actuator even in the absence of an input signal, could exert many
deleterious effects if its activity targets a critical host gene or
mRNA. The consequences of these off-target effects are a major
limitation in the development of new gene therapies, and so current
therapies are restricted to targeting certain mRNAs, such as viral
RNAs in virally infected cells, to minimize off-target effects and
maximize safety.
[0006] The engineered RNAs described in the present disclosure are
specific, being unlikely to exert any activity in the absence of
input signal. A major benefit of this increased specificity is that
if they reliably exert activity only in cells containing the input
signal, that activity can be directed towards the most effective
target, even if that target is a gene that is essential for
cellular replication. In the treatment of virally infected cells,
for example, engineered RNAs such as the ones described in the
present disclosure are not limited to targeting viral mRNAs, but
may also target genes or mRNAs encoding host factors that are
essential for viral replication. This increased specificity allows
the engineered RNAs described in the present disclosure to target
more genes or mRNAs, improving their therapeutic efficacy without
compromising safety.
[0007] In some aspects, the present disclosure provides an
engineered RNA comprising: (i) an effector portion; and (ii) a
responder sequence, wherein the effector portion comprises a coding
sequence for a functional RNA, wherein, in the absence of a input
signal, the engineered RNA forms a first secondary structure in
which the engineered RNA is not capable of being recognized by an
actuator; and wherein, in the presence of the input signal, the
responder sequence is capable of responding to the input signal
such that the engineered RNA forms a second secondary structure,
not at its lowest energy state, in which the engineered RNA is
capable of being recognized by the actuator.
[0008] In some embodiments, the effector portion comprises the
coding sequence for a pre-microRNA (pre-miRNA).
[0009] In some embodiments, the engineered RNA comprises parts
T-d-f-e-b-S-a-c, wherein the coding sequence for a pre-miRNA
comprises parts b-S-a; wherein the responder sequence comprises
parts T-d-f-e; wherein the actuator is Drosha; wherein, in the
absence of an input signal which comprises an input RNA that is
completely or partially complementary to parts T-d-f, the
engineered RNA forms a first secondary structure in which part d
completely or partially hybridizes to part b, part e completely or
partially hybridizes to part f, part a partially hybridizes to part
c, and parts a and b are incapable of hybridizing with each other
such that the engineered RNA is not capable of being recognized by
Drosha; wherein, in the presence of the input RNA that is
completely or partially complementary to parts T-d-f, the
engineered RNA forms a secondary structure in which parts T-d-f
form a double strand with the input RNA, thereby releasing part e
from part f and part b from part d, and in which part a partially
hybridizes to part b to form a Drosha recognizable cleavage site
not at its lowest energy state.
[0010] In some embodiments, the engineered RNA comprises, parts
T-f-d-c-a-S-b-e, wherein the coding sequence for a pre-miRNA
comprises parts b-S-a; wherein the responder sequence comprises
parts T-f-d and e; wherein the actuator is Drosha; wherein, in the
absence of an input signal which comprises an input RNA that is
completely or partially complementary to parts T-f-d, the
engineered RNA forms a first secondary structure in which part d
completely or partially hybridizes to part b, part e completely or
partially hybridizes to part f, part a partially hybridizes to part
c, and parts a and b are incapable of hybridizing with each other
such that the engineered RNA is not capable of being recognized by
Drosha; wherein, in the presence of the input RNA that is
completely or partially complementary to parts T-f-d, the
engineered RNA forms a secondary structure in which parts T-f-d
form a double strand with the input RNA, thereby releasing part e
from part f and part b from part d, and in which part a partially
hybridizes to part b to form a Drosha recognizable cleavage site
not at its lowest energy state.
[0011] In some embodiments, the engineered RNA comprises parts 5'
hairpin-toehold-antisense-ribozyme-stem-seed-sense-3' hairpin,
wherein the coding sequence for a pre-miRNA comprises parts
stem-seed-sense; wherein the responder sequence comprises parts
toehold-antisense-ribozyme; wherein the actuator is Drosha;
wherein, in the absence of an input signal which comprises an input
RNA that is completely or partially complementary to parts
toehold-antisense-ribozyme, the engineered RNA forms a first
secondary structure in which part 5' hairpin completely or
partially hybridizes to itself, part antisense completely or
partially hybridizes to part sense, part ribozyme completely or
partially hybridizes to itself, part stem completely or partially
hybridizes to itself, part seed completely or partially hybridizes
to itself, part 3' hairpin completely or partially hybridizes to
itself, and part stem is incapable of hybridizing to part sense,
such that the engineered RNA is not capable of being recognized by
Drosha; wherein, in the presence of the input RNA that is
completely or partially complementary to parts
toehold-sense-ribozyme, the engineered RNA forms a secondary
structure in which parts toehold-antisense-ribozyme hybridize with
the input RNA, resulting in ribozyme-mediated cleavage that
releases an RNA waste product comprising the input RNA hybridized
to parts 5' hairpin-toehold-sense and a portion of part ribozyme of
the engineered RNA, wherein, following the release of the RNA waste
product, the remaining portion of the engineered RNA forms a
secondary structure in which part stem partially or completely
hybridizes to part sense to form a Drosha recognizable cleavage
site not at its lowest energy state.
[0012] In some embodiments, miRNA is therapeutic miRNAs selected
from the group consisting of miR-16, miR-29, miR-34, miR-143,
miR-145, and miR-200 family.
[0013] In some embodiments, the effector portion comprises the
coding sequence for a single guide RNA (sgRNA).
[0014] In some embodiments, the engineered RNA is an engineered
sgRNA comprising: parts S-g-a-c-T-d-f-e-b-h; wherein the coding
sequence for sgRNA comprises part S-g-a and b-h, wherein the
responder sequence comprises parts c-T-d-f-e, wherein the actuator
is a Cas protein, wherein, in the absence of an input signal which
comprises an input RNA that is completely or partially
complementary to parts T-d-f, the engineered RNA forms a first
secondary structure in which part d partially hybridizes to part b,
part e completely or partially hybridizes to part f, part a
completely or partially hybridizes to part c, part g hybridizes to
part h, and parts a and b are incapable of hybridizing with each
other; wherein, in the presence of the input RNA that is completely
or partially complementary to parts T-d-f, the engineered RNA forms
a second secondary structure in which parts T-d-f form a double
strand with the input RNA, thereby releasing part e from part f and
part b from part d, and in which part a partially hybridizes to
part b to form a Cas protein binding site not in its lowest energy
state.
[0015] In some embodiments, the engineered RNA is an engineered
sgRNA comprising, comprising: parts S-g-b-e-f-d-T-c-a-h; wherein
the coding sequence for sgRNA comprises part S-g-b and a-h; wherein
the responder sequence comprises parts e-f-d-T-c, wherein the
actuator is a Cas protein, wherein, in the absence of an input
signal which comprises an input RNA that is completely or partially
complementary to parts T-d-f, the engineered RNA forms a first
secondary structure in which part d completely or partially
hybridizes to part b, part e completely or partially hybridizes to
part f, part a completely or partially hybridizes to part c, part g
hybridizes to part h, and parts a and b are incapable of
hybridizing with each other; wherein, in the presence of the input
RNA that is completely or partially complementary to parts T-d-f,
the engineered RNA forms a second secondary structure in which
parts T-d-f form a double strand with the input RNA, thereby
releasing part e from part f and part b from part d, and in which
part a partially hybridizes to part b to form a Cas protein binding
site not at its lowest energy state. In some embodiments, the
engineered RNA further comprises a nexus and hairpins.
[0016] In some embodiments, the Cas protein selected from a group
consisting of Cas9, saCas9, CjCas9, xCas9, Cas13a/C2c2, Cas13b,
Cpf1 and variants thereof. In some embodiments, the Cas protein is
a Cas9 fusion protein selected from a group consisting of
dCas9-transcription factor, dCas9-VP64, dCas9-VPR, dCas9-Suntag,
dCas9-P300, dCas9-VP160, dCas9VP192, dCas9-KRAB and its derivative,
dCas9- MXI1, dCas9-SID4X, dCas9-LSD1, dCas9-CIB1, dCas9-GFP, and
dCas9-RFP.
[0017] In some aspects, the present disclosure also provides an
engineered nucleic acid, comprising a promoter operably linked to a
nucleotide sequence encoding the engineered RNA described
herein.
[0018] In some aspects, the present disclosure also provides a
recombinant virus comprising: a viral capsid containing a promoter
operably linked to a nucleotide sequence encoding the engineered
RNA described herein. In some embodiments, the recombinant virus is
a recombinant AAV (rAAV). In some embodiments, the recombinant
virus is a recombinant lentivirus, adeno virus, or a
bacteriophage.
[0019] In some aspects, the present disclosure also provides a host
cell, comprising the engineered RNA, the engineered nucleic acid,
or the recombinant virus, as described herein. In some embodiments,
the host cell is a prokaryotic cell. In some embodiments, the
prokaryotic cell is a bacterial cell. In some embodiments, the host
cell is a eukaryotic cell. In some embodiments, the eukaryotic cell
is a fungal cell, a plant cell, an insect cell, or a mammalian
cell. In some embodiments, the mammalian cell is a human cell. In
some embodiments, the host cell is a diseased cell. In some
embodiments, the host cell is from a specific tissue. In some
embodiments, the host cell comprises the input signal. In some
embodiments, the host cell is capable of processing the engineered
RNA of described herein to produce the functional RNA.
[0020] In some aspects, the present disclosure also provides a
pharmaceutical composition, comprising the engineered RNA, the
engineered nucleic acid, the recombinant virus, or the cell
described herein. In some embodiments, the pharmaceutical
composition further comprises a pharmaceutically acceptable
carrier.
[0021] In some aspects, the present disclosure also provides a
method comprising delivering the engineered RNA, the engineered
nucleic acid, the recombinant virus, the cell, or the
pharmaceutical composition described herein to a subject in need
thereof.
[0022] In some aspects, the present disclosure also provides a
method for delivering a functional RNA to a cell in a subject in
need thereof, comprising administering to the subject an effective
amount of the engineered RNA, the engineered nucleic acid, the
recombinant virus of any one, the cell of, or the pharmaceutical
composition described herein.
[0023] In some aspects, the present disclosure also provides A
method for treating a disease in a subject in need thereof,
comprising administering to the subject an effective amount of the
engineered RNA, the engineered nucleic acid, the recombinant virus
of any one, the cell of, or the pharmaceutical composition
described herein. In some embodiments, the subject is a human or a
non-human mammal. In some embodiments, the subject has or is at
risk of having Sickle Cell Disease, X-linked severe combined
immunodeficiency (SCID-X1), Hurler Syndrome, Gaucher Disease,
Wiskot-Aldrich syndrome, human immunodeficiency virus (HIV),
Hepatitis B, human papillomavirus (HPV), Herpesviruses, Cystic
Fibrosis, B-thalassemia, Retinitis Pigmentosa, amyotrophic lateral
sclerosis (ALS), BEST disease, Parkinson's Disease, Schizophrenia,
or severe combined immunodeficiency (SCID).
[0024] In some embodiments, wherein the engineered RNA is an
engineered sgRNA, further comprising delivering Cas protein prior
to or concurrently with the engineered RNA. In some embodiments,
wherein the Cas protein is saCas9, and wherein the saCas9 is
delivered by a rAAV comprising an rAAV capsid enclosing a promoter
operably linked to a saCas9 coding sequence, the promoter and the
saCas9 coding sequence being flanked by AAV ITRs. In some
embodiments, the Cas protein is not saCas9, and wherein the Cas
protein is delivered by a first rAAV comprising an rAAV capsid
enclosing a promoter operably linked to a first portion of saCas9
coding sequence, the promoter and the first portion of the saCas9
coding sequence being flanked by AAV ITRs; and a second rAAV
comprising an rAAV capsid enclosing a promoter operably linked to a
second portion of saCas9 coding sequence, the promoter and the
second portion of the saCas9 coding sequence being flanked by AAV
ITRs
BRIEF DESCRIPTION OF DRAWINGS
[0025] FIGS. 1A-1F show the basic concept of an RNA having a
portion that interacts with the input signal, and another portion
that interacts with an actuator. FIG. 1A: B1=folding state of B
when A-B is at the lowest energy state; B1*=folding state of B
immediately after input interact with A. B2=folding state of B at
the lowest energy when the input interact with A. FIG. 1B: B1, and
B2 represent different RNA folding of the same RNA strand, the one
processed by the actuator. FIGS. 1C-1D show an improved design of
RNA folding: engineering an energy gap to reduce unwanted side
reactions. FIG. 1C: B1=folding state of B when A-B is at the lowest
energy state; B2=folding state of B at the lowest energy when the
input interact with A; B3=folding state of B that can interact with
the actuator. FIG. 1D shows a design with B3 at an energy state
higher than the lowest. B1, B2 and B3 represent different RNA
folding of the same RNA strand, the one processed by the actuator.
FIG. 1E shows the possibility of the engineered energy gap being
too large for the actuator to interact with B3 in the presence of
the input. FIG. 1F shows the possibility of the engineered energy
gap being too low to impede actuator to interact with B3 in the
absence of the input.
[0026] FIG. 2 shows strand displacement: input, gate and the
toehold mediated reaction.
[0027] FIG. 3A-3G shows a design of engineering trans-activated
miRNA by the use of the strand displacement reaction. FIG. 3A shows
one of the designs for trans-activated miRNA by the use of strand
displacement. FIG. 3B shows identifying the maximum energy gap in
transactivating miRNA. X axis=Energy gap (Kcal/mol) between the
lowest energy state (the one characterizing B in the B2 folding
state, which is its lowest energy state after the interaction with
the input), which Drosha cannot process, and the energy state
accessible by Drosha (the one characterizing B in B3 folding
state). FIGS. 3C-3D show detained design of one of the
trans-activated miRNA. FIG. 3E-3F shows additional designs of the
trans-activating RNA. FIG. 3G shows trans-activated miRNA by the
use of the strand displacement reaction: experimental validation in
mammalian cells. 30 folds activation at high transfection marker. A
trans activated miRNA can be delivered through AAV without the use
of exogenous proteins.
[0028] FIG. 4A-4F shows a design of engineering trans-activated
sgRNA by the use of the strand displacement reaction. FIG. 4A shows
one of the designs for trans-activated sgRNA by the use of strand
displacement. FIGS. 4B-4C show trans-activated gRNA for CAS9:
Energy gap for the gRNA. An actual gRNA sequence is shown in the
conformation B2 (FIG. 4B) and B3 (FIG. 4C). The folding is computed
with mFold. FIG. 4D-4E shows two different designs of the
trans-activating sgRNA. FIG. 4F shows trans-activated sgRNA by the
use of the strand displacement reaction: experimental validation in
mammalian cells.
[0029] FIG. 5A-5H shows the position of different RNA domains on an
exemplary trans-activated miRNA, including 5' hairpin sequence
(FIG. 5A), toehold sequence (FIG. 5B), antisense sequence (FIG.
5C), ribozyme sequence (FIG. 5D), stem sequence (FIG. 5E), seed
sequence (FIG. 5F), sense sequence (FIG. 5G), 3' hairpin sequence
(FIG. 5H). In each, bases shown in dark color correspond to the
named domain, while bases shown in lighter color correspond to the
rest of the RNA. In each, the 5' guanine base is circled in blue,
while the 3' uracil base is circled in red.
[0030] FIG. 6A-6D shows the possible secondary structures of an
exemplary trans-activated miRNA. FIG. 6B shows the structure of the
RNA in the absence of input signal, which is the most stable
conformation and corresponds to the lowest energy state B1 in FIG.
1. FIG. 6C-6D shows two possible folding states of the RNA that may
result after hybridization with an input RNA sequence, the release
of an RNA waste product by the ribozyme domain of the RNA, and
refolding of the remaining portion of the RNA molecule. The folding
state shown in FIG. 6C is more stable and corresponds to the lower
energy state B2 in FIG. 1, which cannot be processed by Drosha. The
folding state shown in FIG. 6D is less stable and corresponds to
the higher energy state B3 in FIG. 1, which can be processed by
Drosha.
[0031] FIG. 7 shows the elements of a trans-activated miRNA that
can be manipulated to change the dynamics of RNA folding and
stability, which include hairpin length and GC content, loop
length, stem length and GC content, and number of base
mismatches.
DETAILED DESCRIPTION
[0032] The present disclosure, at least in part, relates to an
engineered RNA (e.g., microRNA and sgRNA), in the absence of an
input signal, that is engineered to have a large enough energy gap
between the formations of a first secondary structure at its lowest
energy state, which is unrecognizable by an actuator, and a second
secondary structure, which is recognizable by an actuator (e.g.,
Drosha and Cas protein). Such design provides the benefit of
decreasing unwanted activation of the engineered RNA when the input
signal is absent. When the input signal is present, it induces a
conformational change of the engineered RNA molecule, such that the
engineered RNA forms the second secondary structure, not at its
lowest energy state, which is recognizable by an actuator.
I. ENGINEERED TRANS-ACTIVATING RNA
[0033] RNA transcripts fold into secondary structures via intricate
patterns of base pairing. These secondary structures impart
catalytic, ligand binding, and scaffolding functions to a wide
array of RNAs, forming a critical node of biological regulation.
Among their many functions, RNA structural elements modulate
epigenetic marks, alter mRNA stability and translation, regulate
alternative splicing, transduce signals, and scaffold large
macromolecular complexes. It is of crucial importance that an RNA
molecule folds into a correct secondary confirmation to elicit its
intended function. Of the many factors that may affect the
formation of an RNA secondary structure, thermodynamics is a major
determinant. Normally, an RNA favors a secondary structures that
requires the lowest free energy. Such secondary structure can be
defined as a secondary structure at its lowest energy state. In
response to various input signals, the RNA molecule is also capable
of overcoming the free energy barrier (energy gap) to form various
higher energy state secondary structures that requires more free
energy. Sometimes, the energy gap between the lowest energy state
to the higher energy state is not great enough, and the RNA is
capable of forming the higher energy state second structure in the
absence of the input signal. This phenomenon poses significant
issues in engineered RNA circuit in mammalian cells. The present
disclosure provides an engineered RNA molecule for the purpose of
increasing the energy gap between the lowest energy state structure
and the higher energy state, such that the confirmation switch
would only happen when the input signal is present.
[0034] Trans-activated engineered RNA, as used herein, refers to
engineered RNA molecules that is triggered either by biological
processes or by artificial means, through the presence of an input
signal. As used herein, the term "engineered RNA" and
"trans-activated engineered RNA" are used interchangeably.
[0035] In some aspects, the present disclosure provides an
engineered RNA comprising (i) an effector portion; and (ii) a
responder sequence. In some embodiments, the effector portion
comprises a coding sequence for a functional RNA; in the absence of
a input signal, the engineered RNA forms a first secondary
structure in which the engineered RNA is not capable of being
recognized by an actuator; and in the presence of the input signal,
the responder sequence is capable of responding to the input signal
such that the engineered RNA forms a second secondary structure,
not at its lowest energy state, in which the engineered RNA is
capable of being recognized by the actuator. Any RNA that owes its
function to the secondary structure of the RNA can be engineered,
and is within the scope of the present disclosure. Non-limiting
examples of such RNAs are: microRNA, small interference RNA
(siRNA), small hairpin RNA (shRNA), ribozymes, transfer RNA (tRNA),
or single guide RNA (sgRNA). In some embodiments, the engineered
RNA is an engineered pre-miRNA sequence. In some embodiments, the
engineered RNA is an engineered single guide RNA.
[0036] An effector portion of the engineered RNA, as used herein,
refers to the portion of the RNA that can be processed into a
function RNA. In some embodiments, the effector portion comprises
the coding sequence of a pre-microRNA (pre-miRNA). In some
embodiments, the effector portion comprises the coding sequence of
a single guide RNA (sgRNA).
[0037] A responder sequence, as used herein, refers to the sequence
that is capable of interacting with the input signal, and induces
the conformational change of the engineered RNA.
[0038] An input signal, as used herein, refers to a signal that is
provided to the engineered RNA in order to induce its
conformational change. Non-limiting examples of an input signal is
a oligonucleotide sequence (e.g., DNA and RNA), a protein (e.g.,
RNA binding protein), or a small molecule (e.g., a small molecule
that binds to RNA). In some embodiments, the input signal is an
oligonucleotide sequence. In some embodiments, the input signal is
an RNA. In some embodiments, the input signal is an RNA that is
partially or completely complementary to the responder sequence of
the engineered RNA. In some embodiments, the input signal is an
endogenous signal produced by the cell. In some embodiments, the
input signal is an exogenous signal supplied to the cell.
[0039] An actuator, as used herein, refers to the molecule that is
capable of recognizing the secondary structure of the engineered
RNA such that the RNA can elicit its downstream function.
Non-limiting examples of an actuator is a oligonucleotide sequence
(e.g., DNA and RNA), a protein (e.g., endoribonucleases), or a
small molecule (e.g., a small molecule that binds to RNA), In some
embodiments, the actuator is a protein. In some embodiments, the
actuator is an endogenous protein. In some embodiments, the
actuator is a protein that is involved in miRNA biogenesis. In some
embodiments, the actuator is Dicer. In some embodiments, the
actuator is Drosha, a Class 2 ribonuclease III enzyme that is
encoded by the DROSHA gene in humans. Drosha is a nuclear dsRNA
ribonuclease that processes of pri-miRNA to pre-miRNA. In other
embodiments, the actuator is an exogenous protein that needs to be
supplied with the input signal to the cell. In some embodiments,
the actuator is a Cas protein.
[0040] In some embodiments, the input signal interacts with the
responder sequence of the engineered RNA and induces the
conformational change by toehold mediated strand displacement.
Strand displacement, as used herein, refers to an enzyme-free
molecular tool to exchange one strand of DNA or RNA (output) with
another strand (input). It is based on the hybridization of two
complementary strands of DNA or RNA via Watson-Crick base pairing
(A-T/U and C-G) and makes use of a process called branch migration.
(Yurke et al., A DNA-fuelled molecular machine made of DNA, Nature.
406 (6796): 605-8.). Originally, the toehold-mediated strand
displacement reaction has been used in cell free settings. There,
an input (e.g., single strand DNA or RNA) interacts with a double
stranded DNA or RNA. As shown in FIG. 2, the input domain T*
(Toehold), anneal by Watson and Crick base pairing to the
complementary T domain on the double stranded DNA or RNA, and then
the domain Y2* displaces Y2. In some embodiments, the input can
itself be the output of an upstream system, and the output of the
strand displacement reaction can be the input of a system
downstream. Toehold strand displacement in the use of RNA
nanotechnology based on thermodynamics has been previous described.
(See, e.g., Sulc et al., "Modelling Toehold-Mediated RNA Strand
Displacement." Biophys J. 2015 Mar. 10; 108(5): 1238-1247.), the
entire contents of which is incorporated herein by reference.
(i) Engineered RNA for miRNA Processing
[0041] In some embodiments, the engineered RNA described herein,
can be designed to control the biogenesis of an miRNA in response
to an input signal.
[0042] Mature microRNAs (miRNAs) are small single-stranded,
non-coding RNAs (about 22 nucleotides in length), which play
significant regulatory roles in various biological processes of
animals, plants and viruses. There are two other forms of miRNAs:
primary miRNAs (pri-miRNAs) and precursor microRNAs (pre-miRNAs).
Mature miRNAs are cleaved from.about.90nt pre-miRNAs which are
derived from the processing of a long pri-miRNA by a ribonucluease.
In some embodiments, the engineered RNA comprises a pre-miRNA
sequence and additional flanking sequence, including the responder
sequence, at both the 5' and 3' end. In some embodiments,
engineered RNA forms secondary structure that is not recognizable
by Drosha at its lowest energy state in the absence of an input
signal. In some embodiments, when the input signal (e.g., an RNA)
is present, the engineered RNA is promoted to form a secondary
structure that resembles a pri-miRNA, which is recognizable by
Drosha. Such secondary structure is formed not at its lowest energy
state. In some embodiments, formation of such secondary structure
enables Drosha to cleave the pri-miRNA off of the engineered RNA,
thereby activating the biogenesis of the encoded miRNA. In some
embodiments, the input signal triggers the conformational change by
hybridizing to the responder sequence, thereby displacing the
responder sequence from the sequence it originally hybridized to,
which allows the secondary structure recognizable by Drosha to
form. In some embodiments, the input signal triggers a
conformational change by hybridizing to the responder sequence,
thereby activating the ribozyme domain of the responder sequence,
which causes the release of an RNA waste product and allows the
secondary structure recognizable by Drosha to form.
[0043] In some embodiments, the engineered RNA comprises parts
T-d-f-e-b-S-a-c. In some embodiments, the coding sequence for a
pre-miRNA comprises parts b-S-a; the responder sequence comprises
parts T-d-f-e; and the actuator is Drosha. In some embodiments, in
the absence of an input signal which comprises an input RNA that is
completely or partially complementary to parts T-d-f, the
engineered RNA forms a first secondary structure in which part d
completely or partially hybridizes to part b, part e completely or
partially hybridizes to part f, part a partially hybridizes to part
c, and parts a and b are incapable of hybridizing with each other
such that the engineered RNA is not capable of being recognized by
Drosha. In some embodiments, in the presence of the input RNA that
is completely or partially complementary to parts T-d-f, the
engineered RNA forms a secondary structure in which parts T-d-f
form a double strand with the input RNA, thereby releasing part e
from part f and part b from part d, and in which part a partially
hybridizes to part b to form a Drosha recognizable cleavage site
not at its lowest energy state. (FIG. 3E, top panel). In some
embodiments, the engineered RNA, from 5' to 3', comprises
T-d-f-e-b-S-a-c. In other embodiments, the engineered RNA, from 5'
to 3', comprises c-a-S-b-e-f-d-T.
[0044] In other embodiments, the engineered RNA comprises parts
T-f-d-c-a-S-b-e. In some embodiments, the coding sequence for a
pre-miRNA comprises parts b-S-a; the responder sequence comprises
parts T-f-d and e; and the actuator is Drosha. In some embodiments,
in the absence of an input signal which comprises an input RNA that
is completely or partially complementary to parts T-f-d, the
engineered RNA forms a first secondary structure in which part d
completely or partially hybridizes to part b, part e completely or
partially hybridizes to part f, part a partially hybridizes to part
c, and parts a and b are incapable of hybridizing with each other
such that the engineered RNA is not capable of being recognized by
Drosha. In some embodiments, in the presence of the input RNA that
is completely or partially complementary to parts T-f-d, the
engineered RNA forms a second secondary structure in which parts
T-f-d form a double strand with the input RNA, thereby releasing
part e from part f and part b from part d, and in which part a
partially hybridizes to part b to form a Drosha recognizable
cleavage site not at its lowest energy state. (FIG. 3E, bottom
panel). In some embodiments, the engineered RNA, from 5' to 3',
comprises T-f-d-c-a-S-b-e. In other embodiments, the engineered
RNA, from 5' to 3', comprises e-b-S-a-c-d-f-T.
[0045] As used herein, the term "hybridize" or "hybridization"
means annealing of a single-stranded deoxyribonucleic acid (DNA) or
ribonucleic acid (RNA) molecule to a complementary DNA or RNA or to
a complementary portion of itself to form a partially
double-stranded molecule. In molecular biology, "complementary" or
"complementarity" describes a relationship between two structures
each following the lock-and-key principle. Complementarity is
achieved by distinct interactions between pairs of nucleobases:
adenine and thymine (uracil in RNA); and guanine and cytosine.
Adenine and guanine are purines, while thymine, cytosine and uracil
are pyrimidines. Purines are larger than pyrimidines. Both types of
molecules complement each other and can only base pair with the
opposing type of nucleobase. In nucleic acid, nucleobases are held
together by hydrogen bonding, which only works efficiently between
adenine (A) and thymine (T) or uracil (U), and between guanine (G)
and cytosine (C). The base pair A=T (or A=U) shares two hydrogen
bonds, while the base pair G.ident.C has three hydrogen bonds. All
other configurations between nucleobases would hinder
hybridization. DNA strands are oriented in opposite directions,
they are said to be antiparallel. The degree of complementarity
between two nucleic acid strands may vary, from complete
complementarity (each nucleotide is across from its opposite),
partially complementary, to no complementarity (each nucleotide is
not across from its opposite) and determines the stability of the
sequences to be together. Generally speaking, the level of
complementarity and the percentage of G.ident.C pair affect the
stability of the double strand, and may require higher free energy
to separate the double strand.
[0046] Any known miRNA can be engineered based on the principle
described herein. Non-limiting examples of such microRNAs are: FF4,
FF5, hsa-let-7a-2-3p, hsa-let-7a-3p, hsa-let-7a-5p, hsa-let-7b-3p,
hsa-let-7b-5p, hsa-let-7c-5p, hsa-let-7d-3p, hsa-let-7d-5p,
hsa-let-7e-3p, hsa-let-7e-5p, hsa-let-7f-1-3p, hsa-let-7f-2-3p,
hsa-let-7f-5p, hsa-let-7g-3p, hsa-let-7g-5p, hsa-let-7i-5p,
hsa-miR-1, hsa-miR-1-3p, hsa-miR-1-5p, hsa-miR-100-3p,
hsa-miR-100-5p, hsa-miR-101-3p, hsa-miR-101-5p, hsa-miR-103a-2-5p,
hsa-miR-103a-3p, hsa-miR-105-3p, hsa-miR-105-5p, hsa-miR-106a-3p,
hsa-miR-106a-5p, hsa-miR-106b-3p, hsa-miR-106b-5p, hsa-miR-107,
hsa-miR-10a-3p, hsa-miR-10a-5p, hsa-miR-10b-3p, hsa-miR-10b-5p,
hsa-miR-1185-1-3p, hsa-miR-1185-2-3p, hsa-miR-1185-5p,
hsa-miR-122a-5p, hsa-miR-1249-3p, hsa-miR-1249-5p, hsa-miR-124a-3p,
hsa-miR-125a-3p, hsa-miR-125a-5p, hsa-miR-125b-1-3p,
hsa-miR-125b-2-3p, hsa-miR-125b-5p, hsa-miR-126-3p, hsa-miR-126-5p,
hsa-miR-127-3p, hsa-miR-1271-3p, hsa-miR-1271-5p, hsa-miR-1278,
hsa-miR-128-1-5p, hsa-miR-128-2-5p, hsa-miR-128-3p,
hsa-miR-1285-3p, hsa-miR-1285-5p, hsa-miR-1287-3p, hsa-miR-1287-5p,
hsa-miR-129-1-3p, hsa-miR-129-2-3p, hsa-miR-129-5p,
hsa-miR-1296-3p, hsa-miR-1296-5p, hsa-miR-1304-3p, hsa-miR-1304-5p,
hsa-miR-1306-3p, hsa-miR-1306-5p, hsa-miR-1307-3p, hsa-miR-1307-5p,
hsa-miR-130a-3p, hsa-miR-130b-3p, hsa-miR-130b-5p, hsa-miR-132-3p,
hsa-miR-132-5p, hsa-miR-133a-3p, hsa-miR-133a-5p, hsa-miR-133b,
hsa-miR-134-3p, hsa-miR-134-5p, hsa-miR-135a-3p, hsa-miR-135a-5p,
hsa-miR-135b-3p, hsa-miR-135b-5p, hsa-miR-136-3p, hsa-miR-136-5p,
hsa-miR-138-1-3p, hsa-miR-138-5p, hsa-miR-139-3p, hsa-miR-139-5p,
hsa-miR-140-3p, hsa-miR-140-5p, hsa-miR-141-3p, hsa-miR-141-5p,
hsa-miR-142-3p, hsa-miR-142-5p, hsa-miR-143-3p, hsa-miR-143-5p,
hsa-miR-144-3p, hsa-miR-144-5p, hsa-miR-145-5p, hsa-miR-146a-3p,
hsa-miR-146a-5p, hsa-miR-147a, hsa-miR-148a-3p, hsa-miR-148a-5p,
hsa-miR-148b-3p, hsa-miR-148b-5p, hsa-miR-149-3p, hsa-miR-144-3p,
hsa-miR-150-3p, hsa-miR-150-5p, hsa-miR-151a-3p, hsa-miR-151a-5p,
hsa-miR-152-3p, hsa-miR-152-5p, hsa-miR-154-3p, hsa-miR-154-5p,
hsa-miR-155-3p, hsa-miR-155-5p, hsa-miR-15a-3p, hsa-miR-15a-5p,
hsa-miR-15b-3p, hsa-miR-15b-5p, hsa-miR-16-1-3p, hsa-miR-16-2-3p,
hsa-miR-16-5p, hsa-miR-17-3p, hsa-miR-17-5p, hsa-miR-181a-3p,
hsa-miR-181a-5p, hsa-miR-181b-2-3p, hsa-miR-181b-5p,
hsa-miR-181c-5p, hsa-miR-181d-3p, hsa-miR-181d-5p, hsa-miR-182-3p,
hsa-miR-182-5p, hsa-miR-183-3p, hsa-miR-183-5p,hsa-miR-185-3p,
hsa-miR-185-5p, hsa-miR-186-3p, hsa-miR-186-5p, hsa-miR-188-3p,
hsa-miR-188-5p, hsa-miR-18a-3p, hsa-miR-18a-5p, hsa-miR-18b-5p,
hsa-miR-1908-3p, hsa-miR-1908-5p, hsa-miR-190a-3p, hsa-miR-190a-5p,
hsa-miR-191-3p, hsa-miR-191-5p, hsa-miR-1910-3p, hsa-miR-1910-5p,
hsa-miR-192-3p, hsa-miR-192-5p, hsa-miR-193a-3p, hsa-miR-193a-5p,
hsa-miR-193b-3p, hsa-miR-193b-5p, hsa-miR-194-3p, hsa-miR-194-5p,
hsa-miR-195-3p, hsa-miR-195-5p, hsa-miR-196a-3p, hsa-miR-196a-5p,
hsa-miR-196b-3p, hsa-miR-196b-5p, hsa-miR-197-3p, hsa-miR-197-5p,
hsa-miR-199a-3p, hsa-miR-199a-5p, hsa-miR-199b-3p, hsa-miR-199b-5p,
hsa-miR-19a-3p, hsa-miR-19a-5p, hsa-miR-19b-1-5p, hsa-miR-19b-2-5p,
hsa-miR-19b-3p, hsa-miR-200a-3p, hsa-miR-200a-5p, hsa-miR-200b-3p,
hsa-miR-200b-5p, hsa-miR-200c-3p, hsa-miR-200c-5p, hsa-miR-202-3p,
hsa-miR-202-5p, hsa-miR-203a-3p, hsa-miR-203a-5p, hsa-miR-204-5p,
hsa-miR-208b-3p, hsa-miR-208b-5p, hsa-miR-20a-3p, hsa-miR-20a-5p,
hsa-miR-20b-3p, hsa-miR-20b-5p, hsa-miR-21-5p, hsa-miR-210-3p,
hsa-miR-210-5p, hsa-miR-211-3p, hsa-miR-211-5p, hsa-miR-2116-3p,
hsa-miR-2116-5p, hsa-miR-212-3p, hsa-miR-214-3p, hsa-miR-215-5p,
hsa-miR-217, JG_miR-218-1-3p, hsa-miR-218-5p, hsa-miR-219a-1-3p,
hsa-miR-219a-2-3p, hsa-miR-219a-5p, hsa-miR-219b-3p,
hsa-miR-219b-5p, hsa-miR-22-3p, hsa-miR-22-5p, hsa-miR-221-3p,
hsa-miR-221-5p, hsa-miR-222-3p, hsa-miR-222-5p, hsa-miR-223-3p,
hsa-miR-223-5p, hsa-miR-23a-3p, hsa-miR-23a-5p, hsa-miR-23b-3p,
hsa-miR-24-1-5p, hsa-miR-25-3p, hsa-miR-25-5p, hsa-miR-26a-1-3p,
hsa-miR-26a-2-3p, hsa-miR-26a-5p, hsa-miR-26b-5p, hsa-miR-27a-3p,
hsa-miR-27a-5p, hsa-miR-27b-3p, hsa-miR-27b-5p, hsa-miR-28-3p,
hsa-miR-28-5p, hsa-miR-296-3p, hsa-miR-296-5p, hsa-miR-299-3p,
hsa-miR-299-5p, hsa-miR-29a-3p, hsa-miR-29a-5p, hsa-miR-29b-1-5p,
hsa-miR-29b-3p, hsa-miR-29c-3p, hsa-miR-301a-3p, hsa-miR-301a-5p,
hsa-miR-301b-3p, hsa-miR-301b-5p, hsa-miR-302a-3p, hsa-miR-302a-5p,
hsa-miR-302b-5p, hsa-miR-302c-3p, hsa-miR-302c-5p, hsa-miR-3065-3p,
hsa-miR-3065-5p, hsa-miR-3074-3p, hsa-miR-3074-5p, hsa-miR-30a-3p,
hsa-miR-30a-5p, hsa-miR-30b-3p, hsa-miR-30b-5p, hsa-miR-30c-1-3p,
hsa-miR-30c-2-3p, hsa-miR-30c-5p, hsa-miR-30d-3p, hsa-miR-30d-5p,
hsa-miR-30e-3p, hsa-miR-30e-5p, hsa-miR-31-3p, hsa-miR-31-5p,
hsa-miR-3130-3p, hsa-miR-3130-5p, hsa-miR-3140-3p, hsa-miR-3140-5p,
hsa-miR-3144-3p, hsa-miR-3144-5p, hsa-miR-3158-3p, hsa-miR-3158-5p,
hsa-miR-32-3p, hsa-miR-32-5p, hsa-miR-320a, hsa-miR-323a-3p,
hsa-miR-323a-5p, hsa-miR-324-3p, hsa-miR-324-5p, hsa-miR-326,
hsa-miR-328-3p, hsa-miR-328-5p, hsa-miR-329-3p, hsa-miR-329-5p,
hsa-miR-330-3p, hsa-miR-330-5p, hsa-miR-331-3p, hsa-miR-331-5p,
hsa-miR-335-3p, hsa-miR-335-5p, hsa-miR-337-3p, hsa-miR-337-5p,
hsa-miR-338-3p, hsa-miR-338-5p, hsa-miR-339-3p, hsa-miR-339-5p,
hsa-miR-33a-3p, hsa-miR-33a-5p, hsa-miR-33b-3p, hsa-miR-33b-5p,
hsa-miR-340-3p, hsa-miR-340-5p, hsa-miR-342-3p, hsa-miR-342-5p,
hsa-miR-345-3p, hsa-miR-345-5p, hsa-miR-34a-3p, hsa-miR-34a-5p,
hsa-miR-34b-3p, hsa-miR-34b-5p, hsa-miR-34c-3p, hsa-miR-34c-5p,
hsa-miR-3605-3p, hsa-miR-3605-5p, hsa-miR-361-3p, hsa-miR-361-5p,
hsa-miR-3613-3p, hsa-miR-3613-5p, hsa-miR-3614-3p, hsa-miR-3614-5p,
hsa-miR-362-3p, hsa-miR-362-5p, hsa-miR-363-3p, hsa-miR-363-5p,
hsa-miR-365a-3p, hsa-miR-365a-5p, hsa-miR-365b-3p, hsa-miR-365b-5p,
hsa-miR-369-3p, hsa-miR-369-5p, hsa-miR-370-3p, hsa-miR-370-5p,
hsa-miR-374a-3p, hsa-miR-374a-5p, hsa-miR-374b-3p, hsa-miR-374b-5p,
hsa-miR-375, hsa-miR-376a-2-5p, hsa-miR-376a-3p, hsa-miR-376a-5p,
hsa-miR-376c-3p, hsa-miR-376c-5p, hsa-miR-377-3p, hsa-miR-377-5p,
hsa-miR-378a-3p, hsa-miR-378a-5p, hsa-miR-379-3p, hsa-miR-379-5p,
hsa-miR-381-3p, hsa-miR-381-5p, hsa-miR-382-3p, hsa-miR-382-5p,
hsa-miR-409-3p, hsa-miR-409-5p, hsa-miR-411-3p, hsa-miR-411-5p,
hsa-miR-412-3p, hsa-miR-421, hsa-miR-423-3p, hsa-miR-423-5p,
hsa-miR-424-3p, hsa-miR-424-5p, hsa-miR-425-3p, hsa-miR-425-5p,
hsa-miR-431-3p, hsa-miR-431-5p, hsa-miR-432-5p, hsa-miR-433-3p,
hsa-miR-433-5p, hsa-miR-449a, hsa-miR-449b-5p, hsa-miR-450a-1-3p,
hsa-miR-450a-2-3p, hsa-miR-450a-5p, hsa-miR-450b-3p,
hsa-miR-450b-5p, hsa-miR-451a, hsa-miR-452-3p, hsa-miR-4524a-3p,
hsa-miR-4524a-5p, hsa-miR-4536-3p, hsa-miR-4536-5p, hsa-miR-454-3p,
hsa-miR-454-5p, hsa-miR-4707-3p, hsa-miR-4707-5p, hsa-miR-4755-3p,
hsa-miR-4755-5p, hsa-miR-4787-3p, hsa-miR-4787-5p, hsa-miR-483-3p,
hsa-miR-483-5p, hsa-miR-484, hsa-miR-485-3p, hsa-miR-485-5p,
hsa-miR-487b-3p, hsa-miR-487b-5p, hsa-miR-488-3p, hsa-miR-488-5p,
hsa-miR-489-3p, hsa-miR-490-3p, hsa-miR-490-5p, hsa-miR-491-3p,
hsa-miR-491-5p, hsa-miR-493-3p, hsa-miR-493-5p, hsa-miR-494-3p,
hsa-miR-494-5p, hsa-miR-495-3p, hsa-miR-495-5p, hsa-miR-497-3p,
hsa-miR-497-5p, hsa-miR-498, hsa-miR-5001-3p, hsa-miR-5001-5p,
hsa-miR-500a-3p, hsa-miR-500a-5p, hsa-miR-5010-3p, hsa-miR-5010-5p,
hsa-miR-503-3p, hsa-miR-503-5p, hsa-miR-504-3p, hsa-miR-504-5p,
hsa-miR-505-3p, hsa-miR-505-5p, hsa-miR-506-3p, hsa-miR-506-5p,
hsa-miR-508-3p, hsa-miR-508-5p, hsa-miR-509-3-5p, hsa-miR-509-3p,
hsa-miR-509-5p, hsa-miR-510-3p, hsa-miR-510-5p, hsa-miR-512-5p,
hsa-miR-513c-3p, hsa-miR-513c-5p, hsa-miR-514a-3p, hsa-miR-514a-5p,
hsa-miR-514b-3p, hsa-miR-514b-5p, hsa-miR-516b-5p, hsa-miR-518c-3p,
hsa-miR-518f-3p, hsa-miR-5196-3p, hsa-miR-5196-5p, hsa-miR-519a-3p,
hsa-miR-519a-5p, hsa-miR-519c-3p, hsa-miR-519e-3p, hsa-miR-520c-3p,
hsa-miR-520f-3p, hsa-miR-520g-3p, hsa-miR-520h, hsa-miR-522-3p,
hsa-miR-525-5p, hsa-miR-526b-5p, hsa-miR-532-3p, hsa-miR-532-5p,
hsa-miR-539-3p, hsa-miR-539-5p, hsa-miR-542-3p, hsa-miR-542-5p,
hsa-miR-543, hsa-miR-545-3p, hsa-miR-545-5p, hsa-miR-548a-3p,
hsa-miR-548a-5p, hsa-miR-548ar-3p, hsa-miR-548ar-5p,
hsa-miR-548b-3p, hsa-miR-548d-3p, hsa-miR-548d-5p, hsa-miR-548e-3p,
hsa-miR-548e-5p, hsa-miR-548h-3p, hsa-miR-548h-5p, hsa-miR-548j-3p,
hsa-miR-548j-5p, hsa-miR-548o-3p, hsa-miR-548o-5p, hsa-miR-548v,
hsa-miR-551b-3p, hsa-miR-551b-5p, hsa-miR-552-3p, hsa-miR-556-3p,
hsa-miR-556-5p, hsa-miR-561-3p, hsa-miR-561-5p, hsa-miR-562,
hsa-miR-567, hsa-miR-569, hsa-miR-570-3p, hsa-miR-570-5p,
hsa-miR-571, hsa-miR-574-3p, hsa-miR-574-5p, hsa-miR-576-3p,
hsa-miR-576-5p, hsa-miR-577, hsa-miR-579-3p, hsa-miR-579-5p,
hsa-miR-582-3p, hsa-miR-582-5p, hsa-miR-584-3p, hsa-miR-584-5p,
hsa-miR-589-3p, hsa-miR-589-5p, hsa-miR-590-3p, hsa-miR-590-5p,
hsa-miR-595, hsa-miR-606, hsa-miR-607, hsa-miR-610, hsa-miR-615-3p,
hsa-miR-615-5p, hsa-miR-616-3p, hsa-miR-616-5p, hsa-miR-617,
hsa-miR-619-5p, hsa-miR-624-3p, hsa-miR-624-5p, hsa-miR-625-3p,
hsa-miR-625-5p, hsa-miR-627-3p, hsa-miR-627-5p, hsa-miR-628-3p,
hsa-miR-628-5p, hsa-miR-629-3p, hsa-miR-629-5p, hsa-miR-630,
hsa-miR-633, hsa-miR-634, hsa-miR-635, hsa-miR-636, hsa-miR-640,
hsa-miR-642a-3p, hsa-miR-642a-5p, hsa-miR-643, hsa-miR-645,
hsa-miR-648, hsa-miR-6503-3p, hsa-miR-6503-5p, hsa-miR-651-3p,
hsa-miR-651-5p, hsa-miR-6511a-3p, hsa-miR-6511a-5p, hsa-miR-652-3p,
hsa-miR-652-5p, hsa-miR-653-5p, hsa-miR-654-3p, hsa-miR-654-5p,
hsa-miR-657, hsa-miR-659-3p, hsa-miR-660-3p, hsa-miR-660-5p,
hsa-miR-664b-3p, hsa-miR-664b-5p, hsa-miR-671-3p, hsa-miR-671-5p,
hsa-miR-675-3p, hsa-miR-675-5p, hsa-miR-7-1-3p, hsa-miR-7-5p,
hsa-miR-708-3p, hsa-miR-708-5p, hsa-miR-744-3p, hsa-miR-744-5p,
hsa-miR-758-3p, hsa-miR-758-5p, hsa-miR-765, hsa-miR-766-3p,
hsa-miR-766-5p, hsa-miR-767-3p, hsa-miR-767-5p, hsa-miR-769-3p,
hsa-miR-769-5p, hsa-miR-802, hsa-miR-873-3p, hsa-miR-873-5p,
hsa-miR-874-3p, hsa-miR-874-5p, hsa-miR-876-3p, hsa-miR-876-5p,
hsa-miR-885-3p, hsa-miR-885-5p, hsa-miR-887-3p, hsa-miR-887-5p,
hsa-miR-9-3p, hsa-miR-9-5p, hsa-miR-92a-1-5p, hsa-miR-92a-2-5p,
hsa-miR-92a-3p, hsa-miR-92b-3p, hsa-miR-92b-5p, hsa-miR-93-3p,
hsa-miR-93-5p, hsa-miR-941, hsa-miR-942-3p, hsa-miR-942-5p,
hsa-miR-96-3p, hsa-miR-96-5p, hsa-miR-98-3p, hsa-miR-98-5p,
hsa-miR-99a-3p, hsa-miR-99a-5p, hsa-miR-99b-3p, and
hsa-miR-99b-5p.
(ii) Engineered RNA for sgRNA Biogenesis
[0047] In some embodiments, the engineered RNA described herein,
can be design to control the biogenesis of a single guide (sgRNA)
in response to an input signal.
[0048] sgRNA, as used herein, refers to an sgRNA is a single RNA
molecule that contains both the custom-designed short crRNA
sequence fused to the scaffold tracrRNA sequence. sgRNA can be
synthetically generated or made in vitro or in vivo from a DNA
template. It is known in the art that the sgRNA forms a secondary
structure that facilitates the binding and the endonuclease
activity of Cas protein. An sgRNA includes the following structural
components: spacer sequence, low stem, bulge, upper stem, nexus,
and hairpins. Individual functional modules of the sgRNA was
described in Briner et al., 2014, Guide RNA functional modules
direct Cas9 activity and orthogonality, Mol Cell. 2014 Oct. 23;
56(2):333-339. doi: 10.1016/j.molcel.2014.09.019. Epub 2014 Oct.
16. The spacer sequence dictates Cas protein localization within
the genome. The lower stem is formed by the duplex between the
CRISPR repeat sequence from the crRNA and the region of
complementarity in the tracrRNA. Cas protein interacts with the
upper and lower stems in a sequence-independent manner, whereas the
bulge interactions with Cas protein appear to be
sequence-dependent. The nexus contains both sequence and structural
features necessary for DNA cleavage and lies at the center of the
sgRNA: Cas protein interactions. The nexus also forms a junction
between the sgRNA and both Cas protein and the target DNA. The
terminal hairpins assist in stabilizing the sgRNA and supports
stable complex formation with SpCas9. In some embodiments, the
sgRNA can be engineered to form a secondary structure that is
unrecognizable by Cas protein at its lowest energy state in the
absence of an input signal. In some embodiments, the lower stem,
the bulge, the upper stem, the nexus or the hairpins can be
engineered such that the engineered sgRNA does not form the
secondary structure that is recognizable by Cas protein at its
lowest energy state. In some embodiments, the upper stem and the
bulge are engineered to incorporate additional sequences, including
the responder sequence, that would hinder the formation of the
upper stem and the bulge at its lowest energy state in the absence
of an input signal. In some embodiments, when the input signal
(e.g., an RNA) is present, the input signal triggers a
conformational change of the engineered sgRNA such that it forms
the secondary structure that is recognizable by the Cas protein not
at its lowest energy state. In some embodiments, the input signal
triggers the conformational change by hybridizing to the responder
sequence, thereby displacing the responder sequence from the
sequence it originally hybridized to, which allows the secondary
structure recognizable by Cas protein to form.
[0049] In some embodiments, the engineered RNA is an engineered
sgRNA comprising: parts S-g-a-c-T-d-f-e-b-h (FIG. 4D, left panel).
In some embodiments, the coding sequence for sgRNA comprises part
S-g-a and b-h. In some embodiments, the responder sequence
comprises parts c-T-d-f-e. In some embodiments, the actuator is a
Cas protein. In some embodiments, in the absence of an input signal
which comprises an input RNA that is completely or partially
complementary to parts T-d-f, the engineered RNA forms a first
secondary structure in which part d partially hybridizes to part b,
part e completely or partially hybridizes to part f, part a
completely or partially hybridizes to part c, part g hybridizes to
part h, and parts a and b are incapable of hybridizing with each
other. In some embodiments, in the presence of the input RNA that
is completely or partially complementary to parts T-d-f, the
engineered RNA forms a second secondary structure in which parts
T-d-f form a double strand with the input RNA, thereby releasing
part e from part f and part b from part d, and in which part a
partially hybridizes to part b to form a Cas protein binding site
not in its lowest energy state.
[0050] In other embodiments, the engineered RNA is an engineered
sgRNA comprising, comprising: parts S-g-b-e-f-d-T-c-a-h (FIG. 4E,
left panel). In other embodiments, herein the coding sequence for
sgRNA comprises part S-g-b and a-h. In other embodiments, the
responder sequence comprises parts e-f-d-T-c. In other embodiments,
the actuator is a Cas protein. In other embodiments, in the absence
of an input signal which comprises an input RNA that is completely
or partially complementary to parts T-d-f, the engineered RNA forms
a first secondary structure in which part d completely or partially
hybridizes to part b, part e completely or partially hybridizes to
part f, part a completely or partially hybridizes to part c, part g
hybridizes to part h, and parts a and b are incapable of
hybridizing with each other. In other embodiments, in the presence
of the input RNA that is completely or partially complementary to
parts T-d-f, the engineered RNA forms a second secondary structure
in which parts T-d-f form a double strand with the input RNA,
thereby releasing part e from part f and part b from part d, and in
which part a partially hybridizes to part b to form a Cas protein
binding site not at its lowest energy state.
[0051] It can be appreciated that any Cas protein or Cas protein
variant can be employed herein. In some embodiments, the Cas
protein is a Cas9 domain, for example a nuclease active Cas9, a
Cas9 nickase (nCas9), or a nuclease inactive Cas9 (dCas9). Examples
of Cas protein include, without limitation, Cas9 (e.g., dCas9 and
nCas9), CasX, CasY, Cpf1, C2c1, C2c2, C2C3, saCas9, CjCas9, xCas9,
Cas13a/C2c2, Cas13b, Cpf1 and variants thereof. Other Cas proteins
are also within the scope of this disclosure, though they may not
be specifically listed in this disclosure.
[0052] A Cas9 or Cas9 domain refers to an RNA-guided nuclease
comprising a Cas9 protein, or a fragment thereof (e.g., a protein
comprising an active, inactive, or partially active DNA cleavage
domain of Cas9, and/or the gRNA binding domain of Cas9). A Cas9
nuclease is also referred to sometimes as a casnl nuclease or a
CRISPR (clustered regularly interspaced short palindromic
repeat)-associated nuclease. CRISPR is an adaptive immune system
that provides protection against mobile genetic elements (viruses,
transposable elements and conjugative plasmids). CRISPR clusters
contain spacers, sequences complementary to antecedent mobile
elements, and target invading nucleic acids. CRISPR clusters are
transcribed and processed into CRISPR RNA (crRNA). In type II
CRISPR systems correct processing of pre-crRNA requires a
trans-encoded small RNA (tracrRNA), endogenous ribonuclease 3 (rnc)
and a Cas9 protein. The tracrRNA serves as a guide for ribonuclease
3-aided processing of pre-crRNA. Subsequently, Cas9/crRNA/tracrRNA
endonucleolytically cleaves linear or circular dsDNA target
complementary to the spacer. The target strand not complementary to
crRNA is first cut endonucleolytically, then trimmed 3'-5'
exonucleolytically. In nature, DNA-binding and cleavage typically
requires protein and both RNAs. However, single guide RNAs
("sgRNA", or simply "gRNA") can be engineered so as to incorporate
aspects of both the crRNA and tracrRNA into a single RNA species.
See, e.g., Jinek M., Chylinski K., Fonfara I., Hauer M., Doudna
J.A., Charpentier E. Science 337:816-821(2012), the entire contents
of which is hereby incorporated by reference. Cas9 recognizes a
short motif in the CRISPR repeat sequences (the PAM or protospacer
adjacent motif) to help distinguish self versus non-self. Cas9
nuclease sequences and structures are well known to those of skill
in the art (see, e.g., "Complete genome sequence of an M1 strain of
Streptococcus pyogenes." Ferretti et al., J.J., McShan W.M., Ajdic
D.J., Savic D.J., Savic G., Lyon K., Primeaux C., Sezate S.,
Suvorov A.N., Kenton S., Lai H.S., Lin S.P., Qian Y., Jia H.G.,
Najar F.Z., Ren Q., Zhu H., Song L., White J., Yuan X., Clifton
S.W., Roe B.A., McLaughlin R.E., Proc. Natl. Acad. Sci. U.S.A.
98:4658-4663(2001); "CRISPR RNA maturation by trans-encoded small
RNA and host factor RNase III." Deltcheva E., Chylinski K., Sharma
C.M., Gonzales K., Chao Y., Pirzada Z.A., Eckert M.R., Vogel J.,
Charpentier E., Nature 471:602-607(2011); and "A programmable
dual-RNA-guided DNA endonuclease in adaptive bacterial immunity."
Jinek M., Chylinski K., Fonfara I., Hauer M., Doudna J.A.,
Charpentier E. Science 337:816-821(2012), the entire contents of
each of which are incorporated herein by reference). Cas9 orthologs
have been described in various species, including, but not limited
to, S. pyogenes and S. thermophilus. Additional suitable Cas9
nucleases and sequences will be apparent to those of skill in the
art based on this disclosure, and such Cas9 nucleases and sequences
include Cas9 sequences from the organisms and loci disclosed in
Chylinski, Rhun, and Charpentier, "The tracrRNA and Cas9 families
of type II CRISPR-Cas immunity systems" (2013) RNA Biology 10:5,
726-737; the entire contents of which are incorporated herein by
reference. In some embodiments, a Cas9 nuclease has an inactive
(e.g., an inactivated) DNA cleavage domain, that is, the Cas9 is a
nickase. Additional suitable nuclease-inactive dCas9 domains will
be apparent to those of skill in the art based on this disclosure
and knowledge in the field, and are within the scope of this
disclosure.
[0053] A nuclease-inactivated Cas9 protein may interchangeably be
referred to as a "dCas9" protein (for nuclease-"dead" Cas9).
Methods for generating a Cas9 protein (or a fragment thereof)
having an inactive DNA cleavage domain are known (See, e.g., Jinek
et al., Science. 337:816-821(2012); Qi et al., "Repurposing CRISPR
as an RNA-Guided Platform for Sequence-Specific Control of Gene
Expression" (2013) Cell. 28;152(5):1173-83, the entire contents of
each of which are incorporated herein by reference). For example,
the DNA cleavage domain of Cas9 is known to include two subdomains,
the HNH nuclease subdomain and the RuvC1 subdomain. The HNH
subdomain cleaves the strand complementary to the gRNA, whereas the
RuvC1 subdomain cleaves the non-complementary strand. Mutations
within these subdomains can silence the nuclease activity of Cas9.
For example, the mutations D10A and H840A completely inactivate the
nuclease activity of S. pyogenes Cas9 (Jinek et al., Science.
337:816-821(2012); Qi et al., Cell. 28;152(5):1173-83 (2013)). In
some embodiments, proteins comprising Cas9 or fragments thereof are
referred to as "Cas9 variants." A Cas9 variant shares homology to
Cas9, or a fragment thereof.
[0054] In some embodiments, the Cas protein may be a fusion protein
comprising a dCas9 domain and a functional protein. Non-limiting
examples of Cas9 fusion proteins are dCas9-transcription factor,
dCas9-VP64, dCas9-VPR, dCas9-Suntag, dCas9-P300, dCas9-VP160,
dCas9VP192, dCas9-KRAB and its derivative, dCas9-MXI1, dCas9-SID4X,
dCas9-LSD1, dCas9-CIB1, dCas9-GFP, and dCas9-RFP. Additional
suitable Cas9 fusion proteins will be apparent to those of skill in
the art based on this disclosure and knowledge in the field, and
are within the scope of this disclosure.
[0055] Also within the scope of the present disclosure is an
engineered nucleic acid that encodes the engineered RNA described
herein. A "nucleic acid" is at least two nucleotides covalently
linked together, and in some instances, may contain phosphodiester
bonds (e.g., a phosphodiester "backbone"). A nucleic acid may be
DNA, both genomic and/or cDNA, RNA or a hybrid, where the nucleic
acid contains any combination of deoxyribonucleotides and
ribonucleotides (e.g., artificial or natural), and any combination
of bases, including uracil, adenine, thymine, cytosine, guanine,
inosine, xanthine, hypoxanthine, isocytosine and isoguanine.
Nucleic acids of the present disclosure may be produced using
standard molecular biology methods (see, e.g., Green and Sambrook,
Molecular Cloning, A Laboratory Manual, 2012, Cold Spring Harbor
Press).
[0056] In some embodiments, the engineered nucleic acids comprise a
promoter operably linked to a nucleotide sequence encoding the
engineered RNA described herein. A "promoter" refers to a control
region of a nucleic acid sequence at which initiation and rate of
transcription of the remainder of a nucleic acid sequence are
controlled. A promoter drives expression or drives transcription of
the nucleic acid sequence that it regulates. A promoter may also
contain sub-regions at which regulatory proteins and molecules may
bind, such as RNA polymerase and other transcription factors.
Promoters may be constitutive, inducible, activatable, repressible,
tissue-specific or any combination thereof. A promoter is
considered to be "operably linked" when it is in a correct
functional location and orientation in relation to a nucleic acid
sequence it regulates to control ("drive") transcriptional
initiation and/or expression of that sequence.
[0057] A promoter may be one naturally associated with a gene or
sequence, as may be obtained by isolating the 5' non-coding
sequences located upstream of the coding segment of a given gene or
sequence. Such a promoter can be referred to as "endogenous."
[0058] In some embodiments, a nucleic acid sequence may be
positioned under the control of a recombinant or heterologous
promoter, which refers to a promoter that is not normally
associated with the encoded sequence in its natural environment.
Such promoters may include promoters of other genes; promoters
isolated from any other cell; and synthetic promoters or enhancers
that are not "naturally occurring" such as, for example, those that
contain different elements of different transcriptional regulatory
regions and/or mutations that alter expression through methods of
genetic engineering that are known in the art. In addition to
producing nucleic acid sequences of promoters and enhancers
synthetically, sequences may be produced using recombinant cloning
and/or nucleic acid amplification technology, including polymerase
chain reaction (PCR) (see U.S. Pat. No. 4,683,202 and U.S. Pat. No.
5,928,906).
[0059] In some embodiments, a promoter is a constitutive promoter.
Examples of constitutive promoters include, without limitation, the
retroviral Rous sarcoma virus (RSV) LTR promoter (optionally with
the RSV enhancer), the cytomegalovirus (CMV) promoter (optionally
with the CMV enhancer) [see, e.g., Boshart et al., Cell, 41:521-530
(1985)], the SV40 promoter, the dihydrofolate reductase promoter,
the .beta.-actin promoter, the phosphoglycerol kinase (PGK)
promoter, and the EF1.alpha. promoter [Invitrogen]. In some
embodiments, a promoter is an enhanced chicken (3-actin promoter.
In some embodiments, a promoter is a U6 promoter.
[0060] In some embodiments, a promoter is an "inducible promoter,"
which refer to a promoter that is characterized by regulating
(e.g., initiating or activating) transcriptional activity when in
the presence of, influenced by or contacted by an inducer signal.
An inducer signal may be endogenous or a normally exogenous
condition (e.g., light), compound (e.g., chemical or non-chemical
compound) or protein that contacts an inducible promoter in such a
way as to be active in regulating transcriptional activity from the
inducible promoter. Thus, a "signal that regulates transcription"
of a nucleic acid refers to an inducer signal that acts on an
inducible promoter. A signal that regulates transcription may
activate or inactivate transcription, depending on the regulatory
system used. Activation of transcription may involve directly
acting on a promoter to drive transcription or indirectly acting on
a promoter by inactivation a repressor that is preventing the
promoter from driving transcription. Conversely, deactivation of
transcription may involve directly acting on a promoter to prevent
transcription or indirectly acting on a promoter by activating a
repressor that then acts on the promoter. An inducible promoter of
the present disclosure may be induced by (or repressed by) one or
more physiological condition(s), such as changes in light, pH,
temperature, radiation, osmotic pressure, saline gradients, cell
surface binding, and the concentration of one or more extrinsic or
intrinsic inducing agent(s). An extrinsic inducer signal or
inducing agent may comprise, without limitation, amino acids and
amino acid analogs, saccharides and polysaccharides, nucleic acids,
protein transcriptional activators and repressors, cytokines,
toxins, petroleum-based compounds, metal containing compounds,
salts, ions, enzyme substrate analogs, hormones or combinations
thereof.
[0061] Inducible promoters of the present disclosure include any
inducible promoter described herein or known to one of ordinary
skill in the art. Examples of inducible promoters include, without
limitation, chemically/biochemically-regulated and
physically-regulated promoters such as alcohol-regulated promoters,
tetracycline-regulated promoters (e.g., anhydrotetracycline
(aTc)-responsive promoters and other tetracycline-responsive
promoter systems, which include a tetracycline repressor protein
(tetR), a tetracycline operator sequence (tetO) and a tetracycline
transactivator fusion protein (tTA)), steroid-regulated promoters
(e.g., promoters based on the rat glucocorticoid receptor, human
estrogen receptor, moth ecdysone receptors, and promoters from the
steroid/retinoid/thyroid receptor superfamily), metal-regulated
promoters (e.g., promoters derived from metallothionein (proteins
that bind and sequester metal ions) genes from yeast, mouse and
human), pathogenesis-regulated promoters (e.g., induced by
salicylic acid, ethylene or benzothiadiazole (BTH)),
temperature/heat-inducible promoters (e.g., heat shock promoters),
and light-regulated promoters (e.g., light responsive promoters
from plant cells).
[0062] Also within the scope of the present disclosure are cells
comprising the engineered RNA described herein and the engineered
nucleic acid encoding the same described herein. The cell can be
any cell suitable for expressing the engineered RNA described
herein. In some embodiments, the cells are prokaryotic cells. In
some embodiments, the cells are bacteria cells. In other
embodiments, the cells are eukaryotic cells. In some embodiments,
the cells are mammalian cells. In other examples, the cells are
human cells or non-human cells. Non-limiting example for non-human
cells can be non-human mammalian cells, plant cells, insect cells,
bacterial cells or fungal cells (including yeast cells). In some
embodiments, the cell is a specific cell type in tissue. In some
embodiments, the cell is a specific diseased cell. In some
embodiments, the cell comprises the input signal necessary to
trigger the conformational change of the engineered RNA. In some
embodiments, the cell is a disease cell. In some embodiments, the
cell is from a specific tissue. Non-limiting examples of the
tissues are lung tissue, skin tissue, breast tissue, connective
tissue, brain tissue, gastrointestinal tissue, heart tissue, kidney
tissue, etc. Non-limiting examples for specific cell types are
epithelial cells, endothelial cells, fibroblasts, immune cells,
etc. Non-limiting examples of a diseased cells are neo-plastic
cells, infected cells, cells harboring genetic mutations, fibro
genetic cells, etc. The engineered RNA described herein, the
engineered nucleic acid and/or the vectors can be delivered to the
cells by methods known in the art. Non-limiting methods of delivery
is transfection (e.g., electroporation, or liposome), viral
particles (e.g., adeno-associated virus), nanoparticles (e.g.,
lipid nanoparticles), or genomic integration. In some embodiments,
the engineered nucleic acid described herein is integrated into the
genomic DNA of the cell. Genomic integration of the present
engineered nucleic acid can be done by methods known in the art. In
some embodiments, the genomic integration of the present engineered
nucleic acid can be achieved by viral transduction (e.g., including
but not limited to lentiviral vectors, retroviral vectors, PiggyBac
transposon vector and SleepingBeauty transposon vector) and
introduced into host immune cells using conventional recombinant
technology. Sambrook et al., Molecular Cloning, A Laboratory
Manual, 3rd Ed., Cold Spring Harbor Laboratory Press.
[0063] Also provided herein are organisms comprising the engineered
RNA, the engineered nucleic acid encoding the same, the vector
and/or cells described herein. Exemplary organisms can be
prokaryotic organisms or eukaryotic organisms. In some embodiments,
the prokaryotic organism is a bacteria. In some embodiments, the
eukaryotic organism is an animal, a plant, or a fungus. In some
embodiments, the eukaryotic organism is an animal. In some further
examples, the animal is a non-human animal. Non-limiting examples
of non-human animals are mice, chickens, goats, rabbits, pigs,
donkeys, cows, or camels.
II. RECOMBINANT VIRUSES FOR DELIVERY OF THE ENGINEERED RNA
[0064] Also within the scope of the present disclosure are the
delivery of the engineered nucleic acid encoding the engineered RNA
described herein by recombinant viruses. Non-limiting examples of
such recombinant viruses are adeno-associated viruses, lentivirus,
alphavirus, adeno virus, or bacteriophage.
[0065] In some embodiments, the engineered nucleic acid encoding
the engineered RNA described herein are delivered by
adeno-associated viruses (AAV). The engineered nucleic acid
encoding the engineered RNA described herein may be recombinant
adeno-associated virus (AAV) vectors (rAAV vectors). In some
embodiments, an engineered nucleic acid encoding the engineered RNA
described herein as described by the disclosure comprises a first
adeno-associated virus (AAV) inverted terminal repeat (ITR) and a
second AAV ITR, or a variant thereof. The isolated nucleic acid
(e.g., the recombinant AAV vector) may be packaged into a capsid
protein and administered to a subject and/or delivered to a
selected target cell. "Recombinant AAV (rAAV) vectors" are
typically composed of, at a minimum, a transgene and its regulatory
sequences, and 5' and 3' AAV inverted terminal repeats (ITRs). The
engineered RNA described herein coding sequence may also comprise a
region encoding, for example, a protein and/or an expression
control sequence (e.g., a poly-A tail), as described elsewhere in
the disclosure.
[0066] Generally, ITR sequences are about 145 bp in length.
Preferably, substantially the entire sequences encoding the ITRs
are used in the molecule, although some degree of minor
modification of these sequences is permissible. The ability to
modify these ITR sequences is within the skill of the art. (See,
e.g., texts such as Sambrook et al., "Molecular Cloning. A
Laboratory Manual", 2d ed., Cold Spring Harbor Laboratory, New York
(1989); and K. Fisher et al., J Virol., 70:520 532 (1996)). An
example of such a molecule employed in the present invention is a
"cis-acting" plasmid containing the engineered RNA described herein
coding sequence, in which the selected the engineered RNA described
herein coding sequence and associated regulatory elements are
flanked by the 5' and 3' AAV ITR sequences. The AAV ITR sequences
may be obtained from any known AAV, including presently identified
mammalian AAV types. In some embodiments, the isolated nucleic acid
(e.g., the rAAV vector) comprises at least one ITR having a
serotype selected from AAV1, AAV2, AAV5, AAV6, AAV6.2, AAV7, AAV8,
AAV9, AAV10, AAV11, and variants thereof. In some embodiments, the
isolated nucleic acid comprises a region (e.g., a first region)
encoding an AAV2 ITR.
[0067] In addition to the major elements identified above for the
recombinant AAV vector, the vector also includes conventional
control elements which are operably linked with elements of the
transgene in a manner that permits its transcription, translation
and/or expression in a cell transfected with the vector or infected
with the virus produced by the invention. As used herein, "operably
linked" sequences include both expression control sequences that
are contiguous with the gene of interest and expression control
sequences that act in trans or at a distance to control the gene of
interest. Expression control sequences include appropriate
transcription initiation, termination, promoter and enhancer
sequences; efficient RNA processing signals such as splicing and
polyadenylation (polyA) signals; sequences that stabilize
cytoplasmic mRNA; and when desired, sequences that enhance
secretion of the encoded product. A number of expression control
sequences, including promoters which are native, constitutive,
inducible and/or tissue-specific, are known in the art and may be
utilized. In some embodiments, the engineered RNA described herein
coding sequence is operably linked to a suitable promoter described
herein above.
[0068] In some aspects, the disclosure provides isolated AAVs
(e.g., rAAVs encoding the engineered RNA described herein). As used
herein with respect to AAVs, the term "isolated" refers to an AAV
that has been artificially produced or obtained. Isolated AAVs may
be produced using recombinant methods. Such AAVs are referred to
herein as "recombinant AAVs." Recombinant AAVs (rAAVs) preferably
have tissue-specific targeting capabilities, such that a nuclease
and/or transgene of the rAAV will be delivered specifically to one
or more predetermined tissue(s). The AAV capsid is an important
element in determining these tissue-specific targeting
capabilities. Thus, an rAAV having a capsid appropriate for the
tissue being targeted can be selected. In some embodiments, an rAAV
expressing the engineered RNA described herein is capable of
increasing tissue or cell specificity such that the engineered RNA
described herein can only function in the cells having the input
signal that the rAAV can infect.
[0069] Methods for obtaining recombinant AAVs (e.g., encoding the
engineered RNA described herein) having a desired capsid protein
are well known in the art. (See, for example, US 2003/0138772), the
contents of which are incorporated herein by reference in their
entirety). Typically the methods involve culturing a host cell
which contains a nucleic acid sequence encoding an AAV capsid
protein; a functional rep gene; a recombinant AAV vector composed
of, AAV inverted terminal repeats (ITRs) and a transgene; and
sufficient helper functions to permit packaging of the recombinant
AAV vector into the AAV capsid proteins. In some embodiments,
capsid proteins are structural proteins encoded by the cap gene of
an AAV. AAVs comprise three capsid proteins, virion proteins 1 to 3
(named VP1, VP2 and VP3), all of which are transcribed from a
single cap gene via alternative splicing. In some embodiments, the
molecular weights of VP1, VP2 and VP3 are respectively about 87
kDa, about 72 kDa and about 62 kDa. In some embodiments, upon
translation, capsid proteins form a spherical 60-mer protein shell
around the viral genome. In some embodiments, the functions of the
capsid proteins are to protect the viral genome, deliver the genome
and interact with the host. In some aspects, capsid proteins
deliver the viral genome to a host in a tissue specific manner.
[0070] In some embodiments, the rAAV (e.g., encoding the engineered
RNA described herein) comprises an AAV capsid protein is of an AAV
serotype selected from the group consisting of AAV2, AAV3, AAV4,
AAV5, AAV6, AAV8, AAVrh8, AAV9, and AAV10.
[0071] The components to be cultured in the host cell to package a
rAAV vector in an AAV capsid may be provided to the host cell in
trans. Alternatively, any one or more of the required components
(e.g., recombinant AAV vector, rep sequences, cap sequences, and/or
helper functions) may be provided by a stable host cell which has
been engineered to contain one or more of the required components
using methods known to those of skill in the art. Most suitably,
such a stable host cell will contain the required component(s)
under the control of an inducible promoter. However, the required
component(s) may be under the control of a constitutive promoter.
Examples of suitable inducible and constitutive promoters are
provided herein, in the discussion of regulatory elements suitable
for use with the transgene. In still another alternative, a
selected stable host cell may contain selected component(s) under
the control of a constitutive promoter and other selected
component(s) under the control of one or more inducible promoters.
For example, a stable host cell may be generated which is derived
from 293 cells (which contain E1 helper functions under the control
of a constitutive promoter), but which contain the rep and/or cap
proteins under the control of inducible promoters. Still other
stable host cells may be generated by one of skill in the art.
[0072] The recombinant AAV vector, rep sequences, cap sequences,
and helper functions required for producing the rAAV of the
disclosure may be delivered to the packaging host cell using any
appropriate genetic element (vector). The selected genetic element
may be delivered by any suitable method, including those described
herein. The methods used to construct any embodiment of this
disclosure are known to those with skill in nucleic acid
manipulation and include genetic engineering, recombinant
engineering, and synthetic techniques. See, e.g., Sambrook et al.,
Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press,
Cold Spring Harbor, N.Y. Similarly, methods of generating rAAV
virions are well known and the selection of a suitable method is
not a limitation on the present disclosure. See, e.g., K. Fisher et
al., J. Virol., 70:520-532 (1993) and U.S. Pat. No. 5,478,745.
[0073] In some embodiments, recombinant AAVs may be produced using
the triple transfection method (described in detail in U.S. Pat.
No. 6,001,650). Typically, the recombinant AAVs are produced by
transfecting a host cell with an recombinant AAV vector (comprising
a transgene) to be packaged into AAV particles, an AAV helper
function vector, and an accessory function vector. An AAV helper
function vector encodes the "AAV helper function" sequences (i.e.,
rep and cap), which function in trans for productive AAV
replication and encapsidation. Preferably, the AAV helper function
vector supports efficient AAV vector production without generating
any detectable wild-type AAV virions (i.e., AAV virions containing
functional rep and cap genes). Non-limiting examples of vectors
suitable for use with the present disclosure include pHLP19,
described in U.S. Pat. No. 6,001,650 and pRep6cap6 vector,
described in U.S. Pat. No. 6,156,303, the entirety of both
incorporated by reference herein. The accessory function vector
encodes nucleotide sequences for non-AAV derived viral and/or
cellular functions upon which AAV is dependent for replication
(i.e., "accessory functions"). The accessory functions include
those functions required for AAV replication, including, without
limitation, those moieties involved in activation of AAV gene
transcription, stage specific AAV mRNA splicing, AAV DNA
replication, synthesis of cap expression products, and AAV capsid
assembly. Viral-based accessory functions can be derived from any
of the known helper viruses such as adenovirus, herpesvirus (other
than herpes simplex virus type-1), and vaccinia virus.
[0074] In some aspects, the disclosure provides transfected host
cells. The term "transfection" is used to refer to the uptake of
foreign DNA by a cell, and a cell has been "transfected" when
exogenous DNA has been introduced inside the cell membrane. A
number of transfection techniques are generally known in the art.
See, e.g., Graham et al. (1973) Virology, 52:456, Sambrook et al.
(1989) Molecular Cloning, a laboratory manual, Cold Spring Harbor
Laboratories, New York, Davis et al. (1986) Basic Methods in
Molecular Biology, Elsevier, and Chu et al. (1981) Gene 13:197.
Such techniques can be used to introduce one or more exogenous
nucleic acids, such as a nucleotide integration vector and other
nucleic acid molecules, into suitable host cells. A "host cell"
refers to any cell that harbors, or is capable of harboring, a
substance of interest. Often a host cell is a mammalian cell. A
host cell may be used as a recipient of an AAV helper construct, an
AAV minigene plasmid, an accessory function vector, or other
transfer DNA associated with the production of recombinant AAVs.
The term includes the progeny of the original cell which has been
transfected. Thus, a "host cell" as used herein may refer to a cell
which has been transfected with an exogenous DNA sequence. It is
understood that the progeny of a single parental cell may not
necessarily be completely identical in morphology or in genomic or
total DNA complement as the original parent, due to natural,
accidental, or deliberate mutation. As used herein, the term "cell
line" refers to a population of cells capable of continuous or
prolonged growth and division in vitro. Often, cell lines are
clonal populations derived from a single progenitor cell. It is
further known in the art that spontaneous or induced changes can
occur in karyotype during storage or transfer of such clonal
populations. Therefore, cells derived from the cell line referred
to may not be precisely identical to the ancestral cells or
cultures, and the cell line referred to includes such variants.
[0075] In some embodiments, an rAAV described herein (e.g.,
encoding the engineered RNA described herein) is a single stranded
rAAV. An ssAAV, as used herein, refers to a rAAV with the coding
sequence and complementary sequence of the transgene expression
cassette on separate strands and are packaged in separate viral
capsids. In some embodiments, the rAAV (e.g., encoding the
engineered RNA described herein) is a self-complementary AAV
(scAAV). A scAAV, as used herein, refers to an rAAV with both the
coding and complementary sequence of the transgene expression
cassette are present on each plus-and minus-strand genome. The
coding region of a scAAV was designed to form an intra-molecular
double-stranded DNA template. Upon infection, rather than waiting
for cell mediated synthesis of the second strand, the two
complementary halves of scAAV will associate to form one double
stranded DNA (dsDNA) unit that is ready for immediate replication
and transcription.
[0076] In some embodiments, when the engineered RNA is an sgRNA,
the Cas protein is also provided to the cell by an rAAV. In some
embodiments, the Cas protein is saCas9. In some embodiments, the
saCas9 is delivered to the cell by a single rAAV. In some
embodiments, the Cas protein is not saCas9. In some embodiments,
the Cas protein can be delivered to the cell by a dual AAV system.
In some embodiments, a first rAAV delivers a portion of the Cas
protein, and a second rAAV delivers a second portion of the Cas
protein. A full length Cas protein coding sequence can be produced
by trans-splicing or by homologous recombination of the two AAV
genome.
III. PHARMACEUTICAL COMPOSITIONS
[0077] In some aspects, the present disclosure, at least in part,
relates to a pharmaceutical composition, comprising engineered RNA
described herein, the engineered nucleic acid, the recombinant
virus, the cells, as described herein. The pharmaceutical
composition described herein may further comprise a
pharmaceutically acceptable carrier (excipient) to form a
pharmaceutical composition for use in treating a target disease.
"Acceptable" means that the carrier must be compatible with the
active ingredient of the composition (and preferably, capable of
stabilizing the active ingredient) and not deleterious to the
subject to be treated. Pharmaceutically acceptable excipients
(carriers) including buffers, which are well known in the art. See,
e.g., Remington: The Science and Practice of Pharmacy 20th Ed.
(2000) Lippincott Williams and Wilkins, Ed. K. E. Hoover.
[0078] The pharmaceutical compositions to be used for in vivo
administration must be sterile. This is readily accomplished by,
for example, filtration through sterile filtration membranes. The
pharmaceutical compositions described herein may be placed into a
container having a sterile access port, for example, an intravenous
solution bag or vial having a stopper pierceable by a hypodermic
injection needle.
[0079] In other embodiments, the pharmaceutical compositions
described herein can be formulated for intra-muscular injection,
intravenous injection, intratumoral injection or subcutaneous
injection.
[0080] The pharmaceutical compositions described herein to be used
in the present methods can comprise pharmaceutically acceptable
carriers, buffer agents, excipients, salts, or stabilizers in the
form of lyophilized formulations or aqueous solutions. See, e.g.,
Remington: The Science and Practice of Pharmacy 20th Ed. (2000)
Lippincott Williams and Wilkins, Ed. K. E. Hoover). Acceptable
carriers, excipients, or stabilizers are nontoxic to recipients at
the dosages and concentrations used, and may comprise buffers such
as phosphate, citrate, and other organic acids; antioxidants
including ascorbic acid and methionine; preservatives (such as
octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride;
benzalkonium chloride, benzethonium chloride; phenol, butyl or
benzyl alcohol; alkyl parabens such as methyl or propyl paraben;
catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low
molecular weight (less than about 10 residues) polypeptides;
proteins, such as serum albumin, gelatin, or immunoglobulins;
hydrophilic polymers such as polyvinylpyrrolidone; amino acids such
as glycine, glutamine, asparagine, histidine, arginine, or lysine;
monosaccharides, disaccharides, and other carbohydrates including
glucose, mannose, or dextrans; chelating agents such as EDTA;
sugars such as sucrose, mannitol, trehalose or sorbitol;
salt-forming counter-ions such as sodium; metal complexes (e.g.,
Zn-protein complexes); and/or non-ionic surfactants such as
TWEEN.TM., PLURONICS.TM. or polyethylene glycol (PEG).
[0081] In some examples, the pharmaceutical composition described
herein comprises lipid nanoparticles which can be prepared by
methods known in the art, such as described in Epstein, et al.,
Proc. Natl. Acad. Sci. USA 82:3688 (1985); Hwang, et al., Proc.
Natl. Acad. Sci. USA 77:4030 (1980); and U.S. Pat. Nos. 4,485,045
and 4,544,545. Liposomes with enhanced circulation time are
disclosed in U.S. Pat. No. 5,013,556. Particularly useful liposomes
can be generated by the reverse phase evaporation method with a
lipid composition comprising phosphatidylcholine, cholesterol and
PEG-derivatized phosphatidylethanolamine (PEG-PE). Liposomes are
extruded through filters of defined pore size to yield liposomes
with the desired diameter.
[0082] In other examples, the pharmaceutical composition described
herein can be formulated in sustained-release format. Suitable
examples of sustained-release preparations include semipermeable
matrices of solid hydrophobic polymers containing the engineered
RNA described herein, the nucleic acid encoding the same, the
recombinant virus encoding the same or the cell comprising the
same, which matrices are in the form of shaped articles, e.g.,
films, or microcapsules. Examples of sustained-release matrices
include polyesters, hydrogels (for example,
poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)),
polylactides (U.S. Pat. No. 3,773,919), copolymers of L-glutamic
acid and 7 ethyl-L-glutamate, non-degradable ethylene-vinyl
acetate, degradable lactic acid-glycolic acid copolymers such as
the LUPRON DEPOTTM (injectable microspheres composed of lactic
acid-glycolic acid copolymer and leuprolide acetate), sucrose
acetate isobutyrate, and poly-D-(-)-3-hydroxybutyric acid.
[0083] Suitable surface-active agents include, in particular,
non-ionic agents, such as polyoxyethylenesorbitans (e.g., TWEEN.TM.
20, 40, 60, 80 or 85) and other sorbitans (e.g., SPAN.TM. 20, 40,
60, 80 or 85). Compositions with a surface-active agent will
conveniently comprise between 0.05 and 5% surface-active agent, and
can be between 0.1 and 2.5%. It will be appreciated that other
ingredients may be added, for example mannitol or other
pharmaceutically acceptable vehicles, if necessary.
[0084] The pharmaceutical compositions described herein can be in
unit dosage forms such as tablets, pills, capsules, powders,
granules, solutions or suspensions, or suppositories, for oral,
parenteral or rectal administration, or administration by
inhalation or insufflation.
[0085] For preparing solid compositions such as tablets, the
principal active ingredient can be mixed with a pharmaceutical
carrier, e.g., conventional tableting ingredients such as corn
starch, lactose, sucrose, sorbitol, talc, stearic acid, magnesium
stearate, dicalcium phosphate or gums, and other pharmaceutical
diluents, e.g., water, to form a solid preformulation composition
containing a homogeneous mixture of a compound of the present
invention, or a non-toxic pharmaceutically acceptable salt thereof.
When referring to these preformulation compositions as homogeneous,
it is meant that the active ingredient is dispersed evenly
throughout the composition so that the composition may be readily
subdivided into equally effective unit dosage forms such as
tablets, pills and capsules. This solid preformulation composition
is then subdivided into unit dosage forms of the type described
above containing from 0.1 to about 500 mg of the active ingredient
of the present invention. The tablets or pills of the novel
composition can be coated or otherwise compounded to provide a
dosage form affording the advantage of prolonged action. For
example, the tablet or pill can comprise an inner dosage and an
outer dosage component, the latter being in the form of an envelope
over the former. The two components can be separated by an enteric
layer that serves to resist disintegration in the stomach and
permits the inner component to pass intact into the duodenum or to
be delayed in release. A variety of materials can be used for such
enteric layers or coatings, such materials including a number of
polymeric acids and mixtures of polymeric acids with such materials
as shellac, cetyl alcohol and cellulose acetate.
[0086] Suitable emulsions may be prepared using commercially
available fat emulsions, such as INTRALIPID.TM., LIPOSYN.TM.,
INFONUTROL.TM., LIPOFUNDIN.TM. and LIPIPHYSAN.TM.. The active
ingredient may be either dissolved in a pre-mixed emulsion
composition or alternatively it may be dissolved in an oil (e.g.,
soybean oil, safflower oil, cottonseed oil, sesame oil, corn oil or
almond oil) and an emulsion formed upon mixing with a phospholipid
(e.g., egg phospholipids, soybean phospholipids or soybean
lecithin) and water. It will be appreciated that other ingredients
may be added, for example glycerol or glucose, to adjust the
tonicity of the emulsion. Suitable emulsions will typically contain
up to 20% oil, for example, between 5 and 20%. The fat emulsion can
comprise fat droplets having a suitable size and can have a pH in
the range of 5.5 to 8.0.
[0087] Pharmaceutical compositions for inhalation or insufflation
include solutions and suspensions in pharmaceutically acceptable,
aqueous or organic solvents, or mixtures thereof, and powders. The
liquid or solid compositions may contain suitable pharmaceutically
acceptable excipients as set out above. In some embodiments, the
compositions are administered by the oral or nasal respiratory
route for local or systemic effect.
[0088] Compositions in preferably sterile pharmaceutically
acceptable solvents may be nebulized by use of gases. Nebulized
solutions may be breathed directly from the nebulizing device or
the nebulizing device may be attached to a face mask, tent or
intermittent positive pressure breathing machine. Solution,
suspension or powder compositions may be administered, preferably
orally or nasally, from devices which deliver the formulation in an
appropriate manner.
IV. THERAPEUTIC APPLICATIONS
[0089] The engineered RNAs, the engineered nucleic acids, the
recombinant viruses, the host cells and the pharmaceutical
compositions described herein can be used to treat various diseases
(e.g., disease cells having the input signal).
[0090] To practice the method disclosed herein, an effective amount
of any of the engineered RNAs, the engineered nucleic acids, the
recombinant viruses, the host cells, or the pharmaceutical
compositions described herein can be administered to a subject
(e.g., a human) in need of the treatment via a suitable route, such
as intratumoral administration, by intravenous administration,
e.g., as a bolus or by continuous infusion over a period of time,
by intramuscular, intraperitoneal, intracerebrospinal,
subcutaneous, intra-articular, intrasynovial, intrathecal, oral,
inhalation or topical routes. Commercially available nebulizers for
liquid formulations, including jet nebulizers and ultrasonic
nebulizers are useful for administration. Liquid formulations can
be directly nebulized and lyophilized powder can be nebulized after
reconstitution. Alternatively, pharmaceutical composition described
herein can be aerosolized using a fluorocarbon formulation and a
metered dose inhaler, or inhaled as a lyophilized and milled
powder. In some examples, the pharmaceutical composition described
herein is formulated for intratumoral injection. In particular
examples, the pharmaceutical composition may be administered to a
subject (e.g., a human patient) via a local route, for example,
injected to a local site such as a tumor site or an infectious
site.
[0091] As used herein, "an effective amount" refers to the amount
of each active agent required to confer therapeutic effect on the
subject, either alone or in combination with one or more other
active agents. For example, the therapeutic effect can be reduced
tumor burden, reduction of cancer cells, increased immune activity,
reduction of a mutated protein, reduction of over-active immune
response. Determination of whether an amount of engineered RNA
described herein achieved the therapeutic effect would be evident
to one of skill in the art. Effective amounts vary, as recognized
by those skilled in the art, depending on the particular condition
being treated, the severity of the condition, the individual
patient parameters including age, physical condition, size, gender
and weight, the duration of the treatment, the nature of concurrent
therapy (if any), the specific route of administration and like
factors within the knowledge and expertise of the health
practitioner. These factors are well known to those of ordinary
skill in the art and can be addressed with no more than routine
experimentation. It is generally preferred that a maximum dose of
the individual components or combinations thereof be used, that is,
the highest safe dose according to sound medical judgment.
[0092] Empirical considerations, such as the half-life, generally
will contribute to the determination of the dosage. Frequency of
administration may be determined and adjusted over the course of
therapy, and is generally, but not necessarily, based on treatment
and/or suppression and/or amelioration and/or delay of a target
disease/disorder. Alternatively, sustained continuous release
formulations of pharmaceutical composition described herein may be
appropriate. Various formulations and devices for achieving
sustained release are known in the art.
[0093] In some embodiments, the treatment is a single injection of
the engineered RNAs, the engineered nucleic acids, the recombinant
viruses, the host cells or the pharmaceutical compositions
described herein. In some embodiments, the method described herein
comprises administering to a subject in need of the treatment
(e.g., a human patient) one or multiple doses of the engineered
RNAs, the engineered nucleic acids, the recombinant viruses, the
host cells or the pharmaceutical compositions described herein.
[0094] In some example, dosages for an engineered RNA, engineered
nucleic acid, recombinant virus, host cell or pharmaceutical
composition described herein (each a "therapeutic comprising the
engineered RNA described herein") may be determined empirically in
individuals who have been given one or more administration(s) of
such a therapeutic. Individuals are given incremental dosages of
the engineered RNA, engineered nucleic acid, recombinant virus,
host cell or pharmaceutical composition described herein. To assess
efficacy of a therapeutic comprising the engineered RNA described
herein, an indicator of the disease/disorder can be followed. For
repeated administrations over several days or longer, depending on
the condition, the treatment is sustained until a desired
suppression of symptoms occurs or until sufficient therapeutic
levels are achieved to alleviate a target disease or disorder, or a
symptom thereof.
[0095] In some embodiments, dosing frequency is once every week,
every 2 weeks, every 4 weeks, every 5 weeks, every 6 weeks, every 7
weeks, every 8 weeks, every 9 weeks, or every 10 weeks; or once
every month, every 2 months, or every 3 months, or longer. The
progress of this therapy is easily monitored by conventional
techniques and assays. The dosing regimen of the therapeutic
comprising the engineered RNA described herein used can vary over
time.
[0096] For the purpose of the present disclosure, the appropriate
dosage of the therapeutic comprising the engineered RNA described
herein will depend on the specific miRNA signature of the cell and
the miRNA to be expressed, the type and severity of the
disease/disorder, the pharmaceutical composition described herein
is administered for preventive or therapeutic purposes, previous
therapy, the patient's clinical history and response to the
engineered RNA described herein, and the discretion of the
attending physician. A clinician may administer a therapeutic
comprising the engineered RNA described herein, until a dosage is
reached that achieves the desired result. Methods of determining
whether a dosage resulted in the desired result would be evident to
one of skill in the art. Administration of one or more therapeutic
comprising the engineered RNA described herein can be continuous or
intermittent, depending, for example, upon the recipient's
physiological condition, whether the purpose of the administration
is therapeutic or prophylactic, and other factors known to skilled
practitioners. The administration of a therapeutic comprising the
engineered RNA described herein may be essentially continuous over
a preselected period of time or may be in a series of spaced dose,
e.g., either before, during, or after developing a target disease
or disorder.
[0097] As used herein, the term "treating" refers to the
application or administration of a composition including one or
more active agents to a subject, who has a target disease or
disorder, a symptom of the disease/disorder, or a predisposition
toward the disease/disorder, with the purpose to cure, heal,
alleviate, relieve, alter, remedy, ameliorate, improve, or affect
the disorder, the symptom of the disease, or the predisposition
toward the disease or disorder.
[0098] Alleviating a target disease/disorder includes delaying the
development or progression of the disease, or reducing disease
severity. Alleviating the disease does not necessarily require
curative results. As used therein, "delaying" the development of a
target disease or disorder means to defer, hinder, slow, retard,
stabilize, and/or postpone progression of the disease. This delay
can be of varying lengths of time, depending on the history of the
disease and/or individuals being treated. A method that "delays" or
alleviates the development of a disease, or delays the onset of the
disease, is a method that reduces probability of developing one or
more symptoms of the disease in a given time frame and/or reduces
extent of the symptoms in a given time frame, when compared to not
using the method. Such comparisons are typically based on clinical
studies, using a number of subjects sufficient to give a
statistically significant result.
[0099] "Development" or "progression" of a disease means initial
manifestations and/or ensuing progression of the disease.
Development of the disease can be detectable and assessed using
standard clinical techniques as well known in the art. However,
development also refers to progression that may be undetectable.
For purpose of this disclosure, development or progression refers
to the biological course of the symptoms. "Development" includes
occurrence, recurrence, and onset. As used herein "onset" or
"occurrence" of a target disease or disorder includes initial onset
and/or recurrence.
[0100] The subject to be treated by the methods described herein
can be a mammal, such as a human, farm animals, sport animals,
pets, primates, horses, dogs, cats, mice and rats. In one
embodiment, the subject is a human.
[0101] In some embodiments, the subject may be a human patient
having, suspected of having, or at risk for a disease. Non-limiting
examples of diseases that are suitable for treatment with the
therapeutics comprising the engineered RNA described herein
include: Alpha-1 antitrypsin deficiency, Hypercholesterolemia,
Hepatitis B infection, Liver adenoma due to HIV infection,
Hepatitis C virus infection, Ornithine transcarbamylase deficiency,
Hepatocellular carcinoma, Amyotrophic lateral sclerosis,
Spinocerebellar ataxia type 1, Huntington's disease, Parkinson
disease, Spinal and Bulbar muscular atrophy, Pyruvate dehydrogenase
deficiency, Hyperplasia, obesity, facioscapulohumeral muscular
dystrophy (FSHD), Nerve Injury-induced Neuropathic Pain,
Age-related macular degeneration, Retinitis pigmentosa, heart
failure, cardiomyopathy, cold-induced cardiovascular dysfunction,
Asthma, Duchenne muscular dystrophy, infectious diseases, or
cancer.
[0102] Non limiting examples of cancers include melanoma, squamous
cell cancer, small-cell lung cancer, non-small cell lung cancer,
adenocarcinoma of the lung, squamous carcinoma of the lung, cancer
of the peritoneum, hepatocellular cancer, gastrointestinal cancer,
pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer,
liver cancer, bladder cancer, hepatoma, breast cancer, colon
cancer, colorectal cancer, endometrial or uterine carcinoma,
salivary gland carcinoma, kidney cancer, prostate cancer, vulval
cancer, thyroid cancer, hepatic carcinoma, gastric cancer, and
various types of head and neck cancer, including squamous cell head
and neck cancer. In some embodiments, the cancer can be melanoma,
lung cancer, colorectal cancer, renal-cell cancer, urothelial
carcinoma, or Hodgkin's lymphoma.
[0103] A subject having a target disease or disorder (e.g., cancer
or an infectious disease) can be identified by routine medical
examination, e.g., laboratory tests, organ functional tests, CT
scans, or ultrasounds. A subject suspected of having any of such
target disease/disorder might show one or more symptoms of the
disease/disorder. A subject at risk for the disease/disorder can be
a subject having one or more of the risk factors associated with
that disease/disorder. Such a subject can also be identified by
routine medical practices.
[0104] In some embodiments, a therapeutic comprising the engineered
RNA described herein may be co-used with another suitable
therapeutic agent (e.g., an anti-cancer agent an anti-viral agent,
or an anti-bacterial agent) and/or other agents that serve to
enhance effect of an engineered RNA described herein. In such
combined therapy, the therapeutic comprising the engineered RNA
described herein, and the additional therapeutic agent (e.g., an
anti-cancer therapeutic agent or others described herein) may be
administered to a subject in need of the treatment in a sequential
manner, i.e., each therapeutic agent is administered at a different
time. Alternatively, these therapeutic agents, or at least two of
the agents, are administered to the subject in a substantially
simultaneous manner. Combination therapy can also embrace the
administration of the therapeutic comprising the engineered RNA
described herein in further combination with other biologically
active ingredients (e.g., a different anti-cancer agent) and
non-drug therapies (e.g., surgery).
V. GENERAL TECHNIQUES
[0105] The practice of the present invention will employ, unless
otherwise indicated, conventional techniques of molecular biology
(including recombinant techniques), microbiology, cell biology,
biochemistry and immunology, which are within the skill of the art.
Molecular Cloning: A Laboratory Manual, second edition (Sambrook,
et al., 1989) Cold Spring Harbor Press; Oligonucleotide Synthesis
(M. J. Gait, ed., 1984); Methods in Molecular Biology, Humana
Press; Cell Biology: A Laboratory Notebook (J. E. Cellis, ed.,
1998) Academic Press; Animal Cell Culture (R. I. Freshney, ed.,
1987); Introduction to Cell and Tissue Culture (J. P. Mather and P.
E. Roberts, 1998) Plenum Press; Cell and Tissue Culture: Laboratory
Procedures (A. Doyle, J. B. Griffiths, and D. G. Newell, eds.,
1993-8) J. Wiley and Sons; Methods in Enzymology (Academic Press,
Inc.); Handbook of Experimental Immunology (D. M. Weir and C. C.
Blackwell, eds.); Gene Transfer Vectors for Mammalian Cells (J. M.
Miller and M. P. Calos, eds., 1987); Current Protocols in Molecular
Biology (F. M. Ausubel, et al., eds., 1987); PCR: The Polymerase
Chain Reaction, (Mullis, et al., eds., 1994); Current Protocols in
Immunology (J. E. Coligan et al., eds., 1991); Short Protocols in
Molecular Biology (Wiley and Sons, 1999); Immunobiology (C. A.
Janeway and P. Travers, 1997); Antibodies (P. Finch, 1997);
Antibodies: a practical approach (D. Catty., ed., IRL Press,
1988-1989); Monoclonal antibodies: a practical approach (P.
Shepherd and C. Dean, eds., Oxford University Press, 2000); Using
antibodies: a laboratory manual (E. Harlow and D. Lane (Cold Spring
Harbor Laboratory Press, 1999); The Antibodies (M. Zanetti and J.
D. Capra, eds., Harwood Academic Publishers, 1995). Without further
elaboration, it is believed that one skilled in the art can, based
on the above description, utilize the present invention to its
fullest extent. The following specific embodiments are, therefore,
to be construed as merely illustrative, and not limitative of the
remainder of the disclosure in any way whatsoever. All publications
cited herein are incorporated by reference for the purposes or
subject matter referenced herein.
[0106] Without further elaboration, it is believed that one skilled
in the art can, based on the above description, utilize the present
invention to its fullest extent. The following specific embodiments
are, therefore, to be construed as merely illustrative, and not
limitative of the remainder of the disclosure in any way
whatsoever. All publications cited herein are incorporated by
reference for the purposes or subject matter referenced herein.
EXAMPLES
Example 1: Gene Therapy in Mammalian Cells by the Use of the Strand
Displacement Reaction
[0107] In recent years, the use of RNA technologies has been
steadily increasing. A general framework for some of these
RNA-based technologies is the one where an RNA molecule interacts
with an input, changes conformation/folding state due to this
interaction and then, as consequence of that, it interacts with an
actuator. According to the specific design, the input and actuator
can be proteins, protein complexes, RNAs or also small
molecules.
[0108] While this framework is robust for its use in cell free
settings, in some cases, it has not been possible to use it in
genetically encoded circuits in mammalian cells. Here, the folding
of the RNA after the transcription from DNA can lead the RNA in a
conformation state that allows the RNA to interact with the
actuator even in case the input is absent. These unwanted side
reactions must be avoided to use this framework in genetically
encode circuits for gene therapy.
[0109] The technology disclosed herein provides clear design
principles to avoid these side reactions. It is based on the
general idea that an RNA strand can still interact with its related
actuator when the conformation of the RNA needed for this
interaction is at an energy state that is far from the lowest
one.
[0110] As an application of this technology, the use of the strand
displacement reaction in genetically encoded constructs in
mammalian cells was reliably and robustly enabled. Indeed, a
genetically encoded trans-activated gate-miRNA and a genetically
encoded trans-activated gate-gRNA (for cas9) were engineered;
importantly, the same design principles can be applied to other
CRISPR-based technologies. In these applications the inputs are
RNAs and the actuators are respectively the Drosha complex and
Cas9. The inputs interact with the gates through the strand
displacement reaction, but this is not limited to. Indeed, the
technology disclosed herein can be used with any technology that
uses the above-mentioned general framework.
[0111] The trans-activated gate-miRNA and the trans-activated
gate-gRNA disclosed herein are currently the only genetically
encoded constructs that allow respectively the downregulation of an
endogenous gene and DNA editing only in response to a specific RNAs
biomarker signature. This will finally allow the conditional
expression of therapeutic agents in cells that are characterized by
RNAs biomarkers instead of miRNAs biomarkers. Additionally, both
genetic constructs require a small DNA footprint and can be
successfully delivered in vivo for gene therapy by the use of the
AAV virus, which is considered among the safest viral vectors for
the delivery of exogenous DNA in vivo. Finally, the trans-activated
gate-miRNA does not use any exogenous protein and thus can be used
in gene therapy drastically reducing the likelihood of unwanted and
dangerous immune responses.
[0112] RNA is a versatile molecule that can be engineered in order
to have RNA-RNA, Protein-RNA or small molecule-RNA interactions. A
general framework for these interactions is shown in FIG. 1A. In
this framework, an RNA strand can be seen as made by two parts, A
and B. This RNA strand is usually designed such that, at its lowest
energy level, part A can interact with an input, which according to
the design can be another RNA, a protein or a protein complex, or a
small molecule, while part B cannot interact with an actuator. This
happens because B is designed to be in a conformation B1 that
impedes the interaction to happen. After the interaction with the
input, B changes folding state going from B1 to B2. After that, the
actuator, which according to the design can be another RNA, a
protein or a protein complex, can interact with B2. According to
this general framework, the action of the actuator is triggered
only in the presence of the input.
[0113] In cell free settings, technologies based on this framework
have worked well. Usually, in these settings, first the RNA is let
to fold at its lowest energy state and then is added to the samples
containing the other components of the system like the input and
actuator. Another way to use these technologies has been the
delivery of an already folded RNA to living cells. Currently, the
main obstacle to overcome for the use of some of these technologies
in genetically encoded circuits is a drastic reduction of the side
effects due to the folding of the RNA in living cells after its
transcription from DNA. Indeed, when the RNA strand folds (FIG. 1B)
the strand can fold in the A-B2 conformation, with can allow the
actuator to interact with B2 even in the case the input is not
present. In a scenario where the actuator should be triggered just
when a specific biomarker signature is present, this is something
that must be avoided.
[0114] This technology drastically reduces these side reactions and
it is based on the idea that B can still interact and trigger the
activator in case it is designed to have this interaction at an
energy state that is not its lowest (FIG. 1C); let's call B3 this
conformation of B. As consequence of that, the energy barrier to
overcome for the formation of B3 during the folding process will be
higher (FIG. 1D) than the barrier in the case of B designed to
interact with the actuator at its lowest energy state (FIG. 1D). A
higher energy barrier makes it more unlikely for the RNA to form in
the A-B3 conformation which in turn makes it more unlikely for the
actuator to be triggered in the absence of the input (it is
important to highlight that B at its lowest energy not necessarily
is the same as B1, which is the conformation of the RNA strand B
when A-B is at its lowest energy state before the interaction of
the input with A).
[0115] Currently, modeling of the RNA folding dynamic is not
sophisticated enough to predict whether an energy gap between B3
and B2 is too high to allow the interaction of B3 with the actuator
after the input interacts with A. In the following, engineering
design principles are disclosed that will allow the introduction of
an energy gap between B2 and B3 and the experimental identification
of the maximum of that gap. Those design principles are general and
do not apply just to the strand displacement reaction.
[0116] In the strand displacement reaction, the input is another
RNA strand while the actuator can be another RNA strand, a protein
or a protein complex.
[0117] Using this technology in strand displacement reactions,
genetically encoded trans-activated miRNA and trans-activated gRNA
for cas9 have been engineered, where the activation is triggered by
RNAs. This in turn will finally enable gene therapy for those
diseases that are characterized by RNAs biomarkers signatures.
[0118] More in general, this technology can be used each time an
endogenously transcribed RNA can interact with an actuator only in
case an input triggers a change in conformation/folding of that
RNA.
Example 2: Strand Displacement
[0119] Originally, the toehold-mediated strand displacement
reaction has been used in cell free settings. There, a single
strand DNA (or RNA) that will be called input (FIG. 2) interacts
with a double stranded DNA (or RNA) that will be called gate. In
this interaction, the input domain T*, that will be called toehold,
anneal by Watson and Crick base pairing to the complementary T
domain on the gate and then the domain Y2* displaces Y2. The input
can itself be the output of an upstream system (FIG. 2), and the
output of the strand displacement reaction can be the input of a
system downstream. The gate is designed in its lowest energy state
NOT to interact with the downstream system, whereas the output is
usually designed to interact with the downstream system at the
output's lowest energy state. According to the downstream system,
the domain Y1 and X1 may or may not be present, can form secondary
structures or have domains that bind to each other or change the
way they interact with each other after the strand displacement
happens. It is also common to have a gate where the X strand is 5'
X2-X1 3' instead of 5' X1-X2 3' (still with X2 binding with Y2 by
Watson and Crick base pairing). The output of the gate can be
either the X1-X2 strand or the entire gate in its new conformation
after the strand displacement reaction happens.
[0120] To avoid unwanted side reactions, in cell free settings, the
gates are formed separately from each other, and then, all the
parts of the system are put together. Each gate is usually formed
by annealing two or more strands, but it can also be made by just
one strand. For the same reason, for application in living
mammalian cells, the gate are first formed separately in cell free
settings and then transfected to cells.
[0121] The paper Sulc et al., "Modelling Toehold-Mediated RNA
Strand Displacement. Biophys J. 2015 Mar. 10; 108(5):1238-47."
Present a mathematical model for kinetic and thermodynamic of the
strand displacement reaction.
[0122] This technology provides a way to express endogenously
transcribed engineered RNAs in mammalian cells, avoiding unwanted
side reactions between the gate and the downstream system during
the RNA folding process of the gate itself. This enables the use of
the strand-displacement-based technology in genetically encoded
logic circuits. Currently, in mammalian cells, the strand
displacement reaction has been reliably used only in those cases
where either the inputs or the gates were NOT endogenously
transcribed. Indeed, when the gate is endogenously transcribed,
because of the side reactions due to the RNA folding process, the
background noise becomes too high. Endogenously transcribed inputs
cannot reach a concentration high enough to trigger the gate beyond
the noise level. So far, this has impeded the use of strand
displacement in gene therapy. In order to use strand displacement
as reliable tool in gene therapy, it is necessary to have
endogenously transcribed gates that can interact with endogenously
transcribed inputs. This would allow the possibility to sense
basically any RNA biomarkers of choice. The technology disclosed
herein allows the design of genetically encoded logic gates that
respond to RNAs biomarkers that are signature of diseases in
mammalian cells, but this is not limited to. Currently, without
this technology, this would not be possible.
[0123] For instance, in the article Guo et al., "Recent advances in
molecular machines based on toehold-mediated strand displacement
reaction," the authors show systems where gates are endogenously
transcribed in mammalian cells but not the inputs. The paper
written by Wu et al., "A Survey of Advancements in Nucleic
Acid-based Logic Gates and Computing for Applications in
Biotechnology and biomedicine" show just a genetically encoded
system that process miRNAs but not RNAs. Additionally, the gates of
the system based on strand displacement are not endogenously
transcribed. The paper written by Chen et al., "A DNA logic gate
based on strand displacement reaction and rolling circle
amplification, responding to multiple low-abundance DNA fragment
input signals, and its application in detecting miRNAs" and the one
written by Deng et al., "DNA Logic Gate Based on Metallo-Toehold
Strand Displacement" show systems were the gates are not
endogenously transcribed.
Example 3: Design of Trans-Activated Engineered RNA by Strand
Displacement
[0124] Currently, this technology is the only one that can allow
the use of the strand displacement reaction in mammalian cells in
genetically encoded circuits. As consequence, this is the only
technology that can allow to engineer these circuits for gene
therapy in diseases that are characterized by RNAs biomarkers
signature instead of miRNAs signatures.
[0125] This technology is based on the idea that the output of a
strand displacement reaction can still interact with its downstream
target when this interaction is designed to happen with the output
strand not at its lowest energy state On the other hand, the gate
is still designed such that, at its lowest energy state, it does
not interact with the downstream system in the absence of the
strand displacement reaction with the input. This design
drastically reduces unwanted side reactions between the output
strand and the downstream system during the folding of the
RNA-transcript-gate in genetically encoded logic circuits. Indeed,
the output signal now interacts with the downstream system far from
its lowest energy state. Consequently, it will be more unlikely
that, during the folding of the gate-RNA-transcript, the part of
this transcript that correspond to the output will reach that
energy state that is far from its lowest one.
[0126] In order to tune the energy states at which the output
interacts with the downstream system, in the following design
principles are provided and used to engineer a trans-activated
sgRNA for Cas9 and a trans-activated miRNA.
[0127] One possible way to tune the energy state at which the
output signal interacts with the downstream system is the
following: First, a RNA strand domain is identified in the output,
which will be called `a` that has to bind to another RNA strand
domain of the output, that will be called `b`, for the output of
the strand displacement reaction to interact with the downstream
system (the actuator). After that, it can be inserted in the output
a new strand domain, that will be called `c`, which binds with `a`
more strongly than `a` binding to `b`. At the same time, the RNA
structure formed by `c` binding `a` should not allow the output to
interact with the downstream system. By tuning the binding energy
between `c` and `a` it is possible to tune the energy gap between
the lowest energy state of the output and the one that allows the
output to interact with the downstream system.
[0128] Then, the maximum value of that energy gap that still allows
B3 to interact with the actuator is experimentally determined. It
is possible for instance to reduce the number of mismatches between
`c` and `a` to increase the energy gap. The B3 conformation strand
that will be no more able to interact with the actuator will set an
upper limit for that energy gap. The chosen energy gap will be the
one just below that limit. The same idea can be used in case the
downstream system requires an RNA strand domain in the output to be
single stranded (like when the downstream system is another gate to
be strand displaced. Here, the single stranded RNA domain can be
the toehold, but this is not limited to). Calling again `a` this
single stranded RNA domain, it is possible to insert in the output
a new strand domain, that again will be called `c`, where `a` and
`c` binds each other. Again in this case, by tuning the binding
energy between `a` and `c`, it is possible to tune the energy gap
between the lowest energy of the output and the one at which the
output can interact with the downstream system.
Trans-Activated miRNA
[0129] miRNAs can downregulate coding genes. One way to genetically
encode miRNAs, but this is not limited to, is to endogenously
transcribe pre-miRNA that are later processed by Drosha, which in
turn starts the miRNA biogenesis. In the left column of FIG. 3A,
the substrate for Drosha processing is depicted. Drosha recognizes
in the nucleus the RNA secondary structures depicted in orange. The
Green part is later processed by the Dicer in the cytosol and,
after that, one of the two red strands (usually their length is
around 22nts) is loaded into the RISC complex that will then
downregulate the expression of the target gene. The following is an
excerpt from "The current state and future directions of RNAi-based
therapeutics" Setten et al., Nature Reviews I Drug Discovery 2019:
`To date, there is not yet a published system that can reliably
couple cellular RNA inputs to RNAi outputs in mammalian
cells.`.
[0130] The technology disclosed herein enables exactly that. This
is the only technology to allow to engineer a genetically encoded
miRNA that is activated only when an endogenous RNA is transcribed.
Importantly, the system does not use exogenous proteins and has a
small DNA footprint, because of that it can be used with AAV to
easily deliver it in vivo and to reduce unwanted immune reactions.
Here, the design is provided for an endogenously transcribed
trans-activated miRNA triggered by the toehold-mediated strand
displacement reaction due to an endogenously transcribed RNA input
strand. The input sequence is orthogonal to the sequence loaded in
the RISC complex. Additionally, the RNAs secondary structures
recognized by Drosha are loosely constrained by specific sequences
and so they do not constraint the choice of the input sequence.
[0131] A similar design could be used to engineer a
Dicer-trans-activated miRNA.
[0132] In left column of FIG. 3A, the design of the one-strand-gate
with the output of the strand displacement reaction that interact
with Drosha (which is the downstream system) at its lowest energy
state (center column of FIG. 3A). This design requires that, after
the strand displacement reaction, the strand `a` binds to the
strand `b` in order for the processing by Drosha to take place. By
expressing this gate in living cells, during the folding process of
the gate-RNA-transcript, the Drosha-substrate can be processed by
Drosha even in the absence of the input. This makes the detection
of the input not possible. Because of that and according to the
design principles mentioned before, a new strand `c` was introduced
that binds with `a` more strongly than `a` binding with `b` (right
column of FIG. 3A). This still allows the gate, in its lowest
energy state, to avoid interaction with the downstream system
(Drosha). However, this time, after the strand displacement
reaction happens (right column of FIG. 3A), the output is processed
by Drosha when the output is far from its lowest energy state.
[0133] In FIG. 3B, the experimental results related to the
investigation needed to find the upper limit of the energy gap.
Thirteen different energy gaps were tested to identify what the
limit was for Drosha to process the RNA. An energy gap between B2
and B3 less than 8.5 Kcal/mol allowed B3 to be still processed by
Drosha. The energy gap was computed for each design using the
software mFold (unafold.rna.albany.edu/?q=mfold). The introduction
of this energy gap, drastically reduces the processing of the
Drosha-substrate by Drosha during the folding of the
gate-RNA-transcript, and so, it allows the detection of the input
(it is more unlikely for A-B3 to form in the absence of the input).
In FIGS. 3C-3D, the actual sequence of the trans-activated miRNA,
while, in FIG. 3G, the experimental results in HEK293FT after
transient transfection of the DNA encoding the different parts.
FIGS. 3E-3F provides additional designs that adopts the same
concept.
[0134] The difference in miRNA activation between the expression of
an endogenously transcribed input and scramble is 30 folds, as
shown in FIG. 3G.
[0135] The different domains of a trans-activated miRNA, including
5' hairpin, toehold, antisense, ribozyme, stem, seed, sense, and 3'
hairpin, are shown in FIGS. 5A-5H. A schematic of this
trans-activated miRNA interacting with input signal RNA is shown in
FIG. 6A. In its native conformation, Drosha cannot interact with
this RNA (FIG. 6B), but an input signal, such as an RNA associated
with a disease state like viral infection, may hybridize to the
trans-activated miRNA, resulting in the release of an RNA waste
product by the ribozyme domain (FIG. 6A). Following this release,
the trans-activated miRNA may undergo another conformational
change, forming one of multiple possible structures, which may or
may not be able to interact with Drosha and release pre-miRNA. The
trans-activated miRNAs described herein are designed such that the
more stable conformation cannot be processed by Drosha (FIG. 6C),
but the less stable conformation can be processed by Drosha (FIG.
6D). These trans-activated miRNAs are thus less likely to interact
with Drosha in the absence of input signal, which limits the
release of pre-miRNA and biogenesis of miRNA against the
therapeutic target to cells in which the input signal is
present.
[0136] A major benefit of this increased specificity is that if
they reliably exert activity only in cells containing the input
signal, that activity can be directed towards the most effective
target, even if that target is a gene that is essential for
cellular replication. In the treatment of virally infected cells,
for example, engineered RNAs such as the ones described in the
present disclosure are not limited to targeting viral mRNAs, but
may also target genes or mRNAs encoding host factors that are
essential for viral replication. This increased specificity allows
the engineered RNAs described in the present disclosure to target
more genes or mRNAs, improving their therapeutic efficacy without
compromising safety.
Trans-Activated sgRNA
[0137] sgRNAs are small non-coding RNAs (FIG. 4) that can bind the
protein cas9 for gene editing or other cas9-enabled applications.
After the binding of cas9 to the sgRNA, cas9 is able to bind to a
DNA sequence that is complementary to the spacer, which in turn
triggers the cas9-enabled applications. Here, the design is
provided of an endogenously transcribed trans-activated sgRNA by
toehold mediated strand displacement due to an input that is
endogenously transcribed as well.
[0138] Currently, this is the only technology to allow to engineer
in mammalian cells a sgRNA that become active only when an RNA
biomarker signature is detected. This in turn will enable DNA gene
editing only in cells that express that RNA and thus will
drastically reduce unwanted side reactions. Importantly, the system
has a small DNA footprint, because of that, it can be used with AAV
virus for easy delivery in vivo.
[0139] The input sequence is orthogonal with the spacer sequence.
Additionally, the choice of the upper stem (FIG. 4A, left and
center columns) for the engineering of the strand displacement
reactions does not constraint the input sequence. This same idea
can of course be applied to other stems of the sgRNA and to gRNA
related to other-than-cas9 CRISPR proteins. In the center column of
FIG. 4A, the design of a one-strand-gate with the output of the
strand displacement reaction that interact with Cas9 (which is the
downstream system) at its lowest energy state (center column of
FIG. 4A). This design requires that, after the strand displacement
reaction, the strand `a` binds to the strand `b` for the processing
by Cas9 to take place. By endogenously transcribing this gate in
living cells, during the folding process of the
gate-RNA-transcript, unfortunately the sgRNA can be processed by
Cas9 even in the absence of the input. This makes the detection of
the input not possible. Because of that and according to the design
principles mentioned before, new strand `c` was introduced that
binds with `a` more strongly than `a` binding with `b` (right
column of FIG. 4A). This still allows the gate to avoid interaction
with the downstream system (Cas9) in its lowest energy state but
this time, after the strand displacement reaction happens (right
column of FIG. 4A) the output is processed by Cas9 when it is far
from its lowest energy state.
[0140] FIGS. 4B-4C show the activated gRNA in the conformation B2
and B3. In this case as well, mFold was used to compute the energy
state. The procedure is the same as for the pre-miRNA; by
decreasing the number of mismatches between `c` and `a` the energy
gap increases. This will lead to different designs to test
experimentally. The upper limit for the energy gap will be the one
where the gRNA in the B3 conformation will not be able anymore to
be processed by cas9. The introduction of the energy gap
drastically reduces the processing of the sgRNA by Cas9 during the
folding of the gate-RNA-transcript and so it allows the detection
of the input. FIGS. 4D and 4E provides additional designs of the
trans-activated sgRNA based on the same concept
[0141] In FIG. 4F, the experimental results in HEK293FT after
transient transfection of the DNA encoding the different parts. The
difference in Cas9 activation between the expression of an
endogenously transcribed input and scramble is 8 folds.
OTHER EMBODIMENTS
[0142] All of the features disclosed in this specification may be
combined in any combination. Each feature disclosed in this
specification may be replaced by an alternative feature serving the
same, equivalent, or similar purpose. Thus, unless expressly stated
otherwise, each feature disclosed is only an example of a generic
series of equivalent or similar features.
[0143] From the above description, one skilled in the art can
easily ascertain the essential characteristics of the present
invention, and without departing from the spirit and scope thereof,
can make various changes and modifications of the invention to
adapt it to various usages and conditions. Thus, other embodiments
are also within the claims.
EQUIVALENTS
[0144] While several inventive embodiments have been described and
illustrated herein, those of ordinary skill in the art will readily
envision a variety of other means and/or structures for performing
the function and/or obtaining the results and/or one or more of the
advantages described herein, and each of such variations and/or
modifications is deemed to be within the scope of the inventive
embodiments described herein. More generally, those skilled in the
art will readily appreciate that all parameters, dimensions,
materials, and configurations described herein are meant to be
exemplary and that the actual parameters, dimensions, materials,
and/or configurations will depend upon the specific application or
applications for which the inventive teachings is/are used. Those
skilled in the art will recognize, or be able to ascertain using no
more than routine experimentation, many equivalents to the specific
inventive embodiments described herein. It is, therefore, to be
understood that the foregoing embodiments are presented by way of
example only and that, within the scope of the appended claims and
equivalents thereto, inventive embodiments may be practiced
otherwise than as specifically described and claimed. Inventive
embodiments of the present disclosure are directed to each
individual feature, system, article, material, kit, and/or method
described herein. In addition, any combination of two or more such
features, systems, articles, materials, kits, and/or methods, if
such features, systems, articles, materials, kits, and/or methods
are not mutually inconsistent, is included within the inventive
scope of the present disclosure.
[0145] All definitions, as defined and used herein, should be
understood to control over dictionary definitions, definitions in
documents incorporated by reference, and/or ordinary meanings of
the defined terms.
[0146] All references, patents and patent applications disclosed
herein are incorporated by reference with respect to the subject
matter for which each is cited, which in some cases may encompass
the entirety of the document.
[0147] The indefinite articles "a" and "an," as used herein in the
specification and in the claims, unless clearly indicated to the
contrary, should be understood to mean "at least one." The phrase
"and/or," as used herein in the specification and in the claims,
should be understood to mean "either or both" of the elements so
conjoined, i.e., elements that are conjunctively present in some
cases and disjunctively present in other cases. Multiple elements
listed with "and/or" should be construed in the same fashion, i.e.,
"one or more" of the elements so conjoined. Other elements may
optionally be present other than the elements specifically
identified by the "and/or" clause, whether related or unrelated to
those elements specifically identified. Thus, as a non-limiting
example, a reference to "A and/or B", when used in conjunction with
open-ended language such as "comprising" can refer, in one
embodiment, to A only (optionally including elements other than B);
in another embodiment, to B only (optionally including elements
other than A); in yet another embodiment, to both A and B
(optionally including other elements); etc.
[0148] As used herein in the specification and in the claims, "or"
should be understood to have the same meaning as "and/or" as
defined above. For example, when separating items in a list, "or"
or "and/or" shall be interpreted as being inclusive, i.e., the
inclusion of at least one, but also including more than one, of a
number or list of elements, and, optionally, additional unlisted
items. Only terms clearly indicated to the contrary, such as "only
one of" or "exactly one of," or, when used in the claims,
"consisting of," will refer to the inclusion of exactly one element
of a number or list of elements. In general, the term "or" as used
herein shall only be interpreted as indicating exclusive
alternatives (i.e. "one or the other but not both") when preceded
by terms of exclusivity, such as "either," "one of," "only one of,"
or "exactly one of." "Consisting essentially of," when used in the
claims, shall have its ordinary meaning as used in the field of
patent law.
[0149] As used herein in the specification and in the claims, the
phrase "at least one," in reference to a list of one or more
elements, should be understood to mean at least one element
selected from any one or more of the elements in the list of
elements, but not necessarily including at least one of each and
every element specifically listed within the list of elements and
not excluding any combinations of elements in the list of elements.
This definition also allows that elements may optionally be present
other than the elements specifically identified within the list of
elements to which the phrase "at least one" refers, whether related
or unrelated to those elements specifically identified. Thus, as a
non-limiting example, "at least one of A and B" (or, equivalently,
"at least one of A or B," or, equivalently "at least one of A
and/or B") can refer, in one embodiment, to at least one,
optionally including more than one, A, with no B present (and
optionally including elements other than B); in another embodiment,
to at least one, optionally including more than one, B, with no A
present (and optionally including elements other than A); in yet
another embodiment, to at least one, optionally including more than
one, A, and at least one, optionally including more than one, B
(and optionally including other elements); etc.
[0150] It should also be understood that, unless clearly indicated
to the contrary, in any methods claimed herein that include more
than one step or act, the order of the steps or acts of the method
is not necessarily limited to the order in which the steps or acts
of the method are recited.
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