U.S. patent application number 17/597351 was filed with the patent office on 2022-08-11 for methods for the treatment of epilepsy.
The applicant listed for this patent is CENTRE NATIONAL DE LA RECHERCHE SCIENTIFIQUE, INSERM (INSTITUT NATIONAL DE LA SANT ET DE LA RECHERCHE MEDICALE), REGENXBIO INC., UNIVERSITE D'AIX MARSEILLE, UNIVERSITE DE BORDEAUX. Invention is credited to Celine BOILEAU, Valerie CREPEL, Olivier DANOS, Severine DEFORGES, Julie MASANTE, Andrew MERCER, Christophe MULLE, Angelique PERET.
Application Number | 20220251567 17/597351 |
Document ID | / |
Family ID | |
Filed Date | 2022-08-11 |
United States Patent
Application |
20220251567 |
Kind Code |
A1 |
CREPEL; Valerie ; et
al. |
August 11, 2022 |
METHODS FOR THE TREATMENT OF EPILEPSY
Abstract
The present disclosure relates to gene therapy targeting GluK2
subunit that can be used to inhibit epileptiform discharges. Short
interfering RNA sequences against the human Grik2 gene sequence are
described which are efficient in decreasing the expression of
GluK2-containing KARs in neurons engineered to express the
equivalent shRNA or miRNA. Using a tissue culture model of TLE, the
examples remarkably demonstrate that viral expression of shRNA or
miRNA inhibits the frequency of epileptiform discharges. Therefore,
RNA therapeutics aimed at decreasing the expression of
GluK2-containing KARs in neurons can remarkably prevent spontaneous
epileptiform discharges in TLE. In particular, the present
disclosure relates to a recombinant antisense oligonucleotide that
targets a Grik2 mRNA. The present disclosure also relates to a
method for treating epilepsy in a subject in need thereof, wherein
the method comprises: administering an effective amount of a vector
comprising an oligonucleotide encoding an inhibitory RNA that binds
(e.g., hybridizes) specifically to Grik2 mRNA and inhibits
expression of Grik2 in the subject.
Inventors: |
CREPEL; Valerie; (Marseille,
FR) ; MULLE; Christophe; (Pessac, FR) ;
BOILEAU; Celine; (Marseille, FR) ; MASANTE;
Julie; (Saint Jean D'illac, FR) ; DEFORGES;
Severine; (Bruges, FR) ; PERET; Angelique;
(Marseille, FR) ; DANOS; Olivier; (Rockville,
MD) ; MERCER; Andrew; (Rockville, MD) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
INSERM (INSTITUT NATIONAL DE LA SANT ET DE LA RECHERCHE
MEDICALE)
UNIVERSITE D'AIX MARSEILLE
UNIVERSITE DE BORDEAUX
REGENXBIO INC.
CENTRE NATIONAL DE LA RECHERCHE SCIENTIFIQUE |
Paris
Marseille
Bordeaux
Rockville
Paris |
MD |
FR
FR
FR
US
FR |
|
|
Appl. No.: |
17/597351 |
Filed: |
July 10, 2020 |
PCT Filed: |
July 10, 2020 |
PCT NO: |
PCT/EP2020/069610 |
371 Date: |
January 4, 2022 |
International
Class: |
C12N 15/113 20060101
C12N015/113; A61P 25/08 20060101 A61P025/08; C12N 7/00 20060101
C12N007/00; C12N 15/86 20060101 C12N015/86 |
Foreign Application Data
Date |
Code |
Application Number |
Jul 10, 2019 |
EP |
19185533.7 |
Claims
1. A recombinant antisense oligonucleotide comprising a guide
sequence that targets a Grik2 mRNA, wherein the guide sequence
comprises a polynucleotide with at least 85% sequence identity to
the nucleic acid sequence of SEQ ID NOs: 14, 15, 18, or 19.
2. The antisense oligonucleotide of claim 1, wherein the
polynucleotide has at least 90% sequence identity to the nucleic
acid sequence of SEQ ID NOs: 14, 15, 18, or 19.
3. The antisense oligonucleotide of claim 2, wherein the
polynucleotide has at least 95% sequence identity to the nucleic
acid sequence of SEQ ID NOs: 14, 15, 18, or 19.
4. The antisense oligonucleotide of claim 3, wherein the
polynucleotide has the nucleic acid sequence of SEQ ID NOs: 14, 15,
18, or 19.
5. The antisense oligonucleotide of any one of claims 1 to 4,
further comprising a passenger sequence, wherein the passenger
sequence has at least 85% sequence identity to the nucleic acid
sequence of SEQ ID NOs: 2, 3, 16, or 17, wherein optionally the
polynucleotide has at least 90% or 95% sequence identity to the
nucleic acid sequence of SEQ ID NOs: 2, 3, 16, or 17, wherein
optionally, the polynucleotide has the nucleic acid sequence of SEQ
ID NOs: 2, 3, 16, or 17.
6. The antisense oligonucleotide of claim 1, wherein the guide
sequence is fully or partially complementary to the nucleic acid
sequence of SEQ ID NOs: 2, 3, 16, or 17.
7. The antisense oligonucleotide of claim 1, wherein the antisense
oligonucleotide is capable of reducing the amount of GluK2
containing kainate receptors in neurons.
8. An expression vector comprising a polynucleotide with at least
85% sequence identity to the nucleic acid sequence of SEQ ID NOs:
2, 3, 16, or 17.
9. The expression vector of claim 8, further comprising the
antisense oligonucleotide of any one of claims 1-7.
10. The expression vector of claim 8, wherein the vector is a
mammalian, bacterial, or viral vector.
11. The expression vector according to claim 10, wherein the viral
vector is an adeno-associated viral (AAV) vector, lentiviral
vector, or retroviral vector.
12. The expression vector of claim 10, wherein the viral vector is
an AAV vector.
13. The expression vector of claim 12, wherein the AAV vector is an
AAV9 or AAVrh10 vector.
14. The expression vector of claim 12, wherein the AAV vector
comprises (i) an expression cassette comprising a transgene
operably linked to one or more regulatory elements and flanked by
ITRs, and (ii) an AAV capsid.
15. The expression vector of claim 14, wherein the one or more
regulatory elements comprise a promoter sequence, enhancer
sequence, transcription termination sequence, and/or
polyadenylation signal.
16. An expression cassette comprising a polynucleotide comprising:
(a) a stem-loop sequence comprising, from 5' to 3': (i) a 5'
stem-loop arm comprising a guide sequence having a nucleic acid
sequence of SEQ ID NOs: 14, 15, 18, or 19; (ii) a loop region,
wherein the loop region comprises a miR-30 loop sequence; (iii) a
3' stem-loop arm comprising a passenger sequence having a nucleic
acid sequence of SEQ ID NOs: 2, 3, 16, or 17 (b) a first flanking
region located 5' to the guide sequence; and (c) a second flanking
region located 3' to the passenger sequence.
17. The expression cassette of claim 16, wherein the stem-loop
sequence comprises a polynucleotide having at least 85% sequence
identity to the nucleic acid sequence of SEQ ID NO: 20.
18. The expression cassette of claim 17, wherein the expression
cassette comprises a polynucleotide having at least 85% sequence
identity to the nucleic acid sequence of SEQ ID NO: 21.
19. An expression cassette comprising a polynucleotide comprising:
(a) a stem-loop sequence comprising, from 5' to 3': (i) a 5'
stem-loop arm comprising a passenger sequence having a nucleic acid
sequence of SEQ ID NOs: 2, 3, 16, or 17; (ii) a loop region,
wherein the loop region comprises a miR-30 loop sequence; (iii) a
3' stem-loop arm comprising guide sequence having a nucleic acid
sequence of SEQ ID NOs: 14, 15, 18, or 19; (b) a first flanking
region located 5' to the guide sequence; and (c) a second flanking
region located 3' to the passenger sequence.
20. The expression cassette of claim 19, wherein the stem-loop
sequence comprises a polynucleotide having at least 85% sequence
identity to the nucleic acid sequence of SEQ ID NO: 22.
21. The expression cassette of claim 20, wherein the expression
cassette comprises a polynucleotide having at least 85% sequence
identity to the nucleic acid sequence of SEQ ID NO: 23.
22. The expression cassette of claim 16 or claim 19, wherein the
first flanking region and the second flanking regions are miR-30
flanking regions.
23. The expression cassette of claim 16 or claim 19, wherein the
first flanking region comprises a polynucleotide having at least
85% sequence identity to the nucleic acid sequence of SEQ ID NO:
24.
24. The expression cassette of claim 16 or claim 19, wherein the
second flanking region comprises a polynucleotide having at least
85% sequence identity to the nucleic acid sequence of SEQ ID NO:
26.
25. The expression cassette of claim 16 or claim 19, wherein the
miR-30 loop sequence comprises a polynucleotide having at least 70%
sequence identity to the nucleic acid sequence of SEQ ID NO:
25.
26. The expression cassette of claim 16 or claim 19, wherein the
passenger sequence is fully or partially complementary to the guide
sequence.
27. The expression cassette of claim 16 or claim 19, wherein the
expression cassette comprises a promoter.
28. The expression cassette of claim 27 wherein the promoter is a
Pol II, Pol III promoter, or a neuron-specific promoter.
29. The expression cassette of claim 27, wherein the promoter is an
hSyn promoter, CaMKII promoter, U6 promoter, or CAG promoter.
30. A pharmaceutical composition comprising the antisense
oligonucleotide of any one of claims 1-7, the expression vector of
any one of claims 8-15, or the expression cassette of any one of
claims 16-29, wherein the pharmaceutical composition further
comprises a pharmaceutically acceptable carrier, excipient, or
diluent.
31. The pharmaceutical composition of claim 30 for use in treating
a disorder in a subject in need thereof.
32. The pharmaceutical composition for use according to claim 31,
wherein the disorder is an epilepsy.
33. The pharmaceutical composition for use according to claim 32,
wherein the epilepsy is temporal lobe epilepsy, a chronic epilepsy,
and/or a drug-resistant epilepsy.
34. The pharmaceutical composition for use according to any one of
claims 31-33, wherein the subject is a human.
35. A method for treating a disorder in a subject in need thereof
comprising administering the pharmaceutical composition of claim
30.
36. The method of claim 35, wherein the disorder is an
epilepsy.
37. The method of claim 36, wherein the epilepsy is temporal lobe
epilepsy, a chronic epilepsy, and/or a drug-resistant epilepsy.
38. The method of any one of claims 35-37, wherein the subject is a
human.
Description
SEQUENCE LISTING
[0001] The instant application contains a Sequence Listing which
has been submitted electronically in ASCII format and is hereby
incorporated by reference in its entirety. Said ASCII copy, created
on Jul. 6, 2020, is named
"51460-002WO2_Sequence_Listing_7.6.20.txt" and is 66,840 bytes in
size.
FIELD OF THE DISCLOSURE
[0002] The disclosure is in the field of epilepsy, more
particularly the disclosure relates to methods and compositions for
treating epilepsy.
BACKGROUND OF THE DISCLOSURE
[0003] Temporal Lobe Epilepsy (TLE) is the most common form of
partial epilepsy in adults (30-40% of all forms of epilepsies). It
is well established that the hippocampus plays a key role in the
pathophysiology of TLE. In human patients and animal models of TLE,
an important rewiring of neuronal circuits occurs. One of the best
examples of network reorganization ("reactive plasticity") is the
sprouting of recurrent mossy fibers (rMF) that establish novel
aberrant glutamatergic synapses onto dentate granule cells (DGCs)
in the hippocampus (Tauck and Nadler, 1985; Represa et al., 1989a,
1989b; Sutula et al., 1989; Gabriel et al., 2004) leading to a
recurrent excitatory circuit. As a key element with regards to this
disclosure, these recurrent synapses operate through ectopic
kainate receptors (KARs) (Epsztein et al., 2005; Artinian et al.,
2011, 2015). In a collaborative work, Valerie Crepel (INMED,
Marseille) and Christophe Mulle (IINS, Bordeaux) have explored the
pathophysiological implications of ectopic synaptic KARs in chronic
seizures in a mouse model of TLE. KARs tetrameric glutamate
receptors assembled from the GluK1-GluK5 subunits. In heterologous
expression systems, GluK1, GluK2, and GluK3 may form homomeric
receptors, while GluK4 and GluK5 form heteromeric receptors in
conjunction with GluK1-3 subunits. Native KARs are widely
distributed in the brain with high densities of receptors found in
the hippocampus (Carta et al, 2016, EJN), a key structure featuring
in TLE. Valerie Crepel and Christophe Mulle demonstrated that
epileptic activities including interictal spikes and ictal
discharges were markedly reduced in mice lacking the GluK2 KAR
subunit. Moreover, epileptiform activities were also strongly
reduced following the use of pharmacological antagonists of
GluK2/GluK5-containing KARs, which block ectopic synaptic KARs
(Peret et al., 2014). These data have established that KARs
ectopically expressed at rMFs in DGCs play a major role in chronic
seizures in TLE. Therefore, aberrant ectopic KARs expressed in DGCs
and composed of GluK2/GluK5 represent a promising target for the
treatment of intractable TLE.
[0004] While hypothetical RNA interference (RNAi) strategies have
been proposed for many disease targets, RNAi molecules capable of
ameliorating disease are rare. For example, for over a decade, the
huntingtin (Htt) gene seemed a likely candidate to knockdown in
order to augment the outcome of Huntington's Disease. At least two
thousand short interfering RNA (siRNA) sequences were proposed
before identifying a strategy for targeting both mutant and
non-mutant Htt genes with no apparent detrimental effect (see e.g.,
WO2005105995; WO2008134646 and U.S. Pat. No. 10,174,321, each of
which is hereby incorporated by reference in its entirety). A
further analogy relates to Parkinson's Disease (PD) which is linked
to a hereditary single-point mutation in the .alpha.-synuclein
(.alpha.-syn) gene as well as genetic duplication or triplication
of .alpha.-syn (Hardy et al., 2010). Some studies targeting
.alpha.-syn expression revealed RNAi as a potential therapeutic
approach to PD, however, conflicting results were reported
(Boudreau et al., 2011; Sapru, et al., 2006). Prediction of
susceptible off-target domains to inform RNA design, variable in
vivo gene silencing efficiencies, and avoiding off-target effects
especially where complex gene expression patterns exist, such as in
CNS (central nervous system) regions, are just a few of the
challenges in choosing an RNA therapeutic.
[0005] Therefore, there exists an unmet need for the treatment of
epileptic disorders, such as, e.g., TLE (e.g., drug-resistant
TLE).
SUMMARY OF THE DISCLOSURE
[0006] The disclosure relates to gene therapy targeting an mRNA
sequence encoding a GluK2 receptor subunit that can be used to
inhibit epileptiform discharges. A siRNA sequence (e.g., SEQ ID NO:
14, 15, 18, or 19) that targets and binds (e.g., hybridizes) to a
corresponding region of the human Grik2 mRNA (e.g., SEQ ID NO: 2,
3, 16, or 17) is described, which is efficient in decreasing the
expression of GluK2-containing kainate receptors (KARs) in neurons
engineered to express the equivalent shRNA or miRNA. Using a tissue
culture model of TLE, the examples remarkably demonstrate that
viral expression of shRNA or miRNA containing an antisense sequence
of the disclosure inhibits the frequency of epileptiform
discharges.
[0007] While not wishing to be bound to any theory, aberrant
recurrent mossy fiber-dentate granule cell (rMF-DGC) synapses,
which operate via ectopic GluK2-containing KARs (Epsztein et al.,
2005; Artinian et al., 2011, 2015) may play a key role in chronic
seizures in TLE (Peret et al., 2014). For example, interictal
spikes and ictal events were reduced in transgenic mice lacking the
GluK2 subunit, or in the presence of a pharmacological agent
inhibiting GluK2/GluK5 receptors (Peret et al., 2014; Crepel and
Mulle, 2015). While knockdown or silencing of GluK2 in transgenic
animal models designed to test these theories is feasible,
designing an inhibitor selective for the GluK2 subunit of the KAR
in humans is challenging. The GluK subunits are structurally
conserved and their nucleotide coding sequences share significant
homologies. The complex gene expression pattern in the brain with
respect to homomeric and heteromeric ionotropic and metabotropic
glutamate receptors further complicates any therapeutic strategy.
In accordance with the disclosure, RNA therapeutics aimed at
decreasing the expression of GluK2-containing KARs in neurons are
described that can remarkably prevent spontaneous epileptiform
discharges in TLE.
[0008] In a first aspect, the disclosure provides a recombinant
antisense oligonucleotide including a guide sequence that targets a
Grik2 mRNA, wherein the guide sequence is a polynucleotide having
at least 85% (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%,
97%, 98%, 99%, or more) sequence identity to the nucleic acid
sequence of SEQ ID NO: 14, 15, 18, or 19. In some embodiments, the
polynucleotide has at least 90% (e.g., at least 90%, 91%, 92%, 93%,
94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to a
nucleic acid sequence of SEQ ID NOs: 14, 15, 18, or 19. In another
embodiment, the polynucleotide has at least 95% (e.g., at least
95%, 96%, 97%, 98%, 99%, or more) sequence identity to a nucleic
acid sequence of SEQ ID NOs: 14, 15, 18, or 19. In another
embodiment, the polynucleotide has a nucleic acid sequence of SEQ
ID NOs: 14, 15, 18, or 19.
[0009] In some embodiments, the antisense oligonucleotide further
includes a passenger sequence, wherein the passenger sequence is a
polynucleotide having at least 85% (e.g., at least 90%, 91%, 92%,
93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to a
nucleic acid sequence of SEQ ID NOs: 2, 3, 16, or 17. In some
embodiments, the polynucleotide has at least 90% (e.g., at least
90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence
identity to a nucleic acid sequence of SEQ ID NOs: 2, 3, 16, or 17.
In some embodiments, the polynucleotide has at least 95% (e.g., at
least 95%, 96%, 97%, 98%, 99%, or more) sequence identity to a
nucleic acid sequence of SEQ ID NOs: 2, 3, 16, or 17. In some
embodiments, the polynucleotide has a nucleic acid sequence of SEQ
ID NOs: 2, 3, 16, or 17.
[0010] In some embodiments, the guide sequence is fully or
partially complementary to the nucleic acid sequence of SEQ ID NOs:
2, 3, 16, or 17.
[0011] In some embodiments, the antisense oligonucleotide is
capable of reducing the amount of GluK2 containing kainate
receptors in neurons.
[0012] In another aspect, the disclosure provides an expression
vector including a polynucleotide having at least 85% (e.g., at
least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more)
sequence identity to the nucleic acid sequence of SEQ ID NOs: 2, 3,
16, or 17.
[0013] In some embodiments, the expression vector includes an
antisense oligonucleotide of the foregoing aspect and
embodiments.
[0014] In some embodiments, the expression vector is a mammalian,
bacterial, or viral vector. In some embodiments, the viral vector
is an adeno-associated viral (AAV) vector, lentiviral vector, or
retroviral vector. In some embodiments, the viral vector is an AAV
vector. In some embodiments, the AAV vector is an AAV9 or AAVrh10
vector. In some embodiments, wherein the AAV vector includes (i) an
expression cassette containing a transgene (e.g., a polynucleotide
encoding an antisense oligonucleotide of the disclosure) operably
linked to one or more regulatory elements and flanked by ITRs, and
(ii) an AAV capsid. In some embodiments, the one or more regulatory
elements include a promoter sequence, enhancer sequence,
transcription termination sequence, and/or polyadenylation
signal.
[0015] In another aspect, the disclosure provides an expression
cassette including a polynucleotide containing: (a) a stem-loop
sequence including from 5' to 3': (i) a 5' stem-loop arm comprising
a guide sequence having a nucleic acid sequence of SEQ ID NOs: 14,
15, 18, or 19 or a variant thereof having at least 85% (e.g., at
least 90%, 91% 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more)
sequence identity to the nucleic acid sequence of SEQ ID NOs: 14,
15, 18, or 19; (ii) a loop sequence, such as, e.g., a microRNA
(miR) loop sequence (e.g., a miR-30 loop sequence, such as, e.g., a
human, non-human primate, rat, or mouse miR-30 loop sequence); and
(iii) a 3' stem-loop arm including a passenger sequence having a
nucleic acid sequence of SEQ ID NOs: 2, 3, 16, or 17 or a variant
there of having at least 85% (e.g., at least 90%, 91%, 92%, 93%,
94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to the
nucleic acid sequence of SEQ ID NOs: 2, 3, 16, or 17; (b) a first
flanking region located 5' to the guide sequence; and (c) a second
flanking region located 3' to the passenger sequence.
[0016] In some embodiments, the stem-loop sequence includes a
polynucleotide having at least 85% (e.g., at least 90%, 91%, 92%,
93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to
the nucleic acid sequence of SEQ ID NO: 20.
[0017] In some embodiments, the expression cassette includes a
polynucleotide having at least 85% (e.g., at least 90%, 91%, 92%,
93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to
the nucleic acid sequence of SEQ ID NO: 21.
[0018] In another aspect, the disclosure provides an expression
cassette including a polynucleotide containing: (a) a stem-loop
sequence including from 5' to 3': (i) a 5' stem-loop arm comprising
a passenger sequence having a nucleic acid sequence of SEQ ID NOs:
2, 3, 16, or 17 or a variant thereof having at least 85% (e.g., at
least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more)
sequence identity to the nucleic acid sequence of SEQ ID NOs: 14,
15, 18, or 19; (ii) a loop sequence, such as, e.g., a miR loop
sequence (e.g., a miR-30 loop sequence, such as, e.g., a human,
non-human primate, rat, or mouse miR-30 loop sequence); and; and
(iii) a 3' stem-loop arm comprising guide sequence having a nucleic
acid sequence of SEQ ID NOs: 14, 15, 18, or 19 or a variant there
of having at least 85% (e.g., at least 90%, 91%, 92%, 93%, 94%,
95%, 96%, 97%, 98%, 99%, or more) sequence identity to the nucleic
acid sequence of SEQ ID NOs: 2, 3, 16, or 17; (b) a first flanking
region located 5' to the guide sequence; and (c) a second flanking
region located 3' to the passenger sequence.
[0019] In some embodiments, the stem-loop sequence includes a
polynucleotide having at least 85% (e.g., at least 90%, 91%, 92%,
93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to
the nucleic acid sequence of SEQ ID NO: 22.
[0020] In some embodiments, the expression cassette includes a
polynucleotide having at least 85% (e.g., at least 90%, 91%, 92%,
93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to
the nucleic acid sequence of SEQ ID NO: 23.
[0021] In some embodiments, the first flanking region and the
second flanking regions are miR-30 flanking regions. In some
embodiments, the first flanking region comprises a polynucleotide
having at least 85% (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%,
96%, 97%, 98%, 99%, or more) sequence identity to the nucleic acid
sequence of SEQ ID NO: 24. In some embodiments, the second flanking
region comprises a polynucleotide having at least 85% (e.g., at
least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more)
sequence identity to the nucleic acid sequence of SEQ ID NO:
26.
[0022] In some embodiments, the miR-30 loop sequence comprises a
polynucleotide having at least 70% (e.g., at least 75%, 80%, 85%,
90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence
identity to the nucleic acid sequence of SEQ ID NO: 25.
[0023] In some embodiments, the passenger sequence is fully or
partially complementary to the guide sequence.
[0024] In some embodiment, the expression cassette includes a
promoter. In some embodiments, the promoter is a Pol II, Pol III,
or a neuron-specific promoter. In some embodiments, the promoter is
a human synapsin (hSyn) promoter (e.g., SEQ ID NO: 27 or SEQ ID NO:
28), calcium/calmodulin-dependent protein kinase II (CaMKII)
promoter (e.g., any one of SEQ ID NOs: 30-34), U6 promoter (e.g.,
SEQ ID NO: 29), or CAG promoter (e.g., SEQ ID NO: 35).
[0025] In another aspect, the present disclosure provides a
pharmaceutical composition including the antisense oligonucleotide
of the foregoing aspects and embodiments, the expression vector of
the foregoing aspects and embodiments, or the expression cassette
of the foregoing aspects and embodiments, wherein the
pharmaceutical composition further includes a pharmaceutically
acceptable carrier, excipient, or diluent. In some embodiments, the
pharmaceutical composition is for use in treating a disorder in a
subject in need thereof.
[0026] In some embodiments, the disorder is an epilepsy. In some
embodiments, the epilepsy is temporal lobe epilepsy, a chronic
epilepsy, and/or a drug-resistant (i.e., refractory) epilepsy. In
some embodiments, the subject is a human.
[0027] In another aspect, the disclosure provides a method for
treating a disorder in a subject in need thereof, the method
including administering an effective amount of at least one
antisense oligonucleotide of the foregoing aspects and embodiments,
the expression vector of the foregoing aspects and embodiments, or
the expression cassette of the foregoing aspects and embodiments,
or the pharmaceutical composition of the foregoing aspect and
embodiments.
[0028] In some embodiments, the disorder is an epilepsy. In some
embodiments, the epilepsy is temporal lobe epilepsy, a chronic
epilepsy, and/or a drug-resistant epilepsy. In some embodiments,
the subject is a human.
DETAILED DESCRIPTION
[0029] The present disclosure is based, in part, on the inventors'
discovery that gene therapy targeting the GluK2 subunit can be used
to inhibit epileptiform discharges. They have identified an RNAi
sequence against the human Grik2 gene sequence (e.g., SEQ ID NOs:
2, 3, 16, or 17), which is efficient in decreasing the expression
of GluK2-containing kainate receptors in neurons infected with the
equivalent shRNA or miRNA. Using an in vitro model recapitulating
epileptic network in the hippocampus as described in TLE, they
demonstrate that viral expression of shRNA or miRNA inhibits the
frequency of epileptiform discharges.
Inhibitory Nucleic Acid Sequences
[0030] Accordingly, an object of the present disclosure relates to
isolated, synthetic or recombinant antisense oligonucleotide
targeting Grik2 gene. The oligonucleotide of the disclosure may be
of any suitable type.
[0031] In some embodiments, the oligonucleotide is an RNA
oligonucleotide. In some embodiments, the oligonucleotide is a DNA
oligonucleotide.
[0032] The term "about" refers to an amount that is .+-.10% of the
recited value and may be .+-.5% of the recited value or .+-.2% of
the recited value.
[0033] The term "nucleotide" is defined as a modified or naturally
occurring deoxyribonucleotide or ribonucleotide. Nucleotides
typically include purines and pyrimidines, which include thymidine,
cytidine, guanosine, adenosine and uridine. The term
"oligonucleotide" is defined as an oligomer of the nucleotides
defined above. The term "oligonucleotide" refers to a nucleic acid
sequence, 3'-5' or 5'-3' oriented, which may be single- or
double-stranded. The oligonucleotide used in the context of the
disclosure may in particular be DNA or RNA. The term also includes
"oligonucleotide analog" which refers to an oligonucleotide having
(i) a modified backbone structure, e.g., a backbone other than the
standard phosphodiester linkage found in natural oligo- and
polynucleotides, and (ii) optionally, modified sugar moieties,
e.g., morpholino moieties rather than ribose or deoxyribose
moieties. Oligonucleotide analogs support bases capable of hydrogen
bonding by Watson-Crick base pairing to standard polynucleotide
bases, where the analog backbone presents the bases in a manner to
permit such hydrogen bonding in a sequence-specific fashion between
the oligonucleotide analog molecule and bases in a standard
polynucleotide (e.g., single-stranded RNA or single-stranded DNA).
Particularly, analogs are those having a substantially uncharged,
phosphorus containing backbone. A substantially uncharged,
phosphorus containing backbone in an oligonucleotide analog is one
in which a majority of the subunit linkages, e.g., between 50-100%,
typically at least 60% to 100% or 75% or 80% of its linkages, are
uncharged, and contain a single phosphorous atom.
[0034] The term "oligonucleotide" also refers to an oligonucleotide
sequence that is inverted relative to its normal orientation for
transcription and so corresponds to an RNA or DNA sequence that is
complementary to a target gene mRNA molecule expressed within the
host cell (e.g., it can hybridize to the target gene mRNA molecule
through Watson-Crick base pairing).
[0035] An antisense strand may be constructed in a number of
different ways, provided that it is capable of interfering with the
expression of a target gene. For example, the antisense strand can
be constructed by reverse-complementing the coding region (or a
segment thereof) of the target gene relative to its normal
orientation for transcription to allow the transcription of its
complement, (e.g., RNAs encoded by the antisense and sense gene may
be complementary). In some embodiments, the oligonucleotide need
not have the same intron or exon pattern as the target gene, and
noncoding segments of the target gene may be equally effective in
achieving antisense suppression of target gene expression as coding
segments such as antisense oligonucleotide (ASO). In some
embodiments, the oligonucleotide has the same exon pattern as the
target gene such as siRNA and antisense oligonucleotide (ASO).
[0036] The terms, "guide strand," or "guide sequence" refer to a
component of a stem-loop RNA structure/sequence (e.g., an shRNA or
microRNA) or its DNA equivalent positioned on either the 5' or the
3' stem-loop arm, also referred to as the -5p or -3p arm, of the
stem-loop structure/sequence, in which the guide strand/sequence
includes a Grik2 mRNA antisense sequence (e.g., SEQ ID NOs: 14, 15,
18, or 19 or a variant thereof with at least 85% (e.g., at least
90%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to the
nucleic acid sequence of SEQ ID NOs: 14, 15, 18, or 19) that is
capable of binding (e.g., hybridizing) to a Grik2 mRNA and
inhibiting the expression of a GluK2 protein. Without wishing to be
bound by any theory, inhibition of expression of the GluK2 protein
may occur as a result of diverse cellular mechanisms, such as,
e.g., mRNA degradation or translational repression. The guide
sequence may be complementary to or substantially complementary
(e.g., having no more than 5, 4, 3, 2, or 1 mismatches) to a
passenger strand/sequence of the stem-loop RNA structure or its DNA
equivalent.
[0037] The terms "passenger strand" and "passenger sequence" refer
to a component of a stem-loop RNA structure/sequence (e.g., an
shRNA or microRNA) or its DNA equivalent positioned on either the
5' or the 3' stem-loop arm, also referred to as the -5p or -3p arm,
of the stem-loop structure/sequence that includes a sequence
complementary to or substantially complementary (e.g., having no
more than 5, 4, 3, 2, or 1 mismatches) to Grik2 mRNA antisense
sequence (e.g., SEQ ID NOs: 14, 15, 18, or 19 or a variant thereof
with at least 85% (e.g., at least 90%, 95%, 96%, 97%, 98%, 99%, or
more) sequence identity to the nucleic acid sequence of SEQ ID NOs:
14, 15, 18, or 19). The passenger sequence may be complementary to
or substantially complementary (e.g., having no more than 5, 4, 3,
2, or 1 mismatches) to a guide strand/sequence of the stem-loop RNA
structure or its DNA equivalent.
[0038] The term "target" or "targeting" refers to an
oligonucleotide able to specifically bind (e.g., hybridize) to a
Grik2 gene or a Grik2 mRNA encoding a Grik2 gene product. In
particular, it refers to an oligonucleotide able to inhibit said
gene or said mRNA by the methods known to the skilled in the art
(e.g., antisense, RNA interference).
[0039] The term "corresponding region" refers to a target region of
a Grik2 mRNA that is substantially complementary (e.g., having no
more than 5, such as, e.g., no more than 4, 3, 2, or 1 mismatches)
with the antisense oligonucleotide of the disclosure. Accordingly,
a corresponding region of a Grik2 mRNA (e.g., any one of SEQ ID
NOs: 2, 3, 16, or 17) refers to a region that is targeted and bound
by the antisense oligonucleotide of the disclosure (e.g.,
oligonucleotide encoded by any one of SEQ ID NOs: 14, 15, 18, or
19).
[0040] The term "stem-loop" (also known as a hairpin or hairpin
loop) refers to a secondary RNA structure containing a "stem" and a
"loop region." The stem region is formed by hybridization of two
regions of the same RNA strand (e.g., two stem-loop arms, such as,
e.g., a 5' stem-loop arm and a 3' stem-loop arm) via complementary
base pairing. The "loop" region corresponds to a short (e.g., 3-8
bp) RNA sequence that covalently links the 3' end of the 5'
stem-loop arm and the 5' end of the 3' stem-loop arm. Generally,
the loop region is excised within a cell by the endonuclease Dicer
to form a stem structure containing only the 5' stem-loop arm and
the 3' stem-loop arm. Within the context of the present disclosure,
the term "stem-loop" may refer to the secondary RNA structure
described above or an RNA or cDNA sequence encoding the same.
[0041] The term "stem-loop arm" refers to an RNA sequence that
forms part of the stem region of a stem-loop structure by
complementary base pairing with a second stem-loop arm. In the
context of the present disclosure, the stem-loop arm may comprise a
guide sequence or a passenger sequence disclosed herein.
[0042] According to the disclosure, the antisense oligonucleotide
of the present disclosure targets an mRNA encoding Grik2 gene
product and is capable of reducing the amount of Grik2 expression
in cells.
[0043] That is to say, the antisense oligonucleotide comprises a
sequence that is at least partially complementary, particularly
perfectly complementary, to a region of the sequence of said mRNA,
said complementarity being sufficient to yield specific binding
(e.g., hybridization) under intra-cellular conditions. As
immediately apparent to the skilled in the art, by a sequence that
is "perfectly complementary to" a second sequence is meant the
reverse complement counterpart of the second sequence, either under
the form of a DNA molecule or under the form of an RNA molecule. A
sequence is "partially complementary to" a second sequence if there
are one or more mismatches. The antisense oligonucleotide of the
present disclosure that targets an mRNA encoding GluK2 receptor
subunit (e.g., GluK2 protein comprising any one of SEQ ID NO: 4,
SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO:
9 or SEQ ID NO: 10, or GluK2 protein comprising at least amino
acids 1 to 509 of SEQ ID NO: 4) may be designed by using the
sequence of said mRNA as a basis, e.g., using bioinformatic tools.
GluK2 (Grik2) mRNA sequences may be found in NCBI Gene ID NO: 2898.
In another example, a polynucleotide sequence encoding SEQ ID NO:
4, a polynucleotide sequence encoding contiguous amino acids 1 to
509 of SEQ ID NO: 4, or a polynucleotide sequence encoding the
GluK2 amino acid sequence of any one of SEQ ID NO: 4 (UniProtKB
Q13002-1), SEQ ID NO: 5 (UniProtKB Q13002-2), SEQ ID NO: 6
(UniProtKB Q13002-3), SEQ ID NO: 7 (UniProtKB Q13002-4), SEQ ID NO:
8 (UniProtKB Q13002-5), SEQ ID NO: 9 (UniProtKB Q13002-6) and SEQ
ID NO: 10 (UniProtKB Q13002-7) can be used as a basis for designing
nucleic acids that target an mRNA encoding GluK2 receptor.
Polynucleotide sequences encoding GluK2 receptor may be selected
from SEQ ID NO: 11, SEQ ID NO: 12 and/or SEQ ID NO: 13.
TABLE-US-00001 >(UniProt Q13002-1; GRIK2_HUMAN Glutamate
receptor ionotropic, kainate 2) (SEQ ID NO: 4)
MKIIFPILSNPVFRRTVKLLLCLLWIGYSQGTTHVLRFGGIFEYVESGPMGAEELAFRFAVNTINRNRTLLPNT-
TLTY
DTQKINLYDSFEASKKACDQLSLGVAAIFGPSHSSSANAVQSICNALGVPHIQTRWKHQVSDNKDSFYVSLYPD-
F
SSLSRAILDLVQFFKWKTVTVVYDDSTGLIRLQELIKAPSRYNLRLKIRQLPADTKDAKPLLKEMKRGKEFHVI-
FDC
SHEMAAGILKQALAMGMMTEYYHYIFTTLDLFALDVEPYRYSGVNMTGFRILNTENTQVSSIIEKWSMERLQAP-
P
KPDSGLLDGFMTTDAALMYDAVHVVSVAVQQFPQMTVSSLQCNRHKPVVRFGTRFMSLIKEAHVVEGLTGRITF-
N KTNGLRTDFDLDVISLKEEGLEKIGTVVDPASGLNMTESQKGK
PANITDSLSNRSLIVTTILEEPYVLFKKSDKPLYGNDRFEGYCIDLLRELSTILGFTYEIRLVEDGKYGAQDDA-
NGQ
VVNGMVRELIDHKADLAVAPLAITYVREKVIDFSKPFMTLGISILYRKPNGTNPGVFSFLNPLSPDIWMYILLA-
YLGV
SCVLFVIARFSPYEVVYNPHPCNPDSDVVENNFTLLNSFWFGVGALMQQGSELMPKALSTRIVGGIVVWFFTLI-
IIS
SYTANLAAFLTVERMESPIDSADDLAKQTKIEYGAVEDGATMTFFKKSKISTYDKMWAFMSSRRQSVLVKSNEE
GIQRVLTSDYAFLMESTTIEFVTQRNCNLTQIGGLIDSKGYGVGTPMGSPYRDKITIAILQLQEEGKLHMMKEK-
W
VVRGNGCPEEESKEASALGVQNIGGIFIVLAAGLVLSVFVAVGEFLYKSKKNAQLEKRSFCSAMVEELRMSLKC-
Q RRLKHKPQAPVIVKTEEVINMHTFNDRRLPGKETMA >(UniProt Q13002-2;
GRIK2_HUMAN Isoform 2 of Glutamate receptor ionotropic, kainate 2)
(SEQ ID NO: 5)
MKIIFPILSNPVFRRTVKLLLCLLWIGYSQGTTHVLRFGGIFEYVESGPMGAEELAFRFAVNTINRNRTLLPNT-
TLTY
DTQKINLYDSFEASKKACDQLSLGVAAIFGPSHSSSANAVQSICNALGVPHIQTRWKHQVSDNKDSFYVSLYPD-
F
SSLSRAILDLVQFFKWKTVTVVYDDSTGLIRLQELIKAPSRYNLRLKIRQLPADTKDAKPLLKEMKRGKEFHVI-
FDC
SHEMAAGILKQALAMGMMTEYYHYIFTTLDLFALDVEPYRYSGVNMTGFRILNTENTQVSSIIEKWSMERLQAP-
P
KPDSGLLDGFMTTDAALMYDAVHVVSVAVQQFPQMTVSSLQCNRHKPVVRFGTRFMSLIKEAHVVEGLTGRITF-
N
KTNGLRTDFDLDVISLKEEGLEKIGTWDPASGLNMTESQKGKPANITDSLSNRSLIVTTILEEPYVLFKKSDKP-
LYG
NDRFEGYCIDLLRELSTILGFTYEIRLVEDGKYGAQDDANGQVVNGMVRELIDHKADLAVAPLAITYVREKVID-
FSK
PFMTLGISILYRKPNGTNPGVFSFLNPLSPDIWMYILLAYLGVSCVLFVIARFSPYEVVYNPHPCNPDSDVVEN-
NFT
LLNSFWFGVGALMQQGSELMPKALSTRIVGGIVVWFFTLIIISSYTANLAAFLTVERMESPIDSADDLAKQTKI-
EYGA
VEDGATMTFFKKSKISTYDKMWAFMSSRRQSVLVKSNEEGIQRVLTSDYAFLMESTTIEFVTQRNCNLTQIGGL-
I
DSKGYGVGTPMGSPYRDKITIAILQLQEEGKLHMMKEKVVWRGNGCPEEESKEASALGVQNIGGIFIVLAAGLV-
LS VFVAVGEFLYKSKKNAQLEKESSIWLVPPYHPDTV >(UniProt Q13002-3;
GRIK2_HUMAN Isoform 3 of Glutamate receptor ionotropic, kainate 2)
(SEQ ID NO: 6)
MKIIFPILSNPVFRRTVKLLLCLLWIGYSQGTTHVLRFGGIFEYVESGPMGAEELAFRFAVNTINRNRTLLPNT-
TLTY
DTQKINLYDSFEASKKACDQLSLGVAAIFGPSHSSSANAVQSICNALGVPHIQTRWKHQVSDNKDSFYVSLYPD-
F
SSLSRAILDLVQFFKWKTVTVVYDDSTGLIRLQELIKAPSRYNLRLKIRQLPADTKDAKPLLKEMKRGKEFHVI-
FDC
SHEMAAGILKQALAMGMMTEYYHYIFTTLDLFALDVEPYRYSGVNMTGFRILNTENTQVSSIIEKWSMERLQAP-
P
KPDSGLLDGFMTTDAALMYDAVHVVSVAVQQFPQMTVSSLQCNRHKPVVRFGTRFMSLIKEAHVVEGLTGRITF-
N
KTNGLRTDFDLDVISLKEEGLEKIGTWDPASGLNMTESQKGKPANITDSLSNRSLIVTTILEEPYVLFKKSDKP-
LYG
NDRFEGYCIDLLRELSTILGFTYEIRLVEDGKYGAQDDANGQWNGMVRELIDHKADLAVAPLAITYVREKVIDF-
SK PFMTLGISILYRKPNGTNPGVFSFLNPLSPDIWMYILLAYLGVSCVLFVIARF
>(UniProt Q13002-4; GRIK2_HUMAN Isoform 4 of Glutamate receptor
ionotropic, kainate 2) (SEQ ID NO: 7)
MKIIFPILSNPVFRRTVKLLLCLLWIGYSQGTTHVLRFGGIFEYVESGPMGAEELAFRFAVNTINRNRTLLPNT-
TLTY
DTQKINLYDSFEASKKACDQLSLGVAAIFGPSHSSSANAVQSICNALGVPHIQTRWKHQVSDNKDSFYVSLYPD-
F
SSLSRAILDLVQFFKWKTVTVVYDDSTGLIRLQELIKAPSRYNLRLKIRQLPADTKDAKPLLKEMKRGKEFHVI-
FDC
SHEMAAGILKQALAMGMMTEYYHYIFTTLDLFALDVEPYRYSGVNMTGFRILNTENTQVSSIIEKWSMERLQAP-
P
KPDSGLLDGFMTTDAALMYDAVHVVSVAVQQFPQMTVSSLQCNRHKPVVRFGTRFMSLIKEAHVVEGLTGRITF-
N
KTNGLRTDFDLDVISLKEEGLEKIGTWDPASGLNMTESQKGKPANITDSLSNRSLIVTTILEEPYVLFKKSDKP-
LYG
NDRFEGYCIDLLRELSTILGFTYEIRLVEDGKYGAQDDANGQVVNGMVRELIDHKADLAVAPLAITYVREKVID-
FSK
PFMTLGISILYRKPNGSELMPKALSTRIVGGIVVWFFTLIIISSYTANLAAFLTVERMESPIDSADDLAKQTKI-
EYGAV
EDGATMTFFKKSKISTYDKMWAFMSSRRQSVLVKSNEEGIQRVLTSDYAFLMESTTIEFVTQRNCNLTQIGGLI-
D
SKGYGVGTPMGSPYRDKITIAILQLQEEGKLHMMKEKVVWRGNGCPEEESKEASALGVQNIGGIFIVLAAGLVL-
SV
FVAVGEFLYKSKKNAQLEKRSFCSAMVEELRMSLKCQRRLKHKPQAPVIVKTEEVINMHTFNDRRLPGKETMA
>(UniProt Q13002-5; GRIK2_HUMAN Isoform 5 of Glutamate receptor
ionotropic, kainate 2) (SEQ ID NO: 8)
MKIIFPILSNPVFRRTVKLLLCLLWIGYSQGTTHVLRFGGIFEYVESGPMGAEELAFRFAVNTINRNRTLLPNT-
TLTY
DTQKINLYDSFEASKKACDQLSLGVAAIFGPSHSSSANAVQSICNALGVPHIQTRWKHQVSDNKDSFYVSLYPD-
F
SSLSRAILDLVQFFKWKTVTVVYDDSTGLIRLQELIKAPSRYNLRLKIRQLPADTKDAKPLLKEMKRGKEFHVI-
FDC
SHEMAAGILKQALAMGMMTEYYHYIFTTLDLFALDVEPYRYSGVNMTGFRILNTENTQVSSIIEKWSMERLQAP-
P
KPDSGLLDGFMTTDAALMYDAVHVVSVAVQQFPQMTVSSLQCNRHKPVVRFGTRFMSLIKEAHVVEGLTGRITF-
N
KTNGLRTDFDLDVISLKEEGLEKIGTWDPASGLNMTESQKGKPANITDSLSNRSLIVTTILEEPYVLFKKSDKP-
LYG
NDRFEGYCIDLLRELSTILGFTYEIRLVEDGKYGAQDDANGQWNGMVRELIDHKADLAVAPLAITYVREKVIDF-
SK
PFMTLGISILYRKPNGTNPGVFSFLNPLSPDIVVMYILLAYLGVSCVLFVIARFSPYEVVYNPHPCNPDSDVVE-
NNFT
LLNSFWFGVGALMQQGSELMPKALSTRIVGGIVVWFFTLIIISSYTANLAAFLTVERMESPIDSADDLAKQTKI-
EYGA
VEDGATMTFFKKSKISTYDKMWAFMSSRRQSVLVKSNEEGIQRVLTSDYAFLMESTTIEFVTQRNCNLTQIGGL-
I
DSKGYGVGTPMGSPYRDKITIAILQLQEEGKLHMMKEKVVWRGNGCPEEESKEASALGVQNIGGIFIVLAAGLV-
LS VFVAVGEFLYKSKKNAQLEKRAKTKLPQDYVFLPILESVSISTVLSSSPSSSSLSSCS
>(UniProt Q13002-6; GRIK2_HUMAN Isoform 6 of Glutamate receptor
ionotropic, kainate 2) (SEQ ID NO: 9)
MKIIFPILSNPVFRRTVKLLLCLLWIGYSQGTTHVLRFGGIFEYVESGPMGAEELAFRFAVNTINRNRTLLPNT-
TLTY
DTQKINLYDSFEASKKACDQLSLGVAAIFGPSHSSSANAVQSICNALGVPHIQTRWKHQVSDNKDSFYVSLYPD-
F
SSLSRAILDLVQFFKWKTVTVVYDDSTGLIRLQELIKAPSRYNLRLKIRQLPADTKDAKPLLKEMKRGKEFHVI-
FDC
SHEMAAGILKQALAMGMMTEYYHYIFTTLDLFALDVEPYRYSGVNMTGFRILNTENTQVSSIIEKWSMERLQAP-
P
KPDSGLLDGFMTTDAALMYDAVHVVSVAVQQFPQMTVSSLQCNRHKPVVRFGTRFMSLIKEAHVVEGLTGRITF-
N
KTNGLRTDFDLDVISLKEEGLEKIGTWDPASGLNMTESQKGKPANITDSLSNRSLIVTTILEEPYVLFKKSDKP-
LYG
NDRFEGYCIDLLRELSTILGFTYEIRLVEDGKYGAQDDANGQVVNGMVRELIDHKSKISTYDKMWAFMSSRRQS-
V
LVKSNEEGIQRVLTSDYAFLMESTTIEFVTQRNCNLTQIGGLIDSKGYGVGTPMGSPYRDKITIAILQLQEEGK-
LHM
MKEKVVVVRGNGCPEEESKEASALGVQNIGGIFIVLAAGLVLSVFVAVGEFLYKSKKNAQLEKESSIVVLVPPY-
HPDTV >(UniProt Q13002-7; GRIK2_HUMAN Isoform 7 of Glutamate
receptor ionotropic, kainate 2) (SEQ ID NO: 10)
MKIIFPILSNPVFRRTVKLLLCLLWIGYSQGTTHVLRFGGIFEYVESGPMGAEELAFRFAVNTINRNRTLLPNT-
TLTY
DTQKINLYDSFEASKKACDQLSLGVAAIFGPSHSSSANAVQSICNALGVPHIQTRWKHQVSDNKDSFYVSLYPD-
F
SSLSRAILDLVQFFKWKTVTVVYDDSTGLIRLQELIKAPSRYNLRLKIRQLPADTKDAKPLLKEMKRGKEFHVI-
FDC
SHEMAAGILKQALAMGMMTEYYHYIFTTLDLFALDVEPYRYSGVNMTGFRILNTENTQVSSIIEKWSMERLQAP-
P
KPDSGLLDGFMTTDAALMYDAVHVVSVAVQQFPQMTVSSLQCNRHKPVVRFGTRFMSLIKEAHVVEGLTGRITF-
N
KTNGLRTDFDLDVISLKEEGLEKIGTWDPASGLNMTESQKGKPANITDSLSNRSLIVTTILEEPYVLFKKSDKP-
LYG
NDRFEGYCIDLLRELSTILGFTYEIRLVEDGKYGAQDDANGQVVNGMVRELIDHKSVLVKSNEEGIQRVLTSDY-
AF
LMESTTIEFVTQRNCNLTQIGGLIDSKGYGVGTPMGSPYRDKITIAILQLQEEGKLHMMKEKVVVVRGNGCPEE-
ES
KEASALGVQNIGGIFIVLAAGLVLSVFVAVGEFLYKSKKNAQLEKRAKTKLPQDYVFLPILESVSISTVLSSSP-
SSSS LSSCS >(RefSeq NM_021956.4:294-3020 Homo sapiens glutamate
ionotropic receptor kainate type subunit 2 (GRIK2), transcript
variant 1, mRNA) (SEQ ID NO: 11)
ATGAAGATTATTTTCCCGATTCTAAGTAATCCAGTCTTCAGGCGCACCGTTAAACTCCTGCTCTGTTTACTGT
GGATTGGATATTCTCAAGGAACCACACATGTATTAAGATTTGGTGGTATTTTTGAATATGTGGAATCTGGCCC
AATGGGAGCTGAGGAACTTGCATTCAGATTTGCTGTGAACACAATTAACAGAAACAGAACATTGCTACCCAA
TACTACCCTTACCTATGATACCCAGAAGATAAACCTTTATGATAGTTTTGAAGCATCCAAGAAAGCCTGTGAT
CAGCTGTCTCTTGGGGTGGCTGCCATCTTCGGGCCTTCACACAGCTCATCAGCAAACGCAGTGCAGTCCAT
CTGCAATGCTCTGGGAGTTCCCCACATACAGACCCGCTGGAAGCACCAGGTGTCAGACAACAAAGATTCCT
TCTATGTCAGTCTCTACCCAGACTTCTCTTCACTCAGCCGTGCCATTTTAGACCTGGTGCAGTTTTTCAAGTG
GAAAACCGTCACGGTTGTGTATGATGACAGCACTGGTCTCATTCGTTTGCAAGAGCTCATCAAAGCTCCATC
AAGGTATAATCTTCGACTCAAAATTCGTCAGTTACCTGCTGATACAAAGGATGCAAAACCCTTACTAAAAGAA
ATGAAAAGAGGCAAGGAGTTTCATGTAATCTTTGATTGTAGCCATGAAATGGCAGCAGGCATTTTAAAACAG
GCATTAGCTATGGGAATGATGACAGAATACTATCATTATATCTTTACCACTCTGGACCTCTTTGCTCTTGATG
TTGAGCCCTACCGATACAGTGGTGTTAACATGACAGGGTTCAGAATATTAAATACAGAAAATACCCAAGTCT
CCTCCATCATTGAAAAGTGGTCGATGGAACGATTGCAGGCACCTCCGAAACCCGATTCAGGTTTGCTGGAT
GGATTTATGACGACTGATGCTGCTCTAATGTATGATGCTGTGCATGTGGTGTCTGTGGCCGTTCAACAGTTT
CCCCAGATGACAGTCAGTTCCTTGCAGTGTAATCGACATAAACCCTGGCGCTTCGGGACCCGCTTTATGAG
TCTAATTAAAGAGGCACATTGGGAAGGCCTCACAGGCAGAATAACTTTCAACAAAACCAATGGCTTGAGAAC
AGATTTTGATTTGGATGTGATCAGTCTGAAGGAAGAAGGTCTAGAAAAGATTGGAACGTGGGATCCAGCCA
GTGGCCTGAATATGACAGAAAGTCAAAAGGGAAAGCCAGCGAACATCACAGATTCCTTATCCAATCGTTCTT
TGATTGTTACCACCATTTTGGAAGAGCCTTATGTCCTTTTTAAGAAGTCTGACAAACCTCTCTATGGTAATGA
TCGATTTGAAGGCTATTGCATTGATCTCCTCAGAGAGTTATCTACAATCCTTGGCTTTACATATGAAATTAGA
CTTGTGGAAGATGGGAAATATGGAGCCCAGGATGATGCCAATGGACAATGGAATGGAATGGTTCGTGAACT
AATTGATCATAAAGCTGACCTTGCAGTTGCTCCACTGGCTATTACCTATGTTCGAGAGAAGGTCATCGACTT
TTCCAAGCCCTTTATGACACTTGGAATAAGTATTTTGTACCGCAAGCCCAATGGTACAAACCCAGGCGTCTT
CTCCTTCCTGAATCCTCTCTCCCCTGATATCTGGATGTATATTCTGCTGGCTTACTTGGGTGTCAGTTGTGT
GCTCTTTGTCATAGCCAGGTTTAGTCCTTATGAGTGGTATAATCCACACCCTTGCAACCCTGACTCAGACGT
GGTGGAAAACAATTTTACCTTGCTAAATAGTTTCTGGTTTGGAGTTGGAGCTCTCATGCAGCAAGGTTCTGA
GCTCATGCCCAAAGCACTGTCCACCAGGATAGTGGGAGGCATTTGGTGGTTTTTCACACTTATCATCATTTC
TTCGTATACTGCTAACTTAGCCGCCTTTCTGACAGTGGAACGCATGGAATCCCCTATTGACTCTGCTGATGA
TTTAGCTAAACAAACCAAGATAGAATATGGAGCAGTAGAGGATGGTGCAACCATGACTTTTTTCAAGAAATC
AAAAATCTCCACGTATGACAAAATGTGGGCCTTTATGAGTAGCAGAAGGCAGTCAGTGCTGGTCAAAAGTAA
TGAAGAAGGAATCCAGCGAGTCCTCACCTCTGATTATGCTTTCCTAATGGAGTCAACAACCATCGAGTTTGT
TACCCAGCGGAACTGTAACCTGACACAGATTGGCGGCCTTATAGACTCTAAAGGTTATGGCGTTGGCACTC
CCATGGGTTCTCCATATCGAGACAAAATTACCATAGCAATTCTTCAGCTGCAAGAGGAAGGCAAACTGCATA
TGATGAAGGAGAAATGGTGGAGGGGCAATGGTTGCCCAGAAGAGGAGAGCAAAGAGGCCAGTGCCCTGG
GGGTTCAGAATATTGGTGGCATCTTCATTGTTCTGGCAGCCGGCTTGGTGCTTTCAGTTTTTGTGGCAGTGG
GAGAATTTTTATACAAATCCAAAAAAAACGCTCAATTGGAAAAGAGGTCCTTCTGTAGTGCCATGGTAGAAG
AATTGAGGATGTCCCTGAAGTGCCAGCGTCGGTTAAAACATAAGCCACAGGCCCCAGTTATTGTGAAAACA
GAAGAAGTTATCAACATGCACACATTTAACGACAGAAGGTTGCCAGGTAAAGAAACCATGGCATAA
>(RefSeq NM_175768.3:294-2903 Homo sapiens glutamate ionotropic
receptor kainate type subunit 2 (GRIK2), transcript variant 2,
mRNA) (SEQ ID NO: 12)
ATGAAGATTATTTTCCCGATTCTAAGTAATCCAGTCTTCAGGCGCACCGTTAAACTCCTGCTCTGTTTACTGT
GGATTGGATATTCTCAAGGAACCACACATGTATTAAGATTTGGTGGTATTTTTGAATATGTGGAATCTGGCCC
AATGGGAGCTGAGGAACTTGCATTCAGATTTGCTGTGAACACAATTAACAGAAACAGAACATTGCTACCCAA
TACTACCCTTACCTATGATACCCAGAAGATAAACCTTTATGATAGTTTTGAAGCATCCAAGAAAGCCTGTGAT
CAGCTGTCTCTTGGGGTGGCTGCCATCTTCGGGCCTTCACACAGCTCATCAGCAAACGCAGTGCAGTCCAT
CTGCAATGCTCTGGGAGTTCCCCACATACAGACCCGCTGGAAGCACCAGGTGTCAGACAACAAAGATTCCT
TCTATGTCAGTCTCTACCCAGACTTCTCTTCACTCAGCCGTGCCATTTTAGACCTGGTGCAGTTTTTCAAGTG
GAAAACCGTCACGGTTGTGTATGATGACAGCACTGGTCTCATTCGTTTGCAAGAGCTCATCAAAGCTCCATC
AAGGTATAATCTTCGACTCAAAATTCGTCAGTTACCTGCTGATACAAAGGATGCAAAACCCTTACTAAAAGAA
ATGAAAAGAGGCAAGGAGTTTCATGTAATCTTTGATTGTAGCCATGAAATGGCAGCAGGCATTTTAAAACAG
GCATTAGCTATGGGAATGATGACAGAATACTATCATTATATCTTTACCACTCTGGACCTCTTTGCTCTTGATG
TTGAGCCCTACCGATACAGTGGTGTTAACATGACAGGGTTCAGAATATTAAATACAGAAAATACCCAAGTCT
CCTCCATCATTGAAAAGTGGTCGATGGAACGATTGCAGGCACCTCCGAAACCCGATTCAGGTTTGCTGGAT
GGATTTATGACGACTGATGCTGCTCTAATGTATGATGCTGTGCATGTGGTGTCTGTGGCCGTTCAACAGTTT
CCCCAGATGACAGTCAGTTCCTTGCAGTGTAATCGACATAAACCCTGGCGCTTCGGGACCCGCTTTATGAG
TCTAATTAAAGAGGCACATTGGGAAGGCCTCACAGGCAGAATAACTTTCAACAAAACCAATGGCTTGAGAAC
AGATTTTGATTTGGATGTGATCAGTCTGAAGGAAGAAGGTCTAGAAAAGATTGGAACGTGGGATCCAGCCA
GTGGCCTGAATATGACAGAAAGTCAAAAGGGAAAGCCAGCGAACATCACAGATTCCTTATCCAATCGTTCTT
TGATTGTTACCACCATTTTGGAAGAGCCTTATGTCCTTTTTAAGAAGTCTGACAAACCTCTCTATGGTAATGA
TCGATTTGAAGGCTATTGCATTGATCTCCTCAGAGAGTTATCTACAATCCTTGGCTTTACATATGAAATTAGA
CTTGTGGAAGATGGGAAATATGGAGCCCAGGATGATGCCAATGGACAATGGAATGGAATGGTTCGTGAACT
AATTGATCATAAAGCTGACCTTGCAGTTGCTCCACTGGCTATTACCTATGTTCGAGAGAAGGTCATCGACTT
TTCCAAGCCCTTTATGACACTTGGAATAAGTATTTTGTACCGCAAGCCCAATGGTACAAACCCAGGCGTCTT
CTCCTTCCTGAATCCTCTCTCCCCTGATATCTGGATGTATATTCTGCTGGCTTACTTGGGTGTCAGTTGTGT
GCTCTTTGTCATAGCCAGGTTTAGTCCTTATGAGTGGTATAATCCACACCCTTGCAACCCTGACTCAGACGT
GGTGGAAAACAATTTTACCTTGCTAAATAGTTTCTGGTTTGGAGTTGGAGCTCTCATGCAGCAAGGTTCTGA
GCTCATGCCCAAAGCACTGTCCACCAGGATAGTGGGAGGCATTTGGTGGTTTTTCACACTTATCATCATTTC
TTCGTATACTGCTAACTTAGCCGCCTTTCTGACAGTGGAACGCATGGAATCCCCTATTGACTCTGCTGATGA
TTTAGCTAAACAAACCAAGATAGAATATGGAGCAGTAGAGGATGGTGCAACCATGACTTTTTTCAAGAAATC
AAAAATCTCCACGTATGACAAAATGTGGGCCTTTATGAGTAGCAGAAGGCAGTCAGTGCTGGTCAAAAGTAA
TGAAGAAGGAATCCAGCGAGTCCTCACCTCTGATTATGCTTTCCTAATGGAGTCAACAACCATCGAGTTTGT
TACCCAGCGGAACTGTAACCTGACACAGATTGGCGGCCTTATAGACTCTAAAGGTTATGGCGTTGGCACTC
CCATGGGTTCTCCATATCGAGACAAAATTACCATAGCAATTCTTCAGCTGCAAGAGGAAGGCAAACTGCATA
TGATGAAGGAGAAATGGTGGAGGGGCAATGGTTGCCCAGAAGAGGAGAGCAAAGAGGCCAGTGCCCTGG
GGGTTCAGAATATTGGTGGCATCTTCATTGTTCTGGCAGCCGGCTTGGTGCTTTCAGTTTTTGTGGCAGTGG
GAGAATTTTTATACAAATCCAAAAAAAACGCTCAATTGGAAAAGGAATCTTCTATTTGGTTAGTGCCACCATA
CCATCCAGACACTGTTTAG >(RefSeq NM_001166247.1:294-2972 Homo
sapiens glutamate ionotropic receptor kainate type subunit 2
(GRIK2), transcript variant 3, mRNA) (SEQ ID NO: 13)
ATGAAGATTATTTTCCCGATTCTAAGTAATCCAGTCTTCAGGCGCACCGTTAAACTCCTGCTCTGTTTACTGT
GGATTGGATATTCTCAAGGAACCACACATGTATTAAGATTTGGTGGTATTTTTGAATATGTGGAATCTGGCCC
AATGGGAGCTGAGGAACTTGCATTCAGATTTGCTGTGAACACAATTAACAGAAACAGAACATTGCTACCCAA
TACTACCCTTACCTATGATACCCAGAAGATAAACCTTTATGATAGTTTTGAAGCATCCAAGAAAGCCTGTGAT
CAGCTGTCTCTTGGGGTGGCTGCCATCTTCGGGCCTTCACACAGCTCATCAGCAAACGCAGTGCAGTCCAT
CTGCAATGCTCTGGGAGTTCCCCACATACAGACCCGCTGGAAGCACCAGGTGTCAGACAACAAAGATTCCT
TCTATGTCAGTCTCTACCCAGACTTCTCTTCACTCAGCCGTGCCATTTTAGACCTGGTGCAGTTTTTCAAGTG
GAAAACCGTCACGGTTGTGTATGATGACAGCACTGGTCTCATTCGTTTGCAAGAGCTCATCAAAGCTCCATC
AAGGTATAATCTTCGACTCAAAATTCGTCAGTTACCTGCTGATACAAAGGATGCAAAACCCTTACTAAAAGAA
ATGAAAAGAGGCAAGGAGTTTCATGTAATCTTTGATTGTAGCCATGAAATGGCAGCAGGCATTTTAAAACAG
GCATTAGCTATGGGAATGATGACAGAATACTATCATTATATCTTTACCACTCTGGACCTCTTTGCTCTTGATG
TTGAGCCCTACCGATACAGTGGTGTTAACATGACAGGGTTCAGAATATTAAATACAGAAAATACCCAAGTCT
CCTCCATCATTGAAAAGTGGTCGATGGAACGATTGCAGGCACCTCCGAAACCCGATTCAGGTTTGCTGGAT
GGATTTATGACGACTGATGCTGCTCTAATGTATGATGCTGTGCATGTGGTGTCTGTGGCCGTTCAACAGTTT
CCCCAGATGACAGTCAGTTCCTTGCAGTGTAATCGACATAAACCCTGGCGCTTCGGGACCCGCTTTATGAG
TCTAATTAAAGAGGCACATTGGGAAGGCCTCACAGGCAGAATAACTTTCAACAAAACCAATGGCTTGAGAAC
AGATTTTGATTTGGATGTGATCAGTCTGAAGGAAGAAGGTCTAGAAAAGATTGGAACGTGGGATCCAGCCA
GTGGCCTGAATATGACAGAAAGTCAAAAGGGAAAGCCAGCGAACATCACAGATTCCTTATCCAATCGTTCTT
TGATTGTTACCACCATTTTGGAAGAGCCTTATGTCCTTTTTAAGAAGTCTGACAAACCTCTCTATGGTAATGA
TCGATTTGAAGGCTATTGCATTGATCTCCTCAGAGAGTTATCTACAATCCTTGGCTTTACATATGAAATTAGA
CTTGTGGAAGATGGGAAATATGGAGCCCAGGATGATGCCAATGGACAATGGAATGGAATGGTTCGTGAACT
AATTGATCATAAAGCTGACCTTGCAGTTGCTCCACTGGCTATTACCTATGTTCGAGAGAAGGTCATCGACTT
TTCCAAGCCCTTTATGACACTTGGAATAAGTATTTTGTACCGCAAGCCCAATGGTACAAACCCAGGCGTCTT
CTCCTTCCTGAATCCTCTCTCCCCTGATATCTGGATGTATATTCTGCTGGCTTACTTGGGTGTCAGTTGTGT
GCTCTTTGTCATAGCCAGGTTTAGTCCTTATGAGTGGTATAATCCACACCCTTGCAACCCTGACTCAGACGT
GGTGGAAAACAATTTTACCTTGCTAAATAGTTTCTGGTTTGGAGTTGGAGCTCTCATGCAGCAAGGTTCTGA
GCTCATGCCCAAAGCACTGTCCACCAGGATAGTGGGAGGCATTTGGTGGTTTTTCACACTTATCATCATTTC
TTCGTATACTGCTAACTTAGCCGCCTTTCTGACAGTGGAACGCATGGAATCCCCTATTGACTCTGCTGATGA
TTTAGCTAAACAAACCAAGATAGAATATGGAGCAGTAGAGGATGGTGCAACCATGACTTTTTTCAAGAAATC
AAAAATCTCCACGTATGACAAAATGTGGGCCTTTATGAGTAGCAGAAGGCAGTCAGTGCTGGTCAAAAGTAA
TGAAGAAGGAATCCAGCGAGTCCTCACCTCTGATTATGCTTTCCTAATGGAGTCAACAACCATCGAGTTTGT
TACCCAGCGGAACTGTAACCTGACACAGATTGGCGGCCTTATAGACTCTAAAGGTTATGGCGTTGGCACTC
CCATGGGTTCTCCATATCGAGACAAAATTACCATAGCAATTCTTCAGCTGCAAGAGGAAGGCAAACTGCATA
TGATGAAGGAGAAATGGTGGAGGGGCAATGGTTGCCCAGAAGAGGAGAGCAAAGAGGCCAGTGCCCTGG
GGGTTCAGAATATTGGTGGCATCTTCATTGTTCTGGCAGCCGGCTTGGTGCTTTCAGTTTTTGTGGCAGTGG
GAGAATTTTTATACAAATCCAAAAAAAACGCTCAATTGGAAAAGAGAGCCAAGACTAAGTTACCTCAAGACTA
TGTATTCCTCCCTATTTTGGAGTCAGTTTCCATTTCTACAGTGTTGTCATCATCACCATCTTCATCATCATTAT
CATCATGTTCTTAA
[0044] Therefore, the present disclosure contemplates antisense
oligonucleotides that, when bound to one or more corresponding
regions of a Grik2 mRNA, forms a duplex structure with the Grik2
mRNA of that is between 7-25 (e.g., 7, 8, 9, 10, 11, 12, 13, 14,
15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25) nucleotides in
length. For example, the duplex structure between the antisense
oligonucleotide and the Grik2 mRNA may be 7 nucleotides in length.
In another example, the duplex structure between the antisense
oligonucleotide and the Grik2 mRNA may be 8 nucleotides in length.
In another example, the duplex structure between the antisense
oligonucleotide and the Grik2 mRNA may be 9 nucleotides in length.
In another example, the duplex structure between the antisense
oligonucleotide and the Grik2 mRNA may be 10 nucleotides in length.
In another example, the duplex structure between the antisense
oligonucleotide and the Grik2 mRNA may be 11 nucleotides in length.
In another example, the duplex structure between the antisense
oligonucleotide and the Grik2 mRNA may be 12 nucleotides in length.
In another example, the duplex structure between the antisense
oligonucleotide and the Grik2 mRNA may be 13 nucleotides in length.
In another example, the duplex structure between the antisense
oligonucleotide and the Grik2 mRNA may be 14 nucleotides in length.
In another example, the duplex structure between the antisense
oligonucleotide and the Grik2 mRNA may be 15 nucleotides in length.
In another example, the duplex structure between the antisense
oligonucleotide and the Grik2 mRNA may be 16 nucleotides in length.
In another example, the duplex structure between the antisense
oligonucleotide and the Grik2 mRNA may be 17 nucleotides in length.
In another example, the duplex structure between the antisense
oligonucleotide and the Grik2 mRNA may be 18 nucleotides in length.
In another example, the duplex structure between the antisense
oligonucleotide and the Grik2 mRNA may be 19 nucleotides in length.
In another example, the duplex structure between the antisense
oligonucleotide and the Grik2 mRNA may be 20 nucleotides in length.
In another example, the duplex structure between the antisense
oligonucleotide and the Grik2 mRNA may be 21 nucleotides in length.
In another example, the duplex structure between the antisense
oligonucleotide and the Grik2 mRNA may be 22 nucleotides in length.
In another example, the duplex structure between the antisense
oligonucleotide and the Grik2 mRNA may be 23 nucleotides in length.
In another example, the duplex structure between the antisense
oligonucleotide and the Grik2 mRNA may be 24 nucleotides in length.
In yet another example, the duplex structure between the antisense
oligonucleotide and the Grik2 mRNA may be 25 nucleotides in
length.
[0045] According to the disclosed methods and compositions, the
duplex structure formed by an antisense oligonucleotide (e.g., any
one of the antisense oligonucleotides disclosed herein, such as,
e.g., any one of SEQ ID NOs: 14, 15, 18, or 19 or a variant thereof
having at least 85% (at least 86%, 87%, 88%, 89%, 90%, 91%, 92%,
93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to
the nucleic acid sequence of any one of SEQ ID NOs: 14, 15, 18, or
19) and one or more corresponding regions of a Grik2 mRNA may
include at least one (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,
11, 12, 13, 14, or 15) mismatch. For example, the duplex structure
may contain 1 mismatch. In another example, the duplex structure
contains 2 mismatches. In another example, the duplex structure
contains 3 mismatches. In another example, the duplex structure
contains 4 mismatches. In another example, the duplex structure
contains 5 mismatches. In another example, the duplex structure
contains 6 mismatches. In another example, the duplex structure
contains 7 mismatches. In another example, the duplex structure
contains 8 mismatches. In another example, the duplex structure
contains 9 mismatches. In another example, the duplex structure
contains 10 mismatches. In another example, the duplex structure
contains 11 mismatches. In another example, the duplex structure
contains 12 mismatches. In another example, the duplex structure
contains 13 mismatches. In another example, the duplex structure
contains 14 mismatches. In yet another example, the duplex
structure contains 15 mismatches.
[0046] Particularly, the antisense oligonucleotide according to the
disclosure is capable of reducing the amount of GluK2-containing
kainate receptors in neurons. Methods for determining whether an
oligonucleotide is capable of reducing the amount of GluK2 receptor
in cells are known to those skilled in the art. This may for
example be done by analyzing Grik2 RNA expression such as by
RT-qPCR, in situ hybridization or GluK2 protein expression such as
by immunohistochemistry, Western blot, and by comparing GluK2
protein expression or GluK2 functional activity in the presence and
in the absence of the antisense oligonucleotide to be tested.
[0047] In other embodiments, the oligonucleotide is targeted to a
translation initiation site (AUG codon), sequences in the coding
region (e.g., one or more exons), 5'-untranslated region or
3'-untranslated region of an mRNA. The aim is to interfere with the
processing and expression of the mRNA, such as, e.g., translocation
of the mRNA to the site for protein translation, actual translation
of protein from the mRNA, splicing or maturation of the pre-mRNA
and possible independent catalytic activity which may be performed
by the RNA. The overall effect of such interference with the RNA
function is to cause interference with protein expression.
[0048] In some embodiments, the oligonucleotide of the present
disclosure has a length from 15 to 25 nucleotides. In particular,
the oligonucleotide of the present disclosure has a length of 16,
17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides.
[0049] In some embodiments, the oligonucleotide of the present
disclosure is further modified, particularly chemically modified,
in order to increase the stability and/or therapeutic efficiency in
vivo. The one skilled in the art can easily provide some
modifications that will improve the efficacy of the oligonucleotide
such as stabilizing modifications and modifications avoiding the
RNase H activation in order to avoid degradation of the targeted
transcript (C. Frank Bennett and Eric E. Swayze, RNA Targeting
Therapeutics: Molecular Mechanisms of Antisense Oligonucleotides as
a Therapeutic PlatformAnnu. Rev. Pharmacol. Toxicol.
2010.50:259-293; Juliano RL. The delivery of therapeutic
oligonucleotides. Nucleic Acids Res. 2016 Aug. 19; 44
(14):6518-48). In particular, the oligonucleotide used in the
context of the disclosure may comprise modified nucleotides.
Chemical modifications may occur at three different sites: (i) at
phosphate groups, (ii) on the sugar moiety, and/or (iii) on the
entire backbone structure of the oligonucleotide. Typically,
chemical modifications include backbone modifications, heterocycle
modifications, sugar modifications, and conjugation strategies.
[0050] For example the oligonucleotide may be selected from the
group consisting of oligodeoxyribonucleotides,
oligoribonucleotides, small regulatory RNAs (sRNAs), U7- or
U1-mediated ASOs or conjugate products thereof such as
peptide-conjugated or nanoparticle-complexed ASOs, chemically
modified oligonucleotide by backbone modifications such as
morpholinos, phosphorodiamidate morpholino oligomers
(Phosphorodiamidate morpholinos, PMO), peptide nucleic acid (PNA),
phosphorothioate (PS) oligonucleotides, stereochemically pure
phosphorothioate (PS) oligonucleotides, phosphoramidates modified
oligonucleotides, thiophosphoramidate-modified oligonucleotides,
and methylphosphonate modified oligonucleotides; chemically
modified oligonucleotide by heterocycle modifications such as
bicycle modified oligonucleotides, Bicyclic Nucleic Acid (BNA),
tricycle modified oligonucleotides, tricyclo-DNA-antisense
oligonucleotides (ASOs), nucleobase modifications such as 5-methyl
substitution on pyrimidine nucleobases, 5-substituted pyrimidine
analogues, 2-Thio-thymine modified oligonucleotides, and purine
modified oligonucleotides; chemically modified oligonucleotide by
sugar modifications such as Locked Nucleic Acid (LNA)
oligonucleotides, 2',4'-Methyleneoxy Bridged Nucleic Acid (BNA),
ethylene-bridged nucleic acid (ENA), constrained ethyl (cEt)
oligonucleotides, 2'-Modified RNA, 2'- and 4'-modified
oligonucleotides such as 2'-O-Me RNA (2'-OMe), 2'-O-Methoxyethyl
RNA (MOE), 2'-Fluoro RNA (FRNA), and 4'-Thio-Modified DNA and RNA;
chemically modified oligonucleotide by conjugation strategies such
as N-acetyl galactosamine (GaINAc) oligonucleotide conjugates such
as 5'-GaINAc and 3'-GaINAc ASO conjugates, lipid oligonucleotide
conjugates, cell penetrating peptides (CPP) oligonucleotide
conjugates, targeted oligonucleotide conjugates,
antibody-oligonucleotide conjugates, polymer-oligonucleotide
conjugate such as with PEGylation and targeting ligand; and
chemical modifications and conjugation strategies described for
example in Bennett and Swayze, 2010 (RNA targeting therapeutics:
molecular mechanisms of antisense oligonucleotides as a therapeutic
platform. Annu Rev Pharmacol Toxicol. 2010; 50:259-93); Wan and
Seth, 2016 (The Medicinal Chemistry of Therapeutic
Oligonucleotides. J Med Chem. 2016 Nov. 10; 59 (21):9645-9667);
Juliano, 2016 (The delivery of therapeutic oligonucleotides.
Nucleic Acids Res. 2016 Aug. 19; 44 (14):6518-48); Lundin et al.,
2015 (Oligonucleotide Therapies: The Past and the Present. Hum Gene
Ther. 2015 August; 26 (8):475-85); and Prakash, 2011 (An overview
of sugar-modified oligonucleotides for antisense therapeutics. Chem
Biodivers. 2011 September; 8 (9):1616-41). Indeed, for use in vivo,
the oligonucleotide may be stabilized. A "stabilized"
oligonucleotide refers to an oligonucleotide that is relatively
resistant to in vivo degradation (e.g., via an exo- or
endo-nuclease). Stabilization can be a function of length or
secondary structure. In particular, oligonucleotide stabilization
can be accomplished via phosphate backbone modifications,
phosphodiester modifications, phosphorothioate (PS) backbone
modifications, combinations of phosphodiester and phosphorothioate
modifications, thiophosphoramidate modifications, 2' modifications
(2'-O-Me, 2'-O-(2-methoxyethyl) (MOE) modifications and 2'-fluoro
modifications), methylphosphonate, methylphosphorothioate,
phosphorodithioate, p-ethoxy, and combinations thereof.
[0051] For example, the oligonucleotide may be employed as
phosphorothioate derivatives (replacement of a non-bridging
phosphoryl oxygen atom with a sulfur atom), which have increased
resistance to nuclease digestion. 2'-methoxyethyl (MOE)
modification (such as the modified backbone commercialized by IONIS
Pharmaceuticals) is also effective. Additionally or alternatively,
the oligonucleotide of the present disclosure may comprise
completely, partially or in combination, modified nucleotides which
are derivatives with substitutions at the 2' position of the sugar,
in particular with the following chemical modifications: O-methyl
group (2'-O-Me) substitution, 2-methoxyethyl group (2'-O-MOE)
substitution, fluoro group (2'-fluoro) substitution, chloro group
(2'-Cl) substitution, bromo group (2'-Br) substitution, cyanide
group (2'-CN) substitution, trifluoromethyl group (2'-CF3)
substitution, OCF3 group (2'-OCF3) substitution, OCN group (2'-OCN)
substitution, O-alkyl group (2'-O-alkyl) substitution, S-alkyl
group (2'-S-alkyl) substitution, N-alkyl group (2'-N-akyl)
substitution, O-alkenyl group (2'-O-alkenyl) substitution,
S-alkenyl group (2'-S-alkenyl) substitution, N-alkenyl group
(2'-N-alkenyl) substitution, SOCH3 group (2'-SOCH3) substitution,
SO2CH3 group (2'-SO2CH3) substitution, ONO2 group (2'-ONO2)
substitution, NO2 group (2'-NO2) substitution, N3 group (2'-N3)
substitution and/or NH2 group (2'-NH2) substitution. Additionally
or alternatively, the oligonucleotide of the present disclosure may
comprise completely or partially modified nucleotides wherein the
ribose moiety is used to produce locked nucleic acid (LNA), in
which a covalent bridge is formed between the 2' oxygen and the 4'
carbon of the ribose, fixing it in the 3'-endo configuration. These
molecules are extremely stable in biological medium, able to
activate RNase H such as when LNA are located to extremities
(gapmer) and form tight hybrids with complementary RNA and DNA.
[0052] In some embodiments, the oligonucleotide used in the context
of the disclosure comprises modified nucleotides selected from the
group consisting of LNA, 2'-OMe analogs, 2'-O-Met,
2'-O-(2-methoxymethyl) (MOE) oligomers, 2'-phosphorothioate
analogs, 2'-fluoro analogs, 2'-Cl analogs, 2'-Br analogs, 2'-CN
analogs, 2'-CF3 analogs, 2'-OCF3 analogs, 2'-OCN analogs,
2'-O-alkyl analogs, 2'-S-alkyl analogs, 2'-N-alkyl analogs,
2'-O-alkenyl analogs, 2'-S-alkenyl analogs, 2'-N-alkenyl analogs,
2'-SOCH3 analogs, 2'-SO2CH3 analogs, 2'-ONO2 analogs, 2'-NO2
analogs, 2'-N3 analogs, 2'-NH2 analogs, tricyclo (tc)-DNAs, U7
short nuclear (sn) RNAs, tricyclo-DNA-oligoantisense molecules and
combinations thereof (U.S. Provisional Patent Application Ser. No.
61/212,384 For: Tricyclo-DNA Antisense Oligonucleotides,
Compositions and Methods for the Treatment of Disease, filed Apr.
10, 2009, the complete contents of which is hereby incorporated by
reference).
[0053] In a particular embodiment, the oligonucleotide according to
the disclosure is a LNA oligonucleotide. The term "LNA" (Locked
Nucleic Acid) (or "LNA oligonucleotide") refers to an
oligonucleotide containing one or more bicyclic, tricyclic or
polycyclic nucleoside analogues also referred to as LNA nucleotides
and LNA analogue nucleotides. LNA oligonucleotides, LNA nucleotides
and LNA analogue nucleotides are generally described in
International Publication No. WO 99/14226 and subsequent
applications; International Publication Nos. WO 00/56746, WO
00/56748, WO 00/66604, WO 01/25248, WO 02/28875, WO 02/094250, WO
03/006475; U.S. Pat. Nos. 6,043,060, 6,268,490, 6,770,748,
6,639,051, and U.S. Publication Nos. 2002/0125241, 2003/0105309,
2003/0125241, 2002/0147332, 2004/0244840 and 2005/0203042, all of
which are incorporated herein by reference. LNA oligonucleotides
and LNA analogue oligonucleotides are commercially available from,
for example, Proligo LLC, 6200 Lookout Road, Boulder, Colo. 80301
USA.
[0054] Other forms of oligonucleotides of the present disclosure
are oligonucleotide sequences coupled to small nuclear RNA
molecules such as U1 or U7 in combination with a viral transfer
method based on, but not limited to, lentivirus or adeno-associated
virus (Denti, M A, et al, 2008; Goyenvalle, A, et al, 2004).
[0055] Other forms of oligonucleotides of the present disclosure
are peptide nucleic acids (PNA). In peptide nucleic acids, the
deoxyribose backbone of oligonucleotides are replaced with a
backbone more akin to a peptide than a sugar. Each subunit, or
monomer, has a naturally occurring or non-naturally occurring base
attached to this backbone. One such backbone is constructed of
repeating units of N-(2-aminoethyl)glycine linked through amide
bonds. Because of the radical deviation from the deoxyribose
backbone, these compounds were named peptide nucleic acids (PNAs)
(Dueholm et al., New J. Chem., 1997, 21, 19-31). PNA binds both DNA
and RNA to form PNA/DNA or PNA/RNA duplexes. The resulting PNA/DNA
or PNA/RNA duplexes are bound with greater affinity than
corresponding DNA/DNA, DNA/RNA or RNA/RNA duplexes as determined by
Tm's. This high thermal stability might be attributed to the lack
of charge repulsion due to the neutral backbone in PNA. The neutral
backbone of the PNA also results in the Tm's of PNA/DNA(RNA) duplex
being practically independent of the salt concentration. Thus, the
PNA/DNA(RNA) duplex interaction offers a further advantage over
DNA/DNA, DNA/RNA or RNA/RNA duplex interactions which are highly
dependent on ionic strength. Homopyrimidine PNAs have been shown to
bind complementary DNA or RNA in an anti-parallel orientation
forming (PNA)2/DNA(RNA) triplexes of high thermal stability (see,
e.g., Egholm, et al., Science, 1991, 254, 1497; Egholm, et al., J.
Am. Chem. Soc., 1992, 114, 1895; Egholm, et al., J. Am. Chem. Soc.,
1992, 114, 9677). In addition to increased affinity, PNA has also
been shown to bind to DNA or RNA with increased specificity. When a
PNA/DNA duplex mismatch is melted relative to the DNA/DNA duplex
there is seen an 8 to 20.degree. C. drop in the Tm. This magnitude
of a drop in Tm is not seen with the corresponding DNA/DNA duplex
with a mismatch present. The binding of a PNA strand to a DNA or
RNA strand can occur in one of two orientations. The orientation is
said to be anti-parallel when the DNA or RNA strand in a 5' to 3'
orientation binds to the complementary PNA strand such that the
carboxyl end of the PNA is directed towards the 5' end of the DNA
or RNA and amino end of the PNA is directed towards the 3' end of
the DNA or RNA. In the parallel orientation the carboxyl end and
amino end of the PNA are just the reverse with respect to the 5'-3'
direction of the DNA or RNA. A further advantage of PNA compared to
oligonucleotides is that their polyamide backbones (having
appropriate nucleobases or other side chain groups attached
thereto) is not recognized by either nucleases or proteases and are
not cleaved. As a result, PNAs are resistant to degradation by
enzymes unlike nucleic acids and peptides. WO92/20702 describes a
peptide nucleic acid (PNA) compounds which bind complementary DNA
and RNA more tightly than the corresponding DNA. PNA have shown
strong binding affinity and specificity to complementary DNA
(Egholm, M., et al., Chem. Soc., Chem. Commun., 1993, 800; Egholm,
M., et.al., Nature, 1993, 365, 566; and Nielsen, P., et.al. Nucl.
Acids Res., 1993, 21, 197). Furthermore, PNA's show nuclease
resistance and stability in cell-extracts (Demidov, V. V., et al.,
Biochem. Pharmacol., 1994, 48, 1309-1313). Modifications of PNA
include extended backbones (Hyrup, B., et.al. Chem. Soc., Chem.
Commun., 1993, 518), extended linkers between the backbone and the
nucleobase, reversal of the amida bond (Lagriffoul, P. H., et.al.,
Biomed. Chem. Lett., 1994, 4, 1081), and the use of a chiral
backbone based on alanine (Dueholm, K. L, et.al., BioMed. Chem.
Lett., 1994, 4, 1077). Peptide Nucleic Acids are described in U.S.
Pat. No. 5,539,082 and U.S. Pat. No. 5,539,083. Peptide Nucleic
Acids are further described in U.S. patent application Ser. No.
08/686,113.
[0056] Typically, the oligonucleotides of the present disclosure
are obtained by conventional methods well known to those skilled in
the art. For example, the oligonucleotide of the disclosure can be
synthesized de novo using any of a number of procedures well known
in the art. For example, the b-cyanoethyl phosphoramidite method
(Beaucage et al., 1981); nucleoside H-phosphonate method (Garegg et
al., 1986; Froehler et al., 1986, Garegg et al., 1986, Gaffney et
al., 1988). These chemistries can be performed by a variety of
automated nucleic acid synthesizers available in the market. These
nucleic acids may be referred to as synthetic nucleic acids.
Alternatively, oligonucleotide can be produced on a large scale in
plasmids (see Sambrook, et al., 1989). Oligonucleotide can be
prepared from existing nucleic acid sequences using known
techniques, such as those employing restriction enzymes,
exonucleases or endonucleases. Oligonucleotide prepared in this
manner may be referred to as isolated nucleic acids.
[0057] The one skilled in the art can easily provide some
approaches and modifications for enhancing the delivery and the
efficacy of oligonucleotides such as chemical modification of the
oligonucleotides, lipid- and polymer-based nanoparticles or
nanocarriers, ligand-oligonucleotide conjugates by linking
oligonucleotides to targeting agents such as carbohydrates,
peptides, antibodies, aptamers, lipids or small molecules and small
molecules that improve oligonucleotide delivery such as described
in Juliano RL. The delivery of therapeutic oligonucleotides.
Nucleic Acids Res. 2016 Aug. 19; 44 (14):6518-48. Lipophilic
conjugates and lipid conjugates include fatty acid-oligonucleotide
conjugates; sterol-oligonucleotide conjugates and
vitamin-oligonucleotide conjugates.
[0058] In some embodiments, the oligonucleotide of the present
disclosure is modified by substitution at the 3' or the 5' end by a
moiety comprising at least three saturated or unsaturated,
particularly saturated, linear or branched, particularly linear,
hydrocarbon chains comprising from 2 to 30 carbon atoms,
particularly from 5 to 20 carbon atoms, more particularly from 10
to 18 carbon atoms as described in WO2014195432.
[0059] In some embodiments, the oligonucleotide of the present
disclosure is modified by substitution at the 3' or the 5' end by a
moiety comprising at least one ketal functional group, wherein the
ketal carbon of said ketal functional group bears two saturated or
unsaturated, particularly saturated, linear or branched,
particularly linear, hydrocarbon chains comprising from 1 to 22
carbon atoms, particularly from 6 to 20 carbon atoms, in particular
10 to 19 carbon atoms, and even more particularly from 12 to 18
carbon atoms as described in WO2014195430.
[0060] In a particular embodiment, the oligonucleotide of the
present disclosure is conjugated to a second molecule. Typically
said second molecule is selected from the group consisting of
aptamers, antibodies or polypeptides. For example, the
oligonucleotide of the present disclosure may be conjugated to a
cell penetrating peptide. Cell penetrating peptides are well known
in the art and include for example the TAT peptide (Bechara C,
Sagan S. Cell-penetrating peptides: 20 years later, where do we
stand? FEBS Lett. 2013 Jun. 19; 587 (12):1693-702).
[0061] In some embodiments, the oligonucleotide of the present
disclosure is associated with a carrier or vehicle, e.g., liposomes
or micelles, although other carriers could be used, as would be
appreciated by one skilled in the art. Liposomes are vesicles made
of a lipid bilayer having a structure similar to biological
membranes. Such carriers are used to facilitate the cellular uptake
or targeting of the oligonucleotide, or improve the
oligonucleotide's pharmacokinetic or therapeutic properties. For
example, the oligonucleotide of the present disclosure may also be
administered encapsulated in liposomes, pharmaceutical compositions
wherein the active ingredient is contained either dispersed or
variously present in corpuscles consisting of aqueous concentric
layers adherent to lipidic layers. The oligonucleotide, depending
upon solubility, may be present both in the aqueous layer and in
the lipidic layer, or in what is generally termed a liposomal
suspension. The hydrophobic layer, generally but not exclusively,
comprises phospholipids such as lecithin and sphingomyelin,
steroids such as cholesterol, more or less ionic surfactants such
as diacetylphosphate, stearylamine, or phosphatidic acid, or other
materials of a hydrophobic nature. The diameters of the liposomes
generally range from about 15 nm to about 5 microns. The use of
liposomes as drug delivery vehicles offers several advantages.
Liposomes increase intracellular stability, increase uptake
efficiency and improve biological activity. Liposomes are hollow
spherical vesicles composed of lipids arranged in a similar fashion
as those lipids, which make up the cell membrane. They have an
internal aqueous space for entrapping water-soluble compounds and
range in size from 0.05 to several microns in diameter. Several
studies have shown that liposomes can deliver nucleic acids to
cells and that the nucleic acids remain biologically active. For
example, a liposome delivery vehicle originally designed as a
research tool, such as Lipofectin, can deliver intact nucleic acid
molecules to cells. Specific advantages of using liposomes include
the following: they are non-toxic and biodegradable in composition;
they display long circulation half-lives; and recognition molecules
can be readily attached to their surface for targeting to tissues.
Finally, cost-effective manufacture of liposome-based
pharmaceuticals, either in a liquid suspension or lyophilized
product, has demonstrated the viability of this technology as an
acceptable drug delivery system.
[0062] In some embodiments, the oligonucleotide of the present
disclosure is complexed with a complexing agent to increase
cellular uptake of oligonucleotides. An example of a complexing
agent includes cationic lipids. Cationic lipids can be used to
deliver oligonucleotides to cells. The term "cationic lipid"
includes lipids and synthetic lipids having both polar and
non-polar domains and which are capable of being positively charged
at or around physiological pH and which bind to polyanions, such as
nucleic acids, and facilitate the delivery of nucleic acids into
cells. In general, cationic lipids include saturated and
unsaturated alkyl and alicyclic ethers and esters of amines,
amides, or derivatives thereof. Straight-chain and branched alkyl
and alkenyl groups of cationic lipids can contain, e.g., from 1 to
about 25 carbon atoms. Particularly, straight chain or branched
alkyl or alkene groups have six or more carbon atoms. Alicyclic
groups include cholesterol and other steroid groups. Cationic
lipids can be prepared with a variety of counterions (anions)
including, e.g., Cl--, Br--, I--, F--, acetate, trifluoroacetate,
sulfate, nitrite, and nitrate. Examples of cationic lipids include:
polyethylenimine, polyamidoamine (PAMAM) starburst dendrimers,
Lipofectin (a combination of DOTMA and DOPE), Lipofectase,
Lipofectamine, DOPE, Cytofectin (Gilead Sciences, Foster City,
Calif.), and Eufectins (JBL, San Luis Obispo, Calif.). Cationic
liposomes may comprise the following:
N-[1-(2,3-dioleoloxy)-propyl]-N,N,N-trimethylammonium chloride
(DOTMA), N-[1-(2,3-dioleoloxy)-propyl]-N,N,N-trimethylammonium
methylsulfate (DOTAP),
3p-[N-(N',N'-dimethylaminoethane)carbamoyl]cholesterol (DC-Chol),
2,3,-dioleyloxy-N-[2(sperminecarboxamido)ethyl]-N,N-dimethyl-1-propanamin-
ium trifluoroacetate (DOSPA),
1,2-dimyristyloxpropyl-3-dimethy-1-hydroxyethyl ammonium bromide;
and dimethyldioctadecylammonium bromide (DDAB). The cationic lipid
N-(1-(2,3-dioleyloxy)propyI)-N,N,N-trimethylammonium chloride
(DOTMA), for example, was found to increase 1000-fold the antisense
effect of a phosphorothioate oligonucleotide (Vlassov et al., 1994,
Biochimica et Biophysica Acta 1197:95-108). Oligonucleotides can
also be complexed with, e.g., poly(L-lysine) or avidin and lipids
may, or may not, be included in this mixture (e.g.,
steryl-poly(L-lysine). Cationic lipids have been used in the art to
deliver oligonucleotides to cells (see, e.g., U.S. Pat. Nos.
5,855,910; 5,851,548; 5,830,430; 5,780,053; 5,767,099; Lewis et al.
1996. Proc. Natl. Acad. Sci. USA 93:3176; Hope et al. 1998.
Molecular Membrane Biology 15:1). Other lipid compositions which
can be used to facilitate uptake of the instant oligonucleotides
can be used in connection with the claimed methods. In addition to
those listed supra, other lipid compositions are also known in the
art and include, e.g., those taught in U.S. Pat. Nos. 4,235,871;
4,501,728; 4,837,028; 4,737,323.
[0063] The term "ionotropic glutamate receptors" comprise members
of the NMDA (N-methyl-D-aspartate), AMPA
(.alpha.-amino-3-hydroxy-5-methyl-4-isoxazoleproprionic acid) and
kainate receptor classes. Functional kainate receptors can be
assembled into tetrameric assemblies from the homomeric or
heteromeric combination of five subunits named GluK1, GluK2, GluK3,
GluK4 and GluK5 subunits (Reiner et al., 2012). The targets of the
disclosure are, in some instances, kainate receptor complexes
composed of GluK2 and GluK5. Inhibiting the expression of Grik2
gene is sufficient to abolish GluK2/GluK5 kainate receptor
function, given the observation that GluK5 by itself does not form
functional homomeric channels.
[0064] The term "GluK2", also known as "GluR.sub.6", "GRIK2",
"MRT6", "EAA4", or "GluK6", refers to the glutamate ionotropic
receptor kainate type subunit 2, as named in the currently used
IUPHAR nomenclature (Collingridge, G. L., Olsen, R. W., Peters, J.,
Spedding, M., 2009. A nomenclature for ligand-gated ion channels.
Neuropharmacology 56, 2-5). The terms GluK2 containing kainate
receptor, GluK2 receptor, and GluK2 subunit may be used
interchangeably throughout and generally refer to the protein
encoded by or expressed by a Grik2 gene.
[0065] In some embodiments, the oligonucleotide of the present
disclosure is a GluK2 inhibitor.
[0066] In one embodiment, the GluK2 inhibitor of the disclosure is
also known as a Grik2 expression inhibitor.
[0067] The term "expression" when used in the context of expression
of a gene or nucleic acid refers to the conversion of the
information, contained in a gene, into a gene product. A gene
product can be the direct transcriptional product of a gene (e.g.,
mRNA, tRNA, rRNA, antisense RNA, ribozyme, structural RNA or any
other type of RNA) or a protein produced by translation of a mRNA.
Gene products also include messenger RNAs, which are modified, by
processes such as capping, polyadenylation, methylation, and
editing, and proteins (e.g., GluK2) modified by, for example,
methylation, acetylation, phosphorylation, ubiquitination,
SUMOylation, ADP-ribosylation, myristilation, and
glycosylation.
[0068] Inhibiting the expression of GluK2 also inhibits the levels
of Gluk2/GluK5 heteromeric receptors (Ruiz et al, J Neuroscience
2005). While not wishing to be bound to any theory, the disclosure
is based on the principle that sufficient removal of GluK2 alone
should remove all GluK2/GluK5 heteromers.
[0069] An "inhibitor of expression" refers to a natural or
synthetic compound that has a biological effect to inhibit or
decrease the expression of a gene, e.g., the Grik2 gene. It will be
understood to those persons of skill in the relevant art that
inhibiting expression of a gene, e.g., the Grik2 gene, typically
results in a decrease or even abolition of the gene product
(protein, e.g., GluK2 protein) in target cells or tissues, although
various levels of inhibition may be achieved. Inhibiting or
decreasing expression is typically referred to as knockdown
[0070] In one embodiment, the GluK2 inhibitor of the disclosure is
an antisense nucleic acid.
[0071] Grik2 expression inhibitors for use in the present
disclosure may be based on antisense oligonucleotide constructs.
Anti-sense oligonucleotides, including antisense RNA molecules and
antisense DNA molecules, would act to directly block the
translation of Grik2 mRNA by binding (e.g., hybridizing) thereto
and thus preventing protein translation or increasing mRNA
degradation, thus decreasing the level of GluK2 proteins, and thus
activity, in a cell. For example, antisense oligonucleotides of at
least about 15 bases and complementary to unique regions of the
mRNA transcript sequence encoding GluK2 can be synthesized, e.g.,
by conventional phosphodiester techniques and administered by e.g.,
intravenous injection or infusion. Methods for using antisense
techniques for specifically alleviating gene expression of genes
whose sequence is known are well known in the art (e.g., see U.S.
Pat. Nos. 6,566,135; 6,566,131; 6,365,354; 6,410,323; 6,107,091;
6,046,321; and 5,981,732, each of which is incorporated by
reference herein in its entirety).
[0072] In one embodiment, the GluK2 inhibitor of the disclosure is
a siRNA.
[0073] Small inhibitory RNAs, also referred to as short interfering
RNAs (siRNAs) can also function as GluK2 expression inhibitors for
use in the present disclosure. Grik2 gene expression can be reduced
by contacting the subject or cell with a small double stranded RNA
(dsRNA), or a vector or construct causing the production of a small
double stranded RNA, such that Grik2 expression is specifically
inhibited (i.e., RNA interference or RNAi) by degradation of mRNAs
in a sequence specific manner. Methods for selecting an appropriate
dsRNA or dsRNA-encoding vector are known in the art for genes whose
sequence is known (e.g., see Tuschl, T. et al. (1999); Elbashir, S.
M. et al. (2001); Hannon, G J. (2002); McManus, M T. et al. (2002);
Brummelkamp, T R. et al. (2002); U.S. Pat. Nos. 6,573,099 and
6,506,559; and International Patent Publication Nos. WO 01/36646,
WO 99/32619, and WO 01/68836, each of which is incorporated by
reference herein in its entirety).
[0074] In one embodiment, the GluK2 inhibitor of the disclosure is
a shRNA.
[0075] Short hairpin RNAs (shRNA) can also function as Grik2
expression inhibitors for use in the present disclosure. A short
hairpin RNA (shRNA) is a sequence of RNA that makes a tight hairpin
turn that can be used to silence gene expression via RNA
interference. shRNA is generally expressed using a vector
introduced into cells, wherein the vector utilizes the U6 promoter
(e.g., SEQ ID NO: 29) or another Pol III promoter to ensure that
the shRNA is always expressed. In some embodiments, the vector
(e.g., a lentiviral vector) may be passed on to daughter cells
following cell division, allowing the gene silencing nucleic acids
to be inherited. The shRNA hairpin structure is cleaved by the
cellular machinery into an siRNA, which is then bound to the
RNA-induced silencing complex (RISC). This complex binds to and
cleaves mRNAs that are fully or partially complementary to the
siRNA sequence to which it is bound.
[0076] In one embodiment, the GluK2 inhibitor of the disclosure is
a miRNA.
[0077] MicroRNAs (miRNAs) can also function as Grik2 expression
inhibitors for use in the present disclosure. MicroRNA refers to
antisense RNA molecules that are generally 21 to 22 nucleotides in
length, even though lengths of 19 and up to 25 nucleotides have
been reported, and suppress translation of targeted mRNAs. MicroRNA
biogenesis generally involves transcription of a non-protein-coding
gene or a non-coding region (e.g., intron or UTR) of a
protein-coding gene into a primary transcript (i.e., pri-miRNA),
which is then processed by the Microprocessor complex in the
nucleus to form another precursor microRNA molecule ("precursor
miRNA" or "pre-miRNA"). Pre-miRNA is subsequently translocated out
of the nucleus to be further processed into mature miRNA. The
pre-miRNA have two regions of complementarity that enable them to
form a stem-loop- or fold-back-like structure, which is cleaved in
animals by a ribonuclease III-like nuclease enzyme called Dicer.
The processed miRNA is typically a part of the stem. The processed
miRNA (also referred to as "mature miRNA") becomes part of a
complex to downregulate, e.g., repress translation, of a particular
target gene. Within the context of the present disclosure,
pri-miRNA refers to a microRNA precursor containing, in the 5' to
3' direction:
[0078] a 5' flanking sequence, a stem-loop sequence containing a
guide sequence, a loop sequence (e.g., a miRNA loop sequence, such
as, e.g., a miR-30 loop sequence), and a passenger sequence, and a
3' flanking sequence. Additionally, a pri-miRNA may refer to a
microRNA precursor containing, in the 5' to 3' direction: a 5'
flanking sequence, a stem-loop sequence containing a passenger
sequence, a loop sequence (e.g., a miRNA loop sequence, such as,
e.g., a miR-30 loop sequence), and a guide sequence, and a 3'
flanking sequence. In particular examples, the pri-miRNA may be an
RNA equivalent of the DNA sequence of SEQ ID NO: 21 or SEQ ID NO:
23. According to the present disclosure, pre-miRNA refers to a
microRNA precursor having a stem-loop sequence containing a guide
sequence, a loop sequence (e.g., a miRNA loop sequence, such as,
e.g., a miR-30 loop sequence), and a passenger sequence. In
particular examples, the pre-miRNA may be an RNA equivalent of the
DNA sequence of SEQ ID NO: 20 or SEQ ID NO: 22.
[0079] In one embodiment, the GluK2 inhibitor of the disclosure is
a miRNA-adapted shRNA (shmiRNA). shmiRNA agents refer to chimeric
molecules that incorporate antisense sequences within the -5p or
the -3p arm of a microRNA scaffold (e.g., a miR-30 scaffold)
containing microRNA flanking and loop sequences. Compared to an
shRNA, shmiRNA generally has a longer stem-loop structure based on
microRNA-derived sequences, with the -5p and the -3p arm exhibiting
full or partial complementarity (e.g., mismatches, G:U wobbles).
Owing to their longer sequences and processing requirements,
shmiRNAs are generally expressed from a Pol II promoter. These
constructs have also been shown to exhibit reduced toxicity as
compared to shRNA-based agents.
[0080] Multiple miRNAs may be employed to knockdown Grik2 (and
subsequently its gene product, GluK2). The miRNAs may be
complementary to different target transcripts or different binding
sites of a target transcript. Polycistronic transcripts may also be
utilized to enhance the efficiency of target gene knockdown. In
some embodiments, multiple genes encoding the same miRNAs or
different miRNAs may be regulated together in a single transcript,
or as separate transcripts in a single vector cassette. In one
embodiment, the vector is a viral vector, including but not limited
to recombinant adeno-associated viral (rAAV) vectors, lentiviral
vectors, retroviral vectors and retrotransposon-based vector
systems.
[0081] The antisense RNA that is complementary to the sense target
sequence is encoded by a nucleic acid sequence for the production
of any of the foregoing inhibitors (e.g., antisense, siRNAs,
shRNAs, miRNAs, or shmiRNA). The polynucleotide encoding double
stranded RNA of interest is incorporated into a gene cassette,
e.g., an expression cassette in which transcription of the DNA is
controlled by a promoter.
[0082] In some embodiments, the antisense nucleic acid of the
disclosure targets and binds (e.g., hybridizes) to a nucleic acid
sequence comprising or consisting of the sequence
AAARCAGGCATTAGCTATG (SEQ ID NO: 1), wherein "R" represents an
adenine or a guanine.
[0083] In some embodiments, the antisense nucleic acid of the
disclosure targets and binds (e.g., hybridizes) to a nucleic acid
sequence comprising or consisting of a sequence SEQ ID NO: 2 or SEQ
ID NO: 16, which can correspond to a passenger sequence of a
nucleic acid construct of the disclosure. This oligonucleotide is
able to bind to and hybridize (e.g., by way of complementary base
pairing) with an antisense sequence (e.g., SEQ ID NO: 14 or SEQ ID
NO: 18) targeting the Grik2 gene or Grik2 mRNA of multiple species,
including human and rat.
[0084] In some embodiments, the antisense nucleic acid of the
disclosure targets and binds to a nucleic acid sequence comprising
or consisting of the sequence SEQ ID NO: 3 or SEQ ID NO: 17, which
can correspond to a passenger sequence of a nucleic acid construct
of the disclosure. This oligonucleotide is able to hybridize with
an antisense sequence (e.g., SEQ ID NO: 15 or SEQ ID NO: 19)
targeting the Grik2 gene or Grik2 mRNA of the mouse.
[0085] The foregoing gene sequences are represented as DNA (e.g.,
cDNA) sequences that can be incorporated into a vector of the
disclosure; however, these sequences may also be represented as
corresponding RNA sequences (e.g., a gene coding RNA) that are
synthesized from the vector within the cell (e.g., any one of SEQ
ID NOs: 16-19). One skilled in the art would understand that the
cDNA sequence is equivalent to the RNA (e.g., a gene coding RNA)
sequence, except for the substitution of uridines with thymidines,
and can be used for the same purpose herein, i.e., the generation
of an antisense oligonucleotide for inhibiting the expression of
Grik2 mRNA. In the case of DNA vectors (e.g., AAV), the
polynucleotide containing the antisense nucleic acid is a DNA
sequence. In the case of RNA vectors, the expression cassette
incorporates the RNA equivalent of the antisense DNA sequences
described herein.
[0086] In other embodiments, each of SEQ ID NOs: 1-3 correspond to
a cDNA sequence of a corresponding region of a Grik2 mRNA (e.g.,
SEQ ID NO: 11) targeted by the antisense oligonucleotides of the
disclosure. In further embodiments, each of SEQ ID NOs: 1-3, 16, or
17 correspond to a passenger sequence of a 5' arm or a 3' arm of a
stem-loop RNA or its DNA equivalent sequence containing a guide
sequence (e.g., any one of SEQ ID NOs: 14, 15, 18, or 19) that is
fully or partially complementary to the passenger sequence and a
loop sequence operably linking the 5' or 3' end of the 3' or 5' end
of the guide sequence or the passenger sequence, wherein the
stem-loop RNA or its DNA equivalent sequence is incorporated into a
nucleic acid expression vector (e.g., an AAV or lentiviral vector)
for heterologous expression in one or more target cells (e.g.,
neurons or glial cells).
[0087] In some embodiments, the antisense nucleic acid of the
disclosure comprises or consists of the sequence SEQ ID NO: 14 or
SEQ ID NO: 18. This oligonucleotide is able to target the Grik2
gene or Grik2 mRNA of multiple species, including human and rat. In
some embodiments, SEQ ID NO: 14 or SEQ ID NO: 18 is a guide
sequence that is fully or partially complementary to a passenger
sequence (e.g., SEQ ID NO: 2 or SEQ ID NO: 16). In some
embodiments, the antisense sequence of SEQ ID NO: 14 is transcribed
within the cell into an RNA sequence of SEQ ID NO: 18.
[0088] In some embodiments, the antisense nucleic acid of the
disclosure comprises or consists of the sequence SEQ ID NO:15 or
SEQ ID NO: 19. This oligonucleotide is able to target a murine
Grik2 gene or Grik2 mRNA. In some embodiments, SEQ ID NO: 15 or SEQ
ID NO: 19 is a guide sequence that is fully or partially
complementary to a passenger sequence (e.g., SEQ ID NO: 3 or SEQ ID
NO: 17). In some embodiments, the antisense sequence of SEQ ID NO:
15 is transcribed within the cell into an RNA sequence of SEQ ID
NO: 19.
[0089] In some embodiments, the antisense nucleic acid of the
disclosure comprises or consists of a nucleic acid sequence having
at least 70% identity to the antisense nucleic acid of the
disclosure.
[0090] According to the disclosure a first nucleic acid sequence
having at least 70% sequence identity with a second nucleic acid
sequence means that the first sequence has 70%; 71%; 72%; 73%; 74%;
75%; 76%; 77%; 78%; 79%; 80%; 81%; 82%; 83%; 84%; 85%; 86%; 87%;
88%; 89%; 90%; 91%; 92%; 93%; 94%; 95%; 96%; 97%; 98%; or 99%
identity to the second nucleic acid sequence. Nucleic acid sequence
identity is particularly determined using a suitable sequence
alignment algorithm and default parameters, such as BLASTn (Karlin
and Altschul, Proc. Natl Acad. Sci. USA 87 (6):2264-2268
(1990)).
Nucleic Acid Vectors
[0091] In a particular embodiment, oligonucleotides (e.g.,
antisense nucleic acid) of the disclosure may be delivered in vivo
alone or in association with a vector. In its broadest sense, a
"vector" is any vehicle capable of facilitating the transfer of the
oligonucleotide of the disclosure to the cells. In some examples,
the vector disclosed herein may directly transport the mature
antisense nucleic acid sequences to cells with reduced degradation
relative to the extent of degradation that would result in the
absence of the vector. In other cases, the vector disclosed herein
delivers a transgene (e.g., a heterologous polynucleotide
containing the antisense nucleic acid sequence) that is
subsequently transcribed (e.g., in the case of an AAV) or reverse
transcribed (e.g., in the case of a retroviral vector) within the
cell. In general, the vectors useful in the disclosure include, but
are not limited to, naked plasmids, non-viral delivery systems
(cationic transfection agents, liposomes, lipid nanoparticles, and
the like), phagemids, viruses, other vehicles derived from viral or
bacterial sources that have been manipulated by the insertion or
incorporation of the oligonucleotide sequences. Viral vectors
include, but are not limited to nucleic acid sequences from the
following viruses: RNA viruses such as a retrovirus (as for example
Moloney murine leukemia virus and lentiviral derived vectors),
Harvey murine sarcoma virus, murine mammary tumor virus, and Rous
sarcoma virus; adenovirus, adeno-associated virus (AAV); SV40-type
viruses; polyoma viruses; Epstein-Barr viruses; papilloma viruses;
herpes virus; vaccinia virus; polio virus. One can readily employ
other vectors not named but known to the art.
[0092] In some embodiment, the present disclosure relates a vector
for delivery of a heterologous nucleic acid, wherein the nucleic
acid encodes an inhibitory RNA that specifically binds (e.g.,
hybridizes) to Grik2 mRNA and inhibits expression of Grik2 in a
cell.
[0093] Accordingly, an object of the disclosure relates to a vector
comprising an antisense sequence of a corresponding region of the
GluK2 receptor, or variants thereof.
[0094] In another embodiment, the vector of the disclosure may
comprise any variant of the antisense sequence of a corresponding
region of the GluK2 receptor.
[0095] In another embodiment, the vector of the disclosure may
comprise any variant of the antisense sequence of any variant of
the corresponding region of the GluK2 receptor.
[0096] Accordingly, an object of the disclosure relates to a vector
comprising an antisense sequence that targets a corresponding
region of the GluK2 receptor, or variants thereof.
[0097] Accordingly, an object of the disclosure relates to a vector
comprising a shRNA sequence that targets a corresponding region of
the GluK2 receptor, or variants thereof.
[0098] Accordingly, an object of the disclosure relates to a vector
comprising a miRNA sequence that targets a corresponding region of
the GluK2 receptor, or variants thereof.
[0099] Accordingly, an object of the disclosure relates to a vector
comprising a shmiRNA sequence that targets a corresponding region
of the GluK2 receptor, or variants thereof.
[0100] Accordingly, an object of the disclosure relates to a vector
comprising the sequence SEQ ID NO: 1 that encodes a corresponding
region of the GluK2 receptor, or variants thereof.
[0101] Accordingly, an object of the disclosure relates to a vector
comprising the sequence SEQ ID NO: 2 or SEQ ID NO: 16 that encodes
a corresponding region of the GluK2 receptor, or variants
thereof.
[0102] Accordingly, an object of the disclosure relates to a vector
comprising the sequence SEQ ID NO: 3 or SEQ ID NO 17 that encodes a
corresponding region of the GluK2 receptor, or variants
thereof.
[0103] In some embodiments, the sequences of SEQ ID NOs: 1-3, 16,
or 17 are oriented in the sense direction with respect to a
corresponding region of a Grik2 mRNA sequence (any one of the Grik2
mRNA sequences disclosed herein).
[0104] Accordingly, an object of the disclosure relates to a vector
comprising the sequence SEQ ID NO: 14 or SEQ ID NO: 18 or a variant
thereof having at least 85% (e.g., at least 90%, 91%, 92%, 93%,
94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to the
nucleic acid sequence of SEQ ID NO: 14 or SEQ ID NO: 18 that binds
(e.g., hybridizes) to a corresponding region of the mRNA sequence
encoding a GluK2 receptor, or variants thereof. In some
embodiments, the oligonucleotide of SEQ ID NO: 14 is transcribed
from the vector (e.g., an AAV) into an RNA sequence of SEQ ID NO:
18. In some embodiments, the vector (e.g., a retroviral vector,
such as, e.g., a lentiviral vector) comprises the RNA sequence of
SEQ ID NO: 18.
[0105] Accordingly, an object of the disclosure relates to a vector
comprising the sequence SEQ ID NO: 15 or SEQ ID NO: 19 or a variant
thereof having at least 85% (e.g., at least 90%, 91%, 92%, 93%,
94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to the
nucleic acid sequence of SEQ ID NO: 15 or SEQ ID NO: 19 that binds
(e.g., hybridizes) to a corresponding region of the mRNA sequence
encoding a GluK2 receptor, or variants thereof. In some
embodiments, the oligonucleotide of SEQ ID NO: 15 is transcribed
from the vector (e.g., an AAV) into an RNA sequence of SEQ ID NO:
19. In some embodiments, the vector (e.g., a retroviral vector,
such as, e.g., a lentiviral vector) comprises the RNA sequence of
SEQ ID NO: 19.
[0106] In some embodiments, SEQ ID NOs: 14, 15, 18, or 19 are
oriented in the antisense direction with respect to (i.e., are a
reverse complement of) a corresponding region of a Grik2 mRNA
sequence (any one of the Grik2 mRNA sequences disclosed
herein).
[0107] In another embodiment, the vector of the disclosure may
comprise any variant of the sequence SEQ ID NO: 1 that encodes a
corresponding region of the GluK2 receptor, or variants
thereof.
[0108] In another embodiment, the vector of the disclosure may
comprise any variant of the sequence SEQ ID NO: 2 or SEQ ID NO: 16
or a variant thereof having at least 85% (e.g., at least 90%, 91%,
92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity
to the nucleic acid sequence of SEQ ID NO: 2 or SEQ ID NO: 16 that
encodes a corresponding region of the GluK2 receptor, or variants
thereof.
[0109] In another embodiment, the vector of the disclosure may
comprise any variant of the sequence SEQ ID NO: 3 or SEQ ID NO: 17
or a variant thereof having at least 85% (e.g., at least 90%, 91%,
92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity
to the nucleic acid sequence of SEQ ID NO: 3 or SEQ ID NO: 17 that
encodes a corresponding region of the GluK2 receptor, or variants
thereof.
[0110] In another embodiment, the vector of the disclosure may
comprise any variant of the sequence SEQ ID NO: 14 or SEQ ID NO: 18
or a variant thereof having at least 85% (e.g., at least 90%, 91%,
92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity
to the nucleic acid sequence of SEQ ID NO: 14 or SEQ ID NO: 18 that
binds (e.g., hybridizes) to a corresponding region of the mRNA
sequence encoding a GluK2 receptor, or variants thereof.
[0111] In another embodiment, the vector of the disclosure may
comprise any variant of the sequence SEQ ID NO: 15 or SEQ ID NO: 19
or a variant thereof having at least 85% (e.g., at least 90%, 91%,
92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity
to the nucleic acid sequence of SEQ ID NO: 15 or SEQ ID NO: 19 that
binds (e.g., hybridizes) to a corresponding region of the mRNA
sequence encoding a GluK2 receptor, or variants thereof.
[0112] Accordingly, an object of the disclosure relates to a vector
comprising an antisense sequence of a corresponding region of the
GluK2 receptor, or variants thereof and an hSyn promoter.
[0113] Accordingly, an object of the disclosure relates to a vector
comprising an antisense sequence of a corresponding region of the
GluK2 receptor, or variants thereof and a hSyn promoter.
[0114] Accordingly, an object of the disclosure relates to a vector
comprising a miRNA sequence that targets a corresponding region of
the GluK2 receptor, or variants thereof and an hSyn promoter.
[0115] Accordingly, an object of the disclosure relates to a vector
comprising a shRNA sequence that targets a corresponding region of
the GluK2 receptor, or variants thereof and an hSyn promoter.
[0116] Accordingly, an object of the disclosure relates to a vector
comprising a shmiRNA sequence that targets a corresponding region
of the GluK2 receptor, or variants thereof and an hSyn
promoter.
[0117] Accordingly, an object of the disclosure relates to a vector
comprising an antisense sequence of a corresponding region of the
GluK2 receptor, or variants thereof and a
calcium/calmodulin-dependent kinase II (CaMKII) promoter (e.g., any
one of SEQ ID NOs: 31-35).
[0118] Accordingly, an object of the disclosure relates to a vector
comprising a miRNA sequence that targets a corresponding region of
the GluK2 receptor, or variants thereof and a CaMKII promoter.
[0119] Accordingly, an object of the disclosure relates to a vector
comprising a shRNA sequence that targets a corresponding region of
the GluK2 receptor, or variants thereof and a CaMKII promoter.
[0120] Accordingly, an object of the disclosure relates to a vector
comprising a shmiRNA sequence that targets a corresponding region
of the GluK2 receptor, or variants thereof and a CaMKII
promoter.
[0121] Accordingly, an object of the disclosure relates to a vector
comprising the sequence SEQ ID NO: 1 and a promoter. In some
embodiments, the vector comprises the sequence set forth in SEQ ID
NO: 1 and an hSyn promoter (e.g., SEQ ID NO: 27 or SEQ ID NO: 28),
CaMKII promoter (e.g., SEQ ID NOs: 31-35), U6 promoter (e.g., SEQ
ID NO: 29), or Pol III promoter.
[0122] The U6 promoter may be a polynucleotide having a nucleic
acid sequence of SEQ ID NO: 29, or a variant thereof having at
least 85% (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,
98%, 99%, or more) sequence identity to the nucleic acid sequence
of SEQ ID NO: 29, as is shown below.
TABLE-US-00002 (SEQ ID NO: 29)
ATCCGACGCCGCCATCTCTAGGCCCGCGCCGGCCCCCTCGCACAGACTTGTGGGAGAAGCTCGGCTACTC
CCCTGCCCCGGTTAATTTGCATATAATATTTCCTAGTAACTATAGAGGCTTAATGTGCGATAAAAGACAGATA
ATCTGTTCTTTTTAATACTAGCTACATTTTACATGATAGGCTTGGATTTCTATAAGAGATACAAATACTAAATT
ATTATTTTAAAAAACAGCACAAAAGGAAACTCACCCTAACTGTAAAGTAATTGTGTGTTTTGAGACTATAAATA
TCCCTTGGAGAAAAGCCTTGTT
[0123] The CaMKII promoter may be a polynucleotide having a nucleic
acid sequence of any one of SEQ ID NOs: 31-35, or a variant thereof
having at least 85% (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%,
96%, 97%, 98%, 99%, or more) sequence identity to the nucleic acid
sequence of any one of SEQ ID NOs: 31-35, as is shown below.
TABLE-US-00003 CaMKII promoter, alpha subunit (RefSeq NM_171825, H.
sapiens) (SEQ ID NO: 30)
GATGCTGACGAAGGCTCGCGAGGCTGTGAGCAGCCACAGTGCCCTGCTCAGAAGCCCCGG CaMKII
promoter, beta 1 subunit (RefSeq NM_172084, H. sapiens) (SEQ ID NO:
31) GTCTCCCGCGCCCGCGCCCGTGTCGCCGCCGTGCCCGCGAGCGGGAGCCGGAGTCGCCGC
CaMKII promoter, beta 2 subunit (RefSeq NM_172084, H. sapiens) (SEQ
ID NO: 32)
CGTGTGCAGATGCAGGGCGCCGGTGCCCTGCGGGTGCGGGTGCAGGAGCAGCGTGTGCAG CaMKII
promoter, delta subunit (RefSeq NM_172115, H. sapiens) (SEQ ID NO:
33) CCCCACGCCACCCTTTCTGGTCATCTCCCCTCCCGCCCCGCCCCTGCGCACACTCCCTCG
CaMKII promoter, gamma subunit (RefSeq NM_172171, H. sapiens) (SEQ
ID NO: 34)
TCTCCCCGGTAAAGTCTCGCGGTGCTGCCGGGCTCAGCCCCGTCTCCTCCTCTTGCTCCC
[0124] Additional CaMKII promoters may include the human alpha
CaMKII promoter sequence described in Wang et al. (Mol. Biol. Rep.
35 (1): 37-44, 2007), the disclosure of which is incorporated in
its entirety herein as it relates to the CaMKII promoter
sequence.
[0125] The CAG promoter may be a polynucleotide having the nucleic
acid of SEQ ID NO: 35 or a variant thereof having at least 85%
(e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%,
or more) sequence identity to the nucleic acid sequence of SEQ ID
NO: 35, as is shown below.
TABLE-US-00004 (SEQ ID NO: 35)
GACATTGATTATTGACTAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTT
CCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCA
ATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGG
TAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGT
AAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTA
TTAGTCATCGCTATTACCATGGTCGAGGTGAGCCCCACGTTCTGCTTCACTCTCCCCATCTCCCCCCCCTCC
CCACCCCCAATTTTGTATTTATTTATTTTTTAATTATTTTGTGCAGCGATGGGGGCGGGGGGGGGGGGGGG
GCGCGCGCCAGGCGGGGCGGGGCGGGGCGAGGGGCGGGGCGGGGCGAGGCGGAGAGGTGCGGCGGC
AGCCAATCAGAGCGGCGCGCTCCGAAAGTTTCCTTTTATGGCGAGGCGGCGGCGGCGGCGGCCCTATAAA
AAGCGAAGCGCGCGGCGGGCGGGAGTCGCTGCGCGCTGCCTTCGCCCCGTGCCCCGCTCCGCCGCCGC
CTCGCGCCGCCCGCCCCGGCTCTGACTGACCGCGTTACTCCCACAGGTGAGCGGGCGGGACGGCCCTTC
TCCTCCGGGCTGTAATTAGCGCTTGGTTTAATGACGGCTTGTTTCTTTTCTGTGGCTGCGTGAAAGCCTTGA
GGGGCTCCGGGAGGGCCCTTTGTGCGGGGGGAGCGGCTCGGGGGGTGCGTGCGTGTGTGTGTGCGTGG
GGAGCGCCGCGTGCGGCTCCGCGCTGCCCGGCGGCTGTGAGCGCTGCGGGCGCGGCGCGGGGCTTTGT
GCGCTCCGCAGTGTGCGCGAGGGGAGCGCGGCCGGGGGCGGTGCCCCGCGGTGCGGGGGGGGCTGCG
AGGGGAACAAAGGCTGCGTGCGGGGTGTGTGCGTGGGGGGGTGAGCAGGGGGTGTGGGCGCGTCGGTC
GGGCTGCAACCCCCCCTGCACCCCCCTCCCCGAGTTGCTGAGCACGGCCCGGCTTCGGGTGCGGGGCTC
CGTACGGGGCGTGGCGCGGGGCTCGCCGTGCCGGGCGGGGGGTGGCGGCAGGTGGGGGTGCCGGGCG
GGGCGGGGCCGCCTCGGGCCGGGGAGGGCTCGGGGGAGGGGCGCGGCGGCCCCCGGAGCGCCGGCG
GCTGTCGAGGCGCGGCGAGCCGCAGCCATTGCCTTTTATGGTAATCGTGCGAGAGGGCGCAGGGACTTCC
TTTGTCCCAAATCTGTGCGGAGCCGAAATCTGGGAGGCGCCGCCGCACCCCCTCTAGCGGGCGCGGGGC
GAAGCGGTGCGGCGCCGGCAGGAAGGAAATGGGCGGGGAGGGCCTTCGTGCGTCGCCGCGCCGCCGTC
CCCTTCTCCCTCTCCAGCCTCGGGGCTGTCCGCGGGGGGACGGCTGCCTTCGGGGGGGACGGGGCAGG
GCGGGGTTCGGCTTCTGGCGTGTGACCGGCGGCTCTAGAGCCTCTGCTAACCATGTTCATGCCTTCTTCTT
TTTCCTACAGCTCCTGGGCAACGTGCTGGTTATTGTGCTGTCTCATCATTTTGGCAAAG
[0126] Accordingly, an object of the disclosure relates to a vector
comprising the sequence SEQ ID NO: 2 or SEQ ID NO: 16 and a
promoter. In some embodiments, the vector comprises the sequence
set forth in SEQ ID NO: 2 or SEQ ID NO: 16 or a variant thereof
having at least 85% (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%,
96%, 97%, 98%, 99%, or more) sequence identity to the nucleic acid
sequence of SEQ ID NO: 2 or SEQ ID NO: 16 and an hSyn, CaMKII
promoter, U6 promoter, or Pol III promoter.
[0127] Accordingly, an object of the disclosure relates to a vector
comprising the sequence SEQ ID NO: 3 or SEQ ID NO: 17 and a
promoter. In some embodiments, the vector comprises the sequence
set forth in SEQ ID NO: 3 or SEQ ID NO: 17 or a variant thereof
having at least 85% (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%,
96%, 97%, 98%, 99%, or more) sequence identity to the nucleic acid
sequence of SEQ ID NO: 3 or SEQ ID NO: 17 and an hSyn promoter,
CaMKII promoter, U6 promoter, or Pol III promoter.
[0128] Accordingly, an object of the disclosure relates to a vector
comprising the sequence SEQ ID NO: 14 or SEQ ID NO: 18 and a
promoter. In some embodiments, the vector comprises the sequence
set forth in SEQ ID NO: 14 or SEQ ID NO: 18 or a variant thereof
having at least 85% (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%,
96%, 97%, 98%, 99%, or more) sequence identity to the nucleic acid
sequence of SEQ ID NO: 14 or SEQ ID NO: 18 and an hSyn promoter,
CaMKII promoter, U6 promoter, or Pol III promoter.
[0129] Accordingly, an object of the disclosure relates to a vector
comprising the sequence SEQ ID NO: 15 or SEQ ID NO: 19 and a
promoter. In some embodiments, the vector comprises the sequence
set forth in SEQ ID NO: 15 or SEQ ID NO: 19 or a variant thereof
having at least 85% (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%,
96%, 97%, 98%, 99%, or more) sequence identity to the nucleic acid
sequence of SEQ ID NO: 15 or SEQ ID NO: 19 and an hSyn promoter,
CaMKII promoter, U6 promoter, or a Pol III promoter.
[0130] Accordingly, an object of the disclosure relates to a vector
comprising an antisense sequence of a corresponding region of the
GluK2 receptor, or variants thereof and a CAG promoter (e.g., SEQ
ID NO: 35).
[0131] Accordingly, an object of the disclosure relates to a vector
comprising an antisense sequence of a corresponding region of the
GluK2 receptor, or variants thereof and a CAG promoter.
[0132] Accordingly, an object of the disclosure relates to a vector
comprising a miRNA sequence that targets a corresponding region of
the GluK2 receptor, or variants thereof and a CAG promoter or a Pol
II promoter.
[0133] Accordingly, an object of the disclosure relates to a vector
comprising a shRNA sequence that targets a corresponding region of
the GluK2 receptor, or variants thereof and a U6 (e.g., SEQ ID NO:
29) promoter or a Pol III promoter.
[0134] Accordingly, an object of the disclosure relates to a vector
comprising a shmiRNA sequence that targets a corresponding region
of the GluK2 receptor, or variants thereof and a U6 promoter or a
Pol III promoter.
[0135] Accordingly, an object of the disclosure relates to a vector
comprising the sequence SEQ ID NO: 1 and a CAG promoter.
[0136] Accordingly, an object of the disclosure relates to a vector
comprising the sequence SEQ ID NO: 2 or SEQ ID NO: 16 or a variant
thereof having at least 85% (e.g., at least 90%, 91%, 92%, 93%,
94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to the
nucleic acid sequence of SEQ ID NO: 2 or SEQ ID NO: 16 and a CAG
promoter.
[0137] Accordingly, an object of the disclosure relates to a vector
comprising the sequence SEQ ID NO: 3 or SEQ ID NO: 17 or a variant
thereof having at least 85% (e.g., at least 90%, 91%, 92%, 93%,
94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to the
nucleic acid sequence of SEQ ID NO: 3 or SEQ ID NO: 17 and a CAG
promoter.
[0138] Accordingly, an object of the disclosure relates to a vector
comprising the sequence SEQ ID NO: 14 or SEQ ID NO: 18 or a variant
thereof having at least 85% (e.g., at least 90%, 91%, 92%, 93%,
94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to the
nucleic acid sequence of SEQ ID NO: 14 or SEQ ID NO: 18 and a CAG
promoter.
[0139] Accordingly, an object of the disclosure relates to a vector
comprising the sequence SEQ ID NO: 15 or SEQ ID NO: 19 or a variant
thereof having at least 85% (e.g., at least 90%, 91%, 92%, 93%,
94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to the
nucleic acid sequence of SEQ ID NO: 15 or SEQ ID NO: 19 and a CAG
promoter.
[0140] Accordingly, an object of the disclosure relates to a vector
comprising the sequence SEQ ID NO: 1 and a CaMKII promoter (e.g.,
any one of SEQ ID NOs: 30-34).
[0141] Accordingly, an object of the disclosure relates to a vector
comprising the sequence SEQ ID NO: 2 or SEQ ID NO: 16 or a variant
thereof having at least 85% (e.g., at least 90%, 91%, 92%, 93%,
94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to the
nucleic acid sequence of SEQ ID NO: 2 or SEQ ID NO: 16 and a CaMKII
promoter.
[0142] Accordingly, an object of the disclosure relates to a vector
comprising the sequence SEQ ID NO: 3 or SEQ ID NO: 17 or a variant
thereof having at least 85% (e.g., at least 90%, 91%, 92%, 93%,
94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to the
nucleic acid sequence of SEQ ID NO: 3 or SEQ ID NO: 17 and a CaMKII
promoter.
[0143] Accordingly, an object of the disclosure relates to a vector
comprising the sequence SEQ ID NO: 14 or SEQ ID NO: 18 or a variant
thereof having at least 85% (e.g., at least 90%, 91%, 92%, 93%,
94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to the
nucleic acid sequence of SEQ ID NO: 14 or SEQ ID NO: 18 and a
CaMKII promoter.
[0144] Accordingly, an object of the disclosure relates to a vector
comprising the sequence SEQ ID NO: 15 or SEQ ID NO: 19 or a variant
thereof having at least 85% (e.g., at least 90%, 91%, 92%, 93%,
94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to the
nucleic acid sequence of SEQ ID NO: 15 or SEQ ID NO: 19 and a
CaMKII promoter.
[0145] In particular examples, the expression vector or
polynucleotide may include a nucleic acid sequence that encodes a
stem and a loop which form a duplex stem-loop structure. For
example, the expression vector or polynucleotide may include a
nucleic acid sequence that encodes a loop region, in which the loop
region may be derived in whole or in part from wild type microRNA
sequence gene (e.g., miR-30) or be completely artificial. In a
particular example, the loop region may be an miR-30 loop sequence.
Furthermore, the stem-loop structure may include a guide sequence
(e.g., an antisense RNA sequence, such as, e.g., any one of SEQ ID
NOs: 14, 15, 18, or 19) and a passenger sequence (e.g., any one of
SEQ ID NOs: 2, 3, 16, or 17) that is complementary to all or part
of the guide sequence. For example, the passenger sequence may be
complementary to all of the nucleotides of the guide sequence
except for 10, 9, 8, 7, 6, 5, 4,3 2, or 1 nucleotide(s) of the
guide sequence or the passenger sequence may be complementary to
any one of SEQ ID NOs: 14, 15, 18, or 19.
[0146] Pre-miRNA or pri-miRNA scaffolds that include guide (i.e.,
antisense) sequences of the disclosure may be used in conjunction
with the compositions and methods disclosed herein, for example,
for use in construction of shmiRNA antisense agents. A pri-miRNA
scaffold includes a pre-miRNA scaffold, and pri-miRNA may be 50-800
nucleotides in length (e.g., 50-800, 75-700, 100-600, 150-500,
200-400, or 250-300 nucleotides). In particular examples, the
pre-miRNA may be 50-100 nucleotides (e.g., between 50-60, 60-70,
70-80, 80-90, or 90-100 nucleotides), 100-200 nucleotides (e.g.,
between 110-120, 120-130, 130-140, 140-150, 150-160, 160-170,
170-180, 180-190, or 190-200 nucleotides), 200-300 nucleotides
(e.g., between 200-210, 210-220, 220-230, 230-240, 240-250,
250-260, 260-270, 270-280, 280-290, or 290-300 nucleotides),
300-400 nucleotides (e.g., between 300-310, 310-320, 320-330,
330-340, 340-350, 350-360, 360-370, 370-380, 380-390, or 390-400
nucleotides), 400-500 nucleotides (e.g., between 400-410, 410-420,
420-430, 430-440, 440-450, 450-460, 460-470, 470-480, 480-490, or
490-500 nucleotides), 500-600 nucleotides (e.g., between 500-510,
510-520, 520-530, 530-540, 540-550, 550-560, 560-570, 570-580,
580-590, or 590-600 nucleotides), 600-700 nucleotides (e.g.,
between 600-610, 610-620, 620-630, 630-640, 640-650, 650-660,
660-670, 670-680, 680-690, or 690-700 nucleotides), or 700-800
nucleotides (e.g., between 700-710, 710-720, 720-730, 730-740,
740-750, 750-760, 760-770, 770-780, 780-790, or 790-800
nucleotides). These engineered scaffolds allow processing of the
pre-miRNA into a double stranded RNA comprising a guide sequence
and a passenger sequence. As such, pre-miRNA includes a 5' arm
including the sequence encoding a guide (i.e., antisense) RNA
(e.g., any one of SEQ ID NOs: 14, 15, 18, or 19), a loop sequence
usually derived from a wild-type miRNA (e.g., miR-30) and a 3' arm
including a sequence encoding a passenger (i.e., sense) strand
(e.g., any one of SEQ ID NOs: 2, 3, 16, 17) which is substantially
complementary to the guide sequence. Pre-miRNA "stem-loop"
sequences are generally longer than 50 nucleotides, e.g. 50-150
nucleotides (e.g., 50-60, 60-70, 70-80, 80-90, 90-100, 100-110,
110-120, 120-130, 130-140, or 140-150 nucleotides), 50-110
nucleotides (e.g., 50-60, 60-70, 70-80, 80-90, 90-100, 100-110
nucleotides), or 50-80 nucleotides (e.g., 50-60, 60-70, 70-80
nucleotides) in length. Pri-miRNA further includes 5' flanking and
3' flanking sequences, flanking the 5' and 3' arms, respectively.
Flanking sequences are not necessarily contiguous with other
sequences (the arm region or the guide sequence), are unstructured,
unpaired regions, and may also be derived, in whole or in part,
from one or more wild-type pri-miRNA scaffolds (e.g., pri-miRNA
scaffolds derived, in whole or in part, from, e.g., miR-30).
Flanking sequences are each at least 4 nucleotides in length, or up
to 300 nucleotides or more in length (e.g., 4-300, 10-275, 20-250,
30-225, 40-200, 50-175, 60-150, 70-125, 80-100, or 90-95
nucleotides). Spacer sequences may be present as intervening
between the aforementioned sequence structures, and in most
instances provide linking polynucleotides, e.g., 1-30 nucleotides
(e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,
18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides),
to provide flexibility without interfering with functionality to
the overall pre-miRNA structure. The spacer may be derived from a
naturally occurring linking group from a naturally occurring RNA, a
portion of a naturally occurring linking group, a poly-A or
poly-U/T, or a random sequence of nucleotides, so long as the
spacer does not interfere with the processing of the double
stranded RNA, nor does the spacer interfere with the
binding/interaction of the guide RNA with the target mRNA
sequence.
[0147] In some embodiments, the pre-miRNA may include a
polynucleotide having a nucleic acid sequence encoded by any one of
SEQ ID NOs: 20 or SEQ ID NO: 22 or a variant thereof having at
least 85% (e.g., at least 90%, 95%, 96%, 97%, 98%, 99%, or more)
sequence identity to the nucleic acid sequence of SEQ ID NOs: 20 or
SEQ ID NO: 22.
[0148] In some embodiments, the pri-miRNA may include a
polynucleotide having a nucleic acid sequence encoded by any one of
SEQ ID NOs: 21 or SEQ ID NO: 23 or a variant thereof having at
least 85% (e.g., at least 90%, 95%, 96%, 97%, 98%, 99%, or more)
sequence identity to the nucleic acid sequence of SEQ ID NOs: 21 or
SEQ ID NO: 23.
[0149] According to the methods and compositions disclosed herein,
the expression vector or polynucleotide may encode (i) a 5'
stem-loop arm including a guide (e.g., antisense) strand (e.g., any
one of SEQ ID NOs: 14, 15, 18, or 19) and, optionally, a 5' spacer
sequence upstream (i.e., 5') relative to the 5' stem-loop arm; a
loop region/sequence, such as, e.g., a miR-30 loop sequence (e.g.,
SEQ ID NO: 23); and (ii) a 3' stem-loop arm including a passenger
(e.g., sense) strand (e.g., any one of SEQ ID NOs: 2, 3, 16, or 17)
and optionally a 3' spacer sequence downstream (i.e., 3') relative
to the 3' stem-loop arm. In another example, the expression vector
or polynucleotide including a nucleotide sequence may further
encode (i) a 5' stem-loop arm including a passenger sequence (e.g.,
any one of SEQ ID NO: 2, 3, 16, or 17) and, optionally, a 5' spacer
sequence upstream (i.e., 5') relative to the 5' stem-loop arm; a
loop region/sequence; (ii) a loop region/sequence, such as, e.g., a
miR-30 loop sequence (e.g., SEQ ID NO: 25); and (ii) a 3' stem-loop
arm including a guide sequence (e.g., any one of SEQ ID NOs: 14,
15, 18, or 19) and optionally a 3' spacer sequence downstream
(i.e., 3') relative to the 3' stem-loop arm. In a further example,
the expression vector or polynucleotide includes a leading 5'
flanking region (e.g., SEQ ID NO: 24) upstream of the guide
sequence (e.g., any one of SEQ ID NOs: 14, 15, 18, or 19) and the
flanking region may be of any length and may be derived in whole or
in part from wild type microRNA sequence, may be heterologous or
derived from a miRNA of different origin from the other flanking
regions or the loop, or may be completely artificial. A 3' flanking
region (e.g., SEQ ID NO: 26) may mirror the 5' flanking region in
size and origin and the 3' flanking region may be downstream (i.e.,
3') of the guide sequence. In yet another example, one or both of
the 5' flanking sequence and the 3' flanking sequences are
absent.
[0150] The expression vector or polynucleotide may include a
nucleotide sequence that further encodes a first flanking region
(e.g., a miR-30 flanking region), said first flanking region
includes a 5' flanking sequence (e.g., SEQ ID NO: 24) and,
optionally, a 5' spacer sequence. In a particular example, the
first flanking region is located upstream (i.e., 5') to said
passenger sequence. In another example, the expression vector or
polynucleotide including a nucleotide sequence encodes a second
flanking region (e.g., SEQ ID NO: 26), said second flanking region
includes a 3' flanking sequence and, optionally, a 3' spacer
sequence. In a particular example, the first flanking region is
located 5' to the guide sequence.
[0151] According to the methods and compositions disclosed herein,
the expression vector may include a polynucleotide sequence that
encodes:
[0152] (a) a stem-loop sequence (e.g., SEQ ID NO: 20) including,
from 5' to 3': [0153] (i) a 5' stem-loop arm including a guide
nucleotide sequence which is at least 85% (e.g., at least 85%, 90%,
95%, 96%, 97%, 98%, 99%, or more) identical to SEQ ID NO: 14, 15,
18, or 19; [0154] (ii) a microRNA loop region, in which the loop
region includes a microRNA loop sequence (e.g. a microRNA loop
sequence (e.g., miR-30 loop sequence of SEQ ID NO: 25); [0155]
(iii) a 3' stem-loop arm including a passenger nucleotide sequence
(e.g., SEQ ID NO: 2, 3, 16, or 17) that is complementary or
substantially complementary to the guide sequence,
[0156] (b) a first flanking region (e.g., a miR-30 flanking region
of SEQ ID NO: 24 or a variant thereof having at least 85% (e.g., at
least 90%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to
the nucleic acid sequence of SEQ ID NO: 24) located 5' to the guide
sequence, in which the first flanking region includes a 5' flanking
sequence and, optionally, a 5' spacer sequence; and
[0157] (c) a second flanking region (e.g., a miR-30 flanking region
of SEQ ID NO: 26 or a variant thereof having at least 85% (e.g., at
least 90%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to
the nucleic acid sequence of SEQ ID NO: 26) located 3' to the
passenger sequence, in which the second flanking region includes a
3' flanking sequence and, optionally, a 3' spacer sequence.
[0158] In some embodiments, the expression construct includes a
polynucleotide having a sequence of SEQ ID NO: 20 or a variant
thereof having at least 85% (e.g., at least 90%, 95%, 96%, 97%,
98%, 99%, or more) sequence identity to the nucleic acid sequence
of SEQ ID NO: 20, as is shown below.
TABLE-US-00005 (SEQ ID NO: 20) TAAAACAGGCATTAGCTATGGGTAGTGAAGCCACAG
ATGCCCATAGCTAATGCCTGTTTTA
[0159] In some embodiments, the expression construct includes a
polynucleotide having a sequence of SEQ ID NO: 21 or a variant
thereof having at least 85% (e.g., at least 90%, 95%, 96%, 97%,
98%, 99%, or more) sequence identity to the nucleic acid sequence
of SEQ ID NO: 21, as is shown below.
TABLE-US-00006 (SEQ ID NO: 21)
TCGACTAGGGATAACAGGGTAATTGTTTGAATGAGGCTTCAGTACTTTACAGAATCGTTGCCTGCACATCTT
GGAAACACTTGCTGGGATTACTTCTTCAGGTTAACCCAACAGAAGGCTCGAGAAGGTATATTGCTGTTGACA
GTGAGCGCTAAAACAGGCATTAGCTATGGGTAGTGAAGCCACAGATGCCCATAGCTAATGCCTGTTTTATTG
CCTACTGCCTCGGAATTCAAGGGGCTACTTTAGGAGCAATTATCTTGTTTACTAAAACTGAATACCTTGCTAT
CTCTTTGATACATTTTTACAAAGCTGAATTAAAATGGTATAAATTATCAC
[0160] In another example, the expression vector or polynucleotide
includes a nucleotide sequence that encodes:
[0161] (a) a stem-loop sequence (e.g., SEQ ID NO: 22) including,
from 5' to 3': [0162] (i) a 5' stem-loop arm including a passenger
nucleotide sequence (e.g., SEQ ID NO: 2, 3, 16, or 17) which is
complementary or substantially complementary to the guide
nucleotide sequence; [0163] (ii) a microRNA loop sequence (e.g. a
miR-30 loop sequence of SEQ ID NO: 25); [0164] (iii) a 3' stem-loop
arm including a guide nucleotide sequence which is at least 85%
(e.g., at least 90%, 95%, 96%, 97%, 98%, 99%, or more) identical to
SEQ ID NO: 14, 15, 18, or 19;
[0165] (b) a first flanking region (e.g., a miR-30 flanking region
of SEQ ID NO: 24 or a variant thereof having at least 85% (e.g., at
least 90%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to
the nucleic acid sequence of SEQ ID NO: 24) located 5' to the guide
sequence; and
[0166] (c) a second flanking region (e.g., a miR-30 flanking region
of SEQ ID NO: 26 or a variant thereof having at least 85% (e.g., at
least 90%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to
the nucleic acid sequence of SEQ ID NO: 26) located 3' to the
passenger sequence, in which the second flanking region includes a
3' flanking sequence and, optionally, a 3' spacer sequence.
[0167] In some embodiments, the expression construct includes a
polynucleotide having a sequence of SEQ ID NO: 22 or a variant
thereof having at least 85% (e.g., at least 90%, 95%, 96%, 97%,
98%, 99%, or more) sequence identity to the nucleic acid sequence
of SEQ ID NO: 22, as is shown below.
TABLE-US-00007 (SEQ ID NO: 22) CCCATAGCTAATGCCTGTTTTATAGTGAAGCCACAG
ATGTAAAACAGGCATTAGCTATGGG
[0168] In some embodiments, the expression construct includes a
polynucleotide having a sequence of SEQ ID NO: 23 or a variant
thereof having at least 85% (e.g., at least 90%, 95%, 96%, 97%,
98%, 99%, or more) sequence identity to the nucleic acid sequence
of SEQ ID NO: 23, as is shown below.
TABLE-US-00008 (SEQ ID NO: 23)
TCGACTAGGGATAACAGGGTAATTGTTTGAATGAGGCTTCAGTACTTTACAGAATCGTTGCCTGCACATCTT
GGAAACACTTGCTGGGATTACTTCTTCAGGTTAACCCAACAGAAGGCTCGAGAAGGTATATTGCTGTTGACA
GTGAGCGCCCCATAGCTAATGCCTGTTTTATAGTGAAGCCACAGATGTAAAACAGGCATTAGCTATGGGTTG
CCTACTGCCTCGGAATTCAAGGGGCTACTTTAGGAGCAATTATCTTGTTTACTAAAACTGAATACCTTGCTAT
CTCTTTGATACATTTTTACAAAGCTGAATTAAAATGGTATAAATTATCAC
[0169] Exemplary microRNA flanking and loop sequences suitable for
use with the present disclosure are provided in Table 6, as is
shown below.
TABLE-US-00009 TABLE 6 MicroRNA sequences for use with expression
constructs of the disclosure MicroRNA SEQ ID sequence NO cDNA
sequence miR-30-A 5' 24
TCGACTAGGGATAACAGGGTAATTGTTTGAATGAGGCTTCAGTACT flanking
TTACAGAATCGTTGCCTGCACATCTTGGAAACACTTGCTGGGATTA region/sequence
CTTCTTCAGGTTAACCCAACAGAAGGCTC miR-30-A loop 25 TAGTGAAGCCACAGATG
region/sequence miR-30-A 26
AAGGGGCTACTTTAGGAGCAATTATCTTGTTTACTAAAACTGAATA 3' flanking
CCTTGCTATCTCTTTGATACATTTTTACAAAGCTGAATTAAAATGGT region/sequence
ATAAATTATCAC
[0170] The length of the aforementioned guide sequence and
passenger sequence may be between 19-25 (e.g., 19, 20, 21, 22, 23,
24, or 25) nucleotides in length. In a particular example, the
length of the guide sequence is 19 nucleotides. In another example,
the length of the guide sequence is 20 nucleotides. In another
example, the length of the guide sequence is 21 nucleotides. In
another example, the length of the guide sequence is 22
nucleotides. In another example, the length of the guide sequence
is 23 nucleotides. In another example, the length of the guide
sequence is 24 nucleotides. In another example, the length of the
guide sequence is 25 nucleotides. In a particular example, the
length of the passenger sequence is 19 nucleotides. In another
example, the length of the passenger sequence is 20 nucleotides. In
another example, the length of the passenger sequence is 21
nucleotides. In another example, the length of the passenger
sequence is 22 nucleotides. In another example, the length of the
passenger sequence is 23 nucleotides. In another example, the
length of the passenger sequence is 24 nucleotides. In another
example, the length of the passenger sequence is 25
nucleotides.
[0171] Accordingly, the guide sequence (e.g., any one of SEQ ID
NOs: 14, 15, 18, or 19) and passenger sequence (e.g., any one of
SEQ ID NOs: 2, 3, 16, or 17) may fully or partially hybridize to
form a stem-loop duplex that is between 7-25 (e.g., 7, 8, 9, 10,
11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25)
nucleotides in length. For example, the stem-loop structure between
the guide sequence and the passenger sequence may be 7 nucleotides
in length. In another example, the stem-loop structure between the
guide sequence and the passenger sequence may be 8 nucleotides in
length. In another example, the stem-loop structure between the
guide sequence and the passenger sequence may be 9 nucleotides in
length. In another example, the stem-loop structure between the
guide sequence and the passenger sequence may be 10 nucleotides in
length. In another example, the stem-loop structure between the
guide sequence and the passenger sequence may be 11 nucleotides in
length. In another example, the stem-loop structure between the
guide sequence and the passenger sequence may be 12 nucleotides in
length. In another example, the stem-loop structure between the
guide sequence and the passenger sequence may be 13 nucleotides in
length. In another example, the stem-loop structure between the
guide sequence and the passenger sequence may be 14 nucleotides in
length. In another example, the stem-loop structure between the
guide sequence and the passenger sequence may be 15 nucleotides in
length. In another example, the stem-loop structure between the
guide sequence and the passenger sequence may be 16 nucleotides in
length. In another example, the stem-loop structure between the
guide sequence and the passenger sequence may be 17 nucleotides in
length. In another example, the stem-loop structure between the
guide sequence and the passenger sequence may be 18 nucleotides in
length. In another example, the stem-loop structure between the
guide sequence and the passenger sequence may be 19 nucleotides in
length. In another example, the stem-loop structure between the
guide sequence and the passenger sequence may be 20 nucleotides in
length. In another example, the stem-loop structure between the
guide sequence and the passenger sequence may be 21 nucleotides in
length. In another example, the stem-loop structure between the
guide sequence and the passenger sequence may be 22 nucleotides in
length. In another example, the stem-loop structure between the
guide sequence and the passenger sequence may be 23 nucleotides in
length. In another example, the stem-loop structure between the
guide sequence and the passenger sequence may be 24 nucleotides in
length. In yet another example, the stem-loop structure between the
guide sequence and the passenger sequence may be 25 nucleotides in
length.
[0172] According to the disclosed methods and compositions, the
stem-loop structure (e.g., SEQ ID NO: 20 or SEQ ID NO: 22) formed
by the guide sequence (e.g., any one of SEQ ID NOs: 14, 15, 18, or
19) and passenger sequence (e.g., any one of SEQ ID NOs: 2, 3, 16,
or 17) may include at least one (e.g., at least 1, 2, 3, 4, 5, 6,
7, 8, 9, 10, 11, 12, 13, 14, or 15) mismatch. For example, the
stem-loop structure may contain 1 mismatch. In another example, the
stem-loop structure contains 2 mismatches. In another example, the
stem-loop structure contains 3 mismatches. In another example, the
stem-loop structure contains 4 mismatches. In another example, the
stem-loop structure contains 5 mismatches. In another example, the
stem-loop structure contains 6 mismatches. In another example, the
stem-loop structure contains 7 mismatches. In another example, the
stem-loop structure contains 8 mismatches. In another example, the
stem-loop structure contains 9 mismatches. In another example, the
stem-loop structure contains 10 mismatches. In another example, the
stem-loop structure contains 11 mismatches. In another example, the
stem-loop structure contains 12 mismatches. In another example, the
stem-loop structure contains 13 mismatches. In another example, the
stem-loop structure contains 14 mismatches. In yet another example,
the stem-loop structure contains 15 mismatches.
[0173] The variants include, for instance, naturally-occurring
variants due to allelic variations between individuals (e.g.,
polymorphisms), alternative splicing forms, etc. The term variant
also includes genes sequences of the disclosure from other sources
or organisms. Variants are preferably substantially homologous to
sequences according to the disclosure, e.g., exhibit a nucleotide
sequence identity of typically at least about 75%, preferably at
least about 85%, more preferably at least about 90%, more
preferably at least about 95% with sequences of the disclosure.
Variants of the genes of the disclosure also include nucleic acid
sequences, which hybridize to a sequence as defined above (or a
complementary strand thereof) under stringent hybridization
conditions. Typical stringent hybridization conditions include
temperatures above 30.degree. C., preferably above 35.degree. C.,
more preferably in excess of 42.degree. C., and/or salinity of less
than about 500 mM, preferably less than 200 mM. Hybridization
conditions may be adjusted by the skilled person by modifying the
temperature, salinity and/or the concentration of other reagents
such as SDS, SSC, etc.
[0174] In one embodiment, the vector use according to the
disclosure is a non-viral vector or a viral vector.
[0175] In a particular embodiment, the non-viral vector may be a
plasmid comprising a polynucleotide that encodes an antisense
sequence that hybridizes to a corresponding region of an mRNA
encoding the GluK2 receptor.
[0176] In another particular embodiment, the vector may be a viral
vector.
[0177] Gene delivery viral vectors useful in the practice of the
present disclosure can be constructed utilizing methodologies well
known in the art of molecular biology. Typically, viral vectors
carrying transgenes are assembled from polynucleotides encoding the
transgene, suitable regulatory elements and elements necessary for
production of viral proteins which mediate cell transduction.
[0178] The term "transgene" refers to the antisense oligonucleotide
of the disclosure.
[0179] The terms "gene transfer" or "gene delivery" refer to
methods or systems for reliably inserting foreign DNA into host
cells. Such methods can result in transient expression of
non-integrated transferred DNA, extrachromosomal replication and
expression of transferred replicons (e.g., episomes), or
integration of transferred genetic material into the genomic DNA of
host cells.
[0180] Such recombinant viruses may be produced by techniques known
in the art, such as by transfecting packaging cells or by transient
transfection with helper plasmids or viruses. Typical examples of
virus packaging cells include PA317 cells, PsiCRIP cells, GPenv+
cells, 293 cells, etc. Detailed protocols for producing such
replication-defective recombinant viruses may be found for instance
in WO95/14785, WO96/22378, U.S. Pat. Nos. 5,882,877, 6,013,516,
4,861,719, 5,278,056 and WO94/19478.
[0181] In a particular embodiment, the viral vector may be an
adenoviral, a retroviral, a lentiviral, a herpesvirus or an
adeno-associated virus (AAV) vectors.
[0182] In one embodiment, adeno-associated viral (AAV) vectors are
employed.
[0183] Accordingly, an object of the disclosure relates to an
adeno-associated virus (AAV) vector comprising an antisense
sequence that encodes a corresponding region of the GluK2 receptor,
or variants thereof.
[0184] In another embodiment, the adeno-associated virus (AAV)
vector of the disclosure may comprise any variant of the antisense
sequence of a corresponding region of the GluK2 receptor.
[0185] Accordingly, an object of the disclosure relates to an
adeno-associated virus (AAV) vector comprising an antisense
sequence of a corresponding region of the GluK2 receptor, or
variants thereof.
[0186] Accordingly, an object of the disclosure relates to an
adeno-associated virus (AAV) vector comprising a shRNA sequence
that targets a corresponding region of the GluK2 receptor, or
variants thereof.
[0187] Accordingly, an object of the disclosure relates to an
adeno-associated virus (AAV) vector comprising a miRNA sequence
that targets a corresponding region of the GluK2 receptor, or
variants thereof.
[0188] Accordingly, an object of the disclosure relates to an
adeno-associated virus (AAV) vector comprising a shmiRNA sequence
that targets a corresponding region of the GluK2 receptor, or
variants thereof.
[0189] Accordingly, an object of the disclosure relates to an
adeno-associated virus (AAV) vector comprising the sequence SEQ ID
NO: 1 that encodes a corresponding region of the GluK2 receptor, or
variants thereof.
[0190] Accordingly, an object of the disclosure relates to an
adeno-associated virus (AAV) vector comprising the sequence SEQ ID
NO: 2 that encodes a corresponding region of the GluK2 receptor, or
variants thereof.
[0191] Accordingly, an object of the disclosure relates to an
adeno-associated virus (AAV) vector comprising the sequence SEQ ID
NO: 3 that encodes a corresponding region of the GluK2 receptor, or
variants thereof.
[0192] In another embodiment, the adeno-associated virus (AAV)
vector of the disclosure may comprise any variant of the sequence
SEQ ID NO: 1 which encodes a corresponding region of the GluK2
receptor, or variants thereof.
[0193] In another embodiment, the adeno-associated virus (AAV)
vector of the disclosure may comprise any variant of the sequence
SEQ ID NO: 2 which encodes a corresponding region of the GluK2
receptor, or variants thereof.
[0194] In another embodiment, the adeno-associated virus (AAV)
vector of the disclosure may comprise any variant of the sequence
SEQ ID NO: 3 which encodes a corresponding region of the GluK2
receptor, or variants thereof.
[0195] Accordingly, an object of the disclosure relates to an
adeno-associated virus (AAV) vector comprising the sequence SEQ ID
NO: 14 or a variant thereof having at least 85% (e.g., at least
90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence
identity to the nucleic acid sequence of SEQ ID NO: 14 that is
complementary (i.e., antisense) to a corresponding region of the
mRNA sequence encoding a GluK2 receptor, or variants thereof.
[0196] Accordingly, an object of the disclosure relates to an
adeno-associated virus (AAV) vector comprising the sequence SEQ ID
NO: 15 or a variant thereof having at least 85% (e.g., at least
90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence
identity to the nucleic acid sequence of SEQ ID NO: 15 that is
complementary (i.e., antisense) to a corresponding region of the
mRNA sequence encoding a GluK2 receptor, or variants thereof.
[0197] Accordingly, an object of the disclosure relates to an
adeno-associated virus (AAV) vector comprising an antisense
sequence of a corresponding region of the GluK2 receptor, or
variants thereof and an hSyn promoter (e.g., SEQ ID NO: 27 or SEQ
ID NO: 28).
[0198] Accordingly, an object of the disclosure relates to an
adeno-associated virus (AAV) vector comprising an antisense
sequence of a corresponding region of the GluK2 receptor, or
variants thereof and an hSyn promoter.
[0199] Accordingly, an object of the disclosure relates to an
adeno-associated virus (AAV) vector comprising a miRNA sequence
that targets a corresponding region of the GluK2 receptor, or
variants thereof and an hSyn promoter.
[0200] Accordingly, an object of the disclosure relates to an
adeno-associated virus (AAV) vector comprising a shRNA sequence
that targets a corresponding region of the GluK2 receptor, or
variants thereof and an hSyn promoter.
[0201] Accordingly, an object of the disclosure relates to an
adeno-associated virus (AAV) vector comprising a shmiRNA sequence
that targets a corresponding region of the GluK2 receptor, or
variants thereof and an hSyn promoter.
[0202] Accordingly, an object of the disclosure relates to an
adeno-associated virus (AAV) vector comprising the sequence SEQ ID
NO: 1 and an hSyn promoter.
[0203] Accordingly, an object of the disclosure relates to an
adeno-associated virus (AAV) vector comprising the sequence SEQ ID
NO: 2 and an hSyn promoter.
[0204] Accordingly, an object of the disclosure relates to an
adeno-associated virus (AAV) vector comprising the sequence SEQ ID
NO: 3 and an hSyn promoter.
[0205] Accordingly, an object of the disclosure relates to an
adeno-associated virus (AAV) vector comprising the sequence SEQ ID
NO: 14 or a variant thereof having at least 85% (e.g., at least
90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence
identity to the nucleic acid sequence of SEQ ID NO: 14 and an hSyn
promoter.
[0206] Accordingly, an object of the disclosure relates to an
adeno-associated virus (AAV) vector comprising the sequence SEQ ID
NO: 15 or a variant thereof having at least 85% (e.g., at least
90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence
identity to the nucleic acid sequence of SEQ ID NO: 15 and an hSyn
promoter.
[0207] Accordingly, an object of the disclosure relates to an AAV
vector comprising an antisense sequence of a corresponding region
of the GluK2 receptor, or variants thereof and a CaMKII promoter
(e.g., any one of SEQ ID NOs: 30-34).
[0208] Accordingly, an object of the disclosure relates to an AAV
vector comprising an antisense sequence that targets a
corresponding region of the GluK2 receptor, or variants thereof and
a CaMKII promoter.
[0209] Accordingly, an object of the disclosure relates to an AAV
vector comprising a miRNA sequence that targets a corresponding
region of the GluK2 receptor, or variants thereof and a CaMKII
promoter.
[0210] Accordingly, an object of the disclosure relates to an AAV
vector comprising a shRNA sequence that targets a corresponding
region of the GluK2 receptor, or variants thereof and a CaMKII
promoter.
[0211] Accordingly, an object of the disclosure relates to an AAV
vector comprising a shmiRNA sequence that targets a corresponding
region of the GluK2 receptor, or variants thereof and a CaMKII
promoter.
[0212] Accordingly, an object of the disclosure relates to an AAV
vector comprising the sequence SEQ ID NO: 1 and a CaMKII
promoter.
[0213] Accordingly, an object of the disclosure relates to an AAV
vector comprising the sequence SEQ ID NO: 2 and a CaMKII
promoter.
[0214] Accordingly, an object of the disclosure relates to an AAV
vector comprising the sequence SEQ ID NO: 3 and a CaMKII
promoter.
[0215] Accordingly, an object of the disclosure relates to an AAV
vector comprising the sequence SEQ ID NO: 14 or a variant thereof
having at least 85% (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%,
96%, 97%, 98%, 99%, or more) sequence identity to the nucleic acid
sequence of SEQ ID NO: 14 and a CaMKII promoter.
[0216] Accordingly, an object of the disclosure relates to an AAV
vector comprising the sequence SEQ ID NO:
[0217] 15 or a variant thereof having at least 85% (e.g., at least
90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence
identity to the nucleic acid sequence of SEQ ID NO: 15 and a CaMKII
promoter.
[0218] Accordingly, an object of the disclosure relates to an
adeno-associated virus (AAV) vector comprising an antisense
sequence of a corresponding region of the GluK2 receptor, or
variants thereof and a CAG promoter (e.g., SEQ ID NO: 35).
[0219] Accordingly, an object of the disclosure relates to an
adeno-associated virus (AAV) vector comprising an antisense
sequence that targets a corresponding region of the GluK2 receptor,
or variants thereof and a CAG promoter.
[0220] Accordingly, an object of the disclosure relates to an
adeno-associated virus (AAV) vector comprising a miRNA sequence
that targets a corresponding region of the GluK2 receptor, or
variants thereof and a CAG promoter.
[0221] Accordingly, an object of the disclosure relates to an
adeno-associated virus (AAV) vector comprising a shRNA sequence
that targets a corresponding region of the GluK2 receptor, or
variants thereof and a CAG promoter.
[0222] Accordingly, an object of the disclosure relates to an
adeno-associated virus (AAV) vector comprising a shmiRNA sequence
that targets a corresponding region of the GluK2 receptor, or
variants thereof and a CAG promoter.
[0223] Accordingly, an object of the disclosure relates to an
adeno-associated virus (AAV) vector comprising the sequence SEQ ID
NO: 1 and a CAG promoter.
[0224] Accordingly, an object of the disclosure relates to an
adeno-associated virus (AAV) vector comprising the sequence SEQ ID
NO: 2 and a CAG promoter.
[0225] Accordingly, an object of the disclosure relates to an
adeno-associated virus (AAV) vector comprising the sequence SEQ ID
NO: 3 and a CAG promoter.
[0226] Accordingly, an object of the disclosure relates to an
adeno-associated virus (AAV) vector comprising the sequence SEQ ID
NO: 14 or a variant thereof having at least 85% (e.g., at least
90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence
identity to the nucleic acid sequence of SEQ ID NO: 14 and a CAG
promoter.
[0227] Accordingly, an object of the disclosure relates to an
adeno-associated virus (AAV) vector comprising the sequence SEQ ID
NO: 15 or a variant thereof having at least 85% (e.g., at least
90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence
identity to the nucleic acid sequence of SEQ ID NO: 15 and a CAG
promoter.
[0228] In one embodiment, the AAV vector is AAV1, AAV2, AAV3, AAV4,
AAS, AAV6, AAV7, AAV8, AAV9, AAV10, AAVrh.10 or any other serotypes
of AAV that can infect human, rodents, monkeys or other
species.
[0229] In a more embodiment, the AAV vector is an AAV9.
[0230] By an "AAV vector" is meant a vector derived from an
adeno-associated virus serotype, including without limitation,
AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV6, AAV7, AAV8, AAV9, AAV10,
AAVrh.10, etc. AAV vectors can have one or more of the AAV
wild-type genes deleted in whole or part, e.g., the rep and/or cap
genes, but retain functional flanking ITR sequences. Functional ITR
sequences are necessary for the rescue, replication and packaging
of the AAV virion. Thus, an AAV vector is defined herein to include
at least those sequences required in cis for replication and
packaging (e.g., functional ITRs) of the virus. ITRs do not need to
be the wild-type polynucleotide sequences, and may be altered,
e.g., by the insertion, deletion or substitution of nucleotides, so
long as the sequences provide for functional rescue, replication
and packaging. AAV expression vectors are constructed using known
techniques to at least provide as operatively linked components in
the direction of transcription, control elements including a
transcriptional initiation region, the DNA of interest (i.e., the
nucleic acid sequences of the disclosure) and a transcriptional
termination region.
[0231] In certain embodiments the viral vectors utilized in the
compositions and methods of the disclosure are recombinant
adeno-associated virus (rAAV). The rAAV may be of any serotype,
modification, or derivative, known in the art, or any combination
thereof (e.g., a population of rAAV that comprises two or more
serotypes, e.g., comprising two or more of rAAV2, rAAV8, and rAAV9)
known in the art. In some embodiments, the rAAV are rAAV1, rAAV2,
rAAV3, rAAV4, rAAV5, rAAV6, rAAV7, rAAV8, rAAV9, rAAV10, rAAV-11,
rAAV-12, rAAV-13, rAAV-14, rAAV-15, rAAV-16, rAAV.rh8, rAAV.rh10,
rAAV.rh20, rAAV.rh39, rAAV.Rh74, rAAV.RHM4-1, AAV.hu37, rAAV.Anc80,
rAAV.Anc80L65, rAAV.7m8, rAAV.PHP.B, rAAV2.5, rAAV2tYF, rAAV3B,
rAAVIK03, rAAV.HSC1, rAAV.HSC2, rAAV.HSC3, rAAV.HSC4, rAAV.HSCS,
rAAV.HSC6, rAAV.HSC7, rAAV.HSC8, rAAV.HSC9, rAAV.HSC10, rAAV.HSC11,
rAAV.HSC12, rAAV.HSC13, rAAV.HSC14, rAAV.HSC15, or rAAV.HSC16, or
other rAAV, or combinations of two or more thereof.
[0232] In some embodiments, the rAAV used in the compositions and
methods of the disclosure comprise a capsid protein from an AAV
capsid serotype selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6,
AAV7, AAV8, AAV9, AAV10, AAV-11, AAV-12, AAV-13, AAV-14, AAV-15,
AAV-16, AAV.rh8, AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74,
AAV.RHM4-1, AAV.hu37, AAV.Anc80, AAV.Anc80L65, AAV.7m8, AAV.PHP.B,
AAV2.5, AAV2tYF, AAV3B, AAV.LK03, AAV.HSC1, AAV.HSC2, AAV.HSC3,
AAV.HSC4, AAV.HSCS, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9,
AAV.HSC10, AAV.HSC11, AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15,
or AAV.HSC16, or a derivative, modification, or pseudotype thereof.
In some embodiments, the rAAV comprise a capsid protein at least
80% or more identical, e.g., 85%, 85%, 87%, 88%, 89%, 90%, 91%,
92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, etc., i.e., up to
100% identical, to e.g., vp1, vp2 and/or vp3 sequence of an AAV
capsid serotype selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6,
AAV7, AAV8, AAV9, AAV10, AAV-11, AAV-12, AAV-13, AAV-14, AAV-15,
AAV-16, AAV.rh8, AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74,
AAV.RHM4-1, AAV.hu37, AAV.Anc80, AAV.Anc80L65, AAV.7m8, AAV.PHP.B,
AAV2.5, AAV2tYF, AAV3B, AAV.LK03, AAV.HSC1, AAV.HSC2, AAV.HSC3,
AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9,
AAV.HSC10, AAV.HSC11, AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15,
or AAV.HSC16.
[0233] In certain embodiments, the AAV that is used in the methods
described herein is Anc80 or Anc80L65, as described in Zinn et al.,
2015: 1056-1068, which is incorporated by reference in its
entirety. In certain embodiments, the AAV that is used in the
methods described herein comprises one of the following amino acid
insertions: LGETTRP (SEQ ID NO: 14 of '956, '517, '282, or '323) or
LALGETTRP (SEQ ID NO: 15 of '956, '517, '282, or '323), as
described in U.S. Pat. Nos. 9,193,956; 9,458,517; and 9,587,282 and
US patent application publication no. 2016/0376323, each of which
is incorporated herein by reference in its entirety. In certain
embodiments, the AAV that is used in the methods described herein
is AAV.7m8, as described in U.S. Pat. Nos. 9,193,956; 9,458,517;
and 9,587,282 and US patent application publication no.
2016/0376323, each of which is incorporated herein by reference in
its entirety. In certain embodiments, the AAV that is used in the
methods described herein is any AAV disclosed in U.S. Pat. No.
9,585,971, such as AAV-PHP.B. In certain embodiments, the AAV that
is used in the methods described herein is any AAV disclosed in
U.S. Pat. No. 9,840,719 and WO 2015/013313, such as AAV.Rh74 and
RHM4-1, each of which is incorporated herein by reference in its
entirety. In certain embodiments, the AAV that is used in the
methods described herein is any AAV disclosed in WO 2014/172669,
such as AAV rh.74, which is incorporated herein by reference in its
entirety. In certain embodiments, the AAV that is used in the
methods described herein is AAV2/5, as described in Georgiadis et
al., 2016, Gene Therapy 23: 857-862 and Georgiadis et al., 2018,
Gene Therapy 25: 450, each of which is incorporated by reference in
its entirety. In certain embodiments, the AAV that is used in the
methods described herein is any AAV disclosed in WO 2017/070491,
such as AAV2tYF, which is incorporated herein by reference in its
entirety. In certain embodiments, the AAV that is used in the
methods described herein is AAVLKO3 or AAV3B, as described in Puzzo
et al., 2017, Sci. Transl. Med. 29 (9): 418, which is incorporated
by reference in its entirety. In certain embodiments, the AAV that
is used in the methods described herein is any AAV disclosed in
U.S. Pat. Nos. 8,628,966; 8,927,514; 9,923,120 and WO 2016/049230,
such as HSC1, HSC2, HSC3, HSC4, HSCS, HSC6, HSC7, HSC8, HSC9,
HSC10, HSC11, HSC12, HSC13, HSC14, HSC15, or HSC16, each of which
is incorporated by reference in its entirety.
[0234] In certain embodiments, the AAV that is used in the methods
described herein is an AAV disclosed in any of the following
patents and patent applications, each of which is incorporated
herein by reference in its entirety: U.S. Pat. Nos. 7,282,199;
7,906,111; 8,524,446; 8,999,678; 8,628,966; 8,927,514; 8,734,809;
9,284,357; 9,409,953; 9,169,299; 9,193,956; 9,458,517; and
9,587,282; US patent application publication nos. 2015/0374803;
2015/0126588; 2017/0067908; 2013/0224836; 2016/0215024;
2017/0051257; and International Patent Application Nos.
PCT/US2015/034799; PCT/EP2015/053335. In some embodiments, the rAAV
have a capsid protein at least 80% or more identical, e.g., 85%,
85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,
99%, 99.5%, etc., i.e., up to 100% identical, to the vp1, vp2
and/or vp3 sequence of an AAV capsid disclosed in any of the
following patents and patent applications, each of which is
incorporated herein by reference in its entirety: U.S. Pat. Nos.
7,282,199; 7,906,111; 8,524,446; 8,999,678; 8,628,966; 8,927,514;
8,734,809; 9,284,357; 9,409,953; 9,169,299; 9,193,956; 9,458,517;
and 9,587,282; US patent application publication nos. 2015/0374803;
2015/0126588; 2017/0067908; 2013/0224836; 2016/0215024;
2017/0051257; and International Patent Application Nos.
PCT/US2015/034799; PCT/EP2015/053335.
[0235] In some embodiments, the rAAV has a capsid protein disclosed
in Intl. Appl. Publ. No. WO 2003/052051 (see, e.g., SEQ ID NO: 2 of
'051), WO 2005/033321 (see, e.g., SEQ ID NOs: 123 and 88 of '321),
WO 03/042397 (see, e.g., SEQ ID NOs: 2, 81, 85, and 97 of '397), WO
2006/068888 (see, e.g., SEQ ID NOs: 1 and 3-6 of '888), WO
2006/110689, (see, e.g., SEQ ID NOs: 5-38 of '689) WO2009/104964
(see, e.g., SEQ ID NOs: 1-5, 7, 9, 20, 22, 24 and 31 of '964), WO
2010/127097 (see, e.g., SEQ ID NOs: 5-38 of '097), and WO
2015/191508 (see, e.g., SEQ ID NOs: 80-294 of '058), and U.S. Appl.
Publ. No. 20150023924 (see, e.g., SEQ ID NOs: 1, 5-10 of '924), the
contents of each of which is herein incorporated by reference in
its entirety, such as, e.g., an rAAV vector having a capsid protein
that is at least 80% or more identical, e.g., 85%, 85%, 87%, 88%,
89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or
more to the vp1, vp2 and/or vp3 amino acid sequence of an AAV
capsid disclosed in Intl. Appl. Publ. No. WO 2003/052051 (see,
e.g., SEQ ID NO: 2 of '051), WO 2005/033321 (see, e.g., SEQ ID NOs:
123 and 88 of '321), WO 03/042397 (see, e.g., SEQ ID NOs: 2, 81,
85, and 97 of '397), WO 2006/068888 (see, e.g., SEQ ID NOs: 1 and
3-6 of '888), WO 2006/110689 (see, e.g., SEQ ID NOs: 5-38 of '689)
WO2009/104964 (see, e.g., SEQ ID NOs: 1-5, 7, 9, 20, 22, 24 and 31
of '964), WO 2010/127097 (see, e.g., SEQ ID NOs: 5-38 of '097), and
WO 201 5/1 91 508 (see, e.g., SEQ ID NOs: 80-294 of '508), and U.S.
Appl. Publ. No. 20150023924 (see, e.g., SEQ ID NOs: 1, 5-10 of
'924).
[0236] Nucleic acid sequences of AAV based viral vectors and
methods of making recombinant AAV and AAV capsids are taught, for
example, in U.S. Pat. Nos. 7,282,199; 7,906,111; 8,524,446;
8,999,678; 8,628,966; 8,927,514; 8,734,809; 9,284,357; 9,409,953;
9,169,299; 9,193,956; 9,458,517; and 9,587,282; US patent
application publication nos. 2015/0374803; 2015/0126588;
2017/0067908; 2013/0224836; 2016/0215024; 2017/0051257;
International Patent Application Nos. PCT/US2015/034799;
PCT/EP2015/053335; WO 2003/052051, WO 2005/033321, WO 03/042397, WO
2006/068888, WO 2006/110689, WO2009/104964, WO 2010/127097, and WO
2015/191508, and U.S. Appl. Publ. No. 20150023924.
[0237] In additional embodiments, the rAAV comprise a pseudotyped
rAAV. In some embodiments, the pseudotyped rAAV are rAAV2/8 or
rAAV2/9 pseudotyped rAAV. Methods for producing and using
pseudotyped rAAV are known in the art (see, e.g., Duan et al., J.
Virol., 75:7662-7671 (2001); Halbert et al., J. Virol.,
74:1524-1532 (2000); Zolotukhin et al., Methods 28:158-167 (2002);
and Auricchio et al., Hum. Molec. Genet. 10:3075-3081, (2001).
[0238] In additional embodiments, the rAAV comprise a capsid
containing a capsid protein chimeric of two or more AAV capsid
serotypes. In some embodiments, the capsid protein is a chimeric of
2 or more AAV capsid proteins from AAV serotypes selected from
AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10,
AAV-11, AAV-12, AAV-13, AAV-14, AAV-15, AAV-16, AAV.rh8, AAV.rh10,
AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu37, AAV.Anc80,
AAV.Anc80L65, AAV.7m8, AAV.PHP.B, AAV2.5, AAV2tYF, AAV3B, AAV.LK03,
AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6,
AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10, AAV.HSC11, AAV.HSC12,
AAV.HSC13, AAV.HSC14, AAV.HSC15, or AAV.HSC16.
[0239] In certain embodiments, a single-stranded AAV (ssAAV) can be
used. In certain embodiments, a self-complementary vector, e.g.,
scAAV, can be used (see, e.g., Wu, 2007, Human Gene Therapy, 18
(2):171-82, McCarty et al, 2001, Gene Therapy, Vol. 8, Number 16,
Pages 1248-1254; and U.S. Pat. Nos. 6,596,535; 7,125,717; and
7,456,683, each of which is incorporated herein by reference in its
entirety).
[0240] In certain embodiments, the recombinant AAV vector used for
delivering the transgene (e.g., a heterologous polynucleotide
encoding an antisense oligonucleotide of the disclosure) have a
tropism for cells in the CNS, including but not limited to neurons
and/or glial cells. Such vectors can include non-replicating
"rAAV", particularly those bearing an AAV9 or AAVrh10 capsid are
preferred. In certain embodiments, the viral vectors provided
herein are AAV9 or AAVrh10 based viral vectors. In certain
embodiments, the AAV9 or AAVrh10 based viral vectors provided
herein retain tropism for CNS cells. AAV variant capsids can be
used, including but not limited to those described by Wilson in
U.S. Pat. No. 7,906,111 which is incorporated by reference herein
in its entirety, with AAV/hu.31 and AAV/hu.32 being particularly
preferred; as well as AAV variant capsids described by Chatterjee
in U.S. Pat. No. 8,628,966, 8,927,514 and Smith et al. (Mol Ther
22: 1625-1634, 2014), each of which is incorporated by reference
herein in its entirety.
[0241] In some embodiment, the present disclosure relates to a
recombinant adeno-associated virus (rAAV) comprising (i) an
expression cassette containing a transgene under the control of
regulatory elements and flanked by ITRs, and (ii) an AAV capsid,
wherein the transgene encodes an inhibitory RNA that specifically
binds (e.g., hybridizes) to Grik2 mRNA and inhibits expression of
Grik2 in a cell.
[0242] Provided in particular embodiments are AAV9 vectors
comprising an artificial genome comprising (i) an expression
cassette containing the transgene under the control of regulatory
elements and flanked by ITRs; and (ii) a viral capsid that has the
amino acid sequence of the AAV9 capsid protein or is at least 95%,
96%, 97%, 98%, 99% or 99.9% identical to the amino acid sequence of
the AAV9 capsid protein while retaining the biological function of
the AAV9 capsid. In certain embodiments, the encoded AAV9 capsid
has the sequence of SEQ ID NO: 123 set forth in U.S. Patent No.
7,906,111 which is incorporated by reference herein in its
entirety, with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,
16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 amino
acid substitutions and retaining the biological function of the
AAV9 capsid.
[0243] Provided in particular embodiments are AAVrh10 vectors
comprising an artificial genome comprising (i) an expression
cassette containing the transgene (e.g., a heterologous
polynucleotide encoding an antisense oligonucleotide of the
disclosure) under the control of regulatory elements and flanked by
ITRs; and (ii) a viral capsid that has the amino acid sequence of
the AAVrh10 capsid protein or is at least 95%, 96%, 97%, 98%, 99%
or 99.9% identical to the amino acid sequence of the AAVrh10 capsid
protein while retaining the biological function of the
AAVrh10capsid. In certain embodiments, the encoded AAVrh10 capsid
has the sequence of SEQ ID NO: 81 set forth in U.S. Pat. No.
9,790,427 which is incorporated by reference herein in its
entirety, with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,
16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 amino
acid substitutions and retaining the biological function of the
AAVrh10 capsid.
[0244] The control elements are selected to be functional in a
mammalian cell. The resulting construct which contains the
operatively linked components is flanked by (5' and 3') functional
AAV ITR sequences. By "adeno-associated virus inverted terminal
repeats" or "AAV ITRs" is meant the art-recognized regions found at
each end of the AAV genome which function together in cis as
origins of DNA replication and as packaging signals for the virus.
AAV ITRs, together with the AAV rep coding region, provide for the
efficient excision and rescue from, and integration of a
polynucleotide sequence interposed between two flanking ITRs into a
mammalian cell genome. The polynucleotide sequences of AAV ITR
regions are known. An "AAV ITR" does not necessarily comprise the
wild-type polynucleotide sequence, but may be altered, e.g., by the
insertion, deletion or substitution of nucleotides. Additionally,
the AAV ITR may be derived from any of several AAV serotypes,
including without limitation, AAV-1, AAV-2, AAV-3, AAV-4, AAV-5,
AAV6, AAV7, AAV8, AAV9, AAV10, AAVrh.10, etc. Furthermore, 5' and
3' ITRs which flank a selected polynucleotide sequence in an AAV
vector need not necessarily be identical or derived from the same
AAV serotype or isolate, so long as they function as intended,
i.e., to allow for excision and rescue of the sequence of interest
from a host cell genome or vector, and to allow integration of the
heterologous sequence into the recipient cell genome when AAV Rep
gene products are present in the cell. Additionally, AAV ITRs may
be derived from any of several AAV serotypes, including without
limitation, AAV-1, AAV-2, AAV-3, AAV-4, AAV 5, AAV6, AAV7, AAV8,
AAV9, AAV10, AAVrh.10, etc. Furthermore, 5' and 3' ITRs which flank
a selected polynucleotide sequence in an AAV expression vector need
not necessarily be identical or derived from the same AAV serotype
or isolate, so long as they function as intended, i.e., to allow
for excision and rescue of the sequence of interest from a host
cell genome or vector, and to allow integration of the DNA molecule
into the recipient cell genome when AAV Rep gene products are
present in the cell.
[0245] Particular embodiments are vectors derived from AAV
serotypes having tropism for and high transduction efficiencies in
cells of the mammalian CNS, particularly neurons. A review and
comparison of transduction efficiencies of different serotypes is
provided in this patent application. In certain examples, AAV2,
AAV5, AAV9 and rh.10 based vectors direct long-term expression of
transgenes (e.g., heterologous polynucleotides encoding an
antisense oligonucleotide of the disclosure) in CNS, preferably
transducing neurons.
[0246] The selected polynucleotide sequence is operably linked to
control elements that direct the transcription or expression
thereof in the subject in vivo. Such control elements can comprise
control sequences normally associated with the selected gene.
[0247] Typically, the vector of the present disclosure comprises an
expression cassette. The term "expression cassette" refers to a
nucleic acid construct comprising nucleic acid elements sufficient
for the expression of the nucleic acid molecule of the present
disclosure. Typically, the nucleic acid molecule encodes a
heterologous gene and may also include suitable regulatory
elements. The heterologous gene refers to a transgene that encodes
an RNA of interest.
[0248] One or more expression cassettes may be employed. Each
expression cassette may comprise at least a promoter sequence
operably linked to a sequence encoding the RNA of interest. Each
expression cassette may consist of additional regulatory elements,
spacers, introns, UTRs, polyadenylation site, and the like. In some
embodiments, the expression cassette is polycistronic with respect
to the transgenes encoding e.g., two or more miRNAs. In other
embodiments the expression cassette comprises a promoter, a nucleic
acid encoding one or more RNA molecules of interest, and a polyA.
In further embodiments, the expression cassette comprises
5'-promoter sequence, a sequence encoding a first RNA of interest,
a sequence encoding a second RNA of interest, and a polyadenylation
sequence-3'.
[0249] In some embodiments, an expression cassette may comprise
additional elements, for example, an intron, an enhancer, a
polyadenylation site, a woodchuck posttranscriptional response
element (WPRE), and/or other elements known to affect expression
levels of the encoding sequence. Typically, an expression cassette
comprises the nucleic acid molecule of the present disclosure
operatively linked to a promoter sequence.
[0250] The term "operatively linked" refers to the association of
two or more nucleic acid fragments on a single nucleic acid
fragment so that the function of one is affected by the other.
[0251] For example, a promoter is operatively linked with a coding
sequence when it is capable of affecting the expression of that
coding sequence (e.g., the coding sequence is under the
transcriptional control of the promoter). Encoding sequences can be
operatively linked to regulatory sequences in sense or antisense
orientation.
[0252] The term "promoter" sequence refers to a polynucleotide
region comprising a DNA regulatory sequence, wherein the regulatory
sequence is derived from a gene which is capable of binding RNA
polymerase and initiating transcription of a downstream
(3'-direction) coding sequence. Transcription promoters can include
"inducible promoters" (where expression of a polynucleotide
sequence operably linked to the promoter is induced by an analyte,
cofactor, regulatory protein, etc.), "repressible promoters" (where
expression of a polynucleotide sequence operably linked to the
promoter is induced by an analyte, cofactor, regulatory protein,
etc.), and "constitutive promoters".
[0253] In some embodiments, the promoter is a heterologous
promoter. The term "heterologous promoter" refers to a promoter
that is not found to be operatively linked to a given encoding
sequence in nature.
[0254] Useful heterologous control sequences generally include
those derived from sequences encoding mammalian or viral genes.
Examples include, but are not limited to, the phosphoglycerate
kinase (PGK) promoter, CAG (composite of the (CMV) cytomegalovirus
enhancer the chicken beta actin promoter (CBA) and the rabbit beta
globin intron), U6 promoter, neuronal promoters (Human synapsin 1
(hSyn) promoter, NeuN promoters, CaMKII promoter, promoter of
Dopamine-1 receptor and Dopamine-2 receptor), the SV40 early
promoter, mouse mammary tumor virus LTR promoter; adenovirus major
late promoter (Ad MLP); a herpes simplex virus (HSV) promoter, a
CMV promoter such as the CMV immediate early promoter region
(CMV-IE), Rous sarcoma virus (RSV) promoter, synthetic promoters,
hybrid promoters, and the like. In addition, sequences derived from
non-viral genes, such as the murine metallothionein gene, will also
find use herein. Such promoter sequences are commercially available
from, e.g., Stratagene (San Diego, Calif.). Examples of CNS
specific promoters include those isolated from the genes of neuron
specific enolase (NSE). Example of dentate gyrus selective
promoters include the promoter of the C1ql2, POMC and prox1
genes.
[0255] For purposes of the present disclosure, both heterologous
promoters and other control elements, such as CNS-specific and
inducible promoters, enhancers and the like, will be of particular
use.
[0256] An "enhancer" is a polynucleotide sequence that can
stimulate promoter activity and may be an innate element of the
promoter or a heterologous element inserted to enhance the level or
tissue-specificity of a promoter. In some embodiments, the promoter
is derived in its entirety from a native gene. In some embodiments,
the promoter is composed of different elements derived from
different naturally occurring promoters. In some embodiments, the
promoter comprises a synthetic polynucleotide sequence. It will be
understood by those skilled in the art that different promoters
will direct the expression of a gene in different tissues or cell
types, or at different stages of development, or in response to
different environmental conditions or to the presence or the
absence of a drug or transcriptional co-factor. Ubiquitous,
cell-type-specific, tissue-specific, developmental stage-specific,
and conditional promoters, for example, drug-responsive promoters
(e.g., tetracycline-responsive promoters) are well known to those
of skill in the art.
[0257] In mammalian systems, three kinds of promoters exist and are
candidates for construction of the expression vectors: Pol I
promoters control transcription of large ribosomal RNAs; Pol II
promoters control the transcription of mRNAs (that are translated
into protein) and small nuclear RNAs (snRNAs); and Pol III
promoters uniquely transcribe small non-coding RNAs. Each has
advantages and constraints to consider when designing the construct
for expression of the RNAs in vivo. For example, Pol III promoters
are useful for synthesizing small interfering RNAs (e.g., shRNAs)
from DNA templates in vivo. For greater control over tissue
specific expression, Pol II promoters are preferred but can only be
used for transcription of miRNAs. When a Pol II promoter is used,
however, it may be preferred to omit translation initiation signals
so that the RNAs function as antisense, siRNA, shRNA, miRNAs,
shmiRNA and are not translated into peptides in vivo.
[0258] The AAV expression vector which harbors the DNA molecule of
interest flanked by AAV ITRs, can be constructed by directly
inserting the selected sequence (s) into an AAV genome which has
had the major AAV open reading frames ("ORFs") excised therefrom.
Other portions of the AAV genome can also be deleted, so long as a
sufficient portion of the ITRs remain to allow for replication and
packaging functions. Such constructs can be designed using
techniques well known in the art (see, e.g., U.S. Pat. Nos. 5,173,
414 and 5,139,941; International Publications Nos. WO 92/01070
(published 23 Jan. 1992) and WO 93/03769 (published 4 Mar. 1993)).
Alternatively, AAV ITRs can be excised from the viral genome or
from an AAV vector containing the same and fused 5' and 3'of a
selected nucleic acid construct that is present in another vector
using standard ligation techniques. AAV vectors which contain ITRs
have been described in, e.g., U.S. Pat. No. 5,139,941. In
particular, several AAV vectors are described therein which are
available from the American Type Culture Collection ("ATCC") under
Accession Numbers 53222, 53223, 53224, 53225 and 53226.
Additionally, chimeric genes can be produced synthetically to
include AAV ITR sequences arranged 5'and 3' of one or more selected
nucleic acid sequences. Preferred codons for expression of the
chimeric gene sequence in mammalian CNS cells can be used, and in
certain embodiments codon optimization of the transgene (e.g., a
heterologous polynucleotide encoding an antisense oligonucleotide
of the disclosure) is performed by well-known methods. The complete
chimeric sequence is assembled from overlapping oligonucleotides
prepared by standard methods. In order to produce AAV virions, an
AAV expression vector is introduced into a suitable host cell using
known techniques, such as by transfection. A number of transfection
techniques are generally known in the art. Particularly suitable
transfection methods include calcium phosphate co-precipitation,
direct microinjection into cultured cells, electroporation,
liposome mediated gene transfer, lipid-mediated transduction, and
nucleic acid delivery using high-velocity microprojectiles.
[0259] For instance, a particular viral vector comprises, in
addition to a nucleic acid sequence of the disclosure, the backbone
of AAV vector plasmid with ITR derived from AAV-2, the promoter,
such as the mouse PGK (phosphoglycerate kinase) gene or the
cytomegalovirus/.beta.-actin hybrid promoter (CAG) consisting of
the enhancer from the CMV immediate gene, the promoter, splice
donor and intron from the chicken .beta.-actin gene, the splice
acceptor from rabbit .beta.-globin, or any neuronal promoter such
as the promoter of Dopamine-1 receptor or Dopamine-2 receptor, or
the synapsin promoter, with or without the wild-type or mutant form
of woodchuck hepatitis virus post-transcriptional regulatory
element (WPRE), and a rabbit beta-globin polyA sequence. The viral
vector may comprise in addition, a nucleic acid sequence encoding
an antibiotic resistance gene such as the genes of resistance
ampicillin (AmpR), kanamycin, hygromycin B, geneticin, blasticidin
S or puromycin.
[0260] In one embodiment, retroviral vectors are employed.
[0261] Retroviruses may be chosen as gene delivery vectors due to
their ability to integrate their genes into the host genome,
transferring a large amount of foreign genetic material, infecting
a broad spectrum of species and cell types and for being packaged
in special cell-lines. In order to construct a retroviral vector, a
nucleic acid encoding a gene of interest is inserted into the viral
genome in the place of certain viral sequences to produce a virus
that is replication-defective. In order to produce virions, a
packaging cell line is constructed containing the gag, pol, and/or
env genes but without the LTR and/or packaging components. When a
recombinant plasmid containing a cDNA, together with the retroviral
LTR and packaging sequences is introduced into this cell line (by
calcium phosphate precipitation for example), the packaging
sequence allows the RNA transcript of the recombinant plasmid to be
packaged into viral particles, which are then secreted into the
culture media. The media containing the recombinant retroviruses is
then collected, optionally concentrated, and used for gene
transfer. Retroviral vectors are able to infect a broad variety of
cell types.
[0262] In another embodiment, lentiviral vectors are employed.
[0263] Accordingly, an object of the disclosure relates to a
lentivirus vector comprising an oligonucleotide sequence that
encoding a corresponding region of the GluK2 receptor, or variants
thereof.
[0264] In another embodiment, the lentivirus vector of the
disclosure may comprise any variant of the antisense sequence of a
corresponding region of the GluK2 receptor.
[0265] In another embodiment, the lentivirus vector of the
disclosure may comprise any variant of the antisense sequence of
for any variant of the GluK2 receptor.
[0266] Accordingly, an object of the disclosure relates to a
lentivirus vector comprising an antisense sequence that targets a
corresponding region of the GluK2 receptor, or variants
thereof.
[0267] Accordingly, an object of the disclosure relates to a
lentivirus vector comprising a shRNA sequence that targets a
corresponding region of the GluK2 receptor, or variants
thereof.
[0268] Accordingly, an object of the disclosure relates to a
lentivirus vector comprising a miRNA sequence that targets a
corresponding region of the GluK2 receptor, or variants
thereof.
[0269] Accordingly, an object of the disclosure relates to a
lentivirus vector comprising a shmiRNA sequence that targets a
corresponding region of the GluK2 receptor, or variants
thereof.
[0270] Accordingly, an object of the disclosure relates to a
lentivirus vector comprising the sequence SEQ ID NO: 1 that encodes
a corresponding region of the GluK2 receptor, or variants
thereof.
[0271] Accordingly, an object of the disclosure relates to a
lentivirus vector comprising the sequence SEQ ID NO: 16 that
encodes a corresponding region of the GluK2 receptor, or variants
thereof having at least 85% (e.g., at least 90%, 91%, 92%, 93%,
94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to the
nucleic acid sequence of SEQ ID NO: 16.
[0272] Accordingly, an object of the disclosure relates to a
lentivirus vector comprising the sequence SEQ ID NO: 17 that
encodes a corresponding region of the GluK2 receptor, or variants
thereof having at least 85% (e.g., at least 90%, 91%, 92%, 93%,
94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to the
nucleic acid sequence of SEQ ID NO: 17.
[0273] Accordingly, an object of the disclosure relates to a
lentivirus vector comprising the sequence SEQ ID NO: 18 that
hybridizes to a sequence encoding a corresponding region of the
GluK2 receptor, or variants thereof having at least 85% (e.g., at
least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more)
sequence identity to the nucleic acid sequence of SEQ ID NO:
18.
[0274] Accordingly, an object of the disclosure relates to a
lentivirus vector comprising the sequence SEQ ID NO: 19 that
hybridizes to a sequence encoding a corresponding region of the
GluK2 receptor, or variants thereof having at least 85% (e.g., at
least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more)
sequence identity to the nucleic acid sequence of SEQ ID NO:
19.
[0275] In another embodiment, the lentivirus vector of the
disclosure may comprise any variant of the sequence SEQ ID NO: 1
which encodes a corresponding region of the GluK2 receptor.
[0276] In another embodiment, the lentivirus vector of the
disclosure may comprise any variant of the sequence SEQ ID NO: 16
which encodes for any variant of a corresponding region of the
GluK2 receptor or variants thereof having at least 85% (e.g., at
least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more)
sequence identity to the nucleic acid sequence of SEQ ID NO:
16.
[0277] In another embodiment, the lentivirus vector of the
disclosure may comprise any variant of the sequence SEQ ID NO: 17
which encodes for any variant of a corresponding region of the
GluK2 receptor or variants thereof having at least 85% (e.g., at
least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more)
sequence identity to the nucleic acid sequence of SEQ ID NO:
17.
[0278] Accordingly, an object of the disclosure relates to a
lentivirus vector comprising an antisense sequence of a
corresponding region of the GluK2 receptor, or variants thereof and
an hSyn promoter (e.g., SEQ ID NO: 27 or SEQ ID NO: 28).
[0279] Accordingly, an object of the disclosure relates to a
lentivirus vector comprising an antisense sequence that targets a
corresponding region of the GluK2 receptor, or variants thereof and
an hSyn promoter.
[0280] Accordingly, an object of the disclosure relates to a
lentivirus vector comprising a miRNA sequence that targets a
corresponding region of the GluK2 receptor, or variants thereof and
an hSyn promoter.
[0281] Accordingly, an object of the disclosure relates to a
lentivirus vector comprising a shRNA sequence that targets a
corresponding region of the GluK2 receptor, or variants thereof and
an hSyn promoter.
[0282] Accordingly, an object of the disclosure relates to a
lentivirus vector comprising a shmiRNA sequence that targets a
corresponding region of the GluK2 receptor, or variants thereof and
an hSyn promoter.
[0283] Accordingly, an object of the disclosure relates to a
lentivirus vector comprising an antisense sequence of a
corresponding region of the GluK2 receptor, or variants thereof and
a CaMKII promoter (e.g., any one of SEQ ID NOs: 30-34).
[0284] Accordingly, an object of the disclosure relates to a
lentivirus vector comprising an antisense sequence that targets a
corresponding region of the GluK2 receptor, or variants thereof and
a CaMKII promoter.
[0285] Accordingly, an object of the disclosure relates to a
lentivirus vector comprising a miRNA sequence that targets a
corresponding region of the GluK2 receptor, or variants thereof and
a CaMKII promoter.
[0286] Accordingly, an object of the disclosure relates to a
lentivirus vector comprising a shRNA sequence that targets a
corresponding region of the GluK2 receptor, or variants thereof and
a CaMKII promoter.
[0287] Accordingly, an object of the disclosure relates to a
lentivirus vector comprising a shmiRNA sequence that targets a
corresponding region of the GluK2 receptor, or variants thereof and
a CaMKII promoter.
[0288] Accordingly, an object of the disclosure relates to a
lentivirus vector comprising the sequence SEQ ID NO: 1 and a CaMKII
promoter.
[0289] Accordingly, an object of the disclosure relates to a
lentivirus vector comprising the sequence SEQ ID NO: 16 or a
variant thereof having at least 85% (e.g., at least 90%, 91%, 92%,
93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to
the nucleic acid sequence of SEQ ID NO: 16 and a CaMKII
promoter.
[0290] Accordingly, an object of the disclosure relates to a
lentivirus vector comprising the sequence SEQ ID NO: 17 or a
variant thereof having at least 85% (e.g., at least 90%, 91%, 92%,
93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to
the nucleic acid sequence of SEQ ID NO: 17 and a CaMKII
promoter.
[0291] Accordingly, an object of the disclosure relates to a
lentivirus vector comprising the sequence SEQ ID NO: 18 or a
variant thereof having at least 85% (e.g., at least 90%, 91%, 92%,
93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to
the nucleic acid sequence of SEQ ID NO: 18 and a CaMKII
promoter.
[0292] Accordingly, an object of the disclosure relates to a
lentivirus vector comprising the sequence SEQ ID NO: 19 or a
variant thereof having at least 85% (e.g., at least 90%, 91%, 92%,
93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to
the nucleic acid sequence of SEQ ID NO: 19 and a CaMKII
promoter.
[0293] Accordingly, an object of the disclosure relates to a
lentivirus vector comprising a shRNA sequence that targets a
corresponding region of the GluK2 receptor, or variants thereof and
a U6 promoter (e.g., SEQ ID NO: 29).
[0294] Accordingly, an object of the disclosure relates to a
lentivirus vector comprising a miRNA sequence that targets a
corresponding region of the GluK2 receptor, or variants thereof and
a CaMKII promoter.
[0295] Accordingly, an object of the disclosure relates to a
lentivirus vector comprising a shmiRNA sequence that targets a
corresponding region of the GluK2 receptor, or variants thereof and
a CaMKII promoter.
[0296] Accordingly, an object of the disclosure relates to a
lentivirus vector comprising the sequence SEQ ID NO: 1 and a U6
promoter (e.g., SEQ ID NO: 29).
[0297] Accordingly, an object of the disclosure relates to a
lentivirus vector comprising the sequence SEQ ID NO: 16 or a
variant thereof having at least 85% (e.g., at least 90%, 91%, 92%,
93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to
the nucleic acid sequence of SEQ ID NO: 16 and a U6 promoter.
[0298] Accordingly, an object of the disclosure relates to a
lentivirus vector comprising the sequence SEQ ID NO: 17 or a
variant thereof having at least 85% (e.g., at least 90%, 91%, 92%,
93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to
the nucleic acid sequence of SEQ ID NO: 17 and a U6 promoter.
[0299] Accordingly, an object of the disclosure relates to a
lentivirus vector comprising the sequence SEQ ID NO: 18 or a
variant thereof having at least 85% (e.g., at least 90%, 91%, 92%,
93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to
the nucleic acid sequence of SEQ ID NO: 18 and a U6 promoter.
[0300] Accordingly, an object of the disclosure relates to a
lentivirus vector comprising the sequence SEQ ID NO: 19 or a
variant thereof having at least 85% (e.g., at least 90%, 91%, 92%,
93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to
the nucleic acid sequence of SEQ ID NO: 19 and a U6 promoter.
[0301] Accordingly, an object of the disclosure relates to a
lentivirus vector comprising an antisense sequence of a
corresponding region of the GluK2 receptor, or variants thereof and
a CAG promoter (e.g., SEQ ID NO: 35).
[0302] Accordingly, an object of the disclosure relates to a
lentivirus vector comprising an antisense sequence that targets a
corresponding region of the GluK2 receptor, or variants thereof and
a CAG promoter.
[0303] Accordingly, an object of the disclosure relates to a
lentivirus vector comprising a miRNA sequence that targets a
corresponding region of the GluK2 receptor, or variants thereof and
a CAG promoter.
[0304] Accordingly, an object of the disclosure relates to a
lentivirus vector comprising a shRNA sequence that targets a
corresponding region of the GluK2 receptor, or variants thereof and
a CAG promoter.
[0305] Accordingly, an object of the disclosure relates to a
lentivirus vector comprising a shmiRNA sequence that targets a
corresponding region of the GluK2 receptor, or variants thereof and
a CAG promoter.
[0306] Accordingly, an object of the disclosure relates to a
lentivirus vector comprising the sequence SEQ ID NO: 1 and a CAG
promoter.
[0307] Accordingly, an object of the disclosure relates to a
lentivirus vector comprising the sequence SEQ ID NO: 16 or a
variant thereof having at least 85% (e.g., at least 90%, 91%, 92%,
93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to
the nucleic acid sequence of SEQ ID NO: 16 and a CAG promoter.
[0308] Accordingly, an object of the disclosure relates to a
lentivirus vector comprising the sequence SEQ ID NO: 17 or a
variant thereof having at least 85% (e.g., at least 90%, 91%, 92%,
93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to
the nucleic acid sequence of SEQ ID NO: 17 and a CAG promoter.
[0309] Accordingly, an object of the disclosure relates to a
lentivirus vector comprising the sequence SEQ ID NO: 18 or a
variant thereof having at least 85% (e.g., at least 90%, 91%, 92%,
93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to
the nucleic acid sequence of SEQ ID NO: 18 and a CAG promoter.
[0310] Accordingly, an object of the disclosure relates to a
lentivirus vector comprising the sequence SEQ ID NO: 19 or a
variant thereof having at least 85% (e.g., at least 90%, 91%, 92%,
93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to
the nucleic acid sequence of SEQ ID NO: 19 and a CAG promoter.
[0311] Lentiviruses are complex retroviruses, which, in addition to
the common retroviral genes gag, pol, and env, contain other genes
with regulatory or structural function. The higher complexity
enables the virus to modulate its life cycle, as in the course of
latent infection. Some examples of lentivirus include the Human
Immunodeficiency Viruses (HIV1, HIV2) and the Simian
Immunodeficiency Virus (SIV). Lentiviral vectors have been
generated by multiply attenuating the HIV virulence genes, for
example, the genes env, vif, vpr, vpu and nef are deleted making
the vector biologically safe. Lentiviral vectors are known in the
art, see, e.g., U.S. Pat. Nos. 6,013,516 and 5,994,136, both of
which are incorporated herein by reference. In general, the vectors
are plasmid-based or virus-based, and are configured to carry the
essential sequences for incorporating foreign nucleic acid, for
selection and for transfer of the nucleic acid into a host cell.
The gag, pol and env genes of the vectors of interest also are
known in the art. Thus, the relevant genes are cloned into the
selected vector and then used to transform the target cell of
interest. Recombinant lentivirus capable of infecting a
non-dividing cell wherein a suitable host cell is transfected with
two or more vectors carrying the packaging functions, namely gag,
pol and env, as well as rev and tat is described in U.S. Pat. No.
5,994,136, incorporated herein by reference. This describes a first
vector that can provide a nucleic acid encoding a viral gag and a
pol gene and another vector that can provide a nucleic acid
encoding a viral env to produce a packaging cell. Introducing a
vector providing a heterologous gene into that packaging cell
yields a producer cell which releases infectious viral particles
carrying the foreign gene of interest. The env preferably is an
amphotropic envelope protein which allows transduction of cells of
human and other species. Typically, the nucleic acid molecule or
the vector of the present disclosure include "control sequences",
which refers collectively to promoter sequences, polyadenylation
signals, transcription termination sequences, upstream regulatory
domains, origins of replication, internal ribosome entry sites
("IRES"), enhancers, and the like, which collectively provide for
the replication, transcription and translation of a coding sequence
in a recipient cell. Not all of these control sequences need always
be present so long as the selected coding sequence is capable of
being replicated, transcribed and translated in an appropriate host
cell.
[0312] In a particular embodiment, the vector of the disclosure
contains a nucleic acid sequence that encodes GluK2 receptor,
including GluK2 isoforms 1 to 7 (e.g., SEQ ID NOs: 4 to 10,
respectively).
Modulation of GluK2 Receptor Activity
[0313] An ionotropic glutamate receptor activity that exhibits fast
gating by glutamate, acts by opening a cation channel permeable to
sodium and potassium, and for which kainate is an agonist. Kainate
selective receptor complexes can form when several subunits,
multimers e.g., heteromers or homomers, of kainate receptors
assemble to form a structure with an extracellular N-terminus and a
large loop that together form the ligand binding domain. The
C-terminus of such a complex is intracellular. The ionotropic
glutamate receptor complex itself acts as a ligand gated ion
channel, and upon binding glutamate, charged ions pass through a
channel in the receptor complex. Kainate receptors are multimeric
assemblies of GluK1, 2 and/or 3 (also called GluR5, R6 and R7),
GluK4 (KA1) and GluK5 (KA2) subunits (Collingridge,
Neuropharmacology. 2009 January; 56 (1):2-5). GluK2 containing
kainate receptors (which terms may be used interchangeably in most
cases with GluK2 receptor and GluK2 subunit to generally refer to
the protein encoded by or expressed by a Grik2 gene) are targets
for modulation of ionotropic glutamate receptor activity and
subsequently amelioration of symptoms related to
epileptogenesis.
[0314] Epileptogenesis, which leads to the establishment of
epilepsy, may appear latent while cellular and molecular changes
that lead to neuronal network reorganization occur. Because plastic
responses of the CNS seem to depend on both the developmental state
and the regional susceptibility, not all subjects with brain
injuries develop epilepsy. The hippocampus, including the dentate
gyrus, has been identified as an epileptic brain region susceptible
to damage, is associated with temporal lobe epilepsy (TLE), and, in
some instances, has been attributed with refractory epilepsy
(resistant to treatment) (Jarero-Basulto, J. J., et al.
Pharmaceuticals, 2018, 11, 17; doi:10.3390/ph11010017). An
amplification of excitatory glutamatergic components may facilitate
spontaneous epileptiform seizures (Kuruba, et al. Epilepsy Behay.
2009, 14 (Suppl. 1), 65-73). Chemical glutamate inhibitors
(antagonists), for example NMDA receptor antagonists, have been
shown to block or reduce neuronal death by Glu-mediated
excitotoxicity and acute seizure generation, however, have poor
efficacy in TLE (Foster, A C, and Kemp, J A. Curr. Opin. Pharmacol.
2006, 6, 7-17). The siRNAs disclosed herein, however, have been
shown in the examples, to decrease the expression of
GluK2-containing KARs in neurons and remarkably prevent spontaneous
epileptiform discharges in a model of TLE.
[0315] In one embodiment, the oligonucleotide encoding a
corresponding region of the Grik2 gene, or variants thereof,
decreases or inhibits epileptiform discharges, or decreases the
frequency of epileptiform discharges. In another embodiment, a
vector comprising an oligonucleotide encoding a corresponding
region of the Grik2 gene, or variants thereof.
[0316] In some embodiments, the oligonucleotide encoding a
corresponding region of the Grik2 gene, or variants thereof, or a
vector comprising the oligonucleotide, is capable of reducing the
expression level of GluK2 in hippocampal cells, including cells of
the dentate gyrus.
[0317] In other embodiments, a method is provided for reducing
epileptiform discharges in a CNS cell comprising providing to the
cell an effective amount of a synthetic RNA molecule encoded by a
nucleic acid that targets and binds (e.g., hybridizes) to a nucleic
acid sequence comprising or consisting of any one of SEQ ID NOs: 2,
3, 16, or 17.
[0318] In other embodiments, a method is provided for reducing
epileptiform discharges in a CNS cell comprising providing to the
cell an effective amount of a synthetic RNA molecule encoded by a
nucleic acid comprising SEQ ID NO: 14 or SEQ ID NO: 18 or a variant
thereof having at least 85% (e.g., at least 90%, 91%, 92%, 93%,
94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to the
nucleic acid sequence of SEQ ID NO: 14 or SEQ ID NO: 18.
[0319] In other embodiments, a method is provided for reducing
epileptiform discharges in a CNS cell comprising providing to the
cell an effective amount of a synthetic RNA molecule encoded by a
nucleic acid comprising SEQ ID NO: 15 or SEQ ID NO: 19 or a variant
thereof having at least 85% (e.g., at least 90%, 91%, 92%, 93%,
94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to the
nucleic acid sequence of SEQ ID NO: 15 or SEQ ID NO: 19.
[0320] Short-term neuronal synaptic plasticity generally involves
increasing or decreasing synaptic sensitivity, and may encompass a
process that modulates the ability of neuronal synapses to change
in the short-term. Long-term neuronal plasticity generally involves
increasing or decreasing numbers of synapses, and may encompass a
process that modulates the ability of neuronal synapses to change
in the long-term. In some embodiments, the antisense
oligonucleotide that binds (e.g., hybridizes) to a corresponding
region of the Grik2 mRNA or variants thereof, or a vector
comprising the oligonucleotide, is capable of reducing the neuronal
expression level of GluK2 leading to the reduction in the frequency
of epileptiform discharges in cortical structures including (but
not restricted to) the hippocampus (including the dentate
gyrus).
Method for Treating Epilepsy
[0321] Accordingly, an object of the present disclosure relates to
a method for treating epilepsy in a subject in need thereof,
wherein the method comprises: administering an effective amount of
a vector comprising an oligonucleotide encoding an inhibitory RNA
that binds (e.g., hybridizes) specifically to Grik2 mRNA and
inhibits expression of Grik2 in the subject. In other words, the
disclosure relates to a vector comprising an oligonucleotide
encoding an inhibitory RNA that binds (e.g., hybridizes)
specifically to Grik2 mRNA and inhibits expression of Grik2 for use
in the treatment of epilepsy.
[0322] Accordingly, an object of the present disclosure relates to
a method of treating epilepsy disease in a subject in need thereof
comprising administering to the subject a therapeutically effective
amount of an antisense agent according to the disclosure. In other
words, the disclosure provides an antisense agent for use in the
treatment of an epilepsy disease. In a particular example, the
antisense agent comprises or consists of SEQ ID NO: 14 or SEQ ID
NO: 18 or a variant thereof having at least 85% (e.g., at least
90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence
identity to the nucleic acid sequence of SEQ ID NO: 14 or SEQ ID
NO: 18. In another example, the antisense agent comprises or
consists of SEQ ID NO: 15 or SEQ ID NO: 19 or a variant thereof
having at least 85% (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%,
96%, 97%, 98%, 99%, or more) sequence identity to the nucleic acid
sequence of SEQ ID NO: 15 or SEQ ID NO: 19.
[0323] Accordingly, an object of the present disclosure relates to
a method of treating epilepsy disease in a subject in need thereof
comprising administering to the subject a therapeutically effective
amount of a vector comprising an antisense agent according to the
disclosure.
[0324] In particular, the disclosure relates to a method of
treating epilepsy disease in a subject in need thereof comprising
administering to the subject a therapeutically effective amount of
a vector comprising an antisense agent according to the disclosure
and a promoter according to the disclosure.
[0325] The term "subject" denotes a mammal, such as a rodent, a
feline, a canine, and a primate. Particularly, the subject
according to the disclosure is a human, a mouse or a rat. More
particularly, the subject according to the disclosure has or is
susceptible to epilepsy. The term "subject" encompasses
"patient".
[0326] The term "epilepsy" can be classified according the
electroclinical syndromes following the Classification and
Terminology of the International League Against Epilepsy (ILAE)
[Berg et al., 2010]. These syndromes can be categorized by age at
onset, distinctive constellations (surgical syndromes), and
structural-metabolic causes: (A) age at onset: (i) neonatal period
includes Benign familial neonatal epilepsy (BFNE), Early myoclonic
encephalopathy (EME), Ohtahara syndrome. (ii) Infancy period
includes Epilepsy of infancy with migrating focal seizures, West
syndrome, Myoclonic epilepsy in infancy (MEI), Benign infantile
epilepsy, Benign familial infantile epilepsy, Dravet syndrome,
Myoclonic encephalopathy in nonprogressive disorders. (iii)
Childhood period includes Febrile seizures plus (FS+),
Panayiotopoulos syndrome, Epilepsy with myoclonic atonic
(previously astatic) seizures, Benign epilepsy with centrotemporal
spikes (BECTS), Autosomal-dominant nocturnal frontal lobe epilepsy
(ADNFLE), Late onset childhood occipital epilepsy (Gastaut type),
Epilepsy with myoclonic absences, Lennox-Gastaut syndrome,
Epileptic encephalopathy with continuous spike-and-wave during
sleep (CSWS), Landau-Kleffner syndrome (LKS), Childhood absence
epilepsy (CAE). (iv) Adolescence-Adult period includes Juvenile
absence epilepsy (JAE) Juvenile myoclonic epilepsy (JME), Epilepsy
with generalized tonic-clonic seizures alone, Progressive myoclonus
epilepsies (PME), Autosomal dominant epilepsy with auditory
features (ADEAF), Other familial temporal lobe epilepsies. (v)
Variable age onset includes Familial focal epilepsy with variable
foci (childhood to adult), Reflex epilepsies. (B) Distinctive
constellations (surgical syndromes) include Mesial Temporal Lobe
Epilepsy (MTLE), Rasmussen syndrome, Gelastic seizures with
hypothalamic hamartoma, Hemiconvulsion-hemiplegia-epilepsy. (C)
Epilepsies attributed to and organized by structural-metabolic
causes include Malformations of cortical development
(hemimegalencephaly, heterotopias, etc.), Neurocutaneous syndromes
(tuberous sclerosis complex, Sturge-Weber, etc.), Tumor, Infection,
Trauma, Angioma, Perinatal insults, Stroke, Etc.
[0327] In another embodiment, the epilepsy may be a benign Rolandic
epilepsy, a frontal lobe epilepsy, an infantile spasms, a juvenile
myoclonic epilepsy, a juvenile absence epilepsy, a childhood
absence epilepsy (pyknolepsy), a hot water epilepsy, a
Lennox-Gastaut syndrome, a Landau-Kleffner syndrome, a Dravet
syndrome, a progressive myoclonus epilepsies, a reflex epilepsy, a
Rasmussen's syndrome, a temporal lobe epilepsy, a limbic epilepsy,
a status epilepticus, an abdominal epilepsy, a massive bilateral
myoclonus, a catamenial epilepsy, a Jacksonian seizure disorder, a
Lafora disease or photosensitive epilepsy.
[0328] In a particular embodiment, the epilepsy is a temporal lobe
epilepsy.
[0329] In one embodiment, the epilepsy is a chronic epilepsy.
[0330] In another embodiment, the epilepsy can be a drug-resistant
(i.e., refractory) epilepsy.
[0331] The term "refractory epilepsy" denotes an epilepsy which is
refractory to current pharmaceutical treatment; that is to say that
current pharmaceutical treatment does not allow an effective
treatment of patients' disease (see for example Dario J. Englot et
al., 2013).
[0332] In a particular embodiment, the refractory epilepsy is a
chronic refractory epilepsy.
[0333] The term "Temporal Lobe Epilepsy" or "TLE" denotes a chronic
neurological condition characterized by chronic and recurrent
seizures (epilepsy) which originate in the temporal lobe of the
brain. This disease is different from acute seizures in naive brain
tissue since in TLE morpho-functional reorganization of neuronal
network and sprouting of hippocampal mossy fibers appears whereas
in acute seizures in naive tissue such reorganization is not
present.
[0334] The term "treatment" or "treat" refer to both prophylactic
or preventive treatment as well as curative or disease modifying
treatment, including treatment of patient at risk of contracting
the disease or suspected to have contracted the disease as well as
patients who are ill or have been diagnosed as suffering from a
disease or medical condition, and includes suppression of clinical
relapse. The treatment may be administered to a subject having a
medical disorder or who ultimately may acquire the disorder, in
order to prevent, cure, delay the onset of, reduce the severity of,
or ameliorate one or more symptoms of a disorder or recurring
disorder, or in order to prolong the survival of a subject beyond
that expected in the absence of such treatment. By "therapeutic
regimen" is meant the pattern of treatment of an illness, e.g., the
pattern of dosing used during therapy. A therapeutic regimen may
include an induction regimen and a maintenance regimen. The phrase
"induction regimen" or "induction period" refers to a therapeutic
regimen (or the portion of a therapeutic regimen) that is used for
the initial treatment of a disease. The general goal of an
induction regimen is to provide a high level of drug to a patient
during the initial period of a treatment regimen. An induction
regimen may employ (in part or in whole) a "loading regimen", which
may include administering a greater dose of the drug than a
physician would employ during a maintenance regimen, administering
a drug more frequently than a physician would administer the drug
during a maintenance regimen, or both. The phrase "maintenance
regimen" or "maintenance period" refers to a therapeutic regimen
(or the portion of a therapeutic regimen) that is used for the
maintenance of a patient during treatment of an illness, e.g., to
keep the patient in remission for long periods of time (months or
years). A maintenance regimen may employ continuous therapy (e.g.,
administering a drug at a regular intervals, e.g., weekly, monthly,
yearly, etc.) or intermittent therapy (e.g., interrupted treatment,
intermittent treatment, treatment at relapse, or treatment upon
achievement of a particular predetermined criteria (e.g., disease
manifestation, etc.)).
[0335] Electroencephalography (EEG) assesses electrical brain
function and is complementary to the neuroimaging techniques, e.g.,
functional MRI (fMRI), single-photon emission computed tomography
(SPECT), and positron emission tomography (PET), that can assess
anatomical brain changes. EEG provides a continuous measure of
cortical function with time resolution, and detection of interictal
(period between seizures) epileptiform discharges is also
informative in a diagnostic setting.
[0336] For the treatment of epilepsy, and to ameliorate the
symptoms of seizures and epileptiform discharges as discussed
supra, a useful transgene may be deployed by a vector which encodes
a functional RNA, e.g., shRNA, miRNA, or shmiRNA that inhibits the
expression of Grik2.
[0337] Methods of delivery of vectors to neurons and/or astrocytes
of the subject includes generally any method suitable for delivery
vectors to the neurons and/or astrocytes such that at least a
portion of cells of a selected synaptically connected cell
population is transduced. Vectors may be delivered to any cells of
the central nervous system, or both. Generally, the vector is
delivered to the cells of the central nervous system, including for
example cells of the spinal cord, brainstem (medulla, pons, and
midbrain), cerebellum, diencephalon (thalamus, hypothalamus),
telencephalon (corpus striatum, cerebral cortex, or, within the
cortex, the occipital, temporal, parietal or frontal lobes), or
combinations thereof, or preferably any suitable subpopulation
thereof. Further preferred sites for delivery include the ruber
nucleus, corpus amygdaloideum, entorhinal cortex and neurons in
ventralis lateralis, or to the anterior nuclei of the thalamus.
[0338] In a particular embodiment, vectors of the disclosure are
delivered by stereotactic injections or microinjections directly in
the brain. In other embodiments, the vectors of the disclosure may
be administered by intravenous injection, for example in the
context of vectors that exhibit tropism for CNS tissues, including
but not limited to AAV9 or AAVrh10.
[0339] To deliver vectors of the disclosure specifically to a
particular region and to a particular population of cells of the
CNS, vectors may be administered by stereotaxic microinjection. For
example, subjects have the stereotactic frame base fixed in place
(screwed into the skull). The brain with stereotactic frame base
(MRI compatible with fiducial markings) is imaged using high
resolution MRI. The MRI images are then transferred to a computer
which runs stereotactic software. A series of coronal, sagittal and
axial images are used to determine the target (site of AAV vector
injection or lentivirus vector injection) and trajectory. The
software directly translates the trajectory into 3 dimensional
coordinates appropriate for the stereotactic frame. Holes are
drilled above the entry site and the stereotactic apparatus
positioned with the needle implanted at the given depth. The AAV
vector or the lentivirus vector are then injected at the target
sites. Since the AAV vector or the lentivirus vector integrate into
the target cells, rather than producing viral particles, the
subsequent spread of the vector is minor, and mainly a function of
passive diffusion from the site of injection and of course the
desired transsynaptic transport, prior to integration. The degree
of diffusion may be controlled by adjusting the ratio of vector to
fluid carrier.
[0340] Additional routes of administration may also comprise local
application of the vector under direct visualization, e.g.,
superficial cortical application, or other non-stereotactic
application. The vector may be delivered intrathecally, in the
ventricles or by intravenous injection.
[0341] In one example, the method of the disclosure comprises
intracerebral administration through stereotaxic injections.
However, other known delivery methods may also be adapted in
accordance with the disclosure. For example, for a more widespread
distribution of the vector across the CNS, it may be injected into
the cerebrospinal fluid, e.g., by lumbar puncture. To direct the
vector to the peripheral nervous system, it may be injected into
the spinal cord or into the peripheral ganglia, or the flesh
(subcutaneously or intramuscularly) of the body part of interest.
In certain situations, the vector can be administered via an
intravascular approach. For example, the vector can be administered
intra-arterially (carotid) in situations where the blood-brain
barrier is disturbed or not disturbed. Moreover, for more global
delivery, the vector can be administered during the "opening" of
the blood-brain barrier achieved by infusion of hypertonic
solutions including mannitol.
[0342] Vectors used herein may be formulated in any suitable
vehicle for delivery. For instance, they may be placed into a
pharmaceutically acceptable suspension, solution or emulsion.
Suitable mediums include saline and liposomal preparations. More
specifically, pharmaceutically acceptable carriers may include
sterile aqueous of non-aqueous solutions, suspensions, and
emulsions. Examples of non-aqueous solvents are propylene glycol,
polyethylene glycol, vegetable oils such as olive oil, and
injectable organic esters such as ethyl oleate. Aqueous carriers
include water, alcoholic/aqueous solutions, emulsions or
suspensions, including saline and buffered media. Intravenous
vehicles include fluid and nutrient replenishers, electrolyte
replenishers (such as those based on Ringer's dextrose), and the
like.
[0343] Preservatives and other additives may also be present such
as, for example, antimicrobials, antioxidants, chelating agents,
and inert gases and the like.
[0344] A colloidal dispersion system may also be used for targeted
gene delivery. Colloidal dispersion systems include macromolecule
complexes, nanocapsules, microspheres, beads, and lipid-based
systems including oil-in-water emulsions, micelles, mixed micelles,
and liposomes.
[0345] Accordingly, an object of the present disclosure relates to
a composition comprising an antisense agent according to the
disclosure.
[0346] In particular, the present disclosure relates to a
composition comprising a vector comprising an antisense according
to the disclosure.
[0347] In particular, the present disclosure relates to a
composition comprising a vector comprising an antisense agent
according to the disclosure and a promoter according to the
disclosure.
[0348] In some embodiments, the present disclosure relates to a
pharmaceutical composition comprising an adeno-associated viral
(AAV) vector comprising:
[0349] (a) a viral capsid; and
[0350] (b) an artificial genome comprising an expression cassette
flanked by AAV inverted terminal repeats (ITRs), wherein the
expression cassette comprises a transgene encoding an
oligonucleotide that binds (e.g., hybridizes) to Grik2 mRNA,
operably one or more regulatory sequences that control expression
of the transgene (e.g., a heterologous polynucleotide encoding an
antisense oligonucleotide of the disclosure) in CNS cells.
[0351] In some embodiments, the expression cassette has a general
structure including the following elements oriented in a 5' to 3'
direction:
[0352] (i) A 5' ITR sequence;
[0353] (ii) a promoter sequence (e.g., a promoter having a sequence
of any one of the promoters disclosed herein);
[0354] (iii) a 5' flanking sequence (a miR-30 5' flanking sequence;
e.g., SEQ ID NO: 24);
[0355] (iv) a stem-loop sequence (e.g., SEQ ID NO: 20 or SEQ ID NO:
22) that includes in the 5' to 3' direction: [0356] a. a stem-loop
5' arm, in which the stem-loop 5' arm includes a guide sequence
containing at least a nucleic acid sequence of SEQ ID NOs: 14, 15,
18, or 19, or a passenger sequence (e.g., having a sequence of SEQ
ID NOs: 2, 3, 16, or 17) that is fully or partially complementary
(e.g., containing no more than 5, no more than 4, no more than 3,
no more than 2, or no more than 1 mismatched nucleotides) to the
nucleic acid sequence of SEQ ID NOs: 14, 15, 18, or 19; [0357] b. a
loop sequence (e.g., a miR-30 loop sequence; e.g., SEQ ID NO: 25);
and [0358] c. a stem-loop 3' arm, in which the stem-loop 3' arm
includes a guide sequence containing at least a nucleic acid
sequence of SEQ ID NOs: 14, 15, 18, or 19 or a passenger sequence
(e.g., having a sequence of SEQ ID NOs: 2, 3, 16, or 17) that is
fully or partially complementary (e.g., containing no more than 5,
no more than 4, no more than 3, no more than 2, or no more than 1
mismatch) to the nucleic acid sequence of SEQ ID NOs: 14, 15, 18,
or 19;
[0359] (v) a 3' flanking sequence (a miR-30 3' flanking sequence;
e.g., SEQ ID NO: 26);
[0360] (vi) optionally, a WPRE sequence;
[0361] (vii) a polyA sequence; and
[0362] (viii) a 3' ITR sequence.
[0363] In a particular example, the passenger sequence (e.g., SEQ
ID: NOs: 2, 3, 16, or 17) that is fully or partially complementary
to the nucleic acid sequence of the guide sequence (e.g., SEQ ID:
NO: 14, 15, 18, or 19) has no more than 5 (e.g., no more than 5, 4,
3, 2, or 1) mismatched nucleotides (i.e., mismatches) relative to
the nucleic acid sequence of SEQ ID NOs: 14, 15, 18, or 19. In
another example, the passenger sequence (e.g., SEQ ID: NOs: 2, 3,
16, or 17) that is fully or partially complementary to the nucleic
acid sequence of the guide sequence (e.g., SEQ ID: NO: 14, 15, 18,
or 19) has no more than 4 (e.g., no more than 4, 3, 2, or 1)
mismatched nucleotides (i.e., mismatches) relative to the nucleic
acid sequence of SEQ ID NOs: 14, 15, 18, or 19. In another example,
the passenger sequence (e.g., SEQ ID: NOs: 2, 3, 16, or 17) that is
fully or partially complementary to the nucleic acid sequence of
the guide sequence (e.g., SEQ ID: NO: 14, 15, 18, or 19) has no
more than 3 (e.g., no more than 3, 2, or 1) mismatched nucleotides
(i.e., mismatches) relative to the nucleic acid sequence of SEQ ID
NOs: 14, 15, 18, or 19. In another example, the passenger sequence
(e.g., SEQ ID: NOs: 2, 3, 16, or 17) that is fully or partially
complementary to the nucleic acid sequence of the guide sequence
(e.g., SEQ ID: NO: 14, 15, 18, or 19) has no more than 2 (e.g., no
more than 2 or 1) mismatched nucleotides (i.e., mismatches)
relative to the nucleic acid sequence of SEQ ID NOs: 14, 15, 18, or
19. In yet another example, the passenger sequence (e.g., SEQ ID:
NOs: 2, 3, 16, or 17) that is fully or partially complementary to
the nucleic acid sequence of the guide sequence (e.g., SEQ ID: NO:
14, 15, 18, or 19) has no more than 1 mismatched nucleotide (i.e.,
mismatches) relative to the nucleic acid sequence of SEQ ID NOs:
14, 15, 18, or 19.
[0364] The antisense agent as described above may be combined with
pharmaceutically acceptable excipients, and optionally
sustained-release matrices, such as biodegradable polymers, to form
pharmaceutical compositions. "Pharmaceutically" or
"pharmaceutically acceptable" refer to molecular entities and
compositions that do not produce an adverse, allergic or other
untoward reaction when administered to a mammal, especially a
human, as appropriate. A pharmaceutically acceptable carrier or
excipient refers to a non-toxic solid, semi-solid or liquid filler,
diluent, encapsulating material or formulation auxiliary of any
type.
[0365] The pharmaceutical compositions of the present disclosure
for oral, sublingual, subcutaneous, intramuscular, intravenous,
transdermal, local or rectal administration, the active principle,
alone or in combination with another active principle, can be
administered in a unit administration form, as a mixture with
conventional pharmaceutical supports, to subjects, such as animals
and human beings. Suitable unit administration forms comprise
oral-route forms such as tablets, gel capsules, powders, granules
and oral suspensions or solutions, sublingual and buccal
administration forms, aerosols, implants, subcutaneous,
transdermal, topical, intraperitoneal, intramuscular, intravenous,
subdermal, transdermal, intrathecal and intranasal administration
forms and rectal administration forms. Typically, the
pharmaceutical compositions contain vehicles which are
pharmaceutically acceptable for a formulation capable of being
injected. These may be in particular isotonic, sterile, saline
solutions (monosodium or disodium phosphate, sodium, potassium,
calcium or magnesium chloride and the like or mixtures of such
salts), or dry, especially freeze-dried compositions which upon
addition, depending on the case, of sterilized water or
physiological saline, permit the constitution of injectable
solutions.
[0366] The pharmaceutical forms suitable for injectable use include
sterile aqueous solutions or dispersions; formulations including
sesame oil, peanut oil or aqueous propylene glycol; and sterile
powders for the extemporaneous preparation of sterile injectable
solutions or dispersions. In all cases, the form must be sterile
and must be fluid to the extent that easy syringability exists. It
must be stable under the conditions of manufacture and storage and
must be preserved against the contaminating action of
microorganisms, such as bacteria and fungi. Solutions comprising
compounds of the disclosure as free base or pharmacologically
acceptable salts can be prepared in water suitably mixed with a
surfactant, such as hydroxypropylcellulose. Dispersions can also be
prepared in glycerol, liquid polyethylene glycols, and mixtures
thereof and in oils. Under ordinary conditions of storage and use,
these preparations contain a preservative to prevent the growth of
microorganisms.
[0367] The polypeptide (or nucleic acid encoding thereof) can be
formulated into a composition in a neutral or salt form.
Pharmaceutically acceptable salts include the acid addition salts
(formed with the free amino groups of the protein) and which are
formed with inorganic acids such as, for example, hydrochloric or
phosphoric acids, or such organic acids as acetic, oxalic,
tartaric, mandelic, and the like. Salts formed with the free
carboxyl groups can also be derived from inorganic bases such as,
for example, sodium, potassium, ammonium, calcium, or ferric
hydroxides, and such organic bases as isopropylamine,
trimethylamine, histidine, procaine and the like. The carrier can
also be a solvent or dispersion medium containing, for example,
water, ethanol, polyol (for example, glycerol, propylene glycol,
and liquid polyethylene glycol, and the like), suitable mixtures
thereof, and vegetables oils. The proper fluidity can be
maintained, for example, by the use of a coating, such as lecithin,
by the maintenance of the required particle size in the case of
dispersion and by the use of surfactants. The prevention of the
action of microorganisms can be brought about by various
antibacterial and antifungal agents, for example, parabens,
chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In
many cases, it will be preferable to include isotonic agents, for
example, sugars or sodium chloride.
[0368] Prolonged absorption of the injectable compositions can be
brought about by the use in the compositions of agents delaying
absorption, for example, aluminum monostearate and gelatin. Sterile
injectable solutions are prepared by incorporating the active
polypeptides in the required amount in the appropriate solvent with
several of the other ingredients enumerated above, as required,
followed by filtered sterilization. Generally, dispersions are
prepared by incorporating the various sterilized active ingredients
into a sterile vehicle which contains the basic dispersion medium
and the required other ingredients from those enumerated above. In
the case of sterile powders for the preparation of sterile
injectable solutions, the preferred methods of preparation may be
vacuum-drying and freeze-drying techniques which yield a powder of
the active ingredient plus any additional desired ingredient from a
previously sterile-filtered solution thereof.
[0369] Upon formulation, solutions will be administered in a manner
compatible with the dosage formulation and in such amount as is
therapeutically effective. The formulations are easily administered
in a variety of dosage forms, such as the type of injectable
solutions described above, but drug release capsules and the like
can also be employed. For parenteral administration in an aqueous
solution, for example, the solution should be suitably buffered if
necessary and the liquid diluent first rendered isotonic with
sufficient saline or glucose. These particular aqueous solutions
are especially suitable for intravenous, intramuscular,
subcutaneous and intraperitoneal administration. In this
connection, sterile aqueous media which can be employed will be
known to those of skill in the art in light of the present
disclosure. For example, one dosage could be dissolved in 1 ml of
isotonic NaCl solution and either added to 1000 ml of
hypodermoclysis fluid or injected at the proposed site of infusion.
Some variation in dosage will necessarily occur depending on the
condition of the subject being treated. The person responsible for
administration will, in any event, determine the appropriate dose
for the individual subject.
Further embodiments are listed in the following:
[0370] 1. A recombinant antisense oligonucleotide that targets a
Grik2 mRNA.
[0371] 2. An oligonucleotide comprising or consisting of a
nucleotide sequence selected from SEQ ID NO: 1, SEQ ID NO: 2, and
SEQ ID NO: 3.
[0372] 3. The antisense oligonucleotide according to embodiment 1,
wherein the antisense oligonucleotide is capable of reducing the
amount of GluK2 containing kainate receptors in neurons.
[0373] 4. A vector for delivery of a heterologous nucleic acid,
wherein the nucleic acid encodes an inhibitory RNA that
specifically binds Grik2 mRNA and inhibits expression of Grik2 in a
cell.
[0374] 5. The vector according to embodiment 3, wherein the vector
is a recombinant adeno-associated viral (rAAV) vector, lentiviral
vector, or retroviral vector.
[0375] 6. A recombinant adeno-associated virus (rAAV) comprising
(i) an expression cassette containing a transgene under the control
of regulatory elements and flanked by ITRs, and (ii) an AAV capsid,
wherein the transgene encodes an inhibitory RNA that specifically
binds Grik2 mRNA and inhibits expression of Grik2 in a cell.
[0376] 7. The rAAV according to embodiment 5, wherein the transgene
comprises an antisense sequence complimentary to mRNA encoding
GluK2 receptor.
[0377] 8. A recombinant adeno-associated virus (rAAV) comprising
the nucleic acid sequence set forth in SEQ ID NO: 1.
[0378] 9. The rAAV according to any one of embodiments 5 to 7,
wherein the transgene is under the control of a promoter.
[0379] 10. The rAAV according to any one of embodiments 5 to 9,
wherein the AAV capsid is AAV9 or AAVrh10.
[0380] 11. A method for treating epilepsy in a subject in need
thereof, wherein the method comprises: administering an effective
amount of a vector comprising an oligonucleotide encoding an
inhibitory RNA that binds specifically to Grik2 mRNA and inhibits
expression of Grik2 in the subject.
[0381] 12. The method according to embodiment 11, wherein the
oligonucleotide encodes a siRNA, shRNA, antisense RNA or miRNA.
[0382] 13. The method according to embodiment 11, wherein the
vector comprises a nucleotide sequence selected from SEQ ID NO: 1,
SEQ ID NO: 2, and SEQ ID NO: 3.
[0383] 14. The method according to any one of embodiments 11 to13,
wherein the epilepsy is temporal lobe epilepsy, a chronic epilepsy,
and/or a refractory epilepsy.
[0384] 15. A pharmaceutical composition comprising an
adeno-associated viral (AAV) vector comprising:
[0385] a) a viral capsid; and
[0386] b) an artificial genome comprising an expression cassette
flanked by AAV inverted terminal repeats (ITRs), wherein the
expression cassette comprises a transgene encoding an
oligonucleotide that binds Grik2 mRNA, operably one or more
regulatory sequences that control expression of the transgene in
CNS cells.
[0387] 16. The composition according to embodiment 14, wherein the
viral capsid is encoded by a nucleic acid sequence that encodes a
capsid protein that is at least 95% identical to an AAV9 or AAVrh10
capsid amino acid sequence.
[0388] The above embodiments relate to gene therapy targeting GluK2
subunit that can be used to inhibit epileptiform discharges. An RNA
sequence (e.g. aaarcaggcattagctatg, wherein "r" represents an
adenine or a guanine; SEQ ID NO: 1) is described.
[0389] The disclosure will be further illustrated by the following
figures and examples. However, these examples and figures should
not be interpreted in any way as limiting the scope of the present
disclosure.
BRIEF DESCRIPTION OF THE FIGURES
[0390] FIGS. 1A-1D illustrate knockdown of GluK2 protein in rat
hippocampal neurons with lentivirally-encoded antisense sequences
against human Grik2 mRNA. (FIG. 1A) Plasmid map of an LV-control
vector (LV104) encoding a scrambled control sequence. (FIG. 1B)
Plasmid map of an LV vector encoding a Grik2 antisense sequence
(SEQ ID NO: 14) as an shRNA (LV173). (FIG. 1C) Plasmid map of an LV
vector encoding a Grik2 antisense sequence (SEQ ID NO: 14) as a
microRNA (LV178) (FIG. 1D) Bar graphs illustrating the relative
GluK2 expression (mean.+-.s.e.m.) of protein lysates harvested from
rat primary hippocampal cultures following treatment with different
viral vectors compared with control condition without infection
(p-value versus control).
[0391] FIG. 2: Bar graphs illustrating the effect of LV137 and
LV178 compared with control conditions (LV104, LV180-scramble,
respectively) on the frequency of epileptiform discharges in mouse
organotypic hippocampal slices. Note the similar effects observed
in slices treated with LV137, LV178 compared with GluK2-/- slices.
*, ** and *** denote significance of P<0.05, P<0.01 and
P<0.001.
[0392] FIGS. 3A-3B illustrate the effect of AAV vectors on
epileptiform discharges in rodent disinhibited cortical slices.
(FIG. 3A) Plasmid map of an AAV9-hu1010 vector including an
expression cassette that contains from 5' to 3': a 5' inverted
terminal repeat (ITR), human synapsin (hSyn) promoter (SEQ ID NO:
27 or SEQ ID NO: 28), a mir-30 5' flanking sequence (SEQ ID NO:
24), 5' stem-loop arm containing an antisense guide sequence (SEQ
ID NO: 14), a mir-30 loop sequence (SEQ ID NO: 25), a 3' stem-loop
arm containing a sense passenger sequence (SEQ ID NO: 2), a 3'
flanking sequence (SEQ ID NO: 26), a rabbit globin polyA signal, a
stuffer DNA, and a 3' ITR. (FIG. 3B) Bar graphs illustrating the
effect of AAV9-hu1010 compared with control conditions
(AAV9-scramble) on the frequency of epileptiform discharges in
mouse organotypic hippocampal slices. Note the similar effects
observed in slices treated with AAV9-hu1010 compared with GluK2-/-
slices. The symbols *, ** and *** denote significance of P<0.05,
P<0.01 and P<0.001.
EXAMPLES
Example 1
Material & Methods
[0393] Ethics
[0394] All the procedures were conducted in accordance with the
guidelines of the University of Bordeaux/CNRS Animal Care or
approved by the Institut National de la Sante et de la Recherche
Medicale (INSERM) animal care and use agreement (B-13-055-19) and
the European community council directive (2010/63/UE).
[0395] Primary Pippocampal Cultures
[0396] Primary hippocampal cultures were prepared from 18-day
embryonic Sprague-Dawley rats. Briefly, hippocampi were dissected
and collected in HBSS containing Penicillin-Streptomycin (PS) and
HEPES. Tissues were dissociated with Trypsin-EDTA/PS/HEPES and
neurons were plated in minimum essential medium supplemented with
10% horse serum on coverslips coated with 1 mg/mL poly-Llysine
(PLL) in 6-well plates at a density of 550.000 cells, for
transfection, per dish. Following neuronal attachment to the
surface, Ara was added to prevent the growth of glial cells. Cells
were maintained at 36.5.degree. C. with 5% CO2.
[0397] In Vitro Models of Temporal Lobe Epilepsy
[0398] Swiss mice were used. They had access to food and water ad
libitum and were housed under a 12 h light/dark cycle at
22-24.degree. C. Hippocampal organotypic slices (350 .mu.m) were
prepared from mice (P8-9) using a McIlwain tissue chopper. Slices
were placed on mesh inserts (Millipore) inside culture dishes
containing 1 ml of the following medium: MEM 50%, HS 25%, HBSS 25%,
HEPES 15 mM, glucose 6.5 mg/ml and insulin 0.1 mg/ml. Culture
medium was changed every 2-3 days and slices maintained in an
incubator at 37.degree. C./5% CO2. Pilocarpine (0.5 .mu.M) was
added to the medium at 5 D.I.V and removed at 7 D.I.V; slices were
recorded for experiments from 13 D.I.V. to 15 D.I.V. When slices
were treated with lentivirus or adeno-associated virus (AAV), the
infection were performed at 0 D.I.V.
[0399] Electrophysiological Recordings and Analysis
[0400] Organotypic slices were individually transferred to a
recording chamber maintained at 30-32.degree. C. and continuously
perfused (2 ml/min) with oxygenated ACSF containing the following
(in mM): 126 NaCl, 3.5 KCl, 1.2 NaH2PO4, 26 NaHCO3, 1.3 MgCl2, 2.0
CaCl2, and 10 D-glucose, pH 7.4. Experiments were performed in the
presence of 5 .mu.M SR-95531 (gabazine, Sigma). Local field
potentials were recorded in the granule cell layer of the dentate
gyrus with an insulated tungsten electrode (diameter 50 .mu.m)
using a DAM-80 amplifier (low filter, 1 Hz; highpass filter, 3 KHz;
World Precision Instruments, Sarasota, Fla.). Signals were analyzed
off-line using Clampfit 10.7 (PClamp) and MiniAnalysis 6.0.1
(Synaptosoft, Decatur, Ga.).
[0401] RNAi and Viral Vectors
[0402] We designed RNAi sequences using Smart selection design
(Birmingham et al., A protocol for designing siRNAs with high
functionality and specificity, Nature Methods., August 2007; 9:
2068-2078.) We compared the efficiency of RNAi sequences (RNAi#h,
RNAi#r, RNAi#m) either as shRNAs, or folded as a short hairpin
micro RNA adapted (shmiRNA), and finally as a microRNA using the
miR30 structure. To express RNAi sequences (RNAi#h, RNAi#r,
RNAi#m), we used viral vectors, in order to promote more efficient
transfection than with plasmids for DNA expression. In a first
series of experiments, RNAi were delivered by lentiviral vectors
(Table 3). We selected RNAi#h sequence as an efficient sequence to
downregulate the levels of GluK2 in infected primary cultures of
rat neurons by Western blotting. We next changed to AAVs which are
commonly used viral vectors for gene therapy (Table 3). These AAV
were produced by REGENXBIO, Inc. (Rockville, Md.; see exemplary AAV
vector map of FIG. 3A).
[0403] The selected human RNAi (RNAi#h) sequence was compared with
rat and mouse sequences (Table 1):
TABLE-US-00010 TABLE 1 Oligonucleotides encoding RNAi sequences
targeting human, mouse, and rat Grik2 mRNA SEQ cDNA encoding a SEQ
cDNA encoding a ID passenger strand ID guide strand Name Species NO
sequence NO sequence RNAi#h H. sapiens 2 taaaacaggcattagctatggg 14
cccatagctaatgcctgtttta RNAi#r R. norvegicus 2
taaaacaggcattagctatggg 14 cccatagctaatgcctgtttta RNAi#m M. musculus
3 taaagcaggcattagctatggg 15 cccatagctaatgcctgcttta
[0404] Table 2 below describes RNA sequences encoded by vectors of
the disclosure.
TABLE-US-00011 TABLE 2 RNA sequences encoded by vectors of the
disclosure SEQ RNA sequence SEQ RNA sequence ID corresponding to ID
corresponding to Name Species NO passenger strand NO guide strand
RNAi#h H. sapiens 16 uaaaacaggcauuagcuauggg 18
cccauagcuaaugccuguuuua RNAi#r R. norvegicus 16
uaaaacaggcauuagcuauggg 18 cccauagcuaaugccuguuuua RNAi#m M. musculus
17 uaaagcaggcauuagcuauggg 19 cccauagcuaaugccugcuuua
[0405] Lentivirus or AAV9 coding for miRNAih were used; miRNAih was
expressed under CAG or human synapsin (hSyn) promoters. The
promoter sequence for the hSyn promoter used in conjunction with
lentiviral vectors is provided in SEQ ID NO: 27, as is shown
below.
TABLE-US-00012 (SEQ ID NO: 27)
CTGCAGAGGGCCCTGCGTATGAGTGCAAGTGGGTTTTAGGACCAGGATGAGGCGGGGTGGGGGTGCCTA
CCTGACGACCGACCCCGACCCACTGGACAAGCACCCAACCCCCATTCCCCAAATTGCGCATCCCCTATCAG
AGAGGGGGAGGGGAAACAGGATGCGGCGAGGCGCGTGCGCACTGCCAGCTTCAGCACCGCGGACAGTG
CCTTCGCCCCCGCCTGGCGGCGCGCGCCACCGCCGCCTCAGCACTGAAGGCGCGCTGACGTCACTCGCC
GGTCCCCCGCAAACTCCCCTTCCCGGCCACCTTGGTCGCGTCCGCGCCGCCGCCGGCCCAGCCGGACCG
CACCACGCGAGGCGCGAGATAGGGGGGCACGGGCGCGACCATCTGCGCTGCGGCGCCGGCGACTCAGC
GCTGCCTCAGTCTGC
[0406] The promoter sequence for the hSyn promoter used in
conjunction with the AAV9 vectors is provided in SEQ ID NO: 28, as
is shown below.
TABLE-US-00013 (SEQ ID NO: 28)
CTGCAGAGGGCCCTGCGTATGAGTGCAAGTGGGTTTTAGGACCAGGATGAGGCGGGGTGGGGGTGCCTA
CCTGACGACCGACCCCGACCCACTGGACAAGCACCCAACCCCCATTCCCCAAATTGCGCATCCCCTATCAG
AGAGGGGGAGGGGAAACAGGATGCGGCGAGGCGCGTGCGCACTGCCAGCTTCAGCACCGCGGACAGTG
CCTTCGCCCCCGCCTGGCGGCGCGCGCCACCGCCGCCTCAGCACTGAAGGCGCGCTGACGTCACTCGCC
GGTCCCCCGCAAACTCCCCTTCCCGGCCACCTTGGTCGCGTCCGCGCCGCCGCCGGCCCAGCCGGACCG
CACCACGCGAGGCGCGAGATAGGGGGGCACGGGCGCGACCATCTGCGCTGCGGCGCCGGCGACTCAGC
GCTGCCTCAGTCTGCCAATTGCAGCGGAGGAGTCGTGTCGTGCCTGAGAGCGCAG
[0407] Some constructs were hybrid constructs also expressing
fluorescent reporter genes.
TABLE-US-00014 TABLE 3 List of viral constructs used to knockdown
the expression of Grik2 by RNAi Construct Sequence Construct Viral
titer/mL LV180 Scramble LV.hSyn.TdTomato.U6.shRNAscramble 4.6E+08
Infectious particles LV173 Human LV.hSyn.TdTomato.U6.shRNAi#h
7.9E+08 Infectious particles LV137 Human
LV.CAG.tGFP.IRES.shmiRNAi#h 2.7E+08 Infectious particles LV104 --
LV.hSyn.GFP 8E+08 Infectious particles LV178 Human
LV.hSyn.TdTomato.miRNAi#h 3.0E+09 Infectious particles AAV9-control
-- AAV9.CAG.GFP 2.0E+13 Gene copies AAV9-scramble Scramble
AAV9.hSyn.GFP.Scr2 9E+12 Gene copies AAV9-hu1010 Human
AAV9.hSyn.miR30.miRNAi#h 9E+12 (SEQ ID NO: 25) Gene copies
[0408] Statistics Analyses
[0409] All values are given as means+SEM. Statistical analyses were
performed using Graphpad Prism Graphpad Prism 7 (GraphPad Software,
La Jolla, CA). For between-group comparisons, raw data were
analyzed by a Mann-Whitney test. The level of significance was set
at P<0.05.
Results
[0410] Firstly, cell culture experiments were performed in primary
embryonic rat neurons to evaluate the effect of RNA interference
strategy on the levels of the endogenous GluK2 protein level. By
Western blotting, we observed a significant reduction of the GluK2
level with the RNAi#1h (SEQ ID NO: 14) (FIG. 1 and corresponding
Table 4). The results showed also the importance of the RNAi
processing: the stabilization of RNAi#1h by mir30 structure (LV178)
increased the efficacy in comparison to the shRNAi (LV173) despite
the fact that these two constructs significantly reduce GluK2 level
expression in rat neurons, respectively by 64.2.+-.1.5%
(p<0.0001) and by 59.5.+-.10.9% (p<0.005) compared to the
control without infection.
TABLE-US-00015 TABLE 4 GluK2 levels in cultured cortical neurons
infected with LV constructs encoding RNAi sequences GluK2 levels
LV173 LV137 LV178 Mean 40.6 61.2 35.8 S.E.M. 10.9 12.8 1.5 P-value*
0.005 0.038 <0.0001 *Statistically significant differences in
GluK2 levels were measured between GluK2 levels in cells treated
with RNAi sequences and a control condition in which cortical
neurons were not infected.
[0411] Secondly, reliable stereotyped spontaneous epileptiform
discharges were recorded in organotypic slices in the presence of 5
.mu.M gabazine as previously described (Peret et al., 2014). In
this condition, we observed a striking reduction of the frequency
of epileptiform discharges in treated slices with LV137
(LV.CAG.tGFP.IRES.shmiRNAi#h), LV178 (LV.hSyn.TdTomato.miRNAi#h)
and AAV9-hu1010 (AAV9.hSyn.miR30.miRNAi#h), compared with control
conditions (LV.hSyn.GFP, LV.hSyn.TdTomato.U6.shRNAscramble
(TTTGTGAGGGTCTGGTC; SEQ ID NO: 36) and AAV9.CAG.GFP, respective;
Tables 5 and 6 and FIGS. 2 and 3). Remarkably, the reduction of the
frequency of epileptiform discharges observed in the presence of
viral vectors was similar to the one observed in hippocampal
organotypic slices from mice lacking the GluK2 (GluK2-/-) subunit
(Peret et al., 2014) (Table 5 and 6 and FIGS. 2 and 3).
TABLE-US-00016 TABLE 5 Effect of LV137 and LV178 on the frequency
of epileptiform discharges WT.sup.(a) GluK2.sup.-/-(a) LV104 LV137
LV180 LV178 Mean 0.057 0.029 0.065 0.032 0.067 0.043 S.E.M. 0.0068
0.0046 0.0149 0.0098 0.0099 0.0060 n 28 25 6 11 12 23 P-value*
***0.0001 *0.0449 *0.0348 *Statistically significant differences in
epileptiform discharges were measured between LV137 or LV178 and
control treatments (LV104 and LV180-scramble, respectively).
.sup.(a)From Peret et al. Cell Rep. 8(2): 347-54, 2014.
TABLE-US-00017 TABLE 6 Effect of AAV9-hu1010 compared with control
conditions (AAV9- scramble) on the frequency of epileptiform
discharges. AAV9- AAV9- AAV9- WT.sup.(a) GluK2.sup.-/-(a) control
scramble hu1010 Mean 0.057 0.029 0.077 0.080 0.029 S.E.M. 0.0068
0.0046 0.0127 0.0146 0.0085 n 28 25 11 7 17 P-value* ***0.0001
**0.0031 *Statistically significant differences in epileptiform
discharges were measured between AAV9-hu1010 and control vector
(AAV9-scramble; UAAUGUUAGUCAUGUCCACCG; SEQ ID NO: 37) treatment
groups. .sup.(a)From Peret et al. Cell Rep. 8(2): 347-54, 2014.
Conclusion
[0412] In conclusion, our data demonstrated that GluK2 gene (Grik2)
silencing, using lentivirus or AAV vectors carrying a RNAi sequence
targeting Grik2 (e.g., miRNAi1h), is an efficient strategy to
prevent spontaneous epileptiform discharges in TLE.
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Sequence CWU 1
1
37119DNAArtificial SequenceSynthetic Constructmisc_feature(4)..(4)R
is A or G 1aaarcaggca ttagctatg 19219DNAHomo sapiens 2aaaacaggca
ttagctatg 19319DNAMus musculus 3aaagcaggca ttagctatg 194908PRTHomo
sapiens 4Met Lys Ile Ile Phe Pro Ile Leu Ser Asn Pro Val Phe Arg
Arg Thr1 5 10 15Val Lys Leu Leu Leu Cys Leu Leu Trp Ile Gly Tyr Ser
Gln Gly Thr 20 25 30Thr His Val Leu Arg Phe Gly Gly Ile Phe Glu Tyr
Val Glu Ser Gly 35 40 45Pro Met Gly Ala Glu Glu Leu Ala Phe Arg Phe
Ala Val Asn Thr Ile 50 55 60Asn Arg Asn Arg Thr Leu Leu Pro Asn Thr
Thr Leu Thr Tyr Asp Thr65 70 75 80Gln Lys Ile Asn Leu Tyr Asp Ser
Phe Glu Ala Ser Lys Lys Ala Cys 85 90 95Asp Gln Leu Ser Leu Gly Val
Ala Ala Ile Phe Gly Pro Ser His Ser 100 105 110Ser Ser Ala Asn Ala
Val Gln Ser Ile Cys Asn Ala Leu Gly Val Pro 115 120 125His Ile Gln
Thr Arg Trp Lys His Gln Val Ser Asp Asn Lys Asp Ser 130 135 140Phe
Tyr Val Ser Leu Tyr Pro Asp Phe Ser Ser Leu Ser Arg Ala Ile145 150
155 160Leu Asp Leu Val Gln Phe Phe Lys Trp Lys Thr Val Thr Val Val
Tyr 165 170 175Asp Asp Ser Thr Gly Leu Ile Arg Leu Gln Glu Leu Ile
Lys Ala Pro 180 185 190Ser Arg Tyr Asn Leu Arg Leu Lys Ile Arg Gln
Leu Pro Ala Asp Thr 195 200 205Lys Asp Ala Lys Pro Leu Leu Lys Glu
Met Lys Arg Gly Lys Glu Phe 210 215 220His Val Ile Phe Asp Cys Ser
His Glu Met Ala Ala Gly Ile Leu Lys225 230 235 240Gln Ala Leu Ala
Met Gly Met Met Thr Glu Tyr Tyr His Tyr Ile Phe 245 250 255Thr Thr
Leu Asp Leu Phe Ala Leu Asp Val Glu Pro Tyr Arg Tyr Ser 260 265
270Gly Val Asn Met Thr Gly Phe Arg Ile Leu Asn Thr Glu Asn Thr Gln
275 280 285Val Ser Ser Ile Ile Glu Lys Trp Ser Met Glu Arg Leu Gln
Ala Pro 290 295 300Pro Lys Pro Asp Ser Gly Leu Leu Asp Gly Phe Met
Thr Thr Asp Ala305 310 315 320Ala Leu Met Tyr Asp Ala Val His Val
Val Ser Val Ala Val Gln Gln 325 330 335Phe Pro Gln Met Thr Val Ser
Ser Leu Gln Cys Asn Arg His Lys Pro 340 345 350Trp Arg Phe Gly Thr
Arg Phe Met Ser Leu Ile Lys Glu Ala His Trp 355 360 365Glu Gly Leu
Thr Gly Arg Ile Thr Phe Asn Lys Thr Asn Gly Leu Arg 370 375 380Thr
Asp Phe Asp Leu Asp Val Ile Ser Leu Lys Glu Glu Gly Leu Glu385 390
395 400Lys Ile Gly Thr Trp Asp Pro Ala Ser Gly Leu Asn Met Thr Glu
Ser 405 410 415Gln Lys Gly Lys Pro Ala Asn Ile Thr Asp Ser Leu Ser
Asn Arg Ser 420 425 430Leu Ile Val Thr Thr Ile Leu Glu Glu Pro Tyr
Val Leu Phe Lys Lys 435 440 445Ser Asp Lys Pro Leu Tyr Gly Asn Asp
Arg Phe Glu Gly Tyr Cys Ile 450 455 460Asp Leu Leu Arg Glu Leu Ser
Thr Ile Leu Gly Phe Thr Tyr Glu Ile465 470 475 480Arg Leu Val Glu
Asp Gly Lys Tyr Gly Ala Gln Asp Asp Ala Asn Gly 485 490 495Gln Trp
Asn Gly Met Val Arg Glu Leu Ile Asp His Lys Ala Asp Leu 500 505
510Ala Val Ala Pro Leu Ala Ile Thr Tyr Val Arg Glu Lys Val Ile Asp
515 520 525Phe Ser Lys Pro Phe Met Thr Leu Gly Ile Ser Ile Leu Tyr
Arg Lys 530 535 540Pro Asn Gly Thr Asn Pro Gly Val Phe Ser Phe Leu
Asn Pro Leu Ser545 550 555 560Pro Asp Ile Trp Met Tyr Ile Leu Leu
Ala Tyr Leu Gly Val Ser Cys 565 570 575Val Leu Phe Val Ile Ala Arg
Phe Ser Pro Tyr Glu Trp Tyr Asn Pro 580 585 590His Pro Cys Asn Pro
Asp Ser Asp Val Val Glu Asn Asn Phe Thr Leu 595 600 605Leu Asn Ser
Phe Trp Phe Gly Val Gly Ala Leu Met Gln Gln Gly Ser 610 615 620Glu
Leu Met Pro Lys Ala Leu Ser Thr Arg Ile Val Gly Gly Ile Trp625 630
635 640Trp Phe Phe Thr Leu Ile Ile Ile Ser Ser Tyr Thr Ala Asn Leu
Ala 645 650 655Ala Phe Leu Thr Val Glu Arg Met Glu Ser Pro Ile Asp
Ser Ala Asp 660 665 670Asp Leu Ala Lys Gln Thr Lys Ile Glu Tyr Gly
Ala Val Glu Asp Gly 675 680 685Ala Thr Met Thr Phe Phe Lys Lys Ser
Lys Ile Ser Thr Tyr Asp Lys 690 695 700Met Trp Ala Phe Met Ser Ser
Arg Arg Gln Ser Val Leu Val Lys Ser705 710 715 720Asn Glu Glu Gly
Ile Gln Arg Val Leu Thr Ser Asp Tyr Ala Phe Leu 725 730 735Met Glu
Ser Thr Thr Ile Glu Phe Val Thr Gln Arg Asn Cys Asn Leu 740 745
750Thr Gln Ile Gly Gly Leu Ile Asp Ser Lys Gly Tyr Gly Val Gly Thr
755 760 765Pro Met Gly Ser Pro Tyr Arg Asp Lys Ile Thr Ile Ala Ile
Leu Gln 770 775 780Leu Gln Glu Glu Gly Lys Leu His Met Met Lys Glu
Lys Trp Trp Arg785 790 795 800Gly Asn Gly Cys Pro Glu Glu Glu Ser
Lys Glu Ala Ser Ala Leu Gly 805 810 815Val Gln Asn Ile Gly Gly Ile
Phe Ile Val Leu Ala Ala Gly Leu Val 820 825 830Leu Ser Val Phe Val
Ala Val Gly Glu Phe Leu Tyr Lys Ser Lys Lys 835 840 845Asn Ala Gln
Leu Glu Lys Arg Ser Phe Cys Ser Ala Met Val Glu Glu 850 855 860Leu
Arg Met Ser Leu Lys Cys Gln Arg Arg Leu Lys His Lys Pro Gln865 870
875 880Ala Pro Val Ile Val Lys Thr Glu Glu Val Ile Asn Met His Thr
Phe 885 890 895Asn Asp Arg Arg Leu Pro Gly Lys Glu Thr Met Ala 900
9055869PRTHomo sapiens 5Met Lys Ile Ile Phe Pro Ile Leu Ser Asn Pro
Val Phe Arg Arg Thr1 5 10 15Val Lys Leu Leu Leu Cys Leu Leu Trp Ile
Gly Tyr Ser Gln Gly Thr 20 25 30Thr His Val Leu Arg Phe Gly Gly Ile
Phe Glu Tyr Val Glu Ser Gly 35 40 45Pro Met Gly Ala Glu Glu Leu Ala
Phe Arg Phe Ala Val Asn Thr Ile 50 55 60Asn Arg Asn Arg Thr Leu Leu
Pro Asn Thr Thr Leu Thr Tyr Asp Thr65 70 75 80Gln Lys Ile Asn Leu
Tyr Asp Ser Phe Glu Ala Ser Lys Lys Ala Cys 85 90 95Asp Gln Leu Ser
Leu Gly Val Ala Ala Ile Phe Gly Pro Ser His Ser 100 105 110Ser Ser
Ala Asn Ala Val Gln Ser Ile Cys Asn Ala Leu Gly Val Pro 115 120
125His Ile Gln Thr Arg Trp Lys His Gln Val Ser Asp Asn Lys Asp Ser
130 135 140Phe Tyr Val Ser Leu Tyr Pro Asp Phe Ser Ser Leu Ser Arg
Ala Ile145 150 155 160Leu Asp Leu Val Gln Phe Phe Lys Trp Lys Thr
Val Thr Val Val Tyr 165 170 175Asp Asp Ser Thr Gly Leu Ile Arg Leu
Gln Glu Leu Ile Lys Ala Pro 180 185 190Ser Arg Tyr Asn Leu Arg Leu
Lys Ile Arg Gln Leu Pro Ala Asp Thr 195 200 205Lys Asp Ala Lys Pro
Leu Leu Lys Glu Met Lys Arg Gly Lys Glu Phe 210 215 220His Val Ile
Phe Asp Cys Ser His Glu Met Ala Ala Gly Ile Leu Lys225 230 235
240Gln Ala Leu Ala Met Gly Met Met Thr Glu Tyr Tyr His Tyr Ile Phe
245 250 255Thr Thr Leu Asp Leu Phe Ala Leu Asp Val Glu Pro Tyr Arg
Tyr Ser 260 265 270Gly Val Asn Met Thr Gly Phe Arg Ile Leu Asn Thr
Glu Asn Thr Gln 275 280 285Val Ser Ser Ile Ile Glu Lys Trp Ser Met
Glu Arg Leu Gln Ala Pro 290 295 300Pro Lys Pro Asp Ser Gly Leu Leu
Asp Gly Phe Met Thr Thr Asp Ala305 310 315 320Ala Leu Met Tyr Asp
Ala Val His Val Val Ser Val Ala Val Gln Gln 325 330 335Phe Pro Gln
Met Thr Val Ser Ser Leu Gln Cys Asn Arg His Lys Pro 340 345 350Trp
Arg Phe Gly Thr Arg Phe Met Ser Leu Ile Lys Glu Ala His Trp 355 360
365Glu Gly Leu Thr Gly Arg Ile Thr Phe Asn Lys Thr Asn Gly Leu Arg
370 375 380Thr Asp Phe Asp Leu Asp Val Ile Ser Leu Lys Glu Glu Gly
Leu Glu385 390 395 400Lys Ile Gly Thr Trp Asp Pro Ala Ser Gly Leu
Asn Met Thr Glu Ser 405 410 415Gln Lys Gly Lys Pro Ala Asn Ile Thr
Asp Ser Leu Ser Asn Arg Ser 420 425 430Leu Ile Val Thr Thr Ile Leu
Glu Glu Pro Tyr Val Leu Phe Lys Lys 435 440 445Ser Asp Lys Pro Leu
Tyr Gly Asn Asp Arg Phe Glu Gly Tyr Cys Ile 450 455 460Asp Leu Leu
Arg Glu Leu Ser Thr Ile Leu Gly Phe Thr Tyr Glu Ile465 470 475
480Arg Leu Val Glu Asp Gly Lys Tyr Gly Ala Gln Asp Asp Ala Asn Gly
485 490 495Gln Trp Asn Gly Met Val Arg Glu Leu Ile Asp His Lys Ala
Asp Leu 500 505 510Ala Val Ala Pro Leu Ala Ile Thr Tyr Val Arg Glu
Lys Val Ile Asp 515 520 525Phe Ser Lys Pro Phe Met Thr Leu Gly Ile
Ser Ile Leu Tyr Arg Lys 530 535 540Pro Asn Gly Thr Asn Pro Gly Val
Phe Ser Phe Leu Asn Pro Leu Ser545 550 555 560Pro Asp Ile Trp Met
Tyr Ile Leu Leu Ala Tyr Leu Gly Val Ser Cys 565 570 575Val Leu Phe
Val Ile Ala Arg Phe Ser Pro Tyr Glu Trp Tyr Asn Pro 580 585 590His
Pro Cys Asn Pro Asp Ser Asp Val Val Glu Asn Asn Phe Thr Leu 595 600
605Leu Asn Ser Phe Trp Phe Gly Val Gly Ala Leu Met Gln Gln Gly Ser
610 615 620Glu Leu Met Pro Lys Ala Leu Ser Thr Arg Ile Val Gly Gly
Ile Trp625 630 635 640Trp Phe Phe Thr Leu Ile Ile Ile Ser Ser Tyr
Thr Ala Asn Leu Ala 645 650 655Ala Phe Leu Thr Val Glu Arg Met Glu
Ser Pro Ile Asp Ser Ala Asp 660 665 670Asp Leu Ala Lys Gln Thr Lys
Ile Glu Tyr Gly Ala Val Glu Asp Gly 675 680 685Ala Thr Met Thr Phe
Phe Lys Lys Ser Lys Ile Ser Thr Tyr Asp Lys 690 695 700Met Trp Ala
Phe Met Ser Ser Arg Arg Gln Ser Val Leu Val Lys Ser705 710 715
720Asn Glu Glu Gly Ile Gln Arg Val Leu Thr Ser Asp Tyr Ala Phe Leu
725 730 735Met Glu Ser Thr Thr Ile Glu Phe Val Thr Gln Arg Asn Cys
Asn Leu 740 745 750Thr Gln Ile Gly Gly Leu Ile Asp Ser Lys Gly Tyr
Gly Val Gly Thr 755 760 765Pro Met Gly Ser Pro Tyr Arg Asp Lys Ile
Thr Ile Ala Ile Leu Gln 770 775 780Leu Gln Glu Glu Gly Lys Leu His
Met Met Lys Glu Lys Trp Trp Arg785 790 795 800Gly Asn Gly Cys Pro
Glu Glu Glu Ser Lys Glu Ala Ser Ala Leu Gly 805 810 815Val Gln Asn
Ile Gly Gly Ile Phe Ile Val Leu Ala Ala Gly Leu Val 820 825 830Leu
Ser Val Phe Val Ala Val Gly Glu Phe Leu Tyr Lys Ser Lys Lys 835 840
845Asn Ala Gln Leu Glu Lys Glu Ser Ser Ile Trp Leu Val Pro Pro Tyr
850 855 860His Pro Asp Thr Val8656584PRTHomo sapiens 6Met Lys Ile
Ile Phe Pro Ile Leu Ser Asn Pro Val Phe Arg Arg Thr1 5 10 15Val Lys
Leu Leu Leu Cys Leu Leu Trp Ile Gly Tyr Ser Gln Gly Thr 20 25 30Thr
His Val Leu Arg Phe Gly Gly Ile Phe Glu Tyr Val Glu Ser Gly 35 40
45Pro Met Gly Ala Glu Glu Leu Ala Phe Arg Phe Ala Val Asn Thr Ile
50 55 60Asn Arg Asn Arg Thr Leu Leu Pro Asn Thr Thr Leu Thr Tyr Asp
Thr65 70 75 80Gln Lys Ile Asn Leu Tyr Asp Ser Phe Glu Ala Ser Lys
Lys Ala Cys 85 90 95Asp Gln Leu Ser Leu Gly Val Ala Ala Ile Phe Gly
Pro Ser His Ser 100 105 110Ser Ser Ala Asn Ala Val Gln Ser Ile Cys
Asn Ala Leu Gly Val Pro 115 120 125His Ile Gln Thr Arg Trp Lys His
Gln Val Ser Asp Asn Lys Asp Ser 130 135 140Phe Tyr Val Ser Leu Tyr
Pro Asp Phe Ser Ser Leu Ser Arg Ala Ile145 150 155 160Leu Asp Leu
Val Gln Phe Phe Lys Trp Lys Thr Val Thr Val Val Tyr 165 170 175Asp
Asp Ser Thr Gly Leu Ile Arg Leu Gln Glu Leu Ile Lys Ala Pro 180 185
190Ser Arg Tyr Asn Leu Arg Leu Lys Ile Arg Gln Leu Pro Ala Asp Thr
195 200 205Lys Asp Ala Lys Pro Leu Leu Lys Glu Met Lys Arg Gly Lys
Glu Phe 210 215 220His Val Ile Phe Asp Cys Ser His Glu Met Ala Ala
Gly Ile Leu Lys225 230 235 240Gln Ala Leu Ala Met Gly Met Met Thr
Glu Tyr Tyr His Tyr Ile Phe 245 250 255Thr Thr Leu Asp Leu Phe Ala
Leu Asp Val Glu Pro Tyr Arg Tyr Ser 260 265 270Gly Val Asn Met Thr
Gly Phe Arg Ile Leu Asn Thr Glu Asn Thr Gln 275 280 285Val Ser Ser
Ile Ile Glu Lys Trp Ser Met Glu Arg Leu Gln Ala Pro 290 295 300Pro
Lys Pro Asp Ser Gly Leu Leu Asp Gly Phe Met Thr Thr Asp Ala305 310
315 320Ala Leu Met Tyr Asp Ala Val His Val Val Ser Val Ala Val Gln
Gln 325 330 335Phe Pro Gln Met Thr Val Ser Ser Leu Gln Cys Asn Arg
His Lys Pro 340 345 350Trp Arg Phe Gly Thr Arg Phe Met Ser Leu Ile
Lys Glu Ala His Trp 355 360 365Glu Gly Leu Thr Gly Arg Ile Thr Phe
Asn Lys Thr Asn Gly Leu Arg 370 375 380Thr Asp Phe Asp Leu Asp Val
Ile Ser Leu Lys Glu Glu Gly Leu Glu385 390 395 400Lys Ile Gly Thr
Trp Asp Pro Ala Ser Gly Leu Asn Met Thr Glu Ser 405 410 415Gln Lys
Gly Lys Pro Ala Asn Ile Thr Asp Ser Leu Ser Asn Arg Ser 420 425
430Leu Ile Val Thr Thr Ile Leu Glu Glu Pro Tyr Val Leu Phe Lys Lys
435 440 445Ser Asp Lys Pro Leu Tyr Gly Asn Asp Arg Phe Glu Gly Tyr
Cys Ile 450 455 460Asp Leu Leu Arg Glu Leu Ser Thr Ile Leu Gly Phe
Thr Tyr Glu Ile465 470 475 480Arg Leu Val Glu Asp Gly Lys Tyr Gly
Ala Gln Asp Asp Ala Asn Gly 485 490 495Gln Trp Asn Gly Met Val Arg
Glu Leu Ile Asp His Lys Ala Asp Leu 500 505 510Ala Val Ala Pro Leu
Ala Ile Thr Tyr Val Arg Glu Lys Val Ile Asp 515 520 525Phe Ser Lys
Pro Phe Met Thr Leu Gly Ile Ser Ile Leu Tyr Arg Lys 530 535 540Pro
Asn Gly Thr Asn Pro Gly Val Phe Ser Phe Leu Asn Pro Leu Ser545 550
555 560Pro Asp Ile Trp Met Tyr Ile Leu Leu Ala Tyr Leu Gly Val Ser
Cys 565 570 575Val Leu Phe Val Ile Ala Arg Phe 5807832PRTHomo
sapiens 7Met Lys Ile Ile Phe Pro Ile Leu Ser Asn Pro Val Phe Arg
Arg Thr1 5 10 15Val Lys Leu Leu Leu Cys Leu Leu Trp Ile Gly Tyr Ser
Gln Gly Thr 20 25 30Thr His Val Leu Arg Phe Gly Gly Ile Phe Glu Tyr
Val Glu Ser Gly 35 40 45Pro Met Gly Ala Glu Glu Leu Ala Phe Arg Phe
Ala Val Asn Thr Ile 50 55
60Asn Arg Asn Arg Thr Leu Leu Pro Asn Thr Thr Leu Thr Tyr Asp Thr65
70 75 80Gln Lys Ile Asn Leu Tyr Asp Ser Phe Glu Ala Ser Lys Lys Ala
Cys 85 90 95Asp Gln Leu Ser Leu Gly Val Ala Ala Ile Phe Gly Pro Ser
His Ser 100 105 110Ser Ser Ala Asn Ala Val Gln Ser Ile Cys Asn Ala
Leu Gly Val Pro 115 120 125His Ile Gln Thr Arg Trp Lys His Gln Val
Ser Asp Asn Lys Asp Ser 130 135 140Phe Tyr Val Ser Leu Tyr Pro Asp
Phe Ser Ser Leu Ser Arg Ala Ile145 150 155 160Leu Asp Leu Val Gln
Phe Phe Lys Trp Lys Thr Val Thr Val Val Tyr 165 170 175Asp Asp Ser
Thr Gly Leu Ile Arg Leu Gln Glu Leu Ile Lys Ala Pro 180 185 190Ser
Arg Tyr Asn Leu Arg Leu Lys Ile Arg Gln Leu Pro Ala Asp Thr 195 200
205Lys Asp Ala Lys Pro Leu Leu Lys Glu Met Lys Arg Gly Lys Glu Phe
210 215 220His Val Ile Phe Asp Cys Ser His Glu Met Ala Ala Gly Ile
Leu Lys225 230 235 240Gln Ala Leu Ala Met Gly Met Met Thr Glu Tyr
Tyr His Tyr Ile Phe 245 250 255Thr Thr Leu Asp Leu Phe Ala Leu Asp
Val Glu Pro Tyr Arg Tyr Ser 260 265 270Gly Val Asn Met Thr Gly Phe
Arg Ile Leu Asn Thr Glu Asn Thr Gln 275 280 285Val Ser Ser Ile Ile
Glu Lys Trp Ser Met Glu Arg Leu Gln Ala Pro 290 295 300Pro Lys Pro
Asp Ser Gly Leu Leu Asp Gly Phe Met Thr Thr Asp Ala305 310 315
320Ala Leu Met Tyr Asp Ala Val His Val Val Ser Val Ala Val Gln Gln
325 330 335Phe Pro Gln Met Thr Val Ser Ser Leu Gln Cys Asn Arg His
Lys Pro 340 345 350Trp Arg Phe Gly Thr Arg Phe Met Ser Leu Ile Lys
Glu Ala His Trp 355 360 365Glu Gly Leu Thr Gly Arg Ile Thr Phe Asn
Lys Thr Asn Gly Leu Arg 370 375 380Thr Asp Phe Asp Leu Asp Val Ile
Ser Leu Lys Glu Glu Gly Leu Glu385 390 395 400Lys Ile Gly Thr Trp
Asp Pro Ala Ser Gly Leu Asn Met Thr Glu Ser 405 410 415Gln Lys Gly
Lys Pro Ala Asn Ile Thr Asp Ser Leu Ser Asn Arg Ser 420 425 430Leu
Ile Val Thr Thr Ile Leu Glu Glu Pro Tyr Val Leu Phe Lys Lys 435 440
445Ser Asp Lys Pro Leu Tyr Gly Asn Asp Arg Phe Glu Gly Tyr Cys Ile
450 455 460Asp Leu Leu Arg Glu Leu Ser Thr Ile Leu Gly Phe Thr Tyr
Glu Ile465 470 475 480Arg Leu Val Glu Asp Gly Lys Tyr Gly Ala Gln
Asp Asp Ala Asn Gly 485 490 495Gln Trp Asn Gly Met Val Arg Glu Leu
Ile Asp His Lys Ala Asp Leu 500 505 510Ala Val Ala Pro Leu Ala Ile
Thr Tyr Val Arg Glu Lys Val Ile Asp 515 520 525Phe Ser Lys Pro Phe
Met Thr Leu Gly Ile Ser Ile Leu Tyr Arg Lys 530 535 540Pro Asn Gly
Ser Glu Leu Met Pro Lys Ala Leu Ser Thr Arg Ile Val545 550 555
560Gly Gly Ile Trp Trp Phe Phe Thr Leu Ile Ile Ile Ser Ser Tyr Thr
565 570 575Ala Asn Leu Ala Ala Phe Leu Thr Val Glu Arg Met Glu Ser
Pro Ile 580 585 590Asp Ser Ala Asp Asp Leu Ala Lys Gln Thr Lys Ile
Glu Tyr Gly Ala 595 600 605Val Glu Asp Gly Ala Thr Met Thr Phe Phe
Lys Lys Ser Lys Ile Ser 610 615 620Thr Tyr Asp Lys Met Trp Ala Phe
Met Ser Ser Arg Arg Gln Ser Val625 630 635 640Leu Val Lys Ser Asn
Glu Glu Gly Ile Gln Arg Val Leu Thr Ser Asp 645 650 655Tyr Ala Phe
Leu Met Glu Ser Thr Thr Ile Glu Phe Val Thr Gln Arg 660 665 670Asn
Cys Asn Leu Thr Gln Ile Gly Gly Leu Ile Asp Ser Lys Gly Tyr 675 680
685Gly Val Gly Thr Pro Met Gly Ser Pro Tyr Arg Asp Lys Ile Thr Ile
690 695 700Ala Ile Leu Gln Leu Gln Glu Glu Gly Lys Leu His Met Met
Lys Glu705 710 715 720Lys Trp Trp Arg Gly Asn Gly Cys Pro Glu Glu
Glu Ser Lys Glu Ala 725 730 735Ser Ala Leu Gly Val Gln Asn Ile Gly
Gly Ile Phe Ile Val Leu Ala 740 745 750Ala Gly Leu Val Leu Ser Val
Phe Val Ala Val Gly Glu Phe Leu Tyr 755 760 765Lys Ser Lys Lys Asn
Ala Gln Leu Glu Lys Arg Ser Phe Cys Ser Ala 770 775 780Met Val Glu
Glu Leu Arg Met Ser Leu Lys Cys Gln Arg Arg Leu Lys785 790 795
800His Lys Pro Gln Ala Pro Val Ile Val Lys Thr Glu Glu Val Ile Asn
805 810 815Met His Thr Phe Asn Asp Arg Arg Leu Pro Gly Lys Glu Thr
Met Ala 820 825 8308892PRTHomo sapiens 8Met Lys Ile Ile Phe Pro Ile
Leu Ser Asn Pro Val Phe Arg Arg Thr1 5 10 15Val Lys Leu Leu Leu Cys
Leu Leu Trp Ile Gly Tyr Ser Gln Gly Thr 20 25 30Thr His Val Leu Arg
Phe Gly Gly Ile Phe Glu Tyr Val Glu Ser Gly 35 40 45Pro Met Gly Ala
Glu Glu Leu Ala Phe Arg Phe Ala Val Asn Thr Ile 50 55 60Asn Arg Asn
Arg Thr Leu Leu Pro Asn Thr Thr Leu Thr Tyr Asp Thr65 70 75 80Gln
Lys Ile Asn Leu Tyr Asp Ser Phe Glu Ala Ser Lys Lys Ala Cys 85 90
95Asp Gln Leu Ser Leu Gly Val Ala Ala Ile Phe Gly Pro Ser His Ser
100 105 110Ser Ser Ala Asn Ala Val Gln Ser Ile Cys Asn Ala Leu Gly
Val Pro 115 120 125His Ile Gln Thr Arg Trp Lys His Gln Val Ser Asp
Asn Lys Asp Ser 130 135 140Phe Tyr Val Ser Leu Tyr Pro Asp Phe Ser
Ser Leu Ser Arg Ala Ile145 150 155 160Leu Asp Leu Val Gln Phe Phe
Lys Trp Lys Thr Val Thr Val Val Tyr 165 170 175Asp Asp Ser Thr Gly
Leu Ile Arg Leu Gln Glu Leu Ile Lys Ala Pro 180 185 190Ser Arg Tyr
Asn Leu Arg Leu Lys Ile Arg Gln Leu Pro Ala Asp Thr 195 200 205Lys
Asp Ala Lys Pro Leu Leu Lys Glu Met Lys Arg Gly Lys Glu Phe 210 215
220His Val Ile Phe Asp Cys Ser His Glu Met Ala Ala Gly Ile Leu
Lys225 230 235 240Gln Ala Leu Ala Met Gly Met Met Thr Glu Tyr Tyr
His Tyr Ile Phe 245 250 255Thr Thr Leu Asp Leu Phe Ala Leu Asp Val
Glu Pro Tyr Arg Tyr Ser 260 265 270Gly Val Asn Met Thr Gly Phe Arg
Ile Leu Asn Thr Glu Asn Thr Gln 275 280 285Val Ser Ser Ile Ile Glu
Lys Trp Ser Met Glu Arg Leu Gln Ala Pro 290 295 300Pro Lys Pro Asp
Ser Gly Leu Leu Asp Gly Phe Met Thr Thr Asp Ala305 310 315 320Ala
Leu Met Tyr Asp Ala Val His Val Val Ser Val Ala Val Gln Gln 325 330
335Phe Pro Gln Met Thr Val Ser Ser Leu Gln Cys Asn Arg His Lys Pro
340 345 350Trp Arg Phe Gly Thr Arg Phe Met Ser Leu Ile Lys Glu Ala
His Trp 355 360 365Glu Gly Leu Thr Gly Arg Ile Thr Phe Asn Lys Thr
Asn Gly Leu Arg 370 375 380Thr Asp Phe Asp Leu Asp Val Ile Ser Leu
Lys Glu Glu Gly Leu Glu385 390 395 400Lys Ile Gly Thr Trp Asp Pro
Ala Ser Gly Leu Asn Met Thr Glu Ser 405 410 415Gln Lys Gly Lys Pro
Ala Asn Ile Thr Asp Ser Leu Ser Asn Arg Ser 420 425 430Leu Ile Val
Thr Thr Ile Leu Glu Glu Pro Tyr Val Leu Phe Lys Lys 435 440 445Ser
Asp Lys Pro Leu Tyr Gly Asn Asp Arg Phe Glu Gly Tyr Cys Ile 450 455
460Asp Leu Leu Arg Glu Leu Ser Thr Ile Leu Gly Phe Thr Tyr Glu
Ile465 470 475 480Arg Leu Val Glu Asp Gly Lys Tyr Gly Ala Gln Asp
Asp Ala Asn Gly 485 490 495Gln Trp Asn Gly Met Val Arg Glu Leu Ile
Asp His Lys Ala Asp Leu 500 505 510Ala Val Ala Pro Leu Ala Ile Thr
Tyr Val Arg Glu Lys Val Ile Asp 515 520 525Phe Ser Lys Pro Phe Met
Thr Leu Gly Ile Ser Ile Leu Tyr Arg Lys 530 535 540Pro Asn Gly Thr
Asn Pro Gly Val Phe Ser Phe Leu Asn Pro Leu Ser545 550 555 560Pro
Asp Ile Trp Met Tyr Ile Leu Leu Ala Tyr Leu Gly Val Ser Cys 565 570
575Val Leu Phe Val Ile Ala Arg Phe Ser Pro Tyr Glu Trp Tyr Asn Pro
580 585 590His Pro Cys Asn Pro Asp Ser Asp Val Val Glu Asn Asn Phe
Thr Leu 595 600 605Leu Asn Ser Phe Trp Phe Gly Val Gly Ala Leu Met
Gln Gln Gly Ser 610 615 620Glu Leu Met Pro Lys Ala Leu Ser Thr Arg
Ile Val Gly Gly Ile Trp625 630 635 640Trp Phe Phe Thr Leu Ile Ile
Ile Ser Ser Tyr Thr Ala Asn Leu Ala 645 650 655Ala Phe Leu Thr Val
Glu Arg Met Glu Ser Pro Ile Asp Ser Ala Asp 660 665 670Asp Leu Ala
Lys Gln Thr Lys Ile Glu Tyr Gly Ala Val Glu Asp Gly 675 680 685Ala
Thr Met Thr Phe Phe Lys Lys Ser Lys Ile Ser Thr Tyr Asp Lys 690 695
700Met Trp Ala Phe Met Ser Ser Arg Arg Gln Ser Val Leu Val Lys
Ser705 710 715 720Asn Glu Glu Gly Ile Gln Arg Val Leu Thr Ser Asp
Tyr Ala Phe Leu 725 730 735Met Glu Ser Thr Thr Ile Glu Phe Val Thr
Gln Arg Asn Cys Asn Leu 740 745 750Thr Gln Ile Gly Gly Leu Ile Asp
Ser Lys Gly Tyr Gly Val Gly Thr 755 760 765Pro Met Gly Ser Pro Tyr
Arg Asp Lys Ile Thr Ile Ala Ile Leu Gln 770 775 780Leu Gln Glu Glu
Gly Lys Leu His Met Met Lys Glu Lys Trp Trp Arg785 790 795 800Gly
Asn Gly Cys Pro Glu Glu Glu Ser Lys Glu Ala Ser Ala Leu Gly 805 810
815Val Gln Asn Ile Gly Gly Ile Phe Ile Val Leu Ala Ala Gly Leu Val
820 825 830Leu Ser Val Phe Val Ala Val Gly Glu Phe Leu Tyr Lys Ser
Lys Lys 835 840 845Asn Ala Gln Leu Glu Lys Arg Ala Lys Thr Lys Leu
Pro Gln Asp Tyr 850 855 860Val Phe Leu Pro Ile Leu Glu Ser Val Ser
Ile Ser Thr Val Leu Ser865 870 875 880Ser Ser Pro Ser Ser Ser Ser
Leu Ser Ser Cys Ser 885 8909682PRTHomo sapiens 9Met Lys Ile Ile Phe
Pro Ile Leu Ser Asn Pro Val Phe Arg Arg Thr1 5 10 15Val Lys Leu Leu
Leu Cys Leu Leu Trp Ile Gly Tyr Ser Gln Gly Thr 20 25 30Thr His Val
Leu Arg Phe Gly Gly Ile Phe Glu Tyr Val Glu Ser Gly 35 40 45Pro Met
Gly Ala Glu Glu Leu Ala Phe Arg Phe Ala Val Asn Thr Ile 50 55 60Asn
Arg Asn Arg Thr Leu Leu Pro Asn Thr Thr Leu Thr Tyr Asp Thr65 70 75
80Gln Lys Ile Asn Leu Tyr Asp Ser Phe Glu Ala Ser Lys Lys Ala Cys
85 90 95Asp Gln Leu Ser Leu Gly Val Ala Ala Ile Phe Gly Pro Ser His
Ser 100 105 110Ser Ser Ala Asn Ala Val Gln Ser Ile Cys Asn Ala Leu
Gly Val Pro 115 120 125His Ile Gln Thr Arg Trp Lys His Gln Val Ser
Asp Asn Lys Asp Ser 130 135 140Phe Tyr Val Ser Leu Tyr Pro Asp Phe
Ser Ser Leu Ser Arg Ala Ile145 150 155 160Leu Asp Leu Val Gln Phe
Phe Lys Trp Lys Thr Val Thr Val Val Tyr 165 170 175Asp Asp Ser Thr
Gly Leu Ile Arg Leu Gln Glu Leu Ile Lys Ala Pro 180 185 190Ser Arg
Tyr Asn Leu Arg Leu Lys Ile Arg Gln Leu Pro Ala Asp Thr 195 200
205Lys Asp Ala Lys Pro Leu Leu Lys Glu Met Lys Arg Gly Lys Glu Phe
210 215 220His Val Ile Phe Asp Cys Ser His Glu Met Ala Ala Gly Ile
Leu Lys225 230 235 240Gln Ala Leu Ala Met Gly Met Met Thr Glu Tyr
Tyr His Tyr Ile Phe 245 250 255Thr Thr Leu Asp Leu Phe Ala Leu Asp
Val Glu Pro Tyr Arg Tyr Ser 260 265 270Gly Val Asn Met Thr Gly Phe
Arg Ile Leu Asn Thr Glu Asn Thr Gln 275 280 285Val Ser Ser Ile Ile
Glu Lys Trp Ser Met Glu Arg Leu Gln Ala Pro 290 295 300Pro Lys Pro
Asp Ser Gly Leu Leu Asp Gly Phe Met Thr Thr Asp Ala305 310 315
320Ala Leu Met Tyr Asp Ala Val His Val Val Ser Val Ala Val Gln Gln
325 330 335Phe Pro Gln Met Thr Val Ser Ser Leu Gln Cys Asn Arg His
Lys Pro 340 345 350Trp Arg Phe Gly Thr Arg Phe Met Ser Leu Ile Lys
Glu Ala His Trp 355 360 365Glu Gly Leu Thr Gly Arg Ile Thr Phe Asn
Lys Thr Asn Gly Leu Arg 370 375 380Thr Asp Phe Asp Leu Asp Val Ile
Ser Leu Lys Glu Glu Gly Leu Glu385 390 395 400Lys Ile Gly Thr Trp
Asp Pro Ala Ser Gly Leu Asn Met Thr Glu Ser 405 410 415Gln Lys Gly
Lys Pro Ala Asn Ile Thr Asp Ser Leu Ser Asn Arg Ser 420 425 430Leu
Ile Val Thr Thr Ile Leu Glu Glu Pro Tyr Val Leu Phe Lys Lys 435 440
445Ser Asp Lys Pro Leu Tyr Gly Asn Asp Arg Phe Glu Gly Tyr Cys Ile
450 455 460Asp Leu Leu Arg Glu Leu Ser Thr Ile Leu Gly Phe Thr Tyr
Glu Ile465 470 475 480Arg Leu Val Glu Asp Gly Lys Tyr Gly Ala Gln
Asp Asp Ala Asn Gly 485 490 495Gln Trp Asn Gly Met Val Arg Glu Leu
Ile Asp His Lys Ser Lys Ile 500 505 510Ser Thr Tyr Asp Lys Met Trp
Ala Phe Met Ser Ser Arg Arg Gln Ser 515 520 525Val Leu Val Lys Ser
Asn Glu Glu Gly Ile Gln Arg Val Leu Thr Ser 530 535 540Asp Tyr Ala
Phe Leu Met Glu Ser Thr Thr Ile Glu Phe Val Thr Gln545 550 555
560Arg Asn Cys Asn Leu Thr Gln Ile Gly Gly Leu Ile Asp Ser Lys Gly
565 570 575Tyr Gly Val Gly Thr Pro Met Gly Ser Pro Tyr Arg Asp Lys
Ile Thr 580 585 590Ile Ala Ile Leu Gln Leu Gln Glu Glu Gly Lys Leu
His Met Met Lys 595 600 605Glu Lys Trp Trp Arg Gly Asn Gly Cys Pro
Glu Glu Glu Ser Lys Glu 610 615 620Ala Ser Ala Leu Gly Val Gln Asn
Ile Gly Gly Ile Phe Ile Val Leu625 630 635 640Ala Ala Gly Leu Val
Leu Ser Val Phe Val Ala Val Gly Glu Phe Leu 645 650 655Tyr Lys Ser
Lys Lys Asn Ala Gln Leu Glu Lys Glu Ser Ser Ile Trp 660 665 670Leu
Val Pro Pro Tyr His Pro Asp Thr Val 675 68010687PRTHomo sapiens
10Met Lys Ile Ile Phe Pro Ile Leu Ser Asn Pro Val Phe Arg Arg Thr1
5 10 15Val Lys Leu Leu Leu Cys Leu Leu Trp Ile Gly Tyr Ser Gln Gly
Thr 20 25 30Thr His Val Leu Arg Phe Gly Gly Ile Phe Glu Tyr Val Glu
Ser Gly 35 40 45Pro Met Gly Ala Glu Glu Leu Ala Phe Arg Phe Ala Val
Asn Thr Ile 50 55 60Asn Arg Asn Arg Thr Leu Leu Pro Asn Thr Thr Leu
Thr Tyr Asp Thr65 70 75 80Gln Lys Ile Asn Leu Tyr Asp Ser Phe Glu
Ala Ser Lys Lys Ala Cys 85 90 95Asp Gln Leu Ser Leu Gly Val Ala Ala
Ile Phe Gly Pro Ser His Ser 100 105 110Ser Ser Ala Asn Ala Val Gln
Ser Ile Cys Asn Ala Leu Gly Val Pro
115 120 125His Ile Gln Thr Arg Trp Lys His Gln Val Ser Asp Asn Lys
Asp Ser 130 135 140Phe Tyr Val Ser Leu Tyr Pro Asp Phe Ser Ser Leu
Ser Arg Ala Ile145 150 155 160Leu Asp Leu Val Gln Phe Phe Lys Trp
Lys Thr Val Thr Val Val Tyr 165 170 175Asp Asp Ser Thr Gly Leu Ile
Arg Leu Gln Glu Leu Ile Lys Ala Pro 180 185 190Ser Arg Tyr Asn Leu
Arg Leu Lys Ile Arg Gln Leu Pro Ala Asp Thr 195 200 205Lys Asp Ala
Lys Pro Leu Leu Lys Glu Met Lys Arg Gly Lys Glu Phe 210 215 220His
Val Ile Phe Asp Cys Ser His Glu Met Ala Ala Gly Ile Leu Lys225 230
235 240Gln Ala Leu Ala Met Gly Met Met Thr Glu Tyr Tyr His Tyr Ile
Phe 245 250 255Thr Thr Leu Asp Leu Phe Ala Leu Asp Val Glu Pro Tyr
Arg Tyr Ser 260 265 270Gly Val Asn Met Thr Gly Phe Arg Ile Leu Asn
Thr Glu Asn Thr Gln 275 280 285Val Ser Ser Ile Ile Glu Lys Trp Ser
Met Glu Arg Leu Gln Ala Pro 290 295 300Pro Lys Pro Asp Ser Gly Leu
Leu Asp Gly Phe Met Thr Thr Asp Ala305 310 315 320Ala Leu Met Tyr
Asp Ala Val His Val Val Ser Val Ala Val Gln Gln 325 330 335Phe Pro
Gln Met Thr Val Ser Ser Leu Gln Cys Asn Arg His Lys Pro 340 345
350Trp Arg Phe Gly Thr Arg Phe Met Ser Leu Ile Lys Glu Ala His Trp
355 360 365Glu Gly Leu Thr Gly Arg Ile Thr Phe Asn Lys Thr Asn Gly
Leu Arg 370 375 380Thr Asp Phe Asp Leu Asp Val Ile Ser Leu Lys Glu
Glu Gly Leu Glu385 390 395 400Lys Ile Gly Thr Trp Asp Pro Ala Ser
Gly Leu Asn Met Thr Glu Ser 405 410 415Gln Lys Gly Lys Pro Ala Asn
Ile Thr Asp Ser Leu Ser Asn Arg Ser 420 425 430Leu Ile Val Thr Thr
Ile Leu Glu Glu Pro Tyr Val Leu Phe Lys Lys 435 440 445Ser Asp Lys
Pro Leu Tyr Gly Asn Asp Arg Phe Glu Gly Tyr Cys Ile 450 455 460Asp
Leu Leu Arg Glu Leu Ser Thr Ile Leu Gly Phe Thr Tyr Glu Ile465 470
475 480Arg Leu Val Glu Asp Gly Lys Tyr Gly Ala Gln Asp Asp Ala Asn
Gly 485 490 495Gln Trp Asn Gly Met Val Arg Glu Leu Ile Asp His Lys
Ser Val Leu 500 505 510Val Lys Ser Asn Glu Glu Gly Ile Gln Arg Val
Leu Thr Ser Asp Tyr 515 520 525Ala Phe Leu Met Glu Ser Thr Thr Ile
Glu Phe Val Thr Gln Arg Asn 530 535 540Cys Asn Leu Thr Gln Ile Gly
Gly Leu Ile Asp Ser Lys Gly Tyr Gly545 550 555 560Val Gly Thr Pro
Met Gly Ser Pro Tyr Arg Asp Lys Ile Thr Ile Ala 565 570 575Ile Leu
Gln Leu Gln Glu Glu Gly Lys Leu His Met Met Lys Glu Lys 580 585
590Trp Trp Arg Gly Asn Gly Cys Pro Glu Glu Glu Ser Lys Glu Ala Ser
595 600 605Ala Leu Gly Val Gln Asn Ile Gly Gly Ile Phe Ile Val Leu
Ala Ala 610 615 620Gly Leu Val Leu Ser Val Phe Val Ala Val Gly Glu
Phe Leu Tyr Lys625 630 635 640Ser Lys Lys Asn Ala Gln Leu Glu Lys
Arg Ala Lys Thr Lys Leu Pro 645 650 655Gln Asp Tyr Val Phe Leu Pro
Ile Leu Glu Ser Val Ser Ile Ser Thr 660 665 670Val Leu Ser Ser Ser
Pro Ser Ser Ser Ser Leu Ser Ser Cys Ser 675 680 685112727DNAHomo
sapiens 11atgaagatta ttttcccgat tctaagtaat ccagtcttca ggcgcaccgt
taaactcctg 60ctctgtttac tgtggattgg atattctcaa ggaaccacac atgtattaag
atttggtggt 120atttttgaat atgtggaatc tggcccaatg ggagctgagg
aacttgcatt cagatttgct 180gtgaacacaa ttaacagaaa cagaacattg
ctacccaata ctacccttac ctatgatacc 240cagaagataa acctttatga
tagttttgaa gcatccaaga aagcctgtga tcagctgtct 300cttggggtgg
ctgccatctt cgggccttca cacagctcat cagcaaacgc agtgcagtcc
360atctgcaatg ctctgggagt tccccacata cagacccgct ggaagcacca
ggtgtcagac 420aacaaagatt ccttctatgt cagtctctac ccagacttct
cttcactcag ccgtgccatt 480ttagacctgg tgcagttttt caagtggaaa
accgtcacgg ttgtgtatga tgacagcact 540ggtctcattc gtttgcaaga
gctcatcaaa gctccatcaa ggtataatct tcgactcaaa 600attcgtcagt
tacctgctga tacaaaggat gcaaaaccct tactaaaaga aatgaaaaga
660ggcaaggagt ttcatgtaat ctttgattgt agccatgaaa tggcagcagg
cattttaaaa 720caggcattag ctatgggaat gatgacagaa tactatcatt
atatctttac cactctggac 780ctctttgctc ttgatgttga gccctaccga
tacagtggtg ttaacatgac agggttcaga 840atattaaata cagaaaatac
ccaagtctcc tccatcattg aaaagtggtc gatggaacga 900ttgcaggcac
ctccgaaacc cgattcaggt ttgctggatg gatttatgac gactgatgct
960gctctaatgt atgatgctgt gcatgtggtg tctgtggccg ttcaacagtt
tccccagatg 1020acagtcagtt ccttgcagtg taatcgacat aaaccctggc
gcttcgggac ccgctttatg 1080agtctaatta aagaggcaca ttgggaaggc
ctcacaggca gaataacttt caacaaaacc 1140aatggcttga gaacagattt
tgatttggat gtgatcagtc tgaaggaaga aggtctagaa 1200aagattggaa
cgtgggatcc agccagtggc ctgaatatga cagaaagtca aaagggaaag
1260ccagcgaaca tcacagattc cttatccaat cgttctttga ttgttaccac
cattttggaa 1320gagccttatg tcctttttaa gaagtctgac aaacctctct
atggtaatga tcgatttgaa 1380ggctattgca ttgatctcct cagagagtta
tctacaatcc ttggctttac atatgaaatt 1440agacttgtgg aagatgggaa
atatggagcc caggatgatg ccaatggaca atggaatgga 1500atggttcgtg
aactaattga tcataaagct gaccttgcag ttgctccact ggctattacc
1560tatgttcgag agaaggtcat cgacttttcc aagcccttta tgacacttgg
aataagtatt 1620ttgtaccgca agcccaatgg tacaaaccca ggcgtcttct
ccttcctgaa tcctctctcc 1680cctgatatct ggatgtatat tctgctggct
tacttgggtg tcagttgtgt gctctttgtc 1740atagccaggt ttagtcctta
tgagtggtat aatccacacc cttgcaaccc tgactcagac 1800gtggtggaaa
acaattttac cttgctaaat agtttctggt ttggagttgg agctctcatg
1860cagcaaggtt ctgagctcat gcccaaagca ctgtccacca ggatagtggg
aggcatttgg 1920tggtttttca cacttatcat catttcttcg tatactgcta
acttagccgc ctttctgaca 1980gtggaacgca tggaatcccc tattgactct
gctgatgatt tagctaaaca aaccaagata 2040gaatatggag cagtagagga
tggtgcaacc atgacttttt tcaagaaatc aaaaatctcc 2100acgtatgaca
aaatgtgggc ctttatgagt agcagaaggc agtcagtgct ggtcaaaagt
2160aatgaagaag gaatccagcg agtcctcacc tctgattatg ctttcctaat
ggagtcaaca 2220accatcgagt ttgttaccca gcggaactgt aacctgacac
agattggcgg ccttatagac 2280tctaaaggtt atggcgttgg cactcccatg
ggttctccat atcgagacaa aattaccata 2340gcaattcttc agctgcaaga
ggaaggcaaa ctgcatatga tgaaggagaa atggtggagg 2400ggcaatggtt
gcccagaaga ggagagcaaa gaggccagtg ccctgggggt tcagaatatt
2460ggtggcatct tcattgttct ggcagccggc ttggtgcttt cagtttttgt
ggcagtggga 2520gaatttttat acaaatccaa aaaaaacgct caattggaaa
agaggtcctt ctgtagtgcc 2580atggtagaag aattgaggat gtccctgaag
tgccagcgtc ggttaaaaca taagccacag 2640gccccagtta ttgtgaaaac
agaagaagtt atcaacatgc acacatttaa cgacagaagg 2700ttgccaggta
aagaaaccat ggcataa 2727122610DNAHomo sapiens 12atgaagatta
ttttcccgat tctaagtaat ccagtcttca ggcgcaccgt taaactcctg 60ctctgtttac
tgtggattgg atattctcaa ggaaccacac atgtattaag atttggtggt
120atttttgaat atgtggaatc tggcccaatg ggagctgagg aacttgcatt
cagatttgct 180gtgaacacaa ttaacagaaa cagaacattg ctacccaata
ctacccttac ctatgatacc 240cagaagataa acctttatga tagttttgaa
gcatccaaga aagcctgtga tcagctgtct 300cttggggtgg ctgccatctt
cgggccttca cacagctcat cagcaaacgc agtgcagtcc 360atctgcaatg
ctctgggagt tccccacata cagacccgct ggaagcacca ggtgtcagac
420aacaaagatt ccttctatgt cagtctctac ccagacttct cttcactcag
ccgtgccatt 480ttagacctgg tgcagttttt caagtggaaa accgtcacgg
ttgtgtatga tgacagcact 540ggtctcattc gtttgcaaga gctcatcaaa
gctccatcaa ggtataatct tcgactcaaa 600attcgtcagt tacctgctga
tacaaaggat gcaaaaccct tactaaaaga aatgaaaaga 660ggcaaggagt
ttcatgtaat ctttgattgt agccatgaaa tggcagcagg cattttaaaa
720caggcattag ctatgggaat gatgacagaa tactatcatt atatctttac
cactctggac 780ctctttgctc ttgatgttga gccctaccga tacagtggtg
ttaacatgac agggttcaga 840atattaaata cagaaaatac ccaagtctcc
tccatcattg aaaagtggtc gatggaacga 900ttgcaggcac ctccgaaacc
cgattcaggt ttgctggatg gatttatgac gactgatgct 960gctctaatgt
atgatgctgt gcatgtggtg tctgtggccg ttcaacagtt tccccagatg
1020acagtcagtt ccttgcagtg taatcgacat aaaccctggc gcttcgggac
ccgctttatg 1080agtctaatta aagaggcaca ttgggaaggc ctcacaggca
gaataacttt caacaaaacc 1140aatggcttga gaacagattt tgatttggat
gtgatcagtc tgaaggaaga aggtctagaa 1200aagattggaa cgtgggatcc
agccagtggc ctgaatatga cagaaagtca aaagggaaag 1260ccagcgaaca
tcacagattc cttatccaat cgttctttga ttgttaccac cattttggaa
1320gagccttatg tcctttttaa gaagtctgac aaacctctct atggtaatga
tcgatttgaa 1380ggctattgca ttgatctcct cagagagtta tctacaatcc
ttggctttac atatgaaatt 1440agacttgtgg aagatgggaa atatggagcc
caggatgatg ccaatggaca atggaatgga 1500atggttcgtg aactaattga
tcataaagct gaccttgcag ttgctccact ggctattacc 1560tatgttcgag
agaaggtcat cgacttttcc aagcccttta tgacacttgg aataagtatt
1620ttgtaccgca agcccaatgg tacaaaccca ggcgtcttct ccttcctgaa
tcctctctcc 1680cctgatatct ggatgtatat tctgctggct tacttgggtg
tcagttgtgt gctctttgtc 1740atagccaggt ttagtcctta tgagtggtat
aatccacacc cttgcaaccc tgactcagac 1800gtggtggaaa acaattttac
cttgctaaat agtttctggt ttggagttgg agctctcatg 1860cagcaaggtt
ctgagctcat gcccaaagca ctgtccacca ggatagtggg aggcatttgg
1920tggtttttca cacttatcat catttcttcg tatactgcta acttagccgc
ctttctgaca 1980gtggaacgca tggaatcccc tattgactct gctgatgatt
tagctaaaca aaccaagata 2040gaatatggag cagtagagga tggtgcaacc
atgacttttt tcaagaaatc aaaaatctcc 2100acgtatgaca aaatgtgggc
ctttatgagt agcagaaggc agtcagtgct ggtcaaaagt 2160aatgaagaag
gaatccagcg agtcctcacc tctgattatg ctttcctaat ggagtcaaca
2220accatcgagt ttgttaccca gcggaactgt aacctgacac agattggcgg
ccttatagac 2280tctaaaggtt atggcgttgg cactcccatg ggttctccat
atcgagacaa aattaccata 2340gcaattcttc agctgcaaga ggaaggcaaa
ctgcatatga tgaaggagaa atggtggagg 2400ggcaatggtt gcccagaaga
ggagagcaaa gaggccagtg ccctgggggt tcagaatatt 2460ggtggcatct
tcattgttct ggcagccggc ttggtgcttt cagtttttgt ggcagtggga
2520gaatttttat acaaatccaa aaaaaacgct caattggaaa aggaatcttc
tatttggtta 2580gtgccaccat accatccaga cactgtttag 2610132679DNAHomo
sapiens 13atgaagatta ttttcccgat tctaagtaat ccagtcttca ggcgcaccgt
taaactcctg 60ctctgtttac tgtggattgg atattctcaa ggaaccacac atgtattaag
atttggtggt 120atttttgaat atgtggaatc tggcccaatg ggagctgagg
aacttgcatt cagatttgct 180gtgaacacaa ttaacagaaa cagaacattg
ctacccaata ctacccttac ctatgatacc 240cagaagataa acctttatga
tagttttgaa gcatccaaga aagcctgtga tcagctgtct 300cttggggtgg
ctgccatctt cgggccttca cacagctcat cagcaaacgc agtgcagtcc
360atctgcaatg ctctgggagt tccccacata cagacccgct ggaagcacca
ggtgtcagac 420aacaaagatt ccttctatgt cagtctctac ccagacttct
cttcactcag ccgtgccatt 480ttagacctgg tgcagttttt caagtggaaa
accgtcacgg ttgtgtatga tgacagcact 540ggtctcattc gtttgcaaga
gctcatcaaa gctccatcaa ggtataatct tcgactcaaa 600attcgtcagt
tacctgctga tacaaaggat gcaaaaccct tactaaaaga aatgaaaaga
660ggcaaggagt ttcatgtaat ctttgattgt agccatgaaa tggcagcagg
cattttaaaa 720caggcattag ctatgggaat gatgacagaa tactatcatt
atatctttac cactctggac 780ctctttgctc ttgatgttga gccctaccga
tacagtggtg ttaacatgac agggttcaga 840atattaaata cagaaaatac
ccaagtctcc tccatcattg aaaagtggtc gatggaacga 900ttgcaggcac
ctccgaaacc cgattcaggt ttgctggatg gatttatgac gactgatgct
960gctctaatgt atgatgctgt gcatgtggtg tctgtggccg ttcaacagtt
tccccagatg 1020acagtcagtt ccttgcagtg taatcgacat aaaccctggc
gcttcgggac ccgctttatg 1080agtctaatta aagaggcaca ttgggaaggc
ctcacaggca gaataacttt caacaaaacc 1140aatggcttga gaacagattt
tgatttggat gtgatcagtc tgaaggaaga aggtctagaa 1200aagattggaa
cgtgggatcc agccagtggc ctgaatatga cagaaagtca aaagggaaag
1260ccagcgaaca tcacagattc cttatccaat cgttctttga ttgttaccac
cattttggaa 1320gagccttatg tcctttttaa gaagtctgac aaacctctct
atggtaatga tcgatttgaa 1380ggctattgca ttgatctcct cagagagtta
tctacaatcc ttggctttac atatgaaatt 1440agacttgtgg aagatgggaa
atatggagcc caggatgatg ccaatggaca atggaatgga 1500atggttcgtg
aactaattga tcataaagct gaccttgcag ttgctccact ggctattacc
1560tatgttcgag agaaggtcat cgacttttcc aagcccttta tgacacttgg
aataagtatt 1620ttgtaccgca agcccaatgg tacaaaccca ggcgtcttct
ccttcctgaa tcctctctcc 1680cctgatatct ggatgtatat tctgctggct
tacttgggtg tcagttgtgt gctctttgtc 1740atagccaggt ttagtcctta
tgagtggtat aatccacacc cttgcaaccc tgactcagac 1800gtggtggaaa
acaattttac cttgctaaat agtttctggt ttggagttgg agctctcatg
1860cagcaaggtt ctgagctcat gcccaaagca ctgtccacca ggatagtggg
aggcatttgg 1920tggtttttca cacttatcat catttcttcg tatactgcta
acttagccgc ctttctgaca 1980gtggaacgca tggaatcccc tattgactct
gctgatgatt tagctaaaca aaccaagata 2040gaatatggag cagtagagga
tggtgcaacc atgacttttt tcaagaaatc aaaaatctcc 2100acgtatgaca
aaatgtgggc ctttatgagt agcagaaggc agtcagtgct ggtcaaaagt
2160aatgaagaag gaatccagcg agtcctcacc tctgattatg ctttcctaat
ggagtcaaca 2220accatcgagt ttgttaccca gcggaactgt aacctgacac
agattggcgg ccttatagac 2280tctaaaggtt atggcgttgg cactcccatg
ggttctccat atcgagacaa aattaccata 2340gcaattcttc agctgcaaga
ggaaggcaaa ctgcatatga tgaaggagaa atggtggagg 2400ggcaatggtt
gcccagaaga ggagagcaaa gaggccagtg ccctgggggt tcagaatatt
2460ggtggcatct tcattgttct ggcagccggc ttggtgcttt cagtttttgt
ggcagtggga 2520gaatttttat acaaatccaa aaaaaacgct caattggaaa
agagagccaa gactaagtta 2580cctcaagact atgtattcct ccctattttg
gagtcagttt ccatttctac agtgttgtca 2640tcatcaccat cttcatcatc
attatcatca tgttcttaa 26791422DNAHomo sapiens 14cccatagcta
atgcctgttt ta 221522DNAMus musculus 15cccatagcta atgcctgctt ta
221622RNAHomo sapiens 16uaaaacaggc auuagcuaug gg 221722RNAMus
musculus 17uaaagcaggc auuagcuaug gg 221822RNAHomo sapiens
18cccauagcua augccuguuu ua 221922RNAMus musculus 19cccauagcua
augccugcuu ua 222061DNAHomo sapiens 20taaaacaggc attagctatg
ggtagtgaag ccacagatgc ccatagctaa tgcctgtttt 60a 6121339DNAHomo
sapiens 21tcgactaggg ataacagggt aattgtttga atgaggcttc agtactttac
agaatcgttg 60cctgcacatc ttggaaacac ttgctgggat tacttcttca ggttaaccca
acagaaggct 120cgagaaggta tattgctgtt gacagtgagc gctaaaacag
gcattagcta tgggtagtga 180agccacagat gcccatagct aatgcctgtt
ttattgccta ctgcctcgga attcaagggg 240ctactttagg agcaattatc
ttgtttacta aaactgaata ccttgctatc tctttgatac 300atttttacaa
agctgaatta aaatggtata aattatcac 3392261DNAHomo sapiens 22cccatagcta
atgcctgttt tatagtgaag ccacagatgt aaaacaggca ttagctatgg 60g
6123339DNAHomo sapiens 23tcgactaggg ataacagggt aattgtttga
atgaggcttc agtactttac agaatcgttg 60cctgcacatc ttggaaacac ttgctgggat
tacttcttca ggttaaccca acagaaggct 120cgagaaggta tattgctgtt
gacagtgagc gccccatagc taatgcctgt tttatagtga 180agccacagat
gtaaaacagg cattagctat gggttgccta ctgcctcgga attcaagggg
240ctactttagg agcaattatc ttgtttacta aaactgaata ccttgctatc
tctttgatac 300atttttacaa agctgaatta aaatggtata aattatcac
33924121DNAHomo sapiens 24tcgactaggg ataacagggt aattgtttga
atgaggcttc agtactttac agaatcgttg 60cctgcacatc ttggaaacac ttgctgggat
tacttcttca ggttaaccca acagaaggct 120c 1212517DNAHomo sapiens
25tagtgaagcc acagatg 1726105DNAHomo sapiens 26aaggggctac tttaggagca
attatcttgt ttactaaaac tgaatacctt gctatctctt 60tgatacattt ttacaaagct
gaattaaaat ggtataaatt atcac 10527429DNAHomo sapiens 27ctgcagaggg
ccctgcgtat gagtgcaagt gggttttagg accaggatga ggcggggtgg 60gggtgcctac
ctgacgaccg accccgaccc actggacaag cacccaaccc ccattcccca
120aattgcgcat cccctatcag agagggggag gggaaacagg atgcggcgag
gcgcgtgcgc 180actgccagct tcagcaccgc ggacagtgcc ttcgcccccg
cctggcggcg cgcgccaccg 240ccgcctcagc actgaaggcg cgctgacgtc
actcgccggt cccccgcaaa ctccccttcc 300cggccacctt ggtcgcgtcc
gcgccgccgc cggcccagcc ggaccgcacc acgcgaggcg 360cgagataggg
gggcacgggc gcgaccatct gcgctgcggc gccggcgact cagcgctgcc 420tcagtctgc
42928469DNAHomo sapiens 28ctgcagaggg ccctgcgtat gagtgcaagt
gggttttagg accaggatga ggcggggtgg 60gggtgcctac ctgacgaccg accccgaccc
actggacaag cacccaaccc ccattcccca 120aattgcgcat cccctatcag
agagggggag gggaaacagg atgcggcgag gcgcgtgcgc 180actgccagct
tcagcaccgc ggacagtgcc ttcgcccccg cctggcggcg cgcgccaccg
240ccgcctcagc actgaaggcg cgctgacgtc actcgccggt cccccgcaaa
ctccccttcc 300cggccacctt ggtcgcgtcc gcgccgccgc cggcccagcc
ggaccgcacc acgcgaggcg 360cgagataggg gggcacgggc gcgaccatct
gcgctgcggc gccggcgact cagcgctgcc 420tcagtctgcc aattgcagcg
gaggagtcgt gtcgtgcctg agagcgcag 46929313DNAMus musculus
29atccgacgcc gccatctcta ggcccgcgcc ggccccctcg cacagacttg tgggagaagc
60tcggctactc ccctgccccg gttaatttgc atataatatt tcctagtaac tatagaggct
120taatgtgcga taaaagacag ataatctgtt ctttttaata ctagctacat
tttacatgat 180aggcttggat ttctataaga gatacaaata ctaaattatt
attttaaaaa acagcacaaa 240aggaaactca ccctaactgt aaagtaattg
tgtgttttga gactataaat atcccttgga 300gaaaagcctt gtt 3133060DNAHomo
sapiens 30gatgctgacg aaggctcgcg aggctgtgag cagccacagt gccctgctca
gaagccccgg 603160DNAHomo sapiens 31gtctcccgcg cccgcgcccg
tgtcgccgcc gtgcccgcga gcgggagccg gagtcgccgc 603260DNAHomo sapiens
32cgtgtgcaga tgcagggcgc cggtgccctg cgggtgcggg tgcaggagca gcgtgtgcag
603360DNAHomo sapiens 33ccccacgcca ccctttctgg tcatctcccc tcccgccccg
cccctgcgca cactccctcg 603460DNAHomo sapiens 34tctccccggt aaagtctcgc
ggtgctgccg ggctcagccc cgtctcctcc tcttgctccc 60351725DNAHomo sapiens
35gacattgatt attgactagt tattaatagt aatcaattac ggggtcatta gttcatagcc
60catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca
120acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg
ccaataggga 180ctttccattg acgtcaatgg gtggagtatt tacggtaaac
tgcccacttg gcagtacatc 240aagtgtatca tatgccaagt acgcccccta
ttgacgtcaa tgacggtaaa tggcccgcct 300ggcattatgc ccagtacatg
accttatggg actttcctac ttggcagtac atctacgtat 360tagtcatcgc
tattaccatg gtcgaggtga gccccacgtt ctgcttcact ctccccatct
420cccccccctc cccaccccca attttgtatt tatttatttt ttaattattt
tgtgcagcga 480tgggggcggg gggggggggg gggcgcgcgc caggcggggc
ggggcggggc gaggggcggg 540gcggggcgag gcggagaggt gcggcggcag
ccaatcagag cggcgcgctc cgaaagtttc 600cttttatggc gaggcggcgg
cggcggcggc cctataaaaa gcgaagcgcg cggcgggcgg 660gagtcgctgc
gcgctgcctt cgccccgtgc cccgctccgc cgccgcctcg cgccgcccgc
720cccggctctg actgaccgcg ttactcccac aggtgagcgg gcgggacggc
ccttctcctc 780cgggctgtaa ttagcgcttg gtttaatgac ggcttgtttc
ttttctgtgg ctgcgtgaaa 840gccttgaggg gctccgggag ggccctttgt
gcggggggag cggctcgggg ggtgcgtgcg 900tgtgtgtgtg cgtggggagc
gccgcgtgcg gctccgcgct gcccggcggc tgtgagcgct 960gcgggcgcgg
cgcggggctt tgtgcgctcc gcagtgtgcg cgaggggagc gcggccgggg
1020gcggtgcccc gcggtgcggg gggggctgcg aggggaacaa aggctgcgtg
cggggtgtgt 1080gcgtgggggg gtgagcaggg ggtgtgggcg cgtcggtcgg
gctgcaaccc cccctgcacc 1140cccctccccg agttgctgag cacggcccgg
cttcgggtgc ggggctccgt acggggcgtg 1200gcgcggggct cgccgtgccg
ggcggggggt ggcggcaggt gggggtgccg ggcggggcgg 1260ggccgcctcg
ggccggggag ggctcggggg aggggcgcgg cggcccccgg agcgccggcg
1320gctgtcgagg cgcggcgagc cgcagccatt gccttttatg gtaatcgtgc
gagagggcgc 1380agggacttcc tttgtcccaa atctgtgcgg agccgaaatc
tgggaggcgc cgccgcaccc 1440cctctagcgg gcgcggggcg aagcggtgcg
gcgccggcag gaaggaaatg ggcggggagg 1500gccttcgtgc gtcgccgcgc
cgccgtcccc ttctccctct ccagcctcgg ggctgtccgc 1560ggggggacgg
ctgccttcgg gggggacggg gcagggcggg gttcggcttc tggcgtgtga
1620ccggcggctc tagagcctct gctaaccatg ttcatgcctt cttctttttc
ctacagctcc 1680tgggcaacgt gctggttatt gtgctgtctc atcattttgg caaag
17253617DNAArtificial SequenceSynthetic Construct 36tttgtgaggg
tctggtc 173721RNAArtificial SequenceSynthetic Construct
37uaauguuagu cauguccacc g 21
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