U.S. patent application number 17/532123 was filed with the patent office on 2022-03-10 for compositions and methods for hemoglobin production.
The applicant listed for this patent is THE CHILDREN'S HOSPITAL OF PHILADELPHIA, COLD SPRING HARBOR LABORATORY, THE TRUSTEES OF THE UNIVERSITY OF PENNSYLVANIA. Invention is credited to Gerd Blobel, Jeremy Grevet, Junwei Shi, Christopher Vakoc.
Application Number | 20220071981 17/532123 |
Document ID | / |
Family ID | |
Filed Date | 2022-03-10 |
United States Patent
Application |
20220071981 |
Kind Code |
A1 |
Blobel; Gerd ; et
al. |
March 10, 2022 |
COMPOSITIONS AND METHODS FOR HEMOGLOBIN PRODUCTION
Abstract
Methods and compositions for producing fetal hemoglobin and
treating a hemoglobinopathy or thalassemia are disclosed.
Inventors: |
Blobel; Gerd; (Bala Cynwyd,
PA) ; Grevet; Jeremy; (Philadelphia, PA) ;
Shi; Junwei; (Philadelphia, PA) ; Vakoc;
Christopher; (Cold Spring Harbor, NY) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
THE CHILDREN'S HOSPITAL OF PHILADELPHIA
THE TRUSTEES OF THE UNIVERSITY OF PENNSYLVANIA
COLD SPRING HARBOR LABORATORY |
Philadelphia
Philadelphia
Cold Spring Harbor |
PA
PA
NY |
US
US
US |
|
|
Appl. No.: |
17/532123 |
Filed: |
November 22, 2021 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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16478651 |
Jul 17, 2019 |
11179379 |
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PCT/US2018/015918 |
Jan 30, 2018 |
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17532123 |
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62452069 |
Jan 30, 2017 |
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62465283 |
Mar 1, 2017 |
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International
Class: |
A61K 31/454 20060101
A61K031/454; A61P 7/06 20060101 A61P007/06; A61K 31/517 20060101
A61K031/517; C12N 15/113 20060101 C12N015/113 |
Goverment Interests
[0002] This invention was made with government support under Grant
Nos. R37DK058044, R01DK054937, and R01HL119479 awarded by National
Institutes of Health. The government has certain rights in the
invention.
Claims
1. A method of increasing the level of fetal hemoglobin in a cell
or subject, the method comprising administering at least one
eukaryotic translation initiation factor 2-alpha kinase 1
(eIF2.alpha.K1) inhibitor to the cell or subject.
2. The method of claim 1, wherein the subject has a .beta.-chain
hemoglobinopathy.
3. The method of claim 1, wherein the subject has thalassemia.
4. The method of claim 1, wherein the subject has sickle cell
anemia.
5. The method of claim 1, wherein the eIF2.alpha.K1 inhibitor is a
small molecule.
6. The method of claim 1, wherein the eIF2.alpha.K1 inhibitor is a
kinase domain inhibitor or a heme binding domain inhibitor.
7. The method of claim 1, wherein the cell is an erythroid
cell.
8. The method of claim 1, further comprising administering at least
one fetal hemoglobin inducer to the cell or subject.
9. The method of claim 8, wherein said fetal hemoglobin inducer is
pomalidomide.
10. The method of claim 8, wherein said fetal hemoglobin inducer is
a histone methyltransferase (HMT) inhibitor.
11. The method of claim 10, wherein said HMT inhibitor is
UNC0638.
12. A method of treating a hemoglobinopathy in a subject in need
thereof, the method comprising administering a composition
comprising at least one eukaryotic translation initiation factor
2-alpha kinase 1 (eIF2.alpha.K1) inhibitor and a pharmaceutically
acceptable carrier to the subject.
13. The method of claim 12, wherein the subject has a .beta.-chain
hemoglobinopathy.
14. The method of claim 12, wherein the subject has
thalassemia.
15. The method of claim 12, wherein the subject has sickle cell
anemia.
16. The method of claim 12, wherein the eIF2.alpha.K1 inhibitor is
a small molecule.
17. The method of claim 12, wherein the eIF2.alpha.K1 inhibitor is
a kinase domain inhibitor or a heme binding domain inhibitor.
18. The method of claim 12, further comprising administering at
least one fetal hemoglobin inducer to the subject.
19. The method of claim 18, wherein said fetal hemoglobin inducer
is pomalidomide.
20. The method of claim 18, wherein said fetal hemoglobin inducer
is a histone methyltransferase (HMT) inhibitor.
21. The method of claim 20, wherein said HMT inhibitor is
UNC0638.
22. The method of claim 12, wherein the eIF2.alpha.K1 inhibitor is
contained within a cell administered to the subject.
Description
[0001] This application is a continuation application of U.S.
patent application Ser. No. 16/478,651, filed Jul. 17, 2019, which
is a .sctn. 371 application of PCT/US2018/015918, filed Jan. 30,
2018, which claims priority under 35 U.S.C. .sctn. 119(e) to U.S.
Provisional Patent Application No. 62/452,069, filed Jan. 30, 2017
and U.S. Provisional Patent Application No. 62/465,283, filed Mar.
1, 2017. The foregoing applications are incorporated by reference
herein.
FIELD OF THE INVENTION
[0003] The present invention relates to the field of hematology.
More specifically, the invention provides compositions and methods
for the production of various forms of hemoglobin, including adult
and fetal type hemoglobin.
BACKGROUND OF THE INVENTION
[0004] Several publications and patent documents are cited
throughout the specification in order to describe the state of the
art to which this invention pertains. Each of these citations is
incorporated herein by reference as though set forth in full.
[0005] Sickle cell disease and thalassemia cause significant
worldwide morbidity and mortality (Modell et al. (2008) Bull. World
Health Org., 86:480-487). However, effective drugs do not exist for
these illnesses. One goal in the treatment of these diseases is to
reactivate fetal hemoglobin (HbF). HbF reduces the propensity of
sickle cell disease red blood cells to undergo sickling. Indeed,
high fetal globin levels are associated with improved outcomes for
sickle cell anemia patients (Platt et al. (1994) N. Engl. J. Med.,
330:1639-1644). Elevating HbF also reduces the globin chain
imbalance in certain thalassemias, thereby improving symptoms.
There is an enormous unmet need to identify compounds that
ameliorate the course of these diseases.
SUMMARY OF THE INVENTION
[0006] In accordance with the present invention, compositions and
methods are provided for increasing hemoglobin levels (e.g., fetal
hemoglobin) in a cell or subject. In a particular embodiment, the
method comprises administering at least one eIF2.alpha.K1 inhibitor
to the cell or subject. In a particular embodiment, the subject has
a hemoglobinopathy such as sickle cell disease or thalassemia. In a
particular embodiment, the cell is an erythroid cell. In a
particular embodiment, the eIF2.alpha.K1 inhibitor is a small
molecule. The eIF2.alpha.K1 inhibitor may be, for example, a kinase
domain inhibitor or a heme binding domain inhibitor. The
eIF2.alpha.K1 inhibitor may be a CRISPR based or siRNA/shRNA based
inhibitor of the eIF2.alpha.K1 gene. The method may further
comprise delivering at least one fetal hemoglobin inducer to the
cell or subject. The method may further comprise the administration
of other fetal hemoglobin inducing methods (e.g., pharmacologic
compounds or various forms of gene therapy) in order to produce
additive or synergistic effects.
[0007] In accordance with another aspect of the instant invention,
methods of inhibiting, treating, and/or preventing a
hemoglobinopathy (e.g., sickle cell disease or thalassemia) in a
subject are provided. In a particular embodiment, the method
comprises administering at least one eIF2.alpha.K1 inhibitor to a
subject in need thereof. The eIF2.alpha.K1 inhibitor may be in a
composition with a pharmaceutically acceptable carrier. In a
particular embodiment, the subject has a .beta.-chain
hemoglobinopathy. In a particular embodiment, the subject has
sickle cell anemia. In a particular embodiment, the eIF2.alpha.K1
inhibitor is a small molecule. The eIF2.alpha.K1 inhibitor may be,
for example, a kinase domain inhibitor or a heme binding domain
inhibitor. The eIF2.alpha.K1 inhibitor may be a CRISPR based or
siRNA/shRNA based inhibitor of the eIF2.alpha.K1 gene. The method
may further comprise delivering at least one other fetal hemoglobin
inducer to the subject.
BRIEF DESCRIPTIONS OF THE DRAWINGS
[0008] FIG. 1A is a graph of the percentage of cells that are
positive for fetal hemoglobin (HbF) expression. Human umbilical
cord blood-derived erythroid progenitor cells (HUDEP-2) expressing
Cas9 were transduced with one of the six guideRNAs targeting
eIF2.alpha.K1; a guideRNA targeting BCL11A exon 2 (positive
control; BCL11A is a repressor of HbF expression); one of two
guideRNAs targeting death associated protein kinase 2 (DAPK2); one
of two guideRNAs targeting transforming growth factor beta receptor
2 (TGFBR2); or a negative control guideRNA. FIG. 1B is a graph of
the gamma globin mRNA levels in the cells of FIG. 1A. A negative
control of untreated cells is also provided. FIG. 1C is a
representative HbF FACS analysis for HUDEP-2 cells expressing sgRNA
#5 or non-targeting sgRNA. FIG. 1D is a graph of the percentage of
HUDEP-2 cells that are positive for HbF expression. Mean is
shown.+-.standard deviation from biological replicates (n=2). FIG.
1E is a Western blot analysis of various proteins in HUDEP-2 cells
transduced with BCL11A exon 2 sgRNA, sgRNA #3, sgRNA #5, or
non-targeting sgRNA.
[0009] FIG. 2 provides the results of a whole proteome mass
spectrometry analysis of cells transduced with a guideRNA targeting
eIF2.alpha.K1 compared to a negative control guideRNA. Left panel
is for cells transduced with eIF2.alpha.K1 sgRNA #3 and right panel
is for cells transduced with eIF2.alpha.K1 sgRNA #5. Black ovals
with arrow indicate fetal globin expression. White ovals--zeta
globin; ovals with vertical lines--adult .beta.-globin; ovals with
horizontal lines--adult .alpha.-globin. Vertical dashed lines
indicate a 2 fold change. Horizontal dashed line indicates a
p-value of 0.01.
[0010] FIG. 3 is a graph of the percentage of cells positive for
HbF expression. HUDEP-2 expressing Cas9 cells were transduced with
eIF2.alpha.K1 sgRNA #3, eIF2.alpha.K1 sgRNA #5, a gRNA targeting
BCL11A exon 2, or a negative control gRNA. Cells were then
incubated with or without pomalidomide. n=3.
[0011] FIG. 4 provides graphs of the expression of HbF in human
primary cells treated with eIF2.alpha.K1 shRNA, BCL11A shRNA, or
scrambled shRNA (negative control).
[0012] FIG. 5A provides representative HbF flow cytometry on day 14
of CD34+ erythroid differentiation. FIG. 5B provides a graph of HbF
flow cytometry experiments. Error bars: SEM from 3 independent
donors (* denotes p-value <0.05, ** denotes p-value <0.01
from unpaired student T-tests). FIG. 5C shows .gamma.-globin mRNA
as fraction of .gamma.-globin+.beta.-globin by RT-qPCR on day 12.
Error bars: SEM from 3 independent donors (* denotes p-value
<0.05, ** denotes p-value <0.01 from unpaired student
T-tests). FIG. 5D shows the RNA-seq for CD34+ cells on day 12.
R-value denotes Pearson correlation coefficient. Data was obtained
from one patient donor. FIG. 5E shows HPLC analysis of samples from
cell expressing annotated shRNAs, at day 14 of differentiation.
HbA: hemoglobin A (adult form). HbF: fetal hemoglobin. Error bars:
standard deviation from 2 independent donors. FIG. 5F provides
images of sickle cell patient-derived erythroid cultures. Arrow
heads mark cells with sickle-like morphology. Bar graph: summary of
blindly counted sickle shaped cells from 3 fields from one patient
with sickle cell disease.
[0013] FIG. 6A provides a Western blot with indicated antibodies in
cells uninfected, or infected with virus expressing scrambled or
HRI shRNAs on day 14 of differentiation (left). BCL11A mRNA levels
in HRI knock down cells as fraction of SC shRNA expressing cells is
also shown (right). Error bars standard deviation from two
biological replicates. FIG. 6B shows RT-qPCR for .gamma.-globin as
a fraction of .gamma.-globin+.beta.-globin in a BCL11A cDNA rescue
in HRI depleted HUDEP2 clonal cell line. Error bars: standard
deviation from two biological replicates.
[0014] FIG. 7A provides an example of an amino acid sequence (SEQ
ID NO: 19) of eIF2.alpha.K1 and FIGS. 7B and 7C provide an example
of a nucleotide sequence (SEQ ID NO: 20) encoding
eIF2.alpha.K1.
DETAILED DESCRIPTION OF THE INVENTION
[0015] A major goal in the treatment of sickle cell disease and
thalassemia is the reactivation of fetal type globin expression in
cells of the adult red blood lineage. In an unbiased genetic
screen, the protein kinase eIF2.alpha.K1 (eukaryotic translation
initiation factor 2-alpha kinase 1; also known as Heme-Regulated
Inhibitor (HRI)) was identified as a strong regulator of fetal
globin production. eIF2.alpha.K1 (see, e.g., PubMed GeneID: 27102;
see, e.g., GenBank Accession Nos. NP_055228, NP_001127807,
NM_001134335, and NM_014413) is a protein kinase that
phosphorylates the translation initiation factor eIF2a and
regulates protein translation (Klann et al. (2004) Nat. Rev.
Neurosci., 5:931-942).
[0016] The activation of the pathway controlled by eIF2.alpha.K1
has been associated with a modest increase in HbF (Hahn et al.,
Blood (2013) 122(4):477-85; Han et al., EMBO J. (2001)
20:6909-6919). However, in complete contrast, it is shown herein
that the inhibition of eIF2.alpha.K1 raises fetal hemoglobin
levels. Indeed, the genetic screen described herein for HbF
inducers in human cells indicates that the loss of eIF2.alpha.K1
function increases HbF levels. Indeed, additional experiments show
that the loss of eIF2.alpha.K1 increases fetal hemoglobin
production in human erythroid cells, including primary cells.
Without being bound by theory, the mechanism by which this occurs
likely involves transcriptional and translational upregulation of
fetal hemoglobin production. Thus, the role of eIF2.alpha.K1 in the
suppression of fetal and, to a lesser extent, embryonic globin
production has been shown herein. This role is exploited herein to
treat hemoglobinopathies such as sickle cell anemia and
thalassemia.
[0017] As a protein kinase, eIF2.alpha.K1 has a kinase domain that
can be inhibited (e.g., by a small molecule). Additionally,
eIF2.alpha.K1 can be inhibited by its natural ligand heme. This
inhibition can be exploited for designing new inhibitors of
eIF2.alpha.K1 (e.g., small molecule ligands that mimic heme). The
kinase domain inhibitors and heme binding domain inhibitors can be
used individually or in combination. Inasmuch as eIF2.alpha.K1 is
expressed almost exclusively in erythroid cells, its inhibition
will have little or no impact on other tissues or cells. Indeed,
mice in which eIF2.alpha.K1 is deleted are fertile, appear normal,
and do not present any gross abnormalities (Han et al., EMBO J.
(2001) 20:6909-6919).
[0018] In accordance with the instant invention, compositions and
methods are provided for increasing hemoglobin production in a cell
or subject. In a particular embodiment, the method increases fetal
hemoglobin and/or embryonic globin expression, particularly fetal
hemoglobin. The method comprises administering at least one
eIF2.alpha.K1 inhibitor to the cell, particularly an erythroid
precursor cell or erythroid cell, or subject. In a particular
embodiment, the subject has a hemoglobinopathy such as sickle cell
disease or thalassemia. In a particular embodiment, the subject has
sickle cell anemia. The eIF2.alpha.K1 inhibitor may be administered
in a composition further comprising at least one pharmaceutically
acceptable carrier. The methods of the instant invention may
comprise administering at least two different eIF2.alpha.K1
inhibitors (e.g., two different mechanisms of action), particularly
one kinase domain inhibitor and at least one heme binding domain
inhibitor. In a particular embodiment, the method further comprises
any means by which to induce fetal hemoglobin, such as
administering at least one other fetal hemoglobin inducer. Fetal
hemoglobin inducers include, without limitation, a lysine-specific
demethylase 1 (LSD1) inhibitor (e.g., RN-1 (Cui et al. (2015) Blood
126(3):386-96) and tranylcypromine (TCP) (Sun et al. (2016) Reprod.
Biol. Endocrinol., 14:17)), pomalidomide, hydroxyurea, and
5-azacytidine, sodium butyrate, activators of the Foxo3 pathway
(e.g., metformin, phenformin, or resveratrol), histone
methyltransferase (HMT) inhibitors (e.g., a histone lysine
methyltrasnferase inhibitor, euchromatic histone-lysine
N-methyltransferase 2 (EHMT2; G9a) inhibitor, euchromatic
histone-lysine N-methyltransferase 1 (EHMT1; G9a-like protein
(GLP)) inhibitor, UNC0638
(2-cyclohexyl-N-(1-isopropylpiperidin-4-yl)-6-methoxy-7-.beta.-(pyrrolidi-
n-1-yl)propoxy) quinazolin-4-amine), chaetocin, BIX-01294, UNC
0224, UNC 0642, UNC 0631, UNC 0646, A-366 (Sweis et al. (2014) ACS
Med. Chem. Lett., 5(2):205-209), etc.), and histone deacetylase
(HDAC) inhibitors. In a particular embodiment, the fetal hemoglobin
inducer is a histone methyltransferase (HMT) inhibitor,
particularly UNC0638. In a particular embodiment, the fetal
hemoglobin inducer is pomalidomide or hydroxyurea, particularly
pomalidomide. The eIF2.alpha.K1 inhibitor and the fetal hemoglobin
inducer can be delivered to the cell or subject sequentially or
consecutively (e.g., in different compositions) and/or at the same
time (e.g., in the same composition).
[0019] In accordance with another aspect of the instant invention,
compositions and methods for inhibiting (e.g., reducing or
slowing), treating, and/or preventing a hemoglobinopathy or
thalassemia in a subject are provided. In a particular embodiment,
the methods comprise administering to a subject in need thereof a
therapeutically effective amount of at least one eIF2.alpha.K1
inhibitor. The eIF2.alpha.K1 inhibitor may be administered in a
composition further comprising at least one pharmaceutically
acceptable carrier. In a particular embodiment, the
hemoglobinopathy is .beta.-thalassemia or sickle cell anemia. In a
particular embodiment, the subject has sickle cell anemia. The
methods of the instant invention may comprise administering at
least two different eIF2.alpha.K1 inhibitors (e.g., two different
mechanisms of action), particularly one kinase domain inhibitor and
at least one heme binding domain inhibitor. In a particular
embodiment, the method further comprises administering at least one
other fetal hemoglobin inducer to the subject. Fetal hemoglobin
inducers include, without limitation, a lysine-specific demethylase
1 (LSD1) inhibitor (e.g., RN-1 (Cui et al. (2015) Blood
126(3):386-96) and tranylcypromine (TCP) (Sun et al. (2016) Reprod.
Biol. Endocrinol., 14:17)), pomalidomide, hydroxyurea, and
5-azacytidine, sodium butyrate, activators of the Foxo3 pathway
(e.g., metformin, phenformin, or resveratrol), histone
methyltransferase (HMT) inhibitors (e.g., a histone lysine
methyltrasnferase inhibitor, euchromatic histone-lysine
N-methyltransferase 2 (EHMT2; G9a) inhibitor, euchromatic
histone-lysine N-methyltransferase 1 (EHMT1; G9a-like protein
(GLP)) inhibitor, UNC0638
(2-cyclohexyl-N-(1-isopropylpiperidin-4-yl)-6-methoxy-7-(3-(pyrrolidin-1--
yl)propoxy) quinazolin-4-amine), chaetocin, BIX-01294, UNC 0224,
UNC 0642, UNC 0631, UNC 0646, A-366 (Sweis et al. (2014) ACS Med.
Chem. Lett., 5(2):205-209), etc.), and histone deacetylase (HDAC)
inhibitors. In a particular embodiment, the fetal hemoglobin
inducer is a histone methyltransferase (HMT) inhibitor,
particularly UNC0638. In a particular embodiment, the fetal
hemoglobin inducer is pomalidomide or hydroxyurea, particularly
pomalidomide. The eIF2.alpha.K1 inhibitor and the fetal hemoglobin
inducer can be administered to the subject sequentially or
consecutively (e.g., in different compositions) and/or at the same
time (e.g., in the same composition).
[0020] eIF2.alpha.K1 inhibitors are compounds which reduce
eIF2.alpha.K1 activity (particularly its kinase activity) and/or
the expression of eIF2.alpha.K1. In a particular embodiment, the
eIF2.alpha.K1 inhibitor is specific to eIF2.alpha.K1. In a
particular embodiment, the eIF2.alpha.K1 inhibitor reduces
eIF2.alpha.K1 activity and/or expression to greater levels than
other eukaryotic translation initiation factor 2-alpha kinases
(e.g., eIF2.alpha.K2, eIF2.alpha.K3, or eIF2.alpha.K4). Examples of
eIF2.alpha.K1 inhibitors include, without limitation, proteins,
polypeptides, peptides, antibodies, small molecules, and nucleic
acid molecules. In a particular embodiment, the eIF2.alpha.K1
inhibitor is a kinase domain inhibitor or heme binding domain
inhibitor. In another embodiment, the eIF2.alpha.K1 inhibitor is an
inhibitory nucleic acid molecule, such as an antisense, siRNA, or
shRNA molecule (or a nucleic acid molecule encoding the inhibitory
nucleic acid molecule). In a particular embodiment, the
eIF2.alpha.K1 inhibitor is a CRISPR based targeting of the
eIF2.alpha.K1 gene (e.g., with a guide RNA targeting the
eIF2.alpha.K1 gene). In a particular embodiment, the eIF2.alpha.K1
inhibitor is a small molecule.
[0021] A variety of eIF2.alpha.K1 inhibitors are known in the art.
As stated hereinabove, heme is an inhibitor of eIF2.alpha.K1
activity. Heme metabolites and heme synthesis intermediates have
also been shown to inhibit eIF2.alpha.K1 activity (Miksanova et al.
(2006) Biochem., 45:9894-9905). More specifically, Fe(III)-hemin,
Fe(II)-heme, protoporphyrin IX, bilirubin, biliverdin, and
uroporphyrin have been shown to inhibit eIF2.alpha.K1 activity
(Miksanova et al. (2006) Biochem., 45:9894-9905). Quercetin has
also been identified as an ATP-competitive kinase inhibitor of
eIF2.alpha.K1 (Srivastava et al. (1986) Prog. Clin. Biol. Res.,
213:315-8; Kanelakis et al. (2009) Adv. Hematol., 251915). A series
of aminopyrazoloindanes have also been shown to be effective
inhibitors of eIF2.alpha.K1 (Rosen et al. (2009) Bioorg. Med. Chem.
Lett., 19(23):6548-51; incorporated herein by reference for the
aminopyrazoloindane inhibitors of eIF2.alpha.K1, particularly those
in Tables 2, 3, and 4). In a particular embodiment, the
eIF2.alpha.K1 inhibitor is a synthetic or non-natural compound.
[0022] Clustered, regularly interspaced, short palindromic repeat
(CRISPR)/Cas9 (e.g., from Streptococcus pyogenes) technology and
gene editing are well known in the art (see, e.g., Sander et al.
(2014) Nature Biotech., 32:347-355; Jinek et al. (2012) Science,
337:816-821; Cong et al. (2013) Science 339:819-823; Ran et al.
(2013) Nature Protocols 8:2281-2308; Mali et al. (2013) Science
339:823-826; addgene.org/crispr/guide/). The RNA-guided CRISPR/Cas9
system involves expressing Cas9 along with a guide RNA molecule
(gRNA). When coexpressed, gRNAs bind and recruit Cas9 to a specific
genomic target sequence where it mediates a double strand DNA
(dsDNA) break. The binding specificity of the CRISPR/Cas9 complex
depends on two different elements. First, the binding
complementarity between the targeted genomic DNA (genDNA) sequence
and the complementary recognition sequence of the gRNA (e.g.,
.about.18-22 nucleotides, particularly about 20 nucleotides).
Second, the presence of a protospacer-adjacent motif (PAM)
juxtaposed to the genDNA/gRNA complementary region (Jinek et al.
(2012) Science 337:816-821; Hsu et al. (2013) Nat. Biotech.,
31:827-832; Sternberg et al. (2014) Nature 507:62-67). The PAM
motif for S. Pyogenes Cas9 has been fully characterized, and is NGG
or NAG (Jinek et al. (2012) Science 337:816-821; Hsu et al. (2013)
Nat. Biotech., 31:827-832). Other PAMs of other Cas9 are also known
(see, e.g., addgene.org/crispr/guide/#pam-table). Guidelines and
computer-assisted methods for generating gRNAs are available (see,
e.g, CRISPR Design Tool (crispr.mit.edu/); Hsu et al. (2013) Nat.
Biotechnol. 31:827-832; addgene.org/CRISPR; and CRISPR gRNA Design
tool--DNA2.0 (dna20.com/eCommerce/startCas9)). Typically, the PAM
sequence is 3' of the DNA target sequence in the genomic
sequence.
[0023] In a particular embodiment, the method comprises
administering at least one Cas9 (e.g., a nucleic acid molecule
encoding Cas9) and at least one gRNA (e.g., a nucleic acid molecule
encoding the gRNA) to the cell or subject. In a particular
embodiment, the Cas9 is S. pyogenes Cas9. In a particular
embodiment, the targeted PAM is in the 5'UTR, promoter, or first
intron. When present, a second gRNA is provided which targets
anywhere from the 5'UTR to the 3'UTR of the gene, particularly
within the first intron. The nucleic acids of the instant invention
may be administered consecutively (before or after) and/or at the
same time (concurrently). The nucleic acid molecules may be
administered in the same composition or in separate compositions.
In a particular embodiment, the nucleic acid molecules are
delivered in a single vector (e.g., a viral vector).
[0024] In a particular embodiment, the nucleic acid molecules of
the instant invention are delivered (e.g., via infection,
transfection, electroporation, etc.) and expressed in cells via a
vector (e.g., a plasmid), particularly a viral vector. The
expression vectors of the instant invention may employ a strong
promoter, a constitutive promoter, and/or a regulated promoter. In
a particular embodiment, the nucleic acid molecules are expressed
transiently. Examples of promoters are well known in the art and
include, but are not limited to, RNA polymerase II promoters, the
T7 RNA polymerase promoter, and RNA polymerase III promoters (e.g.,
U6 and H1; see, e.g., Myslinski et al. (2001) Nucl. Acids Res.,
29:2502-09). Examples of expression vectors for expressing the
molecules of the invention include, without limitation, plasmids
and viral vectors (e.g., adeno-associated viruses (AAVs),
adenoviruses, retroviruses, and lentiviruses).
[0025] In a particular embodiment, the guide RNA of the instant
invention may comprise separate nucleic acid molecules. For
example, one RNA may specifically hybridize to a target sequence
(crRNA) and another RNA (trans-activating crRNA (tracrRNA))
specifically hybridizes with the crRNA. In a particular embodiment,
the guide RNA is a single molecule (sgRNA) which comprises a
sequence which specifically hybridizes with a target sequence
(crRNA; complementary sequence) and a sequence recognized by Cas9
(e.g., a tracrRNA sequence; scaffold sequence). Examples of gRNA
scaffold sequences are well known in the art (e.g., 5'-GUUUUAGAGC
UAGAAAUAGC AAGUUAAAAU AAGGCUAGUC CGUUAUCAAC UUGAAAAAGU GGCACCGAGU
CGGUGCUUUU; SEQ ID NO: 18). As used herein, the term "specifically
hybridizes" does not mean that the nucleic acid molecule needs to
be 100% complementary to the target sequence. Rather, the sequence
may be at least 80%, 85%, 90%, 95%, 97%, 99%, or 100% complementary
to the target sequences (e.g., the complementary between the gRNA
and the genomic DNA). The greater the complementarity reduces the
likelihood of undesired cleavage events at other sites of the
genome. In a particular embodiment, the region of complementarity
(e.g., between a guide RNA and a target sequence) is at least about
10, at least about 12, at least about 15, at least about 17, at
least about 20, at least about 25, at least about 30, at least
about 35, or more nucleotides. In a particular embodiment, the
region of complementarity (e.g., between a guide RNA and a target
sequence) is about 15 to about 25 nucleotides, about 15 to about 23
nucleotides, about 16 to about 23 nucleotides, about 17 to about 21
nucleotides, about 18 to about 22 nucleotides, or about 20
nucleotides. In a particular embodiment, the guide RNA targets a
sequence or comprises a sequence (inclusive of RNA version of DNA
molecules) as set forth in the Example provided herein (see, e.g.,
the sequences provided in Table 1 (e.g., gRNA1, gRNA2, gRNA3,
gRNA4, gRNA5, or gRNA6, particularly gRNA3 or gRNA5)). In a
particular embodiment, the guide RNA targets a sequence or
comprises a sequence which has at least 80%, 85%, 90%, 95%, 97%,
99%, or 100% homology or identity to a sequence set forth in the
Example (e.g., Table 1 provided herein; (e.g., gRNA1, gRNA2, gRNA3,
gRNA4, gRNA5, or gRNA6, particularly gRNA3 or gRNA5)). The
sequences may be extended or shortened by 1, 2, 3, 4, or 5
nucleotides at the end of the sequence opposite from the PAM (e.g.,
at the 5' end). When the sequence is extended the added nucleotides
should correspond to the genomic sequence.
[0026] The above methods also encompass ex vivo methods. For
example, the methods of the instant invention can comprise
isolating hematopoietic cells (e.g., erythroid precursor cells) or
erythroid cells from a subject, delivering at least one
eIF2.alpha.K1 inhibitor to the cells, and administering the treated
cells to the subject. The isolated cells may also be treated with
other reagents in vitro, such as at least one fetal hemoglobin
inducer, prior to administration to the subject. In a particular
embodiment, the cells are not fully mature, anucleated
erythrocytes.
[0027] The methods of the instant invention may further comprise
monitoring the disease or disorder in the subject after
administration of the composition(s) of the instant invention to
monitor the efficacy of the method. For example, the subject may be
monitored for characteristics of low hemoglobin or a
hemoglobinopathy.
[0028] When an inhibitory nucleic acid molecule is delivered to a
cell or subject, the inhibitory nucleic acid molecule may be
administered directly or an expression vector may be used. In a
particular embodiment, the inhibitory nucleic acid molecules are
delivered (e.g., via infection, transfection, electroporation,
etc.) and expressed in cells via a vector (e.g., a plasmid),
particularly a viral vector. The expression vectors of the instant
invention may employ a strong promoter, a constitutive promoter,
and/or a regulated promoter. In a particular embodiment, the
inhibitory nucleic acid molecules are expressed transiently. In a
particular embodiment, the promoter is cell-type specific (e.g.,
erythroid cells). Examples of promoters are well known in the art
and include, but are not limited to, RNA polymerase II promoters,
the T7 RNA polymerase promoter, and RNA polymerase III promoters
(e.g., U6 and H1; see, e.g., Myslinski et al. (2001) Nucl. Acids
Res., 29:2502-09). Examples of expression vectors for expressing
the molecules of the invention include, without limitation,
plasmids and viral vectors (e.g., adeno-associated viruses (AAVs),
adenoviruses, retroviruses, and lentiviruses).
[0029] As explained hereinabove, the compositions of the instant
invention are useful for increasing hemoglobin production and for
treating hemoglobinopathies and thalassemias. A therapeutically
effective amount of the composition may be administered to a
subject in need thereof. The dosages, methods, and times of
administration are readily determinable by persons skilled in the
art, given the teachings provided herein.
[0030] The components as described herein will generally be
administered to a patient as a pharmaceutical preparation. The term
"patient" or "subject" as used herein refers to human or animal
subjects. The components of the instant invention may be employed
therapeutically, under the guidance of a physician for the
treatment of the indicated disease or disorder.
[0031] The pharmaceutical preparation comprising the components of
the invention may be conveniently formulated for administration
with an acceptable medium (e.g., pharmaceutically acceptable
carrier) such as water, buffered saline, ethanol, polyol (for
example, glycerol, propylene glycol, liquid polyethylene glycol and
the like), dimethyl sulfoxide (DMSO), oils, detergents, suspending
agents or suitable mixtures thereof. The concentration of the
agents in the chosen medium may be varied and the medium may be
chosen based on the desired route of administration of the
pharmaceutical preparation. Except insofar as any conventional
media or agent is incompatible with the agents to be administered,
its use in the pharmaceutical preparation is contemplated.
[0032] The compositions of the present invention can be
administered by any suitable route, for example, by injection
(e.g., for local (direct) or systemic administration), oral,
pulmonary, topical, nasal or other modes of administration. The
composition may be administered by any suitable means, including
parenteral, intramuscular, intravenous, intraarterial,
intraperitoneal, subcutaneous, topical, inhalatory, transdermal,
intrapulmonary, intraareterial, intrarectal, intramuscular, and
intranasal administration. In a particular embodiment, the
composition is administered directly to the blood stream (e.g.,
intravenously). In general, the pharmaceutically acceptable carrier
of the composition is selected from the group of diluents,
preservatives, solubilizers, emulsifiers, adjuvants and/or
carriers. The compositions can include diluents of various buffer
content (e.g., Tris HCl, acetate, phosphate), pH and ionic
strength; and additives such as detergents and solubilizing agents
(e.g., polysorbate 80), anti oxidants (e.g., ascorbic acid, sodium
metabisulfite), preservatives (e.g., Thimersol, benzyl alcohol) and
bulking substances (e.g., lactose, mannitol). The compositions can
also be incorporated into particulate preparations of polymeric
compounds such as polyesters, polyamino acids, hydrogels,
polylactide/glycolide copolymers, ethylenevinylacetate copolymers,
polylactic acid, polyglycolic acid, etc., or into liposomes. Such
compositions may influence the physical state, stability, rate of
in vivo release, and rate of in vivo clearance of components of a
pharmaceutical composition of the present invention. See, e.g.,
Remington: The Science and Practice of Pharmacy, 21st edition,
Philadelphia, Pa. Lippincott Williams & Wilkins. The
pharmaceutical composition of the present invention can be
prepared, for example, in liquid form, or can be in dried powder
form (e.g., lyophilized for later reconstitution).
[0033] As used herein, "pharmaceutically acceptable carrier"
includes any and all solvents, dispersion media and the like which
may be appropriate for the desired route of administration of the
pharmaceutical preparation, as exemplified in the preceding
paragraph. The use of such media for pharmaceutically active
substances is known in the art. Except insofar as any conventional
media or agent is incompatible with the molecules to be
administered, its use in the pharmaceutical preparation is
contemplated.
[0034] Pharmaceutical compositions containing a compound of the
present invention as the active ingredient in intimate admixture
with a pharmaceutical carrier can be prepared according to
conventional pharmaceutical compounding techniques. The carrier may
take a wide variety of forms depending on the form of preparation
desired for administration, e.g., intravenous. Injectable
suspensions may be prepared, in which case appropriate liquid
carriers, suspending agents and the like may be employed.
Pharmaceutical preparations for injection are known in the art. If
injection is selected as a method for administering the therapy,
steps should be taken to ensure that sufficient amounts of the
molecules reach their target cells to exert a biological
effect.
[0035] A pharmaceutical preparation of the invention may be
formulated in dosage unit form for ease of administration and
uniformity of dosage. Dosage unit form, as used herein, refers to a
physically discrete unit of the pharmaceutical preparation
appropriate for the patient undergoing treatment. Each dosage
should contain a quantity of active ingredient calculated to
produce the desired effect in association with the selected
pharmaceutical carrier. Procedures for determining the appropriate
dosage unit are well known to those skilled in the art. Dosage
units may be proportionately increased or decreased based on the
weight of the patient. Appropriate concentrations for alleviation
of a particular pathological condition may be determined by dosage
concentration curve calculations, as known in the art. The
appropriate dosage unit for the administration of the molecules of
the instant invention may be determined by evaluating the toxicity
of the molecules in animal models. Various concentrations of
pharmaceutical preparations may be administered to mice with
transplanted human tumors, and the minimal and maximal dosages may
be determined based on the results of significant reduction of
tumor size and side effects as a result of the treatment.
Appropriate dosage unit may also be determined by assessing the
efficacy of the treatment in combination with other standard
therapies.
[0036] The pharmaceutical preparation comprising the molecules of
the instant invention may be administered at appropriate intervals,
for example, at least twice a day or more until the pathological
symptoms are reduced or alleviated, after which the dosage may be
reduced to a maintenance level. The appropriate interval in a
particular case would normally depend on the condition of the
patient.
Definitions
[0037] The singular forms "a," "an," and "the" include plural
referents unless the context clearly dictates otherwise.
[0038] The terms "isolated" is not meant to exclude artificial or
synthetic mixtures with other compounds or materials, or the
presence of impurities that do not interfere with the fundamental
activity, and that may be present, for example, due to incomplete
purification, or the addition of stabilizers.
[0039] "Pharmaceutically acceptable" indicates approval by a
regulatory agency of the Federal or a state government or listed in
the U.S. Pharmacopeia or other generally recognized pharmacopeia
for use in animals, and more particularly in humans.
[0040] A "carrier" refers to, for example, a diluent, adjuvant,
preservative (e.g., Thimersol, benzyl alcohol), anti-oxidant (e.g.,
ascorbic acid, sodium metabisulfite), solubilizer (e.g.,
polysorbate 80), emulsifier, buffer (e.g., Tris HCl, acetate,
phosphate), antimicrobial, bulking substance (e.g., lactose,
mannitol), excipient, auxilliary agent or vehicle with which an
active agent of the present invention is administered.
Pharmaceutically acceptable carriers can be sterile liquids, such
as water and oils, including those of petroleum, animal, vegetable
or synthetic origin. Water or aqueous saline solutions and aqueous
dextrose and glycerol solutions are preferably employed as
carriers, particularly for injectable solutions. Suitable
pharmaceutical carriers are described in Remington: The Science and
Practice of Pharmacy, (Lippincott, Williams and Wilkins); Liberman,
et al., Eds., Pharmaceutical Dosage Forms, Marcel Decker, New York,
N.Y.; and Rowe, et al., Eds., Handbook of Pharmaceutical
Excipients, Pharmaceutical Pr.
[0041] The term "treat" as used herein refers to any type of
treatment that imparts a benefit to a patient suffering from an
injury, including improvement in the condition of the patient
(e.g., in one or more symptoms), delay in the progression of the
condition, etc.
[0042] As used herein, the term "prevent" refers to the
prophylactic treatment of a subject who is at risk of developing a
condition and/or sustaining an injury, resulting in a decrease in
the probability that the subject will develop conditions associated
with the hemoglobinopathy or thalassemia.
[0043] A "therapeutically effective amount" of a compound or a
pharmaceutical composition refers to an amount effective to
prevent, inhibit, or treat a particular injury and/or the symptoms
thereof. For example, "therapeutically effective amount" may refer
to an amount sufficient to modulate the pathology associated with a
hemoglobinopathy or thalassemia.
[0044] As used herein, the term "subject" refers to an animal,
particularly a mammal, particularly a human.
[0045] A "vector" is a genetic element, such as a plasmid, cosmid,
bacmid, phage or virus, to which another genetic sequence or
element (either DNA or RNA) may be attached so as to bring about
the replication and/or expression of the attached sequence or
element. A vector may be either RNA or DNA and may be single or
double stranded. A vector may comprise expression operons or
elements such as, without limitation, transcriptional and
translational control sequences, such as promoters, enhancers,
translational start signals, polyadenylation signals, terminators,
and the like, and which facilitate the expression of a
polynucleotide or a polypeptide coding sequence in a host cell or
organism.
[0046] As used herein, the term "small molecule" refers to a
substance or compound that has a relatively low molecular weight
(e.g., less than 4,000, less than 2,000, particularly less than 1
kDa or 800 Da). Typically, small molecules are organic, but are not
proteins, polypeptides, amino acids, or nucleic acids.
[0047] The phrase "small, interfering RNA (siRNA)" refers to a
short (typically less than 30 nucleotides long, particularly 12-30
or 20-25 nucleotides in length) double stranded RNA molecule.
Typically, the siRNA modulates the expression of a gene to which
the siRNA is targeted. Methods of identifying and synthesizing
siRNA molecules are known in the art (see, e.g., Ausubel et al.,
Current Protocols in Molecular Biology, John Wiley and Sons, Inc).
Short hairpin RNA molecules (shRNA) typically consist of short
complementary sequences separated by a small loop sequence wherein
one of the sequences is complimentary to the gene target. shRNA
molecules are typically processed into an siRNA within the cell by
endonucleases. Exemplary modifications to siRNA molecules are
provided in U.S. Application Publication No. 20050032733. For
example, siRNA and shRNA molecules may be modified with nuclease
resistant modifications (e.g., phosphorothioates, locked nucleic
acids (LNA), 2'-O-methyl modifications, or morpholino linkages).
Expression vectors for the expression of siRNA or shRNA molecules
may employ a strong promoter which may be constitutive or
regulated. Such promoters are well known in the art and include,
but are not limited to, RNA polymerase II promoters, the T7 RNA
polymerase promoter, and the RNA polymerase III promoters U6 and H1
(see, e.g., Myslinski et al. (2001) Nucl. Acids Res., 29:2502
09).
[0048] "Antisense nucleic acid molecules" or "antisense
oligonucleotides" include nucleic acid molecules (e.g., single
stranded molecules) which are targeted (complementary) to a chosen
sequence (e.g., to translation initiation sites and/or splice
sites) to inhibit the expression of a protein of interest. Such
antisense molecules are typically between about 15 and about 50
nucleotides in length, more particularly between about 15 and about
30 nucleotides, and often span the translational start site of mRNA
molecules. Antisense constructs may also be generated which contain
the entire sequence of the target nucleic acid molecule in reverse
orientation. Antisense oligonucleotides targeted to any known
nucleotide sequence can be prepared by oligonucleotide synthesis
according to standard methods. Antisense oligonucleotides may be
modified as described above to comprise nuclease resistant
modifications.
[0049] The following example is provided to illustrate various
embodiments of the present invention. It is not intended to limit
the invention in any way.
Example
[0050] A CRISPR screening strategy (Shi et al. (2015) Nat.
Biotechnol., 33(6):661-7) was employed to identify regulators of
fetal globin expression. Clustered, regularly interspaced, short
palindromic repeat (CRISPR)/Cas9 technology is well known in the
art (see, e.g., Sander et al. (2014) Nature Biotech., 32:347-355;
Jinek et al. (2012) Science, 337:816-821; Cong et al. (2013)
Science 339:819-823; Ran et al. (2013) Nature Protocols
8:2281-2308; Mali et al. (2013) Science 339:823-826). Cas9
possesses two nuclease domains, a RuvC-like nuclease domain and a
HNH-like nuclease domain, and is responsible for the destruction of
the target DNA (Jinek et al. (2012) Science, 337:816-821;
Sapranauskas et al. (2011) Nucleic Acids Res. 39:9275-9282). The
two nucleases generate double-stranded breaks. The double-stranded
endonuclease activity of Cas9 requires a target sequence (e.g.,
.about.20 nucleotides, see above) and a short conserved sequence
(.about.2-5 nucleotides; e.g., 3 nucleotides) known as
protospacer-associated motif (PAM), which follows immediately
3'--of the CRISPR RNA (crRNA) complementary sequence (Jinek et al.
(2012) Science, 337:816-821; Nishimasu et al. (2014) Cell
156(5):935-49; Swarts et al. (2012) PLoS One, 7:e35888; Sternberg
et al. (2014) Nature 507(7490):62-7). The double strand break can
be repaired by non-homologous end joining (NHEJ) pathway yielding
an insertion and/or deletion or, in the presence of a donor
template, by homology-directed repair (HDR) pathway for replacement
mutations (Overballe-Petersen et al. (2013) Proc. Natl. Acad. Sci.
U.S.A. 110:19860-19865; Gong et al. (2005) Nat. Struct. Mol. Biol.
12:304-312). The RNA-guided CRISPR/Cas9 system involves expressing
Cas9 along with a guide RNA molecule (gRNA). When coexpressed,
gRNAs bind and recruit Cas9 to a specific genomic target sequence
where it mediates a double strand DNA (dsDNA) break and activates
the dsDNA break repair machinery. Specific DNA fragments can be
deleted when two gRNA/Cas9 complexes generate dsDNA breaks at
relative proximity, and the genomic DNA is repaired by
nonhomologous end joining.
[0051] Briefly, cells from a human umbilical cord blood-derived
erythroid cells progenitor cell line (HUDEP2) expressing Cas9 were
transduced with a lentiviral library of guideRNAs (gRNAs). The
library targeted over 500 different kinase domains with 6 guides
per domain (i.e., over 3000 independent guides). Cells were then
stained with fetal hemoglobin antibody and positive cells were
sorted. The results showed that all 6 guides for eIF2.alpha.K1
resulted in enriched fetal globin expression.
[0052] These results were confirmed in HUDEP-2 cells by
individually expressing each one of the six gRNAs targeting
eIF2.alpha.K1, a gRNA targeting BCL11A exon 2 (positive control;
BCL11A is a repressor of HbF expression), one of two gRNAs
targeting death associated protein kinase 2 (DAPK2), one of two
gRNAs targeting transforming growth factor beta receptor 2
(TGFBR2), or a negative control gRNA.
[0053] Table 1 provides sequences of the gRNAs
fluorescence-activated cell sorting (FACS) with an HbF antibody
shows that the eIF2.alpha.K1 gRNAs significantly increased
expression of HbF compared to the other gRNAs and negative controls
(FIG. 1A). A quantitative reverse transcription PCR (RT-qPCR)
analysis was also performed on the above cells to measure mRNA
levels. As seen in FIG. 1B, the eIF2.alpha.K1 gRNAs significantly
increased gamma globin RNA levels compared to the other gRNAs and
negative controls. FIG. 1C provides a representative FACS analysis
for sgRNA #5 showing increased HbF production. FIG. 1D provides a
graph of HbF flow cytometry of cells expressing indicated sgRNAs
over two experiments. Western blot analysis confirmed the decreased
expression of eIF2.alpha.K1 (HRI) and increased expression of
.gamma.-globin with sgRNA #3 and sgRNA #5.
TABLE-US-00001 TABLE 1 gRNA sequences. CRISPR sgRNA name Target
Sequence SEQ ID NO BCL11A Exon2 TGAACCAGACCACGGCCCGT 1 sgRNA
eIF2.alpha.K1 sgRNA1 TCTGGAAGTGCTCTCCGACC 2 eIF2.alpha.K1 sgRNA2
GTATCCACCTTTTCCTAAGA 3 eIF2.alpha.K1 sgRNA3 TTTTAGTTGCACCCTTAATC 4
eIF2.alpha.K1 sgRNA4 TTGTTGGCTATCACACCGCG 5 eIF2.alpha.K1 sgRNA5
ATAGTCGAGAGAAACAAGCG 6 eIF2.alpha.K1 sgRNA6 TGGTGAACTTGAGTCGACCC 7
Neg02 sgRNA GACCGGAACGATCTCGCGTA 8
[0054] In addition to the above, a whole proteome mass spectrometry
analysis of cells transduced with a gRNA targeting eIF2.alpha.K1
was performed. As seen in FIG. 2, fetal globin expression increased
more than two fold upon transduction with eIF2.alpha.K1 gRNA #3 or
gRNA #5.
[0055] Combination experiments with the immunomodulatory
pomalidomide, which is an HbF inducer, were also performed. If
eIF2.alpha.K1 inhibition works at least in part by altering protein
translation, additive effects may be observed when using compounds
that predominantly affect transcription of fetal globin genes.
Briefly, HUDEP-2 expressing Cas9 cells were transduced with
eIF2.alpha.K1 gRNA #3, eIF2.alpha.K1 gRNA #5, a gRNA targeting
BCL11A exon 2, or a negative control gRNA. The cells were then
incubated with or without pomalidomide. As seen in FIG. 3, the
combination of eIF2.alpha.K1 inhibition and pomalidomide greatly
increased the number of HbF positive cells. Indeed, the results
with pomalidomide in combination with eIF2.alpha.K1 inhibition were
very strong in three independent experiments, even stronger than
those observed with Bcl11A inhibition and pomalidomide. This
demonstrates that a compound that inhibits eIF2.alpha.K1 can be
combined with pomalidomide or other HbF inducers to achieve a
greater therapeutic index.
[0056] In additional experiments, eIF2.alpha.K1 was knocked down in
primary human erythroid cells using shRNAs, as editing was not
efficient enough in primary cells. Fetal hemoglobin was found to be
upregulated in a manner comparable to or exceeding that of Bcl11A
depletion (positive control). As seen in FIG. 4, two of the four
shRNA (#1 and #4) dramatically increased HbF production in human
primary cells. Table 2 provides the sequences of the shRNA
molecules.
TABLE-US-00002 TABLE 2 shRNA sequences. shRNA name Target Sequence
SEQ ID NO ZBTB7A shRNA2 AAGTCGATCTCGTACACGTTC 9 ZBTB7A shRNA4
AATAGGTTTGTGTCTCAGTGG 10 BCL11A shRNA1 AATCCATGAGTGTTCTGTGCG 11
BCL11A shRNA2 TAAACAATCGTCATCCTCTGG 12 eIF2.alpha.K1 shRNA1
TTAACACCACATTGCTCTCTG 13 eIF2.alpha.K1 shRNA2 ATTAAGTGAGTAATAGCTCTG
14 eIF2.alpha.K1 shRNA3 TAAACGTCTGGCAAAGTAGC 15 eIF2.alpha.K1
shRNA4 TAAACCTGTTAGAACTTCTGC 16 SC shRNA AAATTATTAGCGCTATCGCGC
17
[0057] FIG. 5A provides a representative HbF flow cytometry on day
14 of CD34+ erythroid differentiation. LRF shRNA and shRNA 1 and 2
increased HbF expression. Leukemia/lymphoma-related factor (LRF),
as with BCL11A, represses expression of fetal hemoglobin (Masuda et
al. (2016) Science 351(6270): 285-289). FIG. 5B provides a summary
of HbF flow cytometry experiments. FIG. 5C provides .gamma.-globin
mRNA as fraction of .gamma.-globin+.beta.-globin by RT-qPCR on day
12. FIG. 5D provides RNA-seq for CD34+ cells on day 12. FIG. 5E
shows an HPLC analysis of samples from cell expressing annotated
shRNAs, at day 14 of differentiation. FIG. 5F provides images of
sickle cell patient-derived erythroid cultures showing that
eIF2.alpha.K1 shRNA1 and eIF2.alpha.K1 shRN2 decreased the number
of sickle cells.
[0058] Further experiments demonstrate that HRI regulates BCL11A
protein levels. Western blot analyses were performed with indicated
antibodies (FIG. 6A) in cells uninfected, or infected with virus
expressing scrambled or HRI shRNAs on day 14 of differentiation.
BCL11A expression is decreased with HRI shRNAs. FIG. 6A also shows
BCL11A mRNA levels in HRI knock down cells as fraction of SC shRNA
expressing cells. FIG. 6B shows BCL11A cDNA rescue in HRI depleted
HUDEP2 clonal cell lines.
[0059] While certain of the preferred embodiments of the present
invention have been described and specifically exemplified above,
it is not intended that the invention be limited to such
embodiments. Various modifications may be made thereto without
departing from the scope and spirit of the present invention, as
set forth in the following claims.
Sequence CWU 1
1
20120DNAArtificial SequenceTarget Sequence 1tgaaccagac cacggcccgt
20220DNAArtificial SequenceTarget Sequence 2tctggaagtg ctctccgacc
20320DNAArtificial SequenceTarget Sequence 3gtatccacct tttcctaaga
20420DNAArtificial SequenceTarget Sequence 4ttttagttgc acccttaatc
20520DNAArtificial SequenceTarget Sequence 5ttgttggcta tcacaccgcg
20620DNAArtificial SequenceTarget Sequence 6atagtcgaga gaaacaagcg
20720DNAArtificial SequenceTarget Sequence 7tggtgaactt gagtcgaccc
20820DNAArtificial SequenceTarget Sequence 8gaccggaacg atctcgcgta
20921DNAArtificial SequenceTarget Sequence 9aagtcgatct cgtacacgtt c
211021DNAArtificial SequenceTarget Sequence 10aataggtttg tgtctcagtg
g 211121DNAArtificial SequenceTarget Sequence 11aatccatgag
tgttctgtgc g 211221DNAArtificial SequenceTarget Sequence
12taaacaatcg tcatcctctg g 211321DNAArtificial SequenceTarget
Sequence 13ttaacaccac attgctctct g 211421DNAArtificial
SequenceTarget Sequence 14attaagtgag taatagctct g
211520DNAArtificial SequenceTarget Sequence 15taaacgtctg gcaaagtagc
201621DNAArtificial SequenceTarget Sequence 16taaacctgtt agaacttctg
c 211721DNAArtificial SequenceTarget Sequence 17aaattattag
cgctatcgcg c 211880RNAArtificial SequenceScaffold Sequence
18guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc cguuaucaac uugaaaaagu
60ggcaccgagu cggugcuuuu 8019630PRTHomo Sapiens 19Met Gln Gly Gly
Asn Ser Gly Val Arg Lys Arg Glu Glu Glu Gly Asp1 5 10 15Gly Ala Gly
Ala Val Ala Ala Pro Pro Ala Ile Asp Phe Pro Ala Glu 20 25 30Gly Pro
Asp Pro Glu Tyr Asp Glu Ser Asp Val Pro Ala Glu Ile Gln 35 40 45Val
Leu Lys Glu Pro Leu Gln Gln Pro Thr Phe Pro Phe Ala Val Ala 50 55
60Asn Gln Leu Leu Leu Val Ser Leu Leu Glu His Leu Ser His Val His65
70 75 80Glu Pro Asn Pro Leu Arg Ser Arg Gln Val Phe Lys Leu Leu Cys
Gln 85 90 95Thr Phe Ile Lys Met Gly Leu Leu Ser Ser Phe Thr Cys Ser
Asp Glu 100 105 110Phe Ser Ser Leu Arg Leu His His Asn Arg Ala Ile
Thr His Leu Met 115 120 125Arg Ser Ala Lys Glu Arg Val Arg Gln Asp
Pro Cys Glu Asp Ile Ser 130 135 140Arg Ile Gln Lys Ile Arg Ser Arg
Glu Val Ala Leu Glu Ala Gln Thr145 150 155 160Ser Arg Tyr Leu Asn
Glu Phe Glu Glu Leu Ala Ile Leu Gly Lys Gly 165 170 175Gly Tyr Gly
Arg Val Tyr Lys Val Arg Asn Lys Leu Asp Gly Gln Tyr 180 185 190Tyr
Ala Ile Lys Lys Ile Leu Ile Lys Gly Ala Thr Lys Thr Val Cys 195 200
205Met Lys Val Leu Arg Glu Val Lys Val Leu Ala Gly Leu Gln His Pro
210 215 220Asn Ile Val Gly Tyr His Thr Ala Trp Ile Glu His Val His
Val Ile225 230 235 240Gln Pro Arg Ala Asp Arg Ala Ala Ile Glu Leu
Pro Ser Leu Glu Val 245 250 255Leu Ser Asp Gln Glu Glu Asp Arg Glu
Gln Cys Gly Val Lys Asn Asp 260 265 270Glu Ser Ser Ser Ser Ser Ile
Ile Phe Ala Glu Pro Thr Pro Glu Lys 275 280 285Glu Lys Arg Phe Gly
Glu Ser Asp Thr Glu Asn Gln Asn Asn Lys Ser 290 295 300Val Lys Tyr
Thr Thr Asn Leu Val Ile Arg Glu Ser Gly Glu Leu Glu305 310 315
320Ser Thr Leu Glu Leu Gln Glu Asn Gly Leu Ala Gly Leu Ser Ala Ser
325 330 335Ser Ile Val Glu Gln Gln Leu Pro Leu Arg Arg Asn Ser His
Leu Glu 340 345 350Glu Ser Phe Thr Ser Thr Glu Glu Ser Ser Glu Glu
Asn Val Asn Phe 355 360 365Leu Gly Gln Thr Glu Ala Gln Tyr His Leu
Met Leu His Ile Gln Met 370 375 380Gln Leu Cys Glu Leu Ser Leu Trp
Asp Trp Ile Val Glu Arg Asn Lys385 390 395 400Arg Gly Arg Glu Tyr
Val Asp Glu Ser Ala Cys Pro Tyr Val Met Ala 405 410 415Asn Val Ala
Thr Lys Ile Phe Gln Glu Leu Val Glu Gly Val Phe Tyr 420 425 430Ile
His Asn Met Gly Ile Val His Arg Asp Leu Lys Pro Arg Asn Ile 435 440
445Phe Leu His Gly Pro Asp Gln Gln Val Lys Ile Gly Asp Phe Gly Leu
450 455 460Ala Cys Thr Asp Ile Leu Gln Lys Asn Thr Asp Trp Thr Asn
Arg Asn465 470 475 480Gly Lys Arg Thr Pro Thr His Thr Ser Arg Val
Gly Thr Cys Leu Tyr 485 490 495Ala Ser Pro Glu Gln Leu Glu Gly Ser
Glu Tyr Asp Ala Lys Ser Asp 500 505 510Met Tyr Ser Leu Gly Val Val
Leu Leu Glu Leu Phe Gln Pro Phe Gly 515 520 525Thr Glu Met Glu Arg
Ala Glu Val Leu Thr Gly Leu Arg Thr Gly Gln 530 535 540Leu Pro Glu
Ser Leu Arg Lys Arg Cys Pro Val Gln Ala Lys Tyr Ile545 550 555
560Gln His Leu Thr Arg Arg Asn Ser Ser Gln Arg Pro Ser Ala Ile Gln
565 570 575Leu Leu Gln Ser Glu Leu Phe Gln Asn Ser Gly Asn Val Asn
Leu Thr 580 585 590Leu Gln Met Lys Ile Ile Glu Gln Glu Lys Glu Ile
Ala Glu Leu Lys 595 600 605Lys Gln Leu Asn Leu Leu Ser Gln Asp Lys
Gly Val Arg Asp Asp Gly 610 615 620Lys Asp Gly Gly Val Gly625
630204465DNAHomo Sapiens 20ccccagtgaa ggtgcgcgcg tggcggaggc
tggttttccg tctggtgagg ggttacttcc 60gggtcggacg gcgctagctg cagcatcgga
gtgtggcagt gctgggctgg ccggcgggct 120gggctgcggc ccgcgcgcgg
ccggcgatgc aggggggcaa ctccggggtc cgcaagcgcg 180aagaggaggg
cgacggggct ggggctgtgg ctgcgccgcc ggccatcgac tttcccgccg
240agggcccgga ccccgaatat gacgaatctg atgttccagc agaaatccag
gtgttaaaag 300aacccctaca acagccaacc ttcccttttg cagttgcaaa
ccaactcttg ctggtttctt 360tgctggagca cttgagccac gtgcatgaac
caaacccact tcgttcaaga caggtgttta 420agctactttg ccagacgttt
atcaaaatgg ggctgctgtc ttctttcact tgtagtgacg 480agtttagctc
attgagacta catcacaaca gagctattac tcacttaatg aggtctgcta
540aagagagagt tcgtcaggat ccttgtgagg atatttctcg tatccagaaa
atcagatcaa 600gggaagtagc cttggaagca caaacttcac gttacttaaa
tgaatttgaa gaacttgcca 660tcttaggaaa aggtggatac ggaagagtat
acaaggtcag gaataaatta gatggtcagt 720attatgcaat aaaaaaaatc
ctgattaagg gtgcaactaa aacagtttgc atgaaggtcc 780tacgggaagt
gaaggtgctg gcaggtcttc agcaccccaa tattgttggc tatcacaccg
840cgtggataga acatgttcat gtgattcagc cacgagcaga cagagctgcc
attgagttgc 900catctctgga agtgctctcc gaccaggaag aggacagaga
gcaatgtggt gttaaaaatg 960atgaaagtag cagctcatcc attatctttg
ctgagcccac cccagaaaaa gaaaaacgct 1020ttggagaatc tgacactgaa
aatcagaata acaagtcggt gaagtacacc accaatttag 1080tcataagaga
atctggtgaa cttgagtcga ccctggagct ccaggaaaat ggcttggctg
1140gtttgtctgc cagttcaatt gtggaacagc agctgccact caggcgtaat
tcccacctag 1200aggagagttt cacatccacc gaagaatctt ccgaagaaaa
tgtcaacttt ttgggtcaga 1260cagaggcaca gtaccacctg atgctgcaca
tccagatgca gctgtgtgag ctctcgctgt 1320gggattggat agtcgagaga
aacaagcggg gccgggagta tgtggacgag tctgcctgtc 1380cttatgttat
ggccaatgtt gcaacaaaaa tttttcaaga attggtagaa ggtgtgtttt
1440acatacataa catgggaatt gtgcaccgag atctgaagcc aagaaatatt
tttcttcatg 1500gccctgatca gcaagtaaaa ataggagact ttggtctggc
ctgcacagac atcctacaga 1560agaacacaga ctggaccaac agaaacggga
agagaacacc aacacatacg tccagagtgg 1620gtacttgtct gtacgcttca
cccgaacagt tggaaggatc tgagtatgat gccaagtcag 1680atatgtacag
cttgggtgtg gtcctgctag agctctttca gccgtttgga acagaaatgg
1740agcgagcaga agttctaaca ggtttaagaa ctggtcagtt gccggaatcc
ctccgtaaaa 1800ggtgtccagt gcaagccaag tatatccagc acttaacgag
aaggaactca tcgcagagac 1860catctgccat tcagctgctg cagagtgaac
ttttccaaaa ttctggaaat gttaacctca 1920ccctacagat gaagataata
gagcaagaaa aagaaattgc agaactaaag aagcagctaa 1980acctcctttc
tcaagacaaa ggggtgaggg atgacggaaa ggatgggggc gtgggatgaa
2040agtggactta acttttaagg tagttaactg gaatgtaaat ttttaatctt
tattagggta 2100tagttggtac aatgcttcgt tgtatttagt aagcctttac
aagacttgtt aaagatgtca 2160gagtgcccca agctgccgtt ccttcccttc
ctgccccaca agctcctttt cctgaatttc 2220ctacctaaat attaaccata
tgcctagtct ctgaaactaa aaacttggac ctcatcctca 2280attattttct
cctttcaact ctgttgaccc tctgtctggt cttcctctag aaggtaccgc
2340agaaattgat gtgtgctccc tgccctcgtc actgcccaag cccgggcctg
cacatactca 2400ctggactgtt ccagttttga cagctgccag tcttcctgcc
cctttcacac tgcagctgaa 2460gttcattacc tgaaggacgc ctcatcattt
cattccttgg ctccaaacct tctgctgcct 2520ctaagataaa agctcaactt
cttaacagtg tacagtgtgc aacttccaac ctttttatct 2580gttctctcca
ccttcagttt agcgtcattc caaaaccaca cccttgcaaa gctttgtact
2640ccgcacccca gatgatctcc aggcagctca gatctctttc ctgcctttgc
cctgcactgt 2700tccccggtac ttcctccttt attgtagcac tcagctcccc
agccaatctg tacatccctc 2760agaggcagcg atctgatgaa ttggtttttg
aatcccagaa agggtctgcc atggagttgg 2820cagtcatcac ggtagatggc
gtatgatttt gctgaatttt aaataaaatg aaaaccataa 2880attacatgat
gcttttattg acacttgaca actggcctaa ataaaaagac tctgactcta
2940atacaagtcc ccttactgat aataggcatg aaagagcacc attcttaaaa
tctaaaccct 3000ttaaaatcag ttacggcaat tcacttaagg agcttgaggg
ccgtgttaaa aggagccagg 3060ttttcacaag acctcatcca cctctgcaca
ttggctggca ctgtcacact gcagcctccg 3120atctgctgga gtacagacca
cagcaccacg tctgctacgg tgagttcatt cccagcgagc 3180caagggctct
tcccaagagc agagttcatg gagcggaaaa cagcggcttt ttctttactg
3240cttccctctt ttaactgaaa aatcgcaata tctacccagc tatctataag
ggttgcgttg 3300acagcattat gcttctggcc aaacagagag aacaagaaac
gtgcaatgtt cccttcgcct 3360tcgatggggc acatcgtctg gatgctgaat
ttcatctgcg tcttcggcac tgaaaagaaa 3420aaacacgcca ggtagcatca
ccagagcagc cctgagggaa gtactcgcac agtggggaag 3480gggacagtca
ccaaacacca cagactgatg acatgggaaa gggtgttttc aagaagaaca
3540tcacattttc agtctgtctc taagatgctt actactgaaa agggtttaag
tgcaaactat 3600gttaaaaaag taaccaaaga gtcattcctg gtatttgatt
atgacacttc tgaagctaga 3660agggacctca gaccacctgc tccaatccct
gctgcctaag gttttggggg ctgagggctg 3720gaaacccagg agctgcatct
cccaggctct tggcctcata atcgccttct gtgagactga 3780gggcaattaa
gatatgaatg acatgaactc gcttatctcc atatcaacat aaaccagaag
3840aggcatacat ctgtgggata gtcaatacat aacatcctat ctttatatgg
ccatatgaga 3900agataacttc agtgtcccta tgatggtaag ttcttgaatg
tgagggcaag atggctgtga 3960tagccatgac tccccgcttt gcctggttgg
acagaacccc cttccccaaa tgctcacagt 4020cccccccttg agctgtgtct
cactcagaga ccctaaattt tgcagacgaa caagggcatc 4080tttgcgttgc
ttcatctctg ctcctcctct agaactatcc cacaggcttt ctagacttag
4140aatatctgac ctgatgcaaa tcgctatgtg gccagtatgc cacagaatgt
cctaaaccct 4200tgctgcctct tatcaaaacc atgttgcaca tctcatcata
tagtacagct aacccttgaa 4260cagtgggggg gttagggaca ctgacccccg
actgctgtgc agtcgaaaat ccacataaaa 4320cttctgactc cttcaaaact
taagcactaa tagcctacta ttcatgggaa gccttaccca 4380tcacagttga
ttaacacgtt ttgtatggtc tgtatattat atgctatatt cttacaataa
4440acttgagaaa atgttaagaa aataa 4465
* * * * *