NZ726792B2 - Compositions and methods to treating hemoglobinopathies - Google Patents
Compositions and methods to treating hemoglobinopathies Download PDFInfo
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- NZ726792B2 NZ726792B2 NZ726792A NZ72679215A NZ726792B2 NZ 726792 B2 NZ726792 B2 NZ 726792B2 NZ 726792 A NZ726792 A NZ 726792A NZ 72679215 A NZ72679215 A NZ 72679215A NZ 726792 B2 NZ726792 B2 NZ 726792B2
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Abstract
Embodiment herein provide specially designed synthetic BCL11A-targeting microRNAs for RNA polymerase II expression, and methods o use to treat hemoglobinopathies such as sickle cell disease or thalassemia by increasing the expression levels of fetal hemoglobin levels. In particular illustrative embodiment, the present invention provides, in part, improved compositions and methods for achieving gene therapy in hematopoietic cells and hematopoietic precursor cells, including erythrocytes, erythroid progenitors, and embryonic stem cells. The invention further provides improved gene therapy methods for treating hematoppictic-related disorders. diment, the present invention provides, in part, improved compositions and methods for achieving gene therapy in hematopoietic cells and hematopoietic precursor cells, including erythrocytes, erythroid progenitors, and embryonic stem cells. The invention further provides improved gene therapy methods for treating hematoppictic-related disorders.
Description
COMPOSITIONS AND METHODS TO TREATING OBINOPATHIES
CROSS REFERENCE TO RELATED ATION
This application is an International Application which claims the benefit under 35
U.S.C. § 119(e) of U.S. Provisional Application No. 61/984,247, filed on April 25, 2014, and
U.S. Provisional Application No. 62/066,783, filed on October 21, 2014 the contents of each
application are incorporated herein by nce in its entirety.
SEQUENCE LISTING
The t application contains a Sequence Listing which has been submitted
electronically in ASCII format and is hereby incorporated by nce in its entirety. Said
ASCII copy, created on May 7, 2015, is named 701039PCT_SL.txt and is 81,264 bytes
in size.
GOVERNMENT SUPPORT
This invention was made with Government support under Grant No.:
5U01HL117720-03 awarded by the National Institutes of Health. The Government has certain
rights in the invention.
TECHNICAL FIELD
Embodiments disclosed herein relate to compositions and methods for the
treatment of hemoglobinopathies. More particularly, the embodiments relate to compositions
and methods of increasing fetal hemoglobin in a cell by ive knockdown of the endogenous
BCL11A.
BACKGROUND
Hemoglobinopathies, including sickle cell disease/anemia (SCD) and thalassemia
(THAL), are the most prevalent inherited monogenic disorders in the human. Approximately
% of the world’s population carries a globin gene on. The World Health Organization
tes that each year about 300,000 infants are born with major hemoglobin disorders. SCD
has segregated to populations from sub-Saharan Africa, India, Saudi Arabia, and Mediterranean
countries, where up to 2% of all children are born with the condition, due to the survival
advantage against malarial transmission conferred by a heterozygous sickle in (βS)
mutation (WHO Report on Sickle-cell anaemia - A59.9. Fifty-ninth World Health ly –
Provisional agenda item 114: United Nations; 2006:1-5). Due to ic and/or recent
migration, increasing numbers of patient populations can now be found in developed countries,
and public health ations of SCD are significant (Kauf et al., American Journal of
Hematology. 2009;84:323-327; Amendah et al., American l of Preventive Medicine.
2010;38:S550-556). In the United States of America, median survival of patients having a
hemoglobinopathy was estimated in 1994 to be 42 years for men and 48 years for women (Platt
et al., New England Journal of ne. 1994; 330:1639-1644). At a molecular level, SCD was
the first disease to be linked to a molecular alteration (Pauling et al., Science. 1949;110:543-
548). A single tide mutation results in glutamic acid to valine substitution by at position 6
of the β-globin protein. This cation s in the polymerization of the molecule in
deoxygenated ions, and subsequent “sickling” of the erythrocyte ultimately leading to
anemia by sis and acute and chronic vaso-occlusive and ischemic complications ing
multiple organs, including kidney, brain, lung, and others). Although preventive measures
(including the chemoprophylactic agent hydroxyurea) have led to moderate reduction in the
burden of selected patient groups, at present, the only available curative therapy for SCD is
allogeneic hematopoietic stem cell transplantation (HSCT) (Hsieh et al., New England Journal
of Medicine. 2009;361:2309-2317; Hsieh et al., Blood; Electronic pre-publication June 31,
2011). HSCT has unetly been associated in the SCD and THAL setting wmith significant
mortality and morbidity, which is due in part to pre-HSCT transfusion-related iron overload,
graft-versus-host disease, and high doses of chemotherapy/radiation required for pre-transplant
conditioning of the host, among others.
New molecular therapies are being developed. For example, U.S. Patent No.:
8,383,604 describes that the BCL11A as a key regulator of the globin genes during
development. In particular, BCL11A promotes the transitional switch from the expression of
fetal obin genes to the sion of adult hemoglobin genes during fetal development.
Supression of BCL11A s this transitional switch and maintains a significantly higher
expression of the fetal obin genes post fetal development. The higher amount of fetal
hemoglobin genes expressed ameliorates the symptoms associated with various
hemoglobinopathies.
SUMMARY
In particular illustrative embodiments, disclosed are, in part, improved
compositions and methods for ing gene therapy in hematopoietic cells and hematopoietic
precursor cells, including erythrocytes, erythroid progenitors, and embryonic stem cells. Also
described are improved gene therapy methods for treating hematopoietic-related disorders.
The goal is to efficiently knock-down BCL11A in cells derived from transduced,
engraftable hematopoietic stem cells. Success at induction of γ-globin and thus simultaneous
increase in HbF and reduction in mutant HbS s on the quantitative reduction of BCL11A
transcript and n. The inventors have embedded a BCL11A shRNA in a mir223 loop. This
approach allows the BCL11A shRNA to be transcribed via rase II (PolII) promoters
instead of the polymerase III promoters. This allows exploitation of the microRNA-biogenesis
pathway to generate siRNAs that target BCL11A sion in engraftable HSCs. Lentiviral
transgenes are engineered to s shRNAs that mimic primary NAs (pri-miRNAs)
and are sequentially processed by the endogenous Microprocessor and Dicer complexes to
generate small interfering RNAs (siRNAs) with sequence complementarity to the BCL11A
messenger RNA (mRNA).
In a first aspect the present invention provides a synthetic BCL11A microRNA
comprising;
a) a first BCL11A segment, a loop segment; and
b) a second BCL11A t arranged in tandem in a 5' to 3' direction,
n the loop segment is between and directly linked to the first and second BCL11A
segments, and
wherein the second BCL11A segment is complementary to the first BCL11A segment so
that the first and second BCL11A segments base pair to form a hairpin loop with the loop
segment forming the loop portion of the hairpin loop thus formed; and the first BCL11A
segment starts with a -GCGC- at the 5' end and the second BCL11A segment ends with a -
GCGC- at the 3' end.
In a second aspect the present invention es an ed nucleic acid molecule
comprising the nucleotide sequence set forth in any one of SEQ ID NOS: 34, 35, 36, 37, 38, 39,
40, 41 and 42.
In a third aspect the present invention provides a vector comprising the isolated nucleic
acid molecule of the second aspect.
In a fourth aspect the present invention provides an ex vivo host cell comprising the
vector of the third aspect, wherein the cell is a hematopoietic stem cell or its progeny.
In a fifth aspect the present invention provides a bacterium comprising the isolated
nucleic acid molecule of the second aspect.
In a sixth aspect the present invention provides a composition comprising a tic
BCL11A microRNA of the first aspect; an isolated nucleic acid molecule of the second aspect, a
vector of the third aspect, or a host cell of the fourth aspect.
In a seventh aspect the present invention provides a use of a composition of the sixth
aspect in the manufacture of a medicament for the treatment or for reducing a risk of developing,
a hemoglobinopathy in a subject.
In an eighth aspect the t invention provides a use of a composition of the sixth
aspect in the manufacture of a medicament for increasing the fetal obin levels expressed
by a cell.
Also described are itions and methods that efficiently down
BCL11A in cells derived from transduced, engraftable hematopoietic stem cells. In one
embodiment, a quantitative ion of BCL11A transcript and protein induces γ-globin
production, and thus simultaneous increase in HbF and reduction in mutant HbS.In a particular
embodiment, a BCL11A shRNA is embedded in a mir223 loop. In particular embodiments, a
lentivirus is engineered to express shRNAs that mimic pri-miRNAs that are sequentially
sed by the endogenous Microprocessor and Dicer complexes to generate siRNAs with
sequence complementarity to the BCL11A mRNA.
Accordingly, in various illustrative embodiments, the present specification
describes, in part, a synthetic BCL11A microRNA comprising a first BCL11A segment, a loop
segment, and a second BCL11A segment arranged in tandem in a 5' to 3' direction, wherein the
loop segment is between and directly linked to the first and second BCL11A segments, and
n the second BCL11A segment is complementary to the first BCL11A segment such that
the first and second BCL11A segments base pair to form a hairpin loop with the loop segment
forming the loop portion of the hairpin loop thus formed.
In one embodiment of any one of the synthetic BCL11A microRNA described
herein, the first and second BCL11A segments are about 18 to 25 nucleotides long. The first
BCL11A t is derived from a BCL11A ce and gives rise to the passenger strand
during shRNA processing to a duplex siRNA and the second BCL11A t is
complementary to first BCL11A segment, wherein the second BCL11A segment gives rise to
the guide strand that is incorporated into the RNA Interference Specificity Complex (RISC) for
RNA interference or BCL11A gene silencing.
In one embodiment of any one of the synthetic BCL11A microRNA described
herein, the first and second BCL11A segments contain sequences that are derived from BCL11A
mRNA sequence.
In one embodiment of any one of the synthetic BCL11A microRNA described
herein, the first BCL11A segment starts with a –GCGC- at the 5’ end and the second BCL11A
segment ends with a -GCGC- at the 3’ end.
In one embodiment of any one of the synthetic BCL11A microRNA described
herein, the first BCL11A segment further consist a –GCGC- at the 5’ end and the second
BCL11A segment ends with a -GCGC- at the 3’ end.
In one ment of any one of the tic BCL11A microRNA described
herein, the first BCL11A segment starts with a -GCGA- , -TCTG-, or -TG- at the 5’ end and the
second BCL11A segment is complementary to first BCL11A t.
In one embodiment of any one of the synthetic BCL11A NA described
herein, the first BCL11A segment further t a -GCGA- , , or -TG- at the 5’ end.
In one embodiment of any one of the synthetic BCL11A microRNA described
herein, the second BCL11A segment ends with a -TTTT- at the 3’ end.
In one embodiment of any one of the synthetic BCL11A microRNA described
herein, the synthetic BCL11A NA comprise a nucleotide ce selected from the
group consisting of SEQ ID NOS:1-10,13-18, 25-44.
In one embodiment of any one of the synthetic BCL11A microRNA described
herein, the synthetic BCL11A microRNA comprises a nucleotide sequence or a segment
therefrom described in this disclosure.
In one embodiment of any one of the synthetic BCL11A microRNA described
herein, the synthetic BCL11A microRNA consists of a nucleotide sequence ed from the
group consisting of SEQ ID 10,13-18, 25-44.
In one embodiment of any one of the synthetic BCL11A microRNA described
herein, the synthetic BCL11A microRNA consist essentially of a nucleotide sequence selected
from the group ting of SEQ ID NOS:1-10,13-18, 25-44.
In one embodiment of any one of the synthetic BCL11A microRNA described
herein, the first BCL11A segment is selected from the group consisting of
CGCACAGAACACTCATGGATT (SEQ. ID. NO: 46; derived from BCL11A miR1 oligo
described herein), CCAGAGGATGACGATTGTTTA (SEQ. ID. NO: 47; d from
BCL11A miR2 oligo described herein), TCGGAGACTCCAGACAATCGC (SEQ. ID. NO: 48;
derived from BCL11A E3 oligo or shRNA1 or E3 described herein),
CCTCCAGGCAGCTCAAAGATC, (SEQ. ID. NO: 49; derived from shRNA2 or B5 described
herein), TCAGGACTAGGTGCAGAATGT (SEQ. ID. NO: 50; derived from shRNA4 or B11
described herein), TTCTCTTGCAACACGCACAGA (SEQ. ID. NO: 51; derived from BCL11A
D8 oligo or shRNA3 or D8 described herein), GATCGAGTGTTGAATAATGAT (SEQ. ID.
NO: 52; derived from shRNA5 or 50D12 ol D12 described herein),
CAGTACCCTGGAGAAACACAT (SEQ. ID. NO: 53; derived from shRNA5 or 50A5
bed herein), CACTGTCCACAGGAGAAGCCA (SEQ. ID. NO: 54; derived from
shRNA7 or 50B11 described herein), ACAGTACCCTGGAGAAACACA (SEQ. ID. NO: 55;
derived from BCL11A XLC4, shRNA8 and 50C4 described herein),
GATGAAGAGCACCAA (SEQ. ID. NO: 56; derived from BCL11A rgeting
oligos described herein), gcgcCGCACAGAACACTCATG (SEQ. ID. NO: 57; derived from
miR1G5 oligo described herein), GCGCTCGGAGACTCCAGACAA (SEQ. ID. NO: 58;
derived from E3G5 or E3 mod oligo or shRNA1mod described herein),
gcgcCCTCCAGGCAGCTCAAA (SEQ. ID. NO: 59; derived from B5G5 or mod
described herein); gcgcTCAGGACTAGGTGCAGA (SEQ. ID. NO: 60; derived from B11G5 or
mod described herein); gcgcGATCGAGTGTTGAATAA (SEQ. ID. NO: 61; derived
from 50D12G5, D12G4 or shRNA5mod described ); gcgcCAGTACCCTGGAGAAAC
(SEQ. ID. NO: 62; derived from 50A5G5or shRNA6mod described herein);
CTGTCCACAGGAGAA (SEQ. ID. NO: 63; derived from 50B11G5 or shRNA7mod
described herein); GCGCTTCTCTTGCAACACGCA (SEQ. ID. NO: 64; derived from BCL11A
D8G5 or D8 mod or shRNA3mod described herein), GCGCACAGTACCCTGGAGAAA (SEQ.
ID. NO: 65; derived from BCL11A C4G5, or C4 mod or shRNA8mod described ),
CGCACAGAACACTCATGGATT (SEQ. ID. NO: 66; derived from BCL11A D12G5-2
described herein), and ACGCTCGCACAGAACACTCATGGATT (SEQ. ID. NO: 67; derived
from BCL11A D12G5-2 described herein).
In one embodiment of any one of the synthetic BCL11A microRNA described
herein, the loop segment is derived from a microRNA. In one ment, the microRNA is a
hematopoietic specific microRNA. For examples, miR-142, miR-155, 1 and miR-223.
In one embodiment of any one of the synthetic BCL11A microRNA described
herein, the microRNA is miR223.
In one embodiment of any one of the synthetic BCL11A microRNA described
herein, the loop segment is ctccatgtggtagag (SEQ ID .
Accordingly, in one aspect, the present specification bes an isolated nucleic
acid molecule comprising a nucleotide sequence selected from the group consisting of SEQ ID
NOS:1-10,13-18, 25-44, or a synthetic BCL11A microRNA described herein..
Accordingly, in one aspect, the t specification describes a composition
comprising at least one nucleic acid molecule comprising a nucleotide sequence selected from
the group ting of SEQ ID NOS:1-10,13-18, 25-44, or a synthetic BCL11A microRNA
described .
Accordingly, in one aspect, the present specification describes a composition
comprising at least a vector comprising a nucleic acid molecule comprising a nucleotide
sequence selected from the group consisting of SEQ ID NOS:1-10,13-18, 25-44, or a synthetic
BCL11A microRNA bed herein.
In one embodiment of any isolated nucleic acid molecule described, the molecule
comprises the nucleotide sequence of SEQ ID NO:1.
In one embodiment of any isolated c acid molecule described, the molecule
comprises the nucleotide sequence of SEQ ID NO:2.
In one ment of any isolated nucleic acid le described, the molecule
comprises the nucleotide sequence of SEQ ID NO:3.
In one embodiment of any isolated nucleic acid le described, the molecule
comprises the nucleotide sequence of SEQ ID NO:4.
In one embodiment of any ed nucleic acid molecule described, the molecule
comprises the nucleotide sequence of SEQ ID NO:5.
In one embodiment of any isolated nucleic acid molecule described, the molecule
comprises the nucleotide sequence of SEQ ID NO:6.
In one embodiment of any isolated nucleic acid molecule described, the molecule
comprises the nucleotide sequence of SEQ ID NO:7.
In one embodiment of any isolated nucleic acid le described, the molecule
comprises the nucleotide sequence of SEQ ID NO:8.
In one embodiment of any isolated nucleic acid molecule described, the molecule
ses the nucleotide sequence of SEQ ID NO:9.
In one embodiment of any isolated nucleic acid molecule described, the molecule
comprises the nucleotide sequence of SEQ ID NO:10.
In one embodiment of any isolated nucleic acid molecule described, the molecule
comprises the tide sequence of SEQ ID NO:13.
In one embodiment of any isolated nucleic acid molecule described, the molecule
ses the nucleotide sequence of SEQ ID NO:14.
In one embodiment of any isolated c acid molecule described, the molecule
comprises the nucleotide sequence of SEQ ID NO:15.
In one embodiment of any isolated nucleic acid molecule described, the molecule
comprises the nucleotide sequence of SEQ ID NO:16.
In one embodiment of any isolated nucleic acid molecule described, the
le comprises the nucleotide sequence of SEQ ID NO:17.
In one embodiment of any isolated nucleic acid molecule described, the molecule
ses the nucleotide sequence of SEQ ID NO:18.
In one embodiment of any isolated nucleic acid molecule described, the molecule
comprises the nucleotide sequence of SEQ ID NO:25.
In one embodiment of any ed nucleic acid molecule described, the molecule
comprises the nucleotide sequence of SEQ ID NO:26.
In one embodiment of any isolated nucleic acid molecule described, the molecule
comprises the nucleotide ce of SEQ ID NO:27.
In one embodiment of any isolated nucleic acid molecule described, the molecule
comprises the nucleotide sequence of SEQ ID NO:28.
In one embodiment of any isolated nucleic acid molecule described, the molecule
comprises the nucleotide sequence of SEQ ID NO:29.
In one ment of any isolated nucleic acid molecule described, the molecule
comprises the nucleotide sequence of SEQ ID NO:30.
In one embodiment of any ed nucleic acid molecule described, the molecule
comprises the nucleotide sequence of SEQ ID NO:31.
In one embodiment of any isolated nucleic acid molecule described, the molecule
comprises the nucleotide sequence of SEQ ID NO:32.
In one embodiment of any ed nucleic acid molecule described, the molecule
comprises the nucleotide sequence of SEQ ID NO:33.
In one ment of any isolated nucleic acid molecule described, the molecule
comprises the nucleotide sequence of SEQ ID NO:34.
In one embodiment of any isolated nucleic acid molecule described, the molecule
comprises the nucleotide sequence of SEQ ID NO:35.
In one embodiment of any isolated nucleic acid molecule described, the molecule
comprises the nucleotide sequence of SEQ ID NO:36.
In one ment of any isolated nucleic acid molecule described, the molecule
comprises the nucleotide sequence of SEQ ID NO:37.
In one ment of any isolated nucleic acid molecule bed, the molecule
comprises the nucleotide sequence of SEQ ID NO:38.
In one embodiment of any isolated nucleic acid molecule described, the
molecule comprises the nucleotide ce of SEQ ID NO:39.
In one embodiment of any isolated nucleic acid molecule described, the molecule
comprises the nucleotide sequence of SEQ ID NO:40.
In one embodiment of any isolated c acid molecule described, the molecule
comprises the nucleotide sequence of SEQ ID NO:41.
In one embodiment of any isolated nucleic acid molecule described, the molecule
comprises the nucleotide sequence of SEQ ID NO:42.
In one embodiment of any isolated nucleic acid molecule described, the molecule
comprises the nucleotide sequence of SEQ ID NO:43.
In one embodiment of any isolated nucleic acid molecule described, the molecule
comprises the nucleotide sequence of SEQ ID NO:44.
In one aspect, the present specification describes a vector comprising at least one
nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of
SEQ ID 10,13-18, 25-44 or a synthetic BCL11A microRNA described herein.
In one embodiment of any vector described, the vector further ses a spleen
focus-forming virus er, a tetracycline-inducible er, or a β-globin locus control
region and a β-globin promoter. The promoter provide for targeted expression of the nucleic
acid molecule therein or the synthetic BCL11A microRNA therein.
In one , the present specification describes a host cell comprising a vector
which comprises at least one nucleic acid molecule sing a nucleotide sequence selected
from the group consisting of SEQ ID NOS:1-10,13-18, 25-44, or a tic BCL11A
microRNA described herein.
In one ment of any host cell described , the host cell is an
embryonic stem cell, a somatic stem cell, a progenitor cell, a bone marrow cell, a hematopoietic
stem cell, or a hematopoietic progenitor cell. In one embodiment, the host cell is isolated from a
t. In one embodiment, the host cell is isolated from a subject who has been diagnosed with
a hemoglobinopathy. Diagnosis can be made by any method known in the art. For example, by
genetic testing, by RT-PCR, and by blood cytology.
In one embodiment of any host cell described herein, the host cell is an
erythrocyte.
In one aspect, the present specification describes a host cell sing a vector
or a bacterium which comprises at least one nucleic acid molecule comprising a nucleotide
sequence selected from the group consisting of SEQ ID NOS:1-10,13-18, 25-44, or a synthetic
BCL11A microRNA described herein.
In one aspect, the present specification describes a host cell comprising a virus
which comprises at least one nucleic acid molecule comprising a nucleotide sequence selected
from the group consisting of SEQ ID NOS:1-10,13-18, 25-44, or a synthetic BCL11A
microRNA described herein.
In one embodiment of any virus or vector described , the virus is a
lentivirus.
In one embodiment of any vector or virus described , the lentivirus is
selected from the group consisting of: human immunodeficiency virus type 1 (HIV-1), human
immunodeficiency virus type 2 (HIV-2), e arthritis-encephalitis virus (CAEV), equine
infectious anemia virus (EIAV), feline immunodeficiency virus (FIV), bovine immune
deficiency virus (BIV), and simian immunodeficiency virus (SIV).
Accordingly, one aspect of , the present specification describes s for
increasing fetal hemoglobin levels expressed by a cell, comprising the steps of contacting an
embryonic stem cell, a somatic stem cell, a progenitor cell, a bone marrow cell, a hematopoietic
stem cell, or a hematopoietic progenitor cell with an effective amount of a composition
described herein or an effective amount of at least ed nucleic acid le described
herein, whereby fetal hemoglobin sion is increased in the cell, or its progeny, relative to
the cell prior to such contacting. In some embodiments, the composition comprises at least one
vector or cell sing at least one nucleic acid molecule comprising the nucleotide sequence
selected from the group consisting of SEQ ID NOS:1-10,13-18, 25-44, or a synthetic BCL11A
microRNA described herein. In one embodiment, the method further comprises providing a
sample of stem or itor cells for the contacting. In one embodiment, the sample of cells
comprises CD34+ ed cells. In one embodiment, the ition comprises a mixture of
the nucleotide sequences ed from the group ting of SEQ ID NOS:1-10,13-18, 25-44.
For e, the composition has 2-5 different nucleotide sequences selected from the group
consisting of SEQ ID NOS:1-10,13-18, 25-44. For example, the composition comprises SEQ ID
NOS: 34, 37, 39, 41 and 43.
In one aspect, the present specification describes methods of treating, or reducing
a risk of developing, a obinopathy, e.g., SCD and THAL, in a subject. The s can
include selective knockdown of the BCL11A gene in the poietic stem cells of subjects or
individuals. These subjects are at risk of developing, a hemoglobinopathy.
In one embodiment of any method described, the selective knockdown of the
BCL11A gene in the hematopoietic stem cells comprises using an isolated nucleic acid molecule
comprising a nucleotide sequence of SEQ ID NOS:1-10,13-18, 25-44 or using a vector (e.g. a
viral vector) comprising a nucleic acid molecule comprising any one of the tide sequence
of SEQ ID 10,13-18, 25-44, or a synthetic BCL11A microRNA described herein.
In one embodiment of any method described, the selective knockdown of the
BCL11A gene in the hematopoietic stem cells ses contacting the hematopoietic stem cells
with a composition which comprises at least an isolated nucleic acid molecule sing the
nucleotide sequence of SEQ ID NOS:1-10,13-18, 25-44, or with a composition which ses
at least a vector (e.g. a viral vector) sing a nucleic acid molecule comprising any one of
the nucleotide sequence of SEQ ID NOS:1-10,13-18, 25-44, or a synthetic BCL11A microRNA
described herein. In one embodiment, the hematopoietic stem cells are isolated prior the
contacting.
In one embodiment of any method described, the selective knockdown of the
BCL11A gene in the hematopoietic stem cells occurs in vivo, in vitro, or ex vivo. In a further
embodiment, the hematopoietic progenitor cell being targeted for selective knockdown is of the
erythroid lineage.
In one embodiment of any method described, the contacting of the hematopoietic
stem cells with any of the composition described herein occurs in vivo, in vitro, or ex vivo. In a
further embodiment, the hematopoietic progenitor cell being contacted is of the oid
lineage.
In one embodiment of any method described, the contacting of the hematopoietic
stem cells with any of the composition described herein occurs in vivo, in vitro, or ex vivo.
In other embodiments of any method described, selective knockdown of the
BCL11A gene occurs in an embryonic stem cell, a somatic stem cell, a progenitor cell, a bone
marrow cell in addition to a hematopoietic stem cell, or a poietic progenitor cell. In one
embodiment, an nic stem cell, a somatic stem cell, a progenitor cell, or a bone marrow
cell is contacted with the described composition. The embryonic stem cell, the somatic stem cell,
the itor cell, or the bone marrow cell is ed prior the contacting. In one ment,
the contacting of the embryonic stem cell, the somatic stem cell, the progenitor cell, or the bone
marrow cell with any of the composition described herein occurs in vivo, in vitro, or ex vivo.
In other embodiments of any method described, the hematopoietic stem cells are
collected from peripheral blood, cord blood, chorionic villi, amniotic fluid, placental blood, or
bone marrow.
In other ments of any method described, the embryonic stem cell, somatic
stem cell, progenitor cell, or bone marrow cell is collected from peripheral blood, cord blood,
chorionic villi, amniotic fluid, placental blood, or bone marrow.
In one aspect, the present specification describes a method of treating, or
reducing a risk of developing, a hemoglobinopathy in a subject, the method comprising:
administering to the subject a therapeutically effective amount of one or more isolated nucleic
acid molecule bed herein, a virus or a vector described herein, or a cell described herein,
thereby treating, or reducing the risk of developing, the hemoglobinopathy in the subject,
wherein the virus, the vector or cell comprises at least one nucleic acid molecule comprising the
nucleotide sequence selected from the group ting of SEQ ID NOS:1-10,13-18, 25-44, or a
synthetic BCL11A microRNA described . For example, the ive amount of one or
more isolated c acid molecule described herein, a virus or a vector described herein, or a
cell described herein are injected directly into the bone marrow of the subject.
In one aspect, the present specification describes a method of treating, or
reducing a risk of developing, a hemoglobinopathy in a subject, the method sing
contacting a population of hematopoietic stem cells in vitro or ex vivo with a composition
described herein or with at least one or more isolated nucleic acid molecule described herein, a
virus or a vector described herein, and implanting or administering the contacted hematopoietic
stem cells or the progeny cells thereof to the subject. In one embodiment, the contacted
hematopoietic stem cells or the y cells engrafts in the subject. In one embodiment, the
contacted hematopoietic stem cells or the progeny cells thereof are implanted with prostaglandin
E2 and/or antioxidant N-acetyl-L-cysteine (NAC) to promote the engraftments of the contacted
cells.
In one aspect, the present ication describes a method of treating, or
reducing a risk of developing, a hemoglobinopathy in a subject, the method sing
sing at least one synthetic BCL11A microRNA described herein in an embryonic stem
cell, a somatic stem cell, a progenitor cell, a bone marrow cell, a hematopoietic stem cell, or a
hematopoietic progenitor cell of the subject n the expression is ex vivo or in vitro, and
implanting or administering the cell into the subject.
In one aspect, the present specification describes a method for sing fetal
obin levels expressed by a cell, the method comprising expressing at least one synthetic
BCL11A microRNA described herein in an embryonic stem cell, a somatic stem cell, a
progenitor cell, a bone marrow cell, a hematopoietic stem cell, or a hematopoietic progenitor cell
of a subject wherein the sion is ex vivo or in vitro or in vivo. In one embodiment, the
sion is by contacting the cells with an effective amount of a composition described herein
or an effective amount of at least isolated nucleic acid molecule described herein.
In one aspect, the present specification describes a method for decreasing
BCL11A levels expressed by a cell, the method comprising expressing at least one synthetic
BCL11A microRNA described herein in an embryonic stem cell, a somatic stem cell, a
itor cell, a bone marrow cell, a hematopoietic stem cell, or a hematopoietic progenitor cell
of a t wherein the expression is ex vivo or in vitro or in vivo. In one embodiment, the
expression comprises the steps of contacting an embryonic stem cell, a somatic stem cell, a
progenitor cell, a bone marrow cell, a hematopoietic stem cell, or a hematopoietic progenitor cell
with an effective amount of a composition described herein or an effective amount of at least
isolated nucleic acid molecule described herein, whereby fetal hemoglobin expression is
increased in the cell, or its progeny, relative to the cell prior to such contacting. In some
embodiments, the composition comprises at least one vector or cell comprising at least one
nucleic acid molecule sing the nucleotide sequence selected from the group consisting of
SEQ ID NOS:1-10,13-18, 25-44, or a synthetic BCL11A microRNA described herein.
In a further embodiment of any s described herein, the hematopoietic stem
cell or hematopoietic progenitor cell being contacted is of the erythroid lineage.
In one embodiment of any methods described herein, the hematopoietic stem cell
or hematopoietic progenitor cell is collected from peripheral blood, cord blood, nic villi,
amniotic fluid, placental blood, or bone marrow.
In a further embodiment of any methods described herein, the recipient subject is
treated with chemotherapy and/or radiation prior to tation of the contacted or transfected
cells.
In one ment, the chemotherapy and/or radiation is to reduce endogenous
stem cells to facilitate engraftment of the implanted cells.
In one , the present specification describes a method of treating, or
reducing a risk of developing a hemoglobinopathy in a subject, the method sing
providing hematopoietic stem cells from the subject, contacting the hematopoietic stem cells in
vitro or ex vivo with a composition described herein or with at least one or more isolated c
acid molecule described , a virus or a vector described herein, and implanting or readministering
the contacted hematopoietic stem cells back into the same subject. In one
embodiment, the ted hematopoietic stem cells or the progeny cells engrafts in the subject.
In one aspect of any method, the contacted hematopoietic stem cells, embryonic
stem cells, somatic stem cells, progenitor cells, bone marrow cells, or the progeny cells f
are treated ex vivo with prostaglandin E2 and/or antioxidant N-acetyl-L-cysteine (NAC) to
promote subsequent tment in a recipient subject.
In one aspect of any method, the population of hematopoietic stem cells or host
cells is ed from a subject at risk of developing a hemoglobinopathy or has been diagnose
with a hemoglobinopathy.
In one aspect of any method, the population of hematopoietic stem cells is
autologous or allogeneic to the subject.
In one aspect of any method, the population of hematopoietic stem cells or host
cells is ex vivo expanded in culture prior to contacting with a composition described herein or
with at least one or more isolated nucleic acid molecule described herein, a virus or a vector
described herein.
In one aspect of any method, the population of poietic stem cells or host
cells is ex vivo expanded in culture after to contacting with a composition bed herein or
with at least one or more isolated nucleic acid molecule described herein, a virus or a vector
described herein.
In one aspect of any method, the contacted population of hematopoietic stem
cells or host cells is fferentiated ex vivo in culture prior to implanting into a subject.
In one aspect of any method, the contacted hematopoietic stem cells are expanded
in vitro or ex vivo prior to administering into the subject. In one aspect of any method, the
contacted hematopoietic stem cells are cryopreserved prior to administering into the subject. In
another aspect of any method, the contacted hematopoietic stem cells are expanded in vitro or ex
vivo and eserved prior to administering into the subject. In another aspect of any ,
the ted hematopoietic stem cells are expanded in vitro or ex vivo after cryopreservation
prior to administering into the t.
In one aspect of any method, the subject is a human. In one aspect of any method,
the subject is diagnosed with a hemoglobinopathy.
In one aspect of any method, the method further comprises of selecting a subject
diagnosed with a hemoglobinopathy or a subject at risk of developing a obinopathy.
In one aspect of any method, the hemoglobinopathy is sickle cell disease (SCD)
or thalassemia (THAL). For example, β-thalassemias.
In one aspect of the method, the method r comprising administering to the
subject a therapy comprising oxygen, hydroxyurea, folic acid, or a blood transfusion.
In one , the present specification describes a method of treating, or
reducing a risk of developing, a hemoglobinopathy in a subject, the method comprising
expressing in vivo at least one synthetic BCL11A microRNA described herein in the subject.
In one aspect of any method, the in vivo expression occurs in an embryonic stem
cell, a somatic stem cell, a progenitor cell, a bone marrow cell, a hematopoietic stem cell, or a
hematopoietic progenitor cell.
In one aspect of any method, the nic stem cell, somatic stem cell,
progenitor cell, bone marrow cell, hematopoietic stem cell, or hematopoietic progenitor cell is
autologous or allogeneic to the subject.
In one aspect of any method, the embryonic stem cell, somatic stem cell,
progenitor cell, bone marrow cell, hematopoietic stem cell, or hematopoietic progenitor cell
sing the at least one tic BCL11A microRNA described herein is expanded in vitro
or ex vivo prior to administering into the subject. In a further embodiment, the progenitor cell,
bone marrow cell, hematopoietic stem cell and hematopoietic progenitor cell is of the erythroid
lineage.
In one aspect of any method, the embryonic stem cell, somatic stem cell,
progenitor cell, bone marrow cell, hematopoietic stem cell, or hematopoietic progenitor cell
expressing the at least one synthetic BCL11A microRNA described herein is cryopreserved prior
to administering into the subject.
In another aspect of any method, the embryonic stem cell, somatic stem cell,
progenitor cell, bone marrow cell, hematopoietic stem cell, or hematopoietic progenitor cell
expressing the at least one synthetic BCL11A microRNA described herein is ed in vitro
or ex vivo and cryopreserved prior to administering into the subject.
In another aspect of any method, the embryonic stem cell, somatic stem cell,
progenitor cell, bone marrow cell, hematopoietic stem cell, or hematopoietic progenitor cell
expressing the at least one tic BCL11A microRNA described herein is expanded in vitro
or ex vivo after cryopreservation prior to administering into the t.
In one aspect of any method, the at least one tic BCL11A microRNA is
ly linked to a promoter and constructed in a vector for expression in a eukaryotic cell.
In one aspect of any method, the at least one tic BCL11A microRNA is
expressed from a RNA II polymerase.
In one aspect of any method, the at least one synthetic BCL11A microRNA is not
expressed from a RNA III polymerase.
In one aspect of any method, the promoter is ed from a group ting of
a spleen forming virus promoter, a tetracycline-inducible er, or a in locus
control region and a β-globin promoter, or a hematopoietic specific promoter.
In one aspect of any method, the vector is a virus.
In one aspect of any method, the virus is a lentivirus.
In one aspect of any method, the lentivirus is selected from the group consisting
of: human immunodeficiency virus type 1 (HIV-1), human immunodeficiency virus type 2
(HIV-2), caprine arthritis-encephalitis virus (CAEV), equine infectious anemia virus (EIAV),
feline immunodeficiency virus (FIV), bovine immune deficiency virus (BIV), and simian
immunodeficiency virus (SIV).
In one aspect of any method, the subject is an animal, human or non-human, and
rodent or non-rodent. For example, the subject can be any mammal, e.g., a human, other
primate, pig, rodent such as mouse or rat, rabbit, guinea pig, hamster, cow, horse, cat, dog, sheep
or goat, or a non-mammal such as a bird.
In one aspect of any method, the method comprises obtaining a sample or a
population of embryonic stem cells, somatic stem cells, progenitor cells, bone marrow cells,
hematopoietic stem cells, or hematopoietic progenitor cells from the subject.
In one embodiment, the embryonic stem cells, somatic stem cells, progenitor
cells, bone marrow cells, hematopoietic stem cells, hematopoietic progenitor cells are isolated
from the host subject, transfected, cultured (optional), and transplanted back into the same host,
i. e. an autologous cell lant. In another ment, the embryonic stem cells, c
stem cells, itor cells, bone marrow cells, hematopoietic stem cells, or hematopoietic
itor cells are isolated from a donor who is an HLA-type match with a host (recipient) who
is diagnosed with or at risk of developing a hemoglobinopathy. recipient antigen typematching
is well known in the art. The HLA-types include HLA-A, HLA-B, HLA-C, and HLAD.
These represent the minimum number of cell surface antigen matching required for
transplantation. That is the transfected cells are transplanted into a different host, i.e., allogeneic
to the recipient host subject. The donor’s or subject’s embryonic stem cells, somatic stem cells,
itor cells, bone marrow cells, hematopoietic stem cells, or hematopoietic itor cells
can be transfected with a vector or nucleic acid comprising the nucleic acid molecule described
herein, the transfected cells are culture expanded, and then transplanted into the host subject. In
one embodiment, the transplanted cells engrafts in the host subject. The transfected cells can
also be cryopreserved after ected and stored, or cryopreserved after cell expansion and
stored.
As used herein, treating or reducing a risk of developing a hemoglobinopathy in a
t means to ameliorate at least one symptom of hemoglobinopathy. In one aspect,
described are methods of treating, e.g., reducing ty or progression of, a hemoglobinopathy
in a subject. In another aspect, the s can also be used to reduce a risk of developing a
hemoglobinopathy in a subject, delaying the onset of symptoms of a hemoglobinopathy in a
subject, or increasing the longevity of a subject having a hemoglobinopathy. In one aspect, the
methods can include selecting a subject on the basis that they have, or are at risk of developing,
a hemoglobinopathy, but do not yet have a hemoglobinopathy, or a subject with an underlying
hemoglobinopathy. Selection of a subject can include detecting symptoms of a
hemoglobinopathy, a blood test, genetic testing, or clinical recordings. If the results of the
test(s) indicate that the subject has a obinopathy, the methods also include administering
the compositions described herein, thereby treating, or reducing the risk of developing, a
obinopathy in the subject. For example, a subject who is diagnosis of SCD with genotype
HbSS, HbS/β0 thalassemia, HbSD, or HbSO, and/or HbF <10% by electrophoresis.
As used herein, the term “hemoglobinopathy” refers to a condition involving the
presence of an abnormal hemoglobin molecule in the blood. Examples of obinopathies
include, but are not limited to, SCD and THAL. Also included are obinopathies in which
a combination of abnormal hemoglobins is present in the blood (e.g., sickle cell/Hb-C disease).
An exemplary example of such a disease includes, but is not limited to, SCD and THAL. SCD
and THAL and their symptoms are well-known in the art and are described in further detail
below. Subjects can be diagnosed as having a hemoglobinopathy by a health care er,
medical caregiver, physician, nurse, family member, or acquaintance, who recognizes,
appreciates, acknowledges, determines, concludes, opines, or s that the subject has a
hemoglobinopathy.
The term “SCD” is defined herein to include any symptomatic anemic condition
which results from sickling of red blood cells. Manifestations of SCD include: anemia; pain;
and/or organ dysfunction, such as renal failure, retinopathy, acute-chest syndrome, ischemia,
priapism, and stroke. As used herein the term “SCD” refers to a variety of clinical problems
attendant upon SCD, especially in those ts who are homozygotes for the sickle cell
substitution in HbS. Among the constitutional manifestations referred to herein by use of the
term of SCD are delay of growth and development, an increased tendency to p serious
infections, particularly due to coccus, marked impairment of splenic function,
preventing effective clearance of circulating bacteria, with recurrent infarcts and eventual
destruction of splenic tissue. Also included in the term “SCD” are acute episodes of
musculoskeletal pain, which affect primarily the lumbar spine, abdomen, and femoral shaft, and
which are similar in mechanism and in severity. In adults, such attacks commonly manifest as
mild or moderate bouts of short on every few weeks or months persed with agonizing
attacks lasting 5 to 7 days that strike on average about once a year. Among events known to
trigger such crises are acidosis, hypoxia, and dehydration, all of which potentiate intracellular
polymerization of HbS (J. H. Jandl, Blood: ok of Hematology, 2nd Ed., Little, Brown and
y, Boston, 1996, pages 544-545).
As used herein, “THAL” refers to a hereditary disorder characterized by defective
production of hemoglobin. In one embodiment, the term encompasses hereditary s that
occur due to mutations affecting the synthesis of hemoglobins. In other ments, the term
includes any symptomatic anemia resulting from thalassemic conditions such as severe or βthalassemia
, thalassemia major, thalassemia intermedia, α-thalassemias such as hemoglobin H
disease. assemias are caused by a mutation in the in chain, and can occur in a major
or minor form. In the major form of β-thalassemia, children are normal at birth, but develop
anemia during the first year of life. The mild form of β-thalassemia produces small red blood
cells. Αlpha-thalassemias are caused by deletion of a gene or genes from the globin chain.
By the phrase “risk of developing e” is meant the relative probability that a
t will develop a hemoglobinopathy in the future as ed to a control subject or
population (e.g., a healthy subject or population). For example, an individual carrying the
c mutation associated with SCD, an A to T mutation of the β-globin gene, and r the
individual in heterozygous or homozygous for that mutation increases that individual’s risk.
The term “inhibitory RNA” is meant to include a nucleic acid molecule that
contains a sequence that is complementary to a target nucleic acid (e.g., a target microRNA) that
mediates a decrease in the level or activity of the target nucleic acid. Non-limiting examples of
inhibitory RNAs include interfering RNA, shRNA, siRNA, ribozymes, antagomirs, and
antisense oligonucleotides. Methods of making tory RNAs are described herein.
Additional s of making inhibitory RNAs are known in the art. In one embodiment, the
BCL11A microRNA described herein is an inhibitory RNA that cause a decrease in the activity
of BCL11A mRNA.
As used herein, “an interfering RNA” refers to any double stranded or single
stranded RNA sequence, capable -- either directly or indirectly (i.e., upon conversion) of
inhibiting or down-regulating gene expression by mediating RNA interference. Interfering RNA
includes, but is not limited to, small interfering RNA (“siRNA”) and small hairpin RNA
(“shRNA”). “RNA interference” refers to the selective degradation of a sequence-compatible
ger RNA transcript.
As used herein “an shRNA” (small hairpin RNA) refers to an RNA molecule
comprising an nse , a loop portion and a sense region, wherein the sense region has
complementary nucleotides that base pair with the antisense region to form a duplex stem.
ing post-transcriptional sing, the small hairpin RNA is ted into a small
interfering RNA by a cleavage event mediated by the enzyme Dicer, which is a member of the
RNase III family. As used herein, the phrase “post-transcriptional processing” refers to mRNA
processing that occurs after transcription and is ed, for example, by the enzymes Dicer
and/or Drosha.
A “small interfering RNA” or “siRNA” as used herein refers to any small RNA
molecule capable of inhibiting or down regulating gene expression by mediating RNA
interference in a sequence specific manner. The small RNA can be, for example, about 18 to 21
nucleotides long. Each siRNA duplex is formed by a guide strand and a passenger strand. The
endonuclease Argonaute 2 (Ago 2) catalyzes the unwinding of the siRNA duplex. Once
unwound, the guide strand is incorporated into the RNA Interference Specificity Complex
(RISC), while the ger strand is ed. RISC uses the guide strand to find the mRNA
that has a complementary sequence leading to the endonucleolytic cleavage of the target mRNA.
iruses are RNA viruses that utilize reverse transcriptase during their
replication cycle. The term “retrovirus” refers to any known irus (e.g., type c retroviruses,
such as Moloney murine sarcoma virus (MoMSV), Harvey murine sarcoma virus (HaMuSV),
murine mammary tumor virus (MuMTV), gibbon ape leukemia virus (GaLV), feline leukemia
virus (FLV), irus, Friend, Murine Stem Cell Virus (MSCV) and Rous Sarcoma Virus
(RSV)). “Retroviruses” of the invention also include human T cell leukemia s, HTLV-1
and HTLV-2, and the lentiviral family of retroviruses, such as Human Immunodeficiency
Viruses, HIV-1, HIV-2, simian immunodeficiency virus (SW), feline immonodeficiency virus
(FIV), equine immunodeficiency virus (EIV), and other classes of retroviruses.
The retroviral c RNA is converted into double-stranded DNA by reverse
transcriptase. This double-stranded DNA form of the virus is capable of being integrated into
the some of the infected cell; once integrated, it is referred to as a “provirus.” The
provirus serves as a template for RNA polymerase II and directs the expression of RNA
les which encode the structural proteins and enzymes needed to produce new viral
particles.
At each end of the provirus are structures called “long terminal repeats” or
“LTRs.” The term “long terminal repeat (LTR)” refers to domains of base pairs located at the
ends of retroviral DNAs which, in their natural sequence context, are direct repeats and contain
U3, R, and U5 s. LTRs generally provide functions fundamental to the expression of
retroviral genes (e.g., promotion, initiation and polyadenylation of gene transcripts) and to viral
replication. The LTR contains numerous regulatory signals including transcriptional control
elements, enylation signals and sequences needed for replication and integration of the
viral genome. The viral LTR is divided into three regions called U3, R and U5. The U3 region
contains the enhancer and promoter elements. The U5 region is the sequence between the
primer binding site and the R region and contains the polyadenylation sequence. The R (repeat)
region is flanked by the U3 and U5 regions. The LTR composed of U3, R, and U5 s,
appears at both the both the 5' and 3' ends of the viral genome. In one embodiment described
herein, the er within the LTR, including the 5' LTR, is replaced with a heterologous
promoter. Examples of heterologous promoters that can be used include, for example, a spleen
forming virus (SFFV) promoter, a tetracycline-inducible (TET) promoter, a β-globin locus
control region and a β-globin promoter (LCR), and a cytomegalovirus (CMV) promoter.
The term “lentivirus” refers to a group (or genus) of retroviruses that give rise to
slowly developing disease. Viruses included within this group include HIV (human
immunodeficiency virus; including HIV type 1, and HIV type 2), the etiologic agent of the
human acquired immunodeficiency syndrome (AIDS); visna-maedi, which causes encephalitis
) or pneumonia (maedi) in sheep, the caprine arthritis-encephalitis virus, which causes
immune deficiency, tis, and encephalopathy in goats; equine infectious anemia virus,
which causes autoimmune hemolytic anemia, and encephalopathy in horses; feline
immunodeficiency virus (FIV), which causes immune deficiency in cats; bovine immune
deficiency virus (BIV), which causes denopathy, cytosis, and possibly central
nervous system infection in cattle; and simian immunodeficiency virus (SIV), which cause
immune deficiency and encephalopathy in sub-human primates. es caused by these
viruses are characterized by a long incubation period and protracted course. Usually, the viruses
latently infect monocytes and macrophages, from which they spread to other cells. HIV, FIV,
and SIV also readily infect T lymphocytes, i.e., T-cells.
The term “R region” refers to the region within retroviral LTRs beginning at the
start of the capping group (i.e., the start of transcription) and ending immediately prior to the
start of the poly A tract. The R region is also defined as being flanked by the U3 and U5
regions. The R region plays an important role during reverse transcription in permitting the
transfer of nascent DNA from one end of the genome to the other.
The term “promoter/enhancer” refers to a segment of DNA which contains
sequences e of providing both promoter and enhancer functions. For example, the long
terminal repeats of retroviruses contain both er and enhancer ons. The
enhancer/promoter may be “endogenous,” “exogenous,” or “heterologous.” An “endogenous”
enhancer/promoter is one which is lly linked with a given gene in the genome. An
“exogenous” or “heterologous” enhancer/promoter is one which is placed in osition to a
gene by means of genetic manipulation (i.e., lar biological techniques) such that
transcription of that gene is directed by the linked enhancer/promoter.
Unless otherwise defined, all technical terms used herein have the same meaning
as commonly understood by one of ordinary skill in the art to which this invention belongs.
Methods and als are described herein for use in the present invention; other, suitable
s and materials known in the art can also be used. The materials, methods, and examples
are illustrative only and not intended to be limiting. All ations, patent applications,
patents, and other references mentioned herein are incorporated by reference in their entirety. In
case of conflict, the present ication, including definitions, will control. Other es and
advantages of the invention will be apparent from the following detailed description and figures,
and from the claims.
DESCRIPTION OF DRAWINGS
is a diagram of two embodiments of the disclosed synthetic BCL11A
micro RNA: BCL11A miR1 and BCL11A miR2 oligonucleotides. The stem/loop structure is
generated by the complementary sequences of the BCL11A targeting sequences (in bold upper
case nucleotide bases) in the ucleotides. The BCL11A targeting sequences are the
BCL11A ts. The stem/loop structure is then cloned in to a miR-223/miR-30 background
(micro RNA background). The entire miRNA/shRNA structure is then cloned into a
SFFV/LCR/TET te containing SIN lentiviral vector containing a transgene er
(Venus).
is a schematic diagram of lentiviral vector proviruses with SFFV, TET and
LCR ers.
is a panel of two bar graphs showing that SFFV-LV efficiently knocks
down BCL11A and induces εγ-globin expression.
is a panel of two bar graphs depicting that LCR/TET-LV efficiently
knocks down BCL11A and induces εγ-globin expression.
is a panel of photomicrographs and graphs showing that transduced
CD34+ HSC differentiate ex vivo into erythrocytes and express HbF.
is a panel of scatter plots depicting LCR-LV transduced CD34+ HSCs
from ts with SCD transplanted into NSG mice.
is a panel of a photomicrograph and graphs showing the study of ial
toxicity of BCL11A in lymphoid development.
FIGS. 8A-8E show the screening and tion of shRNAs targeting BCL11A
in pol III and pol II expression systems.
. Schematic representation of RNA polymerase III (pol III, U6 promoter,
left side) and RNA polymerase II (pol II, SFFV promoter, right side) driven shRNA and
miRNA(223) embedded shRNA cassettes, respectively. Both expression cassettes were
engineered into lentivirus vectors. The various boxes represent the passenger strand, the guide
strand, and the loop structure as indicated. The miRNA223 scaffold is represented with dotted
line box. Different shRNA sequences targeting BCL11A were expressed in these two backbones
and evaluated for knockdown ency.
. High-throughput screening of multiple shRNA sequences targeting
s regions in BCL11A mRNA (XL/L-shared isoform sequences, que coding
sequences and the 3'-UTR of XL isoform, as indicated ) for knockdown efficiency using pol III-
based lentivirus vectors. Both induction of ε-γ by qPCR and induction of mCherry er by
FACS (as a surrogate for n-y induction in a reporter cell line) were used as a functional
readout for BCL11A knockdown. Normalized expression of ε-γ mRNA relative to non-targeting
control is plotted on y-axis and mean florescence intensity (MFI) of mCherry expression relative
to nsduced l is plotted on x-axis. The 11 shRNAs that were further tested are marked
with circles.
FIGS. 8C and 8D. Comparison of knockdown efficiency of selected shRNAs in
pol III-based and pol II-based systems. MEL cells were transduced with LKO vector or with
LEGO vector to express the indicated shRNAs and the transduced cells were selected either in
the presence of puromycin (LKO) or sorted for Venus expression (LEGO). miR1 shRNA
previously reported by Sankaran et al. (). BCL11A protein levels are shown () by
immunoblot with β-actin as control. XL and L show the position of each m of BCL11A
protein. () Band intensity was analyzed using ImageJ re.
. Fold induction of normalized expression of ε-γ compared to nontargeting
control is measured by qPCR. Non-targeting shRNA transduced MEL cells were used
and expression set to 1. Data represent mean ± SD from a representative experiment of three
independent experiments conducted in triplicates. * P<0.05, **P< 0.01, ***P< 0.001.
FIGS. 9A-9F are data collected from small RNA sequencing analysis which
reveals differential processing between pol III vs pol II ripts.
FIGS. 9A and 9B. Total RNA was isolated from transduced, sorted or puromycin
selected MEL cells expressing either miR1 or C4 shRNA. The resulting RNA was then
subjected to RNA deep-sequencing. Processed final guide and passenger strand sequence
ribed from () pol III (LKO) or () from pol II (LEGO) are represented on
the x-axis and corresponding number of reads per n of total reads of each strand are plotted
on the y-axis.
FIGS. 9C-9F. The sequences of processed variant guide strands of miR1
ribed from () pol III promoter or () pol II promoter are plotted on the yaxis
with the number of total reads plotted on the x-axis. The sequence of processed variant
guide strand species of C4 transcribed from () pol III er or () pol II
promoter are plotted on y-axis with the number of total reads plotted on x-axis.
FIGS. 10A-10D show that modification of shRNA sequences leads to increased
knockdown and improved guide vs passenger strand ratio.
A. mIR1 and C4 shRNAs were modified such that four 5’ bases were
d and GCGC was added on 3’ end to yield modified shRNA termed miR1 G5 and C4G5.
B. Comparison of own efficiency of modified and parent shRNA
sequences. MEL cells were transduced with LEGO to express the indicated shRNAs via pol II
er and transduced cells were sorted for Venus expression. BCL11A protein levels were
measured by immunoblot with β-actin as a loading l. XL and L indicate position of these
isoforms of BCL11A protein.
C. Immunoblot band intensity was analyzed using ImageJ software.
D. Fold induction of normalized expression compared to non-targeting
l of ε-γ by modified/ unmodified shRNA sequences measured by qPCR. Data represent
mean ± SD from a representative experiment of three independent experiments conducted in
triplicate showing similar results. *P<0.05, **P< 0.01.
FIGS.11A-11C show the RNA cing analysis of four base-pairs ed
shRNAs exhibit faithful sing.
A. Total small RNA was isolated from uced, sorted MEL
expressing modified miR1 and modified C4 shRNAs and sequenced. Frequency distribution of
processed guide strand species of modified miR1 (miR1-G5 and C4G5) transcribed from pol II
promoter are plotted on the x-axis with the proportion of reads per million total reads plotted on
y-axis.
FIGS. 11B and 11C. The sequence of processed variant guide strand species of
mIR1-G5 and C4-G5 are displayed on the y-axis and the frequency of reads are shown on the xaxis.
A. Candidates from the shRNA screen targeting BCL11A using pLKO
vector.
B. Guide strand sequence composition and distribution in PLKO. With
pLKO constructs there is always a shift at the 5’ end which may be due to extension of T rich
sequence at the 3’ end. The added T's are part of the pol III ation sequence. This shift in
mature shRNA sequence indicates that during Dicer-mediated processing the 3’ counting rule is
dominant, meaning cleavage of the shRNA is initiated 21nt from the 3'-end. This results in a 3 or
4 basepair shift at the 5’end and also in an identically shifted seed-region (bases 2-7 of the guide
strand) which is for target recognition.
. Guide strand ce composition and distribution in LEGO. Small
RNA deep sequencing analysis reveals differential processing between pol III vs pol II
transcripts.With lego constructs there is no shift at the 5’ end and the guide strand is faithfully
processed by dicer which results in the predicted product. ingly the final guide strand
differs n pol III and pol II driven ucts.
A. Design of new shRNAs to mimic mature guide strands produced in
pLKO vector. All shRNAs were ed such that four bases on the 5’ were deleted and GCGC
was added on 3’ end to yield modified shRNA termed miR1G5, E3G5, B5G5, D8G5, B11G5,
50D12G5, 50B11G5, 50A5G5, 50C4G5. With incorporation of this shift, significant
improvement was observed with E3G5, D8G5 and C4G5 regarding the BCL11A knockdown
and epsilon-gamma induction. The “xxxx” ents the on of the 4-base pair (bp) frame
shift that results in the 4-bases removed from the unmodified miR1, E3, B5, D8, B11, 50D12
(also refered to as D12), 50B11, 50A5 (also refered to as A5), and 50C4 (also refered to as C4).
B. Guide strand sequence composition and distribution in modified
LEGO. RNA deep sequencing analysis of modified shRNAs shows faithful processing with a
4bp shift, which indicates that by introducing the shift we are able to perfectly mimic the
product of pLKO-vectors. As pLKO vectors were used for screening of ive shRNAs, this
modification mimics the precise mature product guide sequence when transferring the shRNA
cassette into pol II driven backbones.
. Comparison of BCL11A knockdown with modified Guide sequences.
Comparison of knockdown efficiency of modified and parent shRNA sequences. Western blot
showing BCL11A expression (XL and L-isoforms, top panel). Red circles indicate shRNAs
where an ed BCL11A knockdown was ed upon introduction of a 4 bp shift.
Bottom panel: Fold induction of normalized expression of ε-γ by modified/ unmodified ShRNA
ces were compared to nontargeting control as measured by qPCR.
. Comparison of miR expression with modified Guide sequences.
tent with the increase in knockdown efficiency and epsilon-y induction, the guide strand
expression was high (which leads to increase in knockdown efficiency) when northern was
performed in modified constructs compared to unmodified especially with E3G5, D8G5 and
C4G5.
. BCL11A knockdown efficiency and εγ induction with LEGO vectors.
ison of knockdown efficiency of selected shRNAs in pLKO pol III-based and pLEGO
pol II-based systems. MEL cells were transduced with ted shRNAs either in pLKO vector
or with pLEGO vector and the transduced cells were selected either in the presence of
puromycin (pLKO) or sorted for Venus expression (pLEGO). BCL11A protein levels were
measured by immunoblot with β-actin as control. Fold ion of normalized ε-γ ed to
non-targeting control is measured by qPCR. Non-targeting shRNA transduced MEL cells were
used as negative controls. Frame shift has strong effect on both knockdown ency and εγ
induction. shRNAs targeting XL isoform alone have strong effect on εγ induction. Data
represent mean ± SD from three independent experiments, each conducted in triplicates. *
shows the differential processing in pol-III shRNA vectors and pol-II
microRNA adapted shRNA vectors.
FIGS. 19A-19D show the screening and evaluation of shRNAs targeting
BCL11A in pol III and pol II expression systems.
A. Schematic representation of -BCL11A-shRNA (left side) and
LEGO-SFFV-BCL11A-shRNAmiR (right side). Both expression cassettes were engineered into
lentiviral vectors as described in Material and Methods. The light grey boxes represent the sense
strand; white boxes represent the antisense strand; dark grey boxes represent the loop structure
and the miRNA223 scaffold is indicated by a dotted line. The hairpin structures are shown
below. Different shRNA sequences targeting BCL11A were sed in these two backbones
and ted for knockdown efficiency.
B. High-throughput screening of multiple shRNA sequences targeting
BCL11A mRNA for knockdown efficiency using pol III-based lentivirus vectors. Both
induction of Hbb-y mRNA by qRT-PCR and ion of mCherry reporter by FACS (as a
surrogate for ε -γ induction in a reporter cell line) were used as a functional readout for BCL11A
knockdown. Normalized expression of Hbb-y mRNA relative to non-targeting control is plotted
on y-axis and fold ion of mCherry expression (by mean fluorescence intensity, MFI)
relative to non-transduced l is plotted on x-axis. The eight best performing shRNAs
isolated from the screen were further tested and are labeled as 1 h 8.
C. Comparison of knockdown efficiency of selected shRNAs in pol III
(U6)- and pol II (SFFV) -based systems. MEL cells were transduced with U6- (top panel) or
with SFFV- (bottom panel) s to express the indicated shRNAs and the transduced cells
were selected either in the presence of puromycin (pol III) or sorted for Venus expression (pol
II). BCL11A protein levels are shown by immunoblot with β-actin as control. XL and L on left
of panel denote the position of each isoform of BCL11A protein.
D. Fold induction of normalized expression of Hbb-y compared to geting
control measured by qPCR. Expression in non-targeting (NT) shRNA transduced MEL
cells was set 1. Black bars represent the relative expression by U6 promoter driven shRNAs and
white bars represent SFFV promoter driven shRNAs. Data represent mean ± SD from a
representative experiment of three independent experiments ted in triplicates. * P<0.05.
FIGS. 20A and 20B shows the small RNA sequencing analysis reveals
differential processing between pol III versus pol II transcripts. Small RNA sequencing results
of MEL cells transduced with U6-shRNAs and SFFV-shRNAmiRs1, 2, 3, 4, 7, or 8. The RNA
sequences were aligned to the corresponding reference guide strand sequence, shown at the top
of each panel in bold and the flanking sequences in grey. ent variants of guide strands
produced from (Fig. 20A) U6-shRNAs or (Fig. 20B) SFFV-shRNAmiRs are plotted on the yaxis.
The relative % contribution of each variant is ted on the x-axis ated based on
the total number of reads ng the reference shRNA ce.
FIGS. 21A-21E show the modification of shRNA sequences leads to increased
own and improved guide vs. passenger strand ratio in MEL cells.
A. SFFV-shRNAmiRs were modified by deleting the first four bases from
the guide sequence and the addition of GCGC to the 3’ end (shRNA modified)
B. Comparison of knockdown efficiency of modified and parental
shRNAmiR ces sed from a SFFV-pol II promoter in MEL cells. BCL11A protein
levels were measured in FACS sorted transduced cells by immunoblot with β-actin as a loading
control. XL and L on the left of top panel indicate the position of these isoforms of BCL11A
protein. PIII: pol III promoter vector; PII: pol II promoter vector; PIIM: pol II promoter vector
containing modified shRNAmiR sequences.
C. Fold induction of Hbb-y compared to the non-targeting control by
unmodified (white bars) and modified (shaded bars) shRNAmiR sequences measured by qRTPCR.
Data represent mean ± SD. **P< 0.01.
D. Northern blot analysis of total RNA extracted from cells transduced
with le shRNAs and shRNAmiRs. Probes (20nt) complementarity to the guide and
passenger strands from positions 1 to 20 of shRNAs and iRs were utilized to measure
the abundance of processed small RNAs. A probe complementary to 5S RNA was used as an
internal control to determine RNA g. PIII: pol III promoter vector; PII: pol II promoter
vector; PIIM: pol II promoter vector containing ed iR sequences.
E. RNA-sequencing results of homogeneous tions of uced
MEL cells expressing shRNA1, 2, 3, 4, 7, or 8. The sequences of these RNAs were aligned to
the corresponding reference guide strand sequence shown at the top of each panel. The
sequences of different guide strand species are displayed on the y-axis and the frequency as
percentage of aligned reads are shown on the .
FIGS. 22A-22E show the modified shRNAmiRs lead to increased BCL11A
knockdown efficiency and gamma globin induction in human CD34+ derived erythroid cells.
A. CD34+ cells transduced with pol III or pol II vectors expressing
different shRNAs with and t modification were selected either in the presence of
puromycin (pol III) or sorted for Venus expression (pol II and pol II modified). BCL11A
expression was measured by immunoblot with β-actin as a loading control on day 11 of
differentiation.
B. Induction of γ-globin mRNA was determined on day 18 of
differentiation by qRT-PCR. Data represents the percentage of γ-globin of total β-locus output (γ
+ β-globin) for pol III (black bars), pol II (white bars), and modified pol II (grey bars). * p<0.05;
*** p<0.001.
C. Quantification and statistical analysis of erythroid differentiation
markers (CD71, GpA) and enucleation were assessed by flow cytometry. CTRL: control vectors
SFFV-shRNAmiRNT and SEW; PIII: pol III vectors; PII: pol II vectors; PIIM: pol II vectors
ning ed shRNAmiR ces. Data represents mean ± SD from three independent
experiments. *** p<0.001.
D. Hemoglobin F of cell lysates was measured by HPLC on day 18 of
differentiation. The arrow indicates the HbF peaks and the percentage of HbF of total
hemoglobin is shown below the chromatogram.
E. Correlation graph of γ-globin mRNA sion assessed by qRT-PCR
versus HbF by HPLC. Black circles represent pol III vectors, open and grey circles represent pol
II or modified shRNAmiRs, respectively. Correlation coefficient (r2) is shown for all data.
FIGS. 23A-23I show the negative impact of BCL11A knockdown on HSCs in
vivo is prevented by restricting expression to erythroid cells.
A. Lineage negative bone marrow cells isolated from β-YAC mice
2) were transduced ex vivo with LeGO vectors expressing iR* targeting
BCL11A or a non-targeting control vector (SFF-shRNAmiRNT) and transplanted into lethally
irradiated BoyJ recipients (CD45.1). Untransduced control cells were transplanted as control.
Engraftment analysis was performed 4, 8 and 12 weeks post transplantation in peripheral blood
and bone marrow, respectively. (n=4 mice per group)
B. The on of gene modified cells (Venus+ cells) in these mice was
determined 4, 8 and 12 weeks post transplantation in peripheral blood and bone marrow.
C. Competitive transplants were performed using CD45.1 and CD45.2
donor cells uced with the indicated vectors and transplanted into CD45.1/2 heterozygous
mice (top panels). Alternatively a neutral vector encoding blue fluorescent protein (SFFV-BFP)
was used to identify the competitor population in a CD45.1 donor into CD45.2 recipient setting
(lower panels). Shown are representative dot blots of different mixed tions used for
transplantation three days post transduction. The two competing s are indicated above
each panel, the first one indicates the CD45.2 or FP transduced populations,
respectively.
D. The bution of gene ed cells in competitively repopulated
mice was analyzed at 4 and 8 weeks post transplantation in peripheral blood (PB) or at week 12
in bone marrow (BM) and spleen (Spl). The relative contribution of gene modified cells
transduced with the two competing vectors is shown. The first vector mentioned dominated the
poietic output. Each dot represents an individual recipient mouse.
E. A pairwise comparison of the bone marrow B-cell fraction within the
transduced fraction of cells n BCL11A targeting vectors versus control vectors (SFF-
shRNAmiRNT and SFFV-BFP, left panel). Similarly, the LSK t within transduced cell
fractions was analyzed. Each dot represents an dual recipient. * and ** indicate p-values ≤
0.05 and 0.01, respecitvely.
F. Configuration of the LCR-shRNAmiR vector used for erythroid
specific expression (details in text).
G. The in vivo expression profile of the LCR-vector was analyzed in
various hematopoietic lineages 12 weeks after transplantation. The percentages of Venus+ cells
in each mouse were normalized to CD71+/Ter119+ erythroid cells (n=4).
H. A competitive transplantation experiment as described in c and d was
performed using the LCR or SFFV vectors expressing shRNAmiR*. Each dot represents an
individual recipient.
I. Mobilized peripheral blood CD34+ cells were transduced with LCR-
shRNAmiR*, 3 and 8 or a SFFV-GFP mock vector and subjected to erythroid differentiation in
vitro. At day 7 after transduction the promoter activity of SFFV-GFP and LCR-vectors in
different erythroid subpopulation was assessed. Representative flow diagrams are shown. Error
bars in all s = SD. Statistical analysis: t-test.
FIGS. 24A-24F show the lineage ic BCL11A knockdown and gamma
globin induction by modified shRNAmiRs.
A. CD34+ HSPCs transduced with LCR-shRNAmiR 3, 8 or the SFFVGFP
mock vector were FACS-sorted for fluorescent er expression and BCL11A
expression was measured by immunoblot with β-actin as a loading control on day 11 of
differentiation.
B. Induction of γ-globin mRNA was ined on day 18 of
differentiation by qRT-PCR. Data represents the tage of γ/(γ+β) globin.
C. Quantification and statistical analysis of erythroid differentiation
markers (CD71, GpA) and enucleation by flow cytometric is. CTRL: SFFV-GFP control
vector; LCRM: Modified iRs shown in Fig. 23A expressed via LCR promoter. Data
represents mean ± SD from three independent experiments.
D. HbF level of cell lysates was measured by HPLC on day 18 of
differentiation. Arrows indicate the HbF peaks and the percentage of HbF of total hemoglobin is
shown below the chromatogram.
E. Correlation graph of γ-globin induction by qRT-PCR versus HbF by
HPLC. Error bars te ± SD from three independent experiments.
F. Bone marrow CD34+ HSPCs were transduced with LCR-shRNAmiR3
or NT and transplanted into hally irradiated NSG-mice (n=3 per group). Untransduced
cells were used as a l. Fourteen weeks later CD34 cells were isolated from the bone
marrow of transplanted animals and subjected to erythroid entiation in vitro for 14 days.
Expression of γ-globin and β-globin was assessed in cells sorted for Venus reporter expression.
shows the deep sequencing of 247 processed TRC shRNA products in
four cell lines.
shows the in vivo expression profile of the LCR-shRNAmiR vector.
A is a Western blot of in vitro differentiated erythroid cells derived from
transduced CD34+ cells from healthy donors showing BCL11A isoforms (L and XL) and ß-
ACTIN as loading control and demonstrating ive knock-down of BCL11A XL. VCN
determined by DNA PCR is show below each lane.
B shows quantification of BCL11A knock down in erythroid cells. Data
is d from Western blots as shown in A. Data summarizes three independent
ments using cells from a single donor. (Error bars: SD)
C shows induction of gamma globin in erythroid cells as assessed by RT-
qPCR and hemoglobin (HbF) assessed by HPLC. (Error bars: SD)
shows induction of gamma globin in erythroid cells as assessed by RT-
qPCR . The amount of gamma globin induction in the erythroid cells is a measure of the in vivo
BCL11A knockdown in the cells. Error bars: SD. Data from three transplanted animals per
group is shown.
A shows Western blots showing of BCL11A (L and XL isoforms) and β-
ACTIN as loading control and demonstrates effective knock-down of BLC11A-XL. Each panel
(labeled 1-6 below the lane) represents an independent experiment using cells from a single
donor.
B shows quantification of BCL11A knock down in erythroid cells. Data
is d from Western blots shown in A. (Error bar: SD)
C shows resulting induction of HbF by HPLC. (Error bars: SD)
shows the sequences used in both SFFV and LCR backbones for the
knockdown of BCL11A in CD34+ differentiated erythroid cells.
shows Western blots of the BCL11A knockdown in CD34+ entiated
erythroid cells.
DETAILED DESCRIPTION
The disclosure described herein is based, in part, on development of lentiviral
gene therapy vectors that selectively express the BCL11A-targeting shRNA in progeny of
hematopoietic stem cells (HSC). ingly, the disclosure encompasses novel methods for
the regulation of γ-globin expression in oid cells. More specifically, these activities can be
harnessed in methods for the treatment of hemoglobinopathies, including SCD and THAL, by
induction of γ-globin via tion of the BCL11A gene product. In ular embodiments,
lentiviral gene therapy vectors that selectively express the BCL11A-targeting shRNA in y
of HSCs, hematopoietic progenitor cells, or other stem cells such as embryonic cells are
described.
Normal adult hemoglobin comprises four globin proteins, two of which are alpha
(α) proteins and two of which are beta (β) proteins. During mammalian fetal development,
particularly in humans, the fetus produces fetal hemoglobin, which comprises two gamma (γ)-
globin proteins instead of the two β-globin proteins. At some point during fetal development, a
globin fetal switch occurs at which point erythrocytes in the fetus switch from making
predominantly γ-globin to making predominantly β-globin. The developmental switch from
production of predominantly fetal hemoglobin or HbF (α2γ2) to production of adult hemoglobin
or HbA (α2β2) begins at about 28 to 34 weeks of gestation and continues shortly after birth until
HbA becomes inant. This switch results primarily from decreased transcription of the n
genes and increased transcription of β-globin genes. On average, the blood of a normal
adult contains only about 2% HbF, though residual HbF levels have a ce of over d in
healthy adults (Atweh, Semin. Hematol. 38:367-73 (2001)).
Hemoglobinopathies ass a number of anemias of c origin in which
there is a decreased production and/or increased destruction ysis) of red blood cells
(RBCs). These also include genetic defects that result in the tion of abnormal
hemoglobins with a concomitant impaired ability to in oxygen concentration. Some such
disorders involve the e to produce normal β-globin in sufficient amounts, while others
involve the failure to produce normal β-globin entirely. These disorders associated with the βglobin
protein are referred to generally as hemoglobinopathies. For example, SCD results from
a point mutation in the β-globin structural gene, leading to the production of abnormal (sickled)
hemoglobin (HbS). HbS RBCs are more fragile than normal RBCs and undergo hemolysis more
readily, leading eventually to anemia (Atweh, Semin. Hematol. 38(4):367-73 (2001)). THAL
results from a partial or complete defect in the expression of the β-globin gene, leading to
deficient or absent HbA.
The search for treatment aimed at reduction of globin chain imbalance in patients
with hemoglobinopathies has focused on the pharmacologic lation of fetal HbF. The
therapeutic potential of such approaches is demonstrated by observations that certain
populations of adult patients with β chain abnormalities and higher than normal levels of HbF
ence a milder al course of disease than patients with normal adult levels of HbF. For
example, a group of Saudi Arabian sickle cell anemia patients who express 20-30% HbF have
only mild clinical manifestations of the e (Pembrey, et al., Br. J. Haematol. 40: 415-429
(1978)). It is now accepted that hemoglobinopathies, such as SCD and THAL, are ameliorated
by increased HbF production (Jane et al., Br. J. Haematol. 102: 415-422 (1998); Bunn, N. Engl.
J. Med. 328: 129-131 (1993)).
The transcriptional repressor BCL11A represents a therapeutic target for βhemoglobinopathies.
RNA interference was applied using pol III promoter-expressed short
hairpin RNAs (shRNAs) to reduce BCL11A sion in hematopoietic cells. own of
BCL11A in murine hematopoietic stem cells (HSCs) impaired long-term engraftment. To avoid
HSC toxicity, the sion of BCL11A in erythroid cells was selectively suppressed via pol II
promoter expressed microRNA adapted shRNAs miRs). With identical target matched
sequences, markedly reduced knockdown was observed using pol II vectors due to 3-5 nt
differences in the guide s between the s that strongly influence target knockdown.
A corresponding 4 nt shift was engineered into guide s of shRNAmiRs that singly
and unexpectedly improved the knockdown of BCL11A and derepression of Hbb-y, a functional
homolog of the human γ-globin gene in a murine erythroid cell line. The modified shRNAmiRs
were sed in an erythroid-specific fashion to circumvented the e s on murine
HSC engraftment, and this led to efficient BCL11A knockdown and high levels of HbF in
human CD34-derived erythroid cells. A strategy was developed for the prospective design of
shRNAmiRs derived from pol III-expressed shRNA screens. This strategy constitutes an
improved approach to genetic therapy in hemoglobinopathies and other es requiring
lineage-specific expression of gene ing sequences.
Retroviral and Lentiviral s
In some embodiments, described are improved compositions and methods for
treating hemoglobinopathies using retrovirus-based, e.g., lentivirus-based, gene delivery vectors
that achieve sustained, high-level expression of transferred therapeutic genes in eythroid cells or
erythroid sor cells. In one embodiment described herein, the vector comprises an artificial
miRNA comprising targeting sequences to BCL11A cloned into the stem loop of the
endogenouse miR-223 sequence (Amendola et al., Mol Ther 17:1039-52, 2009). The stem/loop
structure of the present vectors are generated by complementary sequences of the
oligonucleotides of SEQ ID NOs:1-18 and 25-44 disclosed herein. See FIGS. 1, 12A, 14A, 21A,
and EXAMPLE 11. This stem/loop structure was cloned into a miR-223/miR-30 background.
The entire miRNA/shRNA structure was then cloned into a cassette with a SFFV, TET, or LCR
promoter containing self-inactivating (SIN) vector. Particular lentiviral vectors described herein
are described by Pawliuk et al. (2001) Science 294:2368 and Imren et al. (2002) PNAS
80, incorporated by reference herein.
Accordingly, in one aspect, described herein is a synthetic BCL11A microRNA
comprising a first BCL11A segment, a loop segment, and a second BCL11A segment arranged
in tandem in a 5' to 3' direction, wherein the loop t is between and directly linked to the
first and second BCL11A segments, and wherein the second BCL11A t is
complementary to the first BCL11A segment such that the first and second BCL11A segments
base pair to form a hairpin loop with the loop segment forming the loop portion of the n
loop thus formed.
In one embodiment of any one of the synthetic BCL11A microRNA described
herein, the first and second BCL11A segments are about 18 to 25 nucleotides long. The first
BCL11A segment is derived from a BCL11A sequence and gives rise to the ger strand
during shRNA processing to a duplex siRNA and the second BCL11A segment is
complementary to first BCL11A segment, wherein the second BCL11A segment gives rise to
the guide strand that is orated into the RNA Interference Specificity Complex (RISC) for
RNA interference or BCL11A gene silencing.
In one embodiment of any one of the synthetic BCL11A microRNA described
herein, the first and second BCL11A segments are derived from BCL11A mRNA sequence.
In one embodiment of any one of the synthetic BCL11A microRNA described
herein, the first BCL11A segment starts with a -GCGC- at the 5' end and the second BCL11A
segment ends with a -GCGC- at the 3' end.
In one ment of any one of the synthetic BCL11A microRNA described
herein, the first BCL11A segment further consist a –GCGC- at the 5’ end and the second
BCL11A segment ends with a –GCGC- at the 3’ end.
In one embodiment of any one of the synthetic BCL11A microRNA described
herein, the first BCL11A segment starts with a –GCGA- , -TCTG-, or –TG- at the 5’ end and the
second BCL11A segment is mentary to first BCL11A segment.
In one embodiment of any one of the synthetic BCL11A microRNA described
herein, the first BCL11A segment further consist a –GCGA- , , or –TG- at the 5’ end.
In one embodiment of any one of the synthetic BCL11A microRNA described
herein, the second BCL11A segment ends with a –TTTT- at the 3’ end.
In one embodiment of any one of the synthetic BCL11A microRNA described
herein, the tic BCL11A microRNA comprise a nucleotide sequence selected from the
group ting of SEQ ID NOS:1-10,13-18, 25-44.
In one embodiment of any one of the synthetic BCL11A microRNA described
herein, the synthetic BCL11A microRNA consists of a nucleotide sequence selected from the
group consisting of SEQ ID NOS:1-10,13-18, 25-44.
In one embodiment of any one of the synthetic BCL11A microRNA described
herein, the synthetic BCL11A microRNA consist essentially of a nucleotide sequence selected
from the group consisting of SEQ ID 10,13-18, 25-44.
In one embodiment of any one of the synthetic BCL11A microRNA bed
herein, the first BCL11A segment is selected from the group consisting of
CGCACAGAACACTCATGGATT (SEQ. ID. NO: 46; d from BCL11A miR1 oligo
described herein), CCAGAGGATGACGATTGTTTA (SEQ. ID. NO: 47; derived from
BCL11A miR2 oligo described herein), TCGGAGACTCCAGACAATCGC (SEQ. ID. NO: 48;
derived from BCL11A E3 oligo or shRNA1 or E3 described ),
CCTCCAGGCAGCTCAAAGATC, (SEQ. ID. NO: 49; derived from shRNA2 or B5 described
herein), TCAGGACTAGGTGCAGAATGT (SEQ. ID. NO: 50; derived from shRNA4 or B11
described herein), TTCTCTTGCAACACGCACAGA (SEQ. ID. NO: 51; derived from BCL11A
D8 oligo or shRNA3 or D8 described herein), GATCGAGTGTTGAATAATGAT (SEQ. ID.
NO: 52; derived from shRNA5 or 50D12 ol D12 described herein),
CAGTACCCTGGAGAAACACAT (SEQ. ID. NO: 53; derived from shRNA5 or 50A5
described herein), CCACAGGAGAAGCCA (SEQ. ID. NO: 54; d from
shRNA7 or 50B11 described ), CCCTGGAGAAACACA (SEQ. ID. NO: 55;
derived from BCL11A XLC4, shRNA8 and 50C4 described herein),
CAACAAGATGAAGAGCACCAA (SEQ. ID. NO: 56; derived from BCL11A Non-targeting
oligos described herein), gcgcCGCACAGAACACTCATG (SEQ. ID. NO: 57; derived from
miR1G5 oligo described herein), GCGCTCGGAGACTCCAGACAA (SEQ. ID. NO: 58;
derived from E3G5 or E3 mod oligo or shRNA1mod described herein),
gcgcCCTCCAGGCAGCTCAAA (SEQ. ID. NO: 59; derived from B5G5 or shRNA2mod
described herein); AGGACTAGGTGCAGA (SEQ. ID. NO: 60; derived from B11G5 or
shRNA4mod described herein); gcgcGATCGAGTGTTGAATAA (SEQ. ID. NO: 61; derived
from 50D12G5, D12G4 or shRNA5mod described herein); gcgcCAGTACCCTGGAGAAAC
(SEQ. ID. NO: 62; derived from 50A5G5or shRNA6mod described );
gcgcCACTGTCCACAGGAGAA (SEQ. ID. NO: 63; d from 50B11G5 or shRNA7mod
described herein); GCGCTTCTCTTGCAACACGCA (SEQ. ID. NO: 64; derived from BCL11A
D8G5 or D8 mod or shRNA3mod described herein), GCGCACAGTACCCTGGAGAAA (SEQ.
ID. NO: 65; derived from BCL11A C4G5, or C4 mod or shRNA8mod described herein),
GAACACTCATGGATT (SEQ. ID. NO: 66; derived from BCL11A D12G5-2
described herein), and ACGCTCGCACAGAACACTCATGGATT (SEQ. ID. NO: 67; derived
from BCL11A D12G5-2 described herein).
In one embodiment of any one of the tic BCL11A NA described
herein, the loop segment is derived from a microRNA. In one embodiment, the NA is a
hematopoietic specific microRNA. For examples, 2, miR-155, miR-181 and miR-223.
In one embodiment of any one of the synthetic BCL11A microRNA described
herein, the microRNA is .
In one embodiment of any one of the synthetic BCL11A microRNA bed
herein, the loop segment is ctccatgtggtagag (SEQ ID NO:68).
In one aspect, the present specification describes an isolated nucleic acid
molecule comprising a nucleotide ce selected from the group consisting of SEQ ID
NOS:1-18, 25-44, or a synthetic BCL11A microRNA described herein.
Accordingly, in one aspect, the present specification describes a composition
comprising at least one nucleic acid molecule comprising a nucleotide sequence selected from
the group consisting of SEQ ID NOS:1-10,13-18, 25-44, or a synthetic BCL11A microRNA
described herein.
Accordingly, in one aspect, the present specification describes a composition
comprising at least a vector or a bacterium comprising a nucleic acid molecule comprising a
nucleotide sequence selected from the group consisting of SEQ ID NOS:1-10,13-18, 25-44, or a
synthetic BCL11A microRNA described herein.
In one , the present specification describes a host cell comprising a vector
or virus which comprises at least one nucleic acid molecule comprising a tide sequence
selected from the group consisting of SEQ ID 10,13-18, 25-44, or a synthetic BCL11A
microRNA described .
In one aspect, the present specification describes a host cell comprising a vector,
virus or a bacterium which comprises at least one nucleic acid molecule comprising a nucleotide
sequence selected from the group consisting of SEQ ID 10,13-18, 25-44, or a synthetic
BCL11A microRNA described .
In one embodiment, the vector is a viral vector or a virus.
RNA interference (RNAi) mediated by short interfering RNAs (siRNA) or
microRNAs (miRNA) is a powerful method for post-transcriptional regulation of gene
expression. RNAi has been extensively used for the study of biological processes in mammalian
cells and could constitute a therapeutic approach to human diseases in which selective
modulation of gene expression would be desirable. Depending on the degree of complementarity
between miRNA and target mRNA sequences, loss of gene expression occurs by inducing
degradation of the cognate mRNA or by translational ation. Endogenous miRNAs are
transcribed as primary transcripts and subsequently processed by the RNAse III enzyme
Drosha,(1) to create a stem loop ure. Nuclear export and cleavage by Dicer generates a
mature short double ed molecule ) that is ted into guide and passenger
strands. The guide strand is loaded into the RNA induced silencing x (RISC), the effector
complex mediating cleavage of target mRNAs with the functional guide strand binding to RISC
proteins (2) while the passenger strand is degraded [reviewed in (3)]. The loading of guide
versus passenger strands into RISC largely depends on the 5’ end stability of the siRNA, with
the less stable strand preferentially incorporated into RISC (4, 5), although the exact regulation
in mammalian cells is incompletely understood. The 5’ end of the guide strand contains the
“seed region,” which is critical for target identification (6, 7). Precise ge by Drosha and
Dicer is critical for the generation of guide RNAs with defined seed regions that mediate
ent binding to the appropriate target mRNAs. rate processing results in g to
off-target molecules but a shift in ge sites also alters the nucleotide composition of duplex
ends, which may have a profound effect on strand loading into RISC (8).
The inhibiting the expression of selected target polypeptides is through the use of
RNA interference agents. RNA interference (RNAi) uses small interfering RNA )
duplexes that target the messenger RNA encoding the target polypeptide for selective
degradation. siRNA-dependent post-transcriptional silencing of gene expression involves
cleaving the target messenger RNA molecule at a site guided by the siRNA. RNAi is an
evolutionally conserved process whereby the expression or introduction of RNA of a sequence
that is identical or highly similar to a target gene results in the sequence ic degradation or
specific post-transcriptional gene silencing (PTGS) of messenger RNA (mRNA) transcribed
from that targeted gene (see Coburn, G. and Cullen, B. (2002) J. gy 76(18):9225), thereby
inhibiting expression of the target gene. In one ment, the RNA is double stranded RNA
(dsRNA). This process has been described in plants, invertebrates, and mammalian cells. In
nature, RNAi is initiated by the dsRNA-specific endonuclease Dicer, which promotes processive
cleavage of long dsRNA into double-stranded fragments termed siRNAs. siRNAs are
incorporated into a n complex (termed “RNA induced silencing complex,” or “RISC”) that
recognizes and cleaves target mRNAs. RNAi can also be initiated by introducing nucleic acid
molecules, e.g., synthetic siRNAs or RNA interfering agents, to t or silence the expression
of target genes. As used herein, “inhibition of target gene expression” es any decrease in
expression or protein activity or level of the target gene or protein encoded by the target gene as
compared to a situation wherein no RNA interference has been induced. The decrease will be of
at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at
least 80%, at least 90%, at least 95%, at least 99%, or more as compared to the expression of a
target gene or the activity or level of the protein encoded by a target gene which has not been
targeted by an RNA interfering agent.
The terms “RNA interference agent” and “RNA interference” as they are used
herein are intended to ass those forms of gene silencing mediated by double-stranded
RNA, regardless of whether the RNA interfering agent comprises an siRNA, miRNA, shRNA or
other double-stranded RNA molecule. siRNA is defined as an RNA agent which ons to
inhibit expression of a target gene, e.g., by RNAi. An siRNA may be chemically synthesized,
may be produced by in vitro transcription, or may be produced within a host cell. In one
embodiment, siRNA is a double stranded RNA ) molecule of about 15 to about 40
nucleotides in length, preferably about 15 to about 28 tides, more preferably about 19 to
about 25 nucleotides in , and more preferably about 19, 20, 21, 22, or 23 nucleotides in
length, and may contain a 3' and/or 5' overhang on each strand having a length of about 0, 1, 2,
3, 4, or 5 nucleotides. The length of the overhang is independent between the two strands, i.e.,
the length of the overhang on one strand is not dependent on the length of the overhang on the
second strand. ably the siRNA is capable of promoting RNA interference through
degradation or ic post-transcriptional gene silencing (PTGS) of the target messenger RNA
(mRNA).
siRNAs also include small hairpin (also called stem loop) RNAs (shRNAs). In
one embodiment, these shRNAs are composed of a short (e.g., about 19 to about 25 nucleotide)
antisense strand, followed by a nucleotide loop of about 5 to about 9 nucleotides, and the
analogous sense strand. Alternatively, the sense strand may precede the nucleotide loop
structure and the nse strand may follow. These shRNAs may be contained in plasmids,
retroviruses, and iruses and expressed from, for example, the pol III U6 promoter, or
another promoter (see, e.g., Stewart, et al. (2003) RNA April; 9(4):493-501, incorporated by
reference herein in its entirety). The target gene or sequence of the RNA interfering agent may
be a cellular gene or c sequence, e.g., the BCL11A sequence. An siRNA may be
substantially homologous to the target gene or genomic sequence, or a fragment thereof. As
used in this context, the term “homologous” is defined as being ntially identical,
sufficiently complementary, or r to the target mRNA, or a fragment thereof, to effect RNA
interference of the target. In on to native RNA molecules, RNA suitable for ting or
interfering with the expression of a target sequence e RNA derivatives and s.
Preferably, the siRNA is identical to its target. The siRNA ably targets only one sequence.
Each of the RNA interfering agents, such as siRNAs, can be ed for potential off-target
effects by, for example, expression profiling. Such methods are known to one skilled in the art
and are described, for example, in Jackson et al. Nature Biotechnology 6:635-637, 2003. In
addition to expression profiling, one may also screen the ial target sequences for similar
ces in the sequence ses to identify potential sequences which may have off-target
effects. For example, 15, or perhaps as few as 11 contiguous nucleotides, of sequence identity
are sufficient to direct silencing of non-targeted transcripts. ore, one may initially screen
the proposed siRNAs to avoid ial off-target silencing using the sequence identity analysis
by any known ce comparison methods, such as BLAST. siRNA sequences are chosen to
maximize the uptake of the antisense (guide) strand of the siRNA into RISC and thereby
maximize the ability of RISC to target BCL11A mRNA for degradation. This can be
accomplished by scanning for sequences that have the lowest free energy of binding at the 5'-
terminus of the antisense strand. The lower free energy leads to an enhancement of the
unwinding of the 5'-end of the antisense strand of the siRNA duplex, thereby ensuring that the
antisense strand will be taken up by RISC and direct the sequence-specific cleavage of the
human BCL11A mRNA. siRNA molecules need not be limited to those molecules containing
only RNA, but, for example, further encompasses chemically modified nucleotides and nonnucleotides
, and also include molecules wherein a ribose sugar molecule is tuted for
another sugar molecule or a molecule which performs a similar function. Moreover, a nonnatural
linkage between nucleotide residues can be used, such as a phosphorothioate e.
The RNA strand can be derivatized with a reactive functional group of a reporter group, such as
a fluorophore. Particularly useful derivatives are modified at a terminus or termini of an RNA
strand, typically the 3' terminus of the sense strand. For example, the 2'-hydroxyl at the 3'
terminus can be readily and selectively derivatizes with a variety of groups. Other useful RNA
tives incorporate nucleotides having modified carbohydrate moieties, such as 2'O-
alkylated residues or 2'-O-methyl ribosyl derivatives and 2'-O-fluoro ribosyl derivatives. The
RNA bases may also be modified. Any modified base useful for inhibiting or interfering with
the expression of a target sequence may be used. For e, halogenated bases, such as 5-
racil and 5-iodouracil can be incorporated. The bases may also be alkylated, for
example, 7-methylguanosine can be incorporated in place of a guanosine residue. Non-natural
bases that yield successful inhibition can also be incorporated. The most preferred siRNA
modifications include 2'-deoxy-2'-fluorouridine or locked c acid (LNA) nucleotides and
RNA duplexes containing either phosphodiester or varying numbers of phosphorothioate
linkages. Such cations are known to one skilled in the art and are described, for example,
in Braasch et al., Biochemistry, 42: 7967-7975, 2003. Most of the useful modifications to the
siRNA molecules can be introduced using chemistries established for antisense ucleotide
technology. Preferably, the modifications involve minimal 2'-O-methyl modification, preferably
excluding such modification. Modifications also preferably exclude modifications of the free 5'-
yl groups of the siRNA. The Examples herein provide specific examples of RNA
interfering agents, such as shRNA molecules that ively target BCL11A mRNA.
rase (pol) III driven short hairpin RNAs (shRNAs) are most commonly
used in biological experimental settings. shRNAs mimic the structure of miRNA precursor
intermediates, and thus bypass the first cleavage step ed by Drosha. shRNAs can be
abundantly expressed to provide efficient knockdown. However at high multiplicities of
infection (MOI), oversaturation of the nous RNAi machinery has been reported in some
cases to be ated with cytotoxic effects due to the dysregulation of endogenous miRNAs (9-
11). Two components of microRNA processing, Exportin5 and Ago2, seem to limit the capacity
of this pathway, and overexpression of these proteins results in increased knockdown capacity
). Additionally, activation of innate immune responses triggered by small RNAs in a
sequence ic as well as non-specific manner may mediate cytotoxic side effects (16, 17),
reviewed in (18). These effects have resulted in increased mortality in mice in some
experimental transgenic model s reportedly as a direct side effect of shRNA overexpression
(14, 19).
For clinical translation of RNAi based therapeutics, alternative expression
systems utilizing polymerase II promoters will likely be required. This class of promoters allows
for utilization of appropriate regulatory elements for lineage or even cell-type specific
expression. It also could provide lower levels of expression compared to pol III promoters,
which may obviate over-saturation of the processing machinery that have been reported in cells
transduced at high MOIs). Complicating the use of pol II ers for shRNA expression,
requires embedding of the shRNA sequences into flanking sequences usually derived from
endogenous miRNA precursors for efficient processing. shRNAs flanked by a miRNA ld
mimic the structure of endogenous miRNAs (10, 20). To date, flanking regions derived from
human miRNA-30 and miRNA-223 have been widely used for incorporation of recombinant
shRNAs for expression in mammalian cells, and there have been numerous efforts to better
understand and to improve this expression gy (21). The latter miRNA has been shown to be
particularly effective when used as scaffold for shRNA sion in hematopoietic cells and
mediates substantial knockdown of target mRNAs as a result of efficient processing and low
passenger strand activity in several hematopoietic cell types (21, 22).
In this disclosure, the inventors utilized BCL11A as a target to study the
processing and optimization of shRNAmiRs for potential therapeutic applications. BCL11A is a
validated therapeutic target for reactivation of in gene and therefore HbF expression in the
major hemoglobinopathies, sickle cell disease (SCD) and β-thalassemias. Down modulation or
c deletion of BCL11A relieves γ-globin repression (23) and inactivation of BCL11A in the
erythroid lineage prevents SCD phenotype and organ toxicities in genetically engineered mice
(24). The mouse embryonic Hbb-y gene is a functional homolog of the human γ-globin gene,
and therefore serves as a convenient ate for assessment of the effect of BCL11A
knockdown in murine erythroleukemia (MEL) cells. Initially we observed a markedly reduced
efficiency of knockdown of BCL11A upon expression of shRNA using pol II-based as
compared with pol III-based vectors. Pol III and pol II shRNAmiR designs typically incorporate
21 base target site d sequences within the palindromic hairpin stem, but the ripts
from these two types of expression cassettes are expected to be processed differently (25). The
pol II shRNAmiR transcripts enter the RNAi processing pathway upstream of Drosha
processing, whereas the much shorter pol III products are expected to enter the pathway
downstream of Drosha and to be cleaved only at the loop end by Dicer. Based upon the
sequences of processed small RNAs derived from pol III and pol II promoters we ed that
pol III shRNA cassettes and pol II shRNAmiR cassettes yielded ent processed shRNAs
with respect to the relative positioning of the 21 base target-matched sequences. Redesigned
shRNAmiRs that ed the mature guide strand sequences produced by effective pol III-
driven shRNAs led to enhancement in processing efficiency and tion of the target mRNA.
Incorporation of these modifications into an erythroid-specific mammalian sion vector led
to significant knockdown of BCL11A protein and re-induction of fetal hemoglobin. This
gy also avoided ty in the hematopoietic stem cell and B cell lineage compartments
that accompanied pan-hematopoietic shRNA expression. In summary, the data demonstrate
critical features of RNA processing relevant to the use of shRNA in different vector contexts,
and also provide a strategy for lineage-specific gene knockdown that circumvents adverse
consequences of widespread sion. Our gs have ant implications for design of
microRNA embedded shRNAs and their application in RNAi based gene therapy approaches.
In one embodiment, the RNA interference agent is delivered or administered in a
pharmaceutically acceptable carrier. Additional r agents, such as mes, can be added
to the pharmaceutically acceptable carrier. In another embodiment, the RNA interference agent
is delivered by a vector encoding small or short hairpin RNA (shRNA) in a pharmaceutically
acceptable carrier to the cells in an organ of an individual. The shRNA is converted by the cells
after transcription into siRNA capable of targeting, for example, BCL11A.
In one embodiment, the RNA interference agent is a nucleic acid molecule
comprising the nucleotide sequence selected from the group consisting of SEQ ID NOS: 1-18,
and 25-44, or a synthetic BCL11A microRNA described herein.
In one embodiment, the vector is a regulatable vector, such as tetracycline
inducible vector. Methods described, for example, in Wang et al. Proc. Natl. Acad. Sci. 100:
106, using pTet-On vectors (BD Biosciences Clontech, Palo Alto, Calif.) can be used. In
one embodiment, the RNA interference agents used in the methods described herein are taken up
actively by cells in vivo ing intravenous ion, e.g., hydrodynamic injection, without
the use of a vector, illustrating efficient in vivo delivery of the RNA interfering . One
method to deliver the siRNAs is catheterization of the blood supply vessel of the target organ.
Other strategies for delivery of the RNA erence agents, e.g., the siRNAs or shRNAs used
in the methods described herein, may also be ed, such as, for example, delivery by a
vector, e.g., a plasmid or viral , e.g., a lentiviral vector. Such vectors can be used as
described, for example, in Xiao-Feng Qin et al. Proc. Natl. Acad. Sci. U.S.A., 100: 183-188.
Other delivery methods include delivery of the RNA ering agents, e.g., the siRNAs or
shRNAs described herein, using a basic peptide by conjugating or mixing the RNA ering
agent with a basic peptide, e.g., a fragment of a TAT peptide, mixing with cationic lipids or
formulating into particles. The RNA interference agents, e.g., the siRNAs targeting BCL11A
mRNA, may be delivered singly, or in combination with other RNA interference agents, e.g.,
siRNAs, such as, for e siRNAs directed to other cellular genes. BCL11A siRNAs may
also be administered in ation with other pharmaceutical agents which are used to treat or
prevent diseases or disorders associated with oxidative stress, especially respiratory diseases,
and more especially asthma. Synthetic siRNA molecules, including shRNA molecules, can be
obtained using a number of techniques known to those of skill in the art. For example, the
siRNA molecule can be chemically synthesized or recombinantly ed using methods
known in the art, such as using appropriately protected cleoside oramidites and a
conventional DNA/RNA synthesizer (see, e.g., Elbashir, S. M. et al. (2001) Nature 411:494-498;
Elbashir, S. M., W. Lendeckel and T. Tuschl (2001) Genes & Development 15:188-200;
Harborth, J. et al. (2001) J. Cell Science 114:4557-4565; Masters, J. R. et al. (2001) Proc. Natl.
Acad. Sci., USA 98:8012-8017; and Tuschl, T. et al. (1999) Genes & Development 13:3191-
3197). Alternatively, l commercial RNA synthesis suppliers are available including, but
not limited to, Proligo (Hamburg, y), Dharmacon ch (Lafayette, Colo., USA),
Pierce Chemical (part of Perbio Science, Rockford, Ill., USA), Glen Research (Sterling, Va.,
USA), ChemGenes (Ashland, Mass., USA), and Cruachem (Glasgow, UK). As such, siRNA
molecules are not overly difficult to synthesize and are readily provided in a quality suitable for
RNAi. In on, dsRNAs can be expressed as stem loop structures encoded by plasmid
s, retroviruses and lentiviruses (Paddison, P. J. et al. (2002) Genes Dev. 16:948-958;
McManus, M. T. et al. (2002) RNA 8:842-850; Paul, C. P. et al. (2002) Nat. Biotechnol. 20:505-
508; Miyagishi, M. et al. (2002) Nat. hnol. 20:497-500; Sui, G. et al. (2002) Proc. Natl.
Acad. Sci., USA 99:5515-5520; Brummelkamp, T. et al. (2002) Cancer Cell 2:243; Lee, N. S., et
al. (2002) Nat. Biotechnol. 20:500-505; Yu, J. Y., et al. (2002) Proc. Natl. Acad. Sci., USA
99:6047-6052; Zeng, Y., et al. (2002) Mol. Cell. 9:1327-1333; Rubinson, D. A., et al. (2003)
Nat. Genet. 33:401-406; Stewart, S. A., et al. (2003) RNA 9:493-501). These vectors generally
have a polIII promoter upstream of the dsRNA and can express sense and antisense RNA strands
separately and/or as a hairpin structures. Within cells, Dicer processes the short hairpin RNA
) into effective siRNA. The targeted region of the siRNA le described herein can
be selected from a given target gene sequence, e.g., a BCL11A coding sequence, beginning from
about 25 to 50 nucleotides, from about 50 to 75 nucleotides, or from about 75 to 100 nucleotides
downstream of the start codon. Nucleotide sequences may contain 5' or 3' UTRs and regions
nearby the start codon. One method of designing a siRNA molecule described herein es
identifying the 23 nucleotide sequence motif and selecting hits with at least 25%, 30%, 35%,
40%, 45%, 50%, 55%, 60%, 65%, 70% or 75% G/C content. Alternatively, if no such sequence
is found, the search may be extended using the motif NA(N21), where N can be any nucleotide.
In this situation, the 3' end of the sense siRNA may be converted to TT to allow for the
generation of a symmetric duplex with respect to the sequence composition of the sense and
antisense 3' overhangs. The antisense siRNA molecule may then be synthesized as the
complement to nucleotide ons 1 to 21 of the 23 nucleotide sequence motif. The use of
symmetric 3' TT overhangs may be advantageous to ensure that the small interfering
ribonucleoprotein particles s) are formed with approximately equal ratios of sense and
nse target RNA-cleaving siRNPs (Elbashir et al., (2001) supra and Elbashir et al., 2001
supra). Analysis of sequence databases, including but not limited to the NCBI, BLAST,
Derwent, and GenSeq as well as cially available oligosynthesis companies such as
OLIGOENGINE®, may also be used to select siRNA sequences against EST libraries to ensure
that only one gene is targeted.
Lentiviral vectors described herein e, but are not limited to, human
immunodeficiency virus (e.g., HIV-1, HIV-2), feline immunodeficiency virus (FIV), simian
immunodeficiency virus (SIV), bovine immunodeficiency virus (BIV), and equine infectious
anemia virus (EIAV). These s can be ucted and engineered using art-recognized
techniques to increase their safety for use in therapy and to include suitable sion elements
and therapeutic genes, such as those described below, which encode siRNAs for treating
conditions including, but not d to, hemoglobinopathies.
In consideration of the potential toxicity of lentiviruses, the vectors can be
designed in different ways to increase their safety in gene therapy applications. For example,
the vector can be made safer by separating the ary lentiviral genes (e.g., gag and pol) onto
separate s as described, for example, in U.S. Patent No. 6,365,150, the contents of which
are incorporated by reference herein. Thus, recombinant retrovirus can be constructed such that
the retroviral coding sequence (gag, pol, env) is replaced by a gene of interest rendering the
retrovirus replication defective. The replication defective retrovirus is then packaged into
virions through the use of a helper virus or a packaging cell line, by standard techniques.
Protocols for producing recombinant retroviruses and for infecting cells in vitro or in vivo with
such viruses can be found in Current ols in Molecular Biology, Ausubel, F. M. et al. (eds.)
Greene Publishing Associates, (1989), Sections 9.10-9.14 and other standard laboratory
manuals.
A major prerequisite for the use of viruses as gene delivery vectors is to ensure
the safety of their use, particularly with regard to the possibility of the spread of wild-type virus
in the cell tion. The pment packaging cell lines, which produce only replicationdefective
retroviruses, has increased the utility of retroviruses for gene therapy, and defective
retroviruses are well characterized for use in gene transfer for gene therapy purposes (for a
review see Miller, A. D. (1990) Blood 76:271). Accordingly, in one embodiment described
herein, packaging cell lines are used to propagate vectors (e.g., lentiviral vectors) described
herein to increase the titer of the vector virus. The use of ing cell lines is also considered
a safe way to propagate the virus, as use of the system reduces the likelihood that recombination
will occur to generate wild-type virus. In addition, to reduce toxicity to cells that caused by
expression of packaging proteins, packaging systems can be use in which the plasmids encoding
the packaging functions of the virus are only transiently transfected by, for example, al
means.
In another embodiment, the vector can be made safer by replacing certain
iral sequences with non-lentiviral sequences. Thus, lentiviral vectors of the present
disclosure may contain partial (e.g., split) gene lentiviral ces and/or non-lentiviral
sequences (e.g., ces from other retroviruses) as long as its function (e.g., viral titer,
infectivity, integration and ability to confer high levels and duration of therapeutic gene
expression) are not substantially reduced. ts which may be cloned into the viral vector
include, but are not limited to, er, packaging signal, , polypurine , and a
reverse response element (RRE).
In one embodiment of the disclosure, the LTR region is modified by replacing the
viral LTR promoter with a heterologous promoter. In one ment, the promoter of the 5'
LTR is replaced with a heterologous promoter. Examples of heterologous promoters which can
be used include, but are not limited to, a spleen focus-forming virus (SFFV) promoter, a
tetracycline-inducible (TET) promoter, a β-globin locus control region and a β-globin promoter
(LCR), and a cytomegalovirus (CMV) promoter.
In some embodiments, the lentiviral vectors of the disclosure also include vectors
which have been modified to improve upon safety in the use of the vectors as gene delivery
agents in gene y. In one embodiment described herein, an LTR , such as the 3'
LTR, of the vector is modified in the U3 and/or U5 regions, wherein a SIN vector is created.
Such modifications contribute to an increase in the safety of the vector for gene delivery
purposes. In one embodiment, the SIN vector described herein comprises a deletion in the 3'
LTR wherein a portion of the U3 region is replaced with an insulator element. The insulator
prevents the enhancer/promoter sequences within the vector from influencing the expression of
genes in the nearby , and vice/versa, to t the nearby genomic sequences from
influencing the expression of the genes within the . In a further embodiment described
herein, the 3' LTR is modified such that the U5 region is replaced, for example, with an ideal
) sequence. It should be noted that modifications to the LTRs such as modifications to
the 3' LTR, the 5' LTR, or both 3' and 5' LTRs, are also included in the present disclosure.
The promoter of the lentiviral vector can be one which is naturally (i.e., as it
occurs with a cell in vivo) or turally associated with the 5' flanking region of a particular
gene. Promoters can be derived from eukaryotic genomes, viral genomes, or synthetic
sequences. Promoters can be selected to be non-specific e in all tissues) (e.g., SFFV),
tissue specific (e.g., (LCR), regulated by natural regulatory processes, regulated by exogenously
d drugs (e.g., TET), or regulated by specific physiological states such as those promoters
which are activated during an acute phase response or those which are activated only in
replicating cells. miting examples of promoters bed herein include the spleen focusforming
virus promoter, a tetracycline-inducible promoter, a β-globin locus control region and a
β-globin promoter (LCR), a cytomegalovirus (CMV) promoter, retroviral LTR promoter,
galovirus immediate early promoter, SV40 promoter, and dihydrofolate reductase
promoter. The promoter can also be ed from those shown to specifically express in the
select cell types which may be found associated with ions including, but not limited to,
hemoglobinopathies. In one embodiment described herein, the promoter is cell specific such
that gene expression is restricted to red blood cells. Erythrocyte-specific expression is achieved
by using the human in promoter region and locus control region (LCR).
d practitioners will recognize that selection of the promoter to express the
cleotide of interest will depend on the vector, the nucleic acid cassette, the cell type to be
targeted, and the desired biological effect. Skilled practitioners will also recognize that in the
selection of a promoter, the parameters can include: achieving sufficiently high levels of gene
expression to achieve a physiological effect; maintaining a al level of gene expression;
achieving temporal regulation of gene expression; achieving cell type ic expression;
achieving pharmacological, endocrine, paracrine, or autocrine regulation of gene expression; and
preventing inappropriate or rable levels of expression. Any given set of selection
requirements will depend on the conditions but can be readily determined once the specific
requirements are ined. In one embodiment described herein, the promoter is cell-specific
such that gene expression is restricted to red blood cells. Erythrocyte-specific expression is
achieved by using the human β-globin promoter region and locus control region (LCR).
Standard techniques for the construction of expression s suitable for use
herein are well-known to those of ordinary skill in the art and can be found in such publications
as Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual, 2nd Ed. Cold Spring
Harbor, N.Y. A variety of strategies are ble for ligating fragments of DNA, the choice of
which depends on the nature of the termini of the DNA fragments and which choices can be
readily made by the skilled artisan.
Gene therapy vectors described herein, such as the ing lentiviral vectors,
can be used to s a variety of therapeutic siRNAs in transformed erythroid cells. In one
embodiment, the siRNA of interest to be sed in the vector is derived from a gene that can
be used to treat a hemoglobinopathy, such as an siRNA to BCL11A.
Particular gene therapy constructs described herein include, but are not limited to,
those shown in The three lentiviral vectors bed herein are schematically shown
with a stem of the shRNA containing BCL11A mRNA targeting sequence, while the loop is
-specific. All contain a fluorochrome marker (Venus) and are built into a selfinactivating
(SIN) delta-U3 LEGO backbone (Ferhse Lab, Germany). A constitutive knockdown
lentivirus, where the targeting shRNA is expressed via the very potent, ubiquitously
sed SFFV promoter, was used to assess functionality and toxicity of the targeting shRNA.
An inducible knock-down lentivirus, where the shRNA is expressed via a PGK ycline
inducible promoter, was used to assess functional, dose- and schedule-dependent s of the
targeting shRNA. A lineage-specific lentivirus, where the shRNA is expressed via a β-globin
LCR promoter landscape (HS2/3 DNA ensitive sites, Naldini Lab, Italy) is a therapeutic
option to validate in in vivo systems.. The LTR regions further comprise a U3 and U5 region, as
well as an R region. The U3 and U5 regions can be modified together or independently to create
a vector which is self-inactivating, thus increasing the safety of the vector for use in gene
delivery. The U3 and U5 regions can further be modified to comprise an insulator element.
The step of facilitating the tion of infectious viral particles in the cells may
be carried out using conventional techniques, such as rd cell culture growth techniques. If
desired by the skilled practitioner, lentiviral stock solutions may be prepared using the vectors
and methods described herein. Methods of preparing viral stock solutions are known in the art
and are illustrated by, e.g., Y. Soneoka et al. (1995) Nucl. Acids Res. 23:628-633, and N. R.
Landau et al. (1992) J. Virol. 66:5110-5113. In the method of producing a stock solution
described herein, lentiviral-permissive cells red to herein as producer cells) are transfected
with the vector system described herein. The cells are then grown under suitable cell culture
conditions, and the lentiviral particles collected from either the cells themselves or from the cell
media as described above. Suitable producer cell lines include, but are not limited to, the human
embryonic kidney cell line 293, the equine dermis cell line NBL-6, and the canine fetal thymus
cell line Cf2TH.
The step of collecting the infectious virus particles also can be carried out using
conventional techniques. For example, the infectious particles can be ted by cell lysis, or
collection of the supernatant of the cell culture, as is known in the art. Optionally, the collected
virus particles may be purified if desired. Suitable purification techniques are well known to
those skilled in the art.
Other methods relating to the use of viral s in gene therapy can be found in,
e.g., Kay, M. A. (1997) Chest 111(6 Supp.):1385-1425; Ferry, N. and Heard, J. M. (1998) Hum.
Gene Ther. -81; Shiratory, Y. et al. (1999) Liver 19:265-74; Oka, K. et al. (2000) Curr.
Opin. Lipidol. 11:179-86; Thule, P. M. and Liu, J. M. (2000) Gene Ther. 7:1744-52; Yang, N. S.
(1992) Crit. Rev. Biotechnol. 12:335-56; Alt, M. (1995) J. Hepatol. 23:746-58; Brody, S. L. and
Crystal, R. G. (1994) Ann. N.Y. Acad. Sci. -101; Strayer, D. S. (1999) Expert Opin.
Investig. Drugs 8:2159-2172; Smith-Arica, J. R. and tt, J. S. (2001) Curr. Cardiol. Rep.
3:43-49; and Lee, H. C. et al. (2000) Nature 408:483-8.
Retroviral vectors, including lentiviral vectors, as described above or cells
comprising the same, can be administered in vivo to ts by any suitable route, as is well
known in the art. The term “administration” refers to the route of introduction of a formulated
vector into the body. For example, administration may be intravascular, intraarterial,
intravenous, intramuscular, topical, oral, or by gene gun or hypospray mentation. Thus,
administration can be direct to a target tissue or through systemic delivery. Administration can
be direct injection into the bone marrow. Administration directly to the target tissue can involve
needle ion, hypospray, electroporation, or the gene gun. See, e.g., WO 93/18759, which is
incorporated by reference herein.
Alternatively, the iral vectors bed herein can be administered ex vivo
or in vitro to cells or tissues using standard transfection techniques well known in the art.
In one embodiment, the retroviral vectors described herein can also be transduced
into host cells, including embryonic stem cells, somatic stem cells, or progenitor cells.
Examples of progenitor host cells which can be transduced by the retroviral vectors described
herein include precursors of erythrocytes and hematopoietic stem cells. In another embodiment,
the host cell is an erythrocyte. uced host cells can be used as a method of achieving
oid-specific expression of the gene of interest in the treatment of hemoglobinopathies.
r aspect described herein pertains to pharmaceutical compositions of the
lentiviral vectors described herein. In one embodiment, the ition includes a lentiviral
vector in a therapeutically effective amount sufficient to treat or reduce the risk of developing
(e.g. ameliorate the symptoms of a hemoglobinopathy) and a pharmaceutically acceptable
carrier. A “therapeutically effective ” refers to an amount effective, at dosages and for
periods of time necessary, to achieve the desired therapeutic result, such as treatment or
prevention of a hemoglobinopathic condition. A therapeutically effective amount of lentiviral
vector may vary according to factors such as the disease state, age, sex, and weight of the
individual, and the ability of the lentiviral vector to elicit a desired response in the dual.
Dosage regimens may be adjusted to provide the m therapeutic response. A
therapeutically effective amount is also one in which any toxic or detrimental effects of the
lentiviral vector are outweighed by the eutically beneficial effects. The potential toxicity
of the iral vectors described herein can be d using cell-based assays or art
recognized animal models and a therapeutically effective modulator can be selected which does
not exhibit significant toxicity. In a preferred embodiment, a therapeutically effective amount of
a lentiviral vector is sufficient to treat a hemoglobinopathy.
Sterile injectable solutions can be prepared by orating lentiviral vector in
the required amount in an appropriate t with one or a combination of ingredients
enumerated above, as required, followed by filtered sterilization. Generally, sions are
prepared by incorporating the active compound into a sterile vehicle which ns a basic
dispersion medium and the required other ingredients from those enumerated above. In the case
of e s for the preparation of sterile injectable solutions, the preferred methods of
preparation are vacuum drying and freeze-drying which yields a powder of the active ingredient
plus any additional desired ingredient from a previously sterile-filtered solution thereof.
It is to be noted that dosage values may vary with the severity of the condition to
be alleviated. It is to be further understood that for any particular subject, specific dosage
regimens can be adjusted over time according to the individual need and the professional
judgment of the person administering or ising the administration of the compositions, and
that dosage ranges set forth herein are exemplary only and are not intended to limit the scope or
practice of the claimed composition. In one embodiment, the dosage is ranges from 103-108 viral
particles / 50 kg weight. In other embodiments, the dosage is ranges from 5 viral particles
/ 50 kg weight, 104-106 viral particles / 50 kg weight, 105-107 viral particles / 50 kg weight, 103-
108 viral particles / 50 kg weight. In one ment, the dosage is about 104 viral particles / 50
kg weight.
The amount of viral vector in the composition may vary according to factors such
as the disease state, age, sex, and weight of the individual. Dosage regimens may be adjusted to
provide the optimum eutic response. For example, a single bolus may be administered,
several divided doses may be administered over time or the dose may be proportionally reduced
or increased as indicated by the exigencies of the therapeutic situation. It is especially
advantageous to formulate parenteral compositions in dosage unit form for ease of
administration and uniformity of dosage. Dosage unit form as used herein refers to physically
discrete units suited as unitary dosages for the mammalian subjects to be treated; each unit
containing a predetermined quantity of active compound calculated to produce the desired
therapeutic effect in association with the ed pharmaceutical carrier. The ication for
the dosage unit forms described herein are dictated by and directly dependent on (a) the unique
characteristics of the active compound and the particular eutic effect to be achieved, and
(b) the limitations inherent in the art of compounding such an active nd for the treatment
of sensitivity in individuals. However, for any given case, an appropriate “effective amount”
can be determined by one of ordinary skill in the art using only routine experimentation.
The present sure contemplates, in particular embodiments, cells genetically
modified to express the therapeutic polypeptides and inhibitory RNAs contemplated herein, for
use in the treatment of hemoglobinopathies. As used herein, the term "genetically engineered"
or "genetically modified" refers to the addition, deletion, or modification of the genetic material
in a cell. The terms, "genetically modified cells," "modified cells," and, "redirected cells," are
used interchangeably. In particular embodiments, cells transduced with vectors contemplated
herein are genetically modified. As used , the term "gene therapy" refers to the
introduction of extra c material in the form of DNA or RNA into the total genetic material
in a cell that restores, corrects, or modifies the cell's logy to provide a desired eutic
outcome.
In various embodiments, the genetically modified cells contemplated herein are
transduced in vitro or ex vivo with vectors described herein, and ally expanded ex vivo.
The transduced cells are then administered to a subject in need of gene therapy.
Cells suitable for transduction and administration in the gene y methods
contemplated herein include, but are not limited to stem cells, progenitor cells, and differentiated
cells. In certain embodiments, the transduced cells are embryonic stem cells, bone marrow stem
cells, cal cord stem cells, placental stem cells, mesenchymal stem cells, hematopoietic
stem cells, erythroid progenitor cells, and erythroid cells.
Hematopoietic stem cells (HSCs) give rise to committed hematopoietic
progenitor cells (HPCs) that are capable of generating the entire repertoire of mature blood cells
over the lifetime of an organism. The term "hematopoietic stem cell" or "HSC" refers to
multipotent stem cells that give rise to the all the blood cell types of an organism, including
d (e.g., monocytes and macrophages, neutrophils, basophils, eosinophils, ocytes,
megakaryocytes/platelets, dendritic cells), and lymphoid lineages (e.g., T-cells, B-cells, NK-
cells), and others known in the art (See Fei, R., et al., U.S. Patent No. 5,635,387; McGlave, et
al., U.S. Patent No. 5,460,964; Simmons, P., et al., U.S. Patent No. 5,677,136; Tsukamoto, et al.,
U.S. Patent No. 5,750,397; tz, et al., U.S. Patent No. 5,759,793; DiGuisto, et al., U.S.
Patent No. 5,681,599; Tsukamoto, et al., U.S. Patent No. 5,716,827). When transplanted into
lethally ated animals or , hematopoietic stem and progenitor cells can repopulate
the erythroid, neutrophil-macrophage, megakaryocyte and lymphoid hematopoietic cell pool.
In some embodiments, the transduced cells are hematopoietic stem and/or
progenitor cells isolated from bone marrow, umbilical cord blood, or peripheral circulation. In
ular embodiments, the transduced cells are hematopoietic stem cells isolated from bone
, umbilical cord blood, or peripheral circulation.
In one embodiment, the hematopoietic cells are CD34+ cells.
In one embodiment, the hematopoietic cells are erythroid itor cells.
In one embodiment, the hematopoietic cells are oid cells.
Cells described herein can be autologous/autogeneic ("self") or non-autologous
("non-self," e.g., allogeneic, syngeneic or xenogeneic). "Autologous," as used herein, refers to
cells from the same subject. "Allogeneic," as used herein, refers to cells of the same species that
differ genetically to the cell in comparison. "Syngeneic," as used herein, refers to cells of a
different subject that are genetically identical to the cell in comparison. "Xenogeneic," as used
, refers to cells of a different species to the cell in comparison. In preferred embodiments,
the cells described herein are allogeneic. An ted cell" refers to a cell that has been
obtained from an in vivo tissue or organ and is substantially free of extracellular matrix.
Illustrative examples of cally modified cells suitable for cell-based
therapies contemplated herein include, but are not limited to: embryonic stem cells, bone
marrow stem cells, umbilical cord stem cells, placental stem cells, mesenchymal stem cells,
hematopoietic stem cells, hematopoietic progenitor cells, myeloid progenitors, erythroid
progenitors, and other oid cells.
In preferred embodiments, cells suitable for cell-based therapies contemplated
herein include, but are not limited to: hematopoietic stem or progenitor cells, proerythroblasts,
ilic erythroblasts, polychromatic erythroblasts, orthochromatic erythroblasts,
polychromatic erythrocytes, and erythrocytes (RBCs), or any combination thereof.
Methods of Treating, or ng a Risk of Developing, a Hemoglobinopathy
Also described are improved compositions and methods for increasing HbF
production in a cell, by administering vectors that inhibit expression of BCL11A. The data
demonstrate that inhibition of BCL11A leads to increased expression from the γ-globin genes.
As disclosed herein, it is an object of the present ion to provide compositions and methods
for increasing fetal hemoglobin levels in a cell. In some embodiments, the cell is an embryonic
stem cell, a somatic stem cell, a progenitor cell, a bone marrow cell, a hematopoietic stem cell, a
hematopoietic progenitor cell or a progeny thereof.
Accordingly, one aspect bed herein es methods for increasing fetal
hemoglobin levels expressed by a cell, comprising the steps of ting an embryonic stem
cell, a somatic stem cell, a progenitor cell, a bone marrow cell, a hematopoietic stem cell, or a
hematopoietic with an effective amount of a composition comprising at least a virus or vector
comprising a nucleic acid molecule described herein, whereby the sion of BCL11A is
reduced and the fetal hemoglobin expression is increased in the cell, or its progeny, relative to
the cell prior to such contacting. In one embodiment, the vector or virus expresses an RNA
interference agent which is a BCL11A microRNA which inhibits BCL11A, thereby reducing the
expression of BCL11A.
In connection with contacting a cell with an inhibitor of BCL11A, “increasing the
fetal obin levels” in a cell indicates that HbF is at least 5% higher in populations treated
with a BCL11A inhibitor, than in a comparable, control population, wherein no BCL11A
inhibitor is present. It is preferred that the percentage of HbF expression in a BCL11A inhibitor
treated population is at least 10% , at least 20% higher, at least 30% , at least 40%
higher, at least 50% higher, at least 60% higher, at least 70% , at least 80% higher, at least
90% higher, at least 1-fold , at least 2-fold higher, at least 5-fold higher, at least 10 fold
higher, at least 100 fold higher, at least 1000-fold higher, or more than a control treated
population of able size and e conditions. The term “control treated population” is
used herein to describe a tion of cells that has been treated with identical media, viral
induction, nucleic acid sequences, temperature, confluency, flask size, pH, etc., with the
exception of a non-targeting oligonucleotide.
In some embodiments of any of the methods described herein, the subject is
suspected of , is at risk of having, or has a hemoglobinopathy, e.g., SCD or THAL. It is
well within the skills of an ry tioner to recognize a subject that has, or is at risk of
developing, a hemoglobinopathy.
The subjects can also be those undergoing any of a variety of onal therapy
treatments. Thus, for example, subjects can be those being d with oxygen, hydroxyurea,
folic acid, or a blood transfusion.
Methods of delivering RNA erence agents, e.g., an siRNA, or vectors
containing an RNA interference agent, to the target cells, e.g., erythrocytes or other desired
target cells, for uptake include injection of a composition containing the RNA interference
agent, e.g., an siRNA, or directly ting the cell, e.g., a erythrocyte, with a composition
comprising an RNA interference agent, e.g., an siRNA. In r embodiment, RNA
interference agent, e.g., an siRNA may be injected directly into any blood vessel, such as vein,
artery, venule or arteriole, via, e.g., hydrodynamic injection or catheterization. Administration
may be by a single injection or by two or more injections. The RNA interference agent is
delivered in a pharmaceutically acceptable carrier. One or more RNA interference agent may be
used simultaneously. In one preferred ment, only one siRNA that targets human
BCL11A is used. In one embodiment, specific cells are targeted with RNA interference,
limiting potential side effects of RNA interference caused by non-specific ing of RNA
interference. The method can use, for example, a complex or a fusion molecule comprising a
cell targeting moiety and an RNA interference g moiety that is used to deliver RNA
interference ively into cells. For example, an antibody-protamine fusion protein when
mixed with siRNA, binds siRNA and selectively delivers the siRNA into cells expressing an
antigen ized by the antibody, resulting in silencing of gene expression only in those cells
that express the antigen. The siRNA or RNA interference-inducing molecule binding moiety is
a protein or a nucleic acid g domain or fragment of a protein, and the binding moiety is
fused to a portion of the targeting moiety. The location of the targeting moiety can be either in
the carboxyl-terminal or amino-terminal end of the construct or in the middle of the fusion
protein. A viral-mediated delivery mechanism can also be employed to deliver siRNAs to cells
in vitro and in vivo as described in Xia, H. et al. (2002) Nat Biotechnol 20(10):1006). Plasmidor
viral-mediated delivery mechanisms of shRNA may also be employed to deliver shRNAs to
cells in vitro and in vivo as described in Rubinson, D. A., et al. ) Nat. Genet. 33:401-406)
and Stewart, S. A., et al. ((2003) RNA 9:493-501). The RNA erence agents, e.g., the
siRNAs or shRNAs, can be introduced along with ents that perform one or more of the
following activities: enhance uptake of the RNA interfering agents, e.g., siRNA, by the cell, e.g.,
lymphocytes or other cells, inhibit annealing of single strands, stabilize single strands, or
ise facilitate ry to the target cell and increase inhibition of the target gene, e.g.,
BCL11A. The dose of the particular RNA interfering agent will be in an amount necessary to
effect RNA interference, e.g., post translational gene silencing, of the particular target gene,
y leading to inhibition of target gene expression or tion of activity or level of the
protein encoded by the target gene.
In one embodiment of any methods described herein, the embryonic stem cell,
somatic stem cell, progenitor cell, bone marrow cell, or hematopoietic progenitor cell, or
hematopoietic stem cell (HSC) is contacted ex vivo or in vitro. In a specific embodiment, the
cell being contacted is a cell of the erythroid e. In one embodiment, the composition
ts BCL11A expression.
In one embodiment of any methods described herein, the embryonic stem cell,
somatic stem cell, progenitor cell, bone marrow cell, or hematopoietic progenitor cell, or HSC is
isolated from the subject prior to contacting with the composition described herein or contacting
with the virus or vector carrying a nucleic acid le comprising a nucleic acid sequence
selected from a group consisting of SEQ ID NOS:1-10,13-18, 25-44, or contacting with the virus
or vector sing a synthetic BCL11A microRNA described herein.
Mature blood cells have a finite lifespan and must be continuously replaced
throughout life. Blood cells are produced by the proliferation and differentiation of a very small
population of pluripotent hematopoietic stem cells (HSCs) that also have the ability to replenish
themselves by self-renewal. HSCs are multipotent, self-renewing progenitor cells that develop
from rmal hemangioblast cells. HSCs are the blood cells that give rise to all the other
blood cells, that includes all the differentiated blood cells from the erythroid, lymphoid and
myeloid lineages. HSCs are located in the adult bone marrow, peripheral blood, and umbilical
cord blood.
During entiation, the progeny of HSCs progress through various
ediate maturational stages, generating multi-potential hematopoietic progenitor cells and
lineage-committed hematopoietic progenitor cells, prior to reaching maturity. Bone marrow
(BM) is the major site of hematopoiesis in humans and, under normal conditions, only small
numbers of HSCs and hematopoietic progenitor cells can be found in the peripheral blood (PB).
Treatment with cytokines (in particular granulocyte colony-stimulating factor; G-CSF),
myelosuppressive drugs used in cancer treatment, and compounds that disrupt the interaction
between hematopoietic cells and BM stromal cells can rapidly mobilize large numbers of stem
and progenitor cells into the circulation.
“Hematopoietic progenitor cell” as the term is used herein, refers to cells of a
hematopoietic stem cell lineage that give rise to all the blood cell types including the myeloid
(monocytes and macrophages, neutrophils, basophils, eosinophils, erythrocytes,
ryocytes/platelets, dendritic cells), and the id lineages (T-cells, B-cells, NK-
cells). A “cell of the erythroid lineage” indicates that the cell being ted is a cell that
undergoes erythropoeisis such that upon final differentiation it forms an erythrocyte or red blood
cell (RBC). Such cells belong to one of three es, erythroid, lymphoid, and myeloid,
originating from bone marrow hematopoietic progenitor cells. Upon exposure to specific
growth factors and other components of the hematopoietic microenvironment, hematopoietic
progenitor cells can mature through a series of intermediate differentiation ar types, all
ediates of the erythroid e, into RBCs. Thus, cells of the “erythroid lineage,” as the
term is used herein, comprise hematopoietic progenitor cells, rubriblasts, prorubricytes,
erythroblasts, metarubricytes, reticulocytes, and erythrocytes.
In some embodiment of any methods described herein, the hematopoietic
progenitor cell has at least one of the cell surface marker characteristic of hematopoietic
progenitor cells: CD34+, CD59+, Thy1/CD90+, /-, and C-kit/CD117+. Preferably, the
hematopoietic progenitor cells have several of these s.
In some embodiment of any s bed herein, the hematopoietic
progenitor cells of the erythroid lineage have the cell surface marker characteristic of the
erythroid lineage: CD71 and Ter119.
In some embodiment of any s described herein, the HSC has at least one
of the cell surface marker characteristic of hematopoietic progenitor cells: CD34+, CD59+,
Thy1/CD90+, CD38lo/-, and CD117+.
The HSCs, similar to the hematopoietic progenitor cells, are capable of
proliferation and giving rise to more itor cells having the ability to generate a large
number of mother cells that can in turn give rise to differentiated or differentiable daughter cells.
The daughter cells themselves can be induced to proliferate and produce progeny that
subsequently differentiate into one or more mature cell types, while also retaining one or more
cells with parental developmental potential. The term “stem cell” refers then, to a cell with the
capacity or potential, under particular circumstances, to differentiate to a more specialized or
differentiated phenotype, and which retains the capacity, under certain circumstances, to
proliferate without substantially entiating. In one embodiment, the term progenitor or stem
cell refers to a generalized mother cell whose descendants (progeny) specialize, often in
different ions, by differentiation, e.g., by acquiring completely individual characters, as
occurs in progressive diversification of embryonic cells and s. Cellular differentiation is a
complex process lly occurring through many cell divisions. A differentiated cell may
derive from a multipotent cell which itself is derived from a multipotent cell, and so on. While
each of these otent cells may be ered stem cells, the range of cell types each can
give rise to may vary considerably. Some differentiated cells also have the capacity to give rise
to cells of greater developmental potential. Such capacity may be natural or may be induced
artificially upon treatment with various factors. In many biological instances, stem cells are also
potent” because they can produce progeny of more than one distinct cell type, but this is
not required for “stem-ness.” Self-renewal is the other classical part of the stem cell definition,
and it is essential as used in this document. In theory, self-renewal can occur by either of two
major isms. Stem cells may divide asymmetrically, with one daughter ing the stem
state and the other daughter expressing some distinct other specific on and phenotype.
Alternatively, some of the stem cells in a population can divide symmetrically into two stems,
thus maintaining some stem cells in the population as a whole, while other cells in the
population give rise to differentiated progeny only. Generally, “progenitor cells” have a ar
phenotype that is more primitive (i.e., is at an r step along a pmental pathway or
progression than is a fully differentiated cell). Often, progenitor cells also have significant or
very high proliferative potential. Progenitor cells can give rise to multiple distinct entiated
cell types or to a single differentiated cell type, depending on the developmental pathway and on
the environment in which the cells develop and differentiate.
In one embodiment of any methods described herein, the hematopoietic stem cell
or hematopoietic progenitor cell is collected from peripheral blood, cord blood, chorionic villi,
amniotic fluid, placental blood, or bone marrow.
In one embodiment of any methods described herein, the embryonic stem cell,
somatic stem cell, progenitor cell, or bone marrow cell is collected from peripheral blood, cord
blood, chorionic villi, amniotic fluid, placental blood, or bone marrow.
Peripheral blood itor cells (PBPC) have become the preferred source of
hematopoetic progenitor cells for neic and autologous transplantation because of technical
ease of collection and shorter time required for engraftment. Traditionally, ocyte-colony
stimulating factor (G-CSF) has been used to stimulate more PBPC and release of hematopoetic
progenitor cells from the bone marrow. Although regimens using G-CSF usually succeed in
collecting adequate numbers of PBPC from healthy donors, 5%-10% will ze stem cells
poorly and may require multiple large volume apheresis or bone marrow harvesting.
In some embodiments of any methods described herein, the embryonic stem cell,
somatic stem cell, progenitor cell, bone marrow cell, or hematopoietic itor cell, or HSC is
selected for the CD34+ surface marker prior to the contacting.
Accordingly, in one embodiment of any methods bed herein, the isolated
CD34+ embryonic stem cell, isolated CD34+ somatic stem cell, isolated CD34+ progenitor cell,
isolated CD34+ bone marrow cell, isolated CD34+ hematopoietic progenitor cell, or isolated
CD34+ HSC is contacted with the composition described herein or contacted with the virus or
vector carrying a nucleic acid le comprising a nucleic acid sequence selected from a
group consisting of SEQ ID NOS:1-10,13-18, 25-44, or ted with the virus or vector
expressing a synthetic BCL11A microRNA described herein.
In one embodiment of any s described herein, the embryonic stem cell,
c stem cell, itor cell, bone marrow cell, or hematopoietic progenitor cell, or HSC is
cryopreserved prior to any contacting with the composition described herein or contacting with
the virus or vector carrying a nucleic acid molecule comprising a c acid sequence selected
from a group consisting of SEQ ID NOS:1-10,13-18, 25-44, or ting with the virus or
vector expressing a synthetic BCL11A microRNA described herein.
In one embodiment of any methods described herein, the contacting is in vitro, ex
vivo or in vivo.
In one embodiment of any methods described , the contacting is repeated at
least once. That is, after the initial first contacting of the embryonic stem cell, somatic stem cell,
progenitor cell, bone marrow cell, or hematopoietic progenitor cell, or HSC with the
composition described herein or contacting with the virus or vector carrying a nucleic acid
molecule comprising a nucleic acid sequence selected from a group consisting of SEQ ID
NOS:1-10,13-18, 25-44, or ting with the virus or vector expressing a synthetic BCL11A
microRNA described herein, the cell is washed, and the washed cell is then contacted for a
second time with the composition described herein or contacted with the virus or vector carrying
a nucleic acid molecule comprising a c acid sequence selected from a group consisting of
SEQ ID NOS:1-10,13-18, 25-44, or contacted with the virus or vector expressing a synthetic
BCL11A microRNA described herein.
In other embodiments, the contacting is repeated at least twice after the l
first contacting.
In one embodiment of any methods described herein, after the contacting, the
contacted embryonic stem cell, somatic stem cell, progenitor cell, bone marrow cell, or
hematopoietic itor cell, or HSC is cryopreserved prior to use, for example, ex vivo
expansion and/or implantation into a subject.
In one embodiment of any methods bed herein, after the contacting, the
contacted nic stem cell, somatic stem cell, progenitor cell, bone marrow cell, or
hematopoietic progenitor cell, or HSC is culture expanded ex vivo prior to use, for example,
cryopreservation, and/or implantation/engraftment into a subject.
In one embodiment of any s described herein, after the contacting, the
ted embryonic stem cell, somatic stem cell, progenitor cell, bone marrow cell, or
hematopoietic progenitor cell, or HSC is differentiated in culture ex vivo prior to use, for
example, cryopreservation, and/or implantation/engraftment into a subject.
In the context of cell ontogeny, the adjective “differentiated,” or “differentiating”
is a relative term. A “differentiated cell” is a cell that has progressed r down the
developmental pathway than the cell it is being ed with. Thus, stem cells can
differentiate to lineage-restricted precursor cells (such as a poietic progenitor cell), which
in turn can differentiate into other types of precursor cells further down the pathway (such as an
erthyrocyte precursor), and then to an end-stage differentiated cell, such as an erthyrocyte, which
plays a characteristic role in a certain tissue type, and may or may not retain the capacity to
proliferate further.
In one embodiment, the inhibitor of BCL11A expression is a BCL11A specific
RNA interference agent, or a vector encoding said BCL11A specific RNA interference agent. In
one specific embodiment, the RNA interference agent comprises one or more of the nucleotide
sequences of SEQ ID NOS:1-10,13-18, 25-44.
A “nucleic acid,” as described herein, can be RNA or DNA, and can be single or
double stranded, and can be selected, for example, from a group ing: nucleic acid
encoding a protein of interest, oligonucleotides, c acid analogues, for example peptidenucleic
acid (PNA), pseudo-complementary PNA (pc-PNA), and locked nucleic acid (LNA).
Such nucleic acid sequences include, for example, but are not limited to, nucleic acid sequence
encoding proteins, for example that act as transcriptional repressors, antisense molecules,
ribozymes, small inhibitory nucleic acid sequences, for example but are not limited to RNAi,
shRNAi, siRNA, microRNAi (miRNA), and antisense oligonucleotides.
As disclosed herein, it is an object of the present invention to provide a method
for sing fetal hemoglobin levels in a t.
Accordingly, one aspect described herein includes a method for increasing fetal
hemoglobin levels in a t in need f, the method comprising the step of contacting a
hematopoietic progenitor cell or a HSC in the subject with an effective amount of a composition
sing an inhibitor of , whereby HbF expression is increased, ve to
expression prior to such contacting. In one embodiment, the inhibitor of BCL11A is an RNA
interference agent which comprises one or more of the nucleotide sequences of SEQ ID NOS:1-
,13-18, 25-44, or a synthetic BCL11A microRNA described herein.
In connection with contacting a cell in a subject with an inhibitor of BCL11A,
“increasing HbF levels in a subject” indicates that HbF in the subject is at least 5% higher in
populations treated with a BCL11A inhibitor, than a comparable, control population, wherein no
BCL11A inhibitor is present. It is preferred that the fetal hemoglobin expression in a BCL11A
inhibitor treated subject is at least 10% higher, at least 20% higher, at least 30% higher, at least
40% higher, at least 50% higher, at least 60% higher, at least 70% higher, at least 80% higher, at
least 90% higher, at least 1-fold higher, at least 2-fold higher, at least 5-fold , at least 10
fold higher, at least 100 fold higher, at least 1000-fold higher, or more than a comparable control
d subject. The term “comparable control treated subject” is used herein to be a
subject that has been d identically, with the exception of the addition of a non-targeting
oligonucleotide.
Accordingly, in one ment, the subject has been diagnosed with a
hemoglobinopathy. In a r ment, the hemoglobinopathy is a SCD. As used herein,
SCD can be sickle cell anemia, sickle-hemoglobin C disease (HbSC), sickle beta-plusthalassaemia
(HbS/β+), or sickle beta-zero-thalassaemia (HbS/β0). In another preferred
embodiment, the hemoglobinopathy is THAL.
The treatment described herein ameliorates one or more ms associated
with the disorder by increasing the amount of fetal hemoglobin in the dual. Symptoms
typically associated with a hemoglobinopathy, include for example, anemia, tissue hypoxia,
organ dysfunction, abnormal hematocrit values, ineffective erythropoiesis, abnormal reticulocyte
(erythrocyte) count, abnormal iron load, the presence of ring sideroblasts, splenomegaly,
hepatomegaly, impaired peripheral blood flow, dyspnea, increased hemolysis, jaundice, anemic
pain crises, acute chest syndrome, splenic sequestration, priapism, stroke, hand-foot syndrome,
and pain such as angina pectoris.
In one ment, the hematopoietic progenitor cell or HSC is contacted ex
vivo or in vitro, and the cell or its progeny is administered to the subject. In a r
embodiment, the hematopoietic progenitor cell is a cell of the erythroid e.
In one embodiment, the hematopoietic progenitor cell or HSC is contacted with a
ition comprising of an tor of BCL11A and a pharmaceutically acceptable carrier or
diluent. In one embodiment, the composition is administered by injection, infusion, instillation,
or ingestion. In one embodiment, the composition is administered by direct injection into the
bone marrow.
In one embodiment of any one method described, the gene therapy method is
used to treat, prevent, or ameliorate a hemoglobinopathy is selected from the group ting
of: hemoglobin C disease, hemoglobin sickle cell disease (SCD), sickle cell anemia, hereditary
anemia, thalassemia, β-thalassemia, thalassemia major, thalassemia intermedia, α-thalassemia,
and hemoglobin H disease.
In various embodiments of any one method described, the retroviral vectors are
stered by direct injection to a cell, , or organ of a subject in need of gene therapy, in
vivo. In various other ments of any one method described, cells are transduced in vitro or
ex vivo with vectors bed herein, and optionally expanded ex vivo. The transduced cells are
then administered to a subject in need of gene therapy.
A "subject," as used herein, includes any animal that ts a symptom of a
nic disease, disorder, or condition that can be treated with the gene therapy vectors, cellbased
therapeutics, and methods disclosed elsewhere herein. In preferred embodiments, a
subject includes any animal that exhibits symptoms of a disease, disorder, or condition of the
hematopoietic , e.g., a hemoglobinopathy, that can be treated with the gene therapy
vectors, cell-based therapeutics, and s contemplated herein. Suitable ts (e.g.,
patients) include laboratory animals (such as mouse, rat, rabbit, or guinea pig), farm animals,
and domestic animals or pets (such as a cat or dog). Non-human primates and, preferably,
human patients, are included. Typical subjects include animals that t aberrant s
(lower or higher amounts than a "normal" or "healthy" subject) of one or more physiological
activities that can be modulated by gene therapy.
In one ment, as used herein "treatment" or "treating," includes any
cial or desirable effect on the symptoms or pathology of a disease or pathological
condition, and may include even minimal reductions in one or more measurable markers of the
disease or condition being treated. In another embodiment, treatment can involve optionally
either the reduction or amelioration of symptoms of the disease or ion, or the ng of
the progression of the disease or ion. "Treatment" does not arily indicate complete
eradication or cure of the disease or condition, or associated symptoms thereof.
In one embodiment, as used herein, "prevent," and similar words such as
"prevented," "preventing" etc., te an approach for preventing, inhibiting, or reducing the
likelihood of the occurrence or recurrence of, a disease or condition. In another embodiment,
the term refers to delaying the onset or ence of a e or condition or delaying the
occurrence or ence of the symptoms of a disease or ion. In another embodiment, as
used herein, ntion" and similar words includes reducing the intensity, effect, symptoms
and/or burden of a e or condition prior to onset or recurrence of the disease or condition.
In one embodiment of any one method described, the method further comprises
selecting a subject in need of the gene therapy described. For example, a subject exhibiting
symptoms or cytology of a hemoglobinopathy is selected from the group consisting of
hemoglobin C disease, hemoglobin sickle cell disease (SCD), sickle cell anemia, hereditary
anemia, thalassemia, β-thalassemia, thalassemia major, thalassemia intermedia, α-thalassemia,
and hemoglobin H disease. Alternatively, the subject carrys a genetic mutation that is associated
with a hemoglobinopathy, a genetic mutation described herein. For example, a subject diagnosis
of SCD with genotype HbSS, HbS/β0 thalassemia, HbSD, or HbSO, and/or with HbF <10% by
electrophoresis.
In various embodiments of any one method bed, a subject in need of gene
therapy is administered a population of cells comprising an ive amount of genetically
modified cells contemplated herein. That is a genetically modified cells that express one or more
of the nucleotide sequences of SEQ ID NOS:1-10,13-18, 25-44, or a synthetic BCL11A
microRNA described herein.
As used herein, the term "amount" refers to "an amount effective" or "an ive
amount" of a virus or transduced therapeutic cell to achieve a beneficial or desired lactic
or therapeutic result, including clinical results.
A ylactically effective amount" refers to an amount of a virus or
transduced therapeutic cell effective to achieve the desired prophylactic result. Typically but not
necessarily, since a prophylactic dose is used in subjects prior to or at an earlier stage of disease,
the prophylactically effective amount is less than the therapeutically ive amount.
A "therapeutically effective amount" of a virus or transduced therapeutic cell may
vary according to factors such as the disease state, age, sex, and weight of the individual, and the
ability of the stem and progenitor cells to elicit a desired response in the individual. A
therapeutically effective amount is also one in which any toxic or detrimental effects of the virus
or transduced therapeutic cells are outweighed by the therapeutically beneficial effects. The
term "therapeutically effective amount" includes an amount that is effective to "treat" a subject
(e.g., a patient).
In one embodiment, described is a method of ing a transduced cell to a
subject that comprises administering, e.g., parenterally, one or more cells transduced with a
vector contemplated herein into the subject. In one embodiment, the vector is one that carrys
one or more of the nucleotide sequences of SEQ ID NOS:1-10,13-18, 25-44, or a tic
BCL11A microRNA described herein.
In a particular embodiment, a method of ting, rating, or treating a
hemoglobinopathy in a subject is described. The method comprises administering a population
of cells comprising hematopoietic cells transduced with a vector contemplated . In one
embodiment, the vector is one that carrys one or more of the nucleotide sequences of SEQ ID
NOS:1-10,13-18, 25-44, or a synthetic BCL11A microRNA described herein.
In ular embodiments, a population of cells administered to a subject
comprises hematopoietic stem or progenitor cells, proerythroblasts, basophilic erythroblasts,
polychromatic erythroblasts, orthochromatic erythroblasts, polychromatic erythrocytes, and
erythrocytes (RBCs), or any combination thereof, and any proportion of which may be
genetically ed by the vectors contemplated herein. In one embodiment, the vector is one
that carrys one or more of the tide ces of SEQ ID NOS:1-10,13-18, 25-44, or a
synthetic BCL11A microRNA described herein.
The genetically modified cells may be administered as part of a bone marrow or
cord blood transplant in an individual that has or has not undergone bone marrow ablative
therapy. In one embodiment, cally modified cells contemplated herein are stered in
a bone marrow transplant to an individual that has undergone chemoablative or radioablative
bone marrow therapy.
In one embodiment, a dose of genetically modified cells is delivered to a subject
intravenously. In one embodiment, genetically modified hematopoietic cells are intravenously
administered to a t.
In particular embodiments, patients e a dose of genetically modified cells,
e.g., hematopoietic stem cells, of about 1 x 105 cells/kg, about 5 x 105 cells/kg, about 1 x 106
cells/kg, about 2 x 106 cells/kg, about 3 x 106 cells/kg, about 4 x 106 kg, about 5 x 106
cells/kg, about 6 x 106 cells/kg, about 7 x 106 kg, about 8 x 106 cells/kg, about 9 x 106
cells/kg, about 1 x 107 kg, about 5 x 107 cells/kg, about 1 x 108 cells/kg, or more in one
single intravenous dose. In certain embodiments, patients receive a dose of genetically modified
cells, e.g., hematopoietic stem cells, of at least 1 x 105 cells/kg, at least 5 x 105 cells/kg, at least 1
x 106 cells/kg, at least 2 x 106 cells/kg, at least 3 x 106 cells/kg, at least 4 x 106 cells/kg, at least 5
x 106 cells/kg, at least 6 x 106 cells/kg, at least 7 x 106 cells/kg, at least 8 x 106 cells/kg, at least 9
x 106 cells/kg, at least 1 x 107 kg, at least 5 x 107 cells/kg, at least 1 x 108 cells/kg, or more
in one single intravenous dose.
In an additional embodiment, patients receive a dose of genetically modified
cells, e.g., hematopoietic stem cells, of about 1 x 105 kg to about 1 x 108 cells/kg, about 1 x
106 kg to about 1 x 108 cells/kg, about 1 x 106 cells/kg to about 9 x 106 cells/kg, about 2 x
106 cells/kg to about 8 x 106 cells/kg, about 2 x 106 cells/kg to about 8 x 106 kg, about 2 x
106 cells/kg to about 5 x 106 cells/kg, about 3 x 106 cells/kg to about 5 x 106 cells/kg, about 3 x
106 cells/kg to about 4 x 108 cells/kg, or any intervening dose of cells/kg.
In various ments, the methods described herein provide more robust and
safe gene y than existing methods and comprise administering a population or dose of
cells comprising about 5% transduced cells, about 10% transduced cells, about 15% transduced
cells, about 20% transduced cells, about 25% transduced cells, about 30% transduced cells,
about 35% transduced cells, about 40% transduced cells, about 45% transduced cells, or about
50% uced cells, to a subject.
In one embodiment, described are genetically modified cells, such as a stem cell,
e.g., hematopoietic stem cell, with the potential to expand or increase a population of erythroid
cells. In ular embodiments, hematopoietic stem cells are transduced with a vector
described herein and administered to an individual in need of therapy for hemoglobinopathy.
Hematopoietic stem cells are the origin of erythroid cells and thus, are preferred. In one
embodiment, the vector is one that carrys one or more of the nucleotide sequences of SEQ ID
NOS:1-10,13-18, 25-44, or a synthetic BCL11A microRNA described herein.
As used , the term “pharmaceutically acceptable,” and grammatical
variations thereof, as they refer to compositions, carriers, ts and reagents, are used
interchangeably and represent that the materials are capable of administration to or upon a
subject without the production of undesirable physiological effects such as nausea, ess,
gastric upset, and the like. Each carrier must also be “acceptable” in the sense of being
compatible with the other ingredients of the formulation. A pharmaceutically acceptable carrier
will not promote the raising of an immune response to an agent with which it is admixed, unless
so desired. The preparation of a pharmacological ition that contains active ingredients
dissolved or dispersed therein is well understood in the art and need not be d based on
formulation. The pharmaceutical formulation contains a nd described herein in
combination with one or more pharmaceutically acceptable ingredients. The carrier can be in
the form of a solid, semi-solid or liquid diluent, cream or a capsule. Typically such
compositions are prepared as injectable either as liquid solutions or suspensions, however, solid
forms suitable for on, or suspensions, in liquid prior to use can also be prepared. The
preparation can also be fied or ted as a liposome ition. The active
ingredient can be mixed with excipients which are pharmaceutically acceptable and compatible
with the active ingredient and in amounts suitable for use in the therapeutic methods described
herein. Suitable excipients are, for example, water, saline, dextrose, glycerol, ethanol or the like
and combinations thereof. In addition, if desired, the composition can contain minor s of
auxiliary substances such as wetting or emulsifying , pH buffering agents and the like
which enhance the effectiveness of the active ingredient. The therapeutic composition described
herein can include pharmaceutically acceptable salts of the components therein.
Pharmaceutically acceptable salts include the acid addition salts (formed with the free amino
groups of the ptide) that are formed with inorganic acids such as, for example,
hydrochloric or phosphoric acids, or such organic acids as acetic, ic, mandelic, and the like.
Salts formed with the free yl 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, 2-ethylamino ethanol, histidine, procaine and the like.
Physiologically tolerable carriers are well known in the art. Exemplary liquid carriers are sterile
aqueous solutions that contain no als in addition to the active ingredients and water, or
contain a buffer such as sodium phosphate at physiological pH value, physiological saline or
both, such as phosphate-buffered saline. Still further, s carriers can contain more than
one buffer salt, as well as salts such as sodium and potassium chlorides, dextrose, polyethylene
glycol and other solutes. Liquid compositions can also contain liquid phases in addition to and
to the exclusion of water. ary of such additional liquid phases are in, vegetable
oils such as cottonseed oil, and water-oil emulsions. The amount of an active agent used herein
that will be effective in the treatment of a particular disorder or condition will depend on the
nature of the disorder or condition, and can be determined by standard clinical techniques. The
phrase “pharmaceutically acceptable carrier or diluent” means a pharmaceutically acceptable
material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, solvent or
ulating material, involved in carrying or transporting the subject agents from one organ,
or portion of the body, to another organ, or n of the body.
In one embodiment of any methods described, as used herein, “administered”
refers to the placement of an inhibitor of BCL11A into a subject by a method or route which
results in at least partial localization of the inhibitor at a d site. An agent which inhibits
BCL11A can be administered by any appropriate route which results in ive treatment in the
subject, i.e., administration results in delivery to a desired on in the subject where at least a
portion of the composition delivered, i.e., at least one agent, which inhibits , is active in
the desired site for a period of time. The period of time the inhibitor is active depends on the
half-life in vivo after administration to a subject, and can be as short as a few hours, e.g., at least
1 hour, at least 2 hours, at least 3 hours, at least 4 hours, at least 5 hours, at least 6 hours, at least
8 hours, at least 10 hours, at least 12 hours, at least 18 hours, at least 24 hours, to a few days, to
as long as several years. Modes of administration e injection, infusion, instillation, or
ingestion. “Injection” includes, without limitation, intravenous, intramuscular, intraarterial,
intrathecal, intraventricular, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal,
transtracheal, subcutaneous, subcuticular, rticular, sub capsular, subarachnoid, intraspinal,
erebro spinal, and intrasternal injection and infusion.
In one ment, the composition described herein, or the virus or vector
carrying a nucleic acid molecule comprising a nucleic acid sequence selected from a group
consisting of SEQ ID NOS:1-10,13-18, 25-44, or the virus or vector expressing a synthetic
BCL11A microRNA bed herein, is injected into the bone .
In one embodiment, the hematopoietic progenitor cell or HSC from a subject
needing treatment is contacted with a composition that inhibits BCL11A expression. In other
embodiments, the composition comprises a virus or vector carrying a nucleic acid molecule
comprising a nucleic acid sequence selected from a group consisting of SEQ ID NOS:1-10,13-
18, 25-44, or a virus or vector expressing a synthetic BCL11A microRNA described herein. The
subject needing treatment is one diagnosed with a hemoglobinopathy such as SCD or THAL.
By its BCL11A expression” is meant that the amount of expression of
BCL11A is at least 5% lower in populations treated with a BCL11A inhibitor, than a
comparable, control population, wherein no BCL11A tor is present. It is preferred that the
percentage of BCL11A expression in a BCL11A inhibitor treated population is at least 10%
lower, at least 20% lower, at least 30% lower, at least 40% lower, at least 50% lower, at least
60% lower, at least 70% lower, at least 80% lower, at least 90% lower, at least 1-fold lower, at
least 2-fold lower, at least 5-fold lower, at least 10 fold lower, at least 100 fold lower, at least
1000-fold lower, or more than a able control treated population in which no BCL11A
inhibitor is added.
In one embodiment, the nucleic acid is a BCL11A ic RNA erence
agent or a vector encoding the RNA interference agent. In oneembodiment, the RNA
interference agent comprises one or more of the nucleotide sequences of SEQ ID 10,13-
18, 25-44.
As an example of a method of treatment of a subject or reducing the risk of
developing a hemoglobinopathy in a subject, the method comprises administering to the subject
a composition comprising modified engineered cells that se a vector carrying a nucleic
acid sequence selected from the group consisting of SEQ ID NOS:1-10, 13-18 and 25-44, or a
BCL11A microRNA described herein. In one embodiment, the method further comprises
indentifying a subject having a obinopathy or is at risk of developing a
hemoglobinopathy. In another embodiment, the method further comprises ing the
identified subject having a hemoglobinopathy or is at risk of ping a hemoglobinopathy.
As another example of a method of treatment of a subject or reducing the risk of
developing a hemoglobinopathy in a subject, the method comprises the following steps:
mobilize the hematopoietic stem and hematopoietic progenitor cells in a subject; harvest and
collect peripheral blood from the subject, positive selection of CD34+ cells from the peripheral
blood, transduce or transfect the CD34+ selected cells in vitro with a vector carrying a nucleic
acid sequence selected from the group consisting of SEQ ID NOS:1-10, 13-18 and 25-44, or a
BCL11A microRNA described herein; wash the transduced CD34+ selected cells; and
administer the cells into the t. In one embodiment, the method further comprises
indentifying a t having a hemoglobinopathy or is at risk of developing a
hemoglobinopathy. In one embodiment, the method further comprises selecting the subject
having a hemoglobinopathy or is at risk of developing a hemoglobinopathy. In another
embodiment, the method further comprises expanding in culture the washed, transduced CD34+
selected cells in vitro prior to administering to the subject. In another embodiment, the method
further comprises differentiating in culture the washed, transduced CD34+ selected cells in vitro
prior to administering to the subject.
As another e of a method of treatment of a subject or reducing the risk of
ping a hemoglobinopathy in a subject, the method comprises the following steps:
mobilize the hematopoietic stem and hematopoietic progenitor cells in a donor t; harvest
and collect peripheral blood from the donor subject, positive selection of CD34+ cells from the
peripheral blood, transduce or transfect the CD34+ ed cells in vitro with a vector carrying
a nucleic acid ce selected from the group consisting of SEQ ID NOS:1-10, 13-18 and 25-
44, or a BCL11A microRNA described herein; wash the transduced CD34+ selected cells; and
administer the cells into a recepient t. In one embodiment, the method further comprises
selecting a recepient subject having a hemoglobinopathy or is at risk of developing a
hemoglobinopathy. In another embodiment, the method further comprises expanding in e
the washed, transduced CD34+ selected cells in vitro prior to administering to the recepient
subject. In another ment, the method further comprises differentiating in culture the
washed, transduced CD34+ selected cells in vitro prior to administering to the recepient subject.
As another example of a method of treatment of a subject or reducing the risk of
ping a obinopathy in a subject, the method ses the following steps: harvest
and t the blood from the bone marrow of a subject, positive selection of CD34+ cells from
the bone marrow blood, transduce or transfect the CD34+ selected cells in vitro with a vector
carrying a nucleic acid sequence selected from the group ting of SEQ ID NOS:1-10, 13-18
and 25-44, or a BCL11A microRNA bed herein; wash the transduced CD34+ selected
cells; and administer the cells into the subject. In one embodiment, the method further
comprises indentifying a subject having a hemoglobinopathy or is at risk of developing a
hemoglobinopathy. In one embodiment, the method further ses ing the subject
having a hemoglobinopathy or is at risk of developing a hemoglobinopathy. In another
embodiment, the method further comprises expanding in culture the washed, transduced CD34+
selected cells in vitro prior to administering to the subject. In another embodiment, the method
further comprises differentiating in culture the washed, transduced CD34+ selected cells in vitro
prior to administering to the subject.
As another example of a method of treatment of a subject or reducing the risk of
developing a hemoglobinopathy in a subject, the method comprises the ing steps: harvest
and collect the blood from the bone marrow of a donor subject, positive selection of CD34+
cells from the bone marrow blood, transduce or ect the CD34+ selected cells in vitro with a
vector carrying a nucleic acid sequence selected from the group consisting of SEQ ID NOS:1-
, 13-18 and 25-44, or a BCL11A microRNA described herein; wash the transduced CD34+
ed cells; and ster the cells into a recepient t. In one embodiment, the method
further comprises indentifying a recepient subject having a hemoglobinopathy or is at risk of
developing a hemoglobinopathy. In one embodiment, the method further comprises selecting a
recepient subject having a hemoglobinopathy or is at risk of developing a hemoglobinopathy. In
another embodiment, the method r ses expanding in culture the washed, transduced
CD34+ selected cells in vitro prior to administering to the ent subject. In another
embodiment, the method further comprises differentiating in culture the washed, transduced
CD34+ selected cells in vitro prior to administering to the recepient subject.
In one ment, described is a modified engineered cell comprising a nucleic
acid sequence selected from the group consisting of SEQ ID NOS:1-10, 13-18 and 25-44, or a
BCL11A NA bed herein.
In one embodiment, described is a modified ered cell that has been
transduced or transfected with a vector comprising a nucleic acid sequence selected from the
group consisting of SEQ ID NOS:1-10, 13-18 and 25-44, or a BCL11A microRNA described
herein. In one embodiment, the vector is a lentivirus.
In one embodiment, decribed is a method of treatment of a subject or reducing
the risk of developing a obinopathy in a subject, the method comprises administering a
modified engineered cell that has been transduced or transfected with a vector comprising a
nucleic acid sequence selected from the group consisting of SEQ ID NOS:1-10, 13-18 and 25-
44, or a BCL11A microRNA described herein. In one embodiment, the vector is a lentivirus.
In one embodiment, described is a method of treatment of a subject or reducing
the risk of developing a hemoglobinopathy in a subject, the method comprises stering a
modified engineered cell comprising a nucleic acid sequence selected from the group consisting
of SEQ ID NOS:1-10, 13-18 and 25-44, or a BCL11A microRNA described herein.
In one ment, the modified engineered cell is an embryonic stem cell, a
somatic stem cell, a progenitor cell, a bone marrow cell, a hematopoietic stem cell, or a
hematopoietic progenitor cell.
In one embodiment, the modified engineered cell is a cell that has been
differentiated from an embryonic stem cell, a somatic stem cell, a progenitor cell, a bone marrow
cell, a hematopoietic stem cell, or a hematopoietic itor cell.
In one embodiment, the modified engineered cell is a cell that has been
differentiated into the erythroid lineage.
In one embodiment, the modified engineered cell is a cell that has been
differentiated into an erythrocyte.
In one embodiment, the modified engineered cell is a CD34+ cell.
The present invention can be defined in any of the following numbered
paragraphs.
A synthetic BCL11A microRNA sing a first BCL11A t, a loop
segment; and a second BCL11A segment arranged in tandem in a 5' to 3' ion,
wherein the loop segment is between and directly linked to the first and second BCL11A
segments, and wherein the second BCL11A segment is complementary to the first
BCL11A segment so that the first and second BCL11A segments base pair to form a
hairpin loop with the loop segment forming the loop portion of the hairpin loop thus
formed.
The synthetic BCL11A microRNA of paragraph 1, wherein the first and second
BCL11A segments are about 18 to 25 nucleotides long.
The synthetic BCL11A NA of paragraph 1 or 2, wherein the first
BCL11A segment contains a sequence d from a BCL11A mRNA sequence.
The synthetic BCL11A microRNA of any one of paragraphs 1-3, n the
first BCL11A segment is complementary to the second BCL11A segment.
The tic BCL11A microRNA of any one of paragraphs 1-4, n the
first BCL11A segment starts with a -GCGC- at the 5' end and the second BCL11A
t ends with a -GCGC- at the 3' end.
The synthetic BCL11A microRNA of any one of paragraphs 1-5, wherein the
first BCL11A segment is selected from the group consisting of
CGCACAGAACACTCATGGATT (SEQ. ID. NO: 46; derived from BCL11A miR1
oligo described herein), CCAGAGGATGACGATTGTTTA (SEQ. ID. NO: 47; derived
from BCL11A miR2 oligo described herein), TCGGAGACTCCAGACAATCGC (SEQ.
ID. NO: 48; derived from BCL11A E3 oligo or shRNA1 or E3 described herein),
CCTCCAGGCAGCTCAAAGATC, (SEQ. ID. NO: 49; derived from shRNA2 or B5
described herein), TCAGGACTAGGTGCAGAATGT (SEQ. ID. NO: 50; d from
shRNA4 or B11 described herein), TTCTCTTGCAACACGCACAGA (SEQ. ID. NO:
51; derived from BCL11A D8 oligo or shRNA3 or D8 described herein),
GATCGAGTGTTGAATAATGAT (SEQ. ID. NO: 52; derived from shRNA5 or 50D12
ol D12 described herein), CAGTACCCTGGAGAAACACAT (SEQ. ID. NO: 53;
derived from shRNA5 or 50A5 described ), CACTGTCCACAGGAGAAGCCA
(SEQ. ID. NO: 54; derived from shRNA7 or 50B11 described herein),
ACAGTACCCTGGAGAAACACA (SEQ. ID. NO: 55; derived from BCL11A XLC4,
shRNA8 and 50C4 described ), CAACAAGATGAAGAGCACCAA (SEQ. ID.
NO: 56; derived from BCL11A rgeting oligos described herein),
CACAGAACACTCATG (SEQ. ID. NO: 57; derived from miR1G5 oligo
described ), GCGCTCGGAGACTCCAGACAA (SEQ. ID. NO: 58; derived from
E3G5 or E3 mod oligo or shRNA1mod described herein),
gcgcCCTCCAGGCAGCTCAAA (SEQ. ID. NO: 59; derived from B5G5 or
mod described herein); gcgcTCAGGACTAGGTGCAGA (SEQ. ID. NO: 60;
derived from B11G5 or shRNA4mod described herein);
gcgcGATCGAGTGTTGAATAA (SEQ. ID. NO: 61; derived from 50D12G5, D12G4 or
mod described herein); gcgcCAGTACCCTGGAGAAAC (SEQ. ID. NO: 62;
derived from 50A5G5or shRNA6mod described herein);
gcgcCACTGTCCACAGGAGAA (SEQ. ID. NO: 63; derived from 50B11G5 or
shRNA7mod described herein); GCGCTTCTCTTGCAACACGCA (SEQ. ID. NO: 64;
derived from BCL11A D8G5 or D8 mod or shRNA3mod described herein),
GCGCACAGTACCCTGGAGAAA (SEQ. ID. NO: 65; derived from BCL11A C4G5, or
C4 mod or shRNA8mod described herein), CGCACAGAACACTCATGGATT (SEQ.
ID. NO: 66; derived from BCL11A D12G5-2 bed herein), and
ACGCTCGCACAGAACACTCATGGATT (SEQ. ID. NO: 67; derived from BCL11A
D12G5-2 described herein).
The synthetic BCL11A microRNA of any one of paragraphs 1-6, n the
loop segment is derived from a microRNA.
The synthetic BCL11A microRNA of paragraph 7, wherein the microRNA is a
hematopoietic specific microRNA.
The synthetic BCL11A microRNA of paragraph 8, wherein the microRNA is
miR223.
The synthetic BCL11A microRNA of paragraph 9, wherein the loop segment is
ctccatgtggtagag.
The synthetic BCL11A microRNA of any one of paragraphs 1- 10, wherein the
microRNA comprising a nucleotide sequence selected from the group consisting of SEQ
ID NOS:1-10, 13-18 and 25-44.
A method of treating, or reducing a risk of developing, a hemoglobinopathy in a
subject, the method comprising expressing in vivo at least one synthetic BCL11A
microRNA of any one of paragraphs 1-11 in the subject.
The method of paragraph 12, wherein the in vivo expression occurs in an
embryonic stem cell, a somatic stem cell, a progenitor cell, a bone marrow cell, a
hematopoietic stem cell, or a hematopoietic progenitor cell in the subject.
A method of treating, or ng a risk of developing, a hemoglobinopathy in a
subject, the method comprising expressing at least one synthetic BCL11A microRNA of
any one aphs 1-11 in an embryonic stem cell, a somatic stem cell, a progenitor
cell, a bone marrow cell, a hematopoietic stem cell, or a hematopoietic progenitor cell of
the subject wherein the expression is ex vivo, and implanting the cell into the subject.
A method of sing fetal hemoglobin levels sed by a cell sing
sing at least one synthetic BCL11A microRNA of any one paragraphs 1-11 in a
cell, wherein the cell is an embryonic stem cell, a somatic stem cell, a progenitor cell, a
bone marrow cell, a hematopoietic stem cell, or a hematopoietic progenitor cell.
The method of any one paragraphs 12-15, wherein the at least one synthetic
BCL11A microRNA is operably linked to a promoter and constructed in a vector for
expression in a eukaryotic cell.
The method of any one paragraphs 12-16, wherein the at least one synthetic
BCL11A microRNA is expressed from a RNA II polymerase.
The method of any one paragraphs 12-17, wherein the at least one synthetic
BCL11A microRNA is not expressed from a RNA III rase.
The method of paragraph 18, wherein the promoter is selected from a group
ting of a spleen focus-forming virus promoter, a ycline-inducible promoter,
or a β-globin locus control region and a β-globin promoter.
The method of any one paragraphs 16-19, n the vector is a virus.
The method of paragraph 20, wherein the virus is a lentivirus.
The method of aph 21, wherein the lentivirus is selected from the group
consisting of: human immunodeficiency virus type 1 (HIV-1), human immunodeficiency
virus type 2 (HIV-2), caprine arthritis-encephalitis virus (CAEV), equine infectious
anemia virus (EIAV), feline immunodeficiency virus (FIV), bovine immune deficiency
virus (BIV), and simian immunodeficiency virus (SIV).
An isolated nucleic acid molecule comprising the nucleotide sequence selected
from the group consisting of SEQ ID NOS: 1-10, 13-18, and 25-44.
The isolated nucleic acid le of paragraph 23, wherein the molecule
comprises the nucleotide sequence of SEQ ID NO: 1.
The isolated nucleic acid molecule of paragraph 23, wherein the molecule
comprises the nucleotide sequence of SEQ ID NO: 2.
The ed nucleic acid molecule of paragraph 23, n the molecule
comprises the nucleotide sequence of SEQ ID NO: 3.
The isolated nucleic acid molecule of paragraph 23, n the molecule
comprises the tide sequence of SEQ ID NO: 4.
The isolated nucleic acid molecule of aph 23, wherein the molecule
comprises the nucleotide sequence of SEQ ID NO: 5.
The isolated nucleic acid molecule of paragraph 23, wherein the molecule
comprises the nucleotide sequence of SEQ ID NO: 6.
The isolated nucleic acid molecule of paragraph 23, wherein the molecule
comprises the tide sequence of SEQ ID NO: 7.
The isolated nucleic acid molecule of paragraph 23, wherein the molecule
comprises the nucleotide sequence of SEQ ID NO: 8.
The isolated nucleic acid molecule of paragraph 23, wherein the molecule
comprises the tide sequence of SEQ ID NO: 9.
The ed nucleic acid molecule of paragraph 23, wherein the molecule
ses the nucleotide sequence of SEQ ID NO: 10.
The isolated nucleic acid molecule of paragraph 23, wherein the molecule
comprises the nucleotide sequence of SEQ ID NO: 13.
The isolated nucleic acid le of paragraph 23, wherein the molecule
comprises the nucleotide sequence of SEQ ID NO: 14.
The isolated nucleic acid molecule of paragraph 23, n the molecule
ses the nucleotide sequence of SEQ ID NO: 15.
The isolated nucleic acid molecule of paragraph 23, wherein the molecule
comprises the nucleotide sequence of SEQ ID NO: 16.
The isolated nucleic acid molecule of paragraph 23, wherein the molecule
comprises the nucleotide sequence of SEQ ID NO: 17.
The isolated nucleic acid molecule of paragraph 23, wherein the molecule
comprises the nucleotide sequence of SEQ ID NO: 18.
The ed nucleic acid molecule of paragraph 23, n the molecule
comprises the nucleotide sequence of SEQ ID NO: 25.
The isolated nucleic acid molecule of aph 23, wherein the molecule
comprises the nucleotide sequence of SEQ ID NO: 26.
The isolated nucleic acid molecule of paragraph 23, wherein the molecule
comprises the nucleotide sequence of SEQ ID NO: 27.
The isolated nucleic acid molecule of paragraph 23, wherein the molecule
comprises the tide sequence of SEQ ID NO: 28.
The isolated nucleic acid molecule of paragraph 23, wherein the molecule
ses the nucleotide ce of SEQ ID NO: 29.
The isolated nucleic acid le of paragraph 23, wherein the molecule
comprises the nucleotide sequence of SEQ ID NO: 30.
The isolated nucleic acid molecule of paragraph 23, wherein the molecule
comprises the nucleotide sequence of SEQ ID NO: 31.
The isolated nucleic acid molecule of paragraph 23, wherein the molecule
comprises the nucleotide sequence of SEQ ID NO: 33.
The isolated nucleic acid molecule of paragraph 23, wherein the molecule
comprises the nucleotide sequence of SEQ ID NO: 34.
The isolated nucleic acid molecule of paragraph 23, wherein the molecule
comprises the nucleotide ce of SEQ ID NO: 35.
The isolated nucleic acid le of paragraph 23, wherein the le
ses the nucleotide sequence of SEQ ID NO: 36.
The isolated c acid molecule of paragraph 23, wherein the molecule
comprises the tide sequence of SEQ ID NO: 37.
The isolated nucleic acid molecule of paragraph 23, wherein the molecule
comprises the nucleotide sequence of SEQ ID NO: 38.
The isolated nucleic acid molecule of paragraph 23, wherein the molecule
comprises the nucleotide sequence of SEQ ID NO: 39.
The ed nucleic acid molecule of paragraph 23, wherein the molecule
comprises the nucleotide sequence of SEQ ID NO: 40.
The isolated nucleic acid molecule of paragraph 23, wherein the le
comprises the nucleotide sequence of SEQ ID NO:41.
The isolated nucleic acid molecule of paragraph 23, wherein the le
comprises the tide sequence of SEQ ID NO: 42.
The ed nucleic acid le of paragraph 23, wherein the molecule
comprises the nucleotide sequence of SEQ ID NO: 43.
The isolated nucleic acid molecule of paragraph 23, wherein the molecule
comprises the nucleotide sequence of SEQ ID NO: 44.
A vector comprising the isolated nucleic acid molecule of paragraph 23.
The vector of paragraph 59, n the vector further comprises a spleen focusforming
virus promoter, a tetracycline-inducible promoter, or a β-globin locus control
region and a β-globin promoter.
A host cell comprising the vector of paragraph 59 or 60.
The cell of paragraph 61, wherein the cell is an embryonic stem cell, a somatic
stem cell, a itor cell, a bone marrow cell, a hematopoietic stem cell, or a
hematopoietic progenitor cell.
The cell of paragraph 61, wherein the cell is an erythrocyte.
A bacterium comprising the isolated nucleic acid molecule of paragraph 23.
A virus comprising the ed nucleic acid molecule of paragraph 23.
The virus of paragraph 65, wherein the virus is a lentivirus.
The virus of paragraph 66, wherein the lentivirus is selected from the group
consisting of: human immunodeficiency virus type 1 (HIV-1), human immunodeficiency
virus type 2 (HIV-2), caprine arthritis-encephalitis virus (CAEV), equine infectious
anemia virus (EIAV), feline deficiency virus (FIV), bovine immune deficiency
virus (BIV), and simian immunodeficiency virus (SIV).
A composition comprising an isolated nucleic acid molecule of any one of
paragraphs 1-58, a vector of aphs 59 or 60, a host cell of any one of paragraphs 61-
63, or a virus of any one of paragraphs 65-67.
A composition comprising a vector of paragraphs 59 or 60, a host cell of any one
of paragraphs 61-63, or a virus of any one of paragraphs 65-67.
The composition of paragraph 68 or 69, further comprising a pharmaceutically
acceptable carrier or t.
A composition of any one of paragraphs 68-70 for use in the treatment or for
reducing a risk of developing a hemoglobinopathy in a subject.
A composition of any one of paragraphs 68-70 for use in the manufacture of
medicament in treatment or for reducing a risk of developing, a hemoglobinopathy in a
subject.
A composition of any one of paragraphs 68-70 for use in increasing the fetal
hemoglobin levels expressed by a cell.
The composition of aph 73, n the cell is an embryonic stem cell, a
somatic stem cell, a progenitor cell, a bone marrow cell, a hematopoietic stem cell, or a
hematopoietic progenitor cell.
A method of treating, or ng a risk of developing, a hemoglobinopathy in a
subject, the method comprising: administering to the subject a therapeutically effective
amount of an isolated nucleic acid le of any one of paragraphs 1-58, a vector of
aphs 59 or 60, a host cell of any one of paragraphs 61-63, or a virus of any one of
paragraphs 65-67 to the subject, thereby treating, or reducing the risk of ping, the
hemoglobinopathy in the subject.
A method of treating, or reducing a risk of developing, a obinopathy in a
subject, the method comprising: administering to the subject a therapeutically effective
amount of a composition of any one of paragraphs 68-74 into the subject, thereby
treating, or reducing the risk of developing, the hemoglobinopathy in the subject.
A method of treating, or reducing a risk of developing a hemoglobinopathy in a
subject, the method comprising increasing fetal hemoglobin levels expressed by a cell in
the subject.
The method of any one of paragraphs 75-77, the method further comprising
selecting a subject having a hemoglobinopathy or is at risk of developing a
obinopathy.
The method of claim 78, wherein the hemoglobinopathy is sickle cell disease or
thalassemia.
The method of any one of paragraphs 75-80, the method further comprising
administering to the subject a therapy comprising , hydroxyurea, folic acid, or a
blood transfusion.
The invention is further described in the following es, which do not limit
the scope of the invention bed in the claims.
EXAMPLES
Materials and methods
The typical PCR reaction conditions are as follows: 1x reaction buffer (consist of
MgCl2 at 1.5mM; 3.0mM; 4.5mM (final concentration)); 0.2 mM of each of dATP, dCTP, dGTP
and dTTP; 25 pmol each ; 50 ng template DNA; 3-10% (v/v) DMSO to melt ure
(this is al) in a total volume of 100 µl.
The following is the typical on conditions or setting on thermal cycler for
the PCR reaction : 94°C for 3-5min, during this time add 1U DNA polymerase or set up reaction
on ice and then put tubes into PCR machine when it gets up to 94oC; followed by 25 cycles of
94°C for 1min; 60°C for 1min, and 70°C for 1min; and end with 4°C till PCR samples are used.
EXAMPLE 1
Manufacturing synthetic miRs
Three different synthetic miRs were constructed, two which target BCL11A at
different sites and a third non-targeting to act as a control. Each of these miRs was inserted into
a constitutive expressing vector, a TET-inducible vector, and an erythroid specific vector.
miRs are made by annealing complimentary oligonucleotides, which have 4 base
pair 5’ overlaps corresponding to the sticky end left by a restriction digest with BbsI.
BCL11A miR1 oligos:
Sense
ACGCTCGCACAGAACACTCATGGATTctccatgtggtagagAATCCATGAGTG
TTCTGTGCGAG (SEQ ID NO:1)
Anti-sense
CGCACTCGCACAGAACACTCATGGATTctctaccacatggagAATCCATGAGT
GTTCTGTGCGA (SEQ ID NO:2)
BCL11A miR2 oligos:
Sense
ACGCTCCAGAGGATGACGATTGTTTActccatgtggtagagTAAACAATCGTC
ATCCTCTGGag (SEQ ID NO:3)
Anti-sense
CGCActCCAGAGGATGACGATTGTTTActctaccacatggagTAAACAATCGTC
ATCCTCTGGa (SEQ ID NO:4)
Non-targeting oligos:
Sense
ACGCTCAACAAGATGAAGAGCACCAActccatgtggtagagTTGGTGCTCTTC
ATCTTGTTGAG (SEQ ID NO:11)
Anti-sense
CGCACTCAACAAGATGAAGAGCACCAActctaccacatggagTTGGTGCTCTT
GTTGA (SEQ ID NO:12)
Oligonucleotide pairs were denatured and then re-annealed (as for oligo te
in LM-PCR protocol) following which the cassette was purified using microcentrifuge
concentration devices. In the me, plasmid KS(miR223) was digested with BbsI
and purified by running out on an agarose gel (no treatment with alkaline phosphatase). Each
oligo cassette was then ligated into the digested O.6.pBKS construct and transformed into
ent bacteria (Stbl3). Bacterial clones were picked and mini-prepped to prepare isolated
vectors. The synthetic miRs were sequenced using primers miR223 SEQ FOR and miR223
SEQ REV using DMSO to melt structure.
miR223 SEQ FOR TAAGCTTGATATCGAATTCC (SEQ ID NO:19)
miR223 SEQ REV GCTCTAGAACTAGTGGATCC (SEQ ID NO:20)
EXAMPLE 2
Manufacture of constitutive miR vectors
Each miR was cloned into the LeGO-V2 lentiviral backbone such that VenusmiR
expression is driven by the constitutive SFFV promoter.
Modification of the Venus cDNA. The Venus cDNA will be amplified via PCR
to add a NaeI restriction site to the 5’ end (as well as maintain a good Kozak consensus
sequence) and a NotI site to the 3’ end.
Venus NaeI FOR: TTgccggcATGGTGAGCAAGGGCGAGG (SEQ ID
NO:21)
Venus NotI REV: TAgcggccgcTTACTTGTACAGCTCGTCC (SEQ ID
NO:22)
The PCR products were run out on an agarose gel and then purified. The purified
PCR product were ned into vector PCR 2.1 TOPO (INVITROGEN™) using the TA
cloning kit. Bacterial clones were picked and DNA repped. Using restriction digest
analysis, clones were selected that a) contain the Venus PCR product (EcoRI digest) and b)
contain the clone in an orientation where the NotI that was added is next to the NotI site in the
polylinker (i.e., so that a NotI digest does not excise the PCR nt, but instead just
linearises the vector). These clones were then sequenced using M13Forward and Reverse
primers.
Insertion of the miR sequences into the Venus-PCR 2.1 TOPO plasmid. The
Venus –PCR 2.1 TOPO plasmid was ed with NotI, treated with calf inal alkaline
phosphatase, then run out on an agarose gel and purified. The synthetic miR constructs were
excised from the O.6.pBKS plasmid by double digest with NotI and PspOMI, following by
purification by agarose gel extraction. The digested miR inserts were ligated into the Venus-
PCR 2.1 TOPO d and the ligation t was used to transform competent bacteria
). Individual bacterial clones were picked and mini-prepped. Plasmids that contain the
miR insert in the correct orientation (i.e., yield the full fragment when digested with NotI and
NaeI) were selected.
Insertion of the Venus-miR cassette into LeGO-V2. The Venus-miR cassette was
excised from PCR 2.1 TOPO by double digestion with NotI and NaeI, followed by treatment
with Klenow large fragment to blunt the NotI overhang. This te was ed by agarose
gel extraction. LeGO-V2 or LeGO G2 was ed with BamHI and EcoRI, which released the
Venus/eGFP cDNA. This linearized vector was treated with Klenow large fragment to blunt the
EcoRI and BamHI overhangs, followed by purification of the vector by agarose gel
electrophoresis. The purified Venus-miR cassette and the LeGO vector were ligated together,
and the product was used to transform competent bacteria. Individual bacterial clones were
picked and DNA mini-prepped. Clones that contain the insert in the correct orientation were
selected and grown up and used in maxi preps to manufacture viral supernatant.
EXAMPLE 3
cture of erythroid-specific miR vectors
A polyadenylation signal was attached to the Venus-miR cassettes manufactured
described above. The resulting Venus-miR-PolyA cassettes were inserted in the anti-sense
orientation into the erythroid specific S3-HS2-B-globin lentiviral vector provided by
Guilianna Ferrari.
Modification of the BGH polyadenylation signal. The BGH polyA signal was
amplified via PCR to maintain the PspOMI restriction site at the 5’ end and add NaeI and NotI
sites to the 3’ end.
BGHpA PspOMI FOR: AGCATGCATCTAGAGG (SEQ ID
NO:23)
BGHpA NaeI/NotI REV:
TTgcggccgccggcCGCGCTTAATGCGCCGCTACAG (SEQ ID NO:24)
The PCR products were run out on an agarose gel and then purified. The purified
PCR product was TA-cloned into vector PCR 2.1 TOPO (INVITROGEN™) using the TA
cloning kit. Bacterial clones were picked and DNA mini-prepped. Using restriction digest
analysis, clones were selected that contain the BGHpA PCR product (EcoRI digest and/or
NotI/PspOMI double digest). These clones were sequenced using M13Forward and Reverse
primers.
Insertion of the BGHpA sequence into the Venus-miR-PCR 2.1 TOPO plasmids
manufactured described above. The BGHpA cassette was excised from PCR 2.1 TOPO by
digestion with PspOMI and NotI following which the insert was purified by agarose gel
extraction. The Venus-miR-PCR 2.1 TOPO constructs manufactured in step B2 above were
digested with NotI and uently d with calf intestinal alkaline phosphatase. The
linearized Venus-miR-PCR 2.1 TOPO vector was purified by running out on an agarose gel.
The BGHpA insert was ligated into the Venus-miR-PCR 2.1 TOPO vector and the product used
to transform competent bacteria (Stbl3). Individual bacterial clones will be picked and miniprepped.
Plasmids that contain the BGHpA sequence inserted in the correct orientation (yields
the whole insert upon ion with NaeI) were selected.
Insertion of the miR-BGHpA cassette into pRRL-HS3-HS2-B-globin
vector. The Venus-miR-BGHpA cassettes were excised from PCR 2.1 TOPO by digestion with
NaeI. These inserts were purified by agarose gel electrophoresis. The pRRL-HS3-HS2-B-
globin vector was digested with EcoRV and treated with calf intestinal alkaline phosphatase.
The linearized vector was purified by agarose gel ophoresis. The Venus-miR-BGHpA
tes were ligated into pRRL-HS3-HS2-B-globin and the ligation product used to transform
ent bacteria. Individual bacterial clones were picked and mini-prepped. Plasmids that
contain the miR-BGHpA cassettes in the correct orientation in the pRRL-HS3-HS2-B-
globin vector were grown up for maxi prep in order that they can be used to generate lentiviral
supernatant.
EXAMPLE 4
In vitro cell RNA interference experiments are performed as follows.
Murine erythroleukemia cells kept in culture in IMDM with FCS were uced
on fibronectin with SFFV-LVs (NT=scrambled shRNA, miR-2=targeting shRNA) at MOI=2
and sorted for Venus fluorescence. Timepoint analyzed after transduction was day 7. Cells
were >95% Venus positive and 106 cells were collected and RNA extracted, cDNA was obtained
by e ription and real-time qPCR was performed for BCL11A and epsi-gamma globin
mRNAs with Gapdh as an internal control transcript (. A rd curve method was
employed to quantify expression.
In vivo RNA interference experiments in mice are performed as s.
BojJ donor derived LSK HSCs were transplanted into lethally irradiated
C57/BL6 mice after transduction on ectin with SFFV-LVs (NT=scrambled shRNA, miR-
l=targeting shRNA) at MOI=2. Injected cell dose was 100,000 cells per mouse. Venus positive
WBC carrying animals at 4 months were pooled (n=2) and bone marrow sorted for Venus
fluorescence after viability stain (7-AAD) (. RNA extraction and qPCR was performed
as above.
EXAMPLE 5
LCR-LV
Murine oleukemia cells kept in culture in IMDM with FCS were transduced
on fibronectin with LCR-LVs (NT=scrambled shRNA, targeting shRNA) at MOI=2 and
MOI=100 and sorted for Venus fluorescence. Timepoint analyzed after transduction was day 7.
Cells were >95% Venus positive and 106 cells were collected and RNA extracted, cDNA was
ed by reverse transcription and real-time qPCR was performed for BCL11A and epsigamma
globin mRNAs with Gapdh as an internal control ript (. A standard curve
method was employed to quantify expression.
TET-LV
Murine erythroleukemia cells kept in culture in IMDM with FCS were transduced
on fibronectin with TET-LVs (NT=scrambled shRNA, miR-l=targeting shRNA) at MOI=2 and
sorted for Venus fluorescence after exposure to doxycycline at differential concentrations.
Timepoint ed after transduction was day 7. Cells were >95% Venus positive and 106 cells
were collected and RNA extracted, cDNA was obtained by reverse transcription and real-time
qPCR was performed for epsi-gamma globin mRNA with Gapdh as an internal control transcript
(. A standard curve method was employed to quantify expression.
EXAMPLE 6
eral blood SCD-patient derived CD34+ circulating HSC were fractionated
from discarded apheresis material (approximately 200 ml, 106 CD34+ cells). Cells were
transduced with SFFV-LVs (NT=scrambled shRNA, miR-1=targeting shRNA) at MOI=2 on
fibronectin and entiated as ed from Giarratana et al. (Nat Biotech 2005). Cells were
analyzed maturational acquisition of erythroid surface markers (GPA, CD71) by flow cytometry.
Erythroid cells sequentially e erythroblast and erythocyte morpholosy and express Venus
fluorescence. Cells are collected at terminal differentiation stage and RNA ted and qPCR
analysis performed to te gamma-globin mRNA induction by miR-1 SFFV-IV compared to
scrambled (NT) control (.
EXAMPLE 7
Peripheral blood SCD-patient derived CD34+ circulating HSC were fractionated
from discarded apheresis material (approximately 200 ml, 106 CD34+ cells). Cells were
transduced with LCR-LVs (NT=scrambled shRNA, miR-1=targeting shRNA) at MOI=2 on
fibronectin and injected at 30,000 cells/animal into sub-lethally irradiated NSG mice without
prior sorting. Animals were bled at 4 weeks post-injection and RBCs fixed and permeabilized.
HbF stain was performed and identified a LCR-LV-miR-1 animal with human HbF levels at
% (.
EXAMPLE 8
Cord blood d CD34+ human HSCs were transduced on fibronectin with
SFFV-LVs (NT=scrambled shRNA, miR-l=targeting shRNA) at MOI=2 and sorted for Venus
fluorescence. Cells were also visualized by fluorescent microscopy on MS-5 stroma. The cells
were differentiated along the B-lymphopoietic path by methods modified from Luo et al. (Blood
2009). Cells were analyzed weekly for the acquisition of mature B-lymphocyte surface markers
and loss of immature progenitor markers to identify a block in entiation caused by the
knock-down of BCL11A via SFFV-LVs. Cells were collected at weekly timepoints, and RNA
ted to verify BCL11A mRNA own by shRNA targeting via V-miR-1 (FIG.
EXAMPLE 9
Optimization of lentivirus vector RNA polymerase II driven microRNA embedded shRNAs for
enhanced processing and efficient knockdown of BCL11A for ion of fetal hemoglobin
in erythroid cells.
RNA interference (RNAi) technology using short hairpin RNAs (shRNAs)
expressed via pol III promoters has been widely exploited to te gene expression in a
variety of mammalian cell types. To achieve lineage-specific targeting of mRNAs, expression of
shRNAs via pol II promoters is required, necessitating embedding the shRNA in mammalian
microRNA (shRNAmiR) sequences for expression and processing. Here, in order to achieve
knockdown of the BCL11A transcription factor in hematopoietic cells, which has direct
ational application in hemoglobinapathies, we compared the efficiency of mRNA
modulation via pol III vs pol II based lentiviral vectors. Repression of the BCL11A protein
could represent a therapeutic target for sickle cell disease and assemias, as its knock-down
has been shown to induce the expression of the fetal HBG (γ-globin) gene ultimately leading to
enhance levels of the fetal obin tetramer (HbF, α2γ2). In the mouse, BCL11A is a key
repressor of murine Hbb-y gene representing a murine HBG homolog. The inventors
demonstrate up to 100-1000 fold lower Hbb-y induction due to reduced BCL11A knockdown
efficiency using shRNAmiR vs pol III mediated shRNA vector backbones. In order to
understand the molecular basis for these ences, the inventors ted small RNA
sequence analysis of cells transduced by multiple shRNA–shRNAmiR pairs. The inventors show
that shRNAs expressed via a U6 pol III promoter yield guide strand sequences that differ by a
4bp shift compared to pol lI driven (shRNAmiR) mature guide strand sequences. RNA
sequencing demonstrated that the stretch of uridines making up part of the pol III termination
signal is transcribed and included at the 3’ end of the mature shRNA in a pol III vector
backbone. The absence of these additional sequences is ated with a corresponding shift in
the dicer cleavage site, thereby ting a ent mature shRNA with an alternate seed
sequence influencing the efficacy of target gene knockdown in pol II based vectors. In addition,
both the absolute abundance and the ratio of guide to ger strand are significantly different
in cells transduced with either pol II or pol llI based vectors. Incorporating a 4bp shift in the
guide strand of shRNAmiR resulted in a faithfully processed (a mature guide strand sequence
identical to U6-driven sh-RNAs) shRNA sequence and ed knock-down efficiency of
BCL11A by 50-70% at the protein level and was ated with a 100fold enhancement of
Hbb-y ion in murine erythroleukemia cells. The inventors have discovered a modified
strategy for the prospective design of shRNAmiR vector nes to achieve lineage-specific
regulation of target genes.
EXAMPLE 10
Optimization of miRNA-embedded shRNAs for lineage-specific BCL11A knockdown and
hemoglobin F ion.
Materials and methods
Design and screening of shRNAs
U6 promoter-driven lentiviral vectors (pol III-puro) expressing different shRNAs
targeting BCL11A/BCL11A mRNA were obtained from the Broad Institute (Cambridge, MA).
The pol III-puro has hPGK promoter driven puromycin selection marker. More than 100
shRNAs targeting either both XL/L forms or only XL form and 3’UTR region were screened in
MEL cells in a 96 well format using a Qiagen Turbocapture plate and with a multiplexed
Taqman qRT-PCR reaction measuring Gapdh and Hbb-y.
uction of shRNAmiR constructs
The shRNAmiR, vectors were constructed by cloning the shRNA sequences with
flanking mir223 sequences into the lentiviral 2 vector containing a riven Venus
er (28). The shRNAmiR sequences with mir223 loop were synthesized by genscript USA
Inc. (NJ, USA) and the resulting shRNAmiRs were cloned into the pol II ne ream
of the Venus coding sequence using Xba1 and BamH1 sites. All the sequences of shRNAs are
listed in A. A non-targeting control shRNA sequence was designed and named as SFFV-
iRNT or NT in short form. The SFFV-GFP vector, not containing any shRNA cassette
and expressing GFP via an SFFV promoter, was used as a mock control (33).
Virus production and titration
Lentiviral vector supernatants were generated by co-transfecting 10μg of
lentiviral transfer vectors, 10 μg of gag-pol, 5 μg of rev and 2 μg of VSVG packaging plasmids
into HEK293T cells in a 10 cm culture dish using calcium phosphate reagent
ROGEN™). Supernatants were collected at 24 h and 48 h after transfection, filtered
through a 0.4 micron membrane (CORNING®, NY, USA) and uently concentrated by
entrifugation at 23000 rpm for 2 h in a Beckmann XL-90 centrifuge using SW-28 swinging
buckets. To determine the titer, NIH3T3 cells were infected with the virus in the presence of
polybrene (8 µg/ml) and analyzed 48 h post-transduction by FACS for Venus expression (pol II
constructs) or by puromycin selection (1 mg/ml, pol III constructs).
Cell culture
3T3, 293T and MEL cells were maintained in DULBECCO’s modified Eagle's or
RPMI medium supplemented with 10% fetal calf serum, 2% penicillin-streptomycin and 2 mM
glutamine, respectively.
In vitro oid differentiation e
Frozen stocks of primary human CD34+ cells were obtained from mobilized
peripheral blood of healthy donors r of Excellence in Molecular Hematology at Fred
Hutchinson Cancer Research Center, e or the Flow Core at Boston Children’s al)
according a protocol approved by the BCH Institutional Review Board. Erythroid differentiation
protocol used is based on a 3-phase protocol adapted from (48). The cells were cultured in
erythroid differentiation medium (EDM) based on IMDM e modified DULBECCO’s
medium), (CELLGRO®) supplemented with stabilized glutamine, 330 μg/mL holo- human
transferrin (SIGMA®), 10 μg/mL inant human insulin (SIGMA®), 2 IU/mL heparin
Choay (SIGMA®) and 5% solvent/detergent virus vated (S/D) plasma. During the first
phase of expansion (days 0 to 7), CD34+cells were cultured in EDM (erythroid entiated
) in the presence of 10-6 M hydrocortisone (HC) (SIGMA®),100 ng/mL SCF (R&D
SYSTEMS™), 5 ng/mL IL-3 (R&D SYSTEMS™), ) and 3 IU/mL Epo (AMGEN®). On day 4,
cells were resuspended in EDM containing SCF, IL-3, Epo and HC. In the second phase (days 7
to 11), the cells were ended in EDM supplemented with SCF and Epo. In the third phase
(day 11 to day 18), the cells were cultured in EDM supplemented with Epo alone. The cultures
were maintained at 37°C in 5% CO2 in air.
uction and flow cytometry for in vitro culture
MEL and CD34+cells were uced with lentiviral vectors expressing U6-
shRNA or SFFV-shRNAmiR in the presence of polybrene (8 µg/ml) (SIGMA-ALDRICH®
Corp. St. Louis, MO, US) for MEL cells and 10 µM prostaglandin E2 and 2µg/ml polybrene for
CD34+ cells rand centrifuged for two hours at (2000r pm) at room temperature. Live cells were
either sorted for Venus expression (pol II vectors) 48h post transduction by using BD FACS Aria
II (BD BIOSCIENCES®) or cells were selected in the presence of puromycin (1 mg/ml, pol III
constructs). For FACS analysis 7AAD (INVITROGEN™) was included as dead cell marker.
CD34+ cells were labeled with Allophycocyanin (APC), phycoerythrin (PE), and PE-Cyanine7
conjugated antibodies. Anti-CD235 (glycophorin A) -PE, anti-CD71-APC, or anti-CD71- PECyanine7
antibodies and DRAQ-5 (all EBIOSCIENCE®) were used for phenotyping. Analyses
were performed on LSR-II flow cytometer (BECTON DICKINSON®) using Diva or FloJoX
(TREESTAR™) software.
Isolation, transduction and flow cytometry for mouse transplantation experiments
Lineage negative mouse bone marrow cells were isolated by flushing femur, tibia
and hip of CD45.1 BoyJ (B6.SJL-Ptprca Pepcb/BoyJ) and CD45.2 B6 mice (C57BL/6J)
followed by lineage depletion using the Mouse Lineage Cell ion kit (Miltenyi,Biotec Inc.,
San Diego, USA). Cells were cultured at a density of 0.2-1x106 cells/ml in 100 ng/ml mSCF, 20
ng/ml mIL-3 ( both PEPROTECH®, Rocky Hill, USA), 100 ng/ml hFlt3-L and 100 ng/ml hTPO
(both CELLGENIX®, Portsmouth, USA) in STEMSPAN™ SFEM medium (STEMCELL®
Technologies, Vancouver, CA). ing 24 h pre-stimulation cells were transduced at a
density of 1x106 cells/ml at an MOI of 40 and transplanted into lethally ated (7+4Gy, split
dose) recipients three days after isolation. For competitive repopulation experiments, equal
numbers of cells from different transduction groups were mixed prior to transplantation into
CD45.2 or heterozygous CD45.1/CD45.2 double positive recipients (0.4 – 1x106 per animal).
Cell mixtures were analyzed via flow cytometry to confirm equal contributions of both
competitor cell fractions, and readjusted if required. Analysis of peripheral blood, bone marrow
and spleen was performed at multiple time points using the following antibodies: ,
CD45.2, B220, CD11b, CD3, CD71, Ter119 and fixiable ity dye EFLUOR780®. For
analysis of the oid lineage red blood cell lysis was d. Analyses were performed on
LSR-II or LSRFortessa flow cytometers (BECTON DICKINSON®) and Diva or FloJoX
(Treestar™) software. Data analyses and tics were done using Excel (MICROSOFT®) and
GRAPHPAD PRISM® 5.
For transplantation of hCD34 cells ~10 week old female NSG-mice (NOD/LtSzscid
Il2rg-/-) (Jackson Laboratory, Bar Harbor, ME) were irradiated with 2.7Gy followed by
ion of ~106 cells per animal three days post isolation. Irradiated mice were fed BAYTRIL®
supplemented water for 14 days.
RNA extraction and qRT-PCR
Total RNA was extracted from MEL cells 7 days after sorting/ post ion with
puromycin, or freshly sorted cells on day 18 of erythroid differentiation of CD34+cells, using
the QIAGEN® RNA Plus micro kit cia, CA). CDNA was generated using random
hexamer primers and superscript III (INVITROGEN™, Carlsbad, CA). Quantitative PCR was
performed using SYBR® Green PCR master mix (APPLIED BIOSYSTEMS®, Warrington UK)
with intron ng mouse Hbb-y and Gapdh primers (Hbb-y forward 5’-
TGGCCTGTGGAGTAAGGTCAA-3’, reverse 5’-GAAGCAGAGGACAAGTTCCCA-3’(SEQ.
ID. NO:69)), (Gapdh forward 5’-TCACCACCATGGAGAAGGC-3’ (SEQ. ID. NO:70), reverse
’-GCTAAGCAGTTGGTGGTGCA-3’ (SEQ. ID. NO:71)) and human HBG, HBB and GAPDH
s (HBG forward 5’-TGGATGATCTCAAGGGCAC-3’ (SEQ. ID. NO:72), reverse 5’-
TCAGTGGTATCTGGAGGACA-3’ (SEQ. ID. NO:73)) (HBB forward 5’-
CTGAGGAGAAGTCTGCCGTTA-3’ (SEQ. ID. NO:74), reverse 5’-
AGCATCAGGAGTGGACAGAT-3’ (SEQ. ID. NO:75)) and GAPDH forward 5’-
ACCCAGAAGACTGTGGATGG-3’ (SEQ. ID. NO:76), reverse 5’-
TTCAGCTCAGGGATGACCTT-3’ (SEQ. ID. NO:77)). The PCR amplification conditions
were: 95°C for 10 min, ed by 40 cycles of 15 sec at 95°C and 1 min at 60°C. The qPCRs
were performed on a ABI® 7500 machine (APPLIED BIOSYSTEMS®, Foster City, CA). A
standard curve using serial dilutions of cDNAs was used to determine the precise amplification
efficacy for each reaction. Hbb-y and γ-globin expression levels were normalized to GAPDH as
an internal control, and relative gene expression (ΔΔCt method) was used for is of PCR
data, ing correction for differential amplification efficiencies.
Northern blot analysis
MEL cells transduced with U6-shRNAs and SFFV-shRNAmiRs were sorted and
collected after puromycin selection culturing for 7 days. Total RNA was isolated using 1 ml
TRIZOL® reagent (AMBION®), and 15 µg were resolved on a 15% acrylamide gel. Small
transcript sizes were determined using the Decade Ladder (AMBION®, , TX). RNA was
transferred to ™-XL membrane (AMERSHAM™, Piscataway, NJ) and UV-
crosslinked. Blots were pre-hybridized using yb-Oligo (AMBION®, , Tx) at 35 °C,
probed with γ-32P-labeled oligonucleotides (4 polynucleotide kinase; AMERSHAM™,
Piscataway, NJ) at 37 °C for one hour, washed in 2× sodium citrate, 0.1% sodium dodecyl
sulphate at 30–35 °C, and exposed to film. Probe sequences for detecting mature miRNA were
as follows: shRNA1, 5’ CGGAGACTCCAGACAATCGC 3’ (SEQ. ID. NO:78); shRNA2, 5’
CTCCAGGCAGCTCAAAGATC 3’ (SEQ. ID. NO:79); shRNA3, 5’
TCTCTTGCAACACGCACAGA 3’ (SEQ. ID. NO:80); shRNA4, 5’
CAGGACTAGGTGCAGAATGT 3’ (SEQ. ID. NO:81); shRNA5, 5’
ATCGAGTGTTGAATAATGAT 3’ (SEQ. ID. NO:82); shRNA6, 5’
GTACCCTGGAGAAACACAT 3’ (SEQ. ID. NO:83); , 5’
ACTGTCCACAGGAGAAGCCA 3’ (SEQ. ID. NO:84); , 5’
CAGTACCCTGGAGAAACACA 3’ (SEQ. ID. NO:85).
Western blot analysis
Transduced MELs and CD34+ cells were lysed in lysis buffer (RIPA) with
protease (ROCHE®) and phosphatase inhibitors (SANTA CRUZ BIOTECHNOLGY®),
pepstatin and leupeptin (SIGMA). n lysates were estimated by BCA n assay
(THERMO SCIENTIFIC). 25μg of protein was suspended in 2X Laemmli sample buffer, boiled
and loaded on to a 8% SDS-poly-acrylamide gel and subsequently transferred to a
Polyvinylidene fluoride (PVDF) membrane (MILLIPORE®). Following blocking in PBS with
0.1% Triton-X100 and 5% nonfat dry milk, the PVDF membrane was incubated with a
monoclonal mouse CL11A antibody (ABCAM®) or mouse -actin (SIGMA®). Antimouse
IgG nked secondary antibody (CELL SIGNALING®) was used for detection by
chemiluminescence 20X LUMIGLO® Reagent and 20X Peroxide (CELL SIGNALING®).
HPLC analysis
Hemolysates were prepared from cells on day 18 of differentiation using osmotic
lysis in water and three rapid freeze-thaw cycles. Hemoglobin electrophoresis with cellulose
acetate and high performance liquid chromatography (HPLC) were carried out with the s,
in the clinical laboratories of the Brigham and Women’s Hospital using clinically-calibrated
standards for the human hemoglobins.
RNA Sequencing and analysis
Small RNAs were extracted from 6x106 MEL cells using mirVana miRNA
isolation kit (INVITROGEN™) according to the manufacturer’s instructions and sent out for
deep RNA sequencing using ILLUMINA® Hiseq2000. A eveloped PERL script was used
to remove the adaptor sequence, and 19-25nt small RNAs were used for further analysis. The
BOWTIE software (obtained from the internet website at bowtie-bio period forge period
net) was used for alignment, and 1 ch was permitted. Expression level of small RNAs
was normalized to one million of total reads of each library for comparison n different
samples. For the experiment with 250 TRC shRNAs in 4 cell lines, lentivirus was prepared by
the Broad Institute using a high-throughput virus preparation protocol and cells were infected at
high MOI with a single shRNA per well in 96 well plates (the protocols are obtained from the
et website of the Broad Institute at Cambridge, MA, USA, at the RNAi public resources
section under “puromycin”) was added at 1 day post-infection and cells were lysed in TRIZOL®
at 4 days post-infection. All lysates were pooled for each cell line, followed by total RNA
extraction and small RNA library preparation (49). ILLUMINA® reads were d, collapsed
to unique reads (>17nt) with counts, and mapped to TRC shRNA expression vector sequences
allowing no mismatches. Mature shRNA sequence distributions were calculated for each shRNA
before averaging across shRNAs.
Statistical analysis
The GRAPHPAD PRISM® 5.0 software package was used for statistical
analysis. Results are expressed as mean ± standard deviation (SD). Statistical significance was
assessed by t-test.
Results
Decreased knockdown efficiency of BCL11A by shRNAs embedded in a microRNA scaffold
(shRNAmiR) compared to simple stem-loop shRNAs
To identify candidate shRNAs mediating effective knockdown of BCL11A, a
iral library of 118 shRNAs targeting coding ces of BCL11A mRNA conserved
between humans and mice was screened in MEL cells. ShRNAs were expressed from a pol III
based U6 promoter (A, left panel) in the LKO lentivirus backbone (26) containing a
puromycin resistance gene for selection named LKO-U6-BCL11A-shRNAmiR (hereafter U6-
shRNA). MEL cells, a commonly used cell line for the study of globin gene regulation, were
transduced with the lentivirus vectors expressing shRNAs at a multiplicity of infection (MOI) of
2. The ized expression of embryonic mouse Hbb-y mRNA, which serves as a functional
homolog of the human in gene (27) es an indirect readout of BCL11A knockdown
(B, y-axis). As a second readout, the shRNA pool was also screened using a MEL-
er cell line harboring a mCherry knock-in at the Hbb-y locus (D. Bauer, unpublished).
Fluorescent reporter induction was analyzed by flow cytometry (B, ). Eight
shRNAs (labeled and named as shRNA1 through 8 in B) that tently induced Hbb-y
and mCherry reporter expression in MEL cells were cloned into human microRNA223 (miR-
223) ng and loop sequences to create synthetic microRNAs (shRNAmiR) with the goal of
developing lineage-specific expression s for knockdown of BCL11A. For l analysis,
this cassette was incorporated in the pLeGO iral vector (28) (Fig.19A, right panel) into the
3’ untranslated region of the Venus fluorescent reporter under control of the strong and
ubiquitously expressed spleen focus forming virus (SFFV) promoter/enhancer named LEGOSFFV-BCL11A-shRNAmiR
(hereafter SFFV-shRNAmiR).
The knockdown efficacy of shRNAs that orated the same 21-base targetmatching
sequences was directly compared , but in the context of the pol III and pol II
expression cassettes (i.e. NAs vs SFFV-shRNAmiRs.) in MEL cells using a nontargeting
(NT) shRNA as negative control. BCL11A protein was detected by immunoblot in cell
lysates from MEL-cells transduced at an MOI of 2 (C). Knockdown of BCL11A was
consistently less efficient in cells expressing SFFV-shRNAmiR compared to U6-shRNAs (C). To confirm the functional significance of this ence, the inventors measured induction
of Hbb-y mRNA levels by qRT-PCR (D) in homogeneous populations of transduced
cells obtained either by puromycin selection or by fluorescence-activated cell sorting (FACS).
The reduced knockdown efficiency of SFFV-shRNAmiRs as compared to U6-shRNAs (see C) translated into significantly less ion of Hbb-y by hRNAmiRs (D) and
the ences in Hbb-y induction appear more pronounced than the differences in BCL11A
knockdown.
SFFV-shRNAmiR and U6-shRNAs give rise to different mature guide strand sequences
To understand the molecular basis for these differences, sequencing of small
RNAs from cells transduced with various U6-shRNAs were med and their corresponding
SFFV-shRNAmiR rparts. It was esized that significant differences in Hbb-y
induction were due to the production of different mature guide strands from the distinct shRNA-
containing transcripts that are produced from the pol II and pol III contexts. The sed guide
strand sequences from both the U6-shRNAs and SFFV-shRNAmiR contexts were therefore
assessed (A and B). The most ntly found mature guide RNAs ed from
SFFV-shRNAmiRs y corresponded to the in silico predicted mature sequence (B).
In contrast, most of the U6-shRNAs yielded mature guide strand sequences that match the
predicted Dicer product consisting of ~22nt of the 3’ end of the pol III transcript, including a
stretch of 3-5 nt derived from the pol III termination signal, but lacking a corresponding number
of nucleotides of the target matched sequence at the 5’ end. A similar distribution of processed
products were observed in a large scale screen of 247 different NAs in A549, MCF7,
Jurkat and U937 cell lines, in which the predominant guide strand sequence has an average
length of 22nt with its 5’ end starting 4 bases from the constant loop sequence. Deep sequencing
of 247 processed TRC shRNA products in these four cell lines were performed (). The
results indicate that the predominant mature guide strand starts at position 4 of the antisense
sequence of the shRNA and includes four 3'-terminal U residues. Processing was lly
tent among cell lines and among different shRNA sequences. The average read frequency
for each mature sequence is weighted equally across shRNAs, although some shRNAs generated
>1,000-fold more reads than others. The semiquantitative nature of small RNA sequencing, due
to strong RNA ligase biases during library preparation, make comparisons of relative expression
or sing levels impossible, but consistent trends across cell lines and shRNAs demonstrate
the likely predomonant guide strand identity. Sense strand reads were also detected (<30% per
shRNA on average), with the vast majority starting with 'GG' and extending 20nt into the sense
strand sequence. These mature sequences are exactly consistent with a Dicer product of the
primary hU6 shRNA transcript, with no need to invoke a Drosha/DCGR8 processing step. Taken
together with, these findings indicate the importance of considering the sing events that
generate mature sequences from pol II shRNAmiR and pol III shRNA transcripts when
transferring shRNA sequences between vectors. The very similar distributions of mature
sequences observed for the four cell types that were studied ts that these processing
ns will generalize across different cellular contexts. The differences in the mature guide
strand sequence generated in pol III vs pol II based vectors contribute substantially to
differential BCL11A down regulation observed with U6-shRNAs compared to SFFV-
shRNAmiRs. These data suggest predicted conversions between pol III and pol II vectors may
be possible by considering the Drosha and Dicer cleavage of pol II shRNAs compared to the
Dicer cleavage of pol III shRNAs.
Modification of shRNA ces in a pol II based vector leads to improved own
efficiency
Based on these findings, the inventors hypothesized that using the predicted
mature sequence from pol III shRNA vectors when transferring sequences into SFFV-
shRNAmiR would lead to enhanced knockdown efficiency. Therefore, a set of SFFV-
shRNAmiRs ning a 4-nucleotide shift in the 5’ end of the guide strand sequence were
designed (A). At the 3’-end the nucleotides GCGC were added to achieve higher 3’-end
thermodynamic stability in the siRNA duplex which should promote preferential RISC-loading
of the intended guide strand. The effect of cations on knockdown efficiency and Hbb-y
induction was evaluated in MEL cells by immunoblot and qRT-PCR, respectively. Improved
knockdown efficiency of BCL11A n was observed with hRNAmiR1, 3 and 8 (B). The enhanced knockdown correlated with a 200 to 400 fold increased induction of Hbb-y
ripts (C). The other SFFV-shRNAmiRs did not show an appreciable increase in
knockdown efficiency. To understand more fully the mechanism underlying the improved
efficiency of modified SFFV-shRNAmiRs 1, 3 and 8 were analyzed the nce of guide and
passenger strand small RNAs and their ratios by Northern blot. First, a higher nce of
guide strand was seen for pol III versus pol II vectors in all cases. Furthermore, particularly for
modified SFFV-shRNAmiRs 1 and 3 a higher abundance and higher guide to passenger strand
ratios were found versus the unmodified shRNAmiRs, while these parameters were not ed
for SFFV-shRNA8 (D). Deep sequencing of small RNAs was performed to evaluate the
impact of the modification on guide strand ces and to correlate it with the changes
observed in BCL11A knockdown. Generally, the resulting processed sequences reflected the
introduced 4 nt shift, resulting in a guide strand with seed regions r to the sequences
obtained from pol III shRNAs expressed in the LKO backbone. For SFFV-shRNAmiRs 1, 3 and
8, a single dominant sequence was found, which contrasts the less effective SFFV-shRNAmiRs
which showed a broader distribution of sequences.
Effect of shRNAmiR modification on BCL11A knockdown and γ-globin induction in primary
human CD34+ derived erythroid cells
Reactivation of fetal globin with BCL11A knockdown has therapeutic ial
for the treatment of sickle cell disease and β-thalassemia. To evaluate the effect of modified
SFFV-shRNAmiR on knockdown efficiency of BCL11A and induction of γ-globin and HbF
expression in primary human cells, G-CSF mobilized peripheral blood (mPB) CD34+ HSPCs
from healthy volunteers were transduced with vectors expressing U6-shRNAs, SFFV-
shRNAmiR and modified SFFV-shRNAmiR and then subjected to erythroid differentiation.
After eleven days in culture, BCL11A levels were determined via n blot (A).
Consistent with findings in MEL cells, enhanced knockdown was observed with modified
SFFV-shRNAmiRs 1, 3, and 8, which also led to increased induction of γ-globin transcripts
(B). The status of erythroid differentiation was assessed at day 18 of e by flow
cytometric analysis for surface expression of CD71 and GpA and enucleation. No significant
differences were observed between SFFV-shRNAmiRs and control vector transduced samples
(C). In contrast, U6-shRNAs led to mild delay in the ition of differentiation
markers during the later phases of maturation, which could indicate toxicity due to U6-promoter
mediated shRNA overexpression. The observations of high γ-globin mRNA induction were
confirmed by increased HbF n measured by high performance liquid chromatography
(HPLC). All three tested modified shRNAmiRs yielded increased HbF output compared to
unmodified SFFV-shRNAmiRs (D), where between 40-50% of total obin in the
erythroid cells was HbF. The correlation between in mRNA and HbF protein was high
(r2=0.96), ting the reliability of the analyses (E).
In summary, the inventors have demonstrated that shRNAs embedded into a
miRNA ld and expressed via pol II promoters are processed to yield ing mature
siRNAs in uced cells compared with siRNA expressed from the U6 promoter. The targetmatched
sequence in the mature shRNA derived from the pol III promoter uct is uniformly
shifted 3’ by 3-5 nt and this difference was associated with significant differences in knockdown
efficiency of the target transcript. In the case of , a potential therapeutic target, this led
to appreciable differences in the vation of γ-globin expression. These data demonstrate the
importance of design optimization when transferring shRNA ces into a microRNA
scaffold to allow for pol II mediated expression.
Ubiquitous knockdown of BCL11A in hematopoietic stem and itor cells (HSPCs) impairs
hematopoietic reconstitution and can be circumvented by targeting shRNAmiR expression to
erythroid cells
The impact of SFFV-shRNAmiR expression in vivo was assessed in a mouse
model of HSPC transduction and transplantation. Lineage-negative (lin-) HSPCs from the bone
marrow of β-YAC mice expressing the CD45.2 cell surface maker were transduced ex vivo with
SFFV-shRNAmiR vectors or a non-targeting vector (SFFV-shRNAmiRNT) and transplanted
into lethally irradiated CD45.1 BoyJ-recipient mice. Untransduced cells were lanted in a
control group. β-YAC mice harbor the human β-globin locus as a transgene that is
developmentally regulated in the mouse nment, showing ential sion of fetal
and adult β-globin genes. For validation purposes and for better comparison with previously
published data a well described shRNA (23, 27, 29) (here termed shRNAmiR*) embedded into
miRNA223 flanking sequences was employed. At 4, 8 and 12 weeks after transplantation, donor
cell engraftment was determined based on CD45.1 and CD45.2 chimerism (A). Donor
cell engraftment followed the expected pattern with near complete engraftment in peripheral
blood (PB) and bone marrow (BM) after 8 weeks. However, unexpectedly, the fraction of gene
ed cells steeply declined over time (B). Despite initial transduction rates of ~40%
using the BCL11A knockdown vector, gene marking at week 12 was only 2-3% of total donor
derived CD45 cells. Overexpression of the SFFV-shRNAmiR NT was also ated with
reduced engraftment of gene modified cells but to a lesser extent, indicating both sequencespecific
and non-specific toxicity in the engrafting HSPC cells. The timing of the loss of donor
cells expressing shRNAs suggests an effect on the more primitive poietic stem cell
compartment.
To further investigate the negative impact on hematopoietic reconstitution,
quantitative competitive repopulation experiments were performed (C-23F). Lineage
negative cells from CD45.1 (BoyJ) and CD45.2 (Bl\6) donor s were transduced with
s vectors expressing SFFV-shRNAmiRs against BCL11A, a shRNAmiRNT or only a blue
fluorescent n (BFP) reporter under control of the ubiquitously expressed SFFV-promoter
(SFFV-BFP). Cells were transplanted into congenic CD45.1/CD45.2 animals, allowing for
identification of both donor populations and the ent cells. In experiments in which the
SFFV-BFP vector was employed, CD45.1 donor cells were transplanted into CD45.2 animals
and the transduced donor cell populations were identified and ed based on fluorescence.
Prior to transplantation, equal numbers of cells of the two populations transduced with
competing vectors were mixed. The final ratio of gene modified cells obtained with both vectors
in the transplanted population was ed via flow cytometry which confirmed comparable
transduction rates ranging from 55-70% (C). The contribution of gene modified cells
was assessed in transplanted animals in peripheral blood, bone marrow and spleen 4, 8 and 12
weeks after transplantation (D) and minor differences in the ratio of the infused
transduced cells were taken into account for this analysis. In all instances and at each time point,
cells transduced with vectors ing BCL11A were outcompeted by cells transduced with the
NT or FP vector, indicating a selective disadvantage upon BCL11A knockdown. No
significant differences in reconstitution of hematopoietic cells compared to the ratio of the
initially transplanted population was observed when two BCL11A targeting vectors competed
against each other. Consistent with the findings in B, the overexpression of
shRNAmiRNT also had a negative impact on hematopoietic reconstitution, as this group was
outcompeted by cells transduced by a vector expressing only SFFV-BFP and not expressing a
shRNA. The inventors performed a more detailed analysis of the B lymphocyte and more
primitive HSC compartment within the transduced on of bone marrow cells (E). As
anticipated from previous studies showing an absence of B cells in BCL11A-/- mice (30, 31) the
number of B220 positive B-cells was significantly reduced upon BCL11A knockdown. Although
not reaching significance, there was a trend toward loss of more primitive lin-, Sca-1+, c-kit+
(LSK) cells that include the ting HSC compartment.
oid ic knockdown of BCL11A could potentially vent the
adverse effect of BCL11A knockdown on HSCs and B cells, while maintaining the therapeutic
effect of γ-globin ion in erythroid cells. To direct knockdown ively to erythroid cells,
a lentivirus vector was generated in which the shRNAmiR te and the Venus fluorescent
reporter is expressed under the control of the minimal β-globin proximal promoter linked to
hypersensitive sites 2 and 3 (HS2 and HS3) of the in locus control region (LCR) (32)
(F) named LV-LCR-BCL11A-shRNAmiR (hereafter LCR-shRNAmiR). The expression
profile of the Venus reporter transgene in the engrafted poietic cell populations in vivo
was first ed in mice transplanted with transduced HSPC (G and ). In , lineage negative cells were transduced using the LCR-shRNAmiR vector and engrafted into
lethally irradiated recipient mice. Twelve weeks later donor cells and different hematopoietic
cell types were identified using surface markers. Shown here is a representative gating scheme
and histogram blots showing Venus expression in various es. Numbers in blots indicate
ther percentage of venus positive cells and mean fluorescence ities (MFI). sion of
the transgene was tightly regulated; with no detectable expression in LSK and B cell fractions,
very low levels of expression in T-cells and low levels of expression in myeloid cells in some
s. In contrast, transgene expression was strongly upregulated during erythroid
entiation, ing in CD71+/Ter119- cells representing erythroid itors and throblasts
and peaking in the CD71+/Ter119+ double ve stage, representing basophilic
erythroblasts. During final stages of erythroid tion, a large fraction of CD71-/Ter119+
cells representing reticulocytes and mature erythrocytes expressed the reporter at a similar
percentage compared to CD71+/Ter119+ cells.
Next, to determine whether use of the LCR-vector circumvents the reconstitution
defect observed upon ubiquitous SFFV- iR overexpression, a competitive
transplantation experiment was performed using LCR-shRNAmiR and hRNAmiR (H). As the LCR-vector is transcriptionally silent in lin- cells, an aliquot of cells to be used for
lantation was subjected to in vitro erythroid differentiation and the ratio of Venus+ cells
measured in the transcriptional permissive CD71+/Ter119+ population and used for
normalization of the ratio of SFFV vs. ansduced cells. Venus sion in transplanted
animals was compared in erythroid cells, as this is the only population that is equally permissive
for expression from both vectors. Reconstitution of transplanted mice demonstrated a clear
dominance of cells derived from HSPC transduced with the LCR-vector, suggesting less toxicity
in the HSPCs associated with erythroid lineage specific expression of the LCR-shRNAmiR
(H). In summary, these data demonstrate that the adverse effect of BCL11A knockdown
on HSC engraftment/function may be circumvented by erythroid specific miRNA expression.
LCR-vector mediated erythroid specific knockdown of BCL11A using modified shRNAmiRs
yields high levels of HbF in human erythroid cells
To test the efficacy of LCR-mediated erythroid specific knockdown of BCL11A
in a human experimental system, CD34+ cells were transduced with LCR-shRNAmiR vectors
containing modified shRNAmiRs 3 or 8 (F). The inventors first confirmed the
expression profile of several LCR-shRNAmiR vectors (LCR-shRNA*, LCRshRNAmiR3 and 8)
in human cells during in vitro erythroid differentiation of human mPB CD34+ cells. Venus
expression by the LCR-vectors and a SFFV-driven control vector without iR-cassette
(SFFV-GFP) (33) was evaluated at different stages of erythroid maturation, as defined by CD71
and GpA staining (I). Consistent with the findings in mouse cells shown in G,
low levels of expression were ed in CD71-/GpA- immature erythroid cells. There was a
strong upregulation of expression in CD71+ single positive cells with the highest level of
transgene expression in the more mature CD71+/GpA+ double positive cells. As expected, the
SFFV-GFP control drove high level constitutive expression in all ulations. Following the
previously described entiation protocol, BCL11A protein levels were ed on day 11
of culture and compared with non-targeting and mock control vectors (LCR-shRNAmiRNT and
SFFV-GFP). Significant reduction in BCL11A was observed in the cells expressing the modified
shRNAmiR compared to the cells expressing the non-targeting (NT) and control vector (SEW)
(A). Gamma globin mRNA constituted 40 and 70% of total β-like globins in cells
derived from CD34+ cells transduced with vectors expressing shRNAmiR3 and 8, respectively
(B). No differences in cell growth were observed between cells transduced with LCR-
shRNAmiRs or control vectors. Erythroid differentiation, as evaluated by surface expression of
CD71, GpA and by enucleation was indistinguishable from controls (C), suggesting no
negative impact of BCL11A knockdown upon lineage-specific sion of the BCL11A
shRNAmiRs. Strong correlation was observed between the levels of γ-globin mRNA (qRT-PCR)
and HbF as assessed by HPLC (D). HbF buted to 35% and 55% of total
hemoglobin in cells transduced with LCR-shRNAmiR3 and LCR-shRNAmiR8, representing
levels comparable to SFFV-promoter mediated expression (D and E). Finally, to
show in proof of ple that LCR-shRNAmiR mediated knockdown allows for efficient
engraftment of hCD34+ cells and induction of γ-globin, transplantation of bone marrow derived
CD34+ HSPCs transduced with LCR-shRNAmiR3 or NT vectors were performed into sublethally
irradiated NSG-mice. Due to poor development of human erythroid cells in this
xenograft model, CD34+ HSPCs were isolated from the bone marrow of transplanted animals 14
weeks after transplantation and subjected to oid differentiation in vitro. Venus+ cells were
enriched by FACS and expression of γ- and β-globin was ined by RT-PCR (F).
Consistent with previous data, the on of γ-globin of total β-globin locus output was 44.9%
± 5.5% for cells transduced with LCR-shRNAmiR3, compared to ~9% ± 0.5% in the two control
groups consisting of sduced or LCR-shRNAmiRNT transduced cells.
ShRNAs have been used extensively to analyze gene functions in biological
studies, and there is increasing interest in the use of RNAi for therapeutic purposes. BCL11A
ents an tive therapeutic target for RNAi based modulation. BCL11A is a repressor of
γ-globin expression and thus acts as a major tor of the fetal to adult hemoglobin switch in
erythroid cells. Importantly high levels of fetal hemoglobin are ated with milder disease
phenotypes in sickle cell disease (SCD) and β-thalassemias and lineage-specific knockout of
BCL11A has been validated as a therapeutic strategy in models of SCD. In the studies reported
here, our goal was to develop a clinically applicable vector to reactivate fetal hemoglobin
expression by RNAi mediated suppression of BCL11A. Using an optimized lentiviral vector
containing a miRNA adapted shRNA (shRNAmiR) expressed from an erythroid lineage ic
pol II promoter the inventors achieved HbF levels of >50% of total hemoglobin in primary
erythroid cells d from transduced CD34+ HSPCs. This level of HbF induction is likely to
be clinically ive and compares favorably with previously published s (23, 27, 29)
ing pol III driven expression cassettes that lack lineage specificity and the safety e of
SIN lentivirus vectors reported here.
Curative treatment for SCD can be ed with hematopoietic stem cell
transplantation (HSCT). Favorable outcomes in SCD are largely dependent on the bility of
matched sibling donors. Fewer than 10% of SCD patients have cted HLA-matched sibling
potential donors (34). Gene therapy for hemoglobinopathies offers the clear advantage of
eliminating the risk of GVHD by the use of gous cells. The long-term aim of our studies is
to modulate the hemoglobin switch, leading to the endogenous and physiologic induction of the
protective HbF and suppression of the sickle globin. The inventors esize this dual
manipulation of expression will be the most effective therapeutic approach to prevent toxicities
in SCD including hemolysis and end organ damage of the mutant, polymerizing hemoglobin. To
realize the goal of therapeutic benefit, sufficient knockdown of BCL11A and induction of HbF
on a per cell basis must occur and sufficient numbers of gene modified long-lived HSC must
engraft in order for ism of the red cell compartment to attenuate the e phenotype.
Thus optimization of BCL11A knockdown and vation of reconstitution capacity of
transduced HSCP as shown here is critical to the long-term success of genetic therapy in SCD.
As relates to the second point, the inventors believe this is currently attainable, as previous data
from allogeneic transplants resulting in mixed chimerism have demonstrated that as low as 10%
chimerism of the myeloid compartment is associated with peripheral blood red cell chimerism of
80-100% (35). The skewing of red cell mass after engraftment is most likely attributable to the
enhanced survival of normal red cells ed with sickled cells. This level of marking of
long-lived myeloid cells has recently been attained in a human trials ing lentivirus vector
(36-38), including in βe-thalassemia (39).
Pol III driven shRNAs are the most commonly utilized vector systems to effect
gene knockdown by RNAi, but these vectors mediate ubiquitous expression that may be
associated with both non-specific toxicities from high expression levels and sequence-specific
toxicities in n cell types. Here the inventors demonstrate that BCL11A knockdown in
HSCs impairs engraftment of these cells in transplant settings and B cell development in vivo.
Although reduced engraftment in the absence of BCL11A is an rted phenomenon, the
data reported here are tent with known expression of BCL11A in early HSPCs and with
the report of a ~two-fold reduced HSC content in mice upon genetic deletion of BCL11A (31,
40, 41). The negative impact of BCL11A knockdown on engrafting HSCs may be more evident
in the assays reported here due to increased selective pressure present in this mental
g. Limiting numbers of HSCs are generally present following ex vivo culture and
transduction of these cells and competition with control HSCs utilized in the assays used here
may enhance the detection of toxicity at the HSC level. Within the erythroid lineage BCL11A is
sable (24). In the data reported here, use of the erythroid specific LCR-vector, containing
tory sequences derived from the β-globin locus (32, 42) circumvented the negative effects
of BCL11A knockdown on HSC engraftment. The ctor displayed a high degree of
lineage fidelity in expression of the shRNAmiR ing BCL11A. In addition, this vector
architecture has been demonstrated to reduce the risk of transactivation of neighboring ar
genes when used to express other enes (43), an important feature for clinical translation.
Thus, riptional targeting of shRNAmiRs appears critical in the case of BCL11A,
underscoring the importance of developing effective pol II based knockdown vectors. This
approach bypasses the negative impact of knockdown of BCL11A on HSPCs and also lymphoid
cell development (30, 31), avoids ty related to shRNA overexpression (9, 11, 19) and
improves the safety profile of the vector system, while maintaining the therapeutic efficacy.
The use of pol II ers for shRNA expression itates embedding the
shRNA in microRNA sequences. As the majority of previously validated ive shRNA
sequences are d from analyses performed using pol III promoters and the majority of
commercially available knockdown systems are based on pol III promoters, conversion of
shRNA sequences into a pol II configuration is important. In spite of significant research in this
area, guidelines for conversion of shRNA sequences derived from effective pol III based vectors
into pol II based shRNAmiR vectors are lacking. Here by comparing the results of RNA
processing from cells transduced with both types of vectors in parallel the inventors confirmed
that different small RNA products are generated with respect to the target matched sequences
resulting in a markedly reduced efficiency of target knockdown via pol II based vectors. The
mature guide strand sequences produced from pol II versus pol III systems containing identical
target mRNA matched sequences are generally shifted by 3-5 nt relative to each other. Addition
of 3-5 U-residues from the pol III ation signal to the 3’ end of the shRNA transcript leads
to a corresponding shift of the Dicer cleavage site, proving the dominant role of the 3’-counting
rule for Dicer cleavage (44, 45). The shift of the guide strand in pol III versus pol II has a major
impact on knockdown efficiency, as the seed region is altered and the thermodynamical
properties and terminal nucleotide identity of the small RNA duplex changes, thereby impacting
guide strand incorporation into the RISC-effector complex (4, 5, 46, 47). Re-engineering
shRNAmiRs to mimic the mature guide strand sequences produced by pol III-driven shRNAs
led to enhanced processing and ed knockdown of the target mRNA. This approach should
be applicable for the pment of vectors targeting other genes using pol II promoters,
including other lineage specific expression cassettes.
In y, the data demonstrate critical features of RNA processing nt to
the use of shRNA in different vector contexts, and also provide a strategy for lineage-specific
gene knockdown that circumvents e uences of widespread expression. The findings
have important implications for design of NA embedded shRNAs and their application in
RNAi based gene therapy approaches.
EXAMPLE 11
Efficacy studies of transduction of BCL11A shRNAmiR in health donor human CD34+ cells.
The transcriptional repressor BCL11A represents a therapeutic target for βhemoglobinopathies.
The selectively suppression of BCL11A in erythroid cells via pol II
promoter expressed microRNA adapted shRNAs (shRNAmiRs) resulted in effective knockdown
of BCL11A in both murine and human cells. Expressing the modified shRNAmiRs in an
erythroid-specific fashion circumvented the adverse effects on murine HSC engraftment and B
cell development (see EXAMPLE 10 supra) and led to efficient BCL11A knockdown and high
levels of HbF in primary human CD34-derived oid cells and in human erythroid cells
differentiated in vitro after full engraftment of modified CD34+ cells in murine xenografts. The
inventors also demonstrated effective ion of HbF in erythroid cells derived from
transduced CD34 cells ed from a donor with sickle cell disease.
In a series of experiments, GCSF zed CD34 from healthy donors were
transduced with a vector sing a non-targeting shRNA (LCR-NT) or BMS11-D12G5, and
subjected to erythroid in vitro differentiation.
BCL11A D12G5-2 shRNA: Sense
ACGCTCGCACAGAACACTCATGGATTctccatgtggtagagAATCCATGAGTGTTC
TGTGCGAG (SEQ. ID. NO:43)
Anti-sense
CGCACTCGCACAGAACACTCATGGATTctctaccacatggagAATCCATGAGTGTT
CTGTGCGA (SEQ. ID. NO:44)
A is a Western blot of in vitro differentiated erythroid cells derived from
uced CD34 cells showing BCL11A isoforms (L and XL) and ß-ACTIN as loading control
and demonstrating effective knock-down of BCL11A XL. B shows quantification of
BCL11A knock down in erythroid cells. Data is derived from Western blots as shown in A. Data summarizes three independent experiments using cells from a single donor. (Error
bars: SD)
C shows induction of gamma globin in erythroid cells as assessed by RT-
qPCR and obin (HbF) assessed by HPLC.
EXAMPLE 12
Quantification of BCL11A knock down in erythroid cells.
The engraftment of the transduced CD34+ cells into NSG immunodeficient mice
were studied, including the effectiveness of the in vivo knockdown of the BCL11A expression.
Human CD34 were transduced with LCR-NT or BMS11-D8G5 and ed into sublethally
irradiated cipient mice. Bone marrow CD34+ were isolated 14 weeks later and subjected
to erythroid in vitro differentiation. shows ion of gamma globin in erythroid cells
as assessed by RT-qPCR.
EXAMPLE 13
Knockdown of BCL11A and induction of fetal hemoglobin in oid cells derived from
transduced CD34 cells from a sickle cell patient.
Bone marrow CD34 were isolated from a SCD-patient which received HU
treatment and had high baseline HBF. The cells were tranduced with LCR-NT or LCR-D12G5
and subjected to erythroid in vitro differentiation.
BCL11A knock-down was studied in sickle cell patient cells. Bone marrow CD34
were ed from a sickle cell t and the cells were transduced with LCR-NT or LCRD12G5-2
(untransduced cells used as an additional control) and subjected to erythroid
differentiation in vitro. A shows Western blots showing of BCL11A (L and XL isoforms)
and β-ACTIN as loading control and demonstrates effective knock-down of BLC11A-XL. Each
panel (labeled 1-6 below the lane) represents an independent ment using cells from a
single donor. B shows quantification of BCL11A knockdown in erythroid cells. Data is
derived from Western blots shown in A. C shows resulting induction of HbF by
HPLC. This t was receiving hydroxyurea treatment which accounts for the high baseline
Hb F level.
EXAMPLE 14
An embodiment of a treatment Protocol
Initial evaluation
ts will undergo rd work-up for autologous bone marrow
transplantation according to institutional guidelines, and then undergo two bone marrow
harvests at a minimum of 4 weeks apart that will be used for a back-up marrow um of 2
x 106 CD34+ cells/kg) and for a harvest of gous bone marrow for gene transfer (target of
x 106 CD34+cells/kg with a minimum of 4 x 106 CD34+ cells/kg).
t of a back-up autologous graft
Hematopoietic cells will be collected from the patient in advance of the treatment,
to serve as a salvage procedure (“back-up graft”), should there be no hematopoietic recovery
observed 6 weeks following the injection of genetically-manipulated cells, or should lated
cells fail to meet e criteria. Bone marrow (up to 20 cc/kg) will be harvested from the patient
under general anesthesia from the posterior iliac crests on both sides by multiple punctures at a
minimum of 4 weeks prior to gene therapy. A n of the bone marrow containing 2 x 106
CD34+ cells/kg will be frozen and stored unmanipulated in liquid nitrogen vapors (162ºC and -
180ºC) according to rd clinical procedures for autologous bone marrow collection to
constitute the back-up graft. The remainder of the harvest will be selected for CD34+ cells
(described below) and utilized for gene modification (described below).
Bone marrow harvest
[jnnn] The remainder of the first bone marrow harvest in excess of the needed back up
marrow will be utilized with a second bone marrow harvest for gene transfer. The second
harvest will occur no sooner than n weeks after the initial harvest (described above). For
the second harvest, bone marrow will again be harvested from the patient under general
anesthesia from the ior iliac crests on both sites by multiple punctures. The amount
of marrow collected will be up to lj ml/kg of body weight. This will give a total nucleated
cell count of greater than ~n x kjr cells/kg. This in turn should yield a CDmn+ cell dose of
greater than n x kjp cells/kg after CDmn+ cell selection.
[jnno] Subjects from whom the estimated CDmn+ count of both harvests is < n x kjp
cells/kg will not receive conditioning. After a period of at least p weeks, if the subject
wishes to remain on study, he may be harvested again. If the subject does not wish to be
harvested again, he will be withdrawn from the study.
[jnnp] Subjects withdrawn from the study prior to administration of transduced
CDmn+ cells will resume normal clinical care (supportive care and/or allogeneic HSCT).
Efficacy and safety assessments will not be carried out from the point of withdrawal and
data will not be collected for the database.
CD34+ cell isolation, pre-stimulation, and transduction
CD34+ cell purification.
[jnnq] To allow sufficient time for clearance of conditioning agents and minimize
the time of pre-stimulation and culture, whole bone marrow will be held overnight. All the
manufacturing steps are performed in the Connell & ly Families Cell Manipulation
Core Facility at the DFCI. The bone marrow will be red cell-depleted by density gradient
centrifugation. CDmn+ cells will be positively selected from the bone marrow mononuclear
cells using the CliniMACS reagent and instrument. Quality control (QC) samples are taken
to assess purity and ity. Purified cells will be immediately sed for prestimulation
and uction.
CD34+ pre-stimulation and uction
[jnnr] Transduction will be carried out on one or both harvests. Transduction of
cells in excess of the back-up marrow target from the first harvest will be transduced and
frozen for future use. The second harvest will be used for gene transfer in its entirety and
the transduced t of the second harvest will be infused with the thawed transduced
cells from the first harvest after ioning.
[jnns] Purified CDmn+ cells are seeded in closed culture bags at a density of j.o-k x
kjp/ml in serum-free medium supplemented with growth s (IL-m, SCF, FLTmL, TPO)
and placed in an incubator at mqºC, o% COl. After ln-mj hours, cells are harvested and
counted. Additional QC testing includes cell viability, and Colony Forming Unit (CFU)
assay. Cells are transferred to a new e bag and treated with lentiviral supernatant. For
this first round of transduction, cells are ted for kr-ln hours. Cells are then
harvested, counted, and transferred to a new bag, with lentiviral supernatant for a second
round of transduction.
Final harvest and formulation
[jnoj] After the second round of transduction, cells are harvested, washed in
plasmalyte and resuspended in their final formulation (PLASMALYTE, k%HSA) in a
volume of oj-kjj mL. All cells available after l of the QC samples will be infused
into the patient. QC includes cell count, viability, sterility on wash supernatant,
Mycoplasma, Endotoxin on supernatant, phenotype, CFU, RCL (samples taken and
archived), insertional analysis, and average vector copy number by qPCR (cultured cells).
A sample for Gram stain is taken from the product immediately before delivery to the
patient.
Testing prior to subject re-infusion
[jnok] Samples are collected during and at the end of the procedure for cell count
and viability (trypan blue exclusion or equivalent), sterility, mycoplasma, transduction
efficiency (vector copy number), Gram stain, endotoxin and RCL testing. Of these only
cell ity, sterility (in process, ql hours), Gram stain and endotoxin measurements will
be available prior to infusion.
[jnol] If microbiological cultures reveal ent bacterial contamination, by Gram
stain or positive culture at ql hours, Cell lation Core Facility staff will contact the
PI, the assistant medical director and attending physician to decide whether to infuse the
p harvest or infuse the product with antibiotic coverage. If p harvest is
infused, the subject will be withdrawn from the protocol. If the cell ity is <qj%,
sterility testing is positive, or endotoxin is > o hr, the cells will not be ed,
p harvest will be infused and the subject will be withdrawn from the protocol.
[jnom] If viable cell count from both harvests/transductions is greater than or equal
to n x kjp CDmn+ cells/kg at the end of uction, cells will be d. If viable cell
count from both harvests/transductions is less than n x kjp CDmn+ cells/kg at the end of
transduction, cells will not be infused and back-up harvest will be infused nr hours later.
Subject conditioning regimen
Subjects will receive myeloablative conditioning with Busulfan (~4mg/kg
intravenously daily, adjusted for weight, (given over 3 hours once daily) administered on days -4
to -2, prior to infusion of transduced cells. Conditioning will occur concurrent with purification
and transduction of bone marrow cells. Busulfan levels will be drawn on all 3 days of
administration, and levels on days 1 and 2 will be used to adjust the area under the curve target.
Infusion of transduced cells
Cells will be infused intravenously over 30-45 s after standard
ration and premedication according to Boston Children’s al Hematopoietic Stem
Cell Transplantation Unit standard guidelines. This standard requires that the patient be on
continuous cardiac, respiratory and oxygen saturation monitor throughout the infusion and for
minutes afterwards. Vital signs will be measured and recorded ansfusion, 15 minutes
into transfusion, every hour for duration of infusion, and end of transfusion. The RN will stay
with the patient for the first 5 minutes of the transfusion. If two transduction products are
administered, the second transduced product will be administered without delay after the first.
It is to be understood that while the invention has been described in ction
with the ed description thereof, the foregoing description is intended to illustrate and not
limit the scope of the invention, which is defined by the scope of the appended claims. Other
s, advantages, and modifications are within the scope of the following claims.
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List of synthetic miR oligonucleotides
BCL11A miR1 oligos:
Sense ACGCTCGCACAGAACACTCATGGATTctccatgtggtagagAATCCATGAGTGTTCTGTGCGag (SEQ
ID NO:1)
Anti-sense CGCActCGCACAGAACACTCATGGATTctctaccacatggagAATCCATGAGTGTTCTGTGCGa
(SEQ ID NO:2)
BCL11A miR2 oligos:
Sense ACGCTCCAGAGGATGACGATTGTTTActccatgtggtagagTAAACAATCGTCATCCTCTGGag (SEQ
ID NO:3)
Anti-sense CGCActCCAGAGGATGACGATTGTTTActctaccacatggagTAAACAATCGTCATCCTCTGGa
(SEQ ID NO:4)
BCL11A E3 oligos:
Sense
ACGCTTCGGAGACTCCAGACAATCGCctccatgtggtagagGCGATTGTCTGGAGTCTCCGAag (SEQ ID
NO:5)
ense CGCActTCGGAGACTCCAGACAATCGCctctaccacatggagGCGATTGTCTGGAGTCTCCGAa
(SEQ ID NO:6)
BCL11A D8 oligos:
Sense
TCTCTTGCAACACGCACAGActccatgtggtagagTCTGTGCGTGTTGCAAGAGAAag (SEQ ID
NO:7)
Anti-sense CGCActTTCTCTTGCAACACGCACAGActctaccacatggagTCTGTGCGTGTTGCAAGAGAAa
(SEQ ID NO:8)
BCL11A XLC4 or C4 oligos:
Sense
ACGCTACAGTACCCTGGAGAAACACActccatgtggtagagTGTGTTTCTCCAGGGTACTGTag (SEQ ID
NO:9)
Anti-sense CGCActACAGTACCCTGGAGAAACACActctaccacatggagTGTGTTTCTCCAGGGTACTGTa
(SEQ ID NO:10)
Non-targeting oligos:
Sense ACGCTCAACAAGATGAAGAGCACCAActccatgtggtagagTTGGTGCTCTTCATCTTGTTGag (SEQ
ID NO:11)
Anti-sense CGCActCAACAAGATGAAGAGCACCAActctaccacatggagTTGGTGCTCTTCATCTTGTTGa
(SEQ ID NO:12)
BCL11A E3G5 or E3 mod : (modified version)
Sense
ACGCTGCGCTCGGAGACTCCAGACAActccatgtggtagagTTGTCTGGAGTCTCCGAGCGCag(SEQ ID
NO:13)
Antisense
CGCActGCGCTCGGAGACTCCAGACAActctaccacatggagTTGTCTGGAGTCTCCGAGCGC a(SEQ ID
NO:14)
BCL11A D8G5 or D8 mod oligos: (modified version)
Sense
ACGCTGCGCTTCTCTTGCAACACGCActccatgtggtagagTGCGTGTTGCAAGAGAAGCGCag(SEQ ID
NO:15)
Antisense
CGCActGCGCTTCTCTTGCAACACGCActctaccacatggagTGCGTGTTGCAAGAGAAGCGC a(SEQ ID
NO:16)
BCL11A XLC4G5 oligos: (modified version)
Sense
ACGCTGCGCACAGTACCCTGGAGAAActccatgtggtagagTTTCTCCAGGGTACTGTGCGCag(SEQ ID
NO:17)
Antisense
CGCActGCGCACAGTACCCTGGAGAAActctaccacatggagTTTCTCCAGGGTACTGTGCGCa(SEQ ID
NO:18)
mIR1 CGCACAGAACACTCATGGATTctccatgtggtagagAATCCATGAGTGTTCTGTGCG (SEQ ID NO:25)
(shRNA1 or E3) TCGGAGACTCCAGACAATCGCctccatgtggtagagGCGATTGTCTGGAGTCTCCGA (SEQ ID
NO:26)
(shRNA2 or B5 ) CCTCCAGGCAGCTCAAAGATCctccatgtggtagagGATCTTTGAGCTGCCTGGAGG (SEQ ID
NO:27)
(shRNA3 or D8) TTCTCTTGCAACACGCACAGActccatgtggtagagTCTGTGCGTGTTGCAAGAGAA (SEQ ID
NO:28)
(shRNA4 or B11) TCAGGACTAGGTGCAGAATGTctccatgtggtagagACATTCTGCACCTAGTCCTGA (SEQ ID
NO:29)
(shRNA5 or 50D12 or D12) GATCGAGTGTTGAATAATGATctccatgtggtagagATCATTATTCAACACTCGATC
(SEQ ID NO:30)
(shRNA6 or 50A5 or A5) CCTGGAGAAACACATctccatgtggtagagATGTGTTTCTCCAGGGTACTG
(SEQ ID NO:31)
(shRNA7 or 50B11) CACTGTCCACAGGAGAAGCCActccatgtggtagagTGGCTTCTCCTGTGGACAGTG (SEQ
ID NO:32)
(shRNA8 or 50C4) ACAGTACCCTGGAGAAACACActccatgtggtagagTGTGTTTCTCCAGGGTACTGT (SEQ ID
NO:33)
mIR1G5 gcgcCGCACAGAACACTCATGctccatgtggtagagCATGAGTGTTCTGTGCGgcgc (SEQ ID
NO:34)
(shRNA1mod or E3G5) gcgcTCGGAGACTCCAGACAActccatgtggtagagTTGTCTGGAGTCTCCGAgcgc
(SEQ ID NO:35)
(shRNA2mod or B5G5) gcgcCCTCCAGGCAGCTCAAActccatgtggtagagTTTGAGCTGCCTGGAGGgcgc
(SEQ ID NO:36)
3mod or D8G5) gcgcTTCTCTTGCAACACGCActccatgtggtagagTGCGTGTTGCAAGAGAAgcgc
(SEQ ID NO:37)
(shRNA4mod or B11G5)
gcgcTCAGGACTAGGTGCAGActccatgtggtagagTCTGCACCTAGTCCTGAgcgc (SEQ ID NO:38)
5mod or 50D12G5 or D12G5)
gcgcGATCGAGTGTTGAATAActccatgtggtagagTTATTCAACACTCGATCgcgc (SEQ ID NO:39)
(shRNA6mod or 50A5G5)
gcgcCAGTACCCTGGAGAAACctccatgtggtagagGTTTCTCCAGGGTACTGgcgc (SEQ ID NO:40)
(shRNA7mod or 50B11G5)
gcgcCACTGTCCACAGGAGAActccatgtggtagagTTCTCCTGTGGACAGTGgcgc (SEQ ID NO:41)
(shRNA8mod or 50C4G5 or C4G5)
gcgcACAGTACCCTGGAGAAActccatgtggtagagTTTCTCCAGGGTACTGTgcgc (SEQ ID NO:42)
(BCL11A D12G5-2 shRNA): Sense
ACGCTCGCACAGAACACTCATGGATTctccatgtggtagagAATCCATGAGTGTTCTGTGCGAG (SEQ. ID.
NO:43)
(BCL11A D12G5-2 shRNA): Anti-sense
CGCACTCGCACAGAACACTCATGGATTctctaccacatggagAATCCATGAGTGTTCTGTGCGA (SEQ. ID.
NO:44)
’-CCGGCGCACAGAACACTCATGGATTCTCGAGAATCCATGAGTGTTCTGTGCGTTTTT-
. ID. NO:86)
’CGCTCGCACAGAACACTCATGGATTctccatgtggtagagAATCCATGAGTGTTCTGTGCG
AGTG-3’(SEQ. ID. NO:87)
’CGCTGCGCCGCACAGAACACTCATGctccatgtggtagagCATGAGTGTTCTGTGCGGCGCA
GTG-3’(SEQ. ID. NO:88)
’-CCGGACAGTACCCTGGAGAAACACACTCGAGTGTGTTTCTCCAGGGTACTGTTTTTT-
3’(SEQ. ID. NO:89)
’-CGCTACAGTACCCTGGAGAAACACActccatgtggtagagTGTGTTTCTCCAGGGTACTGT
AGTG-3’(SEQ. ID. NO:90)
’CGCTGCGCACAGTACCCTGGAGAAActccatgtggtagagTTTCTCCAGGGTACTGTGCGCA
GTG-3’(SEQ. ID. NO:91)
’-CCGGTTCTCTTGCAACACGCACAGACTCGAGTCTGTGCGTGTTGCAAGAGAATTTTT-
3’(SEQ. ID. NO:92)
TTCTCTTGCAACACGCACAGActccatgtggtagagTCTGTGCGTGTTGCAAGAGAAA
GTG-3’(SEQ. ID. NO:93)
’CGCTGCGCTTCTCTTGCAACACGCActccatgtggtagagTGCGTGTTGCAAGAGAAGCGCA
GTG-3’(SEQ. ID. NO:94)
’-CCGGGATCGAGTGTTGAATAATGATCTCGAGATCATTATTCAACACTCGATCTTTTT-
3’(SEQ. ID. NO:95)
TGATCGAGTGTTGAATAATGATctccatgtggtagagATCATTATTCAACACTCGATC
AGTG-3’(SEQ. ID. NO:96)
’CGCTGCGCGATCGAGTGTTGAATAActccatgtggtagagTTATTCAACACTCGATCGCGCA
GTG-3’(SEQ. ID. NO:97)
New name Old name
shRNA1 E3
shRNA2 B5
shRNA3 D8
shRNA4 B11
shRNA5 50D12 or D12
shRNA6 50A5 or A5
shRNA7 50B11
shRNA8 50C4
mod E3G5
shRNA2mod B5G5
shRNA3mod D8G5
shRNA4mod B11G5
shRNA5mod 50D12G5 or
D12G5
shRNA6mod 50A5G5
shRNA7mod 50B11G5
shRNA8mod 50C4G5 or C4G5
Claims (32)
1. A synthetic BCL11A microRNA comprising; a) a first BCL11A segment, a loop segment; and b) a second BCL11A segment arranged in tandem in a 5' to 3' ion, wherein the loop segment is between and directly linked to the first and second BCL11A segments, and wherein the second BCL11A segment is complementary to the first BCL11A segment so that the first and second BCL11A segments base pair to form a n loop with the loop segment forming the loop n of the hairpin loop thus formed; and the first BCL11A segment starts with a -GCGC- at the 5' end and the second BCL11A segment ends with a - GCGC- at the 3' end.
2. The synthetic BCL11A microRNA of claim 1, wherein the first and second BCL11A segments are about 18 to 25 nucleotides long.
3. The synthetic BCL11A microRNA of claim 1 or 2, wherein the first BCL11A segment contains a ce derived from a BCL11A mRNA
4. The synthetic BCL11A microRNA of any one of claims 1-3, wherein the first BCL11A t comprises the nucleotide sequence set forth in any one of SEQ ID NOS: 57, 58, 59, 60, 61, 62, 63, 64, and 65.
5. The synthetic BCL11A microRNA of any one of claims 1-3, wherein the loop segment is derived from a microRNA.
6. The synthetic BCL11A NA of claim 5, wherein the microRNA is a hematopoietic specific microRNA.
7. The synthetic BCL11A microRNA of claim 6, wherein the microRNA is miR223.
8. The synthetic BCL11A microRNA of claim 7, wherein the loop segment is ctccatgtggtagag.
9. The synthetic BCL11A microRNA of any one of claims 1-8, wherein the microRNA comprises a nucleotide sequence set forth in any one of SEQ ID NOS: 34, 35, 36, 37, 38, 39, 40, 41 and 42.
10. An isolated nucleic acid molecule comprising the nucleotide sequence set forth in any one of SEQ ID NOS: 34, 35, 36, 37, 38, 39, 40, 41 and 42.
11. A vector sing the isolated c acid molecule of claim 10.
12. The vector of claim 11, wherein the vector further comprises a spleen focusforming virus promoter, a tetracycline-inducible promoter, or a β-globin locus control region and a β- globin promoter.
13. The vector of claim 11, wherein the vector is a viral vector.
14. The vector of claim 13, n the viral vector is a iral vector.
15. The vector of claim 14, n the lentiviral vector is ed from a lentivirus selected from the group consisting of: human immunodeficiency virus type 1 (HIV-1), human immunodeficiency virus type 2 (HIV-2), caprine arthritis-encephalitis virus , equine infectious anemia virus (EIAV), feline immunodeficiency virus (FIV), bovine immune deficiency virus (BIV), and simian immunodeficiency virus (SIV).
16. An ex vivo host cell comprising the vector of any one of claims 11-15, wherein the cell is a hematopoietic stem cell or its progeny.
17. The cell of claim 16, wherein the cell is an erythroid progenitor cell or its progeny.
18. The cell of claim 16, wherein the cell is an erythrocyte.
19. A bacterium comprising the isolated nucleic acid molecule of claim 10.
20. A composition comprising a synthetic BCL11A microRNA of any one of claims 1-9; an isolated nucleic acid molecule of claim 10, a vector of any one of claims 11-15, or a host cell of any one of claims 16-18.
21. A composition comprising a vector of any one of claims 11-15, or a host cell of any one of claims 16-18.
22. The composition of claim 20 or 21, further comprising a pharmaceutically acceptable carrier or diluent.
23. A use of a ition of any one of claims 20-22 in the manufacture of a medicament for the treatment or for reducing a risk of developing, a hemoglobinopathy in a subject.
24. A use of a composition of any one of claims 20-22 in the manufacture of a medicament for increasing the fetal hemoglobin levels expressed by a cell.
25. The use of claim 24, n the cell is an erythroid progenitor cell or its progeny.
26. A synthetic BCL11A microRNA as claimed in any one of claims 1-9 substantially as herein described and with reference to any example thereof.
27. An isolated nucleic acid as d in claim 10 substantially as herein described and with reference to any example thereof.
28. A vector as claimed in any one of claims 11-15 substantially as herein described and with nce to any example thereof.
29. A host cell as d in any one of claims 16-18 substantially as herein described and with reference to any example thereof.
30. A bacterium as claimed in claim 19 substantially as herein described and with nce to any example thereof.
31. A composition as claimed in any one of claims 20-22 substantially as herein described and with reference to any example thereof.
32. A use as claimed in any one of claims 23-25 substantially as herein described and with reference to any example thereof. O C\ i . = o 'Ji ^1 C/i -4 ■0 n H C c/) u ACGCTCGCACAGAACACTCATGGATTctCCatgtggtagagAATCCATGAGTGTTCTGTGGGAG ESr gAATCCATGAGTGTTCTGTGCGA CGCACTCGCACAGAACACTCATGGATTctctaccaca t gga ACGCTCGAGAGGATGACGATTGTTTActCCatgtggtagagTAAACAATCGTCATCCTCTGGag tctaccacatggagTAAACAATCGTCATCCTCTGGa shRNA a r»- m J3-globiii P forming shRNA *-1 for JFGK Verus Venus shRNA shRNA tmm TxTelO-rme sequence Venus CCAGAGGATGACGATTGTTTAc r* CGCAc t (synthetic) SFFV evaluation) evaluation) RRE cPPT SA ends RRE cPPT SA RRE cPPT SA toxicty dosage 1) 2) 3) 4) shRNA loop BCL11A targeting down >i> SO V SO V SD ■ = Cloning ID. NO: ID. NO: NO: ID. NO: ID. = bases {functional and Inducibie knock-down (functional and Constitutive Lineage-specific knock-down (therapeutic option) (SEQ. bases case oligonucleotides: (SEQ. (SEQ. (SEQ. upper miR2 oligonucleotides: Bcllla miRl Sense Anti-sense Bcllla Sense Anti-sense ized lower Bold and Underlined bases 4-
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US201461984247P | 2014-04-25 | 2014-04-25 | |
US61/984,247 | 2014-04-25 | ||
US201462066783P | 2014-10-21 | 2014-10-21 | |
US62/066,783 | 2014-10-21 | ||
PCT/US2015/027527 WO2015164750A2 (en) | 2014-04-25 | 2015-04-24 | Compositions and methods to treating hemoglobinopathies |
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NZ726792B2 true NZ726792B2 (en) | 2021-01-06 |
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