U.S. patent application number 17/694841 was filed with the patent office on 2022-06-30 for viral vectors for prophylaxis and therapy of hemoglobinopathies.
The applicant listed for this patent is The Children's Hospital of Philadelphia, CORNELL UNIVERSITY. Invention is credited to Gerd Blobel, Laura Breda, Wulan Deng, Alisa Dong, Stefano Rivella.
Application Number | 20220202958 17/694841 |
Document ID | / |
Family ID | 1000006207789 |
Filed Date | 2022-06-30 |
United States Patent
Application |
20220202958 |
Kind Code |
A1 |
Rivella; Stefano ; et
al. |
June 30, 2022 |
Viral Vectors for Prophylaxis and Therapy of Hemoglobinopathies
Abstract
Provided are compositions and methods for inducing expression of
human beta-globin in erythrocytes for use in prophylaxis and/or
therapy of a hemoglobinopathy in an individual. The method
generally entails introducing into CD34+ cells a polynucleotide
encoding: i) a 5' long terminal repeat (LTR) and a
self-inactivating 3' LTR; ii) at least one polyadenylation signal;
iii) at least one promoter; iv) a globin gene locus control region
(LCR); v) an ankyrin insulator element (Ank); vi) a Woodchuck
Post-Regulatory Element (WPRE) configured such that the WPRE does
not integrate into a target genome; and vii) a sequence that is a
reverse complement of a sequence encoding human beta-globin, and
can include beta-globin that has a .beta.T87Q mutation. Intron 2 of
the beta globin gene can be a complete intron. Modified erythrocyte
progenitor cells, recombinant vectors and virions comprising
recombinant polynucleotides, and methods of making the vectors and
virions are included.
Inventors: |
Rivella; Stefano; (New York,
NY) ; Breda; Laura; (New York, NY) ; Dong;
Alisa; (New York, NY) ; Blobel; Gerd; (Bala
Cynwyd, PA) ; Deng; Wulan; (Ashburn, VA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
CORNELL UNIVERSITY
The Children's Hospital of Philadelphia |
Ithaca
Philadelphia |
NY
PA |
US
US |
|
|
Family ID: |
1000006207789 |
Appl. No.: |
17/694841 |
Filed: |
March 15, 2022 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15545159 |
Jul 20, 2017 |
11311632 |
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PCT/US16/14269 |
Jan 21, 2016 |
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17694841 |
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62105829 |
Jan 21, 2015 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12N 2830/50 20130101;
C12N 15/867 20130101; C12N 2740/15043 20130101; A61K 48/005
20130101; C12N 2830/40 20130101; C12N 2830/52 20130101; C12N
2830/48 20130101; C12N 15/86 20130101 |
International
Class: |
A61K 48/00 20060101
A61K048/00; C12N 15/86 20060101 C12N015/86; C12N 15/867 20060101
C12N015/867 |
Goverment Interests
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH
[0002] This invention was made with government support under
contract no. 1R01HL102449 awarded by the National Institutes of
Health. The government has certain rights in the invention.
Claims
1. A method for inducing expression of human beta-globin in
erythrocytes for use in prophylaxis and/or therapy of a
hemoglobinopathy in an individual comprising introducing into
erythrocyte progenitor cells a polynucleotide encoding: i) a 5'
long terminal repeat (LTR) and a self-inactivating 3' LTR; ii) at
least one polyadenylation signal; iii) at least one promoter; iv) a
globin gene locus control region (LCR); v) an ankyrin insulator
element (Ank); vi) a Woodchuck Post-Regulatory Element (WPRE)
configured such that the WPRE does not integrate into a target
genome; and vii) a sequence that is a reverse complement of a
sequence encoding modified human beta-globin comprising a (3T87Q
mutation (B-globinM), wherein the sequence encoding the B-globinM
comprises a first intron (intron 1) between exon 1 and exon 2, and
a second intron (intron 2) between exon 2 and exon 3 of said
B-globinM sequence, wherein intron 2 comprises more than 476
nucleotides of human B-globinM intron 2 sequence; wherein,
subsequent to the introducing, the erythrocyte progenitor cells
differentiate into erythrocytes, wherein the erythrocytes are
present in the individual and the erythrocytes produce more human
beta-globin than a control.
2. The method of claim 1, wherein the control comprises a human
beta-globin value obtained from control cells, wherein the control
cells comprise erythrocytes from an individual who has the
hemoglobinopathy, wherein the erythrocytes are progeny of cells
into which a control viral vector is introduced, the control viral
vector comprising the 5' LTR, the 3' LTR, the at least one
polyadenylation signal, the at least one promoter, the LCR, the
Ank, a WPRE, and the sequence encoding the B-globinM, but wherein
the sequence encoding the B-globinM in the control viral vector
comprises 476 or fewer nucleotides of the human B-globinM intron 2
sequence.
3. The method of claim 1, wherein the lentiviral vector further
comprise a sequence encoding a fusion of an Ldb1 transcription
factor and a zinc finger (ZF) domain.
4. The method of claim 1, wherein the lentiviral vector further
comprises a sequence encoding an RNA polynucleotide that is a
reverse complement of mRNA encoding transferrin receptor 1, and
wherein the RNA polynucleotide comprises a microRNA or an shRNA
sequence and is capable of decreasing transferrin receptor 1 mRNA
in an RNAi-mediated process.
5. The method of claim 1, wherein the erythrocyte progenitor cells
comprise CD34+ cells, wherein the CD34+ cells are separated from
the individual before the introducing the lentiviral vector to the
cells, and wherein the CD34+ cells are introduced into the
individual subsequent to the introducing of the lentiviral
vector.
6. The method of claim 1, wherein the intron 2 comprises 851
nucleotides of the adult human B-globinM intron 2 sequence.
7. The method of claim 1, wherein the increased human beta-globin
comprises adult hemoglobin, fetal hemoglobin, B-globinM, or a
combination thereof.
8. The method of claim 7, wherein the hemoglobinopathy comprises
sickle-cell anemia (SCA) or beta-thalassemia.
9. A lentiviral vector for inducing expression of human beta-globin
in erythrocytes and/or erythrocyte progenitor cells for use in
prophylaxis and/or therapy of a hemoglobinopathy in an individual,
the lentiviral vector comprising: i) a 5' long terminal repeat
(LTR) and a self-inactivating 3' LTR; ii) at least one
polyadenylation signal; iii) at least one promoter; iv) a globin
gene locus control region (LCR); v) an ankyrin insulator element
(Ank); vi) a Woodchuck Post-Regulatory Element (WPRE) configured
such that the WPRE does not integrate into a target genome; vii) a
sequence that is a reverse complement of a sequence encoding
modified adult human beta-globin comprising a .beta.T87Q mutation
(B-globinM), wherein the sequence encoding the B-globinM comprises
a first intron (intron 1) between exon 1 and exon 2, and a second
intron (intron 2) between exon 2 and exon 3 of said B-globinM
sequence, wherein intron 2 comprises more than 476 nucleotides of
adult human B-globinM intron 2 sequence.
10. The lentiviral vector of claim 9, wherein the lentiviral vector
further comprise a sequence encoding a fusion of an Ldb1
transcription factor and a zinc finger (ZF) domain.
11. The lentiviral vector of claim 9, wherein the lentiviral vector
further comprises a sequence encoding a RNA polynucleotide that a
reverse complement of mRNA encoding transferrin receptor 1, and
wherein the RNA polynucleotide comprises a microRNA or a shRNA
sequence and is capable of decreasing transferrin receptor 1 mRNA
in a RNAi-mediated process.
12. The lentiviral vector claim 9, wherein the lentiviral vector is
present in CD34+ cells, wherein the CD34+ cells have been separated
from an individual who has a hemoglobinopathy.
13. The lentiviral vector of claim 12, wherein the lentiviral
vector is present in a virion.
14. A pharmaceutical composition comprising viral particles,
wherein the viral particles each comprise a RNA strand which
comprises a lentiviral vector of claim 9.
15. A method of making a viral particle preparation for use in
prophylaxis and/or therapy for one or more hemoglobinopathies
comprising introducing a plasmid encoding a lentiviral vector of
claim 1 into packaging cells which comprise a DNA packaging plasmid
which encodes at least one virion protein, and a DNA envelope
plasmid which encodes an envelope protein, and allowing expression
of the virion protein and the envelope protein such that viral
particles form, and separating the viral particles from the
packaging cells.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional application of U.S. patent
application Ser. No. 15/545,159, filed Jul. 20, 2017, which is a
.sctn. 371 application of PCT/US2016/014269, filed Jan. 21, 2016,
which claims priority to U.S. Provisional application No.
62/105,829, filed on Jan. 21, 2015. The foregoing applications are
incorporated by reference herein.
FIELD
[0003] The present disclosure related to compositions and methods
useful for prophylaxis and/or therapy of hemoglobinopathies.
BACKGROUND
[0004] There are a variety of hemoglobinopathies that affect large
portions of the human population. For example, sickle-cell anemia
(SCA) is a blood related disorder that affects the structure of the
hemoglobin molecules. In SCA the hemoglobin molecule is defective,
causing the entire blood cell to change shape (Steinberg, M. H.,
Forget, B. G., Higgs, D. R. & Nagel, R. L. Disorders of
hemoglobin: Genetics, Pathophysiology and Clinical Management,
(Cambridge University Press, Cambridge, UK, 2001)). After these
abnormal hemoglobin molecules release oxygen in circulation, they
may cluster together and form long, rod-like structures, which
become rigid and assume sickle shape. Unlike healthy red blood
cells, which are usually smooth and donut-shaped, sickled red blood
cells cannot squeeze through small blood vessels. Instead, they
stack up and cause blockages that deprive organs and tissues of
oxygen-carrying blood. This produces periodic episodes of pain and
ultimately can damage tissues and vital organs and lead to other
serious medical problems. Normal red blood cells live about 120
days in the bloodstream, but sickled red cells die after about 10
to 20 days. Because they cannot be replaced fast enough, the blood
is chronically short of red blood cells, leading to anemia.
[0005] SCA affects millions throughout the world. It is
particularly common among people whose ancestors come from
Sub-Saharan Africa, South America, Cuba, Central America, Saudi
Arabia, India, and Mediterranean countries such as Turkey, Greece,
and Italy. In the Unites States, it affects around 72,000 people,
most of whose ancestors come from Africa. The disease occurs in
about 1 in every 500 African-American births and 1 in every 1000 to
1400 Hispanic-American births. About 2 million Americans, or 1 in
12 African Americans, carry the sickle cell allele.
[0006] Beta-thalassemia is one of the two the most common
congenital anemias and is due to partial or complete lack of
synthesis of beta-globin chains and hemoglobin. Patients affected
by beta-thalassemia have mutations in the beta-globin gene. The
World Health Organization (WHO) estimates that 50,000 to 100,000
children are born with symptomatic features of beta-thalassemia
every year. Cooley's anemia, also known as beta-thalassemia major,
the most severe form of this disease, is characterized by
ineffective erythropoiesis (IE) and extra medullary hematopoiesis
(EMH) requiring regular blood transfusions to sustain life.
[0007] In beta-thalassemia intermedia, where a greater number of
beta-globin chains are synthesized, the clinical picture is milder
and patients do not require frequent transfusions (Musallam, K. M.,
et al. Non-transfusion-dependent thalassemias. Haematologica 98,
833-844 (2013); Rivella, S. The role of ineffective erythropoiesis
in non-transfusion-dependent thalassemia. Blood reviews 26 Suppl 1,
S12-15 (2012); Ginzburg, Y. & Rivella, S. beta-thalassemia: a
model for elucidating the dynamic regulation of ineffective
erythropoiesis and iron metabolism. Blood 118, 4321-4330 (2011)).
However, hemoglobin levels often decrease over time, splenomegaly
appears, and patients suffer from progressive iron overload due to
increased gastrointestinal iron absorption. Current disease
management includes prenatal diagnosis, transfusion therapy, iron
chelation and allogeneic bone marrow transplantation (BMT), which
is limited by finding a compatible bone marrow donor and presents
many risks and complications. Beta-thalassemia or Cooley's anemia
has a serious impact on the life of those afflicted, as well as on
society in general. Therefore, potential gene therapy approaches
that provide therapy to these patients would be highly relevant. In
this regard, mutations leading to Cooley's anemia can be classified
as beta0, such as beta0-39, in which a single point mutation
creates a stop codon and no beta-globin protein is produced, or
beta+, such as beta+-IVS1-110, where a mutation in the first intron
results in alternative splicing and insufficient beta-globin chain
synthesis (Musallam, K. M., et al. Non-transfusion-dependent
thalassemias. Haematologica 98, 833-844 (2013); Rivella, S. The
role of ineffective erythropoiesis in non-transfusion-dependent
thalassemia. Blood reviews 26 Suppl 1, S12-15 (2012); Ginzburg, Y.
& Rivella, S. beta-thalassemia: a model for elucidating the
dynamic regulation of ineffective erythropoiesis and iron
metabolism. Blood 118, 4321-4330 (2011)). Previous research has
shown that it is possible to rescue beta-thalassemia in mouse
models by lentiviral-mediated beta-globin gene transfer (May, C.,
et al. Successful treatment of murine beta-thalassemia intermedia
by transfer of the human beta-globin gene. Blood 99, 1902-1908.
(2002); May, C., et al. Therapeutic hemoglobin synthesis in
beta-thalassaemic mice expressing lentivirus-encoded human
beta-globin. Nature 406, 82-86 (2000); Rivella, S., et al. A novel
murine model of Cooley anemia and its rescue by lentiviral-mediated
human beta-globin gene transfer. Blood 101, 2932-2939 (2003)).
However, these animals are characterized by a complete deletion of
the mouse beta-globin gene. Additional hemoglobinopathies are
characterized by mutations in the beta-globin gene that lead to
additional aberrant beta-globin chains and patho-physiological
sequelae similar to sickle cell anemia and/or beta-thalassemia.
Thus, there is an ongoing and unmet need for improved compositions
and methods for treating hemoglobinopathies. The present disclosure
is pertinent to this need.
SUMMARY
[0008] The present disclosure provides compositions and methods for
prophylaxis and/or therapy of hemoglobinopathies. In general,
hemoglobinopathies comprise disorders that are characterized by
reduced synthesis or abnormal structure of the hemoglobin molecule.
Thalassemias are considered a type of hemoglobinopathy, with the
understanding that certain forms of hemoglobinopathies are
typically inherited single-gene disorders that result in abnormal
structure of one of the globin chains of the hemoglobin molecule,
whereas thalassemias are considered other forms of
hemoglobinopathies associated with underproduction of normal globin
proteins, such as those cases that arise due to mutations in
regulatory genes. Thus, the disclosure is pertinent to individuals
in need of treatment for disorders that include but are not
necessarily limited to those characterized by altered hemoglobin
structure, such as in hemoglobin C disease, hemoglobin S-C disease,
sickle cell anemia, as well as in various types of thalassemia that
are well known in the art, including but not necessarily limited to
beta-thalassemia. This is also pertinent to patients that have
mixed hemoglobinopathy features, such as hemoglobin S/thalassemia,
for instance.
[0009] In one aspect the disclosure provides a method for inducing
expression of human beta-globin in erythrocytes for use in
prophylaxis and/or therapy of a hemoglobinopathy in an individual.
The method generally comprises introducing into erythrocyte
progenitor cells, typically CD34+ cells, a polynucleotide encoding:
i) a 5' long terminal repeat (LTR) and a self-inactivating 3' LTR
(self-inactivating meaning the 3' LTR comprises a deletion relative
to its native sequence, and thus results is replication
incompetent); ii) at least one polyadenylation signal; iii) at
least one promoter; iv) a globin gene locus control region (LCR);
v) an ankyrin insulator element (Ank); vi) a Woodchuck
Post-Regulatory Element (WPRE) configured such that the WPRE does
not integrate into a target genome; and vii) a sequence that is a
reverse complement to a sequence encoding modified human
beta-globin comprising a .beta.T87Q mutation (B-globinM). The
sequence of human B-globinM is included with this disclosure. The
B-globinM is expressed from integrated DNA that is derived from the
viral genome. Thus, the B-globinM sequence in the RNA genome of the
modified lentiviral vectors of this disclosure is configured so
that the B-globinM is expressed in the context of the double
stranded DNA that is from integration of the modified lentiviral
genome. The expression can in certain embodiments be exclusive to
certain cell types, such as erythrocytes. It will be recognized in
certain instances that DNA sequences are provided, but those
skilled in the art can readily envision the RNA equivalent of the
DNA sequence, such as when a DNA sequence is used to illustrate
features of a lentiviral genome.
[0010] The sequence encoding the B-globinM comprises a first intron
(intron 1) between exon 1 and exon 2, and a second intron (intron
2) between exon 2 and exon 3. In certain embodiments intron 2
comprises more than 476 nucleotides of the human B-globinM intron 2
sequence, and may comprise up to the full intron 2 sequence. After
introducing the lentiviral construct into erythrocyte progenitor
cells the lentiviral construct integrates into one or more
chromosomes in the progenitor cells, and the progenitor cells
differentiate into erythrocytes. The erythrocytes derived from the
erythrocyte progenitor cells produce the beta-globin protein.
[0011] The disclosure provides for modified erythrocytes that
produce more human beta-globin than a suitable control. In one
embodiment the control comprises a human beta-globin value obtained
from control cells. In one non-limiting approach the control cells
comprise erythrocytes from an individual who has the
hemoglobinopathy, wherein the erythrocytes are progeny of
progenitor cells into which a control viral vector was introduced.
The control viral vector can comprise, for example, the 5' LTR, the
3' LTR, the at least one polyadenylation signal, the at least one
promoter, the LCR, the Ank, a WPRE, and the sequence encoding the
B-globinM, but the sequence encoding the B-globinM in the control
viral vector comprises 476 or fewer nucleotides of the human
B-globinM intron 2 sequence, and thus comprises an intron 2 that
contains a deletion. In one embodiment, the modified erythrocytes
produce increased adult hemoglobin, fetal hemoglobin, B-globinM, or
a combination thereof, relative to a control.
[0012] In one embodiment the lentiviral vectors can further
comprise a sequence encoding a fusion of an Ldb1 transcription
factor and a zinc finger (ZF) domain. In certain embodiments the
lentiviral vector can comprise a sequence encoding an RNA
polynucleotide that is at has reverse complementarity to mRNA
encoding transferrin receptor 1. The RNA polynucleotide is capable
of decreasing transferrin receptor 1 mRNA by, for example, an
RNAi-mediated process.
[0013] In certain approaches the disclosure includes modifying
erythrocyte progenitor cells that are CD34+ cells. The CD34+ cells
can be separated from the individual before the lentiviral vector
is introduced into them. The CD34+ cells can be introduced into the
individual subsequent to the introduction of the lentiviral vector.
The lentiviral vector can be introduced using any suitable
approach. In one embodiment the erythrocyte progenitor cells are
infected with viral particles comprising a lentiviral vector, such
as a recombinant+strand viral polynucleotide. In certain
embodiments, approximately 50 infection particles (IP) per cell are
used. In certain embodiments the cells into which the vector is
introduced can be enriched for a certain cell type, such as CD34+
cells. In certain approaches the cells are held for a period of
from 2-3 days after infection prior to introducing into an
individual in need thereof In embodiments the cells can be frozen
and introduced into the patients after they are thawed. The
lentiviral vectors can be introduced into a patient using any
suitable method. In one approach they are administered by
intravenous infusion, which may be performed subsequent to any
other technique, such as after depletion of bone marrow cells,
i.e., myeloablation. Those skilled in the art will recognize that a
cells characterized by a suitable vector copy number (VCN) can be
used. In one embodiment the VCN is approximately 1 VCN per
cell.
[0014] The disclosure includes the lentiviral vectors themselves,
the components of which are as described above for use in a method
of the disclosure. Lentiviral vectors comprise modified,
recombinant polynucleotides, and can comprise RNA or DNA
polynucleotides. In certain embodiments a lentiviral vector of this
disclosure comprises an isolated polynucleotide, or an isolated
preparation of virions that comprise the lentiviral vector. In an
embodiment a recombinant lentiviral vector of the disclosure is
present in CD34+ cells, wherein the CD34+ cells have been separated
from an individual.
[0015] Also provided is a method of making a viral particle
preparation for use in prophylaxis and/or therapy for one or more
hemoglobinopathies. This approach comprises introducing a plasmid
encoding a lentiviral vector as described above into packaging
cells, wherein the packaging cells comprise a DNA packaging plasmid
which encodes at least one virion protein, and wherein the
packaging cells comprises a DNA envelope plasmid which encodes an
envelope protein. The packaging and envelope plasmids express their
respective proteins, which facilitate formation of virions which
comprise an RNA lentiviral vector of this disclosure.
DESCRIPTION OF THE FIGURES
[0016] FIGS. 1A-1C. The level of HbF/GFP expressing cells increases
proportionally to the amount of molecules of GG1-SA vector
integrated. Lentiviral construct carrying the bicistronic cassette
that expresses the Ldb1 SA domain under the ankyrin promoter (Ank
Pr.) and the green fluorescence protein (GFP) through an internal
ribosomal entry site (IRES). The Woodchuck hepatitis virus
Posttrascriptional Regulatory Element (WPRE) increases RNA
stability and protein yield. FIG. 1A: Percentage of F, S and
A.sub.2 hemoglobins measured by HPLC in differentiated untreated
erythroid cells (left) or with up to 0.7 copies of ZF-Ldb1-viral
molecules integrated on average (middle and right). Increase in HbF
(FIG. 1B) and GFP (FIG. 1C) expressing cells measured by flow
cytometry in cells untreated or with 0.26 and 0.67 copies/cell of
ZF-Ldb1-viral molecules integrated.
[0017] FIGS. 2A-2C. pCL-ZF-Ldb1 supports high levels of
.gamma.-globin/hemoglobin F induction and concurrently reduces
sickle globin levels in sickle cell disease (SCD) erythroblasts.
FIG. 2A: .beta.-like globin mRNA content (.gamma., left, and
.beta.s, middle) was measured by Q-PCR and normalized by endogenous
GAPDH expression. .beta.-like globin expression changes were
further normalized by .alpha.-globin expression (which should
remain unchanged and is a measure of differentiation across
samples). Right: transgenic ZF-Ldb1 mRNA expression in treated
samples is plotted against integration of pCL-ZF-Ldb1. FIG. 2B: HbF
(left), HbS (middle) and HbA2 (right) changes in all SCD samples
treated with pCL-ZF-Ldb1 LV. FIG. 2C: Net HbF % increase and HbS
%-A2% decrease.
[0018] FIGS. 3A-3B. Quantification of tetrameric Hbs and single
globin chains in SCD cells untreated and treated with pCL-ZF-Ldb1.
FIG. 3A: Content of HbF increase (left) and all Hbs (right) in
erythroid cells without or with pCL-ZF-Ldb1. FIG. 3B: Single globin
quantification by liquid chromatography in denaturing conditions.
On left, the quantity of single .beta.- and .gamma.-globin chains
(.mu.g) is calculated over the quantity of single .alpha.-globin
chain. On right, means of .gamma.A+G chains/all .beta.-like chains
area indicated. .mu.g of Hbs or single globin chains in hemolysates
were extrapolated from calibration curves obtained with standard
samples with known Hb concentration.
[0019] FIGS. 4A-4D. pCL-ZF-Ldb1-transduced SCD cells expressing HbF
have a reduced expression of HbS compared to untransduced cells.
FIG. 4A: The number of GFP expressing SCD erythroblasts after
pCL-ZF-Ldb1 integration (right) is compared to untransduced
erythroblast (left). FIG. 4B: Percentage of HbF producing cells in
permeabilized erythroblasts (left, no anti-HbF Ab), untransduced
(center) and pCL-ZF-Ldb1 transduced erythroblasts (right). FIG. 4C:
.beta.-globin expressing cells within the HbF positive populations
from B (center and right) are compared to permeabilized cells
(left, no anti-HbB Ab). FIG. 4D: Quantification of HbF of SCD
samples (from B) analyzed by HPLC.
[0020] FIGS. 5A-5C. Expression of y-globin repressor genes in
healthy cells, sickle cells untreated or treated with pCL-ZF-Ldb1
LV. Bcl11A and C-Myb, (FIG. 5A) and SOX6 and KLF1 (FIG. 5B) genes'
expression is expressed in function of the level of cell
differentiation marker glycophorin A (GPA) and normalized by GAPDH
expression. The expression of KEL (FIG. 5C, left), the gene that
encodes Kell, a blood group antigen, was chosen as internal
control. Expression of transgenic Ldb1 in transduced samples (FIG.
5C, right) was confirmed in the same samples analyzed for the other
messenger RNAs.
[0021] FIGS. 6A-6B. Hemoglobin F increase/Sickle hemoglobin
decrease in CD34+-derived SCD erythroid cells treated with
pCL-ZF-Ldb1 LV and with HbF inducers in vitro. FIG. 6A: (Top) Net
increase of HbF % and (bottom) net decrease of HbS % in SCD
erythroblasts treated with the HbF inducers decitabine,
tranylcypromine, hydroxyurea, pomalidomide, butyrate or with the LV
carrying ZF-Ldb1. FIG. 6B: Erythroid cell count (hemoglobinized
cells, or benzidine+stained cells) for each treatment has been
normalized over the untreated sample. In A-B n=11, except for Hu
and pCL-ZF-Ldb1 in which n=10, while for But n=9.
[0022] FIGS. 7A-7C. Differential single globin chain synthesis in
cells treated with pCL-ZF-Ldb1. FIG. 7A: Chromatographic profile of
representative sickle cells hemolysate untreated (UT) or treated
with pCL-ZF-Ldb1. FIG. 7B: Calibration curve obtained using known
hemoglobin concentration of blood from SCD transgenic mouse. FIG.
7C: Areas under peak of single globin chains extrapolated from the
calibration curve (from B) in samples untreated or after treatment
with pCL-ZF-Ldb1 (n=5).
[0023] FIG. 8. Trend of moderately elevated HbF levels in
CD34+-derived SCD erythroid cells at steady state. Comparative HPLC
assessment of HbF% between healthy and SCD erythroid samples in
which .gamma.-globin repressors BCl11a, c-Myb, KLF1 and SOX6 were
quantified
[0024] FIG. 9. Cytotoxicity effect and dose/response calibration.
Erythroid cells count measurements (benzidine plus count) of cells
(N=2) at different doses of drugs in comparison with integration of
.about.1 copy/cell of GG1-SA. The arrows in black indicate the dose
of each drug chosen for the bulk of the experiments.
[0025] FIG. 10. Variation of HbF % SCD erythroid cells treated with
pCL-ZF-Ldb1 vector and with HbF inducers in vitro at different
times. Net increase of HbF % in SCD erythroblasts treated with the
HbF inducers 5-aza-cytidine or with the LV carrying ZF-Ldb1,
according to FIG. 16(L) and FIG. 17(E). Kruskal-Wallis with Dunn's
multiple comparison test.
[0026] FIG. 11. Graphical maps of vectors. Topmost vector map is
pCL-ZF-Ldb1, also referred to as pCL20cAnkyrinGG1DDiGFP. Second
from vector map is ALS-10. Third from top vector map is CT9Ank.
Bottom vector map is ALS-10T. Each vector comprises a 5' and a 3'
self-inactivating long terminal repeat (5'LTR and 3' SinLTR,
respectively). Also shown is "B-globinM" which is a mutant
beta-globin is known as the "(3T87Q" form. The B-globinM is
configured in the vector such that it is expressed from an
integrated DNA that is derived from the RNA genome via a well-known
process. "Ank" is an Ankyrin insulator. "IRES" is an internal
ribosomal entry site. "P" designates a promoter. "LCR" is a Locus
control region. "GFP" is enhanced green fluorescence protein. "pA"
is a polyadenylation signal. "WPRE" is the Woodchuck
Post-Regulatory Element. "fI1" is a full beta-globin gene intron 1.
"fI2" is a full beta-globin gene intron 2. "I1S" is a modified
beta-globin gene intron 1 comprising a microRNA targeting the
transferrin receptor. "SV40 oriR-pA" is an origin of replication
and polyadenylation signal. Beta-globin gene exons 1, 2 and 3 are
labeled accordingly.
[0027] FIG. 12 provides a graphical summary of results comparing
adult hemoglobin values (HbA) obtained from .beta.0/+ or .beta.0/0
erythroblasts patient CD34+ cells into which the AnkT9W and ALS10
vectors were introduced, as labeled.
[0028] FIG. 13 provides an annotated polynucleotide and encoded
protein sequence of the ZF-Ldb1 vector and the DNA equivalent of
the RNA. The nucleotide sequence in FIG. 13 is SEQ ID NO:1. The
ZF-LDB1 AA sequence (shown as GG1=ZF and DDi=LDB1, with the HA
terminal amino acid sequence) is SEQ ID NO:2. Vector features
labeled in boxes are shown above the pertinent sequences.
[0029] FIG. 14 provides an annotated polynucleotide sequence of the
ALS-10 vector as the DNA equivalent of the RNA and encoded protein
sequence. The nucleotide sequence shown in FIG. 14 is provided as
SEQ ID NO:3. Vector features in boxes are shown above the pertinent
sequences. The location of the initiating .beta.T87Q beta globin
methionine codon in its antiparallel configuration is the "CAT"
triplet to the immediate left of the beta globin 5' UTR, reading in
the 5' to 3' direction.
[0030] FIG. 15 provides an annotated sequence of the non-mutated
beta-globin cDNA. The cDNA sequence is SEQ ID NO:4. Also shown is
the beta-globin amino acid sequence which is provided in SEQ ID
NO:5. The .beta.T87Q mutation occurs at the Threonine which is
shown in the sequence at position 88. The mutation is referred to
as .beta.T87Q according to convention wherein the first Methionine
at position 1 is not included in the amino acid numbering. In the
.beta.T87Q mRNA, the codon for the Threonine at position 88 is
replaced by a codon encoding Glutamine.
[0031] FIG. 16. (Top) Experimental flow for expansion,
differentiation and treatment of human SCD CD-34+ cells with
pCL-ZF-Ldb1 and/or HbF pharmacological inducers. (Bottom) Type of
HbF inducer, biological activity and dose used in the study.
[0032] FIG. 17. Experimental flow for expansion, differentiation
and treatment of human SCD CD-34+ cells with pCL-ZF-Ldb1 or HbF
pharmacological inducers.
DETAILED DESCRIPTION
[0033] The present disclosure provides compositions and methods for
prophylaxis and/or therapy for hemoglobinopathies. In this regard,
and as is well known in the art, a few months after birth children
start producing red cells containing adult hemoglobin, which is the
oxygen carrier molecule made of alpha- and beta-globin chains,
encoded, respectively, by the alpha- and beta-globin genes. In many
hemoglobinopathies, mutations in the gene encoding the beta-globin
chain impair the synthesis of adult hemoglobin or lead to the
production of abnormal adult hemoglobin. This leads to limited
production of red cells or synthesis of abnormal red cells. For
these reasons, patients require blood transfusion for survival.
Before birth and during the first months of life, children express
fetal hemoglobin, which comprises gamma- and alpha-chains.
Generally, after birth the gene encoding the gamma-chain is
silenced, while the beta-globin gene is activated, switching the
production of hemoglobin from fetal to adult. In rare cases,
individuals that do not silence the gamma-globin gene and also
carry mutations in the beta-globin gene are spared from the disease
associated with reduced or abnormal production of adult hemoglobin.
Therefore, reactivation of fetal hemoglobin might be therapeutic in
ameliorating diseases associated with mutations in the beta-globin
gene. Activation and silencing of the gamma-globin gene depends on
the proximity of a genomic region called locus control region
(LCR), which maps .about.40 to 60 kilobases upstream of the gamma-
and beta-globin genes and is associated with many factors that
activate transcription. This region is required to "loop" and bind
the promoter of the gene that needs to be activated. When the
gamma-globin gene is expressed, the LCR loops and binds on the
gamma-globin promoter. After birth, the LCR moves away from the
gamma-globin promoter and loops and binds the beta-globin promoter
leading to silencing of the gamma gene and activation of the
globin-gene. The transcription co-factor Ldb1 is involved in the
LCR looping to the promoters of the gamma- and beta-globin genes.
Ldb1 alone does not promote the binding of the LCR to the promoter,
but requires additional factors. Artificial zinc-finger (ZF)
proteins have the ability of binding specific sequences on the DNA.
Ldb1 has been fused to a specific ZF protein that binds the
gamma-globin promoter. In transgenic mice, this protein promotes
the looping and binding of the LCR to the promoter of the gamma
globin gene, activating its expression (Deng, W., et al.
Controlling long-range genomic interactions at a native locus by
targeted tethering of a looping factor. Cell 149, 1233-1244
(2012)).
[0034] An alternative way to approach therapy of hemoglobinopathies
associated with mutation in the beta-globin gene is by inserting in
the bone marrow cells (and in particular in the hematopoietic stem
cells or HSCs) a functional beta-globin gene. Therefore, gene
transfer of the non-mutated form of the beta-globin gene by
lentiviral vectors could potentially restore production of the
human beta-globin protein in patients and be utilized for gene
therapy trials. Lentiviral vectors are well characterized for their
ability to infect and insert the human beta-globin gene into HSCs.
In this regard, the present disclosure provides novel viral vectors
that are designed for prophylaxis and/or therapy of
hemoglobinopathies. The viral vectors include but are not
necessarily limited to lentiviral vectors. In various embodiments
an isolated mammalian hematopoietic progenitor cell or an isolated
mammalian stem cell comprising a recombinant lentiviral vector is
provided.
[0035] Various embodiments of lentiviral vectors that are subjects
of this disclosure are shown in FIG. 11. Features of these vectors
include but are not necessarily limited to the capability to, when
introduced to an appropriate cell: 1) reactivate expression of
fetal hemoglobin, and/or 2) express a novel transgene adult
hemoglobin and/or, concurrently, 3) inactivate the expression of
mutant hemoglobin. Certain features of the vectors are known in the
art, and FIGS. 13 and 14 provide representative sequences of them.
In particular, any suitable IRES sequence can be used, and those
skilled in the art will recognize, given the benefit of this
disclosure, which promoter sequences can be adapted for use in
embodiments of the invention. Likewise, the LCR sequence is known
in the art, as are suitable polyadenylation signals that can
function, for example, in erythrocytes. The representative
sequences of this disclosure, such as those shown in FIGS. 13 and
14, can be altered according to well-known parameters, so long as
they impart to erythrocytes the ability to produced therapeutically
effective amounts of elevated globin. In certain cases
polynucleotide sequences can be identical to those presented
herein, or they can have least 80, 81, 82, 83, 84, 85, 86, 87, 88,
89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity across a
contiguous segment of the sequences.
[0036] It is expected that lentiviral vectors of this disclosure
will result in the expression of therapeutic levels of hemoglobins
where previous vectors have failed to do so. In embodiments, these
vectors can be specifically adapted to optimize production of
beta-globin plus gamma-globin mRNA, and protein levels in presence
of different mutations associated with beta-thalassemia, sickle
cell anemia and other hemoglobinopathies. Certain aspects of this
disclosure involve combinations of genomic elements, specific
embodiments of which are depicted schematically in FIG. 11. The
disclosure includes each and every polynucleotide sequence
disclosed herein, the RNA equivalent of every DNA polynucleotide
(i.e., where uracil replaces thymine) and every DNA equivalent of
every RNA, and the complementary sequence and the reverse
complement of every polynucleotide sequence. The disclosure
includes every amino acid sequence, and all polynucleotide
sequences encoding the amino acid sequences. Contiguous segments of
polynucleotides and polypeptides sequences are also included. In
non-limiting examples, sequences of this disclosure can comprise or
consist of any one or any combination of the contiguous segments of
each vector construct disclosed herein, including but not limited
to the segments and sequences depicted in FIGS. 11, 13, 14 and 15,
the RNA equivalents thereof, and the DNA and RNA reverse
complements thereof. In aspects of the disclosure integration of a
lentiviral vector of the disclosure results in an integrated
functional globin gene, the expression of which produces a globin
molecule that is effective to a participate in providing a
therapeutic benefit to an individual with a globinopathy. The gene
may exhibit conditional expression, such as being expressed in
erythrocytes, but not being expressed in for example, hematopoietic
stem cells comprising an integrated DNA segment derived from the
lentiviral construct, wherein the stem cells differentiate into the
erythrocytes that express the globin molecule. The disclosure also
includes cells, which comprise the recombinant polynucleotides.
[0037] The present disclosure includes a demonstration of using the
ALS10 lentiviral vector depicted in FIG. 11 to elevate HbA
production in cells from hemoglobinopathy patients. In particular,
the disclosure includes a demonstration that the ALS10 vector is
superior to previously available approaches for improving HbA
production in the most severe thalassemic specimens, i.e., those
individuals with .beta.0/0 phenotype. In this regard, the
disclosure provides a demonstration of using ALS10 to modify CD34+
cells from .beta.0/0 phenotype patients such that HbA production is
improved relative to a suitable control, and those skilled in the
art will recognize suitable controls given the benefit of this
disclosure. In embodiments, the control can be a single value or a
range of values. For example, a control can be a standardized curve
or an area on a graph. In one embodiment the control comprises the
increase in HbA produced using a construct described in Breda, L.,
et al., Therapeutic hemoglobin levels after gene transfer in
beta-thalassemia mice and in hematopoietic cells of
beta-thalassemia and sickle cells disease patients. PLoS One, 2012.
7(3): p. e32345, which is known in the art as "AnkT9W."
[0038] In more detail, and without intending to be constrained by
any particular theory, the approach of the current disclosure was
taken in part in an effort to increase adult hemoglobin in
thalassemic and SCD cells in a tissue specific manner, and
proportionally to the number of viral molecules inserted. To
attempt to reduce genome toxicity believed to be caused by random
integration of viral vectors, we strived to maintain viral
integration within an average of 2 copies/cell.
[0039] It has been demonstrated in thalassemic specimens with
moderate to medium range of HbA reduction (.beta.+/+ and .beta.+/0,
respectively) 0.6 copies of AnkT9W were sufficient to generate HbA
levels comparable to those detected in healthy or carrier cells,
which is about 80-90% (Breda et al, Plos One, 2012). However, in
specimens with the most severe phenotype, .beta.0/0, in which no
adult HbA is observed, 0.6 copies of AnkT9W could not meet the
level of adult hemoglobin observed in healthy or carrier cells,
which was a therapeutic threshold that we strived to achieve in the
presently provided approach. In connection with this we modified
AnkT9W to generate ALS10. In ALS10, the Woodchuck Post-Regulatory
Element (WPRE) was eliminated from the integrating sequence to
increase the safety of the vector. The WPRE was part of the
integrated portion of AnkT9W since it was placed between the LCR
and the 3' LTR. The original purpose of the WPRE was to increase
the titer of the lentivirus as it had been previously shown to have
that effect, but we have now determined that it is dispensable in
the current position. WPRE is a viral sequence and is not required
for the expression of the beta-globin gene. It is considered safer
to limit as much as possible the amount of viral sequences that are
integrated in the genome of patient cells. For this reason, we
moved this sequence in the vector to the non-integrating region.
This preserves the WPRE's ability to produce high titers of viral
particles but excludes the WPRE from the genome of patient cells.
In order to preserve the ability of WPRE to increase viral titers
without having it in the integrating sequence, we removed the WPRE
from the integrating portion (before the 3'LTR) and added it back
after the 3'LTR so that it is positioned outside the sequence that
is integrated in the chromosome(s) of target cells. We also added a
strong bovine growth hormone polyA tail after the WPRE region
(outlined in FIG. 11). Our data indicate that the modifications do
not decrease viral titer during production of compositions of this
disclosure. However, we also modified the segment of the construct
that results in expression of HbA in erythrocytes that are derived
from CD34+ cells into which the lentiviral vector is introduced. In
particular, the portion of the beta-globin gene intron 2 that is
deleted in the AnkT9W construct is annotated in FIG. 14. As can be
seen from that annotation, the beta-globin gene intron 2
encompasses nucleotides 4772-5621, inclusive, and is thus 851
nucleotides in length. However, the intron 2 in AnkT9W comprises a
deletion of the 375 nucleotides spanning nucleotides 5,164 through
5,537, inclusive (as designated in FIG. 14). As a consequence of
that deletion, the AnkT9W beta-globin gene intron 2 is 476
nucleotides in length. In contrast, and while not intending to be
bound by any particular theory, it is considered that including a
longer segment of intron 2 in the context of the ALS10 construct is
at least partially responsible for the unexpected and statistically
significant increase in HbA in the .beta.0/0 patient cells. In this
regard, when compared to AnkT9W, ALS10 showed significant
improvement and in particular leads to much higher level of HbA in
the most severe thalassemic patient specimens, namely, the
.beta.0/0 phenotype. It is therefore reasonable expect that the
present approach will benefit patients with hemoglobinopathies that
are not necessarily due to a .beta.0/0 phenotype, such as SCD. A
summary of the results demonstrating this advantage of ALS10 is
presented in FIG. 12. The disclosure thus includes lentiviral
vectors and cells comprising them, and the integrated construct,
wherein the beta-globin gene comprises an intron 2 of more than 476
nucleotides in length, and up to 851 nucleotides in length. The
intron is accordingly between 477-875 nucleotides, inclusive, and
including all integers and ranges of integers there between. The
intron 2 can be thus comprise or consist of 477, 478, 479, 480,
481, 482, 483, 484, 485, 486, 487, 488, 489, 490, 491, 492, 493,
494, 495, 496, 497, 498, 499, 500, 501, 502, 503, 504, 505, 506,
507, 508, 509, 510, 511, 512, 513, 514, 515, 516, 517, 518, 519,
520, 521, 522, 523, 524, 525, 526, 527, 528, 529, 530, 531, 532,
533, 534, 535, 536, 537, 538, 539, 540, 541, 542, 543, 544, 545,
546, 547, 548, 549, 550, 551, 552, 553, 554, 555, 556, 557, 558,
559, 560, 561, 562, 563, 564, 565, 566, 567, 568, 569, 570, 571,
572, 573, 574, 575, 576, 577, 578, 579, 580, 581, 582, 583, 584,
585, 586, 587, 588, 589, 590, 591, 592, 593, 594, 595, 596, 597,
598, 599, 600, 601, 602, 603, 604, 605, 606, 607, 608, 609, 610,
611, 612, 613, 614, 615, 616, 617, 618, 619, 620, 621, 622, 623,
624, 625, 626, 627, 628, 629, 630, 631, 632, 633, 634, 635, 636,
637, 638, 639, 640, 641, 642, 643, 644, 645, 646, 647, 648, 649,
650, 651, 652, 653, 654, 655, 656, 657, 658, 659, 660, 661, 662,
663, 664, 665, 666, 667, 668, 669, 670, 671, 672, 673, 674, 675,
676, 677, 678, 679, 680, 681, 682, 683, 684, 685, 686, 687, 688,
689, 690, 691, 692, 693, 694, 695, 696, 697, 698, 699, 700, 701,
702, 703, 704, 705, 706, 707, 708, 709, 710, 711, 712, 713, 714,
715, 716, 717, 718, 719, 720, 721, 722, 723, 724, 725, 726, 727,
728, 729, 730, 731, 732, 733, 734, 735, 736, 737, 738, 739, 740,
741, 742, 743, 744, 745, 746, 747, 748, 749, 750, 751, 752, 753,
754, 755, 756, 757, 758, 759, 760, 761, 762, 763, 764, 765, 766,
767, 768, 769, 770, 771, 772, 773, 774, 775, 776, 777, 778, 779,
780, 781, 782, 783, 784, 785, 786, 787, 788, 789, 790, 791, 792,
793, 794, 795, 796, 797, 798, 799, 800, 801, 802, 803, 804, 805,
806, 807, 808, 809, 810, 811, 812, 813, 814, 815, 816, 817, 818,
819, 820, 821, 822, 823, 824, 825, 826, 827, 828, 829, 830, 831,
832, 833, 834, 835, 836, 837, 838, 839, 840, 841, 842, 843, 844,
845, 846, 847, 848, 849, 850, 851 nucleotides. The polynucleotide
sequence of the human beta globin gene is depicted in FIG. 13. It
includes the 3' enhancer element, the 3' untranslated region (UTR),
the polyA signal, exon 3, intron 2, exon 2, intron 1, exon 1, the
5' UTR, and the beta globin promoter segment. It is shown in the
3'-5' orientation because, as is well known in the art, it is
anti-parallel to the depicted remainder of the construct and is
expressed only after integration and in the context of a double
stranded DNA region wherein one strand of the DNA comprises the
beta globin gene sequence presented in FIG. 14
[0040] In ALS10 the sequence of the beta-globin gene is also
modified to increase its ability to prevent the sickling of the
sickle-beta-globin chains (referred to as "B-globinM" in FIG. 11).
This mutant beta-globin is known as the .beta.T87Q form, due to its
amino acid substitution at the 87th position. .beta.T87Q has been
used to improve hematological parameters of the SAD and BERK mouse
models of SCA (Pawliuk, R., et al. Correction of sickle cell
disease in transgenic mouse models by gene therapy. Science 294,
2368-2371. (2001)). This form was also used in the first successful
clinical trial to correct a patient with .beta.0/.beta.E
thalassemia (Cavazzana-Calvo, M., et al. Transfusion independence
and HMGA2 activation after gene therapy of human beta-thalassaemia.
Nature 467, 318-322 (2010)).
[0041] The ankyrin insulator that is well known in the art and was
present in AnkT9W is also present in ALS10. ALS10 has the ankyrin
insulator in the 3'LTR, while the vector AnkT9W has the ankyrin
insulator between the promoter and the LCR.
[0042] In one aspect of this disclosure, an intron of the
beta-globin gene, or other locations such as the 3'UTR, can be
modified to include a polynucleotide that targets and decreases the
synthesis of the transferrin receptor 1 (TR1). The rational of this
approach is based on our observations that decreased erythroid iron
intake can be beneficial in beta-thalassemia and, potentially other
hemoglobinopathies, because this decreases formation of heme
molecules. Heme is normally included in hemoglobin molecules but in
many hemoglobinopathies, due to the reduction in synthesis of
beta-globin chains, there is an excess of heme not bound to
hemoglobin, and these molecules are toxic to red cells. An excess
of heme is responsible for apoptosis of erythroblasts and for
altering the intracellular content, production of reactive oxygen
species (ROS), and for reducing membrane stability and plasticity
of erythrocytes, leading to their reduced lifespan, hemolysis
and/or sickling. A combination vector that expressed gamma-globin
and concurrently knocked-down sickle beta-globin via small hairpin
RNA has been described (Samakoglu, S., et al. A genetic strategy to
treat sickle cell anemia by coregulating globin transgene
expression and RNA interference. Nature biotechnology 24, 89-94
(2006)) and this approach can be incorporated in embodiments of the
present disclosure (i.e., FIG. 11, in ALS-10T, signified by "I1S").
With respect to polynucleotides that target TR1 and that can be
included in vectors of this disclosure, shRNA is one non-limiting
example of an RNAi-mediated approach. But RNAi-based inhibition can
be achieved using any suitable RNA polynucleotide that is targeted
to TR1 mRNA. With respect to shRNAs, they are known in the art to
adopt a typical hairpin secondary structure that contains a paired
sense and antisense portion, and a short loop sequence between the
paired sense and antisense portions. shRNA is delivered to the
cytoplasm where it is processed by DICER into siRNAs. siRNA is
recognized by RNA-induced silencing complex (RISC), and once
incorporated into RISC, siRNAs facilitate cleavage and degradation
of targeted mRNA. In embodiments, an shRNA polynucleotide segment
or DNA segment encoding it included for use in suppressing TR1
expression can comprise or consist of between 45-100 nucleotides,
inclusive, and including all integers between 45 and 100. The
portion of the shRNA that has reverse complementarity to the TR1
mRNA mRNA can be from 21-29 nucleotides, inclusive, and including
all integers between 21 and 29. In another approach, a ribozyme
that can specifically cleave TR1 mRNA can be included. In another
embodiment, microRNA (.mu.RNA) targeted to the TR1 mRNA can be
used.
[0043] The present disclosure provides an illustration that vector
encoding a fusion of an Ldb1 transcription factor and a zinc finger
(ZF) domain (ZF-Ldb1) is useful for approaching certain
hemoglobinopathies. A ZF-Lbd1 gene can be incorporated into vectors
of this disclosure in several configurations. In one non-limiting
approach using ALS-10 in FIG. 11 as a representative example, the
ZF-Ldb1 gene can be positioned such that the vector comprises
consecutively (with the same stranded-ness as the beta-globin
coding sequence) the 5'LTR, a polyadenylation signal, the ZF-Ldb1
region and a either a promoter that can drive expression of a
separate mRNA encoding the ZF-Ldb1 protein, or an IRES so that the
mRNA encoding the ZF-Ldb1 protein can be made as a distinct
protein, but from the same mRNA that encodes the beta-globin
protein. The rational for including ZF-Ldb1 into ALS is twofold: 1)
the ZF-Ldb1 will move the LCR from the promoter of the mutant
beta-globin gene to that of the gamma-globin gene. In this way the
production of mutant RNA will be reduced or shut down, while
expression of the functional gamma-globin gene will be activated:
2) The expression of transgenic beta-globin gene (considered to be
a potentially curative gene carried by the lentiviral vector) will
not be affected in the presence of ZF-Ldb1. Without intending to be
constrained by theory this is expected to lead to an additive or
synergistic effect by the production of both hemoglobins: fetal
hemoglobin (HbF, .alpha.2.gamma.2, from the endogenous locus), and
adult hemoglobin (HbA or .alpha.2.beta.2, from the therapeutic
vector). As hemoglobinopathies in humans are characterized by more
than 300 mutations, it is reasonable to predict that the many or
all hemoglobinopathies could be improved by a vector of this
disclosure. In this regard, we demonstrated that a lentiviral
vector encoding the ZF-Ldb1 cassette (pCL-ZF-Ldb1) increases
synthesis of fetal hemoglobin (HbF, .alpha.2.gamma.2) in
CD34-derived erythroid cells from normal individuals and SCA
patients. As HbF increased, the synthesis of adult hemoglobin (HbA)
or sickle hemoglobin (HbS or .alpha.2.beta.s2) diminished,
respectively in cells derived from normal individuals or SCA
patients. We also investigated the number of vectors integrated per
cell, on average. This number is indicated as number of integration
per cell or vector copy number (VCN). We observed that the increase
in VCN was associated with an increase of HbF both in normal and
SCA cells. The number of HbF positive cells measured by flow
cytometry also increased proportionally to the VCN. .beta.,
.gamma.- and .alpha.-globin mRNA levels measured by quantitative
PCR also showed that the relative ratio .gamma./.alpha. increased
while the .beta./.alpha. or .beta.S/.alpha. were reduced in the
samples treated with the ZF-Ldb1 vector. Taken together these data
suggest that the ZF-Ldb1 can redirect the LCR enhancing effect from
the .beta.-globin promoter to the .gamma.-globin promoter. In SCA
this effect is particularly beneficial given the toxicity caused by
the accumulation of HbS. Examples 1-6 further confirm potential
usefulness of including ZF-Ldb1 in ALS10. In particular, these
Examples demonstrate infection of hematopoietic stem cells isolated
from blood of SCD patients with a lentivirus expressing the ZF-Ldb1
transgene and differentiation of them into mature erythroid cells
in vitro. HbF synthesis induced by ZF-Ldb1 was compared to that
obtained in specimens treated with hydroxyurea and various
additional HbF inducers. ZF-Ldb1 increased HbF synthesis and
simultaneously reduced sickle Hb (HbS), establishing a balanced
synthesis between .alpha.- and functional .beta.-like globins. The
induction of HbF in cells treated with ZF-Ldb1 was roughly three
times higher (+34%), than that observed using decitabine and
pomalidomide; tranylcypromine had an intermediate effect, while
butyrate and hydroxyurea showed marginal HbF induction. Notably,
erythroid cell differentiation and viability remained unaltered in
ZF-Ldb1 expressing cells. Thus, lentiviral-mediated ZF-Ldb1 gene
transfer appears superior to existing drug regimens for affecting
SCD erythroid cells and it is reasonable to expect that combining
ZF- to an ALS10 vector will provide an effective approach to
prophylaxis and/or therapy of a variety of hemoglobinopathies. The
Examples provide also provide a demonstration of the effectiveness
of ALS10 in .beta.0/0 patient cells.
[0044] Compositions comprising recombinant lentiviral vectors are
provided. In certain approaches pharmaceutical compositions are
provided and can be prepared by mixing, for example, virions
comprising a lentiviral vector of this disclosure and any suitable
pharmaceutically acceptable carriers, excipients and/or
stabilizers. Some examples of compositions suitable for mixing with
IL-8 can be found in: Remington: The Science and Practice of
Pharmacy (2005) 21st Edition, Philadelphia, Pa. Lippincott Williams
& Wilkins. In certain approaches.
[0045] The disclosure includes methods of making a virion
preparations for use in prophylaxis and/or therapy of
hemoglobinopathies. In one embodiment this method comprises
introducing a plasmid encoding a lentiviral vector of this
disclosure into packaging cells. The packaging cells comprise a DNA
packaging plasmid, which encodes at least one virion protein, and a
DNA envelope plasmid, which encodes a suitable viral envelope
protein. The packaging and envelope plasmids express the respective
proteins, which facilitate formation of virions which comprise a
recombinant RNA lentiviral vector of this disclosure. Suitable
packaging systems that can be adapted to produce virions of this
disclosure are commercially available, such as from Addgene
(Australia).
[0046] The following Examples are intended to illustrate various
aspects of the present disclosure but are not meant to be limiting
in any way.
EXAMPLE 1
[0047] This Example demonstrates that lentivirally expressed
ZF-Ldb1 raises HbF levels in CD34+-derived sickle erythroblasts. To
obtain the data presented here, we used the lentiviral vector
pCL-ZF-Ldb1, (FIG. 11, top vector map), which carries a zinc-finger
protein that specifically binds the .gamma.-globin promoters, fused
to the self-association (SA) domain of Ldb1, as well as a green
fluorescent protein under the control of the erythroid specific
ankyrin promoter. The effects of ZF-Ldb1 expression on chromatin
contacts within the .beta.-globin locus have been shown using 3C
experiments in healthy adult erythroblasts. ZF-Ldb1 expression
promotes the juxtaposition of the .gamma.-globin promoters with the
LCR leading to transcription [Deng, W., et al., Reactivation of
developmentally silenced globin genes by forced chromatin looping.
Cell, 2014. 158(4): p. 849-60]. This is accompanied by a
concomitant reduction in the expression of adult globin genes,
compatible with a mechanism in which the fetal and adult promoters
compete for LCR enhancer activity. This example describes an
attempt to improve the amount of functional hemoglobin in CD34+
cells isolated from sickle cell patients. Sickle CD34+ cells
produce predominantly HbS (.alpha.2.beta.s2) once differentiated
into erythroblasts in vitro. Other hemoglobins, as HbF
(.alpha.2.gamma.2) and HbA2 (.alpha.2.beta.2) are also produced to
a lower degree (FIG. 1A, left). Following infection with
pCL-ZF-Ldb1 vector, sickle cells dramatically increased HbF
synthesis in a manner proportional to the copy number of viral
integrants. In a representative experiment the same SCD derived
erythroid cells (shown in FIG. 1A, left) transduced with 0.26 or
0.67 viral molecules on average per cell (VCN or vector copy
number) produce 22% or 44% (FIG. 1A, middle and right,
respectively) more HbF than the control sample. Concomitantly, with
progressively higher viral integration the number of HbF (FIG. 1B)
and GFP (FIG. 1C) expressing erythroblasts increased, as determined
by flow cytometry measurements. The gain in the fraction of GFP+
cells is somewhat lower, likely the result of reduced translation
efficiency that is often observed downstream of the internal
ribosomal entry site (IRES) between the ZF-Ldb1 cassette and the
GFP gene.
EXAMPLE 2
[0048] This Example demonstrates that transgenic ZF-Ldb1 supports
high levels of fetal globin induction and concurrently reduces
sickle globin levels in SCD erythroblasts. In particular, we
analyzed the mRNA and protein content of erythroblasts derived from
10 SCD subjects. CD34+ cells isolated from peripheral blood
mononuclear cells (PBMCs) were frozen and used for independent
replicate experiments. Infection with pCL-ZF-Ldb1 was performed on
pro-erythroblast within the first 10 days of the expansion phase.
At this time cells still express high levels of CD1117 and CD44
markers and do not express glycophorin A (data not shown) (Breda,
L., et al., Therapeutic hemoglobin levels after gene transfer in
beta-thalassemia mice and in hematopoietic cells of
beta-thalassemia and sickle cells disease patients. PLoS One, 2012.
7(3): p. e32345).
[0049] To assess the ability of ZF-Ldb1 to reactivate HbF we
measured the amount of .gamma.-globin mRNA expressed in cells
untreated or after transduction. These values were normalized to
GAPDH and to .alpha.-globin gene expression whose expression is
directly proportional to the erythroid differentiation stage in the
cells. Cells with 1.1 copies of pCL-ZF-Ldb1 produced on average a
3-fold increase of the .gamma./.alpha. globin ratio (from
0.2.+-.0.11 to 0.6.+-.0.33), compared to untreated cells and,
simultaneously, a reduction of the .beta./.alpha. ratio (from
0.38.+-.0.08 to 0.29.+-.0.16) (FIG. 2A, left and center),
confirming the ability of the ZF-Ldb1 construct to partially
redirect the LCR from the .beta.- to the .gamma.-globin promoter.
These changes were observed only in cells expressing transgenic
Ldb1, whose expression was proportional to the level of ZF-Ldb1
integration (FIG. 2A, right).
[0050] On average, cells treated with pCL-ZF-Ldb1 produced nearly
40% more HbF (63.10%.+-.14.01) compared to untreated cells
(27.27%.+-.16.29) (FIG. 2B, left) and lowered production of HbS
(-35.65%) and HbA2 (-5.18%) (FIG. 2B, center and right). Reduction
of HbA2 is likely the result of reduced contact frequencies between
.quadrature.-globin and the LCR in the presence of ZF-Ldb1. A
summary of net HbA increase and HbS and HbA2 decrease is provided
in FIG. 3C.
EXAMPLE 3
[0051] To establish the effect of ZF-Ldb1 treatment on the total
amount of cellular Hb we measured absolute Hb content per
differentiated cell. Importantly, in spite of significant shifts in
the ration of fetal to adult globin, total Hb synthesis changes
(HbF+HbS+HbA2) remained essentially unaltered (FIG. 3A, right). To
corroborate this evidence is the fact that .beta.S chains are
diminished while both .gamma.A+.gamma.G chains are increased in
specimens treated with ZF-Ldb1, analyzed by reversed-phase liquid
chromatography (FIG. 3B and S1) which allows for the quantification
of single globin chains rather than to tetrameric hemoglobin
abundance. The reduced amount of HbS was confirmed by flow
cytometry (FIG. 4). ZF-Ldb1-expressing SCD erythroblasts, which can
be tracked by GFP expression (FIG. 4A), have a greater fraction of
HbF positive cells (FIG. 4B), and within the HbF positive
population, a lower fraction of HbS compared to untransduced SCD
erythroblasts (FIG. 4C). Untreated baseline HbF positive
erythroblasts (FIG. 4B, center) were less frequent and contained
less than half the HbF/erythroid cell when compared to the ZF-Ldb1
expressing cells (FIG. 4D).
EXAMPLE 4
[0052] This Example demonstrates that y-globin gene repressors SOX6
and KLF1 are down regulated in sickle erythroblasts. In particular,
BCL11A, SOX6, C-MYb and KLF1 have emerged as salient repressors of
.gamma.-globin during erythroid differentiation. We investigated
the impact of pCL-ZF-Ldb1 on transcription of these repressors in
both wild type and sickle cell disease derived erythroid cells.
RT-qPCR analyses indicates that differentiated erythroblasts
obtained from SCD patients present a different pattern of
expression for certain negative regulators of .gamma.-globin gene
expression, compared to erythroblasts obtained from healthy
individuals. BCL11A and C-MYB messenger RNAs present comparable
level of expression (FIG. 5A), whereas both KLF1 and SOX6 show
significant down regulation in SCD specimens (0.17.+-.0.05 and
4.49.+-.0.76, respectively) compared to healthy ones (0.55.+-.0.43
and 8.29.+-.3.14, respectively) (FIG. 5B). All samples analyzed
present similar expression of KEL, an internal control mRNA that
increases with level of differentiation. These trends are
independent from the level of transgene expression (FIGS. 5 and 5C,
right). These data could indicate a more permissive chromatin state
of the .gamma.-globin gene in SCD cells in culture, as suggested by
a trend of higher content of HbF at steady state in these cells
compared to healthy ones (FIG. 7).
EXAMPLE 5
[0053] This Example demonstrates that HbF induction in SCD
erythroblasts ZF-Ldb1-mediated is greater than induction mediated
by pharmacological inducers. A scheme of the experimental
procedures is illustrated in FIG. 16. Briefly, erythroid progenitor
SCD cells were infected with pCL-ZF-Ldb1 within days 9-11 in
expansion phase, or treated at days 1 and 3 of differentiation
phase with pharmacological inducers 5-aza-2'-deoxy-cytidine (0.5
.mu.M), tranyl-cypromine (1.5 .mu.M), hydroxyurea (150 .mu.M),
pomalidomide (30 .mu.M), and butyrate (100 .mu.M), These
concentrations were determined through evaluation of efficacy (net
increase of HbF) versus toxicity (cell death); the original scaling
dosage was extrapolated from the recent literature (FIG. 9)
[Watanapokasin, Y., et al., In vivo and in vitro studies of fetal
hemoglobin induction by hydroxyurea in beta-thalassemia/hemoglobin
E patients. Exp Hematol, 2005. 33(12): p. 1486-92; Moutouh-de
Parseval, L. A., et al., Pomalidomide and lenalidomide regulate
erythropoiesis and fetal hemoglobin production in human CD34+cells.
J Clin Invest, 2008. 118(1): p. 248-58; Shi, L., et al.,
Lysine-specific demethylase 1 is a therapeutic target for fetal
hemoglobin induction. Nat Med, 2013. 19(3): p. 291-4]. Studies were
performed on day 8 of differentiation phase, at the
orthochromatophilic stage when high amounts of hemoglobin were
accumulated. As measured by HPLC, pCL-ZF-Ldb1 had the most robust
increase of HbF and decrease in HbS. Specifically, on average the
net HbF increase in SCD erythroblast treated with the lentivirus
was 34.2%.+-.12.53, versus 15.19%.+-.12.77 with 5-aza-cytidine
(p<0.01), 8.08%.+-.6.28 with tranyl-cypromine (p<0.001),
2.39%.+-.2.13 with hydroxyurea (p<0.001), 11.84%.+-.9.02 with
pomalidomide (p<0.01), and 4.84%.+-.5.03 with butyrate
(p<0.001). ZF-Ldb1 expressing cells presented HbF significantly
higher than untreated cells (p<0.001) (FIG. 6A, top).
Conversely, on average the net HbS decrease in SCD erythroblast
treated with the lentivirus was -31.36%.+-.11.61, versus
-17.30%.+-.15.04 with 5-aza-cytidine, -6.93%.+-.6.64 with
tranyl-cypromine, -3.48%.+-.4.68 with hydroxyurea, 11.61-%.+-.9.27
with pomalidomide, and -4.90%.+-.7.94 with butyrate (FIG. 6A,
bottom).
[0054] Along with recovery of greater levels of functional Hb,
cells expressing ZF-Ldb1 did not show significant changes in
viability compared to untreated samples, whereas cells treated with
pomalidomide, butyrate and hydroxyurea showed reduced viability
(FIG. 6B). These differences were confirmed in a subset of samples
treated with pharmacological inducers at earlier time points (FIG.
17) to exclude biases due to a delay to drug response (FIG. 10).
Taken together, pCL-ZF-Ldb1 was superior to all tested compounds in
augmenting HbF and F-Cell levels and importantly was associated
with minimal toxicity.
[0055] It will be apparent from the foregoing that a lentiviral
vector carrying the SA Ldb1 domain linked to a ZF protein, which
selectively binds the .gamma.-globin promoters, significantly
increased HbF synthesis and exceeds previously described
pharmacological inducers. It is therefore reasonable that adding an
SA Ldb1 domain linked to a ZF protein to an ALS10 vector of this
disclosure will likely enhance beneficial properties of the
combined vectors.
EXAMPLE 6
[0056] This Example provides a description of the materials and
methods used to obtain the results described in Examples 1-5.
[0057] Human and Animal Ethics
[0058] Peripheral blood samples from SCD patients were obtained
during automated red cell exchange as part of their routine
clinical care at Montefiore Medical Center. Since the samples were
unlinked and de-identified medical waste, the Montefiore Medical
Center Institutional Review Board deemed them to be IRB exempt.
[0059] Construct
[0060] The ZFs targeting HS2 of the human .gamma.-globin promoters
are known in the art. The SA domain containing amino acids 1-200 of
Ldb1 was inserted C-terminal to the ZF. The SA domain was attached
in C-terminal to GG1 tagged with HA.
[0061] Vector Production and Titration
[0062] Viral stocks were generated by co-transfection of the gene
transfer plasmid (pCL-ZF-Ldb1) together with the envelope plasmid
(VSV-G), the packaging plasmid (pMDLg/p RRE), and the pRSV-REV
plasmid into 293T cells. An aliquot (5.times.10.sup.6) of 293T
cells was seeded into cell culture dishes (10 cm) 24 hours prior to
transfection in Iscove's modification of Eagle's medium (DMEM,
Cellgro, Manassas, Va.) with 10% fetal bovine serum, 100 U/ml
penicillin, and 100 mg/ml streptomycin, at 37.degree. C. under 5%
CO2. The culture medium was changed 2 hours prior to transfection.
The precipitate was formed by adding the plasmids to 450 uL of
0.1.times.TE (0.1.times.TE is 10 mM Tris plus 1 mM EDTA) and 50
.mu.L of 2 M CaCl2, then adding 500 .mu.L of 2.times.
HEPES-buffered saline (281 mM NaCl, 100 mM HEPES, 1.5 mM Na2HPO4)
drop wise after which the precipitate was vortexed and immediately
added to the cultures. The medium (10 ml) was replaced after 16
hours. Viral supernatants were collected at 24 and 48 hours,
cleared by low speed centrifugation, and filtered through cellulose
acetate (0.2 .mu.m). Following ultracentrifugation, serial
dilutions of concentrated virus (5; 0.5 and 0.05 .mu.L,
respectively) were used to infect 1.times.10.sup.5 NIH 3T3 cells
(ATCC, Manassas, Va.) in 1 mL of transfection buffer complemented
with polybrene (Millipore, Billerica, Ma.) at a final concentration
of 8 .mu.g/mL. Genomic DNA was extracted after 3 days (Qiagen kit,
Valencia, Calif.). The multiplicity of infection (MOI) was
calculated using the following formula: number of cells
(1.times.10.sup.5).times.dilution factor (1 mL/.mu.L viral
preparation).times.VCN (measured via real-time PCR, using oligos
for WPRE element and ID gene, see PCR and Real Time PCR).
[0063] Real Time (RT)-PCR
[0064] Retrotranscription of total mRNA was done using the
SuperScript.TM. II First Strand Kit (Invitrogen, Carlsbad, Calif.).
Q-PCR reactions were performed using the ABI Prism 7700 Sequence
Detection System (Applied Biosystems, Foster City, Calif.), with
either TaqMan (TaqMan PCR 2.times. Master mix; Applied Biosystems)
or SYBR Green (iTaq.TM. SYBR.RTM. Green Supermix, Bio-Rad
Laboratories, Hercules, Calif.) chemistry. Quantitative real-time
PCR assays of globin and GAPDH transcripts were carried out using
gene-specific double fluorescently labeled probes. The following
primer and probe sequences were used (forward, reverse and probe,
when used, of each gene, respectively): .beta.: Fw:
5'-CAAGAAAGTGCTCGGTGCCT-3' (SEQ ID NO:6); Rev:
5'-GCAAAGGTGCCCTTGAGGT-3' (SEQ ID NO:7);
5'-FAM-TAGTGATGGCCTGGCTCACCTGGAC-TAMRA-3' (SEQ ID NO:8); .alpha.:
Fw: 5'-TCCCCACCACCAAGACCTAC-3' (SEQ ID NO:9); Rev:
5'-CCTTAACCTGGGCAGAGCC-3' (SEQ ID NO:10);
5'-FAM-TCCCGCACTTCGACCTGAGCCA-TAMRA-3' (SEQ ID NO:11); .gamma.: Fw:
5'-TGGCAAGAAGGTGCTGACTTC-3' (SEQ ID NO:12); Rev:
5'-TCACTCAGCTGGGCAAAGG (SEQ ID NO:13); 5'-FAM-TGG
GAGATGCCATAAAGCACCTGG-TAMRA-3 (SEQ ID NO:14); BCL11A: Fw:
5'-TGATGTGTGTCCATTGGTGTGAGC-3' (SEQ ID NO:15); Rev:
5'-TGCGAACTTGAACGTCAGGAGTCT, SOX-6 (SEQ ID NO:16): Fw:
5'-AGCTGCTTTCGGCTTTCTCCCTTA-3' (SEQ ID NO:17); Rev:
5'-CCTTTGCATTTGCAGCAGTTCAGC-3' (SEQ ID NO:18); C-MYB: Fw:
5'-TCAACCGATCATCCCTCACACTCT-3' (SEQ ID NO:19); Rev:
5'-AATCAGCAGCGCTTCCATTCAAGG-3' (SEQ ID NO:20), KLF-1: Fw:
5'-GCTGCCTCCACCCAAGTG-3' (SEQ ID NO:21); Rev:
5'-ACCAACTCTGGGCAGTCACAT-3' (SEQ ID NO:22), Kell: Fw:
5'-AGCAACCACCCATGCCTGCC-3' (SEQ ID NO:23); Rev:
5'-CTCGGGCCAAAGGCCTCACG-3' SEQ ID NO:24). For real-time PCR of the
reference genes, we used as an endogenous control the human
glyceraldehyde-3-phosphate dehydrogenase (GAPDH) kit, in which the
probe is fluorescently labeled with VIC (Applied Biosystems). The
number of integrations (VCN) was quantified by Q-PCR using oligos
(Fw: 5'-CGGCTGTTGGGCACTGA-3' SEQ ID NO:25); Rev:
5'-GGAAGGTCCGCTGGATTGA-3' SEQ ID NO:26)) and a probe
(5'-FAM-ATGGCTGCTCGCCTGTGTTGCC-TAMRA-3' SEQ ID NO:27) for a
specific sequence present in the vector (WPRE) and compared it to
an endogenous control present in two copies within the genome (ID-1
Fw: 5'-AAGGTGAGCAAGGTGGAGATTC-3 SEQ ID NO:28); Rev:
5'-TTCCGAGTTCAGCTCCAACTG-3' SEQ ID NO:29).
[0065] Two-phase liquid cultures, benzidine staining and
transduction
[0066] CD34+ cells selection from blood samples was performed by
immunomagnetic separation, using the CD34 microbeads kit (Miltenyi
Biotec Inc., Auburn, Calif.). These cells were then expanded
following a modified version of the protocol described by
Leberbauer and colleagues. Cells were seeded in 5 mL of serum-free
StemSpan with 50 .mu.L of StemSpan CC-100 cytokine cocktail (both
from Stemcell Technologies, Vancouver, BA, Canada), 2 U/mL
Erytrhopoietin (Amgen, Thousand Oaks, Calif.), 10-6 M dexamethasone
(Sigma) and 1% penicillin streptomycin. CD34+ cultures were kept
undifferentiated by refreshing the medium twice a week and density
gradient centrifugation was used to remove both dead and
spontaneously differentiating cells. At this stage cells were
either frozen (with 50% characterized Hyclone FBS, 10% DMSO, Sigma,
and 40% Iscove's Modified DMEM, Cellgro), or used for experiments.
After 10 days in phase I, cells were transferred into phase II
media containing a-modified essential medium supplemented with 30%
fetal calf serum and 10-5 M .beta.-mercaptoethanol. Erythropoietin
was added (5 U/mL) to stimulate erythroid differentiation. Cells
were infected with serial dilutions of the virus. Cells were
collected on day 7-10 of phase II for all analyses. The level of
differentiation was assessed by benzidine staining. At this stage,
cells were analyzed by flow cytometry for GFP, HbF and HbB
expression.
[0067] Treatment with HbF Inducers
[0068] We treated peripheral blood derived CD34+ cells (see
procedure) with HbF inducer drugs: Hydroxyurea (HU), 5-azacytidine,
pomalidomide, sodium butyrate, Tranyl-cypromine (TCP).
Tranyl-cypromine (TCP), 5-aza-2'-deoxy-cytidine, pomalidomide and
sodium butyrate. We first titrated the drugs to find the most
efficient and less toxic concentration according to published data.
Hydroxyurea (HU; Sigma-Aldrich), dissolved in water, was added to
the culture medium at final concentrations of 150 .mu.M.
Tranylcypromine (Sigma-Aldrich) was dissolved in water and added to
the culture medium at final concentrations 1.5 .mu.M. 5-azacytidine
(DAC; Sigma-Aldrich) was dissolved in water and added to the
culture medium at final concentrations of 0.5 .mu.M. Pomalidomide
(Sigma-Aldrich) was dissolved in DMSO and was added to the culture
medium at final concentrations of 30 .mu.M. Sodium butyrate
(Sigma-Aldrich) was dissolved in water and added to the culture
medium at final concentration of 100 .mu.M. The treatment timeline
is shown in FIG. 16.
[0069] Tetrameric and single chain analysis by high performance
liquid chromatography (HPLC)
[0070] Red cell pellets were lysed with HPLC-grade water, and the
resulting membrane-free hemolysates loaded into a System Gold 126
Solvent Module instrument (Beckman Coulter, Fullerton, Calif.).
Hemoglobins were separated on a weak cation-exchange PolyCAT A
column (PolyLC, Inc, Columbia, Md.), and detected at a wavelength
of 415 nm. The Hbs were bound to the column with mobile phase A (20
mmol/L Bis-Tris, 2 mmol/L KCN, pH 6.96) and eluted with mobile
phase B (20 mmol/L Bis-Tris, 2 mmol/L KCN, 200 mmol/L NaCl, pH
6.55). Single chain quantification was assessed via reversed-phase
HPLC. Hb samples in this case were injected on a Hitachi D-7000 HSM
Series apparatus (Hitachi Instruments, San Jose, Calif.) using a
Zorbax 5 .mu.m 300SB-C8 300 .ANG., LC 150.times.2.1 mm column
(Agilent Technologies, Santa Clara, Calif.) and a gradient from 20%
to 60% acetonitrile in 0.1% trifluoroacetic acid in 25 minutes,
with UV detection at 215 nm. Serial dilutions of a solution with
known concentrations of HbA and HbF (Analytical Control System,
Inc, Fishers, Ind.) were used to generate a calibration curve,
where the peak areas were plotted against the concentration values.
Types and relative quantity of Hbs in samples were assessed by
comparison to standard hemoglobin controls.
EXAMPLE 7
[0071] This Example demonstrates that introducing the lentiviral
vector ALS10 into CD34+ cells from .beta.0/0 phenotype samples, and
thus the most severe thalassemic specimens, results in
statistically significantly elevated levels of HbA produced by
erythrocytes derived from the modified CD34+ cells. The elevation
in HbA is relative to a previously describe construct, which is
used in this Example as a comparison control (AnkT9W, from Breda et
al, Plos One, 2012), which did not include a complete intron 2.
Thus, when compared to the previously described construct, ALS10
showed significant and unexpected improvement, as demonstrated by
the results depicted in FIG. 12. To obtain the results presented in
FIG. 12, the following materials and methods were used.
[0072] Vector Production and Titration
[0073] Viral stocks were generated by co-transfection of the gene
transfer plasmid (GG1-SA) together with the envelope plasmid
(VSV-G), the packaging plasmid (pMDLg/p RRE), and the pRSV-REV
vector into 293T cells. An aliquot (5.times.10.sup.6) of 293T cells
was seeded into cell culture dishes (10 cm) 24 hours prior to
transfection in Iscove's modification of Eagle's medium (DMEM,
Cellgro, Manassas, Va.) with 10% fetal bovine serum, 100 U/ml
penicillin, and 100 mg/ml streptomycin, at 37.degree. C. under 5%
CO2. The culture medium was changed 2 hours prior to transfection.
The precipitate was formed by adding the plasmids to 450 uL of
0.1.times.TE (0.1.times.TE is 10 mM Tris plus 1 mM EDTA) and 50
.mu.L of 2 M CaCl2, then adding 500 .mu.L of 2.times.
HEPES-buffered saline (281 mM NaCl, 100 mM HEPES, 1.5 mM Na2HPO4)
dropwise after which the precipitate was vortexed and immediately
added to the cultures. The medium (10 ml) was replaced after 16
hours. Viral supernatants were collected at 24 and 48 hours,
cleared by low speed centrifugation, and filtered through cellulose
acetate (0.2 .mu.m). Following concentration by
ultracentrifugation, serial dilutions of concentrated virus (5; 0.5
and 0.05 .mu.L, respectively) were used to infect 1.times.10.sup.5
NIH 3T3 cells (ATCC, Manassas, Va.) in 1 mL of transfection buffer
complemented with polybrene (Millipore, Billerica, Mass.) at a
final concentration of 8 .mu.g/mL. Genomic DNA was extracted after
3 days (Qiagen kit, Valencia, Calif.). The multiplicity of
infection (MOI) was calculated using the following formula: number
of cells (1.times.10.sup.5).times.dilution factor (1 mL/.mu.L viral
preparation)'VCN (measured via real-time PCR, using oligos for Psi
element and ID gene, by Real Time PCR).
[0074] Two-Phase Liquid Cultures, Benzidine Staining and
Transduction
[0075] Consented patients with .beta.0/0 and healthy individuals
donated between 20 and 30 mL of peripheral blood; alternatively 30
to 60 mL of peripheral blood from .beta.0/0 patients were obtained
from the discarded blood from the red cell exchange therapeutic
procedure. We selected CD34+ cells by immunomagnetic separation,
using the CD34 microbeads kit (Miltenyi Biotec Inc., Auburn,
Calif.) and then expanded these cells following a modified version
of the protocol described by Leberbauer and colleagues. Cells were
seeded in 5 mL of serum-free StemSpan with 50 .mu.L of StemSpan
CC-100 cytokine cocktail (both from Stemcell Technologies,
Vancouver, BA, Canada), 2 U/mL Erytrhopoietin (Amgen, Thousand
Oaks, Calif.), 10-6 M dexamethasone (Sigma) and 1% penicillin
streptomycin. CD34+ cultures were kept undifferentiated by
refreshing the medium twice a week and density gradient
centrifugation was used to remove both dead and spontaneously
differentiating cells. At this stage cells were either frozen (with
50% characterized Hyclone FBS, 10% DMSO, Sigma, and 40% Iscove's
Modified DMEM, Cellgro), or used for experiments. After 10 days in
phase I, cells were transferred into phase II media containing
.alpha.-modified essential medium supplemented with 30% fetal calf
serum and 10-5 M .beta.-mercaptoethanol. Erythropoietin was added
(5 U/mL) to stimulate erythroid differentiation. Cells were
infected with serial dilutions of the virus. Cells were collected
on day 7-10 of phase II for all analyses. The level of
differentiation was assessed by benzidine staining.
[0076] High Performance Liquid Chromatography (HPLC)
[0077] Cell pellets were lysed with HPLC-grade water and loaded
into a System Gold 126 Solvent Module instrument (Beckman Coulter,
Fullerton, Calif.). Hemoglobins were separated on a PolyCAT A
column (PolyLC, Inc, Columbia, Md.), which is packed with
silica-based material with a bonded coating of polyaspartic acid,
and detected at a wavelength of 415 nm. The Hbs were bound to the
column with mobile phase A (20 mmol/L Bis-Tris, 2 mmol/L KCN, pH
6.96) and eluted with mobile phase B (20 mmol/L Bis-Tris, 2 mmol/L
KCN, 200 mmol/L NaCl, pH 6.55). Serial dilutions of a solution with
known concentrations of HbA and HbF (Analytical Control System,
Inc, Fishers, Ind.) were used to generate a calibration curve,
where the absorbance detected at 415 nm was plotted against the
concentration values. Types and quantity of hemoglobins in samples
were assessed by comparison to standard hemoglobin controls.
[0078] While the invention has been described through specific
embodiments, routine modifications will be apparent to those
skilled in the art and such modifications are intended to be within
the scope of the present invention.
Sequence CWU 1
1
2912875DNAartificial sequenceZF-Ldb1 vector as DNA equivalent of
RNA lentivirus 1cgcgttcgaa ggggcaacca ggggtccgcg cgccgaggcc
tggggagcgg ggcctcctgg 60ggttggggga ggaggtgctc ttgtaatctg cggtggtccc
caggcgggcg ccacccctcc 120gcccgcccgt gccgggagcg cccggcccga
cagcaagcgc ctctggggcc gataaggccc 180tcgggggcct ggcccgcacg
tcacaggccc cgcagaggct gcggtgagtc cgccagcccc 240agctgctcct
cctcaagccc ccaaggccct tcggcggcaa ttcccaccgg tcgccaccat
300ggcccaggcg gccttggagc ccggagagaa accctacaaa tgcccggagt
gtggaaagtc 360cttcagcgac tgtagggacc tggcacgcca ccagcgaacc
cacacaggcg agaagcccta 420taaatgtcca gagtgtggaa agagcttctc
tagaaacgat gcactgactg agcaccaacg 480cacccatacc ggggaaaaac
cttataaatg tcctgagtgt ggtaaaagtt tttctcaatt 540ggctcatctc
cgcgctcacc aacgcacgca tactggtgag aagccctata agtgccccga
600atgcggcaag agtttttctc aaagcgggga ccttagaaga caccaaagaa
cccataccgg 660cgaaaaacct tacaagtgtc ccgagtgcgg aaaatctttt
tcacgcaaag acaatttgaa 720gaaccaccag cggacacaca ccggtgaaaa
gccttacaaa tgtcccgaat gtggcaagtc 780attctcagac cccggagccc
tggtgcgcca tcagcgcact cacactggca agaagactag 840cggccaggcc
ggccaggcta gcccgaaaaa gaaacgcaaa gttgggcgcg ccctggatcg
900ggatgtgggc ccaactccca tgtacccacc tacatacctg gagcctggga
tcgggaggca 960cacaccatat ggtaaccaaa ccgactatag aatatttgag
cttaacaaac ggctacagaa 1020ctggacagag gagtgtgaca atctctggtg
ggatgctttc acaactgagt tctttgaaga 1080tgacgccatg ctgaccatca
ctttctgctt ggaggatgga ccaaagagat ataccattgg 1140ccggaccctg
ataccacgct acttccgaag catttttgag gggggtgcca cagagctgta
1200ctacgtgctc aagcacccca aggaggcatt ccacagcaac ttcgtgtccc
tcgactgtga 1260ccagggcagc atggtgaccc agcacggcaa acccatgttt
acccaggtgt gtgtggaagg 1320ccggttgtac ctggagttca tgtttgacga
catgatgcgg ataaagacgt ggcacttcag 1380catccggcaa cacagagagc
tcatccccag aagtatcctg gccatgcacg cccaggaccc 1440ccagatgctg
gatcagctgt ccaaaaacat tacccggtgt gggctgtcct taattaacta
1500cccgtacgac gttccggact acgcttcttg aatcggtagg aattaattct
gcagcggccg 1560cggatccgcc cctctccctc ccccccccct aacgttactg
gccgaagccg cttggaataa 1620ggccggtgtg cgtttgtcta tatgttattt
tccaccatat tgccgtcttt tggcaatgtg 1680agggcccgga aacctggccc
tgtcttcttg acgagcattc ctaggggtct ttcccctctc 1740gccaaaggaa
tgcaaggtct gttgaatgtc gtgaaggaag cagttcctct ggaagcttct
1800tgaagacaaa caacgtctgt agcgaccctt tgcaggcagc ggaacccccc
acctggcgac 1860aggtgcctct gcggccaaaa gccacgtgta taagatacac
ctgcaaaggc ggcacaaccc 1920cagtgccacg ttgtgagttg gatagttgtg
gaaagagtca aatggctctc ctcaagcgta 1980ttcaacaagg ggctgaagga
tgcccagaag gtaccccatt gtatgggatc tgatctgggg 2040cctcggtgca
catgctttac atgtgtttag tcgaggttaa aaaaacgtct aggccccccg
2100aaccacgggg acgtggtttt cctttgaaaa acacgatgat aatatggcca
caaccatggt 2160gagcaagggc gaggagctgt tcaccggggt ggtgcccatc
ctggtcgagc tggacggcga 2220cgtaaacggc cacaagttca gcgtgtccgg
cgagggcgag ggcgatgcca cctacggcaa 2280gctgaccctg aagttcatct
gcaccaccgg caagctgccc gtgccctggc ccaccctcgt 2340gaccaccctg
acctacggcg tgcagtgctt cagccgctac cccgaccaca tgaagcagca
2400cgacttcttc aagtccgcca tgcccgaagg ctacgtccag gagcgcacca
tcttcttcaa 2460ggacgacggc aactacaaga cccgcgccga ggtgaagttc
gagggcgaca ccctggtgaa 2520ccgcatcgag ctgaagggca tcgacttcaa
ggaggacggc aacatcctgg ggcacaagct 2580ggagtacaac tacaacagcc
acaacgtcta tatcatggcc gacaagcaga agaacggcat 2640caaggtgaac
ttcaagatcc gccacaacat cgaggacggc agcgtgcagc tcgccgacca
2700ctaccagcag aacaccccca tcggcgacgg ccccgtgctg ctgcccgaca
accactacct 2760gagcacccag tccgccctga gcaaagaccc caacgagaag
cgcgatcaca tggtcctgct 2820ggagttcgtg accgccgccg ggatcactct
cggcatggac gagctgtaca agtaa 28752410PRTartificial sequenceZF-LDB1
fusion 2Met Ala Gln Ala Ala Leu Glu Pro Gly Glu Lys Pro Tyr Lys Cys
Pro1 5 10 15Glu Cys Gly Lys Ser Phe Ser Asp Cys Arg Asp Leu Ala Arg
His Gln 20 25 30Arg Thr His Thr Gly Glu Lys Pro Tyr Lys Cys Pro Glu
Cys Gly Lys 35 40 45Ser Phe Ser Arg Asn Asp Ala Leu Thr Glu His Gln
Arg Thr His Thr 50 55 60Gly Glu Lys Pro Tyr Lys Cys Pro Glu Cys Gly
Lys Ser Phe Ser Gln65 70 75 80Leu Ala His Leu Arg Ala His Gln Arg
Thr His Thr Gly Glu Lys Pro 85 90 95Tyr Lys Cys Pro Glu Cys Gly Lys
Ser Phe Ser Gln Ser Gly Asp Leu 100 105 110Arg Arg His Gln Arg Thr
His Thr Gly Glu Lys Pro Tyr Lys Cys Pro 115 120 125Glu Cys Gly Lys
Ser Phe Ser Arg Lys Asp Asn Leu Lys Asn His Gln 130 135 140Arg Thr
His Thr Gly Glu Lys Pro Tyr Lys Cys Pro Glu Cys Gly Lys145 150 155
160Ser Phe Ser Asp Pro Gly Ala Leu Val Arg His Gln Arg Thr His Thr
165 170 175Gly Lys Lys Thr Ser Gly Gln Ala Gly Gln Ala Ser Pro Lys
Lys Lys 180 185 190Arg Lys Val Gly Arg Ala Leu Asp Arg Asp Val Gly
Pro Thr Pro Met 195 200 205Tyr Pro Pro Thr Tyr Leu Glu Pro Gly Ile
Gly Arg His Thr Pro Tyr 210 215 220Gly Asn Gln Thr Asp Tyr Arg Ile
Phe Glu Leu Asn Lys Arg Leu Gln225 230 235 240Asn Trp Thr Glu Glu
Cys Asp Asn Leu Trp Trp Asp Ala Phe Thr Thr 245 250 255Glu Phe Phe
Glu Asp Asp Ala Met Leu Thr Ile Thr Phe Cys Leu Glu 260 265 270Asp
Gly Pro Lys Arg Tyr Thr Ile Gly Arg Thr Leu Ile Pro Arg Tyr 275 280
285Phe Arg Ser Ile Phe Glu Gly Gly Ala Thr Glu Leu Tyr Tyr Val Leu
290 295 300Lys His Pro Lys Glu Ala Phe His Ser Asn Phe Val Ser Leu
Asp Cys305 310 315 320Asp Gln Gly Ser Met Val Thr Gln His Gly Lys
Pro Met Phe Thr Gln 325 330 335Val Cys Val Glu Gly Arg Leu Tyr Leu
Glu Phe Met Phe Asp Asp Met 340 345 350Met Arg Ile Lys Thr Trp His
Phe Ser Ile Arg Gln His Arg Glu Leu 355 360 365Ile Pro Arg Ser Ile
Leu Ala Met His Ala Gln Asp Pro Gln Met Leu 370 375 380Asp Gln Leu
Ser Lys Asn Ile Thr Arg Cys Gly Leu Ser Leu Ile Asn385 390 395
400Tyr Pro Tyr Asp Val Pro Asp Tyr Ala Ser 405
410313550DNAartificial sequenceALS10 as DNA equilvalent of RNA
lentiviral vector 3ctgtcagacc aagtttactc atatatactt tagattgatt
taaaacttca tttttaattt 60aaaaggatct aggtgaagat cctttttgat aatctcatga
ccaaaatccc ttaacgtgag 120ttttcgttcc actgagcgtc agaccccgta
gaaaagatca aaggatcttc ttgagatcct 180ttttttctgc gcgtaatctg
ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt 240tgtttgccgg
atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg
300cagataccaa atactgtcct tctagtgtag ccgtagttag gccaccactt
caagaactct 360gtagcaccgc ctacatacct cgctctgcta atcctgttac
cagtggctgc tgccagtggc 420gataagtcgt gtcttaccgg gttggactca
agacgatagt taccggataa ggcgcagcgg 480tcgggctgaa cggggggttc
gtgcacacag cccagcttgg agcgaacgac ctacaccgaa 540ctgagatacc
tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg
600gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga
gcttccaggg 660ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc
acctctgact tgagcgtcga 720tttttgtgat gctcgtcagg ggggcggagc
ctatggaaaa acgccagcaa cgcggccttt 780ttacggttcc tggccttttg
ctggcctttt gctcacatgt tctttcctgc gttatcccct 840gattctgtgg
ataaccgtat taccgccttt gagtgagctg ataccgctcg ccgcagccga
900acgaccgagc gcagcgagtc agtgagcgag gaagcggaag agcgcccaat
acgcaaaccg 960cctctccccg cgcgttggcc gattcattaa tgcagctggc
acgacaggtt tcccgactgg 1020aaagcgggca gtgagcgcaa cgcaattaat
gtgagttagc tcactcatta ggcaccccag 1080gctttacact ttatgcttcc
ggctcgtatg ttgtgtggaa ttgtgagcgg ataacaattt 1140cacacaggaa
acagctatga ccatgattac gccaagcgcg caattaaccc tcactaaagg
1200gaacaaaagc tggagctgca agcttggcca ttgcatacgt tgtatccata
tcataatatg 1260tacatttata ttggctcatg tccaacatta ccgccatgtt
gacattgatt attgactagt 1320tattaatagt aatcaattac ggggtcatta
gttcatagcc catatatgga gttccgcgtt 1380acataactta cggtaaatgg
cccgcctggc tgaccgccca acgacccccg cccattgacg 1440tcaataatga
cgtatgttcc catagtaacg ccaataggga ctttccattg acgtcaatgg
1500gtggagtatt tacggtaaac tgcccacttg gcagtacatc aagtgtatca
tatgccaagt 1560acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct
ggcattatgc ccagtacatg 1620accttatggg actttcctac ttggcagtac
atctacgtat tagtcatcgc tattaccatg 1680gtgatgcggt tttggcagta
catcaatggg cgtggatagc ggtttgactc acggggattt 1740ccaagtctcc
accccattga cgtcaatggg agtttgtttt ggcaccaaaa tcaacgggac
1800tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa tgggcggtag
gcgtgtacgg 1860tgggaggtct atataagcag agctcgttta gtgaaccggg
gtctctctgg ttagaccaga 1920tctgagcctg ggagctctct ggctaactag
ggaacccact gcttaagcct caataaagct 1980tgccttgagt gcttcaagta
gtgtgtgccc gtctgttgtg tgactctggt aactagagat 2040ccctcagacc
cttttagtca gtgtggaaaa tctctagcag tggcgcccga acagggacct
2100gaaagcgaaa gggaaaccag agctctctcg acgcaggact cggcttgctg
aagcgcgcac 2160ggcaagaggc gaggggcggc gactggtgag tacgccaaaa
attttgacta gcggaggcta 2220gaaggagaga gatgggtgcg agagcgtcag
tattaagcgg gggagaatta gatcgcgatg 2280ggaaaaaatt cggttaaggc
cagggggaaa gaaaaaatat aaattaaaac atatagtatg 2340ggcaagcagg
gagctagaac gattcgcagt taatcctggc ctgttagaaa catcagaagg
2400ctgtagacaa atactgggac agctacaacc atcccttcag acaggatcag
aagaacttag 2460atcattatat aatacagtag caaccctcta ttgtgtgcat
caaaggatag agataaaaga 2520caccaaggaa gctttagaca agatagagga
agagcaaaac aaaagtaaga ccaccgcaca 2580gcaagcggcc gctgatcttc
agacctggag gaggagatat gagggacaat tggagaagtg 2640aattatataa
atataaagta gtaaaaattg aaccattagg agtagcaccc accaaggcaa
2700agagaagagt ggtgcagaga gaaaaaagag cagtgggaat aggagctttg
ttccttgggt 2760tcttgggagc agcaggaagc actatgggcg cagcctcaat
gacgctgacg gtacaggcca 2820gacaattatt gtctggtata gtgcagcagc
agaacaattt gctgagggct attgaggcgc 2880aacagcatct gttgcaactc
acagtctggg gcatcaagca gctccaggca agaatcctgg 2940ctgtggaaag
atacctaaag gatcaacagc tcctggggat ttggggttgc tctggaaaac
3000tcatttgcac cactgctgtg ccttggaatg ctagttggag taataaatct
ctggaacaga 3060tttggaatca cacgacctgg atggagtggg acagagaaat
taacaattac acaagcttaa 3120tacactcctt aattgaagaa tcgcaaaacc
agcaagaaaa gaatgaacaa gaattattgg 3180aattagataa atgggcaagt
ttgtggaatt ggtttaacat aacaaattgg ctgtggtata 3240taaaattatt
cataatgata gtaggaggct tggtaggttt aagaatagtt tttgctgtac
3300tttctatagt gaatagagtt aggcagggat attcaccatt atcgtttcag
acccacctcc 3360caaccccgag gggacccgac aggcccgaag gaatagaaga
agaaggtgga gagagagaca 3420gagacagatc cattcgatta gtgaacggat
ctcgacggta tcggttaact tttaaaagaa 3480aaggggggat tggggggtac
agtgcagggg aaagaatagt agacataata gcaacagaca 3540tacaaactaa
agaattacaa aaacaaatta caaaaattca aaattttatc gataagcttg
3600ggagttccgc gtgttggggg tggaccatcc tctaggtatt gaataagaaa
aatgaagtta 3660aggtggttga tggtaacact atgctaataa ctgcagagcc
agaagcacca taagggacat 3720gataagggag ccagcagacc tctgatctct
tcctgaatgc taatcttaaa catcctgagg 3780aagaatggga cttccatttg
gggtgggcct atgatagggt aataagacag tagtgaatat 3840caagctacaa
aaagccccct ttcaaattct tctcagtcct aacttttcat actaagccca
3900gtccttccaa agcagactgt gaaagagtga tagttccggg agactagcac
tgcagattcc 3960gggtcactgt gagtggggga ggcagggaag aagggctcac
aggacagtca aaccatgccc 4020cctgtttttc cttcttcaag tagacctcta
taagacaaca gagacaacta aggctgagtg 4080gccaggcgag gagaaaccat
ctcgccgtaa aacatggaag gaacacttca ggggaaaggt 4140ggtatctcta
agcaagagaa ctgagtggag tcaaggctga gagatgcagg ataagcaaat
4200gggtagtgaa aagacattca tgaggacagc taaaacaata agtaatgtaa
aatacagcat 4260agcaaaactt taacctccaa atcaagcctc tacttgaatc
cttttctgag ggatgaataa 4320ggcataggca tcaggggctg ttgccaatgt
gcattagctg tttgcagcct caccttcttt 4380catggagttt aagatatagt
gtattttccc aaggtttgaa ctagctcttc atttctttat 4440gttttaaatg
cactgacctc ccacattccc tttttagtaa aatattcaga aataatttaa
4500atacatcatt gcaatgaaaa taaatgtttt ttattaggca gaatccagat
gctcaaggcc 4560cttcataata tcccccagtt tagtagttgg acttagggaa
caaaggaacc tttaatagaa 4620attggacagc aagaaagcga gcttagtgat
acttgtgggc cagggcatta gccacaccag 4680ccaccacttt ctgataggca
gcctgcactg gtggggtgaa ttctttgcca aagtgatggg 4740ccagcacaca
gaccagcacg ttgcccagga gctgtgggag gaagataaga ggtatgaaca
4800tgattagcaa aagggcctag cttggactca gaataatcca gccttatccc
aaccataaaa 4860taaaagcaga atggtagctg gattgtagct gctattagca
atatgaaacc tcttacatca 4920gttacaattt atatgcagaa atatttatat
gcagaaatat tgctattgcc ttaacccaga 4980aattatcact gttattcttt
agaatggtgc aaagaggcat gatacattgt atcattattg 5040ccctgaaaga
aagagattag ggaaagtatt agaaataaga taaacaaaaa agtatattaa
5100aagaagaaag cattttttaa aattacaaat gcaaaattac cctgatttgg
tcaatatgtg 5160tacacatatt aaaacattac actttaaccc ataaatatgt
ataatgatta tgtatcaatt 5220aaaaataaaa gaaaataaag tagggagatt
atgaatatgc aaataagcac acatatattc 5280caaatagtaa tgtactaggc
agactgtgta aagttttttt ttaagttact taatgtatct 5340cagagatatt
tccttttgtt atacacaatg ttaaggcatt aagtataata gtaaaaattg
5400cggagaagaa aaaaaaagaa agcaagaatt aaacaaaaga aaacaattgt
tatgaacagc 5460aaataaaaga aactaaaacg atcctgagac ttccacactg
atgcaatcat tcgtctgttt 5520cccattctaa actgtaccct gttacttctc
cccttcctat gacatgaact taaccataga 5580aaagaagggg aaagaaaaca
tcaagggtcc catagactca ccctgaagtt ctcaggatcc 5640acgtgcagct
tgtcacagtg cagctcactc agtgtggcaa aggtgccctt gaggttgtcc
5700aggtgagcca ggccatcact aaaggcaccg agcactttct tgccatgagc
cttcacctta 5760gggttgccca taacagcatc aggagtggac agatccccaa
aggactcaaa gaacctctgg 5820gtccaagggt agaccaccag cagcctaagg
gtgggaaaat agaccaatag gcagagagag 5880tcagtgccta tcagaaaccc
aagagtcttc tctgtctcca catgcccagt ttctattggt 5940ctccttaaac
ctgtcttgta accttgatac caacctgccc agggcctcac caccaacttc
6000atccacgttc accttgcccc acagggcagt aacggcagac ttctcctcag
gagtcaggtg 6060caccatggtg tctgtttgag gttgctagtg aacacagttg
tgtcagaagc aaatgtaagc 6120aatagatggc tctgccctga cttttatgcc
cagccctggc tcctgccctc cctgctcctg 6180ggagtagatt ggccaaccct
agggtgtggc tccacagggt gaggtctaag tgatgacagc 6240cgtacctgtc
cttggctctt ctggcactgg cttaggagtt ggacttcaaa ccctcagccc
6300tccctctaag atatatctct tggccccata ccatcagtac aaattgctac
taaaaacatc 6360ctcctttgca agtgtattta cgtaatattt ggaatcacag
cttggtaagc atattgaaga 6420tcgttttccc aattttctta ttacacaaat
aagaaattga tgcactaaaa gtggaagagt 6480tttgtctacc ataattcagc
tttgggatat gtagatggat ctcttcctgc gtctccagaa 6540tatgcaaaat
acttacagga cagaatggat gaaaactcta cctcagttct aagcatatct
6600tctccttatt tggattaaaa ccttctggta agaaaagaaa aaaaatatat
atatatatgt 6660gtatatatac acacatacat atacatatat atgcattcat
ttgttgttgt ttttcttaat 6720ttgctcatgg tatatgtgta tatatatata
tatatattca ggaaataata tattctagaa 6780tatgtcacat tctgtctcag
gcatccattt tctttatgat gccgtttgag gtggagtttt 6840agtcaggtgg
tcagcttctc cttttttttg ccatctgccc tgtaagcatc ctgctgggga
6900cccagatagg agtcatcact ctaggctgag aacatctggg cacacaccct
aagcctcagc 6960atgactcatc atgactcagc attgctgtgc ttgagccaga
aggtttgctt agaaggttac 7020acagaaccag aaggcggggg tggggcactg
accccgacag gggcctggcc agaactgctc 7080atgcttggac tatgggaggt
cactaatgga gacacacaga aatgtaacag gaactaagga 7140aaaactgaag
cttatttaat cagagatgag atgctggaag ggatagaggg agctgagctt
7200gtaaaaagta tagtaatcat tcagcaaatg gttttgaagc acctgctgga
tgctaaacac 7260tattttcagt gcttgaatca taaataagaa taaaacatgt
atcttattcc ccacaagagt 7320ccaagtaaaa aataacagtt aattataatg
tgctctgtcc cccaggctgg agtgcagtgg 7380cacgatctca gctcactgca
acctccgcct cccgggttca agcaattctc ctgcctcagc 7440caccctaata
gctgggatta caggtgcaca ccaccatgcc aggctaattt ttgtactttt
7500tgtagaggca gggtatcacc atgttgtcca agatggtctt gaactcctga
gctccaagca 7560gtccacccac ctcagcctcc caaagtgcta tctgcggccg
cctatctgta ccactagtct 7620cgagaagctt tcattaaaaa aagtctaacc
agctgcattc gactttgact gcagcagctg 7680gttagaaggt tctactggag
gagggtccca gcccattgct aaattaacat caggctctga 7740gactggcagt
atatctctaa cagtggttga tgctatcttc tggaacttgc ctgctacatt
7800gagaccactg acccatacat aggaagccca tagctctgtc ctgaactgtt
aggccactgg 7860tccagagagt gtgcatctcc tttgatcctc ataataaccc
tatgagatag acacaattat 7920tactcttact ttatagatga tgatcctgaa
aacataggag tcaaggcact tgcccctagc 7980tgggggtata ggggagcagt
cccatgtagt agtagaatga aaaatgctgc tatgctgtgc 8040ctcccccacc
tttcccatgt ctgccctcta ctcatggtct atctctcctg gctcctggga
8100gtcatggact ccacccagca ccaccaacct gacctaacca cctatctgag
cctgccagcc 8160tataacccat ctgggccctg atagctggtg gccagccctg
accccacccc accctccctg 8220gaacctctga tagacacatc tggcacacca
gctcgcaaag tcaccgtgag ggtcttgtgt 8280ttgctgagtc aaaattcctt
gaaatccaag tccttagaga ctcctgctcc caaatttaca 8340gtcatagact
tcttcatggc tgtctccttt atccacagaa tgattccttt gcttcattgc
8400cccatccatc tgatcctcct catcagtgca gcacagggcc catgagcagt
agctgcagag 8460tctcacatag gtctggcact gcctctgaca tgtccgacct
taggcaaatg cttgactctt 8520ctgagctcag tcttgtcatg gcaaaataaa
gataataata gtgttttttt atggagttag 8580cgtgaggatg gaaaacaata
gcaaaattga ttagactata aaaggtctca acaaatagta 8640gtagatttta
tcatccatta atccttccct ctcctctctt actcatccca tcacgtatgc
8700ctcttaattt tcccttacct ataataagag ttattcctct tattatattc
ttcttatagt 8760gattctggat attaaagtgg gaatgagggg caggccacta
acgaagaaga tgtttctcaa 8820agaagccatt ctccccacat agatcatctc
agcagggttc aggaagataa aggaggatca 8880aggtcgaagg taggaactaa
ggaagaacac tgggcaagtg gatcctgagc cccttttcct 8940ctaactgaaa
gaaggaaaaa aaaaatggaa cccaaaatat tctacatagt ttccatgtca
9000cagccagggc tgggcagtct cctgttattt cttttaaaat aaatatatca
tttaaatgca 9060taaataagca aaccctgctc gggaatggga gggagagtct
ctggagtcca ccccttctcg 9120gccctggctc tgcagatagt gctatcaaag
ccctgacaga gccctgccca ttgctgggcc 9180ttggagtgag tcagcctagt
agagaggcag ggcaagccat ctcatagctg ctgagtggga 9240gagagaaaag
ggctcattgt ctataaactc aggtcatggc tattcttatt ctcacactaa
9300gaaaaagaat gagatgtcta catataccct gcgtcccctc ttgtgtactg
gggcccccaa 9360gagctctcta aaagtgatgg caaagtcatt gcgctagatg
ccatcccatc tattataaac 9420ctgcatttgt ctccacacac cagtcatgga
caataaccct cctcccaggt ccacgtgctt 9480gtctttgtat aatactcaag
taatttcgga aaatgtattc
tttcaatctt gttctgttat 9540tcctgtttca atggcttagt agaaaaagta
catacttgtt ttcccataaa ttgacaatag 9600acaatttcac atcaatgtct
atatgggtcg ttgtgtttgc tgtgtttgca aaaactcaca 9660ataactttat
attgttacta ctctaagaaa gttacaacat ggtgaataca agagaaagct
9720attacaagtc cagaaaataa aagttatcat cttgaggcct cagctttcta
ggaataatat 9780caatattaca aaattaatct aacaattatg aacagcaatg
agataatatg tacaaagtac 9840ccagacctat gtggtagagc atcaaggaag
cgcattgcgg agcagttttt tgtttgtttg 9900tttttgtatt ctgtttcgtg
aggcaaggtt tcactctgct gtccaggctg gagtgcagtg 9960gcaagatcat
gtctcactgc agccttgaca ctacacgtgc tttaagacca atgacttaca
10020aggcagctgt agatcttagc cactttttaa aagaaaaggg gggactggaa
gggctaattc 10080actcccaacg aagacaagat ctgctttttg ctagcgcggc
cgctctagac tagtggggcc 10140cgtgcaattg aagccggctg gcgccaagct
tctctgcagg atgtgcgggc caggcccccg 10200agggccttat cggccccaga
ggcgcttgct gtcgggccgg gcgctcccgg cacgggcggg 10260cggaggggtg
gcgcccgcct ggggaccgca gattacaaga gcacctcctc ccccaacccc
10320aggaggcccc gctccccagg cctcggccgg cgcggacccc tggttgcccc
ggatgtacag 10380gcatgcgtcg acctcgaggg ggggcccggt accgctagca
ctgggtctct ctggttagac 10440cagatctgag cctgggagct ctctggctaa
ctagggaacc cactgcttaa gcctcaataa 10500agcttgcctt gagtgcttca
agtagtgtgt gcccgtctgt tgtgtgactc tggtaactag 10560agatccctca
gaccctttta gtcagtgtgg aaaatctcta gcaggtcgac aatcaacctc
10620tggattacaa aatttgtgaa agattgactg gtattcttaa ctatgttgct
ccttttacgc 10680tatgtggata cgctgcttta atgcctttgt atcatgctat
tgcttcccgt atggctttca 10740ttttctcctc cttgtataaa tcctggttgc
tgtctcttta tgaggagttg tggcccgttg 10800tcaggcaacg tggcgtggtg
tgcactgtgt ttgctgacgc aacccccact ggttggggca 10860ttgccaccac
ctgtcagctc ctttccggga ctttcgcttt ccccctccct attgccacgg
10920cggaactcat cgccgcctgc cttgcccgct gctggacagg ggctcggctg
ttgggcactg 10980acaattccgt ggtgttgtcg gggaagctga cgtcctttcc
atggctgctc gcctgtgttg 11040ccacctggat tctgcgcggg acgtccttct
gctacgtccc ttcggccctc aatccagcgg 11100accttccttc ccgcggcctg
ctgccggctc tgcggcctct tccgcgtctt cgccttcgcc 11160ctcagacgag
tcggatctcc ctttgggccg cctccccgcc tggaattcga gctcggtacc
11220tgatcagcct cgactgtgcc ttctagttgc cagccatctg ttgtttgccc
ctcccccgtg 11280ccttccttga ccctggaagg tgccactccc actgtccttt
cctaataaaa tgaggaaatt 11340gcatcgcatt gtctgagtag gtgtcattct
attctggggg gtggggtggg gcaggacagc 11400aagggggagg attgggaaga
caatagcagg catgctgggg atgcggtggg ctctatggct 11460tctgaggaaa
gaaccagctg gggctcgaga tccactagtt ctagcctcga ggctagagcg
11520gccgccaccg cggtagtagt tcatgtcatc ttattattca gtatttataa
cttgcaaaga 11580aatgaatatc agagagtgag aggaacttgt ttattgcagc
ttataatggt tacaaataaa 11640gcaatagcat cacaaatttc acaaataaag
catttttttc actgcattct agttgtggtt 11700tgtccaaact catcaatgta
tcttatcatg tctggctcta gctatcccgc ccctaactcc 11760gcccagttcc
gcccattctc cgccccatgg ctgactaatt ttttttattt atgcagaggc
11820cgaggccgcc tcggcctctg agctattcca gaagtagtga ggaggctttt
ttggaggcct 11880aggcttttgc gtcgagacgt acccaattcg ccctatagtg
agtcgtatta cgcgcgctca 11940ctggccgtcg ttttacaacg tcgtgactgg
gaaaaccctg gcgttaccca acttaatcgc 12000cttgcagcac atcccccttt
cgccagctgg cgtaatagcg aagaggcccg caccgatcgc 12060ccttcccaac
agttgcgcag cctgaatggc gaatggcgcg acgcgccctg tagcggcgca
12120ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc
cagcgcccta 12180gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca
cgttcgccgg ctttccccgt 12240caagctctaa atcgggggct ccctttaggg
ttccgattta gtgctttacg gcacctcgac 12300cccaaaaaac ttgattaggg
tgatggttca cgtagtgggc catcgccctg atagacggtt 12360tttcgccctt
tgacgttgga gtccacgttc tttaatagtg gactcttgtt ccaaactgga
12420acaacactca accctatctc ggtctattct tttgatttat aagggatttt
gccgatttcg 12480gcctattggt taaaaaatga gctgatttaa caaaaattta
acgcgaattt taacaaaata 12540ttaacgttta caatttccca ggtggcactt
ttcggggaaa tgtgcgcgga acccctattt 12600gtttattttt ctaaatacat
tcaaatatgt atccgctcat gagacaataa ccctgataaa 12660tgcttcaata
atattgaaaa aggaagagta tgagtattca acatttccgt gtcgccctta
12720ttcccttttt tgcggcattt tgccttcctg tttttgctca cccagaaacg
ctggtgaaag 12780taaaagatgc tgaagatcag ttgggtgcac gagtgggtta
catcgaactg gatctcaaca 12840gcggtaagat ccttgagagt tttcgccccg
aagaacgttt tccaatgatg agcactttta 12900aagttctgct atgtggcgcg
gtattatccc gtattgacgc cgggcaagag caactcggtc 12960gccgcataca
ctattctcag aatgacttgg ttgagtactc accagtcaca gaaaagcatc
13020ttacggatgg catgacagta agagaattat gcagtgctgc cataaccatg
agtgataaca 13080ctgcggccaa cttacttctg acaacgatcg gaggaccgaa
ggagctaacc gcttttttgc 13140acaacatggg ggatcatgta actcgccttg
atcgttggga accggagctg aatgaagcca 13200taccaaacga cgagcgtgac
accacgatgc ctgtagcaat ggcaacaacg ttgcgcaaac 13260tattaactgg
cgaactactt actctagctt cccggcaaca attaatagac tggatggagg
13320cggataaagt tgcaggacca cttctgcgct cggcccttcc ggctggctgg
tttattgctg 13380ataaatctgg agccggtgag cgtgggtctc gcggtatcat
tgcagcactg gggccagatg 13440gtaagccctc ccgtatcgta gttatctaca
cgacggggag tcaggcaact atggatgaac 13500gaaatagaca gatcgctgag
ataggtgcct cactgattaa gcattggtaa 135504754DNAhuman 4cggctgtcat
cacttagacc tcaccctgtg gagccacacc ctagggttgg ccaatctact 60cccaggagca
gggagggcag gagccagggc tgggcataaa agtcagggca gagccatcta
120ttgcttacat ttgcttctga cacaactgtg ttcactagca acctcaaaca
gacaccatgg 180tgcacctgac tcctgaggag aagtctgccg ttactgccct
gtggggcaag gtgaacgtgg 240atgaagttgg tggtgaggcc ctgggcaggc
tgctggtggt ctacccttgg acccagaggt 300tctttgagtc ctttggggat
ctgtccactc ctgatgctgt tatgggcaac cctaaggtga 360aggctcatgg
caagaaagtg ctcggtgcct ttagtgatgg cctggctcac ctggacaacc
420tcaagggcac ctttgccaca ctgagtgagc tgcactgtga caagctgcac
gtggatcctg 480agaacttcag gctcctgggc aacgtgctgg tctgtgtgct
ggcccatcac tttggcaaag 540aattcacccc accagtgcag gctgcctatc
agaaagtggt ggctggtgtg gctaatgccc 600tggcccacaa gtatcactaa
gctcgctttc ttgctgtcca atttctatta aaggttcctt 660tgttccctaa
gtccaactac taaactgggg gatattatga agggccttga gcatctggat
720tctgcctaat aaaaaacatt tattttcatt gcaa 7545147PRThuman 5Met Val
His Leu Thr Pro Glu Glu Lys Ser Ala Val Thr Ala Leu Trp1 5 10 15Gly
Lys Val Asn Val Asp Glu Val Gly Gly Glu Ala Leu Gly Arg Leu 20 25
30Leu Val Val Tyr Pro Trp Thr Gln Arg Phe Phe Glu Ser Phe Gly Asp
35 40 45Leu Ser Thr Pro Asp Ala Val Met Gly Asn Pro Lys Val Lys Ala
His 50 55 60Gly Lys Lys Val Leu Gly Ala Phe Ser Asp Gly Leu Ala His
Leu Asp65 70 75 80Asn Leu Lys Gly Thr Phe Ala Thr Leu Ser Glu Leu
His Cys Asp Lys 85 90 95Leu His Val Asp Pro Glu Asn Phe Arg Leu Leu
Gly Asn Val Leu Val 100 105 110Cys Val Leu Ala His His Phe Gly Lys
Glu Phe Thr Pro Pro Val Gln 115 120 125Ala Ala Tyr Gln Lys Val Val
Ala Gly Val Ala Asn Ala Leu Ala His 130 135 140Lys Tyr
His145620DNAartificial sequencePrimer 6caagaaagtg ctcggtgcct
20719DNAartificial sequencePrimer 7gcaaaggtgc ccttgaggt
19825DNAartificial sequencePrimer 8tagtgatggc ctggctcacc tggac
25920DNAartificial sequencePrimer 9tccccaccac caagacctac
201019DNAartificial sequencePrimer 10ccttaacctg ggcagagcc
191122DNAartificial sequencePrimer 11tcccgcactt cgacctgagc ca
221221DNAartificial sequencePrimer 12tggcaagaag gtgctgactt c
211319DNAartificial sequencePrimer 13tcactcagct gggcaaagg
191424DNAartificial sequencePrimer 14tgggagatgc cataaagcac ctgg
241524DNAartificial sequencePrimer 15tgatgtgtgt ccattggtgt gagc
241624DNAartificial sequencePrimer 16tgcgaacttg aacgtcagga gtct
241724DNAartificial sequencePrimer 17agctgctttc ggctttctcc ctta
241824DNAartificial sequencePrimer 18cctttgcatt tgcagcagtt cagc
241924DNAartificial sequencePrimer 19tcaaccgatc atccctcaca ctct
242024DNAartificial sequencePrimer 20aatcagcagc gcttccattc aagg
242118DNAartificial sequencePrimer 21gctgcctcca cccaagtg
182221DNAartificial sequencePrimer 22accaactctg ggcagtcaca t
212320DNAartificial sequencePrimer 23agcaaccacc catgcctgcc
202420DNAartificial sequencePrimer 24ctcgggccaa aggcctcacg
202517DNAartificial sequencePrimer 25cggctgttgg gcactga
172619DNAartificial sequencePrimer 26ggaaggtccg ctggattga
192722DNAartificial sequencePrimer 27atggctgctc gcctgtgttg cc
222822DNAartificial sequencePrimer 28aaggtgagca aggtggagat tc
222921DNAartificial sequencePrimer 29ttccgagttc agctccaact g 21
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