U.S. patent application number 17/239780 was filed with the patent office on 2021-11-11 for nuclease-mediated regulation of gene expression.
The applicant listed for this patent is Children's Medical Center Corporation, Sangamo Therapeutics, Inc.. Invention is credited to Stuart H. Orkin, Andreas Reik, Fyodor Urnov.
Application Number | 20210348143 17/239780 |
Document ID | / |
Family ID | 1000005738767 |
Filed Date | 2021-11-11 |
United States Patent
Application |
20210348143 |
Kind Code |
A1 |
Orkin; Stuart H. ; et
al. |
November 11, 2021 |
NUCLEASE-MEDIATED REGULATION OF GENE EXPRESSION
Abstract
The present disclosure is in the field of genome engineering,
particularly targeted modification of the genome of a hematopoietic
cell.
Inventors: |
Orkin; Stuart H.; (Boston,
MA) ; Reik; Andreas; (Brisbane, CA) ; Urnov;
Fyodor; (Brisbane, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Children's Medical Center Corporation
Sangamo Therapeutics, Inc. |
Boston
Brisbane |
MA
CA |
US
US |
|
|
Family ID: |
1000005738767 |
Appl. No.: |
17/239780 |
Filed: |
April 26, 2021 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14540729 |
Nov 13, 2014 |
11021696 |
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17239780 |
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62042075 |
Aug 26, 2014 |
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61903823 |
Nov 13, 2013 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 48/00 20130101;
C12N 15/113 20130101; C07K 2319/81 20130101; A61K 35/28 20130101;
C07K 14/4702 20130101; C12N 2310/20 20170501; A61K 35/12 20130101;
C12N 15/85 20130101; A61K 38/465 20130101; C12N 9/22 20130101; C12N
2310/10 20130101 |
International
Class: |
C12N 9/22 20060101
C12N009/22; C12N 15/85 20060101 C12N015/85; A61K 35/12 20060101
A61K035/12; C12N 15/113 20060101 C12N015/113; C07K 14/47 20060101
C07K014/47; A61K 38/46 20060101 A61K038/46; A61K 35/28 20060101
A61K035/28 |
Claims
1-19. (canceled)
20. A DNA-binding protein comprising a TALE-effector protein
(TALE), wherein the TALE protein comprises a plurality of TALE
repeat units, each repeat unit comprising a hypervariable diresidue
region (RVD), wherein the RVDs of the TALE repeats units are shown
in a single row of Table 1, Table 2 or Table 4.
21. A fusion protein comprising a TALE protein of claim 20 and a
wild-type or engineered cleavage domain or cleavage
half-domain.
22. A polynucleotide encoding one or more proteins of claim 20.
23. An isolated cell comprising one or more proteins according to
claim 20.
24. An isolated cell comprising one or more polynucleotides
according to claim 22.
25. The cell of claim 23, wherein the cell is a hematopoietic stem
cell.
26. A kit comprising a protein according to claim 20.
27. A method of altering globin gene expression in a cell, the
method comprising: introducing, into the cell, one or more
polynucleotides according to claim 22, under conditions such that
the one or more proteins are expressed and expression of the globin
gene is altered.
28. The method of claim 27, wherein expression of the globin gene
is increased.
29. The method of claim 28, wherein the globin gene is a gamma
globin or beta globin gene.
30. The method of claim 27, further comprising integrating a donor
sequence into the genome of the cell.
31. The method of claim 30, wherein the donor sequence is
introduced to the cell using a viral vector, as an oligonucleotide
or on a plasmid.
32. The method of claim 27, wherein the cell is selected from the
group consisting of a red blood cell (RBC) precursor cell and a
hematopoietic stem cell.
33. The method of claim 30, wherein the donor sequence comprises a
transgene under the control of an endogenous or exogenous
promoter.
34-39. (canceled)
40. A method of treating a patient in need of an increase in globin
gene expression, the method comprising administering to the patient
the pharmaceutical preparation of claim 19 in an amount sufficient
to increase the globin gene expression in the patient.
41. The method of claim 40, wherein the patient is known to have,
is suspected of having, or is at risk of developing a
globinopathy.
42. The method of claim 41, wherein the globinopathy is a
thalassemia or sickle cell disease.
43. The method of claim 42, wherein the thalassemia is
.beta.-thalassemia.
44. A pharmaceutical composition comprising the cell of claim
23.
45. The cell of claim 25, wherein the hematopoietic stem cell is a
CD34+ cell.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] The present application claims the benefit of U.S.
Provisional Application No. 61/903,823, filed Nov. 13, 2013 and
U.S. Provisional Application No. 62/042,075, filed Aug. 26, 2014,
the disclosures of which are hereby incorporated by reference in
their entireties.
SEQUENCE LISTING
[0002] The instant application contains a Sequence Listing which
has been submitted electronically in ASCII format and is hereby
incorporated by reference in its entirety. Said ASCII copy, created
on Apr. 23, 2021, is named 717317_SA9-601DIV_ST25.txt, and is
58,688 bytes in size.
TECHNICAL FIELD
[0003] The present disclosure is in the field of genome
engineering, particularly targeted modification of the genome of a
hematopoietic cell.
BACKGROUND
[0004] When one considers that genome sequencing efforts have
revealed that the human genome contains between 20,000 and 25,000
genes, but fewer than 2000 transcriptional regulators, it becomes
clear that a number of factors must interact to control gene
expression in all its various temporal, developmental and tissue
specific manifestations. Expression of genes is controlled by a
highly complex mixture of general and specific transcriptional
regulators and expression can also be controlled by cis-acting DNA
elements. These DNA elements comprise both local DNA elements such
as the core promoter and its associated transcription factor
binding sites as well as distal elements such as enhancers,
silencers, insulators and locus control regions (LCRs) (see Maston
et al (2006) Ann Rev Genome Hum Genet 7: 29-50).
[0005] Enhancer elements were first identified in the SV40 viral
genome, and then found in the human immunoglobulin heavy chain
locus. Now known to play regulatory roles in the expression of many
genes, enhancers appear to mainly influence temporal and spatial
patterns of gene expression. It has also been found that enhancers
function in a manner that is not dependent upon distance from the
core promoter of a gene, and is not dependent on any specific
sequence orientation with respect to the promoter. Enhancers can be
located several hundred kilobases upstream or downstream of a core
promoter region, where they can be located in an intron sequence,
or even beyond the 3' end of a gene.
[0006] Various methods and compositions for targeted cleavage of
genomic DNA have been described. Such targeted cleavage events can
be used, for example, to induce targeted mutagenesis, induce
targeted deletions of cellular DNA sequences, and facilitate
targeted recombination at a predetermined chromosomal locus. See,
e.g., U.S. Pat. Nos. 8,623,618; 8,034,598; 8,586,526; 6,534,261;
6,599,692; 6,503,717; 6,689,558; 7,067,317; 7,262,054; 7,888,121;
7,972,854; 7,914,796; 7,951,925; 8,110,379; 8,409,861; U.S. Patent
Publications 20030232410; 20050208489; 20050026157; 20060063231;
20080159996; 201000218264; 20120017290; 20110265198; 20130137104;
20130122591; 20130177983, 20130177960 and 20150056705, the
disclosures of which are incorporated by reference in their
entireties for all purposes. These methods often involve the use of
engineered cleavage systems to induce a double strand break (DSB)
or a nick in a target DNA sequence such that repair of the break by
an error born process such as non-homologous end joining (NHEJ) or
repair using a repair template (homology directed repair or HDR)
can result in the knock out of a gene or the insertion of a
sequence of interest (targeted integration). This technique can
also be used to introduce site specific changes in the genome
sequence through use of a donor oligonucleotide, including the
introduction of specific deletions of genomic regions, or of
specific point mutations or localized alterations (also known as
gene correction). Cleavage can occur through the use of specific
nucleases such as engineered zinc finger nucleases (ZFN),
transcription-activator like effector nucleases (TALENs), or using
the CRISPR/Cas system with an engineered crRNA/tracr RNA (`single
guide RNA`) to guide specific cleavage. Further, targeted nucleases
are being developed based on the Argonaute system (e.g., from T.
thermophilus, known as `TtAgo`, see Swarts et al (2014) Nature
507(7491): 258-261), which also may have the potential for uses in
genome editing and gene therapy.
[0007] Red blood cells (RBCs), or erythrocytes, are the major
cellular component of blood. In fact, RBCs account for one quarter
of the cells in a human. Mature RBCs lack a nucleus and many other
organelles in humans, and are full of hemoglobin, a metalloprotein
that functions to carry oxygen to the tissues as well as carry
carbon dioxide out of the tissues and back to the lungs for
removal. This protein makes up approximately 97% of the dry weight
of RBCs and it increases the oxygen carrying ability of blood by
about seventy fold. Hemoglobin is a heterotetramer comprising two
alpha (.alpha.)-like globin chains and two beta (.beta.)-like
globin chains and 4 heme groups. In adults the .alpha.2.beta.2
tetramer is referred to as Hemoglobin A (HbA) or adult hemoglobin.
Typically, the alpha and beta globin chains are synthesized in an
approximate 1:1 ratio and this ratio seems to be critical in terms
of hemoglobin and RBC stabilization. In a developing fetus, a
different form of hemoglobin, fetal hemoglobin (HbF), is produced
which has a higher binding affinity for oxygen than Hemoglobin A
such that oxygen can be delivered to the baby's system via the
mother's blood stream. Fetal hemoglobin also contains two .alpha.
globin chains, but in place of the adult .beta.-globin chains, it
has two fetal gamma (.gamma.)-globin chains (i.e., fetal hemoglobin
is .alpha.2.gamma.2). At approximately 30 weeks of gestation, the
synthesis of gamma globin in the fetus starts to drop while the
production of beta globin increases. By approximately 10 months of
age, the newborn's hemoglobin is nearly all .alpha.2.beta.2
although some HbF persists into adulthood (approximately 1-3% of
total hemoglobin). The regulation of the switch from production of
gamma- to beta-globin is quite complex, and primarily involves a
down-regulation of gamma globin transcription with a simultaneous
up-regulation of beta globin transcription.
[0008] Genetic defects in the sequences encoding the hemoglobin
chains can be responsible for a number of diseases known as
hemoglobinopathies, including sickle cell anemia and thalassemias.
In the majority of patients with hemoglobinopathies, the genes
encoding gamma globin remain present, but expression is relatively
low due to normal gene repression occurring around parturition as
described above.
[0009] It is estimated that 1 in 5000 people in the U.S. have
sickle cell disease (SCD), mostly in people of sub-Saharan Africa
descent. There appears to be a benefit for heterozygous carriers of
the sickle cell mutation for protection against malaria, so this
trait may have been positively selected over time, such that it is
estimated that in sub-Saharan Africa, one third of the population
has the sickle cell trait. Sickle cell disease is caused by a
mutation in the .beta. globin gene as a consequence of which valine
is substituted for glutamic acid at amino acid #6 (a GAG to GTG at
the DNA level), where the resultant hemoglobin is referred to as
"hemoglobinS" or "HbS." Under lower oxygen conditions, a
conformational shift in the deoxy form of HbS exposes a hydrophobic
patch on the protein between the E and F helices. The hydrophobic
residues of the valine at position 6 of the beta chain in
hemoglobin are able to associate with the hydrophobic patch,
causing HbS molecules to aggregate and form fibrous precipitates.
These aggregates in turn cause the abnormality or `sickling` of the
RBCs, resulting in a loss of flexibility of the cells. The sickling
RBCs are no longer able to squeeze into the capillary beds and can
result in vaso-occlusive crisis in sickle cell patients. In
addition, sickled RBCs are more fragile than normal RBCs, and tend
towards hemolysis, eventually leading to anemia in the patient.
[0010] Treatment and management of sickle cell patients is a
life-long proposition involving antibiotic treatment, pain
management and transfusions during acute episodes. One approach is
the use of hydroxyurea, which exerts its effects in part by
increasing the production of gamma globin. Long term side effects
of chronic hydroxyurea therapy are still unknown, however, and
treatment gives unwanted side effects and can have variable
efficacy from patient to patient. Despite an increase in the
efficacy of sickle cell treatments, the life expectancy of patients
is still only in the mid to late 50's and the associated
morbidities of the disease have a profound impact on a patient's
quality of life.
[0011] Thalassemias are also diseases relating to hemoglobin and
typically involve a reduced expression of globin chains. This can
occur through mutations in the regulatory regions of the genes or
from a mutation in a globin coding sequence that results in reduced
expression or reduced levels or functional globin protein. Alpha
thalassemias are mainly associated with people of Western Africa
and South Asian descent, and may confer malarial resistance. Beta
thalassemia is mainly associated with people of Mediterranean
descent, typically from Greece and the coastal areas of Turkey and
Italy. Treatment of thalassemias usually involves blood
transfusions and iron chelation therapy. Bone marrow transplants
are also being used for treatment of people with severe
thalassemias if an appropriate donor can be identified, but this
procedure can have significant risks.
[0012] One approach that has been proposed for the treatment of
both SCD and beta thalassemias is to increase the expression of
gamma globin with the aim to have HbF functionally replace the
aberrant adult hemoglobin. As mentioned above, treatment of SCD
patients with hydroxyurea is thought to be successful in part due
to its effect on increasing gamma globin expression. The first
group of compounds discovered to affect gamma globin reactivation
activity were cytotoxic drugs. The ability to cause de novo
synthesis of gamma-globin by pharmacological manipulation was first
shown using 5-azacytidine in experimental animals (DeSimone (1982)
Proc Nat'l Acad Sci USA 79(14):4428-31). Subsequent studies
confirmed the ability of 5-azacytidine to increase HbF in patients
with .beta.-thalassemia and sickle cell disease (Ley, et al.,
(1982) N. Engl. J. Medicine, 307: 1469-1475, and Ley, et al.,
(1983) Blood 62: 370-380). In addition, short chain fatty acids
(e.g. butyrate and derivatives) have been shown in experimental
systems to increase HbF (Constantoulakis et al., (1988) Blood
72(6):1961-1967). Also, there is a segment of the human population
with a condition known as `Hereditary Persistence of Fetal
Hemoglobin` (HPFH) where elevated amounts of HbF persist in
adulthood (10-40% in HPFH heterozygotes (see Thein et al (2009)
Hum. Mol. Genet 18 (R2): R216-R223). This is a rare condition, but
in the absence of any associated beta globin abnormalities, is not
associated with any significant clinical manifestations, even when
100% of the individual's hemoglobin is HbF. When individuals that
have a beta thalassemia also have co-incident HPFH, the expression
of HbF can lessen the severity of the disease. Further, the
severity of the natural course of sickle cell disease can vary
significantly from patient to patient, and this variability, in
part, can be traced to the fact that some individuals with milder
disease express higher levels of HbF.
[0013] One approach to increase the expression of HbF involves
identification of genes whose products play a role in the
regulation of gamma globin expression. One such gene is BCL11A,
first identified because of its role in lymphocyte development.
BCL11A encodes a zinc finger protein that is thought to be involved
in the developmental stage-specific regulation of gamma globin
expression. BCL11A is expressed in adult erythroid precursor cells
and down-regulation of its expression leads to an increase in gamma
globin expression. In addition, it appears that the splicing of the
BCL11A mRNA is developmentally regulated. In embryonic cells, it
appears that the shorter BCL11A mRNA variants, known as BCL11A-S
and BCL11A-XS are primary expressed, while in adult cells, the
longer BCL11A-L and BCL11A-XL mRNA variants are predominantly
expressed. See, Sankaran et al (2008) Science 322 p. 1839. The
BCL11A protein appears to interact with the beta globin locus to
alter its conformation and thus its expression at different
developmental stages. Use of an inhibitory RNA targeted to the
BCL11A gene has been proposed (see, e.g., U.S. Patent Publication
20110182867) but this technology has several potential drawbacks,
namely that complete knock down may not be achieved, delivery of
such RNAs may be problematic and the RNAs must be present
continuously, requiring multiple treatments for life.
[0014] Targeting of BCL11A enhancer sequences may provide a
mechanism for increasing HbF. For example, genome wide association
studies have identified a set of genetic variations at BCL11A that
are associated with increased HbF levels. These variations are a
collection of SNPs found in non-coding regions of BCL11A that
function as a stage-specific, lineage-restricted enhancer region.
Further investigation revealed that this BCL11A enhancer is
required in erythroid cells for BCL11A expression, but is not
required for its expression in B cells (see Bauer et al, (2013)
Science 343:253-257). The enhancer region was found within intron 2
of the BCL11A gene, and three areas of DNAseI hypersensitivity
(often indicative of a chromatin state that is associated with
regulatory potential) in intron 2 were identified. These three
areas were identified as "+62", "+58" and "+55" in accordance with
the distance in kilobases from the transcription start site of
BCL11A. These enhancer regions are roughly 350 (+55); 550 (+58);
and 350 (+62) nucleotides in length (Bauer 2013, ibid).
[0015] Thus, there remains a need for additional methods and
compositions that can utilize these genome wide association studies
for genome editing and the alteration of gene expression for
example to treat hemoglobinopathies such as sickle cell disease and
beta thalassemia.
SUMMARY
[0016] The present invention describes compositions and methods for
use in gene therapy and genome engineering. Specifically, the
methods and compositions described relate to inactivating (e.g., by
completely or partially abolishing its expression) a gene, for
example a gene that acts as regulator of one or more additional
genes. In particular, the invention describes methods and
compositions for interfering with enhancer function in a BCL11A
gene to diminish or knock out its activity in specific cell
lineages. Additionally, the invention provides methods and
compositions for interfering with BCL11A enhancer functions wherein
the enhancer sequences are not located within the BCL11A gene. The
resulting down-regulation of the BCL11A gene in these circumstances
in turn results in increased expression of gamma globin.
[0017] In some aspects, the invention comprises delivery of at
least one nuclease (e.g., a nuclease that binds to a BCL11A
enhancer sequence) to a human stem cell or precursor cell (HSC/PC)
for the purpose of genome engineering. In certain embodiments, the
nuclease recognizes a target sequence comprising at least 9 (e.g.,
9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 or even more)
contiguous base pairs of SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:3).
Exemplary target sequences are shown in Tables 1, 2, 3, 4 and 6. In
certain embodiments, the nuclease comprises a DNA-binding domain
comprising A DNA-binding protein comprising a zinc finger protein
comprising 4, 5 or 6 zinc finger domains comprising a recognition
helix region, for example, the recognition helix regions in the
order shown in a single row of Table 3 or Table 6. In other
embodiments, the nuclease comprises a TALE protein comprising a
plurality of TALE repeat units, each repeat unit comprising a
hypervariable diresidue region (RVD), for example the RVDs of the
TALE repeats units are shown in a single row of Table 1, Table 2 or
Table 4. The nuclease(s) as described herein may further comprise a
linker (e.g., between the DNA-binding domain and the cleavage
domain), for example a linker as shown in FIGS. 14 and 17.
[0018] In some embodiments, the nuclease is delivered as a peptide,
while in others it is delivered as a nucleic acid encoding the at
least one nuclease. In some embodiments, more than one nuclease is
used. In some preferred embodiments, the nucleic acid encoding the
nuclease is an mRNA, and in some instances, the mRNA is protected.
In some aspects, the mRNA may be chemically modified (See e.g.
Kormann et al, (2011) Nature Biotechnology 29(2):154-157). In other
aspects, the mRNA may comprise an ARCA cap (see U.S. Pat. Nos.
7,074,596 and 8,153,773). In further embodiments, the mRNA may
comprise a mixture of unmodified and modified nucleotides (see U.S.
Patent Publication 2012-0195936). The nuclease may comprise a zinc
finger nuclease (ZFN), a TALE-nuclease (TALEN) or a CRISPR/Cas
nuclease system or a combination thereof. In a preferred
embodiment, the nucleic acid encoding the nuclease(s) is delivered
to the HSC/PC via electroporation. In some embodiments, the
nuclease cleaves at or near the binding site of transcription
factor. In some aspects, the transcription factor is GATA-1.
[0019] In some embodiments comprising a nuclease system that
utilizes a nucleic acid guide (e.g. CRISPR/Cas; TtAgo), the cell
can be contacted with the nucleic acid guide at the same time as it
is contacted with the nuclease, prior to contact with the nuclease,
or after contact with the nuclease. The cell can be contacted where
the nuclease is provided as a polypeptide, a mRNA or an vector
(including a viral vector) capable of expression of the gene
encoding the nuclease. The guide nucleic acid maybe provided as an
oligonucleotide (for TtAgo) or RNA (CRISPR/Cas). Further, guide RNA
may be provided via an expression system for expression of the
guide RNA within the cell. In some aspects, more than one guide RNA
is provided (see Mandal et al (2014) Cell Stem Cell 15:643). In
some embodiments, two guide RNAs are provided, while in others,
more than two (e.g. three, four, five, six, seven, eight, nine, ten
or more than ten) are provided. In some aspects, truncated guide
RNAs are used to increase specificity (Fu et al (2014) Nature
Biotechnol 32(3): 279). Also see U.S. Patent Publication No.
20150056705.
[0020] In one aspect, the invention comprises mutated Cas nucleases
specific for a BCL11A enhancer. In some embodiments, these mutant
Cas nucleases are Cas9 nucleases, and have altered functionality.
In some embodiments, the Cas9 protein is mutated in the HNH domain,
rendering it unable to cleave the DNA strand that is complementary
to the guide RNA. In other embodiments, the Cas9 is mutated in the
Rvu domain, making it incapable of cleaving the non-complimentary
DNA strand. These mutations can result in the creation of Cas9
nickases. In some embodiments, two Cas nickases are used with two
separate guide RNAs to target a DNA, which results in two nicks in
the target DNA at a specified distance apart. In other embodiments,
both the HNH and Rvu endonuclease domains are altered to render a
Cas9 protein which is unable to cleave a target BCl11A enhancer
DNA.
[0021] In another aspect, the methods and compositions of the
invention comprise truncations of the Cas9 protein. In one
embodiment, the Cas9 protein is truncated such that one or more of
the Cas9 functional domains are removed. In one embodiment, the
removal of part or one of the nuclease domains renders the Cas
nuclease a nickase. In one embodiment, the Cas9 comprises only the
domain responsible for interaction with the crRNA or sgRNA and the
target DNA.
[0022] In still further aspects, the methods and compositions of
the invention also comprise fusion proteins wherein the Cas9
protein, or truncation thereof, is fused to a functional domain. In
some aspects, the functional domain is an activation or a
repression domain. In other aspects, the functional domain is a
nuclease domain. In some embodiments, the nuclease domain is a FokI
endonuclease domain (e.g. Tsai (2014) Nature Biotech
doi:10.1038/nbt.2908). In some embodiments, the FokI domain
comprises mutations in the dimerization domain.
[0023] In other aspects, the invention comprises a cell or cell
line in which an endogenous BCL11A enhancer sequence is modified,
for example as compared to the wild-type sequence of the cell. The
cell or cell lines may be heterozygous or homozygous for the
modification. The modifications may comprise insertions, deletions
and/or combinations thereof. In some preferred embodiments, the
insertions, deletions and/or combinations thereof result in the
destruction of a transcription factor binding site. In certain
embodiments, the BCL11A enhancer sequence is modified by a nuclease
(e.g., ZFN, TALEN, CRISPR/Cas system, Ttago system, etc.). In
certain embodiments, the BCL11A enhancer is modified anywhere
between exon 2 and exon 3. In other embodiments, the BCL11A
enhancer is modified in the regions shown in SEQ ID NO:1, SEQ ID
NO:2 or SEQ ID NO:3 (FIG. 11). In certain embodiments, the
modification is at or near the nuclease(s) binding and/or cleavage
site(s), for example, within 1-300 (or any value therebetween) base
pairs upstream or downstream of the site(s) of cleavage, more
preferably within 1-100 base pairs (or any value therebetween) of
either side of the binding and/or cleavage site(s), even more
preferably within 1 to 50 base pairs (or any value therebetween) on
either side of the binding and/or cleavage site(s). In certain
embodiments, the modification is at or near the "+58" region of the
BCL11A enhancer, for example, at or near a nuclease binding site
shown in any of SEQ ID NOs:4 to 80 and 276. In other embodiments,
the modification is at or near the "+55" region of the BCL11A
enhancer, for example, at or near a nuclease site shown in any of
SEQ ID NOs:143 to 184 and 232-251. In still further embodiments,
the modification occurs at other BCL11A enhancer sequences. Any
cell or cell line may be modified, for example a stem cell
(hematopoietic stem cell). Partially or fully differentiated cells
descended from the modified stem cells as described herein are also
provided (e.g., RBCs or RBC precursor cells). Any of the modified
cells or cell lines disclosed herein may show increased expression
of gamma globin. Compositions such as pharmaceutical compositions
comprising the genetically modified cells as described herein are
also provided.
[0024] In other aspects, the invention comprises delivery of a
donor nucleic acid to a target cell. The donor may be delivered
prior to, after, or along with the nucleic acid encoding the
nuclease(s). The donor nucleic acid may comprise an exogenous
sequence (transgene) to be integrated into the genome of the cell,
for example, an endogenous locus. In some embodiments, the donor
may comprise a full length gene or fragment thereof flanked by
regions of homology with the targeted cleavage site. In some
embodiments, the donor lacks homologous regions and is integrated
into a target locus through homology independent mechanism (i.e.
NHEJ). The donor may comprise any nucleic acid sequence, for
example a nucleic acid that, when used as a substrate for
homology-directed repair of the nuclease-induced double-strand
break, leads to a donor-specified deletion to be generated at the
endogenous chromosomal locus (e.g., BCL11A enhancer region) or,
alternatively (or in addition to), novel allelic forms of (e.g.,
point mutations that ablate a transcription factor binding site)
the endogenous locus to be created. In some aspects, the donor
nucleic acid is an oligonucleotide wherein integration leads to a
gene correction event, or a targeted deletion.
[0025] In other aspects, the nuclease and/or donor is(are)
delivered by viral and/or non-viral gene transfer methods. In
preferred embodiments, the donor is delivered to the cell via an
adeno-associated virus (AAV). In some instances, the AAV comprises
LTRs that are of a heterologous serotype in comparison with the
capsid serotype.
[0026] In some aspects, the methods and compositions of the
invention comprise one or more nucleases (e.g., ZFNs and/or TALENs)
targeted to specific regions in the BCL11A enhancer region. In some
embodiments, the one or more pairs of nucleases target sequences
that result in the modification of the enhancer region by deletion
of it in its entirety, while in other embodiments, subsections of
the enhancer are deleted. In some embodiments, the deletion
comprises one or more of the +55, +58 and/or +62 DNAseI
hypersensitivity regions of the enhancer region. In other
embodiments, a subset (less than all) of the hypersensitive regions
is deleted. In some embodiments, only the +55, only the +58 or only
the +62 region is deleted. In other embodiments, two of the regions
are deleted (e.g., +55 and +58; +58 and +62; or +55 and +62).
[0027] In some aspects, deletions comprising regions within the
DNAseI hypersensitive regions of the enhancer are made. These
deletions can comprise from about 1 nucleotide to about 551
nucleotides. Thus, the deletions can comprise, 1, 5, 10, 15, 20,
25, 30, 40, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550
nucleotides, or any value therebetween. In some embodiments, the
deletions comprise binding regions for one or more transcription
factors. In some preferred embodiments, the deletions comprise a
GATA-1 binding site, or the binding site for GATA-1 in combination
with other factors.
[0028] Some aspects of the invention relate to engineered
(non-natural) DNA binding proteins that bind to the BCL11A enhancer
sequence(s) but do not cleave it. In some embodiments, the Cas9
nuclease domain in a CRISPR/Cas system can be specifically
engineered to lose DNA cleavage activity ("dCAS"), and fused to a
functional domain capable of modulating gene expression (see
Perez-Pimera (2013) Nat Method 10(10):973-976) to create a
CRISPR/dCas-TF. In some instances, the engineered DNA binding
domains block interaction of the transcription factors active in
enhancer activity from binding to their cognate enhancer
sequences.
[0029] In some embodiments, the DNA binding domains are fused to a
functional domain. Some aspects include fusion of the DNA binding
domains with domains capable of regulating the expression of a
gene. In some embodiments, the fusion proteins comprise a DNA
binding domain (zinc finger, TALE, CRISPR/dCas, TtaGo or other DNA
binding domains that can be engineered for binding specificity)
fused to a gene expression modulatory domain where the modulator
represses gene expression.
[0030] In some embodiments, the HSC/PC cells are contacted with the
nucleases and/or DNA binding proteins of the invention. In some
embodiments, the nucleases and/or DNA binding proteins are
delivered as nucleic acids and in other embodiments, they are
delivered as proteins. In some embodiments, the nucleic acids are
mRNAs encoding the nucleases and/or DNA binding proteins, and in
further embodiments, the mRNAs may be protected. In some
embodiments, the mRNA may be chemically modified, may comprise an
ARCA cap and/or may comprise a mixture of unmodified and modified
nucleotides.
[0031] In some aspects, the HSC/PC are contacted with the nucleases
and/or DNA binding proteins of the inventions ex vivo, following
apheresis of the HSC/PC from a subject, or purification from
harvested bone marrow. In some embodiments, the nucleases cause
modifications within the BCL11A enhancer regions. In further
embodiments, the HSC/PC containing the BCL11A enhancer region
modifications are introduced back into the subject. In some
instances, the HSC/PC containing the BCL11A enhancer region
modifications are expanded prior to introduction. In other aspects,
the genetically modified HSC/PC are given to the subject in a bone
marrow transplant wherein the HSC/PC engraft, differentiate and
mature in vivo. In some embodiments, the HSC/PC are isolated from
the subject following G-CSF- and/or plerixafor-induced
mobilization, and in others, the cells are isolated from human bone
marrow or human umbilical cords. In some aspects, the subject is
treated to a mild myeloablative procedure prior to introduction of
the graft comprising the modified HSC/PC, while in other aspects,
the subject is treated with a vigorous myeloablative conditioning
regimen. In some embodiments, the methods and compositions of the
invention are used to treat or prevent a hemoglobinopathy. In some
aspects, the hemoglobinopathy is a beta thalassemia, while in other
aspects, the hemoglobinopathy is sickle cell disease.
[0032] In some embodiments, the HSC/PC are further contacted with a
donor molecule. In some embodiments, the donor molecule is
delivered by a viral vector. The donor molecule may comprise one or
more sequences encoding a functional polypeptide (e.g., a cDNA or
fragment thereof), with or without a promoter. Additional sequences
(coding or non-coding sequences) may be included when a donor
molecule is used for inactivation, including but not limited to,
sequences encoding a 2A peptide, SA site, IRES, etc.
[0033] In one aspect, the methods and compositions of the invention
comprise methods for contacting the HSC/PC in vivo. The nucleases
and/or DNA binding proteins are delivered to HSC/PC in situ by
methods known in the art. In some embodiments, the nucleases and/or
DNA binding proteins of the invention comprise a viral particle
that is administered to the subject in need, while in others, the
nucleases and/or DNA binding proteins comprise a nanoparticle (e.g.
liposome). In some embodiments, the viral particles and/or
nanoparticles are delivered to the organ (e.g. bone marrow) wherein
the HSC/PC reside.
[0034] In another aspect, described herein are methods of
integrating a donor nucleic acid into the genome of a cell via
homology-independent mechanisms. The methods comprise creating a
double-stranded break (DSB) in the genome of a cell and cleaving
the donor molecule using a nuclease, such that the donor nucleic
acid is integrated at the site of the DSB. In certain embodiments,
the donor nucleic acid is integrated via non-homology dependent
methods (e.g., NHEJ). As noted above, upon in vivo cleavage the
donor sequences can be integrated in a targeted manner into the
genome of a cell at the location of a DSB. The donor sequence can
include one or more of the same target sites for one or more of the
nucleases used to create the DSB. Thus, the donor sequence may be
cleaved by one or more of the same nucleases used to cleave the
endogenous gene into which integration is desired. In certain
embodiments, the donor sequence includes different nuclease target
sites from the nucleases used to induce the DSB. DSBs in the genome
of the target cell may be created by any mechanism. In certain
embodiments, the DSB is created by one or more zinc-finger
nucleases (ZFNs), fusion proteins comprising a zinc finger binding
domain, which is engineered to bind a sequence within the region of
interest, and a cleavage domain or a cleavage half-domain. In other
embodiments, the DSB is created by one or more TALE DNA-binding
domains (naturally occurring or non-naturally occurring) fused to a
nuclease domain (TALEN). In yet further embodiments, the DSB is
created using a CRISPR/Cas nuclease system where an engineered
single guide RNA or its functional equivalent is used to guide the
nuclease to a targeted site in a genome.
[0035] In one aspect, the donor may encode a regulatory protein of
interest (e.g. ZFP TFs, TALE TFs or a CRISPR/Cas TF) that binds to
and/or modulates expression of a gene of interest. In one
embodiment, the regulatory proteins bind to a DNA sequence and
prevent binding of other regulatory factors. In another embodiment,
the binding of a the regulatory protein may modulate (i.e. induce
or repress) expression of a target DNA.
[0036] In some embodiments, the transgenic HSC/PC cell and/or
animal includes a transgene that encodes a human gene. In some
instances, the transgenic animal comprises a knock out at the
endogenous locus corresponding to exogenous transgene, thereby
allowing the development of an in vivo system where the human
protein may be studied in isolation. Such transgenic models may be
used for screening purposes to identify small molecules or large
biomolecules or other entities which may interact with or modify
the human protein of interest. In some aspects, the transgene is
integrated into the selected locus (e.g., safe-harbor) into a stem
cell (e.g., an embryonic stem cell, an induced pluripotent stem
cell, a hematopoietic stem cell, etc.) or animal embryo obtained by
any of the methods described herein, and then the embryo is
implanted such that a live animal is born. The animal is then
raised to sexual maturity and allowed to produce offspring wherein
at least some of the offspring comprise edited endogenous gene
sequence or the integrated transgene.
[0037] In another aspect, provided herein is a method of altering
gene expression (e.g., BCL11a and/or a globin gene) in a cell, the
method comprising: introducing, into the cell, one or more
nucleases as described herein, under conditions such that the one
or more proteins are expressed and expression of the gene is
altered. In certain embodiments, expression of a globin gene (e.g.,
gamma globin or beta globin) is altered (e.g., increased). Any of
the methods described herein may further comprise integrating a
donor sequence (e.g., transgene or fragment thereof under the
control of an exogenous or endogenous promoter) into the genome of
the cell, for example integrating a donor at or near the site of
nuclease cleavage in the BCL11a gene. The donor sequence is
introduced to the cell using a viral vector, as an oligonucleotide
and/or on a plasmid. The cell in which gene expression is altered
may be, for example, a red blood cell (RBC) precursor cell and/or a
hematopoietic stem cell (e.g., CD34+ cell).
[0038] In other embodiments, provided herein is a method of
producing a genetically modified cell comprising a genomic
modification within an endogenous BCL11a enhancer sequence, the
method comprising the steps of: a) contacting a cell with a
polynucleotide (e.g. DNA or mRNA) encoding a zinc finger nuclease
comprising 4, 5, or 6 zinc finger domains in which each of the zinc
finger domains comprises a recognition helix region in the order
shown in a single row of Table 3 or Table 6; b) subjecting the cell
to conditions conducive to expressing the zinc finger protein from
the polynucleotide; and c) modifying the endogenous BCL11A enhancer
sequence with the expressed zinc finger protein sufficient to
produce the genetically modified cell. In certain embodiments, the
cells are stimulated with at least one cytokine (e.g., prior to
step (a)). The polynucleotide may be contacted with the cell using
any suitable method, including but not limited, via transfection,
using a non-viral vector, using a viral vector, by chemical means
or by exposure to an electric field (e.g., electroporation).
[0039] Also provided is a method of treating a patient in need of
an increase in globin gene expression, the method comprising
administering to the patient the pharmaceutical preparation as
described herein in an amount sufficient to increase the globin
gene expression in the patient. In certain embodiments, the patient
is known to have, is suspected of having, or is at risk of
developing a thalassemia or sickle cell disease.
[0040] A kit, comprising the nucleic acids, proteins and/or cells
of the invention, is also provided. The kit may comprise nucleic
acids encoding the nucleases, (e.g. RNA molecules or ZFN, TALEN or
CRISPR/Cas system encoding genes contained in a suitable expression
vector), or aliquots of the nuclease proteins, donor molecules,
suitable stemness modifiers, cells, buffers, and/or instructions
(e.g., for performing the methods of the invention) and the
like.
[0041] The invention therefore includes, but is not limited to the
following embodiments:
[0042] 1. A genetically modified cell comprising a genomic
modification made by a nuclease, wherein the genomic modification
is within an endogenous BCL11a enhancer sequence, and further
wherein the genomic modification is selected from the group
consisting of insertions, deletions and combinations thereof.
[0043] 2. The genetically modified cell of embodiment 1, wherein
the genomic modification is within one or more of the sequences
shown in SEQ ID NO:1, 2 or 3.
[0044] 3. The genetically modified cell of embodiment 2, wherein
the genomic modification is within at least 9 contiguous base pairs
of SEQ ID NO:1, 2 or 3.
[0045] 4. The genetically modified cell of embodiment 2, wherein
the genomic modification is within the +55 BCL11A enhancer sequence
(SEQ ID NO:1).
[0046] 5. The genetically modified cell of embodiment 4, wherein
the genomic modification is at or near any of the sequences shown
as SEQ ID Nos. 143 to 184 and 232-251.
[0047] 6. The genetically modified cell of embodiment 2, wherein
the genomic modification is within the +58 BCL11A enhancer sequence
(SEQ ID NO:2).
[0048] 7. The genetically modified cell of embodiment 6, wherein
the genomic modification is at or near any of the sequences shown
as SEQ ID Nos. 4 to 80 and 276.
[0049] 8. The genetically modified cell of embodiment 2, wherein
the genomic modification is within the +62 BCL11A enhancer sequence
(SEQ ID NO:3)
[0050] 9. The genetically modified cell of any of embodiments 1 to
8, wherein the cell is a stem cell.
[0051] 10. The genetically modified cell of embodiment 9, wherein
the stem cell is a hematopoietic stem cell.
[0052] 11. The genetically modified cell of embodiment 10, wherein
the hematopoietic stem cell is a CD34+ cell.
[0053] 12. A genetically modified differentiated cell descended
from the stem cell of any of embodiments 1 to 11.
[0054] 13. The genetically modified cell of embodiment 12, wherein
the cell is a red blood cell (RBC).
[0055] 14. The genetically modified cell of any of embodiments 1 to
13, wherein the nuclease comprises at least one zinc finger
nuclease (ZFN) or TALEN.
[0056] 15. The genetically modified cell of any of embodiments 1 to
14, wherein the nuclease is introduced into the cell as a
polynucleotide.
[0057] 16. The genetically modified cell of any of embodiments 1 to
15, wherein the insertion comprises integration of a donor
polynucleotide encoding a transgene.
[0058] 17. The genetically modified cell of any of embodiments 14
to 16, wherein the nuclease comprises a zinc finger nuclease, the
zinc finger nuclease comprising 4, 5, or 6 zinc finger domains
comprising a recognition helix and further wherein the zinc finger
proteins comprise the recognition helix regions in the order shown
in a single row of Table 3 or Table 6.
[0059] 18. The genetically modified cell of any of embodiments 14
to 16, wherein the nuclease comprises a TALEN, the TALEN comprising
a plurality of TALE repeat units, each repeat unit comprising a
hypervariable diresidue region (RVD), wherein the RVDs of the TALE
repeats units are shown in a single row of Table 1, Table 2 or
Table 4.
[0060] 19. A pharmaceutical composition comprising the genetically
modified cell of any of embodiments 1 to 18.
[0061] 20. A DNA-binding protein comprising a zinc finger protein
or a TALE-effector protein (TALE), wherein
[0062] (i) the zinc finger protein comprises 4, 5 or 6 zinc finger
domains comprising a recognition helix region, wherein the zinc
finger proteins comprise the recognition helix regions in the order
shown in a single row of Table 3 or Table 6; and
[0063] (ii) the TALE protein comprising a plurality of TALE repeat
units, each repeat unit comprising a hypervariable diresidue region
(RVD), wherein the RVDs of the TALE repeats units are shown in a
single row of Table 1, Table 2 or Table 4.
[0064] 21. A fusion protein comprising a zinc finger protein or
TALE protein of embodiment 20 and a wild-type or engineered
cleavage domain or cleavage half-domain.
[0065] 22. A polynucleotide encoding one or more proteins of
embodiment 20 or embodiment 21.
[0066] 23. An isolated cell comprising one or more proteins
according to embodiment 20 or embodiment 21.
[0067] 24. An isolated cell comprising one or more polynucleotides
according to embodiment 22.
[0068] 25. The cell of embodiment 23 or embodiment 24, wherein the
cell is a hematopoietic stem cell.
[0069] 26. A kit comprising at least one of: i) a polynucleotide
encoding the protein according to embodiment 20 or embodiment 21 or
ii) a protein according to embodiment 20 or embodiment 21.
[0070] 27. A method of altering globin gene expression in a cell,
the method comprising:
[0071] introducing, into the cell, one or more polynucleotides
according to embodiment 22, under conditions such that the one or
more proteins are expressed and expression of the globin gene is
altered.
[0072] 28. The method of embodiment 27, wherein expression of the
globin gene is increased.
[0073] 29. The method of embodiment 27 or embodiment 28, wherein
the globin gene is a gamma globin or beta globin gene.
[0074] 30. The method of any of embodiments 27 to 29, further
comprising integrating a donor sequence into the genome of the
cell.
[0075] 31. The method of embodiment 30, wherein the donor sequence
is introduced to the cell using a viral vector, as an
oligonucleotide or on a plasmid.
[0076] 32. The method of any of embodiments 27 to 31, wherein the
cell is selected from the group consisting of a red blood cell (RB
C) precursor cell and a hematopoietic stem cell.
[0077] 33. The method of any of embodiments 30 to 32, wherein the
donor sequence comprises a transgene under the control of an
endogenous or exogenous promoter.
[0078] 34. A method of producing a genetically modified cell
comprising a genomic modification within an endogenous BCL11a
enhancer sequence, the method comprising the steps of:
[0079] a) contacting a cell with a polynucleotide encoding a fusion
protein comprising a zinc finger nuclease comprising 4, 5, or 6
zinc finger domains in which each of the zinc finger domains
comprises a recognition helix region in the order shown in a single
row of Table 3 or Table 6,
[0080] b) subjecting the cell to conditions conducive to expressing
the fusion protein from the polynucleotide; and
[0081] c) modifying the endogenous BCL11A enhancer sequence with
the expressed fusion protein sufficient to produce the genetically
modified cell.
[0082] 35. The method of embodiment 34, wherein the method further
comprises stimulating the cells with at least one cytokine.
[0083] 36. The method of embodiment 34 or embodiment 35, wherein
the method further comprises the step of delivering the
polynucleotide inside the cell.
[0084] 37. The method of embodiment 36, wherein the delivery step
comprises use of at least one of a non-viral delivery system, a
viral delivery system, and a delivery vehicle.
[0085] 38. The method of any of embodiments 34 to 37, wherein the
delivery step further comprises subjecting the cells to an electric
field.
[0086] 39. A kit for performing the method of any of embodiments 34
to 37, the kit comprising:
[0087] a) at least one polynucleotide encoding a fusion protein
comprising a zinc finger nuclease comprising 4, 5, or 6 zinc finger
domains in which each of the zinc finger domains comprises a
recognition helix region in the order shown in a single row of
Table 3 or Table 6,
[0088] b) at least one polynucleotide encoding a TALE protein
comprising a plurality of TALE repeat units, each repeat unit
comprising a hypervariable diresidue region (RVD), wherein the RVDs
of the TALE repeats units are shown in a single row of Table 1,
Table 2 or Table 4; and optionally,
[0089] c) directions for using the kit.
[0090] 40. A method of treating a patient in need of an increase in
globin gene expression, the method comprising administering to the
patient the pharmaceutical preparation of embodiment 19 in an
amount sufficient to increase the globin gene expression in the
patient.
[0091] 41. The method of embodiment 40, wherein the patient is
known to have, is suspected of having, or is at risk of developing
a globinopathy.
[0092] 42. The method of embodiment 41, wherein the globinopathy is
a thalassemia or sickle cell disease.
[0093] 43. The method of embodiment 42, wherein the thalassemia is
.beta.-thalassemia.
[0094] These and other aspects will be readily apparent to the
skilled artisan in light of disclosure as a whole.
BRIEF DESCRIPTION OF THE DRAWINGS
[0095] FIG. 1 depicts a diagram of the BCL11A coding region,
indicating the position of the introns and of the enhancer regions.
The derivations of the differing splicing products are also
indicated (see Liu et al. (2006) Molecular Cancer 5:18).
[0096] FIG. 2 depicts the genomic region that encodes the various
Bcl11a isoforms (University of California Santa Cruz genome
browser, coordinates listed in the hg19 assembly of the human
genome), the enhancer region in the BCL11A intron 2 (coordinates
listed in the hg19 assembly of the human genome), and defines the
three subregions of the enhancer region as represented by DNAse I
hypersensitive sites (listed in kb by approximate distance from the
transcription start site): +55, +58 and +62. Nuclease target
locations within the three subregions are indicated as follows:
nucleases were designed to cleave on the left end of each subregion
(the `L` sites), in the middle of each (the `M` sites) and on the
right end (the `R` sites). Cleavage of the locus in vivo with a
pair of nucleases results in a deletion of the intervening region
in a significant fraction of the cells.
[0097] FIG. 3 depicts the results of in-cell cleavage by the TALEN
pair sets (indicated in the table in the upper panel of the figure)
in the BCL11A enhancer region as gauged by a PCR-based assay for
deletion of various regions of the enhancer. Pairs of TALENs used
were designed to create deletions in human HSPCs either within the
+55 region ("55L-R"), within the +62 region ("62L-R), or within the
+58 region ("58L-R", "58M-R"). The gel shows PCR products produced
by isolating genomic DNA from human HSPCs transfected with
expression constructs encoding the indicated TALENs and amplifying
the region surrounding the target region. These data demonstrate
the generation of deletions (band indicated by the symbol A) in the
targeted region following cleavage by the TALEN pair sets
indicated.
[0098] FIG. 4 is a graph showing results from a real-time RT-qPCR
("Taqman.RTM.") analysis designed to detect a change in expression
of fetal gamma-globin mRNA following targeted editing of the BCL11A
enhancer. Following electroporation of CD34 cells from healthy
human volunteers with mRNAs encoding the designated nucleases (see
FIG. 3), erythrocytes were generated in vitro, after which total
RNA was harvested. The relative levels of alpha globin and gamma
globin mRNA for each sample were determined in an RT-PCR
Taqman.RTM. analysis, and the relative ratio of gamma globin
mRNA/alpha globin mRNA was plotted. Thus, increasing gamma globin
expression in the nuclease-treated samples leads to an increase in
the normalized gamma/alpha ratio compared to the controls. The
Figure displays the results from treating CD34 cells with single
TALEN pairs and for the TALEN pair sets described in FIGS. 2 and 3.
The level of gamma/alpha is increased for the 58L-R and 58M-R pair
sets. Note that the ratio in the GFP transfection control was 3.4
in this experiment.
[0099] FIG. 5 is a graph showing results from a real-time RT-qPCR
("Taqman.RTM.") analysis designed to detect a change in expression
of fetal gamma-globin mRNA following targeted editing of the BCL11A
enhancer. Following electroporation of CD34 cells from healthy
human volunteers with mRNAs encoding the designated nucleases (see
FIG. 3), erythrocytes were generated in vitro, after which total
RNA was harvested. The relative levels of beta globin and gamma
globin mRNA for each sample were determined in an RT-PCR
Taqman.RTM. analysis, and the relative ratio of gamma globin
mRNA/beta globin mRNA was plotted. The results demonstrate that
while the specific single TALEN pairs used in these experiments
were not able to induce a change in gamma globin expression,
creation of deletions by use of the pair sets caused an increase in
relative gamma expression, corrected by adult beta globin
expression in this instance. In particular, deletion of the DNA
sequenced encompassed by the +55 or of the +58 DNAse I
hypersensitive elevated gamma globin, while such elevation
following a deletion of the sequence encompassed by the +62 DNAse I
hypersensitive site was not detected in this experiment. Note that
the ratio in the GFP transfection control was 0.5 in this
experiment.
[0100] FIG. 6 shows the results from a Taqman.RTM. analysis for
fetal globin levels as described in FIG. 4. In this set of
experiments, the levels of beta globin and gamma globin mRNA were
measured, and thus the data depicted is the ratio of gamma to beta
and compared to the same gamma/beta ratio in control treated cells.
A series of single TALEN pairs were used to "walk across" the +58
region of the BCL11A enhancer. In contrast to the earlier
experiments where a deletion of 400-900 base pairs was required to
see an increase in gamma expression, the results depicted in this
experiment demonstrated a single site (indicated by an arrow) that
when cleaved caused that relative increase. Results from the
deletion pairs are included in this graph for comparison. See FIG.
7 for location of cleavage sites of the TALEN pairs across the
region of interest.
[0101] FIG. 7 shows the results from a Taqman.RTM. analysis as
described in FIG. 4 using ZFN pairs targeted to the +58 enhancer
region. The levels of beta globin and gamma globin were
characterized and used to express the ratio of gamma to beta-globin
compared to the same ratio in control treated cells. The data
demonstrates that ZFN-driven disruption of the same region
identified in the TALEN screen (FIG. 6 and see FIG. 8 below))
resulted in increased gamma globin expression. Note that the ratio
in the GFP transfection control was 2.6 in this experiment.
[0102] FIG. 8 shows a representation of the binding sites of the
+58 enhancer region specific TALENs (102852 and 102853) and ZFNs
(45843 and 45844), use of which in human HSPCs increases the
relative expression of gamma globin following in vitro
erythropoiesis. The sequence shown is the double stranded form of
the DNA sequence encompassing the +58 region of the BCL11A enhancer
(SEQ ID NO:264), and the numbering system relates to the +58
itself. Also indicated in the figure is the location of a match to
the binding site of the GATA-1-transcription factor (sequence of
locus, gtGATAAag, consensus GATA-1 site--swGATAAvv). Additionally,
the cleavage sites of the TALEN pairs used in the +58 "walk" are
indicated where the numbers correspond to the samples used in the
data sets presented in FIG. 6.
[0103] FIG. 9 shows the results from a Taqman.RTM. analysis as
described in FIG. 4 where a series of TALENs were made to target
the +55 region of the BCL11A enhancer. In this set of experiments,
the levels of beta globin and gamma globin were characterized, and
thus the data depicted is the ratio of gamma to beta and compared
to the same ratio in control treated cells (which, in this
experiment, was 0.8). The data confirm that mutations generated at
specific positions within the +55 region can increase relative
gamma globin expression (see arrows).
[0104] FIG. 10 is a representation of the cleavage sites of the +55
enhancer region specific TALENs as shown in FIG. 9. The sequence
shown is the double stranded form of the DNA sequence encompassing
the +55 region of the BCL11A enhancer (SEQ ID NO:254). The numbers
that highlight short regions of nucleotides indicate the likely
cleavage sites induced by the TALEN samples listed in FIG. 10. Also
indicated in FIG. 10 are two matches to the consensus binding site
for the GATA-1 transcription factor.
[0105] FIGS. 11A to 11C display the DNA sequence of the three DNAse
I hypersensitive sites within the BCL11A enhancer sequence. Because
their identification was performed by probing regions of accessible
chromatin in cells (see Bauer et al, (2013) ibid), the exact
boundaries of the regions are not known and approximate boundaries
are shown. FIG. 11A shows the sequence of the +55 region (SEQ ID
NO:1), FIG. 11B shows the sequence of the +58 region (SEQ ID NO:2)
and FIG. 11C shows the sequence of the +62 region (SEQ ID
NO:3).
[0106] FIGS. 12A to 12C demonstrate that ZFN-driven cleavage in
cells closer to the core of the GATA-1 consensus elevates fetal
globin levels to an even greater extent than cleavage closer to the
3' end of the motif. FIG. 12A displays a diagram depicting the
binding sites of the Bcl11A-specific ZFN pairs in relation to the
GATA-1 consensus sequence (FIG. 12A) and depicts a DNA sequence
within the +58 region comprising the GATA-1 consensus sequence (SEQ
ID NO:255). Bars above and below the DNA sequence indicate the
binding sites of the ZFNs. FIG. 12B shows the relative expression
of gamma globin and beta globin as measured by mRNA expression
following of human HSPCs with mRNA encoding the indicated ZFNs (see
FIG. 12A), followed by in vitro erythropoiesis and measurement of
levels of fetal globin (see, FIG. 4). The ratio observed when a GFP
expressing mRNA was transfected into the CD34+ cells was 0.97. FIG.
12C represents in "pie chart" form the allelic forms of the BCL11A
enhancer (specifically, the region cleaved by the ZFNs shown in
FIG. 12A) found in human HSPCs following electroporation with the
indicated ZFNs. While comparable levels of unmodified (wild-type)
chromatids are observed in the two samples, the sample treated with
ZFNs that cut closer to the GATA-1 motif contain a greater number
of chromatids that eliminate the GATA-1 consensus (e.g., the "-15"
allele, which represents a deletion of 15 base pairs). The data
demonstrates that cleavage by the two ZFN pairs that are closer to
the center of the GATA-1 consensus sequence (pairs 46801/46880 and
46923/46999) is associated with increased gamma globin
expression.
[0107] FIG. 13 demonstrates that altering the linker between the
zinc finger and FokI moiety in ZFNs used for genome editing of the
BCL11A enhancer affects fetal globin levels following in vitro
erythropoiesis despite comparable levels disrupted chromatids.
Human HSPCs were electroporated with the indicated ZFNs (the linker
used in each ZFN monomer is indicated in parentheses), and
immediately prior and following in vitro erythroid differentiation,
the % of disrupted alleles was measured (shown below each sample in
"X/Y" form, with the first number corresponding to % of
non-wild-type indels following electroporation, and the second
number show results following 14 days of in vitro erythroid
differentiation). Whole mRNA was harvested and the levels of fetal
globin (normalized to alpha globin) were measured.
[0108] FIG. 14 depicts the amino acid and DNA sequences for four
linkers (L0 (SEQ ID NO:256 and 257), L7a (SEQ ID NO:258 and 259),
L7c5 (SEQ ID NO:260 and 261) and L8c5 (SEQ ID NO:262 and 263) used
in the ZFP designs. Sequences with a solid underline indicate the
carboxy terminal region of the ZFP DNA binding domain, while
sequences indicated with the dashed underline indicate the amino
terminal region of the Fok I nuclease domain. Sequences in bold
indicate the novel sequences added to the standard L0 linker.
[0109] FIGS. 15A and 15B depict the percent of cells of human
origin, and targeted genetic modification at the nuclease target
site in these human cells, respectively found in the peripheral
blood of mice following edited human CD34+ cell transplant. FIG.
15A is a graph depicting the percent human cells in the mouse
periphery following transplantation of human CD34+ cells that had
been edited with two different sets of ZFN 4 weeks post-transplant.
FIG. 15B depicts the level of indels detected in those human cells.
Each symbol represents data obtained from an individual mouse.
[0110] FIGS. 16A through 16D, depict the percentage of indels
induced by the nucleases in human cells that differentiated from
the original transplanted CD34+ cells 16 weeks post
transplantation. FIG. 16A shows the level of indel activity in
pan-myeloid cells, identified by the presence of the CD33 marker.
FIG. 16B shows the activity in CD19+ B cells. FIG. 16C shows the
activity in glyA+ or erythroid cells, while FIG. 16D shows the
activity in stem cells. Each symbol represents data obtained from
an individual mouse.
[0111] FIG. 17 shows a series of linker sequences (SEQ ID NOS
265-275, respectively, in order of appearance). These linkers can
serve to link the zinc finger DNA binding domain to the FokI
nuclease domain.
DETAILED DESCRIPTION
[0112] Disclosed herein are compositions and methods for genome
engineering for the modulation of BCL11A and/or gamma globin
expression and for the treatment and/or prevention of
hemoglobinopathies. In particular, nuclease-mediated (i.e. ZFN,
TALEN or CRISPR/Cas or TtAgo system) targeted deletion of specific
sites in a BCL11A enhancer region is efficiently achieved in HSC/PC
and results in a change in relative gamma globin expression during
subsequent erythropoiesis. This modulation of BCL11A and gamma
globin expression is particularly useful for treatment of
hemoglobinopathies (e.g., beta thalassemias, sickle cell disease)
wherein there is insufficient beta globin expression or expression
of a mutated form of beta-globin. Using the methods and
compositions of the invention, the complications and disease
related sequelae caused by the aberrant beta globin can be overcome
by alteration of the expression of gamma globin in erythrocyte
precursor cells.
General
[0113] Practice of the methods, as well as preparation and use of
the compositions disclosed herein employ, unless otherwise
indicated, conventional techniques in molecular biology,
biochemistry, chromatin structure and analysis, computational
chemistry, cell culture, recombinant DNA and related fields as are
within the skill of the art. These techniques are fully explained
in the literature. See, for example, Sambrook et al. MOLECULAR
CLONING: A LABORATORY MANUAL, Second edition, Cold Spring Harbor
Laboratory Press, 1989 and Third edition, 2001; Ausubel et al.,
CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, New
York, 1987 and periodic updates; the series METHODS IN ENZYMOLOGY,
Academic Press, San Diego; Wolfe, CHROMATIN STRUCTURE AND FUNCTION,
Third edition, Academic Press, San Diego, 1998; METHODS IN
ENZYMOLOGY, Vol. 304, "Chromatin" (P. M. Wassarman and A. P.
Wolffe, eds.), Academic Press, San Diego, 1999; and METHODS IN
MOLECULAR BIOLOGY, Vol. 119, "Chromatin Protocols" (P. B. Becker,
ed.) Humana Press, Totowa, 1999.
Definitions
[0114] The terms "nucleic acid," "polynucleotide," and
"oligonucleotide" are used interchangeably and refer to a
deoxyribonucleotide or ribonucleotide polymer, in linear or
circular conformation, and in either single- or double-stranded
form. For the purposes of the present disclosure, these terms are
not to be construed as limiting with respect to the length of a
polymer. The terms can encompass known analogues of natural
nucleotides, as well as nucleotides that are modified in the base,
sugar and/or phosphate moieties (e.g., phosphorothioate backbones).
In general, an analogue of a particular nucleotide has the same
base-pairing specificity; i.e., an analogue of A will base-pair
with T.
[0115] The terms "polypeptide," "peptide" and "protein" are used
interchangeably to refer to a polymer of amino acid residues. The
term also applies to amino acid polymers in which one or more amino
acids are chemical analogues or modified derivatives of a
corresponding naturally-occurring amino acids.
[0116] "Binding" refers to a sequence-specific, non-covalent
interaction between macromolecules (e.g., between a protein and a
nucleic acid). Not all components of a binding interaction need be
sequence-specific (e.g., contacts with phosphate residues in a DNA
backbone), as long as the interaction as a whole is
sequence-specific. Such interactions are generally characterized by
a dissociation constant (K.sub.d) of 10.sup.-6 M.sup.-1 or lower.
"Affinity" refers to the strength of binding: increased binding
affinity being correlated with a lower K.sub.d.
[0117] A "binding protein" is a protein that is able to bind to
another molecule. A binding protein can bind to, for example, a DNA
molecule (a DNA-binding protein), an RNA molecule (an RNA-binding
protein) and/or a protein molecule (a protein-binding protein). In
the case of a protein-binding protein, it can bind to itself (to
form homodimers, homotrimers, etc.) and/or it can bind to one or
more molecules of a different protein or proteins. A binding
protein can have more than one type of binding activity. For
example, zinc finger proteins have DNA-binding, RNA-binding and
protein-binding activity.
[0118] A "zinc finger DNA binding protein" (or binding domain) is a
protein, or a domain within a larger protein, that binds DNA in a
sequence-specific manner through one or more zinc fingers, which
are regions of amino acid sequence within the binding domain whose
structure is stabilized through coordination of a zinc ion. The
term zinc finger DNA binding protein is often abbreviated as zinc
finger protein or ZFP.
[0119] A "TALE DNA binding domain" or "TALE" is a polypeptide
comprising one or more TALE repeat domains/units. The repeat
domains are involved in binding of the TALE to its cognate target
DNA sequence. A single "repeat unit" (also referred to as a
"repeat") is typically 33-35 amino acids in length and exhibits at
least some sequence homology with other TALE repeat sequences
within a naturally occurring TALE protein.
[0120] Zinc finger and TALE binding domains can be "engineered" to
bind to a predetermined nucleotide sequence, for example via
engineering (altering one or more amino acids) of the recognition
helix region of a naturally occurring zinc finger or TALE protein.
Therefore, engineered DNA binding proteins (zinc fingers or TALEs)
are proteins that are non-naturally occurring. Non-limiting
examples of methods for engineering DNA-binding proteins are design
and selection. A designed DNA binding protein is a protein not
occurring in nature whose design/composition results principally
from rational criteria. Rational criteria for design include
application of substitution rules and computerized algorithms for
processing information in a database storing information of
existing ZFP and/or TALE designs and binding data. See, for
example, U.S. Pat. Nos. 6,140,081; 6,453,242; 6,534,261 and
8,585,526; see also WO 98/53058; WO 98/53059; WO 98/53060; WO
02/016536 and WO 03/016496.
[0121] A "selected" zinc finger protein or TALE is a protein not
found in nature whose production results primarily from an
empirical process such as phage display, interaction trap or hybrid
selection. See e.g., U.S. Pat. Nos. 5,789,538; 5,925,523;
6,007,988; 6,013,453; 6,200,759; 8,586,526; WO 95/19431; WO
96/06166; WO 98/53057; WO 98/54311; WO 00/27878; WO 01/60970 WO
01/88197, WO 02/099084.
[0122] "TtAgo" is a prokaryotic Argonaute protein thought to be
involved in gene silencing. TtAgo is derived from the bacteria
Thermus thermophilus. See, e.g., Swarts et al, ibid, G. Sheng et
al., (2013) Proc. Natl. Acad. Sci. U.S.A. 111, 652). A "TtAgo
system" is all the components required including, for example,
guide DNAs for cleavage by a TtAgo enzyme.
[0123] "Recombination" refers to a process of exchange of genetic
information between two polynucleotides, including but not limited
to, donor capture by non-homologous end joining (NHEJ) and
homologous recombination. For the purposes of this disclosure,
"homologous recombination (HR)" refers to the specialized form of
such exchange that takes place, for example, during repair of
double-strand breaks in cells via homology-directed repair
mechanisms. This process requires nucleotide sequence homology,
uses a "donor" molecule to template repair of a "target" molecule
(i.e., the one that experienced the double-strand break), and is
variously known as "non-crossover gene conversion" or "short tract
gene conversion," because it leads to the transfer of genetic
information from the donor to the target. Without wishing to be
bound by any particular theory, such transfer can involve mismatch
correction of heteroduplex DNA that forms between the broken target
and the donor, and/or "synthesis-dependent strand annealing," in
which the donor is used to resynthesize genetic information that
will become part of the target, and/or related processes. Such
specialized HR often results in an alteration of the sequence of
the target molecule such that part or all of the sequence of the
donor polynucleotide is incorporated into the target
polynucleotide.
[0124] In the methods of the disclosure, one or more targeted
nucleases as described herein create a double-stranded break (DSB)
in the target sequence (e.g., cellular chromatin) at a
predetermined site. The DSB may result in deletions and/or
insertions by homology-directed repair or by non-homology-directed
repair mechanisms. Deletions may include any number of base pairs.
Similarly, insertions may include any number of base pairs
including, for example, integration of a "donor" polynucleotide,
optionally having homology to the nucleotide sequence in the region
of the break. The donor sequence may be physically integrated or,
alternatively, the donor polynucleotide is used as a template for
repair of the break via homologous recombination, resulting in the
introduction of all or part of the nucleotide sequence as in the
donor into the cellular chromatin. Thus, a first sequence in
cellular chromatin can be altered and, in certain embodiments, can
be converted into a sequence present in a donor polynucleotide.
Thus, the use of the terms "replace" or "replacement" can be
understood to represent replacement of one nucleotide sequence by
another, (i.e., replacement of a sequence in the informational
sense), and does not necessarily require physical or chemical
replacement of one polynucleotide by another.
[0125] In any of the methods described herein, additional pairs of
zinc-finger proteins or TALEN can be used for additional
double-stranded cleavage of additional target sites within the
cell.
[0126] Any of the methods described herein can be used for
insertion of a donor of any size and/or partial or complete
inactivation of one or more target sequences in a cell by targeted
integration of donor sequence that disrupts expression of the
gene(s) of interest. Cell lines with partially or completely
inactivated genes are also provided.
[0127] In any of the methods described herein, the exogenous
nucleotide sequence (the "donor sequence" or "transgene") can
contain sequences that are homologous, but not identical, to
genomic sequences in the region of interest, thereby stimulating
homologous recombination to insert a non-identical sequence in the
region of interest. Thus, in certain embodiments, portions of the
donor sequence that are homologous to sequences in the region of
interest exhibit between about 80 to 99% (or any integer
therebetween) sequence identity to the genomic sequence that is
replaced. In other embodiments, the homology between the donor and
genomic sequence is higher than 99%, for example if only 1
nucleotide differs as between donor and genomic sequences of over
100 contiguous base pairs. In certain cases, a non-homologous
portion of the donor sequence can contain sequences not present in
the region of interest, such that new sequences are introduced into
the region of interest. In these instances, the non-homologous
sequence is generally flanked by sequences of 50-1,000 base pairs
(or any integral value therebetween) or any number of base pairs
greater than 1,000, that are homologous or identical to sequences
in the region of interest. In other embodiments, the donor sequence
is non-homologous to the first sequence, and is inserted into the
genome by non-homologous recombination mechanisms.
[0128] "Cleavage" refers to the breakage of the covalent backbone
of a DNA molecule. Cleavage can be initiated by a variety of
methods including, but not limited to, enzymatic or chemical
hydrolysis of a phosphodiester bond. Both single-stranded cleavage
and double-stranded cleavage are possible, and double-stranded
cleavage can occur as a result of two distinct single-stranded
cleavage events. DNA cleavage can result in the production of
either blunt ends or staggered ends. In certain embodiments, fusion
polypeptides are used for targeted double-stranded DNA
cleavage.
[0129] A "cleavage half-domain" is a polypeptide sequence which, in
conjunction with a second polypeptide (either identical or
different) forms a complex having cleavage activity (preferably
double-strand cleavage activity). The terms "first and second
cleavage half-domains;" "+ and - cleavage half-domains" and "right
and left cleavage half-domains" are used interchangeably to refer
to pairs of cleavage half-domains that dimerize.
[0130] An "engineered cleavage half-domain" is a cleavage
half-domain that has been modified so as to form obligate
heterodimers with another cleavage half-domain (e.g., another
engineered cleavage half-domain). See, also, U.S. Patent
Publication Nos. 2005/0064474, 20070218528, 20080131962 and
20110201055, incorporated herein by reference in their
entireties.
[0131] The term "sequence" refers to a nucleotide sequence of any
length, which can be DNA or RNA; can be linear, circular or
branched and can be either single-stranded or double stranded. The
term "donor sequence" refers to a nucleotide sequence that is
inserted into a genome. A donor sequence can be of any length, for
example between 2 and 100,000,000 nucleotides in length (or any
integer value therebetween or thereabove), preferably between about
100 and 100,000 nucleotides in length (or any integer
therebetween), more preferably between about 2000 and 20,000
nucleotides in length (or any value therebetween) and even more
preferable, between about 5 and 15 kb (or any value
therebetween).
[0132] "Chromatin" is the nucleoprotein structure comprising the
cellular genome. Cellular chromatin comprises nucleic acid,
primarily DNA, and protein, including histones and non-histone
chromosomal proteins. The majority of eukaryotic cellular chromatin
exists in the form of nucleosomes, wherein a nucleosome core
comprises approximately 150 base pairs of DNA associated with an
octamer comprising two each of histones H2A, H2B, H3 and H4; and
linker DNA (of variable length depending on the organism) extends
between nucleosome cores. A molecule of histone H1 is generally
associated with the linker DNA. For the purposes of the present
disclosure, the term "chromatin" is meant to encompass all types of
cellular nucleoprotein, both prokaryotic and eukaryotic. Cellular
chromatin includes both chromosomal and episomal chromatin.
[0133] A "chromosome," is a chromatin complex comprising all or a
portion of the genome of a cell. The genome of a cell is often
characterized by its karyotype, which is the collection of all the
chromosomes that comprise the genome of the cell. The genome of a
cell can comprise one or more chromosomes.
[0134] An "episome" is a replicating nucleic acid, nucleoprotein
complex or other structure comprising a nucleic acid that is not
part of the chromosomal karyotype of a cell. Examples of episomes
include plasmids and certain viral genomes.
[0135] An "accessible region" is a site in cellular chromatin in
which a target site present in the nucleic acid can be bound by an
exogenous molecule which recognizes the target site. Without
wishing to be bound by any particular theory, it is believed that
an accessible region is one that is not packaged into a nucleosomal
structure. The distinct structure of an accessible region can often
be detected by its sensitivity to chemical and enzymatic probes,
for example, nucleases.
[0136] A "target site" or "target sequence" is a nucleic acid
sequence that defines a portion of a nucleic acid to which a
binding molecule will bind, provided sufficient conditions for
binding exist.
[0137] An "exogenous" molecule is a molecule that is not normally
present in a cell, but can be introduced into a cell by one or more
genetic, biochemical or other methods. "Normal presence in the
cell" is determined with respect to the particular developmental
stage and environmental conditions of the cell. Thus, for example,
a molecule that is present only during embryonic development of
muscle is an exogenous molecule with respect to an adult muscle
cell. Similarly, a molecule induced by heat shock is an exogenous
molecule with respect to a non-heat-shocked cell. An exogenous
molecule can comprise, for example, a functioning version of a
malfunctioning endogenous molecule or a malfunctioning version of a
normally-functioning endogenous molecule.
[0138] An exogenous molecule can be, among other things, a small
molecule, such as is generated by a combinatorial chemistry
process, or a macromolecule such as a protein, nucleic acid,
carbohydrate, lipid, glycoprotein, lipoprotein, polysaccharide, any
modified derivative of the above molecules, or any complex
comprising one or more of the above molecules. Nucleic acids
include DNA and RNA, can be single- or double-stranded; can be
linear, branched or circular; and can be of any length. Nucleic
acids include those capable of forming duplexes, as well as
triplex-forming nucleic acids. See, for example, U.S. Pat. Nos.
5,176,996 and 5,422,251. Proteins include, but are not limited to,
DNA-binding proteins, transcription factors, chromatin remodeling
factors, methylated DNA binding proteins, polymerases, methylases,
demethylases, acetylases, deacetylases, kinases, phosphatases,
integrases, recombinases, ligases, topoisomerases, gyrases and
helicases.
[0139] An exogenous molecule can be the same type of molecule as an
endogenous molecule, e.g., an exogenous protein or nucleic acid.
For example, an exogenous nucleic acid can comprise an infecting
viral genome, a plasmid or episome introduced into a cell, or a
chromosome that is not normally present in the cell. Methods for
the introduction of exogenous molecules into cells are known to
those of skill in the art and include, but are not limited to,
lipid-mediated transfer (i.e., liposomes, including neutral and
cationic lipids), electroporation, direct injection, cell fusion,
particle bombardment, calcium phosphate co-precipitation,
DEAE-dextran-mediated transfer and viral vector-mediated transfer.
An exogenous molecule can also be the same type of molecule as an
endogenous molecule but derived from a different species than the
cell is derived from. For example, a human nucleic acid sequence
may be introduced into a cell line originally derived from a mouse
or hamster. Methods for the introduction of exogenous molecules
into plant cells are known to those of skill in the art and
include, but are not limited to, protoplast transformation, silicon
carbide (e.g., WHISKERS.TM.) Agrobacterium-mediated transformation,
lipid-mediated transfer (i.e., liposomes, including neutral and
cationic lipids), electroporation, direct injection, cell fusion,
particle bombardment (e.g., using a "gene gun"), calcium phosphate
co-precipitation, DEAE-dextran-mediated transfer and viral
vector-mediated transfer.
[0140] By contrast, an "endogenous" molecule is one that is
normally present in a particular cell at a particular developmental
stage under particular environmental conditions. For example, an
endogenous nucleic acid can comprise a chromosome, the genome of a
mitochondrion, chloroplast or other organelle, or a
naturally-occurring episomal nucleic acid. Additional endogenous
molecules can include proteins, for example, transcription factors
and enzymes.
[0141] As used herein, the term "product of an exogenous nucleic
acid" includes both polynucleotide and polypeptide products, for
example, transcription products (polynucleotides such as RNA) and
translation products (polypeptides).
[0142] A "fusion" molecule is a molecule in which two or more
subunit molecules are linked, preferably covalently. The subunit
molecules can be the same chemical type of molecule, or can be
different chemical types of molecules. Examples of the first type
of fusion molecule include, but are not limited to, fusion proteins
(for example, a fusion between a ZFP or TALE DNA-binding domain and
one or more activation domains) and fusion nucleic acids (for
example, a nucleic acid encoding the fusion protein described
supra). Examples of the second type of fusion molecule include, but
are not limited to, a fusion between a triplex-forming nucleic acid
and a polypeptide, and a fusion between a minor groove binder and a
nucleic acid.
[0143] Expression of a fusion protein in a cell can result from
delivery of the fusion protein to the cell or by delivery of a
polynucleotide encoding the fusion protein to a cell, wherein the
polynucleotide is transcribed, and the transcript is translated, to
generate the fusion protein. Trans-splicing, polypeptide cleavage
and polypeptide ligation can also be involved in expression of a
protein in a cell. Methods for polynucleotide and polypeptide
delivery to cells are presented elsewhere in this disclosure.
[0144] A "gene," for the purposes of the present disclosure,
includes a DNA region encoding a gene product (see infra), as well
as all DNA regions which regulate the production of the gene
product, whether or not such regulatory sequences are adjacent to
coding and/or transcribed sequences. Accordingly, a gene includes,
but is not necessarily limited to, promoter sequences, terminators,
translational regulatory sequences such as ribosome binding sites
and internal ribosome entry sites, enhancers, silencers,
insulators, boundary elements, replication origins, matrix
attachment sites and locus control regions.
[0145] "Gene expression" refers to the conversion of the
information, contained in a gene, into a gene product. A gene
product can be the direct transcriptional product of a gene (e.g.,
mRNA, tRNA, rRNA, antisense RNA, ribozyme, structural RNA or any
other type of RNA) or a protein produced by translation of an mRNA.
Gene products also include RNAs which are modified, by processes
such as capping, polyadenylation, methylation, and editing, and
proteins modified by, for example, methylation, acetylation,
phosphorylation, ubiquitination, ADP-ribosylation, myristilation,
and glycosylation.
[0146] "Modulation" of gene expression refers to a change in the
activity of a gene. Modulation of expression can include, but is
not limited to, gene activation and gene repression. Genome editing
(e.g., cleavage, alteration, inactivation, random mutation) can be
used to modulate expression. Gene inactivation refers to any
reduction in gene expression as compared to a cell that does not
include a ZFP, TALE or CRISPR/Cas system as described herein. Thus,
gene inactivation may be partial or complete.
[0147] A "protected" mRNA is one in which the mRNA has been altered
in some manner to increase the stability or translation of the
mRNA. Examples of protections include the use of replacement of up
to 25% of the cytodine and uridine residues with 2-thiouridine
(s2U) and 5-methylcytidine (m5C). The resulting mRNA exhibits less
immunogenicity and more stability as compared with its unmodified
counterpart. (see Kariko et al. ((2012), Molecular Therapy, Vol.
16, No. 11, pages 1833-1844). Other changes include the addition of
a so-called ARCA cap, which increases the translationability of the
in vitro produced mRNA (see U.S. Pat. No. 7,074,596).
[0148] A "region of interest" is any region of cellular chromatin,
such as, for example, a gene or a non-coding sequence within or
adjacent to a gene, in which it is desirable to bind an exogenous
molecule. Binding can be for the purposes of targeted DNA cleavage
and/or targeted recombination. A region of interest can be present
in a chromosome, an episome, an organellar genome (e.g.,
mitochondrial, chloroplast), or an infecting viral genome, for
example. A region of interest can be within the coding region of a
gene, within transcribed non-coding regions such as, for example,
leader sequences, trailer sequences or introns, or within
non-transcribed regions, either upstream or downstream of the
coding region. A region of interest can be as small as a single
nucleotide pair or up to 2,000 nucleotide pairs in length, or any
integral value of nucleotide pairs.
[0149] "Eukaryotic" cells include, but are not limited to, fungal
cells (such as yeast), plant cells, animal cells, mammalian cells
and human cells (e.g., T-cells).
[0150] The terms "operative linkage" and "operatively linked" (or
"operably linked") are used interchangeably with reference to a
juxtaposition of two or more components (such as sequence
elements), in which the components are arranged such that both
components function normally and allow the possibility that at
least one of the components can mediate a function that is exerted
upon at least one of the other components. By way of illustration,
a transcriptional regulatory sequence, such as a promoter, is
operatively linked to a coding sequence if the transcriptional
regulatory sequence controls the level of transcription of the
coding sequence in response to the presence or absence of one or
more transcriptional regulatory factors. A transcriptional
regulatory sequence is generally operatively linked in cis with a
coding sequence, but need not be directly adjacent to it. For
example, an enhancer is a transcriptional regulatory sequence that
is operatively linked to a coding sequence, even though they are
not contiguous.
[0151] With respect to fusion polypeptides, the term "operatively
linked" can refer to the fact that each of the components performs
the same function in linkage to the other component as it would if
it were not so linked. For example, with respect to a fusion
polypeptide in which a ZFP, TALE or Cas DNA-binding domain is fused
to an activation domain, the ZFP, TALE or Cas DNA-binding domain
and the activation domain are in operative linkage if, in the
fusion polypeptide, the ZFP, TALE of Cas DNA-binding domain portion
is able to bind its target site and/or its binding site, while the
activation domain is able to upregulate gene expression. When a
fusion polypeptide in which a ZFP, TALE or Cas DNA-binding domain
is fused to a cleavage domain, the ZFP, TALE or Cas DNA-binding
domain and the cleavage domain are in operative linkage if, in the
fusion polypeptide, the ZFP, TALE or Cas DNA-binding domain portion
is able to bind its target site and/or its binding site, while the
cleavage domain is able to cleave DNA in the vicinity of the target
site.
[0152] A "functional fragment" of a protein, polypeptide or nucleic
acid is a protein, polypeptide or nucleic acid whose sequence is
not identical to the full-length protein, polypeptide or nucleic
acid, yet retains the same function as the full-length protein,
polypeptide or nucleic acid. A functional fragment can possess
more, fewer, or the same number of residues as the corresponding
native molecule, and/or can contain one or more amino acid or
nucleotide substitutions. Methods for determining the function of a
nucleic acid (e.g., coding function, ability to hybridize to
another nucleic acid) are well-known in the art. Similarly, methods
for determining protein function are well-known. For example, the
DNA-binding function of a polypeptide can be determined, for
example, by filter-binding, electrophoretic mobility-shift, or
immunoprecipitation assays. DNA cleavage can be assayed by gel
electrophoresis. See Ausubel et al., supra. The ability of a
protein to interact with another protein can be determined, for
example, by co-immunoprecipitation, two-hybrid assays or
complementation, both genetic and biochemical. See, for example,
Fields et al. (1989) Nature 340:245-246; U.S. Pat. No. 5,585,245
and PCT WO 98/44350.
[0153] A "vector" is capable of transferring gene sequences to
target cells. Typically, "vector construct," "expression vector,"
and "gene transfer vector," mean any nucleic acid construct capable
of directing the expression of a gene of interest and which can
transfer gene sequences to target cells. Thus, the term includes
cloning, and expression vehicles, as well as integrating
vectors.
[0154] The terms "subject" and "patient" are used interchangeably
and refer to mammals such as human patients and non-human primates,
as well as experimental animals such as rabbits, dogs, cats, rats,
mice, and other animals. Accordingly, the term "subject" or
"patient" as used herein means any mammalian patient or subject to
which the or stem cells of the invention can be administered.
Subjects of the present invention include those that have been
exposed to one or more chemical toxins, including, for example, a
nerve toxin.
[0155] "Stemness" refers to the relative ability of any cell to act
in a stem cell-like manner, i.e., the degree of toti-, pluri-, or
oligopotentcy and expanded or indefinite self-renewal that any
particular stem cell may have.
Nucleases
[0156] Described herein are compositions, particularly nucleases,
that are useful for in vivo cleavage of a donor molecule carrying a
transgene and nucleases for cleavage of the genome of a cell such
that the transgene is integrated into the genome in a targeted
manner. In certain embodiments, one or more of the nucleases are
naturally occurring. In other embodiments, one or more of the
nucleases are non-naturally occurring, i.e., engineered in the
DNA-binding domain and/or cleavage domain. For example, the
DNA-binding domain of a naturally-occurring nuclease may be altered
to bind to a selected target site (e.g., a meganuclease that has
been engineered to bind to site different than the cognate binding
site). In other embodiments, the nuclease comprises heterologous
DNA-binding and cleavage domains (e.g., zinc finger nucleases;
TAL-effector domain DNA binding proteins; meganuclease DNA-binding
domains with heterologous cleavage domains).
[0157] A. DNA-Binding Domains
[0158] In certain embodiments, the composition and methods
described herein employ a meganuclease (homing endonuclease)
DNA-binding domain for binding to the donor molecule and/or binding
to the region of interest in the genome of the cell.
Naturally-occurring meganucleases recognize 15-40 base-pair
cleavage sites and are commonly grouped into four families: the
LAGLIDADG (SEQ ID NO: 287) family, the GIY-YIG family, the His-Cyst
box family and the HNH family. Exemplary homing endonucleases
include I-SceI, I-CeuI, PI-PspI, PI-Sce, I-SceIV, I-CsmI, I-PanI,
I-SceII, I-PpoI, I-SceIII, I-CreI, I-TevI, I-TevII and I-TevIII.
Their recognition sequences are known. See also U.S. Pat. Nos.
5,420,032; 6,833,252; Belfort et al. (1997) Nucleic Acids Res.
25:3379-3388; Dujon et al. (1989) Gene 82:115-118; Perler et al.
(1994) Nucleic Acids Res. 22, 1125-1127; Jasin (1996) Trends Genet.
12:224-228; Gimble et al. (1996) J. Mol. Biol. 263:163-180; Argast
et al. (1998) J. Mol. Biol. 280:345-353 and the New England Biolabs
catalogue.
[0159] In certain embodiments, the methods and compositions
described herein make use of a nuclease that comprises an
engineered (non-naturally occurring) homing endonuclease
(meganuclease). The recognition sequences of homing endonucleases
and meganucleases such as I-SceI, I-CeuI, PI-PspI, PI-Sce, I-SceIV,
I-CsmI, I-PanI, I-SceII, I-PpoI, I-SceIII, I-CreI, I-TevI, I-TevII
and I-TevIII are known. See also U.S. Pat. Nos. 5,420,032;
6,833,252; Belfort et al. (1997) Nucleic Acids Res. 25:3379-3388;
Dujon et al. (1989) Gene 82:115-118; Perler et al. (1994) Nucleic
Acids Res. 22, 1125-1127; Jasin (1996) Trends Genet. 12:224-228;
Gimble et al. (1996) J. Mol. Biol. 263:163-180; Argast et al.
(1998) J. Mol. Biol. 280:345-353 and the New England Biolabs
catalogue. In addition, the DNA-binding specificity of homing
endonucleases and meganucleases can be engineered to bind
non-natural target sites. See, for example, Chevalier et al. (2002)
Molec. Cell 10:895-905; Epinat et al. (2003) Nucleic Acids Res.
31:2952-2962; Ashworth et al. (2006) Nature 441:656-659; Paques et
al. (2007) Current Gene Therapy 7:49-66; U.S. Patent Publication
No. 20070117128. The DNA-binding domains of the homing
endonucleases and meganucleases may be altered in the context of
the nuclease as a whole (i.e., such that the nuclease includes the
cognate cleavage domain) or may be fused to a heterologous cleavage
domain.
[0160] In other embodiments, the DNA-binding domain of one or more
of the nucleases used in the methods and compositions described
herein comprises a naturally occurring or engineered (non-naturally
occurring) TAL effector DNA binding domain. See, e.g., U.S. Pat.
No. 8,586,526, incorporated by reference in its entirety herein.
The plant pathogenic bacteria of the genus Xanthomonas are known to
cause many diseases in important crop plants. Pathogenicity of
Xanthomonas depends on a conserved type III secretion (T3S) system
which injects more than 25 different effector proteins into the
plant cell. Among these injected proteins are transcription
activator-like (TAL) effectors which mimic plant transcriptional
activators and manipulate the plant transcriptome (see Kay et al
(2007) Science 318:648-651). These proteins contain a DNA binding
domain and a transcriptional activation domain. One of the most
well characterized TAL-effectors is AvrBs3 from Xanthomonas
campestgris pv. Vesicatoria (see Bonas et al (1989) Mol Gen Genet
218: 127-136 and WO2010079430). TAL-effectors contain a centralized
domain of tandem repeats, each repeat containing approximately 34
amino acids, which are key to the DNA binding specificity of these
proteins. In addition, they contain a nuclear localization sequence
and an acidic transcriptional activation domain (for a review see
Schornack S, et al (2006) J Plant Physiol 163(3): 256-272). In
addition, in the phytopathogenic bacteria Ralstonia solanacearum
two genes, designated brg11 and hpx17 have been found that are
homologous to the AvrBs3 family of Xanthomonas in the R.
solanacearum biovar 1 strain GMI1000 and in the biovar 4 strain
RS1000 (See Heuer et al (2007) Appl and Envir Micro 73(13):
4379-4384). These genes are 98.9% identical in nucleotide sequence
to each other but differ by a deletion of 1,575 base pairs in the
repeat domain of hpx17. However, both gene products have less than
40% sequence identity with AvrBs3 family proteins of Xanthomonas.
See, e.g., U.S. Pat. No. 8,586,526, incorporated by reference in
its entirety herein.
[0161] Specificity of these TAL effectors depends on the sequences
found in the tandem repeats. The repeated sequence comprises
approximately 102 base pairs (bp) and the repeats are typically
91-100% homologous with each other (Bonas et al, ibid).
Polymorphism of the repeats is usually located at positions 12 and
13 and there appears to be a one-to-one correspondence between the
identity of the hypervariable diresidues (RVD) at positions 12 and
13 with the identity of the contiguous nucleotides in the
TAL-effector's target sequence (see Moscou and Bogdanove, (2009)
Science 326:1501 and Boch et al (2009) Science 326:1509-1512).
Experimentally, the natural code for DNA recognition of these
TAL-effectors has been determined such that an HD sequence at
positions 12 and 13 leads to a binding to cytosine (C), NG binds to
T, NI to A, C, G or T, NN binds to A or G, and ING binds to T.
These DNA binding repeats have been assembled into proteins with
new combinations and numbers of repeats, to make artificial
transcription factors that are able to interact with new sequences
and activate the expression of a non-endogenous reporter gene in
plant cells (Boch et al, ibid). Engineered TAL proteins have been
linked to a FokI cleavage half domain to yield a TAL effector
domain nuclease fusion (TALEN) exhibiting activity in a yeast
reporter assay (plasmid based target). See, e.g., U.S. Pat. No.
8,586,526; Christian et al ((2010)<Genetics epub
10.1534/genetics.110.120717). In certain embodiments, TALE domain
comprises an N-cap and/or C-cap as described in U.S. Pat. No.
8,586,526.
[0162] In certain embodiments, the DNA binding domain of one or
more of the nucleases used for in vivo cleavage and/or targeted
cleavage of the genome of a cell comprises a zinc finger protein.
Preferably, the zinc finger protein is non-naturally occurring in
that it is engineered to bind to a target site of choice. See, for
example, See, for example, Beerli et al. (2002) Nature Biotechnol.
20:135-141; Pabo et al. (2001) Ann. Rev. Biochem. 70:313-340;
Isalan et al. (2001) Nature Biotechnol. 19:656-660; Segal et al.
(2001) Curr. Opin. Biotechnol. 12:632-637; Choo et al. (2000) Curr.
Opin. Struct. Biol. 10:411-416; U.S. Pat. Nos. 6,453,242;
6,534,261; 6,599,692; 6,503,717; 6,689,558; 7,030,215; 6,794,136;
7,067,317; 7,262,054; 7,070,934; 7,361,635; 7,253,273; and U.S.
Patent Publication Nos. 2005/0064474; 2007/0218528; 2005/0267061,
all incorporated herein by reference in their entireties.
[0163] An engineered zinc finger binding domain can have a novel
binding specificity, compared to a naturally-occurring zinc finger
protein. Engineering methods include, but are not limited to,
rational design and various types of selection. Rational design
includes, for example, using databases comprising triplet (or
quadruplet) nucleotide sequences and individual zinc finger amino
acid sequences, in which each triplet or quadruplet nucleotide
sequence is associated with one or more amino acid sequences of
zinc fingers which bind the particular triplet or quadruplet
sequence. See, for example, co-owned U.S. Pat. Nos. 6,453,242 and
6,534,261, incorporated by reference herein in their
entireties.
[0164] Exemplary selection methods, including phage display and
two-hybrid systems, are disclosed in U.S. Pat. Nos. 5,789,538;
5,925,523; 6,007,988; 6,013,453; 6,410,248; 6,140,466; 6,200,759;
and 6,242,568; as well as WO 98/37186; WO 98/53057; WO 00/27878; WO
01/88197 and GB 2,338,237. In addition, enhancement of binding
specificity for zinc finger binding domains has been described, for
example, in co-owned WO 02/077227.
[0165] In addition, as disclosed in these and other references,
zinc finger domains and/or multi-fingered zinc finger proteins may
be linked together using any suitable linker sequences, including
for example, linkers of 5 or more amino acids in length. See, also,
U.S. Pat. Nos. 6,479,626; 6,903,185; and 7,153,949 for exemplary
linker sequences 6 or more amino acids in length. The proteins
described herein may include any combination of suitable linkers
between the individual zinc fingers of the protein.
[0166] Selection of target sites; ZFPs and methods for design and
construction of fusion proteins (and polynucleotides encoding same)
are known to those of skill in the art and described in detail in
U.S. Pat. Nos. 6,140,081; 5,789,538; 6,453,242; 6,534,261;
5,925,523; 6,007,988; 6,013,453; 6,200,759; WO 95/19431; WO
96/06166; WO 98/53057; WO 98/54311; WO 00/27878; WO 01/60970 WO
01/88197; WO 02/099084; WO 98/53058; WO 98/53059; WO 98/53060; WO
02/016536 and WO 03/016496.
[0167] Nearly any linker (spacer) may be used between one or more
of the components of the DNA-binding domain (e.g., zinc fingers),
between one or more DNA-binding domains and/or between the
DNA-binding domain and the functional domain (e.g., nuclease).
Non-limiting examples of suitable linker sequences include U.S.
Pat. Nos. 8,772,453; 7,888,121; 6,479,626; 6,903,185; and
7,153,949; U.S. Publication Nos. 20090305419 and 20150064789. Thus,
the proteins described herein may include any combination of
suitable linkers between the individual DNA-binding components
and/or between the DNA-binding domain and the functional domain of
the compositions described herein.
[0168] The CRISPR (clustered regularly interspaced short
palindromic repeats) locus, which encodes RNA components of the
system, and the cas (CRISPR-associated) locus, which encodes
proteins (Jansen et al., 2002. Mol. Microbiol. 43: 1565-1575;
Makarova et al., 2002. Nucleic Acids Res. 30: 482-496; Makarova et
al., 2006. Biol. Direct 1: 7; Haft et al., 2005. PLoS Comput. Biol.
1: e60) make up the gene sequences of the CRISPR/Cas nuclease
system. CRISPR loci in microbial hosts contain a combination of
CRISPR-associated (Cas) genes as well as non-coding RNA elements
capable of programming the specificity of the CRISPR-mediated
nucleic acid cleavage.
[0169] The Type II CRISPR is one of the most well characterized
systems and carries out targeted DNA double-strand break in four
sequential steps. First, two non-coding RNA, the pre-crRNA array
and tracrRNA, are transcribed from the CRISPR locus. Second,
tracrRNA hybridizes to the repeat regions of the pre-crRNA and
mediates the processing of pre-crRNA into mature crRNAs containing
individual spacer sequences. Third, the mature crRNA:tracrRNA
complex directs Cas9 to the target DNA via Watson-Crick
base-pairing between the spacer on the crRNA and the protospacer on
the target DNA next to the protospacer adjacent motif (PAM), an
additional requirement for target recognition. Finally, Cas9
mediates cleavage of target DNA to create a double-stranded break
within the protospacer. Activity of the CRISPR/Cas system comprises
of three steps: (i) insertion of alien DNA sequences into the
CRISPR array to prevent future attacks, in a process called
`adaptation`, (ii) expression of the relevant proteins, as well as
expression and processing of the array, followed by (iii)
RNA-mediated interference with the alien nucleic acid. Thus, in the
bacterial cell, several of the so-called `Cas` proteins are
involved with the natural function of the CRISPR/Cas system and
serve roles in functions such as insertion of the alien DNA
etc.
[0170] In certain embodiments, Cas protein may be a "functional
derivative" of a naturally occurring Cas protein. A "functional
derivative" of a native sequence polypeptide is a compound having a
qualitative biological property in common with a native sequence
polypeptide. "Functional derivatives" include, but are not limited
to, fragments of a native sequence and derivatives of a native
sequence polypeptide and its fragments, provided that they have a
biological activity in common with a corresponding native sequence
polypeptide. A biological activity contemplated herein is the
ability of the functional derivative to hydrolyze a DNA substrate
into fragments. The term "derivative" encompasses both amino acid
sequence variants of polypeptide, covalent modifications, and
fusions thereof. Suitable derivatives of a Cas polypeptide or a
fragment thereof include but are not limited to mutants, fusions,
covalent modifications of Cas protein or a fragment thereof. Cas
protein, which includes Cas protein or a fragment thereof, as well
as derivatives of Cas protein or a fragment thereof, may be
obtainable from a cell or synthesized chemically or by a
combination of these two procedures. The cell may be a cell that
naturally produces Cas protein, or a cell that naturally produces
Cas protein and is genetically engineered to produce the endogenous
Cas protein at a higher expression level or to produce a Cas
protein from an exogenously introduced nucleic acid, which nucleic
acid encodes a Cas that is same or different from the endogenous
Cas. In some case, the cell does not naturally produce Cas protein
and is genetically engineered to produce a Cas protein.
[0171] In some embodiments, the DNA binding domain is part of a
TtAgo system (see Swarts et al, ibid; Sheng et al, ibid). In
eukaryotes, gene silencing is mediated by the Argonaute (Ago)
family of proteins. In this paradigm, Ago is bound to small (19-31
nt) RNAs. This protein-RNA silencing complex recognizes target RNAs
via Watson-Crick base pairing between the small RNA and the target
and endonucleolytically cleaves the target RNA (Vogel (2014)
Science 344:972-973). In contrast, prokaryotic Ago proteins bind to
small single-stranded DNA fragments and likely function to detect
and remove foreign (often viral) DNA (Yuan et al., (2005) Mol. Cell
19, 405; Olovnikov, et al. (2013) Mol. Cell 51, 594; Swarts et al.,
ibid). Exemplary prokaryotic Ago proteins include those from
Aquifex aeolicus, Rhodobacter sphaeroides, and Thermus
thermophilus.
[0172] One of the most well-characterized prokaryotic Ago protein
is the one from T. thermophilus (TtAgo; Swarts et al. ibid). TtAgo
associates with either 15 nt or 13-25 nt single-stranded DNA
fragments with 5' phosphate groups. This "guide DNA" bound by TtAgo
serves to direct the protein-DNA complex to bind a Watson-Crick
complementary DNA sequence in a third-party molecule of DNA. Once
the sequence information in these guide DNAs has allowed
identification of the target DNA, the TtAgo-guide DNA complex
cleaves the target DNA. Such a mechanism is also supported by the
structure of the TtAgo-guide DNA complex while bound to its target
DNA (G. Sheng et al., ibid). Ago from Rhodobacter sphaeroides
(RsAgo) has similar properties (Olivnikov et al. ibid).
[0173] Exogenous guide DNAs of arbitrary DNA sequence can be loaded
onto the TtAgo protein (Swarts et al. ibid.). Since the specificity
of TtAgo cleavage is directed by the guide DNA, a TtAgo-DNA complex
formed with an exogenous, investigator-specified guide DNA will
therefore direct TtAgo target DNA cleavage to a complementary
investigator-specified target DNA. In this way, one may create a
targeted double-strand break in DNA. Use of the TtAgo-guide DNA
system (or orthologous Ago-guide DNA systems from other organisms)
allows for targeted cleavage of genomic DNA within cells. Such
cleavage can be either single- or double-stranded. For cleavage of
mammalian genomic DNA, it would be preferable to use of a version
of TtAgo codon optimized for expression in mammalian cells.
Further, it might be preferable to treat cells with a TtAgo-DNA
complex formed in vitro where the TtAgo protein is fused to a
cell-penetrating peptide. Further, it might be preferable to use a
version of the TtAgo protein that has been altered via mutagenesis
to have improved activity at 37 degrees Celsius. Ago-RNA-mediated
DNA cleavage could be used to affect a panopoly of outcomes
including gene knock-out, targeted gene addition, gene correction,
targeted gene deletion using techniques standard in the art for
exploitation of DNA breaks.
[0174] Thus, the nuclease comprises a DNA-binding domain in that
specifically binds to a target site in any gene into which it is
desired to insert a donor (transgene).
[0175] B. Cleavage Domains
[0176] Any suitable cleavage domain can be operatively linked to a
DNA-binding domain to form a nuclease. For example, ZFP DNA-binding
domains have been fused to nuclease domains to create ZFNs--a
functional entity that is able to recognize its intended nucleic
acid target through its engineered (ZFP) DNA binding domain and
cause the DNA to be cut near the ZFP binding site via the nuclease
activity. See, e.g., Kim et al. (1996) Proc Nat'l Acad Sci USA
93(3):1156-1160. More recently, ZFNs have been used for genome
modification in a variety of organisms. See, for example, United
States Patent Publications 20030232410; 20050208489; 20050026157;
20050064474; 20060188987; 20060063231; and International
Publication WO 07/014275. Likewise, TALE DNA-binding domains have
been fused to nuclease domains to create TALENs. See, e.g., U.S.
Publication No. 20110301073.
[0177] As noted above, the cleavage domain may be heterologous to
the DNA-binding domain, for example a zinc finger DNA-binding
domain and a cleavage domain from a nuclease or a TALEN DNA-binding
domain and a cleavage domain, or meganuclease DNA-binding domain
and cleavage domain from a different nuclease. Heterologous
cleavage domains can be obtained from any endonuclease or
exonuclease. Exemplary endonucleases from which a cleavage domain
can be derived include, but are not limited to, restriction
endonucleases and homing endonucleases. See, for example, 2002-2003
Catalogue, New England Biolabs, Beverly, Mass.; and Belfort et al.
(1997) Nucleic Acids Res. 25:3379-3388. Additional enzymes which
cleave DNA are known (e.g., S1 Nuclease; mung bean nuclease;
pancreatic DNase I; micrococcal nuclease; yeast HO endonuclease;
see also Linn et al. (eds.) Nucleases, Cold Spring Harbor
Laboratory Press, 1993). One or more of these enzymes (or
functional fragments thereof) can be used as a source of cleavage
domains and cleavage half-domains.
[0178] Similarly, a cleavage half-domain can be derived from any
nuclease or portion thereof, as set forth above, that requires
dimerization for cleavage activity. In general, two fusion proteins
are required for cleavage if the fusion proteins comprise cleavage
half-domains. Alternatively, a single protein comprising two
cleavage half-domains can be used. The two cleavage half-domains
can be derived from the same endonuclease (or functional fragments
thereof), or each cleavage half-domain can be derived from a
different endonuclease (or functional fragments thereof). In
addition, the target sites for the two fusion proteins are
preferably disposed, with respect to each other, such that binding
of the two fusion proteins to their respective target sites places
the cleavage half-domains in a spatial orientation to each other
that allows the cleavage half-domains to form a functional cleavage
domain, e.g., by dimerizing. Thus, in certain embodiments, the near
edges of the target sites are separated by 5-8 nucleotides or by
15-18 nucleotides. However any integral number of nucleotides or
nucleotide pairs can intervene between two target sites (e.g., from
2 to 50 nucleotide pairs or more). In general, the site of cleavage
lies between the target sites.
[0179] Restriction endonucleases (restriction enzymes) are present
in many species and are capable of sequence-specific binding to DNA
(at a recognition site), and cleaving DNA at or near the site of
binding. Certain restriction enzymes (e.g., Type IIS) cleave DNA at
sites removed from the recognition site and have separable binding
and cleavage domains. For example, the Type IIS enzyme Fok I
catalyzes double-stranded cleavage of DNA, at 9 nucleotides from
its recognition site on one strand and 13 nucleotides from its
recognition site on the other. See, for example, U.S. Pat. Nos.
5,356,802; 5,436,150 and 5,487,994; as well as Li et al. (1992)
Proc. Natl. Acad. Sci. USA 89:4275-4279; Li et al. (1993) Proc.
Natl. Acad. Sci. USA 90:2764-2768; Kim et al. (1994a) Proc. Natl.
Acad. Sci. USA 91:883-887; Kim et al. (1994b) J. Biol. Chem.
269:31,978-31,982. Thus, in one embodiment, fusion proteins
comprise the cleavage domain (or cleavage half-domain) from at
least one Type IIS restriction enzyme and one or more zinc finger
binding domains, which may or may not be engineered.
[0180] An exemplary Type IIS restriction enzyme, whose cleavage
domain is separable from the binding domain, is FokI. This
particular enzyme is active as a dimer. Bitinaite et al. (1998)
Proc. Natl. Acad. Sci. USA 95: 10,570-10,575. Accordingly, for the
purposes of the present disclosure, the portion of the Fok I enzyme
used in the disclosed fusion proteins is considered a cleavage
half-domain. Thus, for targeted double-stranded cleavage and/or
targeted replacement of cellular sequences using zinc finger-Fok I
fusions, two fusion proteins, each comprising a FokI cleavage
half-domain, can be used to reconstitute a catalytically active
cleavage domain. Alternatively, a single polypeptide molecule
containing a zinc finger binding domain and two Fok I cleavage
half-domains can also be used. Parameters for targeted cleavage and
targeted sequence alteration using zinc finger-Fok I fusions are
provided elsewhere in this disclosure.
[0181] A cleavage domain or cleavage half-domain can be any portion
of a protein that retains cleavage activity, or that retains the
ability to multimerize (e.g., dimerize) to form a functional
cleavage domain.
[0182] Exemplary Type IIS restriction enzymes are described in
International Publication WO 07/014275, incorporated herein in its
entirety. Additional restriction enzymes also contain separable
binding and cleavage domains, and these are contemplated by the
present disclosure. See, for example, Roberts et al. (2003) Nucleic
Acids Res. 31:418-420.
[0183] In certain embodiments, the cleavage domain comprises one or
more engineered cleavage half-domain (also referred to as
dimerization domain mutants) that minimize or prevent
homodimerization, as described, for example, in See, e.g., U.S.
Pat. Nos. 7,914,796; 8,034,598 and 8,623,618, the disclosures of
all of which are incorporated by reference in their entireties
herein. Amino acid residues at positions 446, 447, 479, 483, 484,
486, 487, 490, 491, 496, 498, 499, 500, 531, 534, 537, and 538 of
FokI are all targets for influencing dimerization of the FokI
cleavage half-domains.
[0184] Exemplary engineered cleavage half-domains of FokI that form
obligate heterodimers include a pair in which a first cleavage
half-domain includes mutations at amino acid residues at positions
490 and 538 of FokI and a second cleavage half-domain includes
mutations at amino acid residues 486 and 499.
[0185] Thus, in one embodiment, a mutation at 490 replaces Glu (E)
with Lys (K); the mutation at 538 replaces Iso (I) with Lys (K);
the mutation at 486 replaced Gln (Q) with Glu (E); and the mutation
at position 499 replaces Iso (I) with Lys (K). Specifically, the
engineered cleavage half-domains described herein were prepared by
mutating positions 490 (E.fwdarw.K) and 538 (I.fwdarw.K) in one
cleavage half-domain to produce an engineered cleavage half-domain
designated "E490K:I538K" and by mutating positions 486 (Q.fwdarw.E)
and 499 (I.fwdarw.L) in another cleavage half-domain to produce an
engineered cleavage half-domain designated "Q486E:I499L". The
engineered cleavage half-domains described herein are obligate
heterodimer mutants in which aberrant cleavage is minimized or
abolished. See, e.g., U.S. Patent Publication No. 2008/0131962, the
disclosure of which is incorporated by reference in its entirety
for all purposes. In certain embodiments, the engineered cleavage
half-domain comprises mutations at positions 486, 499 and 496
(numbered relative to wild-type FokI), for instance mutations that
replace the wild type Gln (Q) residue at position 486 with a Glu
(E) residue, the wild type Iso (I) residue at position 499 with a
Leu (L) residue and the wild-type Asn (N) residue at position 496
with an Asp (D) or Glu (E) residue (also referred to as a "ELD" and
"ELE" domains, respectively). In other embodiments, the engineered
cleavage half-domain comprises mutations at positions 490, 538 and
537 (numbered relative to wild-type FokI), for instance mutations
that replace the wild type Glu (E) residue at position 490 with a
Lys (K) residue, the wild type Iso (I) residue at position 538 with
a Lys (K) residue, and the wild-type His (H) residue at position
537 with a Lys (K) residue or a Arg (R) residue (also referred to
as "KKK" and "KKR" domains, respectively). In other embodiments,
the engineered cleavage half-domain comprises mutations at
positions 490 and 537 (numbered relative to wild-type FokI), for
instance mutations that replace the wild type Glu (E) residue at
position 490 with a Lys (K) residue and the wild-type His (H)
residue at position 537 with a Lys (K) residue or a Arg (R) residue
(also referred to as "KIK" and "KIR" domains, respectively. See,
e.g., U.S. Pat. Nos. 7,914,796; 8,034,598 and 8,623,618. In other
embodiments, the engineered cleavage half domain comprises the
"Sharkey" and/or "Sharkey'" mutations (see Guo et al, (2010) J.
Mol. Biol. 400(1):96-107).
[0186] Engineered cleavage half-domains described herein can be
prepared using any suitable method, for example, by site-directed
mutagenesis of wild-type cleavage half-domains (Fok I) as described
in U.S. Patent Publication Nos. 20050064474; 20080131962; and
20110201055.
[0187] Alternatively, nucleases may be assembled in vivo at the
nucleic acid target site using so-called "split-enzyme" technology
(see, e.g. U.S. Patent Publication No. 20090068164). Components of
such split enzymes may be expressed either on separate expression
constructs, or can be linked in one open reading frame where the
individual components are separated, for example, by a
self-cleaving 2A peptide or IRES sequence. Components may be
individual zinc finger binding domains or domains of a meganuclease
nucleic acid binding domain.
[0188] Nucleases can be screened for activity prior to use, for
example in a yeast-based chromosomal system as described in WO
2009/042163 and 20090068164. Nuclease expression constructs can be
readily designed using methods known in the art. See, e.g., United
States Patent Publications 20030232410; 20050208489; 20050026157;
20050064474; 20060188987; 20060063231; and International
Publication WO 07/014275. Expression of the nuclease may be under
the control of a constitutive promoter or an inducible promoter,
for example the galactokinase promoter which is activated
(de-repressed) in the presence of raffinose and/or galactose and
repressed in presence of glucose.
[0189] The Cas9 related CRISPR/Cas system comprises two RNA
non-coding components: tracrRNA and a pre-crRNA array containing
nuclease guide sequences (spacers) interspaced by identical direct
repeats (DRs). To use a CRISPR/Cas system to accomplish genome
engineering, both functions of these RNAs must be present (see Cong
et al, (2013) Sciencexpress 1/10.1126/science 1231143). In some
embodiments, the tracrRNA and pre-crRNAs are supplied via separate
expression constructs or as separate RNAs. In other embodiments, a
chimeric RNA is constructed where an engineered mature crRNA
(conferring target specificity) is fused to a tracrRNA (supplying
interaction with the Cas9) to create a chimeric cr-RNA-tracrRNA
hybrid (also termed a single guide RNA). (see Jinek ibid and Cong,
ibid).
Target Sites
[0190] As described in detail above, DNA domains can be engineered
to bind to any sequence of choice. An engineered DNA-binding domain
can have a novel binding specificity, compared to a
naturally-occurring DNA-binding domain. In certain embodiments, the
DNA-binding domains bind to a sequence within a BCL11A enhancer
sequence, for example a target site (typically 9, 10, 11, 12, 13,
14, 15, 16, 17, 18, 19, 20, 21 or even more base pairs) is between
exon 2 and exon 3 of BCL11A, including DNA-binding domains that
bind to a sequence within a DNAseI hypersensitive site in the
BCL11A enhancer sequence (e.g., +55, +58, +62; see FIG. 11).
Engineering methods include, but are not limited to, rational
design and various types of selection. Rational design includes,
for example, using databases comprising triplet (or quadruplet)
nucleotide sequences and individual zinc finger amino acid
sequences, in which each triplet or quadruplet nucleotide sequence
is associated with one or more amino acid sequences of zinc fingers
which bind the particular triplet or quadruplet sequence. See, for
example, co-owned U.S. Pat. Nos. 6,453,242 and 6,534,261,
incorporated by reference herein in their entireties. Rational
design of TAL-effector domains can also be performed. See, e.g.,
U.S. Publication No. 20110301073.
[0191] Exemplary selection methods applicable to DNA-binding
domains, including phage display and two-hybrid systems, are
disclosed in U.S. Pat. Nos. 5,789,538; 5,925,523; 6,007,988;
6,013,453; 6,410,248; 6,140,466; 6,200,759; and 6,242,568; as well
as WO 98/37186; WO 98/53057; WO 00/27878; WO 01/88197 and GB
2,338,237. In addition, enhancement of binding specificity for zinc
finger binding domains has been described, for example, in co-owned
WO 02/077227.
[0192] Selection of target sites; nucleases and methods for design
and construction of fusion proteins (and polynucleotides encoding
same) are known to those of skill in the art and described in
detail in U.S. Patent Application Publication Nos. 20050064474 and
20060188987, incorporated by reference in their entireties
herein.
[0193] In addition, as disclosed in these and other references,
DNA-binding domains (e.g., multi-fingered zinc finger proteins)
and/or fusions of DNA-binding domain(s) and functional domain(s)
may be linked together using any suitable linker sequences,
including for example, linkers of 5 or more amino acids. U.S. Pat.
Nos. 8,772,453; 7,888,121 (e.g., "ZC" linker); U.S. Pat. Nos.
6,479,626; 6,903,185; and 7,153,949; U.S. Publication Nos.
20090305419 and 20150064789. The proteins described herein may
include any combination of suitable linkers between the individual
DNA-binding domains of the protein. See, also, U.S. Pat. No.
8,586,526.
Donors
[0194] In certain embodiments, the present disclosure relates to
nuclease-mediated targeted integration of an exogenous sequence
into the genome of a cell using the BCL11A enhancer region-binding
molecules described herein. As noted above, insertion of an
exogenous sequence (also called a "donor sequence" or "donor" or
"transgene"), for example for deletion of a specified region and/or
correction of a mutant gene or for increased expression of a
wild-type gene. It will be readily apparent that the donor sequence
is typically not identical to the genomic sequence where it is
placed. A donor sequence can contain a non-homologous sequence
flanked by two regions of homology to allow for efficient HDR at
the location of interest or can be integrated via non-homology
directed repair mechanisms. Additionally, donor sequences can
comprise a vector molecule containing sequences that are not
homologous to the region of interest in cellular chromatin. A donor
molecule can contain several, discontinuous regions of homology to
cellular chromatin, and, for example, lead to a deletion of a
Bcl11a enhancer region (or a fragment thereof) when used as a
substrate for repair of a DBS induced by one of the nucleases
described here. Further, for targeted insertion of sequences not
normally present in a region of interest, said sequences can be
present in a donor nucleic acid molecule and flanked by regions of
homology to sequence in the region of interest.
[0195] Polynucleotides for insertion can also be referred to as
"exogenous" polynucleotides, "donor" polynucleotides or molecules
or "transgenes." The donor polynucleotide can be DNA or RNA,
single-stranded and/or double-stranded and can be introduced into a
cell in linear or circular form. See, e.g., U.S. Patent Publication
Nos. 20100047805 and 20110207221. The donor sequence(s) are
preferably contained within a DNA MC, which may be introduced into
the cell in circular or linear form. If introduced in linear form,
the ends of the donor sequence can be protected (e.g., from
exonucleolytic degradation) by methods known to those of skill in
the art. For example, one or more dideoxynucleotide residues are
added to the 3' terminus of a linear molecule and/or
self-complementary oligonucleotides are ligated to one or both
ends. See, for example, Chang et al. (1987) Proc. Natl. Acad. Sci.
USA84:4959-4963; Nehls et al. (1996) Science 272:886-889.
Additional methods for protecting exogenous polynucleotides from
degradation include, but are not limited to, addition of terminal
amino group(s) and the use of modified internucleotide linkages
such as, for example, phosphorothioates, phosphoramidates, and
O-methyl ribose or deoxyribose residues. If introduced in
double-stranded form, the donor may include one or more nuclease
target sites, for example, nuclease target sites flanking the
transgene to be integrated into the cell's genome. See, e.g., U.S.
Patent Publication No. 20130326645.
[0196] A polynucleotide can be introduced into a cell as part of a
vector molecule having additional sequences such as, for example,
replication origins, promoters and genes encoding antibiotic
resistance. Moreover, donor polynucleotides can be introduced as
naked nucleic acid, as nucleic acid complexed with an agent such as
a liposome or poloxamer, or can be delivered by viruses (e.g.,
adenovirus, AAV, herpesvirus, retrovirus, lentivirus and integrase
defective lentivirus (IDLV)).
[0197] In certain embodiments, the double-stranded donor includes
sequences (e.g., coding sequences, also referred to as transgenes)
greater than 1 kb in length, for example between 2 and 200 kb,
between 2 and 10 kb (or any value therebetween). The
double-stranded donor also includes at least one nuclease target
site, for example. In certain embodiments, the donor includes at
least 2 target sites, for example for a pair of ZFNs or TALENs.
Typically, the nuclease target sites are outside the transgene
sequences, for example, 5' and/or 3' to the transgene sequences,
for cleavage of the transgene. The nuclease cleavage site(s) may be
for any nuclease(s). In certain embodiments, the nuclease target
site(s) contained in the double-stranded donor are for the same
nuclease(s) used to cleave the endogenous target into which the
cleaved donor is integrated via homology-independent methods.
[0198] The donor is generally inserted so that its expression is
driven by the endogenous promoter at the integration site, namely
the promoter that drives expression of the endogenous gene into
which the donor is inserted (e.g., globin, AAVS1, etc.). However,
it will be apparent that the donor may comprise a promoter and/or
enhancer, for example a constitutive promoter or an inducible or
tissue specific promoter.
[0199] The donor molecule may be inserted into an endogenous gene
such that all, some or none of the endogenous gene is expressed. In
other embodiments, the transgene (e.g., with or without globin
encoding sequences) is integrated into any endogenous locus, for
example a safe-harbor locus. See, e.g., US patent publications
20080299580; 20080159996 and 201000218264.
[0200] Furthermore, although not required for expression, exogenous
sequences may also include transcriptional or translational
regulatory sequences, for example, promoters, enhancers,
insulators, internal ribosome entry sites, sequences encoding 2A
peptides and/or polyadenylation signals.
[0201] The transgenes carried on the donor sequences described
herein may be isolated from plasmids, cells or other sources using
standard techniques known in the art such as PCR. Donors for use
can include varying types of topology, including circular
supercoiled, circular relaxed, linear and the like. Alternatively,
they may be chemically synthesized using standard oligonucleotide
synthesis techniques. In addition, donors may be methylated or lack
methylation. Donors may be in the form of bacterial or yeast
artificial chromosomes (BACs or YACs).
[0202] The double-stranded donor polynucleotides described herein
may include one or more non-natural bases and/or backbones. In
particular, insertion of a donor molecule with methylated cytosines
may be carried out using the methods described herein to achieve a
state of transcriptional quiescence in a region of interest.
[0203] The exogenous (donor) polynucleotide may comprise any
sequence of interest (exogenous sequence). Exemplary exogenous
sequences include, but are not limited to any polypeptide coding
sequence (e.g., cDNAs), promoter sequences, enhancer sequences,
epitope tags, marker genes, cleavage enzyme recognition sites and
various types of expression constructs. Marker genes include, but
are not limited to, sequences encoding proteins that mediate
antibiotic resistance (e.g., ampicillin resistance, neomycin
resistance, G418 resistance, puromycin resistance), sequences
encoding colored or fluorescent or luminescent proteins (e.g.,
green fluorescent protein, enhanced green fluorescent protein, red
fluorescent protein, luciferase), and proteins which mediate
enhanced cell growth and/or gene amplification (e.g., dihydrofolate
reductase). Epitope tags include, for example, one or more copies
of FLAG, His, myc, Tap, HA or any detectable amino acid
sequence.
[0204] In a preferred embodiment, the exogenous sequence
(transgene) comprises a polynucleotide encoding any polypeptide of
which expression in the cell is desired, including, but not limited
to antibodies, antigens, enzymes, receptors (cell surface or
nuclear), hormones, lymphokines, cytokines, reporter polypeptides,
growth factors, and functional fragments of any of the above. The
coding sequences may be, for example, cDNAs.
[0205] For example, the exogenous sequence may comprise a sequence
encoding a polypeptide that is lacking or non-functional in the
subject having a genetic disease, including but not limited to any
of the following genetic diseases: achondroplasia, achromatopsia,
acid maltase deficiency, adenosine deaminase deficiency (OMIM No.
102700), adrenoleukodystrophy, aicardi syndrome, alpha-1
antitrypsin deficiency, alpha-thalassemia, androgen insensitivity
syndrome, apert syndrome, arrhythmogenic right ventricular,
dysplasia, ataxia telangictasia, barth syndrome, beta-thalassemia,
blue rubber bleb nevus syndrome, canavan disease, chronic
granulomatous diseases (CGD), cri du chat syndrome, cystic
fibrosis, dercum's disease, ectodermal dysplasia, fanconi anemia,
fibrodysplasiaossificans progressive, fragile X syndrome,
galactosemis, Gaucher's disease, generalized gangliosidoses (e.g.,
GM1), hemochromatosis, the hemoglobin C mutation in the 6.sup.th
codon of beta-globin (HbC), hemophilia, Huntington's disease,
Hurler Syndrome, hypophosphatasia, Klinefleter syndrome, Krabbes
Disease, Langer-Giedion Syndrome, leukocyte adhesion deficiency
(LAD, OMIM No. 116920), leukodystrophy, long QT syndrome, Marfan
syndrome, Moebius syndrome, mucopolysaccharidosis (MPS), nail
patella syndrome, nephrogenic diabetes insipdius,
neurofibromatosis, Neimann-Pick disease, osteogenesis imperfecta,
porphyria, Prader-Willi syndrome, progeria, Proteus syndrome,
retinoblastoma, Rett syndrome, Rubinstein-Taybi syndrome,
Sanfilippo syndrome, severe combined immunodeficiency (SCID),
Shwachman syndrome, sickle cell disease (sickle cell anemia),
Smith-Magenis syndrome, Stickler syndrome, Tay-Sachs disease,
Thrombocytopenia Absent Radius (TAR) syndrome, Treacher Collins
syndrome, trisomy, tuberous sclerosis, Turner's syndrome, urea
cycle disorder, von Hippel-Landau disease, Waardenburg syndrome,
Williams syndrome, Wilson's disease, Wiskott-Aldrich syndrome,
X-linked lymphoproliferative syndrome (XLP, OMIM No. 308240).
[0206] Additional exemplary diseases that can be treated by
targeted integration include acquired immunodeficiencies, lysosomal
storage diseases (e.g., Gaucher's disease, GM1, Fabry disease and
Tay-Sachs disease), mucopolysaccahidosis (e.g. Hunter's disease,
Hurler's disease), hemoglobinopathies (e.g., sickle cell diseases,
HbC, .alpha.-thalassemia, .beta.-thalassemia) and hemophilias.
[0207] In certain embodiments, the exogenous sequences can comprise
a marker gene (described above), allowing selection of cells that
have undergone targeted integration, and a linked sequence encoding
an additional functionality. Non-limiting examples of marker genes
include GFP, drug selection marker(s) and the like.
[0208] Additional gene sequences that can be inserted may include,
for example, wild-type genes to replace mutated sequences. For
example, a wild-type Factor IX gene sequence may be inserted into
the genome of a stem cell in which the endogenous copy of the gene
is mutated. The wild-type copy may be inserted at the endogenous
locus, or may alternatively be targeted to a safe harbor locus.
[0209] Construction of such expression cassettes, following the
teachings of the present specification, utilizes methodologies well
known in the art of molecular biology (see, for example, Ausubel or
Maniatis). Before use of the expression cassette to generate a
transgenic animal, the responsiveness of the expression cassette to
the stress-inducer associated with selected control elements can be
tested by introducing the expression cassette into a suitable cell
line (e.g., primary cells, transformed cells, or immortalized cell
lines).
[0210] Furthermore, although not required for expression, exogenous
sequences may also transcriptional or translational regulatory
sequences, for example, promoters, enhancers, insulators, internal
ribosome entry sites, sequences encoding 2A peptides and/or
polyadenylation signals. Further, the control elements of the genes
of interest can be operably linked to reporter genes to create
chimeric genes (e.g., reporter expression cassettes).
[0211] Targeted insertion of non-coding nucleic acid sequence may
also be achieved. Sequences encoding antisense RNAs, RNAi, shRNAs
and micro RNAs (miRNAs) may also be used for targeted
insertions.
[0212] In additional embodiments, the donor nucleic acid may
comprise non-coding sequences that are specific target sites for
additional nuclease designs. Subsequently, additional nucleases may
be expressed in cells such that the original donor molecule is
cleaved and modified by insertion of another donor molecule of
interest. In this way, reiterative integrations of donor molecules
may be generated allowing for trait stacking at a particular locus
of interest or at a safe harbor locus.
Delivery
[0213] The nucleases, polynucleotides encoding these nucleases,
donor polynucleotides and compositions comprising the proteins
and/or polynucleotides described herein may be delivered in vivo or
ex vivo by any suitable means into any cell type.
[0214] Suitable cells include eukaryotic (e.g., animal) and
prokaryotic cells and/or cell lines. Non-limiting examples of such
cells or cell lines generated from such cells include COS, CHO
(e.g., CHO-S, CHO-K1, CHO-DG44, CHO-DUXB11, CHO-DUKX, CHOK1SV),
VERO, MDCK, WI38, V79, B14AF28-G3, BHK, HaK, NSO, SP2/0-Ag14, HeLa,
HEK293 (e.g., HEK293-F, HEK293-H, HEK293-T), and perC6 cells as
well as insect cells such as Spodoptera fugiperda (Sf), or fungal
cells such as Saccharomyces, Pichia and Schizosaccharomyces. In
certain embodiments, the cell line is a CHO, MDCK or HEK293 cell
line. Suitable cells also include stem cells such as, by way of
example, embryonic stem cells, induced pluripotent stem cells,
hematopoietic stem cells, neuronal stem cells and mesenchymal stem
cells.
[0215] Methods of delivering nucleases as described herein are
described, for example, in U.S. Pat. Nos. 6,453,242; 6,503,717;
6,534,261; 6,599,692; 6,607,882; 6,689,558; 6,824,978; 6,933,113;
6,979,539; 7,013,219; and 7,163,824, the disclosures of all of
which are incorporated by reference herein in their entireties.
[0216] Nucleases and/or donor constructs as described herein may
also be delivered using vectors containing sequences encoding one
or more of the ZFN(s), TALEN(s) or CRIPSR/Cas systems. Any vector
systems may be used including, but not limited to, plasmid vectors,
retroviral vectors, lentiviral vectors, adenovirus vectors,
poxvirus vectors; herpesvirus vectors and adeno-associated virus
vectors, etc. See, also, U.S. Pat. Nos. 6,534,261; 6,607,882;
6,824,978; 6,933,113; 6,979,539; 7,013,219; and 7,163,824,
incorporated by reference herein in their entireties. Furthermore,
it will be apparent that any of these vectors may comprise one or
more of the sequences needed for treatment. Thus, when one or more
nucleases and a donor construct are introduced into the cell, the
nucleases and/or donor polynucleotide may be carried on the same
vector or on different vectors (DNA MC(s)). When multiple vectors
are used, each vector may comprise a sequence encoding one or
multiple nucleases and/or donor constructs. Conventional viral and
non-viral based gene transfer methods can be used to introduce
nucleic acids encoding nucleases and/or donor constructs in cells
(e.g., mammalian cells) and target tissues. Non-viral vector
delivery systems include DNA or RNA plasmids, DNA MCs, naked
nucleic acid, and nucleic acid complexed with a delivery vehicle
such as a liposome or poloxamer. Suitable non-viral vectors include
nanotaxis vectors, including vectors commercially available from
InCellArt (France). Viral vector delivery systems include DNA and
RNA viruses, which have either episomal or integrated genomes after
delivery to the cell. For a review of in vivo delivery of
engineered DNA-binding proteins and fusion proteins comprising
these binding proteins, see, e.g., Rebar (2004) Expert Opinion
Invest. Drugs 13(7):829-839; Rossi et al. (2007) Nature Biotech.
25(12):1444-1454 as well as general gene delivery references such
as Anderson, Science 256:808-813 (1992); Nabel & Felgner,
TIBTECH 11:211-217 (1993); Mitani & Caskey, TIBTECH 11:162-166
(1993); Dillon, TIBTECH 11:167-175 (1993); Miller, Nature
357:455-460 (1992); Van Brunt, Biotechnology 6(10):1149-1154
(1988); Vigne, Restorative Neurology and Neuroscience 8:35-36
(1995); Kremer & Perricaudet, British Medical Bulletin
51(1):31-44 (1995); Haddada et al., in Current Topics in
Microbiology and Immunology Doerfler and Bohm (eds.) (1995); and Yu
et al., Gene Therapy 1:13-26 (1994).
[0217] Methods of non-viral delivery of nucleic acids include
electroporation, lipofection, microinjection, biolistics,
virosomes, liposomes, immunoliposomes, polycation or lipid:nucleic
acid conjugates, naked DNA, artificial virions, and agent-enhanced
uptake of DNA. Sonoporation using, e.g., the Sonitron 2000 system
(Rich-Mar) can also be used for delivery of nucleic acids.
[0218] Additional exemplary nucleic acid delivery systems include
those provided by Amaxa Biosystems (Cologne, Germany), Maxcyte,
Inc. (Rockville, Md.), BTX Molecular Delivery Systems (Holliston,
Mass.) and Copernicus Therapeutics Inc., (see for example U.S. Pat.
No. 6,008,336). Lipofection is described in e.g., U.S. Pat. Nos.
5,049,386; 4,946,787; and 4,897,355) and lipofection reagents are
sold commercially (e.g., Transfectam.TM. and Lipofectin.TM.).
Cationic and neutral lipids that are suitable for efficient
receptor-recognition lipofection of polynucleotides include those
of Felgner, WO 91/17424, WO 91/16024.
[0219] The preparation of lipid:nucleic acid complexes, including
targeted liposomes such as immunolipid complexes, is well known to
one of skill in the art (see, e.g., Crystal, Science 270:404-410
(1995); Blaese et al., Cancer Gene Ther. 2:291-297 (1995); Behr et
al., Bioconjugate Chem. 5:382-389 (1994); Remy et al., Bioconjugate
Chem. 5:647-654 (1994); Gao et al., Gene Therapy 2:710-722 (1995);
Ahmad et al., Cancer Res. 52:4817-4820 (1992); U.S. Pat. Nos.
4,186,183, 4,217,344, 4,235,871, 4,261,975, 4,485,054, 4,501,728,
4,774,085, 4,837,028, and 4,946,787).
[0220] Additional methods of delivery include the use of packaging
the nucleic acids to be delivered into EnGeneIC delivery vehicles
(EDVs). These EDVs are specifically delivered to target tissues
using bispecific antibodies where one arm of the antibody has
specificity for the target tissue and the other has specificity for
the EDV. The antibody brings the EDVs to the target cell surface
and then the EDV is brought into the cell by endocytosis. Once in
the cell, the contents are released (see MacDiarmid et al (2009)
Nature Biotechnology 27(7):643).
[0221] The use of RNA or DNA viral based systems for the delivery
of nucleic acids encoding engineered ZFPs, TALEs and/or CRISPR/Cas
systems take advantage of highly evolved processes for targeting a
virus to specific cells in the body and trafficking the viral
payload to the nucleus. Viral vectors can be administered directly
to patients (in vivo) or they can be used to treat cells in vitro
and the modified cells are administered to patients (ex vivo).
Conventional viral based systems for the delivery of ZFPs include,
but are not limited to, retroviral, lentivirus, adenoviral,
adeno-associated, vaccinia and herpes simplex virus vectors for
gene transfer. Integration in the host genome is possible with the
retrovirus, lentivirus, and adeno-associated virus gene transfer
methods, often resulting in long term expression of the inserted
transgene. Additionally, high transduction efficiencies have been
observed in many different cell types and target tissues.
[0222] The tropism of a retrovirus can be altered by incorporating
foreign envelope proteins, expanding the potential target
population of target cells. Lentiviral vectors are retroviral
vectors that are able to transduce or infect non-dividing cells and
typically produce high viral titers. Selection of a retroviral gene
transfer system depends on the target tissue. Retroviral vectors
are comprised of cis-acting long terminal repeats with packaging
capacity for up to 6-10 kb of foreign sequence. The minimum
cis-acting LTRs are sufficient for replication and packaging of the
vectors, which are then used to integrate the therapeutic gene into
the target cell to provide permanent transgene expression. Widely
used retroviral vectors include those based upon murine leukemia
virus (MuLV), gibbon ape leukemia virus (GaLV), Simian
Immunodeficiency virus (SW), human immunodeficiency virus (HIV),
and combinations thereof (see, e.g., Buchscher et al., J. Virol.
66:2731-2739 (1992); Johann et al., J. Virol. 66:1635-1640 (1992);
Sommerfelt et al., Virol. 176:58-59 (1990); Wilson et al., J.
Virol. 63:2374-2378 (1989); Miller et al., J. Virol. 65:2220-2224
(1991); PCT/US94/05700).
[0223] In applications in which transient expression is preferred,
adenoviral based systems can be used. Adenoviral based vectors are
capable of very high transduction efficiency in many cell types and
do not require cell division. With such vectors, high titer and
high levels of expression have been obtained. This vector can be
produced in large quantities in a relatively simple system.
Adeno-associated virus ("AAV") vectors are also used to transduce
cells with target nucleic acids, e.g., in the in vitro production
of nucleic acids and peptides, and for in vivo and ex vivo gene
therapy procedures (see, e.g., West et al., Virology 160:38-47
(1987); U.S. Pat. No. 4,797,368; WO 93/24641; Kotin, Human Gene
Therapy 5:793-801 (1994); Muzyczka, J. Clin. Invest. 94:1351
(1994). Construction of recombinant AAV vectors are described in a
number of publications, including U.S. Pat. No. 5,173,414;
Tratschin et al., Mol. Cell. Biol. 5:3251-3260 (1985); Tratschin,
et al., Mol. Cell. Biol. 4:2072-2081 (1984); Hermonat &
Muzyczka, PNAS 81:6466-6470 (1984); and Samulski et al., J. Virol.
63:03822-3828 (1989).
[0224] At least six viral vector approaches are currently available
for gene transfer in clinical trials, which utilize approaches that
involve complementation of defective vectors by genes inserted into
helper cell lines to generate the transducing agent.
[0225] pLASN and MFG-S are examples of retroviral vectors that have
been used in clinical trials (Dunbar et al., Blood 85:3048-305
(1995); Kohn et al., Nat. Med. 1:1017-102 (1995); Malech et al.,
PNAS 94:22 12133-12138 (1997)). PA317/pLASN was the first
therapeutic vector used in a gene therapy trial. (Blaese et al.,
Science 270:475-480 (1995)). Transduction efficiencies of 50% or
greater have been observed for MFG-S packaged vectors. (Ellem et
al., Immunol Immunother. 44(1):10-20 (1997); Dranoff et al., Hum.
Gene Ther. 1:111-2 (1997).
[0226] Recombinant adeno-associated virus vectors (rAAV) are a
promising alternative gene delivery systems based on the defective
and nonpathogenic parvovirus adeno-associated type 2 virus. All
vectors are derived from a plasmid that retains only the AAV 145 bp
inverted terminal repeats flanking the transgene expression
cassette. Efficient gene transfer and stable transgene delivery due
to integration into the genomes of the transduced cell are key
features for this vector system. (Wagner et al., Lancet 351:9117
1702-3 (1998), Kearns et al., Gene Ther. 9:748-55 (1996)). Other
AAV serotypes, including AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7,
AAV8, AAV9 and AAVrh.10 and any novel AAV serotype can also be used
in accordance with the present invention.
[0227] Replication-deficient recombinant adenoviral vectors (Ad)
can be produced at high titer and readily infect a number of
different cell types. Most adenovirus vectors are engineered such
that a transgene replaces the Ad E1a, E1b, and/or E3 genes;
subsequently the replication defective vector is propagated in
human 293 cells that supply deleted gene function in trans. Ad
vectors can transduce multiple types of tissues in vivo, including
nondividing, differentiated cells such as those found in liver,
kidney and muscle. Conventional Ad vectors have a large carrying
capacity. An example of the use of an Ad vector in a clinical trial
involved polynucleotide therapy for antitumor immunization with
intramuscular injection (Sterman et al., Hum. Gene Ther. 7:1083-9
(1998)). Additional examples of the use of adenovirus vectors for
gene transfer in clinical trials include Rosenecker et al.,
Infection 24:1 5-10 (1996); Sterman et al., Hum. Gene Ther. 9:7
1083-1089 (1998); Welsh et al., Hum. Gene Ther. 2:205-18 (1995);
Alvarez et al., Hum. Gene Ther. 5:597-613 (1997); Topf et al., Gene
Ther. 5:507-513 (1998); Sterman et al., Hum. Gene Ther. 7:1083-1089
(1998).
[0228] Packaging cells are used to form virus particles that are
capable of infecting a host cell. Such cells include 293 cells,
which package adenovirus, and w2 cells or PA317 cells, which
package retrovirus. Viral vectors used in gene therapy are usually
generated by a producer cell line that packages a nucleic acid
vector into a viral particle. The vectors typically contain the
minimal viral sequences required for packaging and subsequent
integration into a host (if applicable), other viral sequences
being replaced by an expression cassette encoding the protein to be
expressed. The missing viral functions are supplied in trans by the
packaging cell line. For example, AAV vectors used in gene therapy
typically only possess inverted terminal repeat (ITR) sequences
from the AAV genome which are required for packaging and
integration into the host genome. Viral DNA is packaged in a cell
line, which contains a helper plasmid encoding the other AAV genes,
namely rep and cap, but lacking ITR sequences. The cell line is
also infected with adenovirus as a helper. The helper virus
promotes replication of the AAV vector and expression of AAV genes
from the helper plasmid. The helper plasmid is not packaged in
significant amounts due to a lack of ITR sequences. Contamination
with adenovirus can be reduced by, e.g., heat treatment to which
adenovirus is more sensitive than AAV.
[0229] In many gene therapy applications, it is desirable that the
gene therapy vector be delivered with a high degree of specificity
to a particular tissue type. Accordingly, a viral vector can be
modified to have specificity for a given cell type by expressing a
ligand as a fusion protein with a viral coat protein on the outer
surface of the virus. The ligand is chosen to have affinity for a
receptor known to be present on the cell type of interest. For
example, Han et al., Proc. Natl. Acad. Sci. USA 92:9747-9751
(1995), reported that Moloney murine leukemia virus can be modified
to express human heregulin fused to gp70, and the recombinant virus
infects certain human breast cancer cells expressing human
epidermal growth factor receptor. This principle can be extended to
other virus-target cell pairs, in which the target cell expresses a
receptor and the virus expresses a fusion protein comprising a
ligand for the cell-surface receptor. For example, filamentous
phage can be engineered to display antibody fragments (e.g., FAB or
Fv) having specific binding affinity for virtually any chosen
cellular receptor. Although the above description applies primarily
to viral vectors, the same principles can be applied to nonviral
vectors. Such vectors can be engineered to contain specific uptake
sequences which favor uptake by specific target cells.
[0230] Gene therapy vectors can be delivered in vivo by
administration to an individual patient, typically by systemic
administration (e.g., intravenous, intraperitoneal, intramuscular,
subdermal, or intracranial infusion) or topical application, as
described below. Alternatively, vectors can be delivered to cells
ex vivo, such as cells explanted from an individual patient (e.g.,
lymphocytes, bone marrow aspirates, tissue biopsy) or universal
donor hematopoietic stem cells, followed by reimplantation of the
cells into a patient, usually after selection for cells which have
incorporated the vector.
[0231] Vectors (e.g., retroviruses, adenoviruses, liposomes, etc.)
containing nucleases and/or donor constructs can also be
administered directly to an organism for transduction of cells in
vivo. Alternatively, naked DNA can be administered. Administration
is by any of the routes normally used for introducing a molecule
into ultimate contact with blood or tissue cells including, but not
limited to, injection, infusion, topical application and
electroporation. Suitable methods of administering such nucleic
acids are available and well known to those of skill in the art,
and, although more than one route can be used to administer a
particular composition, a particular route can often provide a more
immediate and more effective reaction than another route.
[0232] Vectors suitable for introduction of polynucleotides (e.g.
nuclease-encoding and/or double-stranded donors) described herein
include non-integrating lentivirus vectors (IDLV). See, for
example, Ory et al. (1996) Proc. Natl. Acad. Sci. USA
93:11382-11388; Dull et al. (1998) J. Virol. 72:8463-8471; Zuffery
et al. (1998) J. Virol. 72:9873-9880; Follenzi et al. (2000) Nature
Genetics 25:217-222; U.S. Patent Publication No 2009/0117617.
[0233] Pharmaceutically acceptable carriers are determined in part
by the particular composition being administered, as well as by the
particular method used to administer the composition. Accordingly,
there is a wide variety of suitable formulations of pharmaceutical
compositions available, as described below (see, e.g., Remington's
Pharmaceutical Sciences, 17th ed., 1989).
[0234] It will be apparent that the nuclease-encoding sequences and
donor constructs can be delivered using the same or different
systems. For example, the nucleases and donors can be carried by
the same DNA MC. Alternatively, a donor polynucleotide can be
carried by a MC, while the one or more nucleases can be carried by
a standard plasmid or AAV vector. Furthermore, the different
vectors can be administered by the same or different routes
(intramuscular injection, tail vein injection, other intravenous
injection, intraperitoneal administration and/or intramuscular
injection. The vectors can be delivered simultaneously or in any
sequential order.
[0235] Thus, the instant disclosure includes in vivo or ex vivo
treatment of diseases and conditions that are amenable to insertion
of a transgenes encoding a therapeutic protein. The compositions
are administered to a human patient in an amount effective to
obtain the desired concentration of the therapeutic polypeptide in
the serum or the target organ or cells. Administration can be by
any means in which the polynucleotides are delivered to the desired
target cells. For example, both in vivo and ex vivo methods are
contemplated. Intravenous injection to the portal vein is a
preferred method of administration. Other in vivo administration
modes include, for example, direct injection into the lobes of the
liver or the biliary duct and intravenous injection distal to the
liver, including through the hepatic artery, direct injection in to
the liver parenchyma, injection via the hepatic artery, and/or
retrograde injection through the biliary tree. Ex vivo modes of
administration include transduction in vitro of resected
hepatocytes or other cells of the liver, followed by infusion of
the transduced, resected hepatocytes back into the portal
vasculature, liver parenchyma or biliary tree of the human patient,
see e.g., Grossman et al., (1994) Nature Genetics, 6:335-341.
[0236] The effective amount of nuclease(s) and donor to be
administered will vary from patient to patient and according to the
therapeutic polypeptide of interest. Accordingly, effective amounts
are best determined by the physician administering the compositions
and appropriate dosages can be determined readily by one of
ordinary skill in the art. After allowing sufficient time for
integration and expression (typically 4-15 days, for example),
analysis of the serum or other tissue levels of the therapeutic
polypeptide and comparison to the initial level prior to
administration will determine whether the amount being administered
is too low, within the right range or too high. Suitable regimes
for initial and subsequent administrations are also variable, but
are typified by an initial administration followed by subsequent
administrations if necessary. Subsequent administrations may be
administered at variable intervals, ranging from daily to annually
to every several years. One of skill in the art will appreciate
that appropriate immunosuppressive techniques may be recommended to
avoid inhibition or blockage of transduction by immunosuppression
of the delivery vectors, see e.g., Vilquin et al., (1995) Human
Gene Ther., 6:1391-1401.
[0237] Formulations for both ex vivo and in vivo administrations
include suspensions in liquid or emulsified liquids. The active
ingredients often are mixed with excipients which are
pharmaceutically acceptable and compatible with the active
ingredient. Suitable excipients include, for example, water,
saline, dextrose, glycerol, ethanol or the like, and combinations
thereof. In addition, the composition may contain minor amounts of
auxiliary substances, such as, wetting or emulsifying agents, pH
buffering agents, stabilizing agents or other reagents that enhance
the effectiveness of the pharmaceutical composition.
Cells
[0238] Also described herein are cells and/or cell lines in which
an endogenous BCL11A enhancer sequence is modified. The
modification may be, for example, as compared to the wild-type
sequence of the cell. The cell or cell lines may be heterozygous or
homozygous for the modification. The modifications to the BCL11A
sequence may comprise insertions, deletions and/or combinations
thereof.
[0239] The BCL11A enhancer sequence may be modified by a nuclease
(e.g., ZFN, TALEN, CRISPR/Cas system, Ttago system, etc.), for
example a nuclease as described herein. In certain embodiments, the
BCL11A enhancer is modified anywhere between exon 2 and exon 3. In
other embodiments, the BCL11A enhancer is modified in the regions
shown in SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:3 (FIG. 11). The
modification is preferably at or near the nuclease(s) binding
and/or cleavage site(s), for example, within 1-300 (or any value
therebetween) base pairs upstream or downstream of the site(s) of
cleavage, more preferably within 1-100 base pairs (or any value
therebetween) of either side of the binding and/or cleavage
site(s), even more preferably within 1 to 50 base pairs (or any
value therebetween) on either side of the binding and/or cleavage
site(s). In certain embodiments, the modification is at or near the
"+58" region of the BCL11A enhancer, for example, at or near a
nuclease binding site shown in any of SEQ ID NOs:4 to 80 and 276.
In other embodiments, the modification is at or near the "+55"
region of the BCL11A enhancer, for example, at or near a nuclease
site shown in any of SEQ ID NOs:143 to 184 and 232-251.
[0240] Any cell or cell line may be modified, for example a stem
cell, for example an embryonic stem cell, an induced pluripotent
stem cell, a hematopoietic stem cell, a neuronal stem cell and a
mesenchymal stem cell. Other non-limiting examples of cells as
described herein include T-cells (e.g., CD4+, CD3+, CD8+, etc.);
dendritic cells; B-cells. A descendent of a stem cell, including a
partially or fully differentiated cell, is also provided (e.g., a
RBC or RBC precursor cell). Non-limiting examples other cell lines
including a modified BCL11A sequence include COS, CHO (e.g.,
CHO--S, CHO-K1, CHO-DG44, CHO-DUXB11, CHO-DUKX, CHOK1SV), VERO,
MDCK, WI38, V79, B14AF28-G3, BHK, HaK, NSO, SP2/0-Ag14, HeLa,
HEK293 (e.g., HEK293-F, HEK293-H, HEK293-T), and perC6 cells as
well as insect cells such as Spodoptera fugiperda (Sf), or fungal
cells such as Saccharomyces, Pichia and Schizosaccharomyces.
[0241] The cells as described herein are useful in treating and/or
preventing a disorder, for example, by ex vivo therapies. The
nuclease-modified cells can be expanded and then reintroduced into
the patient using standard techniques. See, e.g., Tebas et al
(2014) New Eng J Med 370(10):901. In the case of stem cells, after
infusion into the subject, in vivo differentiation of these
precursors into cells expressing the functional transgene also
occurs. Pharmaceutical compositions comprising the cells as
described herein are also provided. In addition, the cells may be
cryopreserved prior to administration to a patient.
[0242] Any of the modified cells or cell lines disclosed herein may
show increased expression of gamma globin. Compositions such as
pharmaceutical compositions comprising the genetically modified
cells as described herein are also provided
Applications
[0243] The methods and compositions disclosed herein are for
modifying expression of protein, or correcting an aberrant gene
sequence that encodes a protein expressed in a genetic disease,
such as a sickle cell disease or a thalassemia. Thus, the methods
and compositions provide for the treatment and/or prevention of
such genetic diseases. Genome editing, for example of stem cells,
can be used to correct an aberrant gene, insert a wild type gene,
or change the expression of an endogenous gene. By way of
non-limiting example, a wild type gene, e.g. encoding at least one
globin (e.g., .alpha. and/or .beta. globin), may be inserted into a
cell (e.g., into an endogenous BCL11a enhancer sequence using one
or more nucleases as described herein) to provide the globin
proteins deficient and/or lacking in the cell and thereby treat a
genetic disease, e.g., a hemoglobinopathy, caused by faulty globin
expression. Alternatively or in addition, genomic editing with or
without administration of the appropriate donor, can correct the
faulty endogenous gene, e.g., correcting the point mutation in
.alpha.- or .beta.-hemoglobin, to restore expression of the gene
and/or treat a genetic disease, e.g. sickle cell disease and/or
knock out or alteration (overexpression or repression) of any
direct or indirect globin regulatory gene (e.g. inactivation of the
.gamma. globin-regulating gene BCL11A or the BCL11A-regulator
KLF1). Specifically, the methods and compositions of the invention
have use in the treatment or prevention of hemoglobinopathies.
[0244] The nucleases of the invention are targeted to the BCL11A
enhancer region, known to be required for the expression of BCL11A,
and hence the down regulation of gamma globin expression.
Modification of this enhancer region may result in erythrocytes
with increased gamma globin expression, and thus may be helpful for
the treatment or prevention of sickle cell disease or beta
thalassemia.
[0245] The following Examples relate to exemplary embodiments of
the present disclosure in which the nuclease comprises a zinc
finger nuclease (ZFN) or TALEN. It will be appreciated that this is
for purposes of exemplification only and that other nucleases can
be used, for example TtAgo and CRISPR/Cas systems, homing
endonucleases (meganucleases) with engineered DNA-binding domains
and/or fusions of naturally occurring of engineered homing
endonucleases (meganucleases) DNA-binding domains and heterologous
cleavage domains and/or fusions of meganucleases and TALE
proteins.
EXAMPLES
Example 1: Assembly of Zinc Finger Nucleases and TALEN
Nucleases
[0246] ZFNs were assembled against the human BCL11A gene and were
tested by CEL1 assays as described in Miller et al. (2007) Nat.
Biotechnol. 25:778-785. TALENs were assembled as described in
Miller et al (2011) Nature Biotechnology 29 (2): 143-151.
Additionally, see co-owned U.S. Patent Publication No. 20140093913
and U.S. Pat. No. 8,586,526. The TALENs were assembled with the +63
architecture.
Example 2: Introduction of Deletions in the +55, +58 and +62 BCL11A
Enhancer Regions
[0247] To test which regions of the BCL11A intron 2 (FIG. 1)
enhancer region were required for repression of gamma globin in
during erythropoiesis, a series of TALENs were made to target
sections of these regions (FIG. 2). The TALEN pairs are shown below
in Table 1. Nucleotides in the target site that are contacted by
the nuclease are indicated in uppercase letters; non-contacted
nucleotides indicated in lowercase.
TABLE-US-00001 TABLE 1 TALENs targeted to the BCL11A enhancer
region # of SEQ ID Sample SBS# target 5'.fwdarw.3' RVDs NO:
N.fwdarw.C RVD Sequence 55R 102740 ctACATAGAGGCCCTTCCTgc 17 276
NI-HD-NI-NG-NI-NN-NI-NN-NN-HD- HD-HD-NG-NG-HD-HD-NG 102741
gtGGAGGGGATAACTGGGTca 17 4 NN-NN-NI-NN-NN-NN-NN-NI-NG-
NI-NI-HD-NG-NN-NN-NN-NG 55M 102736 ttGTGTGCTTGGTCGGCACtg 17 5
NN-NG-NN-NG-NN-HD-NG-NG-NN- NN-NG-HD-NN-NN-HD-NI-HD 102737
gtGCCGACAACTCCCTACCgc 17 6 NN-HD-HD-NN-NI-HD-NI-NI-HD-
NG-HD-HD-HD-NG-NI-HD-HD 58R 102756 gtGCCGACAACTCCCTACCgc 17 6
HD-NG-NG-NN-NN-NG-NN-NI-NG- NN-NN-NI-NN-NI-NI-NG-NG 102757
atTATTTCATTCCCATTGAga 17 7 NG-NI-NG-NG-NG-HD-NI-NG-NG-
HD-HD-HD-NI-NG-NG-NN-NI 58M 102752 atAGGCCAGAAAAGAGATAtg 17 8
NI-NN-NN-HD-HD-NI-NN-NI-NI-NI- NI-NN-NI-NN-NI-NG-NI 102753
ctGGTGTGTTATGTCTAAGag 17 9 NN-NN-NG-NN-NG-NN-NG-NG-NI-
NG-NN-NG-HD-NG-NI-NI-NK 58L 102750 ctAGTTTATAGGGGGTTCTac 17 10
NI-NN-NG-NG-NG-NI-NG-NI-NN- NN-NN-NN-NN-NG-NG-HD-NG 102751
atAGCACCCAAGGTCCATCag 17 11 NI-NN-HD-NI-HD-HD-HD-NI-NI-NN-
NN-NG-HD-HD-NI-NG-HD 62R 102775 atTCAACAAATAGCATATAaa 17 12
NG-HD-NI-NI-HD-NI-NI-NI-NG-NI- NN-HD-NI-NG-NI-NG-NI 102774
ctTCCCTTTTAGGAAGGTAaa 17 13 NG-HD-HD-HD-NG-NG-NG-NG-NI-
NN-NN-NI-NI-NN-NN-NG-NI 62L 102795 atGCCAGAGGGCAGCAAACat 17 14
NN-HD-HD-NI-NN-NI-NN-NN-NN- HD-NI-NN-HD-NI-NI-NI-HD 102794
ctTAATAGCTGAAGGGGGCca 17 15 NG-NI-NI-NG-NI-NN-HD-NG-NN-NI-
NI-NN-NN-NN-NN-NN-HD
[0248] Human K562 cells were cultured in DMEM supplemented with 10%
FBS and 200,000 cells were transfected with 800 ng of plasmid DNA
encoding the TALENs by Amaxa Nucleofector.RTM. following the
manufacturer's instructions. The Cel-I assay (Surveyor.TM.,
Transgenomics) as described in Perez et al. (2008) Nat. Biotechnol.
26: 808-816 and Guschin et al. (2010) Methods Mol Biol.
649:247-56), was used to detect TALEN-induced modifications of the
target gene. In this assay, PCR-amplification of the target site
was followed by quantification of insertions and/or deletions
(indels) using the mismatch detecting enzyme Cel-I (Yang et al.
(2000) Biochemistry 39: 3533-3541) which provided a lower-limit
estimate of DSB frequency. Deep sequencing on the Illumina platform
("miSEQ") was used according to the manufacturer's instructions to
measure editing efficiency as well as nature of editing-generated
alleles. To detect deletions following cell treatment with more
than one nuclease pair, a PCR-based assay was used in which bulk
genomic DNA is amplified with primers that flank the region to be
deleted, and a gel is used to separate the PCR product derived from
the wild-type allele and the one derived from the deletion-bearing
allele. All designs shown in Table 1 were active.
[0249] Three days following transfection of the TALEN expression
vector at standard conditions (37.degree. C.) genomic DNA was
isolated from K562 cells using the DNeasy kit (Qiagen) or
QuickExtract (Epicentre) and subject to PCR amplification.
[0250] The results from the Cel-I assay demonstrated that the
TALENs were capable of inducing cleavage at their respective target
sites.
[0251] To test the effect on relative gamma globin expression, the
mRNAs encoding the TALEN pairs were introduced into CD34+ cells
(obtained from healthy donor volunteers) by BTX nucleofection
according to manufacturer's instructions. The cells were then
differentiated into erythrocytes. Briefly, CD34+ cells were
purified using Ficoll-Paque (GE Healthcare) and CD34.sup.+
microbeads (MiltenyiBiotec) according to the manufacturers'
instructions. CD34.sup.+ cells were cultured in Iscove's MDM with
BIT 95000 (StemCell Technologies) in the presence of growth
factors. Cells were differentiated toward the erythroid lineage
using a 3 step liquid culture model. During the first 6 days (first
phase), CD34.sup.+ cells were expanded with SCF (100 ng/ml), Flt3-L
(100 ng/ml), and IL-3 (20 ng/ml). Expanded cells were then
committed and differentiated toward the erythroid lineage (second
phase) with Epo (2 U/ml) and SCF (50 ng/ml). See, Giarratana et al.
(2011) Blood 118(19):5071-9.
[0252] To analyze relative gamma globin expression, the ratios of
mRNAs encoding gamma globin and beta globin following TALEN
treatment were determined at 14 days following TALEN introduction
by Taqman.RTM. analysis. The analysis was done by standard
Taqman.RTM. analysis, following the protocol and using gene
specific assays supplied by the manufacturer (Applied Biosystems)
and the primer sets supplied. The relative levels of gamma globin
were normalized by the level of alpha or beta globin expression
where the ratio was compared to the alpha/beta or gamma/beta ratio
in untreated cells. The results (FIGS. 3, 4 and 5) demonstrated
that deletions of regions within the +58 and +55 BCL11A DNAseI
hypersensitive site resulted in an increase in the relative levels
of gamma globin expression in these experiments.
Example 3: TALEN "Walk Across" the +58 DNAse I Hypersensitive Site
in BCL11A
[0253] To further define the area required for enhancer activity in
the +58 region, a series of TALENs were made to create a series of
DSBs and deletions across this stretch of DNA. The TALENs used in
this experiment are shown below in Table 2.
TABLE-US-00002 TABLE 2 TALENs used in the +58 enhancer walk # of
SEQ ID Sample SBS# target 5'.fwdarw.3' RVDs NO: N.fwdarw.C RVD
Sequence 1 102830 gtGTGCATAAGTAAGAGCAga 17 16
NN-NG-NN-HD-NI-NG-NI-NI- NN-NG-NI-NI-NN-NI-NN-HD- NI 102831
ctGTATGGACTTTGCACTGga 17 17 NN-NG-NI-NG-NN-NN-NI-HD-
NG-NG-NG-NN-HD-NI-HD-NG- NK 2 102832 gtAAGAGCAGATAGCTGATtc 17 18
NI-NI-NN-NI-NN-HD-NI-NN- NI-NG-NI-NN-HD-NG-NN-NI- NG 102833
atGTTATTACCTGTATGGAct 17 19 NN-NG-NG-NI-NG-NG-NI-HD-
HD-NG-NN-NG-NI-NG-NN-NN- NI 3 102834 atAGCTGATTCCAGTGCAAag 17 20
NI-NN-HD-NG-NN-NI-NG-NG- HD-HD-NI-NN-NG-NN-HD-NI- NI 102835
ttTTCTGGCCTATGTTATTac 17 21 NG-NG-HD-NG-NN-NN-HD-
HD-NG-NI-NG-NN-NG-NG-NI- NG-NG 4 102836 gtGCAAAGTCCATACAGGTaa 17 22
NN-HD-NI-NI-NI-NN-NG-HD- HD-NI-NG-NI-HD-NI-NN-NN- NG 102837
atGCCATATCTCTTTTCTGgc 17 23 NN-HD-HD-NI-NG-NI-NG-HD-
NG-HD-NG-NG-NG-NG-HD- NG-NK 5 102838 atACAGGTAATAACATAGGcc 17 24
NI-HD-NI-NN-NN-NG-NI-NI- NG-NI-NI-HD-NI-NG-NI-NN-NK 102839
ctAAGAGTAGATGCCATATct 17 25 NI-NI-NN-NI-NN-NG-NI-NN-
NI-NG-NN-HD-HD-NI-NG-NI- NG 6 102840 atAACATAGGCCAGAAAAGag 17 26
NI-NI-HD-NI-NG-NI-NN-NN- HD-HD-NI-NN-NI-NI-NI-NI-NK 102841
gtGTTATGTCTAAGAGTAGat 17 27 NN-NG-NG-NI-NG-NN-NG-HD-
NG-NI-NI-NN-NI-NN-NG-NI- NK 7 102842 ctCTTAGACATAACACACCag 17 28
HD-NG-NG-NI-NN-NI-HD-NI- NG-NI-NI-HD-NI-HD-NI-HD-HD 102843
ctAGACTAGCTTCAAAGTTgt 17 29 NI-NN-NI-HD-NG-NI-NN-HD-
NG-NG-HD-NI-NI-NI-NN-NG- NG 8 102844 atAACACACCAGGGTCAATac 17 30
NI-NI-HD-NI-HD-NI-HD-HD-NI- NN-NN-NN-NG-HD-NI-NI-NG 102845
gtTAGCTTGCACTAGACTAgc 17 31 NG-NI-NN-HD-NG-NG-NN-HD-
NI-HD-NG-NI-NN-NI-HD-NG- NI 9 102846 gtCAATACAACTTTGAAGCta 17 32
HD-NI-NI-NG-NI-HD-NI-NI-HD- NG-NG-NG-NN-NI-NI-NN-HD 102847
atAAAAGCAACTGTTAGCtt 17 33 NI-NI-NI-NI-NN-HD-NI-NI-HD-
NG-NN-NG-NG-NI-NN-HD 10 102848 ttGAAGCTAGTCTAGTGCAag 17 34
NN-NI-NI-NN-HD-NG-NI-NN- NG-HD-NG-NI-NN-NG-NN-HD- NI 102849
ctGGAGCCTGTGATAAAAGca 17 35 NN-NN-NI-NN-HD-HD-NG-NN-
NG-NN-NI-NG-NI-NI-NI-NI-NK 11 102850 ctAGTCTAGTGCAAGCTAac 17 36
NI-NN-NG-HD-NG-NI-NN-NG- NN-HD-NI-NI-NN-HD-NG-NI 102851
ctTCCTGGAGCCTGTGATAaa 17 37 NG-HD-HD-NG-NN-NN-NI-NN-
HD-HD-NG-NN-NG-NN-NI-NG- NI 12 102852 gtGCAAGCTAACAGTTGCTtt 17 38
NN-HD-NI-NI-NN-HD-NG-NI- NI-HD-NI-NN-NG-NG-NN-HD- NG 102853
atCAGAGGCCAAACCCTTCct 17 39 HD-NI-NN-NI-NN-NN-HD-HD-
NI-NI-NI-HD-HD-HD-NG-NG- HD 13 102854 ctAACAGTTGCTTTTATCAca 17 40
NI-NI-HD-NI-NN-NG-NG-NN- HD-NG-NG-NG-NG-NI-NG-HD- NI 102855
ctAATCAGAGGCCAAACCCtt 17 41 NI-NI-NG-HD-NI-NN-NI-NN-
NN-HD-HD-NI-NI-NI-HD-HD- HD 14 102856 atCACAGGCTCCAGGAAGGgt 17 42
HD-NI-HD-NI-NN-NN-HD-NG- HD-HD-NI-NN-NN-NI-NI-NN- NK 102857
ctACCCCACCCACGCCCCCac 17 43 NI-HD-HD-HD-HD-NI-HD-HD-
HD-NI-HD-NN-HD-HD-HD-HD- HD 15 102858 ctCCAGGAAGGGTTTGGCCtc 17 44
HD-HD-NI-NN-NN-NI-NI-NN- NN-NN-NG-NG-NG-NN-NN- HD-HD 102859
ctACCCCACCCACGCCCCCac 17 45 NI-HD-HD-HD-HD-NI-HD-HD-
HD-NI-HD-NN-HD-HD-HD-HD- HD 16 102860 ttGGCCTCTGATTAGGGTGgg 17 46
NN-NN-HD-HD-NG-HD-NG- NN-NI-NG-NG-NI-NN-NN-NN- NG-NK 102861
ctGCCAGTCCTCTTCTACCcc 17 47 NN-HD-HD-NI-NN-NG-HD-HD-
NG-HD-NG-NG-HD-NG-NI-HD- HD 17 102862 atTAGGGTGGGGGCGTGGGtg 17 48
NG-NI-NN-NN-NN-NG-NN- NN-NN-NN-NN-HD-NN-NG- 102863
atGGAGAGGTCTGCCAGTCct 17 49 NN-NN-NK NN-NN-NI-NN-NI-NN-NN-NG-
HD-NG-NN-HD-HD-NI-NN-NG- HD 18 102864 gtGGGGTAGAAGAGGACTGgc 17 50
NN-NN-NN-NN-NG-NI-NN-NI- NI-NN-NI-NN-NN-NI-HD-NG- NK 102865
ctGGGCAAACGGCCACCGAtg 17 51 NN-NN-NN-HD-NI-NI-NI-HD-
NN-NN-HD-HD-NI-HD-HD-NN- NI 19 102866 ctGGCAGACCTCTCCATCGgt 17 52
NN-NN-HD-NI-NN-NI-HD-HD- NG-HD-NG-HD-HD-NI-NG-HD- NK 102867
ctTCCGAAAGAGGCCCCCCtg 17 53 NG-HD-HD-NN-NI-NI-NI-NN-
NI-NN-NN-HD-HD-HD-HD-HD- HD 20 102868 atCGGTGGCCGTTTGCCCag 16 54
HD-NN-NN-NG-NN-NN-HD- HD-NN-NG-NG-NG-HD 102869
atCACCAAGAGAGCCTTCCga 17 55 HD-NI-HD-HD-NI-NI-NN-NI-
NN-NI-NN-HD-HD-NG-NG-HD- HD 21 102870 gtTTGCCCAGGGGGGCCTCtt 17 56
NG-NG-NN-HD-HD-HD-NI-NN- NN-NN-NN-NN-NN-HD-HD- NG-HD 102871
atTCTCCATCACCAAGAGAgc 17 57 NG-HD-NG-HD-HD-NI-NG-HD-
NI-HD-HD-NI-NI-NN-NI-NN-NI 22 102872 ttGCCCAGGGGGGCCTCTTtc 17 58
NN-HD-HD-HD-NI-NN-NN-NN- NN-NN-NN-HD-HD-NG-HD- NG-NG 102873
atAAAATCCAATTCTCCATca 17 59 NI-NI-NI-NI-NG-HD-HD-NI-NI-
NG-NG-HD-NG-HD-HD-NI-NG 23 102874 ctTTCGGAAGGCTCTCTTGgt 17 60
NG-NG-HD-NN-NN-NI-NI-NN- NN-HD-NG-HD-NG-HD-NG- NG-NK 102875
atTGAGAAATAAAATCCAAtt 17 61 NG-NN-NI-NN-NI-NI-NI-NG-
NI-NI-NI-NI-NG-HD-HD-NI-NI 58R 102756 ctCTTGGTGATGGAGAATTgg 17 62
HD-NG-NG-NN-NN-NG-NN-NI- NG-NN-NN-NI-NN-NI-NI-NG- NG 102757
atTATTTCATTCCCATTGAga 17 7 NG-NI-NG-NG-NG-HD-NI-NG-
NG-HD-HD-HD-NI-NG-NG-NN- NI 58M 102752 atAGGCCAGAAAAGAGATAtg 17 8
NI-NN-NN-HD-HD-NI-NN-NI- NI-NI-NI-NN-NI-NN-NI-NG-NI 102753
ctGGTGTGTTATGTCTAAGag 17 9 NN-NN-NG-NN-NG-NN-NG-
NG-NI-NG-NN-NG-HD-NG-NI- NI-NK 58L 102750 ctAGTTTATAGGGGGTTCTac 17
10 NI-NN-NG-NG-NG-NI-NG-NI- NN-NN-NN-NN-NN-NG-NG- HD-NG 102751
atAGCACCCAAGGTCCATCag 17 11 NI-NN-HD-NI-HD-HD-HD-NI-
NI-NN-NN-NG-HD-HD-NI-NG- HD
[0254] In this table, `Sample` refers to the samples shown in FIG.
6. The results demonstrate that the TALEN pair 102853/102852
(indicated by the arrow in the figure) was able to increase
relative gamma expression. Further, large deletions introduced by
some pair sets of the TALENs (Sample 24: pair from Sample 22+pair
from Sample 6; Sample 25: pair from Sample 16+pair from Sample 6;
Sample 26: pair from Sample 22+pair from Sample 16) were also able
to increase relative gamma expression. The TALENs were engineered
in this study to probe throughout the +58 region (see FIG. 8
depicting the enhancer sequence and the TALEN cleavage sites). All
designs shown in Table 2 were active.
Example 4: ZFNs Targeted to the +58 Enhancer Region of BCL11A
[0255] In parallel to the TALEN pairs described in Example 3, ZFN
pairs were made to target the +58 region. The ZFNs used are shown
below in Table 3. The nucleases are identified by their "SBS"
number, a unique numeric identifier for each protein.
TABLE-US-00003 TABLE 3 ZFN pairs specific for +58 BCL11A enhancer
region SBS # (target site, 5'- Design 3') F1 F2 F3 F4 F5 F6 45796
RSDNLSE TRSPLRN RSDDLTR QKSNLSS QSAHRKN DSSHRTR atGGCTGAAA (SEQ ID
(SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID AGCGATACAG NO: 81) NO: 82)
NO: 83) NO: 84) NO: 85) NO: 86) ggctggct (SEQ ID NO: 63) 45795
DSSDRKK DRSNRTT TNSNRKR QSGDLTR LKDTLRR N/A tcACTACAGA (SEQ ID (SEQ
ID (SEQ ID (SEQ ID (SEQ ID TAACTCCcaa NO: 87) NO: 88) NO: 89) NO:
90) NO: 91) gtcctgtc (SEQ ID NO: 64) 45802 GYCCLRD TSGNLTR QSGDLTR
QRTHLKA QSGALAR QSANRTK caTAAGTAAG (SEQ ID (SEQ ID (SEQ ID (SEQ ID
(SEQ ID (SEQ ID AGCAGATAGC NO: 92) NO: 93) NO: 90) NO: 94) NO: 95)
NO: 96) tgattcca (SEQ ID NO: 65) 45800 DSSDRKK QNAHRKT QSGDLTR
RSDHLSR QQWDRKQ N/A caCCTGGGGC (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ
ID AtAGAGCCag NO: 87) NO: 97) NO: 90) NO: 98) NO: 99) ccctgtat (SEQ
ID NO: 66) 45812 RSDYLSK TSSVRTT TNQNLTV TSGHLSR RSADLTR TNQNRIT
tcCATACAGG (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID
TAATaACATA NO: 100) NO: 101) NO: 102) NO: 103) NO: 104) NO: 105)
Ggccagaa (SEQ ID NO: 67) 45813 QSGALAR RLDWLPM QSGDLTR HKWVLRQ N/A
N/A acTTTGCACT (SEQ ID (SEQ ID (SEQ ID (SEQ ID GGAAtcagct NO: 95)
NO: 106) NO: 90) NO: 107) atctgctc (SEQ ID NO: 68) 45816 GYCCLRD
TSGNLTR QSGDLTR QRTHLKA QSGALAR N/A aaGTAAGAGC (SEQ ID (SEQ ID (SEQ
ID (SEQ ID (SEQ ID AGATAGCtga NO: 92) NO: 93) NO: 90) NO: 94) NO:
95) ttccagtg (SEQ ID NO: 69) 45815 DSSDRKK QNAHRKT LKQNLDA RSAHLSR
RSDVLST DTRNLRA caCACCTGGG (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID
(SEQ ID GCATAGAGCC NO: 87) NO: 97) NO: 108) NO: 109) NO: 110) NO:
111) agccctgt (SEQ ID NO: 70) 45844 LRHHLTR RRDNLHS RSDHLSN DSRSRIN
DRSHLTR QSGTRKT tcACAGGCTC (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID
(SEQ ID CAGGAAGGGT NO: 112) NO: 113) NO: 114) NO: 115) NO: 116) NO:
117) ttggcctc (SEQ ID NO: 71) 45843 DQSNLRA RPYTLRL TGYNLTN TSGSLTR
QHQVLVR QNATRTK aaGCAACTGT (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID
(SEQ ID TAGCTTGCAC NO: 118) NO: 119) NO: 120) NO: 121) NO: 122) NO:
123) tagactag (SEQ ID NO: 72) 45849 TSGSLSR RSDHLTQ QSGHLAR QKGTLGE
QSSDLSR RRDNLHS caCAGGCTCC (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID
(SEQ ID AGGAAGGGTT NO: 124) NO: 125) NO: 126) NO: 127) NO: 128) NO:
113) tggcctct (SEQ ID NO: 73) 45848 DQSNLRA RPYTLRL TGYNLTN TSGSLTR
DQSNLRA AQCCLFH aaAGCAACtG (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID
(SEQ ID TTAGCTTGCA NO: 118) NO: 119) NO: 120) NO: 121) NO: 118) NO:
129) Ctagacta (SEQ ID NO: 74) 45872 QSGALAR RSDHLSR TSGHLSR RSDALAR
DRSHLTR RSDHLSR gtGGGGGCGT (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID
(SEQ ID GGGTGGGGTA NO: 95) NO: 98) NO: 103) NO: 130) NO: 116) NO:
98) gaagagga (SEQ ID NO: 75) 45871 QSNDLSN RSHHLKA RSDNLSE TSSNRKT
N/A N/A ctAATCAGAG (SEQ ID (SEQ ID (SEQ ID (SEQ ID GCCAaaccct NO:
131) NO: 132) NO: 81) NO: 133) tcctggag (SEQ ID NO: 76) 45881
DRSHLTR RSDHLSR RSDNLSE ASKTRKN TSGSLSR QWKSRAR tgGCCGTTtG (SEQ ID
(SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID CCCAGGGGGG NO: 116) NO: 98)
NO: 81) NO: 134) NO: 124) NO: 135) Cctctttc (SEQ ID NO: 77) 45880
DRSALSR QSGDLTR RSDVLSE TSGHLSR RSANLAR RSDALTQ cgATGGAGaG (SEQ ID
(SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID GTCTGCCAGT NO: 136) NO: 90)
NO: 137) NO: 103) NO: 138) NO: 139) Cctcttct (SEQ ID NO: 78) 45889
DRSHLTR RSDHLSR RSDNLSE ASKTRKN N/A N/A ttGCCCAGGG (SEQ ID (SEQ ID
(SEQ ID (SEQ ID GGGCctcttt NO: 116) NO: 98) NO: 81) NO: 134)
cggaaggc (SEQ ID NO: 79) 45888 DRSALSR RSDNLTR QSGHLSR TSGNLTR
DLTTLRK N/A ccACCGATGG (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID
AGAGGTCtgc NO: 136) NO: 140) NO: 141) NO: 93) NO: 142) cagtcctc
(SEQ ID NO: 80) 48117 DQSNLRA RPYTLRL SGYNLEN TSGSLTR DQSNLRA
AQCCLFH aaAGCAACtG (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID
TTAGCTTGCA NO: 118) NO: 119) NO: 253) NO: 121) NO: 118) NO: 129)
Ctagacta (SEQ ID NO: 74) 48037 DQSNLRA RPYTLRL SGYNLEN TSGSLTR
DQSNLRA AQCCLFH aaAGCAACtG (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID
(SEQ ID TTAGCTTGCA NO: 118) NO: 119) NO: 253) NO: 121) NO: 118) NO:
129) Ctagacta (SEQ ID NO: 74)
[0256] These ZFNs were used to cleave the BCL11A +58 enhancer
region in CD34 cells and transfected cells were analyzed for
relative gamma expression after erythrocyte differentiation. One
ZFN pair, 45844/45843 was identified that caused an increase in
relative gamma expression as compared to cells treated with a GFP
transduction control, or cells treated with other ZFNs. FIG. 8
shows that the site targeted by this ZFN pair partially overlap
that targeted by the most active TALEN pair described in Example 3.
Closer inspection of the sequence that is cleaved reveals that it
contains a "GATA-1" consensus site (A/T GATA A/G), known to be one
of the sequences bound by the GATA1 and related transcription
factors. See, e.g., Martin and Orkin (1990) Genes Dev 4:1886-1898;
Fujiwara et al. (2009) Molecular Cell 36:667-681; Tijssen et al.
(2011) Developmental Cell 20:597-609; May et al. (2013) Cell Stem
Cell 13:1-15. All designs shown in Table 3 were active.
[0257] A region comprising the cleavage site was amplified by PCR,
and following amplification, the PCR product was sequenced via
MiSeq high throughput sequencing analysis according to
manufacturer's instructions (Ilumina) for both the ZFN 45843/45844
and TALEN 102852/102853 pairs. The 5 most common genotypes are
shown below in Tables 5a and 5b, show cleavage at or near the
nuclease target sites and reveal a nuclease-mediated loss of the
GATA-1 consensus sequence (in box) in both instances (Table 5a
shows SEQ ID NOS 227-281 from top to bottom; Table 5b shows SEQ ID
NOS 282-286 from top to bottom). The TALEN pair cleaves slightly
downstream of the consensus sequence, potentially resulting in a
lesser incidence of the knock out of this sequence.
TABLE-US-00004 TABLE 5a MiSeq analysis of deletion region for ZFN
45843/45844 pair Length % Seq Vs over- Count Length Amplicon all
Alignment 18669 123 NA 0 53.565 ##STR00001## CTCCAGGAAGG 2187 108
shor- -15 6.275
ACACACCAGGGTCAATACAACTTTGAAGCTAGTCTAGTGAAAGCTAACAG---------------
ter GCTCAAGGAAGG 1415 110 shor- -13 4.060
ACACACCAGGGTCAATACAACTTTGAAGCTAGTCTAGTGCAAGCTAACAGTTGCT----------
ter ---CCAGGAAGG 1374 122 shor- -1 3.942
ACACACCAGGGTCAATACAACTTTGAAGCTAGTCTAGTGCAAGCTAACAGTTGCTTT-ATCACAG
ter GCTCCGGGAAGG 818 118 shor- -5 2.347
ACACACCAGGGTCAATACAACTTTGGAGCTAGTCTAGTGCAAGCTAACAGTTGCC-----CACAG
ter GCTCCAGGAAGG
TABLE-US-00005 TABLE 5b MiSeq analysis of deletion region for TALEN
102852/102853 pair Length % Seq Vs over- Count Length Amplicon all
Alignment 19238 123 NA 0 68.511 ##STR00002## CAGGAAGG 1265 115
shor- -8 4.505
ACACACCAGGGTCAATACAACTTTGAAGCTAGTCTAGTGCAAGCTACCAGTTGCTTTTATC--------
ter CAGGAAGG 893 110 shor- -13 3.180
ACACACCAGGGTCAATACAACTTTGAAGCTAGTCTAGTGCAAGCTACCAGTTGCT-------------C
ter CAGGAAGG 524 121 shor- -2 1.866
ACACACCAGGGTCAATACAACTTTGAAGCTAGTCTAGTGCAAGCTAACAGTTGCTTTCATCA--GGCTC
ter CAGGAAGG 449 122 shor- -1 1.599
ACACACCAGGGTCAATACAACTTTGAAGCTAGTCTAGTGCAAGCTAACAGTTGCTTTTGTC-CAGGCTC
ter CAGGAAGG
Example 5: TALEN Walk Across the +55 DNAseI Hypersensitive Site in
the BCL11A Enhancer Region
[0258] To further refine the area required for enhancer activity in
the +55 region, a series of TALEN pairs were made to create a
series of mutations across this stretch of DNA. The TALEN pairs
made in this experiment are shown below in Table 4.
TABLE-US-00006 TABLE 4 TALEN pairs that recognize the +55 BCL11A
enhancer region # of SEQ ID Sample SBS# target 5'.fwdarw.3' RVDs
NO: N.fwdarw.C RVD Sequence 1 102876 atAATGAATGTCCCAGGCCaa 17 143
NI-NI-NG-NN-NI-NI- NG-NN-NG-HD-HD- HD-NI-NN-NN-HD- HD 102877
ctGCCCCATACCCACTTCcc 16 144 NN-HD-HD-HD-HD- NI-NG-NI-HD-HD-HD-
NI-HD-NG-NG-HD 2 102878 atTCTAGGAAGGGAAGTGGgt 17 145
NG-HD-NG-NI-NN- NN-NI-NI-NN-NN- NN-NI-NI-NN-NG- NN-NK 102879
gtACCAGGAAGGCAATGGGct 17 146 NI-HD-HD-NI-NN-NN- NI-NI-NN-NN-HD-NI-
NI-NG-NN-NN-NK 3 102880 gtGGGTATGGGGCAGCCCAtt 17 147
NN-NN-NN-NG-NI- NG-NN-NN-NN-NN- HD-NI-NN-HD-HD- HD-NI 102881
atTGCATCATCCTGGTACca 16 148 NG-NN-HD-NI-NG- HD-NI-NG-HD-HD-
NG-NN-HD 4 102882 ctTCCTGGTACCAGGATGAtg 17 149 NG-HD-HD-NG-NN-
NN-NG-NI-HD-HD-NI- NN-NN-NI-NG-NN-NI 102883 gtGGGGAGCTCACAGCCTCca
17 150 NN-NN-NN-NN-NI- NN-HD-NG-HD-NI- HD-NI-NN-HD-HD- NG-HD 5
102884 atGATGCAATGCTTGGAGGct 17 151 NN-NI-NG-NN-HD- NI-NI-NG-NN-HD-
NG-NG-NN-NN-NI- NN-NK 102885 gtGTGCCCTGAGAAGGTGGgg 17 152
NN-NG-NN-HD-HD- HD-NG-NN-NI-NN- NI-NI-NN-NN-NG- NN-NK 6 102886
atGCTTGGAGGCTGTGAGCtc 17 153 NN-HD-NG-NG-NN- NN-NI-NN-NN-HD-
NG-NN-NG-NN-NI- NN-HD 102887 atCACAGGGTGTGCCCTGAga 17 154
HD-NI-HD-NI-NN-NN- NN-NG-NN-NG-NN- HD-HD-HD-NG-NN- NI 7 102888
ctCCCCACCTTCTCAGGGCac 17 155 HD-HD-HD-HD-NI- HD-HD-NG-NG-HD-
NG-HD-NI-NN-NN- NN-HD 102889 ctGGACAGAGGGGTCCCACaa 17 156
NN-NN-NI-HD-NI- NN-NI-NN-NN-NN- NN-NG-HD-HD-HD- NI-HD 8 102890
ctTCTCAGGGCACACCCTGtg 17 157 NG-HD-NG-HD-NI- NN-NN-NN-HD-NI-
HD-NI-HD-HD-HD- NG-NK 102891 ctGGGCTGGACAGAGGGGTc 17 158
NN-NN-NN-HD-NG- c NN-NN-NI-HD-NI- NN-NI-NN-NN-NN- NN-NG 9 102892
ctGTGATCTTGTGGGACCcc 16 159 NN-NG-NN-NI-NG- HD-NG-NG-NN-NG-
NN-NN-NN-NI-HD- HD 102893 atGCACACCCAGGCTGGGct 16 160
NN-HD-NI-HD-NI-HD- HD-HD-NI-NN-NN- HD-NG-NN-NN-NK 10 102894
ctTGTGGGACCCCTCTGTCca 17 161 NG-NN-NG-NN-NN- NN-NI-HD-HD-HD-
HD-NG-HD-NG-NN- NG-HD 102895 gtGCCGACCAAGCACACAAga 17 162
NN-HD-HD-NN-NI- HD-HD-NI-NI-NN-HD- NI-HD-NI-HD-NI-NI 11 102896
ctGTCCAGCCCAGCCTGGGtg 17 163 NN-NG-HD-HD-NI- NN-HD-HD-HD-NI-
NN-HD-HD-NG-NN- NN-NK 102897 atCAGTGCCGACCAAGCACac 17 164
HD-NI-NN-NG-NN- HD-HD-NN-NI-HD- HD-NI-NI-NN-HD-NI- HD 12 102898
ctGGGTGTGCATCTTGTGTgc 17 165 NN-NN-NN-NG-NN- NG-NN-HD-NI-NG-
HD-NG-NG-NN-NG- NN-NG 102899 ctACCGCGACCCCTATCAGtg 17 166
NI-HD-HD-NN-HD- NN-NI-HD-HD-HD- HD-NG-NI-NG-HD-NI- NK 13 102902
gtAGGGAGTTGTCGGCACAca 17 167 NI-NN-NN-NN-NI- NN-NG-NG-NN-NG-
HD-NN-NN-HD-NI- HD-NI 102903 ttGGGGACCGCTCACAGGAca 17 168
NN-NN-NN-NN-NI- HD-HD-NN-HD-NG- HD-NI-HD-NI-NN-NN- NI 14 102904
ctGCTGCATGTCCTGTGAgc 16 169 NN-HD-NG-NN-HD- NI-NG-NN-NG-HD-
HD-NG-NN-NG-NN- NI 102905 ctGAAGGCTGGGCACAGCCtt 17 170
NN-NI-NI-NN-NN- HD-NG-NN-NN-NN- HD-NI-HD-NI-NN-HD- HD 15 102906
gtCCCCAAGGCTGTGCCCAgc 17 171 HD-HD-HD-HD-NI-NI- NN-NN-HD-NG-NN-
NG-NN-HD-HD-HD- NI 102907 ctGTCAGAAGAGGCCCTGGac 17 172
NN-NG-HD-NI-NN- NI-NI-NN-NI-NN-NN- HD-HD-HD-NG-NN- NK 16 102912
ttCTGACAGGCCCTGCTGGtt 17 173 HD-NG-NN-NI-HD-NI- NN-NN-HD-HD-HD-
NG-NN-HD-NG-NN- NK 102913 gtGGTGCGTGGAGATAATGcc 17 174
NN-NN-NG-NN-HD- NN-NG-NN-NN-NI- NN-NI-NG-NI-NI-NG- NK 17 102914
ctGCTGGTTATCACTGTTGgc 17 175 NN-HD-NG-NN-NN- NG-NG-NI-NG-HD-NI-
HD-NG-NN-NG-NG- NK 102915 ctGGGCACAGAAGTGGTGCgt 17 176
NN-NN-NN-HD-NI- HD-NI-NN-NI-NI-NN- NG-NN-NN-NG-NN- HD 18 102916
ttGGCATTATCTCCACGCAcc 17 177 NN-NN-HD-NI-NG- NG-NI-NG-HD-NG-
HD-HD-NI-HD-NN- HD-NI 102917 gtGACCCAGCAGCCCTGGGca 17 178
NN-NI-HD-HD-HD- NI-NN-HD-NI-NN-HD- HD-HD-NG-NN-NN- NK 19 102918
atCTCCACGCACCACTTCTgt 17 179 HD-NG-HD-HD-NI- HD-NN-HD-NI-HD-
HD-NI-HD-NG-NG- HD-NG 102919 ctCCTTAAGGTGACCCAGCag 17 180
HD-HD-NG-NG-NI-NI- NN-NN-NG-NN-NI- HD-HD-HD-NI-NN-HD 20 102920
gtGCCCAGGGCTGCTGGGTca 17 181 NN-HD-HD-HD-NI- NN-NN-NN-HD-NG-
NN-HD-NG-NN-NN- NN-NG 102921 ctATGTAGACGGGTGTGTGgc 17 182
NI-NG-NN-NG-NI- NN-NI-HD-NN-NN- NN-NG-NN-NG-NN- NG-NK 21 102922
gtCACCTTAAGGAGCCACAca 17 183 HD-NI-HD-HD-NG- NG-NI-NI-NN-NN-NI-
NN-HD-HD-NI-HD-NI 102923 gtCAGACCCCAAGCAGGAAgg 17 184
HD-NI-NN-NI-HD-HD- HD-HD-NI-NI-NN-HD- NI-NN-NN-NI-NI
[0259] The TALENs were introduced into CD34+ cells as described
above and the cells were induced to differentiate into erythroid
cells as described above. Taqman.RTM. analysis was performed as
described and several sites identified that caused an increase in
relative gamma expression (FIG. 9). The cleavage sites are
displayed in FIG. 10. All designs shown in Table 4 were active.
Interestingly, one of the TALEN pairs which drove an increase in
gamma globin mRNAs cleaves at another GATA-1 consensus sequence
(cleavage site 17 on FIG. 10).
Example 6: ZFNs Directed to the +55 DNAse I Hypersensitive Site in
BCL11A
[0260] Similar to Example 4, a set of ZFNs were made to probe the
+55 DNAse I hypersensitive region. The ZFNs are shown below in
Table 6.
TABLE-US-00007 TABLE 6 +55 enhancer region specific ZFNs: designs
and targets SBS # (target site, 5'- Design 3') F1 F2 F3 F4 F5 F6
46156 TSSNRKT AACNRNA WKCQLPI DRSNLTR RSDHLSQ DSSTRKK (tgGCCTGGG
(SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID ACATTCATTA NO: 133)
NO: 185) NO: 186) NO: 187) NO: 188) NO: 189) Tttagccac; SEQ ID NO:
232) 46158 QSGALAR RKYYLAK RSDNLSV RSAHLSR QSGNLAR ARWSLGK
(tcTAGGAAG (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID
GGAAGTGGGT NO: 95) NO: 190) NO: 191) NO: 109) NO: 192) NO: 193)
Atggggcag; SEQ ID NO: 233) 46163 WKCQLPI DRSNLTR RSDHLSQ DSSTRKK
RPYTLRL QSGNLAR (taGAATTGG (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID
(SEQ ID CCTGGGACAT NO: 186) NO: 187) NO: 188) NO: 189) NO: 119) NO:
192) Tcattattt; SEQ ID NO: 234) 46164 RSAHLSR RSDALTQ TSGHLSR
RSDALAR QSGNLAR RQEHRVA (gaAGGGAAG (SEQ ID (SEQ ID (SEQ ID (SEQ ID
(SEQ ID (SEQ ID TGGGTATGGG NO: 109) NO: 139) NO: 103) NO: 130) NO:
192) NO: 194) Gcagcccat; SEQ ID NO: 235 46180 DRSHLTR QSGNLAR
RSDSLSA DNSNRIK RSDVLSE SPSSRRT (tcATCCTGg (SEQ ID (SEQ ID (SEQ ID
(SEQ ID (SEQ ID (SEQ ID TACCAGGAAG NO: 116) NO: 192) NO: 195) NO:
196) NO: 137) NO: 197) GCaatgggc; SEQ ID NO: 236) 46181 RSDNLAR
WQSSLIV DRSHLTR QSGHLSR QSSDLSR LKWNLRT (gcAATGCTt (SEQ ID (SEQ ID
(SEQ ID (SEQ ID (SEQ ID (SEQ ID GGAGGCTGTG NO: 198) NO: 199) NO:
116) NO: 141) NO: 128) NO: 200) AGctcccca; SEQ ID NO: 237) 46188
PCRYRLD RSANLTR RSDHLSR TSGHLSR QSGNLAR QKPWRTP (ccTGAGAAG (SEQ ID
(SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID GTGGGGAGCT NO: 201) NO:
202) NO: 98) NO: 103) NO: 192) NO: 203) Cacagcctc; SEQ ID NO: 238)
46189 QSSHLTR RSDALAR YRSSLKE TSGNLTR RSDTLSA DKSTRTK (acACCCTGt
(SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID GATCTTGTGG NO: 204)
NO: 130) NO: 205) NO: 93) NO: 206) NO: 207) GAcccctct; SEQ ID NO:
239) 46208 DRSALAR RSDHLSR QGAHLGA QSSHLTR QSSDLTR N/A (ggGCTGGAc
(SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID AGAGGGGTCc NO: 208) NO: 98)
NO: 209) NO: 204) NO: 210) cacaagatc; SEQ ID NO: 240) 46209 RSDSLLR
SASARWW TQSNLRM RNASRTR DRSHLTR RLDWLPM (gcCTGGGTG (SEQ ID (SEQ ID
(SEQ ID (SEQ ID (SEQ ID (SEQ ID TGCATCTTGT NO: 211) NO: 212) NO:
213) NO: 214) NO: 116) NO: 106) Gtgcttggt; SEQ ID NO: 241) 46216
RSDHLSR QGAHLGA QSSHLTR QSSDLTR RSDHLSQ DSSHRTR (caGGCTGGG (SEQ ID
(SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID CTGGAcAGAG NO: 98) NO: 209)
NO: 204) NO: 210) NO: 188) NO: 86) GGgtcccac; SEQ ID NO: 242) 46217
RSDHLSQ RRSDLKR RSDSLLR SASARWW TQSNLRM RNASRTR (gtGTGCATC (SEQ ID
(SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID TTGTGtGCTT NO: 188) NO:
215) NO: 211) NO: 212) NO: 213) NO: 214) GGtcggcac; SEQ ID NO: 243)
46226 RSDNLST DNSNRIN QSGDLTR QSGNLHV DRSDLSR DSSTRRR (gtGCCGACC
(SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID AAGCACACAA NO: 216)
NO: 217) NO: 90) NO: 218) NO: 219) NO: 220) Gatgcacac; SEQ ID NO:
244) 46228 LKQNLDA RSAHLSR QSGALAR RSDDLTR LKQNLDA RSHHLKA
(atAGGGGTc (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID
GCGGTAGGGA NO: 108) NO: 109) NO: 95) NO: 83) NO: 108) NO: 132)
GTtgtcggc; SEQ ID NO: 245) 46229 QSGDLTR QSGNLHV DRSDLSR DSSTRRR
RSDNLSE TSSNRKT (ccTATCAGt (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID
(SEQ ID GCCGACCAAG NO: 90) NO: 218) NO: 219) NO: 220) NO: 81) NO:
133) CAcacaaga; SEQ ID NO: 246) 46230 DRSHLSR DRSALAR TSGSLSR
QAGHLAK QSGALAR RSDDLTR (tcGCGGTAg (SEQ ID (SEQ ID (SEQ ID (SEQ ID
(SEQ ID (SEQ ID GGAGTTGTCG NO: 221) NO: 208) NO: 124) NO: 222) NO:
95) NO: 83) GCacacact; SEQ ID NO: 247) 46240 RSDSLSV QSGDLTR
QSGDLTR TSHNRNA RSDHLSQ DNSNRIN (ctCACAGGa (SEQ ID (SEQ ID (SEQ ID
(SEQ ID (SEQ ID (SEQ ID CATGCAGCAG NO: 223) NO: 90) NO: 90) NO:
224) NO: 188) NO: 217) TGtgtgccg; SEQ ID NO: 248) 46241 DRSNLSS
RSHSLLR QSSDLSR RSDNLSV DNRDRIK N/A (tcCCCAAGG (SEQ ID (SEQ ID (SEQ
ID (SEQ ID (SEQ ID CTGTGCCCag NO: 225) NO: 226) NO: 128) NO: 191)
NO: 227) ccttcagtg; SEQ ID NO: 249) 46246 ASKTRTN RNASRTR RSDNLSV
YSSTRNS QSSDLSR RSDALAR (gtGTGGCTc (SEQ ID (SEQ ID (SEQ ID (SEQ ID
(SEQ ID (SEQ ID CTTAAGGTGA NO: 228) NO: 214) NO: 191) NO: 229) NO:
128) NO: 130) CCcagcagc; SEQ ID NO: 250) 46247 RSDNLAR QSTPRNT
WPDYLPT DRSALAR N/A N/A (ccGTCTACA (SEQ ID (SEQ ID (SEQ ID (SEQ ID
TAGAGgccct NO: 198) NO: 230) NO: 231) NO: 208) tcctgcttg; SEQ ID
NO: 251)
[0261] The +55 region specific ZFNs were tested for cleavage
activity using the Cel-I assay and found to be active in K562
cells. All designs shown in Table 6 were active and the ZFN-induced
gene modification, described as % NHEJ (non-homologous end joining)
found for each pair is listed below in Table 7.
TABLE-US-00008 TABLE 7 Cleavage activity of BCL11A +55 enhancer
region specific ZFN in K562 cells ZFN 1 ZFN 2 (SBS#) (SBS#) % NHEJ
46158 46156 30.36 46164 46163 21.90 46181 46180 15.21 46189 46188
12.76 46209 46208 27.96 46217 46216 16.63 46228 46226 37.79 46230
46229 37.23 46241 46240 24.97 46247 46246 17.30 GFP Transduction
control 0.00
[0262] The CD34+ cells were transfected with the ZFN pairs as
described for Example 4, and then differentiated into erythrocytes
as above. The ZFNs shown in 6 bound to and modified the BCL11A +55
enhancer region.
Example 7: Increasing Activity of +58 Specific ZFN Pairs
[0263] The ZFNs targeting the +58 region were further refined by
shifting the target sequences, altering finger identity and using
alternate linkers between the zinc finger DNA binding domain and
the FokI cleavage domains.
[0264] ZFN pairs were made to target a sequence very close to the
cleavage site of the 45843/45844 ZFN pair. The pairs are shown
below in Table 8, and the location of their binding sites are shown
in FIG. 12A.
TABLE-US-00009 TABLE 8 ZFNs targeting the +58 enhancer region SBS #
(target site, 5'- Design 3') F1 F2 F3 F4 F5 F6 linker 46880 RSDHLTQ
QSGHLAR QKGTLGE QSSDLSR RRDNLHS N/A L7a caCAGGCTC (SEQ ID (SEQ ID
(SEQ ID (SEQ ID (SEQ ID CAGGAAGGg NO: 125) NO: 126) NO: 127) NO:
128) NO: 113) tttggcctc t (SEQ ID NO: 73) 47923 RSDHLTQ QSGHLAR
QKGTLGE QSSDLSR RRDNLHS N/A L0 caCAGGCTC (SEQ ID (SEQ ID (SEQ ID
(SEQ ID (SEQ ID CAGGAAGGg NO: 125) NO: 126) NO: 127) NO: 128) NO:
113) tttggcctc t (SEQ ID NO: 73) 50679 RSDHLTQ QSGHLAR QKGTLGE
QSSDLSR RRDNLHS N/A L0[-1] caCAGGCTC (SEQ ID (SEQ ID (SEQ ID (SEQ
ID (SEQ ID CAGGAAGGg NO: 125) NO: 126) NO: 127) NO: 128) NO: 113)
tttggcctc t (SEQ ID NO: 73) 50680 RSDHLTQ QSGHLAR QKGTLGE QSSDLSR
RRDNLHS N/A L0[-2] caCAGGCTC (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ
ID CAGGAAGGg NO: 125) NO: 126) NO: 127) NO: 128) NO: 113) tttggcctc
t (SEQ ID NO: 73) 46923 QKGTLGE QSGSLTR TGYNLTN TSGSLTR QHQVLVR
QNATRTK L7e4 aaGCAACTG (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ
ID TTAGCttGC NO: 127) NO: 252) NO: 120) NO: 121) NO: 122) NO: 123)
ACTAgacta g (SEQ ID NO: 72) 46999 RSDHLTQ QSGHLAR QKGTLGE QSSDLSR
RRDNLHS N/A L7c5 caCAGGCTC (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID
CAGGAAGGg NO: 125) NO: 126) NO: 127) NO: 128) NO: 113) tttggcctc t
(SEQ ID NO: 73) 45844 LRHHLTR RRDNLHS RSDHLSN DSRSRIN DRSHLTR
QSGTRKT L7a tcACAGGCT (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ
ID CCAGGAAGG NO: 112) NO: 113) NO: 114) NO: 115) NO: 116) NO: 117)
GTttggcct c (SEQ ID NO: 71) 47021 LRHHLTR RRDNLHS RSDHLSN DSRSRIN
DRSHLTR QSGTRKT L7c5 tcACAGGCT (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ
ID (SEQ ID CCAGGAAGG NO: 112) NO: 113) NO: 114) NO: 115) NO: 116)
NO: 117) GTttggcct c (SEQ ID NO: 71) 45843 DQSNLRA RPYTLRL TGYNLTN
TSGSLTR QHQVLVR QNATRTK L7a aaGCAACTG (SEQ ID (SEQ ID (SEQ ID (SEQ
ID (SEQ ID (SEQ ID TTAGCTTGC NO: 118) NO: 119) NO: 120) NO: 121)
NO: 122) NO: 123) ACtagacta g (SEQ ID NO: 72) 46801 DQSNLRA RPYTLRL
SGYNLEN TSGSLTR DQSNLRA AQCCLFH L7a aaAGCAACt (SEQ ID (SEQ ID (SEQ
ID (SEQ ID (SEQ ID (SEQ ID GTTAGCTTG NO: 118) NO: 119) NO: 253) NO:
121) NO: 118) NO: 129) CACtagact a (SEQ ID NO: 74) 46786 DQSNLRA
RPYTLRL SGYNLEN TSGSLTR DQSNLRA AQCCLFH L0 aaAGCAACt (SEQ ID (SEQ
ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID GTTAGCTTG NO: 118) NO: 119) NO:
253) NO: 121) NO: 118) NO: 129) CACtagact a (SEQ ID NO: 74) 46934
DQSNLRA RPYTLRL SGYNLEN TSGSLTR DQSNLRA AQCCLFH L7c5 aaAGCAACt (SEQ
ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID GTTAGCTTG NO: 118) NO:
119) NO: 253) NO: 121) NO: 118) NO: 129) CACtagact a (SEQ ID NO:
74) 46816 DQSNLRA RPYTLRL SGYNLEN TSGSLTR DQSNLRA AQCCLFH L8c4
aaAGCAACt (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID GTTAGCTTG
NO: 118) NO: 119) NO: 253) NO: 121) NO: 118) NO: 129) CACtagact a
(SEQ ID NO: 74) 50670 DQSNLRA RPYTLRL SGYNLEN TSGSLTR DQSNLRA
AQCCLFH L0[+9] aaAGCAACt (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID
(SEQ ID GTTAGCTTG NO: 118) NO: 119) NO: 253) NO: 121) NO: 118) NO:
129) CACtagact a (SEQ ID NO: 74) 50671 DQSNLRA RPYTLRL SGYNLEN
TSGSLTR DQSNLRA AQCCLFH L0[+7] aaAGCAACt (SEQ ID (SEQ ID (SEQ ID
(SEQ ID (SEQ ID (SEQ ID GTTAGCTTG NO: 118) NO: 119) NO: 253) NO:
121) NO: 118) NO: 129) CACtagact a (SEQ ID NO: 74) 50672 DQSNLRA
RPYTLRL SGYNLEN TSGSLTR DQSNLRA AQCCLFH L0[+5] aaAGCAACt (SEQ ID
(SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID GTTAGCTTG NO: 118) NO: 119)
NO: 253) NO: 121) NO: 118) NO: 129) CACtagact a (SEQ ID NO: 74)
48117 DQSNLRA RPYTLRL SGYNLEN TSGSLTR DQSNLRA AQCCLFH L7c5
aaAGCAACt (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID GTTAGCTTG
NO: 118) NO: 119) NO: 253) NO: 121) NO: 118) NO: 129) CACtagact a
(SEQ ID NO: 74) 50674 DQSNLRA RPYTLRL SGYNLEN TSGSLTR DQSNLRA
AQCCLFH L0[+11] aaAGCAACt (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID
(SEQ ID GTTAGCTTG NO: 118) NO: 119) NO: 253) NO: 121) NO: 118) NO:
129) CACtagact a (SEQ ID NO: 74) 50676 DQSNLRA RPYTLRL SGYNLEN
TSGSLTR DQSNLRA AQCCLFH L0[+7] aaAGCAACt (SEQ ID (SEQ ID (SEQ ID
(SEQ ID (SEQ ID (SEQ ID GTTAGCTTG NO: 118) NO: 119) NO: 253) NO:
121) NO: 118) NO: 129) CACtagact a (SEQ ID NO: 74) 48037 DQSNLRA
RPYTLRL SGYNLEN TSGSLTR DQSNLRA AQCCLFH L7a aaAGCAACt (SEQ ID (SEQ
ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID GTTAGCTTG NO: 118) NO: 119) NO:
253) NO: 121) NO: 118) NO: 129) CACtagact a (SEQ ID NO: 74)
[0265] As can be seen in FIG. 12, the binding site of the new pairs
is located one base pair closer to the center of the GATA-1
consensus sequence. All pairs were active for binding and cleaving
their targets in the genome as gauged by an initial screen in K562
cells. mRNAs encoding the ZFNs were electroporated into CD34+ cells
and then the cells were differentiated into the erythroid lineage
as described in Example 2. To analyze relative gamma globin
expression, the ratios of mRNAs encoding gamma globin and beta
globin following ZFN treatment were determined by Taqman.RTM.
analysis at 14 days following ZFN introduction. The results (FIG.
12B) demonstrated that the ZFN pairs targeting the shifted binding
sites had a greater influence on the expression of gamma
globin.
[0266] Next, the proteins were made with an alternate linker types
to test the effect on the Bcl11a proteins. Similar sets of ZFNs
were made that comprised the same helices in the same fingers but
where each contained different linkers between the ZFP DNA binding
domain and the FokI nuclease. For example, ZFNs 46801, 46786, 46816
and 46934 have the same ZFP DNA binding domain, but are linked to
the nuclease domain using the L7a, L0, L8c4 and L7c5 linkers
respectively. Similarly, ZFNs 45844 and 47021 have the same DNA
binding domain, but 45844 has the L7a linker while 47021 uses the
L7c5 linker. In addition, 46880, 47923, 50679 and 50680 have the
same DNA binding domains, but 46880 uses the L7a linker; 47923 has
the L7c5 linker; 50679 uses L0[-1] and 50680 has L0[-3]. The
linkers are shown in FIG. 14 and FIG. 17.
[0267] As shown in FIG. 13, the ZFNs were tested in various pairs
where one set of pairs targeted the binding site typified by the
one targeted by the 45843/45844 pair, and then a second set
typified by the one targeted by 46801/46880. The pairs tested are
shown below in Table 9 as follows along with the percent cleavage
(NHEJ) as measured by sequence analysis at either Day 0 (DO) or Day
14 (D14). Each data point is a mean of three replicates.
TABLE-US-00010 TABLE 9 Effect of Linker on ZFN cleavage activity
HGB/ Target NHEJ, NHEJ, HBA Pair Linkers type D 0 D 14 ratio
45843/45844 L7a/L7a 45843/45844 78% 73% 7.65 45843/47021 L7a/L7c5
45843/45844 82% 72% 7.57 46801/46880 L7a/L7a 46801/46880 87% 64%
8.33 46801/47923 L7a/L0 46801/46880 82% 65% 12.07 46786/46880
L0/L7a 46801/46880 71% 40% 6.06 46934/47923 L7c5/L0 46801/46880 80%
58% 83 GFP 2.71
[0268] Additional tests were designed to measure the number of
indel containing edits that destroyed the GATA site in the target.
Shown in Table 10 below are combinations of ZFNs with various
linkers and the effect the linkers have in increasing the percent
of indels overall and the increase in indels that result in the
loss of the GATA binding site.
TABLE-US-00011 TABLE 10 Exemplary linker activity % indel, no % of
no Left ZFN Right ZFN GATA % GATA/total SBS# Linker Type SBS#
Linker Type left total_indels indel 50670 L0 [+9] 47923 L0 28.0
30.8 0.91 50671 L0 [+7] 24.5 26.7 0.92 50672 L0 [+5] 30.3 33.0 0.92
46801 L7a 27.6 30.4 0.91 46816 L8c4 27.7 29.9 0.93 46934 L7c5 43.8
47.8 0.92 50670 L0 [+9] 50679 L0 [-1] 30.1 32.6 0.92 50671 L0 [+7]
23.3 25.3 0.92 50672 L0 [+5] 31.1 34.2 0.91 46801 L7a 16.1 18.0
0.89 46816 L8c4 33.8 36.2 0.93 46934 L7c5 23.7 25.4 0.94 50670 L0
[+9] 50680 L0 [-2] 16.9 18.1 0.94 50671 L0 [+7] 16.9 17.9 0.94
50672 L0 [+5] 23.2 24.9 0.93 46801 L7a 24.2 26.0 0.93 46816 L8c4
21.3 23.2 0.92 46934 L7c5 25.1 27.0 0.93 46801 47923 16.5 19.1 0.86
GFP 1.4 0.8 1.64
[0269] As can be seen in the table above, refining of the original
pair, 46801 (L7a)/47923 (L0), whose activity was measured in this
experiment to be 19% overall indel formation, with 86% of those
measured indels having a destroyed GATA binding site, can lead to
an overall increased in cleavage (indel) activity, and an overall
increase in the percent of indels that lead to destruction of GATA.
See for example 46934(L7c5)/47923 (L0) where 47.8% total indels
were observed and 92% of those indels had a destroyed GATA site.
These and other linkers described (see FIG. 17) may be incorporated
into the ZFNs to increase and/or refine activity.
[0270] Thus, alternate linkers may be used with the ZFN pairs
described herein to cleave Bcl11a and increase in gamma hemoglobin
relative to alpha hemoglobin.
Example 8: In Vivo Administration
[0271] Compositions including cells (e.g., HSCs and/or RBC
precursor cells), proteins (e.g., nucleases) and/or polynucleotides
(e.g., encoding nucleases) as described herein are administered to
a subject, for example a subject with a hemoglobinopathy,
essentially as described in U.S. Pat. Nos. 7,837,668; 8,092,429;
U.S. Patent Publication No. 20060239966; U.S. Pat. Nos. 6,180,613;
6,503,888 and/or U.S. Pat. Nos. 6,998,118 and 7,101,540 to provide
therapy for a subject in need thereof.
[0272] In addition, the cells are studied for use in large scale
production of edited LT-HSC. Bulk CD34+ cells are pre-stimulated
with cytokines comprising Stemspan.TM. CC110, Flt-3 ligand, SCF,
and TPO and all combinations thereof in concentrations from 10
ng/mL to 1000 ng/mL. Pre-stimulation may require exposure times of
24, up to 48 and up to 72 hours. For clinical-scale HSPC
transfection, any high capacity system may be used (e.g. Maxcyte GT
Flow Transfection System).
[0273] For ex vivo therapies, edited cells (e.g., HSCs) are
subjected to colony forming assays in methylcellulose medium to
confirm the frequency of pluripotent cells and to verify that the
colonies possess the desired genetic editing at the expected
frequencies. The methylcellulose studies are carried out using
methods known in the art (see for example Keller et al (1993) Mol
Cell Bio 13(1):473).
[0274] To further ensure the engraftability of the BLC11a-edited
cells (e.g., HSC), the cells are engrafted into a relevant mouse
model and/or a non-human primate model. Engraftment in these
animals is done according to methods known in the art. See, for
example Holt et al. (2010) Nat Biotech 28, 839-847, Mo et al (2009)
Retrovirology 6:65 and Peterson et al (2013) J. Med Primatol 42:
237. Engraftment with (1020 cGy irradiation) or without (200 cGy
irradiation) myeloablative preconditioning is used to investigate
optimum engraftment and expansion conditions for stem cell
transplantation.
Example 9: In Vivo Administration and Engraftment
[0275] As described above, CD34+ human cells were treated with
mRNAs encoding the +55 enhancer specific ZFNs and then engrafted
into NSG mice. CD34+ cells were obtained from healthy human
volunteers. In some cases, CD34+ mobilization strategies were done,
using either G-CSF (Neupogen.RTM.) or G-CSF+Plerixafor
(Mozobil.RTM.) prior to apheresis. The G-CSF was administered daily
for the four days prior to apheresis according to manufacturer's
instructions, and if Plerixafor was used, it was administered on
the final evening prior to harvest, again according to
manufacturer's instructions. The apheresis was performed by
standard methods. CD34+ cells were enriched from the mobilized PBMC
leukopaks using a Miltenyi CliniMACs system by standard methods and
according to manufacturer's instructions.
[0276] Capped and poly-adenylated mRNAs encoding the ZFNs were
synthesized using Ambion mMessage mMachine.RTM. T7 ultra kit as
instructed by the manufacturer and then electroporated into the
CD34+ cells using either a Maxcyte GT system or a BTX ECM830
electroporator, both according to manufacturer's instructions.
[0277] NOD.Cg-Prkdc.sup.scid Il2rg.sup.tw1Wjl/SzJ mice were used to
receive the CD34+ transplant. One day (16-24 hours) prior to
implantation, the mice were subject to sublethal irradiation (300
RAD). The ZFN-treated CD34+ cells from above were transplanted into
the irradiated mice through a tail vein injection, where 1 million
cells in 0.5 mL PBS-0.1% BSA were given per mouse.
[0278] For this experiment, CD34+ cells were electroporated with
mRNAs encoding either the 45843/45844 pair (electroporation #1) or
the 46801/46880 pair (electroporation #2). In both cases, GFP was
used as a control. Following transplantion into the mice, samples
were taken at either 4 or 16 weeks post-transplant to observe the
level of marking in cells. At week 4, up to approximately 4% of the
cells in the peripheral blood were human cells from both
electroporations (see FIG. 15A). Genome editing (indels) in these
cells was about 30-40% (FIG. 15B).
[0279] At week 16 post transplantation, the level of editing was
measured in human pan-myeloid, B-cells, erythroid and stem cells in
the mice. In these experiments, the cells from both
electroporations were pooled. The data (FIG. 16) indicated that
40-50% gene editing was detected in all human cell populations
analyzed. This experiment demonstrates that transplanted CD34+
cells are maintained and differentiate while maintaining the gene
editing at the BCL11A locus.
[0280] All patents, patent applications and publications mentioned
herein are hereby incorporated by reference in their entirety.
[0281] Although disclosure has been provided in some detail by way
of illustration and example for the purposes of clarity of
understanding, it will be apparent to those skilled in the art that
various changes and modifications can be practiced without
departing from the spirit or scope of the disclosure. Accordingly,
the foregoing descriptions and examples should not be construed as
limiting.
Sequence CWU 1
1
2871366DNAHomo sapiens 1caattctagg aagggaagtg ggtatggggc agcccattgc
cttcctggta ccaggatgat 60gcaatgcttg gaggctgtga gctccccacc ttctcagggc
acaccctgtg atcttgtggg 120acccctctgt ccagcccagc ctgggtgtgc
atcttgtgtg cttggtcggc actgataggg 180gtcgcggtag ggagttgtcg
gcacacactg ctgcatgtcc tgtgagcggt ccccaaggct 240gtgcccagcc
ttcagtgtcc agggcctctt ctgacaggcc ctgctggtta tcactgttgg
300cattatctcc acgcaccact tctgtgccca gggctgctgg gtcaccttaa
ggagccacac 360acccgt 3662553DNAHomo sapiens 2gaggtactga tggaccttgg
gtgctattcc tgtgataagg aaggcagcta gacaggactt 60gggagttatc tgtagtgaga
tggctgaaaa gcgatacagg gctggctcta tgccccaggt 120gtgcataagt
aagagcagat agctgattcc agtgcaaagt ccatacaggt aataacatag
180gccagaaaag agatatggca tctactctta gacataacac accagggtca
atacaacttt 240gaagctagtc tagtgcaagc taacagttgc ttttatcaca
ggctccagga agggtttggc 300ctctgattag ggtgggggcg tgggtggggt
agaagaggac tggcagacct ctccatcggt 360ggccgtttgc ccaggggggc
ctctttcgga aggctctctt ggtgatggag aattggattt 420tatttctcaa
tgggaatgaa ataatttgta tgccatgccg tgtggactcc caaaattgta
480aaggaggtga agcttcccct gtctgcactc tcccctcctc ataattgtcc
atttttcatc 540tgtcgggctg tcc 5533352DNAHomo sapiens 3cgtttttaga
acttagcttt ttgcattgag gatgcgcagg tggctgagac taacttcttt 60gcagatgacc
atggttgaaa gtcagctata gagttgcaca accacgtagt tgggcttcac
120atatagaaga tgttgtcatt ttttggtaac tctgtcagac tttaccaacc
tggcgcacag 180tctggttggc acataaactt cacatttgct cttctccagg
gtgtggggtg gctgtttaaa 240gagggtggat attcatgcta atctttgtgt
agcataacat gttactgcaa cttgcttttt 300tttttttatc tgaaagttca
agtagatatc agaagggaaa tgtttgtggg tg 352421DNAHomo sapiens
4gtggagggga taactgggtc a 21521DNAHomo sapiens 5ttgtgtgctt
ggtcggcact g 21621DNAHomo sapiens 6gtgccgacaa ctccctaccg c
21721DNAHomo sapiens 7attatttcat tcccattgag a 21821DNAHomo sapiens
8ataggccaga aaagagatat g 21921DNAHomo sapiens 9ctggtgtgtt
atgtctaaga g 211021DNAHomo sapiens 10ctagtttata gggggttcta c
211121DNAHomo sapiens 11atagcaccca aggtccatca g 211221DNAHomo
sapiens 12attcaacaaa tagcatataa a 211321DNAHomo sapiens
13cttccctttt aggaaggtaa a 211421DNAHomo sapiens 14atgccagagg
gcagcaaaca t 211521DNAHomo sapiens 15cttaatagct gaagggggcc a
211621DNAHomo sapiens 16gtgtgcataa gtaagagcag a 211721DNAHomo
sapiens 17ctgtatggac tttgcactgg a 211821DNAHomo sapiens
18gtaagagcag atagctgatt c 211921DNAHomo sapiens 19atgttattac
ctgtatggac t 212021DNAHomo sapiens 20atagctgatt ccagtgcaaa g
212121DNAHomo sapiens 21ttttctggcc tatgttatta c 212221DNAHomo
sapiens 22gtgcaaagtc catacaggta a 212321DNAHomo sapiens
23atgccatatc tcttttctgg c 212421DNAHomo sapiens 24atacaggtaa
taacataggc c 212521DNAHomo sapiens 25ctaagagtag atgccatatc t
212621DNAHomo sapiens 26ataacatagg ccagaaaaga g 212721DNAHomo
sapiens 27gtgttatgtc taagagtaga t 212821DNAHomo sapiens
28ctcttagaca taacacacca g 212921DNAHomo sapiens 29ctagactagc
ttcaaagttg t 213021DNAHomo sapiens 30ataacacacc agggtcaata c
213121DNAHomo sapiens 31gttagcttgc actagactag c 213221DNAHomo
sapiens 32gtcaatacaa ctttgaagct a 213320DNAHomo sapiens
33ataaaagcaa ctgttagctt 203421DNAHomo sapiens 34ttgaagctag
tctagtgcaa g 213521DNAHomo sapiens 35ctggagcctg tgataaaagc a
213620DNAHomo sapiens 36ctagtctagt gcaagctaac 203721DNAHomo sapiens
37cttcctggag cctgtgataa a 213821DNAHomo sapiens 38gtgcaagcta
acagttgctt t 213921DNAHomo sapiens 39atcagaggcc aaacccttcc t
214021DNAHomo sapiens 40ctaacagttg cttttatcac a 214121DNAHomo
sapiens 41ctaatcagag gccaaaccct t 214221DNAHomo sapiens
42atcacaggct ccaggaaggg t 214321DNAHomo sapiens 43ctaccccacc
cacgccccca c 214421DNAHomo sapiens 44ctccaggaag ggtttggcct c
214521DNAHomo sapiens 45ctaccccacc cacgccccca c 214621DNAHomo
sapiens 46ttggcctctg attagggtgg g 214721DNAHomo sapiens
47ctgccagtcc tcttctaccc c 214821DNAHomo sapiens 48attagggtgg
gggcgtgggt g 214921DNAHomo sapiens 49atggagaggt ctgccagtcc t
215021DNAHomo sapiens 50gtggggtaga agaggactgg c 215121DNAHomo
sapiens 51ctgggcaaac ggccaccgat g 215221DNAHomo sapiens
52ctggcagacc tctccatcgg t 215321DNAHomo sapiens 53cttccgaaag
aggcccccct g 215420DNAHomo sapiens 54atcggtggcc gtttgcccag
205521DNAHomo sapiens 55atcaccaaga gagccttccg a 215621DNAHomo
sapiens 56gtttgcccag gggggcctct t 215721DNAHomo sapiens
57attctccatc accaagagag c 215821DNAHomo sapiens 58ttgcccaggg
gggcctcttt c 215921DNAHomo sapiens 59ataaaatcca attctccatc a
216021DNAHomo sapiens 60ctttcggaag gctctcttgg t 216121DNAHomo
sapiens 61attgagaaat aaaatccaat t 216221DNAHomo sapiens
62ctcttggtga tggagaattg g 216328DNAHomo sapiens 63atggctgaaa
agcgatacag ggctggct 286428DNAHomo sapiens 64tcactacaga taactcccaa
gtcctgtc 286528DNAHomo sapiens 65cataagtaag agcagatagc tgattcca
286628DNAHomo sapiens 66cacctggggc atagagccag ccctgtat
286728DNAHomo sapiens 67tccatacagg taataacata ggccagaa
286828DNAHomo sapiens 68actttgcact ggaatcagct atctgctc
286928DNAHomo sapiens 69aagtaagagc agatagctga ttccagtg
287028DNAHomo sapiens 70cacacctggg gcatagagcc agccctgt
287128DNAHomo sapiens 71tcacaggctc caggaagggt ttggcctc
287228DNAHomo sapiens 72aagcaactgt tagcttgcac tagactag
287328DNAHomo sapiens 73cacaggctcc aggaagggtt tggcctct
287428DNAHomo sapiens 74aaagcaactg ttagcttgca ctagacta
287528DNAHomo sapiens 75gtgggggcgt gggtggggta gaagagga
287628DNAHomo sapiens 76ctaatcagag gccaaaccct tcctggag
287728DNAHomo sapiens 77tggccgtttg cccagggggg cctctttc
287828DNAHomo sapiens 78cgatggagag gtctgccagt cctcttct
287928DNAHomo sapiens 79ttgcccaggg gggcctcttt cggaaggc
288028DNAHomo sapiens 80ccaccgatgg agaggtctgc cagtcctc
28817PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 81Arg Ser Asp Asn Leu Ser Glu1 5827PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 82Thr
Arg Ser Pro Leu Arg Asn1 5837PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 83Arg Ser Asp Asp Leu Thr
Arg1 5847PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 84Gln Lys Ser Asn Leu Ser Ser1 5857PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 85Gln
Ser Ala His Arg Lys Asn1 5867PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 86Asp Ser Ser His Arg Thr
Arg1 5877PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 87Asp Ser Ser Asp Arg Lys Lys1 5887PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 88Asp
Arg Ser Asn Arg Thr Thr1 5897PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 89Thr Asn Ser Asn Arg Lys
Arg1 5907PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 90Gln Ser Gly Asp Leu Thr Arg1 5917PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 91Leu
Lys Asp Thr Leu Arg Arg1 5927PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 92Gly Tyr Cys Cys Leu Arg
Asp1 5937PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 93Thr Ser Gly Asn Leu Thr Arg1 5947PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 94Gln
Arg Thr His Leu Lys Ala1 5957PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 95Gln Ser Gly Ala Leu Ala
Arg1 5967PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 96Gln Ser Ala Asn Arg Thr Lys1 5977PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 97Gln
Asn Ala His Arg Lys Thr1 5987PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 98Arg Ser Asp His Leu Ser
Arg1 5997PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 99Gln Gln Trp Asp Arg Lys Gln1 51007PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 100Arg
Ser Asp Tyr Leu Ser Lys1 51017PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 101Thr Ser Ser Val Arg Thr
Thr1 51027PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 102Thr Asn Gln Asn Leu Thr Val1
51037PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 103Thr Ser Gly His Leu Ser Arg1
51047PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 104Arg Ser Ala Asp Leu Thr Arg1
51057PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 105Thr Asn Gln Asn Arg Ile Thr1
51067PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 106Arg Leu Asp Trp Leu Pro Met1
51077PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 107His Lys Trp Val Leu Arg Gln1
51087PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 108Leu Lys Gln Asn Leu Asp Ala1
51097PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 109Arg Ser Ala His Leu Ser Arg1
51107PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 110Arg Ser Asp Val Leu Ser Thr1
51117PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 111Asp Thr Arg Asn Leu Arg Ala1
51127PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 112Leu Arg His His Leu Thr Arg1
51137PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 113Arg Arg Asp Asn Leu His Ser1
51147PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 114Arg Ser Asp His Leu Ser Asn1
51157PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 115Asp Ser Arg Ser Arg Ile Asn1
51167PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 116Asp Arg Ser His Leu Thr Arg1
51177PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 117Gln Ser Gly Thr Arg Lys Thr1
51187PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 118Asp Gln Ser Asn Leu Arg Ala1
51197PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 119Arg Pro Tyr Thr Leu Arg Leu1
51207PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 120Thr Gly Tyr Asn Leu Thr Asn1
51217PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 121Thr Ser Gly Ser Leu Thr Arg1
51227PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 122Gln His Gln Val Leu Val Arg1
51237PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 123Gln Asn Ala Thr Arg Thr Lys1
51247PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 124Thr Ser Gly Ser Leu Ser Arg1
51257PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 125Arg Ser Asp His Leu Thr Gln1
51267PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 126Gln Ser Gly His Leu Ala Arg1
51277PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 127Gln Lys Gly Thr Leu Gly Glu1
51287PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 128Gln Ser Ser Asp Leu Ser Arg1
51297PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 129Ala Gln Cys Cys Leu Phe His1
51307PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 130Arg Ser Asp Ala Leu Ala Arg1
51317PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 131Gln Ser Asn Asp Leu Ser Asn1
51327PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 132Arg Ser His His Leu Lys Ala1
51337PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 133Thr Ser Ser Asn Arg Lys Thr1
51347PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 134Ala Ser Lys Thr Arg Lys Asn1
51357PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 135Gln Trp Lys Ser Arg Ala Arg1
51367PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 136Asp Arg Ser Ala Leu Ser Arg1
51377PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 137Arg Ser Asp Val Leu Ser Glu1
51387PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 138Arg Ser Ala Asn Leu Ala Arg1
51397PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 139Arg Ser Asp Ala Leu Thr Gln1
51407PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 140Arg Ser Asp Asn Leu Thr Arg1
51417PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 141Gln Ser Gly His Leu Ser Arg1
51427PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 142Asp Leu Thr Thr Leu Arg Lys1 514321DNAHomo
sapiens 143ataatgaatg tcccaggcca a
2114420DNAHomo sapiens 144ctgccccata cccacttccc 2014521DNAHomo
sapiens 145attctaggaa gggaagtggg t 2114621DNAHomo sapiens
146gtaccaggaa ggcaatgggc t 2114721DNAHomo sapiens 147gtgggtatgg
ggcagcccat t 2114820DNAHomo sapiens 148attgcatcat cctggtacca
2014921DNAHomo sapiens 149cttcctggta ccaggatgat g 2115021DNAHomo
sapiens 150gtggggagct cacagcctcc a 2115121DNAHomo sapiens
151atgatgcaat gcttggaggc t 2115221DNAHomo sapiens 152gtgtgccctg
agaaggtggg g 2115321DNAHomo sapiens 153atgcttggag gctgtgagct c
2115421DNAHomo sapiens 154atcacagggt gtgccctgag a 2115521DNAHomo
sapiens 155ctccccacct tctcagggca c 2115621DNAHomo sapiens
156ctggacagag gggtcccaca a 2115721DNAHomo sapiens 157cttctcaggg
cacaccctgt g 2115821DNAHomo sapiens 158ctgggctgga cagaggggtc c
2115920DNAHomo sapiens 159ctgtgatctt gtgggacccc 2016020DNAHomo
sapiens 160atgcacaccc aggctgggct 2016121DNAHomo sapiens
161cttgtgggac ccctctgtcc a 2116221DNAHomo sapiens 162gtgccgacca
agcacacaag a 2116321DNAHomo sapiens 163ctgtccagcc cagcctgggt g
2116421DNAHomo sapiens 164atcagtgccg accaagcaca c 2116521DNAHomo
sapiens 165ctgggtgtgc atcttgtgtg c 2116621DNAHomo sapiens
166ctaccgcgac ccctatcagt g 2116721DNAHomo sapiens 167gtagggagtt
gtcggcacac a 2116821DNAHomo sapiens 168ttggggaccg ctcacaggac a
2116920DNAHomo sapiens 169ctgctgcatg tcctgtgagc 2017021DNAHomo
sapiens 170ctgaaggctg ggcacagcct t 2117121DNAHomo sapiens
171gtccccaagg ctgtgcccag c 2117221DNAHomo sapiens 172ctgtcagaag
aggccctgga c 2117321DNAHomo sapiens 173ttctgacagg ccctgctggt t
2117421DNAHomo sapiens 174gtggtgcgtg gagataatgc c 2117521DNAHomo
sapiens 175ctgctggtta tcactgttgg c 2117621DNAHomo sapiens
176ctgggcacag aagtggtgcg t 2117721DNAHomo sapiens 177ttggcattat
ctccacgcac c 2117821DNAHomo sapiens 178gtgacccagc agccctgggc a
2117921DNAHomo sapiens 179atctccacgc accacttctg t 2118021DNAHomo
sapiens 180ctccttaagg tgacccagca g 2118121DNAHomo sapiens
181gtgcccaggg ctgctgggtc a 2118221DNAHomo sapiens 182ctatgtagac
gggtgtgtgg c 2118321DNAHomo sapiens 183gtcaccttaa ggagccacac a
2118421DNAHomo sapiens 184gtcagacccc aagcaggaag g
211857PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 185Ala Ala Cys Asn Arg Asn Ala1
51867PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 186Trp Lys Cys Gln Leu Pro Ile1
51877PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 187Asp Arg Ser Asn Leu Thr Arg1
51887PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 188Arg Ser Asp His Leu Ser Gln1
51897PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 189Asp Ser Ser Thr Arg Lys Lys1
51907PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 190Arg Lys Tyr Tyr Leu Ala Lys1
51917PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 191Arg Ser Asp Asn Leu Ser Val1
51927PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 192Gln Ser Gly Asn Leu Ala Arg1
51937PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 193Ala Arg Trp Ser Leu Gly Lys1
51947PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 194Arg Gln Glu His Arg Val Ala1
51957PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 195Arg Ser Asp Ser Leu Ser Ala1
51967PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 196Asp Asn Ser Asn Arg Ile Lys1
51977PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 197Ser Pro Ser Ser Arg Arg Thr1
51987PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 198Arg Ser Asp Asn Leu Ala Arg1
51997PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 199Trp Gln Ser Ser Leu Ile Val1
52007PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 200Leu Lys Trp Asn Leu Arg Thr1
52017PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 201Pro Cys Arg Tyr Arg Leu Asp1
52027PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 202Arg Ser Ala Asn Leu Thr Arg1
52037PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 203Gln Lys Pro Trp Arg Thr Pro1
52047PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 204Gln Ser Ser His Leu Thr Arg1
52057PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 205Tyr Arg Ser Ser Leu Lys Glu1
52067PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 206Arg Ser Asp Thr Leu Ser Ala1
52077PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 207Asp Lys Ser Thr Arg Thr Lys1
52087PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 208Asp Arg Ser Ala Leu Ala Arg1
52097PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 209Gln Gly Ala His Leu Gly Ala1
52107PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 210Gln Ser Ser Asp Leu Thr Arg1
52117PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 211Arg Ser Asp Ser Leu Leu Arg1
52127PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 212Ser Ala Ser Ala Arg Trp Trp1
52137PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 213Thr Gln Ser Asn Leu Arg Met1
52147PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 214Arg Asn Ala Ser Arg Thr Arg1
52157PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 215Arg Arg Ser Asp Leu Lys Arg1
52167PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 216Arg Ser Asp Asn Leu Ser Thr1
52177PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 217Asp Asn Ser Asn Arg Ile Asn1
52187PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 218Gln Ser Gly Asn Leu His Val1
52197PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 219Asp Arg Ser Asp Leu Ser Arg1
52207PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 220Asp Ser Ser Thr Arg Arg Arg1
52217PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 221Asp Arg Ser His Leu Ser Arg1
52227PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 222Gln Ala Gly His Leu Ala Lys1
52237PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 223Arg Ser Asp Ser Leu Ser Val1
52247PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 224Thr Ser His Asn Arg Asn Ala1
52257PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 225Asp Arg Ser Asn Leu Ser Ser1
52267PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 226Arg Ser His Ser Leu Leu Arg1
52277PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 227Asp Asn Arg Asp Arg Ile Lys1
52287PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 228Ala Ser Lys Thr Arg Thr Asn1
52297PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 229Tyr Ser Ser Thr Arg Asn Ser1
52307PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 230Gln Ser Thr Pro Arg Asn Thr1
52317PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 231Trp Pro Asp Tyr Leu Pro Thr1 523228DNAHomo
sapiens 232tggcctggga cattcattat ttagccac 2823328DNAHomo sapiens
233tctaggaagg gaagtgggta tggggcag 2823428DNAHomo sapiens
234tagaattggc ctgggacatt cattattt 2823528DNAHomo sapiens
235gaagggaagt gggtatgggg cagcccat 2823628DNAHomo sapiens
236tcatcctggt accaggaagg caatgggc 2823728DNAHomo sapiens
237gcaatgcttg gaggctgtga gctcccca 2823828DNAHomo sapiens
238cctgagaagg tggggagctc acagcctc 2823928DNAHomo sapiens
239acaccctgtg atcttgtggg acccctct 2824028DNAHomo sapiens
240gggctggaca gaggggtccc acaagatc 2824128DNAHomo sapiens
241gcctgggtgt gcatcttgtg tgcttggt 2824228DNAHomo sapiens
242caggctgggc tggacagagg ggtcccac 2824328DNAHomo sapiens
243gtgtgcatct tgtgtgcttg gtcggcac 2824428DNAHomo sapiens
244gtgccgacca agcacacaag atgcacac 2824528DNAHomo sapiens
245ataggggtcg cggtagggag ttgtcggc 2824628DNAHomo sapiens
246cctatcagtg ccgaccaagc acacaaga 2824728DNAHomo sapiens
247tcgcggtagg gagttgtcgg cacacact 2824828DNAHomo sapiens
248ctcacaggac atgcagcagt gtgtgccg 2824928DNAHomo sapiens
249tccccaaggc tgtgcccagc cttcagtg 2825028DNAHomo sapiens
250gtgtggctcc ttaaggtgac ccagcagc 2825128DNAHomo sapiens
251ccgtctacat agaggccctt cctgcttg 282527PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 252Gln
Ser Gly Ser Leu Thr Arg1 52537PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 253Ser Gly Tyr Asn Leu Glu
Asn1 5254494DNAHomo sapiens 254tatttgctat taaaatttga aaaacaaatg
caggtgccag aggtggctaa ataatgaatg 60tcccaggcca attctaggaa gggaagtggg
tatggggcag cccattgcct tcctggtacc 120aggatgatgc aatgcttgga
ggctgtgagc tccccacctt ctcagggcac accctgtgat 180cttgtgggac
ccctctgtcc agcccagcct gggtgtgcat cttgtgtgct tggtcggcac
240tgataggggt cgcggtaggg agttgtcggc acacactgct gcatgtcctg
tgagcggtcc 300ccaaggctgt gcccagcctt cagtgtccag ggcctcttct
gacaggccct gctggttatc 360actgttggca ttatctccac gcaccacttc
tgtgcccagg gctgctgggt caccttaagg 420agccacacac ccgtctacat
agaggccctt cctgcttggg gtctgaccca gttatcccct 480ccacacctcc attc
49425559DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 255agtctagtgc aagctaacag ttgcttttat
cacaggctcc aggaagggtt tggcctctg 5925612PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 256Gly
Ser Gln Leu Val Lys Ser Glu Leu Glu Glu Lys1 5 1025736DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotideCDS(1)..(36) 257gga tcc cag ctg gtg aag agc gag ctg
gag gag aag 36Gly Ser Gln Leu Val Lys Ser Glu Leu Glu Glu Lys1 5
1025819PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 258Gly Ser Gln Leu Val Lys Ser Lys Ser Glu Ala
Ala Ala Arg Glu Leu1 5 10 15Glu Glu Lys25957DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotideCDS(1)..(57) 259gga tcc cag ctg gtg aag agc aag agc
gag gcc gct gcc cgc gag ctg 48Gly Ser Gln Leu Val Lys Ser Lys Ser
Glu Ala Ala Ala Arg Glu Leu1 5 10 15gag gag aag 57Glu Glu
Lys26018PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 260Gly Ser Ile Ser Arg Ala Arg Pro Leu Asn Pro
His Pro Glu Leu Glu1 5 10 15Glu Lys26154DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotideCDS(1)..(54) 261gga tcc atc agc aga gcc aga cca ctg
aac ccg cac ccg gag ctg gag 48Gly Ser Ile Ser Arg Ala Arg Pro Leu
Asn Pro His Pro Glu Leu Glu1 5 10 15gag aag 54Glu
Lys26219PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 262Gly Ser Tyr Ala Pro Met Pro Pro Leu Ala Leu
Ala Ser Pro Glu Leu1 5 10 15Glu Glu Lys26357DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotideCDS(1)..(57) 263gga tcc tac gct cca atg cca ccc ctg
gct ctg gct tcc cca gag ctg 48Gly Ser Tyr Ala Pro Met Pro Pro Leu
Ala Leu Ala Ser Pro Glu Leu1 5 10 15gag gag aag 57Glu Glu
Lys264677DNAHomo sapiens 264cacaacaggc agagaatgtc tgcaccccac
cctggaaaac agcctgactg tgccccatgg 60gcaaaccaga ctagtttata gggggttcta
ctctgaggta ctgatggacc ttgggtgcta 120ttcctgtgat aaggaaggca
gctagacagg acttgggagt tatctgtagt gagatggctg 180aaaagcgata
cagggctggc tctatgcccc aggtgtgcat aagtaagagc agatagctga
240ttccagtgca aagtccatac aggtaataac ataggccaga aaagagatat
ggcatctact 300cttagacata acacaccagg gtcaatacaa ctttgaagct
agtctagtgc aagctaacag 360ttgcttttat cacaggctcc aggaagggtt
tggcctctga ttagggtggg ggcgtgggtg 420gggtagaaga ggactggcag
acctctccat cggtggccgt ttgcccaggg gggcctcttt 480cggaaggctc
tcttggtgat ggagaattgg attttatttc tcaatgggaa tgaaataatt
540tgtatgccat gccgtgtgga ctcccaaaat tgtaaaggag gtgaagcttc
ccctgtctgc 600actctcccct cctcataatt gtccattttt catctgtcgg
gctgtccacc catccatcac 660atataggcac ctatcag 67726529PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 265Leu
Arg Gly Ser Gln Phe Val Ile Pro Asn Arg Gly Val Thr Lys Gln1 5 10
15Leu Val Lys Ser Glu Leu Glu Glu Lys Lys Ser Glu Leu 20
2526627PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 266Leu Arg Gly Ser Val Ile Pro Asn Arg Gly Val
Thr Lys Gln Leu Val1 5 10 15Lys Ser Glu Leu Glu Glu Lys Lys Ser Glu
Leu 20 2526725PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 267Leu Arg Gly Ser Pro Asn Arg Gly Val
Thr Lys Gln Leu Val Lys Ser1 5 10 15Glu Leu Glu Glu Lys Lys Ser Glu
Leu 20 2526823PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 268Leu Arg Gly Ser Arg Gly Val Thr Lys
Gln Leu Val Lys Ser Glu Leu1 5 10 15Glu Glu Lys Lys Ser Glu Leu
2026921PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 269Leu
Arg Gly Ser Val Thr Lys Gln Leu Val Lys Ser Glu Leu Glu Glu1 5 10
15Lys Lys Ser Glu Leu 2027025PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 270Leu Arg Gly Ser Gln Leu
Val Lys Ser Lys Ser Glu Ala Ala Ala Arg1 5 10 15Glu Leu Glu Glu Lys
Lys Ser Glu Leu 20 2527125PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 271Leu Arg Gly Ser Tyr Ala
Pro Met Pro Pro Leu Ala Leu Ala Ser Pro1 5 10 15Glu Leu Glu Glu Lys
Lys Ser Glu Leu 20 2527224PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 272Leu Arg Gly Ser Ile Ser
Arg Ala Arg Pro Leu Asn Pro His Pro Glu1 5 10 15Leu Glu Glu Lys Lys
Ser Glu Leu 2027318PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 273Leu Arg Gly Ser Gln Leu Val Lys Ser
Glu Leu Glu Glu Lys Lys Ser1 5 10 15Glu Leu27417PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 274Leu
Arg Gly Ser Leu Val Lys Ser Glu Leu Glu Glu Lys Lys Ser Glu1 5 10
15Leu27516PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 275Leu Arg Gly Ser Val Lys Ser Glu Leu Glu Glu
Lys Lys Ser Glu Leu1 5 10 1527621DNAHomo sapiens 276ctacatagag
gcccttcctg c 2127778DNAArtificial SequenceDescription of Artificial
Sequence Synthetic polynucleotide 277acacaccagg gtcaatacaa
ctttgaagct agtctagtgc aagctaacag ttgcttttat 60cacaggctcc aggaaggg
7827862DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 278acacaccagg gtcaatacaa ctttgaagct
agtctagtga aagctaacag gctcaaggaa 60gg 6227964DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 279acacaccagg gtcaatacaa ctttgaagct agtctagtgc
aagctaacag ttgctccagg 60aagg 6428076DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 280acacaccagg gtcaatacaa ctttgaagct agtctagtgc
aagctaacag ttgctttatc 60acaggctccg ggaagg 7628172DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 281acacaccagg gtcaatacaa ctttggagct agtctagtgc
aagctaacag ttgcccacag 60gctccaggaa gg 7228277DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 282acacaccagg gtcaatacaa ctttgaagct agtctagtgc
aagctaacag ttgcttttat 60cacaggctcc aggaagg 7728369DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 283acacaccagg gtcaatacaa ctttgaagct agtctagtgc
aagctaccag ttgcttttat 60ccaggaagg 6928464DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 284acacaccagg gtcaatacaa ctttgaagct agtctagtgc
aagctaccag ttgctccagg 60aagg 6428575DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 285acacaccagg gtcaatacaa ctttgaagct agtctagtgc
aagctaacag ttgctttcat 60caggctccag gaagg 7528676DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 286acacaccagg gtcaatacaa ctttgaagct agtctagtgc
aagctaacag ttgcttttgt 60ccaggctcca ggaagg
762879PRTUnknownDescription of Unknown cleavage site 287Leu Ala Gly
Leu Ile Asp Ala Asp Gly1 5
* * * * *